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COMMUNAUTE FRANCAISE DE BELGIQUE ACADEMIE UNIVERSITAIRE WALLONIE-EUROPE
UNIVERSITE DE L IEGE – GEMBLOUX AGRO-BIO TECH
“ Rhizosphere-driven Lipopeptide Production by Different
Strains of Bacillus spp. as Mechanism Involved in
Biological Control of Plant Pathogens”
Venant NIHORIMBERE
Dissertation originale présentée en vue de l’obtention du grade de Docteur en sciences agronomiques et ingénierie biologique
Promoteurs: Pr. Philippe THONART
Dr. Marc ONGENA
2010
COMMUNAUTE FRANCAISE DE BELGIQUE ACADEMIE UNIVERSITAIRE WALLONIE-EUROPE
UNIVERSITE DE L IEGE – GEMBLOUX AGRO-BIO TECH
“ Rhizosphere-driven Lipopeptide Production by Different
Strains of Bacillus spp. as Mechanism Involved in
Biological Control of Plant Pathogens”
Venant NIHORIMBERE
Dissertation originale présentée en vue de l’obtention du grade de Docteur en sciences agronomiques et ingénierie biologique
Promoteurs: Pr. Philippe THONART
Dr. Marc ONGENA
2010
Copyright. Aux termes de la loi belge du 30 juin 1994, sur le droit d'auteur et les droits voisins, seul l'auteur a le droit de reproduire partiellement ou complètement cet ouvrage de quelque façon et forme que ce soit ou d'en autoriser la reproduction partielle ou complète de quelque manière et sous quelque forme que ce soit. Toute photocopie ou reproduction sous autre forme est donc faite en violation de la dite loi et de des modifications ultérieures.
Members of the jury
Mme Marianne SINDIC (president), ULg, Gbx AGRO-BIO TECH
M. Philippe THONART (promotor), ULg, Gbx AGRO-BIO TECH
M. Marc ONGENA (co-promotor), ULg, Gbx AGRO-BIO TECH
M. Daniel PORTETELLE, ULg, Gbx AGRO-BIO TECH
M. Georges LOGNAY, ULg, Gbx AGRO-BIO TECH
Mme Magali DELEU, ULg, Gbx AGRO-BIO TECH
M. Philippe JACQUES, Université des Sciences et Technologies de Lille, France
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Summary/ Résumé Nihorimbere Venant (2010). “Rhizosphere-driven lipopeptide production by different strains of Bacillus spp. as mechanism involved in biological control of plant pathogens” (Ph.D. Thesis). Belgium, University of Liège - Gembloux Agro-Bio Tech, 151 p., 9 Tabl., 28 Fig. Summary: Some plant-beneficial bacteria with biopesticide potential produce antimicrobial compounds that are tightly involved in multitrophic interactions occurring in the phytosphere among which antagonism toward a diverse range of phytopathogens. However, there is a crucial need for an accurate assessment of antibiotic production rate in this environment colonized by these bacteria. In this context, we conducted the present thesis work with the scope to better understand how two different Bacillus genus strains produce lipopeptides in the rhizosphere of plants. In the first part, one of the strains (B. amyloliquefaciens S499) was selected out of other isolates for its technological traits and in vitro inhibition growth activity of plant pathogenic fungi. We further demonstrated its biocontrol potential in tomato open-field experiments where plantings have been devastated by a local fungus preliminary identified as Fusarium semitectum. In a second part, we combined two mass spectrometry-based approaches (electrospray ionization and imaging) to analyze the pattern of surfactin, iturin and fengycin lipopeptide families produced in planta by strain S499. Our results show that rhizosphere conditions are conducive for surfactin synthesis but not for other types of lipopeptides and that the lipopeptide pattern can be markedly influenced by nutritional factors, biofilm formation and oxygen availability. In a last part, surfactin gene expression (srfA) level was evaluated in situ on tomato root using the reporter gene (LacZ) inserted in B. subtilis strain BGS3. Results showed effective expression of srfA and production of surfactin in biologically important level quantities upon establishment of bacterial population on roots. Our results also demonstrate that BGS3 developing in colonies, efficiently utilizes the main substrates from plant exudates to produce surfactins. The production may also be favored in bacteria growing slowly in the rhizosphere. Globally, this work contributes to better appreciate the impact of some environmental factors on the in situ biosynthesis of lipopeptides by strains of Bacillus which is probably an essential step for improving the level and reliability of their efficacy as biological agents for the control of plant diseases. Nihorimbere Venant (2010). “ Production des lipopeptides dans la rhizosphère par Bacillus en tant que mécanisme impliqué dans le contrôle biologique des agents pathogènes des plantes”(Thèse de Doctorat en anglais). Belgique, Université de Liège - Gembloux Agro-Bio Tech, 151 p., 9 Tabl., 28 Fig. Résumé: Certaines bactéries bénéfiques des plantes à fort potentiel biopesticide produisent des composés antimicrobiens qui sont largement impliqués dans les interactions multi-trophiques qui ont lieu dans la phytosphère et notamment l’antagonisme envers une gamme de phytopathogènes. Actuellement, il est crucial d’évaluer avec précision les taux de productions de ces antibiotiques dans la phytosphère. Dans ce contexte, nous avons réalisé cette thèse ayant comme objectif de mieux comprendre la production de lipopeptides par deux souches de Bacillus dans la rhizosphère. Dans un premier temps, l’une des souches (B. amyloliquefaciens S499) a été sélectionnée parmi une collection d’isolats sur base de son potentiel technologique et de sa capacité à inhiber in vitro la croissance de divers champignons phytopathogènes. De plus, nous avons démontré le potentiel de cette souche en tant qu’agent de biocontrôle lors d’essais en plein champs. Ces derniers ont été réalisés sur des cultures de tomates contaminées par un pathogène local identifié comme étant potentiellement une souche de Fusarium semitectum. Dans un second temps, nous avons combiné deux approches basées sur la spectrométrie de masse (ionisation par électrospray et «imaging») afin d’analyser les profils en surfactines, iturines et fengycines produits par la souche S499 in planta. Nos résultats indiquent que les conditions de la rhizosphère permettent la production de surfactines mais pas celle des autres familles de lipopeptides. De plus, nous avons également montré que la production de ces trois familles d’antibiotiques est fortement influencée par les nutriments disponibles, la formation de biofilm et la pression en oxygène. Dans une dernière partie, l’expression in situ du gène de la surfactine (srfA) dans la rhizosphère de plants de tomate a été évaluée grâce au gène rapporteur (LacZ) inséré dans la souche B. subtilis BGS3. Nos résultats montrent que le gène srfA est effectivement exprimé et entraine la production de quantités biologiquement significatives de surfactine après la colonisation des racines par la bactérie. Nous avons également démontré que BGS3 exploite efficacement les principaux substrats présents dans les exsudats de tomates afin de produire de la surfactine. Les résultats obtenus laissent également supposer que la production de surfactine pourrait être favorisée par la croissance lente de la bactérie dans la rhizosphère. Globalement, ce travail contribue à une meilleure compréhension des facteurs environnementaux influençant la production in situ de lipopeptides par les souches de Bacillus. Ceci est probablement une étape essentielle dans l’amélioration de la fiabilité et l’augmentation de l’intensité de la protection associée à ces agents biologiques de contrôle des phytopathogènes.
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Scientific communications
Publications Venant Nihorimbere, Patrick Fickers, Philippe Thonart and Marc Ongena (2009). Ecological
fitness of Bacillus subtilis BGS3 regarding production of the surfactin lipopeptide in the rhizosphere. Environmental Microbiology Reports, 1: 124-130.
Venant Nihorimbere, Marc Ongena, Hélène Cawoy, Yves Brostaux, Pascal Kakana, Emmanuel Jourdan and Philippe Thonart (2010). Beneficial effects of Bacillus subtilis on field-grown tomato in Burundi: Reduction of local Fusarium disease and growth promotion. African Journal of Microbiology Research 4: 1135-1142.
Venant Nihorimbere, Marc Ongena, Maïté Smargiassi and Philippe Thonart (2010). Beneficial effect of the rhizosphere microbial community for plant growth and health. In press to be published in the next issue in the journal of Biotechnology Agronomy Sociology and Environment (BASE). Review.
Venant Nihorimbere, Hélène Cawoy, Alexandre Seyer, Alain Brunelle, Philippe Thonart and Marc Ongena (2010). Bacillus life in soil: impact of rhizosphere factors on lipopeptide signature from the plant beneficial strain B. amyloliquefaciens S499. Submitted in the Journal of International Society for Microbial Ecology.
Nihorimbere Venant, Ongena Marc and Thonart Philippe (2010). In situ production of Bacillus antibiotics related to biocontrol: more evidences needed. In preparation for submission as a review.
Venant Nihorimbere, Marc Ongena, Hélène Cawoy, Yves Brostaux, Pascal Kakana and Philippe Thonart (2010). Bacillus-based biocontrol of Fusarium disease on tomato cultures in Burundi. Communications in Agricultural and Applied Biological Science journal, 74: 645-649.
Marc Ongena, Venant Nihorimbere, Patrick Fickers, Philippe Thonart (2009). Influence of rhizosphere-specific parameters on surfactin production by Bacillus subtilis. IOBC/wprs Bulletin, 43: 317-320.
Marc Ongena, Hélène Cawoy, Maïté Smargiassi, Venant Nihorimbere, Emmanuel Jourdan and Philippe Thonart (2010). Modulation of the lipopeptide pattern secreted by Bacillus subtilis upon colonization of different plant roots. IOBC bulletin, in Press.
Symposium & Congress 61th International Symposium on Crop Protection. Gent, Belgium, May 19th 2009. Bacillus-
based biocontrol of Fusarium disease on tomato cultures in Burundi (Oral presentation).
Bioforum, Liège, Belgium, October 11th 2007. Ecological fitness of plant beneficial Bacillus subtilis strains in soil: influence of rhizosphere-specific parameters on surfactin synthesis (Poster).
9th International Congress of Plant Pathology. Torino, Italy, 24-29 August 2008. Ecological fitness of plant-beneficial Bacillus subtilis strains in soil: Influence of rhizosphere-specific parameters on surfactin synthesis (Poster).
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Acknowledgements / Remerciements
La thèse constitue une expérience intense, passionnante et marquante. Elle m’a donné l’occasion de
côtoyer de nombreuses personnes pour qui je ne saurais en quelques lignes exprimer mes profonds
sentiments. First of all, I would like to thank the persons who helped me the most in achieving this
thesis. Je remercie très vivement le Professeur Philippe THONART qui m’a admis dans son
laboratoire de Bioindustries et qui, par la suite, m’a confié à travailler sur le thème de cette thèse de
doctorat. Ses paroles et conseils à l’occasion de nos rencontres sont, on ne peut plus, très
encourageants. Je suis extrêmement reconnaissant au Docteur Pascal KAKANA qui m’a introduit
auprès du Professeur THONART pour qu’il soit promoteur de ma thèse.
Cette thèse a été de plus agréable, vivante, et excitante grâce au Dr. Marc ONGENA. Marc,
thanks very much for all your guidance. You’re very direct, which can be quite confronting. We
discussed regularly the many options in my project, and by thinking in terms of what is required for a
Ph.D. thesis, we achieved a great output. We had a very good collaboration, and fruitful conversations,
also made possible because you also concern somebody’s personal life. Every day you make your
round in the lab to ask how everything was going, and you always noticed when something was not
going okay so well, even when I did not tell you. Not always you would ask about it further, I would
tell you in the end anyway, even when it was something personal. We also had lots of fun if there was
time; we enjoyed and exchanged lots sometimes after lab in the bar. I here want to thank you for being
such a good co-supervisor; also by your enthusiasm, optimism and humor. You are a perfectionist!!!
Uufff, pas encore fatigué, il m’est un honneur et un grand plaisir de t’exprimer ma profonde
reconnaissance et mes sincères remerciements.
Mes remerciements les plus sincères sont aussi à ceux qui m’ont fait l’honneur d’être dans le
comité de lecture de ma thèse en tant que rapporteurs: Prof. Philippe JACQUES, Dr. Magali DELEU,
qui ont bien voulu me faire-part de leurs pertinentes critiques et suggestions, et à ceux qui m’ont fait
l’honneur d’être dans mon jury: Prof. Daniel PORTETELLE, Prof. Georges LOGNAY pour leurs
disponibilités et conseils tout le long des quatres années de mon doctorat. Je remercie également
madame la présidente du Jury Prof. Marianne SINDIC pour sa disponibilité.
Si l’Unité de Bioindustries est un lieu à la fois convivial et pratique, c’est avant tout au Prof.
THONART, qu’elle le doit : je ne serais ni le premier ni le dernier à le reconnaître, mais c’est sincère.
Mais aussi, cet état est plus enthousiamé par les Drs. Jacqueline DESTAIN, Marc ONGENA et Franck
DELVIGNE. Les secrétaires, Mmes CHANET et DEMI, jouant un rôle très essentiel.
Je tiens également à remercier toutes les personnes que j’ai appris à connaître et à apprécier au
cours de ces années passées dans l'Unité de Bioindustries. Merci pour la bonne ambiance et les
échanges, scientifiques et autres. Plus particulièrement, mes remerciements s’adressent à mon groupe
de recherche «Plant-Bacillus-Lipopeptides». Que vous, Hélène, Guillaume et Manu, trouviez ici le
fruit de vos efforts particuliers déployés for the last chechs and finishing touches of the the lay-out of
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the thesis. A Michel et Tambi (merci pour le thé du Burundi partagé chaque soir vers 18 h 30 min, où
j'ai peut-être trouvé une partie de l'inspiration pour rédiger cette thèse !!),…et à tous techniciens, les
étudiants, doctorants, stagiaires de notre unité croisés au labo ou dans les bureaux.
I would also like to acknowledge the financers of this Ph.D. project, CTB/BTC, for giving me
all the support required for that. Je n'oublie pas ma famille pour leur soutien constant au cours de
toutes ces années d’études… Of course, last but not least, un merci et un bisou à mes enfants Reine et
Théo, pour tout le courage que vous avez su me donner pour finaliser cette thèse dans les meilleurs
délais (dommage, vous êtes encore petits pour ne pas réaliser ça. You were born respectively in the
first and third year of this thesis (2007 & 2009)! Enfin, un merci tout particulier à toi ma très chère
épouse Gaudence, pour toute la confiance que tu as toujours su mettre en moi.
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Contents
PREFACE ............................................................................................................................... 1
INTRODUCTION .................................................................................................................... 3
Chapter 1. Beneficial effect of the rhizosphere microbial community for plant growth and
health ..................................................................................................................... 4
Chapter 2. Bacillus lipopeptides: structure, regulation and roles in biocontrol................ 25
Chapter 3. In situ production of Bacillus antibiotics involved in the biocontrol of plant
diseases: more evidences needed ........................................................................ 40
OBJECTIVES OF THE THESIS ......................................................................................... 57
RESULTS ............................................................................................................................. 59
Chapter 4. Beneficial effects of Bacillus subtilis on field-grown tomato in Burundi:
Reduction of local Fusarium disease and growth promotion ............................. 61
Chapter 5. Bacillus life in soil: impact of rhizosphere factors on lipopeptide signature
from the plant beneficial strain B. amyloliquefaciens S499................................ 82
Chapter 6. Ecological fitness of Bacillus subtilis BGS3 regarding production of the
surfactin lipopeptide in the rhizosphere ............................................................ 116
GENERAL DISCUSSION AND FUTURE PROSPECTS............................................... 131
1
PREFACE
The rhizosphere is a very complex environment in which the effects of the plant on soil
microorganisms and the effects of the microorganisms on the plant are interacting and
interdependent. Plant root exudates and breakdown products attracts microbes and feed them.
In turn, the plant may clearly benefits from some microbes. Such interaction may be indeed
essential for plant health not only in view of nutritional requirements and enhancement of
growth but also to control certain plant pathogens. An overview of the fundamental processes
governing rhizosphere microbial ecology is presented in Chapter 1.
On another hand, some microorganisms cause serious diseases to plants leading to
considerable losses in agricultural production. Therefore, plant diseases need to be controlled
to maintain the quality and abundance of food produced by growers to mitigate the problem
of food security faced by the increasing world population. Nowadays, growers still heavily
rely on chemical fertilizers and pesticides to prevent, or control plant diseases. However,
environmental, societal and ethical problems are rising up because of excessive use or misuse
of these agrochemicals. Significant pollution of soils and ground water reservoirs,
accumulation of undesirable chemical residues on products and subsequently in the food
chain, emergence of fungicide-resistant strains of pathogens and, last but not least, very
harmful health concerns for growers. This situation calls for alternative strategies to control
plant infection. Among these alternatives are those related to biological control defined as the
use of living organisms or natural substances isolated thereof to combat plant disease.
Therefore, several groups of scientists are actively engaged in research pertaining to the
identification of biocontrol agents against specific plant pathogens, with special focus on their
use as biopesticides at a commercial scale. Over the past decades, research has repeatedly
demonstrated that phylogenetically diverse microorganisms can act as natural antagonists of
various plant pathogens. In that context, some microorganisms have been successfully
commercialized and are currently marketed as biopesticides including bacteria belonging to
the genera Agrobacterium, Bacillus, Pseudomonas, and Streptomyces and fungi belonging to
the genera Ampelomyces, Candida, Coniothyrium, and Trichoderma.
Among these genera, Bacillus strains are among the best candidates for use as biological
control agents. This is first due to their great potential to produce several different types of
insecticidal and antimicrobial compounds. Secondly, Bacillus species are able to produce
spores that allow them to resist adverse environmental conditions and permit easy formulation
and storage of the commercial products. Among the large panoply of antibiotics synthesized
2
by Bacillus, lipopeptides are more specifically studied in this work because they are tightly
related to the biocontrol activity of the producing strain. The structural traits and cellular
biosynthesis pathways of these molecules are presented in Chapter 2 along with their
biological activities in the context of plant-Bacillus-pathogen interactions. To connect with
the objectives of the thesis, the last chapter of the literature review deals with the
environmental parameters that may influence lipopeptide production in situ by Bacillus spp.
and presents the techniques and limitations for their detection in the rhizosphere.
It should be noted that an important part of this work is devoted to the study of one
particular Bacillus isolate named S499. For better clarity, we want to inform the reader here
that this strain was formerly identified as B. subtilis based on basic bacteriological tests but
further and recent genetic characterization showed that it most probably belongs to the closely
related B. amyloliquefaciens species rather than B. subtilis (see Chapter 5 for details).
3
INTRODUCTION
Introduction-Chapter 1
4
Chapter 1. Beneficial effect of the rhizosphere microbial community for plant growth and health
In Press in Biotechnologie, Agronomie, Société et Environnement Journal
Venant Nihorimbere (1,3†), Marc Ongena (1†), Maïté Smargiassi (1), Philippe Thonart (1,2)
1 Université de Liège, Gembloux Agro-Bio Tech, Centre Wallon de Biologie Industriel,
Passage des déportés, 2, B-5030 Gembloux, Belgique. 2 Université de Liège, Service de Technologie Microbienne, Boulevard du Rectorat, 29, B-40
Liège, Belgique. 3 University of Burundi, Faculty of Agricultural Sciences, PO Box 1550, Bujumbura, Burundi.
†Both authors equally contributed to this article.
Introduction-Chapter 1
5
Beneficial effect of the rhizosphere microbial community for plant growth and health
Abstract
Plant rhizosphere is the soil nearest to the plant root system where roots release large quantity
of metabolites from living root hairs or fibrous root systems. These metabolites act as
chemical signals for motile bacteria to move to the root surface but also represent the main
nutrient sources available to support growth and persistence in the rhizosphere. Some of the
microbes that inhabit this area are bacteria that are able to colonize very efficiently the roots
or the rhizosphere soil of crop plants. These bacteria are referred to as plant growth promoting
rhizobacteria (PGPR). They fulfil important functions for plant growth and health by various
manners. Direct plant growth promotion may result either from improved nutrient acquisition
and/or from hormonal stimulation. Diverse mechanisms are involved in the suppression of
plant pathogens, which is often indirectly connected with plant growth. This paper describes
the different mechanisms commonly used by most PGPR in their natural habitats to influence
plant-growth and health.
Keywords: Rhizosphere, PGPR, Root exudation, Plant-microbe interaction
Effet bénéfique de la communauté microbienne de la rhizosphère sur la croissance et la santé des plantes
Résumé
La rhizosphère est le volume du sol situé au voisinage immédiat des racines des plantes et qui
se caractérise par la présence d’exsudats racinaires (rhizodépôts). Ces exsudats sont utilisés
par la microflore endémique en tant que signaux chimiques en plus d’être un substrat nutritif
disponible pour la croissance et le développement de ces microorganismes dans la
rhizosphère. Certaines de ces bactéries du sol, appelées PGPRs (Plant Growth Promoting
Rhizobacteria), sont capables de coloniser les racines ou bien encore la rhizosphère, mais à la
différence des autres bactéries rhizosphériques elles ont, en retour, un effet bénéfique sur la
plante. Cet effet bénéfique peut être direct, ou indirect. La promotion directe de la croissance
est le résultat du pouvoir d’acquisition des nutriments ou de la stimulation des hormones de la
plante. D’autres mécanismes indirects, mais le plus souvent liés à la croissance des plantes,
sont impliqués dans la réduction/suppression des pathogènes des plantes. Cet article décrit les
différents mécanismes mis en jeu par les PGPRs dans leur environnement naturel pour
influencer favorablement la croissance et la santé des plantes.
Mots-clés: Rhizosphère, PGPR, Exsudation racinaire, Interaction plantes-microorganismes
Introduction-Chapter 1
6
Introduction
According to a general view the rhizosphere includes plant roots and the surrounding soil.
This is a wide and wise definition, already coined more than hundred years ago by Hiltner
(1904). In that particular environment, very important and intensive interactions take place
between the plant, soil, and microfauna. Biochemical interactions and exchanges of signal
molecules between plants and soil microbes have been described and reviewed (Pinton et al.,
2007). The rhizosphere inhabiting microorganisms compete for water, nutrients and space and
sometimes improve their competitiveness by developing an intimate association with plant
(Hartmann et al., 2009). These microorganisms play important roles in the growth and
ecological fitness of their host. An understanding of the basic principles of rhizosphere
microbial ecology, including the function and diversity of microorganisms that reside there, is
necessary before soil microbial technology can be applied in the rhizosphere. Here we review
different mechanisms commonly used by the beneficial rhizosphere bacteria to influence
plant-growth and health in the natural environment.
The rhizosphere effect
During seed germination and seedling growth, the developing plant interacts with a range of
microorganisms present in the surrounding soil. As seeds germinate and roots grow through
the soil, the release of organic material provides the driving force for the development of
active microbial populations in a zone that includes plant root and surrounding soil in a few
mm of thickness. This phenomenon is referred as the rhizosphere effect (Morgan and Whipps,
2001).
Broadly, there are three distinct components recognized in the rhizosphere; the
rhizosphere per se (soil), the rhizoplane, and the root itself. The rhizosphere is thus the zone
of soil influenced by roots through the release of substrates that affect microbial activity. The
rhizoplane is the root surface, including the strongly adhering soil particles. The root itself is a
part of the system, because certain endophytic microorganisms are able to colonize inner root
tissues (Bowen and Rovira, 1999). The rhizosphere effect can thus be viewed as the creation
of a dynamic environment where microbes can develop and interact.
Introduction-Chapter 1
7
Main characteristics of the root exudation process
Root exudation is the release of organic compounds from living plant roots into the
surrounding soil; it is a ubiquitous phenomenon (Moszer et al., 2002). Roots release
compounds via at least two potential mechanisms, and the rates of exudation sensu stricto
vary widely among species and environmental conditions (Kochian et al., 2005). Exudates are
transported across the cellular membrane and secreted into the surrounding rhizosphere. Plant
products are also released from roots border cells and root border-like cells which separate
from border as they grow (Bais et al., 2006). However, it is important to note that it is very
difficult to identify root exudates with respect to the chemical composition and the
concentration in the soil because of methodological difficulties (Stolp, 1988). At the moment
of exudation and thereafter, the organic materials are subject to microbial attack, and thus
cannot be enriched and separated from the roots in the natural environments. Data on the
nature and quantity of root exudates have been obtained from sterile hydroponic cultures; but
the results, however, are difficult to extrapolate to the natural conditions (Stolp, 1988). In this
context, root exudation has been quantified by measuring the production of labelled CO2 in
the rhizosphere of 14C-labelled plants, and it has been estimated that 12-40% of the total
amount of carbohydrates produced by photosynthesis is released into the soil surrounding
roots (Davis et al., 1999). Root exudates are mainly composed of water soluble sugars,
organic acids, and amino acids, but also contain hormones, vitamins, amino compounds,
phenolics and sugar phosphate esters (Uren, 2001).
Release of these low molecular weight compounds is a passive process along the steep
concentration gradient which usually exists between the cytoplasm of intact root cells
(millimolar range) and the external (soil) solution (micromolar range). Direct or passive
diffusion through the lipid bilayer of the plasma membrane is determined by membrane
permeability, which depends on the physiological state of the root cell and on the polarity of
the compounds, facilitating the permeation of lipophilic exudates (Rudrappan et al., 2007).
The efficiency of the exudation process may thus be enhanced by stress factors affecting
membrane integrity such as nutrient deficiency, temperature extremes, or exudation stress
(Ratnayale et al., 1978).
It is assumed that both the qualitative and quantitative compositions of root exudates are
affected by various environmental factors, including pH, soil type, oxygen status, light
intensity, soil temperature, nutrient availability and the presence of microorganisms. These
Introduction-Chapter 1
8
factors may have a greater impact on root exudation than differences due to the plant species
(Singh and Mukerji, 2006).
The proportion of C released from roots has been estimated to as much as 50% in the
young plants (Whipps, 1990) but less in plants grown to maturity in the field (Jensen, 1993).
The nature of exudates may also vary according to the growth stage of the plant. For instance,
there are more carboxylates and root mucilage at the six leaf stage than earlier. On the other
hand, N is also of considerable importance to nutrient cycling, usually as NH4+, NO3-
(Wacquant et al., 1989), amino acids (Boulter et al., 1966), cell lysates, sloughed roots, and
other root-derived debris. It is estimated at the maturity that the rhizodeposition of N
amounted to 20% of the total plant N (Jensen, 1996). Root exudation is also largely dependent
on the nutritional status of the plant regarding oligoelements. Low concentrations of some
nutrients such as K+, Na+ and Mg++ readily stimulate the activity of major enzymes of the
glycolytic pathway, namely phosphofructokinase and pyruvate kinase, which together
regulate glycolysis in plant cells (Plaxton, 1996). Individual micronutrients are similarly
important components of major enzymes, which regulate all biological processes in plants. It
is clear from these considerations that low nutrient availability can constraint plant growth in
many environments of the world, especially the tropics where soils are extremely deficient in
these oligoelement nutrients (Pinton et al., 2007). Some species typically exude organic acid
anions in response to P and Fe deficiency or phytosiderophores due to Fe and Zn deficiency
(Haynes, 1990).
The rhizosphere-inhabiting microflora
Diversity
The rhizosphere microflora include bacteria, fungi, nematodes, protozoa, algae and
microarthrops (Raaijmakers and Weller, 2001). Of the soil microbes, 98% cannot be cultured.
Their identification, characterization and the description of their role are therefore particularly
difficult. Recently, nucleic acid based techniques including analysis of DNA and rRNA
molecules from soil samples have revealed enormous diversity in the rhizosphere inhabiting
microbial flora (Suzuki et al., 2006). The molecular methods used for soil microbial diversity
are covered in the review by Nannipieri and collaborators (2003). The number of microbial
species present in soil may vary from thousands to millions. Many studies indeed suggest that
the Proteobacteria and the Actinobacteria form the most common of the dominant populations
(>1%, usually much more) found in the rhizosphere of many different plant species (Singh et
al., 2007). These groups contain many ‘cultured’ members. They are the most studied of the
Introduction-Chapter 1
9
rhizobacteria, and as such, contain the majority of the organisms investigated, both as
beneficial microbial inoculants and as pathogens.
The specific content of root exudates may create a niche that influences which
microorganisms are to colonize the rhizosphere, thereby altering the composition and
diversity of microorganisms colonizing the rhizosphere in a plant specific manner (Grayston
et al., 1998). Plant species, plant developmental stage and soil type have thus been indicated
as major factors determining the composition of rhizosphere microbial communities
(Broeckling et al., 2008). That said, the extent to which the above-cited factors contribute to
microbial communities is not fully understood and there are several contrasting reports in the
literature indicating either plant or soil type as dominant factor (Nunan et al., 2005). Owing to
the above statement, it can be generalized that the diversity and predominance of rhizosphere
microbial population depend on a number of abiotic and biotic factors prevailing in that
particular ecological niche (Figure 1).
Population level
Studies based on the use of growth media steadily showed that bacterial populations residing
in the rhizosphere are several orders of magnitude larger than those residing in bulk soils.
Rhizosphere bacteria concentration can reach between 1010 and 1012 cells per gram of soil
(Foster, 1988), and they are transferred to various associated environments including plants,
foods, animals, marine and freshwater habitats (Buée et al., 2009). Only few groups of these
bacteria are considered to be soilborne, probably because non-spore forming bacteria cannot
survive well in soil for long periods.
Introduction-Chapter 1
10
Plant species
Root colonization
Root exudates
Humidity
Temperature variationMicrofauna
soil type, pH, …
Photoperiod
Rhizobacteria
Figure 1. Ecological factors influencing the root exudation process and thereby rhizosphere colonization by beneficial rhizobacteria – Facteurs écologiques influençant le processus d’exsudation racinaire et par conséquent la colonisation de la rhizosphère par les rhizobactéries du sol.
Introduction-Chapter 1
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The effect of root exudates depends on the distance that they can diffuse away from
rhizoplane (Gupta and Mukerji, 2002). Bacterial communities are not uniformly distributed
along root axes, and differ between root zones. Distinct bacterial community compositions are
obtained by molecular fingerprints in different root zones, like those of emerging roots and
root tips, elongating roots, sites of emergence of lateral roots, and older roots (Yang and
Crowley, 2000). It has been proposed that populations residing in the rhizosphere oscillate
along root axes in a wave-like fashion (Semenov et al., 1999). Accordingly, bacterial
communities temporarily profit from the nutrients released by younger roots in the root hair
zones, and wave-like fluctuations in bacterial cell numbers can be explained by death and
lysis of bacterial cells upon starvation when nutrients become depleted, followed by cell
divisions in surviving and thus viable populations as promoted by the release of nutrients
from dead and decaying cells (Semenov et al., 1999). Bacterial communities in rhizosphere
soils are thus not static, but will fluctuate over time in different root zones.
The rhizosphere as a battle field
The number and diversity of microorganisms are related to the quantity and quality of the
exudates but also to the outcome of the microbial interactions that occur in the rhizosphere
(Somers et al., 2004). Soil biota (bacteria, fungi, micro-fauna and the plant root) are
themselves embedded in food webs and thus interactions with consumers or predators in the
microbial as well as macro- and mesofaunal world are important to understand rhizosphere
processes. A high number of soil microbes attained properties enabling them to interact more
efficiently with roots and withstand the quite challenging conditions of rhizosphere life. The
rhizosphere inhabiting microorganisms compete each other for water, nutrients and space and
sometimes improve their competitiveness by developing an intimate association with plant.
This process can be regarded as an ongoing process of micro-evolution in low-nutrient
environments, which are quite common in natural ecosystems (Schloter et al., 2000).
Plant-bacteria interactions in the rhizosphere
Microorganisms present in the rhizosphere play important roles in ecological fitness of their
plant host. Important microbial processes that are expected to occur in the rhizosphere include
pathogenesis and its counterpart, plant protection/growth promotion, as well as the production
of antibiotics, geochemical cycling of minerals and plant colonization (Kent and Triplett,
2002). Plant-microbe interactions may thus be considered beneficial, neutral, or harmful to
the plant, depending on the specific microorganisms and plants involved and on the prevailing
environmental conditions (Bais et al., 2006). Exploring these microorganisms by unravelling
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their possible relationships with plants has launched a new and fascinating area of
investigations in the rhizosphere research.
Pathogenic interactions
Roots exudates can attract beneficial organisms (see below), but they can also be equally
attractive to pathogenic populations (Schroth and Hildebrand, 1964), that many express
virulence on only a limited number of host species. Many pathogenic organisms, bacteria as
well as fungi, have coevolved with plants and show a high degree of host specificity
(Raaijmakers et al., 2009). In nature however, plant disease is the exception rather than the
rule because the conditions that are optimized for the plant growth may not be favourable for
pathogens (Paulitz and Bélanger, 2001).
Plants are not defenceless. In fact, it is estimated that only about 2% of the known
fungal species are able to colonize plants and cause disease (Buchanan et al., 2000). Even
though plants are in permanent contact with potential pathogens such as fungi, bacteria or
viruses, successful infection is rarely established. Such a general resistance against most
pathogens has been named “horizontal resistance” or “non-host-resistance” (Heath, 1981).
This reflects the fact that the plant is not a suitable target for infection by a specific pathogen
due to preformed, passive resistance mechanisms resulting in “basic incompatibility”. These
resistance mechanisms comprise structural barriers and toxic compounds that are present in
the unaffected, healthy plant and limit successful infection to specialized pathogens that have
the ability to overcome these factors and therefore exhibit “basic compatibility”. If contact is
nevertheless established with the plant tissue, pathogens are often confronted with preformed
chemical components named phytoanticipins (van Etten et al., 1994). This term comprises a
variety of compounds produced by different biosynthetic pathways which possess
antimicrobial properties. These low molecular weight secondary metabolites are mainly
stored in inactive form in the vacuoles or organelles and are released upon destruction of the
cells. Since destroying the integrity of the plant tissue is part of the colonization strategy by
fungi, phytoanticipins represent an important resistance mechanism against these pathogens.
However, in some instances, pathogens can overcome the pre-formed barriers and
develop virulent infection processes leading to plant disease. Plant diseases play a direct role
in the destruction of natural resources in agriculture. In particular, soilborne pathogens cause
important losses, fungi being the most aggressive. The extent of their harmful effects ranges
from mild symptoms to catastrophes where large fields planted with agricultural crops are
destroyed. Thus, they are major and chronic threats to food production and ecosystem
Introduction-Chapter 1
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stability worldwide. Common and well investigated bacterial agents include Gram-negative
bacteria Erwinia carotovora, Pseudomonas, Ralstonia spp. and the Gram-positive bacterium
Streptomyces scabies. The fungal and oomycete phytopathogens include members of
Fusarium, Phytophthora, Pythium, Rhizopus, Rhizoctonia and Verticillium (Tournas and
Katsoudas, 2005). From the forest pathogens, among the most important are the filamentous
fungi Heterobasidion and Armillariella (Asiegbu and Nahalkova, 2005), and Phytophthora
spp. (Rizzo et al., 2005).
Beneficial microorganisms and modes of action
Plant-beneficial microbial interactions can be roughly divided into three categories. First,
those microorganisms that, in association with plants, are responsible for its nutrition (i.e.,
microorganisms that can increase the supply of mineral nutrients to the plant). In this case,
while most may not directly interact with the plant, their effects on soil biotic and abiotic
parameters certainly have an impact on plant growth. Second, there is a group of
microorganisms that stimulate plant growth indirectly by preventing the growth or activity of
pathogens. Such microorganisms are referred to as biocontrol agents, and they have been well
documented. A third group involves those microorganisms responsible for direct growth
promotion, for example, by production of phytohormones. There has been a large body of
literature describing potential uses of plant associated bacteria as agents stimulating plant
growth and managing soil and plant fitness (Welbaum et al., 2004). On another hand,
apparently neutral interactions are found extensively in the rhizosphere of all crop plants.
Saprophytic microorganisms are responsible for many vital soil processes, such as
decomposition of organic residues in soil and associated soil nutrient mineralization or
turnover processes. Whereas these organisms do not appear to benefit or harm the plant
directly (hence the term neutral), their presence is obviously vital for soil dynamic, and their
absence would clearly influence plant health and productivity (Brimecombe et al., 2007).
Rhizosphere-living bacteria that exert a global beneficial effect on plant growth are
referred as plant growth promoting rhizobacteria (PGPR) (Kloepper and Schroth, 1978). The
number of bacterial species identified as PGPR increased recently as a result of the numerous
studies covering a wider range of plant species and because of the advances made in bacterial
taxonomy and the progress in our understanding of the different mechanisms of action of
PGPR. Presently, PGPR include representatives from very diverse bacterial taxa (Lucy et al.,
2004) and in the following sections we are not giving a thorough description of all the genera
and species of PGPR, but rather a few examples to illustrate the diversity and modes of action
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14
of these beneficial bacteria. Diverse PGPR strains have been used successfully for crop
inoculations. These comprise members of the bacterial genera Azospirillum (Cassán and
García Salamone, 2008), Bacillus (Jacobsen et al., 2004), Pseudomonas (Loper and Gross,
2007), Rhizobium (Long, 2001), Serratia (De Vleeschauwer and Höfte, 2007),
Stenotrophomonas (Ryan et al., 2009), and Streptomyces (Schrey and Tarkka, 2008). Some
fungi belonging to the genera Ampelomyces, Coniothyrium, and Trichoderma have also been
described to be beneficial for the host plant (Harman et al., 2004). The modes of action of
PGPR involve complex mechanisms to promote plant growth, development and protection.
Important among them are biofertilization (increasing the availability of nutrients to plant),
phytostimulation (plant growth promoting, usually by the production of phytohormones) and
biocontrol (controlling diseases, mainly by the production of antibiotics and antifungal
metabolites, lytic enzymes and induction of plant defense responses). Pseudomonas and
Bacillus genera are the most commonly investigated PGPR, and often the dominating
bacterial groups in the rhizosphere (Morgan et al., 2005). One has to mention that, in many
cases of individual beneficial plant–microbe interactions, several mechanisms are involved
(Müller et al., 2009). Ad planta, direct mechanisms of plant growth promotion are difficult to
differentiate from disease suppression and the relative importance on a specific mechanism
can vary within different pathosystems (Chet and Chernin, 2002).
Colonization
In all successful plant–microbe interactions, the competence to colonize plant habitats is
important (Lugtenberg et al., 2002; Kamilova et al., 2005). Single bacterial cells can attach to
surfaces and, after cell division and proliferation, form dense aggregates commonly referred
to as macrocolonies or biofilms. Steps of colonization include attraction, recognition,
adherence, invasion (only endophytes and pathogens), colonization and growth, and several
strategies to establish interactions. Plant roots initiate crosstalk with soil microbes by
producing signals that are recognized by the microbes, which in turn produce signals that
initiate colonization (Berg, 2009). PGPR reach root surfaces by active motility facilitated by
flagella and are guided by chemotactic responses (Pinton et al., 2007). This implies that
PGPR competence highly depends either on their abilities to take advantage of a specific
environment or on their abilities to adapt to changing conditions or plant species. As an
example, when strain S499 of Bacillus subtilis was applied to plant seedlings, it showed more
distinct but effective colonization of the root system of two distinct plants (Figure 2). In most
cases, the population of many PGPR inoculants actually declines progressively in time after
Introduction-Chapter 1
15
inoculation from 107-109 cells per gram dry soil to 105-106 cells per gram dry soil after 2-3
weeks (DeFlaun and Gerba, 1993). Nevertheless this population threshold is often sufficient
to provide beneficial effects (Raaijmakers et al., 2002). Rhizosphere competence of
biocontrol agents thus involves effective root colonization combined with the ability to
survive and proliferate along growing plant roots over a large time period, in the presence of
the indigenous microflora (Weller, 1988; Lugtenberg and Dekkers, 1999).
a b c d
Figure 2. Root colonization by strain S499 of Bacillus subitilis - Colonisation racinaire par la souche de Bacillus subtilis S499. Microscopy visualization of root of – Visualisation par microscopie des racines de, a: treated salad – salade traitée, b: untreated salad – salade non traitée, c: treated tomato – tomate traitée, and d: untreated tomato – tomate non traitée. The arrow in a and c indicates the biofilm formation and the cross-surrounded in b and d indicates absence of biofilm formation by the strain on the root system – La flèche en a et c indique la formation de biofilm et la croix entourée en b et d indique l’absence de formation de biofilm sur le système racinaire par la souche. Surface-sterilized tomato and salad seeds were suspended in bacterial suspension of 108 cells ml-1 and germinated for two weeks in the conditioned chamber and in gelified sterile plant nutrient medium as defined by Murashige and Skoog (1962) – Les graines de tomate et de salade stérilisées sont suspendues dans une solution bactérienne de 108 cellules par ml et sont mises à germer pendant deux semaines dans une chambre d’air conditionnée sur un milieu nutritive pour plantes mis au point par Murashige et Skoog (1962). Source: results obtained in our laboratory - Source: résultats obtenus dans notre laboratoire.
Pathogen inhibition
Bacteria and fungi live around roots and feed on root exudates and dead root cells.
Competition between microbial species in this area is stiff. In the battle for establishment and
persistence in the niche, bacteria use several strategies.
Antagonism
Root colonization not only results in high PGPR population densities on the root system, it
also functions as the delivery system of antagonistic metabolites that are involved in direct
inhibition of plant pathogens (Shoda, 2000; Raaijmakers et al., 2002). It includes antibiosis
i.e. the inhibition of microbial growth by diffusible antibiotics and volatile organic
compounds, toxins, and biosurfactants, and parasitism that may involve production of
extracellular cell wall-degrading enzymes such as chitinases and β-1,3-glucanase (Compant et
al., 2005; Haas and Défago, 2005). The degradation of pathogenicity factors of the pathogen
such as toxins by the beneficial organism has also been reported as protective mechanism
(Haas and Défago, 2005). To demonstrate the role of antibiotics in biocontrol, mutants
impaired in biosynthesis or over-producing mutants have been used together with, in some
Introduction-Chapter 1
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cases, the use of reporter genes or probes to show efficient production of the compound in the
rhizosphere. As example, Bacillus subtilis strains produce a variety of powerful antifungal
metabolites, e.g., zwittermicin-A, kanosamine and lipopeptides from the surfactin, iturin and
fengycin families (Emmert and Handelsman, 1999; Ongena and Thonart, 2006). Dunne and
collaborators (2000) showed that overproduction of extracellular protease in the mutant
strains of Stenotrophomonas maltophilia W81 resulted in improved biocontrol of Pythium
ultimum. Excretion of chitinases and glucanases by species of Trichoderma and Streptomyces
has also been shown to play an important role in mycoparasitism of phytopathogenic fungi
(Whipps, 2001).
Competition
Competition for resources such as nutrients and oxygen occurs generally in soil between soil-
inhabiting organisms. For biocontrol purpose, it occurs when the antagonist directly competes
with pathogens for these resources. Root inhabiting microorganisms compete for suitable sites
at the root surfaces. Competition for nutrients, especially for carbon, is assumed to be
responsible for the well-known phenomenon of fungistasis characterizing the inhibition of
fungal spore germination in soil (Alabouvette et al., 2006). Given the relative abundance of
substrates in the rhizosphere, the efficiency of nutrient uptake and catabolism by bacteria is a
key factor in competitiveness (Chin-A-Woeng et al., 2003). The capacity for rapid growth
when substrates are encountered is not the only factor affecting rhizosphere competence, as
rhizobacteria deploy many other metabolic strategies. For example, the capacity for
extracellular conversion of glucose to gluconic acid and 2-ketogluconic acid enables some
bacteria, including several species of Pseudomonas to sequester glucose effectively and gives
a competitive advantage over microorganisms that lack the ability to utilise these compounds
(Gottschalk, 1986).
Competition for trace elements, such as iron, copper, zinc, manganese etc., also occurs
in soils. For example, iron is an essential growth element for all living organisms and the
scarcity of its bio-available form in soil habitats results in a furious competition (Loper and
Henkels, 1997). Siderophores, low molecular weight compounds with high iron affinity, are
produced by some microorganisms (also by most biocontrol agents) to solubilize and
competitively acquire ferric ion under iron-limiting conditions, thereby making iron
unavailable to other soil microorganisms which cannot grow for lack of it (Loper and
Henkels, 1997; Haas and Défago, 2005). The bacterium that originally synthesized the
siderophores takes up the iron siderophore complex by using a receptor that is specific to the
Introduction-Chapter 1
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complex and is located in the outer cell membrane. Suppression of soilborne plant pathogens
by siderophore producing Pseudomonads has been reported in some instances (Loper, 1988;
Weger et al., 1988; Buysens et al., 1996).
Induced resistance
Plant-associated bacteria can reduce the activity of pathogenic microorganisms not only
through microbial antagonisms, but also by activating the plant to better defend itself, a
phenomenon termed “induced systemic resistance” (ISR) (Shoda, 2000; van Loon, 2007).
Sometimes, the mechanism of ISR elicited by PGPR overlaps partly with that of pathogen-
induced systemic acquired resistance (SAR). Both ISR and SAR represent a state of enhanced
basal resistance of the plant that depends on signalling compounds such as jasmonic acid,
ethylene and salicylic acid (van Loon, 2007). Expression of natural defense reaction against
stresses from biotic or abiotic origin is exhibited by all plants, such as (i) physical stresses
(heat or frost), (ii) inoculation by pathogenic or non-pathogenic organisms, (iii) chemical
molecules from natural or synthetic origins (Alabouvette et al., 2006). Early recognition of
the aggressor by the plant is one of the mechanisms involved in elicitation of plant defense
reactions (Lugtenberg et al., 2002). Recognition of the aggressor immediately initiates a
cascade of molecular signals and the transcription of many genes, which eventually results in
the production of defence molecules by the host plant (van Loon, 2000). Such defence
molecules include phytoalexins, pathogenesis-related (PR) proteins (such as chitinases, β-1,3-
glucanases, proteinase inhibitors, etc.) and lignin for reinforcement of cell walls (van Loon,
2000). In fact, cell wall thickenings, wall appositions or rapid death of the injured plant cells
resulting in necrosis of the immediate adjacent tissues are barriers which cut the pathogen off
its nutrients and contribute to slowing down of the fungus progressive invasion (Lugtenberg
et al., 2002; Alabouvette et al., 2006).
Plant growth promotion
Phytostimulation
Phytostimulation enhances plant growth in a direct way. In the processes of plant growth,
phytohormones (e.g., production of indole-3-acetic acid (IAA), auxins, cytokinins, and
gibberellins) play an important role. These hormones can be synthesized by the plant
themselves but also by their associated microorganisms such as Azospirillum spp., besides
having nitrogen-fixing ability (Steenhoudt and Vanderleyden, 2000). Species of Pseudomonas
and Bacillus can produce as yet not well characterized phytohormones or growth regulators
that cause crops to have greater amounts of fine roots which have the effect of increasing the
Introduction-Chapter 1
18
absorptive surface of plant roots for uptake of water and nutrients. The phytohormones they
produce include indole-acetic acid, cytokinins, gibberellins and inhibitors of ethylene
production. Indole-3-acetic acid is a phytohormone which is known to be involved in root
initiation, cell division, and cell enlargement (Salisbury, 1994). This hormone is very
commonly produced by PGPRs (Barazani and Friedman, 2001). Auxins are quantitatively the
most abundant phytohormones secreted by Azospirillum, and it is generally agreed that their
production, rather than nitrogen-fixation, is the major factor responsible for the stimulation of
rooting and, hence, enhanced plant growth (Bloemberg and Lugtenberg, 2001). Furthermore,
plant-associated bacteria can influence the hormonal balance of the plant. Ethylene is an
important example to show that the balance is most important for the effect of hormones: at
low levels, it can promote plant growth in several plant species including Arabidopsis
thaliana, while it is normally considered as an inhibitor of plant growth and known as a
senescence hormone (Pierik et al., 2006).
The general effect on the plant can be direct, that is through plant growth promotion, or
indirect, that is through improving plant nutrition via the better development of the roots, and
it is difficult to distinguish between them. The elevation of root IAA level in lodgepole pine
plantlets, inoculated with Paenibacillus polymyxa, and, of dihydroxyzeatin riboside root
concentration in plants inoculated with Pseudomonas fluorescens (Fuentes-Ramirez and
Caballero-Mellado, 2005), might be attributed to the induction of plant hormone synthesis by
the bacteria. However, the uptake of bacterial synthesized phytohormones can not be
excluded, since both P. polymyxa and Pseudomonas produce IAA and cytokinins in vitro
(Fuentes-Ramirez and Caballero-Mellado, 2005).
Biofertilization
The mechanisms by which PGPR increase crop performance is not well understood. There are
several PGPR inoculants currently commercialized that seem to promote growth through at
least one mechanism; suppression of plant disease (termed bioprotectants), phytohormone
production (termed biostimulants), or improved nutrient acquisition (termed biofertilizers).
The mode of action of PGPR by biofertilizers act either, directly by helping to provide
nutrient to the host plant, or indirectly by positively influencing root growth and morphology
or by aiding other beneficial symbiotic relationships (Vessey, 2003). The most prominent
example is bacterial nitrogen fixation. The symbiosis between rhizobia and its legume host
plants is an important example for plant growth-promoting rhizobacteria (PGPR). Bacteria of
this group metabolize root exudates (carbohydrates) and in turn provide nitrogen to the plant
Introduction-Chapter 1
19
for amino acid synthesis. The ability to fix nitrogen also occurs in free-living bacteria like
Azospirillum, Burkholderia, and Stenotrophomonas (Dobbelare et al., 2003). Biofertilization
accounts for approximately 65% of the nitrogen supply to crops worldwide (Bloemberg and
Lugtenberg, 2001). Another nutrient is sulfate, which can be provided to the plant via
oxidation by bacteria (Banerjee and Yesmin, 2002). Bacteria may contribute to plant nutrition
by liberating phosphorous from organic compounds such as phytates and thus indirectly
promote plant growth (Unno et al., 2005). Azospirillum treatment resulted in enhancement of
root growth and activities (e.g., acidification of the root surroundings) that increases
phosphorous and other macroelements and microelements uptake (Dobbelaere and Okon,
2007). Recently, De Werra and collaborators (2009) showed that the ability of Pseudomonas
fluorescens CHA0 to acidify its environment and to solubilize mineral phosphate is strongly
dependent on its ability to produce gluconic acid.
Conclusions
The rhizosphere is the zone of soil surrounding a plant root where the biology and chemistry
of the soil are influenced by the root. As plant roots grow through soil they mostly release
water soluble compounds such as amino acids, sugars and organic acids that supply food for
the microorganisms. High levels of exudates in the rhizosphere attract a plethora of
microorganisms to a larger extend than elsewhere in the soil. The composition and pattern of
root exudates affect microbial activity and population numbers. Plant species, plant
developmental stage and soil type have been indicated as major factors determining the
composition of rhizosphere microbial communities (Broeckling et al., 2008). As shown in
many studies, there is no general decision about the key player: the diversity and
predominance of rhizosphere microbial population depend on a number of abiotic and biotic
factors of a particular ecological niche.
A better understanding of the basic principles of the rhizosphere ecology, including the
function and diversity of inhabiting microorganisms is on the way but further knowledge is
necessary to optimize soil microbial technology to the benefit of plant-growth and health in
the natural environment. In sum, this can constitute overwhelming evidence indicating that an
ever exploitation of plant growth promoting rhizobateria (PGPR) can be a true success story
in sustainable agriculture. As a consequence, current production methods in agriculture, e.g.,
the improper use of chemical pesticides and fertilizers creating a long list of environmental
and health problems, will reduce.
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20
Acknowledgements Venant Nihorimbere is recipient of a grant from the Belgian Technical Cooperation (BTC/CTB). Marc Ongena is Research Associate at the F.R.S.-FNRS (National Funds for Scientific Research, Belgium).
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Chapter 2. Bacillus lipopeptides: structure, regulation and roles in biocontrol
Introduction-Chapter 2
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Abstract
Lipopeptides (LPs) from plant-associated bacteria display versatile functions in the ecology of
the producing strains and notably in interactions with co-existing organisms, including
bacteria, fungi, oomycetes, protozoan predators and plants. The proposed primary mode of
action of LPs is pore formation in membranes leading to an imbalance in transmembrane ion
fluxes and cell death. LPs have demonstrated to play a crucial role in the antagonism
developed towards phytopathogens sharing the same microenvironment and thus in the
biocontrol potential of the strains. Beside their antimicrobial properties, LPs are also involved
in root colonization as well as in the systemic stimulation of host plant immune system. We
thus summarized here the current knowledge of LPs biocontrol activities.
A variety of structures
Cyclic lipopeptides (LPs) are synthesized by non-ribosomal peptide synthetases (NRPS) or
hybrid polyketide synthases/non-ribosomal peptide synthetases (PKS/NRPS). These modular
proteins are megaenzymes organized in iterative functional units called modules that catalyze
the different reactions leading to polyketide or peptide transformation (Finking and Marahiel,
2004). Such biosynthetic systems lead to a remarkable heterogeneity among the LPs products
which vary in the type and sequence of amino acid residues, the nature of the peptide
cyclisation and in the length and branching of the fatty acid chain (Ongena and Jacques,
2008). LPs are synthesized by various genera of plant-associated bacterial such as
Streptomyces, Pseudomonas and Bacillus (Pirri et al., 2009).
Surfactins Iturins Fengycins
Figure 1: Structures of representative members of the three families of Bacillus LPs. Source: Raaijmakers et al.
(2010).
Introduction-Chapter 2
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The three main Bacillus LP families are surfactin, iturin and fengycin. They encompass
structural variants depending on the genetic background of the considered strain and in
several case environmental growth conditions (Figure 1 and Table 1).
Table 1: Updated list of Bacillus LPs identified so far*. Amino-acid residues in bold are involved in cyclisation.
Number
of aa Fatty acids Peptidic sequence
surfactin 7 C12-C14 Glu Leu Leu Val Asp Leu Leu
surfactin 7 C12-C14 Glu Leu Leu Val Asp Leu Val
surfactin 7 C12-C14 Glu Leu Leu Val Asp Leu Ile
bamylocin 7 C13 Glu Leu Met Leu Pro Leu Leu
pumilacidin A 7 C12-C14 Glu Leu Leu Leu Asp Leu Val
pumilacidin B 7 C12-C14 Glu Leu Leu Leu Asp Leu Ile
lichenysin 7 C12-C14 Gln Leu Leu Val Asp Leu Val
lichenisyn 7 C12-C14 Gln Leu Leu Val Asp Leu Ile
kurstakin 7 C10-C12 Thr Gly Ala Ser His Gln Gln
iturin A 7 C14-C16 Asn Tyr Asn Gln Pro Asn Ser
iturin C 7 C14-C16 Asp Tyr Asn Gln Pro Asn Ser
mycosubtilin 7 C16-C17 Asn Tyr Asn Gln Pro Ser Asn
bacillomycin D 7 C14-C16 Asn Tyr Asn Pro Glu Ser Thr
bacillomycin F 7 C16-C17 Asn Tyr Asn Gln Pro Asn Thr
bacillomycin L 7 C14-C16 Asp Tyr Asn Ser Gln Ser Thr
fengycin A 10 C15-C17 Glu Orn Tyr aThr Glu Ala Pro Gln Tyr Ile
fengycin B 10 C15-C17 Glu Orn Tyr aThr Glu Val Pro Gln Tyr Ile
plipastatin A 10 C15-C17 Glu Orn Tyr aThr Glu Ala Pro Gln Tyr Ile
plipastatin B 10 C15-C17 Glu Orn Tyr aThr Glu Val Pro Gln Tyr Ile
polymyxin 10 Dad Thr Dab Dab Dab Phe Leu Dab Dab Thr
*: Table adapted from works of Fernando et al. (2005) and others.
Surfactins are heptapeptides interlinked with a β-hydroxy fatty acid to form a cyclic lactone
ring structure. The group of iturins encompasses 7 variants including bacillomycins and
mycosubtilin. All are heptapeptides linked to a β-amino fatty acid chain with a length from
C14 to C17. The third family comprises fengycins A and B, which are also called plipastatins.
These molecules are lipodecapeptides with an internal lactone ring in the peptidic moiety and
with a β-hydroxy fatty acid chain (C14 to C18) that can be saturated or unsaturated. Beside
these three main families, other classes of bioactive lipopeptides synthesized by Bacillus
species have been identified such as kurstakins isolated from B. thuringiensis (Hathout et al.,
Introduction-Chapter 2
28
2000), polymyxins, the dodecacyclopeptide bacitracins and the surfactin-ressembling
bamylocin A (Lee et al., 2007).
Cellular regulatory network of LP biosynthesis
According to Duitman and collaborators (2007), there is no auto-induction process in the
biosynthesis of surfactins or iturins. However, there is relatively few information on the
regulatory aspects at the molecular level of the biosynthesis of Bacillus LPs compared to the
well-characterized structural and functional organization of their biosynthetic genes.
Regulation of srfA is typically multi-component and take part of a complex regulatory
cascade involved in multiple basal cellular processes that governs several differentiation
pathways in B. subtilis. This regulation at the cellular level is schematized in Figure 2A.
Transcription of the srfA gene is under the primary control of the quorum-sensing pheromone
ComX which is secreted with the help of ComQ in the culture medium. ComP senses the
accumulation of the ComX and at critical ComX concentration phosphorylates itself.
Subsequently autophosphorylated ComP phosphorylates ComA. ComA-P activates
expression of surfactin operon and ComS which is an open reading frame encoded within de
srfA mRNA. ComS leads to the autoactivation of ComK. In addition, several activators such
as ComK itself and DegU and repressors including CodY, AbrB and Rok are involved in
ComK activation. By analogy with the ComA/ComP regulon, DegU is a two-component
response regulator that controls many cellular processes in Bacillus (Dahl et al., 1992; Steil et
al. 2003). The second type of pheromones involved in surfactin expression is the group of Phr
peptides: PhrC, PhrF, PhrG and PhrH. They are probably first synthesized as small proteins
which are secreted, processed in pentapeptides and then internalised by an oligopeptide
permease (Opp). The phrH gene constitutes an operon with a rapH gene. Boths genes are thus
activated by ComK. Products of phr and rap genes are interacting regulators factors that
modulate the phosphorylation state of DNA specific response regulators. The phrC, phrF and
phrG genes need the sigmaH (Spo0H) form of RNA polymerase to be transcribed. RapC,
RapF and, in certain circumstances RapG and RapH, are inhibitors of the DNA binding of
ComA-P. ComA-P activates rapC and rapF. PhrC, PhrF, PhrG and PhrH inhibit their cognate
Rap proteins. RapG and RapH inhibit DNA binding of DegU. RghR is a rapG, rapH and
phrH repressor.
Expression of the mycosubtilin genes is under the influence of AbrB, one of the main
transition state regulators in B. subtilis but additional regulators may be involved (Figure 2B).
Introduction-Chapter 2
29
Figure 2. Regulation of surfactin (A) and iturin (B) biosynthesis in Bacillus. Source: Jacques (2011).
As for surfactin, the pheromone PhrC is seemingly involved in the regulation of mycosubtilin
biosynthesis (Duitman et al., 2007). For bacillomycin D, another member of the iturin family,
Introduction-Chapter 2
30
the synthesis is activated by interaction of the promoter with DegU in the early stationary
growth phase and DegQ is also involved (Koumoutsi et al., 2007). To our knowledge, little
information is available about the regulation of fengycin biosynthesis, although it was
demonstrated that expression of the plipastatin operon ppsABCDE requires at least degQ in
addition to the sfp gene (Tsuge et al., 2007).
A variety of biocontrol-related functions
Involvement in root colonization
The ability of B. subtilis to efficiently colonize surfaces of plant roots is a prerequisite for
phytostimulation. The first step of translocation on surfaces like roots, the spreading, can be
achieved through several ways. The probably most studied is the cell motility in colonies,
swarming, which involves differentiation of vegetative cells into hyperflagellated “swarmer
cells” (Fraser and Hughes, 1999). This swarming process allows an easier colony spreading
but also an improved antimicrobial substances resistance. The second step for rhizosphere
competence is linked to the capability to form sessile, highly structured and antimicrobial
resistant multicellular communities. Microbial populations such as plant-associated bacteria
evolve and behave as structured communities called biofilms (on solid surfaces) or pellicles
(at air/liquid interfaces) that could adhere to root and on soil particle surfaces (Danhorn and
Fuqua, 2007). Several studies showed that LPs could be involved at different levels in the
complex network linking motility and biofilms/pellicles/fruiting-bodies formation.
Surfactin is thought to act by the aggregation of the cells into dendrites and by the
coordination of their advance throughout the swarm front (Julkowska et al., 2005). The LPs
surfactin and mycosubtilin have been shown to be implicated in a flagella-independent
surface motility of B. subtilis (Kinsinger et al., 2003; Leclère et al., 2006). This surfactant-
induced spreading is most likely due to a reduction of frictions between cells and surface in
combination with a surface tension-driven flow. A low surface tension which could be
reached with strong surfactants such as surfactin and mycosubtilin was demonstrated to be
sufficient to facilitate microbial colonization (Leclère et al., 2006). This could explain why, in
some strains but not all (Figure 3), surfactin production is necessary but not sufficient for
swarming, in which other factors like genes swrABC and efp are additionally involved
(Kearns et al., 2004). Results obtained with B. subtilis A1/3 and B. amyloliquefaciens FZB42
(Figure 3) have shown that surfactin but no other lipopeptides produced by the strain was
required for the formation of biofilms and pellicles indicating that surfactins may still serve
specific developmental functions (Hofemeister et al., 2004). More conclusively in the view of
Introduction-Chapter 2
31
biocontrol, the production of surfactin was demonstrated to be essential for biofilm formation
and colonization of Arabidopsis roots by the strain B. subtilis 6051 and that biocontrol
exhibited against P. syringae is linked to the formation of this antibiotic at the root surface
(Bais et al., 2004).
FZB42 wtSrf+
Fen+
Bac+
AK3 deriv Srf+
Fen-
Bac-
CH1 deriv Srf-
Fen+
Bac-
CH2 derivSrf-
Fen-
Bac+
Figure 3: Illustration of the importance of surfactin for biofilm formation (up, as revealed by the blue coloration of cell aggregates in microtiter plate wells) and for motility (down, as revealed by the spreading ability on soft agar plates) of B. amyloliquefaciens FZB42. Source: Raaijmakers et al. (2010).
Finally, genes that mediate production of surfactin (srfAA and sfp) were shown to be required
for the erection of fruiting-bodies (Branda et al., 2001), at least in part by their ability to lower
the surface tension of water.
Involvement in direct antagonism
Each family of Bacillus LPs displays specific antibiotic activities and may thus be
differentially involved in the antagonism of the various plant pathogens. Reports suggesting a
role for LPs in antagonism based on in vitro studies (such as illustrated in Figure 4A) are
numerous but there are only few studies that associate biocontrol activity with LPs production
in planta. In the case of soilborne diseases, iturin A produced by B. subtilis RB14 was
involved in damping-off of tomato (a seedling disease) caused by Rhizoctonia solani (Asaka
and Shoda, 1996). Overexpression of mycosubtilin in B. subtilis ATCC 6633 also led to a
significant reduction of seedling infection by Pythium aphanidermatum (Leclère et al., 2005).
As examples in control of phyllosphere diseases, a contribution of both iturins and fengycins
was recently shown in the antagonism of B. subtilis toward Podosphaera fusca infecting
melon leaves (Figure 4B) (Romero et al., 2007). This was notably demonstrated by showing
the strong inhibitory effect of these LPs on P. fusca conidia germination, and by recovering
LPs from bacterial-treated leaves and using LP-deficient transformants. In the protection of
Introduction-Chapter 2
32
post harvest diseases, the strain Bacillus subtilis strain GA1 which efficiently produces LPs
from the three families and notably a wide variety of fengycins, protected wounded apple
fruits against gray mold disease caused by Botrytis cinerea. The role of fengycins was
demonstrated by the very effective disease control provided by treatment of fruits with LPs-
enriched extracts and by in situ detection of fengycins in inhibitory amounts (Touré et al.,
2004). To further illustrate the broad range of fungal targets, fengycins were also reported for
their antagonistic activity against Fusarium graminearum (Wang et al., 2007), and iturins for
their inhibitory effect towards the anthracnose-causing agent Colletotrichum demiatium
(Hiradate et al., 2002), Penicillium roqueforti (Chitara et al, 2003), Aspergillus flavus (Moyne
et al., 2001), Rhizoctonia solani (Yu et al., 2002), wood-staining fungi (Velmurugan et al.,
2009) and nematophagous fungi (Li et al., 2007). In some instances, the fungitoxic activity
was clearly related to the permeabilization of spore/conidia therefore inhibiting germination
or alternatively to hyphal cell perturbation.
- LP + LP
- LP + LP
A
B
C
s s
s s
Figure 4: Antagonistic effect of LPs at various stages of the development of phytopathogenic fungi illustrated by global growth reduction on solid medium (up), hyphal-induced swelling and collapse and conidia lysis (down) occurring within minutes after addition of LPs. Results obtained in our laboratory.
As revealed by transmission electron miscroscopy techniques, both phenomena most probably
result from membrane damaging by the LPs (Chitarra et al., 2003, Romero et al., 2007;
Etchegaray et al., 2008). LPs from Pseudomonas such as viscosinamide, massetolide,
Introduction-Chapter 2
33
putisolvin and orfamide also have significant impact on pathogenic fungi and oomycetes
either through their ability to lyse zoospores (Figure 4C) or to inhibit hyphal growth
(Raaijmakers et al., 2010). A few studies have revealed some insecticide activity of LPs from
B. subtilis. Surfactin and iturin were described for their antagonistic effect against fruit fly
Drosophila melanogaster (Assie et al., 2002) and LPs contained in a crude extract were
efficient at inhibiting the development of larvae of the mosquito Culex quinquefasciatus (Das
and Mukherjee, 2006). Thought that active doses are quite high (approx. 200 µM) and that
mechanisms underpinning such biocidal effect have not been investigated,, treatment with
LPs are presented as possible alternative to the use of the endotoxin producer B. thuringenesis
in the biocontrol of insect disease for which this bacterium is not efficient.
Like other antimicrobial peptides, LPs are not only membrane disruptive but can also
directly or indirectly act on intracellular targets and inhibit some functions such as enzymatic
activity. An example is the inhibition of phospholipase A2 in target fungal cells
(Vanittanakom et al., 1986). Whether this inhibition results from a direct interference with
enzyme structure/stability within the plasma membrane or from some repression of associated
gene expression is not clear. However, that may have some important metabolic consequences
since products released by this enzyme from membrane phospholipids are involved in
multiple cellular processes among which the synthesis of bioactive products (Köhler et al.,
2006). In the recent report on inhibition of toxic fumonisin B1 synthesis in Fusarium
verticillioides, it has been suggested that some products deriving from phospholipase A2
activity may be somewhere involved in the down-regulation of toxin genes transcriptions
observed upon treatment with fengycin (Hu et al., 2009). Whether the action of fengycin in
the inhibition of toxin production by Fusarium is direct or not, results from such studies allow
to extend application of LPs or their producing strains to the food safety sector. Such potential
has also recently been illustrated by the involvement of iturin in the inhibition of aflatoxin
producing Aspergillus isolates (Cho et al., 2009).
Bacterial LPs as elicitors of plant defense mechanisms
Another well established way for some beneficial rhizobacteria to provide plant protective
effect is through the stimulation of the plant immune system (Bakker et al., 2007). These
isolates are indeed able to reduce disease through the stimulation of a priming state in host
plant which allows an accelerated activation of defense responses upon pathogen attack,
leading to an enhanced resistance to the attacker encountered (Conrath et al., 2006). This
induced systemic phenomenon (ISR) can be globally viewed as a three-step process involving
Introduction-Chapter 2
34
sequentially, i) the perception by plant cells of elicitors produced by the inducing agents that
initiates the phenomenon, ii) signal transduction that is needed to propagate the induced state
systemically through the plant and iii) expression of defense mechanisms stricto sensu that
limit or inhibit pathogen penetration into the host tissues.
The potential of LPs as plant resistance inducers was demonstrated in 2007 for two
different molecules synthesized by Pseudomonas and Bacillus. Tran and collaborators
showed that massetolide A produced by Pseudomonas fluorescens retains ISR-eliciting
activity in tomato plants for the control of Phytophthora infestans, the causal agent of late
blight. It was demonstrated by testing the purified compound and by testing mutants impaired
in their production, in typical ISR assays where the inducing agent and the pathogen remain
spacially separated (Figure 5).
C LPs
Figure 5: Illustration of the ISR phenomenon based on disease reduction observed on leaves of various plants after treatment at the root level with lipopeptide (over)producing Bacillus isolates. Source: results obtained in our laboratory. C: control plants, LPs: treated plants. Results obtained in our labortory.
In bean, pure surfactins and fengycins to a lower extent but not iturins provided a significant
induced protective effect similar to the one induced by living cells of the producing strain (B.
amyloliquefaciens S499). In a complementary approach, experiments conducted on bean and
tomato showed that overexpression of both surfactin and fengycin biosynthetic genes in the
naturally poor producer B. subtilis strain 168 was associated with a significant increase in the
potential of the derivatives to induce resistance (Ongena et al., 2007). Moreover, the
macroscopic disease reduction induced by the surfactin overproducer was associated with
Introduction-Chapter 2
35
defense-related metabolic changes in the host plant tissues (Ongena et al., 2007). Treatment
of tobacco cell suspensions with surfactin but not fengycin nor iturin, also induced some
defence-related early events such as phosphorylation, Ca2+-dependent extracellular
alkalinisation and oxidative burst without causing any significant cell death (Jourdan et al.,
2009). The same study also showed that perception of surfactin by tobacco cells resulted in
the stimulation of defense pathways initiated by the enzymes phenylalanine ammonia lyase
(PAL) and lipoxygenase (LOX). It is thus clear that the three lipopeptide families retain
differential abilities to stimulate defense reactions in different plant species.
Other applications for Bacillus LPs
Because of their potential to disturb the integrity of the biological membranes, cyclic
lipopeptides secreted by Bacillus and Pseudomonas are potentially toxic for auxiliary
microflora and higher microorganisms. However, based on some recent data reported here, it
is now obvious that LPs cannot be simply viewed as molecular pneumatic drills creating
irreversible pores, cytosolic leakage and cell death. Despite similar global structures and
apparent physico-chemical traits, not all LPs do provoke lysis of a given cell target and it also
became clear that some lipopeptides may induce some modifications in basic cell processes
without causing any detrimental leakage in the plasma membrane.
These compounds thus retain other natural functions beside those directly involved in
biocontrol of plant diseases. These properties schematized in Figure 6 not only favour the
developmental processes of the producing isolates in their natural habitat in terms of growth
and niche colonization but may also strongly influence the interactions with the other
organisms sharing the microenvironment.
Besides plants, surfactins could also interfere with several stages of the immune
processes in animals. They indeed display lipopolysaccharide-binding and neutralizing
activities (Hwang et al., 2005; Takahashi et al., 2006) and antitumor activity. This activity is
based on apoptosis induction via cellular Ca2+ augmentation, induction of MAPK and related
up/down regulation of protein factors associated with apoptosis (Wang et al., 2007). Another
in depth study demonstrated anti-proliferative activity of surfactin on human colon carcinoma
cell line related to induction of apoptosis and cell cycle arrest (Kim et al., 2007). Surfactin
also retains some anti-inflammatory (Huang et al., 2007) properties and can also inhibit
platelet aggregation (Kim et al., 2006). Finally these LPs could inhibit AMPc
phosphodiesterase, alkaline phosphatase activities (Bortolato et al., 1997) and either inhibit or
Introduction-Chapter 2
36
enhance phospholipases A2 depending on their origin (Kim et al., 1998). These activities
open the way to interesting biomedical and pharmaceutical applications (Pirri et al., 2009).
Colonization, biofilm
Antiviral
Antiprotozoal
Antibacterial
Antioomycete Antifungal
Chelation,solubilization
MotilityVirulence,Immunization
Figure 6. Natural functions of lipopeptides secreted by plant-associated bacteria with special emphasis on those involved in biological control of plant diseases (in bold). Source: figure adapted from Raaijmakers et al. (2010).
Finally, some of these surface-active LPs are also considered as among the most
powerful biosurfactants isolated so far. They retain exceptional emulsifying/foaming
properties and high potential for the solubilization of hydrophobic compounds. LPs have
thereby received considerable attention for various environmental applications such as
bioremediation of soils polluted by oils or recalcitrant compounds.
Introduction-Chapter 2
37
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Chapter 3. In situ production of Bacillus antibiotics involved in the biocontrol of plant diseases: more evidences
needed
Nihorimbere Venant, Thonart Philippe and Ongena Marc
To be submitted as review paper after including our contribution to the topic (Chapters 5 and 6)
Introduction-Chapter 3
41
Abstract
Interest in biological control of plant pathogens has been stimulated in recent years by trends
in agriculture towards greater sustainability and public concern about the use of hazardous
pesticides. There is now unequivocal evidence that antimicrobial compounds play a key role
in the suppression of various soilborne plant pathogens by antagonistic microorganisms. The
significance of antibiotics in biocontrol, often has been questioned because of the indirect
nature of the supporting evidence and the perceived constraints to antibiotic production in
rhizosphere environments. Reporter gene systems and bio-analytical techniques have
demonstrated that antibiotics are effectively produced in the rhizosphere of a variety of host
plants, although in limited quantities. Several abiotic factors such as oxygen, temperature,
specific carbon and nitrogen sources, and microelements have been shown to influence
antibiotic production by bacteria biocontrol agents. Among the biotic factors that may play a
determinative role in antibiotic production are the plant host, the pathogen, the indigenous
microflora, and the cell density of the producing strain. Alternatives to antimicrobials, such as
biocontrol agents of Bacillus spp., are being developed and marketed but slowly because of
their higher production cost. A discussion of how and why these antibiotics are produced in
rhizosphere by soil bacterial biocontrol agents is given.
Bacillus as biopesticide
Biological control through the use of natural antagonistic microorganisms has emerged as a
promising alternative to chemical pesticides for more rational and safe crop management
(Montesinos, 2007). There is a large body of literature reporting the beneficial effect of
rhizosphere-associated bacteria for the stimulation of plant growth and for the reduction of
diseases caused by phytopathogens (Lucy et al., 2004; Lugtenberg and Kamilova, 2009).
Most of the bacterial strains exploited to date as biopesticides belong to the genera
Agrobacterium, Bacillus and Pseudomonas (Flavel, 2005). By taking benefits from the
nutrients exuded by the host plant, beneficial strains efficiently colonize root systems and the
surrounding soil layer (rhizosphere). In turn, they beneficially influence the plant. From a
very global point of view, improvement in plant health/productivity may be mediated via two
different mechanisms e.g. protection against pest and pathogens and promotion of host
nutrition and growth. The beneficial protective effect of these bacterial agents may rely on
different mechanisms. Antibiosis through the production of antifungal metabolites and
antibiotics is probably the best known and most important mechanism used by biocontrol
bacteria to limit pathogen invasion in host plant tissues. Beside this direct growth inhibitory
Introduction-Chapter 3
42
activity, other widely recognized mechanisms involved in biocontrol mediated by rhizosphere
bacteria are: competition for an ecological niche/substrate, production of inhibitory
allelochemicals, and induction of systemic resistance (ISR) in host plants to a broad spectrum
of pathogens (Figure 1) (Mukerji et al., 2006).
Direct antagonism through the production of highly active antibiotics is supposed to
play a pivotal role in the multiptrophic interactions taking place in the phytosphere and
thereby in the global protective effect against phytopathogens (Stallings, 1954; Thomashow et
al., 2008). The exploitation of these natural antagonistic interactions has been a driving force
in research on the biological control of plant pathogens over the past century. Some species of
the Bacillus genus such as B. subtilis or B. amyloliquefaciens retain the potential to produce
many different antibiotics (Koumousti et al., 2004; Stein, 2005). Several strains belonging to
these species were reported effective for the biocontrol of multiple plant diseases caused by
soilborne (Chen et al., 2009) or post-harvest pathogens (Kotan et al., 2009). The production
of antimicrobial agents thus appears to be an important criterion in the evaluation of the
potential use of Bacillus strains as biocontrol.
Figure 1. Beneficial effects of Bacillus subtilis on plants
They are numerous reports suggesting a role for some specific antibiotics in antagonism
based only on in vitro antimicrobial activity displayed by the producing strain. However, it
just highlights some intrinsic potential of that strain to synthesize antibiotics under laboratory
conditions. One needs to consider at least two other approaches to provide evidence for the
direct involvement of antibiotic production in beneficial bacteria-mediated disease
Introduction-Chapter 3
43
suppression. First by using either biosynthesis suppressed mutants that are no longer able to
prevent phytopathogens to cause damage in plants or antibiotic-overproducer isolates that
protect plants against pathogens more effectively than the wild type strain. Second, by
showing that the antibiotic applied in a pure form is also effective at reducing disease.
Furthermore, providing evidence for an efficient in situ production is a third approach that has
to be combined with the others to unambiguously demonstrate the involvement of a particular
compound in the global biocontrol activity of the producing strain. In soil, rhizosphere-
specific factors may strongly impact the physiology of the biocontrol strain developing on
roots. It is thus crucial to demonstrate that the antibiotic is actually secreted and may
accumulate in the microenvironment in biologically relevant quantities.
This review aims at showing that only a very limited number of studies have
successfully linked antibiotic-based biocontrol with efficient in planta production of the
compound. Despite many recent conceptual and technical advances, the investigation of
antibiotic gene expression during interaction with plants and of their secretion by Bacillus
isolates growing in the phytosphere is still in its infancy. This is however crucial for our
global knowledge of Bacillus fitness in its ecological niche regarding antibiotics synthesis.
More particularly, it is also important to understand the biotrophic interactions occurring in
the micro-environment which is pivotal for optimizing biocontrol strategies using these
organisms.
The Bacillus antibiotic arsenal to combat plant diseases
In the case of Bacillus, members of multiple species such as B. amyloliquefaciens, B. subtilis,
B. cereus, B. licheniformis, B. megaterium, B. mycoides, and B. pumilus have been reported
for developing antibiotic-based antagonism toward a number of phytopathogens (Choudhary
and Jorhi, 2009). Bacillus subtilis has an average of 4-5% of its genome devoted to antibiotic
synthesis and has the potential to produce more than two dozen structurally diverse
antimicrobial compounds (Stein, 2005; Ongena and Jacques, 2008). In strain FZB42, which is
proposed as paradigm for plant-associated Bacillus amyloliquefaciens, an even larger part of
the genome (~8%) is seemingly involved in antibiotic synthesis.
Among the vast array of biologically active molecules synthesized by Bacillus, some
have been reported for their inhibitory activity against plant pathogens (Figure 2). B. cereus
UW85 produces two fungistatic antibiotics, zwittermicin A and kanosamine, that are
suggested to contribute to the suppression of damping-off disease of alfalfa caused by
Introduction-Chapter 3
44
Phytophthora medicaginis (Silo-Suh et al., 1994). Zwittermicin A may also control the fruit
rot of cucumber (Smith et al., 1993) and suppress other plant diseases (Silo-Suh et al., 1998).
A B C
D
Figure 2: Some antibiotics (other than lipopeptides) from Bacillus with proven or putative biocontrol-related activity. A: 3,3-Neotrehalosadiamine, B: Bacilysin, C: Amicoumacin, D: Ericin S. The figure reproduced from Stein (2005).
Polyketides are synthesized via PKS modularly organized assembly lines that require specific
domains and follows the same logic as in NRP systems involved in the synthesis of peptidic
compounds (Chapter 2). Bacillaene, difficidin and macrolactin are polyketides displaying
broad spectrum antibacterial activities and that may be involved in the biocontrol activity of
the producing strain such as in the case of fire blight, a serious disease of orchard trees caused
by Erwinia amylovora (Chen et al., 2009). The phosphono-oligopeptide rhizocticin produced
by B. subtilis displays some antifungal and nematicidal but no bactericidal properties.
Peptide compounds represent the predominant class of Bacillus antibiotics. They are of
various sizes, may be composed entirely of amino acids but some contain other residues. They
are synthesized either by ribosomal or non-ribosomal mechanisms. Cyclic or linear
oligopeptides, basic peptides and aminoglycoside antibiotics usually occur (Stein, 2005). Low
molecular weight and hydrophobic or cyclic structures, with unusual constituents like D-
amino acids, are also common characteristics of peptide antibiotics normally synthesized by
Bacillus. Moreover, they are generally resistant to hydrolysis by peptidases and proteases of
animal and plant origins (Katz and Demain, 1977). Bacillus brevis (Brevibacillus brevis) and
Bacillus polymyxa (PaeniBacillus polymyxa) produce gramicidin S and polymyxin B peptide
antibiotics that strongly inhibited in vitro the phytopathogen Botrytis cinerea at the various
stages of germination, growth and extra-cellular enzyme production. These antibiotics also
Introduction-Chapter 3
45
exhibited significant biocontrol activity under natural field conditions against the Botrytis
grey mould on strawberry (Haggag, 2008). More simple molecules such as the dipeptide
bacilysin (L-Ala linked to the non-proteinogenic amino acid L-anticapsin) also retain strong
bactericidal effect and are seemingly involved in the control of some phytopathogens (Chen et
al., 2009). Another group of peptide antibiotics usually produced by Bacillus subtilis is
composed by lanthionines with inter-residual thioether bonds that are formed through post-
translational modification of ribosomally synthesized precursor peptides (Stein, 2005). These
compounds display strong antibacterial properties mostly against gram-positive bacteria but
their involvement in the biocontrol activity of plant-associated Bacillus isolates has not been
clearly demonstrated so far. However, some lantibiotics (subtilin and ericin) also display other
functions than pure antibiotic activity. They are autoregulated via two-component regulatory
systems and function as pheromones for quorum-sensing in some B. subtilis strains (Stein,
2005).
Lipopeptides as crucial ingredients for biocontrol
As described in Chapter 2, a major class of peptide antibiotics in the context of biological
control are lipopeptides (LPs) of the surfactin, iturin and fengycin families. According to our
current knowledge, these lipopeptides are by far the most important antibiotic compounds
involved in multitrophic interactions occurring in the plant microenvironment and thereby
they are probably the most investigated for their role in the global biocontrol activity of
specific strains from Bacillus species. They are composed of a lipid tail linked to a cyclised or
partially cyclised oligopeptide and can be synthesized by various species of Bacillus. Some of
these surface-active compounds are considered as among the most powerful biosurfactants
isolated so far. LPs actually display versatile functions in the ecology of the producing strains
in the phytosphere and notably in interactions with co-existing organisms, including bacteria,
fungi, oomycetes, protozoan, predators and plants. When produced by plant beneficial
rhizobacteria, LPs have thus demonstrated to play a crucial role in the antagonism developed
towards phytopathogens and thus in the biocontrol potential of the strains (Chapter 2). In this
case, the proposed primary mode of action of LPs is pore formation in membranes leading to
an imbalance in transmembrane ion fluxes and cell death (Baltz, 2009). Beside their
antimicrobial properties, LPs are also involved in root colonization as well as in the systemic
stimulation of host plant immune system. The current knowledge of LPs activities directly
involved in biocontrol is summarized in Figure 3. Beside these traits, LPs may also have
different roles in the development and survival of B. subtilis strains in their natural habitat.
Introduction-Chapter 3
46
These include increasing of the surface area and bioavailability of hydrophobic water-
insoluble substrates, heavy metal binding, bacterial pathogenesis, quorum-sensing, motility
and biofilm formation (Mulligan, 2005; Ron and Rosenberg, 2001; Raajmakers et al., 2010).
Surfactins Iturins Fengycins
Powerful biosurfactants,
Virucidal activity,
Bactericidal activity,
Strong fungitoxic activity (yeastand filamentous fungi),
Strong fungitoxic activity(filamentous fungi)
Root colonization(biofilm formation, surface motility)
Direct antagonism toward phytopathogens
Involvement in plant systemic resistance,elicitation
Figure 3: Specific biocontrol-related activities for the three families of Bacillus lipopeptides.
Linking basic cellular regulatory processes and environmental conditions affecting lipopeptide production Even if few information is currently available concerning regulation of fengycin biosynthesis,
it has become clear that LP production by Bacillus is modulated at the gene level by complex
regulatory events (presented in more details in Chapter 2 for surfactin and iturin). As a global
picture, regulation of surfactin genes is typically multi-component and takes part of a complex
regulatory cascade involved in multiple basal cellular central processes and that governs
several differentiation pathways in B. subtilis. LP production is growth-phase dependent and
may be linked to population-driven responses such as quorum-sensing via the ComX
pheromone in the case of surfactin. Two-component response regulators that control many
cellular processes like transition state are also involved in fine tuning the expression of
biosynthesis genes for both three families.
On another hand, plants roots usually release specific soluble carbon compounds as well
as pH and redox-modulating factors, complexing agents (siderophores, phenols,
carboxylates), antimicrobials (antibiotics, quorum-sensing inhibitors) and specific stimulatory
compounds. They may also physically shape specific habitat conditions and the combination
Introduction-Chapter 3
47
of all these events results in the so-called rhizosphere effect. In other words, plants usually
create a specific environment for microbial development in the soil surrounding roots
(Chapter 1). This may results in a selection of particular microbial populations and/or in a
drastic modification of the cellular physiology of established species associated with roots
(Hartmann et al., 2009). This rhizosphere effect depends both qualitatively and quantitatively
on the plant species, cultivar and even developement stage. Other environmental factors
inherent to the soil ecology are the co-existence of an auxiliary microflora sharing the
ecosystem, the soil type and heterogeneity and other abiotic parameters such as humidity, pH,
temperature or dissolved oxygen availability. All these rhizosphere factors may potentially
impact on the expression of biocontrol traits by plant beneficial strains and corollary on the
biosynthesis of specific metabolites responsible for these activities like Bacillus lipopeptides.
The influence of some environmental factors may be quite easily estimated under well-
controlled laboratory conditions in flask-based cultures or by using bioreactors in fed-batch or
chemostat modes. By contrast with batch culture where the exhaustion of a specific nutrient
terminates the exponential growth phase, biomass concentration in the chemostat culture is
usually controlled and maintained stable in concentration by the permanent limitation of a
single defined nutrient (soluble iron in this case). It allows the study of metabolite synthesis
upon fixed growth rate and constant cellular physiological state. Here are summarized some
works performed in vitro which provided some valuable information about the impact of
rhizosphere-specific factors on the secretion of Bacillus LPs.
Carbon and energy resources are scarce in bulk soil and, consequently, metabolic levels
in microorganisms are low. Microorganisms in the phytosphere depend on substrates liberated
from the root or shoot for their growth and consequently, the host plant greatly influences the
quantity and composition of indigenous microorganism populations as well as the expression
of antibiotic biosynthetic genes. This was illustrated by Milner and collaborators (1996) who
showed that production of the antibiotic kanosamine in Bacillus cereus was enhanced by
more than 300% by the addition of alfalfa seedling exudates to the culture medium. The
surfactin-apparented lychenysin also accumulates differentially in the culture broth upon
growth in various media differing in C source (Li et al., 2008). Akpa and collaborators (2001)
have also demonstrated the influence of the nature of carbon source on the pattern of
surfactins but not mycosubtilins produced by B.subtilis. This low flexibility in the
mycosubtilin homologue eventail could be related to the rather high selectivity of the domain
responsible for fixation and loading of the fatty acid in the mycosubtilin synthetase (Hansen et
al., 2007). Nutrient status may also be viewed in terms of availability of some essential
Introduction-Chapter 3
48
oligoelements and improvement of surfactin production was obtained upon elevated Mg, K,
Mn as well as Fe cations concentrations in the medium (Wei and Chu, 2002; Wei et al., 2004;
2007).
At least for surfactin, a dependence of Bacillus LP synthesis to the nitrogen metabolism
has been evidenced as the production clearly increased under dissimilatory nitrate metabolism
compared to the aerobic conditions of ammonium consumption (Davis et al., 1999). This
study also showed the influence of oxygen concentration in the surrounding medium on LP
production rate since nitrate may be assimilated in both aerobic and anaerobic conditions.
However, the question remains open in how far these two parameters (N status and O2) are
indissociable in Bacillus cells to globally influence LP production through the form of
nitrogen metabolism or alternatively if they may trigger their own regulatory processes
independently. Nevertheless, a positive influence of O2 limitation on production of surfactin
has also been demonstrated by modifying the oxygenation method (Lee et al., 2007) or by
modulating the oxygen transfer rate in bioreactor (Yeh et al., 2006). Such a process appears
not to be adapted to Bacillus subtilis S499, which produced higher surfactin yield in better
aeration conditions (Hbid et al., 1996; Jacques et al., 1999). Production of lichenysin by
Bacillus licheniformis was also conducted in anaerobic conditions. By contrast, mycosubtilin
expression is oxygen dependent (Guez et al., 2008). These results should be considered with
the view that a low oxygen status prevails in the rhizosphere of many soils (Hojberg et al.,
1999).
On another hand, root-colonizing rhizobacteria such as Bacillus or Pseudomonas
typically develop in microcolonies or form biofilms and their actual physiology is thus
probably not related to a planktonic state. Few studies have accurately evaluated the
consequences of growing on solid support on LP synthesis by B. subtilis but it is clear that at
least surfactin and iturin productions are efficient in swarming/spreading or colony-forming
cells (Leenders et al., 1999; Debois et al., 2008). In another approach, Gancel and
collaborators (2009) have also demonstrated an enhancement of surfactin production upon
cell immobilization on polymer particles.
In the same context of basic cellular processes, Bacillus cells on roots most obviously
evolve in a nutrient-starved state that would drive cell physiology to slow growth rate. Such a
slow-growth-imposed state may be more favorable for surfactin synthesis compared to higher
growth rates based on the expression level of the surfactin srfA biosynthetic genes in Bacillus
subtilis (Kakana, 2005).
Introduction-Chapter 3
49
Other physico-chemical parameters such as soil moisture, temperature and pH may also
drastically influence LP synthesis in Bacillus (Georgakopoulos et al., 1994; Cosby et al.,
1998; Fickers et al., 2009). Higher temperatures (37 °C) favoured surfactin synthesis of
Bacillus subtilis RB14 isolated from compost (Ohno et al., 1995) and ATCC6633, but not of
Bacillus subtilis S499 (Jacques et al., 1999). A 30-fold increase in mycosubtilin production
was observed when the temperature was decreased from 37 °C to 25 °C. This was observed
for both strain ATCC6633 and its derivative BBG100, which is a constitutive mycosubtilin
overproducer. However, the results suggest that the observed phenotype might originate from
a higher mycosubtilin synthetase turnover at lower temperature rather than reflecting changes
in the regulatory mechanisms per se (Fickers et al., 2008). The relative abundance of the
various co-produced mycosubtilin homologues was also affected by temperature. Surfactin
gene expression is pH-dependent.
Many rhizosphere-related abiotic parameters may thus impact the biosynthesis of
lipopeptides from Bacillus spp. This is certainly due to the complex regulation of lipopeptide
operon expression involving several pleiotropic regulators. However, intracellular pools of
synthetase substrates (amino acid residues and fatty chain) and turn-over of the enzymes have
also to be taken into account.
Evidence for in situ lipopeptide production by Bacillus: so few examples!
Studies conducted in vitro may thus be useful to evaluate the influence of a number of factors
prevailing in the natural environment. However, some conditions such as very specific soil
characteristics or exogenous micro- or macroflora with putative competing effect, are very
difficult to re-create in artificial systems. The influence of some parameters inherent to the
rhizosphere is thus merely not possible to assess under artificial conditions without
introducing some bias. The study of antibiotic production by Bacillus in natural conditions for
plant cultivation is thus necessary and retains the advantage of being conducted in an
integrated system where all the parameters evoked above are interplaying. Results presented
here deal almost exclusively with lipopeptides, illustrating our inability to find any work
reporting consistent study of in situ production of other Bacillus antibiotics in the
phytosphere.
Direct detection
As an illustration of the analytical power of the most recent and optimized mass spectrometry
technologies, matrix assisted laser desorption ionization-time of flight MS was used to detect
and identify the whole array of Bacillus LPs and their biosynthetic intermediates, not only in
Introduction-Chapter 3
50
culture filtrate but also at the whole cell level in a very rapid and sensitive way (Vater et al.,
2002; 2009). A qualitative and quantitative image of the surfactin homologue patterns
produced by Bacillus subtilis under various experimental conditions has also been obtained by
coupling HPLC with electrospray ionization MS (Li et al., 2008). Other studies that used
MALDI-TOF mass spectrometry to very precisely compare the lipopeptide products
synthesized by various B. subtilis strains also confirm that the potential to produce surfactins
and iturins is widespread in this species (Athukorala et al., 2009; Stein, 2008; Price et al.,
2007). Also time of flight secondary ion mass spectrometry (TOF-SIMS) imaging appears to
be a yet emerging but extremely powerful technology to study the production of lipidic
molecules by microbes developing at the surface of solid supports. It was recently used by
Debois and collaborators (2008) to study the heterogeneity of surfactin secretion in a
swarming pattern of Bacillus cells.
HPLC and mass spectrometry or a combination thereof have also been used successfully
for identification and quantification of antibiotics directly in rhizosphere extracts. However,
by contrast with the multiple examples available for antibiotics from other bacterial genera
such as phenazines or DAPG formed by Pseudomonas sp., only few biocontrol-related
antibiotics synthesized by Bacillus species have been studied for their in situ production.
Surfactin and/or iturin were recovered in biologically relevant quantities from cucumber roots
surrounding soil inoculated with Bacillus strain QST173 (Kinsela et al., 2009) and from
tomato rhizosphere inoculated with strain BGS3 (Ongena et al., 2007) and strain RB14-C
(Asaka and Shoda, 1996). Fengycins and iturins were also occasionally detected in the
phyllosphere and on fruits, illustrating that growth conditions on these plant tissues are
conducive for their biosynthesis at least by some strains (Romero et al., 2007; Touré et al.,
2004). In all cases, accumulation of LPs in or around plant tissues was actually correlated
with disease suppression.
For Bacillus isolates, there is a competitive advantage for efficient co-production of
surfactin, fengycin and iturin with specific roles and targets in the context of biocontrol of
plant diseases (Figure 2) (Ongena and Jacques, 2008; Raaijmakers et al., 2010). Several
strains efficiently co-produces the three lipopeptide families under well defined and optimized
in vitro conditions. However, almost nothing is known about the actual physiology of Bacillus
cells developing on plant and how far it may impact on the lipopeptide signature secreted in
situ. Also, whether in situ productivities may generate rhizospheric LP concentrations
sufficient to provide any biological effect remain questionable. These molecules are generally
active at 2-30 µM depending on the role and target organism considered. Interestingly,
Introduction-Chapter 3
51
quantities corresponding to micromolar concentrations of both surfactin and iturin were
detected in the cucumber rhizosphere colonized by strain QST713 (Kinsella et al., 2009) but
additional examples of consistent in vivo LPs secretion by other beneficial Bacillus strains are
required.
Inherent difficulties for evaluation of actual antibiotic concentration in soil
The limited number of reports on in situ production of bacterial antibiotics may reflect the
inherent difficulties in detecting or quantifying these small-size compounds in complex
environments like rhizosphere. This is particularly true for molecules such as LPs that can
tightly be adsorbed on the surface of soil particles because of their amphiphilic character or
because of unspecific, rapid and irreversible embedment in lipid structures at the surface or in
the cytosol of target and non-target organisms present in the diffusion zone. Moreover, the
stability of LPs and other peptide antibiotics in complex environments is questionable. The
cyclic nature and the alternance of L/D configured amino acids not only provide some
conformational advantages but should also enhance the level of resistance to degradation by
proteolytic enzymes from other organisms sharing the same niche. However, several works
obviously suggest that such decreased susceptibility to enzymatic degradation is only limited.
For Bacillus, Asaka and Shoda (1996) showed that iturin levels declined within 25 days in
sterile soil, while surfactin levels remained stable. The authors suggested that iturin may have
been degraded by microorganisms or leached from soil by watering, or was not detectable due
to irreversible binding to soil particles (Asaka and Shoda, 1996). It was also shown that an
endoprotease from Staphylococcus aureus hydrolyzed surfactin, thereby generating a linear
peptide (Grangemard et al., 1999). In addition, Nitschke and Pastore (2004) suggested that
protease activity contributed to a decrease of the surfactin levels in culture medium of B.
subtilis strain 21332. It globally means that antibiotic amounts that can be recovered from the
phytosphere may be limited and one needs to optimize the extraction procedures and use the
most powerful and sensitive detection methods for an accurate estimation of the quantities
that may be actually secreted by root colonizing Bacillus.
Gene expression
On another hand, the detection of antibiotic biosynthetic genes provides insight into the
distribution of antibiotic producers in nature and serves as a first indication that antibiotics
may be present (Thomashow et al., 2008). However, PCR-based molecular detection of such
antibiotic producers has inherent limitations keeping in mind that those genes may be present
but not all readily expressed under in vitro or natural conditions (Athukorala et al., 2009;
Introduction-Chapter 3
52
Joshi and McSpadden Gardener, 2006). In that context, the demonstration of an efficient
expression of biosynthetic genes in the environment is more informative and must also be
considered in support to direct detection of the antibiotic to provide conclusive evidence for in
situ production. Reporter gene systems are widely used as a marker to monitor populations of
introduced strains and occasionally to provide information on the transcriptional activity of
specific biocontrol-related antibiotic genes (Loper and Lindow, 1997; Thomashow et al.,
2008). However, it was mainly done for other bacterial genera and to our best knowledge,
there was no published work concerning Bacillus using some gene reporter system to provide
information on the transcriptional activity of genes coding for LPs on roots yet tools do exist.
Unstable variants of green fluorescent protein (gfp) have been used successfully as reporters,
and the corresponding gfp genes are fused to bacterial genes of interest, allowing gene
expression to be observed in situ (Leveau and Lindow, 2002; Haas and Keel, 2003). In
contrast to conventional reporters such as the stable enzyme β-galactosidase, which can only
monitor increases in gene expression, unstable gfp can report both “on” and “off” states. All
together, these molecular approaches are fundamentally indirect and subject to limitations, but
they can provide a degree of sensitivity not achieved by direct analysis.
Next to these genetic approaches, immunological assays have been proposed as sensitive
approach to detect and quantify antibiotics in situ, especially in plant associated environments
where relatively low amounts are produced and where plant-derived compounds may interfere
with chemical detection. For example, immunological detection of Bacillus LPs is still in its
infancy, but has been successfully adopted to monitor in situ production of the LP
syringopeptin by Pseudomonas pv. lachrymans, the causal agent of angular leaf spot. The
competitive ELISA assay developed by Fogliano and colleagues appeared to be
approximatively 100 times more sensitive than HPLC analysis and did not require extraction
of plant material with organic solvents (Fogliano et al., 1999). Specific antibodies will also be
highly instrumental to study the localization of LPs or other antibiotics in complex
environments or to monitor their fate and stability after application to soil, plant tissues or
other habitats.
Conclusion
The overall lack of information on LP gene expression during plant interaction is in part due
to the fact that Bacillus has been primarily studied for its biochemical and genetic traits. By
contrast with some other plant beneficial bacteria such as Pseudomonas, research on Bacillus
as a biocontrol agent has focussed mainly on the protective effect under practical use in the
Introduction-Chapter 3
53
field or greenhouse. Several Bacillus strains have great potential for biological control of
various plant diseases. However, there is still crucial lack of data on the level of antibiotic
production in the natural environments. This would be also helpful for a better understanding
of the complex biotrophic interactions occurring in the soil micro-environment but also more
globally, for Bacillus agricultural fitness.
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Yeh M.S., Wei Y.H. & Chang J.S. (2006). Bioreactor design for enhanced carrier-assisted surfactin production with Bacillus subtilis. Process Biochem. 41: 1799-1805.
57
OBJECTIVES OF THE THESIS
58
OBJECTIVES OF THE THESIS
As stated in the introducive chapters, Bacillus has been primarily studied for its biochemical
and genetic traits. By contrast with some other plant beneficial bacteria such as Pseudomonas,
research on Bacillus as a biocontrol agent has focussed mainly on the efficacy of the
protective effect under practical use in the field or greenhouse. However, despite many recent
conceptual and technical advances, the investigation of antibiotic gene expression during
interaction with plants and of their secretion by Bacillus isolates growing in the phytosphere
is still in its infancy. Thus, there is still a crucial lack of data on the antibiotics production by
these bacteria in the natural environment. The main objective of our thesis is to contribute at
filling this gap of knowledge.
In a first part, we wanted to evaluate the potential of strain S499, selected out of more
than forty other soil isolates, as a microbial biocontrol agent. To that end, we tested S499 for
its ability to control an important disease under field conditions in Burundi (Chapter 4 in
Results section). In the next part of this work, we wanted to gain some insight into how
lipopeptide patterns of surfactin, iturin and fengycin families are produced by B.
amyloliquefaciens strain S499 developing on roots of tomato plants and assert environmental
factors that inherently influence their production (Chapter 5). Further exploring surfactin gene
expression and production upon growth of Bacillus on tomato roots under greenhouse
conditions was our last objective. Another strain, Bacillus subtilis BGS3, containing a
reporter gene system that helps strain discrimination among many other bacteria, was used.
59
RESULTS
Results-Chapter 4
60
Foreword on Chapter 4
Tomato is one of the most important crops food worldwide. However, this plant productivity
is very low in Burundi because of, amongst other factors, primitive agricultural practices most
of which are meant for subsistence farming, led to continuous reduction in yields. This
situation is worsened by increased infection by phytopathogenic fungi such as Fusarium,
Pythium spp. and others causing root rot in a wide variety of crops especially tomato. Efforts
to combat these problems using fungicides and chemical fertilizers were not only
unsuccessful but are also unaffordable to the local farmers. In contrast to research on the
suppression of diseases with chemical pesticides, to date no study has yet addressed the
introduction of antagonistic microorganisms in the plant rhizosphere in Burundi.
The first objective of our thesis was thus to test selected rhizobacteria, with potential
antimicrobial activity as biocontrol agents to protect tomato. In this context, we first screened
for Bacillus-like strains isolated from tomato rhizosphere samples collected in local fields in
Burundi. Their antagonistic potential against some fungi well known for their severe
pathogenicity towards many economically important crops was compared to the one of B.
subtilis/amyloliquefaciens strain S499. However, none of them displayed greater potential and
we decided to test S499 for its biocontrol activity in field trials because of the scientific and
technological background in our laboratory. More specifically, the main part of this chapter
will describe the beneficial effect of tomato treatment with spores suspensions from strain
S499 on plant growth and tolerance to an uncommon fungal isolate under agricultural field
conditions in Burundi.
Results-Chapter 4
61
Chapter 4. Beneficial effects of Bacillus subtilis on field-grown tomato in Burundi: Reduction of local Fusarium
disease and growth promotion
Published in African Journal of Microbiology Research (2010) 4 (11): 1135-1142.
(except data concerning Bacillus strain screening in the first section of Material and
method and in second section of the Results)
Venant Nihorimbere1,3†, Marc Ongena1†, Hélène Cawoy1, Yves Brostaux2, Pascal Kakana3 and
Philippe Thonart1
1 Walloon Centre of Industrial Biology, Gembloux Agricultural University, Passage des
déportés, 2, B-5030 Gembloux, Belgium. 2 Statistics, informatics and applied mathematics, Gembloux Agricultural University, Passage
des déportés, 2, B-5030 Gembloux, Belgium. 3 Faculty of Agronomy, University of Burundi, PO Box 1550, Bujumbura, Burundi.
†Both authors equally contributed to this article.
Results-Chapter 4
62
Abstract
In this work, we first determined the in vitro antagonistic activity of 44 native strains of
Bacillus spp. isolated from the tomato rhizosphere against some fungi well known for their
severe pathogenicity towards many economically important crops throughout the world. The
bacterial isolates showed differential levels of antagonistic activity against the
phytopathogenic fungi tested in vitro on PDA medium. Four of them were selected to be
stronger inhibitors of at least two fungi but globally, none displayed a superior potential
compared to strain S499 which is commonly used in our laboratory. The biocontrol potential
of this S499 strain was thus further evaluated on tomato in open field sites in low altitude area
of the plain of Imbo in Burundi. This strain was tested in order to reduce the impact of an
important fungal disease giving rise to large losses in local plantings. The causing pathogen
was isolated from diseased leaves at different locations in the fields and identified as
Fusarium most probably related to the semitectum species according to the fermentation
profile, morphology and gene homology. Results of assays performed in two successive years
on the same site indicate that bacterial treatment on seeds significantly increased growth and
fruit yield of tomato plants and also provided a high level of protection against the disease
caused by the Fusarium pathogen. This is the first reported study on this disease and based on
the collected data, Bacillus subtilis S499 may represent an effective solution as biocontrol
agent where other, chemical, options have failed.
Keywords: Bacillus subtilis, biocontrol, Fusarium disease, tomato plants
Results-Chapter 4
63
Introduction
Root colonizing bacteria that exert beneficial effects on plant development through direct or
indirect mechanisms have been defined as Plant Growth-Promoting Rhizobacteria (PGPR).
These bacteria include several genera such as Acinetobacter, Agrobacterium, Arthrobacter,
Azospirillum, Bacillus, Bradyrhizobium, Frankia, Pseudomonas, Rhizobium, Serratia, and
Thiobacillus (Flavel, 2005) but the most well-known and commonly-used strains belong to
the Pseudomonas and Bacillus genera (Kloepper et al., 2004; Haas and Défago, 2005). In
addition to promoting plant growth, PGPR are also employed to control plant pathogens,
enhance efficiency of fertilizers, and degrade xenobiotic compounds (Clayet-Marcel et al.,
2001; Choudary and Johri, 2008; Berg, 2009).
Some Bacillus strains with PGPR activity are among the most exploited bacteria as
biocontrol agents against plant diseases (Flavel, 2005). Bacillus isolates are considered to be
safe microorganisms and hold the remarkable abilities of synthesizing a vast array of
beneficial substances (Stein, 2005) for agronomic and industrial purposes. They produce
endospores which warrant their prevalence under different environmental conditions and
facilitate its long-term storage and easy development of reliable formulations. One of the most
commonly used and well-studied organisms, the rhizobacterium Bacillus subtilis, has an
average of 4-5% of its genome devoted to antibiotic synthesis and has the potential to produce
more than two dozen structurally diverse antimicrobial compounds (Stein, 2005). Among
these antimicrobial compounds, cyclic lipopeptides (LPs) of the surfactin, iturin and fengycin
(or plipastatin) families have well-recognized potential uses in biotechnology and
pharmaceutical applications because of their surfactant properties (Ongena and Jacques,
2008). Several mechanisms have been postulated to explain how these beneficial
rhizobacteria stimulate plant growth. Some strains secrete antibiotics that can directly inhibit
the growth of fungal, oomycete, and bacterial plant pathogens (Mckeen et al., 1986; Touré et
al., 2004; Romero et al., 2007). Some other strains of B. subtilis suppress diseases by
inducing host defenses (Kloepper et al., 2004). However, it is likely that the most effective
biological control strains act via multiple mechanisms. For example, B. subtilis strain S499
can inhibit fungal pathogens directly through antibiosis, but it also was found to induce
resistance to foliar pathogens when it was applied to the plant root (Ongena et al., 2005a; b).
Such induction of enhanced defensive capacity can be systemic as seed-treatment with
bacteria at the time of seeding was shown to trigger protective effects on above-ground parts
(van Loon et al., 1998). The induced resistance constitutes an increase on the level of basal
Results-Chapter 4
64
resistance to several pathogens simultaneously, which is one of the benefits under natural
conditions where multiple pathogens exist (van Loon and Glick, 2004).
Tomato is intensively cultivated in Burundi, mainly in low-altitude regions of the
country (800 to 1200 m), which yields three quarters of the national production of tomato
fruits. Recently, however, this plant has been experiencing significant losses from pathogenic
fungal attacks. The causal agent(s) in this case adversely affects plant survival and growth
before flowering. The resulting crop losses are increasing and the global fruit production in
the last five years only represents 52% of the average yield recorded in the preceding five-
year period (1997-2002) (Bitoga J.P., Institut des Sciences Agronomiques du Burundi,
Burundi, unpublished). The disease symptoms in this case begin with a change in the colour
of the leaves from green to yellowish brown. When the infection progresses, defoliation
occurs as does the darkening of the vessels followed by plant death. This disease was not
known in Burundi tomato cultures. In the region, the control of tomato diseases has been most
exclusively based on the application of chemical pesticides, but there is no real efficient
solution for local farmers to reduce disease impact. In that context and according to present
and future regulations on the use of many chemical fungicides, and considering that phyto-
pharmaceutical treatment must prevent environment pollution, the use of biological agents to
essentially control the fungus that devastates tomato plantings in Burundi can be assayed.
In the work reported here, we have isolated the most predominant fungal pathogen from
diseased tomato plants and have performed an initial putative characterization of this isolate.
The Bacillus subtilis strain S499 has then been selected among other Bacillus isolates based,
first on its high antagonistic activity against the growth of the fungus and second, on its
known biocontrol potential. Indeed, strain S499 efficiently colonizes plant roots and has been
already demonstrated to induce resistance in tomato and other plants under greenhouse
conditions (Ongena et al., 2005a; b). However, no studies have been attempted to date in
order to determine its biocontrol potential on plants cultivated in the open field. In this
context, the objective of the study was to evaluate the beneficial effect of tomato seed
treatment with an aqueous suspension of spores from B. subtilis strain S499 on plant growth
and tolerance to an uncommon fungal isolate under agricultural field conditions in Burundi.
Results-Chapter 4
65
Materials and Methods
In vitro primary screening for antagonistic activities against phytopathogens *1
Screening for in vitro antagonism was performed on Potato Dextrose Agar (PDA) medium.
Bacillus isolates were tested for their ability to inhibit the growth of fungal pathogens:
Botrytis cinerea, Pythium ultimum, Fusarium oxysporum, Cladosporium cucumerinum and
Aspergillus niger. A bacterial culture at an approximate concentration of 108 cfu/ml was
spotted with a sterile toothpick on two edges opposites one of another of the Petri dish, and a
small mycelial section of each fungus comprised of only a few mycelial sections (6-mm
diameter) was deposited at the centre at approx. 3 cm from the isolate, ± 24 h in advance
when needed depending on the fungus growth-rate. After 4 days of incubation at 25-28 °C,
the inhibition effect on fungal growth was evaluated. The radii of the fungal colony towards
and away from the bacterial colony were assessed after the incubation period. All in vitro
antagonism assays were made in duplicates. To make a selection of the best biocontrol
isolatespreference was given to the isolats with inhibition abilities against more than one
pathogen.
Microbial strains and inoculum preparation
B. subtilis strain S499 was isolated from soil in Congo by Dr. L. Delcambe (Centre National
de Production et d’Etude de Substances d’Origine Microbienne, Liège, Belgium). The
bacterium was maintained on plate count agar (PCA medium; Becton, Dickinson and Co., Le
pont de Claix, France) at 4 °C before experimental use. For long-term storage, it was
conserved at −80 °C in cryotubes, according to the manufacturer’s recommendations
(Microbank, Prolab Diagnostic). Bacterial spore inoculum used in this study was provided by
the society Artechno S.A. after growing the S499 strain under optimized industrial conditions
in a 2000-L bioreactor. The fermentation was stopped at the time of almost full sporulation,
centrifuged and lyophilized to yield a highly concentrated stable powder. This product was
resuspended in sterile distilled water to obtain the final desired spore concentration to
inoculate tomato seeds.
The fungal pathogen of tomato plants was isolated from different fields in the intensive
agricultural zone in the plain of Imbo (Bujumbura-Burundi). The isolate was obtained from
infected parts of leaves and tested for characterization firstly by the laboratory of mycology at
the University of Louvain-La-Neuve (Belgium) and secondly by Scientific Institute for Public
Health in Brussels (Belgium). The technique of slide culture was used (Rodrigues and
* 1 Unpublished section
Results-Chapter 4
66
Menezes, 2005), which allows the direct microscopic observation of morphological structures
of taxonomic value. This technique consisted of inoculating the fungus at the sides of small
cube of agar (1 cm2) maintained in the center or at the edge of a slide and covered with a glass
cover. The slide cultures were kept on a support to avoid direct contact with the humid base of
the Petri dish. After incubation for 48 hours, the microcultures were examined in preparation
with Amman blue. For short-term maintenance, a piece of agar with mycelium measuring
about 1 cm2 was transferred top-down to a new plate approximately every three weeks under
sterile conditions. The plates were sealed with parafilm and kept at room temperature in the
dark for several days until the medium was completely covered by the fungus. The plates
were then stored at 4 °C. The pathogen inoculum was prepared by harvesting both micro- and
macroconidia from 10-day-old cultures in sterile peptone water (1 g/l Bactopeptone, 9 g/l
NaCl, 0.02% Tween 80). After removing mycelial debris by filtration through cheese cloth,
the suspension was centrifuged for 5 min at 5,000 g and the conidia were resuspended in an
adequate volume of sterile distilled water to obtain the desired final concentration of 6x105
conidia/ml, determined microscopically by the use of a Bürker counting cell.
Biocontrol experiments
Field experiments were conducted during the dry tomato-growing season from April to
September for two consecutive years. Tomato seeds (Lycopersion esculentum L. cv Merveille
des Marchés) were dipped in the various bacterial spore suspensions or in distilled water
(control) for twenty minutes immediately before sowing. In addition, for each treatment, 100
ml of a spore suspension at the same concentration as the one used to treat seeds was poured
on the soil surface surrounding each seed in every planting. Four week-old tomato plants were
pathogen-inoculated by depositing three drops of the conidia suspension on three different
leaves of each plant. Two replications (parcels), each consisting of 30 tomato plants, were
used for every bacterial treatment with specific spore concentration and arranged in a
randomized design (single row plot). Two weeks after leaf inoculation, plants in each
treatment were rated for disease severity on the basis of necrosis zones (lesion diameter).
The different treatments were compared by ANOVA (P < 0.05, Minitab software) and
data from experiments with the same set-up in the two different years were pooled for
analysis, as interactions between experiment and treatment were not significant. Means from
the different treatments were compared using Newman and Keuls' test (least significant
difference at α = 0.05).
Results-Chapter 4
67
In vitro antagonism
Antifungal activities of multiple Bacillus strains were tested in plate bioassays. Inhibition of
pathogen growth was estimated on PDA medium poured into 9.0-cm Petri dishes. Mycelial
plugs (5.0 mm) of the fungus were deposited in the center of the plates, approximately 3.5 cm
from bacterial colonies. The antagonism developed by strain S499 was tested by streaking the
bacterium on the edge of the plates (Figure 2). Plates were incubated at 25 °C and fungal
growth inhibition was rated after 4 days.
Results
Fusarium isolate identification
Six different isolates of Fusarium–resembling fungal pathogens were isolated from diseased
leaves of tomato plants cultivated in different fields of the agricultural zone in the plain of
Imbo (Bujumbura-Burundi). One of these isolates was recovered from almost all tomato
plantings and was submitted to preliminary characterization after single spore isolation on
PDA. On this medium, the fungus grows rapidly, with abundant aerial mycelium that turns
from a white/ yellowish coloration to dark brown as the culture ages. In two separate
experiments, the fungal isolate was grown at different temperatures in vitro for four days by
placing mycelium discs (6 mm diameter) on PDA. The optimal temperature for mycelium
growth is between 20-25 °C and no or very little growth of mycelium was observed at 15 °C
and 35 °C.
A first identification of the fungal isolate was made in collaboration with the laboratory
of mycology at the University of Louvain-la-Neuve (Belgium). Conidiophores and conidia are
produced after 9-11 days of incubation at 19-22 °C in darkness. Microscopic observation
revealed two types of conidia. The primary ones formed in aerial mycelium on conidiophores
in polyphialides. These conidia were slightly sickle-shaped, thin-walled and without a
pedicellate basal cell. They had three to six septate. The second type of conidia were
abundant, mostly with no septate, ellipsoidal to cylindrical, slightly curved or straight,
occurring in false head (Figure 1).
Results-Chapter 4
68
Figure 1 Microscopic observations of 10-day old conidia of Fusarium isolated from leaves of diseased tomato plants in Burundi field. a: macroconidia, b: microconidia. The bar is equal to 5µm.
The fungal isolate causes blight symptoms and foliar necrosis. Under appropriate conditions,
infected leaves turn brown about 10 days after inoculation with a conidial suspension (Figure
3A). As the disease symptoms progress, host plants generally die before the flouring stage.
Morphology, fermentation profile and a first set of gene sequencing (calmodulin and
elongation factor) indicated that this isolate probably belongs to the genus Fusarium. It may
correspond to the semitectum species but this should be confirmed by further genetic
characterization.
Selection of Bacillus strain S499 as antagonist used for field experiments *2
A total of forty-four isolates from the rhizosphere of tomato plant that were cultivated in the
plain of Imbo (Burundi) were selected as Bacillus spp. on glucose yeast peptone agar
medium. According to preliminary characterization, all these isolates may belong to Bacillus
genus based on the Bergey’s Manual of determinative Bacteriology (Holt, 1994). Microscopic
studies revealed that the cells were rod-like, motile, Gram-positive, catalase positive, and
spore-forming under aerobic conditions.
To carry out the antagonistic activity test in vitro in the primary screening, the isolates
were assayed on two media viz. LB and PDA agar, and growth was almost similar but PDA
agar medium was chosen for further assays because its white color allows more clarity of the
inhibition zone. The strength of fungal growth inhibition depends on the rhizobacterial isolate
antimicrobial activity. To allow comparisons, growth alterations (expressed in mm) were
evaluated for all fungus-bacterium combinations on the fourth day of growth (not shown).
The determination of the strains’antagonistic behaviour towards fungi crucially depends upon
the dual bacterial-fungus simultaneous growth rate. To determine a procedure for assessing
the antagonism between these organisms, many attempts were assayed to first inoculate either
the fungus or the antagonistic agent a few days early or late. Therefore, the bacterial isolates
* 2Unpublished section
Results-Chapter 4
69
were inoculated on the medium 24h before the strain of Pythium ultimum, 60 h after Botrytis
cinerea, 48 h after Fusarium oxysporum, 24 h after Cladosporium cucumerinum and
simultaneously with Aspergillus niger. The antagonistic isolates were clearly discerned by
limited growth or complete absence of fungal mycelium in the inhibition zone surrounding a
bacterial colony. All the investigated isolates showed individual inhibition patterns and were
able to inhibit, even weakly, at least one of the tested fungi (Table 1). Then, we further
selected the most efficient strains based on the criterion of isolates showing capacity of
mycelia inhibition of at least two fungi with a 3 mm clearance zone towards and away from
the bacterial colony. Among the 44 bacterial isolates, only four of them (Table 2) behaved
very comparably to the strain S499 of B. subtilis considered in our case as antifungal model
because previous studies had shown this strain to exhibit strong antagonizing potential against
some pathogenic fungi (Ongena et al., 2007; Nihorimbere et al., 2009). In the present study,
the bacterial isolates inhibited the fungi growth at different degrees from 3 to 8 mm (Table 2).
Such variation in the extent of mycelial inhibition can be influenced by the nature of bacterial
isolates. Landa and collaborators (1997), for example, indicated that the ability of four
bacteria isolates to inhibit different strains of F. oxysporum differed significantly. This
suggests that taxonomically distinct isolates may exert their antifungal potential through
different modes of action and/or produce different types of antifungal metabolites (William
and Ash, 1996). Here, the modes of action were not elucidated. However, early works have
correlated the production of glucanolytic and proteolytic enzymes with antagonism in
PaeniBacillus polymyxa, B. pumilis and Bacillus spp. (Nielsen and Sorensen, 1997). The
production of HCN, siderophores, ammonia, lipase and chitinase in growth medium by
Pseudomonas corrugata was considered contributing to the antagonistic activity of the
bacterium against Alternaria alternate and Fusarium oxysporum (Trivedi et al., 2008). It is
likely that the above proposed mechanisms be involved in antagonistic behaviour of the
strains, but we believe that some other mechanisms might be operating behind antagonism.
The isolates that most effectively inhibited fungal growth on the dual culture experiment
resulted in such a major inhibition zone that there was no physical contact with the pathogens
suggesting that the rhizobacteria could be producing certain metabolites (Montealegre et al.,
2003; Ahmed et al., 2007). Many of these specialized compounds, such as antibiotics, are
either liquid or solid at room temperature, and little is known about volatiles (with molecular
masses less than 300 Da, low polarity, and a high vapor pressure) that can act as antibiotics
and cause growth inhibition or have more deleterious effects on organisms. Indeed, the
microbial world synthesizes and emits many volatile compounds that can inhibit pathogen
Results-Chapter 4
70
growth (Dickschat et al., 2005). At present, the biologically active volatiles causing the
inhibition are not known because many volatiles have not been detected or identified (Kai et
al., 2007).
Table 1. Bacterial isolates screening for fungal growth inhibition
Bacillus isolates
Otrytis cinerea
Pythium ultimum
Fusarium oxysporum
Cladosporium cucumerinum
Aspergilus niger
BG152 + - - + + BG2511 - - - + + BG2512 - - - + + BG252 + - - + +
BG2571a + + - + + BG257b + + - + - BG311 + + + - + BG312 - - - - + BG3361 - - - + - BG3362 + - - - + PD2511 + - - + + PD2512 + - - + - PD31 - - + + + PD32 - - - + - PD35a + - - - - PD35b - - - + - PD35c + + - - - PD35d - + - - - PD35e - - - - + PD36 - - - - - PD361 + - - + - PD362 - - - + + PD6571 + - - + + PD6572 + - - + + PD6573 + + + - + PD661 - - - + - PD662 + + - + +
PD6652a + + - - - PD6652b + - - - +
PD71 + - - - - PD72 + - - - - PD76 - - - + - PD81 - - - + - PD82 - - - + - PD9 - - - + -
KD15 + - - + - KD151 - - - + - KD152 - - - + + KD161 - - - + - KD162 + + + - + KD25 + - - - + KD261 + - - - - KD35 - - - - +
KD4621 - - - + - KD1622 - - - + -
S499 (ctrl)* + - - + + +: Halo formation; -: No halo formation; *: control strain
Antifungal effects of organic volatiles were previously shown to inhibit germination or
mycelial growth of S. sclerotiorum (Fernando et al., 2005), and unidentified compounds from
Results-Chapter 4
71
Bacillus subtilis cause structural deformation of pathogenic fungi (Chaurasia et al., 2005).
The approach herein investigated (phytopathogens’inhibition) provided an opportunity to
select effective biocontrol strains capable of antagonizing tested fungi that are susceptible to
damage crops in the same environment. We believe that the isolates showing the highest
ability to reduce the pathogens’ growths in the in vitro dual assay can also be efficient
antagonists against phytopathogens of many crops in the in vivo biological control tests.
However, it is known that in vitro assays have certain limitations in that biocontrol
efficiencies may not be equally expressed under gnotobiotic (axenic) and in vivo conditions
(Inam-ul-Haq et al., 2003). Tests based on in vitro mycelial inhibition and root colonization
do not always correlate with biocontrol efficacy under natural conditions (Williams and
Asher, 1996).
Table 2. In vitro assay of bacterial isolates most inhibiting towards some fungi
Isolates Botrytis cinerea
Pythium ultimum
Fusarium oxysporum
Cladosporium cucumerinum
Aspergilus niger
BG311 + + + + + BG2571a + +++ - ++ + PD6572 ++ - - + + BG252 + - - + ++
S499(ctrl)* ++ - - +++ ++ Clearance zone: +3 mm; ++: 5 mm; +++: 8 mm; _: negative reaction; *: ctrl: control strain
Dual assay method allowed determination of the antifungal activity of the native
isolates, and showed higher activity of four isolates among 44 isolated. These isolates should
be further identified and later investigated for their efficacy in greenhouse and in field. On the
basis of this preliminary study on biocontrol, it is deduced that the rhizosphere of tomato in
the plain of Imbo harbours beneficial microorganisms with the potential to suppress soilborne
diseases caused by some common fungi especially encountered in tropical regions such as
Fusarium, Pythium, Apergillus, Botrytis spp. and possibly other soilborne pathogens. We thus
selected the strain S499 for the next experimentations because it presents a little bit high
antagonistic activity as compared to the four local strains which are potent fungal growth
inhibitors.
Fusarium disease reduction upon treatment with B. subtilis
As a first step, a dozen of Bacillus strains either isolated from local soil or originating from
suppressive soils in other regions of central Africa, were tested for their in vitro antagonistic
potential towards growth of the pathogen. The B. subtilis strain S499 from Congo was among
the best with this respect (Figure 2) and was selected because of its high spore-forming
capability and based on its previously well established biocontrol activity against tomato,
Results-Chapter 4
72
bean and cucumber diseases. A formulated powder containing high concentration of viable
endospores was obtained from strain S499 by pilot-scale fermentation and lyophilisation.
Using this product, biocontrol assays were performed in two successive years on the same site
by treating tomato seeds with spore suspensions of S499 before sowing.
Figure 2 In vitro growth inhibition of the pathogen Fusarium semitectum caused by Bacillus subtilis S499 on PDA medium. The bacterium and the fungus were inoculated at the same time and the antagonism was scored after incubation of the plates for 4 days at 25 °C.
The disease was introduced artificially by inoculating the Fusarium pathogen on the leaves of
21-day-old plants. Disease incidence in control and Bacillus-treated plants was compared on
the basis of the diameter of spreading lesions observed two weeks later. In these assays, we
also evaluated the dose-efficacy relationship for the biocontrol agent. Four spore
concentrations were used to inoculate tomato seeds in separate parcels. Control plants were
not inoculated and as these control plants did not present any necrosis or symptoms at the
time of assessment, we assume that there has been no cross contamination of the fungus
between parcels. Based on the diameter of lesions on leaves, plants inoculated with strain
S499 showed significantly lower infection rates compared to the non-treated, inoculated
plants (Figure 3). Interestingly, the four inoculum concentrations were all efficient at the same
level to reduce the fungal pathogen ingress and in all cases, a disease reduction of up to 65-
70% was observed.
Colonization of the rhizosphere is required for a PGPR strain to consistently influence
plant growth and health (Chin-A-Woeng et al., 2003). In order to support these biocontrol
data, the evolution of S499 populations was evaluated on roots of tomato plants grown under
these fields' conditions. For the four different inocula tested, the population level was assessed
by agar plate count 30, 49 and 73 days after inoculation.
Results-Chapter 4
73
0
1
2
3
4
5
6
7
C 10exp5 10exp6 10exp7 10exp8
Me
an
dia
me
ter
of
ne
cro
sis
(cm
)
Bacterial inoculum concentration (CFU/ml)
a b c A
B
Figure 3 A, Disease symptoms on infected leaves of untreated tomato plants collected from the field two weeks after pathogen inoculation (b) compared to plants treated with strain S499 prior to infection(c) and to treated, uninfected control plants (a). B, Disease incidence rated as mean diameter of necrosis (cm) measured two weeks after fungal inoculation on leaves of plants germinated from bacterized and non-bacterized seeds with various bacterial inoculum concentrations and seeded in separate parcels. Infection rate data from two parcels per treatment with 30 plants each were submitted to statistical analysis. For infection, the first three leaves of each plant were infected with three drops of a fungal spore suspension containing 6 x 105 cfu/ml. As the analysis of variance and the multiple comparison tests did not reveal any statistical difference between results from two successive years for similar treatments, these data were pooled and the results are thus means and standards errors calculated from two independent experiments.
Discrimination between B. subtilis S499 and soil microflora was based on colony
morphology. Populations observed for the different inocula were found to progressively
converge and decrease, stabilizing around 2x106 (Table 3).
Table 3 Root colonization of tomato plants grown under field conditions in separate parcels from seeds treated with different spore suspension concentrations of B. subtilis S499. Values represent the mean and standard error calculated from at least three plants randomly collected in each parcel.
Inoculum (cfu/g) 30 days 49 days 73 days
105 3.9 ±4.2 x 107 5.3 ±1.4 x 106 1.8 ±0.6 x 106
106 2.3 ±2.2 x 107 7.3 ±14.6 x 106 2.6 ±2.6 x 106
107 4.8 ±0.6 x 107 1.6 ±15 x 107 2.1 ±12 x 106
108 8.4 ±1.4 x 106 4.5 ±0.6 x 106 1.6 ±1.0 x 106
Results-Chapter 4
74
Effect of B. subtilis on tomato plant growth and fruit yield
In parallel to plant protection assays, some additional parcels were used to evaluate the plant
growth-promoting effect of strain S499. To this end, we only used non-treated tomatoes and
plants treated with various concentrations of Bacillus but without pathogen inoculation. The
first parameter used to evaluate plant growth promotion was the height of the plants at 62
days after sowing. Our results show that treatment with B. subtilis S499 significantly
increases plant height at this stage. At 62 days, water control plants had only reached an
average size of 44 cm while plants inoculated with the bacterium suspensions reached an
average height of 70 cm representing an increase in shoot height of more than 59%. Plants
inoculated with a suspension at 107 CFU/ml were significantly higher than those inoculated
with other inoculum concentrations, but with only less than 5 cm difference. No statistical
difference was recorded between plants inoculated with suspensions at 105, 106 and 108
CFU/ml (Figure 4A). These results indicate that the inoculation of tomato seeds with a spore
suspension of S499 at the low 105 CFU/ml concentration is sufficient to significantly increase
plant growth.
To further evaluate the growth stimulation potential of B. subtilis S499, fruit yield was
measured. At 72 days post-sowing, we first determined the percentage of plants bearing three
to four fruits in the various parcels treated with different inoculum concentrations. At least
60% of the tomato plants bacterized with the various inoculum densities had three to four
fruits but only five percents of the untreated control plants had reached this stage (Figure 4B).
A few days later, the total fruit yield per plant was also compared. Weight measurements of
fruits on treated plants were much higher (at least 14-fold increase) compared to controls
(Figure 4C). Concerning both the quantity and weight of fruits, these highly significant
statistical differences in comparison to untreated tomatoes was observed regardless of the
inoculum concentration tested.
Results-Chapter 4
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A
B
C
Figure 4 Effect of strain S499 on tomato growth under field conditions. This effect was evaluated within each treatment by (A) Plant shoot height measured 62 days after sowing, (B) Percentage of tomato plants bearing at least three fruits 72 days after sowing; (C) Tomato fruit weight (g) 78 days after sowing. Plants were sown in separate parcels with two parcels per treatment and 30 plants per parcel. Seed-treatments were performed with various concentrations of S499 spores and compared to untreated control plants (C). Data were treated statistically as described earlier and mean values and standard errors were calculated from 10 plants selected randomly among 30 in every parcel.
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Discussion
Six fungal isolates were obtained from diseased tomato plants from different fields in the
plain of Imbo (Bujumbura-Burundi). One isolate stood out by having the greatest frequency,
with 98.7% in all the tomato plantings. Although further work is required, microscopic
observation, cultural characteristics and preliminary gene homology analyses indicated that
the fungus is of the Fusarium genus and may belong to the semitectum species (Bokshi et al.,
2003). Fusarium is relatively common within subtropical and tropical environments and some
species can be pathogenic to several plant species (Nelson et al., 1983; Bokshi et al., 2003;
Kloepper et al., 2004). In this work, the fungus was isolated from leaves of tomato plants
displaying brown symptoms and foliar necrosis. It is possibly this same pathogen which
caused great losses by devastating young plants and subsequently affected the local farming
economy. This isolate has not been previously described as an infectious agent attacking
aerial parts of tomato plantings in Burundi. By contrast, F. semitectum is well known as a
major cause of seedling diseases on cotton and other crops in many subtropical African
countries (Abd-Elsalam et al., 2003; Kloepper et al., 2004) and was also shown to be
moderately virulent on Acacia Koa seedlings (Dudley et al., 2007). Its high pathogenicity
may be due to the fact that it produces some mycotoxins that may adversely affect plants
(Logrieco et al., 2002). It is also interesting to note that this strain we isolated grows well in
vitro at 20-25 °C. This optimal temperature range corresponds to the average values
prevailing locally in the natural conditions in Burundi.
Strain S499 was selected out of a range of B. subtilis isolates and tested for its efficacy
at controlling the disease caused by this locally important fungal isolate. Results revealed a
highly significant disease reduction following the treatment of seeds with bacterial spore
suspensions in comparison with the control plants. Tomato plants germinated from bacterized
seeds show strong resistance of about five-fold less necrosis diameter as compared with
controls (Figure 3). Strain S499 has been extensively studied in our laboratory and
demonstrated to be effective as biocontrol agent in greenhouse conditions (Ongena et al.,
2005a; b). This strain efficiently produces antibiotic compounds, among which lipopeptides
that may act through direct antagonism toward various fungi and oomycetes and/or by
enhancing the host plant defensive capacity (Ongena and Jacques, 2008). Various bacterial
spore suspensions were used and their effects on disease suppression were shown to be
similar suggesting a low importance of the inoculum concentrations used. This may be
explained by the fact that the population levels corresponding to the four inocula tested
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became almost similar after a few weeks post-inoculation. These results also illustrate the
colonization potential and thus, the rhizosphere fitness, of strain S499 that maintained a
certain steady level of population for a prolonged period even if some decrease in cell
densities were observed over time in a prior phase. Such progressive decline of PGPR
populations after introduction at the root level has already been reported and the following
stable phase probably corresponds to a resident phase where the population size is restricted
by space and/or nutrients availability (Bashan, 1998; Di Mattia et al., 2002; Espinosa-Urgel et
al., 2002). From a more practical point of view, the use of low inoculum densities (105 cfu ml-
1) is also economically relevant for treating this kind of disease in open fields.
Possible colonization of leaf tissues by S499 or migration of the strain through the plant
resulting from seed treatment was also assessed by counting colonies on the basis of their
typical morphology. Bacillus cells were not detected, suggesting that the bacterium remained
within the root system and demonstrating that the disease suppression was due to the
induction of resistance in the host plant as the beneficial strain and the pathogen are localized
on different plant organs. Moreover, the bacterial populations established on the roots are in
the range of the threshold commonly required to trigger plant systemic resistance. The ISR
triggering potential of strain S499 has already been observed on tomato and other plants
(Ongena et al., 2005a; b) but the experiments conducted in this work are the first
demonstration of ISR-based biocontrol activity by S499 under field conditions. In previous
works, it has also been evidenced that lipopeptides of the surfactin and fengycin families do
play a crucial role in the elicitation of this induced systemic resistance phenomenon (Ongena
et al., 2007; Jourdan et al., 2009). Treatment with pure compounds and/or with overproducing
derivatives triggered ISR in bean and tomato plants (Ongena et al, 2007). We have also
recently demonstrated that surfactin genes in B. subtilis are readily expressed in vitro
(Nihorimbere et al., 2009) and that the compound is efficiently produced by S499 cells
developing in the rhizosphere of tomato plants (V. Nihorimbere, University of Liège,
unpublished results). On the basis of these data, the production of lipopeptides may also be
involved in ISR-based disease protection by S499 under field conditions.
In parallel to plant protection, some additional parcels were reserved to evaluate the
strain for its plant growth promoting effect sensu stricto. Results show that tomato shoot
heights were markedly increased by approx. 59% upon seed-treatment with all the S499
inoculum densities tested (Figure 4A). Based on two additional parameters, trial assessment
approx. 70 days after sowing also revealed a clearly enhanced fruit yield for treated-plants as
compared with the controls. All the inoculum concentrations tested improved similarly the
Results-Chapter 4
78
number of fruits as well as the total fruit weight per plant (Figures 4B and C). The beneficial
effect of rhizobacteria in general and of Bacillus species in particular, on plant development
putatively rely on diverse mechanisms that may be involved concomitantly (Ping and Boland,
2004; Berg, 2009). Plant growth may be directly stimulated through the bacterial production
of auxins, cytokinins, gibberellins or hormone-like compounds. The hormonal balance of the
plant can also be influenced indirectly by associated microorganisms as exemplified by those
producing ACCdeaminase able to degrade the precursor of ethylene that plays a pivotal role in
various developmental and stress tolerance functions (Glick, 2005). Besides these
mechanisms, plant-associated bacteria can also improve nutrient acquisition by the plant,
especially via nitrogen fixation or via solubilization of phosphorous, iron and other elements.
Other compounds, such as the volatile acetoin and butandiol emitted by some Bacillus strains
are also involved in the stimulation of plant growth (Ryu et al., 2003). We don’t know yet
what traits are involved in the case of S499 but experiments are being performed to evaluate
the potential of the strain at producing phytohormones, volatile organics, siderophores and
specific enzyme activities such as ACCdeaminase and phytase with the aim to better
understand its broad-spectrum growth promotion activity as it was also observed with oat,
broad bean and maize (data not shown).
In conclusion, this work illustrates the effectiveness of one particular B. subtilis strain at
providing beneficial effects on health of tomato plants cultivated in open fields in Burundi.
Based on the level of both growth-promoting effect and disease reduction, our results suggest
that a formulated B. subtilis product can be utilized as inoculum at low spore concentration to
help local farmers at combating a new Fusarium disease devastating their tomato fields. The
efficacy of inocula at low spore concentrations used for seed treatment, leads to conclude that
such a Bacillus-based product may represent for local growers a low cost solution or
alternative to the use of chemicals for the control of Fusarium diseases in this region.
Acknowledgements V. Nihorimbere is recipient of a grant from the Belgian Technical Cooperation (BTC/CTB). M. Ongena and E. Jourdan are respectively Research Associate and Post-doctoral Researcher at the Fonds de la Recherche Scientifique–FNRS in Belgium. H. Cawoy’s Ph.D. thesis is supported by a grant from the Fonds pour la formation à la recherche dans l’industrie et dans l’agriculture (F.R.I.A.). The authors thank the society Artechno s.a. for provinding the B. subtilis powder.
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Foreword on chapter 5
Altogether, the results presented in Chapter 4 demonstrated the effectiveness of S499 strain at
controlling disease in the tomato open field in Burundi. On another hand, it is known that
lipopeptide antibiotics are the main antimicrobial components produced by this strain, and
these molecules may play an important role in the global antagonism developed towards
phytopathogens thereby contributing to the efficacy of some specific strains at reducing plant
disease incidence. The objective of the work reported within Chapter 5 is to evaluate whether
S499 strain (re-identified as B. amyloliquefaciens) produces or not these antibiotics of the
lipopeptides families of surfactins, iturins, and fengycins in situ on tomato plant root and in
vitro versus different parameters known to have influence on the cell culture development.
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Chapter 5. Bacillus life in soil: impact of rhizosphere factors on lipopeptide signature from the plant beneficial strain
B. amyloliquefaciens S499
Submitted in International Society M icrobial Ecology Journal Venant Nihorimbere1, Hélène Cawoy1, Alexandre Seyer2, Alain Brunelle2, Philippe Thonart1, Marc Ongena1 1 Walloon Center for Industrial Biology, University of Liège/Gembloux Agro-Bio Tech, Gembloux B-5030, Belgium 2 Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, F-91198 Gif-sur-Yvette, France Running title: Bacillus lipopeptide produced in planta
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Abstract
Through the so-called rhizosphere effect, plants usually create a specific environment in the
soil surrounding roots thereby influencing the production of biologically active compounds by
plant-associated microorganisms. In that context, lipopeptides represent a major class of
antibiotics involved in multiple facets of the tritrophic interactions between plant, pathogen
and beneficial Bacillus strains. In this work, we combined two mass spectrometry-based
approaches (electrospray ionization and imaging) to analyze the pattern of surfactin, iturin and
fengycin families produced in vivo by B. amyloliquefaciens strain S499. Our results show that
rhizosphere conditions are conducive for surfactin synthesis but not for other types of
lipopeptides that are actually secreted upon laboratory growth in artificial optimized medium.
We also show that the LP pattern secreted by S499 can be markedly influenced by nutritional
factors, biofilm formation and oxygen availability. From a qualitative point of view, it also
clearly appears that the relative proportions of homologues with long fatty acid chains are
higher in LP pattern produced in planta by strain S499. This is of biological relevance since
these LP forms were proven to be biologically more active regarding their haemolytic,
antiviral and antimicrobial activities as well as their potential to trigger plant defense
reactions. Globally, this work contributes to better appreciate the impact of some
environmental factors on the in situ biosynthesis of lipopeptides by strains of Bacillus which
is probably an essential step for improving the level and reliability of their efficacy as
biological agents for the control of plant diseases.
Keywords: Bacillus/ in situ/ lipopeptides/ production
Introduction
Bacterial lipopeptides (LPs) are composed of a lipid tail linked to a short linear or cyclic
oligopeptide and can be synthesized by various genera and species such as Streptomyces,
Pseudomonas and Bacillus. LPs are synthesized by non-ribosomal peptide synthetases or
hybrid polyketide synthases/non-ribosomal peptide synthetases. These modular proteins are
megaenzymes organized in iterative functional units called modules that catalyze the different
reactions leading to polyketide or peptide transformation (Finking and Marahiel, 2004). Such
biosynthetic systems lead to a remarkable heterogeneity among the LPs products which vary
in the type and sequence of amino acid residues, the nature of the peptide cyclisation and in
the nature, length and branching of the fatty acid chain (Raaijmakers et al., 2006; Ongena and
Jacques, 2008). Cyclic lipopeptides secreted by Bacillus strains (LPs) also encompass
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structural variants depending on the genetic background of the considered strain. Although
other classes have been identified recently (Hagelin et al., 2007; Lee et al., 2007), Bacillus
LPs may be classified in three main families, surfactins, iturins and fengycins (Ongena and
Jacques, 2008). Surfactins are heptapeptides interlinked with a β-hydroxy fatty acid to form a
cyclic lactone ring structure. The group of iturins encompasses 7 variants including
bacillomycins and mycosubtilin. Iturins are also heptapeptides but linked to a β-amino fatty
acid chain with a length that usually varies from C14 to C17. The third family comprises
fengycins A and B, which are also called plipastatins. These molecules are lipodecapeptides
with an internal lactone ring in the peptidic moiety and with a β-hydroxy fatty acid chain (C14
to C18) that can be saturated or unsaturated.
Some of these bacterial LPs are considered as among the most powerful biosurfactants
isolated so far. They combine high effects on surface tension and low critical micellar
concentrations and thus retain exceptional emulsifying/foaming properties and high potential
for the solubilization of hydrophobic compounds. LPs have also received considerable
attention for their antimicrobial, cytotoxic, antitumor and immunosuppressant properties.
There is thus a growing interest in the study and application of these molecules for various
environmental and pharmaceutical applications (Pirri et al., 2009). LPs also display versatile
functions in the ecology of the producing strains and notably in interactions with co-existing
organisms, including bacteria, fungi, oomycetes, protozoan predators and plants in the case of
soil isolates (reviewed in Raaijmakers et al., 2010). When produced by plant beneficial
rhizobacteria, LPs may play an important role in the global antagonism developed towards
phytopathogens thereby contributing to the efficacy of some specific strains at reducing plant
disease incidence. Such a biocontrol potential mostly relies on efficient root colonization,
direct pathogen inhibition and possible stimulation of the host plant immune system. As a
matter of fact, LPs from Bacillus have been demonstrated to be involved in these three
mechanisms (Ongena and Jacques, 2008; Raaijmakers et al., 2010).
They are numerous reports suggesting a role for LPs in biocontrol relying on in vitro
antimicrobial activity. However, additional approaches are necessary to correlate biocontrol
activity with an efficient production of LPs since rhizosphere conditions can greatly modulate
antibiotic gene expression compared to laboratory. These approaches include the use of non-
producing or over-producing derivatives with respective loss or increase in the biocontrol
activity and/or the study of in situ LP gene expression (fusion of promoters of LP operons to
reporter genes such as lacZ, xylE, gusA, luxAB or gfp). Also necessary is the detection at
active concentrations of the antibiotic compound in the microenvironment which has been
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inoculated with the producer strain. However, very few studies have been devoted to
investigate LP gene expression during interaction with plants and only a very limited number
of works have successfully detected LPs secreted by Bacillus isolates growing in the
phytosphere.
In that context, the present study was initiated with the first aim to perform an in-depth
characterization of the LP pattern produced by B. amyloliquefaciens strain S499 developing
on roots of tomato plants. Strain S499 has been selected because this natural soil isolate co-
produces in vitro both three LP families in relatively high amounts under optimized in vitro
conditions and retains some biocontrol activity in various pathosystems. On that basis, our
main goal is to better appreciate to what extent its LP production may be modulated both
qualitatively and quantitatively by some rhizosphere-specific factors.
Materials and methods
Strain identification
Strain identification was performed in the laboratory of microbiology at the University of
Gent in Belgium. Beside basic bacteriological tests, fatty acid analysis was performed on
whole-cells by gas-chromatography following the recommendation of the commercial
identification system MIDI (Microbial identification System, Inc., Delaware U.S.A.). The
profile was compared with the MIDI identification database TSBA50 V.5.0 and with the in-
house database containing several profiles of reference strains.
The applicability of the gyrA sequence for species discrimination within Bacillus subtilis
complex has been reported by Chun and Bae (2000). Total DNA was prepared according to
the protocol of Niemann and collaborators (1997). The gyrA gene was amplified by PCR
using the following primers: gyrA-forward, CAG TCA GGA AAT GCG TAG GTC CTT
Sequence (5´-3´) and gyrA-reverse, CAA GGT AAT GCT CCT GGC ATT GCT. The gyrA
fragment corresponds to B. subtilis gyrA numbering 43-1070. The PCR amplified gyrA was
purified using the NucleoFast® Terminator 96 PCR Clean-up Kit (Machinerey-Nagel, Düren,
Germany). Sequencing reactions were performed using the BigDye® Terminator Cycle
Sequencing Kit (Applied Biosystes, Foster City, CA, USA) and purified using montage ™
SEQ96 Sequencing Reaction Cleanup Kit (Millipore, Bedford, MA, USA). Sequencing was
performed using an ABI prism® 3100 Genetic Analyser (Applied Biosystems, Foster City,
CA, USA).
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Extraction and LC-MS analyses of lipopeptides from tomato hydroponic cultures
Surface-sterilized tomato seeds (treated or not with 10 µl of Bacillus cell suspension prepared
as described below) were germinated for 5 days at room temperature in the dark and on
gelified sterile nutrient medium containing 5mM Ca(NO3)2, 5 mM KNO3, 2 mM MgSO4, 1
mM KH2PO4 and micronutrients. Germinated seedlings were transferred in sterilized 50-ml
tubes filled with nutrient solution, inoculated with strain S499 and incubated at 25 ± 2 °C in
the greenhouse with a 16 h-photoperiod. For inoculum preparation, B. amyloliquefaciens
strain S499 was grown on gelified Luria Bertani (LB) medium for 24 h. Cells were
resuspended in NaCl 0.85% and the concentration was determined by measuring turbidity at
600 nm. A final concentration of 5x107 cells/ml in the nutrient solution was used.
To evaluate in situ production of LPs, we used plantlets of approx. four weeks old.
Fresh roots (approx. 2 g RFW) were carefully separated from plants and cut into 2-3 cm
segments. The tissues were immersed in 10 ml of 90%ACN, 0.1% HCl, 0.9% Triton-X 100,
ultrasonicated for 2 min and extracted for 2h under regular vortexing. This extract was
combined with the hydroponic liquid which was collected from same plant after 4 weeks and
filtered on Whatman Paper Grade 1 to remove macroscopic insoluble material. This mix was
loaded on C18 solid-phase extraction cartridges (900 mg, Alltech), washed with 20% ACN
and lipopeptides were desorbed with 1 ml 100% ACN. The material was then concentrated 5
times using speed vacuum.
The resulting samples were analyzed by reverse phase HPLC (HPLC Waters Alliance
2695/ diode array detector) coupled with single quad mass spectrometer, (Waters SQD mass
analyser) on a X-terra MS (Waters) 150*2.1 mm, 3.5 mm column. Surfactins were eluted in
the isochratic mode (78% acetonitrile in water acidified with 0.1% formic acid) at a constant
flow rate of 0.5 ml min -1 and 40 °C. Iturins and fengycins were selectively desorbed by using
ACN gradients from 35% to 65% in 35 min and from 40% to 60% in 40 min, respectively. In
both cases, a lag time of 5 min was set after injection of the samples on the column.
Compounds were first identified on the basis of their retention times compared with authentic
standards (supplied by AIBI asbl). The identity of each LP homologue in every family was
confirmed on the basis of the masses detected in the SQD by setting electrospray ionization
source conditions in the MS as source temperature, 130 °C; desolvation temperature, 250 °C
and nitrogen flow at 500 l h-1. Specific cone voltages were used for each family and were 70
V, 60 V and 100 V for surfactins, iturins and fengycins, respectively. The positive ion mode
was used for analysis of both three families because also in the case of surfactins, a higher
signal/background ration was obtained compared to negative ion recording. Depending on the
Results-Chapter 5
87
experiment and purpose, LP samples were analyzed either in the SIR (Single Ion Recording)
or the SCAN mode. In the first case, masses of both proton, sodium and potassium adducts of
the various LP forms were used for optimal detection and in the second case, the scanned
mass range was limited to 150 mu spanning over all the masses of homologues within each
family. The amounts were calculated on the basis of the corresponding peak area in the SIR or
SCAN traces. We used 95%-pure standards as methanolic solutions with serial dilutions to
0.1 µg/ml.
LP analyses by imaging MS
Sterilized tomato seeds were immersed in S499 strain suspension at concentration of 108 cfu
for 5 minutes. The seeds were gently harvested from the suspension to be transferred on agar
plates containing sterile plant nutrient solution (PNS) as described above. The plates were
incubated at 30 °C for approx. two weeks.
Globally, the method used for sample processing is the same as described by Debois and
collaborators (2008), as well as detailed characteristics and settings for the mass spectrometer.
Briefly, lipopeptide material present at the surface of the plate was directly transferred onto
polished silicon wafer by applying a manual press for 30 s. The wafer was then allowed to dry
for 30 min under vacuum at room temperature before introducing in the mass spectrometer. A
TOF-SIMS IV (Ion-TOF, Münster, Germany) reflectron-type TOF mass spectrometer was
used for the MS imaging experiments. The primary ion source is a bismuth LMIG which
delivers Bi3+ cluster ions. The secondary ions are accelerated to a kinetic energy of 2 keV and
are postaccelerated to 10 keV before hitting a hybrid detector. Details concerning image
processing are given in the legend of Figure 3. Methanolic solutions (1 mg/ml or serial
dilutions of 0.3 mg/ml) of authentic and 95%-pure standards (AIBI asbl) were prepared and
10-µl drops were deposited on the same gelified medium as the one used for plantlet grow and
colonization. After incubation at room temperature for 2 h, the plates were processed for
imaging as described for those used for analyzing LP production by S499. Core sampling in
gelified medium were performed with a Pasteur Pipette and lipopeptides contained in the 200
µl agar piece were then extracted by adding 100 µl acidified acetonitrile/water 80/20 v/v. The
mixture was then ultrasonicated for one minute, extracted under regular stirring for 30 min
and incubated overnight at – 20 °C. Extracts were then centrifuged at 14000 r.p.m. for 10
minutes and the supernatant was analysed in LC-MS as described above.
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88
Growth conditions for LP production by S499 cells in liquid cultures
All the S499 cultures were performed in 12-well microplates by dispensing 2.5 ml of medium
per well. Plates were incubated under agitation on a rotary shaker for 72h at 28 °C. In all
cases, the pH of the different media was adjusted to 6.8 before filter-sterilization. Over the
incubation time, bacterial growth was monitored 24 h and 48 h after inoculation by measuring
turbidity (OD) at 600 nm. At the end, cultures were centrifuged (25 min, 13000 g) and
lipopeptides were extracted from supernatant samples using C18 solid-phase extraction
cartridges before analysis by LC-MS as described above. Three wells per plate were used for
each medium but corresponding supernatants were combined to yield a unique sample
submitted to extraction and LC-MS analysis. However, the experiments were repeated at least
three times allowing to calculate means values and standard deviations mentioned in the
results.
To test LP production in the presence of natural exudates, we collected the nutrient
solution of non-inoculated tomato plants grown for four weeks in the greenhouse. This
medium was supplemented with 2 g/l of (NH4)2SO4 to compensate for the lack of available
nitrogen. In order to evaluate the production of LPs in various carbon sources, the individual
substrates were added at a final concentration of 5 g l -1 to a minimal medium composed by
MgSO4 7H2O 0.5 g l -1, K2HPO 4 1.0 g l -1, KCl 0.5 g l -1, Yeast extract 1.0 g l -1, Fe2(SO4)3
1.2 mg l -1, MnSO4 0.4 mg l -1, CuSO4 1.6 mg l -1, (NH4)2SO4 1 g l -1.
LP production in function of oxygen concentration
These experiments were conducted in 2-L bioreactor using a medium described in
Nihorimbere and colleagues (2009) and recomposed on the basis of tomato root exudate
composition as described by Kamilova and collaborators (2006). The fermentor is equipped
with automated pH control maintained with H3PO4 (3N) and KOH 50% at a value of 6.8. The
fermentor working volume of 1.5 l was inoculated with a 16 h old preculture (25 ml) prepared
in the same medium. The temperature was maintained at 30 °C. For aerobic conditions,
aeration rate and agitation were fixed respectively at 0.5 V.V.M. (volume of air per volume of
medium per minute) and 150 r.p.m (rotation per minute). In these conditions, the oxygen
concentration transiently decreased to zero during a few hours corresponding to the mid-/late
exponential growth phase but oxygen was not limiting for the remaining part of the culture.
Oxygen-depleted condition was created by replacing air with nitrogen for flushing the reactor
through the sparger until pO2 value reached zero. This nearly-anaerobic condition was
maintained over the culture time by subsurface injection of nitrogen at a low flow rate.
Results-Chapter 5
89
Results
Strain identification
The Bacillus strain S499 used in this study was formerly identified as B. subtilis based on
basic bacteriological tests (cell morphology, gram stain, oxidase and catalase reactions) and
on API fermentation profile. The whole-cell fatty acid profiling confirmed that strain S499
belongs to the Bacillus subtilis complex but gyrA sequencing revealed that its closest relatives
are two strains of B. amyloliquefaciens, LMG 12384 and LMG 17599 with respectively
99.8% and 98.1% of sequence similarity (supplementary data, Figure 1S). These data show
that strain S499 most probably belongs to the closely related B. amyloliquefaciens species
rather than B. subtilis.
LC-MS determination of LP pattern produced by strain S499 in vitro
The first objective of the work was to obtain an in-depth characterization of the lipopeptide
pattern synthesized by strain S499 upon growth in erlenmeyer flasks in a culture medium
defined by Jacques and collaborators (1999) for high LP productivity (OM medium). To that
end, we have optimized an LC-MS method providing both sensitive detection and reliable
identification/quantification of the three families of LPs secreted in the medium. As detailed
in the Material and Methods section, HPLC parameters and settings of the electrospray source
in the mass spectrometer were adapted to allow the resolution and optimal detection of most
of the peaks corresponding to the various homologues within each LP family produced by the
strain (Figure 1). By using the SCAN mode of detection (over mass range of 950 mu to 1150
mu for iturin and surfactin and from 1400 mu to 1550 mu for fengycin) in the spectrometer,
detection limits for surfactin, iturin and fengycin were respectively 2.5 ng, 10 ng and 25 ng
injected on the HPLC column. This sensitivity could be approx. 5 times enhanced by using
the single ion recording (SIR) mode which allows to markedly reducing the signal to
background noise ratio. In most cases, specific LC-MS methods were thus used for surfactin,
iturin and fengycin analyses. In some instances, a single method allowing the simultaneous
measurement of both three families was used as it.
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90
I
F
S DAD
MS
MS
MS
DAD
DAD
I1
I3I2
I4
S1
S2S3
S4
S5
S6
S7
S8 S9S10
I5
F3
F7F5
F6F8
F9F10
F13
F19F4F14
F15 F16
Figure 1. Typical LC-MS separation and identification of the three lipopeptide families (I, iturins; F, fengycins; S,surfactins) produced by strain S499 in optimized medium. DAD, HPLC chromatograms obtained with diode arraydetection of material eluted under family-specific conditions. MS, corresponding signal from the mass spectrometerupon Single Ion Recording detection using masses specific for each homologue within each family.
As shown in Figure 1, the various surfactin homologues could be easily identified on the
basis of their retention times in the UV-Vis trace. However, an accurate identification of iturin
and fengycin homologues simply based on the chromatogram generated by the diode array
detector is not possible due to contaminating material produced by the strain that co-eluates.
Coupling with mass spectrometry is thus necessary for peak identification but also to
significantly improve the sensitivity. Homologues corresponding to the peaks in Figure 1 are
listed in Table 1. No additional MS signal in the range 900-1550 mu were recorded upon LC-
MS analysis of crude S499 supernatant strongly suggesting that other LP variants putatively
Results-Chapter 5
91
synthesized by Bacillus (Ongena and Jacques, 2008) are not formed by the strain (data not
shown).
Table 1. Assignement of peaks detected by LC-ESI-MS in Figure 1 and corresponding to surfactins, fengycins and iturins produced by strain S499 in optimized medium. Surfactin (A, Leu in position 7) and fengycin (A, Ala-7 and B, Val-7) variants were identified on the basis of typical MS fragments and amino-acid composition (data not shown). However our data do not allow to discriminate between iturin A and mycosubtilin which only vary in the order of the last two Asn and Ser residues of the peptide. Only protonated forms are mentioned but in most cases, sodium and potassium adducts were also detected for the various homologues. n1 means that there is a double bond in the fatty acid chain of the fengycin homologue.
Peak m/z [M+H]+ Assignement S1 994.8 Surfactin A C12 S2 1008.8 Surfactin A C13 S3 994.8 Surfactin A C12 S4 1022.8 Surfactin A C14 S5 1022.8 Surfactin A C14 S6 1008.8 Surfactin A C13 S7 1036.8 Surfactin A C15 S8 1022.8 Surfactin A C14 S9 1050.8 Surfactin A C16 I1 1043.7 Iturin A/mycosubtilin C14 I2 1057.7 Iturin A/mycosubtilin C15 I3 1057.7 Iturin A/mycosubtilin C15 I4 1071.7 Iturin A/mycosubtilin C16 I5 1071.7 Iturin A/mycosubtilin C16 F4 1435.9 Fengycin A C14 F5 1449.9 Fengycin A C15 F6 1449.9 Fengycin A C15 F7 1463.9 Fengycin B C14 F8 1463.9 Fengycin A C16 F9 1477,9 Fengycin B C15 F10 1477.9 Fengycin A C17 F13 1491,7 Fengycin B C16 F14 1477.9 Fengycin A C17 F15 1505,7 Fengycin B C17 F16 1467.8 Fengycin A C15 n1 F19 1491,7 Fengycin A C18
LP pattern of strain S499 developing on roots of hydroponic plants
In a first approach to investigate in situ LP production, experiments were conducted on
tomato plants grown in a hydroponic culture mode to facilitate colonization study and
quantification of the metabolites secreted into the surrounding environment. Efficient root
colonization is a key event required for optimal expression of biocontrol activity of beneficial
rhizobacteria acting through competition for substrate, antibiosis or plant resistance triggering
(Lugtenberg et al., 2001). Rhizobacterial populations must reach a threshold level sufficient
to produce the active metabolites in sufficient amounts. Plate counts of bacterial cells
adhering to tomato roots were performed 22 days after inoculation and Bacillus colonies were
detected on the basis of their typical morphology at concentrations up to 5x105 cfu/g root.
Results-Chapter 5
92
However, S499 populations could not be accurately quantified due to the lack of natural
antibiotic resistance or antibiotic-resistant derivative.
At the same time, lipopeptide recovery was performed by combining material extracted
from the nutrient solution with the one recovered by extraction of root tissues using detergent.
This is justified by the fact that surfactins secreted by colonizing Bacillus cells may readily
insert into the membrane structure of the plant cells as suggested by the rapid disappearance
of the lipopeptide from the medium when supplemented in hydroponically-grown tomatoes or
when added to tobacco cell suspension where more than 90% of the added surfactin
embedded within minutes into the plasma membrane (data not shown). LC-MS analyses using
specific methods in the SIR mode were performed on 100-times concentrated extracts in order
to determine their precise content in surfactins, iturins and fengycins. A high variability was
observed in the LP quantities recovered from one plant to another and data presented in
Figure 2 are means calculated from 4 independent cultures using each time the material
collected from three plants as a single sample. These data show that members of the surfactin
family were mainly detected while iturins were present but in low concentrations and
fengycins are seemingly not produced under these conditions. In this work, we could not
determine precisely the S499 population on roots but considering a biomass of 5x105 cell/g, it
corresponds to surfactin and iturin productivities of 26 µg/108 cells and 1.7 µg/108 cells,
respectively. It also corresponds to surfactin and iturin concentrations in the hydroponic
medium of approx. 120 nM and 8 nM, respectively.
Results-Chapter 5
93
itu
srf
fen
Rel
ativ
e ab
und
ance
Elution time
Figure 2. Representative ESI mass spectrometric signal recorder during LC-MS analysis of lipopeptidesproduced byB. amyloliquefacienscolonizing roots of hydroponically-grown tomato plants. Detection wasperformed in the SCAN mode (m/z range 900-1550). Elution time periods for every LP family are indicated.Every single peak represents a specific homologue (srf, surfactin; itu, iturin; fen, fengycin). The relativeproportions of total surfactins, iturins and fengycins were calculated using authentic standards and are shownin the boxed graph. Data and standard errors were calculatedfrom 4 samples obtained in 4 differentexperiments but each consisting of approx.2 g fresh weight of combined root materialcoming from 3 plants.
Mass spectrometry imaging of LP pattern
Recent developments in Time-Of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS)
imaging allow the two-dimensional analysis of some lipidic compounds in various biological
samples and it was successfully applied for determining the distribution of surfactins secreted
by B. subtilis in a swarming pattern (Debois et al., 2008). In a second approach to evaluate in
situ LP production by strain S499, we thus wanted to use this imaging technique to analyze
the lipopeptides secreted by cells developing on roots of tomato plantlets grown in a
gnotobiotic system. Disinfected tomato seeds were germinated on gelified medium, bacterized
and colonization was observed after a two-week incubation period. As shown in Figure 3,
S499 readily colonizes the root system by forming macro-colonies that cover many root hairs.
Mean value for S499 population established on roots at that time was 2.3 x 107 CFU/g root
FW. Under these conditions, no significant root-associated microflora was observed on
control plantlets after two weeks of incubation (detection limit of 102 CFU/g root FW).
Lipopeptides secreted by these S499 cells and present at the surface of the gelified
medium were then analyzed by TOF-SIMS after transfer onto a silicon wafer. Scanning over
Results-Chapter 5
94
a small area around S499-coated root hair tips revealed the relative amounts of LP from the
three families (Figure 3d).
a b c
S I F
d
e
*
*
*
*
**
****
f
S C12 S C13 S C15
Figure 3. Imaging MS of LPs produced by strain S499 colonizing roots of tomato plantlets grown on gelified medium in Petriplates. A, microscope image of the main root and root hairs colonized by S499 cells forming macrocolonies. B, microscopeimage of a part of the transferred colonization pattern transferred onto silicon wafer. C, v ideo image of the scanned zonerecorded with an integrated camera inside the TOF-SIMS apparatus. D, High-definition scan of the lipopeptide distribution atthe extremity of biofilm-embedded root hairs. TOF-SIMS images of the sum of surfactin (S), iturins (I) and fengycins (F)ionsare shown. The colour scale indicates the relative amounts of total surfactin/iturin/fengycin ions detected. E, Relativedistribution of surfactin homologues in the same pattern. F, corresponding partial mass spectra of surfactins/iturins (left) andfengycins (right). Similar images and results were obtained by analyzing two other root zones from plantlets inoculated andgrown independently.
Results-Chapter 5
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Based on total counts in the MS detector corresponding to the more abundant homologues of
the three families (surfactin C14, iturin C15 and fengycin C16), the quantity of surfactin is
respectively 11 times and 34 times higher than iturin and fengycin (Figure 3f). Analysis of
concentrated solutions of authentic standards showed a detection limit of approx. 3 µg/ml.
Almost the same results were obtained upon scanning a similar small area around the
extremity of root hairs in an independent experiment (data not shown). TOF-SIMS imaging
over a larger surface (5 x 5 mm) of the colonized-root system also revealed that surfactins are
produced in much higher quantities compared to iturins and fengycins, respectively 14 times
and 84 times (supplementary data, Figure 2S). In all cases, surfactins are much more abundant
in the periphery than on biofilm suggesting that these hydrophobic LP do not accumulate on
or around the producing cells but readily diffuse into the gelified medium as demonstrated for
swarming cells (Debois et al., 2008).
TOF-SIMS imaging thus provides useful information to study in situ production of LPs
but this technique does not allow quantitative measurements and only molecules present at the
surface are detected. Moreover, the analysis of pure LP solutions (10 µl-drop deposit on the
same gelified medium) in the same conditions also revealed that fengycin detection is 5 times
less sensitive either because they are less efficiently ionized in the source of the mass
spectrometer or because they are less efficiently extracted from the surface of the medium to
adsorb onto the silicium wafer or both. In order to better determine the actual concentrations
of the three types of LPs, we have extracted the compounds contained in agar cylinders (5
mm diameter) collected by core-sampling from the medium in the close vicinity of S499-
colonized roots. Analysis of these extracts by LC-ESI-MS also revealed much higher relative
quantities for surfactin compared to the two other LP families as mentioned in Table 2. It is
not possible to express the quantities measured in term of productivity but it corresponds to
quite high concentrations that were may be generated by S499 macrocolonies locally around
the roots. Surfactins are dominant but fengycins and iturins are also present in concentrations
from 2 to 9 µM (supplementary data, Figure 3S).
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Table 2: Comparison of the lipopeptide patterns produced by strain S499 in various conditions. RZ (root zone), LPs produced by cells colonizing roots of tomato plantlets grown in Petri plates and extracted from the gelified medium surrounding roots after core sampling. NE, RE, OM, LPs quantified from liquid cultures in natural exudates, recomposed exudates (Kamilova et al., 2006) and optimized medium (Jacques et al., 1999) respectively. OM, b is LP production under conditions conducive for biofilm formation (growth in 96-well plates) where most of the cells are in the pellicle by contrast with OM, p which means LP production by planktonic cells in agitated flask. O2+ and O2- mean LP production following growth in well aerated and in nitrogen flushed and oxygen depleted bioreactor respectively.
Surfactin Iturin Fengycin Total
LPs
Conc.
(mg/l)
Rel. prop.
(%)
Conc.
(mg/l)
Rel. prop.
(%)
Conc.
(mg/l)
Rel. prop.
(%)
Prod.
µg/108
cfu
RZ* 58 83 9 12 3 4 nc
NE 32 ± 7 58 15 ± 8 27 8 ± 6 14 147
RE 364 ± 112 61 175 ± 65 27 70 ± 24 11 304
OM, b 179 ± 46 80 27 ± 18 13 17 ± 11 7 nc
OM, p 628 ± 108 51 321 ± 55 26 272 ± 71 22 436
RE, O2 +* 556 66 212 25 73 8 341
RE, O2 -* 252 67 108 29 10 2 740
Data are means and standard errors calculated from at least four repeats of the experiment otherwise stated (*) representing average values from two independent assays. nc: means non calculated due to non accurate biomass quantification.
LPs secreted upon growth in the presence of root exudates
In order to test more specifically the effect of the nutritional status imposed by the plant on
LP production, the S499 strain was grown in agitated liquid culture in the presence of root
exudates as sole carbon source (NE medium). These exudates were collected from hydroponic
tomato plants after 23-25 days of growth in nutrient solution under controlled conditions.
S499 growth was obviously substrate-limited since low biomass levels (OD600 between 0.35
and 0.45) were observed. It resulted in much lower concentrations of surfactins, iturins and
fengycins in the medium compared to those observed in OM (Table 2). From a quantitative
point of view, the secretion thus appears much less efficient than in optimized conditions.
However, in term of productivity, LP synthesis efficiency in natural exudates corresponds to
the third of the one observed in OM and thus globally, these data confirm that exudate
components are conducive for LP synthesis. Qualitatively, the relative proportion of iturins
compared to surfactins is similar to the one observed in optimized medium but the formation
of fengycins appears to be significantly less efficient.
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LPs formed in the presence of specific carbon sources
Strain S499 was also cultivated in a minimal medium (RE medium) containing as sole carbon
sources the sugars, organic acids and amino acids typically found in the tomato exudates and
added in the proportions determined by Kamilova and collaborators (2006). Increased levels
of biomass and LP synthesis (Table 2) compared to natural exudates can be explained by
higher concentrations of nutrients in the medium. The relative proportions of the three LP
families produced in this medium are similar to those observed in NE.
The main representatives of these substrates were tested individually for their potential
to support growth of strain S499 and lipopeptide synthesis. Most of these C sources supported
growth to an almost similar level (data not shown) but it clearly appears from Figure 4 that
surfactin synthesis is significantly higher in the presence of organic acids as unique C sources
compared to sugars. Thought it may vary in function of various plant factors, succinic and
citric acids are the more abundant substrates released by uninoculated tomato roots and also
represent adequate nutrient sources utilizable by S499 for surfactin production (Figure 4A).
By contrast, iturin productivity is slightly higher upon growth in the presence of sugars
compared to organic acids (Figure 4B) and fengycins that are only poorly produced in the
presence of citrate, succinate or malate (Figure 4C).
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A
C
B
Figure 4. Lipopeptide productivities in the presence of various carbon sources typically found in tomato exudates. A, surfactins; B, iturins; C, fengycins. Data are mean values and standard errors calculated from five independent experiments.
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LP production upon biofilm formation and oxygen limitation
When grown in the same optimized medium as used for flask cultures but in 96-well plates,
strain S499 readily forms pellicle at the surface of the liquid medium. We wanted to evaluate
the effect of such a pellicle formation on the pattern of LP produced since the macro-colonies
developing on roots may be considered as biofilm-related structures (Bais et al., 2004; Ramey
et al., 2004). The results presented in Table 2 show that surfactin synthesis in S499 cells
forming pellicles is very effective compared to iturins and fengycins. The relative proportion
of the three LP families produced under these conditions is similar to the one observed upon
root colonization. However, it significantly differs from the one secreted upon growth of S499
as planktonic cells in optimized liquid medium where iturins and fengycins are secreted in
much higher amounts corresponding to approx. half of the surfactin produced (Table 2).
The effect of oxygen concentration was evaluated by cultivating the strain in a
bioreactor upon two different aeration rates. Final cell densities were much lower in the non-
aerated culture but a significant biomass was measured, showing that strain S499 could
develop under limited availability or absence of oxygen molecular as a terminal electron
acceptor. Some Bacillus strains can utilize nitrate as an alternative electron acceptor (Mach et
al., 1988; Glaser et al., 1995) or are able to grow anaerobically on minimal media in the
absence of terminal electron acceptors (Hoffmann et al., 1988; Nakano et al., 1988; Ramos et
al., 2000). As shown in Table 2, strain S499 produces iturin, surfactin and fengycin
lipopeptides upon growth in the two conditions of oxygen availability tested. In terms of total
productivity, LPs are more efficiently produced under oxygen-starved conditions but the
relative production rate of the three families is similar in both conditions of aeration.
Influence of temperature and pH on LP production
To test the effect of temperature, cells were grown until late stationary phase in Erlenmeyer
agitated flaks in RE medium and LP concentrations are determined by LC-MS (Table 3). The
production of total amount of iturins and surfactins increased with temperature while the
production of fengycins is higher at 25 °C compared to 20 °C but then decreased beyond that
temperature. It appears that cell population was not significantly different among the tested
temperatures (not shown).
The effect of pH on LP production was tested on B. amyloliquefaciens S499 cultured in
fermentor with automatic regulation allowing the medium to be maintained at different
specific pH values (Table 3). Not surprisingly, optimal growth of the strain was observed at
pH 7 but lipopeptide production under this condition was very low and only traces of
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100
surfactin could be detected. Very low biomass level was observed by growing S499 at a
controlled pH value of 8 and no LP were detected. Significant amounts of surfactin and iturin
could only be measured in culture supernatant after growth at pH continuously adjusted to 5.7
but the quantities recovered were still fairly reduced compared to those observed upon growth
in bioreactor in the same conditions but without pH control (RE, O2 + in Table 1).
Table 3. Lipopeptide productivities (µg/108 cfu) in function of temperature and pH. Each value is an average of results obtained from two independent experiments with similar results. OD: Optical density, Srf: surfactins, Itu: Iturins, Fen: Fengycins, ND: not detected.
Parameters OD 600nm Srf Itu Fen 20 °C 1.4 510 245.46 98.46 25 °C 1.15 862 367.31 231 30 °C 1.32 1295.2 683.17 84.8 5.7 0.43 187 58 ND 7.0 2.75 1.2 ND ND 8.0 0.17 ND ND ND
Changes in the relative proportions of LP homologues
Under laboratory growth conditions in OM medium, strain S499 usually produces a
mixture of C12/C13/C14/C15/C16 surfactin homologues in the proportion of 7/17/48/32/1 in %.
Interestingly, the proportion of C15 tremendously increased in the pattern of surfactins that are
secreted by the strain evolving on roots of hydroponic tomato plants (Figure 5). As a matter of
fact, higher levels of C15 surfactins were also detected after growth in liquid cultures in the
presence of natural root exudates. Globally, SC14 and SC15 together represent more than
80% of the total surfactins produced in vivo which is in agreement with imaging data showing
much higher MS signals corresponding to C15 surfactins compared to SC12 and SC13 (Figure
3e). However, this increase is difficult to interpret on a nutritional basis since organic acids as
sole C substrates are only slightly more conducive for C15 synthesis than sugars (Figure 5).
On another hand, higher proportions of long chain fengycins (mainly C18) are also seemingly
produced by the strain upon growth in the presence of natural exudates compared to those
formed in the OM medium but globally, these changes are much less obvious than in the case
of surfactins and these minor differences have to be confirmed in additional experiments.
Concerning iturins, the C16 homologue is usually produced in most of the media tested in
laboratory conditions but was not detected in LP extracts prepared from S499-colonized
plants. In these in vivo samples, iturin C15 represents more than 85% of the total and thus
much higher quantities compared to the other conditions (Figure 5). However, given the small
amounts detected in the hydroponic systems, other assays are also required. In bioreactor
cultures performed to evaluate the influence of aeration rate, the relative proportions of
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101
C12/C13/C14/C15/C16 surfactin homologues were respectively 2/45/14/28/11 and 6/9/62/19/2 in
aerobic and anaerobic conditions.
S
I
F
OM In situ NE Glu Malic
Figure 5. Distribution of homologues varying in the length of the fatty acid chains within the three families of lipopeptides(surfactins (S), iturins (I) and fengycins (F)) produced byS499 after growth upon various conditions and as determinedby LC-ESI-MS. OM., LPs produced in optimized medium (Jacqueset al. 1999);In situ, in hydroponic cultures; NE, in natural exudates;Glu and Malic, in the presence of glucose and malic acid as sole carbon sources. Data are mean values calculated from 3independent cultures.
Thus higher quantities of C15 and C16 homologues were observed upon aerated conditions but
the global proportions of surfactins with fatty-acid chains higher than C13 is greater upon
growth under oxygen-depleted conditions (Figure 6B). There was no significant difference in
homologues of iturins and fengycins produced by the strain in different aeration conditions
(results not shown).
Concerning surfactins and iturins, there is no marked differences between the relative
proportions of individual homologue produced considering the temperature of growth. By
contrast, the fengycin homologues with short chain were abundant at 20 °C whereas at 25 °C
and 30 °C, C16 and C18 fengycins dominate with 50% and 33% of total amounts, respectively
(Figure 6A).
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20 C 25 C 30 C
S
I
F
S
O2 O2 d
Figure 6. Relative proportions of homologues varying in length of fatty acid chain within the threefamilies of LPs vis-à-vis temperature of growth (20 C, 25 C, 30 C) and oxygen availability (O2:oxygen supplied, O2d: oxygen depleted). Data are mean values obtained from two replications of oneconclusive experiment.
A
B
Discussion
Through the so-called rhizosphere effect, plants create a specific environment for microbial
development in the soil surrounding roots. The influence of rhizosphere conditions on the
inhabiting microflora is being extensively studied at the population level because of the
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interest in engineering this environment in order to improve the efficacy of beneficial
microbes as biocontrol agents. Much less is known about the influence of rhizosphere factors
on the biosynthesis of specific metabolites responsible for biocontrol activity of these
beneficial strains. In this context, the present work was initiated to accurately identify and
quantify lipopeptides produced by Bacillus in situ. The combination of HPLC and mass
spectrometry techniques allowed us to determine the whole pattern of LPs from the surfactin,
iturin and fengycin families produced by the biocontrol strain B amyloliquefaciens S499
evolving on roots. Multiple studies used optimized mass spectrometry techniques to
characterize or compare the LP patterns produced by Bacillus isolates under laboratory
conditions (Vater et al., 2002; Price et al., 2007; Stein, 2008; Athukorala et al., 2009; Vater et
al., 2009). However, only few works published up to now deled with LPs secreted in the
natural environment in general and in the rhizosphere in particular. Surfactin and/or iturins
were recovered from cucumber roots surrounding soil inoculated with Bacillus strain QST713
(Kinsella et al., 2009) and from tomato rhizosphere inoculated with strain BGS3 (Ongena et
al., 2007; Nihorimbere et al., 2009) and strain RB14-C (Asaka and Shoda, 1996). Fengycins
and Iturins were also occasionally detected in the phyllosphere and on fruits, (Touré et al.,
2004; Romero et al., 2007). However, in all these studies, only some LP variants or even
homologues were analyzed. This work is, to our knowledge, the first report dealing with
determination of the whole array of surfactin, iturin and fengycin homologues that may be co-
produced in the soil by a natural strain of Bacillus.
B. amyloliquefaciens S499 retain biocontrol potential and efficiently co-produces the
three lipopeptide families under well defined and optimized in vitro conditions. However, LC-
ESI-MS analyses of the LPs secreted by S499 cells colonizing tomato roots revealed much
higher relative quantities of surfactins. Fengycins and iturins were detected in much smaller
amounts or only traces (Figure 1). This strongly suggests that the physiology of S499 cells
developing on plant tissues is conducive for surfactin synthesis but not for an efficient
production of the two other types of lipopeptides. We previously used the LacZ reporter
system in B. subtilis strain BGS3 to provide a first evidence that surfactin genes may be
readily expressed under rhizosphere conditions (Nihorimbere et al., 2009). However, BGS3
derives from the B. subtilis 168 Marburg strain which can no longer be viewed as natural
isolate since it has been acclimated to laboratory conditions for several decades and exposed
to X-rays in the mid-1940’s.
Such in vivo lipopeptide signature is of biological relevance since the three LPs families
actually display distinct functions in the ecology of the producing strains in the phytosphere
Results-Chapter 5
104
and notably in interactions with co-existing organisms, including bacteria, fungi, oomycetes,
protozoan predators and plants (Ongena and Jacques, 2008; Raaijmakers et al., 2010). LPs
play a crucial role in the antagonism developed towards fungal pathogens but it mainly
concerns iturins and fengycins while surfactins has been demonstrated to be mostly
antibacterial and antiviral. Beside their antimicrobial properties, surfactin and to a lower
extent iturins, are also involved in root colonization and surfactin has been identified as
elicitor of the systemic stimulation of host plant immune system (Ongena and Jacques, 2008).
Beside these biocontrol-related traits, LPs may also have different roles in the development
and survival of B. subtilis strains in their natural habitat. It includes increasing of the surface
area and bioavailability of hydrophobic water-insoluble substrates, heavy metal binding,
bacterial pathogenesis, quorum-sensing, motility and biofilm formation (Ron and Rosenberg,
2001; Mulligan, 2005; Raaijmakers et al., 2010). Intrinsically, there is a competitive
advantage for Bacillus isolates to co-produce surfactin, fengycin and iturin with specific roles
and targets in the context of biocontrol of plant diseases. However, our results strongly
suggest that in situ expression of the related biosynthesis genes may considerably differ from
one family to another. This could not be demonstrated experimentally because we were
unable to generate reporter derivatives of strain S499 which is highly recalcitrant to
transformation like many natural B. subtilis/amyloliquefaciens isolates. Investigating the
differential expression of srf, fen and myc genes under natural soil conditions in S499 or in
another natural isolate co-producing the three families, is one of our main future prospects.
Such a surfactin-enriched LP pattern produced by strain S499 in the tomato rhizosphere
under non sterile conditions was also observed by TOF-SIMS imaging after root colonization
of tomato plantlets on gelified medium (Figure 3). Under such sterile conditions, S499 cells
can readily grow without having to compete with auxiliary microflora. Seemingly, the
presence of other microorganisms sharing the same ecological niche does not significantly
impact on the LP pattern secreted by S499 since surfactins again represent the large majority
of the lipopeptidic compounds produced in the gnotobiotic system. From a technological
point of view, this imaging MS technique appears to be a yet emerging but extremely
powerful method to study the production of lipidic molecules by microbes developing on
solid supports. It was successfully used by Debois and collaborators (2008) to study the
heterogeneity of surfactin secretion in a swarming pattern of Bacillus cells. In that study,
fentomole amounts of C15 surfactin could be detected. Surfactin as major LP produced in
planta by S499 is a trend that was also observed in preliminary experiments with corn and
lettuce. However, the LP signature may also be strongly influenced by the host plant species
Results-Chapter 5
105
since almost no production of surfactin was observed upon growth on cucumber and soy
(Ongena et al., unpublished data).
It is clear from our results (Figure 4) that the S499 LP pattern can be markedly
influenced by nutritional factors such as carbon source. The proportions of
surfactin/iturin/fengycin readily differed in the presence of sugars or organic acids as sole
carbon source. Much lower relative quantities of iturins and fengycins are produced by S499
in the presence of the last substrates. It may partly explain the lower production levels of
these two LPs in planta because organic acids are the main components of tomato exudates
(Kamilova et al., 2006). However, the proportions of iturins and fengycins secreted upon
growth in natural exudates collected from hydroponically-grown tomatoes were higher (Table
2). So, the nutritional context in term of carbon source created by the plant exudation may
favour surfactin synthesis to some extent but is not sufficient by itself to explain the very high
proportion of this LP secreted in situ compared with the two other families. The effect of the
non-polar fraction of natural exudates (hydrophobic material eluted from C18 cartridge with
pure methanol and potentially containing phenolics, flavonoides, etc...) on LP production was
also tested. However, supplementing the RE medium with a concentrated extract of this non-
polar fraction had no effect on the pattern of LPs both qualitatively and quantitatively (data
not shown).
The oxygen status can also affect the physiology of both the host plant and the
colonizing bacterium and thus alter the interactions. In agreement with previous works (Hbid
et al., 1996; Jacques et al., 1999), final LP concentrations in the supernatant were higher in
well aerated cultures. However, results from the present study show that productivities for
both three LP families were quite higher under oxygen starvation conditions (Table 2).
Oxygen depletion is common in many soils and especially in the rhizosphere, notably due to
root and microbe-associated consumption (Højberg et al., 1999). However, the bacterium can
obviously adapt to such conditions and our results suggest that strain S499 can well develop
and produce LPs on roots where oxygen is not abundant. It agrees with previous work
performed with another strain demonstrating that srfA expression and surfactin production
still remain very effective in oxygen-starved culture conditions (Nihorimbere et al., 2009). A
positive influence of O2 limitation on surfactin production has also been demonstrated by
modifying the oxygenation method (Lee et al., 2001) or by modulating the oxygen transfer
rate in bioreactor (Yeh et al., 2006). The introduction of oxygen limitation, which redirects
the energy flux into product synthesis, has led to the highest productivity of surfactin by
Bacillus subtilis C9 (Kim et al., 1997). That said, our bioreactor experiments also revealed
Results-Chapter 5
106
that the level of oxygen availability during growth of S499 has no marked effect on the
relative proportions of the three LP families (Table 2). Therefore, this parameter does not
deserve further consideration for explaining the lipopeptide signature in planta.
By contrast, our results showed that surfactin synthesis in S499 cells forming biofilm is
very effective compared to iturins and fengycins with relative proportions of the three
families similar to the ones observed in rhizosphere extracts (Table 2). In situ, root-colonizing
rhizobacteria such as Bacillus typically develop in macrocolonies or form biofilms and their
actual physiology is thus probably not related to a planktonic state. Few studies have
accurately evaluated the consequences of growing on solid support on LP synthesis but some
clues arose from mass spectrometry studies of LPs produced by B. subtilis cells developing on
gelified media showing that patterns of surfactins and fengycins may be qualitatively and
quantitatively modified compared to cells living freely in liquid cultures (Leenders et al.,
1999; Nihorimbere et al., 2009). In another approach, Gancel and collaborators (2009) have
also demonstrated an enhancement of surfactin production upon cell immobilization on
polymer particles. So living in root-adhering macrocolonies may readily influence the pattern
of LPs secreted by Bacillus cells.
In batch cultures, the synthesis of the three LP families occurs at different growth
stages. Surfactin typically accumulates in the medium during the late exponential phase while
the syntheses of fengycins and iturins are delayed to the stationary phase. It means that
expression of the corresponding genes is somewhere related to the growth rate of the
bacterium. Such effect of growth rate on surfactins’ synthesis was confirmed by previous
experiments performed in chemostat bioreactors which showed that srfA expression
significantly decreased with increasing µ (Nihorimbere et al., 2009). A similar relationship
between µ and srfA expression was observed with another Bacillus subtilis isolate derived
from the surfactin over-producing strain JH642 (Kakana, 2005). Interestingly, the
development of established bacterial populations in the rhizosphere is actually restricted by
nutrient availability due to low root exudation rate (Lugtenberg et al., 2001; Bais et al., 2006).
Bacillus cells on roots can thus be in a nutrient-starved state driving cell physiology to slow
growth rate which is apparently conducive for surfactin but not fengycin or iturin synthesis.
The effect of temperature on LP production by Bacillus has not been studied
extensively. It seems globally that the expression of synthesis genes is not tremendously
influenced by this parameter (Ohno et al., 1995; Fickers et al., 2008) but, changes in
production level were actually observed suggesting that protein turn-over or some
downstream regulatory processes may be affected (Fickers et al., 2008). Higher temperatures
Results-Chapter 5
107
(37 °C) favoured surfactin production by B. subtilis strains RB14 (Ohno et al., 1995) and
ATCC6633 but not by strain S499 for which increasing temperature from 30 °C to 40 °C is
detrimental for LP production (Jacques et al., 1999). In our experiments, surfactin and iturin
productions are higher at 30 °C than 20 °C but we did not test higher temperatures. This is
however contrasting with the trends observed for two others Bacillus strains for which iturin
production clearly decreased with higher temperatures (Fickers et al., 2008). So the incidence
of temperature on LP synthesis seems to be strain-specific and may be related to the synthetic
pathways of fatty acids forming cellular membranes. The fatty acid composition of these
membranes is altered by varying the growth temperature to maintain the proper membrane
rigidity at a given temperature (Phae et al., 1990). It has been reported that the relative
amount of long-chain fatty acids increases with increasing temperature for five Bacillus
strains (Kaneda, 1991) as well as Clostridia (Huang et al., 1993), mainly because the longer
the chain of a fatty acid is, the higher the melting temperature of the fatty acid becomes.
Lipopeptide production by strain S499 in bioreactor under controlled pH was very low.
Significant amounts of surfactins but not iturins or fengycins, could be detected only at pH
5.7 and these results have to be taken with care. However it is in agreement with the fact that
surfactin gene expression is pH-dependent and also agrees with a previous study conducted
with the same strain showing a clear negative effect of raising the pH value from 6 to 8 on
biosurfactant production (Akpa et al., 2001). More generally, it has been reported that many
soil-bacterial strains are known to produce metabolites at maximum when they are cultured at
pH below neutrality (Abushady et al., 2005). For instance, rhamnolipids production by
Pseudomonas sp. was at its maximum in a pH range of 6.0-6.5 and decreased sharply above
pH 7 (Guerra–Santos et al., 1984). Globally, it appears from these data that an efficient
synthesis of LPs may be favoured in acidic to neutral environments, which is often the case
for rhizosphere due to multiple processes related notably to plant exudation and microflora
activity (Hinsinger et al., 2009).
Whether in situ productivities may generate rhizospheric LP concentrations sufficient to
provide any biological effect remains questionable. LPs are generally active at 2-30 µM
depending on the role and target organism considered but data mentioned in Figure 2
correspond to approx. 0.12 µM surfactins and 0.008 µM iturins in the nutrient solution. In the
previous study conducted with B. subtilis BGS3, we observed a LP concentration of 1.8 µM
in the hydroponic medium surrounding tomato roots (Nihorimbere et al., 2009). Even higher
quantities of both surfactin and iturin were detected in the cucumber rhizosphere colonized by
strain QST713 (Kinsella et al., 2009). Possible enzymatic degradation by auxiliary microflora
Results-Chapter 5
108
and incomplete extraction may contribute to underestimate the actual abundance of LPs in the
vicinity of the root system in the soil on the basis of results presented here. Another
possibility is that S499 retains only poor ability to produce LPs while developing on roots.
However, under in vitro conditions, this isolate produces a quite high level of surfactin
compared to other B. subtilis/amyloliquefaciens strains (data not shown). Moreover, LC-ESI-
MS measurements of LPs secreted into the gelified medium by S499 cells in plate
experiments (Table 2) revealed that surfactin concentration may locally reach 50 µM
concentrations within the diffusion zone close to the colonized roots. Because LP destruction
by other organisms cannot occur and dilution effect is reduced under these conditions
compared to the hydroponic system, such 50 µM concentration probably better reflects the
amounts of surfactin that may be actually secreted by S499 evolving on tomato roots.
Concentration of surfactins in the tomato rhizosphere may thus be compatible with biological
activity as inducer of resistance in the host plant (2-10 µM) (Ongena et al., 2007; Jourdan et
al., 2009) but obviously, iturin and fengycin amounts by S499 are not sufficient for antifungal
activity against soilborne phytopathogens.
From a qualitative point of view, it also clearly appears that the relative proportions of
the different homologues within the surfactin and iturin families may drastically vary in
function of the growth conditions. For instance, the C15 homologue by itself represents more
than 75% of the total surfactins produced by cells colonizing tomato roots. It largely differs
from the one observed upon growth of S499 as planktonic cells in the optimized laboratory
culture medium (Figure 5). Such an in vivo LP signature is interesting since, in some
instances, homologues with the longest fatty acid chains were proven to be biologically more
active. This does not only concern their potential to trigger plant defense reactions (Jourdan et
al., 2009), but also its haemolytic, antiviral and antimicrobial activities (Kracht et al., 1999;
Bonmatin et al., 2003; Dufour et al., 2005). Such an important role for the length of the acyl
chain may be related to the fact that it readily inserts into phospholipid bilayers (Carrillo et
al., 2003; Eeman et al., 2006; Heerklotz and Seelig, 2007). Synthesis of C15 surfactins is
seemingly favoured in the presence of organics acids compared to sugars (Figure 5). Akpa et
al. (2001) have also demonstrated some influence of the nature of carbon source on the
pattern of surfactins produced by B.subtilis. The surfactin-apparented lychenisin also
accumulates differently in the culture broth upon growth in various media differing in C
source (Li et al., 2008).
In conclusion, the success of some isolates of the Bacillus genus as biopesticides
certainly rely on the large part of their genome devoted to antibiotic synthesis (Stein, 2005).
Results-Chapter 5
109
We show here that lipopeptides may be readily produced by Bacillus colonizing roots. This
reinforces the importance of these compounds for rhizosphere fitness and biocontrol activity.
However, the in planta lipopeptide signature does not reflect the broad panoply of LPs that
may be produced in vitro in an appropriate medium. It means that promising strains selected
on the basis of an efficient co-production of surfactins, fengycins and iturins with specific
roles and targets, have to be further tested for their potential to secrete the same LPs pattern
under natural rhizosphere conditions. This would provide a competitive advantage for such
Bacillus isolates with potential multi-faceted biocontrol activity.
Acknowledgements This work received financial support from the Program F.R.F.C. n° 2.4.628.10F and Crédit aux chercheurs n° 1.5.192.08F (National Funds for Scientific Research, F.R.S.-FNRS, Belgium). V. Nihorimbere is a recipient of a grant from the Belgian Technical Cooperation (BTC/CTB). M. Ongena is a Research Associate at the National Funds for Scientific Research and H. Cawoy's PhD thesis is supported by a grant from the Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture (F.R.I.A.).
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Supplementary data
Figure 1S
Figure 2S
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114
Surfactin
1 mg/mL
Iturine
1 mg/mL
Fengycin
1 mg/mL
Figure 3S
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Foreword on Chapter 6
In Chapter 5, we have studied the pattern of production of the three families of lipopeptides
by strain S499 of B. amyloliquefaciens cultured in situ on tomato plants in greenhouse and in
vitro versus different environmental parameters. This strain showed to produce the three
families of lipopeptides but in very different relative amounts; and, surfactins were by far
more efficiently secreted compared to iturins and fengycins in the conditions assayed.
Moreover, surfactins are probably the Bacillus LPs displaying the largest eventail of
biocontrol-related activities. It is why we demonstrated sensu stricto its potential factories
involved in biological activities. We wanted to further study in situ production of this
lipopeptide by Bacillus by coupling direct detection of the compound and genetic expression
(Chapter 6). To that end, we had to use another Bacillus strain BGS3 that contains a reporter
gene system LacZ which helps to evaluate the surfactin gene expression. BGS3 strain was
constructed by integrating into the strain BC25 of Bacillus amyloliquefaciens the promoter of
surfactins (psrfA) obtained from B. amyloliquefaciens S499 genomic DNA.
Results-Chapter 6
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Chapter 6. Ecological fitness of Bacillus subtilis BGS3 regarding production of the surfactin lipopeptide in the
rhizosphere
Published in Environmental Microbiology Report (2009) 1(2), 124-130.
Nihorimbere Venanta, Fickers Patrickb, Thonart Philippea, and Ongena Marca
a Centre Wallon de Biologie Industrielle, Unité de Bioindustries, Gembloux University of
Agricultural Sciences, B-5030 Gembloux. b Centre d'Ingénierie des Protéines, University of Liège, B-4000 Liège, Belgium.
Running title: Bacillus lipopeptide synthesis in the rhizosphere
The first two authors contributed equally to the paper.
Results-Chapter 6
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Abstract
Cyclic lipopeptides and particularly surfactins produced by Bacillus species retain
antibacterial, antiviral, biofilm-forming and plant resistance-inducing activities. In most cases,
their role in biological control of plant diseases was evoked on the basis of in vitro assays or
by using non-producing/ overproducing mutants but there is a need for more direct evidence
of an efficient lipopeptide biosynthesis in the rhizosphere. In this work, we coupled LC-MS
quantification of the lipopeptides secreted by cells colonizing tomato plants with the use of
psrfA–lacZ reporter system integrated within the BGS3 chromosome to study the expression
of the surfactin operon in planta. Results showed that a higher level of psrfA induction was
observed upon the establishment of a stable BGS3 population on roots and surfactins
extracted from the rhizosphere were produced in biologically significant quantities. Our
results also demonstrate that BGS3 efficiently utilizes the main substrates from plant exudates
to produce surfactins. This synthesis is also efficient in cells forming colonies and the
production may be favoured in bacteria developing slowly in the rhizosphere. This provides a
first understanding of how environmental factors may influence lipopeptide production by
beneficial Bacillus strains.
Introduction
Among the panoply of antimicrobial compounds that can be produced by Bacillus species,
cyclic lipopeptides of the surfactin, iturin and fengycin families retain potential
biotechnological applications (Mulligan, 2005; Mukherjee et al., 2006) but are also tightly
involved in most of the mechanisms described to date that explain the beneficial effect of
specific strains to control plant diseases (Bais et al., 2004; Ongena and Jacques, 2008). They
can facilitate root colonization, act as antagonists by directly inhibiting phytopathogens and
reinforce the resistance potential of the host plant via the stimulation of the so-called induced
systemic resistance (ISR) phenomenon. This broad range of activities is due to the physico-
chemical properties of these non-ribosomally synthesized amphiphilic molecules. The srf
operon encodes genes involved in surfactin biosynthesis. Members of this group are
heptapeptides interlinked with a b-hydroxy fatty acid to form a cyclic lactone ring structure.
The length of this fatty acid chain may vary from C12 to C15 and different homologous
compounds are thus usually co-produced (Peypoux et al., 1999). Plant roots exude a large
range of potentially valuable small-molecular-weight compounds into the rhizosphere that
creates a highly dynamic front for interactions between roots and soil microflora (Bais et al.,
2006). Some of these exuded molecules act as chemical signals for motile bacteria to move to
Results-Chapter 6
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the root surface but also represent the main nutrient sources available to support growth and
persistence of rhizobacteria (Hirsch et al., 2003). The host plant thus imposes a specific
nutritional context but the complexity of microbial communities sharing the ecosystem and
the variability of physico-chemical parameters inherent to the soil such as mineral content,
pH, temperature and oxygen availability may also drastically influence rhizobacterial growth
and production of biocontrol determinants as demonstrated with various pseudomonads
(Duffy and Défago, 1999; Van Rij et al., 2004; Dubern and Bloemberg 2006; Ongena et al.,
2008). Understanding which and how environmental factors may modulate the production of
biocontrol determinants is a key to improve the level and reliability of plant disease reduction
through the use of rhizobacteria. However, in the case of Bacillus antibiotics and particularly
lipopeptides, most of the data suggesting their role in biocontrol derive from antagonism
assays performed in vitro or by using non-producing or overproducing derivatives and
correlation with respective loss or increase in the biocontrol activity (Ongena and Jacques,
2008). Comparatively, studies providing direct evidence through the demonstration of an
efficient lipopeptide production in situ are scarce (Asaka and Shoda, 1996; Touré et al., 2004;
Romero et al., 2007). In this context, the study reported here was conducted with the first
objective to demonstrate efficient expression of biosynthetic genes and surfactin secretion in
the rhizosphere and second to evaluate the influence of some rhizosphere-specific parameters
on lipopeptide synthesis. To this end, we used the LacZ gene reporter system in Bacillus
subtilis BGS3, not only to provide information on the transcriptional activity of specific
antibiotics in situ but also to easily monitor populations of the introduced strain among the
whole root-associated microflora. The BGS3 derivative was generated from the surfactin-
overproducing isolate BC25 previously demonstrated to provide a protective effect on tomato
via the stimulation of systemic resistance in the host plant (Ongena et al., 2007).
Results and discussion
Colonization and surfactin production in the rhizosphere
Expression of biosynthetic genes and surfactin secretion by the BGS3 derivative was first
verified during growth in an optimized medium in batch-type bioreactor with control of
fermentation parameters. Surfactins production clearly occurred within the 5-6 next hours and
reached a maximum after 18 h of cultivation corresponding to the transition from exponential
to stationary growth phase which is in agreement with results from other studies (Figure S1 in
Supporting information) (Lin et al., 1998; Koumoutsi et al., 2004). Biomass and surfactins
concentrations were similar to the parental BC25 strain showing that the genetic
Results-Chapter 6
119
transformation did not significantly affect the basal metabolic functions or those related to the
synthesis of surfactins (not shown).
0
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Relative ββ ββ-gal activity U
/105
cells)
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ls x
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oot
Days post-inoculation
colonization BGS3 relative srfA gene expression
Figure 1. Evolution of population and expression of surfactin operon in B.subtilis BGS3 cells colonizing tomato roots. Bacillus subtilis BGS3 was constructed as follow: psrfA promoter was PCR amplified from B. subtilis S499 genomic DNA using primer MO01b (5′-CCT CAT GCC TAT TCT TGA AGC CAT GTA TG-3′) and MO02b (5′-GGA TCC TAT TTC CAT ATT GTC ATA CCT CCC CTA ATC-3′) and cloned into pGEM-T Easy vector (Promega, Madisson, WI) to generate pMO01. After correct sequence verification with universal primer T7 and SP6, psrfA promoter was rescued from pMO01 by BamHI–EcoRI digestion and cloned into pDG1663 (Guérout-fleury et al., 1996) at the corresponding site to yield pMO02. This final construct was then integrated into B. subtilis BC25 (Ongena et al., 2007) at the thrc locus to give rise to strain BGS3. Ectopic integration was verified by threonine auxothrophy on minimal medium, resistance to erythromycine and sensibility to spectinomycin as described elsewhere (Guérout-fleury et al., 1996). Surface-sterilized tomato seeds were germinated for 5 days at room temperature in the dark and in gelified sterile nutrient medium consisting of 5 mM Ca(NO3)2, 5 mM KNO3, 2 mM MgSO4, 1 mM KH2PO4 and micronutrients. They were transferred in sterilized 50 ml tubes filled with nutrient solution, inoculated with strain BGS3 and incubated at 25 ± 2 °C in the greenhouse with a 16 h photoperiod alternating sunlight and fluorescent light. For inoculum preparation, the bacterium was grown on solidified Luria–Bertani (LB) medium for 24 h and harvested cells were re-suspended in NaCl 0.85% and the concentration was determined by measuring turbidity at 600 nm. BGS3 cells were inoculated at a final concentration of 108 cells ml -1 nutrient solution. Root colonization by the BGS3 strain was determined by plate counts on LB medium on the basis of typical morphology of the colonies and blue coloration upon growth on X-Gal-supplemented medium. The use of both criteria was necessary to reliably discriminate BGS3 cells from the contaminating bacterial microflora. Three plants were used to obtain values at each time point and data represent mean values from two experiments. In situ surfactin gene expression was evaluated on samples of approximately 0.2 g of root material that was suspended in buffer Z containing lysozyme for lysis of root-adhering cells and further incubated for 60 min at 40 °C at pH 7 in the presence of β-gal substrate. srfA expression was determined by A 420 after 60 min of incubation at 40 °C. For determination of both colonization and srfA promoter gene expression (on the same plants), root samples from three different plants were used at each time point in one assay and data mentioned in the figure represent mean values calculated from two independent experiments that yielded similar results. Relative β-galactosidase activity was obtained by subtracting values obtained for untreated control plants from those measured for BGS3-inoculated tomatoes.
In vivo experiments were conducted on tomato plants grown in a hydroponic culture
mode to facilitate colonization study and quantification of surfactins secreted into the
surrounding environment. The colonization process observed for BGS3 was quite similar to
Results-Chapter 6
120
the one reported for other plant beneficial rhizobacteria (Figure 1) (Rainey, 1999; Espinosa-
Urgel et al., 2002) with a first step of bacterial cell attraction/adhesion followed by an
efficient colonization phase and by a further decrease of the population to reach a stable level
at approx. 5 x 106 cell/g roots (Figure 1). Such steady-state phase most probably corresponds
to a resident phase where the population size is restricted by space and/or nutrients
availability and is thus limited by plant growth and root exudation rate.
0
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batch 0,1 0,21 0,33
ββ ββ-gal activity (U)C
ells
x 1
08/m
l
biomass srfA gene expression
µ= µ= µ=
Figure 2. Influence of growth rate of BGS3 cells on expression of surfactin synthetase gene (srfA) and biomass production. Chemostat cultures were performed in a 2 l laboratory glass bioreactor with a working volume of 1.5 l and equipped with automated pH control, a dissolved oxygen tension electrode and a magnetic coupled stirrer (BiostatB, B. Braun Biotech. International, Melsungen, Germany). The temperature was maintained at 30 °C and pH was regulated at 7 by addition of H3PO4 (3N). Aeration rate and agitation were fixed respectively at 0.5 V.V.M. (volume of air per volume of medium per minute) and 600 r.p.m.. By contrast with batch culture where the exhaustion of a specific nutrient terminates the exponential growth phase, biomass concentration in the chemostat culture is usually controlled by the permanent limitation of a single defined nutrient (peptone as source of threonine in this case). It allows the study of metabolite synthesis upon fixed growth rate and constant cellular physiological state. Fresh medium (containing per liter bacto-peptone 1 g, (NH4)2SO4 1 g, sucrose 10g, KH2PO4 1.9g, CuSO4 0.001 mg, FeCl3 0.005 mg, NaMoO4 0.004 mg, MnSO4 3.6 mg, KI 0.002 mg, MgSO4 3.6 mg, ZnSO4 0.014 mg, H3BO3 0.01 mg, citric acid 10 mg) was fed into the reactor using a peristaltic pump to control the dilution rate. The volume of the culture was maintained constant by the use of an overflow device. Cultures were inoculated with a 2% volume of 16 h-subcultures realized in the same medium and the bioreactor was switched to continuous mode after an initial batch start-up. Silicone antifoam was added to control extensive foam formation. The different growth rates (µ) of 0.1 h-1, 0.21 h-1, 0.33 h-1, were respectively considered as low, intermediate and relatively high in this medium on the basis of the growth kinetic observed in batch culture in the same conditions. Samples were harvested at steady state for cell density determination (OD600 nm) and β-galactosidase activity measurement on cell extract prepared by lysozyme treatment and centrifugation (Fickers et al., 2008). One unit of β-galactosidase activity was defined as the amount of enzyme that produces 1 nmol of o-nitrophenol min-1 at 37 °C per OD 600 unit. Data are average values (± standard error) obtained from surfactin concentrations in three independent cultures performed for the study of one specific µ. Surfactin concentration in every independent culture was calculated on the basis of three measurements at 1 h intervals during steady state.
The level of srfA gene expression in root-colonizing BGS3 cells remained almost
stable during the first four days after inoculation, but sharply increased from day 10 to reach a
maximum at day 21 (Figure 1). A high and constant relative expression of srfA gene per cell
unit in the rhizosphere was then observed until the end of the experiment corresponding to the
period where BGS3 cells maintained a stable population. The development of established
Results-Chapter 6
121
bacterial populations in the rhizosphere is actually restricted by nutrient availability owing to
limited root exudation rate (Lugtenberg et al., 2001; Bais et al., 2006). Bacillus cells on roots
can thus be in a nutrient-starved state that would drive cell physiology to slow growth rate
that is apparently conducive for surfactin synthesis. Such effect of growth rate on surfactin
synthesis was confirmed by experiments in chemostat bioreactors that allows the study of
metabolite synthesis upon fixed growth rate (µ) and constant cellular physiological state.
Biomass levels obtained at steady state in the different conditions of growth rates were similar
at approx. 2.5 x 108 cells/ml (Figure 2). In contrast, srfA expression significantly decreased
with increasing µ and the residual β-galactosidase activity measured at µ=0.33 h-1 represented
only 6% of the activity observed at µ=0.1 h-1 (Figure 2). A similar relationship between µ and
srfA expression was observed with another B. subtilis isolate derived from the surfactin over-
producing JH642 strain (not shown).
In parallel to the gene expression determination, a reliable recovery and quantification
of lipopeptides secreted in the microenvironment by the root-colonizing strain is essential. To
this end, we had to combine material extracted from the nutrient solution with the one
recovered by repeated extraction of root tissues [1 g of samples extracted with 4 ml of
MeOH/H2O/Triton X-100, 50/50/0.9 (v/v/v) solution]. This could be explained by the fact
that secreted surfactins are rapidly embedded in the membrane structure of the target
organism as suggested by the rapid disappearance of the lipopeptides from the nutrient
solution when supplemented in hydroponically grown tomatoes (not shown). In two different
experiments, rhizosphere samples were collected after a period of 20 days post inoculation
and LC-MS analyses (see footnote of Table 1 for details) of their content in surfactin revealed
a mean value of 320 ± 80 µg/108 cells. In our conditions, considering an average biomass of
7.5x106 cell/g of root (Figure 1) and 2 g of root tissue per plant, it corresponds to approx. 1.8
µM in the nutrient solution. Importantly, such a surfactin concentration is in the range of the
one necessary to induce ISR on the same plant (5 µM) (Ongena et al., 2007). It indicates that
the lipopeptide may be produced in biologically significant quantities in the rhizosphere of the
host plant.
Results-Chapter 6
122
Surfactin biosynthesis in tomato exudates
We further tested srfA expression and surfactins production upon growth of strain BGS3 in
the root-exudates collected from tomato plantlets after 23-25 days of growth in nutrient
solution under sterile conditions. Freshly collected exudates supported growth of strain BGS3
(Table 1). The srfA gene expression and lipopeptides secretion into the medium were also
effective under these conditions (Table 1), confirming that BGS3 cells can efficiently use
exsudate components for surfactin biosynthesis. This was also demonstrated by cultivating
BGS3 in a minimal medium containing as sole carbon sources the sugars, organic acids and
amino acids typically found in the tomato exudates and added in the proportions established
by Kamilova and colleagues (2006) (Table 1). Increased levels of biomass and surfactin
synthesis compared to natural exudates can be explained by higher concentrations of nutrients
in the medium.
Table 1. Surfactin biosynthesis upon growth of strain BGS3 in tomato root exudatesa.
Biomass
(cells x 108/ml) srfA expressionb
(β-gal U) Surfactin prod.c (µg/108 cells)
Natural exudatesd, liquide 0.34 ± 0.09 17 ± 3 14 ± 4
Recomposed exudatesf, liquid 1.40 ± 0.07 25 ± 8 34 ± 9
Natural exudates, solide 0.37 ± 0.05 14 ± 4 4 ± 2
Recomposed exudates, solid 1.28 ± 0.09 18 ± 7 5 ± 1 a Cultures were realized in 150 ml agitated erlenmeyer flasks (130 r.p.m., 28 °C) filled with 50 ml of medium. In all cases, data are mean values and standard error from four independent experiments. b See legend of Figure 2 for methods of quantification. Cell densities are those corresponding to the maximal OD measured 48 h after incubation. Values for gene expression also correspond to the maximal β-gal activities measured during the culture, usually 24 h after inoculation. c Supernatant samples were collected after 48 h of growth, loaded on C18 solid-phase extraction cartridges (900 mg, Alltech) and surfactins were desorbed with 100% ACN. The resulting samples were analyzed by reverse phase HPLC coupled with single quad mass spectrometer (HPLC Waters Alliance 2695/ diode array detector, coupled with waters SQD mass analyser) on a X-terra MS (Waters) 150*2.1 mm, 3.5 mm column. Surfactins were eluted in the isochratic mode (78% acetonitrile in water acidified with 0.1% formic acid) at a constant flow rate of 0.3 ml min -1 and 40 °C. Compounds were first identified on the basis of their retention times compared with purified standards and the amounts were calculated on the basis of the corresponding peak area (max plot). The identity of each homologue was confirmed on the basis of the masses detected in the SQD by setting electrospray ionization (positive ion) conditions in the MS as source temperature, 130 °C; desolvation temperature, 250 °C; nitrogen flow, 500 l h-1; cone voltage, 70 V. d Filter-sterilized tomato exudates freshly collected from non-inoculated plants grown for 23 days in the greenhouse (see legend of Figure 1). These natural exudates were supplemented with 2 g l-1 (NH4)2SO4 to compensate for the lack of available nitrogen. e Production by BGS3 upon growth as planktonic cells in liquid cultures or as colonies on the same but solidified media (bactoagar at 1.5%). f Medium composed of exudates artificially recomposed [containing 10 g l-1 glucose equivalent in (i) sugars 5 g l-1: glucose 34%, fructose 57%, maltose 8%, ribose 0.75%; (ii) organic acids 4.5 g l-1: citrate 77%, succinate 19%, malate 2%, fumarate 0.5%; (iii) amino acids 0.5 g l-1: glutamate 23%, aspartate 18%, leucine 12%, isoleucine 10%, lysine 9%, (serine + aspartame) 5.5%, (arginine + threonine) 5.5%, (glutamate + glycine) 5.5%, histidine 5.5%, phenylalanine 5.5%] on the basis of data from Kamilova and colleagues (2006).
Results-Chapter 6
123
When tested individually, the main representatives of typical root exudates supported
growth to an almost similar (data not shown) but srfA gene expression and surfactin secretion
were significantly higher in the presence of organic acids and amino acids as unique C
sources (Figure 3A).Aspartate and glutamate are the major amino acid residues found in
tomato exudates but quantitatively, they represent minor components. Although it may vary in
function of various plant factors, succinic and citric acids are the more abundant substrates in
tomato roots and also represent adequate nutrient sources utilizable by BGS3 for surfactin
production.
Rhizosphere-colonizing bacteria typically aggregate in microcolonies on the root
surface (Ramey et al., 2004) and their actual physiology is probably not related to a
planktonic state. We therefore performed BGS3 cultures in 12-well microplates on the various
media described above but gelified by adding 1.5% bactoagar in order to test for lipopeptide
synthesis by immobilized cells. Growth on X-Gal-supplemented media containing either
natural or recomposed exudates resulted in both cases in the formation of blue colonies with
b-galactosidase-positive phenotype (not shown). This first visual assessment was supported
by measurements of the gene expression within the cells scrapped off the wells. The values
obtained for surfactin gene expression are in the range of those measured in liquid cultures in
the presence of both kinds of exudates (Table 1). In contrast, we observed reduced surfactin
production rates. The secreted amounts could be underestimated due to diffusion limitation of
the lipopeptide throughout the biofilm into the environment or because of a less efficient
recovery from the gelified matrix than from the liquid medium. On the other hand, differential
surfactin biosynthesis observed following growth on solid media with individual carbon
sources correlates well with those from liquid cultures with higher levels of gene expression
in the presence of organic and amino acids than the three sugars tested (Figure 3B). Globally,
these results demonstrate that surfactin production is also readily effective in BGS3 cells
developing on solid surface according to the fact that it actually occurs in biofilm-related
structures formed on roots (Bais et al., 2004; Ramey et al., 2004).
Results-Chapter 6
124
0
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4
5
6
7
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30
Glu Fru Malt Succ Cit Asp Glu ExR
Surfactin µg/10
8cellsββ ββ-
gal a
ctiv
ity (
U)
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10
20
30
40
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60
70
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Glu Fru Mal Suc Cit Asp Glu ExR
Surfactin µg/10
8cellsββ ββ-
gal a
ctiv
ity (
U)
A
B
0
20
40
60
80
100
Glu Fru Mal Succ Cit Asp Glu ExR
Rel
ativ
e pr
opor
tion
(%)
C12 C13 C14 C15C
Glu Fru Malt Suc Cit Asp Glut ExR
Glu Fru Malt Suc Cit Asp Glut ExR
Glu Fru Malt Suc Cit Asp Glut ExR
Figure 3. Surfactin synthesis in the presence of various carbon sources typically found in tomato exudates. Lipopeptide gene expression (black columns) and production (white columns) were determined both in liquid cultures (A) in flasks under the conditions described in Table 1 and on the same gelified media (B). The proportions of the various homologues differing in the length of the fatty acid chain from C12 to C15 and produced in the liquid cultures are shown in (C). The substrates (glucose 10 g l -1, fructose 10 g l -1, maltose 5 g l -1, succinate 15 g l -1, citrate 10 g l -1, aspartame 10 g l -1, glutamate 15 g l -1, recomposed exudates containing 10 g l -1 glucose equivalent, for details see footnote of Table 1) were tested by adding a concentrated solution to a minimal medium composed by MgSO4 7H2O 0.5 g l -1, K 2 HPO 4 1.0 g l -1, KCl 0.5 g l -1, Yeast extract 1.0 g l -1, Fe2(SO4)3 1.2 mg l -1, MnSO4 0.4 mg l -1, CuSO4 1.6 mg l -1, (NH4)2SO4 1 g l -1. In all cases, pH was adjusted to 7 ± 0.2 with NaOH or HCl 0.1 N before sterilization. Cultures on solid media were performed in 12-well
Results-Chapter 6
125
microplates in a volume of 1.5 ml per well. Three wells per plate were used for each carbon source. Bacterial growth in the presence of the various substrates was monitored 24 and 48 h after inoculation by measuring turbidity (OD) at 600 nm after scrapping cells for the surface of the medium and re-suspension to homogeneity. The same cells were used for β-gal activity determination. Surfactin produced in each well was extracted from the gelified medium by adding the same volume of HCl 4 N (1.5 ml) and heating for 10 min at 80 °C. The resulting solution was diluted to 10 ml with distilled water and extracted on C18 cartridge to yield semi-purified surfactins in pure methanol (1 ml) used for LC-MS analysis (see footnote of Table 1). In every experiment and for each specific substrate, surfactin concentration and β-gal activity were determined in three replicates (wells) in the same plate and first means were calculated from these data. Results presented in the figure are average values (± standard error) calculated from those first means obtained in three independent experiments.
On another hand, qualitative analyses of lipopeptide patterns by LC-MS revealed that
the C14 and C15 homologues together represent more than 80% of the total surfactins produced
in most media. It also clearly appeared that the relative proportions of the different
homologues may vary in function of the C source and the synthesis of C15 surfactins is
seemingly favoured in the presence of organic acids and amino acids compared to the sugar
group (Figure 3C). The C15 homologue is also by far (58%) the main form produced by BGS3
in natural exudates. This is of biological relevance since surfactins with the longest fatty acid
chains appear to be the more active, not only for antimicrobial activity (Kracht et al., 1999)
but also for their potential to trigger plant defense reactions (Jourdan et al., 2009). It partly
relies on the fact that a more hydrophobic aliphatic chain in the molecule induces greater
surface active properties and membrane destabilization/perturbation (Eeman et al, 2006).
These results suggest that the nutritional status encountered in the rhizosphere could also
somewhat influence qualitatively the production of surfactin homologues by B. subtilis.
Influence of immobilization*3
These results with planktonic cells provide usefull indications but have to be considered in the
context of soil where rhizobacteria aggregate in microcolonies on the root surface. We
therefore performed BGS3 cultures in twelve-well microplates on the various media described
above but gelified by adding 1.5% bactoagar in order to test for lipopeptide synthesis by
immobilized cells. Growth on X-Gal-supplemented media containing either natural or
recomposed exudates resulted in both cases in the formation of blue colonies with β-gal
positive phenotype (not shown). This first visual assessment was supported by measurements
of the gene expression within the cells scrapped-off the wells. The values obtained for
surfactin gene expression are in the range of those measured in liquid cultures in the presence
of both kinds of exudates (Table 1). By contrast, we observed reduced surfactin production
rates suggesting some unidentified regulatory process at the post-transcriptional level. The
secreted amounts could also be underestimated due to diffusion limitation of the lipopeptide
3 Unpublished results
Results-Chapter 6
126
throughout the biofilm into the environment or because of a less efficient recovery from the
gelified matrix than from the liquid medium. On the other hand, differential surfactin
biosynthesis observed following growth on solid media with individual carbon sources
correlate well with those from liquid cultures with higher levels of gene expression in the
presence of organic and amino acids than the three sugars tested (Figure 3B). Globally, these
results demonstrate that surfactin production is also readily effective in BGS3 cells
developing in macrocolonies on solid support thereby confirming that it actually occurs in
biofilm-related structures formed on roots (Bais et al., 2004).
Influence of aeration*4
Oxygen availability is among the physico-chemical parameters inherent to the soil that can
influence the fitness of PGPRs notably regarding the production of biocontrol metabolites in
the rhizosphere. The effect of oxygen concentration in the growth medium on the synthesis of
surfactin was investigated by comparing srfA gene expression and lipopeptide production by
strain BGS3 upon grown in different aerated conditions. Results presented in Table 1 show
that the strain readily adapts its respiratory metabolism to very low oxygen content of the
medium and the final biomass was not drastically affected upon growth in sealed and nitrogen
flushed flask compared with aerated cultures (Table 2). BGS3 cells may thus use nitrate as an
alternative electron acceptor like source of nutrients to grow (Ghiglione et al., 2000).
Interestingly, srfA expression and surfactin production are lower but still remain very
effective in such oxygen-starved culture conditions suggesting that the low oxygen
concentrations in the rhizosphere is not detrimental to lipopeptide synthesis by BGS3 cells
evolving on roots. This observation is relevant in the context of rhizosphere where decreasing
oxygen concentration gradient occurs in poorly aerated soils due to root and microbial
respiration (Højberg et al., 1999).
4 Unpublished results
Results-Chapter 6
127
Table 2. Effect of aeration on surfactin production by strain BGS3 in an optimized medium (Jacques et al.
1999).
Biomass
(cells x 108/ml) srfA expression (β-gal /108 cells)
Surfactin prod. (µg/108 cells)
Bioreactora 5.1 44 550
Aerated flaskb 5.6 ± 0.3 31 ± 9 660 ± 70
Anaerobic flaskc 3.7 ± 0.5 18 ± 4 480 ± 40 a Batch cultures in 2-liter bioreactor with aeration rate and agitation fixed respectively at 1 VVM and 300 rpm. b Erlenmeyers (150 ml filled with 50 ml of medium) with cotton wool that normally allows oxygen exchange. The second flask was simply closed without pushing air out. This allows the culture to start in the presence of oxygen that will be fast limited by the lack of oxygen exchange. This was named sealed flask. c Anaerobic culture conditions created in flasks closed hermetically and medium flushed with nitrogen to eliminate oxygen. Data are from three independent replicate cultures.
Article-supporting information : Gene expression and production of surfactin in artificial medium
Figure S1. Gene expression and production of surfactin in artificial medium. Time-course profiles of cell growth, dissolved oxygen level, srfA expression and surfactin production during batch fermentation of Bacillus subtilis BGS3 under an aeration rate of 1.0 V.V.M. and an agitation rate of 300 r.p.m.
Results-Chapter 6
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Conclusion
Previous in vitro experiments have provided some insights on the effect of various sugars and
nitrogen substrates and of some abiotic conditions such as temperature and pH on lipopeptide
synthesis by Bacillus strains (Peypoux et al., 1999; Kim, et al., 2004; Guez et al., 2008).
However, the study of antimicrobial compound synthesis in the rhizosphere is more
challenging. To our knowledge, this work provides a first demonstration of efficient
lipopeptide gene expression in the rhizosphere during Bacillus-plant interaction that is
associated with relevant surfactin production in the context of biocontrol both quantitatively
and qualitatively. Our data show that important rhizosphere-specific factors such as particular
nutritional status, slow growth rate and development in aggregated and immobilized
structures are conducive for surfactin synthesis. In other experiments, we also observed that
srfA expression and surfactin production still remain very effective in oxygen-starved culture
conditions (V. Nihorimbere, unpublished) suggesting that the low oxygen status prevailing in
the rhizosphere (Højberg et al., 1999) is not detrimental to lipopeptide synthesis by BGS3 cells
evolving on roots. Reduced O2 availability and slow growth were also reported to stimulate
the production of the cyclic lipopeptide putisolvin by Pseudomonas putida (Dubern and
Bloemberg, 2006). Although the influence of many other factors deserves further
investigation and other Bacillus strains have to be studied, these results contribute to fill the
gap in the global knowledge of Bacillus fitness in the rhizosphere regarding antibiotic
synthesis, a crucial point for optimizing biocontrol strate gies using this organism.
Acknowledgments This work received financial support from the Program F.R.F.C. No. 2.4624.06 and Crédit aux chercheurs No.1.5.192.08F (National Funds for Scientific Research, F.R.S.-FNRS, Belgium). V. Nihorimbere is recipient of a grant from the Belgian Technical Cooperation (BTC/CTB). M. Ongena and P. Fickers are respectively research associate and post-doctoral researcher at the F.R.S.-FNRS.
Results-Chapter 6
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Guez, J.S., Muller, C.H., Danze, P.M., Buchs, J., and Jacques, P. (2008) Respiration activity monitoring system (RAMOS), an efficient tool to study the influence of the oxygen transfer rate on the synthesis of lipopeptide by Bacillus subtilis ATCC6633. J Biotechnol 134: 121–126.
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GENERAL DISCUSSION AND FUTURE PROSPECTS
General discussion and future prospects
132
GENERAL DISCUSSION AND FUTURE PROSPECTS
B. amyloliquefaciens strain S499 as biocontrol agent
During the past decades, microbial biopesticides have gained increased attention and interest
for developing environmentally friendly approaches to control plant diseases by reducing the
use of chemicals (Copping and Menn, 2000). In that context, isolation of new bacterial strains
with promising biocontrol potential is necessary to enlarge the still limited range of
marketable products. In the first part of this thesis (Chapter 4), we have selected Bacillus
amyloliquefaciens strain S499 out of more than forty other isolates to conduct open-field
experiments in order to evaluate its efficacy to control a disease that is devastating tomato
plantings in Burundi. This disease is caused by a fungal isolate belonging to the Fusarium
semitectum species based on preliminary characterization. This fungus has not been
previously described as an infectious agent attacking aerial parts of tomato plantings but is
well known as a major cause of seedling diseases on cotton and other crops in many
subtropical african countries (Zhang et al., 1996; Abd-Elsalam et al., 2003). Our results
showed that soil treatment with strain S499 significantly reduced leaf infection rates. Fungal
disease reduction by strain S499 has already been observed on tomato and other plants in
greenhouse system (Jourdan et al., 2009) but our experiments are the first demonstration of
biocontrol activity for that strain against Fusarium infection under field conditions.
In addition, the same treatments significantly increased growth and fruit yield of tomato,
strongly suggesting that S499 strain may also retain plant growth promoting activity besides
disease reduction. A better understanding of such growth promoting activity of the bacterium
deserves further investigation but it may arise from various phenomena such as enhancement
of nutrient (essential oligoelements) availability, production of hormone-like substances or
inhibition of the endogenous soil microflora that is not pathogenic per se but may trigger
adverse effects on plant growth (McKellar and Nelson, 2003; McSpadden Gardener, 2010).
The last phenomenon may be explained by the secretion of antimicrobials by S499 that
directly affect the development of auxiliary microflora or by competition for the limited
sources of nutrients provided through root exudation. For example, diverse seed-colonizing
bacteria can consume nutrients that are released into the soil during germination thereby
suppressing pathogen germination and growth (Pal and McSpadden Gardener, 2006). In both
cases, these effects can only be supported by the fact that S499 strain successfully colonized
the plant root system and rhizosphere. Accurate cell counts on root samples could not be
performed during these field experiments but other results obtained in our research (Chapter
General discussion and future prospects
133
6) together with those obtained in the laboratory have actually illustrated the good root-
colonizing potential of S499 (Ongena et al., 2002; Ongena et al., 2007; Ongena et al., 2010).
The success of an introduced biocontrol agent in suppressing disease can be influenced
by the conduciveness of the environment to the disease but also by the successful
establishment and proliferation of the antagonist in the root zone (Landa, 2004; Chatterton
and Punja, 2010). In our assays, the use of inoculum densities as low as 105 cfu ml-1 showed
to be relevant to sufficiently reduce the disease. It is clear that biocontrol was not significantly
improved by using higher Bacillus inoculum concentrations suggesting that populations tend
to equilibrate after a certain time. It is important since the success of biocontrol also depends
on the preparation of appropriate formulations of the antagonist and from the commercial
side, it is also crucial to use the active ingredient at the lowest concentration.
Also regarding growth and antibiotic lipopeptide synthesis, strain B. amyloliquefaciens
S499 is well adapted to soil temperature range between 20 °C and 25 °C (Chapter 5)
prevailing in (sub)tropical regions from which it was isolated. Additional experiments are
obviously required but taken together; our results suggest that this strain can be proposed for
biological product to be used in Burundi and other subtropical regions, not only for tomato
but also for other crops.
Strain S499 acting via ISR
Some threshold in root colonization rate by the introduced biocontrol agent is also a
prerequisite for disease control activity. Thought that pathogen inhibition might be attributed
to other mechanisms such as the production of enzymes, antibiotics or siderophores,
geographic separation of the beneficial strain and the pathogen in our field trials strongly
suggests that disease suppression was due to induction of resistance in the host plant. Such an
ISR-triggering potential of strain S499 has already been observed on tomato and other plants
in greenhouse systems (Ongena et al., 2002; Ongena et al., 2005b; Ongena et al., 2007;
Choudhary and Johri, 2009; Jourdan et al., 2009). More globally, systemic induction of plant
defenses is currently viewed as an important mechanism by which antagonistic strains, among
which Bacillus isolates inoculated in soil diminish foliar diseases (Harman et al., 2004; Liu et
al., 2008; Huang et al., 2010). ISR-based biocontrol strategies are promising and our trials
may be added to the limited number of those that were successfully performed under field
conditions using Bacillus as inducing agent.
General discussion and future prospects
134
Linking ISR potential with surfactin production in vivo
Although a lot still needs to be discovered, some information is available concerning the
molecular mechanisms underlying ISR triggered by beneficial rhizobacteria. For instance, a
previous work realized in the laboratory has demonstrated that surfactin and to a lower extent
fengycin lipopeptides act as signals emitted by Bacillus to trigger ISR (Ongena et al., 2007).
Surfactin plays a predominant role and represent the main ingredient secreted by strain S499
retaining plant defense eliciting potential. These molecules can thus be considered as a novel
class of microbial-associated molecular patterns perceived by plant cells to activate immune
reaction (Jourdan et al., 2009). Together with the volatile compound 2,3-butandiol (Ryu et al.,
2004), these lipopeptides are the sole determinants for ISR elicitation identified from Bacillus
spp so far.
That said, this ISR eliciting activity of surfactin has been demonstrated by treating
plants with the pure compound and by using over-producing mutants (Ongena et al., 2007).
However, the demonstration of an efficient surfactin production by S499 cells colonizing
roots is crucial to provide conclusive and direct evidence for the implication of this LP in ISR
triggering. In that context, the main goal of this thesis was to better evaluate whether these
lipopeptides are actually produced in the rhizosphere of plants colonized at the root level by
Bacillus. This is a challenging issue because of the small amounts of LPs potentially secreted
and/or the difficulties to quantify them in a complex mixture relative to other organic
compounds present in the environment. Moreover, a major challenge is the reproducibility of
the extraction/detection efficacy protocol. Nevertheless, in this thesis work, we combined
different approaches based on electrospray and imaging mass spectrometry techniques to
demonstrate that surfactin is actually secreted by strain S499 evolving in the tomato
rhizosphere (Chapter 5). Efficient synthesis of this LP in planta was further supported by
using a psrfA-LacZ reporter system inserted in strain BGS3 which is a surfactin overproducer
that protected bean and tomato plants against Botrytis disease via ISR (Ongena et al. 2007).
By contrast with S499, the BGS3 isolate derives from the laboratory “acclimated” isolate B.
subtilis 168 and therefore, is no longer considered as “natural”. It is nevertheless a nice model
strain and results showed that a significant level of psrfA induction was observed upon the
establishment of a stable BGS3 population on roots and surfactins extracted from the
rhizosphere were produced in biologically important quantities (Chapter 6). From both studies
with S499 and BGS3, it also appears that not only the nature of tomato root exudates but also
several other physiological and environmental parameters inherent to Bacillus life in the
rhizosphere are conducive for surfactin biosynthesis. More precisely, oxygen limitation,
General discussion and future prospects
135
adhesion on solid surface, biofilm formation or reduced growth rate are absolutely not
detrimental to surfactin production.
Surfactin in the rhizosphere: the right form at the right concentration
From a qualitative point of view, our results also revealed that the relative proportions of
surfactin homologues produced by cells colonizing tomato roots is clearly different compared
to those observed upon growth of S499 as planktonic cells in an optimized laboratory culture
medium (Chapter 5). The C15 form represents more than 75% of the total surfactins secreted
by S499 in planta and long-fatty acid chain surfactins are also predominantly produced by
BGS3 colonizing roots (Chapter 6). Such an in vivo LP signature is interesting in the context
of ISR since homologues with the longest fatty acid chains were proven to be the more active
regarding their potential to trigger plant defense reactions (Jourdan et al., 2009). Such an
important role for the length of the acyl chain is most probably related to the fact that it
readily inserts into the plasma membrane of plant cells. However, this insertion is limited and
does not lead to pore-formation and phytotoxicity at concentrations below 20µM (Jourdan et
al., 2009; Henry et al., unpublished results).
Whether in situ productivities may generate rhizospheric surfactin concentrations
sufficient to provide any biological effect remains questionable. This LP is active in the range
2-10 µM for inducing plant defense reactions and triggering ISR. However, quantities
recovered from the hydroponic medium surrounding colonized tomato roots in our
experiments ranged from approx. 0.12 µM to 1.8 µM (Chapters 5 and 6). Possible enzymatic
degradation by auxiliary microflora and/or incomplete extraction may contribute to
underestimate the actual abundance of these LPs in the vicinity of the root system. In a more
artificial but gnotobiotic system, measurements of LPs secreted into the surrounding medium
by S499 cells colonizing tomato plantlets revealed that surfactin concentration may locally
reach 50 µM (Chapter 5). Such micromolar concentrations probably better reflects the
amounts of surfactin that may be actually secreted by Bacillus evolving on roots and it may
thus be compatible with biological activity as inducer of resistance in the host plant (Ongena
et al., 2007; Jourdan et al., 2009). This is supported by the higher surfactin quantities that
were detected in the cucumber rhizosphere colonized by strain QST713 (Kinsella et al.,
2009).
General discussion and future prospects
136
Surfactin production in the rhizosphere: other advantages!
Several studies showed that surfactins could be involved in motility and biofilm formation
that are essential traits for plant-associated bacteria in rhizosphere competence and root
colonization process (Danhorn and Fuqua, 2007). For instance, surfactin is necessary but not
always sufficient for surface motility of B. subtilis (Kinsinger et al., 2003; Kearns et al., 2004;
Julkowska et al., 2005). Results obtained with at least two different B. subtilis and B.
amyloliquefaciens strains have shown that surfactin but no other lipopeptides was required for
the formation of biofilms and pellicles (Hofemeister et al., 2004). The production of surfactin
was also demonstrated to be essential for biofilm formation and colonization of Arabidopsis
roots by the strain B. subtilis 6051 (Bais et al., 2004). The same study also showed that
surfactin may also play some role in direct antagonism developed towards bacterial plant
pathogens and the molecule also retains some antiviral activity. Occasionally, surfactin was
also described for its antagonistic effect against insect larvae (Assie et al., 2002).
Additionally, this LP may act as signalling molecule in quorum-sensing and in inducing
cannibalism and matrix formation in biofilms. Beside biocontrol-related traits, surfactins thus
also retain different roles in the development and survival of B. subtilis strains in their natural
habitat (see Raaijmakers et al., 2010 for recent review).
Global pattern of lipopeptides produced in vivo by S499
In vitro, B. amyloliquefaciens S499 co-produces efficiently the three families of
lipopeptides retaining distinct biological activities (Chapter 2). From a practical point of view,
the strain should therefore display some multi-faceted biocontrol activity in different
environmental sites against many plant pathogens. However, only surfactin is produced in
biologically significant amounts in planta (Chapter 5). Iturins and fengycins are also secreted
by S499 colonizing tomato roots but in much lower amounts that are probably not sufficient
to support consistent antifungal activity. It means that these two LPs would not play a crucial
role in antagonism that has to be potentially developed at the root level towards fungal
pathogens. As surfactins are mostly antibacterial and antiviral, our results strongly suggest
that strain S499 would not be very efficient at protecting tomato plants against diseases
caused by soilborne fungal/oomycete phytopathogens.
Such a differential production of LP families strongly suggests that in situ expression of
the related biosynthesis genes may also considerably differ. This could not be demonstrated
experimentally because of the lack of reporter derivatives of strain S499 which is highly
recalcitrant to transformation like many natural B. subtilis/amyloliquefaciens isolates.
General discussion and future prospects
137
Investigating the differential expression of srf, fen and myc genes under natural soil conditions
in S499 or in another natural isolate co-producing the three families, should represent one of
the main future prospects.
Whether such differential production of the three lipopeptide families is specific for
S499 or not is difficult to establish given the rather limited information available in the
literature concerning in situ synthesis of these compounds by other Bacillus isolates.
Mizumoto and Shoda (2007) have also demonstrated that iturins in comparison with
surfactins are less associated to young seedlings in cucumber. By contrast, a few other studies
have shown that lipopeptides from the three families were occasionally detected in the
phytosphere, illustrating that natural conditions are conducive for their biosynthesis, at least in
some cases (Ongena and Jacques, 2008). However, these studies were conducted with
different Bacillus strains on different crops (and organs) and a more consistent information
can only be obtain by comparing LP production by various isolates colonizing the same
model plant in the same conditions of growth. This is clearly one of the main perspectives of
this work.
Another appealing perspective of this thesis is to establish whether the surfactin-
enriched LP pattern secreted by S499 in situ is conserved upon colonization of other plants
than tomato. Preliminary experiments suggest that a similar trend occurs with corn and lettuce
but the LP signature may also be strongly influenced by the host plant species since almost no
production of surfactin was observed upon growth on cucumber and soy (Ongena et al.,
unpublished data). The influence of many other rhizosphere-related factors such as the
auxiliary microflora, the soil type and heterogeneity or the plant developmental stage, on
Bacillus LP signature also deserves further investigation.
Concluding comments
In light of the initial great optimism (Copping and Menn, 2000), and in spite of
extensive research efforts, it is paradoxical that progress in achieving commercial, large-scale
usage of biological control has been slow. The reasons for this are partly of commercial and
legislative character. Cost-effective production, good storage shelf-life, worked out
application procedures, marketing or legislative requirements for registration may explain
limitations in product’s distribution and selling. Another serious constraint is that, in
comparison to the conventional pesticides that usually have simple knockout effects, the field
efficacy of many biological products, which most often are more dependent on environmental
and other circumstances, is inherently more variable. Because many successful antagonists
General discussion and future prospects
138
exert their activity by secreting antibiotic metabolites, an obvious approach is to optimize this
process of antibiotic production in the field. To maximize it, a fundamental understanding of
their production in their natural niche is imperative.
In this work, we have inoculated Bacillus strains in situ on tomato cultivated in
greenhouse in hydroponic mode. We first assumed that root exudates represent food-web for
bacterial fitness in the rhizosphere regarding antibiotic production. Our second hypothesis
was that the lipopeptides biosynthesis in situ may be modulated by multiple rhizosphere-
related parameters in addition to the substrates. The influence of all these environmental
factors on the lipopeptide pattern production has been evaluated qualitatively and
quantitatively. Globally, it was shown that the nutritive and physico-chemical conditions of
bacterial cells growing in the rhizosphere on the root system are conducive for efficient
surfactin lipopeptide synthesis. Thus, these results provide a first demonstration of efficient
related-gene expression and surfactin production in the rhizosphere during Bacillus-plant
interaction.
From a more general and ecological point of view, it is often speculated that the frequent
occurrence of B. subtilis in its natural environment might be due to the selective advantage
conferred by the panoply of bioactive metabolites that it may produce (Stein, 2005). However,
even if some B. subtilis or amyloliquefaciens strains are well equipped genetically to produce
a vast array of antibiotics (Chen et al., 2009), our results suggest that only a limited part of
this antibiotic-devoted genetic background may be readily expressed in situ. Thus the results
from this work also substantially contribute to enhance our knowledge of Bacillus fitness in
natural living conditions regarding its antibiotic producing potential.
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