from production to application of arbuscular mycorrhizal fungi in

Proceedings

of

COST 870 meeting

From production to application of arbuscular mycorrhizal fungi in agricultural systems: a multidisciplinary approach

Meeting in working group 1 and 3 and Management Committee

Meeting

Denmark, May 27-30, 2008

University of Aarhus Faculty of Agricultural Sciences

Department of Integrated Pest Management Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark

Organising committee Sabine Ravnskov Mauritz Vestberg Meriel Jones Yoram Kapulnik Jacqueline Baar John Larsen Sonja Graugaard

Proceedings of COST 870 Meeting, May 27-20, 2008 Proofreading, graphic preparation and production Administrative case officer Sonja Graugaard and senior administrative assistent Henny Ras-mussen, University of Aarhus, Faculty of Agricultural Sciences Composition, reproduction and printing Frederiksberg Bogtrykkeri A/S, DK-2000 Fredriksberg, Denmark ISBN: 978-87-91949-11-1

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Contents Foreword ................................................................................................................................... 5 WG3 - “Bottlenecks for implementation of AM in plant production” Molecular ecology of arbuscular mycorrhizal fungi in an agricultural context ......................... 9 Tim Daniell Raspberry biotisation for quality plant production ................................................................. 11 Armelle Gollotte, Louis Mercy, Benjamin Secco, Julie Laurent, Michel Prost, Silvio Gianinazzi & Marie-Claude Lemoine Use of photosynthesis parameters in optimization of Arnica montana cultivation and selection of AM inoculum........................................................................................................ 15 K. Turnau, P. Ryszka & T. Anielska Genotypic variation in mycorrhizal root colonisation in spring barley ................................... 21 Peter Schweiger Performance of AM fungi in peat substrates in greenhouse and field studies ......................... 25 Mauritz Vestberg &, Sanna Kukkonen Novel approaches to enhance application of arbuscular mycorrhizal fungi for the development of sustainable agricultural and landscape systems: experiences from The Netherlands........................................................................................... 27 Jacqueline Baar, Fraukje Steffen, Huig Bergsma & Bert Carpay Transformation of soil organic matter: a background for growth and functioning of arbuscular mycorrhizal fungi ................................................................................................... 29 Milan Gryndler Characterisation of Central European soils by their compositions of arbuscular mycorrhizal fungi .................................................................................................................... 31 Fritz Oehl, Ewald Sieverding, Endre Laczko, Arno Bogenrieder, Karl Stahr, Robert Bösch, Sue Furler, Kurt Ineichen & Andres Wiemken Evaluation of two different mycorrhzal inocula at Mediteranean agricultural fields ............. 33 M. Orfanoudakis., D. Alifragis & A. Papaioannou

WG1 - “Plant breeding and colonisation by AM fungi” Molecular genetics of AMF as a tool for improving symbiosis and plant growth................... 41 Ian R. Sanders, Daniel Croll & Caroline Angelard The microbial ecology of an upland grassland ecosystem....................................................... 43 Nicholas Clipson

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A novel gene-candidate approach of socio-economic interest? – Breeding on efficient plant genotype – mycorrhiza interaction ................................................................................. 47 Birgit Arnholdt-Schmitt Breeding stress-tolerant maize: the role of arbuscular mycorrhiza.......................................... 51 Shawn Kaeppler The role of mycorhizzas in nutrient acquisition by European and African maize varieties .... 55 Stephen Rolfe, Derek Wright, David Read & Julie Scholes Plant genes involved in AM symbiosis .................................................................................... 61 Franziska Krajinski Posters Different transgenic Bt corn genotypes affect mycorrhizal colonization................................. 67 Alessandra Turrini, Cristiana Sbrana & Manuela Giovannetti Soil and inoculum infectivity evaluated during the early stage of mycorrhizal Establishment ........................................................................................................................... 69 Luciano Avio, Stefano Bedini, Elisa Pellegrino & Manuela Giovannetti

The effect of atmospheric change on arbuscular mycorrhizal fungi in agricultural systems... 71 T.E. Anne Cotton, Thorunn Helgason & Alastair Fitter Potential of dual purpose intercrops for the management of plant-parasitic nematodes and beneficial mycorrhizal fungi in banana-based cropping systems............................................. 73 L. Van der Veken, P.P. Win, M. Lin, A. Elsen, R. Swennen & D. De Waele Mycorrhizal colonisation on Allium schoenoprasum L. in peat-based substrates ................... 79 Siri Caspersen & Mats Kron The influence of biofumigation on mycorrhizal fungi and growth of strawberries ................. 81 Darinka Koron Agroecological management of soil borne diseases in tropical horticultural crops by biocidal plants: hypothesis and chosen methodologies............................................................ 85 Paula Fernandes, Peninna Deberdt, Alain Soler, Christian Chabrier & Laurent Thuries List of participants ................................................................................................................. 87

COST 870 Meeting “From production to application of arbuscular mycorrhizal fungi in agricultural systems: a multidisciplinary approach”, Denmark, May 27-30, 2008

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Foreword The meeting within the COST Action 870 “From production to application of arbuscular my-corrhizal fungi in agricultural systems: a multidisciplinary approach” (http://www.cost870.eu/) took place in Køge, Denmark at Hotel Niels Juel on May 27-30 2008. The meeting was initiated by the COST 870 management committee and organised by the Department of Integrated Pest Management, Faculty of Agricultural Sciences, University of Aarhus. The themes of this joint working group 1 and 3 meeting were “Plant breeding and colonisa-tion of AM fungi” (WG1) and “Bottlenecks for implementation of AM in plant production” (WG3). The meeting had participants from 21 countries (see page 87) with 15 oral presentations and 7 poster presentations. I would like to thank to Sonja Graugaard and Henny Rasmussen for their excellent work on these proceedings and the members of the organising committee for good collaboration. Additional copies of this report can be obtained by contacting Sonja Graugaard – [email protected] Sabine Ravnskov Head of organising committee Organising committee Sabine Ravnskov Mauritz Vestberg Meriel Jones Yoram Kapulnik Jacqueline Baar Local organising committee Sabine Ravnskov John Larsen Sonja Graugaard


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WG3

“Bottlenecks for implementation of AM in plant production”


870 report (2008), page 9-10. 9

Molecular ecology of arbuscular mycorrhizal fungi in an agricultural context Tim Daniell Environmental Plant Interactions Programme, SCRI, Dundee, DD2 5DA, United Kingdom, [email protected] Arable practice has been shown to result in a reduction in diversity of arbuscular mycorrhizal (AM fungi) compared to nearby semi-natural habitat. (Daniell et al., 2001; Helgason et al., 1998). Hijri et al. (2006) confirmed the observed low diversity in high intensity plots but showed that the diversity of AM fungi in reduced fertilisation/pesticide treatments plots was higher suggesting a role of agro-chemicals in reduced diversity. There are a number of possi-ble reasons for this observed reduction including physical disturbance, growth of monocul-tures and fertilisation The low representation of, for example, Scutellospora or Gigaspora species types in arable fields may be due to a reduced ability to colonise in disturbed systems (Klironomos and Hart, 2002). There is evidence to suggest a degree of host preference in the AM symbiosis (eg (Bidartondo et al., 2002; Vandenkoornhuyse et al., 2002). If this effect is evident in intensive arable systems it may also, in part, explain observed low diversity due to selection of types within a field or crop dependant on the rotation cycle.

We established a field experiment on the SCRI site to assess if host preference was de-tectable in an arable context. The site had been under standard rotation (conventional or high intensity farming practice) for an extended period as part of the SCRI farm. A range of mono and dicotyledonous crop plants were sown in a replicated strip design within a single field. Root samples were taken over a time course and colonisation assessed by molecular methods. There were significant effects of time, plant species and interactions between them. There was no clear pattern to suggest linkage between classical plant taxonomy or land practice and the AM fungal community structures observed although the high physical disturbance associated with sowing of potato may explain the early separation of these samples. This experiment provides information suggesting that host preference is present within arable species indicat-ing that crop monoculture may have effects on the AM fungal community structure in subse-quent years.

One aspect of the success of any given AM fungal inoculum will be the ability for the AM fungal type or types within the inoculum to establish within the target system. To test this we established a field-plot based experiment testing the efficiency and in-field diversity pat-terns of two forms of inocula, applied singly or in combination, to a Vigna radiata (mung bean) crop grown under standard arable practice at NIBGE (Pakistan). One inoculum was a mix of bacteria (Biopower), a selection of three bacterial types including rhizobial symbionts of the host. The other was a mycorrhizal inoculum generated from previous growth of Allium cepa (onion) in the same field, but spatially separated. Above ground biomass was not signifi-cantly increased with any treatment but yield was increased with inoculation although only significantly in treatments with Biopower. There was greater variability in yield in samples with AM fungal inoculation. Both inoculation and time significantly altered AM fungal com-munity structure associated with the mung bean crop although this effect was reduced with dual inoculation.

Molecular based approaches to understand intra-radicle AM fungal community structure by their nature rely on the presence of nuclei within the plant root. The development of real time PCR applications has allowed the comparison of morphological and molecular methods

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to assess root colonisation. We performed a pot experiment featuring time-course sampling of plant roots colonized by a natural community of AM fungi. The roots were sampled from the same replicate pots with a weekly interval. The root samples were divided in two: part was stained for microscopic assessment of fungal colonization; the other part was used for quantitative real-time PCR. The results showed no overall relation between microscopic and molecular assessment of root-inhabiting fungal biomass. However, there was a clear relation between DNA-based and microscopic measurements of one sampling point, where the coloni-zation levels, as seen microscopically, had reached the maximum. We conclude that the mi-croscopy and DNA-based assessments of intra-radicle AM fungal biomass describe different aspects of the fungal colonization dynamics. The lack of clear relationship can be explained by an uneven and dynamic distribution of fungal nuclei in the intra-radicle mycelium.

The last 10 years have seen the development of a range of techniques that have allowed us to assess the community structure of AM fungi in the field with the high sample size re-quired to answer questions relating to the ecology of this important group of fungi. The chal-lenge over the current and next period will be to develop protocols that allow the assessment of the functionality of AM fungi in a field context. Such tools will allow the accurate assess-ment of the AM fungal types either from natural or artificial inocula both in an ecological and physiological sense.

References Bidartondo ML, Redecker D, Hijri I, Wiemken A, Bruns TD, Dominguez L et al. 2002. Epi-

parasitic plants specilised on arbuscular mycorrhizal fungi. Nature 419, 389-392. 2002. Daniell TJ, Husband R, Fitter AH & Young JPW. 2001. Molecular diversity of arbuscular

mycorrhizal fungi colonising arable crops. FEMS Micobiol ecol 36: 203-209. Helgason T, Daniell TJ, Husband R, Fitter AH & Young JPW. 1998. Ploughing up the wood-

wide web? Nature 394: 431. Hijri I, Sykorova Z, Oehl F, Ineichen K, Mader P, Wiemken A & Redecker D. 2006. Com-

munities of arbuscular mycorrhizal fungi in arable soils are not necessarily low in diver-sity. Molecular Ecology 15, 2277-2289. 2006.

Klironomos JN & Hart MM. 2002. Colonisation of roots by arbuscular mycorrhizal fungi using different sources of inoculum. Mycorrhiza 12, 181-184. 2002.

Vandenkoornhuyse P, Husband R, Daniell TJ, Watson IJ, Duck JM, Fitter AH & Young JPW. 2002. Arbuscular mycorrhizal community composition associated with two plant species in a grassland system. Molecular Ecology 11, 1555-1564. 2002.


870 report (2008), page 11-14.

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Raspberry biotisation for quality plant production Armelle Gollotte1, Louis Mercy1, Benjamin Secco1, Julie Laurent1, Michel Prost2, Silvio Gianinazzi3, Marie-Claude Lemoine1 1CRITT AgroEnvironnement, 17 rue Sully, 21065 Dijon Cedex, France, [email protected]. 2LARA Spiral, 3 rue des Mardors, 21560 Couternon, France. 3UMR Plante-Microbe-Environnement, INRA/CMSE, 17 rue Sully, 21065 Dijon Cedex, France The raspberry market is growing following medical studies indicating that regular consuming of this red fruit has a protective effect against cancer, cardiovascular diseases, diabetes, inflammation and age-related degenerative diseases (Juranic et al., 2005). This benefit results from the action of antioxidant molecules (AOM) such as vitamins and polyphenols which are particularly abundant in raspberry (Beekwilder et al., 2005). However, AOM synthesis in raspberry depends on several factors including plant varieties, climatic conditions and agricul-tural practices. Biotisation is a biotechnology consisting in the inoculation of young plants with beneficial micro-organisms (bacteria and/or fungi) in order to increase plant survival and growth (Gianinazzi et al., 2003). Biotisation using arbuscular mycorrhizal (AM) fungi has actually been shown to increase polyphenol content in grapevine and sweet basil (Krishna et al., 2005; Toussaint et al., 2007). In the case of raspberry, recent results indicate that biotisa-tion induces an increase in AOM content in leaves whilst fertilisation with a nutrient solution rich in phosphorus has an inhibitory effect (unpublished results). In addition, conventional cultural practices make use of fungicides generating potentially noxious food residues but not always controlling plant diseases. In particular, root rot due to Phytophthora fragariae var. rubi which is the main cause of raspberry plant death is not ef-fectively controlled by chemicals. Only prophylactic measures can be adopted by planting healthy raspberry plants in non-contaminated soils. The contamination of young plants during their multiplication has been a major source of spreading for this disease. For quality insur-ance, it is therefore important to produce plants which are healthy and devoid of P. fragariae var rubi contamination. There is a European directive (92/34/EEC) to ensure that purchasers throughout the Community receive propagating material and plants which are healthy and of good quality. In France, controls involved in the certification process include the identifica-tion of varieties by morphological characters, verification of good plant labelling practices and absence of important viruses. Producers now would like to include absence of P. fra-gariae in this certification scheme as it is already in place in the UK. Therefore production of raspberry plants of high quality in terms of AOM content, ab-sence of P. fragariae and reduced use of pesticides is important for this evolving market. Experiments of biotisation of young raspberry plants either in the acclimatisation or post-acclimatisation phases have proved the usefulness of this biotechnology for improving plant survival and growth and increasing tolerance to Phytophthora fragariae var rubi (Lemoine et al., 2000). These promising results have prompted producers to develop routine application of biotisation for raspberry plants. However, it appears that transferring this technology is not easy and that positive effects are not always reproducible or only transient. There are several reasons for this, including quality of the inoculum used and lack of compatibility of growing conditions with biotisation. In particular, some critical factors have to be taken into considera-tion such as step of plant inoculation, growth substrate and fertilisation regime. Finally, once the plants are transferred to the field, persistence of mycorrhizal fungi in roots depends on nature of the soil, fertilisation and pesticide applications.

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Our aim is therefore to optimise biotisation of raspberry in order to gain a long-term positive effect on plant quality in terms of growth, health and AOM content. Assessing inoculum quality More and more commercial mycorrhizal inocula are becoming available on the market. How-ever, as there is no systematic quality/legal control of inoculum production at present, there is a great variation in quality depending on the producers. Several technologies have been de-veloped for assessing inoculum quality (Gianinazzi & Vosatka, 2004). The most probable number (MPN) method is often used for the estimation of the number of viable propagules in inocula. However, this technique is relatively expensive, as it requires greenhouse space and the result is not obtained before several weeks. Molecular methods would be useful in order to obtain results in a few days and to reduce the analysis cost. Recent results based on PCR for the detection of AM fungi in inocula by using a series of DNA dilutions are really promising for solving these problems (unpublished results). Step of plant inoculation Raspberry plants are often multiplied in vitro, acclimatised in multi-pot trays and then trans-planted in larger pots before their transfer to the field. They can therefore be inoculated at different steps. We have shown that it is possible to inoculate raspberry plants with spores during the multiplication phase in vitro and obtain good mycorrhizal levels. However, this technology has not been applied to a large scale so far as it requires large quantities of spores in axenic culture. Inoculation at the acclimatisation phase is easy, requires relatively low quantities of inoculum and has been proved beneficial to plant growth and survival at this critical step of plant culture. It is also possible to inoculate raspberry plants during their trans-fer into pots or their transplanting in the field. However, this requires larger amounts of inocu-lum and may be less efficient for the establishment of the symbiosis and for improving plant growth. Growing conditions Biotisation has been shown to be efficient in improving plant growth and survival but has often a transient effect. This shows that growing conditions following biotisation have to be carefully optimised in order to obtain a sustained effect of mycorrhization during plant cul-ture. The choice of growth substrate and fertilisation regime is crucial for establishing and maintening plant mycorrhization. Substrates which are rich in organic matter or phosphorus content are known to inhibit root colonisation by AM fungi. In our hands, alkalinised moss peat was shown to be a good substrate for sustaining both plant and fungal growth. This growing medium is however low in nutrients and an appropriate fertilisation regime has to be adopted. For this, organic fertilisers or nutrient solutions containing low levels of phosphorus can be used. In this purpose, tests have to be run in order to find the concentrations compati-ble with biotisation and which have a positive effect on plant growth. Persistence of mycorrhization in the field Biotised plants present the advantage of surviving and growing faster after transplanting into the field. However, this effect is often transient and it would be interesting to retain this bene-

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ficial effect longer. For this, diverse practices have to be taken into consideration. As for growing plants in the greenhouse, the fertilisation regime has to be adapted by using phospho-rus- poor fertilisers or organic fertilisers. In addition, the use of pesticides has to be avoided. Some fungicides inhibit AM fungal growth whilst they are not efficient in preventing dis-eases. It would therefore be important to limit fungicide applications and find alternatives, such as natural elicitors of plant defence responses, for example. In this context, raspberry growth would probably benefit from biotisation in organic farming systems (Harrier & Wat-son, 2004). A constraint in field applications is the lack of information concerning persistence of inoculated AM fungi in the field and their competition with natural populations of AM fungi. The development of molecular tools based on the detection of ribosomal DNA sequences by PCR has enabled to study the persistence of inoculated AM fungi in roots of sweet potato in the field (Farmer et al., 2007). Such studies have not been conducted for raspberry yet. How-ever, we have been able to show that Glomus intraradices which was inoculated to strawberry plants in the field was still present 2 months after inoculation. Assessing plant sanitary status In order to implement a certification system for biotised raspberry plants, it is important to show that they are really biotised and that they are not contaminated by pathogenic fungi and

Figure 1. Detection of Glomus intraradices (A) and Phytophthora fragariae (B) by nested PCR either in pure culture (spores of G. intraradices or mycelium of P. fragariae) or in roots of plants produced in the greenhouse or in the field.

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In conclusion, producing plants with a sustained biotised status requires adapted growing conditions at the different steps of culture with controlled growing media, fertilisation regime and use of pesticides. This should result in healthier plants with interesting properties, such as increased levels of antioxidant molecules which are important for human health. References Beekwilder J, Hall RD & de Vos CHR. 2005. Identification and dietary relevance of antioxi-

dants from raspberry. BioFactors 23: 197-205. Farmer MJ, Li X, Feng G, Zhao B, Chatagnier O, Gianinazzi S, Gianinazzi-Pearson V & van

Tuinen D. 2007. Molecular monitoring of field-inoculated AMF to evaluate persistence in sweet potato crops in China. Appl Soil Ecol 35: 599-609.

Gianinazzi S, Oubaha L, Chahbandar M, Blal B & Lemoine MC. 2003. Biotization of mi-croplants for improved performance. In : “Proceedings of the XXVI International Horti-cultural Congress: Biotechnology in Horticultural crop Improvement : achievement, op-portunities and limitations”, Hammerschlag FA, Saxena P (eds). ISHS, Acta Horticul-turae, Belgium, 625: 165-172.

Gianinazzi S & Vosatka M. 2004. Inoculum of arbuscular mycorrhizal fungi for production systems: science meets business. Can J Bot 82: 1264-1271.

Gollotte A, van Tuinen D & Atkinson D. 2004. Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species Agrostis capillaris and Lolium perenne in a field ex-periment. Mycorrhiza 14: 111-117.

Harrier LA & Watson CA. 2004. The potential role of arbuscular mycorrhizal (AM) fungi in the bioprotection of plants against soil-borne pathogens in organic and/or sustainable farming systems. Pest Management 60: 149-157.

Juranic Z & Zizak Z. 2005. Biological activities of berries: from antioxidant capacity to anti-cancer effects. BioFactors: 207-211.

Krishna H, Singh SK, Sharma RR, Khawale RN, Grover M & Patel VB. 2005. Biochemical changes in micropropagated grape (Vitis vinifera L.) plantlets due to arbuscular-mycorrhizal fungi (AMF) inoculation during ex vitro acclimatization. Scientia Horticul-turae 106: 554-567.

Lemoine MC, Cordier C, Gianinazzi S, Jaubertie JP & Alabouvette C. 2000. Biological con-trol in raspberry: promising results and portential use to control Phytophthora fragariae var. rubi. L'arboriculture fruitière 538: 19-23.

Toussaint JP, Smith FA & Smith SE. 2007. Arbuscular mycorrhizal fungi can induce the pro-duction of phytochemicals in sweet basil irrespective of phosphorus nutrition. My-corrhiza 17: 291-297.


870 report (2008), page 15-19.

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Use of photosynthesis parameters in optimization of Arnica montana cultivation and selection of AM inoculum K. Turnau, P. Ryszka, T. Anielska Institute of Environmental Sciences of the Jagiellonian University, ul Gronostajowa 7, 30-387 Kraków, Poland, [email protected] Abstract: Arnica montana was found to be always mycorrhizal under field condition. Although it is possible to grow nonmycorrhizal A. montana under culture conditions, it is extremely susceptible to a variety of stresses such as drought, excess of N and P. Differences between the selected fungal isolates were found concerning their ability to stimulate plant photosynthesis. The most effective under the given culture conditions were G. geosporum and G. constrictum. Parameters describing plant performance were found to be negatively correlated with arbuscular richness, while others (such as ETo/TRo, ABS/RC) were correlated positively. Introduction Arnica montana L. (Asteraceae) is a rare plant under strict protection in several European countries (Ellenberger, 1998). Extracts of this plant stimulate blood flow, promote healing and soothe arthritic pains. The species is cultured for industrial purposes, but its culture is difficult and non-profitable; therefore, the participation of cultured plants in the overall mate-rial pool is almost negligible. It is known that mycorrhiza is of basic importance for both in-dividual plants and for entire plant communities (Van der Heijden et al., 1998). The interac-tion of AMF and plants is beneficial for both partners (Hayman, 1983), although in rare cases negative effects can be observed. Mycorrhiza was found to influence the level of plant secon-dary metabolites (Strack et al., 2003). It is highly probable that the level of the produced ac-tive compounds also depends on colonization by mycorrhizal fungi (Abu-Zeyad et al., 1999; Fester et al., 1999). The main aim of the present study was to optimize the culture methods of A. montana, supported by mycorrhizal fungi. Parameters of photosynthesis and mycorrhiza were monitored to select the best inoculum and soil compostion. Material and Methods Seeds of A. montana obtained from Planta-Naturalis (Markvartice, Czech Republic; http://www.plantanaturalis.com) were germinated on wet filter paper in Petri dishes. A few weeks old seedlings were transferred into containers with sterile substratum composed of a mixture of garden soil (Rolex substratum containing peat, sand, pine bark, compost enriched with organic fertilizers and dolomite), sand and expanded clay using the following rates 5:1:1, 5:4:1 and 5:8:1 (v:v:v). The substrata differed mainly in N content (as determined with Kjeldahl method). The highest N level was found in the first case (5:1:1) and was 0.7% while in the others it was 0.3 to 0.4%. Phosphorus content (vanadian method) was less diverse, from 0.04 to 0.1%. The following inocula were prepared: 1. no inoculum (NM); 2. Glomus geospo-rum UNIJAG PL 12-2; 3. G. constrictum 265-5 Walker; 4. G. intraradices BEG 140; 5. G. intraradices UNIJAG.PL24-1; 6. a mixture composed of G. geosporum UNIJAG PL 12-2, Glomus intraradices BEG 140, G. intraradices UNIJAG.PL24-1, G. constrictum 265-5 Walker and G. mosseae BEG 12; 7. inoculum obtained from samples collected in Kurpie; and 8. from Jaworowa Łąka.

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The pots with plants were kept in Sun bags (Sigma-Aldrich, Poland) under greenhouse condi-tions under the following light regime: 100–110 µmol×m–2×s–1 PAR, 12/12 h.

Chlorophyll a fluorescence transients of intact leaves were measured with a Plant Effi-ciency Analyser (PEA) fluorimeter (Hansatech Instruments, GB). The transients, induced by a red light of 600 W×m-2, were recorded for 1 s, starting 50 µs after the onset of illumination. The data was acquired every 10 µs for the first 2 ms and every 1 ms thereafter as described by Strasser et al. (1995). Each transient was analysed according to the OJIP-test (Strasser et al., 1995; Strasser et al., 2000). For the characterization of PSII behaviour the following bio-physical parameters (referring to time point zero) were calculated: specific energy fluxes per reaction centre for absorption (ABS/RC), electron trapping (TR0/RC), electron dissipation (DI0/RC) and electron transport (T0/RC); the flux ratios or yields, i.e. the maximum quantum yield of primary photochemistry (φPo= TR0/ABS), the efficiency with which a trapped exciton can move an electron into the electron transport chain further than QA (ψ0=ET0/TR0), the quantum yield of electron transport (φEo= ET0/ABS) and Sm – total electron carriers per RC of PSII.

Plants (9 month after planting into pots), following the photosynthesis measurements, were harvested, stained and examined for AM colonisation according to the previously de-scribed Trouvelot method (Trouvelot et al., 1986).

Data obtained from measurements of chlorophyll a fluorescence were transformed with decimal logarithms prior to the analysis, to meet statistical assumptions and compared using GLM models using the STATISTICA ver. 7.0 software (Statsoft, USA) (P < 0.05). For my-corrhizal evaluation the data were analyzed with the Kruskal-Wallis test. Comparison of sec-ondary metabolites in nonmycorrhizal plants with plants inoculated with different AM fungi were compared using t-test. Results Culturing plants in pots filled with Rolex soil, sand and expanded clay in 5:1:1 (v:v:v), char-acterized by 0.7% of N resulted in very poor development of mycorrhizal fungi and no differ-ences between plants were visible. When the substratum was 5 times diluted with sand and the N content was down to 0.3%, while P was at 0.04% the mycorrhizal plants were develop-ing well, while nonmycorrhizal plants were a few times smaller and dark green. Their survival strongly decreased with time as they were very sensitive to drought and changes of tempera-ture. In the following experiment the plants were cultivated on a substratum mix of 5:4:1 (v:v:v) soil: sand: expanded clay. In this case N level was 0.3% and P level 0.2%. There were no statistically important differences in shoot and root biomass of differently inoculated plants and those noninoculated. In this case mycorrhizal colonization of A. montana was the highest in the case of Glomus intraradices (both strains used), Glomus geosporum and inoculum originating from Jaworowa Łąka. The lowest parameters were observed in the case of G. con-strictum and inoculum from Kurpie. Except for G. constrictum, the frequency of mycorrhizal colonization was mostly 100%.

The parameters of photosynthesis measured in plants grown under greenhouse condition in pots filled with substratum that was too rich in N were very similar and no statistically sig-nificant differences were found between samples. Similar results were obtained after 9 months of cultivation. Statistically significant differences between treatments were observed only in the case of plants cultivated on the most diluted substratum, where the mycorrhiza was well developed in inoculated plants and nonmycorrhizal plants were a few times smaller and the growth was arrested. Nonmycorrhizal plants had at least two times higher performance index PI, higher driving force of photosynthesis Df, ET0/TR0, ET0/ABS, and ET0/RC.

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Among plants forming mycorrhiza with different AMF isolates, the highest PI was found in those that were inoculated with G. geosporum, G.constrictum and the mixture of strains. Lower PI was found in the case of G. intraradices and inoculum from Jaworowa Łąka.

When the plants were cultivated on substratum at the proportion 5:4:1, the situation was different. The highest PI was found in the case of plants that were strongly mycorrhizal, while the lowest PI was found in case of Kurpie and nonmycorrhizal plants.

The level of mycorrhizal colonization was found to be correlated to several photosyn-thethic parameters. VJ was usually negatively correlated with mycorrhiza colonization, while a positive correlation was found in the case of ET0/TR0, ET0/ABS, ABS/RC and ET0/RC.

In the case of inoculum originating from soil samples from Kurpie and Jaworowa Łąka and G. intraradices a negative correlation was found between the plant performance index and arbuscule richness (A% and a%).

Discussion A. montana plants cultivated under laboratory conditions were very susceptible to stresses such as drought. The most critical for the development of the mycorrhiza and efficient stimu-lation of plant photosynthesis was properly prepared substratum with the right ratio of P and N. A high content of N prevents the formation of mycorrhiza. Too low nutrient levels resulted even in negative effect of mycorrhiza on photosynthesis parameters such as PI, ET0/RC, ABS/RC etc. Despite of this mycorrhizal ones were actually more stress-resistant than those that were nonmycorrhizal. Lower photosynthesis parameters in the case of nonmycorrhizal versus mycorrhizal plants were obtained in plants grown at appropriate N levels. The experi-ment in which the garden soil, sand and clay were mixed in a ratio of 5:4:1 allowed to culti-vate plants with and without mycorrhiza. These results confirm that the interaction of the plant with the mycorrhizal fungus depends not only on plant species or cultivar and on the fungal strain, but also on the conditions under which the plant is cultivated. HandyPEA proved to be very useful for the selection of proper ratio of components within the substratum, what confirmed the data obtained by the Strasser group (eg. Corrêa et al., 2006). This approach offers broader information concerning plant adaptation to the given site and is not invasive. As shown by Corrêa et al. (2006) it is also useful in evaluation of symbiosis impact, and physiological parameters such as plant height show correlation with the performance index when comparing mycorrhizal and nonmycorrhizal plants. In the early trials with A. montana when the substratum with too high N content was used and no my-corrhiza was formed (despite inoculation with AMF), the photosynthesis of both inoculated and non inoculated plants was equal. This means that by measuring photosynthesis we could see that either there was something wrong with the experiment or that photosynthesis is not reacting to inoculation. The mycorrhizal status of these plants was checked confirming the fact that simply the lack of formation of mycorrhiza was the reason that photosynthesis was not affected. After realizing that we can use the photosynthesis parameters to non-invasively check the performance of the plants and development of the mycorrhiza we repeated the ex-periment with other substrata. PI was reduced after mycorrhiza introduction if the plants were grown on substrata with too low nutrient levels. At low-nutrient conditions, also photosyn-thetic parameters were decreased after mycorrhiza was established. In non-mycorrhizal plants the plant performance index was very high, but the plants were unable to grow due to low nutrient levels. On the same substratum the mycorrhizal plants were able to grow, although the lack of nutrient caused the lower plant performance. In literature it was already known that the PI value can be lower (even up to 50%) in mycorrhizal plants while the mycorrhiza is developing actively, at the beginning of the cultivation and also if the nutrients are the limit-

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ing factor (Calantzis et al., 2000). In the first case this tendency is reversed when the plants start to show a positive response to mycorrhizal colonization. This can give information on the rate of establishing of positive interaction and on how fast the balance between symbionts is reached. In all cases, differences in parameters were detectable prior to effects visible in plant growth. It enables the selection of the inoculum based on the photosynthesis measurements. Differences between selected fungal isolates were found concerning the effectiveness of stimulation of photosynthesis. The most effective under the given culture conditions were G. geosporum and G. constrictum. These fungi were less efficient in developing mycorrhizal structures such as arbuscules. On the contrary, G. intraradices, an aggresive symbiont, was less effective in increasing plant performance. In this paper for the first time the correlation between the mycorrhizal parameters, especially arbuscular richness, with photosynthetic pa-rameters was shown, as the mycorrhizal colonization was estimated in the individual plants previously subjected to measurements of the activity of photosynthesis. Plant performance was found to be negatively correlated with arbuscular richness while others (such as ET0/TR0, ABS/RC) were correlated positively. Acknowledgements This work was supported by the Polish Ministry of Science and Higher Education (Grant No. 2 P04G 109 28) in years 2005–2007. References Abu-Zeyad R, Khan AG & Khoo C. 1999. Occurrence of arbuscular mycorrhiza in Castano-

spermum australe A. Cunn. & C. Fraser and effects on growth and production of cas-tanospermine. Mycorrhiza 9:111–117.

Calantzis C, Trouvelot S, Van Tuinen D, Gianinazzi-Pearson V, Gianinazzi S & Strasser R. 2000. A non destructive method for evaluating the mycorrhizal status of micropropa-gated vine root-stock. Bulletin SGPW/SSA 14:8.

Corrêa A, Strasser RJ & Martins-Loução MA. 2006. Are Mycorrhiza Always Beneficial? Plant Soil 279:65–73.

Ellenberger A. 1998. Assuming responsibility for a protected plant: WELEDA’s endeavour to secure the firm’s supply of Arnica montana. In: TRAFFIC-Europe, edg. medicinal Plant Trade in Europe: conservation and supply. Proceedings of the First International Sym-posium on the Conservation of Medicinal Plants in Trade in Europe. TRAFFIC-Europe, Brussels, pp 127–130.

Fester T, Maier W & Strack D. 1999. Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria. Mycorrhiza 8:241–246.

Hayman DS. 1983. The physiology of vesicular-arbuscular endomycorrhizal symbiosis. Can J Bot 61:944–963.

Strasser RJ, Srivastava A & Govindjee. 1995. Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem. Photobiol. 61:32–42.

Strasser RJ, Srivastava A & Tsimilli-Michael M. 2000. The fluorescence transient as a tool to characterise and screen photosynthetic samples. In Yunus M, Pathre U, Mohanty P (eds) Probing photosynthesis: mechanisms, regulation and adaptation. Taylor & Francis, Lon-don, pp 445–483.

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Trouvelot A, Kough JL & Gianinazzi-Pearson V. 1986. Mesure du taux de mycorhization VA d’un systeme radiculaire. Recherche de methodes d’estimation ayant une signification fonctionnelle. In: Gianinazzi-Pearson V, Gianinazzi S(eds) Physiological and Genetical aspects of Mycorrhizae. INRA, Paris, pp 217–221.

Van Der Heijden MGA, Klironomos JN, Ursic M, Moutogolis P, Streitwolf-Engel R, Boller T, Wiemken A & Sanders IR. 1998b. Mycorrhizal fungal diversity determines plant bio-diversity, ecosystem variability and productivity. Nature 396:69–72.


870 report (2008), page 21-23.

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Genotypic variation in mycorrhizal root colonisation in spring barley Peter Schweiger Bio Forschung Austria, Rinnböckstraße 15, A-1110 Vienna, Austria, [email protected] Introduction Organic arable farming systems are generally characterised by a lower soil P availability than conventional systems. An improved plant P uptake efficiency is therefore of considerable in-terest for organic agriculture. P uptake efficiency has been shown to vary genetically in nu-merous crop plants. Plant traits that determine P uptake efficiency include root morphological and physiological characters but also root colonisation by arbuscular mycorrhizal fungi (AMF). The plant growth enhancing effect of these fungi has been shown to vary between crop plant genotypes. This is at least partly due to genotypic variation in the degree of AMF root colonisation, which has been extensively studied in wheat. But also barley genotypes have been reported to vary in AMF root colonisation. In the present study, the genotypic variation in AMF root colonisation was examined for barley cultivars most widely grown by organic farmers in Austria. Based on the obtained re-sults, an assessment was to be made, whether breeding efforts in barley specifically aimed at developing genotypes for organic growing conditions need to include increasing AMF root colonisation as a breeding target. Material and Methods In a first screening, a field experiment was conducted with twelve barley cultivars currently recommended in Austria for use in organic farming. The cultivars were grown in three repli-cates in a randomised block design. The soil at the site has a near-neutral pH (pHCaCl2 = 6.3) and an average P-fertility (PCAL = 59 mg kg-1). Barley seed was sown at standard within and between row spacings into 1.2 by 8 m plots. At shooting, three soil cores of 3 cm diameter from each plot were pooled and all roots within washed free of soil. Obvious weed roots within the sample were removed. A random subsample of the roots was cleared and stained for AMF root colonisation using standard procedures (Giovannetti & Mosse, 1980). Stained roots were finally mounted on slides. The percentage of root length colonised by AMF was determined by the method of McGonigle et al. (1990). In a follow-up pot experiment, an increased number of barley genotypes (n = 16) were screened under controlled environmental conditions for potential root colonisation by a mix-ture of AMF from a commercial inoculum (Symbivit). The basic growth substrate was a mix-ture of a sieved (<5 mm) sandy loam (pHCaCl2 = 7.2; PCAL = 44 mg kg-1) with builders sand and perlite. Four pots of each genotype with three individual plants per pot were kept in a glass-house arranged in blocks. Plants were harvested at early heading. A representative sub-sample of the complete root system from each pot was treated and analysed as described above for the field root samples. Results AMF root colonisation in field-grown barley varied from 20 to 70% of total root length colo-nised. Average root colonisation of all cultivars was in the range of 40-50%. Data were very

22

variable, and as a result, no significant genotypic variation was observed in this character. A major factor contributing to the observed variability was the inhomogeneity of the soil at the experimental site. This was shown by a significant contribution of the factor block to the overall variability of the data (p<0.01). A weak but non-significant effect was obtained for the block x cultivar interaction (p<0.1).

Figure 1. Relative AMF root colonisation of pot-grown barley cultivars. AMF root colonisation of pot-grown plants was comparatively low, with an average of 20 and a maximum of 40% of the total root length being colonised. Arrangement of the pots in the glasshouse affected root colonisation, nonetheless, some genotypes differed significantly in this character (p<0.05). Figure 1 shows the relative level of colonisation for the different cul-tivars with the overall experimental mean given as 100. Discussion The formation of an effective arbuscular mycorrhiza is essential for efficient plant nutrient (mainly phosphorus) uptake. In good agreement with work by for example Zhu et al. (2003), barley cultivars were found to differ in the extent of AMF formation. The differences were however not very pronounced and were not confirmed under field conditions. Different levels of root colonisation did not result in plant growth differences as observed in some studies (Zhu et al., 2003). Such results were for barley mainly obtained in pot studies with only very few field studies showing growth-enhancing effects of AMF (Dekkers & van der Werff, 2001). This has most often been explained by the P status of especially conventionally man-aged agricultural soils. P supply to barley is thus not limiting growth. As a result, barley has sometimes not been found to derive any quantifiable benefit from AMF root colonisation (Khaliq & Sanders, 2000). Based on literature data and the obtained results, breeding barley with increased AMF root colonisation was not assessed as a breeding target with high prior-ity.

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References

Dekkers TBM & van der Werff PA. 2001. Mutualistic functioning of indigenous arbuscular mycorrhizae in spring barley and winter wheat after cessation of long-term phosphate fertilization. Mycorrhiza 10, 195-201.

Giovannetti M & Mosse B. 1980. An evaluation of techniques for measuring vesicular arbus-cular mycorrhizal infection in roots. New Phytologist 84, 489-500.

Khaliq A & Sanders FE. 2000. Effects of vesicular-arbuscular mycorrhizal inoculation on the yield and phosphorus uptake of field-grown barley. Soil Biology & Biochemistry 32, 1691-1696.

McGonigle TP, Miller MH, Evans DG, Fairchild GL & Swan DJA. 1990. A new method which gives an objective measure of colonization of roots by vesicular—arbuscular my-corrhizal fungi. New Phytologist 115, 495-501.

Zhu Y-G, Smith FA & Smith SE. 2003. Phosphorus efficiencies and responses of barley (Hordeum vulgare L.) to arbuscular mycorrhizal fungi grown in highly calcareous soil. Mycorrhiza 13, 93-100.


870 report (2008), page 25-26.

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Performance of AM fungi in peat substrates in greenhouse and field studies Mauritz Vestberg, Sanna Kukkonen MTT Agrifood Research Finland, Plant Production Research, Antinniementie 1, FI-41330 Vihtavuori, Finland, [email protected] Growing media based on light peat are commonly used in horticultural production. Although peat possesses a range of positive properties, it may also incur problems. Structural problems are common in long-term use. Light peat has a very low anion exchange capacity resulting in leaching of nitrate and phosphorus. Peat lacks beneficial symbiotic arbuscular mycorrhizal fungi (AMF) which are important to crops in sustainable systems. However, several studies have shown a negative impact of peat on AMF. Peat has been found to inhibit AMF colonisa-tion, but this effect could be reduced by adding 25% of soil or sand to the substrate. Hypnum peat has been less inhibitory to AMF than Sphagnum peat. In a study including several peat types, an interaction between peat type and AMF strain was also observed. The real reasons behind the incompatibility between peat and AMF are still not known in detail, but biological or chemical properties have been suggested as possible reasons for the phenomenon. The im-pacts of peat on mycorrhizal traits has been studied in several field and greenhouse experi-ments at MTT Agrifood Research Finland. Material and Methods Field studies Mycorrhizal traits were studied in two large field experiments conducted at MTT Agrifood Research Finland, Laukaa, Central Finland. Mycorrhizal effectiveness, in terms of ability of AMF to increase growth, was determined in a bioassay. AMF spores were extracted from soil, and species richness and diversity were determined. The Shannon-Wiener index (SWI), which combines both species richness and evenness, was calculated. Alongside with the AMF traits, a large number of other soil properties were also studied. One of the field experiments was a long-term cropping system experiment, while the other one was a short-term preceding crop study. The cropping system experiment established in 1982 included conventional and organic plots. During the study period 2000-2002, strawberry was grown on the whole ex-perimental area. In the other experiment, we studied the impact of three years of cultivation of eight crops with different degrees of mycotrophy, including mycorrhizal and non-mycorrhizal hosts. In both experiments the impact of amendment with highly humified peat (H8-9 on von Post’s scale) on mycorrhizal traits were studied. Greenhouse studies The effects of light and dark Sphagnum peat and dark Carex Bryales peat, as well as their mixtures with minerals like sand, pumice, vermiculite, perlite, clay or zeolite on AMF func-tion were studied in three short-term experiments carried out in 2006 and 2007 with daisy as a host plant. In all experiments, a commercial AMF inoculum “Myko-Ymppi” (from MTT) was mixed with the various substrates at a rate of 5%. At harvest, shoot fresh and dry weight as well as AMF root colonisation were estimated. Results In the field experiments, AMF traits were affected by cropping systems and preceding crops

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but also by peat amendment. Peat had a negative impact on mycorrhizal effectiveness and AMF spore numbers, but did not affect species frequency or the Shannon-Wiener diversity index. In the pot experiments, light Sphagnum peat clearly suppressed the function of AMF, resulting in less growth in AMF-inoculated plants and low AMF root colonisation. In mix-tures with clay, AMF root colonisation was higher, but the effect of AMF on growth was still negative. However, AMF did not usually affect plant growth negatively when decomposed Sphagnum or Carex Bryales peat was used. AMF root colonisation of daisy was also very high in these peat quatities. Compared with peat alone, the functioning of AMF was im-proved, especially in peat mixtures with clay or pumice. In mixtures of light and dark peat the effect of AMF inoculation was very similar to that obtained in light peat alone. Conclusions Negative interactions between peat and AMF were noticed after use of peat in mineral field soil as well as in potting mixes with peat for use in greenhouse cultures. This negative phe-nomenon can at least partly be overcome by using decomposed peats and potting mixes of peat and mineral components, in particular clay. Since peat is commonly used in horticulture, and will probably be so for a long time, this incompatibility phenomenon is one of the real bottle necks for utilization of AMF in greenhouse and nursery production. It stands clear that deeper studies are needed for finding the reasons behind the phenomenon. The COST Action 870, WG3, could provide a unique possibility for organising a joint study in this matter.


870 report (2008), page 27.

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Novel approaches to enhance application of arbuscular mycorrhizal fungi for the development of sustainable agricultural and landscape systems: experiences from The Netherlands Jacqueline Baar, Fraukje Steffen, Huig Bergsma, Bert Carpay ARCADIS Netherlands, The Netherlands, [email protected] In the last few decades, we have gained considerably amount of knowledge on arbuscular mycorrhizal (AM) fungi. A large number of research projects on AM fungi have been carried out which has resulted in numerous scientific publications. Previous COST Actions on AM fungi have largely contributed to this in Europe. The increase in knowledge on AM fungi has resulted in a growing interest in the produc-tion of these fungi for application purposes. During the last decade, several SME’s in Europe have started commercial production of these fungi for application. These companies produce inocula for different markets ranging from agriculture, horticulture, landscaping to gardening. However, AM fungal inocula are still not applied at a large scale in Europe. In countries as The Netherlands, there is still hesitance applying these fungi to achieve enhanced sustainabil-ity in agriculture or in landscape development. During the last year, we looked into possible reasons why the interest in the application of AM fungi is still low for The Netherlands. This resulted in the following insights:

• Firstly, the majority of soils in The Netherlands are highly enriched by nutrients (ni-trogen (N), phosphate (P)) by heavy fertilization due to intensive high production ag-riculture. This has resulted in extremely high nutrient levels of soils and acidified soil conditions over the last few decades. Particularly, enrichment of P in combination with lowered soil pH is considerable causing a major problem for the development of either sustainable agricultural fields with a good soil biological environment or natural fields with high above- and below-ground biodiversity. The transition process of P-enriched systems into low level nutrient systems is hampered by the high levels of P in the soil and low pH. The development of moist to wet agricultural or natural fields is even more troublesome, because of release of the immobilized P under acidified con-ditions. Therefore, at ARCADIS we have developed a novel sustainable methodology reduc-ing high P levels in the soil and increasing soil pH. Application of this novel method-ology results in a more suitable soil environment for application of arbuscular my-corrhizal inocula.

• Secondly, we noticed a lack of knowledge about convincing results of projects with mycorrhizal fungi. Therefore, we are taking an approach that we reach potential users in agriculture and natural landscape development. Moreover, we make scientific knowledge and results of projects accessible to users. The first results indicate that in-terest in mycorrhizal fungi is growing.

Our approaches for The Netherlands will be presented and discussed in relation to possibili-ties for other countries in Europe.


870 report (2008), page 29.

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Transformation of soil organic matter: a background for growth and functioning of arbuscular mycorrhizal fungi Milan Gryndler Institute of Microbiology ASCR, v.v.i., Videnska 1083, CZ 14220, Prague, Czech Republic, [email protected] When the interactions between mycorrhizal fungi and decomposing organic matter, including cellulose, are studied, the experimental system is often treated as a black box, with no ambi-tion to describe the details of links between the growth of mycorrhizal fungi and decomposi-tion products. This situation is becoming an anachronism since the development of advanced analytical tools described below provides an opportunity to study the interaction between transformation of soil organic matter and mycorrhizal fungi in detail. These works begun in the frame of collaboration between Department of Integrated Pest Management, Faculty of Ag-ricultural Science, University of Aarhus, DK, and Institute of Microbiology ASCR, v.v.i., Prague, CZ. In-vitro growth of mycorrhizal hyphae was evaluated in the extracts of soils in which the decomposing organics were incubated. The same soil samples were pyrolysed, the products of pyrolysis were separated using gas chromatography and identified on the basis of mass-spectroscopic analysis of fragmentation products. Microbial community was characterized on the basis of whole cell signature fatty acids extracted from soil. In-vitro growth of mycorrhizal hyphae behaved differently than root colonization, being stimulated by organic amendment and independent of mineral N and P availability. Root my-corrhizal colonization and plant growth parameters were stimulated by available mineral nu-trients. Among the pyrolysis products, 3,4,5-substituted benzyl structures showed strongest cor-relation with the growth of mycorrhizal hyphae. These structures correlate with growth of mycorrhizal hyphae if purified cellulose is decomposing but when plant necromass rich in mineral nutrients is used in the experiment, the correlation becomes insignificant. Fatty acid analysis revealed that the abundance of 3,4,5-substituted benzyl structures cor-relates with some components of soil microbial community rather than with the community as a whole. This is the first attempt to find out the relationships between chemistry of soil organic matter and growth of arbuscular mycorrhizal fungi. The results indicate that relatively small amounts of organic matter can affect arbuscular mycorrhizal fungal growth and that 3,4,5-substituted benzyl structures may be important for the development of mycorrhizal fungi. If the method of soil pyrolysis will be widely used in future, it might, for example, pro-vide the data explaining the preference of particular mycorrhizal fungi for particular soils with important practical applications: it might predict the suitability of a particular fungal isolate for a given soil (plant cultivation substrate) on the basis of its detailed chemical analysis.


870 report (2008), page 31-32.

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Characterisation of Central European soils by their compositions of arbuscular mycorrhizal fungi Fritz Oehl1, Ewald Sieverding2, Endre Laczko3, Arno Bogenrieder4, Karl Stahr5, Robert Bösch1, Sue Furler1, Kurt Ineichen1, Andres Wiemken1

1Zurich Basel Plant Science Center, Institute of Botany, University of Basel, Hebelstrasse 1, CH-4056 Basel, Switzerland, [email protected]. 2Institute of Plant Production and Agroecology in the Tropics and Subtropics, University of Stuttgart Hohenheim, Garbenstr. 13, D-70593 Stuttgart-Hohenheim, Germany. 3Functional Genomics Center Zurich, Winter-thurerstrasse 180, CH-8057 Zürich, Switzerland. 4Institute of Biologie II, Research Area Ge-obotany, Albert-Ludwigs-University of Freiburg, Schänzlestr. 1, D-79104 Freiburg, Ger-many. 5Institute of Soil Science, University of Hohenheim, Emil-Wolff-Strasse 27, D-70599 Stuttgart, Germany Previous ecological studies in Central European Regosol and Luvisol soils have shown that the diversity of arbuscular mycorrhizal fungi (AMF) is strongly influenced by the agricultural land use intensity: it is low in intensively managed soils and high in natural grasslands. The objective of the present study was to define whether soil types dominant in Central Europe can be characterized by their AMF population. Representative soil samples were taken from Cambisols (5 locations), a Fluvisol (one location) and Leptosols (two locations). For each of the soils, locations were selected with contrasting agricultural land use systems – arable land and grassland – so that in total samples from 16 sites were investigated. AMF spores were isolated and counted directly from field soils and after reproduction in trap cultures during 20 months. Spore morphology was used to taxonomically identify AMF species. Regardless of the soil type, AMF species numbers decreased with increasing land use intensity. However, the AMF species diversity and community structure depended strongly on the soil type. Whereas in the calcareous Leptosols (pH 6.9-7.2) Glomus spp. comprised 95-99% of the AMF spore populations, 20-35 % of the spores belonged to other AMF genera than Glomus in the siliceous Cambisols and Fluvisols (pH 4.2-5.4). In the Cambisols the number of Acau-lospora and Scutellospora spp. was higher than in the Leptosol. Also, Gigaspora margarita was only detected in the siliceous soils. Some Glomus spp., alone or in combination with other species, could be defined as character species for specific soils under specific agricul-tural land use. While G. caledonium was typical for the cultivated Cambisols, G. mosseae was much more prominent in cultivated Leptosols. Higher spore abundance of G. macrocarpum and A. paulinae was typical for the Cambisol and Fluvisol grasslands, while in the Leptosol grasslands G. constrictum dominated together with the sporocarpic species G. rubiforme and G. sinuosum. The plant species compositions (either natural or planted) may have only a sub-ordinate influence on the occurrence, diversity and composition of AMF species, In conclu-sion, specific AMF species or their compositions are characteristic for Cambisols and Lepto-sols and their land use intensity in Central Europe. References Herrmann L & Stahr K. 1998. Environment and soilscapes of South-West Germany. Tour

guide excursion B 6, 16th World Congress of Soil Science, Montpellier, France, In: E Kandeler, M Kaupenjohann, K Stahr (eds.) Hohenheimer Bodenkundliche Hefte, vol. 47, Stuttgart, Germany.

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Oehl F & Sieverding E. 2004. Pacispora, a new vesicular arbuscular mycorrhizal fungal ge-nus in the Glomeromycetes. Journal of Applied Botany and Food Quality 78, 72-82.

Oehl F, Redecker D & Sieverding E. 2005a. Glomus badium, a new sporocarpic mycorrhizal fungal species from European grasslands with higher soil pH. Journal of Applied Botany and Food Quality 79, 38–43.

Oehl F, Sieverding E, Ineichen K, Mäder P, Boller T & Wiemken A. 2003. Impact of land use intensity on the species diversity of arbuscular mycorrhizal fungi in agroecosystems of Central Europe. Applied and Environmental Microbiology 69, 2816-2824.

Oehl F, Sieverding E, Ineichen K, Ris EA, Boller T & Wiemken A. 2005. Community struc-ture of arbuscular mycorrhizal fungi at different soil depths in extensively and intensively managed agroecosystems. New Phytologist 165, 273-283.

Oehl F, Sieverding E, Mäder P, Ineichen K, Dubois D, Boller T & Wiemken A. 2004. Impact of long-term conventional and organic farming on the diversity of arbuscular mycorrhizal fungi. Oecologia, 138, 574-583.

Oehl F, Wiemken A & Sieverding E. 2003. Glomus aureum, a new sporocarpic arbuscular mycorrhizal fungal species from European grasslands. Journal of Applied Botany 77, 111-115.

Oehl F, Wiemken A & Sieverding E. 2003. Glomus spinuliferum, a new ornamented species in the Glomales. Mycotaxon 86, 157-162.

Sieverding E & Oehl F. 2006. Revision of Entrophospora and description of Kuklospora and Intraspora, two new genera in the arbuscular mycorrhizal Glomeromycetes. Journal of Applied Botany and Food Quality 80, 69-81.

Spain JL, Sieverding E & Oehl F. 2006. Appendicispora, a new genus in the arbuscular my-corrhizal-forming Glomeromycetes, with a discussion of the genus Archaeospora. Myco-taxon 97, 163-182.

Stahr K & Fleck W. 2004. Soils, landscapes and environmental problems. Excursion guide book of the International conference Eurosoil 2004, Freiburg, Germany, Forstwirtschaftliche Versuchsanstalt Baden-Württemberg.

Stahr K & Jahn R. 1989. Southern Black Forest In: Stahr K and Jahn R (eds). Development of soil minerals in relation to parent material and environmental conditions in the Black Forest and Upper Rhine Graben, Southwest Germany. 9th Int. Clay Conference, Stras-bourg, France, August 28- September 2, 1989. Guide book, p. 46-51.

Walker C. 2008. Ambispora and Ambisporaceae resurrected. Mycological Research 112, 297-298.


870 report (2008), page 33-38.

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Evaluation of two different mycorrhzal inocula at Mediteranean agricultural fields M. Orfanoudakis, D. Alifragis, A. Papaioannou Forest Soil Laboratory, School of Forestry and Natural Environment, Aristotle University of Thessaloniki PO Box 271, 54124, Thessaloniki Greece, [email protected] Abstract: Two agricultural fields at 30m altitudes located in N. Greece at the east and west side of the city of Thessaloniki, were selected in order to compare the efficacy of two differ-ent AMF inocula. Leeks originated from Israel Volcany research institute were selected as the host plant. The results varied due to different soil conditions that occurred. The leeks inocu-lated with commercial inocula, originated from France, were with more biomass production when planted on Alluvial deposits with low salinity. Glomus intraradices inoculums from Volcany Research Institute were more effective on biomass when leeks planted on Marl soil. Despite the differences on size leeks inoculated with the Israeli inoculum were better in ap-pearance promoting an easier market for them. Results suggesting that the efficiency of my-corrhizal inoculum on agricultural plant growth may vary due to differences on soil properties and to the interactions with the indigenous fungi. Introduction Modern agriculture change the face of the land field radically making possible for large land sites to be in crop system production in order to satisfy the needs in feeding of an expanding world population (Tinker, 2000; Atkinson & Watson, 2004). The agricultural systems become more depended upon agrochemicals; fertilisers and gradually become more independent from the soil processes. The limited availability of nitrogen and phosphate is probably the impor-tant factor most limits the development of soil based crop systems. Those needs were covered by the industrialised agriculture. Intensive agricultural systems achieved maximum food pro-duction. However, those agricultural systems lead to agriculture with significant impact upon the environment. Fertiliser application is usually based upon the crop and the good knowledge of soils and the previous cropping history; requiring a good scientific analysis of the fields soil, often an expensive practice. In order to avoid the high cost of the soil analysis farmers often choose practises were leading to bad fertilisation managing system, with significant loss of the fertiliser to the air (N) or the soil water via the water flow or by the soil degrada-tion (N and P). Mediterranean regions are facing increased loss of fertilisers due to bad land management (Atkinson et al., 1996; Alifragis, 2008). Those problems could gradually in-crease the cost of farming while keeping the production at intense levels could be problem-atic. Gradually the agricultural practises are coming to point were the production cost could not be covered by the income, particularly when the size of individual farms are small. Such problems of the modern agriculture are creating the need for mew farming practices in sustainable agriculture systems, aiming to for efficient use of the natural resources. Sus-tainable systems however, are difficult to maintain the productivity while the environmental impact is reduced. Those systems should involve greater control of both microbial processes and optimised crop production. AMF has an important role as provides resources to the plant crop. The symbiosis of AM fungi and plant roots is critical to the plant growth and it is regard as a norm. Plants without AMF are incomplete. AMF importance to the host for absorbing nutrients from soils with low nutrient availability was clearly documented (Koide, 1991). There, the colonisation is more effective and should be expected to result an enhanced

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growth. With this point of view, the mycorrhizal application could fit with the conventional agricultural systems. However, the role of AMF only as a nutrient provider under estimates the role of the symbiont. Colonised roots could alter the chemical compounds released via the root system and the hyphae to the soil, creating specific conditions to the mycorrhizosphere. Those are the basis on the symbiosis establishment, and the initiation of defence mechanisms against pathogens. Additionally colonised plants could modify their physiology and manage the plant water potential more effectively providing an advance on drought stress. Mycorrhi-zal management is under consideration in modern sustainable agricultural systems. There is particular interest about the use of AMF products at the Mediterranean region, and in particular, in Greece were the size of field sites o doesn’t permit large industrialised agricultural applications. These conditions seem ideal for AMF applications were the concept of robust environmental systems with efficient use of the natural soil resources is essential. Material and Methods Different agricultural fields were selected as experimental sites. The climatic conditions were similar since they are both at the same geographical region and with the same elevation. The selected sites were located at the East and West side of Thessaloniki. Both field sites were selected since from the antiquities were related with the agricultural production supporting the population lived in the city. The soil conditions were different since the west side was influ-enced by a river. The site to the west was loam-sandy with pH=7.8 while the soil to the East was on marls with pH= 7.9. The extractable P of the field soil was 7.92 mg/100 g and 1.03 mg/100 g at the marl and alluvial soil respectively. Both Fields were planted with Leeks origi-nated from Israel. One third of the plants used were previously inoculated with Glomus intra-radices at the Volcany Research Institute, one third was inoculated with Gigaspora marga-rita, while one third remained not inoculated. Soil analysis was conducted to both sites were the C% and the organic matter was esti-mated (Nelson and Sommers 1982), the organic N%, the NaHCO3 extractable P (Olsen and Sommers 1982), and the exhalable Ca, Mg, K, and Na (Grant 1982). Pant tissue analysis was also conducted were the N%, P, Mg,Ca,K,Na were measured. Mycorrhizal colonisation was estimated with the grid line intersect method. Randomly selected plants were used in order to measure the effects of the indigenous AMF population. The root system of the selected plants was isolated from the rest of the soil via a nylon mesh (20μm) at a ratio of 5cm from the steam. The soil from the proximity of the mesh collected and the AMF spores were counted from the outer soil layer. Results Inoculation with an aggressive fungus such as G. intraradices resulted to an increased root branching. Plants inoculated with G. intraradices at the Marl soil remained with high levels of colonisation >85% to the harvest day, while there was no significant interaction with the in-digenous AMF population. In the contrary, the colonisation levels at the Alluvial soils with the moderate extractable P level were lower (68-83%). Indigenous mycorrhizal fungi at both sites were Gigaspora species. The biomass production on both sites was increased at inocu-lated plants (Figure 1). The best overall production occurred at leeks inoculated with the French inoculum at the alluvial soils. Inoculated plants and non-inoculated were with the same nitrogen level at the plant tissues except from those inoculated with the French inocu-lum and grown at the Marl field (Figure 2). The phosphate levels at the plant tissues were also increased at inoculated plants up to 50% particularly at the alluvial soil (Figure 3).

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Figure 3. Leeks Phosphate content after inoculation with G. intraradices (lines), G. marga-rita (filed), and with no AMF (empty). Plants were grown on two different soil conditions Figures are means of 50 replicated plants. Data with the same later are with no significant difference from each other (P<0.05).

Discussion These data clearly demonstrate that AMF contribution upon field agricultural application is significant. The results however vary according the type of the inoculum used and the soil properties were the inocula applied. Soil properties presumably have the key role upon the variation of the results. Different fungal inoculum is more efficient at different soil condi-tions. The colonisation levels at the Alluvial soils with the moderate extractable P level were lower (75-85%), while indigenous fungi were at the rhizosphere in small numbers. The extensive root colonisation by the Israeli inoculum could possibly provide a good defence against other soil micro organisms. Along with the extensive root growth measured, both effects fitting with the scope of the modern agriculture were more efficient use of the natural resources in essential. However the effective symbiosis didn’t achieve to increase crop production significantly, at one of the soils applied. The contrast on growth at the two differ-ent field applications could be related with the soil environment; that includes the soil proper-ties, indigenous AMF and soil bacterial. The low P conditions at the Marl soil environment the symbiotic partners interact in favour of the root system in search of more resources. The extensive root branching could be support this hypothesis. In addition, the relative harsh soil environment at the Marl field also resulted to a more extensive fungal growth. Extensive root growth with extensive hyphal network along with spore production could lead to unbalanced plant growth. The host simply needs more roots and hyphae to reach more resources. Changes to root growth as a result of colonisation by AMF and the likely consequences for function have been reviewed (Berta et al., 2002). Hypotheses have been proposed for mechanisms through which AMF modify (usually increase) root growth, including plant hormones, nutri-ents and changes to cell cycles (see Hooker and Atkinson, 1996). Colonisation by AMF can change allocation of resources to the root system of plants in a more sophisticated way, caus-ing alterations to the spatial pattern of root branching and significant modifications, including changes to root mortality (Atkinson et al., 2003; Hooker et al., 1995) The increased allocation

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of resources to the root that were observed are not unusual and are measured frequently in studies where plants are inoculated with AMF. Increased internal hyphal network could pre-vent the indigenous fungi to colonize the crop root. Indigenous fungi should expect to be more adapted to the local soil conditions. However the control leek plats left to be colonised by the indigenous fungal population, develop poor colonisation levels. Possibly the non my-corrhizal Leeks originated from Volcany Research Institute in Israel and the poor capacity of the soil in indigenous AMF inoculum due to the antifungal products use of the previous years. Also the occurrence of Gigaspora species at the indigenous mycorrhizal population although didn’t achieve a detectable colonisation to any of the Leek roots examined, it is possible to form an interaction with the fungi used at the inoculated plants. Leek growth at the alluvial soils with the moderate P soil conditions achieved good crop production levels. There the best plant growth appeared after inoculation with Gigaspora margarita. The moderate P soil conditions were possibly the reason for lower colonisation levels than the leek planted at the Marls soil. The more balanced growth resulted to better crop production at the harvest day. Soil is a significant parameter at mycorrhizal application in the field. The results pre-sented here suggest that the combination of soil-mycorrhiza is essential and could result to important variations on the production. The crops production does not reach the possible ca-pacity of the conventional agriculture. Despite that smaller size of the leeks produced, the consumers were convinced that they were good organic products even when their size was far smaller that the conventional leeks. The beneficial effect of mycorrhizal inoculation in agri-culture should not always be linked with the biomass production but rather as a part of a sus-tainable ecological system. References Alifragis D. 2008. “The soil” 247-356 Aivazis editions Thessaloniki 2004. Atkinson D, Black KE, Forbes P J, Hooker JE, Baddeley JA & Watson CA. 2003. The influ-

ence of arbuscular mycorrhizal colonisation and environment on root development in soil. European Journal of Soil Science 54: 751-757.

Atkinson D & Watson CA. 1996. The environmental impact of intensive systems of animal production in the lowlands. Animal Science 65:353-361.

Atkinson D & Watson CA. 2004. A revised agricultural perspective: working in harmony with the soil. Proceedings of the 10th Soil conference of the Greek soil Science society 313-324. Volos 2004.

Berta G, Fusconi A & Hooker JE. 2002. Arbuscular mycorrhizal modifications to plant root systems: scale, mechanisms and consequences. In Gianinazzi S, Schuepp H, Barea JM, Haselwandter K, eds. Mycorrhiza Technology in Agriculture: from genes to bioproducts . Birkhauser, 71-85.

Grant EG. 1982. Exchangeable Cations. In: A.L. Page (ed) Amer. Soc. Of Agronomy and Soil Sci.Soc. of America, Madison, Wisconsin, USA, pp:159-164.

Hooker JE & Atkinson D. 1996. Arbuscular mycorrhizal fungi-induced alteration to tree-root architecture and longevity. Pflanzen. Bod. 159: 229-234.

Hooker JE, Black KE, Perry RL & Atkinson D. 1995. Arbuscular mycorrhizal fungi induced alterations to root longevity of poplar. Plant Soil 172: 327-329.

Nelson DW & Sommers LE. 1982. Total carbon, organic carbon and organic matter require-ment. In Methods of Soil Analysis. Part 2. A L. Page (ed) Amer. Soc. of Agronomy and Soil Sci.Soc. of America, Madison, Wisconsin, USA, pp:539-577.

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Olsen SR & Sommers LE. 1982. Phosphorous. In: Methods of Soil Analysis. Part 2.A.L. Page (ed) Amer. Soc. Of Agronomy and Soil Sci.Soc. of America, Madison, Wisconsin, USA, pp: 403-427.

Tinker PB. 2000. Shades of Green. A review of UK Farming Systems, RASE, Stoneleigh, UK.


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WG1

“Plant breeding and colonisation by AM fungi”


870 report (2008), page 41.

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Molecular genetics of AMF as a tool for improving symbiosis and plant growth Ian R. Sanders, Daniel Croll, Caroline Angelard Department of Ecology & Evolution, University of Lausanne, 1015 Lausanne, Switzerland, [email protected] Molecular genetic studies of a population of the AMF G. intraradicies show that a very large amount of genetic variation exists within 1 AMF species, even within a very small field (Koch et al., 2004). Given that these genetic differences result in variation in plant growth (Koch et al., 2006), such variation could potentially be used in a genetic-based “breeding pro-gram” on the fungi for enhancing plant growth. Such programs have never been attempted on AMF because of the assumption of clonality, hence, no recombination and no genetic ex-change. Over the past few years we have taken a molecular genetics approach to understand-ing whether genetic drift, recombination and genetic exchange occur in AMF. However, until recently, our basic knowledge of AMF genetics has been limited because of poor molecular markers and limited within-population isolate numbers. We have, therefore, developed 11 simple sequence repeat (or microsatellite) markers from the nuclear genome of G. intra-radicies that allow reliable estimates of genetic variation in populations of this fungus (Croll et al., 2008) and to study AMF genetics. I will present results from experiments showing how this genetic variation from the field can be used to create new genetically different lines of the fungi (either through exchange or drift) and how such changes result in changes in the fungal phenotype in the root and plant growth. Our results show that manipulation of the genetic variation in one AMF species from one field is already enough to induce beneficial, and sometimes detrimental changes, in the symbiosis, even in plants that normally show little growth response following AMF colonization. Our results demonstrate that recombination, genetic exchange and drift occur in these fungi and that such processes can potentially be har-nessed to enhance AMF effectiveness on plant growth. References Croll D et al. 2008. New Phytologist (in press, published online). Koch AM et al. 2004. PNAS 101, 2369-2374. Koch AM et al. 2006. Ecology Letters 9, 103-110.


870 report (2008), page 43-46.

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The microbial ecology of an upland grassland ecosystem Nicholas Clipson Environmental Microbiology Group, School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland, [email protected] This paper reviews studies over the last decade of the microbial ecology of soil communities from an acidic upland grassland in Co. Wicklow, Ireland. The focus of this work has been to determine how environmental change, in particular changes in soil nutrient status brought about by agricultural practice, has impacted on the microbial component of these ecosytems. Agricultural intensification (a process to increase agricultural productivity) of grasslands has been widespread in upland areas, with fields previously used for rough grazing being fer-tilized and limed to increase plant and animal productivity. Intensification processes have led to diminished floristic diversity in grasslands, with a shift from semi-natural, unimproved species-rich grasslands to species-poor but agriculturally high-yielding (improved) plant communities. In Ireland and the UK, the predominant grass species of upland acidic grass-lands is Agrostis capillaris, with Nardus stricta, Festuca ovina, F. rubra and Holcus lanatus occurring at lower abundances as part of a highly diverse plant assemblage (Rodwell, 1992). Such semi-natural acidic grasslands classify as U4 types within the UK National Vegetation Classification (Rodwell, 1992), and are converted under improvement to much less diverse mesotrophic forms, principally MG6 or MG7, dominated by Lolium perenne and Trifolium repens. Loss in floristic diversity resulting from intensification has been a cause for concern, particularly as little is known about the possible resulting impacts on below-ground diversity including soil microbial communities. Changes in soil biodiversity may affect important soil ecosystem processes such as biogeochemical cycling and decomposition, and may be influen-tial in determining plant species composition. At present, there is no clear view of the factors determining below-ground microbial di-versity, with data indicating both plant-species-related influences and the influences of changes in soil chemical status. Several possible factors driving shifts in microbial commu-nity composition during intensification have been postulated, including soil physico-chemical variables such as pH and nitrogen concentration, the influence of shifts in floristic composi-tion during improvement, physical factors such as changes in soil structure and tillage, and grazing. pH has been found to have a significant impact on microbial diversity in soils, with the addition of nitrogen also affecting the structure of soil microbial communities. Soil type, encompassing mineral composition (percentage silt, sand, clay), chemical characteristics (pH, carbon, nitrogen, phosphorus), and management practices (tilling) can also be significant fac-tors affecting soil microbial community structure. Another significant factor may be the change in plant species that occurs during grassland improvement. Some studies have indi-cated that rhizosphere community composition may be plant-species specific. The advent of molecular techniques over the last decade that can assess soil microbial community structures has been key to elucidating effects of environmental influences on these systems. Conventional microbial culturing techniques probably identify less than 1% of soil microbial diversity (Torsvik & Lise, 2002), so that culture-based studies substantially bias any view of community structure. Environmental nucleic acids can now be successfully extracted from most environments and PCR amplification with appropriate phylogenetic or functional primers can effectively identify most components of microbial diversity. The complexity of resulting PCR amplicon mixtures has led to the development of techniques to separate indi-vidual amplicons. Where high quality phylogenetic information is required, cloning is typi-

44

cally used; whereas for an overall community view, more rapid techniques such as denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment polymorphism (TRFLP) or automated rbisomal intergenic spacer analysis (ARISA) are used (Gleeson et al., 2007). The complexity of resulting community fingerprints generally means that multivariate ap-proaches are necessary to facilitate comparisons between samples. The Wicklow Mountains in south-eastern Ireland are one of the main areas of acidic up-land grasslands in Ireland and are an area of extensive sheep grazing. Microbial studies have largely been centred on a site at Longhill, Kilmacanogue in the northern part of these moun-tains. The site is at 300m, is underlaid by a granite/quartzite bedrock and has relatively high rainfall (2000 – 2800 mm p.a.). The soil is a peaty podzol, with a pH of 3.9. Chemical analy-sis showed low levels of phosphate and nitrate, with nitrogen largely held in reduced forms probably as organic N. A portion of the site has been improved by ploughing, reseeding and annual addition of fertilizer (150, 25, 50 kg N P K, p.a.), together with liming, resulting in a soil pH of 6.3, and substantially higher levels of soil phosphate and nitrate. Vegetation analy-sis indicated that the unimproved grassland contained 25 plant species with Agrostis capillaris and Festuca ovina being the most abundant grass species, and classifying as U4a under the UK National Vegetation Classification. Improvement has resulted in a great reduction in plant diversity, with just 6 species being present, dominated by the grass Lolium perenne, and clas-sifying as MG7b (Brodie et al., 2002). Two experimental approaches have been used to determine influences on microbial community structure. The site (together with a number of similar sites across the Wicklow Mountains) has been sampled to assess in situ effects of season, locality and transition be-tween the two vegetational forms at Longhill. To try to separate individual environmental factors as determinants of microbial community structure, ex situ microcosms have also been used. In general, both bacterial and fungal components of microbial diversity have been as-sessed, generally using 16S rRNA primers for bacteria, and ITS-based primers for fungi. Ini-tially, DGGE was used to separate amplicons, although this is not a particularly sensitive community fingerprinting approach. More recently TRFLP or ARISA has been employed using an automated DNA sequencer to give much higher resolution. Initially, microbial community structure was assessed between the two grassland types across a zone of transition. Culturable bacterial numbers and microbial activity were highest in the MG7b grassland (Brodie et al., 2002), but with fungal biomass substantially higher in the U4a grassland (Brodie et al., 2003). Bacterial ribotype number (a measure of bacterial richness) was significantly (87 as compared to 55) higher in the improved MG7b grassland. Fungal ribotype numbers were generally considerably lower (13 – 17) with no significant dif-ferences between grassland type. A simple similarity index based upon terminal restriction size (bp) or band position on DGGE gels revealed a 21% and a 28% similarity, for both bacte-rial and fungal ribotypes respectively. This data revealed that U4a grassland is more fungally dominated than MG7b. Bacteria predominate in MG7b, both in terms of biomass and diver-sity. Interestingly, the level of plant diversity was not linked to the extent of microbial diver-sity, particularly with substantially higher bacterial diversity in the plant-species-poor MG7b grassland. The membership of microbial communities between the two grasslands was also substantially different. It is difficult to use field observations to identify environmental factors influencing community structure. In this case, it was suggested that certainly in terms of bac-terial diversity, plant species composition had little effect, and environmental factors such as soil nitrogen or phosphate status may be more important determinants of community compo-sition. A number of studies were carried out to observe effects of season and locality on these grasslands (Kennedy et al., 2005a, b; 2006). Bacterial and fungal community structure was

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assessed for two unimproved grassland types (U4a and U4b) at five sites across the Wicklow Mountains, chosen so that underlying geology and vegetational compositions were similar. The original hypothesis was that if geology and vegetation were very similar between sites, then microbial community structure would select to be broadly the same. In reality, microbial communities varied substantially between both sites and grassland types, perhaps indicating that a compendium of factors relating to site (both biotic and abiotic) is the determinant of microbial community structure. Seasonal based sampling at the Longhill site indicated that community structure did show some changes when winter months were compared to summer months. Field based studies indicated that soil environmental conditions might be particularly important in influencing microbial community structures; to explore this a number of micro-cosm experiments were set up to explore effects of single environmental factors such as soil ph, N or P status. Seven perennial plant species typical of both U4a and MG7b grasslands (Agrostis capillaris, Nardus stricta, Festuca ovina, F. rubra, Holcus lanatus, Lolium perenne and Trifolium repens) were either left unamended or treated with lime, nitrogen, or lime plus nitrogen in a 75-day pot-based microcosm in a glasshouse (Kennedy et al., 2005; 2005). Lime and nitrogen amendments were shown to have a greater effect on microbial activity, biomass and bacterial ribotype number than plant species. Liming increased soil pH, microbial activity and biomass, while decreasing bacterial ribotype number. Nitrogen addition decreased soil pH, microbial activity and bacterial ribotype number. Addition of lime plus nitrogen had in-termediate effects, which appeared to be driven more by lime than nitrogen. TRFLP analysis revealed that lime and nitrogen addition altered soil bacterial community structure, while plant species had little effect. These results were further confirmed by multivariate redun-dancy analysis, and suggested that soil lime and nitrogen status were more important control-lers of bacterial community structure than plant rhizosphere effects. Although different plant species were associated with some changes in fungal biomass, this did not result in significant differences in fungal community structure between plant species. Addition of lime alone caused no changes in fungal biomass, ribotype number or community structure. Overall, fun-gal community structure appeared to be more significantly affected through interactions be-tween plant species and chemical treatments, as opposed to being directly affected by changes in individual improvement factors. These were in contrast to those found for bacterial com-munities, which changed substantially in response to chemical (lime and nitrogen) additions. In upland grasslands, soil nitrogen addition occurs mostly via animal excreta, with sheep’s urine probably being the most significant N input. A microcosm where different con-centrations of synthetic sheep’s urine (SSU) were applied showed that SSU increased micro-bial activity at higher concentrations but did not affect microbial biomass (Rooney et al., 2006). Effects on bacterial community composition depended upon plant species present. Where A.capillaris (upland grass species) was present, a high SSU concentration affected communities only in the period after application (10 days); this was in contrast with L.perenne (improved grassland species) where effects were seen only after 50 days. When urea is added to soils it is generally hydrolysed to increase soil ammonium pools, which both increases soil pH and soil N status. Grasslands are typically very patchy in their diversity, perhaps influenced by the randomness of urea inputs via sheep urination. SSU application had much less effect on fungal community structures than on bacterial structures. A micro-cosm varying soil phosphate contents has also recently been performed (Rooney and Clipson, 2008) In conclusion, both field based and microcosm studies have reinforced the view that mi-crobial community structures in soils are determined by a complex of factors. In this grass-land, bacteria do appear to be more responsive to external influences than fungi, at least in the

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short term. The relationships between plant diversity and microbial diversity appear to be complex, with no apparently direct relationship. It is likely that these relationships function through interactions with other environmental factors. Soil physico-chemical factors did ap-pear in some cases to have direct effects on bacterial community structure. It should be noted that the molecular approaches used profiled whole communities, without focusing on specific phylogenetic or functional groups. It remains to be seen whether such relationships hold for microbial groups more implicitly linked to plant activity through symbiosis, such as nitrogen fixation or mycorhizal association. Acknowledgements I would like to thank Eoin Brodie, Nabla Kennedy, Deidre Rooney, Deirdre Gleeson, Suzanne Edwards and Ann Kathrin Liliensiek for their great contribution to these studies over the last decade. I acknowledge Enterprise Ireland, the Irish EPA and Science Foundation Ireland for financial support. References Brodie E, Edwards S & Clipson N. 2002. Microbial Ecology 44, 260-270. Brodie E, Edwards S & Clipson N. 2003. FEMS Microbiology Ecology 45, 105-114. Gleeson DB, McDermott PF & Clipson N. 2007. Advances in Applied Microbiology, 62, 81-

104. Kennedy N, Brodie E, Connolly J & Clipson N. 2004. Environmental Microbiology 6, 1070-

76. Kennedy N, Connolly J & Clipson N. 2005. Environmental Microbiology 7, 780-788. Kennedy N, Gleeson D, Connolly J & Clipson N. 2005. FEMS Microbiology Ecology 53,

329-338. Kennedy N, Edwards S & Clipson N. 2005. Microbial Ecology. 50, 463-473. Kennedy N, Brodie E, Connolly J & Clipson N. 2006. Canadian Journal of Microbiology 52,

689-694. Rooney D, Kennedy N, Deering L, Gleeson D & Clipson N. 2006. Applied and Environ-

mental Microbiology, 72, 7231-37. Rooney D & Clipson N. 2008. Microbial Ecology (in press). Rodwell JS. 1992. British Plant Communities: Grasslands and Montane Communities. Cam-

bridge. Torsvik V & Lise O. 2002. Curr Opin Microbiol 5: 240–245.


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A novel gene-candidate approach of socio-economic interest? – Breeding on efficient plant genotype – mycorrhiza interaction Birgit Arnholdt-Schmitt EU Marie Curie Chair/ICAM, University of Évora, Apart. 94, 7002-554 Évora, Portugal, [email protected] Introduction Plant breeding of agricultural crops is used to consider the complexity of whole plant systems on the background of diversely interacting environments. This challenge is complicated by the dynamics of both systems through plant development and the spatially as well as tempo-rally variable importance of interfering environmental factors. As a consequence, conven-tional breeding traditionally determines heritability and the significance of the environment for a given trait through testing genotypes in different years and at various locations. Plant breeding has a long tradition. It is therefore much more advanced in handling and understand-ing the complexity of plant production systems than more recent disciplines, such as molecu-lar biology and biotechnology. This includes plant genetics as well as phenotyping in relation to various quantitative and qualitative traits. However, in current years, interdisciplinary re-search approaches are increasingly important to meet the requirements of more sophisticated advances through molecular plant breeding. Complementing disciplines, such as eco-physiology, molecular biology, biotechnology and breeding are converging (Arnholdt-Schmitt, 2005a, b). Molecular plant breeding is expected to help improving yield stability. To be successful, it must integrate available experiences and knowledge on plant and production systems and consider also current progress in agricultural management practices, such as or-ganic and precision farming. Plant breeding and breeding research requires investments from the private and public sector. Thus, appropriate breeding strategies and well-justified selection of candidate marker genes for crop improvement are crucial to minimize the risk of mis-investment. Investing in breeding on efficient genotype-mycorrhiza interaction is especially difficult. It has to take into account the high complexity of plant – AM fungi networking in the rhizosphere. This space is characterized through variable physical and chemical soil char-acteristics and multi-organismal interference. Varying seasonal and annual conditions have strong impact on regional ecosystems and contribute highly to yield instability. Breeding on efficient AM symbiosis The scheme next page demonstrates recommended criteria and steps of decision-making for breeding on efficient genotype – mycorrhiza interaction:

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Importance of crop – mycorrhiza association in relation to non-mycorrhiza crop improvement?

Arguments in favour of breeding on efficient AM symbioses: - combining nutrient and water acquisition efficiency - combining abiotic and biotic stress tolerance - enhancing the diversity of germplasms for stress tolerance

1.

Genetic variability for efficient genotype – mycorrhiza interaction?

Measures: Differences in the adaptive regulation of fungal colonization (cost/benefit calculation)!! Plant nutrient efficiency (nutrient uptake and exchange) Plant drought tolerance (water uptake and exchange) Biotic plant stress tolerance

2.

3. Traditional or functional marker-assisted breeding methodology?

if functional marker development:

4. Top-down hypothesis-driven research strategy: candidate gene Molecular-physiological analysis of plant-fungus interaction

Functional analysis through reverse genetics and RNAi

If genetic variability is confirmed:

5. Screening for gene polymorphisms and variable expression; phenotyp-ing and association studies

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If hypothesis verified:

If candidate gene linkage: functional marker-assisted breeding

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AOX - a potential gene candidate for adaptive fungal colonisation regulation Our group is studying the role of the alternative oxidase (AOX) in stress-induced cell-reprogramming in diverse species, including also carrot (Daucus carota L.). We are less in-terested to advance understanding the biochemistry behind, but we want preferentially under-stand, whether AOX may serve as a functional marker for cell reprogramming. Thus, correla-tive analyses are in this sense more important for us than functional validation. Carrot is well-known to produce high amounts of strigolactones and to be a host of AM fungi. In the presen-tation, I will speculate about a potential role of AOX in AM fungi interaction. AOX is nuclear encoded by a small multigene family that consists typically of three to five genes belonging either to the AOX1 or AOX2 subfamily. We have currently identified diverse forms of poly-morphic AOX gene sequences in carrot and other species as a potential for functional marker development. RNAi silenced carrot genotypes will be produced for functional analysis. Alter-native oxidase is recently of high interest in stress research. It is linked to all kinds of abiotic and biotic stress reactions in various organisms, including plants as well as fungi. Our hy-pothesis focus on the importance of AOX for adaptive carbohydrate turnover during stress-induced cell reprogramming to enable required structure-building and to modulate growth and development under avoidance of oxidative stress (Arnholdt-Schmitt et al., 2006a). Root in-duction is important for plant – AM interaction. We have preliminary hints that AOX may play a role in root induction in olive tree shoot cuttings (Santos Macedo et al. unpublished, Arnholdt-Schmitt et al., 2006b). At the meeting, I will propose collaboration to study the rela-tionship between strigolactone production and growth control/root induction in carrots and AOX activities. Further, it was shown that AOX of fungus may help its survival in situations, where the cytochrome pathway for respiration is blocked (Crop Protection, 2003). Tamasloukht et al. (2003) speculated on the role of alternative respiration in the pre-symbiotic phase to enable survival of AM fungi spores. We would like to initiate working on the hy-pothesis that the regulation of AOX from plants and fungus is connected during the pre-symbiotic phase and plays a crucial role in mycorrhiza colonisation. Investing in AM preparations for commercial use? Real-time crop management is getting increasingly important in modern agricultural practice. Precision farming with intelligent automatic supplies is under debate. However, sophisticated technology application needs to consider regional and even micro eco-systemic differences that may strongly influence the necessities for stable plant production. Crop-specific designed AM preparations could be of commercial importance and may importantly contribute to precision farming. In this sense, a more targeted breeding approach on functional marker development considering specific plant – fungus interaction could be helpful for future applications. However, genetic characterization of AM fungal diversity is still in the beginning. Additionally, AM fungi infection needs effective low-cost strategies to monitor rapidly the nutritional status of plants and related soil characteristics. The practicabil-ity and the relation to input costs for farmers needs to be discussed independently from com-mercial interests before initiating long-term breeding strategies. Acknowledgements I appreciate discussions with my colleagues Mário Carvalho, Department of Plant Production, University of Évora, Benvindo Maçãs and Manuel Tavares de Sousa, former National Station of Plant Breeding (ENMP), INRB, Portugal.

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References

Arnholdt-Schmitt B. 2005a. Functional markers and a ‘systemic strategy’: convergency be-tween plant breeding, plant nutrition and molecular biology. Plant Physiology and Bio-chemistry 43, 817-820.

Arnholdt-Schmitt B. 2005b. Efficient cell reprogramming as a target for functional-marker strategies? Towards new perspectives in applied plant-nutrition research. J. Plant Nutr. Soil Sci. 168:617-624.

Arnholdt-Schmitt B, Costa JH & Fernandes de Melo D. 2006a. AOX – A functional marker for efficient cell reprogramming under stress? Trends in Plant Science 11:281-287.

Arnholdt-Schmitt B, Santos Macedo E, Peixe A, Cardoso HCG & Cordeiro AM. 2006b. AOX – A potential functional marker for efficient rooting in olive shoot cuttings. Proceed. Second International Seminar Olivebioteq, Marsala – Mazara del Vallo, Italy, November 5th -10th, p. 249-254.

Crop Protection. 2003. Resistance and the alternative oxidase pathway. 26. April, 38-39. Tamasloukht et al. 2003. Root factors induce mitochondrial-related gene expression and fun-

gal respiration during the developmental switch from asymbiosis to presymbiosis in the arbuscular mycorrhizal fungus Gigaspora rosea. Plant PHysiol, 131, 1468-1478.


870 report (2008), page 51-54.

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Breeding stress-tolerant maize: the role of arbuscular mycorrhiza

Shawn Kaeppler Department of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison, WI, USA 53706, [email protected] Abstract: Widely adapted stress-tolerant varieties of annual commodity crops such as maize are a breeding goal of private companies and international breeding centers. Our research on P tolerance in maize has included studies on variation among maize genotypes for their relative performance under P stress and with adequate fertility in the presence and absence of arbuscu-lar mycorrhizal fungi. Maize inbred lines vary dramatically in their response to mycorrhiza when grown under P stress, with the least responsive genotypes being those which are most P efficient in the absence of the fungus. Species and isolates of mycorrhizal fungi varied in their ability to promote growth under P stress. The issue of how to incorporate the mycorrhi-zal symbiosis into a selection program aimed at producing stress-tolerant maize remains un-clear. Two factors have led us to focus more of our efforts on P efficiency in the absence of mycorrhiza. First, early season nutrient stress is an important, although transient, limitation to maize growth at our latitude. At this time of the year, temperatures are cool minimizing soil microbial activity. In addition, tillage used to plant annual crops disrupts existing hyphal networks. Therefore, the mycorrhizal symbiosis seems to offer a relatively small potential benefit for this critical growth period. Second, the density and composition of mycorrhizal populations is highly variable across locations, and varies at specific sites across years due to climactic conditions and crop rotations. Varieties which are insensitive to microbial popula-tions – that is, inherently nutrient efficient – seem likely to be more stable across diverse envi-ronments. However, important questions remain such as the physiological cost of nutrient efficiency, and whether depending on mycorrhizal symbiosis would allow more partitioning of carbohydrate to aerial tissues and thereby increase yield. Interpretation Our research has focused on genes and genotypes of maize that have potential utility in im-proving P stress tolerance. This research has included the analysis of genetic variation for root morphology, physiological processes of roots, and interactions with arbuscular mycorrhiza (Kaeppler et al., 2000; Mickelson & Kaeppler, 2005; Pletsch-Rivera & Kaeppler, 2007; Riggs et al., 2001; Yun & Kaeppler, 2001; Zhu et al., 2006; Zhu et al., 2005 a, b; Zhu et al., 2005). Motivated, in part, by research such as Hetrick et al. (Hetrick et al., 1992), we character-ized variation among 28 diverse Midwestern maize genotypes for their interaction with arbus-cular mycorrhiza (Kaeppler et al., 2000). This study was conducted in a glasshouse. The me-dium was field-soil from a plot in Hancock, WI that had not received fertilizer, including ma-nure, for 19 years at the time the soil was collected. Plants were grown for 6 weeks in 500 ml pots. The low P treatment, which was sterilized field soil, was 17 ppm Bray P1, and amended soil had a Bray P1 value of 87 ppm. There was substantial variation among genotypes for P efficiency in the absence of mycorrhiza, biomass accumulation across all treatments, and re-sponse to mycorrhizal inoculation and to P fertilization relative to low P. An important observation from this study was that responsiveness was accounted for, in large part, by the relative P efficiency of genotypes in the absence of inoculation. That is, genotypes which were highly sensitive to P stress generally showed a large response to my

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corrhizal inoculation when grown at low P, while stress-tolerant genotypes showed little re-sponse (Figure 1). This result led us to conclude that a large proportion of the agronomically relevant variation among this germplasm was for P efficiency in the absence mycorrhizal in-fection. It should be noted that genotypes such as B73, CMD5, and Pa36 demonstrate that there is also substantial variation for responsiveness among P stress-susceptible genotypes that is also worthy of consideration. Sawers et al. provide a useful discussion of potential dif-ferences in genetic variation for P uptake by plants versus via mycorrhiza (Sawers et al., 2008).

Figure 1. Genotypes which are most responsive to mycorrhiza when grown at low P are least P efficient. Scatter plot of 28 maize inbreds comparing aerial biomass accumulation at low P versus response to mycorrhizal infection at low P. In a companion study (Mickelson & Kaeppler, 2005), we evaluated the ability of six my-corrhizal isolates to promote the growth of 7 maize genotypes grown at low P. This study indicated that there was a differential benefit of the isolates in promoting maize growth. In addition, our research provided evidence of a genotype by mycorrhizal strain interaction indi-cating that diverse maize genotypes may not respond uniformly to the same strain of inocu-lant. Our recent research has focused more on inherent P efficiency of maize genotypes rather than on genetic variation for the interaction of maize and mycorrhizal fungi. This decision was based on the following argument. Widely adapted genotypes of annual commodity crops such as maize are attractive to companies because they have a low probability of failure and simplify inventories. They are also desirable to international breeding centers because breed-ing programs can have a broader impact if varieties can be used in diverse environments. Broad adaptation requires nutrient efficiency across a range of environments, soil types, and microbial communities. Yield potential is also maximized in genotypes that are able to grow well early in the season, maximizing photosynthate accumulation, and avoiding late season water stress. Genotypes which depend on mycorrhizal symbiosis face several challenges in the context of broad adaptation. Microbial communities are highly variable across environments and sea-sons, and may be very low in certain conditions such as following fallow or flooding. Fur-thermore, in latitudes further from the equator such as Wisconsin, cold temperatures early in the season limit microbial activity and transient nutrient stress in seedlings is often observed.

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Finally, tillage required for annual crop planting can disrupt hyphal networks and microbial communities. Therefore, it is my opinion, that broadly adapted annual cultivars – at least in the context of P stress – need to be inherently efficient and rather than rely on symbiosis or interactions with spatially variable microbial communities for nutrient acquisition. There are, however, likely additional considerations. Nutrient efficient genotypes which also maintain a productive mycorrhizal symbiosis may realize additional stress tolerance under unique environments, or due to benefits in the context of other biotic and abiotic stresses. Another unknown is the relative physiological cost of a P efficient root system. If inherent P efficiency requires a substantial carbon drain to build and maintain the root system and through release of metabolites such as organic acids, then it may be beneficial in efforts to maximize yield to partition resources to the grain rather than the roots, relying on mycorrhizal symbiosis for P acquisition. Paszkowski and Boller (Paszkowski & Boller, 2002) made the interesting observation that mycorrhizal symbiosis can rescue a maize root mutant grown at low P. By analogy, perhaps a maize genotype with a relatively small root system which is still sufficient for anchorage and basic root services, could be highly productive under limit-ing P if it participated in a productive mycorrhizal symbiosis. Continued research is necessary to better clarify basic processes underlying the symbio-sis between plants and arbuscular mycorrhiza and to determine how to most efficiently har-ness this interaction in production agriculture. References Kaeppler SM, Parke JL, Mueller SM, Senior L & Stuber C. 2000. Variation among maize

inbred lines and detection of quantitative trait loci for growth at low phosphorus and re-sponsiveness to mycorrhizal fungi. Crop Sci. 40:358-364.

Mickelson, SM & Kaeppler SM. 2005. Evaluation of six mycorrhizal isolates for their ability to promote growth of maize genotypes under phosphorus deficiency. Maydica 50:137-146.

Pletsch-Rivera LA & Kaeppler SM. 2007. Investigation of xenia effect on phosphorus accu-mulation in the maize grain (Zea mays L.). Maydica 52:151-157.

Riggs PJ, Chelius MK, Iniguez AL, Kaeppler SM & Triplett EW. 2001. Enhanced maize productivity by inoculation with diazotrophic bacteria. Aust. J. Plant Physiol. 28:829-836.

Yun SJ & Kaeppler SM. 2001. Induction of maize acid phosphatase activities under phospho-rus starvation. Plant and Soil 237:109-115.

Zhu J, Mickelson SM, Kaeppler SM & Lynch JP. 2006. Detection of quantitative trait loci for seminal root traits in maize (Zea mays L.) seedlings grown under differential phosphorus levels. Theor Appl Genet 113:1-10.

Zhu J, Kaeppler SM & Lynch JP. 2005. Mapping of QTL for lateral root branching and length in maize under differential phosphorus supply. Theor Appl Genet 111:688-695.

Zhu J, Kaeppler SM & Lynch JP. 2005. Topsoil Foraging and Phosphorus Acquisition Effi-ciency in Maize (Zea mays L.). Functional Plant Biology 32:749-762.

Zhu J, Lynch JP & Kaeppler SM. 2005. Mapping of QTL controlling root hair length in maize (Zea mays L.) under phosphorus deficiency. Plant and Soil 274:299-310.

Hetrick BAD, Wilson GWT & Cox TS. 1992. Mycorrhizal dependence of modern wheat va-rieties, landraces, and ancestors. Can. J. Bot. 70:2032-204010.

Sawers RJH, Gutjahr C & Paszkowski U. 2008. Cereal mycorrhiza: an ancient symbiosis in modern agriculture. Trends Plant Sci. 13:93-97.

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Paszkowski U & Boller T. 2002. The growth defect of lrt1, a maize mutant lacking lateral roots, can be complemented by symbiotic fungi or high phosphate nutrition. Planta 214:584-590.


870 report (2008), page 55-60.

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The role of mycorhizzas in nutrient acquisition by European and African maize varieties Stephen Rolfe, Derek Wright, David Read, Julie Scholes Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, United Kingdom, [email protected] Introduction Globally, maize (Zea mays) is one of the most important cereal crops whose products are di-rectly consumed by humans. Most countries in Africa are not self-sufficient in food produc-tion and therefore import grain. This has significant implications particularly given the recent worldwide increase in cereal costs resulting from changing climatic conditions, increased en-ergy costs incurred during production and the diversion of grain from food use to biofuel pro-duction (Food and Agriculture Organisation, United Nations, 2008). In developed countries agricultural production is sustained by the application of fertilis-ers. However, in the low input, subsistence agricultural systems which dominate developing countries in tropical Africa, soil P availability represents a major limiting factor for crop pro-duction. Maize cultivars differ in their intrinsic ability to take up P when grown axenically. However when P supply is limited, the ability of field-grown cereal plants to capture P from the soil is facilitated by the formation of symbioses with arbuscular mycorrhizal (AM) fungi (Smith and Read, 1997). For Z. mays growing in acidic, tropical soils up to 60% of the total P requirement of the plant can be supplied by the AM fungus Glomus intraradices (Nurlaeny et al., 1996). The effectiveness of P acquisition, whether directly from soil or via AM fungal associa-tions, differs widely between plant species and even between modern and older cultivars or wild accessions of the same species. It has also been recognised that modern plant breeding programmes designed to maximise yield in high nutrient input agriculture may have led to a reduction in responsiveness to AM colonisation and compromised the effectiveness of the symbiotic acquisition of soil nutrients (Zhu et al., 2001). Under the conditions of low P sup-ply that are prevalent in tropical subsistence agriculture, this would be a severe disadvantage. In the present study, we have compared the responsiveness of various maize cultivars to infection by the AM fungi Glomus mossae and G. intraradices. Growth analyses of mycorrhi-zal and non-mycorrhizal plants were performed for the cultivar River, which is grown in high-input agricultural systems of Western Europe, and cultivars H511, H662 and Katamuni which are grown extensively in subsistence agricultural systems in African countries, notably Kenya and Tanzania. An intensive comparative analysis was then performed for two contrasting cul-tivars, River and H511, when non-mycorrhizal or colonised by G. intraradices, to determine whether cultivars of diverse origins responded differently to mycorrhizal colonisation. The growth responses, rates of photosynthesis, carbohydrate concentrations of the leaves and roots of mycorrhizal (M) and non-mycorrhizal (NM) plants of both cultivars were determined when grown at six different soluble P concentrations ranging from 0 to 65 µM. In addition we iso-lated phosphate and monosaccharide transporter genes from maize and investigated how the expression of these genes varied in these two cultivars in response to P supply and mycorrhi-zal colonisation. We hypothesise that mycorrhizal responsiveness to colonisation by G. intra-radices and the underlying alterations in the expression of genes potentially involved in nutri-ent exchange may be different in these two maize cultivars, bred for production in very differ-ent agricultural systems.

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Results and Discussion A screen of the responsiveness to mycorrhizal infection by G. mossae and G. intraradices was performed on greenhouse-grown plants grown in sand supplemented daily with Long Ashton’s solution containing 65 µM soluble P as KHPO4. Growth was measured as the height to the youngest ligule (Figure 1) and at the end of the experiment plants were harvested and dry matter accumulation in roots, stem and leaves determined (Figure 2).

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Figure 1. The effect of mycorrhizal colonisation on the growth of maize cultivars. Plants were grown in sand supplemented with Long Ashton’s solution and growth (the height to the youngest ligule) determined. Plants were non-mycorrhizal (non-myc) or infected with G. mossae or G. intraradices. Results are the mean +/- S.E.

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Figure 2. The effect of mycorrhizal colonisation on dry matter accumulation of maize culti-vars. Plants were harvested after 48 days growth and the dry matter accumulation in roots, shoots and leaves determined. Plants were non-mycorrhizal (NM) or infected with G. mossae or G. intraradices. Results for Katamuni are omitted due to poor plant survival.

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The cultivars River and H511 grew well under the conditions tested attaining a final harvest ligule height of approx 300 mm. M plants were slightly shorter than NM plants presumably due to diversion of photosynthate to the fungal partner. Cultivars H622 and particularly Katamuni grew less well and did not exhibit a M-induced reduction in height (Figure 1). Analysis of biomass partitioning 48 days after planting indicated that both River and H511 allocated significantly less biomass to roots when mycorrhizal (Figure 2). There was no sig-nificant difference in M colonisation between cultivars with over 75% of the root colonised in each treatment (data not shown).

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Figure 3. The effect of mycorrhizal colonisation on biomass partitioning in mycorrhizal and non-mycorrhizal River and H511 cultivars. Cultivars River and H511 were selected for further analysis and partitioning of biomass measured at 3 points during the growth period in an independent experiment (Figure 3). Again, a reduction in total biomass was observed in the final harvest of M plants as a result of a reduction in root biomass. This was apparent 42 days after planting with both cultivars in-fected with either G. mossae or G. intraradices. However, the reduction in root biomass of M River plants was also significant 28 days after planting. River and H511 cultivars were then grown, with and without infection by G. intraradi-ces, at soluble P concentrations ranging from 0 to 65 µM. Under these conditions, NM plants were completely reliant on seed reserves and Pi supplied in the nutrient medium for P, whereas M plants could access additional insoluble Pi in the sand substrate. NM River plants grew poorly when supplied with 0-5 µM Pi but better at higher supply rates (Figure 4). NM H511 plants grew in a similar manner to River at 0-0.1 µM Pi supply rates, outperformed River at 1-5 µM Pi, but did not show such an increase in growth at the highest supply rates. Clearly the sensitivity of the two cultivars to soluble Pi supply differed. NM H511 grew better than River under low nutrient conditions, but was unable to make use of the highest Pi supplies to increase biomass. When infected with G. intraradices, River grew well at all soluble Pi supply rates demonstrating that it could efficiently access insoluble Pi in the sand. In contrast, although H511 showed some stimulation in growth when M, growth was still influenced by soluble Pi at most supply rates tested. M River outperformed M H511 at 1-5 µM Pi, reversing the relative performance observed in NM plants.

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Figure 4. The effect of soluble Pi supply rate on the growth of M and NM River and H511. Dry matter (a,b) and the root:shoot ratio (c,d) were measured for M and NM River and H511 grown at different soluble Pi. Results are mean +/- S.E. There was a marked difference in the root:shoot ratio (RSR) between the two cultivars. There was little impact of soluble Pi supply or M infection on the RSR of River (except at 0 µM soluble Pi), whilst the RSR of H511 was strongly influenced by both factors. These differences in growth were reflected in the specific P uptake of the two cultivars when M or NM (Figure 5). M plants had a significantly higher specific P uptake at all supply rates tested, but that of River generally exceeded that of H511, particularly at 65 µM Pi. The maximum rate of photosynthesis of the youngest fully expanded leaf was strongly influenced by Pi supply in both cultivars. For NM plants, the rate of photosynthesis of H511 exceeded that of River by 8-64%, with the most marked differences at 20 µM Pi and below. Photosynthesis was strongly stimulated in M plants but did not differ significantly between the cultivars. These results are consistent with H511 being better adapted to low Pi conditions than River when NM, particularly at supply rates of 1-10 µM Pi.

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Figure 6. Rates of photosynthesis. The uptake of Pi from the soil is facilitated by specific membrane-localised, high-affinity Pi transport proteins. Genes encoding these transport proteins (ZmPT1-3) were isolated from River and their expression in response to soluble Pi and mycorrhizal infection determined in both cultivars (Figure 7). The relative expression of each transporter gene in the absence of supplied soluble Pi (0 µM) was then plotted against the root total P content (Figure 8).

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Figure 7. Northern blot analysis of phosphate transporter gene expression. ZMPT1-3 are spe-cific probes for maize phosphate transporters. GiTS is a probe specific to G. intraradices. The expression of each P transporter gene declined in the roots of NM maize as the concentra-tion of soluble Pi in the nutrient medium increased, such that expression in the highest treat-ment (65 µM) was barely detectable (Figure 7). Although the overall pattern was the same in both cultivars, expression was maintained at higher Pi concentrations in H511 compared to River. In M roots of River, ZmPT1-3 expression was very low (less than 10% of that observed in NM roots grown in the absence of soluble Pi). Although expression was also repressed with increasing soluble Pi concentrations in H511, expression was maintained somewhat in the M

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roots of H511 grown with soluble Pi concentrations of 1 µM or less – expression levels in M roots grown in the absence of added soluble Pi were 34 to 51% of that of NM roots grown under the same conditions. When the expression of each P transporter was plotted as a func-tion of root P content it was apparent that expression was maintained at higher levels in the roots of H511 than in River as root P content increased, both in the M and NM condition (Figure 8). We propose that the decline in expression results from the improved P nutrition of M roots. However, repression of P transporter gene expression in H511 was inherently less sensitive to internal or external P signals compared to River indicating a general alteration in P responsiveness.

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Conclusions Although the European cultivar River has been developed for growth in high nutrient input agriculture, and as such will normally be grown in the NM condition, it still retained the ca-pacity to form efficient symbiotic associations with the Glomus intraradices and, in fact, per-formed better (in terms of biomass accumulation) than the African variety H511 when my-corrhizal. H511 only out-performed River when NM at intermediate Pi (1 to 5 µM) supply rates. A major difference between these cultivars was the responsiveness of the RSR to Pi supply and M infection. Clearly, a large root system, as found in H511, will have beneficial roles in water acquisition as well as nutrient foraging which is likely to be important in Afri-can agriculture. However, the specific P uptake of River was always significantly greater than that of H511, and P transporter gene expression more sensitive to Pi repression under all con-ditions tested, indicating that River may have beneficial traits which may improve the per-formance of cultivars such as H511 in low nutrient input agriculture systems. This work was supported by The Leverhulme Trust (Grant number: F/118/AT). Details of methods employed can be found in Wright et al. (2005) New Phytologist (2005) 167: 881–896.


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Plant genes involved in AM symbiosis Franziska Krajinski Max Planck Institute of Molecular Plant Physiology, Potsdam Golm, Germany, [email protected] Phosphorous (P) is second to nitrogen as the most limiting element for plant growth. Not only it is a major component of fundamental macromolecules, also it plays an important role in energy transfer, regulating enzymatic reactions and different metabolic pathways. P is taken up by plant roots as phosphate (Pi), which is one of the least available nutrients in the soil. Hence, the majority of agricultural systems depend on the application of P-fertilizers, pro-duced from nonrenewable resources, which could be depleted as early as by 2060 according to recent estimations. Plants have evolved a variety of adaptive strategies to improve their Pi-acquisition, in-volving altered root morphology, exudation of organic acids, phosphatases and nucleases in order to solubilize Pi from organic resources and the establishment of a symbiosis with arbus-cular mycorrhizal (AM) fungi (Harrison, 1999; Hause and Fester, 2005). AM fungi are pre-sent in nearly all terrestrial ecosystems, forming mutualistic symbiosis with the roots of 80% of vascular plants. They often increase Pi uptake and plant growth. The ability of AM fungal hyphae to grow beyond the root’s Pi-depletion zone and deliver Pi to the host plant is thought to be the main basis for their positive effects on Pi-uptake (Smith & Read, 1997). Despite the positive influence on plant P-nutrition and growth, little is known about the molecular mecha-nism of the AM symbiosis. While the development of the AM symbiosis is likely to be controlled by a specific ge-netic program of the plant, for several reasons, little information is available on how plants control the symbiosis development. The major difficulty in analysing AM associations is the obligate biotrophic nature of the AM fungi, which so far makes them nearly inaccessible for genetic and genomic characterisations. Moreover, the colonisation of a root system by AM fungi is a non-synchronous process including re-infections of the root system, a limited life span of arbuscules and re-infections of cortical cells. Important insight in the analysis of mo-lecular events during AM symbiosis has been achieved recently by the identification of plant Pi-transporters which either are specifically induced in response to AM symbiosis (Harrison et al., 2002; Paszkowski et al., 2002; Glassop et al., 2005; Nagy et al., 2005) or strongly upregulated in mycorrhizal roots but having a basal expression in non-mycorrhizal roots (Rausch et al., 2001; Maeda et al., 2006; Chen et al., 2007). In order to identify further AM-specific or AM-induced plant genes, M. truncatula is applied as model plant. M. truncatula, which has a relatively small, completely sequenced genome of 500 Mb (Sato et al., 2007), has evolved as a reference legume species for func-tional genomic research on legume biology and root symbioses. Since the adoption of M. truncatula as a model plant, a number of useful tools and resources have been developed, as a variety of different mutant populations and tools and protocols for transcriptome, but also for proteome, and metabolome analysis. In order to identify AM-exclusive gene inductions of Medicago truncatula, which neither occur during other plant root-microbe interactions nor are due to a better phosphate nutrition, we used a pool of different RNA samples as subtractor population in a suppressive subtractive hybridization (SSH) experiment. This approach resulted in the identification of a number of new AM-regulated genes. None of these genes were expressed in non-mycorrhiza roots or leaves. Electronic data obtained by comparison of the cDNA sequences to EST sequences

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from a wide range of cDNA libraries in the M. truncatula EST database (Gene Index, MtGI) support the mycorrhiza specificity of the corresponding genes, as the tentative consensus (TC) and EST sequences in the MtGI that were found to match the identified SSH-cDNA se-quences are originating exclusively from AM cDNA libraries. The promoter of one of those genes, MtGst1, showing similarities to plant glutathione-S-transferase (GST) encoding genes, was cloned and used in reporter gene studies. In contrast to studies with the potato GST-gene PRP, MtGst 1 promoter activity was detected in all zones of the root cortex colonized by Glomus intraradices, but no elsewhere. In order to identify novel AM-induced genes, which have not been listed in other data-bases before, 5646 ESTs were generated from two Medicago truncatula cDNA libraries: a random cDNA library (MtAmp) and a suppression subtractive hybridization (SSH) library (MtGim), the latter being designed to enhance the cloning of mycorrhiza-upregulated genes. In silico expression analysis was applied to identify those tentative consensus sequences (TCs) of the TIGR M. truncatula Gene Index (MtGI) that are composed exclusively of ESTs deriving from the MtGim- or MtAmp-library, but not from any other cDNA-library of the MtGI. This search revealed 115 MtAmp/MTGim-specific TCs. For the majority of these TCs with sequences similarities to plant genes, the AM-specific expression was verified by quanti-tative reverse transcription-PCR. Annotation of the novel genes induced in mycorrhizal roots suggested their involvement in different transport as well as signaling processes and revealed a novel family of AM-specific lectin genes. The expression of reporter gene fusions in trans-genic roots revealed an arbuscule-related expression of two members of the lectin gene family indicating a role for AM-specific lectins during arbuscule formation or functioning. The recent identification of AM specific genes revealed first insight in the molecular background of AM symbiosis but moreover these genes can be used as diagnostic markers for studying phosphate homeostasis in AM-symbiosis. The recently gained knowledge on the mechanism of Pi- signaling in plants provides the possibility to study Pi-signaling in relation to AM symbiosis development in order to identify possible links of these two pathways that lead a systemic suppression of AM development under high Pi conditions. Since most studies of Pi-signaling have been varied out in A. thaliana, a non AM-capable plant, nothing is known so far about a correlation of Pi-signaling and AM symbiosis development. Currently we have identified putative Medicago orthologs of Pho2 and Phr1, two key regulators of Pi-starvation response. Moreover, we established different diagnostic markers for Pi-starvation using specific primer pairs for either Phr1–dependent or Phr1–independent Medicago genes induced after Pi-deprivation. The systemic effect of internal Pi levels on AM development was confirmed using split root systems were plants were subjected simultaneously to low (20 µM) and high (up to 2 mM) phosphate concentration applied to one compartment. The second halfs of all split systems contained G. intraradices inoculum and were treated with low Pi concentration. As described several times in the literature, a clear negative correlation be-tween phosphorous fertilization in the first compartment and AM colonization in the my-corrhizal, low Pi-compartment was observed after staining for mycorrhizal structures and es-timation of mycorrhization. The phosphorous content of the shoots of the split root plants was estimated and correlated clearly to the fertilization treatments. qRT-PCR was applied to measure the expression levels of diagnostic markers for mycorrhization (MtGst1; MtPt4, Glomus rDNA) and phosphate starvation (PlD), as well as key regulators of Pi-signaling (399c, Mt4, Pho2). The Glomus RNA primer pair binds to G. intraradices rRNA genes, hence observed amplification using this primer pair is not a direct marker for a mycorrhiza symbio-sis but rather for the presence of G. intraradices in the root compartment. Comparative ex-pression levels were determined after normalization to a set of housekeeping genes (MtEf1; MtUbi, MtPdf2) and expression ratios of different phosphate treatments versus the lowest

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phosphate treatment (0.02 mM) were calculated. In conformity with the estimation of my-corrhization after root staining, clearly decreased expression of plant mycorrhiza markers was observed in mycorrhizal compartments of split systems where the other compartments were subjected to 1 or 2 mM phosphate fertilization as compared to low Pi-treated plants. Expres-sion levels of mycorrhiza markers were negatively correlated to phosphate concentrations in the fertilization solution as a consequence of AM suppression. The expression pattern of the diagnostic marker for Pi-stress as well as of the Pi-signalling key regulators correlated to their described functions. These data deliver preliminary, but reliable data to support the hypothesis that the plant phosphorous status systemically influences AM development and suggest a link between plant Pi-signalling and signalling leading to AM formation.

References Benedito VA, Torres-Jerez I, Murray JD, Andriankaja A, Allen S, Kakar K, Wandrey M,

Verdier J, Zuber H, Ott T, Moreau S, Niebel A, Frickey T, Weiller G, He J, Dai X, Zhao PX, Tang Y & Udvardi MK. 2008. A gene expression atlas of the model legume Medi-cago truncatula. Plant J.

Chen A, Hu J, Sun S & Xu G. 2007. Conservation and divergence of both phosphate- and mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species. New Phytol 173: 817-831.

Farag MA, Huhman DV, Lei Z & Sumner LW. 2007. Metabolic profiling and systematic identification of flavonoids and isoflavonoids in roots and cell suspension cultures of Medicago truncatula using HPLC-UV-ESI-MS and GC-MS. Phytochemistry 68: 342-354.

Glassop D, Smith SE & Smith FW. 2005. Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots. Planta 222: 688-698.

Harrison MJ. 1999. Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis [Review]. Annual Review of Plant Physiology & Plant Molecular Biology 50: 361-389.

Harrison MJ, Dewbre GR & Liu J. 2002. A Phosphate Transporter from Medicago truncatula Involved in the Acquisition of Phosphate Released by Arbuscular Mycorrhizal Fungi. Plant Cell 14: 2413-2429.

Hause B & Fester T. 2005. Molecular and cell biology of arbuscular mycorrhizal symbiosis. Planta 221: 184-196.

Kuster H, Hohnjec N, Krajinski F, El YF, Manthey K, Gouzy J, Dondrup M, Meyer F, Kali-nowski J, Brechenmacher L, van Tuinen D, Gianinazzi-Pearson V, Puhler A, Gamas P & Becker A. 2004. Construction and validation of cDNA-based Mt6k-RIT macro- and mi-croarrays to explore root endosymbioses in the model legume Medicago truncatula. J Biotechnol 108: 95-113.

Maeda D, Ashida K, Iguchi K, Chechetka SA, Hijikata A, Okusako Y, Deguchi Y, Izui K & Hata S. 2006. Knockdown of an Arbuscular Mycorrhiza-inducible Phosphate Transporter Gene of Lotus japonicus Suppresses Mutualistic Symbiosis. Plant Cell Physiol 47: 807-817.

Nagy R, Karandashov V, Chague V, Kalinkevich K, Tamasloukht M, Xu G, Jakobsen I, Levy AA, Amrhein N & Bucher M. 2005. The characterization of novel mycorrhiza-specific phosphate transporters from Lycopersicon esculentum and Solanum tuberosum uncovers functional redundancy in symbiotic phosphate transport in solanaceous species. Plant J 42: 236-250.

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Paszkowski U, Kroken S, Roux C & Briggs SP. 2002. Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci U S A 99: 13324-13329.

Rausch C, Daram P, Brunner S, Jansa J, Laloi M, Leggewie G, Amrhein N & Bucher M. 2001. A phosphate transporter expressed in arbuscule-containing cells in potato. Nature 414: 462-470.

Sato S, Nakamura Y, Asamizu E, Isobe S & Tabata S. 2007. Genome sequencing and genome resources in model legumes. Plant Physiol 144: 588-593.

Schliemann W, Ammer C & Strack D. 2008. Metabolite profiling of mycorrhizal roots of Medicago truncatula. Phytochemistry 69: 112-146.

Smith SE & Read DJ. 1997. Mycorrhizal symbiosis. In, Ed 2. Academic Press, London, pp 347-376.

Udvardi MK, Kakar K, Wandrey M, Montanari O, Murray J, Andriankaja A, Zhang JY, Benedito V, Hofer JM, Chueng F & Town CD. 2007. Legume transcription factors: global regulators of plant development and response to the environment. Plant Physiol 144: 538-549.

Urbanczyk-Wochniak E & Sumner LW. 2007. MedicCyc: a biochemical pathway database for Medicago truncatula. Bioinformatics 23: 1418-1423.

Zhang K, McKinlay C, Hocart CH & Djordjevic MA. 2006. The Medicago truncatula small protein proteome and peptidome. J Proteome Res 5: 3355-3367.


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Posters


870 report (2008), page 67-68.

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Different transgenic Bt corn genotypes affect mycorrhizal colonization

Alessandra Turrini1, Cristiana Sbrana2, Manuela Giovannetti1 1Department of Crop Plant Biology, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy. 2Institute of Biology and Agricultural Biotechnology, CNR, Via del Borghetto 80, Pisa, Italy. [email protected]; [email protected]; [email protected] Introduction Plants genetically modified for resistance to pests represent a potential environmentally safe alternative to pesticides, decreasing chemical contamination of groundwater, soil and air. However, since the introduction of this new agrotechnology may pose risks to natural and agricultural ecosystems many studies were carried out to evaluate their impact on animal and microbial communities (Castaldini et al., 2005; Zangerl et al., 2001). A few data are available on interactions of transgenic plants with associated beneficial fungi, such as arbuscular mycorrhizal (AM) symbionts, fundamental for soil fertility, plant nutrition and ecosystems functioning (Smith & Read, 1997). Recent studies reported that both the transgenes and the transformation events may differentially affect the establishment of mycorrhizal symbiosis (Medina et al., 2003; Vierheilig et al., 1995). With the aim of developing an experimental model system for the evaluation of plant-fungus interactions, we assayed transgenic corn plant genotypes for their responsiveness to the AM fungus Glomus mosseae. Materials and Methods Three different stages of host-symbiont interaction were studied in two transgenic corn lines (Bt 11 and Bt 176) and in a non transgenic line using microcosm experimental systems (Cas-taldini et al., 2005): pre-symbiotic host recognition responses, formation of infection struc-tures and root colonization. Results and Discussion No significant differences were observed in differential hyphal morphogenesis and in total number of appressoria developed by Glomus mosseae in the presence of both Bt and control plants. However, many appressoria produced on Bt corn roots were not able to form infection units (Figure 1). Consistently, both transgenic plants showed decreased mycorrhizal colonization, with respect to control plants, after five, eight and ten weeks of culture (Figure 2). Data obtained showed that plant transformation events Bt 11 and Bt 176 affect the estab-lishment of mycorrhizal symbiosis and suggest that such “defective” transgenic plants may represent a useful tool for the study of the interactions between plants and their mycorrhizal symbionts.

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Figure 1. Abortive appressoria developed on Bt 176 corn roots.

weeks of culture

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Figure 2. Reduction of colonisation by the AM fungus Glomus mosseae in roots of transgenic corn plants, with respect to control. References Castaldini M et al. 2005. Appl. Environ. Microbiol. 71: 6719-6729. Medina MJH et al. 2003. Plant Sci. 164: 993-998. Smith SE & Read DJ. 1997. Mycorrhizal symbiosis. 2nd edn, Academic Press, London. Vierheilig H et al. 1995. Appl. Environ. Microbiol. 61: 3031-3034. Zangerl AR et al. 2001. Proc. Nat. Acad. Sci. 98: 11908-11912.


870 report (2008), page 69.

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Soil and inoculum infectivity evaluated during the early stage of mycorrhizal establishment

Luciano Avio1, Stefano Bedini2, Elisa Pellegrino2, Manuela Giovannetti2

1Institute of Biology and Agricultural Biotechnology, U.O. Pisa, C. N. R., Via del Borghetto 80, 56124 Pisa, Italy; 2Department of Crop Plant Biology, University of Pisa, Via del Bor-ghetto 80, 56124 Pisa, Italy, [email protected]; [email protected]; [email protected]; [email protected] Soil microbial community represents a vital factor for the functioning of agroecosystems. Among soil microbes, arbuscular mycorrhizal fungi (AMF) are fundamental for plant nutri-tion and health and their beneficial effects, including the enhanced P uptake by the extraradi-cal mycelium, are crucial for ecosystem productivity. Crop genotypes may show a high level of variation in AM fungal colonization and, because of the large genetic variability of AMF, the development of the symbiosis, in terms of extent and pattern of root colonization, depends on the different plant-fungal combinations (Turrini et al., 2004; Avio et al., 2006). Therefore, a rapid method to screen the compatibility of different varieties of crops with both indigenous AM fungal populations and introduced inocula may be useful for successful low-input agri-cultural systems. Here, we show preliminary results on a rapid test to evaluate fungal infectivity of soils, to be used in studies of plant-fungal compatibility. We assessed AMF root colonization and number of entry points during the early stages of symbiosis establishment in two orticultural crops: Cichorium intybus L. and Lactuca sativa L. The infectivity of organic, low-input and conventionally managed soils and of single and mixed AMF inocula was analysed 7 and 14 days after plant germination. Root colonization after 1 week’s growth was always lower than 0.3% for agricultural soils and lower than 4.2% for single or mixed inocula. After 2 week’s growth, colonization always increased to 4-12%, depending on soils. Entry points did not in-crease significantly from the first to the second week of growth and were always less than 4.5 cm-1 plant root in C. intybus and L. sativa. The number of entry points after one week’s growth revealed a high reliability for the evaluation of soil and infectivity and may be use to test large numbers of crop varieties for their ability to be colonized by AMF. References Turrini A et al. 2004. Plant and Soil 226: 69-75. Avio L et al. 2006. New Phytologist 172: 347-357.


870 report (2008), page 71.

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The effect of atmospheric change on arbuscular mycorrhizal fungi in agricultural systems T.E. Anne Cotton, Thorunn Helgason, Alastair Fitter Department of Biology, University of York, PO Box 373, York, YO10 5YW, United Kingdom, [email protected] Anthropogenic increases in atmospheric ozone and carbon dioxide (CO2) concentrations will affect crop root development patterns in time and space and the allocation to roots of photo-synthates on which arbuscular mycorrhizal (AM) fungi rely. These changes may alter AM fungal communities if different AM fungal species have different phenological or spatial pat-terns or carbon requirements. Any such shifts in community diversity and composition would have implications for crop growth if there were functional differences among AM fungal spe-cies. They are also likely to affect the success and suitability of using plants bred to form ef-fective AM symbioses and AM fungal inocula in agricultural systems in the future. At the SoyFACE experiment, Illinois, soybeans (Glycine max) are grown in normal agri-cultural conditions in the field but are exposed to predicted atmospheric conditions expected in 2050. Four different atmospheric treatments are applied to the crops: elevated CO2 (550ppm) with elevated ozone (1.2× ambient levels), elevated CO2 with ambient ozone, am-bient CO2 (380ppm) with elevated ozone and ambient CO2 with ambient ozone. Terminal restriction fragment length polymorphisms (TRFLPs) are being used to assess the community diversity and composition of AM fungi in the roots of these plants. Preliminary results from this experiment suggest that there is a change in the composition of the AM fungal commu-nity in response to the atmospheric treatments at SoyFACE. For example the O3 treatment appears to produce a shift towards more generalist AM fungal taxa. A larger study is now being conducted to further investigate the nature and direction of these changes.


870 report (2008), page 73-78.

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Potential of dual purpose intercrops for the management of plant-parasitic nematodes and beneficial mycorrhizal fungi in banana-based cropping sys-tems L. Van der Veken, P.P. Win, M. Lin, A. Elsen, R. Swennen, D. De Waele Laboratory for Tropical Crop Improvement, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 13, B-3001 Heverlee, Belgium, [email protected] Introduction When damage thresholds are exceeded, the plant-parasitic nematodes, Radopholus similis and Meloidogyne spp. can cause yield losses up to 70 % to bananas and plantains in poor soil con-ditions. Whereas chemical nematicide use is non-existing in subsistence cropping systems (87% of the world banana production), it is currently being restricted in commercial produc-tion systems due to serious environmental and health concerns. Therefore, further research on alternative sustainable nematode management strategies is becoming increasingly important. Though intercropping is common practice in subsistence banana-based cropping systems, knowledge about its effects on nematode field populations is rather scarce. Even less is known about the effects of the used intercrops on arbuscular mycorrhizal fungal (AMF) field inoculum. AMF are known to promote plant growth and provide a bio-protective effect against plant parasitic nematodes in banana(1). To study the potential of banana intercrops, 7 leguminous intercrops (pigeon pea, sunn hemp, Grant’s rattlebox, soybean, hairy indigo, common bean and cowpea) were screened individually for their nematode susceptibility and AMF compatibility in greenhouse conditions with inclusion of a susceptible and compatible banana cultivar (Musa AAA Grande Naine) as a reference crop. After identifying the promis-ing intercrops (nematode resistance and/or AMF compatibility), 3 leguminous intercrops with different levels of nematode susceptibility were studied in an interaction experiment and mixed greenhouse set-ups to study their effect on nematode population build-up and AMF inoculum potential. As such, promising intercrops for reduction of nematode population and enhancement of AMF field inoculum potential were identified. By including AMF-colonized intercrops we ultimately intend to address both biotic (nematodes) and abiotic (poor soil con-ditions) stress in the often low input banana-based cropping-systems. Materials and Methods Biological materials Seven leguminous intercrops (pigeon pea, sunn hemp, Grant’s rattlebox, soybean, hairy in-digo, common bean, cowpea) were selected based on their potential AMF compatibility, nematode susceptibility and prevalence in banana-based cropping systems. A susceptible ba-nana cultivar (Musa AAA Grande Naine) was included as a reference crop. All intercrops were grown for six weeks in 1-l pots in a 2:1 sand:potting soil mixture before colonising with AMF or inoculating with nematodes. After 8 weeks plants were analysed for AMF colonisa-tion or nematode susceptibility. A Glomus mosseae isolate, originating from a banana field in the Canary Islands, was used to determine the mycorrhizal response of the intercrops. Mycorrhizal inoculum consisted of a 50 g mixture of soil and roots collected from a 6-month old well-established AMF-sorghum pot culture.

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1000 vermiform stages of Radopholus similis (Ugandan population) originating from monox-enic carrot disc culture (Pinochet et al., 1995) were used to inoculate the intercrops in 1-l pots. Rhizobium etli CNPAF 512 (good nodulator of common bean), was cultured for 2 to 3 days at 28°C on a solid TY medium (Bittinger and Handelsman, 2000). After sterilizing the media, 1% of a sterilized 10 mM CaCl2.H2O solution was added to promote growth of the bacterial cell wall. This was followed by 1 day on a liquid TY medium at 28°C and 200 rpm. The optical density of the bacterial suspension was determined by measuring its absorbance at 600 nm wavelength using a spectrophotometer. The suspension was diluted in a sterile 10 mM MgSO4 solution to obtain a bacterial inoculum containing about 106 cfu/ml colony forming units per ml. One ml of this sus-pension was poured over the 5-day old tap rootlets, allowing any excess to drip into the planting hole. Nodule formation was assessed upon uprooting. Methods Pot screenings for the individual intercrops were performed in greenhouse conditions. Eight weeks after colonization or inoculation, the plants were uprooted, the roots washed with tap water and weighed. For microscopic observation, a 5 g root sub sample was stained using the ink and vinegar technique (Vierheilig et al., 1998) and ten 1-cm root pieces were mounted on a glass slide. Two slides per root were scored for frequency (F%) and intensity (I%) of mycorrhizal coloni-zation. F% was calculated as the percentage of root segments colonized by either hyphae or arbuscules or vesicles. I% was estimated as the abundance of hyphae, arbuscules and vesicles in each mycorrhizal root segment (Plenchette and Morel, 1996). Nematodes were extracted from a 5-g root sub sample using the maceration sieving tech-nique (Hooper et al., 2005). Two 2-ml sub samples were poured in a counting dish and the number of males, females, juveniles and eggs counted under a light microscope. Interaction experiments with common bean and three other intercrops were performed in greenhouse conditions. The four treatments (16 replicates in each case) were control, rhizobial (RHIZ+), AMF (AMF+) and dual colonization (RHIZ+/AMF+). Six weeks after colonization with Rhizobium sp. and/or AMF, 8 plants per treatment were inoculated with 1000 vermiform nematodes (NEM+) and 8 were left to grow in the absence of R. similis (NEM-). Results AMF compatibility Seven leguminous intercrops were tested for their AMF compatibility together with banana Grande Naine as a compatible reference crop (Table 1). Banana cv. Grande Naine was very well colonized (Table 1) with a very high F% (100) and intermediate I% (29). Two groups can be distinguished based on F% and I%: a group with intermediate mycorrhizal compatibility including soybean, Grant’s rattlebox, common bean, pigeon pea and sunn hemp (F%= 36-55 and I%= 18-24) and a group with higher my-corrhizal compatibility including hairy indigo and cowpea (F% > 70 and I% > 20).

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Table 1. Frequency (F%) and intensity (I%) of mycorrhizal colonization in 7-leguminous intercrops with different nematode susceptibility 8 weeks after colonization with Glomus mosseae, Banana cv. Grande Naine was included as a reference crop.

n F%+SE (%) I%+SE (%) Leguminous intercrops (experiment 2) Soybean (Glycine max) 8 36+0.7 a 18+0.3 c Grant’s rattlebox (Crotalaria virgulata) 7 44+1.0 b 24+0.4 e Common bean (Phaseolus vulgaris) 6 49+0.9 bc 16+0.2 b Pigeon pea (Cajanus cajan) 4 51+1.2 cd 11+0.2 a Sunn hemp (Crotalaria juncea) 8 55+0.6 d 24+0.3 e Hairy indigo (Indigofera hirsuta) 8 73+1.7 e 20+0.5 d Cowpea (Vigna unguiculata) 8 83+0.3 e 31+0.3 g Banana (Musa) Grande Naine 8 100+0.0 f 29+0.3 f

R. similis susceptibility Seven leguminous intercrops with intermediate AMF compatibility were tested for their nematode susceptibility (table 2). All intercrops were less susceptible to R. similis than banana Grande Naine. As such, the banana Grande Naine can be classified as a good host of R. similis. The susceptibility for R. similis differed among the intercrops tested. Hairy indigo was a non-host (Rr < 0.1) of R. similis. The Rr of Grant’s rattlebox, soybean and sunn hemp was lower than 1 and these intercrops can be classified as poor hosts of R. similis. Rr observed in cowpea, common bean and pigeon pea was significantly (P < 0.05) lower than in banana Grande Naine and these crops can be classified as intermediate hosts. Generally, intercrops with the lowest number of R. similis g-1 fresh root and Rr also had the lowest root necrosis index, while inter-crops with the highest number of R. similis g-1 fresh root and Rr had the highest root necrosis index. Table 2. Susceptibility for R. similis of seven leguminous intercrops, 8 weeks after inocula-tion with 1000 vermiform nematodes. Banana cv. Grande Naine was included as a reference crop.

n: number of replicates. Rr: Reproductive ratio (final nematode population/initial population). Plants followed by different letters in the same column differ significantly (P < 0.05) according to the Tukey test. a Root necrosis was scored as follows; 0: absence of lesions; 1: sporadic lesions and traces of infections in up to 5% of the root; 2: bigger coalesced lesions in 5-10% of the root; 3: necrotic root parts present in < 25% of the root; 4: 25% of necrotic root and clear lesions in the other roots, 5: 25-50% of necrotic root, 6: 50-75% of necrotic root and 7: >75% of necrotic root.

n No. R. similis g-1 fresh roots Rr Root necrosisa

Intercrops

Hairy indigo (Indigofera hirsuta) 7 17 a 0.05 a 1

Grant’s rattlebox (Crotalaria virgulata) 8 32 ab 0.13 a 2

Soybean (Glycine max) 8 32 ab 0.20 ab 1

Sunn hemp (Crotalaria juncea) 8 69 ab 0.50 ab 2

Cowpea (Vigna unguiculata) 8 33 ab 1.02 b 2

Common bean (Phaseolus vulgaris) 8 133 bc 1.30 b 3

Pigeon pea (Cajanus cajan) 7 85 bc 1.40 b 3

Banana (Musa) Grande Naine 8 229 c 10.10 c 3

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Interaction experiments Fresh shoot and root weight of the common bean plants from the different treatments was recorded 8 weeks after nematode inoculation, at harvest (14 weeks of plant growth). AMF colonized (AMF+) or dual colonized bean (RHIZ+/AMF+) plants grew the best followed by the rhizobial (RHIZ+) and control treatment. The 50% growth reduction in the control treatment due to nematode inoculation was absent in any other treatment. Overall AMF colonization in common bean was good (F% 99-100% and I% > 20%), with presence of arbuscles and vesicles in most of the observed root parts (Figure 1). For common bean a rela-tive mycorrhizal dependency1 (RMD) of (65.28%) was observed.

Figure 1. Phaseolus vulgaris roots colonized with Glomus mosseae (left to right: hyphae and spores, arbuscules and vesicles). The effect of rhizobial and AMF colonization on the R. similis population build-up, 8 weeks after nematode inoculation is depicted in Figure 2.

Mean R.similis density (nem/g)

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Figure 2. Radopholus similis density 8 weeks after inoculation in rhizobial (RHIZ+), AMF (AMF+) or dual inoculated (RHIZ+/AMF+) bean plants. Means with the same letter did not differ significantly according to Tukey’s Equal HSD test (P≤0.05) performed on log(x+1) transformed data. 1 RMD is determined by expressing the difference between the dry weight of the mycorrhizal plant and the dry weight of the non-mycorrhizal plant as a percentage of the dry weight of the mycorrhizal plants at specific soil conditions.

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b ab a

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When compared to the control treatment, a reduction in nematode population was accom-plished by single or dual colonization with beneficial soil organisms (BCO’s). However, no extra bio-protective effect was obtained with dual inoculation when compared to AMF colo-nization. No synergistic nor antagonistic effect from dual inoculation with Rhizobium etli and Glomus mosseae on the nematode density could be detected. Discussion All retained intercrops showed an intermediate AMF compatibility, when compared to banana Grande Naine. Since RMD can have a strong influence on the role of the AMF in mediating com-petition in an intercropping system, intercrops with intermediate RMD tend to improve the com-petitive advantageous for plant growth for the highly compatible crop (banana in our case) than when combing with highly AMF compatible intercrops (Yao, 2005). Therefore, we expect the intercrops with intermediate RMD to be suitable in our mixed banana-based cropping systems. However, further research in mixed set-ups is currently ongoing. Both BCO’s (Rhizobium and AMF) in the interaction experiment showed a bio-protective effect towards the migratory endoparasite Radopholus similis. However, once the common bean plants are colonized by AMF, no significant additional bio-protective effect could be detected from dual inoculation. The same was observed for plant growth parameters. Although the important role of rhizobacteria role in nitrogen acquisition in legumes is widely studied, the importance of AMF colonization for their growth promotion and bio-protective effect should not be neglected and deserves our further interest. Prospectives Research on the effect of integrating AMF colonized intercrops in mixed set-ups on the sys-tem’s AMF field inoculum is currently ongoing. In a second phase the effect on nematode population build up will be tested. Acknowledgements This research was made possible thanks to a VLIR scholarship to P.P. Win and M. Lin and a FWO postdoctoral fellowship to A. Elsen. We also like to thank all the seed suppliers and the Centre of Microbial and Plant Genetics (CMPG) of the Katholieke Universiteit Leuven (K.U.Leuven) for providing us with the intercrop seeds and the different bacterial strains. References Bittinger MA & Handelsman J. 2000. Identification of genes in the Rosr regulon of Rhizo-

bium etli. Journal of Bacteriology 6, 1706-1713. Hooper DJ, Hallman J & Subbotin S. 2005. Methods for extraction, processing and detection

of plant and soil nematodes. In: Plant-parasitic Nematodes in Subtropical and Tropical Agriculture, 2nd edition. Luc, M., Sikora, R.A. and Bridge, J.(eds). CABI Publishing, Oxfordshire 53-86.

Pinochet J, Fernandez C & Sarah JL. 1995. Influence of temperature on in vitro reproduction of Pratylenchus coffeae, P. goodeyi and Radopholus similis. Fundamental and Applied Nematology 18, 391-392.

Plenchette C & Morel C. 1996. External phosphorus requirements of mycorrhizal and non-mycorrhizal barley and soybean plants. Biology and Fertility of Soils 21, 303–308.

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Vierheilig H, Coughlan AP, Wyss U & Piche Y. 1998. Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Applied and Environmental Microbiology 64, 5004-5007.

Yao Q, Zhu HH, Chen JZ & Christie P. 2005. Influence of an arbuscular mycorrhizal fungus on competition for phosphorus between sweet orange and a leguminous herb. Journal of Plant Nutrition, 28, 2179-2192.


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Mycorrhizal colonisation on Allium schoenoprasum L. in peat-based substrates Siri Caspersen1, Mats Kron2 1Department of Horticulture, Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden, [email protected], 2Bara Mineraler, S-230 40 Bara, Sweden Peat-based substrates are commonly used for greenhouse production of vegetable transplants. Inoculation with AM fungi during transplant production may be desirable to improve plant nutrient uptake and establishment after transplantation to the field. Mycorrhizal formation, however, is sometimes low when plants are grown in peat-based substrates. The objective of the present experiment was to develop a substrate conducive to mycorrhizal establishment on chives (Allium schoenoprasum L.) seedlings. A factorial experiment with four substrate mixes, two phosphorus levels, two arbuscular mycorrhizal inoculants and four replicates was conducted. The peat was a lightly humified block peat and the substrate mixes contained sand:pumice 50:50 (v:v) or peat:pumice in the proportions 50:50, 75:25 or 87:13 (v:v). The AMF inoculants were mixed into the substrates at 5 vol%. The plants were grown in the greenhouse with additional light. When the plants were harvested at eight weeks after sowing, the roots were colonised with AM fungi in all substrates. Colonisation levels and plant growth will be presented and the differences between the substrates will be discussed.


870 report (2008), page 81-84.

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The influence of biofumigation on mycorrhizal fungi and growth of strawberries Darinka Koron Agricultural Institute of Slovenia, Hacquetova 17, 1000 Ljubljana, Slovenia, [email protected] Summary Green manure from Brassica plants reduced the micro organisms in soil due to biocidal ef-fect. Biofumigation with glucosinolates from Brassica juncea, Sinapis alba and Eruca sativa only partially influenced the mycorrhizal fungi of the strawberry cv. ‘Marmolada’. In the field trial the differences in mycorrhizal parameters were not statistically significant between treatments (Brassica juncea, Sinapis alba, Eruca sativa, dazomet, control). The highest fre-quency of roots with mycorrhizal fungi (F) was observed in the chemical treatment with da-zomet (50.3%). In the control, F was 49.2% and it was the lowest in the B. juncea treatment (43.4%). In pot trials, the average F was found in B. juncea, 57.9%, followed by S. alba, 54.9%, E. sativa, 66.8%, dazomet, 2.9%, and control, 60.8%. The average intensity of fungi infection (M) was found in B. juncea, 7.4%, then in S. alba, 9.8%, E. sativa, 7.5%, dazomet, 0.0%, and control, 7.1%. Differences in yield observed in the field trial were not statistically significant. In pot trials the number of fruits per plant was the highest in E. sativa (8.1), S. alba (7.0), B. juncea (6.9), control (3.7) and dazomet (1.8). Increase of the yield at the treat-ment with biocidal plants was the result of incorporation of organic matter into soil and undis-turbed activity of mycorrhizal fungi. Additional keywords: strawberry, biofumigation, Brassica, mycorrhiza, yield Introduction Biofumigation is the name to describe the effect of chemicals produced by Brassica green manure crop. It is a type of allelopathy, chemical inhibition of one species by another (McGuire, 2003). By incorporating green manure we increase organic mass in the soil and also add substances (glucosinolates) which influence the soilborne plant pathogens (fungi, nematodes) and weeds. Plants of the Brassica family influence in different ways the pathogen fungi Pythium (Charron and Sams, 1999; Gengotti et al., 2001; Bates and Rothrock, 2006), Rhizoctonia solani (Charron and Sams, 1999; Bates and Rothrock, 2006), Verticillium, (Gen-gotti et al., 2001), Phytophthora (Gengotti et al., 2001), Thielaviopsis basicola (Bates and Rothrock, 2006) etc. The effect of biofumigation is ranged between the effect of methyl bro-mide and that of the control. Biofumigation is one of the best alternative methods of fumiga-tion. The effect is dependent on the release of isothiocyanates (ITC) into the soil. Experiments with biofumigation were performed on a small number of soil microorganisms (Noling, 2002). Arbuscular mycorrhiza is an important part of microflora in soil. It is the link between plants and soil. Mycorrhizal fungi increase the efficiency of mineral uptake, especially of the immobile element such as phosphorus, they increase the water uptake, reduce plant stresses and disease response to plant pathogens due to some morphological or physiological changes in the plant. Mycorrhizal fungi induce changes in carbon and phytohormons (Linderman, 1986). Branzanti et al. (2002) measured mycorrhizal infection of roots of strawberries under

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different cultivation techniques (organic farming, low input, green manure of barley, green manure of B. juncea, fumigation with methyl bromide and the control). Mycorrhizal struc-tures (hyphae, arbuscules, vesicles) developed in all treatments, except in chemical fumiga-tion. In our project we observed the effect of biocid plants on the mycorrhizal infection of strawberry roots and growth and development of plants. Materials and Methods The project with strawberry cv. ‘Marmolada’ is based on a field trial and three consecutive pot trials. The field trial was conducted in the experimental field of Agricultural Institute of Slovenia at Brdo pri Lukovici. Pot trials were conducted in growing chamber, screen house and greenhouse of the same Institute. The field trial was conducted on heavy soil well sup-plied with potassium and nitrogen and poor with phosphorus. Organic matter was high, pH was optimal (pH - 6.0; P2O5 - 4.1 mg/100 g soil; K2O - 25.9 mg/100 g soil; organic matter - 3.4%; N - 0.27%). Field trial In the field trial we planted frigo plants. The trial was set in three random blocks with ten plants in each treatment. In spring (April), plots were sawn with biocid plants from Brassica family (Brassica juncea, Sinapis alba, Eruca sativa). Two weeks before planting strawber-ries, biocid plants were chopped and incorporated into the soil. After cultivating we prepared raised beds with black foil. Strawberries were planted on July 22, 2002. Chemical fumiga-tion with dazomet (Basamid -50 g/m2), was done 7 weeks before planting. Dazomet was in-corporated and the plots were covered with black foil. After two weeks the plots were uncov-ered. After tillage and cultivating the control plots were left to overgrow. The yield was measured every two to three days. In the first year (2003) the yield was picked between May 19 and June 20 and in the second year (2004) between June 1 and July 5. The intensity of mycorrhizal infection was evaluated by the Trouvelot method (1986) which is based on the estimation and disposition of roots in classes on the basis of typical mycorrhi-zal structures. The presence of fungus in roots was estimated by the frequency of root parts with mycorrhiza (F %), as intensity of infection in the root tissue and intensity of infection in colonized parts of root (M %, m %), as arbuscular frequency in the root tissue and arbuscular frequency in colonized parts of root (A %, a %). Pot trials In pot trials we planted micropropagated plants. Prior to the start of the trials (transplanting plants with green manure) plants were acclimatized in peat substrata for one month. Pot trials were conducted in three successive years (2003, 2004, 2005). The trials were set up in random groups with 15 plants in each treatment. They included B. juncea, S. alba and E. sativa, which were sawn in April 2003. The plants were cut before flowering in the phase when the quantity of glucosinolates was the highest. They were frozen (-20°C) and milled before adding to sub-strata. We planted plants in pots of 10 cm diameter. The substrata were made of 250 g of soil from field trial and 25 g of frozen biocid plant. With green manure 90 g/kg biomass was added. In the dazomet treatment we added 275 g fumigated soil (50 g/m2). The control con-tained soil from field trial. At the third pot trial (2005) the dazomet dose was 25 g/m2. My-corhization was evaluated in the same manner as in field trial. In the pot trials we valued the mycorrhization of ten plants in each treatment. We took samples of roots 7 weeks after plant-ing the trials. The growth potential of plants was measured with the number of fruits.

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Results and Discussion In the field trial the mycorrhizal infection in different treatments was not statistically differ-ent. The frequency of root parts with the fungus (F) was the highest in the chemical fumiga-tion with dazomet (50.3%). In the control it was 49.2% and it was the lowest in the treatment with B. juncea (43.4%). The number of pathogenic fungi per sample was the biggest in dazo-met (1.2), in B. juncea and in the control (0.1). There were no other differences between other parameters of mycorrhizal infection. From the results it can be concluded that in nature the biocid plants have no negative effect on the mycorrhizal fungi. In the pot trials the results were comparable only between the first two trials. In the third pot trial there were no differences in mycorrhizal parameters except in F where the trial with dazomet was statistically different than trials with biocid plants. We can conclude that plants after being frozen for a long time lost their biocid activity or that biocid plants really had no effect on mycorrhizal fungi. In the first two trials the differences in mycorrhizal parameters were bigger. In both trials mycorrhizal parameter in the treatment with dazomet deviates from others, except in the number of vesicles and in the number of pathogen fungi per sample. In the first pot trial con-sisting of tretaments with biocid plants F was higher than 90%, in the second pot trial F was 59% in B. juncea, in S. alba it was 47.3% and in E. sativa 80.3%. In the control in the first trial F was 82.3% and in the second pot trial it was 84.3%. In the first trial M was in biocid plants between 16.5% and 24.4% and in the control it was 10.7%. In the second pot trial M was between 3.5% and 4.4% and in the control it was 9.5%. Part of arbuscules at treatments with biocid plants in the first trial was between 4% and 12.9%, and in the control it was 5.5%. In the second trial the part of arbuscules at treatments with biocid plants was between 1.9% and 3.3% and in the control it was 7.6%. The average F in pot trials was 57.9% in B. juncea, 54.9% in S. alba, 66.8% in E. sativa, 2.9% in dazomet and 60.8% in the control. The average M was 7.4% in B. juncea, 9.8% in S. alba, 7.5% in E. sativa, 0.0% in dazomet and 7.1% in the control. The reason for differences in mycorrhizal parameters could be the result of different time of planting. The first pot trial was planted on July 15 2003. The samples of roots for my-corrhiza were taken in September. The second pot trial was planted in December 10, 2003. Samples were taken in February. Even though the plants grew in green house the season could influence the mycorrhizal infection of the plants. Because of deviations between trials we can not exactly determine which biocid plant was successful against pathogen fungi and tolerant against mycorrizal fungi. In 2002, Branzanti with co-workers found out that the highest mycorrhizal infection in strawberries (50% - 70%) was reached in May. Arbuscules (A) was the highest at the time of flowering (25%). At the same time M was about 42%. At the harvesting time M was the high-est (64%) and A was lower than at flowering (20%). Mycorrhizal infection of strawberries oscillates in different seasons, independent of technology, except fumigation, which obstructs the development of mycorrhizal fungi. After harvesting the value of A and M begin to fall. With regard to the results of Branzanti in our field trial we took the samples for mycorrhizal infection too late. They compared different types of technologies and they found out that M at organic production was about 25%, at low fertilization it was 6%, at green manure with barley 14% and at green manure with B. juncea 8%. In the treatment with chemical fumigation there was no mycorrhizal infection. In the field trial there was no difference in yield between treatments. In both years the highest yield was obtained in the treatment with S. alba. In the first pot trial the highest yield was reached in the treatment with S. alba and in the second and third pot trial the yield was

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the highest in the treatment with E. sativa. In the first and second pot trial in the treatments with dazomet we almost had no fruits, but in the third pot trial with dazomet we had higher yield than in the control. In the average of all pot trials, the highest yield was achieved in the treatments with E. sativa. It was two times higher than in the control. In experiment of Particka and Hancoock (2005) on plots treated with methyl bromide the yield of strawberries was between 33 and 46% higher. Also, the number of crowns was higher. But the mass of fruit was higher on non-fumigated plots, but only for 10%. Lazzeri with co-workers (1999) conducted an experiment with biocid plants. They found out that the yield was the highest with methyl bromide (472 g/plant) followed by B. juncea (406 g/plant), barley (334 g/plant) and control (228 g/plant). The reason for high yield in our pot trials is also high organic mass which was incorpo-rated in soil with biocid plants. In the field trial the effect of organic mass may have been lower due to very intensive natural microbiological activity. Also, the incorporated mass in the field trial was three times lower than in the pot trials. Biomass in the first pot trials did not affect the number but the mass of strawberry leaves, which was bigger in the treatments with biocid plants than in the control. Trials conducted in frame of our project indicate the importance of organic mass with regard to plants and microorganisms in the soil. Glucosinolates released from B. juncea, S. alba and E. sativa had no negative or small effect on microorganisms in the soil. The final effect of green manure of biocid plants was indicated in intensive growth, higher yield and in healthier strawberry plants. References Bates GD & Rothrock CS. 2006. Use of high glucosinolate Indian mustard cover crops to

suppress soilborne pathogens of cotton, Phytopathology 96 (6), S10. Branzanti MB, Gentili M, Perini R, Neri D & Cozzolino E. 2002. The Mycorrhizal Status of

Strawberry Plants under Different Pre Planting and Cultivation Techniques in the Field. Acta Horticulturae 567, 495-489.

Charron CS & Sams CE. 1999. Inhibition of Pythium ultimum and Rhizoctonia solani by Shredded Leaves of Brassica Species. J.Amer.Soc.Hort.Sci. 124 (5), 462-467.

Gengotti S, Tisselli V, Lucchi C & Nasolini T. 2001. La coltivazione della fragola in biologico, L’Informatore Agrario 27, 45-48.

Lazzeri L, Manici LM, Baruzzi G, Malaguti L, De Paoli E & Antoniacci L. 1999. Primi risultati sull’azione dei sovesci di piante biocidi nella coltura della fragola, Frutticoltura 6, 20-26.

Linderman 1986. Managing rhizosphere microorganisms in the production of horticultural crops. HortScience 21, 1299-1302.

McGuire A. 2003. Green Manuring with Mustard, http://www.aenews.wsu.edu/June03AENews/June03AENews.htm

Noling JW. 2002. The Practical Realities of Alternatives to Methyl Bromide: Concluding Remarks. Phytopathology 12, Vol. 92, 1373-1375.

Particka CA & in Hancock JF. 2005. Field Evaluation of Strawberry Genotypes for Tolerance to Back Root Rot on Fumigated and Nonfumigated Soil, J.Amer.Soc.Hort.Sci. 130(5), 688-693.

Trouvelot A, Kough JL & Gianinazzi-Pearson V. 1986. Measure du taux de mycorrhiazaon VA d’un système radicularie. Recherche de Mèthodes d’estimation ayant une significa-tion fonctionnelle. In Physiological and Genetical Aspects of Mycorrhizae (Gianinazzi-Pearson, V., Gianinazzi, S., eds. INRA, Paris, 217-221.


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Agroecological management of soil borne diseases in tropical horticultural crops by biocidal plants: hypothesis and chosen methodologies Paula Fernandes1, Peninna Deberdt1, Alain Soler2, Christian Chabrier2, Laurent Thuries3 1CIRAD – Persyst Horticulture – BP 214 – 97285 Lamentin cedex 2 – Martinique, France [email protected], 2CIRAD – Persyst Bananes Plantains Ananas – BP 214 – 97285 Lamentin cedex 2 – Martinique, France, 3CIRAD – Persyst Recyclage et risque - Station de la Bretagne - BP 20 - 97408 Saint-Denis Messagerie Cedex 9 – Réunion, France Summary In Martinique, horticultural crops are likely to be attacked by many soil borne diseases among which major ones are emerging strains of Ralstonia solanacearum that generate high eco-nomical losses in cucurbits and solanaceae crops. Simultaneously, nematodes like Meloi-dogyne sp, Pratylenchus coffeae and Rotylenchulus reniformis constitute the second group of soil borne pests that impact vegetables (tomato, pepper, cucumber, melon, watermelon…), root and tuber crops (yam, carrots) and pineapple. Because of the absence of sustainable chemical solutions, and as horticultural farms ex-tend usually on small areas, long term rotations with non susceptible crops are not adoptable by local farmers. An enquiry showed that introducing “service” crops with biocidal properties could be an adoptable alternative, provided that these plants fulfil some conditions (easy to install and to destroy, short term cycle…). In order to obtain an agroecological method to control these soil borne diseases, a re-search program is established to evaluate the efficiency of biocidal plants to be introduced in agroecological cropping systems. The main hypothesis is that by emphasising the cultivated biodiversity through these plants, we could (i) reduce the populations of the targeted soil borne pathogens with biocidal compounds that exudate from roots during the growing phase, and (ii) stimulate other microbial communities that could be antagonists. Both phenomenons may lead to the acquisition of general suppressiveness, mainly during the phase of decompo-sition of the service crop. It is however necessary to verify the non-toxicity of these plants and their compounds on useful microbial populations.


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List of participants Birgit Arnholdt-Schmitt University of Évora ICAM, 7002-554 Évora, Portugal [email protected] Luciano Avio CNR-IBBA U.O. di Pisa c/o Dipartimento di Biologia delle Piante Agrarie-Microbiologia Via del Borghetto, 80 - 56124 Pisa, Italy [email protected] Jacqueline Baar ARCADIS Nederland BV Beaulieustraat 22, 6814 DV Arnhem, The Netherlands [email protected] Stefano Bedini Department of Crop Plant Biology Via del Borghetto, 80, 56124-Pisa, Italy [email protected] Siri Caspersen Swedish University of Agricultural Sci-ences, Horticulture, Box 44, SE 230 53 Alnarp, Sweden [email protected] Nicholas Clipson University College Dublin, School of Biology and Environmental Science, Ardmore House, Belfield, Dublin 4, Ireland [email protected]

Anne Cotton University of York, Department of Biol-ogy, Area 14, PO Box 373, YO10 5YW York, United Kingdom [email protected] Tim Daniell Soil Molecular Ecologist SCRI, Invergowrie Dundee, DD2 5DA, United Kingdom [email protected] Victoria Estaun IRTA Protecció Vegetal, Ctra de Cabrils, km2, E-08348 Cabrils Spain [email protected] Paula Fernandes PRAM-CIRAD Horticulture BP214 97232 Lamentin cedex 2 Martinique, FWI, France [email protected] Armelle Gollotte INRA, GRITT Agro-Environment 17 rue de Sully, 21065 Dijon Cedex, France [email protected] Sonja Graugaard University of Aarhus Faculty of Agricultural Sciences, Depart-ment of Integrated Pest Management, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark [email protected]


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Milan Gryndler Institute of Microbiology ASCR, Division of Ecology, v.v.i., Videnska 1083, CZ14220, Prague 4, Czech Republic [email protected] Mette Grønlund Technical University of Denmark Biosystems Department, Risø National Laboratory for Sustainable Energy, Frederiksborgvej 399, Building 330, PO Box 49, DK-4000 Roskilde, Denmark [email protected] Meriel G. Jones University of Liverpool School of Biological Sciences, Biosciences Building, Liverpool L69 7ZB United Kingdom [email protected] Shawn Kaeppler University of Wisconsin Department of Agronomy 455 Moore Hall, 1575 Linden Drive Madison, WI 53706, USA [email protected] Yoram Kapulnik Agronomy and Natural Resources, Bet Dagan, 50250, Israel [email protected] Darinka Koron Agricultural Institute of Slovenia Hacquetova 17, 1000 Ljubljana, Slovenia [email protected] Franziska Krajinki MPI of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam, Germany [email protected]

John Larsen University of Aarhus Faculty of Agricultural Sciences, Depart-ment of Integrated Pest Management, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark [email protected] Peter Moutoglis BioSyneterra Solutions Inc., 801 route 344, PO Box 3158, J5W 4M9, L’Assomption, Quebec, Canada [email protected] Fritz Oehl University of Basel, Institute of Botany, Hebelstrasse 1, 4056 Basel, Switzerland [email protected] Michail Orfanoudakis Aristotele University of Thessaloniki, School of Forestry and Natural Environ-ment, Forest Soil Lab., PO Box 271, 54124 Thessaloniki, Grece [email protected] Sabine Ravnskov University of Aarhus Faculty of Agricultural Sciences, Depart-ment of Integrated Pest Management, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark [email protected] Stephen Rolfe University of Sheffield Department of Animal and Plant Sciences, Sheffield, S10 2TN, United Kingdom [email protected] Ian R. Sanders University of Lausanne Department of Ecology and Evolution Biophore Building, 1015 Lausanne, Switzerland [email protected]


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Carolin Schneider Institut für Pflanzenkultur Solkau 2, 29465 Schnega, Germany [email protected] Peter Schweiger Bio Forschung Austria Rinnböckstr.15, 1110 Wien, Austria [email protected] Fraukje Steffen ARCADIS Nederland BV Beaulieustraat 22, 6814 DV Arnhem, The Netherlands [email protected] Katarzyna Turnau Jagiellonian University Department of Ecological Microbiology Institute of Environmental Sciences Gronostajowa 7, 30-387 Krakow, Poland [email protected] Lieselot Van der Veken Faculty of Bioscience Engineering, De-partment of Biosystems – Division of Crop Biotechnics, Laboratory of Tropical Crop Improvement, Kasteelpark Arenberg 13 Bus 2455, BE-3001 Heverlee, Belgium [email protected] Mauritz Vestberg MTT Agrifood Research Finland Plant Production Research, Laukaa Antinniementie 1 FI-41330 Vihtavuori Finland [email protected]

1 COST870, May 2008, Køge, Denmark

A report of the Working Group 1 sessions at the COST 870 meeting Denmark 28 – 30 May 2008.

Plant Breeding and Colonisation by AM Fungi

The chair of the sessions was Yoram Kapulnik. During the two sessions (Wednesday afternoon, Friday morning) there were five lectures from invited speakers following by a discussion. The objective was to obtain current views on mycorrhiza in plant breeding and prospects for further developments. Nicholas Clipson from University College, Dublin, Ireland, set the scene for the transition from natural to agricultural systems through reviewing the impact of molecular techniques on the otherwise undetectable soil microbial communities. The change from reliance on culture-based studies to ones utilising environmental nucleic acids has caused profound changes to microbial ecology. Using primarily examples from the work of his own research group, he illustrated how several molecular analysis techniques contribute to community fingerprints including denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment polymorphism (TRFLP) and automated ribosomal intergenic spacer analysis (ARISA) as well as cloning and sequencing. The change from a diverse natural flora to the more limited biodiversity in arable or agricultural grassland affects the soil microflora. The soil nutrient status is also affected by agricultural practices with significant consequences for the microbial community. Taking unimproved and improved grassland in the Wicklow Mountains, Ireland, as a case study, studies from his group into both bacterial and fungal communities showed a complex diversity. At present, the relative roles of soil and plant species diversity in determining this underground community are unclear but are of obvious importance in the context of applications of AM fungi within agriculture. Birgit Arnholdt-Schmitt, University of Évora, Portugal approached the question of breeding for an efficient plant genotype – mycorrhizal interaction from a candidate gene approach. She also gave a very thoughtful evaluation of whether the whole question of breeding for efficient AM relationships was sensible. She considered that it was a valuable approach because plants with this characteristic were also likely to contain diverse other attributes for abiotic stress tolerance such as more root hairs, exudates and improved phosphate transport. An important agronomic aim will always be to maintain yield stability through robust, stress tolerant plants. Molecular breeding requires suitable markers, and hypotheses derived from ecophysiological and physiological studies can suggest relevant genes. The research of her group focuses on plant cell reprogramming under stress, and especially the role of the alternative oxidase (AOX) as an important functional marker because of its potential for energy re-direction. Expression of members of this gene family in plants is known to be responsive to development and stress, and the alternative oxidation pathway is also important in fungi. One line of research being


followed by her research group is therefore the role of AOX in mycorrhizal colonisation. Shawn Kaeppler from the University of Wisconsin, USA, gave a plant breeder’s viewpoint of the role of arbuscular mycorrhiza within breeding for stress-tolerant maize. The commercial objective with this commodity crop is to produce widely-adapted genotypes that can be grown across the diverse climates, microbial communities and soil-types of the USA and, ideally, the world. Unlike wheat, inbred populations of maize have only been available from the 1930s and there is therefore still much more genetic diversity within commercial varieties. His research has concentrated on genes and genotypes with potential utility in improving phosphate stress tolerance and has thus included consideration of root processes and architecture, and interactions with arbuscular mycorrhiza. There is a physiological cost to root architecture. Deep rooted maize varieties are generally more resistant to drought and mycorrhiza may assist with phosphate provision. Shallower-rooted varieties have better access to mineralisation and the microbial community in the warm surface layers early in the season but are more susceptible to drought. Using results from a study of maize genotypes and five species of mycorrhizal fungi, he showed that mycorrhiza can increase maize growth in low phosphate soil while has little effect at high phosphate. These overall results conceal substantial varietal variation in phosphate efficiency and mycorrhizal responsiveness that are especially apparent at low phosphate. There has not been selection against AM responsiveness in maize and varieties with high responsiveness are in the pedigrees of important US commercial cultivars. One very striking feature was that there was no correlation between the mycorrhizal colonisation of the maize plants and their responsiveness. This implies that there is no need to maximise fungal colonisation when selecting for plants with efficient mycorrhizal association. Investigating the fungal component further showed that different species, and indeed different isolates of one fungal species, had different growth-promoting effects. Stephen Rolfe from the University of Sheffield, UK, continued discussion of maize with an analysis of the role of mycorrhizas in nutrient acquisition by European and African maize varieties. He pointed out the financial value of world trade in cereals and particularly the need for maize imports to the world’s poorest countries, where it is also grown in low input subsistence farming. Important European and African varieties might therefore be expected to differ in their nutrient use characteristics and in response to infection by arbuscular mycorrhizal fungi. Work from his group has shown they do not, but there are traits in both that might be advantageous under different nutrient conditions. The experimental design provided plants with a fresh small supply of phosphate every day and compared growth in the presence and absence of AM fungi using morphological, physiological and gene expression measures. Rather surprisingly, the European variety River accumulated more biomass than the African variety H511 when mycorrhizal. The most significant difference between the two varieties, however, was in the responsiveness of root-shoot ratio to phosphate supply and AM infection. To continue an idea from the previous speaker, the mycorrhizal infection may ‘rescue’ plants with low phosphate supply. The overinvestment in a large root system of H511 is beneficial in water as well as nutrient supply while specific


phosphorous uptake is greater in River, although it is also more sensitive to repression. These indicate traits that might be advantageous under low phosphate conditions if they could be brought together. Franziska Krajinski from the Max Planck Institute of Molecular Plant Physiology, Germany, brought discussion to the molecular level with an outline of the current state of knowledge of plant genes involved in AM symbiosis. Her research group uses Medicago trunculatula as a model for global studies using microarrays. She outlined technical difficulties with these studies, including the currently intractable nature of the Glomeromycota genome and the non-synchronous nature of AM root infections. Nevertheless, her group has successfully obtained large data-sets of AM-induced genes through a combination of methods. Many have unknown functions and the future goal is to investigate these, especially if they are transcription factors. The approaches will include the effect of RNAi knockouts, monitoring the time-course of expression and reporter-gene studies. The links between phosphate signalling and mycorrhizal development are also receiving attention and microRNAs are now know to be involved in the signalling pathways, adding a further layer of regulatory complexity. Another research focus of her group uses the fungal pathogen Aphanomyces euteiches. Although this causes a root rot disease resulting in significant losses in pea and clover production, it is biotrophic during early infection. The mycorrhizal fungus Glomus intraradices has a bioprotective effect and reduces root loss. Proteomic analyses indicated that a group of PR10 proteins correlated specifically with the infection level. Counter-intuitively, knock-down of members of this family increased plant tolerance of A. euteiches. Following these presentations, a Discussion, involving the speakers and members of WG1 and 3, brought together themes, centring on the following points:

• Is it easy to pick a plant trait that correlates with mycorrhization? o We need to view the whole process to get real improvements o Plant breeders usually cannot consider component traits because of the

need for varieties with an excellent performance across many seasons and locations

o We do not yet have good tools to study the fungal side of interactions o The development of good functional markers for effective fungal

involvement in mycorrhiza is important o Plants and fungi have substantial developmental plasticity, so there is

undoubtedly more than one solution to developing or selecting plants with efficient mycorrhization

o Regulatory requirements regarding phosphate use, and regulatory or market-place driven requirements for sustainable agriculture could impact on plant breeding trends

• Should fungal inocula provide one or many strains or species? o There is substantial variability from place to place in the field o Microbial communities contain bacteria as well as fungi, and in the

field situation all are probably important


o Root exudates provide nutrition for the microbial communities and are an under-researched area

o The persistence of fungi within soil also warrants more attention and is being addressed by WG2.

• A ‘bottom-up’ approach via molecular biology can also lead to useful information.

o The relevance and importance of molecular studies can be difficult to understand for those seeking ‘real-world’ applications.

o Bridges between molecular and ‘whole organism’ approaches need to be fostered. This meeting was an excellent example of this.

o There also needs to be an exchange of ideas and understanding between plant breeders and researchers and fungal biologists. The sessions in WG1, WG3 and the field visit, were valuable in bringing together researchers who would not otherwise meet so readily.


Report from the Working Group 3 sessions at the COST 870 meeting Denmark 28 – 30 May 2008. Bottlenecks for implementation of AM in plant production Chair and reporter of the session was respectively, Mauritz Vestberg and John Larsen. During the session nine lectures were presented by invited speakers, which were followed up by a general discussion. The objective of the session was to identify important bottlenecks for implementation of AM in plant production and prospects for further developments. Tim Daniell (SCRI Dundee, Scotland) gave a presentation on impact of agricultural practices on AMF communities and within this context presented results from field experiments on host preference of AMF and effects of AMF inoculation on AMF communities. In addition, different methods to quantify AMF were compared including molecular, biochemical and conventional staining techniques. T-RFLP analyses based on two AMF primers were employed to measure AMF communities in roots. Results from field experiments at SCRI showed that AMF communities differ between crops, but differently at a seasonal time scale. The differences obtained were due only to a few T-RF types and driven by relative abundance not presence-absence. Results from another field experiment at NIBGE in Pakistan showed that AMF inoculum (field produced) and a bacterial inoculum (Biopower) affected the AMF community when applied to mung bean. However, dual inoculation counteracted this effect. Also seasonal effects were found. A RT-PCR based approach to quantify AMF in roots was presented. RT-PCR quantification of AMF showed no correlation neither with conventional percent root length colonization nor amount of the biomarker fatty acid 16:1w5. RT-PCR based quantification of AMF was suggested to reflect the level of the active part of the AMF root colonization. Armelle Gollotte (CRITT AgroEnvironment, France) presented results from a series of experimental work all aiming at integrating AMF in raspberry production. Application of AMF biotechnology in raspberry may improve plant quality including increased levels of health promoting antioxidants and reduce the development of important root diseases such as Phytophthora fragariae. A prospect for AMF application was presented including: assessment of inoculum quality, when to inoculate, how to optimise growth conditions for AMF and persistence of AMF. Also a PCR based method to assess plant sanitary status including both AMF and root pathogens were presented. PCR based detection of AMF seems promising as a tool to assess quality of AMF inoculum. Using nested PCR it was possible to detect both AMF and the root pathogen P. fragaraie, which is also useful in assessment of plant sanitary status, before transplanting to the field. Inoculation with AMF can be performed at different phases in the production cycle both at the in-vitro micropropagation stage, when transplanted for the acclimatization phase or when transplanted to the field. It is suggested to inoculate transplants for the acclimatization phase using commercial pot-culture based AMF inocula. Alkalinized moss peat amended with organic matter using with low P levels was compatible with AMF. Persistence of AMF in the field was found to be at least two month after transplanting strawberry with AMF inoculum to the field.


Katarzyna Turnau (Jagiellonian University, Poland) presented results on the possible use of photosynthesis parameters in optimization of Arnica montana cultivation and selection of AM inoculum. Photosynthesis parameters were used as a sensitive response measure of A. montana when inoculated with different AMF species (both single and mixed) and different combinations of soil, sand and expanded clay in the growth media. In the first experiment the mycorrhiza inoculation failed and coincided with no response in the measured photosynthesis parameters. High level of N and P reduced mycorrhiza establishment. The tested AMF differed in their ability to increase plant photosynthesis. The AMF Glomus geosporum and G. constrictum gave the highest increase in photosynthesis, whereas G. intraradices less effective in increasing plant performance. Arbuscule richness, in general, correlated negative and positively, with plant performance and photosynthesis parameters, respectively. Overall, both nutrient levels in the growth substrate and AMF species/isolate were presented as important parameters when optimizing A. montana cultivation. Peter Schweiger (BioForschung Austria) presented results from a pot experiement on the compatibility between spring barley varieties and field populations of AMF in terms of root colonization. The work was prsented in an organic farming context with the research question whether there is a need to consider AMF in organic breeding programmes. Results from a field experiment with different varieties of spring barley gave no difference in AMF root colonization, but the site was highly inhomogeneous why a pot experiment with field soil was conducted showing a high variation in AMF root colonization between barley varieties. It was concluded that variation in AMF root colonisation of barley do exist but that priority of increasing AMF root colonisation as a breeding target was considered small. Furthermore, barley is a generally ‘low-responsive‘ AM plant so should AMF in barley be ignored? Mauritz Vestberg (MTT Agrifood, Finland) gave a summary from several projects on the performance of AMF in peat substrates. In two field experiments with different crops peat amendment reduced mycorrhiza performance in terms of both root colonization and number of spores in soil, but the diversity of AMF was unaffected. Peat reduced the effectiveness of indigenous AMF on growth of strawberry in organic soil but in the opposite way in conventionally managed soil. Mycorrhizal effectiveness was slightly increased due to peat application in rye and buckwheat, of which the latter is a non-mycorrhizal crop. Results from a series of pot experiments with daisy as the host plant examining performance of AMF in different peat types and peat additives were presented. AMF root colonization was low in light peat except in combination with a clay additive. In general, AMF root colonization was highest in dark peat. It was concluded that peat is inhibiting performance of AMF, but the reason remains unclear. However, different ideas were presented including, high P levels, antagonistic microflora and toxic chemical components. Jacqueline Baar (ARCADIS, Netherlands) gave a presentation on new approaches to improve AMF applications in sustainable agriculture and landscape restoration. High P levels in most agricultural soils in the Netherlands were identified as one of the major constraints of commercial application of AMF and also high P levels is hampering the transition process of P-enriched systems into low-level nutrient systems. ARCADIS has developed a novel methodology aiming at reducing soil P levels and increase soil pH. Also ARCADIS consider knowledge transfer to the potential users of AMF and the first results suggest a growing interest in AMF.


Milan Gryndler (Czech Academy of Science, Czech Republic) gave a presentation on how AMF are affected by organic matter transformation. Pyrolysis products from the decomposition process from different types of organic matter both simple (cellulose) and complex (plant necromass) affected AMF performance. AM hyphal growth correlated positively with several of the pyrolysis products and especially 3,4,5-substituted benzyl structures gave high correlations. The results show that a relative small amount of organic matter can affect growth of AMF. It was concluded that the soil organic matter complex is important for the performance of AMF and that pyrolysis may be a useful method to study preference of particular AMF for particular soils. Such information may be useful to predict the suitability of a particular AMF for a certain soil or substrate. Fritz Oehl (University of Basel, Switzerland) presented results from a project studying whether Central European agricultural soils can be characterized by their AMF population. Soil samples were collected from different geographical locations differing in soil type and land use intensity (agricultural land and natural grass land). AMF spores from the soil samples were identified based on morphology. It was concluded that both AMF communities and certain AMF species are characteristic for certain soil types and land use management. Michail Orfanoudakis (Aristole University of Thessaloniki, Grece) gave a presentation based on results from a field experiment evaluating two different commercial mycorrhzal inocula at Mediteranean agricultural fields using leek as the test plant. The results showed that a Glomus intraradices inoculum increased yield in Marl soil, whereas Gigaspora margarita based inoculum gave an increased yield in alluvial soil. It was concluded that the efficacy of AMF inoculum on plant growth vary with soil properties both chemical/physical and biological. General discussion Following the above-mentioned presentations the speakers and members of WG1 and WG3 had a joint discussion on bottlenecks for implementation of AMF in plant production. During the discussion several bottlenecks were identified and general ideas and take home messages are listed below: Proof of concept The apparent lack of a proof of concept in AMF application seems to be one of the main bottlenecks. In order to achieve a proof of concept more focus should be given on validation and demonstration projects at the respective application areas both agricultural and horticultural. Proof of concept is important both when managing natural field populations of AMF and when applying AMF inoculum. It is important to give firm evidence for claimed beneficial traits of AMF in plant production. Phosphorous High phosphorous levels in soil and horticultural growth substrates were considered as one of the major constraints of AMF application. Different approaches to reduce P levels were discussed. Also it was noted that in some cases inoculation with AMF in high P soils have resulted in increased yield. In addition if the desired trait is plant protection the perhaps high P is not a problem as long as AMF root colonization can be achieved.


Peat Peat clearly reduces AMF performance and seems to be an important bottleneck for implementation of AMF in horticulture. However, it is important to note that even a low AMF root colonization can be sufficient to obtain the desired AMF traits. Clearly more research is needed to understand the suppressive nature of peat on AMF. Screening for plant beneficial functional traits of AMF Most research on AMF functional traits such as plant growth promotion and plant protection against stress only include a few species and isolates. Also at the commercial scale use of screening strategies are limited. This is in strong contrast to other commercial biological products where a high number of microorganisms are included in the screening programmes. However, the biotrophic nature of AMF makes screening difficult. New in-vitro whole plant model systems may help to overcome this problem. Plant growth promotion is the key functional trait of AMF, but AMF may have a marked concerning other beneficial traits such as content of bioactive compounds in the host plant, biotic and abiotic stress alleviation, flowering etc. New name From a sales point of view the name of a product is important and “arbuscular mycorrhizal fungi” is a difficult name. An alternative popular name may improve interest in AMF and marked access. AMF as a part of a broader concept AMF are not expected to solve all problems in plant production, land restoration etc., but AMF could be presented as one of the key components in a multiple integrated approach in biological management of plant production systems including both agricultural and environmental aspects. Knowledge transfer Another important bottleneck seems to be transfer of knowledge from science to growers. In some countries grower associations have consultants to support such knowledge transfer. Other possibilities could be to make “growers mycorrhiza schools” and to focus more on writing papers for applied popular local journals.

from production to application of arbuscular mycorrhizal fungi in

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