microscopy techniques to analyse plant-pathogen ...formatex.info/microscopy4/1108-1114.pdf ·...

Microscopy techniques to analyse plant-pathogen interaction: Fusarium

graminearum as pathogen of wheat.

Teresa M. Alconada Magliano and Gisele E. Kikot

Research and Development Center for Industrial Fermentations (CINDEFI), UNLP; CCT-La Plata, CONICET,

School of Science, La Plata National University, La Plata, Argentina

When a pathogenic fungus colonizes and infects a plant, a series of biologic processes begins, being modified the integrity

of the plant, as a result of the hyphal growth through cells, tissue and organs. From wild type or genetically modified

organisms, several aspects of infection can be studied, such as hyphal growth, morphogenesis in germinating spores,

visualization of enzymatic hydrolysis, production of mycotoxins, inhibition of the hyphal growth by antagonist

microorganisms, use of natural substances or by modification of the host resistance, as well as transcriptoma analysis and

expression of genes that regulate the infection. Three of the world’s most important crops, wheat, rice and maize, are

susceptible to a number of Fusarium species. Fusarium head blight (FHB) on small-grain cereals is one of the most

devastating diseases. Severe epidemics have occurred all over the world, altering quality parameters of grains, their

weight, carbohydrate and protein composition and they can result in contamination with fungal toxins such as

deoxynivalenol (DON). Several species can cause head blight, though Fusarium graminearum is the predominant

pathogen in most regions. F. graminearum is one of the most intensively studied fungal plant pathogens, therefore

different aspects have been studied: pathogenicity, population genetics, evolution and genomics. Diverse methodologies

are applied to analyze these aspects, being microscopic techniques the tools that complement other methodologies,

allowing the observation from bio-images of the changes produced. In this report, a review of microscopy techniques

applied in the interpretation of interaction F. graminearum-wheat crop will be carried out.

Keywords: Pathogenic fungi, Fusarium graminearum, wheat crops, disease, microscopy techniques.

Introduction

Fusarium head blight (FHB) of wheat (Triticum aestivum) is one of the most destructive diseases in humid and semi-

humid wheat-growing areas. Epidemics of FHB result in a significant yield reduction and low quality of grains, but also

in contamination of grains with mycotoxins produced by pathogens [1-3]. The mycotoxins have proved to be harmful to

human and animal health. Different Fusarium species may cause this disease, but the predominant species is F.

graminearum [4]. F. graminearum is one of the most intensively studied fungal plant pathogens [5]. In the study of the

interaction F. graminearum-wheat, diverse aspects have been investigated to understand the progress of the infection

and to control the disease, such as morphogenesis in germinating macroconidia, degradation of cell wall, production of

enzymes and mycotoxins, defense response of wheat and control of disease by natural substances and antagonist

microorganisms [6-11]. It is worth mentioning that microscopy is a valuable and essential tool for the analysis of

different approaches that involve the study of the plant-pathogen interactions. In this report, microscopic techniques

applied to the study of relevant issues involved in the infection of wheat by F. graminearum are detailed.

Fungal infection and disease progression

FHB is a floral disease, being able to infect wheat florets from the time of anthesis to the soft dough stage of kernel

development [12]. The ability of F. graminearum macroconidia to adhere and germinate on host tissue presumably

plays an important role in the localized dissemination of FHB. The infected spikelet spreads the infection to adjacent

spikelets throughout the rachis [13]. The hyphae of the pathogen extending inter- and intracellularly in the parenchyma

and vascular tissues of the lemma, glume, ovary and rachis cause severe damage to the host tissues. Cell wall degrading

enzymes are involved in the colonization of host tissues by fungal hyphae [7]. During penetration and growth, the

hyphae produce toxins that finally contaminate the grain or cariopse. Several defense responses against the infection

result from the genes induction [14-16].

1. Infection and colonization

F. graminearum forms multicellular macroconidia or spores that play an important role in dissemination of the disease.

Germ tubes preferentially emerge from the apical cells in a bipolar pattern that appear to be common to filamentous

fungi. Chitin deposition occurs at two locations: the spore apices and cortical regions of macroconidial cells that

subsequently produce a germ tube. The spatial pattern of morphogenesis requires the presence of functional

microtubules, which may be responsible for the transport of key polarity factors to specific sites. These observations

Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.)

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suggest that F. graminearum possesses a regulatory system that marks germ tube emergence sites. Perturbation of this

system may represent an effective approach for inhibiting colonization of host plant surfaces. The spatial pattern of

morphogenesis in germinating macroconidia was analyzed by Harris [6] with fluorescent microscopy. To monitor

nuclear division and septation, cover slips with adherent macroconidia and germlings were stained with calcofluor and

apropiate molecular probes. To characterize patterns of cell wall deposition, germinating macroconidia on cover slips

were incubated appropriately with wheat germ agglutinin or concavalin A. The images were captured by a photometrics

camera and processed through computational software. Distinct patterns of chitin deposition associated with

germination were observed, with demarcation between chitin-rich and chitin-poor region of new germ tube. The pattern

suggests the existence of a mechanism that marks sites of germ tube emergence. Furthermore proteins that designate

these sites may play an important role in the interaction with the host surface.

2. Cell wall and storage substances degrading enzymes

Diverse cytological studies were carried out to elucidate the importance of cell wall degrading enzymes (CWDE) during

infection process and pathway colonization of wheat spikes by Fusarium spp. In these cases, the microscopy was

relevant for clarifying the mechanism studied, employing light microscopy, scanning electron microscopy (SEM) and

transmission electron microscopy (TEM) [7, 17, 18]. In some cases enzyme-gold and immune-gold labeling techniques

were utilized for microscopic visualization to elucidate the importance of cell wall degrading enzymes such as

cellulases, xylanases and pectinases [7, 18]. These results allowed confirming that F. graminearum penetrates and

invades its hosts with the help of secreted CWDE.

By scanning electron microscopy, Pristch et al. [14] observed from inoculated glumes that the fungus penetrated,

grew sub-cuticullarly and colonized glume parenchyma cells, and sporulated within 2 to 3 days. Good results were

obtained for infection visualization when the tissue infected was stained with calcofluor and observed by

epifluorescence microscopy with ultraviolet irradiation. Calcofluor is a fluorochrome of the diamino-stilbenedisulfonate

type that stains fungi in culture and during plant infection [14].

By scanning electron micrographs it was also observed that few hours after inoculation of single spikelets with

macroconidia of F. graminearum, the fungus germinated, forming several germ tubes and developed a dense hyphal

network in the cavity of the spikelets. Then, the fungus hyphae invaded the ovary and inner surface of the lemma and

palea. When the grains were infected, the starch granules of the endosperm were extensively degraded (pitted) [19] and

the storage protein matrix that surrounded the starch granules was absent according to SEM observation [20, 21]. These

microscopic inspections were confirmed by transmission electron microscopy, in this case, the studies revealed that the

fungi extended inter- and intracellularly in the ovary, lemma and rachis causing considerable damage and alterations on

the host cell walls.

The components of the cell walls of plants: cellulose, xylan, pectin and structural proteins are reduced in the contact

areas between the fungi and the cells of the infected tissue, resulting from the action of extracellular enzymes of the

phytopatogen [7, 13]. Once the fungi colonize and infect the tissues, they produce micotoxins, which spread to the

surrounding tissue, and can move to the rest of the plant through xylem and phloem.

Using immuno-gold and enzyme-gold labeling techniques followed by transmission electron microscopy, the

production of CWDE such as pectinase, cellulase and xylanase was demonstrated during infection and colonization of

infected wheat spikes by Fusarium sp. Localization of cellulose, xylan and pectin showed that host cell walls which

were in direct contact with the pathogen surface had reduced gold labeling compared to considerable higher labeling

densities of walls distant from the pathogen–host interface or in non-colonized tissues. The reduced gold labeling

densities in the infected host cell walls indicate that these polysaccharide degrading enzymes might be important

pathogenic factors of F. graminearum during infection of wheat spikes. [7, 17]. The production and secretion of CWDE

enable F. graminearum to spread in the host tissues and to obtain its nutritional needs during the infection process.

3. Visualization of enzymatic hydrolysis

In the agricultural soil, the degradation of the crop rubble takes place, this resulting from the release of extracellular

enzymes by Fusarium sp. and other present fungi. In industry, commercial enzymes are used for hydrolysis of native

cellulose from plant fibers, such as wood and agricultural residues; besides they are a key step for converting biomass

into biofuels through biochemical means. To understand the process of enzymatic hydrolysis of cellulose, it is necessary

to directly investigate and visualize the degradation or modification of microfibrils through combined actions of several

enzymes. Several microscopy techniques such as scanning electron microscopy (SEM), transmission electron

microscopy (TEM), and quantitative fluorescence microscopy (QFM) [22, 23] have been frequently used to visualize

the ultrastructure of cellulose microfibrils as well as the bound cellulases [2, 24] One limitation of these techniques is

that it cannot provide detailed 3-dimensional information, such as height and roughness. Many efforts have been made

to observe the surface topographies of cellulosic substrates by atomic force microscopy (AFM). Liu et al. [25] used

moderate tapping force mode to produce an AFM phase image to visualize the fragmentation of cellulose microfibrils


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from enzymatic hydrolysis based on the discontinuity of material surface mechanical properties. This technique

produces sharper images that can distinguish enzyme particles from cellulose microfibrils or fragments; besides distinct

material features (hard or soft, hydrophobic or hydrophilic) can be revealed using TM-AFM.

4. Contamination by mycotoxins

F. graminearum produces mycotoxins during grain colonization, and in some cases, during storage. Grains

contaminated by mycotoxins are unsuitable for human and animal consumption because they may cause numerous

biological disturbances [8]. The mycotoxin produces inhibition of protein synthesis, electrolyte losses, cytoplasm

convolution and disintegration of organelles [26, 27]. Studies about infection process and localization of mycotoxins in

infected wheat tissue by Fusarium sp. were carried out by Kang and Buchenauer [28]. They localized the DON-

mycotoxin (deoxynivalenol) produced by Fusarium sp. in host and fungus cell by inmunegold –labeling methods

followed by electron microscopy.

Detection and monitoring of DON-mycotoxins in cereal grains is evaluated by analytical methods such as high

performance liquid chromatography (HPLC) and gas chromatography (GC), having determined three categories of

infection according to mycotoxin content (slight, moderate and severe). The grains are examined also by visual

inspection recording characteristics such as their normal color and weights. Differences in degree of fungal invasion

among kernels determined by DON contents are in relation with visual and microscopic observations. The light and

electron microscopy showed that the severely infected grains had the greatest amounts of hyphae and little structure of

the original wheat kernel was recognizable. The micrographs clearly showed that the fungus was distributed through the

kernel with generally the highest concentration of hyphae in pericarp tissues. The presence of hyphae in endosperm

tissues, especially in slightly and moderately infected grains, may prove unlikely to avoid DON contamination. When

the grains were severely infected, most of the protein storage, cell walls and starch granules were severely damaged

[29].

By fluorescent microscopy, Alm [30] observed the damage caused in pig oocyes when the animals had a diet with

Fusarium-toxin contaminated wheat. This contamination could impair growth and reproductive efficiency. Different

fertility parameters were analyzed, such as size of follicles, oocyte chromatin status and oocyte maturation. Based on

their chromatin configuration observed by fluorescence the oocytes were classified into different categories according

to chromatin configuration or degenerated chromatin spread throughout the oocyte. At the molecular level, DON

disrupts normal cell function by inhibiting protein synthesis via binding to the ribosome, and also by critical activation

of cellular kinases involved in signal transduction related to proliferation, differentiation, and apoptosis [30].

Doyle et al. [31] observed that low concentrations of DON modulated the innate immune system resulting in an

increase of animal susceptibility to infections with bacteria. Besides, it was determined that mycotoxins produced

alteration in proteins, which could be visualized by westernblotting, and by fluorescent confocal microscopy for further

validation.

5. Disease control

5.1 Inhibition by natural substances

The continuous use of fungicides has caused the appearance of fungicide resistant strains in F. graminearum and these

resistant populations are becoming dominant [32]. For this reason, the development of novel fungicides, highly safe and

efficient, has become a primary topic in crop protection research. There is an increasing interest in finding natural

substances that could inhibit the fungal growth, having studied the inhibitory effect of farnesol and osthol on F.

graminearum.

To address the specificity of farnesol as an inducer of fungal apoptosis, a series of experiments testing the effects of

natural and synthetic farnesol analogs were carried out by Semighini et al. [9]. Apoptosis markers were observed using

a fluorescent microscope and individual images were captured by a photometrics camera. Samples were processed for

nuclear staining, terminal deoxynucleotidyl transferase biotin dUTP nick end labeling (Tunel assay), detection of ROS

(reactive oxygen species) and Evan´s Blue staining. The microscopic observations demonstrated that farnesol treatment

induced condensation of nuclei with chromatin fragmentation, inhibited the germination of macroconidia, induced lysis

of hyphal cells and decreased viability. These results suggest that this isoprenoid or its derivatives may be valuable as

antifungal agents that limit infection of plants or humans by Fusarium species.

The effects of osthol, a plant coumarin, on morphology, sugar uptake and cell wall components of F. graminearum

were examined by transmission electron microscopy, 14C-labelling and enzyme activity detection. The results revealed

that osthol could inhibit the hypha growth of F. graminearum by decreasing hyphal absorption to reducing sugar. The

morphological observations with TEM showed cytoplasmic vacuolation and blurring of organelles and cell walls after

treatment with osthol. Besides, some enzymatic activities increased when hyphae were treated with osthol. Bioassay

demonstrated that osthol showed a high activity against F. graminearum, breaking the hyphae in fragments, which



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could develop in colonies. This indicated that the treatment with osthol inhibited the growth of fungi, but was not lethal

to them. For TEM analysis the samples were contrasted with uranyl acetate and lead citrate. The recorded images

clearly showed that the cell wall blurring after being treated with osthol was mainly caused by the hydrolyzation of

chitin in the wall cell [33].

5.2 Biocontrol

Biological control through antagonistic microbes to reduce the use of chemical pesticides in a system of integrated plant

disease management offers a powerful alternative to control plant diseases [10, 34, 35]. Wang et al. [36] studied the

inhibitory activity of a Bacillus subtilis strain against the Fusarium head blight disease fungus. The major inhibitory

compound found was purified from the culture by anion exchange, hydrophobic interaction, and reverse phase high-

performance liquid chromatography (RPHPLC) steps. The compound was structurally characterized as fengycin

according to mass spectrometry analyses and its effect on F. graminearum was studied with fluorescence microscope.

Fengycin is a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. The

fluorescence reagents fluorescein diacetete (FDA, molecular probes) and propidium iodide (PI, molecular probes) were

applied to assess the viability of F. graminearum hyphae by fluorescence microscopic analysis. F. graminearum hyphae

could be stained with FDA but not with PI in the absence of fengycin A. In contrast, the treated hyphae could not be

stained with FDA, whereas they were stained with PI. This indicated that fengycin A permeabilized the membranes of

F. graminearum hyphae.Viability test of F. graminearum in PDA media containing different concentrations of the

purified lipopeptide showed that the pathogen was unable to grow in the existence of 8 µg/ml of the inhibitory

compound [36].

To distinguish the introduced microbia from the indigenous population, a traceable marker is necessary [37]. The

green fluorescent protein (GFP) was reported as a potentially valuable tool for tracking the survival and localization of

microbial cells [38] and for monitoring gene expression [39]. Two biocontrol agents (BCAs) Brevibacillus brevis ZJY-1

and Bacillus subtilis ZJY-116, on the spikes of cereal and their effect on suppression of FHB were analyzed by Xin et

al. [10]. They constructed a plasmid that expressed the green fluorescent protein (GFP) for monitoring the survival and

colonization of biocontrol agent. The study revealed that plasmid pRP22-GFP was stably maintained in the bacterial

strains without selective pressure. Furthermore, both biocontrol strains gave significant protection against FHB on

cereal spikes in fields. The GFP fluorescence from all transformants was easily observed under UV radiation by

fluorescence microscopy, allowing making suitable ecological studies even under normal field environments.

In order to elucidate mechanisms for the efficient bio-control agent Clonostachys rosea, strain IK726 against cereal

seed pathogens, various molecular tools were used together with bio-imaging. The action modes of C. rosea are not

well understood but enzymatic activity, mycoparasitism, substrate competition, antibiosis and induced resistance are

thought to play a role [40]. The utilization of transformed microbes with appropriate gene, encoding a fluorescent

protein, facilitates the analysis of plant-pathogen interactions using confocal laser and scanning microscopy [41]. In

addition to C. rosea antagonistic capability against Fusarium spp., it has been found that this has an enzyme, a

lactonodehydrogenase "zeralenone-dehydrogenase" that can convert zeralenone mycotoxin produced by Fusarium spp.

into a non-toxic product [42].

6. Resistance to FHB and Genetic analysis

Plants exhibit a variety of resistance mechanisms when attacked by a pathogen. The common generalized downstream

responses include activation of defense response genes, oxidative burst, programmed cell death, phenylpropanoid

pathway induction, and pathogen walling off [43, 44]. During F. graminearum infection on wheat, a set of defense

response genes in the form of pathogenesis-related (PR) genes and oxidative burst-related genes such as peroxidase are

activated locally and systemically [14, 15, 45]. Increased production of PR proteins encoded by chitinase and β-1,3-

glucanase genes was correlated with resistance in wheat [45]. Temporal patterns of fungus development and transcript

accumulation of defense response genes have been studied in F. graminearum- inoculated wheat spikes. The host

response to Fusarium spp. infection on wheat is complex and combines an assortment of defense responses, including

activation of defense response genes and lignin deposition. Besides, the genetic resistance to FHB in wheat and other

crops is partial and quantitatively controlled by many loci [11]. Boddu et al. [11] determined a clear relationship

between disease development, DON concentration, and host gene transcript accumulation from analytical and molecular

methodologies along with microscopy. Histological evaluation of disease development by microscopy and quantitative

analysis of DON accumulation widened the gene transcript information. By light and scanning electron microscopy,

they examined lemma tissue spikes in order to follow the progression of F. graminearum at different times. Characters

such as spore germination, hyphal growth, putative penetration site and increase in the density of hyphal growth were

observed. The micrographs showed subcuticular hyphal growth with an increase of lignin content on the cell walls of

resistant cultivars during infection. Jansen et al. [46] analyzed the infection patterns in wheat spikes inoculated with

wild-type and trichodiene synthase gene disrupted F. graminearum, using a constitutively green fluorescence protein-

expressing Fusarium wild-type strain, and its knockout mutant, preventing trichothecene synthesis. Infection of the



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developing kernel occurs through the epicarp, successively destroying the layers of the grain coat and finally the starch

and protein accumulating endosperm. Hyphae reaching the rachis continue to apically located developing kernels. The

occurrence of GFP (green fluorescence protein) fluorescence in different tissues of the caryopsis was recorded by

confocal laser scanning microscopy (CLSM). The tissue samples were excited by a specific laser line for detection of

GFP fluorescence and autofluorescence of cell walls. Confocal micrographs of the infection in the rachis and

intracellular movement of the fungus through vascular bundles of xylem and phloem were recorded. The confocal

micrograph of rachis showed the heavily increased thickness of the cell walls in wheat product of infection.

Wheat spikelets inoculated with the wild type or ftl1 mutant strains were examined under confocal laser scanning

microscope with excitation and emission wavelengths of 488 nm and 510 to 530 nm and transmission electron

microscopy. In the microarray molecular analysis, the expression levels of all the F. graminearum genes that are known

to be required for pathogenicity or full virulence were normal in the ftl1 mutant strain. Based on microscopic

examination, loss of full virulence in the ftl1 mutant was related to its failure in penetrating and infecting wheat tissues.

The ftl1 mutant was defective in the colonization of filaments and in spreading from infected anthers to ovaries. The ftl1

mutant failed to penetrate and grew intracellularly in the cortical layer and was defective in the colonization of palea or

seed coat cells. Since the colonization of vascular tissues of the rachis may be a critical factor in the development and

spreading of wheat head blight, it is important to determine the function of FTL1 in regulating the penetration of plant

cells (intracellular growth) and colonization of vascular tissues. Besides, it was observed that 14 hydrolytic enzyme

genes had reduced expression levels in the ftl1 mutant [47].

Gale et al. [48] carried out cytological observation of mitotic chromosomes in populations created by crossing F.

graminearum mutant strains from the germ tube burst methods (GTBM) [49] by visualization via fluorescence

microscopy. Chromosomes were stained with a fluorescence solution to yield clear fluorescent images of mitotic

metaphase chromosomes. Chromosome counting showed that the genome of F. graminearum consisted of four

chromosomes, which was congruent with the combined result from physical and genetic mapping. Cytological analysis

unambiguously confirmed the conclusion from the genetic linkage analysis for chromosome number, showing the

power of cytology in combination with genetic mapping for resolving chromosome number. The construction of

physical and genetic mapping provides valuable information for analyzing the relation plant-pathogen.

Remarked aspects

The use of microscopic techniques as research tools in various fields of investigation has increased due to development

of technology with higher resolution and complexity which in combination with analytical methods and appropriate

molecular techniques have allowed to approach the study plant-pathogen interactions more efficiently. Thus, the

integration of complementary techniques is neccesary for elucidating the mechanisms involved in the infection process

allowing the improvement of knowledge and providing tools that could be used in an integrated disease control.

Acknowledgements. The support by Universidad Nacional de La Plata (Grant 11/X417) and CONICET (Grant PIP 1422) is

gratefully acknowledged.

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