Early Events in the Fusarium verticillioides-Maize Interaction Characterized by Using a Green Fluorescent Protein-Expressing Transgenic Isolate

The infection of maize by Fusarium verticillioides can resultin highly variable disease symptoms ranging from asymptomaticplants to severe rotting and wilting. We produced F. verticillioidesgreen fluorescent protein-expressing transgenic isolates andused them to characterize early events in the F. verticillioides-maizeinteraction that may affect later symptom appearance. Plantsgrown in F. verticillioides-infested soil were smaller and chlorotic.The fungus colonized all of the underground parts of a plantbut was found primarily in lateral roots and mesocotyl tissue.In some mesocotyl cells, conidia were produced within 14 to21 days after infection. Intercellular mycelium was detected,but additional cells were not infected until 21 days after planting.At 25 to 30 days after planting, the mesocotyl and main rootswere heavily infected, and rotting developed in these tissues.Other tissues, including the adventitious roots and the stem,appeared to be healthy and contained only a small number ofhyphae. These results imply that asymptomatic systemic infectionis characterized by a mode of fungal development that includesinfection of certain tissues, intercellular growth of a limitednumber of fungal hyphae, and reproduction of the fungus in afew cells without invasion of other cells. Development of visiblyrotted tissue is associated with massive production of fungalmycelium and much less organized growth.

INTRODUCTION

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Introduction

Fusarium verticillioides (synonym, Fusarium moniliforme Sheldon;teleomorph, Gibberella moniliformis [synonym, Gibberella fujikuroimating population A]) is the most commonly reported fungal speciesinfecting maize (26, 36, 37). F. verticillioides can be foundin plant residues in almost every maize field at harvest, yetthe disease symptoms vary widely and range from asymptomaticinfection to severe rotting of all plant parts. In many casesdiseased and asymptomatic plants occur in the same field plantedwith a genetically uniform host. Environmental conditions, wateravailability (8, 9, 28, 29, 35), and the genetic backgroundof the plant and the pathogen (2, 19, 25, 45) may all be importantfactors in disease development, but it is not known why thedisease does not occur during asymptomatic infections or whatcauses rotting and wilting in the diseased tissues.

Infection of maize by F. verticillioides can occur via severalroutes. The most commonly reported method of kernel infectionis through airborne conidia that infect the silks (15, 31, 32).After invasion through the silks, the fungus infects the kernels,but usually only a small percentage of the infected kernelsbecome symptomatic (33). Another proposed infection pathwayis systemically through the seed (12). Systemic infection canstart from fungal conidia or mycelia that are either carriedinside the seeds or on the seed surface. The fungus developsinside the young plant, moving from the roots to the stalk andfinally to the cob and kernels. Movement of the fungus frominfected seeds to the stalk and kernels has been establishedin a number of studies. Kedera et al. (20) used vegetative compatibility(vegetative compatibility groups) as a marker to track specificstrains and obtained evidence that the fungus moves from theseeds to kernels by recovering the isolate inoculated into theseeds from approximately 10% of the kernels. Similar findingswere reported by Munkvold and coworkers, who also used vegetativecompatibility groups to track fungal movement in the plant (32,33). These researchers also showed that although infected seedsmay contribute to kernel infection, local infection via silksis the main pathway of kernel infection. Systemic infectionalso may result from inoculum that survives in crop residuesin the soil; however, the relative importance of soilborne andseed-borne inocula as the cause of systemic infections is notknown (25, 39).

F. verticillioides produces several toxins that have potentialtoxicity for humans and domesticated animals. The most significantof these toxins produced by F. verticillioides are the fumonisins(7, 37). Since fumonisins can be detected in symptomatic andasymptomatic maize kernels, control of fumonisin contaminationin maize has become a priority area in food safety research(3), and some guidelines for maximum fumonisin levels in humanfood and animal feeds have been issued (13). The presence offumonisins in asymptomatic grain means that a better understandingof the asymptomatic endophytic portion of the life cycle ofF. verticillioides is badly needed. Work with genetically tractablestrains has suggested that the limiting step in the movementof the fungus from the seeds to the upper parts of the plantis the transition of the fungus from the seedling crown to thestalk (19, 33). However, the data generated by such analysesare limited to isolation of the fungus from different tissuesand do not provide a clear picture of the colonization of theplant by the fungus. Moreover, not all experiments have producedthe same results (45).

Light microscopy and electron microscopy have been used to obtainmore detailed information on the infection of maize by F. verticillioides(1, 34, 45). These methods provide information on events suchas seed infection and root penetration, but the data are limitedto particular time points. More continuous information on inplanta fungal development may be obtained by tracing transgenicisolates that express reporter genes. Work with ß-glucuronidase-expressingisolates has provided some insight into the F. verticillioides-maizeinteraction (3, 46); however, the tissue sectioning and otherexperimental manipulations required to monitor ß-glucuronidaseactivity limit its utility in such analyses. Recently, the greenfluorescent protein (GFP) has become a commonly used tool inthe analysis of fungus-plant interactions (27). Spores and hyphaeof GFP-expressing fungal isolates can be identified by fluorescencemicroscopy in intact tissues or tissue sections without extensivemanipulation and provide highly informative data on processesof plant colonization (10, 11, 16, 23, 30).

In the present study we generated F. verticillioides GFP-expressingtransgenic isolates and used them to characterize early stagesof the F. verticillioides-maize interaction. By using GFP-expressingtransgenic isolates we directly monitored development of thefungus in planta during early stages of the interaction anddetermined the spread of the fungus to upper parts of the plant,including leaves and cobs, at different stages of plant growth.These analyses provided new information on the disease, whichcan help explain the differences between aggressive and asymptomaticinteractions.

MATERIALS AND METHODS

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Materials and Methods

Fungi, plants, and growth conditions.
F. verticillioides strain A-00149 (= FGSC 7600) was providedby J. F. Leslie. This strain was first isolated from maize inCalifornia. It has been used as a standard mating type testerstrain for this species, and it is one of the progenitors ofthe mapping population of F. verticillioides (21, 22). Wild-typestrain A-00149 and its derivative transgenic strains were usedin all experiments. The fungus was maintained at 28°C onCzapek-Dox medium (40) or was stored as a conidial suspensionin 20% glycerol at -70°C (21). For mycelium and spore productionthe fungus was grown in shake cultures at 28°C, and themycelium was collected by filtration.

Sweet maize cultivar Jubilee was used for plant inoculationexperiments. This commercial line is widely grown and is highlysusceptible to F. verticillioides (17). Plants were grown inpots of various sizes. Greenhouse experiments were conductedin 3- or 10-liter pots containing a mixture of 40% peat, 40%loam, 10% vermiculite, and 10% compost. The greenhouse temperaturewas 25 to 27°C, and daylight was supplemented with lightfrom fluorescent tubes to provide 14 h of continuous light.The light intensities in the greenhouse during the day werebetween 120 and 200 microeinsteins m^-2. The plants used in greenhouseexperiments were irrigated daily with a hand sprinkler. Experimentsin the nursery were conducted in 10-liter pots containing soilfrom fields in which wilted plants were observed. The plantswere drip irrigated, and the soil moisture content was maintainedat 70% of the soil water capacity to provide optimal water conditions.Soil disinfestation was performed with Basamid G (BASF, Munich,Germany) used according to the manufacturer's instructions.After disinfestation the soil was washed for 24 h with water(drip irrigation, 2 liters h^-1) to remove traces of Basamid.An absence of Basamid residual activity was verified by thedevelopment of healthy, asymptomatic seedlings after maize seedswere planted.

Seed inoculation.
Before seeds were inoculated or planted, all the seeds weresoaked for 30 min in a 0.2% solution of the fungicide Octav(AgrEvo UK, Cambridge, England) to prevent infection from aseed-borne inoculum. No colonies developed when Octav-treatedseeds were plated on the isolation medium. Octav-treated seedswere rinsed with water and then placed in a 0.2% Tween 20 solutioncontaining 5 x 10⁹ conidia ml^-1. After 30 min, the seeds wereremoved from the spore suspension and dried in a laminar flowhood overnight.

Soil inoculation.
The soil inoculum consisted of 3-day-old mycelium that was inoculatedonto dry maize leaves. Fresh mycelium (25 g) was homogenizedwith a blender and suspended in 200 ml of water. Chopped maizeleaves (10 g) were mixed with 50 ml of the original or dilutedmycelial suspension, depending on the treatment. The top 12cm of soil was removed from six pots (either 3- or 10-literpots) and mixed with the inoculum, and then the inoculated soilwas distributed among the pots so that the upper 12 cm in eachpot contained inoculated soil. Seeds were planted immediatelyafter inoculation of the soil.

Low-light experiment.
Experiments to test the effect of low light on disease developmentwere conducted in 50-liter containers in a growth room. Coolwhite fluorescent tubes supplied a light intensity of 20 microeinsteinsm^-2. The temperature was maintained at 25 ± 2°C.

Fungal isolation.
We used the methods described by Burgess et al. (4) for fungalisolation. Plant parts were surface sterilized by submergingand shaking them in 1% sodium hypochlorite-10% ethanol for 30s. The tissues were then washed with sterile water and driedin a laminar flow hood. Three pieces of sterilized tissue weresectioned with a razor (0.5-cm-thick stalk disks, 5-cm-longroots) and placed on peptone-pentachloronitrobenzene (PCNB)medium (4).

Plasmids and fungal transformation.
A DNA fragment was amplified by PCR from F. verticillioidesgenomic DNA by using the decamer primer 3'-GGGTGCAGTT-5'. A500-bp PCR amplification product was cloned into plasmid pUC57(MBI-Fermentas, Hanover, Md.) and sequenced. No putative openreading frame was found. The fragment was cloned into the vectorgGFP (30) at the HindIII and XbaI restriction sites to producethe transformation vector fgGFP.

Protoplast preparation and transformation were performed asdescribed by Tudzynski et al. (44), with some modifications.A mycelial suspension (1 ml) was added to 100 ml of completemedium (44) and cultured for 16 to 18 h at 200 rpm on an orbitalshaker at 28°C. The mycelium was filtered and washed with0.8 N NaCl, and 0.5 g of the mycelium was suspended in an enzymesolution (0.5% Novozyme [InterSpex Products, Inc., Foster City,Calif.], 0.1% driselase [Sigma, Rehovot, Israel], 0.01% chitinase[Sigma]) for 2 to 3 h with shaking at 100 rpm until protoplastswere released. The protoplasts were washed with cold 0.8 N NaCl,and the final concentration was adjusted to 5 x 10⁷ protoplastsml^-1 in STC. Linear plasmid (10 µg) was added to 50 µlof the protoplast solution together with 50 µl of polyethyleneglycol 6000 (Sigma). The mixture was incubated on ice for 25min, an additional 2 ml of polyethylene glycol 6000 was addedto each of the tubes, and the preparations were incubated foranother 10 min at room temperature before dilution with 4 mlof STC. The protoplasts were incorporated into 100 ml of regenerationmedium containing (per liter of water) 145.7 g of mannitol (Merck),4 g of yeast extract (Difco, Detroit, Mich.), 1 g of solublestarch (Merck), and 16 g of agar; hygromycin B was added toa final concentration of 100 µg ml^-1, and the medium wasthen distributed into six petri dishes. The plates were incubatedat 28°C, and colonies were subcultured 4 to 8 days later.

Microscopy.
Fluorescence microscopy was performed with a Leica DMRBE epifluorescencemicroscope (Leica, Deerfield, Ill.). Each of the tissues examinedwas placed on a microscope slide, submerged in a water droplet,and covered with a glass coverslip. The K3/L4 filter cube withpeak transmission for excitation at 470 to 490 nm and peak transmissionfor suppression at 515 to 560 nm was used for GFP detection.Light microscopy was performed with the same microscope withoutfilters. Confocal microscopy was performed with an invertedlaser scanning confocal microscope (CLSM 510; Zeiss, Cberkochen,Germany) with excitation and emission at 488 and 510 nm, respectively.GFP expression in entire colonies was visualized with an OlympusSZx12 fluorescence stereoscope by using an eGFP filter.

Experimental procedures.
The nursery and greenhouse inoculation experiments were conductedin six replications, with six plants per pot. The growth chamberexperiment at a low light intensity was conducted in three replications,with 20 plants per container. To determine plant weight or rootcolonization, one plant from each replication was sampled atevery time point; the fresh weight of the plants was determined,and tissue samples were used for colony isolation.

RESULTS

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Results

GFP-expressing strains.
Transgenic isolates were obtained at a rate of two transformantsper microgram of plasmid DNA. Only 60% of the hygromycin B-resistantisolates expressed GFP, a value which is similar to values reportedfor other fungi (23, 41). GFP expression was generally strongand uniform in all fungal tissues and remained high and stableafter successive transfers on Czapek-Dox medium with and withouthygromycin B. Southern analysis showed that in all cases theplasmid integrated into chromosomal DNA in one or more locationsand that tandem integration of two or more copies was common(data not shown). We analyzed 20 isolates and selected one,gf12, to study in planta fungal development. This isolate expressedthe highest level of GFP, produced abundant conidia, and hadwild-type colony morphology. Inoculation of plants with gf12and recovery on semiselective medium showed that gf12 was asinfective as the wild type and that it could be easily detectedby observation with the fluorescence stereoscope (Fig. 1).

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FIG. 1. Isolation of GFP-expressing F. verticillioides from infected maize roots. Seeds were inoculated with the transgenic isolate gf12 and planted in disinfested soil. Roots of 14-day-old plants were excised, rinsed in water, and plated on peptone-PCNB semiselective medium. Colonies emerged after 4 to 6 days (a), and GFP expression was detected with a fluorescence stereoscope (b) (the white halo fluoresces in green).

Plant infection by using seed and soil inocula.
Nursery experiments were conducted with the wild-type isolateto compare the effects of soil inoculation and seed inoculationon infection of maize plants. Seed inoculation had no effecton the weight of the plants grown in untreated soil comparedto the weight of control plants that developed under similarconditions (Table 1). Growth enhancement was observed in thedisinfested soil compared to the growth in untreated soil. Althoughseed inoculation did not suppress seedling growth, high ratesof root colonization were detected even at the first samplingtime after 20 days (Table 1). Soil inoculation reduced plantgrowth, with greater reductions occurring at higher inoculumlevels, and the number of colonies isolated from the roots wasproportional to the amount of the soil inoculum (Fig. 2).

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TABLE 1. Effect of seed inoculation with F. verticillioides on seedling fresh weight and root colonization

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FIG. 2. Effect of soil inoculation with F. verticillioides on seedling fresh weight and root colonization. Disinfested soil was inoculated with an F. verticillioides mycelium homogenate. Plant fresh weight and root colonization were determined 20 days after planting. The error bars indicate the standard errors.

Early events in plant colonization.
We detected only trace amounts of fluorescent mycelium duringmicroscopic examination of plants that developed from inoculatedseeds, although the fungus was readily recovered from the rootsof these plants by plating on peptone-PCNB medium (Fig. 1).Soil inoculation resulted in more pronounced infection (Fig.3). The transgenic fungus was easily detected on the surfacesof underground organs, such as seeds, roots, and hypocotyl (Fig.3a and b). During the first few days after planting, the fungusgrew both on the surface and inside lateral roots (Fig. 3c andd). Only single hyphae were detected growing along the root,and there was no branching or change in the direction of growth.Thickened organelles were observed in hyphae that developedon the surfaces of lateral roots. These organelles resembledconidia; however, the true nature of these organs and theirrole are unclear. Such organs were not observed in the hyphaethat developed inside the roots.

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FIG. 3. Early events during plant colonization by F. verticillioides. Seeds were planted in soil that was inoculated with a transgenic isolate gf12 mycelium homogenate. The photographs were obtained with a fluorescence microscope unless otherwise indicated. (a) The organs and tissues examined included the root, lateral roots and root hairs, mesocotyl (arrow), seed pericarp, and lower part of the shoot. (b) Adherence of mycelium and direct penetration of the mesocotyl 3 days after planting. (c and d) Hyphae growing on the surface of a lateral root (c) and inside a lateral root (d) 7 days after planting. (e to i) Colonization and reproduction of the fungus in mesocotyl cells. Single cells accumulated fluorescing material after 7 days (e), but fungal growth was restricted to the cell wall spaces (f). After 14 days, conidia started to differentiate (g), and more developed mycelium was detected in the intercellular wall spaces (h). Hyphae grew out of the cells in which conidia were formed, but through specific points of contact with other cells and without rupturing the cells (g, arrows). After 21 days conidia matured (i) but were still retained in the cells in which they were formed. (j) After 21 days, the development of the mycelium on the root intensified, with hyphae growing in the intercellular spaces along the root. (k) Macroconidia attached to the mucilage layer on a root hair 21 days after planting. (l and m) Large amounts of mycelium developed after 30 days and infected the entire mesocotyl tissue (l) and the main root (m) (confocal microscopy).

After 7 days, the fungus also was detected in the mesocotyltissue but not in the tissues above the mesocotyl. The mesocotylis defined as the intermediate tissue between the root and thehypocotyl (Fig. 3a). A very organized pattern of developmentwas observed: most cells in the mesocotyl were unaffected, buta few cells developed yellow-green fluorescence (Fig. 3e). Theemission spectrum of this fluorescence was different from theGFP emission spectrum, indicating that the cells accumulatedautofluorescing material. Very little mycelium could be foundin the mesocotyl at this stage, and the mycelia that we diddetect were retained at the cell boundaries without cell penetration(Fig. 3f).

Green fluorescing fungal material composed of undefined roundedorganelles that filled single cells in the mesocotyl was detectedafter 14 days (Fig. 3g). The adjacent cells were unaffected,but mycelium could be detected in wall connections between aninfected cell and the surrounding cells (Fig. 3g). More myceliumwas observed in the cell wall spaces at this stage (Fig. 3h).This mycelium contained thick organelles that looked like conidiophoresor aerial branching.

After 21 days, the infected cells were filled with mature conidia(Fig. 3i). Conidia were found only inside a cell in which aninfection was established, while the surrounding cells appearedto be normal. A large number of hyphae that developed alongthe root axis (Fig. 3j) and green-fluorescing macroconidia (Fig.3k) were detected on root hairs. Disease symptoms were not observedat this stage.

The initial phase of infection and establishment in the rootsand mesocotyl was followed by a second phase 25 to 35 days afterinoculation, in which the fungus proliferated in the mesocotyland in the main root (Fig. 3l and m). Large amounts of myceliumwere produced in these organs and caused rotting of the mesocotyland the main root. No fluorescing hyphae were observed in thestem by fluorescence or confocal microscopy; however, a smallnumber of green fluorescent, transgenic colonies could be isolatedfrom the stems and even leaves by plating on selective medium(data not shown).

The amount of mycelium that developed in the mesocotyl was largerat high soil inoculum rates. More mycelium was detected in themesocotyl tissue of plants that developed in soil containing0.6 g of inoculum kg of soil^-1 than in plants growing in soilcontaining 0.1 g of inoculum kg of soil^-1 (Fig. 4a and b). Theseresults are consistent with the results of the nursery experimentsperformed with the wild-type strain, in which the number ofcolonies that were isolated from roots increased as the sizeof the soil inoculum increased (Fig. 2).

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FIG. 4. Effects of inoculum levels and light conditions on inoculation of maize plants by F. verticillioides. (a and b) Effect of two levels of inoculum on the amount of mycelium that developed in the mesocotyl 30 days after seeds were planted in inoculated soil. The inocula used were 0.1 g (fresh weight) per kg of soil (a) and 0.6 g (fresh weight) per kg of soil (b). (c and d) Development of mycelium in roots (c) and mesocotyls (d) of plants grown under low-light conditions (20 microeinsteins m^-2) 14 days after inoculated seeds were planted in disinfested soil.

Infection of plants grown under low-light conditions.
Two-week-old plants that were grown under low-light conditions(20 microeinsteins m^-2) had intense rotting, and large amountsof fungal mycelium were detected on and inside the plants. Themycelia grew without any special order, infecting the entireroot system, the mesocotyl, and the stem (Fig. 4a and d). Themassive infection resulted in rapid rotting and seedling mortality2 weeks after planting (Table 2). The aggressive fungal developmentunder low-light conditions was in sharp contrast to developmentof mycelium in tissues of plants that were grown with higherlight intensities; under these conditions the myclelium didnot invade the cells and developed mainly along the cell walls.

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TABLE 2. Disease development in F. verticillioides-infected maize seedlings grown under low-light conditions

DISCUSSION

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Discussion

Systemic movement of F. verticillioides from infected seedsto roots, crowns, stalks, and ears has been documented by usingvarious methods (3, 31, 33, 34, 38, 45). Although systemic infectioncan cause rotting in all plant organs, it is considered a majorcause of stalk rot, while the significance of this pathway inkernel infection appears to be low (9, 31, 33, 38). Munkvoldet al. (33) reported that transmission of the fungus from seedlingcrown tissue to stalk tissue appeared to be the rate-limitingfactor in fungal movement from infected seeds to the upper partsof the plants. These results are consistent with previous findingsof Nelson (36) and Lawrence et al. (24), who suggested thatseed-transmitted strains do not advance to above-ground partsof a plant until after pollination. However, various reportshave indicated that the fungus has been isolated from all partsof young (7- to 14-day-old) seedlings (2, 45). The differentresults may be explained by different genetic backgrounds ofthe plant and the fungus (5, 7, 15, 18, 19, 35), by differencesin the inoculation methods and the experimental conditions (9,28, 29, 33, 38), or by differences in the sensitivities of thedetection methods (3, 31, 34, 45). In the present study, weisolated the transgenic fungus from stems of 14-day-old seedlingsby plating stem sections on semiselective medium (Fig. 1). However,the fungus was not detectable in planta by fluorescence microscopyat this stage, which indicates that there were no more thantrace amounts of fungal mycelium in the above-ground tissuesat this time. These findings confirm that the fungus is capableof moving to above-ground parts early in the interaction, butrelatively little fungal biomass which does not cause rottingor other disease symptoms develops during these early infectionstages. This growth pattern may explain why the fungus was detectedin the upper parts in some studies but not in other studies(2, 31, 36, 45).

The levels of soil inoculum and root colonization correlatedwith reduced seedling growth, which was more pronounced at theearly growth stages, while seed inoculation did not suppressplant growth and may even have enhanced plant development (Fig.2 and 3). Enhanced growth of Fusarium-infected plants has beenreported previously and was attributed to the endophytic natureof the fungus (25, 45). Our results suggest that the same strainmay cause growth enhancement and growth retardation, dependingon the level of plant colonization, which is influenced by theamount of fungal inoculum in the soil and seed. Although F.verticillioides can survive in crop residue in the soil, thisinoculum source has not been considered a major source of plantinfection (25, 31). Our results suggest that soil inoculum,in addition to infected seeds, may be important in initiationof F. verticillioides systemic infection. Furthermore, in ourstudy soil inoculum resulted in more diseased plants than seedinoculation, suggesting that problems associated with standestablishment attributed to Fusarium are more likely to resultfrom soilborne inoculum than from seed-borne inoculum.

GFP-expressing transgenic isolates can be visualized in livingtissues without any processing or manipulation of the samples.This property makes GFP extremely useful for analysis of inplanta fungal development (27). In the present study, we usedGFP-expressing transgenic isolates to obtain detailed informationon the interaction between F. verticillioides and maize plants.The main sites of plant penetration were the lateral roots andthe mesocotyl, which is the tissue connecting the emerging rootwith the emerging shoot. The fungus was detected in these tissuesas early as 72 h after the seeds were planted in infested soil.These findings support previous findings of Murillo et al. (34),who showed that there was direct penetration of the seed pericarpand root epidermal cells by F. verticillioides 3 days afterinoculated maize seeds were planted. These authors also foundthat fungal growth at this stage was mainly intercellular. Lagopodiet al. (23) used GFP-expressing transgenic Fusarium oxysporumto study tomato root colonization and infection. They foundthat the contact between the fungus and the host was initiatedpredominantly at the root hairs, while colonization of the crownwas predominant after the initial contact. These findings aresimilar to our results and suggest that there is a common modeof plant colonization at the early stages, in which the pathogensfirst attach to the lateral roots and root hairs and then penetratedirectly through the cuticle and epidermis of these tissues.

After penetration the fungus develops in the intercellular spacesfor various periods of time (23, 34, 45). In the F. oxysporum-tomatointeraction, the mycelium continued to grow along the junctionsof the plant epidermal cells and covered large areas of theroots after 4 days. Necrosis started to appear 4 to 5 days afterplanting and was associated with a dense hyphal network thatcovered the necrotic tissues (23). In the F. verticillioides-maizeinteraction only single hyphae growing along the roots wereobserved after 7 to 14 days (Fig. 4c and d). A larger numberof hyphae were detected after 21 days, but even these hyphaedid not cover the entire root but rather retained the preexistinghyphal network architecture (Fig. 3j). Rotting associated withthe massive growth of mycelium in these tissues was observed3 to 4 weeks after planting and was restricted to the mesocotyland the root (Fig. 3l and m).

These differences in fungal development in maize and tomatomay reflect differences in the growth rates and aggressivenessof the highly virulent pathogen F. oxysporum and the less virulentpathogen F. verticillioides. These two fungi appear to use similarroutes to penetrate and colonize their hosts, and the differencesin disease appearance result from differences in the rates offungal growth within the plant. In the more virulent pathogenF. oxysporum, the whole process proceeds much faster, and thetransition from the initial symptomless phase to the necrotrophicphase occurs within a few days. The less aggressive pathogenF. verticillioides develops more slowly, leaving the plant moretime to respond and restrict fungal growth. Indeed, activationof defense responses, such as deposition of lignins (45) andaccumulation of PR proteins (34), by F. verticillioides-infectedmaize has been reported. Moreover, development of the fungusin physiologically weakened plants (e.g., plants grown withlow light intensities) was much more aggressive, and large amountsof mycelium that caused severe rotting and seedling mortalitywere observed on and in the roots, mesocotyls, crowns, and stemsof 2-week-old plants (Fig. 4 and 2).

Our data suggest that the well-orchestrated development of F.verticillioides inside maize tissues may represent a delicatebalance between the fungus and the plant. Under conditions thatfavor symptomless infection, fungal growth may be restrictedto specific tissues, where it penetrates only specific cellswithin which it reproduces without damaging the surroundingcells (Fig. 3e to i). The only rotting observed at this stage,in the mesocotyl and the primary root, was compensated for bythe development of adventitious roots above the mesocotyl tissue,which were unaffected by the fungus (data not shown). This responsemay explain the initial slower growth of seedlings in the soilinoculation experiments (Table 1) and their later recovery andresumption of normal growth.

Under conditions that favor pathogenic development more myceliumdevelops, and the fungus switches to a more aggressive phasethat probably involves secretion of hydrolytic enzymes and toxins(6, 7, 14, 18, 37, 42, 43). The magnitude and time of appearanceof symptoms may vary, from no symptoms during the entire lifecycle of the plant to seedling blight and severe rotting ofthe entire plant, depending in part on the physiological conditionsof the plant during the early stages of colonization and laterdevelopmental stages that may favor disease outbreak.

ACKNOWLEDGMENTS

We thank Shimon Sharon for his assistance in figure preparation.

This work was supported in part by Israeli Ministry of Agriculturegrant 891-0148-00.

FOOTNOTES

* Corresponding author. Mailing address: Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972 3 640 6741. Fax: 972 3 640 5498. E-mail: amirsh{at}tauex.tau.ac.il.

REFERENCES

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