Chapter 14

Metarhizium robertsii , aRhizosphere-Competent InsectPathogen*

Monica Pava-RipollDepartment of Entomology, University of Maryland, USA

14.1 INTRODUCTION

The insect pathogen Metarhizium robertsii J.F. Bisch.,Rehner & Humber (Hypocreales: Clavicipitaceae) is anatural inhabitant of the soil (Roddam and Rath, 1997;Zimmermann, 2007). The occurrence and abundance ofdiverse Metarhizium species and strains in soils dependupon environmental factors (i.e., temperature and humid-ity), soil conditions (i.e., pH and organic matter content),and habitat type (Bidochka et al., 1998; Quesada-Moraga et al., 2007; St Leger, 2008; Bruck, 2010;Fisher et al., 2011; Schneider et al., 2012). M. robertsiiARSEF 2575 (formerly known as Metarhizium anisopliaevar. anisopliae; Bischoff et al., 2009) has been shown tobe rhizosphere competent with roles in plant protectionand plant growth (St Leger, 2008; Garcıa et al., 2011).This may explain the persistence of the conidia ofM. robertsii in soils (unlike other insect pathogens) forlong periods of time in the absence of an insect host(Bidochka et al., 2001). However, large populations ofinsects in soils (i.e., white grubs) may also influencethe presence of M. robertsii (St Leger, 2008). As aresult, the soil/root interface is a place where insects,

∗Microarray data have been deposited in the NCBI’s Gene ExpressionOmnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/),accession number GSE16848.

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

plants, and microbes interact to affect Metarhiziumpopulations. This interaction also suggests that Metarhiz-ium may be subject to two different selective pressures:one for insect colonization and the other for soil sur-vivability (Prior, 1992; Wang et al., 2005; St Leger,2008).

Rhizosphere–soil interactions have been widelystudied with the best known rhizosphere-competent fun-gus Trichoderma spp. This fungus establishes symbioticrelationship with the roots of some plants increasing plantgrowth and productivity and has the ability to parasitizefungal plant pathogens (Harman, 2006; see Chapter 54).The complex processes between multiple strains of Tri-choderma, the roots of plants, and other plant pathogensdemonstrate that there is an established molecular crosstalk between them (Woo et al., 2006). However, thegenetic and physiological factors controlling rhizospherecompetence on Trichoderma are little understood (StLeger, 2007).

Likewise, while a lot of research has been performedon the entomopathogenic lifestyle of M. robertsii, itssaprophytic lifestyle has received little consideration.While a set of functionally related genes could becommonly expressed as this fungus adapts to differentsurroundings (insect cuticle, insect blood, and plantroot exudates), there might also exist a different subsetof genes active in each environment (Wang et al.,2005).

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150 Chapter 14 Metarhizium robertsii , a Rhizosphere-Competent Insect Pathogen

This study evaluated the capability of Metarhiziumstrains to germinate in root exudates (RE) and discernedthe gene expression by M. robertsii 2575 growing onplant RE in laboratory conditions over a time course.Increasing knowledge of the genes that are involvedin the rhizospheric lifestyle of M. robertsii will be thefirst step in elucidating pathways that are used by thisfungus for soil survivability and adaptability. A deeperunderstanding of the mechanistic basis of rhizospherecompetency could enable us to identify genes that canbe used to develop M. robertsii as a comprehensiveplant symbiont or at least improve persistence and conse-quently provide greater long-term protection against insectpests.

14.2 METHODS

Plant RE was obtained from black pea seeds (Vignaunguiculata subsp. unguiculata). The seeds were dis-infected and placed on wet, sterile paper for initialgermination. Seedlings were subsequently transferred tocontainers with sterile distilled water and kept for about1 week connected to an aquarium air pump until rootswere formed (Fig. 14.1). RE was then collected, freezedried, prepared in a stock solution of 40 mg/ml, andfiltered sterilized before storing at −20 ◦C.

The germination rate of 13 fungal strains wasperformed at 5 concentrations of RE (1, 2.5, 5, 10,and 20 mg/ml of RE). Evaluated fungi included 11entomopathogenic fungal strains from the genera Beau-veria and Metarhizium (with broad and narrow insecthost ranges) and 2 soil saprophytic fungi: Aspergillusniger ATCC 10574 (non-rhizospheric) and Trichodermaharzianum strain T22 (rhizosphere competent). The ger-mination rate of T. harzianum and M. robertsii was alsoevaluated at concentrations of RE less than 1 mg/ml (0.01,0.05, 0.1, and 0.5 mg/ml). Table 14.1 lists the fungalstrains selected for this study, their host range, geographicarea, and the insect host (or substrate) where they wereoriginally found. Broad host range insect pathogens (alsoknown as generalists) can infect multiple insect species,whereas narrow host range (also known as specialists)show specificity for certain insect species and are unableto infect other insects (i.e., Metarhizium acridum 324 isspecific to locusts and related grasshoppers [Orthoptera:Acrididae]).

Fungal strains were grown on Sabouraud dextroseagar (SDA) (Fisher Scientific, Pittsburgh, PA) for 2 weeksat 27 ◦C. Fungal spores were collected, suspended in0.01% Tween 20 (Sigma Chemical Co., St Louis, MO),and prepared to a final concentration of 104 spores/ml.One hundred microliters of spore suspension were added

to sterile tubes containing 900 µl of the selected concen-tration of RE. Positive and negative controls were alsoprepared for each fungal strain by adding fungal spores to0.1% of yeast extract (Fisher Scientific, Pittsburgh, PA)and sterile distilled water, respectively. Samples wereincubated at 27 ◦C with shaking at 250 rpm for 24 h. Thepercentage spore germination was determined on glassslides using light microscopy and recording the numberof germinated spores.

Three replicates were performed for each treatmentcombination (fungal strains and RE concentrations). Datawere analyzed using one-way analysis of variance (SASInstitute Inc., 2006). A P value <0.05 determined the sig-nificance level.

For microarray analysis, M. robertsii mycelia grownfor 30 h on Sabouraud dextrose broth (SDB) (Fisher Sci-entific, Pittsburgh, PA) were suspended in 20 ml of steriledistilled water. A small portion of the mycelium was sepa-rated for total RNA extraction (time zero). Five millilitersof the mycelium suspension were transferred to four 50-mlflask, each containing 5 ml of 10 mg/ml of RE, thus givinga final concentration of 5 mg/ml of RE. This final concen-tration of RE was selected as it provided the fungus withsufficient nutrients during the 12 h course of the experi-ment, avoiding changes on gene expression due to nutrientdeprivation. Samples were incubated in a shaker/incubatorat 27 ◦C/220 rpm (New Brunswick Scientific, Edison, NJ)and mycelium was collected from individual flasks after1, 4, 8, and 12 h.

Total RNA was immediately extracted from the sam-ples using the Qiagen RNeasy Mini kit (Valencia, CA,USA) according to manufacturer’s instructions. RNA sam-ples were treated with Qiagen DNase I.

A loop design was used to compare differential geneexpression of M. robertsii growing on 5 mg/ml RE whereevery time point was compared to the previous (time 0 vs1 h, 1 h vs 4 h, 4 h vs 8 h, 8 h vs 12 h, and 12 h vs time0). Information on experimental design and hybridization,labeling and scan protocols, as well as data processing andnormalization are available from NCBI Gene ExpressionOmnibus (GEO) database GSE16848.

Microarray slides were customized to contain threereplicates of 1748 M. robertsii unigenes previouslyobtained from three expressed sequence tag (EST)libraries (870 genes from insect cuticle, 276 genes frominsect blood, and 602 genes from RE) (Wang et al., 2005).Blanks using water and saline-sodium citrate (SSC) bufferwere also included per triplicate per slide as negativecontrols. The genes were organized in nine groups ofrelated functionality and this classification was used tocompare the Log2 ratio of expressed genes. Two biologi-cal replicates were performed. Competitive hybridizationof the second biological replicate was performed using areverse dye assignment to eliminate bias on the efficiency

14.2 Methods 151

Germination of beans

Seedling roots inwater

Collection of rootexudates

Figure 14.1 Obtention of bean rootexudates (RE) from black pea seeds(Vigna unguiculata subsp. unguiculata) inthe laboratory.

of dye incorporation (Cy5/Cy3 for replicate 1 andCy3/Cy5 for replicate 2).

To detect differentially expressed genes across timepoints, normalized Log2 ratios were inputted to the

Bayesian Analysis of Time Series (BATS) microarrayexperiments software (version 1.0) (http://www.na.iac.cnr.it/bats/) (Angelini et al., 2008). To account for the limitednumber of time instants, a maximum expected degree of

Table 14.1 List of fungal strains used in this study

Fungi Collection Strain Insect Host/Substrate Geographic Origin Host Range

Metarhizium anisopliae ARSEF 2105 Diptera: Ephydridae Java, Indonesia BroadMetarhizium robertsii ARSEF 2575 Coleoptera: Curculionidae South Caroline, USA BroadMetarhizium anisopliae ARSEF 549 Lepidoptera: Galacticidae Brazil BroadMetarhizium anisopliae ARSEF 1080 Lepidoptera: Noctuidae Florida, USA BroadMetarhizium acridum ARSEF 324 Orthoptera: Acrididae Queensland, Australia NarrowMetarhizium anisopliae ARSEF 4600 Diptera: Stratiomyidae Tasmania, Australia Not knownMetarhizium anisopliae ARSEF 4620 Diptera: Stratiomyidae Tasmania, Australia Not knownMetarhizium brunneum ARSEF 2974 Diptera: Culicidae Buenos Aires, Argentina NarrowBeauveria bassiana ARSEF 3113 Soil Iowa, USA Not knownBeauveria bassiana CENICAFE 9205 Lepidoptera: Pyralidae Valle, Colombia Not knownBeauveria bassiana CENICAFE 9112 Lepidoptera: Geometridae Caldas, Colombia Not knownAspergillus niger ATCC 10574 Culture plate contaminant Egham, England —Trichoderma harzianum ATCC T22-20847 Soil Not known —

Data on fungal collection, strain identification, host/substrate affiliation, geographic area of origin, and insect host range are included.

ARSEF, Agriculture Research Service Entomopathogenic Fungal Collection; CENICAFE, National Centre of Coffee Research; ATCC, AmericanType Culture Collection.

152 Chapter 14 Metarhizium robertsii , a Rhizosphere-Competent Insect Pathogen

polynomial equal to 3 and a lambda for the truncatedPoisson prior probability equal to 4 (corresponding toan expected polynomial degree about 2) were used. TheBayesian Multiple Testing Procedure with the binomialprior was also selected to account for multiple compar-isons. Default values were used for the other parameters.Differentially expressed genes were ranked according totheir Bayes factors (BF <0.006). The fold change was cal-culated by subtracting the Log2 expression ratio of othertime points to time 0.

The nucleotide sequences of the genes that werefound to be differentially expressed were comparedagainst the NCBI non-redundant protein database(BLASTX) and re-organized into previously classifiedgroups of functionally related genes (Wang et al., 2005).

The validation of differentially expressed genes wasperformed through reverse transcription polymerase chainreaction (RT-PCR). Total RNA extracted for microarrayanalysis was used for RT-PCR using the Verso cDNAKit (Thermo Fisher Scientific Inc., Waltham, MA)according to manufacturer’s recommended protocol usingoligo-dT primers. The cDNA product was subsequentlyamplified through PCR reaction using gene-specificprimers. The glyceraldehyde-3-phosphate dehydrogenasegene (gpdA) was used as a control.

14.3 RESULTS

14.3.1 Fungal Spore Germinationon Bean Root ExudatesGermination of spores from all fungal strains evaluatedwas observed at 1 mg/ml RE. T. harzianum and M. aniso-pliae 2105 showed the highest germination rates (rang-ing from 96.8% to 99.3%) at the five concentrations ofRE evaluated with no statistical differences between them(Fig. 14.2a). The broad range insect pathogens M. robertsii2575 and M. anisopliae strains 549 and 1080 showed ger-mination rates ranging from 76.6% to 93.9% at 1 mg/ml,from 91.6% to 95.2% at 2.5 mg/ml, and from 87% to 93%at 5 mg/ml of RE. These germination rates were not statis-tically different from those of the rhizosphere-competentT. harzianum at the same concentrations. However, at 10and 20 mg/ml of RE, the germination rate of M. robertsii2575 was significantly lower from that of T. harzianum(Fig. 14.2a).

The narrow host range insect pathogen M. acridum324 showed its highest germination rate (47.3%) at1 mg/ml of RE and its lowest germination rate (24.7%)at 20 mg/ml of RE. Germination rates of M. acridum 324statistically differed from both the rhizosphere-competentT. harzianum and the nonrhizospheric A. niger at allconcentrations evaluated (Fig. 14.2a). At 1 mg/ml,

M. anisopliae 4620 and 4600 and M.brunneum 2974presented the lowest germination rates (22.5%, 6.5%,and 12.5%, respectively) and were not statically differentfrom the germination rate of A. niger (19.3%) at thatsame concentration. However, germination rates of theseMetarhizium strains increased with increasing concen-tration of RE (except at 20 mg/ml when M. anisopliae4620 and M. brunneum 2974 showed slightly reducedgermination rates) and became significantly differentfrom the germination rate of A. niger that declined withincreasing concentration of RE: 19% (at 1 mg/ml), 14%(at 2.5 mg/ml), 13% (at 5 mg/ml), 2% (at 10 mg/ml), and0% (at 20 mg/ml) (Fig. 14.2a).

Three strains of Beauveria bassiana were includedamong the fungal strains tested. B. bassiana 9205and 9112 were originally isolated from lepidopteranhosts, whereas B. bassiana 3113 was isolated from soil(Table 14.1) and has been reported as a fungal endophytein corn providing protection against lepidopteran pestssuch as Ostrinia nubilalis (Pingel and Lewis, 1996;Wagner and Lewis, 2000). At 1 mg/ml of RE, germi-nation rates of B. bassiana 9205, 9112, and 3113 were73%, 50.5%, and 64%, respectively. These rates didnot differ statistically from each other but they werestatistically higher than M. anisopliae 4620 and 4600,the narrow insect host range M. brunneum 2974, and thenon-rhizospheric A. niger (Fig. 14.2a). Germination ratesof B. bassiana strains 9112 and 3113 were statisticallylower than the germination rates of T. harzianum and allbroad host range Metarhizium strains. Germination ratesof B. bassiana 9112 and 9205 did not differ statisticallyat ≤10 mg/ml RE but germination rate by B. bassiana9112 statistically decreased from B. bassiana 9205 at20 mg/ml RE. Germination rates of B. bassiana 3113gradually declined at concentrations greater than 1 mg/mland had diverged significantly from all other fungalstrains at 10 mg/ml RE (Fig. 14.2a).

Germination rates of T. harzianum and generalistsM. anisopliae 2105 and M. robertsii 2575 were also evalu-ated at 0.01, 0.05, 0.1, and 0.5 mg/ml of RE. Figure 14.2bshows that at concentrations ≤0.1 mg/ml of RE, the ger-mination rate of M. robertsii 2575 was statistically higherthan those of T. harzianum and M. anisopliae 2105.

14.3.2 Microarray Data AnalysisFifty genes (out of 1748 genes, 2.9%) were automaticallydetected by BATS as differentially expressed across alltime points evaluated. These genes belong to the follow-ing functional groups: hypothetical proteins (predictedsequences that lack experimental evidence of in vivoexpression): 13/413 (3.1%); protein metabolism: 11/172(6.4%); cell structure and function: 10/231 (4.3%); cellmetabolism: 7/335 (2.1%); cell cycle, division, and

14.3 Results 153

(a)

(b)

Figure 14.2 Germination of spores of entomopathogenic fungi (genus Metarhizium, Ma, Mr, and Mb and Beauveria, Bb), Trichodermaharzianum (rhizospheric), and Aspergillus niger (non-rhizospheric) at different concentrations of root exudates (RE). (a) Concentrations of REfrom 1 to 20 mg/ml. (b) concentrations of RE ≤ 1 mg/ml. Averages with the same letter are not significantly different (P ≤ 0.05). Reprintedfrom Pava-Ripoll et al. (2011), with permission from the Society for General Microbiology.

growth: 3/93 (3.2%); stress response and defense: 3/107(2.8%); energy metabolism: 2/64 (3.1%); and unknownproteins (orphan sequences with no homologous in databases): 1/236 (0.4%) (Fig. 14.3a).

Figure 14.3b shows the percentage of M. robertsiigenes that were differentially expressed in each ESTlibrary. Although the majority of expressed genes arefrom the RE library (54%), it is clear that genes specificfor penetration of insect cuticle and growth in insectblood are also involved in adaptability of M. robertsii torhizospheric conditions.

Out of the 50 genes that were differentially expressedin RE, 29 (58%) were up-regulated and 21 (42%)were down-regulated across all time points. Nine outof 11 genes (18%) and 14 out of 19 genes (28%)were up-regulated after 4 and 8 h, respectively. Mostdown-regulated genes (28%) were expressed after 12 h inRE (Fig. 14.3c). Figure 14.3d shows the linear expressionmap (LEM) representing the average expression of genesclassified by functional groups at each time point. Thenumber of up-regulated genes/total number of differen-tially expressed genes per functional group is included inthe figure.

Figure 14.4a–j shows the threshold of the Log2expression ratios through time of the 50 genes ofM. robertsii 2575 that were differentially expressed underRE conditions. Figure 14.4a–f shows up-regulated genes,whereas Figure 14.4g–j shows down-regulated genes.Up-regulated hypothetical genes showed the greatest foldincreases through time. Maximum fold increases wereobserved in genes AJ273764 (3.7-fold) and CN809209(3.3-fold) after 8 h and in gene CN808927 (3.3-fold)after 4 h. Hypothetical genes CN808884 (55% similarto a M. anisopliae conserved protein), CN809514, andAJ274093 also increased by 3.0-, 2.5-, and 2.5-fold,respectively, after 8 h in RE (Fig. 14.4a). Figure 14.4bshows an increased expression of the subtilisin-like serineprotease (Pr1A) gene (CN808958) with a maximum3.3-fold increase after 8 h. The stress response heatshock protein (CN808235) also increased by 2.9-foldafter 4 h. Genes involved in energy metabolism, Dihy-drolipoyl dehydrogenase (AJ273762) and oxidoreductase(CN808777) increased their expression by 2.9- and2.0-fold after 8 and 12 h, respectively (Fig. 14.4c). Theexpression of two transport proteins, an ABC trans-porter ATP-binding protein (CN809103) and a GABA

154 Chapter 14 Metarhizium robertsii , a Rhizosphere-Competent Insect Pathogen

(a)

(c) (d)

(b)

Figure 14.3 Fifty genes differentially expressed by Metarhizium robertsii 2575 during growth in root exudate (RE) in a time course (0, 1, 4,8, and 12 h). (a) Differentially expressed genes compared with the total number of genes in each functional group. (b) Percentage of genesdifferentially expressed across all time points in each expressed sequence tag (EST) M. robertsii library. (c) Number of up-regulated anddown-regulated M. robertsii genes at each time point in RE. (d) Linear expression map (LEM) representing the average expression ratio ofgenes organized according to functional groups at each time point. The gradient color of the arrows represents the mean expression ratio and redmarks represent the functional groups which average was up-regulated. Figures 14.3a and 14.3d are reprinted from Pava-Ripoll et al. (2011),with permission from the Society for General Microbiology.

(gamma-amino-N-butyrate) permease (CN808046)increased by 2.4- and 0.8-fold, respectively, after 12 hin RE (Fig. 14.4d). The expression of genes requiredfor the metabolism of carbohydrates such as glycosylhydrolase (CN808813), β-glucosidase (AJ273623), andferulic acid esterase A (faeA) (AJ273114) also increased2.2-fold (after 8 h), 1.9-fold (after 4 h), and 1.3-fold(after 4 h), respectively (Fig. 14.4d). Genes associatedwith extracellular matrix and cell wall proteins, thehydrophobin-like protein precursor (AJ274156) and thecell wall protein (AJ273845), were up-regulated andthey both reached a maximum 2.1-fold increase after8 h and 12 h, respectively (Fig. 14.4e). Also, the genesrequired by M. robertsii to adhere to plant surfaces,the cell wall adhesin MAD2 (CN809626), and its closehomolog CN809322 reached their maximum expression

(1.9- and 1.6-fold increase, respectively) after 4 h in RE(Fig. 14.4e). Genes involved in DNA synthesis (mediatorof replication checkpoint 1, CN809288), lipid metabolism(diacylglycerol O-acyltransferase, DgaT, CN808018),sexual development (EsdC protein, CN809127), andcofactor and vitamins (amidase protein, AJ273042)increased their expression between 0.8- and 2-fold after8 h in RE (Fig. 14.4f).

Expression of ribosomal proteins decreased between2.4- and 1.0-fold after 12 h (Fig. 14.4g). Modulatorsof translation genes, translation elongation factor 1-alpha (CN809122), eukaryotic translation initiationfactor (AJ2737433), and elongation factor 1-gamma(CN809664), along with the pyridoxine biosynthesis pro-tein gene (CN808729) and the reverse transcriptase gene(CN809546) also decreased their expression between

14.3 Results 155

Figure 14.4 Threshold of the Log2 expression ratio of differentially expressed genes Metarhizium robertsii 2575 while growing on RE on atime course (0, 1, 4, 8, and 12 h). Parts (a) through (f) show the threshold of up-regulated genes. Parts (g) through (j) show the threshold ofdown-regulated genes.

156 Chapter 14 Metarhizium robertsii , a Rhizosphere-Competent Insect Pathogen

0.5- and 2.1-fold after 12 h (Fig. 14.4h). Genes belongingto other functional groups such as stress response(cold acclimation induced protein 2–1, CN808997),transport proteins (carrier protein ADP–ATP translocase,CN809461), sexual cycle (sexual development EsdC pro-tein, CN808640), and the cupin family protein, AJ272794(previously classified as a hypothetical protein) graduallydecreased expression within the initial 4 h but presenteda 0.1- to 0.7-fold increase at 8 h (Fig. 14.4i). Expressionof hydrophobin genes (AJ273847 and CN809178) wasalso initially down-regulated but recovered so that by 8 h,these genes were up-regulated 1.5- and 1.6-fold, respec-tively (Fig. 14.4i). The C-3 sterol dehydrogenase gene(AJ274219) involved in lipid metabolism, the potassiumchannel transport protein (CN808889), and the pyridinenucleotide-disulfide protein of the oxidoreductase family(CN808746) (also previously classified as a hypotheticalprotein) slightly increased their expression ratio 0.4-fold,0.5-fold, and 0.7-fold, respectively, during the initial 4 hin RE indicating that these genes were still be requiredby M. robertsii, probably for degradation of the lipidspresent in RE and for the intake of nutrients into the cells(Fig. 14.4j).

14.3.3 RT-PCR Verification ofDifferentially Expressed GenesFive genes (predicted protein-AJ273764, subtilisin-likeserine protease PR1A-CN808958, hydrophobin-likeprotein-AJ274156, adhesin protein Mad2-CN809626,and a ribosomal protein-CN809270) were selected forvalidation of microarray analysis through RT-PCR.Expression patterns of these genes were consistent withmicroarray results (Fig. 14.5).

14.4 DISCUSSION

The symbiotic association between fungi and plant roots(mycorrhiza) occurs in more than 80% of terrestrialplants and represents positive plant–microbe interactions(Bais et al., 2006). While the fungus can access lipidsand carbohydrates from the roots, the plant benefitsfrom the increased uptake of water and nutrients thatinduce plant growth (Harman and Shoresh, 2007). Thecomponents of RE are explained in detail in Chapter 22.Although the function of all RE compounds has not beendetermined yet, they do play important roles in biologicalprocesses. For example, a plant signaling molecule, thebranch-inducing factor, play a key role in stimulatingthe germination and hyphal branching of arbuscularmycorrhizal (AM) fungal spores (Bais et al., 2006,see also Chapter 43), hence it is not surprising if they

gdpa

0 h 1 h 4 h 8 h 12 h

AJ273764

CN808958

AJ274156

CN809626

CN809270

Figure 14.5 Reverse transcriptase polymerase chain reaction(RT-PCR) used to validate microarray gene expression ofMetarhizium anisopliae 2575 growing in root exudates. Theglyceraldehyde-3-phosphate dehydrogenase (gpda) gene was used asthe reference gene. Reprinted from Pava-Ripoll et al. (2011), withpermission from the Society for General Microbiology.

also play an important role for entomopathogenic fungiadapting to soil environments.

Here, it was demonstrated that broad host rangefungal pathogens, M. robertsii 2575 and M. aniso-pliae 2105, 549, and 1080 germinated as well in REas the rhizosphere-competent fungus T. harzianumT22, particularly at concentrations less than 5 mg/ml.However, germination rates of M. robertsii 2575 andM. anisopliae 2105 statistically differed at ≤0.1 mg/mlRE, indicating the hypersensitivity of M. robertsii 2575to RE (Fig. 14.2b). M. anisopliae strains 4600 and 4620and M. brunneum 2974 showed reduced rhizospherecompetence as they need higher RE concentrations toincrease their germination rates. The narrow host rangeM. acridum 324 showed decreased germination at allRE concentrations when compared with the broad hostrange strains (Fig. 14.2a). About 7% of M. robertsii2575 genes are absent or highly divergent in M. acridum324, including genes involved in toxin biosynthesis andcarbohydrate metabolism (Wang et al., 2009), henceit is clear that narrow host range M. acridum haveevolved independently to adapt to alternative hostsrather than saprophytic environments. Additionally,whole-genome comparison between M. anisopliae andM. acridum revealed that M. anisopliae has more genes(i.e., 733 more protein coding genes, 199 more puta-tive pathogen–host interaction (PHI) genes, 48 moreprocessed and fragmented pseudogenes, 43 more trans-porters) and a much greater potential for the productionof secondary metabolites, thus facilitating its ability toadapt to rhizospheric environments (Gao et al., 2011).

14.4 Discussion 157

Plant REs regulate the microbial community in therhizosphere, help plants withstand herbivory, encouragebeneficial symbioses, and contains a mixture of inhibitoryas well as stimulatory chemicals that effect fungal strainsdifferently (Walker et al., 2003; Bais et al., 2004). Thenonrhizospheric A. niger was the most sensitive to theinhibitors in RE indicating that it is not adapted toresist antimicrobial exudate components (Fig. 14.2a).Fungi capable of rhizosphere competence presumablyneed to evolve resistance to these compounds, whichare thought to have a role in defending the rhizosphereagainst pathogens (Bais et al., 2006). Like Metarhizium,B. bassiana is an ubiquitous insect pathogen and a plantendophyte capable of colonizing plant roots (Fuller-Schaefer et al., 2005; Vega et al., 2008). Even though theendophytic B. bassiana 3113 and strains 9205 and 9112were much less responsive to RE than M. robertsii 2575,their germination rates oscillated between those of broadand narrow host range Metarhizium strains. Further stud-ies to evaluate the germination of additional B. bassianastrains in RE would allow a better understanding of theirsurvival strategies in soils.

Although Wang et al. (2005) showed that only a fewM. robertsii genes were sharply up-regulated after 24 hat 0.1 mg/ml RE, results from this study demonstratethat M. robertsii 2575 contains a set of differentiallyexpressed genes that can rapidly (mainly at 4 and 8 h)adapt to nutrients present in RE (Fig. 14.3c). The highestpercentage of up-regulated genes (41.3%) were in thecategory of hypothetical/unknown proteins, indicatingthat many previously uncharacterized M. robertsii genesmay have functions related to saprophytic survival. Someof these genes have orthologs in plant pathogens andother soil fungi so whatever role they play in adapting tosoil conditions may be conserved. In fact, whole-genomesequencing of the broad host range M. anisopliae (strainARSEF 23) revealed a notable large proportion of genesencoding secreted proteins (17.6%) when compared toplant pathogens (5–10%). However, no functionallycharacterized homologs were found in 30% of thesegenes, suggesting previously unsuspected interactionsnot just between fungal pathogens and insects but alsobetween fungal pathogens and rhizospheric environments(Gao et al., 2011).

M. robertsii Pr1A gene, CN808958, was up-regulatedwithin the first 4 h in RE and maintained high expressionlevels throughout the time course. Wang et al. (2005)reported the up-regulation of the Pr1A gene at 24 h ofincubation in RE, indicating the long-term involvementof this protease in utilizing RE. Metarhizium anisopliaehas 132 more genes encoding secreted proteases thanother sequenced fungi (Gao et al., 2011). Althoughthe function of some Metarhizium proteases have beenelucidated (i.e., subtilisins), the function of some others

(i.e., aspartyl proteases, AP) has not been demonstratedyet (Gao et al., 2011) but their role on plant colonizationshould be further studied. Interestingly, two AP (papAand papB) were found highly expressed in a Trichodermastrain with roles in root attachment and mycoparasitism(Viterbo et al., 2004; Lorito et al., 2010).

M. robertsii genes involved in carbohydratemetabolism (glycosyl hydrolase, CN808813; β-gluco-sidase, AJ273623; and ferulic acid esterase, AJ273114)were also up-regulated during the first 4 h in RE(Fig. 14.4d), probably to degrade low-molecular weightcompounds such as sugars that are present in REs of awide variety of plants (Rovira, 1965).

The MAD2 adhesin protein (CN809626) was 1.9-foldup-regulated in the first 4 h of incubation in RE. Thisprotein is responsible for the attachment of M. robertsiito plant surfaces to effectively colonize roots andplants as an endophyte (Sasan and Bidochka, 2012).M. robertsii Mad2 disrupted strain (�Mad2) showed a90% reduction in adherence to plant cells (Wang and StLeger, 2007). Although expressed at slightly lower levelsthan CN809626 (1.6-fold), the MAD2 close homologCN809322 has an identical pattern of gene expressionin RE (Fig. 14.4e), suggesting that is also involved inattachment to plant surfaces and it is perhaps responsiblefor the 10% of Mad2 mutant spores that are still adher-ent. Field experiments releasing M. robertsii �Mad2expressing the green fluorescent protein (�Mad2-GFP)also confirmed the ability of this protein to adhereto root surfaces as mutant populations decreased overtime, regardless of the presence of insect hosts in soils(Wang et al., 2011).

The rhizosphere-competent Trichoderma spp. is alsoan effective biological control agent of plant diseasescaused by soil-borne fungi such as Rhizoctonia solani,Pytium spp., and Sclerotium rolfsii (Hadar et al., 1979;Elad et al., 1980; Harman et al., 1980; Aziz et al., 1997;see Chapter 54). Likewise, M. anisopliae has antag-onistic effects on plant pathogenic fungi, includingFusarium oxysporum, Botrytis cinerea, and Alternariasolani (Kang et al., 1996). Thus, Metarhizium has beenshown to be not just a potent insect pathogen but alsoa rhizosphere-competent fungus with multiple rolesin plant protection and plant productivity. Knowledgeof the genes involved as Metarhizium adapts to soilenvironments is important when considering the potentialcommercial use of as a biological control agent againstfoliar and soil-borne insect pests and soil-borne plantpathogens. A 4-year study on the long-term geneticadaptations and evolution of Metarhizium in cultivatedsoils (turf plots) demonstrated the capability of thisfungus to maintain populations on plant roots and insectswhile posing little risk from gene flow or from evolutionof host virulence, thus alleviating some of the concerns

158 Chapter 14 Metarhizium robertsii , a Rhizosphere-Competent Insect Pathogen

raised for the release of transgenic strains into the field(Wang et al., 2011). The comprehensive knowledgeof the short- and long-term below-ground interactionsbetween Metarhizium, insects, plants, and microbialsoil populations are revealing ecological links that willhelp us to understand the molecular cross talk betweenthem that could ultimately be exploited to benefit plantgrowth and productivity. Further research is also neededto discern the function of the array of M. robertsiihypothetical/unknown proteins that were differentiallyexpressed in RE in order to elucidate their possibleroles in the saprophytic lifestyle of this biological insectpathogen and possibly other fungi.

ACKNOWLEDGMENTS

Special thanks to Raymond St Leger, Francisco J. Posada,Weiguo Fang, Sibao Wang, and Claudia Angelini for col-laboration with experiments and data analysis.

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