Metarhizium Isolates Research Articles

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5′ strains by sequencing of the 59′ end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10–12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.

Metarhizium seed treatment mediates fungal dispersal via roots and induces infections in insects

The study aimed to evaluate the extent to which Metarhizium spp. applied as conidia to seeds will disperse with the growing root system and maintain pathogenicity after dispersion. Tenebrio molitor larvae were exposed to roots of wheat plants that had been grown from seeds inoculated with conidia of either M. brunneum (KVL 04-57 and KVL 12-37) or M. robertsii (ARSEF 2575 and KVL 12-35) in both laboratory and greenhouse settings. All four Metarhizium isolates tested maintained pathogenicity towards T. molitor larvae for up to 4 weeks after being dispersed by roots through both an artificial growth substratum and non-sterile soil. Based on these results we propose that a plant–root association benefits entomopathogenic fungi with mobility in the soil and an increased likelihood of encountering a susceptible insect host.

Heat-induced post-stress growth delay: A biological trait of many Metarhizium isolates reducing biocontrol efficacy?

The habitats of many pest insects have fluctuating climatic conditions. To function effectively, the pathogens of these pests must be capable of infecting and developing disease at a wide range of temperatures. The current study examines ten Metarhizium spp. isolates as to their ability to recover normal metabolic activity after exposure to high temperature for several hours daily; and whether such recovery, with at least some isolates, requires a temporary repair (“retooling”) period. Fungal colonies were exposed to 40°C for 4h or 8h followed by 20h or 16h at 28°C, respectively, for three consecutive days. Growth rates during treatments were compared to control plates (constant 28°C) and to plates with growth stoppage by cold treatment (4h or 8h at 5°C per day). All ten isolates survived 3days of cycled heat treatment and resumed normal growth afterward; some isolates however, were considerably more negatively affected by heat-cycling than others. In fact, some isolates underwent greatly reduced growth not only during 8h heating, but also some hours after cessation of heat treatment. This phenomenon is labeled in the current study as “post-stress growth delay” (PSGD). In contrast, all isolates stopped growing during 8h cold treatments, but immediately recommenced growing on return to 28°C. The delay in recommencing growth of some isolates after heat treatment amplifies the effect of this stress. In addition to the studies on the effects of heat cycling on fungal cultures, the effects of imposing such temperature cycling on fungal infection of insects was documented in the laboratory. Three Metarhizium isolates were bioassayed using Galleria mellonella larvae. Treated insects were placed at daily temperature regimes matching those used for the in vitro fungus rate-of-growth study, and insect mortality recorded daily. For all three isolates the levels of insect mortality at the highest-heat dose (40°C at 8h daily) significantly reduced infection. Fluctuating temperatures are likely to be a factor in most pest–insect habitats; therefore, the presence and level of PSGD of each isolate should be a primary consideration in selecting field-appropriate fungal isolates.

Specific diversity of the entomopathogenic fungi Beauveria and Metarhizium in Mexican agricultural soils

Prior knowledge of the local population structure of entomopathogenic fungi is considered an important requisite when developing microbial control strategies against major pests of crops such as white grubs. An extensive survey in the estate of Guanajuato, one of the main agricultural regions of Mexico, was carried out to determine the abundance and diversity of entomopathogenic fungi in soil. Soil collected from 11 locations was baited for entomopathogenic fungi using Galleria mellonella. In addition, all isolates were morphologically identified and selected isolates of Beauveria and Metarhizium isolates identified using Bloc and ITS or Elongation Factor 1-α and ITS sequence information respectively. Genotypic diversity was then studied using microsatellite genotyping. The proportion of isolates belonging to each genus varied amongst all locations. The species Beauveria bassiana, B. pseudobassiana and Metarhizium robertsii were found, with B. bassiana being the most abundant and widely distributed. Microsatellite genotyping showed that the 36 B. bassiana isolates were grouped in 29 unique haplotypes, but with no separation according to geographical origin.

Pathogenicity of conidia-based preparations of entomopathogenic fungi against the greenhouse pest aphids Myzus persicae, Aphis gossypii, and Aulacorthum solani (Hemiptera: Aphididae)

Seeking new isolates of entomopathogenic fungi with greater virulence against greenhouse aphid pests than those currently registered in North America for control of these insects, single-dose screening assays of 44 selected fungal isolates and 4 commercially available strains were conducted against first-instar nymphs of Myzus persicae and Aphis gossypii. The assays identified a number of Beauveria and Metarhizium isolates with virulence equal to or greater than that of the commercial strains against the nymphal aphids, but none exhibited exceptionally high virulence. Virulence of Isaria isolates was unexpectedly low (<31% mortality at doses>1000conidia/mm2). In dose–response assays, Beauveria ARSEF 5493 proved most virulent against M. persicae and A. gossypii; however, LC50s of this isolate did not differ significantly from those of B. bassiana commercial strain JW-1. Dose–response assays were also conducted with Aulacorthum solani, the first reported evaluations of Beauveria and Metarhizium against this pest. The novel isolate Metarhizium 5471 showed virulence⩾that of Beauveria 5493 in terms of LC25 and LC50, but 5493 produced a steeper dose response (slope). Additional tests showed that adult aphids are more susceptible than nymphs to fungal infection but confirmed that infection has a limited pre-mortem effect on aphid reproduction. Effects of assay techniques and the potential of fungal pathogens as aphid-control agents are discussed.

Cuticle degrading enzyme production by some isolates of the entomopathogenic fungus, Metarhizium anisopliae (Metsch.)

Context: Entomopathogenic fungi have been recognized as viable alternate options to chemicals in insect pest control. Unlike other potential biocontrol agents, fungi do not have to be ingested to infect their hosts but invade directly through the cuticle. Entry into the host involves both enzymic degradation of the cuticle barrier and mechanical pressure. Production of a range of cuticle degrading enzymes is an important event in the interaction of entomopathogenic fungi and host. Enzyme secretion is believed to be a key contributor for the virulence of a fungal isolate. Objectives: The potentiality of nine isolates of M. anisopliae were tested to produce to produce three important cuticle degrading enzymes, viz., chitinase, protease and lipase. Materials and Methods: Nine isolates of M. anisopliae were evaluated for chitinase, protease and lipase enzyme production by determining the enzyme index and activities. Results: Chitinase index of these isolates were ranged from 1.5 to 2.2 and chitinolytic activity from 0.525 to 1.560 U/ml. The isolates showed protease index in the range of 1.2 to 3.3 and the activity ranged from 0.020 to 0.114 U/ml. Lipase index ranged from 1.15 to 7.0 and the enzyme activity ranged from 0.153 to 0.500 U/ml. A strong relationship was observed between virulence of the isolates and cuticle degrading enzyme production as increased enzyme production was observed for virulent isolates. Conclusion: In the present study three isolates as (MIS2, MIS7 and MIS13) demonstrated cuticle degrading enzyme (CDE) that indicate higher virulence based on the bioassay conducted earlier by the authors as strongly substantiating the role of CDEs is considered the virulence of Metarhizium isolates. So, these isolates may be as ecofriendly insect-pest control agent in future. DOI: http://dx.doi.org/10.3329/jbs.v20i0.17648 J. bio-sci. 20: 25-32, 2012

Comparison of the germination rates of Metarhizium spp. conidia from Japan at high and low temperatures

The knowledge of the growth characteristics of entomopathogenic fungi at different temperatures is very important for understanding their ecology and field efficacy as biological control agents. However, the relationships between their DNA-based phylogenetic tree classifications and growth characteristics at different temperatures have not been adequately investigated. In this study, we compared the phylogenetic relationships between Metarhizium anisopliae sensu lato and M.flavoviride isolates obtained from soils in various environments in Japan and the germination rates of their conidia on agar medium in hot and cold conditions. The results showed that the 89 Japanese isolates belonged to the clade of eight species, according to the molecular phylogenetic analysis. The germination rates of isolates belonging to the M.brunneum and M.flavoviride var. pemphigi clades were higher at lower temperatures (9·7-11·0°C) and lower at higher temperatures (34·3-35·2°C) compared with the other six species. The isolates of these two species originated from different geographical regions in Japan, despite their uniform germination characteristics. This study detected the clear interspecific differences in the in vitro germination characteristics of the Japanese isolates of Metarhizium spp. at two different temperature regimes. The relationships between the growth characteristics and phylogenetic placements have not been adequately investigated in species of the entomopathogenic fungus Metarhizium. This study determined the relationships between the germination rates in hot and cold conditions and the phylogenetic placements of 89 Japanese soil isolates of Metarhizium spp. Fourteen isolates each of M.brunneum and M.flavoviride var. pemphigi, identified by molecular phylogenetic analysis, showed relatively high germination rates at lower temperatures and low germination rates at higher temperatures compared with isolates, which were identified as six other species. This study detected a strong relationship between the phylogenetic placements of Japanese Metarhizium spp. isolates and their in vitro germination characteristics.

Metabolomics Reveals the Heterogeneous Secretome of Two Entomopathogenic Fungi to Ex Vivo Cultured Insect Tissues

Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

Genetic Diversity and Pathogenicity of Metarhizium spp. Associated with the White Grub Phyllophaga capillata (Blanchard) (Coleoptera: Melolonthidae) in a Soybean Field

Phyllophaga capillata (Blanchard) was recently described as a soybean pest in Brazil. The occurrence of Metarhizium spp. associated with adults and their pathogenicity were investigated. Natural prevalence of Metarhizium anisopliae was 0.51% in adults collected by light trap. Most Metarhizium isolates were identified as Metarhizium robertsii, although M. anisopliae sensu stricto and a few unidentified isolates were also found. Bioassays with representative isolates selected from different genetic groups resulted in ≤5.6% confirmed mortality against larvae and adults, suggesting low potential of microbial control of this pest by use of indigenous Metarhizium isolates.

VIRULENSI BERBAGAI ISOLAT JAMUR ENTOMOPATOGEN METARHIZIUM SPP. TERHADAP HAMA PENGGEREK BUAH KAKAO CONOPOMORPHA CRAMERELLA SNELL. (LEPIDOPTERA: GRACILLARIIDAE)

Virulence of various Metarhizium spp. (Deuteromycotina: Hyphomycetes) isolates on cocoa pod borrer pest, Conopomorpha cramerella Snell. (Lepidoptera: Gracillariidae). In Indonesia, Conopomorpha cramerella Snell. (Lepidoptera : Gracillariidae) is a serious pest of cocoa. Entomopathogens such as Metarhizium spp. (Deuteromycotina: Hyphomycetes) can be used to control this pest as an alternative to chemical insecticides. The purpose of this research was to study the virulence of isolates of Metarhizium spp. to pupae of C. cramerella. Nine isolates of Metarhizium spp each with 107 conidia/ml rate were used in the experiment. Pupal mortality was recorded daily. The results showed that isolate MetApCi from chili rhizosphere had the highest virulence causing 96.67% mortality of C. cramerella pupae and with an LT50 of 3.04 days.

Endophytic colonization of Vicia faba and Phaseolus vulgaris (Fabaceae) by fungal pathogens and their effects on the life-history parameters of Liriomyza huidobrensis (Diptera: Agromyzidae)

Abstract Ten fungal isolates belonging to the genera Beauveria (3), Hypocrea (1), Gibberella (1), Metarhizium (2), Trichoderma (1) and Fusarium (2) were evaluated in the laboratory to determine whether they could become endophytic in two pea leafminer (Liriomyza huidobrensis) host plants (Vicia faba and Phaseolus vulgaris), and to assess their possible negative effects on leafminers. Beauveria (ICIPE279), Hypocrea, Gibberella, Fusarium and Trichoderma isolates colonized roots, stems and leaves of both host plant species. Beauveria isolates G1LU3 and S4SU1 colonized roots, stems, and leaves of P. vulgaris but only the root and stem of V. faba. Isolates of Metarhizium failed to colonize the two host plants. The effects of endophytically colonized fungal pathogens on mortality, oviposition, emergence and longevity of L. huidobrensis were investigated after endophytic colonization of V. faba plants. All the fungal isolates that succeeded in colonizing the host plant were pathogenic to L. huidobrensis, causing 100 % mortality within 13.2 ± 0.7–15.0 ± 0.6 d. However, Hypocrea outperformed the other isolates (p

Use ofMetarhizium anisopliaeChitinase Genes for Genotyping and Virulence Characterization

Virulence is the primary factor used for selection of entomopathogenic fungi (EPF) for development as biopesticides. To understand the genetic mechanisms underlying differences in virulence of fungal isolates on various arthropod pests, we compared the chitinase genes, chi2 and chi4, of 8 isolates of Metarhizium anisopliae. The clustering of the isolates showed various groups depending on their virulence. However, the analysis of their chitinase DNA sequences chi2 and chi4 did not reveal major divergences. Although their protein translates have been implicated in fungal virulence, the predicted protein structure of chi2 was identical for all isolates. Despite the critical role of chitin digestion in fungal infection, we conclude that chi2 and chi4 genes cannot serve as molecular markers to characterize observed variations in virulence among M. anisopliae isolates as previously suggested. Nevertheless, processes controlling the efficient upregulation of chitinase expression might be responsible for different virulence characteristics. Further studies using comparative “in vitro” chitin digestion techniques would be more appropriate to compare the quality and the quantity of chitinase production between fungal isolates.

Characterization of Metarhizium anisopliae using amplifed ribossomal DNA restriction analysis (ARDRA) and internal transcribed spacer (ITS) sequence analysis

Molecular methods have revolutionized systematic entomology in the genus Metarhizium . In Brazil, the importance of these entomopathogenic fungi in agriculture is critical to the control of the sugar cane spittlebug ( Mahanarva posticata ) ( Homoptera: Cercopidae ). Other important factors for the selection of isolates of Metarhizium anisopliae for use in the biological control of particular pests are virulence, reproductive characteristics and the media of artificial cultures. In addition, the genetic variability of these entomopathogenic fungi indicates a great potential for pest control and a lack of a link between isolates from specific host sites and fungal virulence. We studied five Metarhizium hosts including the sugar cane spittlebug (IPA213, IPA215, IPA219 and IPA216) and the grasshopper (IPA217). It is noteworthy that these five isolates are used as biological control for growing sugar cane on an industrial scale for the Brazilian Northeast. Hence, the importance to genetically characterize these isolates of Metarhizium cannot be over emphasized. Amplifed ribossomal DNA restriction analysis (ARDRA) did not differentiate any of the isolates that were studied following digestion with three endonuclease namely; HindIII, HinfI and HpaI . The data that were generated using ITS1 and ITS4 markers revealed that although these isolates infect the same host, they are genetically distinct perhaps due to the unknown origin of the hosts. However, the isolate IPA217, which was isolated from the grasshopper, did not behave differently from the others. These results show that the M. anisopliae complex is monophyletic and it was concluded that the use of markers and morphological studies yielded results that may corroborate the idea that investigation of the phenotypic and molecular characteristics of this genus may indentify a new species of Metarhizium . Key words : Cordyceps brittebankisoides, genetic identity, ITS1, ITS4, phylogeny, sugar cane spittlebug.

Diversity and Genetic Population Structure of Fungal Pathogens Infecting White Grub Larvae in Agricultural Soils

White grub larvae are important soil-dwelling pests in many regions of Mexico as they attack many important crops such as maize. The use of synthetic chemicals is currently the main control strategy, but they are not always effective; thus, other alternatives are needed. Microbial control using entomopathogenic fungi represents an important alternative strategy, and species within the genera Beauveria and Metarhizium are considered amongst the most promising candidates. Seventeen Beauveria spp. and two Metarhizium spp. isolates were obtained in surveys of white grub larvae from different regions of Guanajuato, Mexico. All isolates were capable of infecting healthy larvae of the white grub Phyllophaga polyphilla in laboratory assays, but mortality never exceeded 50 %. Isolates were identified using morphological and molecular methods. Based on elongation factor1-α and ITS partial gene sequence data, all Beauveria isolates were identified as Beauveria pseudobassiana. Elongation factor1-α and β-tubulin sequence data identified the Metarhizium isolates to be Metarhizium pingshaense. In contrast, three additional Metarhizium isolates obtained the previous year in the same region were identified as M. pingshaense, Metarhizium anisopliae and Metarhizium robertsii. Microsatellite genotyping showed that all B. pseudobassiana isolates were the same haplotype. Enterobacterial Repetitive Intergenic Consensus fingerprinting information confirmed no significant variation amongst the B. pseudobassiana isolates. The ecological role of these isolates and their impact on white grub larvae populations are discussed.

Occurrence of Metarhizium spp. in Central Brazilian soils

The biodiversity of entomopathogenic fungi in tropical ecosystems is still little investigated, and the objective of this study was to isolate and identify fungi of the entomopathogenic genus Metarhizium (Hypocreales: Clavicipitaceae) present in undisturbed soils of the Central Brazilian Cerrado. A total of 107 Metarhizium isolates was obtained from soils collected from Cerrado sites in the state of Goiás; gene sequences from 63 of these were obtained and compared. Among these, one was confirmed to be M. anisopliae sensu stricto; 53 were very closely allied to M. anisopliae but require more extensive genetic characterization to determine if they might represent a new taxon in the M. anisopliae species complex. Eight of these Cerrado isolates were referable to M. robertsii, and the remaining isolate is the first South American (and Southern Hemisphere) collection of M. flavoviride var. pemphigi. These findings underline the need for better characterization of the diversity of these widely distributed fungi in Brazil.

Susceptibility of different developmental stages of large pine weevil Hylobius abietis (Coleoptera: Curculionidae) to entomopathogenic fungi and effect of fungal infection to adult weevils by formulation and application methods

The large pine weevil, Hylobius abietis, is a major pest in European conifer forests causing millions of Euros of damage annually. Larvae develop in the stumps of recently felled trees; the emerging adults feed on the bark of seedlings and may kill them. This study investigated the susceptibility of different developmental stages of H. abietis to commercial and commercially viable isolates of entomopathogenic fungi, Metarhizium and Beauveria. All the developmental stages of H. abietis can be killed by Metarhizium robertsii, Metarhizium brunneum, and Beauveria bassiana. The most virulent isolate of M. robertsii ARSEF4556 caused 100% mortality of pupae, larvae and adults on day 4, 6 and 12, respectively. This strain was further tested against adult weevils in different concentrations (105–108 conidia cm−2 or ml−1) using two types of fungal formulation: ‘dry’ conidia and ‘wet’ conidia (suspended in 0.03% aq. Tween 80) applied on different substrates (tissue paper, peat and Sitka spruce seedlings). ‘Dry’ conidia were more effective than ‘wet’ conidia on tissue paper and on spruce or ‘dry’ conidia premixed in peat. The LC50 value for ‘dry’ conidia of isolate ARSEF4556 was three folds lower than ‘wet’ conidia on tissue paper. This study showed that ‘dry’ conidia are more effective than ‘wet’ conidia, causing 100% adult mortality within 12days. Possible strategies for fungal applications are discussed in light of the high susceptibility of larvae and pupae to fungal pathogen.

Evaluation of indigenous fungal isolates and Metarhizium anisopliae var. acriidum against adult lesser wax moth, Achroia grisella (l) (Pyralidae: Lepidoptera)

The lesser wax moth (LWM) is a widely distributed and devastating insect pest to the honey production sector in Ethiopia. The present study aimed at investigating the potential of native fungal isolates against the lesser wax moth and assessing non target effect of one isolate of Beauveria (IITA 18) and five isolates of Metarhizium (IMI 330189, DLCO-AA83, DLCO-AA 109, DLCO-AA5, DLCO-AA14) via inoculating Ethiopian honeybee race, Apis mellifera bandasii . The effects of these six fungal isolates were evaluated in the laboratory for their pathogenicity to adult lesser wax moths. Spore dilutions were prepared in 0.5% Tween 80. Adults were treated by spraying 2 ml of conidial suspensions ranging from 2x10 4 to 2x10 7 conidia/ml. Adult lesser wax moths were found to be susceptible to all isolates and concentrations used. Comparison of post-treatment mortality of adult, lesser wax moth (LWM) at the lowest concentration (2x10 4 conidia/ml) revealed that infection of > 90% could be achieved by day 8 post inoculation. Investigation into effects of mycosis on percentage emergence of adults from fungal treated last instar larvae of the lwm showed no significant difference (P>0.05) between the treated and untreated controls. Laboratory based experiments on the host specificity of the six fugal isolates had no effect in which only 1 individual honeybee (0.5%) was infected with isolate IITA 18 (Beauveria spp.) while isolate DLCO-AA83 (Metarhizium spp.), caused infection in 2 individual honeybees.

Activity of Metarhizium spp. and Isaria spp. from the Central Brazilian Cerrado against Triatoma infestans nymphs

Entomopathogenic fungi attack Chagas disease vectors, and there is a need of effective isolates for development of biological control. Ten isolates of Metarhizium spp., four of Isaria cateniannulata and two of an Isaria sp., all isolated with Triatoma infestans as a bait insect from soils collected in Central Brazil, and tested under laboratory conditions were effective against third instar nymphs of T. infestans. This is the first report of pathogenicity of M. flavoviride var. pemphigi, M. robertsii and I. cateniannulata against T. infestans. M. robertsii IP 34 and I. cateniannulata IP 141 are among the most virulent fungi reported, and both have potential for integrated control of triatomine vectors.

Phylogenetic analysis of Metarhizium spp. isolated from soil in Japan

As a result of analyzing the internal transcribed spacer (ITS) and 5′ end of the EF-1α sequence of 145 isolates of Metarhizium spp. isolated from soil in Japan using selective agar medium, eight species were identified. ITS sequence analysis divided the isolates into three clades. Two were identified as M. flavoviride var. pemphigi and M. lepidiotae, respectively. EF-1α sequence analysis identified the other clades as six species: M. anisopliae, M. brunneum, M. guizhouense, M. majus, M. pingshaense and M. robertisii. The distribution of M. flavoviride var. pemphigi was restricted to forest or wood soil, and conidial sizes of M. guizhouense and M. majus were incongruent with the phylogeny based on the sequence of the 5′ end of EF-1α.

Comparison of volatile blends and gene sequences of two isolates of Metarhizium anisopliae of different virulence and repellency toward the termite Macrotermes michaelseni

Previously, we reported an interesting relationship between virulence and repellency of different isolates of the fungus Metarhizium anisopliae towards the termite Macrotermes michaelseni: the higher the virulence of a given isolate, the greater its repellency. In the present study, we compared the volatile profiles of two isolates, one that was more virulent (and repellent) and one that was less virulent (and repellent) to the termite. The prominent components of the two blends were characterized by gas chromatography-mass spectrometry and authenticated by gas chromatography co-injections with synthetic standards. There were both qualitative and quantitative differences between the two blends. The repellencies of synthetic blends of 10 prominent constituents of the volatiles of the two isolates were compared and that of the more virulent isolate was found to be significantly more repellent. Subtractive bioassays were carried out with one of the constituents of each of the two 10-component blends missing at a time to determine its relative contribution to the overall repellency. The results indicated that the repellency of the volatiles of each isolate was primarily due to synergistic effects of a smaller number of constituents. Intraspecific differences between the two isolates were also reflected in their nucleotide sequences.