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.     

Metarhizium species in soil from Brazilian biomes: a study of diversity,distribution, and association with natural and agricultural environments

Metarhizium is a diverse genus of fungi adapted for different ecologies, including soil saprotrophs, entomopathogens, and endophytes. We characterized the genetic diversity and distribution of Metarhizium species in soils found in native and agricultural landscapes within Brazilian biomes (Amazon, Cerrado, Atlantic Forest, Caatinga, and Pampa). Current species limits were determined with 5′-TEF, and the genetic diversity discerned using MzIGS3 sequences. Metarhizium robertsii, Metarhizium anisopliae, Metarhizium pingshaense, and three other lineages that lie beyond currently recognized species were found. Only soils from the Amazon contained all the species. The diversity of Metarhizium species associated with native vegetation was greater than that identified in annual and perennial crops. M. robertsii was the most abundant species (65%), followed by Metarhizium sp. indet. 1, which exhibited the highest haplotype and nucleotide diversities. Metarhizium sp. indet. 3 was found predominantly in the Caatinga biome. This information increases the knowledge about diversity and belowground species composition of Metarhizium in Brazil.     

Susceptibility of Phyllophaga polyphylla and Anomala cincta larvae to Beauveria bassiana and Metarhizium anisopliae isolates, and the interaction with soil properties

The white grub species Phyllophaga polyphylla and Anomala cincta (Coleoptera: Melolonthidae) are economically important species that affect many crops in Mexico. A series of experiments to study the pathogenic interaction between isolates of Beauveria bassiana and Metarhizium anisopliae and these two insect species were undertaken. First, the susceptibility of third instar P. polyphylla larvae to each of seven isolates representing both species of fungus was evaluated by dipping the insects in 1?×?10⁸ conidia?ml^?1 suspensions. A second study examined the differences in the susceptibility of P. polyphylla and A. cincta larvae to two selected isolates for each of the fungal species. Finally, the susceptibility of A. cincta larvae to one M. anisopliae isolate when incubated in soil collected from four different sites was assessed. No significant differences in proportion of infection of P. polyphylla larvae were observed amongst the fungal isolates tested and mortality due to fungal infection was never greater than 20% after 36?days incubation. Anomala cincta larvae were more susceptible than P. polyphylla larvae, with greater than 90% infection when inoculated with isolates of M. anisopliae whereas mortalities of only 20% where achieved against P. polyphylla larvae. The soil type in which A. cincta were incubated following inoculation with M. anisopliae affected their susceptibility to infection. The results demonstrated that there is a complex interaction amongst entomopathogenic fungi, white grub larvae and soil properties, and points to the need of further investigation of this system in order to optimize the efficacy of entomopathogenic fungi against these insect species.     

Exploring virulence of new and less studied species of <Emphasis Type="Italic">Metarhizium</Emphasis> spp. from Brazil for two-spotted spider mite control

The two-spotted spider mite Tetranychus urticae is an important pest of strawberry crops in Brazil and many other countries. Focus for biocontrol studies involving entomopathogenic fungi has been on three species from the genus Metarhizium: M. anisopliae sensu stricto (s.s.), M. brunneum and M. robertsii. Also, the species Beauveria bassiana has been studied for spider mite control and one isolate (ESALQPL63) is commercially available in Brazil. New and undescribed Metarhizium species have been found recently in Brazil and provide a pool of isolates with potential for biocontrol in Brazil and probably also elsewhere. The mortality of adult females of T. urticae when exposed to four new Brazilian species of Metarhizium was compared to the mortality when exposed to M. anisopliae s.s., M. brunneum, M. pingshaense, M. robertsii and Beauveria bassiana ESALQPL63. Fungal suspensions were sprayed onto mites at 10⁷ conidia/mL with 0.05% Tween 80 in laboratory bio-assays. We measured total mortality and percentage sporulating cadavers 10 days after exposure and calculated median lethal time (LT₅₀). The lowest LT₅₀ (4.0 ± 0.17) was observed for mites treated with Metarhizium sp. Indet. 1 (ESALQ1638), which also performed well with respect to mortality after 10 days and capacity to sporulate from cadavers. Among the other little studied species tested, M. pingshaense (ESALQ3069 and ESALQ3222) and Metarhizium Indet. 2 (ESALQ1476) performed well and were comparable to B. bassiana (ESALQPL63). The new Metarhizium isolates and species thus showed potential for biological control.     

Diversity of indigenous Beauveria and Metarhizium spp. in a commercial banana field and their virulence toward Cosmopolites sordidus (Coleoptera: Curculionidae)

Metarhizium anisopliae and Beauveria bassiana sensu lato were isolated, from 7 and 41 % of soil samples from a commercial banana field, with average fungal density of 4.3 × 10³ and 8.2 × 10³ CFU g^?1 soil, respectively. Twenty-one morphologically distinct B. bassiana and four M. anisopliae sensu lato isolates from different plots within the field were further characterized. ISSR fingerprinting revealed six different clusters for B. bassiana, whereas gene sequencing revealed three M. anisopliae sensu stricto and one unclassified Metarhizium sp. Bioassays with one or more representative isolates from each Metarhizium species and B. bassiana cluster showed that all indigenous isolates had lower virulence and significantly greater ST₅₀s than reference (exotic) isolates. The data suggest that the low virulence of most indigenous isolates toward Cosmopolites sordidus adults and their relatively low density in soil samples, may help explain the low occurrence of epizootics caused by entomopathogenic fungi in populations of this pest, also known to burrow under the soil surface in banana plantations.     

Pathogenicity of hyphomycetous fungi against <Emphasis Type="Italic">Cyclocephala signaticollis</Emphasis>

Susceptibility of the white grub Cyclocephala signaticollis Burmeister (Coleoptera: Scarabaeidae: Dynastinae) larvae to seven isolates of Beauveria bassiana (Balsamo) Vuillemin, five of Metarhizium anisopliae (Metschnikoff) Sorokin and two of Paecilomyces lilacinus (Thom) Samson (Deuteromycotina: Hyphomycetes) was investigated. Among 14 fungal isolates screened the most virulent was a B. bassiana isolate (Bb 53) that caused 70% mortality of third instar larvae in 40 days after inoculation at 1 × 10⁸ conida/ml. Strains of M. anisopliae and P. lilacinus showed low efficacy or no virulence to the target host.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Pathogenicity of Beauveria bassiana,metarhizium anisopliae, and Paecilomyces farinosus to larvae and adults of S. scolytus

The entomogenous fungi Beauveria bassiana (nine isolates), Metarhizium anisopliae (seven isolates), and Paecilomyces farinosus (four isolates) were tested as pathogens of larvae of the elm bark beetle, Scolytus scolytus. Single isolates of B. bassiana and M. anisopliae were also tested against adult beetles. Of the 21 isolates tested as conidial suspensions against larvae, all proved pathogenic. The three most and least virulent isolates were, respectively, isolates of B. bassiana and M. anisopliae. The other isolates fell between these two extremes, with the four P. farinosus isolates all moderately virulent. Spore retention on larvae following inoculation was estimated by washing conidia off the larvae. From the results it was possible to relate larval mortality to the approximate spore dose causing infection at different spore concentrations. Thus, application of spores of the three pathogens at a concentration of 10³ spores/ml resulted in limited mortality. At this concentration, an average of only a single spore was recovered from the inoculated larva. Adult bark beetles also proved susceptible to infection by isolates of B. bassiana and M. anisopliae. They were exposed to discs of elm bark dipped in a conidial suspension. It was estimated that a dose of less than 100 spores could cause infection of beetles following feeding on the elm bark discs.     

Comparative gene expression and genomics reflect geographical divergence in the plant symbiotic and entomopathogenic fungal genus Metarhizium

Several species within the fungal genus Metarhizium can both infect insects and colonize plant roots. In Brazil, a specific subgroup within Metarhizium anisopliae s.str. named “subclade Mani 2” is frequently observed infecting above-ground insects, whereas sympatric M. robertsii and M. brunneum predominantly occur in the soil environment. Genotypic variability within the genus may be linked to adaptations to these different habitats. We present a comparative analysis of the complete genomes and the adhesin genes Mad1 and Mad2 of 14 Metarhizium isolates representing M. anisopliae Mani 2 (n = 6), M. robertsii (n = 5) and M. brunneum (n = 3). In addition, the relative gene expression of six selected target genes was compared in root exudate solution and insect cuticle suspension. We hypothesized that M. anisopliae Mani 2 is adapted to insect-pathogenicity in the above-ground environment, reflected by higher relative expression of pathogenicity-related genes. In contrast, M. robertsii and M. brunneum are adapted to the soil environment, hence hypothesized to have a higher expression of genes related to plant associations. Phylogenomic and adhesin phylogenetic trees revealed species differences but also intraspecific variability associated with the geographic origin of isolates. Differences in relative gene expression were observed, with one pathogenicity-related gene (Pr1) being higher expressed in M. anisopliae. The insect adhesion Mad1 gene was more conserved than the plant adhesion Mad2 and similarly expressed in exudate solution, while Mad2 was highly expressed by all Brazilian isolates in both exudate and cuticle conditions. The variabilities observed correlated with different habitats and lifestyles, demonstrating the importance of selecting a diverse collection of isolates in genomic and gene expression studies.     

Interaction between entomopathogenic nematodes and entomopathogenic fungi applied to third instar southern masked chafer white grubs,Cyclocephala lurida (Coleoptera: Scarabaeidae), under laboratory and greenhouse conditions

Four entomopathogenic nematode (EPN) species (Heterorhabditis bacteriophora Poinar, Heterorhabditis megidis Poinar, Jackson & Klein, Steinernema feltiae Filipjev and Steinernema riobrave Cabanillas, Poinar & Raulston) were tested for virulence against 3^rd instar southern masked chafer white grubs, Cyclocephala lurida Bland. H. bacteriophora and H. megidis, being the most virulent, were selected to evaluate the interaction with an entomopathogenic fungus (EPF), Beauveria bassiana (Balsamo) Vuillemin strain GHA or Metarhizium anisopliae (Metsch.) Sorokin strain F-52, under laboratory and greenhouse conditions. Nematodes and fungi were either applied alone or in combination, with nematodes added to fungi at different times. When applied alone, B. bassiana and M. anisopliae did not reduce grub numbers. Under laboratory conditions, additive interactions were found between H. megidis and B. bassiana, and between H. bacteriophora and B. bassiana or M. anisopliae in most combinations against chafer grubs; a few treatments showed synergism or antagonism. The combined effect did not differ significantly for nematode and fungal applications made simultaneously or at different times. Nematode infection and infective juveniles (IJs) production in grub carcasses were not significantly affected by the presence of a fungus. Efficacies of H. bacteriophora and M. anisopliae were affected by temperature, with grub mortality increasing at higher temperatures. Under greenhouse conditions, additive or synergistic interaction was found between H. bacteriophora and B. bassiana or M. anisopliae in different formulations in simultaneous applications or when the nematode was applied 4 weeks after the fungi, except between B. bassiana ES and H. bacteriophora. The impact of H. bacteriophora alone or in combination with M. anisopliae or B. bassiana on 3^rd instar C. lurida was comparable to that of an imidacloprid insecticide used as curative applications. More virulent fungal strains or species may be required to achieve a stronger interaction with nematodes in the management of C. lurida.     

Genetic diversity of Metarhizium anisopliae var. anisopliae in southwestern British Columbia

The abundance and genetic diversity of the entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, in southwestern British Columbia (BC) and southern Alberta was examined. The fungus was found to be widespread in soil throughout southwestern BC, and was recovered from 56% of 85 sample sites. In contrast to southwestern BC, no M. anisopliae isolates were recovered in southern Alberta. An automated fluorescent amplified fragment length polymorphism (AFLP) method was used to examine genetic diversity. In excess of 200 isolates were characterized. The method identified 211 polymorphic amplicons, ranging in size from ≈92 to 400 base pairs, and it was found to be reproducible with a resolution limit of 86.2% similarity. The AFLP method distinguished Metarhizium from other entomopathogenic fungal genera, and demonstrated considerable genetic diversity (25 genotypes) among the reference strains of M. anisopliae isolates examined (i.e. recovered from various substrates and geographical locations). Although 13 genotypes of M. anisopliae var. anisopliae were recovered from southwestern BC soils, the vast majority of isolates (91%) belonged to one of two closely-related genotypes. Furthermore, these two genotypes predominated in urban, agricultural and forest soils. The reasons for the limited diversity of M. anisopliae var. anisopliae in southwestern BC are uncertain. However, findings of this study are consistent with island biogeography theory, and have significant implications for the development of this fungus for microbial control of pest insects.     

Isolation and characterization of entomopathogenic fungi from hazelnut-growing region of Turkey

Although Turkey is the first among all hazelnut-producing countries, yield per unit area of this crop is low in comparison to other countries, mainly because many insect species seriously damage hazelnut trees and their fruit. To find effective and safe biocontrol agents, we conducted a survey study to isolate entomopathogenic fungi from the hazelnut-growing region of Turkey and characterized the isolated strains in detail. In addition, we determined the effectiveness of seven selected strains from this region against Melolontha melolontha (Coleoptera: Scarabaeidae) which is one of the most serious pests of hazelnut. In 2006 and 2007, 301 soil samples were collected randomly and analyzed for presence of entomopathogenic fungi using the Galleria bait method. Entomopathogenic fungi were found to occur in 20.59% of the soil samples studied. Based on morphology, ITS sequence and partial sequencing of the 18S (SSU rDNA) and EF1-α genes, the isolates were identified as Metarhizium anisopliae var. anisopliae, Metarhizium sp., Beauveria bassiana, Beauveria cf. bassiana, Isaria fumosorosea and Evlachovaea sp. Metarhizium anisopliae var. anisopliae was isolated from 34 sites and was the most frequent and abundant entomopathogenic species recovered. All the isolates tested were pathogenic to M. melolontha. M. anisopliae var. anisopliae KTU-27 and Evlachovaea sp. KTU-36 produced the highest insecticidal activity (86.6%) within 15 days after inoculation. Our results suggest that entomopathogenic fungi could be good biocontrol agents against M. melolontha, and are discussed with respect to ecology of fungi in relation to habitat in order to evaluate biocontrol potential of these isolates. This is the first study of the distribution of entomopathogenic fungi in the hazelnut-growing region of Turkey and of their pathogenicities against M. melolontha.     

Effect of culture medium on the production and virulence of submerged spores of Metarhizium anisopliae and Beauveria bassiana against larvae and adults of Aedes aegypti (Diptera: Culicidae)

Three Metarhizium anisopliae and three Beauveria bassiana isolates were cultivated in media containing casamino acids, soybean flour or sunflower seed flour and were shaken for three days. M. anisopliae presented similar yields of around 10⁶ submerged spores/ml without significant differences among them, whereas B. bassiana produced yields of around 10⁸ spores/ml, of which GHA strain produced more submerged spores in the casamino acids medium. The other two strains showed no significant difference in the production of submerged spores in the three media used. Differences in mortality on Aedes aegypti larvae were observed with the submerged spores of Metarhizium depending on isolate and medium used. M. anisopliae 2157 caused significantly higher mortality (40%) when cultivated in casamino acids medium. It presented an LC₅₀ of 8.93 × 10⁵ submerged spores/ml water against mosquito larvae five days after application, whereas it caused 27% mortality in Ae. aegypti adults 10 days after application. In conclusion, fungal nutrition affected virulence of some isolates of M. anisopliae against Ae. aegypti larvae while such an effect was not noted for B. bassiana isolates.     

A PCR-based tool for cultivation-independent detection and quantification of Metarhizium clade 1

The entomopathogenic fungus Metarhizium anisopliae and sister species are some of the most widely used biological control agents for insects. Availability of specific monitoring and quantification tools are essential for the investigation of environmental factors influencing their environmental distribution. Naturally occurring as well as released Metarhizium strains in the environment traditionally are monitored with cultivation-dependent techniques. However, specific detection and quantification may be limited due to the lack of a defined and reliable detection range of such methods. Cultivation-independent PCR-based detection and quantification tools offer high throughput analyses of target taxa in various environments. In this study a cultivation-independent PCR-based method was developed, which allows for specific detection and quantification of the defined Metarhizium clade 1, which is formed by the species Metarhizium majus, Metarhizium guizhouense, Metarhizium pingshaense, Metarhizium anisopliae, Metarhizium robertsii and Metarhiziumbrunneum, formerly included in the M. anisopliae cryptic species complex. This method is based on the use of clade-specific primers, i.e. Ma 1763 and Ma 2097, that are positioned within the internal transcribed spacer regions 1 and 2 of the nuclear ribosomal RNA gene cluster, respectively. BLAST similarity searches and empirical specificity tests performed on target and non-target species, as well as on bulk soil DNA samples, demonstrated specificity of this diagnostic tool for the targeted Metarhizium clade 1. Testing of the primer pair in qPCR assays validated the diagnostic method for specific quantification of Metarhizium clade 1 in complex bulk soil DNA samples that significantly correlated with cultivation-dependent quantification. The new tool will allow for highly specific and rapid detection and quantification of the targeted Metarhizium clade 1 in the environment. Habitat with high Metarhizium clade 1 densities can then be analyzed for habitat preferences in greater detail using cultivation-dependent techniques and genetic typing of isolates.     

Species of the Metarhizium anisopliae complex with diverse ecological niches display different susceptibilities to antifungal agents

Species of the Metarhizium anisopliae complex are globally ubiquitous soil-inhabiting and predominantly insect-pathogenic fungi. The Metarhizium genus contains species ranging from specialists, such as Metarhizium acridum that only infects acridids, to generalists, such as M. anisopliae, Metarhizium brunneum, and Metarhizium robertsii that infect a broad range of insects and can also colonize plant roots. There is little information available about the susceptibility of Metarhizium species to clinical and non-clinical antifungal agents. We determined the susceptibility of 16 isolates comprising four Metarhizium species with different ecological niches to seven clinical (amphotericin B, ciclopirox olamine, fluconazole, griseofulvin, itraconazole, tebinafine, and voriconazole) and one non-clinical (benomyl) antifungal agents. All isolates of the specialist M. acridum were clearly more susceptible to most antifungals than the isolates of the generalists M. anisopliae sensu lato, M. brunneum, and M. robertsii. All isolates of M. anisopliae, M. brunneum, and M. robertsii were resistant to fluconazole and some were also resistant to amphotericin B. The marked differences in susceptibility between the specialist M. acridum and the generalist Metarhizium species suggest that this characteristic is associated with their different ecological niches, and may assist in devising rational antifungal treatments for the rare cases of mycoses caused by Metarhizium species.     

Diversity of rhizosphere associated entomopathogenic fungi of perennial herbs, shrubs and coniferous trees

Understanding habitat selection of fungal entomopathogens is critical to improve the efficacy, persistence and cost of these fungi as microbial insecticides. This study sought to determine the prevalence of Metarhizium and Beauveria spp. isolated from the rhizosphere of strawberry, blueberry, grape and Christmas tree crops in the Willamette Valley of Oregon. Entomopathogenic fungi were assigned to thirteen species based on molecular phylogenetic criteria. Four species of Metarhizium were isolated including Metarhizium brunneum, Metarhizium guizhouense, Metarhizium robertsii, and Metarhizium flavoviride var. pemphigi. Nine Beauveria species were isolated including, Beauveria brongniartii, an undescribed species referred to as Clade C and seven phylogenetic species of Beauveria bassiana. Strawberries and blueberries were significantly associated with M. brunneum and Christmas trees with M. guizhouense and M. robertsii. Grapes were significantly associated with B. bassiana phylogenetic species Bbas-16. All of the Metarhizium isolates screened were pathogenic to Otiorhynchus sulcatus larvae in laboratory bioassays but only M. brunneum and M. robertsii caused significant levels of infection. The study results suggest that certain species of Metarhizium and Beauveria are significantly associated with the strawberry, blueberry and Christmas tree rhizosphere and could potentially provide better control of O. sulcatus.     

Microsclerotia production of Metarhizium spp. for dual role as plant biostimulant and control of Spodoptera frugiperda through corn seed coating

The fungal genus Metarhizium comprises entomopathogenic species capable of producing overwintering structures known as microsclerotia. These structures offer many advantages in pest control due to the formation of infective conidia in situ and their persistence in the environment under adverse conditions. In addition, the in vitro production of Metarhizium microsclerotia under controlled liquid fermentation is faster and with greater process control than the production of aerial conidia. However, the potential of Metarhizium microsclerotia to control pests from the orders Lepidoptera and Hemiptera is unexplored. In this study, we examined the ability of Metarhizium spp. microsclerotia to promote corn growth and to provide plant protection against Spodoptera frugiperda (Lepidoptera: Noctuidae) and Dalbulus maidis (Hemiptera: Cicadellidae), through seed coating using microsclerotial granules. A screening to find higher microsclerotia producers was conducted by culturing 48 native Brazilian isolates of Metarhizium spp. (Metarhizium anisopliae, Metarhizium robertsii, Metarhizium humberi and Metarhizium sp. indeterminate). The best microsclerotia producers, M. anisopliae ESALQ1814, M. robertsii ESALQ2450 and M. humberi ESALQ1638 improved the leaf area, plant height, root length, and dry weight of plants compared to un-inoculated plants. Significant reduction in S. frugiperda survival (mortality > 55% after 7 days) was observed when larvae were fed on corn plants treated with any of the three Metarhizium species. Conversely, survival of D. maidis adults were unaffected by feeding on fungus-inoculated plants. Our results suggest that microsclerotia of Metarhizium spp. may act as biostimulants and to provide protection against S. frugiperda in corn through seed coating, thus adding an innovative strategy into the integrated management of this major worldwide pest.     

Factors affecting the occurrence and distribution of entomopathogenic fungi in natural and cultivated soils

Factors affecting the occurrence and distribution of entomopathogenic fungi in 244 soil samples collected from natural and cultivated areas in Spain were studied using an integrated approach based on univariate and multivariate analyses. Entomopathogenic fungi were isolated from 175 of the 244 (71.7 %) soil samples, with only two species found, Beauveria bassiana and Metarhizium anisopliae. Of the 244 soil samples, 104 yielded B. bassiana (42.6 %), 18 yielded M. anisopliae (7.3 %), and 53 soil samples (21.7 %) harboured both fungi. Log-linear models indicated no significant effect of habitat on the occurrence of B. bassiana, but a strong association between M. anisopliae and soils from cultivated habitats, particularly field crops. Also, irrespective of habitat type, B. bassiana predominated over M. anisopliae in soils with a higher clay content, higher pH, and lower organic matter content. Logistic regression analyses showed that pH and clay content were predictive variables for the occurrence of B. bassiana, whereas organic matter content was the predictive variable for M. anisopliae. Also, latitude and longitude predicted the occurrence of these same species, but in opposite directions. Altitude was found to be predictive for the occurrence of B. bassiana. Using principal component analysis, four factors (1 to 4) accounted for 86 % of the total variance; 32.8, 22.9, 19.6 and 10.4 % of the cumulative variance explained, respectively. Factor 1 was associated with high positive weights for soil clay and silt content and high negative weights for soil sand content. Factor 2 was associated with high positive weights for soil organic matter content and high negative weights for soil pH. Factor 3 was associated with high positive weights for latitude and longitude of the sampled localities and factor 4, had high positive weights only for the altitude. Bi-plot displays representing soil samples were developed for different factor combinations and indicated that, irrespective of geographical location, absence of both fungal species was determined by alkaline sandy soils with low organic matter content, whereas heaviness of soil texture, acidity and increasing organic matter content led to progressively higher percentages of samples harbouring entomopathogenic fungi. These results could aid decision-making as to whether or not a particular cultivated or natural soil is suitable for using entomopathogenic fungi as a pest control measure and for selecting the fungal species best suited to a particular soil.     

Molecular characterization and estimation of cellulolytic potential of gut bacteria isolated from four white grub species native to Indian Himalayas

An investigation was carried out to isolate, identify and molecularly characterize the cellulose-degrading bacterial isolates from the guts of four white grub species (Anomala bengalensis, Brahmina coriacea, Holotrichia longipennis and Holotrichia setticollis) native to Uttarakhand, Himalayas through 16S rRNA sequencing. A total of 178 bacterial strains were isolated from different gut compartments of selected white grub species, of which 95 bacterial isolates showed cellulose metabolizing activities in the CMC assay. Maximum degraders i.e., 38 were isolated from A. bengalensis, of which 18 were isolated from the fermentation chamber. The value of cellulolytic index ranged between 0.05 and 16 showing a variable cellulolytic activity by degraders. A total of 25 potent strains of cellulose-degrading bacteria recording cellulolytic activity > 1 were isolated and sequenced for 16S rRNA gene. Bacillus stratosphericus strain CBG4MG1 (10.78 ± 4.18), Bacillus cereus strain CBG2FC1 (10.33 ± 3.53), Bacillus sp. strain CBG3MG2 (7.28 ± 0.16) and Paenibacillus ginsengagri strain CBG1FC2 (5.66 ± 2.67) were the most potent cellulose-degrading bacteria isolated from the gut of B. coriacea, H. longipennis, H. setticollis and A. bengalensis, respectively. Thus, the cellulolytic bacteria isolated from the gut of selected white grub species may be good sources for profiling novel isolates for industrial use besides identifying eco-friendly solutions for agro-waste management.     

Genetic diversity of the Metarhizium anisopliae complex in Colima,Mexico, using microsatellites

Metarhizium anisopliae is a complex of cryptic species with wide geographical distribution and versatile lifestyles. In this study, 45 isolates of the Metarhizium genus harbored in the “Colección de Hongos Entomopatógenos” of the “Centro Nacional de Referencia de Control Biológico” from different substrates, insect-host, and localities from Colima, Mexico, were phylogenetically identified using the 5′end of translation elongation factor 1-α (5′TEF) and intergenic nuclear region MzFG543igs. Seven species were recognized, M. acridum (n = 26), M. pemphigi (n = 1), and within the PARB and MGT clades: M. anisopliae (N = 7; sensu stricto: n = 2; sensu lato: n = 5), M. brunneum (n = 2), M. guizhouense (n = 2), M. pingshaense (n = 2), and M. robertsii (n = 5). Twenty-nine SSR markers were developed for M. acridum; according to the analysis of 12 polymorphic SSR loci, M. acridum showed low genetic diversity, revealing five genotypes with a dominant one (n = 21). Based on the analysis of 13 specific SSR loci, 14 genotypes were identified within the PARB and MGT clades. This study contributes to generating valuable information about the community structure and genotypic diversity of Metharhizum species in the state of Colima, Mexico.     

Phylogenetic analysis of <Emphasis Type="Italic">Metarhizium</Emphasis> 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.