Metarhizium Guizhouense Research Articles

Frequent horizontal chromosome transfer between asexual fungal insect pathogens

Entire chromosomes are typically only transmitted vertically from one generation to the next. The horizontal transfer of such chromosomes has long been considered improbable, yet gained recent support in several pathogenic fungi where it may affect the fitness or host specificity. To date, it is unknown how these transfers occur, how common they are, and whether they can occur between different species. In this study, we show multiple independent instances of horizontal transfers of the same accessory chromosome between two distinct strains of the asexual entomopathogenic fungus Metarhizium robertsii during experimental co-infection of its insect host, the Argentine ant. Notably, only the one chromosome-but no other-was transferred from the donor to the recipient strain. The recipient strain, now harboring the accessory chromosome, exhibited a competitive advantage under certain host conditions. By phylogenetic analysis, we further demonstrate that the same accessory chromosome was horizontally transferred in a natural environment between M. robertsii and another congeneric insect pathogen, Metarhizium guizhouense. Hence, horizontal chromosome transfer is not limited to the observed frequent events within species during experimental infections but also occurs naturally across species. The accessory chromosome that was transferred contains genes that may be involved in its preferential horizontal transfer or support its establishment. These genes encode putative histones and histone-modifying enzymes, as well as putative virulence factors. Our study reveals that both intra- and interspecies horizontal transfer of entire chromosomes is more frequent than previously assumed, likely representing a not uncommon mechanism for gene exchange.

Hyphal Growth and Conidia Germination Are Induced by Phytohormones in the Root Colonizing and Plant Growth Promoting Fungus Metarhizium guizhouense.

Beneficial associations are very important for plants and soil-dwelling microorganisms in different ecological niches, where communication by chemical signals is relevant. Among the chemical signals, the release of phytohormones by plants is important to establish beneficial associations with fungi, and a recently described association is that of the entomopathogenic ascomycete fungus Metarhizium with plants. Here, we evaluated the effect of four different phytohormones, synthetic strigolactone (GR24), sorgolactone (SorL), 3-indolacetic acid (IAA) and gibberellic acid (GA3), on the fungus Metarhizium guizhouense strain HA11-2, where the germination rate and hyphal elongation were determined at three different times. All phytohormones had a positive effect on germination, with GA3 showing the greatest effect, and for hyphal length, on average, the group treated with synthetic strigolactone GR24 showed greater average hyphal length at 10 h of induction. This work expands the knowledge of the effect of phytohormones on the fungus M. guizhouense, as possible chemical signals for the rapid establishment of the fungus-plant association.

Isolation and identification of Metarhizium guizhouense Xct1 and its pathogenicity to Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

BackgroundThe fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is one of the most destructive invasive pests causing serious damage to maize crops in China. At present, the use of chemical pesticides is the main method to prevent and control S. frugiperda. However, the excessive use of chemical pesticides not only causes toxicity in humans and animals but also pollutes the environment and causes public harm. In this context, the present study aimed to isolate, identify and evaluate the pathogenicity of the native strain of Metarhizium guizhouense against eggs, larvae and pupae of S. frugiperda under laboratory conditions.ResultsThe fungal isolate was identified as M. guizhouense (Xct1) based on its morphological and molecular characteristics by sequencing ITS, β-tubulin and EF1-α gene. Meanwhile, the fungal isolate was highly pathogenic to eggs of the S. frugiperda, at the highest concentration of 1 × 108 spores/ml, the cumulative egg hatching rate was only 8.33% at 10 days post exposure. After 10 days of exposure to the highest concentration of 1 × 108 spores/ml, the LC50 values for the first–fifth larval instars of S. frugiperda were 8.12 × 104, 1.11 × 105, 5.55 × 105, 3.87 × 106 and 1.43 × 107 spores/ml, respectively.ConclusionsThe pathogenicity of M. guizhouense Xct1 to S. frugiperda larvae gradually decreased with increasing the developmental stages. Metarhizium guizhouense Xct1 was more pathogenic to egg and younger larvae of S. frugiperda. This provides a basis for determining the optimal period of control.

Natural occurrence of Metarhizium in agricultural avocado soils (Persea americana Mill.) in Colima, Mexico

Avocado (Persea americana Mill.) has become one of the most important fruits in the world, being Mexico the main producer and exporter worldwide, it has led to an accelerated growth in the establishment of monoculture avocado plantations, promoting the increase of insect and mite pests, which has led to the need for biological control strategies for integrated pest management. The objective of this study was to isolate entomopathogenic fungi from the soil of a conventional avocado orchard and identify them by translation elongation factor 1-α (5’TEF). During the sampling, a total of 34 isolates were obtained, belonging to the genus Metarhizium, 23 isolates identified as Metarhizium robertsii and 11 as Metarhizium guizhouense. These results show the low diversity of entomopathogenic fungi species associated with the rhizosphere of the avocado crop and suggest that their occurrence could be associated with their ability to survive agronomic practices that induce their endophytic activity. It is necessary to understand the role of Metarhizium in the rhizosphere, this will allow the development of biological control programs specific for each crop, and thus guarantee its success in pest control.

The infection efficacy of Metarhizium strains (Hypocreales: Clavicipitaceae) against the Queensland fruit fly Bactrocera tryoni (Diptera: Tephritidae).

The Queensland fruit fly (Qfly), Bactrocera tryoni Froggatt, is a devastating pest of Australia's commercial fruit systems. Fruit fly mitigation is heavily centered around the use chemical insecticides, with limited investigation into microbial control alternatives. The wet tropics of northern Queensland is a highly biodiverse ecosystem containing many species of insect pathogenic fungi, but it is unclear whether any of these entomopathogens could contribute to Qfly management programs. In laboratory trials, we investigated the potential for Qfly microbial control by 3 locally sourced strains of entomopathogenic fungi comprising 2 species, Metarhizium guizhouense (Chen and Guo) and Metarhizium lepidiotae (Driver and Milner). Additionally, we evaluated 2 different inoculation methods to derive the most effective way to expose the flies to conidia-either through dry conidia or in a conidial suspension. All 3 strains were successful in causing Qfly mortality. Metarhizium lepidiotae resulted in the highest mean mortality over the trials, while M. guizhouense resulted in the highest mortality in a single replicate. Laboratory experiments revealed exposure through dry conidia to be the most effective method to inoculate the flies. These results suggest that fungal entomopathogens could be a viable pathway to Qfly suppression.

Isolation of Metarhizium guizhouense and Metarhizium robertsii Strains from Soil-Exposed Amblyomma americanum (Acarina: Ixodidae) from Northwest Arkansas, USA

The lone star tick, Amblyomma americanum (L.) (Acarina: Ixodidae), is the most abundant tick species found in Arkansas and is involved in the transmission of pathogens of medical and veterinary importance. When not feeding, most non-nidicolous tick species shelter in the soil and leaf litter where they may be exposed to and potentially infected with entomopathogenic fungi that reside naturally in the soil. Entomopathogenic fungi in the genus Metarhizium Sorokīn (Hypocreales: Clavicipitaceae) have shown promise as biological control agents of ticks. Here, the first study to isolate and identify Arkansas-derived isolates of Metarhizium from A. americanum ticks is presented. We exposed 320 ticks artificially to native soil from Savoy. Of these soil exposed ticks, 2.5% of adults and 1.5% of nymphs displayed signs of infection with Metarhizium. Of the infected Savoy adults, 3.3% were females and 1.7% were males. Similarly exposed ticks from West Fork resulted in only 2.4% of nymphal ticks being infected with this fungus. Eight isolates of Metarhizium were cultured from infected ticks exposed to soil from these locations. Four of these Metarhizium isolates (3 from Savoy and 1 from West Fork) were identified to species by sequencing of the ITS locus and the EF1-α genes. Three Savoy strains (P10N1, P10AF1, and P2AM1) had identical sequences and were identified as Metarhizium robertsii (Bischoff, Rehner & Humber) (Hypocreales: Clavicipitaceae). The strain from West Fork (P9N2) was identified as Metarhizium guizhouense (Chen & Guo) (Hypocreales: Clavicipitaceae). The ITS and the EF1-α sequences of the Savoy strains showed 100% similarity to M. robertsii strains ARSEF 2575 and ART 500, respectively. The ITS and EF1-α sequences of the West Fork strain showed 99% similarity to M. guizhouense strains ARSEF 977 and CBS 258.90, respectively. This study demonstrates that entomopathogenic fungi M. guizhouense and M. robertsii are pathogenic to and can be isolated from A. americanum. Furthermore, the EF1-α genetic marker was shown to be a very effective tool for distinguishing different species of Metarhizium from ticks when used in conjunction with ITS sequence data. Standardizing the use of ticks in soil exposure methods for isolating entomopathogenic fungi could be useful for obtaining isolates that are highly virulent to A. americanum. The isolation and identification of Metarhizium spp. from A. americanum in Arkansas indicates that further exploration of entomopathogenic fungi as biological agents to control A. americanum is warranted.

Land-Use Type Drives Soil Population Structures of the Entomopathogenic Fungal Genus Metarhizium.

Species of the fungal genus Metarhizium are globally distributed pathogens of arthropods, and a number of biological control products based on these fungi have been commercialized to control a variety of pest arthropods. In this study, we investigate the abundance and population structure of Metarhizium spp. in three land-use types—arable land, grassland, and forest—to provide detailed information on habitat selection and the factors that drive the occurrence and abundance of Metarhizium spp. in soil. At 10 sites of each land-use type, which are all part of the Swiss national soil-monitoring network (NABO), Metarhizium spp. were present at 8, 10, and 4 sites, respectively. On average, Metarhizium spp. were most abundant in grassland, followed by forest and then arable land; 349 Metarhizium isolates were collected from the 30 sites, and sequence analyses of the nuclear translation elongation factor 1α gene, as well as microsatellite-based genotyping, revealed the presence of 13 Metarhizium brunneum, 6 Metarhizium robertsii, and 3 Metarhizium guizhouense multilocus genotypes (MLGs). With 259 isolates, M. brunneum was the most abundant species, and significant differences were detected in population structures between forested and unforested sites. Among 15 environmental factors assessed, C:N ratio, basal respiration, total carbon, organic carbon, and bulk density significantly explained the variation among the M. brunneum populations. The information gained in this study will support the selection of best-adapted isolates as biological control agents and will provide additional criteria for the adaptation or development of new pest control strategies.

Impact of temperature and relative humidity on effectiveness of Metarhizium guizhouense PSUM02 against longkong bark eating caterpillar Cossus chloratus Swinhoe under laboratory and field conditions

The longkong bark eating caterpillar, Cossus chloratus Swinhoe (Cossidae: Lepidoptera), is a serious pest of longkong trees Lansium domesticum Corrêa that affects quality and quantity of the fruit. For eco-friendly management of this pest, Metarhizium guizhouense PSUM02, an entomopathogenic fungus, was tested under laboratory and field conditions of Thailand. Relative humidity (RH) more than 93% provides the highest virulence to M. guizhouense PSUM02 by reducing the survival of C. chloratus larvae to 0.0–18.0%. The average survival time (AST) of C. chloratus larvae infected with M. guizhouense PSUM02 at 100% RH showed the lowest AST of 3.9 ± 0.2 days. The optimum temperature for M. guizhouense PSUM02 to infect C. chloratus larvae was 25–30 °C with 2–20% survival after 10 days of treatment. The AST of C. chloratus larvae infected with M. guizhouense PSUM02 at 25 °C or 30 °C was 5.8 ± 0.6 or 3.4 ± 0.2 days, respectively, which was significantly lower than the AST observed at 20 °C and 35 °C temperatures. M. guizhouense PSUM02 showed effectiveness under field conditions by reducing the number of larvae of longkong bark eating caterpillar C. chloratus in treated longkong tree. The application of M. guizhouense PSUM02 could be an effective option for the eco-friendly management of longkong bark eating caterpillars in the longkong orchards of Thailand and other longkong producing countries.

Xuefeng Cordyceps:Insights into Species Diversity, Life Cycle and Host Association

Cordyceps (= Cordyceps sensu lato) fungi are rather well known as entomopathogens and also for their medicinal importance in curing many diseases. Many cordyceps species with widespread distribution throughout China have been recorded earlier. However, their taxonomy, hosts, life cycles and the plants on which the host insects reside in are poorly understood, and have sometimes led to confusion in the proper identification of the species. In this study, Xuefeng Cordyceps collected from Xuefeng Mountain, Hunan, China are identified and an assessment of their life cycles, proper hosts and plants they are associated with is made through morphological and DNA sequence- based analyses. Morphological characterization reveals that Ophiocordyceps xuefengensis from this region as commonly accepted by mycologists and local people. Species collected also include Beauveria bassiana (sexual and asexual), Metacordyceps taii (syn. of Metarhizium guizhouense ), Ophiocordyceps macroacicularis and Ophiocordyceps ramosissimum . Our result challenges previous taxonomic arrangement with regard to host identity. The hosts, previously mistaken for larvae of Endoclita nodus , are corrected herein as larvae of Endoclita davidi (Lepidoptera: Hepialidae). Ribosomal DNA sequence analyses based on the ITS regions also confirm that Clerodendrum cyrtophyllum (Lamiaceae) is the plant on which the host insects reside. Morphological illustrations, where appropriate, are given for the cordyceps species, hosts and plants. The infection mechanism and life cycle of the cordyceps are also outlined.

Biocontrol potential and genetic diversity of Metarhizium anisopliae lineage in agricultural habitats.

To assess phylogenetic and genotypic diversity of Metarhizium anisopliae lineage within diverse agroecosystems in the Karnataka State of India and to compare their chitinase activity and pathogenicity against insect pest of field crops subterranean termite, Odontotermes obesus. Three phylogenetic and 27 microsatellite markers were used to assess the genetic diversity of M. anisopliae lineage within multiple agroecosystems. Multilocus phylogeny of the Metarhizium isolates identified two species: Metarhizium pingshaense and Metarhizium guizhouense. Multilocus phylogeny and microsatellite markers resolved two phylogenetic species of M. pingshaense, Mp_1 and Mp_2, and one phylogenetic species of M. guizhouense, Mg_1. Phylogenetic species, Mp_2 and Mg_1, were detected with one genotype each and Mp_1 with eleven genotypes. Metarhizium pingshaense GKVK 02_16 isolate caused significantly high mortality of O. obesus in bioassays and detected with high chitinase activity. The study revealed phylogenetic and genotypic diversity of M. anisopliae lineage in agroecosystems of Karnataka State. Findings of pathogenicity and chitinase activity suggest that M. pingshaense GKVK 02_16 isolate provides effective control of O. obesus. The investigation provided an understanding of the genetic diversity and biocontrol efficiency of M. anisopliae lineage in agroecosystem. These data will serve as a resource in the future pest management strategies at a regional scale.

Biocontrol of Meloidogyne incognita by Metarhizium guizhouense and its protease

Crude extracts of the fungus Metarhizium guizhouense PSUM02 grown on protein- enriched medium showed high proteolytic activity and produced one main protein indicated as fungal protease with 33 kDa molecular mass. The protease activity had high stability when incubated at 10 or −20 °C for 6 months. The use of Metarhizium spores (1 × 104 spore mL−1) in combination with its protease (354.1 ± 2.2 U mL−1) completely inhibited hatching of the root-knot nematode Meloidogyne incognita within 24 h. Moreover, the mortality of juveniles increased to 100% at 48 h post treatment. The results were significantly different (p < 0.05) from Metarhizium spores or protease individually, as well as from extraction buffer and distilled water controls. Metarhizium spores suspended in crude protease rapidly germinated in 24 h and then mycelia invaded the immature eggs of M. incognita, while the eggs containing juveniles and the hatched juveniles were resistant. SEM observations showed extensive damage to eggshell and cuticle when treated with Metarhizium spore and protease, either individually or in combination. This is the first report on biocontrol potential of M. guizhouense against M. incognita. The combination of Metarhizium spores and protease has enhanced potential to develop into a safe and reliable method for plant parasitic nematode control.

Effect of Metarhizium guizhouense infection on mating competition and mate choice of Bactrocera latifrons (Diptera: Tephritidae)

Metarhizium guizhouense PSUM02 treated males of Bactrocera latifrons were investigated for the mating competition among males and mating choice by female flies to develop an auto-dissemination for the control of B. latifrons. In the present study, on day 1–4 of experiment, M. guizhouense–treated male flies were equally competitive with the normal male flies as we did not observe any differences in mating by treated and normal male flies of B. latifrons. Further, mating competitiveness were found low in treated adult male B. latifrons than normal male B. latifrons from 5th days of treatment until death. Kaplan-Meier survival analysis of treated male flies gave average survival times (AST) of 4.3 ± 0.1 days, while the healthy female and male flies in the same cage showed AST of 9.3 ± 0.3 and 8.3 ± 0.4 days, respectively. The AST of untreated flies in control experiment ranged from 14.2–14.5 days. In mating preference experiment, M. guizhouense–treated male flies were chosen by virgin female than gravid female flies for mating. The treated male flies caused mortality in both virgin and gravid female flies in the same cage with AST of 4.4 ± 0.1, 5.6 ± 0.1 and 7.4 ± 0.2 days, respectively, while untreated flies showed AST ranged from 13.9–14.3 days in control. The treated male flies could transmit the fungus infection to both untreated female and male flies as well as in virgin and gravid female flies by mating and contact. Our experiments showed the potentiality of M. guizhouense PSUM02 in management of B. latifrons by auto-dissemination with treated male flies, which transmit the fungus to a healthy population to reduce insect pest infestations.

Induction of defense responses in longkong fruit (Aglaia dookkoo Griff.) against fruit rot fungi by Metarhizium guizhouense

The defense responses of longkong fruit were induced by an entomopathogenic fungus, Metarhizium guizhouense. The longkong fruits were dipped in a spore suspension of M. guizhouense PSUM04 at concentration of 104 spore mL−1 for 5s, then an enzyme assay, a protein assay and polyacrylamide gel electrophoresis (PAGE) were done. It was found that the longkong fruit presented small necrotic spots on their peels within 24h. β-1,3-glucanase and chitinase were detected in peel extracts at 1.33±0.09 and 0.12±0.004UmL−1, respectively. The sodium dodecyl sulphate (SDS)-PAGE showed protein bands that emerged after the fungal treatment when compared to untreated control. These proteins were a 25–27kDa group of chitinase isoforms, and β-1,3-glucanase at 43kDa. From gel diffusion method, crude extracted from the fruit peel inhibited the mycelial growth of Botrytis sp. and Fusarium sp. as 34.9±3.1 and 29.3±5.0%, respectively. Moreover, the observation from scanning electron microscope (SEM) showed abnormal shapes, twisting and cracking in both fungal mycelia and less dense mycelial growth for Fusarium sp. Thus, M. guizhouense PSUM04 was not only a bio insecticide but it was also be the biotic inducer of plant defense responses against pathogenic fungi.

Species of Metarhizium anisopliae complex implicated in human infections: retrospective sequencing study

ObjectivesFungi belonging to the Metarhizium anisopliae complex comprise ubiquitous arthropod pathogenic moulds used as mycopesticides. Rare cases of human infections due to M. anisopliae have been reported. We hypothesize misidentifications of fungal strains implicated in these cases or used in mycopesticides. MethodsA review of the literature was conducted to identify previously published cases. We collected some of these previous described strains and reported new cases, and a French mycopesticide containing M. anisopliae. All identifications were performed based on elongation factor-1α gene sequencing. ResultsWe report eight new cases of Metarhizium infection in humans (three from France and five from Australia). The strains isolated from these cases, and three others from already published cases and reported as M. anisopliae, were molecularly identified based on elongation factor-1α (Ef1-α) gene sequencing as follows: Metarhizium robertsii (six), Metarhizium guizhouense (three), Metarhizium brunneum (one) and Metarhizium pingshaense (one). ConclusionsIn this study, we report new human cases of Metarhizium infections, and, based on Ef-1α gene sequencing, we demonstrate the misidentification of species in case reports. We also correct the species identification of a strain reported as M. anisopliae used in a commercially available mycopesticide. According to our results, none of the strains from the human infection reports reviewed belongs to the species M. anisopliae.

The phenotypic and metabolic properties of Metarhizium guizhouense on Corcyra cephalonica

The phenotypic and metabolic properties of Metarhizium guizhouense on Corcyra cephalonica

Confirmation of Two Undescribed Fungal Species from Dokdo of Korea Based on Current Classification System Using Multi Loci

Using dilution plating method, 47 fungal isolates were obtained from a soil sample collected from Dokdo in the East Sea of Korea in 2013. In this study, two fungal isolates, EML-MFS30-1 and EML-DDSF4, were confirmed as undescribed species, Metarhizium guizhouense and Mortierella oligospora in Korea based on current classification system using multi loci including rDNA internal transcribed spacer, large subunit, small subunit, and β-tubulin (BTUB) genes. Herein, detailed morphological descriptions on characters of the undescribed fungal species as well as their molecular phylogenetic status are provided with comparisons to related species.

Effects of autodisseminated Metarhizium guizhouense PSUM02 on mating propensity and mating competitiveness of Bactrocera cucurbitae (Diptera: Tephritidae)

We investigated the effects of inoculation by Metarhizium guizhouense PSUM02 on mating propensity and mating competitiveness of Bactrocera cucurbitae, with a view on pest management. On day 4 postinoculation, the M. guizhouense-treated male flies had significantly lowered mating propensity and mating competitiveness, while the treated female flies had reduced mating propensity on day 4 and reduced mating competitiveness on day 5. The mating propensity and competitiveness of treated male and female flies then further declined until death. Kaplan–Meier survival analysis of treated male and female flies gave average survival times (AST) of 6.2 ± 0.2 and 5.4 ± 0.3 days in the mating propensity assay, and about 5.0 ± 0.1 and 4.4 ± 0.2 days in the mating competitiveness assay. The AST of untreated flies ranged from 12.8 ± 0.1 to 14.7 ± 0.2 days for comparison (observation up to 15 days). Untreated flies had decreased AST and mating characteristics when exposed to contact with treated male flies, indicating transmission of the fungal infection by such contact also to untreated male flies. Surprisingly, contact with treated female flies did not affect the AST of untreated males or females in the same cage. These results corroborate the potential for pest control by autodissemination with treated male flies, which transmit the fungus to a healthy population better than the treated female flies.

Three sympatrically occurring species of Metarhizium show plant rhizosphere specificity

Here we tested the hypothesis that species of the soil-inhabiting insect-pathogenic fungus Metarhizium are not randomly distributed in soils but show plant-rhizosphere-specific associations. We isolated Metarhizium from plant roots at two sites in Ontario, Canada, sequenced the 5' EF-1α gene to discern Metarhizium species, and developed an RFLP test for rapid species identification. Results indicated a non-random association of three Metarhizium species (Metarhizium robertsii, Metarhizium brunneum and Metarhizium guizhouense) with the rhizosphere of certain types of plant species (identified to species and categorized as grasses, wildflowers, shrubs and trees). M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense. Supporting this, in vitro experiments showed that M. robertsii conidia germinated significantly better in Panicum virgatum (switchgrass) root exudate than did M. brunneum or M. guizhouense. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, predominantly Acer saccharum (sugar maple), while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types.

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 Metarhizium brunneum, 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.

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.