Metarhizium Species Research Articles

Metarhizium fight club: Within-host competitive exclusion and resource partitioning.

Both Metarhizium robertsii ARSEF 2575 (Mr2575) and Metarhizium anisopliae ARSEF 549 (Ma549) infect a range of insects whilst also interacting with plants; however, little is known about the traits that affect the competitive ability of different strains. We examined the interactions between Mr2575 and Ma549 in culture and during co-infection of plants (Arabidopsis thaliana) and insects. Mr2575 outcompetes Ma549 under nutrient-limiting conditions, including root exudates, giving it a priority advantage on Arabidopsis roots. However, during co-infection of Manduca sexta or Drosophila melanogaster, Ma549's higher blastospore production enhanced its competitive ability within the host. In large M. sexta (fifth instar), blastospores facilitate dispersal, suppress host melanization and prevent Mr2575 from spreading from infection sites, reducing conidia production. However, colonization of smaller hosts such as first instar M. sexta and D. melanogaster did not provide Ma549 with a competitive advantage, as conidial production was dependent on retaining control of the cuticle through which conidiating hyphae emerge. Unexpectedly, Ma549 and Mr2575 segregate within hosts, suggesting resource partitioning with Mr2575 predominating in the thoraxes of Drosophila, especially in females, and Ma549 in the abdomen. In fifth instar M. sexta, Mr2575 was most prevalent around spiracles and the front end of segments, despite Ma549 and Mr2575 having similar susceptibility to hypoxia. Dispersing conidia homogeneously into the hemocoel of fifth instar M. sexta eliminated the blastospore production advantage, making Ma549 and Mr2575 equally competitive, with strict partitioning of Mr2575 at the anterior and Ma549 at the posterior ends of segments. As Metarhizium species have multiple roles in natural ecosystems and agroecosystems these discoveries are relevant to understanding their impact on maintaining biodiversity and for exploiting them to enhance food security.

Metarhizium caribense sp. nov., a Novel Species of Entomopathogenic Metarhizium Fungi Associated with Weevils Impairing Coffee, Sugar Cane and Sweet Potato Cultivation.

(1) Background: Insect pathogenic fungi of the genus Metarhizium are under study and in application as highly solicited, more eco-system friendly substitutes for chemical insecticides in many countries and in different agricultural contexts. In Cuba and Florida, Metarhizium strains have previously been isolated from economically important coffee and sugar cane pests. (2) Methods: Unambiguous species delineation within the Metarhizium anisopliae species complex is methodologically challenging. Recently, a species-discriminating PCR approach has been developed based on ribosomal intergenic spacer (rIGS) sequences that covered the prominent four "PARB" species within the complex. This approach is combined here with further genetic markers and is extended to a further species. (3) Results: Metarhizium isolates from Cuba, found to be more naturally associated with the coffee berry borer, Hypothenemus hampei, were morphologically, microscopically and molecular taxonomically characterized. Multilocus sequence analysis based on 5TEF, MzIGS3 and rIGS markers delineated these weevil-associated strains from all previously established Metarhizium species. (4) Conclusions: The isolates under study represent a new fungal taxon proposed to be designated Metarhizium caribense. The rIGS-based species-discriminating diagnostic PCR is a suitable tool for the identification of new Metarhizium species and can be productively combined to approaches using further genetic markers.

Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis

IntroductionOchratoxins (OTs) are worldwide regulated mycotoxins contaminating a variety of food-environment and agro-environment. Several Aspergillus and Pencillium species synthesize OTs from a six-gene biosynthetic gene cluster (BGC) to produce the highly toxic final product OTA. Although many studies on OTA-degrading enzymes were performed, high efficiency enzymes with strong stability are extremely needed, and the OTA degrading mechanism is poorly understood. ObjectivesThe study aimed to explore the OT-degradation enzyme and investigate its degradation mechanisms in Metarhizium, which contain an OT biosynthetic gene cluster. MethodsPhylogenomic relationship combined with RNA expression analysis were used to explore the distribution of OT BGC in fungi. Bioactivity-guided isolation and protein mass spectrometry were conducted to trace the degrading enzymes in Metarhizium spp., and the enzymes were heterologously expressed in E. coli and verified by in vitro assays. Structure prediction and point mutation were performed to reveal the catalytic mechanism of MbAmh1. ResultsBeyond Aspergillus and Pencillium species, three species of the distant phylogenetic taxon Metarhizium contain an expressed OT-like BGC but lack an otaD gene. Unexpectedly, no OT BGC products were found in some Metarhizium species. Instead, Metarhizium metabolized both OTA and OTB to their non-toxic degradation products. This activity of M. brunneum was attributed to an intracellular hydrolase MbAmh1, which was tracked by bioactivity-guided proteomic analysis combined with in vitro reaction. Recombinant MbAmh1 (5 μg/mL) completely degraded 1 μg/mL OTA within 3 min, demonstrating a strong degrading ability towards OTA. Additionally, MbAmh1 showed considerable temperature adaptability ranging from 30 to 70 °C and acidic pH stability ranging from 4.0 to 7.0. Identification of active sites supported the crucial role of metal iron for this enzymatic reaction. ConclusionThese findings reveal different patterns of OT synthesis in fungi and provide a potential OTA degrading enzyme for industrial applications.

Transfection of entomopathogenic Metarhizium species with a mycovirus confers hypervirulence against two lepidopteran pests

Although most known viruses infecting fungi pathogenic to higher eukaryotes are asymptomatic or reduce the virulence of their host fungi, those that confer hypervirulence to entomopathogenic fungus still need to be explored. Here, we identified and studied a novel mycovirus in Metarhizium flavoviride, isolated from small brown planthopper (Laodelphax striatellus). Based on molecular analysis, we tentatively designated the mycovirus as Metarhizium flavoviride partitivirus 1 (MfPV1), a species in genus Gammapartitivirus, family Partitiviridae. MfPV1 has two double-stranded RNAs as its genome, 1,775 and 1,575 bp in size respectively, encapsidated in isometric particles. When we transfected commercial strains of Metarhizium anisopliae and Metarhizium pingshaense with MfPV1, conidiation was significantly enhanced (t test; P-value < 0. 01), and the significantly higher mortality rates of the larvae of diamondback moth (Plutella xylostella) and fall armyworm (Spodoptera frugiperda), two important lepidopteran pests were found in virus-transfected strains (ANOVA; P-value < 0.05). Transcriptomic analysis showed that transcript levels of pathogenesis-related genes in MfPV1-infected M. anisopliae were obviously altered, suggesting increased production of metarhizium adhesin-like protein, hydrolyzed protein, and destruxin synthetase. Further studies are required to elucidate the mechanism whereby MfPV1 enhances the expression of pathogenesis-related genes and virulence of Metarhizium to lepidopteran pests. This study presents experimental evidence that the transfection of other entomopathogenic fungal species with a mycovirus can confer significant hypervirulence and provides a good example that mycoviruses could be used as a synergistic agent to enhance the biocontrol activity of entomopathogenic fungi.

Mycophagous Mite, Tyrophagus putrescentiae, Prefers to Feed on Entomopathogenic Fungi, except Metarhizium Generalists.

(1) Background: The mycophagous mite, Tyrophagus putrescentiae, was found to feed on entomopathogenic fungi (EPF) in our previous experiments, which seriously impacted the culture and preservation of fungal strains. Therefore, it is necessary to investigate the biological characteristics of the occurrence and damage to EPF. (2) Methods: The mite's growth and development and feeding preference were surveyed by comparative culture and observation; also, optical and electronic microscopies were employed. (3) Results: T. putrescentiae could survive normally after being fed on seven EPF species, including Purpureocillium lilacinum, Marquandii marquandii, Cordyceps fumosorosea, Beauveria bassiana, Metarhizium flavoviride, Lecanicillium dimorphum, and Metacordyceps chlamydosporia. The first four fungi were the mite's favorites with their greater feeding amount and shorter developmental duration. Interestingly, the mite could also feed on Metarhizium anisopliae and Metarhizium robertsii, but this led to the mite's death. After feeding on M. anisopliae and M. robertsii, the mites began to die after 24 h, and the mortality rate reached 100% by 72 h. Observation under optical microscopy and scanning electron microscopy revealed that the conidia of M. anisopliae and M. robertsii adhered to the mite's surface, but there was no evidence of penetration or invasion. However, dissection observation indicated that the two Metarhizium species germinate and grow within the mite's digestive tract, which implies that Metarhizium generalists with broad-spectrum hosts and the production of destruxins have acaricidal activity toward the mycophagous mites.

Utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by the fungus Metarhizium robertsii

Plant-derived sugars and lipids are key nutritional sources for plant associated fungi. However, the relationship between utilization of host-derived sugars and lipids during development of the symbiotic association remains unknown. Here we show that the fungus Metarhizium robertsii also needs plant-derived lipids to develop symbiotic relationship with plants. The fatty acid binding proteins FABP1 and FABP2 are important for utilization of plant-derived lipids as the deletion of Fabp1 and Fabp2 significantly reduced the ability of M. robertsii to colonize rhizoplane and rhizosphere of maize and Arabidopsis thaliana. Deleting Fabp1 and Fabp2 increased sugar utilization by upregulating six sugar transporters, and this explains why deleting the monosaccharide transporter gene Mst1, which plays an important role in utilization of plant-derived sugars, had no impact on the ability of the double-gene deletion mutant ΔFabp1::ΔFabp2 to colonize plant roots. FABP1 and FABP2 were also found in other plant-associated Metarhizium species, and they were highly expressed in the medium using the tomato root exudate as the sole carbon and nitrogen source, suggesting that they could be also important for these species to develop symbiotic relationship with plants. In conclusion, we discovered that utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by M. robertsii.

Metarhizium spp. isolates effective against Queensland fruit fly juvenile life stages in soil.

Queensland fruit fly, Bactrocera tryoni, Froggatt (Diptera: Tephritidae) is Australia's primary fruit fly pest species. Integrated Pest Management (IPM) has been adopted to sustainably manage this polyphagous species with a reduced reliance on chemical pesticides. At present, control measures are aimed at the adult stages of the fly, with no IPM tools available to target larvae once they exit the fruit and pupate in the soil. The use of entomopathogenic fungi may provide a biologically-based control method for these soil-dwelling life stages. The effectiveness of fungal isolates of Metarhizium and Beauveria species were screened under laboratory conditions against Queensland fruit fly. In bioassays, 16 isolates were screened for pathogenicity following exposure of third-instar larvae to inoculum-treated vermiculite used as a pupation substrate. The best performing Metarhizium sp. isolate achieved an average percentage mortality of 93%, whereas the best performing Beauveria isolate was less efficient, with an average mortality of 36%. Susceptibility to infection during different development stages was investigated using selected fungal isolates, with the aim of assessing all soil-dwelling life stages from third-instar larvae to final pupal stages and emerging adults. Overall, the third larval instar was the most susceptible stage, with average mortalities between 51-98% depending on the isolate tested. Moreover, adult mortality was significantly higher when exposed to inoculum during pupal eclosion, with mortalities between 56-76% observed within the first nine days post-emergence. The effect of temperature and inoculum concentration on insect mortality were assessed independently with candidate isolates to determine the optimum temperature range for fungal biological control activity and the rate required for application in field conditions. Metarhizium spp. are highly efficacious at killing Queensland fruit fly and have potential for use as biopesticides to target soil-dwelling and other life stages of B. tryoni.

Genetic diversity of Metarhizium sp. isolated from various hosts in East Dumoga, North Sulawesi, Indonesia

Abstract. Siahaan P, Mangais RER, Kolondam B, Tangapo A, Mambu S. 2023. Genetic diversity of Metarhizium sp. isolated from various hosts in East Dumoga, North Sulawesi, Indonesia. Biodiversitas 24: 6888-6896. Metarhizium fungus is an entomopathogenic fungus known to kill many insect pests. This means that Metarhizium sp. has an important ecological role in the ecosystem, especially in controlling insect populations and recycling nutrients. Studying the genetic diversity of Metarhizium species and their relationships with insect hosts provides insight into pest management and the study of their taxonomy. The study aimed to investigate the genetic variability among Metarhizium fungi by examining their respective host types, which can be used as basic data for taxonomic studies, germplasm conservation efforts, and pest management. Exploration results showed that three insect species were infected with the Metarhizium sp fungus, namely Scotinophara coarctata, Nilaparvata lugens, and Recilia dorsalis. The results of a phylogenetic analysis has shown that the S. coarctata isolate was in the same group as four comparable Metarhizium anisopliae isolates from GenBank with a similarity level of 100%, while the N. lugens and R. dorsalis isolates were in the same group as four Metarhizium huainamdangense isolates with a similarity level. 99% and three isolates of Metarhizium flavoviride with 98% similarity. Molecular analysis confirmed that the Metarhizium isolated from S. coarctata was the M. anisopliae species, while those isolated from N. lugens and R. dorsalis were the M. huainamdangense species. Differences in host insects have been proven to provide genetic variation to the Metarhizium sp. fungus.

The evolution of complex metarhizium-insect-plant interactions

Metarhizium species interact with plants, insects, and microbes within a diffuse co-evolutionary framework that benefits soil health, biodiversity, and plant growth. The insect host ranges of these fungi vary greatly. Specialization to a narrow host range usually occurs in the tropics with its stable insect populations, and is characterized by the rapid evolution of existing protein sequences, sexual recombination, and small genomes. Host-generalists are associated with temperate regions and ephemeral insect populations. Their mutualistic plant-colonizing lifestyle increases survival when insects are rare, while facultative entomopathogenicity feeds both the fungi and plants when insects are common. Host-generalists have lost meiosis and associated genome defense mechanisms, enabling gene duplications to diversify functions related to plant colonization and host exploitation. Horizontal gene transfer events via transposons have also contributed to host range changes, while parasexuality combines beneficial mutations within individual clones of host-generalists. There is also a lot of genetic variation in insect populations and an increasing understanding that both pathogen virulence and insect immunity are linked with stress responses. Thus, susceptibility to host-generalists can vary due to non-specific resistance to multiple stressors, multipurpose physical and chemical barriers, and heterogeneity in physiological and behavioral factors, such as sleep.

Utilization of Metarhizium as an insect biocontrol agent and a plant bioinoculant with special reference to Brazil.

Brazil has a long history of using biological control and has the largest program in sugarcane agriculture to which a biocontrol program has been applied. This achievement is at least partly due to the utilization of the entomopathogenic fungus Metarhizium. This well-known fungal genus exhibits pathogenicity against a broad range of arthropod hosts and has been used globally as a biocontrol agent. This fungus is also a root symbiont, and in this capacity, it is a plant growth promoter. However, this feature (i.e., as a plant symbiont) has yet to be fully explored and implemented in Brazil, although the number of reports demonstrating Metarhizium's utility as a plant bioinoculant is increasing. The Brazilian bioproduct industry targets agricultural pests, and is limited to two Metarhizium species represented by four fungal isolates as active ingredients. Entomopathogenic fungi have also been successful in controlling arthropods of public health concern, as shown in their control of mosquitoes, which are vectors of diseases. The isolation of new indigenous Metarhizium isolates from a variety of substrates such as soil, insects, and plants shows the wide genetic diversity within this fungal genus. In this review, we emphasize the significance of Metarhizium spp. for the biological control of insects in Brazil. We also suggest that the experience and success of biological control with fungi in Brazil is an important resource for developing integrated pest management and sustainable strategies for pest control worldwide. Moreover, the future implementation prospects of species of Metarhizium being used as bioinoculants and possible new advances in the utility of this fungus are discussed.

A Drosophila melanogaster model shows that fast growing Metarhizium species are the deadliest despite eliciting a strong immune response

ABSTRACT We used Drosophila melanogaster to investigate how differences between Metarhizium species in growth rate and mechanisms of pathogenesis influence the outcome of infection. We found that the most rapid germinators and growers in vitro and on fly cuticle were the fastest killers, suggesting that pre-penetration competence is key to Metarhizium success. Virulent strains also induced the largest immune response, which did not depend on profuse growth within hosts as virulent toxin-producing strains only proliferated post-mortem while slow-killing strains that were specialized to other insects grew profusely pre-mortem. Metarhizium strains have apparently evolved resistance to widely distributed defenses such as the defensin Toll product drosomycin, but they were inhibited by Bomanins only found in Drosophila spp. Disrupting a gene (Dif), that mediates Toll immunity has little impact on the lethality of most Metarhizium strains (an exception being the early diverged M. frigidum and another insect pathogen Beauveria bassiana). However, disrupting the sensor of fungal proteases (Persephone) allowed rapid proliferation of strains within hosts (with the exception of M. album), and flies succumbed rapidly. Persephone also mediates gender differences in immune responses that determine whether male or female flies die sooner. We conclude that some strain differences in growth within hosts depend on immune-mediated interactions but intrinsic differences in pathogenic mechanisms are more important. Thus, Drosophila varies greatly in tolerance to different Metarhizium strains, in part because some of them produce toxins. Our results further develop D. melanogaster as a tractable model system for understanding insect-Metarhizium interactions.

A Rapid Method for Measuring In Vitro Growth in Entomopathogenic Fungi.

Quantifying the growth of entomopathogenic fungi is crucial for understanding their virulence and pathogenic potential. Traditional methods for determining growth, such as biomass determination or colony growth area, are time-consuming and quantitatively and spatially limited in scope. In this study, we introduce a high-throughput method for rapidly measuring fungal growth using spectrophotometry in small-volume, liquid media cultures in 96-well microplates. Optical density (OD) changes were directly correlated with dry weight of samples for six isolates from three species of the genus Metarhizium to validate spectrophotometric growth measurements, and investigate species- and isolate-specific effects. We quantified fungal biomass from the microcultures by extracting, drying, and weighing mycelial mats. From the relationship established between OD and biomass, we generated standard curves for predicting biomass based on the OD values. The OD measurements clearly distinguished growth patterns among six isolates from three Metarhizium species. The logistic growth phase, as captured by the OD measurements, could be accurately assessed within a span of 80 h. Using isolates of M. acridum, M. brunneum, and M. guizhouense, this technique was demonstrated to be an effective, reproducible, and simple method for rapidly measuring filamentous fungal growth with high precision. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi and investigating the factors that influence their growth.

Blastospores from Metarhizium anisopliae and Metarhizium rileyi Are Not Always as Virulent as Conidia Are towards Spodoptera frugiperda Caterpillars and Use Different Infection Mechanisms.

Infective conidia from entomopathogenic fungi are widely used to control insect pests. Many entomopathogenic fungi also produce yeast-like cells called blastospores under specific liquid culture conditions that can directly infect insects. However, little is known about the biological and genetic factors that allow blastospores to infect insects and make them potentially effective for biological control in the field. Here, we show that while the generalist Metarhizium anisopliae produces a higher number of and smaller blastospores, the Lepidoptera specialist M. rileyi produces fewer propagules with a higher cell volume under high-osmolarity conditions. We compared the virulence of blastospores and conidia of these two Metarhizium species towards the economically important caterpillar pest Spodoptera frugiperda. Conidia and blastospores from M. anisopliae were equally infectious, but acted slower, and killed fewer insects than M. rileyi conidia and blastospores did, where M. rielyi conidia had the highest virulence. Using comparative transcriptomics during propagule penetration of insect cuticles, we show that M. rileyi blastospores express more virulence-related genes towards S. frugiperda than do M. anisopliae blastospores. In contrast, conidia of both fungi express more virulence-related oxidative stress factors than blastospores. Our results highlight that blastospores use a different virulence mechanism than conidia use, which may be explored in new biological control strategies.

Plant Growth-Promoting Potential of Entomopathogenic Fungus Metarhizium pinghaense AAUBC-M26 under Elevated Salt Stress in Tomato

Entomopathogenic fungi Metarhizium species are generally employed to manage the soil-dwelling stage of insect pests, and are known for their rhizocompetency property. Since this fungus is typically recommended for use in soil, it could potentially be investigated as a bioinoculant to reduce abiotic stress, such as salinity, along with improved plant growth promotion. Salt stress tolerance potential of native Metarhizium isolates was evaluated based on mycelial fresh weight, dry weight, and spore yield. All the isolates were found to tolerate NaCl concentrations (50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 300 mM) supplemented in the culture medium. Metarhizium anisopliae (AAUBC-M15) and Metarhizium pinghaense (AAUBC-M26) were found to be effective at tolerating NaCl stress up to 200 mM NaCl. These two isolates were analyzed in vitro for plant growth-promoting traits at elevated salt concentrations (100 and 200 mM NaCl). No significant effect on IAA production was reported with the isolate M. pinghaense (AAUBC-M26) (39.16 µg/mL) or in combination with isolate M. anisopliae (AAUBC-M15) (40.17 µg/mL) at 100 mM NaCl (38.55 µg/mL). The salinity stress of 100 mM and 200 mM NaCl had a significant influence on the phosphate solubilization activity, except in the co-inoculation treatment at 100 mM NaCl. The isolates were positive for ACC deaminase enzyme activity. An increase in salt concentration was accompanied by a steady and significant increase in chitinase enzyme activity. Total phenolics (149.3 µg/mL) and flavonoids (79.20 µg/mL) were significantly higher in the culture filtrate of Metarhizium isolates at 100 mM NaCl, and gradual decline was documented at 200 mM NaCl. M. pinghaense (AAUBC-M26) proved to be promising in reducing the salt stress in tomato seedlings during the nursery stage. In the pot culture experiment, the treatment comprising soil application + seedling root dip + foliar spray resulted in improved growth parameters of the tomato plant under salt stress. This study shows that Metarhizium, a fungus well known for controlling biotic stress brought on by insect pests, can also help plants cope with abiotic stress, such as salinity.

Impacts of repeated liquid culture on entomopathogenic fungi

ABSTRACT Production of entomopathogenic fungi (EPF) for biopesticides relies on in vitro production of fungal propagules as the active ingredients. Repeated culturing or sub culturing may select for fungi with reduced spore production and virulence. We completed 60 cycles of repeated liquid culture on six isolates representing four genera of EPF. Measurements of the spent media recorded at T2, T15, T30, T45 and T60 cycles included blastospore concentrations, dry matter accumulation as hyphae, glucose concentration remaining, and final pH. Insecticidal activity (LC50 of Trichoplusia ni neonates) was compared between initial and final cultures for conidia grown on nutrient agar. Virulence of Beauveria bassiana GHA was 3× lower at T60 (LC50 ratio = 0.308) and consumption of glucose increased with additional culture cycles. Two Cordyceps javanica (MBC 177 and Apopka 97) had fewer blastospores, higher pH, and altered mycelia dry weight at the T15 sample, but maintained similar values from T15 through T60. MBC 177 and Apopka 97 lost virulence with LC50 ratios of 0.345 and 0.016, respectively. Metarhizium robertsii and M. brunneum F52 isolates failed to produce conidia by plating T30, T45, and T60 cultures on nutrient agar. When comparing T0 with T15 cultures, M. robertsii conidia had increased virulence while M. brunneum had decreased virulence (LC50 ratios of 1.746 and 0.740, respectively). These differences between the two Metarhizium species demonstrate that the direction and level of impact imposed by repeated culture is at least species dependent.

Isolation of a highly virulent Metarhizium strain targeting the tea pest, Ectropis obliqua.

Tea is one of the most widely consumed beverages around the world. Larvae of the moth, Ectropis obliqua Prout (Geometridae, Lepidoptera), are one of the most destructive insect pests of tea in China. E. obliqua is a polyphagus insect that is of increasing concern due to the development of populations resistant to certain chemical insecticides. Microbial biological control agents offer an environmentally friendly and effective means for insect control that can be compatible with "green" and organic farming practices. To identify novel E. obliqua biological control agents, soil and inset cadaver samples were collected from tea growing regions in the Fujian province, China. Isolates were analyzed morphologically and via molecular characterization to identity them at the species level. Laboratory and greenhouse insect bioassays were used to determine the effectiveness of the isolates for E. obliqua control. Eleven isolates corresponding to ten different species of Metarhizium were identified according to morphological and molecular analyses from soil and/or insect cadavers found on tea plants and/or in the surrounding soil sampled from eight different regions within the Fujian province, China. Four species of Metarhizium including M. clavatum, M. indigoticum, M. pemphigi, and M. phasmatodeae were documented for the first time in China, and the other species were identified as M. anisopliae, M. brunneum, M. lepidiotae, M. majus, M. pinghaense, and M. robertsii. Insect bioassays of the eleven isolates of Metarhizium revealed significant variation in the efficacy of each isolate to infect and kill E. obliqua. Metarhizium pingshaense (MaFZ-13) showed the highest virulence reaching a host target mortality rate of 93% in laboratory bioassays. The median lethal concentration (LC50) and median lethal time (LT50) values of M. pingshaense MaFZ-13 were 9.6 × 104 conidia/mL and 4.8 days, respectively. Greenhouse experiments and a time-dose-mortality (TDM) models were used to further evaluate and confirm the fungal pathogenic potential of M. pingshaense MaFZ-13 against E. obliqua larvae. Isolation of indigenous microbial biological control agents targeting specific pests is an effective approach for collecting resources that can be exploited for pest control with lowered obstacles to approval and commercialization. Our data show the presence of four different previously unreported Metarhizium species in China. Bioassays of the eleven different Metarhizium strains isolated revealed that each could infect and kill E. obliqua to different degrees with the newly isolated M. pingshaense MaFZ-13 strain representing a particularly highly virulent isolate potentially applicable for the control of E. obliqua larvae.

Effect of visible light and ultraviolet light on the pathogenicity of entomopathogenic fungi to false codling moth, Thaumatotibia leucotreta (Lepidoptera: Tortricidae) larvae

Entomopathogenic fungi (EPF) are effective and environment-friendly insect biological control agents. Ultraviolet (UV) light is known to have an effect on the survival of fungal conidia, and natural sunlight is potentially one of the most damaging factors undermining EPF persistence and pathogenicity. This study aimed to test the infection potential of an isolate of Beauveria bassiana and five Metarhizium species after exposure to different light treatments, on soil and leaf surfaces under laboratory and field conditions, using Thaumatotibia leucotreta (Lepidoptera: Tortricidae) as the test host. Conidia were exposed either to growth light alone, which emits the same visible light as the sun, but excluding UV light, or directly exposed to UV light for 12 h. The results indicated no negative effect on the infection potential of the conidia of most species tested. The conidia of the two Metarhizium pinghaense (5HEID and TH149) isolates showed the greatest tolerance to visible light and UV radiation exposure on both soil and leaf surfaces. Exposure of M. pinghaense isolates to visible light on soil surfaces showed pathogenicity of &gt; 80% for both isolates, and of between 58% and 88% after exposure to UV light. On leaf surfaces, three Metarhizium isolates, M. pinghaense (5HEID and TH149) and M. majus (TH153) had &gt; 90% pathogenicity following exposure to UV light, and M. pinghaense (TH149) and M. robertsii (6EIKEN) showed greater tolerance of &gt; 70%, under laboratory conditions. However, the pathogenicity of the EPF isolates was very low in field trials, indicating that further trials on the use of formulations and adjuvants with the isolates are needed to improve long-term persistence and efficacy under field conditions.

Tolerance to UV-B radiation of the entomopathogenic fungus Metarhizium rileyi

Soybean, corn, and cotton crops are afflicted by several noctuid pests and the development of bioinsecticides could help control these pests. The fungus Metarhizium rileyi has the greatest potential because its epizootics decimate caterpillar populations in the absence of insecticide applications. However, insect-pathogenic fungi when used for insect control in agriculture have low survival mainly due to the deleterious effects of ultraviolet radiation and heat from solar radiation. In this study, fourteen isolates of M. rileyi were studied and compared with isolates ARSEF 324 and ARSEF 2575 of Metarhizium acridum and Metarhizium robertsii, respectively, whose sensitivity to UV-B radiation had previously been studied. Conidia were exposed at room temperature (ca. 26 °C) to 847.90 mWm−2 of Quaite-weighted UV-B using two fluorescent lamps. The plates containing the conidial suspensions were irradiated for 1, 2, and 3 h, providing doses of 3.05, 6.10, and 9.16 kJ m2, respectively. A wide variability in conidial UV-B tolerance was found among the fourteen isolates of M. rileyi. Isolate CNPSo-Mr 150 was the most tolerant isolate (germination above 80% after 2 h exposure), which was comparable to ARSEF 324 (germination above 90% after 2 h exposure), the most tolerant Metarhizium species. The least tolerant isolates were CNPSo-Mr 141, CNPSo-Mr 142, CNPSo-Mr 156, and CNPSo-Mr 597. Nine M. rileyi isolates exhibited similar tolerance to UV-B radiation as ARSEF 2575 (germination above 50% after 2 h exposure). In conclusion, the majority of M. rileyi isolates studied can endure 1 or 2 h of UV-B radiation exposure. However, after 3 h of exposure, the germination of all studied isolates reduced below 40%, except for CNPSo-Mr 150 and ARSEF 324.

Root colonization of industrial hemp (Cannabis sativa L.) by the endophytic fungi Metarhizium and Pochonia improves growth

Metarhizium is an endophytic, insect pathogenic fungus. Here we investigated the time-course of systemic and rhizospheric colonization by Metarhizium and its influence on the growth of industrial hemp (Cannabis sativa L.). Fungal colonization of hemp root, stem and leaf as well as rhizospheric soil, and agronomic parameters of hemp, were tested in four Metarhizium species with broad- (Metarhizium robertsii, Metarhizium anisopliae, Metarhizium brunneum) and narrow-insect (Metarhizium acridum) host ranges, as well as Pochonia chlamydosporia, a phylogenetically related endophytic fungus that is a nematode pathogen. Root and rhizosphere colonization were found in all Metarhizium species and Pochonia at 10, 20 and 30 days post-inoculation. Pochonia showed greatest root colonization compared to that of all Metarhizium species. Endophytic root colonization by Metarhizium was confirmed by confocal fluorescence microscopy using green fluorescent protein-expressing fungal strains. The genes, cat, mpl and EF1α were tested using a qPCR assay for Metarhizium and hemp. Root colonization by Metarhizium and Pochonia positively influenced shoot length, stem weight and root weight of hemp during the early stages of colonization. Our results suggest the potential for the application of Metarhizium and Pochonia as biocontrol and plant growth promoter agents during hemp colonization.

The potential of using Metarhizium anisopliae and Metarhizium humberi to control the Asian soybean rust caused by Phakopsora pachyrhizi

ABSTRACT Phakopsora pachyrhizi causes Asian soybean rust, resulting in severe losses of soybean yield. This study assessed the biocontrol efficacy of cell-free culture filtrates and conidial suspensions of Metarhizium anisopliae (MABR-01) and M. humberi (MHBR-03) against P. pachyrhizi in vitro and in planta. A 50% concentration of culture filtrates of M. anisopliae and M. humberi inhibited the germination of P. pachyrhizi urediniospores by 85 and 96%, respectively, compared to the 50% potato-dextrose broth (PDB) control. The conidial suspensions caused no inhibition of P. pachyrhizi in vitro, but the conidial suspension of M. anisopliae controlled the rust disease in plants with a 51% efficiency. Soybean plants colonised by M. anisopliae MABR-01 and exposed to P. pachyrhizi showed less severe disease in the shoots compared to plants not colonised by M. anisopliae; the disease was reduced by 40%. Spraying plants with 50 and 75% culture filtrates of M. humberi and a 75% concentration of M. anisopliae significantly reduced rust disease, by an average of 86%. Plants pretreated with Metarhizium culture filtrates showed a 30% reduction in P. pachyrhizi colonisation based on qPCR quantification of the pathogen genomic DNA. These results indicate a high biotechnological and sustainable potential of these two Metarhizium species for control of Asian soybean rust.