Metarhizium Robertsii Research Articles

Isolation, morphological characterization, and screening a virulence of Beauveria bassiana and Metarhizium robertsii as bioagents

Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of pests.

The immunostimulatory role of an Enterococcus-dominated gut microbiota in host protection against bacterial and fungal pathogens in Galleria mellonella larvae.

Understanding the intricate interplay between the gut microbiota and the immune response in insects is crucial, given its diverse impact on the pathogenesis of various microbial species. The microbiota's modulation of the host immune system is one such mechanism, although its complete impact on immune responses remains elusive. This study investigated the tripartite interaction between the gut microbiota, pathogens, and the host's response in Galleria mellonella larvae reared under axenic (sterile) and conventional (non-sterile) conditions. The influence of the microbiota on host fitness during infections was evaluated via two different routes: oral infection induced by Bacillus thuringiensis subsp. galleriae (Btg), and topical infection induced by Metarhizium robertsii (Mr). We observed that larvae without a microbiota can successfully fulfill their life cycle, albeit with more variation in their developmental time. We subsequently performed survival assays on final-instar larvae, using the median lethal dose (LD50) of Btg and Mr. Our findings indicated that axenic larvae were more vulnerable to an oral infection of Btg; specifically, a dose that was calculated to be half-lethal for the conventional group resulted in a 90%-100% mortality rate in the axenic group. Through a dual-analysis experimental design, we could identify the status of the gut microbiota using 16S rRNA sequencing and assess the level of immune-related gene expression in the same group of larvae at basal conditions and during infection. This analysis revealed that the microbiota of our conventionally reared population was dominated entirely by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two antimicrobial peptides (AMPs)-gallerimycin and gloverin-in the conventional larvae compared with the axenic larvae. Furthermore, Enterococcus mundtii, isolated from the gut of conventional larvae, showed inhibition activity against Btg in vitro. Lastly, other immune effectors, namely, phenoloxidase activity in the hemolymph and total reactive oxygen/nitrogen species (ROS/RNS) in the gut, were tested to further investigate the extent of the stimulation of the microbiota on the immune response. These findings highlight the immune-modulatory role of the Enterococcus-dominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host's response to oral and topical infections.

MrCreC, a carbon catabolite repression gene, is required for the growth, conidiation, stress tolerance and virulence of Metarhizium robertsii

As a key component of carbon source metabolism in fungi, CreC WD40 repeat protein is regulated by carbon catabolite repression (CCR). However, the understanding of the functions of CreC in entomopathogenic fungi is currently limited. Here, CreC in Metarhizium robertsii (MrCreC) was identified, and its roles in fungal development, conidiation, environmental stress response, and insecticidal virulence were explored. MrCreC is localized to cytoplasm, and MrCreC deletion affects fungal growth on various nutrients. Compared to the wild type, the sporulation of ΔMrCreC strain was significantly decreased by 60.3%. Further qPCR analysis found that deletion of MrCreC resulted in repression of sporulation-related genes such as AbaA, FlbA, Flbc, MedA, FlbD, FluG, and wetA. In addition, MrCreC loss did not alter heat stress tolerance but resulted in enhanced tolerance to UV-B. Interestingly, bioassays showed that the virulence following exposures to topical applications or injection of conidial suspensions of both infection and injection was impaired compared with that of the wild type. Further analysis showed that the adhesion and cuticle penetration genes in ΔMrCreC was down-regulated during infection, and the appressorial formation rate was significantly reduced. A deletion of MrCreC significantly also reduced immune escape and nutrient utilization genes in insect hemocoel. In conclusion, MrCreC is involved in the growth, development and virulence of M. robertsii. These findings advance our understanding of the function of CCR pathway-related genes.

Only one of three hydrophobins (Hyd1–3) contributes to conidial hydrophobicity and insect pathogenicity of Metarhizium robertsii

Class I/II hydrophobins constitute a family of small amphiphilic proteins that mediate cell hydrophobicity and adhesion to host or substrata and have pleiotropic effects in filamentous fungi. Here we report that only class I Hyd1 is essential for conidial hydrophobicity and insect pathogenicity among three hydrophobins (Hyd1–3) characterized in Metarhizium robertsii, an insect-pathogenic fungus. Aerial conidiation levels of three Δhyd1 mutants were much more reduced in 5-day-old cultures than in 7-day-old cultures, which were wettable (hydrophilic), but restored to a wild-type level in 15-day-old cultures. The Δhyd1 mutants were compromised in conidial quality, including significant decreases in hydrophobicity (58%), adhesion to insect cuticle (36%), insect pathogenicity via normal cuticle infection (37%), UVB resistance (20%), and heat tolerance (10%). In contrast, none of all examined phenotypes were affected in the null mutants of hyd2 and hyd3. Intriguingly, micromorphology and integrity of hydrophobin rodlet bundles on conidial coat were not affected in all mutant and wild-type strains, but the rodlet bundles were disordered in the absence of hyd1, suggesting a link of the disorder to the decreased hydrophobicity. Therefore, Hyd1 mediates the fungal hydrophobicity and plays an important role in conidial quality control and insect-pathogenic lifecycle. Class I Hyd2 and class II Hyd3 seem functionally redundant in M. robertsii.

Optimization of the Fermentation Conditions of Metarhizium robertsii and Its Biological Control of Wolfberry Root Rot Disease.

Fusarium solani is the main pathogenic fungus causing the root rot of wolfberry (Lycium barbarum). The endophytic fungus Metarhizium robertsii has been widely used for the biocontrol of plant pathogenic fungi, but the biocontrol effects of this fungus on wolfberry root rot and its antifungal mechanism against F. solani have not been reported. In this study, the antagonism of endophytic fungus M. robertsii against F. solani was verified. Further, we optimized the fermentation conditions of M. robertsii fermentation broth based on the inhibition rate of F. solani. In addition, the effects of M. robertsii fermentation broth on the root rot of wolfberry and its partial inhibition mechanism were investigated. The results showed that M. robertsii exhibited good antagonism against F. solani. Glucose and beef extracts were the optimal carbon and nitrogen sources for the fermentation of M. robertsii. Under the conditions of 29 °C, 190 rpm, and pH 7.0, the fermentation broth of M. robertsii had the best inhibition effect on F. solani. Furthermore, the fermentation broth treatment decreased the activities of superoxide dismutase, catalase, and peroxidase of F. solani; promoted the accumulation of malondialdehyde; and accelerated the leakage of soluble protein and the decrease in soluble sugar. In addition, inoculation with M. robertsii significantly reduced the decay incidence and disease index of wolfberry root rot caused by F. solani. These results indicate that M. robertsii could be used as a biological control agent in wolfberry root rot disease management.

Divergent roles of Rad4 and Rad23 homologs in Metarhizium robertsii's resistance to solar ultraviolet damage.

The anti-ultraviolet (UV) role of a Rad4-Rad23-Rad33 complex in budding yeast relies on nucleotide excision repair (NER), which is mechanistically distinct from photorepair of DNA lesions generated under solar UV irradiation but remains poorly known in filamentous fungi. Here, two nucleus-specific Rad4 paralogs (Rad4A and Rad4B) and nucleocytoplasmic shuttling Rad23 ortholog are functionally characterized by multiple analyses of their null mutants in Metarhizium robertsii, an entomopathogenic fungus lacking Rad33. Rad4A was proven to interact with Rad23 and contribute significantly more to conidial UVB resistance (90%) than Rad23 (65%). Despite no other biological function, Rad4A exhibited a very high activity in photoreactivation of UVB-impaired/inactivated conidia by 5-h light exposure due to its interaction with Rad10, an anti-UV protein clarified previously to have acquired a similar photoreactivation activity through its interaction with a photolyase in M. robertsii. The NER activity of Rad4A or Rad23 was revealed by lower reactivation rates of moderately impaired conidia after 24-h dark incubation but hardly observable at the end of 12-h dark incubation, suggesting an infeasibility of its NER activity in the field where nighttime is too short. Aside from a remarkable contribution to conidial UVB resistance, Rad23 had pleiotropic effect in radial growth, aerial conidiation, antioxidant response, and cell wall integrity but no photoreactivation activity. However, Rad4B proved redundant in function. The high photoreactivation activity of Rad4A unveils its essentiality for M. robertsii's fitness to solar UV irradiation and is distinct from the yeast homolog's anti-UV role depending on NER. IMPORTANCE Resilience of solar ultraviolet (UV)-impaired cells is crucial for the application of fungal insecticides based on formulated conidia. Anti-UV roles of Rad4, Rad23, and Rad33 rely upon nucleotide excision repair (NER) of DNA lesions in budding yeast. Among two Rad4 paralogs and Rad23 ortholog characterized in Metarhizium robertsii lacking Rad33, Rad4A contributes to conidial UVB resistance more than Rad23, which interacts with Rad4A rather than functionally redundant Rad4B. Rad4A acquires a high activity in photoreactivation of conidia severely impaired or inactivated by UVB irradiation through its interaction with Rad10, another anti-UV protein previously proven to interact with a photorepair-required photolyase. The NER activity of either Rad4A or Rad23 is seemingly extant but unfeasible under field conditions. Rad23 has pleiotropic effect in the asexual cycle in vitro but no photoreactivation activity. Therefore, the strong anti-UV role of Rad4A depends on photoreactivation, unveiling a scenario distinct from the yeast homolog's NER-reliant anti-UV role.

Isolation, morphological characterization, and screening virulence of Beauveria bassiana and Metarhizium robertsii fungal isolates on Galleria mellonella.

Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions. In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x10 7 spores ml -1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day -1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x10 8 conidial ml -1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.

Effects of passages through an insect or a plant on virulence and physiological properties of the fungus Metarhizium robertsii.

Species of the genus Metarhizium are characterized by a multitrophic lifestyle of being arthropod parasites, rhizosphere colonizers, endophytes, and saprophytes. The process of adaptation to various organisms and substrates may lead to specific physiological alterations that can be elucidated by passaging through different hosts. Changes in virulence and cultivation properties of entomopathogenic fungi subcultured on different media or passaged through a live insect host are well known. Nevertheless, comparative in-depth physiological studies on fungi after passaging through insect or plant organisms are scarce. Here, virulence, plant colonization, hydrolytic enzymatic activities, toxin production, and antimicrobial action were compared between stable (nondegenerative) parent strain Metarhizium robertsii MB-1 and its reisolates obtained after eight passages through Galleria mellonella larvae or Solanum lycopersicum or after subculturing on the Sabouraud medium. The passaging through the insect caused similar physiological alterations relative to the plant-based passaging: elevation of destruxin A, B, and E production, a decrease in protease and lipase activities, and lowering of virulence toward G. mellonella and Leptinotarsa decemlineata as compared to the parent strain. The reisolates passaged through the insect or plant showed a slight trend toward increased tomato colonization and enhanced antagonistic action on tomato-associated bacterium Bacillus pumilus as compared to the parental strain. Meanwhile, the subculturing of MB-1 on the Sabouraud medium showed stability of the studied parameters, with minimal alterations relative to the parental strain. We propose that the fungal virulence factors are reprioritized during adaptation of M. robertsii to insects, plants, and media.

Insect fungal pathogens secrete a cell wall-associated glucanase that acts to help avoid recognition by the host immune system.

Fungal insect pathogens have evolved diverse mechanisms to evade host immune recognition and defense responses. However, identification of fungal factors involved in host immune evasion during cuticular penetration and subsequent hemocoel colonization remains limited. Here, we report that the entomopathogenic fungus Beauveria bassiana expresses an endo-β-1,3-glucanase (BbEng1) that functions in helping cells evade insect immune recognition/ responses. BbEng1 was specifically expressed during infection, in response to host cuticle and hemolymph, and in the presence of osmotic or oxidative stress. BbEng1 was localized to the fungal cell surface/ cell wall, where it acts to remodel the cell wall pathogen associated molecular patterns (PAMPs) that can trigger host defenses, thus facilitating fungal cell evasion of host immune defenses. BbEng1 was secreted where it could bind to fungal cells. Cell wall β-1,3-glucan levels were unchanged in ΔBbEng1 cells derived from in vitro growth media, but was elevated in hyphal bodies, whereas glucan levels were reduced in most cell types derived from the BbEng1 overexpressing strain (BbEng1OE). The BbEng1OE strain proliferated more rapidly in the host hemocoel and displayed higher virulence as compared to the wild type parent. Overexpression of their respective Eng1 homologs or of BbEng1 in the insect fungal pathogens, Metarhizium robertsii and M. acridum also resulted in increased virulence. Our data support a mechanism by which BbEng1 helps the fungal pathogen to evade host immune surveillance by decreasing cell wall glucan PAMPs, promoting successful fungal mycosis.

Trade-offs between immunity and competitive ability in fighting ant males

BackgroundFighting disease while fighting rivals exposes males to constraints and trade-offs during male-male competition. We here tested how both the stage and intensity of infection with the fungal pathogen Metarhizium robertsii interfere with fighting success in Cardiocondyla obscurior ant males. Males of this species have evolved long lifespans during which they can gain many matings with the young queens of the colony, if successful in male-male competition. Since male fights occur inside the colony, the outcome of male-male competition can further be biased by interference of the colony’s worker force.ResultsWe found that severe, but not yet mild, infection strongly impaired male fighting success. In late-stage infection, this could be attributed to worker aggression directed towards the infected rather than the healthy male and an already very high male morbidity even in the absence of fighting. Shortly after pathogen exposure, however, male mortality was particularly increased during combat. Since these males mounted a strong immune response, their reduced fighting success suggests a trade-off between immune investment and competitive ability already early in the infection. Even if the males themselves showed no difference in the number of attacks they raised against their healthy rivals across infection stages and levels, severely infected males were thus losing in male-male competition from an early stage of infection on.ConclusionsMales of the ant C. obscurior have a well-developed immune system that raises a strong immune response very fast after fungal exposure. This allows them to cope with mild pathogen exposures without compromising their success in male-male competition, and hence to gain multiple mating opportunities with the emerging virgin queens of the colony. Under severe infection, however, they are weak fighters and rarely survive a combat already at early infection when raising an immune response, as well as at progressed infection, when they are morbid and preferentially targeted by worker aggression. Workers thereby remove males that pose a future disease threat by biasing male-male competition. Our study thus reveals a novel social immunity mechanism how social insect workers protect the colony against disease risk.

Isolation, morphological characterization, and screening a virulence of Beauveria bassiana and Metarhizium robertsii as bioagents

Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of pests.

Assessment of bio-formulations of indigenous strains of Bacillus thuringiensis, Metarhizium robertsii and Metarhizium majus for management of the rhinoceros beetle, Oryctes rhinoceros L., in field

BackgroundAmong all the coconut pests, rhinoceros beetle causes acute and serious damage to coconut palm. Management of this pest is very difficult due to its nocturnal activity, and also, it damages the emerging leaf inside the bud. Management of rhinoceros beetle using entomopathogens will be of great importance as it is economical and ecofriendly. Studies were carried out to decipher the biocontrol potential of indigenous entomopathogenic bacterium (Bacillus thuringiensis) and entomopathogenic fungi (Metarhizium robertsii and M. majus) against Oryctes rhinoceros in the field, individually as well as in combination, by soil drench as well as by topical spray method.ResultsThe study showed that B. thuringiensis strain NBAIR-BTAN4 showed 24.8% mortality at 5th week in soil drench method and 24% mortality in topical spray method. M. robertsii showed 24% mortality at 5th week in soil drench method as well as topical spray method. Similarly, M. majus showed 24% mortality in soil drench method and 23.2% in topical spray method at 5th week. In combination, NBAIR-BTAN4 + M. robertsii showed 40.8 and 44% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. majus showed 44.8 and 40.8% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. robertsii + M. majus showed 52.8 and 57.6% mortality at 5th week in soil drench and topical spray method, respectively.ConclusionThe present study showed application B. thuringiensis in combination with M. robertsii and M. majus is effective in management rhinoceros beetle in coconut orchard. Study also indicated that soil drench method is more promising strategy than topical spray method in managing larval population of the beetles and also confirmed that B. thuringiensis, M. robertsii and M. majus are compatible and they seemed to have a synergistic effect in controlling the pest in coconut orchard.

The occurrence of entomopathogenic fungi in apple orchards and their biocontrol potential against Eriosoma lanigerum

Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae), woolly apple aphid (WAA) is an important pest on apples globally and a key pest of apple production in South Africa. The aphid has developed some level of resistance against several chemical insecticides. Entomopathogenic fungi (EPF) have been identified as promising biological control agents against a wide array of insect pests. The main aim of this study was to conduct a survey in local apple orchards in the Western Cape province for EPF and to use isolates to test the susceptibility of WAA under optimum laboratory conditions. Soil samples were collected from apple orchards and EPF, baited and isolated using larvae of Galleria mellonella and Tenebrio molitor. Six EPF species: Beauveria bassiana, Cordyceps fumosorosea, Metarhizium brunneum, M. pinghaense, M. robertsii and Purpureocillium lilacinum were identified from the soil samples. The results from bioassays showed that Metarhizium robertsii and M. pinghaense isolates were the most effective with an average percentage mortality of > 90%. Metarhizium brunneum also proved to be effective when used against the insect with an average percentage mortality of > 80%, while B. bassiana and C. fumosorosea were the least effective with average percentage mortality of 52% and 48%, respectively. The LT50 and LT90 of M. robertsii (2.12; 4.19) and M. pinghaense (2.05; 4.45) showed to require similar mortality time in days of E. lanigerum. The results obtained in the study have provided an insight into the diversity of EPF species across apple orchards of the Western Cape and shown the efficacy of the Metarhizium isolates as potential biological control agents of the WAA.

Congo red induces trans-priming to UV-B radiation in Metarhizium robertsii

Metarhizium spp. is used as a biocontrol agent but is limited because of low tolerance to abiotic stress. Metarhizium robertsii is an excellent study model of fungal pathogenesis in insects, and its tolerance to different stress conditions has been extensively investigated. Priming is the time-limited pre-exposure of an organism to specific stress conditions that increases adaptive response to subsequent exposures. Congo red is a water-soluble azo dye extensively used in stress assays in fungi. It induces morphological changes and weakens the cell wall at sublethal concentrations. Therefore, this chemical agent has been proposed as a stressor to induce priming against other stress conditions in entomopathogenic fungi. This study aimed to evaluate the capacity of Congo red to induce priming in M. robertsii. Conidia were grown on potato dextrose agar with or without Congo red.The tolerance of conidia produced from mycelia grown in these three conditions was evaluated against stress conditions, including osmotic, oxidative, heat, and UV-B radiation. Conidia produced on medium supplemented with Congo red were significantly more tolerant to UV-B radiation but not to the other stress conditions assayed. Our results suggest that Congo red confers trans-priming to UV-B radiation but not for heat, oxidative, or osmotic stress.

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.

Understanding how diet and temperature affect survival and subsequent sporulation in a major rangeland grasshopper pest, Melanoplus sanguinipes, infected with the entomopathogenic fungus, Metarhizium robertsii

Behavioral fever is well-described in insects as an effective response to pathogens, but recent research also shows that the balance of macronutrients is important. Australian plague locusts (Chortoicetes terminifera) infected with Metarhizium acridum, a fungal entomopathogen, had longer survival by increasing carbohydrate and decreasing protein consumption. Our research tested the effects of Metarhizium robertsii (strain DWR2009) on the dietary macronutrient balance (Intake Target, IT) of the migratory grasshopper (Melanoplus sanguinipes, one of the most pestiferous grasshoppers in the United States), with and without elevated temperatures, to determine if pathogens significantly influence diet selection in this species. We also tested the effects of diet on survival under a M. robertsii inoculation. We found no significant difference in the ITs across all treatment groups; all treatments selected the same carbohydrate-biased IT of 1:2 protein:carbohydrate (p:c). In the prescribed diet experiments, inoculated grasshoppers from both the carbohydrate-biased (7p:35c) and protein-biased (35p:7c) diet treatment groups survived longer than those fed the balanced (21p:21c) diet. However, grasshoppers with access to elevated temperatures were able to completely rescue themselves from the pathogen. In correlation with our results, post-mortem Metarhizium growth was greatest on grasshoppers fed the balanced (21p:21c) diet and minimal on grasshoppers eating the other two diets. Eating the balanced 21p:21c diet either did not support the host to mount an effective immune response, provided a nutritionally optimal environment for pathogen growth, or both. Eating a protein-biased diet potentially supported an effective immune response, whereas eating a carbohydrate-biased diet starved the pathogen of protein and/or supported an immune response via different pathways.

A 1-aminocyclopropane-1-carboxylate deaminase MrACCD from Metarhizium robertsii is associated with plant growth promotion for Metarhizium spp

Besides killing insects, Metarhizium spp. have shown another ecological role as plant associates. Partial genra and groups of these entomopathogenic fungi act as plant growth promoters during root colonization. Here, we report that Metarhizium robertsii produces a 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD encoded by MracdS, MrACCD), which is involved in promoting early vegetative growth in wheat, while Metarhizium acridum lacks a functional ACCD, although a gene encoding for a putative ACCD has been identified in its genome. MracdS expression was up-regulated by a max 10.7-fold with 3 mM ACC and high ACCD enzymatic activities were induced by either ACC (7.5-fold) or wheat root (3.2-fold). In contrast, no ACCD activity was detected in M. acridum in the presence of both inducers. In pot assay, wheat seeds were treated with wild-type M. robertsii (Mr23), wild-type M. acridum (Mac324), MracdS disruption mutant (ΔMracdS) and M. acridum transformant harboring heterologous MracdS (Mac324-MracdS). Relative to the control seeds treated with heat-killed conidia, Mr23, ΔMracdS and Mac324-MracdS increased root length (by 66.2, 31.8 and 40.2%), and plant biomass (by 56.6, 42.1 and 40.9%). Nevertheless, ΔMracdS deficient in ACCD activity heavily impaired its capability of wheat growth promotion by decrease of 20.7% in root length relative to Mr23. In addition, Mr23 and Mac324-MracdS also increased shoot growth (by 42.3, and 42.7%) while ΔMracdS failed. Mac324 showed no effect on plant growth during the test. These data suggest a role for ACCD in the plant growth promotion effect by M. robertsii, which is irrelevant to Metarhizium colonization of roots since rhizosphere competency of both Mr23 and Mac324 are unaffected by the change of ACCD activity.

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.

Transcriptomic dissection of termite gut microbiota following entomopathogenic fungal infection.

Termites are social insects that live in the soil or in decaying wood, where exposure to pathogens should be common. However, these pathogens rarely cause mortality in established colonies. In addition to social immunity, the gut symbionts of termites are expected to assist in protecting their hosts, though the specific contributions are unclear. In this study, we examined this hypothesis in Odontotermes formosanus, a fungus-growing termite in the family Termitidae, by 1) disrupting its gut microbiota with the antibiotic kanamycin, 2) challenging O. formosanus with the entomopathogenic fungus Metarhizium robertsii, and finally 3) sequencing the resultant gut transcriptomes. As a result, 142531 transcripts and 73608 unigenes were obtained, and unigenes were annotated following NR, NT, KO, Swiss-Prot, PFAM, GO, and KOG databases. Among them, a total of 3,814 differentially expressed genes (DEGs) were identified between M. robertsii infected termites with or without antibiotics treatment. Given the lack of annotated genes in O. formosanus transcriptomes, we examined the expression profiles of the top 20 most significantly differentially expressed genes using qRT-PCR. Several of these genes, including APOA2, Calpain-5, and Hsp70, were downregulated in termites exposed to both antibiotics and pathogen but upregulated in those exposed only to the pathogen, suggesting that gut microbiota might buffer/facilitate their hosts against infection by finetuning physiological and biochemical processes, including innate immunity, protein folding, and ATP synthesis. Overall, our combined results imply that stabilization of gut microbiota can assist termites in maintaining physiological and biochemical homeostasis when foreign pathogenic fungi invade.

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.