Entomopathogenic Fungi Research Articles

Discovery of Gibellula floridensis from Infected Spiders and Analysis of the Surrounding Fungal Entomopathogen Community.

Characterization of fungal spider pathogens lags far behind their insect counterparts. In addition, little to nothing is known concerning the ecological reservoir and/or fungal entomopathogen community surrounding infection sites. Five infected spider cadavers were identified in the neo-tropical climate of north-central Florida, USA, from three of which viable cultures were obtained. Multi-locus molecular phylogenetic and morphological characterization identified one isolate as a new Gibellula species, here named, Gibellula floridensis, and the other isolates highly similar to Parengyodontium album. The fungal entomopathogen community surrounding infected spiders was sampled at different habitats/trophic levels, including soil, leaf litter, leaf, and twig, and analyzed using ITS amplicon sequencing. These data revealed broad but differential distribution of insect-pathogenic fungi between habitats and variation between sites, with members of genera belonging to Metarhizium and Metacordyceps from Clavicipitaceae, Purpureocillium and Polycephalomyces from Ophiocordyceps, and Akanthomyces and Simplicillium from Cordycipitaceae predominating. However, no sequences corresponding to Gibellula or Parengyodontium, even at the genera levels, could be detected. Potential explanations for these findings are discussed. These data highlight novel discovery of fungal spider pathogens and open the broader question regarding the environmental distribution and ecological niches of such host-specific pathogens.

Entomopathogenic potential of indigenous Simplicillium subtropicum (Hypocreales: Cordycipitaceae) isolates from Tamil Nadu, India, against the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae)

BackgroundAphis gossypii Glover (Hemiptera: Aphididae) is an economically important polyphagous pest species infesting many agricultural and horticultural crops causing severe yield losses. In order to develop biopesticides for the management of aphids in an environmentally safe manner, 6 indigenous entomopathogenic fungi Simplicillium subtropicum were isolated from soils of Tamil Nadu and their pathogenicity was tested against A. gossypii under laboratory conditions after characterization.ResultsPhenotypic and molecular characterization was performed for all the isolates for species identification. Results of the pathogenicity test showed that the isolate S6 (PP446637) performed well among other isolates causing 58.33% mortality on 7 days after treatment (DAT) @ 1 × 108 spores/ml. The LC50 and LT50 values were found to be 2.36 × 107 spores/ml at 7 DAT and 160.20 h @ 1 × 108 spores/ml, respectively. SEM images of A. gossypii treated with the isolate S6 (PP446637) revealed that initial phase of infection, i.e., adhesion, formation of germ tube and appressoria starts within 24 h post-infection (hpi), hyphal development and penetration of cuticle occurred within 48 hpi and extrusion of mycelium and conidiogenesis occurred within 72 hpi.ConclusionThis study reveals the entomopathogenic potential of indigenous S. subtropicum isolates against A. gossypii and suggests their use for the eco-friendly management of aphids.

Transcriptional reprogramming in the entomopathogenic fungus Metarhizium brunneum and its aphid host Myzus persicae during the switch between saprophytic and parasitic lifestyles

BackgroundThe fungus Metarhizium brunneum has evolved a remarkable ability to switch between different lifestyles. It develops as a saprophyte, an endophyte establishing mutualistic relationships with plants, or a parasite, enabling its use for the control of insect pests such as the aphid Myzus persicae. We tested our hypothesis that switches between lifestyles must be accompanied by fundamental transcriptional reprogramming, reflecting adaptations to different environmental settings.ResultsWe combined high throughput RNA sequencing of M. brunneum in vitro and at different stages of pathogenesis to validate the modulation of genes in the fungus and its host during the course of infection. In agreement with our hypothesis, we observed transcriptional reprogramming in M. brunneum following conidial attachment, germination on the cuticle, and early-stage growth within the host. This involved the upregulation of genes encoding degrading enzymes and gene clusters involved in synthesis of secondary metabolites that act as virulence factors. The transcriptional response of the aphid host included the upregulation of genes potentially involved in antifungal activity, but antifungal peptides were not induced. We also observed the induction of a host flightin gene, which may be involved in wing formation and flight muscle development.ConclusionsThe switch from saprophytic to parasitic development in M. brunneum is accompanied by fundamental transcriptional reprogramming during the course of the infection. The aphid host responds to fungal infection with its own transcriptional reprogramming, reflecting its inability to express antifungal peptides but featuring the induction of genes involved in winged morphs that may enable offspring to avoid the contaminated environment.

Single-cell polymer coating improves the desiccation tolerance of Metarhizium brunneum blastospores

Equipping cells with artificial shells or coats has been explored throughout the last decade, with goals such as immunomasking, in vivo tracing, and imparting tolerance to various biotic and abiotic stressors. One stressor, however, drying, has curiously been overlooked. In an industrial setting, the drying of cells is relevant when a satisfactory product shelf life must be achieved at a low cost. The drying of entomopathogenic organisms for biocontrol is a prime example of this. Here, the thin-walled blastospores of the entomopathogenic fungus Metarhizium brunneum are a great model organism for testing whether thin-cell surface polyelectrolyte layers may increase desiccation tolerance. In this study, we coated single M. brunneum blastospores with alternating layers of chitosan and alginate and assessed their effects on blastospore viability after drying. The desiccation tolerance improved with increasing layer numbers from 6.9% to a maximum of 27.5%. In addition, as the polymer chain length decreased, the desiccation tolerance further increased to 33.1%. Furthermore, we provided visual proof of the coating surrounding the blastospores via the use of fluorescent polymers and scanning electron microscopy. Finally, an investigation of differences in water absorption into coated and uncoated cells revealed that water absorbed faster into coated cells when alginate was on the surface of the structure but slower when the outermost layer was composed of chitosan. We conclude that, via polyelectrolyte multilayering on thin-walled blastospores, desiccation tolerance can be significantly increased, but a deeper understanding is necessary to extract the full potential from this technique.Graphical

Study on the role of RPL23 gene in active immunity of termite Reticulitermes chinensis against Metarhizium anisopliae

Ribosomal proteins are considered to be involved in the immunity of different animals against pathogens. The protein level of RPL23 increased after fungal infection in termites, but how it influence active immunity in termites is unknown. The role of RPL23 gene was studied to evaluate its impact on active immunity of termite Reticulitermes chinensis against entomopathogenic fungus (EPF) Metarhizium anisopliae. The RPL23 gene fragment (414 bp) was cloned and phylogenetic analysis revealed that it’s very close to termite Coptotermes formosanus. Expression of RPL23 gene was significantly higher in abdomen as compared to thorax and head. Silencing RPL23 gene had no significant impact on the frequency and time of allogrooming towards fungus exposed termites from nestmates, which showed that nestmates acquired spores from infected termites through allogrooming. Expression of immune genes (GNBP1, GNBP2 and phenoloxidase) and apoptosis related genes (TNF-α, caspase 1, caspase 3 and caspase 8) decreased significantly in nestmates of fungus-treated termites after silencing of RPL23 gene as compared to control. Antifungal activity and survival of RPL23 silenced nestmates of fungus-treated termites also decreased. To sum up, this study found that silencing of RPL23 gene broke the active immunity against M. anisopliae infection, reduced the antifungal activity of termites, weakened cell apoptosis, and led to increased mortality of termites, which may help to find a potential alternative for chemical insecticides to control termites.

Formulation and bio-efficacy of different isolates of Beauveria bassiana against adults and third nymphal instar of desert locust (Schistocerca gregaria Forskål)

Recently, the Horn of Africa witnessed a swift increase in the incidence of desert locust (Schistocerca gregaria) invasion. During outbreaks, pesticides are applied through aerial or ground spraying to kill the insects, and/or to prevent their spread to new grounds. However, after decades of extensive use, many drawbacks such as contamination of the environment, toxicity to non-target organisms, harmful residues on food, pest resistance, and bioaccumulation in the food chains emerged. Entomopathogenic fungi offer viable alternatives to chemical pesticides against many insect invasions, but few studies have tested their bio-efficacy in desert locusts. Therefore, the current study aimed at isolating, formulating local isolates 231, 334, 333, 341, 349, 351, 339 of Beauveria bassiana, and testing their bio-efficacy against larval and adult desert locusts. The 21-day experiment was conducted under controlled conditions in a greenhouse. Soil samples were collected from two agroecological zones in Isiolo and Laikipia Counties in Kenya. B. bassiana was isolated from the soil samples using the Galleria bait method and cultivated in Sabouraud Dextrose Agar Yeast (SDAY). The isolates were identified based on molecular techniques (DNA and PCR amplification). The conidia of the isolates were screened and bioassays on 30 locusts was conducted for 14 days. The best isolates eliciting over 90 % mortality during screening were used for formulations using three carrier materials (liquid paraffin, Diatomaceous Earth, and whey) which were again tested against adult and 3rd nymphal instars of the locusts. The stability of the formulations was also tested after 1 and 2 months. All the tested isolates of B. bassiana significantly outperformed the control and thus pathogenic to the adults and 3rd nymphal instars of S. gregaria under laboratory conditions. They caused mortality ranging from 57.8–100 % after 14 days post-incubation. The isolates 341, 231, and, 334 elicited 50 % mortality responses at concentrations 1.1 × 105 conidia/ml, 2.5 × 105 conidia/ml and 1.7 × 106 conidia/ml respectively in adults and 1.1 × 105 conidia/ml, 2.5 × 105 conidia/ml and, 1.7 × 106 conidia/ml respectively in 3rd nymphal instars. Formulations with 341–1, 341–2, and, 334–1 had the highest efficacy (>99 %) against the adult locusts. There was a significant 3-way interaction (P < 0.05) of isolate for the formulation, carrier material and, time in determining the Cfu of the B. bassiana formulations. After 1 month, the best Cfu occurred in formulation with isolates 231 and 341 formulated using Diatomaceous Earth, while the highest Cfu was observed in formulation with isolate 334 formulated with either liquid paraffin or whey. After 2 months, the highest Cfu occurred in formulation with isolates 231, 341, and, 334 formulated using liquid paraffin. This study provides encouraging data for the development of potential biopesticides formulated with different carrier materials against desert locusts.

A randomized controlled trial of Tickoff® (Metarhizium anisopliae ICIPE 7) for control of tick infestations and transmission of tick-borne infections in extensively grazed zebu cattle in coastal Kenya

The entomopathogenic fungus Metarhizium anisopliae isolate ICIPE 7 is being developed as an eco-friendly alternative to chemical acaricides in managing natural tick infestation on livestock. Its impact on tick infestation and tick-borne infections in cattle under natural conditions are yet unclear. We conducted a randomized controlled field trial to assess the safety and effects of Tickoff® (a formulation of M. anisopliae isolate ICIPE 7) and the chemical acaricide Triatix® on tick infestation and incidence of Anaplasma marginale and Theileria parva in extensively grazed zebu cattle in coastal Kenya. A total of 217 eligible herds comprising 1459 intent-to-treat zebu cattle were enrolled from 12 villages. The herds were randomly assigned in a 1:1:1 ratio to Tickoff®, Triatix®, or Tickoff® excipients. Tick counts, treatment administrations, and adverse events were registered every two weeks for seven months. The mortality of ticks collected from treated cattle was monitored in vitro. Infections with A. marginale and T. parva were monitored every two months. No adverse events were reported in either treatment group. Tickoff® did not significantly affect tick infestation (p = 0.869) or infection incidence (p > 0.05) compared to excipients. Triatix® significantly reduced tick infestation (p < 0.001) and incidence of T. parva (p = 0.042), but not A. marginale (p = 0.509) compared to the reference Tickoff®. In ticks that were removed from cattle, Tickoff® demonstrated significant pathogenicity in vitro relative to excipients (hazard ratio: 8.50, 95 % CI: 4.67–15.47). Fungus growth and sporulation were also observed on tick cadavers from Tickoff®, but not from excipients. While Tickoff® did not impact tick counts, its delayed, but significant effect on tick mortality may hinder onward pathogen transmission and give rise to indirect (i.e., to untreated animals) epidemiological effects, that were not picked up with this study design. Additionally, adverse environmental conditions resulted in low tick abundance and pathogen circulation towards the end of the study period, reducing the power of the study. This work re-emphasizes the challenges of randomized controlled field trials and the complexity of assessing the impact of vector control products on both direct and indirect impacts on pathogen transmission.

The synergistic potential of polyethylene glycol 400 for the control of Hyphantria cunea larvae by Beauveria bassiana

The efficacy of entomopathogenic fungi as pest control agents is constrained by both their physiological state and external environmental factors. This study identified synergists capable of enhancing the insecticidal activity of Beauveria bassiana (Bb) and investigated the underlying synergistic mechanisms. Our results found that among 6 potential synergists, polyethylene glycol 400 (PEG) and trehalose significantly improved Bb's lethality against Hyphantria cunea larvae, with PEG demonstrating the most pronounced effect. PEG treatment markedly increased Bb spore adhesion and germination rates, while spore hydrophobicity and growth rates remained unaffected. Moreover, PEG-treated spores exhibited higher thermal tolerance compared to untreated ones. In the Bb + PEG treatment group, the hemocyte count, encapsulation and melanization activities, and the expression of related regulatory genes were significantly lower than those in the Bb treatment group. Additionally, pathogen recognition, signal transduction, and humoral immunity effector genes expression were markedly suppressed in the Bb + PEG group. A significant reduction in the content of total amino acids, free fatty acids, glucose, and trehalose, alongside decreased expression of key regulatory genes in the tricarboxylic acid cycle and glycolysis pathways, was observed in the Bb + PEG treatment group. Furthermore, PEG enhanced Bb-induced apoptosis in H. cunea larvae, as evidenced by the upregulation of apoptosis-related genes. Notably, PEG alone did not significantly impact the innate immunity, energy metabolism, or apoptosis in H. cunea larvae. Overall, PEG exhibits considerable potential in amplifying Bb's insecticidal activity by directly optimizing spore performance and indirectly modulating the larvae's innate immunity, energy metabolism, and apoptosis.

Integrative approaches to improve litchi (Litchi chinensis Sonn.) plant health using bio-transformations and entomopathogenic fungi

Bio-transformations refer to the chemical modifications made by an organism on a chemical compound that often involves the interaction of plants with microbes to alter the chemical composition of soil or plant. Integrating bio-transformations and entomopathogenic fungi into litchi cultivation can enhance symbiotic relationships, microbial enzymatic activity in rhizosphere, disease suppression and promote overall plant health. The integration of biological formulations and entomopathogenic fungi can significantly influence growth, nutrient dynamics, physiology, and rhizosphere microbiome of air-layered litchi (Litchi chinensis Sonn.) saplings. Biological modifications included, K-mobilizers, AM fungi, Pseudomonas florescence and Azotobacter chroococcum along with Metarhizium, entomopathogenic fungi have been used. The treatments included, T1-Litchi orchard soil + sand (1:1); T2-Sand + AM fungi + Azotobacter chroococcum (1:2:1); T3-Sand + Pseudomonas florecence + K-mobilizer (1:1:1); T4- AM fungi + K-mobilizers (1:1); T5, P. Florecence + A. chroococcum + K-mobilizer (1:1:1); T6-Sand + P. florecence (1:2) and T7-Uninoculated control for field performance. Treatments T4-T6 were further uniformly amended with drenching of Metarrhizium in rhizosphere. T2 application significantly increased resident microbe survival, total chlorophyll content and root soil ratio in seedlings. A. chroococcum, Pseudomonas, K-mobilizers and AM fungi increased in microbial biomass of 2.59, 3.39, 2.42 and 2.77 times, respectively. Acidic phosphatases, dehydrogenases and alkaline phosphatases were increased in rhizosphere. Leaf nutrients reflected through DOP were considerably altered by T2 treatment. Based on Eigen value, PCA-induced changes at biological modifications showed maximum total variance. The study inferred that the bio-transformations through microbial inoculants and entomopathogenic fungi could be an encouraging strategy to enhance the growth of plants, health and productivity. Such practices align well with the goals of sustainable agriculture through biological means by reducing dependency on chemical inputs. By delving into these aspects, the research gaps including microbial processes, competitive and symbiotic relationships, resistance in microbes and how complex interactions among bio-transformations, entomopathogenic fungi and microbes can significantly impact the health and productivity of litchi. Understanding and harnessing these interactions can lead to more effective and sustainable farming practices.

The secretory protein COA1 enables Metarhizium robertsii to evade insect immune recognition during cuticle penetration.

The interplay between the insect immune system and entomopathogenic fungi during cuticle penetration is not yet fully understood. Here, we show that a secretory protein COA1 (coat of appressorium 1) from Metarhizium robertsii, an entomopathogenic fungus causing diseases in a wide range of insects, is required to avoid host immune recognition during cuticle penetration. COA1 is highly expressed on the cuticle and translocated to the cell surface, where it directly binds with and masks carbohydrates of the fungal cell wall to avoid provoking the host's intense immune response. Deletion of Coa1 results in a robust immune response, leading to a reduction in bacterial load in both the gut and hemocoel and ultimately attenuating fungal virulence. Our work reveals a novel cell surface protein indispensable for fungal pathogenicity via masking cell wall carbohydrates to avert a hypersensitive response from the host.

Bio-insecticidal Efficacy of Beauveria bassiana Vuill. Strains against Tettigonia viridissima L. in Tobacco

The green grasshopper (Tettigonia viridissima L.) is a species which attacks tobacco plants. The purpose of the present study was to establish the bio-insecticidal efficacy of entomopathogenic fungi Beauveria bassiana Vuill. strains 214, 644, and 733 on the growth and development of T. viridissima in tobacco. The research was conducted under laboratory conditions. The study found insecticidal effects of the B. bassiana strains 214, 644, and 733 against T. viridissima. Surviving of larvae was monitored at 5-day intervals until the 15th day after treatment with a spore suspension of the tested Beauveria strains. The highest virulence of the tested fungi was shown by strain 644, followed by strains 214 and 733. On the 5th day after treatment, no efficacy against T. viridissima larvae was detected. On the 10th day post-treatment strain 644 showed 66.7% efficacy, while efficacy against larvae of grasshoppers for the other two strains 214 and 733 was significantly lower: 33.3%. Efficacy gradually increased and on the 15th day post-treatment 100% mortality of T. viridissima was observed for all isolates. The results obtained from the virulence test of B. bassiana 214, 644, and 733 ascertain their application as biocontrol agents in tobacco production under field conditions.

How to control nasty scarabs? Effectiveness of the generalist entomopathogenic fungus Metarhizium brunneum against garden chafer larvae

Several scarab beetles (Scarabaeidae) cause major damage in agriculture, horticulture, and forestry. Especially root feeding scarab larvae cause substantial economic losses on crops, forage plants and recreational areas. In Europe, the entomopathogenic fungi (EPF) Beauveria brongniartii and Metarhizium brunneum are applied against the most problematic, native scarabs, the common cockchafer (Melolontha melolontha) and the garden chafer (Phyllopertha horticola). While the control of cockchafer larvae with B. brongniartii is well-researched, conclusive results from field applications for the control of the garden chafer with M. brunneum remain elusive. We therefore assessed the performance of commercially available fungal strains of M. brunneum against garden chafer larvae in pot and large-scale field experiments.The application of M. brunneum significantly increased the abundance of fungal propagules in the soil by approximately a factor of ten, irrespective of high levels of naturally occurring Metarhizium spp. Furthermore, the applied strains infected and propagated on the larvae and the mortality of garden chafer larvae was slightly increased due to the fungal treatments. We found three other EPF species frequently infecting garden chafer larvae (mean infection rates: 13–25%), including B. brongniartii which is considered to be a specific pathogen of the cockchafer. Thus, the applied fungal strains were only part of a consortium of natural enemies which reduces garden chafer populations strongly as a whole. Hence, we suggest that the application of EPF may be advisable on areas with reduced natural enemies such as golf courses but is probably redundant on meadows harboring a diverse consortium of antagonists.

Mutual interaction of the entomopathogenic and endophytic fungus Metarhizium anisopliae with zearalenone as a native component of crude Fusarium extract

The present study revealed the consequences of the interaction of a widely used bioinsecticide and endophyte Metarhizium anisopliae with the hazardous mycotoxin zearalenone (ZEN) as a pure substance and with ZEN as a native component of a crude Fusarium extract. In the environment, microorganisms encounter a mixture of metabolites secreted by other organisms living in the same area, not single substances. The obtained results suggest that M. anisopliae, exposed to a variety of active substances produced by Fusarium graminearum, is able to eliminate ZEN. Within 14 days, M. anisopliae biotransformed 90.8% and 85.8% of ZEN as a pure substance and ZEN as a native component of the F. graminearum extract from Rice Medium (E-Fg-RM), respectively, through reduction predominantly to α-epimers of zearalenols and zearalanols, considered more estrogenic than ZEN, which can raise concerns. Compared to pure ZEN, E-Fg-RM significantly affected the production of Metarhizium secondary metabolites by increasing the destruxins amount by approximately 20–25% and reducing the swainsonine content by 96.2%. All these findings provide a possible picture of the interaction of M. anisopliae with ZEN in the wild, mainly as a result of the use of crude extract from Fusarium, which contained a mixture of different metabolites.

Biotransformation of Xanthohumol by Entomopathogenic Filamentous Fungi.

Xanthohumol (1) is a major prenylated flavonoid in hops (Humulus lupulus L.) which exhibits a broad spectrum of health-promoting and therapeutic activities, including anti-inflammatory, antioxidant, antimicrobial, and anticancer effects. However, due to its lipophilic nature, it is poorly soluble in water and barely absorbed from the gastrointestinal tract, which greatly limits its therapeutic potential. One method of increasing the solubility of active compounds is their conjugation to polar molecules, such as sugars. Sugar moiety introduced into the flavonoid molecule significantly increases polarity, which results in better water solubility and often leads to greater bioavailability. Entomopathogenic fungi are well known for their ability to catalyze O-glycosylation reactions. Therefore, we investigated the ability of selected entomopathogenic filamentous fungi to biotransform xanthohumol (1). As a result of the experiments, one aglycone (2) and five glycosides (3-7) were obtained. The obtained (2″E)-4″-hydroxyxanthohumol 4'-O-β-D-(4‴-O-methyl)-glucopyranoside (5) has never been described in the literature so far. Interestingly, in addition to the expected glycosylation reactions, the tested fungi also catalyzed chalcone-flavanone cyclization reactions, which demonstrates chalcone isomerase-like activity, an enzyme typically found in plants. All these findings undoubtedly indicate that entomopathogenic filamentous fungi are still an underexploited pool of novel enzymes.

Cordyceps militaris fruit body activates myeloid dendritic cells via a Dectin-1-mediated pathway

Cordyceps militaris, an entomopathogenic fungus, has been traditionally used in East Asian medicine. Recent research indicates that the fruit bodies of C. militaris are rich in bioactive compounds, such as polysaccharides and nucleosides, which may offer health benefits. However, the specific components responsible for its immunostimulatory effects and the mechanisms involved remain unclear. This study explored the immunomodulatory activity of a fruit body extract from C. militaris, named Ryukyu-kaso (RK), and examined the effect of the β-glucan receptor Dectin-1 on bone marrow-derived dendritic cells (BMDCs). Our results demonstrated that RK, which contains 1,3-β-glucan, effectively stimulated BMDCs to secrete pro-inflammatory and immunoregulatory cytokines and upregulated surface markers indicative of maturation and activation. Notably, these immunostimulatory effects were completely absent in BMDCs derived from Dectin-1-knockout mice, confirming that Dectin-1 is crucial for RK-induced immunomodulation. These findings provide new insights into the immunostimulatory mechanisms of C. militaris and underscore the potential of RK as a natural immunomodulatory agent for various therapeutic applications.

How does Neoseiulus californicus McGregor respond to sublethal doses of entomopathogenic fungus Beauveria bassiana (Hyp.: Cordycipitaceae)?

Two-spotted spider mite, Tetranychus urticae Koch (Acari: Prostigmata), is one of the most economically important mite species, mainly controlled by chemical acaricides. Natural enemies have been assessed as reliable alternatives for management of this phytophagous mite. In the current project, demographic characteristics of Neoseiulus californicus McGregor (Acari: Phytoseiidae) to sublethal concentrations (LC10 = 6.76 × 102, LC20 = 8.74 × 103 and LC30 = 55.38 × 103 conidia ml-1) of entomopathogenic fungus, Beauveria bassiana (Bals.) Vuill. TV strain were investigated under laboratory conditions at 25 ± 2°C, 70 ± 5% RH and a photoperiod of 16:8 (L:D) h. Our results indicated that when adult predators were exposed to LC20 and LC30 of B. bassiana, the oviposition period was significantly reduced compared with other treatments. Neoseiulus californicus fecundity was significantly greater in the control (37 eggs) than in LC30 (24 eggs). Life table analysis revealed that the net reproductive rate (R0) declined as the sublethal concentrations of B. bassiana increased. The most striking result to emerge from the data is that not only intrinsic (r); but also, finite rate of increase (λ) was not significantly affected by different concentrations of B. bassiana. Our findings revealed some potential interactions of B. bassiana and N. californicus during their combinations for managing T. urticae that may be helpful for optimising control of this important pest.

The SUMO gene MrSmt3 is involved in SUMOylation, conidiation and stress response in Metarhizium robertsii.

Smt3, as a small ubiquitin-like modifier (SUMO), play an essential role in the regulation of protein SUMOylation, and thus this process can affect various important biological functions. Here, we investigated the roles of MrSmt3 (yeast SUMO/Smt3 homologs) in the entomopathogenic fungus Metarhizium robertsii. Our results of subcellular localization assays demonstrated that MrSmt3 was present in the cytoplasm and nucleus, whereas MrSmt3 was largely localized in the nucleus during oxidative stress. Importantly, disruption of MrSmt3 significantly decreased the level of protein SUMOylation under heat stress. Deletion of MrSmt3 led to a significant decrease in conidial production, and increased sensitivity to various stresses, including heat, oxidative, and cell wall-disturbing agents. However, bioassays of direct injection and topical inoculation demonstrated that deletion of MrSmt3 did not affect fungal virulence. Furthermore, RNA-seq analysis identified 1,484 differentially expressed genes (DEGs) of the WT and ΔMrSmt3 during conidiation, including 971 down-regulated DEGs and 513 up-regulated DEGs, and further analysis showed that the expression level of several classical conidiation-associated genes, such as transcription factor AbaA (MAA_00694), transcription factor bZIP (MAA_00888) and transcription factor Ste12 (MAA_10450), was down-regulated in the ΔMrSmt3 mutant. Specifically, the major downregulated DEGs were mainly associated with a variety of metabolic regulatory processes including metabolic process, organic substance metabolic process and primary metabolic process. Collectively, our findings highlight the important roles of the SUMO gene MrSmt3 in modulating SUMOylation, conidiation and stress response in M. robertsii.

Acaricidal Efficacy of Abamectin against Tetranychus urticae Populations When Combined with Entomopathogenic Fungi

Tetranychus urticae (Acari: Tetranychidae) is a widespread and serious mite pest that infests tomato plants and causes economic losses worldwide. We investigated the acaricidal efficacy of two isolates of entomopathogenic fungi (EPF) Metarhizium robertsii (WG-7) and Beauveria bassiana (WG-12) alone and in combination with abamectin when applied topically to tomato leaf discs in the laboratory against T. urticae. We also evaluated the establishment and proliferation of T. urticae mite life stages on tomato plants in the greenhouse after application of each of the above treatments. The combination of abamectin with each EPF caused 100% mortality in T. urticae immatures after 2 days while each EPF or abamectin alone caused moderate mortality, not exceeding 74.2% 3 days post-exposure. Complete (100%) mortality of adults was observed after 5 days in leaf discs treated with M. robertsii plus abamectin whereas B. bassiana plus abamectin caused 100% mortality after 7 days. The mean number of eggs, emerged immatures, and adults were significantly reduced on both sides of the leaves (i.e., abaxial and adaxial sides) after using the combined application of M. robertsii or B. bassiana plus abamectin, compared to abamectin alone and controls. Our results reveal that the acaricidal efficacy of abamectin combined with either EPF was significantly better in managing the T. urticae life stages than either treatment alone under greenhouse conditions.

Rho4 interacts with BbGDI and is essential for the biocontrol potential of Beauveria bassiana by maintaining intracellular redox homeostasis

Rho4 is a member of the Rho-family small GTPases. In this study, we revealed the function of Rho4 and explored its mechanism involved in intracellular redox homeostasis in Beauveria bassiana, one of the most widely utilized filamentous entomopathogenic fungi. The disruption of Rho4 in B. bassiana resulted in significant phenotypic changes, such as fungal virulence, growth rate on different media, thermotolerance, germination, and conidiation. Integrated analysis of proteomic and transcriptomic data unveiled differential expression patterns of various redox-related genes and proteins in Δrho4, including the down-regulation of GST shown in proteomic and transcriptomic data, and the down-regulated gene expression levels of NOX, SOD, CAT, and GR in the transcriptome. Based on the bi-omics analysis, we focused on the impact of Rho4 in maintaining intracellular redox homeostasis. A decreased ROS content observed in Δrho4 might be attributed to the reduced NOX activity, which subsequently affects the GSH-producing/consuming metabolisms, with the attenuated activities of GR and GST. The imbalanced redox homeostasis also resulted in the reduced enzyme activities of SOD and CAT. Exogenous oxides could partially complement the ROS level and rescue the growth defect in Δrho4 to a certain extent. Besides, BbGDI was initially identified as an interacting protein of Rho4 in entomopathogenic fungi. Our results provide a comprehensive understanding of the function and regulating mechanism of Rho4 in B. bassiana.

Pyricularia oryzae enhances Streptomyces griseus growth via non-volatile alkaline metabolites.

Chemical compounds that affect microbial interactions have attracted wide interest. In this study, Streptomyces griseus showed enhanced growth when cocultured with the rice blast fungus Pyricularia oryzae on potato dextrose agar (PDA) medium. An improvement in S. griseus growth was observed before contact with P. oryzae, and no growth-promoting effect was observed when the growth medium between the two microorganisms was separated. These results suggested that the chemicals produced by P. oryzae diffused through the medium and were not volatile. A PDA plate supplemented with phenol red showed that the pH of the area surrounding P. oryzae increased. The area with increased pH promoted S. griseus growth, suggesting that the alkaline compounds produced by P. oryzae were involved in this growth stimulation. In contrast, coculture with the soilborne plant pathogen Fusarium oxysporum and entomopathogenic fungus Cordyceps tenuipes did not promote S. griseus growth. Furthermore, DL-α-Difluoromethylornithine, a polyamine biosynthesis inhibitor, prevented the increase in pH and growth promotion of S. griseus by P. oryzae. These results indicated that P. oryzae increased pH by producing a polyamine.