Pathogenic fungi synergistically cooperate with Serratia marcescens to increase cockroach mortality.

The abuse of chemical insecticides has led to strong resistance in cockroaches, and biopesticides with active ingredients based on insect pathogens have good development prospects; however, their slow effect has limited their practical application, and improving their effectiveness has become an urgent problem. In this study, the interaction between Serratia marcescens and Metarhizium anisopliae enhanced their virulence against Blattella germanica and exhibited a synergistic effect. The combination of S. marcescens and M. anisopliae caused more severe tissue damage and accelerated the proliferation of the insect pathogen. The results of high-throughput sequencing demonstrated that the gut microbiota was dysbiotic, the abundance of the opportunistic pathogen Weissella cibaria increased, and entry into the hemocoel accelerated the death of the German cockroaches. In addition, the combination of these two agents strongly downregulated the expression of Imd and Akirin in the IMD pathway and ultimately inhibited the expression of antimicrobial peptides (AMPs). S. marcescens released prodigiosin to disrupted the gut homeostasis and structure, M. anisopliae released destruxin to damaged crucial organs, opportunistic pathogen Weissella cibaria overproliferated, broke the gut epithelium and entered the hemocoel, leading to the death of pests. These findings will allow us to optimize the use of insect pathogens for the management of pests and produce more effective biopesticides.

Two Iron(II), α-Ketoglutarate-Dependent Enzymes Encoded by the PPZ Gene Cluster of Metarhizium majus Enable Production of 8-Hydroxyperamine.

Entomopathogenic fungus Metarhizium majus contains the nine-gene PPZ cluster, with ppzA, encoding a peramine-producing nonribosomal peptide synthetase, as the central component. In this work, the roles of two α-ketoglutarate, iron-dependent oxygenases encoded by the PPZ genes ppzC and ppzD were elucidated. PpzD was found to produce both trans-4-hydroxy-l-proline and trans-3-hydroxy-l-proline in a 13.1:1 ratio, yielding a key precursor for peramine biosynthesis. PpzC was found to act directly on peramine, yielding the novel analogue 8-hydroxyperamine.

Pathogenicity of Metarhizium rileyi (Hypocreales: Clavicipitaceae) against Tenebrio molitor (Coleoptera: Tenebrionidae).

Tenebrio molitor L., also known as the mealworm, is a polyphagous insect pest that infests various stored grains worldwide. Both the adult and larval stages can cause significant damage to stored grains. The present study focused on isolating entomopathogenic fungi from an infected larval cadaver under environmental conditions. Fungal pathogenicity was tested on T. molitor larvae and pupae for 12 days. Entomopathogenic fungi were identified using biotechnological methods based on their morphology and the sequence of their nuclear ribosomal internal transcribed spacer (ITS). The results of the insecticidal activity indicate that the virulence of fungi varies between the larval and pupal stages. In comparison to the larval stage, the pupal stage is highly susceptible to Metarhizium rileyi, exhibiting 100% mortality rates after 12 days (lethal concentration 50 [LC50] = 7.8 × 106 and lethal concentration 90 (LC90) = 2.1 × 1013 conidia/mL), whereas larvae showed 92% mortality rates at 12 days posttreatment (LC50 = 1.0 × 106 and LC90 = 3.0 × 109 conidia/mL). The enzymatic analyses revealed a significant increase in the levels of the insect enzymes superoxide dismutase (4.76-10.5 mg-1) and glutathione S-transferase (0.46-6.53 mg-1) 3 days after exposure to M. rileyi conidia (1.5 × 105 conidia/mL) compared to the control group. The findings clearly show that M. rileyi is an environmentally friendly and effective microbial agent for controlling the larvae and pupae of T. molitor.

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.

Conidial mass production of entomopathogenic fungi and tolerance of their mass-produced conidia to UV-B radiation and heat.

We investigated conidial mass production of eight isolates of six entomopathogenic fungi (EPF), Aphanocladium album (ARSEF 1329), Beauveria bassiana (ARSEF 252 and 3462), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium anisopliae sensu lato (ARSEF 2341), Metarhizium pingshaense (ARSEF 1545), and Simplicillium lanosoniveum (ARSEF 6430 and 6651) on white or brown rice at four moisture conditions (75-100%). The tolerance of mass-produced conidia of the eight fungal isolates to UV-B radiation and heat (45 °C) were also evaluated. For each moisture content compared, a 20-g sample of rice in a polypropylene bag was inoculated with each fungal isolate in three replicates and incubated at 28 ± 1 °C for 14 days. Conidia were then harvested by washing the substrate, and conidial concentrations determined by haemocytometer counts. Conidial suspensions were inoculated on PDAY with 0.002% benomyl in Petri plates and exposed to 978 mW m-2 of Quaite-weighted UV-B for 2 h. Additionally, conidial suspensions were exposed to 45 °C for 3 h, and aliquots inoculated on PDAY with benomyl. The plates were incubated at 28 ± 1 °C, and germination was assessed at 400 × magnification after 48 h. Conidial production was generally higher on white rice than on brown rice for all fungal species, except for L. aphanocladii ARSEF 6433, regardless of moisture combinations. The 100% moisture condition provided higher conidial production for B. bassiana (ARSEF 252 and ARSEF 3462) and M. anisopliae (ARSEF 2341) isolates, while the addition of 10% peanut oil enhanced conidial yield for S. lanosoniveum isolate ARSEF 6430. B. bassiana ARSEF 3462 on white rice with 100% water yielded the highest conidial production (approximately 1.3 × 1010 conidia g-1 of substrate). Conidia produced on white rice with the different moisture conditions did not differ in tolerance to UV-B radiation or heat. However, high tolerance to UV-B radiation and heat was observed for B. bassiana, M. anisopliae, and A. album isolates. Heat-treated conidia of S. lanosoniveum and L. aphanocladii did not germinate.

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.

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.

Field efficacy of Metarhizium anisopliae oil formulations against Rhipicephalus microplus ticks using a cattle spray race.

Rhipicephalus microplus tick is the main ectoparasite of cattle in Brazil. The exhaustive use of chemical acaricides to control this tick has favored the selection of resistant tick populations. Entomopathogenic fungi, as Metarhizium anisopliae, has been described as a potential biocontroller of ticks. Therefore, the aim of this study was to evaluate the in vivo efficacy of two oil based formulations of M. anisopliae for the control of the cattle tick R. microplus under field conditions using a cattle spray race as a method of treatment. Initially, in vitro assays were carried out with an aqueous suspension of M. anisopliae, using mineral oil and/or silicon oil. A potential synergism between oils and fungus conidia for tick control was demonstrated. Additionally, the usefulness of silicon oil in order to reduce mineral oil concentration, while improving formulation efficacy was illustrated. Based on the in vitro results, two formulations were selected for use in the field trial: MaO1 (107 conidia/mL plus 5% mineral oil) and MaO2 (107 conidia/mL plus 2.5% mineral oil and 0.01% silicon oil). The adjuvants concentrations (mineral and silicon oils) were chosen since preliminary data indicate that higher concentrations caused significant mortality in adult ticks. For this, 30 naturally infested heifers were divided into three groups based on previous tick counts. The control group did not receive treatment. The selected formulations were applied on animals using a cattle spray race. Subsequently, tick load was evaluated weekly by counting. The MaO1 treatment significantly reduced the tick count only on day +21, reaching approximately 55% efficacy. On the other hand, MaO2 showed significantly lower tick counts on days +7, +14, and +21 after treatment, with weekly efficacy achieving 66%. The results showed a substantial reduction of tick infestation, up to day +28, using a novel formulation of M. anisopliae based in the mixture of two oils. Moreover, we have shown, for the first time, the feasibility of employing formulations of M. anisopliae for large-scale treatment methods, such as a cattle spray race, which in turn, may increase the use and adhesion to biological control tools among farmers.

The Conserved Cysteine-Rich Secretory Protein MaCFEM85 Interacts with MsWAK16 to Activate Plant Defenses.

Metarhizium anisopliae is an entomopathogenic fungus which may enhance plant growth and resistance when acting as an endophyte in host plants. However, little is known about the protein interactions nor their activating mechanisms. Common in fungal extracellular membrane (CFEM) proteins have been identified as plant immune regulators that suppress or activate plant resistance responses. Here, we identified a CFEM domain-containing protein, MaCFEM85, which was mainly localized in the plasma membrane. Yeast two-hybrid (Y2H), glutathione-S-transferase (GST) pull-down, and bimolecular fluorescence complementation assays demonstrated that MaCFEM85 interacted with the extracellular domain of a Medicago sativa (alfalfa) membrane protein, MsWAK16. Gene expression analyses showed that MaCFEM85 and MsWAK16 were significantly upregulated in M. anisopliae and M. sativa, respectively, from 12 to 60 h after co-inoculation. Additional yeast two-hybrid assays and amino acid site-specific mutation indicated that the CFEM domain and 52th cysteine specifically were required for the interaction of MaCFEM85 with MsWAK16. Defense function assays showed that JA was up-regulated, but Botrytis cinerea lesion size and Myzus persicae reproduction were suppressed by transient expression of MaCFEM85 and MsWAK16 in the model host plant Nicotiana benthamiana. Collectively, these results provide novel insights into the molecular mechanisms underlying interactions of M. anisopliae with host plants.

PULMONARY AND COELOMIC MYCOSES DUE TO METARHIZIUM AND BEAUVERIA SPECIES IN REPTILES.

This report documents cases of fatal pulmonary mycosis caused by entomopathogenic fungi in the genera Metarhizium and Beauveria (Order Hypocreales) in a loggerhead sea turtle (Caretta caretta), a Chinese alligator (Alligator sinensis), two gopher tortoises (Gopherus polyphemus), a Cuvier's dwarf caiman (Paleosuchus palpebrosus), a false gharial (Tomistoma schlegelii), a green sea turtle (Chelonia mydas), and a Kemp's ridley sea turtle (Lepidochelys kempii), and a case of granulomatous coelomitis in a hawksbill sea turtle (Eretmochelys imbricata). Fungi identified in these cases included Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae, Metarhizium robertsii, and one case of infection by a novel Metarhizium species. The animals were either housed at zoos or brought into rehabilitation from the wild. Although the majority of animals had comorbidities, the fungal infections were believed to be the primary cause of death. Fungal susceptibility testing was performed on two Beauveria spp. isolates, and revealed lower minimum inhibitory concentrations for itraconazole and voriconazole when compared to terbinafine and fluconazole. This case series demonstrates that a variety of reptile species from different orders are vulnerable to infection with Metarhizium, and multiple species of sea turtle are susceptible to infection with Beauveria.

Role of MicroRNA-Like RNAs in the Regulation of Spore Morphological Differences in the Entomopathogenic Fungus Metarhizium acridum.

Metarhizium acridum is an important microbial pesticide. Conidia (CO) and blastospores (BS) are two types of spores that occur in different patterns in the M. acridum life cycle and exhibit significant differences in cell morphology, structure, and activity. It may suggest that the fungus has a complex gene regulation mechanism. While previous studies on the differences between CO and BS have mainly focused on cell structure and application, little is known regarding the differences between CO and BS in fungi on the transcriptome levels. MicroRNAs (miRNAs) are small noncoding RNAs crucial to gene regulation and cell function. Understanding the miRNA-like RNAs (milRNA) and mRNA expression profiles related to cell growth and cellular morphological changes would elucidate the roles of miRNAs in spore morphological differences. In this study, 4,646 differentially expressed genes (DEGs) were identified and mainly classified in the GO terms cell, cell part, biological process, and catalytic activity. The KEGG annotation suggested that they were enriched in amino acid biosynthesis, carbohydrate metabolism, ribosome, and oxidative phosphorylation and might be involved in cell activity and structure. There were 113 differentially expressed milRNAs (DEMs), targeting 493 DEGs. Target gene functional analysis revealed that the target genes were mainly enriched in RNA transport, purine metabolism, and the cell cycle. In addition, we identified essential genes from milRNA-mRNA pairs that might participate in cell budding growth and cell membrane and wall integrity, including adenosine deaminase, glycosyl hydrolase, and G-patch domain protein (dno-miR-328-3p), WD repeat-containing protein pop1 (age-miR-127), and GPI-anchored wall transfer protein (cgr-miR-598). MilRNAs might therefore play a crucial role in cell growth and cellular morphological changes as transcriptional and post-transcriptional regulators.

Entomopathogenic fungi and Schinus molle essential oil: The combination of two eco-friendly agents against Aedes aegypti larvae.

Aedes aegypti transmits arbovirus, which is a public health concern. Certain filamentous fungi have the potential to control the disease. Here, the effects of Metarhizium anisopliae s.l. CG 153, Beauveria bassiana s.l. CG 206 and Schinus molle L. were investigated against Aedes aegypti larvae. In addition, the effect of essential oil on fungal development was analyzed. Fungal germination was assessed after combination with essential oil at 0.0025 %, 0.0075 %, 0.005 %, or 0.01 %; all of the oil concentrations affected germination except 0.0025 % (v/v). Larvae were exposed to 0.0025 %, 0.0075 %, 0.005 %, or 0.01 % of the essential oil or Tween 80 at 0.01 %; however, only the essential oil at 0.0025 % achieved similar results as the control. Larvae were exposed to fungi at 107 conidia mL-1 alone or in combination with the essential oil at 0.0025 %. Regardless of the combination, M. anisopliae reduced the median survival time of mosquitoes more than B. bassiana. The cumulative survival of mosquitoes exposed to M. anisopliae alone or in combination with essential oil was 7.5 % and 2 %, respectively, and for B. bassiana, it was 75 % and 71 %, respectively. M. anisopliae + essential oil had a synergistic effect against larvae, whereas B. bassiana + essential oil was antagonistic. Scanning and transmission electron microscopy, and histopathology confirmed that the interaction of M. anisopliae was through the gut and hemocoel. In contrast, the mosquito's gut was the main route for invasion by B. bassiana. Results from gas chromatography studies demonstrated sabinene and bicyclogermacrene as the main compounds of S. molle, and the in-silico investigation found evidence that both compounds affect a wide range of biological activity. For the first time, we demonstrated the potential of S. molle and its interaction with both fungal strains against A. aegypti larvae. Moreover, for the first time, we reported that S. molle might be responsible for significant changes in larval physiology. This study provides new insights into host-pathogen interplay and contributes to a better understanding of pathogenesis in mosquitoes, which have significant consequences for biological control strategies.

Identification of entomopathogenic fungus Metarhizium rileyi infested in fall armyworm in the cornfield of Korea, and evaluation of its virulence.

The fall armyworm (FAW) Spodoptera frugiperda is an important invasive pest in Africa and Asia. It is a polyphagous pest with at least 353 recorded host plant species, including corn. Chemical control of this pest is unsuccessful because of a developed resistance and harmful effects on the environment. Entomopathogenic fungi are potential biological control agents for FAW. In this study, the native strain of Metarhizium rileyi (KNU-Ye-1), collected from a cornfield at Yeongcheon, Korea, was identified by morphological and molecular characterization. The susceptibility of the fourth-instar larvae of FAW to the native strain M. rileyi was examined in the laboratory. The results showed that the Korean strain of M. rileyi (KNU-Ye-1) was highly virulent to FAW larvae, causing 89% mortality 7 days posttreatment. Therefore, M. rileyi (KNU-Ye-1) identified in this study is highly valuable for the biological control of FAW in the field.

Enhanced Cd efflux capacity and physiological stress resistance: The beneficial modulations of Metarhizium robertsii on plants under cadmium stress.

Due to the high migration capacity in agricultural soil-crop systems, cadmium (Cd) is accumulated in various crops and severely inhibits plant growth. In this study, we showed that, under Cd stress, the plant-symbiotic fungus Metarhizium robertsii reduced Cd accumulation in Arabidopsis thaliana shoots and roots by 21.8 % and 23.8 %, respectively. This is achieved by M. robertsii colonization-induced elevation of Cd efflux capacity via upregulation of three PCR genes, which is confirmed by the fact that the extent to which M. robertsii reduced Cd accumulation in the WT plants was greater than the inactivating mutants of the PCR genes. M. robertsii also alleviated Cd-induced leaf etiolation in A. thaliana by increasing the chlorophyll amount and modified plant physiological status to increase Cd stress tolerance via increasing production of catalase, peroxidase and glutathione and upregulating multiple HIPP proteins involved in sequestration of Cd. Notably, consistent with that in A. thaliana, the colonization of M. robertsii also reduced the Cd accumulation in Oryza sativa seedlings by upregulating the PCR gene OsPCR1, and increased chlorophyll amount and alleviated oxidative stress. Therefore, M. robertsii colonization reduced Cd accumulation in plants, and promoted plant growth and health by elevating Cd efflux capacity and modifying physiological status.

Metarhizium robertsii as a promising microbial agent for rice in situ cadmium reduction and plant growth promotion.

The toxic chemical element cadmium (Cd) in paddy fields triggered increasing problems of growth inhibition and food security in rice consistently. In this study, we found Metarhizium robertsii, which is widely used as a bioinsecticide and biofertilizer in agriculture and recently found to be resistant to Cd, developed intraradical and extraradical symbiotic hyphae in rice seedlings, and successfully colonized in the rice rhizosphere soil to more than 103 CFUs g-1 soil at harvesting. M. robertsii colonization significantly reduced Cd accumulations in both hydroponically cultured seedlings and the matured rice cultured in Cd contaminated potting soil (2 ppm). Notably, Cd accumulation reduction of the roots, stems, leaves, husks and grains of the matured rice induced by the fungus were 44.3%, 32.1%, 35.3%, 31.9% and 24.7%, respectively. It was caused by the M. robertsii-induced suppression of Cd intake transporter gene osNramp5 in the rice roots, and the chemical stabilizing of Cd to the residual fraction in the rhizosphere soil. In addition, the colonization of M. robertsii significantly promoted the growth characters and the photosynthesis of the rice plants. This is achieved by the increase of endogenous hormone levels of indole-3-acetic, gibberellin A3 and brassinolide induced by M. robertsii. Furthermore, the fungus enhanced the antioxidative capacities via increasing enzyme activities of catalase, peroxidase and the production of glutathione, ascorbic acid, proline in the rice plants. Our work provides theoretical basis for expanding the use of M. robertsii as in situ Cd accumulation reduction and detoxification agents for rice in contaminated paddy fields.

Biological control potential of entomopathogenic fungal strains against peach Fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae).

The peachfruit fly, Bactrocera zonata (Saunders) is a polyphagous pest in nature, belonging to order, Diptera and their respective family is Tephritidae. It mostly feeds on different crops, vegetables and fruits. Different traditional chemical insecticides have been used to control this notorious pest. Excessive consumption of pesticides has become a major threat to the fresh fruits trade since many importing countries refused to accept the shipments due to public health and environmental concerns. There is a growing trend to control these pests using the most effective biological control methods and other preventive measures have been adopted for reducing their attacks. Fungal agents have been used as biological agents to manage the attack of different insects pest through biological means. The present study was conducted to assess the virulence of three entomopathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii, against Bactrocera zonata stages under different laboratory conditions. The results showed that B. bassiana and M. anisopliae were more effective in pathogenicity and potentially kill at all stages of B. zonata as compared to V. lecanii. The highest mortality rate for the third larval instar and the pupal stage were recorded after exposure to the 1 × 1010 conidia/ml concentrations, B. bassiana, with 68.67% and 89.67%, respectively. Adult B. zonata flies were the most susceptible to all entomopathogenic fungi. However, M. anisopliae was more virulent against B. zonata adult flies than B. bassiana and V. lecanii at 1 × 1010 conidial concentration. Therefore, the entomopathogenic fungi B. bassiana and M. anisopliae can be used as an cost effective bio-insecticide in the integrated pest management programs to control B. zonata. This study will be helpful to overcome this pest through biological control means.

Variation of TNF modulates cellular immunity of gregarious and solitary locusts against fungal pathogen Metarhizium anisopliae.

Changes in population density lead to phenotypic differentiation of solitary and gregarious locusts, which display different resistance to fungal pathogens; however, how to regulate their cellular immune strategies remains unknown. Here, our stochastic simulation of pathogen proliferation suggested that humoral defense always enhanced resistance to fungal pathogens, while phagocytosis sometimes reduced defense against pathogens. Further experimental data proved that gregarious locusts had significantly decreased phagocytosis of hemocytes compared to solitary locusts. Additionally, transcriptional analysis showed that gregarious locusts promoted immune effector expression (gnbp1 and dfp) and reduced phagocytic gene expression (eater) and the cytokine tumor necrosis factor (TNF). Interestingly, higher expression of the cytokine TNF in solitary locusts simultaneously promoted eater expression and inhibited gnbp1 and dfp expression. Moreover, inhibition of TNF increased the survival of solitary locusts, and injection of TNF decreased the survival of gregarious locusts after fungal infection. Therefore, our results indicate that the alerted expression of TNF regulated the immune strategy of locusts to adapt to environmental changes.

Inductive Production of the Iron-Chelating 2-Pyridones Benefits the Producing Fungus To Compete for Diverse Niches.

Diverse 2-pyridone alkaloids have been identified with an array of biological and pharmaceutical activities, including the development of drugs. However, the biosynthetic regulation and chemical ecology of 2-pyridones remain largely elusive. Here, we report the inductive activation of the silent polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) (tenS) gene cluster for the biosynthesis of the tenellin-type 2-pyridones in the insect-pathogenic fungus Beauveria bassiana when cocultured with its natural competitor fungus Metarhizium robertsii. A pathway-specific transcription factor, tenR, was identified, and the overexpression of tenR well expanded the biosynthetic mechanism of 15-hydroxytenellin (15-HT) and its derivatives. In particular, a tandemly linked glycosyltransferase-methyltransferase gene pair located outside the tenS gene cluster was verified to mediate the rare and site-specific methylglucosylation of 15-HT at its N-OH residue. It was evident that both tenellin and 15-HT can chelate iron, which could benefit B. bassiana to outcompete M. robertsii in cocultures and to adapt to iron-replete and -depleted conditions. Relative to the wild-type strain, the deletion of tenS had no obvious negative effect on fungal virulence, but the overexpression of tenR could substantially increase fungal pathogenicity toward insect hosts. The results of this study well advance the understanding of the biosynthetic machinery and chemical ecology of 2-pyridones. IMPORTANCE Different 2-pyridones have been identified, with multiple biological activities but unclear chemical ecology. We found that the silent tenS gene cluster was activated in the insect pathogen Beauveria bassiana when the fungus was cocultured with its natural competitor Metarhizium robertsii. It was established that the gene cluster is regulated by a pathway-specific regulator, tenR, and the overexpression of this transcription factor expanded the biosynthetic machinery of the tenellin 2-pyridones. It was also found that the paired genes located outside the tenS cluster contribute to the site-specific methylglucosylation of the main compound 15-hydroxytenellin. Both tenellin and 15-hydroxytenellin can chelate and sequester iron to benefit the producing fungus to compete for different niches. This study well advances the biosynthetic mechanism and chemical ecology of 2-pyridones.

Timing and duration of light exposure during conidia development determine tolerance to ultraviolet radiation.

Metarhizium is an important genus of soil-inhabiting fungi that are used for the biological control of insects. The efficiency of biocontrol is dependent on the maintenance of inoculum viability under adverse field conditions such as solar ultraviolet (UV) radiation. Therefore, increasing the tolerance of Metarhizium to UV radiation is necessary. It was previously established that, in mycelium, exposure to visible light increases tolerance to UV radiation. Similarly, growth under visible light for 14 days induces the production of tolerant conidia. However, a study evaluating if and how visible light affects conidia and their relationship with UV radiation was never performed. Here, we report that a relatively short and timed exposure to light around the time of conidiation is sufficient to induce the production of conidia with increased photoreactivating capacity and UV tolerance in Metarhizium acridum. Conidia produced by this method retain their characteristic higher tolerance even after many days of being transferred to the dark. Furthermore, we show that mature conidia of M. acridum and Metarhizium brunneum can still answer to light and regulate UV tolerance, suggesting that gene expression is possible even in dormant spores. Being able to respond to light in the dormant stages of development is certainly an advantage conferring improved environmental persistence to Metarhizium.

Single cell encapsulation in a Pickering emulsion stabilized by TiO2 nanoparticles provides protection against UV radiation for a biopesticide.

A new formulation for biological pest control with significant UV protection capability has been developed in this research. The formulation is based on individual encapsulation of fungal conidia in an oil/water Pickering emulsion. The droplets size of the emulsions was tuned to meet the demands of single conidia encapsulation in the oil droplets. The emulsions are stabilized by amine-functionalized TiO2 (titania) nanoparticles (NPs). The droplet size, stability, and structure of the emulsions were investigated at different TiO2 contents and oil/water phase ratios. Most of the emulsions remained stable for 6 months. The structural properties of the Pickering emulsions were characterized by confocal microscopy and high-resolution cryogenic scanning electron microscopy (cryo-HRSEM). The presence of the TiO2 particles at the interface was confirmed by both confocal microscopy and cryo-HRSEM. Metarhizium brunneum-7 (Mb7) conidia were added to the emulsions. The successful encapsulation of individual conidia in the oil droplets was confirmed by confocal microscopy. The individual encapsulation of the conidia in the emulsions was significantly improved by dispersing the conidia in a 0.02 % Triton X-100 solution prior to emulsification. In addition, the bioassay results have shown, that exposure of the encapsulated conidia to natural UV light did not change their germination rates, however, the unprotected conidia demonstrated a dramatic decrease in their germination rates. These results confirm the UV protection capability of the studied emulsions.