Optimization of granular formulations of Metarhizium humberi microsclerotia with humectants.

Granular microsclerotial formulations of entomopathogenic fungi deserve attention because of their post-application, in situ production of new conidia that enhance and prolong mycoinsecticidal efficacy against a target pest insect. Because high ambient moisture is a crucial condition to induce fungal development and conidiogenesis on granules, we tested the impacts of the additions of three humectants-glycerin, propylene glycol, and polyethylene glycol 400-on water absorption by pellets incorporating microsclerotia of Metarhizium humberi IP 46 with microcrystalline cellulose or vermiculite carriers, and on the production of infective conidia of IP 46 microsclerotia in ambient humidities suboptimal for routine conidiogenesis. Glycerin facilitated greater and faster absorption of water than the other humectants. Microcrystalline cellulose absorbed low quantities of water without any added humectant whereas vermiculite did not. IP 46 did not grow or sporulate on pellets prepared with or without glycerin at 86% relative humidity (RH) or on control pellets without glycerin at 91% RH; conidial production on pellets prepared with vermiculite or microcrystalline cellulose and 10% glycerin reached 1.1 × 105 conidia/mg and 1 × 105 conidia/mg, respectively, after 20 days of exposure at 91% RH. Hence, these results strongly support glycerin as a suitable humectant for granular microsclerotial formulations of this fungus.

[Effect of the combination of Metarhizium anisopliae and Gliocladium virens on the oviposition, hatching and emergency of Aedes aegypti]. / Efecto de la combinación de Metarhizium anisopliae y Gliocladium virens sobre la oviposición, eclosión y emergencia de Aedes aegypti.

OBJECTIVE: To evaluate the effect of the combination of Metarhizium anisopliae and Gliocladium virens, both with Aqua Reslin Super, on the oviposition, hatching and emergence of Aedes aegypti. MATERIALS AND METHODS: Evaluations were carried out to determine the effect of treatments impregnated on filter paper and exposed within plastic containers on the oviposition, hatching and emergency of Aedes aegypti. RESULTS: The results indicated that the fungus and insecticide combinations did not affect the oviposition behavior, but if the hatching of the eggs and the adult's emergency. CONCLUSIONS: With the results it can be concluded that the combination of fungi + insecticide can be a good option to be applied in oviposition sites with a view to the development of a lethal ovitrap.

OBJETIVO: Evaluar el efecto de la combinación de Metarhizium anisopliae y Gliocladium virens, ambos con Aqua Reslin Super, sobre oviposición, eclosión y emergencia de Aedes aegypti. MATERIAL Y MÉTODOS: Se realizaron evaluaciones para determinar el efecto de los tratamientos impregnados en papel filtro y expuestos dentro de recipientes de plástico sobre la oviposición, eclosión y emergencia de Aedes aegypti. RESULTADOS: Los resultados indicaron que las combinaciones hongo e insecticida no afectaron el comportamiento de oviposición, pero sí la eclosión de los huevos y la emergencia del adulto. CONCLUSIONES: Con los resultados se puede concluir que la combinación de hongos + insecticida puede ser una buena opción para aplicarse en sitios de oviposición con miras al desarrollo de una ovitrampa letal.

Horizontal transmission of Metarhizium anisopliae between Spoladea recurvalis (Lepidoptera: Crambidae) adults and compatibility of the fungus with the attractant phenylacetaldehyde.

The compatibility of the entomopathogenic fungus Metarhizium anisopliae ICIPE 30 which was proved to be pathogenic to adult Spoladea recurvalis, and phenylacetaldehyde (PAA) floral attractant for lepidopteran moths, was investigated under laboratory and field conditions through spatial and temporal separations. Horizontal transmission of M. anisopliae ICIPE 30 between adult S. recurvalis and the number of conidia picked up by a single moth from the autoinoculation device were also determined under laboratory tests. When freshly emerged moths were inoculated with fungal conidia ("donors") and maintained together with an equal number of untreated freshly emerged moths ("recipients") in the laboratory, they were able to transmit infection to untreated moths resulting to 76.9% mortality with an LT50 value of 6.9 days. The quantity of conidia a moth could acquire and retain from the autoinoculation device in the laboratory was assessed at 0, 24, 48, and 72 h post-inoculation. The overall mean number of conidia acquired by a single moth was significantly higher immediately after exposure (0 h) (14.3 ±â€¯2.5 × 105) than at 24, 48, and 72 h after inoculation (F = 10.26, Df = 3,8, P = 0.003), though a single moth still retained 4.6 ±â€¯0.9 × 105 conidia 72 h post inoculation. Laboratory results showed that PAA completely inhibited the germination of the conidia 8 days post exposure, while the conidial viability was not affected in the control treatment without PAA. Under field conditions, the inhibitory effects of PAA on conidial germination was minimized by placing it at a distance of 5-10 cm from M. anisopliae isolate ICIPE 30 conidia. There was no significant difference in conidial germination in the control treatment and in treatments where PAA was placed at 5 cm and 10 cm away from M. anisopliae isolate ICIPE 30. Conidial germination was low in the autoinoculation device that had PAA directly exposed to the fungus. PAA is therefore compatible with M. anisopliae ICIPE 30 for use in integrated management of S. recurvalis, if spatially separated 5 cm away from the fungus and could thus be combined in an autocontamination devices for the control of S. recurvalis.

Riboflavin induces Metarhizium spp. to produce conidia with elevated tolerance to UV-B, and upregulates photolyases, laccases and polyketide synthases genes.

AIMS: The effect of nutritional supplementation of two Metarhizium species with riboflavin (Rb) during production of conidia was evaluated on (i) conidial tolerance (based on germination) to UV-B radiation and on (ii) conidial expression following UV-B irradiation, of enzymes known to be active in photoreactivation, viz., photolyase (Phr), laccase (Lac) and polyketide synthase (Pks). METHODS AND RESULTS: Metarhizium acridum (ARSEF 324) and Metarhizium robertsii (ARSEF 2575) were grown either on (i) potato dextrose agar medium (PDA), (ii) PDA supplemented with 1% yeast extract (PDAY), (iii) PDA supplemented with Rb (PDA+Rb), or (iv) PDAY supplemented with Rb (PDAY+Rb). Resulting conidia were exposed to 866·7 mW m-2 of UV-B Quaite-weighted irradiance to total doses of 3·9 or 6·24 kJ m-2 . Some conidia also were exposed to 16 klux of white light (WL) after being irradiated, or not, with UV-B to investigate the role of possible photoreactivation. Relative germination of conidia produced on PDA+Rb (regardless Rb concentration) or on PDAY and exposed to UV-B was higher compared to conidia cultivated on PDA without Rb supplement, or to conidia suspended in Rb solution immediately prior to UV-B exposure. The expression of MaLac3 and MaPks2 for M. acridum, as well as MrPhr2, MrLac1, MrLac2 and MrLac3 for M. robertsii was higher when the isolates were cultivated on PDA+Rb and exposed to UV-B followed by exposure to WL, or exposed to WL only. CONCLUSIONS: Rb in culture medium increases the UV-B tolerance of M. robertsii and M. acridum conidia, and which may be related to increased expression of Phr, Lac and Pks genes in these conidia. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhanced UV-B tolerance of Metarhizium spp. conidia produced on Rb-enriched media may improve the effectiveness of these fungi in biological control programs.

Endogenous arabitol and mannitol improve shelf life of encapsulated Metarhizium brunneum.

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6 ± 0.2 and 2.1 ± 0.2 µg/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1 ± 4.4 to 55.0 ± 6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5 ± 3.3% at 5 °C followed by 54.5 ± 1.6% at 18 °C and 19.4 ± 1.3% at 25 °C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3 ± 7.6 to 98.0 ± 1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.

Secretome Analysis of Metarhizium anisopliae Under Submerged Conditions Using Bombyx mori Chrysalis to Induce Expression of Virulence-Related Proteins.

The entomopathogenic fungus Metarhizium anisopliae is used to control insect pests. This species is specialized for the secretion of an enzymatic complex consisting of proteases, lipases, and chitinases related to pathogenicity and virulence. In this context, the secretomes of strains IBCB 167 and IBCB 384 of M. anisopliae var. anisopliae, grown under submerged fermentation in the presence of chrysalis as an inducer, were analyzed. Analysis of two-dimensional gels showed qualitative and quantitative differences between secreted proteins in both isolates. Around 102 protein spots were analyzed, and 76 % of the corresponding proteins identified by mass spectrometry were grouped into different classes (hydrolases, oxidases, reductases, isomerases, kinases, WSC domains, and hypothetical proteins). Thirty-three per cent of all the proteins analyzed were found to be common in both strains. Several virulence-related proteins were identified as proteases and mannosidases. Endo-N-acetyl-ß-D-glucosaminidase expression was observed to be 10.14-fold higher for strain IBCB 384 than for strain IBCB 167, which may be an important contributor to the high virulence of IBCB 384 in Diatraea ssaccharalis. These results are important for elucidation of the host-pathogen relationship and the differences in virulence observed between the two strains.

Intracellular siderophore but not extracellular siderophore is required for full virulence in Metarhizium robertsii.

Efficient iron acquisition mechanisms are fundamental for microbial survival in the environment and for pathogen virulence within their hosts. M. robertsii produces two known iron-binding natural products: metachelins, which are used to scavenge extracellular iron, and ferricrocin, which is strictly intracellular. To study the contribution of siderophore-mediated iron uptake and storage to M. robertsii fitness, we generated null mutants for each siderophore synthase gene (mrsidD and mrsidC, respectively), as well as for the iron uptake transcriptional repressor mrsreA. All of these mutants showed impaired germination speed, differential sensitivity to hydrogen peroxide, and differential ability to overcome iron chelation on growth-limiting iron concentrations. RT-qPCR data supported regulation of mrsreA, mrsidC, and mrsidD by supplied iron in vitro and during growth within the insect host, Spodoptera exigua. We also observed strong upregulation of the insect iron-binding proteins, transferrins, during infection. Insect bioassays revealed that ferricrocin is required for full virulence against S. exigua; neither the loss of metachelin production nor the deletion of the transcription factor mrsreA significantly affected M. robertsii virulence.

An ENA ATPase, MaENA1, of Metarhizium acridum influences the Na(+)-, thermo- and UV-tolerances of conidia and is involved in multiple mechanisms of stress tolerance.

In fungi, ENA ATPases play key roles in osmotic and alkaline pH tolerance, although their functions in thermo- and UV-tolerances have not been explored. Entomopathogenic fungi are naturally widespread and have considerable potential in pest control. An ENA ATPase gene, MaENA1, from the entomopathogenic fungus Metarhizium acridum was functionally analyzed by deletion. MaENA1-disruption strain (ΔMaENA1) was less tolerant to NaCl, heat, and UV radiation than a wild-type strain (WT). Digital Gene Expression profiling of conidial RNAs resulted in 281 differentially expressed genes (DEGs) between the WT and ΔMaENA1 strains. Eighty-five DEGs, 56 of which were down-regulated in the ΔMaENA1 strain, were shown to be associated with heat/UV tolerance, including six cytochrome P450 superfamily genes, 35 oxidoreductase genes, 24 ion-binding genes, seven DNA repair genes, and five other genes. In addition, eight genes were components of stress responsive pathways, including the Ras-cAMP PKA pathway, the RIM101 pathway, the Ca(2+)/calmodulin pathway, the TOR pathway, and the HOG/Spc1/Sty1/JNK pathway. These results demonstrated that MaENA1 influences fungal tolerances to Na(+), heat, and UV radiation in M. acridum, and is involved in multiple mechanisms of stress tolerance. Therefore, MaENA1 is required for the adaptation and survival of entomopathogenic fungi in stressful conditions in the environment and in their hosts.

Severe fungal sclerokeratitis caused by Metarhizium anisopliae: a case report and literature review.

To date, there has been only one published report on the infectious sclerokeratitis caused by Metarhizium anisopliae, which is an entomopathogenic fungus. Regarding corneal infection, three reports have been published to date. Although the prognoses of the corneal infections are favourable, prognosis when scleral infection is involved is very poor. A 76-year-old patient presented with foreign body sensation in the left eye. Microscopic examination with Fungi Flora Y staining of the corneal scraping revealed fungal infection. The conjunctiva was melted by the infection over a wide area. Although intensive medications were administered, an emergency surgery was necessary because scleral thinning, corneal perforation and lens prolapse occurred. The fungal isolate was identified as M. anisopliae by sequencing the internal transcribed spacer region. Herein, we report the second known case worldwide of M. anisopliae sclerokeratitis, and we review the literature related to the ocular infections.

The physiological effects of multi-walled carbon nanotubes (MWCNTs) on conidia and the development of the entomopathogenic fungus, Metarhizium anisopliae (Metsch.) Sorok.

The aim of the study was an in vitro evaluation of the effect of MWCNTs on the conidia of two strains of entomopathogenic fungus, Metarhizium anisopliae. The study made use of water suspensions of MWCNTs (concentration ∼ 3 mg·mL(-1)) made from commercial nanotubes and centrifuged. The conidia were placed in contact with nanotubes for 240 h. An assessment of MWCNT influence on conidia was performed after 1, 24, 72 and 240 h and focused on the linear growth of vegetative mycelium derived from these conidia, mycelium sporulation in subcultures and pathogenicity. Using TEM imaging, it was demonstrated that carbon nanotubes are able to damage cell membranes of the examined fungi conidia. However, the absence was noted of a significantly fungistatic effect of both MWCNT suspensions on the examined strains with respect to the physiological features in question. The increase in vegetative mycelium effected by spores after contact with MWCNTs was characterized by a slight modification in relation to the control. There was no strong trend (inhibition - stimulation), in relation to the effect of the tested suspension of carbon nanotubes, on the development of the vegetative mycelium in in vitro culture. Sporulation of the mycelium after completion of the culture only occurred in one case (strain Ma73F and culture of spores after 24-h contact with MWCNTs) significantly more intensely than in the controls. With respect to pathogenicity for test insects compared to the control strain, Ma73F spores grown from the longest contact with nanotubes suspensions performed significantly better. On the basis of the calculated of mycelium index growth rates and the time of death of the test insects (LT50), it was found that the adverse effects of water suspension MWCNTs on the spores of M. anisopliae were applied after a short contact with biological material. This indicates unfavorable physical rather than chemical effects on the tested cell. Over time, nanotube aggregation in water suspensions led to changes in their influence on the cells under examination.

Physiological response of Metarhizium rileyi with linoleic acid supplementation.

Metarhizium rileyi has a broad biocontrol spectrum but is highly sensitive to abiotic factors. A Colombian isolate M. rileyi Nm017 has shown notorious potential against Helicoverpa zea. However, it has a loss of up to 22 % of its conidial germination after drying, which limits its potential as a biocontrol agent and further commercialization. Conidial desiccation resistance can be enhanced by nutritional supplements, which promotes field adaptability and facilitates technological development as a biopesticide. In this study, the effect of culture medium supplemented with linoleic acid on desiccation tolerance in Nm017 conidia was evaluated. Results showed that using a 2 % linoleic acid-supplemented medium increased the relative germination after drying by 41 % compared to the control treatment, without affecting insecticidal activity on H. zea. Also, the fungus increased the synthesis of trehalose, glucose, and erythritol during drying, independently of linoleic acid use. Ultrastructural analyses of the cell wall-membrane showed a loss of thickness by 22 % and 25 %, in samples obtained from 2 % linoleic acid supplementation and the control, respectively. Regarding its morphological characteristics, conidia inner area from both treatments did not change after drying. However, conidia from the control had a 24 % decrease in length/width ratio, whereas there was no alteration in conidia from acid linoleic. The average value of dry conidia elasticity coefficient from linoleic acid treatment was 200 % above the control. Medium supplementation with linoleic acid is a promising fermentation strategy for obtaining more tolerant conidia without affecting production and biocontrol parameters, compatible solutes synthesis, or modifying its cell configuration.

Expansion of the antimicrobial peptide repertoire in the invasive ladybird Harmonia axyridis.

The harlequin ladybird beetle Harmonia axyridis has emerged as a model species in invasion biology because of its strong resistance against pathogens and remarkable capacity to outcompete native ladybirds. The invasive success of the species may reflect its well-adapted immune system, a hypothesis we tested by analysing the transcriptome and characterizing the immune gene repertoire of untreated beetles and those challenged with bacteria and fungi. We found that most H. axyridis immunity-related genes were similar in diversity to their counterparts in the reference beetle Tribolium castaneum, but there was an unprecedented expansion among genes encoding antimicrobial peptides and proteins (AMPs). We identified more than 50 putative AMPs belonging to seven different gene families, and many of the corresponding genes were shown by quantitative real-time RT-PCR to be induced in the immune-stimulated beetles. AMPs with the highest induction ratio in the challenged beetles were shown to demonstrate broad and potent activity against Gram-negative bacteria and entomopathogenic fungi. The invasive success of H. axyridis can therefore be attributed at least in part to the greater efficiency of its immune system, particularly the expansion of AMP gene families and their induction in response to pathogens.

Nest sanitation through defecation: antifungal properties of wood cockroach feces.

The wood cockroach Cryptocercus punctulatus nests as family units inside decayed wood, a substrate known for its high microbial load. We tested the hypothesis that defecation within their nests, a common occurrence in this species, reduces the probability of fungal development. Conidia of the entomopathogenic fungus, Metarhizium anisopliae, were incubated with crushed feces and subsequently plated on potato dextrose agar. Relative to controls, the viability of fungal conidia was significantly reduced following incubation with feces and was negatively correlated with incubation time. Although the cockroach's hindgut contained abundant ß-1,3-glucanase activity, its feces had no detectable enzymatic function. Hence, these enzymes are unlikely the source of the fungistasis. Instead, the antifungal compound(s) of the feces involved heat-sensitive factor(s) of potential microbial origin. When feces were boiled or when they were subjected to ultraviolet radiation and subsequently incubated with conidia, viability was "rescued" and germination rates were similar to those of controls. Filtration experiments indicate that the fungistatic activity of feces results from chemical interference. Because Cryptocercidae cockroaches have been considered appropriate models to make inferences about the factors fostering the evolution of termite sociality, we suggest that nesting in microbe-rich environments likely selected for the coupling of intranest defecation and feces fungistasis in the common ancestor of wood cockroaches and termites. This might in turn have served as a preadaptation that prevented mycosis as these phylogenetically related taxa diverged and evolved respectively into subsocial and eusocial organizations.

A novel dodine-free selective medium based on the use of cetyl trimethyl ammonium bromide (CTAB) to isolate Beauveria bassiana, Metarhizium anisopliae sensu lato and Paecilomyces lilacinus from soil.

This study evaluated the quaternary ammonium compound cetyl trimethyl ammonium bromide (CTAB) as an alternative to the chemically related dodecylguanidine (dodine) for the selective isolation of entomopathogenic fungi. Oatmeal agar (OA) with chloramphenicol was used as basal medium, and three concentrations of CTAB (0.5, 0.6, 0.7 g/L) were evaluated and compared against OA + 0.46 g/L dodine. Selective isolation and growth studies were performed with the entomopathogens Beauveria bassiana, Metarhizium anisopliae s.l. and Paecilomyces lilacinus and five common non-entomopathogenic non-target species. The three entomopathogenic fungi sporulated earlier on OA + 0.6 g/L CTAB than on OA + 0.46 g/L dodine, while none of the non-target fungi sporulated on OA + 0.6 g/L CTAB. All entomopathogenic fungal isolates grew on OA + 0.6 g/L CTAB, despite some intra-species variation, whereas non-target fungi showed no growth or sporulation. OA + 0.6 g/L CTAB resulted in an efficient medium to isolate B. bassiana, M. anisopliae s. l. and P. lilacinus from soil samples. Results of our study suggest that OA + 0.6 g/L CTAB is a suitable, simple and inexpensive to prepare medium to replace OA + 0.46 g/L dodine for the selective isolation of these fungi.

A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella.

BACKGROUND: The larvae of the greater wax moth Galleria mellonella are increasingly used (i) as mini-hosts to study pathogenesis and virulence factors of prominent bacterial and fungal human pathogens, (ii) as a whole-animal high throughput infection system for testing pathogen mutant libraries, and (iii) as a reliable host model to evaluate the efficacy of antibiotics against human pathogens. In order to compensate for the lack of genomic information in Galleria, we subjected the transcriptome of different developmental stages and immune-challenged larvae to next generation sequencing. RESULTS: We performed a Galleria transcriptome characterization on the Roche 454-FLX platform combined with traditional Sanger sequencing to obtain a comprehensive transcriptome. To maximize sequence diversity, we pooled RNA extracted from different developmental stages, larval tissues including hemocytes, and from immune-challenged larvae and normalized the cDNA pool. We generated a total of 789,105 pyrosequencing and 12,032 high-quality Sanger EST sequences which clustered into 18,690 contigs with an average length of 1,132 bases. Approximately 40% of the ESTs were significantly similar (E ≤ e-03) to proteins of other insects, of which 45% have a reported function. We identified a large number of genes encoding proteins with established functions in immunity related sensing of microbial signatures and signaling, as well as effector molecules such as antimicrobial peptides and inhibitors of microbial proteinases. In addition, we found genes known as mediators of melanization or contributing to stress responses. Using the transcriptomic data, we identified hemolymph peptides and proteins induced upon immune challenge by 2D-gelelectrophoresis combined with mass spectrometric analysis. CONCLUSION: Here, we have developed extensive transcriptomic resources for Galleria. The data obtained is rich in gene transcripts related to immunity, expanding remarkably our knowledge about immune and stress-inducible genes in Galleria and providing the complete sequences of genes whose primary structure have only partially been characterized using proteomic methods. The generated data provide for the first time access to the genetic architecture of immunity in this model host, allowing us to elucidate the molecular mechanisms underlying pathogen and parasite response and detailed analyses of both its immune responses against human pathogens, and its coevolution with entomopathogens.

Lethal and sublethal effects of synthetic and bio-insecticides on Trichogramma brassicae parasitizing Tuta absoluta.

The tomato leaf miner (TLM), Tuta absoluta (Meyrick), is an invasive tomato pest found worldwide. Sustainable control strategies aimed at increasing biological control approaches and decreasing chemical inputs are required, due to the tendency to develop insecticide resistance. In this study, the lethal and sublethal effects of four chemical insecticides (abamectin, indoxacarb, chlorantraniliprole, and spinosad) and the sublethal effects of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) on a widespread TLM egg parasitoid, Trichogramma brassicae Bezdenko, were estimated. Concentration mortality response bioassays enabled the estimation of lethal concentrations of the tested insecticides for the parasitoids, with chlorantraniliprole having the lowest LC50 and indoxacarb the highest. The LC25 and LC50 of the tested insecticides on the TLM were sprayed on eggs and then offered at three time intervals to the parasitoids. The fertility and other life table parameters of the individuals emerging from the treated eggs were estimated. All of the chemical insecticides, but not the fungus, had harmful effects on T. brassicae. The insecticide applications caused a 3.84-5.17 times reduction in the net reproductive rate (R0) compared with the control. No parameters were affected by spraying the fungus in the 0h treatment, but effects were recorded at 24 and/or 48h, except for the gross reproduction rate (GRR). The value of the intrinsic rate of increase (rm) also decreased to 0.528-0.617 after the insecticide treatments. The doubling time (DT) increased in all treatments compared to the control. Nevertheless, the generation time (T) was only very slightly affected. In addition, in the combination experiments, M. anisopliae showed a remarkable synergism with T. brassicae in controlling TLM eggs. These results indicate that low levels of lethal effects on key biological control agents should be considered in the choice of insecticides to be included in sustainable TLM control packages.

Fungal tolerance to Congo red, a cell wall integrity stress, as a promising indicator of ecological niche.

Differential sensitivities to the cell wall stress caused by Congo red (CR) have been observed in many fungal species. In this study, the tolerances and sensitivities to CR was studied with an assorted collection of fungal species from three phylogenetic classes: Sordariomycetes, Dothideomycetes, and Eurotiomycetes, three orders, and eight families. These grouped into different ecological niches, such as insect pathogens, plant pathogens, saprotrophs, and mycoparasitics. The saprotroph Aspergillus niger and the mycoparasite Trichoderma atroviride stood out as the most resistant species to cell wall stress caused by CR, followed by the plant pathogenic fungi, a mycoparasite, and other saprotrophs. The insect pathogens had low tolerance to CR. The insect pathogens Metarhizium acridum and Cordyceps fumosorosea were the most sensitive to CR. In conclusion, Congo red tolerance may reflect ecological niche, accordingly, the tolerances of the fungal species to Congo red were closely aligned with their ecology.

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus.

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38µM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 108 conidia mL-1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.

Preservation of aerial conidia and biomasses from entomopathogenic fungi Beauveria brongniartii and Metarhizium anisopliae during lyophilization.

In this study, we assessed the stability provided by different formulations to aerial conidia or biomasses (conidia, blastospores, and mycelia) of Beauveria brongniartii and Metarhizium anisopliae subjected to lyophilization. First, the impact of the freezing and drying processes on spore survival was evaluated. Whereas unprotected B. brongniartii spores showed high cryosensitivity, those of M. anisopliae were markedly harmed by the drying process. Then, the protective efficiency of 14 excipients was systematically evaluated and optimized regarding required concentrations. Fructose, glucose, and saccharose significantly enhanced viabilities for B. brongniartii and M. anisopliae spores following lyophilization, especially as a result of their cryoprotective effects. In addition, the effect of various bulking agents on spore survival was studied and dextran 4 was selected to enhance the physical properties of the lyophilized products. The combination of fructose and dextran 4 was further applied to prepare lyophilized biomasses of both fungi. In comparison to freshly harvested biomasses, the lyophilized products showed similar growth rates and a comparable production of virulent secondary metabolites such as destruxin A, destruxin B, or oosporein, suggesting their applicability as biological control agents.

Debilitation in conidia of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae and implication with respect to viability determinations and mycopesticide quality assessments.

Germination of Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) conidia determined from a fast-rehydration (FR) protocol were compared to those obtained when dry conidia were subjected to slow rehydration (SR) by holding under high humidity conditions prior to aqueous suspension. Differences in viability estimates obtained using the FR vs. SR protocols increased markedly after conidia were exposed to various stress factors in storage (high a(w), temperature, and O(2) concentrations), with the SR protocol producing higher estimates of viability in all cases. After Bb conidia were stored under moist conditions for 21 days at 25 degrees C, the SR estimate of viability was >21% greater than the FR estimate. In jars flushed with different O(2) concentrations and stored at 50 degrees C for 34 days, proportional differences between protocols varied, depending on water activity, from 18-44% in jars flushed with 0% O(2) (100% N(2)) to as high as 63-93% when treated with 21-22% O(2). For conidia stored over a broad range of moderate to high temperatures in the absence of O(2), SR-FR differences were