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BACKGROUND: The hemolymph and insect gut together have an essential role in the immune defense against microorganisms, including the production of antimicrobial peptides (AMP). AMPs are mainly induced by two specific signaling pathways, Toll and immune deficiency (IMD). Here, we characterize the expression profile of four genes from both pathways and describe the importance of AgraRelish in the immune defense of Anthonomus grandis against the entomopathogenic fungus Metarhizium anisopliae by RNA interference (RNAi). RESULTS: To characterize the pathway that is activated early during the A. grandis-M. anisopliae interaction, we assessed the expression profiles of AgraMyD88 and AgraDorsal (Toll pathway), AgraIMD and AgraRelish (IMD pathway), and several AMP genes. Interestingly, we found that IMD pathway genes are upregulated early, and Toll pathway genes are upregulated just 3 days after inoculation (DAI). Furthermore, nine AMPs were upregulated 24 h after fungus inoculation, including attacins, cecropins, coleoptericins, and defensins. AgraRelish knockdown resulted in a reduction in median lethal time (LT50 ) for M. anisopliae-treated insects of around 2 days compared to control treatments. In addition, AgraRelish remained knocked down at 3 DAI. Finally, we identified that AgraRelish knockdown increased fungal loads at 2 DAI compared to control treatments, possibly indicating a faster infection. CONCLUSIONS: Our data indicate the influence of the IMD pathway on the antifungal response in A. grandis. Combining biocontrol and RNAi could significantly improve cotton boll weevil management. Hence, AgraRelish is a potential target for the development of biotechnological tools aimed at improving the efficacy of M. anisopliae against A. grandis.

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A strain within the Metarhizium anisopliae species complex was isolated in 2009 from a soil sample in a banana plantation in the municipality of Quixeré, Northeastern region of Brazil. Previous studies showed that this insect-pathogenic strain does not fit with any current taxon within the M. anisopliae species complex, as determined by both genomic and by mass spectrometric analyses. In the present study, CG1123 (=ARSEF 13308) is shown to be morphologically indistinguishable from most species in this cosmopolitan species complex, whereas multilocus phylogeny confirmed its uniqueness and supports its recognition as a new species, Metarhizium alvesii, in honor of Sérgio Batista Alves, one of the founders of insect pathology in Brazil.

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Meloidogyne enterolobii is one of the most important root-knot nematode in tropical regions, due to its ability to overcome resistance mechanisms of a number of host plants. The lack of new and safe active ingredients against this nematode has restricted control alternatives for growers. Egg-parasitic fungi have been considered as potential candidates for the development of bionematicides. In tissue culture plates, Pochonia chlamydosporia (var. catenulata and chlamydosporia) and Purpureocillium lilacinum strains were screened for their ability to infect eggs of the root-knot nematode M. enterolobii on water-agar surfaces. Reduction in the hatching of J2 varied from 13% to 84%, depending on strain. The more efficacious strains reduced hatchability of J2 by 57% to 84% when compared to untreated eggs, but average reductions were only 37% to 55% when the same strains were applied to egg masses. Combinations of fungal isolates (one of each species) did not increase the control efficacy in vitro. In experiments in which 10,000 nematode eggs were inoculated per plant, reductions in the number of eggs after 12 months were seen in three of four treatments in banana plants, reaching 34% for P. chlamydosporia var. catenulata. No significant reductions were seen in tomato plants after 3 mon. In another experiment with tomato plants using either P. chlamydosporia var. catenulata or P. lilacinum, the number of eggs was reduced by 34% and 44%, respectively, when initial infestation level was low (500 nematode eggs per plant), but tested strains were not effective under a moderate infestation level (5,000 eggs per plant). Under all infestation levels tested in this work, gall and egg mass indexes (MI) did not differ from the untreated controls, bringing concerns related to the practical adoption of this control strategy by farmers. In our opinion, if the fungi P. chlamydosporia and P. lilacinum are to be used as biocontrol tools toward M. entorolobii, they should focus on agricultural settings with low soil infestation levels and within an IPM approach.

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The substantial negative impact of some parameters on the germination of low-quality conidia (high proportion of slow-germinating propagules) was demonstrated, whereas for high-quality batches their effect was small or even absent. Germination was increased as the initial hydration status of conidia immediately prior to suspension preparation was increased, being ca. 33% and 80% for dehydrated Metarhizium anisopliae propagules (water activity ≤0.314) from low- or high-quality batches after an 18 h incubation period, respectively, and 63% and 95% for hydrated propagules (water activity = 0.933). Germination of low-quality propagules also increased as the time dry conidia were kept in aqueous suspension prior to inoculation onto culture media (15 min, 3 or 24 h) or the incubation time at 25°C before counts (18, 48 or 72 h) was increased. Depending on treatment conditions, average germination of low-quality conidia varied from 53% to 98%. On the other hand, germination for high-quality conidia was always ≥94%. Regarding the relative humidity (RH) of the incubation atmosphere, the average germination rates for low-quality conidia on Potato Dextrose Agar (PDA) in Petri plates was 49%, while germination of these conidia on PDA blocks kept under lower RH inside plastic boxes was ≤23%. Use of lactophenol-staining and/or use of coverslips had a negative effect when germination assessment was performed for low-quality conidia, resulting in distorted counts or increased standard deviations compared to high-quality conidial batches. The occurrence of dislodged conidia (ungerminated conidia outside the inoculation zone due to hydraulic pressure exercised by addition of stains and/or coverslips added to the substrate by the time germination is assessed) was common place, whereas dislodged conidia were not seen in treatments with high-quality batches. This work underscores the importance of a number of parameters that anyone working with low-quality fungi needs to be cognizant of in their research.

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