Isolation, fermentation, and formulation of entomopathogenic fungi virulent against adults of Diaphorina citri.

BACKGROUND: Mycopesticides are important for integrated management of the Asian citrus psyllid, Diaphorina citri. However, there are few reports on the fermentation and formulation for mycopesticides with high virulence against D. citri. RESULTS: From four different locations in South China, 12 fungal strains were isolated and classified into Beauveria bassiana (two isolates), Fusarium fujikuroi (five isolates), and Cordyceps javanica (five isolates) based on the phylogenetic analysis of ITS1-5.8S rDNA-ITS4 and morphology of colonies and conidia. Based on the highest initial virulence (86.52 ± 2.35%) and best growth characteristics, F-HY002-ACPHali was further optimized for biphasic fermentation (7.85 ± 2.62 × 109 g-1 dry substrate) with soybean meal + cottonseed flour (1:1) as the solid substrate and full light as the light/dark cycle. Furthermore, the oil-based conidial formulation was optimized with sesame oil as an effective carrier, which significantly enhanced conidial shelf life up to 16 weeks at 26 °C and 4 °C, thermo-stress (50 °C) and UV-B stress resistance, and virulence against adults of D. citri with increased mortalities and decreased LT50 in comparison to that of unformulated conidia. CONCLUSION: Our results not only provide a valuable native strain with high virulence against adults of D. citri, but also imply the soybean meal + cottonseed flour as valuable solid substrate, full-light exposure as environmental stimuli for solid-state fermentation, and the sesame oil as an effective carrier for the formulation of the C. javanica. These findings will facilitate the development of a potential mycopesticide for the biological control of Asian citrus psyllid.

Gut microbiota promotes host resistance to low-temperature stress by stimulating its arginine and proline metabolism pathway in adult Bactrocera dorsalis.

Gut symbiotic bacteria have a substantial impact on host physiology and ecology. However, the contribution of gut microbes to host fitness during long-term low-temperature stress is still unclear. This study examined the role of gut microbiota in host low-temperature stress resistance at molecular and biochemical levels in the oriental fruit fly Bactrocera dorsalis. The results showed that after the gut bacteria of flies were removed via antibiotic treatment, the median survival time was significantly decreased to approximately 68% of that in conventional flies following exposure to a temperature stress of 10°C. Furthermore, we found that Klebsiella michiganensis BD177 is a key symbiotic bacterium, whose recolonization in antibiotic treated (ABX) flies significantly extended the median survival time to 160% of that in the ABX control, and restored their lifespan to the level of conventional flies. Notably, the relative levels of proline and arginine metabolites were significantly downregulated by 34- and 10-fold, respectively, in ABX flies compared with those in the hemolymph of conventional flies after exposure to a temperature stress of 10°C whereas recolonization of ABX flies by K. michiganensis BD177 significantly upregulated the levels of proline and arginine by 13- and 10- fold, respectively, compared with those found in the hemolymph of ABX flies. qPCR analysis also confirmed that K. michiganensis-recolonized flies significantly stimulated the expression of transcripts from the arginine and proline metabolism pathway compared with the ABX controls, and RNAi mediated silencing of two key genes Pro-C and ASS significantly reduced the survival time of conventional flies, postexposure low-temperature stress. We show that microinjection of L-arginine and L-proline into ABX flies significantly increased their survival time following exposure to temperature stress of 10°C. Transmission electron microscopy (TEM) analysis further revealed that low-temperature stress caused severe destruction in cristae structures and thus resulted in abnormal circular shapes of mitochondria in ABX flies gut, while the recolonization of live K. michiganensis helped the ABX flies to maintain mitochondrial functionality to a normal status, which is important for the arginine and proline induction. Our results suggest that gut microbiota plays a vital role in promoting the host resistance to low-temperature stress in B. dorsalis by stimulating its arginine and proline metabolism pathway.