Comprehensive Insights into the Remarkable Function and Regulatory Mechanism of FluG during Asexual Development in Beauveria bassiana.

Asexual development is the main propagation and transmission mode of Beauveria bassiana and the basis of its pathogenicity. The regulation mechanism of conidiation and the key gene resources for utilization are key links to improving the conidia yield and quality of Beauveria bassiana. Their clarification may promote the industrialization of fungal pesticides. Here, we compared the regulation of morphology, resistance to external stress, virulence, and nutrient utilization capacity between the upstream developmental regulatory gene fluG and the key genes brlA, abaA, and wetA in the central growth and development pathway. The results showed that the ΔbrlA and ΔabaA mutants completely lost the capacity to conidiate and that the ΔwetA mutant had seriously reduced conidiation capacity. Although the deletion of fluG did not reduce the conidiation ability as much as deletions of brlA, abaA, and wetA, it significantly reduced the fungal response to external stress, virulence, and nutrient utilization, while the deletion of the three other genes had little effect. Via transcriptome analysis and screening the yeast nuclear system library, we found that the differentially expressed genes in the ΔfluG mutants were concentrated in the signaling pathways of ABC transporters, propionate metabolism, tryptophan metabolism, DNA replication, mismatch repair, and fatty acid metabolism. FluG directly acted on 40 proteins that were involved in various signaling pathways such as metabolism, oxidative stress, and cell homeostasis. The analysis indicated that the regulatory function of fluG was mainly involved in DNA replication, cell homeostasis, fungal growth and metabolism, and the response to external stress. Our results revealed the biological function of fluG in asexual development and the responses to several environmental stresses as well as its influence on the asexual development regulatory network in B. bassiana.

Germicide Fenaminosulf Promots Gall Formation of Zizania latifolia without directly affecting the growth of endophytic fungus Ustilago esculenta.

Zizania latifolia is a popular aquatic vegetable in China because of its enlarged edible stems resulting from persistent infection by a fungal endophyte, Ustilago esculenta. Fenaminosulf (FM) is a germicide that can be used to improve agricultural crop yields. In Z. latifolia fields, appropriate spraying of FM not just controls diseases, but also promotes an earlier harvest of Z. latifolia. In this study, we show that the timing of gall formation was advanced and the plant's yield was increased significantly under a high concentration treatment of FM. Yet FM had a strong inhibitory effect on the growth of U. esculenta in vitro, while the transcript levels of mating-type alleles, cell metabolism-related genes and chitin synthase genes were all substantially downregulated. Through a transcriptome analysis, we investigated changes in gene expression of the host Z. latifolia and fungal endophyte U. esculenta in response to FM. FM directly affected the growth of Z. latifolia by altering the expression level of genes involved in plant-pathogen interactions, plant hormone signal transduction and some metabolism pathways. By contrast, FM had little effect on U. esculenta growing inside of Z. latifolia. Collectively, our results provide a more in-depth understanding of the molecular processes that promote gall formation in Z. latifolia, while also identifying potential targets for genetic manipulation to improve the yield and quality of Z. latifolia, in a safer and more effective way.

Effect of synergists on susceptibility to chlorantraniliprole in field populations of Chilo suppressalis (Lepidoptera: Pyralidae).

The Asiatice stem borer, Chilo suppressalis (Walker), an important rice insect pest in China, has developed resistances to several classes of insecticides. To control C. suppressalis, chlorantraniliprole has been introduced as a novel insecticide in rice field since 2008. It is an anthranilic diamide insecticide that binds and activates ryanodine receptors (RyR). The susceptibility of field populations of C. suppressalis to chlorantraniliprole was determined in this study. The hypotheses of equality and parallelism showed that regression lines in the tested five populations were neither equal nor parallel. The Ruian, Cangnan, and Liangyungang populations (RA12, CN12, and LYG12) collected in 2012 had lower LD50 values, whereas the Zhuji populations (ZJ12 and ZJ13) collected in 2012 and 2013 were approximately 15 times more tolerant than the RA12 population. To determine the potential mechanisms involved in this tolerance variation, synergism bioassays were performed. Significant differences in susceptibility were found between without synergist and with synergist for the three populations (RA12, LYG12, and ZJ13), based on the tests for the hypotheses of equality. In RA12 and LYG12 populations with chlorantraniliprole, the addition of piperonyl butoxide (PBO) significantly synergized the activity, with synergism ratios of 2.68- and 2.33-fold, respectively, whereas addition of S,S,S-tributyl phosphorotrithioate (DEF) and diethyl maleate (DEM) had no synergist effect. In ZJ13 population with chlorantraniliprole, the addition of PBO and DEF increased synergism ratios by 12.43- and 6.31-fold, respectively, whereas addition of DEM had no significant effect on the toxicity against ZJ13 larvae. These susceptibility and synergism data suggested that detoxification enzymes might be involved in the tolerance variation to chlorantraniliprole in field populations of C. suppressalis.

Genome-Wide Identification of SNARE Family Genes and Functional Characterization of an R-SNARE Gene BbSEC22 in a Fungal Insect Pathogen Beauveria bassiana.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating cell membrane fusion and intracellular vesicular trafficking in eukaryotic cells, and have been well-documented to play critical roles in growth, development, and pathogenesis in the filamentous fungal plant pathogens. However, little is known about the contributions of SNAREs to the physiology and biocontrol potential in entomopathogenic filamentous fungi. Here, a genome-wide analysis of SNARE genes was performed taking advantage of the available whole genome sequence of Beauveria bassiana, a classical entomopathogenic fungus. Based on the compared genomic method, 22 genes encoding putative SNAREs were identified from the whole genome of B. bassiana, and were classified into four groups (7 Qa-, 4 Qb-, 6 Qc-, and 5 R-SNAREs) according to the conserved structural features of their encoding proteins. An R-SNARE encoding gene BbSEC22 was further functionally characterized by gene disruption and complementation. The BbSEC22 null mutant showed a fluffy appearance in mycelial growth and an obvious lag in conidial germination. The null mutant also exhibited significantly increased sensitivity to oxidative stress and cell wall perturbing agents and reduced the yield of conidia production by 43.1% compared with the wild-type strain. Moreover, disruption of BbSEC22 caused a significant decrease in conidial virulence to Spodoptera litura larvae. Overall, our results provide an overview of vesicle trafficking in B. bassiana and revealed that BbSec22 was a multifunctional protein associated with mycelial growth, sporulation, conidial germination, stress tolerance, and insecticidal virulence.

Regression analysis of dynamics of insecticide resistance in field populations of Chilo suppressalis (Lepidoptera: Crambidae) during 2002-2011 in China.

To understand the evolution of insecticide resistance in the Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), in field, regression analysis based on a linear or nonlinear model was adopted for analyzing resistance dynamics to six insecticides of two field populations of the Lianyungang (LYG) and Ruian (RA) populations during 2002-2011. For the low-level resistance population, LYG population, sustained susceptibilities to abamectin and fipronil were seen for 10 yr; a polynomial curve regression model showed an increase in resistance to chlorpyrifos; exponential growth models fit to the resistance dynamics to triazophos and deltamethrin, and a sigmoidal growth curve for monosultap. For the high-level multiple resistance population, RA population, a slight increase from susceptible to a minor resistance to abamectin could be modeled by a polynomial cubic equation; an exponential growth model fit to the increase of resistance to fipronil from 8.7-fold to 33.6-fold; a sine waveform model fit to the vibrating tendency of resistance to chlorpyrifos; the dynamics of resistance to triazophos could be modeled by two combined curves, with a polynomial growth model and a sine waveform model; the high level of resistance to monosultap could be modeled with a sine waveform model; and a significant linear growth relationship of the resistance to deltamethrin of the RA population over years was found. Then, the relationship between dynamics of resistance development to insecticides among the field populations of C. suppressalis and the application history of pesticides for controlling rice borers was discussed.

Influence of pymetrozine on feeding behaviors of three rice planthoppers and a rice leafhopper using electrical penetration graphs.

Pymetrozine reportedly inhibits feeding of plant sap-sucking insects, such as aphids and brown planthopper (Nilaparvata lugens (Stål)). By using electrical penetration graph (EPG), this study was conducted to investigate any differential effect of pymetrozine on the feeding behaviors of four major rice sap-sucking insect species, 1) N. lugens, 2) white-backed planthopper (Sogatella furcifera (Horváth)), 3) small brown planthopper (Laodelphax striatellus (Fallen)), and 4) green rice leafhopper (Nephotettix cincticeps Uhler). On pymetrozine-free TN1 rice plants, white-backed planthopper and small brown planthopper showed a significantly less activity in the phloem phases than brown planthopper or green rice leafhopper while green rice leafhopper engaged in relatively more xylem ingestion than brown planthopper, white-backed planthopper, and small brown planthopper. On the plants treated with 100 mg liter(-1) of pymetrozine, all four insect species showed significant increases, in total duration of nonprobing and significant decreases in the activities in phloem tissue, while all species showed similar feeding behavior during the pathway and xylem phases. This study revealed that, regardless of whether the insects on untreated plants spent more time feeding on phloem than xylem (brown planthopper) or more time on xylem than phloem (green rice leafhopper) or similar times on phloem and xylem (white-backed planthopper and small brown planthopper), their feeding behavior was disturbed by pymetrozine and exhibited similar patterns of sharp decline in activity in the phloem tissue and a significant increase the nonprobing.

Gut microbial communities associated with phenotypically divergent populations of the striped stem borer Chilo suppressalis (Walker, 1863).

Chilo suppressalis (Walker, 1863) is a serious stem borer of rice and water-oat plants, and has phenotypically diverged into rice and water-oat populations. Insect gut microbiota plays an important role in the host life and understanding the dynamics of this complicated ecosystem may improve its biological control. The effect of diet and gut compartments on the gut microflora of divergent populations of C. suppressalis is not fully clear. Herein, we characterized the gut microbiota of C. suppressalis populations fed on two hosts (i.e., water-oats fruit pulps and rice seedlings), by sequencing the V3-V4 hypervariable region of the 16S rRNA gene using the Illumina MiSeq platform. Gut bacterial communities showed variation in relative abundance among C. suppressalis populations fed on water-oats fruit pulps or rice seedlings. Proteobacteria and Firmicutes became the predominant phyla, and Enterobacteriaceae, Enterococcaceae and Halomonadaceae were the predominant family in all C. suppressalis populations. The highest bacteria diversity was found in the midgut of the rice population fed on water-oat fruit pulps. Bacterial communities in the midgut were more diverse than those in the hindgut. The bacterial genera distribution showed great differences due to diet types and gut compartments among populations. Our results demonstrated that the host plants tested had a considerable impact on gut bacterial composition of C. suppressalis populations. Additionly, the unique gut morphology and physiological conditions (viz., oxygen content, enzymes) also contributed to variation in microbiomes. In conclusion, our study provided an important insight into investigation of insect-bacteria symbioses, and biocontrol of this species and other related lepidopterans.

Comparative transcriptome analysis reveals host-associated differentiation in Chilo suppressalis (Lepidoptera: Crambidae).

The striped stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae), is one of the most serious rice pests. Besides attacking rice, it also feeds on an economically important vegetable crop, water-oat Zizania latifolia. The species feeding on water-oat has higher growth and survival rate than those on rice, suggesting their success in adaptation to the new host plant. However, little is known about the molecular mechanisms of host plant adaptation. Here we investigated the midgut transcriptome responses of C. suppressalis larvae reared on rice and water-oat. A total of 1,633 differentially expressed genes were identified, with a greater number up-regulated on the more delicious new host. The up-regulation of most digestive and detoxification-related genes may be the result of adaptation to the changes in nutritional requirements and toxic chemicals during host shift. In contrast, down-regulation of ribosomal genes may be related to their better development performance when feeding on the new host. In conclusion, our results suggest that transcriptional regulation of genes related to digestion, detoxification and ribosome may play an important role in adaptation of C. suppressalis to a new host plant.

Electrical penetration graph evidence that pymetrozine toxicity to the rice brown planthopper is by inhibition of phloem feeding.

BACKGROUND: Pymetrozine is a valuable novel insecticide for control of sucking insects, including the brown planthopper Nilaparvata lugens (Stål), one of the most serious pests on rice. This study was conducted to elucidate the action mechanisms of pymetrozine on the feeding behaviour of the planthopper. RESULTS: The activity test showed that pymetrozine primarily functioned as an antifeedant that caused starvation and death in N. lugens, rather than having neurotoxicity. Pymetrozine-treated insects died at a significantly slower speed than insects treated with starvation. Electrical penetration graph (EPG) data indicated that pymetrozine significantly increased the duration of non-probing periods and had a strong inhibition to phloem ingestion. The inhibition was strongly dose dependent, resulting in a complete suppression of the activity in the phloem region when the pymetrozine concentration was increased to 400 mg L(-1) . Starvation caused by inhibition of phloem ingestion might be a major toxicity mechanism of pymetrozine. EPG data also showed that pymetrozine had no significant effect on stylet movement and duration of xylem sap ingestion. CONCLUSION: The study revealed that pymetrozine disturbed the feeding behaviour of N. lugens mainly by increasing the non-probe period and inhibiting phloem ingestion. The inhibition resulted in a slow death similar to starvation.