Isolation and Identification of Culturable Bacteria from South China Seawater and Preliminary Screening of Marine Biocontrol Bacteria.

Marine microorganisms have evolved special metabolic pathways to produce numerous bioactive substances with novel structures and unique functions. This study analyzed the diversity of culturable bacteria in marine water samples from the South China Sea and screened the isolated bacteria with pathogenic fungi. A total of 200 culturable strains of 72 different bacteria were obtained from 56 water samples from the South China Sea. They belonged to three phyla and four classes, namely Gammaproteobacteria, Alphaproteobacteria, Bacilli and Actinomycetia. Bacilli was the dominant class, comprising up to 59.72%, followed by Gammaproteobacteria (20.83%). Bacillus, Pseudomonas, Paenibacillus and Rhizobium were the most dominant genera. Among these strains, HY-88 and HY-91 encoding BamC, FenB and PKSI genes were selected and identified as Bacillus subtilis. The respective inhibition rates of the HY-88 caused by plate confrontation against Magnaporthe grisea, Fusarium oxysporum, Botrytis cinerea, anthrax and Botrytis cinerea were 90.91%, 54.29%, 52.17% and 51.72%, in comparison with HY-91 86.36%, 48.57%, 47.83% and 34.48%. In addition, the supernatant of HY-88 showed a lesion inhibition rate of 74.5%, which was significantly higher than HY-91 (60.55%). In addition, HY-88 and HY-91 showed strong antifungal activity to Colletotrichum viniferum on detached Shine Muscat grapes. Tolerance tests showed that the HY-88 and HY-91 grew at 10-40 °C, 7-10% NaCl and pH 3-11. HY-88 and HY-91 could inhibit various fungal plant diseases, which lays a foundation for the development of new biopesticides.

Isolation and genomic characterization of a cypovirus from Clanis bilineata.

Clanis bilineata Walker, soybean hawkmoth, belongs to the subfamily Ambulicinae (Sphingidae, Lepidoptera) and is an edible insect that usually grows on soybean leaves. In this study, we isolated a new cypovirus from naturally diseased Clanis bilineata larvae (named CbCPV), scanned its structure, sequenced its genome, and studied its phylogenetic relationship to other cypoviruses. Microscopy showed that CbCPV polyhedral occlusion bodies were about 1.878 µm on average and contained many virions in the ultrathin sections. The complete genome sequence of CbCPV is 22,812 bp comprising 10 segmented double-stranded RNAs. Apart from segment 1 containing one open reading frame (ORF) and one sub-ORF, the other nine segments all contain one open reading frame and encoded one putative protein. The non-coding regions contained conserved sequences at 5' termini (AGUCAAA) and 3' termini (AGC), except segment 4 containing a different 5' termini (AUGUUUA). The whole sequence of the polyhedrin gene in CbCPV contained 892 nucleotides, encoding a protein of 246 amino acids. Based on amino acid sequences of polyhedrin or RNA dependent RNA polymerase (RdRp), the phylogenetic analysis indicated that CbCPV was closely related to DnCPV-23. The putative function of all segments differed from each other, but the most closely related species of segments were DnCPV-23 with 98.2-99.8% nucleotide identity. Overall, the evidence of morphology, protein analysis and nucleic acids (genomic pattern) showed that CbCPV is a new isolate in the cypovirus-23 type and can be termed Clanis bilineata cypovirus type 23 (CbCPV-23).

Talaromyces sp. Ethyl Acetate Crude Extract as Potential Mosquitocide to Control Culex pipiens quinquefasciatus.

Vector control is considered an effective approach to controlling diseases spread by mosquito bites. Entomopathogenic fungi are widely used in agriculture to control insect pests, and fungal metabolites can potentially be developed as effective mosquitocides. In this study, a high-throughput screening method was used to search for potential mosquitocides in the Global Fungal Extract Library (GFEL). We tested the larvicidal activity of 264 fungal ethyl acetate crude extracts against Culex pipiens quinquefasciatus. Nine fungal extracts caused moderate to high mortality rates (>50%), with two fungal extracts (58A7 and 101H12) causing a 100% mortality rate. The lethal concentrations for 50% of the population (LC50) were 44.27 mg/L and 31.90 mg/L, respectively. Fraction 14 had a high mortality rate, with an LC50 value of 12.13 mg/L, and was isolated from 58A7 (Fractions 1-11) and 101H12 (Fractions 12-15). Further analyses showed that Fraction 14 was made up of vermistatin and dihydrovermistatin. In a Cx. p. quinquefasciatus larvicidal bioassay, vermistatin (LC50 = 28.13 mg/L) was more toxic than dihydrovermistatin (LC50 = 83.87 mg/L). Our findings suggested that the active fungal extract 101H12 from Talaromyces sp. and its compound vermistatin could be developed as mosquitocides.

Transcriptional responses of Daphnis nerii larval midgut to oral infection by Daphnis nerii cypovirus-23.

BACKGROUND: Daphnis nerii cypovirus-23 (DnCPV-23) is a new type of cypovirus and has a lethal effect on the oleander hawk moth, Daphnis nerii which feeds on leave of Oleander and Catharanthus et al. After DnCPV-23 infection, the change of Daphnis nerii responses has not been reported. METHODS: To better understand the pathogenic mechanism of DnCPV-23 infection, 3rd-instar Daphnis nerii larvae were orally infected with DnCPV-23 occlusion bodies and the transcriptional responses of the Daphnis nerii midgut were analyzed 72 h post-infection using RNA-seq. RESULTS: The results showed that 1979 differentially expressed Daphnis nerii transcripts in the infected midgut had been identified. KEGG analysis showed that protein digestion and absorption, Toll and Imd signaling pathway were down-regulated. Based on the result, we speculated that food digestion and absorption in insect midgut might be impaired after virus infection. In addition, the down-regulation of the immune response may make D. nerii more susceptible to bacterial infections. Glycerophospholipid metabolism and xenobiotics metabolism were up-regulated. These two types of pathways may affect the viral replication and xenobiotic detoxification of insect, respectively. CONCLUSION: These results may facilitate a better understanding of the changes in Daphnis nerii metabolism during cypovirus infection and serve as a basis for future research on the molecular mechanism of DnCPV-23 invasion.

FOXA transcriptional factor modulates insect susceptibility to Bacillus thuringiensis Cry1Ac toxin by regulating the expression of toxin-receptor ABCC2 and ABCC3 genes.

Cry toxins produced by Bacillus thuringiensis (Bt) are insecticidal proteins widely used in insect control. Recently, it was shown that ATP-binding cassette transporter proteins (ABC) such as ABCC2, ABCC3, ABCG1 and ABCA2 are implicated in the insecticidal action of Cry toxins as putative receptors. However, the transcriptional regulators involved in the expression of ABC transporter genes remain unknown. Sequence analysis of promoter regions of ABCC2 gene from Helicoverpa armigera and ABCC3 gene from Spodoptera litura Sl-HP cultured cells, revealed the potential participation of Forkhead box protein A (FOXA), a transcription factor that regulates the expression of genes through remodeling chromatin. To determine if FOXA was involved in regulating expression of ABCC2 and ABCC3 genes, the expression of FOXA, ABCC2 and ABCC3 was compared in Sl-HP cells that are sensitive to Cry1Ac toxin with those in S. frugiperda Sf9 cells that are not sensitive to the toxin. Expression levels of those genes were significantly higher in Sl-HP than in Sf9 cells. Transient expression of FOXA in Sf9 cells activated ABCC2 and ABCC3 transcription, which directly correlated with enhanced Cry1Ac-susceptibility in these cells. Silencing of FOXA gene expression by RNAi in H. armigera larvae resulted in a decreased expression of ABCC2 and ABCC3 without affecting expression of other Cry toxin receptor genes such as alkaline phosphatase, aminopeptidase or cadherin. Silencing of FOXA gene expression also resulted in a Cry1Ac-tolerant phenotype since lower mortality and higher pupation rate were observed in diet containing Cry1Ac protoxin in comparison with the control group. These results demonstrate that FOXA up-regulates expression of the Cry1Ac-toxin receptor ABCC2 and ABCC3 genes, and that lower FOXA expression correlates with tolerance to Cry toxin in cell lines and in lepidopteran larvae.

Endogenous expression of a Bt toxin receptor in the Cry1Ac-susceptible insect cell line and its synergistic effect with cadherin on cytotoxicity of activated Cry1Ac.

Although many insect cell lines derived from various tissues are available, it is unclear whether endogenous receptors of Bacillus thuringiensis (Bt) crystal toxins are expressed in these cell lines. In the present study, we demonstrated that the ovaries-derived Spodoptera litura Sl-HP cell line was susceptible to activated Cry1Ac although larvae of S. litura are not susceptible to the toxin. Assays of the transcriptome revealed that thirteen ATP-binding cassette transporter genes (ABC) were expressed at different levels in this cell line. Of these, the SlABCC3 shared 52-55% amino acid sequence identity with the known Bt toxin receptor ABCC2. RNAi-mediated knockdown targeting SlABCC3 significantly decreased the susceptibility of Sl-HP cells to activated Cry1Ac. Over-expression of the gene strongly increased the susceptibility of Trichoplusia ni Hi5 cells to the toxin. Not only was SlABCC3 comparable to the heterologously expressed Helicoverpa armigera Hacadherin on the receptor-mediated cytotoxicity of activated Cry1Ac to Hi5 cells, but also SlABCC3 and Hacadherin had a strong synergistic effect on cytotoxicity of activated Cry1Ac. These results suggested that Bt toxin receptors-expressing insect cell lines can be used as an alternative model for evaluating cytotoxicity of Bt toxins and studying their mechanisms of action.

Establishment and characterization of a novel cell line from midgut tissue of Helicoverpa armigera (Lepidoptera: Noctuidae).

The midgut of lepidopteran larvae serves as a target for many pathogens such as Bacillus thuringiensis (Bt). Cell lines originating from midgut tissues will be very helpful tools in many research fields. However, to date, no Bt-susceptible midgut-derived cell lines are available. Here, we reported that a novel cell line, designated as HNU-Ha-MG1, was established from midgut tissue of the fourth instar larvae of Helicoverpa armigera. This cell line grew well in Grace's insect cell culture medium supplemented with 10-15% fetal bovine serum. The shape of the most cells was round or polygonal, and some tended to aggregate to form multiple cell masses. The size of the cells was 13.8 ± 1.8 µm in diameter, and the maximum density reached (2.40 ± 0.15) × 10(6) cells/ml. The population doubling time during logarithmic growth phase was 58.6 ± 7.0 h at 28°C. The number of chromosomes was about 90-130, which exhibited typical chromosome characteristics of lepidopteran cell lines. The patterns of random amplified polymorphic DNA of the cell line were different from those of Sl-HP and Hi5 cell lines which were frequently used in our laboratory. 20-Hydroxyecdysterone induced apoptosis in a very small part of cells at 2 µg/ml but did not affect expression of autophagy-related protein 8 (Atg8) and its lipidation at 36 h post-treatment. The cell line was permissive to Autographa californica nuclear polyhedrosis virus (AcMNPV) and H. armigera nuclear polyhedrosis virus (HaSNPV). This cell line was found to be susceptible to activated Cry1C at the final concentration of 0.5-1.0 µg/ml but not to the activated Cry1Ac.

Characterization of Streptomyces padanus JAU4234, a producer of actinomycin X2, fungichromin, and a new polyene macrolide antibiotic.

Strain JAU4234, identified as Streptomyces padanus, was isolated from soil collected in Jiangxi Province, China. It produced actinomycin X2, fungichromin, and a new polyene macrolide compound with antifungal activity, antifungalmycin 702. Antifungalmycin 702 had good general antifungal activity and may have potential future agricultural and/or clinical applications.