Weiguo Miao

Bactericidal metabolites from Phellinus noxius HN-1 against Microcystis aeruginosa

Harmful algal blooms cause serious problems worldwide due to large quantities of cyanotoxins produced by cyanobacteria in eutrophic water. In this study, a new compound named 2-(3, 4-dihydroxy-2-methoxyphenyl)-1, 3-benzodioxole-5-carbaldehyde (Compound 1), together with one known compound, 3, 4-dihydroxybenzalacetone (DBL), was purified from Phellinus noxius HN-1 (CCTCC M 2016242). Compound 1 and DBL displayed activity against the cyanobacteria Microcystis aeruginosa with a half maximal effective concentration of 21 and 5 μg/mL, respectively. Scanning electron and transmission electron microscopic observations showed that the compounds caused serious damage and significant lysis to M. aeruginosa cells. qRT-PCR assay indicated that compound 1 and DBL exposure up-regulated the expression of gene mcyB and down-regulated the expression of genes ftsZ, psbA1, and glmS in M. aeruginosa. This study provides the first evidence of bactericidal activity of a new compound and DBL. In summary, our results suggest that compound 1 and DBL might be developed as naturally-based biocontrol agents.

A new cyclic lipopeptide isolated from Bacillus amyloliquefaciens HAB-2 and safety evaluation

Bacillus is the most widely studied biocontrol agent and has been extensively used in the development of biopesticides and fungicides. In this study, a new cyclic lipopeptide was isolated from Bacillus amyloliquefaciens HAB-2 by column chromatography on silica gel and Sephadex LH-20, and its structures was elucidated on the basis of spectroscopic analysis. This compound is a bacillomycin d-like compound, named as bacillomycin DC. The activity of bacillomycin DC was evaluated against C. gloeosporioides Penz. The median inhibitory concentration of bacillomycin DC was 1.21μg/mL. In addition, bacillomycin DC may have low toxicity to aquatic species as indicated by its 96h half maximum lethal concentration of 22.20μg/mL to zebrafish (Danio rerio). Our current study further provides evidence that bacillomycin DC is a potent fungicide against C. gloeosporioides Penz.

Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display.

Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant–pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew.

Complete genomic sequence of a novel macluravirus, alpinia oxyphylla mosaic virus (AloMV), identified in Alpinia oxyphylla

A macluravirus, tentatively named alpinia oxyphylla mosaic virus (AloMV), was identified in Alpinia oxyphylla, and its complete genomic sequence determined. The positively single-stranded RNA genome is comprised of 8213 nucleotides excluding the poly (A) tail, and contains one large open reading frame encoding a polyprotein of 2,626 amino acids. Blastp search showed that the polyprotein of AloMV shared 48%~68% aa sequence identities with other reported macluraviruses. Phylogenetic analysis based on the nucleotide sequence of the polyprotein showed that AloMV, together with all other macluraviruses, clustered into the same group most closely related to cardamom mosaic virus, sharing 66.3% nt and 68% aa sequence identities, respectively. These data above suggest that AloMV represents an isolate of a putative new member within the genus Macluravirus.

Antifungal activity of the bioactive substance from Bacillus atrophaeus strain HAB-5 and its toxicity assessment on Danio rerio

The prevalence of pathogen inhibitors bacteria has motivate the study for antimicrobial compounds. Bioactive fungicide have always received considerable attention. A bacterial isolated strain HAB-5 showed antifungal activity against plant fungi. Based on morphological, physiological, biochemical and 16SrDNA sequence analysis, the strain was identified to be a Bacillus atrophaeus. This strain possessed a broad spectrum antifungal activity against various plant pathogenic fungi. Extraction of antifungal substance was performed and the crude extract had potent antifungal ability and showed great potential for swelling and inhibiting spore germination. This antifungal displayed heat stability and active in a wide pH range 5.0-10.0. Moreover no reduction was found in its activity after enzyme treatment. The toxicity test was evaluated in Danio rerio. The acute toxicity test indicated that the 24, 48, 72, 96h LC50 values of UMTLS to the zebrafish were 14.4, 13.8, 13.4, and 12.9%, respectively. Based on the results obtained in this study, antifungal substance was not toxic to zebra. Analyses of disease suppression showed that HAB-5 was effective to reduce the incidence of anthracnose symptoms on mango fruits, also prevent disease infection and protect tobacco seedling from Phytophtora nicotianae. The bioactive substance from Bacillus atrophaeus HAB-5 could be a candidate in the generation of new antifungal agents in crop.

Developmental toxicity and inhibition of the fungicide hymexazol to melanin biosynthesis in zebrafish embryos

Hymexazol is an efficacious and widely used fungicide. However, its environmental toxicological assessment has not been well documented. It had no report of its toxicity to fish embryo. Fish embryo acute toxicity tests are highly predictive of aquatic embryotoxicity outcome. In this study, zebrafish (Danio rerio) embryos were exposed to hymexazol at varying concentrations for the study of the developmental toxicity, melanin biosynthesis, biochemical and transcriptional endpoints. The embryotoxicity tests indicated that the 96h LC50 value of hymexazol was 649mg/L with a 95% confidence interval range of 632-667mg/L. Hymexazol at concentrations of 417-738mg/L decreased the heart rate and increased the voluntary swing. Hymexazol inhibited normal development at concentrations above 554mg/L. the 96h EC50 was 411mg/L. Hymexazol in a concentration range of 417-738mg/L induced cardiac edema and yolk sac edema. Exposure of hymexazol at such concentrations to zebrafish embryos for 48h decreased the pigment area density compared with the no hymexazol control. Tyrosinase activity was inhibited by hymexazol relative to the untreated control. The P53 mRNA expression level in embryos upon exposure to 480mg/L or greater of hymexazol was significantly higher than that of the control. The results indicated that hymexazol has quite low acute toxicity and low embryotoxicity to zebrafish.

The signal peptide-like segment of hpaXm is required for its association to the cell wall in transgenic tobacco plants.

Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum—a synonym of X. campestris pv. malvacearum (Smith 1901–1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1–45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall.

Characterization of harpinXoo induced hypersensitive responses in non host plant, tobacco

Harpin proteins encoded by hrp genes are bacterial protein elicitors that can stimulate hypersensitive response (HR) in non-host plants. HR-related pathogen resistance involves a complex form of programmed cell death (PCD). It is increasingly viewed as a key component of the hypersensitive disease response of plants. Currently, the evidence of harpin proteins-induced PCD is deficient though it exhibits phenotypic parallels with HR, and the mechanism of harpin proteins-induced PCD is not well understood. In this study, we demonstrate that harpinXoo protein from Xanthomonas oryzae pv. oryzae of rice bacterial blight expressed and isolated from bacterial cells acted as an agent to induce PCD in infiltrated tobacco plants. Treatment of tobacco leaves with harpinXoo induced typical PCD-related morphological and biochemical changes including cell shrinkage and nuclear DNA degradation. We further analyzed the expression of several genes in signal transduction pathway of PCD in tobacco plants by real-time qRT-PCR analysis using EF-1α as an endogenous control. Our results showed that the expression of NtDAD1 was down-regulated and the expression of BI-1, tpa1 and aox1 was up-regulated following the infiltration of harpinXoo into tobacco leaves. Our data suggest that harpinXoo can induce PCD with the coordination of PCD-related genes in infiltrated tobacco leaves, providing evidence to further investigate the signal transduction pathways of HR and PCD.

First Report of Leaf Anthracnose Caused by Colletotrichum alatae on Water Yam (Dioscorea alata) in China

Water yam (Dioscorea alata [Dioscoreaceae]) is a vegetatively propagated tuberous food crop cultivated widely in the tropics and subtropics (Mignouna et al., 2003). It is the most widely distributed species of yam in China and eaten mainly as a vegetable. During July 2016, anthracnose-like lesions were observed on the leaves of cultivar Da56 at a plantation in Danzhou City, Hainan Province. Leaf symptoms began as small, brown, pinpoint spots ＜1 mm in diameter and enlarged to dark brown to almost black, irregular blotches ＞10 mm diameter with yellow haloes. Approximately 35% of the plants showed the same symptoms on their lower leaves. Symptomatic leaves were collected randomly from different parts of the field. Tissue was removed from the margin of three lesions per leaf. It was then surface sterilized in 75% ethanol, air-dried, plated on potato dextrose agar plates (PDA) and incubated at 28℃ and a 12-h photoperiod. Colonies with similar characteristics were produced after 4 days. Four isolates (DA1605-16...

Evaluation of antifungal metabolites activity from bacillus licheniformis OE-04 against Colletotrichum gossypii

Anthracnose disease in the cotton plant caused by fungal pathogen Colletotrichum gossypii. It is supposed to be most critical diseases in the cotton crop as it causes infection and leads to complete damaging of the cotton crop by infecting the leaves, stems, and bolls in the field. The disease control is challenging due to the absence of an effective fungicide without damaging the farmer health and environment. So the series of experiments were designed to assess the antagonistic activity of biosurfactant released by strain Bacillus licheniformis OE-04 against the anthracnose causing agent in cotton and this strain was screened out from forty eight strain of rhizobacteria. We also estimated the heat stability and pH range and toxicity of biosurfactant produced by strain 0E-04. The results showed that biosurfactant has maximum antifungal activity against C. gossypii. In vitro study concluded that the biosurfactant can reduce fungal activity by inhibiting the spore germination of C. gossypii. Moreover, the biosurfactant also has wide pH and temperature range. We observed Antifungal activity of biosurfactant at 5 to 10 pH range and temperature range was also wide from room temperature to 100 °C. We also observed the toxicity of biosurfactant produced by Bacillus licheniformis against zebra fish (Danio rerio). We were noticed that biosurfactant have least harmful effect with maximum concentration. The study confirmed that biosurfactant of Bacillus licheniformis have high pH and heat stability range with least harmful effects so it can be a good replacement of chemical pesticides for cotton anthracnose control.