Juntao Feng

Sensitivity and biochemical characteristics of Sclerotinia sclerotiorum to propamidine

Propamidine is an aromatic diamidine compound. In the current study, baseline sensitivity of Sclerotinia sclerotiorum to propamidine was determined using 78 strains collected from the oilseed rape fields without a previous history of propamidine usage. The median effective concentration (EC50) values for propamidine inhibiting mycelial growth ranged from 0.406 to 3.647μg/mL, with a mean of 1.616±0.217μg/mL. There was no correlation between sensitivity to propamidine and sensitivity to dimethachlon or carbendazim. After treated with propamidine, mycelia were thinner with irregular distortion and more branches; cell wall became thicker with uneven distribution of cytoplasm than untreated control. In addition, sclerotia production, cell membrane permeability and oxalic acid content significantly decreased. On detached oilseed rape leaves, propamidine exhibited better control efficacy than carbendazim at the same concentration whether the leaves were inoculated with carbendazim-sensitive or resistant strains. All the results showed that propamidine exhibited strong antifungal activity and potential application in controlling S. sclerotiorum. Importantly, these data will provide more information on understanding the mode of action of propamidine against S. sclerotiorum and should be valuable for development of new antifungal drugs.

Purification and characterization of a novel glycoprotein from Streptomyces sp. ZX01.

A novel glycoprotein GP-1 with antiviral activity against plant virus was isolated from the fermentation broth of the actinomycete Streptomyces sp. ZX01. MALDI-TOF-MS proved that molecular weight of GP-1 approximately was 8.5 kDa. GP-1 was a heat-sensitive glycoprotein with decreasing antiviral activity after treated from 80 °C to 100 °C for 30 min. GP-1 contained 40.23% carbohydrate with N-linked and O-linked glycan. FT-IR and NMR spectra proved that GP-1 contained protein and carbohydrate portions with α-D-(1,6)-glucose residues. Circular dichroism revealed that GP-1 was a glycoprotein with a large unordered content. Moreover, protein sequencing was predicted by using MALDI-TOF-MS and Mascot search. These results suggested that glycoprotein GP-1 could be used as a novel natural antiviral agent in agricultural industry.

Antifungal Activity and Biochemical Response of Cuminic Acid against Phytophthora capsici Leonian.

Phytophthora blight of pepper caused by Phytophthora capsici Leonian is a destructive disease throughout the world. Cuminic acid, extracted from the seed of Cuminum cyminum L., belongs to the benzoic acid chemical class. In this study, the sensitivity and biochemical response of P. capsici to cuminic acid was determined. The mean EC50 (50% effective concentration) values for cuminic acid in inhibiting mycelial growth and zoospore germination of the 54 studied P. capsici isolates were 14.54 ± 5.23 μg/mL and 6.97 ± 2.82 μg/mL, respectively. After treatment with cuminic acid, mycelial morphology, sporangium formation and mycelial respiration were significantly influenced; cell membrane permeability and DNA content increased markedly, but pyruvic acid content, adenosine triphosphate (ATP) content, and ATPase activity decreased compared with the untreated control. In pot experiments, cuminic acid exhibited both protective and curative activity. Importantly, POD and PAL activity of the pepper leaves increased after being treated with cuminic acid. These indicated that cuminic acid not only showed antifungal activity, but also could improve the defense capacity of the plants. All the results suggested that cuminic acid exhibits the potential to be developed as a new phytochemical fungicide, and this information increases our understanding of the mechanism of action of cuminic acid against Phytophthora capsici.

Antiviral activity of glycoprotein GP-1 isolated from Streptomyces kanasensis ZX01

Plant virus diseases have seriously damaged global food security. However, current antiviral agents are not efficient enough for the requirement of agriculture production. So, developing new efficient and nontoxic antiviral agents is imperative. GP-1, from Streptomyces kanasensis ZX01, is a new antiviral glycoprotein, of which the antiviral activity and the mode of action against Tobacco mosaic virus (TMV) were investigated in this study. The results showed that GP-1 could fracture TMV particles, and the infection and accumulation of TMV in host plants were inhibited. Moreover, GP-1 could induce systematic resistance against TMV in the host, according to the results of activities of defensive enzymes increasing, MDA decreasing and overexpression of pathogenesis-related proteins. Furthermore, GP-1 could promote growth of the host plant. In conclusion, GP-1 showed the ability to be developed as an efficient antiviral agent and a fertilizer for agriculture.

Optimization of Fermentation Conditions and Bench-Scale for Improvement of a Novel Glycoprotein GP-1 Production by Streptomyces kanasenisi ZX01

GP-1 is a novel glycoprotein produced by Streptomyces kanasenisi ZX01 that was isolated from soil near Kanas Lake with significant bioactivity against tobacco mosaic virus. However, extremely low fermentation production has largely hindered further research and market applications on glycoprotein GP-1. In this study, response surface methodology was used to optimize fermentation conditions in a shake flask for higher glycoprotein GP-1 production. When the optimized fermentation conditions were inoculum volume of 6%, initial pH of 6.5, and rotating speed of 150 rpm, glycoprotein GP-1 production could reach 0.9253 mg/L, which was increased by 52.14% compared to the original conditions. In addition, scale-up fermentation was conducted in a 5-L bioreactor to preliminarily explore the feasibility for mass production of glycoprotein GP-1 in a large fermentor, obtaining GP-1 production of 2.54 mg/L under the same conditions, which was 2.75 times higher than the production obtained from a shake flask of 0.9253 mg/L. This work will be helpful to improve GP-1 production on a large scale and lay the foundations for developing it as a novel agent against plant virus.

Baseline sensitivity and biochemical responses of Valsa mali to propamidine

In the current study, baseline sensitivity of Valsa mali to propamidine was determined using 80 strains collected from apple orchards in Shaanxi Province, China. The median effective concentration (EC50) values for propamidine inhibiting mycelial growth ranged from 0.086 to 0.852 μg/mL, with a mean of 0.405 ± 0.137 μg/mL. After treated with propamidine, mycelia were contorted with an increased number of branches, loss of fruiting body production, and decreased cell membrane permeability. Moreover, the enzyme activities of the complexes I, II, IV and ATPase in the mitochondrial respiratory chain were increased significantly, while the enzyme activities of complexes III decreased. Importantly, both on detached leaves and branches of apple trees, propamidine applied at 100 μg/mL exhibited over 75% protective and curative efficacies, which were even better than the efficacies obtained by carbendazim at the same concentration. These results indicated that propamidine could be used as an alternative compound in controlling Valsa canker and mitochondrial respiratory chains might be correlated with the action mode of propamidine. This study encourages further investigation for the action mechanism of propamidine against plant pathogens and the information could be valuable for synthesis of new antifungal drugs with novel modes of action.

Effects of Agitation, Aeration and Temperature on Production of a Novel Glycoprotein GP-1 by Streptomyces kanasenisi ZX01 and Scale-Up Based on Volumetric Oxygen Transfer Coefficient

The effects of temperature, agitation and aeration on glycoprotein GP-1 production by Streptomyces kanasenisi ZX01 in bench-scale fermentors were systematically investigated. The maximum final GP-1 production was achieved at an agitation speed of 200 rpm, aeration rate of 2.0 vvm and temperature of 30 °C. By using a dynamic gassing out method, the effects of agitation and aeration on volumetric oxygen transfer coefficient (kLa) were also studied. The values of volumetric oxygen transfer coefficient in the logarithmic phase increased with increase of agitation speed (from 14.53 to 32.82 h−1) and aeration rate (from 13.21 to 22.43 h−1). In addition, a successful scale-up from bench-scale to pilot-scale was performed based on volumetric oxygen transfer coefficient, resulting in final GP-1 production of 3.92, 4.03, 3.82 and 4.20 mg/L in 5 L, 15 L, 70 L and 500 L fermentors, respectively. These results indicated that constant volumetric oxygen transfer coefficient was appropriate for the scale-up of batch fermentation of glycoprotein GP-1 by Streptomyces kanasenisi ZX01, and this scale-up strategy successfully achieved 100-fold scale-up from bench-scale to pilot-scale fermentor.

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors—temperature, enzyme lysis time, and concentration of the lysing enzyme—were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46–54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

Evaluating the Potential Value of Natural Product Cuminic Acid against Plant Pathogenic Fungi in Cucumber

Fusarium wilt and anthracnose are two major diseases which limit the yield and quality of cucumber worldwide. Cuminic acid was extracted from the seed of Cuminum cyminum L. The mean EC50 values of cuminic acid for inhibiting mycelial growth and zoospore germination of five Fusarium oxysporum f. sp. cucumerinum strains were 25.66 ± 3.02 μg/mL and 15.99 ± 2.19 μg/mL, and of four Colletotrichum lagenarium (Pass.) Ellis and Halsted strains were 29.53 ± 3.18 μg/mL and 18.41 ± 2.78 μg/mL, respectively. In greenhouse experiments, cuminic acid at 2000 μg/mL exhibited 70.77% protective and 62.63% curative efficacies against F. oxysporum, and 65.43% protective and 55.46% curative efficacies against C. lagenarium. Moreover, the translocation behavior of cuminic acid, determined by high performance liquid chromatography (HPLC), showed that it could be readily absorbed and transported upward and downward in cucumber. Importantly, superoxide dismutase (SOD) and pyphenol oxidase (PPO) activities of cucumber leaves treated with cuminic acid increased significantly. All results indicated that cuminic acid showed antifungal activity, and could be used as a botanical fungicide in disease management. This study encourages further investigation on the mechanism of action of cuminic acid and the development of alternative antifungal drugs.

Genome-Wide Analysis Reveals the Secondary Metabolome in Streptomyces kanasensis ZX01

Streptomyces kanasensis ZX01 produces some antibiotics and a glycoprotein with antiviral activity. To further evaluate its biosynthetic potential, here we sequenced the 7,026,279 bp draft genome of S. kanasensis ZX01 and analyzed all identifiable secondary gene clusters for controlling natural products. More than 60 putative clusters were found in S. kanasensis ZX01, the majority of these biosynthetic loci are novel. In addition, the regulators for secondary metabolism in S. kanasensis ZX01 were abundant. The global regulator nsdA not only controls biosynthesis of some antibiotics, but also enhances production of glycoprotein GP-1 with antiviral activity. This study importantly reveals the powerful interplay between genomic analysis and studies of traditional natural product purification/production increasing.