Effect of Combining Wuyiencin and Pyrimethanil on Controlling Grape Gray Mold and Delaying Resistance Development in Botrytis cinerea.

By screening the compounding combination of Wuyiencin and chemical agents, this study aims to delay the emergence of chemical agent resistance, and provide a technical reference for scientific and rational fungicides technology. This study investigated the impacts of the antibiotic wuyiencin derived from Streptomyces albulus var. wuyiensis and its combination with pyrimethanil on the inhibition of Botrytis cinerea. Treatment with wuyiencin (≥80 µg mL-1) strongly inhibited the pathogenicity of B. cinerea and activated the plant defense response against B. cinerea. Application of 80-100 µg mL-1 wuyiencin effectively controlled grape gray mold (by 57.6-88.1% on leaves and 46.7-96.6% on fruits). Consequently, the application of 80-100 µg mL-1 wuyiencin effectively mitigated grape gray mold incidence, leading to a substantial reduction in disease symptoms to nearly imperceptible levels. When wuyiencin (at the median effective concentration [EC50]) was combined with pyrimethanil (EC50) at a ratio of 7:3, it exhibited the highest efficacy in inhibiting B. cinerea growth. This combination was significantly more potent (p < 0.05) than using wuyiencin or pyrimethanil alone in controlling gray mold on grape leaves and fruits. Furthermore, the combination effectively delayed resistance development in gray mold. The experimental results show that wuyiencin can delay resistance development by affecting the expression of methionine biosynthesis genes and reducing the activity of the cell wall-degrading enzyme activity.

Potential Biocontrol Microorganisms Causing Attenuated Pathogenicity in Plasmopara viticola.

A phenomenon of pathogenicity attenuation of Plasmopara viticola was consistently observed during its subculture on grape. To clarify the causes of attenuated pathogenicity of P. viticola, culturable microbes were isolated from the P. viticola mass (mycelia, sporangiophores, and sporangia) in each generation and tested for their biocontrol efficacies on grape downy mildew (GDM). The results showed that the incidence of GDM decreased with the increase in the number of subculture times on both vineyard-collected leaves and grape leaves from in vitro-grown seedlings. The number of culturable microbial taxa on the surface of P. viticola decreased, whereas the population densities of four specific strains (i.e., K2, K7, P1, and P5) increased significantly with the increase in subculture times. Compared with the control, the biocontrol efficacies of the bacterial strain K2 reached 87.5%, and those of both fungal strains P1 and P5 reached 100.0%. Based on morphological characteristics and molecular sequences, strains K2, P1, and P5 were identified as Curtobacterium herbarum, Thecaphora amaranthi, and Acremonium sclerotigenum, respectively, and these three strains survived very well and multiplied on the surface of P. viticola. As the number of times P. viticola was subcultured increased, all three of these strains became the predominant strains, leading to greater P. viticola inhibition, attenuated P. viticola pathogenicity, and effective GDM biological control. To the best of our knowledge, this is the first report of C. herbarum and T. amaranthi having biological control activity against GDM.

Identification of endophytes with biocontrol potential from Ziziphus jujuba and its inhibition effects on Alternaria alternata, the pathogen of jujube shrunken-fruit disease.

Endophytic strains were isolated from different parts of a healthy "Dongzao" jujube (Ziziphus jujuba Mill. 'Dongzao') to find biocontrol agents against jujube shrunken-fruit disease caused by Alternaria alternata. The strains were screened using A. alternata strain CN193 as the target pathogen. The nutrient competition for all isolates was studied using the dual culture, and their inhibitive capability was tested by measuring the inhibition width of filter paper disks with filtrate. Influence of filtrate from the selected strains with strong inhibition of mycelial growth on spore germination was studied with hanging drop method on concavity slides. Colonization in the jujube leaves was assayed using a rifampicin-resistant mutant of strain St-zn-34 as the screening marker. Strains were identified based on their morphological, physiological, and biochemical characteristics, 16S rDNA sequencing, and phylogenetic analysis. A total of 81 endophytic strains were isolated from the stems, leaves, flowers, and fruits of winter jujube. Among these isolates, 14 strains showed strong antagonism against A. alternata. Further study showed that the filtrate of strains St-zn-9 and St-zn-34 could inhibit the mycelial growth of A. alternata, and the widths of their inhibition zone reached 6.14±0.03 mm and 8.27±0.09 mm, respectively. However, strain St-zn-34 showed stronger inhibition on spore germination than strain St-zn-9. St-zn-34 could significantly reduce the spore germination rate of A. alternata, and the spore did not germinate at all or the germ tube was very short. A rifampicin resistant-derivative of wild-type strain St-zn-34, which was designated as St-zn-34r, was obtained by transferring the strains to media with stepwise-increased rifampicin. Colonization assays indicated that St-zn-34r could colonize in jujube leaves, and the population of St-zn-34r was 1.2×103 CFU/g FW after inoculation for 30 days. Except for its salt tolerance, St-zn-34 was the closest to those of Bacillus subtilis. Thus, the strain was identified as B. subtilis.

[Effect of repeated freeze-thaw on inhibition of Bacillus subtilis strain St-zn-34 to Alternaria alternate].

Objective: The effects of repeated freeze-thaw cycles(F-Tc) on cell shape of fermented Bacillus subtilis and the inhibitory activity to Alternaria alternata, the primary infection pathogen of jujube fruit shrunken disease, were determined using an endophytic B. subtilis strain St-zn-34 isolated from winter jujube. Methods: The fermented broth was freeze-thawed after batch fermentation of the test strain. The population of living bacteria and bacterial endospores were determined by dilution methods of plate counting, and the inhibitory activity to A. alternata was tested by filter paper disks with filtrate of fermented broth. The shape of B. subtilis with different freeze-thaw cycles was observed under environment scanning electron microscope. Results: Changes of pH, living bacteria, bacterial endospore counts in the fermentation broth and the inhibitory activity of filtrate at different time generally increased first and decreased afterwards. The inhibitory activity at 60 h was higher than other time points. The bacterial cells fermented for 60 h were treated with F-Tc, and the living bacteria count and inhibitory activity decreased gradually when cells of B. subtilis were freeze-thawed for 3 cycles, but it had no significant difference (P>0.05) after being freeze-thawed for more than 3 times. With the increase of freeze-thawing times, the bacteria cells became smaller than the normal cells, the surface twisted with one or more depressions, and the jelly flowed out under environment scanning electron microscope. The inhibitory activity of filtrate was determined and it showed a broad-spectrum inhibitive capability against twelve species of plant pathogens. The treatment of different temperature and proteinase to the filtrate showed that the temperature below 60℃ did not affect the inhibitive activity and had no significant difference compared with that of the control. However, the inhibition decreased with increasing of the temperature above 80℃ and was significantly lower than that of control. The inhibitory activity of filtrate to A. alternata decreased by treatment with proteinase K. Conclusion: Repeated freeze-thaw cycles could affect the shape of B. subtilis and reduced the inhibitory activity of filtrate to A. alternata.