Phyllosphere microbial communities are highly diverse and have important ecological implications; in that context, bacterial identification based on 16S rRNA genes is an important research issue. In studies of phyllosphere microbial communities, microporous filtration and centrifugation are used to collect microorganism samples, but it is unclear which one has a better collection efficiency. In this study, we compared these two microorganism collection methods and investigated the effects of the DNA extraction process on the estimation of microbial community composition and organization. The following four treatments were examined: (A) filtration, resuspension, and direct PCR; (B) filtration, DNA isolation, and PCR; (C) centrifugation, resuspension, and direct PCR; (D) centrifugation, DNA isolation, and PCR. Our results showed that the percentage of chloroplast sequence contaminants was affected by the DNA extraction process. The bacterial compositions clearly differed between treatments A and C, suggesting that the collection method has an influence on the determination of community structure. Compared with treatments B and D, treatments A and C resulted in higher Shannon index values, indicating that the DNA extraction process might reduce the observed phyllosphere microbial alpha diversity. However, with respect to community structure, treatments B and D yielded very similar results, suggesting that the DNA extraction process erases the effect of the collection method. Our findings provide key information to ensure accurate estimates of diversity and community composition in studies of phyllosphere microorganisms.

Bacillus thuringiensis is a well-known entomopathogenic bacterium that produces vegetative insecticidal proteins (Vips, including Vip1, Vip2, Vip3, and Vip4) during the vegetative phase. Here, we purified Vip1 and Vip2 from B. thuringiensis and characterized the insecticidal effects of these protoxins. Bioassay results showed that a 1:1 mixture of Vip1Ad and Vip2Ag, purified by ion-affinity chromatography independently, exhibited insecticidal activity against Holotrichia parallela larvae, with a 50% lethal concentration value of 2.33 μg/g soil. The brush border membrane (BBM) in the midgut of H. parallela larvae was destroyed after feeding the Vip1Ad and Vip2Ag mixture. Vacuolization of the cytoplasm and slight destruction of BBM were detected with Vip2Ag alone, but not with Vip1Ad alone. Notably, Vip1Ad bound to BBM vesicles (BBMVs) strongly, whereas Vip2Ag showed weak binding; however, binding of Vip2Ag to BBMV was increased when Vip1Ad was added. Ligand blotting showed that Vip2Ag did not bind to Vip1Ad but bound to Vip1Ad-t (Vip1Ad was activated by trypsin), suggesting the activation of Vip1Ad was important for their binary toxicity. Thus, our findings suggested that Vip1Ad may facilitate the binding of Vip2Ag to BBMVs, providing a basis for studies of the insecticidal mechanisms of Vip1Ad and Vip2Ag.

Rhizosphere bacteria play a crucial role in promoting plant health and development. A full understanding of the bacterial communities in rhizosphere soil and their relationship with those in bulk soil is important for supporting plant growth. Some beneficial bacteria are recruited to the root-zone when plants experience different stresses. However, it is unknown whether the rhizosphere of pear trees enriches some beneficial microorganisms that can resist pathogen infections in natural ecosystems. In this study, we found a higher proportion of antagonistic strains in the rhizosphere of pear trees compared to bulk soil under natural growth conditions. By deep sequencing the V3 and V4 hypervariable regions of 16S rRNA genes, we characterized the bacterial communities in the rhizosphere soil of ‘Yuluxiang’ pear trees and the adjacent bulk soil. Our analysis revealed that the diversity of the bacterial community in the rhizosphere soil was lower than that in the bulk soil, but their compositions differed between the two soil types. We found that 12 phyla, 97 families and 130 genera contributed to these differences. Proteobacteria was the dominant phylum and its relative abundance in the rhizosphere soil was significantly higher than that in the bulk soil. Notably, the two genera, Bacillus and Pseudomonas , were more abundant in the rhizosphere soil. Compared with the bulk soil, the pear rhizosphere soil existed a higher proportion of beneficial bacteria with antagonistic activities against Fusarium oxysporum . These findings indicated that the pear tree rhizosphere can selectively assemble beneficial bacteria with specific antagonistic activities to address threats from pathogens. The distinct bacterial community structure in the rhizosphere of plants reflects a spontaneous ecological adaptation mechanism of plants to their environment.