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Toxicity of Bacillus thuringiensis Strains Derived from the Novel Crystal Protein Cry31Aa with High Nematicidal Activity against Rice Parasitic Nematode Aphelenchoides besseyi.

ABSTRACT: The plant parasitic nematode, Aphelenchoides besseyi, is a serious pest causing severe damage to various crop plants and vegetables. The Bacillus thuringiensis (Bt) strains, GBAC46 and NMTD81, and the biological strain, FZB42, showed higher nematicidal activity against A. besseyi, by up to 88.80, 82.65, and 75.87%, respectively, in a 96-well plate experiment. We screened the whole genomes of the selected strains by protein-nucleic acid alignment. It was found that the Bt strain GBAC46 showed three novel crystal proteins, namely, Cry31Aa, Cry73Aa, and Cry40ORF, which likely provide for the safe control of nematodes. The Cry31Aa protein was composed of 802 amino acids with a molecular weight of 90.257 kDa and contained a conserved delta-endotoxin insecticidal domain. The Cry31Aa exhibited significant nematicidal activity against A. besseyi with a lethal concentration (LC₅₀) value of 131.80 μg/mL. Furthermore, the results of in vitro experiments (i.e., rhodamine and propidium iodide (PI) experiments) revealed that the Cry31Aa protein was taken up by A. besseyi, which caused damage to the nematode's intestinal cell membrane, indicating that the Cry31Aa produced a pore-formation toxin. In pot experiments, the selected strains GBAC46, NMTD81, and FZB42 significantly reduced the lesions on leaves by up to 33.56%, 45.66, and 30.34% and also enhanced physiological growth parameters such as root length (65.10, 50.65, and 55.60%), shoot length (68.10, 55.60, and 59.45%), and plant fresh weight (60.71, 56.45, and 55.65%), respectively. The number of nematodes obtained from the plants treated with the selected strains (i.e., GBAC46, NMTD81, and FZB42) and A. besseyi was significantly reduced, with 0.56, 0.83., 1.11, and 5.04 seedling mL^-1 nematodes were achieved, respectively. Moreover, the qRT-PCR analysis showed that the defense-related genes were upregulated, and the activity of hydrogen peroxide (H₂O₂) increased while malondialdehyde (MDA) decreased in rice leaves compared to the control. Therefore, it was concluded that the Bt strains GBAC46 and NMTD81 can promote rice growth, induce high expression of rice defense-related genes, and activate systemic resistance in rice. More importantly, the application of the novel Cry31Aa protein has high potential for the efficient and safe prevention and green control of plant parasitic nematodes.

SUBMITTER: Liang Z

PROVIDER: S-EPMC9331774 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Plant kinases containing the LysM domain play important roles in pathogen recognition and self-defense reactions. And it could recognize microbe-associated molecules including chitin and other polypeptides. The white tip nematode Aphelenchoides besseyi is a migratory parasitic nematode that infects plant shoots. It is distributed over almost all rice-producing areas and causes up to 50% economic losses. The rice OsRLK3 gene was a defense-related LysM kinase gene of rice. This study showed that the rice LysM kinase OsRLK3 could be induced by flg22, jasmonic acid, salicylic acid, and chitin. An interaction gene, Ab-atps from A. besseyi, was identified by screening the interaction between the rice gene OsRLK3 and an A. besseyi cDNA library using yeast two-hybrid screening. Ab-atps is a novel ATP synthase gene with a full length of 1341 bp, coding for 183 amino acids. The mRNA of Ab-atps was located in the esophagus and reproductive system of A. besseyi. The expression of Ab-atps was assessed at different developmental stages of the nematode and found to be the highest in the juvenile, followed by the egg, female, and male. Reproduction was significantly decreased in nematodes treated with Ab-atps double-stranded RNA (dsRNA) (p < 0.05). Transient expression experiments showed that Ab-ATPS-GFP was distributed in the nucleus, cytoplasm, and cell membrane, and Ab-ATPS-GFP triggered plant cell death. OsRLK3 was expressed significantly higher at 0.5 day and 1 day (p < 0.05) in rice plants inoculated with nematodes treated with Ab-atps dsRNA and gfp dsRNA for 0.5-7 days, respectively. Further, OsRLK3 expression under Ab-atps dsRNA treatment was significantly lower than with gfp dsRNA treatment at 0.5 day (p < 0.05) and significantly higher than with gfp dsRNA treatment at 1 day (p < 0.05). These results suggest that rice OsRLK3 could interact with A. besseyi Ab-atps, which plays an important role in growth, reproduction, and infection of the nematode. Our findings provide a theoretical basis to further understand the parasitic strategy of A. besseyi and its interaction mechanism with host plants, suggesting new ideas and targets for controlling A. besseyi.

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Toxicity of Bacillus thuringiensis Strains Derived from the Novel Crystal Protein Cry31Aa with High Nematicidal Activity against Rice Parasitic Nematode Aphelenchoides besseyi.

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