黄瓜南方根结线虫病生防芽孢杆菌的筛选鉴定及防效

Screening, identification, and efficacy evaluation of Bacillus strains for biocontrol of cucumber root-knot nematodes

摘要:

根结线虫(Meloidogyne spp.)分布范围广泛、破坏能力强，给农业生产带来巨大的经济损失。生物防治被认为是一种防治该病害的有效措施。目的 发掘高效稳定、绿色安全防治根结线虫病的生防资源至关重要。方法 采用系列稀释法从土壤中分离芽孢杆菌，以南方根结线虫为靶标，通过离体试验筛选对二龄幼虫具有较强毒杀性的芽孢杆菌。通过形态学、生理生化特征和分子生物学方法对菌株进行鉴定，并通过抑制卵孵化试验、溶磷解钾及产酶活性能力测定、拮抗广谱性评价、温室盆栽和田间试验进一步评价菌株的生防潜力。结果 从16份土壤样品中分离获得189株细菌，筛选获得2株对南方根结线虫二龄幼虫具有较强毒杀性的细菌Sneb2550和Sneb2556，其24 h校正死亡率分别为95.64%和95.36%。通过形态学特征、生理生化特性结合16S rRNA基因和gyrB序列分析，鉴定菌株Sneb2550和Sneb2556分别为解蛋白芽孢杆菌(Bacillus proteolyticus)和解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。菌株Sneb2550具有产蛋白酶的能力，菌株Sneb2556具有产蛋白酶、淀粉酶和溶磷的能力。菌株Sneb2550对甜瓜果腐病病原菌、苹果霉心病病原菌、番茄早疫病病原菌具有显著抑制作用；菌株Sneb2556对甜瓜果腐病病原菌、马铃薯早疫病病原菌、苹果轮纹病病原菌、苹果霉心病病原菌、辣椒炭疽病病原菌和番茄早疫病病原菌具有显著抑制作用。相比之下，芽孢杆菌Sneb2550和Sneb2556不影响黄瓜种子萌发，且都显著促进黄瓜种子胚根生长。盆栽结果表明，芽孢杆菌Sneb2550和Sneb2556能显著减少黄瓜植株根结数，根结减退率分别为56.02%和50.19%，并且促进黄瓜生长。田间试验结果表明，芽孢杆菌Sneb2550和Sneb2556灌根处理黄瓜幼苗后能有效防治黄瓜南方根结线虫病，防效分别为60.90%和52.63%，且促进黄瓜植株生长。结论 芽孢杆菌Sneb2550和Sneb2556能够有效防治黄瓜南方根结线虫病并促进黄瓜植株生长，为南方根结线虫病的生物防治提供新的潜在资源。

Abstract:

Root-knot nematodes (Meloidogyne spp.) are widely distributed and highly destructive, causing substantial economic losses in agricultural production. Biocontrol has been considered as an effective measure for managing these pathogens.Objective To explore efficient and eco-friendly biocontrol resources for controlling root-knot nematodes.Methods Bacillus strains were isolated from soil via the serial dilution method. Strains with strong nematicidal activity against Meloidogyne incognita second-stage juveniles (J2) were screened by in vitro bioassays. The selected strains were identified based on morphological, physiological, and biochemical characteristics, as well as molecular biological evidence. The biocontrol potential of these strains was further evaluated by egg hatching inhibition assays, phosphorus/potassium solubilization tests, enzymatic activity profiling, and antagonistic spectrum analysis. Additionally, pot and greenhouse experiments were conducted to validate the biocontrol efficacy of strains.Results Among 189 bacterial strains isolated from 16 soil samples, strains Sneb2550 and Sneb2556 demonstrated strong nematicidal activity against M. incognita J2, inducing the corrected mortality rates of 95.64% and 95.36%, respectively, after 24 h. Based on morphological features, physiological and biochemical characteristics, and 16S rRNA gene and gyrB sequences, strains Sneb2550 and Sneb2556 were identified as Bacillus proteolyticus and B. amyloliquefaciens, respectively. Functional characterization showed that strain Sneb2550 produced protease and inhibited Fusarium asiaticum, Trichothecium roseum, and Alternaria solani. Strain Sneb2556 produced protease and amylase, solubilized phosphate, and suppressed F. asiaticum, Aternaria alternata, Botryosphaeria dothidea, T. roseum, Colletotrichum gloeosporioides, and A. solani. Moreover, both strains did not adversely affect cucumber seed germination and significantly promoted the radicle growth. Under pot conditions, Bacillus Sneb2550 and Sneb2556 significantly reduced root galls formation, with the reduction rates of 56.02% and 50.19%, respectively, while promoting plant growth. Field experiments showed that root irrigation with Bacillus Sneb2550 and Sneb2556 effectively controlled cucumber root-knot nematodes, with the control effects of 60.90% and 52.63%, respectively, while promoting plant growth.Conclusion Bacillus Sneb2550 and Sneb2556 effectively controlled cucumber root-knot nematodes and promoted plant growth, providing new potential resources for the biocontrol of root-knot nematodes.