内生假单胞菌NWSUAF303对棉花黄萎病的生物防治作用及其机制

Biocontrol efficacy and mechanism of endophytic Pseudomonas sp. NWSUAF303 against cotton Verticillium wilt

摘要:

棉花黄萎病是由大丽轮枝菌(Verticillium dahliae)引起的一种重要的土传真菌病害，严重影响全球棉花产量。目的 探究棉花内生假单胞菌(Pseudomonas sp.) NWSUAF303对棉花黄萎病的防治作用及其作用机制，为棉花土传病害的生物防治提供新资源。方法 通过16S rRNA基因系统进化分析及生物学特性检测对该菌种进行分类鉴定；采用平板对峙法及对扣试验检测该菌株的抑菌谱；结合顶空固相微萃取气质联用(headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry HS-SPME-GC-MS)技术分析该菌株分泌的挥发性有机化合物(volatile organic compounds, VOCs)；通过盆栽试验评估该菌株对棉花黄萎病的防治效果；基于RT-qPCR和酶活检测解析其抗棉花黄萎病的作用机制。结果 NWSUAF303菌株被鉴定为阿尔万德假单胞菌(Pseudomonas alvandae)，具有固氮、溶解有机磷和分泌吲哚乙酸等促生特性。该菌株的非挥发性代谢物对6种病原真菌具有抑制作用，而其VOCs对7种植物病原真菌具有显著的抑制效果，其中对核盘菌(Sclerotinia sclerotiorum)的抑制率>95.00%，对大丽轮枝菌(V. dahliae) 592的抑制率达89.27%。该菌株的VOCs可显著下调大丽轮枝菌毒力基因VdPR1、Vdpf和VdGAL4的表达(P<0.05)。鉴定出2,3-丁二酮(2,3-butanedione)、2-壬醇(2-nonanol)和6-甲基-2-庚醇(6-methyl-2-heptanol) 3种关键抑菌VOCs，其中2,3-丁二酮对大丽轮枝菌的抑制作用为首次报道。盆栽试验显示该菌株对黄萎病的防治效果达54.40%，与多菌灵相当。机制研究表明，NWSUAF303菌株可激活水杨酸(salicylic acid, SA)和茉莉酸(jasmonic acid, JA)信号通路，显著上调棉花防御相关基因GhPAL、Gh4CL和GhCHI等的表达，并显著提高过氧化物酶(peroxidase, POD)、多酚氧化酶(polyphenol oxidase, PPO)和超氧化物歧化酶(superoxide dismutase, SOD)等棉花防御酶的活性，从而增强植物的抗病能力。结论 本研究报道了P. alvandae NWSUAF303通过产生新型抑菌VOCs和激活SA/JA信号通路及防御酶协同增强棉花抗病性系统抗性双重机制防治黄萎病，兼具广谱抑菌和促生功能，为棉花黄萎病的绿色防控提供了优良菌种资源。

Abstract:

Cotton Verticillium wilt, caused by the soil-borne fungal pathogen Verticillium dahliae, is a devastating disease that severely impacts global cotton production.Objective To investigate the biocontrol potential and mechanism of endophytic Pseudomonas sp. NWSUAF303 against cotton Verticillium wilt and provide novel microbial resources for managing soil-borne diseases in cotton.Methods The strain was identified by phylogenetic analysis based on 16S rRNA gene sequences and phenotypic characterization. Its antifungal spectrum was evaluated via dual-culture and volatile organic compounds (VOCs) inhibition assays. VOCs were detected by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Pot experiments were carried out to assess the disease control efficacy of the strain. RT-qPCR and enzymatic activity assays were employed to elucidate the resistance mechanism of the strain against cotton Verticillium wilt.Results Strain NWSUAF303 was identified as Pseudomonas alvandae, exhibiting plant growth-promoting properties including nitrogen fixation, phosphate solubilization, and indole-3-acetic acid (IAA) production. Its non-volatile metabolites inhibited six phytopathogenic fungi, whereas VOCs demonstrated broader antifungal spectrum against seven pathogens, showing the inhibition rates >95% against Sclerotinia sclerotiorum and of 89.27% against V. dahliae 592. The VOCs of this strain downregulated the expression of virulence genes (VdPR1, Vdpf, and VdGAL4) in V. dahliae (P<0.05). Three key antifungal VOCs were identified, including 2,3-butanedione, 2-nonanol, and 6-methyl-2-heptanol, with the inhibitory effect of 2,3-butanedione on V. dahliae being first reported. Pot experiments revealed the control efficacy of 54.40% against Verticillium wilt, which was comparable to that of carbendazim. Strain NWSUAF303 activated the salicylic acid/jasmonic acid (SA/JA) signaling pathway, upregulating the expression of defense-related genes GhPAL, Gh4CL, and GhCHI (P<0.01), while enhancing the activities of peroxidase (POD), polyphenol oxidase (PPO), and superoxide dismutase (SOD).Conclusion P. alvandae NWSUAF303 combats Verticillium wilt through dual mechanisms: producing novel antifungal VOCs and activating systemic resistance via SA/JA signaling and defense enzyme coordination. With broad-spectrum antifungal activity and plant growth-promoting properties, this strain represents a promising biocontrol agent for sustainable management of cotton Verticillium wilt.