南疆绿洲区冰核细菌多样性及高效拮抗菌的筛选——兼论其对植物抗冻性的提升机理

Diversity of Ice Nucleation-Active（INA）Bacteria and Screening of High-Efficiency Antagonistic Bacteria in the Southern Xinjiang Oasis Area—Also on the Mechanism of Enhancing Plant Frost Resistance

摘要

低温霜冻是制约农作物安全生产的关键逆境因子，而冰核细菌作为重要生物诱发因子，可显著加剧植物冻害程度，对南疆绿洲区农业生态系统构成严重威胁。本研究以南疆绿洲区植物-土壤微生态系统为研究对象，系统开展冰核活性细菌（INA细菌）分离鉴定、拮抗菌筛选及抗霜冻机制研究，旨在挖掘本地特色微生物资源，建立环境友好型植物霜冻生物防控技术体系。采用KB培养基平板稀释法分离INA细菌，结合小液滴冻结法鉴定其冰核活性；以无菌番茄幼苗为模式植物，在可控低温条件下（4 ℃），探究INA细菌对宿主抗冻生理指标的影响；通过双层平板法与平板对峙法筛选高效拮抗菌。从60份样品中分离鉴定出8株INA细菌，涵盖嗜金属寡养单胞菌、潮汐藤黄色单胞菌等8个类群，其中嗜金属寡养单胞菌与潮汐藤黄色单胞菌冰核活性最强（-5 ℃下30 s内诱导结冰）；同步筛选获得14株拮抗菌，枯草芽孢杆菌A-2对莱比托泉游动球菌I-4的抑菌圈直径达3.37±0.09 mm；生理指标检测显示，接种INA细菌可使番茄幼苗过氧化氢含量提高2.1~3.8倍，丙二醛含量增加18%~47%，明确INA细菌加剧植物冻害的生理机制。本研究首次系统探究南疆绿洲区INA细菌种群特征及拮抗菌资源潜力，揭示INA细菌对植物霜冻胁迫的诱发机制，为研发靶向抑菌与抗冻剂协同的霜冻防控技术提供重要理论支撑与微生物资源储备。

Abstract

Low-temperature frost is a critical abiotic stress factor that restricts the safe production of crops．As important biological inducers，ice nucleation-active bacteria can significantly aggravate the degree of plant freezing injury，posing a serious threat to the agricultural ecosystem in the Southern Xinjiang Oasis Area．This study takes the plant-soil microecosystem in the Southern Xinjiang Oasis Area as the research object，and systematically conducts studies on the isolation and identification of INA bacteria，screening of antagonistic bacteria，and research on antifrost mechanisms．Its purpose is to explore local characteristic microbial resources and establish an environmentally friendly biological control technology system for plant frost damage．INA bacteria were isolated using the gradient dilution method on KB medium plates，and their ice nucleation activity was identified by combining with the droplet freezing assay．Using sterile tomato seedlings as the model plant，the effects of INA bacteria on the frost-resistant physiological indices of the host were investigated under controlled low-temperature conditions （4 ℃）．High-efficiency antagonistic bacteria were screened through the double-layer plate method and plate confrontation assay．8 strains of INA bacteria were isolated and identified from 60 samples，covering 8 taxa including Stenotrophomonas chelatiphaga and Luteimonas aestuarii．Among them，Stenotrophomonas chelatiphaga and Luteimonas aestuarii exhibited the strongest ice nucleation activity，inducing freezing within 30 seconds at -5 ℃．Meanwhile，14 strains of antagonistic bacteria were obtained through screening，and the diameter of the inhibition zone of Bacillus subtilis A-2 against Planococcus rifietoensis I-4 reached 3.37±0.09 mm．Detection of physiological indices showed that inoculation with INA bacteria increased the hydrogen peroxide content in tomato seedlings by 2.1~3.8 folds and the malondialdehyde content by 18%~47%，which clarified the physiological mechanism by which INA bacteria exacerbate plant freezing injury．This study is the first to systematically clarify the population characteristics of INA bacteria and the resource potential of antagonistic bacteria in the Southern Xinjiang Oasis Area，reveal the induction mechanism of INA bacteria on plant frost stress，and provide important theoretical support and microbial resource reserves for the development of frost prevention and control technologies that combine targeted bacteriostasis and antifreeze agents．

关键词

冰核活性细菌 / 假单胞菌 / 霜冻损伤 / 拮抗细菌 / 16S rDNA

Key words

ice nucleus active bactena / pseadomonas / frost damage / antagonistic bacteria / 16S rDNA

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基金资助

兵团科技计划项目资助(2024DA009)