甘蔗耐旱内生菌的筛选、鉴定及功能研究

Screening,Identification,and Functional Study on Drought-tolerant Endophytes in Sugarcane

【目的】筛选耐旱性较强的甘蔗内生菌,并对其功能进行测定,以丰富甘蔗耐旱性内生菌资源,并用于提高甘蔗的抗旱性。【方法】以云南农业大学甘蔗研究所分离的329株甘蔗内生菌为供试菌株,采用PEG6000模拟干旱的方法进行耐旱菌株的筛选;通过0~45%PEG6000的胁迫处理,对筛选得到的目标菌株进行耐旱性分析;结合形态观察、生理生化测定、16sDNA等方法对其进行鉴定;并研究了不同培养条件对菌株生长特性的影响,以及菌株固氮和分泌IAA的能力。【结果】筛选得到两株耐旱性较强的菌株,其中K5菌株鉴定为考克氏菌(Kocuria sp.),登录号为OP737815;ZM菌株为芽孢杆菌(Bacillus sp.),登录号为OP740361。K5菌株和ZM菌株都能耐受40%PEG6000的胁迫,其最适初始pH分别为5.0和6.0,而且两株菌株均能在50℃的条件下稳定生长,具有较广的初始pH和温度适应范围,能耐受100 g·L^(-1)的氯化钠胁迫。此外,两株菌株均具有固氮和分泌生长素的能力;其中ZM菌株的固氮能力强于K5菌株,在一定的时间范围内,两株菌株分泌IAA的能力随着培养时间的增加而增强。【结论】K5、ZM菌株耐旱能力较强,且具有固氮和分泌IAA的能力,间接推测其对于提高甘蔗抗旱性具有较大的应用潜力。

【Objective】Endophytes in sugarcane were screened for drought resistance to serve as microbial and germplasm resources.【Methods】Drought-tolerant microbes were isolated from a collection of 329 sugarcane endophytes at the Sugarcane Research Institute,Yunnan Agricultural University.In a simulated experiment,target strains were screened under the artificial draught condition created by using 0-45%PEG6000.Potential candidates were identified based on the morphological,physiological,and biochemical characteristics as well as 16sDNA sequences of the microbes.Optimal culture conditions for the growth,nitrogen fixation ability,and IAA secretion of the identified isolates were determined.【Results】Two strains that exhibited significant drought-tolerance were identified.K5 was a Kocuria sp.with an accession number of OP737815,and ZM a Bacillus sp.with an accession number of OP740361.Withstood the stress exerted by 40%PEG6000 or 100 g·L^(-1) sodium chloride,they could grow stably at 50℃in wide ranges of pHs and temperatures but optimally under an initial pH at 5.0 for K5 and 6.0 for ZM.ZM was stronger than K5 in fixing nitrogen.Within a limited timeframe,the IAA secretion of the two strains increased with prolonged culture.【Conclusion】Both K5 and ZM displayed significant drought-resistance with the abilities of nitrogen fixation and IAA secretion.They could be potential candidates as a microbial agent,and their carrier sugarcane a valuable germplasm.