甘蔗耐旱内生菌的筛选、鉴定及功能研究

罗艳菊; 谢林艳; 吴昊; 刘鲁峰; 何丽莲; 李富生

doi:10.19303/j.issn.1008-0384.2023.07.014

甘蔗耐旱内生菌的筛选、鉴定及功能研究

doi: 10.19303/j.issn.1008-0384.2023.07.014

云南农业大学农学与生物技术学院，云南　昆明　650201

基金项目: 云南省重大科技专项（202202AE090021）；云南省作物生产与智慧农业重点实验室专项（202105AG070007）；云南省教育厅科学研究基金项目（2022J0286）

详细信息

作者简介:
罗艳菊（1998 —），女，硕士研究生，主要从事作物种质资源评价与利用研究，E-mail：1833912990@qq.com

通讯作者:
李富生（1968 —），男，教授，主要从事甘蔗资源研究与利用研究，E-mail：Lfs810@sina.com

中图分类号: S182
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出版历程
- 收稿日期: 2022-11-03
- 修回日期: 2023-04-11
- 网络出版日期: 2023-07-16
- 刊出日期: 2023-07-28

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

College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, Yunnan　650201, China

摘要

摘要: 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。
- 甘蔗 /
- 内生菌 /
- 耐旱 /
- 筛选 /
- 鉴定
Abstract: 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.
- Sugarcane /
- endophyte /
- drought tolerance /
- screening /
- identification

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图 1 不同质量分数PEG6000胁迫下菌株的生长情况

图中不同小写字母表示差异显著（P＜0.05），下同。

Figure 1. Growth of strains under stresses imposed by different mass fractions of PEG6000

Datas with different lowercase letters indicate significant differences (P＜0.05). Same for below.

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图 2 不同温度对菌株生长的影响

Figure 2. Effects of temperatures on growth of isolated strains

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图 3 不同pH对菌株生长的影响

Figure 3. Effects of pH on growth of isolated strains

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图 4 不同氯化钠浓度对菌株生长的影响

Figure 4. Effects of sodium chloride concentration on growth of isolated strains

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图 5 不同氮源对菌株生长的影响

Figure 5. Effects of nitrogen source on growth of isolated strains

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图 6 菌株的生长曲线

Figure 6. Growth curves of isolated strains

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图 7 菌株菌落形态及革兰氏染色结果

Figure 7. Colony morphology and gram staining results

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图 8 基于16sDNA序列的系统发育树

Figure 8. Phylogenetic tree based on 16s DNA sequence

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图 9 菌株在不同条件下分泌IAA的能力

Figure 9. IAA secretion abilities of isolated strains under varied conditions

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表 1 20%PEG6000胁迫条件下菌株的耐旱情况分析

Table 1. Drought-tolerance of strains under 20% PEG6000 stress

OD₆₀₀	菌株数 Strain number
0＜OD₆₀₀≤0.5	33
0.5＜OD₆₀₀≤1.0	91
1.0＜OD₆₀₀≤1.5	120
1.5＜OD₆₀₀≤2.0	65
＞2.0	20

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表 2 30%PEG6000胁迫条件下菌株耐旱情况分析

Table 2. Drought-tolerance of strains under 30% PEG6000 stress

菌株 Strains	OD₆₀₀	菌株 strains	OD₆₀₀
YC89-j-10	0.222±0.014 g	ZM-g-1	1.029±0.023 a
YC89-j-2	0.470±0.016 d	YC89-g-2	1.032±0.014 a
YC89-g-12	0.077±0.060 i	D01-58-g-3	0.921±0.036 b
D01-47-g-5	0.163±0.010 h	YZ-g-2	0.069±0.004 ij
D01-47-g-14	0.067±0.003 ij	YC89-g-18	0.029±0.007 l
D01-47-g-6	0.437±0.012 d	D01-47-g-7	0.470±0.022 d
YT93-g-10	0.772±0.028 c	D01-47-g-17	0.164±0.025 h
YT86-g-1	0.035±0.006 jk	D04-14-8	0.068±0.013 ij
GT-g-8	0.130±0.013 h	YT86-g-4	0.271±0.076 f
河-g-6	0.044±0.010 ijk	GT-j-9	0.390±0.008 e
不同小写字母表示差异显著（P＜0.05），下同。 Datas with different lowercase letters indicate significant difference (P＜0.05). Same for below.

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表 3 菌株的部分生理结果

Table 3. Partial physiological and biochemical properties of isolated strains

生理生化指标 Physiological and biochemical indexes	K5菌株 K5 strain	ZM菌株 ZM strain
酪素水解试验 Casein hydrolysis test	−	+
硝酸还原酶试验 Nitrate reduction test	−	+
接触酶试验 Contactase test	+	−
淀粉水解试验 Starch hydrolysis test	+	+
甲基红试验 Methyl red test	−	+
V-P试验 V-P test	−	+
“+”阳性，“−”阴性。 “+” Positive reaction，“−” The negative reaction.

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表 4 菌株固氮能力分析

Table 4. Nitrogen fixation abilities of isolated strains

菌株 Strains	透明圈直径D Transparent ring diameter D /cm	菌落直径d Colony diameter d /cm	可溶性指数（D/d） Solubility index (D/d)
K5	0.695±0.397 b	0.6	1.158±0.072 b
ZM	1.575±0.299 a	0.6	2.625±0.498 a

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