多花黄精产吲哚乙酸内生菌的分离筛选及其对黄精种子萌发的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-0078

摘要/Abstract

摘要：

从健康多花黄精根部分离筛选对黄精种子萌发具促生作用的内生细菌，以期为解决黄精资源短缺问题奠定理论基础。采用传统组织培养法分离纯化内生菌，沙尔科夫斯基反应（Salkowski法）筛选产IAA能力的促生菌；通过形态学观察、生理生化特征检测及16S rDNA序列分析确定目标菌株的分类地位；温室纸床实验验证目标菌株对黄精种子发芽的促生效果。从多花黄精中共分离纯化得到34株内生菌，其中细菌25株，真菌9株；从中筛选出两株具有稳定高产IAA的内生细菌HNC2和HPB1，其产IAA能力分别高达4.833 mg/L、4.608 mg/L；菌种鉴定结果表明菌株HNC2和HPB1分别为芽孢杆菌（Bacillus sp.）和葡萄球菌（Staphylococcus sp.）；菌种对种子萌发影响的实验结果显示，与无菌水和培养基对照组比，促生菌Bacillus sp. HNC2和Staphylococcus sp. HPB1均显著提高黄精种子的发芽势与发芽率，且对萌发幼苗的生长具有不同程度的促生功效。多花黄精具有丰富的内生菌资源，Bacillus sp. HNC2和Staphylococcus sp. HPB1有望进一步开发为微生物菌肥以提高黄精种子的繁殖率。

关键词: 多花黄精, 内生菌, 筛选, 吲哚乙酸, 促生作用

Abstract:

The endophytic bacteria that can promote the seed germination of Polygonatum were isolated and screened from the healthy roots of Polygonatum cyrtonema Hua.，in order to lay a theoretical foundation for solving the shortage of Polygonatum resources. The endophytes were isolated and purified by the traditional tissue culture method，and the IAA-producing bacteria were screened by the Salkowski reaction（Salkowski method）. The taxonomic status of the target strains was determined by morphological observation，physiological and biochemical characterization，and 16S rDNA sequence analysis. The germination-promoting effect of the target strains on the seeds of Polygonatum was verified by greenhouse paper-bed experiments. The results showed that 34 endophytes were isolated and purified from Polygonatum cyrtonema Hua.，including 25 endophytic bacteria and 9 endophytic fungi. Two endophytic bacteria HNC2 and HPB1 with stable and high content of IAA were screened out，and their IAA production capacity was as high as 4.833 mg/L and 4.608 mg/L，respectively. The results of bacterial identification showed that strains HNC2 and HPB1 were Bacillus sp. and Staphylococcus sp.，respectively. The experimental results regarding the effect of the strains on seed germination showed that both of the growth-promoting bacteria Bacillus sp. HNC2 and Staphylococcus sp. HPB1 significantly increased the germination potential and germination rate of Polygonatum seeds compared to the sterile water and medium controls，and had different degrees of growth-promoting effects on the growth of seedlings. Polygonatum cyrtonema Hua. harbors abundant endophytic bacteria，of which Bacillus sp. HNC2 and Staphylococcus sp. HPB1 are expected to be further developed as microbial fertilizer to increase the germination rate of Polygonatum seeds.

Key words: Polygonatum cyrtonema Hua., endophytes, screening, indole acetic acid, plant-growth-promoting effect

杜佳慧, 徐伟芳, 杨晓冬, 谭松, 尹登科, 刘园旭. 多花黄精产吲哚乙酸内生菌的分离筛选及其对黄精种子萌发的影响[J]. 生物技术通报, 2022, 38(12): 223-232.

DU Jia-hui, XU Wei-fang, YANG Xiao-dong, TAN Song, YIN Deng-ke, LIU Yuan-xu. Isolation and Screening of Endophytes Producing Indole Acetic Acid from Polygonatum cyrtonema Hua. and Its Effect on Seed Germination of Polygonatum[J]. Biotechnology Bulletin, 2022, 38(12): 223-232.

图/表 10

表1 多花黄精内生菌分离情况统计

Table 1 Statistics of endophytes isolated from Polygonatum cyrtonema Hua.

培养基 Medium	内生细菌 Endophytic bacteria	内生真菌 Endophytic fungi
PDA培养基 PDA medium	10	7
LB培养基 LB medium	6	0
NA培养基NA medium	8	0
GA培养基GA medium	1	1
CZA培养基 CZA medium	0	1

图2 多花黄精内生真菌在PDA培养基上的形态图左图：正面菌落形态；右图：反面菌落形态

Fig. 2 Morphology of endophytic fungi isolated from Pol-ygonatum cyrtonema Hua. on PDA medium Left：Colony morphology on the front plate；Right：colony morphology on the reverse plate

图1 部分多花黄精内生细菌在LB培养基上的形态

Fig. 1 Morphology of some endophytic bacteria isolated from Polygonatum cyrtonema Hua. on LB medium

表2 产IAA能力的多花黄精内生菌初筛统计

Table 2 Statistics of IAA-producing ability of endophytic bacteria isolated from Polygonatum cyrtonema Hua.

菌株编号 Strain No.	产IAA强度 IAA production strength
IAA标准品 IAA standard substance（30 mg·L^-1）	+++
HNC2、HPB1	++
HPB3、HPC2、HPC4、HPA3、HLB3	+
HLB2、HNC1、HNB3、HGC1、HPB2、HNC3、HLA3、HPA2、HNA3、HPA1、HLA1、HLA4、HNB1、HNB2、HNA2、HPC3、HPC1、HLA2	-

图3 多花黄精内生细菌产IAA能力的定性检测

Fig. 3 Qualitative detection of IAA-production ability of endophytic bacteria isolated from Polygonatum cyrtonema Hua.

图4 IAA标准曲线（A）与多花黄精内生菌分泌IAA的含量随时间的变化趋势（B）图中误差线表示标准偏差。不同小写字母代表存在显著性差异（P<0.05）

Fig. 4 IAA standard curve（A）and trends of the content of IAA secreted by endophytic bacteria of Polygona-tum cyrtonema Hua. over time（B） The error line in the figure refers to the standard deviation. Different lowercase letters represent significant differences at 0.05 level

图5 HNC2和HPB1菌株的菌落及菌体形态特征 A：HNC2菌株的菌落形态；B：HNC2菌株的革兰氏染色结果；C：HNC2菌株的芽孢染色结果；D：HPB1菌株的菌体形态；E：HPB1菌株的革兰氏染色结果

Fig. 5 Colony and morphological characteristics of HNC2 strain and HPB1 strain A：Colony morphology of HNC2 strain. B：Gram stain result of HNC2 strain. C：Spore stain result of HNC2 strain. D：Colony morphology of HPB1 strain. E：Gram stain result of HPB1 strain

表3 HNC2和HPB1菌株的生理生化特征

Table 3 Physiological and biochemical characteristics of HNC2 strain and HPB1 strain

生理生化测试项目 Test item	结果 Results
生理生化测试项目 Test item	HNC2	HPB1
葡萄糖 Glucose	+	-
乳糖 Lactose	-	-
麦芽糖 Maltose	+	-
甘露醇 Mannitol	-	-
蔗糖 Sucrose	+	-
靛基质试验 Indole test	+	+
甲基红试验Methyl Red（MR）	+	-
乙酰甲基甲醇试验 Voges-Proskaurer（VP）	+	+
西蒙氏柠檬酸盐 Simon’s citrate	+	+
硫化氢 Hydrogen sulphide	-	-
尿素 Urea	-	+
半固体琼脂 Semi-solid agar	+	+

图6 HNC2菌株（A）与HPB1菌株（B）基于16S rDNA序列的系统进化树括号中序号为GenBank的登录号；分支点上的数字代表Bootstrap值；标尺0.005和0.02分别代表0.5%和2%的核苷酸差异

Fig. 6 Phylogenetic tree of HNC2 strain（A）and HPB1 strain（B）based on 16S rDNA sequence Number in parentheses is the GenBank accession numbers. Number at the nodes indicates the Bootstrap value. The scale bar indicates 0.5% and 2% nucleotide substitution，respectively

表4 内生细菌HNC2和HPB1对黄精种子发芽的影响

Table 4 Effects of endophytic bacteria HNC2 and HPB1 on the germination of Polygonatum seeds

处理方法 Treatment	发芽势 Germination potential/%	发芽率 Germination rate/%	胚芽长 Germ length/mm	胚根长 Radicle length/mm	叶宽 Leaf width/mm
无菌水Sterile water	6.67±1.67 cd	11.67±1.67 cd	19.47±3.73	6.83±1.15 b	1.73±0.39
LB液体培养基LB liquid medium	5.00±0.00 d	10.00±2.89 d	22.73±5.25	14.13±2.72 ab	2.83±0.12
IAA标准品IAA standard substance	15.00±0.00 abc	21.67±4.41 abc	21.17±2.81	21.13±7.09 a	2.73±1.23
HNC2发酵液HNC2 supernatant	23.33±1.67 a	26.67±1.67 a	19.70±0.74	14.43±0.93 ab	2.23±0.12
HNC2菌悬液HNC2 cell suspension	18.33±3.33 ab	25.00±5.77 ab	20.10±1.19	14.60±1.85 ab	2.73±0.42
HPB1发酵液HPB1 supernatant	11.67±1.67 bcd	15.00±2.89 bcd	16.80±3.02	15.10±2.66 ab	3.03±1.80
HPB1菌悬液HPB1 cell suspension	21.67±6.01 a	28.33±1.67 a	18.20±0.61	12.23±0.79 ab	2.53±0.23

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