Crops ›› 2016, Vol. 32 ›› Issue (4): 112-117.doi: 10.16035/j.issn.1001-7283.2016.04.018

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Isolation and Identification of Endophytic Bacteria from Roots of Four Inbred Lines in Maize

Xu Yanrui1,Fang Zhijun2,Lu Xiaoping1,Mu Chunhua2,Fan Qiuping1,Hao Lujiang1   

  1. 1 Shandong Provincial Key Laboratory of Microbial Engineering,Qilu University of Technology,Jinan 250353,Shandong,China
    2 Maize Research Institute,Shandong Academy of Agricultural Sciences,Jinan 250100,Shandong,China
  • Received:2016-04-18 Revised:2016-05-29 Online:2016-08-15 Published:2018-08-26
  • Contact: Lujiang Hao

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Abstract:

The endophytic bacteria strains were isolated and characterized from 4 maize (Zea mays L.) inbred lines (PH4CV,PH6WC,Zheng 58 and Chang 7-2),which provided beneficial bacterial strains for further research.On the basis of morphological,physiological,biochemistry and 16S rDNA sequence homology analysis,23 endophytic bacteria were identified to be Bacillussp.,Pseudomonas sp.,Stenotrophomonas sp.,Enterobacter sp.,Variovorax sp.and Sphingobacterium sp..Of them,Bacillus sp.was predominant,distributed in 4 maize inbred lines.The comparative analysis of endophytic bacteria in different maize inbred lines indicated that the population number of different bacterial species was varied.And the bacteria abundance was associated with maize genotypes as the following order,PH6WC>Zheng 58>PH4CV>Chang 7-2.These results indicate that entophytic bacteria populations and their abundance are associated with maize genotypes.

Key words: Maize roots, Endophytic bacteria, 16S rDNA, Isolation and identification

Table 1

PCR reaction system"

组成Ingredient 用量Dosage(μL)
10×PCR缓冲液 4.0
Mg2+ 2.4
dNTP(25mmol/L) 0.8
上游引物GM5F(25pmol/μL) 2.0
下游引物907R(25pmol/μL) 2.0
模板DNA 2.0
Taq酶(5U/μL） 0.8
ddH2O 26.0

Table 2

Morphological characteristics of strains"

菌落形态Colony morphology 革兰氏染色Gram stain
菌株编号
Stain code		 形状
Shape		 颜色
Color		 表面
Surface		 边缘特征
Border		 透明度
Transparency		 迁移性
Migratory		 结果
Result		 菌体形状
Bacteria shape
PH4CV-1 圆形 白略带黄 隆起 毛状 不透明 无 G+ 杆状
PH4CV-2 圆形 黄 隆起 圆整 不透明 无 G- 杆状
PH4CV-3 圆形 白 隆起 毛状 不透明 无 G+ 杆状
PH6WC-1 圆形 白 隆起小 圆整 不透明 无 G+ 杆状
PH6WC-2 圆形 白 隆起 圆整 不透明 无 G+ 球形
PH6WC-3 圆形 乳黄 隆起 圆整 半透明 无 G+ 杆状
PH6WC-4 圆形 白 不隆起 圆整 不透明 无 G+ 杆状
PH6WC-5 圆形 乳黄 隆起 不规则 不透明 无 G+ 球形
PH6WC-6 圆形 灰 隆起 圆整 不透明 无 G+ 杆状
PH6WC-7 圆形 白 不明显 丝状 不透明 无 G+ 杆状
Zh58-1 圆形 乳黄 隆起小 圆整 不透明 无 G+ 杆状
Zh58-2 圆形 白 隆起 圆整 不透明 无 G+ 杆状
Zh58-3 圆形 白略带黄 隆起 圆整 不透明 无 G- 杆状
菌落形态Colony morphology 革兰氏染色Gram stain
菌株编号
Stain code		 形状
Shape		 颜色
Color		 表面
Surface		 边缘特征
Border		 透明度
Transparency		 迁移性
Migratory		 结果
Result		 菌体形状
Bacteria shape
Zh58-4 圆形 白略带黄 隆起 圆整 不透明 无 G+ 球形
Zh58-5 圆形 白 隆起 较圆整 不透明 无 G+ 杆状
Zh58-6 圆形 白 不明显 丝状 不透明 迁移 G- 杆状
Ch7-a 圆形 黑 隆起 圆整 不透明 无 G+ 杆状
Ch7-b 圆形 灰白色 隆起 圆整 不透明 无 G+ 杆状
Ch7-c 圆形 灰 隆起 圆整 不透明 无 G+ 杆状
Ch7-d 圆形 黄 隆起 圆整 不透明 无 G+ 球形
Ch7-e 圆形 白略带黄 隆起 毛状 不透明 无 G+ 杆状
Ch7-f 片状 白 隆起 触手状 不透明 无 G+ 杆状
Ch7-g 圆形 黄带白毛 不明显 丝状 不透明 无 G- 杆状

Table 3

Physiological and biochemical characteristics of strains"

菌株编号Strain code 淀粉水解试验
Starch hydrolysis		 甲基红试验
Methyl red		 硫化氢试验
Hydrogen sulfide		 硝酸盐还原试验
Nitrate reduction		 吲哚试验
Indole		 V-P试验
V-P		 柠檬酸盐利用试验
Citrate utilization		 明胶液化试验
Gelatin liquefaction
PH4CV-1 - + + - + + - -
PH4CV-2 - + - - + + + -
PH4CV-3 - + + + + - + +
PH6WC-1 - + + - - + + -
PH6WC-2 - - + - + - + -
PH6WC-3 - + + - - + + -
PH6WC-4 - + + - - + + -
PH6WC-5 - - + - + - + -
PH6WC-6 + - + + + - - -
PH6WC-7 - - + + + - + -
Zh58-1 - - - + + - - +
Zh58-2 - - - + + - + -
Zh58-3 - - - + + - - +
Zh58-4 - - - - + - + -
Zh58-5 - - + + + - + +
Zh58-6 - - + - - - - +
Ch7-a + - + + + - - -
Ch7-b + - + + + - - -
Ch7-c + - + + + - - -
Ch7-d - - + - + - + -
Ch7-e - - - + + - - +
Ch7-f + + - - - + + -
Ch7-g - + + - + + + -

Fig.1

A profile of genomic DNA extracted from 8 bacterial strains M:λ-DNA/Hind III,Lanes 1-8: bacterial strains"

Fig.2

A profile of 16S rDNA PCR amplification of 8 bacterial strains M: DNA Marker G,Lanes 1-8: the 16S rDNA amplification products from different strains"

Table 4

Results of 23 bacterial strains on 16S rDNA analysis"

菌株编号Strain code 序列长度Sequence length(bp) 相似度Similarity(%) GenBank number 相似菌株Similar strains
PH4CV-1 552 99 KP233828 Stenotrophomonassp.OUC_Est10
PH4CV-2 552 99 JX174251.1 Enterobacter sp.2374
PH4CV-3 554 99 HM055978 Bacillus pumilus strain MB-36
PH6WC-1 549 99 FJ577964 Enterobacter sp.T1
PH6WC-2 550 99 EU373423 Sphingobacterium siyangensis strain YRL14
PH6WC-3 564 99 LN650480 Bacillus aerophilus
PH6WC-4 593 99 JX393000 Stenotrophomonas maltophilia strain W8-1
PH6WC-5 568 98 DQ103761 Pseudomonas aeruginosa
PH6WC-6 564 99 JF893458 Bacillus sp.NIOC27
PH6WC-7 560 99 DQ658992 Bacillus sp.JZDN42
Zh58-1 549 99 JX133197 Stenotrophomonas maltophilia strain UI3
Zh58-2 559 99 HF584815.1 Pseudomonas sp.
Zh58-3 558 99 KJ472791 Variovorax paradoxusstrain ZR 1-3
Zh58-4 550 99 HM055957 Bacillus pumilus strain MB-15
Zh58-5 564 99 KC236781 Pseudomonas sp.B1009
Zh58-6 568 99 KM287518.1 Pseudomonas sp.FRT-Slr-28
Ch7-a 549 99 GQ392047.1 Bacillus sp.HCYL05
Ch7-b 550 100 FJ944650 Bacillus megateriumstrain KUDC1027
Ch7-c 563 99 KC845545 Bacillus sp.DX3
Ch7-d 562 99 HM055961 Bacillus pumilus strain MB-19
Ch7-e 564 99 HM055978 Bacillus pumilus strain MB-36
Ch7-f 548 100 DQ649444 Bacillus sp.YACN-9
Ch7-g 562 99 GQ392044 Bacillus anthracis strain HCYL02

Table 5

The separation frequency of endophytic bacteria from 4 maize inbred lines"

自交系Inbreds Bacillussp. Pseudomonassp. Enterobactersp. Variovoraxsp. Stenotrophomonassp. Sphingobacteriumsp. 合计
PH4CV 1/3 - 1/3 - 1/3 - 3
PH6WC 3/7 1/7 1/7 - 1/7 1/7 5
郑58 Zheng 58 1/6 3/6 - 1/6 1/6 - 4
昌7-2 Ch7-2 4/4 - - - - - 1

Fig.3

Phylogenetic tree of bacterial 16S rDNA from 23 typical strains"

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