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作物杂志,2018, 第6期: 68–75 doi: 10.16035/j.issn.1001-7283.2018.06.011

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

一株甘蔗内生菌鉴定及其溶磷能力的研究

狄义宁1,刘鲁峰1,谢林艳1,李咏梅2,何鹏飞2,崔文艳2,李富生1,何丽莲1,3   

  1. 1 云南农业大学农学与生物技术学院,650201,云南昆明
    2 云南农业大学植物保护学院,650201,云南昆明
    3 云南农业大学甘蔗研究所,650201,云南昆明
  • 收稿日期:2018-06-22 修回日期:2018-09-13 出版日期:2018-12-15 发布日期:2018-12-06
  • 作者简介:狄义宁,硕士研究生,研究方向为植物资源评价与利用
  • 基金资助:
    云南省现代农业甘蔗产业技术体系建设专项(2017-2018);福建农林大学国家甘蔗工程技术研究中心开放课题(NER02018.6.1)

Identification and Characterization of a Phosphate-Solubilizing Endophyte from Sugarcane

Di Yining1,Liu Lufeng1,Xie Linyan1,Li Yongmei2,He Pengfei2,Cui Wenyan2,Li Fusheng1,He Lilian1,3   

  1. 1 College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan,China
    2 College of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan, China
    3 Sugarcane Research Institute, Yunnan Agricultural University, Kunming 650201, Yunnan, China
  • Received:2018-06-22 Revised:2018-09-13 Online:2018-12-15 Published:2018-12-06

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摘要：

采用形态观察、生理生化测定以及16S rDNA、gyrB和rpoB基因片段比对,对分离自甘蔗品种粤糖86-368茎部内的一株D5内生菌进行分类鉴定。在此基础上,使用平板溶磷圈法和液体培养法研究其溶磷能力,并通过盆栽试验探究其对玉米幼苗生长的影响。结果表明：D5菌株属于Pseudomonas extremorientalis;D5菌株对不同磷酸盐的溶磷能力表现不同,表现为磷酸三钙>植酸钙>磷酸铁,溶磷量分别为0.939±0.012、0.655±0.016、0.125±0.005mg/mL,同时,在溶磷过程中,液体培养基的pH均有不同程度的降低,活菌数除了在磷酸铁培养基中一直下降外,其他磷源培养基中均是先升后降的趋势。施用菌剂后,处理组（D5+P）比对照组（CK+P）玉米幼苗的总鲜重、总干重、苗株总磷含量分别增加45.92%、32.65%、45.01%。因此,D5菌株具有制备微生物菌肥的潜力。

关键词: 甘蔗, 内生菌, 分子鉴定, 溶磷

Abstract:

An endophytic D5 strain was isolated from the stem of Yuetang 86-368 which came from one of the sugarcane cultivars.Colony morphology characteristics, physiological and biochemical tests, 16S rDNA, gyrB and rpoB sequence analyses were carried out. Phosphate-solubilizing capability of the strain was studied by methods of phosphorus hole and liquid culture, and the effect on the growth of maize seedling was studied by a pot experiment. The results showed that the D5 strain belonged to Pseudomonas extremorientalis. Its phosphate-solubilizing capability to different phosphate was different which followed the trend: tricalcium phosphate > phytic acid calcium > iron phosphate. The phosphate-solubilizing capacity was 0.939±0.012, 0.655±0.016, 0.125±0.005mg/mL, respectively. The pH value of the liquid media decreased to different extents and the numbers of living bacterium followed a trend of initial rise and then fall in all phosphorus source in the culture medium except in the iron phosphate cultivation showing a straight decline of dissolved phosphorus. Compared with the control (CK+P), the total fresh weight, total dry weight and total phosphorus content of maize seedlings in the D5 strain were increased by 45.92%, 32.65% and 45.01%, respectively. Therefore, it is an excellent candidate for microbial fertilizer.

Key words: Sugarcane, Endophyte, Molecular identification, Phosphate solubilizing

表1

平板溶磷圈法测量的菌株溶磷能力（7d）"

培养基类型Medium type 菌株Strain 菌落颜色Colony color 菌落直径Colony diameter (d) 溶磷圈直径Phosphorus ring diameter (D) D/d
PVK (Pikovskaya) 1-2 褐红 0.475 1.544 3.25
D5 砖红 1.100 1.650 1.50
NBRIP 1-2 黑蓝 0.850 1.800 2.12
D5 蓝 0.490 1.150 2.88
植酸钙Calcium phytate 1-2 淡紫 0.624 1.224 1.96
D5 淡黄 0.508 1.240 2.44

图1

培养液中可溶性磷含量随时间的变化 PVK（A）、NBRIP（B）、磷酸铁（C）、植酸钙（D）,下同"

图2

培养液中活菌数随时间的变化"

图3

培养液中pH随时间的变化"

表2

接种D5菌对玉米幼苗生长影响"

处理
Treatment		 地上鲜重(g)
Aboveground fresh weight		 地上干重(g)
Aboveground dry weight		 地下鲜重(g)
Underground fresh weight		 地下干重(g)
Underground dry weight		 总鲜重(g)
Total fresh weight		 总干重(g)
Total dry weight
D5+P 16.78 1.65 10.64 0.95 27.42 2.60
D5 8.73 0.94 8.59 0.74 17.32 1.68
CK+P 11.97 1.12 6.82 0.84 18.79 1.96
CK 6.77 0.73 7.43 0.65 14.20 1.38
CK2 20.18 1.77 10.23 1.09 30.41 2.86

图4

盆栽试验各处理10株苗全磷总含量与盆栽长势"

表3

D5菌株16S rDNA,gyrB,rpoB基因片段在NCBI数据库中的比对结果"

菌株Strain 类群Species 属Genus 长度Length (bp) 同源菌株Homologous bacteria 序列号GenBank 一致性Indentities(%)
D5 Pseudomonas 16S rDNA 1 436 Pseudomonas extremorientalisCNU082017 KF979139.1 100
gyrB 1 171 Pseudomonas extremorientalis BS2774 LT629708.1 99
rpoB 1 552 Pseudomonas extremorientalisDSM15824T KX186863.1 99

图5

溶磷性内生菌株16S rDNA 序列系统发育树 系统发育树分支上的数值表示大于50%的自举值,标尺指核苷酸替代数,下同"

图6

溶磷性内生菌株gyrB序列系统发育树"

图7

溶磷性内生菌株rpoB序列系统发育树"

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