Crops ›› 2018, Vol. 34 ›› Issue (6): 68-75.doi: 10.16035/j.issn.1001-7283.2018.06.011

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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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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

Table 1

Halo of phosphate-solubilization microorganism in solid media (7d) cm"

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

Fig.1

Variation available phosphorus content in culture medium during solubilizing period PVK (A), NBRIP (B), ironic phosphate (C) and calcium phytate (D), the same below"

Fig.2

Active bacteria number in culture medium during solubilizing period"

Fig.3

pH in culture medium during solubilizing period"

Table 2

Effects of D5 inoculation on maize seedling growth and plant phosphorus content"

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

Fig.4

Total phosphorus content and pot growth of 10 seedlings in pot experiment"

Table 3

Comparison of 16S rDNA, gyrB and rpoB gene fragments of strain D5 in NCBI database"

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

Fig.5

Phylogenetic tree generated by the neighbor-joining method based on 16S rDNA sequences of selected strains Bootstrap values (1000 replicates) above 50% are indicated above the branches. Scale bar indicates 0.05% substitution of nucleotide. The same below"

Fig.6

Phylogenetic tree generated by the neighbor-joining method based on gyrB sequences of selected strains"

Fig.7

Phylogenetic tree generated by the neighbor-joining method based on rpoB sequences of selected strains"

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