两株PGPR菌株的花生定殖及对根际细菌群落结构的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-1577

摘要/Abstract

摘要：

为探讨2株根际促生菌耐酪氨酸束村氏菌P9和吡咯伯克霍尔德氏菌P10对花生的促生机制。利用GFP及利福平对2个菌株进行标记、结合扫描电镜观察,追踪了2株PGPR菌株在花生组织中的定殖动态;并通过16S rRNA全长测序对菌株接种花生根际土壤的细菌多样性进行分析。结果表明,利福平标记的P9和P10菌株具有良好的遗传稳定性,其生长和促生特性与原始菌株基本一致。GFP标记菌株可在花生的根尖及其分生区定殖;利福平标记菌株可稳定定殖在土壤及花生的根、茎部,且接菌30 d后定殖数仍保持在10⁴CFU/g数量级。与未接菌植株根际土壤相比,P9、P10及混合菌株接种组的细菌群落相似性更高;接菌组的Flavihumibacter、unidentified_Rhizobiaceae的相对丰度显著增加,芽孢杆菌属、链霉菌属等的丰度较CK有不同程度增加,溶杆菌属、无色杆菌属及假黄单胞菌属等的丰度降低。2株PGPR菌株均可通过直接定殖在植株组织中、间接影响土壤细菌群落结构而发挥对花生的促生作用,混合菌株接种效果更优。研究结果明析了2株促生菌的促生机制,并为菌株的应用提供了科学依据。

关键词: 耐酪氨酸束村氏菌, 吡咯伯克霍尔德氏菌, 花生, 定殖, 细菌群落结构

Abstract:

This work aims to investigate the growth-promoting mechanism of two plant growth-promoting rhizobacteria（PGPR）strains,Tsukamurella tyrosinosolvens P9 and Burkholderia pyrrocinia P10 on peanut. The two strains were labeled with GFP and rifampicin,respectively,and colonization dynamics of 2 strains in peanut tissues were studied,combined with scanning electron microscopy. Bacterial diversity of rhizosphere soil inoculated with 2 strains was analyzed by 16S rRNA whole-length sequencing. It showed that rifampicin-labeled strains P9 and P10 had good genetic stability,and their growth and growth-promoting characteristics were consistent with original strains,respectively. Two GFP-labeled strains colonized in the root tip and meristematic region of peanut. Rifampicin-labeled strains were stabilized in the roots and stems of peanut plants,and the colonization number remained 10⁴ CFU/g after 30 d of inoculation. In addition,the bacterial communities in inoculation groups of P9 and P10 and mixed strains were more similar than those in the un-inoculated control group. In three inoculation groups,the relative abundance of Flavihumibacter and unidentified_Rhizobiaceae increased significantly,and that of Bacillus and Streptomyces increased in different degrees compared with CK,while the abundance of Lysobacter,Achromobacter and Pseudoxanthomonas decreased. The two PGPR strains could not only directly colonize in peanut tissues,but also indirectly affect soil bacterial community structure to promote the growth of peanut. The inoculation of mixed strains had better growth promotion effect on peanut. The results clearly elucidate the growth-promoting mechanism of the two PGPR strains,and provide scientific basis for the further application of the strains.

Key words: Tsukamurella tyrosinosolvens, Burkholderia pyrrocinia, peanut, colonization, bacterial community structure

李颖, 龙长梅, 蒋标, 韩丽珍. 两株PGPR菌株的花生定殖及对根际细菌群落结构的影响[J]. 生物技术通报, 2022, 38(9): 237-247.

LI Ying, LONG Chang-mei, JIANG Biao, HAN Li-zhen. Colonization on the Peanuts of Two Plant-growth Promoting Rhizobacteria Strains and Effects on the Bacterial Community Structure of Rhizosphere[J]. Biotechnology Bulletin, 2022, 38(9): 237-247.

图/表 10

图1 GFP转化菌株阳性转化子的PCR产物验证 M：1 000 bp marker;1：阳性对照;2、4：分别为P9和P10原始菌株;3、5：分别为GFP转化的P9和P10阳性转化子

Fig.1 PCR verification of positive transformants of GFP-labeled strains M：1 000 bp marker;1：positive control;2 and 4：original strain of P9 and P10,respectively;3 and 5：positive transformants of GFP-labeled P9 and P10,respectively

图2 两株GFP标记的PGPR菌株在花生根尖的分布 A：CK组花生根尖;B、C：CK组根尖分生区;D：P9处理花生根尖;E、F：P9处理根尖分生区;G：P10处理花生根尖;H、I：P10处理花生根尖分生区。除B、E、H为物镜20倍下的视野,其余均为物镜40倍下的视野

Fig.2 Distribution of two GFP-labeled PGPR strains on the root tips of peanut A：Root tip of CK;B and C：meristem region of root tip of CK;D：root tip of P9 treated group;E and F：meristem region of P9 treated group;G：root tip of P10 treated group;H and I：meristem region of P10 treated group. All are the images of microscope objectives 40,except that B,E and H are of objectives 20

图3 两株利福平标记PGPR菌株的菌落形态及细胞形态 A,B分别为利福平LB平板上的P9r和P10r菌落;C,D分别为扫描电镜下的P9r和P10r细胞形态

Fig.3 Colony morphology and cell morphology of two rifampicin-labeled PGPR strains A and B are the colonies of P9r and P10r on rifampicin LB plate respectively;C and D are the cell morphologies of P9r and P10r under scanning electron microscopy,respectively

图4 两株PGPR菌株和利福平标记菌株的生长曲线 P9和P10为原始菌株,P9r和P10r为利福平标记菌株,下同

Fig. 4 Growth curves of two PGPR strains and their rifampicin-labeled strains P9 and P10 are original strains,P9r and P10r are rifampicin-labeled strains,the same below

表1 两株PGPR菌株和利福平标记菌株的促生特性

Table 1 Growth-promoting characteristics of two PGPR strains and their rifampicin-labeled strains

菌株 Strain	可溶磷含量 Soluble phorsphorus content/（µg·mL^-1）	IAA含量 IAA content /（mg·L^-1）	ACC脱氨酶活性 ACC deaminase activity/（μmol·（h·mg）^-1）	铁载体相对含量 Siderophore relative content/%
P9	307.84±10.81a	38.72±0.52a	1.00±0.09a	83.40±0.40a
P9r	297.28±1.40a	36.35±1.46a	0.99±0.02a	83.20±0.50a
P10	74.09±3.71b	44.91±1.3b	26.31±1.53b	83.80±1.60a
P10r	67.26±0.53b	40.31±1.13bc	25.96±1.69b	81.90±2.10a

图5 两株利福平标记PGPR菌株在花生土壤及根、茎中的定殖动态

Fig.5 Colonization dynamics of two rifampicin-labeled PGPR strains in peanut soils,roots and stems

图6 扫描电镜观察两株利福平标记菌株在花生根、茎表面的定殖 A：未接菌花生根;B：P9r处理花生根;C：P10r处理花生根;D：P9r+P10r处理花生根;E：未接菌花生茎;F：P9r处理花生茎;G：P10r处理花生茎;H：P9r+P10r处理花生茎

Fig.6 Colonizaiton of two rifampicin-labeled strains on root and stem surface observed by scanning electron microscope A：Un-inoculated root of peanut;B：P9r treated root;C：P10r treated root;D：P9r+P10r treated root;E：uninoculated stems;F：P9r treated stems;G：P10r treated stems;and H：P9r+P10r treated stems

表2 两株利福平标记菌株及PGPR菌株对花生生长的影响

Table 2 Effects of two rifampicin-labeled and their original PGPR strains on the growth of peanuts

处理 Treatment	鲜重 Fresh weight/g	株高 Plant height /cm	总根长 Total root length /cm	根重 Root weight /g	叶绿素含量 Cholorphyll content /（mg.g^-1FW）
CK	2.91±0.14a	8.92±0.37a	7.48±0.37a	0.524±0.05a	3.72±0.20a
P9r	3.23±0.28ab	11.68±0.22b	11.78±1.23b	0.580±0.06a	4.39±0.39a
P10r	3.03±0.09b	12.53±0.31bc	10.88±0.18b	0.524±0.05a	4.35±0.31a
P9r+P10r	4.20±0.32c	14.28±0.54d	12.08±0.45bc	0.718±0.06bc	4.37±0.26a
P9	3.84±0.16bc	13.18±0.30cd	11.37±0.50b	0.624±0.03ab	4.65±0.14a
P10	3.79±0.27bc	14.23±0.22d	13.83±0.54c	0.739±0.02bc	4.38±0.39a
P9+P10	4.92±0.23d	16.78±0.61e	12.60±0.27bc	0.843±0.01c	4.62±0.36a

图7 花生根际土壤多样本的香农曲线和PCA分析左图为香农曲线,右图为PCA分析;A,B,C,D分别代表CK、P9接种组、P10接种组、P9+P10接种组

Fig.7 Multi-samples Shannon curves and principal component analysis（PCA）of bacterial communities in peanut rhizosphere soil Left：Shannon curves. Right：PCA analysis. A,B,C,D represents CK,P9-inoculated group,P10-inoculated group,P9+P10 inoculated group,respectively

图8 不同处理组花生根际土壤细菌群落在门水平和属水平上的相对丰度 A：门水平;B：属水平

Fig. 8 Relative abundances of bacteria communities at phy-lum and genus level in rhizosphere soils of different treated peanuts A：Phylum level. B：Genus level

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