乌头内生细菌JY-3-1R的鉴定及其生防和促生能力研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0590

摘要/Abstract

摘要：

分离筛选具生防和促生潜力的乌头内生细菌，为乌头土传病害防治提供优质微生物菌种。采用纯培养法从健康乌头分离内生细菌，对峙试验检测菌株拮抗病原菌能力，多位点持家基因序列鉴定菌株分类地位，平板法检测菌株产酶和铁载体能力，比色法检测菌株产IAA能力，大田试验检测菌株对乌头生长的影响和白绢病的生防潜力。从111株乌头内生细菌中筛选到1株对白绢病和根腐病病原菌有显著抑制作用的内生细菌JY-3-1R，该菌鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。JY-3-1R无细胞发酵滤液对齐整小核菌（Sclerotium rolfsii）菌丝生长和菌核萌发抑制率达100%。该菌具产IAA、铁载体、蛋白酶、纤维素酶和葡聚糖酶的能力，同时包含合成伊枯草菌素、丰原素、表面活性素、芽孢菌霉素、杆菌溶素、杆菌烯和大环内酯的功能基因。大田条件下，JY-3-1R接种降低乌头白绢病发病率高达40%，生防效率为61.53%-84.61%，生防能力持续30 d，同时植株茎、主根和子根干重分别提高了34.34%、82.59%和56.08%。JY-3-1R兼具生防和促生能力，具有开发作为乌头生物肥料和生防材料的潜力。

关键词: 内生细菌, 生防, 促生, 白绢病

Abstract:

In order to provide promising microbial resource for the control of soil-borne diseases in Aconitum carmichaelii Debx., endophytic bacterial strains with biocontrol and plant growth promoting potential were isolated and screened from healthy A. carmichaelii plants. Pure culture method was used to isolate endophytic bacteria, and dual-culture method was to check the antagonistic activity of candidate strains against pathogens. Multi-locus housekeeping gene sequence analysis was to investigate phylogeny of the strain. Enzyme- and siderophore-producing capacities of bacterial strain were measured on plates. Salkowski method was applied to measure indoleacetic acid（IAA）-producing ability of the strain. Field experiment was employed to demonstrate plant growth promoting ability and biocontrol potential against southern blight in A. carmichaelii. One strain JY-3-1R screened from 111 A. carmichaelii endophytic bacteria showed significant antagonistic activity against Sclerotium rolfsii and Fusarium oxysporum in vitro. The JY-3-1R was identified as Bacillus amyloliquefaciens. Cell-free culture filtrate of JY-3-1R presented 100% inhibition on the hyphal growth and sclerotia germination of S. rolfsii. JY-3-1R was able to produce IAA, siderophore, protease, cellulase, glucanase, also had the functional genes of synthesizing iturin, fengyin, surfactin, bacillomycin, bacilysin, bacillaene, and macrolactin. Under field conditions, JY-3-1R inoculation reduced around 40% of southern blight occurrence, whose biocontrol rates ranged from 61.53% to 84.61%. This biocontrol efficiency lasted at least for 30 d. Meanwhile, JY-3-1R inoculation promoted 34.34%, 82.59% and 56.08% of stem, main root and lateral root dry weight, respectively of A. carmichaelii. Altogether, JY-3-1R demonstrated both plant growth promoting and biocontrol potential, and could be used as good candidate of biofertilizer and biocontrol agent of A. carmichaelii.

Key words: endophytic bacteria, biocontrol, plant growth promotion, southern blight

邹兰, 王茜, 李慕仪, 叶坤浩, 黄晶. 乌头内生细菌JY-3-1R的鉴定及其生防和促生能力研究[J]. 生物技术通报, 2023, 39(10): 246-255.

ZOU Lan, WANG Qian, LI Mu-yi, YE Kun-hao, HUANG Jing. Identification, Biocontrol and Plant Growth-promoting Potential of Endophytic Bacterial Strain JY-3-1R from Aconitum carmichaelii Debx.[J]. Biotechnology Bulletin, 2023, 39(10): 246-255.

图/表 9

表1 持家基因扩增用引物及反应程序

Table 1 Primers and reaction procedure for housekeeping genes

基因Gene	引物Primer（5'-3'）	反应程序Reaction procedure
16S rRNA	27F: AGAGTTTGATCCTGGCTCAG 1492R: TACGGCTACCTTGTTACGACTT	94℃ for 3 min, 30 cycles of 94℃ for 1 min, 56℃ for 1 min, 72℃ for 2 min, final extension for 72℃ 10 min
atpD	atpDF: RTAATYGGMGCSGTRGTNGAYGT’ atpDR:TCATCCGCMGGWACRTAWAYNGC’	98℃ for 2 min, 35 cycles of 98℃ for 30 s, 56℃ for 1 min, 72℃ for 45 s, final extension for 72℃ 10 min
gyrA	gyrA2F: ATGAGCGATCTGGCCAGAGA gyrA9R: CGCGCCTTGTTCACCTGATA	98℃ for 2 min, 35 cycles of 98℃ for 30 s, 57℃ for 1 min, 72℃ for 45 s, final extension for 72℃ 10 min
rpoB	rpoBF: AGGTCAACTAGTTCAGTATGGAC rpoBR: AAGAACCGTAACCGGCAACTT	94℃ for 3 min, 30 cycles of 94℃ for 30 s, 54℃ for 45 s, 72℃ for 30 min, final extension for 72℃ 10 min

图1 JY-3-1R对病原真菌的抑制效果

Fig. 1 Inhibition effects of JY-3-1R against pathogenic fungi

表2 JY-3-1R对病原真菌的抑制效果

Table 2 Inhibition effects of JY-3-1R against pathogenic fungi

菌株 Strain	PDA平板抑菌率 Inhibition rate on PDA plate /%					乌头切片抑菌率 Inhibition rate on A. carmichaelii root slice/%
菌株 Strain	S. rolfsii-1	S. rolfsii-2	F. oxysporum-1	F. oxysporum-2		S. rolfsii-1	S. rolfsii-2	F. oxysporum-1	F. oxysporum-2
JY-3-1 R	53.13±0.39	51.30±1.09	52.07±0.21	44.58±0.18		16.32±9.76	49.68±3.05	42.19±0.09	46.30±3.27

图2 JY-3-1R基于16S rRNA（A）和gyrA-rpoB-atpD多位点持家基因（B）的系统发育研究括号中为序列或菌株基因组信息在Genbank数据库中的登录号。T表示标准菌株

Fig. 2 Phylogenetic analysis of JY-3-1R based on 16S rRNA gene（A）and multi-locus sequence analysis of gyrA, rpoB and atpD genes（B） Accession numbers of either gene or whole genome of reference strains are indicated in brackets. T indicates type strain

图3 JY-3-1R无细胞发酵滤液对齐整小核菌菌丝生长和菌核萌发的影响

Fig. 3 Effects of cell-free culture filtrate of JY-3-1R on the hyphal growth and sclerotia germination of S. rolfsii

图4 JY-3-1R功能基因PCR产物凝胶电泳

Fig. 4 Gel electrophoresis of JY-3-1R functional genes by PCR amplification

图5 JY-3-1R产IAA、铁载体和分泌酶类能力从左至右依次为JY-3-1R分泌IAA、铁载体、蛋白酶、纤维素酶和葡聚糖酶的表型特征

Fig. 5 Abilities of JY-3-1R producing IAA, siderophore, and secretases Pictures from left to right showed the phenotypes of IAA, siderophore， protease,cellulase and glucanase producing capacity of JY-3-1R, respectively

图6 JY-3-1R对乌头白绢病的田间防病效果 *表示差异显著(P<0.05 ),ns表示差异不显著

Fig. 6 Biocontrol potential of.JY-3-1R against southern blight by field experiment P*indicates significant difference (P<0.05 ), and ns indicates not significant

表3 JY-3-1R对乌头生长的影响

Table 3 Plant growth promoting effect of JY-3-1R on A. carmichaelii

测定指标 Character/（g·plant^-1）	处理 Treatment
测定指标 Character/（g·plant^-1）	JY-3-1R	CK
茎鲜重 Stem fresh weight	37.68±2.91 a	27.98±3.25 b
茎干重Stem dry weight	9.35±0.70 a	6.96±0.90 b
乌头鲜重Main root fresh weight	28.43±2.43 a	19.44±1.02 b
乌头干重Main root dry weight	5.77±0.78 a	3.16±0.11 b
子根鲜重Lateral root fresh weight	77.17±7.80 a	48.54±5.95 b
子根干重Lateral root dry weight	20.01±1.94 a	12.82±1.91 a

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