生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 246-255.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0590
收稿日期:
2023-06-21
出版日期:
2023-10-26
发布日期:
2023-11-28
通讯作者:
黄晶,女,博士,教授,研究方向:中药材生态栽培;E-mail: huang.jing@swust.edu.cn作者简介:
邹兰,女,博士,讲师,研究方向:植物病害生物防治;E-mail: zoulan@swust.edu.cn
基金资助:
ZOU Lan1(), WANG Qian1, LI Mu-yi1, YE Kun-hao2, HUANG Jing1()
Received:
2023-06-21
Published:
2023-10-26
Online:
2023-11-28
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。
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.
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 |
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 |
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/% | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
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 |
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/% | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
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
测定指标 Character/(g·plant-1) | 处理 Treatment | ||
---|---|---|---|
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 |
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 | ||
---|---|---|---|
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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