生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 211-220.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0522
杨东亚(), 祁瑞雪, 李昭轩, 林薇, 马慧, 张雪艳()
收稿日期:
2022-04-27
出版日期:
2023-02-26
发布日期:
2023-03-07
作者简介:
杨东亚,女,硕士研究生,研究方向:设施蔬菜生理与生态;E-mail:基金资助:
YANG Dong-ya(), QI Rui-xue LI, Zhao-xuan , LIN Wei, MA Hui, ZHANG Xue-yan()
Received:
2022-04-27
Published:
2023-02-26
Online:
2023-03-07
摘要:
茄病镰刀菌(Fusarium solani)引起的根腐病严重影响黄瓜产业的可持续发展,为获得高效拮抗黄瓜根腐病的芽孢杆菌并明确其促生效果,从黄瓜根际分离芽孢杆菌,筛选高抗茄病镰刀菌的菌株,并对其进行形态、生理生化、遗传特性及植株促生特性的评价。结果表明,菌株XY-1、XY-13、XY-53抑制茄病镰刀菌效果显著,抑菌率分别为65.90%、66.13%、60.83%,鉴定XY-1、XY-13为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),XY-53为枯草芽孢杆菌(B. subtilis),3个菌株均具有释磷、固氮能力,产蛋白酶和ACC脱氨酶。幼苗促生试验中,接种菌株XY-1、XY-13、XY-53有效促进黄瓜幼苗生长,与对照相比幼苗株高分别增加了37.17%、27.14%、34.94%,叶绿素含量分别增加了31.37%、40.48%、43.43%,地上部鲜重分别增加了33.03%、38.81%、51.52%,地上部干重分别增加了28.66%、29.03%、46.27%。盆栽防效试验中,接种菌株XY-1、XY-13和XY-53 不同浓度发酵液10 d后,108 CFU/mL防治效果最优,对黄瓜根腐病防效分别为65.12%、72.09%和82.86%,此外,病原菌侵染下菌株XY-1、XY-13、XY-53促进了幼苗株高、叶绿素含量的增加。总之,XY-1、XY-13和XY-53可作为防控黄瓜苗期茄病镰刀菌引起的根腐病、促进幼苗生长有潜力的生物防治资源用于黄瓜可持续高效生产。
杨东亚, 祁瑞雪, 李昭轩, 林薇, 马慧, 张雪艳. 黄瓜茄病镰刀菌拮抗芽孢杆菌的筛选、鉴定及促生效果[J]. 生物技术通报, 2023, 39(2): 211-220.
YANG Dong-ya, QI Rui-xue LI, Zhao-xuan , LIN Wei, MA Hui, ZHANG Xue-yan. Screening, Identification and Growth-promoting Effect of Antagonistic Bacillus spp. Against Cucumber Fusarium solani[J]. Biotechnology Bulletin, 2023, 39(2): 211-220.
抑菌率Inhibition rate/% | 菌株数目Number of strains |
---|---|
60-70 | 5 |
50-60 | 59 |
40-50 | 36 |
30-40 | 51 |
30以下 | 192 |
表1 分离菌株对黄瓜茄病镰刀菌的抑菌情况
Table 1 Bacteriostasis of isolated strains against Fusarium solani of cucumber
抑菌率Inhibition rate/% | 菌株数目Number of strains |
---|---|
60-70 | 5 |
50-60 | 59 |
40-50 | 36 |
30-40 | 51 |
30以下 | 192 |
菌株Strain | 菌落直径Colony diameter/mm | 抑菌率Inhibition rate/% |
---|---|---|
XY-1 | 24.67±0.17b | 65.90±0.23a |
XY-13 | 24.50±0.29b | 66.13±0.40a |
XY-53 | 28.33±0.17a | 60.83±0.23b |
表2 菌株培养上清液对黄瓜茄病镰刀菌的抑制效果
Table 2 Inhibition rate of bacterial culture supernatant against Fusarium solani of cucumber
菌株Strain | 菌落直径Colony diameter/mm | 抑菌率Inhibition rate/% |
---|---|---|
XY-1 | 24.67±0.17b | 65.90±0.23a |
XY-13 | 24.50±0.29b | 66.13±0.40a |
XY-53 | 28.33±0.17a | 60.83±0.23b |
菌株 Strain | 颜色 Color | 菌落形状 Colony shape | 边缘 Edge | 表面光滑凸起与否 Surface is smooth and bump or not | 表面干湿 Surface is dry or wet | 色素产生 Pigment |
---|---|---|---|---|---|---|
XY-1 | 白色 | 圆形 | 光滑 | 表面粗糙不规则,隆起有褶皱 | 干燥 | 无色素产生 |
XY-13 | 白色 | 圆形 | 光滑 | 表面隆起有褶皱 | 湿润 | 无色素产生 |
XY-53 | 乳白色 | 圆形 | 光滑 | 表面凸起 | 湿润 | 无色素产生 |
表3 拮抗菌的形态及特征
Table 3 Morphology and characteristics of antagonistic bacteria
菌株 Strain | 颜色 Color | 菌落形状 Colony shape | 边缘 Edge | 表面光滑凸起与否 Surface is smooth and bump or not | 表面干湿 Surface is dry or wet | 色素产生 Pigment |
---|---|---|---|---|---|---|
XY-1 | 白色 | 圆形 | 光滑 | 表面粗糙不规则,隆起有褶皱 | 干燥 | 无色素产生 |
XY-13 | 白色 | 圆形 | 光滑 | 表面隆起有褶皱 | 湿润 | 无色素产生 |
XY-53 | 乳白色 | 圆形 | 光滑 | 表面凸起 | 湿润 | 无色素产生 |
试验项目Test project | 结果 Result | ||||
---|---|---|---|---|---|
XY-1 | XY-13 | XY-53 | |||
生理生化特征 Physiological and biochemical characteristics | 甲基红 Methyl red | - | - | - | |
接触酶 Catalase | + | + | + | ||
氧化酶Oxidase | + | + | + | ||
淀粉水解 Amylum hydrolysis | + | + | - | ||
明胶液化Gelatin experiment | + | + | + | ||
乙酰甲基甲醇(V-P)Voges-Proskauer | + | + | + | ||
葡萄糖氧化发酵Oxidation-fermentation of glucose | + | + | + | ||
抗病促生特性 Antagonistic growth-promoting characteristics | 溶磷能力 Ability of releasing phosphorus | + | + | + | |
固氮能力 Nitrogen-fixing ability | + | + | + | ||
ACC脱氨酶活性 ACC deaminase activity | + | + | + | ||
解钾能力Ability of releasing potassium | - | - | - | ||
铁载体 Siderophore | - | - | - | ||
蛋白酶 Protease | + | + | + | ||
几丁质酶 Chitinase | - | - | - | ||
果胶酶 Pectinase | - | - | - | ||
纤维素酶 Cellulase | - | - | - |
表4 XY-1、XY-13、XY-53的生理生化特征及抗病促生特性测试结果
Table 4 Test results of physiological and biochemical and antagonistic growth-promoting characteristics of XY-1, XY-13, and XY-53
试验项目Test project | 结果 Result | ||||
---|---|---|---|---|---|
XY-1 | XY-13 | XY-53 | |||
生理生化特征 Physiological and biochemical characteristics | 甲基红 Methyl red | - | - | - | |
接触酶 Catalase | + | + | + | ||
氧化酶Oxidase | + | + | + | ||
淀粉水解 Amylum hydrolysis | + | + | - | ||
明胶液化Gelatin experiment | + | + | + | ||
乙酰甲基甲醇(V-P)Voges-Proskauer | + | + | + | ||
葡萄糖氧化发酵Oxidation-fermentation of glucose | + | + | + | ||
抗病促生特性 Antagonistic growth-promoting characteristics | 溶磷能力 Ability of releasing phosphorus | + | + | + | |
固氮能力 Nitrogen-fixing ability | + | + | + | ||
ACC脱氨酶活性 ACC deaminase activity | + | + | + | ||
解钾能力Ability of releasing potassium | - | - | - | ||
铁载体 Siderophore | - | - | - | ||
蛋白酶 Protease | + | + | + | ||
几丁质酶 Chitinase | - | - | - | ||
果胶酶 Pectinase | - | - | - | ||
纤维素酶 Cellulase | - | - | - |
图2 菌株XY-1、XY-13及XY-53的系统发育树和特异性引物PCR电泳图 A:基于16S rDNA 基因序列构建XY-1、XY-13及XY-53菌株系统发育树;B:XY-1、XY-13及XY-53菌株的ropA、gyrA基因序列的凝胶电泳;C:基于gyrA基因序列构建XY-1菌株系统发育树;D:基于ropA基因序列构建XY-13、XY-53菌株系统发育树
Fig. 2 Phylogenetic tree of strain XY-1, XY-13 and XY-53 and PCR electropherogram with specific primer A: Construction of phylogenetic tree of XY-1, XY-13 and XY-53 strains based on 16S rDNA gene sequences; B: gel electrophoresis of ropA and gyrA gene sequences of XY-1, XY-13 and XY-53 strains; C: construction of phylogenetic tree of XY-1 strain based on gyrA gene sequence; D: construction of phylogenetic tree of XY-13 and XY-53 strains based on ropA gene sequence
处理 Treatment | 株高 Stem length/cm | 茎粗 Stem diameter/mm | 叶绿素含量Chlorophyll content | 鲜重Fresh weight/g | 干重Dry weight/g | ||||
---|---|---|---|---|---|---|---|---|---|
地上部 Above ground | 地下部 Below ground | 地上部 Above ground | 地下部 Below ground | ||||||
CK1 | 5.38±0.22b | 4.07±0.09b | 37.3±1.33c | 2.007±0.060c | 0.664±0.028a | 0.154±0.014b | 0.061±0.002 7b | ||
XY-1 | 7.38±0.23a | 4.34±0.07a | 49.00±1.11b | 2.670±0.114b | 0.767±0.026a | 0.198±0.012ab | 0.069±0.002 7b | ||
XY-13 | 6.84±0.17a | 4.26±0.08ab | 52.4±1.33ab | 2.786±0.048b | 0.782±0.049a | 0.199±0.018ab | 0.075±0.006 2ab | ||
XY-53 | 7.26±0.14a | 4.39±0.07a | 53.5±1.53a | 3.041±0.026a | 0.779±0.080a | 0.225±0.021a | 0.084±0.006 5a |
表5 菌株XY-1、XY-13、XY-53对黄瓜幼苗生长的影响
Table 5 Effects of strain XY-1, XY-13 and XY-53 on the growth of cucumber seedlings
处理 Treatment | 株高 Stem length/cm | 茎粗 Stem diameter/mm | 叶绿素含量Chlorophyll content | 鲜重Fresh weight/g | 干重Dry weight/g | ||||
---|---|---|---|---|---|---|---|---|---|
地上部 Above ground | 地下部 Below ground | 地上部 Above ground | 地下部 Below ground | ||||||
CK1 | 5.38±0.22b | 4.07±0.09b | 37.3±1.33c | 2.007±0.060c | 0.664±0.028a | 0.154±0.014b | 0.061±0.002 7b | ||
XY-1 | 7.38±0.23a | 4.34±0.07a | 49.00±1.11b | 2.670±0.114b | 0.767±0.026a | 0.198±0.012ab | 0.069±0.002 7b | ||
XY-13 | 6.84±0.17a | 4.26±0.08ab | 52.4±1.33ab | 2.786±0.048b | 0.782±0.049a | 0.199±0.018ab | 0.075±0.006 2ab | ||
XY-53 | 7.26±0.14a | 4.39±0.07a | 53.5±1.53a | 3.041±0.026a | 0.779±0.080a | 0.225±0.021a | 0.084±0.006 5a |
处理 Treatment | XY-1 | XY-13 | XY-53 | ||||||
---|---|---|---|---|---|---|---|---|---|
病情指数 Disease index | 防病效果 Control efficacy/% | 病情指数 Disease index | 防病效果 Control efficieny/% | 病情指数 Disease index | 防病效果 Control efficieny/% | ||||
CK2 | 95.96±1.36a | - | 95.96±1.36a | - | 95.96±1.36a | - | |||
T1 | 33.33±2.55c | 65.12±2.65a | 26.67±0.96c | 72.09±1.00a | 13.33±2.55d | 82.86±2.66a | |||
T2 | 43.33±3.33c | 54.66±3.47a | 70.00±5.36b | 26.75±5.58b | 56.67±2.89c | 40.70±3.01b | |||
T3 | 60.00±4.41b | 37.21±4.59b | 66.67±2.89b | 30.23±3.01b | 83.33±3.47b | 12.80±3.62c |
表6 菌株XY-1、XY-13、XY-53对黄瓜根腐病的防治效果
Table 6 Control efficiency of strain XY-1, XY-13, and XY-53 on cucumber root rot
处理 Treatment | XY-1 | XY-13 | XY-53 | ||||||
---|---|---|---|---|---|---|---|---|---|
病情指数 Disease index | 防病效果 Control efficacy/% | 病情指数 Disease index | 防病效果 Control efficieny/% | 病情指数 Disease index | 防病效果 Control efficieny/% | ||||
CK2 | 95.96±1.36a | - | 95.96±1.36a | - | 95.96±1.36a | - | |||
T1 | 33.33±2.55c | 65.12±2.65a | 26.67±0.96c | 72.09±1.00a | 13.33±2.55d | 82.86±2.66a | |||
T2 | 43.33±3.33c | 54.66±3.47a | 70.00±5.36b | 26.75±5.58b | 56.67±2.89c | 40.70±3.01b | |||
T3 | 60.00±4.41b | 37.21±4.59b | 66.67±2.89b | 30.23±3.01b | 83.33±3.47b | 12.80±3.62c |
处理 Treatment | 株高 Stem length/cm | 茎粗 Stem diameter/mm | 叶绿素含量 Chlorophyll content | 鲜重 Fresh weight/g | 干重 Dry weight/g | ||||
---|---|---|---|---|---|---|---|---|---|
地上部 Above ground | 地下部 Below ground | 地上部 Above ground | 地下部 Below ground | ||||||
CK2 | 3.85±0.13b | 3.42±0.11b | 33.80±1.15b | 1.68±0.09b | 0.66±0.05a | 0.148±0.01a | 0.024±0.003b | ||
T1 | 5.37±0.23a | 4.01±0.16a | 40.57±1.67a | 1.93±0.06ab | 0.89±0.16a | 0.177±0.01a | 0.026±0.002b | ||
T2 | 5.58±0.26a | 3.81±0.22ab | 41.68±1.18a | 1.91±0.06ab | 0.66±0.09a | 0.155±0.01a | 0.079±0.032a | ||
T3 | 5.77±0.28a | 3.94±0.13a | 39.52±1.76a | 1.98±0.12a | 0.68±0.11a | 0.156±0.02a | 0.028±0.002b |
表7 菌株XY-1、XY-13和XY-53对黄瓜的促生效果
Table 7 Growth-promoting effects of strain XY-1, XY-13 and XY-53 on cucumber
处理 Treatment | 株高 Stem length/cm | 茎粗 Stem diameter/mm | 叶绿素含量 Chlorophyll content | 鲜重 Fresh weight/g | 干重 Dry weight/g | ||||
---|---|---|---|---|---|---|---|---|---|
地上部 Above ground | 地下部 Below ground | 地上部 Above ground | 地下部 Below ground | ||||||
CK2 | 3.85±0.13b | 3.42±0.11b | 33.80±1.15b | 1.68±0.09b | 0.66±0.05a | 0.148±0.01a | 0.024±0.003b | ||
T1 | 5.37±0.23a | 4.01±0.16a | 40.57±1.67a | 1.93±0.06ab | 0.89±0.16a | 0.177±0.01a | 0.026±0.002b | ||
T2 | 5.58±0.26a | 3.81±0.22ab | 41.68±1.18a | 1.91±0.06ab | 0.66±0.09a | 0.155±0.01a | 0.079±0.032a | ||
T3 | 5.77±0.28a | 3.94±0.13a | 39.52±1.76a | 1.98±0.12a | 0.68±0.11a | 0.156±0.02a | 0.028±0.002b |
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