Isolation and identification of Burkholderia contaminans strain GD1-1 and its bio-control potential against Fusarium wilt of banana
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摘要:目的
从香蕉根际土壤中筛选出对香蕉枯萎病具有良好防治效果的生防菌。
方法采用对峙培养法，从健康香蕉根际土壤中筛选对尖孢镰刀菌古巴专化型热带4号小种(Fusarium oxysporum f. sp. cubense tropical race 4，Foc TR4)具有拮抗作用的生防菌株，根据菌落形态和生理生化特征并利用分子生物学技术对生防菌株进行鉴定。通过显微观察分析该菌株对Foc TR4菌丝生长和分生孢子萌发的影响，通过对峙培养法分析其抑菌谱，通过盆栽试验分析该菌株对香蕉枯萎病的防治效果和促生潜力。
结果筛选出了1株对Foc TR4具有显著抑制作用的拮抗菌GD1−1，鉴定为洋葱伯克霍尔德菌Burkholderia contaminans。菌株GD1−1对Foc TR4菌丝生长的抑制率为72.5%，对分生孢子萌发的抑制率为99.8%，并可以导致Foc TR4菌丝膨大变粗、畸形等。菌株GD1−1具有解钾、解磷、产蛋白酶和产铁载体的能力，对10种植物病原真菌均具有较好的抑制效果，表现出广谱抑菌作用。盆栽试验结果表明，菌株GD1−1对香蕉枯萎病的防效为55.6%，且对香蕉植株具有良好的促生作用。
结论洋葱伯克霍尔德菌GD1−1对Foc TR4具有明显的抑制作用，对香蕉枯萎病具有良好的防治效果，对香蕉植株具有促生作用。作为优质的生防菌源，菌株GD1−1具有一定的开发应用潜力。
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关键词:
- 香蕉枯萎病 /
- 尖孢镰刀菌古巴专化型 /
- 洋葱伯克霍尔德菌 /
- 拮抗作用 /
- 生物防治
Abstract:ObjectiveTo isolate and screen antagonistic bacteria against Fusarium wilt disease from healthy banana rhizosphere soil.
MethodsThe bacterial strain antagonizing Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) was screened from banana rhizosphere soil using the plate confrontational culture method. The antagonistic strain was identified by colony morphology, physiological and biochemical characteristics, and phylogenetic analysis of multiple genes including 16S rDNA, GyrB, AtpD and GltB. The effect of this strain on Foc TR4 hyphae growth and spore germination was analyzed by microscopic observation, the antifungi spectrum was determined by the plate confrontation culture method, the biological control and growth-promoting efficiency were tested by banana plant inoculation assay in greenhouse.
ResultAn antagonistic strain of GD1-1 was screened with the inhibition rate of 72.5% on Foc TR4 hyphal growth and the inhibition rate of 99.8% on Foc TR4 spore germination, which was identified as Burkholderia contaminans. The strain GD1-1 could cause hyphal distortion, enlargement and malformation of Foc TR4 under microscope observation. The strain GD1-1 possessed the broad-spectrum antibacterial activity with a good inhibitory effect on the selected ten phytopathogenic fungi. The results of pot inoculation experiment showed that the strain GD1-1 successfully promoted the growth of banana seedlings and suppressed the incidence of banana Fusarium wilt with the biocontrol efficacy of 55.6%.
ConclusionThe strain GD1-1 has the significant inhibitory effect on Foc TR4 with a good control effect on banana wilt disease, and promotes the growth of banana plant. As a high-quality source of biocontrol bacteria, the strain GD1-1 has certain potential for development and application.
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图 1 菌株GD1−1对Foc TR4的抑制作用
Figure 1. The antifungal activity of strain GD1-1 against Foc TR4
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图 2 拮抗菌D1−1的菌落形态
A: NA培养基；B和C: 扫描电镜。
Figure 2. Colony morphology of antagonistic strain GD1−1
A: NA medium; B and C: Scanning electron micrograph.
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图 3 基于16S rDNA、GyrB、AtpD和GltB基因序列构建的菌株GD1−1系统发育树
Figure 3. Phylogentic tree of strain GD1-1 based on concatenated sequences of 16S rDNA, GyrB, AtpD and GltB genes
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图 4 菌株GD1−1在NA培养基中的生长曲线
Figure 4. Growth curve of the strain GD1-1 in NA culture medium
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图 5 菌株GD1−1抑制Foc TR4菌丝生长
A：Foc TR4正常菌丝形态；B：菌株GD1−1导致Foc TR4菌丝畸形。
Figure 5. Inhibition of hyphal growth of Foc TR4 by the strain GD1-1
A: Normal mycelia of Foc TR4; B: Deformed mycelia of Foc TR4 treated with strain GD1-1.
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图 6 菌株GD1−1抑制Foc TR4分生孢子萌发
柱子上不同小写字母表示处理间的差异显著(P<0.05，Duncan’s 法)。
Figure 6. Inhibition of conidial germination of Foc TR4 by the strain GD1-1
Different lowercase letters on the pillar indicated significant difference between treatments (P<0.05, Duncan’s method).
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图 7 菌株GD1−1对不同植物病原真菌的平板对峙
Figure 7. Antagonistic effects of strain GD1-1 on different phytopathogenic fungi
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图 9 香蕉枯萎病病情指数
柱子上不同小写字母表示差异显著(P<0.05，Duncan’s 法)。
Figure 9. Disease index of banana Fusarium wilt
Different lowercase letters on the pillar indicated significant difference (P<0.05, Duncan’s method).
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图 11 香蕉苗鲜质量(A)和球茎直径(B)
柱子上不同小写字母表示差异显著(P<0.05，Duncan’s 法)。
Figure 11. Seedling fresh weight (A) and corm diameter (B) of banana
Different lowercase letters on the pillar indicated significant difference (P<0.05, Duncan’s method).
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图 12 菌株GD1−1的促生潜力
A：NKA培养基；B：NPA培养基；C：牛奶培养基；D：苯胺蓝-PDA培养基；E：CAS培养基。
Figure 12. The growth promoting potential of strain GD1-1
A: NKA medium; B: NPA medium; C: Milk medium; D: Aniline blue-PDA medium; E: CAS medium.
表 1 本文所用引物
Table 1 Primers used in this study
引物名称
Primer
name引物序列(5′→3′)
Primer sequence (5′→3′)参考
文献
Reference27-F AGAGTTTGATCCTGGCTCAG [12] 1492 -RGGTTACCTTGTTACGACTT GyrB-F ACCGGTCTGCAYCACCTCGT [12] GyrB-R YTCGTTGWARCTGTCGTTCCACTGC AtpD-F ATGAGTACTRCTGCTTTGGTAGAAGG [12] AtpD-R CGTGAAACGGTAGATGTTGTCG GltB-F CTGCATCATGATGCGCAAGTG [13] GltB-R CTTGCCGCGGAARTCGTTGG 
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表 2 菌株GD1−1对不同植物病原真菌的抑制作用
Table 2 Inhibition of different phytopathogenic fungi by the strain GD1-1
病原菌
Pathogen抑制率1)/%
Inhibition rate病原菌
Pathogen抑制率1)/%
Inhibition rate尖孢镰刀菌番茄专化型 Fol 78.0±0.3a 香蕉枯萎病菌1号小种 Foc1 72.0±0.5bc 香蕉炭疽病菌 C. musae 77.9±0.3a 尖孢镰刀菌苦瓜专化型 Fom 71.3±0.3bc 链格孢菌 A. alternata 76.8±1.1a 变红镰刀菌 F. incarnatum 69.9±0.5cd 腐皮镰刀菌 F. solani 76.4±0.9a 多主棒孢菌 C. cassiicola 67.9±0.2de 尖孢镰刀菌冬瓜专化型 Fob 73.5±0.4b 禾谷镰刀菌 F. graminearum 66.1±2.6e 1)表中数据为平均值±标准误，不同小写字母表示差异显著(P<0.05，Duncan’s 法)。
1) Values were the means ± SE and different lowercase letters indicated significant difference (P<0.05, Duncan’s method).
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