黄瓜枯萎病菌共生细菌的分离鉴定及功能分析

doi:10.16420/j.issn.0513-353x.2023-0001

摘要/Abstract

摘要：

以黄瓜枯萎病菌（Fusarium oxysporum f. sp. cucumerium）为研究对象，利用荧光核酸染色技术和电子显微镜确定了菌丝外部及内部存在共生细菌；用平板培养法成功分离出共生细菌，并通过16S rDNA测序对菌株进行鉴定，确定分离菌株属于无色杆菌属（Achromobacter spp.）；碳源利用分析表明，与无共生细菌相比，存在共生细菌的黄瓜枯萎病菌的Shannon指数和反Simpson指数分别显著增加了2.18%和1.67%，且对糖类、羧酸类、胺类、聚合物和其他碳源的利用能力较强，但对氨基酸类物质的利用能力较弱。研究结果明确了黄瓜枯萎病菌存在无色杆菌属的共生细菌，并且共生细菌会影响宿主真菌的生长和代谢能力。

关键词: 黄瓜, 黄瓜枯萎病菌, 共生细菌, 分离鉴定, 碳源利用

Abstract:

In this experiment，Fusarium oxysporum f. sp. cucumerium was used as the research object. The presence of symbiotic bacteria outside and inside the hyphae of F. oxysporum f. sp. cucumerium was determined by fluorescent nucleic acid staining and electron microscopy. The symbiotic bacteria were successfully isolated by plate culture method and dentified by 16S rDNA sequencing. It was determined that it belonged to Achromobacter spp. The analysis of carbon source utilization showed that compared with F. oxysporum f. sp. cucumerium without symbiotic bacteria，the Shannon index and anti-Simpson index of F. oxysporum f. sp. cucumerium with symbiotic bacteria were significantly increased by 2.18% and 1.67% respectively，and the utilization ability of sugars，carboxylic acids，amines，polymers and other carbon sources was strong，while the utilization ability of amino acids was weak. The results showed that there was symbiotic bacteria of Achromobacter spp. in F. oxysporum f. sp. cucumerium，and it may affect the growth and metabolic capacity of the host fungus.

Key words: cucumber, Fusarium oxysporum f. sp. cucumerium, symbiotic bacteria, isolation and identification, carbon source utilization

刘梦, 贾惠婷, 周新刚. 黄瓜枯萎病菌共生细菌的分离鉴定及功能分析[J]. 园艺学报, 2024, 51(3): 643-655.

LIU Meng, JIA Huiting, ZHOU Xingang. Isolation，Identification and Functional Analysis of Symbiotic Bacteria of Fusarium oxysporum f. sp. cucumerium[J]. Acta Horticulturae Sinica, 2024, 51(3): 643-655.

图/表 10

图1 黄瓜枯萎病菌共生细菌存在/不存在的菌株（A、C、E/B、D、F）的显微观察 A、B：菌丝外部形态；C、D：菌丝的LIVE/DEAD细菌活性试剂盒染色；E、F：菌丝内部形态。红色箭头指外共生菌；绿色、黄色箭头指内共生菌。H代表菌丝。

Fig. 1 Microscopic observation of strains with or without symbiotic bacteria of Fusarium oxysporum f. sp. cucumerium（A，C，E/B，D，F） A，B：Scanning electron microscope observation of the external morphology of fungal hyphae；C，D：Fungal hyphae stained with LIVE/DEAD bacterial activity kit，observation by laser confocal microscope；E，F：transmission electron microscope observation of the internal morphology of fungal hyphae. Green and yellow arrows indicate viable endosymbiont bacterium，and the red arrow indicates ectosymbiont bacterium. H represents for hyphal.

图2 黄瓜枯萎病菌共生细菌存在/不存在的菌株（A/B）的16S rDNA片段及阴性对照（no DNA）扩增结果

Fig. 2 Amplification results of 16S rDNA fragment of Fusarium oxysporum f. sp. cucumerium strains with or without symbiotic bacteria（A/B）and negative control（no DNA）

表1 黄瓜枯萎病菌内共生菌16S rDNA片段克隆文库序列分析

Table 1 Sequence analysis of 16S rDNA clone library of Fusarium oxysporum f. sp. cucumerium symbiotic bacteria

编号 No．	对应克隆子数 The number of clones	NCBI数据库中最相似菌种（登录号） The strains which have the highest identity from NCBI（Accession No. ）	相似度/% Similarity	系统分类 Phylogenetic classification
OTU1	20	耐氧梭菌Clostridium aerotolerans DSM 5434（T）（X76163）	99.72	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU2	17	解木聚糖梭菌Clostridium xylanolyticum ATCC 49623（T）（X71855）	99.66	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU3	9	耐氧梭菌Clostridium aerotolerans DSM 5434（T）（X76163）	99.65	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU4	1	约氏不动杆菌Acinetobacter johnsonii CIP 64.6（T）（APON01000005）	98.42	变形菌门Proteobacteria，γ‐变形菌纲Gammaproteobacteria假单胞菌目Pseudomonadales，莫拉氏菌科Moraxellaceae，不动杆菌属Acinetobacter
OTU5	1	解木聚糖梭菌Clostridium xylanolyticum ATCC 49623（T）（X71855）	99.24	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU6	1	耐氧梭菌Clostridium aerotolerans DSM 5434（T）（X76163）	99.30	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU7	1	马德普拉塔无色杆菌 Achromobacter marplatensis B2（T）（EU150134）	99.78	变形菌门Proteobacteria，β‐变形菌纲Betaproteobacteria，伯克氏菌目Burkholderiales，产碱菌科Alcaligenaceae，无色杆菌属Achromobacter

表1 黄瓜枯萎病菌内共生菌16S rDNA片段克隆文库序列分析

Table 1 Sequence analysis of 16S rDNA clone library of Fusarium oxysporum f. sp. cucumerium symbiotic bacteria

编号 No．	对应克隆子数 The number of clones	NCBI数据库中最相似菌种（登录号） The strains which have the highest identity from NCBI（Accession No. ）	相似度/% Similarity	系统分类 Phylogenetic classification
OTU1	20	耐氧梭菌Clostridium aerotolerans DSM 5434（T）（X76163）	99.72	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU2	17	解木聚糖梭菌Clostridium xylanolyticum ATCC 49623（T）（X71855）	99.66	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU3	9	耐氧梭菌Clostridium aerotolerans DSM 5434（T）（X76163）	99.65	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU4	1	约氏不动杆菌Acinetobacter johnsonii CIP 64.6（T）（APON01000005）	98.42	变形菌门Proteobacteria，γ‐变形菌纲Gammaproteobacteria假单胞菌目Pseudomonadales，莫拉氏菌科Moraxellaceae，不动杆菌属Acinetobacter
OTU5	1	解木聚糖梭菌Clostridium xylanolyticum ATCC 49623（T）（X71855）	99.24	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU6	1	耐氧梭菌Clostridium aerotolerans DSM 5434（T）（X76163）	99.30	厚壁菌门Firmicutes，梭菌纲Clostridia，梭菌目Clostridiales，梭菌科Clostridiaceae，梭菌属Clostridium
OTU7	1	马德普拉塔无色杆菌 Achromobacter marplatensis B2（T）（EU150134）	99.78	变形菌门Proteobacteria，β‐变形菌纲Betaproteobacteria，伯克氏菌目Burkholderiales，产碱菌科Alcaligenaceae，无色杆菌属Achromobacter

图3 黄瓜枯萎病菌可培养共生细菌A1的菌株形态

Fig. 3 Colony morphology of culturable symbiotic bacteria A1 of Fusarium oxysporum f. sp. cucumerium

图4 黄瓜枯萎病菌可培养共生细菌A1的同源性比对

Fig. 4 Homology comparison of culturable symbiotic bacteria A1 of Fusarium oxysporum f. sp. cucumerium

图5 黄瓜枯萎病菌可培养共生细菌A1的16S rDNA片段与克隆文库OTU序列的系统发育树

Fig. 5 Phylogenetic tree of 16S rDNA fragment and clone library OTU sequence of culturable symbiotic bacteria A1 of Fusarium oxysporum f. sp. cucumerium

图6 黄瓜枯萎病菌共生细菌存在/不存在（FOC/FOC−）对其菌丝生长、产孢和干质量的影响

Fig. 6 Effect of Fusarium oxysporum f. sp. cucumerium with or without symbiotic bacteria（FOC/FOC−）on fungal growth，sporulation and dry weight

表2 黄瓜枯萎病菌共生细菌存在/不存在的菌株（FOC/FOC−）的碳源利用和代谢多样性指数

Table 2 Carbon source utilization and metabolic diversity index of FOC/FOC−

菌株 Strain	碳源利用 Carbon source utilization	Shannon指数 H	反Simpson指数 D
FOC	0.983 ± 0.0595 a	4.304 ± 0.0100 a	0.488 ± 0.0014 a
FOC⁻	0.901 ± 0.0495 b	4.212 ± 0.0122 b	0.480 ± 0.0016 b

图7 黄瓜枯萎病菌共生细菌存在/不存在的菌株（FOC/FOC−）的碳源相对利用率（A）和碳源利用强度（B）

Fig. 7 Relative utilization（A）and intensity of carbon source utilization（B）of FOC/FOC−

图8 黄瓜枯萎病菌共生细菌存在/不存在的菌株（FOC/FOC−）的碳源利用多样性的主成分分析

Fig. 8 Principal component analysis of carbon source utilization diversity of FOC/FOC−

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