盘龙参内生真菌胞内细菌7-2H的分离鉴定和促生特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0038

摘要/Abstract

摘要：

真菌内生细菌是一类特殊细菌，在寄主真菌体内及生态系统中发挥重要作用。为发掘盘龙参内生真菌内具有促生功能的内生细菌资源，并探索其生物学功能，采用荧光原位杂交（fluorescence in situ hybridization, FISH）检测高粱附球菌Epicoccum sorghinum菌丝中内生细菌的存在后，利用菌丝组织研磨法分离内生细菌，通过菌落形态、生理生化特性及分子特征对其进行鉴定。运用特异性引物验证细菌的内生性，采用Salkowski比色法、CAS比色法以及钼锑抗比色法对菌株的促生特性进行测定，通过水稻种子促生长实验初步验证内生细菌的促生长能力，并对内生细菌进行全基因组测序和促生相关功能基因分析。真菌E. sorghinum菌丝内能观察到与荧光素标记的单链核酸探针杂交后的细菌，证实其菌丝内含有细菌，从菌丝内分离出一株内生栖稻根瘤菌Rhizobium oryzihabitans 7-2H，具有产吲哚乙酸（IAA）、铁载体和溶磷能力，可显著提高水稻幼苗的茎长、根长、鲜重和干重。菌株7-2H与标准菌株R. oryzihabitans M15基因组之间的平均核酸一致性（average nucleotide identity, ANI）值为96.98%，通过对基因组分析发现，菌株7-2H含有与产IAA、铁载体及溶磷能力相关的基因。分离得到一株真菌内生栖稻根瘤菌，该菌具有良好的促进植物生长特性，可作为后续研发微生物菌肥的菌种资源。

关键词: 盘龙参, 内生真菌, 高粱附球菌, 真菌内生细菌, 栖稻根瘤菌, 促生特性, 全基因组测序

Abstract:

Endohyphal bacteria are a special class of bacteria that play an essential role in host fungi and ecosystems. To explore the endohyphal bacteria resources with growth-promoting functions in endophytic fungi of Spiranthes sinensis and explore their biological functions，the endohyphal bacteria were isolated by mycelial tissue grinding and identified by colony morphology, physiological and biochemical properties and molecular characteristics, after detecting the presence of endohyphal bacteria in the mycelium of Epicoccum sorghium by fluorescence in situ hybridization（FISH）. Then the endogeneity of the bacteria was verified by specific primers, and the growth-promoting characteristics were determined by Salkowski colorimetry, CAS colorimetry and Mo-Sb colorimetry. The growth-promoting ability of endohyphal bacteria was preliminarily verified by rice seed growth-promoting experiments, and the whole genome sequencing and growth-promoting functional gene analysis of endohyphal bacteria were performed. The presence of bacteria within the mycelium of the fungus E. sorghinum was observed after hybridization with the fluorescein-labeled single-stranded nucleic acid probe, which might confirm the mycelium contains bacteria. An endohyphal Rhizobium oryzihabitans was isolated from the mycelium and named as 7-2H, which had the ability of producing indole-3-acetic（IAA）and siderophore as well as solublizing phosphate, and significantly increased the shoot length, root length, fresh weight and dry weight of rice seedlings. The average nucleotide identity（ANI）value between the genome of strain 7-2H and the standard strain Rhizobium oryzihabitans M15 was 96.98%. In the genome of strain 7-2H, genes related to IAA production, siderophore and phosphate solubilization were revealed. An endohyphal Rhizobium oryzihabitans is isolated, which has fine plant growth-promoting property and has a potential to be developed to be microbial fertilizer.

Key words: Spiranthes sinensis, endophytic fungus, Epicoccum sorghinum, endohyphal bacteria, Rhizobium oryzihabitans, growth-promoting characteristics, whole genome sequencing

方澜, 黎妍妍, 江健伟, 成胜, 孙正祥, 周燚. 盘龙参内生真菌胞内细菌7-2H的分离鉴定和促生特性研究[J]. 生物技术通报, 2023, 39(8): 272-282.

FANG Lan, LI Yan-yan, JIANG Jian-wei, CHENG Sheng, SUN Zheng-xiang, ZHOU Yi. Isolation, Identification and Growth-promoting Characteristics of Endohyphal Bacterium 7-2H from Endophytic Fungi of Spiranthes sinensis[J]. Biotechnology Bulletin, 2023, 39(8): 272-282.

图/表 13

表1 PCR扩增程序

Table 1 PCR amplification procedure

检测基因 Detected gene	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	预变性 Initial denaturation	变性Denaturation	退火Annealing	延伸Extension	最终延伸 Final extension
检测基因 Detected gene	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	预变性 Initial denaturation	30 cycles			最终延伸 Final extension
16S rDNA	27F	AGAGTTTGATCCTGGCTCAG	94℃, 2 min	94℃, 30 s	55℃, 30 s	72℃, 90 s	72℃, 5 min
16S rDNA	1492R	TACGGCTACCTTGTTACGACTT			55℃, 30 s	72℃, 90 s
ChvE	ChvE-F101	GCTGGTCTTCCTTGTAGTAA			56℃, 30 s	72℃, 60 s
ChvE	ChvE-R653	AACGCCTTCTTCTTCTATGA			56℃, 30 s	72℃, 60 s

图 1 荧光原位杂交（FISH）检测E. sorghinum菌丝中的细菌

Fig. 1 Detection of bacteria in the mycelia of E. sorghinum by fluorescence in situ hybridization

图 2 菌株7-2H的菌落形态特征 A：菌落形态图；B：扫描电镜图；C：革兰氏染色图

Fig. 2 Colony morphological characteristics of strain 7-2H A: Colony morphology. B: Scanning electron microscopic morphology. C: Gram staining

表2 7-2H 的各项生理生化测定

Table 2 Physiological and biochemical determination of 7-2H

试验名称 Test name	结果 Result	试验名称 Test name	结果 Result
淀粉水解Starch hydrolysis	-	葡萄糖 Glucose	+
纤维素降解 Cellulose degradation	-	乳糖 Lactose	+
蛋白降解 Protein degradation	-	甘露醇 Mannitol	+
几丁质降解 Chitin degradation	-	接触酶 Catalase	+
硝酸盐还原 Nitrate reduction	+	甲基红 Methyl red	-

图3 基于16S rDNA序列构建的菌株7-2H系统进化树

Fig. 3 Phylogenetic tree of strain 7-2H based on 16S rDNA gene sequence

图4 菌株7-2H基于特异性引物的内生验证 M：2 000 bp maker；1：真菌内生细菌7-2H；2：真菌E. sorghinum

Fig. 4 Endogenous verification of strain 7-2H based on specific primers M: 2 000 bp maker. 1: Endohyphal bacteria 7-2H. 2: E. sorghinum

图5 菌株7-2H的产IAA能力测定 A：菌株7-2H产IAA能力的定性检测；B：菌株7-2H在不同培养时间下IAA含量

Fig. 5 Determination of IAA-producing ability of strain 7-2H A: Qualitative detection of IAA-producing ability of strain 7-2H. B: IAA contents by strain 7-2H at different culture time

图6 菌株7-2H的产铁载体能力测定 A：菌株7-2H在CAS检测平板上生长5 d的效果；B：菌株7-2H在不同培养时间下铁载体相对含量

Fig. 6 Determination of siderophore-producing ability of strain 7-2H A: Effect of strain 7-2H growing on CAS detection plate for 5 d. B: Siderophores relative contents by strain 7-2H at different culture time

图7 菌株7-2H的溶磷能力测定 A：菌株7-2H在NBRIP无机磷平板上生长3 d的效果；B：菌株7-2H在不同培养时间下可溶性磷含量

Fig. 7 Determination of phosphate-solubilizing ability of strain 7-2H A: Effect of strain 7-2H on NBRIP inorganic phosphorus plate for 3 d. B: Soluble phosphorus contents by strain 7-2H at different culture time

图8 菌株7-2H对水稻种子幼苗生长指标的影响 A：茎长；B：根长；C：鲜重；D：干重；E：生长情况。****：极显著性差异（P<0.01）；*：显著性差异（P<0.05）

Fig. 8 Effects of strain 7-2H on the growth indexes of rice seed seedlings A: Shoot length. B: Root length. C: Fresh weight. D: Dry weight. E: Growth situation. ****: Extremely significant difference（P<0.01）; *: significant difference（P<0.05）

图9 菌株7-2H的基因组圈图

Fig. 9 Genome circle of strain 7-2H

图10 菌株7-2H基因组GO功能注释

Fig. 10 GO functional annotation on the genome of strain 7-2H

表3 菌株7-2H基因功能注释数据库分布情况

Table 3 Database distribution of gene functional annotation from the strain 7-2H

数据库 Database	基因数量 Gene number	基因注释比例 Gene annotation ratio/%
Uniprot	3 081	58.12
Pfam	4 501	84.91
Refseq	5 087	95.96
Nr	3 014	56.86
Tigrfam	5 127	96.72
GO	2 961	55.86
KEGG	2 663	50.24
COG	2 107	39.75

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