Isolation, Identification and Growth-promoting Characteristics of Endohyphal Bacterium 7-2H from Endophytic Fungi of Spiranthes sinensis

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

Abstract

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

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.

Figures/Tables 13

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

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

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

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	-

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

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

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

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

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

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）

Fig. 9 Genome circle of strain 7-2H

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

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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