地黄根腐病拮抗类芽胞杆菌的筛选、鉴定和全基因组分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0491

摘要/Abstract

摘要：

目的分离筛选抗地黄根腐病的生防菌株，为地黄根腐病的生物防治提供菌株资源。方法从感染轮纹病的地黄根际土中分离纯化微生物，采用平板对峙法筛选拮抗菌株，并通过盆栽试验进一步明确菌株对地黄根腐病的防治效果；结合形态观察、生理生化及分子生物学等方法确定菌株的分类地位；并对该菌株开展全基因组测序，分析基因组信息，探究其抗病机制。结果根据平板对峙结果，筛选到一株对植物病原真菌有广谱拮抗活性的菌株QH-1，其对地黄根腐病原菌的抑菌率接近90%。经鉴定，QH-1为一株多粘类芽胞杆菌（Paenibacillus polymyxa），为革兰氏阳性菌，产花生状芽孢。其在10-55 ℃，pH 6-8，NaCl浓度为0-5%的条件下均能生长。碳氮源利用实验表明，其可以利用多种氮源，而仅能利用麦芽糖和山梨醇两种碳源。另外，该菌株能产生过氧化氢酶、淀粉酶、酯酶、明胶酶、凝乳酶和蛋白酶。QH-1菌株的基因组信息表明其由一条环状染色体（5 692 874 bp）和一个环状质粒（37 590 bp）组成，（G+C）%含量为45.4%；antiSMASH结果显示基因组中含有17个生物合成基因簇，其中6个生物合成基因簇与6种抗菌化合物（fusaricidin B、paenibacillin、paenilan、tridecaptin、polymyxin和paenicidin A）的生物合成基因簇的相似性为100%，说明菌株QH-1具有产生以上抗菌活性化合物的潜力。结论菌株QH-1是一株抗地黄根腐病的多粘类芽胞杆菌，其基因组信息显示其能产生多种抗菌物质，为进一步研发抗地黄根腐病的微生物菌剂和天然抗菌物质提供了良好的理论和物质基础。

关键词: 地黄根腐病, 多粘类芽胞杆菌, 微生物分离, 平板对峙, 菌种鉴定, 全基因组

Abstract:

Objective Strains resistant to Rehmannia glutinosa root rot were screened to provide a material basis and strain resources for the biological control of R. glutinosa root rot. Method The rhizosphere microorganism of ring rot-infected R. glutinosa were isolated, and strains with antagonistic activities against pathogenic fungi were screened by using plate confrontation assay. Besides, a pot experiment was carried out to explore the active bacteria in preventing and controlling the root rot disease of R. glutinosa. The strain was identified through colonial morphology observation, physiological and biochemical, and molecular biology analysis. The strain was also sequenced throughout its entire genome, its genomic information was analyzed, and its disease resistance mechanism was explored. Result Based on the plate confrontation results, a strain QH-1 with broad-spectrum antagonistic activity against plant pathogenic fungi was screened, showing an inhibition rate of nearly 90% against the pathogen of R. glutinosa root rot. Strain QH-1 was identified as Paenibacillus polymyxa, it was a Gram⁺ bacterium and produced peanut-like endospores that grew at 10-55 ℃, pH 6-8, the concentration of NaCl 0-5%. Carbon and nitrogen source utilization tests showed that it utilized multiple nitrogen sources but only two carbon sources that were maltose and sorbitol. Additionally, the strain produced catalase, amylase, lipoidase, gelatinase, and proteinase. The genome of this strain consisted of a circular chromosome and a circular plasmid, with a size of 5 692 874 bp and 37 590 bp, and a content of (G+C)% of 45.4%. Total 17 biosynthesis gene clusters were explored by using antiSMASH. Of these 17 biosynthesis gene clusters, 6 biosynthesis gene clusters had 100% similarities with known clusters including fusaricidin B, paenibacillin, paenilan, tridecaptin, polymyxin and paenicidin. Thus, strain QH-1 has the potential of producing these antibacterial compounds like mentioned above. Conclusion Strain QH-1 is a P. polymyxa strain resistant to R. glutinosa root rot. Its genomic information indicates the ability of producing multiple antimicrobial substances, providing a solid theoretical and material foundation for further development of microbial agents and natural antimicrobial compounds against R. glutinosa root rot.

Key words: root rot of Rehmannia glutinosa, Paenibacillus polymyxa, plate confrontation, microbe isolation, species identification, whole genome

赵逸凡, 王彤, 叶岚, 赵乐, 张宝, 杜鹏强, 何海荣. 地黄根腐病拮抗类芽胞杆菌的筛选、鉴定和全基因组分析[J]. 生物技术通报, 2025, 41(12): 328-341.

ZHAO Yi-fan, WANG Tong, YE Lan, ZHAO Le, ZHANG Bao, DU Peng-qiang, HE Hai-rong. Screening， Identification and Whole Genome Analysis of a Paenibacillus Strain Resistant to Root Rot of Rehmannia glutinosa[J]. Biotechnology Bulletin, 2025, 41(12): 328-341.

图/表 15

参考文献 24

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病原真菌 Pathogenic fungi	抑菌率 Fungal inhibition rate （%）
病原真菌 Pathogenic fungi	QH-1	QH-9	QH-16	QH-33	QH-39	QH-41
褐斑病菌 C. cassiicola	59.07±5.45^cd	45.73±0.84^b	40.13 1.75^e	35.7±2.65^d	49.63±3.65^c	NA
茎基腐病菌 F. graminearum Schw	88.84±3.49^b	NA	35.76±2.78^d	38.65±2.07^c	NA	47.55±3.98^b
炭疽病菌 C. orbiculare	51.44±3.50^d	65.51±3.03^a	NA	NA	49.78±4.97^d	40.20±3.69^c
枯萎病菌 F. oxysporum	15.47±5.11^e	NA	20.36±1.06^c	NA	NA	NA
叶斑病菌 F. solani	80.28±5.00^c	NA	NA	NA	NA	55.65±6.01^d
黄萎病菌 V. dahliae Kleb	51.80±3.78^d	NA	42.76±6.7^b	38.95±4.88^b	NA	NA
轮纹病菌 P. herbarum	89.27±5.25^c	34.77±4.32^c	NA	58.76±5.68^a	42.65±2.09^a	NA
根腐病菌 Fusarium sp. Rf7	89.57±2.97^a	NA	60.23±3.54^a	NA	75.23±6.35^a	65.30±5.07^a

病原真菌 Pathogenic fungi	抑菌率 Fungal inhibition rate （%）
病原真菌 Pathogenic fungi	QH-1	QH-9	QH-16	QH-33	QH-39	QH-41
褐斑病菌 C. cassiicola	59.07±5.45^cd	45.73±0.84^b	40.13 1.75^e	35.7±2.65^d	49.63±3.65^c	NA
茎基腐病菌 F. graminearum Schw	88.84±3.49^b	NA	35.76±2.78^d	38.65±2.07^c	NA	47.55±3.98^b
炭疽病菌 C. orbiculare	51.44±3.50^d	65.51±3.03^a	NA	NA	49.78±4.97^d	40.20±3.69^c
枯萎病菌 F. oxysporum	15.47±5.11^e	NA	20.36±1.06^c	NA	NA	NA
叶斑病菌 F. solani	80.28±5.00^c	NA	NA	NA	NA	55.65±6.01^d
黄萎病菌 V. dahliae Kleb	51.80±3.78^d	NA	42.76±6.7^b	38.95±4.88^b	NA	NA
轮纹病菌 P. herbarum	89.27±5.25^c	34.77±4.32^c	NA	58.76±5.68^a	42.65±2.09^a	NA
根腐病菌 Fusarium sp. Rf7	89.57±2.97^a	NA	60.23±3.54^a	NA	75.23±6.35^a	65.30±5.07^a

处理 Treatment	病情指数 Disease index	发病率 Incidence （%）	防病效果 Prevention effect （%）
CK（无菌水处理） Treatment with sterile water	10.12 ± 3.16^c	15.56 ± 4.71^c	46.82 ± 11.75
T1（Rf7处理） Treatment with pathogen Rf7	54.32 ± 4.45^a	74.07 ± 9.69^a
T2（Rf7+QH-1处理） Treatment with Rf7 and QH-1	28.64 ± 5.82^b	49.63 ± 5.88^b

处理 Treatment	病情指数 Disease index	发病率 Incidence （%）	防病效果 Prevention effect （%）
CK（无菌水处理） Treatment with sterile water	10.12 ± 3.16^c	15.56 ± 4.71^c	46.82 ± 11.75
T1（Rf7处理） Treatment with pathogen Rf7	54.32 ± 4.45^a	74.07 ± 9.69^a
T2（Rf7+QH-1处理） Treatment with Rf7 and QH-1	28.64 ± 5.82^b	49.63 ± 5.88^b

特征Characteristic	QH-1	P. polymyxa ATCC 842^T	P. ottowii MS2379^T	P. kribbensis AM49^T
pH	6-8	ND	6-10	5-8
温度 Temperature （℃）	10-55	ND	10-45	10-44
NaCl耐受 NaCl tolerance （%）	5	2	3	4
硝酸盐还原 Nitrate reduction	+	+	+	+
过氧化氢酶 Catalase	+	ND	+	-
淀粉酶 Amylase	+	+	+	+
吐温80 Tween 80	+	-	ND	+
牛奶胨化 Milk peptonization	+	+	+	+
明胶液化 Gelatin liquefaction	+	+	+	+
脲酶 Urease	-	-	-	-