Screening， Identification and Whole Genome Analysis of a Paenibacillus Strain Resistant to Root Rot of Rehmannia glutinosa

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

Abstract

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

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.

Figures/Tables 15

References 24

[25]	Liu HW, Li JY, Carvalhais LC, et al. Evidence for the plant recruitment of beneficial microbes to suppress soil-borne pathogens [J]. New Phytol, 2021, 229(5): 2873-2885.
[26]	Gao M, Xiong C, Gao C, et al. Disease-induced changes in plant microbiome assembly and functional adaptation [J]. Microbiome, 2021, 9(1): 187.
[27]	Yuan HB, Yuan MJ, Shi BK, et al. Biocontrol activity and action mechanism of Paenibacillus polymyxa strain Nl4 against pear Valsa canker caused by Valsa pyri [J]. Front Microbiol, 2022, 13: 950742.
[28]	Taheri E, Tarighi S, Taheri P. Characterization of root endophytic Paenibacillus polymyxa isolates with biocontrol activity against Xanthomonas translucens and Fusarium graminearum [J]. Biol Control, 2022, 174: 105031.
[29]	Weselowski B, Nathoo N, Eastman AW, et al. Isolation, identification and characterization of Paenibacillus polymyxa CR1 with potentials for biopesticide, biofertilization, biomass degradation and biofuel production [J]. BMC Microbiol, 2016, 16(1): 244.
[30]	Jung WJ, An KN, Jin YL, et al. Biological control of damping-off caused by Rhizoctonia solani using chitinase-producing Paenibacillus illinoisensis KJA-424 [J]. Soil Biol Biochem, 2003, 35(9): 1261-1264.
[31]	Zhai Y, Zhu JX, Tan TM, et al. Isolation and characterization of antagonistic Paenibacillus polymyxa HX-140 and its biocontrol potential against Fusarium wilt of cucumber seedlings [J]. BMC Microbiol, 2021, 21(1): 75.
[32]	Liu Y, Wang RH, Cao YH, et al. Identification and antagonistic activity of endophytic bacterial strain Paenibacillus sp. 5L8 isolated from the seeds of maize (Zea mays L., Jingke 968) [J]. Ann Microbiol, 2016, 66(2): 653-660.
[33]	Xu SJ, Kim BS. Evaluation of Paenibacillus polymyxa strain SC09-21 for biocontrol of Phytophthora blight and growth stimulation in pepper plants [J]. Trop Plant Pathol, 2016, 41(3): 162-168.
[1]	国家药典委员会. 中华人民共和国药典-一部2015年版 [M]. 北京: 中国医药科技出版社, 2015.
	National Pharmacopoeia Committee. People’s republic of China (PRC) pharmacopoeia-No.1 department [M]. Beijing: China Medical Science Press, 2015.
[2]	Bian ZY, Zhang R, Zhang X, et al. Extraction, structure and bioactivities of polysaccharides from Rehmannia glutinosa: a review [J]. J Ethnopharmacol, 2023, 305: 116132.
[3]	吴廷娟, 杜权, 王玉星, 等. 地黄根腐病病原菌的分离鉴定及其防治 [J]. 江苏农业科学, 2021, 49(22): 132-136.
	Wu TJ, Du Q, Wang YX, et al. Isolation, identification and control of root rot pathogen of Rehmannia glutinosa [J]. Jiangsu Agric Sci, 2021, 49(22): 132-136.
[4]	Lim JD, Yun SJ, Chung IM, et al. Resveratrol synthase transgene expression and accumulation of resveratrol glycoside in Rehmannia glutinosa [J]. Mol Breed, 2005, 16(3): 219-233.
[5]	Wu LK, Wang JY, Huang WM, et al. Plant-microbe rhizosphere interactions mediated by Rehmannia glutinosa root exudates under consecutive monoculture [J]. Sci Rep, 2015, 5: 15871.
[6]	Cao LL, Kang QY, Tian Y. Pesticide residues: Bridging the gap between environmental exposure and chronic disease through omics [J]. Ecotoxicol Environ Saf, 2024, 287: 117335.
[7]	Mei LC, Chen HM, Dong AY, et al. Pesticide informatics platform (PIP): an international platform for pesticide discovery, residue, and risk evaluation [J]. J Agric Food Chem, 2022, 70(22): 6617-6623.
[8]	Pan XY, Yue Y, Zhao FJ, et al. Rhizosphere microbes facilitate the break of chlamydospore dormancy and root colonization of rice false smut fungi [J]. Cell Host Microbe, 2025, 33(5): 731-744.e5.
[9]	Wang X, Xu Q, Hu K, et al. A coculture of Enterobacter and Comamonas species reduces cadmium accumulation in rice [J]. Mol Plant Microbe Interactions^® , 2023, 36(2): 95-108.
[10]	Wang LW, Zhang YB, Wang Y, et al. Inoculation with Penicillium citrinum aids ginseng in resisting Fusarium oxysporum by regulating the root and rhizosphere microbial communities [J]. Rhizosphere, 2022, 22: 100535.
[11]	Kamoun S, Furzer O, Jones JDG, et al. The Top 10 oomycete pathogens in molecular plant pathology [J]. Mol Plant Pathol, 2015, 16(4): 413-434.
[12]	李文均, 刘兰, 焦建宇, 等. 细菌古菌系统分类学原理及鉴定技术 [M]. 北京: 高等教育出版社, 2024.
	Li WJ, Liu L, Jiao JY, et al. Systematic taxonomy principle and identification technology of bacteria and archaea [M]. Beijing: Higher Education Press, 2024.
[13]	He HR, Huang JR, Zhao ZZ, et al. Whole genome analysis of Streptomyces sp. RerS4, a Rehmannia glutinosa rhizosphere microbe producing a new lipopeptide [J]. Heliyon, 2023, 9(9): e19543.
[14]	郭芳芳, 谢镇, 卢鹏, 等. 一株多粘类芽胞杆菌的鉴定及其生防促生效果初步测定 [J]. 中国生物防治学报, 2014, 30(4): 489-496.
	Guo FF, Xie Z, Lu P, et al. Identification of a novel Paenibacillus polymyxa strain and its biocontrol and plant growth-promoting effects [J]. Chin J Biol Control, 2014, 30(4): 489-496.
[15]	杨剑锋, 张园园, 王娜, 等. 苜蓿根腐病病原菌的分离鉴定及苜蓿品种的抗性评价 [J]. 中国草地学报, 2020, 42(3): 52-60.
	Yang JF, Zhang YY, Wang N, et al. Isolation and identification of pathogens causing root rot disease in alfalfa and the evaluation of resistant ability of alfalfa varieties to Fusarium equiseti and F. tricinctum [J]. Chin J Grassland, 2020, 42(3): 52-60.
[16]	陈逢玲, 孙卓, 林红梅, 等. 关防风根腐病拮抗细菌筛选与鉴定 [J]. 微生物学通报, 2022, 49(8): 3192-3204.
	Chen FL, Sun Z, Lin HM, et al. Screening and identification of the antagonistic bacteria against root rot of Saposhnikovia divaricata [J]. Microbiol China, 2022, 49(8): 3192-3204.
[17]	黄家锐, 杜鹏强, 李文均, 等. 地黄内生放线菌的分离及地黄轮纹病拮抗菌Streptomyces folium leaf-16的新种鉴定 [J]. 微生物学报, 2022, 62(12): 4953-4963.
	Huang JR, Du PQ, Li WJ, et al. Isolation of endophytic actinomycetes from Rehmannia glutinosa and identification of a new species Streptomyces folium leaf-16 resistant to Phoma herbarum [J]. Acta Microbiol Sin, 2022, 62(12): 4953-4963.
[18]	Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees [J]. Mol Biol Evol, 1987, 4(4): 406-425.
[19]	Blin K, Shaw S, Kloosterman AM, et al. antiSMASH 6.0: improving cluster detection and comparison capabilities [J]. Nucleic Acids Res, 2021, 49(W1): W29-W35.
[20]	Liu K, Newman M, McInroy JA, et al. Selection and assessment of plant growth-promoting rhizobacteria for biological control of multiple plant diseases [J]. Phytopathology^® , 2017, 107(8): 928-936.
[21]	Dobrzyński J, Naziębło A. Paenibacillus as a biocontrol agent for fungal phytopathogens: is P. polymyxa the only one worth attention? [J]. Microb Ecol, 2024, 87(1): 134.
[22]	Luo CH, He YJ, Chen YP. Rhizosphere microbiome regulation: Unlocking the potential for plant growth [J]. Curr Res Microb Sci, 2025, 8: 100322.
[23]	Liu HW, Brettell LE, Qiu ZG, et al. Microbiome-mediated stress resistance in plants [J]. Trends Plant Sci, 2020, 25(8): 733-743.
[24]	Wen T, Zhao ML, Yuan J, et al. Root exudates mediate plant defense against foliar pathogens by recruiting beneficial microbes [J]. Soil Ecol Lett, 2021, 3(1): 42-51.

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	-	-	-	-