Screening and Biocontrol Effect of Antagonistic Bacteria against Soybean Root Rot

doi:10.13560/j.cnki.biotech.bull.1985.2024-0070

Abstract

Abstract:

【Objective】 Fusarium root rot is one of the most devastating soil-borne diseases in soybean planting areas worldwide. This study aims to obtain antagonistic bacteria effectively against Fusarium graminearum. 【Method】 Antagonistic strains were isolated from the rhizosphere soil of healthy soybean plants and screened by plate confrontation method. The morphological characteristics, physiological and biochemical properties, extracellular enzyme activity and growth-promoting characteristics of strains were identified and evaluated. The biocontrol and growth promotion effect of the strain was determined by pot experiment. 【Result】 Four Bacillus strains and 1 Pseudomonas strain screened were effective against F. graminearum, with the inhibition rates above 60%. Besides, the strains also had certain inhibitory effect on F. oxysporum, F. solani, F. longifundum and F. equiseti. The fermentation liquid and volatile metabolites of the strains affected the growth of F. graminearum. And all strains demonstrated proteolytic, cellulolytic and glucanolytic activity, and were able to solve phosphorus and potassium, as well as possessed nitrogen fixation and iron carrier. Based on the above test results, strain 20-8 had stronger antifungal effect and soybean growth-promoting effect, and it was identified as Bacillus siamensis based on morphology characteristics and 16S rRNA sequencing. The fermentation supernatant of the strain destroyed the mycelium structure of F. graminearum, and the cell-free fermentation supernatant significantly inhibited the spore germination. Moreover, the diluted fermentation solution of the strain enhanced the resistance of soybean to F. graminearum, with biocontrol efficiency of 46.08%, and promoted the growth of soybean seedling. 【Conclusion】 These results indicated that the screeded and identified B. siamensis 20-8 possesses the ability of solving phosphorus solubilization and potassium, fixing nitrogen and producing iron carrier as well as a variety function of extracellular enzymes. Its diluted fermentation solution has strong antifungal activity and soybean growth promotion ability. Therefore, strain 20-8 may be used as a biocontrol strain against soybean root rot caused by F. graminearum.

Key words: soybean root rot, antagonistic bacteria, isolation and identification, biological control

WANG Fang, YU Lu, QI Ze-zheng, ZHOU Chang-jun, YU Ji-dong. Screening and Biocontrol Effect of Antagonistic Bacteria against Soybean Root Rot[J]. Biotechnology Bulletin, 2024, 40(7): 216-225.

Figures/Tables 11

Fig. 1 Inhibitory effects of antagonistic bacteria on different Fusarium species A: Inhibitory effect of antagonistic bacteria on Fusarium strains. B: Inhibition rate colony diameter of F. graminearum. Different lowercase letters indicate significant differences at the level of 0.05, the same below

Fig. 2 Colony morphology of antagonistic bacteria

Table 1 Physiological and biochemical characteristics of antagonistic bacteria

菌株 Strain	革兰氏染色 Gram stain	接触酶试验Contact enzyme test	氧化酶试验 Oxidase test	甲基红试验 Methyl red	V-P试验 V-P test	明胶液化 Gelatin liquefaction	吲哚试验 Indole test	淀粉水解Starch hydrolysis test	苯丙氨酸脱氨酶试验 Phenylalanine deaminase test
20-8	+	-	+	+	-	-	-	+	-
A54-1	+	-	+	+	-	+	-	+	-
A54-5	+	-	+	+	-	-	-	+	-
A54-6	+	-	+	+	-	+	-	+	-
P-7	+	+	+	+	-	+	-	+	-

Table 2 Extracellular enzyme activity of antagonistic bacteria

菌株 Strain	蛋白酶 Proteinase	纤维素酶 Cellulase	葡聚糖酶 Glucanase	几丁质酶 Chitinase
20-8	+	+	+	-
A54-1	+	+	+	-
A54-5	+	+	+	-
A54-6	+	+	+	-
P-7	+	+	+	-

Table 3 Growth-promoting characteristics of antagonistic bacteria

菌株 Strain	溶磷Phosphorus dissolution	溶钾Potassium dissolution	固氮Nitrogen fixation	载铁 Iron carrier
20-8	+	+	+	+
A54-1	+	+	+	+
A54-5	+	+	+	+
A54-6	+	+	+	+
P-7	+	+	+	+

Fig. 3 Inhibitory effects of antagonistic bacteria on the growth of F. graminearum A: Morphology of F. graminearum colony treated with fermentation broth. B: Colony diameter of F. graminearum. C: Morphology of F. graminearum colony treated with volatile metabolite. D: Colony diameter of F. graminearum

Fig. 4 16S rRNA phylogenetic tree of strain 20-8

Fig. 5 Effects of strain 20-8 fermentation solution on the mycelium morphology of F. graminearum A: Mycelium untreated with fermentation solution. B-C: Mycelium treated by fermentation solution for 24 h

Fig. 6 Effects of strain 20-8 cell-free fermentation supernatant at different concentrations on spore germination of F. graminearum A: Microscopic observation of spore germination. B: Spore germination rate

Fig. 7 Control effect of strain 20-8 on the soybean root rot caused by F. graminearum

Table 4 Effects of strain 20-8 on the growth-promoting of soybean seedlings and root rot prevention

处理 Treatment	株高Plant height/cm	根长Root length/cm	鲜重Green weight/g	干重Dry weight/g	病情指数 Disease index/%	死亡率 Mortality rate/%	防治效果 Prevention effect/%
CK	12.33±1.13^ab	15.42±2.53^ab	2.54±0.11^ab	0.27±0.03^ab
20-8	13.56±0.71^a	16.68±3.12^a	2.92±0.19^a	0.31±0.02^a
20-8+ F.graminearum	11.82±0.41^ab	11.71±1.96^ab	2.10±0.13^b	0.22±0.01^bc	38.19	18.75	46.08
F.graminearum	9.94±0.2⁵b	8.05±0.71^b	1.30±0.11^c	0.19±0.02^c	70.83	71.43

References 35

[1]	Peng DL, Jiang R, Peng H, et al. Soybean cyst nematodes: a destructive threat to soybean production in China[J]. Phytopathol Res, 2021, 3(1): 19.
[2]	Chang XL, Dai H, Wang DP, et al. Identification of Fusarium species associated with soybean root rot in Sichuan Province, China[J]. Eur J Plant Pathol, 2018, 151(3): 563-577.
[3]	Chang XL, Yan L, Naeem M, et al. Maize/soybean relay strip intercropping reduces the occurrence of Fusarium root rot and changes the diversity of the pathogenic Fusarium species[J]. Pathogens, 2020, 9(3): 211.
[4]	Chang XL, Naeem M, Li HJ, et al. First report of Fusarium asiaticum as a causal agent for seed decay of soybean(Glycine max)in Sichuan, China[J]. Plant Dis, 2020, 104(5): 1542.
[5]	Hafez M, Abdelmagid A, Aboukhaddour R, et al. Fusarium root rot complex in soybean: molecular characterization, trichothecene formation, and cross-pathogenicity[J]. Phytopathology, 2021, 111(12): 2287-2302.
[6]	Broders KD, Lipps PE, Paul PA, et al. Evaluation of Fusarium graminearum associated with corn and soybean seed and seedling disease in Ohio[J]. Plant Dis, 2007, 91(9): 1155-1160. doi: 10.1094/PDIS-91-9-1155 pmid: 30780657
[7]	Chandra NS, Wulff EG, Udayashankar AC, et al. Prospects of molecular markers in Fusarium species diversity[J]. Appl Microbiol Biotechnol, 2011, 90(5): 1625-1639. doi: 10.1007/s00253-011-3209-3 pmid: 21494869
[8]	Ellis ML, Arias MMD, Cruz Jimenez DR, et al. First report of Fusarium commune causing damping-off, seed rot, and seedling root rot on soybean(Glycine max)in the United States[J]. Plant Dis, 2013, 97(2): 284.
[9]	王爽, 李新民, 刘春来, 等. 引起黑龙江省大豆根腐病的镰刀菌种类鉴定及致病分析[J]. 植物病理学报, 2023, 53(1): 126-128.
	Wang S, Li XM, Liu CL, et al. Identification and pathogenicity of Fusarium species causing soybean root rot in Heilongjiang Province[J]. Acta Phytopathol Sin, 2023, 53(1): 126-128.
[10]	Naeem M, Munir M, Li HJ, et al. Transcriptional responses of Fusarium graminearum interacted with soybean to cause root rot[J]. J Fungi, 2021, 7(6): 422.
[11]	Zhang J, Xia MC, Xue BG, et al. First report of Fusarium pseudograminearum causing root rot on soybean(Glycine max)in Henan, China[J]. Plant Dis, 2018, 102(7): 1454.
[12]	Hartman G, Chang HX, Leandro L. Research advances and management of soybean sudden death syndrome[J]. Crop Prot, 2015, 73: 60-66.
[13]	李柯, 郑素娇, 王晓莉, 等. 251份大豆品种(系)对大豆疫霉及多种镰孢菌的抗性评价[J]. 南京农业大学学报, 2022, 45(2): 261-268.
	Li K, Zheng SJ, Wang XL, et al. Identification of resistance of 251 soybean cultivars(lines)to Phytophthora sojae and Fusarium spp.[J]. J Nanjing Agric Univ, 2022, 45(2): 261-268.
[14]	Xi XD, Fan JL, Yang XY, et al. Evaluation of the anti-oomycete bioactivity of rhizosphere soil-borne isolates and the biocontrol of soybean root rot caused by Phytophthora sojae[J]. Biol Contr, 2022, 166: 104818.
[15]	Jiang CH, Wu F, Yu ZY, et al. Study on screening and antagonistic mechanisms of Bacillus amyloliquefaciens 54 against bacterial fruit blotch(BFB)caused by Acidovorax avenae subsp. citrulli[J]. Microbiol Res, 2015, 170: 95-104.
[16]	Chen K, Tian ZH, He H, et al. Bacillus species as potential biocontrol agents against citrus diseases[J]. Biol Contr, 2020, 151: 104419.
[17]	Mishra J, Arora NK. Secondary metabolites of fluorescent pseudomonads in biocontrol of phytopathogens for sustainable agriculture[J]. Appl Soil Ecol, 2018, 125: 35-45.
[18]	Rodovikov SA, Churakov AA, Popova NM, et al. Bacterial strains for biological control of Fusarium root rot of soybean in Siberia[J]. IOP Conf Ser: Earth Environ Sci, 2020, 548(4): 042054.
[19]	Hashem A, Tabassum B, Fathi Abd Allah E. Bacillus subtilis: a plant-growth promoting rhizobacterium that also impacts biotic stress[J]. Saudi J Biol Sci, 2019, 26(6): 1291-1297. doi: 10.1016/j.sjbs.2019.05.004 pmid: 31516360
[20]	Hazarika DJ, Goswami G, Gautom T, et al. Lipopeptide mediated biocontrol activity of endophytic Bacillus subtilis against fungal phytopathogens[J]. BMC Microbiol, 2019, 19(1): 71.
[21]	褚睿, 李昭轩, 张学青, 等. 黄瓜枯萎病拮抗芽孢杆菌的筛选、鉴定及其生防潜力[J]. 生物技术通报, 2023, 39(8): 262-271. doi: 10.13560/j.cnki.biotech.bull.1985.2022-1574
	Chu R, Li ZX, Zhang XQ, et al. Screening and identification of antagonistic Bacillus spp. against cucumber Fusarium wilt and its biocontrol effect[J]. Biotechnol Bull, 2023, 39(8): 262-271.
[22]	金艳丽, 姚拓, 兰晓君, 等. 燕麦根腐病拮抗菌的筛选及抑菌特性研究[J]. 草地学报, 2022, 30(5): 1095-1101. doi: 10.11733/j.issn.1007-0435.2022.05.009
	Jin YL, Yao T, Lan XJ, et al. Screening of antagonistic bacteria of oat root rot and studies of antibacterial properties[J]. Acta Agrestia Sin, 2022, 30(5): 1095-1101.
[23]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001.
	Dong XZ, Cai MY. Handbook of identification of common bacterial systems[M]. Beijing: Science Press, 2001.
[24]	布坎南. 伯杰细菌鉴定手册: 第八版[M]. 8版. 北京: 科学出版社, 1984.
	Buchanan RE. Bergey's manual of determinative bacteriology[M]. 8th ed. Beijing: Science Press, 1984.
[25]	张武, 杨树, 项鹏, 等. 哈茨木霉拌种对大豆生长及大豆根腐病的影响[J]. 黑龙江农业科学, 2023(7): 41-46.
	Zhang W, Yang S, Xiang P, et al. Effects of seed dressing treatment with Trichoderma harzianum on soybean growth and root rot disease[J]. Heilongjiang Agric Sci, 2023(7): 41-46.
[26]	Mohanram S, Kumar P. Rhizosphere microbiome: revisiting the synergy of plant-microbe interactions[J]. Ann Microbiol, 2019, 69(4): 307-320. doi: 10.1007/s13213-019-01448-9
[27]	You WJ, Ge CH, Jiang ZC, et al. Screening of a broad-spectrum antagonist-Bacillus siamensis, and its possible mechanisms to control postharvest disease in tropical fruits[J]. Biol Contr, 2021, 157: 104584.
[28]	He HT, Zhai QH, Tang YN, et al. Effective biocontrol of soybean root rot by a novel bacterial strain Bacillus siamensis HT1[J]. Physiol Mol Plant Pathol, 2023, 125: 101984.
[29]	Karim H, Azis AA, Jumadi O. Antagonistic activity and characterization of indigenous soil isolates of bacteria and fungi against onion wilt incited by Fusarium sp.[J]. Arch Microbiol, 2021, 204(1): 68.
[30]	杨东亚, 祁瑞雪, 李昭轩, 等. 黄瓜茄病镰刀菌拮抗芽孢杆菌的筛选、鉴定及促生效果[J]. 生物技术通报, 2023, 39(2): 211-220. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0522
	Yang DY, Qi RX, Li ZX, et al. Screening, identification and growth-promoting effect of antagonistic Bacillus spp. against cucumber Fusarium solani[J]. Biotechnol Bull, 2023, 39(2): 211-220.
[31]	朱杰, 程亮, 姚强, 等. 枸杞根腐致病菌及拮抗菌的分离鉴定[J]. 西北农业学报, 2023, 32(7): 1120-1130.
	Zhu J, Cheng L, Yao Q, et al. Isolation and identification of pathogenic fungi and antagonistic bacteria from Lycium barbarum root rot[J]. Acta Agric Boreali Occidentalis Sin, 2023, 32(7): 1120-1130.
[32]	Elsherbiny EA, Dawood DH, Safwat NA. Antifungal action and induction of resistance by β-aminobutyric acid against Penicillium digitatum to control green mold in orange fruit[J]. Pestic Biochem Physiol, 2021, 171: 104721. doi: 10.1016/j.pestbp.2020.104721 pmid: 33357543
[33]	贾方方, 许跃奇, 阎海涛, 等. 烟草镰刀菌根腐病(Fusarium spp.)拮抗菌L210的鉴定及生防潜力评价[J]. 烟草科技, 2023, 56(10): 40-48.
	Jia FF, Xu YQ, Yan HT, et al. Identification and biocontrol potential evaluation of antagonistic bacterial strain L210 against Fusarium spp. of tobacco root rot[J]. Tob Sci Technol, 2023, 56(10): 40-48.
[34]	Agbodjato NA, Noumavo PA, Adjanohoun A, et al. Synergistic effects of plant growth promoting rhizobacteria and chitosan on in vitro seeds germination, greenhouse growth, and nutrient uptake of maize(Zea mays L.)[J]. Biotechnol Res Int, 2016, 2016: 7830182.
[35]	Liu YP, Shu X, Chen L, et al. Plant commensal type VII secretion system causes iron leakage from roots to promote colonization[J]. Nat Microbiol, 2023, 8(8): 1434-1449. doi: 10.1038/s41564-023-01402-1 pmid: 37248429