大豆镰刀菌根腐病拮抗菌的筛选及生防效果

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

摘要/Abstract

摘要：

【目的】 镰刀菌根腐病是世界范围内大豆生产上最具破坏性的土传病害之一，为获得对禾谷镰刀菌Fusarium graminearum具有较好拮抗效果的生防菌株。【方法】 从健康大豆根际土壤分离细菌，平板对峙法筛选拮抗菌株，通过形态观察、生理生化特性、胞外酶活性及促生特性对菌株进行鉴定分析，采用盆栽试验进一步测定菌株生防及促生效果。【结果】 筛选出的4株芽孢杆菌和1株假单胞杆菌对F. graminearum的抑菌率均达到60%以上，对F. oxysporum，F. solani，F. longifundum以及 F. equiseti也均有一定的抑制作用，其发酵液及挥发代谢物均会影响F. graminearum的生长。5株拮抗菌具有产蛋白酶、纤维素酶以及葡聚糖酶的活性，解磷、解钾、固氮以及产铁载体的能力。综合以上测试结果，菌株20-8具有较强的抑菌及大豆促生效果。根据形态特征及16S rRNA序列分析，将菌株20-8鉴定为暹罗芽孢杆菌（Bacillus siamensis）。该菌株的发酵上清液可以破坏F. graminearum菌丝体结构，无细胞发酵上清液可以显著抑制孢子萌发。室内盆栽防效测定结果表明，20-8的稀释发酵液对F. graminearum引起大豆根腐病的防效可达46.08%，并且促进大豆植株生长。【结论】 筛选鉴定的暹罗芽孢杆菌20-8具有解磷、解钾、固氮以及产铁载体及多种胞外酶功能，其稀释发酵液具有较强的抗真菌活性及大豆促生能力，菌株20-8可以用于防治F. graminearum引起的大豆根腐病。

关键词: 大豆根腐病, 拮抗细菌, 分离鉴定, 生物防治

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

王芳, 于璐, 齐泽铮, 周长军, 于吉东. 大豆镰刀菌根腐病拮抗菌的筛选及生防效果[J]. 生物技术通报, 2024, 40(7): 216-225.

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.

图/表 11

图1 拮抗菌对不同镰刀菌的抑制作用 A：拮抗菌对镰刀菌抑制作用；B：F. graminearum的生长直径。不同小写字母表示在P < 0.05 水平差异显著，下同

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

图2 拮抗菌菌落形态

Fig. 2 Colony morphology of antagonistic bacteria

表1 拮抗菌的生理生化鉴定结果

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	+	+	+	+	-	+	-	+	-

表2 拮抗菌胞外酶活性

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	+	+	+	-

表3 拮抗菌促生特性鉴定

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	+	+	+	+

图3 拮抗菌对F. graminearum生长的抑制作用 A：发酵液处理下的F. graminearum 菌落形态；B：F. graminearum生长直径；C：挥发代谢物处理下的F. graminearum 菌落形态；D：F. graminearum生长直径

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

图4 菌株20-8的16S rRNA基因序列系统发育树

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

图5 菌株20-8发酵液对F. graminearum菌丝形态影响 A：未经发酵液处理的菌丝； B-C：菌株20-8处理24 h的菌丝

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

图6 菌株20-8不同浓度的无细胞发酵液对F. graminearum孢子萌发影响 A：孢子萌发显微镜观察；B：孢子萌发率

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

图7 菌株20-8对F. graminearum大豆根腐病的防治效果

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

表4 菌株20-8对大豆幼苗的促生效果及根腐病防治效果

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

参考文献 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