解淀粉芽孢杆菌YK3对沃柑溃疡病的防效及叶际细菌群落相关性的影响

doi:10.13560/j.cnki.biotech.bull.1985.2024-0370

摘要/Abstract

摘要：

【目的】 探究解淀粉芽孢杆菌YK3对沃柑溃疡病的防治效果及对沃柑叶际细菌网络关系的影响，为微生物防治沃柑溃疡病提供实际参考。【方法】 采用平板抑菌法分离沃柑溃疡病的拮抗菌，离体生测和盆栽实验研究拮抗菌对沃柑溃疡病的防治效果，并通过高通量测序技术研究接种拮抗菌对沃柑叶际细菌群落组成、相对丰度以及细菌互作关系网络的影响。【结果】 分离获得一株发酵液和发酵过滤液均对病原菌有强烈抑制作用的解淀粉芽孢杆菌YK3，该菌株在以有机氮作为氮源时才会产生抑菌物质，不能与铜类农药同时使用。生防测试实验结果表明，仅接种病原菌的病情指数为96.67%，同时接种病原菌和YK3的病情指数为33.33%，YK3对沃柑溃疡病的相对防治效果为65.56%。YK3可以降低沃柑叶际Xanthomonas的相对丰度。仅接种YK3的沃柑叶际细菌群落正相关关系最复杂，其次为同时接种病原菌和YK3处理，接种病原菌处理的沃柑叶际细菌群落正相关关系最弱。同时接种病原菌和YK3处理中Xanthomonas、Bacillus与沃柑叶际土著细菌群落均为负相关关系，但是Xanthomonas和Bacillus与沃柑叶际土著细菌群落的关系网不存在重叠，Xanthomonas和Bacillus独自面对不同的土著细菌的竞争作用。【结论】 解淀粉芽孢杆菌YK3对沃柑溃疡病有较好的防治效果，会改变沃柑叶际细菌多样性和结构，使沃柑叶际土著细菌群落的正相关性网络增强，负相关性网络减弱，互作关系网络更密集。

关键词: 沃柑, 溃疡病, 生物防治, 解淀粉芽孢杆菌, 拮抗作用, 叶际, 细菌群落多样性, 网络互作

Abstract:

【Objective】 To study the effects of Bacillus amyloliquefaciens YK3 on the control of Orah canker and its influence on the network of phyllosphere bacteria may provide practical reference for microbial prevention and control of Orah canker. 【Method】 The flat plate antibacterial method was used to isolate the antagonistic bacteria against the Orah canker. In vitro bioassay and potted experiments were used to study the control effect of antagonistic bacteria on Orah canker. The effects of inoculated biocontrol bacteria on bacterial community composition, abundance and bacterial interaction network were studied by high-throughput sequencing technology. 【Result】 A strain of Bacillus amyloliquefaciens YK3 was isolated, which had strong inhibitory effect on pathogenic bacteria. This strain produced bacteriostatic substances only when organic nitrogen was used as a nitrogen source and could not be used together with copper pesticides. The results of biocontrol test showed that the incidence rate of inoculation with pathogen alone was 96.67%, the incidence rate of inoculation with pathogen and YK3 was 33.33%. The control effect of YK3 on Orah canker was 65.56%. YK3 reduced the relative abundance of phyllosphere Xanthomonas. The positive correlation of phyllosphere bacterial community inoculated with only YK3 was the most complex, followed by simultaneous inoculation of pathogenic bacteria and YK3. The positive correlation of phyllosphere bacterial community inoculated with pathogenic bacteria was the weakest. Simultaneously inoculated with pathogens and YK3, Xanthomonas and Bacillus were negatively correlated with the indigenous bacterial community in phyllosphere. However, there was no overlap between Xanthomonas and Bacillus and the indigenous bacterial community in phyllosphere. Xanthomonas and Bacillus faced competition from different indigenous bacteria alone. 【Conclusion】 Bacillus amyloliquefaciens YK3 has a good control effect on Orah canker and can change the diversity and structure of phyllosphere bacteria in Orah. YK3 enhances the positive correlation network, weakens the negative correlation network and improved the interaction network.

Key words: Citrus reticulata cv. Orah, canker, biological control, Bacillus amyloliquefaciens, antagonistic effect, phyllosphere, bacterial community diversity, network interaction

叶柳健, 贺愉岚, 王小虎, 韦圣博, 何双, 朱绮霞, 卢洁, 周礼芹. 解淀粉芽孢杆菌YK3对沃柑溃疡病的防效及叶际细菌群落相关性的影响[J]. 生物技术通报, 2024, 40(11): 248-258.

YE Liu-jian, HE Yu-lan, WANG Xiao-hu, WEI Sheng-bo, HE Shuang, ZHU Qi-xia, LU Jie, ZHOU Li-qin. Effects of Bacillus amyloliquefaciens YK3 on the Control of Citrus reticulata cv. Orah Canker and Its Influence on the Network of Phyllosphere Bacteria[J]. Biotechnology Bulletin, 2024, 40(11): 248-258.

图/表 10

表1 试验所用培养基配方

Table 1 Formula of culture medium used in the experiment

培养基名称 Name of culture medium	培养基的配方 Formula of culture medium/（g·L^-1）
NA	牛肉膏3.00，胰蛋白胨5.00，琼脂粉15.00
LB	胰蛋白胨10.00，酵母粉5.00，氯化钠10.00，琼脂粉15.00
NB	胰蛋白胨10.00，牛肉膏3.00，氯化钠5.00
CD	硝酸钠2.00，磷酸二氢钾0.70，磷酸氢二钾0.30，氯化钾0.50，硫酸镁0.50，硫酸亚铁0.01，蔗糖30.00
YPD	酵母粉10.00，胰蛋白胨20.00，葡萄糖20.00
PDB	马铃薯200.00，葡萄糖20.00
SPA	胰蛋白胨10.00，磷酸氢二钾0.50，硫酸镁0.25，蔗糖20.00
KB	胰蛋白胨20.00，甘油10.00，磷酸氢二钾1.50，硫酸镁1.50
BP	牛肉膏3.00，胰蛋白胨10.00，氯化钠5.00

图1 病原菌的分离及其对沃柑叶片的致病作用 A：沃柑溃疡病的病原菌K32；B：病原菌对沃柑叶片的致病作用

Fig. 1 Isolation of pathogenic bacteria and their pathogenic effects on the Orah leaf A: Pathogen K32 of Orah canker disease. B: Pathogenic effects of pathogens on the Orah leaf

图2 拮抗菌的分离及其对病原菌的拮抗效果 A：拮抗菌YK2；B：拮抗菌YK3；C：拮抗菌YK2和YK3在平板上对病原菌的拮抗效果；图上两孔分别为YK2发酵液和发酵过滤液，图下两孔分别为YK3发酵液和发酵过滤液；D：拮抗菌YK3在沃柑叶片上对病原菌的拮抗效果，图片边缘两列为Cu制剂，中间两列为拮抗菌YK3发酵过滤液

Fig. 2 Isolation of antagonistic bacteria and their antagonistic effects on pathogenic bacteria A: Antagonistic bacteria YK2. B: Antagonistic bacteria YK3. C: Antagonistic effects of YK2 and YK3 on pathogenic bacteria on agar plates. The two holes above indicate the YK2 fermentation broth and the filtrate of the fermentation broth, while the two holes below indicate the YK3 fermentation broth and the filtrate of the fermentation broth, respectively. D: The antagonistic effect of YK3 on the pathogenic bacteria on the Orah leaf. The two columns at the edge of the image show Cu preparations, while the middle two columns show the filtrate of the antagonistic bacteria YK3 fermentation broth

图3 K32的生物学特性 NB：营养肉汤培养基；LB：LB培养基；CD：察氏培养基；YPD：酵母浸出粉胨葡萄糖培养基；PDB：马铃薯葡萄糖肉汤培养基

Fig. 3 Biological characteristics of K32 NB: Nutrient broth medium. LB: Luria-Bertani medium. CD: Czapek-Dox medium. YPD: Yeast extract peptone dextrose medium. PDB: Potato dextrose broth medium

表2 YK3的生物学特性

Table 2 Biological characteristics of YK3

检测指标Detection index	反应Response	检测指标Detection index	反应Response	检测指标Detection index	反应Response
氨苄青霉素Ampicillin	+	氯霉素Chloramphenicol	-	卡纳霉素Kanamycin	-
链霉素Streptomycin	+	庆大霉素Gentamicin	-	盐浓度Salt concentration	+
溶解钙 Dissolved calcium	-	溶解有机磷Dissolved organic phosphorus	-	溶解无机磷Dissolved inorganic phosphorus	+
纤维素Cellulose	-	柠檬酸盐Citrate	+	还原硝酸盐Reduced nitrate	+
铁载体Siderophore	+	吲哚Indole	+	淀粉酶Diastase	+
蛋白酶Protease	+	脲酶Urease	+	H₂O₂接触酶H₂O₂ Catalase	+
V-P试验V-P test	+	甲基红试验Methyl red test	-

图4 YK3产抑菌物质的发酵条件优化 LB：LB培养基；SPA：胰蛋白胨蔗糖培养基；KB：金氏B培养基；BP：牛肉膏蛋白胨培养基；PDB：马铃薯葡萄糖肉汤培养基；Gl：葡萄糖；Su：蔗糖；Fr：果糖；Ma：麦芽糖；Ss：可溶性淀粉；La：乳糖；Sp：大豆蛋白胨；Tr：胰蛋白胨；Ye：酵母粉；Ca：酪蛋白；Be：牛肉膏；As：硫酸铵；K：磷酸氢二钾；Na：氯化钠；Mg：硫酸镁；Fe：硫酸亚铁；Mn：硫酸锰；Ca：氯化钙；Cu：硫酸铜

Fig. 4 Optimization of fermentation conditions for producing bacteriostatic substances in YK3 LB: Luria-Bertani medium. SPA: Tryptone sucrose medium. KB: King’s B medium. BP: Beef-protein medium. PDB: Potato dextrose broth medium. Gl: Glucose. Su: Sucrose. Fr: Fructose. Ma: Maltose. Ss: Soluble starch. La: Lactose. Sp: Soy peptone. Tr: Tryptone. Ye: Yeast extract. Ca: Casein. Be: Beef extract. As: Ammonium sulfate. K: Dipotassium hydrogen phosphate. Na: Sodium chloride. Mg: Magnesium sulphate. Fe: Ferrous sulfate. Mn: Manganese sulfate. Ca: Calcium chloride. Cu: Copper sulphate

表3 拮抗菌YK3对沃柑溃疡病的防治效果

Table 3 Effects of antagonistic bacterium YK3 on the control of Orah canker

分组 Group	病情指数 Disease index/%	相对防治效果 Relative control efficiency/%
CK	0 ± 0	-
T1	96.67 ± 4.71	-
T2	0 ± 0	100 ± 0
T3	33.33 ± 4.71	65.56 ± 4.16

表4 不同处理对沃柑叶际细菌群落α多样性的影响

Table 4 Effects of different treatments on α diversity of phyllospheric bacterial communities of Orah

分组 Group	Chao1	Shannon
CK	1 599.33 ± 386.47a	7.43 ± 1.05a
T1	626.14 ± 218.99b	2.38 ± 0.30b
T2	549.14 ± 145.68b	2.20 ± 1.06b
T3	496.00 ± 150.29b	1.96 ± 0.29b

图5 不同分组的沃柑叶际细菌群落组成（属水平）

Fig. 5 Composition of phyllospheric bacterial communities of Orah in different groups（genus level）

图6 不同分组的沃柑叶际细菌群落的相关性网络（属水平）球体节点代表物种，球体大小代表丰度，球体颜色代表物种所属不同的门水平。线条代表两物种间相关，线的粗细代表相关性的强弱，红色线代表正相关，绿色线代表负相关

Fig. 6 Correlation networks of phyllospheric bacterial communities of Orah in different groups（Genus level） Sphere nodes indicate species, sphere size indicates abundance, and sphere color represents different phylum levels to which species belong. The line indicates the correlation between the two species, the thickness of the line indicates the strength of the correlation, the red line indicates the positive correlation, and the green line indicates the negative correlation

参考文献 38

[1]	雷能刚. 广西沃柑栽培技术要点[J]. 南方农业, 2023, 17(5): 145-147, 151.
	Lei NG. Key points of cultivation techniques for Citrus reticulata Blanco in Guangxi[J]. South China Agric, 2023, 17(5): 145-147, 151.
[2]	Li Q, Xian BH, Yu QY, et al. The CsAP2-09-CsWRKY25-CsRBOH2 cascade confers resistance against citrus bacterial canker by regulating ROS homeostasis[J]. Plant J, 2024, 118(2): 534-548.
[3]	邱发发, 潘贞珍, 李鸾翔, 等. 沃柑叶片响应柑橘溃疡病菌侵染的转录组分析[J]. 果树学报, 2022, 39(4): 631-643.
	Qiu FF, Pan ZZ, Li LX, et al. Transcriptome analysis of Orah leaves in response to citrus canker infection[J]. J Fruit Sci, 2022, 39(4): 631-643.
[4]	Angelotti-Mendonça J, de Oliveira PN, Ansante NF, et al. Expression of the Citrus sinensis EDS5 gene, MATE family, in Solanum lycopersicum L. cv. Micro-Tom enhances resistance to tomato spot disease[J]. Trop Plant Pathol, 2022, 47(2): 287-297.
[5]	Long YF, Luo RF, Xu Z, et al. A fluorescent reporter-based evaluation assay for antibacterial components against Xanthomonas citri subsp. citri[J]. Front Microbiol, 2022, 13: 864963.
[6]	Machado FJ, da Silva Marin TG, Canôas F, et al. Timing of copper sprays to protect mechanical wounds against infection by Xanthomonas citri subsp. citri, causal agent of citrus canker[J]. Eur J Plant Pathol, 2021, 160(3): 683-692.
[7]	Ferreira DH, Moreira RR, Silva Junior GJ, et al. Copper rate and spray interval for joint management of citrus canker and citrus black spot in orange orchards[J]. Eur J Plant Pathol, 2022, 163(4): 891-906.
[8]	Sarrocco S. Biological disease control by beneficial(micro)organisms: selected breakthroughs in the past 50 years[J]. Phytopathology, 2023, 113(4): 732-740.
[9]	Abdelaziz AM, Hashem AH, El-Sayyad GS, et al. Biocontrol of soil borne diseases by plant growth promoting rhizobacteria[J]. Trop Plant Pathol, 2023, 48(2): 105-127.
[10]	Dutilloy E, Arias AA, Richet N, et al. Bacillus velezensis BE2 controls wheat and barley diseases by direct antagonism and induced systemic resistance[J]. Appl Microbiol Biotechnol, 2024, 108(1): 64.
[11]	段娇, 刘阳, 冯广达, 等. 柑橘溃疡病及其微生物防治研究进展[J]. 微生物学报, 2023, 63(5): 1944-1958.
	Duan J, Liu Y, Feng GD, et al. Research progress on citrus canker disease and its microbial control[J]. Acta Microbiol Sin, 2023, 63(5): 1944-1958.
[12]	Poveda J, Roeschlin RA, Marano MR, et al. Microorganisms as biocontrol agents against bacterial citrus diseases[J]. Biol Contr, 2021, 158: 104602.
[13]	Nugroho YA, Suharjono S, Widyaningsih S. Biological control of citrus canker pathogen Xanthomonas citri subsp. citri using Rangpur lime endophytic bacteria[J]. Egypt J Biol Pest Contr, 2022, 32(1): 63.
[14]	Zhou JY, Zhang Y, Xu WP, et al. Tug of war-who is the winner? Canker disease restructures the endophytic bacterial community of Citrus[J]. Hortic Sci Technol, 2023, 41(5): 605-616.
[15]	余沁涵. 柑橘溃疡病菌分离鉴定、全基因组测序及防治药剂筛选[D]. 湛江: 广东海洋大学, 2020.
	Yu QH. Isolation, identification, whole genome sequencing and bactericides screening of Xanthomonas citri subsp. citri[D]. Zhanjiang: Guangdong Ocean University, 2020.
[16]	Nauman M, Mushtaq S, Fahad Khan M, et al. Morphological, biochemical, and molecular characterization of Xanthomonas citri subsp. citri, cause of citrus canker disease in Pakistan[J]. Pak J Bot, 2023, 55(6): 2409-2421.
[17]	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.
[18]	Yang Q, Cahn JKB, Piel J, et al. Marine sponge endosymbionts: structural and functional specificity of the microbiome within Euryspongia arenaria cells[J]. Microbiol Spectr, 2022, 10(3): e0229621.
[19]	Wang JJ, Wang JN, Wu SG, et al. Global geographic diversity and distribution of the myxobacteria[J]. Microbiol Spectr, 2021, 9(1): e0001221.
[20]	Gao PF, Ma C, Sun Z, et al. Feed-additive probiotics accelerate yet antibiotics delay intestinal microbiota maturation in broiler chicken[J]. Microbiome, 2017, 5(1): 91.
[21]	Ali S, Hameed A, Muhae-Ud-Din G, et al. Citrus canker: a persistent threat to the worldwide citrus industry—an analysis[J]. Agronomy, 2023, 13(4): 1112.
[22]	王鹤. 柑橘溃疡病生防菌株筛选及药剂减量化研究[D]. 沈阳: 沈阳化工大学, 2022.
	Wang H. Screening of biocontrol strains against citrus canker and study on pesticide reduction[D]. Shenyang: Shenyang University of Chemical Technology, 2022.
[23]	赵洪涛, 陈东奎, 陈香玲, 等. 沃柑溃疡病病原菌分离鉴定及防治药剂筛选[J]. 南方农业学报, 2019, 50(12): 2703-2712.
	Zhao HT, Chen DK, Chen XL, et al. Pathogen identification and bactericide screening of Orah citrus canker[J]. J South Agric, 2019, 50(12): 2703-2712.
[24]	黄观荣, 邓志明, 张艳珍, 等. 柑橘溃疡病气象条件等级预测模型构建与应用[J]. 广东气象, 2023, 45(2): 70-73.
	Huang GR, Deng ZM, Zhang YZ, et al. Construction and application of meteorological condition grade prediction model for citrus canker[J]. Guangdong Meteorol, 2023, 45(2): 70-73.
[25]	Wang X, Liang LQ, Shao H, et al. Isolation of the novel strain Bacillus amyloliquefaciens F9 and identification of lipopeptide extract components responsible for activity against Xanthomonas citri subsp. citri[J]. Plants, 2022, 11(3): 457.
[26]	Qian JL, Zhang T, Tang S, et al. Biocontrol of citrus canker with endophyte Bacillus amyloliquefaciens QC-Y[J]. Plant Prot Sci, 2020, 57(1): 1-13.
[27]	Ahsan T, Zang CQ, Yu SY, et al. Screening, and optimization of fermentation medium to produce secondary metabolites from Bacillus amyloliquefaciens, for the biocontrol of early leaf spot disease, and growth promoting effects on peanut(Arachis hypogaea L.)[J]. J Fungi, 2022, 8(11): 1223.
[28]	Murniasih T, Mardiana NA, Untari F, et al. Optimization of carbon and nitrogen source to enhance antibacterial activity from a sponge-derived Bacillus tequilensis[J]. Turk J Fish Aquat Sci, 2023, 24(2): 24222.
[29]	Chaudhry V, Runge P, Sengupta P, et al. Shaping the leaf microbiota: plant-microbe-microbe interactions[J]. J Exp Bot, 2021, 72(1): 36-56.
[30]	Gong TY, Xin XF. Phyllosphere microbiota: community dynamics and its interaction with plant hosts[J]. J Integr Plant Biol, 2021, 63(2): 297-304.
[31]	Xiong Q, Yang J, Ni SY. Microbiome-mediated protection against pathogens in woody plants[J]. Int J Mol Sci, 2023, 24(22): 16118.
[32]	Carvalho CR, Dias AC, Homma SK, et al. Phyllosphere bacterial assembly in citrus crop under conventional and ecological management[J]. PeerJ, 2020, 8: e9152.
[33]	Jiang HB, Luo JY, Liu QH, et al. Rice bacterial leaf blight drives rhizosphere microbial assembly and function adaptation[J]. Microbiol Spectr, 2023, 11(6): e0105923.
[34]	Huang F, Ling JF, Zhu CY, et al. Canker disease intensifies cross-Kingdom microbial interactions in the endophytic microbiota of Citrus phyllosphere[J]. Phytobiomes J, 2023, 7: 365-374.
[35]	Ginnan NA, Dang T, Bodaghi S, et al. Disease-induced microbial shifts in citrus indicate microbiome-derived responses to huanglongbing across the disease severity spectrum[J]. Phytobiomes J, 2020, 4(4): 375-387.
[36]	Xiao ZF, Lu CY, Wu ZY, et al. Continuous cropping disorders of eggplants(Solanum melongena L.)and tomatoes(Solanum lycopersicum L.) in suburban agriculture: Microbial structure and assembly processes[J]. Sci Total Environ, 2024, 909: 168558.
[37]	Ahmed W, Dai ZL, Zhang JH, et al. Plant-microbe interaction: mining the impact of native Bacillus amyloliquefaciens WS-10 on tobacco bacterial wilt disease and rhizosphere microbial communities[J]. Microbiol Spectr, 2022, 10(4): e0147122.
[38]	Flores-Núñez VM, Camarena-Pozos DA, Chávez-González JD, et al. Synthetic communities increase microbial diversity and productivity of Agave tequilana plants in the field[J]. Phytobiomes J, 2023, 7(4): 435-448.