Effects of Bacillus amyloliquefaciens YK3 on the Control of Citrus reticulata cv. Orah Canker and Its Influence on the Network of Phyllosphere Bacteria

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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

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	-

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

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

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

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

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

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