Isolation and Identification of Apple Brown Rot Pathogen in Parts of Gansu and Screening of Antagonistic Bacteria

doi:10.13560/j.cnki.biotech.bull.1985.2023-0234

Abstract

Abstract:

This work aims to clarify the pathogenic species of apple brown rot in Jingning county and Yuzhong county of Gansu province and to screen high-quality pathogen antagonistic bacteria for providing resources for biological control of apple postharvest diseases. The pathogens were isolated and purified by conventional tissue isolation method, verified by Koch's postulates, and identified by molecular biology method combined with morphology. The antagonistic bacteria were screened by plate confrontation, identified by morphological, physiological, biochemical and molecular biological methods, and the disease control effects of the antagonistic bacteria were determined by in vitro fruits. The results showed that the pathogens causing apple brown rot in Jingning county and Yuzhong county of Gansu province were Monilia yunnanensis. The optimal antagonistic bacterium X2-2 was Bacillus amyloliquefaciens, which inhibited（81.29±0.57）% of the mycelium of M. yunnanensis and 98.8% of the control effect of the antagonistic bacteria on the isolated fruits. It significantly inhibited the expansion rate of M. yunnanensis lesion spots of apple fruits. M. yunnanensis was the main pathogen of apple brown rot in some areas of Gansu province. The endogenous antagonistic bacterium X2-2 screened in this study had significant antibacterial effect and had the potential of being a high-quality biocontrol strain.

Key words: apple brown rot, Monilia yunnanensis, Bacillus amyloliquefaciens

JIANG Jing-jing, ZHOU Zhao-xu, DU Hui, LYU Zhao-long, WANG Chun-ming, GUO Jian-guo, ZHANG Xin-rui, LI Ji-ping. Isolation and Identification of Apple Brown Rot Pathogen in Parts of Gansu and Screening of Antagonistic Bacteria[J]. Biotechnology Bulletin, 2023, 39(10): 209-218.

Figures/Tables 10

Fig. 1 Disease characteristics of brown rot of apple and symptoms of pathogenic inoculation A-B: Disease symptom（A: ‘Qinguan apple’. B: ‘Red Fuji’ apple）. C, E: Fungus cake inoculation for 5 d（C: ‘Qinguan’ apple. E: ‘Red Fuji’ apple）. D, F: Spore suspension inoculation for 5 d（D: ‘Qinguan’ apple. F: ‘Red Fuji’ apple）

Fig. 2 Colony and conidial morphology of apple brown rot bacteria A: The front of the colony. B: The back of the colony. C: Conidiophores（10×40）. D: The conidium（10×40）. Scale bar=20 μm

Fig. 3 Phylogenetic tree constructed using the neighbour-joining based on rDNA-ITS sequences

Table 1 Inhibition effect of antagonistic bacterium on M. yunnanensis

菌株编号 Strain No.	抑菌率 Inhibition rate/%	抑菌带 Inhibition bands/mm
X2-2	81.29±0.57a	7.42±0.17a
Zyx-3	80.08±0.66b	5.42±0.51a
X1-5	74.64±0.97c	4.25±0.38a
X1-6	71.29±0.62d	4.11±0.42a
P-sy-3	70.88±0.52d	3.20±0.55a

Fig. 4 Effect of antagonistic bacteria X2-2 against M. yun-nanensis

Fig. 5 Effect of inoculation time on lesion area and control effect A: Inoculated with the M. yunnanensis first, and then inoculated with the X2-2 bacterial suspension after 24 h. B: M. yunnanensis + bacterial suspension. C: First inoculated with X2-2 bacterial suspension, then inoculated with M. yunnanensis after 24 h. Lowercase letters indicate significant difference at P<0.05 level

Fig. 6 Inhibition effect of X2-2 bacterial suspension on the lesion expansion of M. yunnanensis A: Inoculated with the M. yunnanensis first, and then inoculated with the X2-2 bacterial suspension after 24 h. B: M. yunnanensis + bacterial suspension. C: First inoculated with X2-2 bacterial suspension, then inoculated with M. yunnanensis after 24 h

Fig. 7 Colony（A）and Gram stain（B）of antagonistic bacterium X2-2

Table 2 Physiological and biochemical identification results of antagonistic bacterium X2-2

指标 Index	结果 Result	指标 Index	结果 Result
革兰氏染色 Gram staining	+	V.P试验 V.P test	+
淀粉水解 Amylolysis	+	甲基红试验 Methyl red test	+
明胶液化 Gelatin liquefaction	+	柠檬酸盐 Citrate utilization	+
酪蛋白水解 Caseinhydrolyzation	-	硫化氢试验 Hydrogen sulfide test	-
7% NaCl	+	接触酶 Catalase	+

Fig. 8 Phylogenetic tree of antagonistic bacterium X2-2 based on 16S rRNA (A) and gyrA (B) sequences

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