Identification, Biological Characteristics and Fungicide Screening of Tobacco Leaf Spot Disease Pathogen Epicoccum latusicollum

doi:10.3969/j.issn.1000-2561.2022.12.016

Abstract

Abstract:

Leaf spot disease is one of the main tobacco diseases in the field period, which is mainly caused by fungal infection. It is easy to happen and spread, and has a great impact on the quality and yield of tobacco. In this study, the strain was isolated and purified from the leaves with typical leaf spot diseases of tobacco variety ‘K326’ in Guangxi Zhuang Autonomous Region by tissue separation, and through pathogenicity determination combined with morphological characteristics and molecular biological methods to identify pathogens. The mycelium growth rate method was used to measure the mycelium growth of pathogens in different media, temperature, pH, carbon source, nitrogen source, light, lethal temperature treatment and 10 common chemical conditions. In the early period of tobacco leaf spot disease, the typical symptoms of the leaf spot disease are gray white small round spots. As the disease worsens, the round spot gradually diffuses into irregular shape, and the center of the spot forms a perforation, the color is gray white, the edge is brown, and accompanied by a yellow halo. We isolated and purified two strains and named them as HZFC36 and HZZS76. The pathogenicity test results showed that both strains could cause disease spots on the leaves of healthy ‘K326’ tobacco plant under the condition of no leaf injury. Epicoccum latusicollum was identified as the pathogen causing tobacco leaf spot disease in Guangxi, based on morphological characteristics of pathogen and phylogenetic analysis of multiple loci (LSU, ITS, RPB2 and TUB2). The mycelia of the pathogen grew the fastest on CMA medium, and the optimal growth temperature was 28℃, the optimal pH was 6, the optimal carbon was sucrose and nitrogen source was beef extract powder. 24 h continuous light is favorable to the growth of the mycelia, and the mycelia did not grow after being continuously treated in water bath at 47℃ for 10 min. Preliminary screening results of chemical fungicides in the laboratory showed that 50% boscalid and 25% pyraclostrobine had the best inhibitory effect on the pathogen with EC₅₀ value 4.752×10^-2 mg/L and 4.989×10^-2 mg/L respectively. The results of the study would provide a theoretical basis for the field control of tobacco leaf spot caused by E. latusicollum.

Key words: leaf spot disease, identification of pathogenic bacteria, biological characteristics, fungicide screening

CLC Number:

S435.72

AN Xuanxian, SANG Weijun, LU Yanhui, KONG Fei, WANG Wuquan, LI Haoxi, YANG Jiangmin, PENG Lijuan, YANG Maofa. Identification, Biological Characteristics and Fungicide Screening of Tobacco Leaf Spot Disease Pathogen Epicoccum latusicollum[J]. Chinese Journal of Tropical Crops, 2022, 43(12): 2534-2544.

Figures/Tables 7

References 39

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药剂Fungicide	生产厂家Manufacture factory	浓度 Concentration/(mg·L^-1)
50%啶酰菌胺WG	巴斯夫植物保护（江苏）有限公司	3.704、1.235、0.412、0.137、0.046、0.015
25%吡唑醚菌酯SC	河北中保绿农作物科技有限公司	11.111、3.704、1.235、0.412、0.137、0.046
50%咯菌腈FS	先正达南通作物保护有限公司	3.704、1.235、0.412、0.137、0.046、0.015
450 g/L咪鲜胺EW	上海沪联生物药业（夏邑）股份有限公司	11.111、3.704、1.235、0.412、0.137、0.046
10%苯醚甲环唑ME	河北中保绿农作物科技有限公司	11.111、3.704、1.235、0.412、0.137、0.046
500 g/L异菌脲SC	苏州富美实植物保护剂有限公司	11.111、3.704、1.235、0.412、0.137、0.046
430 g/L戊唑醇SC	山西德威本草生物科技有限公司	100、33.334、11.111、3.704、1.235、0.412
40%腈菌唑SC	山西禾益化工股份有限公司	100、33.334、11.111、3.704、1.235、0.412
80%代森锰锌WP	兴农股份有限公司	300、100、33.334、11.111、3.704、1.235
250 g/L嘧菌酯SC	先正达南通作物保护有限公司	900、300、100、33.334、11.111、3.704

药剂Fungicide	生产厂家Manufacture factory	浓度 Concentration/(mg·L^-1)
50%啶酰菌胺WG	巴斯夫植物保护（江苏）有限公司	3.704、1.235、0.412、0.137、0.046、0.015
25%吡唑醚菌酯SC	河北中保绿农作物科技有限公司	11.111、3.704、1.235、0.412、0.137、0.046
50%咯菌腈FS	先正达南通作物保护有限公司	3.704、1.235、0.412、0.137、0.046、0.015
450 g/L咪鲜胺EW	上海沪联生物药业（夏邑）股份有限公司	11.111、3.704、1.235、0.412、0.137、0.046
10%苯醚甲环唑ME	河北中保绿农作物科技有限公司	11.111、3.704、1.235、0.412、0.137、0.046
500 g/L异菌脲SC	苏州富美实植物保护剂有限公司	11.111、3.704、1.235、0.412、0.137、0.046
430 g/L戊唑醇SC	山西德威本草生物科技有限公司	100、33.334、11.111、3.704、1.235、0.412
40%腈菌唑SC	山西禾益化工股份有限公司	100、33.334、11.111、3.704、1.235、0.412
80%代森锰锌WP	兴农股份有限公司	300、100、33.334、11.111、3.704、1.235
250 g/L嘧菌酯SC	先正达南通作物保护有限公司	900、300、100、33.334、11.111、3.704

基因 Gene	引物 Primer	引物序列（5′-3′） Primer sequence (5′-3′)
LSU	LROR^[22]	GTACCCGCTGAACTTAAGC
LSU	LR7^[22]	TACTACCACCAAGATCT
ITS	ITS1^[23]	TCCGTAGGTGAACCTGCGG
ITS	ITS4^[24]	TCCTCCGCTTATTGATATGC
RPB2	RPB2-5F2^[25]	GGGGWGAYCAGAAGAAGGC
RPB2	fRPB2-7cR^[26]	CCCATRGCTTGYTTRCCCAT
TUB2	TUB2Fd^[27]	GTBCACCTYCARACCGGYCARTG
TUB2	TUB4Rd^[27]	CCRGAYTGRCCRAARACRAAGTTGTC

基因 Gene	引物 Primer	引物序列（5′-3′） Primer sequence (5′-3′)
LSU	LROR^[22]	GTACCCGCTGAACTTAAGC
LSU	LR7^[22]	TACTACCACCAAGATCT
ITS	ITS1^[23]	TCCGTAGGTGAACCTGCGG
ITS	ITS4^[24]	TCCTCCGCTTATTGATATGC
RPB2	RPB2-5F2^[25]	GGGGWGAYCAGAAGAAGGC
RPB2	fRPB2-7cR^[26]	CCCATRGCTTGYTTRCCCAT
TUB2	TUB2Fd^[27]	GTBCACCTYCARACCGGYCARTG
TUB2	TUB4Rd^[27]	CCRGAYTGRCCRAARACRAAGTTGTC

药剂 Fungicide	回归方程 Regression equation		相关系数 Correlation coefficient		EC₅₀/(mg·L^-1)
药剂 Fungicide	HZFC36	HZZS76	HZFC36	HZZS76	HZFC36	HZZS76	均值Average
50%啶酰菌胺WG	y=1.701+2.257x	y=2.754+3.633x	0.933	0.981	4.713×10²	4.792×10^-2	4.752×10^-2
25%吡唑醚菌酯SC	y=0.427+0.534x	y=0.468+0.637x	0.918	0.981	5.616×10^-2	4.362×10^-2	4.989×10^-2
50%咯菌腈FS	y=1.292+1.487x	y=1.197+1.432x	0.991	0.990	1.352×10^-1	1.460×10^-1	1.406×10^-1
450g/L咪鲜胺EW	y=2.315+1.536x	y=1.944+1.378x	0.910	0.889	2.171×10^-1	1.954×10^-1	2.063×10^-1
10%苯醚甲环唑ME	y=1.456+0.542x	y=1.008+0.718x	0.969	0.973	4.244×10^-1	1.937×10^-1	3.090×10^-1
500g/L异菌脲SC	y=1.594+0.010x	y=1.804+0.220x	0.986	0.979	9.858×10^-1	7.548×10^-1	8.703×10^-1
430g/L戊唑醇SC	y=2.037-0.514x	y=2.253-0.521x	0.911	0.922	1.787×10⁰	1.704×10⁰	1.745×10⁰
40%腈菌唑SC	y=-0.679+2.068x	y=-0.814+2.221x	0.959	0.937	2.128×10⁰	2.326×10⁰	2.227×10⁰
80%代森锰锌WP	y=0.881-1.158x	y=1.115-1.059x	0.969	0.991	2.064×10¹	8.908×10⁰	1.477×10¹
250g/L嘧菌酯SC	y=-0.052+0.359x	y=-0.111+0.567x	-0.368	-0.582	8.570×10⁶	1.256×10⁵	4.348×10⁶