Isolation and Identification of a Fungus from Moldy Tobacco Leaf and Study on Its Mold-causing Factors

doi:10.13560/j.cnki.biotech.bull.1985.2024-0773

Abstract

Abstract:

Objective Conducting biological research on mold-causing fungi mold in tobacco leaves is of significantly economic importance to the cigarette industry. Method In this study, the plate separation method was employed to isolate and purify mold-causing fungi from tobacco leaves. Sequencing technology and phylogenetic analysis were used to identify the obtained strains, while a back-crossing test was conducted to confirm their pathogenicity. The mold-causing factors of the strains were determined through growth assays, and model simulations were utilized to assess the relevance of mold-causing factors. 【Resul】 A mold-causing fungus was isolated from the surface of moldy tobacco leaves, and morphological and ITS sequence homology analysis identified the strain as closely related to Penicillium citrinum, and homology was 99.82%. The pathogenicity test confirmed that this strain, under conditions of 90% relative humidity, induced mold growth on tobacco leaves. The analysis of mold-causing factors revealed that the optimal conditions for colony growth and sporulation of this strain were 30℃ and a water activity of 0.99. Gompertz model simulation demonstrated that the onset of mold in tobacco leaves was not significantly correlated with the initial number of fungal spores but was strongly influenced by temperature, water activity, and the interaction between these two factors. Conclusion It is confirmed that P. citrinum CY-H4 possessed mold-causing ability, and temperature and water activity are identified as the primary factors causing mold on tobacco leaves.

Key words: isolation and identification, tobacco leaf mildew, mold-causing, model simulation

XIANG Bo-ka, ZHOU Zuan-zuan, FENG Jia-hui, XIA Chen, LI Qi, CHEN Chun. Isolation and Identification of a Fungus from Moldy Tobacco Leaf and Study on Its Mold-causing Factors[J]. Biotechnology Bulletin, 2025, 41(2): 321-330.

Figures/Tables 8

Fig. 1 Colony growth morphology and phylogenetic tree of the purified isolation CY-H4A: Front view of the petri dish; B: back view of the petri dish; C: microscopic observation photos; D: growth of mold on tobacco leaves at 90% humidity; E: neighbor-joining phylogenetic tree constructed based on ITS sequences (Bootstrap =1 000)

Fig. 2 Effects of different temperatures on the colony areas of P. citrinum with 6 treatments of water activity (Initial inoculation concentration: 104 spore/mL)

Fig. 3 Effects of different temperatures on the colony areas of P. citrinum with 6 treatments of water activity (Initial inoculation concentration: 105 spore/mL)

Fig. 4 Effects of different temperatures on the colony areas of P. citrinum with 6 treatments of water activity (Initial inoculation concentration: 106 spore/mL)

Fig. 5 Variation in spore productions of P. citrinum after 7 d of culture at different initial inoculation concentrations

Table 1 Parameters estimated for the modified Gompertz model separately depicting the parameters related to colony growth rate (temperature and water activity) through colony area

参数 Parameter		Gompertz 模型 ^a Gompertz model ^a
参数 Parameter		平均值±标准误 Mean±SE	t	P
K₁		9.075±0.506	17.95	0.000 1
c₀		7.421±0.429	17.31	0.000 1
c₁		23.92±2.245	10.65	0.000 1
c₂		-1 518.8±99.97	15.19	0.000 1
c₃		-1.793±0.129	13.95	0.000 1
c₄		93.95±5.899	15.93	0.000 1
	ANOVA: r² =0.92, F_{5, 624}=1 367.54, P<0.000 1

Fig. 6 Model prediction of the changes of colony areas of P. citrinum with culture temperature and culture time under different water activity treatments

Fig. 7 Contribution ratio and cumulative contribution ratio of each factor in the mold-causing growth model

References 30

1	罗丽琼, 夏玉珍, 张天顺, 等. 储烟霉变原因及其防控技术研究进展［J］. 贵州农业科学, 2015, 43(8): 118-121.
	Luo LQ, Xia YZ, Zhang TS, et al. Research progress in moldy causes and control technology in stored tobacco [J]. Guizhou Agric Sci, 2015, 43(8): 118-121.
2	Zhou JX, Cheng Y, Yu LF, et al. Characteristics of fungal communities and the sources of mold contamination in mildewed tobacco leaves stored under different climatic conditions [J]. Appl Microbiol Biotechnol, 2022, 106(1): 131-144.
3	Welty RE, Vickroy DG. Evaluations of cigarettes made with mold-damaged and nondamaged flue-cured tobacco [J]. Contrib Tob Res, 1975, 8(2): 102-106.
4	Ross T. Indices for performance evaluation of predictive models in food microbiology [J]. J Appl Bacteriol, 1996, 81(5): 501-508.
5	Yang L, Yang QX, Yang SH, et al. Application of near infrared spectroscopy to detect mould contamination in tobacco [J]. J Infrared Spectrosc, 2015, 23(6): 391-400.
6	Pauly JL, Paszkiewicz G. Cigarette smoke, bacteria, mold, microbial toxins, and chronic lung inflammation [J]. J Oncol, 2011, 2011: 819129.
7	Chen L, Zhang QQ, Huang K, et al. Tumonoic acids K and L, novel metabolites from the marine-derived fungus Penicillium citrinum [J]. Heterocycles, 2012, 85(2): 413.
8	Zhou JX, Yu LF, Zhang J, et al. Characterization of the core microbiome in tobacco leaves during aging [J]. Microbiologyopen, 2020, 9(3): e984.
9	Zhou JX, Liu J, Wang DF, et al. Fungal communities are more sensitive to mildew than bacterial communities during tobacco storage processes [J]. Appl Microbiol Biotechnol, 2024, 108(1): 88.
10	卢灿华, 苏家恩, 盖晓彤, 等. 烤烟霉变病原菌鉴定及其生防菌筛选 [J]. 中国烟草科学, 2022, 43(2): 45-51.
	Lu CH, Su JN, Gai XT, et al. Causal pathogen identification and antagonistic bacteria screening of tobacco leaf mold [J]. Chinese Tobacco Science, 2022, 43(2):45-51.
11	Gao JN, Uwiringiyimana E, Zhang D. Microbial composition and diversity of the tobacco leaf phyllosphere during plant development [J]. Front Microbiol, 2023, 14: 1199241.
12	Zhang MZ, Guo DF, Wang HQ, et al. Analyzing microbial community and volatile compound profiles in the fermentation of cigar tobacco leaves [J]. Appl Microbiol Biotechnol, 2024, 108(1): 243.
13	王林, 王宇栋, 朱宝, 等. 仓储片烟中优势霉菌的分离鉴定及其霉变挥发性代谢产物研究 [J]. 河南农业科学, 2023, 52(3): 101-108.
	Wang L, Wang YD, Zhu B, et al. Isolation and identification of dominant mold and its moldy volatile metabolites in tobacco strips during storage [J]. J Henan Agric Sci, 2023, 52(3): 101-108.
14	潘祖贤, 蔡永占, 何鹏飞, 等. 烘烤期烟叶霉烂病病原菌的分离、鉴定及生物学特性 [J]. 中国烟草科学, 2019, 40(4): 42-47.
	Pan ZX, Cai YZ, He PF, et al. Isolation, identification and characterization of the pathogen of tobacco leaf mold during flue-curing [J]. Chin Tob Sci, 2019, 40(4): 42-47.
15	刘亚兵, 何腊平, 高泽鑫, 等. 食品微生物生长预测模型的研究 [J]. 食品工业, 2016, 37(11): 159-164.
	Liu YB, He LP, Gao ZX, et al. Research on predictive model of food microorganism growth [J]. Food Ind, 2016, 37(11): 159-164.
16	Kim JY, Jeon EB, Song M, et al. Development of predictive growth models of Aeromonas hydrophila on raw tuna Thunnus orientalis as a function of storage temperatures [J]. LWT, 2022, 156: 113052.
17	Pei PG, Xiong K, Wang XY, et al. Predictive growth kinetic parameters and modelled probabilities of deoxynivalenol production by Fusarium graminearum on wheat during simulated storing conditions [J]. J Appl Microbiol, 2022, 133(2): 349-361.
18	Mousa W, Ghazali FM, Jinap S, et al. Modelling the effect of water activity and temperature on growth rate and aflatoxin production by two isolates of Aspergillus flavus on paddy [J]. J Appl Microbiol, 2011, 111(5): 1262-1274.
19	Feng JH, Zhou ZZ, Xiong QY, et al. Exploration on the mildew conditions of Cladosporium asperulatum CY-H1, a pathogenic fungus on tobacco leaves [J]. Annu Res Rev Biol, 2024, 39: 48-57.
20	White TJ, Bruns T, Lee S, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics [M]//PCR Protocols. Amsterdam: Elsevier, 1990: 315-322.
21	Berger RD. Comparison of the gompertz and logistic equations to describe plant disease progress [J]. Phytopathology, 1981, 71(7):716.
22	唐启义, 冯明光. 实用统计分析及其DPS数据处理系统 [M]. 北京: 科学出版社, 2002.
	Tang QY, Feng MG. DPS data processing system for practical statistics [M]. Beijing: Science Press, 2002.
23	Chen QL, Cai L, Wang HC, et al. Fungal composition and diversity of the tobacco leaf phyllosphere during curing of leaves [J]. Front Microbiol, 2020, 11: 554051.
24	李魁, 李廷生, 王平诸, 等. 我国烟草真菌区系调查及霉变成因的研究 [J]. 郑州工程学院学报, 2003, 24(3): 20-24.
	Li K, Li TS, Wang PZ, et al. Study on mycoflora in tobacco and the cause of its going midewed [J]. J Zhengzhou Inst Technol, 2003, 24(3): 20-24.
25	李跃锋, 王锐亮, 陈河祥, 等. 烟支霉变微生物的鉴定及其生物学特性分析 [J]. 烟草科技, 2017, 50(6): 9-15.
	Li YF, Wang RL, Chen HX, et al. Identification and biological characterization of microorganisms resulted mildew cigarette [J]. Tob Sci Technol, 2017, 50(6): 9-15.
26	黄浦, 肖瑜, 刘以清, 等. 桔青霉降解茶皂素的实验研究 [J]. 桂林理工大学学报, 2016, 36(3): 592-596.
	Huang P, Xiao Y, Liu YQ, et al. Degradation of tea saponin by Penicillium citrinum [J]. J Guilin Univ Technol, 2016, 36(3): 592-596.
27	李文谦, 张恒, 茅燕勇. 桔青霉在小麦中最大比生长速率的温度 - 水分活度复合模型的建立 [J]. 食品科技, 2011, 36(11): 135-139.
	Li WQ, Zhang H, Mao YY. Development of predictive model for combined effect of temperature and water activity on the maximum specific growth rate of Penicillium citrinum on wheat [J]. Food Sci Technol, 2011, 36(11): 135-139.
28	罗云, 陈斌, 李凤丽, 等. 引起贮存期烟叶霉变的深层原因综述 [J]. 昆明学院学报, 2020, 42(6): 13-15, 34.
	Luo Y, Chen B, Li FL, et al. Summary on deeper reasons of moldiness of tobacco leaves during storage [J]. J Kunming Univ, 2020, 42(6): 13-15, 34.
29	徐晓光, 李红娟, 田振伟. 基于HP-Elman-LSSVM模型的仓储烟草霉变预测 [J]. 中国烟草科学, 2015, 36(4): 102-105.
	Xu XG, Li HJ, Tian ZW. Prediction of warehouse tobacco mildew based on HP-Elman-LSSVM model [J]. Chin Tob Sci, 2015, 36(4): 102-105.
30	张利华, 马钧钊, 勒国庆, 等. 基于BP神经网络的仓储烟草霉变预测 [J]. 华东交通大学学报, 2013, 30(3): 71-75.
	Zhang LH, Ma JZ, Le GQ, et al. Mildew prediction of warehousing tobacco based on BP neural network [J]. J East China Jiaotong Univ, 2013, 30(3): 71-75.

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