一株烟叶霉变真菌的分离鉴定及其致霉因素研究

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

摘要/Abstract

摘要：

目的开展烟叶霉变真菌的生物学研究对卷烟产业具有重要经济意义。方法利用平板分离法对致霉真菌进行分离纯化，利用测序技术和遗传进化分析对所得菌株进行鉴定，利用回接试验对所得菌株进行致霉性检测，利用生长检测确定所得菌株的致霉因素，利用模型模拟确定致霉因素的相关性。结果从霉变烟叶表面分离得到一株致霉真菌，经形态学及ITS序列同源性分析确定该菌株与Penicillium citrinum具有99.82%的同源性；致霉性实验证实，该菌株在相对湿度90%条件下可以导致烟叶霉变。致霉因素分析发现，该菌株的最适菌落生长及产孢条件为30℃和水活度为0.99。Gompertz模型模拟和因子分析表明，烟叶发生霉变与烟叶接触到的初始真菌孢子数量无显著的相关性，但与温度、水活度和温度水活度的互作密切相关。结论证实了Penicillium citrinum CY-H4具有致霉能力，温度和水活度是该菌引发烟叶霉变的主要致霉因素。

关键词: 分离鉴定, 烟叶霉变, 致霉, 模型模拟

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

项波卡, 周钻钻, 冯佳卉, 夏琛, 李奇, 陈春. 一株烟叶霉变真菌的分离鉴定及其致霉因素研究[J]. 生物技术通报, 2025, 41(2): 321-330.

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.

图/表 8

图1 分离纯化的霉菌CY-H4的菌落生长形态及其系统发育树A：培养皿正面照片；B：培养皿背面照片；C：显微观察照片；D：霉菌90%湿度下在烟叶上的生长情况；E：基于ITS序列构建的Neighbor-Joining系统发育树（Bootstrap =1 000）

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)

图2 六组水活度处理下不同温度影响橘青霉菌落面积变化（初始接种浓度：104个/mL）

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)

图3 六组水活度处理下不同温度影响橘青霉菌落面积变化（初始接种浓度：105个/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)

图4 六组水活度处理下不同温度影响橘青霉菌落面积变化（初始接种浓度：106个/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)

图5 不同初始接种浓度下橘青霉培养7 d后产孢量的变化A: 104个/mL; B: 105个/mL; C: 106个/mL

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

表1 比生长速度相关参数（温度和水活度）与菌落面积拟合改良型Gompertz模型的参数估计

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

图6 橘青霉不同水活度处理下菌落面积随培养温度、培养时间变化的模型预测A: Aw =0.77; B: Aw =0.83; C: Aw =0.87; D: Aw =0.90; E: Aw =0.95; F: Aw =0.99

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

图7 致霉生长模型中各因素的贡献比例及累计贡献比例

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

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