牡丹叶斑病病原真菌鉴定及生物学特性研究
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摘要
随着我国牡丹种植面积的扩大、老园区种植年限的延长及对外交流频次的增加,发生在牡丹上的老病害为害日益严重,新病害种类不断增加,特别是真菌病害的为害已成为制约我国牡丹产业发展的重要因素之一。目前,我国关于牡丹病害的系统调查和病原鉴定工作研究的较少。本试验选择国家牡丹园、洛阳市国花园、白云山高山牡丹园等主要牡丹种植园、圃,对牡丹叶部真菌病害展开调查,根据病害症状和病原菌的形态特征,并结合分子生物学手段进行病原分类鉴定。
调查发现了牡丹叶部真菌病害10种,以黑斑病、叶霉病、红点病为害最为严重。其中牡丹黑斑病等7种病害的病原经形态学鉴定已经确定,分别是( Alternaria. su?ruticosae、A. su?ruticosicola、A. tenuissima )、芍药尾孢(Cercospora paeoniae)、牡丹枝孢(Cladosporium paeoniae Pass)、牡丹葡萄孢菌(Botrytis paeoniae Oudem)、牡丹白粉菌(Erysiphe paeoniae)、牡丹拟盘多毛孢(Pestalotiopsis paeoniae)、斑点叶点霉(Phyllosticta commonsii)。而牡丹红点病(菌株I)、瘤点病(菌株II)与叶斑病(菌株III)的病原仅靠形态学观察难以确定,需利用分子手段进一步研究。
对分离纯化的菌株I、菌株II、菌株III的rDNA的ITS序列进行了PCR扩增,并对扩增产物进行了克隆和序列分析。结果表明,菌株I、菌株II与GENBANK中编号为AY487094.1的Pilidium concavum ITS区段进行了比对,其同源性分别为100%和99.58%,表明菌株I和菌株II均为Pilidium concavum;菌株III初定为双毛壳孢属真菌Discosia sp.。
对Pilidium concavum生物学特性的研究结果表明,该菌菌丝生长的最适培养基为PDA,分生孢子产生的最适培养基为PYA。在最适培养基上培养,菌丝生长的最适温度为25℃,产孢的最适温度为30℃;在pH值6～11范围内该菌均能生长和产孢,菌丝生长的最适pH值为6,pH值为11时产孢最好,pH值为5时孢子萌发达到最大;光照对菌丝生长、产孢及孢子萌发无显著影响;在供试的碳源中,菌丝生长及产孢均以葡萄糖最好,氮源以酵母膏最好;RH在65%～100%孢子均能萌发,萌发率随湿度增大而升高,在饱和湿度时萌发率最高,达到94.51%;孢子的致死温度为51℃10min。
本试验以形态学为基础,在分子水平上鉴定了牡丹叶斑病病原真菌,发现了Pilidium concavum、Discosia sp.两种国内新病原,探讨了P. concavum的生物学特性,对进一步揭示牡丹真菌病害及其防治具有重要意义。
With the expansion of cultivated area and plant life of Peony in China and the increase in frequency of foreign exchange, new diseases continue to rise and the damage of old diseases, especially fungal diseases, has been a key facter in restricting the industrial development of Peony in China. Currently, the studies on systematic investigation of peony diseases and pathogen identification is very little in our country. In order to investigate the peony leaf fungal diseases, this study selected the country Peony Garden, Luoyang City of Garden, Baiyun Mountain Alpine Peony Garden and other major peony plantations, nurseries to identify pathogens according to symptoms and morphology, combined with molecular biology characteristics.
Ten kinds of fungal diseases were found and black spot, leaf mold and red spot are the most serious diseases. Seven kinds of pathogens included black spot had been identified on morphology, and they were Alternaria su?ruticosae, A. su?ruticosicola, A. tenuissima, Cercospora paeoniae, Cladosporium paeoniae Pass, Botrytis paeoniae Oudem, Erysiphe paeoniae, Pestalotiopsis paeoniae and Phyllosticta commons. However, Peony red spot (strain 1), Peony tumor spot (strain 2) and Peony leaf spot (strain 3) were difficult to determine by their morphological character alone, so further study is needed at the molecular level.
PCR amplification products of ITS of rDNA of the three strains obtained were sequenced. Compared strain1 and strain 2 with the ITS sequnces of Pilidium concavum numbered AY487094.1 of GENBANK, the homologies were 100% and 99.58% seperately. The results showed that both strain 1 and strain 2 were Pilidium concavum, while strain 3 is Discosia sp..
Studies on the biological characteristics of Pilidium concavum showed that the optimum grown was PDA, the optimum sporulation was PYA. The optimum grown temperature was 25℃, the optimum temperature for sporulation was 30℃. The optimum growth pH was 6, sporulation was a maximum of pH 11. Light had no significant effect on mycelial growth and sporulation. Glucose and yeast were the most suitable carbon and nitrogen sources for mycelial growth. Conidia germinated during RH 65%-100% and the germination rate increased with the increase of RH. when RH is saturated, spore reached its maximum germination rate 94.51%. The lethiferous temperature of conidia was 51℃for l0 min.
In this study, based on morphology, pathogenic fungi of Peony leaf spot were further identified at the molecular level. The results indicate that Pilidium concavum and Discosia sp. are new pathogens on Peony in our country. Biological characteristics of P. concavum had a great significance on further revealing the prevention and treatment of fungal diseases of peony.
调查发现了牡丹叶部真菌病害10种,以黑斑病、叶霉病、红点病为害最为严重。其中牡丹黑斑病等7种病害的病原经形态学鉴定已经确定,分别是( Alternaria. su?ruticosae、A. su?ruticosicola、A. tenuissima )、芍药尾孢(Cercospora paeoniae)、牡丹枝孢(Cladosporium paeoniae Pass)、牡丹葡萄孢菌(Botrytis paeoniae Oudem)、牡丹白粉菌(Erysiphe paeoniae)、牡丹拟盘多毛孢(Pestalotiopsis paeoniae)、斑点叶点霉(Phyllosticta commonsii)。而牡丹红点病(菌株I)、瘤点病(菌株II)与叶斑病(菌株III)的病原仅靠形态学观察难以确定,需利用分子手段进一步研究。
对分离纯化的菌株I、菌株II、菌株III的rDNA的ITS序列进行了PCR扩增,并对扩增产物进行了克隆和序列分析。结果表明,菌株I、菌株II与GENBANK中编号为AY487094.1的Pilidium concavum ITS区段进行了比对,其同源性分别为100%和99.58%,表明菌株I和菌株II均为Pilidium concavum;菌株III初定为双毛壳孢属真菌Discosia sp.。
对Pilidium concavum生物学特性的研究结果表明,该菌菌丝生长的最适培养基为PDA,分生孢子产生的最适培养基为PYA。在最适培养基上培养,菌丝生长的最适温度为25℃,产孢的最适温度为30℃;在pH值6～11范围内该菌均能生长和产孢,菌丝生长的最适pH值为6,pH值为11时产孢最好,pH值为5时孢子萌发达到最大;光照对菌丝生长、产孢及孢子萌发无显著影响;在供试的碳源中,菌丝生长及产孢均以葡萄糖最好,氮源以酵母膏最好;RH在65%～100%孢子均能萌发,萌发率随湿度增大而升高,在饱和湿度时萌发率最高,达到94.51%;孢子的致死温度为51℃10min。
本试验以形态学为基础,在分子水平上鉴定了牡丹叶斑病病原真菌,发现了Pilidium concavum、Discosia sp.两种国内新病原,探讨了P. concavum的生物学特性,对进一步揭示牡丹真菌病害及其防治具有重要意义。
With the expansion of cultivated area and plant life of Peony in China and the increase in frequency of foreign exchange, new diseases continue to rise and the damage of old diseases, especially fungal diseases, has been a key facter in restricting the industrial development of Peony in China. Currently, the studies on systematic investigation of peony diseases and pathogen identification is very little in our country. In order to investigate the peony leaf fungal diseases, this study selected the country Peony Garden, Luoyang City of Garden, Baiyun Mountain Alpine Peony Garden and other major peony plantations, nurseries to identify pathogens according to symptoms and morphology, combined with molecular biology characteristics.
Ten kinds of fungal diseases were found and black spot, leaf mold and red spot are the most serious diseases. Seven kinds of pathogens included black spot had been identified on morphology, and they were Alternaria su?ruticosae, A. su?ruticosicola, A. tenuissima, Cercospora paeoniae, Cladosporium paeoniae Pass, Botrytis paeoniae Oudem, Erysiphe paeoniae, Pestalotiopsis paeoniae and Phyllosticta commons. However, Peony red spot (strain 1), Peony tumor spot (strain 2) and Peony leaf spot (strain 3) were difficult to determine by their morphological character alone, so further study is needed at the molecular level.
PCR amplification products of ITS of rDNA of the three strains obtained were sequenced. Compared strain1 and strain 2 with the ITS sequnces of Pilidium concavum numbered AY487094.1 of GENBANK, the homologies were 100% and 99.58% seperately. The results showed that both strain 1 and strain 2 were Pilidium concavum, while strain 3 is Discosia sp..
Studies on the biological characteristics of Pilidium concavum showed that the optimum grown was PDA, the optimum sporulation was PYA. The optimum grown temperature was 25℃, the optimum temperature for sporulation was 30℃. The optimum growth pH was 6, sporulation was a maximum of pH 11. Light had no significant effect on mycelial growth and sporulation. Glucose and yeast were the most suitable carbon and nitrogen sources for mycelial growth. Conidia germinated during RH 65%-100% and the germination rate increased with the increase of RH. when RH is saturated, spore reached its maximum germination rate 94.51%. The lethiferous temperature of conidia was 51℃for l0 min.
In this study, based on morphology, pathogenic fungi of Peony leaf spot were further identified at the molecular level. The results indicate that Pilidium concavum and Discosia sp. are new pathogens on Peony in our country. Biological characteristics of P. concavum had a great significance on further revealing the prevention and treatment of fungal diseases of peony.
引文
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