不同散斑壳真菌致病性比较及产毒条件优化
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摘要
松属植物多为先锋树种,在荒山造林、改善生念环境、提供用材、医用等方面造福人类。散斑壳属Lophodermium spp.真菌引起的松落针病可危害松Pinus、冷杉Abies、云杉Picea、落叶松Larix等属,对育林、造林及林木材积造成巨大损失。作者对四川省四种松落针病原菌(针叶树散斑壳、松针散斑壳、四川散斑壳、二郎山散斑壳)的培养特性、致病物质成分分析、离体产毒条件、毒素基本性质及胞外水解酶活性变化进行了较系统的研究,以期了解散斑壳真菌的致病机理及不同种散斑壳真菌在致病性上的差异,从而为松属植物抗落针病育种及松落针病的防治提供可靠的理论基础。
本研究对四种散斑壳真菌在培养基平板上的生长速度进行比较,发现四种真菌在麦芽汁固体培养基上生长最快,且在不同培养基上生长形态存在差异。四种散斑壳真菌之间在培养基上生长情况表明其亲和性与其遗传相似系数不完全吻合。
用高山松、华山松、云南松、马尾松松针作生测材料对四种散斑壳真菌的代谢产物进行生测,结合对材料细胞膜的电导率测定,结果表明散斑壳真菌的代谢产物对生测材料有毒害作用,导致松针上产生黄色段斑、整个针叶枯死,并且在华山松、云南松及高山松上,还会产生黑色的环状线纹,与自然发病的症状极为相似。其中蛋白质类组份毒性不明显,非蛋白质类组份是致病成份。结合对雪松松针和三种不同科属杂草进行生测,结果表明四种散斑壳真菌的毒素为非专化性毒素。
作者对四种散斑壳真菌的产毒条件进行筛选,得出四种散斑壳病原真菌在pH4.0-6.0的PD培养基中,12hr半光照/d下20-23℃静止培养25d时产毒最佳。这些条件与其菌丝生长、产孢既有一定联系,又有一定差异,表明这四种散斑壳病原真菌的生长与产毒条件不完全一致。
散斑壳真菌毒素粗提液的基本性质研究显示:四种散斑壳真菌毒素对100℃以下的温度有较高的稳定性,随着处理温度的升高,四种毒素对四种寄主松针的伤害率呈下降趋势,但是在121℃下仍具有一定活性。四种毒素粗提液在pH 7.0以上时,毒性会有所下降。不同生测温度下,四种毒素均有毒性,但是在温度交替下对细胞膜的伤害程度较高。四种散斑壳真菌毒素为极性较大的物质。经过筛选,利用氯仿:乙酸乙酯:乙醇=6:3:1进行分离,组分较多,差异性明显,且R_f值较合理,可作为四种散斑壳真菌毒素的最佳展开剂。其中5号针叶树散斑壳和9号松针散斑壳真菌毒素粗提液所分离获得的组分毒性较强,而19号四川散斑壳真菌毒素粗提液所分离的组分最多,但其中一些组分不具有致病性。因此可以推论出四种散斑壳真菌的毒素粗提液在结构和生物活性上存在一定差异性。
四种散斑壳菌在PD半糖培养基中均分泌胞外纤维素酶、半纤维素酶、果胶酶及木质素酶,且分泌能力存在差异性,其中5号菌株分泌胞外水解酶的能力最强。在培养基中添加入松针后,四种散斑壳菌的产酶能力均得到了一定程度的提高。而且不同松针对不同菌种产酶能力的诱导作用具有差异性。四种散斑壳菌分泌的果胶酶随着时间变化一直呈上升趋势,说明了果胶酶在它们侵染松针中起主要作用。
Most of pinus are considered as pioneer species,which benefit mankind via afforestation. improvement of the ecological environment,timber supply,medical purpose and others. Lophodermium spp.fungi can cause needle cast disease to Pinus,Abies Abies,Picea,Larix, and so on,which will result in a great losses on breeding,afforestation and forest timber.In order to gain insight into the pathogenic mechanism of the toxin and pathogenicity differences of various species of fungi,cultural characteristics,toxin component and production conditions in vitro,basic nature and host-specific characteristics of the toxin,and extracellular hydrolytic enzyme activity changes of four Lophodermium spp.fungi(Lophodermium conigenum,L.pinastri,L.sichuanense and L.erlangshanense) isolated in Sichuan Province were systematically studied,expecting to obtain a basic and reliable theory for the resistance breeding of pine to needle cast and prevention and control these kinds of diseases.
By comparing the growth rate of four kinds of Lophodermium spp.fungi on medium plate,all of the four fungi were found to grow fastest in wort solid medium,but display different growth patterns in different media.The growth characteristics of the four fungi in the medium plate showed that the affinity with the genetic similarity coefficient did not fully coincide.
Bioassay of the bio-determination material from the needles of Pinus densata,P.armandi, P yunnanensis,P.massoniana,together with the data from conductivity of the cell membrane, showed that the four kinds of Lophodermium spp.fungal metabolites had toxic effect to the bio-materials,causing yellow spots on pine needles,death of the whole needles,and black ring lines on the Pinus armandi,P.yunnanensis,P.densata,pretty similar with the incidence and the natural symptoms.The toxic components were identified as non-protein,but not protein.The bioassay of Cedrus deodara and three kinds of weeds from different family showed that the four toxins were not host specific.
Toxin production conditions were optimized for the four kinds of Lophodermium spp. fungi.The optimal conditions for toxin production of four pathogenic fungi were pH4.0-6.0, 20-23℃,12h half-light/d,stilling culture for 25d in the PD medium.A little difference observed between toxin production and hyphae growth,spore production conditions,which revealed that toxin production conditions of the four kinds of Lophodermium spp.fungi were not completely consistent with their growth conditions.
Studies of the four kinds of toxin crude extracts traits showed that the four kinds of toxin crude extracts were stronger resistant to temperature under 100℃,and the pathogenicity of these toxin crude extracts decreased with increasing of temperature,but toxin activity was still detected even if temperature reached 121℃.The pathogenicity of the four kinds of toxin crude extracts decreased once pH value is above pH7.0.These toxin crude extracts had toxicity under different bioassay temperatures,but the cell membrane was damaged greater by the four kinds of bio-determination materials under alternating temperature.The four kinds of mycotoxins were larger polar substances.Chloroform:ethyl acetate:ethanol(6:3:1) was the best choice for preparation of toxin crude extracts,due to the best mobile phase with more different compositions and more reasonable Rf value.The mycotoxin components extracted from 5~(th) Lophodermium conigenum and the 9~(th) L.pinastri had more toxicity,and the 19~(th) L. sichuanense mycotoxin extract had the most components of the separation,but some components are not pathogenic.Therefore,we inferred that a certain differences of the four fungal toxin extracts should exist in structure and biological activity.
For all of the four kinds of Lophodermium spp.Fungi,a series of cell wall degrading enzymes(CWDEs) including cellulose,hemicellulose,pectase and ligninase were found in the PD semi-sugar medium;and these enzymes's activity were difference to some degree.The 5~(th) fungi had the highest ability to generate extracellular CWDEs.The enzyme production of the four kinds of fungi was improved by addition of the host pine needles to the medium,and induction effect of the different types of pine needles was difference in different kinds of fungi.The pectase played an important role in the process of pine needles infection by the four kinds of fungi according to the fact of over time increase of the pectase.
本研究对四种散斑壳真菌在培养基平板上的生长速度进行比较,发现四种真菌在麦芽汁固体培养基上生长最快,且在不同培养基上生长形态存在差异。四种散斑壳真菌之间在培养基上生长情况表明其亲和性与其遗传相似系数不完全吻合。
用高山松、华山松、云南松、马尾松松针作生测材料对四种散斑壳真菌的代谢产物进行生测,结合对材料细胞膜的电导率测定,结果表明散斑壳真菌的代谢产物对生测材料有毒害作用,导致松针上产生黄色段斑、整个针叶枯死,并且在华山松、云南松及高山松上,还会产生黑色的环状线纹,与自然发病的症状极为相似。其中蛋白质类组份毒性不明显,非蛋白质类组份是致病成份。结合对雪松松针和三种不同科属杂草进行生测,结果表明四种散斑壳真菌的毒素为非专化性毒素。
作者对四种散斑壳真菌的产毒条件进行筛选,得出四种散斑壳病原真菌在pH4.0-6.0的PD培养基中,12hr半光照/d下20-23℃静止培养25d时产毒最佳。这些条件与其菌丝生长、产孢既有一定联系,又有一定差异,表明这四种散斑壳病原真菌的生长与产毒条件不完全一致。
散斑壳真菌毒素粗提液的基本性质研究显示:四种散斑壳真菌毒素对100℃以下的温度有较高的稳定性,随着处理温度的升高,四种毒素对四种寄主松针的伤害率呈下降趋势,但是在121℃下仍具有一定活性。四种毒素粗提液在pH 7.0以上时,毒性会有所下降。不同生测温度下,四种毒素均有毒性,但是在温度交替下对细胞膜的伤害程度较高。四种散斑壳真菌毒素为极性较大的物质。经过筛选,利用氯仿:乙酸乙酯:乙醇=6:3:1进行分离,组分较多,差异性明显,且R_f值较合理,可作为四种散斑壳真菌毒素的最佳展开剂。其中5号针叶树散斑壳和9号松针散斑壳真菌毒素粗提液所分离获得的组分毒性较强,而19号四川散斑壳真菌毒素粗提液所分离的组分最多,但其中一些组分不具有致病性。因此可以推论出四种散斑壳真菌的毒素粗提液在结构和生物活性上存在一定差异性。
四种散斑壳菌在PD半糖培养基中均分泌胞外纤维素酶、半纤维素酶、果胶酶及木质素酶,且分泌能力存在差异性,其中5号菌株分泌胞外水解酶的能力最强。在培养基中添加入松针后,四种散斑壳菌的产酶能力均得到了一定程度的提高。而且不同松针对不同菌种产酶能力的诱导作用具有差异性。四种散斑壳菌分泌的果胶酶随着时间变化一直呈上升趋势,说明了果胶酶在它们侵染松针中起主要作用。
Most of pinus are considered as pioneer species,which benefit mankind via afforestation. improvement of the ecological environment,timber supply,medical purpose and others. Lophodermium spp.fungi can cause needle cast disease to Pinus,Abies Abies,Picea,Larix, and so on,which will result in a great losses on breeding,afforestation and forest timber.In order to gain insight into the pathogenic mechanism of the toxin and pathogenicity differences of various species of fungi,cultural characteristics,toxin component and production conditions in vitro,basic nature and host-specific characteristics of the toxin,and extracellular hydrolytic enzyme activity changes of four Lophodermium spp.fungi(Lophodermium conigenum,L.pinastri,L.sichuanense and L.erlangshanense) isolated in Sichuan Province were systematically studied,expecting to obtain a basic and reliable theory for the resistance breeding of pine to needle cast and prevention and control these kinds of diseases.
By comparing the growth rate of four kinds of Lophodermium spp.fungi on medium plate,all of the four fungi were found to grow fastest in wort solid medium,but display different growth patterns in different media.The growth characteristics of the four fungi in the medium plate showed that the affinity with the genetic similarity coefficient did not fully coincide.
Bioassay of the bio-determination material from the needles of Pinus densata,P.armandi, P yunnanensis,P.massoniana,together with the data from conductivity of the cell membrane, showed that the four kinds of Lophodermium spp.fungal metabolites had toxic effect to the bio-materials,causing yellow spots on pine needles,death of the whole needles,and black ring lines on the Pinus armandi,P.yunnanensis,P.densata,pretty similar with the incidence and the natural symptoms.The toxic components were identified as non-protein,but not protein.The bioassay of Cedrus deodara and three kinds of weeds from different family showed that the four toxins were not host specific.
Toxin production conditions were optimized for the four kinds of Lophodermium spp. fungi.The optimal conditions for toxin production of four pathogenic fungi were pH4.0-6.0, 20-23℃,12h half-light/d,stilling culture for 25d in the PD medium.A little difference observed between toxin production and hyphae growth,spore production conditions,which revealed that toxin production conditions of the four kinds of Lophodermium spp.fungi were not completely consistent with their growth conditions.
Studies of the four kinds of toxin crude extracts traits showed that the four kinds of toxin crude extracts were stronger resistant to temperature under 100℃,and the pathogenicity of these toxin crude extracts decreased with increasing of temperature,but toxin activity was still detected even if temperature reached 121℃.The pathogenicity of the four kinds of toxin crude extracts decreased once pH value is above pH7.0.These toxin crude extracts had toxicity under different bioassay temperatures,but the cell membrane was damaged greater by the four kinds of bio-determination materials under alternating temperature.The four kinds of mycotoxins were larger polar substances.Chloroform:ethyl acetate:ethanol(6:3:1) was the best choice for preparation of toxin crude extracts,due to the best mobile phase with more different compositions and more reasonable Rf value.The mycotoxin components extracted from 5~(th) Lophodermium conigenum and the 9~(th) L.pinastri had more toxicity,and the 19~(th) L. sichuanense mycotoxin extract had the most components of the separation,but some components are not pathogenic.Therefore,we inferred that a certain differences of the four fungal toxin extracts should exist in structure and biological activity.
For all of the four kinds of Lophodermium spp.Fungi,a series of cell wall degrading enzymes(CWDEs) including cellulose,hemicellulose,pectase and ligninase were found in the PD semi-sugar medium;and these enzymes's activity were difference to some degree.The 5~(th) fungi had the highest ability to generate extracellular CWDEs.The enzyme production of the four kinds of fungi was improved by addition of the host pine needles to the medium,and induction effect of the different types of pine needles was difference in different kinds of fungi.The pectase played an important role in the process of pine needles infection by the four kinds of fungi according to the fact of over time increase of the pectase.
引文
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