两株生防菌株对苹果树腐烂病菌拮抗作用的研究
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摘要
随着苹果树大面积种植和集约化生产的发展，苹果树腐烂病（Apple tree valsacanker）已成为世界各国苹果生产上一种毁灭性的病害，对苹果产业的发展已造成了严重的影响。因此本试验对甘肃省苹果树腐烂病病原、潜育期、诱变菌株致病力和黄腐酸对苹果树腐烂病菌的抑制作用及诱导抗病性进行了鉴定和测定，以及对苹果树腐烂病菌的拮抗菌鉴定、抑制作用进行了研究，取得的结果如下：
1．综合形态学和分子生物学鉴定，确定了引起甘肃省苹果树腐烂病的病原主要为Valsa ceratosperma（无性态为Cytospora mandshurica），其次为C.carphosperma。
2．苹果树腐烂病菌的潜育期随保湿时间增加而缩短、枝龄的增加而增加，温度30℃时，光暗交替潜育期最短。
3．紫外诱变后腐烂病菌5个菌株致病力均增强，其中菌株LZU-5致病力增强幅度最大，其相对增长率为115.94%，菌株LZU-2增强幅度最小，为14.50%；微波诱变后腐烂病菌5个菌株致病力均减弱，其中菌株LZM-2减弱幅度最大，其相对降低率为46.38%，菌株LZM-1减弱幅度最小，为20.29%。
4．黄腐酸对腐烂病菌有诱导抗性作用，经黄腐酸处理后腐烂病组织中各种酶活性较对照明显提高，其中SOD、POD、PAL和CAT均呈现先升高后降低的趋势。
5．通过培养形状、革兰氏染色、16SrDNA序列分析鉴定，将筛选出的拮抗菌LZ-1201菌株初步鉴定为枯草芽孢杆菌（Bacillus subtilis），TS-1203菌株初步鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。
6.两株拮抗菌LZ-1201和TS-1203对腐烂病菌孢子萌发和菌丝生长具有明显的抑制作用，可使腐烂病菌菌丝变细、扭曲、内含物浓缩，末端膨大畸形、颜色加深，菌丝节间内含物溢出、菌丝断裂。
Apple tree valsa canker is one of the most destructive diseases all over the worldwith the development of apple trees planting and intensive production. It has causedseriously affects on the development of apple industry. Therefore, the pathogen ofapple tree valsa canker, the incubation period of apple tree valsa canker pathogen andpathogenicity of induced strains were identified and determined. The inhibitoryeffects and induced disease resistance of fulvic acid, screening and Identification ofantagonistic bacteria, inhibitory effects and mechanism to apple tree valsa cankerwere studied in this experiment. The main results were listed as follows.
1.Morphological and molecular biological identification were used for theidentification of apple valsa canker in Gansu Province. It was made clear that applevalsa canker caused mainly by Valsa ceratosperma(the anamorph was Cytosporamandshurica), followed by C. carphosperma.
2. Incubation period of apple valsa canker decreases with the increase ofmoisture time.While incubation period increased when branch age increased.Incubation period of the alternation of light and darkness every12hours was theshortest at30℃.
3. Five isolates of apple tree valsa canker pathogen were enhanced by UVmutagenesis. Pathogenicity of LZU-5isolate showed maximum enhancement extent,the relative growth rate was115.94%. Enhancement extent of LZU-2isolatepathogenicity was the smallest, the relative growth rate was14.50%. Pathogenicity ofLZM-2isolate showed maximum decreasing extent, the relative decreasing rate was46.38%. Decreasing extent of LZM-1isolate pathogenicity was the smallest, therelative decreasing rate was20.29%.
4．Fulvic acid had induced resistance to apple tree valsa canker pathogen.Enzyme activities of apple tree valsa canker tissue was significantly differentcompared with the control. SOD, POD, PAL and CAT increased at first and thendecreased.
5．Based on properties of morphology, gram stain and16SrDNA sequenceanalysis. LZ-1201isolate and TS-1203isolate were defined respectively as Bacillussubtilis and Bacillus amyloliquefaciens.
6. Two antagonistic bacteria LZ-1201and TS-1203had obvious inhibition tospore germination and mycelial growth of apple tree valsa canker pathogen. Observedby light microscope, Two antagonistic bacteria had teratogenicety to myceliummorphology and cellular structure of apple tree valsa canker pathogen, includingmycelial tapering, twisting, inclusions concentrated, the enlargement deformity of end,color deepened, inclusion overflow of mycelium internode, mycelium break.
1．综合形态学和分子生物学鉴定，确定了引起甘肃省苹果树腐烂病的病原主要为Valsa ceratosperma（无性态为Cytospora mandshurica），其次为C.carphosperma。
2．苹果树腐烂病菌的潜育期随保湿时间增加而缩短、枝龄的增加而增加，温度30℃时，光暗交替潜育期最短。
3．紫外诱变后腐烂病菌5个菌株致病力均增强，其中菌株LZU-5致病力增强幅度最大，其相对增长率为115.94%，菌株LZU-2增强幅度最小，为14.50%；微波诱变后腐烂病菌5个菌株致病力均减弱，其中菌株LZM-2减弱幅度最大，其相对降低率为46.38%，菌株LZM-1减弱幅度最小，为20.29%。
4．黄腐酸对腐烂病菌有诱导抗性作用，经黄腐酸处理后腐烂病组织中各种酶活性较对照明显提高，其中SOD、POD、PAL和CAT均呈现先升高后降低的趋势。
5．通过培养形状、革兰氏染色、16SrDNA序列分析鉴定，将筛选出的拮抗菌LZ-1201菌株初步鉴定为枯草芽孢杆菌（Bacillus subtilis），TS-1203菌株初步鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。
6.两株拮抗菌LZ-1201和TS-1203对腐烂病菌孢子萌发和菌丝生长具有明显的抑制作用，可使腐烂病菌菌丝变细、扭曲、内含物浓缩，末端膨大畸形、颜色加深，菌丝节间内含物溢出、菌丝断裂。
Apple tree valsa canker is one of the most destructive diseases all over the worldwith the development of apple trees planting and intensive production. It has causedseriously affects on the development of apple industry. Therefore, the pathogen ofapple tree valsa canker, the incubation period of apple tree valsa canker pathogen andpathogenicity of induced strains were identified and determined. The inhibitoryeffects and induced disease resistance of fulvic acid, screening and Identification ofantagonistic bacteria, inhibitory effects and mechanism to apple tree valsa cankerwere studied in this experiment. The main results were listed as follows.
1.Morphological and molecular biological identification were used for theidentification of apple valsa canker in Gansu Province. It was made clear that applevalsa canker caused mainly by Valsa ceratosperma(the anamorph was Cytosporamandshurica), followed by C. carphosperma.
2. Incubation period of apple valsa canker decreases with the increase ofmoisture time.While incubation period increased when branch age increased.Incubation period of the alternation of light and darkness every12hours was theshortest at30℃.
3. Five isolates of apple tree valsa canker pathogen were enhanced by UVmutagenesis. Pathogenicity of LZU-5isolate showed maximum enhancement extent,the relative growth rate was115.94%. Enhancement extent of LZU-2isolatepathogenicity was the smallest, the relative growth rate was14.50%. Pathogenicity ofLZM-2isolate showed maximum decreasing extent, the relative decreasing rate was46.38%. Decreasing extent of LZM-1isolate pathogenicity was the smallest, therelative decreasing rate was20.29%.
4．Fulvic acid had induced resistance to apple tree valsa canker pathogen.Enzyme activities of apple tree valsa canker tissue was significantly differentcompared with the control. SOD, POD, PAL and CAT increased at first and thendecreased.
5．Based on properties of morphology, gram stain and16SrDNA sequenceanalysis. LZ-1201isolate and TS-1203isolate were defined respectively as Bacillussubtilis and Bacillus amyloliquefaciens.
6. Two antagonistic bacteria LZ-1201and TS-1203had obvious inhibition tospore germination and mycelial growth of apple tree valsa canker pathogen. Observedby light microscope, Two antagonistic bacteria had teratogenicety to myceliummorphology and cellular structure of apple tree valsa canker pathogen, includingmycelial tapering, twisting, inclusions concentrated, the enlargement deformity of end,color deepened, inclusion overflow of mycelium internode, mycelium break.
引文
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