壶瓶枣褐斑病的病原鉴定和防治研究
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摘要
壶瓶枣褐斑病为近年来山西壶瓶枣发生的一种严重的果实病害,在田间发病初期主要在枣果的果顶或果肩部位出现红色的病斑,病斑凹陷,后期变为红褐色,病斑的边缘清晰,除病斑外的其他部位的果肉既不腐烂也不变色。该病严重发生时可使果实发病率高达50%以上,给红枣的产量和品质造成很大影响。本实验通过分离病原菌,用形态学和分子生物学方法对病菌进行了鉴定,并筛选出对病菌有抑制作用的拮抗菌和化学药剂,为此病害的田间防治奠定了基础,主要研究结果如下:
1病原菌的分离和鉴定
以壶瓶枣病果为材料,分离了30个枣果的150个病组织块,分离获得135个菌株,根据菌落特征发现各菌株没有明显差异,为同一种类型的真菌。通过室内和田间致病性测定以及人工接种后再分离,证明编号为CN535的真菌菌株为该病的致病菌。该病菌在PDA上培养7 d菌落直径达69.2～73.5 mm,基内菌丝和气生菌丝均发达,具明显的浅灰与墨绿色的同心轮纹;分生孢子单生或短链生,具纵横隔膜和短喙,大小为22.5～40.0×8.0～13.5μm,为典型的Alternaria属真菌特征。在PDA和PCA培养基上进行产孢培养,结果发现在细长分生孢子梗的上部形成特征性的具短分枝的孢子链,支链一般长1～5个孢子,似小树丛状,为A. alternata的特征。其rDNA-ITS序列分析结果表明该菌与A. alternata、A. tenuissima、A. longipes、A. mali和A. citri的同源性均为100%。用两对链格孢菌的特异性引物AAF2/AAR3和Aalt-F/Aalt-R分别扩增出相对应的341 bp和450 bp的片段。综合形态特征和分子分析结果,确定壶瓶枣褐斑病的病原菌为A. alternata (Fries)Keissler。
2内生菌的分离和拮抗菌的室内筛选
在PDA、KB和NA三种培养基上分离枣果的内生菌,纯化后,根据形态将其确定为同一种菌,在KB上菌落呈黄绿色,边缘平滑。从土壤中直接分离和筛选拮抗菌,共得到16个菌株,经过测定这些菌株对病原菌的菌丝生长均无明显的抑制作用。从经过高温预处理的土壤中分离拮抗菌的比例比常温下从土壤中分离拮抗菌的比例高,但是并不呈正比关系。同时得到4个抑菌率90.0%以上的菌株和11个抑菌率在80.0%～90.0%之间的菌株。生防细菌中CHA0r、WCS358::phl和SB对病原菌菌丝生长抑制率分别达到54.7%、51.2%和46.5%。CHA0r、ZN和WCS358::phl的抑菌圈直径分别达到14.3 mm、12.7 mm和12.7 mm,抑菌圈效果明显。生防链霉菌中龟裂链霉菌和武夷菌素链霉菌对菌丝生长抑制率分别达到58.0%和67.3%,抑菌圈直径分别达到30.0 mm和25.3 mm,抑制作用均较明显。而菌株CN120和CN121对褐斑病菌菌丝生长不但没有抑制作用,反而促使其菌落生长。
3化学药剂防治壶瓶枣褐斑病
选用9种常见的杀菌剂室内测定对壶瓶枣褐斑病菌Alternaria alternata的毒力。采用生长速率抑制法和悬滴法分别测定了9种杀菌剂对该病菌的菌丝生长和孢子萌发的抑制作用,并测定了其中6种杀菌剂对该病菌的毒力。同时在室内测定了6种药剂对病原菌的防治效果,将室内防效好的药剂用于田间防效测定。结果表明,异菌脲、代森锰锌、施力科、剑斑和多菌灵对病菌菌丝生长抑制作用较强,抑菌率均达到90.0%以上;代森锰锌、剑斑、多菌灵、炭阻绝和施力科对病菌孢子萌发抑制活性比较强,抑菌率也达到85.0%以上。毒力测定结果表明,异菌脲、炭阻绝、施力科、多菌灵、剑斑和代森锰锌的有效中浓度(EC50)分别为0.2,11.1,14.5,20.8,28.5和149.9 mg/L,其中异菌脲的有效中浓度最低,相对抑制效果最好;施力科和多菌灵次之;代森锰锌最差。室内防效测定,发现施力科、剑斑、代森锰锌和异菌脲的防效较好,而炭阻绝和多菌灵的防效较差。6种药剂的相关系数均在0.90以上,表明药剂浓度与抑制作用呈现较高的正相关。田间防效测定结果表明,异菌脲的田间防治效果最好,防效达到72.9%,施力科和剑斑稍差,代森锰锌的防治效果最差,仅达到25.6%。
Brown spot in Ziziphus jujuba Mill. cv. Hupingzao was a serious fruit disease in Shanxi Province in recent years. The typical symptom was that red spots occurred on the top or shoulder parts of jujube fruits, then the spots changed into brownish and the edge was much clear. Fruit of other part neither rot nor turning color except spot part. The rate of diseased fruit can reached 50 % when it happened seriously. It destroyed yield and quality of Hupingzao. The pathogen was isolated and identificated by the morphological and molecule method. Antagonistic microorganism and fungicides were screened against pathogen. The main results are as follows:
1 Isolation and identification of the pathogen
One hundred and thirty five isolates were obtained from 150 tissue pieces of 30 diaeased fruits and no distinct difference could be observed among them. After pathogenicity tests in lab and field, and pathogen re-isolation, the strain CN535 was determined to be responsible for the disease. The pathogen colony on PAD attained to 69.2~73.5 mm in diameter in seven days, with concentric rings in light-gray and dark-green color. The conidia are obclavate, obpyriform, ellipsoidal in form, and they are formed singly or in short chains, with a conical or cylindrical beak and transverse, longitudinal or oblique septa, 22.5~40.0×8.0~13.5μm, showing the typical morphology of the genus Alternaria. The character of porulation pattern was conidial chain with short branch formed on the top of the conidiophor and the branch chain was 1 to 5 spores which looks like boskage. Its rDNA-ITS sequence has 100% similarity with those of A. alternata, A. tenuissima, A. longipes, A. mali and A. citri searched in GenBank. The specific fragments of 341 bp and 450 bp were amplified with two pairs of sensitive primers AAF2/AAR3 and Aalt-F/Aalt-R. Based on the morphological characteristics and rDNA molecular analysis, the pathogen was finally identified as A. alternata (Fries) Keissler.
2 Isolation of endophytic bacteria and screening of antagonistic microorganisms
Isolated endophytic bacteria of the jujube on the PDA、KB and NA culture medium and culutring clony on the KB culture medium.The clony was in Kelly colour and the edge was smooth. Sixteen strains were obtaind after directly isolating and screening antagonistic microorganism and all these strains can not inhibit the mycelium growth of the pathogen. The rate of antagonistic microorganism isolated from the heated soil was larger than from the unheated, but the rate is not direct ratio to the temperature. At the same time, 4 strains whose inhibition rate were upon 90.0% of and 11 strains whose inhibition rate were upon 80.0% were obtained. Result of screening antagonistic microorganism shows that the mycelium growth inhibition rate of CHA0r、WCS358::phl and SB were 54.7%、51.2% and 46.5%. The inhibition zone diameter were 14.3 mm、12.7 mm and 12.7 mm.The mycelium growth inhibition rate of Wuyiencin Streptomyces and Streptomyces aureofaciens can reach 58.0% and 67.3%. The inhibition zone diameter were 30.0 mm and 25.3 mm. CN120 and CN121 not only inhibit the mycelium growth but also promote it.
3 Chemical controlling of the brwon spot of Ziziphus jujuba Mill. cv. Hupingzao Toxicity test to Alternaria alternata of 9 familiar fungicides was studied. The in vitro inhibition to mycelium growth and spore germination of 9 fungicides against pathogen was tested by using growth inhibition of mycelium on PDA agar plates and spore germination on concave slides. Test control effect of 6 fungicides in lab and put the fungicides in good effect into field. The results indicated that Iprodion, Mancozeb, Propiconazole, Jianban and Carbendazim, could significantly inhibit mycelium growth with inhibiting rates of 90.0% or more, and the fungicides including Mancozeb, Jianban, Carbendazim, Tanzujue, and Propiconazole could greatly suppress spore germination with above 85.0% inhibition rates as well. Results from toxicity test showed that EC50 of Iprodion, Tanzujue, Propiconazole, Carbendazim, Jianban and Mancozeb was 0.2,11.1,14.5,20.8,28.5 and 149.9 mg/L, respectively, and Iprodion was the best with the lowest EC50 Mancozeb was the worst. The correlation coefficients of six fungicides were all above 0.90, indicating a highly positive correlation between fungicide concentration and its inhibition ability. Result of control effect in the lab shows that Propiconazole, Jianban, Mancozeb and Iprodion were good and Tanzujue and Carbendazim were not very well. Result of control effect in the field shows that Iprodion can reach 72.9%, Propiconazole and Jianban were worse, Mancozeb can only reach 25.6%.
1病原菌的分离和鉴定
以壶瓶枣病果为材料,分离了30个枣果的150个病组织块,分离获得135个菌株,根据菌落特征发现各菌株没有明显差异,为同一种类型的真菌。通过室内和田间致病性测定以及人工接种后再分离,证明编号为CN535的真菌菌株为该病的致病菌。该病菌在PDA上培养7 d菌落直径达69.2～73.5 mm,基内菌丝和气生菌丝均发达,具明显的浅灰与墨绿色的同心轮纹;分生孢子单生或短链生,具纵横隔膜和短喙,大小为22.5～40.0×8.0～13.5μm,为典型的Alternaria属真菌特征。在PDA和PCA培养基上进行产孢培养,结果发现在细长分生孢子梗的上部形成特征性的具短分枝的孢子链,支链一般长1～5个孢子,似小树丛状,为A. alternata的特征。其rDNA-ITS序列分析结果表明该菌与A. alternata、A. tenuissima、A. longipes、A. mali和A. citri的同源性均为100%。用两对链格孢菌的特异性引物AAF2/AAR3和Aalt-F/Aalt-R分别扩增出相对应的341 bp和450 bp的片段。综合形态特征和分子分析结果,确定壶瓶枣褐斑病的病原菌为A. alternata (Fries)Keissler。
2内生菌的分离和拮抗菌的室内筛选
在PDA、KB和NA三种培养基上分离枣果的内生菌,纯化后,根据形态将其确定为同一种菌,在KB上菌落呈黄绿色,边缘平滑。从土壤中直接分离和筛选拮抗菌,共得到16个菌株,经过测定这些菌株对病原菌的菌丝生长均无明显的抑制作用。从经过高温预处理的土壤中分离拮抗菌的比例比常温下从土壤中分离拮抗菌的比例高,但是并不呈正比关系。同时得到4个抑菌率90.0%以上的菌株和11个抑菌率在80.0%～90.0%之间的菌株。生防细菌中CHA0r、WCS358::phl和SB对病原菌菌丝生长抑制率分别达到54.7%、51.2%和46.5%。CHA0r、ZN和WCS358::phl的抑菌圈直径分别达到14.3 mm、12.7 mm和12.7 mm,抑菌圈效果明显。生防链霉菌中龟裂链霉菌和武夷菌素链霉菌对菌丝生长抑制率分别达到58.0%和67.3%,抑菌圈直径分别达到30.0 mm和25.3 mm,抑制作用均较明显。而菌株CN120和CN121对褐斑病菌菌丝生长不但没有抑制作用,反而促使其菌落生长。
3化学药剂防治壶瓶枣褐斑病
选用9种常见的杀菌剂室内测定对壶瓶枣褐斑病菌Alternaria alternata的毒力。采用生长速率抑制法和悬滴法分别测定了9种杀菌剂对该病菌的菌丝生长和孢子萌发的抑制作用,并测定了其中6种杀菌剂对该病菌的毒力。同时在室内测定了6种药剂对病原菌的防治效果,将室内防效好的药剂用于田间防效测定。结果表明,异菌脲、代森锰锌、施力科、剑斑和多菌灵对病菌菌丝生长抑制作用较强,抑菌率均达到90.0%以上;代森锰锌、剑斑、多菌灵、炭阻绝和施力科对病菌孢子萌发抑制活性比较强,抑菌率也达到85.0%以上。毒力测定结果表明,异菌脲、炭阻绝、施力科、多菌灵、剑斑和代森锰锌的有效中浓度(EC50)分别为0.2,11.1,14.5,20.8,28.5和149.9 mg/L,其中异菌脲的有效中浓度最低,相对抑制效果最好;施力科和多菌灵次之;代森锰锌最差。室内防效测定,发现施力科、剑斑、代森锰锌和异菌脲的防效较好,而炭阻绝和多菌灵的防效较差。6种药剂的相关系数均在0.90以上,表明药剂浓度与抑制作用呈现较高的正相关。田间防效测定结果表明,异菌脲的田间防治效果最好,防效达到72.9%,施力科和剑斑稍差,代森锰锌的防治效果最差,仅达到25.6%。
Brown spot in Ziziphus jujuba Mill. cv. Hupingzao was a serious fruit disease in Shanxi Province in recent years. The typical symptom was that red spots occurred on the top or shoulder parts of jujube fruits, then the spots changed into brownish and the edge was much clear. Fruit of other part neither rot nor turning color except spot part. The rate of diseased fruit can reached 50 % when it happened seriously. It destroyed yield and quality of Hupingzao. The pathogen was isolated and identificated by the morphological and molecule method. Antagonistic microorganism and fungicides were screened against pathogen. The main results are as follows:
1 Isolation and identification of the pathogen
One hundred and thirty five isolates were obtained from 150 tissue pieces of 30 diaeased fruits and no distinct difference could be observed among them. After pathogenicity tests in lab and field, and pathogen re-isolation, the strain CN535 was determined to be responsible for the disease. The pathogen colony on PAD attained to 69.2~73.5 mm in diameter in seven days, with concentric rings in light-gray and dark-green color. The conidia are obclavate, obpyriform, ellipsoidal in form, and they are formed singly or in short chains, with a conical or cylindrical beak and transverse, longitudinal or oblique septa, 22.5~40.0×8.0~13.5μm, showing the typical morphology of the genus Alternaria. The character of porulation pattern was conidial chain with short branch formed on the top of the conidiophor and the branch chain was 1 to 5 spores which looks like boskage. Its rDNA-ITS sequence has 100% similarity with those of A. alternata, A. tenuissima, A. longipes, A. mali and A. citri searched in GenBank. The specific fragments of 341 bp and 450 bp were amplified with two pairs of sensitive primers AAF2/AAR3 and Aalt-F/Aalt-R. Based on the morphological characteristics and rDNA molecular analysis, the pathogen was finally identified as A. alternata (Fries) Keissler.
2 Isolation of endophytic bacteria and screening of antagonistic microorganisms
Isolated endophytic bacteria of the jujube on the PDA、KB and NA culture medium and culutring clony on the KB culture medium.The clony was in Kelly colour and the edge was smooth. Sixteen strains were obtaind after directly isolating and screening antagonistic microorganism and all these strains can not inhibit the mycelium growth of the pathogen. The rate of antagonistic microorganism isolated from the heated soil was larger than from the unheated, but the rate is not direct ratio to the temperature. At the same time, 4 strains whose inhibition rate were upon 90.0% of and 11 strains whose inhibition rate were upon 80.0% were obtained. Result of screening antagonistic microorganism shows that the mycelium growth inhibition rate of CHA0r、WCS358::phl and SB were 54.7%、51.2% and 46.5%. The inhibition zone diameter were 14.3 mm、12.7 mm and 12.7 mm.The mycelium growth inhibition rate of Wuyiencin Streptomyces and Streptomyces aureofaciens can reach 58.0% and 67.3%. The inhibition zone diameter were 30.0 mm and 25.3 mm. CN120 and CN121 not only inhibit the mycelium growth but also promote it.
3 Chemical controlling of the brwon spot of Ziziphus jujuba Mill. cv. Hupingzao Toxicity test to Alternaria alternata of 9 familiar fungicides was studied. The in vitro inhibition to mycelium growth and spore germination of 9 fungicides against pathogen was tested by using growth inhibition of mycelium on PDA agar plates and spore germination on concave slides. Test control effect of 6 fungicides in lab and put the fungicides in good effect into field. The results indicated that Iprodion, Mancozeb, Propiconazole, Jianban and Carbendazim, could significantly inhibit mycelium growth with inhibiting rates of 90.0% or more, and the fungicides including Mancozeb, Jianban, Carbendazim, Tanzujue, and Propiconazole could greatly suppress spore germination with above 85.0% inhibition rates as well. Results from toxicity test showed that EC50 of Iprodion, Tanzujue, Propiconazole, Carbendazim, Jianban and Mancozeb was 0.2,11.1,14.5,20.8,28.5 and 149.9 mg/L, respectively, and Iprodion was the best with the lowest EC50 Mancozeb was the worst. The correlation coefficients of six fungicides were all above 0.90, indicating a highly positive correlation between fungicide concentration and its inhibition ability. Result of control effect in the lab shows that Propiconazole, Jianban, Mancozeb and Iprodion were good and Tanzujue and Carbendazim were not very well. Result of control effect in the field shows that Iprodion can reach 72.9%, Propiconazole and Jianban were worse, Mancozeb can only reach 25.6%.
引文
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