落叶松枯梢病的生物防治与分子检测初步研究
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摘要
本文研究了长白山、完达山、小兴安岭地区落叶松枯梢病梢栖真菌的群落结构,应用形态学和分子生物学的方法对真菌进行了鉴定,并利用所分离到的真菌筛选出拮抗菌株并进行了林间生物防治试验;以梢栖真菌的序列为基础,针对落叶松枯梢病(Botryosphaeria laricina(Sawada)Y.Z.Shang)设计特异引物,初步探索出了落叶松枯梢病分子检测的方法。
(1)用形态学和分子生物学的方法,对落叶松枯梢病病原真菌(B.laricina)进行鉴定,并获取NCBI基因登录号:EU442284。经DNAman比对小兴安岭地区、长白山地区、完达山地区三个地点所获得的菌株,ITS区域相似度为100%。
(2)从落叶松病/健康梢上共分离到子囊菌3属(Botryosphaeria、Chaetomium和Sordaria),有丝分裂菌15属。夏季梢栖真菌与冬季梢栖真菌相比,多出7种有丝分裂菌。冬季落叶松枯梢病/健康梢上分离率最高的为Alternaria,Pestalotiopsis,以及Epicoccum,分别为32.2%,21.8%和19%:夏季落叶松枯梢病/健康梢上的优势种类为Alternaria,其次为Tolura和Epicoccum,其分离率分别为32.0%,18.O%和14.0%。3年生病梢上种类略为丰富,而1年生健康枝条上种类相对较少。B.laricina主要分布于1年生梢部;健康及感病未发病的2、3年梢部,均有Chaetomium、Sordaria和Trichoderma出现,各个季节病/健康梢均部有分布的种有:Epicoccum、Alternaria、Trichoderma、Chaetomium和Sordaria,共5属。
(3)根据拮抗系数、抑菌率、被覆盖程度和防治预试验的综合评价确定Sordaria fimicola、Trichoderma atroviride和Chaetomium globosum为生防菌。野外防治试验表明,综合比较防治效果为S.fimicola＞代森锰锌400倍液＞T.atroviride＞C.globosum。三种菌剂在50%喷洒浓度时,真菌多样性最大;防治区真菌群落数量明显少于非防治区群落数量;非防治区同期梢栖真菌分离结果显示在健康梢部,分离到大量Trichoderma、Chaetomium,说明在自然界中Trichoderma、Chaetomium本身对B.laricina有较强的抑制作用。
(4)通过对落叶松梢栖真菌ITS序列的分析,设计出特异引物ITSA1与ITSB1。该特异引物可以将落叶松枯梢病病原菌与其它9种(属)落叶松梢栖真菌分别开,具有较好的特异性和较高的灵敏性,初步建立了落叶松枯梢病的分子检测方法。
In this paper, the fungal community structures on the healthy shoot and shoot infected with shoot blight of larch were studied in the Changbai Mountain, Wanda Mountain and Xiaoxing'an Mountain. The morphological and molecular biological methods were applied in the identification of the fungi. Fungi strains with antagonistic abilities were screened and used in the biological control of larch shoot blight. Specific primers were designed to identify the pathogen of larch shoot blight (Botryosphaeria laricina(Sawada) Y.Z. Shang) based on the ITS sequence of the branch-habiting mycoflora. It was proved to be an effective method in the detection of larch shoot blight.
(1) Botryosphaeria laricina, one of the pathogens for larch shoot blight, was identified by the combination of morphological and molecular biological methods. The ITS sequence of B. laricina was deposited in GenBank (accession No. EU442284). Alignments of ITS sequences were analyzed using DNAman among different B. laricina strains isolated from Changbai Mountain, Wanda Mountain and Xiaoxing'an Mountain. The result showed that there was a 100% similarity in the ITS regions of these strains.
(2) Three ascomycetes (Botryosphaeria, Sordaria and Chaetomium) and fifteen mitosporic fungi were isolated from the disease/health shoot. There were seven kinds of mitosporic fungi isolated in summer more than those isolated in winter. The most isolated fungi from the diseased/healthy shoot in winter was Alternaria, followed by Pestalotiopsis and Epicoccum, with separation rates of 32.2%, 21.8% and 19%, respectively; while the dominant species in summer was Alternaria, followed by Tolura and Epicoccum, with separation rates of 32.0%, 18.0% and 14.0%, respectively. There are more kinds of fungi on the 3-year old diseased shoot, and less on the 1-year old healthy shoot. B. laricina was mainly located on the 1-year old shoot. On the healthy shoot, 2 or 3-years old shoot which were infected but not caused disease, there were different degrees on the separation of Chaetomium, Sordaria and Trichoderma. Five genus were isolated on all shoots, which were Epicoccum, Alternaria, Trichoderma, Chaetomium and Sordaria.
(3) Sordaria fimicola, Trichoderma atroviride and Chaetomium globosum were identified as the biocontrol fungi based on the overall evaluation of antagonistic coefficient, inhibitory rate, extent of coverage and indoor infection experiment. The field biocontrol test showed that the comprehensive control effects were S. fimicola> Mancozeb 400 times> T. atroviride> C. globosum. The greatest diversity of fungi was found when the three inocula were sprayed with a concentration of 50%. The species diversity of mycoflora in the controlled zone was significantly less than that in the non-controlled zone. The result showed that a large number of Trichoderma and Chaetomium were isolated from the healthy shoot in the non-controlled during the same period, indicating that both Trichoderma and Chaetomium had an inhibitory effect on B. laricina in nature.
(4) A pair of specific primers: ITSA1 and ITSB1 was designed through the analysis of the ITS sequence of branch-habiting mycoflora of larch. B. laricina could be distinguished from other 9 species of (genus) fungi on shoot of larch by molecular methods using the specific primers. It was proved to be an effective and sensitive method in the molecular detection of larch shoot blight.
(1)用形态学和分子生物学的方法,对落叶松枯梢病病原真菌(B.laricina)进行鉴定,并获取NCBI基因登录号:EU442284。经DNAman比对小兴安岭地区、长白山地区、完达山地区三个地点所获得的菌株,ITS区域相似度为100%。
(2)从落叶松病/健康梢上共分离到子囊菌3属(Botryosphaeria、Chaetomium和Sordaria),有丝分裂菌15属。夏季梢栖真菌与冬季梢栖真菌相比,多出7种有丝分裂菌。冬季落叶松枯梢病/健康梢上分离率最高的为Alternaria,Pestalotiopsis,以及Epicoccum,分别为32.2%,21.8%和19%:夏季落叶松枯梢病/健康梢上的优势种类为Alternaria,其次为Tolura和Epicoccum,其分离率分别为32.0%,18.O%和14.0%。3年生病梢上种类略为丰富,而1年生健康枝条上种类相对较少。B.laricina主要分布于1年生梢部;健康及感病未发病的2、3年梢部,均有Chaetomium、Sordaria和Trichoderma出现,各个季节病/健康梢均部有分布的种有:Epicoccum、Alternaria、Trichoderma、Chaetomium和Sordaria,共5属。
(3)根据拮抗系数、抑菌率、被覆盖程度和防治预试验的综合评价确定Sordaria fimicola、Trichoderma atroviride和Chaetomium globosum为生防菌。野外防治试验表明,综合比较防治效果为S.fimicola＞代森锰锌400倍液＞T.atroviride＞C.globosum。三种菌剂在50%喷洒浓度时,真菌多样性最大;防治区真菌群落数量明显少于非防治区群落数量;非防治区同期梢栖真菌分离结果显示在健康梢部,分离到大量Trichoderma、Chaetomium,说明在自然界中Trichoderma、Chaetomium本身对B.laricina有较强的抑制作用。
(4)通过对落叶松梢栖真菌ITS序列的分析,设计出特异引物ITSA1与ITSB1。该特异引物可以将落叶松枯梢病病原菌与其它9种(属)落叶松梢栖真菌分别开,具有较好的特异性和较高的灵敏性,初步建立了落叶松枯梢病的分子检测方法。
In this paper, the fungal community structures on the healthy shoot and shoot infected with shoot blight of larch were studied in the Changbai Mountain, Wanda Mountain and Xiaoxing'an Mountain. The morphological and molecular biological methods were applied in the identification of the fungi. Fungi strains with antagonistic abilities were screened and used in the biological control of larch shoot blight. Specific primers were designed to identify the pathogen of larch shoot blight (Botryosphaeria laricina(Sawada) Y.Z. Shang) based on the ITS sequence of the branch-habiting mycoflora. It was proved to be an effective method in the detection of larch shoot blight.
(1) Botryosphaeria laricina, one of the pathogens for larch shoot blight, was identified by the combination of morphological and molecular biological methods. The ITS sequence of B. laricina was deposited in GenBank (accession No. EU442284). Alignments of ITS sequences were analyzed using DNAman among different B. laricina strains isolated from Changbai Mountain, Wanda Mountain and Xiaoxing'an Mountain. The result showed that there was a 100% similarity in the ITS regions of these strains.
(2) Three ascomycetes (Botryosphaeria, Sordaria and Chaetomium) and fifteen mitosporic fungi were isolated from the disease/health shoot. There were seven kinds of mitosporic fungi isolated in summer more than those isolated in winter. The most isolated fungi from the diseased/healthy shoot in winter was Alternaria, followed by Pestalotiopsis and Epicoccum, with separation rates of 32.2%, 21.8% and 19%, respectively; while the dominant species in summer was Alternaria, followed by Tolura and Epicoccum, with separation rates of 32.0%, 18.0% and 14.0%, respectively. There are more kinds of fungi on the 3-year old diseased shoot, and less on the 1-year old healthy shoot. B. laricina was mainly located on the 1-year old shoot. On the healthy shoot, 2 or 3-years old shoot which were infected but not caused disease, there were different degrees on the separation of Chaetomium, Sordaria and Trichoderma. Five genus were isolated on all shoots, which were Epicoccum, Alternaria, Trichoderma, Chaetomium and Sordaria.
(3) Sordaria fimicola, Trichoderma atroviride and Chaetomium globosum were identified as the biocontrol fungi based on the overall evaluation of antagonistic coefficient, inhibitory rate, extent of coverage and indoor infection experiment. The field biocontrol test showed that the comprehensive control effects were S. fimicola> Mancozeb 400 times> T. atroviride> C. globosum. The greatest diversity of fungi was found when the three inocula were sprayed with a concentration of 50%. The species diversity of mycoflora in the controlled zone was significantly less than that in the non-controlled zone. The result showed that a large number of Trichoderma and Chaetomium were isolated from the healthy shoot in the non-controlled during the same period, indicating that both Trichoderma and Chaetomium had an inhibitory effect on B. laricina in nature.
(4) A pair of specific primers: ITSA1 and ITSB1 was designed through the analysis of the ITS sequence of branch-habiting mycoflora of larch. B. laricina could be distinguished from other 9 species of (genus) fungi on shoot of larch by molecular methods using the specific primers. It was proved to be an effective and sensitive method in the molecular detection of larch shoot blight.
引文
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