黄连叶斑病病原学及防治技术研究

发布时间：2018-09-14 08:28

【摘要】：黄连(Coptis chinensis Franch.)为毛茛科多年生草本植物,具有降火解毒、清热燥湿以及抗肿瘤等功效,是重要的中药材。湖北省是黄连主产区,近年来黄连叶斑病在湖北省的发病面积和危害程度呈加重趋势,田间发病率在30%左右,但目前尚未见对该病害的病原学以及防治技术的深入研究。本文对黄连叶斑病的病原进行了鉴定,研究了温度、光照、碳源、氮源等对病原菌生长的影响,并测定了10种杀菌剂以及1株生防菌株对该病原菌的防治效果,取得的主要结果如下:1、从湖北省恩施州太山庙黄连种植基地的发病植株上分离到Phoma sp.菌株,通过菌株的菌丝块以及分生孢子悬浮液对黄连叶片的接种,证明了菌株Phoma sp.为病原菌。该菌株在PDA培养基上的菌落为圆形,边缘规则,菌丝呈绒状,生长速率较快;在OA培养基上生长则较慢,菌丝埋生,具分枝;分生孢子器黑褐色,大小为100.5~120μm×175.3~210μm;分生孢子无色,长椭圆形或棍棒状,单胞,大小为2~2.5μm×4.1~5.4μm。该菌株ITS序列系统发育进化分析结果表明,其与Phoma aquilegiicola(HM222537.1)的同源性为100%。因此根据形态学特征、分子鉴定结果以及茎点霉属最新分类研究确定该菌株为耧斗菜茎点霉Calophoma aquilegiicola。2、病原菌生物学特性研究结果表明,其在MA培养基上菌体茂密,菌丝发达,生长5 d后菌落平均直径为6.72 cm;病原菌在pH值4.0~10.0范围内均可生长,在pH值为6.0时生长效果最好;该病原菌在4~25°C内均能生长,最适温度为20°C;不同的光照条件对该病原菌生长的影响无显著性差异;在所有供试碳源中,病原菌对麦芽糖的利用效果最好,对蔗糖和甘露醇的利用效果最差;在所有供试氮源中,对蛋白胨利用最好,对硝酸铵的利用最差;病原菌菌丝生长和分生孢子萌发的致死温度为50°C水浴10 min。3、室内药剂筛选实验表明,在供试的10种药剂中,所有药剂对黄连叶斑病病菌均表现出了一定的抑制作用,但是不同种类药剂的防治效果存在明显的差异。10%苯醚甲环唑对黄连叶斑病病原菌的防治效果最好,EC50值为0.0252μg/ml;其次是206.7 g/LVA酮氟硅唑,其EC50值是0.8016μg/ml;20%甲基立枯磷、70%甲基托布津等杀菌剂抑菌效果相对较差。盆栽试验表明,10%苯醚甲环唑施药5天后病害发病率仅为3.8%,病情指数为0.96,防治效果达96.5%;25%戊唑醇处理后叶片发病率为7.33%,病情指数为1.83,防治效果为93.29%;206.7g/LVA铜氟硅唑处理后对黄连叶斑病的防治效果为92.05%。4、生防菌株ES-89分离自恩施州黄连根系土壤,通过培养性状和菌株形态学观察,结合16S rDNA分子鉴定,确定菌株ES-89为伯克霍尔德氏菌(Burkholdria sp.)。平板对峙实验发现其对黄连叶斑病病原菌的抑菌率在57%以上;ES-89不同浓度的发酵上清液均能够抑制黄连叶斑病菌菌丝生长,当发酵上清液与PDA体积比在1:3时,抑制率达95%以上;离体叶片防治试验结果表明,ES-89发酵上清液处理后防效达80%以上;盆栽防治试验表明,经ES-89菌株发酵液处理的黄连叶片发病率仅为12.5%,平均病情指数为3.1,防治效果达88%。生防菌株ES-89表现出了很好的防病效果和应用前景。
[Abstract]:Coptis chinensis Franch. (Coptis chinensis Franch.) is a perennial herb of Ranunculaceae. It has the functions of reducing fire and detoxification, clearing away heat and dampness, and anti-tumor. It is an important Chinese herbal medicine. Hubei Province is the main producing area of Coptis chinensis Franch. In this paper, the pathogen of Coptis chinensis leaf spot was identified, the effects of temperature, light, carbon and nitrogen sources on the growth of the pathogen were studied, and the control effects of 10 fungicides and one Biocontrol Strain on the pathogen were determined. The main results were as follows: 1. Phoma sp. strain was isolated from diseased plants of Coptis chinensis planting base in Taishan Miao of Enshi Prefecture, and was proved to be a pathogen by inoculating the mycelium block and conidia suspension of the strain. The conidia was dark brown with a size of 100.5-120 micron *175.3-210 micron, and the conidia were colorless, oval or clavate with a size of 2-2.5 micron *4.1-5.4 micron. The ITS sequence of the strain was phylogenetic and evolutionary analysis showed that it had 100% homology with Phoma aquilegiicola (HM222537.1). Therefore, the strain was identified as Calophoma aquilegiicola.2 according to the morphological characteristics, molecular identification results and the latest taxonomic study of the genus Puccinia. The biological characteristics of the pathogen showed that the strain was dense on MA medium with well-developed mycelia. The average diameter of the colony was 6.72 cm after 5 days of growth, and the pH value of the pathogen was 4.0-10.0. The pathogen could grow in the range of 6.0 pH, and the optimum temperature was 20 C. There was no significant difference in the growth of the pathogen under different light conditions. Among all the nitrogen sources tested, peptone was the best, ammonium nitrate was the worst; the lethal temperature of mycelial growth and spore germination was 50 degree C water bath for 10 minutes. The control effect of 10% Difenoconazole was the best, EC50 was 0.0252 ug/ml, followed by 206.7 g/LVA ketone fluorosilazole, whose EC50 was 0.8016 ug/ml, 20% methyl phosphorus, 70% methyl tobrazine and other fungicides had relatively poor bacteriostatic effect. The disease incidence rate was only 3.8%, disease index was 0.96 and control effect was 96.5%. The disease incidence rate was 7.33%, disease index was 1.83 and control effect was 93.29% after treatment with 25% pentazole. The control effect of 206.7 g/LVA copper fluorosilazole was 92.05%. 4, the Biocontrol Strain ES-89 was isolated from Coptis chinensis in Enshi prefecture. In root soil, the strain ES-89 was identified as Burkholdria sp. through the observation of culture characters and strain morphology, and 16S rDNA molecular identification. The inhibition rate of ES-89 to the pathogen of Coptis chinensis was above 57% by plate confrontation test. The supernatant of ES-89 could inhibit the mycelial growth of Coptis chinensis at different concentrations. Long, when the volume ratio of fermentation supernatant to PDA was 1:3, the inhibition rate was over 95%; the control effect of ES-89 fermentation supernatant was over 80%; pot experiment showed that the incidence rate of Coptis chinensis leaves treated with ES-89 fermentation supernatant was only 12.5%, the average disease index was 3.1, and the control effect was 88%. Strain ES-89 showed good disease control effect and application prospect.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.675

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