【摘要】：黃連(Coptis chinensis Franch.)為毛茛科多年生草本植物,具有降火解毒、清熱燥濕以及抗腫瘤等功效,是重要的中藥材。湖北省是黃連主產(chǎn)區(qū),近年來(lái)黃連葉斑病在湖北省的發(fā)病面積和危害程度呈加重趨勢(shì),田間發(fā)病率在30%左右,但目前尚未見(jiàn)對(duì)該病害的病原學(xué)以及防治技術(shù)的深入研究。本文對(duì)黃連葉斑病的病原進(jìn)行了鑒定,研究了溫度、光照、碳源、氮源等對(duì)病原菌生長(zhǎng)的影響,并測(cè)定了10種殺菌劑以及1株生防菌株對(duì)該病原菌的防治效果,取得的主要結(jié)果如下:1、從湖北省恩施州太山廟黃連種植基地的發(fā)病植株上分離到Phoma sp.菌株,通過(guò)菌株的菌絲塊以及分生孢子懸浮液對(duì)黃連葉片的接種,證明了菌株P(guān)homa sp.為病原菌。該菌株在PDA培養(yǎng)基上的菌落為圓形,邊緣規(guī)則,菌絲呈絨狀,生長(zhǎng)速率較快;在OA培養(yǎng)基上生長(zhǎng)則較慢,菌絲埋生,具分枝;分生孢子器黑褐色,大小為100.5~120μm×175.3~210μm;分生孢子無(wú)色,長(zhǎng)橢圓形或棍棒狀,單胞,大小為2~2.5μm×4.1~5.4μm。該菌株ITS序列系統(tǒng)發(fā)育進(jìn)化分析結(jié)果表明,其與Phoma aquilegiicola(HM222537.1)的同源性為100%。因此根據(jù)形態(tài)學(xué)特征、分子鑒定結(jié)果以及莖點(diǎn)霉屬最新分類研究確定該菌株為耬斗菜莖點(diǎn)霉Calophoma aquilegiicola。2、病原菌生物學(xué)特性研究結(jié)果表明,其在MA培養(yǎng)基上菌體茂密,菌絲發(fā)達(dá),生長(zhǎng)5 d后菌落平均直徑為6.72 cm;病原菌在pH值4.0~10.0范圍內(nèi)均可生長(zhǎng),在pH值為6.0時(shí)生長(zhǎng)效果最好;該病原菌在4~25°C內(nèi)均能生長(zhǎng),最適溫度為20°C;不同的光照條件對(duì)該病原菌生長(zhǎng)的影響無(wú)顯著性差異;在所有供試碳源中,病原菌對(duì)麥芽糖的利用效果最好,對(duì)蔗糖和甘露醇的利用效果最差;在所有供試氮源中,對(duì)蛋白胨利用最好,對(duì)硝酸銨的利用最差;病原菌菌絲生長(zhǎng)和分生孢子萌發(fā)的致死溫度為50°C水浴10 min。3、室內(nèi)藥劑篩選實(shí)驗(yàn)表明,在供試的10種藥劑中,所有藥劑對(duì)黃連葉斑病病菌均表現(xiàn)出了一定的抑制作用,但是不同種類藥劑的防治效果存在明顯的差異。10%苯醚甲環(huán)唑?qū)S連葉斑病病原菌的防治效果最好,EC50值為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%甲基托布津等殺菌劑抑菌效果相對(duì)較差。盆栽試驗(yàn)表明,10%苯醚甲環(huán)唑施藥5天后病害發(fā)病率僅為3.8%,病情指數(shù)為0.96,防治效果達(dá)96.5%;25%戊唑醇處理后葉片發(fā)病率為7.33%,病情指數(shù)為1.83,防治效果為93.29%;206.7g/LVA銅氟硅唑處理后對(duì)黃連葉斑病的防治效果為92.05%。4、生防菌株ES-89分離自恩施州黃連根系土壤,通過(guò)培養(yǎng)性狀和菌株形態(tài)學(xué)觀察,結(jié)合16S rDNA分子鑒定,確定菌株ES-89為伯克霍爾德氏菌(Burkholdria sp.)。平板對(duì)峙實(shí)驗(yàn)發(fā)現(xiàn)其對(duì)黃連葉斑病病原菌的抑菌率在57%以上;ES-89不同濃度的發(fā)酵上清液均能夠抑制黃連葉斑病菌菌絲生長(zhǎng),當(dāng)發(fā)酵上清液與PDA體積比在1:3時(shí),抑制率達(dá)95%以上;離體葉片防治試驗(yàn)結(jié)果表明,ES-89發(fā)酵上清液處理后防效達(dá)80%以上;盆栽防治試驗(yàn)表明,經(jīng)ES-89菌株發(fā)酵液處理的黃連葉片發(fā)病率僅為12.5%,平均病情指數(shù)為3.1,防治效果達(dá)88%。生防菌株ES-89表現(xiàn)出了很好的防病效果和應(yīng)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.675

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