摘要
多菌灵和乙霉威是生产上防治多种作物病害的重要杀菌剂,作用靶标均为β-微管蛋白。多菌灵是一种广谱杀菌剂,对子囊菌、多数半知菌有活性,但对鞭毛菌和少数半知菌无效,即这些真菌对其具有耐药性。随着多菌灵的广泛使用,多种作物病害的病原菌对其产生抗药性；乙霉威作为负交互抗药性杀菌剂用于多菌灵抗性菌株的治理。本研究一方面以多主棒孢菌(Corynespora cassiicola)等几种多菌灵耐药性真菌为研究对象,对其β-微管蛋白基因进行克隆及氨基酸序列分析,探究其耐药性分子机理；另一方面多主棒孢菌(Corynespora cassiicola)为对象,对其多菌灵和乙霉威的抗药性进行了系统研究,重要研究结果如下：
1.β-微管蛋白167位氨基酸突变为酪氨酸可能与植物病原真菌对多菌灵的耐药性有关。
(1)明确了链格孢属3个种、匍柄霉属2个种和两种卵菌对多菌灵具有耐药性。
通过测定20个属23种蔬菜和花卉病害病原菌对多菌灵敏感性,发现多菌灵浓度为1μg/ml,对灰葡萄孢(Botrytis cinerea)菌丝生长抑制率已达100%；多菌灵浓度为100μg/ml,对瓜链格孢(Alternaria.cucumerina)、茄链格孢(Alternaria.solani)、链格孢(Alternaria. alternata)、茄匍柄霉(Stemphylium solani),菠菜匍柄霉(Stemphylium spinaciae)辣椒疫霉(Phytophthora. capsici)和德巴利腐霉(Pythium. debaryanum)菌丝生长仍无明显抑制效果,表明上述7种真菌为多菌灵耐药性真菌。
(2)初步明确了β-微管蛋白167位氨基酸突变为酪氨酸是链格孢属3个种、匍柄霉属2个种和两种卵菌对多菌灵具有耐药性的重要原因。
对链格孢属3个种瓜链格孢、茄链格孢和链格孢,匍柄霉属2个种茄匍柄霉和菠菜匍柄霉,2种卵菌辣椒疫霉和德巴利腐霉β-微管蛋白基因进行了克隆并对其氨基酸序列进行分析,其β-微管蛋白167位氨基酸均为酪氨酸(Tyr),而其他对多菌灵敏感的真菌167位均为苯丙氨酸(Phe)。据此推测植物病原真菌β-微管蛋白167位氨基酸由苯丙氨酸(Phe)突变为酪氨酸是对多菌灵产生抗药性的原因。
2.明确了我国多主棒孢菌对多菌灵和乙霉威抗性频率、抗性菌株适合度、交互抗药性及其对多菌灵和乙霉威抗药性分子机制。
(1)我国多主棒孢菌对多菌灵抗性频率极高,而对多菌灵和乙霉威双抗频率较低。对采自我国12个地区,18个寄主的163株多主棒孢菌对多菌灵和乙霉威的敏感性进行分析,145个菌株对多菌灵具有抗药性,抗性频率高达89.0%,18个菌株对多菌灵敏感,占供试菌株的11.0%。24个菌株对多菌灵和乙霉威具有双重抗药性,双抗频率为14.7%。
(2)多菌灵高抗(MBCHR)和多菌灵乙霉威双抗(MBCHRNPCR)菌株的适合度无明显下降。野生菌株(MBCS)菌落直径为55.17mm和50.83mm,产孢量为1.78×105个/ml和2.50×105个/ml,病指为79.80和83.37,多菌灵高抗(MBCHR)和多菌灵乙霉威双抗(MBCHRNPCR)菌株菌落直径在50.17mm-59.83mm之间,产孢量在1.89个/ml-2.94×105个/ml之间,病指在81.33-89.29之间。三种敏感性类型菌株菌丝生长速率、产孢量和致病力并无明显的差别。
(3)多菌灵高抗(MBCHR)和多菌灵乙霉威双抗(MBCHRNPCR)菌株与其它5种类型杀菌剂无交互抗药性。烟酰胺类杀菌剂啶酰菌胺对多主棒孢菌两个野生菌(MBCS)的EC50分别为0.49μg/ml和0.651μg/ml,而对于高抗(MBCHR)和双抗(MBCHRNPCR)菌株EC50在0.36μg/ml-0.71μg/ml之间。苯基吡咯类杀菌剂咯菌腈对多主棒孢菌两个野生菌株(MBCS)的EC50分别为0.26μg/ml和0.20μg/ml,而对于高抗(MBCHR)和双抗(MBCHRNPCR)菌株EC50在0.15μg/ml-0.27μg/ml之间。咪唑类杀菌剂咪鲜胺对多主棒孢菌两个野生菌株(MBCS)的EC50分别为0.14μg/ml和0.191μg/ml,而对于高抗(MBCHR)和双抗菌株(MBCHRNPCR) EC50在0.14μg/ml-0.26μg/ml之间。嘧啶胺杀菌剂嘧菌环胺对多主棒孢菌两个野生菌株(MBCS)的EC50分别为0.7μg/ml和0.75μg/ml,而对于高抗(MBCHR)和双抗(MBCR)菌株EC50在0.67-0.83μg/ml之间。二甲酰亚胺类杀菌剂腐霉利对多主棒孢菌两个野生菌株(MBCS)的EC50分别为0.45μg/ml和0.37μg/ml,而对于高抗(MBCHR)和双抗(MBCHRNPCR)菌株EC50在0.37μg/ml-0.50μg/ml之间。
(4)明确了多主棒孢菌对多菌灵抗药性和多菌灵乙霉威双重抗药性分子机制。通过比较多主棒孢菌敏感、多菌灵高抗(MBCHR)和多菌灵乙霉威双抗(MBCHRNPCR)菌株β-微管蛋白序列,发现198位氨基酸由谷氨酸(Glu)突变为丙氨酸(Ala)是导致其对多菌灵产生高等抗药性的原因。198位氨基酸由谷氨酸突变为赖氨酸(Lys),200位氨基酸由苯丙氨酸(Phe)突变为酪氨酸(Tyr)是其对多菌灵和乙霉威产生双重抗药性的原因。
本研究首次提出植物病原真菌β-微管蛋白167位氨基酸的种类可能与多菌灵对不同属植物病原真菌敏感性有关,从分子水平解释了多菌灵对不同属植物病原真菌的敏感性,为进一步开发基于p-微管蛋白的新型杀菌剂和筛选模型奠定基础；此外,在我国首次报道了多主棒孢菌多菌灵高抗(MBCHR)和多菌灵乙霉威双抗(MBCHRNPCR)菌株及其抗药性的分子机理,上述结果将为我国多主棒孢菌的化学防治,抗性菌株的分子检测等提供有用的信息。
Carbendazim and diethofencarb that target on fungus β-tubulin, are two important fungicides for controlling various crop diseases. Carbendazim shows a broaden-spectrum active efficiency on inhibiting the mycelia expension of ascomycetes and most of deuteromycetes, but exerts no effect on mastigomycotina and few deuteromycetes that might possess tolerant properties to carbendazim.Diethofencarb has a negative cross-resistant spectrum of carbendazim and usually been used to control carbendazim-resistant isolates. In this dissertation, the carbendazim-tolerant mechanism was explored on several carbendazim-resistant fungi by cloning β-tubulin gene, analyzing and comparing the mutant amino acid sequences. Meanwhile, the resistance of Corynespora cassiicola to carbendazim and diethofencarb was also studied systematically. The research progresses were shown as follows:
1.The changes of amino acid at the position167of β-tubulin may be responsible for the sensitivity of plant pathogenic fungi from different genus to carbendazim.
(1)Three Alternaria, two Stemphylium and two-Oomycetes species have been shown carbendazim-tolerant properties.
The sensitivities of23pathogenic fungi from20genus were determined to carbendazim. The results showed that1μg/ml carbendazim inhibited thoroughly the mycelium growth of Botrytis cinerea, but100μg/ml carbendazim revealed no significant effect on Alternaria cucumerina, Alternaria solani, Alternaria Alternate, Stemphylium solani, Stemphylium spinaciae, Phytophthora capsici and Pythium debaryanum, indicating that these fungi were tolerant to carbendazim.
(2)Tyrosine at the position167of P-tubulins in three Alternaria, two Stemphylium, two-Oomycetes species play an important role on these fungi's tolerance to carbendazim.
P-tubulin genes of A. cucumerina, A.solani, A Alternate, S.solani, S.spinaciae, P.capsici, and P.debaryanum were cloned and their amino acid sequences were analysed. The results showed that the amino acid at position167were changed from phenylalanine in carbendazim-sensitive isolate totyrosine in carbendazim-tolerant isolate, indicating that the mutation of the amino acid at positon167of β-tubulins caused the tolerance of plant pathogenic fungi to carbendazim.
2.The resistance mechanisms of Chinese C.cassiicola to carbendazim and diethofencarb have been clarified explicitly. Meanwhile, the characters of the tolerant strains have been elucidated clearly on bases of the resistance frequency, the fitness and the cross-resistant property.
(1)Most of163C.cassiicola isolates revealed a strong resistance to carbendazim and a low resistant frequency to diethofencarb, including145isolates that expressed high resistance to carbendazim with MIC value greater than10μg/ml and18isolates that were sensitive to carbendazim. The resistant frequency among the total isolates was89.0%to carbendazim and14.7%to both carbendazim and diethofencarb.
(2)The fitness of MBCHR and MBCHRNPCR isolates had been reduced insignificantly. Under normal cultured condition, MBCS isolates developed55.17mm-50.83mm of round colony, produced1.78×105/ml-2.50×105/ml of spores, and displayed79.50-82.80of disease index. However, MBCHR and MBCHRNPCR isolates showed50.17mm-59.83mm of round colony,1.89×105/ml and2.94×105/ml of spores,81.40and85.40of disease index, respectively.
(3)MBCHR and MBCHRNPCR isolates exibited no cross resistance to boscalid, fludioxonil, prochloraz, cyprodinil and procymidone. For two MBCS isolates, the EC50values of the above five fungicides were0.49μg/ml and0.65μg/ml,0.26and0.20μg/ml,0.14and0.19μg/ml,0.71and0.75μg/ml, and0.45μg/ml and0.37μg/ml, respectively. For MBCHR and MBCHRNPCR isolates,the EC50values of the five fungicides were about0.36μg/ml-0.71μg/ml,0.15μg/ml-0.27μg/ml,0.14μg/ml-0.26μg/ml,0.67μg/ml-0.83μg/ml and0.37μg/ml-0.50μg/ml, respectively.
(4)The resistance mechanism of C.cassiicola to carbendazim and diethofencarb were taken into in-depth investigated. Sequence comparison revealed that a point mutation at the position198had replaced MBCS alanine with MBCHR glutamic, and the amino acids at the position198and200have been changed from MBCS glutamic and phenylalanine to MBCHRNPCR lysine and tyrosine.
In this dissertation, the amino acid at the position167of β-tubulinwas found to be relative to the sensitivity of plant pathogenic fungi to carbendazim at the first time.The MBCHR and MBCHRNPCR isolates were also first reported in China. The resistance mechanism of C.cassiicola to carbendazim and diethofencarb had been studied systematically. The results laid a foundation todevelop new fungicide and fungicide screening modeland provide better strategy for the chemical control and molecular detection of resistant strains.
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