芒果可可球二孢对多菌灵抗性水平差异机理研究

Stem-end rot disease, caused by Botryodiplodia theobromae Pat., is an important postharvest disease of mango, and using chemical fungicide is main preventive and control measure. In previous study, the applicant isolated B. theobromae field isolates with different resistance to carbendazim-resistance (field MBCR isolates) from mango in Hainan. The study on carbendazim resistance test, biological characteristics and genetic diversity analysis of the strains have been finished. On this basis, the project will investigate the molecular and biochemical mechanisms of carbendazim-resistance of B. theobromae using traditional biochemical and molecular biology methods. The study will focus on: 1) Investigate the biochemical mechanisms of resistance based on systematically analyzing the physiological-biochemical characteristics between B. theobromae field MBCR isolates and sensitive isolates; 2) Analyze the genotypes and regulation mechanisms of B. theobromae isolates with different resistance level to carbendazim based on the sequence and transcriptional level of β-tubulin gene. 3) Identify whether there are any changes in biotic fitness of field MBCR isolates based on biological characteristics; This study will provide theoretic basis for developing rapid diagnostic test and molecular detection technology of B. theobromae resistance to carbendazim, having important application value for resistance management of B. theobromae.

可可球二孢引起的蒂腐病是芒果最严重的采后病害之一，采用杀菌剂进行化学防治是重要防控手段。申请人发现海南芒果可可球二孢群体中存在不同水平的多菌灵抗性菌株，完成可可球二孢多菌灵抗性检测、生物学特性和遗传多样性测定。在此基础上，本项目拟采用传统生物化学和分子生物技术相结合的方法，探明B. theobromae多菌灵抗药性的分子遗传和生理生化机制，内容如下：分析抗感菌株生理生化特性，探索B. theobromae多菌灵抗药性的生化机制；基于β-微管蛋白基因序列及转录水平，分析不同多菌灵抗性水平的基因型及调控机制；基于生物学特性明确B. theobromae对多菌灵抗药性是否导致适合度变异，分析突变后的基因对其他生物学性状的调控作用。该项目成果不仅能够为B. theobromae对多菌灵抗药性的快速诊断和分子检测技术的研发提供理论依据， 对于芒果蒂腐病菌抗药性治理策略的制定还具有重要应用价值。

2016-2018年对海南芒果调查发现蒂腐病自然发病率为27.79%；室内致病力测定表明，芒果蒂腐病菌可可球二孢(Botryodiplodia theobromae)强致病菌株平均为68.3%，中等致病菌株为20.79%，弱致病菌株为10.92%。对多菌灵抗性检测发现：2016与2018年的多菌灵抗性菌株的平均发生频率分别为60.94%，其中高抗菌株均能在1000 mg/L多菌灵PDA培养基上生长，抗性频率为52.4%；中抗菌株抗性频率分别为8.53%；没发现任何低抗菌株。芒果蒂腐病菌B. theobromae田间分离菌株对多菌灵EC50的频率分布呈现典型的双峰型，呈不连续分布，抗性为质量遗传特性，具有抗性水平高、进化快、固有抗性风险高的特点。研究还发现：B. theobromae多药抗性严重，目前已发现对苯并咪唑类、甲氧基丙烯酸酯类及甾醇生物合成抑制剂均有产生抗药性。多菌灵抗性菌株具有抗性遗传稳定性。与敏感菌株相比，田间抗性菌株在菌丝生长、致病力、温度敏感性、渗透压敏感性等方面并没有明显差异，表现出极高的适合度。与抗性菌株相比，在杀菌剂作用下敏感菌株菌丝相对电导率会明显升高，这说明敏感菌株的菌丝细胞膜受药剂的严重破坏导致细胞液的外渗。多菌灵抗性菌株的GST和CAT酶活性均表现出比敏感菌株更高的活性，这表明B. theobromae菌株对杀菌剂抗药性机制与酶活性存在极大的关联。我们推测B. theobromae多菌灵抗药性产生的生化机制与菌丝细胞膜的通透性及相关酶活性有关。另外，针对B. theobromae对吡唑醚菌酯的抗药性展开研究，研究结果进一步验证了抗药性的产生与细胞膜透性及相关酶活性有关。分子检测研究证实β-微管蛋白基因发生点突变是导致病菌产生抗药性的重要机制：多菌灵高抗菌株β-微管蛋白基因第198位从谷氨酸（GAG）转变为丙氨酸（GCG）；多菌灵中抗菌株β-微管蛋白基因第200从苯丙氨酸（TTC）转变为酪氨酸（TAC）。另外，通过qrtPCR技术证实多菌灵抗性菌株的β-微观蛋白基因表达量明显上调。通过克隆、测序、实时PCR等方法，针对抗敏菌株β-微管蛋白基因的DNA序列多态性分析，明确B. theobromae菌株对多菌灵抗性的分子机制。