灰葡萄孢黄色菌系产生的分子机制及流行风险研究

Our previous studies indicated that the populations of Botrytis cinerea evolved three dinstinct types based on sclerotial color: the black-sclerotial type (accounting for 99.4%), the yellow-and-black-sclerotial type (accounting for 0.1%) and the yellow-sclerotial type (accounting for 0.5%). The former two types formed black-colored sclerotia with accumulation of melanin in the rind tissue, whereas the last type forms yellow-colored sclerotia without accumulation of the melanin in the rind tissue. The strains of the all the three types have melanin accumulation both in hyphae and in conidia. Therefore, the yellow-sclerotial strains (YS) of B. cinerea might be formed by suppression of melanin biosynthyesis during sclerotial development. We also found that formation of the yellow-colored sclerotia by the YS strains is a genetically-stable trait and the YS strains had stronger pathogenicity than the black sclerotial strains (BS) of B. cinerea. Therefore, the YS strains of B. cinerea have a epidemic risk in causing diseases. Based on these results, we proposed to elucidate the molecular mechanisms responsible for formation of the YS strains and to evaluate the epidemic risk of the YS strains. The contents for the former aspect include: (i) use of hybridization between the BS and the YS strains of B. cinerea to elucidate the genetic model for formation of the yelllow-colored sclerotia, and (ii) use of molecular techniques such as transcriptome profiling, small RNA (sRNA)-sequencing and gene disruption to screen the genes or the small RNAs (sRNAs) related to development of yellow-colored sclerotia. The contents for the latter aspect include: (i) comparison of the BS strains and the YS strains in development-related biological characteristics, ecological adaptation-related characteristics, competiability and multiple-season epidemics under controlled conditions, and (ii) detection of the natural occurrence frequencies of both BS strains and the YS strains in different geographical regions. The results from the project will advance our knowledge on the evolution and evolution-related mechanisms in populations of B. cinerea, will help us to further understand the role of the sclerotia formed by B. cinerea in the natual life history of this fungus, and will lay a solid foundation for initiation of some novel strategies (or methods) for control of gray mold diseases caused by B. cinerea.

前期研究发现灰葡萄孢群体中出现黑色菌系（占99.4%）、黄黑菌系（0.1%）和黄色菌系（0.5%）的分化。前两者产生黑色菌核，含黑色素，最后一类产生黄色菌核，缺黑色素。三类菌系菌丝体和分生孢子均含有黑色素。可见，黄色菌系产生与菌核黑色素合成受阻有关。黄色菌系遗传稳定，致病力较黑色菌系强，存在流行风险。鉴此，我们提出研究黄色菌系产生的分子机制及流行风险。项目拟采用黄色菌系和黑色菌系杂交的方法研究黄色菌核性状的遗传模式，采用转录组和小RNA分析技术及基因敲除技术挖掘与黄色菌核形成相关的基因和小RNA，以揭示黄色菌系产生的分子机制。项目还将比较黄色菌系和黑色菌系的生物学特性、生态适应性、竞争性致病能力、控制条件下的多季流行效率及自然发生频率，以评估黄色菌系的流行风险。项目的完成将加深对灰葡萄孢群体演化及机制的认识，进一步明确菌核在灰葡萄孢生活史中扮演的角色，为发展防治灰霉病新策略提供理论依据。

灰葡萄孢（Botrytis cinerea）引起的灰霉病危害多种蔬菜、水果、花卉和油料作物。这种真菌产生灰白色至褐色菌丝和分生孢子以及黑色菌核，但对菌核在病害循环中的作用缺乏研究。前期研究发现了产生黄色菌核的黄色菌系，并发现黄色菌系致病力较黑色菌系强。由此提出研究黄色菌系产生的分子机制及流行风险。研究内容包括：（1）黄色菌核性状的遗传稳定性；（2）控制黄色菌核产生的基因；（3）黄色菌系的生态学；（4）黄色菌系流行风险。经过4年的研究，取得了如下研究结果：（1）证明黄色菌系菌株XN-1产生黄色菌核的性状可以通过无性繁殖（产生分生孢子）遗传。通过与黑色菌系杂交试验，证实黄色菌核产生受一个基因控制；（2）发现黑色菌系菌核皮层细胞有黑色素积累，皮层细胞饱满；而黄色菌系菌核皮层细胞缺乏黑色素积累，皮层细胞皱缩、塌陷。分析黑色素合成相关基因（Bcpks12、Bcpks13、Bcygh1、Bcbrn1、Bcbrn2、Bcscd1）的表达量及序列进一步证实黄色菌系产生的原因在于黑色素合成受阻。发现控制菌核黑色素合成的转录因子基因Bcsmr1基因序列出现了一个单碱基缺失，导致产生截短蛋白质BcSmr1，转录激活活性下降，由此下调了黑色素合成基因。将完整的Bcsmr1导入XN-1菌株中，黑色素合成基因表达上调，菌核部分恢复黑色。可见，黄色菌系产生的原因是Bcsmr1中的单碱基缺失。（3）发现黑色菌系和黄色菌系的菌丝和分生孢子存活差异较小，而黄色菌核存活较黑色菌核存活差。（4）发现黄色菌系在自然条件下的出现频率为0.32%。将黄色菌系分生孢子接种在草莓上，第一季引起发病，并检测到黄色菌系的存在，但第二季和第三季则检测不到黄色菌系的存在。可见，黄色菌系流行风险较低。本项目研究结果为理解菌核在灰霉病病害循环中的作用提供了有力试验证据。项目执行期间发表SCI论文4篇，培养博士研究生4名，硕士研究生2名。