由灰葡萄孢（Botrytis cinerea）引起的人参灰霉病近年在我国人参产区大流行，造成人参叶片、茎秆、参籽腐烂脱落，严重影响人参正常生长及种子发育，给人参种植业造成了较为严重的经济损失。目前，人参灰霉病的防治主要依赖化学农药。杀菌剂滥用导致抗（耐）药人参灰霉病菌菌株不断出现，杀菌剂的防病效果显著降低。为了解我国人参灰霉病菌的抗药水平现状，本研究检测了我国人参产区102株灰霉病菌对多菌灵、异菌脲和嘧霉胺的抗性水平，研究结果对于整体了解我国人参灰霉病菌的抗药性水平，指导产区制定科学合理的灰霉病防治策略具有重要参考价值；通过DNA测序对比分析了人参灰霉病菌β-微管蛋白基因和BcOS-1基因的突变位点，结合菌株的抗性表型，为深入了解人参灰霉病菌基因突变与抗（耐）药性的关系提供了理论依据；结合转录组测序技术，研究了抗性和敏感菌株在嘧霉胺胁迫下的基因差异表达情况，为深入研究人参灰霉病菌对嘧霉胺产生抗药性的分子机制奠定了理论基础。

全文研究结果如下：

（1）采用菌丝生长速率法对102株人参灰霉病菌进行抗性水平检测，结果为：640 μg·mL^-1多菌灵对供试菌株的生长抑制率均小于50%，部分菌株的生长未受影响，说明多菌灵对我国当前人参灰霉病菌群体已基本丧失防治效果。异菌脲抗药性检测发现，供试人参灰霉病菌中含异菌脲敏感菌株24株（25.53%），低抗菌株16株（15.69%），中抗菌株62株（60.78%），大部分人参灰霉病菌已对异菌脲产生一定程度的抗药性，但并未发现高抗菌株；另外，不同产区人参灰霉病菌对异菌脲的抗性水平存在差异，吉林通化人参灰霉病菌对异菌脲最敏感（EC₅₀=1.3344 μg·mL^-1），黑龙江逊克人参灰霉病菌对异菌脲抗性最强（EC₅₀=3.1680 μg·mL^-1）。嘧霉胺抗药性检测发现，供试人参灰霉病菌中含嘧霉胺敏感菌株28株（24.75%），中抗菌株3株（2.94%），高抗菌株71株（69.61%）。吉林靖宇人参灰霉病菌对嘧霉胺的抗性最强（EC₅₀=44.8734 μg·mL^-1），黑龙江嘉荫人参灰霉病菌对嘧霉胺最敏感（EC₅₀=1.2229 μg·mL^-1）。相关性分析结果表明，人参灰霉病菌对异菌脲和嘧霉胺无交互抗性。

（2）通过人参灰霉病菌多菌灵抗性相关β-微管蛋白基因和异菌脲抗性相关BcOS-1基因片段扩增以及与敏感型灰霉病菌的序列比对，发现供试人参灰霉病菌β-微管蛋白基因第198位密码子均发生了碱基突变，共检测到3种突变类型，其中E198A突变32株，E198V突变69株，E198K突变1株，并且所有菌株均对多菌灵表现高抗，说明β-微管蛋白基因第198位密码子突变导致人参灰霉病菌对多菌灵产生高抗药性。供试人参灰霉病菌中有2株敏感菌株BcOS-1基因未发生突变，其余100株检测到3种突变类型，I365S突变47株，I365N突变23株，Q369P+N373S突变30株。异菌脲抗性水平检测结果表明，异菌脲抗性菌株的抗药水平和BcOS-1基因突变密切相关，Q369P+N373S突变菌株的EC₅₀均值在三种突变类型中最高。

（3）以嘧霉胺抗性菌株HRG21、敏感菌株FSG43为材料，在嘧霉胺处理2 h和6 h取样，开展转录组测序分析。以相同时间点无嘧霉胺处理为空白对照。测序共获得158.87 Gb的Clean Base。以padj＜0.05且|log₂FoldChange|≥1为条件筛选差异基因，HM2 vs FM2有1357个DEGs，HM6 vs FM6有1962个DEGs，HM2 vs HH2有687个DEGs，HM6 vs HH6有2820个DEGs，HM6 vs HM2有1786个DEGs，FM6 vs FM2有2467个DEGs，FM6 vs FH6有2047个DEGs，FM2 vs FH2比较组在筛选条件下未发现DEGs。生物信息学分析结果表明，嘧霉胺胁迫下大部分BcatrB、BcatrO等ABC转运蛋白基因及Bcmfs1、BcmfsM2等MFS转运蛋白基因在嘧霉胺抗性菌株HRG21中上调表达，而在嘧霉胺敏感菌株FSG43中下调表达。

差异表达基因GO富集分析发现，嘧霉胺抗性菌株HRG21在嘧霉胺胁迫前后的差异表达基因主要富集在辅因子结合、四吡咯结合、铁离子结合、血红素结合和氧化还原酶等通路，而嘧霉胺敏感菌株FSG43在嘧霉胺胁迫前后的差异表达基因主要富集在膜组成、跨膜运输、辅因子结合、辅酶结合和转运蛋白结合等通路。KEGG分析结果表明，各个比较组均有部分差异表达基因参与了氨基酸代谢和核糖体相关途径，并且参与上述代谢途径的基因大多在嘧霉胺处理后下调表达；在HM2 vs HH2、HM2 vs FM2、HM6vsHH6、HM6 vs FM6比较组中参与ABC转运蛋白代谢途径的差异表达基因大部分上调表达；FM6 vs FH6比较组中参与ABC转运蛋白代谢途径的差异表达基因大部分下调表达。

Gray mold caused by Botrytis cinerea has been prevalent in ginseng producing areas in China in recent years, resulting in rotting and shedding of leaves, stems, and seeds in the period of ginseng growing, seriously affecting the normal growth and seed development of ginseng plants, causing serious economic loss to ginseng planting industry. At present, the control of ginseng gray mold mainly depends on chemical pesticides. The abuse of fungicides led to the continuous emergence of fungicide resistance B. cinerea isolates, and the control efficacy of fungicides reduced significantly.

In total, B. cinerea 102 isolates were purified from the main ginseng growing areas (MGGA) in China, the fungicide resistance of them to carbendazim, iprodione and pyrimethanil were examined in the present work. The research results have valuable reference for the overall understanding the fungicide resistance of B. cinerea isolates from MGGA in China and guiding to formulate scientific and reasonable grey mold control strategies.

The mutation sites of β-tubulin gene and BcOS-1 gene in B. cinerea related to carbendazim and iprodione resistance were analyzed by DNA sequencing, respectively. Combined with the resistance phenotype of B. cinerea isolates, all the results providing a theoretical basis for further understanding the relationship between gene mutation and fungicide resistance of B. cinerea isolates. Furthermore, the differential expression of genes in resistant and sensitive isolates under pyrimethanil stress were studied by transcriptome sequencing technology, the results provided a theoretical basis for explaining the molecular mechanism of pyrimethanil resistance in B. cinerea.

The full-text research results were listed as follows:

(1) The fungicide resistance level of all B. cinerea isolates was examined by the mycelial growth rate method. The results showed that, the growth inhibition rate of all B. cinerea isolates on PDA medium containing 640 μg·mL^-1 carbendazim were no more than 50%, and the growth of part B. cinerea isolates even not inhibit, which indicated that carbendazim has lost its control efficacy on the current B. cinerea population from MGGA in China. For the resistance of B. cinerea isolates to iprodione, 24 isolates were sensitive（23.53%）, 16 isolates were low resistance（15.69%）, and 62 isolates were medium resistance（60.78%）, high resistance isolate was not found. There were significant differences in the iprodione resistance of B. cinerea isolates from different MGGA. Iprodione resistance of B. cinerea isolates from Jilin Tonghua was the most sensitive (EC₅₀=1.3344 μg·mL^-1), and those from Xunke was the highest (EC₅₀=3.1680 μg·mL^-1).

For the resistance of B. cinerea isolates to pyrimethanil, 28 isolates were sensitive（27.45%）, 3 isolates were moderate resistant（2.94%）, and 71 isolates were high resistant（69.61%）. The pyrimethanil resistance of B. cinerea isolates from Jingyu, Jilin was the highest (EC₅₀=44.8734 μg·mL^-1), and the pyrimethanil resistance of those from Jiayin of Heilongjiang was the lowest (EC₅₀=1.2229 μg·mL^-1). Correlation analysis showed that, B. cinerea isolates tested have no cross resistance to iprodione and pyrimethanil.

(2) The β-tubulin and BcOS-1 gene related to carbendazim and iprodione resistance were amplified, respectively. Through blast with the nucleotide sequences of fungicide susceptible B. cinerea strain, we found that the codon 198 of β-tubulin gene in all B. cinerea isolates were mutated, and there are three mutation types were detected, including 32 strains of E198A mutation, 69 strains of E198V mutation and 1 strain of E198K mutation. All B. cinerea isolates shown high resistance to carbendazim, indicating that the mutation at codon 198 of β-tubulin gene led to the resistance of B. cinerea to carbendazim.

Except for 2 B. cinerea isolates have no mutation in BcOS-1 gene, three gene mutation types were detected on the other 100 B. cinerea isolates, including 47 strains of I365S mutation, 23 strains of I365N mutation and 30 strains of Q369P+N373S mutation. Results indicated that, the iprodione resistance of B. cinerea isolates was closely related to the mutation of BcOS-1 gene, and the resistance of Q369P+N373S mutant was the highest.

（3）The pyrimethanil resistant B. cinerea strain HRG21 and the pyrimethanil sensitive B. cinerea strain FSG43 treated 2 h (HM2 and FM2, respectively) and 6 h (HM6 and FM6, respectively) by pyrimethanil were performed transcriptome sequencing. B. cinerea strains treated with sterile water were used as blank control (HH2 and HH6, respectively). In total, 158.87 Gb clean base was generated through Illumina sequencing. Under the conditions of padj≤0.05 and |log₂FoldChange|≥1, 1357 differential expression genes (DEGs) in HM2 vs FM2, 1962 DEGs in HM6 vs FM6, 687 DEGs in HM2 vs HH2, 2820 DEGs in HM6 vs HH6, 1786 DEGs in HM6 vs HM2, 2467 DEGs in FM6 vs FM2, and 2047 DEGs in FM6 vs FH6 were identified. However, no DEGs were found in FM2 vs FH2. It was found that, most ABC transporter genes including BcatrB and BcatrO and MFS transporter genes including Bcmfs1 and BcmfsM2 were up-regulated in HRG21 and down-regulated in FSG43 after treatment with pyrimethanil.

GO enrichment analysis results indicated that, the DEGs of resistant strain HRG21 before and after treated by pyrimethanil were mainly enriched in the pathways of cofactor binding, tetrapyrrole binding, iron ion binding, heme binding and oxidoreductase, while the DEGs of sensitive strain FSG43 were mainly enriched in the pathways of membrane composition, transmembrane transport, cofactor binding, coenzyme binding and transporter binding. The results of KEGG analysis showed that, some DEGs in each comparison group were involved in amino acid metabolism and ribosome-related pathways, and most of the DEGs involved in above metabolic pathways were down-regulated when treated by pyrimidine. Most of the DEGs involved in the metabolic pathway of ABC transporters were up-regulated in the HM2 vs HH2, HM2 vs FM2, HM6vsHH6, and HM6 vs FM6. Most of the DEGs involved in ABC transporter pathway in FM6 vs FH6 group were down-regulated.