Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. To reveal the potential resistance mechanism of P. capsici to fungicide metalaxyl, a metalaxyl-resistant mutant strain SD1-9 was obtained under laboratory conditions. The pathogenicity test showed that mutant strain SD1-9 had different pathogenicity to different host plants with or without the treatment of metalaxyl compared with that of the wild type SD1. Comparative transcriptome sequencing of mutant strain SD1-9 and wild type SD1 led to the identification of 3845 differentially expressed genes, among them, 517 genes were upregulated, while 3328 genes were down-regulated in SD1-9 compared to that in the SD1. The expression levels of 10 genes were further verified by real-time RT-PCR. KEGG analysis showed that the differentially expressed genes were enriched in the peroxisome, endocytosis, alanine and tyrosine metabolism. The expression of the candidate gene XLOC_020226 during 10 life history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage and basically showed a gradually increasing trend with increasing infection time in pepper leaves in SD1-9 strain, while its expression gradually increased in the SD1 strain throughout the 10 stages, indicated that XLOC_020226 may be related to the growth and pathogenicity of P. capsici. In summary, transcriptome analysis of plant pathogen P. capsici strains with different metalaxyl resistance not only provided database of the genes involved in the metalaxyl resistance of P. capsici, but also allowed us to gain novel insights into the potential resistance mechanism of P. capsici to metalaxyl in peppers.Microorganisms 2020, 8, 278 2 of 17 and has a single site of action on pathogens, Phytophthora can develop resistance as they are susceptible to mutations [3][4][5]. Metalaxyl resistance is mainly found in Phytophthora species such as P. capsici, P. infestans, and P. parasitica. A metalaxyl-resistant strain of P. infestans was discovered in the Netherlands in 1980 [6]. Subsequently, European and American countries reported metalaxyl-resistant strains of P. capsici [7][8][9]. China also reported resistant P. capsici in the 1960s [10]. To date, metalaxyl-resistant strains of P. capsici have been successively found in Anhui, Gansu, and Yunnan Provinces [10][11][12][13].There are some studies on the resistance mechanism of Phytophthora to metalaxyl. Chen et al.[14] revealed two evolutionary pathways of resistance involving the RPA190 gene. The results of their research indicate that changes in the activity of Phytophthora RNA polymerase are important resistance mechanisms. Similar results were also confirmed in P. infestans. The diversity of the RNA polymerase I large subunit sequence of P. infestans plays a key role in its resistance to metalaxyl [15]. The biolog...