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Overlap-extension PCR is a method for splice of gene segments to produce focused fragments for constructing recombinant plasmid, but its complexity limits its application. To simplify the protocol and to improve the effectiveness, we employed gradient temperatures to replace the single annealing temperature in the thermo-cycling program, and optimize the templates ratio. The concentration of each fragment was adjusted to 10 ng µl . Fragment concentration ratio was the inverse of the fragment size ratio. The products of fused segments were 2000-5000 bp in length using the revised one-step method. This method splices effective two or more fragments to fused gene and produce recombinant plasmid.

Curvularia leaf spot (CuLS), caused by Curvularia lunata, is a devasting foliar disease in the maize-growing regions of China. Resistant varieties were widely planted in these regions in response to CuLS. However, over time, C. lunata has gradually adapted to the selective pressure and, in recent years, the incidence of CuLS has increased. To assess the correlation between virulence and genetic diversity, a total of 111 isolates was collected from 15 maize-growing regions located in nine provinces in China. These isolates were evaluated for virulence on maize using nine differential hosts: Shen135, CN165, Mo17, Luyuan92, 78599, Ye478, B73, E28, and Huangzaosi. To evaluate the genetic diversity, 657 polymorphic amplified fragment length polymorphism markers were generated. Results showed that the isolates could be grouped into three pathotypes according to the phenotypic expression of the differential inbred lines. Isolates were clustered into two genetic diversity groups and further divided into subgroups. However, the correlation between virulence and genetic diversity grouping was low. Also, there was a low correlation observed between pathotype and geographic distribution. The ratio of mating type I to mating type II for all isolates was close to 3:4.

Summary Iron is virtually an essential nutrient for all organisms, to understand how iron contributes to virulence of plant pathogenic fungi, we identified ClFTR1 and ClNPS6 in maize pathogen Curvularia lunata (Cochliobolus lunatus) in this study. Disruption of ClNPS6 significantly impaired siderophore biosynthesis. ClFTR1 and ClNPS6 did mediate oxidative stress but had no significant impact on vegetative growth, conidiation, cell wall integrity and sexual reproduction. Conidial germination delayed and appressoria formation reduced in ΔClftr1 comparing with wild type (WT) CX‐3. Genes responsible for conidial germination, appressoria formation, non‐host selective toxin biosynthesis and cell wall degrading enzymes were also downregulated in the transcriptome of ΔClftr1 and ΔClnps6 compared with WT. The conidial development, toxin biosynthesis and polygalacturonase activity were impaired in the mutant strains with ClFTR1 and ClNPS6 deletion during their infection to maize. ClFTR1 and ClNPS6 were upregulated expression at 12–24 and 48–120 hpi in WT respectively. ClFTR1 positively regulated conidial germination, appressoria formation in the biotrophy‐specific phase. ClNPS6 positively regulates non‐host selective toxin biosynthesis and cell wall degrading enzyme activity in the necrotrophy‐specific phase. Our results indicated that ClFTR1 and ClNPS6 were key genes of pathogen known to conidia development and virulence factors.

The role of NADPH oxidases (NOXs) in pathogenesis and development in the Curvularia leaf spot (CuLS) agent Curvularia lunata remains poorly understood. In this study, we identified C. lunata ClNOX2, which localized to the plasma membrane and was responsible for reactive oxygen species (ROS) generation. Scavenging the ROS production inhibited the conidial germination and appressorial formation. The ClNOX2 and ClBRN1 deletion mutants were defective in 1,8-dihydroxynaphthalene (DHN) melanin accumulation, appressorial formation, and cellulase synthesis and exhibited lower virulence. However, disruption of the ClNOX2 and ClBRN1 genes facilitated hyphal growth, enhanced stress adaptation to cell wall-disrupting agents, and promoted developmental processes such as conidiation, conidial germination, and pseudothecium and ascus formation. Interestingly, loss of ClM1, the cell wall integrity (CWI) MAPK gene in C. lunata led to similar morphology and pathogenicity phenotypes with ClNOX2 and ClBRN1 deletion mutants, like abnormal conidia, fewer appressoria, less melanin, increased hyphal growth and enhanced tolerance to congo red (CR). These results indicated that the ClNOX2 gene plays an important role in C. lunata development and virulence via regulating intracellular DHN melanin biosynthesis. RT-qPCR revealed that the ClNOX2-related ROS signaling pathway and ClM1-mediated CWI signaling pathway are cross-linked in regulating DHN melanin biosynthesis. Our findings provide new insights into how ClNOX2 participates in the pathogenesis and development in hemibiotrophic plant fungal pathogens.