Field screening for PM resistance

A total of 140 genotypes of cucumber were used for PM characterization. Out of these 101 genotypes were collected from local areas of Jammu and Kashmir and India (online Supplementary Table S1), while another 39 genotypes consisting of varieties and advanced lines were imported from Asian Vegetable Research and Development Center (AVRDC), Taiwan and U.S. National Plant Germplasm System (USNPGS), USA (online Supplementary Table S2). It is to state that the landraces were procured from farmers under the Protection of Plant Varieties and Farmers Rights Act (PPVFRA) with access and benefit-sharing provisions to farmers. The experiments were conducted at Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, India, over for 2 years during the summer seasons of 2016 and 2017. The germplasm comprising 101 genotypes was screened for PM resistance under natural epiphytotic conditions of Jammu (online Supplementary Table S1) in a randomized block design with three replications for each of the genotypes. Two genotypes as checks Pusa Barkha (GS13) and Punjab Naveen (GS48) were used as spreader/infector rows to ensure a uniform spread of the disease. The disease severity for PM was recorded on a scale of 0–5 (Ransom et al., Reference Ransom, Briens and Glass1991). Genotypes with no disease were recorded as ‘0’, ‘1’ for genotypes with trace levels of infection, ‘2’ for mildew colonies confluent on some leaves with no visible stem infection, ‘3’ for many (up to 50%) leaves completely covered with mildew and stems infected, ‘4’ for most (>50%) leaves covered with mildew and ‘5’ for all leaves severely affected. Ratings were based on the assessment of five plants per replication using the following scale:

Per cent disease index (PDI) was calculated as per the formula:

$${\rm PDI} = \displaystyle{{{\rm Sum}\,{\rm of}\,{\rm all}\,{\rm disease}\,{\rm ratings} \times 100} \over {{\rm Total}\,{\rm number}\,{\rm of}\,{\rm ratings} \times {\rm Maximum}\,{\rm disease}\,{\rm grade}}}$$

Based on the PDI value, the germplasm in the population was classified into five groups namely highly resistant (HR) (0–15%), moderately resistant (MR) (16–25%), T (26–35%), susceptible (S) (36–50%) and highly susceptible (HS) (>50%).

Artificial screening for PM resistance

The highly and MR genotypes from field screening along with the imported germplasm (59 genotypes) were screened under artificial conditions as well (online Supplementary Table S2). The germplasm imported from USNPGS has been reported to be R to PM (Block and Reitsma, Reference Block and Reitsma2005). The cucumber plants were infected with Spharotheca fuliginea under artificially controlled conditions by a method adopted by Ma et al. (Reference Ma, Cui, Qiang and Sun2002). An isolate of the fungus was obtained from naturally infected field-grown cucumber plants, and identified by observing its morphology and spore structure under a microscope with the aid of available literature. The seeds of cucumber were sown in flowerpots of 30 cm in diameter, which were placed in a greenhouse with a temperature range of 20–25°C. The isolate was maintained on the young plants of Pusa Barkha and Punjab Naveen (checks). The infection was observed visually by leaf disc assay as proposed by Cohen (Reference Cohen1993) as well as using the PDI value on three plants. The discs were inoculated by conidial suspension as well as by dusting of powdery mass on the adaxial side of the discs cut from third and fourth true leaves. The disease severity on discs was recorded 5–8 days after inoculation and rated as 0 = non-infected; 1 = only hyphae present; 2 = hyphae upto 50 conidiophores per disc (heavy sporulation). The sporulating discs ranking 2 and 3 were considered as S and 1 as R. The response of leaf discs to whole plants was also observed. The whole plants were dusted with heavily infected leaves.

SSR genotyping

Genomic DNA was isolated from young green leaves of cucumber following CTAB method (Doyle and Doyle, Reference Doyle and Doyle1990). The quality and concentration of extracted DNA were checked by agarose gel electrophoresis and nanodrop. A total of eight PM-linked SSR markers from previous studies were used for molecular characterization of 19 R/T/S lines selected following field and artificial screening (online Supplementary Table S1 and Table 1) (Sakata et al., Reference Sakata, Kubo, Morishita, Kitadani, Sugiyama and Hirai2006; Hai-ying et al., Reference Hai-ying, Zhen-guo, Ai-jun, Feng, Yong-jian and Yong2008; Xu et al., Reference Xu, Yu, Xu, Shi, Lin, Xu, Qi, Weng and Chen2016). The PCR products were analysed for the presence of alleles linked to previously identified resistance genes/QTLs. PCR amplification was performed in a 15 μl reaction mixture as reported by Dar et al. (Reference Dar, Mahajan, Lay and Sharma2017). The PCR reagents used were procured from PROMEGA, WI USA. The amplifications were carried out in thermal cycler (Make: Eppendorf, nexus GX2) with an initial denaturation of 94°C for 5 min, followed by 35 cycles of 94°C for 15 s, 55–60°C for 30 s and 72°C for 30 s, and a final extension at 72°C for 7 min. Amplified products were separated using 3% agarose gel with 100 bp ladder (Thermo Scientific, SM0241), and gels were documented by DNR Bio-Imaging System MiniLumi.

Table 1. List of reported linked markers used in the present study