Screening lentil (Lens culinaris Medik) Genotypes for Resistance against Pre-Flowering Blight and Identification of Pathogen by ITS Sequencing

Lentil is a popular food legume in eastern India cultivated during cool season followed by kharif rice. Nutritional value of pulses is more in developing countries than other area in terms of energy per unit uptake (Iqbal et al., 2006). At the national level, though central and lower Gangetic areas predominately recorded for higher production, but productivity is high in eastern India. In spite of being higher productivity in eastern Indian states, production is not assured even after optimum availability of water, nutrients due to several hindrances which includes seedling mortality due to collar rot and adult mortality or destruction of foliage due to attack of blight during early flowering stage. Thus, farmers do not show interest in adoption of lentil as legume after rice harvest. Therefore, cultivation in rice fallow areas, in spite of having major potentiality for lentil area expansion becomes difficult. Identification of core set of resistance source has been always based on preliminary screening of a large set of germplasm. Not only in eastern India, in whole south Asia, Collar rot, Alternaria and Stemphylium, are major problem for lentil. Besides these two major diseases in lentil, vascular wilt caused by Fusarium oxysporum is also reported to cause a significant yield loss in lentil (Bayaa et al., 1997). However, Alternaria and Stemphylium blight occurs when plant starts flowering (Kumar, 2007). Most of the popular cultivars are highly susceptible. Several earlier reports of field screening against blight disease of lentil during flowering stage were not confirmed by identification of causal organism (Mandal et al., 2018, Das et al., 2017). Identification of causal organism is critical for lentil because both Alternaria and Stemphylium blight occurs during flowering stage and can grow profusely above 25°C (Mwakutuya, 2010). Only difference is that Stemphylium requires 24-48 hrs wet period where as Alternaria prefers humid environment. In eastern part of India, both higher temperature during flowering stage in addition to a spell of 1-2 days of light rain are common. Therefore, this study aims to identify resistant genotypes from a large set of lentil genotypes against blight disease at flowering stage and confirmation of the causal organism thereof.

A set of 500 genotypes consisting of endogenous and exotic collection was collected from NBPGR, New Delhi, India during 2018-19 (Table 1). Sowing was done on 5th December, 2018 at University Experimental Station, Kalyani (Latitude 22.87° and longitude 88.20°) at new alluvial zone which is 20 days later than the recommended sowing date to assure congenial temperature for disease screening. All 500  genotypes of lentil have been sown in 3 replicated lines of 1m length each keeping local popular variety WBL-77, Ranjan, Asha as a check, after each 50 genotypes. Screening plot was so selected where continuous lentil cultivation and outbreak of blight symptom of consecutive ten years were recorded.
 
Screening methodology
 
Recommended fertilizer dose of N:P:K @ 20:40:20 was applied during final land preparation. Four days before disease scoring, spraying of water was made at bud formation stage to create congenial environment for pathogens for disease screening under natural condition. Screening was done at pre flowering stage for one year during February 2019, using 1-9 scale for each replicated genotype, following methodologies described by Kant et al., (2017). Only 466 genotypes survived up to budding stage and they were considered for scoring against Alternaria tolerance, keeping three check varieties as control in each block of fifty genotypes.
        
Each genotype was sown in replicated manner and scored separately. From replicated disease score data percentage disease incidence (PDI) was worked out using the formula, PDI = [Sum of numerical rating/total number of observations taken × maximum disease score] × 100 (Wheeler, 1969). Classification of genotypes including checks have been done based on mean score using disease rating scale recently used by Kant et al., (2017) for blight disease (Table 1). Mean values have been classified in total nine categories i.e. 1-9. Genotype which showed mean value of around 1 was classified as resistant and genotypes which showed mean value of around 2 was classified as resistant to moderate resistant category etc.
 

 
Isolation of fungal DNA and ITS sequencing
 
Infected plant debris have been collected on the day of disease scoring in a moist polythene packet from ten different locations of the field and transferred to lab for preparation of pure culture. Fungal DNA has been isolated from pure culture following the methodology as described earlier (Guharoy et al., 2006). In brief, 50 mg mycelia were ground adding 0.1% PVP and liquid nitrogen followed by 750 µl extraction buffer comprising of 40 mM Tris-Cl, 50 mM NaCl, 5 mM EDTA and 0.025% SDS. After spin down, lysate has been mixed with 500 µl of mix having Phenol: Chloroform: Isoamyl alcohol in 25:24:1 ratio. Supernatant has been taken and incubated in 37°C water bath after RNase addition. Finally, DNA was precipitated using ice cold isopropanol followed by 70% ethanol wash and re-suspended in TE.  Polymerase chain reaction has been set at 58°C using ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGC TTAT TGATATGC) primer to amplify 550 base pair. Gel purified amplicon was sequenced and compared in NCBI database using BLAST.

Our primary goal was to screen resistance against Stemphylium blight and accordingly screening was made at budding stage only. During screening period, day temperature was between 26°C to 30°C and at night, it was 16°C-19°C. We have also created moist and humid environment suitable for blight disease of lentil. From the infected plant, pure culture of fungus was made. Microscopic observation of conidial structure as isolated from the infected plants indicates the isolated fungal pathogen as Alternaria. To confirm the microscopic observation, ITS was amplified by universal primer pair and sequencing was made. Universal primer pair amplified around 550bp fragment only (Fig 1a). Sequencing was made from both ends of all ten isolates so that recovery of sequencing for the whole length can be confirmed. Multi-alignment with the existing data base showed cent percent similarity (Fig 1b) with partial ITS sequence of the Alternaria genomic data (Accession no. MN394880). Nucleotide sequencing was finally submitted to NCBI data base (Accession number MN336221). ITS sequence of our isolates showed two nucleotide mismatch with Alternaria tenuissima, that was reported as blight causing pathogen in lentil also (Prasad et al., 2017). Mismatch nucleotides were also confirmed by chromatogram (Fig 1c) as shown in boxes. Mismatch sequences as shown in boxes of chromatogram Fig 1c, are reverse complementary to the sequence submitted (Fig 1a), as it was developed by using reverse primer. No deviation was observed for remaining samples. Although, Stemphylium and Alternaria are very closely related fungi but occurrence of Alternaria was confirmed by ITS sequencing during our screening. Any Stemphylium out of ten randomly samples from the entire field was not noted. For successful disease symptom, both the pathogens require above 25°C temperature but Stemphylium requires moisture droplets (Mwakutuya, 2010) in leaves where as Alternaria can grow in presence of humid weather (Bashi and Rotem, 1974) and that may be one of the reasons why Stemphylium was not observed in our experimental field during our screening. However, Alternaria was not considered as widespread disease in eastern part of our country like Stemphylium but its presence observed throughout the field. Symptom of Alternaria is very much similar with Stemphylium as phaeo-dictyosporic conidia produced by Stemphylium, Ulocladium and Alternaria are similar (Thomma, 2003). Strong confirmation study, molecular analysis of rDNA sequence has been reported to be ideal (Pryor and Gilbertson, 2000). So, it is recommended to confirm the causal organism during lentil breeding program, particularly against Stemphylium blight.
 

        
Based on field screening, 466 genotypes were grouped considering three genotypes WBL-77, Asha and Ranjan as check after each fifty lines. Asha and Ranjan were reported to be susceptible genotypes against Stemphylium (Kumar, 2007) whereas WBL77 showed to be moderately susceptible. Our observation also confirms the susceptibility reaction of Asha, Ranjan and WBL77 against Alternaria. Most of the studied genotypes are susceptible as observed from the mean value of around 6 where standard deviation is only 1.86. One exotic and two indigenous collections, viz EC866132, IC267667, IC201778 have been selected as resistant. In second category, resistant to moderately resistant includes total 11 lines viz. ILL2665, ILL1810 (A), ILL1854, IC139817, IC267669, IC201783, IC248966, IC98383, IC201739, IC267530, IC78554. Totally 46 genotype have been categorized as moderately resistant, 41 genotypes as moderately resistant to moderately susceptible moderately susceptible group had total of 83 genotypes, in moderately susceptible to susceptible there were 90 genotypes, 77 genotypes in susceptible group, there was 100 genotypes in susceptible to very susceptible group and in very susceptible group 15 genotype (Table 1). Stemphylium susceptible genotypes like Asha and Ranjan also showed same reaction against Alternaria. Thus susceptibility reaction in lentil for both the pathogens might have a common pathway, confirmation of which requires further detailed study. Although there are several earlier reports of Stemphylium resistance (Das et al., 2017), but this is the first report of occurrence of Alternaria in eastern India as well as its resistance source. Identified genotype can be used as donor parent in future lentil breeding program. 

Authors are thankful to NBPGR for providing the germplasm set and ICARDA for financial support.