Characterization of blast pathogen in finger millet and the biochemical and molecular mechanisms of diseas resistance.

Abstract

The study entitled “Characterization of blast pathogen in finger millet and the biochemical and molecular mechanisms of disease resistance” was carried out at the Department of Plant Pathology, College of Agriculture, Vellayani, during 2023-2025 with the objective of characterization of blast pathogen in finger millet, varietal screening for resistance and evaluation of biochemical and molecular mechanisms of disease resistance. A field survey was conducted across six agro-ecological units (AEUs) of Kerala viz., AEU 3, AEU 8, AEU 11, AEU 15, AEU 16 and AEU 18 covering the districts of Alappuzha, Thiruvananthapuram, Kasaragod, Palakkad, and Idukki. Ten locations were surveyed, and leaf samples showing typical blast symptoms were collected. The symptoms ranged from small brown specks to spindle-shaped lesions with greyish-white centre and yellowish-brown margin, which often coalesced, giving a blighting appearance. Disease incidence and severity were recorded, with the highest incidence (56.33%) and severity (52.60%) observed in Santhanpara region of Idukki district. Correlation analysis revealed a positive relationship between disease parameters and relative humidity, and a negative correlation with maximum temperature. Twelve isolates of the pathogen were obtained from diseased samples, and pathogenicity was confirmed through artificial inoculation on one month old finger millet plants. Cultural and morphological characterization revealed colony colours ranging from greyish-white to black, with flat to raised mycelium and brown to black pigmentation on potato dextrose agar (PDA). The isolates took 13–15 days for full plate coverage (90 mm). Microscopic examination showed hyaline, septate mycelia with two-septate, three- celled, pyriform conidia. Among the isolates, the Vellayani isolate (P4) showed the fastest growth (growth rate of 0.77 cm/day) and highest virulence, producing symptoms within 5 days, with an average of 14.7 lesions per leaf and lesion size of 2.05 × 0.47 cm. Plants inoculated with P4 exhibited the highest disease severity (75.30%), identifying it as the most virulent isolate. Symptom development and sporulation studies with the isolate P4 were conducted and observed that symptoms appeared on 5 days after inoculation (DAI), whereas 135 sporulation began at 10 DAI. Morphological observation showed erect, hyaline conidiophores bearing successively arranged, pale-brown, two-septate, pyriform conidia with a pointed apex and basal hilum. The physiological studies of the pathogen were conducted by growing on different media (PDA, Oatmeal agar, Host extract +2% sucrose agar, Ragi yeast lactose agar and Water agar), temperature (15℃, 20℃, 25℃, 30℃ and 35℃) and light intensity (1000 lux, 2000 lux, 3000 lux, 12 hours darkness and 12 hours light and complete darkness). The results showed highest mycelial growth on PDA medium, at 25 °C, and under complete darkness. Molecular characterization using LSU primers confirmed the identity of the virulent isolate as Pyricularia grisea. The nucleotide sequence was submitted to GenBank under the accession number PX369007. Twenty finger millet accessions (10 indigenous and 10 high-yielding types) were screened against P. grisea. The results revealed that accessions Aduvilanthankudy, Attappady Ragi Millet Mission, Ramapuram, and GPU 28 were resistant; Cherthala South, Pachamutti, Neelikuzhy, Co 15, ATL-1, ML-365, Vakula, Tirumala, and CFMV-1 were moderately resistant; while Kadukumanna, Mattakkavu, Sirukkorai, Palakkini, PPR 1170, and VL 408 were susceptible and KMR 301 was highly susceptible. Aduvilanthankudy recorded the lowest percent disease index (PDI) of 19.24% and a disease score of 1.73, whereas KMR 301 showed the highest susceptibility (PDI 84.73%). Yield assessment revealed that GPU 28 had the highest yield (8.10 g/plant), while Aduvilanthankudy recorded the highest yield among indigenous accessions (7.15 g/plant). Biochemical assays were conducted on resistant (Aduvilanthankudy) and susceptible (KMR 301) accessions at 24, 48, and 72 hours after inoculation (HAI). Defense-related enzymes—peroxidase, polyphenol oxidase, and catalase showed significantly higher activity in the resistant accession, with early peaks at 24 HAI. Phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD) exhibited delayed but sustained maximum activity at 72 HAI in the resistant plants, indicating their role in defense response.

Gene expression analysis using quantitative Real-Time PCR (qRT-PCR) conducted five days after inoculation revealed upregulation of defense-related genes PAL and EIN2 in the resistant accession Aduvilanthankudy, showing relative fold changes of 2.90 and 1.34, respectively, compared to the susceptible KMR 301. The study concludes that the blast pathogen prevalent in Kerala finger millet ecosystems is Pyricularia grisea. The indigenous accession Aduvilanthankudy displayed strong resistance, characterized by early activation of key defense enzymes and higher expression of defense genes. This genotype will be a promising source of resistance for incorporation into breeding programs aimed at developing high-yielding, blast-resistant finger millet accessions adapted to regional climatic conditions and pathogen pressure.