RESUMO
Cenchrus americanus (L.) Morrone (Poaceae), is an important millet crop cultivated mainly in arid and semiarid regions and is a staple food grain for millions of people. During 2021 July surveys in the pearl millet fields in Mysore (12°30'55" N; 76°56'54" E), Karnataka, India, plants showed spathe blight and leaf spot disease with an overall incidence ranging from 5 - 8% in the 15 hectares surveyed. Infected leaves appeared brown, and lesions extended to the sheath. Some spathes were also found infected with similar symptoms. Diseased leaves and spathes were collected (n = 5 each) for pathogen identification. Samples were cut into small pieces (0.5 cm2), sterilized with sodium hypochlorite (2%, v/v), and blotted dried. The associated fungal pathogen was isolated on potato dextrose agar (PDA) medium amended with Streptomycin (40 mg/L) and incubated at 28 ºC for 1 week. Colonies were grey, fluffy, cottony with an irregular margin, undulate and dark brown in the back of the plate. Conidiophores were pale brown, erect, slightly curved, septate, unbranched, verruculose and measured 27.1 - 94 µm in length × 2.3 - 4.5 µm in width (n = 20). Conidiogenous cells were brown, subcylindrical, irregularly shaped, and conidia were straight, mainly elliptical, dark brown smooth, with two to three septa, with measurements of 11.1 - 26.4 µm by 5.7 - 14.3 µm (n = 50). Based on morphological characters, the pathogen was identified as Curvularia sp. Two representative isolates (UOMPM1 & UOMPM2) were molecularly identified. The total genomic DNA was extracted with a CTAB method, and ITS, GAPDH and tef-1α loci were amplified using primers ITS1/ITS4 (White et al., 1990), GPD1/GPD2 (Berbee et al., 1999) and EF1983F/EF-2218R (Schoch et al., 2009) respectively. ITS sequence had 100% similarity (706/706bp) with reference sequence C. spicifera (MH863648; HF934915 & HF934916); tef-1α sequence had 100% (933/933bp) identity with C. spicifera (KM062878, KJ939505), and the GAPDH sequence was 99.8% identical to that of Curvularia sp. (MG979055), and C. spicifera (MH809681). Combined dataset of concatenated sequence (ITS-GAPDH-tef-1α) was used in a phylogenetic analysis and revealed that the isolates were in a common clade with the isolate of Curvularia spicifera (CBS 274.52) thus, confirming the identity of the isolated pathogen as C. spicifera. The sequences obtained in the present study were deposited in the GenBank (ITS: OQ253406, OQ253407; LSU: OQ253429, OQ253430; GAPDH: OQ263372, OQ263373 & TEF: OQ263374, OQ263375). Pathogenicity test was carried out by inoculating (foliar /whole plant spray) 60 healthy pearl millet plants (45-days old), grown in field plot with spore suspension (105 conidia/ml). Control plants (n=20) were treated with sterile water. The experiments were conducted in triplicates and repeated twice. Development of disease symptoms was recorded on 41 plants, and all control plants remained healthy. The identity was confirmed after re-isolation as C. spicifera based on cultural and molecular sequence analysis. To our knowledge, this is the first report of C. spicifera causing a leaf spot and spathe blight disease of pearl millet in India. This disease seriously affects grain production, and effective disease management strategies need to be investigated.
RESUMO
Lipopolysaccharide (LPS) elicitors isolated from Pseudomonas fluorescens UOM SAR 14 effectively induced systemic and durable resistance against pearl millet downy mildew disease caused by the oomycete Sclerospora graminicola. Rapid and increased callose deposition and H2O2 accumulation were evidenced in downy mildew susceptible seeds pre-treated with LPS (SLPS) in comparison with the control seedlings, which also correlated with expression of various other defense responses. Biochemical analysis of enzymes and quantitative real-time polymerase chain reaction data suggested that LPS protects pearl millet against downy mildew through the activation of plant defense mechanisms such as generation of nitric oxide (NO), increased expression, and activities of defense enzymes and proteins. Elevation of NO concentrations was shown to be essential for LPS-mediated defense manifestation in pearl millet and had an impact on the other downstream defense responses like enhanced activation of enzymes and pathogen-related (PR) proteins. Temporal expression analysis of defense enzymes and PR-proteins in SLPS seedlings challenged with the downy mildew pathogen revealed that the activity and expression of peroxidase, phenylalanine ammonia lyase, and the PR-proteins (PR-1 and PR-5) were significantly enhanced compared to untreated control. Higher gene expression and protein activities of hydroxyproline-rich glycoproteins (HRGPs) were observed in SLPS seedlings which were similar to that of the resistant check. Collectively, our results suggest that, in pearl millet-downy mildew interaction, LPS pre-treatment affects defense signaling through the central regulator NO which triggers the activities of PAL, POX, PR-1, PR-5, and HRGPs.