First Report of Coniella granati Associated with Dieback of Rose (Rosa sp.) in India.

Abstract

Rose (Rosa sp.) is an important floricultural crop largely cultivated in the Karnataka state of southern India. A field survey conducted in Devanahalli, Bangalore rural during October, 2019 revealed rose plants (cv. Arka Pride) showing die-back symptoms with a disease incidence of 7% in an area of 30 hectare rose field. Die-back was characterized by the development of necrotic lesions that started at the tip of shoots and progressed downwards leading to the death of young shoots. Pycnidia were found on young shoots showing necrotic lesions. Die-back affected shoots (n=10) were surface sterilized with 2% NaOCl for 2 min, rinsed thrice in sterile distilled water, and plated on potato dextrose agar (PDA) medium amended with chloramphenicol (40mg/L). The plates were incubated at 28±2 °C and pure cultures were obtained by hyphal tipping. Fungal colonies with white aerial mycelia along with pycnidia were observed on eight samples. Colony growth rate was at 4.2 ± 0.4 mm/day with an average colony diameter of 47.2 ± 3.8 mm after 7 days (12/12 h light and dark period). Pycnidia were solitary and globose. Conidia were hyaline, single celled ellipsoid to fusiform, measured 12.4 - 16.78 × 3.2 - 5.1 μm (n=50). Based on cultural and morphological features, the fungus was identified as Coniella granati (Van Niekerk et al., 2004; Mahadevakumar et al., 2019) (Figure S1). Further, two representative isolates (NLD-2021-1 & NLD-2021-2) were used for molecular identification based on barcoding of ITS-rDNA, LSU and tef1 regions amplified using primer pairs described in Alvarez et al., 2016. PCR amplified products were sequenced and the consensus sequence were analyzed through BLASTn search which showed ITS (NLD-2021-1: 611/611bp - MH860368 & NLD-2021-2: 573/573bp - KU147239), LSU (NLD-2021-1: 783/906bp - MH872113 & NLD-2021-2: 844/1172bp - KX833400) and tef1 (NLD-2021-1 & NLD-2021-2: 548/548bp -KX833682) sequences shared 100% sequence similarity with C. granati respectively. The nucleotide sequences were deposited in GenBank with the accession numbers ITS: MW898436, MW898437; LSU: MZ895475, MZ895476 and tef1 MW916275, MW916276 respectively. The phylogenetic tree constructed based on combined ITS-LSU-tef1 loci confirmed that the sequences shared a common clade with C. granati (Figure S2). Further, pathogenicity tests were conducted on conducted twice on healthy rose plants (Arka Pride; 60 days old after grafting) maintained in a greenhouse at 28±2°C and 70% relative humidity. Whole plant inoculation was followed by spraying the conidial suspension (10ml / plant) of C. granati (NLD-2021-1) (3 × 106 conidia/ml, amended with Tween20 at 2%) on 15 plants. A total of 10 healthy plants inoculated with sterile distilled water amended with Tween20 at 2% served as control. Inoculated plants were covered with polythene bags for 48 h to maintain relative humidity. Die-back symptoms were observed on young shoots after 15-18 days of post inoculation (DPI), and control plants remained disease free after 15 DPI. The pathogen was re-isolated on PDA from 10 diseased plants and identity was confirmed through morphological methods. Coniella granati is an important phytopathogen known to cause die-back and twig blight of pomegranate globally and other economically important crops (Cintora-Martinez et al. 2017). No reports are available on the association of C. granati with dieback of rose (Farr & Rossman, 2021), which should help in developing appropriate disease management strategies.