Search for a command to run...
Guava (Psidium guajava) is a commercially grown fruit from the Myrtaceae family. It is primarily cultivated in tropical and subtropical areas around the globe, with India being the world's leading producer (Food and Agriculture Organization of the United Nations 2008). It has nutritional value, including antioxidant properties, and is a rich source of vitamin C, potassium and dietary fibre (Correa et al. 2016). It also possesses numerous medicinal benefits, including anti-inflammatory, anti-neoplastic and anti-allergic properties (Mudliar et al. 2008). During a survey of guava, 12 leaf samples showing symptoms of virus infection (chlorosis, reddening and mosaic patterns) were collected in the Kangra district, Himachal Pradesh, India: five from Punner (32.0507°N, 76.5119°E), two from Palampur (32.1128°N, 76.5403°E), three from Tanda (32.1154°N, 76.3077°E) and two from Shahpur (32.2143°N, 76.1673°E) (Figures 1 and 2). Total RNA was extracted from the samples using the CTAB method (Zeng and Yang 2002). Following this, cDNA was prepared using M-MLV reverse transcriptase (Promega, USA) according to the manufacturer's protocol, which includes primer annealing at 70°C for 5 min and incubation with reaction buffer, dNTPs, RNasin and 200 units enzyme at 42°C for 60 min. The samples were tested for the presence of major apple viruses and a viroid, viz. apple chlorotic leaf spot virus (ACLSV), apple mosaic virus, apple stem grooving virus, apple stem pitting virus and apple scar skin viroid (Kumar et al. 2014). These viruses and the viroid were selected based on an ongoing surveillance programme on low-chilling apple cultivars in the same region using the same multiplex assay. The guava plants sampled were located adjacent to apple orchards previously found infected with these pathogens. Among all the pathogens tested, only ACLSV was detected, being found in seven samples (three from Punner, two from Shahpur and one each from Palampur and Tanda), while in the rest 5 samples, it was not detected (Figure S1). Amplicons from three different areas were selected at random (GPR3, Punner; GC10, Palampur; and GSR4, Shahpur), cloned using a Mighty TA Cloning Kit (Takara Bio, Japan) and sequenced (GenBank Accession Nos. PP035103-PP035105, respectively). Phylogenetic analysis was conducted based on the partial coat protein nucleotide sequences obtained in this study, along with representative ACLSV isolates from GenBank (Figure 3). The evolutionary history was inferred using the Neighbor-Joining method. Evolutionary distances were computed using the Maximum Composite Likelihood model and expressed as the number of base substitutions per site. The analysis included 29 nucleotide sequences, with ambiguous positions removed using the pairwise deletion option. A total of 835 positions were retained in the final dataset. All evolutionary analyses were performed using MEGA11. Phylogenetically, the sequence of these three isolates clustered with Prunus cerasoides isolates from Palampur (FN666579.1), sharing 99.32% (amino acid) and 99.84% (nucleotide) identity (Figure 3). To rule out false-positive results due to possible contamination, additional samples were collected from the same symptomatic guava trees and retested. The presence of ACLSV was independently confirmed by RT-PCR assays targeting two additional genomic regions, namely the replicase (F: 5′-GCCAAAGCGGGGCAGACG-3′; R: 5′-TTCTCCAGTGAAGCTGATGAGGG-3′) and movement protein genes (F: 5′-GTGAGTCCTTTCAAGAGGGCCTC-3′; R: 5′-TCTATTGATATTGACAAAGCCC-3′), thereby validating the initial detection (Figure S2). ACLSV (genus Trichovirus, family Betaflexiviridae) has until now been known to infect only stone and pome fruit species of the Rosaceae family, such as apple, apricot, cherry, pear, plum and peach (Guo et al. 2016). To our knowledge, this is the first report of ACLSV infecting guava, identifying it as a new host. The detection in guava has implications for future disease monitoring and further research is needed to understand how ACLSV is transmitted and infects guava and to assess its impact on yield. Although the presence of greenhouse whitefly (Trialeurodes vaporariorum) was noted, its role as a vector of the virus could not be confirmed. India is the world's largest producer of guava and a new disease could severely affect production. The authors gratefully acknowledge the financial support provided by the Department of Biotechnology, Government of India (Grant No. GAP-0297), and the institutional support from CSIR–Institute of Himalayan Bioresource Technology (CSIR-IHBT), Palampur, for conducting this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.