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Phylogenetic Relationship of Venturia Carpophila, the Causal Agent of Almond Scab from Kashmir Valley As Inferred by ITS Nr DNA

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Phylogenetic Relationship of Venturia Carpophila, the Causal Agent of Almond Scab from Kashmir Valley As Inferred by ITS Nr DNA Int.J.Curr.Microbiol.App.Sci (2019) 8(6): 2913-2919 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 06 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.806.350 Phylogenetic Relationship of Venturia carpophila, the Causal Agent of Almond Scab from Kashmir Valley as Inferred by ITS nr DNA M.S. Dar1*, Mushtaq Ahmad2, M.D. Shah3, Nazir A. Bhat4, M.Anwar Khan5, and Bilal A. Padder3 1Division of Plant Pathology, Faculty of Agriculture, Wadura Sopore, SKUAST-Kashmir 191121, India 2Directorate of Extension, SKUAST-Kashmir, Shalimar, Srinagar, 190025, India 3Plant Virology and Molecular Plant Pathology Laboratory, Division of Plant Pathology, SKUAST-Kashmir, Shalimar Srinagar 190025, India 4Mountain Field Crop Research Centre, Khudwani, Anantnag, 192001, India 5Division of Genetics and Plant Breeding, Faculty of Agriculture, Wadura Sopore, SKUAST-Kashmir 191121, India *Corresponding author ABSTRACT K e yw or ds The fungus Venturia carpophila causes scab in almond. In order to gain insight, an in vitro Almond scab, nr culture of the fungus has been established and its identity confirmed by its nr DNA. DNA, Internal Internal transcribed spacer-ribosomal DNA (ITS-rDNA); the fungal molecular marker was transcribed spacer, used for molecular analysis. The target region of rDNA (ITS1-5.8S-ITS2) of this species Phylogenetics, was amplified using universal fungal primers (ITS1 and ITS4). The sequencing of Venturia carpophila amplified products and their subsequent Basic Local Alignment Search Tool analysis Article Info confirmed the identification of species by comparing the sequence of the species with respective species sequences present in Gen Bank. Phylogenetic analysis also confirmed Accepted: 2 4 May 2019 the identification of fungi belongs to Venturiaceae family having 100% similarity to other Available Online: V. carpophila species. 10 June 2019 Introduction Pradesh with commercial cultivation confined to the state of Jammu and Kashmir, Almond scab caused by the fungus Venturia occupying an area of 5710 ha with an annual carpophila (Fisher) (Fisher, 1961) (anamorph, production of 13109 metric tonnes Fusicladium carpophillum) is the most (Anonymous, 2018). Almond scab is posing common fungal disease on almonds in serious problem to the orchardist of Kashmir Kashmir valley. In India almond is mainly valley from last few years, in which the grown in Jammu and Kashmir and Himachal severity of disease on leaves and twigs ranges 2913 Int.J.Curr.Microbiol.App.Sci (2019) 8(6): 2913-2919 from 24.30 and 26.50 percent respectively 2017) from Kashmir valley. However sexual (Kacho et al., 2017). Symptoms of scab state of V. carpophila has been reported in occurs on shoots, leaves and fruits, first on Australia on apricot trees in 1961 (Fisher, twigs in the first week of May and later on 1961), providing clues about pathogen has leaves in the second week of May and in capacity to undergo genetic recombination. severe cases on fruits. Lesions on twigs, are Based on dominant marker system like RAPD circular to oval with brown centres and and UP-PCR markers, genetic diversity and slightly raised purple margins, while leaves divergence within closely related species has have small, indistinct, somewhat circular, clearly show difference between V. effuse and greenish yellow blotches undersurface. The V. carpophila and also indicated difference lesions later enlarge same reaching 10 mm or between isolates of V. carpophila of peach more in diameter. With the production of and almond (Chen et al., 2014). However, the spores, they take an olivaceous appearance result of dominant markers is un reliable and eventually brownish black. On fruit, spots because of poor reproducibility, artefactual were dark grey to black sooty appearance and variation and limitations in understanding of coalesced into large dark blotches (Fig. 1). population genetics (Novelo et al., 2010). The disease affects fruits and also leads to Some molecular analysis has been used to premature leaf fall resulting in low understand the phylogenetic relationships productivity and poor fruit quality. between V. carpophila and other Venturia Management of almond scab is done with species, by (Schnabel et al., 1999), however dormant treatments of liquid lime sulfur or this is less useful for taxonomic interpretation with copper-agricultural oil mixtures to delay because of Cladosporium caryigenum was and reduce sporulation of twig lesions taken as out group, which is true anamorph of (Forster et al., 2009) and within season Venturiaceae. In Kashmir valley no treatments to prevent new infections. Before phylogenetic analysis has been carried out on the introduction of the quinone outside V. carpophila and preset study objective was inhibitor (Qol) fungicides azoxystrobin, to mine and characterize phylogenetic trifloxystrobin, and pyraclostrobin, multi-site relationships of V. carpophila using ITS mode of action fungicides such as captan, nrDNA with other species of Venturiaceae ziram, maneb, or wet table sulfur were family. applied during the petal fall period during leaf emergence and commonly prior to spring Materials and Methods rains to manage the disease. Additionally, the single-site mode-of-action methyl Sample collection and fungal isolation benzimidazole carbamate fungicides (e.g., benomyl and thiophanatemethyl) were also Samples were collected from leaves and fruits used. Resistance against these latter of almond in 2015 from Kashmir valley. They fungicides developed in the pathogen were first dried with absorbent paper in the populations in different regions (Ogawa and laboratory for isolation of the pathogen and English, 1991). A number of pathogens from stored at -4 °C for further processing. The Venturiaceae family has been reported on mono conidial isolations were carried out on prunus species causing scab like symptoms water agar in Petri plates as described by (Xu such as F. pomi, V. carpophila, F. obducens et al., 2008). A small fragment of an infected and F. cerasi (Schubert et al., 2003). In India, leaf disc was added to a little amount of the pathogen was first reported to be causal distilled water and agitated thoroughly to agent of almond scabin 2017 by (Kacho et al., release conidia. The conidial suspensions 2914 Int.J.Curr.Microbiol.App.Sci (2019) 8(6): 2913-2919 were diluted to 8 × 103 ml–1 conidia and 200 selected by Akaike information criterion μl of the suspension pipetted onto the water (AIC) (Posada and Buckley, 2004) in agar plates and spread evenly. These plates MrModel test 2.3. Bootstrap analysis with were incubated at 20±1ºC for 24 h. The 1000 replicates was used to test the statistical individual germinated spores were excised support of the branches. The nucleotide using a cork borer or scalpel blade under a sequences reported in this paper were compound microscope and transferred to Petri deposited in GenBank and accession number dishes containing MEA under aseptic was obtained as MK482360. conditions. The plates were incubated and maintained at20±1oC. At least 40 isolates Results and Discussion from different plant species were collected and maintained for further studies. During the survey of Kashmir valley, scab like disease on almond which was previously DNA extraction, PCR, sequencing reported by (Kacho et al., 2017) as C. carpophillum on the morphological basis was Total genomic DNA of fungal isolates was collected. Since the pathogen is reported as extracted using modified CTAB anamorph on almond. However, its sexual (Cetyltrimethyl ammonium bromide) method stage has been reported on apricot by Fisher (Murray and Thompson, 1980). The ITS in 1961 from Australia. This pathogen is nrDNA internal transcribed spacer (ITS) with difficult to culture because of slow growth primers ITS-1 and ITS-4 was amplified and rate on culture media which can be masked by sequenced with primers (White et al., 1990). other pathogens. The mycelial colony on Comparisons to other nrDNA sequences were MEA was dark green turning black after 30- conducted with BLAST2.2.24 queries 35 days of incubation in pure culture (Fig. 2). (National Center for Biotechnology Number of isolates was cultured and one Information, National Institute of Health, isolate were sequenced to find its Bethesda, Maryland). Representative phylogenetic relationship with other closely sequence was deposited in GenBank. related species. Sequence alignment and phylogenetic Molecular characterization of V. carpophila analysis To elucidate the relationships between our Sequence generated were analysed with other isolates and other related Venturia species, we sequences obtained from GenBank (Table 1). carried out an analysis of ITS sequences in A BLAST query was performed to find which new sequence data of V. carpophila possible sister groups of the sequenced tax on, was used for present analysis together with and closely related sister groups are included other additional sequences obtained from in the phylogenetic analysis (Table 1). A GenBank at NCBI. The accession number of multiple alignment was conducted in MEGA present isolate is MK482360. The GenBank v. 6.02 (Tamura et al., 2013) and analyses sequences with highest identity to ITS1-5.8 were performed. Prior to phylogenetic rDNA-ITS2 from V. carpophila were analysis, ambiguous sequences at the start and identified using BLASTN analysis as shown the end were deleted and gaps manually in (Table 1). The sequence of V. carpophila adjusted to optimize alignment. ITS rDNA shows 91.22-100% identity with other dataset was analysed in this study. Best-fit Venturia species including V. carpophila. The model of nucleotide evolution (T92+G) was phylogenetic tree was generated from the 2915 Int.J.Curr.Microbiol.App.Sci (2019) 8(6): 2913-2919 ITS-5.8 rDNA-ITS2 of V. carpophila isolate in the family of Venturiaceae, provides with other species of Venturiaceae, using supporting data of the taxonomic position of neighbor-joining method of the MEGA v. almond scab fungus (Fig. 3). The observed 6.02 (Tamura et al., 2013). The V. carpophila distances between V. carpophila and other isolate clustered with another V.
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