ISOLATION, PURIFICATION AND METAGENOMIC CHARACTERIZATION OF BACTERIOPHAGES OF Xanthomonas axonopodis pv. punicae CAUSING BACTERIAL BLIGHT IN POMEGRANATE
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Date
2019-12
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University of Horticultural Sciences, Bagalkot
Abstract
Pomegranate (Punica granatum L.) is an economically important fruit crop of
tropical and subtropical region. Bacterial blight caused by Xanthomonas axonopodis
pv. punicae (Xap) is one of the major constraints in pomegranate cultivation and
production leading to 60-80 percent yield loss. Current disease management practices
have limited efficacy in the management of bacterial blight. Use of bacteriophage that
infect and lyses the bacteria, have received increased research interest for biological
management of bacterial diseases. The present research was emphasized on isolation
of Xap lysing bacteriophages from the bacterial blight infected pomegranate debris
and their rhizosphere. Samples were collected from the 5-7 years old pomegranate
orchards of Bagalkot, Koppal, Hiriyur, Tumkur of Karnataka and Solapur of
Maharashtra states. The isolated phages formed clear spots (phage plaques) and
exhibited lytic activity against the Xap bacterial lawn with varied plaque morphology.
Plaques were irregular to round shape with their size ranging from 1 to 10 mm in
diameter. These plaques exhibited varied phage titer values against Xap with titer
values ranging from 2.25×104 to 1.75×107 pfu/mL, which is on par with the
commercial phage formulations against different phytobacteria. The isolated phages
with high phage titer values were morphologically characterized using transmission
electron microscopy. Based on the morphology, phages were grouped into four
different families; Podoviridae, Siphoviridae, Myoviridae and Inoviridae. Further,
phages were identified by metagenomic analysis by sequencing the nucleic acid
isolated from the soil phages. 1,40,703 nucleotide contigs were assembled using
MetaSPades tools. Phage nucleotides were annotated by BLAST searching against
viruSITE and IMG/VR virome database. Approximately 1% of the contigs were
annotated as phage contigs belonging to the phages that target Xanthomonas sp. such
as X. dyei, Pseudoxanthomonas sp., X. translucens, X. hyacinthi, X. campestris, X.
melonis and X. citri. This research indicates that bacterial blight infected pomegranate
soil has rich diversity of bacteriophages that effectively lyse the bacterial blight
pathogen Xap. Isolation of individual phages, development of phage cocktails with
different combination of phages and development of spray formulations that thrive
well under field conditions would make phage therapy as an effective biological
management of bacterial blight in pomegranate.