Identification and differentiation of strains of xanthomonas axonopodis PV punicae at molecular level
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Date
2015-07-09
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Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani
Abstract
Pomegranate (Punica granatum L.) is an important fruit crop of India and
other subtropical countries. Bacterial blight caused by Xanthomonas axonopodis pv.
punicae (Hingorani and Singh, 1959), is becoming a major threat to pomegranate
production in Maharashtra as well as other pomegranate growing states of India. It
causes yield loss to the extent of 80 - 90 %. Considering importance of this disease,
study regarding assessment identification and differentiation of strains of X. a. pv.
punicae was undertaken.
Based on genetic diversity study of isolates of Xanthomonas axonopodis pv.
Punicae, conducted earlier by kale (2012) five representative samples of each region
have been selected towards assessment of aggressiveness and nucleotide sequences
variation at pathogenicity gene fragment sequence level. A Rifampicin resistant
mutant were developed by continuous growing of bacterial cell on Rifampicin (200
pg/ml) supplemented YGCA medium. During syringe-infiltration of known
concentration of each of five isolates on pomegranate cv. Bhagwa produced confluent
water soaking spots. Based upon appearance of symptoms, there were grouped in to
three categories as highly virulent (Xap5), moderately virulent (Xapl, Xap2 and
Xap4) and less virulent (Xap3). Further aggressiveness was assessed through Inplanta
growth assay.
The highly aggressive isolate Xap5 produced 3.1 X 108 cfu/ml bacterial
colonies while, moderately and lesser virulent isolates were produced bacterial count
of 2 X 107 cfu/ral and 4 X 106 cfu/ml respectively after 5 days of inoculation. The
pathogenicity gene fragment of size 460-490 bp were also exploited for restriction
digestion by enzyme BamHl, Pstl and Smal. The Restriction enzyme analysis
categorized all of them into four groups. Further pathogenicity gene fragment (460-
490 bp) were cloned and sequenced. The BLAST analysis confirmed the identity of
the sequences by showing 93 % homology with pathogenicity gene sequences
available in public domain. Also the multiple sequence analysis revealed the variation
in the sequences of the isolates collected from different regions.
Thus based upon the results comprising variation in aggressiveness test and
nucleotide sequences of pathogenicity gene fragment, is an indicative of existence of
at least three strains among pathogen Xanthomonas axonopodis pv. punicae causing
oily spot disease in pomegranate.
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