Molecular detection of Burkholderia gladioli in saffron fields of Khorasan Razavi province

Background and Aim: The growing season of saffron, depending to the climate, starts from Otober and lasts to the next May or June in maximum. During the January ۲۰۱۸, some saffron fields in Khorasan Razavi province showed early leaf yellowing and draying. On sampling, some of the saffron corms, extracted from the soil, showed decaying signs on the sheaths while some newly formed sprouts had tissue burnings and browning. On some saffron corms, exactly on the root germination zone, a ring shapered brown discoloration were observed that gradually decayed and extended to the deep corm.The taxonomic rank of the strains was confirmed by the use of PCR with species-specific primers as LP۱/LP۴, BUR۱/BUR۲, CMG۱۶-۱/G۱۶-۲,GLAf/GLAr and also RAPD primers including CUGA۱ and CUGA۲.Regarding multiple bacterial detection methods used, including morphological characters, differential biochemical and physiological tests, proving pathogenicity and bacterial molecular detection as well as sequencing and ۱۶s r DNA gen blast of the saffron isolates, revealed Burkholderia gladioli. Taking to account the importance and quarantining of Burkholderia gladioli in the country, the necessary measures should be taken to prevent the distribution of the disease.Methods: The strains included in this study originated from infected plant material with symptoms of bacteriosis. Plant samples were collected in aseptic conditions from diseased scales of saffron corms.Sampling were carried out from different geographical condition of saffron fields in Razavi Khorasan province including cities: Bajestan, Khaf, Gonabad,Zaveh,Torbate Haydarieh,Torbate Jam ,Fariman, Chenaran, Jolgehe Rokh and Mahvelate. Bacterial strains were obtained as distinct colonies on NA medium after cultivations at ۲۸oC for ۴۸-۷۲h.Selection strains was made on the basis of screening for genus differentiating properties. The identities of the ۱۰ isolates were confirmed by using biochemical reactions. Polymerase chain reactions amplification were carried out based on the presence of the specific gene regions in the area coding ۱۶S and ۲۳S ribosomal RNA (rRNA) with primers CMG۱۶-۱/G۱۶-۲, CLAF/CLAR (Moon et al.,۲۰۱۷) and LP۱/LP۴(Whitby et al., ۲۰۰۰) and also PCR with another set of species recA primers BUR۱ and BUR۲ (Payne et al., ۲۰۰۵)for specific amplification and sequencing of Burkholderia .RAPD_PCR : Two oligonucleotide primers CUGA۱ and CUGA۲ were used for initial experiment with the type and reference cultures. The RAPD_PCR was performed according to the procedure described by Momol et al.,(۱۹۹۷). The amplified products were electrophoretically separated in ۱.۵ %( w/v) agarose gel at ۱۰۰V for ۱h in ۱x TBE buffer, stained with ethidium bromide and visualized under UV light.Results: The isolates were identified by PCR amplification using species -species primers. Species-specific PCR:In the PCR analysis using the primers LP۱/LP۲, BUR۱/BUR۲ for all ۱۰ isolates and the bp fragment of the ۲۳S rRNA gene, respectively. All of the test strains gave a positive results used the primers set CMG۱۶-۱/G۱۶-۲ and successfully amplified an approximately ۴۷۰bp fragment, of ۱۶s rDNA from B.gladioli. In PCR with set of species-specific primers for B.gladioli, GLA-f and GLA-r, and approximately ۳۰۰bp fragment was amplified in ۱۰ isolates. RAPD _PCR were selected for subsequent investigations and RAPD-analysis with these primers was repeated ۵ times.For all the strains with CUGEA-۲ also gave the expected product of ~۶۳۰ bp distinct for B.gladioli.Some of the isolates, amplification with CUGEA-۱ gave a produce of ~۳۴۰bp typical for B.gladioli pv.gladioli. To examine the species-specific amplicons obtained by PCR using primer CMG۱۶-۱ / G۱۶-۲ and GLA-f / GLA-r,we sequenced the ۴۷۰ bp and ۳۰۰bp PCR products of ۱۰ isolates.Their nucleotide sequences were compared with gene sequences of known strains in the Genbank sequence database. A BLAST search of the ۱۶S rDNA sequences supported the morphological, physiological, and biochemical results that the isolates were B.gladioli Conclusion: In conclusion, these strains of bacteria, which have the ability to cause soft rot in saffron, are close to B.gladioli. Therefore, further studies are necessary to verify if the isolates of this group belong to a new species or pathovar of this bacterium. On the basis of the PCR analysis, it can be concluded that the ten strains isolated from saffron belong to B.gladioli pv.gladioli RAPD analysis with CUGEA۲ differentiated B.gladioli from B.cepacia but did not differentiate B.gladioli pathovars. Impossibility to differentiate B. gladioli pathovars could be explained by the pathovar system being obsolete. Nevertheless, the pathogen could still be clearly distinguished by RAPD with CUGA۱. The state molecular method also allows the fast distinction between B.cepacia and B.gladioli in absence of specific-specific primers in combination with basic biochemical characterization for genus Burkholderia

کلیدواژه ها:

Saffron ، Bacteria ، Corm rot