African Journal of Biotechnology Vol. 9 (20), pp. 2860-2865, 17 May, 2010 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2010 Academic Journals Full Length Research Paper Isolation and identification of bacterial glum blotch and leaf blight on wheat (Triticum aestivum L.) in Iran Mostafa Niknejad Kazempour1*, Maesomeh Kheyrgoo1, Hassan Pedramfar1 and Heshmat Rahimian2 1Department Plant Pathology, Faculty of Agricultural Sciences, University of Guilan, Rasht – Iran. 2Department Plant Pathology, Faculty of Agricultural Sciences, Mazanderan University, Sary – Iran. Accepted 27 March, 2009 Basal glume blotch and leaf blight of wheat caused by Pseudomonas syringae pv. atrofaciens and P. syringae pv. syringae respectively are the bacterial diseases of wheat in Iran. The disease causes damage on wheat which leads to lots of yield and crop losses in the host plants. During the spring and summer of 2005-2006 different wheat fields in Guilan province (Roodsar, Langrud, Rostamabad, loshan, and Roodbar) were surveyed. Samples were collected from infected wheats with glumes blotch and leaf blight. Infected tissues were washed with sterile distilled water and crushed in peptone water. Then 50 µl of the extract were cultured on King’s B and NA media containing cyclohexamide (50 µg/ml). After 48 to 72 h, bacterial colonies were selected and purified. On the basis of morphological, physiological and biochemical characteristics, pathogenicity and PCR with specific primers, the isolated were placed in two groups. The first group consists of 20 isolates that caused leaf blight, identified as P. syringae pv. Syringae, while the second group is made up of 18 isolates that caused basal glumes blotch identified as P. syringae pv. atrofaciens. This is the first report of the existence of P. syringae pv. atrofaciens on wheat in Iran. Key words: Pseudomonas syringae pv. atrofaciens. P. syringae pv. syringae, wheat, basal glumes blotch, leaf blight. INTRODUCTION Iran produces about 15 million tons of wheat (Triticum ovars (Gardan et al., 1997). In many countries the aestivum L.) annually on about 6 million ha situated in the occurrence of P. syringae pvs., atrofaciens and syringae north, southwest, central west, and southeast regions. has only been reported once (Argentina, Australia, New The wheat is one of the crops in Guilan province. Pseu- Zealand, Italy and Pakistan) or has not been published at domonas that cause the disease known as basal glumes all (Belgium, Ethiopia and Denmark). Consequently, yield blotch are designated as Pseudomonas syringae pv. losses have never been thoroughly assessed. P. syrin- atrofaciens (McCulloch) (Young et al 1978); those gae pv. atrofaciens isolates from Bulgarian and Ukrainian causing leaf blight are grouped under P. syringae pv. wheat belong mainly to serogroups II and IV, respectively syringae van Hall 1902. The pathogens are one of the (Pasichnik et al., 2003). The symptom of basal glumes world-wide spread major bacterial diseases of wheat, blotch is dull brownish-black, discoloured area found at present in Guilan province of Iran. In Germany, losses the base of each glumes covering the kernel. Dark blot- due to P. syringae pv. atrofaciens in an area of marshy ches or streaks are found near base of glumes, streaks soils were estimated to exceed 50% (Toben et al., 1991). may extend more than halfway up glumes (Capparelli et The disease can affect all small grain cereal crops; distri- al., 2005). Leaves affected by this disease organism bution is worldwide (Matveeva et al., 2003). The species show small, dark, water-soaked spots. These spots tend P. syringae is heterogeneous and is divided into 57 path- to enlarge and turn yellow and finally brown as the tissue dies (Vassilev et al., 1997). Symptoms can be confused with those of other bacterial diseases, genetic melanism (false black chaff), septoria blotch, and frost damage. *Corresponding author. E-mail: [email protected]. Leaf symptoms incited by P. syringae pv. Syringae are Kazempour et al. 2861 water-soaked spots expand and become necrotic and The instrument used for inoculating the rice plant with the turn from gray-green to tan-white. Entire leaves may be- bacterial is scissors. Before using the scissors they were sterilized come necrotic. During very wet periods, white droplets of using 70% ethanol. The scissors were dipped in the bacterial sus- pension and are used to cut the inoculated rice plant. Lesions on bacteria may be visible. P. syringae pv. atrofaciens was leaves were observed 7 days after inoculation (Backer, 2002). isolated from 10-48% of symptomless wheat leaves and Individual leaves were ground in 3 ml of sterile distilled water. The glumes (von Kietzell et al., 1997). Typical P. syringae pv. suspensions were then appropriately diluted and 50 µl aliquots atrofaciens isolates were isolated from grassy weeds were spotted on duplicate King’s B plates. Control plants were (Fessehaie, 1993). The fact that P. syringae pv. atrofa- treated with sterile distilled water. ciens and P. syringae pv. syringae populations are always present epiphytically on wheat plant surfaces and Biochemical, biological and physiological tests other hosts indicates that weather conditions are more relevant to disease outbreaks than the presence of inocu- Isolates were characterized on the basis of the following tests: lums. The bacterium survives on host plant residues, in Gram test in 3% KOH (Sulsow et al., 1982), oxidative/fermentative soil and on seed. Wind-blown dust or residue fragments test (Hugh and Leifson, 1953), production of fluorescent pigment on King’s B medium (Sulsow et al., 1982), hypersensitive reaction (HR) carry bacteria to seeds (Capparelli et al., 2005). Seed in tobacco and geranium leaves (Lelliot and Stead, 1987), oxidase infestation can play an important role in disease epide- test, levan formation, catalase, urease, gelatin liquefaction, litmus miology. No control measures for routine application have milk, salt tolerance (5 and 7%) and gas formation from glucose. In as yet been established. However, seed lots from heavily addition, tests were performed for arginine dihydrolase, hydrogen infested fields should not be used for sowing. It is sulfide production from peptone, reduction substance from sucrose, disseminated by splashing rain or by insects, and can be tyrosinase casein hydrolase, nitrate reduction, indole production, 2- keto gluconate oxidation lecitinase, starch hydrolysis, phenylalanine seed borne (Wiese, 1987). Basal glumes blotch usually is deaminase, aesculin and Tween 80 hydrolysis and optimal growth not economically important, but is frequently reported in temperature (Schaad et al., 2001). The presence of DNAse was humid cereal-growing areas (Mamulk et al., 1990). Basal tested on DNA agar (Diagonistic Pasteur, France). Carbohydrate glumes blotch can reduce yields by reducing seed fill utilization using Ayer basal medium was carried out and the results (Capparelli et al., 2005). The objectives of the present were recorded daily up to 2-8 days (Hildebrand, 1998). For each test defined in this study, a representative isolate has been research, isolation of causal agent of basal glumes blotch deposited in the Collection Française de Bactéries Phytopathogèns and leaf blight on wheat in the Guilan province and identi- (CFBP) and UPB (Collection Unité de Phytopathologie, Louvain-La- fication of isolates by biochemical, nutritional, pathogeni- Neuve, Belgium). These references isolates were considered as a city and PCR methods. typical isolates of P. syringae pv. atrofaciens and P. syringae. pv. syringae. MATERIALS AND METHODS DNA extraction Bacterial isolation For bacterial DNA extraction, the isolates were grown overnight, in Wheat samples were collected from fields of wheat in Roodsar, nutrient broth (Merck, Darmstadt, Germany), at 26°C and the DNA Langrud, Rostamabad, loshan and Roodbar during 2005–2006. Iso- was extracted as described by Martins et al. (2005). One tube of lations were made from infected leaves and spikes. From each 1.5 ml was used to centrifuged the cells at 13,000 x g for 5 min and field, four replicatations of 30 wheats were collected at random. the pellet was suspended in 200 µl Tris 0.1 mol L-1 and added with Individual leaves and spikes were ground in 5 ml of sterile distilled 200 µl of lysis solution (NaOH 0.2 N and 1% SDS), mixed and water with a homogenizer (Pro200, Pro Scientific Inc., Monroe, CT, deproteinazed with 700 µl of phenol/chloroform/isoamyl alcohol USA) and 100 µl of homogenate was streaked on King’s B medium, (25:24:1, v/v/v), homogenized and centrifuged 10 min at 13,000 × g containing 50 µg/ml At least 30 samples were tested from each .To precipitate DNA, 700 µl of cold absulate isopropanol was added field. From each infected leaf sample, three single colonies were and spinned, washed in 70% ethanol and centrifuged. Precipitated isolated and one isolate/field was selected as a representative for DNA was dried at room temperature and suspended in 100 µl of this study. For long-term storage, all cultures were stored at 70°C in water. The method described by Ausubel et al. (1996) was perform- nutrient broth containing 60% glycerol. ed comparing 30 isolates. The samples from the both methods were electrophoresed on 1.5% agarose gels, stained with ethidium bromide and photographed under UV light. Pathogenicity test on wheat Seeds of wheat cultivar Shirazy were sown in 30 cm diameter plas- Primers for P. syringae pv. atrofaciens and P. syringae pv. tic pots and were kept for 3 months in greenhouse condition. For syringae inoculations on wheat, bacterial suspensions were prepared in 10 ml of sterile distilled water at 1 × 108 CFU/ml. The 20 oligonucleotid PSF, 5'-AGCCGTAGGGGAACCTGCGG-'3 In the case of pathogenicity test, the isolates (that is 18 isolates and PSR 5'- TGACTGCCAAGGCATCCACC-'3 were designed and of P. syringae pv. atrofaciens and 20 isolates P. syringae pv. syrin- tested for P. syringae pv. syringae (Manceau and Horvais, 1997) gae), plants with fully expanded leaves were inoculated by the leaf- and for P.