Isolation and Identification of Bacterial Glum Blotch and Leaf Blight on Wheat (Triticum Aestivum L.) in Iran

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

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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. syringae pv. atrofaciens, SyD1 5'-CAGCGGCGTTGCG- clipping method (Kauffman et al., 1973) and injection method TCCATTGC-3' and SyD2 5'-TGCCGCCGACGATGTAGACCAGC- (Schaad et al., 2001). 3' (Bultreys and Gheysen, 1999).

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PCR and electrophoresis two or three days, these lesions expand and often coal- esce into large, grayish-green, desiccated areas. These Amplification was carried out in a 25 µl volume in 0.5 ml micro tube symptoms did not occur in the control. The bacteria re- using a Hybaid programmable thermal controller. Each 25 µl PCR reaction mixture contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, covered from these lesions were identified as P. syringae 0.1% Triton X-100, 2.5 mM MgCl2, 0.2 mM of each nucleotide pv. atrofaciens and P. syringae pv. syringae confirming (dATP,dCTP, dGTP, and dTTP), 0.25 µM of each primer, 100 ng Koch’s postulates. DNA and 2 U of Taq DNA polymerase (Promega Corp., Madison, WI). A 25 µl sterile, mineral-oil overlay was added to reduce eva- poration. DNA amplification was carried out in a PTC-100 programmable DNA thermal cycler (MJ Research, Watertown MA). The Detection of P. syringae pv. atrofaciens and P. amplification was performed for P. s. pv. Atrofaciens as follows: syringae pv. syringae by direct PCR initial denaturation at 93°C for 3 min; 37 cycles of 93°C for 1 min, 60°C for 1 min, 72°C for 1 min; and 3 min extension at 72°C. For P. All isolates of P. syringae pv. atrofaciens were identified s. pv. syringae: initial denaturation at 93°C for 1 min; 37 cycles of by specific primers SyD1 and SyD2. On agarose gel elec- 93°C for 1 min, 60°C for 1 min, 72°C for 1 min; and 5 min extension trophoresis 1.5%, isolates produced a band of 558 bp at 72°C. Amplified DNA fragments were examined by horizontal electrophoresis in 2% agarose gel in TBE buffer with 10 L aliquots and all isolates P. syringae pv. syringae were identified of PCR products. Gels were stained with ethidium bromide and by specific primers PSF and PSR, and produced a band were photographed under UV light (312 nm). of 752 bp (expected size). The bands of isolates were similar to those of the isolates standards of UPB 0448 for P. syringae pv. atrofaciens (Figure 1) and CFBP 3077 for RESULTS P. syringae pv. syringae (Figure 2). Based on the phenol- typic pathogenicity and PCR tests, the causal agent of Biochemical and physiological tests leaf blight was identified P. syringae pv. syringae and causal agent of basal glumes blotch was classified as P. All isolates were gram, oxidase, catalyzed negative, and s. pv. atrofaciens. are unable to utilize glucose under anaerobic conditions (Table 1). None of the isolates was able to produce reducing compounds from sucrose or show lecithinase, DISCUSSION arginine dihydrolase activity or produce gas from glucose. None of the isolates were able to hydrolyze Tween 80, Two pathovars of P. syringae induced symptoms on ei- produce indole, reduce nitrate and oxidize 2-keto-gluco- ther leaves or spikes of wheat: P. syringae pv. atrofa- nate. All isolates of P. syringae pv. syringae and P. syrin- ciens and P. syringae pv. syringae were caused by basal gae pv. atrofaciens were able to produce syringomycin glumes blotch on spikes and leaf blight respectively. and showed ice nucleation activity. All isolates were able These pathogens have been reported from nearly all to utilize citrate, L-lysine, lecithinase and produce acid wheat growing areas in temperate and subtropical zones. from manitol, L-arabinose, xylose, D(+)galactose, inositol, Disease outbreaks occur sporadically, usually under maltose, sorbitol, manose and sucrose. None of the iso- extremely humid conditions during spring and summer. lates of P. syringae pv. syringae were capable of utilize L- Significant losses have been recorded only in few cases tartrate. None of the isolates were capable of utilize triha- (Toben et al., 1991). The pathogens damages wheat, lose. The presence of DNAse was tested on DNA agar barley and rye and possess high physiological variability (Diagonistic Pasteur, France). (Kotlyarov et al., 2004). All the isolates of P. syringae pv. atrofaciens and P. syringae pv. syringae produced glum blotch and leaf blight of wheat. No significant differences Pathogenicity test were observed in the degree of disease symptoms in greenhouse conditions. These results suggest that All 18 isolates of P. syringae pv. atrofaciens and 19 iso- strains isolated from different fields did not show any lates of P. syringae pv. syringae caused leaf blight on the differences in their degree of virulence. This is the first surface of wheat leaves after one weeks inoculation. report of basal glumes blotch of wheat in Iran. Although Spike symptoms caused by P. syringae pv. atrofaciens the disease has occurred in several consecutive years, P. are generally referred to as basal glumes blotch. The glu- syringae pv. atrofaciens does not appear to be a wide- mes show a dull, brownish black area at the base. Leaf spread problem on wheat in Iran. The identification of symptoms incited by P. syringae pv. syringae are gene- isolates of P. syringae at the pathovar level poses serious rally referred to as bacterial leaf blight (Sellam and practical problems. Much effort has been invested in Wilcoxson, 1976) or leaf necrosis (Otta, 1974). Initial finding biochemical or genotypic markers for pathovars symptoms appear at booting to early heading in the form so as to avoid the need for host range tests, but most of of numerous, tiny, water soaked spots on the flag leaf these markers have limited utility. Biochemical tests can and the first and second leaves below the flag leaf. Within differentiate clusters that include several, very closely Kazempour et al. 2863

Table 1. Phenotypic characteristics of P. s. pv. atrofaciens and P. s. pv. syringae Isolates tested.

Characteristic Isolates of P. s. pv. Isolates of P. s. pv. atrofaciens syringae Gram reaction - - Oxidative/Fermentative - - Fluorescent pigment + + HR on tobacco + + Ice nucleation + + Growth at 39°C - - Syringomycin production + + Leaf blight on wheat + + Glume blotch on wheat + - Pectinase - - Acetoin - - Argenine - - dihydrolase Levan formation + + Nitrate reduction - - Catalase - - Tween 80 hydrolysis + + Oxidase - - Starch hydrolysis - - Gelatin hydrolysis + + Esculin hydrolysis + + DNase activity + + Indole formation - -

H2S from cysteine - - Casein hydrolysis - - Urease + + MR - - Utilization of L-lysine + + Citrate + + lecithinase - - Growth in 5% NaCl - - Acid from : L-Arabinose + + Myo-Inositol + + Manitol + + Xylose + + Trihalose - - Maltose + + L-tartrate - + D-Galactose + + D-Sorbitol + + Sucrose + + D-Rafinose - - D-Manose + + D-Glucose + +

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heterogeneous P. syringae pv. syringae a few isolates that are very similar to pv. atrofaciens have been found (von Kietzell and Rudolph, 1997). Pseudomonas spp. produces a wide spectrum of phytotoxic compounds (Bender et al., 1999). Syringomycin production and syringopeptin production are not limited to P. syringae pv. syringae. These compounds have been found in P. syringae pv. atrofaciens (Vassilev et al., 1997; Bobrova et al., 2005). Syringomycin E and syringopeptin 25 A were identified in P. syringae pv. atrofaciens culture fluids (Vassilev et al., 1997; Mekki et al., 2007). These pathogens have been reported from nearly all wheat growing areas in temperate and subtropical zones. Disease outbreaks occur sporadically, usually under Figure 1. Agarose gel, stained with ethidium bromide, of DNA extremely humid conditions during spring and summer products extracted from Pseudomonas syringae pv. (Shane and Baumer 1987). Only in a few cases have atrofaciens isolates and amplified by polymerase chain reaction. Lane M, 100-bp ladder; lane 1, P. s. pv. atrofaciens significant losses been recorded. P. syringae pvs. UPB 0448, showing the amplification the approximately 558 syringae and atrofaciens have important epiphytic bp, lanes 2, 3, 5 and 6 isolates of P. s. pv. atrofaciens isolated phases. Populations of both pathovars are always from glume blotch of wheat; lane 4, control negative (distilled present epiphytically on wheat plant surfaces and on water). other hosts. Therefore, weather conditions are more

relevant for disease out-breaks than the mere presence of the inoculum (Toben et al., 1989). Because of the temperate climate in north of Iran, the infection was more severe in May and June with large basal glumes blotch on several varieties of wheat. Further research is needed for elucidating the mecha-nisms eliciting this genetic diversity. An understanding of the ecology of natural microbial communities should lead to a more efficient deployment of bacterial populations for disease management.

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