J Phytopathol 159:377–381 (2011) 2010 Blackwell Verlag GmbH doi: 10.1111/j.1439-0434.2010.01776.x Colegio de Postgraduados, Instituto de Fitosanidad, Texcoco, Me´xico Leaf Stripe and Stem Rot Caused by Burkholderia gladioli, a New Maize Disease in Mexico Adriana driana Gijon ijon-Hernandez ernandez1, Daniel aniel Teliz eliz-Ortiz rtiz1, Dimas imas Mejia ejia-Sanchez anchez2, Rodolfo odolfo De e La a Torre orre-Almaraz lmaraz3, Elizabeth lizabeth Cardenas ardenas-Soriano oriano1, Carlos arlos De e Leon eon1 and Antonio ntonio Mora ora-Aguilera guilera1 AuthorsÕ addresses: 1Colegio de Postgraduados, Instituto de Fitosanidad, Texcoco, México, 2Departamento de Parasitologia Agricola, Universidad Autonoma Chapingo, Texcoco, Mexico, 3UNAM, UBIPRO, FES Iztacala, Mexico (correspondence to D. Teliz-Ortiz. E-mails: [email protected]; [email protected]) Received July 17, 2009; accepted October 31 2010 Keywords: Zea mays, PCR, bacterial disease Abstract A disease in maize plants, not previously observed, appeared in 2003–2004 in Cosoleacaque, Tlalixcoyan and Paso de Ovejas counties, in the state of Veracruz, Mexico. Initial symptoms on leaves were small whiteyellow watery spots, which coalesced into dry necrotic stripes 0.3 wide and up to 8 cm long. Reddening sometimes developed on these leaves. Stems developed a rot in the crown. The flag leaf became rot and necrotic at the base, rolled inwards and dried out. Necrosis developed at the base of the corn ears and their growth was inhibited. These symptoms were initially observed in Asgrow-7573 commercial maize plantings. A bacterium characterized by white colonies, gram negative, aerobic, rod-shape, opaque, round colonies with entire margins on casaaminoacid peptone and glucose media was consistently isolated from diseased maize plants. On KingÕs Medium B, the isolates produced yellow, non-mucoid colonies, with the majority of the strains secreting a diffusible yellow pigment into the media. The bacterium identity was confirmed by PCR amplification and sequencing of 16S and 23S genes rDNA fragments. The bacterium was identified as Burkholderia gladioli. Its pathogenicity on maize plants in Mexico is a new record. Introduction Stem, root and corn ear rots, leaf spots, wilt, blight and stunt are registered maize diseases (De Leon 1984; Quiroga 1995). The most important and common maize bacterial diseases are bacterial wilt (Pantoea stewartii, syn. Erwinia stewartii) and stem rot (Erwinia chrysanthemi pv. zeae, syn. E. carotovora f. sp. zeae) (Shurtleff 1980; De Leon 1984; Quiroga 1995). A disease, not previously described in maize, was observed during 2003–2004 in three important maize producing counties (Paso de Ovejas, Cosoleacaque and Tlalixcoyan) in the State of Veracruz, Mexico, planted with the var. Asgrow-7573, reducing yields from 30 to 70% (Chavero 2004). Several probable aetiological agents were proposed: sugarcane mosaic potyvirus, a geminivirus, corn stunt phytoplasm (Alcantara-Mendoza et al. 2010) and the bacterium Herbaspirillum rubrisubalbicans (Chavero 2004). Due to the recent introduction and socioeconomic importance of this disease, the objective of the present work was to determine the causal agent. Materials and Methods Isolation and identification of the pathogen A total of 480 Asgrow 75–73 maize samples were collected in the counties of Cosoleacaque, Tlalixcoyan and Paso de Ovejas, during 2005–2007. Small pieces of symptomatic leaves and stems were cut from the margins of the advancing lesion, disinfested with 1% sodium hypochlorite for one minute and rinsed three times with sterile distilled water. Disinfested pieces were placed in sterile tubes with 10-ml sterile distilled water for 10 min, and the suspension was streaked on Petri plates containing casaaminoacid peptone and glucose (CPG), KingÕs Medium B (KB) and nutrient agar (NA) (Schaad et al. 2001). Plates were incubated at 28C for 72 h. Individual bacterial colonies were purified and stored at )80C in 15% aqueous glycerol. For all isolates, Gram reactions were determined by the lysis of bacteria in 3% KOH (Gregersen 1978). Cytochrome C oxidase was tested according to Kovacs (1956). Oxidative or fermentation metabolism was evaluated (Schaad et al. 2001). All strains were initially identified based on the Guide for Identification of Plant Pathogenic Bacteria (Schaad et al. 2001). Pathogenicity tests From the 480 leaf and stem samples, 240 bacterial cultures similar in morphology were obtained, from which 10 were selected for characterization and pathogenicity Gijon-Hernandez et al. 378 tests. Thirty healthy 45-day-old maize AS-7573 seedlings were syringe inoculated at the base of the stem with a 1 · 107 CFU⁄ml bacterial suspension. The inoculum was obtained from a bacterial pure culture grown at 30C for 48 h in KB, resuspended in 100 ml m NaCl, 2.7 mm m of of a sterile phosphate buffer (137 mm m de KH2PO4, pH 7.4). KCl, 0.01 m Na2HPO4, 1.8 mm Sterile phosphate buffer was used for control inoculations. The inoculated maize seedlings were covered with plastic bags and kept in a greenhouse (25–30C; 80% HR, with natural light). Symptoms development was followed for 45 days, and the bacteria were reisolated onto KB medium. Hypersensitivity reaction on tobacco plants A 107 CFU⁄ml bacterial suspension was infiltrated in the main vein of tobacco leaves (Nicotiana tabacum var. xhanti) and kept at 28C for 24 h. Control leaves were infiltrated with sterile distilled water. Sequencing 16S rDNA DNA was extracted from the selected bacterial colonies and from the diseased plant material with the Plant DNAZol (Invitrogen life technologies, Carlsbad, CA, USA) commercial protocol. The universal bacterial primers used were forward primer FD1 Eubacteria (5¢-AGAGTTTGATCCTGGCTCAG-3¢) that corresponds to position 8–28 of the Escherichia coli 16S rRNA sequence, and the reverse primer RD1 Eubacteria (5¢-AAGGAGGTGATCCAGCC-3¢) that corresponds to position 1526–1542 of Xylella fastidiosa gyrB gen (Rodrigues et al. 2003). DNA was amplified with m of each primer, 200 lm m dNTP, 1· PCR 25 ll of 0.2 lm m MgCl2, 2 U Taq polymerase (Invitrobuffer, 1.5 mm gen) and 10 ng of genomic DNA. PCR consisted of an initial denaturation at 94C for 3 min; followed of 30 cycles of 94C for 1 min, 55C for 30 s and 72C for 3 min, with a final extension at 72C for 7 min. A 10-ll aliquot was analysed by electrophoresis on a 1% agarose gel (Promega) at 80 V for 90 min in TBE buffer. Gels were stained with ethidium bromide. A 100-Pb ladder (Invitrogen Co.) was used as a size marker. DNA was sequenced in a Genetic Analyzer 3100 (Applied Biosystem Corp., Foster city, CA, USA). Sequences data were aligned, and homology was determined using the National Center for Biotechnology Information Blast Network Server (Altschul et al. 1990). Genus identification by PCR Identification was confirmed using the pair of primers RHG-F (5¢-GGGATTCATTTCCTTAGTAAC-3¢), which corresponds to position 835–851 16S rDNA sequence and RHG-R (5¢-GCGATTACTAGCGATTC CAGC-3¢) which corresponds to position 1324–1345 16S rDNA (LiPuma et al. 1999). Amplification was m of each primer, initiated in 25 ll containing 0.2 lm m dNTP, 1· PCR buffer, 1.5 mm m MgCl2, 2 U 200 lm Taq polymerase (Invitrogen Co.) and 10 ng of genomic DNA. Amplification initiated with denaturation at 95C for 3 min; followed by 35 cycles at 94C for 1 min, 55C for 30 s and 72C for 2 min; with a final extension at 72C for 7 min. All amplified products were visualized by electrophoresis as previously mentioned. A multiple sequence alignment was constructed using clustal x (Conway Inst., UCD Dublin, Ireland). A phylogenetic tree was constructed with the neighbour-joining method from the 16S rDNA sequences, with 10 000 bootstrap repetitions with mega 4 software (Tamura et al. 2007). PCR species specifics The identification of Burkholderia gladioli was confirmed with the pair of primers LP1 (5¢-GGGGGGTC CATTGCG-3¢), which corresponds to position 872– 886, and LP4 (5¢-AGAAGCTCGCGCCACG-3¢) whose position is 1523–1508, directed towards a specific region in the rRNA 23S gene (Whitby et al. 2000). m of Amplification was made with 25 ll containing 0.2 lm m dNTP, 1· buffer, 1.5 mm m MgCl2, each primer, 200 lm 2 U Taq polymerase and 10 ng of genomic DNA. Amplification initiated with denaturation at 95C for 3 min, followed by 35 cycles at 94C for 1 min, 55C for 30 s and 72C for 2 min, with a final extension at 72C for 7 min. All amplified products were visualized by electrophoresis as previously mentioned. RFLPs Ten microlitres of the PCR product was digested with HaeIII (Roche), Taq1 and ApaI (Fermentas) enzymes in a thermocycler (Applied Biosystems 2720), with the following programme: HaeIII and ApaI at 37C for 20 h and Taq1 at 65C for 20 h. Digested products were analysed in a 2% agarose gel. Results Field sampling Initial symptoms on leaves were small white-yellow watery spots which coalesced into dry necrotic stripes 0.3 wide and up to 8 cm long. Stems developed a soft rot in the crown. The flag leaf became rot and necrotic at the base, rolled inwards and dried out. The base of the corn ears was necrotic and did not grow. Isolation and identification of the pathogen From the 480 Asgrow-7573 maize plants collected in the field, 240 yielded a bacterial colony with similar morphology in KB, NA and CPG media. An aerobic, bacillary, gram negative, white bacterium was constantly isolated from the symptomatic plants; the colonies were white, convex, with smooth edges, humid, opaque, and with a yellow diffusible pigment in KB medium. All bacterial colonies had a negative oxidase reaction, grew at pH 4.0 and utilized glucose, mannitol, arabinose, threalose, xylose, sucrose, cellobiose and sorbitol but not l-rhamnose nor maltose. None of the strains produced indol. Starch hydrolysis, levan, gelatin liquefaction and nitrate reduction were positive. Based on the morphology, physiology and biochemical characterization, the 10 selected cultures were identified as B. gladioli (Schaad et al. 2001). Maize Leaf Stripe and Stem Rot Caused by B. gladioli Pathogenicity tests The 30 inoculated AS-7573 maize plants infiltrated with the bacteria in the greenhouse developed symptoms similar to those observed in the field. Symptoms on leaves appeared 8 h after inoculation as soft, whiteyellow spots (Fig. 1a). These spots coalesced into 0.3 by 8-cm necrotic stripes (Fig. 1b), and some leaves developed a reddish colour. A soft rot and necrosis developed in the crown of the stem (Fig. 1c). The stem became necrotic (Fig. 1d), The flag leaf developed a rot and necrosis at the base, rolled inwards and dried out (Fig. 1e). The base of the corn ear became necrotic and did not grow (Fig. 1f). Control plants remained healthy. KochÕs postulates were fulfilled with the reisolation and identification of the bacteria from the inoculated tissues. The bacteria isolated from the maize plants were positive for the hypersensitivity reaction in tobacco leaves; 24 h after infiltrating the main vein, wilting and necrosis developed in the whole leaf (Fig. 2). Sequencing 16S rDNA All bacterial isolates generated a product of approximately 1600 bp when amplified by PCR with FD1 and RD1 primers, which amplify the 16S rDNA gene (data not shown). blast analysis of the 10 16S rDNA sequences aligned with B. gladioli. Genus identification by PCR The confirmation of the genus Burkholderia was made with the specific primers (LiPuma et al. 1999), amplifying a fragment of approximately 500 bp. The identity of the isolates was confirmed by sequence alignment (a) (d) Fig. 1 (a) Maize leaves with initial symptoms of Burkholderia gladioli as white-yellow spots. (b) Stripes on leaves. (c) Soft rot of the stem crown. (d) Stem necrosis. (e) Inward rolling and necrosis of the flag leaf. (f) Maize corn ear with necrosis and inhibited growth 379 using clustal w and Megaling of the DNstar program. Partial 16S rDNA sequences of the 10 suspected Burkholderia strains were obtained and deposited into the GenBank database with accession numbers EU161873 to EU161893. There was 100% similarity between isolates from the three counties and 99% similarity with B. gladioli sequences from the Genbank. The phylogenetic trees generated with the 16S RNA sequences indicated a high homology, despite that the isolates were from three counties. There was no amplification with a strain of Xanthomonas campestris pv. vesicatoria used as negative control (Fig. 3). Species specifics PCR The identity of the isolated bacterial strains was also confirmed using B. gladioli-specific PCR (Whitby et al. 2000). An approximately 700-bp amplified fragment of the 23S rRNA gene was obtained for all B. gladioli strains tested (Fig. 4). No amplified fragment was observed with the Xanthomonas campestris pv. vesicatoria strain used as negative control. The analysis of 16S rDNA and 23S rRNA with universal primers for genus and species confirmed the morphological, physiological and biochemical results and indicated the identity of the bacterial isolations as B. gladioli. RFLPs The amplification products digested with HaeIII enzyme gave fragments of 150, 350, 400 and 500 bp. Taq1 generated three fragments of 150, 300 and 350 bp, while ApaI did not show any restriction cut (data not shown). The restriction fragments originated by the three enzymatic digestions were the same for all (c) (b) (e) (f) Gijon-Hernandez et al. 380 M 1 2 3 4 5 6 7 8 9 10 11 M Fig. 4 Amplification of the 700-bp product from the 23 rDNA gene of Burkholderia gladioli. Lane 1–10 with the 10 selected strains of B. gladioli isolated from maize. Lane 11, Xanthomonas campestris pv. vesicatoria as negative control. M = Invitrogen 1-Kb DNA ladder and 100 bp DNA ladder (left) Fig. 2 Hypersensitive necrosis caused by Burkholderia gladioli on tobacco leaves: necrosis developed in the whole leaf M 1 2 3 4 5 6 7 8 9 10 11 Fig. 3 Amplification of the 500-bp product from the 16S rDNA gene of Burkholderia. Lane 1–10 with the 10 selected strains of Burkholderia isolated from maize. Lane 11, Xanthomonas campestris pv. vesicatoria as negative control. M = Invitrogen 100 bp DNA ladder the bacterial strains tested, which indicates the possibility that the enzyme cutting sites are related with conserved regions for 16S rDNA. Discussion This new disease appeared in maize plantings of the var Asgrow 7573 in 2003–2004 in Tlalixcoyan, Cosoleacaque and Paso de Ovejas counties, in the State of Veracruz, México. Asgrow-7573 is still the only variety grown in that area; growers prefer it for its short cycle. A bacterial strain was constantly isolated from the symptomatic maize tissues which grew as white, convex colonies with smooth margins, humid, bright, and with a yellow diffusible pigment in KB medium. KochÕs postulates were fulfilled with 10 pure cultures, and symptoms on inoculated maize plants were identical to those observed in the field (Fig. 1a–f). The 10 strains were very virulent when inoculated on maize and tobacco plants. Symptoms on inoculated maize leaves appeared as early as 8 h after inoculation as soft, white-yellow spots. Tobacco leaves developed wilting and necrosis of the whole leaf when infiltrated area mid vein. 16S rDNA analysis with universal primers and the partial sequence of that region confirm the biochemical and physiological studies. Using primers suggested by LiPuma et al. 1999 resulted in a 100% similarity with all of our B. gladioli tested strains and 99% similarity with B. gladioli from the GenBank. The final confirmation as B. gladioli is supported by the 23S rDNA analysis with LP1 and LP4 primers (Whitby et al. 2000). Burkholderia gladioli had not been previously reported in maize in Mexico, and the symptoms caused had not been observed before. The State of Veracruz grows mostly rice, sorghum and sugar cane, which could also become infected because they are hosts of B. gladioli. The phylogenetic analysis and the use of restriction enzymes did not show variability between isolates from the three sampled counties, which suggests that this bacterium was not present in Mexico and that it was recently introduced in infected seeds, possibly from a single source. Yield losses caused by this disease have decreased from 30 to 70% in 2003–2004 to approximately 1% in 2007. Burkholderia gladioli causes disease in both humans [cystic fibrosis in pulmonary infections in hospitalassociated infections (Stoyanova et al. 2007); granulomatous disease in respiratory tracts and bacteremia and sterile wound infections in lung transplant patients (http://www.chestjournal.org/cgi/reprint/114/2/ 658.pdf)] and plants notably causing rot of gladiolus corms (McCulloch 1921). Its pathogenicity on maize plants in Mexico is a new record. Maize Leaf Stripe and Stem Rot Caused by B. gladioli Acknowledgements Authors express their appreciation to Dr. Laura Ongay, Unidad de Biologia Molecular del Instituto de Fisiologia Celular, UNAM, Mexico, where DNA sequence was made. References Alcantara-Mendoza S, Teliz-Ortiz D, De Leon C, Cardenas-Soriano E, Hernandez-Anguiano AM, Mejia-Sanchez D, De la TorreAlmaraz R. (2010) Detection and evaluation of Maize bushy stunt phytoplasma in the state of Veracruz, Mexico. Rev Mex Fitopatol 28:34–43. 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