Detection and Characterization of Xanthomonas vasicola pv. vasculorum (Cobb 1894) comb. nov. Causing Bacterial Leaf Streak of Corn in the United States.

Bacterial leaf streak of corn (Zea mays) recently reached epidemic levels in three corn-growing states, and has been detected in another six states in the central United States. Xanthomonas vasicola was identified as the causal agent of this disease. A multilocus sequence alignment of six housekeeping genes and comparison of average nucleotide identity from draft genome sequence were used to confirm phylogenetic relationships and classification of this bacteria relative to other X. vasicola strains. X. vasicola isolates from Nebraska and South Africa were highly virulent on corn and sugarcane and less virulent on sorghum but caused water-soaking symptoms that are typical of X. vasicola infection on the leaves of all three hosts. Based on host range and phylogenetic comparison, we propose the taxonomic designation of this organism to X. vasicola pv. vasculorum ( Cobb 1894 ) comb. nov. Polymerase chain reaction-based diagnostic assays were developed that distinguish X. vasicola pv. vasculorum and X. vasicola pv. holcicola from each other and from other Xanthomonas spp.

First Report of Goss's Bacterial Leaf Blight and Wilt of Corn Caused by Clavibacter michiganensis subsp. nebraskensis in North Dakota.

In August of 2011, the North Dakota State University Plant Diagnostic Lab received a hybrid corn (Zea mays) leaf sample from Burleigh County in south-central North Dakota (ND). The leaf had long, irregular, water-soaked lesions consistent with Goss's leaf blight of corn. Using a light microscope at 10× magnification, bacterial streaming was observed from the excised edge of leaf tissue. A bacterial suspension was created, streaked onto a semi-selective CNS medium (1), and incubated at 22°C. Dark yellow-orange colonies appeared on the medium after 5 days. Single colonies were subcultured onto additional CNS media. To verify the identity of the bacterial isolate, PCR amplification of the 16S ribosomal DNA from this isolate along with a known Clavibacter michiganensis spp. nebraskensis (Cmn) isolate collected in Indiana (4) was performed using the eubacterial universal primers 27f and 1525r (3). The 1,431-bp 16S rDNA region was obtained for each isolate and they were compared with each other and with those deposited in NCBI GenBank. Sequence alignment identified only one nucleotide difference between the ND isolate and the Indiana isolate. BLASTn search against the NCBI database showed the first 100 hits were described as C. michiganensis or unidentified Clavibacter sp. The ND isolate had a two-nucleotide difference with Cmn isolate NCPPB2581 (HE614873), and a three nucleotide difference was found with the C. michiganensis spp. michiganensis isolate NCPPB 382 (AM711867). To satisfy Koch's postulates, eight corn plants (Golden Cross Bantam) were grown in the greenhouse at 22 to 24°C. Four corn plants were inoculated at growth stage V4-V5 using a suspension of approximately 1 × 109 CFU/ml from cultures grown on CNS for 6 days. Wounds were created on the fifth leaf approximately 7 cm from the leaf tip using a tongue-seizing forceps outfitted with a rubber stopper composed of pins (2). Simultaneously, 1 ml of the bacterial suspension was delivered into the wounds through a hole on top of the rubber stopper. Four control plants were inoculated with sterile water in a similar fashion. No symptoms were observed on the control plants. After 6 days, long water-soaked symptoms were observed on leaves inoculated with the bacterial suspension. Using leaves with water-soaked lesions, the pathogen was re-isolated onto CNS media and subjected to PCR amplification, and the resulting amplicons were sequenced as before. The sequence of the amplicon from the re-isolation matched that of the original ND isolate. To our knowledge, this is the first account of Goss's leaf blight and wilt identified in ND. As the corn acreage and no-till production systems in the state have increased, the economic implications of this disease may become more significant. Recognition of symptoms and proper identification of this bacterial disease in the field should help reduce unnecessary foliar fungicide sprays. References: (1) D. C. Gross and A. K. Vidaver. Phytopathology 69:82, 1979. (2) W. A. Hagborg. Can. J. Bot. 48:1135, 1970. (3) X. Li and S. H. DeBoer. Can. J. Microbiol. 41:925, 1995. (4) G. Ruhl et al. Plant Dis. 93:841, 2009.

First Report of Goss's Bacterial Wilt and Leaf Blight (Clavibacter michiganensis subsp. nebraskensis) of Corn in Texas.

In September 2009, the University of Nebraska-Lincoln Plant and Pest Diagnostic Clinic received leaf samples of hybrid corn (Zea mays L.) displaying long, necrotic lesions with wavy margins. The lesions had discontinuous water-soaked spots that are indicative of Goss's bacterial wilt and leaf blight. The symptomatic leaves were submitted from Dallam County, located in the Texas Panhandle (northwest Texas). According to the USDA Farm Service Agency and the National Agricultural Statistics Service, in 2009 Dallam County had 54,025 ha planted to corn. This is approximately 19% of the total corn planted in the 26 counties in the Texas Panhandle and 6% of the total corn planted in the state of Texas. Extracts from the infected leaf tissue tested positive for Clavibacter michiganensis subsp. nebraskensis with a commercially available ELISA test (Neogen Inc., Scotland, UK). Isolation from the infected tissue onto CNS selective media (1) resulted in round, dark orange, mucoid colonies that tested gram positive with the Gram-stain test. BLAST nucleotide sequence alignments of the amplified 500-bp 16S rRNA region of the suspect culture's genome (2) revealed a 96% similarity for C. michiganensis subsp. nebraskensis (NCBI BLAST Accession No. U09381.1). To fulfill Koch's postulates, three sweet corn plants (Golden Cross Bantam) at growth stage V3 to V4 were inoculated in the greenhouse with a suspension of approximately 1 × 109 CFU/ml from suspect cultures grown on CNS for 5 days. Wounds approximately 6.5 cm long were created with sterile scissors on the fifth leaf from the bottom running parallel to the veins on either side of the midrib at the leaf apex. The leaf apex was dipped into 150 ml of the inoculum suspension for 5 s. Approximately 6 days after inoculation, discontinuous, water-soaked spots consistent with the symptoms on the original symptomatic leaves appeared on all the inoculated leaves near the site of infection. Colonies consistent with C. michiganensis subsp. nebraskensis (dark orange, mucoid) were reisolated onto CNS, completing Koch's postulates. To our knowledge, this is the first report of Goss's bacterial wilt and leaf blight on corn in Texas and because it is a residue-borne pathogen, the probability of it becoming a resident disease is relatively high. References: (1) D. C. Gross and A. K. Vidaver. Phytopathology 69:82, 1979. (2) X. Li and S. H. De Boer. 1995. Phytopathology 85:837, 1995.