Target spot symptoms were first observed on dryland and irrigated cotton (Gossypium hirsutum L.) statewide in Alabama in 2011. Leaf spots first appeared in the lower canopy and spread upward through the canopy toward the shoot tips. Individual leaf spots were roughly circular, formed concentric rings of alternating light and dark brown bands, and were up to 10 mm in diameter. Leaves with multiple lesions senesced prematurely. In 2012, target spot symptoms were observed as early as 68 days after planting in Tallapoosa County, Alabama. The possible combination of early disease onset and frequent showers/irrigation triggered rapid premature defoliation in some fields in excess of 75% in susceptible cultivars (Phytogen 499). Estimated yield losses in select cultivars (Deltapine 1050 and Phytogen 499) exceeded 336 kg/ha seed cotton. In 2012, symptomatic leaves were obtained from two separate locations in Alabama (Baldwin and Tallapoosa counties). The fungus was isolated from lesions by single spores plated on antibiotic V8 agar (1) and incubated at 21°C for 2 weeks under 12-h light cycles. Conidiophores arising from the gray, flocculose colonies were simple, erect, cylindrical, brown or olivaceous, unbranched, with two to seven septa. Conidia were borne singly, ranging from subhyaline to olivaceous, obclavate to cylindrical, straight to slightly curved, contained 4 to 15 pseudosepta, and were 50 to 209 μm long and 7 to 15 μm wide. These characteristics were consistent with the original description of Corynespora cassiicola on cotton (2). The internal transcribed spacer region (ITS) of two isolates, one representing each location, was amplified using primers 2234c and 3126t targeting a 550-bp region of the ITS1, 5.8S rRNA gene, and ITS2 (3). Sequences revealed 99% similarity to C. cassiicola in NCBI (Accession Nos. AY238606 and JQ717069). In greenhouse pathogenicity tests, 10 cotton seedlings (Phytogen 499) were inoculated by spraying a fungal suspension (2 × 104 spores/ml) of each of the two isolates prepared from 2-week-old cultures until runoff. Controls were inoculated with sterile water. Cotton seedlings were incubated in a moist chamber at 21°C for 72 h. All plants inoculated with the fungus developed leaf spot symptoms in 6 days. The fungus was reisolated from five inoculated plants. DNA was extracted from each isolate, amplified using primer pair 2234c/3126t, and sequenced. Sequences (550-bp) from all isolates shared 99% similarity to other C. cassiicola sequences in GenBank (Accession Nos. AY238606 and JQ717069). Nucleotide sequence data reported are available in GenBank under Accession Nos. KC544017 to 23. This pathogen has been reported previously to be economically important on a number of other hosts. To our knowledge, this is the first report of C. cassiicola on cotton in Alabama. Given the increasing prevalence of this disease in Alabama, its confirmation is a significant step toward developing management recommendations for growers. References: (1) L. J. Dixon et al. Phytopathology 99:1015, 2009. (2) J. P. Jones. Phytopathology 51:305, 1961. (3) J. Sequerra et al. Mycol. Res. 101:465, 1997.
We determined the structure of a 1.1-kb cytoplasmic polyadenylated RNA transcribed from a region of the bovine herpesvirus 4 (BHV-4) genome not conserved among gammaherpesviruses by sequencing its cDNA, by S1 nuclease analysis, and by primer extension analysis. We found that the RNA consists of a short, approximately 193-nucleotide (nt), 5' exon spliced to a 799-nt 3' exon and contains two short (53 and 57 codons) overlapping open reading frames (ORFs). The 53-codon ORF was previously designated BORFE1. Neither ORF exhibits detectable amino acid sequence homology with ORFs of other gammaherpesviruses. The 1.1-kb RNA promoter-regulatory region was specifically transactivated by the BHV-4 IE2 gene product, a homolog of the Epstein-Barr virus R transactivator, in cotransfection assays. In gel retardation experiments, IE2 protein formed a complex with DNA in a 129-bp fragment between -23 and -151 relative to the transcription start site of the 1.1-kb RNA, and less efficiently with a 57-bp subfragment between -78 and -22. A sequence similar to sequences of IE2-binding fragments of other BHV-4 IE2 responsive promoters was found partly in the 57-bp subfragment, extending into the portion of the 129-bp fragment not found in the 57-bp fragment. The 129-bp fragment, but not the 57-bp fragment, was sufficient for transactivation of a promoterless chloramphenicol acetyl transferase (CAT) reporter gene by IE2. However, the 129-bp fragment did not function efficiently as an IE2-responsive enhancer when inserted approximately 140 base pairs (bp) 5' to the transcription start site of a CAT reporter gene driven by an enhancerless simian virus 40 early promoter. Based on this and other observations, we propose that IE2 functions as a promoter factor rather than an enhancer factor.
Full-length mariner elements were isolated and sequenced from house flies (Musca domestica) and German cockroaches (Blattella germanica). The amino acid sequence of the house fly mariner element (accession number: AF373028) showed 99.5% identity with Mos1 and peach elements, whereas the German cockroach mariner element (accession number: AF355143) showed 98.8% and 99.8% identity, respectively. Sequence analysis revealed that the mariner elements in house flies and German cockroaches differed from the active Mos1 mariner element by seven and 15 nucleotides, respectively. Four essential nucleotide substitutions at positions 64, 154, 305, and 1203, which have been proposed to contribute to the loss of activity of the inactive elements, were detected in the German cockroach mariner element. In contrast, although the mariner element in house flies contained substitutions at positions 64, 154, and 305, it retained T at position 1203, identical to active mariner elements. Mariner is present in approximately eight copies in the German cockroach genome.
Bacterial gall symptoms were observed on Loropetalum chinense (R. Br.) Oliv. in two separate commercial nurseries in South Alabama during the spring of 2012. Limb dieback and plant death was first reported by the growers. Plants with dieback symptoms had galling and irregular dark callus formation on the lower stem and lower branches. Galls were small, 0.2 to 1 cm, inconspicuous, and in some cases girdled the stem causing breakage of the main stem. In both locations, 30 to 40% of the crop was affected. Similar symptoms have been observed on L. chinense in nursery and landscape plantings in central Alabama, North Carolina, and Georgia in previous years. Bacterial colonies were isolated from four plants representing two different locations. Isolates were recovered from surface sterilized symptomatic tissue on nutrient agar and King's medium B (KMB). All isolates were gram-negative and fluoresced blue-green under UV light after 48 h of growth at 28°C on KMB. One representative isolate from each site was identified as Pseudomonas savastanoi based on their fatty acid profiles (similarity index of 0.776; MIS-TSBA, version 4.0, MIDI Inc., Newark, DE) and LOPAT tests (2). The identity was confirmed by sequencing a 900-bp portion of the 16S rDNA gene, which revealed 98% similarity to the P. savastanoi type strain in NCBI (Accession No. AB021402). In greenhouse pathogenicity tests, eight Loropetalum liners were inoculated with a bacterial suspension (107 CFU/ml) of each of the two isolates. Plants were inoculated by injecting the suspension into the lower stem after wounding by puncturing with needles or slicing sections of the bark. Controls were inoculated with water. All plants inoculated with the bacteria developed gall symptoms in 8 weeks under 90% relative humidity at 30°C. The bacteria were reisolated from five inoculated plants. DNA was extracted from each isolate, amplified using primer pair 27F/1492R targeting the 16S rDNA gene (1), and sequenced. Sequences (900 bp) from all isolates shared 98 to 99% similarity to P. savastanoi type strain in GenBank (Accession No. AB021402). Nucleotide sequence data reported are available in GenBank under accessions JX915832 to 37. To our knowledge, this is the first report of bacterial gall of L. chinense caused by P. savastanoi in the United States. Given the increasing prevalence of this disease in South Alabama, its confirmation is a significant step toward management recommendations for growers. References: (1) D. J. Lane. 16S/23S rRNA sequencing. Page 115-175 in: Nucleic Acid Techniques in Bacterial Systematics. E. Stackebrandt and M. Goodfellow, eds. John Wiley and Sons, New York, 1991. (2) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.
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