Genotypic Diversity and Virulence Traits ofStreptococcus mutansIsolated from Carious Dentin after Partial Caries Removal and Sealing

The aim of this study was to compare the genotypic diversity and virulence traits ofStreptococcus mutansisolated from carious dentin before and after partial dentin caries removal (PDR) and sealing. Carious dentin samples were obtained three months before and after the PDR and cavity sealing. Up to seven isolates of each morphological type ofS. mutanswere selected and strain identity was confirmed using gtfB primer. Genotyping was performed by arbitrary primer-PCR (AP-PCR). Acidogenesis and acidurance of the genotypes were evaluated as virulence traits. A pairedt-test and a Wilcoxon test were used to compare the virulence of genotypes. A total of 48 representativeS. mutansisolates were genotyped (31 before and 17 after the sealing). At least one of the genotypes found before the sealing was also found on dentin after the sealing. The number of genotypes found before the sealing ranged from 2 to 3 and after the sealing from 1 to 2 genotypes. No difference was observed in the acidogenesis and acidurance between genotypes isolated before and after the sealing. In conclusion, genotypic diversity ofS. mutansdecreased after the PDR and sealing, but the virulence traits ofS. mutansremained unchangeable.

First Report of Race 3 of Bipolaris zeicola on Corn in China

In Meixian County of Shaanxi Province, China, during the summer of 2002, mature corn plants in a field plot showed severe leaf spot symptoms. The lesions were narrow (3.5 to 18 mm long and 0.4 to 1.5 mm wide), grayish tan, and surrounded by a light- to dark-pigmented border. Leaves wilted when lesions coalesced. From 2002 to 2005, the disease was observed in other Shaanxi Province counties, including Yangling, Wugong, Qianxian, Longxian, and Qianyang, although in most cases, symptom development was less severe than it was in Meixian. Seven isolates from four counties were obtained by isolation from host tissue on potato dextrose agar (PDA), followed by single-spore culturing and incubation on PDA at 25°C in the dark for 7 days. Conidial suspensions were prepared from a single-spored culture on PDA plates. Pathogenicity tests were performed by spraying five corn seedlings (cv. Yuyu 22) at the three- to four-leaf stage in separate 10-cm-diameter pots with 10 ml of a conidial suspension (106 spores per ml) per plant. Each of three isolates was used in separate inoculations that were performed in different weeks. Controls were sprayed with sterile distilled water only. Plants were covered with plastic bags for 48 h and incubated at 23 to 25°C in a chamber. One week after inoculation, leaves on all inoculated plants developed characteristic lesions, whereas untreated controls had no symptoms. The pathogen was reisolated from diseased leaves on PDA after surface sterilization with 2% NaOCl. On PDA, proliferation of conidia usually occurred on all sides of the conidiophore. Conidiophores were cylindrical, simple, smooth, septate, and straight to flexuous. Conidia were 49 to 89 μm long and 11 to 17 μm wide, with 3 to 10 distosepta, straight or moderately curved, dark or olivaceous brown, and the cells on the ends sometimes appeared paler than those in the middle. These characteristics match those of Bipolaris zeicola (Stout) Shoemaker. On the basis of the arbitrary primers selected by Jones et al. (1), random amplified polymorphic DNA (RAPD) analysis was used for species and physiological race determination. A single DNA fragment approximately 1.2 kb, which is characteristic of B. zeicola, was amplified from all seven isolates with arbitrary primer A20 (5′CTTGGATTC3′). Analysis of PCR products obtained with arbitrary primer A03 (5′AGTCAGCCAC3′) showed that all seven isolates lacked 2,700- and 2,300-base bands, and therefore, sorted into B. zeicola race 3. On the basis of pathogenicity, morphology, and RAPD band patterns of primer A20, the fungus was confirmed as B. zeicola. The shape of leaf lesions and RAPD band patterns using primer A03 showed further that the pathogen was race 3 of B. zeicola. Bai et al. (2) reported race 1 and race 2 of B. zeicola in China, but to our knowledge, this is the first report of race 3 in China. References: (1) M. J. Jones and L. D. Dunkle. Phytopathology 83:366, 1993. (2) J. K. Bai et al. Acta Phytopathol. Sin. 12:61, 1982.

A Phylogeny of Pelargonium Based on TRAP Markers

Pelargonium is one of the priority genera collected by the Ornamental Plant Germplasm Center (OPGC). In order to protect future breeders from a loss of genetic diversity, the OPGC collects heirloom cultivars, breeding lines, and wild species. The current Pelargonium collection consists primarily of cultivars originating from P. ×hortorum and P. ×domesticum. Our project was designed to analyze the current collection in order to facilitate the maintenance of a more-diverse core collection. We have expanded our TRAP (Target Region Amplified Polymorphism) analysis from 120 plants with one primer set to include 780 plants with four primer sets. Each primer set consists of a labeled arbitrary primer paired with a gene-specific primer, and two different fluorescent labels were used to allow multiplexed PCR reactions. We scored about 90 markers in each of the first two primer sets and about 60 markers in each of the second two. In comparisons between the phylogeny and the morphology and taxonomy of these plants, we show some matching clusters that may be explained by the breeding history of the plants.

Random amplified polymorphisms between two South American subspecies of rattlesnakes (Crotalus durissus collilineatus and Crotalus durissus terrificus)

The present work was made to determine the suitability of RAPD analysis for the identification of specimens of two subspecies of South American rattlesnakes (Crotalus durissus terrificus and Crotalus durissus collilineatus). The 11 arbitrary primer sequence tested amplified a total of 161 bands, of which 31% were polymorphics when compared with Crotalus specimens. The combining results from different primers allowed the identification of all the specimens under analysis. Several characteristic bands of each subspecies were identified. Dendrogram, based on RAPD markers, show three groups that corresponded to the subspecies of Crotalus, and to Bothrops jararaca specimen.

Identification of Trypanosoma evansi by DNA hybridisation using a non-radioactive probe generated by arbitrary primer PCR: Short communication

A highly reproducible, dominant, monomorphic fragment of 473 base pair (bp) amplified from the genome of Trypanosoma evansi by arbitrary primer — polymerase chain reaction (AP-PCR) was labelled with digoxigenin and investigated for its potential as DNA probe. Dot-blot hybridisation of total genomic DNA with the probe proved useful in detecting bubaline, cameline and equine strains of T. evansi down to 10 pg of parasite template DNA. No cross-hybridisation was seen with Babesia bigemina, Theileria annulata and the bubaline host DNA. This probe may facilitate laboratory identification of T. evansi in developing countries, without the inherent risk associated with radioisotopes.

Optimization of Differential Display of Prokaryotic mRNA: Application to Pure Culture and Soil Microcosms

ABSTRACT The differential display (DD) technique, which is widely used almost exclusively for eukaryotic gene discovery, was optimized to detect differential mRNA transcription from both pure-culture and soil-derived bacterial RNA. A model system which included toluene induction of todC1 in Pseudomonas putida F1 was used to optimize the procedure. At 24-h tod induction was determined to be approximately 8 × 107transcripts/μg or 0.08% of the total mRNA. The primer concentration, primer length, annealing temperature, and template, deoxynucleoside triphosphate, and MgCl2 concentrations were varied to optimize amplification of a todC1 fragment. The limit of detection of todC1 by DD was found to be 0.015 ng of total RNA template or approximately 103 transcripts. Once optimized, a todC1C2 gene fragment from P. putida F1 RNA was detected by using an arbitrary primer for the reverse transcriptase step in conjunction with the same arbitrary primer and a Shine-Dalgarno primer in the PCR. To verify the results, an arbitrary primer was used to detect recovery of a new salicylate-inducible naphthalene dioxygenase in Burkholderia cepacia JS150. The method was then used to detect mRNA induction in both inoculated and uninoculated toluene-induced soil microcosms. Several putative differentially expressed partial gene sequences obtained from the uninoculated microcosms were examined, and one novel fragment was found to be differentially expressed.