First Report of Xylella fastidiosa in Oklahoma.

In Oklahoma, during the late summer of 2004, an elm tree (Ulmus americanus L.) located in the Oklahoma Botanical Gardens near Stillwater showed symptoms of marginal leaf scorch bordered by a yellow band between necrotic and green tissues, indicating possible Xylella fastidiosa infection. Three leaves from the symptomatic tree and one leaf from an asymptomatic nearby elm were sampled. DNA was extracted with the Extract-N-Amp kit (Sigma, St. Louis, MO). Samples were tested for X. fastidiosa using real-time polymerase chain reaction (PCR) with Xylella genus specific primers XfF1/XfR2 and dual-labeled TaqMan probe XfP2 (2). Infected oleander from California was used as a positive control. All three samples from symptomatic leaves and the positive control were PCR positive, and the sample from the asymptomatic tree was PCR negative. Attempts to culture an isolate of the bacteria from petioles and branch tissues on PD3 and PW, media selective for X. fastidiosa, failed. For more detailed molecular characterization of the putative pathogen, DNA from additional symptomatic petioles from the same tree was isolated using the cetyltrimethylammoniumbromide (CTAB) extraction. X. fastidiosa specific primers BBXFOUTF1 (5'-AAGCGCCTCCGTGAGTTATC-3') and BBXFOUTR1 (5'-CCTTCACGCATATCATCACC-3') were used to PCR amplify the gyrB gene. The amplification product was recovered after gel electrophoresis with QIAquick gel extraction kit (Qiagen, Valencia, CA) and was subjected to automated sequencing (Oklahoma State University Recombinant DNA/Protein Resource Facility). BLASTN alignment (1) of the obtained 381 bp sequence revealed 100% identity with the gyrB gene from elm (GenBank Accession No. AF534966) and mulberry (GenBank Accession No. AF534965) isolates of X. fastidiosa. During 2005, petiole samples from the tree were collected and serological diagnosis was confirmed using enzyme-linked immunosorbent assay (Agdia, Inc., Elkhart, IN). Some strains of X. fastidiosa have very wide host ranges and many of the hosts may be asymptomatic. Therefore, the economic importance and implications of the detection of X. fastidiosa in the state of Oklahoma remain to be determined. To our knowledge, this is the first report of X. fastidiosa in Oklahoma. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) N. W. Schaad et al. Phytopathology 92:721, 2002.

Population Structure of Phytophthora cinnamomi in South Africa.

ABSTRACT Phytophthora cinnamomi isolates collected from 1977 to 1986 and 1991 to 1993 in two regions in South Africa were analyzed using isozymes. A total of 135 isolates was analyzed for 14 enzymes representing 20 putative loci, of which four were polymorphic. This led to the identification of nine different multilocus isozyme genotypes. Both mating types of P. cinnamomi occurred commonly in the Cape region, whereas, predominantly, the A2 mating type occurred in the Mpumalanga region of South Africa. A2 mating type isolates could be resolved into seven multilocus isozyme genotypes, compared with only two multilocus isozyme genotypes for the A1 mating type isolates. Low levels of gene (0.115) and genotypic (2.4%) diversity and a low number of alleles per locus (1.43) were observed for the South African P. cinnamomi population. The genetic distance between the Cape and Mpumalanga P. cinnamomi populations was relatively low (D(m) = 0.165), and no specific pattern in regional distribution of multilocus isozyme genotypes could be observed. The genetic distance between the "old" (isolated between 1977 and 1986) and "new" (isolated between 1991 and 1993) P. cinnamomi populations from the Cape was low (D(m) = 0.164), indicating a stable population over time. Three of the nine multilocus isozyme genotypes were specific to the "old" population, and only one multilocus isozyme genotype was specific to the "new" population. Significant differences in allele frequencies, a high genetic distance (D(m) = 0.581) between the Cape A1 and A2 mating type isolates, significant deviations from Hardy-Weinberg equilibrium, a low overall level of heterozygosity, and a high fixation index (0.71) all indicate that sexual reproduction occurs rarely, if at all, in the South African P. cinnamomi population.

Calcium Interference with Zoospore Biology and Infectivity of Phytophthora parasitica in Nutrient Irrigation Solutions.

ABSTRACT Calcium, applied as either CaCl2 or Ca(NO3)2 to water or calcium-free soluble fertilizer solution (Peters 20-10-20 Peat Lite Special), affected several important stages of Phytophthora parasitica zoospore behavior relevant to infection and disease spread. Release of zoospores from sporangia was suppressed by Ca(2+) concentrations in the range of 10 to 50 meq. These concentrations also curtailed zoospore motility; 20 meq of Ca(2+) in fertilizer solution caused all zoospores to encyst within 4 h, whereas 94% of zoospores remained motile in unamended solution. In addition, Ca(2+) in the range of 10 to 30 meq stimulated zoospore cysts to germinate in the absence of an organic nutrient trigger, while suppressing the release of a single zoospore (diplanetism) from cysts that did not germinate. In growth chamber experiments, the amendment of the fertilizer solution with 10 or 20 mM Ca(NO3)2 greatly suppressed infection of flood-irrigated, containerized vinca seedlings in a peat-based mix by motile or encysted zoospores of P. parasitica. These results demonstrate that Ca(2+) amendments interfere with P. parasitica zoospore biology at multiple stages, with compounding effects on epidemiology, and suggest that manipulation of Ca(2+) levels in irrigation water or fertilizer solutions could contribute to management of Phytophthora in recirculating irrigation systems.

Mode of Pisatin Induction: Increased Template Activity and Dye-binding Capacity of Chromatin Isolated from Polypeptide-treated Pea Pods.

Increases in phenylalanine ammonia lyase activity and pisatin synthesis were induced in excised pea pods (a) by basic polypeptides such as protamine, histone, lysozyme, cytochrome c, and ribonuclease; (b) by the polyamines spermine, spermidine, cadaverine, and putrescine, and (c) by the synthetic oligopeptides poly-l-lysine, poly-dl-ornithine, and poly-l-arginine.Poly-l-lysine (1 milligram per milliliter, molecular weight 7,200) was utilized as a model inducer of pisatin and phenylalanine ammonia lyase. The poly-l-lysine-induced responses could be inhibited by adding the RNA synthesis inhibitors cordycepin or alpha-amanitin to the pods prior to or at the time of inducer application. Cordycepin added 1.5 hours after inducer no longer completely inhibited induction. The application of poly-l-lysine was shown to characteristically change the rate of RNA synthesis within 30 minutes. Ultrastructural changes in pea nuclei were detected within 3 hours, and gross changes in nuclear morphology were apparent at 14 hours after inducer application. The physical appearance of uranyl acetate-stained chromatin isolated from poly-l-lysine 2 hours after inducer application differed from that of water-treated tissues. The template properties of chromatin extracted from pods 3 hours after inducer application were consistently superior to control chromatin when assayed with Escherichia coli RNA polymerase (without sigma factor). Chromatin from poly-l-lysine-induced tissue also bound 49% more actinomycin D-(3)H.The DNA-complexing properties of inducer compounds and the induced changes in the template and dye-binding properties of pea chromatin formed the basis for a proposed mode of action for phytoalexin induction.