Virulence Spectrum of Single-Spore and Field Isolates of Plasmodiophora brassicae Able to Overcome Resistance in Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important disease of canola (Brassica napus L.) that is managed mainly by planting clubroot-resistant (CR) cultivars. Field isolates of P. brassicae can be heterogeneous mixtures of various pathotypes, making assessments of the genetics of host-pathogen interactions challenging. Thirty-four single-spore isolates were obtained from nine field isolates of the pathogen collected from CR canola cultivars. The virulence patterns of the single-spore and field isolates were assessed on the 13 host genotypes of the Canadian Clubroot Differential (CCD) set, which includes the differentials of Williams and Somé et al. Indices of disease (IDs) severity of 25, 33, and 50% (±95% confidence interval) were compared as potential thresholds to distinguish between resistant and susceptible reactions, with an ID of 50% giving the most consistent responses for pathotype classification purposes. With this threshold, 13 pathotypes could be distinguished based on the CCD system, 7 on the differentials of Williams, and 3 on the hosts of Somé et al. The highest correlations were observed among virulence matrices generated using the three threshold IDs on the CCD set. Genetically homogeneous single-spore isolates gave a clearer profile of the P. brassicae pathotype structure. Novel pathotypes, not reported in Canada previously, were identified among the isolates. This large collection of single-spore isolates can serve as a reference in screening and breeding for clubroot resistance.

An exo-1,3-ß-glucanase GLU1 contributes to the virulence of the wheat tan spot pathogen Pyrenophora tritici-repentis.

Tan spot, caused by Pyrenophora tritici-repentis, is an important foliar disease of wheat. In the present study, a gene named glucanase gene (GLU1) encoding a putative exo-1,3-ß-glucanase was cloned from a race five isolate of P. tritici-repentis. Transcription profile analysis of the GLU1 gene showed a carbon source control of the accumulation of transcript, which is strongly induced in minimal medium but depressed in rich medium. A time-course study of the infection process of the wild-type isolate on a susceptible wheat genotype revealed that the transcript level of GLU1 increased more than 8000-fold 8 h after inoculation. To study its potential function in pathogenicity, GLU1 was silenced via a sense and antisense mediated silencing mechanism. One transformant named C1 showed significantly reduced growth and sporulation relative to the wild-type. Cytological analysis of the infection revealed that C1 produced significantly lower numbers of germ tubes and appressoria than the wild-type strain on susceptible wheat leaves. This strain, as well as another two transformants, caused significantly less disease symptoms relative to the wild-type after inoculation onto a susceptible wheat genotype. These results indicate that GLU1 contributes to the development and virulence of P. tritici-repentis.

Almond Leaf Scorch Disease Development on Almond Branches High-Grafted on Peach Rootstock.

Development of almond leaf scorch (ALS) disease was monitored on young almond (Prunus dulcis 'NePlus' and 'Peerless') shoots produced from almond scion wood that was high-grafted on peach rootstocks (P. persica 'Queencrest/Nemaguard'), after the almond shoots were mechanically inoculated with Xylella fastidiosa. The objective of this study was to evaluate the potential movement of X. fastidiosa through almond-peach graft unions. ALS symptoms developed on both X. fastidiosa-inoculated and uninoculated almond shoots that were high-grafted on different peach limbs of the same tree in September following inoculations with X. fastidiosa made in June and July 2002, respectively, when the average distance in peach rootstock between the two almond-peach graft unions was 30.5 cm or shorter. No ALS symptoms were observed on uninoculated almond shoots late in the growing season of the second year. The incidence of ALS-affected leaves on shoots inoculated with X. fastidiosa decreased in the second year on the inoculated shoots of Peerless as determined by the number of inoculated shoots showing ALS symptoms in 2002 versus 2003. No visible ALS symptoms were observed in NePlus late in the growing season of the second year, suggesting that survival of X. fastidiosa in NePlus was less than in Peerless. These data demonstrate that movement of X. fastidiosa through two almond-peach graft unions was possible during the year of inoculation when the peach rootstock stem distance between the two almond-peach graft unions was minimal. However, X. fastidiosa may not survive the winter season or survived so poorly as not to be able to produce ALS symptoms in the second growing season on the uninoculated shoots that previously showed ALS symptoms in the fall of the year when an adjacent shoot was inoculated. The results suggest that high-grafting almond scion wood on multiple peach rootstock limbs may provide a means to limit movement of X. fastidiosa from one almond limb to another on the same tree.

Influence of Inoculation Date on the Colonization of Xylella fastidiosa and the Persistence of Almond Leaf Scorch Disease Among Almond Cultivars.

Field grown 2-year-old almond trees (Prunus dulcis cvs. Butte, Carmel, Mission, Ne Plus Ultra, Padre, Peerless, Price, Solano, Sonora, and Thompson) were mechanically inoculated with Xylella fastidiosa in the growing seasons of 2002 and 2003 to study the effect of inoculation date on the movement and colonization of X. fastidiosa and the overwintering persistence of almond leaf scorch disease (ALS) in these cultivars. X. fastidiosa was inoculated into the base of current-season growing shoots in April, May, June, July, August, September, and October. Almond trees inoculated in spring months developed more ALS-symptomatic leaves and more extensive within-plant spread of X. fastidiosa by the end of the current growing season compared with trees inoculated in July, August, September, and October. Trees inoculated in June developed the most severe ALS symptoms during the season in which they were inoculated. Trees inoculated in June and July 2002 had significantly higher disease ratings in 2003 than inoculations made in August and October 2002. Based on disease ratings observed in 2003, 1 year after inoculation, Sonora and Solano were the most ALS susceptible, Mission and Price intermediate, and Carmel, Padre, Ne Plus Ultra, Butte, Peerless, and Thompson were the least susceptible cultivars for allowing X. fastidiosa to overwinter and cause disease the following year. Assessment of all trees in August 2004 indicated that trees inoculated in June and July 2002 had a significantly higher amount of ALS-infected branches than trees inoculated in other months. Butte, Carmel, Padre, and Thompson cultivars had no symptomatic branches, while X. fastidiosa infections persisted or colonized new branches in Sonora, Solano, Peerless, Price, Mission, and Ne Plus Ultra. Based on the 2004 assessment, Sonora was the most susceptible cultivar. Surveys of a diseased orchard in Chico, CA showed large differences in ALS incidence in four almond cultivars. Nonpareil and Peerless had significantly greater incidence of disease than Butte and Carmel over the 2 years surveyed. These data suggest that cultivar susceptibility and the time of X. fastidiosa infection are important factors in determining the persistence of ALS in almond trees.

A proteomic evaluation of Pyrenophora tritici-repentis, causal agent of tan spot of wheat, reveals major differences between virulent and avirulent isolates.

Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat. The fungus produces multiple host-specific toxins, including Ptr ToxB, a chlorosis-inducing protein encoded by the ToxB gene. A homolog of ToxB is also found in avirulent isolates of the fungus. In order to improve understanding of the role of this homolog and evaluate the general pathogenic ability of P. tritici-repentis, we compared the proteomes of avirulent race 4 and virulent race 5 isolates of the pathogen. Western blotting analysis revealed the presence of Ptr ToxB in spore germination and culture fluids of race 5 but not race 4. A comprehensive proteome-level comparison by 2-DE indicated 133 differentially abundant proteins in the secretome (29 proteins) and mycelium (104 proteins) of races 4 and 5, of which 63 were identified by MS/MS. A number of the proteins found to be up-regulated in race 5 have been implicated in microbial virulence in other pathosystems, and included the secreted enzymes alpha-mannosidase and exo-beta-1,3-glucanase, heat-shock and BiP proteins, and various metabolic enzymes. These proteome-level differences suggest a reduced general pathogenic ability in race 4 of P. tritici-repentis, irrespective of toxin production. Such differences may reflect an adaptation to a saprophytic habit.

Molecular Detection of Plasmodiophora brassicae, Causal Agent of Clubroot of Crucifers, in Plant and Soil.

Clubroot of crucifers, caused by Plasmodiophora brassicae, recently has been identified in canola (Brassica napus) fields in Alberta, Canada. An effective strategy for managing the disease is to avoid planting cruciferous crops in P. brassicae-infested soil, because the pathogen produces resting spores that can remain infectious for many years. A simple, one-step polymerase chain reaction (PCR) protocol was developed to detect the pathogen in plant and soil samples. The primers TC1F and TC1R, based on a P. brassicae partial 18S ribosomal RNA (rRNA) gene sequence from GenBank, yielded a 548-bp product in the optimized PCR. A second pair of primers, TC2F and TC2R, which amplified a fragment of the 18S and internal transcribed spacer (ITS) 1 regions of the rDNA repeat, also was tested and produced a 519-bp product. Neither set of primers amplified any DNA fragment from noninfected plant hosts, noninfested soil, or common soil fungi and bacteria tested in this study. Quantities of 100 fg or less of total P. brassicae DNA, or 1 × 103 resting spores per gram of soil, could be detected consistently using these primers and PCR protocol, corresponding to an index of disease of 11% or lower when the soil was bioassayed. The protocol also enabled detection of P. brassicae in symptomless root tissue 3 days after inoculation with the pathogen. Therefore, the PCR assay described in this study could provide a reliable diagnosis for routine detection of P. brassicae in plant and soil materials in a specific and rapid manner.

Influence of Ring Nematode Infestation and Calcium, Nitrogen, and Indoleacetic Acid Applications on Peach Susceptibility to Pseudomonas syringae pv. syringae.

ABSTRACT Two field experiments were conducted to study the effects of added nitrogen, calcium, and indoleacetic acid, in the presence or absence of ring nematodes (Mesocriconema xenoplax), on susceptibility of peach to bacterial canker. When noninfested soil was inoculated with ring nematodes, peach tree susceptibility to bacterial canker infection caused by Pseudomonas syringae pv. syringae was dramatically increased after a period of 2 years. However, no evidence was found that ring nematode infestation increased tree water stress or, in turn, altered plant calcium uptake. Soil fumigation with methyl bromide prior to planting in a commercial orchard significantly reduced both nematode populations and peach tree susceptibility to bacterial canker infection when compared with nonfumigated treatments. In both experiments, tree susceptibility, as measured by canker length following inoculation of stems with P. syringae pv. syringae, was negatively correlated with plant tissue nitrogen content and positively correlated with tissue calcium content. A principal components analysis showed that tissue nitrogen and calcium levels were negatively correlated, and that high-nitrogen, low-calcium tissues were less susceptible to bacterial canker than low-nitrogen, high-calcium tissues. These results indicate that the increased susceptibility of peach to P. syringae pv. syringae under nematode infestation conditions is mediated by both nutritional effects (primarily nitrogen) and nutritional-independent effects, but do not support previous reports of beneficial effects of calcium for reducing bacterial canker.

Interaction Between Nitrogen-Fertilized Peach Trees and Expression of syrB, a Gene Involved in Syringomycin Production in Pseudomonas syringae pv. syringae.

ABSTRACT The in vitro expression of the syrB gene that controls the synthesis of syringomycin, a non-host-specific phytotoxin produced by Pseudomonas syringae pv. syringae van Hall, was studied using aqueous extracts derived from bark tissues collected from nitrogen-fertilized and nonfertilized peach trees. Expression of the syrB gene was quantified as beta- galactosidase activity expressed by P. syringae pv. syringae B3AR-132 containing a syrB::lacZ fusion. Gene expression was significantly less in three of four paired comparisons using extracts derived from fertilized versus nonfertilized trees; however, canker lengths were significantly different in only one of four comparisons. Expression was negatively correlated with plant tissue nitrogen content and positively correlated with a plant carbon/nitrogen ratio. Bark tissue from ring nematodeinfested trees had significantly higher concentrations of total soluble phenolic compounds and carbon/nitrogen ratios than bark samples from trees without nematodes, and canker size was significantly greater in trees growing in ring nematode-infested soil compared with noninfested soil. Nitrogen fertilization significantly decreased the plant carbon/nitrogen ratio, which was positively correlated with the concentration of total soluble phenolic compounds. Canker size developing after bacterial inoculation was positively correlated with higher plant carbon/nitrogen ratios and total soluble phenolic compounds. These results support the hypothesis that one reason why nitrogen fertilization decreases host susceptibility to bacterial canker is by either reducing the amount of plant metabolites that can induce syrB gene expression, or producing or increasing the concentration of compounds that antagonize syrB inducing compounds.