RESUMEN

Host diversification methods such as within-field mixtures (or field mosaics, depending on the spatial scale considered) are promising methods for agroecological plant disease control. We explore disease spread in host mixtures (or field mosaics) composed of two host genotypes (susceptible and resistant). The pathogen population is composed of two genotypes (wild-type and resistance-breaking). We show that for intermediate fractions of resistant hosts, the spatial spread of the disease may be split into two successive fronts. The first front is led by the wild-type pathogen and the disease spreads faster, but at a lower prevalence, than in a resistant pure stand (or landscape). The second front is led by the resistance-breaking type, which spreads slower than in a pure resistant stand (or landscape). The wild-type and the resistance-breaking genotypes coexist behind the invasion fronts, resulting in the same prevalence as in a resistant pure stand. This study shows that host diversification methods may have a twofold effect on pathogen spread compared to a resistant pure stand (or landscape): on the one hand, they accelerate disease spread, and on the other hand they slow down the spread of the resistance-breaking genotype. This work contributes to a better understanding of the multiple effects underlying the performance of host diversification methods in agroecology.

Asunto(s)

RESUMEN

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

Asunto(s)

RESUMEN

KEY MESSAGE: Nitrogen levels can modulate the effectiveness of clubroot resistance in an isolate- and host-specific manner. While the same QTL were detected under high and low nitrogen, their effects were altered. Clubroot, caused by Plasmodiophora brassicae, is one of the most damaging diseases of oilseed rape and is known to be affected by nitrogen fertilization. However, the genetic factors involved in clubroot resistance have not been characterized under nitrogen-limiting conditions. This study aimed to assess the variability of clubroot resistance under different nitrogen levels and to characterize the impact of nitrogen supply on genetic resistance factors. Linkage analyses and a genome-wide association study were conducted to detect QTL for clubroot resistance and evaluate their sensitivity to nitrogen. The clubroot response of a set of 92 diverse oilseed rape accessions and 108 lines derived from a cross between 'Darmor-bzh' (resistant) and 'Yudal' (susceptible) was studied in the greenhouse under high- and low-nitrogen conditions, following inoculation with the P. brassicae isolates eH and K92-16. Resistance to each isolate was controlled by a major QTL and a few small-effects QTL. While the same QTL were detected under both high and low nitrogen, their effects were altered. Clubroot resistance to isolate eH, but not K92-16, was greater under a low-N supply versus a high-N supply. New sources of resistance were found among the oilseed rape accessions under both low and high-N conditions. The results are discussed relative to the literature and from a crop improvement perspective.

Asunto(s)

RESUMEN

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is an economically important disease of canola (Brassica napus) commonly managed by routine application of fungicides. Petal infestation has been demonstrated to be an important stage of the disease cycle in canola and has been the focus of previously developed Sclerotinia stem rot risk assessment methods. Quantitative polymerase chain reaction (qPCR) analysis can provide a more rapid and accurate assessment of petal infestation levels. Primers and a hydrolysis probe were designed to amplify a 70-bp region of an S. sclerotiorum-specific gene, SS1G_00263. A hydrolysis probe-based qPCR assay was developed that had a detection limit of 8.0 × 10-4 ng of S. sclerotiorum DNA and only amplified S. sclerotiorum DNA. Evaluation of petals collected at five sampling points in each of 10 commercial canola fields on each of two sampling dates (corresponding to 20 to 30% bloom and 40 to 50% bloom) revealed S. sclerotiorum DNA infestation levels of 0 to 3.3 × 10-1 ng/petal. This qPCR assay can be used to reliably quantify petal infestation and, with further research, has the potential to serve as the basis for a Sclerotinia stem rot risk assessment tool or as a means to study Sclerotinia stem rot epidemiology.

RESUMEN

Clubroot caused by Plasmodiophora brassicae is an important disease of crucifers worldwide. Isolates of the pathogen can be classified into pathotypes according to their pathogenicity on differential hosts. In this study, the presence or absence of all database-available nonhousekeeping P. brassicae genes (118 in total) were assessed by polymerase chain reaction (PCR) analysis in isolates belonging to five P. brassicae pathotypes (2, 3, 5, 6, and 8 according to Williams' differential set). One gene, designated Cr811, was present exclusively in the isolate of pathotype 5. This was further confirmed by dot blot hybridization and by PCR using alternative DNA preparations and primers. Reverse transcription quantitative PCR analysis indicated that in planta expression of Cr811 was up-regulated during canola infection, especially in the stage of secondary plasmodia. Primers specific to Cr811 could distinguish a field isolate of P. brassicae belonging to pathotype 5 from two other field isolates representing pathotypes 3 and 8. These findings suggest that Cr811 is a gene that is potentially involved in clubroot pathogenesis and that it also might serve as a molecular marker for differentiation of pathotype 5 from other pathotypes.

Asunto(s)

RESUMEN

Root rot is a major disease of dry bean and can cause significant yield reductions due to weakened root systems and poor plant stands. An in-depth study on root rot pathogen identification was conducted in 2011 in three commercial dry bean fields from the major production areas in Manitoba. Ten plants, sampled at each of four random sites within each field, were rated for disease severity. Twenty roots were processed for pathogen isolation and identification in the laboratory. Roots were cut into eight sections (~1 cm) and surface-sterilized in a laminar flow bench. Four root sections were placed on potato dextrose agar plates amended with 0.02% streptomycin sulfate (PDA-Strep) and four root sections were placed on peptone-pentachloronitrobenzene agar amended with 0.1% streptomycin sulfate and 0.012% neomycin sulfate. Afterward, 960 monosporic cultures were obtained representing 320 single spore isolates of potential root rot pathogens per commercial field. Common monosporic cultures from each field were subcultured on PDA-Strep and Spezieller Nährstoffarmer Agar (SNA) media. Based on morphological characteristics, 74 isolates were identified as Fusarium cuneirostrum (1). Colonies grew slowly on PDA-Strep with undulated margins, radial cream-grey mycelia, and conidia pustules with a cream-greyish pigmentation. Sporodochial conidia were falcate, mostly 5-septate, with a wedge shape and slightly protruding basal foot cell (56.3 to 71.8 × 4.6 to 6.2 µm on average). Species identity was confirmed for two isolates by sequencing the translation elongation factor 1 alpha (EF1-α) gene (2), the internal transcribed spacer (ITS) region (4), and the ribosomal intergenic spacer (IGS) (3) (GenBank Accession Nos. KF530848, KF530849, and KF025648 to 51). Sequence homology was compared using BLAST analysis and the FUSARIUM-ID database. The F. cuneirostrum isolates were deposited at the Canadian Collection of Fungal Cultures (DAOM 242540 and 242541). Pathogenicity screenings of two isolates was performed using sterilized seed of navy bean cv. Envoy. Seeds were germinated on moist filter paper for 3 days at 25°C and then inoculated by immersion in a prepared conidial suspension (2.5 × 105 conidia/ml) for 5 min. Seeds of the controls were immersed in sterile water. After inoculation, the germinated seeds were planted in 10-cm diameter pots, filled with sterile soilless mix (Sunshine #3). In the greenhouse, the experiment was arranged as a completely randomized design with three replicates with four germinated seeds per isolate, and was repeated twice. Disease assessment was performed 14 days after inoculation. Infected plants displayed dark brown lesions on the hypocotyl and primary root with a disease severity of 4 scored on a 0 to 5 scale. Fusarium cuneirostrum was re-isolated from roots of symptomatic plants. To our knowledge, this is the first report of F. cuneirostrum causing root rot of dry bean in Canada. It has been previously isolated from mung bean (Vigna radiata) in Ontario (1). References: (1) T. Aoki et al. Mycoscience. 46:162, 2005. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (4) H. Wang et al. J. Clin. Microbiol. 49:1890, 2011. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

RESUMEN

A protocol for genetic transformation of the obligate parasite Plasmodiophora brassicae, causal agent of clubroot of crucifers, was developed. In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted. In two independent experiments, germinating resting spores of P. brassicae were transformed by two fungal expression vectors containing either a green fluorescent protein (gfp) gene or a hygromycin resistance (hph) gene. Putative transformants were produced from both transformations, with ≈50% of the obtained galls containing resting spores from which transforming DNA could be detected by polymerase chain reaction (PCR). PCR, quantitative PCR (qPCR), and genome walking conducted on selected transformants indicated that the transforming DNA was intergraded into the P. brassicae genome. Transcript of hph but not gfp was detected by reverse-transcription qPCR from selected transformants. From all galls produced by transformants, no GFP activity could be identified. Verified transformants were inoculated on canola and new galls were generated. PCR and qPCR analyses based on these galls indicated that transforming DNA was still resident in P. brassicae. This is the first report on genetic transformation of P. brassicae. The information and data generated from this study will facilitate research in multiple areas of the clubroot pathosystem.

Asunto(s)

RESUMEN

Plasmodiophora brassicae, the cause of clubroot of crucifers, is an increasingly important pathogen of canola (Brassica napus) in Alberta, Canada. In response, clubroot-resistant canola genotypes are being deployed to help reduce yield losses. Two experiments were conducted to examine the effect on P. brassicae virulence of repeated exposure of a population and single-spore isolate of the pathogen to the same host. The first experiment examined changes in the index of disease over five cycles of infection on seven Brassica hosts (European Clubroot Differential [ECD] 02, ECD 04, ECD 05, ECD 15, '45H26', '45H29', and 08N823R). The second experiment tested the virulence of five cycled populations ('45H29', 08N823R, ECD 05, and ECD 15) and three cycled single-spore isolates ('45H29', 08N823R, and ECD05) on four resistant canola genotypes ('73-77', '73-67', VT-SD-09, and '9558C'). The results from these experiments clearly demonstrate the ability of both single-spore isolates and populations of P. brassicae to rapidly erode the resistance present in the two canola genotypes, '45H29' and 08N823R. Although the index of disease increased on these two genotypes, the four resistant canola genotypes remained resistant to all the cycled populations and single-spore isolates in the second experiment. These results underscore the importance of crop rotation in the management of clubroot in Alberta.

RESUMEN

Internal fruit rot, caused by Fusarium lactis, is an important disease of sweet pepper (Capsicum annuum) in Canadian greenhouses. Production of the mycotoxins fumonisin B1 (FB1), moniliformin (MON) and beauvericin (BEA) by F. lactis (17 isolates) and the related species F. proliferatum (three isolates) and F. verticillioides (one isolate), which are also associated with internal fruit rot, was evaluated on rice medium. All 21 isolates examined were found to produce BEA, at concentrations ranging from 13.28 to 1674.60 ppm, while 13 of 17 F. lactis isolates and two of three F. proliferatum isolates produced MON (0.23 to 181.85 ppm). Only one isolate of F. lactis produced detectable levels of FB1 in culture, whereas all three F. proliferatum isolates and the F. verticilloides isolate produced this mycotoxin (0.28 to 314 ppm). Production of FB1, MON and BEA was also evaluated in inoculated pepper fruits showing mild or severe symptoms of infection. FB1 could be detected in both lightly and heavily diseased fruit tissue after inoculation with F. lactis, F. proliferatum or F. verticilloides, at concentrations ranging from 0.61 to 8.04 ppm. BEA was also detected in lightly and heavily diseased fruit tissue inoculated with F. lactis, as well as in heavily diseased tissue inoculated with F. proliferatum (3.00 to 19.43 ppm), but not in tissue inoculated with F. verticilloides. MON was detected in all tissues inoculated with F. proliferatum or F. verticilloides, and in heavily diseased tissue inoculated with F. lactis (0.03 to 0.27 ppm). The three mycotoxins were also found in naturally infected sweet pepper fruits exhibiting symptoms of internal fruit rot and collected from a commercial greenhouse. The production of MON, BEA and FB1 alone or in combination by isolates of F. lactis suggests that development of internal fruit rot of sweet pepper is an important food safety concern, and that every effort should be made to cull infected fruit before it makes it to market.

Asunto(s)

RESUMEN

Clubroot of crucifers, caused by Plasmodiophora brassicae, is emerging as an important disease of canola (Brassica napus) in Alberta, Canada. Populations of the pathogen often consist of a mixture of different pathotypes. Therefore, a simple and efficient method to isolate single resting spores of P. brassicae was developed, based on serial dilution of spore suspensions. The virulence of 24 single-spore isolates, representing five populations of the pathogen from Alberta, Ontario, and British Columbia, was characterized on the differentials of Williams and Somé et al. Symptoms were rated 6 weeks after inoculation and Fisher's least significant difference (P < 0.05) was used to differentiate resistant from susceptible host reactions. The pathotype composition of P. brassicae in Canada appeared more diverse when single-spore isolates were examined rather than populations of the pathogen. In Alberta, at least three and possibly four pathotypes were identified among the 14 isolates tested, whereas a maximum of only two pathotypes had been reported previously when populations of the pathogen were examined. Pathotype 3 or P2, as classified on the differentials of Williams and Somé et al., respectively, was found to be predominant in the province. The occurrence of other pathotypes at lower frequencies suggests that caution should be used in any breeding strategy, because rare pathotypes of P. brassicae may quickly become predominant if susceptible host genotypes are continuously grown.

RESUMEN

ABSTRACT Pyrenophora tritici-repentis, causal agent of tan spot, induces necrosis and chlorosis in its wheat host. The tan spot system conforms to the toxin model and three host-specific toxins have been identified (Ptr ToxA, Ptr ToxB, and putative Ptr ToxC). Processing of a collection of isolates, obtained in the Fertile Crescent and Caucasus regions, yielded two new virulence patterns. Isolate Az35-5 combined the virulences of races 2 and 5 and was classified in the new race 7. Isolates TS93-71B and TS93-71F had a virulence pattern that combined those of races 2, 3, and 5 and were grouped in the new race 8. Southern analysis revealed that all three isolates possessed copies of the ToxA and ToxB genes, the first time the genes were found in a common background. The production of Ptr ToxA and Ptr ToxB by the isolates was confirmed by western blotting. Virulence patterns suggested that TS93-71B and TS93-71F may also produce Ptr ToxC, even though it was not present at detectable levels in culture filtrates. The identification of races 7 and 8 complete the theoretical maximum number of races that can be differentiated by three loci in the host (2(3) = 8), assuming a one-to-one relationship. It appears that the wheat/P. tritici-repentis system is a mirror image of the classical gene-for-gene relationship.