Multilocus Sequence Typing of Strains of Bacterial Spot of Lettuce Collected in the United States

Studies on genetic diversity and recombination in bacterial pathogens are providing a better understanding of the mechanisms shaping bacterial diversity, which can affect disease control. Xanthomonas campestris pv. vitians, causal agent of bacterial leaf spot of lettuce, is a threat to the worldwide lettuce industry. We examined the genetic variation within a sample of 83 strains from California, Florida, and Ohio using multilocus sequence typing of six housekeeping genes, totaling 2.7 kb. Additionally, polymorphism in two virulence-related genes, hrpB2 and a putative glycosyl hydrolase, were examined. Based on housekeeping genes, we found three genetic groups of strains that were all able to induce the disease. These included strains collected from weeds and irrigation water that had haplotypes identical to strains from diseased lettuce. High linkage disequilibrium across the sequenced loci indicates that the pathogen is predominantly clonal but recombination has contributed to the observed sequence variation. Although there was significant genetic variation in X. campestris pv. vitians within and among sampled states, identical haplotypes were observed across all three states. This finding suggests that seedborne inoculum may contribute to the diversity of X. campestris pv. vitians in the United States. Knowledge of the genetic structure of the pathogen may be used for developing resistant lettuce varieties.

Prediction of Potato Tuber Damage by Root-Knot Nematodes using Quantitative DNA Assay of Soil

Root-knot nematodes (Meloidogyne fallax and M. hapla) cause significant reductions in potato yield by reducing tuber quality. Concentrations of M. fallax and M. hapla DNA in soil were determined by quantitative polymerase chain reaction following sampling at planting and harvest within 78 fields across 3 years in Australia. Meloidogyne spp. were also detected using a tomato bioassay. M. fallax was more prevalent than M. hapla and DNA concentrations of M. fallax in soil were significantly higher in samples collected at harvest compared with those at planting. In contrast, M. hapla DNA in soil did not significantly change from planting to harvest. Using receiver operating characteristic curve analysis, M. fallax DNA in soil at planting and harvest was a highly accurate predictor of tuber damage at harvest and galling on tomato. Prediction accuracy for tuber damage was highest for M. fallax DNA compared with M. hapla or M. fallax + M. hapla. Both Meloidogyne spp. were detected in the peel of asymptomatic certified seed. For M. fallax, the addition of seedborne inoculum did not improve tuber damage predictions. This suggested that soilborne M. fallax populations contributed most substantially to tuber damage. These findings highlight the utility of this approach for predicting risk of crop damage from nematodes. The use of this technique as a practical management tool is discussed.

Colonization of Spinach by Verticillium dahliae and Effects of Pathogen Localization on the Efficacy of Seed Treatments

Verticillium wilt on spinach (Spinacia oleracea) is caused by the soilborne fungus Verticillium dahliae. The pathogen is seedborne and transmission through seed is a major concern because of the dispersal of the pathogen to areas where fresh and processing spinach crops are grown in rotation with susceptible crops. Reduction in seedborne inoculum minimizes pathogen spread; therefore, knowledge of pathogen localization in seed is critical to develop methods to reduce seedborne inoculum. Spinach seedlings were inoculated with conidial suspensions of a green fluorescent protein-tagged strain of V. dahliae and colonization events were followed through seed production by confocal laser-scanning microscopy. Between 24 to 96 h postinoculation (PI), conidia germinated and formed hyphal colonies on root tips and in root elongation zones. Hyphae colonized root cortical tissues both intra and intercellularly by 2 weeks, and colonized the taproot xylem with abundant mycelia and conidia that led to vascular discoloration coincident with foliar symptom expression by 8 weeks PI. At 10 weeks PI, the xylem of the upper stem, inflorescence, and spinach seed parts, including the pericarp, seed coat, cotyledons, and radicle, had been colonized by the pathogen but not the perisperm (the diploid maternal tissue). Maximum concentration of the fungus was in the seed coat, the outermost layer of the vasculature. Infection of V. dahliae in spinach seed was systemic and transmissible to developing seedlings. Additional analyses indicated that fungicide and steam seed treatments reduced detectable levels of the pathogen but did not eliminate the pathogen from the seed. This information will assist in the development of seed treatments that will reduce the seedborne inoculum transmission to crop production fields.

Epidemics of Ray Blight on Pyrethrum are Linked to Seed Contamination and Overwintering Inoculum of Phoma ligulicola var. inoxydabilis

Ray blight, caused by Phoma ligulicola var. inoxydabilis, is the most damaging disease of pyrethrum (Tanacetum cinerariifolium) in Australia. Data collected from 72 plots in commercial pyrethrum fields since 2001 to 2009 revealed substantial annual variations in isolation frequency of the pathogen during semidormancy of the crop in autumn and winter. Isolation frequency of the pathogen during this time and subsequent outbreaks of ray blight in spring were similar across the eight production regions where sampling was conducted, and isolation frequency of the pathogen was linearly correlated (r = 0.88; P < 0.0001) with subsequent defoliation severity when plants commenced growth in spring. Isolation frequency and defoliation severity also were correlated with the incidence of seed infested with P. ligulicola var. inoxydabilis (r = 0.71 and 0.44, respectively; P < 0.0001 in both correlations). Highly accurate risk algorithms for the occurrence of severe epidemics of ray blight were constructed using logistic regression. A model based solely on isolation frequency of the pathogen over autumn and winter correctly predicted epidemic development in 92% of fields. Another model utilizing the incidence of infested seed and rain–temperature interactions in early autumn (austral March and April) and late winter (austral June and July) had similar predictive ability (92% accuracy). Path analysis modeling was used to disentangle interrelationships among the explanatory variables used in the second logistic regression model. The analysis indicated that seedborne inoculum of P. ligulicola var. inoxydabilis contributes indirectly to ray blight defoliation severity through directly increasing overwintering frequency of the pathogen. Autumn and fall weather variables were modeled to have indirect effects on defoliation severity through increasing overwintering success of the pathogen but also direct effects on defoliation severity. Collectively, the analyses point to several critical stages in the disease cycle that can be targeted to minimize the probability of regional epidemics of ray blight in this perennial pathosystem.

The influence of Tilletia spp. inoculum source and environmental conditions on the frequency of infected wheat spikes

The influence of inoculum source on the incidence of common bunt, caused by fungi from the genus Tilletia, was estimated based on the frequency of bunt infected wheat spikes in our agroecological conditions. The cultivar Novosadska rana 5 was sown in a random split plot design with four replicates at Rimski Sancevi on three sawing dates in 1999/2000 and 2000/2001. The following variables were evaluated: I - control, II - soilborne inoculum (4 g teliospores/1 l soil), III - seedborne inoculum (2 g teliospores/1 kg seeds), IV - seedborne inoculum + soilborne inoculum (4 g teliospores/1 l soil + 2 g teliospores/1 kg seeds). Correlation and regression analysis were used to evaluate the effect of temperature and precipitation on the frequency of infected spikes. The frequency of bunt infected spikes depended on the source of Tilletia spp. inoculum, and difference in infection frequencies between variables II and III, as well as III and IV, were determined for the assessed infection parameters. When teliospores are the only source of inoculum in soil, 60 days after sawing (r>+0.52) is a critical period in which temperature influences the development of infection. The highest number of plants was infected in the first, while less were infected in the second ten days (decade) after sawing (r>0.41), when temperature was the optimal 5.0-6.0?C. The initial 60 days after sawing were also critical for disease development when teliospores on seeds were the only source of inoculum (r>+0.50). The highest number of plants was infected in the third and fewer in the fifth decade after sawing (r>0.41), when temperature was the optimal 5.0-6.0?C. When infection was caused by teliospores on seeds and in soil, the critical period lasted 120 days after sawing (r>0.42), with a maximum frequency of infection found at the optimal temperatures for the period of 4.0- 5.0?C.

Downy Mildew Caused by Peronospora valerianellae on Corn-Salad (Valerianella locusta) in California

Corn-salad or lamb's lettuce (Valerianella locusta) is a specialty leafy green, annual vegetable grown commercially in California as a fresh market commodity used in salads. In the spring (February through April) of 2008, fields in coastal California (Monterey County) showed symptoms and signs of a downy mildew. Initial symptoms consisted of irregularly shaped, light green patches observed on adaxial leaf surfaces; these lesions later turned yellow. As disease progressed, patches became brown and necrotic. The abaxial sides of affected leaves were heavily colonized by an extensive, purplish growth characteristic of a downy mildew pathogen. Symptomatic leaves were unmarketable and diseased portions of plantings were not harvested. Crop loss was estimated to be between 1 and 15% for any one particular planting. The purple growth consisted of hyaline, branched conidiophores that emerged from stomata and had branches ending in slender, curved branchlets that did not have swollen tips. Conidia were slightly brown, ovoid, mostly nonpapillate, and measured 23.7 to 29.4 μm long × 15.9 to 22.2 μm wide. When necrotic leaf tissue was examined microscopically, thick-walled, yellow brown oospores were abundant within leaf tissues. Oospores measured 33.2 to 36.9 μm in diameter. On the basis of disease symptoms and morphology of the organism, the pathogen was identified as Peronospora valerianellae (1,3). To prove pathogenicity on corn-salad, 24 3-week-old seedlings were sprayed until runoff, using a hand-held spray bottle, with a conidial suspension (1.0 × 104 sporangia/ml), incubated for 24 h in a dew chamber (18 to 20°C), and then maintained in a greenhouse (22 to 24°C). Inoculum was obtained from one section of an affected commercial field. After 10 to 12 days, symptoms and signs of downy mildew occurred on inoculated plants, and the pathogen morphology matched that of the pathogen originally observed. Twelve untreated control plants did not develop downy mildew. To test for seedborne inoculum, 10 g of seed of each of two corn-salad cultivars were added to 100 ml of a dilute (0.05%) Tween 20 solution. The suspension was agitated for 3 h, filtered through cheesecloth to remove seed, and centrifuged. The resulting pellet was examined microscopically and found to contain low numbers of oospores that were similar in morphology to those observed in necrotic leaf lesions. To my knowledge, this is the first report of downy mildew caused by P. valerianellae on corn-salad in California and the United States. The pathogen has been reported on corn-salad in England, France, Germany, Scotland, and the Ukraine (1–3). Seedborne oospores of downy mildew have been reported on corn-salad seed tested in France (1). References: (1) R. Champion and H. Mecheneau. Seed Sci. Technol. 7:259, 1979. (2) D. F. Farr et al. Fungal Databases. Systematic Mycology and Microbiology. Online publication. ARS, USDA, 2008. (3) G. Pietrek and V. Zinkernagel. Advances in Downy Mildew Research. Kluwer Academic Publishers, Dordrecht, the Netherlands, 2002.

Relative Contribution of Seed-Transmitted Inoculum to Foliar Populations of Phaeosphaeria nodorum

A marked-isolate, release-recapture experiment was conducted to assess the relative contributions of seed-transmitted (released isolates) versus all other inocula to foliar and grain populations of Phaeosphaeria nodorum in winter wheat rotated with nonsusceptible crops in New York and Georgia, United States. Seed infected with two distinct groups of marked isolates of P. nodorum containing rare alleles (identified by amplified fragment length polymorphisms [AFLPs]) and balanced for mating type were planted in experimental field plots in two locations in each state. Recapture was done by isolating P. nodorum from leaves showing necrotic lesions at spring tillering and flowering stages, and mature grains from spikes showing glume blotch. Isolates from these samples were genotyped by AFLPs and categorized as released or nonreleased to infer sources of inoculum. Both infected seed and other sources of the pathogen contributed significant primary inocula to populations recovered from leaves and harvested grain. Seed-transmitted genotypes accounted for a total of 57% of all isolates recovered from inoculated plots, with a range of 15 to 90% of the populations of P. nodorum collected over the season in individual, inoculated plots at the four locations. Plants in the noninoculated control plots also became diseased and 95% or more of the isolates recovered from these plots were nonreleased genotypes. Although other potential sources of P. nodorum within and adjacent to experimental plots were not ruled out, nonreleased genotypes likely were derived from immigrant ascospores potentially from sources at a considerable distance from the plots. Our results suggest that, although reduction of seedborne inoculum of P. nodorum may delay foliar epidemics, this strategy by itself is unlikely to result in high levels of control in eastern North America because of the additional contribution from alternative sources of inoculum.

Influence of internal seedborne Fusarium semitectum on cotton seedlings

Fusarium semitectum was found to be the major seed colonizing fungus in the commercial acid delinted cotton (Gossypium hirsutum) seed lots. There was no correlation, however, between its incidence and seedling emergence and disease symptoms on the emerged seedlings in autoclaved sand. Inoculation technique simulating internally seedborne nature of the fungus showed that the observed non-correlation might be related to the threshold level of seed coat colonization. The internally seedborne inoculum besides reducing seedling emergence, incited an array of symptoms on the emerged seedlings, which ranged from negative geotropism, leaf tearing, collar rot leading to dry root rot and seedling mortality. The dry root rot continued to develop on the plants surviving the seedling phase. The collar rot symptoms can be confused with those caused by Rhizoctonia solani.