Infection, Survival, and Growth of Clavibacter nebraskensis on Crop, Weed, and Prairie Plant Species.

Clavibacter nebraskensis is the causal agent of Goss's leaf blight and wilt, an important disease of maize in the United States and Canada. The epidemiology and ecology of this bacterial pathogen are poorly understood. Infested maize residue is often considered to be the primary source of inoculum for maize; however, the potential for many other plant species to be infected and serve as inoculum sources is unknown. The goal of this study was to determine if C. nebraskensis could infect, survive, and grow on common weed, crop, and grass species. Seedling leaves of 18 plant species that grow in maize production areas in the United States were inoculated with this pathogen in a controlled environment and in the field. Lesion development, bacterial streaming, and pathogen population size on leaves were then determined and used as criteria to evaluate host-pathogen interactions. Woolly cupgrass (Eriochloa villosa) and the native prairie grasses big bluestem (Andropogon gerardii) and little bluestem (Schizachyrium scoparium) developed lesions and bacterial streaming, identifying them as hosts and susceptible to infection. To our knowledge, this is the first report of these grass species being hosts of C. nebraskensis. Ten other grass species, including wheat and oats, were identified as potential sustaining hosts that maintained epiphytic or endophytic pathogen populations >106 colony forming units per leaf sample but displayed no evidence of infection. Five broadleaf species tested were nonhosts based on the three criteria. This study suggests that multiple plant species support infection and growth of C. nebraskensis and further elucidates the ecology of this pathogen and the epidemiology of Goss's wilt.

Fungal endophyte diversity in soybean.

AIM: To determine the identity and diversity of endophytes in soybean plants using culture-dependent (CD) and culture-independent (CI) methods. METHODS AND RESULTS: Stem samples were collected from three field-grown soybean cultivars grown to a reproductive stage in Minnesota, USA. Samples were surface disinfested, and CD and CI methods were used to assess the endophytes. For the CD method, fungi were isolated and grouped based on colony morphology, and the rDNA ITS region was sequenced to identify the cultures. The most frequently isolated genera were Cladosporium (36%), Alternaria (13%), Diaporthe (9%) and Epicoccum (9%). For the CI method, DNA was extracted from the stems, and the ITS region was amplified, cloned and sequenced for identification. The most prevalent genus detected using CI method was Cladosporium (85%). CONCLUSIONS: Soybean contains a diverse array of endophytic fungi that were identified in this study. The CD method detected greater endophyte diversity (H' = 2·12) than the CI method (H' = 0·66). SIGNIFICANCE AND IMPACT OF THE STUDY: The results improve our understanding of the identity and diversity of endophytic fungi that likely have different kinds of interactions with soybean plants. The results suggest that CD and CI methods should be used to study endophytes in soybean and perhaps other annual crop plants.

Symptomatic and Asymptomatic Host Range of Fusarium virguliforme, the Causal Agent of Soybean Sudden Death Syndrome.

Sudden death syndrome, caused by Fusarium virguliforme, is an important disease of soybean in the United States. Fifteen species of crops, weeds, or prairie plants were evaluated for their potential as hosts of F. virguliforme. Root and foliar symptoms and plant biomass were assessed following greenhouse inoculation studies. Root colonization of F. virguliforme was determined with isolations and with polymerase chain reaction assays. Soybean, alfalfa, pinto and navy bean, white and red clover, pea, and Canadian milk vetch developed root necrosis. Soybean, alfalfa, and red clover also developed foliar symptoms following inoculation. Sugar beet and canola did not develop symptoms but had significant reductions in biomass, suggesting that they are also hosts of F. virguliforme. Corn, wheat, ryegrass, pigweed, and lambsquarters did not develop symptoms. However, these species appeared to be asymptomatic hosts because quantities of pathogen DNA detected in inoculated roots were similar to quantities detected in inoculated soybean roots. These results suggest that the number and diversity of hosts for F. virguliforme are greater than previously reported. The likely broad host range limits the efficacy of crop rotation and indicates that crops other than soybean can be damaged by F. virguliforme and maintain or increase inoculum in soil.

First Report of Clonostachys rosea Causing Root Rot of Soybean in the United States.

Multiple fungal species have been associated with root rot of soybean (Glycine max) in the United States, but root rot in Minnesota (MN) also occurs in plants not known to be infected with previously reported pathogens (1). Soybean plants that lacked foliar symptoms, but exhibited taproot and lateral root necrosis were observed in 15 fields from nine counties in MN during 2007 and 2008. Plants were arbitrarily dug up at the R3 growth stage in July as part of a root rot study. Roots were washed, surface disinfested in 0.5% NaOCl for 3 min, rinsed in deionized water, dried, and embedded in potato dextrose agar (PDA). Thirty isolates with morphological characteristics consistent with those of Clonostachys rosea were recovered in total from necrotic lesions on different plants from all fields (3). For further morphological characterization, cultures were grown on PDA for 1 week at 24°C in the dark. Colonies were 39 to 46 mm in diameter, yellowish-white, and the surface was felty to tomentose with thick aerial hyphae. Primary conidiophores were Verticillium-like with two to three levels. The stipe length measured 65 to 105 µm and the base width was 5 µm. Primary conidia were smooth, hyaline, slightly curved, with an average length and width of 7 to 9 × 2.6 to 3 µm. Secondary conidiophores were penicillate with two or three whorls of phialides. The stipe length measured 50 to 75 µm, base width was 5 µm, and penicillus height was 25 to 35 µm. Secondary conidia were 5 to 6 × 2.5 µm. Perithecia were not produced. The identity of isolates was confirmed by sequencing the internal transcribed spacer (ITS) locus using the primers ITS1F/ITS4. BLAST analysis of the sequences in the NCBI database resulted in a 99.8 to 100% match for both C. rosea and its teleomorph Bionectria ochroleuca (e.g., HM751081, GU256766). Each isolate was tested for pathogenicity on soybean by initially growing it on sterile sorghum grain for 2 weeks at 23°C. Sterile sorghum was used for control plants. Seeds of soybean 'AG2107' were planted in 11.4-cm square pots containing pasteurized potting mix and a 25-cm3 layer of infested or sterile sorghum placed ~1 cm below the seeds. Two replicate pots containing four plants each were used per treatment and the experiment was repeated once. Root rot was assessed 28 days after planting in a greenhouse at 23°C day and 18°C night with a 14-h photoperiod. Twenty-eight of 30 C. rosea isolates caused taproot necrosis on inoculated plants in both experiments, whereas control roots did not exhibit necrosis. Approximately 6% of inoculated plants also developed interveinal chlorosis and marginal necrosis on trifoliates. Isolations were attempted from roots of all plants, and the isolates recovered from inoculated plants were identified as C. rosea based on morphology and ITS sequences. This fungus was not isolated from control plants. C. rosea was also isolated from petioles of symptomatic trifoliates, indicating systemic colonization of the plants. To our knowledge, this is the first report of C. rosea causing root rot of soybean and systemically colonizing soybean. This fungus may have been previously isolated from asymptomatic soybean plants and identified as Gliocladium roseum (2). The impact of this fungus on soybean production is unknown. References: (1) G. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathological Society, St. Paul, MN, 1999. (2) J. D. Mueller and J. B. Sinclair. Trans. Brit. Mycol. Soc. 86:677, 1986. (3) H.-J. Schroers et al. Mycologia 91:365, 1999.

Meta-analysis of yield response of hybrid field corn to foliar fungicides in the U.S. Corn Belt.

The use of foliar fungicides on field corn has increased greatly over the past 5 years in the United States in an attempt to increase yields, despite limited evidence that use of the fungicides is consistently profitable. To assess the value of using fungicides in grain corn production, random-effects meta-analyses were performed on results from foliar fungicide experiments conducted during 2002 to 2009 in 14 states across the United States to determine the mean yield response to the fungicides azoxystrobin, pyraclostrobin, propiconazole + trifloxystrobin, and propiconazole + azoxystrobin. For all fungicides, the yield difference between treated and nontreated plots was highly variable among studies. All four fungicides resulted in a significant mean yield increase relative to the nontreated plots (P < 0.05). Mean yield difference was highest for propiconazole + trifloxystrobin (390 kg/ha), followed by propiconazole + azoxystrobin (331 kg/ha) and pyraclostrobin (256 kg/ha), and lowest for azoxystrobin (230 kg/ha). Baseline yield (mean yield in the nontreated plots) had a significant effect on yield for propiconazole + azoxystrobin (P < 0.05), whereas baseline foliar disease severity (mean severity in the nontreated plots) significantly affected the yield response to pyraclostrobin, propiconazole + trifloxystrobin, and propiconazole + azoxystrobin but not to azoxystrobin. Mean yield difference was generally higher in the lowest yield and higher disease severity categories than in the highest yield and lower disease categories. The probability of failing to recover the fungicide application cost (p(loss)) also was estimated for a range of grain corn prices and application costs. At the 10-year average corn grain price of $0.12/kg ($2.97/bushel) and application costs of $40 to 95/ha, p(loss) for disease severity <5% was 0.55 to 0.98 for pyraclostrobin, 0.62 to 0.93 for propiconazole + trifloxystrobin, 0.58 to 0.89 for propiconazole + azoxystrobin, and 0.91 to 0.99 for azoxystrobin. When disease severity was >5%, the corresponding probabilities were 0.36 to 95, 0.25 to 0.69, 0.25 to 0.64, and 0.37 to 0.98 for the four fungicides. In conclusion, the high p(loss) values found in most scenarios suggest that the use of these foliar fungicides is unlikely to be profitable when foliar disease severity is low and yield expectation is high.

First Report of Fusarium redolens Causing Root Rot of Soybean in Minnesota.

Multiple Fusarium species have been found in association with soybean (Glycine max) plants exhibiting root rot in the United States (3). Soybean plants that lacked apparent foliar symptoms, but exhibited 2- to 5-mm brown, necrotic taproot lesions and lateral root necrosis were observed in Minnesota in one field each in Marshall and Otter Tail counties in July of 2007, as well as in one field in Marshall County in July of 2008. Sampling was conducted as part of a study investigating root rot in major soybean-production areas of Minnesota. Plants were arbitrarily dug up at the R3 growth stage. Root systems were washed, surface disinfested in 0.5% NaOCl for 3 min, rinsed in deionized water, and dried. Fusarium isolates were recovered from root sections with necrotic lesions embedded in modified Nash-Snyder medium (1). One resulting Fusarium colony from one plant per county was transferred to half-strength acidified potato dextrose agar (PDA) and carnation leaf agar (CLA) to examine morphological characteristics (4). Culture morphology on PDA consisted of flat mycelium with sparse white aerial mycelium. On CLA, thick-walled macroconidia with a hooked apical cell and a foot-shaped basal cell were produced in cream-colored sporodochia. Macroconidia ranged from 32.5 to 45.0 µm long. Microconidia were oval to cylindrical with 0 to 1 septa, ranged from 7.5 to 11.25 µm long, and were produced on monophialides. Chlamydospores were produced abundantly in chains that were terminal and intercalary in the hyphae of 4-week-old cultures. Morphological characteristics of the three isolates were consistent with descriptions of F. redolens (2,4). The identity of each isolate was confirmed by sequencing the translation elongation factor 1-α (TEF) locus (4). BLAST analysis of the TEF sequences from each isolate against the FUSARIUM-ID database resulted in a 100% match for 17 accessions of F. redolens (e.g., FD 01103, FD 01369). Each F. redolens isolate was tested for pathogenicity on soybean. Sterile sorghum grain was infested with each isolate and incubated for 2 weeks. Sterile sorghum was used for control plants. Soybean seeds of cv. AG2107 were planted in 11.4-cm pots ~1 cm above a 25-cm3 layer of infested sorghum or sterile sorghum. Two replicate pots containing four plants each were used per treatment and the experiment was repeated once. Root rot was assessed 28 days after planting. Each F. redolens isolate consistently caused taproot necrosis on inoculated plants, whereas control plants did not exhibit root necrosis. Isolations were made from roots of inoculated and control plants and the isolates recovered from inoculated plants were identified as F. redolens based on morphological characteristics and TEF sequences. Fusarium species were not isolated from control plants. To our knowledge, this is the first report of F. redolens causing root rot of soybean; however, it is possible F. redolens has been found previously and misidentified as F. oxysporum (2,4). Results from inoculations suggest that F. redolens may be an important root rot pathogen in Minnesota soybean fields. References: (1) J. C. Bienapfl et al. Acta Hortic. 668:123, 2004. (2) C. Booth and J. M. Waterston. No. 27 in: CMI Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, England, 1964. (3) G. L. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathological Society, St. Paul, MN, 1999. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

Soybean Rust Caused by Phakopsora pachyrhizi Detected in the State of Campeche on the Yucatan Peninsula, Mexico.

Soybean rust caused by Phakopsora pachyrhizi Syd. & P. Syd is a destructive foliar disease of soybean (Glycine max L), which was first confirmed in North America in Louisiana during 2004 (4). Soybean rust (SBR) has also been reported late in the growing season as far north as Illinois, Indiana, and Iowa. SBR was first confirmed in Mexico in 2005 in the state of San Luis Potosi on soybean (3) and subsequently reported in the states of Tamaulipas, Veracruz, and the southwestern coast of Chiapas. Symptoms of SBR were observed on leaves of multiple, nearly mature soybean plants near the city of Campeche (19.72796°N, 90.0771°W) on the Gulf Coast of the Yucatan Peninsula during November 2008. Angular and irregular chlorotic lesions on leaves contained necrotic spots and pale brown, erumpent, cone-like uredinia with a central opening. Ellipsoid to obovoid, echinulate, light tan urediniospores (10 to 13 × 16 to 18 µm) were observed microscopically. DNA was extracted from leaf tissue containing uredinia and from asymptomatic tissue with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA). P. pachyrhizi was confirmed in the symptomatic leaves by a PCR assay with Ppm1/Ppa2 primers, but not from the asymptomatic leaves (1). Subsequently, the DNA extracted from symptomatic and asymptomatic leaf tissues was tested again in another laboratory by a specific quantitative PCR assay (1), and positive results for the presence of soybean rust were obtained only from the symptomatic tissue. As a final confirmatory step, amplified DNA from the PCR assay was sequenced, and the results matched P. pachyrhizi sequences in the GenBank database. To our knowledge, these observations confirm for the first time the presence of P. pachyrhizi in the state of Campeche of southern Mexico. Although it was confirmed on soybean during 2008, it is not known how long the pathogen has been present or which other hosts may be infected there. The presence of SBR on the Yucatan Peninsula is significant because of its potential effects on local plant hosts. In addition, the climate allows possible year-round survival of the pathogen and long-distance transport of urediniospores to the United States. Potential transport of SBR spores from this part of Mexico to the United States has been reported through the application of NOAA's HYSPLIT (Hybrid Single Particle Lagrangian Integrated Transport) model and atmospheric back-trajectory analysis (2). References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) S. V. Krupa et al. Plant Dis. 90:1254, 2006. (3) A. C. Rodriguez et al. Plant Dis. 90:1260, 2006. (4) R. W. Schneider et al. Plant Dis. 89:774, 2005.

Characterizing Reaction of Soybean to Phialophora gregata Using Pathogen Population Density and DNA Quantity in Stems.

Evaluation of soybean germplasm for resistance to brown stem rot (BSR) is typically based on symptom severity. However, this approach may not reflect the level of colonization of soybean by the casual agent, Phialophora gregata. A potentially more accurate method to characterize resistance to BSR is to estimate pathogen quantity. The primary goal of this study was to evaluate soybean accessions for resistance to BSR based on the quantity of pathogen in stems. Plants were collected from experiments in field and controlled environments, and CFU and pathogen DNA quantity were determined using dilution plating techniques and real-time quantitative PCR (qPCR), respectively. In the field, the BSR-susceptible cultivars Corsoy 79 and Century 84 expressed greater than 73% foliar and stem symptom severity and had the highest pathogen population density, with a range from log10 4.3 to 4.7 CFU per gram of stem tissue. The resistant cultivar Bell expressed less than 10% foliar symptom severity, but had a pathogen population density that was not statistically different from the susceptible accessions. CFU measured in Dwight and L84-5873 were consistently lower than CFU in susceptible accessions and several resistant accessions. The amount of pathogen DNA differed among accessions in controlled environments. For example, Corsoy 79 and Century 84 had the highest pathogen DNA quantity, ranging from log10 6.19 to 6.65 copies, whereas the resistant cultivars Bell, Dwight, and L84-5873 had significantly lower DNA quantities, ranging from log10 2.04 to 2.91 copies. PI 437833 and IA2008R expressed low symptom severity but contained high DNA quantities. Pella 86, a highly symptomatic cultivar, had fewer CFU and lower DNA quantity in comparison to two other highly symptomatic cultivars and some cultivars with low symptom severity. These results suggest that some accessions express resistance to both pathogen colonization and symptom development, while others are resistant to symptom development but not to pathogen colonization. Results also indicate that resistant and susceptible accessions can be distinguished based on DNA quantity in controlled environments. In the field, differences between the pathogen population in resistant and susceptible cultivars were less distinct, possibly due to when plants were assayed.

Detection and Quantification of Phialophora gregata in Soybean and Soil Samples with a Quantitative, Real-Time PCR Assay.

Brown stem rot of soybean, caused by the soilborne fungus Phialophora gregata, is a common and widespread disease of soybean (Glycine max) in the midwestern United States. This pathogen is challenging to study due to a long latent period and slow growth. A TaqMan probe-based quantitative, real-time polymerase chain reaction (qPCR) assay was developed for sensitive and specific detection and quantification of genotypes A and B of P. gregata in plant and soil samples. It is sensitive with detection limits of 50 fg of pure genomic DNA, 100 copies of the target DNA sequence, and approximately 400 conidia. The qPCR assay is approximately 1,000 times more sensitive in detecting DNA and conidia of P. gregata, and is more rapid and less sensitive to PCR inhibitors from soybean stems than a standard PCR (sPCR) assay. Using this single-step qPCR assay, low levels of infection were detected in soybean stems at least 1 to 2 weeks prior to symptom development and before P. gregata was detected with sPCR. This assay also was used to detect the pathogen in field-grown plants and in naturally infested field soils. This new qPCR assay is a powerful tool for rapid, specific, and sensitive detection, diagnosis, and quantification of P. gregata in plants and soil, and for advancing studies of the ecology of P. gregata and its interactions with host plants.

Traits of Soybean-Infecting Phytophthora Populations from Illinois Agricultural Fields.

Phytophthora rot caused by Phytophthora sojae is a common and significant disease of soybean (Glycine max) in Illinois and throughout the Midwestern United States. The pathogenic characteristics of P. sojae populations in several Midwestern states have been reported recently, but pathogenicity and fungicide sensitivity traits of populations in Illinois were poorly understood. Isolates (n = 121) of soybean-infecting Phytophthora spp. were baited using susceptible cv. Sloan seedlings from soybean field soils with a history of seedling diseases in 24 counties across Illinois. The pathotype and race of isolates of P. sojae were characterized using 11 differential soybean cultivars in greenhouse tests using a hypocotyl inoculation method. Sensitivity to the fungicidal compounds metalaxyl and mefenoxam was tested with 63 isolates in vitro. Most (96%) of the Phytophthora isolates sampled from Illinois soybean fields were P. sojae, but 4% were an unidentified Phytophthora sp. as determined by phenotypic and genotypic traits. We present a preliminary description of another Phytophthora sp. from soybean fields in a restricted region of Illinois that is pathogenic and capable of killing soybean. Based on eight Rps gene differentials (Rps1a, 1b, 1c, 1d, 1k, 3a, 6, and 7 ), 22 virulence pathotypes of P. sojae were identified and 88% of all isolates were characterized to a defined race. The four most common races, which were 58% of all isolates, were races 1 (21%), 4 (15%), 33 (12%), and 28 (10%). Based on 11 differentials, (those noted above and Rps 2, 4, and 5), 31 virulence pathotypes were identified. The mean virulence complexities, which are the number of susceptible interactions on the sets of 8 and 11 Rps gene differentials, were 3.3 and 3.7, respectively. All isolates tested were sensitive to Apron XL, Allegiance, technical grade mefenoxam, and technical grade metalaxyl at 1.0 µg a.i./ml. The population of P. sojae is diverse and composed of multiple pathotypes and races in Illinois, and the results suggest that pathogen virulence partially explains poor performance of Phytophthora-resistant cultivars in many Illinois soybean fields.

Cultivar Preference and Genotype Distribution of the Brown Stem Rot Pathogen Phialophora gregata in the Midwestern United States.

Brown stem rot (BSR), caused by Phialophora gregata f. sp. sojae, is an important yield-limiting disease of soybean (Glycine max) in the midwestern United States. Midwestern populations of P. gregata are separated into genotypes A and B based on intergenic spacer sequences of nuclear ribosomal DNA. Genotype A causes both leaf and stem symptoms, and genotype B typically causes internal stem symptoms only. Data are limited on the geographic distribution of genotypes A and B. It is not well understood whether cultivars may be infected preferentially by a genotype. Field plots were established at five sites in Illinois, three sites in Wisconsin, and two sites in Minnesota in two different years. Soybean cvs. Bell, BSR101, Dwight, Sturdy, Williams 82, LN92-12033, and LN92-12054 were sown with two to four replications at each field site. From each plot, 5 to 10 stems were harvested arbitrarily at the R8 growth stage and assayed by polymerase chain reaction to detect the A and B genotypes. Both pathogen genotypes were detected at all locations except Urbana, where only genotype A was detected, and St. Paul, where only B was detected. Genotype A was the predominant genotype detected in susceptible cvs. Williams 82 and LN92-12054, with 70 and 78% of infected stems, respectively, positive for A. The other susceptible cultivar, Sturdy, yielded predominantly genotype A at four of the seven Illinois and Wisconsin locations where both pathogen genotypes were present, but yielded predominantly B at the Minnesota location where both genotypes were detected. Genotype B was the predominant type detected in partially resistant cvs. Dwight, LN92-12033, and Bell, with 56, 85, and 99% of the infected stems, respectively, testing positive for B.

Characteristics and Frequency of Aphanomyces euteiches Races 1 and 2 Associated with Alfalfa in the Midwestern United States.

Aphanomyces root rot of alfalfa, caused by Aphanomyces euteiches, kills seedlings and causes decline of established plants in slowly drained soils. Two races of A. euteiches that are pathogenic to alfalfa have been identified. Despite the contribution of race 1 resistance to establishment and yield of alfalfa, race 1-resistant alfalfa cultivars perform poorly in some fields infested with A. euteiches. Many isolates of A. euteiches obtained from the soils of problematic fields are of a race 2 phenotype. The purpose of this study was to determine distribution, frequency, and pathogenic and genotypic characteristics of race 1 (R1) and race 2 (R2) isolates from 21 fields: 13 in Wisconsin, 7 in Minnesota, and 1 in Kentucky. A. euteiches was successfully isolated from the soil of 16 of the 21 fields; 405 isolates were obtained from Wisconsin, 4 from Minnesota, and 48 from Kentucky. Pathogenicity and race phenotype of isolates were characterized on Saranac (susceptible to R1 and R2 isolates) and WAPH-1 (resistant to R1 and susceptible to R2 isolates) alfalfa populations. One Wisconsin field with no recent history of alfalfa production had a high frequency (51%) of R2 isolates, and 43% of all isolates were R2 from fields with a history of alfalfa production. In a location that was planted continuously to pea for 30 years, 27% of the isolates were R2. Random amplified polymorphic DNA (RAPD) analysis of three R1 and three R2 isolates with eight primers generated 43 total polymorphic bands; however, none of the bands were uniquely associated with race phenotype. Cluster analysis based on RAPD bands revealed no consistent genotypic distinctions between R1 and R2 isolates of A. euteiches. Evaluation of eight commercial alfalfa cultivars for resistance to two R1 and two R2 isolates demonstrated that most are susceptible to R2 isolates; however, those selected for R2 resistance express resistance to R2 isolates. The results suggest that R2 isolates represent a widespread risk to alfalfa cultivars having resistance only to R1 isolates in fields with varied cropping histories.

Identification of Pisum sativum Germ Plasm with Resistance to Root Rot Caused by Multiple Strains of Aphanomyces euteiches.

Aphanomyces root rot is a serious disease of pea (Pisum sativum), and additional sources of resistance are needed for development of disease-resistant cultivars. Accessions (n = 123) from the P. sativum Plant Introduction (PI) collection with the highest relative levels of resistance to one strain of Aphanomyces euteiches were previously identified from among approximately 2,500 accessions evaluated. The chosen 123 accessions were evaluated in this study for resistance to root rot caused by multiple strains of this pathogen. Five strains representing different US geographical locations and pathogenicity characteristics were used to evaluate pea seedlings in a greenhouse. Disease severity (DS) and percent loss of fresh biomass (inoculated vs. non-inoculated plants) were determined 15 days after inoculation. Significant differences (P = 0.05) in levels of DS and biomass loss (BL) occurred among the accessions after inoculation individually with the five strains. The relative rank of accessions based on DS and BL varied with the strain of A. euteiches used for inoculations. The 20 accessions with the lowest DS after inoculation with each strain were identified. Based on lowest DS, two accessions were among the 20 identified with all five individual strains, and four other accessions were among the 20 identified with four of the five strains. The results suggest that the P. sativum PI collection contains useful accessions for breeding programs aimed at developing pea varieties with resistance to A. euteiches.

Genotypic and Pathogenic Diversity Among Pea-Infecting Strains of Aphanomyces euteiches from the Central and Western United States.

ABSTRACT Pathogenic and genotypic variability among four populations of Aphanomyces euteiches from individual fields in Minnesota, Wisconsin, and Oregon were investigated using pathogenicity and randomly amplified polymorphic DNA (RAPD) analyses. About 50 strains were isolated from each of two pea fields in Minnesota, and 11 and 6 strains from pea fields in Wisconsin and Oregon, respectively, using pea (Pisum sativum) as a baiting host. Pathogenic variability and host range were evaluated in greenhouse studies with five pea lines or cultivars having different levels of resistance to Aphanomyces root rot and one cultivar each of alfalfa and snap bean. All strains were pathogenic on one or more pea cultivars, and 18 and 14% were pathogenic on alfalfa and bean, respectively. Disease severity incited by different strains varied significantly on individual pea cultivars and on all hosts combined. The percentage of strains pathogenic on different hosts varied among locations. Genotypic variation among all 114 strains was evaluated with RAPD analysis. Ten decanucleotide primers detected 92 polymorphic bands. Cluster and principal coordinates analysis revealed one large group containing 102 of the 114 strains from all locations. Two closely related minor groups of strains (12 strains) were genotypically distinct, with about 55% similarity to the main group of 102 strains. The strains in the minor groups were all isolated from the Minnesota locations and were pathogenic on two disease-resistant pea breeding lines (MN313 and MN314). Estimates of genetic diversity based on RAPD analysis ranged from 0.24 to 0.33 within populations to 0.35 among all strains from all populations. A. euteiches populations were genotypically and phenotypically variable, but no distinct genotypic differences were identified among populations from the four isolated locations.

Variation in Pathogenicity and Genotype Among Single-Zoospore Strains of Aphanomyces euteiches.

ABSTRACT The role of asexual reproduction in the production of pathogenic and genotypic variation in Aphanomyces euteiches was investigated. Variation was studied among three groups of 18 single-zoospore progeny of A. euteiches derived from each of three single-zoospore parental strains. Pathogenicity was assessed by evaluating disease severity (DS) on roots of five pea lines possessing different levels of resistance to Aphanomyces root rot and of a susceptible cultivar of snap bean and alfalfa. None of the single-zoospore progeny incited significantly higher DS levels than their parental strain on any of the seven hosts; however, 3 or 4 of the 18 progeny in each group incited significantly lower DS than their parental strains. The host range of the progeny either decreased or remained the same as compared with parental strains. Genotypic variation was assessed with randomly amplified polymorphic DNA (RAPD) analysis. Polymorphic RAPD markers that distinguished parental and progeny strains were detected within two of the three groups of strains with two of four RAPD primers used. Of 76 total RAPD markers that were detected among all strains in all groups, four (5%) were polymorphic. The polymorphic markers were not associated with the pathogenic variation.

Interaction of Temperature and Moisture on Infection of Wild Rice by Bipolaris oryzae in the Growth Chamber.

Infection of wild rice (Zizania palustris) flag leaves by Bipolaris oryzae was studied at temperatures of 5 to 35°C and wet periods of 2 to 36 h after inoculation. Lesion densities (lesions/cm2) increased with increasing wet periods depending on optimum temperature. High rates of infection occurred at 25 and 30°C and generally increased with continuous wet periods of 16, 18, 24, and 28 h. There were no lesions at 5°C and few at 10 and 35°C. Lesion densities declined when wet periods of 2, 4, or 6 h were interrupted by dry periods of 4, 6, 8, 10, or 12 h followed by a final 14 h of wetness. Lesion densities decreased at all temperatures with increased dry periods regardless of the initial wet period. The interaction of dry period length × wet period length × temperature was significant at the 0.5% level. With continuous wet periods, lesion numbers were highest at 25 to 30°C.

Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria.

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu) strains of the pathogen were isolated. The Cu genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu genes on pXV10A. A probe containing subcloned Cu genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids.

Plasmid-mediated production of the phytotoxin coronatine in Pseudomonas syringae pv. tomato.

Pseudomonas syringae pv. tomato PT23.2 produces the chlorosis-inducing phytotoxin coronatine. Thirty-eight chlorosis-defective mutants of PT23.2 were previously generated by using the transposon Tn5. Five mutants contained Tn5 insertions in the indigenous plasmid pPT23A; the remaining 33 mutants either were missing pPT23A (29 mutants) or contained deletions in this plasmid (4 mutants). These results suggested that pPT23A was involved in coronatine production in strain PT23.2. This plasmid was introduced into P. syringae pv. syringae PS61, which does not produce coronatine. A bioassay for coronatine suggested that PS61(pPT23A) transconjugants were able to make this phytotoxin. In a chemical analysis, organic acids were isolated from PT23.2, PS61, and the transconjugant PS61(pPT23A); these were derivatized to their methyl esters and analyzed by gas chromatography. The derivatized organic acids extracted from PT23.2 and PS61(pPT23A) contained peaks that corresponded to coronafacic acid, coronafacoylvaline, and coronatine, but these were absent in the extracts from the wild-type strain PS61. The identification of these components was confirmed by combined gas chromatography-mass spectrophotometry. Therefore, the acquisition of pPT23A by PS61 resulted in biosynthesis of coronafacic acid, coronafacoylvaline, and coronatine, clearly demonstrating the involvement of pPT23A in coronatine production in P. syringae pv. tomato.