Impact of Arbuscular Mycorrhizal Species on Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines) is a widely occurring pest and the leading cause of soybean yield losses in the U.S.A. There is a need to find additional SCN management strategies as sources of SCN resistance have become less effective in managing SCN populations. Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with roots of most plants including soybean. Research has shown that AMF can reduce disease severity in plants caused by pathogens and pests, including plant parasitic nematodes. The goal of this study was to evaluate the impact of AMF on SCN cyst production, SCN juveniles in roots, and SCN egg hatching. In one experiment, all five AMF species tested (Claroideoglomus claroideum, Diversispora eburnean, Dentiscutata heterogama, Funneliformis mosseae, and Rhizophagus intraradices) reduced (P < 0.05) the number of cysts on soybean roots by 59 to 81%, compared with soybean roots not inoculated with AMF. Inoculation with F. mosseae reduced SCN J2-J3 stage juveniles in soybean roots by 60% at 7 days post inoculation. A separate experiment showed that egg hatch was reduced (P < 0.05) in the presence of F. mosseae spores and their exudates by 27% and 62%, respectively. Further research is needed to evaluate the potential usefulness of AMF in field conditions and to determine the usefulness and potential of the exudates associated with SCN hatching suppression by F. mosseae. Making AMF a more effective biological control agent would provide another management tool to reduce the negative impact of SCN on soybean production.

Effect of Selected Biopesticides in Reducing Soybean Rust (Phakopsora pachyrhizi) Development.

The intensive use of fungicides in controlling soybean rust (SBR), a damaging foliar fungal disease of soybean caused by the obligate fungus Phakopsora pachyrhizi, may have accelerated the insensitivity of P. pachyrhizi populations to fungicides. The objective of this study was to determine the effect of selected biopesticides and their application time on reducing SBR infection. There were differences (P < 0.05) in percent rust reduction values for application times, biopesticide treatments, and their interaction in detached-leaf and whole-plant greenhouse experiments. All application times and nearly all biopesticide treatments reduced (α = 0.05) fungal infection compared with the nonfungicide control. Among the treatments, Bacillus subtilis QST 713 and acibenzolar-S-methyl often reduced fungal sporulation more than the other treatments in detached-leaf and whole-plant greenhouse experiments. The identification of biopesticides effective to P. pachyrhizi may be a valuable alternative or complement to synthetic fungicides and may be useful in integrated pest management programs for SBR control.

Prediction of Short-Distance Aerial Movement of Phakopsora pachyrhizi Urediniospores Using Machine Learning.

Dispersal of urediniospores by wind is the primary means of spread for Phakopsora pachyrhizi, the cause of soybean rust. Our research focused on the short-distance movement of urediniospores from within the soybean canopy and up to 61 m from field-grown rust-infected soybean plants. Environmental variables were used to develop and compare models including the least absolute shrinkage and selection operator regression, zero-inflated Poisson/regular Poisson regression, random forest, and neural network to describe deposition of urediniospores collected in passive and active traps. All four models identified distance of trap from source, humidity, temperature, wind direction, and wind speed as the five most important variables influencing short-distance movement of urediniospores. The random forest model provided the best predictions, explaining 76.1 and 86.8% of the total variation in the passive- and active-trap datasets, respectively. The prediction accuracy based on the correlation coefficient (r) between predicted values and the true values were 0.83 (P < 0.0001) and 0.94 (P < 0.0001) for the passive and active trap datasets, respectively. Overall, multiple machine learning techniques identified the most important variables to make the most accurate predictions of movement of P. pachyrhizi urediniospores short-distance.

Sensitivity of Phakopsora pachyrhizi Isolates to Fungicides and Reduction of Fungal Infection Based on Fungicide and Timing of Application.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, is a damaging foliar fungal disease in many soybean-growing areas of the world. Strategies to manage SBR include the use of foliar fungicides. Fungicide types, the rate of product application, and the number and timing of applications are critical components for successful rust management. The objectives of this study were to determine i) the sensitivity of P. pachyrhizi isolates collected in the U.S. to a range of fungicides and ii) the reduction of fungal infection based on fungicide type and timing of applications on soybean. There were differences (P < 0.05) in effective concentration (EC50) values among the fungicides tested. Azoxystrobin had low EC50 values for both urediniospore germination and fungal sporulation on inoculated leaflets. There were differences (P < 0.05) in fungal sporulation for application times, fungicide treatments, and their interaction when the fungus was inoculated on plants. All application times and nearly all fungicide treatments reduced (α = 0.05) fungal infection compared with the nonfungicide control. Information on fungicide sensitivity of P. pachyrhizi isolates and the preventive and curative effects of different fungicides are important in the management of SBR.

Accessions of Perennial Glycine Species With Resistance to Multiple Types of Soybean Cyst Nematode (Heterodera glycines).

Soybean cyst nematode (SCN; Heterodera glycines; HG) is a widely occurring and damaging pathogen that limits soybean production. Developing resistant cultivars is the most cost-effective method for managing this disease. Genes conferring SCN resistance in soybean have been identified; however, there are SCN populations that overcome known resistance genes. In order to identify additional sources of resistance and potentially new resistance genes, 223 plant introductions (PIs) of G. tomentella and 59 PIs of 12 other perennial Glycine species were inoculated with HG Types 0, HG 2, and HG 1.2.3, and then 36 PIs out of this set were further evaluated with HG Type 1.2.3.4.5.6.7, a population that overcomes all the resistance genes in soybean. Of 223 G. tomentella PIs evaluated, 86 were classified as resistant to three HG types, 69 as resistant to two HG types, and 22 as resistant to one HG type. Of the other 12 perennial Glycine species, all PIs of G. argyrea and G. pescadrensis were resistant to all three HG types. Of the 36 PIs challenged with HG Type 1.2.3.4.5.6.7, 35 were resistant with 16 showing no cyst reproduction. Our study confirms that there are high levels of resistance to SCN among the perennial Glycine species. This represents an untapped resource for use in genetic studies and for improving resistance to SCN in soybean.

Suppression of Soilborne Diseases of Soybean With Cover Crops.

Field trials were conducted from 2010 to 2013 at four locations in Illinois to evaluate the impact of cover crops (cereal rye [Secale cereal], brown mustard [Brassica juncea], winter canola [B. napus], and winter rapeseed [B. napus]) on soybean [Glycine max] stands and yield, diseases, pathogen populations, and soil microbial communities. Cover crops were established in the fall each year and terminated the following spring either by using an herbicide (no-till farms), by incorporation (organic farm), or by an herbicide followed by incorporation (research farm). Although shifts in soilborne pathogen populations and microbial community structure were not detected, cover crops were found to induce general soil suppressiveness in some circumstances. Cereal rye and rapeseed improved soybean stands in plots inoculated with Rhizoctonia solani and decreased levels of soybean cyst nematode in the soil. Cereal rye increased soil suppressiveness to R. solani and Fusarium virguliforme, as measured in greenhouse bioassays. Cereal rye significantly improved yield when Rhizoctonia root rot was a problem.

Virulence Diversity of Phakopsora pachyrhizi Isolates From East Africa Compared to a Geographically Diverse Collection.

Soybean rust, caused by the biotrophic pathogen Phakopsora pachyrhizi, is a highly destructive disease causing substantial yield losses in many soybean producing regions throughout the world. Knowledge about P. pachyrhizi virulence is needed to guide development and deployment of soybean germplasm with durable resistance against all pathogen populations. To assess the virulence diversity of P. pachyrhizi, 25 isolates from eight countries, including 17 isolates from Africa, were characterized on 11 soybean genotypes serving as differentials. All the isolates induced tan lesions with abundant sporulation on genotypes without any known resistance genes and on soybean genotypes with resistance genes Rpp4 and Rpp5b. The most durable gene was Rpp2, where 96% of the isolates induced reddish brown lesions with little or no sporulation. Of the African isolates tested, the South African isolate was the most virulent, whereas those from Kenya, Malawi, and some of the isolates from Tanzania had the lowest virulence. An Argentinian isolate was virulent on most host differentials, including two cultivars carrying multiple resistance genes. Ten distinct pathotypes were identified, four of which comprised the African isolates representing considerable P. pachyrhizi virulence. Soybean genotypes carrying Rpp1b, Rpp2, Rpp3, and Rpp5 resistance genes and cultivars Hyuuga and UG5 were observed to be resistant against most of the African isolates and therefore may be useful for soybean-breeding programs in Africa or elsewhere.

Clustering of clinical and environmental Escherichia coli O104 isolates using the DiversiLab™ repetitive sequence-based PCR system.

The DiversiLab™ rep-PCR system was used to amplify DNA regions of 28 well-characterized Escherichia coli O104 strains to generate a digital DNA fingerprint profile for strain differentiation. E. coli O104 strains from human stools and other sources were examined. The results indicate that this system can cluster similar O104 strains rapidly.

Optimizing Conditions of a Cell-Free Toxic Filtrate Stem Cutting Assay to Evaluate Soybean Genotype Responses to Fusarium Species that Cause Sudden Death Syndrome.

Cell-free toxic culture filtrates from Fusarium virguliforme, the causal fungus of soybean sudden death syndrome (SDS), cause foliar symptoms on soybean stem cuttings similar to those obtained from root inoculations in whole plants and those observed in production fields. The objectives of this study were to (i) optimize the production conditions for F. virguliforme cell-free toxic culture filtrates and the incubation conditions of the stem cutting assay used to test the toxicity of the cell-free toxic culture filtrates, and (ii) use the optimized assay and a whole plant root inoculation assay to compare four SDS-causing isolates on a panel of selected soybean genotypes. Area under the disease progress curve (AUDPC) values were highest (P = 0.05) when cuttings were immersed in culture filtrate of fungus grown in soybean dextrose broth, in filtrate produced from the fungus grown for 18 or 22 days, and when stem cuttings were incubated at 30°C. AUDPC values and shoot dry weights from the whole plant root inoculations and the AUDPC values from the stem cutting assay differed (P < 0.05) among nine soybean genotypes tested with F. virguliforme and F. tucumaniae isolates, and the AUDPC values from the two assays were positively correlated (r = 0.44 at P < 0.0001).

Comparison of Pathogenic Variation among Phakopsora pachyrhizi Isolates Collected from the United States and International Locations, and Identification of Soybean Genotypes Resistant to the U.S. Isolates.

A major constraint in breeding for resistance to soybean rust has been the virulence diversity in Phakopsora pachyrhizi populations. In greenhouse experiments, reactions of 18 soybean genotypes to 24 U.S. isolates from 2007 and 2008 and 4 foreign isolates were compared. Reactions of four differentials (Rpp1 to Rpp4) to these U.S. isolates were also compared with reactions to nine foreign isolates and three U.S. isolates from 2004. Principal component analysis (PCA) of the reaction types grouped the U.S. isolates into a single virulence group, whereas each of the foreign isolates had a unique virulence pattern. In another experiment, reactions of 11 differentials to the 24 U.S. isolates were compared and significant interactions (P < 0.001) were found between the isolates and host genotypes for rust severity and uredinia densities. PCA of these two measures of disease placed the 24 isolates into seven or six aggressiveness groups, respectively. In a third experiment, evaluation of 20 soybean genotypes for resistance to the previously established aggressive groups identified 10 genotypes resistant to isolates representing most of the groups. This study confirmed the pathogenic diversity in P. pachyrhizi populations and identified soybean germplasm with resistance to representative U.S. isolates that can be used in breeding.

First Report of Phakopsora pachyrhizi Causing Rust on Soybean in Malawi.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, has become established in Africa since the first report in Uganda in 1996 (2). The urediniospores, as windborne propagules, have infested new regions of Africa, initiating SBR in many countries, including Ghana and Democratic Republic of the Congo in 2007 (4) and Tanzania in 2014 (3). No refereed reports have been published about rust in Malawi, but some people have indicated that soybean rust may have been observed as early as 2008. Typical symptoms and signs of SBR, including leaf yellowing and tan, sporulating uredinia, were observed on soybean in May 2014 during field surveys in the major soybean-growing areas of Malawi, including the central (Dowa, Mchinji, and Kasungu) and southern (Thyolo) regions in nine out of 12 sites surveyed. When microscopically examined, urediniospores were elliptical, echinulate, and hyaline to pale yellowish brown. Leaves exhibiting sporuliferous uredinia were sent by APHIS permit to the University of Illinois. To confirm the pathogen, symptomatic soybean leaf tissue of approximately 1 cm2 was excised from each of the samples, and DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), with further purification using the MicroElute DNA Clean-up Kit (Omega Bio-Tek, Norcross, GA). The resulting DNA was analyzed by quantitative PCR using published Taqman assays for P. pachyrhizi and P. meibomiae, with a multiplexed exogenous internal control reaction to validate negative results (1). P. pachyrhizi DNA was detected in excess of 180,000 genome equivalents/cm2 in all samples, indicating a substantial infection. P. meibomiae DNA was determined to be absent from all samples, within the limit of quantification of ~2 pg DNA/cm2. Urediniospores dislodged from three leaves and inoculated onto susceptible soybean cultivar Williams 82 produced tan lesions after 2 weeks of incubation in a detached-leaf assay. This is the first confirmed report of P. pachyrhizi causing rust on soybean in Malawi, putting at risk 14,000 ha currently under soybean production. The reports of soybean rust in Malawi and adjoining countries will alter soybean production practices and research interests. In some cases, foliar application of fungicides has increased and planting dates have been changed to avoid conditions that are most conducive for rust development. Efforts to understand the virulence and genetic diversity of the pathogen in the region are needed in order to develop and deploy resistant cultivars. References: (1) J. S. Haudenshield and G. L. Hartman. Plant Dis. 95:343, 2011. (2) R. Kawuki, et al. Afr. Crop Sci. J. 11:301, 2003. (3) H. M. Murithi et al. Plant Dis. 98:1586, 2014. (4) P. S. Ojiambo et al. Plant Dis. 91:1204, 2007.

Characterization and quantification of fungal colonization of Phakopsora pachyrhizi in soybean genotypes.

Soybean rust, caused by the fungus Phakopsora pachyrhizi, is an economically important disease of soybean with potential to cause severe epidemics resulting in significant yield losses. Host resistance is one of the management tools to control this disease. This study compared soybean genotypes exhibiting immunity, complete and incomplete resistance, and susceptibility to an isolate of P. pachyrhizi based on visual assessment of reaction type, other visual traits such as sporulation, quantitative measurements of the amount of fungal DNA (FDNA) present in leaf tissues, and data on infection and colonization levels. Soybean genotype UG5 (immune), and plant introduction (PI) 567102B and PI 567104B (complete resistance) had lower quantities of uredinia and FDNA than four other genotypes with incomplete resistance. Based on microscopic observations, early events of spore germination, appressorium formation, and fungal penetration of the epidermis occurred within 24 h postinoculation and were similar among the tested soybean genotypes. Differences in infection among the genotypes were evident once the hyphae penetrated into the intercellular spaces between the mesophyll cells. At 2 days after inoculation (dai), soybean genotype Williams 82 had a significantly (P < 0.05) higher percentage of hyphae in the mesophyll tissue than other soybean genotypes, with UG5 having significantly (P < 0.05) lower percentages than all of the other soybean genotypes at 3, 4, and 5 dai. The percentage of interaction sites with mesophyll cell death was significantly (P < 0.05) higher in UG5 than other genotypes at 3, 4, and 5 dai. There was a significant positive correlation (r = 0.30, P < 0.001) between quantities of hyphae in the mesophyll cells and FDNA. These results demonstrated that incompatible soybean-P. pachyrhizi interaction results in restricted hyphal development in mesophyll cell tissue, likely due to hypersensitive apoptosis.

Use of Quantitative Traits to Assess Aggressiveness of Phakopsora pachyrhizi Isolates from Nigeria and the United States.

Soybean rust, caused by Phakopsora pachyrhizi, is one of the most important foliar diseases of soybean worldwide. The soybean-P. pachyrhizi interaction is often complex due to genetic variability in host and pathogen genotypes. In a compatible reaction, soybean genotypes produce tan-colored lesions, whereas in an incompatible reaction soybean genotypes produce an immune response (complete resistance) or reddish-brown lesions (incomplete resistance). In this study, in total, 116 and 72 isolates of P. pachyrhizi from Nigeria and the United States, respectively, were compared based on six quantitative traits to assess their aggressiveness on two soybean genotypes. All isolates produced reddish-brown lesions on plant introduction (PI) 462312 and tan lesions on TGx 1485-1D. The number of days after inoculation to first appearance of lesions, uredinia, and sporulation, along with the number of lesions and sporulating uredinia per square centimeter of leaf tissue, and the number of uredinia per lesion, were significantly (P < 0.001) different between the two soybean genotypes for all isolates from each country. The number of days to first appearance of lesions, uredinia, and sporulation were greater on PI 462312 than on TGx 1485-1D for all the test isolates. Similarly, the number of lesions and sporulating uredinia per square centimeter, and the number of uredinia per lesion were lower on PI 462312 than on TGx 1485-1D. For both soybean genotypes, the number of sporulating uredinia per square centimeter significantly (P = 0.0001) increased with an increase in the number of lesions per square centimeter. Although the slope of the regression of sporulating uredinia on number of lesions was greater (P < 0.0001) when TGx 1485-1D was inoculated with Nigerian isolates compared with U.S. isolates, slopes of the regression lines did not differ significantly (P > 0.0675) when PI 46312 was inoculated with Nigerian or U.S. isolates. This is the first study that used a large number of isolates from two continents to assess aggressiveness of P. pachyrhizi using multiple traits in soybean genotypes with contrasting types of disease reaction.

First Report of Phakopsora pachyrhizi on Soybean Causing Rust in Tanzania.

Phakopsora pachyrhizi Syd. was reported on legume hosts other than soybean in Tanzania as early as 1979 (1). Soybean rust (SBR), caused by P. pachyrhizi, was first reported on soybean in Africa in Uganda in 1996 (3), and its introduction into Africa was proposed to occur through urediniospores blowing from western India to the African east coastal areas by moist northeast monsoon winds (4). The fungus rapidly spread and was reported on soybean in South Africa in 2001, in western Cameroon in 2003, and in Ghana and the Democratic Republic of the Congo in 2007 (5). A second species causing SBR on soybean, P. meibomiae, has not been reported in Africa or elsewhere, outside of the Americas. From 2012 to 2014, symptomatic leaf samples were collected in the major soybean growing areas of the Tanzanian Southern Highlands (Iringa, Mbeya, and Ruvuma regions). Symptoms of SBR included yellowing of leaves and tan sporulating lesions. These symptoms were observed at flowering through seed maturity. From fields surveyed in 2012, 2013, and 2014, SBR was observed in 5 of 14, 7 of 11, and 14 of 31 fields, respectively. Some of the leaves sampled had up to 80% of the leaf area affected. When microscopically examined, urediniospores were elliptical, echinulate, and hyaline to pale yellowish brown. In 2014, sporuliferous uredinia were observed on leaf material collected from the Iringa and Ruvuma regions of Tanzania, and a subset of these samples was sent by APHIS permit to the University of Illinois. To confirm the pathogen, symptomatic soybean leaf tissue of approximately 1 cm2 was excised from each of the samples, and DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), with further purification using the MicroElute DNA Clean-up Kit (Omega Bio-Tek, Norcross, GA). The DNA was subjected to quantitative PCR using published Taqman assays for P. pachyrhizi, P. meibomiae, and a multiplexed exogenous internal control reaction to validate negative results (2). P. pachyrhizi DNA was detected in excess of 66,000 genome equivalents/cm2 in all samples, and P. meibomiae DNA was determined to be absent from all samples (limit of quantification ~2 pg DNA/cm2). Free surviving urediniospores were dislodged from 12 samples and inoculated onto susceptible soybean cultivar Williams 82, which produced sporulating SBR lesions after 2 weeks of incubation in a detached-leaf assay. Thus, Koch's postulates were completed. This is the first report of P. pachyrhizi causing rust on soybean in Tanzania. In vivo cultures have been established from most of these samples, and ongoing research includes an evaluation of the P. pachyrizi virulence on a differential set, and characterization of the genetic diversity. References: (1) D. L. Ebbels and D. J. Allen. Phytopath. Pap. 22:1-89. (2) J. S. Haudenshield and G. L. Hartman. Plant Dis. 95:343, 2011. (3) R. Kawuki et al. Afr. Crop Sci. J. 11:301, 2003. (4) C. Levy. Plant Dis. 89:669, 2005. (5) P. S. Ojiambo et al. Plant Dis. 91:1204, 2007.

A Coordinated Effort to Manage Soybean Rust in North America: A Success Story in Soybean Disease Monitoring.

Existing crop monitoring programs determine the incidence and distribution of plant diseases and pathogens and assess the damage caused within a crop production region. These programs have traditionally used observed or predicted disease and pathogen data and environmental information to prescribe management practices that minimize crop loss. Monitoring programs are especially important for crops with broad geographic distribution or for diseases that can cause rapid and great economic losses. Successful monitoring programs have been developed for several plant diseases, including downy mildew of cucurbits, Fusarium head blight of wheat, potato late blight, and rusts of cereal crops. A recent example of a successful disease-monitoring program for an economically important crop is the soybean rust (SBR) monitoring effort within North America. SBR, caused by the fungus Phakopsora pachyrhizi, was first identified in the continental United States in November 2004. SBR causes moderate to severe yield losses globally. The fungus produces foliar lesions on soybean (Glycine max) and other legume hosts. P. pachyrhizi diverts nutrients from the host to its own growth and reproduction. The lesions also reduce photosynthetic area. Uredinia rupture the host epidermis and diminish stomatal regulation of transpiration to cause tissue desiccation and premature defoliation. Severe soybean yield losses can occur if plants defoliate during the mid-reproductive growth stages. The rapid response to the threat of SBR in North America resulted in an unprecedented amount of information dissemination and the development of a real-time, publicly available monitoring and prediction system known as the Soybean Rust-Pest Information Platform for Extension and Education (SBR-PIPE). The objectives of this article are (i) to highlight the successful response effort to SBR in North America, and (ii) to introduce researchers to the quantity and type of data generated by SBR-PIPE. Data from this system may now be used to answer questions about the biology, ecology, and epidemiology of an important pathogen and disease of soybean.

Effect of Fungicide Application and Cultivar on Soybean Green Stem Disorder.

Green stem disorder of soybean (Glycine max) has increasingly become a nuisance for soybean producers. The disorder is distinguished from other manifestations of delayed plant maturity by the delayed senescence of stems only, with normal pod ripening and seed maturation. The primary objective of the first study was to determine whether green stem disorder increased with a fungicide treatment. Field cages to isolate soybean plants to prevent insect interactions were used and treatments included maturity group (MG) II insensitive and sensitive soybean cultivars with or without fungicide applications. A secondary objective was to determine fungi potentially associated with the disorder. The results indicated significant elevation of the incidence of green stem disorder when using a fungicide. Species of Diaporthe or Phomopsis and Macrophomina phaseolina were more frequent in stems without the disorder, whereas species of Colletotrichum were found mostly in stems with the disorder. In another study, field experiments were conducted without cages in replicated field plots to compare the effects of fungicides with different chemistries and timing of fungicide application on incidence of green stem disorder using green stem disorder MG II- and MG III-sensitive and insensitive soybean cultivars. There was a significant increase in percentage of green stem disorder due to fungicide application, depending on fungicide chemistry, timing of application, year, location, and cultivar sensitivity to green stem disorder. Generally, Headline and Headline-Domark applications resulted in higher incidence of green stem disorder than Domark alone or the nonsprayed control, with over 50% incidence in many cases. Higher percent green stem disorder was significantly (P < 0.05) associated with higher yields in 11 of the 28 trials. From the results of this research, soybean producers should be aware of the possible risk that fungicide application may have in increasing incidence of green stem disorder. In addition, producers can help manage green stem disorder by selecting soybean cultivars reported to be consistently insensitive to the disorder.

First Report of Colletotrichum chlorophyti Infecting Soybean Seed in Arkansas, United States.

Colletotrichum chlorophyti was first reported in the United States in 2009 on soybean petioles (Glycine max [L.] Merr.) collected from Alabama, Illinois, and Mississippi (4). This species has not been reported to infect seed, unlike other Colletotrichum spp. (2). From the 2012 growing season, soybean seeds obtained from the National Agricultural Statistics Service representing 151 seed lots from growers' fields in 11 states were assayed by plating them on acidified potato dextrose agar (APDA). Before plating, seeds were surface disinfected by sequential immersion in 50% ethanol for 30 s, 20% commercial bleach for 1 min, two 1 min rinses in sterile distilled water, and kept at 25°C in the dark for 1 week. Infected seeds from one seed lot from Arkansas produced colonies similar to Colletotrichum spp. This seed lot was visually examined and divided into asymptomatic or discolored symptomatic seeds. Because of the limited number of seeds in the seed lot, 20 seeds that asymptomatic and 40 seeds that appeared symptomatic were assayed on APDA as previously described. Asymptomatic seeds did not produce any dark fungal colonies. Among the symptomatic seeds, five appeared to have flecked light gray seed coats with some larger grayish to black and irregular spots where cracks were sometimes formed, and they developed small black fungal masses or became entirely dark on the surface. Five fungal isolates were obtained from these infected seeds. On APDA, the isolates initially produced white to pink smooth-margined colonies, turned black with age, produced no aerial growth, and filled a 9 cm diameter petri dish within 10 days. DNA of one isolate was extracted for PCR and sequencing of the ITS region with ITS1 and ITS4 primers (3). From the BLAST analysis, the sequence was 100% identical to C. chlorophyti isolates, IMI 103806, and CBS 142.79 (Accession Nos. GU227894 and GU227895, respectively). To test for pathogenicity, the fungus was sub-cultured on APDA and eight APDA discs (4 mm diameter) were set into 50 ml potato dextrose broth inside a 250-ml flask and shook at a speed of 100 rpm at room temperature (24 ± 1°C) for 10 days. The mycelium was then weighed, fragmented with a blender, and resuspended in sterile distilled water to a final concentration of ~40 mg/ml. The mycelial suspension was sprayed on soybean seedlings of cv. Williams 82 (two plants/pot) at growth stage V1 to V2 until runoff. The inoculated plants were kept in a moist chamber (>90% relative humidity) for 48 h at 24 ± 1°C in the dark, and then transferred to normal plant growing conditions. At 5 days post-inoculation (dpi), the leaves showed typical symptoms caused by C. chlorophyti, including necrosis on the edge of young leaves and petioles, formation of irregular dark brown lesions, and leaves became scrolled (4). Setose acervuli, curved conidia with tapered ends (21.4 ± 1.1 × 3.8 ± 0.3 µm), and chlamydospores were found on the detached symptomatic leaves after 12 dpi. No perithecia formed. The morphology matched the description of C. chlorophyti (1,4). To our knowledge, this is the first report of C. chlorophyti in Arkansas and the first time that this species has been reported infecting seed of any plant. References: (1) S. Chandra and R. N. Tandon. Curr. Sci. 34:565, 1965. (2) G. L. Hartman et al. Page 13 in: Compendium of Soybean Diseases, APS Press, St. Paul, MN, 1999. (3) T. J. White et al. Page 315 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (4) H.-C. Yang et al. Plant Dis. 96:1699, 2012.

First Report of Orange Rust Caused by Puccinia kuehnii in Sugarcane in Louisiana.

In June 2012, lesions typical of rust were observed on sugarcane cultivar Ho 05-961 (a complex hybrid of Saccharum L. spp.) on a farm near Schriever, Louisiana. Incidence and severity of disease symptoms were low. Two types of pustules were observed on leaves of the infected plants. One pustule type was reddish-brown in color turning brown with age, characteristic of brown rust which has been observed in Louisiana since 1979 (2). The other pustule type was orange and did not turn brown with age. Urediniospore samples from the two pustule types were collected. The morphology of the urediniospores from the reddish-brown pustules was consistent with that described for Puccinia melanocephala Syd. & Syd., the fungus that causes brown rust of sugarcane, while the morphology of the urediniospores from the orange pustules was consistent with those described for P. kuehnii E.J. Butler, the causal organism of orange rust of sugarcane (3). Telia and teliospores were not observed. The identity of the two species of Puccinia causing the brown and orange rust lesions was verified using the species-specific quantitative PCR assays (1). Two DNA samples extracted from the pustules identified as P. kuehnii were independently subjected to PCR amplification using primers Pk1F and Pk1R (1) to yield a product from the rDNA that was then bidirectionally sequenced using the same primers. The resulting 480-nt sequences were identical to each other, and a BLAST search of GenBank revealed 100% identity to 19 previously reported isolates of P. kuehnii but not more than 89% similarity to any isolate of P. melanocephala (4). To our knowledge, this is the first report of orange rust in Louisiana. In the 4 months following the detection of orange rust, observations of the disease have been limited to Ho 05-961. Seed cane increase plots of this newly released cultivar were surveyed, and orange rust symptoms and urediniospores were detected in 17 of 38 (45%) fields. The incidence and severity of the disease remained low, and the distribution appeared to be limited to the southern portion of the Louisiana sugarcane production area. References: (1) N. C. Glynn et al. Plant Pathol. 59:703, 2010. (2) H. Koike. Plant Dis. 64:226, 1980. (3) C. C. Ryan et al. Page 189 in: Diseases of Sugarcane: Major Diseases. C. Ricaud et al., eds. Elsevier, Amsterdam, 1989. (4) E. V. Virtudazo et al. Mycoscience 42:447, 2001.

First Report of Phakopsora pachyrhizi Adapting to Soybean Genotypes with Rpp1 or Rpp6 Rust Resistance Genes in Field Plots in the United States.

During the years following the first detection of soybean rust, caused by Phakopsora pachyrhizi Syd., in the continental United States in November, 2004, soybean (Glycine max [L.] Merr.) genotypes with the Rpp1 or Rpp6 resistance genes exhibited high levels of resistance there (1,2,3). When challenged with 72 different American isolates collected between 2006 and 2009, PI 200492 (source of Rpp1) produced no sporulating lesions (2). In 2011 and 2012, however, field populations of P. pachyrhizi from Gadsden County, FL, caused higher rust severity on plants with Rpp1 or Rpp6 than in previous years. To assess aggressiveness, sporulation ratings were made using a 1 to 5 scale (no sporulation to profuse sporulation) on leaflets collected from field plants at or near the R6 (full seed) stage of development. A dissecting microscope was used to examine 3 replications of 5 leaflets each in 2009 or 2 replications of 10 leaflets each in 2012. The sporulation ratings increased on PI 200492 (from 1.1 ± 0.1 in 2009 to 4.1 ± 0.4 in 2012), PI 567102B (Rpp6; from 1.1 ± 0.1 in 2009 to 2.4 ± 0.2 in 2012), and L85-2378, a 'Williams 82' isoline carrying the Rpp1 gene (from 1.0 ± 0 in 2009 to 4.0 ± 0.3 in 2012). The mean 2009 and 2012 sporulation ratings for susceptible control Williams 82 were 5.0 ± 0 and 4.2 ± 0.1, respectively. Single-uredinium-derived isolates were purified from bulk isolates collected from field plots in 2009 (FL-Q09-1), 2011 (FL-Q11-1), and 2012 (FL-Q12-1). Greenhouse and detached leaflet assays were then used to test the virulence of these isolates under controlled conditions. Detached leaflets from 3-week-old seedlings of Williams 82, PI 200492, PI 567102B, and L85-2378 were inoculated by pipetting 15-µl drops of a 30 to 40 urediniospore µl-1 suspension onto the abaxial side of 3 to 4 leaflets per genotype, which were then sealed in Petri plates and incubated in a growth chamber at 20 to 22°C. Plates were kept in the dark for 12 h following inoculation. For the greenhouse assay, the first trifoliolate leaves of at least 3 seedlings were each sprayed with 1.5 ml of a 40 urediniospore µl-1 suspension and incubated 24 h at 22 to 24°C in a dark mist chamber. The plants were then maintained at 22 to 24°C and 76 to 86% relative humidity in a greenhouse with 10 h of daylight on average. Two weeks after inoculation with FL-Q11-1 or FL-Q12-1, all of the genotypes had developed TAN lesions with abundant sporulation, indicating susceptibility. On leaves inoculated with FL-Q09-1, however, no visible reaction was observed on PI 200492, and PI 567102B developed reddish-brown (RB) lesions associated with incomplete resistance. Although the lesions on Rpp1 and Rpp6 greenhouse seedlings inoculated with the FL-Q11-1 and FL-Q12-1 isolates were slightly darker than those that developed on Williams 82 plants or on detached leaflets, the profuse sporulation that is characteristic of the TAN infection type was observed. The higher virulence of the 2011 and 2012 Florida isolates on two soybean genotypes with Rpp1 and one with Rpp6 confirmed the presence of a P. pachyrhizi pathotype in north-central Florida that is more virulent against these genes than earlier populations from the southeastern United States. References: (1) S. Li. Crop Sci. 49:887, 2009. (2) Twizeyimana and Hartman. Plant Dis. 96:75, 2012. (3) Walker et al. Crop Sci. 51:678, 2011.

A multiplexed immunofluorescence method identifies Phakopsora pachyrhizi Urediniospores and determines their viability.

Soybean rust, caused by Phakopsora pachyrhizi, occurs concomitantly wherever soybean is grown in the tropical and subtropical regions of the world. After reports of its first occurrence in Brazil in 2001 and the continental United States in 2004, research on the disease and its pathogen has greatly increased. One area of research has focused on capturing urediniospores, primarily by rain collection or wind traps, and detecting them either by microscopic observations or by immunological or molecular techniques. This system of detection has been touted for use as a potential warning system to recommend early applications of fungicides. One shortcoming of the method has been an inability to determine whether the spores are viable. Our study developed a method to detect viable P. pachyrhizi urediniospores using an immunofluorescence assay combined with propidium iodide (PI) staining. Antibodies reacted to P. pachyrhizi and other Phakopsora spp. but did not react with other common soybean pathogens or most other rust fungi tested, based on an indirect immunofluorescence assay using fluorescein isothiocyanate-labeled secondary antibodies. Two vital staining techniques were used to assess viability of P. pachyrhizi urediniospores: one combined carboxy fluorescein diacetate (CFDA) and PI, and the other utilized (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide] (FUN 1). Using the CFDA-PI method, viable spores stained green with CFDA and nonviable spores counterstained red with PI. Using the FUN 1 method, cylindrical intravacuolar structures were induced to form within metabolically active urediniospores, causing them to fluoresce bright red to reddish-orange, whereas dead spores, with no metabolic activity, had an extremely diffused, faint fluorescence. An immunofluorescence technique in combination with PI counterstaining was developed to specifically detect viable P. pachyrhizi urediniospores. The method is rapid and reliable, with a potential for application in forecasting soybean rust based on the detection of viable urediniospores.