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.

Characterizing Resistance to Phakopsora pachyrhizi in Soybean.

Resistance in soybean to Phakopsora pachyrhizi, the cause of soybean rust, is characterized by either reddish-brown (RB) lesions or an immune response. The RB type of resistance can be incomplete, as evidenced by the presence of sporulating uredinia within lesions. Susceptibility, on the other hand, is exemplified by tan-colored (TAN) lesions, and can be expressed in gradations of susceptibility or partial resistance that are less well defined. This study evaluated traits associated with incomplete or partial resistance to P. pachyrhizi in soybean by comparing 34 soybean accessions inoculated with four P. pachyrhizi isolates. Six accessions produced RB lesions to all four isolates, while 19 accessions produced TAN lesions, including plant introduction (PI) 200492 (Rpp1) and the susceptible check 'Williams'. Williams had among the largest area under the disease progress curve (AUDPC) values and area under the sporulating uredinia progress curve (AUSUPC) values, while eight accessions had lower AUSUPC values. Of the known sources of single-gene resistance, only PI 230970 (Rpp2), PI 459025B (Rpp4), and PI 594538A (Rpp1b) had lower AUDPC and AUSUPC values than Williams. PI 594538A and PI 561356 had RB lesions and had the lowest AUDPC and AUSUPC values. Of the known sources of single-gene resistance, only PI 230970 (Rpp2) and PI 594538A (Rpp1b) produced fewer and smaller-diameter uredinia than Williams. This study characterized reactions to P. pachyrhizi in 34 accessions based on lesion type and sporulation, and defined incomplete resistance and partial resistance in the soybean-P. pachyrhizi interaction.

Differential Responses of Resistant Soybean Entries to Isolates of Phakopsora pachyrhizi.

Soybean rust, caused by the fungus Phakopsora pachyrhizi, was detected in the continental United States in 2004. Several new sources of resistance to P. pachyrhizi have been identified in soybean (Glycine max); however, there is limited information about their resistance when challenged with additional U.S. and international isolates. Resistance of 20 soybean (G. max) entries was compared after inoculation with 10 P. pachyrhizi isolates, representing different geographic and temporal origins. Soybean entries included 2 universal susceptible cultivars, 4 sources of soybean rust resistance genes (Rpp1-4), and 4 and 10 resistant entries selected from field trials in Paraguay and Vietnam, respectively. Of the known Rpp1-4 sources of resistance, plant introduction (PI) 459025B (Rpp4) produced reddish-brown (RB) lesions in response to all of the P. pachyrhizi isolates, while PI 230970 (Rpp2) produced RB lesions to all isolates except one from Taiwan, in response to which it produced a susceptible tan (TAN) lesion. PI 200492 (Rpp1) and PI 462312 (Rpp3) produced TAN lesions in response to most P. pachyrhizi isolates. The resistant entries selected from Paraguay and Vietnam varied considerably in their responses to the 10 P. pachyrhizi isolates, with M 103 the most susceptible and GC 84058-18-4 the most resistant. The reaction patterns on these resistant entries to the P. pachyrhizi isolates were different compared with the four soybean accessions with the Rpp genes, indicating that they contain novel sources of rust resistance. Among the P. pachyrhizi isolates, TW 72-1 from Taiwan and IN 73-1 from India produced the most susceptible and resistant reactions, respectively, on the soybean entries.

Expression patterns in soybean resistant to Phakopsora pachyrhizi reveal the importance of peroxidases and lipoxygenases.

Soybean rust caused by Phakopsora pachyrhizi Sydow is a devastating foliar disease that has spread to most soybean growing regions throughout the world, including the USA. Four independent rust resistance genes, Rpp1-Rpp4, have been identified in soybean that recognize specific isolates of P. pachyrhizi. A suppressive subtraction hybridization (SSH) complementary DNA (cDNA) library was constructed from the soybean accession PI200492, which contains Rpp1, after inoculation with two different isolates of P. pachyrhizi that result in susceptible or immune reactions. Both forward and reverse SSH were performed using cDNA from messenger RNA pooled from 1, 6, 12, 24, and 48 h post-inoculation. A total of 1,728 SSH clones were sequenced and compared to sequences in GenBank for similarity. Microarray analyses were conducted on a custom 7883 soybean-cDNA clone array encompassing all of the soybean-rust SSH clones and expressed sequence tags from four other soybean cDNA libraries. Results of the microarray revealed 558 cDNA clones differentially expressed in the immune reaction. The majority of the upregulated cDNA clones fell into the functional category of defense. In particular, cDNA clones with similarity to peroxidases and lipoxygenases were prevalent. Downregulated cDNA clones included those with similarity to cell-wall-associated protein, such as extensins, proline-rich proteins, and xyloglucan endotransglycosylases.

New Legume Hosts of Phakopsora pachyrhizi Based on Greenhouse Evaluations.

Phakopsora pachyrhizi, the causal organism of soybean rust, was first found in the continental United States in 2004 and has been found on soybean, kudzu, Florida beggarweed, and three Phaseolus species in the field. The pathogen has been reported to occur on more than 90 legume species worldwide and it is likely to infect native and introduced legume species in the United States. The objective of this study was to determine if 176 species representing 57 genera of legumes, the majority of which are either native or naturalized to soybean-growing areas of the United States, could be hosts of P. pachyrhizi. Between one and three accessions of each species, a total of 264 accessions, were inoculated with a mixture of four isolates of P. pachyrhizi. Severity and sporulation were rated on a 1-to-5 scale at 14 and 28 days after inoculation. P. pachyrhizi was confirmed by the presence of sporulating uredinia and/or immunological assay on 65 new species in 25 genera; 12 of these genera have not been reported previously as hosts. Many of the newly identified hosts grow in the southern United States, and like kudzu, could serve as overwintering hosts for P. pachyrhizi.

Comparative Susceptibilities of Legume Species to Infection by Phakopsora pachyrhizi.

Knowledge of the host range of Phakopsora pachyrhizi is important to agriculture in the United States because of the distinct possibility that economic losses could occur to crops other than soybean. Furthermore, it is possible that alternative hosts could provide a means of overwintering of the pathogen, providing inoculum to initiate epidemics in future years. To clarify the potential importance of soybean rust on nonsoybean legumes and their role in overwintering of the disease, multiple accessions of clover, cowpea, pea, kudzu, lima bean, snap bean, and single accessions of coffee senna, Florida beggarweed, hemp sesbania, hyacinth bean, partridge pea, and showy crotalaria were inoculated under greenhouse conditions with urediniospores of P. pachyrhizi; infected soybean plants served as a control. The four criteria used to assess susceptibility were lesion density, proportion of lesions with sporulating uredinia, average number of uredinia per lesion, and average uredinia diameter, each determined 2 weeks following inoculation. Based on lesion densities, percentage of lesions with sporulation, and average numbers of uredinia per lesion, soybean, kudzu, and pea were the most susceptible species, followed by snap bean. However, because infected pea plants defoliated rapidly, urediniospore production presumably was limited, lessening the potential for epidemics on pea. Cultivars of snap bean produced numerous brown to reddish-brown lesions, many of which sporulated, but numbers of uredinia per lesion were lower than on soybean, kudzu, or pea. The presence of both tan (susceptible) and reddish-brown (resistant) lesions on kudzu demonstrated physiological differentiation on that host. Some kudzu plants appeared to be potentially excellent hosts for overwintering of the disease. The average number of uredinia per lesion appeared to be a valid measurement with which to compare host susceptibilities, and may have epidemiological significance. High susceptibility of a host was characterized by numerous uredinia with a wide range of sizes within individual lesions. In contrast, low susceptibility to rust was characterized by no or a few small uredinia.

Adult Plant Evaluation of Soybean Accessions for Resistance to Phakopsora pachyrhizi in the Field and Greenhouse in Paraguay.

Five hundred thirty soybean accessions from maturity groups (MG) III through IX were evaluated for resistance to Phakopsora pachyrhizi in a replicated field trial at Centro Regional de Investigación Agrícola in Capitán Miranda, Itapúa, Paraguay during the 2005-06 season. Soybean rust severities of individual accessions ranged from 0% (resistant) to 30.0% (susceptible). In MG III and IV, the most resistant accessions were PI 506863, PI 567341, and PI 567351B, with severities less than 1.2%. In MG V, the most resistant accessions were PI 181456, PI 398288, PI 404134B, and PI 507305, with severities less than 0.3%. In MG VI, the most resistant accessions were PI 587886, PI 587880A, and PI 587880B, with severities less than 0.3%. In MG VII and VIII, the most resistant were PI 587905 and PI 605779E, with severities less than 1.0%. In MG IX, the most resistant accessions were PI 594754, PI 605833, PI 576102B, and PI 567104B, with severities less than 1.0%. The resistance in 10 selected accessions from MG VI, VII, VIII, and XI was confirmed in subsequent greenhouse and field experiments where severities of 0.4% or less and reddish-brown lesions with sporulation levels less than 3.0 were observed. These accessions, with low severities in the adult plant field evaluation, may be new sources of resistance to P. pachyrhizi.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi in Ghana.

Nigeria is the only country in West Africa where soybean rust, caused by Phakopsora pachyrhizi, has been officially reported (1). During a disease survey in Ghana during October 2006, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 11 fields in the following districts: Kassena Nankana in the Upper East Region; East Gonja, Central Gonja, and Tolon-Kumbungu in the Northern Region; and Ejisu-Juabeng in the Ashanti Region. Disease incidence in these fields ranged from 50 to 100% and disease severity ranged between 3 and 40% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 14 to 18 µm. Within a week of collection, leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit for verification of pathogen identity. DNA was extracted from leaf pieces containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 11 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Sequence alignment of the internal transcribed spacer (ITS) region 2 further confirmed the identification as P. pachyrhizi (2). Infected leaves from three fields were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves (for each isolate) of susceptible cultivar TGx 1485-1D (3). The abaxial surface of detached leaves was sprayed with 400 µl of spore suspension (1 × 106 spores per ml). A single leaf piece was placed in a 9-cm-diameter petri dish with adaxial side appressed on 1% technical agar amended with 10 µg/ml of kinetin. Lactic acid (1.5 ml/liter) and benomyl (12.5 mg/liter) were added to the agar medium to inhibit growth of saprophytic fungi and bacteria. Petri dishes were incubated at 20°C with a 12-h light/12-h dark cycle. Lesions on inoculated leaves developed 5 to 6 days after inoculation (DAI), and pustules (105 to 120 µm) formed 7 to 8 DAI and erupted 3 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculating another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. The PCR assay, alignment of ITS region 2, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in Ghana. To our knowledge, this is the first report of P. pachyrhizi in Ghana. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

Differential Response of Common Bean Cultivars to Phakopsora pachyrhizi.

Soybean rust (Phakopsora pachyrhizi) has been reported on common bean (Phaseolus vulgaris) in Asia, South Africa, and the United States. However, there is little information on the interaction of individual isolates of Phakopsora pachyrhizi with common bean germplasm. A set of 16 common bean cultivars with known genes for resistance to Uromyces appendiculatus, the causal agent of common bean rust, three soybean accessions that were sources of the single gene resistance to P. pachyrhizi, and the moderately susceptible soybean 'Ina' were evaluated using seedlings inoculated with six isolates of P. pachyrhizi. Among the common bean cultivars, Aurora, Compuesto Negro Chimaltenango, and Pinto 114, were the most resistant to all six P. pachyrhizi isolates, with lower severity, less sporulation, and consistent reddish-brown (RB) lesions associated with resistance in soybean. A differential response was observed among the common bean cultivars, with a cultivar-isolate interaction for both severity and sporulation levels, as well as the presence or absence of the RB lesion type. This differential response was independent of the known genes that condition resistance to U. appendiculatus, suggesting that resistance to P. pachyrhizi was independent of resistance to U. appendiculatus.

First Report of Phakopsora pachyrhizi, Cause of Soybean Rust, on Neonotonia wightii in Paraguay.

Phakopsora pachyrhizi Syd. & P. Syd., the cause of soybean rust, was first observed on soybean (Glycine max (L.) Merr.) in South America in the district of Itapúa in Paraguay during March, 2001 (2). The disease is now widespread in soybean-production areas in South America on soybean and kudzu (Pueraria lobata (Willd.) Ohwi). On 15 March 2006, leaves of the perennial legume Neonotonia wightii (Graham ex Wight & Arn.) Lackey with lesions and rust sori were observed in the Reserva Biológica de Itabó, Departamento Alto Paraná. Lesions were scattered, most contained a single uredinium, mostly hypophyllous, and appeared to be new infections. Lesions with several uredinia, which are indicative of older infections on soybean, were also observed. Sori (Malupa-type) contained hyaline, peripheral, cylindric to clavate paraphyses measuring 24 to 45 × 6 to 13 µm and urediniospores that were hyaline, ovoid to globose, and measuring 20 to 40 × 14 to 25 µm with an echinulate spore wall, characteristics typical of a Phakopsora sp. DNA extracted from sori from leaves of N. wightii was amplified in a real-time fluorescent polymerase chain reaction with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (1). Sequence alignment of the internal transcribed spacer region 2 further confirmed the identification as P. pachyrhizi (1). The host identification was confirmed by J. Kirkbride, USDA/ARS/SBML, using the Smithsonian Institution Department of Botany, U.S. National Herbarium. To our knowledge, this is the first confirmed report of natural infection of P. pachyrhizi on a host other than soybean or kudzu in South America. Voucher specimens were deposited in the herbarium of the Facultad Ciencias Químicas of the Universidad Nacional de Asunción of Paraguay (FCQ) and the National Fungus Collection (Accession No. BPI 875340). References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) W. Morel and J. Yorinori. Bol. Divulg. No. 44. Ministerio de Agricultura y Ganadería, Centro Regional de Investigación Agrícola, Capitán Miranda, Paraguay, 2002.

First Report of Rust Caused by Phakopsora pachyrhizi on Soybean in Democratic Republic of Congo.

Nigeria (1) and Uganda (3) are the closest countries to the Democratic Republic of Congo (DRC) where soybean rust caused by Phakopsora pachyrhizi has been reported. In February 2007, during a disease survey in DRC, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 10 fields in the following areas in Bas Congo Province: Bangu, Kimpese, Kolo-Fuma, Lukala, Mbanza-Ngungu, Mpalukide, Mvuazi, and Ntemo. Rust incidence in these fields ranged from 85 to 100%, while severity ranged between 3 and 35% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 16 to 20 µm. Within a week of collection, infected leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit (FDWSRU) for pathogen identification. DNA was extracted from sections of leaves containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 10 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Infected leaves of cultivar Vuangi collected from one field each in the INERA Research Station, Kimpese-Crawford, and Kimpese-Ceco were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves of susceptible cultivar TGx 1485-1D (4). Lesions on inoculated leaves developed 5 days after inoculation (DAI), and pustules (110 to 130 µm) formed 7 DAI and erupted 2 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculation of another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. Seedlings of cultivar Williams also showed typical rust symptoms when inoculated separately with urediniospores collected from nine fields (i.e., all except Kimpese-Ceco, which was infective in the detached leaf assay). Inoculation and incubation were carried out at the FDWSRU Plant Pathogen Containment Facility at Fort Detrick as described earlier (2). The PCR assay, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in DRC. To our knowledge, this is the first report of P. pachyrhizi infecting soybean in DRC. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) E. Kawuki et al. J. Phytopathol. 151:7, 2003. (4) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

International Fungicide Efficacy Trials for the Management of Soybean Rust.

The efficacy of fungicides in managing soybean rust was evaluated in 12 environments in South America and southern Africa over three growing seasons from 2002 to 2005. There were differences in final soybean rust severity, defoliation, and yield among the treatments at most locations. In locations where soybean rust was not severe, all the fungicides evaluated reduced severity. In locations where soybean rust was severe, applications of triazole and triazole + strobilurin fungicides resulted in lower severity and higher yields compared with other fungicides. The strobilurin fungicides provided the highest yields in many locations; however, severity tended to be higher than that of the triazole fungicides. There also were differences in yield and severity between the trials with two and three applications of several fungicides, with three applications resulting in less severe soybean rust and higher yields. However, the third application of tebuconazole, tetraconazole, and the mixtures containing azoxystrobin and pyraclostrobin was not needed to maintain yield. These fungicides were among the most effective for managing soybean rust and maintaining yield over most locations.

Effects of temperature on urediniospore germination, germ tube growth, and initiation of infection in soybean by phakopsora isolates.

ABSTRACT Temperature is a critical factor in plant disease development. As part of a research program to determine how specific environmental variables affect soybean rust, we determined temperature effects on urediniospore germination and germ tube growth of four isolates of Phakopsora pachyrhizi, one each from Brazil, Hawaii, Taiwan, and Zimbabwe, and an isolate of P. meibomiae from Puerto Rico, collected over a 25-year period. Also compared were the effects of temperature during a night dew period on initiation of disease by the P. pachyrhizi isolates. All variables were fit to a nonlinear beta function with temperature as the independent variable. Minimum, maximum, and optimum temperatures, along with shape parameters of the beta function for each variable, were statistically analyzed. All Phakopsora isolates behaved similarly as to how temperature affected urediniospore germination, germ tube growth, and initiation of disease. The results suggest that P. pachyrhizi has changed little in the past few decades with respect to how it responds to temperature and that previously collected research data continues to be valid, simplifying the development of soybean rust disease models.

Evaluation of Virulence of Phakopsora pachyrhizi and P. meibomiae Isolates.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi and recently discovered for the first time in continental United States, has been of concern to the U.S. agricultural industry for more than 30 years. Since little soybean rust resistance is known, and resistance is often difficult to detect or quantitate, we initiated a project to develop a better, more quantitative, method. The methodology determined the average numbers and diameters of uredinia in lesions that developed on leaves of inoculated plants 14 days after inoculation. It was used to compare virulence of P. pachyrhizi isolates from Asia and Australia and P. meibomiae from Puerto Rico and Brazil, collected as many as 30 years earlier, with isolates of P. pachyrhizi recently collected from Africa or South America. Susceptible reactions to P. pachyrhizi resulted in tan-colored lesions containing 1 to 14 uredinia varying greatly in size within individual lesions. In contrast, on these same genotypes at the same time of year, resistance to other P. pachyrhizi isolates was typified by 0 to 6 small uredinia in reddish-brown to dark-brown lesions. Using appropriate rust resistant and rust susceptible genotypes as standards, examination of uredinia 14 days after inoculation allowed quantitative comparisons of sporulation capacities, one measure of susceptibility or resistance to soybean rust. The study verified the presence and ability to detect all known major genes for resistance to soybean rust in the original sources of resistance. It demonstrated that soybean lines derived from the original PI sources, and presumed to possess the resistance genes, in actuality may lack the gene or express an intermediate reaction to the rust pathogen. We suggest that a determination of numbers and sizes of uredinia will detect both major gene and partial resistance to soybean rust.

An assessment model for rating high-threat crop pathogens.

ABSTRACT Natural, accidental, and deliberate introductions of nonindigenous crop pathogens have become increasingly recognized as threats to the U.S. economy. Given the large number of pathogens that could be introduced, development of rapid detection methods and control strategies for every potential agent would be extremely difficult and costly. Thus, to ensure the most effective direction of resources a list of high-threat pathogens is needed. We address development of a pathogen threat assessment model based on the analytic hierarchy process (AHP) that can be applied world-wide, using the United States as an illustrative example. Previously, the AHP has been shown to work well for strategic planning and risk assessment. Using the collective knowledge of subject matter expert panels incorporated into commercial decision-making software, 17 biological and economic criteria were determined and given weights for assessing the threat of accidental or deliberately introduced pathogens. The rating model can be applied by experts on particular crops to develop threat lists, especially those of high priority, based on the current knowledge of individual diseases.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi on Dry Beans in South Africa.

During April 2004, a 150-m2 dry bean (Phaseolus vulgaris) plot growing adjacent to rust-infected soybean (Glycine max) at Cedara Agricultural Research Farm (29°32'S 30°16'E) was observed to be infected with two distinct rust types. Common bean rust (caused by Uromyces appendiculatus) with reddish brown uredinia and black telia was readily identified. A second rust with smaller sporulating uredinia (1.0 to 1.5 mm2), which were gray in appearance, was also found. Visual rust severity on the dry bean plants, which were in mid pod-fill, was high (approximately 30 to 40% disease incidence). Twenty plants were examined and observed to be infected with both rusts. With microscopic examination of no fewer than 20 leaves per plant, the urediniospores from the smaller lesions were determined to be morphologically similar to Phakopsora pachyrhizi (3). Real-time fluorescent polymerase chain reaction assays on six leaves and sequence analysis of the nuclear ribosomal internal transcribed spacer region 2 (1) verified the identity of the urediniospores as P. pachyrhizi. Although P. vulgaris is a known host of P. pachyrhizi, to our knowledge this is the first time since the arrival of soybean rust in 2001 that P. pachyrhizi has been observed on an alternate host plant in South Africa (2). Since dry beans are grown all year in frost-free areas, the implications are that dry beans may serve as an important overwintering host and source of inoculum for seasonal soybean rust outbreaks. References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) Z. A. Pretorius et al. Plant Dis. 85:1288, 2001. (3) J. B. Sinclair and G. L. Hartman. Soybean Rust. Pages 25-26 in: Compendium of Soybean Diseases, 4th ed. G. L. Hartman et al. eds. The American Phytopathological Society, St. Paul, MN, 1999.

Epidemics of Soybean Rust (Phakopsora pachyrhizi) in Brazil and Paraguay from 2001 to 2003.

In 5 March 2001, a severe rust outbreak was recorded at Pitapó, Paraguay, and the causal organism was determined to be Phakopsora pachyrhizi using polymerase chain reaction (PCR) and DNA sequence analysis. In May, rust surveys showed spread throughout most of Paraguay and into western and northern Parana, Brazil. In the 2001-02 season, rust was widespread in Paraguay, but losses were reduced due to severe drought; however, in Brazil it spread to more than 60% of the soybean acreage, causing field losses estimated at 0.1 million metric tons (MMT). In 2003, the disease was observed in more than 90% of the fields in Brazil, and the projected losses in Mato Grosso and Bahia alone are 2.2 MMT (US$487.3 million). Approximately 80% of the soybean acreage in Brazil was sprayed twice with fungicides at the cost of US$544 million. Differences in efficacy have been observed among the commercial strobilurin and triazol fungicides.

Human utilization of subsurface extraterrestrial environments.

Caves have been used in the ancient past as shelter or habitat by many organisms (including humans). Since antiquity, humans have explored caves for the minerals they contain and sometimes for ceremonial purposes. Over the past century, caves have become the target of increasing exploration, scientific research, and recreation. The use of caves on extraterrestrial bodies for human habitation has been suggested by several investigators. Lunar lava tube bases received early attention because lava tubes were clearly visible in lunar images from the Apollo Era. More recently, Mars Observer Camera data has shown us clear evidence of large tubes visible in a number of volcanic regions on Mars. The budding field of cave geomicrobiology has direct application to questions about subsurface life on other planets. Caves contain many unusual organisms making their living from unlikely materials like manganese, iron, and sulfur. This makes caves and other subsurface habitats prime targets for astrobiological missions to Mars and possibly other bodies. We present the results of a completed Phase I and on-going Phase II NASA Institute for Advanced Concepts (NIAC) study that intensively examines the possibilities of using extraterrestrial caves as both a resource for human explorers and as a highly promising scientific target for both robotic and future human missions to Mars and beyond.

Improved Detection of Tilletia indica Teliospores in Seed or Soil by Elimination of Contaminating Microorganisms with Acidic Electrolyzed Water.

Acidic electrolyzed water (AEW) is a germicidal product of electrolysis of a dilute solution (e.g., 0.4% vol/vol) of sodium chloride. This solution can be used to disinfest wheat seed or soil samples being tested for teliospores of Tilletia indica, causal agent of Karnal bunt, without risk of damaging the teliospores. The AEW used in this study had a pH of 2.5 to 2.8 and oxidation-reduction potential of approximately 1,130 mV. In simulations of routine extractions of wheat seed to detect teliospores of T. indica, the effectiveness of a 30-min AEW treatment was compared with a 2-min 0.4% sodium hypochlorite (NaOCl) treatment to eradicate bacteria and nonsmut fungi. Each treatment reduced bacterial and fungal populations in wheat seed extracts by 6 to 7 log10 units when determined on 2% water agar with antibiotics. Reductions of 5 log10 units or more were observed on other media. NaOCl and AEW also were very effective at eliminating bacteria and fungi from soil extracts. In studies to detect and quantitate T. indica teliospores in soil, AEW was nearly 100% effective at eliminating all nonsmut organisms. Free chlorine levels in AEW were very low, suggesting that compounds other than those with chlorine play a significant role in sanitation by AEW. The low pH of AEW was shown to contribute substantially to the effectiveness of AEW to reduce microorganisms. A standardized protocol is described for a 30-min AEW treatment of wheat seed washes or soil extracts to eliminate contaminating microorganisms. A significant advantage of the use of AEW over NaOCl is that, with AEW, teliospore germination is not reduced and usually is stimulated, whereas teliospore germination declines after contact with NaOCl. The protocol facilitates detection and enumeration of viable teliospores of T. indica in wheat seed or soil and the isolation of pure cultures for identification by polymerase chain reaction. The germicidal effects of AEW, as demonstrated in this study, illustrate the potential of AEW as an alternative to presently used seed disinfestants.

Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons.

The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.