© 2008 Plant Management Network.
Hosts of Phakopsora pachyrhizi Identified in Field Evaluations in Florida
T. L. Slaminko, Department of Crop Sciences, University of Illinois, and M. R. Miles, USDA-ARS, National Soybean Research Center (NSRC), Urbana, IL 61801; J. J. Marois, Department of Plant Pathology, and D. L. Wright, Department of Agronomy, University of Florida, North Florida Research and Education Center, Quincy, FL; and G. L. Hartman, USDA-ARS and Department of Crop Sciences, NSRC, University of Illinois, Urbana, IL 61801
Slaminko, T. L., Miles, M. R., Marois, J. J., and Hartman, G. L. 2008. Hosts of Phakopsora pachyrhizi identified in field evaluations in Florida. Online. Plant Health Progress doi:10.1094/PHP-2008-1103-01-RS.
Phakopsora pachyrhizi, the causal organism of soybean rust, was first found on Glycine max in the continental United States in 2004, and subsequently on Pueraria lobata, Desmodium tortuosum, three Phaseolus species, and Erythrina herbacea in the field. The pathogen has been reported to occur on over 150 legume species worldwide, and it is likely to infect native and introduced legume species in the USA. The objective of this study was to determine if USA-native or -naturalized legume species could become infected with P. pachyrhizi in field conditions. A total of 80 accessions representing 52 species in 29 genera were infected in the field trials. Crotalaria retusa, Lathyrus latifolius, Phaseolus angustissimus, P. polystachios, and Robinia hispida are new hosts. This is the first report showing the broad host range of P. pachyrhizi based on field infections in the USA. Some of these hosts grow in the southern USA, and could, like kudzu, overwinter P. pachyrhizi.
Soybean rust, caused by the fungus Phakopsora pachyrhizi, was first described in Japan in 1903 on Glycine max subsp. soja (8), and was subsequently described in Taiwan in 1914 on Pachyrhizus erosus, or yam bean (19). It is a serious disease on soybean (Glycine max) in Africa, Asia, Australia, and South America (13), but its impact on soybean production in North America since its arrival in 2004 (16) has been limited.
There are over 150 host species of P. pachyrhizi reported (12,14,17,18), all of which belong to the legume family Fabaceae and subfamily Papilionoideae. The subfamily contains an estimated 476 genera and 13,860 species and has a cosmopolitan distribution (2). Approximately 120 of the 150 known host species of P. pachyrhizi can be found in North America (9).
Most reported hosts of P. pachyrhizi were determined in inoculated trials in controlled environments (10,11,14,15,18). Approximately 50 of the reported hosts were observed in the field or in their natural settings around the world (4,14). In the USA, only seven species have been reported as hosts in the field: soybean (16); kudzu, Pueraria lobata (6); Florida beggarweed, Desmodium tortuosum (17); dry bean, Phaseolus vulgaris; lima bean, P. lunatus; scarlet runner bean, P. coccineus (12); and coralbean, Erythrina herbacea (22). P. pachyrhizi was also reported on yam bean in Mexico in January 2007 (3). With the appearance of soybean rust in the continental USA, many legume species not previously exposed to P. pachyrhizi may now become hosts. These new hosts may aid in the overwintering of the fungus and provide a source of primary inoculum to soybean. The hosts that support natural infection may impact the distribution and spread of P. pachyrhizi and may play a role in the management of the disease. The objective of this research was to test previously identified legume hosts (18) in non-inoculated field conditions.
Selection of Hosts for Field Testing and Confirmation of Rust Infection
A total of 263 accessions in the legume subfamily Papilionoideae were tested in 2 years. Most of these species occur in southern or major soybean-producing states in the USA. Entries identified by the presence of rust-like lesions in a greenhouse trial in Ft. Detrick, MD (18) were included in this study. The list of entries differed in the 2 years as some seed were not available the second year and some additional entries were added. Seed was ordered from several repositories in the USDA-ARS National Plant Germplasm System (Table 1).
Table 1. Legume host species and accessions that were infected with Phakopsora pachyrhizi in a field location at Quincy, FL, and the seed source and sporulation information for each species.
v Species names and authorities as found in the GRIN (20) and ILDIS (9) online databases.
w Accession identifiers: Ames = Ames, IA; CU = Champaign-Urbana, IL; DLEG = Desert Legume Program; Guatemala = seed originally from Guatemala; NSL = National Seed Lab; PI = Plant introduction. * Accessions observed with uredinia and urediniospores.
x DLEG = Desert Legume Program, Tucson, AZ; kudzu = kudzukingdom.com; NC7 = North Central Regional Plant Introduction Station, Ames, IA; NPGS = National Center for Genecic Resources Preservation; S9 = Plant Genetic Resources Conservation Unit, Griffin, GA; W6 = Western Regional Plant Introduction Station, Pullman, WA; UIUC = University of Illinois, Urbana, IL.
y "+" = The presence of sporulating uredinia for a given species.
z "+" = confirmation of P. pachyrhizi by qPCR assay. Note: samples were not assayed if the presence of sporulating uredinia confirmed infection by P. pachyrhizi.
Between two and twenty seeds were planted per cell in 6 × 12 peat flats (Jiffy-520, Jiffy Poly Pak) containing soilless medium (Sunshine Mix, LC1, Sun Gro Horticulture Inc., Bellevue, WA) in a rust-free greenhouse at the University of Illinois in Urbana, IL. Five weeks after planting — on 06 Sept 2006 and 30 August 2007 — plants were transplanted into a field at the North Florida Research and Education Center (NFREC) in Quincy, FL. The experiment was a randomized complete block design with a soybean border and spreader rows between replications. There were three replications of 164 entries in 2006 and four replications of 203 entries in 2007; 104 entries overlapped in both years. Soybean and radish were used as susceptible and non-host controls, respectively. The field was located adjacent to experimental plots of infected soybeans. Plants in the trials were not inoculated. The experiments ended prior to a frost event, approximately 12 weeks after transplanting.
Samples were collected from all entries at the end of the trial. Several weed samples were also collected from each replication. Samples were evaluated macroscopically and with the aid of a stereoscope. Approximately 1 cm² of tissue was excised and frozen at -20°C in a 1.5-ml microcentrifuge tube for PCR analysis. Select samples were pressed and dried for herbarium records.
Lesion type and sporulation were assessed for each plant. Lesions were rated as either tan (TAN), reddish-brown (RB), or a mixture of both (1). Lesion types that differed from the typical TAN or RB were noted. These included black, brown, and red lesions as well as necrotic flecks (18). Sporulation was rated as 1 if sporulating uredinia were present and 0 if absent.
Additional confirmation of infection was necessary if morphological features indicative of P. pachyrhizi were absent. For such entries, thawed tissue was disrupted using FastDNA lysing tubes (Lysing Matrix A, Qbiogene, Irving, CA) containing garnet particles and 0.6-cm diameter ceramic beads and buffer AP1 from the DNeasy Plant Mini Kit (Qiagen, Germantown, MD). Tubes were placed in the FastPrep instrument (FP120, Qbiogene) and disrupted on speed 6 (6.0 m/s²) for 40 s after which DNA was extracted using the DNeasy Plant Mini Kit and protocol. Each group of extractions included a plain extraction buffer negative control and a known infected soybean positive control. Extracted DNA eluate was diluted 1/10 prior to PCR analysis.
The quantitative PCR assay, primers, and probe for detection and quantification of P. pachyrhizi were developed in 2002 (5). A modified method was performed on 2.0-µl samples in a total volume of 25 µl per reaction. Each reaction contained Platinum qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA); 7 mM MgCl2; 0.3 µM primer Ppm1; 0.3 µM primer Ppa2; 0.1 µM dual-labeled probe (FAM and TAMRA); 50 nM ROX reference dye; and ultrapure water. A real-time, quantitative PCR (qPCR) instrument (MX3005P, Stratagene, La Jolla, CA) was used with the following cycling conditions: 120 s at 60°C, 120 s at 95°C, and 40 cycles of 15 s at 95°C and 30 s at 60°C. Each lot included at least one positive and negative extraction control, and all extracts were assayed in duplicate. Quantification of DNA was estimated by using serial dilutions of DNA extracted from a known number of spores (7). A sample was considered positive if it had a stronger signal than those of the non-host controls and weed samples collected from the field.
In order to prevent false positives in these trials, samples of non-host weed species were collected and qPCR assayed with the other samples in order to estimate the baseline, or environmental, spore levels. The amount of "background" signal that was detected on non-hosts was used to set the limit for what would be considered positive in the test. Quantification showed that all weed species had less P. pachyrhizi DNA than that of 200 spores per 1-cm² sample.
Confirmation of Previously Reported Hosts and New Hosts of Soybean Rust
A total of 80 accessions representing 52 species in 29 genera were infected in the 2006 and 2007 field trials (Table 1). Of these, 35 species in 21 genera (53 accessions) produced sporulating uredinia. The remaining 17 species had lesions without sporulation and were confirmed infected with P. pachyrhizi by PCR. In 2006, 40 species in 25 genera were infected with P. pachyrhizi with 26 species in 20 genera producing sporulating uredinia. In 2007, 30 species in 18 genera were positive with 20 species in 13 genera producing sporulating uredinia. No infection occurred on 145 entries.
Nearly all accessions that became infected produced RB lesions (Fig. 1). The exceptions were the soybean controls and Desmodium canadense, which had TAN lesions and Lupinus angustifolius, which had black lesions. TAN lesions are considered by some to be a more susceptible reaction than RB lesions (1), but RB is the more common reaction type among alternative hosts. TAN lesions frequently have higher levels of sporulation than RB lesions and they are found commonly on soybean and closely related host species.
Five new species are reported as hosts of P. pachyrhizi: Crotalaria retusa (rattleweed); Lathyrus latifolius (perennial pea); Phaseolus angustissimus (slimleaf bean); P. polystachios (thicket bean); and Robinia hispida (bristly locust). Two new subspecies are also reported: P. polystachios subsp. polystachios and P. polystachios subsp. sinuatus. All new host species and subspecies produced uredinia and urediniospores except C. retusa, which produced RB lesions that were confirmed as soybean rust by PCR assay. Additional information, like geographic distribution, on any specific host of P. pachyrhizi can be found at the USDA PLANTS Database website (21). For example, these five new species all have a broad distribution that includes most of the gulf coast states in the USA, except for P. angustissimus that is limited in geographic range to Texas.
Two of the five new host species, C. retusa and L. latifolius, were also tested in the greenhouse trial (18), but failed to become infected. The failure of some species tested to support the production of uredinia and urediniospores in one location, but not in another could be explained by environmental conditions and/or isolate differences of P. pachyrhizi. A host without urediniospores but with a confirmed P. pachyrhizi lesion should not be discounted as a potentially important host because of the significant role of environment and even microclimate on sporulation. This is different from a host that consistently does not sporulate and often is difficult to infect. These "hosts" are usually more distantly related to soybean (e.g., species of Medicago and Trifolium) and will not likely influence soybean rust spread. Therefore, the importance of infection of these hosts may not be so important in the field or in the overwintering of the pathogen, but may be of interest in plant systematics and evolutionary biology.
Of the 263 accessions tested in the field, 152 of these accessions were positive (81 with sporulating uredinia and 71 by PCR) for P. pachyrhizi infection under controlled greenhouse conditions (18); but only 80 accessions (53 with sporulating uredinia) were positive in the field. Five new host species are reported and all but P. angustissimus have a broad geographic range including most if not all the gulf coast states in the USA. To date, there has been no concerted scouting effort in the USA to determine which host species other than kudzu could be supporting and overwintering the pathogen. This study, in addition to the preceding greenhouse screening (18), provides additional information on the host range of P. pachyrhizi in the USA, nearly doubling the number of known host species. This is important because many of the hosts with sporulating uredinia identified in this study are native or naturalized perennials that occur in areas where they can overwinter P. pachyrhizi. Some examples include various species of Crotalaria, Desmodium, Lathyrus, Macroptilium, Phaseolus, Tephrosia, and Vigna (21). It can be expected that the hosts with sprorulating uredinia in this field trial would be capable of having the same reaction with the fungus if infected in similar conditions. Therefore, these hosts should be monitored in areas where P. pachyrhizi is known to occur.
We thank Todd Steinlage and Dr. David Smith for their help in sample collection and Tristan Mueller for help in field maintenance. We thank Dr. James Haudenshield for the P. pachyrhizi DNA standards used in qPCR. This research was funded in part by the Illinois Soybean Association, the North Central Soybean Research Program, and the United Soybean Board.
Trade and manufacturers’ names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.
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