© 2005 Plant Management Network.
Asian Soybean Rust Caused by Phakopsora pachyrhizi on Soybean and Kudzu in Florida
Philip F. Harmon, University of Florida, IFAS, Department of Plant Pathology, Gainesville 32611; M. Timur Momol, J. J. Marois, and Hank Dankers, University of Florida, IFAS, Department of Plant Pathology, North Florida Research and Education Center, Quincy 32351; and Carrie L. Harmon, Southern Plant Diagnostic Network, University of Florida, IFAS, Department of Plant Pathology, Gainesville 32611
Harmon, P. F., Momol, M. T., Marois, J. J., Dankers, H., and Harmon, C. L. 2005. Asian soybean rust caused by Phakopsora pachyrhizi on soybean and kudzu in Florida. Online. Plant Health Progress doi:10.1094/PHP-2005-0613-01-RS.
Asian soybean rust caused by Phakopsora pachyrhizi was found on soybean and kudzu in Florida in November of 2004. The initial diagnosis of soybean rust was based on observations of symptoms and urediniospores. The two species of Phakopsora that cause rust diseases on soybean, P. pachyrhizi and P. meibomiae, cannot be differentiated with light microscopy. A rapid DNA extraction and PCR amplification protocol discriminated between the two species. The sequence of the amplified DNA product confirmed this first report of P. pachyrhizi in Florida.
Rust diseases of soybean (Glycine max) can be caused by two pathogens. The pathogens cannot be discriminated reliably by visual observations of symptoms or through light microscopy (1). The Asian soybean rust pathogen, Phakopsora pachyrhizi, was first identified in Japan in 1903 and was limited to the Eastern hemisphere until reported in Hawaii in 1994 (3). Phakopsora pachyrhizi was first detected in Paraguay in South America in 2001 and was reported in soybean production areas of Brazil in 2003 (5). In November of 2004, P. pachyrhizi was confirmed for the first time in the continental U.S. near Baton Rouge, LA (7). A less aggressive soybean rust pathogen, P. meibomiae, is present in limited areas in the Western hemisphere, including Puerto Rico (5).
Soybean rust symptoms begin as small tan lesions on lower sides of leaves. Lesions expand and become visible on upper leaf surfaces. One to several erumpent and globose uredinia form in lesions typically on lower leaf surfaces. Lesions are less common on petioles, pods, and stems. Premature defoliation and early senescence occur when disease is severe (5). Urediniospores are obovoid to ellipsoid and colorless to pale yellowish brown (8).
Detection of Soybean Rust
Rust symptoms were observed on soybean plants on Nov. 15, 2004 and kudzu vines (Pueraria lobata) on November 16, 2004 in Gadsden Co., FL. Typical rust lesions described above were observed (Fig. 1). Soybeans were located in research plots at the UF-IFAS North Florida Research and Education Center in Gadsden Co., and kudzu was growing along road-sides nearby. Approximately 90% of soybean plants in the research plot were affected. Disease severity on affected leaves was estimated to be 75%. Approximately 25% of kudzu leaves observed in the roadside frondescence showed rust symptoms. Affected leaves were estimated to have lesions and pustules covering 10% of the leaf surfaces. Uredinia were observed to be more abundant on soybean than kudzu. Urediniospores were colorless to pale yellow in color (Fig. 2) and measured 21 × 31 µm (mean of 15 spores) and 24 × 32 µm (mean of 15 spores) from soybean and kudzu, respectively.
Identification of Pathogen
To confirm the causal agent of rust disease on soybean and kudzu in Florida, total DNA was extracted from symptomatic plant tissues with the Extract-N-Amp Plant PCR Kit (XNAP, Sigma-Aldrich Co., St. Louis, MO). Plant samples were processed within 24 h of collection, because storage at room temperature for longer periods of time was found to increase the likelihood of false negatives (data not shown). The manufacturer’s protocol was modified according to Lartey et al. (4) and Harmon et al. (2). Extractions were performed on excised soybean and kudzu leaf tissue (0.5 cm2) exhibiting sporulating rust pustules. Tissue pieces were placed in 1.5 ml Eppendorf tubes. Sufficient extract solution (provided in the kit) was added to cover the plant material (100 µl), and tubes were incubated at 95°C for 10 min. After incubation, tubes were placed on ice, and an equal volume (100 µl) of dilution solution (provided in kit) was added to the tube before the sample was homogenized with a polypropylene pestle for 30 seconds. An aliquant (30 µl) of the liquid sample extract was diluted 10-fold in sterile distilled water. The remaining sample extract was stored at -20°C for future use.
DNA extracts from symptomatic plant tissues were processed with a polymerase chain reaction (PCR) method utilizing species-specific primers (1). Primer sets Ppa1:Ppa2, specific for P. pachyrhizi, and Pme1:Pme2, specific for P. meibomiae, were selected from Frederick et al. (1). Both primer sets were reported to produce diagnostic amplicons approximately 330 bp in length. Each PCR reaction mix contained sample extract or dilute purified DNA (4 µl), 60 ng of each primer of one of the sets (6 µl), and the PCR reaction mix (10 µl) (provided in the kit). The PCR program consisted of initial denaturation at 94°C for 2 min followed by 35 cycles of 30-s denaturation at 94°C, 30 s of annealing at 65°C, and 30 s of extension at 72°C. Final extension was 10 min at 72°C. Amplification products were subjected to electrophoresis in a 2% agarose gel and stained for 10 min in an ethidium bromide solution (10 µg/ml). The gel was illuminated and DNA bands visualized with UV light. Gel images were captured with a Bio-Rad Gel Doc 2000 imaging system (Bio-Rad Laboratories, Hercules, CA).
Six excised soybean leaf disks and three kudzu leaf disks were processed in PCR reaction mixtures with primer set Ppa1:Ppa2, specific for P. pachyrhizi. A diagnostic DNA band approximately 330 bp in length was detected from five of six soybean leaf extracts and from all 3 kudzu leaf extracts (Fig. 3). In a subsequent PCR reaction, the diagnostic 330 bp band was produced from the extract of the negative soybean sample from the first reaction (data not shown). Purified P. pachyrhizi DNA (80 ng in 4 µl) was amplified as a positive control, and the diagnostic 330 bp band was observed. A faint band approximately 330 bp in size also was observed when P. meibomiae DNA (80 ng in 4 µl) was used as a negative control.
Sample extracts also were processed in PCR reaction mixtures with primer set Pme1:Pme2, specific for P. meibomiae (data not shown). No diagnostic 330 bp band was observed from any of the plant samples processed. Purified P. meibomiae DNA (80 ng in 4 µl) was amplified in a positive control, and the diagnostic 330 bp band was observed. A faint band approximately 330 bp in size also was observed when P. pachyrhizi DNA (80 ng in 4 µl) was used as a negative control. These results indicate that sample extracts should be processed with both primer sets, especially if a faint band is observed, because observation of a faint band produced with Ppa1:Ppa2 primers could indicate little P. pachyrhizi DNA in the sample or non-specific amplification of P. meibomiae DNA.
PCR product in DNA bands produced from amplification of soybean leaf extract was excised from the gel. DNA was extracted, purified (Qiaquick Gel Extraction kit, Qiagen Inc., Valencia, CA), and sent for sequencing to the DNA Sequencing Core Laboratory, University of Florida, Gainesville, FL. The DNA fragment was sequenced bidirectionally (two runs each direction) with the Ppa1:Ppa2 primer set. Results from the four sequencing runs were aligned and combined to produce a sequence of 241 bp. Because of discrepancies in the sequences produced, four nucleotide base pairs could not be resolved.
The nucleotide-nucleotide Blast program (web-based database search tool, National Center for Biotechnology Information, Bethesda, MD) was used to compare the edited sequence of 241 bp obtained to known DNA sequences. The sequence was found to be 98% identical (4 nucleotides could not be determined in our sequence) to a sequence published for P. pachyrhizi isolate MUT Zimbabwe by Frederick (1). The next closest match (not P. pachyrhizi) was 92% identical and was published for P. meibomiae isolate PR also by Frederick (1). These results confirm the rust pathogen on soybean and kudzu observed in Florida in November of 2004 was P. pachyrhizi.
A real-time PCR protocol modified from Frederick et al (1) is used by USDA APHIS labs to confirm Asian soybean rust. The protocol described here differs from the USD APHIS protocol because samples are processed with a rapid DNA extraction kit that does not require liquid nitrogen, different primer sets are used, and the thermocycler program is optimized for traditional (not real-time) PCR. The protocol described here offers labs without real-time PCR equipment an alternative for confirming samples subsequent to the initial USDA APHIS confirmation in a state.
This is the first report of Asian soybean rust in Florida. Florida had been identified as a potential location for the over-winter survival of P. pachyrhizi (6). Leguminous crops, weeds, ornamentals, and forages could potentially serve as alternative hosts. The impact of this pathogen on these potential hosts and the contribution of inoculum produced in Florida to soybean crops in the rest of the U.S. are currently unknown. However, in February and March of 2005, P. pachyrhizi was confirmed with this protocol on kudzu leaf samples collected by the Florida Department of Agriculture and Consumer Services, Division of Plant Industry in Pasco and Hernando Counties in Florida.
The authors wish to thank Reid Frederick, USDA, ARS, NAA for his assistance and for providing purified genomic DNA of P. pachyrhizi and P. meibomiae and the Florida Department of Agriculture and Consumer Services, Division of Plant Industry.
1. Frederick, R. D., Snyder, C., Peterson, G. L., and Bonde, M. R. 2002. Polymerase chain reaction assays for the detection and discrimination of the soybean rust pathogens Phakopsora pachyrhizi and P. meibomiae. Phytopathology 92:217-227.
2. Harmon, P. F., Dunkle, L. D., and Latin, R. 2003. A rapid PCR-based method for the detection of Magnaporthe oryzae from infected perennial ryegrass. Plant Dis. 87:1072-1076
3. Kilgore, E., and Heu, R. 1994. First report of soybean rust in Hawaii. Plant Dis. 78:1216
4. Lartey, R. T., Weiland, J. J., Caesar, T., and Bucklin Comiskey, S. A. 2003. A PCR protocol for rapid detection of Cercospora beticola in infected sugar beet tissues. J. Sugarbeet Res. 40:1-10
6. Pivonia, S., and Yang, X. B. 2004. Assesment of the potential year-round establishment of soybean rust throughout the world. Plant Dis. 88:523-529
7. Schneider, R. W., Hollier, C. A., Whitam, H. K., Palm, M. E., McKemy, J. M., Hernandez, J. R., Levy, L., and DeVries-Paterson, R. 2005. First report of soybean rust caused by Phakopsora pachyrhizi in the continental United States. Plant Dis. (In press).
8. Sinclair, J. B. 1999. Soybean rust. Pages 25-26 in: Compendium of Soybean Diseases, 4th ed. American Phytopathological Society, St. Paul, MN.