C. L. Harmon

Plant Disease Diagnostic Capabilities and Networks

Emerging, re-emerging and endemic plant pathogens continue to challege our ability to safeguard plant health worldwide. Further, globalization, climate change, increased human mobility, and pathogen and vector evolution have combined to increase the spread of invasive plant pathogens. Early and accurate diagnoses and pathogen surveillance on local, regional, and global scales are necessary to predict outbreaks and allow time for development and application of mitigation strategies. Plant disease diagnostic networks have developed worldwide to address the problems of efficient and effective disease diagnosis and pathogen detection, engendering cooperation of institutions and experts within countries and across national borders. Networking maximizes impact in the face of shrinking government investments in agriculture and diminishing human resource capacity in diagnostics and applied pathology. New technologies promise to improve the speed and accuracy of disease diagnostics and pathogen detection. Widespread adoption of standard operating procedures and diagnostic laboratory accreditation serve to build trust and confidence among institutions. Case studies of national, regional, and international diagnostic networks are presented.

Winter Survival of the Soybean Rust Pathogen, Phakopsora pachyrhizi, in Florida

Soybean rust (SBR) survival and host availability (kudzu, Pueraria spp.) were assessed from November 2006 through April 2007 at six sites from the panhandle to southwest Florida. Micro loggers recorded both temperature and relative humidity hourly at each location. Periods of drought and cumulative hours below 0°C correlated with kudzu defoliation. Inoculum potential from detached kudzu leaves was evaluated in vitro under various temperature and relative humidity levels. Kudzu leaves with SBR kept at 4°C produced viable urediniospores with the highest germination at all moisture levels over time. Freezing temperatures (-4 and -20°C) drastically reduced spore germination. However, when leaves were incubated at low (<35%) relative humidity, inoculum potential was prolonged. Results from this study demonstrate that both temperature and relative humidity impact P. pachyrhizi in the field and in vitro, and that detached kudzu leaves have the potential to serve as an inoculum source in kudzu stands.

Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics

BackgroundPlant viruses in the genus Begomovirus, family Geminiviridae often cause substantial crop losses. These viruses have been emerging in many locations throughout the tropics and subtropics. Like many plant viruses, they are often not recognized by plant diagnostic clinics due in large part to the lack of rapid and cost effective assays. An isothermal amplification assay, Recombinase polymerase amplification (RPA), was evaluated for its ability to detect three begomoviruses and for its suitability for use in plant diagnostic clinics. Methods for DNA extraction and separation of amplicons from proteins used in the assay were modified and compared to RPA manufacturer’s protocols. The modified RPA assays were compared to PCR assays for sensitivity, use in downstream applications, cost, and speed.ResultsRecombinase polymerase amplification (RPA) assays for the detection of Bean golden yellow mosaic virus, Tomato mottle virus and Tomato yellow leaf curl virus (TYLCV) were specific, only amplifying the target viruses in three different host species. RPA was able to detect the target virus when the template was in a crude extract generated using a simple inexpensive extraction method, while PCR was not. Separation of RPA-generated amplicons from DNA-binding proteins could be accomplished by several methods, all of which were faster and less expensive than that recommended by the manufacturer. Use of these modifications resulted in an RPA assay that was faster than PCR but with a similar reagent cost. This modified RPA was the more cost effective assay when labor is added to the cost since RPA can be performed much faster than PCR. RPA had a sensitivity approximate to that of ELISA when crude extract was used as template. RPA-generated amplicons could be used in downstream applications (TA cloning, digestion with a restriction endonuclease, direct sequencing) similar to PCR but unlike some other isothermal reactions.ConclusionsRPA could prove useful for the cost effective detection of plant viruses by plant diagnostic clinics. It can be performed in one hour or less with a reagent cost similar to that of PCR but with a lower labor cost, and with an acceptable level of sensitivity and specificity.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi on Phaseolus spp. in the United States

Phakopsora pachyrhizi Syd. & P. Syd., the cause of soybean rust, was first observed in the continental United States in November 2004 (2). During the growing season of 2005, P. pachyrhizi was confirmed on soybean (Glycine max) and/or kudzu (Pueraria montana) in nine states in the southern United States. It is known that P. pachyrhizi has a much larger host range within the Fabaceae family. On 29 September 2005 in Quincy, FL, 45 entries of mostly large-seeded legumes were planted next to soybeans that were infected with P. pachyrhizi. Several seeds of each entry were planted on one hill. Soybean plants growing adjacent to these potential hosts had 15 to 25% of the leaf area affected, 95% incidence, and 73% defoliation on 16 November. On 7 December 2005, all the plants of Phaseolus coccineus L. (scarlet runner bean, PI311827), Phaseolus lunatus L. (lima bean, PI583558), and two Phaseolus vulgaris L. (kidney bean) cvs. Red Hawk and California Early Light Red Kidney (CELRK) were found to have leaves with suspected rust lesions. These plants were at physiological maturity but had not senesced. None of the hosts had been inoculated other than from spores produced by the adjacent rust-infected soybean plants or from unknown locations. On the basis of microscopic examination, suspected infected leaves from plants of the Phaseolus spp. had rust pustules characteristic of P. pachyrhizi uredinia. Uredinia were counted within a randomly selected 2-cm2 area of one leaf of each sample. The mean and range of uredinia per lesion for Phaseolus coccineus was 29 uredinia with a range of 0 to 3 uredinia per lesion, Phaseolus lunatus had 2 uredinia with 0 to 1 uredinium per lesion, Phaseolus vulgaris cv. Red Hawk had 22 uredinia with 0 to 5 uredinia per lesion, and Phaseolus vulgaris cv. CELRK had 43 uredinia with 0 to 4 uredinia per lesion. Polymerase chain reactions using two sets of primers (Ppa1/Ppa2 and Pme1/Pme2) were performed on DNA extracted from leaves of the three species with sporulating rust pustules (1). The results of these tests and further tests conducted by the USDA/APHIS confirmed that P. pachyrhizi was the causal organism for the observed rust. References: (1) P. F. Harmon et al. On-line publication. doi:10.1094/PHP-2005-0613-01-RS. Plant Health Progress, 2005. (2) R. W. Schneider et al. Plant Dis. 89:774, 2005.

Characterization of kudzu (Pueraria spp.) resistance to Phakopsora pachyrhizi, the causal agent of soybean rust.

Kudzu (Pueraria spp.) is an accessory host for soybean rust (SBR) (caused by Phakopsora pachyrhizi) that is widespread throughout the southeastern United States. An expanded survey of kudzu sites was conducted in 2008 to determine the proportion of natural resistance in the north-Florida kudzu population. Of the 139 sites evaluated, approximately 18% were found to be free of SBR infection, while 23% had reduced sporulation. Ten accessions of kudzu from north-central Florida were characterized for their response to challenge by a single isolate of P. pachyrhizi under laboratory conditions. Three outcomes were observed: tan lesions with profuse sporulation (susceptible); reddish-brown lesions with delayed, reduced sporulation (resistant); and an immune response in which no lesions developed (immune). Of the 10 accessions, 6 were susceptible, 3 were immune, and 1 was resistant. Cytological examination revealed that resistant interactions were typified by early onset of a multicell hypersensitive response (HR) while typical immune interactions were the result of cell wall depositions that blocked penetration in combination with early onset of the HR. Quantitative real-time polymerase chain reaction was performed to determine the extent of colonization. After 15 days, there was 10-fold less P. pachyrhizi DNA present in resistant compared with susceptible kudzu, while the amount of P. pachyrhizi DNA present in the immune kudzu was below the detection level. Susceptible kudzu had approximately half the amount of P. pachyrhizi DNA present when compared with a susceptible soybean cultivar.

First Report of Phakopsora pachyrhizi Telia on Kudzu in the United States

Soybean rust caused by Phakopsora pachyrhizi H. Sydow & Sydow was first reported in the continental United States during 2004 (2). By 10 November 2005, the disease was confirmed in eight southern states (Florida, Georgia, Alabama, Mississippi, South Carolina, North Carolina, Louisiana, and Texas). Diagnoses have been based on visual observation of uredinia and urediniospores of the pathogen followed by polymerase chain reaction confirmation. On 10 November 2005, uredinia and telia were identified on leaves of kudzu (Pueraria lobata) in central Florida. Telia first were noted as dark brown-to-black flecks on the abaxial leaf surface intermingled with abundant tan-to-light brown uredinia. Of 200 leaves examined, 143 (72%) had telia. The number of telia ranged from a few (1/cm2) that were scattered to many (73/cm2). Telia were approximately the same diameter as uredinia, but were appressed to the leaf surface and pigmented. Twenty telia were excised from host tissue with the aid of a dissecting microscope and a 20 gauge hypodermic needle. Telia averaged 89 × 100 μm (n = 20, σ = 17 and 16 μm, respectively). Four telia were crushed and five teliospores from each averaged 4.3 × 8.3 μm (n = 20, σ = 0.5 and 0.9 μm, respectively). Pale yellowish brown-to-hyaline teliospores were similar in color to urediniospores. Observations matched descriptions by Ono et al. (1). To our knowledge, this is the first report of the telial stage of P. pachyrhizi in the United States. References: (1) Y. Ono et al. Mycol. Res. 96:825, 1992. (2) R. W. Schneider et al. Plant Dis. 89:774, 2005.

Effects of Shade Intensity and Duration on Asian Soybean Rust Caused by Phakopsora pachyrhizi

Field studies to quantify the effects of shade intensity and duration on soybean rust caused by Phakopsora pachyrhizi were carried out in Florida in 2006 and 2007. Soybean plants at the V4 stage were inoculated with urediniospores at 2100, 0000, and 0200 h. Inoculated plants were either placed in cages that were covered with shade cloths of different mesh sizes allowing 70, 50, or 20% transmission of sunlight or were not covered so that the plants received 100% of sunlight. Plants kept under 20 and 100% sunlight were sampled 12, 18, and 36 h after inoculation to determine the in vivo germination percentage of urediniospores and the percentage of germ tubes that formed appressoria. In separate experiments, inoculated plants were placed under the shade (20% sunlight) and moved to unshaded conditions after 1, 2, and 7 days. For all experiments, soybean rust incidence and severity were rated 12 days after inoculation. Higher levels of disease incidence and severity were detected in plants under shade compared with those under full sunlight. Shade duration greater than 2 days favored disease development. Within 36 h, in vivo germination of urediniospores and formation of appressoria were not significantly affected by the treatments. These results may explain why soybean rust is more severe in the lower canopy and shaded areas in the field.

First report of soybean rust caused by Phakopsora pachyrhizi on kudzu (Pueraria montana var. lobata) in Kentucky.

Phakopsora pachyrhizi, the causal organism of soybean rust, was first observed in the continental United States on 6 November 2004 (2). On 11 November 2005, as part a national soybean rust monitoring effort, 75 leaves of kudzu (Pueraria montana var. lobata) were arbitrarily collected from a patch growing in Princeton, Caldwell County, Kentucky (37.106650°N, 87.886120°W) that had been periodically scouted for the presence of the disease since May 2005. Upon microscopic examination of the nonincubated sample, a small (˜2.0 cm2) area of one leaf exhibited lesions, uredinia, and urediniospores characteristic of those reported for P. pachyrhizi (the Asian species) and P. meibomiae (the New World species) (2). No other infected leaves were observed despite repeated visits to the site and collection and observation of nearly 200 leaves. On 16 November 2005, one-half of the symptomatic tissue was sent by overnight courier to the USDA/APHIS/PPQ/NIS Laboratory, Beltsville, MD and the other half was sent to the Southern Plant Diagnostic Network Laboratory (SPDN), University of Florida, Gainesville. Both laboratories confirmed that the rust was a Phakopsora spp. on the basis of morphological examination. The preliminary polymerase chain reaction (PCR) testing conducted by the SPDN according to Harmon et al. (1) indicated the presence of P. pachyrhizi that was confirmed by the USDA/NPGBL using the validated modified real-time PCR assay described previously (2). The field diagnosis of P. pachyrhizi and preliminary PCR results were officially confirmed by USDA/APHIS on 18 November 2005. To our knowledge, this is the first report of P. pachyrhizi on kudzu or any host in Kentucky, and currently, the northernmost report of soybean rust on any host in the continental United States. References: (1) P. F. Harmon et al. On-line publication, doi:10.1094/PHP-2005-0613-O1-RS. Plant Health Progress, 2005. (2) R. W. Schneider et al. Plant Dis. 89:774, 2005.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi on Erythrina herbacea (Coral Bean)

Soybean rust (SBR), caused by the obligate fungus Phakopsora pachyrhizi Syd. & P. Syd., was initially reported on soybean (Glycine max L.) in Louisiana in 2004 and has since been reported on soybean and/or kudzu (Pueraria lobata (Willd.) Ohwi) in 9 states in 2005, 15 states in 2006, and 19 states in 2007 (1). The host range of P. pachyrhizi includes plants that are all in the Fabaceae or legume family. Six plant species in the United States have been reported as hosts of P. pachyrhizi: soybean, kudzu, Florida beggarweed (Desmodium tortuosum (Sw) DC.), dry bean (Phaseolus vulgaris L.), lima bean (P. lunatus L.), and scarlet runner bean (P. coccineus L.) (4). On 17 April 2008, a rust disease was observed on a weedy legume host with red showy flowers that was growing with kudzu in an overgrown vacant lot in the understory of live oak trees (Quercus virginiana Mill.) in Citra, FL. The discovery was made during routine scouting of this Integrated Pest Management Pest Information Platform for Extension and Education (IPM PIPE) mobile sentinel plot (3). The plant was confirmed by University of Florida botanists to be Erythrina herbaceae L., commonly known as coral bean. Coral bean is native to the southeastern United States and also is planted as a perennial ornamental. A sample of leaves exhibiting rust pustules characteristic of P. pachyrhizi uredinia was collected and examined with a microscope. Brown-to-brick red, angular lesions that were 3 to 11 mm in diameter (average 6.75 mm) were observed on the undersides of the leaves of two trifoliates. Within these lesions, there were several uredinia, some exuding hyaline, echinulate urediniospores (20 × 25 μm). The visual diagnosis and the species of the rust fungus were confirmed to be P. pachyrizi by a real-time PCR protocol (2). The diagnosis on this new host was verified by a USDA, APHIS National Mycologist in Beltsville, MD. Coral bean may serve as an additional overwintering host for P. pachyrhizi in the southeast. To our knowledge, this is the first report of soybean rust caused by P. pachyrhizi on E. herbaceae. References: (1) R. S. C. Christiano and H. Scherm, Phytopathology 97:1428, 2007. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) S. A. Isard et al. Online publication. doi:10.1094/PHP-2006-0915-01-RV. Plant Health Progress, 2006. (4) T. L. Slaminko et al. Plant Dis. 92:767, 2008.

Susceptibility of Latrodectus geometricus (Araneae: Theridiidae) to a Mucor Strain Discovered in North Central Florida, USA

ABSTRACT A population of adult reproductive female brown widow spiders, Latrodectus geometricus Koch (Araneae: Theridiidae) was collected in North Central Florida. Within weeks, the spiders displayed reduced foraging behavior and began to die. A white fungal growth was observed on deceased individuals and others who showed signs of decline. Fungal samples were collected from living spiders as well as from recently deceased specimens. Samples were excised from the spiders and isolated on acidified potato dextrose agar. Morphological characterization was cross referenced with the most recent phenotypic variability studies to confirm probable matches. Molecular identification was rendered using ITS1/5.8S/ITS2 and Mucor fragilis Bainier (Mucorales: Mucoraceae) was indicated with 99% match. Little is documented regarding M. fragilis, and no prior records of pathogenic occurrence are known by the authors. Subsequent bioassay trials supported with 83% mortality that this novel strain of M. fragilis does infect and kill L. geometricus.