Philip F. Harmon

A Rapid PCR-Based Method for the Detection of Magnaporthe oryzae from Infected Perennial Ryegrass

Gray leaf spot caused by Magnaporthe oryzae is a serious disease of perennial ryegrass in the midwestern United States. Symptoms of gray leaf spot can be confused with those caused by other fungal diseases that also are common during periods of high temperatures and ample moisture. Because turf managers must select appropriate fungicides for remedial treatment, accurate and timely identification of the pathogen is essential for efficient and effective disease management. We developed and evaluated a polymerase chain reaction (PCR)-based method to detect M. oryzae in infected perennial ryegrass tissue. The method utilizes a commercially available kit that is used for isolation and amplification of plant DNA from leaf tissue. The kit protocol was modified and found to be reliable for the extraction of M. oryzae DNA from infected perennial ryegrass. Primers were designed to amplify a 687-bp fragment of the Pot2 transposon that is found in multiple copies in the genome of the pathogen. The protocol amplified amounts of purified DNA as low as 5 pg and consistently and specifically detected M. oryzae in single diseased leaf blades as well as in field samples of infected perennial ryegrass. The total time required for detection was approximately 4 to 8 h.

First Report of Downy Mildew Caused by a Peronospora sp. on Basil in Florida and the United States

Basil is grown as a specialty crop in greenhouse and field production in Florida and other regions of the United States. Downy mildew on basil (Ocimum basilicum) was detected from four production sites (Collier, Hendry, Miami-Dade, and Palm Beach counties) in south Florida in the fall of 2007, and within months, it was also found in west-central north Florida (Hillsborough County). Incidence reached nearly 100% on some of the affected crops and caused complete yield losses on basil grown both in the field for fresh market and potted herbs market. Symptoms developed during transit on basil that appeared symptomless at harvest. Symptoms initially appeared as yellowing on the lower leaves that was typically delineated by the veins, although in some cases the entire leaf area of the leaf surface was affected. A gray, fuzzy growth was apparent on the abaxial leaf surface. Microscopic observation detected dichotomous branching, hyaline sporangiophores (220 to 750 × 4 to 9 μm) bearing single sporangia. Sporangia were light brown, ovoid to slightly ellipsoid, and measured 14 to 15 × 15 to 18 μm. Oospores were not observed. Leaves of potted basil plants and coleus (Solenostemon scutellarioides) were inoculated with a suspension containing 1 × 105 sporangia/ml and sprayed till runoff (approximately 15 ml per plant) with a hand-held pressurized aerosol canister. Plants were covered with a plastic bag for 24 h and maintained in the greenhouse under ambient conditions. Noninoculated plants served as controls. After 7 days, symptoms typical of downy mildew occurred only on the inoculated basil plants and sporulation was confirmed microscopically. The internal transcribed spacer regions of an isolate collected in Hendry County were sequenced bidirectionally. The consensus sequence was deposited into GenBank (Accession No. FJ346561). Sequence data matched (100% homology) with a Peronospora sp. reported on sweet basil in Switzerland (GenBank Accession No. AY884605) and was similar (99% homology) to an isolate (GenBank Accession No. DQ523586) reported on coleus, although inoculation to coleus failed to confirm pathogenicity on this host. The sequence data also distinguished the isolate from P. lamii (87% homology) previously reported to occur on basil. The pathogen was identified as a Peronospora sp. based on morphological characteristics and sequencing homology (1-3). References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) S. Francis. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 688. CMI, Kew, England, 1981. (3) A. McLeod et al. Plant Dis. 90:1115, 2006.

Emergence and accumulation of novel pathogens suppress an invasive species

Emerging pathogens are a growing threat to human health, agriculture and the diversity of ecological communities but may also help control problematic species. Here we investigated the diversity, distribution and consequences of emerging fungal pathogens infecting an aggressive invasive grass that is rapidly colonising habitats throughout the eastern USA. We document the recent emergence and accumulation over time of diverse pathogens that are members of a single fungal genus and represent multiple, recently described or undescribed species. We also show that experimental suppression of these pathogens increased host performance in the field, demonstrating the negative effects of emerging pathogens on invasive plants. Our results suggest that invasive species can facilitate pathogen emergence and amplification, raising concerns about movement of pathogens among agricultural, horticultural, and wild grasses. However, one possible benefit of pathogen accumulation is suppression of aggressive invaders over the long term, potentially abating their negative impacts on native communities.

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.

A Rapid Resazurin-Based Microtiter Assay to Evaluate QoI Sensitivity for Alternaria alternata Isolates and Their Molecular Characterization

Chemical management of Alternaria brown spot of citrus is based upon the timely application of site-specific fungicides, many of which are vulnerable to the development of fungicide resistance. A rapid microtiter bioassay based on the colorimetric changes of resazurin (RZ) dye was developed to evaluate the sensitivity of Alternaria alternata to quinone outside inhibitor (QoI) fungicides. Four liquid media (complete medium, minimal medium, potato dextrose broth, and yeast peptone dextrose broth), five conidia concentrations (from 101 to 105 conidia/ ml), and five RZ concentrations (10, 20, 30, 40, and 50 μM) were evaluated. Complete medium at 105 conidia/ml and 40 μM RZ were identified as optimal for measuring RZ reduction. The effective concentration of two QoI fungicides (azoxystrobin and pyraclostrobin) needed to reduce RZ by 50% (EC50) was calculated and compared with those obtained from conidia germination tests on fungicide-amended media. Concordant EC50 values were observed (R2 = 0.923; P < 0.0001) from both methods. Resistant phenotypes were further characterized by the partial sequencing of the cytochrome b gene. Genetic variability associated with the presence or absence of two introns was observed among isolates. The identified resistant isolates had the amino acid substitution G143A, typical of QoI resistance in other fungi.

Evidence for Morphological, Vegetative, Genetic, and Mating-Type Diversity in Sclerotinia homoeocarpa

Morphology, vegetative compatibility groups, and molecular characteristics were compared among 47 isolates of the dollar spot pathogen Sclerotinia homoeocarpa. Isolates were collected from cool- and warm-season turfgrasses in Florida and the northern United States. Mycelial pigment accumulation, substratal stromata formation, and symptom development were used to separate the collection into two distinct morphological types: a common-type (C-type) and a Floridian-type (F-type). Phylogenetic relationships estimated from ITS sequences supported the morphological typing. Identification and characterization of the S. homoeocarpa mating-type locus revealed an idiomorphic organization for both C- and F-types with nearly equal frequencies of each mating types present in both groups. These findings suggest heterothallic control of mating and indicate potential for outcrossing in both groups. Dollar spot disease of turfgrass in Florida is caused by two distinct morphological types of S. homoeocarpa which may be cryptic species. These findings could have implications for disease management.

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.

Winter Survival of the Perennial Ryegrass Pathogen Magnaporthe oryzae in North Central Indiana

Winter survival of Magnaporthe oryzae in north central Indiana was investigated in response to reports and observations of sporadic disease incidence. Survival of the fungus in perennial ryegrass residue was assessed. Time course studies were designed to assess the conidia production potential of infested perennial ryegrass residue exposed to ambient and predetermined treatments. Approximately 50,000 conidia per gram dry weight were produced initially on infested residue. In all years of the study, ambient winter conditions in Lafayette, IN, reduced conidia production on residue to fewer than 60 conidia per gram by spring. Unless residue was dried prior to treatment, storage of residue at all temperatures tested reduced conidia production potential. Airborne M. oryzae conidia, over the plot of perennial ryegrass where the winter survival studies were conducted, were estimated from particles collected with a volumetric air sampler. The stand of perennial ryegrass was inoculated with residue infested with M. oryzae in the summer of 2000 and late fall of 2000 and 2001. The stand of ryegrass was not inoculated in the summers of 200l or 2002. Conidia were captured with the air sampler each year in early July, before outbreaks had been observed in north central Indiana, but capture peaked in September when outbreaks most often occur. Cumulative conidia capture followed disease severity in 2000; however, no disease was observed in 2001 or 2002. Results of this study suggest poor survival of M. oryzae, and inadequate populations of viable pathogen limit disease development in north central Indiana. Disease risk assessment in north central Indiana should include an estimate of viable inoculum.

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.