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In recent years, some golf course superintendents in Florida have reported that the turf health is no longer as great, and nematode responses to fluopyram have decreased. The objective of this research was to determine if the mechanism of the reported reduced efficacy was attributable to either: i ) enhanced degradation accelerating its breakdown in the soil, or ii ) reduced sensitivity to the nematicide in the nematode populations. In a field experiment, soil and nematodes were collected from small plots that had been treated multiple times over four years, for only one year, or never treated. Soil and nematodes were additionally collected from commercial turf sites where either multiple applications of fluopyram had been made for numerous years, or it had never been used. Bioassay experiments found no evidence of enhanced degradation. However, M. graminis collected from small field plots and commercial sites with long-term use of fluopyram were less sensitive to fluopyram in-vitro than those from small plots and commercial sites where fluopyram had not been used. These results indicate that nematicide resistance is a likely cause of reduced fluopyram efficacy on golf-course turf in Florida., (© 2023 Christian L. Kammerer et al., published by Sciendo.)

Watermelon is an important crop in Florida, representing $88.2 million in cash receipts in 2015 (USDA/NASS 2017). In April and May 2021, the UF/IFAS Plant Diagnostic Center in Gainesville, Florida received eight diseased watermelon leaf samples from Alachua, Gilcrest, Levy, and Suwannee counties in Florida. Lesions were round to oblong, light gray to tan with reddish brown margins and white to light gray center, and some were coalescing resulting in about 15% disease severity. Symptomatic leaf tissue (0.5 cm2) was surface sterilized in 0.6% sodium hypochlorite for one minute, rinsed with sterile tap water, plated onto water agar media plates, and incubated at 27°C under 12-h light/dark cycle for 7 days. Characteristic Bipolaris conidia with gray to black brownish cottony mycelial growth were consistently found growing from plated lesions. The pathogen was isolated from two of the eight samples using a 0.5 mm diameter sterile metal needle to transfer a single conidium onto DifcoTM Potato Dextrose Agar (PDA) plates. Three isolates were designated G21-562 from Levy and G21-599a and G21-599b from Alachua County. All three isolates produced curved or straight, cylindrical, obclavate, distoseptate brownish gray conidia with 3 to 8 septa, mostly tapering towards ends with dark brownish to black hilum, that ranged from averaged 62um x 25um (n=30, SD=8 for length and 3 for width). Conidiophores were brownish, septate, smooth, and straight, single or in small groups, simple or branched, and swollen at the upper tip. Internal transcribed spacer region (ITS) and partial glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene sequences were amplified using primers ITS1/ITS4 and GPD-1/GPD-2 (Berbee et al. 1999). Reference sequences (Adhikari et al. 2020 and Manamgoda et al. 2014) were aligned using MUSCLE and trimmed to consistent length. Using concatenated sequence alignments of both loci, a maximum likelihood phylogenetic tree was constructed based on K2+G substitution model selected by BIC using Mega X (Kumar et al. 2018) with 1,000 bootstrap. The ITS and GPDG sequences of G21_599b, G21_599a and G21_562 (GenBank accessions OK614094 to 96, OP297398 to 400) showed 100% identity across 888 nucleotides across both loci to B. sorokiniana isolates CBS_110.14 and CBS_ 120.24 and were distinct from other reference isolates. To fulfill Koch's postulates, all three isolates were grown on PDA at 27°C and 12-h light/dark cycle. After a week, conidia were harvested in sterile water, and the conidial suspensions were adjusted to 105 conidia/ml using a hemocytometer. Each conidial suspension and Tween 20 water control was sprayed onto three seedlings of 'Sugar Baby' watermelon until runoff, and inoculated seedlings were sealed in a plastic bag for 24 hrs. The experiment was done in a greenhouse (20- 25°C) and repeated once. After a week of incubation, the same leaf lesion symptoms described above were observed on seedlings inoculated with conidia, whereas seedlings sprayed with the control were asymptomatic. Isolations from symptomatic tissue produced gray to black mycelia with conidia that were the same as described from field samples. To our knowledge, this is the first report of leaf spot on watermelon caused by B. sorokiniana. B. sorokiniana is a common pathogen of grasses and agronomic crops (Farr and Rossman 2020). The extent to which this emerging disease of Florida watermelon may negatively impact production is unknown and should be the subject of future observation and research.

Stem blight is a major disease of blueberry caused by Botryosphaeriaceae fungi. Chemical and cultural management options are limited, putting emphasis on breeding efforts to identify sources of resistance. The efficacy and durability of host resistance could be impacted by the species composition of the pathogen population in a region and by the isolates employed in the screenings used to identify the resistance. Samples (365) were collected from southern highbush (SHB) and rabbiteye blueberry (REB) cultivars from 28 sites in the southeastern US (AL, FL, GA, NC, and SC). Colony morphology identified 86% of the isolates as Botryosphaeriaceae. Conidia morphology and Maximum Likelihood analysis of the Internal Transcribed Spacer rDNA regions (ITS), translation elongation factor one alpha (tef1-α), and β-tubulin were used to identify isolates at genera or species level. A PCR-restriction fragment length polymorphism (PCR-RFLP) test was used to identify isolates to genus. Neofusicoccum and Lasiodiplodia were the predominant genera. N. kwambonambiense, N. ribis, L. theobromae and L. pseudotheobromae were the most common species isolated. Phylogenies conducted with a limited number of isolates indicated non-clonal and potentially diverse populations occur on blueberry that warrant additional study. Botryosphaeria corticis, B. dothidea, and Diplodia seriata were isolated infrequently., Competing Interests: Declarations of competing interest This scientific article was part of Amanda Wright's dissertation and is not reflective of the USDA-Farm Service Agency (FSA) viewpoints. The USDA/FSA takes no responsibility for any of the work published in this article., (Copyright © 2022 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

In recent decades, increasing attention has been paid to food safety and organic horticulture. Thus, people are looking for natural products to manage plant diseases, pests, and weeds. Essential oils (EOs) or EO-based products are potentially promising candidates for biocontrol agents due to their safe, bioactive, biodegradable, ecologically, and economically viable properties. Born of necessity or commercial interest to satisfy market demand for natural products, this emerging technology is highly anticipated, but its application has been limited without the benefit of a thorough analysis of the scientific evidence on efficacy, scope, and mechanism of action. This review covers the uses of EOs as broad-spectrum biocontrol agents in both preharvest and postharvest systems. The known functions of EOs in suppressing fungi, bacteria, viruses, pests, and weeds are briefly summarized. Related results and possible modes of action from recent research are listed. The weaknesses of applying EOs are also discussed, such as high volatility and low stability, low water solubility, strong influence on organoleptic properties, and phytotoxic effects. Therefore, EO formulations and methods of incorporation to enhance the strengths and compensate for the shortages are outlined. This review also concludes with research directions needed to better understand and fully evaluate EOs and provides an outlook on the prospects for future applications of EOs in organic horticulture production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chang, Harmon, Treadwell, Carrillo, Sarkhosh and Brecht.)

Blueberry ( Vaccinium spp.) plants are exposed to existing and emerging viruses as a result of expanding acreage of blueberry plantations across the world, primarily in North America. Since blueberry is cultivated in areas where there are wild Vaccinium spp., there is increasing risk of virus movement between wild and cultivated blueberries. This is theoretically possible because viruses can spread from commercial cultivars to native species and vice versa causing the spread of existing and new viruses. The occurrence of these viruses in blueberry can be devastating to the industry considering the cost for cultivation and production of this perennial crop. However, the advent of high-throughput sequencing and bioinformatic sequence analysis have allowed for rapid identification of known and novel viruses in any crop including blueberry, thus facilitating proper intervention in response to serious viral diseases. In this paper, we aim to focus on the current status of known and novel viruses emerging in blueberry worldwide, which may impact the blueberry industry.

Southern highbush blueberry (interspecific hybrids of Vaccinium corymbosum L.) is cultivated near wild V. corymbosum as well as closely related species in Florida, USA. The expansion of blueberry cultivation into new areas in Florida and deployment of new cultivars containing viruses can potentially increase the diversity of viruses in wild and cultivated V. corymbosum . In this study, viral diversity in wild and cultivated blueberries ( V. corymbosum ) is described using a metagenomic approach. RNA viromes from V. corymbosum plants collected from six locations (two cultivated and four wild) in North Central Florida were generated by high throughput sequencing (HTS) and analyzed using a bioinformatic analysis pipeline. De novo assembled contigs obtained from viromes of both commercial and wild sites produced sequences with similarities to plant virus species from a diverse range of families ( Amalgaviridae , Caulimoviridae , Endornaviridae , Ophioviridae , Phenuiviridae , and Virgaviridae ). In addition, this study has enabled the identification of blueberry latent virus (BlLV) and blueberry mosaic associated ophiovirus (BlMaV) for the first time in Florida, as well as a tentative novel tepovirus (blueberry virus T) (BlVT) in blueberry. To the best of our knowledge, this is the first study that compares viral diversity in wild and cultivated blueberry using a metagenomic approach.

Infectious diseases and invasive species can be strong drivers of biological systems that may interact to shift plant community composition. For example, disease can modify resource competition between invasive and native species. Invasive species tend to interact with a diversity of native species, and it is unclear how native species differ in response to disease-mediated competition with invasive species. Here, we quantified the biomass responses of three native North American grass species (Dichanthelium clandestinum, Elymus virginicus, and Eragrostis spectabilis) to disease-mediated competition with the non-native invasive grass Microstegium vimineum. The foliar fungal pathogen Bipolaris gigantea has recently emerged in Microstegium populations, causing a leaf spot disease that reduces Microstegium biomass and seed production. In a greenhouse experiment, we examined the effects of B. gigantea inoculation on two components of competitive ability for each native species: growth in the absence of competition and biomass responses to increasing densities of Microstegium. Bipolaris gigantea inoculation affected each of the three native species in unique ways, by increasing (Dichanthelium), decreasing (Elymus), or not changing (Eragrostis) their growth in the absence of competition relative to mock inoculation. Bipolaris gigantea inoculation did not, however, affect Microstegium biomass or mediate the effect of Microstegium density on native plant biomass. Thus, B. gigantea had species-specific effects on native plant competition with Microstegium through species-specific biomass responses to B. gigantea inoculation, but not through modified responses to Microstegium density. Our results suggest that disease may uniquely modify competitive interactions between invasive and native plants for different native plant species., Competing Interests: The authors have declared that no competing interests exist.

Guinea grass is an invasive perennial C4 grass and is a common weed around agricultural crops in Louisiana, Texas, and Hawaii, USA (Overholt and Franck 2019). In November 2018, leaf spots were observed on Guinea grass occurring in an organic garden located in Gainesville, Florida, USA. Lesions were oblong to irregular, dark grey to brownish center with pale-yellow to brownish black margin. Lesions had coalesced, forming necrotic margins that spread from the leaf tip, resulting in leaf blight and collapse of the canopy. Pieces of symptomatic leaf blades (5 sq cm) were surface sterilized (1 min), washed with sterile distilled water and plated onto water agar media plates. Plates were incubated at 27°C under 12-h light/dark for 3 to 5 days. Grey to black cottony mycelium was consistent on all plates and produced conidia characteristic of Bipolaris spp. Conidia were transferred to potato dextrose agar (PDA) plates with a 0.5 mm diameter sterile needle. Three isolates GG1, GG2 and GG3 were successfully grown on PDA. Conidia were black to brown colored, distoseptate with 3 to 8 septa and measured from (60.6- )70-105(-139.8) × (16.0-)17-23(-25.9) μm (avg: 93.3 μm, n=35, SD = 20.6; avg = 21.3 μm, n = 35, SD = 2.89). Conidiophores were in groups or single, brown, smooth and straight, septate and swollen at upper tip. Sigma Extract-N-Amp was used for genomic DNA extraction. Primers ITS1/ITS4 and GPD1/GPD2 (Berbee et al. 1999) were used to amplify and sequence the internal transcribed spacer region (ITS) and partial glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene, respectively. Sequences were aligned using MUSCLE and alignment was trimmed for length. Maximum likelihood phylogenetic trees were constructed with 1,000 bootstrap samples based on the K2+G substitution model, selected by BIC for these two loci using Mega X (Kumar et al. 2018). The ITS and GPDH sequences of GG1, GG2 and GG3 (Genbank accessions MT514518-20, MT576654-56), grouped with B. yamadae isolates CPC_28807 and CBS_202.29 in phylogenetic trees (Marin-Felix et al. 2017). All three isolates from Guinea grass were inoculated on Sach's agar (Luttrell 1958) at 27°C under 12-h light/dark for a week, but no sexual morph was observed, and consistent for two repeated inoculations. To fulfill Koch's postulates, one isolate, GG1, was used. Conidia were harvested from a one-week-old colony grown on PDA incubated at 27°C and 12-h light/dark cycle. The concentration of the conidial suspension was adjusted to 105 conidia/ml using a hemocytometer. Using a Passche H-202S airbrush sprayer, five-week-old seedlings of Guinea grass were sprayed until runoff with the conidia suspension or 0.5% tween water only. Each treatment included four replicates and the experiment was repeated. Leaf spot symptoms were observed on the seedlings inoculated with conidia, whereas seedlings sprayed with water were asymptomatic. Cultures with the expected morphology were isolated from symptomatic leaf blades and absent from control plants. To our knowledge, this is the first report of leaf spot on Guinea grass caused by B. yamadae in Florida, USA. B. yamadae was previously reported from Guinea grass in India, and from other Panicum species in the northern USA (Farr and Rossman 2019). B. yamadae was also isolated from sugarcane in Cuba and China, and corn in Japan (Manamgoda et al. 2014, Raza et al. 2019), which suggests that it has the potential to impact agronomic crops in Florida, such as sugarcane and corn.

Environmentally damaging invasive plants can also serve as reservoir hosts for agricultural pathogens. Microstegium vimineum is an invasive C4 annual grass that is present throughout the midwestern and eastern United States. It can reach high densities in disturbed areas such as crop-forest interfaces, which creates the potential for pathogen spillover from M. vimineum to agricultural crops and native plants. A previous study that surveyed disease on M. vimineum found a large-spored Bipolaris species that was widespread on M. vimineum and also isolated from co-occurring native grasses. Here, we report that the large-spored fungus isolated from M. vimineum and the native grass Elymus virginicus is Drechslera gigantea , based on comparison with published descriptions of morphological traits, and establish that D. gigantea is a pathogen of M. vimineum and E. virginicus . We review the phylogenetic placement and taxonomic history of D. gigantea and propose that it be reassigned to the genus Bipolaris as Bipolaris gigantea.

Non-native invasive plants can establish in natural areas, where they can be ecologically damaging and costly to manage. Like cultivated plants, invasive plants can experience a relatively disease-free period upon introduction and accumulate pathogens over time. Diseases of invasive plant populations are infrequently studied compared to diseases of agriculture, forestry, and even native plant populations. We evaluated similarities and differences in the processes that are likely to affect pathogen accumulation and disease in invasive plants compared to cultivated plants, which are the dominant focus of the field of plant pathology. Invasive plants experience more genetic, biotic, and abiotic variation across space and over time than cultivated plants, which is expected to stabilize the ecological and evolutionary dynamics of interactions with pathogens and possibly weaken the efficacy of infectious disease in their control. Although disease is expected to be context dependent, the widespread distribution of invasive plants makes them important pathogen reservoirs. Research on invasive plant diseases can both protect crops and help manage invasive plant populations.

A growing number of metagenomics-based approaches have been used for the discovery of viruses in insects, cultivated plants, and water in agricultural production systems. In this study, sixteen blueberry root transcriptomes from eight clonally propagated blueberry plants of cultivar 'Emerald' (interspecific hybrid of Vaccinium corymbosum and V. darrowi) generated as part of a separate study on varietal tolerance to soil salinity were analyzed for plant viral sequences. The objective was to determine if the asymptomatic plants harbored the latent blueberry red ringspot virus (BRRV) in their roots. The only currently known mechanism of transmission of BRRV is through vegetative propagation; however, the virus can remain latent for years with some plants of 'Emerald' never developing red ringspot symptoms. Bioinformatic analyses of 'Emerald' transcriptomes using de novo assembly and reference-based mapping approaches yielded eight complete viral genomes of BRRV (genus Soymovirus, family Caulimoviridae). Validation in vitro by PCR confirmed the presence of BRRV in 100% of the 'Emerald' root samples. Sequence and phylogenetic analyses showed 94% to 97% nucleotide identity between BRRV genomes from Florida and sequences from Czech Republic, Japan, Poland, Slovenia, and the United States. Taken together, this study documented the first detection of a complete BRRV genome from roots of asymptomatic blueberry plants and in Florida through in silico analysis of plant transcriptomes.

Petrifilms are dehydrated agar culture plates that have been used to quantify colony forming units (CFU) mL of either aerobic bacteria (Petrifilm-AC) or fungus (Petrifilm-YM), depending on substrate composition. Microbes in irrigation systems can indicate biofilm risk and potential clogging of irrigation emitters. The research objective was to compare counts on Petrifilms versus traditional, hydrated-agar plates using samples collected from recirculated irrigation waters and cultures of isolated known species. The estimated count (in CFU mL) from a recirculated irrigation sample after 7 d of incubation on Petrifilm-YM was only 5.5% of the count quantified using sabouraud dextrose agar (SDA) with chloramphenicol after 14 d. In a separate experiment with a known species, Petrifilm-YM did not successfully culture zoospores of . Isolates of viable zoospores were cultured successfully on potato-dextrose agar (PDA), with comparable counts with a vegetable juice medium supplemented with the antibiotics pimaricin, ampicillin, rifamycin, pentochloronitrobenzene and hymexazol (PARP-H). The quantification of pv. Begoniaceae on Petrifilm-AC was not significantly different ( < 0.05) than on PDA, but was lower than on Reasoner and Goldrich agar (R2A) or with a hemocytometer. The current formulation of Petrifilm-YM is unlikely to be a useful monitoring method for plant pathogens in irrigation water because of the inability to successfully culture oomycetes. However, Petrifilm-AC was an effective method to quantify bacteria and can provide an easy-to-use on-farm tool to monitor biofilm risk and microbial density., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

The complete genome of a variant of the multi-segmented (+) RNA virus blueberry necrotic ring blotch virus (BNRBV), which has not been assigned to a genus, was obtained from foliar red lesions on southern highbush blueberries grown in Alachua Co., Florida. The genome organization of this variant, BNRBV-RL, is the same as that of BNRBV: four genomic segments and seven ORFs (one ORF on each of RNA 1, RNA 2, and RNA 4 and as many as four ORFs on RNA 3). BLAST analysis revealed nucleic acid sequence identities of 89 %, 90 %, 90 % and 86 % to BNRBV RNA 1, RNA 2, RNA 3 and RNA 4, respectively. Phylogenetic analysis of the amino acid sequence of the putative RdRp domain indicated that BNRBV-RL was closely related to BNRBV and less related to citrus leprosis virus type C and three other mite-transmitted viruses. The nucleotide and amino acid sequence differences between BNRBV-RL and BNRBV combined with differences in symptom expression in blueberry would suggest that BNRBV-RL is a strain of BNRBV.

A new disorder was observed on southern highbush blueberries in several south-eastern states in the USA. Symptoms included irregularly shaped circular spots or blotches with green centres on the upper and lower surfaces of leaves. Double-stranded RNA was extracted from symptomatic leaves suggesting the presence of virus(es) possibly involved in the disease. Sequencing revealed the presence of a novel RNA virus with a ~14 kb genome divided into four RNA segments. Sequence analyses showed that the virus, for which we propose the name Blueberry necrotic ring blotch virus (BNRBV), possesses protein domains conserved across RNA viruses in the alpha-virus-like supergroup. Phylogenetic inferences using different genes placed BNRBV in a clade that includes the Bromoviridae, the genus Cilevirus (CiLV) and the recently characterized Hibiscus green spot virus (HGSV). Despite the strong genetic relationships found among BNRBV, Cilevirus and HGSV, the genome of BNRBV contains three features that distinguish it significantly from its closest relatives: (i) the presence of two helicase domains with different evolutionary pathways, (ii) the existence of three conserved nucleotide stretches located at the 3' non-coding regions of each RNA segment and (iii) the conservation of terminal nucleotide motifs across each segment. Furthermore, CiLV and HGSV possess poly(A)-tailed bipartite and tripartite genomes, respectively, whereas BNRBV has a quadra-partite genome lacking a poly(A) tail. Based on these genetic features a new genus is proposed for the classification of BNRBV.

Camelina sativa (L.) Crantz, Brassicaceae, whose common name is Crantz-large-seeded false flax, is an annual oilseed species. It is grown as a forage and biofuel crop in Europe and North America. False flax is an ideal low-input crop for biodiesel production, because of its low requirements for nitrogen fertilizer and pesticides. Production costs of this crop are substantially lower than those of many other oilseed crops such as rapeseed, corn, and soybean. It is an excellent rotation crop and can reduce disease and insect and weed pressure in wheat fields. During the spring of 2011, commercial and research plantings of C. sativa cultivar Calena in Liberty and Gadsden counties in north Florida developed symptoms typical of downy mildew. In spring of 2012, the same symptoms were observed in experimental plantings of false flax. A white downy mold was observed on the upper third portion of the plants, on the upper stem internodes, and on the developing seed. The affected stems exhibited a twisted growth. Conidiophores had main trunks with dichotomous branches terminating in slender curved tips. Conidia were ovoid and 28 to 45 (mean 36) μm long and 22 to 38 (mean 30) μm wide. Conidiophores were branched (three to four branches, each with six to eight curved tips) and ranged from 107 to 236 μm long and 5 to 14 μm wide. Mycelium was obtained directly from diseased plants for DNA extraction (Qiagen DNeasy Plant Mini Kit, Valencia, CA). Primers ITS1 and ITS4 were used for PCR amplification (4). The PCR product was sequenced bidirectionally with the PCR primers. A consensus nucleotide sequence (Accession JQ997103) was compared to those in the NCBI GenBank database using a BLAST search. The sequence was 99% similar to sequence from Hyaloperonospora camelinae (Gäum.) Göker, Voglmayr, Riethm, M. Weiss & Oberw. (Accession AY198249.1) with a 95% query coverage (1). Pathogenicity was established by applying white conidial masses of downy mildew from field samples to stems of 4-week-old plants grown in pots in a greenhouse maintained at 25 ± 2°C. Noninoculated plants maintained under the similar conditions served as control. Symptoms and signs of downy mildew developed after 14 days only on inoculated plants. Downy mildew constitutes a serious threat to the cultivation of C. sativa in Florida because of the humid climate favoring disease development. Diseased plants may reduce yield and disease management would increase production costs. H. camelinae was previously reported on C. sativa in Oregon, Minnesota, Montana (3), and Nebraska (2). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Florida. References: (1) M. Göker et al. Canad. J. Bot. 81:672, 2003. (2) R. M. Harveson et al. Plant Health Progress. 2011. doi: 10.1094/PHP-2011-1014-01-BR. (3) M. L. Putnam et al. Plant Health Progress. 2009. doi: 10.1094/PHP-2009-0910-01-BR. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

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 10 1 to 10 5 conidia/ ml), and five RZ concentrations (10, 20, 30, 40, and 50 μM) were evaluated. Complete medium at 10 5 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% (EC 50 ) was calculated and compared with those obtained from conidia germination tests on fungicide-amended media. Concordant EC 50 values were observed (R 2 = 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.

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.

Seashore paspalum (Paspalum vaginatum) is a saline-tolerant, warm-season turfgrass species popular for golf course use in tropical and subtropical climates. A new variety of Waitea circinata (proposed as W. circinata var. prodigus) is described as the causal agent of basal leaf blight, a novel disease of seashore paspalum. Foliar necrosis and canopy thinning of seashore paspalum were observed on three different golf course fairways in Florida over an 18-month period. Five isolates with profuse, pink to yellow mycelia and small, salmon-colored or yellow to light-brown sclerotia were cultured from diseased turf foliage. Isolates grew rapidly over a temperature range of 25 to 35°C and were initially identified as an uncharacterized variety of W. circinata. Internal transcribed spacer sequences of rDNA from the isolates were compared with sequences from previously described W. circinata varieties. The paspalum isolates formed a phylogenetic clade that was distinct from the other W. circinata varieties. Pathogenicity was confirmed on 'SeaDwarf' and 'SeaIsle Supreme' seashore paspalum, 'Penncross' creeping bentgrass (Agrostis stolonifera), 'Senesta' bermudagrass (Cynodon dactylon), and 'Dark Horse' roughstalk bluegrass (Poa trivialis). The geographical distribution and potential impact of basal leaf blight is unknown. However, the range of potential turfgrass hosts and environmental conditions conducive for disease development suggest that the pathogen may infect other species in addition to seashore paspalum.

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.

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.

Stem blight of southern highbush blueberry (SHB) results in premature plant mortality and has been identified by Florida blueberry growers as the economically most important disease for the industry. In 2007, plants with stem blight and dieback symptoms were sampled at 4-month intervals from two farms located in Alachua and Polk Co., FL. In all, 30 cane samples (stem blight) and 30 crown segments (dieback) were collected at each sample date and each location. In total, 360 samples were collected; fungal species in the family Botryosphaeriaceae were isolated from 85% of the samples. Based on morphology and phylogenetic analysis of the internal transcribed spacer region and elongation factor 1-α (EF1-α) sequences, two dominant species recovered from SHB in Florida were identified: Lasiodiplodia theobromae and Neofusicoccum ribis. Species isolation was independent of location, symptom type, and time of year. Additional samplings are needed to investigate population change over multiple years and in the rest of the southeastern United States. Breeding for resistance and management of stem blight and dieback in Florida should focus on these two fungal species.

In May of 2008, samples of southern highbush blueberry (interspecific Vaccinium corymbosum hybrids) exhibiting marginal leaf necrosis were collected at a farm in Interlachen, FL. The cv. Star showed severe leaf scorch, partial defoliation, and a generally unthrifty growth structure of many thin twigs as has been observed in Georgia (1). The block of Star plants was approximately 10 years old and incidence of the disease was 100%. The grower reported the planting had become increasingly unproductive in the most recent 4 to 5 years. Plants of the cv. Windsor also showed scorch symptoms and yellow-to-red discoloration of leaves. Proportionally fewer Windsor plants showed disease symptoms than Star plants and the disease was not as severe on this cultivar on the basis of visual estimates at the time. Each sample consisted of 5 to 10 cuttings of spring wood with attached leaves showing marginal necrosis taken from a single plant. Three samples, two from Star plants and one from a Windsor plant, were divided into two subsamples each. One subsample was submitted to Agdia for Xylella fastidiosa double-antibody sandwich (DAS)-ELISA assay (TSE XF; Agdia Inc., Elkhart IN). All three samples were reported as positive for X. fastidiosa by DAS-ELISA. A number of asymptomatic plants from this farm and other additional farms were tested in the same manner and results were negative. The other subsample was used for isolation of the causal bacterium. Petioles and main veins from symptomatic leaves were surface disinfested in 1% sodium hypochlorite, cut into segments (0.5 cm), and squeezed with forceps or pliers. Sap that exuded from the segment was blotted directly onto periwinkle wilt medium (2). Bacterial colonies consistent in morphology with X. fastidiosa that were DAS-ELISA positive were obtained from all three samples. One isolate from each sample was inoculated into four Star plants each with the pin-pricking method (3). Leaf scorch symptoms were first observed 8 weeks after inoculation. By 12 weeks after inoculation, all plants inoculated with the three isolates had developed symptoms, including defoliation. Plants inoculated without bacteria showed no symptoms. X. fastidiosa was reisolated from symptomatic plants. Bacterial leaf scorch is an important emerging disease that threatens the southern highbush blueberry industry in the south. On certain cultivars like Star, the potential to reduce yield appears to be great. Differences between cultivars are likely, but have not yet been explored. Additional research is needed into the epidemiology of the disease and potential vectors of pathogen transmission. References: (1) C. Chang et al. HortScience 44:413, 2009. (2) M. Davis et al. Curr. Microbiol. 6:309, 1981. (3) D. L. Hopkins et al. Phytopathology 75:713, 1985.

Stem blight of southern highbush blueberries has been attributed to Botryosphaeria dothidea (2). Symptoms include necrotic branches with attached leaves and brown discoloration of the vasculature extending the length of the affected branch. A 2007 field survey of stem blight in Florida resulted in isolates of the previously reported B. dothidea and Neofusicoccum ribis and isolates of unreported Lasiodiplodia theobromae (2). Isolates of L. theobromae were identified to species level by morphological characterization (3). Identity was confirmed by comparison of rDNA sequences of representative isolates (GenBank Accession No. FJ882072) to reference sequences (99% similarity to Accession No. EF622074) (1). Seven, fresh, pruning wounds on southern highbush blueberries cv. Misty were inoculated with a 10-mm V8 juice agar plug of isolate MixFC-6 taken from the margin of a 3-day-old colony. Seven wounds were inoculated with a sterile agar plug. All plugs were attached to the wounds with Parafilm. Mean lesion length 14 days after inoculation was 8.6 ± 2.4 cm. The pathogen was reisolated from the margin of lesions and identified by colony growth characteristics on potato dextrose agar. No lesions were observed on control plants. To our knowledge, this is the first report that stem blight of southern highbush blueberries in Florida can be caused by L. theobromae. References: (1) A. Alves et al. Fungal Divers. 28:1, 2008. (2) F. L. Caruso and D. C. Ramsdell. Compendium of Blueberry and Cranberry Diseases. The American Phytopathological Society, St. Paul, MN, 1995. (3) P. W. Crous et al. Stud. Mycol. 55:235, 2006.

Brown ring patch was first described as a disease of cool-season turfgrass on creeping bentgrass (Agrostis palustris) (4) in Japan and later reported in California on annual bluegrass (Poa annua) (2). Brown ring patch symptoms were observed beginning in December 2007 through spring 2008 on 6 of 18 putting greens on a golf course in Reston, VA. Symptoms included yellow rings and patches of blighted turfgrass on the mixed stands of creeping bentgrass (A. palustris) and primarily annual bluegrass (Poa annua). Chlorosis and blight occurred predominantly on P. annua. A turfgrass sample was received from a consultant in April 2008, and disease severity on affected greens was estimated to be 40%. After incubating for 2 days in a moist chamber, Rhizoctonia-like aerial mycelia were observed. The pathogen was isolated on water agar and potato dextrose agar amended with thiophanate-methyl (100 mg/L), rifampicin (100 mg/L), and ampicillin (500 mg/L) from P. annua plants that had been surface sterilized with 70% ethanol for 15 s. Colony and sclerotia morphology were consistent with Waitea circinata var. circinata as previously described (2,4). Hyphae were stained with aniline blue and multiple nuclei were observed per cell. The teleomorph was not observed on plant material or in culture. Amplified fragments of rDNA including internal transcribed spacers from the isolate were amplified in three bacterial clones and sequenced bidirectionally (GenBank Accession Nos. FJ154894, FJ154895, and FJ154896) using primers ITS1/ITS4 (2,4). The consensus sequences matched, with 99% homology and 99% sequence overlap, isolate TRGC1.1 of W. circinata var. circinata (GenBank Accession No. DQ900586) (2). Annual bluegrass was not available for use in performing Koch's postulates, but previous studies have shown that W. circinata var. circinata is pathogenic to roughstalk bluegrass (P. trivialis) (1,3). Pots of P. trivialis cv. Cypress that were 1 week postemergence were inoculated with seven wheat grains that had been autoclaved and then infested with the isolate. Plants were incubated at 25°C in a sealed plastic bag with a moist paper towel on the bottom. Hyphae grew from the grains and colonized the grass. Individual plants began to turn chlorotic within 3 days, and more than 80% of the turf in pots was dead after 1 week. Control pots were inoculated with autoclaved wheat seed and showed no disease symptoms after 1 week. Inoculations were repeated twice more with the same results. W. circinata var. circinata was reisolated from affected plants in all replications of the test. To our knowledge, this is the first report of brown ring patch in Virginia. Additional research is needed to assess the prevalence and importance of this disease on golf course putting greens in Virginia. References: (1) C. M. Chen et al. Plant Dis. 91:1687, 2007. (2) K. A. de la Cerda et al. Plant Dis. 91:791, 2007. (3) N. Flor et al. Plant Dis. 92:1586, 2008. (4) T. Toda et al. Plant Dis. 89:536, 2005.

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 × 10 5 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.

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.

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.

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-cm 2 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.

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 cm 2 ) 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.

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/cm 2 ) that were scattered to many (73/cm 2 ). 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.

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.

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.