Reduced Sensitivity to Fluopyram in Meloidogyne graminis following Long-Term Exposure in Golf Turf.

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.

First report of Bipolaris sorokiniana leaf spot disease on watermelon (Citrullus lanatus) in Florida.

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.

Identification of Fungi in the Botryosphaeriaceae Family Associated with Stem Blight of Vaccinium spp. in the Southeastern United States.

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 ß-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.

Known and New Emerging Viruses Infecting Blueberry.

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.

Discovery of Known and Novel Viruses in Wild and Cultivated Blueberry in Florida through Viral Metagenomic Approaches.

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.

Emerging fungal pathogen of an invasive grass: Implications for competition with native plant species.

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.

Biocontrol Potential of Essential Oils in Organic Horticulture Systems: From Farm to Fork.

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.

First report of Bipolaris yamadae leaf spot disease on Guinea grass (Panicum maximum) in Florida.

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.

Large-spored Drechslera gigantea is a Bipolaris species causing disease on the invasive grass Microstegium vimineum.

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.

Disease in Invasive Plant Populations.

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.

Use of Dehydrated Agar to Estimate Microbial Water Quality for Horticulture Irrigation.

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.

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.

Genetic characterization of Blueberry necrotic ring blotch virus, a novel RNA virus with unique genetic features.

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.

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.

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.

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.

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.

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.

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.