Standard Area Diagrams for Pecan Leaf Scab: Effect of Rater Experience, Location, and Leaf Size on Reliability and Accuracy of Visual Estimates.

Assessments of the severity of scab (Venturia effusa), an economically significant disease of pecan, are critical for determining pecan cultivar susceptibility, disease epidemiology, and integrated disease management approaches. We developed a standard area diagram (SAD) set to aid in assessments of pecan leaflet scab. Leaflets with scab lesions were harvested and scanned using a flatbed scanner at 600 dpi, and Fiji (ImageJ) was used to determine the actual percent disease severity. The SADs had 10 leaflets ranging in severity from 0.2 to 48.9%. Forty "small" (1.34 to 7.43 cm2) and 40 "large" (7.67 to 25.9 cm2) leaflet images were randomized for rater assessments. The images were assessed twice by 36 raters, first without and then with the SADs as a guide. Data were subjected to analysis using Lin's concordance correlation coefficient (LCC, pc) to determine the accuracy of ratings and by intraclass correlation coefficient (ICC) analysis to determine interrater reliability. The effects of rater experience, rater location, and leaflet size were also determined. The SADs significantly improved the agreement between raters and the actual values (LCC, pc = 0.70 and 0.84 without and with the SADs, respectively). The reliability of estimates was improved (ICC = 0.54 and 0.82 without and with the SADs, respectively). The effect of rater location on overall concordance was significant without and with the SADs based on an analysis of variance using a generalized linear model and lsmeans separation (P < 0.05). A generalized linear mixed model analysis revealed that there was a significant interaction between rater location, experience, and the use of the SADs, with some raters having greater improvement in generalized bias and concordance. Raters had a significantly better accuracy when rating "small" leaves (LCC, pc = 0.86) compared with "large" leaves (LCC, pc = 0.82) when using the SADs, highlighting the impact of psychophysics on field evaluations of plant disease severity. The proposed SADs will serve as an improved tool for performing pecan leaflet scab assessments by the pecan research community.

Sterile White Basidiomycete Fungus Marasmius graminum: A New Pathogen Causing Seedling Blight in Rice.

In April 2018, damping-off of rice (Oryza sativa L.) seedlings at the 2-to-3-leaf stage was observed in three fields in the counties of Wharton and Matagorda of Texas and Jefferson-Davis Parish of Louisiana. All affected areas were 1 ha or greater, with 10 to 20% of the seedlings showing the symptoms. Infected seedlings showed dark-brown necrotic lesions on the roots and/or mesocotyls where white superficial mycelium was usually present. Symptomatic tissues excised from 10 diseased seedlings of each field were surface sterilized with 1% NaOCl, double rinsed in sterilized distilled water, and plated on potato dextrose agar (PDA). The plates were incubated at 25°C with a 12-h photoperiod in a growth chamber. After 48 h, hyphal tips of fungal colonies were transferred onto PDA and 12 isolates were obtained. Clamp connections and dolipore septa were observed in young hyphae, indicating that these isolates were a basidiomycete fungus. Young hyphal cells were binucleate based on safranin O stain (Bandoni 1979). No fruiting bodies or sclerotia produced on PDA after one month of incubation. Based on these morphological characteristics, these isolates were identified as belonging to sterile white basidiomycetes (SWB) (Howard et al. 1977). To further identify the isolates into the species level, the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of a representative isolate was sequenced with primer ITS1 and ITS4 (Vinnere et al. 2005). The ITS sequence (GenBank acc. no. MT524457) had more than 97% sequence similarity with known Marasmius graminum strains from Denmark (JN943595) (Schoch et al. 2012) and Sweden (MH857692) (Vu et al. 2019). Pathogenicity was tested with three representative isolates in a growth chamber using a modified method (Carling and Leiner 1990). Pots (6.5 cm in diameter x 7.5 cm height) were filled with 100 g of sterilized sand and watered to field capacity. Five PDA plugs (4 mm in diameter) from 5-day-old growing culture were placed on the sand surface of each pot. Pots inoculated with PDA plugs without fungus served as the controls. Five seeds of rice cv. Presidio were planted into each pot and covered with 10 g of sterilized sand. Pots were maintained at 25±2°C in a growth chamber with a 12-h photoperiod for 14 days. There were four replicated pots for each treatment and the experiment repeated twice. After 2 weeks, severe damping-off and associated symptoms similar to those observed in the field appeared in the inoculated pots. No symptoms developed in the control pots. The same fungus was consistently re-isolated from infected plants. Based on morphological characteristics and rDNA-ITS sequencing, these isolates were identified as M. graminum. The SWB fungus was first reported as a causal agent of stem rot of snap bean in Florida (Howard et al. 1977) and Nebraska (Harveson 2002), root or hypocotyl rot of corn, snap bean, squash and peanut in Georgia (Sumner et al. 1979; Bell and Sumner 1984), and crown rot of pigeon pea (Cajanus cajan) in Puerto Rico (Kaiser et al. 1987). Later, the SWB strain (ATCC 28344) causing stem rot of snap bean in Florida was further identified as M. graminum based on nuclear large subunit rRNA gene (Vinnere et al. 2005). Comparing the ITS region of this isolate (AY445120) with our isolate revealed a 99% similarity. To our knowledge, this is the first report that the SWB fungus M. graminum causes seedling blight in rice. Identification of this new disease will help to develop management strategies for control of stand loss in rice.

Curvularia malina sp. nov. incites a new disease of warm-season turfgrasses in the southeastern United States.

A novel species of Curvularia was identified as a foliar pathogen of Cynodon dactylon (bermudagrass) and Zoysia matrella (zoysiagrass), two important warm-season turfgrasses in the southeastern United States. Field symptoms were conspicuous chocolate brown to black spots in turf of both species on golf course putting greens and fairways. Leaves of plants within these spots exhibited prominent, black eyespot lesions from which a darkly pigmented fungus was consistently isolated. The fungus produced gray- to black-olivaceous mycelium within 10 d on potato dextrose agar at 25 C but never produced conidia despite numerous attempts to induce them. Field symptoms were reproduced in inoculated plants of both grasses, and re-isolation of the pathogen from symptomatic tissues confirmed its pathogenicity in fulfillment of Koch's postulates. A phylogenetic analysis was performed using sequence markers of internal nuclear ribosomal transcribed spacer region (ITS), glyceralde-hyde-3-phosphate dehydrogenase (GPD1) and translation elongation factor 1-α (TEF 1). The concatenated phylogenetic tree showed strong support for a new species within Curvularia that is distinctly divergent from other Curvularia spp. Therefore, the darkly pigmented pathogen of warm-season turfgrasses is described and illustrated as a new species, Curvularia malina.

Screening Brassicaceous Plants as Biofumigants for Management of Rhizoctonia solani AG1-IA.

Brassicaceae plants rich in glucosinolates have been used as biofumigants for management of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) and other soilborne pathogens. Efficacy of brassica plant tissue has mainly been attributed to toxic isothiocyanates released upon hydrolysis of glucosinolates. Management of R. solani AG 1-IA, the causal agent of sheath blight in rice (Oryza sativa), using biofumigation, is promising but needs more validation. Biofumigation activity of nine Brassicaceae plants and two other related species were evaluated in vitro with soils from Texas, Arkansas, or Mississippi. All plants evaluated significantly suppressed the mycelium growth of R. solani AG 1-IA. Mustard (Brassica juncea) cultivars ('Brand 199', 'Ruby Streak', 'Florida Broadleaf', and 'Green Wave') consistently provided the greatest (>90%) mycelial inhibition, while sunn hemp (Crotalaria juncea) and Chinese cabbage (B. rapa) had the least suppressive effect. B. juncea 'Red Giant' and 'Sheali Hong', turnip (B. rapa), kale (B. oleracea), and arugula (Eruca sativa) showed intermediate efficacy or were inconsistent. Effects of soil pasteurization and plant tissue amendment rates were examined with B. juncea Brand 199 and Texas soil. Inhibition of mycelial growth became greater with increasing plant amendment rates up to 3.2% (wt/wt) in the soil. Soil pasteurization almost completely suppressed the release of allyl isothiocyanate (AITC). The nonpasteurized soil amended with 0.5% (wt/wt) of the plant material released 96% more AITC than the soil amended with 0.25% (wt/wt) of the plant material. The highest levels of AITC release were observed at 12 and 24 h after soil amendment, with 0.25 and 0.5% (wt/wt) of the plant material, respectively. Antifungal effects of B. juncea are attributed to dose-dependent production of volatile AITC and could be used for managing rice sheath blight caused by R. solani AG 1-IA.

Integration of Brassica Cover Crop with Host Resistance and Azoxystrobin for Management of Rice Sheath Blight.

Sheath blight caused by Rhizoctonia solani is the most important rice disease that can cause significant losses in grain yield and quality in the southern United States. Current management options for sheath blight primarily consist of fungicides, tolerant cultivars, and cultural practices. These options are not always very effective. Brassica plants have been used for soil fumigation to manage a variety of different soilborne pathogens. In this field study, the efficacy of a Brassica juncea cover crop integrated with use of a tolerant rice cultivar and fungicide application was evaluated in 2011, 2012, and 2013. The B. juncea cover crop significantly lowered sheath blight severity in all 3 years and led to a significantly higher grain yield in 2013 as compared with the fallow control. 'Presidio' rice had lower sheath blight severity and higher yield than 'Cocodrie' in 2012 and 2013. Fungicide applications with azoxystrobin at the label rate (0.16 kg a.i./ha) or half the label rate (0.08 kg a.i./ha) significantly reduced sheath blight severity in all 3 years, resulting in a yield increase in 2 of the 3 years. B. juncea along with use of a tolerant rice cultivar and half the label rate of azoxystrobin can be an effective approach for management of sheath blight in rice.

Nematicidal Effects of Silver Nanoparticles on Root-knot Nematode in Bermudagrass.

Certain nematodes are common soilborne organisms found in turfgrass in the United States that cause significant economic damage to golf course turf. One of the most prevalent plant-parasitic nematodes infesting turfgrass are root-knot nematodes (Meloidogyne spp.). Chemical treatment options for root-knot nematodes in turfgrass are limited, and there is a need for new nematicidal active ingredients to address this problem. In this study, we evaluated the use of silver nanoparticles (AgNP) as a potential nematicide in laboratory and field experiments. AgNP was synthesized by a redox reaction of silver nitrate with sodium borohydride using 0.2% starch as a stabilizer. When J2 of M. incognita were exposed to AgNP in water at 30 to 150 µg/ml, >99% nematodes became inactive in 6 hr. When turfgrass and soil composite samples infested with M. graminis were treated with 150 µg/ml AgNP, J2 were reduced in the soil samples by 92% and 82% after 4- and 2-d exposures, respectively, in the treated compared to the nontreated soil samples. Field trials evaluating AgNP were conducted on a bermudagrass (Cynodon dactylon × C. transvaalensis) putting green infested with M. graminis. Biweekly application of 90.4 mg/m(2) of AgNP improved turfgrass quality in one year and reduced gall formation in the roots in two years without phytotoxicity. The AgNP application did not significantly reduce the number of M. graminis J2 in plots during the growing season. The laboratory assays attested to the nematicidal effect of AgNP, and the field evaluation demonstrated its benefits for mitigating damage caused by root-knot nematode in bermudagrass.

Draft genome sequence of Neofusicoccum caryigenum associated with pecan leaf dieback.

Pecan leaf dieback caused by Neofusicoccum caryigenum is an emerging disease in southeastern United States pecan orchards. In this study, a first draft N. caryigenum genome was sequenced and assembled. Genome size was estimated as 42.5 Mbp, and genome completeness was estimated as 97.4%.

Raman Spectroscopy Enables Confirmatory Diagnostics of Fusarium Wilt in Asymptomatic Banana.

Fusarium oxysporum f. sp. cubense (FOC) causes Fusarium wilt, one of the most concerning diseases in banana (Musa spp.), compromising global banana production. There are limited curative management options after FOC infections, and early Fusarium wilt symptoms are similar with other abiotic stress factors such as drought. Therefore, finding a reliable and timely form of early detection and proper diagnostics is critical for disease management for FOC. In this study, Portable Raman spectroscopy (handheld Raman spectrometer equipped with 830 nm laser source) was applied for developing a confirmatory diagnostic tool for early infection of FOC on asymptomatic banana. Banana plantlets were inoculated with FOC; uninoculated plants exposed to a drier condition were also prepared compared to well-watered uninoculated control plants. Subsequent Raman readings from the plant leaves, without damaging or destroying them, were performed weekly. The conditions of biotic and abiotic stresses on banana were modeled to examine and identify specific Raman spectra suitable for diagnosing FOC infection. Our results showed that Raman spectroscopy could be used to make highly accurate diagnostics of FOC at the asymptomatic stage. Based on specific Raman spectra at vibrational bands 1,155, 1,184, and 1,525 cm-1, Raman spectroscopy demonstrated nearly 100% accuracy of FOC diagnosis at 40 days after inoculation, differentiating FOC-infected plants from uninoculated plants that were well-watered or exposed to water deficit condition. This study first reported that Raman spectroscopy can be used as a rapid and non-destructive tool for banana Fusarium wilt diagnostics.

Evidence for Seed Transmission of Xylella fastidiosa in Pecan (Carya illinoinensis).

Pecan bacterial leaf scorch, caused by Xylella fastidiosa subsp. multiplex, is an economically significant disease of pecan with known detrimental effects on the yield of susceptible cultivars. In this study, endosperm was harvested from developing pecan seeds, and direct qPCR and sequencing were used to detect and confirm the presence of X. fastidiosa. DNA was isolated from mature seeds originating from seven trees, revealing a positivity rate up to 90%, and transmission of X. fastidiosa from infected seed to the germinated seedlings was found to be over 80%. Further epidemiological analyses were performed to determine where X. fastidiosa localizes in mature seed and seedlings. The highest concentrations of X. fastidiosa DNA were found in the hilum and outer integument of the seeds and the petioles, respectively. High-, medium-, and low-density seeds were harvested to determine the impact of the bacterium on seed density and seedling growth rate. The growth rate of seedlings originating from low-density seeds was significantly reduced compared to the medium- and high-density seeds. Despite the increased growth and germination rates, the high-density seed group had a greater proportion of samples that tested positive for the presence of X. fastidiosa by qPCR. The results demonstrate the ability of X. fastidiosa to colonize developing seeds and be efficiently transmitted from well-developed seeds to germinated seedlings. Continued research is needed to understand the plant-microbe interactions involved in the colonization of pecan seeds by X. fastidiosa and to develop effective phytosanitary approaches to reduce the risks posed by seed transmission.

Complexity of Gaeumannomyces species causing take-all root rot of St. Augustinegrass in Texas.

Gaeumannomyces graminis var. graminis (Ggg) has been the etiological agent of take-all root rot (TARR) in St. Augustinegrass (Stenotaphrum secundatum) and root decline of the other warm-season turfgrasses. Seventy-five Ggg isolates were obtained from St. Augustinegrass in central and east Texas. Evaluation of colony morphologies on potato dextrose agar (PDA) within 2 wk and follow-up multilocus phylogenic analyses revealed three phenotypic groups associated with different Gaeumannomyces species: (i) G. floridanus, highly melanized with round colony formation; (ii) G. arxii, none to slightly melanized with round colony formation; and (iii) G. graminicola, highly melanized with irregular colony formation. Further examination with representative isolates from each group revealed that their phenotypic characterizations supported the distinctive genetic groups within Ggg associated with St. Augustinegrass TARR. Gaeumannomyces floridanus isolates grew faster at warmer temperature (30 C) than G. arxii or G. graminicola. Pathogenicity assays using rice seedlings indicated that G. floridanus was more aggressive in disease symptom development than G. arxii or G. graminicola. A multilocus phylogeny reconstruction supported that most of Gaeumannomyces isolates tested in this study were separated into three phylogenetically distinct groups: G. floridanus, G. arxii, and G. graminicola. The resolution of intravarietal complexities of causal fungi of TARR is important for proper diagnostics and management strategies for TARR in St. Augustinegrass and other root-decline diseases in warm-season turfgrasses.

Thermal treatment using microwave irradiation for the phytosanitation of Xylella fastidiosa in pecan graftwood.

Pecan bacterial leaf scorch caused by Xylella fastidiosa is an emerging disease for the U.S. and international pecan industries and can be transmitted from scion to rootstock via grafting. With the expanse of global transportation and trade networks, phytosanitation is critical for reducing the spread of economically significant pathogens, such as X. fastidiosa. We developed and evaluated thermal treatments using microwave irradiation and microwave absorbers [sterile deionized water (dH2O) and carbon nanotubes (CNTs)] as novel disinfectant methods for remediating X. fastidiosa in pecan scions. Partial submergence of scions in dH2O or CNT dispersions resulted in the transport of microwave absorbers in the xylem tissue via transpiration but did not compromise plant health. The microwave absorbers effectively transferred heat to the scion wood to reach an average temperature range of 55-65°C. Microwave radiation exposure for 6 sec (3 sec for two iterations) of CNT- or dH2O-treated scions reduced the frequency of X. fastidiosa-positive in pecan scions without negatively affecting plant viability when compared to the control group (dH2O-treated with no microwave). The efficacy of the new thermal treatments based on microwave irradiation was comparable to the conventional hot-water treatment (HWT) method, in which scions were submerged in 46°C water for 30 min. Microwave irradiation can be employed to treat X. fastidiosa-infected scions where the conventional HWT treatment is not feasible. This study is the first report to demonstrate novel thermal treatment methods based on the microwave irradiation and microwave absorbers of dH2O and CNT as an application for the phytosanitation of xylem-inhabiting bacteria in graftwood.

Antifungal Activity of Silver Ions and Nanoparticles on Phytopathogenic Fungi.

Silver in ionic or nanoparticle forms has a high antimicrobial activity and is therefore widely used for various sterilization purposes including materials of medical devices and water sanitization. There have been relatively few studies on the applicability of silver to control plant diseases. Various forms of silver ions and nanoparticles were tested in the current study to examine the antifungal activity on two plant-pathogenic fungi, Bipolaris sorokiniana and Magnaporthe grisea. In vitro petri dish assays indicated that silver ions and nanoparticles had a significant effect on the colony formation of these two pathogens. Effective concentrations of the silver compounds inhibiting colony formation by 50% (EC50) were higher for B. sorokiniana than for M. grisea. The inhibitory effect on colony formation significantly diminished after silver cations were neutralized with chloride ions. Growth chamber inoculation assays further confirmed that both ionic and nanoparticle silver significantly reduced these two fungal diseases on perennial ryegrass (Lolium perenne). Particularly, silver ions and nanoparticles effectively reduced disease severity with an application at 3 h before spore inoculation, but their efficacy significantly diminished when applied at 24 h after inoculation. The in vitro and in planta evaluations of silver indicated that both silver ions and nanoparticles influence colony formation of spores and disease progress of plant-pathogenic fungi. In planta efficacy of silver ions and nanoparticles is much greater with preventative application, which may promote the direct contact of silver with spores and germ tubes, and inhibit their viability.

Thiophanate-Methyl and Propiconazole Sensitivity in Sclerotinia homoeocarpa Populations from Golf Courses in Wisconsin and Massachusetts.

Management of dollar spot, caused by the fungus Sclerotinia homoeocarpa, is dependent upon repeated fungicide applications in intensively managed turfgrass such as golf course putting greens and fairways. Repeated fungicide applications could potentially select for fungicide-resistant isolates and result in a reduction of disease control. The objectives of this study were to determine the degree of S. homoeocarpa in vitro sensitivity to the fungicides thiophanate-methyl and propiconazole using isolates collected from golf course putting greens, fairways, and roughs; and to determine the relationships of golf course age and fungicide history to the frequency of fungicide-insensitive isolates within the population. More than 1,400 S. homoeocarpa isolates were collected from putting greens, fairways, and roughs at six Wisconsin golf courses and one Massachusetts golf course and subjected to in vitro fungicide sensitivity assays with single discriminatory concentrations of thiophanate-methyl and propiconazole. Five of seven pathogen populations from rough areas were not significantly different from one another in propiconazole sensitivity. These populations were collectively the most sensitive to both fungicides and therefore, served as baseline populations for comparison with fungicide-exposed populations from putting greens and fairways. Greater propiconazole insensitivity was observed in populations collected from fairways and putting greens that received more frequent applications of the fungicide than those isolated from the roughs. In nearly all the golf courses, the frequency of thiophanate-methyl insensitivity was higher among isolates of S. homoeocarpa collected from fairways than from roughs regardless of the age of the golf course or history of benzimidazole use. Thus, while the development of resistance to propiconazole can be predicted in part by the relative frequency of demethylation inhibitor fungicide applications, the occurrence of populations resistant to thiophanate-methyl appears to be unrelated to recent use of the benzimidazole class of fungicides.

Host-mediated microbiome engineering (HMME) of drought tolerance in the wheat rhizosphere.

Host-mediated microbiome engineering (HMME) is a strategy that utilizes the host phenotype to indirectly select microbiomes though cyclic differentiation and propagation. In this experiment, the host phenotype of delayed onset of seedling water deficit stress symptoms was used to infer beneficial microbiome-host interactions over multiple generations. By utilizing a host-centric selection approach, microbiota are selected at a community level, therein using artificial selection to alter microbiomes through both ecological and evolutionary processes. After six rounds of artificial selection using host-mediated microbiome engineering (HMME), a microbial community was selected that mediated a 5-day delay in the onset of drought symptoms in wheat seedlings. Seedlings grown in potting medium inoculated with the engineered rhizosphere from the 6th round of HMME produced significantly more biomass and root system length, dry weight, and surface area than plants grown in medium similarly mixed with autoclaved inoculum (negative control). The effect on plant water stress tolerance conferred by the inoculum was transferable at subsequent 10-fold and 100-fold dilutions in fresh non-autoclaved medium but was lost at 1000-fold dilution and was completely abolished by autoclaving, indicating the plant phenotype is mediated by microbial population dynamics. The results from 16S rRNA amplicon sequencing of the rhizosphere microbiomes at rounds 0, 3, and 6 revealed taxonomic increases in proteobacteria at the phylum level and betaproteobacteria at the class level. There were significant decreases in alpha diversity in round 6, divergence in speciation with beta diversity between round 0 and 6, and changes in overall community composition. This study demonstrates the potential of using the host as a selective marker to engineer microbiomes that mediate changes in the rhizosphere environment that improve plant adaptation to drought stress.

Bioprospecting Plant Growth-Promoting Rhizobacteria That Mitigate Drought Stress in Grasses.

This study reports the application of a novel bioprospecting procedure designed to screen plant growth-promoting rhizobacteria (PGPR) capable of rapidly colonizing the rhizosphere and mitigating drought stress in multiple hosts. Two PGPR strains were isolated by this bioprospecting screening assay and identified as Bacillus sp. (12D6) and Enterobacter sp. (16i). When inoculated into the rhizospheres of wheat (Triticum aestivum) and maize (Zea mays) seedlings, these PGPR resulted in delays in the onset of plant drought symptoms. The plant phenotype responding to drought stress was associated with alterations in root system architecture. In wheat, both PGPR isolates significantly increased root branching, and Bacillus sp. (12D6), in particular, increased root length, when compared to the control. In maize, both PGPR isolates significantly increased root length, root surface area and number of tips when compared to the control. Enterobacter sp. (16i) exhibited greater effects in root length, diameter and branching when compared to Bacillus sp. (12D6) or the control. In vitro phytohormone profiling of PGPR pellets and filtrates using LC/MS demonstrated that both PGPR strains produced and excreted indole-3-acetic acid (IAA) and salicylic acid (SA) when compared to other phytohormones. The positive effects of PGPR inoculation occurred concurrently with the onset of water deficit, demonstrating the potential of the PGPR identified from this bioprospecting pipeline for use in crop production systems under drought stress.

Rapid development of fungicide resistance by Sclerotinia homoeocarpa on turfgrass.

Dollar spot, caused by Sclerotinia homoeocarpa, is the most prevalent and economically important turfgrass disease in North America. Increasing levels of fungicide resistance, coupled with tightening environmental scrutiny of existing fungicides, has left fewer options for managing dollar spot. More knowledge about S. homoeocarpa populations is needed to improve dollar spot management strategies, especially with respect to minimizing the development of fungicide resistance. Population diversity of S. homoeocarpa was examined using inter-simple sequence repeat markers and vegetative compatibility assays. Two subgroups were found in S. homoeocarpa field populations on both fairway and putting green turfgrass at a research field in Wisconsin. These subgroups were genetically different, vegetatively incompatible, and had different fungicide sensitivities. The frequency of the two genetic subgroups differed significantly between the fairway and putting green, but was uniform within the fairway or within the green. Population dynamics of S. homoeocarpa in response to two systemic fungicides (thiophanate-methyl and propiconazole) were assessed based on in vitro fungicide sensitivity. Dynamics of S. homoeocarpa populations depended on the presence of fungicide-resistant isolates in the initial populations before fungicide applications and changed rapidly after fungicide applications. Shifting of the population toward propiconazole resistance was gradual, whereas thiophanate-methyl resistance developed rapidly in the population. In conclusion, field populations of S. homoeocarpa containing genetically distinct, vegetatively incompatible groups were different on turfgrass that was managed differently, and they were changed rapidly after exposure to fungicides.

Microalgal cultivation for biofertilization in rice plants using a vertical semi-closed airlift photobioreactor.

Nitrogen (N) is one of the most important limiting factors in conventional rice (Oryza sativa) production, which heavily relies on synthetic fertilizers. In this study, we researched on the development and use of a vertical semi-closed airlift photobioreactor (PBR) for microalgal cultivation and subsequently determined the efficacy of microalgae-based fertilizers to rice plant growth. The PBR system was developed to produce two strains of N2-fixing cyanobacteria (Anabaena sp. UTEX 2576, Nostoc muscorum UTEX 2209S), and a polyculture of Chlorella vulgaris (UTEX 2714) and Scenedesmus dimorphus (UTEX 1237). When these biofertilizers were evaluated for rice under the greenhouse conditions, results showed that the rice plant heights treated with polyculture-based microalgal biomass were similar to or better than the urea treatment. The effects of the inoculation of the N2-fixing cyanobacterial inoculation on seedling growth was not statistically significant. In conclusion, the vertical semi-closed system PBR cultivation method developed in this study proved to be a simple and effective method for cultivating microalgae. Demonstration of the reliable production system for N2-fixing cyanobacteria and chlorophytes at a medium scale could potentially open the future application of microalgal biofertilizers in rice production.

Expression Analysis of Rice Defense-Related Genes in Turfgrass in Response to Magnaporthe grisea.

ABSTRACT Magnaporthe grisea (anamorph = Pyricularia grisea) causes blast on rice (Oryza sativa) and gray leaf spot on turfgrass. Gray leaf spot is a serious disease on St. Augustinegrass (Stenotaphrum secundatum), perennial ryegrass (Lolium perenne), and tall fescue (Festuca arundinacea). Virulence assays performed in this study revealed that M. grisea collected from rice could also cause disease on St. Augustinegrass and tall fescue. One rice isolate, Che86061, caused similar disease reactions on susceptible cultivars of rice and St. Augustinegrass and an incompatible interaction on resistant cultivars of both species. To explore whether similar defense-related genes are expressed in rice and St. Augustinegrass, a rice cDNA library was screened using pooled cDNAs derived from M. grisea-infected St. Augustinegrass. Thirty rice EST (expressed sequence tag) clones showing differential expression in St. Augustinegrass following M. grisea inoculation were identified and classified into six putative functional groups. Northern blot analyses of seven EST clones that collectively represented each putative functional group confirmed that the expression of five out of seven EST clones was similar in both rice and St. Augustinegrass. This study represents one of the first attempts to use a broad-scale genomic approach and resources of a model monocot system to study defense gene expression in St. Augustinegrass following M. grisea infection.

Fungicide Sensitivity of Sclerotinia homoeocarpa from Golf Courses in Ohio.

Managing dollar spot, the most common and chronic disease on intensively cultivated turfgrass, relies on the judicious use of fungicides. The heavy use of fungicides has led to the development of isolates of Sclerotinia homoeocarpa insensitive to several classes of fungicides, including benzimidazoles, demethylation-inhibitors, and dicarboximides. In vitro fungicide sensitivity assays using single discriminatory concentrations of thiophanate-methyl, propiconazole, and iprodione were developed in this study for evaluating field efficacy of these fungicides and the prevalence of fungicide insensitivity within S. homoeocarpa isolated from golf courses throughout Ohio. Discriminatory concentrations for these fungicides were determined to be: thiophanate-methyl = 1,000 µg a.i. ml-1, propiconazole = 0.1 µg a.i. ml-1, and iprodione = 1.0 µg a.i. ml-1. Effective concentration that produces 50% inhibition (EC50) was estimated based on relative mycelial growth of S. homoeocarpa on potato dextrose agar (PDA) versus PDA amended with the discriminatory concentration of each fungicide. Field trials conducted at 3 locations in 2002 and 10 locations in 2003 revealed that the in vitro assays accurately predicted field efficacy for thiophanate-methyl. When used to screen 192 S. homoeocarpa isolates collected previously from 55 golf courses throughout Ohio, the in vitro assays revealed that 34 of the golf courses sampled had S. homoeocarpa resistant to thiophanate-methyl. S. homoeocarpa with reduced in vitro sensitivities was isolated from 18 and 1 golf courses for propiconazole and iprodione, respectively.

Evidence for Genetic Similarity of Vegetative Compatibility Groupings in Sclerotinia homoeocarpa.

Vegetative compatibility groups (VCGs) are determined for many fungi to test for the ability of fungal isolates to undergo heterokaryon formation. In several fungal plant pathogens, isolates belonging to a VCG have been shown to share significantly higher genetic similarity than those of different VCGs. In this study we sought to examine the relationship between VCG and genetic similarity of an important cool season turfgrass pathogen, Sclerotinia homoeocarpa. Twenty-two S. homoeocarpa isolates from the Midwest and Eastern US, which were previously characterized in several studies, were all evaluated for VCG using an improved nit mutant assay. These isolates were also genotyped using 19 microsatellites developed from partial genome sequence of S. homoeocarpa. Additionally, partial sequences of mitochondrial genes cytochrome oxidase II and mitochondrial small subunit (mtSSU) rRNA, and the atp6-rns intergenic spacer, were generated for isolates from each nit mutant VCG to determine if mitochondrial haplotypes differed among VCGs. Of the 22 isolates screened, 15 were amenable to the nit mutant VCG assay and were grouped into six VCGs. The 19 microsatellites gave 57 alleles for this set. Unweighted pair group methods with arithmetic mean (UPGMA) tree of binary microsatellite data were used to produce a dendrogram of the isolate genotypes based on microsatellite alleles, which showed high genetic similarity of nit mutant VCGs. Analysis of molecular variance of microsatellite data demonstrates that the current nit mutant VCGs explain the microsatellite genotypic variation among isolates better than the previous nit mutant VCGs or the conventionally determined VCGs. Mitochondrial sequences were identical among all isolates, suggesting that this marker type may not be informative for US populations of S. homoeocarpa.