Dollar Spot Research Articles

European Turfgrass Society Conference Special Section in Agronomy Journal and Crop Science

The Fifth European Turfgrass Society (ETS) Conference was held in Portugal last June. Professor Carlos Guerrero from the University of Algarve organized this international conference with support from the Portuguese Foundation for Science and Technology, Luso-American Development Foundation, and multiple corporate sponsors. Guerrero also helped organize the publication of a selection of papers from this conference. These papers were coordinately published in the March–April 2017 issues of Crop Science and Agronomy Journal, as a special section. An introductory paper appears in Crop Science. Guerrero shared his thoughts on the special section, and says, “The Organizing Committee is proud of the effort done to carry on with the Fifth ETS Conference, and as a member of the ETS, I am proud to share this ‘drop of knowledge’ in this [special section].” CSA News: What was the overarching theme of the Fifth ETS Conference? Guerrero: The theme of the ETS Conference was “Turfgrass—Towards Sustainability and Perfection for Aesthetic, Recreation and Sports.” Presentations covered many topics, including: turfgrass genetics and breeding, turfgrass for sports, water management, technology advances and turfgrass maintenance (mowing, fertilization, environment, and certification), turfgrass pests (diseases, insects, and weeds), and turfgrass nutrition and physiology. CSA News: Are papers in this special section focused on a single topic, or do they reflect the conference as a whole? Guerrero: The papers cover a wide range of topics from the conference. It is important to note that the papers in this special section reflect approximately 10% of the total work presented. CSA News: Briefly describe the most important findings included in this special section. Guerrero: For this growing industry, there are several important issues addressed such as water use, pesticide use, fertilizer use, and biodiversity. These topics are significantly studied, and research is widely developed all around the world under different climatic, edaphic, and technological arrangements, enhancing and improving solutions for a better use of turfgrass. From a personal point of view, disease control or even disease suppression is one of the most ‘attractive’ research subjects covered. Dollar spot, brown patch, anthracnose, and Microdochium nivale are the most important diseases studied in cool- and warm-season turfgrasses. The turfgrass pathogen causing the most use of fungicides at northern latitudes is Microdochium nivale, and at warmer latitudes, Sclerotinia homoeocarpa and Rhizoctonia solani are the most commons diseases. Researchers are investigating biological control methods to deal with these diseases. Biological control is achieved by using organic amendments or by using specific inoculants. Both strategies have the goal to increase the population of the disease suppressive organism necessary to achieve the desired level of control. Other research focuses on fertility control. Nitrogen, iron, or iron sulfate are management practices that suppress, or control, for example, Sclerotinia homoeocarpa. And increased N fertilization, mowing height, and sand topdressing can suppress Colletotrichum cereale in cool-season turfgrass. Another topic of interest to the turfgrass community is the control of Poa annua in bentgrass greens. There is no registered herbicide for Poa annua invading bentgrass greens. One of the articles published in this special issue shows that paclobutrazol and iron sulfate combined controlled annual bluegrass and moss on putting greens, offering acceptable surface quality.

Antifungal mechanisms of ZnO and Ag nanoparticles to Sclerotinia homoeocarpa

Fungicides have extensively been used to effectively combat fungal diseases on a range of plant species, but resistance to multiple active ingredients has developed in pathogens such as Sclerotinia homoeocarpa, the causal agent of dollar spot on cool-season turfgrasses. Recently, ZnO and Ag nanoparticles (NPs) have received increased attention due to their antimicrobial activities. In this study, the NPs’ toxicity and mechanisms of action were investigated as alternative antifungal agents against S. homoeocarpa isolates that varied in their resistance to demethylation inhibitor (DMI) fungicides. S. homoeocarpa isolates were treated with ZnO NPs and ZnCl2 (25–400 μg ml−1) and Ag NPs and AgNO3 (5–100 μg ml−1) to test antifungal activity of the NPs and ions. The mycelial growth of S. homoeocarpa isolates regardless of their DMI sensitivity was significantly inhibited on ZnO NPs (≥200 μg ml−1), Ag NPs (≥25 μg ml−1), Zn2+ ions (≥200 μg ml−1), and Ag+ ions (≥10 μg ml−1) amended media. Expression of stress response genes, glutathione S-transferase (Shgst1) and superoxide dismutase 2 (ShSOD2), was significantly induced in the isolates by exposure to the NPs and ions. In addition, a significant increase in the nucleic acid contents of fungal hyphae, which may be due to stress response, was observed upon treatment with Ag NPs using Raman spectroscopy. We further observed that a zinc transporter (Shzrt1) might play an important role in accumulating ZnO and Ag NPs into the cells of S. homoeocarpa due to overexpression of Shzrt1 significantly induced by ZnO or Ag NPs within 3 h of exposure. Yeast mutants complemented with Shzrt1 became more sensitive to ZnO and Ag NPs as well as Zn2+ and Ag+ ions than the control strain and resulted in increased Zn or Ag content after exposure. This is the first report of involvement of the zinc transporter in the accumulation of Zn and Ag from NP exposure in filamentous plant pathogenic fungi. Understanding the molecular mechanisms of NPs’ antifungal activities will be useful in developing effective management strategies to control important pathogenic fungal diseases.

Trends in Indian Rupee-Us Dollar Rate: An Empirical Analysis

The paper attempts to identify Indian and US macroeconomic factors and other global factors significantly influencing Indian rupee- US dollar spot rate and also, any long term association among these macroeconomic variables and rupee-dollar spot rates. Time series analysis has been undertaken for ten year period of April, 2005 –March, 2015 using monthly data of these variables. The exogenous variables considered are money stock (India and US), nominal long run bond yield (India and US), commodity price levels (India and US), stock prices (India and US), forward premium in Indian rupee-US dollar forward market, foreign exchange reserves with Reserve Bank of India., foreign institutional investment in India, Indian rupee-euro spot rate, Indian rupee-British pound spot rate and Indian rupee-Japanese yen spot rate. Ordinary least squares regression results indicate that significant non-price variables are foreign exchange reserves and RBI intervention measure, both being negatively related to rupee value. Out of price variables, consumer prices in US, rupee-euro spot rate and rupeepound spot rate are significant variables, all being positively related to rupee value and US 10 year bond yield is negatively related to value of rupee. Johansen’s co-integration analysis indicate that all Indian and US macroeconomic variables and other currency pairs of rupee are in a long run relationship with rupee-dollar spot rate.

Influence of Ferrous Sulfate and Its Elemental Components on Dollar Spot Suppression

Dollar spot (caused by Sclerotinia homoeocarpa F.T. Bennett) is a common disease of Agrostis stolonifera L. and is especially devastating on putting greens. Sequential fungicide applications are often required throughout the growing season for adequate control. This research was conducted to determine if ferrous sulfate (FeSO4) and its elemental components are capable of reducing dollar spot development. Trials were established in situ and in vitro to address this objective. Putting green trials were conducted in Blacksburg, VA, during the summers of 2012 and 2015. Biweekly (May to September) foliar treatments were applied in four randomized complete blocks as: control, FeSO4 (48.8 kg ha−1), sulfur (10.3 kg ha−1), and iron chelate (11.2 kg Fe ha−1). Dollar spot infection centers and visual turf quality were recorded throughout the summer. When disease exceeded a threshold of 75 infection centers per plot, chlorothalonil (2,4,5,6‐tetrachloroisophthalonitrile, 12.5 kg ha−1 a.i.) and nitrogen fertility (12.2 kg ha−1) were applied to encourage recovery. After recovery, inputs were minimized to encourage dollar spot development. Ferrous sulfate reduced dollar spot pressure each year. Turf quality was improved with FeSO4 in 2012, but not in 2015. Iron chelate suppressed dollar spot in 2012, but not in 2015. Sulfur had no impact on dollar spot or turf quality in either year. Direct fungitoxic activity of FeSO4 against the dollar spot pathogen was evaluated in vitro across a range of pH. Pure cultures of S. homoeocarpa obtained from the field test site were plated on 0.25× potato dextrose agar amended with 0, 10, 100, and 1000 mg kg−1 FeSO4. Mycelial growth was enhanced at 10 mg kg−1, was not affected at 100 mg kg−1, and was completely inhibited at 1000 mg kg−1. These data suggest FeSO4 suppresses dollar spot by acting as a fungistat against the pathogen.

Molecular Mechanisms Involved in Qualitative and Quantitative Resistance to the Dicarboximide Fungicide Iprodione in Sclerotinia homoeocarpa Field Isolates.

The dicarboximide fungicide class is commonly used to control Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrass. Despite frequent occurrences of S. homoeocarpa field resistance to iprodione (dicarboximide active ingredient), the genetic mechanisms of iprodione resistance have not been elucidated. In this study, 15 field isolates (seven suspected dicarboximide resistant, three multidrug resistance (MDR)-like, and five dicarboximide sensitive) were used for sequence comparison of a histidine kinase gene, Shos1, of S. homoeocarpa. The suspected dicarboximide-resistant isolates displayed nonsynonymous polymorphisms in codon 366 (isoleucine to asparagine) in Shos1, while the MDR-like and sensitive isolates did not. Further elucidation of the Shos1 function, using polyethylene glycol-mediated protoplast transformation indicated that S. homoeocarpa mutants (Shos1I366N) from a sensitive isolate gained resistance to dicarboximides but not phenylpyrrole and polyols. The deletion of Shos1 resulted in higher resistance to dicarboximide and phenylpyrrole and higher sensitivity to polyols than Shos1I366N. Levels of dicarboximide sensitivity in the sensitive isolate, Shos1I366N, and Shos1 deletion mutants were negatively correlated to values of iprodione-induced expression of ShHog1, the last kinase in the high-osmolarity glycerol pathway. Increased constitutive and induced expression of the ATP-binding cassette multidrug efflux transporter ShPDR1 was observed in six of seven dicarboximide-resistant isolates. In conclusion, S. homoeocarpa field isolates gained dicarboximide resistance through the polymorphism in Shos1 and the overexpression of ShPDR1.

Genes Required for the Anti-fungal Activity of a Bacterial Endophyte Isolated from a Corn Landrace Grown Continuously by Subsistence Farmers Since 1000 BC.

Endophytes are microbes that inhabit internal plant tissues without causing disease. Some endophytes are known to combat pathogens. The corn (maize) landrace Chapalote has been grown continuously by subsistence farmers in the Americas since 1000 BC, without the use of fungicides, and the crop remains highly valued by farmers, in part for its natural tolerance to pests. We hypothesized that the pathogen tolerance of Chapalote may, in part, be due to assistance from its endophytes. We previously identified a bacterial endophyte from Chapalote seeds, Burkholderia gladioli strain 3A12, for its ability to combat a diversity of crop pathogens, including Sclerotinia homoeocarpa, the most important fungal disease of creeping bentgrass, a relative of maize used here as a model system. Strain 3A12 represents a unique opportunity to understand the anti-fungal activities of an endophyte associated with a crop variety grown by subsistence farmers since ancient times. Here, microscopy combined with Tn5-mutagenesis demonstrates that the anti-fungal mode of action of 3A12 involves flagella-dependent swarming toward its pathogen target, attachment and biofilm-mediated microcolony formation. The mutant screen revealed that YajQ, a receptor for the secondary messenger c-di-GMP, is a critical signaling system that mediates this endophytic mobility-based defense for its host. Microbes from the traditional seeds of farmers may represent a new frontier in elucidating host–microbe mutualistic interactions.

Bacterial endophytes from wild and ancient maize are able to suppress the fungal pathogen Sclerotinia homoeocarpa.

The aim of this study was to determine if endophytes from wild and ancient Zea plants (corn family) have anti-fungal activities, specifically against the most important fungal pathogen (Sclerotinia homoeocarpa) of creeping bentgrass, a relative of Zea, used here as a model grass. A library of 190 bacterial endophytes from wild, ancient and modern Zea plants were tested for their ability to suppress S.homoeocarpa invitro, followed by in planta testing of candidates using greenhouse trials. Three endophytes could suppress S.homoeocarpa, originating from wild maize and an ancient Mexican landrace, consistent with our hypothesis. 16S phylogenetic analysis and BOX-PCR DNA fingerprinting suggest that the anti-fungal endophytes are distinct strains of Burkholderia gladioli. One strain (3A12) was confirmed to colonize creeping bentgrass using green fluorescent protein (GFP) tagging. Evans blue vitality staining demonstrated that the bacterial endophytes exhibited fungicidal activities against the pathogen. The endophytes inhibited a wide spectrum of plant-associated fungi including diverse crop pathogens. The results support the hypothesis that wild and ancient Zea genotypes host bacterial endophytes that can control fungal pathogen(s). These results suggest that wild and ancient crops may be an unexplored reservoir of anti-fungal bacterial endophytes.

Draft Genome Sequences of the Turfgrass Pathogen Sclerotinia homoeocarpa.

Sclerotinia homoeocarpa (F. T. Bennett) is one of the most economically important pathogens on high-amenity cool-season turfgrasses, where it causes dollar spot. To understand the genetic mechanisms of fungicide resistance, which has become highly prevalent, the whole genomes of two isolates with varied resistance levels to fungicides were sequenced.

Influence of simulated rainfall on efficacy of fluazinam, chlorothalonil and iprodione for dollar spot control in creeping bentgrass

Efficacy of foliar applied fungicides following simulated rainfall for the control of dollar spot (caused by Sclerotinia homoeocarpa F.T. Bennett) was assessed in a two-year field study on creeping bentgrass (Agrostis stolonifera L.) turf maintained as a golf course fairway. The study was conducted as a randomized complete block design with a factorial arrangement. Fluazinam (0.8 kg a.i. ha−1), chlorothalonil (3.79 kg a.i. ha−1) or iprodione (1.5 kg a.i. ha−1) were applied, then subjected to simulated rainfall (2.54-mm) at intervals of 15-, 30-, 60-min post-application, or no simulated rain. In most cases, simulated rainfall occurring ≤60 min post-application had greater disease than no rain plots; however, few differences occurred among rainfall intervals ≤60 min. Fluazinam provided the greatest dollar spot reduction regardless of simulated rainfall interval throughout the study. Chlorothalonil was most susceptible to losses in efficacy due to simulated rainfall, resulting in the greatest disease incidence of those fungicides evaluated. Iprodione was comparable to chlorothalonil during high disease pressure, although during moderate disease pressure it controlled dollar spot for approximately 7 days before disease increased in plots receiving simulated rain compared to those without simulated rain. These data demonstrate that efficacy of fungicides applied for dollar spot control are affected by rain, and differ in their ability to control disease following post-application rain events. Fluazinam, a recently introduced contact fungicide for use on turfgrass, can provide improved control compared to chlorothalonil or iprodione when rain is eminent.

Relevance of in vitro agar based screens to characterize the anti-fungal activities of bacterial endophyte communities.

BackgroundEndophytes are microbes that inhabit internal plant tissues without causing disease. Plant microbial communities consist of large numbers of endophyte species. Understanding the functions of these endophytes is a major challenge. An important function of some endophytes is to suppress fungal pathogens. Typically, plant associated microbes are screened for anti-fungal activities in vitro using the high-throughput dual culture screen, but it is not clear whether this method correlates with the activities of these microbes in planta. Furthermore, it is not clear whether in vitro screening captures all of the microbes that show this activity inside plants. The objective of this study was to evaluate the relevance of the in vitro dual culture method for screening endophytes with anti-fungal activity.ResultsIn parallel, 190 bacterial endophytes from the corn grass family (Zea) were screened for suppression of two fungal pathogens (Sclerotinia homoeocarpa and Rhizoctonia solani) using the in vitro dual culture method, and in planta using the model plant, creeping bentgrass. All endophytes that showed anti-fungal activity in planta against Sclerotinia homoeocarpa and Rhizoctonia solani (3 or 4 strains, respectively, out of 190), were captured in vitro. The in vitro and in planta screening results strongly correlated (r = 0.81 and r = 0.94 for the two pathogens, respectively).ConclusionsEvidence was gained here that the in vitro dual culture method is a relevant method for high throughput screening of plant endophyte communities for anti-fungal activity. In our study, the method captured all of the microbes that suppressed the corresponding pathogens in planta.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0623-9) contains supplementary material, which is available to authorized users.

Effects of Genotype and Isolate on Expression of Dollar Spot in Seashore Paspalum

Seashore paspalum (Paspalum vaginatum Swartz) is a warm-season turfgrass species primarily used on golf courses and athletic fields, and is often impacted by the disease dollar spot caused by Sclerotinia homoeocarpa F.T. Bennett. Dollar spot is the most common and economically important turfgrass disease in North America, and current management of this disease relies heavily on frequent fungicide applications. An alternate management strategy is host plant resistance, but a better understanding of the interactions between pathogen isolates and the host species is needed to effectively incorporate this resistance into elite seashore paspalum genotypes. The goal of this study was to gather host plant/isolate response data that could be used to develop an effective and efficient screening protocol for resistance to this important disease. Five genotypes of seashore paspalum (‘Aloha’, ‘SeaIsle 2000’, ‘SeaIsle 1’, ‘SeaIsle Supreme’, and 05-1743) varying in dollar spot resistance were inoculated with five isolates of S. homoeocarpa in repeated field studies during 2012 and 2013. Isolates used were from three warm-season and one cool-season turfgrass species. Inoculated plots were evaluated visually and using digital image analysis (DIA) for disease development over time and for number and area of infection centers at two rating dates each year. Statistical differences among the seashore paspalum genotypes and inoculation/isolate treatments were detected for area under the disease progress curve (AUDPC) values, number of infection centers, and infection center area. A significant interaction between seashore paspalum genotype and S. homoeocarpa isolate effects was not observed, indicating that host plant resistance genes are likely not isolate specific. Using this information, breeders should be able to use one highly virulent S. homoeocarpa isolate to screen for host plant resistance in seashore paspalum.

잔디 근권에서 분리된 Actinomycetes균주의 생리학적 특성

Total 443 isolates of actinomycetes were isolated from turfgrass rhizosphere as potential biological control agents. The two isolates (S11 and S4) showed highest cellulase activity with compared to the other isolates that exhibited a clear zone of 1.2 mm around the colony on cellulose agar medium. S12 strain appeared the most active chitin degrading, which exhibited a 1.2 mm of clear zone. The highest proteolytic activity on skim milk agar was which exhibited a 7.5 mm of clear zone by S2 strain. S1 strain from the soli showed siderophore production ability, which exhibited a 0.6 mm of large clear zone on chrome azurol S agar. The antifungal activity of the volatile compound producing by 4 selected actinomycetes was investigated that inhibition rate against Rhizoctonia solani AG2-2 and Sclerotinia homoeocarpa. Growth inhibition effect of S8 isolate against S. homoeocarpa was appeared to 94.8%, S2 to 76.9%, S5 to 46.1% and S12 to 43.5%. The significant inhibition effects on mycelial growth of S. homoeocarpa were shown on media with four strains. The inhibition effect was the highest with S8 strain treatment at 94.8%.

이쑤시개를 이용한 잔디층, 대취층, 및 토양층에서 동전마름병 전염원의 검출

동전마름병은 Sclerotinia homoeocarpa가 원인균으로 주로 한지형 잔디에 발생하는 주요 병이다. 본 연구는 이쑤시개를 이용하여 동전마름병 전염원 검출과 처리물량을 달리하여 약제 살포하였을 때 병원균 검출 특성을 조사하기 위하여 수행하였다. 동전마름병 병원균을 검출한 결과 병반 중심의 잎 부위에서는 100% 검출되었으며, 대취 부분에서는 83.3%가 검출되었으나 토양에서는 전혀 검출되지 않았다(대취층 아래 1, 2와 3 cm). 병반 중심부와 가장자리에서는 100% 검출되었으며 병반으로부터 1.5 cm 떨어진 부분에서는 13% 검출되었으며 병반으로부터 3와 4.5 cm 떨어진 부분에서 병원균의 분리빈도는 각각 3와 0%로 나타났다. 동전마름병 병원균은 병반으로부터 1.5 cm 떨어진 지점의 잎과 대취에서는 73.3%와 20%가 검출되었으며 3 cm 떨어진 부분과 4.5 cm 떨어진 부분에서 각각 46.6%와 13.3% 병원균이 검출되었다. 토양부분에서는 병원균이 검출되지 않았으며 전염원 검출 빈도는 병반으로부터 멀어질수록 낮아지는 경향이었다. 두 가지 약제를 같은 약량으로 2.26와 6.78 L의 물에 희석하여 처리한 결과 병원균 검출빈도는 Chlorothalonil (CH) 처리구보다 Propiconazole (PP) 처리구에서 높게 나타났다. 병반중심부에서 CH의 경우 검출 빈도는 15.5%로 PP의 경우 20.5%로 나타났다. 잎에서는 CH약제 처리구는 각각 15.5%와 2.2%가 검출되었으며 PP 처리구에서 이보다 높은 20.5%와 4.4%가 검출되었다. 대취층에서 전염원은 CH 처리구에서 물 2.26 L와 12.7%로 물 6.78 L가 2.2%로 검출되었다. 가장자리에서는 잎 부분을 검출한 결과 CH의 경우 물 2.26 L 처리구에서는 2.7%로 물 6.78L에서는 검출되지 않았으며 PP의 경우 물 2.26 L에서 9.4%로 물 6.78L에서는 CH 처리구와 같이 검출되지 않은 것으로 나타났다. 이쑤시개 방법은 빠르고 쉽게 동전마름병을 검출하여 전염원의 분포 조사 및 전염원 형성 예측을 할 수 있는 방법으로 제안한다. Dollar spot, caused by Sclerotinia homoeocarpa, is the major disease in cool-season turfgrasses. Understanding the distribution of this pathogen in soil and thatch is important to developing disease control strategies. In this study, toothpicks were used to detect S. homoeocarpa in the turfgrass canopy, thatch, and soil at different distances from dollar spot infection centers. The effect of penetrant and contact fungicide applications with different water volumes on distribution of S. homoeocarpa was also investigated. S. homoeocarpa was detected in 100% of samples taken from the leaf canopy, 83.3% in thatch area, and 0% in the soil from within the infection center. S. homoeocarpa was isolated in 100% of samples taken from the edge of the infection center, but was only detected in 13% of the samples taken at 1.5 cm away from the infection center edge. S. homoeocarpa was isolated at a higher frequency in the propiconazole treated plots than those treated with chlorothalonil and was not detected in leaf canopy samples when either fungicides was applied with 6.78 L of water. In conclusion, the toothpick-aided detection technique has improved our understanding of S. homoeocarpa epidemiology and could be used as a diagnostic tool to detect for fungicide resistance on golf courses.

Effect of watered-in demethylation-inhibitor fungicide and paclobutrazol applications on foliar disease severity and turfgrass quality of creeping bentgrass putting greens

Preventive, watered-in applications of DMI fungicides provide control of several soilborne turfgrass diseases on golf putting greens. The objectives of this two-year field study were to determine the impact of these applications on foliar diseases, and evaluate the impact of application timing of paclobutrazol, a plant growth regulator, on disease severity and turfgrass quality. Triadimefon (1.58 kg a.i. ha−1), tebuconazole (0.82 kg a.i. ha−1), metconazole (0.43 kg a.i. ha−1), and triticonazole (0.64 kg a.i. ha−1), were applied twice in late April and May and immediately watered in to the soil profile with 5 mm of post application irrigation. Paclobutrazol (0.28 kg a.i. ha−1) was applied alone, in a tank-mix with the fungicide application, 7 days or 14 days after the fungicide application. Overall dollar spot severity, assessed as area under the disease progress curve (AUDPC), was significantly lower in fungicide treated plots than non-treated plots in both trial years. The date of threshold symptom observation (≥5 infection centers per plot), however, was not different between fungicide treated and non-treated plots in 2011. Triadimefon treated plots had lower AUDPC values than other fungicide treatments. Brown patch severity was not significantly different among treatments. Paclobutrazol applied alone did not reduce dollar spot or brown patch severity, however, paclobutrazol + fungicide treatments resulted in lower dollar spot severity than plots treated with fungicide alone. Short-lived phytotoxicity (bronze discoloration) was observed in plots treated with triadimefon or paclobutrazol + fungicide. Spring preventive fungicide applications targeted at soilborne disease control also provided residual control of dollar spot in this study.

Information Flow Between Forward and Spot Markets: Evidence From the Chinese Renminbi

We apply a new model selection approach that allows for the joint determination of structural breaks and cointegration to examine the term structure of Chinese Renminbi (RMB)‐U.S. dollar spot and forward exchange rates during the managed‐floating period of 2005–2013. We find that the RMB market has exhibited different dynamic relationships between spot and forward exchange rates over time, apparently due to significant policy changes. Offshore forward rates with either shorter or longer maturities can substantially explain the in‐sample variation of the onshore spot exchange rate at longer horizons, while only the offshore forward rate with a shorter maturity can significantly predict RMB onshore spot rate changes out‐of‐sample. © 2015 Wiley Periodicals, Inc. Jrl Fut Mark 36:695–718, 2016

Characterization and distribution of mating-type genes of the turfgrass pathogen Sclerotinia homoeocarpa on a global scale

Sclerotinia homoeocarpa F.T. Bennett is a filamentous member of Ascomycota that causes dollar spot, the most economically important disease of turfgrass worldwide. We sequenced and characterized the mating-type (MAT) locus of four recently-collected contemporary strains causing dollar spot, four historical type strains used to describe the fungus, and three species of Rutstroemiaceae. Moreover, we developed a multiplex PCR assay to screen 1019 contemporary isolates for mating-type. The organization of the MAT loci of all strains examined could be classified into one of four categories: (1) putatively heterothallic, as exemplified by all contemporary strains and three of four historical type strains; (2) putatively heterothallic with a deleted putative gene in the MAT1-2 idiomorph, as detected in strains from two recently-collected populations in the United Kingdom that show more similarity to historical strains; (3) putatively homothallic with close physical linkage between MAT1-1-1 and MAT1-2-1, as found in one historical type strain of S. homoeocarpa and two strains of Rutstroemia cuniculi; and (4) an unresolved but apparently homothallic organization in which strains contained both MAT1-1-1 and MAT1-2-1 but linkage between these genes and between the two flanking genes could not be confirmed, as identified in R. paludosa and Poculum henningsianum. In contemporary S. homoeocarpa populations there was no significant difference in the frequency of the two mating types in clone-corrected samples when analyzed on regional and local scales, suggesting sex may be possible in this pathogen. However, two isolates from Italy and twenty from California were heterokaryotic for both complete heterothallic MAT idiomorphs. Results from this study contribute to knowledge about mating systems in filamentous fungi and enhance our understanding of the evolution and biology of an important plant pathogen.

Regulation with Trinexapac-ethyl and Dew Removal at the Time of Fungicide Application Did Not Influence Dollar Spot Control

Dollar spot, caused by Sclerotinia homoeocarpa F.T. Bennett, is an important disease of creeping bentgrass (Agrostis stolonifera L.) on golf courses in the northern United States. Canopy moisture in the form of dew plays an important role in the development of dollar spot and routine displacement has been shown to reduce disease severity. The use of plant growth regulators (PGRs) is a common management practice for maintaining creeping bentgrass fairways, but their influence on dollar spot is unclear. The objective of this field study was to elucidate the influence of dew removal at the time of fungicide application on dollar spot control in creeping bentgrass regulated by trinexapac-ethyl (TE). Main factors in the study included three dew removal strategies (non-treated, dew removed–mowed, and dew removed–not mowed) before the application of four fungicide treatments (non-treated, chlorothalonil, propiconazole, and iprodione). All fungicide treatments were applied once to turfgrass previously treated with TE or not treated. The presence or absence of dew at the time of fungicide application generally had no influence on fungicide performance with respect to dollar spot control. Based on the results of this study, dew removal before the application of fungicides targeting dollar spot is unnecessary. Applications of TE before fungicides reduced dollar spot severity in some cases, but reductions in symptom expression were limited and did not result in markedly improved dollar spot control.

First Report of Dollar Spot, Caused by Sclerotinia homoeocarpa, of Creeping Bentgrass in Norway.

In September 2013, symptoms similar to dollar spot caused by Sclerotinia homoeocarpa F.T. Benn., were observed on creeping bentgrass (Agrostis stolonifera L.) fairways at Losby Golf Course, Lørenskog, Akershus County, in Norway (59.8864' N, 10.9862' E). There were small, circular spots and larger irregular patches of sunken, bleached, straw-colored turf. Affected leaves had light-tan lesions with light reddish-brown margins (2). Abundant aerial mycelium was observed in the diseased turf after incubation for 24 h at room temperature in a moist chamber. The mycelium was septate with y-shaped branches. No spores were observed. Diseased leaf segments were washed 30 min in cold running water, surface-sterilized for 60 s using 70% ethanol, placed on water agar, and incubated at room temperature. After 4 days, water agar plugs containing the fungus were transferred to 50% potato dextrose agar (PDA; 19.5 g PDA and 7.5 g agar per 1 liter of media). The fungus colonized the entire 9-cm PDA plates in 6 days. The diameter of the hyphae varied from 2.5 to 12.5 μm. The white, floccose mycelium turned olive green after 7 days and cinnamon brown after 21 days. The cultures became brown from the bottom, forming flat, dark-brown stroma of 0.5 to 5.0 mm in diameter. DNA was extracted from three isolates (from different plants) using a DNeasy Plant Mini Kit (Qiagen). The ribosomal internal transcribed spacer (ITS) region was PCR-amplified using primers ITS1 and ITS4 (3). All three isolates were identical in sequence (GenBank Accession No. KJ775860) and showed up to 97.6% similarity with isolates of S. homoeocarpa of the common type (C-type; e.g., GenBank Accession No. HQ449691) (1). This similarity is considered quite low within a species and indicates that the Norwegian isolates are distinct from other S. homoeocarpa. For Koch's postulates, the fungus was scraped off 21-day-old PDA cultures and chopped, using a sterile scalpel. All three sequenced isolates were pooled and mixed with 200 ml of autoclaved water. Four mature, healthy sod plugs of creeping bentgrass cv. Independence (10-cm-diameter and 10-cm-depth) were taken from an experimental golf green at Landvik, inserted into pots, and inoculated by even distribution of 50 ml of the fungal suspension. Two control pots with creeping bentgrass received 50 ml of sterile water only. All six pots were incubated individually in plastic bags at room temperature and 16-h daylight. After 14 days, 30 to 90% of the inoculated pots of turfgrass exhibited dollar spot symptoms and controls remained healthy. The fungus was recovered from inoculated turf and identified morphologically. This is the first report of dollar spot on any grass species in Norway. For climatic reasons, dollar spot has been considered to be nonexistent in Scandinavia. However, during recent years, symptoms resembling dollar spot have been observed on more than 15 golf courses in Sweden, Denmark, and Norway, and the damage has varied from low to severe. The disease has been given the Norwegian name myntflekk (i.e., coin spot).

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.

Sclerotinia homoeocarpa overwinters in turfgrass and is present in commercial seed.

Dollar spot is the most economically important disease of amenity turfgrasses in the United States, yet little is known about the source of primary inoculum for this disease. With the exception of a few isolates from the United Kingdom, Sclerotinia homoeocarpa, the causal agent of dollar spot, does not produce spores. Consequently, it was assumed that overwintering of this organism in soil, thatch, and plant debris provides primary inoculum for dollar spot epidemics. Overwintering of S. homoeocarpa in roots and shoots of symptomatic and asymptomatic creeping bentgrass turfgrass was quantified over the course of a three-year field experiment. Roots did not consistently harbor S. homoeocarpa, whereas S. homoeocarpa was isolated from 30% of symptomatic shoots and 10% of asymptomatic shoots in the spring of two out of three years. The presence of stroma-like pathogen material on leaf blades was associated with an increase in S. homoeocarpa isolation and colony diameter at 48 hpi. Commercial seed has also been hypothesized to be a potential source of initial inoculum for S. homoeocarpa. Two or more commercial seed lots of six creeping bentgrass cultivars were tested for contamination with S. homoeocarpa using culture-based and molecular detection methods. A viable, pathogenic isolate of S. homoeocarpa was isolated from one commercial seed lot and contamination of this lot was confirmed with nested PCR using S. homoeocarpa specific primers. A sensitive nested PCR assay detected S. homoeocarpa contamination in eight of twelve (75%) commercial seed lots. Seed source, but not cultivar or resistance to dollar spot, influenced contamination by S. homoeocarpa. Overall, this research suggests that seeds are a potential source of initial inoculum for dollar spot epidemics and presents the need for further research in this area.