Plant Wounding and Ophiostoma Mitovirus 3A (Omv3a) Influence Infection of Creeping Bentgrass by Sclerotinia Homoeocarpa Angela M

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln

Papers in Plant Pathology Plant Pathology Department

3-2012 Plant wounding and Ophiostoma mitovirus 3a (OMV3a) influence infection of creeping bentgrass by Sclerotinia homoeocarpa Angela M. Orshinsky Ohio State University, [email protected]

Michael J. Boehm University of Nebraska-Lincoln, [email protected]

Greg J. Boland University of Guelph, [email protected]

Follow this and additional works at: http://digitalcommons.unl.edu/plantpathpapers Part of the Other Plant Sciences Commons, Plant Biology Commons, and the Plant Pathology Commons

Orshinsky, Angela M.; Boehm, Michael J.; and Boland, Greg J., "Plant wounding and Ophiostoma mitovirus 3a (OMV3a) influence infection of creeping bentgrass by Sclerotinia homoeocarpa" (2012). Papers in Plant Pathology. 418. http://digitalcommons.unl.edu/plantpathpapers/418

This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Published in Canadian Journal of Plant Pathology 34:4 (March 2012), pp. 493-506; doi: 10.1080/07060661.2012.678886 Copyright © 2012 The Canadian Phytopathological Society; published by Taylor & Francis. Used by permission. Submitted March 20, 2012, author version online April 4, 2012, published online August 31, 2012. digitalcommons.unl.edu

Plant wounding and Ophiostoma mitovirus 3a (OMV3a) influence infection of creeping bentgrass by Sclerotinia homoeocarpa

Angela M. Orshinsky1, Michael J. Boehm1, and Greg J. Boland2

1 Department of Plant Pathology, 201 Kottman Hall, The Ohio State University, Columbus, Ohio, 43210, USA 2 School of Environmental Sciences, University of Guelph. Guelph, Onatario, N1G 2W1, Canada

Abstract Colonization and lesion development by virulent, asymptomatic and hypovirulent isolates of Sclerotinia homoeocarpa on nonwounded (NW) and wounded (W) leaves of creeping bentgrass were characterized. Hypovirulent and asymptomatic isolates contain the fungal virus, Ophiostoma mitovirus 3a, and virulent isolates are virus-free. On NW leaves, all isolates infected leaves with appressoria along cell walls and through stomata by 48 hours post-inoculation (hpi). Inter- and intracellular hyphae formed on heavily colonized, NW leaves at 96 hpi. Wound-inoculated grass had a colonization front characterized by inter- and intracellular hyphal colonization within nonsymptomatic tissues at 8 hpi by direct infection of the wound site. The colonization front grew ahead of symptom development for 50 hpi on both NW and W leaves. In contrast to virulent and asymptomatic isolates, the hypovirulent isolate sel- dom colonized more than 30% of leaf tissue of the NW or W leaves. Callose accumulation under fungal appressoria, oxalate oxidase activity, and hydrogen peroxide accumulation at lesion borders were also demonstrated. The results indicate that S. homoeocarpa is capable of colonizing creeping bentgrass without causing visible symptoms. In addition, the fungus causes disease more rapidly on W than NW grass, and the etiology of virulent, asymptomatic and hypovirulent isolates was similar. The results of this study contribute to our understanding of the basic biology of S. homoeocarpa and may influence integrated management practices.

Keywords: dollar spot, histopathology, hypovirulence, microscopy, mycovirus, Ophiostoma mitovirus 3a, Sclerotinia homoeocarpa, turfgrass

Résumé L’apparition et la colonisation de lésions produites par des isolats virulents, asymptomatiques et hypovirulents de Sclerotinia homeocarpa sur des feuilles saines et meurtries d’agrostide rampante ont été caractérisées. Les isolats hypovirulents et asymptomatiques contiennent le virus fongique Ophiostoma mitovirus 3a et les isolats virulents n’en contiennent pas. Tous les isolats ont infecté les feuilles saines en pénétrant les parois cellulaires et les stomates avec leurs appresseurs, et ce, en moins de 48 heures après inoculation (hpi). À 96 hpi, les hyphes inter- et intracellulaires se sont formés sur les feuilles saines abondamment colonisées. À 8 hpi, les feuilles meurtries inoculées affichaient un front de colonisation caractérisé par la colonisation hyphale inter- et intracellulaire des tis- sus non symptomatiques, et ce, par infection directe du site de la meurtrissure. Le front de colonisation a devancé l’apparition des symptômes de 50 hpi sur les feuilles saines comme sur les feuilles meurtries. Contrairement aux isolats virulents et asymptomatiques, les isolats hypovirulents colonisaient rarement plus de 30 % du tissu des feuilles saines ou meurtries. L’accumulation de callose sous les appresseurs, l’activité de l’oxalate oxydase et l’accumulation de peroxyde d’hydrogène en périphérie de la lésion ont également été démontrées. Les résultats indiquent que S. homeocarpa peut coloniser l’agrostide rampante sans que des symptômes apparais- sent. De plus, le champignon infecte plus rapidement les feuilles meurtries que les feuilles saines, et les étiologies des isolats virulents, asymptomatiques et hypovirulents se ressemblaient. Les résultats de cette étude contribuent à notre compréhension de la bi- ologie de S. homeocarpa et peuvent influer sur les méthodes de lutte intégrée.

Mots clés: brûlure en plaques, gazon, hypovirulence, histopathologie, microscopie, Ophiostoma mitovirus 3a, Sclerotinia homeocarpa, virus fongique

493 494 Orshinsky et al. in Canadian Journal of Plant Pathology, 34:4 (2012)/

Introduction production and/or role of these compounds during disease development. Sclerotinia homoeocarpa F. T. Bennett is a fungal patho- Research on the physiology of S. homoeocarpa has gen causing dollar spot disease of turfgrass. The fungus been aided by the study of hypovirulent isolates that have is capable of causing disease in at least 40 plant species, been documented as containing the mycovirus, Ophios- but most hosts are classified within the grass plant family toma mitovirus 3a (OMV3a). OMV3a is a virus belonging Poaceae (Walsh et al., 1999). All commonly cultivated turf- to the family Narnaviridae and consists of a 2.6 kb unen- grass species in North America are considered susceptible capsidated dsRNA (Deng et al., 2003). Isolates of S. ho- to this pathogen although there are variations in disease moeocarpa containing OMV3a may also carry a satellite severity and recovery among selected species and culti- RNA; however, the presence of the satellite RNA has not vars (Chakraborty et al., 2006; Bonos et al., 2007). Creep- been associated with any effects on fungal growth (Deng ing bentgrass (Agrostis stolonifera) is a cool-season spe- & Boland, 2004). cialty grass commonly used for golf course putting greens, OMV3a has been implicated as a cause of hypovir- lawn bowling greens and lawn tennis facilities; however, ulence in infected isolates (Zhou & Boland, 1997, 1998; creeping bentgrass is one of the most susceptible hosts Deng et al., 2003). OMV3a can be detected in hypoviru- to S. homoeocarpa (Chakraborty et al., 2006). lent isolates by agarose gel electrophoresis of semi-pu- Symptoms of dollar spot in creeping bentgrass can rified dsRNA extracts. OMV3a can also be detected in vary according to cultural practices and mowing height some fully virulent isolates using reverse transcription- but are typically characterized by hourglass-shaped le- polymerase chain reaction (RT-PCR) (Melzer et al., 2005). sions on individual grass blades, and straw-coloured Virulent isolates that are asymptomatically infected with patches of turf approximately 3 cm in diameter, which OMV3a are termed asymptomatic isolates (Melzer et al., can merge to form larger areas of diseased grass (Walsh 2005). Asymptomatic isolates containing OMV3a appear et al., 1999). Only infertile apothecia of S. homoeocarpa to be common in nature, as 17–100% of isolates in vari- have been studied in North American isolates in vitro (Or- ous locations in eastern Canada contained the virus (Mel- shinsky & Boland, 2011), and the role of conidia or as- zer et al., 2005). Little information is available as to how cospores in the S. homoeocarpa lifecycle has not been OMV3a can cause both symptomatic and asymptomatic demonstrated. S. homoeocarpa transmission is thought infections in S. homoeocarpa; however, asymptomatically to occur through the movement of mycelial propagules infected S. homoeocarpa isolates have been shown to re- via infested or diseased materials, such as grass clippings, vert to a slow-growing, hypovirulent phenotype follow- and through pedestrian traffic and turf maintenance prac- ing storage (Deng & Boland, 2006). Hypovirulent isolates tices such as mowing (Walsh et al., 1999). Sclerotinia ho- infected by OMV3a are characterized by degenerate mi- moeocarpa is thought to overwinter as darkly pigmented tochondria (Boland et al., 2000) and OMV3a infection ap- stromata remaining on the margins of dollar spot lesions pears to interfere with the normal functioning of mito- from previous epidemics (Couch & Bloom, 1960; Walsh et chondria in hypovirulent isolates (Orshinsky & Boland, al., 1999) but may also survive as dormant mycelium in 2010). Hypovirulent, OMV3a-containing isolates of S. ho- infected grass crowns and tissues (Fenstermacher, 1980). moeocarpa have been shown to have biological control Pathogen entry occurs by means of mycelial penetra- activity and reduced severity of dollar spot symptoms by tion through cut leaf tips or stomata but direct penetra- up to 80% and 58% on artificially and naturally infested tion can also occur through the formation of appresso- field plots, respectively (Zhou & Boland, 1998). The suc- ria (Endo, 1963). cessful utilization of hypovirulence and other biocontrol Previous studies on the etiology of dollar spot have strategies will depend on establishing a more thorough found that S. homoeocarpa produces a diffusible sub- understanding of the pathogen lifecycle and dollar spot stance that can cause root browning, necrosis and stunt- disease etiology. ing in the absence of the fungal mycelium (Endo, 1966). The aim of this study was to evaluate S. homoeo- Dgalactose was identified as a putative toxic component carpa colonization and lesion development on host tis- produced by S. homoeocarpa in vitro when it was grown sues over time under laboratory conditions. Defence on oligosaccharides containing lactose; however, galac- responses of the creeping bentgrass host were also mon- tose is not believed to be a toxic compound produced by itored using microscopy and biochemical techniques. S. homoeocarpa in vivo (Malca & Endo, 1965). Phytotoxic The effect of OMV3a on this process was also explored tetranorditerpenoid compounds have also been reported by comparing virulent, asymptomatic and hypoviru- to be produced by S. homoeocarpa (Bandara-Herath et al., lent isolates of the fungus. The results of this study may 2009); however, more research is required to confirm the have implications in fungicide application strategies, the infection of creeping bentgrass by Sclerotinia homoeocarpa 495 development of successful disease prediction models, and 144 and 192 hpi. Lesions were measured from the cen- the improvement of current integrated pest management tre of the culture plug in a single direction. For histo- programmes. logical processing, the leaf blades were cut at the loca- tion of the culture plug. The experiment was conducted three times. Materials and methods Statistical analyses Fungal isolates and turfgrass For all experiments, assumptions of normality and homo- Isolates of S. homoeocarpa were maintained on potato geneity of error were tested using proc univariate (SAS dextrose agar (PDA, Difco, BD-Canada, Oakville, ON). The 9.2, SAS Institute, Cary, NC). Analysis of variance was per- virulent, asymptomatic and hypovirulent isolates used in formed using proc glm of SAS 9.2, and the same proce- this study were Sh80, Sh43 and Sh12B, respectively. Creep- dure was used to estimate the least squared means and ing bentgrass ‘Penncross’(Agrostis stolonifera) (OSC Seeds, standard error for each isolate at each hour postinocula- Waterloo, ON), was grown on Promix PGX seedling mix tion (hpi). (Premier Horticulture, Pickering, ON) in a growth room at 23 ◦C with a 16 h photoperiod and a light intensity of 180 μM · m−2 · s−1. Histological investigations Distinctions between water-soaked lesions, chlorotic le- Inoculation of wounded leaf blades sions and necrotic tissues were recorded. The leaf blades Whatman 9-cm diameter filter papers (Piscataway, New were cleared in a solution of 25% acetic acid and 75% eth- Jersey) were placed in 9-cm diameter Petri dishes and anol for 48–72 h, and then washed in water for 24 h with moistened with 0.8 mL of sterile deionized water. Mycelial 3–4 water changes. Samples were then stained in 0.05% plugs (3 mm in diameter) from 5-day-old cultures on PDA trypan blue (BDH Chemical Ltd., Poole, UK) in water for were placed in the centre of each filter paper. Leaf blades up to 24 h. The leaf blades were destained for 12–24 h in were cut to approximately 4 cm in length and arranged water. Prior to microscopic examination, leaf blades were around the plug so that one cut end (i.e. wounded) of the placed in 100% glycerol overnight and mounted in 50% blades was in contact with the plug. Only one end of the glycerol on slides. Some samples were placed directly on leaf blade was cut. Four blades (i.e. subsamples) per Petri slides and mounted in Hoyer’s mounting medium (con- dish were inoculated per isolate for each incubation time. sisting of 15 g gum arabic, 100 g chloral hydrate, 10 g The blades were incubated at 25 ◦C and under approxi- glycerin and 25 mL water (Agerer, 1991). Mycelial length mately 40 μM · m-2 · s−1 fluorescent light and removed at was measured using calipers while viewing the leaves with 8, 16, 24, 32, 40, 48, 56, 64, 72, 84, 96, 108 and 120 hours a Nikon SMZ 1500 stereomicroscope (Nikon Canada, Mis- post-inoculation (hpi) for all three isolates for measure- sissauga, ON). During the initial stages of fungal infection, ment and processing for histopathology. Additional sub- hyphae were found to infect the leaf at various intervals samples were inoculated with the hypovirulent isolate and along the leaf blade. The formation of appressoria, infec- sampled at 132, 144, 156 and 168 hpi. This extended sam- tion hyphae, infection vesicles, primary hyphae, intracel- pling time was to accommodate for the slower growth lular hyphae and intercellular hyphae were also described of the hypovirulent isolate. Lesion length was measured at 200×–1000× magnification using a Nikon Eclipse 50i along each leaf blade at each sampling time. The exper- microscope (Nikon Canada, Mississauga, Onatario). Pic- iment was conducted three times. Control uninoculated tures of these structures were taken with a Nikon Coolpix leaves were incubated on moistened filter paper in sealed 8400 digital camera. Petri dishes. These detached leaves remained green and healthy in appearance for over 200 h. Fluorescence microscopy To assess plant responses to infection by S. homoeo- Inoculation of nonwounded leaf tissue carpa, aniline blue (Sigma-Aldrich Canada, Oakville, Ona- Nonwounded leaf blades were also inoculated in Petri tario) was used to detect the deposition of callose tissue dishes containing moistened filter paper. For these ex- at infection points on wound- and nonwound-inoculated periments, three grass blades (i.e. subsamples) were cut leaves. Leaves were inoculated as described above on to approximately 6 cm in length and the culture plug both nonwounded and wounded inoculation points. At was placed in the centre of the blade segment. Petri 72 hpi, the leaves were cleared as described previously dishes were incubated at 25 ◦C and sampled at 48, 96, and then stained overnight in a solution of 0.1% aniline 496 Orshinsky et al. in Canadian Journal of Plant Pathology, 34:4 (2012)/ blue in 0.1 M potassium phosphate buffer, pH 8.0, and oxidase detection buffer containing 25 mM succinic acid, −1 washed with 0.1 M K2PO4, pH 8.0 for 30 min. The specific- 3.5 mM EDTA, 2.5 mM oxalic acid, 0.6 mg · ml 4-chloro- ity of aniline blue was tested by observing control leaves 1-naphthol, pH 4.0. Leaves were incubated in the oxalate that were not inoculated with S. homoeocarpa, and by oxidase detection buffer for 10 h then scanned as before observing fungal tissue in the absence of plant material. to acquire images of localization of blue precipitate indi- Under bright-field microscopy, aniline blue stain was ob- cating oxalate oxidase activity. served to lightly stain fungal hyphae and plant cell walls To visualize hydrogen peroxide accumulation, leaves and storage granules. A very faint fluorescence of the ani- were inoculated as described previously, sampled ev- line blue was only visualized on epidermal plant cells with ery 24 h for 120 h, and scanned to acquire lesion im- a 1–3 s camera exposure. In contrast, structures thought ages. Leaves were then immersed in a staining solution to be papillae fluoresced brightly and required only a 50 of 0.01 M MES buffer, 0.01% Triton X-100, and 50 mg ms exposure to observe intense fluorescence. Leaves from mL−1 3’,5’-diaminobenzidine (DAB), pH 5.5. Leaves were each treatment were observed using a Leica DM5500B mi- incubated in the solution for 5 h, washed in water then croscope (Richmond Hill, Onatario) and photos were taken scanned to acquire images of DAB precipitation in areas using a Hamamatsu ORCA-ER digital camera (Bridgewa- of H2O2 accumulation. ter, New Jersey), and with the aid of the Volocity 5.0 (Per- kin Elmer, Woodbridge, Onatario) computer program on a Mac OS operating system. This experiment was conducted twice. Aniline blue bound to the 1,3-ß-Dglucan Results helix of callose fluoresces at aλ max of 500–520 nm when excited with UV light at approximately 377 nm in water Lesion development or glycerol. The fluorescence of aniline blue when tightly complexed to the 1,3-ß-D-glucans, such as callose, is ap- Lesions caused by virulent and asymptomatic isolates on proximately 140 times more intense than that of free an- nonwounded and wounded leaves were similar in appear- iline blue (Thistlewaite et al., 1986). The increased fluo- ance; however, the development of lesions on wounded rescence in the presence of 1,3-ß-D-glucans relative to grass leaves proceeded more rapidly than lesion devel- other ß-glucans present in the cell increases as the poly- opment on nonwounded leaves. Lesions started out as mer takes on an open helical formation which binds the water-soaked, dark, green tissue after which chlorotic lesions began to form at the portion of the lesion distal to dye more tightly (Thistlewaite et al., 1986). Calcofluor white staining of fungal infection was also the inoculation point (Fig. 1). In some cases, the entire leaf conducted using fluorescence microscopy. Leaves of became darker and water-soaked in appearance, and the creeping bentgrass ‘Penncross’ were inoculated as pre- leaf tissues were completely macerated, making it diffi- viously described. Every 24 h, leaves were removed from cult to handle the leaves for processing. Lesions caused incubation, placed in tubes containing 1 mL of 3 : 1 etha- by the hypovirulent isolate started out as barely notice- nol : acetic acid and incubated until cleared. Leaves were able, water-soaked areas that were proximal to the inoc- rinsed in tap water, incubated in distilled water for 30 min, ulation point and quickly became chlorotic lesions with then stained with calcofluor white (1 g L−1) and Evans blue distinct, red-coloured borders (Fig. 1). Small brown spots (0.5 g L−1) (Sigma-Aldrich) directly on microscope slides were sometimes noticeable within the chlorotic zones af- for 5 min prior to viewing using a Nikon Eclipse 80i Epi- ter 144 hpi. fluorescence microscope at 200, 400 and 1000 magnifi- cations using a UV-2E/C filter with 340–380 nm excita- Hyphal growth and colonization of leaf tissue using light tion and a 435–485 bandpass emission filter. Photos were microscopy / taken using RT KE SE Spot digital imaging system (Diag- On nonwounded leaves, hyphae of all three isolates grew nostics Instruments). rapidly along the leaf surface. Superficial hyphae were approximately 4.4 μm wide and grew along the periclinal Detection of oxalate oxidase and hydrogen peroxide cell walls and the edge of the leaf. By 48 hpi, hyphae had Wound- and nonwound-inoculated leaves were sampled infected the leaf by forming appressoria along epider- every 24 h for 120 h as described above. At each sam- mal cell walls and over stomates (Fig. 2a, b, c). In some pling time, leaves were scanned using a CanoScan LiDE cases, superficial hyphae were observed infecting and col- 200 (Newport News, Virginia) to acquire an image of the onizing trichomes on the leaf edges (Fig. 2d). Appressoria resulting lesions. Leaves were then immersed in an oxalate formed from superficial hyphae were non-melanized and infection of creeping bentgrass by Sclerotinia homoeocarpa 497

Figure 1. Lesion development on creeping bentgrass inoculated with Sclerotinia homoeocarpa. a, Leaves inoculated at the wound site with isolate Sh12B. b, Leaves inoculated mid-leaf with isolate Sh12B. c, Leaves inoculated at the wound site with isolate Sh80. d, Leaves inoculated mid-leaf with isolate Sh80. Leaves in each group, left to right: 24, 48, 72, 96 and 120 hpi. 498 Orshinsky et al. in Canadian Journal of Plant Pathology, 34:4 (2012)/

Figure 2. Microscopic characterization of creeping bentgrass colonization by Sclerotinia homoeocarpa. Virulent, asymptomatic and hypovirulent isolates were inoculated onto nonwounded or wounded leaves and incubated for up to 240 h. a, Hyphal penetration into nonwounded tissues by appressoria (A) over stomata and cell walls. b, An appressorium on a cell wall with three infection vesicles (IV) and primary infection hyphae (PH) produced inside the initially infected epidermal cell. c, A bi-lobed appressorium with three infection vesicles (IV) and primary infection hyphae (PH). d, A hypha in the early stages of colonizing a trichome. e, Infection hyphae temporarily restricted to individual epidermal cells prior to infecting the neighbouring cells. f, Early colonization of a vascular bundle by S. homoeocarpa hyphae. g, Late, extensive colonization of a vascular bundle by S. homoeocarpa hyphae seen as a large blue row of mycelia. h, Infection of the mesophyll layer characterized by intercellular hyphae (arrow) growing between mesophyll cells (M). i, Papillae (arrows) seen in epidermal cells inoculated with isolate Sh12B which were yellow-brown in colour. Inset: formation of infection vesicle (IV) from infection peg surrounded by papilla. j, Yellow-brown granules observed below appressoria. Scale bar = 10 μm. infection of creeping bentgrass by Sclerotinia homoeocarpa 499 often had a septum separating the appressorial cell from chitinstaining calcofluor white (Fig. 3a). Clusters of ap- the parent hypha. Appressoria were formed singly or in pressoria forming from one or two main hyphal branches groups (Fig. 2a, b, c). Appressoria produced penetration were observed quite often with all isolates (Fig. 3b). Pri- pegs and infection vesicles either singly, or several per ap- mary infection vesicles and infection hyphae forming af- pressorium (Fig. 2a, b, c). Infection vesicles were spherical ter appressorium-mediated infection of epidermal cells in shape and gave rise to intracellular hyphae. The intra- was observed using the fluorescence microscopy tech- cellular hyphae were inflated, with a granular appearance, niques (Fig. 3c). In addition, the presence of infection and were an average of 6.8 μm wide (Fig. 2b, c). Intracel- cushions reminiscent of those produced by Sclerotinia lular hyphae appeared to be initially restricted by the cell sclerotiorum (Lib.) de Bary (Garg et al., 2010) were de- walls of the plant tissue prior to growth into neighbor- tected (Fig. 3d, e). These infection cushions also resulted ing cells (Fig. 2e). Spherical-shaped infection vesicles and in the formation of infection vesicles and primary hy- granular primary infection hyphae were produced in epi- phae in the epidermal cells below them (Fig. 3e). After dermal cells below appressoria (Fig. 2b, c). Primary infec- substantial colonization of host tissues, hyphae were ob- tion hyphae appeared to be temporarily restricted to the served exiting the stomata (Fig. 3f ). These hyphae could initially infected epidermal cell prior to moving into neigh- be followed to distances several millimetres from the exit boring epidermal cells (Fig. 2e). Sclerotinia homoeocarpa site where they reinfected the leaf tissue (not shown). also preferentially colonized areas surrounding vascular Aniline blue staining revealed bright blue fluorescence tissue. Hyphae initially grew towards the vascular bun- directly beneath the penetration site of appressoria of all dles (Fig. 2f ), which demonstrated dense mycelial growth isolates used in this study (Fig. 3i, j). later in the colonization process than the areas in between vascular bundles (Fig. 2g). Intercellular colonization of the mesophyll layer was observed; however, intracellular hy- Colonization of nonwounded and wounded leaves over phae were never observed inside mesophyll cells (Fig. 2h). time Intercellular hyphae were an average of 3.8 μm wide (Fig. In both nonwounded and wounded leaves, the mycelia h 2 ). Colonization of host tissues by isolate Sh12B resulted of virulent and asymptomatic isolates infected leaf tis- in more pronounced infection pegs and what appeared sues slightly ahead of the lesion until 80 hpi (Figs 4, 5). i to be papillae (Fig. 2 ). The papillae were dark brown and Hypovirulent isolate mycelia infected leaf tissues ahead yellow in colour, and extended well into the epidermal of lesion development until about 100 hpi (Figs 4, 5). Af- cells. However, the papilla were not completely success- ter these times, mycelial colonization and lesion devel- ful in preventing the formation of infection via infection opment appeared to proceed at the same rate (Figs 4, i vesicles (Fig. 2 ). Granules of similar-coloured substances 5). On nonwounded leaves, virulent and asymptomatic were seen accumulating below appressoria, suggesting a isolates did not cause lesions on the entire leaf (about j role in the plant response to infection (Fig. 2 ). 30 mm) until about 240 hpi (Fig. 4). In contrast, these Mycelia of all isolates infected wounded leaves at the same isolates reached 30 mm mycelia colonization length wound site as early as 8 hpi. The initial infection pro- and lesion length on wounded leaves between 72 and gressed as both inter- and intracellular hyphae that en- 90 hpi (Fig. 5). In general, mycelia colonization and dis- a tered the wound site directly (Fig. 3 ). Appressorium ease symptom progression proceeded at a slower rate on formation, the formation of penetration pegs, and the nonwounded leaves. On both nonwounded and wounded development of intracellular infection hyphae were sim- grass, the hypovirulent isolate colonized only up to 20– ilar to those observed on nonwounded leaf tissue. Intra- 25 mm of the leaf even after 168 to 240 hpi (Figs 4, 5). cellular hyphae formed the mycelial colonization front on Both nonwounded and wounded grass experiments re- the wound-inoculated leaves, although superficial hyphae sulted in some anomalous data points. The anomalous were observed to pass over several millimetres of healthy, data points occurred at 192 hpi for asymptomatic and uncolonized tissue to infect the leaf using appressoria. virulent isolates on nonwounded leaves and at 96 hpi for the virulent and 108 hpi for the asymptomatic isolate on Fluorescence microscopy wounded leaves (Figs 4, 5). These data points were notice- Observation of grass leaves inoculated at nonwounded ably lower than those preceding them. The values at these and wounded tissues by virulent and hypovirulent iso- times were consistent in all three experiments. However, lates resulted in more resolved images of complex hy- the lower values are likely an artifact of the destructive phal formations in some cases. It was possible to see di- sampling method employed in this study as lesion length rect hyphal infection of wounded leaf tissue using the should not decrease over these time periods. 500 Orshinsky et al. in Canadian Journal of Plant Pathology, 34:4 (2012)/

Figure 3. Fluorescence microscopy of Sclerotinia homoeocarpa infection process on creeping bentgrass. a, Leaves inoculated at a wound site, with extensive colonization after direct penetration at 24 h, shown by the fluorescing hyphae at the wound. b, Appres- soria formed in clusters along cell walls. c, Appressorium with infection vesicles in an epidermal cell. d, Infection cushions observed using fluorescence microscopy, showing fungal hyphal masses and an infection cushion along a cell wall (arrow). e, Infection cushion in (d) (out of focus) above the cell wall (arrow) with the infection vesicles and primary infection hyphae. f, Hyphae seen exiting the stomata (arrows) after extensive colonization of leaf tissue. g, An appressorium along a cell wall with a protrusion into the plant cell. h, Fluorescence microscopy using aniline blue of the appressorium in (g). The bright blue fluorescence along the protrusion in the plant cell wall demonstrates callose accumulation. Scale bar = 100 μm (a) and 10 μm (b–g).

Oxalate oxidase and hydrogen peroxide detection general, oxalate oxidase activity was detected in similar Oxalate oxidase was detected as early at 48 hpi as a dark areas as hydrogen peroxide accumulation. blue precipitate surrounding the lesions of both isolates Sh12B and Sh80 (Fig. 6). On wounded leaves of both isolates, the oxalate oxidase was detected as a thick band at Discussion the border of lesions (Fig. 6). DAB precipitation was evident in tissues surrounding lesions on wound and non- The results from this study suggest that the availability of S. homoeocarpa wound-inoculated grass leaves of both isolates (Fig. 6). In wound sites for direct infection of results infection of creeping bentgrass by Sclerotinia homoeocarpa 501

Figure 4. Colonization and lesion development of nonwounded tissue over time by virulent, asymptomatic and hypovirulent isolates of Sclerotinia homoeocarpa. Average lesion length (dotted line) and length of mycelia colonization front (internal and external mycelia growth, solid line) of (a) virulent, (b) asymptomatic and (c) hypovirulent isolates inoculated onto nonwounded creeping bentgrass leaves and incubated for 240 h. The bars at each data point represent the standard error for each mean. in more rapid disease development. Leaves with available Recommendations by turf extension groups suggest mow- wound sites were infected directly with interand intracel- ing as a means of reducing leaf wetness, but also warn that lular hyphae, whereas nonwounded leaves were only col- mowing during an active outbreak of dollar spot can result onized by penetration through the formation of appres- in spread of the disease (Latin, 2008; Putman & Kaminiski, soria and infection cushions over stomata and cell walls. 2011). The results of the current study suggest that creating Previous studies have suggested that daily mowing re- wounds through practices like mowing could increase the duces dollar spot severity, but this practice could reduce speed with which dollar spot disease symptoms become dollar spot severity by reducing leaf wetness (Ellram et al., evident. However, the effect of mowing and other envi- 2007) or by removing the diseased grass tissue as it grows. ronmental variables on symptom development is not clear. 502 Orshinsky et al. in Canadian Journal of Plant Pathology, 34:4 (2012)/

Figure 5. Colonization and lesion development of wounded grass tissue over time by virulent, asymptomatic and hypovirulent isolates of Sclerotinia homoeocarpa. Average lesion length (dotted line) and length of mycelia colonization front (internal and external mycelia growth, solid line) of (a) virulent, (b) asymptomatic and (c) hypovirulent isolates inoculated onto wounded ends of creeping bentgrass and incubated for 120 h for virulent and asymptomatic isolates and 168 h for hypovirulent isolates. The bars represent the standard error for each mean.

Sclerotinia homoeocarpa is considered to be a necro- development, and intercellular hyphae were seen grow- trophic pathogen; however, the fungus may not pro- ing around intact mesophyll cells. Symptomless infec- duce necrotic lesions immediately following infection of tion of plant hosts by necrotrophic fungal pathogens has grass hosts. This was clearly demonstrated as S. homoeo- been noted in past studies. For example, Mycosphaerella carpa colonized creeping bentgrass leaves ahead of lesion graminicola (Fuckel) J. Schröt., the necrotrophic fungus infection of creeping bentgrass by Sclerotinia homoeocarpa 503 The characteristic small, hourglass-shaped lesions of S. homoeocarpa may also be limited in expansion due to plant defences. Expansion of dollar spot lesions and diseased areas of turfgrass may occur when environmental conditions reduce plant defence gene expression, as in the B. cinerea–tomato pathosystem, or when environmental conditions favour pathogen virulence. This requires further investigation. The change in lesion morphology from green, dark, water-soaked tissue to brown, necrotic tissue bordered by chlorotic tissue only occurred after there had been dense colonization of the leaf tissue in the affected area. This change in lesion morphology coincided with slowing of mycelial colonization after approximately 75% (30 mm) of the leaf had been colonized. This slowing of mycelial growth occurred with both virulent and asymptomatic isolates at 72 hpi. As mycelial progression through the leaf slowed, lesions appeared to extend beyond the mycelial Figure 6. Oxalate oxidase and hydrogen peroxide accumula- colonization front of virulent and asymptomatic isolates. tion in creeping bentgrass ‘Penncross’ leaves. Oxalate oxidase For example, lesions formed ahead of the mycelial coloni- was detected using 4-chloro-4-naphthol on creeping bentgrass zation front of the asymptomatic isolate on nonwounded leaves inoculated with (a) isolate Sh12B and (b) isolate Sh80. Hy- and wounded leaves after 72 hpi; this occurred in con- drogen peroxide was detected using diaminobenzidine staining junction with a change in lesion morphology from water- on creeping bentgrass leaves inoculated with (c) isolate Sh12B soaked to necrotic. A change in lesion morphology was and (d) isolate Sh80. Each leaf pair shown consists of a creep- also associated with slowed mycelia colonization of the ing bentgrass leaf inoculated at a non-wounded site (left) and a virulent and hypovirulent isolates on nonwounded leaves. leaf inoculated at the wound site (right) at 48 hpi. The formation of lesions ahead of the mycelia colonization front suggests that a diffusible factor may be produced by S. homoeocarpa. Likely candidates for this diffusible toxin causing Septoria tritici leaf blotch of wheat, has a latent include the tetraterpenoid compounds that are produced period of 8–9 days prior to symptom development (Rudd by S. homoeocarpa, several of which demonstrated more et al., 2008; Deller et al., 2011). Botrytis cinerea Pers. is phytotoxic activity than 13 commercial herbicides (Ban- also capable of colonizing roots and leaves of host plants dara-Herath et al., 2009). including strawberry, lettuce and Primula without visible The use of virulent, asymptomatic and hypovirulent symptoms (Braun & Sutton, 1988; Barnes & Shaw, 2003; isolates of S. homoeocarpa in this study demonstrated Sowley et al., 2009). On tomato leaves, B. cinerea has three that OMV3a infection does not change the host coloni- phases of infection and lesion development: a small, ini- zation mechanisms of the fungus or the etiology of dol- tial lesion forms at 24 hpi followed by a 72 h symptom- lar spot disease. Hypovirulent and asymptomatic isolates less phase prior to expansion of the necrotic lesion across were both capable of producing appressoria and intracel- the leaf (Benito et al., 1998). Expansion of the necrotic le- lular infection hyphae. However, the hypovirulent isolates sion occurred more frequently at 4 ◦C than at 20 ◦C, coin- demonstrated a decreased ability to colonize host tissues ciding with a reduction in the expression of plant defence compared with virus-free, virulent isolates and asymptom- transcripts (Benito et al., 1998). Sclerotinia homoeocarpa atic isolates. It is possible that the slow, debilitated growth is capable of growing at temperatures between 4 and 20 of hypovirulent isolates prevented them from overcom- ◦C; however, disease doesn’t develop in the field until tem- ing the plant response to infection. The reduced ability of peratures reach approximately 22 ◦C. The effect of tem- hypovirulent isolates to colonize turfgrass leaves means perature on asymptomatic infection of grass blades by S. that they may not be able to effectively compete as a bi- homoeocarpa requires further investigation. Other factors ological control agent against virulent and asymptomatic that may contribute to the expansion of necrotic lesions isolates under field conditions. This is in contrast to the following symptomless infection by B. cinerea include the ability of isolate Sh12B to control disease severity under age of the leaf (Braun & Sutton, 1988) and the relative hu- field conditions by up to 58 or 80% on naturally or arti- midity in the environment (Eden et al., 1996). ficially infected turf, respectively (Zhou & Boland, 1998). 504 Orshinsky et al. in Canadian Journal of Plant Pathology, 34:4 (2012)/

The hypovirulent isolate used in this study contains both activity in creeping bentgrass leaves. Hypovirulent isolates OMV3a as well as a satellite virus (Deng & Boland, 2004). of S. sclerotiorum have been shown to have delayed or re- Previous studies have documented that the presence of duced levels of oxalic acid production compared with vi- the satellite virus does not affect the growth or virulence rus-free isolates (Zhou & Boland, 1999). In the same study, of isolates containing OMV3a (Deng & Boland, 2004), and oxalic acid production was positively correlated with my- it is unlikely that the satellite virus affected this study in celial dry weight, and after an extended incubation period, any discernible way. Consideration must also be given to most of the slow-growing, hypovirulent isolates were able the fact that the isolates used in the present study are not to produce oxalic acid in amounts equal to that of virus- isogenic, and a better measure of OMV3a effects could be free isolates (Zhou & Boland, 1999). The detection of oxa- made by comparing virulent, asymptomatic and hypovir- late oxidase activity in leaves inoculated with hypovirulent ulent isolates that are isogenic. Instead, the isolates used isolate Sh12B indicates that oxalic acid is likely produced in this study were chosen based on the stability of their by hypovirulent isolates. However, without more quanti- respective phenotype. tative results, a correlation between growth, lesion size The reaction of the creeping bentgrass leaves to S. and oxalic acid production is not possible. In addition, homoeocarpa infection and colonization appeared to in- DAB precipitation at similar locations suggests accumu- volve the formation of callose papillae. Papillae formed lation of the oxalate oxidase byproduct hydrogen perox- underneath both virulent and hypovirulent appressoria ide (Zhou et al., 1998). Hydrogen peroxide is a relatively on leaves inoculated at either the cut edge or at a non- stable reactive oxygen species and is capable of passing wounded site in the middle of a leaf. These plant cell wall through cellular membranes, which assists the molecule modifications were observed under bright field micros- in its role in signalling early defence responses in plants copy as protrusions below penetration pegs and appres- (Wojtaszek, 1997). H2O2 can act as a defence for plants soria. Lignification, callose deposition and papillae for- in several ways, including direct toxicity to invading mi- mation have been associated with induced resistance in croorganisms, oxidative cross-linking of glycoproteins and various host–pathogen interactions (Hammerschmidt, phenolic molecules, induction of systemic acquired re- 1999; DaRoche et al., 2004). Previous studies have also sistance and hypersensitive response (Wojtaszek, 1997; identified lignin deposition as a resistance mechanism of Zhou et al., 1998). Further investigation into the impor- creeping bentgrass in both host and nonhost resistance tance of oxalate oxidase and hydrogen peroxide accumu- responses to S. homoeocarpa and S. sclerotiorum, Collet- lation in creeping bentgrass defence against S. homoeo- otrichum graminicola (Ces.) G.W. Wilson and C. orbiculare carpa is therefore warranted. (Berk. & Mont.) Arx, respectively (DaRoche et al., 2004). The current study used detached leaf blades inocu- Another important creeping bentgrass defence re- lated with mycelia culture plugs to determine the etiol- sponse is the production of oxalate oxidase. Oxalate ox- ogy of virulent, asymptomatic and hypovirulent isolates of idase is an enzyme common to graminaceous monocots S. homoeocarpa. This method has been used in previous such as turfgrasses. The enzyme has been thoroughly studies (Zhou & Boland, 1997; Deng et al., 2002) to deter- characterized in the barley–powdery mildew pathosys- mine the relative virulence of S. homoeocarpa isolates. The tem and has been implicated as a key enzyme in defence methods employed in this study enabled a thorough char- responses as early as 6 hours post-inoculation (Zhou et acterization of the etiology of dollar spot disease when al., 1998). Oxalic acid is produced in large amounts by S. mycelial propagules of S. homoeocarpa are introduced to sclerotiorum, and is a pathogenicity factor for this fun- wounded and nonwounded turfgrass. The use of a con- gus (Chipps et al., 2005). S. homoeocarpa also produces trolled environment enabled a comparison of three iso- oxalic acid (Reddyvari-Channarayappa et al., 2009). How- late types without confounding factors such as environ- ever, in contrast to S. sclerotiorum, the primary hosts of S. ment and phyllosphere competition. However, conclusions homoeocarpa are graminaceous monocots that are capa- based on the results of this study should be confirmed ble of detoxifying oxalic acid via oxalate oxidase. In fact, with studies that more accurately represent field condi- cloning oxalate oxidase into dicot hosts such as sunflower, tions, where S. homoeocarpa is more likely to encounter soybean, canola and tomato renders them relatively re- a more variable environment, different host species and sistant to S. sclerotiorum (Hu, 2003; Lu, 2003; Walz et al., cultivars, and competition with other organisms. 2008). In this study, oxalate oxidase activity was observed Further experiments using intact plants and investigat- as early as 48 hpi on grass leaves inoculated with both ing protein expression and transcription data from both virulent and hypovirulent isolates. There were no observ- S. homoeocarpa and the creeping bentgrass host could able differences in the ability of hypovirulent and virulent clarify the type of defence responses important for the isolates of S. homoeocarpa to promote oxalate oxidase protection of creeping bentgrass from S. homoeocarpa. infection of creeping bentgrass by Sclerotinia homoeocarpa 505 Results from future studies on the suppression of dol- Daroche, A. B., Klepaski, J., & Hammerschmidt, R. (2004). Com- lar spot symptoms by hypovirulent isolates and plant de- mon features between non-host and host resistance of bent- fences will be important for the deployment of hypoviru- grass to Sclerotinia homoeocarpa. Phytopathology, 94, S:157 lent isolates of S. homoeocarpa as a viable biological dollar (Abstr.). Deller, S., Hammond-Kosack, K. E., & Rudd, J. J. (2011). The com- spot management strategy. plex interactions between host immunity and non-biotro- phic fungal pathogens of wheat leaves. J. Plant Physiol., 168, 63–71. Acknowledgments - This study was funded by the Natural Sci- Deng, F. & Boland, G. J. (2004). A satellite virus of Ophiostoma ences and Engineering Research Council of Canada (NSERC). The novoulmi mitovirus 3a in hypovirulent isolates of Sclerotinia authors would like to thank Dr Marc Habash and the Molecular homoeocarpa. Phytopathology, 94, 917–923. and Cellular Imaging Center in Columbus at The Ohio State Uni- Deng, F.,& Boland, G. J. (2006). Attenuation of virulence in Scler- versity for use of the fluorescence microscopes. We would also otinia homoeocarpa during storage is associated with latent like to thank Melody Melzer for technical advice. infection by Ophiostoma Mitovirus 3a. Eur. J. Plant Pathol., 114, 127–137. Deng, F., Melzer, M. S., & Boland, G. J. (2002). Vegetative compat- References ibility and transmission of hypovirulence-associated dsRNA in Sclerotinia homoeocarpa. Can. J. Plant Pathol., 24, 481–488. Deng, F., Xu., R., & Boland, G. J. (2003). Hypovirulence associated Agerer, R. (1991). Characterization of ectomycorrhizae. In: Meth- double-stranded rna from Sclerotinia homoeocarpa is con- ods in Microbiology, Vol. 23. (J. R. Agerer, D. J. Read, and A. specific withOphiostoma Novo-Ulmi mitovirus 3a-Ld. Phyto- K. Varma, editors), pp. 26–70. Academic Press Ltd. San Di- pathology, 93, 1407–1414. ego, Ca. Eden, M. A., Hill, R. A., Beresford, R., & Stewart, A. (1996). The Bandara-Herath, H. M. T., Herath, W. H. M. W., Carvalho, P., influence of inoculum concentration, relative humidity, and Khan, S. I., Tekwani, B. L., Duke, S. O., Tomaso-Peterson, M., temperature on the infection of greenhouse tomatoes by & Nanayakkara, N. P. D. (2009). Biologically active tetranord- Botrytis cinerea. Plant Pathol., 45, 795–806. iterpenoids from the fungus Sclerotinia homoeocarpa causal Ellram, A., Horgan, B., & Hulke, B. (2007). Mowing strategies and agent of dollar spot in turfgrass. J. Nat. Prod., 72, 2091–2097. dew removal to minimize dollar spot on creeping bentgrass. Barnes, S. E., & Shaw, M. W. (2003). Infection of commercial Crop Sci., 47, 2129–2137. hybrid primula seed by Botrytis cinerea and latent disease Endo, R. M. (1963). Influence of temperature on rate of growth spread through the plants. Phytopathology, 93, 573–578. of five fungus pathogens of turfgrass and on rate of disease Benito, E. P.,Have, A. T., Van ’T Klooster, J.W.,&Van Kan, J. A. L. spread. Phytopathology, 53, 857–877. (1998). Fungal and plant gene expression during synchro- Endo, R. M. (1966). Control of dollar spot of turfgrass by nitro- nized infection of tomato leaves by Botrytis cinerea. Eur. J. gen and its probable basis. Phytopathology, 56, 877 (Abstr.). Plant Pathol., 104, 207–220. Fenstermacher, J. M. (1980). Certain features of dollar spot dis- Boland, G. J., Leggett, F., & Kokko, E. (2000). Ultrastructural com- ease and its causal organism Sclerotinia homoeocarpa. In: Ad- parisons of hypovirulent and virulent isolates of Sclerotinia vances in Turf Grass Pathology (P. O. Larsen and B. G. Joyner, homoeocarpa. Can. J. Plant Pathol., 22, 181 (Abstr.). Editors), Pp. 49–53. Harcourt Brace Jovanovich, Duluth, Mn. Bonos, S., Buckley, R., & Clarke, B. B. (2007). An Integrated Ap- Garg, H., Li, H., Sivasithamparam, K., Kuo, J., & Barbetti, M. J. proach to Dollar Spot Disease in Turfgrasses. Rutgers Uni- (2010). The infection processes of Sclerotinia sclerotiorum in versity Cooperative Extension Fact Sheet FS1070. New Bruns- cotyledon tissue of a resistant and a susceptible genotype wick, NJ. of Brassica napus. Ann. Bot., 106, 897–908. Braun, P. G., & Sutton, J. C. (1988). Infection cycles and popula- Hammerschmidt, R. (1999). Induced disease resistance: how do tion dynamics of Botrytis cinerea in strawberry leaves. Can. induced plants stop pathogens? Physiol. Mol. Plant Pathol., J. Plant Pathol., 10, 133–141. 55, 77–84. Chakraborty, N., Chang, T., Casler, M. D., & Jung, G. (2006). Re- Hu, X. (2003). Overexpression of a gene encoding hydrogen per- sponse of bentgrass cultivars to Sclerotinia homoeocarpa iso- oxidegenerating oxalate oxidase evokes defense responses lates representing 10 vegetative compatibility groups. Crop in sunflower. Plant Physiol., 133, 170–181. Sci., 46, 1237–1244. Latin, R. (2008). Turfgrass Disease Profiles: Dollar Spot. Publica- Chipps, T. J., Gilmore, B., Myers, J. R., & Stotz, H. U. (2005). Re- tion Number Bp105-W. Purdue University Cooperative Ex- lationship between oxalate, oxalate oxidase activity, oxalate tension Service. West Lafayette, In. sensitivity, and white mold susceptibility in Phaseolus coccin- Lu, G. (2003). Engineering Sclerotinia sclerotiorum resistance in eus. Phytopathology, 95, 292–299. oilseed crops. Afr. J. Biotechnol., 2, 509–516. Couch, H. B., & Bloom, J. R. (1960). Influence of environment Malca, I.,&Endo, R.M. (1965). Identification of galactose in cul- on diseases of turfgrasses. I. Effect of nutrition, pH, and soil tures of Sclerotinia homoeocarpa as the factor toxic to bent- moisture on Sclerotinia dollar spot. Phytopathology, 257, grass. Phytopathology, 55, 775–780. 761–765. 506 Orshinsky et al. in Canadian Journal of Plant Pathology, 34:4 (2012)/

Melzer, M., Deng, F.,&Boland, G. J. (2005). Asymptomatic infec- Thistlewaite, P., Porter, I., & Evans, N. (1986). Photophysics of tion, and distribution of Ophiostoma mitovirus 3a (omv3a), in the aniline blue 432 fluorophore: a fluorescent probe show- populations of Sclerotinia homoeocarpa. Can. J. Plant Pathol., ing specificity towards (1,3)-ß-d-glucans. J. Phys. Chem., 90, 27, 610–615. 5058–5063. Orshinsky, A. M.,&Boland, G. J. (2010). The influence of Ophios- Walsh, B., Ikeda, S., & Boland, G. J. (1999). Biology and manage- toma mitovirus 3a (omv3a) on the respiration and growth of ment of dollar spot (Sclerotinia homoeocarpa), an important the dollar spot pathogen, Sclerotinia homoeocarpa (Bennett). disease of turfgrass. Hortscience, 34, 13–21. Can. J. Plant Pathol., 32, 431–439. Walz, A., Zingen-Sell, I., Loeffler, M., & Sauer, M. (2008). Expres- Orshinsky, A. M., & Boland, G. J. (2011). Ophiostoma mitovirus 3a, sion of an oxalate oxidase gene in tomato and severity of dis- ascorbic acid, glutathione, and photoperiod affect the devel- ease caused by Botrytis cinerea and Sclerotinia sclerotiorum. opment of stromata and apothecia by Sclerotinia homoeo- Plant Pathol., 57, 453–458. carpa. Can. J. Microbiol., 57, 398–407. Wojtaszek, P. (1997). Oxidative burst: an early plant response to Putman, A., & Kaminski, J. (2011). Mowing frequency and plant pathogen infection. Biochem. J., 322, 681–692. growth regulator effects on dollar spot severity and on du- Zhou, F., Zhang, Z., Gregersen, P. L., Mikkelsen, J. D., De Neer- ration of dollar spot control by fungicides. Plant Dis., 95, gaard, E., Collinge, D. B., & Thordal-Christensen, H. (1998). 1433–1442. Molecular characterization of the oxalate oxidase involved in Reddyvari-Channarayappa, V., Beaulieu, R. A., Graham, T. L., Me- the response of barley to the powdery mildew fungus. Plant dina, A. M.,& Boehm, M. J. (2009). Dollar spot fungus Scler- Physiol., 117, 33–41. otinia homoeocarpa produces oxalic acid. Int. Turfgrass Soc. Zhou, T., & Boland, G. J. (1997). Hypovirulence and double- Res. J., 11, 263–270. stranded RNA in Sclerotinia homoeocarpa. Phytopathology, Rudd, J. J., Keon, J., & Hammond-Kosack, K. E. (2008). The wheat 87, 147–153. mitogen-activated protein kinases tampk3 and tampk6 are Zhou, T., & Boland, G. J. (1998). Suppression of dollar spot by hy- differentially regulated at multiple levels during compatible povirulent isolates of Sclerotinia homoeocarpa. Phytopathol- disease interactions with Mycosphaerella Graminicola. Plant ogy, 88, 788–794. Physiol., 147, 802–815. Zhou, T., & Boland, G. J. (1999). Mycelial growth and production Sowley, E. N. K., Dewey, F. M., & Shaw, M. W. (2009). Persistent, of oxalic acid by virulent and hypovirulent isolates of Sclero- symptomless, systemic, and seed-borne infection of lettuce tinia sclerotiorum. Can. J. Plant Pathol., 21, 93–99. by Botrytis cinerea. Eur. J. Plant Pathol., 126, 61–71.