scholarly journals Detection of Pyricularia grisea Causing Gray Leaf Spot of Perennial Ryegrass Turf by a Rapid Immuno-Recognition Assay

Plant Disease ◽  
2003 ◽  
Vol 87 (7) ◽  
pp. 772-778 ◽  
Author(s):  
W. Uddin ◽  
G. Viji ◽  
G. L. Schumann ◽  
S. H. Boyd

An immuno-recognition assay using a monoclonal antibody was developed to detect Pyricularia grisea, the causal agent of gray leaf spot of perennial ryegrass (Lolium perenne). In vitro assays with isolates of P. grisea from perennial ryegrass, tall fescue (Festuca arundinacea), St. Augustinegrass (Stenotaphrum secundatum), crabgrass (Digitaria sanguinalis), finger millet (Eleusine coracana), wheat (Triticum aestivum), triticale (× Triticosecale rimpaui), and rice (Oryza sativa) showed positive reactions; however, the strength of the reactions differed among isolates. Reactions were more intense with isolates from perennial ryegrass, wheat, and triticale. All P. grisea isolates from perennial ryegrass collected from various regions of the United States showed positive reactions. P. grisea was detected at antigen dilution rates of 0.5×, 0.25×, 0.13×, 0.06×, and 0.03×. Dot-blot assays with Bipolaris sorokiniana, Colletotrichum graminicola, Curvularia lunata, Microdochium nivale, Pythium aphanidermatum, Rhizoctonia solani, or Sclerotinia homoeocarpa isolated from turfgrasses were negative. In vivo assays of symptomatic leaves of perennial ryegrass plants inoculated with P. grisea also showed positive reactions, and those inoculated with B. sorokiniana, P. aphanidermatum, R. solani, or S. homoeocarpa were negative. Intensity of reaction between the monoclonal antibody and P. grisea was proportional to disease severity in perennial ryegrass inoculated with P. grisea; however, there was no reaction in dot blots of leaf tissue collected during the latent period. P. grisea was detected in perennial ryegrass samples from golf course fairways affected by gray leaf spot in Connecticut, Massachusetts, Maine, New Jersey, Pennsylvania, and Rhode Island using this procedure. The monoclonal antibody recognition system is highly sensitive to P. grisea and can be used effectively for the rapid diagnosis of gray leaf spot of perennial ryegrass turf.

Plant Disease ◽  
1999 ◽  
Vol 83 (11) ◽  
pp. 1073-1073 ◽  
Author(s):  
G. L. Schumann ◽  
N. Jackson

Pyricularia grisea (Cooke) Sacc. causing significant damage in perennial ryegrass (Lolium perenne L.) was first observed on a golf course in eastern Maryland in 1985 by P. H. Dernoeden, but there is no published account. The first published report of the problem was from southeastern Pennsylvania in 1991 (1). There were scattered reports of gray leaf spot in several other states in 1991. A more severe and widespread epidemic occurred under similar environmental conditions throughout the mid-Atlantic region in 1995 and has reoccurred to some extent annually since then in an expanding area throughout the United States. This report documents the expansion of the northern range of the epidemic into New England. Samples of perennial rye-grass with gray leaf spot from golf courses in three towns in Connecticut (Norwich, Stratford, and Willamantic) and one in Rhode Island (West Warwick) were submitted to the diagnostic labs at the universities of Massachusetts and Rhode Island beginning on 22 September 1998. Severe gray leaf spot was observed in perennial ryegrass fairways and roughs, especially where new seedlings were present, causing turf loss exceeding 50% in some areas. Diagnosis of this now familiar disease is based on the presence of abundant sporulation of the pathogen on infected tissue, distinctive leaf symptoms, and rapid foliar blighting of only perennial ryegrass in plantings of mixed turfgrass species. Golf course superintendents in New England with perennial ryegrass may have to extend their late-season fungicide applications to accommodate this new and destructive late-summer and fall disease. Reference: (1) P. J. Landschoot and B. F. Hoyland. Plant Dis. 76:1280, 1992.


Plant Disease ◽  
2001 ◽  
Vol 85 (8) ◽  
pp. 817-826 ◽  
Author(s):  
G. Viji ◽  
B. Wu ◽  
S. Kang ◽  
W. Uddin ◽  
D. R. Huff

Gray leaf spot is a serious disease of perennial ryegrass (Lolium perenne) turf in the United States. Isolates of Pyricularia grisea causing the disease in perennial ryegrass were characterized using molecular markers and pathogenicity assays on various gramineous hosts. Genetic relationships among perennial ryegrass isolates were determined using different types of trans-posons as probes. Phylogenetic analysis using Pot2 and MGR586 probes, analyzed with AMOVA (analysis of molecular variance), showed that these isolates from perennial ryegrass consist of three closely related lineages. All the isolates belonged to a single mating type, MAT1-2. Among 20 isolates from 16 host species other than perennial ryegrass, only the isolates from wheat (Triticum aestivum) and triticale (× Triticosecale), showed notable similarity to the perennial ryegrass isolates based on their Pot2 fingerprints. The copy number and fingerprints of Pot2 and MGR586 in isolates of P. grisea from perennial ryegrass indicate that they are genetically distinct from the isolates derived from rice (Oryza sativa) in the United States. The perennial ryegrass isolates also had the same sequence in the internal transcribed spacer (ITS) region of the genes encoding ribosomal RNA as that of the wheat and triticale isolates, and exhibited rice isolate sequence polymorphisms. In pathogenicity assays, all the isolates of P. grisea from Legacy II perennial ryegrass caused characteristic blast symptoms on Marilee soft white winter wheat, Bennett hard red winter wheat, Era soft white spring wheat, and Presto triticale, and they were highly virulent on these hosts. An isolate from wheat and one from triticale (from Brazil) were also highly virulent on perennial ryegrass and Rebel III tall fescue (Festuca arundinacea). None of the isolates from perennial ryegrass caused the disease on Lagrue rice, and vice versa. Understanding the population structure of P. grisea isolates infecting perennial ryegrass and their relatedness to isolates from other gramineous hosts may aid in identifying alternate hosts for this pathogen.


Plant Disease ◽  
2005 ◽  
Vol 89 (4) ◽  
pp. 433-433 ◽  
Author(s):  
F. P. Wong ◽  
W. Gelernter ◽  
L. Stowell

Kikuyugrass (Pennisetum clandestinum) is a warm-season turfgrass that has been adopted for use in fairways and roughs in a number of subtropical areas including southern California, Mexico, Australia, and South Africa. During August 2003, a foliar disease of Kikuyugrass was reported from a number of golf courses in southern California. Examination of diseased plants showed the presence of dark, olive green-to-brown lesions on the foliage. Incubation of these plants in a moist chamber for 12 h led to the production of numerous pyriform conidia from these lesions that were characteristic of Pyricularia grisea. Single-spore isolates of the fungus were obtained from infected kikuyugrass samples by transferring conidia to acidified 1.5% water agar and then transferring single, germinated conidia to one-quarter-strength potato dextrose agar. Colony morphology and conidia production were consistent with that described for P. grisea (1). Koch's postulates were performed separately for two single-spore isolates (OSGC-1 and CCCC-1) obtained from infected kikuyugrass. For each isolate, 2-week-old, glasshouse-grown seedlings of kikuyugrass (cv. ‘AZ-1’) and perennial ryegrass (Lolium perenne) grown in 75-mm pots in soilless media were inoculated with conidia from either OSGC-1 or CCCC-1. For each test, six pots of both kikuyugrass and ryegrass were inoculated, and the tests were conducted three times for each isolate. Conidia were obtained from isolates grown on clarified V8 agar in 100-mm petri plates for 14 days at 25°C. Suspensions were made by adding 10 ml of sterile distilled H2O (sdH2O) to the plates, scraping the surface of the media to dislodge the conidia, filtering the suspension through cheesecloth, and then adjusting the final concentration to 1 × 106 conidia/ml with sdH2O. Seedlings were inoculated with the conidial suspensions with an aerosol applicator, placed in plastic boxes lined with wet paper towels, and sealed to provide adequate moisture for infection. Boxes were incubated at 28°C for 48 h after which time the covers were removed and the plants maintained in ambient glasshouse conditions at approximately 28°C. In all three replicated experiments, kikuyugrass seedlings inoculated with OSGC-1 or CCCC-1 developed symptoms of disease approximately 5 days after inoculation, while inoculated perennial ryegrass did not, even 14 days after inoculation. Symptomatic kikuyugrass leaves were taken randomly from plants from each of the three replicated tests, surface disinfested in 0.3% sodium hypochlorite for 30 s, rinsed with sdH2O, blotted dry, and placed onto acidified water agar in petri plates. Twenty-four hours later, abundant sporulation was observed from symptomatic tissue with conidiophores bearing conidia typical of P. grisea. To our knowledge, this is the first report of gray leaf spot being caused by P. grisea on Pennisetum clandestinum in North America. Reference: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, UK, 1971.


2002 ◽  
Vol 92 (3) ◽  
pp. 245-254 ◽  
Author(s):  
Mark L. Farman

Gray leaf spot of perennial ryegrass (prg) (Lolium perenne), caused by the fungus Pyricularia grisea (teleomorph = Magnaporthe grisea), has rapidly become the most destructive of all turf grass diseases in the United States. Fungal isolates from infected prg were analyzed with several molecular markers to investigate their relationship to P. grisea strains found on other hosts. All of the molecular markers used in this study revealed that isolates from prg are very distantly related to those found on crabgrass. Fingerprinting with MGR586 (Pot3) revealed zero to three copies of this transposon in the prg pathogens, distinguishing them from isolates pathogenic to rice, which typically have more than 50 copies of this element. RETRO5, a newly identified retroelement in P. grisea, was present at a copy number of >50 in isolates from rice and Setaria spp. but only six to eight copies were found in the isolates from prg. The MAGGY retrotransposon was unevenly distributed in the prg pathogens, with some isolates lacking this element, some possessing six to eight copies, and others having 10 to 30 copies. These results indicated that the P. grisea isolates causing gray leaf spot are distinct from those found on crabgrass, rice, or Setaria spp. This conclusion was supported by an unweighted pair-group method with arithmetic average cluster analysis of single-copy restriction fragment length polymorphism haplo-types. Fingerprints obtained with probes from the Pot2 and MGR583 transposons revealed that the prg pathogens are very closely related to isolates from tall fescue, and that they share similarity with isolates from wheat. However, the wheat pathogens had fewer copies of these elements than those found on prg. Therefore, I conclude that P. grisea isolates commonly found on other host plant species did not cause gray leaf spot epidemics on prg. Instead, the disease appears to be caused by a P. grisea population that is specific to prg and tall fescue.


Plant Disease ◽  
2007 ◽  
Vol 91 (5) ◽  
pp. 517-524 ◽  
Author(s):  
Y. Tosa ◽  
W. Uddin ◽  
G. Viji ◽  
S. Kang ◽  
S. Mayama

Gray leaf spot caused by Magnaporthe oryzae is a serious disease of perennial ryegrass (Lolium perenne) turf in golf course fairways in the United States and Japan. Genetic relationships among M. oryzae isolates from perennial ryegrass (prg) isolates within and between the two countries were examined using the repetitive DNA elements MGR586, Pot2, and MAGGY as DNA fingerprinting probes. In all, 82 isolates of M. oryzae, including 57 prg isolates from the United States collected from 1995 to 2001, 1 annual ryegrass (Lolium multiflorum) isolate from the United States collected in 1972, and 24 prg isolates from Japan collected from 1996 to 1999 were analyzed in this study. Hybridization with the MGR586 probe resulted in approximately 30 DNA fragments in 75 isolates (designated major MGR586 group) and less than 15 fragments in the remaining 7 isolates (designated minor MGR586 group). Both groups were represented among the 24 isolates from Japan. All isolates from the United States, with the exception of one isolate from Maryland, belonged to the major MGR586 group. Some isolates from Japan exhibited MGR586 fingerprints that were identical to several isolates collected in Pennsylvania. Similarly, fingerprinting analysis with the Pot2 probe also indicated the presence of two distinct groups: isolates in the major MGR586 group showed fingerprinting profiles comprising 20 to 25 bands, whereas the isolates in the minor MGR586 group had less than 10 fragments. When MAGGY was used as a probe, two distinct fingerprint types, one exhibiting more than 30 hybridizing bands (type I) and the other with only 2 to 4 bands (type II), were identified. Although isolates of both types were present in the major MGR586 group, only the type II isolates were identified in the minor MGR586 group. The parsimony tree obtained from combined MGR586 and Pot2 data showed that 71 of the 82 isolates belonged to a single lineage, 5 isolates formed four different lineages, and the remaining 6 (from Japan) formed a separate lineage. This study indicates that the predominant groups of M. oryzae associated with the recent outbreaks of gray leaf spot in Japan and the United States belong to the same genetic lineage.


Plant Disease ◽  
2006 ◽  
Vol 90 (5) ◽  
pp. 683-683 ◽  
Author(s):  
F. P. Wong ◽  
K. A. de la Cerda

In August of 2005, a golf course in Las Vegas, NV reported turf loss from an unknown disease on perennial ryegrass fairways. Samples from this course were examined, and diseased plants were found covered with lesions and sporulation typical of gray leaf spot as caused by Pyricularia grisea (Cooke) Sacc. With petroleum jelly, sporulating leaves were attached to the inside top surface of 100-mm petri dishes filled with 15 ml of 1.5% water agar. Conidia were allowed to drop onto the agar surface and 24 h later, individual germinating pyriform conidia were transferred to petri dishes containing one-quarter-strength potato dextrose agar (¼-PDA) with the aid of a fine needle and stereomicroscope. Isolates of the fungus were maintained at 28°C with constant fluorescent light. Isolates were examined 7 to 10 days later, and morphology and conidia production were consistent with that described previously for P. grisea (1). Koch's postulates were performed using a single isolate (SSGC-1.1) grown for 14 days on ¼-PDA. The petri dish was flooded with 15 ml of sterile distilled water plus 0.05% Tween 20 and conidia dislodged into the solution with a rubber policeman to obtain a solution of approximately 5 × 103 conidia per ml. Using a modified thin-layer chromatography plate sprayer, the solution was misted onto six pots of 6-week-old perennial ryegrass (a mixture of approximately 33% each of varieties ‘Kokomo’, ‘Cabo’ and ‘Secretaria’), seeded at a density of 2 kg per 93 m2 grown in 4- × 4-cm plastic pots filled with University of California soil mix. As a control treatment, six pots of perennial ryegrass (grown as previously described) were treated with water plus 0.05% Tween 20 only. Pots of plants were placed into closed, translucent, plastic containers lined with wet paper towels to provide a moist environment and held at 30°C for 48 h. Pots of plants were transferred to an incubator set at 30°C and 80% relative humidity with 12 h of alternating light and dark cycles. Four days after inoculation, plants misted with conidia developed symptoms typical of gray leaf spot. Plants were again placed into closed plastic containers lined with wet paper towels for 24 h, at which time, lesions on symptomatic plants developed abundant conidia characteristic of P. grisea. Water-only treated plants did not show any symptoms or signs of disease. P. grisea was reisolated from sporulating leaves as described above. The disease has been spreading in the midwestern and northeastern United States since first reported in 1991 on perennial ryegrass in Pennsylvania. It has only recently been found on turfgrass in California (2), and to our knowledge, this is the first report of this pathogen on perennial ryegrass in Nevada. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) W. Uddin et al. Plant Dis. 86:75, 2002.


Plant Disease ◽  
2002 ◽  
Vol 86 (3) ◽  
pp. 235-240 ◽  
Author(s):  
P. Vincelli ◽  
E. Dixon

In August 2000, azoxystrobin was ineffective in controlling gray leaf spot of perennial ryegrass at a golf course in Lexington, KY and at two golf courses in Illinois. Isolates suspected of being fungicide-resistant (“suspect isolates”) were compared to “baseline” isolates obtained from sites with no known use of quinol-oxidizing inhibitor (QoI) fungicides. Conidial germination of Pyricularia grisea was tested in vitro with 100 μg of salicylhydroxamic acid per ml. For baseline isolates, 50% effective concentration (EC50) values for azoxystrobin and trifloxystrobin were 0.015 to 0.064 and 0.013 to 0.078 μg/ml, respectively; EC50 values for suspect isolates were 2.39 to 44.8 and 0.31 to 111, respectively. All suspect isolates exhibited significantly (P = 0.05) lower sensitivity to QoI fungicides than all baseline isolates. The mean EC50 values for suspect isolates for azoxystrobin and trifloxystrobin were 690 and 827 times higher, respectively, than the means for baseline isolates. In the laboratory, azoxystrobin and trifloxystrobin provided essentially complete control of disease induced by nine baseline isolates in vivo. Azoxystrobin and trifloxystrobin provided poor to no control of disease induced by six of eight suspect isolates; control of disease induced by the remaining two isolates was partial for azoxystrobin and complete for trifloxystrobin. We conclude that one or more biotypes of perennial ryegrass-infecting strains of P. grisea with resistance to QoI fungicides have emerged. This is the first report of resistance to QoI fungicides in P. grisea. Furthermore, this is one of two QoIresistant fungal pathogens collected in the United States during the 2000 growing season, the first instances reported for North America.


Plant Disease ◽  
2004 ◽  
Vol 88 (10) ◽  
pp. 1146-1152 ◽  
Author(s):  
W. Uddin ◽  
M. D. Soika ◽  
A. S. McNitt ◽  
M. Fidanza

Ethofumesate is a widely used herbicide for control of annual bluegrass (Poa annua) in perennial ryegrass (Lolium perenne) fairways on golf courses in the United States. Effect of timing of ethofumesate application on development of gray leaf spot was evaluated on perennial ryegrass turf treated with six classes of fungicide. Two applications of ethofumesate (2.28 kg a.i./ha) were made to perennial ryegrass turf maintained at a 2-cm height, at 4-week intervals, each fall (October and November 1999 and 2000) or spring (April and May 2000 and 2001). In addition, turf was treated with the fungicides, azoxystrobin, chlorothalonil, flutolanil, iprodione, propiconazole, or thiophanate-methyl at the label rates at 14-day intervals. There were significant effects (P ≤ 0.05) of ethofumesate application timing and fungicide regime on gray leaf spot development. There also were significant interactions between the ethofumesate application timing and fungicide. Severity of gray leaf spot was significantly greater in turf plots treated with ethofumesate in spring compared to turf treated in fall or nontreated control plots treated with fungicides, flutolanil, iprodione, and propiconazole that were relatively less effective in control of gray leaf spot. There was no significant difference in disease severity in turf treated with ethofumesate in fall or to turf not treated with herbicide regardless of the fungicide used. Results of this study indicate that spring application of ethofumesate contributes to development of gray leaf spot epidemics, and the application timing interacts with the classes of fungicides. This study suggests that ethofumesate should be applied only in fall for control of P. annua, particularly in golf courses with a chronic gray leaf spot problem, as part of an integrated management of gray leaf spot in perennial ryegrass fairways.


2003 ◽  
Vol 93 (7) ◽  
pp. 891-900 ◽  
Author(s):  
Yun-Sik Kim ◽  
Edward W. Dixon ◽  
Paul Vincelli ◽  
Mark L. Farman

Gray leaf spot caused by Pyricularia grisea is a highly destructive disease of perennial ryegrass turf. Control of gray leaf spot is dependent on the use of preventative fungicide treatments. Strobilurin-based (QoI) fungicides, which inhibit the cytochrome bc1 respiratory complex, have proven to be very effective against gray leaf spot. However, in August 2000, disease was diagnosed in QoI-treated perennial ryegrass turf on golf courses in Lexington, KY, Champaign, IL, and Bloomington, IL. To determine if resistance was due to a mutation in the fungicide target, the cytochrome b gene (CYTB) was amplified from baseline and resistant isolates. Nucleotide sequence analysis revealed an intronless coding region of 1,179 bp. Isolates that were resistant to QoI fungicides possessed one of two different mutant alleles, each of which carried a single point mutation. The first mutant allele had a guanine-to-cytosine transition at nucleotide position +428, resulting in a replacement of glycine 143 by alanine (G143A). Mutant allele two exhibited a cytosine-to-adenine transversion at position +387, causing a phenylalanine-to-leucine change (F129L). Cleavable amplified polymorphic sequence analysis revealed that neither mutation was present in a collection of baseline isolates collected before QoI fungicide use and indicated that suspected QoI- resistant isolates found in 2001 in Indiana and Maryland possessed the F129L mutation. The Pyricularia grisea isolates possessing the G143A substitution were significantly more resistant to azoxystrobin and trifloxystrobin, in vitro, than those having F129L. DNA fingerprinting of resistant isolates revealed that the mutations occurred in just five genetic backgrounds, suggesting that field resistance to the QoI fungicides in Pyricularia grisea is due to a small number of ancestral mutations.


Plant Disease ◽  
2002 ◽  
Vol 86 (1) ◽  
pp. 75-75 ◽  
Author(s):  
W. Uddin ◽  
G. Viji ◽  
L. Stowell

Gray leaf spot of perennial ryegrass (Lolium perenne L.) turf was first reported in the United States in 1991. The disease epidemic was primarily confined to golf course fairways in southeastern Pennsylvania (1). Subsequently, moderate to severe outbreaks of gray leaf spot occurred in perennial ryegrass fairways and roughs in numerous locations throughout the eastern and midwestern United States. In August 2001, a serious decline of perennial ryegrass turf was observed in a bermudagrass (Cynodon dactylon (L.) Pers) baseball field in Dodger Stadium in Los Angeles, CA, that had been overseeded with perennial ryegrass. The bermudagrass turf was not affected. The perennial ryegrass turf developed necrotic lesions that resulted in blighting of leaf blades. In laboratory assays, Pyricularia grisea (Cooke) Sacc., was consistently isolated from symptomatic ryegrass blades from turf samples collected from the site. Of the 12 P. grisea isolates collected from the assayed leaf blades, five isolates were selected for a pathogenicity assay. Twenty-five ‘Legacy II’ perennial ryegrass plants were grown from seeds in 4 × 4 in.-plastic pots, (10 × 10 cm) which were filled to 1 cm below the rim with granular calcine clay medium (Turface MVP, Allied Industrial Material Corp., Buffalo Grove, IL). Three weeks after seeding, plants were fertilized with a water-soluble 20-20-20 N-P-K fertilizer (1.3 g/liter of water) once per week. Treatments (isolates of P. grisea and a control) were arranged as a randomized complete block design with five replications. Five-week-old plants were sprayed with an aqueous suspension of P. grisea conidia (≈5 × 104 conidia per ml of sterilized distilled water with 0.1% Tween 20) using an atomizer until the leaves were completely wet. Plants sprayed with sterilized distilled water served as the control. After inoculation, individual pots were covered with clear polyethylene bags and placed in a controlled environment chamber maintained at 28°C and continuous fluorescent light (88 μE m-2 s-1). Four days after inoculation, necrotic lesions (<2 mm diameter) developed on ryegrass blades inoculated with each isolate of P. grisea. Lesions did not develop on leaves of control plants. Seven days after inoculation, the polyethylene bags were removed, and 50 symptomatic blades from each pot were collected, and disease incidence (percent infected leaves) and severity (index 0 to 10; 0 = none, 10 = >90% of the leaf blade necrotic ) were assessed. P. grisea was isolated from symptomatic leaves of plants inoculated with the fungus. Disease incidence and severity on inoculated plants were 92 to 96% and 8.8 to 10, respectively. There were no significant differences in disease incidence and severity (P = 0.05) among the isolates of P. grisea included in the test. To our knowledge, this is the first report of gray leaf spot of perennial ryegrass turf in California. Reference: (1) P. J. Landschoot and B. F. Hoyland. Plant Dis. 76:1280, 1992.


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