Waitea Circinata Research Articles

DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA

Rhizoctonia blight is a common and serious disease of many turfgrass species. The most widespread causal agent, Thanatephorus cucumeris (anamorph: R. solani), consists of several genetically different subpopulations. In addition, Waitea circinata varieties zeae, oryzae and circinata (anamorph: Rhizoctonia spp.) also can cause the disease. Accurate identification of the causal pathogen is important for effective management of the disease. It is challenging to distinguish the specific causal pathogen based on disease symptoms or macroscopic and microscopic morphology. Traditional methods such as anastomosis reactions with tester isolates are time consuming and sometimes difficult to interpret. In the present study universally primed PCR (UP-PCR) fingerprinting was used to assess genetic diversity of Rhizoctonia spp. infecting turfgrasses. Eighty-four Rhizoctonia isolates were sampled from diseased turfgrass leaves from seven distinct geographic areas in Virginia and Maryland. Rhizoctonia isolates were characterized by ribosomal DNA internal transcribed spacer (rDNA-ITS) region and UP-PCR. The isolates formed seven clusters based on ITS sequences analysis and unweighted pair group method with arithmetic mean (UPGMA) clustering of UP-PCR markers, which corresponded well with anastomosis groups (AGs) of the isolates. Isolates of R. solani AG 1-IB (n = 18), AG 2-2IIIB (n = 30) and AG 5 (n = 1) clustered separately. Waitea circinata var. zeae (n = 9) and var. circinata (n = 4) grouped separately. A cluster of six isolates of Waitea (UWC) did not fall into any known Waitea variety. The binucleate Rhizoctonia-like fungi (BNR) (n = 16) clustered into two groups. Rhizoctonia solani AG 2-2IIIB was the most dominant pathogen in this study, followed by AG 1-IB. There was no relationship between the geographic origin of the isolates and clustering of isolates based on the genetic associations. To our knowledge this is the first time UP-PCR was used to characterize Rhizoctonia, Waitea and Ceratobasidium isolates to their infra-species level.

Susceptibility of tall fescue to Rhizoctonia zeae infection as affected by endophyte symbiosis

AbstractEndophytic fungi belonging to the genus Neotyphodium often form symbiotic associations with grasses. The host plants usually benefit from the association with an endophyte. Presence of the symbiont may increase host resistance to infection by some pathogens. However, the exact mechanism of the lower susceptibility of endophyte‐infected plants to diseases is still unclear. Growth chamber trials were conducted to determine whether (a) tall fescue plants infected with the endophyte Neotyphodium coenophialum (E+) are more resistant to sheath and leaf spot disease caused by Rhizoctonia zeae than endophyte‐free (E−) plants, and (b) R. zeae growth inhibition is associated with endophyte presence. Tall fescue genotypes, each symbiotic with a genetically different native endophyte strain, were inoculated with isolates of R. zeae. The tillers infection by R. zeae, density of endophyte hyphae and content of total phenolic compounds in tillers were studied. Antifungal activity of the N. coenophialum towards R. zeae, Rhizoctonia solani, Bipolaris sorokiniana and Curvularia lunata was also investigated in dual‐culture assays. For Tf3, Tf4, TfA2 and TfA9 tall fescue genotypes, the E+ plants had reduced R. zeae infection. In the Tf9 and Tf8085 genotypes, R. zeae infection was similar for both E+ and E− plants. The strongest effect was observed for the Tf4 endophyte. A strongly positive correlation (r = 0.94) occurred between endophyte hyphal density and disease index across all tall fescue genotypes. Dual‐culture assays showed no inhibitory interaction between the seven endophyte strains and the R. zeae isolates; however, some endophytes inhibited R. solani, B. sorokiniana and C. lunata. Endophyte presence increased the production of phenolic compounds by the host grasses. The level of phenolics also differed significantly depending on the time of analysis after inoculation of plants by R. zeae. The results indicate that N. coenophialum can suppress disease severity caused by R. zeae infection. The mechanism of higher resistance of E+ plants is likely not based on direct inhibition such as antibiosis or competition. Thus, the induction of specific mechanisms in the host plant, for example, production of phenolic compounds, seems to be the main way of providing resistance to the grass by the endophyte.

EXPERIMENTAL STUDIES ON ACTIVE METABOLITES OF CALOTROPIS GIGANTEA FOR EVALUATION OF POSSIBLE ANTIFUNGAL PROPERTIES

The ac tive metabolites like phytochemicals from the medicinal plants were under exploration for the development of novel and biodeg radable effective drugs as an alternative to the ineffective contemporary medicine. In the similar lines, the Calotropis gigantea l eaves, buds and flowers were extracted for the active metabolites using ethanol, methanol, chloroform and aqueous solvents and then these solvent extracts of 100µl (25µg/ml) were tested separately for the in vitro antifungal activity against the test patho genic fungi viz. Aspergillus niger , Rhizoctonia oryzae - sativae , Fusarium solani and Trichoderma viride . In the results ethanol buds extract has shown the highest growth reducing activity (67.82%) on Fusarium solani where the positive control, clotrimazole (10µg/ml) recorded 58% growth reduction . Similarly aqueous buds extract has shown ~66% growth reduction on Rhizoctonia oryzae - sativae where Clotrimazole, the positive control has shown the maximum growth reduction capability (~73%); however all the aqueous extracts exhibited the growth reduction capabilities on all the test fungi . On the contrary methanol leaves extract increased the growth of Aspergillus niger by 85%; and similarly chloroform leaves and buds extracts exhibited the growth supporting activit y on Rhizoctonia oryzae - sativae (32%) and Trichoderma viride (34%) respectively. This study brings out the mixed results of Calotropis gigantean solvent extracts as antifungal drugs and hence makes a caution on the knowledgeable and judicious use of these extracts as a medicine as evident among the folklores. Further it is also advised to make advanced studies in the similar line for the effe ctive utilization of this Calotropis species in either directions to serve the mankind.

Identification of Waitea circinata as a Pathogen of the Moss Bryum argenteum var. argenteum on a Golf Course Fairway.

Waitea circinata Warcup and Talbot (also referred to as W. circinata var. circinata) is an important fungal pathogen of amenity turfgrasses and is especially problematic on Poa annua in putting greens in the late spring or early summer. The pathogen was first identified in 2005 from Japan and has since been seen widely throughout the United States (1,2). On occasion, the pathogen has been observed attacking creeping bentgrass (Agrostis stolonifera) but is typically less virulent. Disease symptoms include prominent yellow rings appearing throughout established turf and moderate leaf necrosis. In the summer of 2012, moss from a section of fairway on a golf course in Edwards, CO was observed to be rapidly killed by a fungal pathogen producing copious amounts of aerial mycelium and appearing similar to Waitea microscopically. Aerial mycelium was transferred to acidified potato dextrose agar (PDA) (1 ml lactic/L). After 1 day at 25°C, mycelia were transferred to PDA. After 2 weeks, plates were covered with white aerial mycelium and separate, spherical, 0.5-mm diameter, salmon-colored sclerotia, which turned dark brown within a few days and were produced submerged throughout the media. Spores were never produced and right-angled branching of mycelia, characteristic of Waitea, was observed in mature cultures. Mycelial plugs were incubated in nutrient broth and DNA was extracted using a MoBio Power Plant DNA extraction kit. Amplification of ribosomal ITS sequences with ITS4 and ITS5 resulted in a 100% identity match with GenBank sequence HM807352, W. circinata var. circinta (3). To demonstrate pathogenicity on Bryum argenteum, unaffected moss from the submitted sample (identified as B. argenteum var. argenteum via 100% sequence identity with the published GenBank sequence GU907062) was removed from the originally submitted sample and placed in separate growth chambers at 95% humidity and 21, 26, and 31°C. An additional experiment employed local B. agenteum plants collected from the URI Kingston, RI campus. Agar plugs from the isolated W. circinata were placed on top of the moss and within 2 days the fungus had caused complete mortality at all three temperatures. The experiment was also undertaken using the same environmental conditions with 5-week-old annual bluegrass (P. annua) and creeping bentgrass cv. A4 grown from seed. Plants were inoculated with infected rye grains at 31, 26, and 21°C. After 1 week, the P. annua plants showed significant mortality at 26 and 31°C with little infection at 21°C and the A. stolonifera plants showed moderate mortality at 26°C and little infection at the other two temperatures. All experiments utilized an additional uninoculated control treatment that showed no moss/turf necrosis or mortality. Experiments were all repeated once and used three replicates per experiment. While moss is not intentionally cultivated on golf courses, it does occur with regularity and often presents itself as a difficult pest to manage. This particular isolate of W. circinata has identical ribosomal and physiological characteristics of the reported P. annua pathogen but can attack one moss species and may be a possible candidate for selective biological control of moss in golf course settings. It is unclear how widespread moss pathogenicity is within W. circinata.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Agrostis stolonifera and Poa pratensis in China.

Brown ring patch, caused by Waitea circinata var. circinata, is a recently described disease of turf grass (1,2). The disease was first reported in Japan in 2005 (2) and then in the United States (1). In late May to early September 2011, large yellow rings (20 to 30 cm in diameter) were observed on creeping bentgrass (Agrostis stolonifera) and Kentucky bluegrass (Poa pratensis) growing at the Qinghe Bay golf course, Beijing, China. Leaf blades turned from yellow to brown as the disease developed, and eventually died. The disease incidence was estimated at 20 to 30%. The rings became continuous on creeping bentgrass and Kentucky bluegrass in several putting greens. The same symptom was observed on the lawn of China Agricultural University. Symptomatic leaves were collected and incubated in high humidity at 25°C until mycelia developed. The leaves were then disinfested in 1% NaClO for 1 min, rinsed with sterile water three times, and placed on potato dextrose agar (PDA). Four isolates were obtained, including one isolate from the lawn of China Agricultural University (cau-1), and three from Qinghe Bay golf course (qhw-1, qhw-2, and qhw-3). The colonies that formed on PDA changed from white to orange over time, and minute orange to brown sclerotia (approx. 2 to 3 mm in diameter) formed after 2 weeks at 25°C. These characteristics were similar to W. circinata var. circinata (1,2). DNA was extracted from each isolate using a CTAB extraction method (3) and the internal transcribed spacer (ITS) regions were amplified with the ITS1/ITS4 universal primers. The ITS sequences of the isolated fungi (Accession Nos. JQ964235 and JQ964236) had 99 to 100% homology with the sequences of W. circinata in GenBank (Accession Nos. EU591763 and HQ207169). Pathogen inocula were prepared by inoculating autoclaved oat grains with strains qhw-1 and cau-1 respectively, followed by 4 days of incubation at 25°C. Each inoculum was placed in five spots in a uniform arrangement (5 g grain inoculum per spot) on soil in a 40 × 60 cm tray, followed by sowing bluegrass seed. In another experiment, 4-week-old bentgrass was transplanted into soil infested with 5 g grain inoculum in the middle of a 20-cm diameter pot (non-colonized grain was used as a control). There were five replicates for each isolate. Plants were then incubated in a growth chamber at 26°C and high relative humidity (>90%). After 5 to 6 days, the grass in the inoculated pots and trays began to turn yellow, and then became chlorotic and necrotic as the disease developed. Orange sclerotia were observed on the bluegrass leaves by the eighth day, and all the bentgrass turned chlorotic by the tenth day. After 2 weeks, brown ring patches formed in the trays with inoculated bluegrass. Waitea circinata var. circinata was reisolated from all inoculated plants and confirmed by morphological observation and the ITS sequences analysis as described above, while no symptoms were observed on the control plants and no isolate was obtained from them. To our knowledge, this is the first report of W. circinata var. circinata infecting turf grass in China.

Pathogenicity and symptom expressions of Rhizoctonia solani, R. oryzae and R. oryzae-sativae to some commercial cultivars of rice in Myanmar

Rhizoctonia complex of rice has been detected in rice growing areas of Myanmar. The primary objective of this study is to study the varietal response of rice to Rhizoctonia complex and to distinguish the symptom expression of rice responses to these pathogens. Myanmar rice cultivars namely Manawthukha, Shwethweyin, Sinthwelatt and Yezinlonthwe were used to inoculate with three isolates of each species of Rhizoctonia solani, Rhizoctonia oryzae and Rhizoctonia oryzae-sativae. The symptoms created by each species of Rhizoctonia were distinguished by the size and colour of the lesion. Variation in lesion length was observed among different isolate-rice cultivar combination. Shwethweyin variety is the most susceptible one to all the tested three species among the four tested varieties.

Detection and assessment of chemical hormesis on the radial growth in vitro of oomycetes and fungal plant pathogens.

Although plant diseases can be caused by bacteria, viruses, and protists, most are caused by fungi and fungus-like oomycetes. Intensive use of fungicides with the same mode of action can lead to selection of resistant strains increasing the risk of unmanageable epidemics. In spite of the integrated use of nonchemical plant disease management strategies, agricultural productivity relies heavily on the use of chemical pesticides and biocides for disease prevention and treatment and sanitation of tools and substrates. Despite the prominent use of fungi in early hormesis studies and the continuous use of yeast as a research model, the relevance of hormesis in agricultural systems has not been investigated by plant pathologists, until recently. A protocol was standardized for detection and assessment of chemical hormesis in fungi and oomycetes using radial growth as endpoint. Biphasic dose-responses were observed in Pythium aphanidermatum exposed to sub-inhibitory doses of ethanol, cyazofamid, and propamocarb, and in Rhizoctonia zeae exposed to ethanol. This report provides an update on chemical hormesis in fungal plant pathogens and a perspective on the potential risks it poses to crop productivity and global food supply.

In vitro symbiotic seed germination and molecular characterization of associated endophytic fungi in a commercially important and endangered Indian orchid Vanda coerulea Griff. Ex Lindl.

The technique of symbiotic seed germination-using fungi to cultivate orchid seedlings in vitro leading to their reintroduction in situhas considerable potential for conservation as evidenced by studies mostly in Australia and North America. However, its use has yet tobe fully realized throughout the world. On the Indian subcontinent, which harbors a considerable number of orchid species, symbioticgermination has been virtually unexplored. In the present studies, we provide a protocol for the symbiotic seed germination and ecorestorationof an endangered orchids Vanda coerulea Griff. ex Lindl., which is a floriculturally significant epiphyte used to progenatea vast variety of hybrids. Seeds were obtained from the mature un-dehisced capsules and sown on oat meal agar medium with thefungus isolated from the roots of mature V. coerulea plants in situ. Using molecular characterization techniques, cultures were assignableto Rhizoctonia zeae with teleomorph Thanatephorus cucumeris. All the seeds germinated within 5 wks of culture and very healthy,dark green protocorms were obtained in 5 month old cultures. Seedlings with 1–2 roots and 2–3 leaves were obtained in 8 months.These were acclimatized in the greenhouse for a year and introduced to their natural habitat at Manipur in North East India. Seedlinggrowth and development was continuously monitored, demonstrating active growth during monsoon season (April–July). Out of 29plants reintroduced, 23 survived and are growing well with the formation of new roots and leaves, observed after twelve months ofreintroduction.

Suppression of Rhizoctonia and Pythium root rot of wheat by new strains of Pseudomonas

This study is part of an effort to identify new biological control agents with broad spectrum activity against pathogenic soilborne fungi and pests. New strains of Pseudomonas spp. were isolated from agricultural soils, river silt, and rhizosphere soils from herbarium specimens. Provisional species assignments based on 16S ribosomal DNA typing were: Pseudomonas borealis, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas marginalis, Pseudomonas poae, Pseudomonas putida, Pseudomonas syringae and Pseudomonas vranovensis. We evaluated the strains for activity against root rot pathogens of wheat, with the view to discovery and deployment of new suppressive activities. Our objectives were to compare the strains relative to: (i) production of antifungal metabolites, (ii) suppression of Rhizoctonia and Pythium damage in greenhouse assays, (iii) plant growth promotion, and (iv) ability to colonize the wheat rhizosphere. Colonization assays showed that 11 strains rapidly established and maintained rhizosphere populations of ⩾log5CFUg−1 root. Strains 14B2R, 15G2R, 29G9R, 39A2R, 48G9R and Wood3R reduced disease symptoms of both Rhizoctonia solani AG-8 and Pythium ultimum, and the latter four also suppressed Rhizoctonia oryzae and Pythium irregulare. Four strains increased seedling shoot length and root weight but these growth promotion effects were correlated to disease suppression only for two strains. Finally, the strains differed in demonstrated and potential antifungal metabolite activities, indicating that no single factor could be correlated to disease suppression. We have identified several strains for genome sequencing and for long-term development of integrated management of soilborne diseases of wheat.

Effect of some plant extracts on Rhizoctonia spp. and Sclerotium hydrophilum

Soilborne phytopathogens affect rice production by inhabiting inoculum permanently in the soil. Pesticides of plant origin are preferred in order to reduce the risks involved in chemical control measure. The present study was conducted to find out bioresource to control some rice pathogens. Sixteen naturally available phytoextracts were tested in vitro for their potential to control phytopathogens of rice, such as Rhizoctonia solani, Rhizoctonia oryzae,Rhizoctonia oryzae-sativae and Sclerotium hydrophilum. All of the tested fungal growths were suppressed 100% by using clove extract. Neem leaf, rosemary and pelargonium extracts were found to give the second best suppression against the tested fungi. Neem leaf extract inhibited the growth of R. solani by 87.5%, R. oryzae by 92.5% and R. oryzae-sativae by 80%. However, the same extract inhibited S. hydrophilum by only 49.1%. Rosemary extract gave an inhibition of 67.7% for R. solani, 88.0% for R. oryzae, 86.0% forR. oryzae-sativae and 73.89% for S. hydrophilum. The inhibitory effect of pelargonium on the tested fungi showed 48.1% for R. solani, 90.8% for R. oryzae, 84.4% for R. oryzae-sativae and 83.3% for S. hydrophilum. Among the tested phytoextracts, cloves, neem leaf, rosemary and pelargonium are potential phytoextracts to control the tested soilborne phytopathogens. Key words: Rice, Rhizoctonia solani, Rhizoctonia oryzae, Rhizoctonia oryzae-sativae,Sclerotium hydrophilum, sheath diseases, phytoextract.

First Report of Basal Leaf Blight of Kikuyugrass Caused by Waitea circinata var. prodigus in Southern California.

Kikuyugrass (Pennisetum clandestinum) is a C4 grass and invasive weed adopted for use as a primary turf species in some golf course fairways and roughs in southern California. In September 2008, a new Rhizoctonia-like fungus was isolated from a diseased kikuyugrass sample received from a golf course fairway in Oceanside, CA. The kikuyugrass was from a mature stand (>20 years old) that was maintained at a height of approximately 1.25 cm. Symptoms on kikyuygrass developed initially as irregular, blighted, chlorotic patches, several centimeters to one meter, which occurred during a period of warm, humid weather (27 to 29°C maximum daytime temperature, 75 to 85% average relative humidity) on a small part of one fairway. Affected areas became brown and necrotic as the disease progressed. Leaf chlorosis and stem rot were observed on affected plants. The organism was isolated by placing symptomatic leaves on acidified one-quarter-strength potato dextrose agar (PDA) (600 μl of 85% lactic acid per liter of medium) in a petri dish (1). A colony of a Rhizoctonia-like fungus with yellow aerial hyphae, multinucleate hyphal cells, and irregularly shaped, golden brown sclerotia (4 to 7 mm) developed within 30 days at 28°C. The rDNA internal transcribed spacer (ITS) sequence was obtained (GenBank Accession No. HQ850254) using PCR amplification with primers ITS1F and ITS4 (1,2), and a BLAST search showed 100% similarity with Waitea circinata var. prodigus (GenBank Accession No. HM597145), which had recently been described as the cause of basal leaf blight of seashore paspalum (Paspalum vaginatum), another C4 grass (3), in Florida. Colony morphology and other physical characteristics were similar but not completely identical to those from Florida, reflecting the reported morphological variation inherent in the pathogen (3). Koch's postulates were performed by growing this isolate on PDA in a petri dish for 7 days, homogenizing the culture with 100 ml of sterilized water, filtering the suspension through two layers of cheesecloth, and pipetting 10 ml of the mycelial suspension onto the foliage and stems of 4-week-old AZ-1 kikuyugrass plants grown in UC-soilless-mix in 7.5-cm-diameter pots (4). Control plants were treated with a homogenized and filtered dish of PDA only. There were three replicate pots for inoculated and noninoculated treatments and the experiment was repeated independently three times. All of the pots were incubated in a moist chamber with a 12-h light period at 28°C. Yellow lesions were observed on leaves and stems of inoculated plants 4 days postinoculation and necrosis developed 8 days later in all experiments. The same organism was isolated from symptomatic plants. The control plants did not exhibit any symptoms. To our knowledge, this is the first report of basal leaf blight caused by W. circinata var. prodigus on kikuyugrass in California and the first report of this pathogen affecting turfgrass in the western United States.

Identification of a New Waitea circinata Variety Causing Basal Leaf Blight of Seashore Paspalum.

Seashore paspalum (Paspalum vaginatum) is a saline-tolerant, warm-season turfgrass species popular for golf course use in tropical and subtropical climates. A new variety of Waitea circinata (proposed as W. circinata var. prodigus) is described as the causal agent of basal leaf blight, a novel disease of seashore paspalum. Foliar necrosis and canopy thinning of seashore paspalum were observed on three different golf course fairways in Florida over an 18-month period. Five isolates with profuse, pink to yellow mycelia and small, salmon-colored or yellow to light-brown sclerotia were cultured from diseased turf foliage. Isolates grew rapidly over a temperature range of 25 to 35°C and were initially identified as an uncharacterized variety of W. circinata. Internal transcribed spacer sequences of rDNA from the isolates were compared with sequences from previously described W. circinata varieties. The paspalum isolates formed a phylogenetic clade that was distinct from the other W. circinata varieties. Pathogenicity was confirmed on 'SeaDwarf' and 'SeaIsle Supreme' seashore paspalum, 'Penncross' creeping bentgrass (Agrostis stolonifera), 'Senesta' bermudagrass (Cynodon dactylon), and 'Dark Horse' roughstalk bluegrass (Poa trivialis). The geographical distribution and potential impact of basal leaf blight is unknown. However, the range of potential turfgrass hosts and environmental conditions conducive for disease development suggest that the pathogen may infect other species in addition to seashore paspalum.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Creeping Bentgrass in Arizona.

A disease resembling brown ring (Waitea) patch was observed on a 'Dominant Extreme' creeping bentgrass (Agrostis stolonifera) green on a golf course in Maricopa County, Arizona in February 2010. The green was 17 months old and built with 95% sand and 5% peat moss. The superintendent reported seeing yellow rings, 12 to 16 cm in diameter, on several greens as early as 3 months postinstallation; the yellow rings developed into brown, necrotic rings. Symptoms started in the cool, cloudy, and moist conditions of December (5.0 to 6.7°C) and became persistent into the spring. Symptoms on the samples appeared to be yellowing of leaves and stems with the development of a dark, water-soaked appearance of the whole plant on older affected portions. The samples were incubated in a moist chamber at 22 to 25°C for 24 h. Foliar mycelium developed on the symptomatic leaves, and upon microscopic examination, the mycelium appeared to have the characteristics of Rhizoctonia spp.; i.e., a right-angled branching pattern, constriction of the hyphal branch near its point of origin, and the presence of a septum near the point of origin. The pathogen was recovered from chlorotic tissue by plating the symptomatic tissue on one-quarter-strength acidified potato dextrose agar (9.90 g of PDA and 11.26 g of granulated agar [Fisher, Lenexa, KS] and 600 ml of lactic acid [Sigma, St. Louis, MO] per liter of water) and incubating at ~27°C in light. A Rhizoctonia-like pathogen emerged from the tissue within 48 h and was tentatively identified as Waitea circinata var. circinata based on colony and bulbil morphology after 10 days of incubation (3). The recovered isolate was used for DNA extraction and subsequent amplification and sequencing of the rDNA internally transcribed spacer (ITS) region using ITS1F and ITS4 primers (2). The recovered sequence (HM807352) was compared with the National Center for Biotechnology Information (NCBI) nucleic acid database and was found to show 100% similarity to W. circinata var. circinata (FJ755879). To confirm pathogenicity, the isolate was used to fulfill Koch's postulates. The isolate was grown on autoclaved sand and corn meal (250 g of sand and 50 g of corn meal) for 4 weeks to produce inoculum. Eight grams of colonized sand and corn meal was broadcast on 4-week-old creeping bentgrass seedlings ('Penncross') planted in a 90:10 peat moss/sand mixture in 10-cm-diameter pots. There were three replications and the experiment was repeated twice. Negative controls consisted of plants inoculated with sand and corn meal only. Pots were maintained at 28 to 33°C in the greenhouse with ambient light. Within 4 days of inoculation, the plants showed chlorosis and necrosis, while noninoculated plants showed no symptoms. The pathogen was successfully reisolated from several plants from each replication using the method described above. This pathogen has been known to cause disease on annual and rough bluegrass (1,2) in the United States, but not confirmed as a pathogen on creeping bentgrass here. To our knowledge, this is the first report of brown ring patch on creeping bentgrass in Arizona.

GENETIC DIVERSITY OF Waitea circinata var. zeae IN SOUTH CAROLINA REVEALED BY AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)

Rhizoctonia zeae (Waitea circinata var. zeae) is pathogenic to rice (Oniki et al., 1985); corn (Sumner and Bell, 1982); onion (Erper et al., 2006); sugarbeet (Kuznia and Windels, 1994); wheat and barley (Ogoshi et al., 1990) and tall fescue (Martin and Lucas, 1983). Waitea circinata var. zeae also causes foliar lesions on bermuda grass, creeping bentgrass and annual bluegrass (Burpee and Martin, 1992; Hsiang and Dean, 2001) during midsummer. Diseases of turfgrass caused by these pathogens occur most frequently during the warm and humid season, at temperatures between 28 and 36°C, inciting leaf and sheath spot (Burpee and Martin, 1992; Smiley et al., 1992). Waitea circinata (Warcup and Talbot) classified into three varieties, W. circinata var. circinata, W. circinata var. oryzae and W. circinata var. zeae based on differences in the colony morphology of the vegetative state (Gunnell, 1986). Waitea circinata var. circinata forms orange to dark brown, globose sclerotia up to 2 mm in diameter; W. circinata var. oryzae forms orange to salmon, irregularly shaped sclerotia; and W. circinata var. zeae forms orange to brown, regularly shaped sclerotia up to 1 mm in diameter (Leiner and Carling, 1994). Rhizoctonia zeae was assigned to Waitea anastomosis group WAG-Z (Oniki et al., 1985). Previous studies have examined genetic variation of these three varieties at molecular level. Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) of rDNA internal transcribed spacer (ITS) region revealed that isolates of W. circinata var. circinata, W. circinata var. oryzae and W. circinata var. zeae separated into individual clusters (Toda et al., 2005). These results confirmed by using sequence analysis of the internal transcribed spacer (ITS) region of rDNA (de la Cerda et al., 2007; Toda et al., 2007). Amplified fragment length polymorphism (AFLP) is a genetic mapping technique based on selective amplification of a subset of restriction enzyme-digested DNA fragments to create a unique fingerprint for a particular genome (Vos et al., 1995). It is highly reproducible and amenable to a wide range of applications and DNA sources. For these reasons, the method has steadily gained popularity in applications, including genetic mapping (Mueller and Wolfenbarger, 1999; Savelkoul et al., 1999), medical diagnostics (Klaassen et al., 2002; Borst et al., 2003; van den Braak et al., 2004), genetic diversity and phylogenetic studies (Tredway et al., 1999; Bakkeren et al., 2000; Doignon-Bourcier et al., 2000; Rademaker et al., 2000; Mougel et al., 2002; Lee et al.,2004) and environmental management studies (Lucchini, 2003). The similarities within each variety of W. circinata were very high, but similarities were significantly lower between varieties by using rDNA-ITS region (Toda et al., 2007). In this study, we used amplified fragment length polymorphism (AFLP) to examine the genetic diversity of field population of W. circinata var. zeae.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua and Agrostis stolonifera in New Jersey.

Waitea circinata var. circinata was first reported as the causal agent of brown ring patch on annual bluegrass (Poa annua L.) in the United States in 2007 (2). In early April to mid-June of 2009, circular to irregularly shaped yellow rings resembling symptoms of this disease were observed on an annual bluegrass putting green at Rutgers University in North Brunswick, NJ. Severely infected foliage eventually turned brown as the disease progressed. During the same time period, similar disease symptoms were observed on creeping bentgrass (Agrostis stolonifera L.) from a golf course in Bedminster Township, NJ. The disease reappeared in both locations in April of 2010. Five additional samples with similar symptoms on creeping bentgrass and annual bluegrass were received at Rutgers Diagnostic Laboratory from Paramus, Madison, Allamuchy, and Farmingdale, NJ between late April and early May of 2010. Portions of diseased leaf and sheath tissue that displayed symptoms of the disease were disinfested for 1 min in 0.5% NaOCl, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA) amended with 50 mg/liter of streptomycin sulfate. At the first sign of fungal growth, single hyphal tips were transferred to PDA. After 1 week at 25°C, white-to-orange mycelial colonies formed in culture and eventually turned brown with age. Minute sclerotia (≤3 mm), which followed the same color development pattern, formed within 10 days. These features are consistent with those described of W. circinata var. circinata (2,3). The internal transcribed spacer (ITS) region of the ribosomal RNA gene was amplified using primer pair ITS1/ITS4 and sequenced with ITS4 (GenBank Accession Nos. HQ166065 to HQ166071). BLASTn analysis of the ITS sequences showed a 99 to 100% similarity to W. circinata var. circinata sequences deposited in GenBank (1,2). Pathogenicity tests were conducted in 2010 using 6-week-old creeping bentgrass seedlings cv. Declaration inoculated with colonized oat grain that had been autoclaved and then infested with the Bedminster Township isolate. Eight colonized oat grains were uniformly spread around the crowns of seedlings grown in 10-cm-diameter pots. Control plants were treated with autoclaved grain. Plants were incubated at 25°C and high humidity maintained by misting the plants three times per day. Within 3 days postinoculation, foliage near infested grain turned chlorotic. All foliage in pots became completely blighted and spherical orange-brown sclerotia were observed on leaf sheaths by the eighth day. W. circinata var. circinata was consistently reisolated from inoculated plants (as confirmed by isolate morphology and ITS sequencing) but not from control plants. The ITS sequence data, morphological characters of the isolates, and pathogenicity tests demonstrate that W. circinata var. circinata is present in New Jersey. To our knowledge, this is the first report of W. circinata var. circinata infecting turfgrass in New Jersey.

Identification and pathogenicity of Rhizoctonia species isolated from bean and soybean plants in Samsun, Turkey

A total of 434 isolates of Rhizoctonia belonging to 10 anastomosis groups were obtained from the roots and rhizosphere soils of bean and soybean plants grown in Samsun, Turkey. AG-4 was found to be the most common group on bean and soybean plants and AG-5, AG-6, binucleate AG-A, AG-B and R. zeae were other groups isolated from the both plant species. AG-1, AG-7 and AG-K from bean and AG-E from soybean were other groups obtained in the study. The pathogenicity tests on bean and soybean seedlings showed that the highest disease severities were caused by AG-4 isolates, whereas AG-1 and AG-6 isolates were moderately pathogenic. Binucleate Rhizoctonia AG-B isolates were also moderately pathogenic, while other binucleate Rhizoctonia were found to be weakly pathogenic. Rhizoctonia zeae isolates caused moderate disease symptoms on bean, but soybean plants were slightly affected by this group of isolates. This is the first reported observation of R. solani AG-6 and AG-7 and binucleate Rhizoctonia AG-B on bean, and R. solani AG-5 and AG-6 and binucleate Rhizoctonia AG-A, AG-B and AG-E on soybean, in Turkey.

First Report of Brown Ring Patch on Poa annua Caused by Waitea circinata var. circinata in West Virginia.

In mid-November 2009, thin, yellow, and irregular-shaped scalloped rings 10 to 25 cm in diameter were observed on 5 to 10% of a golf course putting green in Charles Town, WV. The 20-year-old USGA-specification sand-based green was mowed at 3.1-mm height and consisted of 60% annual bluegrass (Poa annua L.) and 40% creeping bentgrass (Agrostis stoloniferous L. 'Putter'). Minimum and maximum daily air temperature ranged from 2 to 22°C, respectively, with 38 mm of rainfall during the appearance of rings symptoms. Only affected annual bluegrass plants exhibited a peculiar yellow chlorosis of the upper and lower leaves. A single fungal isolate was obtained from active mycelium found within symptomatic annual bluegrass leaves and grown on potato dextrose agar (PDA) amended with chloramphenicol (0.1 g/liter). Fungal colony morphology (i.e., light yellow with irregular-shaped 2- to 4-mm-diameter sclerotia first appearing off-white but progressing to brown by 21 to 28 days in culture) and sequencing of the internal transcribed spacer (ITS) 5.8S rDNA region with primers ITS1 and ITS4 confirmed the isolate as Waitea circinata var. circinata (Warcup & Talbot) with ≥99% sequence identity with GenBank Accession No. FJ755889 (1,2,4). To confirm pathogenicity, a 6-mm-diameter plug of the isolate was removed from the expanding edge of a 4-day-old culture grown on PDA and placed in contact with the lower leaves of 12-week-old annual bluegrass (0.001 g of surface-sterilized seed per cm2) grown in 5- × 5-cm plastic pots of autoclaved 85% sand and 15% potting soil. Six pots were inoculated with the isolate and six pots were inoculated with an isolate-free agar plug and then placed in a moist chamber at 28°C. Leaf chlorosis and aerial mycelium was observed in all six inoculated pots 8 to 10 days after inoculation, and symptoms were similar to those expressed in the field. All noninoculated plants remained healthy and asymptomatic. W. circinata var. circinata was reisolated from symptomatic leaves and again confirmed by colony traits and sequencing of the ITS-5.8S rDNA region and submitted as GenBank Accession No. HM807582. To our knowledge, this is the first report of brown ring patch in West Virginia and could be economically important because of intensive fungicide practices used to maintain high-quality putting greens on golf courses (3).

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua in Wisconsin and Minnesota.

In summer of 2008, two turfgrass samples were submitted to the Turfgrass Diagnostic Lab at the University of Wisconsin-Madison. The samples were from golf courses in Beaver Dam, WI on 12 June and Minneapolis, MN on 14 July. Both samples were collected from 40-year-old native soil putting greens mowed at 3.2 mm that had received annual sand topdressing since 1992. The putting greens were a mixture of approximately 75% annual bluegrass (Poa annua L.) and 25% creeping bentgrass (Agrostis stolonifera L.) Stand symptoms observed in the field were bright yellow, sunken rings that were approximately 5 cm thick and 15 to 35 cm in diameter. Some rings were incomplete, giving a scalloped appearance. Affected plants were severely chlorotic and lacked any discrete lesions or spots. Symptoms were more prominent on annual bluegrass than creeping bentgrass. Upon incubation of samples at room temperature in a moist chamber for 24 h, fungal mycelia with septations and right-angle branching were observed in the foliage and thatch layer. Two isolates were obtained from affected annual bluegrass in each sample. Isolations were performed by washing affected leaves in 0.5% NaOCl solution for 2 min, blotting the tissue dry, and plating the tissue on potato dextrose agar (PDA) amended with chloramphenicol (0.05 g/liter), streptomycin (0.05 g/liter), and tetracycline (0.05 g/liter). After incubation for 2 days at 23°C, isolates were transferred and maintained on PDA. All four isolates had multinucleate hyphae and displayed sclerotial characteristics similar to those reported for Waitea circinata var. circinata (2). Sequencing the ITS1F/ITS4-amplified rDNA internal transcribed spacer (ITS) region confirmed the isolates as W. circinata var. circinata, with ≥99% sequence similarity to published W. circinata var. circinata ITS sequences (GenBank Accession No. FJ755849) (1,2,4). To confirm pathogenicity, isolates were inoculated onto 6-week-old annual bluegrass (True Putt/DW184) grown in 10-cm-diameter pots containing calcined clay (Turface; Profile Products LLC., Buffalo Grove, IL). Two 4-mm-diameter agar plugs for each isolate were removed from the margins of 3-day-old colonies grown on PDA and placed near the soil surface to ensure contact with the lower leaf blades. Each isolate was placed in four separate pots to have four replicated tests per isolate, and four noninfested pots were utilized as negative controls. All pots were placed in moist chambers at 28°C with a 12-h light/dark cycle. Within 4 to 6 days, inoculated plants exhibited severe chlorosis and a minor amount of aerial mycelium was observed. Inoculated plants became necrotic after 15 to 20 days, while the noninoculated plants remained healthy. W. circinata var. circinata was reisolated from inoculated plants and its identity was confirmed by morphological and molecular characteristics. This pathogen was previously reported as a causal agent of brown ring patch of creeping bentgrass in Japan and annual bluegrass in the western United States (2,4). To our knowledge, this is the first report of brown ring patch in Minnesota and Wisconsin. Intensive fungicide practices are needed to control brown ring patch; therefore, this disease could have significant economic impact throughout the Upper Midwest (3).

Brevibacillus laterosporus strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam, India

A bacterial strain designated as BPM3 isolated from mud of a natural hot water spring of Nambar Wild Life Sanctuary, Assam, India, strongly inhibited growth of phytopathogenic fungi ( Fusarium oxysporum f. sp. ciceri, F. semitectum, Magnaporthe grisea and Rhizoctonia oryzae) and gram-positive bacterium ( Staphylococcus aureus). The maximum growth and antagonistic activity was recorded at 30 °C, pH 8.5 when starch and peptone were amended as carbon and nitrogen sources, respectively. In greenhouse experiment, this bacterium (BPM3) suppressed blast disease of rice by 30–67% and protected the weight loss by 35–56.5%. The maximum disease protection (67%) and weight loss protection (56.5%) were recorded when the bacterium was applied before 2 days of the pathogen inoculation. Antifungal and antibacterial compounds were isolated from the bacterium which also inhibited the growth of these targeted pathogens. The compounds were purified and on spectroscopic analysis of a purified fraction having R f 0.22 which showed strong antifungal and antibacterial activity indicated the presence of C–H, carbonyl group, dimethyl group, –CH 2 and methyl group. The bacterium was characterized by morphological, biochemical and molecular approaches and confirmed that the strain BPM3 is Brevibacillus laterosporus.

First Report of Waitea circinata from Turfgrass in British Columbia, Canada

In Canada, Waitea circinata was first identified from buckwheat (Fagopyrum sp.) in 1965 in Ontario (4). In 2004, the fungus was found on diseased putting greens of Poa annua and Agrostis stolonifera near Toronto, Ontario (2). In late July 2009, symptoms on A. stolonifera resembling those of brown ring patch were seen at a golf course in Kelowna, British Columbia. Brown rings with light-colored, cottony growth were observed on a putting green with mixed P. annua and A. stolonifera, originally seeded with A. stolonifera cv. Penncross. Following a short incubation of the diseased grass at 25°C, hyphae of a Rhizoctonia-like fungus, not matching the characteristics of R. cerealis or R. solani, were seen. Symptomatic leaves were surface sterilized in 1% hypochlorite and plated onto potato dextrose agar (PDA) amended with streptomycin. After 1 week at 23°C, the plates contained white colonies that were 5 cm across. DNA was extracted and amplified with primers ITS1/ITS4 and sequenced with ITS1. The 600-bp sequence (deposited in GenBank as GU176409) from the internal transcribed spacer (ITS) region of ribosomal DNA showed a 100% match in the overlapping range with sequence FJ755879 from GenBank, which is annotated as W. circinata var. circinata. Pathogenicity was tested at 23°C by inoculating 3-week-old A. stolonifera 'Penncross' plants grown in Magenta boxes and incubated for 15 days after inoculation with ground wheat seed inoculum of W. circinata. Within 1 week, significant blighting of leaves and sheaths was observed as well as spherical orange brown sclerotia that were 2 to 5 mm in diameter on sheaths. These sclerotial features match characteristics of W. circinata var. circinata (1). Symptomatic leaves were plated on PDA and fungal growth characteristic of W. circinata was recovered. W. circinata was previously reported as the causal agent of brown ring patch on A. stolonifera in Japan (3), as a pathogen of P. annua in the United States (1), and as a pathogen of both species in Ontario, Canada (2). To our knowledge, this is the first report of W. circinata from turfgrass in western Canada.