Infection and Colonization of Several Bermudagrasses by Ophiosphaerella korrae.

Bermudagrass (Cynodon spp.) is the most commonly used turfgrass in the southern United States where it is severely affected by spring dead spot (SDS) caused by Ophiosphaerella herpotricha, O. korrae, and O. narmari. In this study, infection of bermudagrass roots and stolons by O. korrae was characterized using a transformant that expressed the red fluorescent protein tdTomato. Roots of interspecific hybrid cultivars Midlawn and Tifway 419, C. transvaalensis accessions Uganda and 3200, and C. dactylon cultivar U3 were inoculated and observed from 2 to 14 days postinoculation (DPI) while stolons were observed from 2 to 22 DPI. For all five cultivars tested, a similar level of root colonization was observed; however, differences were observed in the rate of necrosis development. Necrosis of Tifway 419 and Midlawn tissues was evident at 2 DPI, in Uganda and 3200 at 8 DPI, and in U3 necrosis was often absent as late as 14 DPI. The fungus rapidly penetrated the root epidermis and colonized the cortex of all cultivars by 4 DPI. Colonization of stele tissues by O. korrae was rare in hybrid cultivars but common in C. transvaalensis and C. dactylon accessions. On intact stolons, the fungus did not penetrate the epidermis 22 DPI though epidermal necrosis was evident on the surface of only the hybrid bermudagrasses. Wounded stolons became necrotic in all cultivars. Infection and colonization of various bermudagrasses by O. korrae was found to be similar to that by O. herpotricha, suggesting that host genetic resistance may be used for effective management of SDS caused by both species.

Highly Sensitive End-Point PCR and SYBR Green qPCR Detection of Phymatotrichopsis omnivora, Causal Fungus of Cotton Root Rot.

Phymatotrichopsis omnivora, the causal pathogen of cotton root rot, is a devastating ascomycete that affects numerous important dicotyledonous plants grown in the southwestern United States and northern Mexico. P. omnivora is notoriously difficult to isolate from infected plants; therefore methods for accurate and sensitive detection directly from symptomatic and asymptomatic plant samples are needed for disease diagnostics and pathogen identification. Primers were designed for P. omnivora based on consensus sequences of the nuclear ribosomal internal transcribed spacer (ITS) region of geographically representative isolates. Primers were compared against published P. omnivora sequences and validated against DNA from P. omnivora isolates and infected plant samples. The primer combinations amplified products from a range of P. omnivora isolates representative of known ITS haplotypes using standard end-point polymerase chain reaction (PCR) methodology. The assays detected P. omnivora from infected root samples of cotton (Gossypium hirsutum) and alfalfa (Medicago sativa). Healthy plants and other relevant root pathogens did not produce PCR products with the P. omnivora-specific primers. Primer pair PO2F/PO2R was the most sensitive in end-point PCR assays and is recommended for use for pathogen identification from mycelial tissue and infected plant materials when quantitative PCR (qPCR) is not available. Primer pair PO3F/PO2R was highly sensitive (1 fg) when used in SYBR Green qPCR assays and is recommended for screening of plant materials potentially infected by P. omnivora or samples with suboptimal DNA quality. The described PCR-based detection methods will be useful for rapid and sensitive screening of infected plants in diagnostic laboratories, plant health inspections, and plant breeding programs.

Development of a rapid, sensitive, and field-deployable razor ex BioDetection system and quantitative PCR assay for detection of Phymatotrichopsis omnivora using multiple gene targets.

A validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection of Phymatotrichopsis omnivora. This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. Because P. omnivora is difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based on P. omnivora nucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection of P. omnivora in infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (∼30-min) on-site assay capability for P. omnivora detection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.

Molecular systematics of the cotton root rot pathogen, Phymatotrichopsis omnivora.

Cotton root rot is an important soilborne disease of cotton and numerous dicot plants in the south-western United States and Mexico. The causal organism, Phymatotrichopsis omnivora (= Phymatotrichum omnivorum), is known only as an asexual, holoanamorphic (mitosporic) fungus, and produces conidia resembling those of Botrytis. Although the corticoid basidiomycetes Phanerochaete omnivora (Polyporales) and Sistotrema brinkmannii (Cantharellales; both Agaricomycetes) have been suggested as teleomorphs of Phymatotrichopsis omnivora, phylogenetic analyses of nuclear small- and large-subunit ribosomal DNA and subunit 2 of RNA polymerase II from multiple isolates indicate that it is neither a basidiomycete nor closely related to other species of Botrytis (Sclerotiniaceae, Leotiomycetes). Phymatotrichopsis omnivora is a member of the family Rhizinaceae, Pezizales (Ascomycota: Pezizomycetes) allied to Psilopezia and Rhizina.

Species composition and seasonal occurrence of Phyllophaga (Coleoptera: Scarabaeidae) infesting intensely managed Bermudagrass in Oklahoma.

Larvae of Phyllophaga spp. (Coleoptera: Scarabaeidae) are important turfgrass pests in many regions of the United States. However, not all of the species associated with turfgrass are known, including species most likely to be of economic concern in Oklahoma turfgrasses, especially Bermuda grass. This study documented the species composition and seasonal occurrence of Phyllophaga associated with high maintenance Bermuda grass turf in Oklahoma over a 2-yr period. In 2005 and 2006, adult Phyllophaga spp. were collected with blacklight traps from selected golf courses throughout Oklahoma Phyllophaga larvae were obtained from Bermuda grass stands at selected sod production facilities adjacent to or near the light traps. We collected 20 species of Phyllophaga beetles in light traps, and nine species of Phyllophaga larvae from turfgrass. Peak flight periods for most species occurred in May and June, but some were captured as early as mid-April and others as late as September. The cytochrome c oxidase I (COI) gene from adults and larvae was amplified using polymerase chain reaction, sequenced, and then used to compare larval DNA against DNA from identified adults. These results confirmed the validity of using COI sequences to identify species of some Phyllophaga larvae. The identifications will aid in optimizing the timing of insecticide applications against Phyllophaga white grubs as discussed.

First Report of Dollar Spot of Buffalograss Caused by Sclerotinia homoeocarpa in Oklahoma.

Buffalograss (Buchloe dactyloides (Nutt.) Engelm.) is a perennial, warm-season grass native to the central plains of North America and a dominant plant over much of the shortgrass prairie ecosystem. Its prostrate growth habit and excellent drought tolerance make it a commercially promising turfgrass species, and numerous turf-type cultivars have been released. In the spring of 2007, the southern plains states experienced prolonged periods of excessive precipitation during which numerous buffalograss swards throughout north-central Oklahoma exhibited symptoms of dollar spot (1). A fungus morphologically identical to Sclerotinia homoeocarpa Bennett was consistently isolated from diseased buffalograss leaves collected from three locations in Oklahoma, two from Payne County and one from Logan County. Thirty-day-old seedlings of B. dactyloides ('Cody' and 'Topgun') and Agrostis stolonifera ('SR1020') were inoculated by placing potato dextrose agar (PDA) plugs, colonized by mycelia of each S. homoeocarpa isolate, onto the seedlings' leaves. Sterile PDA plugs were placed on plants as controls. Leaf lesions developed after 4 days only on inoculated plants, and S. homoeocarpa was reisolated from lesions, satisfying Koch's postulates. The nuclear ribosomal internal transcribed spacer (ITS) region was amplified from DNA extracted from cultures of the three buffalograss isolates and a bentgrass isolate using primers ITS4 and ITS5 (2) and sequenced. Sequences were similar to one another (97 to 99% identical), however, two isolates shared a 420-bp, type I intron in the 18S small subunit rDNA. A search of GenBank at NCBI found the ITS sequences were most similar to the ITS regions of other S. homoeocarpa accessions (97% identical). The ITS sequences from the four isolates were deposited in GenBank (Accession Nos. EU123800-EU123803). To our knowledge, this is the first report of dollar spot on a native, warm-season grass in the United States and the disease appears to be endemic to buffalograss in Oklahoma and Kansas (N. A. Tisserat, personal communication). References: (1) R. W. Smiley et al. Page 22 in: Compendium of Turfgrass Diseases. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2005. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press Inc., New York, 1990.

Influence of Temperature and Time of Year on Colonization of Bermudagrass Roots by Ophiosphaerella herpotricha.

The influence of temperature on the infection of bermudagrass seedlings by Ophiosphaerella herpotricha and colonization of plants in the field was investigated. Bermudagrass seedlings (cv. Jackpot) inoculated with O. herpotricha exhibited dark lesions after 8 days. Root lesion length was greatest at 17°C and was similar for all temperatures examined below 21°C. Seedlings grown at 25 or 30°C had small lesions that remained similar in size when evaluated at 8 and 10 days post inoculation. Colonization of bermudagrass roots from field plots were examined in July, October, and November of 2003 and 2004. In 2003, no differences between sampling dates were observed for plants sampled from the edge of the spring patch in 5.4-cm increments to a total distance of 21.6 cm. In 2004, July and October samples were similar; however, an increase in root colonization was found between the October and November samplings. These studies suggest that infection and colonization of bermudagrass roots by O. herpotricha occurs over a wide range of cool soil temperatures, occurs in the spring, and can be variable in the autumn.

First Report of Ergot of Bermudagrass Caused by Claviceps cynodontis in Oklahoma.

During late June and early July of 2005, signs of bermudagrass ergot were reported from numerous northern and eastern counties in Oklahoma. Signs were observed primarily on forage-type bermudagrass (Cynodon dactylon (L.) Pers.), as well as bermudagrass turf. During the "honeydew" stage, honeydew was frequently observed exuding from most of the ovaries of infected inflorescences. These signs of ergot have been observed previously on bermudagrass in Oklahoma and Texas (1). Sphacelia-type conidia were abundantly produced during the honeydew stage and were single-celled, hyaline, averaged 14 × 5 µm in size, and were reniform to allantoid in shape. When streaked on water agar, conidia produced terminal holoblastic secondary conidia. Single-spore cultures were isolated from the honeydew of bermudagrasses from Logan and Muskogee counties in Oklahoma and grew slowly as white mycelium on potato dextrose agar (PDA). Koch's postulates were fulfilled for these two isolates by spray inoculating four bermudagrass inflorescences at anthesis with mycelium scraped from a PDA plate and homogenized in water. Control plants' inflorescences were sprayed with a water suspension of a similar amount of sterile PDA as inoculated plants. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 22°C (14-h photoperiod) and 20°C (10 h of darkness). After 9 days, honeydew exuded from the inoculated inflorescences, but not from the controls. Single-spore cultures were reisolated from the honeydew, and conidia streaked on water agar formed identical secondary conidia. The complete nuclear ribosomal internal transcribed spacer (ITS) region was amplified from DNA extracted from honeydew and single-spore cultures using the ITS4 and ITS5 primers (4) and sequenced. All sequences were identical and a search of GenBank at NCBI found these sequences were most similar to the ITS regions of Claviceps cynodontis Langdon (100%, Accession No. AJ557074) and C. maximensis Theis (99%, Accession No. AJ133396). The ITS sequence from the Logan County isolate was deposited at Gen-Bank (Accession DQ187312). The morphology, secondary conidiation, and ITS sequences identify the causal fungus as C. cynodontis (2) and differentiate it from C. purpurea (Fr.) Tul., the previously identified cause of bermudagrass ergot (1). To our knowledge, this is the first report of C. cynodontis on bermudagrass in Oklahoma and may represent a recent introduction to the United States (2; S. Pazoutová and M. Flieger, personal communication). A Claviceps sp. isolated from bermudagrass has been shown to produce ergot alkaloids possibly causing "bermudagrass tremors" in cattle (3). In regions where bermudagrass is the predominant forage for livestock, the toxicological significance of bermudagrass ergot caused by C. cynodontis is unclear and requires further research. References: (1) K. E. Conway et al. Plant Dis. 76:1077, 1992. (2) S. Pazoutová et al. Can J. Plant Pathol. 27:541, 2005. (3) J. K. Porter et al. J. Agric. Food Chem. 22:838, 1974. (4) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press Inc., New York, 1990.

ParasiticMeloidogyne and mutualisticAcremonium increase chitinase in tall fescue.

Tall fescue (Festuca arundinacea Schreb.) is a C-3 perennial grass noted for its persistence in harsh environments. Tall fescue persistence is enhanced byAcremonium coenophialum, a mutualistic fungal endophyte that increases resistance to drought, pathogens, and insects. This research was conducted to identify and elicit biochemical mechanism(s) that could account for tall fescue persistence. In initial studies, two cultivars known to differ in persistence were analyzed for chitinase, an antifungal hydrolase associated with disease resistance in other plants.Acremonium-infected Kentucky 31 (KY31), a persistent cultivar, and Johnstone, a nonpersistent cultivar, were inoculated with the parasitic nematode,Meloidogyne marylandi, grown for 50 days, and analyzed at 10-day intervals. Chitinase fluctuated throughout the 50-day period of seedling development, and activity was highest in the persistentAcremonium-infected KY31. In addition, chitinase was elicited by parasiticM. marylandi and expressed systemically. Subsequent studies were conducted to determine whether or not mutualisticAcremonium could increase chitinase activity. Genetically identical KY31, with and withoutAcremonium, were grown for 25 days and analyzed for chitinase at 5-day intervals. After 20 days,Acremonium-infected KY31 expressed more chitinase thanAcremonium-free KY31. We concluded that chitinase is related to tall fescue persistence; it was highest in the most persistent cultivar, increased under pathogen attack, and increased in the presence ofAcremonium, a symbiont known to enhance disease resistance.