Contrasting genetic structure between Magnaporthe grisea populations associated with the golf course turfgrasses Lolium perenne (perennial ryegrass) and Pennisetum clandestinum (kikuyugrass).

Gray leaf spot (GLS) disease of perennial ryegrass (Lolium perenne) and kikuyugrass (Pennisetum clandestinum) in golf courses in California was first noted in 2001 and 2003, respectively, and within 5 years had become well established. The causal agent of the disease is the fungus Magnaporthe grisea, which is known to consist primarily of clonal lineages that are highly host specific. Therefore, our objective was to investigate host specificity and population dynamics among isolates associated primarily from perennial ryegrass and kikuyugrass since the disease emerged at similar times in California. We also obtained isolates from additional hosts (tall fescue, St. Augustinegrass, weeping lovegrass, and rice) and from the eastern United States for comparative purposes. A total of 38 polymorphic amplified fragment length polymorphism makers were scored from 450 isolates which clustered by host with high bootstrap support (71 to 100%). Genetic structure between kikuyugrass and perennial ryegrass isolates differed significantly. Isolates from kikuyugrass were genotypically diverse (n = 34), possessed both mating types, and some tests for random mating could not be rejected, whereas isolates from perennial ryegrass were less genotypically diverse (n = 10) and only consisted of a single mating type. Low genotypic diversity was also found among the other host specific isolates which also only consisted of a single mating type. This is the first study to document evidence for the potential of sexual reproduction to occur in M. grisea isolates not associated with rice (Oryza sativa). Moreover, given the significant host specificity and contrasting genetic structures between turfgrass-associated isolates, the recent emergence of GLS on various grass hosts in California suggests that potential cultural practices or environmental changes have become conducive for the disease and that the primary inoculum may have already been present in the state, despite the fact that two genotypes associated with perennial ryegrass and St. Augustinegrass in California were the same as isolates collected from the eastern United States.

Two Mutations in ß-Tubulin 2 Gene Associated with Thiophanate-Methyl Resistance in Colletotrichum cereale Isolates from Creeping Bentgrass in Mississippi and Alabama.

Turfgrass anthracnose, caused by Colletotrichum cereale (≡C. graminicola), has become a common disease of creeping bentgrass putting greens during the summer in Mississippi and Alabama over the last 15 years. Thiophanate-methyl is a single-site mode-of-action fungicide applied to control C. cereale. In vitro bioassays were performed to evaluate the sensitivity of 103 isolates to thiophanate-methyl concentrations ranging from 0.039 to 10 µg/ml. Eighty-three isolates were collected from creeping bentgrass in Mississippi and Alabama that had been exposed to thiophanate-methyl. An additional 20 isolates were included from nonexposed turfgrasses. Radial colony growth in amended media was relative to nonamended media for all in vitro bioassays. With thiophanate-methyl at 10 µg/ml, relative growth of exposed isolates ranged from 77.5 to 130.7% with a mean of 99.3% compared with nonexposed, baseline isolates that ranged from 0.0 to 48.7% with a mean of 20.4%. A representative sample of thiophanate-methyl-exposed and nonexposed isolates was used to determine the mechanism of resistance by comparing amino acid sequences of the ß-tubulin 2 protein. All of the thiophanate-methyl-exposed isolates that were sequenced had a point mutation resulting in substitutions from glutamic acid to alanine at position 198 or from phenylalanine to tyrosine at position 200 of the ß-tubulin 2 protein. These amino acid substitutions in C. cereale isolates from Mississippi and Alabama appear to confer resistance to thiophanate-methyl and differ from those reported previously for this pathogen.

Geographic Distribution and rDNA-ITS Region Sequence Diversity of Waitea circinata var. circinata Isolated from Annual Bluegrass in the United States.

Waitea circinata var. circinata is the causal agent of brown ring patch, an emergent disease of turfgrass in the United States. Forty-two isolates from annual bluegrass were obtained from California, Connecticut, Idaho, Illinois, Massachusetts, New York, Ohio, Oregon, and Rhode Island. Almost all isolates produced white to orange sclerotia (bulbils), 2 to 5 mm in size, that turned dark brown after 21 days on »-strength potato dextrose agar. The ribosomal DNA internal transcribed spacer regions and 5.8S region (ITS) were analyzed by restriction fragment length polymorphism (RFLP) analysis using MspI and sequencing to attempt identification of the isolates. Some isolates were heterozygous at the MspI restriction site, results not found in previous reports using the RFLP technique for identification. Four additional nucleotide positions were found to be variable within ITS based on sequence analysis, including two indels and two additional heterozygous positions. A total of 17 ITS haplotypes were found, and there was no obvious relationship between ITS haplotype and the geographic distribution of the isolates. Results of this work indicate that W. circinata var. circinata is present in multiple states and provide an initial understanding of the diversity of the pathogen in the United States.

Detection and Characterization of Benzimidazole Resistance in California Populations of Colletotrichum cereale.

Colletotrichum cereale is the causal agent of turfgrass anthracnose, which has become a serious problem on annual bluegrass (Poa annua) and creeping bentgrass (Agrostis palustris) golf course putting greens. Thiophanate-methyl is a benzimidazole (methyl benzimidazole carbamate [MBC]) fungicide used for the management of anthracnose. In this study, we examined 481 isolates from 10 California populations to determine the presence and frequency of MBC resistance. An in vitro methodology was developed to construct a baseline sensitivity distribution using 60 isolates from an unexposed population (TCGC). The 50% effective dose (ED50) values for the baseline sensitivity distribution for thiophanate-methyl ranged from 0.14 to 2.3 µg/ml with a mean of 0.75 µg/ml. For 60 isolates assayed from an exposed population (AHCC), 57 isolates were not responsive to in vitro concentrations of thiophanate-methyl of up to 30 µg/ml. Isolates nonresponsive to thiophanate-methyl were not responsive to benomyl in vitro. Two isolates nonresponsive in vitro to thiophanate-methyl or benomyl were not controlled in vivo on annual bluegrass plants treated preventively with either fungicide at 11 mg/ml, confirming the results of the in vitro testing. The remaining 361 isolates from eight populations were tested using the single discriminatory dose of thiophanate-methyl at 10 µg/ml. A high proportion (>90%) of isolates from six of the populations were resistant to thiophanate-methyl, indicating the presence of practical resistance at these locations. To determine the molecular mechanism of MBC resistance, the two ß-tubulin genes, TUB1 and TUB2, of 12 resistant and 6 sensitive isolates were amplified and sequenced, revealing a glutamic acid to lysine substitution at position 198 of TUB2 that was present in all resistant isolates. This work confirms the presence of MBC resistance in C. cereale populations from California and presents methods and information that can be used to manage resistance to the MBC fungicides and improve anthracnose management programs.

Discovery and Characterization of Waitea circinata var. circinata Affecting Annual Bluegrass from the Western United States.

Waitea circinata var. circinata was identified as the causal agent of a new disease of annual bluegrass (Poa annua) in the United States. This pathogen is also known to cause brown ring patch on creeping bentgrass (Agrostis stolonifera) in Japan, but it had not been reported on any turf species outside of Japan. Symptoms on annual bluegrass caused by this fungus included regular to irregular yellow rings several centimeters to 1 m in diameter, typically at maximum daytime temperatures of 15 to 35°C. A total of 26 isolates were collected from diseased annual bluegrass. Twenty-two of these isolates were multinucleate, grew optimally at 25 to 30°C, and in culture formed irregular sclerotia approximately 2 to 5 mm in size that were white to orange and remained orange or turned brown to dark brown over a 28-day period. The remaining four isolates were characterized as being W. circinata var. zeae (Rhizoctonia zeae), which is a known pathogen of annual bluegrass in the United States. Sequence analysis of the internal transcribed spacer (ITS) regions of rDNA on a subset of isolates confirmed the identifications of W. circinata var. circinata (n = 8) and W. circinata var. zeae (n = 1) based on deposited sequences in GenBank. The identity of the remaining 14 isolates suspected to be W. circinata var. circinata was confirmed by HapII digestion of the amplified rDNA ITS region. Pathogenicity of four W. circinata var. circinata isolates was confirmed on both annual bluegrass and creeping bentgrass. This study is the first morphological and molecular confirmation of the presence of W. circinata var. circinata as a pathogen of turfgrass in the United States.

Occurrence and Distribution of QoI-Resistant Isolates of Colletotrichum cereale from Annual Bluegrass in California.

Turfgrass anthracnose, caused by Colletotrichum cereale (ex. Colletotrichum graminicola), is an important disease of turf used on golf course putting greens. Recent management of the disease has become increasingly difficult, partly due to the possible development of practical resistance to the QoI fungicides. In all, 558 single-conidia isolates of C. cereale were collected from 10 California golf courses, 8 of which had been exposed to QoI fungicides and 2 where no fungicides had been used. Isolates were tested using a mycelial expansion assay on azoxystrobinamended media. For the two nonexposed populations, in vitro 50% effective dose (ED50) values ranged from 0.0060 to 0.089 µg/ml. All isolates from the exposed populations could not be fully inhibited by doses of azoxystrobin as high as 8.0 µg/ml. A subset of these isolates were tested in vitro with the QoI fungicides pyraclostrobin and trifloxystrobin and found to be similar in response, indicating that these isolates were fully cross-resistant to all three fungicides. In greenhouse pot experiments, three isolates nonresponsive to QoI fungicides in vitro were not controlled by label rates of the fungicides. Spore germination assays also were examined; for 10 isolates identified as sensitive by mycelial expansion assays, ED50 values for axoystrobin ranged from 0.0040 to 0.0047 µg/ml; for 25 isolates identified as QoI-resistant, 93 to 100% of the conidia germinated at azoxystrobin concentrations as high as 8.0 µg/ml relative to the nonamended check treatments. Mitochondrial cytochrome b genes from a subset of 15 isolates (12 resistant and 3 sensitive) were partially cloned and sequenced; all resistant isolates had an alanine substitution that corresponded to position 143 of the gene product. These results indicate that QoI resistance is present in California populations of C. cereale and is contributing to the difficulty in controlling this disease.

Sensitivity Distributions of California Populations of Colletotrichum cereale to the DMI Fungicides Propiconazole, Myclobutanil, Tebuconazole, and Triadimefon.

The baseline sensitivity of a California population of Colletotrichum cereale (turfgrass anthracnose) to the sterol demethylation inhibitor (DMI) fungicide propiconazole was determined using an in vitro assay with known reproducibility. The 50% effective dose (ED50) values for propiconazole for a nonexposed, baseline population ranged from 0.025 to 0.35 µg/ml with a mean of 0.14 µg/ml. Examination of two DMI-exposed populations indicated an approximate increase of 6.5× in mean ED50 values. In vivo testing of two isolates with ED50 values of propiconazole at 0.15 and 0.90 µg/ml indicated reduced control for the less sensitive isolate by propiconazole at rates ≤38 µg/ml. It was determined that single discriminatory dose testing in vitro with propiconazole at 0.50 µg/ml could differentiate sensitive and resistant isolates. Using this dose, six additional populations were tested and DMI-exposed populations were found to be three to nine times less sensitive compared with the baseline population. Two populations were assayed for sensitivity to myclobutanil, tebuconazole, and triadimefon. Mean ED50 values for a nonexposed population were 0.72, 0.082, and 5.6 µg/ml, respectively; for a DMI-exposed population, mean ED50 values were 3.8, 0.35, and 18 µg/ml, respectively. This work provides information on the development of DMI resistance in populations of C. cereale in California and methodologies for future resistance monitoring for this pathogen.

Phylogenetic Relationships of Xylella fastidiosa Strains Isolated from Landscape Ornamentals in Southern California.

ABSTRACT Xylella fastidiosa is an insect-borne, xylem-limited pathogenic bacterium that has been associated with a rise in incidence of diseased landscape ornamentals in southern California. The objective of this study was to genetically characterize strains isolated from ornamental hosts to understand their distribution and identity. Strains of X. fastidiosa isolated from ornamentals were characterized using a multiprimer polymerase chain reaction (PCR) system, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR). Based on RAPD-PCR and 16S-23S rDNA ISR, strains isolated from daylily, jacaranda, and magnolia clustered with members of X. fastidiosa subsp. sandyi and caused oleander leaf scorch but not Pierce's disease symptoms in glasshouse assays on oleander and grape, respectively. This demonstrated both that our groupings based on genetic characterization were valid and that strains of X. fastidiosa subsp. sandyi are present in hosts other than oleander. Strains isolated from Spanish broom, cherry, and one strain isolated from western redbud clustered with X. fastidiosa subsp. fastidiosa members. Strains isolated from purple-leafed plum, olive, peach, plum, sweetgum, maidenhair tree, crape myrtle, and another western redbud strain clustered with members of X. fastidiosa subsp. multiplex. All strains isolated from mulberry and one from heavenly bamboo formed a separate cluster that has not yet been defined as a subspecies.

Sensitivity to Azoxystrobin Among Isolates of Uncinula necator: Baseline Distribution and Relationship to Myclobutanil Sensitivity.

Two hundred fifty-six single-conidial chain isolates of Uncinula necator were assayed for their sensitivity to azoxystrobin and myclobutanil. These isolates were collected from two sites in New York in 1999: an "organic" vineyard where no synthetic fungicides have been used (baseline population) and a commercial vineyard having a history of compromised powdery mildew control with myclobutanil (demethylation inhibitor [DMI]-resistant population). Mean coefficients of variance for a leaf disk assay used to test fungicide sensitivities were 31% for azoxystrobin and 41% for myclobutanil. Baseline ED50 values ranged from 0.0037 to 0.028 µg/ml (mean 0.0097µg/ml) for azoxystrobin and from 0.0049 to 0.69 µg/ml (mean 0.075 µg/ml) for myclobutanil. A shift in the mean ED50 value for azoxystrobin to 0.018 µg/ml was observed in the DMI-resistant population; with the strongest shift observed for isolates collected from vines treated exclusively with myclobutanil (0.024 µg/ml). For the 256 tested isolates, there was a moderate, but statistically significant, correlation between azoxystrobin and myclobutanil sensitivities (R2 = 0.36, P < 0.001). Tests with three other strobilurin fungicides (kresoxim-methyl, pyraclostrobin, and trifloxystrobin) indicate clear differences in the intrinsic activity of these compounds against U. necator, and the applicability of the methods developed with azoxystrobin for assays with pyraclostrobin and trifloxystrobin. Isolates from the high and low ends of the azoxystrobin sensitivity distribution (15× difference in mean ED50 values) were equally controlled in planta by protectant or postinfection treatment with azoxystrobin at 250 µg a.i./ml, but postinfection application at lower rates (2.5 and 25 µg a.i./ml) resulted in a 41 and 44% decrease, respectively, in the control of the low-sensitivity isolates versus high-sensitivity isolates. The results of this study document the baseline sensitivity distribution of U. necator to azoxystrobin, provide evidence of partial cross-sensitivity between azoxystrobin and myclobutanil, and illustrate the potential selection for individuals with reduced sensitivity (quantitative range) to azoxystrobin by postinfection application and reduced rates of this fungicide.

Comparative Physical Modes of Action of Azoxystrobin, Mancozeb, and Metalaxyl Against Plasmopara viticola (Grapevine Downy Mildew).

The physical modes of action of azoxystrobin, mancozeb, and metalaxyl were evaluated on grapevine seedlings using Plasmopara viticola as a model pathogen. The protectant, postinfection, postsymptom, translaminar, and vapor activities of azoxystrobin were evaluated at a rate of 250 µg/ml. Azoxystrobin provided 100% disease control when applied 1 to 5 days before inoculation. Postinfection applications of azoxystrobin had little effect on the incidence of disease, but colony area and sporulation from the resultant lesions was reduced by 47 and 96%, respectively, relative to the check treatment when applied up to 5 days after inoculation. Postsymptom applications (6 days after inoculation) of azoxystrobin resulted in an 85% mean reduction of resporulation from diseased tissue relative to the check when seedlings were evaluated 1 to 14 days after treatment. Translaminar activity was greatest when the upper surface of the leaf was treated 7 days before inoculation of the lower leaf surface (94% disease control). In contrast, control was <50% when leaves were similarly inoculated 1 and 3 days after treatment. Vapor activity was not pronounced, providing maximum reductions of 5, 11, and 37%, with regard to incidence, colony area, and sporulation, relative to the check when seedlings were treated 1 to 7 days before inoculating adjacent, untreated leaves. Comparatively, mancozeb (1,790 µg/ml) provided complete control of the disease when applied 1 to 5 days before inoculation, but showed little postinfection activity in reducing disease incidence, although it exhibited moderate to high antisporulant activity when applied in postinfection and postsymptom modes (mean reductions of 38 and 89%, respectively, compared with the check treatments). Metalaxyl (260 µg/ml) also provided complete control of the disease when used in protectant mode, and also when applied 1 day after inoculation. Applications at 3 to 5 days after inoculation provided substantial reductions in disease severity and sporulation (mean reductions of 46 and 94%, respectively, compared with the check treatments), and postsymptom applications resulted in a mean 84% reduction in resporulation. Collectively, the results of this study illustrate the unique physical modes of action for azoxystrobin in comparison to that of two traditional protectant and systemic fungicides, and provide information on how azoxystrobin and other strobilurin fungicides with similar physical modes of action should be best used in disease management programs.

Distribution of Baseline Sensitivities to Azoxystrobin Among Isolates of Plasmopara viticola.

An excised leaf disc assay was developed for determining the sensitivity of isolates of Plasmopara viticola (causal agent of grapevine downy mildew) to the strobilurin fungicide azoxystrobin. Five repeated assays with the same five single-sporangiophore isolates showed that the technique yielded reproducible results; that is, coefficients of variation ranged from 4.0 to 20% (mean 12%) for effective doses for 50% control (ED50 values) based on disease incidence and from 4.4 to 14% (mean 8.1%) for ED50 values based on disease severity. Then, the assay was applied to 81 single-sporangiophore isolates of P. viticola collected from 10 geographically distinct vineyards in western New York, providing a baseline distribution of sensitivities within this population. For disease incidence, individual ED50 values ranged from 0.05 to 0.94 µg/ml (mean 0.40 µg/ml), whereas for disease severity they ranged from 0.04 to 0.78 µg/ml (mean 0.24 µg/ml). When 61 of these isolates were similarly tested at a single discriminatory dose of 0.50 µg/ml, azoxystrobin provided 61% control of disease incidence versus 41 and 1.1% control for trifloxystrobin and kresoximmethyl, respectively; for disease severity (colony diameter), azoxystrobin provided 80% control versus 57 and 1.1% control for trifloxystrobin and kresoximmethyl, respectively. These results provide information that can be utilized in future monitoring of P. viticola resistance to azoxystrobin and indicate differences in the intrinsic activities of the three strobilurin fungicides against this pathogen.