Lane P. Tredway

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 April 2010-31 May 2010

This article documents the addition of 396 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anthocidaris crassispina, Aphis glycines, Argyrosomus regius, Astrocaryum sciophilum, Dasypus novemcinctus, Delomys sublineatus, Dermatemys mawii, Fundulus heteroclitus, Homalaspis plana, Jumellea rossii, Khaya senegalensis, Mugil cephalus, Neoceratitis cyanescens, Phalacrocorax aristotelis, Phytophthora infestans, Piper cordulatum, Pterocarpus indicus, Rana dalmatina, Rosa pulverulenta, Saxifraga oppositifolia, Scomber colias, Semecarpus kathalekanensis, Stichopus monotuberculatus, Striga hermonthica, Tarentola boettgeri and Thermophis baileyi. These loci were cross‐tested on the following species: Aphis gossypii, Sooretamys angouya, Euryoryzomys russatus, Fundulus notatus, Fundulus olivaceus, Fundulus catenatus, Fundulus majalis, Jumellea fragrans, Jumellea triquetra Jumellea recta, Jumellea stenophylla, Liza richardsonii, Piper marginatum, Piper aequale, Piper darienensis, Piper dilatatum, Rana temporaria, Rana iberica, Rana pyrenaica, Semecarpus anacardium, Semecarpus auriculata, Semecarpus travancorica, Spondias acuminata, Holigarna grahamii, Holigarna beddomii, Mangifera indica, Anacardium occidentale, Tarentola delalandii, Tarentola caboverdianus and Thermophis zhaoermii.

Phylogenetic and population genetic divergence correspond with habitat for the pathogen Colletotrichum cereale and allied taxa across diverse grass communities.

Over the past decade, the emergence of anthracnose disease has newly challenged the health of turfgrasses on North American golf courses, resulting in considerable economic loss. The fungus responsible for the outbreaks, Colletotrichum cereale, has also been identified from numerous natural grasses and cereal crops, although disease symptoms are generally absent. Here we utilize phylogenetic and population genetic analyses to determine the role of ecosystem in the advancement of turfgrass anthracnose and assess whether natural grass and/or cereal inhabitants are implicated in the epidemics. Using a four‐gene nucleotide data set to diagnose the limits of phylogenetic species and population boundaries, we find that the graminicolous Colletotrichum diverged from a common ancestor into distinct lineages correspondent with host physiology (C3 or C4 photosynthetic pathways). In the C4 lineage, which includes the important cereal pathogens Colletotrichum graminicola, C. sublineolum, C. falcatum, C. eleusines, C. caudatum and several novel species, host specialization predominates, with host‐associated lineages corresponding to isolated sibling species. Although the C3 lineage —C. cereale— is comprised of one wide host‐range species, it is divided into 10 highly specialized populations corresponding to ecosystem and/or host plant, along with a single generalist population spread across multiple habitat types. Extreme differentiation between the specialized C. cereale populations suggests that asymptomatic nonturfgrass hosts are unlikely reservoirs of infectious disease propagules, but gene flow between the generalist population and the specialized genotypes provides an indirect mechanism for genetic exchange between otherwise isolated populations and ecosystems.

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.

Mating Type Distribution and Fertility Status in Magnaporthe grisea Populations from Turfgrasses in Georgia

Populations of Magnaporthe grisea associated with tall fescue and St. Augustinegrass in Georgia were analyzed for mating type distribution and fertility status in 1999 and 2000. A polymerase chain reaction based assay for mating type was developed to facilitate population analysis. M. grisea populations from St. Augustinegrass in Georgia were dominated by the Mat1-1 mating type, whereas populations from tall fescue were dominated by Mat1-2. The opposite mating type was found in low frequency (0 to 5.7%) associated with each host. The fertility status of isolates from two populations was determined using controlled crosses in vitro. Seventy-eight Mat1-1 isolates from St. Augustinegrass were sterile in test crosses, but a single Mat1-2 isolate from St. Augustinegrass was male fertile. Of 87 Mat1-2 isolates from tall fescue, 47 were male fertile in test crosses, but 19 produced perithecia that were barren. All Mat1-1 isolates from tall fescue were sterile. Although both mating types exist in M. grisea populations from turfgrasses in Georgia, no female fertile isolates were identified in sample populations. The predominance of one mating type in eight sample populations and absence of female fertile isolates in two sample populations indicates that sexual reproduction may not occur with significant frequency in M. grisea populations associated with turfgrasses in Georgia.

Genetic Structure of Magnaporthe grisea Populations Associated with St. Augustinegrass and Tall Fescue in Georgia.

ABSTRACT Amplified fragment length polymorphisms (AFLPs) were used to estimate phylogenetic relationships within Magnaporthe grisea and determine the genetic structure of M. grisea populations associated with tall fescue and St. Augustinegrass in Georgia. Sixteen clonal lineages were identified in a sample population of 948 isolates. Five lineages were isolated from tall fescue (E, G1, G2, G4, and H), with lineage G4 comprising 90% of the population. Isolates from tall fescue were closely related to those from perennial ryegrass, weeping lovegrass, and wheat. Two M. grisea lineages were isolated from St. Augustinegrass (C and K), with lineage C comprising 99.8% of the population. Populations from crabgrass were dominated (98%) by lineage K, but also contained a single lineage C isolate. Haplotype diversity indices ranged from 0.00 to 0.29 in tall fescue populations and from 0.00 to 0.04 in St. Augustinegrass populations. Selection due to host species was the primary factor determining population structure according to analysis of molecular variance; host cultivar and geographical region had no significant effect. The host range of M. grisea lineages from turfgrasses was determined in growth chamber experiments and supports the prominent role of host species in determining the genetic structure of M. grisea populations from turfgrasses in Georgia.

Occurrence and Molecular Identification of Azoxystrobin-Resistant Colletotrichum cereale Isolates from Golf Course Putting Greens in the Southern United States

Turfgrass anthracnose, caused by Colletotrichum cereale (≡C. graminicola), has become a common disease of creeping bentgrass and annual bluegrass putting greens throughout the southern United States. Strobilurin (QoI) fungicides such as azoxystrobin are single-site mode-of-action fungicides applied to control C. cereale. In vitro bioassays with azoxystrobin at 0.031 and 8 μg/ml incorporated into agar were performed to evaluate the sensitivity of 175 isolates collected from symptomatic turfgrasses in Alabama, Mississippi, North Carolina, Tennessee, and Virginia. Three sensitivity levels were identified among C. cereale isolates. Resistant, intermediately resistant, and sensitive isolates were characterized by percent relative growth based on the controls with means of 81, 23, and 4%, respectively, on media containing azoxystrobin at 8 μg/ml. The molecular mechanism of resistance was determined by comparing amino acid sequences of the cytochrome b protein. Compared with sensitive isolates, C. cereale isolates exhibiting QoI resistance had a G143A substitution, whereas isolates expressing intermediate resistance had a F129L substitution. C. cereale isolates displaying azoxystrobin resistance in vitro were not controlled by QoI fungicides in a field evaluation. The dominance of QoI-resistant C. cereale isolates identified in this study indicates a shift to resistant populations on highly managed golf course putting greens.

Aggressiveness of Typhula ishikariensis Isolates to Cultivars of Bentgrass Species (Agrostis spp.) Under Controlled Environment Conditions

Speckled snow mold, caused by Typhula ishikariensis, is one of the most important Typhula snow molds in subarctic zones of the Northern Hemisphere. Nine isolates of three T. ishikariensis varieties (var. ishikariensis, var. canadensis, and var. idahoensis) isolated from infected turfgrasses on golf course fairways throughout Wisconsin were evaluated for their aggressiveness toward nine cultivars of three bentgrass species (three creeping, three colonial, and three velvet cultivars) under controlled environmental conditions. Speckled snow mold severity increased as inoculum concentration of T. ishikariensis was increased. In general, bentgrass susceptibility increased between 9 and 11 weeks after seeding but gradually decreased thereafter, suggesting expression of age-related resistance as plants matured. Significant differences in aggressiveness were detected within and among T. ishikariensis varieties. Significant interactions between T. ishikariensis varieties or isolates and bentgrass species were detected, but there was no interaction between pathogen isolates and bentgrass cultivars. Disease severity evaluations showed significant differences among bentgrass cultivars and species in their response to T. ishikariensis. Since bentgrass species exhibit differential responses to T. ishikariensis varieties, representative isolates of each variety should be employed for screening of bentgrass germplasm for resistance to speckled snow mold.

First Report of Xanthomonas translucens Causing Etiolation on Creeping Bentgrass Turf in Illinois, Kentucky, and North Carolina

Symptoms of etiolation, which is an abnormal elongation and yellowing of tillers, have been observed on creeping bentgrass [Agrostis stolonifera L. (CBG)] putting greens for decades; however, symptoms are typically transient and non-problematic. Reports of etiolation have become more frequent recently and research supports the involvement of bacteria (1). During stressful summer periods in 2011 and 2012, 62 CBG putting green samples were submitted to the NCSU Turf Clinic exhibiting symptoms of etiolation, chlorosis, and/or general decline. Microscopic examination of stem and leaf tissue often showed bacterial streaming from the xylem tissue. Symptomatic tissue was surface disinfested in sodium hypochlorite (10% Clorox) for 5 min, blotted dry, and rinsed in sterile dH2O. Disinfested tissue was placed in a small drop of sterile dH2O on a glass microscope slide and cut to allow bacteria to stream into the water for 2 min. The resulting bacterial suspension was streaked onto three nutrient agar (NA) plates and incubated at 30°C overnight. Bacterial colonies varied in morphology and those present in the greatest number based on morphology were re-streaked to isolate individual colonies. Bacterial isolates were tentatively identified to species using rDNA sequencing of 16S and ITS regions (3). Sequencing results showed isolates obtained from 6 locations (in Illinois, Kentucky, and North Carolina) having a positive match (≥99% 16S and ≥93% ITS) to Xanthomonas translucens (GenBank accessions AY572961, HM181927, JX976312, AY253329, and AB680445). Additional research is needed to confirm pathovar designation as X. translucens isolates were similar to both poae and graminis pathovars. A representative isolate (LW10-12A) was also examined for carbon source utilization using the BIOLOG 3rd Gen Microplate (Biolog Inc., Hayward, CA) resulting in a positive identification of X. translucens. Isolate LW10-12A was used to inoculate 6-week-old seeded creeping bentgrass cv. A1 plants maintained at 1 cm height in 3.5 cm diameter containers. Scissors were dipped in a cell suspension (~109 CFU ml-1 in sterile dH2O) and used to cut healthy CBG plants at 1 cm height and the remaining suspension was applied to the foliage until runoff using an atomizer bottle. Non-inoculated plants were cut and misted using sterile dH2O. After inoculation, plants were placed in a sealed clear plastic Camwear container (Cambro Co., Huntington Beach, CA) for 48 h and then transferred to the growth chamber bench (30°C) receiving irrigation twice daily with dH2O. Etiolation was rated within each of the four replicates by counting the number of etiolated leaves that were easily observed as significantly higher than the rest of the turf canopy. Plants inoculated with X. translucens exhibited etiolation of the youngest leaf within 48 h, whereas the non-inoculated plants did not. Symptoms were similar to observations in the field, as etiolated leaves were chlorotic and easily extracted from the turf surface. Microscopic examination showed bacterial streaming and identification of bacteria, using the previously described methods, was positive for X. translucens. Etiolation symptoms persisted over multiple weeks, but a decline in turf quality was not observed. Etiolation has been previously suggested as a precursor to bacterial wilt, caused by X. translucens pv. poae, on annual bluegrass [Poa annua L. f. reptans (Hausskn) T. Koyama] (2) and Acidovorax avenae has also been shown to produce etiolation on CBG (1). To our knowledge, this is the first confirmation of X. translucens as a cause of etiolation in CBG. References: (1) P. R. Giordano et al. Plant Dis. 96:1736, 2012. (2) N. A. Mitkowski et al. Plant Dis. 89:469, 2005. (3) N. W. Schaad et al. Lab. Guide for Ident. of Plant Path Bac., 2001.

Characterization and distribution of mating-type genes of the turfgrass pathogen Sclerotinia homoeocarpa on a global scale.

Sclerotinia homoeocarpa F.T. Bennett is a filamentous member of Ascomycota that causes dollar spot, the most economically important disease of turfgrass worldwide. We sequenced and characterized the mating-type (MAT) locus of four recently-collected contemporary strains causing dollar spot, four historical type strains used to describe the fungus, and three species of Rutstroemiaceae. Moreover, we developed a multiplex PCR assay to screen 1019 contemporary isolates for mating-type. The organization of the MAT loci of all strains examined could be classified into one of four categories: (1) putatively heterothallic, as exemplified by all contemporary strains and three of four historical type strains; (2) putatively heterothallic with a deleted putative gene in the MAT1-2 idiomorph, as detected in strains from two recently-collected populations in the United Kingdom that show more similarity to historical strains; (3) putatively homothallic with close physical linkage between MAT1-1-1 and MAT1-2-1, as found in one historical type strain of S. homoeocarpa and two strains of Rutstroemia cuniculi; and (4) an unresolved but apparently homothallic organization in which strains contained both MAT1-1-1 and MAT1-2-1 but linkage between these genes and between the two flanking genes could not be confirmed, as identified in R. paludosa and Poculum henningsianum. In contemporary S. homoeocarpa populations there was no significant difference in the frequency of the two mating types in clone-corrected samples when analyzed on regional and local scales, suggesting sex may be possible in this pathogen. However, two isolates from Italy and twenty from California were heterokaryotic for both complete heterothallic MAT idiomorphs. Results from this study contribute to knowledge about mating systems in filamentous fungi and enhance our understanding of the evolution and biology of an important plant pathogen.

Induced overexpression of cytochrome P450 sterol 14α-demethylase gene (CYP51) correlates with sensitivity to demethylation inhibitors (DMIs) in Sclerotinia homoeocarpa.

BACKGROUND The fungus Sclerotinia homoeocarpa causes dollar spot, the most important turfgrass disease worldwide. Demethylation inhibitor (DMI) fungicides have been relied upon heavily to manage this disease. Presently, populations of S. homoeocarpa with reduced sensitivity or resistance to DMIs are widespread in the United States. RESULTS Cytochrome P450 sterol 14α-demethylase (ShCYP51) and its flanking regions were identified and sequenced in 29 isolates of S. homoeocarpa with a range of DMI sensitivities. No modifications were found in the gene coding and upstream regions that were consistently related to DMI sensitivity. In the absence of propiconazole, ShCYP51 was expressed at a similar low level among DMI baseline and resistant isolates. In the presence of propiconazole, DMI-resistant isolates were induced to express ShCYP51 at significantly higher levels than baseline isolates by propiconazole at 5 mg L(-1) for 5 h or at 0.5 mg L(-1) for 72 h. The ShCYP51 expression level after 72 h exposure to 0.5 mg L(-1) of propiconazole was linearly related to EC50 values and ΔRG (the change in relative growth rate over time), with R(2) values equal to 83.7 and 90.0% respectively. CONCLUSION Induced overexpression of ShCYP51 in resistant isolates following DMI exposure is an important factor determining DMI sensitivity in S. homoeocarpa.