Detection and phylogenetic analysis of mating type genes ofOphiosphaerella korrae

Portions of the mating type genes from Ophiosphaerella korrae (J. Walker & A.M. Smith) R.A. Shoemaker (=Leptosphaeria korrae J. Walker & A.M. Smith), a pathogenic fungus of grasses, were examined by PCR (polymerase chain reaction). For nine isolates of O. korrae from North America, both mating type genes were amplified, demonstrating that both MAT idiomorphs are detectable in this homothallic ascomycete. Amplified fragments from three isolates were sequenced, and parsimony analyses of MAT1 nucleotide and protein sequences placed O. korrae in the basal position of a clade of Phaeosphaeriaceae and Pleosporaceae, whereas the MAT2 nucleotide and protein data placed O. korrae in a clade with Pleosporaceae. The internal transcribed spacer (ITS) and 18S ribosomal DNA of O. korrae were also sequenced. The 18S sequences had insufficient variability to resolve the placement of O. korrae, whereas the ITS data placed it in Phaeosphaeriaceae. A total evidence analysis of Dothideomycetes with 18S, ITS, and MAT data placed O. korrae alongside Phaeosphaeria species, with moderate bootstrap support. However, the Kishino–Hasegawa test did not demonstrate this topology to be significantly different from one where O. korrae was placed with Pleosporales. Although O. korrae does not belong in Leptosphaeria based on ITS data, MAT data do not strongly support its placement in Phaeosphaeriaceae.Key words: ascomycetes, mating type genes, ribosomal genes, taxonomy.

Impact of Temperature, Osmotic Potential, and Osmoregulant on the Growth of Three Ectotrophic Root-Infecting Fungi of Kentucky Bluegrass

Two isolates each of Magnaporthe poae, Gaeumannomyces incrustans, and Leptosphaeria korrae were grown at 25°C in liquid shake culture in minimal salts medium (-0.12 MPa) or minimal salts medium adjusted to -0.5 MPa with KCl, MgCl2, or polyethylene glycol (PEG). Fungal dry weight of all three species was greater in minimal salts medium amended to -0.5 MPa with MgCl2 than in nonamended medium, and dry weight in medium amended with PEG was not different from dry weights in nonamended medium or medium amended with KCl. Fungi were incubated at varying temperatures on a minimal salts solid-agar medium (-0.12 MPa) adjusted to osmotic potentials ranging from -0.5 to -5.0 MPa with KCl or MgCl2. Optimum growth of M. poae, G. incrustans, and L. korrae on nonamended medium occurred at 30, 30, and 25°C, respectively. At optimum temperatures for each species, fungal growth was greatest at the higher osmotic potentials tested (-0.5 to -1.0 MPa) and decreased in a linear manner as osmotic potential decreased. In most cases, growth was detected at the lowest osmotic potential measured (-5.0 MPa). The relationship of fungal growth to osmotic potential depended on both temperature and osmoregulant. At temperatures optimal or nearly optimal for fungal development, the growth of all three fungi declined more rapidly with decreasing osmotic potential when grown on medium amended with MgCl2 than on medium amended with KCl. At the highest temperature evaluated for growth of M. poae and L. korrae (35 and 30°C, respectively), growth on medium amended with KCl was curvilinear and peaked at osmotic potentials of -2.5 to -3.0 MPa. Furthermore, between osmotic potentials of -2.0 and -5.0 MPa, M. poae grew best at 35°C. When maintained on nonamended minimal salts medium (-0.12 MPa) in liquid culture at 25°C or on nonamended solid-agar medium at temperatures optimal for growth, M. poae grew at a faster daily rate than L. korrae.

Irrigation Frequency and Fertilizer Type Influence Necrotic Ring Spot of Kentucky Bluegrass

Organic and synthetic fertilizers were evaluated under three irrigation regimes (daily, twice weekly, and rain only) for management of necrotic ring spot (Leptosphaeria korrae Walker & Smith) of Kentucky bluegrass (Poa pratensis L.). Disease severity varied due to fertilizer and irrigation treatments. After 2 years of treatments, daily irrigation reduced disease incidence as compared to twice-weekly irrigation, while organic fertilizer, as a feathermeal-bonemeal-soybeanmeal mixture, when applied monthly at 48.8 kg N/ha, reduced disease incidence as compared to urea fertilizer. For 2 of 3 years, differences in disease incidence were attributable to irrigation treatments, and after 4 years, all N fertilizers reduced disease incidence as compared to no fertilizer.

Detection of Leptosphaeria korrae with the polymerase chain reaction and primers from the ribosomal internal transcribed spacers

Leptosphaeria korrae, the causal agent of necrotic ring spot, is a destructive patch disease of Kentucky bluegrass. To develop a rapid molecular test for the detection of this pathogen, an assay based on the polymerase chain reaction (PCR) was developed utilizing the internal transcribed spacer region 1 (ITS 1) of L. korrae ribosomal DNA. DNA sequence comparison showed 94.8% similarity of the ITS 1 region among L. korrae isolates and only 45–50% similarity between L. korrae and other fungal species. Based on ITS 1 sequence differences, a pair of oligonucleotide primers, LK17S and 5.8SC, were selected. With PCR, the primers specifically amplified L. korrae DNA and did not amplify DNA isolated from 15 other fungal species or healthy Kentucky bluegrass. The assay could also specifically detect L. korrae in diseased turfgrass samples. Key words: detection, ITS 1, Leptosphaeria korrae, necrotic ring spot, polymerase chain reaction, ribosomal DNA.

Fungi Associated with Spring Dead Spot Reduces Freezing Resistance in Bermudagrass

Although the effect of cold winters on the severity of bermudagrass (Cynodon dactylon Pers.) spring dead spot (SDS) has been studied, information is needed concerning the effect of infection by fungi associated with SDS on the host's freezing resistance. A-22 bermudagrass was inoculated with Leptosphaeria korrae J. Walker & A.M. Smith and Ophiospharella herpotricha (Fr.) J. Walker & A.M Smith. Differential thermal analysis was used to monitor exotherm temperatures of healthy and O. herpotricha- and L. korrae-infected A-22 bermudagrass at 10-day intervals during 90 days of acclimation in cold chambers. Healthy bermudagrass crowns supercooled to an average of -6.7C and fungi-infected crowns supercooled to an average of -4.8 and -4.4C, respectively. Healthy crown exotherm temperatures were significantly lower than those of fungi-infected bermudagrass crowns on all nine sampling dates. This result indicates that fungi-infected plants are more susceptible to cold damage.