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Sand topdressing applied to annual bluegrass [Poa annua L. f. reptans (Hausskn.) T. Koyama] putting greens during the spring and summer can reduce anthracnose caused by Colletotrichum cereale Manns sensu lato Crouch, Clarke, and Hillman. However, the effects of autumn topdressing on this disease and the annual quantity of sand required to suppress anthracnose are not known. A 3-yr field study was initiated in 2010 to evaluate a 3 × 3 × 3 factorial of autumn, spring, and summer topdressing on anthracnose severity of annual bluegrass maintained at 2.8 mm. Autumn and spring topdressing was applied at 0, 1.2, or 2.4 L m–² yr–¹ and 0, 0.6, or 1.2 L m–² yr–¹ of sand was applied during summer. Linear and Cate-Nelson models were used to evaluate the response of anthracnose to annual topdressing quantity. Spring topdressing caused the greatest reduction in disease. Autumn topdressing reduced disease early each year following application, while spring topdressing provided consistent disease suppression throughout the growing season. Summer topdressing rates, similar to those applied on golf courses, reduced disease only in 2012 (mid- to late summer). Additionally, modeling the disease response against annual topdressing quantity indicated that increasing topdressing up to 6.0 L m–² yr–¹ produced a linear reduction of anthracnose on annual bluegrass. Golf turf managers should recognize that autumn topdressing will aid in anthracnose suppression; however, spring applications are more effective when anthracnose is the management objective.

Strong creeping red fescue (Festuca rubra subsp. rubra) is a commercially important low-maintenance turfgrass and is often naturally infected with the fungal endophyte Epichlo&euml, festucae. Epichlo&euml, spp. are endophytes of several cool-season grass species, often conferring insect resistance to the grass hosts due to the production of toxic alkaloids. In addition to insect resistance, a unique feature of the strong creeping red fescue/E. festucae symbiosis is the endophyte-mediated disease resistance to the fungal pathogen Clarireedia jacksonii, the causal agent of dollar spot disease. Such disease resistance is not a general feature of other grass/ Epichlo&euml, interactions. E. festucae isolates infecting red fescue have an antifungal protein gene Efe-afpA, whereas most other Epichlo&euml, spp. do not have a similar gene. The uniqueness of this gene suggests it may, therefore, be a component of the unique disease resistance seen in endophyte-infected red fescue. Here, we report the generation of CRISPR-Cas9 Efe-afpA gene knockouts with the goal of determining if absence of the protein in endophyte-infected Festuca rubra leads to disease susceptibility. However, it was not possible to infect plants with the knockout isolates, although infection was possible with the wild type E. festucae and with complemented isolates. This raises the interesting possibility that, in addition to having antifungal activity, the protein is required for the symbiotic interaction. The antifungal protein is a small secreted protein with high expression in planta relative to its expression in culture, all characteristics consistent with effector proteins. If Efe-AfpA is an effector protein it must be specific to certain interactions, since most Epichlo&euml, spp. do not have such a gene in their genomes.

Many cool-season grasses have symbiotic relationships with Epichlo&euml, (Ascomycota, Clavicipitaceae) fungal endophytes that inhabit the intercellular spaces of the above-ground parts of the host plants. The presence of the Epichlo&euml, endophytes is generally beneficial to the hosts due to enhanced tolerance to biotic and abiotic stresses conferred by the endophytes. Many Epichlo&euml, spp. are asexual, and those infections always remain asymptomatic. However, some Epichlo&euml, spp. have a sexual stage and produce a macroscopic fruiting body, a stroma, that envelops the developing inflorescence causing a syndrome termed &ldquo, choke disease&rdquo, Here, we report a fungal and plant gene expression analysis of choke stroma tissue and asymptomatic inflorescence tissue of Epichlo&euml, festucae-infected strong creeping red fescue (Festuca rubra subsp. rubra). Hundreds of fungal genes and over 10% of the plant genes were differentially expressed when comparing the two tissue types. The differentially expressed fungal genes in the choke stroma tissue indicated a change in carbohydrate and lipid metabolism, as well as a change in expression of numerous genes for candidate effector proteins. Plant stress-related genes were up-regulated in the stroma tissue, suggesting the plant host was responding to the epiphytic stage of E. festucae as a pathogen.

Epichloë spp. are naturally occurring fungal endophytic symbionts of many cool-season grasses. Infection by the fungal endophytes often confers biotic and abiotic stress tolerance to their hosts. Endophyte-mediated disease resistance is well-established in the fine fescue grass Festuca rubra subsp. rubra (strong creeping red fescue) infected with E. festucae. Resistance to fungal pathogens is not an established effect of endophyte infection of other grass species, and may therefore be unique to the fine fescues. The underlying mechanism of the disease resistance is unknown. E. festucae produces a secreted antifungal protein that is highly expressed in the infected plant tissues and may therefore be involved in the disease resistance. Most Epichloë spp. do not have a gene for a similar antifungal protein. Here we report the characterization of the E. festucae antifungal protein, designated Efe-AfpA. The antifungal protein partially purified from the apoplastic proteins of endophyte-infected plant tissue and the recombinant protein expressed in the yeast Pichia pastoris was found to have activity against the important plant pathogen Sclerotinia homoeocarpa. Efe-AfpA may therefore be a component of the disease resistance seen in endophyte-infected strong creeping red fescue.

Objective: To report the decreased IVF rate of oocytes and the reduced cleavage rate of pronuclear stage embryos after gas has backflowed from a gas analyzer into an incubator during the calibration of the CO 2 concentration. Design: Case report. Setting: Clinical research unit for reproductive medicine in a hospital. Intervention(s): Backflow of gas from a gas analyzer into an incubator during calibration of the CO 2 concentration. Main Outcome Measure: The IVF rate of oocytes and the cleavage rate of pronuclear stage embryos. Result(s): Thirty-two oocytes were retrieved from four infertile female patients. Nine oocytes were fertilized and four fertilized oocytes were cleaved. In a related animal experiment, the cleavage rate was 57.1% (36/63) in the control group and 25.4% (16/63) in the study group ( P = .00026). Conclusion(s): The backflow of gas from the gas analyzer adversely affected the fertilization of oocytes and the cleavage of pronuclear stage embryos.

The fungus Colletotrichum cereale incites anthracnose disease on Poa annua (annual bluegrass) turfgrass. Anthracnose disease is geographically widespread throughout the world and highly destructive to cool-season turfgrasses, with infections by C. cereale resulting in extensive turf loss. Comprehensive research aimed at controlling turfgrass anthracnose has been performed in the field, but knowledge of the causal organism and its basic biology is still needed. In particular, the lack of a reliable greenhouse-based inoculation protocol performed under controlled environmental conditions is an obstacle to the study of C. cereale and anthracnose disease. Our objective was to develop a consistent and reproducible inoculation protocol for the two major genetic lineages of C. cereale. By adapting previously successful field-based protocols and combining with components of existing inoculation procedures, the method we developed consistently produced C. cereale infection on two susceptible P. annua biotypes. Approximately 7 to 10 days post-inoculation, plants exhibited chlorosis and thinning consistent with anthracnose disease symptomology. Morphological inspection of inoculated plants revealed visual signs of the fungus (appressoria and acervuli), although acervuli were not always present. After stringent surface sterilization of inoculated host tissue, C. cereale was consistently re-isolated from symptomatic tissue. Real-time PCR detection analysis based on the Apn2 marker confirmed the presence of the pathogen in host tissue, with both lineages of C. cereale detected from all inoculated plants. When a humidifier was not used, no infection developed for any biotypes or fungal isolates tested. The inoculation protocol described here marks significant progress for in planta studies of C. cereale, and will enable scientifically reproducible investigations of the biology, infectivity and lifestyle of this important grass pathogen.