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Rusts are destructive fungal diseases that can cause severe thinning and unattractive discoloration of kentucky bluegrass (KBG; Poa pratensis L.). Currently, turfgrass breeding programs rely on field evaluations to screen KBG germplasm for rust resistance; methods that are expensive, labor intensive, and require large turf areas. The availability of a greenhouse-based assay to perform prescreening of KBG germplasm for resistance to rust diseases before field trials would allow breeders to remove the poorest performing plants before field evaluations thus enhancing breeding efficiency. In this study, we set out to develop a reliable, low-cost greenhouse inoculation protocol for the two most common rust pathogens of KBG in temperate growing regions: Puccinia coronata and Puccinia graminis, the causal agents of crown and stem rust, respectively. Using a modified inoculation assay and custom-built plexi-glass chambers adapted from protocols used for cereal rust pathogens, urediniospores of crown and stem rust fungi developed on inoculated plants 10 to 14 days postinoculation. Real-time polymerase chain reaction (PCR) assays, disease symptomology, and morphology of urediniospores confirmed the presence and identity of both rust pathogens from inoculated host tissue. The inoculation protocols described here represent an effective method to accelerate screening of KBG germplasm for resistance to crown and stem rust diseases. Infection of KBG plants in the greenhouse will also allow breeders to maintain populations of crown and stem rust fungi throughout the year, providing a reliable and ongoing source of pathogen inoculum for experimentation and screening in the future. Rust (Puccinia sp.) is a common fungal disease of kentucky bluegrass (KBG; Poa pratensis L.), one of the most commonly used cool-season turfgrasses on athletic fields, recreation areas, sod farms, and residential lawns. Diseased turf typically exhibits an unsightly orange/brown coloration, and severe infections can result in extensive turf loss in susceptible KBG stands (Bonos et al., 2006). In the turfgrass seed production industry, yield loss is not uncommon during rust epidemics, with stem rust reportedly reducing perennial ryegrass seed yields by asmuch as 98% (Pfender, 2009a). In recent years, turfgrass breeders have observed a shift in susceptibility to rust disease (reported as stem rust) among once highly tolerant KBG cultivars. This suggests the possibility that new physiological races may be responsible for the observed shift in susceptibility to rust in KBG (Bonos et al., 2006). Although fungicides can be used to control the disease, multiple applications are often required, resulting in increased costs (Pfender, 2001a). Thus, the preferred method of control on landscape turf is through the use of resistant cultivars. Efforts to introduce rust resistance into KBG have been undertaken since the early 1950s, when it became apparent that leaf spot–resistant cultivars such as Merion were highly susceptible to stem rust disease caused by the fungus P. graminis (Ray, 1953). It is now common practice for turfgrass breeding programs to evaluate new sources of germplasm for susceptibility to stem rust disease in the field (Bonos et al., 2006). However, advances in the development of rust-resistant turfgrass have been hindered by the inability to evaluate turfgrass cultivars in the greenhouse or growth chambers under controlled environmental conditions. At present, turfgrass breeders are reliant on natural field infections to evaluate their germplasm. This can be a complicated and unreliable process, as multiple rust species can infect KBG (Beirn et al., 2011) and environmental conditions do not always favor uniform disease development (Welty and Barker, 1993) making selections for resistant cultivars difficult. In addition, fundamental knowledge of the biology and infectivity of the causal agents of rust diseases is lacking, thus slowing progress in the development of resistance to multiple fungal races or strains. Two primary factors have contributed to the limited advancements in the control and study of turfgrass rust fungi: 1) the biotrophic lifestyle of the fungal rust pathogens precludes the establishment of axenic cultures and production of pathogen inoculum for experiments, and 2) previous inoculation protocols for evaluating plants in the growth chamber or greenhouse have been difficult to reproduce. The obligate nature of rust fungi makes propagation in culture extremely difficult or impossible in the absence of the host plant (Agrios, 2005). Attempts to grow graminicolous rust fungi in culture in the laboratory havemet with varying levels of success, and have frequently produced nonrepeatable results (Huang et al., 1990; Williams et al., 1966, 1967). Detached leaf assays have been used to maintain cultures of the crown rust fungus (P. coronata) from oats (Avena sativa L.); however, it remains to be determined if this technique can be used to screen healthy oat plants for resistance to rust due to the confounding physiological processes associated with leaf senescence (Jackson et al., 2008). The complexities surrounding axenic culturing of graminicolous rust fungi has been overcome in the cereal rust system, where inoculation of cool-season cereal crops, including wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and oats, is now a routine procedure that has been performed to screen germplasm for several decades (Jackson et al., 2008). In the cereal rust system, the procedure entails: 1) spraying a mineral oil suspension of viable urediniospores onto susceptible plants, 2) placing plants in a dew chamber overnight to incubate, 3) moving inoculated plants the following day to a greenhouse where urediniospores are allowed to infect plant tissue, a process that takes 10 d, and 4) selecting a single uredinium to mature once pustules erupt (Carson, 2008). Ultimately, spores from the single pustule are harvested and used to inoculate healthy plant material, thus increasing the inoculum of a single rust isolate (Carson, 2008). This method ensures pathogen homogeneity and produces copious amounts of genetically identical spores that can then be used for advanced molecular analyses or to screen germplasm. A similar procedure has been effectively adapted to other grass systems, such as in Received for publication 15 May 2015. Accepted for publication 2 Sept. 2015. Funding for this research was provided by the Rutgers Center for Turfgrass Science, the U.S. Department of Agriculture (USDA), and the USDA Agricultural Research Service. We gratefully acknowledge Mark Peacos for assistance building the misting chamber and Les Szabo, Matthew Rouse, Jerry Johnson, and Samuel Stoxen for graciously hosting Lisa Beirn and demonstrating inoculation protocols used in cereal crops at the USDA-ARS Cereal Disease Laboratory. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. Corresponding author. E-mail: JoAnne.Crouch@

A Greenhouse-based Inoculation Protocol for Fungi Causing Crown Rust and Stem Rust Diseases of Kentucky Bluegrass Turf