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 as much 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 have met 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 Italian ryegrass (Lolium multiflorum Lam.), where inoculations of the crown rust pathogen were used to study resistance markers in the host plant (Studer et al., 2007). Likewise, the successful inoculation of KBG, perennial ryegrass (Lolium perenne L.), and tall fescue (Festuca arundinacea Schreb.) has also been previously described for the stem rust fungus (Pfender, 2001b, 2003, 2009b); however, the reported protocols have not been successfully used to inoculate turfgrass with multiple rust species simultaneously. In addition to stem rust, at least one additional rust disease, crown rust, is known to occur on KBG in temperate regions (Beirn et al., 2011). Resistance to this disease is now an important target of perennial ryegrass breeding programs (Bonos et al., 2006). At present, there remains no universal inoculation protocol for both crown and stem rust diseases on KBG, although unsuccessful attempts have been made by several laboratories (S.A. Bonos, personal communication). The development of an effective inoculation protocol for crown and stem rust fungi would allow breeders to screen large numbers of KBG selections and cultivars in the greenhouse for resistance to rust species/races in a convenient, reproducible manner before evaluating germplasm in the field, thus reducing reliance on inconsistent natural infection. It would also allow researchers to genetically purify rust strains for pathogen analysis at the molecular level. Therefore, our objective in this study was to develop a reliable inoculation protocol for turfgrass rust fungi by adapting and modifying techniques developed and used in the cereal rust system.
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