Identification of Darkly Pigmented Fungi Associated with Turfgrass Roots by Mycelial Characteristics and RAPD-PCR

Abstract

A select group of darkly pigmented turfgrass pathogens are referred to as ectotrophic root-infecting (ERI) fungi. Identification of ERI fungi, as well as darkly pigmented saprophytes, cannot be rapidly or reliably determined by traditional methods. The objectives of this study were (i) to determine if these darkly pigmented fungi could be presumptively identified by mycelial growth rates and characteristics, and (ii) to definitively identify these fungi based on DNA products generated through random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR). Previously identified isolates of Gaeumannomyces cylindrosporus, G. graminis var. avenae, G. graminis var. graminis, G. incrustans, Magnaporthe poae, Ophiosphaerella herpotricha, O. korrae, and Phialophora graminicola were obtained. The species were separated into two groups based on their growth rate on half strength potato dextrose agar at 25°C. Slower-growing species [X = 2.5 ; range = 1.6 to 3.4 mm per day] were G. cylindrosporus, P. graminicola, O. herpotricha, and O. korrae, whereas fast-growing species [X = 5.5 ; range = 3.2 to 6.6 mm per day] were G. graminis var. avenae, G. graminis var. graminis, G. incrustans, and M. poae. Colony characteristics and growth rate were valuable in presumptively distinguishing most species. For example, O. korrae colonies had distinctively raised or dome-shaped mycelium, whereas O. herpotricha isolates produced a brownish black colored exudate in the center of 2-week-old colonies. Gaeumannomyces graminis var. avenae isolates were separated from G. incrustans and M. poae based on their very slow growth at 30°C. Initially, only single isolate of G. cylindrosporus, G. graminis var. avenae, G. graminis var. graminis, G. incrustans, M. poae, O. herpotricha, O. korrae, and P. graminicola were evaluated against 20 oligonucleotide primers (10-mers) for identifiable and reproducible DNA products. Primers were found that reliably produced bands unique to each species, excluding G. graminis var. graminis. Then, an additional 52 known ERI fungal isolates were screened against the species-specific 10-mers to confirm reproducibility and clarity of the DNA fingerprints. When fungal isolates were obtained from diseased turfgrass roots, clinical identification of ERI fungi was achieved in 2 to 3 weeks.