Quantifying Phytophthora medicaginis in Susceptible and Resistant Alfalfa with a Real-Time Fluorescent PCR Assay

A real-time fluorescent PCR assay using a set of specific primers and a fluorochrome-labelled probe (TaqMan) was developed to quantify the amount of Phytophthora medicaginis DNA in alfalfa plants that were classified as either resistant or susceptible to the pathogen based on visual assessment of disease response. The assay clearly discriminated among three standard check alfalfa populations with different levels of resistance based on the analysis of DNA extracted from the roots of bulked plant samples. In two independent experiments, the Spearman rank correlation between pathogen DNA content and the number of susceptible plants in a bulked sample was greater than 0.89 and highly significant (P<0.0001). Significantly less pathogen DNA was detected in bulked samples of a highly resistant check population than in bulked samples from more susceptible check populations. Analysis of individual plants indicated that significantly less pathogen DNA was detected in resistant plants than in susceptible plants. Applications of the assay are considered for breeding programs and the study of microbial population dynamics in plants simultaneously infected with different pathogens. Introduction Phytophthora medicaginis Hansen et Maxwell (1⁄4Phytophthora megasperma Drechs. f. sp. medicaginis T. Kuan & D. C. Erwin) is an important causal agent of root rot in both alfalfa (Medicago sativa L.) and chickpeas (Cicer arietinum L.). Root rot of alfalfa caused by P. medicaginis occurs in nearly every region of the world where alfalfa is cultivated (Erwin, 1990; Hansen and Maxwell, 1991). The disease can cause complete failure of stand establishment due to damping-off and seedling death, and is most prevalent when newly seeded fields are subjected to flooding (Havey and Grau, 1985; Erwin, 1990). The pathogen also causes disease in mature plants (Erwin, 1966; Leuchen et al., 1976). P. medicaginis produces oospores, which can survive for years in the soil (Erwin, 1990). Phytophthora root rot of alfalfa is best controlled by a combination of practices including cultivation of resistant varieties, water management, and use of the fungicide metalaxyl as a seed treatment (Erwin, 1990). Various studies have indicated that alfalfa cultivars with resistance to P. medicaginis have significantly higher yields than susceptible cultivars when grown in naturally infested soil (Faris and Sabo, 1981; Gray et al., 1988). The percentage of plants exhibiting either adult plant resistance (Barnes and Frosheiser, 1983) or seedling resistance (Faris, 1985) to P. medicaginis can vary considerably among alfalfa cultivars that are considered resistant. A significant positive correlation (r 1⁄4 0.78) has been reported between levels of resistance obtained from a greenhouse seedling assay and resistance ratings obtained from field grown mature plants (Hohrein et al., 1983). Most recently released alfalfa cultivars have at least a moderate degree of resistance to P. medicaginis (Alfalfa Council, 2002). However, the disease still causes considerable losses in alfalfa production because many growers continue to cultivate less expensive older cultivars that lack disease resistance (Munkvold and Carlton, 1995). In addition, P. medicaginis has been implicated in damping-off disease complexes of alfalfa with other soilborne pathogens including the northern root knot nematode (Meloidogyne hapla Chitwood) (Gray et al., 1990; Griffin and Gray, 1994), southern root knot nematode [Meloidogyne incognita (Kofoid & White) Chitwood] (Welty et al., 1980) and Aphanomyces euteiches (Havey and Grau, 1985; Munkvold and Carlton, 1995). Several different mechanisms have been implicated in the expression of resistance in alfalfa to P. medicaginis, including hypersensitive reactions in the root cortical cells of resistant plants (Marks and Mitchell, 1971; Vaziri et al., 1981), and increased production in U. S. Copyright Clearance Centre Code Statement: 0931–1785/2003/15112–0577 $ 15.00/0 www.blackwell.de/synergy J. Phytopathology 151, 577–583 (2003) 2003 Blackwell Verlag, Berlin ISSN 0931-1785 resistant plants of the phytoalexin medicarpin (Vaziri et al., 1981). Hyphal staining of infected roots of both alfalfa (Miller and Maxwell, 1984) and chickpea (Dale and Irwin, 1991) has indicated that colonization by P. medicaginis is more extensive in susceptible plants than in resistant plants. However, it is difficult to use hyphal staining techniques to accurately quantify difference between root systems in the amount of colonization by P. medicaginis. Entire root systems cannot be stained on a single slide, and hyphae do not typically grow in a linear manner, making measurement difficult. Comparisons have not been made between resistant and susceptible alfalfa plants for the number of oospores of P. medicaginis produced in infected roots, but these comparisons have been done for other Phytophthora sp. Significantly more oospores of P. fragariae were produced in roots of susceptible strawberry plants than in resistant plants (Milholland et al., 1989). However, no significant differences were observed in the number of oospores of P. infestans produced on leaves of potato genotypes having different levels of resistance to potato late blight disease (Cohen et al., 1997). Counting oospores is a tedious process, and entire root systems cannot be sampled easily. The development of techniques for real-time detection of fluorescent-labelled DNA fragments amplified using polymerase chain reaction (PCR) may provide another approach for accurately quantifying the amount of P. medicaginis in infected plants (Holland et al., 1991; Livak et al., 1995; Heid et al., 1996; Lie and Petropoulus, 1998). Real-time detection of PCR products (amplicons) employs a chemistry that uses the 5¢ nuclease activity of Taq Polymerase to generate fluorescence in proportion to the number of target amplicons produced (Holland et al., 1991). After an initial period, successive cycles of PCR result in the exponential synthesis of the amplicon, which can be quantified automatically using software that measures increase in fluorescence (Schoen et al., 1996). Recently, a real-time PCR assay was developed for A. euteiches (Vandemark et al., 2002). Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings were greater than 0.75 and highly significant (P<0.0005) for analysis of both individual alfalfa plants and bulked plant samples of resistant and susceptible alfalfa check populations (Vandemark et al., 2002). The highly positive and significant correlations between pathogen DNA quantity and DSI suggested it may be possible to use real-time PCR to select the most resistant plants from among a sample of plants considered to be equally resistant based on a visual assessment of disease severity. Our objectives were: (1) develop a real-time quantitative PCR assay that was specific for P. medicaginis, (2) employ the assay to discriminate between standard check alfalfa populations for resistance to P. medicaginis by analysis of DNA extracted from bulked plant samples, and (3) use the assay to compare differences between resistant and susceptible plants in the amount of colonization of entire root systems by P. medicaginis. Applications of this assay for breeding programs and the study of microbial population dynamics are considered. Materials and Methods Pathogen and plant materials Phytophthora medicaginis isolate WI301 was used in this study for all infectivity assays. The isolate was originally obtained from infected alfalfa plants grown under field conditions in Wisconsin. The isolate was maintained on potato dextrose agar (PDA, Difco Inc., Sparks, MD, USA) at 20–24 C. Three different standard check alfalfa populations were evaluated: the varieties Saranac and Agate, and the germplasm WAPH-1. Saranac is the susceptible check in standard tests for evaluating resistance to P. medicaginis, Agate is a resistant check, and WAPH-1 is the highly resistant check (Nygaard et al., 1995). Inoculations and evaluation of disease severity Phytophthora medicaginis isolate WI301 was used to examine the ability of a real-time fluorescent PCR assay to discriminate check populations for resistance based on the analysis of DNA extracted from the roots of bulked plant samples. The standard test protocol for evaluating seedling resistance in alfalfa to P. medicaginis was closely followed in this study (Nygaard et al., 1995), except that each plant was inoculated with 250 zoospores per plant instead of 50 zoospores. This was done to minimize the likelihood of plants escaping infection. Alfalfa seeds were mixed in a commercial preparation of Sinorhizobium mellioti (LiphaTech Inc., Milwaukee, WI), and planted in plastic greenhouse flats containing vermiculite. A single row of each of the three check alfalfa populations was sown in each flat. Seedlings were grown in the greenhouse with 16 h daylength at 20–24 C. The rows were thinned to approximately 70 seedlings/row 8 days after sowing. Cultures of isolate WI301 were transferred from PDA plates to plates containing V-8 agar and zoospores were produced as previously described (Miller and Maxwell, 1984). At 10 days post-sowing, the vermiculite was saturated with water and each seedling was inoculated with 1 ml of a 250 zoospore/ml suspension by pipetting the suspension to the base of the stem. The flats were flooded for 2 days. Ten days after inoculation, 15 plants were randomly selected from each row and rated for resistance as follows: resistant 1⁄4 vigorously growing plants with slight to no necrosis of tap and secondary roots; hypocotyls area sound with slight to no chlorosis of cotyledons, or susceptible 1⁄4 stunted or dead plants with moderate to severe necrosis of roots, hypocotyls and cotyledons (Nygaard et al., 1995). For each sample of 15 plants the number of resistant and susceptible plants was counted and the entire sample was bulked. DNA was subsequently isolated from each entire bulked plant sample and analyzed by quantitative PCR. 578 Vandemark and Barker