Onion (Allium cepa L.) is an economically important vegetable, accounting for nearly $1 billion in farm gate income annually in the United States [United States Department of Agriculture National Agricultural Statistics Service (USDA NASS)]. In 2006, 44,000 ha of summer onions were grown for storage (USDA NASS). Postharvest rots can contribute to significant losses for growers and the primary storage disease of onion is neck rot. Losses to neck rot during storage can be as high as 35% in some years (Tietjen and Ceponis, 1981; Williams-Woodward, 2001).
Five Botrytis species have been linked to neck rot in onion (Yohalem et al., 2003). Three species, however, are considered exclusively associated with symptoms of neck rot in onion. These are B. aclada (Fresenius), B. allii (Munn), and B. byssoidea (Walker) (Yohalem et al., 2003). Based on restriction fragment length polymorphism analysis, Nielsen et al. (2002) suggested that there were two distinct groups of B. aclada, types AI and AII, with the latter containing B. allii. It has been proposed that all three species are distinct and that B. allii was the result of an interspecific hybridization between B. acalada and B. byssoidea (Nielsen and Yohalem, 2001; Yohalem et al., 2003). Nonetheless, B. aclada and B. allii remain difficult to differentiate as a result of morphological similarities (Chilvers and duToit, 2006). Despite being pathogenic to onion bulbs, B. byssoidea is infrequently detected relative to B. aclada or B. allii (Chilvers et al., 2004; du Toit et al., 2004; Nielsen et al., 2002).
Botrytis aclada can infect onions at any stage during the growing season. Potential sources of B. aclada inoculum include infected seeds, onion debris, and alternate crops (Maude, 1976; Walcott et al., 2004). Culling infected bulbs at harvest is challenging because onion plants may become infected without displaying visual symptoms of neck rot (Kritzman, 1983; Maude, 1990). With a latency period of 8 to 10 weeks, infected bulbs may appear asymptomatic at harvest but develop symptoms during storage, resulting in significant economic loss (Maude, 1990). Because visual inspection of intact bulbs in the field is ineffective, alternative methods have been developed to detect B. aclada in onion bulbs. These include culturing samples on semiselective media, enzyme-linked immunosorbent assay tests, and conventional polymerase chain reaction (PCR) detection (Kritzman and Netzer, 1978; Linfield et al., 1995; Nielsen et al., 2002). Although all of these methods are useful in identifying the presence or absence of B. aclada in bulb tissue, none of them can be used to easily quantify the level of B. aclada inoculum in infected tissue. The ability to quantify inoculum in bulb tissue is of potential importance because it could allow for the objective assessment of disease severity and strain aggressiveness. Although other factors such as plant resistance, storage temperature, and relative humidity interact to determine the severity of neck rot infection, it would be useful to quantify the level of B. aclada inoculum present in bulbs at harvest (Alderman and Lacy, 1984; Bertolini and Tian, 1997). Potentially this could allow for the prediction of storage rot based on inoculum levels at harvest. Conventional diagnostic assays do not have the capacity to reliably quantify mycelial mass in onion tissue; however, quantitative real-time PCR represents one technique by which this can be accomplished.
Unlike traditional PCR, quantitative real-time PCR assays can simultaneously amplify and estimate the concentration of specific template DNA sequences (Wilhelm and Pingoud, 2003). The ability to estimate the amount of sequence-specific template DNA or RNA in a sample allows for the identification and quantification of pathogens in crops of interest. Real-time PCR uses fluorescent dyes that emit light of a specific wavelength during amplification (Schena et al., 2004). Fluorescence can be produced through nonspecific methods such as the fluorescence emitted by SYBR green as it is intercalated into double-stranded DNA or sequence-specific methods such as the light-emitting probes used in TaqMan or Scorpian PCR systems (Wilhelm and Pingoud, 2003). Regardless of the system, the amount of fluorescence increases as the product is amplified. In general, a threshold value for fluorescence is established, below which samples are considered negative. The number of cycles required for a sample to reach the threshold is the cycle threshold (Ct) value. The higher the concentration of template DNA, the fewer the number of amplification cycles required to reach the fluorescence threshold and the lower the Ct value will be for that sample (Wilhelm and Pingoud, 2003). Because the amount of pathogen template DNA should be proportional to the level of pathogen present, real-time PCR can be used to estimate fungal mass, viral load, or bacterial counts in a given sample.
Suarez et al. (2005) developed a TaqMan-based real-time PCR assay for the quantification of B. cinerea in Pelargonium species. This assay facilitated quantification of B. cinerea inoculum over four orders of magnitude. Furthermore, there was a positive relationship between estimates of inoculum concentration and the visible symptoms on leaf discs. Quantitative real-time PCR has been used for a range of pathogens in agronomic and horticultural, crops including Fusarium spp. in wheat, Rhizoctonia spp. in tomato, and Phytopthora ramorum in Quercus spp. (Leivens et al., 2006; Schena, et al., 2006; Schnerr, et al., 2001; Tooley et al., 2006).
Recently Chilvers et al. (2007) developed a real-time PCR assay for the detection Botrytis spp. in onion seed. Using primers developed for the intergenic space region of Botrytis spp., the authors detected Botrytis DNA at levels of 10 fg·μL−1. However, the real-time PCR assay was applied for pathogen detection in onion seed and did not consider estimation of mycelial colonization in bulb tissue (Chilvers et al., 2007).
Our objective in this study was to demonstrate that real-time PCR could be used to reliably quantify the level of B. aclada colonization in inoculatated onion tissue while using a reference gene, serine acetyl transferase (SAT1), from onion to normalize data.
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