Abstract
Bacterial blotch is a group of economically important diseases of the common button mushroom (Agaricus bisporus). Once the pathogens are introduced to a farm, mesophilic growing conditions (that are optimum for mushroom production) result in severe and widespread secondary infections. Efficient, timely and quantitative detection of the pathogens is hence critical for the design of localized control strategies and prediction of disease risk. This study describes the development of real-time TaqManTM assays that allow molecular diagnosis of three currently prevalent bacterial blotch pathogens: “Pseudomonas gingeri,” Pseudomonas tolaasii and (as yet uncharacterized) Pseudomonas strains (belonging to Pseudomonas salomonii and Pseudomonas edaphica). For each pathogen, assays targeting specific DNA markers on two different loci, were developed for primary detection and secondary verification. All six developed assays showed high diagnostic specificity and sensitivity when tested against a panel of 63 Pseudomonas strains and 40 other plant pathogenic bacteria. The assays demonstrated good analytical performance indicated by linearity across calibration curve (>0.95), amplification efficiency (>90%) and magnitude of amplification signal (>2.1). The limits of detection were optimized for efficient quantification in bacterial cultures, symptomatic tissue, infected casing soil and water samples from mushroom farms. Each target assay was multiplexed with two additional assays. Xanthomonas campestris was detected as an extraction control, to account for loss of DNA during sample processing. And the total Pseudomonas population was detected, to quantify the proportion of pathogenic to beneficial Pseudomonas in the soil. This ratio is speculated to be an indicator for blotch outbreaks. The multiplexed assays were successfully validated and applied by routine testing of diseased mushrooms, peat sources, casing soils, and water from commercial production units.
Highlights
Cultivation of the white button mushroom (Agaricus bisporus), represents a global economic value of more than 4.7 billion US dollars (Sonnenberg et al, 2011)
In this study we develop TaqmanTM-qPCR assays diagnostic assays against three causative agents of bacterial blotch, that are currently causing severe disease outbreaks in Western Europe, “P. gingeri,” P. tolaasii and a not fully characterized Pseudomonas species, taxonomically related to P. edaphica and P. salomonii, that was formerly identified as P. tolaasii
Each target-pathogen assay was multiplexed with two additional assays that quantify Xanthomonas campestris pv. campestris (Xcc) (Köhl et al, 2011) as an extraction control, and the genus Pseudomonas (Lloyd-Jones et al, 2005) for the total Pseudomonad population (Pp)
Summary
Cultivation of the white button mushroom (Agaricus bisporus), represents a global economic value of more than 4.7 billion US dollars (Sonnenberg et al, 2011). Bacterial blotch diseases render crops unmarketable by strongly affecting the esthetic quality of the caps pre-harvest and reducing the shelf life of the mushrooms post-harvest. Economic losses from this disease have been reported globally across Europe (Paine, 1919; Olivier et al, 1978), North America (Tolaas, 1915), Middle East (Bashan and Okon, 1981; Özaktan and Bora, 1994), Asia (Guleria, 1976; Chen, 1981; Suyama and Fujii, 1993; Kim et al, 1995), and Australia (Nair, 1969; Fahy, 1981). The casing soil is a 5 cm layer of peat and lime that is placed on top of the compost, to facilitate formation of mushroom pinheads
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