Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR

Abstract

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

Detection and Quantification of Rhizoctonia solani and Rhizoctonia solani AG1-IB Causing the Bottom Rot of Lettuce in Tissues and Soils by Multiplex qPCR

“…This production is threatened by several soilborne diseases. In the spring, pythium stunt caused by Pythium tracheiphilum is the most important disease [2], while bottom rot caused by Rhizoctonia solani is predominant throughout the summer. Rhizoctonia solani (Kühn) is a ubiquitous soilborne plant pathogen capable of infecting the seedlings, roots, leaves, and stems of a multitude of vegetable plants, causing a variety of diseases [3].…”

“…Since the molecular method has the advantage of being rapid and able to be designed specifically for a given R. solani AG, unlike baiting methods, it was used to quantify the R. solani soilborne inoculum in various soil types [32,33]. The implementation of such soilborne inoculum quantitation protocols enables the development of damage thresholds, allowing us to make better decisions regarding field selection [2,35]. Hence, this study aims to (i) identify the predominant R. solani AG responsible for lettuce basal rot in the muck soils of southwestern Québec; (ii) develop qPCR assays specific to R. solani and R. solani AG1-IB in order to detect and quantify them in soil and lettuce tissue samples; (iii) develop a molecular detection threshold for R. solani inoculum in muck soil; and (iv) investigate the relationship between the AG1-IB soil inoculum concentration and disease development under controlled conditions to validate the biological significance of the molecular detection threshold found.…”

A Real-Time PCR Assay for the Quantification of Plasmopara viticola Oospores in Grapevine Leaves

“…There is no information about the epidemiological role of the oospores in the soil and their ability to produce zoospores that can be splash-transported from soil to grape leaves, but it would be useful to determine whether the qPCR assay can be used to estimate oospore numbers in vineyard soil samples. In this regard, researchers have recently developed techniques that overcome some of the problems in using qPCR to quantify pathogen inoculum in soil ( Pavón et al., 2008 ; Schena et al., 2013 ; Hussain et al., 2014 ; Gossen et al., 2019 ; Van der Heyden et al., 2019 ). Another limitation of this research is that it was conducted with diseased senescent leaves that would have soon fallen to the soil surface and become litter rather than with leaf litter.…”

“…15.285 ± 1.790 0.497 ± 0.060 3.828 ± 0.076 0. assay can be used to estimate oospore numbers in vineyard soil samples. In this regard, researchers have recently developed techniques that overcome some of the problems in using qPCR to quantify pathogen inoculum in soil (Pavoń et al, 2008;Schena et al, 2013;Hussain et al, 2014;Gossen et al, 2019;Van der Heyden et al, 2019). Another limitation of this research is that it was conducted with diseased senescent leaves that would have soon fallen to the soil surface and become litter rather than with leaf litter.…”

“…A possible reason is that both microscopy and leaf disc bioassays are too expensive and time-consuming for assessing the high numbers of oospores (as many as several hundred per g of leaf residue) that can be present in the leaf litter. Molecular methods that have been used for the quantification of the inoculum in other pathosystems ( Pavón et al., 2008 ; Li et al., 2010 ; Hussain et al., 2014 ; Van der Heyden et al., 2019 ) could be potentially used for P. viticola . Although polymerase chain reaction (PCR) and real-time PCR ( Valsesia et al., 2005 ; Gindro et al., 2014 ) have been developed for the detection and quantification of P. viticola in fresh grape leaves, molecular methods have not been developed for the detection and quantification of oospores.…”