Monitoring important soil-borne plant pathogens in Swedish crop production using real-time PCR

The global demand for food will increase considerably in the nearest future and among the major constraints to agricultural productivity are biotic stresses caused by microorganisms. In this thesis, the causal agents of four of the most important soil-borne diseases threatening the Swedish production of oilseed rape, sugar beets and red clover were selected as targets for developing diagnostic assays using real-time PCR. The disease risk assessment of clubroot in oilseed rape was improved by developing a real-time PCR assay for quantification of the causal pathogen Plasmodiophora brassicae directly in soil samples. Broad disease risk categories including a threshold level for growing resistant cultivars were established. A real-time PCR assay for quantification of Aphanomyces cochlioides, the oomycete causing root rot in sugar beets, was also developed in the present study. The potential use of this assay as a tool in disease risk assessment was demonstrated for fields with high risk of infection. Real-time PCR was used to monitor the plant- and airborne inoculum of Sclerotinia sclerotiorum, the causal fungus of Sclerotinia stem rot, in spring oilseed rape. We found that determining the presence of S. sclerotiorum on petals was not useful for stem rot risk assessment since (i) the inoculum incidence on petals varied during flowering, (ii) there was no clear relationship between petal infection and stem rot incidence and (iii) spore release and flowering were not synchronized at one of the field experimental sites. Real-time PCR detection of the incidence of S. sclerotiorum DNA on leaves revealing the field-borne inoculum and quantification of the airborne inoculum are likely more reliable tools for predicting the potential risk of disease. The pathogen complex causing red clover root rot was monitored over three years in two field experiments. Fusarium avenaceum, Phoma spp. and Cylindrocarpon destructans were detected in red clover roots early in the seeding year using real-time PCR and the levels of pathogen DNA generally increased during the following years. Significant linear relationships were found between the amount of pathogen DNA and disease severity index

Bestämning av förekomst av patogena svampar i vete med PCR-teknik

Today, the quantification of leaf pathogens on wheat mainly relies on a visual inspection and estimation of the percentage of infected leaf area. However, this is both time consuming, requires skilled personnel with experience in plant pathology and may also be misleading if several pathogens are present on the same leaf or if the symptoms are concealed by abiotic factors. More reliable methods suitable for routine diagnosis are necessary to be able to better evaluate and optimise the use of fungicides and different cultivars. In the SLF-project V0648026 "PCR based detection of plant pathogens in wheat", quantitative real-time PCR assays were developed for detection of the three leaf pathogens in winter wheat; Septoria tritici, Drechslera tritici-repentis (DTR) and Stagonospora nodorum. The amount of plant pathogen DNA was determined using relative quantification and a wheat-specific gene was used as reference. With high specificity, high reproducibility and low detection limit, the PCR methods developed enable an objective measurement of fungal infection levels in wheat. The results show a good correlation between visual grading and relative qPCR measurements. Although the visual grading did not report the presence of S. nodorum, this fungi was frequently detected using the qPCR assay. The results also show that qPCR can be used to study the effect of fungicides on the fungal flora with very small variation between replications. In addition, it was shown that qPCR is a useful tool for quantifying the amount of the three wheat pathogens in field trials where different cultivars are evaluated. Pre-symptomatic detection of the pathogens was possible and results show that the PCR-techniques even could be used to evaluate the presence of a few single spores. For the future the results indicates a possibility to improve the evaluation of plant pathogens and plant production systems

Clubroot, a persistent threat to Swedish oilseed rape production

Brassica oilseed crops have been grown by Swedish farmers since the early 1940s. Years of high market prices for vegetable oils resulted in intensive cultivation of Brassica oilseeds in various regions, which led to problems with soilborne pathogens including Plasmodiophora brassicae. This pathogen most likely was present in Swedish soils prior to the Brassica oilseed boom after World War II. Currently, reports of clubroot disease outbreaks in Sweden are frequent, with a trend of increasing incidence. Since 2012, DNA-based soil analyses for the presence of P. brassicae have been offered to farmers in order to improve their crop rotation planning. Other means of limiting the damage caused by P. brassicae also are presently under study, such as the effects of boron or different sources of nitrogen. The distribution and identification of different pathotypes/races of P. brassicae in Sweden could so far not be verified. To aid in race diagnostics, resistance breeding efforts, and in understanding the biology of this Plasmodiophorid, the sequencing of the P. brassicae genome has been initiated in Sweden

Cigarette Smoking and Colorectal Cancer Risk in the European Prospective Investigation Into Cancer and Nutrition Study

BACKGROUND & AIMS: There has been consistent evidence for a relationship between smoking and colorectal cancer (CRC), although it is not clear whether the colon or rectum is more sensitive to the effects of smoking. We investigated the relationships between cigarette smoking and risk of CRC and tumor location. METHODS: We analyzed data from 465,879 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) study; 2741 developed CRC during the follow-up period (mean, 8.7 years). Cox proportional hazard regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: The risk of colon carcinoma was increased among ever smokers (HR, 1.18; 95% CI, 1.06-1.32) and former cigarette smokers (HR, 1.21; 95% CI, 1.08-1.36), compared with never smokers; the increased risk for current smokers was of borderline significance (HR, 1.13; 95% Cl, 0.98-1.31). When stratified for tumor location, the risk of proximal colon cancer was increased for former (HR, 1.25; 95% CI, 1.04-1.50) and current smokers (HR, 1.31; 95% CI, 1.06-1.64), but the risks for cancers in the distal colon or rectum were not. Subsite analyses showed a nonsignificant difference between the proximal and distal colon (P=.45) for former smokers and a significant difference for current smokers (P=.02). For smokers who had stopped smoking for at least 20 years, the risk of developing colon cancer was similar to that of never smokers. CONCLUSIONS: Ever smokers have an increased risk of colon cancer, which appeared to be more pronounced in the proximal than the distal colon location

Cigarette Smoking and Colorectal Cancer Risk in the European Prospective Investigation Into Cancer and Nutrition Study

BACKGROUND & AIMS: There has been consistent evidence for a relationship between smoking and colorectal cancer (CRC), although it is not clear whether the colon or rectum is more sensitive to the effects of smoking. We investigated the relationships between cigarette smoking and risk of CRC and tumor location. METHODS: We analyzed data from 465,879 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) study; 2741 developed CRC during the follow-up period (mean, 8.7 years). Cox proportional hazard regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: The risk of colon carcinoma was increased among ever smokers (HR, 1.18; 95% CI, 1.06-1.32) and former cigarette smokers (HR, 1.21; 95% CI, 1.08-1.36), compared with never smokers; the increased risk for current smokers was of borderline significance (HR, 1.13; 95% Cl, 0.98-1.31). When stratified for tumor location, the risk of proximal colon cancer was increased for former (HR, 1.25; 95% CI, 1.04-1.50) and current smokers (HR, 1.31; 95% CI, 1.06-1.64), but the risks for cancers in the distal colon or rectum were not. Subsite analyses showed a nonsignificant difference between the proximal and distal colon (P=.45) for former smokers and a significant difference for current smokers (P=.02). For smokers who had stopped smoking for at least 20 years, the risk of developing colon cancer was similar to that of never smokers. CONCLUSIONS: Ever smokers have an increased risk of colon cancer, which appeared to be more pronounced in the proximal than the distal colon location