Real-time PCR quantification and spatio-temporal distribution of airborne inoculum of Puccinia triticina in Belgium

In order to better understand the epidemiology of Puccinia triticina and the relationship between airborne inoculum and disease severity, a method for quantifying airborne inoculum was developed using volumetric Burkard 7-day spore traps and real-time PCR. The method was applied using a spore trap network from 1 March to 30 June over a 5-year period. At one site, the inoculum was quantified continuously over 3 years, during which it showed a seasonal distribution, with the highest quantities and detection frequencies occurring between May and June. High mean daily quantities (65.8–121.2 spores/day) and detection frequencies (±20 % of days) were also reported after harvest from September to December. In the coldest months of the year, almost no detection was recorded (1–6 % of days). The study results indicate that the absence of inoculum in the air when upper leaves are emerging could be a limiting factor for the risk of epidemics. Mean daily quantities of airborne inoculum (0–131.4 spores/day) were measured from the beginning of stem elongation (GS30) to the flag leaf stage (GS39). These values were well correlated with the disease severity levels measured during grain development. A multiple regression analysis showed that total rainfall in late summer and autumn and mean minimum temperature in winter positively influence spore density between GS30 and GS39 in the following spring (R2 = 0.73). This relationship and the patterns of airborne inoculum observed in fields strongly suggest the existence of a ‘green bridge’ phenomenon in Belgium. Our study also showed that the quantification of airborne inoculum or its estimation using a weather-based predictive model could be useful for interpreting disease severity models and avoiding over-estimates of disease risk

Real-time PCR quantification and spatio-temporal distribution of airborne inoculum of Mycosphaerella graminicola in Belgium

Two kinds of propagules play a role in Mycosphaerella graminicola dissemination: splash-dispersed pycnidiospores and airborne sexual ascospores. A method based on real-time polymerase chain reaction (PCR) assay and using Burkard spore traps was developed to quantify M. graminicola airborne inoculum. The method was tested for its reliability and applied in a spore trap network over a 2-year period in order to investigate the spatio-temporal distribution of airborne inoculum in Belgium. At four experimental sites, airborne inoculum was detected in both years. A seasonal distribution was observed, with the highest mean daily quantities (up to 351.0 cDNA) trapped in July and with clusters detected from September to April. The first year of trapping, a mean daily quantity of 15.7 cDNA of M. graminicola airborne inoculum was also detected in the air above a building in a city where the spatio-temporal distribution showed a similar pattern to that in the field. Mean daily quantities of up to 60.7 cDNA of airborne inoculum were measured during the cereal stem elongation and flowering stages, suggesting that it contributes to the infection of upper leaves later in the season. Most detection, however, tended to occur between flowering and harvest, suggesting significant production of pseudothecia during that period. Variations in mean daily quantities from 1.0 to 48.2 cDNA were observed between sites and between years in the patterns of airborne inoculum. After stem elongation, the quantities detected at a site were positively correlated with the disease pressure in the field. Quantities trapped at beginning of the growing season were also well correlated with the disease level the previous year. Multiple regressions revealed that some factors partly explain the daily variations of airborne inoculum

PCR chemotyping of Fusarium graminearum, F. culmorum and F.cerealis isolated from winter wheat in Wallonia, Belgium

Within the pathogen complex responsible for Fusarium head blight (FHB) are some species that can produce mycotoxins that accumulate in the grains, creating a threat to human and animal health. In Europe, type B trichothecenes, especially deoxynivalenol (DON), are frequently found in grain batches. Most of the genes involved in producing these mycotoxins (TRI genes) are grouped in a 12- gene core cluster (TRI cluster). Fusarium graminearum, F. culmorum and F. cerealis possess this cluster, but the presence or absence of certain TRI genes, as well as their functionality, results in a strain capable of producing either nivalenol (NIV) or deoxynivalenol and a related acetylated derivative (3- or 15-ADON). Because of the different levels of toxicity in these secondary metabolites, it is important to have a better knowledge of the population in Belgium in order to estimate the risk posed by Fusarium species occurring in wheat ears. Two multiplex PCR reactions, targeting the TRI3 and TRI13 genes, were used to differentiate the strains of the three species cited above in terms of the possible chemotypes (NIV, 3-ADON and 15-ADON). In all, 105 single-spore strains of F. graminearum, 90 of F. culmorum and 20 of F. cerealis, isolated from winter wheat, were tested. The three chemotypes were identified in the F. graminearum population, with the vast majority of the strains (93%) being of the 15-ADON chemotype. For F. culmorum, the 3-ADON chemotype was prominent (76.6%) and the rest of the strains were of the NIV chemotype. The 20 tested F. cerealis strains could produce only nivalenol. The different proportions of chemotypes in F. graminearum and F. culmorum and the existence mixed-chemotype populations in the field indicate different specificities of the chemotypes in epidemics

Detection of Fusarium langsethiae on wheat in Belgium

In 2010, the populations of Fusarium sp. and Microdochium sp. were monitored in Belgium and 16 strains were identified as Fusarium langsethiae on wheat in Belgium. The other species identified from the sampling were F. poae, F. tritinctum, F. graminearum, F. avenaceum and Microdochium nivale. The pathogenicity potential of the F. langsethiae strains was assessed via an in vitro coleoptile growth rate test on wheat seedlings and compared with strains of F. poae, F. tritinctum, F. graminearum and F. avenaceum known to cause Fusarium head blight. The results showed the ability of F. langsethiae to cause retardation in the wheat coleoptile growth rate, but at a lower rate than F. graminearum, F. avenaceum, F. poae and F. tricinctum. A test for mycotoxin production in vitro showed the ability of the four strains tested to produce T-2 and HT-2 toxins at a rate of up to 290 mg kg−1. This is the first report on the potential pathogenicity of F. langsethiae on wheat in Belgium, a species known to produce T-2 and HT-2 toxins, which are highly toxic for humans and animals

Evaluation of the temporal distribution of Fusarium graminearum airborne inoculum above the wheat canopy and its relationship with Fusarium head blight and DON concentration

With the aim of unravelling the role of airborne Fusarium graminearum inoculum in the epidemic of Fusarium head blight (FHB) caused by this species in wheat spikes, a network of Burkard air samplers was set up in five wheat fields distributed in Belgium from 2011 to 2013. Each year from April to July, the daily amounts of F. graminearum inoculum above the wheat canopy were quantified using a newly developed TaqMan qPCR assay. The pattern of spore trapping observed was drastically different per year and per location with a frequency of detection between 9 and 66% and a mean daily concentration between 0.8 and 10.2 conidia-equivalent/m3. In one location, air was sampled for a whole year. Inoculum was frequently detected from the wheat stem elongation stage until the end of the harvesting period, but high inoculum levels were also observed during the fall. Using a window-pane analysis, different periods of time around wheat flowering (varying in length and starting date) were investigated for their importance in the relation between airborne inoculum and FHB parameters (FHB severity, frequency of F. graminearum infection and DON). For almost all the combinations of variables, strong and significant correlations were found for multiple window lengths and starting times. Inoculum quantities trapped around flowering were highly correlated with F. graminearum infection (up to R = 0.84) and DON (up to R = 0.9). Frequencies of detection were also well correlated with both of these parameters. DON concentrations at harvest could even be significantly associated with the F. graminearum inoculum trapped during periods finishing before the beginning of the anthesis (R = 0.77). Overall, these results highlight the key role of the airborne inoculum in F. graminearum epidemics and underline the importance of monitoring it for the development of disease forecasting tools

Prevalence of the species involved in Fusarium head blight (FHB) in winter wheat in Wallonia, Belgium from 2010 to 2012

Fusarium head blight (FHB) is a common plant disease occurring frequently in winter wheat in Belgium and associated with yield losses and mycotoxin contamination. The pathosystem involves a complex of species in the Fusarium and Microdochium genera. In order to evaluate the occurrence and distribution of the causal species in Wallonia, ears of winter wheat were collected in five experimental fields in 2010, 2011 and 2012. Grains from three positions along the rachis (top, middle and bottom) of each ear were investigated for the presence of FHB pathogens, using a plate isolation technique and monoconidial cultures. The species were identified using morphological and molecular (elongation factor 1-) characterization. Both infection prevalence and species occurrence differed from one location to another and from year to year, with one or more species being predominant each year. Some species were present one year, but absent in the following year. For example, in 2011, F. culmorum was the most predominant species, being present in every location, but it was rarely isolated in 2012 and it was absent in 2010. In each year F. graminearum, F. poae and F. avenaceum were isolated; F. poae was the prevalent species in 2010, and F. graminearum and F. avenaceum were the prevalent species in 2012. A rare and highly toxigenic species, F. langsethiae, was isolated in 2010 and 2012. In each year the proportions of FHB infection differed significantly (p < 0.01) between the three positions along the rachis, with an increasing intensity from top to bottom. The infections caused by F. graminearum, however, did not quite follow this gradient, with a slightly greater amount of infection found at the top than at the bottom. This discrepancy could result from the ability of this species to produce airborne ascospore inoculum. Our results highlight the great variation in the composition of the FHB complex, suggesting that the epidemiological characteristics differ from one species to another. Although the plate isolation technique is time consuming, it allowed a pathogen culture collection to be created that could be used for further characterization of the species and could improve the understanding of the epidemiology as well as disease/mycotoxin prediction

A new method for quantifying Puccinia triticina airborne inoculum in wheat fields

Wheat leaf rust caused by Puccinia triticina is regularly observed in Belgian fields. This fungus produces wind-dispersed urediospores that infect host plants many kilometers from their source plant, but the origin of the primary inoculum and the factors involved in disease occurrence and severity are not well known. In order to better understand the epidemiology of the disease, the dispersion of P. triticina airborne inoculum and the relationship with disease incidence in fields were studied. A network of Burkard 7-day spore-recording traps was set up in wheat fields in the Walloon region, in Belgium, to monitor the airborne inoculum. Total DNA from each fragment of spore trap tape, corresponding to 1 day of sampling, was extracted and the quantity of P. triticina on the tape fragments was assessed using a specific real-time polymerase chain reaction (PCR) assay. The disease incidence was recorded weekly in experimental fields from March 2012 onwards. Initial analysis revealed that the real-time PCR assay was highly specific and repeatable. The detection threshold was determined at 7 spores per m3. This method (spore traps coupled with real-time PCR) allows airborne inoculum to be detected before the appearance of symptoms in fields and could be used in a model for predicting wheat leaf rust epidemics

Four years of monitoring Mycosphaerella graminicola airborne inoculum and its relationship with disease symptoms in the field

Mycosphaerella graminicola is the causal agent of Septoria tritici blotch (STB) in winter wheat. This disease is called a ‘gradient disease’ because it generally progresses from the lower to upper leaf stages. Airborne ascospores originating in stubble or trash are now considered to be the most important source of primary infection in autumn and winter, whereas the subsequent development of STB occurs through the infection of the upper leaves by splash-dispersed pycnidiospores produced on the lower leaves. Recent reports and observations, however, question the importance of ascospores in the progress of STB epidemics after stem elongation in wheat plants. A method based on real-time polymerase chain reaction (PCR) assay and using Burkard spore traps was developed to quantify M. graminicola airborne inoculum. The method was tested for its reliability and applied in a spore-trap network over a 4-year period in order to investigate the spatio-temporal distribution of airborne inoculum in Belgium. A seasonal distribution was observed, with the highest quantities trapped between June and mid-August and with clusters detected from September to April. Depending on the year or site, significant quantities of airborne inoculum were measured during the cereal stem elongation and heading stages, suggesting that ascospores contribute to the infection of upper leaves later in the season. When many peaks of airborne inoculum were detected during this period, the disease gradient due to the vertical progression of splash-dispersed pycnidiospores was not always well marked. The STB incidence observed on an upper leaf layer was sometimes greater than the incidence on a lower leaf layer, suggesting that the airborne inoculum accelerates the progression of the disease on the upper leaves of wheat plant. The exploration of the relationship between disease evolution, airborne inoculum detection and meteorological conditions would provide valuable information for integrated crop protection