Matthew A. Back

Molecular studies to identify the Fusarium species responsible for HT-2 and T-2 mycotoxins in UK oats

High levels of Fusarium mycotoxins HT-2 and T-2 have been detected in UK oats since surveys started in 2002. Fusarium langsethiae and the closely related species F. sporotrichioides have previously been associated with the contamination of cereals with type A trichothecenes HT-2 and T-2 in Nordic countries. Preliminary microbiological analysis of UK oat samples with high concentrations of HT-2 and T-2 detected and isolated F. langsethiae and F. poae but not the other type A trichothecene producing species F. sporotrichioides, F. sibiricum and F. armeniacum. Two hundred and forty oat flour samples with a known mycotoxin profile were selected from a previous four year study (2002-2005) to cover the full concentration range from below the limit of quantification (<20 μg/kg) to 9,990 μg/kg HT-2+T-2 combined. All samples were analysed for the DNA of F. langsethiae, F. poae and F. sporotrichioides based on previously published PCR assays. F. langsethiae was detectable in nearly all samples; F. poae was detected in 90% of samples whereas F. sporotrichioides was not detected in any sample. A real-time PCR assay was developed to quantify F. langsethiae DNA in plant material. The assay could quantify as low as 10(-4)ngF. langsethiae DNA/μl. Based on this assay and a previously published assay for F. poae, both species were quantified in the oat flour samples with known HT-2+T-2 content. Results showed a good regression (P<0.001, r(2)=0.60) between F. langsethiae DNA and HT-2+T-2 concentration. F. poae DNA concentration was not correlated to HT2+T2 concentration (P=0.448) but was weakly correlated to nivalenol concentration (P<0.001, r(2)=0.09). Multiple regression with F. langsethiae and F. poae DNA as explanatory variates identified that both F. langsethiae and F. poae DNA were highly significant (P<0.001) but F. poae DNA only accounted for an additional 4% of the variance and the estimate was negative, indicating that higher concentrations of F. poae DNA were correlated with slightly lower concentrations of HT2+T2 detected. A stronger regression (P<0.001, r(2)=0.77) between F. langsethiae DNA and HT-2+T-2 was obtained after extraction and quantification of DNA and mycotoxins from individual oat grains. The results from this study provide strong evidence that F. langsethiae is the primary, if not sole, fungus responsible for high HT-2 and T-2 in UK oats.

Fusarium langsethiae pathogenicity and aggressiveness towards oats and wheat in wounded and unwounded in vitro detached leaf assays

In vitro detached leaf assays involving artificial inoculation of wounded and unwounded oat and wheat leaves were used to investigate the potential pathogenicity and aggressiveness of F. langsethiae, which was linked recently to the production of type A trichothecenes, HT-2 and T-2 in cereals in Europe. In the first two experiments, two assays compared disease development by F. langsethiae with known fusarium head blight pathogen species each used as a composited inoculum (mixture of isolates) at 10°C and 20°C and found all fungal species to be pathogenic to oat and wheat leaves in the wounded leaf assay. In the unwounded leaf assay, F. langsethiae was not pathogenic to wheat leaves. Furthermore, there were highly significant differences in the aggressiveness of pathogens as measured by lesion length (P < 0.001). In the second two experiments, pathogenicity of individual F. langsethiae isolates previously used in the composite inoculum was investigated on three oat and three wheat varieties. The wounded leaf assay showed that all isolates were pathogenic to all oat and wheat varieties but only pathogenic towards oat varieties in the unwounded assay. Highly significant differences (P < 0.001) in lesion length were found between cereal varieties as well as between isolates in the wounded assay. Significant differences in lesion lengths (P = 0.014) were also observed between isolates in the unwounded assay. Results from the detached leaf assays suggest that F. langsethiae is a pathogen of wheat and oats and may have developed some host preference towards oats.

Development of Fusarium langsethiae in commercial cereal production

A field survey was performed to study the infection and development of Fusarium langsethiae in the growing season of wheat, barley, oats and triticale under commercial (2009 – 2011) production. Sampling was completed over three years from fields within the counties of Shropshire and Staffordshire in the UK. Plants sampled (from tillering to harvest) were divided into roots, leaves, lower stem, upper stem and inflorescence/head sub-samples depending on the growth stage of the cereal. DNA was extracted and F. langsethiae DNA quantified using real-time PCR. Fusarium mycotoxins HT-2 and T-2 were quantified from head samples at harvest. Three years of data showed oat to contain the highest levels of both F. langsethiae biomass and HT-2 + T-2 mycotoxins in harvested heads of the cereals studied. The development of F. langsethiae in all three cereals appeared to be similar. Fusarium langsethiae DNA was not detected in the roots and seedlings of all three cereals suggesting F. langsethiae is not a seedling pathogen of cereals. Head infection if it occurs, is at head emergence but before flowering. Seemingly symptomless heads had high levels of F. langsethiae DNA and HT-2 + T-2, confirming previous suggestions that F. langsethiae is a symptomless pathogen of oats.

Biofumigation with brassica juncea, raphanus sativus and eruca sativa for the management of field populations of the potato cyst nematode globodera pallida

BACKGROUND The viability of potato cyst nematode (PCN) populations (Globodera pallida) was evaluated in three field experiments using Brassica juncea, Raphanus sativus and Eruca sativa amendments. These species were summer cultivated and autumn incorporated in experiment 1; in experiment 2, overwintered brassicaceous cover crops were spring incorporated. Experiment 3 involved determination of effects of metconazole application on biomass/glucosinolate production by B. juncea and R. sativus and on PCN pre- and post-incorporation. Glucosinolate contents were determined before incorporation. Following cover crop incorporation, field plots were planted with susceptible potatoes to evaluate the biofumigation effects on PCN reproduction. RESULTS In experiment 1, PCN population post-potato harvest was reduced (P = 0.03) in B. juncea-treated plots, while R. sativus prevented further multiplication, but in experiment 2 there were no significant effects on PCN reproduction. In experiment 3, B. juncea or R. sativus either untreated or treated with metconazole reduced PCN populations. Glucosinolate concentrations varied significantly between different plant regions and cultivation seasons. Metconazole application increased the sinigrin concentration in B. juncea tissues. Glucosinolate concentrations correlated positively with PCN mortality for summer-cultivated brassicaceous plants. CONCLUSION The results demonstrated that B. juncea and R. sativus green manures can play an important role in PCN management, particularly if included in an integrated pest management scheme.

Interactions between the potato cyst nematodes, Globodera pallida , G. rostochiensis , and soil-borne fungus, Rhizoctonia solani (AG3), diseases of potatoes in the glasshouse and the field

This is the first report of a positive interaction between the nematode Globodera pallida and Rhizoctonia solani diseases of potato, and is an important step in the general understanding of interactions between nematodes and fungal diseases. One glasshouse and two field experiments were performed to investigate interactions between G. pallida or G. rostochiensis and R. solani diseases of potatoes. The glasshouse experiment investigated the independent and combined effects of G. pallida or G. rostochiensis and R. solani on the growth of plants and R. solani disease severity. In the glasshouse experiment the combined effects of G. pallida with R. solani or G. rostochiensis with R. solani showed greater R. solani diseases of potatoes compared with only R. solani. The field experiments examined the development of R. solani diseases on potatoes grown in soil with potato cyst nematode population densities ranging from eight to 140 and four to 50 eggs (g soil)–1 in the years 2006 and 2007, respectively. The 2006 field experiment revealed a clear positive relationship between initial population densities of G. pallida and the incidence of stolons infected by R. solani, subsequent stolon pruning and stem canker. In both field experiments a clear positive relationship was found between densities of nematodes within the potato roots and the incidence of infected stolons, stolon pruning and stem canker. Nematicide applied together with R. solani in the 2007 field experiment reduced the effect of G. pallida on the incidence and severity of diseases caused by R. solani.

Field evaluation of the nematicide fluensulfone for control of the potato cyst nematode Globodera pallida

Abstract BACKGROUND Three field experiments evaluated the performance of the nematicide fluensulfone against the potato cyst nematode Globodera pallida in Shropshire, England. RESULTS Experiments 1 and 2 showed reduced root infection and lowered multiplication of G. pallida following fluensulfone (Nimitz 15G ®) soil treatments at five rates (1.95, 3.00, 4.05 (full rate), 5.05 and 6.00 kg AI ha−1) and Nimitz 480EC ® at the full rate. Experiment 3 demonstrated a positive interaction between the full rate of Nimitz 15G and the potato variety Santé in the reduction of G. pallida. The fluensulfone treatments at the full rate had more consistent effects than the lower rates, and there were no greater effects for the treatments higher than this full rate. Generally, fluensulfone was less efficacious than oxamyl or fosthiazate, which suggests that the treatment may not be reliably integrated within shorter potato rotations. CONCLUSION The data suggest that fluensulfone soil application could make a useful addition to the few available nematicide treatments for the control of G. pallida rather than be a substitute for these treatments.© 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Interactions between field populations of the potato cyst nematode Globodera pallida and Rhizoctonia solani diseases of potatoes under controlled environment and glasshouse conditions.

This research elucidates interactions between Globodera pallida and Rhizoctonia solani (AG3) by comparing disease development in potato plants inoculated with G. pallida and R. solani. The effects of juvenile population density and time of infestation on R. solani disease were investigated in controlled environments. Plants were inoculated with a range of densities of second-stage juveniles (J2) of G. pallida (800, 4000, 10 000, 20 000 and 40 000 J2/plant) and R. solani at 2, 4 and 6 weeks after planting and a single treatment of G. pallida density and R. solani alone. The experiment demonstrated that greatest disease incidence and severity occurred when high population densities of G. pallida were applied at an early stage of plant development. To determine if this effect could explain differences in R. solani disease incidence and severity following interactions with individual field populations of G. pallida, a glasshouse experiment was conducted using two populations of G. pallida with known hatching characteristics (fast and slow hatching). The experiment revealed that a combination of a fast-hatching population with R. solani caused significantly more R. solani disease incidence and severity than a population of G. pallida with slower hatching characteristics.

In vitro nematicidal activity of a garlic extract and salicylaldehyde on the potato cyst nematode, Globodera pallida.

The in vitro nematicidal effects of an aqueous garlic extract, salicylaldehyde, a nonylphenol ethoxylate surfactant and a formulation containing these constituents were evaluated against the potato cyst nematode, Globodera pallida. Newly hatched, infective second-stage juveniles (J2) were placed for 24, 48 and 72 h in solutions containing concentrations of the formulation from 30.080.0 μl l–1 with 20% (v/v) potato root leachate and sterile distilled water controls. The garlic extract, salicylaldehyde and surfactant treatments were assessed at concentrations proportional to their occurrence in the formulation. Hatching assays involved a series of experiments in which G. pallida cysts were incubated for 8 weeks in potato root leachate solution containing different concentrations of the test substances. A second set of experiments involved incubating cysts in different concentrations of the test substances for 2, 4 and 8 weeks prior to hatching in potato root leachate solution to determine how prior exposure to these substances influences hatching and in-egg viability. The formulation caused 100% mortality at 75.0 μl l–1 with an LC50 of 43.6 μl l–1 after 24 h exposure. Salicylaldehyde was the most toxic constituent of the formulation with an LC50 of 6.5 μl l–1 after 24 h, while the garlic extract achieved 50% J2 mortality at 983.0 μl l–1, demonstrating that the formulation and salicylaldehyde are more toxic to G. pallida in vitro than oxamyl but less toxic when compared with aldicarb. The surfactant showed no dose-dependent toxic effects on J2 when compared with the controls. Emergence of J2 from the cysts was significantly reduced by concentrations of the formulation above 688.0 μl l–1 and its equivalent concentration of salicylaldehyde, while concentrations of the formulation above 2752.0 μl l–1 and the corresponding salicylaldehyde concentrations resulted in complete irreversible hatch inhibition. Concentrations of the garlic extract below 137.6 μl l–1 caused 26% more J2 hatch in comparison to the potato root leachate solution. This study has shown that salicylaldehyde is more toxic to nematodes than the garlic extract, and is the first report of a hatch stimulatory effect of a garlic extract on G. pallida under in vitro conditions.

In vitro growth characteristics of Fusarium langsethiae isolates recovered from oats and wheat grain in the UK

Fusarium langsethiae is a fungus that has recently been implicated in the contamination of small-grain cereal crops such as oats, wheat and barley with high levels of HT-2 and T-2 toxins in many European countries. The epidemiology of this fungus is not well known and may therefore be a bigger problem than currently thought to be. A study was carried out investigating the in vitro growth characteristics of F. langsethiae isolates from contaminated oats and wheat at various temperatures; 15, 20, 25 and 30 °C. Results indicated similar growth trends of oats and wheat isolates of F. langsethiae. Wheat isolates grew significantly (p<0.001) faster than oat isolates although this difference may have been confounded by the age of cultures, with oat isolates collected one year earlier. The estimated optimum growth temperature for all isolates was 24 °C. Isolates were macro-morphologically categorized as having lobed or entire colony margins, and either possessing one of the following colony colours: white, orange or purple. Since the estimated optimum growth temperature of F. langsethiae is typical in temperate summers when small-grain cereals are flowering, it is possible that this species can infect, colonise and possibly contaminate the developing grains with HT-2 and T-2 toxins which are of food safety concern.

Aggressiveness of Fusarium langsethiae isolates towards wheat, barley and oats in an in vitro leaf assay

Aggressiveness of Fusarium langsethiae isolates towards wheat, barley and oats in an in vitro leaf assay Fusarium langsethiae has been identified as the primary producer of HT-2 and T-2 in European cereals. HT-2 and T-2 are considered as two of the most potent trichothecenes mycotoxins and a public health concern in Europe. There is currently no legislation on HT-2 and T-2, however, there is a discussion limit of 500 μg kg-1 in unprocessed oats, 200 μg kg-1 for oat products and 50 μg kg-1 for infant food. There are limited data regarding F. langsethiaes pathogenicity and mycotoxin production, but it is evident that its behaviour deviates from traditional trichothecene-producing Fusarium species. This experiment was aimed at assessing the aggressiveness (measured by lesion length) of 20 different F. langsethiae isolates on wheat, barley and oats using an in vitro detached leaf assay. There was a significant (P<0.001) difference between lesion lengths formed by different F. langsethiae isolates used. Isolate Fl/2004/17(a) caused the shortest lesion on all cereals and this was significantly (P<0.001) different from that caused by isolate Fl/0/08/009/1 which caused the longest lesion on all cereals used. A highly significant difference (P<0.001) was also observed between lesions on the different cereals (wheat, barley and oats). Lesions on oats were the longest, followed by barley with wheat showing the shortest lesions.