Wheat-infectingFusarium species in Poland — their chemotypes and frequencies revealed by PCR assay

ThreeFusarium species:F. graminearum, F. culmorum andF. cerealis were identified in laboratory cultures and in sporodochia from spikelets of scabby wheat. SCAR (sequence characterized amplified region) primers were used to identifyFusarium species and nivalenol (NIV) and deoxynivalenol (DON) chemotypes within species in laboratory cultures and field collected heads harvested in 2006. Results from PCR analyses confirmed preliminary identifications of species on the basis of examination of macroconidia under a light microscope and identification of cultures on agar media. NIV and DON (3Ac-DON and 15Ac-DON) chemotypes were identified using PCR assay. Among samples and isolates ofF. graminearum, the 15Ac-DON chemotype dominated, and among those whereF. culmorum was identified, the 3Ac-DON chemotype prevailed. Only 5 of the 41 isolates ofF. graminearum tested, displayed the NIV chemotype. An increase in the frequency ofF. graminearum and a decrease in the frequency ofF. culmorum were found during 1998 to 2006.     

The occurrence of culmorin and hydroxy-culmorins in cereals

Forty-five samples from 1988–1995 of naturally contaminated grain, barley, wheat and oats, three samples of mixed feed, and 16 samples of grain artificially inoculated with Fusarium culmorum during the flowering stage were analysed for deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-acetyl-DON), culmorin and hydroxy-culmorins. These compounds are secondary metabolites produced by the fungal species F. culmorum and F. graminearum. Acetonitrile-water extract of the samples was purified on a Mycosep^TM#225 column, derivetized using pentafluoropropionic anhydride (PFPA) and analysed by gas chromatography-mass spectrometry (GC-MS). The amount of each of culmorin, 5-, 12-, 14 and 15-hydroxy-culmorin and one unknown hydroxy-culmorin were determined relative to the amount of DON plus 3-acetyl DON for each sample. The ratio between the total amount of culmorin compounds and the DON compounds ranged from 0.14 to 1.07 in the samples. This study shows that there is a strong correlation between the amount of DON present in the grain and the amount of culmorin and hydroxy-culmorins present. The ratio of each of the culmorin compounds relative to the amount of DON compounds were in the same range in the grain artificially inoculated by F. culmorum as found in an earlier study for F. culmorum strains cultivated on rice, while the hydroxy-culmorin profile in the naturally contaminated grain was more similar to what was found for the F. graminearum cultures in the same study [1]. These results indicate that F. graminearum may be a relatively important source for DON in grain also in relatively cold areas. This revised version was published online in June 2006 with corrections to the Cover Date.     

Toxigenic Fusarium species infecting wheat heads in Poland

Toxigenic Fusarium species are common pathogens of wheat and other cereals worldwide. In total, 449 wheat heads from six localities in Poland, heavily infected with Fusarium during 2009 season, were examined for Fusarium species identification. F. culmorum was the most common species (72.1% on average) with F. graminearum and F. avenaceum the next most commonly observed, but much less frequent (13.4 and 12.5% respectively). F. cerealis was found in 1.8% of all samples, and F. tricinctum was found only in one sample (0.2%). Subsequent quantification of the three major mycotoxins (deoxynivalenol, zearalenone and moniliformin) in grain and chaff fractions with respect to associated prevailing pathogen species uncovered the following patterns. Moniliformin (MON) was found in low amounts in all samples with F. avenaceum present. In contrast, deoxynivalenol (DON) and zearalenone (ZEA) were the contaminants of F. culmorum- and F. graminearum-infected heads. The highest concentration of DON was recorded in grain sample collected in Radzików (77 µg g^?1). High temperatures in Central Poland during July and August accompanied with high rainfall in July were responsible for this high DON accumulation. Trichothecene, zearalenone, enniatin and beauvericin chemotypes were identified among 21 purified isolates using gene-specific PCR markers.     

Impact of aggressiveness of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">F. culmorum</Emphasis> isolates on yield parameters and mycotoxin production in wheat

Plant-associated isolates from Fusarium graminearum and F. culmorum were inoculated on wheat in field experiments in 2007 and 2008 to ascertain their influence on fungal colonization of the ears, as well as mycotoxin contamination (deoxynivalenol, DON; nivalenol, NIV; zearalenone, ZEA) and yield parameters in the mature crop after inoculation with or without irrigation. The isolates were assigned to four different groups of aggressiveness on the basis of pathogenic symptom development and mycotoxin production in vitro. Increased levels of trichothecene-producing Fusarium DNA in the ears indicated a successful inoculation of the plants, which resulted in increased DON content in the wheat kernels in 2007. Dry conditions at anthesis markedly suppressed fungal colonization as well as mycotoxin accumulation. However, due to precipitation during the ripening period, yield and thousand-kernel weight were similar whether or not irrigation was applied at the time of inoculation. The level of aggressiveness among the isolates as determined in vitro was not reflected in the field experiment. The activity of the extracellular invertase in developing ears increased as a plant response to pathogen infection, especially when the plants were irrigated at the time of inoculation. In 2008, the Fusarium inoculation of wheat heads did not cause fungal growth and mycotoxin contamination in the grain, because of the dry weather conditions that occurred over the entire period of anthesis and ripening. The risk of future mycotoxin contamination in grains was discussed based on climate change prognosis.     

Contribution of the endogeic earthworm species <Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis> to the degradation of deoxynivalenol and <Emphasis Type="Italic">Fusarium</Emphasis> biomass in wheat straw

In arable fields managed by conservation tillage combined with crop residue mulching, plant pathogen repression is an important ecosystem service to prevent cultivated plants from fungal diseases and mycotoxin contamination. A laboratory microcosm study was conducted to investigate the contribution of the endogeic, geophagous earthworm species Aporrectodea caliginosa as a secondary decomposer to the reduction of the phytopathogenic fungus Fusarium culmorum and its mycotoxin deoxynivalenol (DON) in wheat straw residues. After 5 weeks experimental time, the Fusarium biomass and the DON concentration in aboveground straw were reduced considerably to the same extent both in presence and absence of A. caliginosa. Another substantial reduction of Fusarium biomass and DON concentration was found in belowground straw, which A. caliginosa had buried into the soil. Thus, we conclude that the particular contribution of secondary decomposers like A. caliginosa to the degradation of phytopathogenic fungi like Fusarium species and their mycotoxins like DON in the soil systems has to be assessed as minor.     

A study of the correlation between the amount of deoxynivalenol in grain of wheat and triticale and percentage of<Emphasis Type="Italic">Fusarium</Emphasis> damaged kernels

The correlation between the amount of deoxynivalenol (DON) and the percentage ofFusarium damaged kernels (FDK) in samples of wheat and triticale was studied.Samples of naturally infected wheat grain, collected in 1986, 1987 and 1988 and of triticale collected in 1986 were used.Additionally, artificial inoculated wheat samples (10 genotypes inoculated with 3F. Culmorum strains of weak, medium and severe pathogenicity and samples of 10 triticale genotypes inoculated withF. culmorum. andF. graminearun) were studied. Using statistical methods (the variance analysis, method of least significant difference (LSD), orthogonal contrast (OC) and minimum within groups sum of squares criterion (MSSC)), the samples were divided into two groups with respect to the attribute DON/FDK.To the first group belong samples of wheat and triticale, of which the heads were artificially inoculated with severely pathogenic strainsF. culmorum. In the samples of this group the amount of DON in kernels damaged withFusarium increased by 0,46 mg/kg per 1% of FDK.In the second group, consisting of naturally infected samples and samples from artificially inoculated heads the amount of DON increased 0,30 mg DON/kg per 1% of FDK.The equation for the calculation of approximated amount of DON in farm and commercial lots of wheat and triticale after examination of percentage of FDK is given.     

Analysis of Fusarium causing dermal toxicosis in marram grass planters

In the European coastal dunes, marram grass (Ammophila arenaria) is planted in order to control sand erosion. In the years 1986 to 1991, workers on the Wadden islands in the Netherlands planting marram grass showed lesions of skin and mucous membranes, suggesting a toxic reaction. Fusarium culmorum dominated the mycoflora of those marram grass culms that were used for planting. This plant material had been cut and stored for more than one week in the open. The Fusarium toxin deoxynivalenol (DON) was detected in the suspect marram grass culms. Isolated F. culmorum strains were able to produce DON in vitro in liquid culture as well as in experimentally inoculated wheat heads. Pathogenicity tests, toxin test as well as RAPD analysis showed that the F. culmorum strains were not specialized for marram grass but may form part of the West-European F. culmorum population infecting cereals and grasses. Storage on old sand-dunes with plant debris may have led to the high occurrence of F. culmorum and contamination with DON. Marram grass culms should be obtained from young plantings on dunes on the seaward slopes and cut culms should not be stored.     

Fungal diversity and natural occurrence of fusaproliferin,beauvericin, deoxynivalenol and nivalenol in wheat cultivated in Santa Fe Province,Argentina

The Fusarium diversity and the mycobiota associated with moldy wheat kernels from Santa Fe province, Argentine, was assessed. The wheat cultivated area in Santa Fe province is divided according to agrometeorological conditions into two zones: Zone I (north-central) and Zone II (south). The natural occurrence of Fusarium toxins BEA, FUP, DON and NIV was also determined. Cladosporium was the most abundant of the 19 genera identified, followed by Fusarium, Phoma and Alternaria. Zone II shows a predominance of F. graminearum and F. culmorum. In Zone I, DON was present in 13/32 samples (range 0.43–3.60 mg kg⁻¹) and NIV in 6/32 samples (range 0.11–0.40 mg kg⁻¹). In zone II, DON was found in 11/21 samples (range 0.57–9.50 mg kg⁻¹) and NIV in 4/21 samples (range 0.10–0.60 mg kg⁻¹). BEA and FP were not detected in both zones.     

Influence of localities and winter wheat cultivars on deoxynivalenol accumulation and disease damage by <Emphasis Type="Italic">Fusarium culmorum</Emphasis>

Toxin B — trichothecene deoxynivalenol (DON) is the most frequent Fusarium mycotoxin in Fusarium head blight (FHB) disease produced by Fusarium fungi. Thirty-one samples of naturally cultivated winter wheat were collected from different localities in Slovakia and evaluated for DON content, and after an artificial inoculation twelve of winter wheat cultivars were evaluated for FHB, fusarium damaged kernels (FDK) and DON content (resistance Type I and II) during two years. Plants were inoculated at anthesis with a conidial suspension of Fusarium culmorum (W. G. Smith) Sacc. The highest mean contents of DON 1.641 ppm were found in produced potato region (PPR) and 1.654 ppm in produced sugar beet region (PSBR). A positive correlation was found between DON content and rainfall, and a negative correlation was found between content of DON and temperature. Lower positive correlations were found between the contents of DON in 2003 and 2004 in the resistance Type I and Type II in twelve artificially infected cultivars. The significant positive correlations in content of DON were found between resistance Type I and Type II in the years 2003 and 2004. The lowest content of DON was found in the cultivars Alka, Malyska and the highest one in the cultivars Vanda and Boka. The positive correlation between the content of DON and FDK (in %) in head (average 2003 and 2004 years) from artificially infected and analysed cultivars was statistically significant in both resistances Type I and Type II.     

Real-time PCR detection and quantification of Fusarium poae,F. graminearum,F. sporotrichioides and F. langsethiae in cereal grains in Finland and Russia

Abstract

TaqMan real-time quantitative PCR assays were developed for the accurate detection and quantification of DNA from Fusarium poae and F. graminearum species, which are able to produce trichothecenes. These and other PCR assays were used for the quantification of trichothecene-producing Fusarium fungi in cereal grains. A correlation was found between the levels of F. poae DNA and nivalenol and enniatins in barley and between the levels of F. graminearum DNA and deoxynivalenol in oats. The correlations between F. poae DNA and nivalenol and F. graminearum DNA and deoxynivalenol levels were higher than those between these mycotoxins and morphologically determined F. poae and F. graminearum/F. culmorum contamination levels. The use of F. poae specific primers and probe together with F. sporotrichioides/F. langsethiae specific primers and probe in a multiplex qPCR assay yielded results in accordance with those obtained using these primers and probes separately.     

Tissue‐specific and pathogen‐inducible expression of a fusion protein containing a Fusarium‐specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins

Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.‐specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea‐specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea‐specific in transgenic wheat. Single‐floret inoculation of independent transgenic wheat lines of the T₃ to T₆ generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography–mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB‐susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real‐time PCR analysis revealed that the tissue‐specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue‐specific and pathogen‐inducible expression of this Fusarium‐specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain.     

Effects of Field Application of Tebuconazole on Yield, Yield Components and the Mycotoxin Content of Fusarium-infected Wheat Grain

Winter wheat cultivar Basalt was artificially inoculated with Fusarium culmorum at the end of anthesis and treated with the systemic fungicide tebuconazole (Folicur^®) a few days before and/or after inoculation. Check plots remained uninoculated and unsprayed. Head infections, yield, yield components and the percentage of Fusarium‐ infected kernels were determined. Artificial Fusarium inoculation lowered yield significantly by 24.2‐45.0%. Any fungicide treatment saved yield, thousand grain weight and kernel numbers per head. Pre‐infectional application of tebuconazole was superior to application carried out post‐infection. Moreover, the fungicide controlled deoxynivalenol (DON) synthesis in the field to a considerable extent, and enabled good control of Fusarium head blight, glume blotch and the percentage of Fusarium‐infected kernels. The levels of Fusarium kernel infection after harvest clearly reflected the DON content of w heat grain.     

Comparative Analysis of Genetic Chemotyping Methods for Fusarium: Tri13 Polymorphism Does not Discriminate between 3‐ and 15‐acetylated Deoxynivalenol Chemotypes in Fusarium graminearum

Genetic chemotyping is an essential tool for characterizing Fusarium populations causing head blight on wheat and other cereals. Three PCR methods, based on tri cluster polymorphism, were optimized and compared on 94 single‐spore isolates obtained from three continents belonging to F. gramineaurm, F. culmorum, F. poae, F. avenaceum and Microdochium nivale. While the methods based on the tri3, tri7 and tri12 polymorphism correctly identified all the tested strains, the method based on tri13 polymorphism was unable to discriminate between the 3‐ and 15‐acetylated DON forms in F. graminearum. It is advised to avoid the use of tri13 polymorphism for genetic chemotyping of the two acetylated chemotypes.     

Rapid Detection of Colletotrichum lagenarium,Causal Agent of Anthracnose of Cucurbitaceous Crops,by PCR and Real‐time PCR

Rapid and accurate polymerase chain reaction (PCR) and real‐time PCR methods were developed for the detection of Colletotrichum lagenarium, the causal agent of anthracnose, in tissues of squash (Cucurbita moschata), watermelon (Citrullus lanatus), cucumber (Cucumis sativus) and muskmelon (Cucumis melo). PCR assays amplified different internal transcribed spacer sequences from C. lagenarium, so effectively detected this pathogen in infected tissues. PCR analysis with the primer co‐m‐337F1/R1 was able to differentiate C. lagenarium from other fungal pathogens, including Colletotrichum spp., Fusarium spp., Alternaria spp. and Didymella spp. An optimized real‐time PCR assay was developed to detect and monitor C. lagenarium in both infected plant tissues and soil samples. The sensitivity of real‐time PCR can detect down to 1 pg of DNA. Thus, PCR‐based analysis is a useful technique for rapid detection and diagnosis of C. lagenarium in infected plants or infested soils.     

Occurrence of toxigenic strains of Fusarium in maize and barley in Germany

Summary A survey was made of maize and barley in Germany for the occurrence of toxigenic strains of Fusarium and of the mycotoxins produced in culture by these strains.The following 6 species of Fusarium were found: F. avenaceum, F. culmorum, F. equiseti, F.oxysporum, F. poae, and F. tricinctum. The species most commonly isolated from bird-damaged maize ears was F. avenaceum while F. culmorum was consistently isolated from maize stem rot. The predominant species in barley grain was F. poae while F. avenaceum, F. culmorum, and F. tricinctum were also isolated frequently.Cultures on autoclaved maize of all the Fusarium strains were assayed for toxicity by feeding to 1-day-old chickens for 14 days. Some strains of F. avenaceum, F. culmorum, F. equiseti, and F. oxysporum proved to be acutely toxic to chickens and caused mortality as well as marked reductions in weight gain and feed consumption. All the strains of F. poae and F. tricinctum had a low degree of toxicity.Culture material of all the strains were analyzed for the presence of 11 known Fusarium mycotoxins. The following 4 mycotoxins were detected in the strains examined: moniliformin in 9 out of 9 F. avenaceum strains (2 to 760 ppm) and in the single strain of F. oxysporum (1150 ppm); zearalenone in 4 out of 5 F. culmorum strains (320 to 1400 ppm); deoxynivalenol in 3 out of 5 F. culmorum strains.(1 to 15 ppm); and acetyldeoxynivalenol (1 to 2 ppm) in 3 out of 5 F. culmorum strains. This is the first report of moniliformin production by F. avenaceum and F. oxysporum and also the first report of the occurrence of moniliformin-producing Fusarium strains in Europe.     

Molecular identification and determination of the trichothecene chemotypes of fungi causing crown and root in different growth stages of wheat in north of Iran

In order to determine the crown and root agents and their mycotoxins produced in different growth stages of wheat including seedling, tillering and heading, sampling was done in north of Iran, during 2011–2012. From 160 isolates of Fusarium, eight species were obtained including F. graminearum, F. culmorum, F. equiseti, F. nygamai, F. semitectum, F. solani, F. acuminatum and F. oxysporum. Sampling at different growth stages showed that F. graminearum was the predominant causal agent of crown and root at the heading stage, whereas other species of Fusarium were mostly observed at the seedling and tillering stages. Moreover, identification of pathogenic species was confirmed using species-specific primers pairs. In F. graminearum isolates, presence of Tri13 gene, responsible for nivalenol (NIV) and deoxynivalenol (DON) mycotoxins biosynthesis, was detected using specific PCR primers. Finally, the ability of trichothecene production of five F. graminearum isolates was confirmed with high-performance liquid chromatography.     

Response to Fusarium culmorum inoculation in barley

In field tests replicated in 2004 and 2005, 32 cultivars of spring barley were assessed for resistance to Fusarium head blight (FHB) by single floret inoculation and spray inoculation with Fusarium culmorum (W. G. Smith) Sacc. It was found that the weather conditions in individual years affect to a large extent the progression of FHB and production of mycotoxin deoxynivalenol (DON). At the same time, in both years the cultivars reacted to F. culmorum infection similarly with respect to areas under disease progress curve (AUDPC) values and content of mycotoxin DON. Spraying inoculation led to stronger infection. The biggest differences in AUDPC values were observed between the cultivars Brise and Celinka, and weak reaction was found in the cultivars Kompakt and Madonna. The cultivars Kompakt and Tolar were most resistant towards FHB. In both monitored years the variety Ludan contained the lowest amounts of mycotoxin DON. Cultivars with high infection and low DON content (r = 0.78) showed weak positive relationship between resistance to FBH and accumulation of DON (concentration 70–200 mg/kg). This is the first information on FHB and in vivo concentrations of DON in certificated barley cultivars in Slovakia.