Interactions between seed-borne wheat pathogens

Fusarium nivale Auct., Fusarium culmorum (W.G.Sm.) Sacc. and Septoria nodorum Berk. were used to inoculate wheat seed either singly or in mixtures. Data collected from glasshouse and field experiments suggest that less pre-emergence death of seedlings and fewer seedlings with lesions occur when F. nivale and F. culmorum are present on the seed together than when F. culmorum only is present. There is also clear evidence that this reduction in disease cannot be attributed to the halving of the spore concentration of each species in the mixture used.     

Toxinogenicity of<Emphasis Type="Italic">Microdochium nivale (Fusarium nivale)</Emphasis> isolates from cereals in Poland

Microdochium nivale (Fusarium nivale) was found to be frequently occuring in Poland pathogen of small grain cereals heads, causing symptoms similar to those observed after infection ofFusarium species. In consecutive years since 1985 till 1989 the following percentage of wheat and rye ears infected withM. Nivale and withFusarium head blight symptoms was found: 34%, 21%, 42%, 9%, 46% (wheat) and 57%, 43%, 65%, 4%, 47% (rye) heads.However, in naturally infected rye and wheat samples (kernels and chaff), we did not detect toxins usually present in samples infected with fungi of genusFusarium — such as deoxynivalenol and derivatives. TypicalFusarium trichothecene metabolites were also not present in cultures of 11M. nivale strains, growing 3–5 weeks on rice (45% water content) at 20°C. Cultures of two typical isolates on wheat grain (strain KF 1124) and on rice (KF 245) were found to be non toxic to broiler chickens when present in amount 20–40% in their diet. It can be concluded thatM. nivale (F. nivale) representatives in Poland did not produce toxic metabolites neither under laboratory condition nor after cereal ears infection under field conditions.     

Quantitative Fusarium spp. and Microdochium spp. PCR assays to evaluate seed treatments for the control of Fusarium seedling blight of wheat

AIMS: To develop sensitive quantitative PCR assays for the two groups of pathogens responsible for Fusarium seedling blight in wheat: Fusarium group (Fusarium culmorum and Fusarium graminearum) and Microdochium group (Microdochium nivale and Microdochium majus); and to use the assays to assess performance of fungicide seed treatments against each group. METHODS AND RESULTS: Primers conserved between the species within each group were used to develop competitive PCR assays and used to quantify DNA of each group in wheat seed produced from inoculated field plots. Seed was used in seed treatment efficacy field experiments and the amount of DNA of each group was determined in emerged seedlings. The performance of treatments towards each group of pathogens was evaluated by comparison of the reduction in DNA in seedlings emerged from treated seed compared with untreated seed. CONCLUSIONS: DNA from the two groups of pathogens causing Fusarium seedling blight of wheat can be quantified separately using the competitive PCR assays. These assays show improved sensitivity compared with those previously reported for the individual species and allowed the quantification of pathogen DNA in seed and seedlings. Significant reductions in pathogen DNA were evident for each seed treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantification of DNA for each group allows the evaluation of seed treatment performance towards the two components of Fusarium seedling blight disease complex. The approach taken and the assays developed in this study will be of use for the study of other Fusarium disease complexes and their control. Based on the results reported here on the seedling stage of crop development, further studies that examine the control of seed-borne pathogens through fungicide seed treatments throughout the growing season are warranted.     

Production of refusal factors by Fusarium strains on grains.

Corn fermented with strains of Fusarium culmorum NRRL 3288, F. poae NRRL 3287, F. moniliforme NRRL 3197, and F. nivale NRRL 3289 at 28 degrees C for 13 days was refused when fed to 30- to 60-pound (about 13.6- to 27.2-kg) swine. Analyses of the refused corn for trichothecenes (T-2, HT-2, acetyl T-2, fusarenon-X, and vomitoxin) showed that only the corn fermented with F. culmorum contained vomitoxin. None of these five trichothecenes could be detected in the other refused corn.F. culmorum grown on rice at 28 degrees C for 13 days also produced vomitoxin.     

Occurrence and Distribution of Microdochium and Fusarium Species Isolated from Durum Wheat in Northern Tunisia and Detection of Mycotoxins in Naturally Infested Grain

An outbreak of Fusarium Head Blight of durum wheat occurred in 2004 being localized in sub-humid and higher semi-arid region of Northern Tunisia. A mycological survey carried out throughout these regions, revealed that 78% of the prospected fields were infested. Results of the morphological and molecular identification, showed that the most common species isolated from diseased wheat spikes was Microdochium nivale var. nivale (63.5%), followed by Fusarium culmorum (26%), F. pseudograminearum (9%) and F. avenaceum (1.5%). To evaluate mycotoxin content of naturally infected grain, the amounts of trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from 45 fields were quantified by RIDASCREEN DON Enzyme Immunoassay Kit (ELISA) . This study showed that the infection levels in freshly harvested grain were very low and the maximum deoxynivalenol (DON) level of the positive samples was 53 ppb. This is the first report on the natural occurrence of DON in naturally infected wheat grain sampled from Northern Tunisia.     

Detection of Fusarium culmorum in wheat by a surface plasmon resonance-based DNA sensor

A surface plasmon resonance (SPR) sensor based on DNA hybridization has been developed for the detection of Fusarium culmorum, a fungal pathogen of cereals. A 0.57 kbp DNA fragment of F. culmorum was amplified by specific primers and a 25-mer oligonucleotide probe was selected within the sequence of the PCR amplicon. After biotinilation, the probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The effect of denaturating agents (formamide and urea) and ionic strength (NaCl) on hybridization efficiency of double-stranded PCR products with the immobilized probe and the specificity of the probe were investigated. The SPR biosensor was successfully used for the detection of F. culmorum in culture material of different strains and in naturally infected wheat samples. Tested on fungal cultures, it showed a good selectivity for F. culmorum against other species of either Fusarium or other fungal genera. A background signal was observed in wheat samples strictly depending on the DNA amount of the testing matrix. Testing 30 ng of durum wheat DNA the detection limit was 0.06 pg of F. culmorum DNA. The developed PCR-SPR assay allowed to detect F. culmorum with sensitivity and specificity higher than gel-electrophoresis analysis.     

Analysis of Microdochium nivale isolates from wheat in the UK during 1993

A total of 144 isolates of Microdochium nivale from stem bases of winter wheat were taken from 30 sites and 91 isolates from grain were taken from seven sites throughout the UK. Identification by polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region followed by restriction enzyme digestion of the PCR product revealed that 70% of stem base isolates were M. nivale var. majus and 30% var. nivale while 93% grain isolates were var. majus and 7% var. nivale. Almost all isolates were resistant to the benzimidazole fungicide benomyl. Perithecial production in vitro was more common in var. majus isolates and occurred in almost all grain isolates, but was less common in stem base isolates. The implications of the findings in terms of epidemiology and chemical control of this important cereal pathogen are discussed.     

Effects of Fusarium nivale and F. culmorum on the establishment of four species of pasture grass

Cocksfoot cv. Aberystwyth S 26 and timothy cv. Scots were found to be significantly more susceptible to pre-emergence death caused by Fusarium nivale and F. culmorum than were either perennial ryegrass cv. Gremie, or Italian ryegrass cv. RvP. The extent to which each grass was affected by Fusarium at the pre-emergence stage was markedly influenced by both the air temperature and the level of inoculum present in the soil at the time the seeds were sown. Under all conditions, however, cocksfoot and timothy suffered greater reductions in emergence when sown in soil containing either species of Fusarium than did the ryegrasses.
Evidence is presented which suggests that the extreme susceptibility of cocksfoot and timothy to pre-emergence damage is related to the slow germination of the former and the small seed size of the latter.
In addition to their direct effects on seedling numbers both fungi reduced the vigour of those seedlings which did emerge, cocksfoot and timothy being particularly badly affected.     

Pathogenicity of fungi associated with wheat and barley seedling emergence and fungicide efficacy of seed treatment

Presented study focused on the influence of Cochliobolus sativus isolates origin on pathogenicity towards wheat and barley seedlings in comparison with pathogenicity of certain Fusarium species and Microdochium nivale. The efficacy of fungicide seed treatment against C. sativus was estimated. The C. sativus isolates were collected from different locations and were isolated from wheat, barley and sunflower seeds. The pathogenicity of C. sativus, Fusarium species and M. nivale towards germinating seedlings were expressed as germination (GA) retardation and coleoptile growth rate retardation (CGR). Of wheat only, the CGR was significantly influenced by the isolate origin. The C. sativus isolates obtained from sunflower seeds were the most aggressive. Of the barley seeds, the barley isolates were the most aggressive. Barley was significantly more susceptible to damage by C. sativus isolates than wheat. The pathogenicity of tested fungal species declined in the order: F. culmorum, F. graminearum, C. sativus, F. avenaceum, M. nivale, F. poae for both barley and wheat. The results highlighted high pathogenicity potential of C. sativus equal to that of F. avenaceum and M. nivale. The symptoms of C. sativus on coleoptile and roots were very similar or the same as the symptoms caused by Fusarium species and M. nivale, except of white, pink or red colours. Of wheat sprouts, the fungicide efficacy (FE) against C. sativus declined in the order: tebuconazole + thiram, carboxin + thiram, quazatine, difenoconazole, iprodione + triticonazole (in term of GA) and carboxin + thiram, iprodione + triticonazole, tebuconazole + thiram, difenoconazole, quazatine (in term of CGR). In barley, the FE declined in the order: carboxin + thiram, iprodione + triticonazole, tebuconazole + thiram, difenoconazole, quazatine (in term of GA) and carboxin + thiram, tebuconazole + thiram, difenoconazole, iprodione + triticonazole, quazatine (in term of CGR).     

Fusarium culmorum Infection of Barley Seedlings: Correlation between Aggressiveness and Deoxynivalenol Content

Fusarium culmorum is a serious plant pathogen, especially on cereals. The production of deoxynivalenol (DON) by F. culmorum is believed to play a role in pathogenesis. This relationship has been almost exclusively studied in connection with head blight. The present paper reports the first finding of DON in cereal seedlings infected with F. culmorum . A pathogenicity test was performed, including 70 isolates of this pathogen from different sites within northern and central Europe. All isolates caused disease on barley seedlings. For 15 isolates with varying aggressiveness, the DON content in the 19-day-old-barley seedlings was determined. There was a significant correlation between DON concentration and disease index. The aggressiveness of two outlying isolates with very low DON production is discussed. The results indicate that for F. culmorum isolates of the DON chemotype, production of this toxin influences the aggressiveness of the isolates towards barley seedlings.     

Influence of the interactions among ecological variables in the characterization of zearalenone producing isolates of Fusarium spp

To carry out the physiological characterization of Fusarium graminearum and F. culmorum isolates with regard to its zearalenone producing ability, an in-depth experiment with a full factorial design was conducted. The effects and mutual interactions of temperature, moisture, substrate and isolate on the production of the toxin were studied. The study was done with twelve isolates of Fusarium (7 of F. graminearum and 5 of F. culmorum). The analysis of variance shows that there is a complex interaction of all of these factors, which can influence the relative concentrations of the mycotoxin produced, and hence, the correct physiological characterization of the strain. All the tested cultures were susceptible to invasion by Fusarium. The moisture content of grains (water activity values 0.960, 0.970 and 0.980) did not constitute a limiting factor for fungal growth or ZEA production, but incubation temperature (15 degrees C, 20 degrees C, 28 degrees C, and 32 degrees C) affected the rate of zearalenone synthesis. Very low or undetectable ZEA production was observed at 32 degrees C. All tested isolates showed a characteristic behavior concerning the optimum temperature for ZEA production, which was usually 20 degrees C maintained during the whole incubation period. This finding, which does not agree with other reports obtained with strains from different origins, suggests that there are genetic differences that would explain the particular physiological behavior of each isolate related to the optimal production conditions for ZEA. The existence of significant differences regarding the susceptibility of the assayed cereal grains (wheat, corn and rice) used for ZEA production by the different Fusarium species (F. graminearum and F. culmorum) is described for the first time in this paper.     

Conditions and characteristics of the early stages of the sexual interaction of Pichia amethionina var. amethionina and Pichia amethionina var. pachycereana yeast cells

Pichia amethionina varieties have different sensitivity to the acidity of the medium: P. amethionina var. amethionina has the optimum pH 3 for agglutination and 5 for conjugation; P. amethionina var. pachycereana has the optimum pH 4-6 for agglutination and 6 for conjugation. The optimum temperature for the both organisms is 24 degrees C. Under the optimum conditions, P. amethionina var. amethionina has a 98% agglutination and a 28% conjugation; for P. amethionina var. pachycereana, these values are 83 and 55%, respectively. The agglutination of the two varieties does not depend on the presence of glucose. The conjugation of P. amethionina var. pachycereana does not occur in a medium without glucose while the conjugation of P. amethionina var. amethionina is reduced twofold under these conditions. Inositol, biotin and folic acid produce positive effect on the conjugation of P. amethionina var. pachycereana, but only folic acid favours the conjugation of P. amethionina var. amethionina. No differences have been found between the varieties by staining their cells and zygotes with FITC-ConA.     

Comparison of environmental profiles for growth and deoxynivalenol production by Fusarium culmorum and F. graminearum on wheat grain

AIMS: Comparisons were made of the effect of water activity (a(w) 0.99-0.85), temperature (15 and 25 degrees C) and time (40 days) on growth/production of the trichothecene mycotoxin deoxynivalenol (DON) by Fusarium culmorum and Fusarium graminearum on wheat grain. METHODS AND RESULTS: Studies examined colonization of layers of wheat grain for 40 days. Fusarium culmorum grew optimally at 0.98 a(w) and minimally at 0.90 a(w) at 15 and 25 degrees C. Colonization by F. graminearum was optimum at 0.99 a(w) at 25 and 0.98 a(w) at 15 degrees C. Overall, temperature, a(w) and their interactions significantly affected growth of both species. Production of DON occurred over a much narrower range (0.995-0.96 a(w)) than that for growth. Optimum DON was produced at 0.97 and 0.99 a(w) at 15 and 25 degrees C, respectively, by F. culmorum, and at 0.99 a(w) and 15 degrees C and 0.98 a(w) at 25 degrees C for F. graminearum. Statistically, one-, two- and three-way interactions were significant for DON production by both species. CONCLUSIONS: This suggests that the ecological requirements for growth and mycotoxin production by such species differ considerably. The two-dimensional profiles on grain for DON production by these two species have not been examined in detail before. SIGNIFICANCE AND IMPACT OF THE STUDY: This type of information is essential for developing climate-based risk models for determining the potential for contamination of cereal grain with this trichothecene mycotoxin. It will also be useful information for monitoring critical control points in prevention of such toxins entering the wheat production chain.     

Studies on the regeneration-restore and karyotype of protoplast in Metarhizium anisopliae var. majus

Abstract:  The characteristics and regeneration-restore of protoplasts and its karyotype of an insect pathological fungus, Metarhizium anisopliae var. majus were studied. Among the protoplasts, 25.3% were without a nucleus, and 74.7% contained a nucleus. Among the nucleus protoplasts, 53.6% contained a single nucleus. The regeneration-restore of protoplasts was of three distinct shapes. Considering the frequency of regeneration and the growing speed of the colony, 0.7 mol/l glucose was the optimum as osmotic stabilizer of culture medium in the regeneration-restore of the protoplasts. The chromosomal DNA molecules of M. anisopliae var. majus have been separated into seven bands by pulsed-field gel electrophoreses. Using the Schizosaccharomyces pombe chromosomes as size standard, the size of chromosomal DNA was estimated to be 1.1–6.5 Mb and its karyotype exhibited polytypism among strains.     

Fusarium head blight: distribution in wheat in Latvia

Fusarium head blight (FHB) of wheat has, in recent years, been a very important worldwide disease in intensive growing of cereal. The objectives of this study were to evaluate the occurrence of FHB in wheat in Latvia and to identify the Fusarium species involved. This paper describes the distribution of Fusarium species that were isolated from samples representing winter and spring wheat varieties in Latvia, identified both by the classical morphological analyses of J. Leslie and B. Summerell (2006) and by PCR. The FHB incidence range in winter wheat was 1-20%, in spring wheat was 1-42%. The most significant factor affecting the incidence of fusarial head blight in wheat in Latvia was heightened temperature at the time of an thesis of wheat. In winter wheat 9 Fusarium species caused FHB: F. culmorum, F. avenaceum, F. graminearum, F. equiseti, F. poae, F. oxysporum, F. cerealis, F. sporotrichoides and F. verticillioides were identified by morphological characterization, and 5 were confirmed by PCR-analysis. After experience of 5 years, it can be concluded that the most frequent in winter wheat were F. poae and F. culmorum. In spring wheat from F. culmorum was dominant among 8 Fusarium species. Among 13 varieties of spring wheat, three were sensitive ('Chamsin', 'W 166', 'Azurite') and one was resistant ('Granny') to FHB in conditions of high natural infection in 2009. The monitoring surveys demonstrate a significant presence of FHB in spring wheat in conditions of heightened temperature at the time of flowering in Latvia.     

Effects of organomercury treatment of wheat seed on Fusarium seedling disease in inoculated soil

In growth room experiments, treatment of wheat seed with phenyl mercuric acetate significantly reduced seedling disease caused by soil-borne Fusarium culmorum, F. nivale and F. avenaceum in inoculated soil. Seed treatment improved the low germination caused by F. culmorum, but did not affect the reduced seedling vigour caused by F. nivale. Seed treatment was normally as effective in conditions favouring disease as it was in less favourable conditions. Conditions favouring disease in soil inoculated with F. culmorum were high inoculum level, deep sowing and dry soil.     

Screening Selected Fungi for Antagonism towards Pseudocercosporella herpotrichoides (Fron) Deighton,the Cause of Eyespot Disease of Cereals

In the laboratory, a dual culture primary screen using wheat straw agar (WSA) showed that 64 out of 152 selected fungi inhibited the in vitro growth of Pseudocercosporella herpotrichoides by 50% or more. An ‘asymmetric’ species of Penicillium isolated from wheat stubble was the most effective, inhibiting growth of the pathogen by 70%. The results were similar using both W‐type and R‐type isolates of P. herpotrichoides. Other fungi showing in vitro antagonism included: Trichoderma viride, T. koningii, T. harzianum, Chaetomium globosum, Acremonium persicum, Botryotrichum piluliferum, Sordaria alcina, Microdochium bolleyi, Rhizoctonia cerealis and Laetisaria arvalis.

In a glasshouse secondary screen, the 64 fungi showing antagonism in vitro were assessed further for their ability to reduce eyespot disease symptoms of wheat seedlings using a straw collar co‐inoculation technique. Of them, 13 fungi reduced disease symptoms significantly: a Chaetomium sp. from stubble, two Fusarium spp., a F. culmorum isolate T. viride, T. koningii, T. harzianum, B. piluliferum, M. bolleyi, L. arvalis, the ‘asymmetric’ Penicillium sp. and two unidentified fungi from stubble. A F. culmorum isolate was the most effective antagonist on average, giving disease reductions of 54 and 58% for the W‐type and R‐type of P. herpotrichoides respectively. M. bolleyi, T. harzianum and the F. culmorum isolate were the most effective antagonists of the W‐type, all three reducing disease by 54%. T. viride was most effective against the R‐type, reducing disease by 64%.     

Two-dimensional environmental profiles of growth,deoxynivalenol and nivalenol production by Fusarium culmorum on a wheat-based substrate

AIMS: To determine the effect of interacting conditions of water activity (aw, 0.99-0.85), temperature (15, 25 degrees C) and time (40 days) on growth and production of the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) by Fusarium culmorum on a wheat-based agar medium. METHODS AND RESULTS: Fusarium culmorum grew optimally at 0.995aw and minimally at 0.90 at both 15 and 25 degrees C. No growth was observed at <0.90aw. Overall, temperature, aw and their interaction had a statistically significant effect on the growth rate of F. culmorum. Production of both DON and NIV were over a much narrower range (0.995-0.95aw) than that for growth. The highest concentrations of DON and NIV levels were produced at 0.995aw and 0.981aw at 25 degrees C, respectively, after 40 days of incubation. Statistically, aw, temperature and incubation time, and aw x temperature and temperature x incubation time had a statistically significant effect on DON/NIV production. CONCLUSIONS: This is the first detailed report on the two-dimensional environmental profiles for DON/NIV production by F. culmorum in the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of a hazard analysis critical control point (HACCP) approach, this type of information is critical in monitoring critical control points for prevention of DON/NIV entering the wheat production chain.     

The role of chitinese of Serratia marcescens in controlling the production of zearalenone by Fusarium graminearum

An experiment was designed to determine the role of the chitinase of S. marcescens in controlling the production of zearalenone by F. graminearum isolated from diseased wheat plants. Fusarium graminearum, F. avenaceum, F. equiseti, F. culmorum, and F. solani were isolated from diseased wheat plant. F. graminearum culture materials were highly pathogenic for wheat seedlings, toxic to ducklings and produced high levels of zearalenone. S. marcescens was grown on the cell wall of F. graminearum and its components, chitin and laminarin as a sole carbon source. The release of N-acetyl-D-glucosamine from chitin, F. graminearum cell wall and living or drying mycelium indicated substrate degradation. S. marcescens applied to F. graminearum infested wheat kernels decreased greatly the occurrence of zearalenone after 4 weeks of incubation. F. graminearum culture materials treated with S. marcescens proved to be non-toxic to ducklings and wheat seed germination.