Diversity and mycotoxin production by Fusarium temperatum and Fusarium subglutinans as causal agents of pre-harvest Fusarium maize ear rot in Poland.

Maize ear rot is a common disease found worldwide, caused by several toxigenic Fusarium species. Maize ears and kernels infected by Fusarium subglutinans contained significant amounts of beauvericin, fusaproliferin, moniliformin, and enniatins. In 2011, F. subglutinans sensu lato has been divided into two species: Fusarium temperatum sp. nov. and F. subglutinans sensu stricto, showing different phylogeny and beauvericin production within the populations of maize pathogens in Belgium. Isolates of the new species-F. temperatum-were also identified and characterized in Spain, Argentina, Poland, France, and China as one of the most important pathogens of maize. Moreover, F. temperatum was proved to be pathogenic to maize seedlings and stalks. We identified Fusarium isolates obtained from diseased maize ears collected between 2013 and 2016 in Poland (321 isolates). Based on morphological analyses, six Fusarium species were identified. Molecular identification performed on the set of selected isolates (42 isolates) revealed 34 isolates to be F. temperatum and only five to be F. subglutinans. Interestingly, the phylogenetic analysis showed that the population of F. temperatum infecting maize in Poland remained quite uniform for over 30 years with only a few exceptions. For the first time, a single isolate of Fusarium ramigenum was detected from the area of Poland. Significant amounts of BEA were found in Fusarium-damaged kernels. The same kernel samples contained also enniatins A1, A, B1, and B. The results clearly demonstrate the occurrence of F. temperatum as maize pathogen in Poland for over the last three decades.

Kinetics of fumonisin B1 formation in maize ears inoculated with Fusarium verticillioides.

The kinetics of fumonisin B1 (FB1) biosynthesis have been examined in ears of four botanical varieties Zea mays var. indentata, Zea mays var. indurata, Zea mays var. saccharata and Zea mays var. everta inoculated with F. verticillioides isolates at silking stage. The level of mycotoxin accumulated in kernels was correlated with sample harvest time (r = 0.73) and ergosterol content (r = 0.70). FB1 biosynthesis was influenced by amylose, starch and moisture contents, which undergo dynamic changes during grain formation. A comparative analysis of ear infection rates and efficiency of FB1 biosynthesis showed that these are separate characters and their expression could be independent.

Powdery mildew resistance genes in wheat: verification of STS markers.

Five accessions of Aegilops speltoides and 67 European wheat cultivars (winter and spring) originating from the Czech Republic, Germany, Poland, Russia, Slovakia, United Kingdom, and 4 non-European wheat cultivars from Brazil and the USA were examined with molecular Sequence Tagged Site (STS) markers for resistance genes to powdery mildew: Pm 1, Pm 2, Pm 3 and Pm 13. All markers gave clear, repeatable results, although three of them (Pm 1, Pm 2 and Pm 3) appeared as not specific for resistance genes. Comparison of STS analysis results with Pm genes, postulated as the reaction type after inoculation with differential isolates of Erysiphe graminis f.sp. tritici (Blumeria graminis), revealed a high number of disparities. The marker for Pm 13 was not detected in any examined cultivar but was present in five accessions of Aegilops speltoides.

Molecular markers for leaf rust resistance genes in wheat.

Over 100 genes of resistance to rust fungi: Puccinia recondita f. sp. tritici, (47 Lr - leaf rust genes), P. striiformis (18 Yr - yellow rust genes) and P. graminis f. sp. tritici (41 Sr - stripe rust genes) have been identified in wheat (Triticum aestivum L.) and its wild relatives according to recent papers. Sixteen Lr resistance genes have been mapped using restriction fragments length polymorphism (RFLP) markers on wheat chromosomes. More than ten Lr genes can be identified in breeding materials by sequence tagged site (STS) specific markers. Gene Lrk 10, closely linked to gene Lr 10, has been cloned and its function recognized. Available markers are presented in this review. The STS, cleaved amplified polymorphic sequence (CAPS) and sequence characterized amplified regions (SCAR) markers found in the literature should be verified using Triticum spp. with different genetic background. Simple sequence repeats (SSR) markers for Lr resistance genes are now also available.

Genetic determination of variability of barley doubled haploids inoculated with Fusarium culmorum (W.G.Sm.) Sacc. with regard to mycotoxin accumulation and reduction in yield traits.

The genetic determination of variability of barley doubled haploid (DH) lines in regard of their susceptibility to Fusarium head blight caused by Fusarium culmorum was studied. The susceptibility was evaluated in 3-year field experiment on the basis of reduction in yield traits and myotoxin accumulation in infected kernels. The following traits were analysed in inoculated and control plants: kernel number and weight per ear, 1000-kernel weight, percentage of plump kernels (>2.5 mm), deoxynivalenol (DON) content and nivalenol (NIV) content of kernels. On the basis of the obtained data, heritability coefficient (ratio of genotypic to phenotypic variance) was assessed, and genetic parameters as well as the number of effective factors were estimated. Heritability coefficients calculated from two-way analysis of variance, i.e.regarding the influence of years and year x genotype interaction, appeared to be exceptionally low and ranged from 5.2% for the reduction in plump kernels to 38.2% for the reduction in 1000-kernel weight. In the case of mycotoxin accumulation about 60% of the observed variability in NIV concentrations and 30% in DON concentration resulted form genetic differences among lines. Additive effects of genes were important for all the analysed traits. Significant effects of dominance and dominance x dominance were observed for 1000-kernel weight and percentage of plump kernels. Moreover, it was found that the observed variability in yield trait reduction resulted from segregation of 5-6 effective factors, DON contents from 4 factors, while NIV content from 5 factors.

The effects of cereal substrate and temperature on production of beauvericin, moniliformin and fusaproliferin by Fusarium subglutinans ITEM-1434.

One strain of Fusarium subglutinans (ITEM-1434) isolated from maize ear rot in Poland was tested for the ability to synthesize moniliformin (MON), beauvericin (BEA) and fusaproliferin (FP) on six cereal substrates (wheat, rye, barley, oat, maize and rice kernels) for 3 weeks at 25 degrees C and on rice at three different temperatures (20, 25 and 30 degrees C). Most MON (497 micrograms/g) was produced on rice; most BEA (704 micrograms/g) on wheat or rice, and most FP (422 micrograms/g) on rye. When cultured on rice, F. subglutinans produced the highest levels of BEA and FP at 20-25 degrees C, while MON production was best at 30 degrees C.

Beauvericin production by Fusarium species.

Beauvericin is a cyclohexadepsipeptide mycotoxin which has insecticidal properties and which can induce apoptosis in mammalian cells. Beauvericin is produced by some entomo- and phytopathogenic Fusarium species (Fusarium proliferatum, F. semitectum, and F. subglutinans) and occurs naturally on corn and corn-based foods and feeds infected by Fusarium spp. We tested 94 Fusarium isolates belonging to 25 taxa, 21 in 6 of the 12 sections of the Fusarium genus and 4 that have been described recently, for the ability to produce beauvericin. Beauvericin was produced by the following species (with the number of toxigenic strains compared with the number of tested strains given in parentheses): Fusarium acuminatum var. acuminatum (1 of 4), Fusarium acuminatum var. armeniacum (1 of 3), F. anthophilum (1 of 2), F. avenaceum (1 of 6), F. beomiforme (1 of 1), F. dlamini (2 of 2), F. equiseti (2 of 3), F. longipes (1 of 2), F. nygamai (2 of 2), F. oxysporum (4 of 7), F. poae (4 of 4), F. sambucinum (12 of 14), and F. subglutinans (3 of 3). These results indicate that beauvericin is produced by many species in the genus Fusarium and that it may be a contaminant of cereals other than maize.

Occurrence of the mycotoxin moniliformin in maize (Zea mays L.) ears infected by Fusarium subglutinans (Wollenw. & Reinking) Nelson et al.

Fusarium subglutinans has been identified as a prevailing pathogen of maize ears in Poland in the seasons 1985-1991. About 95-100% of ears with Fusarium ear rot symptoms were infected by this species. Moniliformin was present in all 57 ears with pink ear rot symptoms examined. Fusarium-damaged kernels contained an average of 130.9 mg/kg of moniliformin, with large differences between individual samples each year--from 4.2 mg/kg to 530 mg/kg.

Biosynthesis of gibberellic acid (GA3) and mycotoxins by F. moniliforme sheldon and other species of Liseola section.

F. moniliforme and other species of Liseola section, F. culmorum, F. dlamini, and F. nygamai, were examined for their ability to produce gibberellic acid (GA3), fumonisins, trichothecenes, zearalenone, moniliformin, and bikaverin (TLC method). Gibberellic acid was produced by F. moniliforme strains in liquid medium and on rice kernels with a maximum concentration level of 470 mg/dm3 and 1 g/kg, respectively. No strain isolated in Poland produced GA3. High-yielding gibberellic acid strains produced neither trichothecenes and fumonisins nor other tested compounds. Also the rest of strains of examined species did not produce trichothecenes and other mycotoxins except for fumonisins which were found in rice cultures of F. moniliforme, F. proliferatum, and F. subglutinans. Bikaverin was produced by F. moniliforme always together with fumonisins. Filtrates of liquid cultures of gibberellin producing strains were tested for their toxicity to brine shrimps larvae (Artemia salina). It was found that GA3 presence does not increase toxicity of these filtrates.

Fusarium Poae (Peck) Wollenw. - Occurrence in maize ears, nivalenol production and mycotoxin accumulation in cobs.

Fusarium poae (Peck) Wollenw. occurred In maize ears with "pink rot" during 1985-1993 up to 18% of Fusarium isolates, with maximum frequency in 1990. Nivalenol and fusarenone X were produced under laboratory conditions by 13 out of 14 isolates up to 115 µg/g and 13.3 µg/g respectively. The same isolates produced diacetoxyscirpenol (DAS) up to 21.7 µg/g and 15-monoacetoxyscirpenol (MAS) up to 12.3 µg/g. However, In none of the strain cultures were T-2 toxin and HT-2 toxin detected. In samples of naturally Infected maize grain nivalenol was detected at levels of 1.8-32.5 µg/g and fusarenone X was not present. In corresponding axial stems were present both nivalenol (up to 13.5 µg/g) and fusarenone X (up to 2.4 µg/g).

Mycotoxins in cereal grain (part 15). Distribution of deoxynivalenol in naturally contaminated wheat kernels.

The analysis of deoxynivalenol (DON) in naturally infected wheat samples, after having been separated into four fractions through laboratory sieves, showed very low levels of DON in the fraction of largest kernels >2.8 mm (0 up to 1 mg/kg). The highest concentration of DON was found in fractions 2.2 to 2.5 mm and <2.2mm with up to 14mg/kg and 15mg/kg DON, respectively. In two samples (fractions <2.2mm) nivalenol was detected in concentrations up to 1,4mg/kg.

Bikaverin production by Fusarium species.

Production of bikaverin has been examined in 130Fusarium isolates belonging to 21 species. The highest yield of bikaverin was produced on autoclaved rice - up to 2.5g/kg of dry culture. Bikaverin was produced by the following species:F verticillioides, F sacchari varsubglutinans, F proliferatum, F anthophilum, F oxysporum, F dlamini, F nygamai, F napiforme, andF solani. SpeciesF coeruleum, F poae, F sporotrichioides, F tricinctum, F chlamydosporum, F culmorum, F graminearum, F cerealis (F crookwellense), F avenaceum, F acuminatum, andF equiseti did not produce bikaverin.The production of bikaverin determines the colour of the mentionedFusarium species cultures on agar media and on rice. The pigment has indicator properties and changes colour from red in acidic solution to violet-blue in alkaline. The role it plays in fungus metabolism is not elucidated.

A survey of the occurrence of the mycotoxin moniliformin in cereal samples from sources worldwide.

A method is reported for the determination of the Fusarium mycotoxin moniliformin in cereals. The samples after extraction with acetonitrile/water are cleaned-up on a combination of reverse-phase and strong-anion exchange disposable cartridge columns. The extract is then analysed by ion-pair HPLC with UV detection. The method gave recoveries from 81 to 96% and a limit of detection of 0.05 mg/kg. A UK survey of 36 samples of maize products (principally meal and flour) generally showed detectable but low levels of contamination ranging from 0.05 to 0.25 mg/kg (with the exception of three samples where moniliformin levels were less than 0.05 mg/kg). Sixty-four samples of maize from ten different countries showed generally higher levels of moniliformin contamination, with samples from Gambia and South Africa containing 3.16 and 2.73 mg/kg respectively. Field samples of maize, oats, wheat, rye and tricticale that were hand-selected as showing signs of visible fungal damage were obtained from Poland. Moniliformin was consistently present at high levels with amounts ranging from 0.5 to 38.3 mg/kg being associated with F. avenaceum contamination and amounts ranging from 4.2 to 399.3 mg/kg being associated with the presence of F. subglutinans.

Possible sources of ochratoxin A in human blood in Poland.

Samples of plant origin and human and porcine blood samples were screened over a long period for the presence of ochratoxin A. Of 1353 cereal samples, 11.7% contained the mycotoxin; of 1372 samples of feed, 1.5%; of 368 bread samples, 17.2%; of 215 flour samples, 22.3%; of 894 porcine serum samples, 37.4%; and of 1065 human serum samples, 7.2%. Seasonal variations in the natural occurrence of ochratoxin A were observed, with an increased percentage of positive samples in the spring. Individual daily intake of the mycotoxin, estimated on the basis of residues in human serum, was found to be 0.4 ng/g of food consumed.

Biosynthesis and preparation of fiveAlternaria metabolites.

Metabolites ofAlternaria alternata were produced on rice as a solid substrate, chosen out of 6 substrates as the most useful. Optimal methods of extraction, purification, and separation of 5 metabolites were elaborated, using liquid - liquid partition, column chromatography, and preparative TLC. Alternariol, alternariol methyl ether, and copper salt of tenuazonic acid were obtained as crystals, altertoxin and altenuene as a film.

Broiler chickens fusariotoxicosis - A histopathological study of lymphoid organs and testes.

The addition of fiveFusarium species, cultured on wheat grain, to the standart chicken diet DKA starter, caused atrophic changes in the thymus and testes, as observed in the microscopic picture of these organs. The degree of lesions were depended on theFusarium species and its amount added to the standart diet.

Diacetoxyscirpenol production by Fusarium sambucinum strain KF 735 on solid and liquid media.

The yield of diacetoxyscirpenol (DAS) production by F. sambucinum strain No KF 735, isolated from potato tuber with dry rot symptoms, cultured on solid media and on liquid medium, has been examined.The amount of DAS produced within 28 days at 25°C in the cultures grown on solid media (wheat, rye, rice, oats, corn, barley, triticale and malt) reached 238mg/kg±9 to 789±16mg/kg (mean ± standard error; n=3), on potato cubes -55±3mg/kg and on the potato extract -147±5mg/dcm(3).The best substracts for crystalline compound production were malt and barley grain.

Ecology and taxonomy ofFusarium species in Poland.

The paper presents 21Fusarium species occurring in Poland on the field crops /mostly on cereals, maize, potato and Papilionaceae plants/, woody plants, grasses, vegetables and ornamentals as well as on kernels or seeds of these hosts and soils. Additionally the commonly observed symptoms on above-mentioned plants and the informations about regions of the highest disease occurrence are added.However the paper results mainly from authors' investigation on theFusarium species occurrence in Poland, it encloses also the available, post-war literature data on the fusariosis in Poland in the past.Majority ofFusarium species cited had been identified according to Nelson et al, taxonomic system. Comparative listing of the monographs / 1,11, 21/ is presented.

A study of the correlation between the amount of deoxynivalenol in grain of wheat and triticale and percentage ofFusarium 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.