In vitro competition between <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">Epicoccum nigrum</Emphasis> on media and wheat grains

Competitive effects between Fusarium graminearum, causing Fusarium head blight, and the endophyte Epicoccum nigrum, were performed in in vitro competition assays between the two species. Two E. nigrum isolates were isolated from wheat grains and tested as competitors against two F. graminearum isolates. A dual petri dish assay showed that E. nigrum reduced the mycelial growth of F. graminearum and vice versa. A glass slide assay revealed that E. nigrum crude cultural filtrate also had reducing effect on the growth of F. graminearum comparable to that of E. nigrum spore suspensions. Microscopy showed hyphae of F. graminearum and E. nigrum with many side branches when in close proximity, in contrast to pronounced apical hyphal growth when growing alone. Combinations of F. graminearum and E. nigrum on sterilised wheat grains were studied over time by qPCR. F. graminearum biomass was significantly reduced in inoculations applying E. nigrum three days prior to F. graminearum. In conclusion, these results showed competition and mycelial behaviour effects between F. graminearum and E. nigrum and support that E. nigrum may have potential to reduce F. graminearum infections in wheat. Competition experiments should be carried out in planta to study the interaction further.     

Validation of a modified Petri-dish test to quantify aggressiveness of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> in durum wheat

Fusarium graminearum is a common agent causing Fusarium head blight (FHB) on wheat throughout the world. Aggressiveness is crucial for understanding the interaction between host-pathogen in the FHB-wheat system. In this paper, we modified and validated the Petri-dish test originally described by Mesterhazy (Phytopathologische Zeitschrift 93:12–25, 1978) to quantify the aggressiveness of 25 F. graminearum strains using four durum wheat cultivars with different resistance levels for FHB. The results were highly significant and correlated with those obtained using adult plants in the growth chamber and in the field (r = 0.94, P < 0.001 and r = 0.65, P < 0.001, respectively). The Petri-dish test was further investigated for its repeatability and stability in different durum wheat cultivars and highly significant correlation coefficients were obtained (r = 0.90–0.91 (P < 0.001), 0.89–0.95 (P < 0.001), respectively). In this study, we also demonstrated that germination rate reduction and coleoptile length reduction are parameters involved with aggressiveness of F. graminearum. The mean of three disease parameters from the modified Petri-dish method is introduced in this paper as a new parameter for aggressiveness and named “Petri-dish aggressiveness index”. The results obtained reveal that this modified Petri-dish test is rapid, reliable and stable with different durum wheat cultivars, and yields highly significant correlation coefficients with floret and ear inoculations, thus it is suitable to be used for quantification of aggressiveness of F. graminearum.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

Fusarium rot of hyacinth caused by <Emphasis Type="Italic">Gibberella zeae</Emphasis> (anamorph: <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic"> graminearum</Emphasis>)

Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium rot of hyacinth” as a new disease because only the anamorph, F. graminearum, was identified on the diseased host plant. The authors contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively.     

Characterisation of novel <Emphasis Type="Italic">Fusarium graminearum</Emphasis> microsatellite markers in different <Emphasis Type="Italic">Fusarium</Emphasis> species from various countries

Fifteen novel microsatellite markers were isolated from Fusarium graminearum. The level of polymorphism at these novel and 13 previously published microsatellite markers was analysed in 33 F. graminearum strains from Europe, North America, and Nepal. The number of alleles for each of the novel markers ranged from 4 to 20 and gene diversity from 0.417 to 0.962. In comparison with the previously published markers, the resolution for distinguishing among different strains was slightly increased. Twenty-seven markers were also detectable in three F. culmorum strains and one F. crookwellense strain. None of the markers was detected in three F. avenaceum and four F. poae strains, underlining the potential use of these microsatellite markers for species differentiation.     

Simultaneous identification of species and trichothecene chemotypes of <Emphasis Type="Italic">Fusarium asiaticum</Emphasis> and <Emphasis Type="Italic">F. graminearum</Emphasis> sensu stricto by multiplex PCR

A multiplex PCR assay was developed for simultaneous identification of the species and trichothecene chemotypes for Fusarium asiaticum and F. graminearum sensu stricto based on the genes related to trichothecene biosynthesis. PCR was carried out in a single reaction with three pairs of primers designed for the tri6 region and one pair of primers designed for tri3. We confirmed that the multiplex PCR was able to identify species and chemotypes for all tested strains of F. asiaticum and F. graminearum s. str. isolated in Japan. This technique would be a useful and rapid tool for diagnosis, epidemiology, and population structure studies of the F. graminearum complex in Japan.     

Infection process of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> in oats (<Emphasis Type="Italic">Avena sativa</Emphasis> L.)

Fusarium head blight in small grain cereals has emerged as a major problem in the Nordic countries. However, the impact of this disease in oats has been less investigated than in other cereals. For this reason we have studied the infection process (the optimal time of infection and infection pathways) of Fusarium graminearum in oats and its subsequent effects on kernel infection, deoxynivalenol (DON) content and germination capacity. In a field experiment the oat cultivar Morton was spray-inoculated at different developmental stages, and the highest kernel infection and DON content and lowest germination percentage were observed when inoculation took place at anthesis. Field grown oats affected by a natural Fusarium head blight epidemic and spray-inoculated field and greenhouse oats were used to study the infection pathway. Results showed that the fungus entered primarily through the floret apex into the floret cavity, where it could infect via the internal surfaces of the palea, lemma and caryopsis. Both visual symptoms and fungal infections started at the apical portions of the florets and progressed to the basal portions. Hyphae of F. graminearum grew more profusely on the anthers than on other floret parts during initial stages of infection. Disease development within the oat panicle was slow and is primarily by physical contact between adjoining florets, indicating that the long pedicels give Type II resistance in oats.     

Comparison of pathogenicity of the Fusarium crown rot (FCR) complex (<Emphasis Type="Italic">F. culmorum</Emphasis>, <Emphasis Type="Italic">F. pseudograminearum</Emphasis> and <Emphasis Type="Italic">F. graminearum)</Emphasis> on hard red spring and durum wheat

Fusarium species involved in the Fusarium crown rot (FCR) complex affect wheat in every stage of development from seedling to grain fill. This study was designed to compare the aggressiveness of the FCR complex members including F. culmorum, F. pseudograminearum and F. graminearum in causing seedling blight, decreased plant vigour and crown rot. To assess their relative pathogenicity, two hard red spring wheat cultivars and two durum wheat cultivars were inoculated in the field with five isolates from each of the three species for two years. Significant differences in patterns of pathogenicity were identified. In particular, F. culmorum caused greater seedling blight while F. pseudograminearum and F. graminearum caused greater crown rot. Greatest yield reductions were caused by F. pseudograminearum. Cultivar differences were identified with respect to seedling disease and late season crown rot. No interactions were identified between cultivar performance and isolates or species with which they were challenged.     

Genetic structure of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> populations isolated from maize in Argentina

Fusarium verticillioides (sexual stage Gibberella moniliformis) is a common fungal pathogen of maize worldwide that also produces fumonisin mycotoxins. Populations of this fungus can be diverse with respect to neutral and selectable genetic markers. We used vegetative compatibility groups (VCGs) and amplified fragment length polymorphisms (AFLPs) to evaluate the genetic structure of three F. verticillioides populations from commercial maize fields in Argentina. Based on work with similar populations from outside South America, we expected individuals within the populations to be genetically diverse, that genotypic variation would be distributed in a manner consistent with random mating, and that populations from different locations would be genetically indistinguishable from one another. We analysed 62 AFLP loci for 133 fungal isolates. All three populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange occurring between the three subpopulations. There was no evidence for linkage disequilibrium at P = 0.05. The low values of G _ST , the lack of frequent private alleles, and the lack of a systemic pattern of linkage disequilibrium all suggest that sexual reproduction is sufficiently common in F. verticillioides and that the dispersal of strains is sufficiently efficient for the population of F. verticillioides in the main maize growing region to be a single randomly mating population with no detectable genetic subdivision. Thus differences in disease and/or toxin production observed in this region are best attributed to differences other than the genetic composition of the population.     

Evidence for natural resistance towards trifloxystrobin in <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

A collection of 55 Fusarium graminearum (Gibberella zeae) strains isolated between 1969 and 2009 in Belgium, Canada, Germany, Italy, Luxembourg, or the USA belonging to the three known chemotypes (3-acetylated deoxynivalenol, 15-acetylated deoxynivalenol and nivalenol) were screened for their sensitivity towards the fungicide trifloxystrobin using a liquid culture assay. None of the isolates was completely inhibited by trifloxystrobin concentrations up to 3 mM. For comparison, prothioconazole completely inhibited fungal growth of a standard isolate at concentrations as low as 0.007 mM. The maximum level of inhibition, which could be obtained by trifloxystrobin, ranged from 14 to 65% among the strains tested and was not significantly affected by the country of origin or by the chemotype. The absence of significant differences in resistance levels between the countries of origin and chemotypes as well as the fact that strains isolated before the market introduction of strobilurins in 1996 also showed a high level of resistance is evidence that this is largely a case of natural resistance and not primarily related to strobilurin use in agriculture.     

<Emphasis Type="Italic">Fusarium temperatum</Emphasis> isolated from maize in France

The presence of Fusarium temperatum in France was investigated by analyzing 40 Fusarium strains, previously isolated from infected maize ears in 2011 and formerly identified as Fusarium subglutinans based on morphological characteristics. In this study, 26 strains out of the 40 were identified as F. temperatum and 14 as F. subglutinans based on sequencing of the translation elongation factor 1α gene. The phylogenetic analysis showed that the two species represented two clades strongly supported by bootstrap values. The pathogenicity of F. temperatum strains was confirmed on maize ears. This study provides new information about F. temperatum isolated from maize in France.     

<Emphasis Type="Italic">Bacillus pumilus</Emphasis> SG2 chitinases induced and regulated by chitin,show inhibitory activity against <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis>

The possible role of chitinase in in vitro growth inhibition of the wheat pathogens Fusarium graminearum and Bipolaris sorokiniana by Bacillus pumilus SG2 was investigated. B. pumilus SG2, a chitinolytic bacterium producing two different chitinases, was previously isolated from the saline deserts of Iran. When grown in Spizizen salts medium with colloidal chitin, B. pumilus SG2 secreted two chitinases into the medium, resulting in growth inhibition of F. graminearum and abortion of hyphal elongation of B. sorokiniana. In contrast, when glucose was used as the carbon source, the chitinases were not expressed and antifungal activity of the B. pumilus SG2 was completely abolished. These results confirmed that expression of the B. pumilus SG2 chitinases is under the control of two types of regulation, special regulation by chitin and global regulation by glucose. We demonstrated that chitinases are the main components that caused hyphal inhibition activity of B. pumilus SG2. Hyphal inhibition of F. graminearum and B. sorokiniana was stable in agar for a minimum of 14 days.     

Natural phenolic acids from wheat bran inhibit <Emphasis Type="Italic">Fusarium culmorum</Emphasis> trichothecene biosynthesis <Emphasis Type="Italic">in vitro</Emphasis> by repressing <Emphasis Type="Italic">Tri</Emphasis> gene expression

The effect of natural phenolic acids from wheat bran on type B trichothecene biosynthesis by Fusarium culmorum was investigated in vitro. Durum wheat bran contained various monomeric forms of phenolic acids, with ferulic acid being the most abundant. In addition, various oligomeric forms of ferulic acid and mainly dimeric forms were also detected. When liquid cultures of F. culmorum were supplemented with a natural wheat bran extract, trichothecene production was fully inhibited. The exact mechanism by which toxin synthesis is repressed remains to be clarified but we showed that the phenolic acid treatment resulted in a drastic reduction in the expression level of structural trichothecene biosynthetic genes. The inhibitory efficiency of the natural phenolic acid extract was significantly higher than that of a reconstituted mixture containing similar concentrations of monomeric forms. Thus, to elucidate the full repression of type B trichothecene production induced by the natural phenolic acid extract from wheat bran, two hypotheses can be raised: (i) a synergistic impact of monomeric and dimeric forms of phenolic acids, (ii) the occurrence of an unidentified oligomeric form able to efficiently repress toxin yield. As a first attempt to investigate the effect of oligomeric forms, one of the most abundant dimer of ferulic acid, the 8-5′-benzofuran dimer, has been synthesized in vitro and was shown to inhibit trichothecene biosynthesis to the same extent than the monomer of ferulic acid.     

Cloning of the pathogenicity-related gene <Emphasis Type="Italic">FPD1</Emphasis> in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB110097     

Infection conditions of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> in barley are variety specific and different from those in wheat

Fusarium head blight is one of the most noxious cereal diseases. Worldwide, F. graminearum (FG) and the mycotoxin deoxynivalenol (DON) is the most dominant species/mycotoxin in barley and wheat. Barley is often produced as on farm feed and thus routine mycotoxin analyses similar to those of cereals for human consumption are not performed. Hence, an early prediction of mycotoxin levels is important for farmers to minimise the risk of contaminated feed but also of contaminated cereals entering the cereal supply chain. Therefore, climate chamber experiments with artificial FG infection of barley investigating the influence of different temperatures (10 °C, 15 °C, 20 °C) and durations (4 h, 8 h, 12 h) at 99% relative humidity were conducted to accumulate data to develop a forecasting system. An up to three times higher DON contamination in the 15 °C treatments for the feed barley variety Ascona was detected compared with the 10 °C and 20 °C treatments. For the malting barley variety Concerto, the prolonged humidity durations had a stronger effect under all tested temperatures and resulted in up to two times higher DON contaminations. In addition, field experiments where spore deposition during anthesis as well as disease incidence, fungal amount and mycotoxins were observed, showed that the overwintered straw treatment resulted, depending on the year, in a three times higher FG incidence and DON content compared with the control and freshly inoculated straw treatment.     

<Emphasis Type="Italic">Fusarium ershadii</Emphasis> sp. nov., a Pathogen on <Emphasis Type="Italic">Asparagus officinalis</Emphasis> and <Emphasis Type="Italic">Musa acuminata</Emphasis>

Two Fusarium strains, isolated from Asparagus in Italy and Musa in Vietnam respectively, proved to be members of an undescribed clade within the Fusarium solani species complex based on phylogenetic species recognition on ITS, partial RPB2 and EF-1α gene fragments. Macro- and micro-morphological investigations followed with physiological studies done on this new species: Fusarium ershadii sp. nov can be distinguished by its conidial morphology. Both isolates of Fusarium ershadii were shown to be pathogenic to the monocot Asparagus officinalis when inoculated on roots and induced hollow root symptoms within two weeks in Asparagus officinalis seedlings. In comparison mild disease symptoms were observed by the same strains on Musa acuminata seedlings.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

<Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subsp. <Emphasis Type="Italic">michiganens</Emphasis>is strains virulence and genetic diversity. a first study in Argentina

Tomato leaves showing severe leaf spot symptoms have been observed and sampled in the central west and southwest Taiwan during 2015 and 2016. The symptoms were similar to those of bacterial leaf spot/late blight diseases, but only Stemphylium-like fungi were consistently isolated from the diseased tomato. Upon spray inoculation of tomato, Stemphylium-like isolates caused leaf spot symptoms identical to those of naturally infected plants, and the pathogenic isolates were successfully re-isolated from inoculated leaves. The tomato-pathogenic isolates were identified as S. lycopersici based on morphological characterization and molecular identification. S. lycopersici has been previously reported to cause gray leaf spot of tomato in the temperate regions, but the majority of S. lycopersici-caused lesions were black/dark brown rather than gray in our surveillance. Accordingly, it is suggested that S. lycopersici-caused disease of tomato is named Stemphylium leaf spot of tomato more appropriately than tomato gray leaf spot. Moreover, S. lycopersici-caused leaf spot disease on tomato has been distributed in major tomato production regions in Taiwan. The information provided by our study will be important for future breeding of tomato cultivars, especially for tomato producers in Taiwan.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> associated with yield-decline of pyrethrum in Australia

Fusarium wilt, one of the destructive diseases of cucumber can be effectively controlled by using biocontrol agents such as Trichoderma harzianum. However, the mechanisms controlling T. harzianum-induced enhanced resistance remain largely unknown in cucumber plants. Here we screened the potent T. harzianum isolate TH58 that could effectively control F. oxysporum (FO). Glasshouse efficacy trials also showed that TH58 decreased disease incidence by 69.7 %. FO induced ROS over accumulation, while TH58 inoculation suppressed ROS over accumulation and improved root cell viability under F. oxysporum infection. TH58 inoculation could reverse the FO-induced cell division block and regulate the proportional distribution of nuclear DNA content through inducing 2C fraction. Moreover, the expression levels of cell cycle-related genes such as CDKA, CDKB, CycA, CycB, CycD3;1 and CycD3;2 in TH58 - pre-inoculated seedlings were up-regulated compared with those infected with FO alone. Taken together, these results suggest that T. harzianum improved plant resistance against Fusarium wilt disease via alterations in nuclear DNA content and cell cycle-related genes expression that might maintain a lower ROS accumulation and higher root cell viability in cucumber seedlings.