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.     

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.     

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.     

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.     

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 Variability of Central European Isolates of the <Emphasis Type="Italic">Fusarium graminearum</Emphasis> Species Complex

The main causative agents of Fusarium head blight in central Europe are Fusarium graminearum and F. culmorum. We examined the mycotoxin producing ability, aggressiveness and molecular variability of F. graminearum isolates. Altogether twenty-six Hungarian, three Austrian isolates and representatives of eight species identified in the F. graminearum species complex were involved in this study. Mycotoxin producing abilities of the isolates were tested by GC-MS and HPLC. The central European isolates were found to belong to chemotype I (producing deoxynivalenol). Most isolates produced more 15-acetyl-deoxynivalenol than 3-acetyl-deoxynivalenol suggesting that they belong to chemotype Ib. All F. graminearum isolates were found to be highly pathogenic in in vitro aggressiveness tests. Phylogenetic analysis of random amplified polymorphic DNA profiles, and restriction profiles of the intergenic spacer region of the ribosomal RNA gene cluster of the isolates allowed clustering of the central European isolates into 17 and 16 haplotypes, respectively. When RAPD and IGS-RFLP data were combined, almost every single central European F. graminearum isolate could be differentiated (27/29 haplotypes). Sequence analysis of a putative reductase gene of some isolates was also performed. Based on molecular data, the majority of the central European isolates belonged to F. graminearum sensu stricto characteristic to the northern hemisphere, with the exception of one Hungarian isolate, which was not related to any known species of the F. graminearum species complex based on sequence data. The taxonomic assignment of two other Hungarian isolates, previously suggested as belonging to F. boothii based on mitochondrial DNA restriction profiles, was supported by sequence analysis.     

Aggressiveness and mycotoxin production of eight isolates each of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> for ear rot on susceptible and resistant early maize inbred lines

Fusarium graminearum and F. verticillioides are among the most important pathogens causing ear rot of maize in Central Europe. Our objectives were to (1) compare eight isolates of each species on two susceptible inbred lines for their variation in ear rot rating and mycotoxin production across 3 years, and (2) analyse two susceptible and three resistant inbred lines for potential isolate x line interactions across 2 years by silk-channel inoculation. Ear rot rating, zearalenone (ZEA) and deoxynivalenol (DON) concentrations were evaluated for all F. graminearum isolates. In addition, nivalenol (NIV) concentrations were analysed for two NIV producers. Fumonisin (FUM) concentrations were measured for all F. verticillioides isolates. Mean ear rot severity was highest for DON producers of F. graminearum (62.9% of the ear covered by mycelium), followed by NIV producers of the same species (24.2%) and lowest for F. verticillioides isolates (9.8%). For the latter species, ear rot severities differed highly among years (2006: 24%, 2007: 3%, 2008: 7%). Mycotoxin concentrations among isolates showed a broad range (DON: 100–284 mg kg⁻¹, NIV: 15–38 mg kg⁻¹, ZEA: 1.1–49.5 mg kg⁻¹, FUM: 14.5–57.5 mg kg⁻¹). Genotypic variances were significant for isolates and inbred lines in all traits and for both species. Isolate x line interactions were significant only for ear rot rating (P < 0.01) and DON concentration (P < 0.05) of the F. graminearum isolates, but no rank reversals occurred. Most isolates were capable of differentiating the susceptible from the resistant lines for ear rot severity. For resistance screening, a sufficiently aggressive isolate should be used to warrant maximal differentiation among inbred lines. With respect to F. verticillioides infections, high FUM concentrations were found in grains from ears with minimal disease symptoms.     

Trichothecene genotype and genetic variability of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">F. cerealis</Emphasis> isolated from durum wheat in Argentina

Fusarium head blight (FHB) is one of the most important fungal diseases affecting wheat worldwide and it is caused mainly by species within the Fusarium graminearum species complex (FGSC). This study evaluated the presence of FGSC in durum wheat from the main growing area in Argentina and analyzed the trichothecene genotype and chemotype of the strains isolated. Also, the genetic variability of the strains was assayed using ISSR markers. Molecular analysis revealed that among the strains isolated and identified morphologically as F. graminearum, there were 14 strains identified as F. cerealis. Also, it revealed that durum wheat grains were mostly contaminated by F. graminearum, being this the only species reported so far, within the FGSC, affecting durum wheat in Argentina. Analysis of molecular variance (AMOVA) indicated a high genetic variability within rather than between F. graminearum populations. All F. graminearum strains presented 15ADON genotype and were able to produce DON while all F. cerealis strains presented the NIV genotype and most of them were able to produce this toxin. The finding of F. cerealis in durum wheat grains indicates the need for investigating if this fungus is the responsible for the NIV contamination found in wheat in Argentina.     

Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout 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.     

<Emphasis Type="Italic">Fusarium</Emphasis> species and mycotoxin profiles on commercial maize hybrids in Germany

High year-to-year variability in the incidence of Fusarium spp. and mycotoxin contamination was observed in a two-year survey investigating the impact of maize ear rot in 84 field samples from Germany. Fusarium verticillioides, F. graminearum, and F. proliferatum were the predominant species infecting maize kernels in 2006, whereas in 2007 the most frequently isolated species were F. graminearum, F. cerealis and F. subglutinans. Fourteen Fusarium-related mycotoxins were detected as contaminants of maize kernels analyzed by a multi-mycotoxin determination method. In 2006, a growth season characterized by high temperature and low rainfall during anthesis and early grain filling, 75% of the maize samples were contaminated with deoxynivalenol, 34% with fumonisins and 27% with zearalenone. In 2007, characterized by moderate temperatures and frequent rainfall during the entire growth season, none of the 40 maize samples had quantifiable levels of fumonisins while deoxynivalenol and zearalenone were detected in 90% and 93% of the fields, respectively. In addition, 3-acetyldeoxynivalenol, 15-acetyldeoxnivalenol, moniliformin, beauvericin, nivalenol and enniatin B were detected as common contaminants produced in both growing seasons. The results demonstrate a significant mycotoxin contamination associated with maize ear rots in Germany and indicate, with regard to anticipated climate change, that fumonisins-producing species already present in German maize production may become more important.     

Inheritance of resistance in <Emphasis Type="Italic">Solanum nigrum</Emphasis> to <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Solanum nigrum, black nightshade, is a wild non-tuber bearing hexaploid species with a high level of resistance to Phytophthora infestans (Colon et al. 1993), the causal agent of potato late blight, the most devastating disease in potato production. However, the genetic mode of resistance in S. nigrum is still poorly understood. In the present study, two S. nigrum accessions, 984750019 (N19) and #13, resistant (R) and susceptible (S), respectively, to three different isolates of P. infestans, were sexually crossed. The various kinds of progeny including F1, F2, F3, and backcross populations (BC₁; F1 × S), as well as two populations produced by self-pollinating the R parent and S parent, were each screened for susceptibility to P. infestans isolate MP 324 using detached leaf assays. Fifty seedling plant individuals of the F1 progeny were each resistant to this specific isolate, similarly to the seedling plants resulting from self-pollination of the resistant R parent. Thirty seedling plants obtained from self-pollination of the S parent were susceptible. Among a total of 180 F2 plants, the segregation ratio between resistant and susceptible plants was approximately 3: 1. Among the 66 seedling plants of the BC₁ progeny originating from crossing an F1 plant with the susceptible S parent, there were 26 susceptible and 40 resistant plants to P. infestans. The segregation patterns obtained indicated monogenic dominant inheritance of resistance to P. infestans isolate MP 324 in S. nigrum acc. 984750019. This gene, conferring resistance to P. infestans, may be useful for the transformation of potato cultivars susceptible to late blight.     

Cell interactions between a nonpathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain and root tissues of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis>

Nonpathogenic isolates of Fusarium oxysporum can be successful antagonists of pathogenic forms of the same fungal species that commonly attacks crop plants. The characteristics that distinguish nonpathogenic from pathogenic forms are not well understood. In this study, the mode of root colonization of Eucalyptus viminalis seedlings by a nonpathogenic F. oxysporum strain is described at the ultrastructural level. Root systems of E. viminalis plants were inoculated with nonpathogenic F. oxysporum strain Fo47 in an in vitro model system. Changes in the occurrence of nonesterified and methyl-esterified pectins in colonized E. viminalis roots were evaluated by in situ immunolabeling using two monoclonal antibodies, JIM 5 and JIM 7. Modes of penetration and root colonization patterns in E. viminalis seedlings by the nonpathogenic fungus were similar to those described for pathogenic forms of F. oxysporum. However, root interactions differed in that the nonpathogenic fungus did not induce host tissue damage. No papilla-like appositions were observed in host cells in response to invading hyphae, which did not disrupt the host plasma membrane in many cases, suggesting that a biotrophic relationship was established. Root colonization by the nonpathogenic strain did not induce alteration in JIM 7 labeling of methyl-esterified pectin in E. viminalis cell walls, whereas nonesterified pectin was detected to a significantly greater extent in cell walls of roots colonized by the fungus. Pectin components decreased slightly only at points of hyphal contact with host cells. Because nonpathogenic strains utilize pectin in pure culture, host control over enzyme activity or production by the fungi may at least partly explain their compatible interactions with host tissues.     

Chitinolytic <Emphasis Type="Italic">Streptomyces griseorubens</Emphasis> E44G enhances the biocontrol efficacy against <Emphasis Type="Italic">Fusarium</Emphasis> wilt disease of tomato

Streptomyces griseorubens E44G is a chitinolytic bacterium isolated from cultivated soil in Saudi Arabia (a hot, arid climatic region). In vitro, antifungal potential of S. griseorubens E44G was assessed against the phytopathogenic fungus, Fusarium oxysporum f. sp. lycopersici (the causative agent of the Fusarium wilt disease of tomato). An inhibition zone of 24 mm was recorded. The chitinolytic activity of S. griseorubens E44G was proved when the colloidal chitin agar plate method was used. A thermostable chitinase enzyme of 45 kDa molecular weight was purified using gel filtration chromatography. The optimum activity was obtained at 60 °C and pH 5.5. The purified enzyme has shown a very pronounced activity against the phytopathogenic fungus, F. oxysporum. The molecular characterization of the chitinase gene indicated that it consists of 1218 bp encoding 407 amino acids. The phylogentic analysis based on the nucleotide DNA sequence and the deduced amino acids sequence showed high similarity percentages with other chitinases isolated from different Streptomyces species. In the field evaluation, application of both S. griseorubens E44G treatments significantly increased all tested growth and yield parameters and decreased the disease severity compared with the infected-untreated tomato plants suggesting potential as a biocontrol agent.     

Genetic analysis and PCR-based identification of major <Emphasis Type="Italic">Fusarium</Emphasis> species causing head blight on wheat in Japan

Identifying the Fusarium species cause Fusarium head blight (FHB) and produces mycotoxins in wheat and other cereal is difficult and time consuming because of confusing phenotypic classification systems. In Japan, the F. graminearum complex, F. culmorum, F. avenaceum, and Microdochium nivale predominantly cause FHB. The internal transcribed spacer (ITS) and 5.8S of rDNA, a partial sequence of β-tubulin and mitochondrial cytochrome b (cytb) genes of the four species were PCR-amplified and analyzed. On the basis of the ITS, β-tubulin and cytb sequences, F. avenaceum and M. nivale are distinct from the F. graminearum complex and F. culmorum, whereas the F. graminearum complex is closely related to F. culmorum. Moreover, thiophanate–methyl-resistant isolates of the F. graminearum complex and F. culmorum did not have an amino acid substitution at amino acid codon 198 or 200 of β-tubulin. In contrast, very highly or highly thiophanate–methyl-resistant isolates of M. nivale had Glu (GAG) substituted with Ala (GCG) or Lys (AAG) at codon 198, respectively. The allele-specific PCR assay was used to identify the F. graminearum complex and F. culmorum, and these Fusarium species could be distinguished rapidly.     

Development of qPCR assays to monitor the ability of <Emphasis Type="Italic">Gliocladium catenulatum</Emphasis> J1446 to reduce the cereal pathogen <Emphasis Type="Italic">Fusarium graminearum</Emphasis> inoculum in soils

A qPCR approach was developed to specifically monitor in soils Fusarium graminearum, the main agent responsible for Fusarium Head Blight, and the biocontrol agent Gliocladium catenulatum J1446 (Prestop®). For both fungi, the amplification efficacy of standard curves obtained by mixing pure fungal DNA and soil background DNA was high (qPCR efficacy>96% with R²?>?0.97) with a linear range from 10^?3 ng to 10 ng/μL. Our qPCR method allowed quantifying down to 1 μg of F. graminearum and G. catenulatum J1446 mycelium per g of soil. The strong correlation observed between fungal biomass and quantified DNA (R²?=?0.9927 and 0.9356 for F. graminearum and G. catenulatum J1446, respectively) supported the use of the primers to monitor both fungi in soils. Under our experimental conditions, the ability of Prestop® to reduce F. graminearum growth was significantly higher in autoclaved soil compared to living soils, suggesting that there is an antagonistic effect of the soil microbial communities. In contrast, G. catenulatum J1446 growth was mostly not affected by the presence of F. graminearum and was able to persist in both autoclaved and living soils after 15 days of incubation. These results indicate that our qPCR approach may be used to assess the success of soil colonization by a biocontrol agent and its control efficacy by monitoring the dynamics of the BCA and the targeted pathogen in soil.     

Multifarious activity of bioformulated <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> PS1 and biocontrol of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in Indian rapeseed (<Emphasis Type="Italic">Brassica campestris</Emphasis> L.)

PGPR strain Pseudomonas fluorescens PS1 was evaluated to formulate carrier based bioformulations. The viability of P. fluorescens PS1 was monitored at different time intervals during the period of storage at room temperature in different carriers such as soil, charcoal, sawdust and sawdust-soil. Sawdust-soil was found to be the most efficient carrier material for P. fluorescens PS1 followed by other carriers. After 1 year of storage, P. fluorescens PS1 was re-isolated and assayed for its antifungal activity against Sclerotinia sclerotiorum a phytopathogenic fungus causing stem blight in Indian mustard, Brassica campestris. Results of scanning electron microscopy exhibited that P. fluorescens PS1 caused morphological alteration in mycelia of S. sclerotiorum as evident by hyphal perforation, and fragmented lysis. Seed bacterization of B. campestris with P. fluorescens PS1 induced enhanced seed germination, increased overall plant growth as well as reduced stem blight in mustard with improved yield. These findings demonstrate that P. fluorescens PS1 has significant potential to raise disease-free crops due to the presence of a wide array of PGP characteristics.     

Morphological and molecular identification of <Emphasis Type="Italic">Fusarium</Emphasis> species associated with head blight on wheat in East Croatia

Fusarium head blight (FHB) is an important fungal disease of wheat. The aim of this research was to determine the diversity of Fusarium species infecting winter wheat ears in East Croatia. In 2008 wheat kernels were obtained from three locations in the eastern part of Croatia (Tovarnik, Osijek, Pozega), and in 2009 from two additional locations (Slavonski Brod, Nova Gradiska). In total, 498 visually diseased kernels were selected for morphological identification of Fusarium spp. The identity of 226 selected isolates was further investigated by molecular techniques. The predominant species on wheat kernels in East Croatia in 2008 were F. graminearum, isolated and confirmed from more than 80% of sampled wheat kernels, followed by F. avenaceum (8%) and F. culmorum (7%). Incidence of F. poae was less than 2%. The most common species identified in 2009 were F. graminearum (50%), F. culmorum (13%), F. avenaceum (12%) and F. poae (7%). This is the first report on the identification of Fusarium species isolated from naturally infected wheat ears in Croatia.     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.