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.     

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.     

<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.     

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.     

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.     

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.     

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.     

Development of a multiplex RT-PCR detection and identification system for <Emphasis Type="Italic">Potato spindle tuber viroid</Emphasis> and <Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis>

We isolated eight polymorphic microsatellite markers for the basidiomycete Armillaria cepistipes and characterised them by analysing 50 isolates representing two geographically distinct populations from Switzerland and the Ukraine. The number of alleles per locus and population varied from one to eight, resulting in 43 alleles over the eight loci and two populations. In both populations, no significant linkage disequilibrium was observed between pairs of loci. Significant (P < 0.05) deviations from Hardy-Weinberg equilibrium were observed at one locus in the Swiss population and at three loci in the Ukrainian population. Of the eight loci developed for A. cepistipes, six were also polymorphic in A. gallica, four in A. ostoyae, two in A. mellea, and one in A. borealis. Beside the potential to be used for population genetic studies on A. cepistipes, these microsatellites thus represent additional molecular markers for three of the four annulated Armillaria species occurring in Europe.     

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.     

Characterization of <Emphasis Type="Italic">Puccinia psidii</Emphasis> isolates in Hawaii using microsatellite DNA markers

We studied genetic variation in Puccinia psidii, a newly introduced rust fungus in Hawaii, using microsatellite markers. All 36 Hawaiian P. psidii isolates analyzed had the same genotype at 11 microsatellite loci, while three genotypes were identified among four isolates from Florida and 11 genotypes among 18 isolates from Brazil. One of the isolates from Florida had 95% similarity to the Hawaiian isolates. We conclude that the genetically homogeneous P. psidii isolates in Hawaii might have derived from the same strain originally introduced into the Islands and are closely related to one of the strains found in Florida.     

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.     

Diversity in genetic structure and chemotype composition of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> sensu stricto populations causing wheat head blight in individual fields in Germany

Fusarium head blight (FHB) is one of the most destructive diseases of wheat. Twelve small commercial wheat fields (size 1–3 hectares) were sampled in Germany for Fusarium populations at three spots per field with 10 heads each. PCR assays using generic primers confirmed 338 isolates as F.graminearum sensu stricto (s.s.) (64.9%) out of 521 Fusarium spp. that were further analyzed. Populations of F. graminearum s.s. in Germany contain three types of trichothecenes with a dominancy of 15-acetyldeoxynivalenol chemotype (92%) followed by 3-acetyldeoxynivalenol chemotype (6.8%) and a few isolates of nivalenol chemotype (1.2%). All these isolates were genotyped using 19 microsatellite loci. The 12 populations showed a high genetic diversity within the small scale sampling areas resulting in 300 different haplotypes. Genetic diversity within populations (71.2%) was considerably higher than among populations (28.8%) as shown by analysis of molecular variance. Gene flow (Nm) between populations ranged from 0.76–3.16. Composition of haplotypes of one population followed over 2 years changed considerably. No correlation between genetic and geographical distance was found. In conclusion, populations of F. graminearum s.s. in Germany display a tremendous genetic variation on a local scale with a restricted diversity among populations.     

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.     

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.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

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.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

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.