Synergistic effect of amino acid substitutions in CYP51B for prochloraz resistance in Fusarium fujikuroi.

Prochloraz has been used to control Fusarium fujikuroi, the causative pathogen of rice bakanae disease. Linkage analysis of FfCYP51 genes in the progenies obtained from crossing prochloraz moderately resistant and sensitive strains suggested that the FfCYP51B gene is involved in prochloraz resistance. Sequence comparison revealed that the prochloraz-resistant strain had an F511S or S312T/F511S substitution in FfCYP51B compared with the sensitive strains. The contribution of the S312T and F511S substitutions in FfCYP51B to prochloraz resistance was investigated by creating S/F-, T/F-, or T/S- types at 312/511 codons from the S/S-type, which is a natural moderately resistant strain, using a gene-editing technique. T/S exhibited the highest prochloraz resistance, followed by S/S-, T/F-, and S/F-types. These results indicated that the S312T and F511S substitutions in FfCYP51B had a synergistic effect on prochloraz resistance in F. fujikuroi.

A LAMP-Based Toolbox Developed for Detecting the Major Pathogens Affecting the Production and Quality of the Chinese Medicinal Crop Aconitum carmichaelii.

Aconitum carmichaelii Debeaux is a traditional Chinese medicinal herb that has been utilized for approximately 2,000 years. However, as cultivation has increased, there have been more reports of A. carmichaelii infections caused by four major pathogenic fungal species, Fusarium oxysporum, F. solani, Mucor circinelloides, and Sclerotium rolfsii, resulting in increased disease incidences and limited production and quality. To detect these infections, we developed a LAMP-based toolbox in this study. The cytochrome c oxidase subunit 1 (cox1) gene, translation elongation factor-1α (EF-1α), internal transcribed spacer (ITS) regions of rDNA, and alcohol dehydrogenase 1 (ADH1) gene, respectively, were used to design species-specific LAMP primer sets for F. oxysporum, F. solani, S. rolfsii, and M. circinelloides. The results showed that the LAMP-based toolbox was effective at detecting pathogens in soil and plant materials. We also used this toolbox to investigate pathogen infection in the main planting regions of A. carmichaelii. Before harvesting, F. oxysporum, M. circinelloides, and S. rolfsii were commonly found in the planting fields and in infected A. carmichaelii plants. Therefore, the toolbox we developed will be useful for tracking these infections, as well as for disease control in A. carmichaelii.

The Stimulation of Indigenous Bacterial Antagonists by γ-Glutamyl-S-Allyl-l-Cysteine Increases Soil Suppressiveness to Fusarium Wilt.

The development of suppressive soil is an ideal strategy to sustainably combat soilborne diseases. Previously, the cultivation of Allium plants increased antagonistic bacteria populations in soil, alleviating Fusarium wilt of different crops. This study aimed to identify a compound produced by Allium plants that can induce bacteria-mediated soil suppressiveness toward Fusarium wilt. The amendment of soils with γ-glutamyl-S-allyl-l-cysteine (GSAC), a unique dipeptide abundantly detected in the root extract of Welsh onion (Allium fistulosum), significantly suppressed Fusarium wilt diseases, whereas three other commercial dipeptides had no such effects. GSAC application did not suppress the disease in sterilized soil. Furthermore, the suppressiveness of soil amended with GSAC could be transferred to sterilized soil via soil microflora transplantation. This suppressiveness was eliminated by pretreating GSAC-amended soil microflora with antibacterial antibiotics, indicating that the suppressiveness of GSAC-amended soil is generated by the activity of antagonistic bacteria. Amplicon sequencing of the 16S rRNA gene revealed that GSAC application significantly increased the relative abundance of Pseudomonas (OTU224), Burkholderia-Caballeronia-Paraburkholderia (OTU387), and Bdellovibrio (OTU1259) in soils. Surprisingly, the relative abundance of OTU224 was significantly greater in Welsh onion rhizospheres than in noncultivated soil. Pseudomonas strains corresponding to OTU224, isolated from Welsh onion rhizospheres, displayed a remarkable suppressive effect against cucumber Fusarium wilt, implying that OTU224 was involved in GSAC-mediated suppressiveness. This is the first study on the potential of GSAC as a soil microflora-manipulating agent that can enhance soil suppressiveness to Fusarium wilt. IMPORTANCE Methods for increasing soil suppressiveness via soil microflora manipulation have long been explored as an ideal strategy to protect plants from soilborne pathogens. However, viable methods offering consistent disease control effects have not yet been developed. Previously, the cultivation of Allium plants was demonstrated to induce bacteria-mediated soil suppressiveness to Fusarium wilt of different crop plants. This study discovered that the application of γ-glutamyl-S-allyl-l-cysteine, a unique dipeptide synthesized by Welsh onion, to soil enhances Fusarium wilt suppressiveness by increasing the relative abundance of indigenous antagonistic bacteria irrespective of the soil type. This finding will facilitate research supporting the development of environmentally friendly control measures for soilborne diseases.

A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing.

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay.

Development of PCR-RFLP Technique for Identify Several Members of Fusarium incarnatum-equiseti Species Complex and Fusarium fujikuroi Species Complex.

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.

Molecular Diagnosis of Thiophanate-Methyl-Resistant Strains of Fusarium fujikuroi in Japan.

Fusarium fujikuroi is the pathogen of rice bakanae disease and is subclassified into gibberellin and fumonisin groups (G and F groups). Thiophanate-methyl (TM), a benzimidazole fungicide, has been used extensively to control F. fujikuroi. Previous investigation showed that F-group strains are TM sensitive (TMS), whereas most G-group strains are TM resistant (TMR) in Japan. The minimum inhibitory concentration in TMS strains was 1 to 10 µg ml-1, whereas that in TMR strains was >100 µg ml-1. E198K and F200Y mutations in ß2-tubulin were detected in TMR strains. A loop-mediated isothermal amplification-fluorescent loop primer method was developed for diagnosis of these mutations and applied to 37 TMR strains and 56 TMS strains. The results indicated that 100% of TMR strains were identified as having either the E198K mutation (41%) or the F200Y mutation (59%), whereas none of the TMS strains tested showed either mutation. We found one remarkable TMR strain in the F group that had an F200Y mutation. These results suggest that E198K and F200Y mutations in ß2-tubulin contribute to TM resistance in F. fujikuroi.

Lysinibacillus xylanilyticus Strain GIC41 as a Potential Plant Biostimulant.

To identify Lysinibacillus strains with the potential to function as plant biostimulants, we screened 10 previously isolated Lysinibacillus strains from the rhizosphere and soil for their plant growth-promoting (PGP) effects. In vitro tests showed that all strains produced indole-3-acetic acid. In primary screening, the PGP effects of these strains were assessed on spinach seedlings grown on Jiffy-7 pellets; strains GIC31, GIC41, and GIC51 markedly promoted shoot growth. In secondary screening, the PGP efficacies of these three strains were examined using spinach seedlings grown in pots under controlled conditions. Only GIC41 exerted consistent and significant PGP effects; therefore, it was selected for subsequent experiments. The results of 6-week glasshouse experiments revealed that GIC41 markedly increased shoot dry weight by ca. 12-49% over that of the control. The impact of fertilization levels on the PGP efficacy of GIC41 was investigated using pot experiments. The application of a specific level of fertilizer was required for the induction of sufficient PGP effects by this strain. The phylogenetic ana-lysis based on the 16S rDNA sequence identified GIC41 as L. xylanilyticus. Collectively, these results show the potential of strain GIC41 to function as a plant biostimulant.

Pythium intermedium, a species complex consisting of three phylogenetic species found in cool-temperate forest ecosystems.

Pythium intermedium plays a vital role in the carbon cycle of cool-temperate forests and is widely distributed in Japan's forest soils. In this study, we performed a phylogenetic analysis of the P. intermedium species complex using DNA sequences from multiple loci. The study included 35 isolates from cool-temperate forest soils, seven known P. intermedium isolates, and six known Pythium attrantheridium isolates. We also performed morphological observations and mating tests. Our results showed that all the isolates formed one large clade but were divided into three subclades. Furthermore, we observed many mating reactions between isolates from different subclades, including between P. attrantheridium and P. intermedium. Therefore, we suggest that P. intermedium, P. attrantheridium, and another phylogenetic species belong to one species complex. This is the first report of a species complex within P. intermedium and will be helpful in understanding the evolution of Pythium species in natural ecosystems.

Multiplex LAMP Detection of the Genus Phytophthora and Four Phytophthora Species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae, with a Plant Internal Control.

Phytophthora species cause destructive plant diseases worldwide. All Phytophthora species, except for one, are listed as plant quarantine organisms in Japan. The exception, Phytophthora nicotianae is considered to be a domestic species. The injurious pests Phytophthora ramorum, Phytophthora lateralis, and Phytophthora kernoviae are invasive pathogens that cause tree mortality worldwide, mainly in the United States and the United Kingdom. To effectively control Phytophthora diseases, we established detection methods that utilize the loop-mediated isothermal amplification (LAMP) of the genus Phytophthora and the four species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae. LAMP primers for P. ramorum, P. lateralis, and P. kernoviae were newly designed in the present study. Our multiplex assay includes the detection of plant DNA as an internal control. When the optimum ratio between plant and pathogen primers was used in multiplex LAMP assays, 1 pg to 100 fg of pathogen DNA was detected with similar sensitivity to that in simplex LAMP assays. The detection of plant DNA in the absence of pathogens enables us to check for and avoid undesirable negative results caused by enzyme inactivation or the contamination of amplification inhibitors from plant tissues. The total time from sample collection to results is approximately 120| |min, and, thus, our multiplex LAMP assay may be used as an accurate and time-saving detection method for Phytophthora pathogens.

Potential Use of L-arabinose for the Control of Tomato Bacterial Wilt.

The present study aimed to investigate the potential of simple sugars for use as protection agents in the control of tomato bacterial wilt caused by Ralstonia pseudosolanacearum. Based on the sugar assimilation patterns of the pathogen, four unassimilable sugars (L-arabinose, maltose, D-raffinose, and D-ribose) were selected from 10 representative sugars present in tomato root exudates. These sugars were evaluated for their effects on bacterial wilt using a tomato seedling bioassay. The application of 0.25% L-arabinose significantly reduced disease severity and was, thus, selected as a candidate for further evaluations in a pot experiment under glasshouse conditions. The results obtained showed that the disease suppressive effects of L-arabinose slightly increased at higher concentrations; drench treatments at 0.1, 0.25, and 0.5% reduced disease severity by ca. 48, 70, and 87%, respectively. The drench treatment with 0.5% L-arabinose significantly reduced the pathogen population in the rhizosphere and stem tissues of tomato plants without any antibacterial activity. Real-time reverse-transcription PCR revealed that the expression of salicylic acid-dependent and ethylene-dependent defense genes was significantly enhanced in the stem tissues of L-arabinose-treated tomato plants following the pathogen inoculation. These results suggest that soil drenching with L-arabinose effectively suppresses tomato bacterial wilt by preventing pathogen proliferation in the rhizosphere and stem tissues of tomato plants. This is the first study to report the potential of L-arabinose as a safe, eco-friendly, and cost-effective plant protection agent for the control of tomato bacterial wilt.

Biocontrol of Tomato Bacterial Wilt by Foliar Spray Application of a Novel Strain of Endophytic Bacillus sp.

The aim of the present study was to identify a strain of endophytic Bacillus species that control tomato bacterial wilt by foliar spray application. Fifty heat-tolerant endophytic bacteria were isolated from the surface-sterilized foliar tissues of symptomless tomato plants that had been pre-inoculated with the pathogen Ralstonia pseudosolanacearum. In the primary screening, we assessed the suppressive effects of a shoot-dipping treatment with bacterial strains against bacterial wilt on tomato seedlings grown on peat pellets. Bacillus sp. strains G1S3 and G4L1 significantly suppressed the incidence of tomato bacterial wilt. In subsequent pot experiments, the biocontrol efficacy of foliar spray application was examined under glasshouse conditions. G4L1 displayed consistent and significant disease suppression, and, thus, was selected as a biocontrol candidate. Moreover, the pathogen population in the stem of G4L1-treated plants was markedly smaller than that in control plants. A quantitative real-time PCR analysis revealed that the foliar spraying of tomato plants with G4L1 up-regulated the expression of PR-1a and LoxD in stem and GluB in roots upon the pathogen inoculation, implying that the induction of salicylic acid-, jasmonic acid-, and ethylene-dependent defenses was involved in the protective effects of this strain. In the re-isolation experiment, G4L1 efficiently colonized foliar tissues for at least 4| |weeks after spray application. Collectively, the present results indicate that G4L1 is a promising biocontrol agent for tomato bacterial wilt. Furthermore, to the best of our knowledge, this is the first study to report the biocontrol of bacterial wilt by the foliar spraying with an endophytic Bacillus species.

Detection of the Genus Phytophthora and the Species Phytophthora nicotianae by LAMP with a QProbe.

Phytophthora is an oomycete genus with worldwide distribution, and many of its species cause destructive diseases. In Japan, Phytophthora species are listed as quarantine organisms with the exception of Phytophthora nicotianae. For effective quarantine control, we designed a Phytophthora genus-specific loop-mediated isothermal amplification (LAMP) primer set and a P. nicotianae species-specific quenching probe (QProbe) to establish a simultaneous LAMP-based detection method. We confirmed the specificity of the genus-specific primers, and all 161 taxa were detected. No other species in the closely related genera Pythium and Phytopythium gave positive results with the exception of two species, Phytopythium delawarense and Phytopythium fagopyri. These two species gave inconsistent results. We used annealing curve analysis with the QProbe to demonstrate that P. nicotianae could be distinguished from other species. DNA from inoculated and naturally infected plants was extracted using a time-saving extraction kit and subjected to the simultaneous detection method. We confirmed that all Phytophthora DNAs in the plant samples were detected, and P. nicotianae was specifically identified. This simultaneous detection method will make quarantine inspections faster and easier.

Systemic resistance induced by Phoma sp. GS8-3 and nanosilica against Cucumber mosaic virus.

Cucumber mosaic virus (CMV) is a very serious hazard to vegetable production worldwide. This study is focused on evaluation of resistance stimulated by the plant growth-promoting fungus, Phoma sp. GS8-3, or nanosilica against CMV under pot and field conditions. The specific aim was to illustrate the mechanism of resistance stimulated by GS8-3 against CMV using microarray technology. Treatments with GS8-3 as well as nanosilica significantly decreased CMV severity and titer in tobacco and cucumber under pot and field conditions, respectively. Growth characters of tobacco and cucumber were significantly increased due to GS8-3 inoculation followed by nanosilica compared with control and BTH treatments. Microarray results showed highly upregulation of defense-related genes expression specially those related to heat shock proteins. Therefore, GS8-3 as well as nanosilica is suitable to serve as effective inducers against CMV in cucumber plants.

A Natural Variation of Fumonisin Gene Cluster Associated with Fumonisin Production Difference in Fusarium fujikuroi.

Fusarium fujikuroi, a member of the Fusarium fujikuroi species complex, stands out as a rice bakanae disease pathogen with a high production of gibberellic acid. Not all, but some F. fujikuroi strains are known to produce a carcinogenic mycotoxin fumonisin. Fumonisin biosynthesis is dependent on the FUM cluster composed of 16 FUM genes. The FUM cluster was detected not only from a fumonisin producing strain, but also from a fumonisin nonproducing strain that does not produce a detectable level of fumonisin. Genetic mapping indicated the causative mutation(s) of fumonisin nonproduction is present in the FUM cluster of the fumonisin nonproducing strain. Comparative analyses of FUM genes between the fumonisin producing and the nonproducing strains and gene complementation indicated that causative mutation of fumonisin nonproduction is not a single occurrence and the mutations are distributed in FUM21 and FUM7. Our research revealed a natural variation in the FUM cluster involving fumonisin production difference in F. fujikuroi.

Polymorphism and expression level of CYP51 (cytochrome P450) and sensitivity to ipconazole in Fusarium fujikuroi isolates.

The relationship between the nucleotide sequences of CYP51, its expression level and its sensitivity to ipconazole of Fusarium fujikuroi isolates were investigated. Single nucleotide polymorphisms (SNPs) were observed in the CYP51 of isolates with different sensitivities to ipconazole, but no amino acid substitution was detected in the putative amino acid sequence of the CYP51 protein. On the other hand, the expression of CYP51 was enhanced by the presence of ipconazole, and it tended to be higher in isolates with lower sensitivities and no gibberellin productivity. In the presumed promoter region, the upstream nucleotide sequence of CYP51, several common SNPs and insertions of nucleotides were detected in the lower sensitivity isolates. These results suggest that F. fujikuroi isolates consist of 2 different groups in sensitivity and gibberellin productivity, and no amino acid substitution in CYP51 protein may contribute to the stably high efficacy of ipconazole against rice Bakanae disease for more than 25 years.

Microbial basis of Fusarium wilt suppression by Allium cultivation.

Crop rotation and intercropping with Allium plants suppresses Fusarium wilt in various crops. However, the mechanisms underlying this phenomenon have not been fully elucidated. This study was designed to assess the role of microorganisms inhabiting Allium rhizospheres and antifungal compounds produced by Allium roots in Fusarium wilt suppression by Allium cultivation. Suppression of cucumber Fusarium wilt and the pathogen multiplication by Allium (Welsh onion and/or onion)-cultivated soils were eliminated by heat treatment at 60 °C, whereas those by Welsh onion-root extract were lost at 40 °C. The addition of antibacterial antibiotics eliminated the suppressive effect of Welsh onion-cultivated soil on pathogen multiplication, suggesting the contribution of antagonistic gram-negative bacteria to the soil suppressiveness. The Illumina MiSeq sequencing of 16S rRNA gene amplicons revealed that genus Flavobacterium was the predominant group that preferentially accumulated in Allium rhizospheres. Flavobacterium species recovered from the rhizosphere soils of these Allium plants suppressed Fusarium wilt on cucumber seedlings. Furthermore, confocal laser scanning microscopy revealed that Flavobacterium isolates inhibited the multiplication of the pathogen in soil. Taken together, we infer that the accumulation of antagonistic Flavobacterium species plays a key role in Fusarium wilt suppression by Allium cultivation.

LAMP Detection of Four Plant-Pathogenic Oomycetes and Its Application in Lettuce Fields.

In Kagawa Prefecture, Japan, the pathogens Phytophthora pseudolactucae, Pythium irregulare, Pythium uncinulatum, and Pythium spinosum have caused huge losses in lettuce production. We used loop-mediated isothermal amplification (LAMP) to analyze soil and plants in lettuce fields for the presence of these four pathogens. To develop an effective on-site detection method, we contrasted the Plant-LAMP and Plant Culture-LAMP procedures for plant samples, and five soil DNA extraction methods for soil samples. Plant-LAMP and a Soil DNA Isolation kit were selected to analyze three fields for the pathogen species present, infected sites, and level of soil contamination. We found that the same wilting symptoms could be caused by Phytophthora or Pythium, or a mixture of species from both genera. Ph. pseudolactucae infects the pith of the lettuce in aboveground parts, whereas Pythium spp. mainly infect roots. Ph. pseudolactucae and Py. uncinulatum caused disease more frequently than the other two pathogens. Furthermore, not all of the pathogens existed in the soil near infected lettuce plants. Therefore, the LAMP method can be used to diagnose pathogenic oomycetes in the field, and will be useful in the development of control strategies in lettuce production.

Genetic Differentiation Associated with Fumonisin and Gibberellin Production in Japanese Fusarium fujikuroi.

Fusarium fujikuroi is a pathogenic fungus that infects rice. It produces several important mycotoxins, such as fumonisins. Fumonisin production has been detected in strains of maize, strawberry, and wheat, whereas it has not been detected in strains from rice seedlings infested with bakanae disease in Japan. We investigated the genetic relationships, pathogenicity, and resistance to a fungicide, thiophanate-methyl (TM), in 51 fumonisin-producing strains and 44 nonproducing strains. Phylogenetic analyses based on amplified fragment length polymorphism (AFLP) markers and two specific genes (a combined sequence of translation elongation factor 1α [TEF1α] and RNA polymerase II second-largest subunit [RPB2]) indicated differential clustering between the fumonisin-producing and -nonproducing strains. One of the AFLP markers, EATMCAY107, was specifically present in the fumonisin-producing strains. A specific single nucleotide polymorphism (SNP) between the fumonisin-producing and nonproducing strains was also detected in RPB2, in addition to an SNP previously found in TEF1α. Gibberellin production was higher in the nonproducing than in the producing strains according to an in vitro assay, and the nonproducing strains had the strongest pathogenicity with regard to rice seedlings. TM resistance was closely correlated with the cluster of fumonisin-nonproducing strains. The results indicate that intraspecific evolution in Japanese F. fujikuroi is associated with fumonisin production and pathogenicity. Two subgroups of Japanese F. fujikuroi, designated G group and F group, were distinguished based on phylogenetic differences and the high production of gibberellin and fumonisin, respectively.IMPORTANCEFusarium fujikuroi is a pathogenic fungus that causes rice bakanae disease. Historically, this pathogen has been known as Fusarium moniliforme, along with many other species based on a broad species concept. Gibberellin, which is currently known as a plant hormone, is a virulence factor of F. fujikuroi Fumonisin is a carcinogenic mycotoxin posing a serious threat to food and feed safety. Although it has been confirmed that F. fujikuroi produces gibberellin and fumonisin, production varies among strains, and individual production has been obscured by the traditional appellation of F. moniliforme, difficulties in species identification, and variation in the assays used to determine the production of these secondary metabolites. In this study, we discovered two phylogenetic subgroups associated with fumonisin and gibberellin production in Japanese F. fujikuroi.

Population structures of the water-borne plant pathogen Phytopythium helicoides reveal its possible origins and transmission modes in Japan.

The purpose of this study was to clarify the genetic diversity of Phytopythium helicoides and to understand the transmission mode of the pathogen in Japan. In total, 232 P. helicoides isolates were collected from various host plants and geographic origins, including farms and natural environments. We developed 6 novel microsatellite markers for use in the study and found 90 alleles among the 6 markers in the 232 isolates. The analysis of molecular variance suggested that P. helicoides has high variance within individuals and low fixation indices between populations. A phylogenetic analysis revealed that isolates collected from the same hosts and/or geographic origins were often grouped together. For example, several isolates from natural environments were grouped with isolates from nearby agricultural areas. On the other hand, 2 geographically distant populations collected from the same host plant had similar genotypes. Our results suggested that migration of the pathogen could be facilitated naturally via drainage systems or by human activity in the transport of agricultural materials.

Novel Mitoviruses and a Unique Tymo-Like Virus in Hypovirulent and Virulent Strains of the Fusarium Head Blight Fungus, Fusarium boothii.

Hypovirulence of phytopathogenic fungi are often conferred by mycovirus(es) infections and for this reason many mycoviruses have been characterized, contributing to a better understanding of virus diversity. In this study, three strains of Fusarium head blight fungus (Fusarium boothii) were isolated from Ethiopian wheats as dsRNA-carrying strains: hypovirulent Ep-BL13 (>10, 3 and 2.5 kbp dsRNAs), and virulent Ep-BL14 and Ep-N28 (3 kbp dsRNA each) strains. The 3 kbp-dsRNAs shared 98% nucleotide identity and have single ORFs encoding a replicase when applied to mitochondrial codon usage. Phylogenetic analysis revealed these were strains of a new species termed Fusarium boothii mitovirus 1 in the genus Mitovirus. The largest and smallest dsRNAs in Ep-BL13 appeared to possess single ORFs and the smaller was originated from the larger by removal of its most middle part. The large dsRNA encoded a replicase sharing the highest amino acid identity (35%) with that of Botrytis virus F, the sole member of the family Gammaflexiviridae. Given that the phylogenetic placement, large genome size, simple genomic and unusual 3'-terminal RNA structures were far different from members in the order Tymovirales, the virus termed Fusarium boothii large flexivirus 1 may form a novel genus and family under the order.