Spray Application of Nonpathogenic Fusaria onto Rice Flowers Controls Bakanae Disease (Caused by Fusarium fujikuroi) in the Next Plant Generation.

Bakanae disease, caused by Fusarium fujikuroi, is an economically important seed-borne disease of rice. F. fujikuroi is horizontally transmitted to rice flowers and vertically transmitted to the next generation via seeds. The fungus induces typical symptoms such as abnormal tissue elongation and etiolation. Sanitation of seed farms and seed disinfection are the only effective means to control bakanae disease at present; however, the efficacy of these methods is often insufficient. Therefore, alternative and innovative control methods are necessary. We developed a novel method for applying nonpathogenic fusaria as biocontrol agents by spraying spore suspensions onto rice flowers to reduce the incidence of seed-borne bakanae. We visualized the interaction between Fusarium commune W5, a nonpathogenic fusarium, and Fusarium fujikuroi using transformants expressing two different fluorescent proteins on/in rice plants. W5 inhibited hyphal extension of F. fujikuroi on/in rice flowers and seedlings, possibly by competing with the pathogen, and survived on/in rice seeds for at least 6 months.IMPORTANCE We demonstrated that a spray treatment of rice flowers with the spores of nonpathogenic fusaria mimicked the disease cycle of the seed-borne bakanae pathogen Fusarium fujikuroi and effectively suppressed the disease. Spray treatment of nonpathogenic fusaria reduced the degree of pathogen invasion of rice flowers and vertical transmission of the pathogen to the next plant generation via seeds, thereby controlling the bakanae disease. The most promising isolate, F. commune W5, colonized seeds and seedlings via treated flowers and successfully inhibited pathogen invasion, suggesting that competition with the pathogen was the mode of action. Seed-borne diseases are often controlled by seed treatment with chemical fungicides. Establishing an alternative method is a pressing issue from the perspectives of limiting fungicide resistance and increasing food security. This work provides a potential solution to these issues using a novel application technique to treat rice flowers with biocontrol agents.

Sequencing of individual chromosomes of plant pathogenic Fusarium oxysporum.

A small chromosome in reference isolate 4287 of F. oxysporum f. sp. lycopersici (Fol) has been designated as a 'pathogenicity chromosome' because it carries several pathogenicity related genes such as the Secreted In Xylem (SIX) genes. Sequence assembly of small chromosomes in other isolates, based on a reference genome template, is difficult because of karyotype variation among isolates and a high number of sequences associated with transposable elements. These factors often result in misassembly of sequences, making it unclear whether other isolates possess the same pathogenicity chromosome harboring SIX genes as in the reference isolate. To overcome this difficulty, single chromosome sequencing after Contour-clamped Homogeneous Electric Field (CHEF) separation of chromosomes was performed, followed by de novo assembly of sequences. The assembled sequences of individual chromosomes were consistent with results of probing gels of CHEF separated chromosomes with SIX genes. Individual chromosome sequencing revealed that several SIX genes are located on a single small chromosome in two pathogenic forms of F. oxysporum, beyond the reference isolate 4287, and in the cabbage yellows fungus F. oxysporum f. sp. conglutinans. The particular combination of SIX genes on each small chromosome varied. Moreover, not all SIX genes were found on small chromosomes; depending on the isolate, some were on big chromosomes. This suggests that recombination of chromosomes and/or translocation of SIX genes may occur frequently. Our method improves sequence comparison of small chromosomes among isolates.

Rapid sex identification method of spinach (Spinacia oleracea L.) in the vegetative stage using loop-mediated isothermal amplification.

MAIN CONCLUSION: A LAMP-mediated, simple and rapid method for sex identification in spinach was developed. Nutrient compositional analysis showed a higher iron content in male than female plants. Spinach (Spinacia oleracea L.) is a dioecious plant with its sex determined by the XY system. Male and female floral organs differ morphologically, but plants do not differ in the vegetative stage before flowering. PCR with Y chromosome markers has been used to determine the sex of dioecious plants before flowering. In this study, we developed a genotype-specific loop-mediated isothermal amplification (LAMP) for sex identification of individual vegetative-stage spinach plants, using primers designed for the genomic region flanked by male-specific markers. LAMP could specifically detect spinach males. The method was further modified to omit DNA purification and use just an aliquot of crude leaf extract homogenized in water. We compared the nutrient composition of males and females, finding higher amounts of iron in the males. Our method could therefore be used for rapidly discriminating male plants in the field, which is useful for efficient hybrid breeding.

Genome sequence of a novel victorivirus identified in the phytopathogenic fungus Alternaria arborescens.

Strains of the phytopathogenic fungus Alternaria spp. have been found to contain a variety of double-stranded RNA (dsRNA) elements indicative of mycovirus infection. Here, we report the molecular characterization of a novel dsRNA mycovirus, Alternaria arborescens victorivirus 1 (AaVV1), from A. arborescens, the tomato pathotype of A. alternata. Using next-generation sequencing of dsRNA purified from an A. arborescens strain from the United States of America, we found that the AaVV1 genome is 5203 bp in length and contains two open reading frames (ORF1 and 2) that overlap at the tetranucleotide AUGA. Proteins encoded by ORF1 and ORF2 showed significant similarities to the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively, of dsRNA mycoviruses of the genus Victorivirus. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of both CP and RdRp indicated that AaVV1 is a member of a distinct species of the genus Victorivirus in the family Totiviridae.

Genome sequence of a novel mitovirus identified in the phytopathogenic fungus Alternaria arborescens.

The phytopathogenic fungus Alternaria spp. contains a variety of double-stranded RNA (dsRNA) elements of different sizes. Detailed analysis of next-generation sequencing data obtained using dsRNA purified from Alternaria arborescens, from which we had previously found Alternaria arborescens victorivirus 1, revealed the presence of another mycoviral-like dsRNA of approximately 2.5 kbp in length. When using the fungal mitochondrial genetic code, this dsRNA has a single open reading frame that potentially encodes an RNA-dependent RNA polymerase (RdRp) with significant to sequence similarity to those of viruses of the genus Mitovirus. Moreover, both the 5'- and 3'-untranslated regions have the potential to fold into stable stem-loop structures, which is characteristic of mitoviruses. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of RdRp indicated that the virus we identified in A. arborescens is a distinct member of the genus Mitovirus in the family Narnaviridae, designated as "Alternaria arborescens mitovirus 1" (AaMV1).

Detection of Magnaporthe oryzae chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification.

Magnaporthe oryzae chrysovirus 1 (MoCV1) is a mycovirus with a dsRNA genome that infects the rice blast fungus Magnaporthe oryzae and impairs its growth. To date, MoCV1 has only been found in Vietnamese isolates of M. oryzae, and the distribution of this virus in M. oryzae isolates from other parts of the world remains unknown. In this study, using a one-step reverse transcription PCR (RT-PCR) assay, we detected a MoCV1-related virus in M. oryzae in Japan (named MoCV1-AK) whose sequence shares considerable similarity with that of the MoCV1 Vietnamese isolate. To establish a system for a comprehensive survey of MoCV1 infection in the field, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for direct detection of the virus. The sensitivity of the RT-LAMP assay was at least as high as that of the one-step RT-PCR assay. In addition, we detected MoCV1-AK in M. oryzae-infected oatmeal agar plates and lesions on rice leaves using the RT-LAMP assay without dsRNA extraction, by simple sampling with a toothpick. Preliminary screening of MoCV1 in Japanese M. oryzae isolates indicated that MoCV1 is currently distributed in rice fields in Japan. Our results provide a first example of the application of RT-LAMP for the detection of mycoviruses, which will accelerate surveys for mycovirus infection.

The MAT1 locus is required for microconidia-mediated sexual fertility in the rice blast fungus.

Rice blast fungus (Pyricularia oryzae) is a heterothallic ascomycete that causes the most destructive disease in cultivated rice worldwide. This fungus reproduces sexually and asexually, and its mating type is determined by the MAT1 locus, MAT1-1 or MAT1-2. Interestingly, most rice-infecting field isolates show a loss of female fertility, but the MAT1 locus is highly conserved in female-sterile isolates. In this study, we performed a functional analysis of MAT1 using the CRISPR/Cas9 system in female- and male-fertile isolates and female-sterile (male-fertile) isolates. Consistent with a previous report, MAT1 was essential for sexual reproduction but not for asexual reproduction. Meanwhile, deletion mutants of MAT1-1-1, MAT1-1-2, and MAT1-1-3 exhibited phenotypes different from those of other previously described isolates, suggesting that the function of MAT1-1 genes and/or their target genes in sexual reproduction differs among strains or isolates. The MAT1 genes, excluding MAT1-2-6, retained their functions even in female-sterile isolates, and deletion mutants lead to loss or reduction of male fertility. Although MAT1 deletion did not affect microconidia (spermatia) production, microconidia derived from the mutants could not induce perithecia formation. These results indicated that MAT1 is required for microconidia-mediated male fertility in addition to female fertility in P. oryzae .

Characterization of Magnaporthe oryzae chrysovirus 1 structural proteins and their expression in Saccharomyces cerevisiae.

Magnaporthe oryzae chrysovirus 1 (MoCV1), which is associated with an impaired growth phenotype of its host fungus, harbors four major proteins: P130 (130 kDa), P70 (70 kDa), P65 (65 kDa), and P58 (58 kDa). N-terminal sequence analysis of each protein revealed that P130 was encoded by double-stranded RNA1 (dsRNA1) (open reading frame 1 [ORF1] 1,127 amino acids [aa]), P70 by dsRNA4 (ORF4; 812 aa), and P58 by dsRNA3 (ORF3; 799 aa), although the molecular masses of P58 and P70 were significantly smaller than those deduced for ORF3 and ORF4, respectively. P65 was a degraded form of P70. Full-size proteins of ORF3 (84 kDa) and ORF4 (85 kDa) were produced in Escherichia coli. Antisera against these recombinant proteins detected full-size proteins encoded by ORF3 and ORF4 in mycelia cultured for 9, 15, and 28 days, and the antisera also detected smaller degraded proteins, namely, P58, P70, and P65, in mycelia cultured for 28 days. These full-size proteins and P58 and P70 were also components of viral particles, indicating that MoCV1 particles might have at least two forms during vegetative growth of the host fungus. Expression of the ORF4 protein in Saccharomyces cerevisiae resulted in cytological changes, with a large central vacuole associated with these growth defects. MoCV1 has five dsRNA segments, as do two Fusarium graminearum viruses (FgV-ch9 and FgV2), and forms a separate clade with FgV-ch9, FgV2, Aspergillus mycovirus 1816 (AsV1816), and Agaricus bisporus virus 1 (AbV1) in the Chrysoviridae family on the basis of their RdRp protein sequences.

Dysfunctional Pro1 leads to female sterility in rice blast fungi.

Although sexual reproduction is widespread in eukaryotes, some fungal species can only reproduce asexually. In the rice blast fungus Pyricularia (Magnaporthe) oryzae, several isolates from the region of origin retain mating ability, but most isolates are female sterile. Therefore, female fertility may have been lost during its spread from the origin. Here, we show that functional mutations of Pro1, a global transcriptional regulator of mating-related genes in filamentous fungi, is one cause of loss of female fertility in this fungus. We identified the mutation of Pro1 by backcrossing analysis between female-fertile and female-sterile isolates. The dysfunctional Pro1 did not affect the infection processes but conidial release was increased. Furthermore, various mutations in Pro1 were detected in geographically distant P. oryzae, including pandemic isolates of wheat blast fungus. These results provide the first evidence that loss of female fertility may be advantageous to the life cycle of some plant pathogenic fungi.

Structural requirements of virion-associated cholesterol for infectivity, buoyant density and apolipoprotein association of hepatitis C virus.

Our earlier study has demonstrated that hepatitis C virus (HCV)-associated cholesterol plays a key role in virus infectivity. In this study, the structural requirement of sterols for infectivity, buoyant density and apolipoprotein association of HCV was investigated further. We removed cholesterol from virions with methyl ß-cyclodextrin, followed by replenishment with 10 exogenous cholesterol analogues. Among the sterols tested, dihydrocholesterol and coprostanol maintained the buoyant density of HCV and its infectivity, and 7-dehydrocholesterol restored the physical appearance of HCV, but suppressed its infectivity. Other sterol variants with a 3ß-hydroxyl group or with an aliphatic side chain did not restore density or infectivity. We also provide evidence that virion-associated cholesterol contributes to the interaction between HCV particles and apolipoprotein E. The molecular basis for the effects of different sterols on HCV infectivity is discussed.

Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae.

Mycoviruses causing impaired growth and abnormal pigmentation of the host were found in the rice blast fungus, Magnaporthe oryzae. Four dsRNAs, dsRNA 1 (3554 bp), dsRNA 2 (3250 bp), dsRNA 3 (307 bp) and dsRNA 4 (3043 bp), were detected in isolate S-0412-II 1a of M. oryzae. By picking up single conidia of S-0412-II 1a, cured strains of the fungus were isolated that had completely lost the mycovirus. The cured strains had normal mycelial growth and pigmentation, suggesting that this mycovirus modulates host traits. The buoyant densities of isometric virus particles (∼35 nm diameter) containing these dsRNAs in CsCl ranged from 1.37 to 1.40 g cm⁻³. The single ORF (3384 nt) of dsRNA 1 encoded a gene product highly homologous to the viral RNA-dependent RNA polymerase of members of the family Chrysoviridae. It is noteworthy that mycovirus S-0412-II 1a was detected not only in host cells but also in culture supernatant. Furthermore, abnormal aggregation of mycelia was observed after adding the mycovirus-containing culture supernatant to an uninfected strain of M. oryzae and mycoviral dsRNAs were detectable from the aggregated mycelia. This novel dsRNA mycovirus was named Magnaporthe oryzae chrysovirus 1.

Population Structure of Double-Stranded RNA Mycoviruses That Infect the Rice Blast Fungus Magnaporthe oryzae in Japan.

Various viruses infect Magnaporthe oryzae (syn. Pyricularia oryzae), which is a well-studied fungus that causes rice blast disease. Most research has focused on the discovery of new viruses and the hypovirulence-associated traits conferred by them. Therefore, the diversity and prevalence of viruses in wild fungal populations have not been explored. We conducted a comprehensive screening of M. oryzae mycoviruses from various regions in Japan using double-stranded RNA (dsRNA) electrophoresis and RT-PCR assays. We detected three mycoviruses, Magnaporthe oryzae virus 2 (MoV2), Magnaporthe oryzae chrysovirus 1 (MoCV1), and Magnaporthe oryzae partitivirus 1 (MoPV1), among 127 of the 194 M. oryzae strains screened. The most prevalent virus was MoPV1 (58.8%), which often co-infected in a single fungal strain together with MoV2 or MoCV1. MoV2 and MoCV1 were found in 22.7 and 10.8% of strains, respectively, and they were usually distributed in different regions so that mixed-infection with these two mycoviruses was extremely rare. The predominance of MoPV1 in M. oryzae is supported by significant negative values from neutrality tests, which indicate that the population size of MoPV1 tends to increase. Population genetic analyses revealed high nucleotide diversity and the presence of phylogenetically diverse subpopulations among the MoV2 isolates. This was not the case for MoPV1. Furthermore, studies of a virus-cured M. oryzae strain revealed that MoV2 does not cause any abnormalities or symptoms in its host. However, a leaf sheath inoculation assay showed that its presence slightly increased the speed of mycelial growth, compared with virus-free mycelia. These results demonstrate that M. oryzae in Japan harbors diverse dsRNA mycovirus communities with wide variations in their population structures among different viruses.

A novel mycovirus associated with four double-stranded RNAs affects host fungal growth in Alternaria alternata.

Four double-stranded RNAs (dsRNAs), referred to as dsRNA 1 (3617 bp), dsRNA 2 (2794 bp), dsRNA 3 (2576 bp) and dsRNA 4 (1420 bp), were detected in the EGS 35-193 strain of Alternaria alternata at high concentration ( approximately 3 microg/g dried mycelium). This strain had an impaired growth phenotype. By exposing the strain to cycloheximide during hyphal tip isolation, we isolated strains which had normal mycelial growth and pigmentation, in which decreased levels of the dsRNAs were observed ( approximately 0.3 microg/g dried mycelium). These results indicate that this dsRNA mycovirus might be involved in modulating traits of its fungal host, A. alternata. The buoyant density of isometric virus particles (about 33 nm in diameter) containing these dsRNAs in CsCl was 1.35-1.40 g/cm(3) depending on the size of the packaged dsRNAs. The dsRNA 1 encodes a single open reading frame (3447 nt) containing the conserved motifs of viral RNA-dependent RNA polymerase (RdRp), which is related to the ORF encoded by dsRNA 1 of Aspergillus mycovirus 341. It is noteworthy that all of the coding strands of the four dsRNA genomes have 3'-poly (A) tails ranging from 33 to 50 nt in length. We named this novel dsRNA mycovirus in the EGS 35-193 strain A. alternata virus-1 (AaV-1).

Inhibition of histone deacetylase causes reduction of appressorium formation in the rice blast fungus Magnaporthe oryzae.

Post-translational modifications (PTMs) are important for cellular functions. The regulation of histone acetyltransferases (HATs) and histone deacetylases (HDACs) is one of important PTMs for epigenetic control, protein activity and protein stability. The regulation of acetylation of the N-terminal histone tails of core histone affects gene expression. Two class I HDAC genes and two class II HDAC genes have been identified in the Magnaporthe oryzae genome. Treatment with Rpd3/Hda1 family (classical) HDAC inhibitor inhibited the appressorium differentiation of M. oryzae. Treatment with trichostatin A, a classical HDAC inhibitor, also decreased pathogenesis. Furthermore, analyses of HDAC mutants indicated that MoHda1 and MoHos2 were required for vegetative growth and conidiation, and MoHos2 was required for appressorium formation. Disruption MoRPD3 was unsuccessful, as in the case with Aspergillus nidulans RpdA. These data indicated that HDACs have important roles in the asexual differentiation of M. oryzae.

Induction of resistance to diseases in plant by aerial ultrasound irradiation.

Ultrasound, which refers to frequencies above the audible limit of human hearing, is a candidate for inducing resistance to pathogens in plants. We revealed that aerial ultrasound of 40.5 kHz could induce disease resistance in tomatoes and rice when the plants were irradiated with ultrasound of ca. 100 dB for 2 weeks during nursery season and reduced the incidence of Fusarium wilt and blast diseases, respectively, when plants were inoculated with pathogen 0 or 1 week after terminating irradiation. Disease control efficacy was also observed with ultrasound at frequencies of 19.8 and 28.9 kHz. However, cabbage yellows and powdery mildew on lettuce were not suppressed by ultrasound irradiation. No significant positive or negative effect on growth was observed in tomato and rice plants. RT-qPCR showed that the expression of PR1a involved in the salicylic acid (SA) signaling pathway was upregulated in the ultrasound-irradiated tomato.

Magnaporthe oryzae chrysovirus 1 strain D confers growth inhibition to the host fungus and exhibits multiform viral structural proteins.

A Japanese isolate of Magnaporthe oryzae is infected by Magnaporthe oryzae chrysovirus 1-D (MoCV1-D), which is classified in cluster II of the family Chrysoviridae. The genome of MoCV1-D consists of five dsRNAs. dsRNAs 1-4 show high identity with those of related MoCV1 viruses, whereas dsRNA5 shows relatively low identity and is sometimes deleted during virus propagation. MoCV1-D causes growth inhibition of its host fungus, and the protein encoded by its dsRNA4 impairs cell growth when expressed in yeast cells. It also causes abnormal pigmentation and colony albinization, and we showed that these phenotypes are associated with reduced accumulation of the melanin biosynthesis intermediate scylatone. MoCV1-D exhibits multiform viral structural proteins during prolonged culture. The original host isolate is co-infected with MoCV1-D, a victorivirus, and a partitivirus, and these mycoviruses are detected in cell-free supernatant fractions after prolonged liquid culturing. Hyphal fusion experiments demonstrated that MoCV1-D is transmissible via anastomosis.

N-terminal region of cysteine-rich protein (CRP) in carlaviruses is involved in the determination of symptom types.

Plant viruses in the genus Carlavirus include more than 65 members. Plants infected with carlaviruses exhibit various symptoms, including leaf malformation and plant stunting. Cysteine-rich protein (CRP) encoded by carlaviruses has been reported to be a pathogenicity determinant. Carlavirus CRPs contain two motifs in their central part: a nuclear localization signal (NLS) and a zinc finger motif (ZF). In addition to these two conserved motifs, carlavirus CRPs possess highly divergent, N-terminal, 34 amino acid residues with unknown function. In this study, to analyse the role of these distinct domains, we tested six carlavirus CRPs for their RNA silencing suppressor activity, ability to enhance the pathogenicity of a heterologous virus and effects on virus accumulation levels. Although all six tested carlavirus CRPs showed RNA silencing suppressor activity at similar levels, symptoms induced by the Potato virus X (PVX) heterogeneous system exhibited two different patterns: leaf malformation and whole-plant stunting. The expression of each carlavirus CRP enhanced PVX accumulation levels, which were not correlated with symptom patterns. PVX-expressing CRP with mutations in either NLS or ZF did not induce symptoms, suggesting that both motifs play critical roles in symptom expression. Further analysis using chimeric CRPs, in which the N-terminal region was replaced with the corresponding region of another CRP, suggested that the N-terminal region of carlavirus CRPs determined the exhibited symptom types. The up-regulation of a plant gene upp-L, which has been reported in a previous study, was also observed in this study; however, the expression level was not responsible for symptom types.

Novel mating type-dependent transcripts at the mating type locus in Magnaporthe oryzae.

The mating type locus (MAT1) of Magnaporthe oryzae has similar structural organization to MAT in other ascomycetes and encodes the mating type genes MAT1-1-1 with an alpha-box motif and MAT1-2-1 with an HMG-box motif in the MAT1-1 and MAT1-2 idiomorphs, respectively. Sequence and expression analyses of the MAT1 locus indicated a second open reading frame (ORF), MAT1-1-2, in the MAT1-1 idiomorph, and novel mating-type dependent ORFs (MAT1-1-3 and MAT1-2-2) at the locus. The MAT1-1-3 ORF initiated within the MAT1-1 idiomorph while the MAT1-2-2 ORF initiated at the border of the MAT1-2 idiomorph with both ORFs sharing most of their reading frames in the MAT1 flanking region. This suggests that the encoded proteins (MAT1-1-3 and MAT1-2-2) should be similar in their primary structures but can be distinguished by distinct N-termini with amino acids of 1 and 32, respectively, in each mating type. A CT dinucleotide repeat, (CT)n, present in the upstream region of MAT1-1-3, was polymorphic among the isolates.

Chitin-deacetylase activity induces appressorium differentiation in the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium to invade rice cells. In this report, we show that CBP1, which encodes a chitin-deacetylase, is involved in the induction phase of appressorium differentiation. We demonstrate that the enzymatic activity of Cbp1 is critical for appressorium formation. M. oryzae has six CDA homologues in addition to Cbp1, but none of these are indispensable for appressorium formation. We observed chitosan localization at the fungal cell wall using OGA488. This observation suggests that Cbp1-catalysed conversion of chitin into chitosan occurs at the cell wall of germ tubes during appressorium differentiation by M. oryzae. Taken together, our results provide evidence that the chitin deacetylase activity of Cbp1 is necessary for appressorium formation.

FCD1 encoding protein homologous to cellobiose: quinone oxidoreductase in Fusarium oxysporum.

We had cloned and characterized a gene from Fusarium oxysporum designated FCD1, encoding a putative cellobiose: quinone oxidoreductase (CBQ) which is a member of the extracellular redox enzyme family and also a member of glucose-methanol-choline (GMC) oxidoreductases. CBQ is known to be a free flavin domain of a cellobiose dehydrogenase (CDH) generated by proteolysis, but FCD1 gene encodes CBQ directly. In a phylogenetic tree of amino acid sequences of FCD1, GMC oxidoreductases and hypothetical GMC oxidoreductases, FCD1 clustered together with flavin domains (CBQs) of CDHs and putative proteins with unknown function of ascomycetes. FCD1-disruptants showed no reduction in virulence toward tomato and no obvious morphological effects such as production of conidia and mycelial growth as compared to the wild type strain, suggesting that FCD1 is not essential for virulence and vigor in F. oxysporum.