Molecular characterization and host reaction to tomato mottle mosaic virus isolated from sweet pepper seeds in Japan.

Many countries have identified tomato mottle mosaic virus (ToMMV) as a serious threat to tomato production. Here, we constructed and characterized infectious clones of ToMMV isolated from Japanese sweet pepper seeds. The genome of the Japanese isolate is 6399 nucleotides in length and exhibits the highest identity with previously characterized isolates. For example, it is 99.7% identical to that of the Mauritius isolate, which occurs worldwide. Phylogenetic analysis based on complete genome sequences revealed that the Japanese isolates clustered in the same clade as those from other countries. When homozygous tomato cultivars with tobamovirus resistance genes were inoculated with an infectious cDNA clone of ToMMV, the virus systemically infected tomato plants with symptoms typical of Tm-1-carrying tomato cultivars. In contrast, tomato cultivars carrying Tm-2 or Tm-22 showed symptoms only on the inoculated leaves. Furthermore, when commercial cultivars of Tm-22 heterozygous tomato were inoculated with ToMMV, systemic infections were observed in all cultivars, with infection frequencies ranging from 25 to 100%. Inoculation of heterozygous sweet pepper cultivars with tobamovirus resistance genes (L1, L3, and L4) with ToMMV resulted in an infection frequency of about 70%, but most of the infected L1, L3, and L4 cultivars were symptomless, and 10-20% showed symptoms of necrosis and yellowing. Tomato mosaic virus strain L11A, an attenuated virus, did not provide cross-protection against ToMMV and led to systemic infection with typical symptoms. These results suggest that ToMMV might cause extensive damage to existing tomato and sweet pepper cultivars commonly grown in Japan.

Re-discovery of MS-347a as a fungicide candidate through a new drug discovery platform with a multidrug-sensitive Saccharomyces cerevisiae screening system and the introduction of a global secondary metabolism regulator, laeA gene.

We found that the culture broth of fungi showed anti-fungal activity against multidrug-sensitive budding yeast. However, we could not identify the anti-fungal compound due to the small quantity. Therefore, we attempted to increase the productivity of the target compound by the introduction of a global secondary metabolism regulator, laeA to the strain, which led to the successful isolation of 10-folds greater amount of MS-347a (1) than Aspergillus sp. FKI-5362. Compound 1 was not effective against Candida albicans and the detailed anti-fungal activity of 1 remains unverified. After our anti-fungal activity screening, 1 was found to inhibit the growth of broad plant pathogenic fungal species belonging to the Ascomycota. It is noteworthy that 1 showed little insecticidal activity against silkworms, suggesting its selective biological activity against plant pathogenic fungi. Our study implies that the combination strategy of multidrug-sensitive yeast and the introduction of laeA is useful for new anti-fungal drug discovery.

Infectivity of highly pathogenic isolates of potato spindle tuber viroid in dahlia.

Dahlias that are naturally infected with potato spindle tuber viroid (PSTVd) do not exhibit symptoms. Therefore, if PSTVd isolates that are highly pathogenic in tomato plants infect dahlias, there is a significant risk of PSTVd infecting other plants via dahlias. In this study, we found that almost all highly pathogenic isolates were able to infect dahlia plants, but the symptoms varied depending on the cultivar. When mixed inocula composed of dahlia isolates and highly pathogenic isolates were tested, the dahlia isolates dominantly infected dahlia plants; however, the highly pathogenic isolates also coinfected plants. Our results also suggest that seed or pollen transmission from infected dahlia plants does not occur.

Long-term passages of Plantago asiatica mosaic virus alter virulence and multiplication in Nicotiana benthamiana plants.

We demonstrated the infectivity and host adaptation of a viola isolate of Plantago asiatica mosaic virus (PlAMV-Vi) in an asymptomatic host, Nicotiana benthamiana, through long-term serial passages. Serial passaging of a green fluorescent protein-tagged full-length cDNA clone of PlAMV-Vi (PlAMV-ViGFP) in N. benthamiana plants resulted in the appearance of a new virus line inducing leaf-crinkle symptoms, the Leaf Crinkle (LC) line. Virus titers were higher for both in the LC and the 14th passage line(s) of PlAMV-ViGFP compared with the original line. The LC line was found to have seven unique nucleotide mutations that may have contributed to its higher virulence and multiplication rate in N. benthamiana.

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.

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.

Characterization of a new carmovirus isolated from an Adonis plant.

An isometric virus was isolated from a cultivated Adonis plant (A. ramosa). The purified virus particle is 28 nm in diameter and is composed of a single coat protein and a single RNA genome of 3,991 nucleotides. Sequence analysis showed that the virus is closely related to carnation mottle virus. The virus was used to mechanically infect healthy A. ramosa plants, resulting in mosaic and leaf curl symptoms; however, attempts to inoculate carnation plants did not result in infection. We propose the virus as a new carmovirus and have named it adonis mosaic virus (AdMV).

Complete genome sequences of two highly divergent Japanese isolates of Plantago asiatica mosaic virus.

Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range. It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and the other from the eudicot shrub Nandina domestica Thunb. (PlAMV-NJ). Their genomes contain five open reading frames (ORFs), which is characteristic of potexviruses. Surprisingly, the isolates showed only 76.0-78.0 % sequence identity with each other and with other PlAMV isolates, including isolates from Japanese lily and American nandina. Amino acid alignments of the replicase coding region encoded by ORF1 showed that the regions between the methyltransferase and helicase domains were less conserved than other regions, with several insertions and/or deletions. Phylogenetic analyses of the full-length nucleotide sequences revealed a moderate correlation between phylogenetic clustering and the original host plants of the PlAMV isolates. This study revealed the presence of two highly divergent PlAMV isolates in Japan.

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.

Effect of Soil Inoculum Density and Temperature on the Incidence of Cucumber Black Root Rot.

The effects of the density of Phomopsis sclerotioides in soil and other environmental factors on black root rot of cucumber were investigated. Cucumber plants were grown in soil containing P. sclerotioides at 1, 10, 100, and 1000 CFU/g. Wilt incidence from 3 to 7 weeks after transplanting was strongly correlated with P. sclerotioides density in soil (P < 0.05). Root rot of squash rootstock occurred in soil with very low inoculum densities (0.1 CFU/g), and was strongly related to P. sclerotioides density (Y = -0.3x + 1.2, R2 = 0.743, P < 0.05) at 8 weeks after transplanting. Cucumber plants showed wilt symptoms in soil containing 1 CFU/g. Wilt symptoms in cucumber plants occurred 4 to 7 days earlier in soil at 22°C than in soil at 27 or 17°C. Root rot development could be predicted from the density of P. sclerotioides in soil and soil temperature. However, further studies on the effects of other environmental factors are required to test the linear model in commercial fields. This information is essential for determining the threshold pathogen density at which most control techniques, particularly those other than soil disinfection, will be effective.

Total Synthesis of Fusaramin, Enabling Stereochemical Elucidation, Structure-Activity Relationship, and Uncovering the Hidden Antimicrobial Activity against Plant Pathogenic Fungi.

Fusaramin (1) was isolated as a mitochondrial inhibitor. However, the fungal producer stops producing 1, which necessitates us to supply 1 by total synthesis. We proposed the complete stereochemical structure based on the biosynthetic pathway of sambutoxin. We have established concise and robust total synthesis of 1, enabling us to determine the complete stereochemical structure and to elucidate the structure-activity relationship, and uncover the hidden antiplant pathogenic fungal activity.

Genetic diversity of viruses infecting cnidium plants (Cnidium officinale) in Japan.

Cnidium vein yellowing virus (CnVYV), cnidium virus X (CnVX), cucumber mosaic virus (CMV) and cnidium virus 1 (CnV1) were detected at extremely high levels in Cnidium officinale plants showing viral symptoms collected from Iwate and Hokkaido Prefectures, Japan. The complete nucleotide sequence of the newly detected CnVYV and CnV1, and genetic diversity of the cnidium-infecting viruses (CnVYV, CnVX, and CnV1) indicated that South Korean and Japanese cnidium plants had close relationship with each other. All three viruses can infect vegetatively propagated perennials and are vertically transmitted once infection occurs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00835-w.

A Combination Strategy of Multidrug-Sensitive Budding Yeast and Chemical Modifications Enabling to Find a New Overlooked Antifungal Compound, Sakurafusariene, from In-House Fractionated Library.

In this report, we disclose our discovery of a new antifungal natural product, sakurafusariene (1), from an in-house fractionated library of the culture broth of Fusarium sp. FKI-7550 strain by using a combination strategy of multidrug-sensitive yeast and chemical modification. Throughout our investigation, we encountered challenges in the isolation of natural product 1. A chemical modification strategy via alkylation of 1 allowed for removal of the impurities enabling us to elucidate the structure of 1. Furthermore, we synthesized ester derivatives using a method inspired by the isolation study of 1, which gave us valuable information to understand a preliminary structure-activity relationship against Pyricularia oryzae growth inhibitory activity.

Comparison of two highly sensitive methods to detect potato spindle tuber viroid in Dahlia using quantitative-reverse transcription-polymerase chain reaction assays.

Potato spindle tuber viroid (PSTVd) belongs to the Pospiviroidae family and is the type species for the genus Pospiviroid. In 2011, PSTVd was first detected in dahlias in Japan. Since that time, unregistered PSTVd isolates have been identified in seven field-grown dahlia cultivars. None of the infected dahlias showed disease symptoms during the early stages of infection, however, growth suppression occasionally occurred during later stages. Therefore, in dahlia, diagnosing PSTVd by the external appearance of plants is difficult, and the threat of new PSTVd isolates spreading to other susceptible hosts still remains. In this study, we developed an efficient inspection method using several dahlia plant tissues and organs including dried bulbs. This developed method will be useful for inspecting seedlings to prevent the invasion of PSTVd at the border.

Isolation of novel types of Arabidopsis mutants with altered reactions to cadmium: cadmium-gradient agar plates are an effective screen for the heavy metal-related mutants.

We are interested in elucidating the molecular mechanisms underlying plant reactions to the toxic heavy metal cadmium (Cd). To this end, we devised a new screening strategy using agar plates with a gradient of Cd concentrations, termed Cd-gradient agar plates (CGAPs), to isolate Arabidopsis mutants that displayed altered reactions to the metal. Arabidopsis M(2) seeds, derived from ethyl methanesulfonate (EMS) treated seeds, were germinated on the CGAPs such that the primary root of each seedling elongated against increasing concentrations of Cd on the surface of the plate. Under these conditions, the lengths of the primary roots reliably demonstrated the degree of Cd tolerance of individual seedlings. The use of CGAPs also allowed close observation of the root reaction of each seedling to Cd without causing lethal damage. The screen identified three mutant lines, MRC-32, MRC-22 and MRC-26, which showed distinctly different characteristics. MRC-32 plants exhibited enhanced tolerance to Cd and contained Cd at higher concentrations than wild-type (WT) plants treated with the heavy metal. The whole root system of MRC-22 plants showed a Cd-phobic response. MRC-26 plants accumulated less Cd in their aboveground tissues than WT plants, suggesting that they were defective in transporting the heavy metal from roots to aboveground tissues. We also determined the likely chromosomal location of each mutation.

Development of specific primers for Fusarium oxysporum causing damping off of Lilium formolongi.

Primers specific for the hypothetical forma specialis of Fusarium oxysporum were designed to amplify DNA from this pathogenic fungus that infects plants including lilies. The F. oxysporum sequence between the transposal elements han and hop was used for primer design. Three primer pairs designed from this region were confirmed as specific for 24 isolates of F. oxysporum pathogenic to lilies, except for one pathogenic isolates as extraordinary. No amplification was observed from F. oxysporum non-pathogenic to lily, from 12 forma specialis, and 14 fungi and oomycetes concerned with Liliaceae plants. We propose that specific primers designed from this region will be useful to detect isolates of F. oxysporum that are pathogenic to lilies.

Mycovirus-Induced Tenuazonic Acid Production in a Rice Blast Fungus Magnaporthe oryzae.

Fungi are a rich source of natural products with biological activities. In this study, we evaluated viral effects on secondary metabolism of the rice blast fungus Magnaporthe oryzae using an isolate of APU10-199A co-infected with three types of mycoviruses: a totivirus, a chrysovirus, and a partitivirus. Comparison of the secondary metabolite profile of APU10-199A with that of the strain lacking the totivirus and chrysovirus showed that a mycotoxin tenuazonic (TeA) acid was produced in a manner dependent on the mycoviruses. Virus reinfection experiments verified that TeA production was dependent on the totivirus. Quantitative reverse transcription PCR and RNA-sequencing analysis indicated the regulatory mechanism underlying viral induction of TeA: the totivirus activates the TeA synthetase gene TAS1 by upregulating the transcription of the gene encoding a Zn(II)2-Cys6-type transcription factor, TAS2. To our knowledge, this is the first report that confirmed mycovirus-associated regulation of secondary metabolism at a transcriptional level by viral reinfection. Because only treatment with dimethyl sulfoxide has been reported to trigger TeA production in this fungus without gene manipulation, our finding highlights the potential of mycoviruses as an epigenomic regulator of fungal secondary metabolism.

Modeling infection of spring onion by Puccinia allii in response to temperature and leaf wetness.

The influence of temperature and leaf wetness duration on infection of spring onion (Japanese bunching onion) leaves by Puccinia allii was examined in controlled-environment experiments. Leaves of potted spring onion plants (Allium fistulosum cv. Yoshikura) were inoculated with urediniospores and exposed to 6.5, 10, 15, 22, or 27 h of wetness at 5, 10, 15, 20, or 25 degrees C. The lesion that developed increased in density with increasing wetness duration. Relative infection was modeled as a function of both temperature and wetness duration using the modified version of Weibull's cumulative distribution function (R(2) = 0.9369). Infection occurred between 6.5 and 27 h of leaf wetness duration at 10, 15, 20, and 25 degrees C and between 10 and 27 h at 5 degrees C, and increased rapidly between 6.5 and 15 h of wetness at 10, 15, and 20 degrees C. At 25 degrees C, few uredinia developed regardless of the wetness duration. Parameter H, one of eight parameters used in the equation and which controls the asymmetry in the response curve, varied markedly according to the temperature, so that the model could be improved by representing H as a function of wetness duration (R(2) = 0.9501).

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.

Changes in the Proteome of Xylem Sap in Brassica oleracea in Response to Fusarium oxysporum Stress.

Fusarium oxysporum f.sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F. oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change > = 2-fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and 10 of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.