Microbial diversity in an apple orchard cultivated with natural farming practices for over 30 years was compared with conventionally farmed orchards to analyze differences in disease suppression. In this long-term naturally farmed orchard, major apple diseases were more severe than in conventional orchards but milder than in a short-term natural farming orchard. Among major fungal species in the phyllosphere, we found that Aureobasidium pullulans and Cryptococcus victoriae were significantly less abundant in long-term natural farming, while Cladosporium tenuissimum predominated. However, diversity of fungal species in the phyllosphere was not necessarily the main determinant in the disease suppression observed in natural farming; instead, the maintenance of a balanced, constant selection of fungal species under a suitable predominant species such as C. tenuissimum seemed to be the important factors. Analysis of bacteria in the phyllosphere revealed Pseudomonas graminis, a potential inducer of plant defenses, predominated in long-term natural farming in August. Rhizosphere metagenome analysis showed that Cordyceps and Arthrobotrys, fungal genera are known to include insect- or nematode-infecting species, were found only in long-term natural farming. Among soil bacteria, the genus Nitrospira was most abundant, and its level in long-term natural farming was more than double that in the conventionally farmed orchard.

Apple russet ring and apple green crinkle are graft-transmitted diseases first reported more than 60 years ago, but at present, no association between a specific virus (variant) and the disease has been clearly demonstrated. In this study, we conducted the following series of experiments to identify the causal viruses (variants) of these apple diseases; (1) comprehensive analysis by next-generation sequencing of all viruses in each apple tree affected with russet ring or green crinkle disease, (2) amplification of full-length genomic cDNA of viruses using primers containing the T3 promoter and the in vitro transcription of infectious viral RNAs, (3) inoculation of viral RNA transcripts to both herbaceous and apple plants, (4) analysis of sequence variants of viruses present in infected plants, (5) back-inoculation of sequence variants of candidate viruses to apple seedlings combined with the virus-induced flowering technology using the apple latent spherical virus vector to reproduce the symptom on the fruit as soon as possible, and (6) reproduction of symptoms on the fruits of apple trees inoculated with sequence variants and the re-isolation of each virus variant from apples showing fruit symptoms. The results showed that one of the sequence variants of the apple chlorotic leaf spot virus causes a characteristic ring-shaped rust on the fruits of infected apple trees and that a sequence variant of the apple stem pitting virus probably causes green crinkle symptoms on an infected apple fruit. Thus, we were able to fulfill Koch's postulates to prove the viral etiology of both the apple russet ring and green crinkle diseases. We also propose an experimental system that can prove whether a virus found in diseased tissues is the pathogen responsible for the diseases when the etiology is undetermined.

The agent causing bud blight disease (BBD) of sweet cherry ( Prunus avium ), first reported in Yamagata Prefecture, Japan, in the 1990s is graft-transmissible. Previously, little cherry virus 1 (LChV-1), little cherry virus-2 (LChV-2), cherry necrotic rusty mottle virus (CNRMV), cherry green ring mottle virus (CGRMV), and/or cherry virus A (CVA) were all detected from sweet cherry trees with BBD. Here, RNA viruses in sweet cherry trees with BBD were reinvestigated by high-throughput sequencing. Illumina RNA-sequencing of double-stranded RNAs from leaves of diseased cherry trees (S1, S2, S3, and S4) indicated that all trees were infected with at least six known viruses, LChV-1, LChV-2, CNRMV, CGRMV, CVA, prune dwarf virus (PDV), and/or apple chlorotic leaf spot virus. Thus, cherry trees with BBD are all infected with multiple viruses. Moreover, we found an undescribed novel virus of the family Betaflexiviridae (tentatively named cherry virus B, ChVB) in tree S3. The complete genome sequence of ChVB comprises 8806 nt, in which five open reading frames were found similar to the viruses of the genus Foveavirus . Homology search and phylogenetic analysis indicated that ChVB is a new species of the genus Foveavirus .

In 2016, a serious outbreak of apple scab (caused by Venturia inaequalis) in Aomori Prefecture, Japan, resulted from reduced fungal sensitivity to sterol demethylation inhibitors (DMIs). In this study, the lanosterol 14α-demethylase (CYP51A1) gene of V. inaequalis isolates from Aomori Prefecture was analyzed. A nonsynonymous nucleotide polymorphism causing an amino acid substitution from tyrosine to phenylalanine at position 133 (Y133F) in CYP51A1 was closely associated with low sensitivity to DMIs. This is first report of a point mutation in CYP51A1 associated with low sensitivity to DMIs in V. inaequalis.

The toxicity of 29 fungicides was tested against 4 native phytoseiid species, namely, Amblyseius eharai Amitai and Swirski, Amblyseius tsugawai Ehara, Euseius sojaensis（Ehara）, and Typhlodromus vulgaris Ehara（Acari: Phytoseiidae）, which are abundantly found in fruit tree orchards in Japan. Mancozeb and propineb were toxic to the immature individuals of all 4 species and to the fecundity of adult females of E. sojaensis and A. eharai. Thiuram was harmless or slightly toxic to A. eharai, A. tsugawai, and T. vulgaris, whereas it was toxic to E. sojaensis. Sulphur and polyoxin were toxic to the immature individuals of all four species. Benomyl and thiophanate-methyl reduced the fecundity of A. eharai, A. tsugawai, and E. sojaensis. Fluazinam was toxic to immature individuals and the fecundity of adult females of E. sojaensis and moderately toxic to A. eharai and A. tsugawai. Iminoctadine triacetate was toxic to immature individuals of E. sojaensis. Fluoroimide was also moderately toxic to immature individuals of E. sojaensis. On the other hand, basic copper sulfate, oxine-copper, captan, chlorothalonil, iminoctadine tris（albesilate）, dithianon, difenoconazole, hexaconazole, imibenconazole, tebuconazole, fluopyram, inpyrfluxam, isopyrazam, penthiopyrad, pyraziflumid, cyprodinil, kresoxim-methyl, pyribencarb, and trifloxystrobin were harmless to all 4 species.

The causal agent of apple mosaic disease has been previously thought to be solely caused by apple mosaic virus (ApMV). In this study, we report that a novel ilarvirus is also associated with apple mosaic disease. Next-generation sequencing analysis of an apple tree showing mosaic symptoms revealed that the tree was infected with three apple latent viruses (apple stem pitting virus, apple stem grooving virus, and apple chlorotic leaf spot virus) and a novel ilarvirus (given the name apple necrotic mosaic virus (ApNMV)) that is closely related to Prunus necrotic ringspot virus (PNRSV) and ApMV. The genome of ApNMV consists of RNA1 (3378 nt), RNA2 (2767 nt), and RNA3 (1956 nt). A phylogenetic analysis based on the coat protein amino acid sequences indicated that the novel virus belongs to the same subgroup 3 of the genus Ilarvirus as PNRSV and ApMV. The presence of mosaic leaves, which tend to be unevenly distributed in diseased apple trees, was correlated with the internal distribution of ApNMV. RT-PCR detection of mosaic-diseased apple trees in Japan indicated that ApNMV was detected in apple trees introduced from China, whereas ApMV was detected from cultivated apple trees in domestic orchards. Consistent with these findings, a survey of mosaic-diseased apple trees in major apple-producing provinces in China revealed that the majority of apple trees showing mosaic symptoms in China are infected with ApNMV.

Apple dimple fruit viroid was detected from an apple tree (‘Jonagold’) bearing apples with mild dapple apple symptom. The isolates in Japan were distinct from those in apples in Italy and China and in fig in Italy. Graft-inoculation experiments showed that the symptoms were variable depending on the cultivar, and the symptom on ‘Starking Delicious’ was virtually similar to those reported in Italy. Symptoms induced by apple dimple fruit viroid were similar in part to those by apple fruit crinkle viroid or apple scar skin viroid, indicating that they cannot be discriminated by symptoms on any specific variety.

ABSTRACT RNA silencing acts as a defense mechanism against virus infection in a wide variety of organisms. Here, we investigated inductions of RNA silencing against encapsidated double-stranded RNA (dsRNA) fungal viruses (mycoviruses), including a partitivirus (RnPV1), a quadrivirus (RnQV1), a victorivirus (RnVV1), a mycoreovirus (RnMyRV3), and a megabirnavirus (RnMBV1) in the phytopathogenic fungus Rosellinia necatrix . Expression profiling of RNA silencing-related genes revealed that a dicer-like gene, an Argonaute-like gene, and two RNA-dependent RNA polymerase genes were upregulated by RnMyRV3 or RnMBV1 infection but not by other virus infections or by constitutive expression of dsRNA in R. necatrix . Massive analysis of viral small RNAs (vsRNAs) from the five mycoviruses showed that 19- to 22-nucleotide (nt) vsRNAs were predominant; however, their ability to form duplexes with 3′ overhangs and the 5′ nucleotide preferences of vsRNAs differed among the five mycoviruses. The abundances of 19- to 22-nt vsRNAs from RnPV1, RnQV1, RnVV1, RnMyRV3, and RnMBV1 were 6.8%, 1.2%, 0.3%, 13.0%, and 24.9%, respectively. Importantly, the vsRNA abundances and accumulation levels of viral RNA were not always correlated, and the origins of the vsRNAs were distinguishable among the five mycoviruses. These data corroborated diverse interactions between encapsidated dsRNA mycoviruses and RNA silencing. Moreover, a green fluorescent protein (GFP)-based sensor assay in R. necatrix revealed that RnMBV1 infection induced silencing of the target sensor gene (GFP gene and the partial RnMBV1 sequence), suggesting that vsRNAs from RnMBV1 activated the RNA-induced silencing complex. Overall, this study provides insights into RNA silencing against encapsidated dsRNA mycoviruses. IMPORTANCE Encapsidated dsRNA fungal viruses (mycoviruses) are believed to replicate inside their virions; therefore, there is a question of whether they induce RNA silencing. Here, we investigated inductions of RNA silencing against encapsidated dsRNA mycoviruses (a partitivirus, a quadrivirus, a victorivirus, a mycoreovirus, and a megabirnavirus) in Rosellinia necatrix . We revealed upregulation of RNA silencing-related genes in R. necatrix infected with a mycoreovirus or a megabirnavirus but not with other viruses, which was consistent with the relatively high abundances of vsRNAs from the two mycoviruses. We also showed common and different molecular features and origins of the vsRNAs from the five mycoviruses. Furthermore, we demonstrated the activation of RNA-induced silencing complex by mycoviruses in R. necatrix . Taken together, our data provide insights into an RNA silencing pathway against encapsidated dsRNA mycoviruses which is differentially induced among encapsidated dsRNA mycoviruses; that is, diverse replication strategies exist among encapsidated dsRNA mycoviruses.

Watercored (WC) apples, characterized by flesh that has a translucent, water-soaked appearance, have gained wide popularity with Japanese consumers. To understand this preference for WC apples, we performed a combination of sensory analysis and metabolic profiling on apple flavors using 'Fuji and 'Kotoku', cultivars susceptible to watercore. The sensory characteristics of WC 'Fuji were enhanced in aroma intensity, floral, sweet and fruity attributes compared to non-WC apples. Rating of preference was also high in WC apples. In regards to the flavor components of 'Fuji' and 'Kotoku', solubles of sorbitol, sucrose, L-alanine, pyro-glutamate and putrescine, volatiles of methyl and ethyl esters, such as methyl 2-methylbutanoate, ethyl 2-methylbutanoate and ethyl hexanoate, were significantly greater in WC apples. While the degree of sweetness with sugar composition slightly increased in a trial calculation, sweet and floral aroma presenting volatiles were remarkably elevated in the WC apples. Thus, we propose that ethyl and methyl esters, which are anaerobically-biosynthesized in the watercored area, contributed to the high preference for WC apples among Japanese consumers.

The violet root rot fungus, Helicobasidium mompa , is agriculturally important, but development of a genetic transformation system for the fungus has been limited. In this study, H. mompa V17 and V664 isolates were biolistically transformed with a plasmid expressing the hygromycin B phosphotransferase ( hph ) gene. Hygromycin-resistant strains were obtained from both isolates. Various numbers of copies of the hph gene expression cassette were integrated into the H. mompa genome. Most transformants grew stably on selective and nonselective media, suggesting that biolistic transformation is an efficient method to generate mitotically stable H. mompa transformants.

Flavors of "Fuji" apple cultivated with or without synthetic agrochemicals were compared using quantitative descriptive analyses (QDA) and metabolite profiling for 3 seasons. Experimental plots included conventional crops (with agrochemicals) and organic crops (without agrochemicals) at our institute and organic and conventional farms. Additionally, mass market samples were analyzed. Organic apples were weak in sweetness and floral characteristics and had enhanced green and sour flavors. Most esters and sugars were present in lower concentrations in organic than in conventional apples. Close relation of principal component 1 of QDA and metabolite profiles, to ethylene production suggested that ethylene is considerably involved in flavor synthesis. Reduced ethylene associated with immaturity accounted for insufficient flavor synthesis and weak aroma and flavor attributes of organic apples. Furthermore, organic apples from the farm were more flavorsome than those from the institute in 2012, suggesting possible recovery of ethylene production after a long organic cultivation period.

The white root rot fungus, Rosellinia necatrix, damages a wide range of fruit trees. R. necatrix is known to host a variety of mycoviruses, and several of these have potential as biological control agents. RNA interference (RNAi) is a fungal defense mechanism against viral infection, and it is therefore important to understand the RNAi amplification and transmission systems in R. necatrix for effective use of mycoviruses in disease control. In this study, we describe an intriguing RNAi signal transmission phenomenon in R. necatrix. In R. necatrix transformants with autonomously replicating vectors carrying a hairpin structure to induce RNAi, the gene silencing effect was distributed locally and unevenly, based on the vector distribution. This indicates that R. necatrix has no mechanism to propagate silencing signals systemically, unlike Caenorhabditis elegans and Arabidopsis thaliana. Furthermore, the expression of RNA-dependent RNA polymerase homologs was not upregulated during RNAi induction, suggesting that silencing signals are not amplified at sufficient levels to induce systemic RNAi in R. necatrix. Our results also suggest that, in addition to hairpin-induced RNAi, there is either a 5' transitive RNAi or quelling-like gene silencing system in R. necatrix. This is the first study demonstrating that systemic RNAi is not induced by local RNAi in fungi. (C) 2015 Elsevier Inc. All rights reserved.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) is a bi-segmented double-stranded RNA mycovirus that reduces the virulence of the fungal plant pathogen R. necatrix. We isolated strains of RnMBV1 with genome rearrangements (RnMBV1-RS1) that retained dsRNA1, encoding capsid protein (ORF1) and RNA-dependent RNA polymerase (ORF2), and had a newly emerged segment named dsRNAS1, but with loss of dsRNA2, which contains two ORFs of unknown function. Analyses of two variants of dsRNAS1 revealed that they both originated from dsRNA1 by deletion of ORF1 and partial tandem duplication of ORF2, retaining a much shorter 5' untranslated region (UTR). R. necatrix transfected with RnMBV-RS1 virions showed maintenance of virulence on host plants compared with infection with RnMBV1. This suggests that dsRNAS1 is able to be transcribed and packaged, as well as suggesting that dsRNA2, while dispensable for virus replication, is required to reduce the virulence of R. necatrix.

Rosellinia necatrix causes white root rot in a wide range of fruit trees and persists for extended periods as pseudosclerotia on root debris. However, the pathogenesis of this disease has yet to be clarified. The functions of endogeneous target genes have not been determined because of the inefficiency in genetic transformation. In this study, the function of a melanin biosynthetic gene was determined to examine its role in morphology and virulence. A polyketide synthase gene (termed as RnPKS1) in the R. necatrix genome is homologous to the 1,8-dihydroxynaphthalene (DHN) melanin biosynthetic gene of Colletotrichum lagenarium. Melanin-deficient strains of R. necatrix were obtained by RNA interference-mediated knockdown of RnPKS1. The virulence of these strains was not significantly reduced compared with the parental melanin-producing strain. However, knockdown strains failed to develop pseudosclerotia and were degraded sooner in soil than the parental strain. Microscopic observations of albino conidiomata produced by knockdown strains revealed that melanization is involved in synnema integrity. These results suggest that melanin is not necessary for R. necatrix pathogenesis but is involved in survival through morphogenesis. This is the first report on the functional analysis of an endogenous target gene in R. necatrix.

We used reverse transcription quantitative PCR (RT-qPCR) to test for apple latent spherical virus (ALSV) in cotyledons of seeds and true leaves of seedlings from apple trees infected with ALSV to accurately determine the status of infection in seedlings from ALSV-infected trees. The amplification curves of ALSV-RNAs from cotyledons were classified into three patterns: fast (F), slow (S), and no amplification (N/A). Viral RNA was detected in high concentrations (F pattern) in cotyledons from approximately 1 % of the seeds, a proportion broadly consistent with previously reported ALSV seed transmission rates (from zero to a few percent). In the majority of seeds (94.4 %); however, minute amounts of RNA related to the virus were detected (S pattern). Minute amounts of viral-related RNA were also detected by siRNA analysis using a next-generation sequencer. After the seeds germinated, however, no viral RNA was detected in the true leaves, so we concluded that the viral RNA in the seeds lacked the capacity to replicate. No ALSV-RNA at all was detected (N/A pattern) in the remaining 4.6 % of seeds. We concluded that almost all apple seedlings from ALSV-infected trees (approx. 99 %) can be considered as virus-free. Therefore, we think that apple seedlings from the generation obtained after the use of ALSV vector technology can be exempted from restrictions on genetically modified plants after they have been confirmed to be virus-free by virus testing.

RNA silencing is a fundamental antiviral response in eukaryotic organisms. We investigated the counterdefense strategy of a fungal virus (mycovirus) against RNA silencing in the white root rot fungus, Rosellinia necatrix. We generated an R. necatrix strain that constitutively induced RNA silencing of the exogenous green fluorescent protein (GFP) gene, and infected it with each of four unrelated mycoviruses, including a partitivirus, a mycoreovirus, a megabirnavirus, and a quadrivirus. Infection with a mycoreovirus (R. necatrix mycoreovirus 3; RnMyRV3) suppressed RNA silencing of GFP, while the other mycoviruses did not. RnMyRV3 reduced accumulation of GFP-small interfering (si) RNAs and increased accumulation of GFP-double-stranded (ds) RNA; suggesting that the virus interferes with the dicing of dsRNA. Moreover, an agroinfiltration assay in planta revealed that the S10 gene of RnMyRV3 has RNA silencing suppressor activity. These data corroborate the counterdefense strategy of RnMyRV3 against host RNA silencing.

A double-stranded (ds) RNA, approximately 9.5kb in size; was identified in the MVC86 isolate of Valsa ceratosperma. Complete sequence of the dsRNA revealed a 9543-bp segment (excluding the 3' poly-A tail) that is predicted to encode a single large protein (P330). P330 has 63%, 49%, and 55% amino acid sequence identities to the proteins encoded by hypoviruses Cryphonectria hypovirus 3 (CHV3), CHV4, and Sclerotinia sclerotiorum hypovirus 1 (SsHV1), respectively. Like polyproteins encoded by CHV3, CHV4, and SsHV1, P330 comprises four conserved domains, including a papain-like protease, a UDP glucose/sterol glucosyltransferase (UGT), an RNA-dependent RNA polymerase (RdRp), and an RNA helicase. These molecular characteristics suggest that this dsRNA represents a new hypovirus that we tentatively designate Valsa ceratosperma hypovirus 1 (VcHV1). Phylogenetic analysis of the RdRp and RNA helicase domains of VcHV1 revealed that VcHV1, CHV3, CHV4, and SsHV1 clustered together into one clade distinct from that of CHV1 and CHV2, indicating the existence of two lineages in the family Hypoviridae. Comparison of biological properties between VcHV1-infected and VcHV1-free isogenic strains did not reveal differences in colony morphology or fungal virulence under laboratory conditions.

In general, mycoviruses are transmitted through hyphal anastomosis between vegetatively compatible strains of the same fungi, and their entire intracellular life cycle within host fungi limits transmission to separate species and even to incompatible strains belonging to the same species. Based on field observations of the white root rot fungus, Rosellinia necatrix, we found two interesting phenomena concerning mycovirus epidemiology. Specifically, apple trees in an orchard were inoculated with one or two R. necatrix strains that belonged to different mycelial compatibility groups (MCGs), strains W563 (virus-free, MCG139) and NW10 (carrying a mycovirus-like double-stranded (ds) RNA element (N10), MCG442). Forty-two sub-isolates of R. necatrix, which were retrieved 23 years later, were all genetically identical to W563 or NW10: however, 22 of the sub-isolates contained novel dsRNAs. Six novel dsRNAs (S1-S6) were isolated: S1 was a new victorivirus; S2, S3, and S4 were new partitiviruses; and S5 and S6 were novel viruses that could not be assigned to any known mycovirus family. N10 dsRNA was detected in three W563 sub-isolates. These findings indicated that novel mycoviruses, from an unknown source, were infecting strains W563 and NW10 of R. necatrix in the soil, and that N10 dsRNA was being transmitted between incompatible strains, NW10 to W563.