Microbial Diversity in the Phyllosphere and Rhizosphere of an Apple Orchard Managed under Prolonged "Natural Farming" Practices.

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 Diseases: Fulfillment of Koch's Postulates by Virome Analysis, Amplification of Full-Length cDNA of Viral Genomes, in vitro Transcription of Infectious Viral RNAs, and Reproduction of Symptoms on Fruits of Apple Trees Inoculated With Viral RNAs.

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.

Differential Inductions of RNA Silencing among Encapsidated Double-Stranded RNA Mycoviruses in the White Root Rot Fungus Rosellinia necatrix.

UNLABELLED: 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.

Sensory and metabolic profiles of "Fuji" apples (Malus domestica Borkh.) grown without synthetic agrochemicals: the role of ethylene production.

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.

Systemic RNA interference is not triggered by locally-induced RNA interference in a plant pathogenic fungus, Rosellinia necatrix.

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.

Functional analysis of a melanin biosynthetic gene using RNAi-mediated gene silencing in Rosellinia 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.

Genome rearrangement of a mycovirus Rosellinia necatrix megabirnavirus 1 affecting its ability to attenuate virulence of the host fungus.

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.

A mycoreovirus suppresses RNA silencing in the white root rot fungus, Rosellinia necatrix.

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.

Appearance of mycovirus-like double-stranded RNAs in the white root rot fungus, Rosellinia necatrix, in an apple orchard.

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 2-3 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.

Transient and multivariate system for transformation of a fungal plant pathogen, Rosellinia necatrix, using autonomously replicating vectors.

Rosellinia necatrix is a fungus that infects a wide range of host plants and ruins a variety of commercially important crops. DNA fragments can be introduced into R. necatrix using conventional protoplast-PEG transformation and genome-integrating vectors; however, transformation efficiency with this strategy is quite low. Therefore, to establish a more effective transformation system for the studies of R. necatrix, an autonomously replicating vector was constructed using AMA1 sequences derived from Aspergillus nidulans, which is distantly related to R. necatrix. Use of this vector with AMA1 sequences increased transformation efficiency in R. necatrix, and the vector was maintained as a plasmid in the transformants. Transient and multivariate functional analyses in R. necatrix were performed using co-transformation of multiple pAMA-H vectors, which each carried either an expression cassette for eGFP, mOrange2, or a geneticin resistance gene. Furthermore, fluorescent proteins expressed from the autonomously replicating vectors were dispersed throughout fungal colonies even though the vectors themselves were restricted to the center of each colony. This intriguing phenomenon indicated that gene products could move from the center to the margin in a colony of the filamentous fungi via a cell-to-cell transport system.

Molecular characterization of a new hypovirus infecting a phytopathogenic fungus, Valsa ceratosperma.

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.

A novel colony-print immunoassay reveals differential patterns of distribution and horizontal transmission of four unrelated mycoviruses in Rosellinia necatrix.

A colony-print immunoassay (CPIA) using an anti-dsRNA antibody was developed to visualize the distribution of four unrelated mycoviruses with dsRNA genomes, a partitivirus (RnPV1), mycoreovirus (RnMyRV3), megabirnavirus (RnMBV1), and an unidentified virus (RnQV1), in mycelia of the white root rot fungus, Rosellinia necatrix. CPIA revealed different distribution patterns within single colonies for each virus. Both RnPV1 and RnMBV1 were distributed throughout single colonies, RnMyRV3 was absent from some colony sectors, and RnQV1 exhibited varied accumulation levels between sectors. RnMyRV3 and RnQV1 were transmitted to the recipient virus-free colonies of virus-infected and virus-free colony pairs more slowly than were RnPV1 or RnMBV1. The presence of RnMyRV3 in recipient colonies restricted horizontal transmission of RnPV1 and RnMBV1. These results imply that one or more mechanisms are present in host-virus and virus-virus interactions that restrict the spread of viruses within and between colonies.

Extending the fungal host range of a partitivirus and a mycoreovirus from Rosellinia necatrix by inoculation of protoplasts with virus particles.

The potential host range of mycoviruses is poorly understood because of the lack of suitable inoculation methods. Recently, successful transfection has been reported for somatically incompatible fungal isolates with purified virus particles of two mycoviruses, the partitivirus RnPV1-W8 (RnPV1) and the mycoreovirus RnMyRV3/W370 (MyRV3), from the white root rot fungus Rosellinia necatrix (class Sordariomycetes, subclass Xylariomycetidae). These studies examined and revealed the effect of the mycoviruses on growth and pathogenicity of R. necatrix. Here, we extended the experimental host range of these two mycoviruses using a transfection approach. Protoplasts of other phytopathogenic Sordariomycetous fungi-Diaporthe sp., Cryphonectria parasitica, Valsa ceratosperma (Sordariomycetidae), and Glomerella cingulata (Hypocreomycetidae)-were inoculated with RnPV1 and MyRV3 viral particles. The presence of double-stranded RNA viral genomes in regenerated mycelia of Diaporthe sp., C. parasitica, and V. ceratosperma confirmed both types of viral infections in these three novel host species. An established RnPV1 infection was confirmed in G. cingulata but MyRV3 did not infect this host. Horizontal transmission of both viruses from newly infected strains to virus-free, wild-type strains through hyphal anastomosis was readily achieved by dual culture; however, vertical transmission through conidia was rarely observed. The virulence of Diaporthe sp., C. parasitica, and V. ceratosperma strains harboring MyRV3 was reduced compared with their virus-free counterpart. In summary, our protoplast inoculation method extended the experimental host range of RnPV1-W8 and MyRV3 within the class Sordariomycetes and revealed that MyRV3 confers hypovirulence to the new hosts, as it does to R. necatrix.

Mating-type loci of heterothallic Diaporthe spp.: homologous genes are present in opposite mating-types.

Sexual reproduction of fungi is governed by genes located on the mating-type (MAT) locus. To analyze the MAT locus of the genus Diaporthe (anamorph: Phomopsis), a large genera within the ascomycetous class Sordariomycetes, we cloned and sequenced loci MAT1-1 and MAT1-2 from two heterothallic Diaporthe species, designated as Diaporthe W- and G-types (four isolates in total). The mating-type loci structures of Diaporthe W- and G-types were similar; MAT1-1 isolates had a MAT locus containing three genes, MAT1-1-1, MAT1-1-2 and MAT1-1-3, as was the case with other Sordariomycetes, and in contrast to other Sordariomycetes, MAT1-2 isolates had genes homologous to MAT1-1-2 and MAT1-1-3, in addition to MAT1-2-1. Expression analysis by RT-PCR revealed that all the mating-type genes of Diaporthe W-type were transcriptionally active during vegetative growth. The structure of MAT loci of Diaporthe W- and G-types is distinct from that in other heterothallic filamentous ascomycetes, which have dissimilar gene structure in opposite mating-type loci. This unique structure is informative to discussing the evolutionary history and function of mating-type genes of Sordariomycete fungi.

Multiple citrus viroids in citrus from Japan and their ability to produce exocortis-like symptoms in citron.

ABSTRACT Sequential polyacrylamide gel electrophoresis analyses showed many viroid-like RNAs in samples collected from citrus trees in Japan. Reverse transcription polymerase chain reaction and sequencing analyses of the amplified fragments verified that they were derived from variants of six citrus viroids, Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd) including CVd-I-LSS (a distinct variant of CBLVd), Hop stunt viroid, Citrus viroid III, Citrus viroid IV, and Citrus viroid OS. The samples induced symptoms with variable severity in Arizona 861-S1 'Etrog' citrons (Citrus medica L.) likely due to the varying accumulation patterns produced by the different viroids. Some of the symptoms caused by the samples harboring the citrus viroids other than CEVd were as severe as those caused by CEVd. Some source citrus trees showing the severe bark scaling characteristic of exocortis disease in trifoliate orange (Poncirus trifoliata (L.) Raf.) rootstocks contained only citrus viroids other than CEVd in complex. This indicates that certain exocortis-like diseases in Japan were caused by some combination of citrus viroids not including CEVd.