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Aims Integrating multiple soil and disease manage- ment practices may improve crop productivity and disease control, but potential interactions and limita- tions need to be determined. Methods Three different potential disease-suppressive management practices, including a Brassica napus (rapeseed) green manure rotation crop, conifer-based compost amendment, and three biological control or- ganisms (Trichoderma virens, Bacillus subtilis, and Rhizoctonia solani hypovirulent isolate Rhs1A1) were evaluated alone and in combination at sites with both organic and conventional management histories for their effects on soilborne diseases and tuber yield. Results Rapeseed rotation reduced all observed soil- borne diseases (stem canker, black scurf, common scab, and silver scurf) by 10 to 52 % in at least one year at both sites. Compost amendment had variable effects on tuber diseases, but consistently increased yield (by 9 to 15 %) at both sites. Biocontrol effects on disease varied, though Rhs1A1 decreased black scurf at the conventional site and T. virens reduced multiple diseases at the organic site in at least one year. Combining rapeseed rotation with compost amend- ment both reduced disease and increased yield, where- as biocontrol additions produced only marginal addi- tive effects. Conclusions Use of these treatments alone, and in combination, can be effective at reducing disease and increasing yield under both conventional and organic production practices.

Soilborne potato diseases are persistent problems in potato production and alternative management practices are needed. In this research, biocontrol agents (Bacillus subtilis GB03 and Rhizoctonia solani hypovirulent isolate Rhs1A1) and compost amendments (from different source material), were evaluated alone and in combination, for their potential to reduce soilborne diseases and increase tuber yield over three field seasons in Maine. Both biocontrol organisms reduced multiple soilborne diseases, stem and stolon canker by 20–38 %, black scurf by 30–58 %, and common scab by 10–34 % relative to the nontreated control treatment, and the combination treatment of both biocontrol organisms together provided nominally better control than individual treatments. However, biocontrol treatments had no direct effect on tuber yield. Compost amendments from different sources all increased total and marketable tuber yield substantially (11–37 % and 17–51 %, respectively) relative to nontreated controls. However, except for some reduction of Rhizoctonia canker, compost amendments did not reduce soilborne diseases (black scurf and commons scab) in any year, and resulted in increased levels of common scab in some years (20–45 % increase). The combination compost-biocontrol treatment, although did not perform significantly better than individual component treatments, still provided indications of combined beneficial effects from both component treatments. This research demonstrated the usefulness of these approaches and combinations as additional options for reduction of soilborne diseases and increased tuber yield and can be implemented for enhanced sustainability and productivity in potato production systems.

Cultural practices such as organic amendments, rotations, and use of biological control organisms are regularly investigated for their effects on controlling plant diseases but their effects on soil microbial populations are often unexplored. In this study, three different sustainable disease management practices, use of compost amendment, biocontrol organisms, and a potentially disease-suppressive rotation, were established in potato field trials at two sites under different management regimes and histories, and evaluated over three potato cropping seasons for their effects on soil microbial communities. Specific management factors assessed included the presence or absence of a conifer-based compost amendment, addition of one of three different biocontrol organisms (Trichoderma virens, Bacillus subtilis, and Rhizoctonia solani isolate Rhs1A1), and a Brassica napus (rapeseed) green manure rotation crop preceding potato, and treatments were assessed in all factorial combinations. The two farm sites represented organic and conventional potato production practices in Aroostook County, Maine. Compost amendment and rapeseed rotation had the greatest impacts on soil microbial communities, with both treatments increasing total populations of culturable bacteria at both sites over the course of the study, as well as causing detectable shifts in soil microbial community characteristics as determined by sole carbon-source substrate utilization and fatty acid methyl ester (FAME) profiles. Compost amendment generally led to increased utilization of complex substrates and increased levels of Gram-positive bacteria and fungi, and compost effects were more pronounced at the conventional site. Rapeseed rotation often resulted in somewhat different effects at the two different sites. Consistent overall effects were observed with the biocontrol amendments Rhs1A1 and T. virens, including increased microbial activity and bacterial populations. Combined effects of multiple treatments were greater than those of individual treatments and were generally additive. These results indicate that each treatment factor had significant and specific effects on soil microbial communities, and that combined effects tended to be complementary, suggesting the potential of combining multiple compatible management practices and their associated changes in soil microbial communities.

Rhizoctonia solani, teleomorph Thanatephorus cucumeris, is a polyphagous necrotrophic plant pathogen of the Basidiomycete order that is split into 14 different anastomosis groups (AGs) based on hyphal interactions and host range. In this investigation, quantitative real-time PCR (qRT-PCR) techniques were used to determine potential pathogenicity factors of R. solani in the AG1-IA/rice and AG3/potato pathosystems. These factors were identified by mining for sequences of pathogen origin in a library of rice tissue infected with R. solani AG1-IA and comparing these sequences against the recently released R. solani AG3 genome. Ten genes common to both AGs and two specific to AG1-IA were selected for expression analysis by qRT-PCR. Results indicate that a number of genes are similarly expressed by AG1 and AG3 during the early stages of pathogenesis. Grouping of these pathogenicity factors based on relatedness of expression profiles suggests three key events are involved in R. solani pathogenesis: early host contact and infiltration, adjustment to the host environment, and pathogen proliferation through necrotic tissue. Further studies of the pathogenesis-associated genes identified in this project will enable more precise elucidation of the molecular mechanisms that allow for the widespread success of R. solani as a phytopathogen and allow for more targeted, effective methods of management.

We reported previously that a 3.6-kb double-stranded RNA, designated as M2, is associated with hypovirulence in the Rhizoctonia solani isolate Rhs 1A1 and proposed that the M2-encoded putative polypeptide A (pA) might interfere with the regulation of the quinate and shikimate pathways. In this study, Western blot analysis showed that a protein band of the predicted size (83 kDa) binds antibodies specific to a pA epitope and is detectable in M2-containing but not in M2-lacking cultures. A mRNA, associated with Rhs 1A1 polysomes immunoprecipitated with anti-pA antibodies, has a sequence basically identical to that of the sense-strand of M2. The normally inducible quinate pathway was constitutively expressed, whereas the shikimate pathway was down-regulated in the M2-containing, hypovirulent Rhs 1A1. Finally, the relative concentration of phenylalanine, precursor of the virulence determinant phenylacetic acid, was correlated with the degree of pathogenicity in the virulent Rhs 1AP but not in the hypovirulent Rhs 1A1.

The soil fungus Rhizoctonia solani is a pathogen of agricultural crops. Here, we report on the 51,705,945 bp draft consensus genome sequence of R. solani strain Rhs1AP. A comprehensive understanding of the heterokaryotic genome complexity and organization of R. solani may provide insight into the plant disease ecology and adaptive behavior of the fungus.

We have recently shown that acquisition of a 6.4-kb, double-stranded (ds) RNA (M1) by hyphal anastomosis is associated with enhanced vigor and virulence, whereas its removal by hyphal tipping correlates with diminished virulence in the plant-pathogenic basidiomycete Rhizoctonia solani. The M1 dsRNA is not encapsidated by a typical nucleocapsid, has a circular and/or concatemeric form, and is associated with the mitochondrial and cytosolic fractions. M1 possesses six open reading frames (ORFs) the longest of which (ORF 2) is located on the (+) strand, and encodes a putative polypeptide consisting of 1,747 amino acids or 199.4 kDa. This polypeptide has a significant amino acid sequence similarity, including six conserved helicase domains and an ATP/GTP binding motif, with the 1A protein of broad bean mottle virus (BBMV) and other bromoviruses. ORF 5, which is located on the (-) strand of M1 and is complementary to a region of ORF 2, codes for a putative polypeptide that has a significant amino acid sequence similarity with the cytochrome c oxidase assembly factor. This complementarity provides direct evidence suggesting that the long-standing hypothesis of viruses evolving from cellular genes may be valid.

A virulent field isolate of Rhizoctonia solani AG 3, Rhs 1AP, has given rise to three sectors in a period of several years. Subculturing of these sectors resulted in three hypovirulent isolates—Rhs 1A1, Rhs 1A2, and Rhs 1A3. We reported previously that five genetically different double-stranded (ds) RNAs occur in these four isolates, with each isolate containing a unique combination of two or more dsRNAs. We report here that all five dsRNA elements occur in the cytoplasm, and none in the nucleus. The mitochondria contains low to moderate concentrations of the four larger dsRNAs. The four isolates were paired in selected combinations in an attempt to transmit specific dsRNAs to cultures lacking these dsRNAs. This approach generated groups of near-isogenic lines possessing the same dsRNA elements. As many as six cultures possessing the same dsRNA genotype were found to have the same degree of pathogenicity. Consistently, acquisition of a 6.4-kb dsRNA brought about increased virulence, whereas the presence or acquisition of a 3.6-kb dsRNA resulted in diminished virulence in the recipient culture. All cultures had the same mitochondrial DNA haplotype as that of the original field isolate, Rhs 1AP. This, in conjunction with the fact that all cultures in this study had a single nuclear DNA origin (Rhs 1AP), indicates a strong correlation between certain phenotypic characters or changes thereof and dsRNA profiles or acquisition of particular dsRNAs, respectively.

Experimental data showed that multimeric, complementary RNA (cRNA) probes, labelled with non-radioactive digoxigenin (DIG), improved sensitivity of detection of the potato spindle tuber viroid (PSTVd) RNA by 2- to 30-fold as compared with corresponding multimeric cDNA probes. The degree of PSTVd detectability improvement depended upon the type of alkaline phosphatase substrate (colorimetric vs. chemiluminescent) used. Use of hexameric DIG-labelled cRNA probes in combination with chemiluminescent (Lumi-Phos 530) substrate resulted in detection of 0.48 pg of PSTVd RNA. The size of the synthesized cRNA probes corresponded to the size of the respective PSTVd cDNA templates. Interestingly, there was no relationship between the size of the synthesized, DIG-labelled DNA probes and that of the PSTVd cDNA template. This type of anomaly was not observed with other plant viral cDNA templates. Monomeric or multimeric cDNA probes detected both a mild and a severe PSTVd strain in viroid-infected potato leaf extracts diluted 1024 to 2048 times. In comparison, cRNA probes exhibited a much greater dilution end point; PSTVd RNA was detectable in viroid-infected potato leaf tissue diluted up to 16,384 times. Comparable levels of PSTVd sensitivity of detection were obtained with viroid-infected potato tuber tissue.

Many species of fungi have been shown to harbor double-stranded RNA (dsRNA) elements. A single fungal isolate of Rhizoctonia solani may have as many as five different dsRNA elements within them. The presence of specific dsRNA elements influence pathogenicity in host plants.

Many species of fungi have been shown to harbor double-stranded RNA (dsRNA) elements. A single fungal isolate of Rhizoctonia solani may have as many as five different dsRNA elements within them. The presence of specific dsRNA elements influence pathogenicity in host plants.

Isolates from closely related fungi in the Rhizoctonia species complex were examined for the occurrence of the M2 double-stranded RNA (dsRNA) by amplifying a conserved 1000 nucleotide region of the dsRNA with reverse transcription PCR. The M2 dsRNA was detected in representative isolates belonging to three anastomosis groups (AG) of R. solani (AG-1-IA, AG-4 and AG-6; teleomorph = Thanatephorus) and four AGs of binucleate Rhizoctonia (AG-A, AG-F, AG-R and AG-U; teleomorph = Ceratobasidium). Amplified PCR products from the 3' region of the M2 dsRNA from a representative sample of 12 isolates from eight different AGs were sequenced and subjected to parsimony analysis and coalescent simulations to infer ancestral lineages and to reconstruct the ancestral history of haplotypes. Seven dsRNA haplotypes were inferred from the sample of 12 isolates. One haplotype was composed of only isolates of Ceratobasidium belonging to different AGs. The rooted gene genealogies from coalescent simulations suggested that the ancestral M2 dsRNA haplotype most likely evolved in Thanatephorus (anamorph = R. solani AG-1-IA) and has been acquired recently by isolates of Ceratobasidium. Reconstruction of the ancestral history of haplotypes with a parsimony-based approach that assumes both mutation and recombination suggested that four haplotypes recombined before coalescing to their most recent common ancestor, while three haplotypes coalesced without recombination in the recent past. There was no unique association of haplotype within a specific AG of either Ceratobasidium or Thanatephorus to support co-evolution of the M2 dsRNA within the fungal host. To our knowledge this is the first report of a dsRNA occurring in Ceratobasidium that also is present in Thanatephorus.

Field experiments were conducted to determine whether a hypovirulent, anastomosis group (AG) 3 isolate (Rhs 1A1) of R. solani could control rhizoctonia disease of potato caused by a virulent AG-3 isolate (Rhs 27). A uniform amount of Rhs 1A1, Rhs 27, or both was applied to formaldehyde-disinfected potato seed tubers at planting. In plots inoculated with Rhs 1A1 and Rhs 27, the area of infected stem tissue was significantly (P = 0.05) reduced by 56% as compared with that of plots inoculated with Rhs 27 alone. The number of stolons per plant was decreased by isolate Rhs 27 to one-half that of the uninoculated control, but remained unchanged in plots treated with both Rhs 1A1 and Rhs 27. The type of inoculum did not affect the percentage of infected stems or stolons. Plants inoculated with Rhs 1A1 alone exhibited a significantly (P = 0.05) greater growth response, expressed as a 4-fold increase in the dry weight of stolons, a 1.7-fold increase in the dry weight of stems, and reached full bloom approximately one week earlier than their respective control. Tuber yield increases associated with Rhs 1Al-treated plots were not statistically significant. When untreated seed tubers bearing natural inoculum (sclerotia) of R. solani were used, Rhs 1A1 failed to reduce disease severity or sig-nificantly stimulate plant growth.

The quinate pathway is induced by quinate in the wild-type virulent Rhizoctonia solani isolate Rhs 1AP but is constitutive in the hypovirulent, M2 dsRNA-containing isolate Rhs 1A1. Constitutive expression of the quinate pathway results in downregulation of the shikimate pathway, which includes the pentafunctional arom gene in Rhs 1A1. The arom gene has 5,323 bp including five introns as opposed to a single intron found in arom in ascomycetes. A 199-bp upstream sequence has a GC box, no TATAA box, but two GTATTAGA repeats. The largest arom transcript is 5,108 nucleotides long, excluding the poly(A) tail. It contains an open reading frame of 4,857 bases, coding for a putative 1,618-residue pentafunctional AROM protein. A Kozak sequence (GCGCCATGG) is present between +127 and +135. The 5′-end of the arom mRNA includes two nucleotides (UA) that are not found in the genomic sequence, and are probably added post-transcriptionally. Size and sequence heterogeneity were observed at both 5′- and 3′-end of the mRNA. Northern blot and suppression subtractive hybridization analyses showed that presence of a low amount of quinate, inducer of the quinate pathway, resulted in increased levels of arom mRNA, consistent with the compensation effect observed in ascomycetes.

A double-stranded (ds)RNA, designated as M2, is associated with hypovirulence, conversion of the quinic acid pathway from inducible to constitutive and downregulation of the shikimic acid pathway in the Rhizoctonia solani culture Rhs 1A1. In this study, we report that in the virulent, M2-lacking isolate Rhs 1AP, which is isogenic to Rhs 1A1, quinic acid reduces virulence dramatically and induces synthesis of an M2-encoded polypeptide and its respective mRNA. The full-length sense strand of M2 is detected in untreated Rhs 1AP only after a second 30-cycle amplification, using nested primers. Quinate-induced Rhs 1AP contains low concentrations of both full-length sense and complementary strand of M2. The quinic acid-induced hypovirulence in Rhs 1AP cannot be overturned by the end-product of the shikimic acid pathway, chorismic acid, which enhances the virulence of Rhs 1AP dramatically when used alone. In addition to its apparent applications, this study confirms the strong association between the M2 dsRNA and hypovirulence in R. solani.

The bisegmented genome of a double-stranded (ds) RNA virus from the fungusRhizoctonia solaniisolate Rhs 717 was characterized. The larger segment, dsRNA 1, is 2363 bases long whereas the smaller segment, dsRNA 2, has 2206 bases. The 5′ ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved (100% identity over 47 bases), and contain inverted repeats capable of forming stable stem–loop structures. Analysis of the coding potential of each of the two segments showed that dsRNAs 1 and 2 could code for polypeptides of 730 aa (bases 86–2275; molecular mass 86 kDa) and 683 aa (bases 79–2130; molecular mass 76 kDa), respectively. The 86 kDa polypeptide has all the motifs of dsRNA RNA-dependent RNA polymerases (RDRP), and has significant homology with putative RDRPs of partitiviruses fromFusarium poaeandAtkinsonella hypoxylon. The 76 kDa protein shows homology with the putative capsid proteins (CP) of the same viruses. Northern blot analysis revealed no subgenomic RNA species, consistent with the fact that the long open reading frames encoding the putative RDRP and CP cover the entire length of the respective dsRNAs.

M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani . Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica , incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

The advent of molecular biology has played a pivotal role in advancing our understanding of the genetics and biology of filamentous fungi such as Neurospora crassa and Aspergillus nidulans. There exists a great need for similar advances in the molecular genetics of other fungi, especially those causing economically important plant diseases. Rhizoctonia solani Kuhn [teleomorph, Thanatephorus cucumeris (Frank) Donk], is an important soilborne pathogen attacking numerous crops, worldwide. Understanding the genetic structure of field soil populations, the functions of the genes involved in virulence expression and genetic recombination is necessary for the design of Rhizoctonia disease management that will be both effective and environmentally sound.

Full-length complementary DNAs (cDNA) of a mild (KF 5) and a severe (S-PSTVd) isolate of potato spindle tuber viroid (PSTVd) were constructed. DNA sequencing of four KF 5 cDNA clones (M 3, M 4, M 5, and M 7) revealed that KF 5 is comprised of 360 nucleotides. By comparison, all three cDNA clones (S 2, S 9, and S 10) of S-PSTVd possess 359 nucleotides. Sequence microheterogeneity was observed among the KF 5 cDNA clones. Clone M 5 differs from mild PSTVd isolate KF 6 by a U-to-A transversion at position 303 followed by an A addition at the lower half of the “virulence-modulating” (VM) region. These changes modified the PSTVd consensus sequence of the VM region from 5′ UCUAUCU 3′ to 5′ UCAAAUCU 3′. Additionally, clones M 4 and M 7 have a G-to-A transition at position 65 of the pathogenic domain, and M 3 has a G-to-A transition at position 133 of the variable domain. The sequence of the three cDNA clones of S-PSTVd was identical to that of PSTVd isolate 440-1. An improved computer program was used to predict the secondary structure of the above two sequence variants as well as that of other PSTVd variants of which the structure has been reported previously. The data provides support for the hypothesis that increasing thermodynamic instability of the VM region is correlated with increasing virulence of the respective naturally occurring PSTVd isolate.

A viroid was isolated from symptomless Nematanthus wettsteinii plants using the return-PAGE method for analysis of low M(r) nucleic acids. The RNA was transmitted to tomato, three cultivars of potato, and Scopolia sinensis plants by mechanical inoculation or by grafting. Infected solanaceous plants developed symptoms similar to those caused by potato spindle tuber viroid (PSTVd). The Nematanthus viroid consists of 372 nucleotides, 214 G+C, 158 A+U, with a G+C/A+U ratio of 1.35. One of seven cDNA clones showed a sequence heterogeneity (G to A) at position 73. The most stable secondary structure of this viroid has 78 G:C, 37 A:U and 11 G:U base pairs with a minimum free energy of -456.9 kJ. The viroid is closely related to the 370 nucleotide Columnea latent viroid. The Nematanthus viroid possesses regions of 100% sequence identity with six viroids belonging to the PSTVd and apple scar skin viroid groups. The viroid also replicated in tomato plants when mixed with PSTVd. Tomato plants were cross-protected against PSTVd when preinfected with the viroid from N. wettsteinii.

We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and β-NAD + . The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5α. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.

Deletion mutations were generated in four structural domains of a potato spindle tuber viroid (PSTV) complementary DNA (cDNA) clone. Deletions of 3 to 5 nucleotides at the central conserved domain (CCGGG, positions 94 to 98), variable domain (GCCG, positions 146 to 149) and pathogenicity domain (CGA, positions 286 to 288) abolished infectivity of dimeric or trimeric cDNA constructs, or their in vitro transcripts. By contrast, a clone (St4) with a deletion of two nucleotides (UU, positions 339 and 340), located at the left terminal domain, retained infectivity when DNA or in vitro transcribed (+)RNA was used as inoculum. Sequencing of four cDNA clones of such viroid progeny demonstrated that two nucleotides were added at the deletion site. Two of the viroid progeny contained a CG addition. A third clone possessed a GU addition, whereas the fourth clone had a UU addition which represents a true reversion to full-length wild-type PSTV RNA. Ribonuclease protection assay of viroid progeny from St4-infected tomato plants suggested that only a negligible proportion of the St4 progeny were true revertants.© 1992 Academic Press, Inc.

Genomic-size RNA isolated from purified potato carlavirus M (PVM) was translated in both the reticulocyte and the wheat germ cell-free, messenger-dependent systems. The PVM RNA translated the same set of major products in both in vitro systems. The Mr values of the most prominent polypeptides observed consistently were 185 000 (P185), 147 000 (P147), 94 000 (P94), 87 000 (P87), 72 000 (P72), 67 000 (P67), 52 000 (P52), 46 000 (P46), 35 000 (P35) and 25 000 (P25). Relatively low amounts of a translation product of Mr 200 000 (P200) were often detectable in both systems. The P35 polypeptide displayed the same molecular weight and one-dimensional peptide map as the virus coat protein (CP), and was precipitated by antibodies raised against PVM and PVM CP. The kinetics of appearance of the in vitro synthesized polypeptides suggested that primary translation products of high molecular weight undergo post-translational proteolytic cleavage.

Tomato mosaic tobamovirus (ToMV) was detected by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction-blot hybridization (RT-PCR-BH) in cloud samples collected from the summit of Whiteface Mountain, NY, and in fog samples from two collection sites along the coast of Maine. The virus was subsequently transmitted to Chenopodium quinoa from a composite of RT-PCR-BH-positive concentrates. There was no apparent relationship between the presence of ToMV and sample collection date, volume, or pH. The RT-PCR products of three cloud and fog samples and one deionized water sample were identical to one another and to a stream water isolate of ToMV from Whiteface Mountain based on nucleotide sequencing of a 347-bp fragment within the coat protein gene and 99.1 and 72.9% similarity to ToMV-L and tobacco mosaic virus-vulgare, respectivelv.

A tuberborne sclerotium of the plant-pathogenic basidiomycete Rhizoctonia solani gave rise to a culture (Rhs 1AP) that exhibited a sector (Rhs 1A1) with reduced pigmentation and growth rate. Rhs 1AP is virulent on potato, which is the only known host plant for Rhs 1AP. In contrast, Rhs 1A1 is hypovirulent or nonpathogenic on potato. The virulent isolate Rhs 1AP contains two double-stranded RNAs (dsRNAs) of 23 and 6.54 kb, whereas the hypovirulent Rhs 1A1 possesses three dsRNAs in addition to the two found in Rhs 1AP [...]

Summary Isometric virus particles, 33 nm in diameter, were purified to apparent homogeneity from a dsRNA-containing isolate (Rhs 717) of anastomosis group 2 of the basidiomycete Rhizoctonia solani. The dsRNA segments isolated from purified particles had the same M r (1.5 × 106 and 1.4 × 106) as those isolated directly from mycelial tissue of Rhs 717. Purified virus particles had a buoyant density of 1.37 g/ml in CsCl, and an A 260/A 280 ratio of 1.43. The melting curve of the mixture of dsRNA segments from Rhs 717 particles had a sharp thermal transition with a melting temperature of 95 °C in standard saline citrate buffer. The RNA polymerase activity associated with the purified virus was characterized. Enzyme activity was dependent on the presence of Mg2+ and was insensitive to actinomycin D. Virus particles and RNA polymerase activity cosedimented as a single peak in isopycnic CsCl gradients. The major reaction products were two dsRNA segments of M r 1.5 × 106 and 1.4 × 106, which corresponded to those of the two viral dsRNAs. The [32P]UMP-labelled products hybridized to the two viral dsRNAs. The RNA synthesized in vitro remained in association with virus particles subjected to agarose gel electrophoresis.

The enzyme-linked immunosorbent assay (ELISA) was adapted for the detection of potato virus M (PVM). Detectability of PVM was influenced by the stage of plant growth and that of the plant part assayed. In young potato plants, one week prior to flowering, PVM titer was relatively high (0.77 A405nm units) in the basal leaves and barely detectable (0.09 A405 nm units) in the top leaves. In mature plants, two weeks prior to harvesting, top leaves contained more virus (0.34 A405 nm units) than middle (0.24 A405nm units) or basal leaves (0.15 A405 nm units). In rapidly growing tubers PVM content was higher (1.57 A405 nm units) than that in fully grown tubers (0.71 A405nm units) whereas the virus was not detectable in the mother tuber four weeks following planting. PVM was reliably detected when disks from intact leaves were substituted for leaf extracts as the test sample

Potato virus M (PVM), a member of the carlavirus group was found to have a sedimentation coefficient ( S 20,w ) of 157 S, a buoyant density in CsCl of 1.322 g·cm −3 , an extinction coefficient, at 260 nm, of 2.88 cm 2 ·mg −1 , and a nucleic acid content of 6.0%. The PVM genome is a polyadenylate-containing single-stranded RNA with a molecular weight (MW) of 2.5 × 10 6 . Virus-specific, double-stranded RNA is consistently associated with PVM infection. PVM possesses a single capsid polypeptide with a MW of 35.7 × 10 3 .