Population Structure of Colletotrichum tanaceti in Australian Pyrethrum Reveals High Evolutionary Potential.

Colletotrichum tanaceti, the causal agent of anthracnose, is an emerging pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium) in Australia. A microsatellite marker library was developed to understand the spatio-genetic structure over three sampled years and across two regions where pyrethrum is cultivated in Australia. Results indicated that C. tanaceti was highly diverse with a mixed reproductive mode; comprising both sexual and clonal reproduction. Sexual reproduction of C. tanaceti was more prevalent in Tasmania than in Victoria. Little differentiation was observed among field populations likely due to isolation by colonization but most of the genetic variation was occurring within populations. C. tanaceti was likely to have had a long-distance gene and genotype flow among distant populations within a state and between states. Anthropogenic transmission of propagules and wind dispersal of ascospores are the most probable mechanisms of long-distance dispersal of C. tanaceti. Evaluation of putative population histories suggested that C. tanaceti most likely originated in Tasmania and expanded from an unidentified host onto pyrethrum. Victoria was later invaded by the Tasmanian population. With the mixed mode of reproduction and possible long-distance gene flow, C. tanaceti is likely to have a high evolutionary potential and thereby has ability to adapt to management practices in the future.

Comparative genome analysis indicates high evolutionary potential of pathogenicity genes in Colletotrichum tanaceti.

Colletotrichum tanaceti is an emerging foliar fungal pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium). Despite being reported consistently from field surveys in Australia, the molecular basis of pathogenicity of C. tanaceti on pyrethrum is unknown. Herein, the genome of C. tanaceti (isolate BRIP57314) was assembled de novo and annotated using transcriptomic evidence. The inferred putative pathogenicity gene suite of C. tanaceti comprised a large array of genes encoding secreted effectors, proteases, CAZymes and secondary metabolites. Comparative analysis of its putative pathogenicity gene profiles with those of closely related species suggested that C. tanaceti likely has additional hosts to pyrethrum. The genome of C. tanaceti had a high repeat content and repetitive elements were located significantly closer to genes inferred to influence pathogenicity than other genes. These repeats are likely to have accelerated mutational and transposition rates in the genome, resulting in a rapid evolution of certain CAZyme families in this species. The C. tanaceti genome showed strong signals of Repeat Induced Point (RIP) mutation which likely caused its bipartite nature consisting of distinct gene-sparse, repeat and A-T rich regions. Pathogenicity genes within these RIP affected regions were likely to have a higher evolutionary rate than the rest of the genome. This "two-speed" genome phenomenon in certain Colletotrichum spp. was hypothesized to have caused the clustering of species based on the pathogenicity genes, to deviate from taxonomic relationships. The large repertoire of pathogenicity factors that potentially evolve rapidly due to the plasticity of the genome, indicated that C. tanaceti has a high evolutionary potential. Therefore, C. tanaceti poses a high-risk to the pyrethrum industry. Knowledge of the evolution and diversity of the putative pathogenicity genes will facilitate future research in disease management of C. tanaceti and other Colletotrichum spp.

Evidence for Sexual Recombination in Didymella tanaceti Populations, and Their Evolution Over Spring Production in Australian Pyrethrum Fields.

Tan spot, caused by Didymella tanaceti, is one of the most important foliar diseases affecting pyrethrum in Tasmania, Australia. Population dynamics, including mating-type ratios and genetic diversity of D. tanaceti, was characterized within four geographically separated fields in both late winter and spring 2012. A set of 10 microsatellite markers was developed and used to genotype 774 D. tanaceti isolates. Isolates were genotypically diverse, with 123 multilocus genotypes (MLG) identified across the four fields. Fifty-eight MLG contained single isolates and Psex analysis estimated that, within many of the recurrent MLG, there were multiple clonal lineages derived from recombination. Isolates of both mating types were at a 1:1 ratio following clone correction in each field at each sampling period, which was suggestive of sexual recombination. No evidence of genetic divergence of isolates of each mating type was identified, indicating admixture within the population. Linkage equilibrium in two of the four field populations sampled in late winter could not be discounted following clone correction. Evaluation of temporal changes in gene and genotypic diversity identified that they were both similar for the two sampling periods despite an increased D. tanaceti isolation frequency in spring. Genetic differentiation was similar in populations sampled between the two sampling periods within fields or between fields. These results indicated that sexual reproduction may have contributed to tan spot epidemics within Australian pyrethrum fields and has contributed to a genetically diverse D. tanaceti population.

Mycoflora Associated With Pyrethrum Seed and the Integration of Seed Steam Treatment Into Foliar Disease Management Strategies.

A complex of foliar diseases can affect pyrethrum in Australia, but those of greatest importance are ray blight, caused by Stagonosporopsis tanaceti, and tan spot, caused primarily by Didymella tanaceti. Isolation of fungi from pyrethrum seed lots produced over 15 years resulted in recovery of six known pathogens: S. tanaceti, D. tanaceti, Alternaria tenuissima, Colletotrichum tanaceti, Stemphylium botryosum, and Botrytis cinerea. The incidence of S. tanaceti and D. tanaceti isolated from seed varied between 0.9 and 19.5% (mean = 7.7%) and 0 and 24.1% (mean = 5.3%) among years, respectively. Commercial heat treatment of pyrethrum seed via steaming reduced the incidence of D. tanaceti from 10.9 to 0.06% and the incidence of S. tanaceti from 24.6% to nondetectable levels (<0.18%). In a second experiment, both species were reduced to nondetectable levels (<0.20%) from their initial incidences of 22.4 and 2.4%, respectively. In a field study in 2013, colonization of pyrethrum foliage by S. tanaceti was reduced from 21.1 to 14.3% in early winter when heat-treated seed was planted. However, isolation frequency of D. tanaceti was not affected significantly by seed treatment in this year. In a related experiment in 2015, the isolation frequency of D. tanaceti in plots planted from heat-treated seed depended on both prior application of an industry-standard fungicide program and proximity to another pyrethrum field in autumn. The fungus was recovered at a similar frequency in fungicide-treated and nontreated plots located near other pyrethrum fields (13.8 versus 16.3%, respectively), whereas recovery of the pathogen was reduced by fungicide applications in geographically remote pyrethrum fields (6.7 versus 1.4%, respectively). However, these differences in isolation frequency of D. tanaceti in autumn did not obviate the need for later fungicide applications to suppress foliar disease intensity in spring or flower yield in summer, independent of the proximity to other pyrethrum fields. This study suggests that steam treatment of seed can delay development of the foliar disease complex on pyrethrum, although an extremely low level of remaining infected seed or exogenous sources of inoculum necessitates the use of foliar fungicide applications in spring.

Mating-Type Gene Structure and Spatial Distribution of Didymella tanaceti in Pyrethrum Fields.

Tan spot of pyrethrum (Tanacetum cinerariifolium) is caused by the ascomycete Didymella tanaceti. To assess the evolutionary role of ascospores in the assumed asexual species, the structure and arrangement of mating-type (MAT) genes were examined. A single MAT1-1 or MAT1-2 idiomorph was identified in all isolates examined, indicating that the species is heterothallic. The idiomorphs were flanked upstream and downstream by regions encoding pyridoxamine phosphate oxidase-like and DNA lyase-like proteins, respectively. A multiplex MAT-specific polymerase chain reaction assay was developed and used to genotype 325 isolates collected within two transects in each of four fields in Tasmania, Australia. The ratio of isolates of each mating-type in each transect was consistent with a 1:1 ratio. The spatial distribution of the isolates of the two mating-types within each transect was random for all except one transect for MAT1-1 isolates, indicating that clonal patterns of each mating-type were absent. However, evidence of a reduced selection pressure on MAT1-1 isolates was observed, with a second haplotype of the MAT1-1-1 gene identified in 4.4% of MAT1-1 isolates. In vitro crosses between isolates with opposite mating-types failed to produce ascospores. Although the sexual morph could not be induced, the occurrence of both mating-types in equal frequencies suggested that a cryptic sexual mode of reproduction may occur within field populations.

Identification of the MAT1 locus in Stagonosporopsis tanaceti, and exploring its potential for sexual reproduction in Australian pyrethrum fields.

Stagonosporopsis chrysanthemi, S. inoxydabilis, and S. tanaceti are closely related Ascomycetes associated with ray blight of the Asteraceae. To date, only S. tanaceti has been identified in Australia, incurring substantial losses to the pyrethrum industry. In contrast to the homothallic S. chrysanthemi and S. inoxydabilis, a sexual state has not been observed for S. tanaceti. The MAT1 locus in S. tanaceti was identified through de novo assembly of shotgun reads, and was further used to develop primers for amplification of the full-length MAT1/2 locus in S. chrysanthemi and S. inoxydabilis. As expected, S. chrysanthemi and S. inoxydabilis possessed a MAT1/2 locus typical of homothallic Dothideomycetes with two adjacent MAT1-1 and MAT1-2 idiomorphs. However, only MAT1-1 could be detected in the assembled genome of S. tanaceti. Although a sexual mode of reproduction cannot be ruled out for S. tanaceti, evidence so far suggests this is absent or occurring at very low frequency in Australian pyrethrum fields.

Rapid Changes in the Genetic Composition of Stagonosporopsis tanaceti Population in Australian Pyrethrum Fields.

A novel set of microsatellite markers were developed and employed for geographical and temporal population analyses of Stagonosporopsis tanaceti, the cause of ray blight of pyrethrum in Australia. Genotyping of 407 isolates, using 13 markers, suggested an asexual mode of reproduction with significant linkage disequilibrium and high levels of clonality. Low geographical differentiation and widespread distribution of a few multilocus genotypes (MLGs), in the absence of airborne ascospores, suggested the role of human-mediated movement of seed as a major means of long-distance pathogen dispersal. The genetic composition of S. tanaceti was stable for a decade then changed rapidly in only 2 years. Bayesian clustering analyses and minimum spanning networks determined only two major clonal lineages in and prior to 2010. However, in 2012, a previously unobserved cluster of MLGs was detected, which significantly increased in frequency and displaced the historically dominant MLGs by 2013. This rapid change in the genetic composition of S. tanaceti could indicate a second introduction then a selective sweep, or strong selection pressures from recently introduced fungicides or pyrethrum varieties. These results may have serious implications for durability of management strategies for this disease.

Production, purification, and characterization of antifungal metabolite from Pseudomonas aeruginosa SD12, a new strain obtained from tannery waste polluted soil.

A new strain, SD12, was isolated from tannery waste polluted soil and identified as Pseudomonas aeruginosa on the basis of phenotypic traits and by comparison of 16S rRNA sequences. This bacterium exhibited broad-spectrum antagonistic activity against phytopathogenic fungi. The strain produced phosphatases, cellulases, proteases, pectinases, and HCN and also retained its ability to produce hydroxamate-type siderophore. A bioactive metabolite was isolated from P. aeruginosa SD12 and was characterized as 1-hydroxyphenazine ((1-OH-PHZ) by nuclear magnetic resonance (NMR) spectral analysis. The strain was used as a biocontrol agent against root rot and wilt disease of pyrethrum caused by Rhizoctonia solani. The stain is also reported to increase the growth and biomass of Plantago ovata. The purified compound, 1-hydroxyphenazine, also showed broad-spectrum antagonistic activity towards a range of phytopathogenic fungi, which is the first report of its kind.

Epidemics of ray blight on pyrethrum are linked to seed contamination and overwintering inoculum of Phoma ligulicola var. inoxydabilis.

Ray blight, caused by Phoma ligulicola var. inoxydabilis, is the most damaging disease of pyrethrum (Tanacetum cinerariifolium) in Australia. Data collected from 72 plots in commercial pyrethrum fields since 2001 to 2009 revealed substantial annual variations in isolation frequency of the pathogen during semidormancy of the crop in autumn and winter. Isolation frequency of the pathogen during this time and subsequent outbreaks of ray blight in spring were similar across the eight production regions where sampling was conducted, and isolation frequency of the pathogen was linearly correlated (r = 0.88; P < 0.0001) with subsequent defoliation severity when plants commenced growth in spring. Isolation frequency and defoliation severity also were correlated with the incidence of seed infested with P. ligulicola var. inoxydabilis (r = 0.71 and 0.44, respectively; P < 0.0001 in both correlations). Highly accurate risk algorithms for the occurrence of severe epidemics of ray blight were constructed using logistic regression. A model based solely on isolation frequency of the pathogen over autumn and winter correctly predicted epidemic development in 92% of fields. Another model utilizing the incidence of infested seed and rain-temperature interactions in early autumn (austral March and April) and late winter (austral June and July) had similar predictive ability (92% accuracy). Path analysis modeling was used to disentangle interrelationships among the explanatory variables used in the second logistic regression model. The analysis indicated that seedborne inoculum of P. ligulicola var. inoxydabilis contributes indirectly to ray blight defoliation severity through directly increasing overwintering frequency of the pathogen. Autumn and fall weather variables were modeled to have indirect effects on defoliation severity through increasing overwintering success of the pathogen but also direct effects on defoliation severity. Collectively, the analyses point to several critical stages in the disease cycle that can be targeted to minimize the probability of regional epidemics of ray blight in this perennial pathosystem.

Susceptibility of chrysanthemum and Paris daisy varieties to several isolates of Fusarium oxysporum f. sp. chrysanthemi.

Fusarium oxysporum f.sp. chrysonthemi is a pathogen recently reported in Italy on four economically important ornamental crops belonging to the Compositae family: chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens), African daisy (Osteospermum sp.) and gerbera (Gerbera jamesonii). The risk of transmission of the pathogen among these species is high because the hosts are frequently cultivated in the same nursery. The susceptibility of 24 Paris daisy and 12 chrysanthemum cultivars to 10 isolates of F. oxysporum f. sp. chrysanthemi and 3 isolates of F. oxysporum of different origin and to one isolate of F. tracheiphilum from gerbera was tested. Among the tested chrysanthemum cultivars, "Menthise bianco", "Cottonball", "Super Yellow" and "Meribel" were resistant to all the tested strains, while Pingpong gel was resistant to 10 out of 12 isolates. Among the 24 tested cultivars of Paris daisy, only "Sole mio", "Butterfly" and "Maria" were resistant to all isolates of F. oxysporum f.sp. chrysanthemi and to F. tracheiphilum. The results obtained in this work suggest the need of devoting more attention to resistance to Fusarium wilt while developing new varieties of both chrysanthemum and Paris daisy, since only few varieties are resistant to all strains tested.

The degradation of the natural pyrethrins in crop storage.

Prolonged storage of harvested Tasmanian pyrethrum crop from Tanacetum cinerariaefolium has resulted in substantial losses of the pyrethrin esters due to the environmental conditions in the storage shed. The generation of heat, the presence of moisture and oxygen, and the microbial activity were identified as possible causes. A pyrethrum crop sample was divided up and stored in different conditions relating to these variables, and the pyrethrins content was monitored over time using a standard method. Temperature was determined to be a critical factor in the rate of the degradation of the natural pyrethrins. Moisture, oxygen, and microbial activity unexpectedly did not play a major role in the degradation. An initial rapid loss of the natural pyrethrins was observed before the pyrethrins content stabilized at a loss of around 65%. This suggests that the plant structure may provide chemical or physical protection to the pyrethrins. In all cases, the majority of the loss was attributed to the pyrethrin I and pyrethrin II esters.

Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases.

An immunoelectron microscopy technique was applied to label Chrysanthemum leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal antibodies at different dilutions and secondary antimouse antibody conjugated with colloidal gold particles of different sizes were used. The monoclonal antibodies demonstrated their specificity against the antigen; immunocytological methods permitted the precise localization and identification of phytoplasmas in thin sections from infected tissues.

Ti- and cryptic-plasmid-borne virulence of wild-type Agrobacterium tumefaciens strain CNI5 isolated from chrysanthemum (Dendranthema grandiflora Tzvelev).

The genetic similarity between pTiBo542 and pTiCNI5, which are harbored, respectively by the supervirulent Agrobacterium tumefaciens strain A281 and by the highly tumorigenic wild-type strain CNI5 isolated from chrysanthemum was investigated by Southern hybridization. pTiCNI5 and pTiBo542 exhibited highly similar hybridization patterns in both BamHI- and EcoRI-digested plasmids by using four vir-region-specific probes, whereas similarity in these two plasmids was not observed by probing with five TL-DNA-specific probes. The characteristics related to tumor formation of cryptic plasmids carried by strain CNI5 were investigated by using single-plasmid-cured derivatives. pTiCNI5-cured derivatives predictably failed to utilize agropine and mannopine and failed to induce tumors on chrysanthemum and tobacco leaf explants, while pAtCNI5a-, pAtCNI5c- and pAtCNI5d-cured derivatives could utilize these opines similar to the parent strain CNI5. Interestingly, pAtCNI5c- and pAtCNI5d-cured derivatives showed low tumorigenicity in comparison with strain CNI5 or with the pAtCNI5a-cured derivative. These results suggest that the highly virulent phenotype of strain CNI5 may be due to one or more vir genes, which exhibit cartographic similarity to those of pTiBo542. The results also suggest that the gene(s) related to tumor formation of strain CNI5 may exist not only on pTiCNI5 but also on cryptic plasmids pAtCNI5c and pAtCNI5d.

In planta expression of a protein encoded by the extrachromosomal DNA of a phytoplasma and related to geminivirus replication proteins.

A new extrachromosomal DNA, EcOYW1, was cloned from the onion yellows phytoplasma (OY-W). Southern blot and PCR analysis showed that EcOYW1 is not present in the OY-M, a mild symptom line derived from OY-W. We determined the complete nucleotide sequence of EcOYW1; it is a circular dsDNA of 7.0 kbp in length, which contains seven ORFs. ORF1 encoded a homologue of the geminivirus Rep protein. Western immunoblot analysis revealed that this Rep homologue is expressed in OY-W infected plants, suggesting that EcOYW1 replicates via a geminivirus-like rolling-circle replication mechanism. EcOYW1 is the first phytoplasmal extrachromosomal DNA shown to express encoded genes.

Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA.

The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as Pseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.

Effect of delivery method and population size of Trichoderma harzianum on growth response of unrooted chrysanthemum cuttings.

In a previous study, addition of Trichoderma harzianum Rifai isolate T-12 to a propagative medium resulted in improved performance of chrysanthemum cuttings. However, root and shoot growth of one cultivar, 'Dark Bronze Charm', were more responsive to a lower (5 g T-12/kg medium) than higher (25 g T-12/kg medium) rate of fungal propagules, suggesting potential phytotoxicity at higher concentrations. The objectives of this study were to investigate higher rates of T-12 medium amendment for phytotoxicity, and to examine an alternative method of delivering the fungus to the propagative medium in order to obtain a more uniform response from cuttings. Isolate T-12 was added to the propagative medium as either a powdered peat-bran amendment (0, 5, or 50 g T-12/kg medium) or as alginate prills (80 or 800 g T-12/kg medium). There were no differences among treatments on day seven, but by day 21, shoot fresh weight and heights were significantly greater for plants treated with prills at 800 g T-12/kg medium. Both prill treatments resulted in greater shoot height on day 14 and 21 than all other treatments, which were similar to controls. Amendment with T-12 powder at 50 g/kg increased root length, but 80 g/kg medium added as prills decreased root dry weight compared to the control. The highest rate of T-12 (800 g prills/kg medium) had no effect on root growth. This suggests that moderate, rather than high rates of T-12 are more effective in promoting rooting of unrooted chrysanthemum, and that there is a potential for phytotoxic effects on root growth with higher rates.

Highly tumorigenic Agrobacterium tumefaciens strain from crown gall tumors of chrysanthemum.

A wild-type Agrobacterium tumefaciens strain CNI5 isolated from crown gall of chrysanthemum (Dendranthema grandiflora Tzvelev) was characterized. Strain CNI5 was classified into biovar 1, based on physiological and biochemical characteristics, and was resistant to ampicillin. Strain CNI5 induced tumors at a higher frequency and on a larger area of explants in most tested plant species, especially in chrysanthemum cultivars, than the octopine-type strain C58C1cmr (pTiB6S3). Agropine and mannopine were detected in tumors induced by strain CNI5 and were specifically catabolized by this strain. Strain CNI5 harbored five plasmids including one plasmid that shared sequence similarity to TL-DNA of the octopine-type Ti plasmid and four cryptic plasmids.

Novel Ti plasmids in Agrobacterium strains isolated from fig tree and chrysanthemum tumors and their opinelike molecules.

Galls naturally induced on Fig and chrysanthemum plants by strains of Agrobacterium contained, in addition to other well-characterized opines such as nopaline, three tumor-specific opinelike molecules. These molecules were identified as deoxy-fructosyl-glutamine (dfg), deoxy-fructosyl-5-oxo-proline (dfop), and chrysopine (Chilton et al., unpublished). Strains isolated from Fig tree and chrysanthemum tumors harbored different and unrelated Ti plasmids as judged by hybridization with various vir and T-DNA probes. They also exhibited different opine-catabolic properties. The strains isolated from chrysanthemum plants (Chry strains) and Fig trees degraded chrysopine, but only the Chry strains used dfg and dfop. Remarkably, other strains of Agrobacterium catabolized these two molecules: dfg was degraded by most pathogenic and nonpathogenic Agrobacterium strains, and dfop by all Agrobacterium strains degrading the opine agropinic acid. These results have strong ecological and evolutionary inferences which fit previous speculation on the origin of opine-related functions.

[Cloning of Chrysanthium stund virion cDNA in pUC19 plasmid and the use of cloned cDNA for detecting the virion]. / Klonirovanie kDNK viroida karlikovosti khrizantem v plazmide pUC19 i ispol'zovanie klonirovannoi kDNK dliia obnaruzheniia viroida karlikovosti khrizantem.

26 base long deoxyribonucleotide complementary to the lower part of the Central Conserved Region of chrysanthemum stund viroid (CSV) was used for synthesis of the first strand cDNA. The cDNA was cloned into plasmid vector pUC19 and the primary structure was determined. Cloned, full length cDNA was used as hybridisation probe for detection of CSV. It was possible to detect about 26 pg of purified CSV RNA immobilized on nitrocellulose filters using 32P-labeled probe. In the case of biotinylated probe it was possible to detect about 26 pg of purified CSV RNA visualizing results by streptavidin-alkaline phosphatase conjugates. It has been shown that such a cloned cDNA can be used for wide scale detection of CSV.

Chrysanthemum virus B: antibodies to structural protein in mammals and Mg2(+)-dependent reverse-transcriptase activity.

Human plasma has been found to contain antibodies reacting with the structural protein of chrysanthemum virus B (CV-B) and about 10 times less intensively with the structural protein of another carlavirus, the potato virus M. It has been shown that the antibodies bind to CV-B through their F(ab)2 fragments. No reaction with proteins of other plant viruses or retroviruses was observed. Antibodies reacting with CV-B protein are also present in the plasma of green monkey, goat, rabbit, and mouse, their level being somewhat lower than in man. In addition, Mg2(+)-dependent reverse transcriptase activity reaching maximum at 37 degrees C was detected in the CV-B preparations. It is suggested that humans and the mammals in question developed antibodies to CV-B which could enter into some enzymatic reactions at the body temperature.