Distant Serological Relationship Research Articles

Purification and properties of blackberry chlorotic ringspot, a new virus species in subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK

AbstractA mechanically transmissible virus was isolated from Bedford Giant blackberry plants showing chlorotic mottling and ringspot symptoms growing in Scotland. It infected several herbaceous test plants, many of them symptomlessly. This virus was also transmitted to several Rubus species and cultivars by graft inoculation with scions from the field‐infected Bedford Giant plant. Most grafted plants were infected symptomlessly, but Himalaya Giant blackberry and the hybrid berry Tayberry developed symptoms similar to those in the infected Bedford Giant plant. In the sap of infected Chenopodium quinoa, the virus lost infectivity when diluted 10−4 but not 10−3, after 6 h and 48 h when kept at 20°C and 4°C, respectively, but was infective for more than 8 days when kept at −15°C. Preparations of purified virus from infected C. quinoa or spinach sedimented as three major nucleoprotein components and consisted of quasi‐isometric particles that varied in size from 24 to 32 nm in diameter and that were not penetrated by negative stain. Such virus particle preparations contained a major polypeptide of ca 28 kDa and three single‐stranded RNA species of estimated size 3.2, 2.8 and 2.1 kb. The complete sequence of the largest RNA (RNA 1, 3478 nt) and the partial sequence of the other RNAs (1863 and 2102 nt long, respectively) were determined and compared with sequences in databases. These findings, together with the biological and biochemical properties of this virus, indicate that it should be regarded as a distinct species in subgroup 1 of the genus Ilarvirus even though it was serologically unrelated to existing members of this subgroup. The virus showed a very distant serological relationship with prune dwarf virus (PDV) but differed significantly from it in the amino acid sequence of its coat protein, experimental host range and symptomatology and was unrelated to PDV at the molecular level. The virus, tentatively named blackberry chlorotic ringspot virus, is therefore a newly described virus and the first ilarvirus found naturally infecting Rubus in the UK.

Gentian mosaic virus: A New Species in the Genus Fabavirus

ABSTRACT A viral isolate, designated N-1 and obtained from a gentian (Gentiana scabra) plant that exhibited mosaic symptoms, was transmitted mechanically to nine plant species in six families. These plants are known as hosts of fabaviruses. The N-1 isolate was composed of isometric particles 30 nm in diameter and included two RNA molecules of approximately 6.0 and 3.6 kb in length, as estimated by agarose gel electrophoresis. The RNAs were encapsidated separately in two of the three types of particle. Each particle contained two distinct proteins with Mr values of 39.3 x 10(3) and 26.6 x 10(3), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of complete nucleotide sequences of the RNAs suggested that each encoded a single large polyprotein, in which putative functional proteins were arranged in a manner similar to those in Broad bean wilt virus 1 (BBWV-1) and Broad bean wilt virus 2 (BBWV-2), which are members of the genus Fabavirus (family Comoviridae). Analysis of the deduced amino acid sequences of the proteins indicated that those of isolate N-1 shared 38 to 66% identity with those of BBWV-1 and BBWV-2 but only 16 to 42% identity with those of a comovirus, Cowpea mosaic virus. Phylogenetic analysis, based on the amino acid sequences of RNA polymerase, placed isolate N-1 in a separate lineage from BBWV-1 and BBWV-2. In indirect-enzyme-linked immunosorbent assay, isolate N-1 exhibited distant serological relationship to BBWV-1, BBWV-2, and Lamium mild mosaic virus, another fabavirus. Our results suggest that N-1 represents a new species of Fabavirus. We propose the name Gentian mosaic virus for this new species.

Further characterization of 'sweet potato virus 2': a distinct species of the genus Potyvirus.

An incompletely described potyvirus isolate from sweet potato in Taiwan, referred to as 'sweet potato virus 2' (SPV2), was further characterised. Electron microscopy revealed that SPV2 has filamentous particles of 850 nm in length and induces cytoplasmic cylindrical inclusions consisting of pinwheels and scrolls. The virus was mechanically transmitted to several species of the genera Chenopodium, Datura, Nicotiana, and Ipomoea. Two biotypes of Myzus persicae transmitted SPV2 in a non-persistent manner. Decoration titer experiments revealed a distant serological relationship between SPV2 and other potyviruses infecting sweet potato. The 3'-terminal 2006 nucleotides of the viral RNA were determined and shown to be a potyviral genome fragment comprising the coding region for the C-terminal half of the NIb protein, the entire coat protein cistron, and the 3' untranslated region (UTR). Comparison of the capsid protein and 3' UTR sequences of SPV2 with those of other potyviruses demonstrated that it is a distinct member of the genus Potyvirus (family Potyviridae). We propose that SPV2 is named Sweet potato virus Y.

New Record of Passionfruit Woodiness Virus in Japan.

A potyvirus isolated from passionfruit hybrid cultivar Ohdama (Passiflora edulis×P. edulis f. flavicarpa) showing systemic mosaic symptoms on the leaves and producing severely malformed and woody fruits, collected in the Amami main island of Kagoshima Prefecture, was identified as passionfruit woodiness virus (PWV). The isolate, designated as PWV-AO, was transmitted by aphids, Myzus persicae, in a non-persistent manner and induced local infection in Chenopodium amaranticolor, C. quinoa, Solanum melongena and Phaseolus vulgaris and systemic infection in Passiflora caerulea, P. foetida and Sesamum indicum by sap inoculation. P. edulis, P. edulis f. flavicarpa and Ohdama could be infected with the virus only by aphid and graft inoculations. Sap from diseased Ohdama was infective to P. vulgaris cv. Sujinashi Edogawa after dilution to 10-4 but not 10-5, after heating for 10min at 55°C but not at 60°C, and after 2 days at 25°C but not 3 days. The virus particles were filamentous, 787nm long. In ultrathin sections, pinwheel and scroll inclusions were observed in the cytoplasm of infected Ohdama leaf cells. The virus particles contained a single protein species of MW 35, 000. PWV-AO reacted strongly with an antiserum to a PWV-Brazilian isolate (PWV-B) and weakly with antisera to soybean mosaic virus (SbMV) and watermelon mosaic virus-2 (WMV-2), suggesting that PWV-AO was serologically similar to PWV-B and have a distant serological relationship to SbMV and WMV-2.

Prunus necrotic ringspot ilarvirus: nucleotide sequence of RNA3 and the relationship to other ilarviruses based on coat protein comparison

The RNA3 of prunus necrotic ringspot ilarvirus (PNRSV) has been cloned and its entire sequence determined. The RNA3 consists of 1943 nucleotides (nt) and possesses two large open reading frames (ORFs) separated by an intergenic region of 74 nt. The 5' proximal ORF is 855 nt in length and codes for a protein of molecular mass 31.4 kDa which has homologies with the putative movement protein of other members of the Bromoviridae. The 3' proximal ORF of 675 nt is the cistron for the coat protein (CP) and has a predicted molecular mass of 24.9 kDa. The sequence of the 3' non-coding region (NCR) of PNRSV RNA3 showed a high degree of similarity with those of tobacco streak virus (TSV), prune dwarf virus (PDV), apple mosaic virus (ApMV) and also alfalfa mosaic virus (AIMV). In addition it contained potential stem-loop structures with interspersed AUGC motifs characteristic for ilar- and alfamoviruses. This conserved primary and secondary structure in all 3' NCRs may be responsible for the interaction with homologous and heterologous CPs and subsequent activation of genome replication. The CP gene of an ApMV isolate (ApMV-G) of 657 nt has also been cloned and sequenced. Although ApMV and PNRSV have a distant serological relationship, the deduced amino acid sequences of their CPs have an identity of only 51.8%. The N termini of PNRSV and ApMV CPs have in common a zinc-finger motif and the potential to form an amphipathic helix.

A potyvirus isolated from solanaceous hosts

A potyvirus was isolated from Datura stramonium, Lycopersicon esculentum (tomato) and Solanum nigrum in the Yemen. It was transmitted mechanically and by Myzus persicae in a non‐persistent manner. Its flexuous rod‐shaped particles had a mean length of 719 nm and some of its pinwheel inclusion bodies in infected Nicotiana clevelandii leaves were unusual in that they were dichotomously branched. The virus infected various solanaceous species, but the symptoms it induced were distinct from those of pepper veinal mottle (PVMV) and potato Y viruses. Its particles were purified from N. glutinosa and their coat protein had an atypically high molecular mass a potyvirus of 41·5 kDa. They showed a distant serological relationship to those of PVMV and potato virus V in ISEM decoration tests, but did not react with antisera to particles of any other potyvirus tested. The virus has been tentatively named tomato mild mottle virus.

Properties of cocksfoot streak and cocksfoot cryptic, two viruses infecting cocksfoot (Dactylis glomerate) in Scotland

SummaryA Scottish isolate of cocksfoot streak virus (CSV‐S) was found to have flexuous filamentous particles which, in sap of infected cocksfoot plants, had a modal length of 712 nm. It was transmitted from infected to healthy cocksfoot plants in a non‐persistent manner by Myzus persicae and by mechanical inoculation of infective sap extracts containing an anti‐oxidant. Apart from cocksfoot, mechanical inoculation of infective sap succeeded in infecting only four of 22 plant species tested. The infectivity of sap extracts containing 0.2% thioglycerol was lost after heating for 10 min at 55oC but not 50oC, storage at room temperature for 48 but not 24 hours, and after diluting 10‐2 to 10‐3. Highly purified preparations of CSV‐S particles sedimented as a single component with a sedimentation coefficient of 139S and had a buoyant density in rubidium bromide of 1.31 g/cm3. Virus particles were composed of one protein and one ssRNA species with estimated Mr of 31 000 and 3.2 times 106 respectively. In ELISA, an antiserum prepared to CSV‐S detected the virus in all aerial parts of infected cocksfoot plants and, when present in the ratio of 1 infected leaf: 1000 healthy leaves. Both CSV‐S‐infected and ‐uninfected cocksfoot also contained a previously undescribed virus with isometric particles c. 30 nm in diameter. This virus, named cocksfoot cryptic virus (CCV), was seed‐borne in two cvs of cocksfoot tested and its particles contained two dsRNA species of estimated Mt of 1.14 times 106 and 1.27 times 106. Despite the fact that particles of CSV‐S were largely free from CCV particles following exclusion chromatography on agarose beads prior to immunisation, immunoelectron microscopy (IEM) showed that the antiserum prepared to CSV‐S also contained some antibodies to CCV. Evidence from IEM suggested a possible distant serological relationship of CCV to ryegrass and beet (BCV 1 or BCV 2, or both) cryptoviruses, all members of sub‐group A of cryptoviruses.

Properties of cocksfoot streak and cocksfoot cryptic, two viruses infecting cocksfoot (Dactylis glomerata) in Scotland

SummaryA Scottish isolate of cocksfoot streak virus (CSV‐S) was found to have flexuous filamentous particles which, in sap of infected cocksfoot plants, had a modal length of 712 nm. It was transmitted from infected to healthy cocksfoot plants in a non‐persistent manner by Myzus persicae and by mechanical inoculation of infective sap extracts containing an anti‐oxidant. Apart from cocksfoot, mechanical inoculation of infective sap succeeded in infecting only four of 22 plant species tested. The infectivity of sap extracts containing 0.2% thioglycerol was lost after heating for 10 min at 55oC but not 50oC, storage at room temperature for 48 but not 24 hours, and after diluting 10‐2to 10‐3. Highly purified preparations of CSV‐S particles sedimented as a single component with a sedimentation coefficient of 139S and had a buoyant density in rubidium bromide of 1.31 g/cm3. Virus particles were composed of one protein and one ssRNA species with estimated Mr of 31 000 and 3.2 times 106respectively. In ELISA, an antiserum prepared to CSV‐S detected the virus in all aerial parts of infected cocksfoot plants and, when present in the ratio of 1 infected leaf: 1000 healthy leaves. Both CSV‐S‐infected and ‐uninfected cocksfoot also contained a previously undescribed virus with isometric particles c. 30 nm in diameter. This virus, named cocksfoot cryptic virus (CCV), was seed‐borne in two cvs of cocksfoot tested and its particles contained two dsRNA species of estimated Mr of 1.14 times 106and 1.27 times 106. Despite the fact that particles of CSV‐S were largely free from CCV particles following exclusion chromatography on agarose beads prior to immunisation, immunoelectron microscopy (IEM) showed that the antiserum prepared to CSV‐S also contained some antibodies to CCV. Evidence from IEM suggested a possible distant serological relationship of CCV to ryegrass and beet (BCV 1 or BCV 2, or both) cryptoviruses, all members of sub‐group A of crypto viruses.

Characterization of Two Viruses Isolated from Patchouli in Japan

Two sap-transmissible viruses were isolated from patchouli (Pogostemon patchouli) in Japan. The virus source plants showed faint mosaic, mottling, or no symptoms. In electron microscopic examinations, however, elongated (760 nm in length) or spherical (27 nm in diameter) viruslike particles were found. The spherical virus, designated patchouli mild mosaic virus (PaMMV), infected plants in seven families and was found to be serologically related to, but different from, broad bean wilt virus. The elongated virus, named patchouli mottle virus (PaMoV), had a narrower host range and was identified as a member of the Potyviridae on the basis of particle morphology, formation of cytoplasmic inclusions, and a distant serological relationship with turnip mosaic potyvirus

Nucleotide sequence of RNA 3 of peanut stunt cucumovirus

The nucleotide sequence of the RNA 3 of the peanut stunt virus strain J (PSV-J) was determined and compared with those of the cucumber mosaic virus strain Y (CMV-Y, subgroup I) and the tomato aspermy virus strain C (TAV-C) at both the nucleotide and protein levels. The RNA 3 of PSV-1 consists of 2186 nucleotides and has two large open reading frames (ORFs). By analogy to other tripartite plant viruses, it is presumed that the first ORF (867 nucleotides) codes for the 3a protein and the second ORF (654 nucleotides) is the cistron of the coat protein which is expressed from subgenomic RNA 4 of 1010 nt residues. At the nucleotide level, PSV-1 RNA 3 shows 60.6% homology to RNA 3 of CMV-Y and 66.3% to that of TAV-C, much lower than the value between the two CMV subgroups. For the 3a proteins there is 65.4% homology between PSV-J and CMV-Y and 70.2% homology between PSV-J and TAV-C. Although it has been known that PSV shows a distant serological relationship to CMV, the coat protein of PSV-J shows only 50% homology with that of CMV-Y.

Tulip chlorotic blotch virus, a second potyvirus causing tulip flower break

SUMMARYTulip chlorotic blotch virus (TCBV), an apparently undescribed potyvirus found in field grown tulips in Australia, causes symptoms in tulip leaves and flowers identical to those induced by tulip breaking virus (TBV). TCBV was transmitted mechanically to 14 of 34 species in four of 13 families. Nicotiana clevelandii is a suitable propagation host and Chenopodium amaranticolor a local‐lesion assay host. TCBV was transmitted from tulip to tulip and TV. clevelandii by the aphid Myzus persicae. Unlike TBV it was not transmitted to Lilium formosanum either by M. persicae or by manual inoculation.Leaf extracts from TCBV‐containing TV. clevelandii were infective after dilution to l0‐3 but not 10‐4 and after heating for 10 min at 50°C but not 60°C; infectivity and particle recovery were adversely affected by freezing at ‐20°C. TCBV particles were purified (c. 1 mg/100g g N. clevelandii leaf) from tissue extracts in 0·3 M citrate buffer containing 10 mM EDTA and 0·2% (v/v) 2‐mercaptoethanol at pH 7·4 by clarification with 8·5% (v/v) n‐butanol followed by differential centrifugation and sucrose density gradient centrifugation. Purified particles measured c. 720 × 12 nm. Virus particle antigen was readily detected in leaf and tepal extracts of tulip by enzyme‐linked immunosorbent assay. A distant serological relationship was found between particles of TCBV and those of bean yellow mosaic virus but no serological relationship was found to TBV or four other potyviruses.

Serological Studies of Dianthoviruses Using Monoclonal and Polyclonal Antibodies

SUMMARY In comparative serological studies of dianthoviruses, reverse passive haemagglutination inhibition (RPHI) tests distinguished between sweet clover necrotic mosaic virus (SCNMV) and each of a Swedish isolate of red clover necrotic mosaic virus (RCNMV-SW), the clover primary leaf necrosis strain of RCNMV (RCNMV-C) and carnation ringspot virus (CRSV). Twenty-one monoclonal antibodies prepared against SCNMV were highly specific and failed to react with RCNMV-SW, RCNMV-C or CRSV when tested by RPHI. The extent of the differences between these dianthoviruses was assessed by direct ELISA using polyclonal antibodies. No heterologous reaction was detected by direct ELISA, but indirect ELISA revealed a distant serological relationship between CRSV and each of the other viruses. This is the first report of a positive serological relationship between CRSV and other dianthoviruses.

Identification of a Cucumber Mosaic Virus Strain from Naturally Infected Peanuts in China

A virus isolated from an infected groundnut seedling grown from seed collected in Jing County, Liaoning Province was identified as a CMV str. (CMV-CA), on the basis of biological, physical and chemical properties, serology and particle morphology. The str. infected 31 of 36 spp. tested (6 families). Longevity in vitro was 6-7 days, thermal inactivation point 55-60°C and dilution end point 10-2-10-3. Macrosiphum euphorbiae transmitted the virus in a nonpersistent manner. Seed transmission in groundnut was c. 1.3%. Virus particles were spherical (28.7 nm diam.). An antiserum against CMV-CA was produced with titres of 1/256 and 1/64 in microprecipitin and gel diffusion tests, respectively. Serologically, CMV-CA was indistinguishable from CMV-D and distinct from CMV-S. There was a more distant serological relationship between CMV-CA and tomato aspermy virus but no serological relationship between CMV-CA and groundnut stunt virus strs. E, W or T. The S20,w value of the particle and mol. wts. of CMV-CA coat protein and of the 4 RNAs agreed with reported values for CMV.

Pathology and properties of tulip virus X, a new potexvirus

SUMMARYTulip virus X (TVX), a previously undescribed mechanically transmissible virus, causes chlorotic and necrotic lesions in leaves and streaks of intensified pigmentation in tepals of tulip plants. The virus infected 22 of 42 other plant species in 10 of 14 families, but most host species were infected only erratically. TVX is best propagated in Chenopodium quinoa and assayed in C. amaranticolor. Spindleshaped inclusions were observed in epidermal cells of C. amaranticolor leaves. Leaf extracts from C. quinoa contained flexuous filamentous particles measuring c. 495 ×13 nm. The extracts were infective after dilution to 10‐9, after heating for 10 min at 60 °C but not at 65 °C, and after storage at c. 20 °C for 30 days or at ‐20 °C for 6 months.TVX particles were purified (500 μg/g C. quinoa leaf) from tissue extracts in 0.067 M phosphate buffer containing 10 mM EDTA at pH 7, by twice precipitating the virus with 8% polyethylene glycol in 0.2 M NaCl followed by differential centrifugation. The virus particles have a sedimentation coefficient (s20, w) of 102 S. They contain a protein of mol. wt c. 22 500 and a nucleic acid that, when glyoxalated, migrates in agarose gel like single‐stranded RNA of mol. wt 2.05 × 106. TVX particles tend to aggregate, and evidence was obtained that a 118 S component which was consistently observed in purified preparations and in infective sap is an end‐to‐end dimer. A distant serological relationship was found between particles of TVX and those of viola mottle and hydrangea ringspot viruses, but no serological relationship was detected to nine other potexviruses. TVX is considered to be a distinct and definitive member of the potexvirus group.

Viruses detected in Ullucus tuberosus (Basellaceae) from Peru and Bolivia

SUMMARY Ullucus tuberosus (Basellaceae) plants from 12 locations in the Andean highlands of Peru and Bolivia contained complexes of either three or four viruses. Specimens from six sites in Peru contained a potexvirus, a tobamovirus, a potyvirus and a comovirus, but those from another location lacked the potexvirus. All samples from five sites in Bolivia lacked the tobamovirus.The potexvirus (PMV/U) is a strain of papaya mosaic virus differing slightly from the type strain (PMV/T) in inducing milder symptoms in some common hosts and failing to infect a few other species. It symptomlessly infected U. tuberosus, and infected 15 of 29 species from seven of nine other families. PMV/U showed a close serological relationship to PMV/T and to boussingaultia mosaic virus and a distant relationship to commelina virus X, but it is apparently unrelated to any of ten other potexviruses.The tobamovirus (TMV/U) induced symptomless or inconspicuous infection in U. tuberosus, and infected 21 of 30 species from six of eight other families. It showed a very distant serological relationship to some strains of ribgrass mosaic, tobacco mosaic and tomato mosaic viruses, but failed to react with antisera to cucumber green mottle mosaic, frangipani mosaic, odontoglossum ringspot and sunn‐hemp mosaic viruses.The potyvirus, tentatively designated ullucus mosaic virus (UMV), alone in U. tuberosus induced leaf symptoms indistinguishable from the chlorotic mottling and distortion found in naturally infected plants. UMV infected 12 of 20 species from four other families, and was transmitted in the non‐persistent manner by Myzus persicae. It showed a distant serological relationship to only two (bidens mottle and alstroemeria mosaic) of 25 members or possible members of the potyvirus group tested.Some hosts and properties of the comovirus are described in an accompanying paper.None of the four viruses infected potato (Solanum tuberosum) and, with the possible exception of UMV, they differed from viruses reported previously to infect three other vegetatively propagated Andean crops (Oxalis tuberosa, Arracacia xanthorrhiza and Tropaeolum tuberosum).

Cowpea mild mottle, a newly recognized virus infecting cowpeas (Vigna unguiculata) in Ghana.

SUMMARYCowpea mild mottle virus (CMMV), a previously undescribed virus widespread in cowpeas (Vigna unguiculata) in the Eastern Region of Ghana, was seed‐borne in V. unguiculata, Phaseolus vulgaris and Glycine max, but was not transmitted by twelve aphid species including Aphis craccivora, A. fabae, Acyrthosiphon pisum and Myzus persicae. CMMV was transmitted by inoculation of sap to eleven of seventeen members of the Papilionaceae causing very severe diseases in G. max and Arachis hypogaea, and to ten of fifty‐one species within five of nineteen other families; it was best propagated in G. max and Nicotiana clevelandii, and assayed in Chenopodium quinoa. Sap from systemically infected G. max was infective after dilution to 10‐3 but not 10‐4, after 10 min at 65 °C but not at 70 °C, or after 4 days at 18 °C or 16 days at 2 °C. Lyophilized sap was infective after 3 years in vacuo.CMMV has straight to slightly flexuous, fragile filamentous particles, c. 13 × 650 nm which, in sap, are occasionally surrounded by a loose external spiral. About 5 mg of purified virus was obtained from 1 kg of leaf tissue of G. max or N. clevelandii by clarifying leaf extracts in 0.02 m borate buffer (pH 9.5) with chloroform, followed by two or three cycles of differential centrifugation, and density gradient centrifugation. Virus preparations had ultraviolet absorption spectra typical of a nucleoprotein containing c. 5 % nucleic acid, contained numerous particles without external spirals, which sedimented as a single component with a sedimentation coefficient (s°20, w) of 165 × 4S, and contained a single polypeptide species with a molecular weight of 32000–33000. CMMV showed a distant serological relationship to carnation latent virus, but not to ten other morphologically similar viruses; it thus seems to be a distinct member of the carlavirus group, and has the cryptogram: */*:*/(5):E/E:S/*.

Serological relationship and detection of bean common and bean yellow mosaic viruses in agar gel

SUMMARYMicroprecipitin tests demonstrated a distant serological relationship between intact virus particles of BCMV and BYMV. Antiserum titres of 2048–4096 and 16–128 (reciprocals of dilution end‐points) were found for homologous and heterologous antigens, respectively. Cross‐reactivity, however, was not obtained in agar immunodiffusion tests when the antigens, in purified preparations or crude infective sap, were treated with pyrrolidine or when the reactants were placed in agar gels containing sodium dodecyl sulphate. Only homologous antigens produced a precipitin line; homologous antiserum titre was 16.In comparative immunodiffusion trials, single‐radial‐diffusion tests (antiserum incorporated in agar) were much more sensitive than double‐diffusion tests for detecting low virus concentrations.Analyses of virus proteins by polyacrylamide‐gel electrophoresis demonstrated that coat protein of each virus was composed of a single polypeptide chain with a molecular weight of 35000.

The preparation of antigens of Rhizobium meliloti by ultrasonic disruption: an anomaly.

Diffusible external antigens of whole cells of Rhizobium meliloti PDD1322 lost the capacity to precipitate with homologous antibodies in the gel diffusion test, when the cells were disrupted by ultrasonic oscillation. Sonicated cell preparations caused diminution of the homologous precipitin reaction when mixed with suspensions of whole cells. Sonicates contained a new, faster-diffusing antigen component, probably of intracellular origin. The serological inactivity of these sonicates was reversed by boiling. Diffusible antigens of whole cells of R. meliloti SU388, a strain closely related serologically to PDD1322, behaved similarly, whereas the cross-reacting antigens of strain CC8, of distant serological relationship to PDD 1322, were not affected by sonication. It is suggested that the external antigens and some intracellular compound released by sonication form a heat-labile, relatively nonspecific complex which will not take part in the precipitin reaction. Antibodies to sonicates of PDD1322 reacted strongly with the diffusible external antigens of whole cells of this strain. The same antigens of homologous sonicates reacted very feebly, but the new faster-diffusing component was evident.