Biochemical and epigenetic changes in phytoplasma‐recovered periwinkle after indole‐3‐butyric acid treatment

Aim: To elucidate the possible mechanism of phytoplasma elimination from periwinkle shoots caused by indole‐3‐butyric acid (IBA) treatment. Methods and Results: It has been shown that a transfer of in vitro‐grown phytoplasma‐infected Catharanthus roseus (periwinkle) plantlets from medium supplemented with 6‐benzylaminopurine (BA) to one supplemented with IBA can induce remission of symptoms and even permanent elimination of ‘Candidatus Phytoplasma asteris’ reference strain HYDB. Endogenous auxin levels and general methylation levels in noninfected periwinkles, periwinkles infected with two ‘Candidatus Phytoplasma’ species and phytoplasma‐recovered periwinkles were measured and compared. After the transfer from cytokinin‐ to auxin‐containing media, healthy shoots maintained their phenotype, methylation levels and hormone concentrations. Phytoplasma infection caused a change in the endogenous indole‐3‐acetic acid to IBA ratio in periwinkle shoots infected with two ‘Candidatus Phytoplasma’ species, but general methylation was significantly changed only in shoots infected with ‘Ca. P. asteris’, which resulted in the only phytoplasma species eliminated from shoots after transfer to IBA‐containing medium. Both phytoplasma infection and treatment with plant growth regulators influenced callose deposition in phloem tissue, concentrations of photosynthetic pigments and soluble proteins, H₂O₂ levels and activities of catalase (CAT) and ascorbate peroxidase (APX). Conclusion: Lower level of host genome methylation in ‘Ca. P. asteris’‐infected periwinkles on medium supplemented with BA was significantly elevated after IBA treatment, while IBA treatment had no effect on cytosine methylation in periwinkles infected with ‘Candidatus Phytoplasma ulmi’ strain EY‐C. Significance and Impact of the Study: Hormone‐dependent recovery is a distinct phenomenon from natural recovery. As opposed to spontaneously recovered plants in which elevated peroxide levels and differential expression of peroxide‐related enzymes were observed, in hormone‐dependent recovery changes in global host genome, methylation coincide with the presence/absence of phytoplasma.     

Detection and identification of ‘Candidatus Phytoplasma asteris’ in Brassica rapa subsp. pekinensis in Poland

Severe growth abnormalities, including leaf yellowing, sprout proliferation and flower virescence and phyllody, were found on Brassica rapa subsp. pekinensis plants in Poland. The presence of phytoplasma in naturally infected plants was demonstrated by polymerase chain reaction assay employing phytoplasma universal P1/P7 followed by R16F2n/R16R2 primer pairs. The detected phytoplasma was identified using restriction fragment length polymorphism analysis (RFLP) of the 16S rRNA gene fragment with AluI, HhaI, MseI and RsaI endonucleases. After enzymatic digestion, all tested samples showed restriction pattern similar to that of ‘Candidatus phytoplasma asteris’. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Sequences of the 16S rDNA gene fragment of analysed phytoplasma isolates were nearly identical. They revealed high nucleotide sequence identity (>98%) with corresponding sequences of other phytoplasma isolates from subgroup 16SrI‐B, and they were classified as members of ‘Candidatus phytoplasma asteris’. This is the first report of the natural occurrence of phytoplasma‐associated disease in plants of Chinese cabbage.     

‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma aurantifolia’, New Phytoplasma Species Infecting Apple Trees in Iran

During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.     

Auxin‐treatment induces recovery of phytoplasma‐infected periwinkle

Aims: To test the effect of auxin‐treatment on plant pathogenic phytoplasmas and phytoplasma‐infected host. Methods and Results: In vitro grown periwinkle shoots infected with different ‘Candidatus Phytoplasma’ species were treated with indole‐3‐acetic acid (IAA) or indole‐3‐butyric acid (IBA). Both auxins induced recovery of phytoplasma‐infected periwinkle shoots, but IBA was more effective. The time period and concentration of the auxin needed to induce recovery was dependent on the ‘Candidatus Phytoplasma’ species and the type of auxin. Two ‘Candidatus Phytoplasma’ species, ‘Ca. P. pruni’ (strain KVI, clover phyllody from Italy) and ‘Ca. P. asteris’ (strain HYDB, hydrangea phyllody), were susceptible to auxin‐treatment and undetected by nested PCR or detected only in the second nested PCR in the host tissue. ‘Ca. P. solani’ (strain SA‐I, grapevine yellows) persisted in the host tissue despite the obvious recovery of the host plant and was always detected in the direct PCR. Conclusions: Both auxins induced recovery of phytoplasma‐infected plants and affected tested ‘Candidatus Phytoplasma’ species in the same manner, implying that the mechanism involved in phytoplasma elimination/survival is common to both, IAA and IBA. Significance and Impact of the Study: The results imply that in the case of some ‘Candidatus Phytoplasma’ species, IBA‐treatment could be used to eliminate phytoplasmas from in vitro grown Catharanthus roseus shoots.     

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

Suspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.     

Identification of four distinct ‘Candidatus Phytoplasma’ species in pomegranate trees showing witches' broom,little leaf and yellowing in Jordan,and preliminary insights on their putative insect vectors and reservoir plants

During field surveys conducted in northern Jordan from June to November 2020, phytoplasma-like symptoms, including leaf yellowing/reddening and rolling, little leaf and witches' broom were observed in pomegranate. Disease incidence in 22 surveyed orchards ranged from 30% to 65%. Nested PCR-based amplification of 16S rRNA gene detected phytoplasmas in 17% of collected symptomatic pomegranate trees. Amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma solani’, ‘Ca. P. aurantifolia’, ‘Ca. P. asteris’ and ‘Ca. P. ulmi’. These phytoplasmas were found in plants showing specific symptoms and differentially distributed in the considered locations. Additionally, three cicadellids (Macrosteles sexnotatus, Cicadulina bipunctata and Psammotettix striatus) and two non-crop plants (Plantago major and Capsicum annuum) resulted hosting ‘Ca. P. asteris’ strains, and one cicadellid (Balclutha incisa) was carrying a ‘Ca. P. solani’ strain. A new pomegranate disease complex associated with multiple phytoplasmas, including ‘Ca. P. aurantifolia’ and ‘Ca. P. ulmi’, never reported before in this host plant, is described here. Moreover, preliminary indications are provided on its possible epidemiology in Jordan, involving two putative insect vectors (M. sexnotatus, B. incisa) first reported in the Country.     

Niger Seed (Guizotia abyssinica), a New Host of ‘Candidatus Phytoplasma asteris’ in Iran

Shrubs of niger seed with phyllody and internode elongation symptoms suggestive of phytoplasma infections occurred in the central regions of Iran. Phytoplasma was detected by polymerase chain reaction (PCR) and nested PCR amplifications using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2. Using aster yellows group–specific primer pair rp(I)F1A/rp(I)R1A, a fragment of 1212 bp of the rp genes was amplified from DNA samples of infected plants. Random fragment length polymorphism (RFLP) analyses of R16F2n/R16R2‐amplified products using the CfoI restriction enzyme confirmed that Iranian niger seed phyllody phytoplasma is associated with aster yellows group phytoplasmas. Sequence analyses of the partial rp genes fragment indicated that the Iranian niger seed phyllody phytoplasma, which was collected from central regions of Iran, is related to ‘Candidatus Phytoplasma asteris’. This is the first report of a phytoplasma infecting the niger seed plant.     

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

Pear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.     

Feeding behaviour of the Asiatic citrus psyllid,Diaphorina citri,on healthy and huanglongbing‐infected citrus

Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Psyllidae) is a vector of huanglongbing, a disease of citrus that in Asia is caused by ‘Candidatus Liberibacter asiaticus’ (α‐Proteobacteria) (Las). Acquisition of Las by D. citri appears to be variable, and this variability may be due to the suitability of the host plants and their tissues for acquisition. Therefore, this study aimed to determine the effect of symptom severity of the disease on the feeding behaviour of D. citri. Use of an electrical penetration graph showed that the pathway phase of D. citri consisted of four waveforms, A, B, C, and D; waveforms A and B have not been reported for D. citri before. The remaining waveforms, E1, E2, and G, conform to those described before for D. citri. The duration of the non‐penetration period did not differ between healthy or infected plants. However, in moderately and severely symptomatic plants, the duration of the pathway phase increased, whereas the phloem phase was shorter. In all diseased plants, the times to first and sustained salivation in the phloem were longer than those in control plants, with the times being related to symptom severity. As symptom expression increased, the percentage of time spent by psyllids salivating during the phloem phase increased; however, the percentage of time spent in phloem activities reduced gradually from ca. 74% in the control plants to ca. 8% in the severely symptomatic plants. In contrast, the percentage of time spent on xylem activities increased, as did the proportion of psyllids feeding from xylem. The differences in the durations of the E waveforms on plants showing different levels of symptom expression may account for differences in acquisition found amongst studies; therefore, future work on the acquisition and transmission of Las needs to carefully document symptom expression.     

Production and characteristics of antisera to Spiroplasma citri and clover phyllody-associated antigens derived from plants

Antisera were produced to clover phyllody- and Spiroplasma citri-associated antigens partially purified from infected Vinca rosea plants. Separate antisera were made to ‘membrane fraction’ (MF) preparations comprising the resuspended pellet obtained by high speed centrifugation, and to ‘soluble fraction’ (SA) preparations, comprising the supernatant from high speed centrifugation concentrated by freeze-drying. All antisera showed considerable activity against normal plant antigens but after cross-absorption with extracts of healthy plants the MF antisera were used in F(ab')₂based ELISA tests to detect S. citri- or clover phyllody-associated antigens in infected plants. The ‘clover phyllody’ antiserum also reacted specifically with extracts of clover plants with phyllody, and with naturally-infected strawberry plants showing symptoms of green petal disease. Both the ‘clover phyllody’ and S. citri antisera were specific for their respective homologous antigens. No cross-reactions were observed in heterologous tests or between either antiserum and extracts of V. rosea infected with various MLOs obtained from different host plants.     

Flavescence dorée phytoplasma deregulates stomatal control of photosynthesis in Vitis vinifera

Flavescence dorée (FD) is among the major grapevine diseases causing high management costs; curative methods against FD are unavailable. In FD‐infected plants, decrease in photosynthesis is usually recorded, but deregulation in stomatal control of leaf gas exchange during FD infection and recovery is unknown. We measured the seasonal time course of gas exchange rates in two cultivars (‘Barbera’ and ‘Nebbiolo’) during the term of 1 year when grapevines experienced a water stress and another with no drought, with difference in gas exchange rates in response to FD infection and recovery as assessed by symptom observation and phytoplasma detection through PCR analysis. Chlorophyll fluorescence was also evaluated at the time of maximum symptom severity in ‘Barbera’, the cultivar showing the most severe stress response to FD infection, causing the highest damage in vineyards of north‐western Italy. In FD‐infected plants, net photosynthesis and transpiration gradually decreased during the season, more during the no drought year than during drought. During recovery, healthy (PCR negative) plants infected 2 years before, but not those infected an year before, regained the gas exchange performances to the level as measured before infection. The relationships between stomatal conductance and the residual leaf intercellular CO₂ concentration (c_i) discriminated healthy versus FD‐infected and recovered plants; at the same c_i, FD‐infected leaves had higher non‐photochemical quenching than healthy ones. We conclude that metabolic, not stomatal, leaf gas exchange limitation in FD‐infected and recovered grapevines is the basis of plant response to FD disease. In addition, we also suggest that such response is dependent upon water stress, by showing that water stress superimposes on FD infection in terms of stomatal and metabolic non‐stomatal limitations to carbon assimilation.     

The isolation and identification of rhubarb viruses occurring in Britain

Virus-like symptoms were common in British crops of rhubarb. All plants tested of the three main varieties, ‘Timperley Early’, ‘Prince Albert’ and ‘Victoria’, were virus-infected. Turnip mosaic virus and a severe isolate of arabis mosaic virus (AMV) were obtained from ‘Timperley Early’; and ‘Prince Albert’ contained turnip mosaic virus, cherry leaf roll virus (CLRV), a mild isolate of AMV and, infrequently, cucumber mosaic virus (CMV). The main commercial variety ‘Victoria’ contained turnip mosaic virus, CLRV, a mild isolate of AMV and, infrequently, strawberry latent ringspot virus (SLRV). All the viruses were identified serologically. The rhubarb isolates did not differ markedly from other isolates of these viruses in herbaceous host reactions, properties in vitro or particle size and shape. A rhubarb isolate of CLRV was distinguished serologically from a cherry isolate of the virus. Turnip mosaic virus, CLRV and SLRV, were transmitted with difficulty, but AMV isolates were readily transmitted by mechanical inoculation. Turnip mosaic virus was also transmitted to rhubarb by Myzus persicae and Aphis fabae. CLRV was transmitted in 6–8% of the seed of infected ‘Prince Albert’ and ‘Victoria’ rhubarb and in 72% of the seed of infected Chenopodium amaranticolor. Mild isolates of AMV were also transmitted in 10–24% of the seed of infected ‘Prince Albert’ and ‘Victoria’ plants.     

Micro Morphological and Chemical Investigation into the Effects of Fungal Diseases on Melissa officinals L., Mentha×Piperita L. and Salvia officinalis L.

A micro-morphological study of infected tissues and a chemical investigation into the volatile fraction of secretory tissues of healthy and infected plants were carried out. The botanic species were: Melissa officinalis L. (infected by Erysiphe galeopsidis and Septoria melissae); Salvia officinalis L. (infected by Erysiphe salviae); Mentha piperita L. (infected by Puccinia menthae). The results show that pathogenic fungal infection seriously damages the secretory tissues and stomata, causing a decrease in the amount of essential oil contained in infected plants, and modifying the composition of the plant's volatile fraction.     

Peroxidase and Poly-Phenol Oxidase in Brassica juncea Plants Infected with Macrophomina phaseolina (Tassai) Goid. and their Implication in Disease Resistance

Studies on the peroxidase and poly-phenol oxidase activity in relatively resistant and susceptible strains of Brassica juncea at sequential intervals after inoculation with different isolates of Macrophomina phaseolina revealed that, fungus infected tissues had relatively higher activities of these oxidative enzymes in comparison to healthy plants, and tissues inoculated with ‘Weakly Virulent’ isolates always exhibited higher activity in comparison to ‘Moderately’, ‘Highly’ and ‘MostHighly Virulent’ isolates. At the early stages of the infection on 6th day, resistant plants showed higher activities in comparison to their susceptible counterparts. Very low activity of these enzymes could be recorded in healthy plants. Findings of this, study indicate the possible involvement of these oxidative enzymes in the resistance of this disease.     

Asparagus officinalis: A New Host of ‘Candidatus Phytoplasma asteris’

Asparagus officinalis plants with severe fasciation of some spears were observed in southern Bohemia between 1998 and 2007. Nucleic acids extracted from these and asymptomatic plants were assayed with nested polymerase chain reaction (PCR) using the phytoplasma‐specific universal ribosomal primers P1/P7 and R16F2n/R2. The restriction profiles obtained from digestion of the PCR products with five endonucleases (AluI, HhaI, KpnI, MseI and RsaI) were identical in all phytoplasmas infecting asparagus in the Czech Republic and indistinguishable from those of phytoplasmas in the aster yellows group (subgroup 16SrI‐B). Sequence analysis of 1754 bp of the ribosomal operon indicated that the closest related phytoplasmas were those associated with epilobium phyllody and onion yellows. This is the first report of the natural occurrence of ‘Candidatus Phytoplasma asteris’ in A. officinalis.     

Molecular Characterization of Phytoplasmas Associated with Potato Purple Top Disease in Iran

Potato plants with symptoms suggestive of potato purple top disease (PPTD) occurred in the central, western and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7 followed by primer pairs R16F2n/R16R2 and fU5/rU3 for phytoplasma detection. Using primer pairs R16F2n/R16R2 and fU5/rU3 in nested PCR, the expected fragments were amplified from 53% of symptomatic potatoes. Restriction fragment length polymorphism (RFLP) analysis using AluI, CfoI, EcoRI, KpnI, HindIII, MseI, RsaI and TaqI restriction enzymes confirmed that different phytoplasma isolates caused PPTD in several Iranian potato‐growing areas. Sequences analysis of partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma solani’, ‘Ca. Phytoplasma astris’ and ‘Ca. Phytoplasma trifolii’ are prevalent in potato plants showing PPTD symptoms in the production areas of central, western and north‐western regions of Iran, although ‘Ca. Phytoplasma solani’ is more prevalent than other phytoplasmas. This is the first report of phytoplasmas related to ‘Ca. Phytoplasma astris’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ causing PPTD in Iran.     

First Report of Phytoplasma (16SrI) Associated with Yellow Decline Disease of Royal Palms [Roystonea regia (Kunth) O. F. Cook] in Malaysia

Royal Palms (Roystonea regia) with symptoms such as severe chlorosis, stunting, collapse of older fronds and general decline were observed in the state of Selangor, Malaysia. Using polymerase chain reaction (PCR) amplification with phytoplasma universal primer pair P1/P7 followed by R16F2N/R16R2 and fU5/rU3 as nested PCR primer pairs, all symptomatic plants tested positively for phytoplasma. Results of phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences revealed that the phytoplasma associated with Royal Palm yellow decline (RYD) was an isolate of ‘Candidatus Phytoplasma asteris’ belonging to a new 16SrI‐subgroup. These results show that Roystonea regia is a new host for the aster yellows phytoplasma (16SrI). This is the first report on the presence of 16SrI phytoplasma on Royal Palm trees in Malaysia.     

Roditis Leaf Discoloration - A New Virus Disease of Grapevine: Symptomatology and Transmission to Indicator Plants

The symptoms and some characteristics of an unreported disease of grapevine, which was observed during the last years on the cv. ‘Roditis’ in central Greece, are described. The disease is transmissible to V. vinifera‘Mission’ and to some herbaceous test plants. The constant association of a virus to naturally infected plants cv. ‘Roditis’ and chip-budding infected indicator plants cv. ‘Mission’ supports the evidence that the disease is caused by a virus.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

Strains of Prunus necrotic ringspot virus in hop (Humulus lupulus L.)

Purified preparations of Prunus necrotic ringspot virus (NRSV) from hop plants formed two light-scattering zones when centrifuged in sucrose density gradients; the upper and lower zones contained particles 25 mμ and 31 mμ in diameter respectively whose sedimentation coefficients were 79 S and 107 S. NSRV isolates from hop were of two distinct serological types: ‘A’ strains, serologically very closely related to NRSV isolates from apple; and ‘C’ strains more nearly related to NRSV from cherry. The variety Fuggle is tolerant to hop mosaic (not related to NRSV) and different selections of apparently healthy female plants usually contained A strains; but C strains were usually isolated from nettlehead-diseased plants. Either A or C strains occurred in male plants grown with the hop-mosaic tolerant varieties. In mosaic-sensitive varieties (Goldings and Bramlings) apparently healthy female plants tested were usually infected with C strains; either A or C types occurred in mosaic-sensitive male plants. NRSV was not detected in the seventy-four hop seedlings obtained from virus-infected plants. Some varieties developed nettlehead when infected with NRSV (A) or (C) + the hop form of arabis mosaic virus, but not with NRSV (A) or (C) alone. Others developed nettlehead when infected with arabis mosaic virus + NRSV (C) but not with arabis mosaic + NRSV (A). A and C strains can multiply together in the same hop plant. There is evidence of partial antagonism, however, and the fluctuating behaviour of the nettlehead syndrome probably reflects changes in the relative concentration of the two serotypes.