Characterization of an Isolate of Tobacco Necrosis Virus From Zucchini

Tobacco necrosis virus (TNV) was isolated from glasshouse zucchini plants in Liguria, northern Italy, showing yellow spots on young leaves and necrotic symptoms on older leaves, petioles and stems. No other viruses were present in these plants. Experimentally, the virus systemically infected zucchini seedlings grown in naturally infected soil but only local infections were induced when leaves and roots were mechanically inoculated. The virus was identified as serotype D, devoid of satellite particles. This appears to be the first report of TNV in zucchini. Three ELISA procedures for field detection of TNV using polyclonal antibodies for serotype D were compared. Antibodies were conjugated with I) alkaline phosphatase (AP-AB);2) N-Hydroxy-succinimid obiotin (BNHS-Ab); 3) N-biotinyl-6-aminocaproic hydrazide (BACH-Ab). The procedure using BNHS-Ab detected the lowest concentration of the homologous virus, followed by BACH-Ab and AP-Ab. Only with biotinylated antibodies. TNV serotype A was also detected.     

Augusta disease in tulip - a reassessment

In an experiment in which the roots of field-grown tulip were commonly infected with tobacco necrosis virus (TNV), Augusta disease did not develop in the year of infection or when progeny bulbs were grown in the field or glass-house. When tulip bulbs of other stocks, including grades of 11 and 12 cm circumference, were forced, the disease developed sporadically, in some instances as the result of infection with TNV from the soil in which they were planted and in others as a result of infection by bulb-borne virus. The incidence of disease produced by current year infection was increased by warming the plunge bed. Different strains of TNV were obtained from field-grown plants with Augusta disease and different strains of the virus produced the disease when inoculated to tulip. Some, but not all, naturally diseased plants contained satellite virus, which therefore does not cause or prevent disease development. The disease was produced in some plants by TNV transmitted by Olpidium brassicae, but neither a vector nor a non-vector isolate of O. brassicae completed its life cycle in tulip. However, Olpidium-like zoospores were observed in some washings of tulip roots from TNV-infested soils. TNV was not obtained from all tulip plants with necrotic leaf symptoms resembling Augusta disease. Some were infected with tomato bushy stunt virus or cucumber mosaic virus, or with another agent that was transmitted by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and carried by bulbs of up to 11 cm circumference.     

Cherry leaf roll virus in the female gametophyte and seed of birch and its relevance to vertical virus transmission

ELISA detected cherry leaf roll virus (CLRV) in infected birch leaf sap diluted 1/320 in buffer, in extracts of one infected leaf with nine healthy leaves and in leaf sap frozen for 2 wk. Similarly, ELISA detected CLRV in mixtures of one infected bud with four virus-free buds. Intensive bioassays and ELISA showed that in some trees CLRV was restricted to only a few branches whereas in others it occurred throughout the tree. The prevalence of CLRV in unmanaged birch populations in Britain was less (3%) than in Midlands street trees (17%). In CLRV-free birch trees that received pollen from infected ones, ELISA indicated that antigen was introduced into, and multiplied within, the embryos but not the seed coat or the pericarp/wings. In one instance, antigen was detected in a branch of an experimentally pollinated tree but not in those parts of the crown that had been exposed to open pollination. The proportion of seed germinating after crosses in which both parents were CLRV-free was greater than when either or both parents were infected but the largest difference occurred with infection in the female parent. Few embryos seemed to escape invasion with CLRV when the maternal tissue was naturally infected. Overall, seed transmission ranged between 0 and 38% (mean 17%) when only females in a cross were infected, and between 11 and 75% (mean 30%) when only the males were infected. Assuming no selective advantage that would help infected plants to achieve reproductive age, we found that CLRV would be lost from a birch population within two generations if transmitted only through seed. Embryos in seeds from CLRV-infected birch that received CLRV-free pollen differed from their healthy counterparts in being shrunken and suspended in a loosely fibrillar matrix that contained numerous virus-like particles in tubular inclusions. In two trees, CLRV-free pollen tended to fertilise a greater percentage of ovules than did CLRV-infected pollen. Seedlings derived from infected seed and cuttings from naturally-infected trees grew less rapidly than their healthy counterparts. In still air, most birch pollen liberated from a height of 3·5 m fell within 3 m of the drop zone and none was detected 10 m from the source. Field observations on the patterns of virus spread as measured by seedling infection were consistent; about 3% of seedlings from a tree 6·9 m away from the nearest source of inoculum were infected but no infected seedlings were detected in more distant trees, even though each was experimentally infectible with CLRV and pollen from the infected tree germinated on their stigmas.     

Subterranean clover mottle sobemovirus: its host range, resistance in subterranean clover and transmission through seed and by grazing animals

Subterranean clover mottle sobemovirus (SCMV) was transmitted by manual inoculation of sap to 27 cultivars belonging to three sub-species of subterranean clover. The virus readily infected systemically all inoculated plants of five susceptible cultivars of ssp. subterraneum. Ten others showed partial resistance as not all infected plants developed systemic infection; cold winter conditions further delayed or prevented systemic movement in four of them. Two cultivars of spp. brachycalycinum and four of spp. yanninicum failed to develop systemic infection following inoculation and were considered highly resistant. Resistance to SCMV in three of the spp. yanninicum was further confirmed by the failure to establish detectable primary infections in most of the inoculated leaves. Moreover, when the four ssp. yanninicum cultivars were graft-inoculated with SCMV, systemic infection eventually developed in them but the virus concentration was low. SCMV was also transmitted by manual inoculation of sap to a further 23 species of Trifolium, Medicago or Pisum. Three species were non-hosts, five were infected only in inoculated leaves and 18 others developed systemic infection in some or all plants. SCMV reached very high concentrations and was stable in subterranean clover sap. It was transmitted experimentally between subterranean clover plants by brushing infected leaves against healthy ones and in swards was readily transmitted by the trampling and grazing of sheep, but only poorly by mowing. Seed transmission of SCMV to seedlings of five cultivars of subterranean clover was low (0–0.12%). SCMV was not transmitted by Myzus persicae.     

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.     

Physiological changes of Fagus sylvatica seedlings infected with Phytophthora citricola and the contribution of its elicitin "citricolin" to pathogenesis

Beech seedlings were infected with the root rot pathogen Phytophthora citricola to study its impact on leaf physiology and water status. Net photosynthesis rate decreased two days after inoculation in infected seedlings. In contrast, electron quantum yield of photosystem II, leaf water potential, and total water consumption were only slightly impaired until 6 dpi. At the same time, wilt symptoms occurred on leaves. These results indicate the involvement of a mobile signal triggering the early changes in leaf physiology by root infection. As the elicitin gene of P. citricola was induced during root infection, we purified and characterised the elicitin protein and tested its ability to change leaf physiological parameters of beech and tobacco plants. P. citricola produced a single acidic elicitin (citricolin), which caused necrosis and decreased gas exchange of tobacco leaves. Furthermore, it induced an oxidative burst in tobacco cell suspension culture. However, none of these effects were observed in beech.     

Investigations on Net CO2 Assimilation, Transpiration and Root Growth of Fagus sylvatica Infested with Four Different Phytophthora Species

Abstract: In this contribution, we compare the influence of four different Phytophthora species on root development, net CO₂ assimilation and transpiration of beech seedlings and saplings. Some few days after inoculation, photosynthesis and transpiration of seedlings infected with either P. citricola or P. cambivora were strongly reduced. In parallel, about 60 % of their root systems was destroyed compared to control plants. Three weeks after infection, all seedlings were dead, showing severe wilt symptoms on leaves. Remarkably, P. syringae and P. undulata infected seedlings and older beech plants did not differ from controls regarding photosynthesis and transpiration, although the root systems were damaged. However, a significant influence on net CO₂ assimilation and transpiration of P. citricola infected beech saplings was visible after bud break in the following year. Some days before plants started to wilt, photosynthesis and transpiration were reduced to almost zero. Water use efficiency data (WUE) clearly indicated that infected plants suffered from severe drought.     

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

Local infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non-inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants. Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene-releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.     

Systemic Acquired Resistance to Virus Infections and Ethylene Biosynthesis in Asparagus Bean

Systemic acquired resistance (SAR) was induced in asparagus bean following inoculation with tobacco necrosis virus (TNV) or tobacco rattle virus (TRV), viruses that produce a hypersensitive reaction in this plant. SAR was expressed against challenge by TNV as reduction in lesion size, but not as inhibition of viral antigen accumulation. Systemic stimulation of ethylene-forming enzyme (EFA) activity, in the absence of any ethylene increase or 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation, was associated with SAR. Formation of local necrotic lesions was necessary for both induction of SAR and stimulation of EFA, because early removal of inducer leaves prevented both events. SAR was expressed at rather constant level between 7 and 12 days after inducing infection. EFA stimulation declined with time and was no longer detected 7 days after inducing infection. SAR was not expressed against cucumber mosaic virus, that infect asparagus bean systemically. Prior inoculation with TNV or TRV was ineffective to reduce CMV antigen content or to minimize the pathogenic effect of this virus in systemically infected leaves.     

Interference Between Tobacco necrosis virus and Turnip crinkle virus in Nicotiana benthamiana

Mixed infections of Nicotiana benthamiana plants by Tobacco necrosis virus (TNV) and Turnip crinkle virus (TCV) exhibited an interference interaction. Accumulation of TNV (+)RNA as well as capsid protein in mixed infection were considerably lower than that of singly infected plants. There were also a slight reduction in the levels of TCV (+)RNA and capsid protein in doubly infected plants, which displayed the concentration of both viruses decreased in dually infected plants. Tissue immunoblot analysis of systemic N. benthamiana leaves infected by TNV and TCV singly or doubly showed the interference between the two viruses in situ, which exhibited the decrease of both viruses in doubly infected leaves although the distribution of them did not change remarkably. These results were consistent with the hybridization analysis of viral genomic RNA and coat protein. Both cross‐protection test and mixed infection of the two viruses confirmed TCV had relatively stronger interference to the infection of TNV. Interference infection by TNV and TCV induced higher increase in the levels of cytochrome pathway respiration and alternative pathway respiration in host plants, especially the latter. Interference often occurred in different strains of one kind of virus or two different closely related viruses in one genus. Our results showed that interference could also occur in different viruses belonging to different genera.     

Seed transmission of broad bean stain virus and Echtes Ackerbohnenmosaik-Virus in field beans (Viciafaba)

When grown in a glasshouse during spring or autumn field bean (Vicia faba minor) seedlings infected with seed-borne broad bean stain virus (BBSV) or Echtes Ackerbohnenmosaik-Virus (EAMV) usually showed symptoms on some leaves within 4 wk of emergence. Symptoms caused by each virus were indistinguishable. The viruses were transmitted as often through unblemished seeds as through seeds with necrotic patches or stains on the seed coat, and sometimes as often through large as through small seeds. Soaking seeds for 24 h in solutions of 8-azaguanine or polyacrylic acid did not decrease transmission. Both viruses were detected in nearly mature seeds by inoculation to Phaseolus vulgaris but neither virus was detected in fully ripened seeds by inoculation or serological tests. The percentage of seeds from field plots that produced infected seedlings when sown in a glasshouse was closely related to the percentage of parent plants that showed symptoms of BBSV and/or EAMV at the end of flowering. The relationship seemed similar in different cultivars. On average EAMV was transmitted through more seeds than BBSV, probably because more parent plants were infected with EAMV. Inspection of seed crops for symptoms of BBSV and EAMV at the seedling stage and again at the end of flowering is probably the most practicable way of identifying progeny seed lots with little or no infection.     

Factors affecting aphid acquisition and transmission of soybean mosaic virus

Myzus persicae transmitted soybean mosaic virus (SMV) most efficiently following 30 or 60 s acquisition probes on infected plants. There were no differences in susceptibility to SMV infection of soybean plants 1 to 12 wk old, but symptoms were more severe in plants inoculated when young than when old. Soybeans inoculated between developmental stages R3 and R6 only showed yellowish-brown blotching on one or more leaves. There were no observable differences in the time of appearance or type of symptoms shown by soybean seedlings inoculated either by sap or by aphids; infected plants became acquisition hosts for aphids 5–6 days after inoculation. There was no change in the efficiency with which M. persicae transmitted SMV from source plants up to 18 wk after inoculation. M. persicae transmitted SMV from leaves of field-grown soybeans when plants were inoculated at developmental stages V6, R2, and R3 and tested as sources 57–74 days after inoculation but not from plants inoculated at R5 and tested as sources 14 to 32 days after inoculation. M. persicae acquired SMV from soybean buds, flowers, green bean pods, and unifoliolate, trifoliolate, and senescent leaves. Middle-aged and deformed leaves were better sources of the virus than buds, unfolding and old symptomless leaves. The results are being incorporated into a computer model of SMV epidemiology.     

Transmission characteristics and some other properties of bean yellow vein-banding virus, and its association with pea enation

Bean yellow vein-banding virus (BYVBV) has been found occasionally in mixed infection with pea enation mosaic virus (PEMV) in spring-sown field beans (Vicia faba minor) in southern England. Glasshouse tests confirmed that, like PEMV, BYVBV is transmissible by manual inoculation and by aphids in the persistent manner. However, BYVBV can be transmitted by aphids only from plants that are also infected with a helper virus, usually PEMV. Thus after separation from PEMV by passage through Phaseolus vulgaris it was no longer aphid-transmissible. It became aphid-transmissible again only after re-mixing in plants with PEMV or with a substitute helper, bean leaf roll virus (BLRV). It was not transmitted by aphids that fed sequentially on plants singly infected with PEMV and BYVBV. Thus the interaction between BYVBV and PEMV (or BLRV) that enables BYVBV to be transmitted by aphids seems to occur only in doubly infected plants. However, it was not transmitted by aphids from plants doubly infected with BYVBV and broad bean wilt virus (BBWV). BYVBV and PEMV were transmitted more readily by Acyrthosiphon pisum than by Myzus persicae; neither virus was transmitted by Aphis fabae. Phenol extracts of BYVBV-infected leaves were more infective than phosphate buffer or bentonite-clarified extracts and were sometimes infective when diluted to 1/1000. The infectivity of BYVBV in phosphate buffer extracts of leaves singly infected with BYVBV, unlike that in extracts of leaves doubly infected with BYVBV and PEMV (or BLRV), was destroyed by treatment with organic solvents. BYVBV infected 11 of 28 plant species that were inoculated with phenol extracts; seven of the infected species were legumes. No transmission of BYVBV was detected through seed harvested from infected field bean plants. Isometric particles c. 30 nm in diameter were seen in extracts of plants doubly infected with BYVBV and PEMV but not in extracts of plants infected with BYVBV alone. Leaves of plants infected with BYVBV, alone or with PEMV, contained membrane-bound structures c. 50–90 nm in diameter associated with the tonoplast in cell vacuoles. These structures were not found in healthy leaves. BYVBV has several properties in common with other known aphid-borne viruses that are helper-dependent and transmitted in a persistent manner. Possibly, as suggested for some of them, aphid transmission of BYVBV depends on the coating of its nucleic acid with helper virus coat protein.     

Induction of resistance in Phaseolus vulgaris L. cv. Lima against some soil-borne fungal pathogens by pre-inoculation with tobacco necrosis virus (TNV) reacting hypersensitivity

Abstract

Tobacco necrosis virus (TNV) was tested to induce systemic acquired resistance (SAR) in Phaseolus vulgaris cv. Lima against three important soil-borne fungal pathogens viz: Rhizoctonia solani, Macrophomina phaseolina and Fusarium oxysporum. Application of TNV as a local infection of seven-day old primary leaves of Phaseolus vulgaris cv. Lima resulted in reduction of the mean disease rating of root-rot and damping-off caused by the tested fungal pathogens. The pre-inoculated plants with TNV showed a significant enhancement in their content of photosynthetic pigments (chlorophyll a, b and carotenoids) compared to those inoculated with fungal pathogens only. The percentage of cell membrane stability and ion leakage of viral-treated plants were significantly increased confirming the healthy cytological status of the treated plants. Results demonstrated that inoculation of the primary leaves of beans with TNV before infection with the fungal pathogens leads to changes in protein patterns and showed differences compared with control and caused the appearance of at least one new protein band compared with only fungal-infected plants. Also, an increase in peroxidase activity emerged in the thickness of the isozymic pattern in addition to the synthesis of new bands which was observed as a result of TNV application before infection with the three fungal pathogens. Induction of the synthesis of a new protein and increasing peroxidase activity in the inoculated plants enhanced the defense system against the target pathogen. The results greatly supported the successful application of TNV in the induction of systemic acquired resistance in P. vulgaris cv. Lima against the fungal pathogens.     

Further studies on seed transmission of broad bean stain virus and Echtes Ackerbohnenmosaik -Virus in field beans (Vicia faba)

Broad bean stain virus (BBSV) and Echtes Ackerbohnenmosaik-Virus (EAMV) were detected in the seed coat and embryo sac fluid of immature seeds from infected field beans (Viciafaba minor) by inoculation to Phaseolus vulgaris; BBSV was also detected in immature embryos. The proportion of seeds infected with either virus decreased during maturation. The viruses were transmitted to seedlings as often through fully ripened seeds from which the seed coats had been removed as through intact seeds. Both viruses were detected in pollen from infected plants, but in glasshouse tests only BBSV was transmitted through pollen to seeds. Delaying fertilization in plants infected with BBSV or EAMV seemed not to affect seed transmission of either virus. In glasshouse tests BBSV was transmitted more often through seeds from plants that were inoculated before flowering than during flowering, and was not transmitted through seeds from plants inoculated after flowering; EAMV was transmitted only through seeds from plants inoculated before flowering. In tests on seed from naturally infected plants BBSV was transmitted more often through seeds from plants that developed symptoms before flowering than during flowering. Both viruses were seed-borne in all cultivars tested and there was no marked difference in the frequency of transmission of either virus among the spring-sown cultivars most common in Britain. Both viruses persisted in seed for more than 4 yr.     

Tests for transmission of four potato viruses through potato true seed

The Andean potato calico strain of tobacco ringspot virus (TRSV-Ca) was detected in 2–9% of potato seedlings grown from true seed from plants of cv. Cara and clone G5998(6) infected with TRSV-Ca. Similarly, a potato isolate of the oca strain of arracacha virus B (AVB-O) was detected in 4–12% of progeny seedlings of cv. Cara and clone D42/8 infected with AVB-O. Potato virus T (PVT) passed through 33–59% of seed from PVT-infected cv. Cara, but only 0–2% infection was detected in seedlings from seed of PVT-infected clone D42/8. By contrast, no infection was detected in seedlings grown from seed from plants of G5998(6), D42/8 or cv. Cara infected with Andean potato latent virus strains Hu (APLV-Hu) or Caj (APLV-Caj), although both strains passed through seed of Nicotiana clevelandii. AVB-O, PVT and TRSV-Ca were detected in all tests of pollen from flowers of infected potato plants, but APLV-Hu and APLV-Caj were detected less frequently. AVB-O and PVT were transmitted through 2% and 8% respectively, of seed from healthy potato plants pollinated with pollen from infected plants. However, no transmission through seed was detected when pollen from TRSV-Ca infected plants was used. None of the four viruses were transmitted to healthy potato plants pollinated with pollen from infected plants. APLV-Hu caused exceptionally severe symptoms in the cv. Cara plants used for seed production, but the Bolivian strain of PVT induced only mild symptoms rather than the severe systemic necrosis previously reported for the type of strain of PVT in this cultivar. No symptoms developed in potato seedlings infected with TRSV-Ca, AVB-O or PVT through the seed.     

Characterization of Synergy between Cucumber mosaic virus and Tobacco necrosis virus in Nicotiana benthamiana

Mixed infections of Nicotiana benthamiana plants by Cucumber mosaic virus (CMV) and Tobacco necrosis virus (TNV) exhibit a synergistic interaction and result in symptom enhancement. Accumulation of CMV(+) RNA as well as capsid protein (CP) in mixed infection was considerably higher than that of singly‐infected plants. There was also a slight increase in TNV(+) RNA and CP levels in doubly infected plants. Synergistic infection by CMV‐ and TNV‐induced higher increase in the levels of malonyldialdehyde, hydrogen peroxide (H₂O₂) and more decline in the activities of catalase than singly infected ones. Both peroxidase and superoxide dismutase activities increased rapidly for the first 10 days post inoculation (dpi) in doubly‐infected plants and then declined, whereas the enzyme activities continued to increase after 10 dpi in singly infected plants and had higher enzyme activities in the late stages than that of co‐infected plants. These results suggest that synergistic infection by CMV and TNV produced severes oxidative stress in N. benthamiana plants and the synergy between the two viruses was mutual.     

The Association of Brome Mosaic Virus and Wheat Rusts: I. Transmission of BMV by Uredospores of Wheat Stem and Leaf Rust

Both the stem and leaf rust fungi of wheat (Puccinia graminis f. sp. tritici, P. recondita f. sp. tritici) were investigated as potential vectors of brome mosaic virus (BMV), a pathogen of Gramineae. They were found to transmit BMV to healthy plants through uredospore transfer from hosts infected with both the viral and fungal pathogens. Virus was transmitted even to rust-resistant and non-susceptible hosts. Rust-transmitted virus produced atypical symptoms on plants, could not be extracted by normal low pH methods, and multiplied only to low levels in leaf tissue. Extracted virus was shown to be similar to BMV-type strain (ATC) by serology, density gradient centrifugation and immunospecific electron microscopy. The importance of the association of BMV with the rust fungus in natural infections is discussed.     

Arabis mosaic and Prunus necrotic ringspot viruses in hop (Humulus lupulus L.)

Purified virus preparations made from nettlehead-diseased hop plants, or from Chenopodium quinoa, to which the virus was transmitted by inoculation of sap, contained polyhedral virus particles of 30 mμ diameter which were identified serologically as arabis mosaic virus (AMV). There were serological differences between AMV isolates from hop and from strawberry, and also differences in host range and in symptoms caused in C. quinoa and C. amaranticolor. AMV was always associated with nettlehead disease. The nematode Xiphinema diversicaudatum occurred in small numbers in most hop gardens, but was numerous where nettlehead disease was spreading rapidly. Preparations from nettlehead-affected hops also contained a second virus, serologically related to Prunus necrotic ringspot virus (NRSV), in mild and virulent forms which infected the same range of test plants but showed some serological differences. Mild isolates did not protect C. quinoa plants against infection by virulent isolates. Hop seedlings inoculated with virulent isolates of NRSV developed symptoms indistinguishable from those of split leaf blotch disease. Latent infection with NRSV was prevalent in symptomless hop plants. Nettlehead disease is apparently associated with dual infection of AMV and virulent isolates of NRSV. An unnamed virus with rod-shaped particles 650 mμ long was common in hop and was transmitted by inoculation of sap to herbaceous plants. Cucumber mosaic virus was obtained from a single plant of Humulus scandens Merr.