Properties of a resistance-breaking strain of potato virus X

During indexing of a potato germplasm collection from Bolivia, a strain of potato virus X (PVX), X_HB, which failed to cause local lesions in inoculated leaves of Gomphrena globosa was found in 7% of the clones. X_HB was transmitted by inoculation of sap to 56 species from 11 families out of 64 species from 12 families tested. It was best propagated in Nicotiana glutinosa or N. debneyi; Montia perfolia and Petunia hybrida were useful as local lesion hosts. Inoculated leaves of G. globosa plants kept at 10°, 14°, 18°, 22°, or 26 °C after inoculation were always infected symptomlessly. X_HB caused a mild mosaic, systemic chlorotic blotching or symptomless infection in 16 wild potato species and eight Andean potato cultivars, systemic necrotic symptoms in clone A6 and cultivar Mi Peru, and bright yellow leaf markings in cultivar Renacimiento. It caused necrotic local lesions in inoculated leaves of British potato cultivars with the PVX hypersensitivity gene Nb but then invaded the plants systemically without causing further necrosis; with gene Nx systemic invasion occurred but no necrotic symptoms developed. These reactions resemble those of PVX strain group four. X_HB differed from other known strains of PVX in readily infecting PVX-immune clones 44/1016/10, G. 4298.69 and USDA 41956, cultivars Saphir and Saco, and Solanum acaule PI 230554. X_HB had slightly flexuous filamentous particles with a normal length of 516 nm. It was transmitted readily by plant contact and it partially protected G. globosa leaves from infection with X_CP, a group two strain of PVX. Sap from infected N. glutinosa was infective after dilution to 10^--⁶ but not 10^--⁷ after 10 min at 75° but not 80 °C and after 1 yr at 20 °C. X_HB was readily purified from infected N. debneyi leaves by precipitation with polyethylene glycol followed by differential centrifugation. Microprecipitin tests showed that X_HB and X_CP are closely related serologically.     

Host range and some properties of potato mop-top virus

Potato mop-top virus (PMTV) was transmitted by inoculation of sap to twenty-six species in the Solanaceae or Chenopodiaceae and to Tetragonia expansa; species in eleven other plant families were not infected. The virus was cultured in inoculated leaves of Nicotiana tabacum cv. Xanthi-nc or in N. debneyi. Diagnostic local lesions were produced in Chenopodium amaranticolor. In winter, ten solanaceous species were slowly invaded systemically but the first leaves infected were those immediately above inoculated leaves. When transmitted to Arran Pilot potato by the vector Spongospora subterranea, PMTV induced all the main types of shoot and tuber symptoms found in naturally infected plants. Isolates of PMTV from different sources differed considerably in virulence. PMTV-containing tobacco sap lost infectivity when heated for 10 min at 80 °C, diluted to 10^-4, or stored at 20 °C for 14 weeks. Infectivity was partially stabilized by 0·02% sodium azide. When sap was centrifuged for 10 min at 8000 g, infectivity was mainly in the sediment. Infective sap contained straight rod-shaped particles about 20 nm wide, with lengths up to 900 nm and crossbands at intervals of 2·5 nm. Many of the particles were aggregated side-to-side, and the ends of most seemed damaged. The slight infectivity of phenol-treated leaf extracts was abolished by pancreatic ribonuclease. The present cryptogram of PMTV is R/*:*/*:E/E:S/Fu.     

Pepino mosaic virus, a new potexvirus from pepino (Solanum muricatum)

Pepino mosaic virus (PepMV), a previously undescribed virus, was found in fields of pepino (Solanum muricatum) in the Canete valley in coastal Peru. PepMV was transmitted by inoculation of sap to 32 species from three families out of 47 species from nine families tested. It caused a yellow mosaic in young leaves of pepino and either a mild mosaic or symptomless infection in 12 wild potato species, five potato cultivars and potato clone USDA 41956 but S. stoloniferum and potato cultivars Merpata and Revolucion reacted with severe systemic necrotic symptoms. The virus was transmitted by plant contact but not by Myzus persicae. It was best propagated and assayed in Nicotiana glutinosa. Sap from infected N. glutinosa was infective after dilution to 10^-1 but not 10^-6, after 10 min at 65°C but not 70°C and after 3 months at 20°C. PepMV had filamentous particles with a normal length of 508 nm; the ends of some seemed damaged. Ultra-thin sections of infected leaves of N. glutinosa revealed many inclusions containing arrays of virus-like particles some of which were banded or whorled; small aggregates of virus-like particles were also common. The virus was purified by extracting sap from infected leaves in a solution containing 0·065 M disodium tetraborate, 0·435 M boric acid, 0·2% ascorbic acid and 0·2% sodium sulphite at pH 7·8, adding silver nitrate solution to the extract, and precipitating the virus with polyethylene glycol followed by two cycles of differential centrifugation. Particles of PepMV normally yielded two proteins with molecular weights of 26 600 and 23 200, but virus obtained from infective sap aged overnight yielded only the smaller protein suggesting that it was a product of degradation of the larger one. The virus is serologically related to two potexviruses, narcissus mosaic and cactus X and its properties are typical of the potexvirus group.     

Peanut green mosaic virus — a member of the potato virus Y group infecting groundnut (Arachis hypogaea) in India

A virus, now named peanut green mosaic virus (PGMV), was isolated from groundnut (Arachis hypogaea) in India and identified as a member of the potato virus Y group by electron microscopy, aphid transmission, and its chemical properties. It was sap transmissible to 16 species of the Leguminosae, Solanaceae, Chenopodiaceae, Aizoaceae and Pedaliaceae; Phaseolus vulgaris was a good local lesion host. PGMV remained infective in buffered groundnut leaf sap at dilutions of 10^-3 after 3 to 4 days at 25 °C, or heating for 10 min to 55 °C but not 60 °C. PGMV was transmitted in the non-persistent manner by Aphis gossypii and Myzus persicae but was not seed-borne. Purified virus preparations contained flexuous filamentous particles c. 750 nm long which sedimented as a single component with a sedimentation coefficient (S°_20w) of 171S, and contained a single polypeptide (mol. wt 34 500 daltons) and one nucleic acid species (mol. wt 3.25 × 10⁶ daltons). PGMV is serologically unrelated to peanut mottle virus (PMV) and other viruses infecting leguminous crops. Infected leaves contained cylindrical, cytoplasmic inclusions.     

Eggplant mosaic virus, and its relationship to Andean potato latent virus

Eggplant mosaic virus (EMV), obtained from Solanum melongena L. from Trinidad, is readily transmitted by inoculation of sap to several solanaceous and a few non-solanaceous plant species. Purified preparations of EMV contain isometric particles 30 nm in diameter, and with sedimentation co efficients of either 111 or 53 S. The particles have thirty-two major morphological subunits. EMV is closely serologically related to Andean potato latent virus and has a similar host range, but is more virulent. Also, whereas EMV accumulates fastest in Nicotiana clevelandii leaves at 20–24 °C, Andean potato latent virus accumulates fastest at 15 °C, and fails to attain a serologically detectable concentration at 24 °C. A few symptomatologically or serologically distinguishable strains of EMV were obtained. EMV has properties typical of viruses of the Andean potato latent subgroup of the turnip yellow mosaic group of viruses, and its present cryptogram is */*:*/*:S/S:S/Cl.     

Purification and properties of closterovirus-like particles associated with a whitefly-transmitted disease of sweet potato

A virus causing sunken veins on ‘Georgia Jet’ sweet potato, and yellow brittle leaves and stunting on Ipomoea setosa, was purified and a specific antiserum was prepared. Flexuous particles with a normal length of 850 nm and a diameter of 12 nm with an open helical structure typical of closteroviruses were observed. The virus particle protein has an apparent mol. wt of c. 34 kD. Double-stranded RNA isolated from SPSVV-infected I. setosa and subjected to electrophoresis in agarose consisted of one major band with an estimated M_r of 10.5 kbp and two minor bands with M_r of 9.0 and 5.0 kbp. Fibril-containing vesicles in phloem cells were observed in ultrathin sections of infected leaf tissues. The virus was transmitted by the whitefly Bemisia tabaci in a semi-persistent manner and by grafting, but not mechanically. The virus could be transmitted to various Ipomoea species, to Nicotiana clevelandii, N. benthamiana and Amaranthus palmeri. The virus did not react with an antiserum to lettuce infectious yellows virus. Based on particle morphology, serology and symptom expression, the virus appears unique and different from all other reported whitefly-transmitted closteroviruses. We propose it be named “sweet potato sunken vein virus” (SPSVV).     

Targeting of SPCSV-RNase3 via CRISPR-Cas13 confers resistance against sweet potato virus disease

Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co-infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis-related factors (i.e., SPCSV-RNase3) by using the CRISPR-Cas13 technique. First, the RNA targeting activity of four CRISPR-Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV-RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3-targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV-GFP and cucumber mosaic virus CMV-RNase3 co-infection. Moreover, transgenic sweet potato plants carrying an RNase3-targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD-prevalent regions.     

Resistance to sweet potato virus disease (SPVD) in wild East African Ipomoea

Sweet potato virus disease (SPVD), the most harmful disease of sweet potatoes in East Africa, is caused by mixed infection with sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Wild Ipomoea spp. native to East Africa (J cairica, I. hildebrandtii, I. involucra and J wightii) were graft-inoculated with SPVD-affected sweet potato scions. Inoculated plants were monitored for symptom development and tested for SPFMV and SPCSV by grafting to the indicator plant J setosa, and by enzyme-linked immunosorbent assay (ELISA). Virus-free scions of sweet potato cv. Jersey were grafted onto these wild Ipomoea spp. in the field, and scions collected 3 wk later were rooted in the greenhouse and tested for viruses using serological tests and bioassays. In all virus tests, J cairica and J involucra were not infected with either SPFMV or SPCSV. J wightii was infected with SPFMV, but not SPCSV, in the field and following experimental inoculation; J hildebrandtii was infected with SPCSV, but not SPFMV, following experimental inoculation. These data provide the first evidence of East African wild Ipomoea germplasm resistant to the viruses causing SPVD.     

Purification and some properties of Tephrosia symptomless virus

Tephrosia symptomless virus (TSV), isolated from Tephrosia villosa, is widely distributed in coastal districts of Kenya. The virus was readily transmitted by inoculation of sap, but not by Aphis craccivora or Apion sp. (Curculionidae) or through soil. Host range was very restricted and it infected only 10 of 70 species tested in one of nine plant families; susceptible species were confined to five genera within the Papilionaceae. The virus was cultured, propagated and assayed in soybean. TSV remained infective after 10 min at 85°C, 3 wk at 20°C and 26 wk at -12°C; crude infective sap of Glycine max retained infectivity when diluted 10^-6 but not 10^-7. Virus was purified from systemically infected soybean by clarifying sap extracted in 0.06 m phosphate buffer containing 0.001 m EDTA and 0.1% thioglycollic acid (pH 7.5) with equal volumes of 1:1 n-butanol/chloroform followed by two cycles of differential and one of sucrose density gradient centrifugation. Purified preparations contained c. 33 nm isometric particles. TSV contained RNA and one protein of molecular weight 1.53. 10⁶ and c. 42 000, respectively. Analytical centrifugation indicated a single component with a sedimentation coefficient (s.20, w) of 127 S; in Cs₂SO₄ and CsCl isopycnic gradients a single virus band formed; buoyant density in CsCl was 1.361. TSV was not related serologically to any of 44 viruses in nine plant virus groups but it resembled the tombusviruses and other ungrouped viruses such as carnation mottle in some of its properties.     

Pepper veinal mottle virus-a new member of the potato virus Y group from peppers (Capsicum annuum L. and C. frutescens L.) in Ghana

Pepper veinal mottle virus (PVMV), a previously undescribed virus widespread in Capsicum annuum and C. frutescens in the Eastern Region of Ghana, is acquired and inoculated in 2 min feeding periods by aphids (Myzus persicae and Aphis gossypii); it is transmissible by inoculation of sap to eleven of fifteen Solanaceae and to five of forty-six other species within three of seventeen other families. The virus was propagated in Nicotiana clevelandii and Petunia hybrida, and assayed in Chenopodium quinoa, C. amaranticolor and C. murale. Sap from Capsicum annuum was infective after dilution to 10^-3 but not 10^-4, after 10 min at 55 but not 60^oC, and after 7 but not 8 days at 25^oC. Lyophilized sap from P. hybrida was infective after 6 years in vacuo. Yields of 10–25 mg of virus per kg of leaf tissue were consistently obtained from P. hybrida or N. clevelandii by extracting systemically infected leaves in 0.5 M borate (pH 7.8) containing 0.2% mercaptoethanol and chloroform, followed by repeated precipitation with 50 g polyethylene glycol (M.W. 6000) per l, several cycles of differential centrifugation and centrifugation in sucrose density-gradient columns. Virus preparations had ultraviolet absorption spectra typical of a nucleoprotein containing c. 6% nuclei acid (A 260/280 = 1.25; A 260/246 = 1.27) and contained numerous unaggregated and unbroken filamentous particles c. 770 times 12 nm which sedimented as a single component with a sedimentation coefficient (s^o_20,w) of 155 S. PVMV contained RNA (moles %: G = 24, A = 23, C = 27, U = 26), and a single protein species with a molecular weight of 32000–33000 daltons. PVMV was not serologically related to potato virus Y (three strains), or to twelve other morphologically similar viruses, and seems to be a distinct member of the potato virus Y group. The cryptogram of PVMV is R/(I):*/(6):E/E:S/Ap.     

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

Cowpea 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/*.     

The behaviour of potato mop-top virus in soil, and evidence for its transmission by Spongospora subterranea (Wallr.) Lagerh.

Potato mop-top virus (PMTV) was best detected in field soils by air-drying them for more than a week before remoistening and growing seedlings of Nicotiana tabacum or N. debneyi for a 6–10 week period. Infection of N. tabacum was assessed by inoculating sap from roots and shoots to Chenopodium amaranticolor. Similar inoculations from N. debneyi were far less convenient for detecting PMTV than recording leaf symptoms, but slightly more efficient. Air-dry soil retained PMTV infectivity for 9 months, when passed through a 50 μ sieve or when diluted with 10³ but not 10⁴ parts of steamed soil. Tobacco seedlings were not infected when their roots were steeped in PMTV-containing tobacco sap. Infective soils contained Spongospora subterranea, spore balls of which resisted air-drying for more than a year and passed a 50 μ sieve. Roots of susceptible seedlings were infected with PMTV when exposed to spore balls of S. subterranea taken from powdery scabs on PMTV-infected potato tubers, or to suspensions obtained by steeping, in nutrient solution, roots infected with virus-carrying cultures of S. subterranea. Plants in several families were hosts of S. subterranea, but probabilities of infection when exposed to spore balls differed greatly between families and only species of Solanaceae were good hosts. The ten species infected with PMTV when grown in infective soil or when exposed to spore balls of S. subterranea taken from PMTV-infected potato tubers are all members of this family. PMTV seems to be carried internally in S. subterranea spore balls and survived in them for at least a year. PMTV was transmitted by S. subterranea to Arran Pilot potato, causing yellow blotches in some leaves and spraing in many tubers. However, when newly infected with PMTV in the field, not all Arran Pilot tubers developed spraing. Also, although many spraing-affected or symptomless but PMTV-infected tubers carried PMTV-containing spore balls of S. subterranea, powdery scabs were rarely found near the centres of the rings of primary spraing. PMTV became established in virus-free soil when PMTV-infected tubers carrying S. subterranea were planted as ‘seed’ but not when virus-free tubers bearing powdery scabs were used. 5. subterranea seems the main, and possibly the only, vector of PMTV in the soils examined. S. subterranea did not transmit potato aucuba mosaic virus from potato to N. debneyi or Capsicum annuum.     

Host plant reactions, physical properties and serology of three isolates of Andean potato latent virus from Peru

Three isolates of Andean potato latent virus (APLV) (Caj, Hu, Ay) each infected twenty-seven species of plants in the families Amaranthaceae, Chenopodiaceae, Cucurbitaceae and Solanaceae but differed somewhat in the symptoms they induced. Nicotiana bigelovii and N. clevelandii proved the most useful diagnostic hosts. Symptoms were sometimes produced by all three isolates in cultivated and wild potatoes. In sap from systemically infected N. bigelovii and N. clevelandii leaves, all three isolates remained infective when diluted to 10^-6 and when stored at room temperature for at least 3 wk. The thermal inactivation points were 65–70 °C for Hu and Ay, but 75–80 °C for Caj. All three isolates differed serologically from Col, the original isolate of APLV, forming spurs in gel diffusion tests. No serological difference was found between Hu and Ay, but both formed spurs in reciprocal reactions with Caj. The data from light absorption, particle morphology and protein molecular weight for Caj, Hu and Ay are similar to those reported for other tymoviruses. APLV was found widespread in Andean countries.     

Narcissus mosaic virus

Narcissus mosaic virus (NMV) is widespread in British crops of trumpet, large-cupped and double daffodils, but was not found in Narcissus jonquilla or N. tazzeta. Many commercial daffodil cultivars seem totally infected, and roguing or selection is therefore impracticable. Strict precautions by breeders and raisers to prevent infection of new cultivars is recommended. Healthy daffodil seedlings were readily infected with NMV by mechanical inoculation, but the virus was not detected in them until 17 months after inoculation, when a mild mosaic appeared. NMV infected twenty-eight of fifty-three inoculated plant species; only five (Nicotiana clevelandii, Gomphrena globosa, Medicago sativa, Trifolium campestre and T. incarnatum) were infected systemically, and NMV was cultured in these and assayed in Chenopodium amaranticolor and Tetragonia expansa. The virus was not transmitted to and from G. globosa or N. clevelandii by three aphid species, or through the seeds of Narcissus, G. globosa and N. clevelandii but was transmitted by handling. G. globosa sap was infective at a dilution of 10 ^-5 but not at 10^-6, when heated for 10 min. at 70° C. but not at 75° C, and after 12 weeks at 18° C, or 36 weeks at 0–4° C. NMV withstood freezing in infected leaves and sap, and purified preparations and freeze-dried sap remained infective for over 2 years. NMV was precipitated without inactivation by ammonium sulphate (313 g./l.) but was better purified by differential centrifugation of phosphate-buffer extracts treated with n-butanol. Such virus preparations from G. globosa, N. clevelandii, C. amaranticolor and T. expansa were highly infective, serologically active, produced a specific light-scattering zone when centrifuged in density-gradients and contained numerous unaggregated particles with a commonest length of 548–568 mμ. Antisera prepared in rabbits had precipitin tube titres of 1/4096. NMV was detected in three experimental hosts but not in narcissus sap. Unlike some viruses with elongated particles, NMV precipitates with antiserum in agar-gel. Purified preparations reacted with antiserum to a Dutch isolate of NMV but not with antisera to seven other viruses having similar particles and in vitro properties, or to narcissus yellow stripe virus.     

Purification and properties of Australian lucerne latent virus, a seed-borne virus having affinities with nepoviruses

An isolate of Australian lucerne latent virus (ALLV) from lucerne in New Zealand was mechanically transmitted to a few herbaceous hosts. It induced diagnostic symptoms in several species of the Chenopodiaceae, but was symptomless in most other hosts including lucerne and Trifolium subterraneum. It was seed transmitted in lucerne. When assayed to Chenopodium quinoa, infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 55°C and storage for 4 days at 4°C. ALLV was purified from infected C. quinoa or pea plants by extracting sap in 0.1 m borate buffer (pH 7) containing 0.2% 2-mercaptoethanol and clarifying with 15% bentonite suspension, high and low speed centrifugation and sucrose density gradient centrifugation. Purified virus preparations contained isometric particles about 25 nm in diameter and sedimented as three virus components with sedimentation coefficients (s_20-w⁰) of 56 S, 128 S and 133 S. The 56 S component appeared to consist of nucleic acid-free protein shells. Polyacrylamide gel electrophoresis of virus preparations showed that ALLV contained a single protein species of mol. wt 55 000 and two RNA species of mol. wt 2.1 × 10⁶ and 2.4 × 10⁶. An antiserum to ALLV had an homologous titre of 1/256 to purified virus but failed to detect ALLV in infective sap of C. quinoa, pea or lucerne. Purified ALLV failed to react to antisera to 28 distinct isometric plant viruses including those to 10 nepoviruses.     

Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification

 Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival of vitrified sweet potato shoot tips cooled to approximately –208  °C was increased by preculturing with 0.3 M sucrose for 24 h at 22  °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod. The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22  °C followed by dehydration with PVS2 for 16 min at 22  °C. Rapid cooling was used and achieved by the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise for cryopreserving sweet potato shoot tips. Received: 2 March 1999 / Revision received: 21 September 1999 / Accepted: 29 September 1999     

Alcohol fermentation of sweet potato. I. Acid hydrolysis and factors involved

Factors affecting acid hydrolysis of sweet potato powder (SSP) to fermentable sugars were examined. These include HCl concentration, temperature, time, and levels of SPP. Maximum reducing sugar, reported as dextrose equivalent (DE), was detected after 24 min hydrolysis (1% SPP) in 0.034N HCl heated at 154°C. These samples also had 3.43% droxymethylfurfural (HMF) based on dry weight. A high level of HMF (9.2%) was detected in 1% SPP heated at 154° in 0.10N HCl for 18 min. The lowest concentration of HMF formed (1.8%), at maximal DE of 61%, was established in samples containing 5% SPP and heated at 154° in 0.034N HCl for 48 min. Aqueous extracts of uncured SPP, examined by HPLC, contained glucose, fructose and sucrose, but degraded SPP had only glucose and fructose. Products of degraded SPP, under appropriate conditions, could be used for alcohol fermentation.     

Pathology, soil transmission and characterization of cymbidium ringspot, a virus from cymbidium orchids and white clover (Trifolium repens)

Two strains of a virus, designated cymbidium ringspot virus (CyRSV), were isolated from cymbidium orchids and from Trifolium repens respectively in Britain. Experimentally infected cymbidiums developed slight chlorotic ring-mottle; T. repens developed flecks and mottling in the leaves, and slight stunting. Of 101 plant species tested, the cymbidium strain infected sixty-one (thirteen systemically) in twenty-three of thirty-five families; the clover strain infected sixty-four species (eighteen systemically) in twenty-two families. Both strains were propagated in Nicotiana clevelandii and assayed in Chenopodium quinoa. CyRSV was readily transmitted by inoculation of sap, and by foliage contact between plants, but not by the aphids Myzus persicae or Acyrtho-siphon pisum, nor through seed of T. incarnatum, Phaseolus vulgaris or N. clevelandii. Highly infective virus was released into soil from roots of infected N. clevelandii, and acquired by bait seedlings planted in such soil. Similar transmission occurred when purified virus was applied to the surface of sterilized soil containing bait plants; there was no evidence for any living soil vector. The virus was eliminated from 96 % of small cuttings taken from infected N. clevelandii plants grown at 35–37 °C for 9 wk. CyRSV was still infective in sap of N. clevelandii after dilution to 10?⁵-io–⁶ (only 2 × 10^_1 in cymbidium sap), or after 10min at 85–90 °C. It survived at least 10 months at c. 20 °C and more than 12 yr at 2 °C. Lyophilized sap was highly infective after over 13 yr at laboratory temperatures under high vacuum. Purified preparations made by clarification with n-butanol, followed by differential centrifugation and exclusion chromatography on controlled-pore glass beads, contained isometric particles c. 30 nm diam., with s°_20W= 137 S, and had a buoyant density in caesium chloride of 1–36 g/ml. The A 260/A 280 ratio was 1–55, and A max(26o)/A min(242) was 1–17. The virus contained c. 15 % of single-stranded RNA of mol. wt 1–7 × 10⁶; the nucleotide base ratios were: G27'8; A24/9; C2I-3; U26-I. There was one capsid polypeptide of mol. wt 43600. The virus was a good immunogen and a strongly reacting antigen in vitro; in Immunoelectrophoresis, each strain migrated as a single antigenic component towards the cathode. The cymbidium and clover strains were serologically closely related, although spurs were produced in immunodiffusion. No serological relationship was found to forty-three other isometric viruses, including eighteen tombusvirus isolates; CyRSV nevertheless shares many properties with tombusviruses, and we assign it provisionally to this group. The cryptogram is: R/r:1:7/15:S/S:S/O.     

Host range, purification and properties of potato virus T

Potato virus T (PVT) infected nine species of tuber-bearing Solanum, most of them symptomlessly, and as a rule was transmitted through the tubers to progeny plants: two genotypes of S. tuberosum ssp. andigena were not infected. The virus was also transmitted by inoculation with sap to 37 other species in eight plant families. Chenopodium amaranticolor is useful as an indicator host, C quinoa as a source of virus for purification, and Phaseolus vulgaris as a local-lesion assay host; the systemic symptoms in Datura stramonium, Nicotiana debneyi and in these three species are useful for diagnosis. Attempts to transmit PVT by aphids failed, but the virus was transmitted through seed to progeny seedlings of four solanaceous species, and from pollen to seed of S. demissum. PVT was purified by clarifying sap with n-butanol or bentonite, followed by precipitation with polyethylene glycol, differential centrifugation and sedimentation in a sucrose density gradient. Purified preparations had an E260/E280 ratio of 1.18 and contained a single infective component with a sedimentation coefficient of 99 S. This component consisted of flexuous filamentous particles of about 640 times 12 nm that showed a characteristic substructure when stained with uranyl acetate. The virus particles contained a single species of infective single-stranded RNA, of molecular weight 2–2 times 106 daltons, and a single species of polypeptide of molecular weight about 27 000 daltons. PVT is serologically related to apple stem grooving virus but not to four other common potato viruses with flexuous filamentous particles. Apple stem grooving virus and PVT cause similar symptoms in several hosts, but also differ somewhat in host range and symptomatology. Apple stem grooving virus did not infect potato, caused additional symptoms in C. quinoa also infected with PVT, and its particles did not show the structural features specific to PVT. The two viruses are considered to be distinct. The cryptogram of PVT is R/1:2–2/(5): E/E: S/C.