Juice Inoculation Research Articles

Selection of Indigenous Saccharomyces cerevisiae Strains and Exploitation of a Pilot-Plant to Produce Fresh Yeast Starter Cultures in a Winery

The inoculation of grape juice with Saccharomyces cerevisiae strains selected from indigenous yeast populations can be a suitable tool to control alcoholic fermentation, contributing to producing wines with typical flavor and aroma and, hence, the demand for native starter cultures is increasing. However, since low amounts of indigenous yeast biomasses are usually required for local winemaking, the industrial production of these yeasts can be expensive. Therefore, in this study, after selecting an indigenous S. cerevisiae strain based on relevant oenological and technological features, a pilot-plant for easy and rapid production of fresh yeast biomass directly in a winery located in Tuscany, was exploited. The selected yeast strain was used as a starter to carry out 25 and 100 hL fermentations and its enological performance was compared with that of the commercial starter normally used in the winery. Chemical and sensory analysis of the resulting wines showed that they differentiated according to the used yeast strain, with the wines produced by the indigenous S. cerevisiae strain being characterized by a distinctive aromatic and sensory profile. In conclusion, the pilot-plant effectively resulted in producing fresh yeast starter cultures in the winery to be successfully used to carry out alcoholic fermentations.

EFFECTS ON THE MINERAL COMPOSITION OF FLEISCHMANN’S YEAST (Saccharomyces cerevisiae) FERMENTING SUGAR-CANE JUICE CONTAMINATED WITH COBALT

The current work aimed to study the accumulation and the effects of cobalt on themineral composition of Fleischmann’s yeast Saccharomyces cerevisiae fermentingsugarcane juice with controlled contamination, in sub-toxic levels of cobalt. Thesterilized juice (120°C, 20 minutes), with 12% of total reducing sugars (TRS) and fourlevels of pH (3.5; 4.5; 5.5 and 6.5) was added with cobalt chloride salt (CoCl2.6H2O)resulting in six levels of contamination (0.0; 0.1; 0.5; 1.0; 2.0 e 5.0 mg Co L-1) and 24treatments. The juice inoculation was carried out with bread yeast (10% p/p). Afterthe end of the fermentation (6 hours), the fermented media were centrifuged, and thecellular viability, budding rate and levels of cobalt, phosphorus, calcium, magnesium,sulfur, copper, iron, manganese, potassium and zinc were determined (in dry weight– dw). The absorption of cobalt was influenced by the pH. This absorption was higherin pH 3.5, whereas in pH 6.5 we found almost no absorption. The levels ofpotassium, calcium, zinc and phosphorus decreased with the increase of pH whilethe levels of sulfur content increased. No significant differences could be observed inthe fermentative velocity for the several treatments.

Fermentation of alfalfa wet-fractionation liquids to volatile fatty acids by Streptococcus bovis and Megasphaera elsdenii

"Green juice", obtained by squeezing fresh alfalfa leaves inoculated with lactic acid bacteria, was fermented at room temperature for 7-21 d to obtain 12-47 g lactic acid L(-1). Inoculation of green juice with Streptococcus bovis and incubation at 39°C reduced fermentation time to 8-12h. The resulting "brown juice" from either fermentation had a pH of ∼4.5 and a protein precipitate. Upon adjustment to pH 5.2-6.8 and inoculation with Megasphaera elsdenii, brown juice was fermented within 48 h to up to 18 g of mixed volatile fatty acids (VFA) L(-1). Single-stage fermentation of green juice by both species in coculture typically resulted in overgrowth of S. bovis and acid inhibition of M. elsdenii, inhibiting VFA production. Because the juice fermentations are conducted without sterilization or supplemental nutrients, they can potentially contribute to an integrated process featuring protein recovery and fermentation of fractionated solids to VFA and other products.

Combined effects of both bacteria and gastric juice on pneumonia in mice

The effects of a combined inoculation of gastric juice and Streptococcus pneumoniae on the lungs of mice was investigated. Survival rates of mice inoculated with bacteria alone, gastric juice alone, and both bacteria and gastric juice were compared over 18 days. Xanthine oxidase (XO) activities in the lung tissues of mice inoculated with bacteria and gastric juice were measured and injected with a free radical scavenger, pyran-superoxide dismutase (pyran-SOD). A high mortality rate was observed in mice inoculated with both gastric juice and Streptococcus pneumoniae (81%). Mice inoculated with either Streptococcus pneumoniae or gastric juice showed a separate mortality rate of up to 10% during 18 days after inoculation. XO activity in the lung tissue of the mice inoculated with both gastric juice and bacteria was higher than in mice inoculated with either of them separately. The high mortality rate in the group inoculated with both two agents was reduced to 25% by the administration of pyran-SOD. XO activity raised by Streptococcus pneumoniae and gastric juice was significantly reduced by pyran-SOD. Thus, we suggest an important role in the combined effects of gastric juice and bacteria on pneumonia.

Growth of yeasts during wine fermentations

Abstract This article emphasises the importance of making quantitative measurements of the growth of yeast species during wine fermentations. Although such studies confirm Saccharomyces cerevisiae as the principal wine yeast, they show that indigenous species of Kloeckera and Candida make a more significant contribution to the fermentation than previously thought. Inoculation of grape juice with S. cerevisiae does not necessarily suppress growth of these indigenous species, nor does it ensure that the inoculated strain will become dominant over indigenous strains of S. cerevisiae. Factors that affect growth of yeasts during fermentation are examined along with the related aspects of stuck fermentations, killer‐yeast activity and yeast autolysis. The contribution of non‐Saccharomyces yeasts to wine quality warrants more serious consideration.

Detection, spread, and aphid transmission of potato virus S

Seventy-two potato tubers of 106 tested from plants exposed 1 year in a field were found infected with potato virus S (PVS) in different tests. Ninety-three percent of these were detected by tuber juice inoculation to Nicotiana debneyi Domin. and 90% by serology of 30-cm plants grown from an eye of such tubers. Sap inoculation to N. debneyi of the same young plants proved to be 96% efficient in detecting the virus, and serological tests at bloom stage were the most efficient of all the tests compared.Tests done on all tubers from 18 plants currently infected with PVS showed that 103 of 116 (89%) were infected, and virtually all eyes from 68 infected tubers produced infected plants.Three years of field trials at Fredericton on the spread of PVS showed that the virus moved into virus-free varieties independently of potato virus X (PVX). In 1970, leaf tests showed that virus-free Netted Gems became 12% infected with PVS; in 1971, spread into Green Mountain, Kennebec, and Sebago was 57, 19, and 9%, respectively; and in 1972, 14% spread occurred in Green Mountain and none in Kennebec or Sebago.Greenhouse experiments on transmission of PVS to potato by Myzus persicae (Sulz.) resulted in 3 of 87 (3.4%) plants becoming infected. Other tests with potato virus Y (PVY) to tobacco, Nicotiana tabacum L. var. Samsun, resulted in 83% transmission.

Viruses isolated from Narcissus (Narcissus spp.) in Japan V

A virus was isolated from a large cupped narcissus plant which showed yellow stripe symptoms collected in Niigata Prefecture in 1971. The virus was readily transmitted by juice inoculation. Among the test plants of 46 species in 14 families, 37 species in 13 families, namely, Chenopodiaceae, Amaranthaceae, Aizoaceae, Caryophylaceae, Papaveraceae, Cruciferae, Leguminosae, Violaceae, Solanaceae, Pedaliaceae, Cucurbitaceae, Compositae, Amaryllidaceae, were found susceptible to the virus.The virus was not transmitted by Myzus persicae, but was transmitted by nematode (Xiphinema bakeri). An evidence of soil transmission was provided by greenhouse tests using soil which was infested with nematodes including Xiphinema bakeri. Lots of 25 X. bakeri sieved from soil in which infected petunia had been grown for 2 to 3 weeks were placed near the roots of potted healthy petunia. After 6 weeks the virus was detected by juice inoculation and by serological test in all inoculated petunia plants. No infection occurred in control petunia plants grown in soil free from X. bakeri. The virus was proved to be transmitted also through seeds of soybean.The virus in vitro withstood heating at 60C for 10 minutes, but not 65C, and 21 days of storage at 22C, but not 28 days. The virus particles were found to be spherical about 25nm in diameter.Antiserum prepared injecting rabbit with a semi-purified virus preparation showed homologous precipitin tube titre of 1/1024. By agar gel diffusion tests, the virus showed negative reaction to antisera against tomato black ring virus (kindly sent from Dr. B.D. Harrison) and against tobacco ringspot virus and tomato ringspot virus (kindly sent from Dr. R. Stace-Smith), but showed positive reaction to the antiserum against arabis mosaic virus (kindly sent from Dr. B.D. Harrison).From the above results, the virus was identified as arabis mosaic virus, which has hitherto not been reported in Japan.Virus-free narcissus seedling juice-inoculated with the virus remained apparently healthy for 15 months, although the virus was recovered from these plants.

Viruses Isolated from Narcissus (Narcissus spp.) in Japan

A virus was isolated from trumpet narcissus showing symptom of mosaic and brown spots, collected in Shizuoka Prefecture in 1967. The virus was readily transmitted by juice inoculation, but not by Myzus persicae. Among the tested plants of 49 species in 14 families, 37 species in 14 families, namely, Chenopodiaceae, Amaranthaceae, Aizoaceae, Caryophyllaceae, Cruciferae, Leguminosae, Malvaceae, Umbelliferae, Solanaceae, Scrophulariaceae, Cucurbitaceae, Compositae, Liliaceae and Amaryllidaceae, were found susceptible to the virus by juice inoculation. Of these plants, Chenopodium amaranticolor, NewZealand spinach and bean were considered to be useful as differential host plants for the virus. The virus was proved to be transmitted through seed of chickweed and soybean. But the virus was not transmitted by Longidorus martini (nematode).The virus in vitro withstood heating at 60C for 10 minutes, but not 65C, and 14 days of storage at 20C, but not 21 days. The virus particles were found to be spherical about 25nm in diameter.Antiserm prepared by injection to rabbit showed homologous precipitin tube titre of 1/512. By agar gel diffusion tests, the virus showed negative reaction to antisera against arabis mosaic virus supplied by Dr. B.D. Harrison and against tobacco ringspot virus and tomato ringspot virus supplied by Dr. R. Stace-Smith. The antiserum against the tomato black ring virus supplied by Dr. B.D. Harrison only gave positive result. The virus and the antiserum were furthermore sent to Dr. B.D. Harrison, and the above results were confirmed, along with an additional negative results with antisera against cherry leaf roll virus, raspberry ringspot virus and strawberry latent ringspot virus. Furthemore, the virus is more closely related to beet ringspot strain than to potato bouquet strain of tomato black ring virus.From these results, the virus was identified as beet ringspot strain of tomato black ring virus, which has not been reported before in Japan.

Transmission of Mulberry Ringspot Virus by Longidorus martini Merny

When mulberry seedlings were planted in soils brought from mulberry fields where mosaic disease was severe, the seedlings showed ringspot and enation symptoms on the leaves 6 to 17 months after planting, reconfirming Okabe's conclusion (1959) that mosaic is a soil-borne disease.From the leaves showing enation and/or ringspot symptoms of these experimentally infected seedling, a virus was isolated by juice inoculation to cowpea, Chenopodium amaranticolor, C. quinoa, and C. murale. In electronmicroscopy, the virus was found to be spherical, about 22mμ in diameter. The virus reacted with antiserum to mulberry ringspot virus prepared by Dr. T. Tsuchizaki, but not with antiserum to tomato black ring virus sent from Dr. B.D. Harrison, Scotland. The virus was identified as mulberry ringspot virus reported by Tsuchizaki et al. (1970).The infected soil lost its transmissibility when dried for 7 days. Many nematodes belonging to Longidorus were collected by sieving technique from soil around roots of infected mulberry trees in the fields. The species was identified as L. martini Merny by Mr. Y. Oshima.Lots of 10 to 56 L. martini sieved from soil in which diseased mulberry trees had been growing were placed near the roots of 31 potted healthy mulberry plants. After 3 to 12 months, 10 of these plants showed symptoms, and the virus was detected from these 10 plants by juice inoculation to test plants. No infection occurred in 8 control mulberry plants grown in soil free from L. martini.Transmission tests using Xiphinema sp. sieved from soil in which diseased mulberry had been growing gave negative results.The symptoms on mulberry plants incited by the nematode transmission were ringspots, enations and some vein necrosis, and no fern-leaf and yellow leaf symptoms were observed.

Viruses Isolated from Narcissus (Narcissus spp.) in Japan

A virus was isolated from large cupped narcissus showing symptoms of mild mosaic collected in Chiba in 1967. The virus was readily transmitted by juice inoculation, but not by Myzus persicae. Among the tested plants of 46 species in 15 families, 36 species of 13 families, namely, Chenopodiaceae, Amaranthaceae, Aizoaceae, Caryophyllaceae, Cruciferae, Leguminosae, Violaceae, Umbelliferae, Solanaceae, Pedaliaceae, Cucurbitaceae, Compositae, and Amaryllidaceae, were found susceptible to the virus. Of these plants, Chenopodium amaranticolor, bean, tobacco (Bright Yellow), petunia and cucumber were considered to be usefull as differential host plants for the virus. The virus was proved to be transmitted also through seeds of soybean at a very high rate.An evidence of soil transmission was provided by a greenhouse test using soil which surrounded diseased narcissus and was infested with nematodes including Xiphinema americanum. Lots of 1 to 50 X. americanum sieved from soil in which infected petunia had been grown for 2 to 3 weeks were placed near the roots of potted healthy petunia and cucumber plants. After 6 weeks the virus was detected, by juice inoculation and serological tests in 6 of 11 petunia plants, and in all of 4 cucumber plants. No infection occurred in control petunia and cucumber plants grown in soil free from X. americanum.The virus in vitro withstood heating at 55°C for 10 minutes, but not 65°C, dilution to 2×103, but not 2×104, and 7 days of storage at 20°C, but not 14 days. The virus particles were found to be spherical about 25-30mμ in diameter.Antiserum prepared showed homologous precipitin tube titre of 1/64. By agar gel diffusion tests, the virus showed negative reaction to antisera to tomato black ring virus and arabis mosaic virus kindly sent from Dr. B.D. Harrison and to tobacco ringspot virus antiserum kindly sent from Dr. R. Stace-Smith. The tomato ringspot antiserum obtained from Dr. R. Stace-Smith only gave positive result. The virus and the antiserum were furthermore sent to Dr. B.D. Harrison, and the above result was confirmed, along with an additional negative result with antiserum to strawberry latent ringspot virus.From these results, the virus was identified as tomato ringspot virus, which has not been reported before in Japan.Symptoms caused by this virus in narcissus is not marked. Virus-free narcissus seedlings that were juice inoculated, remained symptomless carriers for 17 months.

Cucumber Green Mottle Mosaic Virus (Watermelon Strain) in Watermelon and its Bearing on Deterioration of Watermelon Fruit Known as “Konnyaku” Disease

Many watermelon plants cultivated in the Chiba and Ibaraki prefectures showed symptoms similar to those of mosaic disease in June 1968. In these districts it became evident that apparently healthy fruit that were harvested harboured some form of abnormality. Infected plants were retarded in growth. The young leaves showed mosaic symptoms but in the majority of cases the symptoms were not distinct and often disappeared as the leaves grew older. Fruit stalks of infected plants generally showed necrotic lesions and sometimes the fruit showed mosaic-like dark green elevated areas on the surface. The white peripheral regions of infected fruit which were cut open appeared to be water-soaked and whitish yellow. The inner pulp also turned to water-soaked dirty red and contained crescent-shaped crevices while there was an increase in the yellow fibres.The above symptoms were induced on fruit as well as leaves of healthy watermelon by juice inoculation with a virus isolated from infected plants showing the abnormalities described. It became apparent that the symptoms in watermelon were most marked when inoculated at the time of fruit set.The virus was identified with cucumber green mottle mosaic virus (CGMMV) by mechanical inoculation and observation under electron microscope using the dip method. This virus was, however, distinguishable from that isolated from cucumber in the western part of Japan in 1966, on the basis of differential reaction on Datura stramonium, Chenopodium amaranticolor and from serological comparison. The virus isolated from watermelon was designated as watermelon strain and the one isolated from cucumber in 1966 as cucumber strain.Specimens of fruit exhibiting the fruit pulp symptoms were collected from various localities and virus isolations were made. From most of them CGMMV was isolated, while from certain specimens watermelon mosaic virus (WMV) and cucumber mosaic virus (CMV) also were isolated, and from some no virus was isolated. The fruit abnormality referred to here is certainly caused by CGMMV, but WMV, CMV and some physiological disorder may also cause similer fruit abnormalities. These, however, can be distinguished from the symptoms caused by CGMMV.Bottlegourd, Lagenaria siceraria var. hispida, is generally employed as a root-stock in the cultivation of watermelon. The presence of CGMMV was detected in the seed coat of bottlegourd seeds which were collected in Chiba and Ibaraki prefectures from left-over stocks of one season, while it was not detected in the seeds of watermelon. Also in the Tochigi prefecture where bottlegourd is cultivated on a large scale the crop showed a high percentage of mosaic symptoms, and from these mosaic plants CGMMV was isolated.

Viruses Isolated from Carrot

1) A virus isolated from mosaic carrot plants (Daucus carota) from Nagano Prefecture was identified as celery mosaic virus. The symptoms on carrot were mainly mosaic, but a few plants showed fern leaf symptoms. The virus was transmitted by juice inoculation, and also by Myzus persicae and Semiaphis heraclei in a non-persistent manner.By mechanical inoculation, 8 species in 3 families became infected out of 22 species in 7 families tested. The infected plants were carrot, celery, coriander, parsley, Nicotiana clevelandii, Chenopodium amaranticoler, and C. quinoa. The virus in vitro withstood heating at 40°C for 10 minutes, dilution to 640, and 6 hours at 20°C. In electronmicroscopy using dip method, long flexuous particles of about 750-800mμ in length were observed. This is first record of celery mosaic virus in Japan.2) Viruses isolated from mosaic carrot plants from Chiba and Hiratsuka were identified as cucumber mosaic virus. The host range and symptoms indicated that it belonged to ordinary strain of the same virus. In agar gel diffusion tests, the virus reacted specifically with an antiserum to cucumber mosaic virus. The virus was readily transmitted from carrot to tobacco, C. amaranticolor, etc. by juice inoculation and from carrot to carrot by Myzus persicae, although some isolates were difficult to infect carrot by juice inoculation.

Viruses Isolated from Narcissus (Narcissus spp.) in Japan

A virus was isolated from Narcissus poeticus L. showing yellow stripe using Gomphrena globosa by juice inoculation. The virus was readily transmitted by juice inoculation, but not by Myzus persicae. The inoculated leaves of G. globosa showed white necrotic local lesions 3-5 days after inoculation and became brownish within a few days. Systemic irregular lesions followed after about a week.Of twenty plant species inoculated with the virus, fourteen were infected. Beta vulgaris, Vigna sinensis, Trifolium incarnatum, Nicotiana clevelandii, Sesamum indicum and Narcissus sp. were infected systemically without symptoms. Local necrotic or chlorotic lesions were produced on the inoculated leaves of Chenopodium amaranticolor, C. quinoa, Tetragonia expansa, Pisum sativum and Glycine max. Symptomless infection of the inoculated leaves occurred in Datura stramonium and Lycopersicon esculentum. The following species were not infected: Vicia faba, Cucumis sativus, Nicotiana glutinosa, N. tabacum (Bright Yellow), Allium fistulosum and Lilium formosanum.The virus in vitro withstood heating at 60°C for 10 minutes, but not 65°C, dilution to 10-7 and 8 weeks of storage at 20°C, but not 16 weeks. In electron microscopy using dip method, long flexuous thread-like particles were found to be 450-600mμ in length, the mode being 500-550mμ.Juice from inoculated G. globosa leaves reacted positively with the antiserum diluted to 1:128 in precipitation reaction. The virus, on the other hand, did not react with antisera to white clover mosaic virus, cymbidium mosaic virus, and potato virus X in precipitation reaction.Owing to the similarity in host range, physical properties, particle morphology, and lack of aphid transmission, the virus was identified as narcissus mosaic virus (NMV) (Brunt, A.A.). NMV was detected from 34 of 41 specimens of narcissus showing yellow stripe, mosaic and necrotic flecks, and also from 5 of 12 plants showing no symptoms. Typical symptoms caused by this virus in narcissus are not determined, as yet, since virus-free seedlings for inoculation tests are not available at the moment.

Tobacco Rattle Virus Isolated from Aster Showing Ringspot Syndrome and Its Transmission by Trichodorus minor Colbran

A yellow ringspot disease of aster was noticed in Saitama Prefecture for the first time in 1967. The symptoms were brilliant yellow rings, which were identical with those of aster ringspot disease described by Anderson (1954). From these asters, a virus was isolated by juice inoculation, and was identified to be tobacco rattle virus as designated by Corbett (1967).The virus in electronmicroscopy by dip method showed a bimodal length distribution of rigid rod-like particles. The shorter particles had a normal length of 70-80mμ; the longer particles a normal length of 170-180mμ. It was found that the host plants of the virus were aster, tobacco, cucumber, plantago, torenia, bean, cowpea, spinach, etc. The symptoms on these plants were similar to those described for tobacco rattle virus by many workers. The virus was not transmitted by Myzus persicae, nor through seed, but soil-transmission was obvious. Thermal inactivation point in crude juice was 70-75°C (10min.), dilution end point 1:106, and longevity in vitro 91-98 days at 20°C. This aster isolate of tobacco rattle virus causes different symptoms on aster from those caused by HSN isolate of the same virus isolated from tobacco (Tomaru et al., 1967): the former produces characteristic ringspot, while the latter top necrosis and veinal necrosis.Many nematodes belonging to genus Trichodorus were collected from soils around roots of the infected aster in fields with Seinhorst elutrator and sieving techniques. The species was identified to be Trichodorus minor Colbran, 1956 (≈T. christiei Allen, 1957). Experimental transmission of the virus by T. minor was successfully made, although a comparatively low infection rate (20 out of 225 seedlings tested) was secured.Several plant species were grown in infected fields, and two months later virus isolation test from the plant roots were carried out. The virus was recovered from aster and spinach, but not from pea, cabbage, corn, etc.

Soybean Dwarf, a New Virus Disease

A new virus disease, soybean dwarf, was first noticed on the Tsurunoko variety of soybean in southern areas of Hokkaido in about 1952. Since then it has been widely observed throughout Hokkaido, causing a considerable loss in soybean yields. Soybean plants infected with the virus show dwarfing (stunting), and downward curling, rugosity, and/or interveinal yellowing of the leaves.The virus was transmitted by grafting and by the aphid Aulacorthum solani (Kaltenbach), but not by juice inoculation or through seeds. Three species of aphids, Aphis glycines Matsumura, Aphis craccivora Koch and Myzus persicae (Sulzer), and two species of leafhoppers, Scleroracus flavopictus Ishihara, and Psammotettix striatus (Linné) failed to transmit the virus. The virus was retained in the vector A. solani for 20 days after leaving the source plants.It was found that red clovers and white clovers in the neighborhood of affected soybean fields were symptomless carriers of the virus.

Viruses Causing Mosaic Diseases of Amaryllis in Japan

A virus isolated from a mosaic plant of amaryllis (Hippeastrum hybridum) in Shizuoka is identified as Hippeastrum mosaic virus (HMV), because of its similarity to the description of HMV by Brants et al. (1965) in host range, physical properties, morphology, and lack of aphid transmission. Namely, the virus is readily transmitted by juice inoculation, but not by Myzus persicae. By mechanical inoculation, it produced systemic mosaic in amaryllis and Lilium formosanum, and local lesions in Chenopodium amaranticolor and Gomphrena globosa. New Zealand spinach, Raphanus sativus, bean (Chajiro and Otebo), cowpea (Kurodane), broadbean, Nicotiana glutinosa, N. tabacum (Bright Yellow), petunia and tomato were not susceptible. In electron microscopy using dip method, long flexuous thread-like particles were observed. Majority of the particles were 600∼800mμ in length, but there was no distinct mode. The virus in vitro withstood heating at 65°C for 10 minutes, dilution to 1:100 and 1 day's aging at 20°C.Twenty-three samples of mosaic plants of amaryllis obtained from Shizuoka, Fujinomiya and Tachikawa were tested for HMV and CMV on a series of differential hosts. All samples proved to include HMV, while about one third of them included also CMV simultaneously. The samples from which only HMV were isolated, showed mosaic symptoms having rather coarse dark green areas irregular in size and shape. CMV, isolated from amaryllis, revealed some differences from the ordinary strain of CMV in host range and in symptoms on tomato, potato, N. glutinosa, G. globosa, petunia, etc.

Transfer of Virus by a Direct Leaf-to-leaf Method

IN evaluating sources of tobacco mosaic virus (TMV) resistance in tomato1 to a fruit disorder2, large numbers of mature plants grown in the field and greenhouse were indexed just prior to inoculation with TMV. This was necessary because the presence of related or unrelated viruses has been shown to interfere with TMV multiplication3,4. Since the usual method of indexing by juice inoculation to indicator hosts required considerable time spent in washing mortars and pestles between transfers, a less time-consuming method of inoculation seemed desirable. The leaf-to-leaf method described here is a modification of Yarwood's leaf-disk method5 in which the freshly cut edges of leaf disks are used in the inoculation of labile viruses.

Studies on the viruses of Japanese radish mosaic diseases

1. This paper deals with serological experiments using the radish P virus. The virus used as immunizing antigen was the isolate P0, which was obtained from a mosaic radish plant in Matsudo, Chiba Prefecture, in May 1956, and was maintained in a greenhouse by successive juice inoculation on turnip plants.2. By a combined procedure of salting-out with ammonium sulfate and differential centrifuging, a partially purified virus preparation was obtained from infected turnip leaf juice. Antiserum was prepared by injecting rabbits with this virus preparation. The rabbits were given 7∼8 intravenous injections (equivalent to total leaf juice volume of 150ml), at 2∼7 days intervals. The titer of the antiserum obtained was 1:10, 000∼1:40, 000, in precipitation reaction tests.3. The antiserum reacted specifically with leaf juice of Brassica rapa var. glabra, Raphanus sativus, Brassica pekinensis, Petunia hybrida, Chrysanthemum coronarium and Spinacia oleracea, which were infected with the radish P virus; also with leaf juice of mosaic radish plants collected in the suburbs of Tokyo, which were judged, by host range tests, to be infected with the radish P virus. The antiserum did not react with leaf juice of healthy plants, plants infected with radish Q virus, radish R virus, cucumber mosaic virus, or several other viruses. In absorption tests, the antiserum completely absorbed the radish P virus, but not other viruses.4. Virus dilution end-point was: ×300 in agglutination reaction on slide glasses, and ×2, 000 in precipitation reaction, using turnip leaf juice; 0.002∼0.005mg/ml (virus dry weight basis), using purified virus preparations.5. Out of 231 mosaic radish plants collected in the suburbs of Tokyo, 106 reacted with the antiserum. In host range tests of the viruses involved, radish P virus, radish Q virus, radish R virus, and cucumber mosaic virus were detected. As judged from the combined results of these serological tests and host range tests, 31 out of 76 plants tested were found to be infected with a single virus, the rest being infected with two, three, or four viruses.

Studies on the viruses of Japanese radish mosaic diseases

1. This paper deals with the host range, properties, purification, and electron microscopy of the radish P virus. In a preliminary survey of the viruses of Japanese radish mosaic disease complex in the suburbs of Tokyo, made jointly with Mr. Yasuo Komuro, it was found, that besides the cucumber mosaic virus, three other viruses, which we have termed P, Q, and R, are involved. These viruses occur either singly, or as combinations of two, three, and four. While the virus Q is apparently distinct, both the viruses P and R are considered to belong, in the conventional taxonomy, to the turnip mosaic virus group. There are, however, some indications that these two viruses are rather related remotely with each other. Most of the experiments reported here, unless otherwise stated, were made using a virus isolate P0, which was isolated in May 1956, from a mosaic radish plant in Matsudo, Chiba Prefecture, and was maintained in a greenhouse, by successive juice inoculations on turnip plants.2. Host range. (Mosaic, or mottle): Brassica rapa var. glabra, B. rapa var. pervidis, B. juncea (two subvarieties), B. nigra, B. campestris, Raphanus sativus var. acanthiformis (five subvarieties), Matthiola incana, Spinacia oleracea (subvariety Ujo), Chrysanthemum coronarium, and Petunia hybrida.(Symptomless carriers): B. oleracea var. capitata (subvariety Yoshin), and B. napus.(Necrotic local lesions on inoculated leaves): Gomphrena globosa, and Chenopodium album.(Not susceptible): N. tabacum (subvariety Bright yellow), N. glutinosa, Vigna sesquipedalis, Vicia faba, Cucumis sativus, Beta vulgaris var. flavescens, Datura stramonium, Lycopersicum esculentum, Zinnia elegans, Brassica oleracea var. capitata (subvariety Hachigatsudori), B. oleracea var. botrytis, and Spinacia oleracea (subvariety Nihon). N. glutinosa was found to be infected by the P virus, when inoculated together with cucumber mosaic virus.3. Properties. Dilution end-point in extracted leaf juice was 1:2000∼1:5000. Thermal inactivation point was between 55 and 60°C, in 10 minutes treatment. Longevity of the virus in crude juice was between 4 and 7 days at 25∼26°C, but this period became much longer, when a little KCN was added to the leaf juice. In a frozen-dried preparation of leaf juice, the virus still retained infectivity after 23 months.4. Aphid transmission. The P virus is readily transmitted by the aphid, Myzus persicae, except for the particular virus isolate P0, which has been maintained in a greenhouse for more than two years by successive juice inoculation. Although this isolate P0 was found not transmissible by the aphid, a sub-isolate P'0, which was derived from the same source as P0 but which had been kept in a frozen-dried juice preparation, was found to be transmissible by the aphid.5. Purification. Attempts were made to obtain a purified virus preparation, using diseased turnip (subvariety Kanamachikokabu) or petunia leaves. The final procedure adopted was as follows: (freezing and thawing of leaf tissues or juice) → combined procedures of repeated salting-out by ammonium sulfate and differential centrifuging → (treatment with chloroform). The final product was almost colorless, and the yield was about 10∼15mg per 100ml leaf juice. This purified virus preparation diluted to (2∼3)×107 (dry weight basis) was still capable of infecting 20 per cent of inoculated turnip plants.6. Electron microscopy. Examination of the purified virus preparation revealed the presence of sinuous rod-shaped particles, of about 12∼13mμ in width, with the most frequent length of 650∼700mμ. The preparation was found to be almost free of other particulate matter.

Interactions of sugar-beet curly top virus and an unusual mutant

A virus that produces marked vein yellowing was found in a Turkish to-bacco plant that had recovered from curly top. This virus is designated “yellow-vein virus” to differentiate it from typical curly top virus. Insofar as tested, it has the same host range as curly top virus except that it did not persist in Nicotiana glauca Graham, a host of the curly top virus. The yellow-vein virus was transmitted by the beet leafhopper, Circulifer tenellus (Baker), by four species of Cuscuta, and by graftage. It was not transmitted by juice inoculation or through seeds. The yellow-vein virus has not been separated from typical curly top virus but the two have been transmitted in combination to tobacco, sugar beet, tomato, and other plants. No symptoms were produced by the yellow-vein virus on sugar beet but the virus was recovered from inoculated plants. The presence of the yellow-vein virus apparently results in a marked decrease in the curly top component in tobacco. Concentrations of the two components appear to vary greatly in different plants. If the yellowing component is dominant in tobacco, the plants are yellow and stunted; if the curly top component is dominant, leaves are green, curled, and dwarfed at first, but the plants recover. If the two components are more or less evenly balanced, usually little injury results. When tomato plants were inoculated by means of the beet leafhopper, those infected with curly top virus alone almost invariably died, whereas those infected with both yellow-vein and curly top viruses usually showed symptoms of only moderate severity and soon recovered. Tobacco plants that recovered from curly top appeared to be immune to infection with yellow-vein virus introduced by means of the beet leaf-hopper. They were resistant, but not immune, to infection with yellow-vein virus from diseased scions. Typical vein yellowing was produced on Nicotiana glauca Graham, immune to infection by yellow-vein virus, and Datura meteloides DC., immune to both curly top and yellow-vein viruses, when infected Turkish tobacco scions were in a position to supply carbohydrates and virus to plants of these two species. The virus was lost by the plants of both species in a few days after the tobacco scions were removed. The yellow-vein virus seems to be closely limited to the phloem, and rapid movement appears to be correlated with the transport of carbohydrates. Evidence indicates strongly that the vein-yellowing virus is a strain of curly top virus that probably arose as a mutant in a curly top-infected tobacco plant.