Nucleotide sequencing and an improved diagnostic for screening okra (Abelmoschus esculentus L.) genotypes for resistance to a newly described begomovirus in India

Ten okra (Abelmoschus esculentus L.) plants showing distinct yellow vein mosaic disease (YVMD) symptoms were collected from different fields in Karnataka State, India. The genomic DNA of the isolated viruses was amplified, cloned, and sequenced. Sequence analysis revealed that the DNA-A-like sequences of all ten isolates were identical. Sequence analysis of a representative virus isolate (OYSK2) with other begomovirus sequences available in GenBank showed ≥90% sequence identity with Bhendi yellow vein Maharashtra virus (BYVMaV; EU482411) and ≤89% homology with full-length Bhendi yellow vein mosaic virus (BYVMV) infecting okra on the Indian subcontinent. These results suggested that a new strain of BYVMaV was present in all ten samples collected from the field. A source of resistance to BYVMaV and naturally present virus isolates causing YVMD was identified by screening okra genotypes under artificial and natural inoculation conditions, respectively. None of the genotypes tested showed complete immunity to BYVMaV. However, the okra genotypes ‘Tulasi’ and ‘Trisha’ were only moderately susceptible under glasshouse and field conditions. The new begomovirus strain could be detected by dot-blot hybridisation using a non-radioactive DNA probe in the virus samples collected from both symptomless and symptomatic okra plants.     

The culture of shoot tips of hop (Humulus lupulus L.) to eliminate viruses

The culture of hop shoot tips has proved highly effective in eliminating Prunus necrotic ringspot and hop mosaic virus from eleven varieties of hop. The culture technique was successful even when plants were raised from tips as long as 5 mm.     

Development of expression vectors based on pepino mosaic virus

Background  

Plant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV.     

The Production of Virus-Free Rhubarb by Apical Tip-Culture

The apical tip-culture technique has been used to produce virus-free rhubarb from infected plants of the varieties Timperley Early, Victoria and Prince Albert. Growth on various nutrient media was compared. The survival rate of the tips was directly related to their size within the range 300–2,500 μ (diam.). After initial culture on filter paper in sterile tubes, the young plants were transferred to autoclaved sand watered with mineral nutrient on which they made better growth than on other media.

Turnip mosaic, cherry leaf-roll, strawberry latent ringspot and cucumber mosaic viruses, in various combinations, were eliminated by this method. Arabis mosaic was removed from most plants but a few retained this virus. This retention was not related to the size of the tip taken.     

Biological control of the shoot disease of Amaranthus hybridus caused by Choanephora cucurbitarum with Bacillus subtilis

Summary

Isolates of Bacillus subtilis from soil and ogili (a local food condiment) controlled choanephora shoot disease of the vegetable crop plant, Amaranthus hybridus, in the greenhouse. Disease developed on plants inoculated simultaneously with the pathogen Choanephora cucurbitarum and the ‘ogili’ isolate, but not the soil isolate, of B. subtilis. Application of either bacterial strain to plants one day before a challenge with the pathogen prevented disease development. A single application of either strain prevented disease development from a subsequent challenge with the pathogen at any time over a thirty day period. Viable counts of the microflora on the shoot tips of the treated plants indicated strongly that the inoculated bacteria multiplied and colonized the extending shoot tip whether or not the shoots were covered with polythene bags. Both strains of Bacillus subtilis inhibited mycelial extension growth as well as conidial germination in vitro.     

Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

Experiments on the Control of Brown Rot of Apples and Plums: I. Laboratory Tests

The symptoms and some properties of five viruses isolated from celery crops in Britain are described. Cucumber mosaic and lucerne mosaic viruses were found to be prevalent in celery and other crops. Tomato aspermy virus, though widespread in chrysanthemum and occasional in tomato crops in England, and economically damaging in Continental celery crops, has not so far been reported in celery in Britain. British celery varieties tested were resistant to experimental infection with tomato aspermy virus. These three viruses were readily isolated, purified and identified.

Three viruses, designated celery yellow spot, celery yellow vein and celery ringspot, isolated from affected celery plants, were less readily transmitted from celery to celery, and could not be purified by techniques that succeed with many viruses.

Celery yellow spot was transmitted by sap-rubbing inoculation only when cucumber mosaic virus was also present ; it was tentatively identified with the celery yellow spot virus reported from the U.S.A. Celery ringspot virus showed some similarities to the poison hemlock ringspot virus found in the U.S.A., but none of the critical tests for virus identification could be applied to either of these two viruses. Celery yellow net virus differed from all the viruses so far recorded in celery. The chronic symptoms of the different viruses in celery were often similar, and therefore unreliable for diagnosis. The initial or “ shock ” symptoms, however, were usually distinctive.     

The Use of Thermotherapy and in vitro Meristem Culture to Produce Virus-Free ‘Chancellor’ Grapevines

ABSTRACT

Grapevines (Vitisspp.) are very susceptible to virus diseases. Virus infection reduces fruit yield and quality. The objective of this work was to determine the usefulness of thermotherapy (37.2°C) and in vitromeristem culture to obtain virus-free grapevine plants cv. ‘Chancellor’. Grapevine leafroll-associated virus 1,3(GLRaV-1, 3) infected grapevines were multiplied in vitrofrom two infected mother-plants in half strength Murashige and Skoog medium (1/2MS) supplemented with 0.5 mg/L of BA and the in vitroplants were initially tested by ELISA to confirm their virus status; subsequently, 96 infected in vitroplants were propagated on 1/2MS medium with BA and subjected to 0, 7, 10, 12, 14, 16, 18, 20 or 22 days of exposure at 37.2°C. Afterward, the apical meristems from the plants surviving the thermotherapy treatment were excised and transferred to fresh 1/2MS medium with 0.5 mg/L of BA and grown in a culture room until they developed into entire plants. Control plants and all the plants that survived thermotherapy were assessed for their virus status using both ELISA and RT-PCR. After 20 days of exposure at 37.2°C, 100% of the plants submitted to thermotherapy were found to be virus-free by RT-PCR and ELISA tests. Plants derived from meristems with two or three primordial leaves remained virus infected. However, when meristem culture was combined with thermotherapy (12 or more days of heat treatment), all the meristem-derived plants were virus-free.     

Improved Culture of Apical Tissues for Production of Virus-Free Strawberries

Strawberry plants of fourteen varieties were raised from dissected apices of stolon tips and axillary buds excised at a length of less than 0·80 mm.

Two different media were used. On one of them 50% of tips from non-heat-treated plants developed roots and of these 54% survived to maturity; on the other medium (White’s augmented with coconut milk and sucrose) only 35% developed roots but 80%of these rooted tips grew to mature plants.

Tips excised from heat-treated plants grew more rapidly, and a higher proportion reached maturity, than those grown from untreated plants.

The smaller tips (<0·4 mm.) rooted less readily than larger ones (0·4–0·8 mm.) and grew more slowly to a size suitable for potting.

The plants of nine varieties were tested for virus infection by grafting to Fragaria vesca and F. virginiana 12–18 months after excision. One variety was freed from yellow edge (virus 2), three varieties from vein chlorosis (virus 4), two from crinkle (virus 3), and two from latent A virus.     

Transgene expression for Gladiolus plants grown outdoors and in the greenhouse

Transgene expression was evaluated for Gladiolus plants transformed with either the CaMV 35S, double CaMV 35S, rolD, or Arabidopsis UBQ3 promoter controlling the uidA or bean yellow mosaic virus coat protein gene in either the sense or antisense orientation to determine differences in expression for plants grown in the greenhouse and outdoors for two years. There was more variability in GUS expression when plants were grown outdoors than in the greenhouse for two years. Four of the six transformed plant lines with the UBQ3, rolD, and CaMV 35S promoters grown outdoors showed significant differences in GUS expression from year to year as compared to two of the six lines with the UBQ3 and rolD promoters grown in the greenhouse. When grown the same year, two plant lines with the CaMV 35S and one line with the rolD promoter showed 2–16× higher levels of GUS expression outdoors than in the greenhouse, and one plant line with the UBQ3 promoter had 31× higher GUS expression in the greenhouse instead of outdoors. Three of six plant lines transformed with the bean yellow mosaic virus coat protein gene in either the sense or antisense orientation under control of the double CaMV 35S promoter showed obvious transgene expression as compared to three lines that did not show expression or negligible expression for both years when plants were grown both outdoors and in the greenhouse. This study verified long-term gene expression, rather than silencing, for Gladiolus plants when grown outdoors and in the greenhouse from year to year.     

High GUS expression in protoplasts isolated from immature peach fruits

A protocol for transient expression analysis was developed using protoplast isolated from immature peach fruits. The uid A gene for β-glucuronidase (GUS) was introduced into the protoplast as a reporter gene to evaluate the protoplast activity. Protoplasts isolated from immature fruits at 28–32 days after full bloom (DAFB) showed high GUS activity under the cauliflower mosaic virus 35S promoter. The highest GUS activity was obtained from the protoplasts isolated at 32 DAFB, but it was difficult to isolate protoplasts showing high GUS activity from 34 DAFB. A comparison of the effect of promoter cassette on gene expression showed that the promoter containing the tobacco mosaic virus Ω sequence enhanced GUS activity by at least 10-fold in the peach protoplasts relative to the 35S promoter. The level of GUS activity under the 35S promoter in the peach protoplasts was 0.47-fold as that obtained from maize protoplasts isolated from young greening leaves, indicating that the GUS activity of immature peach protoplast is sufficient for gene expression analysis. Since stable transformation and evaluation of fruit traits in peach transformants are difficult, this transient expression system could be useful for the characterization of genes expressed in peach and other Rosaceae fruit species.     

Three new vectors of papaya viruses

Seven aphids were tested for their ability to transmit 3 viruses of papaya. Mild mosaic virus (MMV) was transmitted by Aphis craccivora, A. gossypii and Myzus persicae. Distortion ringspot virus (DRV) was transmitted by A. nerii, Lipaphis pseudobrassicae and M. persicae. Ringspot virus (RSV) was transmitted only by M. persicae. Specificity of transmission by the aphid vectors of MMV and DRV was demonstrated. A. craccivora, A. nerii and L. pseudobrassicae are reported as new vectors of these viruses.     

Spacing Experiments on Vegetables III. The Growth and Yield of Shallots in Relation to Spacing,Manuring and Size of Planting Material,in Cheshire, 1948

Three varieties of plum were inoculated with the three viruses, plum line pattern (apple mosaic), prune dwarf and plum bark split, either singly or in combinations of line pattern with each of the others. Bark lesions developed more rapidly on Cambridge Gage trees infected with bark split virus alone than on those infected also with line pattern virus. Symptoms characteristic of line pattern virus on trees of Cambridge Gage, Early Laxton and Italian Prune were not affected by infection with the bark split virus, but were suppressed by the prune dwarf isolate. This suppression was apparently due to the presence of a mild strain of line pattern virus contaminating the prune dwarf inoculum.

The occurrence of antagonistic strains of line pattern virus, differing in virulence, precludes any precise definition of diagnostic symptoms.     

四川省攀西地区南瓜、甜菜和辣椒部分病毒种类的ELISA检测

利用马铃薯Y病毒（Potato virus Y,PVY）和马铃薯X病毒（Potato virus X,PVX）的多克隆抗体,以及烟草花叶病毒（Tobacco mosaic virus,TMV）、黄瓜花叶病毒（Cucumber mosaic virus,CMV）、蚕豆萎蔫病毒2号（Broad bean wilt virus 2,BBWV-2）及芜菁花叶病毒（Turnip mosaic virus,TuMV）的单克隆抗体,采用抗原直接包被ELISA法对四川省攀枝花、西昌和德昌地区的南瓜、甜菜和辣椒上采集的病毒病样品进行了检测。结果表明,在87份样品中PVY的侵染最普遍,总检出率达74.71 %|CMV、TMV、TuMV、BBWV-2和PVX的总检出率分别为12.64 %、13.79 %、22.99 %、10.34 %和14.94 %。在87份样品中,仅发现TuMV、PVX及PVY的单独侵染|其余25份样品均检测到2种以上病毒的复合侵染,总检出率为34.72 %。表明攀西地区蔬菜上病毒复合侵染现象普遍,PVY是该地区蔬菜上的优势毒原。     

Rapid propagation of white cabbage by tissue culture

A tissue culture technique has been devised to produce plants of white cabbage (Brassica oleracea v. capitata L.f. alba) from heads stored at 0.5 ± 0.5°C for 8 months. Meristem-tips (0.5–2 mm diameter), excised from heads of 11 accessions, were grown initially on MS medium containing 2.56 mg l^?1 of kinetin and then induced to proliferate shoots on MS medium with 12.8 mg l^?1 kinetin. Subsequent transfer to a kinetin-free medium resulted in root development in 1–2 weeks. Rooted plantlets were readily established in soil. Plantlets obtained in this way from parent cabbages containing turnip mosaic virus remained infected.     

BTH诱导西瓜甜瓜抗病毒病研究

为了考察S-甲基苯基(1,2,3)噻二唑-7-硫代羧酸酯(benzothiodiazole,BTH)诱导西瓜对小西葫芦黄花叶病毒(Zuccini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)与甜瓜对黄瓜花叶病毒(Cucumber mosaic virus,CMV)的抗病效果,分别在接种ZYMV、WMV、CMV前喷施BTH。结果表明:在接种ZYMV、WMV、CMV前3d用25μg/mLBTH处理西、甜瓜,4周后植株的病情指数减少,相对防效分别为55.6%、56.8%、78.2%;进一步研究发现,经BTH处理后,诱导时间的长短对病情指数有一定的影响;BTH对病毒病的防治效果优于病毒A、病毒K、宁南霉素、芸薹素内酯。