Occurrence,distribution and seasonal changes of viruses infecting common bean in northwestern Iran

Many surveys were conducted during 2003–2005 to study the identity, prevalence and fluctuation of bean infecting viruses in northwestern Iran. In total, 649 bean samples with virus- like symptoms were collected and analysed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and tissue-print immunoassay to detect infectious viruses. Serological tests revealed the presence of Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), Bean leaf roll virus (BLRV), Bean pod mottle virus (BPMV) and Southern bean mosaic virus (SBMV), with some co-infection occurred, with prevalence of BCMV, BCMNV and BYMV (17–29% infection rate). The incidence of viruses showed variation in over 3 years of research including more than double increase in CMV from 2004 to 2005 and obvious one-third decrease in AMV from 2003 to 2005. SBMV and BPMV were detected sporadically in the fields and the response of some differential test plants was analysed by mechanical inoculation. Western immunoblotting analysis of SBMV infected bean leaf total proteins using SBMV-specific polyclonal antibody revealed viral CP with molecular mass of 28.5 kDa which confirmed the presence of SBMV as a new threat for bean production.     

Plant proteases as interfering factors in the electron microscopic detection of alfalfa mosaic and bean yellow mosaic viruses inSolanum laciniatum Ait

Plant proteases rapidly destroy virus architecture in sap ofSolanum laciniatum Ait., with yellow mosaic symptoms but even under these conditions the partially digested amorphous nucleoproteins retain their immunospecificity and infectivity for a limited time. Homogenization with phenylmethylsulfonylfluoride inhibits efficiently host serine proteases and fully stabilizes the virus particles in plant sap and in partial purificates. During the electronmicroscopic examination of stabilized infectious saps in 12 out of 14 isolates of yellow mosaic ofS. laciniatum besides alfalfa mosaic virus a poty-virus was disclosed. This was identified serologically and by transmission tests as a weakly virulent type of bean yellow mosaic virus.     

The Use of a Simple Electron Microscope Serology Procedure to Observe Relationships of Seven Potyviruses

The relationships between bean yellow mosaic (BYMV), bean common mosaic (BCMV), clover yellow vein (CYVV), lettuce mosaic (LMV), potato virus Y (PVY), turnip mosaic (TuMV) and celery mosaic (CeMV) viruses were studied in homologous and heterologous reactions, using simple and relatively rapid electron microscope serology decoration tests. The degree of relationship between these viruses was assessed by the intensity of antibody coating when the viruses were decorated by heterologous antibodies. A close relationship was observed between BYMV and CYVV, and between BYMV and LMV but not between CYVV and LMV. CeMV was quite closely related to BYMV and CYVV. Antibodies to BCMV and BYMV intensely decorated different strains of their own virus, but decoration was negligible in cross reactions.     

Resistance to six viruses in the legume goat's rue {Galega orientalis Lam.)

Plants from 2nd to 6th year leys of the legume goat's rue (Galega orientalis Lam.) were tested for infection with bean yellow mosaic (BYMV), bean common mosaic (BCMV), alfalfa mosaic (AMV), broad bean stain (BBSV), red clover mottle (RCMV) and cucumber mosaic (CMV) viruses by enzyme-linked immunosorbent assay (ELISA), electron microscopy, and by sap-inoculation to various test plant species. No virus infections were observed in goat's rue in the field. Glasshouse-grown seedlings of goat's rue were inoculated with the above viruses. No virus was detected in the inoculated plants. The results suggest that goat's rue is extremely resistant to the above six viruses which are important in other forage legumes.     

Serological Studies on Mung Bean Yellow Mosaic Virus

Particles of mung bean yellow mosaic virus (MYMV) were purified by a method that yields up to 3 mg per kg of systemically infected Phaseolus vulgaris“Top Crop” and used to prepare antiserum. MYMV antiserum prepared gave a single precipitin line and had a titre of 1/512 with homologous virus in gel double-diffusion tests. MYMV was shown to be serologically related to other whitefly-transmitted viruses, bean golden mosaic virus, tobacco leaf curl virus and cassava latent virus.     

A Simple Procedure for the Purification and Antiserum Production of Bean Yellow Mosaic Virus

A simple procedure was developed to purify bean yellow mosaic virus from infected faba bean. The procedure included clarification of tissue homogenate by 25% chloroform followed by low-speed centrifugation, virus concentration by polyethylene glycolprecipitation and further purification by agarose-acrylamide gel electrophoresis. The partially purified virus preparation was electrophoresed in 0.5% agarose-2% acrylamide gel for 4 h. Gel bands containing the virus were collected, homogenized, and mixed (1:1) with Freunds adjuvant. Four weekly intramuscular injections and a booster injection four weeks after the fourth injection were given to a rabbit. Antisera collected from the first five bleedings produced high A₄₀₅ readings in ELISA (0.47,5–0.790) with virus-infected faba bean leaves and low readings (0.030–0.065) with healthy tissue. Plates were coated with 5μg/ml of gammaglobulins (IgG) fractionated from the different bleedings of the antiserum prepared and a 1: 1000 dilution ofthe IgG from the third bleeding conjugated to alkaline phosphatase was used.     

Immunogenicity of a killed whole Neospora caninum tachyzoite preparation formulated with different adjuvants

A killed whole Neospora caninum tachyzoite preparation was formulated with various adjuvants and tested for its immunogenicity in cattle. The adjuvants used were: Havlogen, a polymer of acrylic acid cross-linked with polyallylsucrose; Polygen, a non-particulate copolymer; a mixture of Havlogen and Bay R-1005, which is a preparation of free base synthetic glycolipids; and Montanide ISA 773, a water-in-oil emulsion made with a mixture of metabolisable and mineral oils. Immune responses in immunised cattle were compared with those of cattle experimentally infected with culture-derived N. caninum tachyzoites. The overall mean serum IFAT titres were significantly higher (P < 0.05) in experimentally infected cattle compared with all immunised cattle. Nonetheless, the maximum antibody titres of the immunised cattle, which were obtained following the third immunisation, were within the range of titres previously described for naturally infected cattle. The overall mean serum IFAT titres were significantly higher (P < 0.05) in cattle immunised with the killed tachyzoite preparation formulated with Polygen and with the mixture of Havlogen and Bay R-1005, compared with cattle immunised with the Havlogen- and Montanide-based preparations. Two of the four adjuvant preparations were able to induce cell-mediated immune responses similar to those of the experimentally infected cattle. The Havlogen-adjuvanted tachyzoite preparation elicited N. caninum-specific proliferation of peripheral blood mononuclear cells statistically similar (P = 0.095) to that of the infected animals. Peripheral blood mononuclear cells from animals immunised with the Polygen-adjuvanted tachyzoite preparation produced interferon-gamma concentrations of similar magnitude (P = 0.17) to those from the infected animals. Polygen was one of two adjuvants that elicited the highest antibody responses, and was the only adjuvant that induced interferon-gamma levels similar to those of the infected heifers.     

Identification of soybean mosaic, southern bean mosaic and tobacco ringspot viruses from soybean in the People's Republic of China

Samples of soybean plants with virus-like symptoms were collected from several locations in the People's Republic of China in 1981. These samples were used to prepare inocula for mechanical inoculation to soybean. Twenty-one virus cultures were obtained, the identities of which were determined by serology, symptomatology and host range. Sixteen cultures contained only soybean mosaic virus, four of which were more pathogenic than any previously studied; one culture contained only tobacco ringspot virus, another only southern bean mosaic virus, and three other cultures mixed infections of soybean mosaic and southern bean mosaic viruses. This is the first report of the occurrence of tobacco ringspot virus and southern bean mosaic virus in soybean in the People's Republic of China.     

Distribution and impact of virus associated diseases of common bean (Phaseolus vulgaris L.) in northern Iran

Different viral diseases infect common bean crops in Iran. A total of 248 symptomatic samples were collected from common bean fields throughout main growing fields of Guilan province in Iran during the summer of 2006. Eight viruses were detected using double antibody-sandwich – enzyme-linked immunosorbent assay (DAS-ELISA). Bean common mosaic virus – BCMV (1%), Bean leaf roll virus – BLRV (9%), Cowpea mild mottle virus – CpMMV (6%), Southern bean mosaic virus – SBMV (3%), Cucumber mosaic virus – CMV (15%), Bean golden mosaic virus – BGMV (2%), Bean common mosaic necrosis virus – BCMNV (1%) and Bean yellow mosaic virus – BYMV (1%) were detected. Comparatively CMV (15%) was found to be more prevalent in Guilan province. Multiple infections of viruses were recorded in many samples. Weed species belonging to Chenopodiaceae, Solanaceae, Malvaceae and Amaranthaceae families were also found to be infected with the viruses.     

Production of antibody to lipopolysaccharide (LPS) after immunization with a LPS-polymyxin B-agarose immunogen

A method was devised to produce antibodies to lipopolysaccharide (LPS) in guinea-pigs following a single immunization. The antigen was prepared by mixing polymyxin B-agarose with LPS from Escherichia coli O55:B5. Use of the agarose support allowed purification of the complex by simple washing procedures. Twenty-nine days after a single injection of the immunogen mixed with Freund complete adjuvant all animals demonstrated antibody to the LPS portion of the complex. No antibodies were detected to the polymyxin B component. Typical titres of LPS as measured by ELISA were 2(11). After, a booster immunization, titres of LPS antibody were further increased and a greater avidity was noted. In contrast to other methods which have been employed for production of antibody to LPS, use of the polymyxin B-agarose complex has the following advantages: ease of antigen preparation, ready purification of the complex, potent immunostimulation, and under the conditions employed here, LPS-specific antibody production, without accompanying antibody to polymyxin B.     

Comparative immunological properties of enantiomeric peptides

Summary The immunogenicity of a peptide composed of only d-amino acids is compared with that of the corresponding l-peptide enantiomer. Following three administrations of 100 g of individual peptide formulated with different adjuvants (Freund's complete adjuvant, QS21, or alum) to BALB/c mice, guinea pigs and rabbits, the l-peptide elicited strong l-peptide-specific IgG antibody responses in all formulations, whereas the d-peptide-induced d-peptide-specific IgG antibodies in the Freund's complete adjuvant and QS21 formulations, but was nonimmunogenic in the alum formulation. Mouse T-cell lines induced by the d-peptide formulated in Freund's complete adjuvant were found to express significant amounts of IL-2 when they were stimulated by the d-peptide. When an equal amount of both enantiomers was mixed and administered in Freund's complete adjuvant, only an l-peptide-specific IgG antibody response was observed. These results suggest that (i) d-peptide is immunogenic when strong adjuvant is provided; (ii) the immune system has preferential recognition of l-amino acid peptide; and (iii) the d-peptide can elicit d-peptide-specific T-cell responses.     

Preliminary studies on sap-transmissible viruses of red clover (Trifolium pratense L.) in England and Wales

Red clover plants, collected from nine widely separated permanent pastures in England and Wales, were tested for sap-transmissible viruses. Viruses were identified by the symptoms they caused in test plants, by electron microscopy, and by serological tests. Of the 265 plants tested 14% were infected. Only pea mosaic virus was common and widespread; it was found in 8% of the plants, and in seven of the fields. Other viruses isolated were arabis mosaic, bean yellow mosaic, red clover mottle, and red clover vein mosaic; only red clover mottle virus produced diagnostic symptoms in red clover. No viruses were detected in seedlings grown from seed from eighty-nine commercial seed crops. Attempts to transmit red clover mottle virus by the Collembolan Sminthurus viridis L., which is common on red clover, failed.     

Production of antibody to lipopolysaccharide (LPS) after immunization with a LPS-polymyxin B-agarose immunogen

A method was devised to produce antibodies to lipopolysaccharide (LPS) in guineapigs following a single immunization, The antigen was prepared by mixing polymyxin B-agarose with LPS from Escherichia coli O55:B5. Use of the agarose support allowed purification of the complex by simple washing procedures. Twenty-nine days after a single injection of the immunogen mixed with Freund complete adjuvant all animals demonstrated antibody to the LPS portion of the complex. No antibodies were detected to the polymyxin B component. Typical titres of LPS as measured by ELISA were 2¹¹. After, a booster immunization, titres of LPS antibody were further inceased and a greater avidity was noted. In contrast to other methods which have been employed for production of antibody to LPS, use of the polymyxin B-agarose complex has the following advantages: ease of antigen preparation, ready purification of the complex, potent immunostimulation, and under the conditions employed here, LPS-specific antibody production, without accompanying antibody to polymyxin B.     

国家种质库保存大豆和菜豆种质的种传病毒检测

以国家种质中期库提供的300份大豆、100份菜豆种质为材料,分别采用血清学和分子生物学方法,对种传病毒的种类进行了检测。结果表明:在大豆种质中检测出大豆花叶病毒(SMV)、黄瓜花叶病毒(CMV)、苜蓿花叶病毒(AMV)3种病毒,阳性检出率分别为25.33%(76份)、13.67%(41份)和4.67%(14份)。大豆种质中还存在大豆花叶病毒与黄瓜花叶病毒、大豆花叶病毒与苜蓿花叶病毒的复合侵染。在菜豆种质中检测出菜豆普通花叶病毒(BCMV)阳性材料92份,种质带毒率高达92%。这些信息将会对今后采取相关措施提高国家种质库保存的豆类种质的质量提供帮助。     

20. Electro-Extraction of Viruses from Infected Plant Tissue (Applied to Turnip Yellow Mosaic,Tobacco Mosaic,and Maize Streak Viruses)

ABSTRACT

An apparatus is described which was used for rapid extraction of viruses from frozen and thawed infected plant tissues. The novel principle is the establishment of a potential gradient of 15 to 20 volts/cm at approximately 90° across the leaves are surrounded by buffer of low molarity and of the appropriate hydrogen ion concentration. To keep the leaves in the correct orientation they were placed as single layers between coarse rigid plastic gauze. The method, termed electro-extraction, was used as the initial step in the purification of turnip yellow mosaic, tobacco mosaic and maize streak viruses. An electron micrograph of the purified maize streak virus is presented.     

Identification of Peanut Green Mosaic Virus Strains in India*

During field surveys, three peanut green mosaic virus isolates differing in symptomatology on groundnut and a few other hosts were collected. Ultrathin sections of infected groundnut leaflets showed cytoplasmic inclusions with pin wheels and scrolls. In enzyme-linked immunosorbent assay they reacted strongly with antisera to peanut green mosaic and soybean mosaic virus antisera, and moderately with adzuki bean mosaic and peanut stripe virus antisera. All isolates also reacted positively with antisera to peanut eye spot, blackeye cowpea mosaic, pea seed-borne mosaic, potato virus Y and tobacco etch viruses, and did not react with antisera to peanut mottle, bean yellow mosaic, bean common mosaic, clover yellow vein and sugarcane mosaic viruses. SDS-PAGE analysis of purified virus preparations of the three isolates showed a single polypeptide with mol. wt. of 34,500 daltons. Based on these results, the three isolates are identified as biologically distinct strains of peanut green mosaic virus.     

Incidence and severity of bean common mosaic disease and resistance of popular bean cultivars to the disease in western Kenya

The common bean (Phaseolus vulgaris) is a high protein crop and the main legume in the cropping system of western Kenya. Despite its importance, common bean yields are low (<1.0 t/ha) and declining. Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) are the most common and most destructive viruses and can cause a yield loss as high as 100%. In Kenya, a limited number of cultivars and exotic genotypes with resistance to BCMV and BCMNV strains have been reported. This study sought to determine the distribution and screen popular cultivars for resistance to the viruses. In October 2016 and May 2017, two diagnostic surveys for bean common mosaic disease (BCMD) were conducted in seven counties of western Kenya namely Bungoma, Busia, Homa Bay, Nandi, Vihiga, Kakamega and Siaya. Leaf samples showing virus-like symptoms were collected and analysed by ELISA. Sixteen popularly grown bean cultivars together with cowpea (Vigna unguiculata), soybean (Glycine max), green grams (Vigna radiata) and groundnut (Arachis hypogaea) were planted in a greenhouse in a completely randomized block design with three replicates. The plants were inoculated with BCMNV isolate at 3-leaf stage. Data were taken weekly for 3 weeks on type of symptoms expressed and number of plants infected. In total, 270 bean farms were visited. Symptoms of mosaic, downward curling, local lesions, stunting or a combination of these were observed during both surveys. Mean virus incidence was higher in the short rain season (50.2%) than in the long rain season (35.6%). The mean BCMD severity on a scale of 0–3 was highest (2.3) in Kakamega County and lowest (0.5) in Siaya. On variety resistance tests to BCMNV isolate, 10 bean cultivars were susceptible, four tolerant and two resistant. BCMNV is widely distributed across counties probably because of use of uncertified seeds by farmers and inoculum pressure from seed and aphid vector. For improved yields of common bean, farmers should be advised to plant certified seeds for all legumes in the cropping system.     

Detection of Four Seed-borne Plant Viruses by the Enzyme-Linked Immunosorbent Assay (ELISA)

This study was conducted to evaluate the sensitivity of the ELISA technique in detecting four economically important viruses, namely barley stripe mosaic (BSMV), cucumber green mottle mosaic (CGMMV), bean common mosaic (BCMV), and squash mosaic (BSMV) viruses in single seeds as well as in batches of barley, cucumber, bean and squash seeds, respectively. Results indicated the suitability of the technique in detecting the above viruses in single germinated seeds or embryos. Accordingly, seed transmission rates of BSMV, CGMMV, BCMV and SqMV were found to be 67 %, 17%, 17% and 12%, respectively. In artificially contrived mixtures of infected: healthy seeds or embryos, BSMV, CGMMV, BCMV and SqMV were successfully detected at ratios of 1 : 500, 1 : 25, 1 : 10 and 1 : 10, respectively. Sensitivity of detection was increased in the ease of BSMV by using germinated rather than ground dry BSMV-infected barly seeds; and in the case of SqMV, by using whole germinating emybryos rather than coleoptiles only. Trials on re-using the enzyme-γ-globulin conjugate indicated that CGMMV conjugate used once can be re-used with little loss in reactivity.     

Recognition and differentiation of seven whitefly-transmitted geminiviruses from India, and their relationships to African cassava mosaic and Thailand mung bean yellow mosaic viruses

Particles resembling those of geminiviruses were found by immunosorbent electron microscopy in extracts of plants infected in India with bhendi yellow vein mosaic, croton yellow vein mosaic, dolichos yellow mosaic, horsegram yellow mosaic, Indian cassava mosaic and tomato leaf curl viruses. All these viruses were transmitted by Bemisia tabaci whiteflies, all reacted with at least one out of ten monoclonal antibodies to African cassava mosaic virus (ACMV), and all reacted with a probe for ACMV DNA-1, but scarcely or not at all with a full-length probe for ACMV DNA-2. Most of the viruses were distinguished by their host ranges when transmitted by whiteflies, and the rest could be distinguished by their pattern of reactions with the panel of monoclonal antibodies. Horsegram yellow mosaic virus was distinguished from Thailand mung bean yellow mosaic virus by its lack of sap transmissibility, ability to infect Arachis hypogaea, failure to react strongly with the probe for ACMV DNA-2 and its pattern of reactions with the monoclonal antibodies. Structures resembling a ‘string of pearls’, but not geminate particles, were found in leaf extracts containing malvastrum yellow vein mosaic virus. Such extracts reacted with two of the monoclonal antibodies, suggesting that this whitefly-transmitted virus too is a geminivirus. All seven viruses from India can therefore be considered whitefly-transmitted geminiviruses.     

Detection and relationships of cotton leaf curl virus and allied whitefly-transmitted geminiviruses occurring in Pakistan

A stock culture of cotton leaf curl virus from Pakistan (CLCuV-PK), was transmitted by whiteflies (Bemisia tabaci) to seven plant species, including French bean, okra, tobacco and tomato, and caused vein thickening and leaf curl symptoms. It was readily detected in triple antibody sandwich ELISA (TAS-ELIS A) by 11 out of 31 monoclonal antibodies raised against the particles of three other geminiviruses: African cassava mosaic, Indian cassava mosaic and okra leaf curl viruses. Reaction strength was enhanced when the tissue extraction fluid contained sodium sulphite. Minor variations in epitope profile were found among virus isolates from cotton (Gossypium hirsutum) collected from different districts in Pakistan over a 5-year period. These epitope profiles were distinguishable from that of cotton leaf curl virus from G. barbadense in southern India but indistinguishable from the profiles of viruses causing yellow vein disease of okra in India or Pakistan, or leaf curl of okra {Abelmoschus esculentus), Hibiscus tiliaceus, radish or sunflower in Pakistan, suggesting that these plants are putative natural hosts of CLCuV-PK. The viruses in cotton, and in okra with leaf curl or yellow vein symptoms, were also detected by PCR with three pairs of CLCuV-PK-specific primers. Five additional whitefly-transmitted geminiviruses were found among isolates from 11 other naturally-infected species in Pakistan, and were distinguished by their epitope profiles. These viruses were associated, respectively, with tobacco leaf curl, squash yellow blotch, tomato yellow leaf curl, watermelon leaf crinkle and soybean yellow mosaic diseases. The first four of these viruses were detected readily by PCR with geminivirus general primers but only weakly, if at all, with two pairs of CLCuV-PK-specific primers. Pakistani crops are infected with a range of distinguishable but relatively closely related whitefly-transmitted geminiviruses, some of which resemble those found in India.