Characterization of Peanut Stripe Virus Isolates from Soybean in Taiwan

Potyvirus isolates were obtained in Taiwan from soybean showing crinkle, mottle, mosaic or blistering. They were identified as peanut stripe virus (PStV) on the basis of host range, serology, molecular weight of the capsid proteins and morphology of cytoplasmic cylindrical inclusions. PStV was found to be closely related serologically to adzuki bean mosaic virus (AzMV), blackeye cowpea mosaic virus (BICMV), and the bean common mosaic virus (BCMV) strain NY 15. A clear differentiation of PStV from these related viruses was possible on the basis of the cylindrical inclusion morphology. Only the peanut isolate of PStV from the USA and the three soybean isolates of PStV from Taiwan produced pinwheels, scrolls and curved laminated aggregates whereas the other serologically related viruses produced scrolls only. Whilst the peanut isolate of PStV infected all nine peanut cvs tested, the soybean isolate PN of PStV infected two peanut cvs only. AzMV, BICMV and two strains of soybean mosaic virus did not infect any of the peanut cultivars tested. On the other hand, nineteen and three of the 27 soybean cvs were susceptible to the soybean isolate PN and the peanut isolate of PStV, respectively. The capsid proteins of the peanut and the three soybean isolates of PStV and of AzMV appeared to be proteolytically undegraded and to have nearly identical molecular weights of 35 kD. Based upon results of virus surveys in soybean plantings in Taiwan, the incidence of soybean isolates of PStV in soybean is similar to that of soybean mosaic virus, suggesting that these PStV strains might be economically significant to soybean production m Taiwan.     

Peanut stripe virus - a new seed-borne potyvirus from China infecting groundnut (Arachis hypogaea)1

A new virus, peanut stripe (PStV), isolated from groundnut (Arachis hypogaea) in the USA, induced characteristic striping, discontinuous vein banding along the lateral veins, and oakleaf mosaic in groundnut. The virus was also isolated from germplasm lines introduced from the People's Republic of China. PStV was transmitted by inoculation of sap to nine species of the Chenopodiaceae, Leguminosae, and Solanaceae; Chenopodium amaranticolor was a good local lesion host. PStV was also transmitted by Aphis craccivora in a non-persistent manner and through seed of groundnut up to 37%. The virus remained infective in buffered plant extracts after diluting to 10^-3, storage for 3 days at 20°C, and heating for 10 min at 60°C but not 65°C. Purified virus preparations contained flexuous filamentous particles c. 752 nm long, which contained a major polypeptide of 33 500 daltons and one nucleic acid species of 3·1 × 10⁶ daltons. In ELISA, PStV was serologically related to blackeye cowpea mosaic, soybean mosaic, clover yellow vein, and pepper veinal mottle viruses but not to peanut mottle, potato Y, tobacco etch, and peanut green mosaic viruses. On the basis of these properties PStV is identified as a new potyvirus in groundnut.     

Survey of aflatoxin-producing fungi in certain fermented foods and beverages in Thailand

Aflatoxin-producing fungi were found in fermented foods and beverages: fermented rice (kaomak), soybean sauce (taotjo), peanut butter, soy sauce (shoyu), Thai red and white wine, and rice sugar wine. These foods were extracted directly and tested for aflatoxins by thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC).Four strains of aflatoxin-producing fungi were isolated from peanut butter, taotjo, and shoyu. Direct extracts of 10% of the peanut butters tested and 5% of the kaomak tested contained large amounts of aflatoxins. The HPLC procedure used in this experiment utilized chloroform-ethyl acetate (31)     

Peanut Stripe Potyvirus Resistance in Peanut (Arachis Hypogaea L.) Plants Carrying Viral Coat Protein Gene Sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.     

几种常见花生病毒血清学关系的研究

花生是世界上重要的经济作物,中国也是个花生消费大国,但是常有8种重要的花生病毒危害,包括花生斑驳病毒（PMV）、花生条纹病毒（PStV）、花生丛簇病毒（GRV）、花生丛矮病毒（PCV）、著茄斑萎病毒（TSWV）、花生芽枯病毒（BNN）、花生矮化病毒（PSV）和黄瓜花叶病毒（CMV）。70年代以来在全国各地多次爆发花生病毒病,并引起大面积减产,经济损失巨大,因而花生病毒的检测和转基因花生抗毒株的选育变得尤为重要［‘－’］。本研究应用免疫双扩散和酶联免疫法（ELISA）Z4几种花生病毒的血清学关系进行了研究。三材料与方法…     

Phylogenetic analyses of Bradyrhizobium strains nodulating soybean (Glycine max) in Thailand with reference to the USDA strains of Bradyrhizobium.

To elucidate the phylogenetic relationships between Thai soybean bradyrhizobia and USDA strains of Bradyrhizobium, restriction fragment length polymorphism (RFLP) analysis using the nifDK gene probe and sequencing of the partial 16S rRNA gene were performed. In our previous work, Thai isolates of Bradyrhizobium sp. (Glycine max) were separated clearly from Bradyrhizobium japonicum and Bradyrhizobium elkanii based on the RFLP analysis using the nodDYABC gene probe. RFLP analysis using the nifDK gene probe divided 14 Thai isolates and eight USDA strains of B. japonicum into different groups, respectively, but categorized into the same cluster. All of seven strains within these Thai isolates had the same sequence of the partial 16S rRNA gene, and it was an intermediate sequence between those of B. japonicum USDA 110 and B. elkanii USDA 76T. Furthermore, three USDA strains of B. japonicum, USDA of (B. japonicum ATCC 10324T), USDA 115 and USDA 129, had the same partial 16S rRNA gene sequence that seven Thai isolates had. These results suggest that Thai isolates of Bradyrhizobium sp. (Glycine max) are genetically distinct from USDA strains of B. japonicum and B. elkanii, but also indicate a close relationship between Thai isolates and USDA strains of B. japonicum.     

Complete Genome Sequence of Peanut stripe virus Isolated in China

The complete genome sequence of a Laixi isolate of Peanut stripe virus (PStV‐Laixi) from China was determined to be 10, 056 nucleotides in length, excluding the 3′‐terminal poly (A) tail. The viral genome contains a single long open reading frame of 9669 nucleotides encoding a polyprotein of 3222 amino acids. The polyprotein was predicted to be cleaved into ten functional proteins by three viral proteases. An additional protein, termed ‘PIPO’, is also found in the P3 cistron. The complete genome sequence comparison and phylogenetic analysis indicated that PStV‐Laixi was most closely related to three other isolates of PStV (two from USA and one from Taiwan). To our knowledge, this is the first report of the complete sequence of a PStV isolate from China.     

Variation in the Coat Protein Gene of Papaya ringspot Virus Isolates from Multiple Locations of China

The potyvirus Papaya ringspot virus （PRSV） is an important pathogen of papaya that causes severe losses in economic crops for papaya production globally. The coat protein （CP） genes of five PRSV isolates originating from different locations in China were cloned and sequenced. The CP-coding region varied in size from 864-873 nucleotides, encoding proteins of 288-291 amino acids. The five Chinese isolates of PRSV have been characterized as papaya-infecting （PRSV-P）. The CP sequences of the Chinese isolates were compared with those of previously published PRSV isolates originating from different countries at amino acid levels. A number of KE repeat boxes in the N terminus of the PRSV-CP were found in all Chinese isolates. The phylogenetic branching pattern revealed that there was certain extended grouping between geographic locations, and the Asian type probably represents the oldest population of PRSV. The information of CP genes will be useful in designing and developing durable virus resistant-PRSV transgenic papaya in China. Meanwhile broad-spectrum-virus resistant, strongly resistant-PRSV and good safe papaya lines are required.     

Infection of human cells by dengue virus is modulated by different cell types and viral strains

Although prior studies have investigated cellular infection by dengue virus (DV), many have used highly passaged strains. We have reassessed cellular infection by DV type 2 (DV2) using prototype and low-passage isolates representing genotypes from different geographic areas. We observed marked variation in the susceptibility to infection among cell types by different DV2 strains. HepG2 hepatoma cells were susceptible to infection by all DV2 strains assayed. Although the prototype strain generated higher titers of secreted virus than the low-passage isolates, this difference did not correspond to positive- or negative-strand viral RNA levels and thus may reflect variation in efficiency among DV2 isolates to translate viral proteins or package and/or secrete virus. In contrast, human foreskin fibroblasts were susceptible to the prototype and low-passage Thai isolates but not to five Nicaraguan strains tested, as reflected by the absence of accumulation of negative-strand viral RNA, viral antigen, and infectious virus. A similar pattern was observed with the antibody-dependent pathway of infection. U937 and THP-1 myeloid cells and peripheral blood monocytes were infected in the presence of enhancing antibodies by the prototype strain but not by low-passage Nicaraguan isolates. Again, the barrier appeared to be prior to negative-strand accumulation. Thus, depending on the cell type and viral isolate, blocks that limit the production of infectious virus in vitro may occur at distinct steps in the pathway of cellular infection.     

Host Ranges, Serology and Cytopathology of Eggplant and Tomato Strains of Eggplant Severe Mottle Virus, a New Potyvirus from Nigeria

Two potyvirus isolates from the eggplant (Solanum melongena L.) cultivars ‘Ex Benin’ and ‘Ex Jos’, respectively, in Nigeria proved to be almost identical in host range, symptomatology and reactivity with antisera to various potyviruses. In eggplant they caused a severe systemic mottle, blistering and malformation of leaves and an abnormal serration of the leaf margins. A potyvirus isolate from tomato showing mosaic symptoms was similar, but not identical to the eggplant isolates. In the slide, precipitin test the serological differentiation indices were between 1 and 3 for the eggplant and tomato isolates. In the immunoelectron microscopical decoration test all three virus isolates showed some reactivity with antisera to the following potyvir, uses: dioscorea green banding mosaic, groundnut eyespot, a mungbean isolate of peanut stripe, pepper veinal mottle, telfairia mosaic and a tomato isolate from Taiwan. No reactions were observed with antisera to other potyviruses. Cytopathogenic effects w,ere similar for all three isolates in the arrangement of virus particles, the structure of the cylindrical inclusions and the occurrence of clusters of small vesicles. However, other cytological alterations like accumulations of rod-shaped aggregates of,granular material, formation of giant mitochondria, degeneration of mitochondria and occurrence of a nucleolar inclusion differentiated the isolates.     

Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae)

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).     

Structural analysis of hemolymph proteins from Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda)

1. Five different molecular weight polypeptides from serum (cell-free hemolymph) of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata, were examined by two-dimensional 125I-peptide mapping and high performance liquid chromatography (HPLC). 2. Peptide mapping indicated that all five radiolabeled polypeptides within and between the two snail strains had similar migration patterns when cleaved with pepsin or alpha-chymotrypsin, thus revealing a shared structural homology. All peptides chosen for analysis appeared to be structurally similar to the 160 kDa hemoglobin molecule. 3. Separations of the radiolabeled enzyme digests by HPLC confirmed results seen in the mapping experiments since all chromatograms had similar elution patterns. 4. Minor differences in the peptide maps and chromatograms within and between snail strains may be due to quantitative differences in the amount of protein present and/or variations in the primary amino acid sequences of the proteins chosen for analysis.     

Characterisation and serological relationships of strains of Kashmir bee virus

Isolates of novel strains of Kashmir bee virus (KBV) were obtained from field-collected dead adults of Apis mellifera from honey bee colonies in Canada and Spain. They differed from other strains of KBV in their tendency to aggregate in dilute buffer solution and in containing only three proteins when analysed by SDS-polyacrylamide gel electrophoresis compared with five proteins resolved in the type strain of KBV from Apis cerana in India and six proteins in KBV strains from South Australia and New Zealand. Immunodiffusion tests and Western blotting studies indicated that the five virus isolates were serologically related and all were related to acute paralysis virus (APV). The world distribution of KBV strains and their apparent relationship with APV are discussed.     

Genetic diversity in Aspergillus parasiticus population from the peanut agroecosystem in Argentina

AIMS: The aims of this work were to identify potential sources of Aspergillus parasiticus inoculum and to evaluate the sclerotial and toxigenic profiles of this species from the peanut agroecosystem in Argentina. Likewise, the genetic diversity of A. parasiticus population was analysed using vegetative compatibility group (VCG) analysis. METHODS AND RESULTS: The A. parasiticus strains were isolated from soil, debris and peanut seeds in Córdoba Province, Argentina. A. parasiticus was recovered from the three sources analysed. Only 11 of 185 A. parasiticus isolates (5.9%) did not produce aflatoxins, while 57% produced sclerotia. Twenty-four VCG were identified from 63 isolates. The VCG diversity index for A. parasiticus, expressed as the number of groups divided by the total number of isolates, was 0.31. In general, there were significant differences among VCG in aflatoxin production. CONCLUSIONS: The presence of aflatoxigenic strains of A. parasiticus in the three substrates suggests that they may be an important source of aflatoxin in Argentina's peanut agroecosystem. The A. parasiticus population shows a low genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study showed data on inoculum distribution, aflatoxin and sclerotia production and genetic diversity in an A. parasiticus population isolated from the peanut agroecosystem in Argentina.     

Phylogeny and diversity of Bradyrhizobium strains isolated from the root nodules of peanut (Arachis hypogaea) in Sichuan, China.

Twenty-two rhizobial strains isolated from the root nodules of two Chinese peanut cultivars (Arachis hypogaea L. Tianfu no. 3 and a local cultivar) growing at four different sites in the Sichuan province, Southwest China, were characterized by growth rate, rep-PCR, PCR-RFLP of 16S rDNA, partial sequencing of ribosomal genes, and fatty acid-methyl ester analysis (FAME), and compared with strains representing Bradyrhizobium japanicum, B. elkanii and other unclassified Bradyrhizobium sp. All peanut isolates from Sichuan were bradyrhizobia. Dendrograms constructed using the rep-PCR fingerprints grouped the strains mainly according to their geographic and cultivar origin. Based on PCR-RFLP and partial sequence analysis of 16S rDNA it appears that peanut bradyrhizobial strains from Sichuan are similar to peanut strains from Africa and Israel, and closely related to B. japonicum. In contrast, analysis of FAME data using two-dimensional principal component analysis indicated that Bradyrhizobium sp. (Arachis) were similar to, but slightly different from other bradyrhizobia. The presence and level of fatty acid 16:1 w5c was the distinguishing feature. The results of PCR-RFLP of the 16S rRNA gene, the partial sequence analysis of 16S rDNA, and FAME were in good agreement.     

A Study on the Prevalence of Bacteria that Occupy Nodules within Single Peanut Plants

In this study, bacteria hosted in root nodules of single plants of legume Arachis hypogaea L. (peanut) cv Tegua Runner growing at field were isolated. The collection of nodule isolates included both fast and slow growing strains. Their genetic diversity was assessed in order to identify the more frequently rhizobial strain associated to nodules from single plants. Molecular fingerprinting of 213 nodular isolates indicated heterogeneity, absence of a dominant genotype and, therefore, of a unique strains highly competitive. Efficient nitrogen-fixing isolates were identified as Bradyrhizobium sp. by phylogenetic analysis of the sequences of their 16S rRNA genes. The genetic diversity of 68 peanut nodulating isolates from all the collected plants was also analyzed. Considering their ERIC-PCR profiles, they were grouped in eighteen different OTUs for 60% similarity cut-off. Results obtained in this study indicate that the genetic diversity of rhizobia occupying nodules from single plant is very high, without the presence of a dominant strain. Therefore, the identification of useful peanut rhizobia for agricultural purposes requires strongly the selection, among the diverse population, of a very competitive genotype in combination with a high-symbiotic performance.     

A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus

All of 33 samples of dried shrimps, shrimp paste, peanut butter and raw groundnut kernels were contaminated with fungi. Aspergillus and Penicillium spp. were the predominant types in dried shrimps and raw groundnut kernels but no Aspergillus spp. were present in peanut butter or shrimp paste samples. Among 81 Aspergillus isolates obtained from dried shrimps and raw groundnut kernels, 10 were A. flavus/A. parasiticus, of which five were potential aflatoxin-producing A. flavus strains. No aflatoxins were detected in the food samples although some were visibly mouldy and some had high mould counts. The occurrence of aflatoxin-producing strains of A. flavus in dried shrimps and raw groundnut kernels warrants further investigation of these foods and their products as potentially significant sources of aflatoxins.     

Strong immunogenicity of a multicomponent peptide vaccine developed with the branched lysine oligopeptide method for human immunodeficiency virus infection

We synthesized one V3 peptide each from HTLV-III_B, Thai A and Thai B, conjugating then to the T cell epitope of the env region, and we also synthesized a p17 protein peptide of the gag region (HGP-30). These peptide were then coupled to 8-lysine copolymers using N-succinimidyl maleimido carboxylate (M_r = ca 60 000). We designated this the branched lysine oligopeptide method. The large peptide complexes constructed from these four macromolecular peptide were used with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay showed high titres of anti-peptide antibodies to each V3loop peptide and the HGP-30 peptide. Strong inhibition of CD4⁺ dependent cell fusion was obtained with these antisera when III_b, Thai A and Thai B strains of human immunodeficiency virus (HIV) were used. Strong anti-fusion inhibition was also observed with two other HIV strains. In addition, an increase of the anti-HIV effect was observed when we used sera obtained by multicomponent vaccine immunization. The same kind of inhibition was also observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in mice immunized with this vaccine. These results suggest that immunization with macromolecular peptide complexes can result in strong immunogenicity towards HIV-1.