Understanding the inheritance of mungbean yellow mosaic virus (MYMV) resistance in mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek)

Mungbean, Vigna radiata, third in the series of important pulse crops, still suffers from yield loss due to mungbean yellow mosaic disease caused by mungbean yellow mosaic virus (MYMV). Hence, studies on plant-microbe interaction are necessary for understanding the inheritance of resistance. This study concentrated on identification of linked molecular markers for MYMV resistance and to find the genetic inheritance of MYMV resistance in mungbean. A total of 413 germplasm entries in a MYMV hot spot area (Vamban) were subjected to natural field infection and 13 selected resistant lines were subjected to Agrobacterium infection using strains harboring partial genome of two different MYMV isolates, VA221 and VA239. Among the resistant lines, KMG189 showed strain-specific resistance to VA221 and had no symptoms during field trials. Ninety F₂ genotypes were developed from the cross made between KMG189 (MYMV-resistant) and VBN(Gg)2 (MYMV-susceptible), segregated in the Mendelian single cross ratio 3S:1R; susceptibility of all the F₁s to MYMV suggested that the MYMV resistance in mungbean is governed by a single recessive gene. Two SCAR markers CM9 and CM815 were developed through bulk segregant analysis, and the linkage analysis proved CM815 SCAR marker to be linked at 5.56 cM with MYMV resistance gene and SCAR CM9 had nil recombination percentage, suggesting it to be very closely linked to the MYMV resistance gene. SCAR marker CM9 was present in chromosome number 3 of mungbean suggesting novel loci for virus resistance in mungbean. The identified loci can be used for developing varieties resistant to MYMV in mungbean.     

Infectivity analysis of two variable DNA B components of<Emphasis Type="Italic">Mungbean yellow mosaic virus-Vigna</Emphasis> in<Emphasis Type="Italic">Vigna mungo</Emphasis> and<Emphasis Type="Italic">Vigna radiata</Emphasis>

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), aBegomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85·6%) for KA27 DNA B. The Rep-binding domain had three complete 11 -nt (5’-TGTATCGGTGT-3′) iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5’-ATCGGTGT-3’) had a 3-nt deletion. KA27 DNA B, which exhibited 93·9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94·1%) and BC1 (97·6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMV-Vig is an important determinant of host-range betweenV. mungo andV. radiata.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

Stable genetic transformation of<Emphasis Type="Italic"> Vigna mungo</Emphasis> L. Hepper via<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>

Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.     

Stimulation of indoleacetic acid production in a <Emphasis Type="Italic">Rhizobium</Emphasis> isolate of <Emphasis Type="Italic">Vigna mungo</Emphasis> by root nodule phenolic acids

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Molecular mapping of <Emphasis Type="Italic">Rym17</Emphasis>, a dominant and <Emphasis Type="Italic">rym18</Emphasis> a recessive barley yellow mosaic virus (BaYMV) resistance genes derived from <Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.

PK23-2, a line of six-rowed barley (Hordeum vulgare L.) originating from Pakistan, has resistance to Japanese strains I and III of the barley yellow mosaic virus (BaYMV). To identify the source of resistance in this line, reciprocal crosses were made between the susceptible cultivar Daisen-gold and PK23-2. Genetic analyses in the F₁ generation, F₂ generation, and a doubled haploid population (DH45) derived from the F₁ revealed that PK23-2 harbors one dominant and one recessive resistance genes. A linkage map was constructed using 61 lines of DH45 and 127 DNA markers; this map covered 1268.8 cM in 10 linkage groups. One QTL having a LOD score of 4.07 and explaining 26.8% of the phenotypic variance explained (PVE) for resistance to BaYMV was detected at DNA marker ABG070 on chromosome 3H. Another QTL having a LOD score of 3.53 and PVE of 27.2% was located at marker Bmag0490 on chromosome 4H. The resistance gene on chromosome 3H, here named Rym17, showed dominant inheritance, whereas the gene on chromosome 4H, here named rym18, showed recessive inheritance in F₁ populations derived from crosses between several resistant lines of DH45 and Daisen-gold. The BaYMV recessive resistance genes rym1, rym3, and rym5, found in Japanese barley germplasm, were not allelic to rym18. These results revealed that PK23-2 harbors two previously unidentified resistance genes, Rym17 on 3H and rym18 on 4H; Rym17 is the first dominant BaYMV resistance gene to be identified in primary gene pool. These new genes, particularly dominant Rym17, represent a potentially valuable genetic resource against BaYMV disease.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

Cadmium-induced oxidative damage and antioxidative defense mechanisms in <Emphasis Type="Italic">Vigna mungo</Emphasis> L.

Cadmium (Cd)-induced oxidative stress and antioxidant defense mechanisms were analyzed in roots and leaves of Vigna mungo L. Seeds were germinated in perlite-vermiculite and irrigated with Hoagland nutrient solution. At day 6, seedlings were exposed to 40 μM Cd under semi-hydroponic conditions for a period of 12 days. Growth anomalies and abnormal chromatin condensation were observed in Cd-treated plants, in comparison with control ones. Cd accumulation was observed in roots of treated plants. The analyses of antioxidative defense and oxidative parameters in roots, stems and leaves showed different tissue-specific responses. Superoxide dismutase (SOD) and guaiacol peroxidase (GPx) activities and the level of lipid peroxidation (MDA content) decreased in roots. However, they increased in leaves. Catalase activity and chlorophyll content, on the other hand, decreased over exposure to Cd stress. Total glutathione, non-protein thiols, reduced glutathione (GSH) and phytochelatins increased significantly, while oxidized glutathione (GSSG) decreased, as compared with control plants. The present data suggest that the presence of Cd in soil and water can cause oxidative damage that may be detrimental for optimum production of nutritional mung.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

<Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> an environmental host for <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> and <Emphasis Type="Italic">Shigella sonnei</Emphasis>

The interaction between Shigella dysenteriae or Shigella sonnei and Acanthamoeba castellanii was studied by viable counts, gentamicin assay and electron microscopy. The result showed that Shigella dysenteriae or Shigella sonnei grew and survived in the presence of amoebae for more than 3 weeks. Gentamicin assay showed that the Shigella were viable inside the Acanthamoeba castellanii which was confirmed by electron microscopy that showed the Shigella localized in the cytoplasm of the Acanthamoeba castellanii. In conclusion, the relationship between Shigella dysenteriae and Shigella sonnei with Acanthamoeba castellanii is symbiotic, and accordingly free-living amoebae may serve as a transmission reservoir for Shigella in water.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">Hsp90</Emphasis> in <Emphasis Type="Italic">Schizothorax prenanti</Emphasis>

Aquatic animals suffer from various environmental stresses because the aquatic environment is a very complex system. To monitor the health status of fish, Hsp90 a potential early warning marker was determined in Schizothorax prenanti after infection with a bacterium. In this study, we cloned Hsp90 from S. prenanti for the first time. The full-length cDNA sequence of SpHsp90 was 2663 bp, contains an open reading frame of 2181 bp, and has a gene encoding 726 amino acids, an estimated molecular mass of 83.38 kDa, and a theoretical isoelectric point of 4.91. The SpHsp90 amino acid sequence has five conserved HSP90 family signatures and shares 87.0–95.5 % identity with other vertebrates. Phylogenetic analysis and structure comparison indicated that SpHsp90 should be a β isoform of the HSP90 family. SpHsp90 was ubiquitously expressed in all examined tissues, and the highest level of expression was in the kidney. After Streptococcus agalactiae infection, the level of SpHsp90 expression had significant changes (P < 0.05) in the hepatopancreas, spleen, kidney, and blood. The expression increased to the highest level at 6 h in the blood and at 24 h in the hepatopancreas, spleen, and kidney. The results suggested that the SpHsp90 gene could be induced by S. agalactiae in S. prenanti and that SpHsp90 may be involved in resistance to bacterial infection and provide an early warning information. The kidney is the most suitable for detecting SpHsp90 after bacterial infection.     

Molecular characterization the <Emphasis Type="Italic">YABBY</Emphasis> gene family in <Emphasis Type="Italic">Oryza sativa</Emphasis> and expression analysis of <Emphasis Type="Italic">OsYABBY1</Emphasis>

Members of the YABBY gene family have a general role that promotes abaxial cell fate in a model eudicot, Arabidopsis thaliana. To understand the function of YABBY genes in monocots, we have isolated all YABBY genes in Oryza sativa (rice), and revealed the spatial and temporal expression pattern of one of these genes, OsYABBY1. In rice, eight YABBY genes constitute a small gene family and are classified into four groups according to sequence similarity, exon-intron structure, and organ-specific expression patterns. OsYABBY1 shows unique spatial expression patterns that have not previously been reported for other YABBY genes, so far. OsYABBY1 is expressed in putative precursor cells of both the mestome sheath in the large vascular bundle and the abaxial sclerenchyma in the leaves. In the flower, OsYABBY1 is specifically expressed in the palea and lemma from their inception, and is confined to several cell layers of these organs in the later developmental stages. The OsYABBY1-expressing domains are closely associated with cells that subsequently differentiate into sclerenchymatous cells. These findings suggest that the function of OsYABBY1 is involved in regulating the differentiation of a few specific cell types and is unrelated to polar regulation of lateral organ development.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.