Cultural, morphological, pathogenic and molecular characterization of Alternaria mali associated with Alternaria leaf blotch of apple

Alternaria blotch (Alternaria mali) causes severe foliar damage to apple trees in Kashmir. Twenty one (21) isolates of A. mali were collected from different locations and characterized for cultural, morphological, pathogenic and molecular variations. A. mali colonies varied in their cultural behaviour ranging from velvety to cottony, mostly appressed, with regular to irregular margins. Colour of colonies ranged between light to dark olivacious. Isolates impregnated media with colour ranging between grey to brown. Growth rate of isolates was between 5.86 to 8.21 mm/day with fast growth in isolate Am-13 and least in Am-5. Morphological variations in size, shape and septation of hyphae, conidiophore and conidia were observed in the isolates with significant variations in conidiophore and conidial septation. Average conidial size ranged from 21.36 to 31.74 x 8.34 to 14.48 μm. Isolates exhibited variations in incubation period, number and size of the lesions were produced. The dendrogram analysis, based on cultural, morphological and pathogenic studies, revealed variation within A. mali population. At 67% similarity matrix, all the isolates formed 2 clusters with 12 and nine isolates in cluster I and II, respectively. However, dendrogram on molecular (random amplification of polymorphic DNA, RAPD) basis revealed five clusters at 68% Dice similarity coefficient. There was no congruence between RAPD pattern and cultural, morphological and pathogenic characters. Isolates identical for one spectrum were often dissimilar for other spectrum. The results demonstrate existence of considerable variation in cultural, morphological, pathogenic and molecular characters of A. mali isolates prevalent in Kashmir valley.Keywords: Apple, Alternaria mali, variability, cultural, morphological, pathogenic, RAPDAfrican Journal of Biotechnology Vol. 12(4), pp. 370-38

Transgenesis: An efficient tool in mulberry breeding

Genetic engineering is the most potent biotechnological approach dealing with transfer of specially constructed gene assemblies through various transformation techniques. Tools of recombinant DNA technology facilitated development of transgenic plants.  The plants obtained through genetic engineering contain a gene or genes usually from an unrelated organisms, and are known as transgenic plants. The combined use of recombinant DNA technology, gene transfer methods and tissue culture techniques has  led to the efficient transformation and production of transgenics in a wide variety of crop plants. In fact transgenesis has emerged as a novel tool for carrying out “single gene breeding” or transgenic breeding of crop plants. Identification, isolation and cloning of resistant genes is the prerequisite for development of transgenic plants for disease resistance. Identification of resistance genes on the basis of amino acid sequence, conservation enables plant breeder to monitor resistance gene segregation using  appropriate DNA probe intend of testing progeny for disease resistance and susceptibility. Significant developments in plant  genetic modification have been achieved in the last 15 years. Some of the success include herbicide tolerant corn, cotton,  soyabeen and papaya; virus resistant corn, potato, cotton among others. In mulberry, little work has been carried out at Delhi University (south campus). They have developed drought and salinity tolerent transgenic mulberry through Agrobacterium mediated transformation. The overexpression of HVA1 gene from barley generates tolerence to salinity and water stress in  transgenic mulberry (Morus indica).Keywords: Transgenic plant, mulberry, resistance, salinity

Molecular Characterization of Bean Common Mosaic Virus Infecting Phaseolus vulgaris L. in Kashmir

Extensive survey of commercial bean growing areas of Kashmir conducted during kharif 2009 and 2010 revealed only 13 out of 100 surveyed locations free from Bean Common Mosaic Virus (BCMV) disease. The disease incidence was found to range from 0.50 to 85.00 per cent, with the maximum incidence recorded in Srinagar district followed by that in district Anantnag and Bandipora. The samples of BCMV from infected bean plants showing characteristic mosaic, leaf distortion and stunting type of symptoms were categorized into 21 isolates based on the symptomatology and the representative commercial bean growing areas of the Valley. Confirmation of the association of BCMV with each isolate was made by mechanical transmission and DAC-ELISA tests. The test isolates in DAC-ELISA test reacted positively with only BCMV polyclonal antisera, confirming the association of BCMV, which was further established through RT-PCR amplification of coat protein gene using BCMV specific primers. The strain spectrum of BCMV, discerned by inoculation separately of 21 BCMV isolates on international differentials, revealed the existence of three pathogroups - PG I, PG II and PG VII- based on the reaction of isolates on host group 1 to 5 of differential varieties having recessive resistance genes. The pathogroups PG I and PG II were further divisible into four strain groups viz., Ia (i.e., NL-1), Ib (i.e., NL-1n) and IIa (i.e., NL-7), IIb (i.e., NL-7n) , based on the capability to induce veinal necrosis at high temperature (>300C) on differential cultivar ‘Jubila’ possessing dominant gene. The prevalence of pathogroup PG VII (i.e., strain NL4) in the cooler regions of Kashmir recorded during the present studies is the first report of its occurrence in India. In order to characterize viral isolates, molecular characterization of coat protein gene of NL-1, NL-4 and NL-7 strains of BCMV was made using standard methods of molecular biology. RT-PCR of the test strains using specific primer pair amplified a product of ~860 bp. Purified PCR product (DNA) was ligated in PGEM-T easy vector system and transformed in E. coli strain DH5α. The cloned product revealed 860 bp sequences, which were submitted to NCBI gene bank Nucleotide Database under accession number JN692256, JN692258 and JN692257, respectively. The pair-wise homology between the nucleotide sequence of NL-1 and NL-4 was 95 per cent, whereas the homology between NL-1 and NL-4 and between NL-4 and NL-7 was 93 per cent, respectively. The nucleotide sequence of NL-4 from Kashmir showed maximum homology of 96 per cent with BCMV strains EU492546 and AF328753 from India and Mexico, respectively. Phylogenetic analysis of CP sequence of NL-1, NL-4 and NL-7 from Kashmir were most closely related to BCMV strains from H.P. (India), USA and other countries. In all the three strains, DAG motif was present at a/a position 13 of CP which is important for aphid transmission in potyviruses. BCMV specific conserved motif MVWCIDN was also present in all the three strains. Further, eighty-five accessions were evaluated under controlled glass-house conditions against all the three pathogroups (I, II and VI) of BCMV prevalent in Kashmir to find out the sources of resistance against BCMV. Only 17 genotypes were found resistant to the test virus strains. Some of the resistant accessions/cultivars include R-40, R-155, R-3, B-6, Pencil white, SKAU-B-02, SKAU-B-01, Pointed lady, TO, P1207262, TU, KRC-5, R-14, Jubila, Contender and Monroe

Nanotechnology for plant disease management

Each year, 20%–40% of crops are lost due to plant pests and pathogens. Existing plant disease management relies predominantly on toxic pesticides that are potentially harmful to humans and the environment. Nanotechnology can offer advantages to pesticides, like reducing toxicity, improving the shelf-life, and increasing the solubility of poorly water-soluble pesticides, all of which could have positive environmental impacts. This review explores the two directions in which nanoparticles can be utilized for plant disease management: either as nanoparticles alone, acting as protectants; or as nanocarriers for insecticides, fungicides, herbicides, and RNA-interference molecules. Despite the several potential advantages associated with the use of nanoparticles, not many nanoparticle-based products have been commercialized for agricultural application. The scarcity of commercial applications could be explained by several factors, such as an insufficient number of field trials and underutilization of pest–crop host systems. In other industries, nanotechnology has progressed rapidly, and the only way to keep up with this advancement for agricultural applications is by understanding the fundamental questions of the research and addressing the scientific gaps to provide a rational and facilitate the development of commercial nanoproducts

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Not AvailablePotato virus Y (PVY) is a major threat to potato cultivation worldwide. PVY exists as biologically and genetically distinct strains and causes varying degrees of pathogenicity and a wide range of symptoms in potato. Knowledge of the nature of PVY strains is essential for breeding PVY resistant cultivars that are durable against a wide range of strains. We report the complete genome of a PVY potato isolate (JK12) characterised from the potato production areas of Jammu and Kashmir, India. Nucleotide sequence comparisons and phylogenetic analysis with known PVY strains revealed that the isolate belongs to the NTN strain of PVY. At the whole genome sequence level, the JK12 isolate shared the highest identity (99.42%) with PVY-NTN strains reported from Germany, followed by those from United Kingdom (99.34%) and Japan (99.33%). Recombination detection analysis identified two recombination break points and JK12 appeared to have originated from a recombination event between a PVY-N strain from Belgium as a major parent and a PVY-O strain from China as the minor parent. Our results suggest possible mutation and recombination could be the basis for the evolution and the subsequent establishment of NTN in this region. Furthermore, a global evolutionary lineage analysis of all the known PVY strains showed relatively low nucleotide diversity among the PVY-NTN strains. Neutrality tests showed that all the genotypes of PVY are undergoing purifying selection suggesting population expansion of PVY. This is the first report of complete genomic characterization of an NTN strain of PVY isolated from commercial potato fields in India. The implications of the emergence of this strain in the Indian context are discussed.Not Availabl

Image_4_Viral metatranscriptomic approach to study the diversity of virus(es) associated with Common Bean (Phaseolus vulgaris L.) in the North-Western Himalayan region of India.JPEG

Plant viruses are a major threat to legume production worldwide. In recent years, new virus strains have emerged with increasing frequencies in various legume cropping systems, which demands the development of cutting-edge virus surveillance techniques. In this study, we surveyed the common bean fields of Kashmir valley for virus infection using a total of 140 symptomatic and non-symptomatic leaf samples collected from different locations. The genetic diversity of viruses was examined by high-throughput sequencing (HTS) with three viruses being identified, namely, Bean Common Mosaic Virus (BCMV), Bean Common Mosaic Necrosis Virus (BCMNV), and Clover Yellow Vein Virus (ClYVV). BCMNV and ClYVV are new reports from India. De novo assembly of transcriptome constructed near-complete genomes of these viruses. RT-PCR results confirmed the presence of these viruses with an emerge incidence of 56. 4% for BCMV, 27.1% for BCMNV and 16.4 for ClYVV in the valley. Several samples were found to contain multiple virus infections with BCMV being the most predominant. Recombination events were detected in the genomes of BCMV and ClYVV, but not BCMNV. Phylogenetic and pairwise identity matrix evidence suggests viral import from multiple countries. Our results demonstrate that HTS followed by multiplex PCR assay is a simple, rapid, and reliable approach for simultaneous diagnosis of plant viruses.</p

Image_3_Viral metatranscriptomic approach to study the diversity of virus(es) associated with Common Bean (Phaseolus vulgaris L.) in the North-Western Himalayan region of India.TIF

Plant viruses are a major threat to legume production worldwide. In recent years, new virus strains have emerged with increasing frequencies in various legume cropping systems, which demands the development of cutting-edge virus surveillance techniques. In this study, we surveyed the common bean fields of Kashmir valley for virus infection using a total of 140 symptomatic and non-symptomatic leaf samples collected from different locations. The genetic diversity of viruses was examined by high-throughput sequencing (HTS) with three viruses being identified, namely, Bean Common Mosaic Virus (BCMV), Bean Common Mosaic Necrosis Virus (BCMNV), and Clover Yellow Vein Virus (ClYVV). BCMNV and ClYVV are new reports from India. De novo assembly of transcriptome constructed near-complete genomes of these viruses. RT-PCR results confirmed the presence of these viruses with an emerge incidence of 56. 4% for BCMV, 27.1% for BCMNV and 16.4 for ClYVV in the valley. Several samples were found to contain multiple virus infections with BCMV being the most predominant. Recombination events were detected in the genomes of BCMV and ClYVV, but not BCMNV. Phylogenetic and pairwise identity matrix evidence suggests viral import from multiple countries. Our results demonstrate that HTS followed by multiplex PCR assay is a simple, rapid, and reliable approach for simultaneous diagnosis of plant viruses.</p

Table_1_Viral metatranscriptomic approach to study the diversity of virus(es) associated with Common Bean (Phaseolus vulgaris L.) in the North-Western Himalayan region of India.DOCX

Plant viruses are a major threat to legume production worldwide. In recent years, new virus strains have emerged with increasing frequencies in various legume cropping systems, which demands the development of cutting-edge virus surveillance techniques. In this study, we surveyed the common bean fields of Kashmir valley for virus infection using a total of 140 symptomatic and non-symptomatic leaf samples collected from different locations. The genetic diversity of viruses was examined by high-throughput sequencing (HTS) with three viruses being identified, namely, Bean Common Mosaic Virus (BCMV), Bean Common Mosaic Necrosis Virus (BCMNV), and Clover Yellow Vein Virus (ClYVV). BCMNV and ClYVV are new reports from India. De novo assembly of transcriptome constructed near-complete genomes of these viruses. RT-PCR results confirmed the presence of these viruses with an emerge incidence of 56. 4% for BCMV, 27.1% for BCMNV and 16.4 for ClYVV in the valley. Several samples were found to contain multiple virus infections with BCMV being the most predominant. Recombination events were detected in the genomes of BCMV and ClYVV, but not BCMNV. Phylogenetic and pairwise identity matrix evidence suggests viral import from multiple countries. Our results demonstrate that HTS followed by multiplex PCR assay is a simple, rapid, and reliable approach for simultaneous diagnosis of plant viruses.</p

Image_1_Viral metatranscriptomic approach to study the diversity of virus(es) associated with Common Bean (Phaseolus vulgaris L.) in the North-Western Himalayan region of India.JPEG

Plant viruses are a major threat to legume production worldwide. In recent years, new virus strains have emerged with increasing frequencies in various legume cropping systems, which demands the development of cutting-edge virus surveillance techniques. In this study, we surveyed the common bean fields of Kashmir valley for virus infection using a total of 140 symptomatic and non-symptomatic leaf samples collected from different locations. The genetic diversity of viruses was examined by high-throughput sequencing (HTS) with three viruses being identified, namely, Bean Common Mosaic Virus (BCMV), Bean Common Mosaic Necrosis Virus (BCMNV), and Clover Yellow Vein Virus (ClYVV). BCMNV and ClYVV are new reports from India. De novo assembly of transcriptome constructed near-complete genomes of these viruses. RT-PCR results confirmed the presence of these viruses with an emerge incidence of 56. 4% for BCMV, 27.1% for BCMNV and 16.4 for ClYVV in the valley. Several samples were found to contain multiple virus infections with BCMV being the most predominant. Recombination events were detected in the genomes of BCMV and ClYVV, but not BCMNV. Phylogenetic and pairwise identity matrix evidence suggests viral import from multiple countries. Our results demonstrate that HTS followed by multiplex PCR assay is a simple, rapid, and reliable approach for simultaneous diagnosis of plant viruses.</p

Image_2_Viral metatranscriptomic approach to study the diversity of virus(es) associated with Common Bean (Phaseolus vulgaris L.) in the North-Western Himalayan region of India.JPEG

Plant viruses are a major threat to legume production worldwide. In recent years, new virus strains have emerged with increasing frequencies in various legume cropping systems, which demands the development of cutting-edge virus surveillance techniques. In this study, we surveyed the common bean fields of Kashmir valley for virus infection using a total of 140 symptomatic and non-symptomatic leaf samples collected from different locations. The genetic diversity of viruses was examined by high-throughput sequencing (HTS) with three viruses being identified, namely, Bean Common Mosaic Virus (BCMV), Bean Common Mosaic Necrosis Virus (BCMNV), and Clover Yellow Vein Virus (ClYVV). BCMNV and ClYVV are new reports from India. De novo assembly of transcriptome constructed near-complete genomes of these viruses. RT-PCR results confirmed the presence of these viruses with an emerge incidence of 56. 4% for BCMV, 27.1% for BCMNV and 16.4 for ClYVV in the valley. Several samples were found to contain multiple virus infections with BCMV being the most predominant. Recombination events were detected in the genomes of BCMV and ClYVV, but not BCMNV. Phylogenetic and pairwise identity matrix evidence suggests viral import from multiple countries. Our results demonstrate that HTS followed by multiplex PCR assay is a simple, rapid, and reliable approach for simultaneous diagnosis of plant viruses.</p