Colletotrichum spp. cause anthracnose disease in several plant species in tropical and temperate ... more Colletotrichum spp. cause anthracnose disease in several plant species in tropical and temperate regions. In this study, a total of 30 isolates of Colletotrichum spp. were isolated from nine different fruit crops showing anthracnose symptoms. Phylogenetic analysis based on the GAPDH gene divided the population into four primary clades. All C. gloeosporioides species complex grouped together and separated from C. karstii and two different monophyletic lineages i.e. C. brevisporum, C. cliviae. C. gloeosporioides species complex was found dominant within the population containing 7 cryptic species. To study the population diversity of Colletotrichum species, the isolates were grouped into 4 geographical clusters based on their collection site i.e. Delhi, Maharashtra, West Bengal (WB), and North East (NE) states (Assam and Meghalaya). Out of 12 ISSR primers used 11 primers generated scorable polymorphic band of 100-1300 bp having average PIC value of 0.27. The intrapopulation genetic di...
The cultivation of dolichos bean [Lablab purpureus (L.) Sweet] has been severely affected by doli... more The cultivation of dolichos bean [Lablab purpureus (L.) Sweet] has been severely affected by dolichos yellow mosaic virus (DoYMV, Begomovirus) transmitted by whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). We tested the transovarial transmission of DoYMV in next-generation B. tabaci by PCR, real-time PCR, Southern blot hybridization, and biological transmission. The eggs, laid by DoYMV-exposed B. tabaci, carry the virus in a unique pattern. Only the eggs laid in between 3 and 6 days post virus acquisition by a parent B. tabaci were DoYMV positive. When tested individually in real-time PCR, around 31–53% of the eggs carried the virus. The presence of DoYMV in ovaries and F1 eggs was further substantiated by the hybridization of a Cy3-conjugated nucleic acid probe complementary to the viral strand of DoYMV. Viral DNA was also detected in F1 adults and F2 eggs. B. tabaci progenies carried not only the DoYMV DNA but were also infective. The F1 adults transmitted DoYMV to all tested p...
Cardamom bushy dwarf virus (CBDV) causes foorkey disease of large cardamom (Ammomum subulatum Rox... more Cardamom bushy dwarf virus (CBDV) causes foorkey disease of large cardamom (Ammomum subulatum Roxburgh) in the eastern sub-Himalayan mountains. Although the aphid Micromyzus kalimpongensis Basu (Hemiptera: Aphididae) is known as a vector of CBDV, its behavior in dissemination of CBDV has not been investigated. In the present study, M. kalimpongensis was observed to colonize in higher number on CBDV-infected large cardamom plants compared with the healthy plants in the several plantations in Sikkim and Darjeeling hills. The affinity of M. kalimpongensis to the diseased large cardamom plants was further confirmed in a contained field experiment with intact plant as well as in a laboratory bioassay with the plant extract, where significantly higher number of aphids settled on the diseased plants or extracts compared with the healthy counterparts. Aphids grown on CBDV-infected large cardamom plants had shortened nymphal period and increased longevity and fecundity compared with those gr...
ABSTRACT Darjeeling hills of West Bengal (India) are well known for the production of mandarin or... more ABSTRACT Darjeeling hills of West Bengal (India) are well known for the production of mandarin orange (Citrus reticulata). The spread of Citrus tristeza virus (CTV) in this area has threatened the cultivation of citrus. Therefore, a study of the incidence and distribution of CTV in Darjeeling hills was undertaken and a geographical map was prepared. In DAS-ELISA, a high titre of CTV was recorded in the tender shoot bark, leaf petiole and mid-rib of seven Citrus spp. Three citrus species, viz., pomelo, trifoliate and kumquat, were found to be free of CTV infection when tested in double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcription polymerase chain reaction (RT-PCR).
ABSTRACT Darjeeling and Sikkim hills of India are well known for production of mandarin orange (C... more ABSTRACT Darjeeling and Sikkim hills of India are well known for production of mandarin orange (Citrus reticulata). The recent spread of Citrus tristeza virus (CTV) has threatened the citrus cultivation in this region. During a survey in Darjeeling and Sikkim hills, pomelo trees were recorded as CTV free. Since pomelo trees did not show any disease appearance, a study was undertaken to ascertain whether they are resistant to CTV infection or resistant to aphid feeding or both. Toxoptera citricida, the most efficient aphid vector and which is abundantly present in this region, did not feed on pomelo when other Citrus species such as mandarin, kagzi lime and rough lemon were available. Additionally, CTV isolates of Darjeeling and Sikkim hills were not transmissible to pomelo either by T. citricida or grafting. We report for the first time that pomelo is resistant to isolates of CTV present in this region.
Sikkim and Darjeeling hills of India are the largest producer and exporter of large cardamom. The... more Sikkim and Darjeeling hills of India are the largest producer and exporter of large cardamom. The two aphid-transmitted viruses, cardamom bushy dwarf virus (CBDV, genus Babuvirus) and large cardamom chirke virus (LCCV, genus Macluravirus), seriously affect the production of large cardamom. In the present study, three aphid species, Micromyzus kalimpongensis Basu, Pentalonia nigronervosa Coquerel and Aulacorthum solani Kalt. were identified and documented to colonize large cardamom in the different plantations based on survey, morphometrics and COI gene sequence-based phylogeny. A. solani was recorded in large cardamom for the first time in this study. The temporal occurrence and distribution of these three aphid species have been documented. M. kalimpongensis, a root feeding aphid was consistently present in large cardamom plantations throughout the year; predominantly on high altitude (>900 m) plantations. P. nigronervosa was predominant in plantations <1000 m altitude, and the population was higher during the summer season only. The incidence of A. solani was observed during the winter season only. All the aphid species infesting large cardamom were tested for their ability to transmit the two viruses, CBDV, and LCCV. Only, M. kalimpongensis but not the other two species of aphid could transmit CBDV to large cardamom. P. nigronervosa transmitted only LCCV, however, the rate of transmission was low. A. solani could not transmit any one of these viruses.
Darjeeling hills and Dooars of West Bengal (India) are well known for production of mandarin oran... more Darjeeling hills and Dooars of West Bengal (India) are well known for production of mandarin orange and lime. Citrus tristeza virus (CTV) is wide spread in this region. Role of insect vectors in spreading CTV in this region has not been studied so far. Therefore, a study on identification of insect vectors along with their temporal and spatial distribution was undertaken. Five aphid species were identified from citrus orchards of Darjeeling and Dooars viz. Toxoptera citricida, T. aurantii, Aphis gossypii, Myzus persicae and Brachycaudus helichrysi. T. citricida was found predominant in the orchards of lower altitude and was responsible for maximum spread of CTV. T. aurantii was dominant in the citrus orchards at high altitude (N 500 m). Incidence of CTV was higher in the orchards where T. citricida was present either alone or with other species. Under caged conditions, T. citricida was more efficient to transmit CTV than the other aphid species. Occurrences of all aphid species were highly influenced by the advent of new flushes.
Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constr... more Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constrain in large cardamom production in India. Purification of LCCV from large cardamom tissues is difficult and therefore immunodiagnostic reagents are not available. In the present study, we have successfully expressed coat protein (CP) gene of LCCV in Escherichia coli. The purification of expressed protein by Ni-NTA affinity chromatography was inefficient due to precipitation of protein during renatur-ation. We have optimized a simple, inexpensive and efficient method for purification of the expressed CP through gel extraction with 5 % SDS followed by renaturation in Milli-Q water, which resulted in high yield (4.7 mg/ml) and good quality of the protein. A higher titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced, which strongly recognized LCCV in crude leaf extract and showed minimal background reaction with the healthy leaf extract in enzyme linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The sensitivities of the ELISA and DIBA were 5 and 0.1 ng of expressed protein, respectively. Both the ELISA and DIBA were validated with 100 % accuracy in detecting LCCV in field samples. The PAb differentiated Cardamom mosaic virus, another close relative of LCCV. Our study is first to report highly efficient immunodiagnosis with PAb to E. coli expressed recombinant CP of a virus under the genus Macluravirus. The antigen expression construct and PAb developed in the present study will be useful in production of virus free planting materials of large cardamom. Keywords Large cardamom chirke virus Á LCCV Á Coat protein gene expression Á ELISA Á DIBA
Colletotrichum spp. cause anthracnose disease in several plant species in tropical and temperate ... more Colletotrichum spp. cause anthracnose disease in several plant species in tropical and temperate regions. In this study, a total of 30 isolates of Colletotrichum spp. were isolated from nine different fruit crops showing anthracnose symptoms. Phylogenetic analysis based on the GAPDH gene divided the population into four primary clades. All C. gloeosporioides species complex grouped together and separated from C. karstii and two different monophyletic lineages i.e. C. brevisporum, C. cliviae. C. gloeosporioides species complex was found dominant within the population containing 7 cryptic species. To study the population diversity of Colletotrichum species, the isolates were grouped into 4 geographical clusters based on their collection site i.e. Delhi, Maharashtra, West Bengal (WB), and North East (NE) states (Assam and Meghalaya). Out of 12 ISSR primers used 11 primers generated scorable polymorphic band of 100-1300 bp having average PIC value of 0.27. The intrapopulation genetic di...
The cultivation of dolichos bean [Lablab purpureus (L.) Sweet] has been severely affected by doli... more The cultivation of dolichos bean [Lablab purpureus (L.) Sweet] has been severely affected by dolichos yellow mosaic virus (DoYMV, Begomovirus) transmitted by whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). We tested the transovarial transmission of DoYMV in next-generation B. tabaci by PCR, real-time PCR, Southern blot hybridization, and biological transmission. The eggs, laid by DoYMV-exposed B. tabaci, carry the virus in a unique pattern. Only the eggs laid in between 3 and 6 days post virus acquisition by a parent B. tabaci were DoYMV positive. When tested individually in real-time PCR, around 31–53% of the eggs carried the virus. The presence of DoYMV in ovaries and F1 eggs was further substantiated by the hybridization of a Cy3-conjugated nucleic acid probe complementary to the viral strand of DoYMV. Viral DNA was also detected in F1 adults and F2 eggs. B. tabaci progenies carried not only the DoYMV DNA but were also infective. The F1 adults transmitted DoYMV to all tested p...
Cardamom bushy dwarf virus (CBDV) causes foorkey disease of large cardamom (Ammomum subulatum Rox... more Cardamom bushy dwarf virus (CBDV) causes foorkey disease of large cardamom (Ammomum subulatum Roxburgh) in the eastern sub-Himalayan mountains. Although the aphid Micromyzus kalimpongensis Basu (Hemiptera: Aphididae) is known as a vector of CBDV, its behavior in dissemination of CBDV has not been investigated. In the present study, M. kalimpongensis was observed to colonize in higher number on CBDV-infected large cardamom plants compared with the healthy plants in the several plantations in Sikkim and Darjeeling hills. The affinity of M. kalimpongensis to the diseased large cardamom plants was further confirmed in a contained field experiment with intact plant as well as in a laboratory bioassay with the plant extract, where significantly higher number of aphids settled on the diseased plants or extracts compared with the healthy counterparts. Aphids grown on CBDV-infected large cardamom plants had shortened nymphal period and increased longevity and fecundity compared with those gr...
ABSTRACT Darjeeling hills of West Bengal (India) are well known for the production of mandarin or... more ABSTRACT Darjeeling hills of West Bengal (India) are well known for the production of mandarin orange (Citrus reticulata). The spread of Citrus tristeza virus (CTV) in this area has threatened the cultivation of citrus. Therefore, a study of the incidence and distribution of CTV in Darjeeling hills was undertaken and a geographical map was prepared. In DAS-ELISA, a high titre of CTV was recorded in the tender shoot bark, leaf petiole and mid-rib of seven Citrus spp. Three citrus species, viz., pomelo, trifoliate and kumquat, were found to be free of CTV infection when tested in double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcription polymerase chain reaction (RT-PCR).
ABSTRACT Darjeeling and Sikkim hills of India are well known for production of mandarin orange (C... more ABSTRACT Darjeeling and Sikkim hills of India are well known for production of mandarin orange (Citrus reticulata). The recent spread of Citrus tristeza virus (CTV) has threatened the citrus cultivation in this region. During a survey in Darjeeling and Sikkim hills, pomelo trees were recorded as CTV free. Since pomelo trees did not show any disease appearance, a study was undertaken to ascertain whether they are resistant to CTV infection or resistant to aphid feeding or both. Toxoptera citricida, the most efficient aphid vector and which is abundantly present in this region, did not feed on pomelo when other Citrus species such as mandarin, kagzi lime and rough lemon were available. Additionally, CTV isolates of Darjeeling and Sikkim hills were not transmissible to pomelo either by T. citricida or grafting. We report for the first time that pomelo is resistant to isolates of CTV present in this region.
Sikkim and Darjeeling hills of India are the largest producer and exporter of large cardamom. The... more Sikkim and Darjeeling hills of India are the largest producer and exporter of large cardamom. The two aphid-transmitted viruses, cardamom bushy dwarf virus (CBDV, genus Babuvirus) and large cardamom chirke virus (LCCV, genus Macluravirus), seriously affect the production of large cardamom. In the present study, three aphid species, Micromyzus kalimpongensis Basu, Pentalonia nigronervosa Coquerel and Aulacorthum solani Kalt. were identified and documented to colonize large cardamom in the different plantations based on survey, morphometrics and COI gene sequence-based phylogeny. A. solani was recorded in large cardamom for the first time in this study. The temporal occurrence and distribution of these three aphid species have been documented. M. kalimpongensis, a root feeding aphid was consistently present in large cardamom plantations throughout the year; predominantly on high altitude (>900 m) plantations. P. nigronervosa was predominant in plantations <1000 m altitude, and the population was higher during the summer season only. The incidence of A. solani was observed during the winter season only. All the aphid species infesting large cardamom were tested for their ability to transmit the two viruses, CBDV, and LCCV. Only, M. kalimpongensis but not the other two species of aphid could transmit CBDV to large cardamom. P. nigronervosa transmitted only LCCV, however, the rate of transmission was low. A. solani could not transmit any one of these viruses.
Darjeeling hills and Dooars of West Bengal (India) are well known for production of mandarin oran... more Darjeeling hills and Dooars of West Bengal (India) are well known for production of mandarin orange and lime. Citrus tristeza virus (CTV) is wide spread in this region. Role of insect vectors in spreading CTV in this region has not been studied so far. Therefore, a study on identification of insect vectors along with their temporal and spatial distribution was undertaken. Five aphid species were identified from citrus orchards of Darjeeling and Dooars viz. Toxoptera citricida, T. aurantii, Aphis gossypii, Myzus persicae and Brachycaudus helichrysi. T. citricida was found predominant in the orchards of lower altitude and was responsible for maximum spread of CTV. T. aurantii was dominant in the citrus orchards at high altitude (N 500 m). Incidence of CTV was higher in the orchards where T. citricida was present either alone or with other species. Under caged conditions, T. citricida was more efficient to transmit CTV than the other aphid species. Occurrences of all aphid species were highly influenced by the advent of new flushes.
Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constr... more Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constrain in large cardamom production in India. Purification of LCCV from large cardamom tissues is difficult and therefore immunodiagnostic reagents are not available. In the present study, we have successfully expressed coat protein (CP) gene of LCCV in Escherichia coli. The purification of expressed protein by Ni-NTA affinity chromatography was inefficient due to precipitation of protein during renatur-ation. We have optimized a simple, inexpensive and efficient method for purification of the expressed CP through gel extraction with 5 % SDS followed by renaturation in Milli-Q water, which resulted in high yield (4.7 mg/ml) and good quality of the protein. A higher titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced, which strongly recognized LCCV in crude leaf extract and showed minimal background reaction with the healthy leaf extract in enzyme linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The sensitivities of the ELISA and DIBA were 5 and 0.1 ng of expressed protein, respectively. Both the ELISA and DIBA were validated with 100 % accuracy in detecting LCCV in field samples. The PAb differentiated Cardamom mosaic virus, another close relative of LCCV. Our study is first to report highly efficient immunodiagnosis with PAb to E. coli expressed recombinant CP of a virus under the genus Macluravirus. The antigen expression construct and PAb developed in the present study will be useful in production of virus free planting materials of large cardamom. Keywords Large cardamom chirke virus Á LCCV Á Coat protein gene expression Á ELISA Á DIBA
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