Development of conventional and real-time PCR protocols for specific and sensitive detection of Colletotrichum capsici in chilli (Capsicum annuum L.)

Anthracnose, caused by Colletotrichum capsici, is a major disease of chilli (Capsicum annuum L.) affecting both fruit and seed quality. The pathogen is both internally and externally seedborne. However, a rapid and sensitive method for detection of this pathogen in seeds is currently limited. In this study, a polymerase chain reaction (PCR) method based on sequence characterized amplified region (SCAR) marker was developed for specific and sensitive detection of C. capsici in chilli seeds and fruits. The developed SCAR primers were highly specific to C. capsici and resulted in the amplification of an expected 250-bp fragment from genomic DNA of all seven of the C. capsici isolates tested. No amplification occurred when the SCAR primers were tested with genomic DNA from three other fungal isolates and four other Colletotrichum species. The SCAR primers successfully amplified similar sized fragments from DNA derived from C. capsici-infected chilli fruits. The molecular detection sensitivity of C. capsici was 1 pg of purified C. capsici DNA template and 25 ng of DNA from C. capsici-infected chilli fruits. A real-time PCR assay was also developed using SYBR Green chemistry for detection of C. capsici in chilli fruits and seeds. The standard curve obtained showed a linear correlation between copy number of the cloned target DNA sequence of C. capsici and cycle threshold (Ct) values, with R² of 0.98. These PCR-based assays may be highly useful in detection of this important pathogen in chilli seeds and fruits in plant quarantine laboratories.     

Molecular phylogeny in Indian Citrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA

The taxonomy and phylogeny of Indian Citrus is revisited using PCR-RFLP of the trnD-trnT and rbcL-ORF 106 regions as well as sequence data analysis of the trnL-trnF intergenic spacer region of cpDNA. The study was based on 50 accessions of Citrus genotypes, collected from wild, semi-wild and domesticated stocks. Of the 13 restriction enzymes (RE) used for restriction digestion of the polymerase chain reaction (PCR) amplicons, four (Hinf I, Msp I, Alu I, Hae III) generated 47 restriction fragments, of which 24 (51%) were polymorphic. PCR-RFLP data showed a genetic distance ranging from 0 to 0.79 among 50 accessions of Citrus, and a cluster analysis, based on Neighbor-Joining (NJ) method, placed all the accessions in eight major clusters. Analysis of trnL-trnF sequences from 23 representative accessions of Citrus showed a pair-wise sequence divergence rate in the range of 0–0.064. NJ, minimum evolution (ME) and maximum parsimony (MP) analyses of trnL-trnF sequences produced phylogenetic trees, which placed all the 23 accessions in five clusters. PCR-RFLP analysis resulted in a well resolved phylogenetic tree with branches supported by moderate to high bootstrap values, while the trnL-trnF sequence-based trees showed only moderate to low bootstrap support for the internal tree branches, indicating uncertain origin of some Citrus genotypes. This study shows that the trnL-trnF spacer sequence data can detect genetic variation in Indian Citrus genotypes, but the utility of the data in inferring phylogeny at intra and inter-specific levels is limited probably by factors such as hybridization, bud mutations, apomixis and polyploidy. However, PCR-RFLP and trnL-trnF data supported the recognition of C. maxima, C. medica, and C. reticulata as the basal species of edible Citrus.     

Bovine serum panel for evaluating foot-and-mouth disease virus nonstructural protein antibody tests.

A panel of 36 sera has been assembled from experimental cattle that had been infected by inoculation or contact exposure with 4 serotypes of foot-and-mouth disease virus (FMDV) with or without prior vaccination. Virus replication and persistence had been characterized in all of the animals. The proportion of the sera scored positive by 5 tests for antibodies to the nonstructural proteins of FMDV varied, suggesting that the panel can discriminate between the sensitivity with which such tests are able to identify infected cattle. Use of this panel will help in assessment of new tests and quality control of existing methods.     

Assessment of genetic diversity in cabbage cultivars using RAPD and SSR markers

Genetic relationship and diversity among seven cabbage cultivars were analyzed using RAPD and SSR markers. These cultivars are of great commercial value in India and are confirmed for their reaction to black rot caused by Xanthomonas campestris pv. campestris. However, so far the extent of genetic diversity and relatedness has not been studied in these cultivars. A total of 17 selected RAPD primers generated 90 bands, 76 of which were polymorphic (84.44%). In addition, 27 selected SSR primers generated 67 amplified bands with 59 of which were polymorphic (87.6%). Though both the marker techniques were able to discriminate the cultivars effectively, analysis of combined data of markers (RAPD and SSR) resulted in better distinction of cultivars. By combining both the markers, a total of 157 bands were detected of which 135 bands (85.98%) were polymorphic, i.e. an average of 5.95 bands per primer. High level of polymorphism (> 85%) recorded with two different marker systems indicated a high level of genetic variation existing among the cultivars. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pairs of cultivars varied from 0.21 to 0.77 in RAPD, 0.42 to 0.82 in SSR, and 0.43 to 0.89 with combined markers. A high correspondence had been recorded between the values of genetic variations generated by UPGMA, clustering, and scatter plot diagrams. The cultivars ‘January King Sel. Improved’ and ‘Golden Acre’ are highly divergent cultivars as demonstrated by both the marker systems.     

Effect of zinc application methods on apparent utilization efficiency of zinc and phosphorus fertilizers under basmati rice–wheat rotation

To examine the effect of zinc (Zn) application method on the utilization of phosphorus (P) from applied P fertilizer, a field experiment was conducted on basmati rice–wheat rotation with combinations of Zn levels (0, soil application of 2.5 kg Zn ha ^{– 1} and two foliar applications of 2.0 kg Zn ha ^–1) and P levels (0, soil application of 8.7, 17.5 and 26.2 kg P ha ^–1). The highest pooled grain yields of basmati rice and wheat were obtained with soil application of 17.5 kg P ha ^–1 and foliar applications of 2 kg Zn ha ^–1. Foliar applications of Zn increased the P concentration in grain and straw and the total P uptake by basmati rice and the P concentration in flag leaves of wheat significantly, while soil or foliar application of Zn increased the total P uptake of wheat. Phosphorus application increased the Zn concentration in flag leaves, grain and straw of basmati rice and in grain and straw of wheat and the total Zn uptake of both crops. Phosphorus levels up to 17.5 kg P ha ^–1 increased utilization efficiency of soil or foliar application of Zn. Zinc application increased the P utilization efficiency of basmati rice and wheat up to 17.5 kg P ha ^–1 level; foliar Zn application was more effective in a wheat crop than a rice crop.     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.     

Surfactant effects on application of a hydrophobic, fluoropolymer coating to cotton by admicellar polymerization

Various methods have been used previously to impart hydrophobicity to cotton. In this study, thin co-polymer films of trifluoroethyl acrylate (TFEA) and octafluoro pentyl methacrylate (OFPM) were formed on cotton using individual fluorosurfactants and surfactant mixtures through the process of admicellar polymerization. Efficacy of 6 different fluorosurfactant systems and process conditions including surfactant type, concentration, adsorption/adsolubilization time, polymerization time, and monomer concentration have been investigated in order to obtain hydrophobic coatings on woven cotton fabric. Adsolubilization times as short as 2 h and polymerization times as short as 1 h yielded satisfactory performance. Characterization of the hydrophobic polymer films obtained included static contact angles, SEM imaging, elemental analysis and XPS analysis. Treated fabrics exhibited good water repellency for several surfactants and surfactant mixtures with initial supernatant monomer concentrations of 4 mM. Surfactant mixtures were able to outperform the separate individual species in admicellar polymerization.