Molecular characterisation and structural assessment of an RXLR effector from Phytophthora palmivora, the coconut bud rot pathogen

Phytophthora species are phytopathogenic oomycetes that damage a wide variety of crops. Phytophthora delivers effectors, which are secretory proteins, into the host cells. Effectors promote infection by reprogramming the host cellular machinery and are key determinants of oomycete virulence. The major class of Phytophthora effector proteins contains the RXLR motif. In this study, we have carried out the molecular and structural characterisation of an RXLR effector (RXLR6744) from a virulent P. palmivora isolated from bud rot disease-affected coconut palm. The open reading frame (ORF) of the RXLR6744, amplified using RT-PCR, had a length of 411 bp. The gene was found to encode a predicted protein of 136 amino acids and had a molecular weight of 15.52 kDa. Phylogenetic analysis of the amino acid sequence revealed that it was closely related to RXLR proteins from P. palmivora (causing black pod disease in cocoa) and related species P. megakarya. Topology analysis revealed that the protein was composed of six α-helices. The structural prediction was undertaken by computer-aided homology modelling. From the Ramachandran plot analysis, it could be observed that the majority (96.3%) of amino acids were present in the preferred region, 3.7 per cent of amino acid residues were present in the allowed region, and no residues were observed in the disallowed region. The structure showed an average quality of 94.4 per cent, indicating it to be a high-quality structure. This study provides the detailed characterisation of an RXLR effector from P. palmivora. It will aid the elucidation of its role in pathogenesis and facilitate further refined investigations of the structure/function relationships of oomycete effectors

LTTRPred: A tool for prediction of LysR-type transcriptional regulator of pyoluteorin pathway in plant growth promoting Pseudomonas spp.

Plant growth promoting Pseudomonas spp. produce an antifungal compound called pyoluteorin (Plt) that suppress diseases caused by phytopathogenic fungi. The pathway specific regulator PltR, a typical LysR-type transcriptional regulator (LTTR), is responsible for the transcriptional activation of the Plt biosynthetic operon. The LTTR family represents one of the largest classes of bacterial transcriptional regulatory proteins. A large number of LTTRs possess function as global transcriptional activators or repressors of unlinked genes or operons involved in metabolism, quinoline signal, virulence etc. The proposed method, LTTRPred, is an useful tool developed for identifying and predicting the LTTR, which is responsible for the activation of Plt transcription regulators, from whole genomes of various Pseudomonas spp. LTTRPred was developed using support vector machine (SVM) and Waikato Environment for Knowledge Analysis (WEKA) based on the composition of amino acid and amino acid pairs. Modules in SVM were developed using traditional amino acid, dipeptide (n+1) and hybrid amino acid composition modules and an overall accuracy of 100, 100 and 98 per cent respectively, was achieved. Modules in WEKA were also developed using the same modules and an overall accuracy of 100 per cent achieved for all. The performance of the tool was tested using various datasets of LTTR genes from different Pseudomonas spp. The best performing SVM and WEKA modules from the present investigation was implemented as a dynamic web server ‘LTTRPred’, which is freely available and can be accessed online (http://210.212.229.56/lttrpred/). This tool can be used for the functional annotation of the Pseudomonas spp. possessing LTTR genes

Assessment of morphological, physiological and molecular characteristics of cocoa accessions from Central and South America in relation to drought tolerance

Eleven cocoa accessions, representing collections from five central and south American countries, were assessed for their morphological, molecular and physiological parameters. Growth characters were observed in three year old plants and initial pod yields were recorded. Photosynthesis, related parameters and chlorophyll indices, measured during two seasons, showed significant differences between non-stress and stress periods as well as among the genotypes. The transpirational water loss was reduced with increased stomatal closure, which is a favourable drought trait in crops. The results indicated that the genotypes showing higher water potential and Fv/Fm ratio can be considered as drought tolerant. The rank sums of these parameters showed that genotypes JA-1/19, POU-16/A and SC-4 were the most drought-tolerant. Microsatellite markers were used to assess the extent of genetic diversity between clones. The amplification of DNA from the 11 accessions using the 15 microsatellite loci revealed a total of 80 consistent and scorable alleles with an average of 5.33 alleles per locus and all the loci were 100 per cent polymorphic, the most polymorphic locus being mTcCIR33 with 8 alleles. The observed heterozygosity ranged from 0.36 to 0.63 with an average of 0.52. The inbreeding co-efficient (f) ranged from -0.22 (mTcCIR8) to 0.58 (mTcCIR40) with an average of 0.32. The microsatellite marker analysis revealed that the genotypes possess a wide genetic diversity. The drought tolerant types identified in this study viz., JA-1/19, POU-16/A and SC-4 could be used for cultivation in areas with moisture deficient stress and in selective cocoa breeding programs for drought tolerance

Cryopreservation of coconut plumule using droplet vitrification

In the present investigation, four types of explants from mature zygotic embryos of coconut, viz., whole upper cotyledonary region without haustorium, half of the upper cotyledonary region without haustorium, plumule with a portion of radicle and exclusively plumular tissue, were cultured in 12 different media combinations to find a suitable explant which could be regenerated after cryopreservation. Explants were pre-cultured in medium with 0.4 and 0.5 M sucrose for three days followed by dehydration in PVS3 solution for different durations on a sterile aluminum strip after treating with loading solution. Strips were treated with liquid nitrogen inside a cryoflask until bubbling stopped and quickly transferred to a cryovial and stored for a minimum period of 24 hours in liquid nitrogen. It was observed that plumule alone or with a small portion of outer tissue was ideal for fast in vitro growth and recovery of whole plantlets of coconut in a medium supplemented with NAA alone. Addition of glutamine (5 mg L-1), TDZ (1 mg L-1) and NAA (18 mg L-1) aided the vigorous growth of plantlets. In control, the survival rate ranged from 60 to 90 per cent in plumule pre-grown in media containing 0.5 M sucrose after dehydration with PVS3 for various durations, whereas it was 14 to 75 per cent in cryopreserved ones. Considering the high survival (75%) and regrowth (35%) of cryopreserved plumule in the present study, there is much scope for further improvement of the procedure to find the right combination of factors so as to enhance complete recovery of plantlets without much injury to plumules during cooling and rewarming

Sustainability of Farmer Producer Organisations - The case of producer organisations involved in the production and marketing of ‘neera’ in the coconut sector

Neera-the coconut inflorescence sap has been promoted as a potential value-added non-alcoholic beverage with abundant health benefits. The concerted effort of the stakeholder agencies could liberate the product from the policy regulations on its production under the excise act (known as ‘Abkari act’ in Kerala). Subsequently, the coconut producer federations (CPFs) in the State were granted licenses for ‘neera’ production and marketing. However, the majority of the CPFs discontinued the ‘neera’ enterprise, which they have initiated with greater enthusiasm. The present study is a modest attempt to trace the reasons for setbacks experienced in the ‘neera’ sector and also to provide a refined framework for revamping the sector. The details of licenses granted to the CPFs were sourced from the Department of Excise, Government of Kerala. The sample size of the study was 95 (13 CPFs who are still active and 82 CPFs who have discontinued). It was observed that the ‘neera’ value chain is in the evolving stage, and the withdrawal of the institutional support had detrimentally affected the confidence of the CPFs ventured into it. The availability of ‘neera’ technicians (tappers) and the high wage rates have evoked concerns on the profitability as well as the assurance of continuous supply of the product. The study categorically highlights the need for a restructured value chain of ‘neera’ with specific roles assigned to the stakeholders through the creation of functional linkages

Characterization of Kuttiyadi ecotype of coconut (Cocos nucifera L.) using morphological and microsatellite markers

West Coast Tall (WCT) is the most popular coconut cultivar grown by the farmers in Kerala, which occupies over 95 per cent of the area under coconut. The long history of coconut cultivation throughout Kerala state has resulted in the development of many ecotypes of WCT. The present work compares the similarity/diversity of the morphological and molecular characteristics of the Kuttiyadi ecotype growing in the hilly, midland region of Kozhikode District, Kerala with those of the WCT cultivar of the coastal region of Kasaragod District, Kerala, using vegetative, reproductive and fruit component characters and microsatellite markers. Geographically, these two locations show a wide range of variation for soil and climactic factors. The vegetative, reproductive and fruit component characteristics and microsatellite markers showed wide variations between selected WCT palms from Kasaragod and Kuttiyadi. The similarity index based on Dice’s coefficient, obtained after pair-wise comparison of Kuttiyadi and WCT samples with 15 SSR markers, revealed that the percentage similarity varied from the coefficient range 0.20 to 0.97 between the WCT and Kuttiyadi palms. UPGMA clustering clearly distinguished the two populations with WCT and Kuttiyadi forming separate clusters. STRUCTURE analysis was also carried out, which also showed that the two populations studied were distinct

Characterization of gibberellin 2-oxidase isoforms in coconut (Cocos nucifera L.)

Gibberellins (GAs) are plant hormones that are essential for many developmental processes in plants, including seed germination, stem elongation, leaf expansion, trichome development, pollen maturation and the induction of flowering. Gibberellin 2-oxidase (GA2-ox) regulates plant growth by inactivating endogenous bioactive GAs through 2β-hydroxylation. There is no information about GA2-ox encoding genes or their functions in coconut. In this study, we have identified 10 transcripts encoding different isoforms of GA2-ox from coconut leaf transcriptome data. Sequence comparison and phylogenetic analysis revealed that these 10 transcripts represented different types of GA2-ox. The secondary structure, three dimensional structure and active sites of these 10 isoforms were predicted. Docking studies of different active GAs with these isoforms was also carried out

Evaluation of molecular diversity of ex situ conserved germplasm of palmyrah (Borassus flabellifer L.) accessions using RAPD markers

The genetic relationship of 96 palmyrah palms, consisting of 24 indigenous accessions from Tamil Nadu and Andhra Pradesh, was investigated using Random Amplified Polymorphic DNA (RAPD) markers. Hundred and eighty primers were used initially to identify the polymorphic primers in six random samples and 10 polymorphic primers were selected to amplify the 96 palms. These 10 primers produced a total of 112 reproducible bands and out of them, 41 fragments (36.6 %) showed polymorphism.  The number of bands produced with each primer varied from seven to 15 with an average of 11.2 bands per primer.  The percent polymorphism ranged from 7.7 to 71.4 with an average of 37.4 per cent when all the primers were taken collectively.  UPGMA grouped all the accessions into two major clusters at 0.85 similarity value. The highest similarity value (0.96) was observed between the accessions KLKM-8 and THY-54 and the lowest similarity value (0.782) was obtained between ANBI-17 and RCML-11. The relatively low polymorphism suggests a narrow genetic diversity of palmyrah populations from which the present accessions have been derived and maintained over the years

Studies on genetic uniformity of Chowghat Green Dwarf and Malayan Green Dwarf varieties of coconut using molecular and morphometric methods

Two coconut varieties viz., Chowghat Green Dwarf (CGD) and Malayan Green Dwarf (MGD) were subjected to morphometric and molecular studies to assess their genetic uniformity. Since both these varieties possess traits for high yield and resistance to root (wilt) disease, they have already been released for cultivation in the root (wilt) disease prevalent tracts. Forty two CGD palms from ‘disease hotspots’ were analyzed using 43 simple sequence repeat (SSR) primers. Monomorphic bands were detected in all the CGD samples with 41 primers, which is an indication of its genetic uniformity. A single CGD palm showed polymorphism with two SSR primers. Forty eight MGD palms were analyzed using 24 SSR primers. The MGD palms clustered at 62 per cent similarity. Analysis of morphological and fruit component characters of CGD and MGD population revealed that both the populations were phenotypically uniform. Breeding behaviour studies revealed that both CGD and MGD were predominantly self pollinated, like other dwarf varieties of coconut. There was complete overlapping of male and female phases in almost 96 per cent of CGD palms. Almost 100 per cent self-pollination was ensured in these palms as male phase prolonged even after completion of female phase. However, only 60 per cent of the MGD palms showed complete overlapping and in the remaining 40 per cent palms, there was only partial overlapping of male and female phases. From the present study, it is inferred that breeding behavior and genetic uniformity could be highly correlated in coconut. Collection of seed nuts preferably from mother palms with overlapping of male and female phases could possibly ensure production of true to type progenies in dwarf varieties of coconut. Present study also indicated that molecular markers like SSRs may be used to identify genetically pure mother palms for varietal improvement programmes in coconut