Elucidation of antibacterial effect of calcium chloride against Ralstonia pseudosolanacearum race 4 biovar 3 infecting ginger (Zingiber officinale Rosc.).

Bacterial wilt incited by Ralstonia pseudosolanacearum (Rps) race 4 biovar 3 is a serious threat to ginger (Zingiber officinale Rosc.) cultivation throughout the ginger growing tracts and warrants effective remedial measures since most of the strategies failed at field level implementation. After a series of experiments, calcium chloride was found to be effective against Rps both in vitro and in planta and its prophylactic effect has been successfully demonstrated under field conditions. CaCl2 at a concentration of > 2% significantly inhibited Rps under in vitro conditions. Calcium is an important nutritional element imparts a major role in plant disease resistance, and numerous studies have demonstrated the mitigating effect of calcium for disease management. CaCl2 being inhibitory to Rps, the mechanism of inhibition by CaCl2 against Rps was elucidated by a series of in vitro assays including swarming motility and biofilm formation. Direct inhibition was also studied using Scanning Electron Microscopy (SEM). The minimum bactericidal concentration and minimum inhibitory concentration were found to be around 3% while the EC 90 value was found to be 2.25%. The SEM analysis revealed the destruction of cell structure by making perforations on the cell surface. CaCl2 at the targeted concentrations inhibited biofilm formation as well as swarming motility of Rps. These findings suggest that CaCl2 exhibits strong antibacterial activity against Rps and has the potential to be used as an effective bactericide for Rps in managing bacterial wilt in ginger.

Dataset of De Novo hybrid berry transcriptome profiling and characterization of Piper species (Piper nigrum and Piper longum) using Illumina and Nanopore sequencing.

Piper nigrum and Piper longum are the most popular and economically essential spice crops globally valued for their aromatic alkaloids, especially Piperine. However, Piperine synthesis pathway mechanisms are not yet well known. This work was aimed to generate the full-length comparative berry transcriptome analysis dataset of P. nigrum and P. longum by Illumina and Nanopore sequencing platforms. While short-read sequencing technology is widely using to capture transcriptome profiles, there are still some limitations due to the read length. We used Oxford Nanopore technology for long reads and the Illumina sequencing platform for short reads to generate a hybrid transcriptome assembly from half matured and fully matured berries of P. nigrum and P. longum. From P. nigrum and P. longum 37.3 million and 38.1 million raw reads were generated respectively. A total of 308369 contigs from P. nigrum and 267715 contigs from P. longum were obtained and successfully annotated. The transcriptome data revealed gene families involved in piperine and other secondary metabolite biosynthetic pathways. The raw data were uploaded to NCBI database. This dataset shed light on the further exploration of the piperine biosynthetic pathway, its transcriptomic changes, and evolution. Data generated has been submitted to SRA of NCBI with Bio samples accession: (SAMN13981803, SAMN22826456).

Sympatric occurrence of sibling Phytophthora species associated with foot rot disease of black pepper in India.

Foot rot disease caused by Phytophthora capsici is a serious threat to black pepper cultivation in India and globally. High diversity exists among the Phytophthora isolates of black pepper and hence detailed investigations of their morphology and phylogenetic taxonomy were carried out in the present study. In order to resolve the diversity, 182 isolates of Phytophthora, collected from different black pepper-growing tracts of South India during 1998-2013 and maintained in the National Repository of Phytophthora at ICAR-Indian Institute of Spices Research, Kozhikode, were subjected to morphological, molecular and phylogenetic characterization. Morphologically all the isolates were long pedicellate with umbellate/simple sympodial sporangiophores and papillate sporangia with l/b ranging from 1.63 to 2.55 µm. Maximum temperature for the growth was ~ 34 °C. Chlamydospores were observed in "tropicalis" group, whereas they were absent in "capsici" group. Initial molecular studies using internal transcribed spacer (ITS) marker gene showed two clear cut lineages-"capsici-like" and "tropicalis-like" groups among them. Representative isolates from each group were subjected to host differential test, multilocus sequence typing (MLST) and phylogeny studies. MLST analysis of seven nuclear genes (60S ribosomal protein L10, beta-tubulin, elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA and TigA gene fusion protein) clearly delineated black pepper Phytophthora isolates into two distinct species-P. capsici and P. tropicalis. On comparing with type strains from ATCC, it was found that the type strains of P. capsici and P. tropicalis differed from black pepper isolates in their infectivity on black pepper. The high degree of genetic polymorphism observed in black pepper Phytophthora isolates is an indication of the selection pressure they are subjected to in the complex habitat which ultimately may lead to speciation. So based on the extensive analysis, it is unambiguously proved that the foot rot disease of black pepper in India is predominantly caused by two species of Phytophthora, viz. P. capsici and P. tropicalis. Presence of multiple species of Phytophthora in the black pepper agro-ecosystem warrants a revisit to the control strategy being adopted for managing this serious disease. The silent molecular evolution taking place in such an ecological niche needs to be critically studied for the sustainable management of foot rot disease.

Draft Genome Sequence of Highly Virulent Race 4/Biovar 3 of Ralstonia solanacearum CaRs_Mep Causing Bacterial Wilt in Zingiberaceae Plants in India.

The genome of Ralstonia solanacearum CaRs_Mep, a race 4/biovar 3/phylotype I bacterium causing wilt in small cardamom and other Zingiberaceae plants, was sequenced. Analysis of the 5.7-Mb genome sequence will aid in better understanding of the genetic determinants of host range, host jump, survival, pathogenicity, and virulence of race 4 of R. solanacearum.

Genetic analysis of plant endophytic Pseudomonas putida BP25 and chemo-profiling of its antimicrobial volatile organic compounds.

Black pepper associated bacterium BP25 was isolated from root endosphere of apparently healthy cultivar Panniyur-5 that protected black pepper against Phytophthora capsici and Radopholus similis - the major production constraints. The bacterium was characterized and mechanisms of its antagonistic action against major pathogens are elucidated. The polyphasic phenotypic analysis revealed its identity as Pseudomonas putida. Multi locus sequence typing revealed that the bacterium shared gene sequences with several other isolates representing diverse habitats. Tissue localization assays exploiting green fluorescence protein expression clearly indicated that PpBP25 endophytically colonized not only its host plant - black pepper, but also other distantly related plants such as ginger and arabidopsis. PpBP25 colonies could be enumerated from internal tissues of plants four weeks post inoculation indicated its stable establishment and persistence in the plant system. The bacterium inhibited broad range of pathogens such as Phytophthora capsici, Pythium myriotylum, Giberella moniliformis, Rhizoctonia solani, Athelia rolfsii, Colletotrichum gloeosporioides and plant parasitic nematode, Radopholus similis by its volatile substances. GC/MS based chemical profiling revealed presence of Heneicosane; Tetratetracontane; Pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); Tetracosyl heptafluorobutyrate; 1-3-Eicosene, (E)-; 1-Heneicosanol; Octadecyl trifluoroacetate and 1-Pentadecene in PpBP25 metabolite. Dynamic head space GC/MS analysis of airborne volatiles indicated the presence of aromatic compounds such as 1-Undecene;Disulfide dimethyl; Pyrazine, methyl-Pyrazine, 2,5-dimethyl-; Isoamyl alcohol; Pyrazine, methyl-; Dimethyl trisulfide, etc. The work paved way for profiling of broad spectrum antimicrobial VOCs in endophytic PpBP25 for crop protection.

Comparison of the transcriptomes of ginger (Zingiber officinale Rosc.) and mango ginger (Curcuma amada Roxb.) in response to the bacterial wilt infection.

Bacterial wilt in ginger (Zingiber officinale Rosc.) caused by Ralstonia solanacearum is one of the most important production constraints in tropical, sub-tropical and warm temperature regions of the world. Lack of resistant genotype adds constraints to the crop management. However, mango ginger (Curcuma amada Roxb.), which is resistant to R. solanacearum, is a potential donor, if the exact mechanism of resistance is understood. To identify genes involved in resistance to R. solanacearum, we have sequenced the transcriptome from wilt-sensitive ginger and wilt-resistant mango ginger using Illumina sequencing technology. A total of 26387032 and 22268804 paired-end reads were obtained after quality filtering for C. amada and Z. officinale, respectively. A total of 36359 and 32312 assembled transcript sequences were obtained from both the species. The functions of the unigenes cover a diverse set of molecular functions and biological processes, among which we identified a large number of genes associated with resistance to stresses and response to biotic stimuli. Large scale expression profiling showed that many of the disease resistance related genes were expressed more in C. amada. Comparative analysis also identified genes belonging to different pathways of plant defense against biotic stresses that are differentially expressed in either ginger or mango ginger. The identification of many defense related genes differentially expressed provides many insights to the resistance mechanism to R. solanacearum and for studying potential pathways involved in responses to pathogen. Also, several candidate genes that may underline the difference in resistance to R. solanacearum between ginger and mango ginger were identified. Finally, we have developed a web resource, ginger transcriptome database, which provides public access to the data. Our study is among the first to demonstrate the use of Illumina short read sequencing for de novo transcriptome assembly and comparison in non-model species of Zingiberaceae.

Virtual screening and in vitro assay of potential drug like inhibitors from spices against Glutathione-S-Transferase of Meloidogyne incognita.

Glutathione S-transferases (GSTs) enzymes are critical antioxidant and detoxification system responsible for long-term existence of nematodes in host species. Hence, 16 phytochemicals predicted and reported to have potential nematicidal activity have been docked to GST enzyme of Meloidogyne incognita to assess their binding affinity and inhibitory activity. In vitro effects of these phytochemicals from in silico results have been done for validation of docking studies and efficacy in GST inhibition of following compounds such as alpha- pinene, alpha- terpineol, beta- caryophyllene, capsaicin, cinnamic acid, citronellol, curcumin, eugenol, geraniol, isoeugenol, linalool, myristicin, neral, NVA (N-vanillylnonanamide), piperine, vanillin have been revealed. Nematode inhibition in vitro bioassay for selected compounds could conclude that maximum mortality was observed with highest concentrations of beta- caryophyllene (78%) followed by eugenol (61.6%), cinnamic acid (55%) and N-vanillylnonanamide (49%). These findings thus suggest that the above phytochemicals could be potentially developed as nematicidal molecules against M. incognita infections.

Identification of single nucleotide polymorphism in ginger using expressed sequence tags.

UNLABELLED: Ginger (Zingiber officinale Rosc) (Family: Zingiberaceae) is a herbaceous perennial, the rhizomes of which are used as a spice. Ginger is a plant which is well known for its medicinal applications. Recently EST-derived SNPs are a free by-product of the currently expanding EST (Expressed Sequence Tag) databases. The development of high-throughput methods for the detection of SNPs (Single Nucleotide Polymorphism) and small indels (insertion/deletion) has led to a revolution in their use as molecular markers. Available (38139) Ginger EST sequences were mined from dbEST of NCBI. CAP3 program was used to assemble EST sequences into contigs. Candidate SNPs and Indel polymorphisms were detected using the perl script AutoSNP version 1.0 which has used 31905 ESTs for detecting SNPs and Indel sites. We found 64026 SNP sites and 7034 indel polymorphisms with frequency of 0.84 SNPs / 100 bp. Among the three tissues from which the EST libraries had been generated, Rhizomes had high frequency of 1.08 SNPs/indels per 100 bp whereas the leaves had lowest frequency of 0.63 per 100 bp and root is showing relative frequency 0.82/100bp. Transitions and transversion ratio is 0.90. In overall detected SNP, transversion is high when compare to transition. These detected SNPs can be used as markers for genetic studies. AVAILABILITY: The results of the present study hosted in our webserver www.spices.res.in/spicesnip.

JUZBOX: a web server for extracting biomedical words from the protein sequence.

UNLABELLED: The recognition of gene/protein names in literature is one of the pivotal steps in the processing of biological literatures for information extraction or data mining. We have compiled a lexicon of biomedical words (conserved patterns/ potential motifs) which has the combination of only 20 alphabets of amino acids. The remaining 6 letters of the English alphabets (B, J, O, U, X, Z) are treated as invalid amino acid characters (to our context), We have jumbled the 6 letters for the sake of usage and convenience and termed as 'JUZBOX' and these characters were filtered in the biomedical lexicon. Undoubtedly, the generation of biomedical words from protein sequence using JUZBOX have applications specific for functional annotation. AVAILABILITY: JUZBOX is available freely at http://www.spices.res.in/juzbox.

PIR pairwise alignment - a slip up for signal peptides.

The ability to calculate the correct sequence alignment is crucial to many types of studies. The accuracy in alignment is critical in predicting gene ancestry, the number and location of point mutations, evolutionary distance and phylogeny. A study was conducted to test the biological significance of PIR pairwise alignment using 40 N-terminal signal peptides of different taxonomic origin and having various functions. Our results suggest that PIR pairwise alignment is not ideal for some proteins with N-terminal signal peptides, because it produces an erroneous alignment that lacks both statistical and biological significances. This communication discusses the shortcomings in the PIR pairwise alignment tool and calls for a cautious approach while using it for signal peptides.

Tropical soil microflora of spice-based cropping systems as potential antagonists of root-knot nematodes.

Suppression of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach than the toxic chemicals. In a study, soil borne fungi from the rhizosphere of major spice crops were collected from diverse cropping systems prevailing in three southern states of India. A series of in vitro studies were conducted using 73 freshly collected fungal isolates and 76 isolates obtained from other sources. Out of this 67 isolates were not parasitic on females of root-knot nematodes whereas 115 isolates, though colonized the egg masses, did not show any signs of parasitism on nematode eggs. Fifty-nine isolates showed 50-90% inhibition in egg hatch. Pochonia chlamydospora, Verticillium lecanii, Paecilomyces lilacinus, and few isolates of Trichoderma spp. showed >25% parasitism on root-knot nematode eggs. The most promising isolates in this study were one isolate each of Aspergillus (F.45), Fusarium (F.47), and Penicillium (F.59); three each isolates of Trichoderma (F.3, F.52, and F.60) and Pochonia (F.30 and Vc.3) Verticillium (Vl); and two isolates of fungi that could not be identified (F.28 and F.62). Parasitism by Aspergillus tamarii, Aspergillus ustus, Drechslera sp., Humicola sp., and Scopulariopsis sp. on root-knot nematode eggs or females, reported in the present study, are new reports.