Santhosh J. Eapen

Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification and evaluation against Phytophthora capsici

Aim:  To isolate and identify black pepper (Piper nigrum L) associated endophytic bacteria antagonistic to Phytophthora capsici causing foot rot disease.

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.

Pre-plant bacterisation: a strategy for delivery of beneficial endophytic bacteria and production of disease-free plantlets of black pepper (Pipernigrum L.)

Naturally occurring endophytic bacteria from black pepper vines were found to exhibit strong antagonistic activities against Phytophthora capsici and Radopholus similis. In order to deliver these bacterial strains, as well as to produce disease-free plantlets of black pepper, a pre-plant stem and root bacterisation was standardised. Stem bacterisation with endophytic Pseudomonas spp. was found to suppress P. capsici infection (over 90% reduction in lesion length) on cut shoots. Pre-plant root bacterisation with Pseudomonas aeruginosa, Pseudomonas putida and Bacillus megaterium yielded over 60% of plantlets free from P. capsici infection on roots. Curtobacterium luteum and B. megaterium recorded over 70% reduction of nematode population in soil with concomitant production of over 65% of nematode-free plantlets. Besides protecting the plants from the pathogens, the bacteria were also found to enhance the growth of rooted cuttings. The biocontrol potential of the above endophytic bacteria and their exploitation for disease management in the black pepper nursery are discussed.

A new species of entomopathogenic nematode Oscheius gingeri sp. n. (Nematoda: Rhabditidae) from ginger rhizosphere

A new species of entomopathogenic nematode (EPN) of the genus Oscheius was isolated from the rhizosphere of ginger (Zingiber officinale Rosc.) cultivated in Indian Institute of Spices Research Experimental Farm, Peruvannamuzhi, Calicut District, Kerala, India. This species is described as a new species on the basis of morphometrics study. Entomopathogenicity of this nematode was also tested against greater wax moth Gallaria mellonella larvae. O. gingeri sp. n. is diagnosed by the presence of leptoderan bursa and crochet needle-shaped spicules, presence of 10 lateral lines, didelphic female reproductive system, double-flapped epipytigma and nine genital papillae. The characteristics of the new species are: male; body ventrally curved, “C”-shaped upon fixation, leptoderan bursa, tail short rounded conoid and nine pairs of genital papillae, female; body almost straight upon fixation, female reproductive system is didelphic, double-flapped epipytigma present on the vulval opening and tail is short conoid with pointed tip and infective and juvenile; body thin elongate, tail elongate attenuated, gradually tapering at tip and spine-like projection present. It closely resembles with O. carolinensis but differs in smaller length (vs. L = 1728 μm), larger “a” value (vs. a = 18.2); smaller “b” value (vs. b = 7.0); and larger “c” value (vs. c = 11.0), vulval opening (vs. V = 50.3%), number of lateral lines (vs. = 4) and male body posture “C” shaped (vs. elongate). This new species also closely resembles with O. columbiana but differs in higher body length (vs. L = 1288 μm), smaller “b” value (vs. b = 6.5), smaller stoma length (vs. SL = 23) and number of lateral lines (vs. = 4). O. gingeri sp. n. also resembles with O. andrassyi but differs in size of the body (vs. L = 1601 μm), larger “b” value (vs. b = 8.2), smaller “c” value (vs. c = 14.4), vulval opening (vs. V = 50.3%) and number of lateral lines (vs. = 6). This new species is capable to kill ginger shoot borer larva Conogethes punctiferalis and G. mellonella within 24–72 h under laboratory conditions. This opens a new hope of utilising Oscheius gingeri sp. n. as a biopesticide for management of the shoot borer and other insect pests of ginger.

Molecular Basis of Endophytic Bacillus megaterium-induced Growth Promotion in Arabidopsis thaliana: Revelation by Microarray-based Gene Expression Analysis

Black pepper root endophytic Bacillus megaterium BP17 (BmBP17) displayed excellent antagonistic activity against diverse plant pathogens. BmBP17 endophytically colonized plantlets of Arabidopsis with significant growth promotion as exemplified by increased root and shoot length. To elucidate the molecular basis of growth promotion, microarray-based gene expression profiling was performed on interactome of Arabidopsis-BmBP17. A total of 150 genes were found differentially expressed which represented 80 up-regulated and 70 down-regulated plant genes. Key up-regulated Arabidopsis genes were (i) NIR1, AMT1–5, TIP2–3, and SULTR1–2 participating in the transport of nutrients through transmembranes; (ii) SHV3, MMP, RLP44, PROPEP4, AGL42, SCPL30, ANAC010, and KNAT7 involved in cell organization, biogenesis, and transcription; (iii) SRO5, ANNAT7, and DDF1 associated with abiotic stress tolerance like salt stress and water deprivation; and (iv) MYB7, MYB4, MYB49, WRR4, ATHCHIB, and ATOSM34 involved in defense against biotic stress. Strikingly, most of the genes participating in growth and development were up-regulated in diverse plant tissues right from root to seed. The up-regulation of nutrient mobilization and uptake-related genes could be attributed to plant growth promotion. The regulation of endogenous population of BmBP17 could be due to the activation of biotic stress defense-associated genes. The bacterial colonization triggered down-regulation of genes coding for transcription factors of ethylene-responsive genes such as ERF5, ERF71, ERF104, ERF105, TEM1, and RAP2.6 and salicylic acid and jasmonic acid-responsive gene such as BAP1, SIB1, BT4, MKK9, and PLA2A. Our study showed that the plant growth promotion as observed in Arabidopsis thaliana Col 0 could be attributed to the up-regulation of nutrient uptake-associated genes and down-regulation of genes coding for transcription factors of ethylene-responsive genes.

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

ABSTRACT 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.

Research on Plant Pathogenic Fungi in the Genomics Era: From Sequence Analysis to Systems Biology

Recent years have seen the development of a variety of so-called “next-generation” sequencing platforms, with several others anticipated to become available shortly. Second-generation sequencing technologies have recently been exploited in genomics of both prokaryotic and eukaryotic plant pathogens. They are also proving to be useful in diagnostics, especially with respect to viruses. Plant pathology stands to gain from the new sequencing technologies that are already making a big impact in academic research. Since high-throughput approaches generate considerable amounts of data, bioinformatics tools will play an essential role in storing, retrieving, sharing, processing, and analyzing them. The management and analysis of next-generation sequencing data requires the development of informatics tools able to assemble, map, and interpret huge quantities of relatively or extremely short nucleotide sequence data. In this review, we summarize some applications of these high-throughput sequencing methods, useful genomics and bioinformatics resources available to plant pathologists with emphasis on the associated computational and bioinformatics challenges and their solutions. The increasing availability of high-throughput technology and the reduction of costs of these technologies have moved genomics from the sequencing of a few model species to sequencing any organism that is economically important. It has opened new avenues in research, as well as poses new challenges to plant pathologists.