Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability.

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50-80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4 mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75 kDa and 25 kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000 MW cut-off) filter, yielded a prominent band of 25 kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4 µg protein and an IC50 value of 0.59 µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.

Transcriptome-based mining and expression profiling of Pythium responsive transcription factors in Zingiber sp.

Transcription factors (TFs) fine-tune the host defense transcriptome in response to pathogen invasions. No information is available on Zingiber zerumbet (Zz) TFs involved in defense response against Pythium myriotylum. Here, we provide a global identification, characterization, and temporal expression profiling of Zz TFs following an incompatible interaction with P. myriotylum using a transcriptome sequencing approach. We identified a total of 903 TFs belonging to 96 families based on their conserved domains. Evolutionary analysis clustered the Zz TFs according to their phylogenetic affinity, providing glimpses of their functional diversities. High throughput expression array analysis highlighted a complex interplay between activating and repressing transcription factors in fine-tuning Zz defense response against P. myriotylum. The high differential modulation of TFs involved in cell wall fortification, lignin biosynthesis, and SA/JA hormone crosstalk allows us to envisage that this mechanism plays a central role in restricting P. myriotylum proliferation in Zz. This study lays a solid foundation and provides valuable resources for the investigation of the evolutionary history and biological functions of Zz TF genes involved in defense response.

Jiangella endophytica sp. nov., an endophytic actinomycete isolated from the rhizome of Kaempferia elegans.

An endophytic actinobacterium, designated strain KE2-3T, was isolated from surface-sterilised rhizome of Kaempferia elegans. The polyphasic approach was used for evaluating the taxonomic position of this strain. The taxonomic affiliation of this strain at genus level could be confirmed by its chemotaxonomic characteristic, i.e. the presence of ll-diaminopimelic acid in the cell peptidoglycan, MK-9(H4) as the major menaquinone, iso-C16 : 0, anteiso-C15 : 0, iso-C14 : 0 and iso-C15 : 0 as the predominant fatty acids in cells, and the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside in its membranes. Based on 16S rRNA gene sequence analysis, strain KE2-3T was identified as a member of the genus Jiangella and showed the highest similarities to Jiangella muralis DSM 45357T (99.3 %) followed by Jiangella albaDSM 45237T (99.2 %), Jiangella alkaliphilia DSM 45079T (99.0 %), Jiangella gansuensisDSM 44835T (98.8 %) and Jiangella mangrovi3SM4-07T (98.6 %). However, the draft genome sequence of strain KE2-3T exhibited low average nucleotide identity values to the reference strains (85.5-90.2 %), which were well below the 95-96 % species circumscription threshold. The DNA G+C content of genomic DNA was 72.3 mol%. With the differences of physiological, biochemical and genotypic data, strain KE2-3T could be discriminated from its closest neighbour. Thus, strain KE2-3T should be recognised as a novel species of genus Jiangella, for which the name Jiangellaendophytica sp. nov. is proposed. The type strain is KE2-3T (=BCC 66359T=NBRC 110004T).

Jishengella zingiberis sp. nov., isolated from root tissue of Zingiber montanum.

A novel actinomycete, strain PLAI 1-1T, which formed spiny single spore directly on substrate mycelium was isolated from root tissue of Zingiber montanum. The isolate contained meso-diaminopimelic acid and 3-hydroxydiaminopimelic acid in the cell-wall peptidoglycan. The acyl type of the cell-wall muramic acid was glycolyl. The whole-cell sugars of strain PLAI 1-1T were glucose, arabinose, xylose, ribose and a trace amount of mannose. The membrane phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. The major menaquinone was MK-9 (H4). The main cellular fatty acids were iso-C15 : 0 and C17 : 1ω8c. The G+C content of the genomic DNA was 70.6 mol%. 16S rRNA gene sequence analysis revealed that strain PLAI 1-1T was a member of the genus Jishengella and had the highest 16S rRNA gene sequence similarity to Jishengella endophytica DSM 45430T (99.2 %). Based on the data of physiological and biochemical tests, including the result of DNA-DNA hybridization, strain PLAI 1-1T represents a novel species of the genus Jishengella, for which the name Jishengellazingiberis sp. nov. is proposed. The type strain is PLAI 1-1T (=TBRC 7644T=NBRC 113144T).

Actinomycetes: an unexplored microorganisms for plant growth promotion and biocontrol in vegetable crops.

Actinomycetes, a Gram positive bacteria, well reported as a source of antibiotics, also possess potential to control various plant pathogens, besides acting as plant growth promoting agent. Chemicals in different forms are extensively being used in vegetable farming, adversely affecting the environment and consumer health. Microbial agent like actinomycetes can substantially replace these harmful chemicals, and have now started finding a place as an important input in to farming practices. Only selected vegetable crops belonging to 11 different families have been explored with use of actinomycetes as biocontrol and plant growth promoting agent till now. It provides ample opportunities to vegetable researchers, to further explore with use of this very important group of microorganisms, in order to achieve even higher production level of safe vegetables. Mycostop and Actinovate are two actinomycetes based formulations globally available for use in vegetable farming as a substitute for chemical formulations. Present review article has summarized the literature available on use of actinomycetes in vegetable farming. Existing wide gap in knowledge, and potential thrust areas for future research have also been projected.

Cystargamide B, a cyclic lipodepsipeptide with protease inhibitory activity from Streptomyces sp.

We identified a new cyclic lipodepsipeptide, cystargamide B (1), from the mycelial extract of a Kaempferia galanga rhizome-derived actinomycete strain, Streptomyces sp. PB013. The planar structure was elucidated based on high resolution fast-atom bombardment mass spectrometry (HRFABMS) spectroscopy and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopic data. The absolute configurations of the constituent amino acids were determined using advanced Marfey's method. Cystargamide B (1) includes rare structural units: a 5-hydroxytryptophan residue and a 2,3-epoxy fatty acid side chain. Notably, cystargamide B (1) inhibited the protease activity of the NS2B/NS3 complex from dengue virus.

Micromonospora globbae sp. nov., an endophytic actinomycete isolated from roots of Globba winitii C. H. Wright.

A novel endophytic actinomycete, strain WPS1-2T, isolated from a root of Globba winitii C. H. Wright, was characterized taxonomically by using a polyphasic approach. Strain WPS1-2T exhibited identical characteristics to the members of the genus Micromonospora. Single spores were observed directly on substrate mycelia. The cell-wall peptidoglycan of the strain contained meso-diaminopimelic acid and 3-OH-meso-diaminopimelic acid. Whole-cell hydrolysates contained glucose, ribose, arabinose and xylose. The predominant menaquinones were MK-10(H8) and MK-10(H10). The major cellular fatty acids consisted of iso-C15 : 0, iso-C16 : 0 and anteiso-C15 : 0. According to the 16S rRNA gene sequence of the strain, WPS1-2T showed highest similarity to Micromonospora costi CS1-12T (99.02 %). Phylogenetic analysis of the gyrase subunit B (gyrB) gene indicated that the strain was related to M. costi CS1-12T. The DNA G+C content was 73.7 mol%. The strain could be distinguished from closely related type strains by using a combination of morphological, chemotaxonomic, physiological and biochemical data together with DNA-DNA relatedness values. Based on these observations, strain WPS1-2T is considered to represent a novel species of the genus Micromonospora, for which the name Micromonospora globbae sp. nov. is proposed. The type strain is WPS1-2T (=KCTC 39787T=NBRC 112325T=TISTR 2405T).

Nonomuraea stahlianthi sp. nov., an endophytic actinomycete isolated from the stem of Stahlianthus campanulatus.

A novel endophytic actinomycete, designated strain SC1-1T, was isolated from sterilized stem tissue from Stahlianthus campanulatus collected in Udon Thani province, Thailand. The isolate formed short chains of spores on aerial mycelium and presented meso-diaminopimelic acid in the cell wall peptidoglycan. Glucose, madurose, mannose, rhamnose and ribose were observed as sugars in the cells. The cell membrane phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxy-phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and ninhydrin-positive phosphoglycolipids. The major menaquinones were MK-9(H4) and MK-9(H2). The main cellular fatty acids were iso-C16:0, 10-methyl C17 : 0 and C17 : 1ω6c. A high G+C content (70.7 mol%) was present in the genomic DNA. The taxonomic position based on the 16S rRNA gene sequence analysis revealed that strain SC1-1T belonged to the genus Nonomuraea and shared the highest 16S rRNA gene sequence similarity value with Nonomuraea dietziae DSM 44320T (98.82 %), followed by Nonomuraea africana IFO 14745T (98.58 %), Nonomuraea jabiensis A4036T (98.43 %), Nonomuraea endophytica YIM 65601T (98.36 %), Nonomuraea purpurea 1SM4-01T (98.34 %), Nonomuraea angiospora IFO 13155T (98.29 %), Nonomuraea roseola IFO 14685T (98.23 %) and Nonomuraea recticatena IFO 14525T (98.23 %). On the basis of the DNA-DNA hybridization relatedness and including the physiological and biochemical characteristics, strain SC1-1T should be judged as a novel species of the genus Nonomuraea, for which the name Nonomuraea stahlianthi sp. nov. is proposed. The type strain is strain SC1-1T (=BCC 66361T=NBRC 110006T).

Phytohabitans kaempferiae sp. nov., an endophytic actinomycete isolated from the leaf of Kaempferia larsenii.

A novel endophytic actinomycete, designated strain KK1-3T, which formed single spores and long chains of spores (more than 10 spores) was isolated from surface-sterilized Kaempferia larsenii leaf collected from Ubon Ratchathani province, Thailand. The isolate contained l-lysine, meso-diaminopimelic acid and hydroxyl diaminopimelic acid in the cell-wall peptidoglycan. The whole-cell sugars included glucose, mannose, rhamnose, ribose, galactose and xylose. The characteristic phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and phosphoglycolipids. The predominant menaquinones were MK-10(H8), MK-10(H6) and MK-10(H4). The predominant cellular fatty acids were anteiso-C17 : 0 and iso-C16 : 0. The G+C content of the genomic DNA was 71 mol%. Phylogenetic analysis using 16S rRNA gene sequences revealed that strain KK1-3T should be classified as representing a member of the genus Phytohabitans. The similarity values of sequences between this strain and those of the closely related species, Phytohabitans houttuyneae K11-0057T (99.0 %), Phytohabitans suffuscus K07-0523T (98.9 %), Phytohabitans flavus K09-0627T (98.6 %) and Phytohabitans rumicisK11-0047T (98.1 %) were observed. The DNA-DNA hybridization result and some physiological and biochemical properties indicated that KK1-3T could be readily distinguished from its closest phylogenetic relatives. On the basis of these phenotypic and genotypic data, this strain represents a novel species, for which the name Phytohabitans kaempferiae sp. nov. is proposed. The type strain is strain KK1-3T (=BCC 66360T =NBRC 110005T).

Molecular phylogenetics and anti-Pythium activity of endophytes from rhizomes of wild ginger congener, Zingiber zerumbet Smith.

Zingiber zerumbet, a perennial rhizomatous herb exhibits remarkable disease resistance as well as a wide range of pharmacological activities. Towards characterizing the endophytic population of Z. zerumbet rhizomes, experiments were carried out during two different growing seasons viz., early-June of 2013 and late-July of 2014. A total of 34 endophytes were isolated and categorized into 11 morphologically distinct groups. Fungi were observed to predominate bacterial species with colonization frequency values ranging from 12.5 to 50%. Among the 11 endophyte groups isolated, molecular analyses based on ITS/16S rRNA gene sequences identified seven isolate groups as Fusarium solani, two as F. oxysporum and one as the bacterium Rhizobium spp. Phylogenetic tree clustered the ITS sequences from Z. zerumbet endophytes into distinct clades consistent with morphological and sequence analysis. Dual culture assays were carried out to determine antagonistic activity of the isolated endophytes against Pythium myriotylum, an economically significant soil-borne phytopathogen of cultivated ginger. Experiments revealed significant P. myriotylum growth inhibition by F. solani and F. oxysporum isolates with percentage of inhibition (PoI) ranging from 45.17 ± 0.29 to 62.2 ± 2.58 with F. oxysporum exhibiting higher PoI values against P. myriotylum. Using ZzEF8 metabolite extract, concentration-dependent P. myriotylum hyphal growth inhibition was observed following radial diffusion assays. These observations were confirmed by scanning electron microscopy analysis wherein exposure to ZzEF8 metabolite extract induced hyphal deformities. Results indicate Z. zerumbet endophytes as promising resources for biologically active compounds and as biocontrol agents for soft rot disease management caused by Pythium spp.

Essential oils and major compounds of Hedychium coronarium Koenig (Zingiberaceae) against pathogenic yeast of Candida and Cryptococcus genus / Óleos essenciais e compostos majoritários de Hedychium coronarium Koenig (Zingiberaceae) contra leveduras patogênicas dos gêneros Candida e Cryptococcus

Among the major causative agents of invasive fungal infections stands out the opportunistic yeasts of Candida and Cryptococcus. Regarding the problem of the high incidence of infections by these agents and the difculty of treating the low stockpile of antifungal drugs and the high toxicity of most therapies, the search for new antifungal compounds has been highlighted in recent decades. Hedychium coronarium, popularly known as "lírio-do-brejo" or "gengibre-branco" features several previously reported biological activities, including antimicrobial activity. Compound 1.8-cineole is the major compound in essential oil extracted from roots of H. coronarium, while caryophyllene oxide presents itself as the major in essential oil extracted from leaves of this plant. Our data show strong antifungal activity of compounds, against species of Candida albicans, Candida parapsilosis, Candida krusei, Cryptococcus neoformans and Cryptococcus gattii, with minimal inhibitory concentration and minimal fungicidal concentration equal to 0.2 % (v/v) for essential oil extracted from roots, while the essential oil extracted from leaves showed no activity against yeasts. The caryophyllene oxide showed higher antifungal activity for Cryptococcus spp. Thus, our results showed that the essential oil of rhizome is a promising antifungal agent against pathogenic yeasts.(AU)

Candida spp e Cryptococcus spp estão classifcadas entre os maiores causadores de infecções fúngicas invasivas em pacientes imunocomprometidos. Diante a alta incidência destas infecções por estes agentes e a difculdade do sucesso no tratamento, decorrente do baixo arsenal de fármacos antifúngicos e da alta toxicidade presente na maioria dos esquemas terapêuticos, a busca por novos compostos antifúngicos tem sido alvo de diversos estudos nas últimas décadas. Hedychium coronarium, popularmente conhecido como "lírio-do-brejo" ou "gengibre-branco", apresenta diversas atividades biológicas já descritas, entre elas a atividade antimicrobiana. O composto 1.8-Cineol é o composto majoritário presente no óleo essencial extraído de raízes de H. coronarium e o composto óxido de cariofleno é o composto majoritário extraído das folhas desta planta. Nossos resultados mostram que os compostos extraídos de H. coronarium apresentam forte atividade contra Candida albicans, Candida parapsilosis, Candida krusei, Cryptococcus neoformans e Cryptococcus gattii, com valores de concentração inibitória minima e concentração fungicida minima igual a 0,2 % (v/v) para o óleo essencial extraído das raízes, enquanto que o óleo essencial extraído das folhas, não mostrou atividade contras as leveduras. O composto óxido de cariofleno mostrou maior atividade antifúngica para Crytopcoccus spp. Assim, nossos dados mostraram que o óleo essencial extraído das raízes de H. coronarium, é um agente antifúngico promissor contra leveduras patogênicas.(AU)

Wickerhamiella kiyanii f.a., sp. nov. and Wickerhamiella fructicola f.a., sp. nov., two yeasts isolated from native plants of Atlantic rainforest in Brazil.

Two novel species, Wickerhamiella kiyanii f.a., sp. nov. (type strain FB1-1DASP(T) = CBS 12905(T) = CBMAI 1613(T)) and Wickerhamiella fructicola f.a., sp. nov. (type strain H10Y(T) = CBS 12902(T) = CBMAI 1614(T)) are proposed in the Wickerhamiella clade (Saccharomycetes, Saccharomycetales) to accommodate three strains isolated from flowers and fruits typical of the Brazilian Atlantic rainforest. The novel status of these yeast species was established by sequence divergence observed in the D1/D2 domains of the LSU rRNA gene from the most closely related, described species as well as by phylogenetic analysis. Wickerhamiella kiyanii sp. nov. differs from its nearest phylogenetic neighbours W. pagnoccae CBS 12178(T), Candida jalapaonensis CBS 10935(T) and Candida drosophilae CBS 8459(T) by 2.2-4.2% in the D1/D2 sequences. By contrast, a sequence divergence of 13.2-13.8% was observed between W. fructicola sp. nov. and its closest, described phylogenetic relative Candida kazoui JCM 12558(T) and Candida hasegawae JCM 12559(T). Taxonomic descriptions of the two novel species are given.

Metabolic profiling of Zingiber zerumbet following Pythium myriotylum infection: investigations on the defensive role of the principal secondary metabolite, zerumbone.

Induced biosynthesis of bioactive secondary metabolites constitutes one of the mechanisms of plant basal innate immunity to fungal infection. Metabolic changes were studied in rhizomes of Zingiber zerumbet, a wild congener of ginger, after infection with soft rot-causative necrotrophic phytopathogen, Pythium myriotylum, by gas chromatography-mass spectrometry (GC-MS) analysis. Infection triggered a considerable alteration in the relative content of zerumbone and α-caryophyllene (humulene) with enhancement in zerumbone content (81.59%) and that of α-caryophyllene (11.91%) compared to 9.97 and 1.11%, respectively, in uninfected rhizomes. While zerumbone is the principal secondary metabolite in Z. zerumbet, α-caryophyllene is its immediate precursor. Principal component analysis (PCA) identified the correlations between metabolite changes in Z. zerumbet rhizomes and P. myriotylum infection. Radial diffusion assay with zerumbone indicated a concentration-dependent P. myriotylum growth inhibition with 93.75% inhibition observed at 700 µg and 50% maximal effective concentration (EC50) value of 206 µg. Scanning electron microscopy (SEM) analysis revealed that the mechanistic basis of zerumbone's antagonistic action on P. myriotylum growth involved the induction of aberrant morphology including severe hyphal deformities and membrane disruption. Results are discussed highlighting the critical role played by sesquiterpenoid zerumbone in affording resistance in Z. zerumbet and could expedite the development of appropriate strategies for biocontrol of Pythium spp., thus reducing the usage of broad-spectrum fungicides.

Expression analysis of defense-related genes in Zingiber (Zingiberaceae) species with different levels of compatibility to the soft rot pathogen Pythium aphanidermatum.

Ginger (Zingiber officinale Roscoe) cultivars are susceptible to soft rot disease caused by Pythium aphanidermatum. We analyzed changes in transcript levels of 41 genes in the highly susceptible ginger cultivar varada, a less susceptible wild accession (wild ginger), and a Pythium aphanidermatum-resistant relative, Z. zerumbet, following treatment with Pythium aphanidermatum or one of three signaling molecules: salicylic acid (SA), jasmonic acid (JA), or ethylene (ET). The 41 studied genes were chosen because they are known to be involved in the hypersensitive response (HR), cell signaling, or host defense. Expression of most genes peaked within 24 h of Pythium aphanidermatum infection. Interestingly, the level of induction was typically manyfold higher in Z. zerumbet than in wild ginger. However, several HR genes that were significantly induced in wild ginger were not induced in Z. zerumbet. Most of the genes, including those involved in signaling, did not respond to any of the three signaling molecules in Z. zerumbet while several genes responded to all the three signaling molecules in varada. In wild ginger, a large proportion of the genes responded to ET, but not to SA or JA. These results suggest that different mechanisms govern the three pathosystems. Resistance in Z. zerumbet seems to be independent of HR and the tested signaling molecules, whereas both mechanisms appear to be activated in the tolerance reaction of wild ginger. This work revealed potential defense components of this understudied tropical taxa, and will contribute to the design of strategies for transgenic improvement of ginger.

Soil fertility and the impact of exotic invasion on microbial communities in Hawaiian forests.

Exotic plant invasions into Hawaiian montane forests have altered many important nutrient cycling processes and pools. Across different ecosystems, researchers are uncovering the mechanisms involved in how invasive plants impact the soil microbial community-the primary mediator of soil nutrient cycling. We examined whether the invasive plant, Hedychium gardnerianum, altered microbial community composition in forests dominated by a native tree, Metrosideros polymorpha, under varying soil nutrient limitations and soil fertility properties within forest plots of the Hawaii long-term substrate age gradient (LSAG). Microbial community lipid analysis revealed that when nutrient limitation (as determined by aboveground net primary production [ANPP]) and soil fertility were taken into account, plant species differentially altered soil microbial community composition. Microbial community characteristics differed under invasive and native plants primarily when N or P was added to the older, highly weathered, P-limited soils. Long-term fertilization with N or P at the P-limited site led to a significant increase in the relative abundance of the saprophytic fungal indicator (18:2 omega 6c,9c) under the invasive plant. In the younger, N-limited soils, plant species played a minor role in influencing soil microbial community composition. We found that the general rhizosphere microbial community structure was determined more by soil fertility than by plant species. This study indicates that although the aggressive invasion of a nutrient-demanding, rapidly decomposable, and invasive plant into Hawaiian forests had large impacts on soil microbial decomposers, relatively little impact occurred on the overall soil microbial community structure. Instead, soil nutrient conditions were more important determinants of the overall microbial community structure within Hawaii's montane forests.