Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production.

In this study, endophytic bacteria belonging to the Bacillus genus were isolated from in vitro bulblets of Leucojum aestivum and their ability to produce Amaryllidaceae alkaloids was studied. Proton Nuclear Magnetic Resonance (1H NMR)-based metabolomics combined with multivariate data analysis was chosen to compare the metabolism of this plant (in vivo bulbs, in vitro bulblets) with those of the endophytic bacteria community. Primary metabolites were quantified by quantitative 1H NMR (qNMR) method. The results showed that tyrosine, one precursor of the Amaryllidaceae alkaloid biosynthesis pathway, was higher in endophytic extract compared to plant extract. In total, 22 compounds were identified including five molecules common to plant and endophyte extracts (tyrosine, isoleucine, valine, fatty acids and tyramine). In addition, endophytic extracts were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for the identification of compounds in very low concentrations. Five Amaryllidaceae alkaloids were detected in the extracts of endophytic bacteria. Lycorine, previously detected by 1H NMR, was confirmed with LC-MS analysis. Tazettine, pseudolycorine, acetylpseudolycorine, 1,2-dihydro-chlidanthine were also identified by LC-MS using the positive ionization mode or by GC-MS. In addition, 11 primary metabolites were identified in the endophytic extracts such as tyramine, which was obtained by decarboxylation of tyrosine. Thus, Bacillus sp. isolated from L. aestivum bulblets synthesized some primary and specialized metabolites in common with the L.aestivum plant. These endophytic bacteria are an interesting new approach for producing the Amaryllidaceae alkaloid such as lycorine.

Dickeya zeae strains isolated from rice, banana and clivia rot plants show great virulence differentials.

BACKGROUND: Dickeya zeae is the causal agent of maize and rice foot rot diseases, but recently it was also found to infect banana and cause severe losses in China. Strains from different sources showed significant diversity in nature, implying complicated evolution history and pathogenic mechanisms. RESULTS: D. zeae strains were isolated from soft rot banana plants and ornamental monocotyledonous Clivia miniata. Compared with D. zeae strain EC1 isolated from rice, clivia isolates did not show any antimicrobial activity, produced less extracellular enzymes, had a much narrow host ranges, but released higher amount of extracellular polysaccharides (EPS). In contrast, the banana isolates in general produced more extracellular enzymes and EPS than strain EC1. Furthermore, we provided evidence that the banana D. zeae isolate MS2 produces a new antibiotic/phytotoxin(s), which differs from the zeamine toxins produced by rice pathogen D. zeae strain EC1 genetically and in its antimicrobial potency. CONCLUSIONS: The findings from this study expanded the natural host range of D. zeae and highlighted the genetic and phenotypic divergence of D. zeae strains. Conclusions can be drawn from a series of tests that at least two types of D. zeae strains could cause the soft rot disease of banana, with one producing antimicrobial compound while the other producing none, and the D. zeae clivia strains could only infect monocot hosts. D. zeae strains isolated from different sources have diverse virulence characteristics.

Sanger and next generation sequencing in the characterisation of arbuscular mycorrhizal fungi (AMF) in Pancratium maritimum L. (Amaryllidaceae), a representative plant species of Mediterranean sand dunes.

MAIN CONCLUSION: An interesting AMF colonization microcosm has been detected in the roots of Pancratium maritimum (sea daffodil). Both sequencing techniques (Sanger and NGS) have been used for AMF characterisation, showing a balanced trade-off between pros and cons. By Sanger and next generation sequencing of rRNA nuclear molecular markers (SSU-ITS-LSU and ITS2, respectively), the presence of AMF communities in the roots of P. maritimum was evaluated. Our results shed light on the presence of AMF in sea daffodil and the diversity of assemblages of AMF detected after Sanger sequencing of the SSU-ITS-LSU marker is much higher than that determined following NGS sequencing of ITS2 alone.

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.

The Hippeastrum hybridum PepR1 gene (HpPepR1) encodes a functional guanylyl cyclase and is involved in early response to fungal infection.

It is generally known that cyclic GMP widespread in prokaryotic and eukaryotic cells, is involved in essential cellular processes and stress signal transduction. However, in contrast to animals the knowledge about plant guanylyl cyclases (GCs) which catalyze the formation of cGMP from GTP is still quite obscure. Recent studies of plant GCs are focused on identification and functional analysis of a new family of membrane proteins called "moonlighting kinases with GC activity" with guanylyl cyclase catalytic center encapsulated within intracellular kinase domain. Here we report identification and characterization of plasma membrane receptor of peptide signaling molecules - HpPepR1 in Hippeastrum hybridum. Both bioinformatic analysis of amimo acid sequence and in vitro studies revealed that the protein can act as guanylyl cyclase. The predicted amino acid sequence contains highly conserved 14 aa-long search motif in the catalytic center of GCs from lower and higher eukaryotes. Here, we provide experimental evidence to show that the intracellular domain of HpPepR1 can generate cGMP in vitro. Moreover, it was shown that the accumulation of HpPepR1 transcript was sharply increased after Peyronellaea curtisii (=Phoma narcissi) fungal infection, whereas mechanical wounding has no influence on expression profile of studied gene. These results may indicate the participation of cGMP-dependent pathway in rapid, alarm plant reactions induced by pathogen infection.

Fusarium agapanthi sp. nov., a novel bikaverin and fusarubin-producing leaf and stem spot pathogen of Agapanthus praecox (African lily) from Australia and Italy.

This study was conducted to characterize a novel Fusarium species that caused leaf and stem spot on Agapanthus praecox (Agapanthus, African lily) in northern Italy and leaf rot and spot on the same host in Melbourne, Australia. Formally described as Fusarium agapanthi, this pathogen was analyzed using phenotypic, phytopathogenic, secondary metabolite, molecular phylogenetic and genomic data. Five strains were characterized, including one isolated in 1999 from symptomatic A. praecox in Saluzzo, Italy, and four in 2010 from diseased leaf tissue from the same host exhibiting leaf rot and spot symptoms in the Melbourne Gardens, Royal Botanic Gardens Victoria, Australia. Maximum parsimony and maximum likelihood molecular phylogenetic analyses of portions of six individual genes and the combined dataset all strongly supported F. agapanthi either as the earliest diverging genealogically exclusive lineage in the American Clade of the F. fujikuroi species complex, or alternatively a novel monotypic lineage sister to the American Clade. Koch's postulates were completed on dwarf blue- and large white-flowering varieties of A. praecox, where two isolates of F. agapanthi produced slowly spreading necrotic lesions when inoculated onto leaves and flower stems. Fusarium agapanthi is distinguished from other fusaria by the production of densely branched aerial conidiophores with polyphialides throughout the aerial mycelium on synthetic nutrient-poor agar. BLASTn searches of the F. agapanthi NRRL 31653 and NRRL 54464 (= VPRI 41787) genome sequences were conducted to predict sexual reproductive mode and mycotoxin potential. Results indicated that they possessed MAT1-2 and MAT1-1 idiomorphs, respectively, indicating that this species might be heterothallic. Furthermore, based on the presence of homologs of the bikaverin and fusarubin biosynthetic gene clusters in the F. agapanthi genomes, liquid chromatography-mass spectrometry analysis was conducted and confirmed production of these secondary metabolites in rice and corn kernel cultures of the fungus.