Host-specific adaptation governs the interaction of the marine diatom,Pseudo-nitzschia and their microbiota

The association of phytoplankton with bacteria is ubiquitous in nature and the bacteria that associate with different phytoplankton species are very diverse. The influence of these bacteria in the physiology and ecology of the host and the evolutionary forces that shape the relationship are still not understood. In this study, we used the Pseudo-nitzschia–microbiota association to determine (1) if algal species with distinct domoic acid (DA) production are selection factors that structures the bacterial community, (2) if host-specificity and co-adaptation govern the association, (3) the functional roles of isolated member of microbiota on diatom–hosts fitness and (4) the influence of microbiota in changing the phenotype of the diatom hosts with regards to toxin production. Analysis of the pyrosequencing-derived 16S rDNA data suggests that the three tested species of Pseudo-nitzschia, which vary in toxin production, have phylogenetically distinct bacterial communities, and toxic Pseudo-nitzschia have lower microbial diversity than non-toxic Pseudo-nitzschia. Transplant experiments showed that isolated members of the microbiota are mutualistic to their native hosts but some are commensal or parasitic to foreign hosts, hinting at co-evolution between partners. Moreover, Pseudo-nitzschia host can gain protection from algalytic bacteria by maintaining association with its microbiota. Pseudo-nitzschia also exhibit different phenotypic expression with regards to DA production, and this depends on the bacterial species with which the host associates. Hence, the influences of the microbiota on diatom host physiology should be considered when studying the biology and ecology of marine diatoms.     

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus: a chronicle of evolution in action

Background

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation.

Results

Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre.

Conclusions

The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-407) contains supplementary material, which is available to authorized users.     

Proteome analysis of Acetobacter pasteurianus during acetic acid fermentation

Acetic acid bacteria (AAB) are Gram-negative, strictly aerobic microorganisms that show a unique resistance to ethanol (EtOH) and acetic acid (AcH). Members of the Acetobacter and Gluconacetobacter genera are capable of transforming EtOH into AcH via the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes and are used for the industrial production of vinegar.Several mechanisms have been proposed to explain how AAB resist high concentrations of AcH, such as the assimilation of acetate through the tricarboxylic acid (TCA) cycle, the export of acetate by various transporters and modifications of the outer membrane. However, except for a few acetate-specific proteins, little is known about the global proteome responses to AcH.In this study, we used 2D-DIGE to compare the proteome of Acetobacter pasteurianus LMG 1262^T when growing in glucose or ethanol and in the presence of acetic acid. Interesting protein spots were selected using the ANOVA p-value of 0.05 as threshold and 1.5-fold as the minimal level of differential expression, and a total of 53 proteins were successfully identified.Additionally, the size of AAB was reduced by approximately 30% in length as a consequence of the acidity. A modification in the membrane polysaccharides was also revealed by PATAg specific staining.     

Biotechnological Applications of Acetic Acid Bacteria

The acetic acid bacteria (AAB) have important roles in food and beverage production, as well as in the bioproduction of industrial chemicals. In recent years, there have been major advances in understanding their taxonomy, molecular biology, and physiology, and in methods for their isolation and identification. AAB are obligate aerobes that oxidize sugars, sugar alcohols, and ethanol with the production of acetic acid as the major end product. This special type of metabolism differentiates them from all other bacteria. Recently, the AAB taxonomy has been strongly rearranged as new techniques using 16S rRNA sequence analysis have been introduced. Currently, the AAB are classified in ten genera in the family Acetobacteriaceae. AAB can not only play a positive role in the production of selected foods and beverages, but they can also spoil other foods and beverages. AAB occur in sugar- and alcohol-enriched environments. The difficulty of cultivation of AAB on semisolid media in the past resulted in poor knowledge of the species present in industrial processes. The first step of acetic acid production is the conversion of ethanol from a carbohydrate carried out by yeasts, and the second step is the oxidation of ethanol to acetic acid carried out by AAB. Vinegar is traditionally the product of acetous fermentation of natural alcoholic substrates. Depending on the substrate, vinegars can be classified as fruit, starch, or spirit substrate vinegars. Although a variety of bacteria can produce acetic acid, mostly members of Acetobacter, Gluconacetobacter, and Gluconobacter are used commercially. Industrial vinegar manufacturing processes fall into three main categories: slow processes, quick processes, and submerged processes. AAB also play an important role in cocoa production, which represents a significant means of income for some countries. Microbial cellulose, produced by AAB, possesses some excellent physical properties and has potential for many applications. Other products of biotransformations by AAB or their enzymes include 2-keto-L-gulonic acid, which is used for the production of vitamin C; D-tagatose, which is used as a bulking agent in food and a noncalorific sweetener; and shikimate, which is a key intermediate for a large number of antibiotics. Recently, for the first time, a pathogenic acetic acid bacterium was described, representing the newest and tenth genus of AAB.     

Entomopathogenic Nematodes as a Model System for Advancing the Frontiers of Ecology

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have a mutualistic–symbiotic association with enteric γ-Proteobacteria (Steinernema–Xenorhabdus and Heterorhabditis–Photorhabdus), which confer high virulence against insects. EPNs have been studied intensively because of their role as a natural mortality factor for soil-dwelling arthropods and their potential as biological control agents for belowground insect pests. For many decades, research on EPNs focused on the taxonomy, phylogeny, biogeography, genetics, physiology, biochemistry and ecology, as well as commercial production and application technologies. More recently, EPNs and their bacterial symbionts are being viewed as a model system for advancing research in other disciplines such as soil ecology, symbiosis and evolutionary biology. Integration of existing information, particularly the accumulating information on their biology, into increasingly detailed population models is critical to improving our ability to exploit and manage EPNs as a biological control agent and to understand ecological processes in a changing world. Here, we summarize some recent advances in phylogeny, systematics, biogeography, community ecology and population dynamics models of EPNs, and describe how this research is advancing frontiers in ecology.     

Structural and molecular genetic insight into a widespread sulfur oxidation pathway

Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds. Oxidation of this sulfur requires the concerted action of Dsr proteins. However, individual functions and interplay of these proteins are largely unclear. We proved with a ΔdsrE mutant experiment that the cytoplasmic α₂β₂γ₂-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum. The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans. The crystal structure of DsrEFH was determined at 2.5 Å resolution to assist functional assignment in detail. In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria. Conserved Cys78 of A. vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD. TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis. DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon. The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction. Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction. In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage.     

Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus)

Bacterial gut symbiont communities are critical for the health of many insect species. However, little is known about how microbial communities vary among host species or how they respond to anthropogenic disturbances. Bacterial communities that differ in richness or composition may vary in their ability to provide nutrients or defenses. We used deep sequencing to investigate gut microbiota of three species in the genus Bombus (bumble bees). Bombus are among the most economically and ecologically important non-managed pollinators. Some species have experienced dramatic declines, probably due to pathogens and land-use change. We examined variation within and across bee species and between semi-natural and conventional agricultural habitats. We categorized as ‘core bacteria'' any operational taxonomic units (OTUs) with closest hits to sequences previously found exclusively or primarily in the guts of honey bees and bumble bees (genera Apis and Bombus). Microbial community composition differed among bee species. Richness, defined as number of bacterial OTUs, was highest for B. bimaculatus and B. impatiens. For B. bimaculatus, this was due to high richness of non-core bacteria. We found little effect of habitat on microbial communities. Richness of non-core bacteria was negatively associated with bacterial abundance in individual bees, possibly due to deeper sampling of non-core bacteria in bees with low populations of core bacteria. Infection by the gut parasite Crithidia was negatively associated with abundance of the core bacterium Gilliamella and positively associated with richness of non-core bacteria. Our results indicate that Bombus species have distinctive gut communities, and community-level variation is associated with pathogen infection.     

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.     

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.     

Biotechnological applications of acetic acid bacteria

The acetic acid bacteria (AAB) have important roles in food and beverage production, as well as in the bioproduction of industrial chemicals. In recent years, there have been major advances in understanding their taxonomy, molecular biology, and physiology, and in methods for their isolation and identification. AAB are obligate aerobes that oxidize sugars, sugar alcohols, and ethanol with the production of acetic acid as the major end product. This special type of metabolism differentiates them from all other bacteria. Recently, the AAB taxonomy has been strongly rearranged as new techniques using 16S rRNA sequence analysis have been introduced. Currently, the AAB are classified in ten genera in the family Acetobacteriaceae. AAB can not only play a positive role in the production of selected foods and beverages, but they can also spoil other foods and beverages. AAB occur in sugar- and alcohol-enriched environments. The difficulty of cultivation of AAB on semisolid media in the past resulted in poor knowledge of the species present in industrial processes. The first step of acetic acid production is the conversion of ethanol from a carbohydrate carried out by yeasts, and the second step is the oxidation of ethanol to acetic acid carried out by AAB. Vinegar is traditionally the product of acetous fermentation of natural alcoholic substrates. Depending on the substrate, vinegars can be classified as fruit, starch, or spirit substrate vinegars. Although a variety of bacteria can produce acetic acid, mostly members of Acetobacter, Gluconacetobacter, and Gluconobacter are used commercially. Industrial vinegar manufacturing processes fall into three main categories: slow processes, quick processes, and submerged processes. AAB also play an important role in cocoa production, which represents a significant means of income for some countries. Microbial cellulose, produced by AAB, possesses some excellent physical properties and has potential for many applications. Other products of biotransformations by AAB or their enzymes include 2-keto-L-gulonic acid, which is used for the production of vitamin C; D-tagatose, which is used as a bulking agent in food and a noncalorific sweetener; and shikimate, which is a key intermediate for a large number of antibiotics. Recently, for the first time, a pathogenic acetic acid bacterium was described, representing the newest and tenth genus of AAB.     

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus

Background

Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling.

Results

Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster.

Conclusions

Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1763-2) contains supplementary material, which is available to authorized users.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.     

Analysis of r-protein and RNA conformation of 30S subunit intermediates in bacteria

The ribosome is a large macromolecular complex that must be assembled efficiently and accurately for the viability of all organisms. In bacteria, this process must be robust and tunable to support life in diverse conditions from the ice of arctic glaciers to thermal hot springs. Assembly of the Small ribosomal SUbunit (SSU) of Escherichia coli has been extensively studied and is highly temperature-dependent. However, a lack of data on SSU assembly for other bacteria is problematic given the importance of the ribosome in bacterial physiology. To broaden the understanding of how optimal growth temperature may affect SSU assembly, in vitro SSU assembly of two thermophilic bacteria, Geobacillus kaustophilus and Thermus thermophilus, was compared with that of E. coli. Using these phylogenetically, morphologically, and environmentally diverse bacteria, we show that SSU assembly is highly temperature-dependent and efficient SSU assembly occurs at different temperatures for each organism. Surprisingly, the assembly landscape is characterized by at least two distinct intermediate populations in the organisms tested. This novel, second intermediate, is formed in the presence of the full complement of r-proteins, unlike the previously observed RI* particle formed in the absence of late-binding r-proteins in E. coli. This work reveals multiple distinct intermediate populations are present during SSU assembly in vitro for several bacteria, yielding insights into RNP formation and possible antimicrobial development toward this common SSU target.     

Nematodes Associated with Fig Wasps,Pegoscapus spp. (Agaonidae), and Syconia of Native Floridian Figs (Ficus spp.)

Syconia in successive developmental phases from Ficus laevigata Vahl (F. citrifolia Miller sensu DeWolf 1960) (Moraceae) and successive life stages of its fig wasp pollinator, Pegoscapus sp. (P. assuetus (Grandi) sensu Wiebes 1983) (Agaonidae) were dissected to elucidate their association with two undescribed species of nematodes. Parasitodiplogazter sp. (Diplogasteridae) are transported by female Pegoscapus sp. into the cavity of a phase B syconium as third-stage juveniles (J3), where they molt to the J4 stage and greatly increase in size in the hemocoel of the fig wasp after it begins to pollinate and oviposit in female florets. The J4 exit the wasp cadaver in a phase B or early phase C syconium, and molt to adults that mate and lay eggs. New J3 infect the next generation of female or male wasps as they emerge from their galls in phase D figs. Mated entomogenous females of Schistonchus sp. (Aphelenchoididae) are transported in the hemocoel of female wasps to the fig cavity of a phase B syconium. Female Schistonchus sp. exit the wasp and parasitize immature male florets causing an exudate, the development of hypertrophied epidermal cells of the anther filaments and anthers, and aberrations of the anther filament, anthers, and pollen. At least one generation of Schistonchus sp. occurs in the male florets. Entomogenous females appear at about the time that fig wasps molt to adults in their galls in late phase C syconia. Another Schistonchus sp. was recovered from females of P. mexicanus (Ashmead) (P. jimenezi (Grandi) sensu Wiebes 1983) and from the syconia of F. aurea Nuttall and appears to have a life cycle similar to that described for the Schistonchus sp. from F. laevigata.     

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn’t produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6–52 μg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems.     

Characterization of Sialyl and Galactosyl Residues on the Body Wall of Different Plant Parasitic Nematodes

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-β-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.