Concentrated Culture Filtrates Research Articles

One-pot synthesis of stable vitamin C analogue using trans-glycosylating enzyme from Aspergillus carbonarius BTCF 5

Biocatalysis using transglycosidases for one-pot glycosylation of small molecules is a greener alternative to chemical glycosylation. The transglycosylation potential of the crude culture filtrate of Aspergillus carbonarius BTCF 5 was evaluated based on the biotransformation of the target molecule, ascorbic acid (Vitamin-C), to its more stable glycosylated analogue -2-O-α-D-glucopyranosyl ascorbic acid (AA2G), using maltose as the glycosyl donor. Media engineering increased the transglycosylation yield by 64%, whereas the use of concentrated culture filtrate increased the transglycosylation yield by 70%. Protein purification studies identified the candidate transglycosylating enzyme to be a GH 31 family CAZyme (α-transglucosidase). A minimally purified enzyme could enhance the transglycosylation yield by 77% with a total AA2G yield of 91.5 mM (10.32 g/L) and improved the overall conversion rate of ascorbic acid and maltose in the reaction by 16% and 6.3% respectively from the base level. A two-step column chromatography helped in product purification with 46% recovery. The structural characterization of purified AA2G provided a detailed insight into the regioselectivity of the enzyme to glycosylate C2 of ascorbic acid and its ability to form AA2G as the predominant isomer, which is also the most preferred one for end applications.

PaR1 secreted by the type IX secretion system is a protective antigen of Riemerella anatipestifer.

Riemerella anatipestifer mainly infects domestic ducks, geese, turkeys, and other birds, and causes considerable economic losses to the global duck industry. Previous studies have shown that concentrated cell-free culture filtrates of R. anatipestifer induce highly significant protection against homologous challenge. In this study, 12 immunogenic proteins were identified in the culture supernatant of R. anatipestifer strain Yb2 with immunoproteomic analysis. Of these, three immunogenic proteins, AS87_RS06600 (designated "PaR1" in this study), AS87_RS09020, and AS87_RS09965, which appeared in more than three spots on the western-blotted membrane, were expressed in Escherichia coli and purified. Animal experiments showed that the recombinant PaR1 (rPaR1) protein protected 41.67% of immunized ducklings against challenge with virulent Yb2, whereas rAS87_RS09020 or rAS87_RS09965 did not, and that ducklings immunized once with rPaR1 were 20, 40, and 0% protected from challenge with R. anatipestifer strains WJ4 (serotype 1), Yb2 (serotype 2), and HXb2 (serotype 10), respectively. In addition, rPaR1 immunized rabbit serum showed bactericidal activity against strain Yb2 at a titer of 1:8. These results indicate that rPaR1 of strain Yb2 protects against homologous challenge. Amino acid homology analysis show that PaR1 is a non-serotype-specific protein among different R. anatipestifer serotypes. Furthermore, PaR1 is mainly secreted outside the cell through the T9SS. Overall, our results demonstrate that R. anatipestifer PaR1 is a non-serotype-specific protective protein secreted by the T9SS.

Non-targeted metabolomic profiling of filamentous cyanobacteria Aphanizomenon flos-aquae exposed to a concentrated culture filtrate of Microcystis aeruginosa

Microcystis and Aphanizomenon are two toxic cyanobacteria genera, which frequently cause blooms in freshwater lakes. In some cases, succession of these two genera was observed in natural water bodies. Among the diverse factors contributing to such succession of dominant cyanobacterial genera, an allelopathic effect was proposed to be involved after the growth inhibitory effect of several Microcystis species on A. flos-aquae was investigated. However, the response of target species exposed to Microcystis are poorly described. In the present study, we used two toxic cyanobacteria strains, Aphanizomenon flos-aquae (Aph1395) and Microcystis aeruginosa strain 905 (Ma905) as research subjects. Aph1395 was inhibited with a necessarily concentrated culture filtrate of Ma905 (MA905-SPE), and the response of the inhibited Aph1395 cells was explored via non-targeted metabolomic profiling. In total, 3735 features were significantly different in the Aph1395 treated with Ma905-SPE vs. those treated with BG11 medium. Among them, the annotations of 146 differential features were considered to be confident via MS/MS spectrum matching analysis. Based on the reported physiological functions of the annotated differential features, we proposed a putative model that in the growth-inhibited Aph1395, a suite of increased or decreased features with activities in apoptosis, growth inhibition, and stress response processes contributed to, or defended against, the allelopathic effect caused by Ma905. Our findings provide insights into the interaction between the bloom forming cyanobacterial species that share the same ecological environment.

Effect of Aspergillus niger NBC001 on the soybean rhizosphere microbial community in a soybean cyst nematode-infested field

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most important pests causing considerable damage to soybean (Glycine max (L.) Merr.) around the world. Biocontrol provides a strategy for sustainable nematode control. Previously, Aspergillus niger NBC001 was isolated from the cysts of Heterodera spp. and able to control H. glycines and promote the growth of soybean in a pot experiment. In this study, the effects of NBC001 on H. glycines density and on the soybean rhizosphere microbial community in a soybean cyst nematode-infested field were studied. The results showed that NBC001 could suppress H. glycines by 31.7% in the field. High-throughput sequencing analysis showed that NBC001 had no significant influence on soybean rhizosphere microbial community structure, indicating that seed coat-dressing with the concentrated culture filtrate of NBC001 was safe for the soil ecological environment. In addition, high-throughput sequencing results demonstrated that at 10 days post transplantation, NBC001 increased the abundances of Actinobacteria and Acidobacteria, but decreased the abundances of Bacteroidetes and Gemmatimonadetes at the phylum level. Meanwhile, the abundances of Phyllobacterium, Ralstonia and H16 were increased, while the abundances of Adhaeribacter, Gemmatimonas, Sphingomonas, Flavisolibacter were suppressed by application of NBC001. However, at 90 days post transplantation, NBC001 only increased the abundances of Aeromicrobium and RB41 whereas it decreased the abundance of H16. The results indicated that application of NBC001 increased the relative abundances of the beneficial microorganisms such as Actinobacteria, Acidobacteria, Aeromicrobium and Phyllobacterium in the soil. In summary, NBC001 is an eco-friendly biocontrol agent for H. glycines control.

In vitro and in silico approach of fungal growth inhibition by Trichoderma asperellum HbGT6-07 derived volatile organic compounds

The species of Trichoderma are one of the most frequently used natural biocontrol agent. This study, we identified isolate HbGT6-07 of Trichoderma asperellum and evaluated the antimicrobial effects both in vitro and in silico approaches. Tested 10% concentrated culture filtrate of HbGT6-07 inhibited 98% of colony radial growth in B. cinerea (B05.10) as well as 91% of S. sclerotiorum (A367). HbGT6-07 was detected to produce volatile organic compounds (VOCs) with antifungal activity. In in-vitro dish-within-dish method (DwD), The HbGT6-07 VOCs effectively reduced colonial diameter, growth rate and sclerotia production by two virulent fungal pathogens. Moreover, the hyphal fragments of HbGT6-07 demonstrated successful mycelia growth suppression (97%) against infection oilseed rape leaves by hyphae of the two virulent fungal pathogens through competition. The mixed culture assay, exhibited that the isolate T. asperellum HbGT6-07 was significantly reduced the production and weight of sclerotia. The GC-MS analysis identified 32 VOCs derived from HbGT6-07. In addition, VOCs derived from HbGT6-07 were assessed against targeted protein of three fungal species; Aspergillus oryzae, Saccharomyces cerevisiae, Candida albicans via molecular docking. Butylated hydroxytolune and Beta-Cedrene had energy (−5.3 and −5.7 Kcal/mol) for targeted protein of Aspergillus oryzae and (−6.8 and −8.0 Kcal/mol) for Saccharomyces cerevisiae, whereas alpha-bergamotene and Beta-Cedrene exihbit energy (−7.5 and −7.4 Kcal/mol), respectively. The molecular dynamics study confirms the structural stability and rigidity of the docked complex through multiple descriptors from simulation trajectories. The above findings indicated that HbGT6-07 could attain competitive progress via production of VOCs and comprehensive mycelial growth.

Detection and partial characterization of extracellular inducers of persistence in Staphylococcus epidermidis and Staphylococcus aureus.

Introduction. This study describes the identification and partial characterization of persistence-inducing factors (PIFs) from staphylococci.Hypothesis/Gap Statement. Increases in persisters during mid-log phase growth indicate that quorum-sensing factors might be produced by staphylococci.Aim. To identify and partially characterize PIFs from Staphylococcus epidermidis RP62A and Staphylococcus aureus SH1000.Methodology. Others have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCFs) from S. epidermidis and S. aureus were obtained at various OD600s and following incubation at 16 h. The CCFs were used to develop a PIF assay. The PIF assay was used to partially characterize PIF from S. epidermidis and S. aureus for sizing of PIF activity, temperature and protease sensitivity and inter-species communications.Results. The optimal OD600s for S. epidermidis and S. aureus PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis' PIF activity was decreased by storage at 4 oC but not at 20 oC (16 h), 37 oC (1 h) or 100 oC (15 min). S. aureus' PIF activity was decreased following storage at 4 oC (2 weeks) and after boiling at 100 oC for 5 min but not after incubation at 37 oC (1 h). PIF activity from both species went through a 3000 molecular weight cutoff ultrafilter. Proteinase K treatment of S. aureus PIF decreased activity but did not decrease the PIF activity of S. epidermidis. PIF from S. epidermidis did not increase persisters when used to treat S. aureus cells and nor did PIF from S. aureus increase persisters when used to treat S. epidermidis cells.Conclusions. Attempts to discover PIFs for staphylococci were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species produce extracellular, low-molecular-weight inducers of persistence when assayed using an OD600 -based PIF assay.

1194. Identification and Characterization of Extracellular Inducers of Persistence in Staphylococcus epidermidis and Staphylococcus aureus

BackgroundThis study describes the identification and partial characterization of persistence inducing factors (PIF) from Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Persistence is an epigenetic process that results in tolerance of bacterial cells to antibiotic treatment, which can result in chronic human infections.MethodsOthers have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCF) from S. epidermidis RP62A and S. aureus SH1000 were obtained at various OD600’s and following incubation at 16 h. The CCF’s were used to develop a persistence inducing factor (PIF) assay. The PIF assay was used to partially characterize PIF from S. epidermidis RP62A and S. aureus SH1000 for relative molecular weight, temperature and protease sensitivity and inter-species communications.ResultsOptimal OD600’s for the S. epidermidis RP62A and S. aureus SH1000 PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis RP62A’s PIF activity was decreased by storage at 4o C (2 weeks or longer) but not following incubation at 20o C (16 h), 37o C (1 h) or 100o C (15 min). S. aureus SH1000’s PIF activity was decreased following storage at 4o C (2 week or longer) and after boiling at 100oC for 5 min but not after incubation at 37o C (1 h). PIF activity from both species was less than 3,000 Mrr. Proteinase-K treatment of S. aureus SH1000 PIF decreased activity but did not decrease PIF activity of S. epidermidis RP62A. PIF from S. epidermidis RP62A did not increase persister numbers when used to treat S. aureus SH1000 cells nor did PIF from S. aureus SH1000 increase persister numbers in S. epidermidis RP62A cells.ConclusionPrevious attempts to discover PIF’s for staphylococcal species were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species appear to produce unique, extracellular, low-molecular-weight inducers of persistence (PIF) when assayed using an OD600-based PIF assay.Disclosures All Authors: No reported disclosures

A comparison of PTI defense profiles induced in Solanum tuberosum by PAMP and non-PAMP elicitors shows distinct, elicitor-specific responses.

The induction of general plant defense responses following the perception of external elicitors is now regarded as the first level of the plant immune response. Depending on the involvement or not of these molecules in pathogenicity, this induction of defense is called either Pathogen-Associated Molecular Pattern (PAMP) Triggered Immunity or Pattern Triggered Immunity—both abbreviated to PTI. Because PTI is assumed to be a widespread and stable form of resistance to infection, understanding the mechanisms driving it becomes a major goal for the sustainable management of plant-pathogen interactions. However, the induction of PTI is complex. Our hypotheses are that (i) the recognition by the plant of PAMPs vs non-PAMP elicitors leads to specific defense profiles and (ii) the responses specifically induced by PAMPs target critical life history traits of the pathogen that produced them. We thus analyzed, using a metabolomic approach coupled with transcriptomic and hormonal analyses, the defense profiles induced in potato foliage treated with either a Concentrated Culture Filtrate (CCF) from Phytophthora infestans or two non-PAMP preparations, β-aminobutyric acid (BABA) and an Ulva spp. Extract, used separately. Each elicitor induced specific defense profiles. CCF up-regulated sesquiterpenes but down-regulated sterols and phenols, notably α-chaconine, caffeoyl quinic acid and rutin, which decreased spore production of P. infestans in vitro. CCF thus induces both defense and counter-defense responses. By contrast, the Ulva extract triggered the synthesis of a large-spectrum of antimicrobial compounds through the phenylpropanoid/flavonoid pathways, while BABA targeted the primary metabolism. Hence, PTI can be regarded as a heterogeneous set of general and pathogen-specific responses triggered by the molecular signatures of each elicitor, rather than as a uniform, non-specific and broad-spectrum set of general defense reactions.

Isolation and characterization of Aspergillus niger NBC001 underlying suppression against Heterodera glycines

Heterodera glycines is the most pervasive soybean pests worldwide. Biocontrol provides a strategy to sustainably control nematodes. In this study, 22 fungal isolates were obtained and identified from cysts of Heterodera spp. Among them, Aspergillus niger NBC001 showed high nematicidal activity against H. glycines. The 2-fold dilution of NBC001 culture filtrate caused 89% mortality of second-stage juveniles and inhibited more than 98% of egg hatching in vitro. In both pot and field experiments, the numbers of H. glycines cysts in soybean seedlings dressed with the the 5-fold concentrated culture filtrate of NBC001 were significantly reduced by 43% and 28%, respectively. In addition, application of NBC001 remarkably reduced the penetration of nematodes into the roots. Histochemical and fluorometric staining analyses indicate that application of NBC001 stimulated hydrogen peroxide activity in the roots and triggered callose deposition in the leaves and roots. Transcription of the PR1a and EREBP genes in the salicylic acid and ethylene signaling pathways was upregulated in soybean plants treated with NBC001. However, the application of concentrated culture filtrate of NBC001 had no significant impacts on the soil microbial community based on next generation DNA sequencing technology. In summary, NBC001 may be a good biocontrol agent against H. glycines via stimulation of the immunity/defense of the plant host.

The Effectiveness of Induced Defense Responses in a Susceptible Potato Genotype Depends on the Growth Rate of Phytophthora infestans.

Phytophthora infestans causes the devastating potato late blight disease, which is widely controlled with fungicides. However, the debate about chemical control is fueling a promotion toward alternative methods. In this context, the enhancement of natural plant immunity could be a strategy for more sustainable protection. We previously demonstrated that a concentrated culture filtrate (CCF) of P. infestans primes defense reactions in potato. They are genotype-dependent and metabolites produced decrease pathogen growth in vitro but not in vivo on tubers. Induced potato defenses are assumed to affect P. infestans life history traits depending on strains. This assumption was studied in vivo through induced leaflets on a susceptible genotype inoculated with four P. infestans strains differing for lesion growth rate. This study combines both defenses mechanistic analysis and ecological observations. Defense-gene expressions were thus assessed by quantitative reverse transcription-polymerase chain reaction; pathogen development was simultaneously evaluated by measuring necrosis, quantifying mycelial DNA, and counting sporangia. The results showed that CCF pretreatment reduced the pathogenicity differences between slow- and fast-growing strains. Moreover, after elicitation, PR-1, PR-4, PAL, POX, and THT induction was strain-dependent. These results suggest that P. infestans could develop different strategies to overcome plant defenses and should be considered in biocontrol and epidemic management of late blight.

Biolytic extraction of poly(3-hydroxybutyrate) from Bacillus megaterium Ti3 using the lytic enzyme of Streptomyces albus Tia1.

The applicability of Streptomyces sp. cell lytic enzymes for devising a simple and competent biological polyhydroxyalkanoate (PHA) recovery approach from Bacillus megaterium cells was investigated. B. megaterium strain Ti3 produced 50% (w/w) PHA using glucose as carbon source. The intracellular PHA was recovered employing a non-PHA accumulating actinomycetes (Tia1) identified as Streptomyces albus, having potent lytic activity against living and heat inactivated B. megaterium. Interestingly, maximum biomass (2.53 ± 0.6 g/L by 24 h) of the lytic actinomycete was obtained in PHA production medium itself thus circumventing the prior actinomycete acclimatization just by co-inoculation with B. megaterium as an inducer. Maximum lytic activity was observed at pH 6.0, 40 °C, 220 mg of biomass and 33.3 mL of concentrated culture filtrate in a 100 mL reaction mixture. Preliminary biochemical investigations confirmed the proteolytic and caseinolytic nature of the lytic enzyme. PHA yield of 0.55 g/g by co-inoculation extraction approach was comparable with the conventional sodium hypochlorite based extraction method. Interestingly, S. albus also demonstrated a broad spectrum lytic potential against varied Gram-negative and Gram-positive PHA producers highlighting the extensive applicability of this biolytic PHA recovery approach. The lytic enzyme retained almost 100% relative activity on storage at −20 °C upto two months. 1H Nuclear magnetic resonance analysis of the extracted polymer confirmed it as a homopolymer composed of 3-hydroxybutyrate monomeric units. This is the first report on Streptomyces sp. based biological and eco-friendly, intracellular PHA recovery from Bacillus spp.

MANAGEMENT OF PARTHENIUM WEED USING METABOLITES OF Alternaria japonica

ABSTRACT Parthenium (Parthenium hysterophorus) is an alien invasive weed infesting many parts of the world including Pakistan. A number of herbicides have been recommended for control of this weed, but these herbicides have issues regarding human health and environmental safety. In the current study, the herbicidal potential of culture filtrates of a fungal species, namely Alternaria japonica, was evaluated against parthenium weed. The fungal species was grown in malt extract broth (MEB) and potato dextrose broth (PDB) for 15 days. Culture filtrates were obtained by passing the materials through muslin cloth followed by filtration through filter paper and then through Millipore filter paper. In laboratory bioassays, the effect of original (X) and diluted (1/2X) filtrates was studied on seed germination, and shoot and root growth of parthenium. In general, fungal culture filtrates prepared in either of the two growth medium significantly reduced germination and seedling growth of parthenium. However, culture filtrates prepared in potato dextrose broth showed greater herbicidal activity than those prepared in malt extract broth. Foliar spray bioassays were carried out by using culture filtrates of A. japonica prepared in potato dextrose broth. In this experiment, 1-, 2- and 3 week old parthenium seedlings were sprayed 4 times with original (X) and concentrated (2X) fungal culture filtrates, with intervals of 4 days. In general, 1 week old parthenium seedlings were highly susceptible to foliar spray of fungal metabolites. The present study concludes that culture filtrates of A. japonica prepared in potato dextrose broth contain potent herbicidal constituents for management of parthenium.

Hormone signalling pathways are differentially involved in quantitative resistance of potato to Phytophthora infestans

Quantitative resistance may depend on the effectiveness of PAMP‐triggered immunity. This study highlights the diversity of mechanisms involved in the quantitative resistance of potato to Phytophthora infestans. The investigation focused on the implication of the hormone signalling pathways induced in four potato genotypes by a concentrated culture filtrate (CCF) of P. infestans. The genotypes were ranked according to their level of resistance to P. infestans and discriminant analysis of gene expression profiles separated the most resistant genotype from the three others, particularly because of a strong induction of the salicylic acid (SA) pathway. In this genotype, transcripts involved in the SA pathway, EDS1, WRKY1, PR‐1 and PR‐2, were induced by CCF. SA pathway involvement was confirmed by a peak of SA accumulation 12 h after elicitation and by the induction of jaz1 (jasmonate Zim domain protein 1) transcripts, which inhibit defence responses mediated by jasmonic acid (JA). By contrast, neither a significant induction of SA‐mediated responses nor an accumulation of free SA and PR‐2 were observed in the other resistant or two susceptible cultivars. Expression of genes in the ET and JA pathways was either not, or weakly, induced by CCF in potato. Finally, the involvement of signalling pathways was genotype dependent rather than correlating with resistance level.

Identification of Three Elicitins and a Galactan-Based Complex Polysaccharide from a Concentrated Culture Filtrate of Phytophthora infestans Efficient against Pectobacterium atrosepticum

The induction of plant immunity by Pathogen Associated Molecular Patterns (PAMPs) constitutes a powerful strategy for crop protection. PAMPs indeed induce general defense responses in plants and thus increase plant resistance to pathogens. Phytophthora infestans culture filtrates (CCFs) are known to induce defense responses and decrease the severity of soft rot due to Pectobacterium atrosepticum in potato tubers. The aim of this study was to identify and characterize the active compounds from P. infestans filtrate. The filtrate was fractionated by gel filtration, and the protection effects against P. atrosepticum and the ability to induce PAL activity were tested for each fraction. The fraction active in protection (F1) also induced PAL activity, as did the whole filtrate. Three elicitins (INF1, INF4 and INF5) were identified in F1b, subfraction of F1, by MALDI-TOF-MS and MS/MS analyses. However, deproteinized F1b still showed biological activity against the bacterium, revealing the presence of an additional active compound. GC-MS analyses of the deproteinized fraction highlighted the presence of a galactan-based complex polysaccharide. These experiments demonstrate that the biological activity of the CCF against P. atrosepticum results from a combined action of three elicitins and a complex polysaccharide, probably through the activation of general defense responses.

Evaluation of β-1,3-glucanase and β-1,4-glucanase enzymes production in some Trichoderma species

Trichoderma species have become the important means of biological control for fungal diseases. This research was carried on to access the high β-1,3-glucanase and β-1,4-glucanase enzyme producer of Trichoderma species isolates using two different carbon sources for finding a method to obtain more concentrate culture filtrates. Therefore, 14 Trichoderma isolates belonging to species: Trichoderma ceramicum, T. virens, T. pseudokoningii, T. koningii, T. koningiosis, T. atroviridae, T. viridescens, T. asperellum, T. harzianum1, T. orientalis, T. harzianum2, T. brevicompactum, T. viride and T. spirale were cultured in Wiendling’s liquid medium plus 0.5% glycerol or 0.5% Phytophthora sojae-hyphe as the carbon source in shaking and non-shaking (stagnant) statuses. Enzyme activity rate and total protein were evaluated in raw, acetony and lyophilized concentrated culture filtrates and the specific enzyme activity of β-1,3-glucanase and β-1,4-glucanase were measured by milligramme glucose equivalent released per minute per milligramme total protein in culture filtrates. The results showed that using Phytophthora – hyphe in medium increased the enzyme activities as compared to glycerol at all Trichoderma species which suggested that these substrates can also act as inducer for synthesis of lytic enzymes, in addition the most enzymes activity was observed in the lyophilised concentrated culture filtrate. The most successful species in β-1,3-glucanase and β-1,4-glucanase enzymes activities were T. brevicompactum and T. virens and these species can be used for mass production of these enzymes which are supposed to be used in commercial formulation and also will be able to control P. sojae directly.

Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors

Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses. Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.

Some Characters of <i>Cl. perfringens</i> Isolated from Fresh and Marketed Processed Meat

This study was carried out a fresh meat immediately after slaughter and a marketed processed cattlemeat (sausage and minced meat). A total of 530 meat samples were examined for the presence of Cl. perfringens, 423 were from fresh meat obtained immediately after slaughtering (108 cattle meat, 101 sheep meat, 100 camel meat and 114 buffaloe meat) and 107 processed meat (57 from sausage and 50 from minced meat). Cl. perfringens was isolated from 204 (48.2%) of fresh meat samples, 61 (56.5%) from cattle, 53 (52.5%) from sheep meat, 45 (45%) from camel meat and 45 (39.5%) from buffaloe meat. The isolation rate of Cl. perfringens was higher in processed meat, it was isolated from 68 (63.6%) of which 45 (78.9%) from sausage and 23 (46%) in minced meat. The processed meat was found to harbour higher viable count ranging between 4 × 102 - 7 × 106 Cl. perfringens cells/gm meat than that Fresh meat in which the number ranged from 102:5 × 106 cells/gm meat. Typing of isolated strains revealed that the majority of it was of Cl. perfringens type A, 2 of type B, 3 of type C and one type D. Sixty strains of Cl. perfringens type A were randomized and tested for heat resistance at 100℃ and the results were recorded. Production of enterotoxin by 10 strains of Cl. perfringens was performed by ligated ileal loop test in rabbits. It was done by injection of whole culture in skimmed milk, cell extracts and concentrated culture filtrates of the organism in the ileal ligated loop of rabbits and the results were recorded.

Nicotiflorin, rutin and chlorogenic acid: phenylpropanoids involved differently in quantitative resistance of potato tubers to biotrophic and necrotrophic pathogens

Physiological and molecular mechanisms underlying quantitative resistance of plants to pathogens are still poorly understood, but could depend upon differences in the intensity or timing of general defense responses. This may be the case for the biosynthesis of phenolics which are known to increase after elicitation by pathogens. We thus tested the hypothesis that differences in quantitative resistance were related to differential induction of phenolics by pathogen-derived elicitors. Five potato cultivars (Solanum tuberosum, L.) spanning a range of quantitative resistance were treated with a concentrated culture filtrate (CCF) of Phytophthora infestans or purified lipopolysaccharides (LPS) from Pectobacterium atrosepticum. The kinetic of phenolics accumulation was followed and a set of typical phenolics was identified: chlorogenic acid, phenolamides and flavonols including rutin (quercetin-3-O-rutinoside) and nicotiflorin (kaempferol-3-O-rutinoside). Our results showed that CCF but not LPS induced differential accumulation of major phenolics among cultivars. Total phenolics were related with resistance to P. atrosepticum but not to P. infestans. However, nicotiflorin was inversely related with resistance to both pathogens. Rutin, but not nicotiflorin, inhibited pathogen growth in vitro at physiological concentrations. These data therefore suggest that (i) several phenolics are candidate markers for quantitative resistance in potato, (ii) some of these are pathogen specific although they are produced by a general defense pathway, (iii) resistance marker molecules do not necessarily have antimicrobial activity, and (iv) the final content of these target molecules-either constitutive or induced-is a better predictor of resistance than their inducibility by pathogen elicitors.

Preliminary Characterizations of a Carbohydrate from the Concentrated Culture Filtrate from <i>Fusarium solani</i> and Its Role in Benzo[a]Pyrene Solubilization

In order to investigate the mechanism of benzo[a]pyrene uptake by a filamentous fungus Fusarium solani, a biochemical characterization of its concentrated culture filtrate has been conducted. The preparation contained approximately (w/w): 50% of total carbohydrate, 6.5% of uronic acid and 6% protein, as determined by colorimetric tests. Gel filtration and anion-exchange chromatographic profiles indicated that the main product of the culture filtrate was a glycoprotein, which contained mannose, glucose and galactose in an approximate molar ratio of 1.5: 0.8: 1. The polysaccharide fraction of the culture filtrate was prepared by treatment with proteinase K, followed by gel-filtration chromatography. Its chemical structure was studied by methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and Nuclear Magnetic Resonance spectroscopy (NMR). The major carbohydrate was a polymer of β-(1 → 6)-linked galactofuranose units fully branched at positions O-2 by single residues of α-glucopyranose. The Fusarium concentrated culture filtrate increased 4-fold the BaP solubilization in comparison with its aqueous solubility and suggested that the carbohydrate present in this filtrate should probably be involved in this enhancement. Our findings point out the potential role of fungal glycoproteins in PAH microbial bioavaibility, an important step for PAH biodegradation.

Down-Regulation of Platelet Surface CD47 Expression in Escherichia coli O157:H7 Infection-Induced Thrombocytopenia

BackgroundPlatelet depletion is a key feature of hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli (STEC) infection. The mechanism underlying STEC-induced platelet depletion, however, is not completely understood.Methodology/Principal FindingsHere we demonstrated for the first time that platelet surface expression of CD47 was significantly decreased in C57BL6 mice treated with concentrated culture filtrates (CCF) from STEC O157:H7. STEC O157:H7 CCF treatment also led to a sharp drop of platelet counts. The reduction of cell surface CD47 was specific for platelets but not for neutrophil, monocytes and red blood cells. Down-regulation of platelet surface CD47 was also observed in isolated human platelets treated with O157:H7 CCF. Platelet surface CD47 reduction by O157:H7 CCF could be blocked by anti-TLR4 antibody but not anti-CD62 antibody. Down-regulation of platelet surface CD47 was positively correlated with platelet activation and phagocytosis by human monocyte-derived macrophages. Furthermore, the enhanced phagocytosis process of O157:H7 CCF-treated platelets was abolished by addition of soluble CD47 recombinants.Conclusions/SignificanceOur results suggest that platelet CD47 down-regulation may be a novel mechanism underneath STEC-induced platelet depletion, and that the interactions between CD47 and its receptor, signal regulatory protein α (SIRPα), play an essential role in modulating platelet homeostasis.