Bacterial Culture Filtrates Research Articles

Toxigenic potential and heat survival of spore-forming bacteria isolated from bread and ingredients

Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B. cereus group III with high values of log-cycle reductions. In conclusion, our results indicate that spore-forming bacteria contaminating bread ingredients and bread could represent a source of concern for consumer health related to the presence of strains, such as strains of B. cereus group III and single strains of other species, showing the ability to produce toxic substances associated to a thermal resistance enough to survive the bread cooking conditions.

Isolation of Butyl 2,3‐Dihydroxybenzoate From Paenibacillus elgii HOA73 Against Fusarium oxysporum f.sp. Lycopersici

AbstractHere we report for the first time the isolation of butyl 2,3‐dihydroxybenzoate (B2,3DB) from the novel antagonistic bacterium Paenibacillus elgii HOA73 and its activity against Fusarium oxysporum f.sp. lycopersici (FOL). In this study, the bacterial strain P. elgii HOA73 was isolated from soil and identified via 16S rRNA gene sequence analysis. The isolate demonstrated significant antagonism towards several plant pathogens including FOL. Our results showed the bacterial culture filtrate of P. elgii HOA73 to be highly active, inhibiting 86.1% of the growth of FOL at 50% concentration. Similarly, the bacterial crude extract of P. elgii HOA73 at 2 mg significantly inhibited FOL growth by 72.5%. An antifungal compound was purified from the bacterial crude extract of P. elgii HOA73 through different chromatographic techniques and was identif‐ied as butyl 2,3‐dihydroxybenzoate (B2,3DB) based on nuclear magnetic resonance and liquid chromatography‐mass spectrometry analyses. B2,3DB displayed potent antifungal properties, inhibiting FOL growth by 83.2% when used at 0.6 mg. The minimum inhibitory concentration of B2,3DB to inhibit any visible mycelial growth of FOL was 32 μg ml−1. All FOL conidia displayed an absence of germination or degradation when treated with 32 μg ml−1 B2,3DB after 8 or 24 h, respectively. Therefore, our results clearly demonstrated B2,3DB, as well as P. elgii HOA73, as potential biological control agents for the management of FOL.

Use of larvae of the wax moth Galleria mellonella as an in vivo model to study the virulence of Helicobacter pylori.

BackgroundHelicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world’s population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection.ResultsIn the present study, we examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for H. pylori infection. G. mellonella larvae were inoculated with bacterial suspensions or broth culture filtrates from either different wild-type H. pylori strains or their mutants defective in specific virulence determinants, such as VacA, CagA, CagE, the whole pathogenicity island (PAI) cag, urease, and gamma-glutamyl transpeptidase (GGT). We also tested purified VacA cytotoxin. Survival curves were plotted using the Kaplan-Meier method and LD50 lethal doses were calculated. Viable bacteria in the hemocoel were counted at different time points post-infection, while apoptosis in larval hemocytes was evaluated by annexin V staining. We found that wild-type and mutant H. pylori strains were able to survive and replicate in G. mellonella larvae which underwent death rapidly after infection. H. pylori mutant strains defective in either VacA, or CagA, or CagE, or cag PAI, or urease, but not GGT-defective mutants, were less virulent than the respective parental strain. Broth culture filtrates from wild-type strains G27 and 60190 and their mutants replicated the effects observed using their respective bacterial suspension. Also, purified VacA cytotoxin was able to kill the larvae. The killing of larvae always correlated with the induction of apoptosis in hemocytes.ConclusionsG. mellonella larvae are susceptible to H. pylori infection and may represent an easy to use in vivo model to identify virulence factors and pathogenic mechanisms of H. pylori. The experimental model described can be useful to screen a large number of clinical H. pylori strain and to correlate virulence of H. pylori strains with patients’ disease status.

Ovicidal activity of lactic acid produced by Lysobacter capsici YS1215 on eggs of root-knot nematode, Meloidogyne incognita.

Lysobacter capsici YS1215 isolated from soil previously showed nematicidal potential for biological control of the root-knot nematode. In this study, lactic acid, a nematicidal compound, was isolated from culture filtrate of YS1215, and its ovicidal activity was investigated. Purification and identification of lactic acid were performed by a series of column chromatographies and identified by (1)H and (13)C NMR spectra and GC-MS analysis. Our results showed that bacterial culture filtrate containing lactic acid significantly inhibited egg hatching. The lowest egg hatch rate (5.9%) was found at a high concentration (25 μl/ml) of lactic acid at 5 days after incubation, followed by 20 (15.2%), 15 (23.7%), 10 (29.8%), and 5 (36.4%) μl/ml, while egg hatching in the control (sterile distilled water) was 44.5%. This is the first report of lactic acid as an ovicidal compound, and it may be considered as an alternative of chemical pesticide against root-knot nematodes.

Antagonistic mechanisms of endophytic Pseudomonas fluorescens against Athelia rolfsii.

To explore specific mechanisms of endophytic Pseudomonas fluorescens antagonizing Athelia rolfsii, causing southern blight of Atractylodes lancea and to evaluate the potential of this Ps. fluorescens strain to control southern blight. Endophytic Ps. fluorescens strain ALEB 7B isolated from A. lancea can significantly inhibit the growth of A. rolfsii strain SY4. Pre-inoculating A. lancea seedlings with Ps. fluorescens ALEB 7B reduces the southern blight morbidity rate significantly. In situ observation using scanning electron microscopy shows Ps. fluorescens ALEB 7B colonizing the plant cells. Volatile organic compounds (VOCs) from Ps. fluorescens ALEB 7B can kill A. rolfsii SY4 and dimethyl disulphide (DMDS) plays a major role. 2-Piperidinone is a unique substance having antifungal activity in dichloromethane extracts of bacterial cell-free culture filtrates. Other antagonistic mechanisms include ecological niche occupation, antibiotic production and lytic exoenzymes secretion. Specific antagonistic mechanisms of Ps. fluorescens ALEB 7B on A. rolfsii SY4 were detailed, including release of DMDS, production of 2-piperidone, secretion of antibiotics and lytic exoenzymes and competition for spaces and nutrients. This work firstly reports the significant inhibition of VOCs released by Ps. fluorescens on the growth of A. rolfsii. 2-Piperidinone is firstly found synthesized by Ps. fluorescens, having antifungal activity. This work provides an antagonistic bacterium with practical convenience and ecologically amity, which has potential for control to A. rolfsii in vitro.

Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines.

Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

Evaluation of persistence and effectiveness of bacterial cell suspensions and culture filtrates against M. incognita on brinjal

Keeping in view the staid health and ecological apprehensions coupled with the use of pesticides, entomopathogenic nematodes have the potential to supersede pesticides for the management of various pests. Brinjal plants are the most seriously affected by Meloidogyne incognita. The main objective of this study was to evaluate the persistence effectiveness of bacterial cell suspensions (Xenorhabdus and Photorhabdus spp.) and their culture filtrates in soil up to 7, 14 and 21 days and their response against M. incognita as a source of biological control for nematode management. In a life cycle study, Xenorhabdus and Photorhabdus spp., isolated from Steinernema asiaticum and Heterorhabditis bacteriophora, were proved more effective in influencing the life cycle of RKNs. It was found that all the treatments of bacterial cell suspensions and their culture filtrates at all persistent times proved effective in reducing the number of females and egg masses as compared to control. It delayed penetration of nematode juveniles (J2) into host roots. It was concluded that persistence effectiveness of bacteria and their metabolites decreased in soil with time.

The nematicidal potential of local Bacillus species against the root-knot nematode infecting greenhouse tomatoes

Bacillus spp. were isolated from Iranian tomato fields and evaluated for their efficacy against root-knot nematodes, Meloidogyne javanica. The 52 spore-forming bacterium isolates were obtained from tomato rhizosphere following heat treatment. Eight isolates were chosen based on their potency in prevention of M. javanica egg hatch and juvenile mortality and production of proteolytic enzymes in Petri plates. Their ability to form biofilms were also determined in pot experiments. According to phenotypic traits and 16s rDNA sequencing, all selected isolates belonged to Bacillus spp. including B. cereus and B. pumilus. Treatment with bacterial culture filtrates in vitro caused juvenile mortality of 72 to 99% after 48 h. After four days, the percentage of egg hatch ranged from 1.6 to 59% depending on the isolate. Bacillus pumilus (ToIr-MA) and Bacillus sp. (ToIr-10) were found to have significant ability to produce extracellular proteases and to form maximum biofilm, considerably reducing the number of egg masses and root gall index (P = 0.05) in comparison to untreated plants. Application of ToIr-MA and ToIr-10 enhanced the fresh and dry weights of shoot and root systems. There was significant enhancement in dry root weight (45 and 50%) and shoot weight (67 and 75%). Results suggest that these two Bacillus spp. have potential as biocontrol agents against root-knot disease in tomato production.

In Vitro Action of Streptomyces griseolus Proteases as Bio-Control on Fasciola gigantic Eggs

This work evaluated the in vitro bio-control action of actinomycetes isolated from Egyptian soil against Fasciola gigantica eggs. In this respect, the identified Streptomyces griseolus was the best parasite-control agent which gave the highest mortality percent applying three methods; the first method was the application of the grown bacterial disks with the eggs at zero time on the medium (2% w/v water-agar) and incubated for 21 days (75.5% mortality), the second method was the incubation of the bacterial disks using the same medium for 5 days before eggs inoculation, after 21 days of incubation the mortality percent was 80.8%. The application of the third method showed a good effect by using all bacterial culture filtrates. S. griseolus filtrate which had the highest lytic enzyme and proteolytic activities reached 2.0 and 660.7 U/ml, respectively, gave the highest mortality (95.5%) of F. gigantica eggs compared to the other bacterial filtrates. The crude culture filtrate and the diluted culture filtrate (1.33 fold) of S. griseolus gave the highest mortality percent against the parasite´s eggs (95.8 and 94.5%, respectively) compared to the higher diluted filtrates. The culture filtrate of S. griseolus grown on two different media and its partially purified enzyme (precipitated by 60% ammonium sulfate) were tested for their proteolytic activity and inhibitory effect against F. gigantica eggs. Medium no. 2 showed the highest proteolytic activity (1030.3 and 1138.1 U/ml) for the crude and the partially purified enzyme, respectively. While the highest inhibitory effect reached 97.6 and 89.9%, respectively. Finally, the application of crude enzyme filtrate was better than the partially purified one. Streptomyces griseolus was proved as a potential biological control agent for this helminth.

Purification and cloning of nicosulfuron-degrading enzymes from Bacillus subtilis YB1

The nicosulfuron-degrading enzymes from Bacillus subtilis strain YB1 were purified and their genes were cloned. The proteins of bacterial culture filtrate were precipitated with ammonium sulfate or acetone. The extracellular proteins concentrated by acetone were purified from DEAE-Sepharose Fast Flow chromatography. The four protein peaks eluted from DEAE-column were separated and purified by native PAGE. Three components (P1-1, P3-2, P4-3) had nicosulfuron-degrading activity, and component P4-3 degradated 57.5% of this compound. The molecular weights of the components were 33.5, 54.8 and 37.0 kDa, respectively. The amino acid sequences of nicosulfuron-degrading enzymes from B. subtilis YB1 were determined by MALDI-TOF-MS, indicating these enzymes as manganese ABC transporter, vegetative catalase 1 and acetoin dehydrogenase E1, respectively. Using PCR amplification, genes 918, 1428, 1026 bp in size were detected for the enzymes studied.

Nematicidal activity of Lysobacter capsici YS1215 and the role of gelatinolytic proteins against root-knot nematodes

Lysobacter capsici YS1215 is a soil-borne strain that could inhibit the growth of phytopathogenic fungi, including Phytophthora capsici, Rhizoctonia solani and Fusarium oxysporum, as well as root-knot nematodes. The effect of different concentrations of bacterial culture filtrate (BCF) of L. capsici YS1215 on the mortality of second-stage juveniles (J2) of Meloidogyne incognita was studied using 24-well plates. The J2 mortality increased with increasing concentrations of BCF. YS1215 also produces gelatinases in the culture filtrate. To study its role in nematicidal activities, the partial purification and the characterisation of gelatinolytic proteins were done from the culture medium of the YS1215. The partially purified proteins showed three clear bands with molecular weights estimated using zymography to be 255.7, 232.1 and 146.4 kDa. The optimal pH and temperature for the proteins were 8.0 and 40°C, respectively. The activity of the proteins was inhibited by ethylenediaminetetraacetic acid, FeCl3 and 1,10-phenanthroline, whereas it was activated by MnCl2. The proteins may belong to the group of metalloproteases. Moreover, the proteins could hydrolyse skimmed milk, collagen, gelatin and bovine serum albumin (BSA) as substrates, but not casein. The proteins could induce 75% J2 mortality in five days and degrade the J2 bodies. The present study demonstrates the role of the gelatinolytic proteins in the nematicidal potential of L. capsici YS1215.

Purification, structural elucidation and bioactivity of tryptophan containing diketopiperazines, from Comamonas testosteroni associated with a rhabditid entomopathogenic nematode against major human-pathogenic bacteria

The cell free culture filtrate of a Comamonas testosteroni associated with an Entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp. exhibited promising antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain five diketopiperazines or cyclic dipeptides (DKP 1–5). The structure and absolute stereochemistry of the compounds were determined based on extensive spectroscopic analyses (HR-MS, 1HNMR, 13CNMR, 1H–1H COSY, 1H–13C HMBC) and Marfey's method. Based on the spectral data the compounds were identified as Cyclo-(L-Trp-L-Pro) (1), Cyclo-(L-Trp-L-Tyr) (2), Cyclo-(L-Trp-L-Ile) (3), Cyclo-(L-Trp-L-Leu) (4) and Cyclo-(L-Trp-L-Phe) (5), respectively. Three diketopiperazines (DKP 2, 3 and 5) were active against all the ten bacteria tested. The highest activity of 0.5μg/ml by Cyclo-(L-Trp-L-Phe) was recorded against Vibrio cholerae followed by Salmonella typhi (1μg/ml) a human pathogen responsible for life threatening diseases like profuse watery diarrhea and typhoid or enteric fever. The activity of this compound against V. cholerae and S. typhi is more effective than ciprofloxacin and ampicillin, the standard antibiotics. Cyclo-(L-Trp-L-Phe) recorded significant antibacterial activity against all the test bacteria when compared to other compounds. Five diketopiperazines were active against all the test fungi and are more effective than bavistin the standard fungicide. Diketopiperazines recorded no cytotoxicity to FS normal fibroblast and VERO cells (African green monkey kidney) except DKP 3 and 4. To our best knowledge this is the first report of antimicrobial activity of the tryptophan containing diketopiperazines against the human pathogenic microbes. The production of cyclic dipeptides by C. testosteroni is also reported here for the first time. We conclude that the C. testosteroni is promising sources of natural bioactive secondary metabolites against human pathogenic bacteria which may receive great benefit in the field of human medicine in near future.

Cyclic dipeptides from rhabditid entomopathogenic nematode-associated Bacillus cereus have antimicrobial activities

The cell free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain four bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (FABMS, (1)H NMR, (13)C NMR, (1)H-(1)H COSY, (1)H-(13)C HMBC) and Marfey's method. The compounds were identified as cyclic dipeptides (CDPs): cyclo(L-Pro-L-Trp), cyclo(L-Leu-L-Val), cyclo(D-Pro-D-Met), and cyclo(D-Pro-D-Phe), respectively. Compounds recorded significant antibacterial activity against all the test bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant S. aureus) except cyclo(L-Leu-L-Val). Cyclo(L-Leu-L-Val) recorded activity only against Gram positive bacteria. Best antibacterial activity was recorded by cyclo(L-Pro-L-Trp) against S. aureus (4 μg/ml). The four compounds were active against all the five fungi tested (Trichophyton rubrum, Aspergillus flavus, Candida albicans, Candida tropicalis and Cryptococcus neoformans) and the activity was compared with amphotericin B, the standard fungicide. The highest activity of 1 μg/ml by cyclo(L-Pro-L-Trp) was recorded against T. rubrum, a human pathogen responsible for causing athlete's foot, jock itch, and ringworm. The activity of cyclo(L-Pro-L-Trp) against T. rubrum, C. neoformans and C. albicans were better than amphotericin B, the standard antifungal agent. To our knowledge, this is the first report of antifungal activity of CDPs against the human pathogenic fungi T. rubrum and C. neoformans. The four CDPs are nontoxic to healthy human cell line up to 200 μg/ml. We conclude that the bacterium associated with entomopathogenic nematode is promising sources of natural antimicrobial secondary metabolites, which may receive greater benefit as potential sources of new drugs in the pharmaceutical industry.

Isolation and identification of antimicrobial secondary metabolites from Bacillus cereus associated with a rhabditid entomopathogenic nematode

The cell-free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain six bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (LCMS, FABMS, 1H NMR, 13C NMR, 1H −1H COSY, 1H −13C HMBC) and Marfey’s method. The compounds were identified as cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), cyclo (L-Pro-D-Phe), cyclo (L-Pro-L-Val), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene, respectively. Compounds recorded antibacterial activity against all four tested bacteria strains of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. 3,5-dihydroxy-4-isopropylstilbene recorded activity only against Gram-positive bacteria while cyclo(L-Pro-L-Val) recorded no antibacterial activity. Best antibacterial activity was recorded by 3,5-dihydroxy-4-ethyl-trans-stilbene (4 μg/ml) against Escherichia coli. The six compounds recorded significant antifungal activities against five fungal strains tested (Aspergillus flavus, Candida albicans, Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum) and they were more effective than bavistin, the standard fungicide. The activity of cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene against Candida albicans was better than amphotericin B. To the best of our knowledge, this is the first report of antifungal activity of the bioactive compounds against the plant pathogenic fungi Fusarium oxysporum, Rhizoctonia solani, and Penicillium expansum. We conclude that the Bacillus cereus strain associated with entomopathogenic nematode is a promising source of natural bioactive secondary metabolites which may receive great benefit as potential sources of new drugs in the agricultural and pharmacological industry.

Microalgae associated Brevundimonas sp. MSK 4 as the nano particle synthesizing unit to produce antimicrobial silver nanoparticles

The biosynthesis of silver nanoparticles and its antimicrobial property was studied using bacteria isolated from Spirulina products. Isolated bacteria were identified as Bacillus sp. MSK 1 (JX495945), Staphylococcus sp. MSK 2 (JX495946), Bacillus sp. MSK 3 (JX495947) and Brevundimonas sp. MSK 4 (JX495948). Silver nanoparticles (AgNPs) were synthesized using bacterial culture filtrate with AgNO3. The initial syntheses of Ag nanoparticles were characterized by UV–vis spectrophotometer (by measuring the color change to intense brown). Fourier Transform Infrared Spectroscopy (FTIR) study showed evidence that proteins are possible reducing agents and Energy-dispersive X-ray (EDX) study showing the metal silver as major signal. The structure of AgNPs was determined by Scanning electron microscopy (SEM) and X-ray diffraction (XRD). Synthesized Ag nanoparticles with an average size of 40–65nm have antimicrobial property against human pathogens like Proteus vulgaris, Salmonella typhi, Vibrio cholera, Streptococcus sp., Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. Among the isolates Brevundimonas sp. MSK 4 alone showed good activity in both synthesis of AgNPs and antimicrobial activity. This work demonstrates the possible use of biological synthesized silver nanoparticles to combat the drug resistant problem.

Purification and identification of two antifungal cyclic dipeptides from Bacillus cereus subsp. thuringiensis associated with a rhabditid entomopathogenic nematode especially against Fusarium oxysporum

The cell-free culture filtrate of Bacillus cereus subsp. thuringiensis associated with an entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain two cyclic dipeptides (CDPs). The structure and absolute stereochemistry of this compound were determined based on extensive spectroscopic analyses (FABMS, 1H NMR, 13C NMR, 1H--1H COSY, 1H--13C HMBC) and Marfey’s method. The compounds were identified as cyclo(D-Pro-L-Met) and cyclo(D-Pro-D-Tyr). CDPs showed significantly higher activity than the standard fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum. The highest activity of 2 µg/ml by cyclo(D-Pro-D-Tyr) was recorded against F. oxysporum, a plant pathogen responsible for causing fusarium wilt followed by R. solani, a pathogen that causes root rot and P. expansum. To our knowledge, this is the first report on the isolation of these compounds from Rhabditis EPN bacterial strain Bacillus cereus subsp. thuringiensis.

Antagonistic potential of Paenibacillus elgii HOA73 against the root-knot nematode, Meloidogyne incognita

This study was conducted to evaluate the nematicidal potential of Paenibacillus elgii strain HOA73 against the root-knot nematode, Meloidogyne incognita, under both in vitro and in vivo conditions. The novel strain HOA73 (GenBank accession number JQ12069) produced hydrolytic enzymes including gelatinase and chitinase. Exposure of M. incognita to various concentrations (10-30%) of the bacterial culture filtrate (BCF) or 0.05-0.4 mg ml−1 of the crude enzymes produced by P. elgii HOA73 significantly reduced hatch of the second-stage juvenile (J2) and caused substantial mortality under in vitro conditions. The hatch inhibition and J2 mortality rate was enhanced with the increase in the concentration of BCF and the crude enzymes. Similarly, the antagonistic effects also increased significantly over the time for which the treatments were given. Moreover, the crude enzymes partially destroyed cuticle layers of eggs and juveniles at 0.4 mg ml−1 concentration. Furthermore, the pot experiment indicated that addition of HOA73 culture into potted soil significantly reduced the root galling, number of egg masses and nematode population by 62.1%, 69.8% and 53.0%, respectively, as compared to the control at 7 weeks after M. incognita infestation. HOA73 inoculation also promoted tomato plant fresh shoot weight by 17.9% as compared to control. Hence, the present results demonstrated the ability of P. elgii HOA73 as a potential biocontrol candidate against the root-knot nematode as well as a plant growth promoter for tomato.

Flavobacterium hercynium EPB-C313 균주를 이용한 배추 뿌리혹병 생물적 방제

Plasmodiophora brassicae에 의한 배추 뿌리혹병은 배추뿐만 아니라 순무, 양배추에 발병하여 큰 피해를 주는 병원균이다. 배추 뿌리혹병균에 길항효과가 있는 미생물을 선발하기 위하여 배추 조직에서 분리한 Flavobacterium hercynium EPB-C313 균주의 항균활성 검정을 하였고, 포장시험을 통하여 배추 뿌리혹병에 대한 방제효과를 조사하였다. F. hercynium EPB-C313의 균체, 배양액, 배양여액를 배추 뿌리혹병 휴면포자와 혼합 처리하여 24시간 후 조사하였을 때 각각 90.4%, 36.8% 및 26.0%의 휴면포자가 불활성화 되었다. 온실 포트검정에서는 배추의 어린묘를 F. hercynium EPB-C313 배양액에 침지한 후 정식하면 뿌리혹 형성 억제율이 100%로 대조약제 fluazinam액상수 화제의 91.7%보다 우수한 효과를 나타내었다. 배추 뿌리혹병이 50% 이상 발병되었던 재배포장에 F. hercynium EPB-C313 혼합펠렛을 토양혼화 하고, F. hercynium EPBC313 배양액의 유묘관주 및 정식 10일 후 1회 토양관주하면 63.7%의 방제효과를 보였다. 이상의 결과로 볼 때 F. hercynium EPB-C313 균주는 매우 유용한 배추 뿌리혹병 생물적 방제제로 판단된다. Clubroot of Chinese cabbage by Plasmodiophora brassicae, was found to be high virulent to the Chinese cabbage, turnips and cabbage. It this study, the endophytic bacteria Flavobacterium hercynium EPB-C313, which was isolated from tissues of Chinese cabbage, was investigated the antimicrobial activity on the inactivation of resting spores and its control effect on clubroot disease by bioassay. The bacterial cells, culture solutions, and culture filtrates of F. hercynium EPB-C313 inactivate the resting spores of P. brassicae with 90.4, 36.8, and 26.0%, respectively. The clubroot was inhibited with 100% by dipping the seedlings of Chinese cabbage in culture solutions of F. hercynium EPB-C313 before planting, however the chemmical 'fluazinam' was 91.7% in pot tests. Complex treatment were highly enhanced control efficacy with 63.7% at field of 50% diseased plants by soil incorporation with the pellet contains organic matter and F. hercynium EPB-C313, seedling drench of culture solution of F. hercynium EPB-C313 and soil drench with 10 days after planting. These results imply that the F. hercynium EPB-C313 is a very useful biological control agent of clubroot disease of Chinese cabbage.

Identification of antimicrobial compound, diketopiperazines, from a Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode against major plant pathogenic fungi

To purify and characterize antimicrobial compounds from Bacillus sp. strain N associated with rhabditid entomopathogenic nematode (EPN). The cell-free culture filtrate of a bacterium associated with an EPN, Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain three diketopiperazines (DKPs). The structure and absolute stereochemistry of this compound were determined based on extensive spectroscopic analyses (FABMS, (1) H NMR, (13) C NMR, (1) H-(1) H COSY, (1) H-(13) C HMBC) and Marfey's method. The compounds were identified as cyclo(l-Pro-l-Leu), cyclo(d-Pro-l-Leu) and cyclo(d-Pro-l-Tyr), respectively. Three DKPs were active against all the five fungi tested (Aspergillus flavus, Candida albicans, Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum) and are more effective than the standard fungicide bavistin. The highest activity of 4μgml(-1) by cyclo(l-Pro-l-Leu) and cyclo(d-Pro-l-Tyr) was recorded against P.expansum, a plant pathogen responsible for causing postharvest decay of stored apples and oranges. Cyclo(d-Pro-l-Leu) recorded good antibacterial activity against all the four bacteria tested (Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa), and cyclo(l-Pro-l-Leu) and cyclo(d-Pro-l-Tyr) recorded good activity only against Gram-positive bacteria. To our knowledge, this is the first report of antifungal activity of the DKPs against the plant pathogenic fungi F.oxysporum, R.solani and P.expansum. The production of cyclo(l-Pro-l-Leu), cyclo(d-Pro-l-Leu) and cyclo-(d-Pro-l-Tyr) by a bacterium associated with EPN is also reported here for the first time. Isolated DKPs demonstrated high antimicrobial activity against bacteria and fungi, especially against plant pathogenic fungi. We conclude that the bacterium associated with EPN is a promising source of natural bioactive secondary metabolites which may receive great benefit in the field of agriculture. This study is a significant contribution to the knowledge of compounds unique from EPN bacteria as potential sources of new drugs in the agricultural and pharmacological industry.

Antioxidative properties and inhibitory effect of Bifidobacterium adolescentis on melanogenesis

Melanin is a dark pigment produced by melanocytes. Tyrosinase is a key enzyme which catalyzes the rate-limiting step of melanogenesis. However, accumulation of melanin leads to various skin hyperpigmentation disorders. To find a novel skin-whitening agent, the antioxidant capacity of Bifidobacterium adolescentis culture filtrate and inhibitory effect on melanogenesis were investigated. The antioxidant effects of B. adolescentis culture filtrate include 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging capacity, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) radical cation scavenging activity and reducing power were measured spectrophotometrically. The reducing power is a useful index for the evaluation of potential antioxidants which carry out reduction of ferricyanide to ferrocyanide. Furthermore, the inhibitory effects of the bacterial culture filtrate on mushroom tyrosinase, B16F10 intracellular tyrosinase activity and melanin content were also determined. The results revealed that B. adolescentis culture filtrate (2.5, 5.0 and 7.5 %; v/v) effectively scavenged DPPH and ABTS radicals, and lower concentrations of the bacterial culture filtrates (0.5, 1.0 and 1.5 %; v/v) showed potent reducing power in a dose-dependent pattern. Additionally, the bacterial culture filtrate suppressed murine tyrosinase activity and decreased the amount of melanin in a dose-dependent manner. Our results demonstrated that B. adolescentis culture filtrate decreases the melanogenesis process of melanoma cells by inhibiting tyrosinase activity, which we suggest may be mediated through its antioxidant activity.