Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism of action against postharvest fruit pathogens

Two Aureobasidium pullulans strains (L1 and L8), effective against some fruit postharvest pathogens were evaluated for VOCs production as a part of their modes of action towards five pathogens (Botrytis cinerea, Colletotrichum acutatum, Penicillium expansum, Penicillium digitatum and Penicillium italicum). The VOCs were assayed with a double petri dish assay against conidia germination of target pathogens. Results obtained showed that the VOCs generated by the antagonists inhibited significantly the conidia germination of all pathogens compared to the control. In particular, the conidia germination of all Penicillium was completely inhibited by VOCs produced by L1 and L8. In in vivo tests, apples and oranges were artificially inoculated with pathogen conidia and then biofumigated with VOCs emitted by both antagonists. The antagonistic treatment controlled significantly pathogen infection, confirming the results obtained in vitro tests. The best L1 and L8 VOCs activity was observed on apple inoculated with B. cinerea where the lesion diameter reduction observed was greater than the 88%. The compounds emitted by L1 and L8 strains were identified with the solid-phase microextraction (SPME)–gas chromatographic technique. Compounds as 2-phenyl, 1-butanol-3-methyl, 1-butanol-2-methyl and 1-propanol-2-methyl belonging to the group of alcohols were mainly produced for both strains, in the first 96 h of growth. These compounds were confirmed by comparison with standards. The pure compounds of VOCs cited above were used to determine the EC₅₀ values for conidia germination of pathogens. The 1-propanol-2-methyl was the VOC least active against all tested fungi, with the EC₅₀ values over 0.8 μl ml⁻¹, while the 2-phenethyl alcohol was the most active with EC₅₀ values lower than 0.8 μl ml⁻¹, except for the C. acutatum (1.97 μl ml⁻¹). The present study demonstrated, for the first time, that the production of VOCs could play an essential role in the antagonistic activity of two A. pullulans strains against five fruit postharvest pathogens.     

Broad activity against porcine bacterial pathogens displayed by two insect antimicrobial peptides moricin and cecropin B

In response to infection, insects produce a variety of antimicrobial peptides (AMPs) to kill the invading pathogens. To study their physicochemical properties and bioactivities for clinical and commercial use in the porcine industry, we chemically synthesized the mature peptides Bombyx mori moricin and Hyalophora cecropia cecropin B. In this paper, we described the antimicrobial activity of the two AMPs. Moricin exhibited antimicrobial activity on eight strains tested with minimal inhibitory concentration values (MICs) ranging between 8 and 128 μg/ml, while cecropin B mainly showed antimicrobial activity against the Gramnegative strains with MICs ranging from 0.5 to 16 μg/ml. Compared to the potent antimicrobial activity these two AMPs displayed against most of the bacterial pathogens tested, they exhibited limited hemolytic activity against porcine red blood cells. The activities of moricin and cecropin B against Haemophilus parasuis SH 0165 were studied in further detail. Transmission electron microscopy (TEM) of moricin and cecropin B treated H. parasuis SH 0165 indicated extensive damage to the membranes of the bacteria. Insights into the probable mechanism utilized by moricin and cecropin B to eliminate pathogens are also presented. The observations from this study are important for the future application of AMPs in the porcine industry.     

Design,synthesis, characterization,antimicrobial evaluation and molecular modeling studies of some dehydroacetic acid-chalcone-1,2,3-triazole hybrids

A series of new dehydroacetic acid chalcone-1,2,3-triazole hybrids as potential antimicrobial agents was designed, synthesized and characterized by FTIR, NMR and HRMS spectral techniques. All the synthesized compounds were screened in vitro against four bacterial strains (Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa) and two fungal strains (Aspergillus niger and Candida albicans). The antimicrobial results indicated that some of the compounds showed remarkable activities comparable to the standard drugs. Most of the compounds exhibited better efficacy compared to the DHA, which is itself an antimicrobial agent. The synergistic effect in biological activity was observed when DHA, chalcone and 1,2,3-triazole are conjugated. The molecular modeling studies of compound 5j into E. coli topoisomerase II DNA gyrase B were also performed.     

Trypanosoma cruzi: Antimicrobial activity and strain differentiating properties of some five- and six-membered heterocyclic compounds on trypomastigotes

Seventy-eight heterocyclic compounds, which included derivatives of furan, isoxazole, pyridine, pyrrole, and thiophene, were tested in an in vitro system for their antimicrobial activity and their strain differentiating properties against three strains of Trypanosoma cruzi (Brazil, Corpus Christi, Tulahuen) and for their effect, in tissue culture, on African Green Monkey Kidney Cells—strain Vero. Forty-eight compounds were 75–100% effective in immobilizing trypomastigote forms of the three strains tested, and 29 of these were rated nondestructive to African Green Monkey Kidney (AGMK) cells. Of these 29 compounds, 12 gave variable results ranging from complete immobilization of one strain to no apparent effect on the motility of one or two of the other strains. Nine of the compounds rated harmless to AGMK fibroblasts are proposed as a basis for constructing molecules which might be active chemotherapeutic agents against various strains of T. cruzi.     

Production of volatile organic compounds by an antagonistic strain Paenibacillus polymyxa WR-2 in the presence of root exudates and organic fertilizer and their antifungal activity against Fusarium oxysporum f. sp. niveum

The volatile organic compounds (VOCs) produced by antagonistic microbes have great antifungal potential against soil-borne fungal pathogens. The VOCs produced by Paenibacillus polymyxa strain WR-2 in the presence of root exudates and organic fertilizer were identified and their effects on the growth and spore germination of Fusarium oxysporum f. sp. niveum were evaluated. The VOCs produced by WR-2 inhibited the growth of F. oxysporum by 38%, 36% and 40% in agar medium, sterilized soil and natural soil, respectively. This inhibitory effect was increased to 60%, 58% and 64% with the addition of organic fertilizer in agar medium, sterilized soil and natural soil, respectively. The addition of root exudates did not affect the production of antifungal VOCs by WR-2. The VOCs produced by WR-2 completely inhibited the germination of F. oxysporum spores. Out of 42 identified VOCs, seven VOCs; benzothiazole, benzaldehyde, undecanal, dodecanal, hexadecanal, 2-tridecanone and phenol were found to inhibit the growth of F. oxysporum. The results of these experiments suggest another significance of using organic fertilizer as a carrier material with the biocontrol agents to control soil-borne fungal pathogens.     

Design,synthesis, antibacterial evaluation and molecular docking studies of some new quinoxaline derivatives targeting dihyropteroate synthase enzyme

Development of new antimicrobial agents is a good solution to overcome drug-resistance problems. From this perspective, new quinoxaline derivatives bearing various bioactive heterocyclic moieties (thiadiazoles, oxadiazoles, pyrazoles and thiazoles) were designed and synthesized. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against nine bacterial human pathogenic strains using the disc diffusion assay. In general, most of the synthesized compounds exhibited good antibacterial activities. The thiazolyl 11c displayed significant antibacterial activities against P. aeruginosa (MIC, 12.5 µg/mL vs levofloxacin 12.5 µg/mL). Molecular docking studies indicated that the synthesized compounds could occupy both p-amino benzoic acid (PABA) and pterin binding pockets of the dihydropteroate synthase (DHPS), suggesting that the target compounds could act by the inhibition of bacterial DHPS enzyme. The results provide important information for the future design of more potent antibacterial agents.     

Biocontrol of citrus bacterial canker caused by Xanthomonas citri subsp. citri by Bacillus velezensis

Microorganisms with biocontrol capabilities against plant pathogens are considered as one of the most promising approaches for healthy crop management. In this study, ethyl acetate extracts of 25 Bacillus strains were investigated for their antagonistic effect on Xanthomonas citri subsp. citri (Xcc), which causes the citrus bacterial canker (CBC) disease. Among them, 21 strains exerted antibacterial activity against wild-type Xcc strains. Based on the strength of the antibacterial activity, nine Bacillus strains were selected for 16S rRNA analysis. 16S rRNA sequence homology revealed that several strains were closely related to B. velezensis, where strains with no antibacterial activity grouped as the soil-associated community of B. amyloliquefaciens. B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively. Furthermore, B. velezensis Bv-21 strain was tested for biocontrol activity against a streptomycin-resistant XccM4 in detached susceptible citrus leaves. The strain reduced the incidence of CBC by 26.30% and pathogen density of XccM4 by 81.68% over control. The results of the study strongly suggest that B. velezensis can be used as an effective and eco-friendly biocontrol agent either by itself or as an active compound, against both, the wild-type and streptomycin-resistant Xcc.     

贝莱斯芽孢杆菌(Bacillus velezensis) JJYY防控土传病害效果评价及其全基因组测序分析和抗菌成分鉴定

【背景】土传病害是世界农业可持续发展的关键限制因子,在我国蔬菜和中药材上发生日趋严重,引起严重的连作障碍。生物防治对环境和农产品安全,是目前研究的热点和重点。【目的】明确对土传病原菌具有广谱抗菌活性菌株JJYY的种类及其防控效果和主要抗菌产物,为新型生物农药开发奠定基础。【方法】结合扫描电镜观察、生理生化分析和16S rRNA基因扩增测序技术鉴定菌株JJYY。分别利用比浊法和菌丝生长抑制法测定该菌提取物对4种土传病原细菌和5种土传病原真菌的EC₅₀,利用盆栽试验评价对番茄青枯病等病害的防控效果。利用二代Illumina NovaSeq与三代PacBio Sequel相结合测序技术对菌株JJYY进行全基因组测序,使用PGAP等软件进行基因注释等分析,利用反相制备液相色谱和质谱初步分离鉴定抗菌物质。【结果】菌株JJYY是一株贝莱斯芽孢杆菌（Bacillus velezensis）,该菌提取物对4种土传细菌和5种土传真菌的EC₅₀分别为0.940-1.092 mg/mL和2.733-3.678 mg/mL。对番茄青枯病、菊花根腐病和辣椒枯萎病的最高防效在80.00%-87.74%之间,与化学药剂链霉素或噁霉灵无显著差异（P>0.05）。该菌基因组大小为3 929 792 bp,编码3 895个基因,与B.velezensis JS25R和B.subtilis 168具有3 445个和2 997个同源编码基因。预测该基因组共有12个次级代谢产物合成功能基因簇。从该菌株提取物中共分离出9种抗菌组分,其中2个初步判定为已知大环内酯类抗生素macrolactin D和7-O-malonyl-macrolactin A。【结论】菌株JJYY是一株对土传病原细菌和真菌病害均有较高防效的贝莱斯芽孢杆菌,该菌基因组与已知贝莱斯芽孢杆菌不同,并产生多种已知和未知的抗菌物质,继续深入分析鉴定其抗菌物质和抗菌机制将为开发新型高效的生物农药奠定良好基础。     

Assessing the diversity of bacterial communities from marine sponges and their bioactive compounds

Symbiotic bacteria play vital roles in the survival and health of marine sponges. Sponges harbor rich, diverse and species-specific microbial communities. Symbiotic marine bacteria have increasingly been reported as promising source of bioactive compounds. A culturomics-based study was undertaken to study the diversity of bacteria from marine sponges and their antimicrobial potential. We have collected three sponge samples i.e. Acanthaster carteri, Rhytisma fulvum (soft coral) and Haliclona caerulea from north region (Obhur) of Red Sea, Jeddah Saudi Arabia. Total of 144 bacterial strains were isolated from three marine sponges using culture dependent method. Screening of isolated strains showed only 37 (26%) isolates as antagonists against oomycetes pathogens (P. ultimum and P. capsici). Among 37 antagonistic bacteria, only 19 bacterial strains exhibited antibacterial activity against human pathogens (Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 29212). Four major classes of bacteria i.e γ-Proteobacteria, α-Proteobacteria, Firmicutes and Actinobacteria were recorded from three marine sponges where γ-Proteobacteria was dominant class. One potential bacterial strain Halomonas sp. EA423 was selected for identification of bioactive metabolites using GC and LC-MS analyses. Bioactive compounds Sulfamerazine, Metronidazole-OH and Ibuprofen are detected from culture extract of strain Halomonas sp. EA423. Overall, this study gives insight into composition and diversity of antagonistic bacterial community of marine sponges and coral from Red Sea and presence of active metabolites from potential strain. Our results showed that these diverse and potential bacterial communities further need to be studied to exploit their biotechnological significance.     

Novel urea and thiourea derivatives of thiazole-glutamic acid conjugate as potential inhibitors of microbes and fungi

Since discovery and development of effective as well as safe drugs has brought a progressive era in human healthcare that is accompanied by the appearance of drug resistant bacterial strains, there is constant need of new antibacterial agent having novel mechanisms of action to act against the harmful pathogens. In the present study, several N-terminal substituted urea/thiourea derivatives were synthesized by the reaction of glutamic acid and 3-(1-piperazinyl)-1,2-benzisothiazole with various substituted phenyl isocyanates/isothiocyanates. Elemental analysis, IR, ¹H NMR, ¹³C NMR and mass spectral data confirmed the structure of the newly synthesized compounds. The derivatives were investigated for their antibacterial and antifungal activities against various pathogens of human origin by agar well diffusion method and microdilution method. The preliminary antimicrobial bioassay reveals that the compounds containing fluoro and bromo as substituents showed promising antimicrobial activity.     

Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory pathogens

Thymus longicaulis C. Presl is a small aromatic perennial herb used as a traditional remedy for cold, flu and cough. Composition of the essential oil of T. longicaulis from Croatia and its in vitro antimicrobial activity against the most common respiratory pathogens were evaluated. The yield of essential oil obtained by hydrodistillation from aerial plant parts was 1.2%. According to the GC-MS analysis, a total of forty one compounds (99%) were identified. Thymol (46.3%), ??-terpinene (16.2%), thymyl methyl ether (11.4%), and p-cymene (9.4%) were the main components. Antimicrobial activity of the essential oil against six clinically isolated bacterial and yeast strains was determined using standard disc agar diffusion method and microdilution broth assay. The essential oil exhibited antimicrobial activity towards all tested respiratory pathogens. The most sensitive strains were Haemophilus influenzae and Streptococcus pneumoniae(MIC=0.78 mg/mL), while Staphylococcus aureus was the most resistant (MIC>25.00 mg/mL). Our results indicate that T. longicaulis essential oil could be effective against clinically relevant respiratory pathogens which have the ability to develop resistance to antimicrobial drugs.     

Synthesis,antiproliferative and antimicrobial activity of new Mannich bases bearing 1,2,4-triazole moiety

Abstract

This study presents the synthesis, antiproliferative and antimicrobial evaluation of a new series of Mannich base derivatives containing 1,2,4-triazole system. New compounds were prepared by the reaction of 4,5-disubstituted 1,2,4-triazole-3-thiones with formaldehyde and various amines. The structures of the prepared compounds were confirmed by means of ¹H NMR, ¹³C NMR and elemental analyses. Twelve compounds were evaluated for their in vitro antiproliferative activities against six chosen cancer cell lines. All synthesized compounds were screened for their in vitro antimicrobial activity by using the agar dilution technique. For 17 potentially active compounds, their antibacterial activity was confirmed on the basis of MIC (minimal inhibitory concentration) by broth microdilution method using the reference Gram-positive and Gram-negative bacterial strains.     

Synthesis,antimicrobial, cytotoxic and E. coli DNA gyrase inhibitory activities of coumarinyl amino alcohols

Here we report the in vitro antimicrobial activity (minimum inhibitory concentration) of fourteen coumarinyl amino alcohols 2–16 against eight bacterial strains and two fungi. Among these compounds 4, 8, 12, 15 and 16 showed moderate to good microbial inhibition with MIC values varied from 6.25 to 25 μg/mL. The most promising compounds were also evaluated for their in vitro cytotoxic and E. coli DNA gyrase inhibitory activities along with the two 7-oxy-4-methyl coumarinyl amino alcohol derivatives 17 and 18, which were found to be the most potent in in vitro antimicrobial screening in our previous study. All the active compounds, including 17 and 18, were also docked into the E. coli DNA gyrase ATP binding site (PDB ID: 1KZN) to investigate their binding interactions. Of these compound 17 has shown maximum binding energy value of −6.13 kcal/mol.     

Diversity and antifungal activity of endophytic bacteria associated with <Emphasis Type="Italic">Panax ginseng</Emphasis> seedlings

Ginseng (Panax ginseng C.A. Meyer) is a medicinal crop that requires a long culture time before it is ready to harvest, thus generating high economic and environmental costs. Symbiotic bacteria that live within the plant provide the host plant with many advantages in terms of metabolism and disease resistance. Here, we isolated endophytic bacteria from various tissues of P. ginseng seedlings using a culture-dependent method and we compared their tissue distribution. In addition, their antimicrobial activity against two fungal pathogens was investigated. Based on 16S rRNA sequencing, we identified 21 bacterial strains from ginseng seedlings. Leaves and rhizomes showed higher bacterial species diversity than root bodies and tails. While Bacillus strains were detected in all tissues, Xanthomonas and Micrococcaceae strains were specifically isolated from rhizome and leaf tissues, respectively. Fourteen bacterial strains showed antimicrobial activities against Cylindrocarpon destructans and/or Botrytis cinerea, with different activities. Among them, two strains (PgKB29 and PgKB35) showed strong antimicrobial activities against both fungi. Taken together, these results provide a better understanding of endophytic bacteria in P. ginseng seedlings and suggest the possibility of biological control of fungal pathogens using endophytic bacteria.     

Design,synthesis, and biological evaluation of 4-chloro-2H-thiochromenes featuring nitrogen-containing side chains as potent antifungal agents

A series of 4-chloro-2H-thiochromenes featuring nitrogen-containing side chains were designed, synthesized and tested in vitro for their antifungal activities. The results of preliminary antifungal tests showed that most target compounds exhibited good inhibitory activities against Candida albicans, Cryptococcus neoformans, Candida tropicalis. Notably, compounds 10e and 10y showed most potent activity in vitro against a variety of fungal pathogens with low MICs. Meanwhile, low cytotoxicity on mammalian cells has been observed for compounds 10e and 10y in the tested concentrations by the MTT assay. Therefore, the 4-chloro-2H-thiochromenes with nitrogen-containing groups provide new lead structures in the search for novel antifungal agents.     

Characterization and evaluation of two Bacillus strains,SS-12.6 and SS-13.1, as potential agents for the control of phytopathogenic bacteria and fungi

Two strains of Bacillus sp., SS-12.6 and SS-13.1, showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed that both strains have the genes for biosynthesis of iturin, bacillomycin and surfactin. Kinetics of production of antimicrobial substances in these strains showed that synthesis started at the beginning of exponential phase of growth. Maximum of activity was slowly reached at the beginning of stationary growth phase and was maintained until the end of observed period. Ethyl acetate extracts of cell-free supernatants of both strains were particularly active against several postharvest fungal pathogens, in vitro and in vivo, in the experiment with apple fruits. Mass spectrometry analysis of ethyl acetate extract of the supernatant of strain SS-12.6 confirmed the presence of antimicrobial lipopeptide surfactin.     

Using In Vitro Immunomodulatory Properties of Lactic Acid Bacteria for Selection of Probiotics against Salmonella Infection in Broiler Chicks

Poultry is known to be a major reservoir of Salmonella. The use of lactic acid bacteria has become one of successful strategies to control Salmonella in poultry. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties for potential use as probiotics against Salmonella infection in broiler chicks. Among 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties (Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities (Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of lipopolysaccharide-induced TNF-α factor (LITAF), IL-1β, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry.     

Design,synthesis and evaluation of oxime-functionalized nitrofuranylamides as novel antitubercular agents

A series of oxime-functionalized nitrofuranylamides were designed, synthesized and evaluated for their in vitro anti-mycobacterial activities against MTB H37Rv and drug-resistant clinical isolates. Among them, two compounds 7a and 7b exhibited excellent activity against the three tested strains. Both of them were comparable to the first-line anti-TB agents INH and RIF against MTB H37Rv, and were far more potent than INH and RIF against MDR-TB 16833 and 16995 strains. Thus, both of them could act as leads for further optimization.     

Novel isoniazid embedded triazole derivatives: Synthesis,antitubercular and antimicrobial activity evaluation

In the present study, a series of new isoniazid embedded triazole derivatives have been synthesized. These compounds were evaluated for their in vitro antitubercular and antimicrobial activities. Among the screened compounds, six have exhibited potent antitubercular activity against Mycobacterium tuberculosis H37Rv strain with MIC value 0.78 μg/mL, whereas, three compounds have displayed activity with MIC value ranging from 1.56 to 3.125 μg/mL. The cytotoxicity of the active compounds was studied against RAW 264.7 cell line by MTT assay and no toxicity was observed even at 25 μg/mL concentration. The five compounds have displayed good antimicrobial activities. Molecular docking have been performed against mycobacterial InhA enzyme to gain an insight into the plausible mechanism of action which could pave the way for our endeavor to identify potent antitubercular candidates. We believe that further optimization of these molecules may lead to potent antitubercular agents.     

Phylogenetic diversity of bacterial endophytes of Panax notoginseng with antagonistic characteristics towards pathogens of root-rot disease complex

Endophytes play an important role in protection of host plants from infection by phytopathogens. Endophytic bacteria were isolated from five different parts (root, stem, petiole, leaf and seed) of Panax notoginseng and evaluated for antagonistic activity against Fusarium oxysporum, Ralstonia sp. and Meloidogyne hapla, three major pathogens associated with root-rot disease complex of P. notoginseng. From 1000 endophytic bacterial strains evaluated in vitro, 104 strains exhibited antagonistic properties against at least one of these three pathogens. Phylogenetic analyses of their 16S rRNA gene sequences showed that these 104 antagonistic bacteria belong to four clusters: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes/Chlorobi. Members of the Firmicutes, in particular the Bacillus spp., were predominant in all analyzed tissues. The root was the main reservoir for antagonistic bacteria. Of the 104 antagonists, 51 strains showed antagonistic activities to one pathogen only, while 43 and 10 displayed the activities towards two and all three pathogens, respectively. The most dominant species in all tissues were Bacillus amyloliquefaciens subsp. plantarum and Bacillus methylotrophicus, which were represented by eight strains with broad antagonistic spectrum to the all three test pathogens of root-rot disease complex of P. notoginseng.