Influence of carbon and nitrogen sources on antifungal metabolite production by bacterium associated with entomopathogenic nematode against Penicillium expansum

A specific symbiotic Bacillus sp. isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce large number of bioactive compounds. The present study was conducted to determine the effect of carbon and nitrogen sources for the production of antimicrobial substances by Bacillus sp. The yield of the crude antimicrobial substances and antimicrobial activity against the test micro-organism also differed significantly when carbon and nitrogen sources in the fermentation media were changed. The antifungal activity was significantly high in yeast extract plus fructose (46.5?±?2.12?mm) followed by yeast extract plus maltose, beef extract plus fructose and meat infusion plus glucose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention time indicating that they produced different compounds. When the carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced. The results indicate that carbon source in the fermentation media plays a vital role in the production of antifungal substances. It is concluded that yeast extract and fructose as nitrogen and carbon sources produced maximum activity, which can effectively control the blue mould caused by Penicillium expansum in apples and pears.     

Impact of beef extract and six carbon source on antifungal metabolites production by bacterium associated with entomopathogenic nematode against Fusarium oxysporum

A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp., was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different carbon sources in combination with beef extract on the production of antifungal substances by Bacillus sp. The yield of crude antimicrobial substances and antimicrobial activity against the test microorganism also differed significantly when the carbon sources in the fermentation media were changed. The highest yield was recorded for fructose plus beef extract (956?mg/l). The antifungal activity was significantly high in beef extract plus maltose (21?±?1.5?mm) followed by beef extract plus glucose and beef extract plus fructose. Antifungal activity was significantly reduced in beef extract plus lactose and sucrose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation media, the antifungal production was substantially reduced. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Beef extract and maltose as nitrogen and carbon sources in the fermentation medium produced maximum antifungal activity. It is concluded that Beef extract and maltose as nitrogen and carbon sources produced maximum activity which can effectively control the Fusarium oxysporum which causes vascular fusarium wilt in tomato, tobacco, legumes, cucurbits, sweet potatoes, banana, etc.     

假单胞菌YL11对扩展青霉的抑制作用及其机理初探

【背景】苹果青霉病是由扩展青霉引起的一种重要的果实采后病害,影响果实品质导致苹果腐烂从而造成经济损失。【目的】研究假单胞菌YL11对扩展青霉的抑制作用和苹果采后青霉病的防治效果,并对抑菌机理进行初步探讨。【方法】以扩展青霉为供试菌株,研究不同浓度的假单胞菌YL11无菌发酵液对扩展青霉菌落直径、孢子萌发率、菌丝体干重、苹果损伤接种病斑直径扩展的影响,利用对电导率、核酸及蛋白释放量、AKP含量、SDH活性、ATP酶活性和ATP含量的影响对抑菌机理进行探究。【结果】假单胞菌YL11无菌发酵液能有效抑制扩展青霉生长,抑菌圈直径为22.33±0.27 mm,抑菌效价为71.67 mm/mL;能有效抑制孢子萌发,100%无菌发酵液对孢子萌发抑制率达到80.2%;对扩展青霉的生物量也有一定抑制作用,体积分数为100%时,菌丝体干重为4.7mg/mL,抑制率达到39.74%;无菌发酵液处理能有效抑制苹果青霉病病斑的扩展,3d时对病斑扩展的抑制率最大,达到47.1%;无菌发酵液处理均能引起电导率升高、胞内核酸和蛋白释放量增大、胞外AKP含量升高、SDH活性降低、ATP酶活性和ATP含量均降低,且随着发酵液浓度的增加效果越明显。【结论】假单胞菌YL11能显著抑制扩展青霉的生长,破坏细胞膜结构、降低能量代谢酶活性,从而扰乱扩展青霉的正常生长,对苹果青霉病有较好的生防效果,具有潜在的开发价值。     

Alkylresorcinols isolated from rye bran by supercritical fluid of carbon dioxide and suspended in a food-grade emulsion show activity against Penicillium expansum on apples

Apple cultivars are attacked by many fungal diseases, both on the tree and during storage. One of the most serious is blue mould, caused by Penicillium expansum. In this study, 5-(n)-alkylresorcinols (AR) were isolated from rye bran by the supercritical fluid of carbon dioxide and were used for the preparation of bioactive emulsions. These emulsions were applied to harvested fruit of five apple varieties to determine the levels of antifungal activity. A significant inhibition of disease symptoms was obtained after spraying some of the prepared AR emulsions on fruits that had been experimentally infected with Penicillium expansum. The most effective emulsions consisted of 0.025% (m/v) ARs, 0.1% (m/v) xanthan gum, 0.5% (m/v) Synperonic 91/6 or PDMs-copolymer, 0.2% (m/v) Tween 20, 1% (m/v) Trioleate, 2% (m/v) oleylalcohol, 2% (m/v) PEG 400, 5% (m/v) CaCl₂ or NaCl suspended in water.     

Carbon,nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ～7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.     

Bioactive stilbenes from a Bacillus sp. N strain associated with a novel rhabditid entomopathogenic nematode

Aims: The aim of the present study was to purify and characterize a natural antimicrobial compound from Bacillus sp. strain N associated with a novel rhabditid entomopathogenic nematode. Methods and Results: The cell‐free culture filtrate of a bacterium associated with a novel entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by column chromatography, and two bioactive compounds were isolated and their chemical structures were established based on spectral analysis. The compounds were identified as 3,4′,5‐trihydroxystilbene (1) and 3,5‐dihydroxy‐4‐isopropylstilbene (2). The presence of 3,4′,5‐trihydroxystilbene (resveratrol) is reported for the first time in bacteria. Compound 1 showed antibacterial activity against all the four test bacteria, whereas compound 2 was effective against the Gram‐positive bacteria only. Compounds 1 and 2 were active against all the five fungi tested and are more effective than bavistin, the standard fungicide. The antifungal activity of the compounds against the plant pathogenic fungi, Rhizoctonia solani is reported for the first time. Conclusions: Cell‐free extract of the bacterium and isolated stilbenes demonstrated high antibacterial activity against bacteria and fungi especially against plant pathogenic fungi. We conclude that the bacterium‐associated EPN are promising sources of natural bioactive secondary metabolites. Significance and Impact of the Study: Stilbene compounds can be used for the control of fungi and bacteria.     

Isolation of proline-based cyclic dipeptides from Bacillus sp. N strain associated with rhabitid entomopathogenic nematode and its antimicrobial properties

Entomopathogenic nematodes (EPN) are well-known as biological control agents and are found to have associated bacteria which can produce a wide range of bioactive secondary metabolites. We report herewith isolation of six proline containing cyclic dipeptides cyclo(d-Pro-l-Leu), cyclo(l-Pro-l-Met), cyclo(d-Pro-l-Phe), cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Tyr) and cyclo(l-Pro-d-Tyr) from ethyl acetate extract of the Luria Broth (LB) cell free culture filtrate of Bacillus sp. strain N associated with a new EPN Rhabditis sp. from sweet potato weevil grubs collected from Central Tuber Crops Research Institute farm. Antimicrobial studies of these 2,5-diketopiperazines (DKPs) against both medicinally and agriculturally important bacterium and fungi showed potent inhibitory values in the range of μg/mL. Cyclic dipeptides showed significantly higher activity than the commercial fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani, and Pencillium expansum. The highest activity of 2 μg/mL by cyclo(l-Pro-l-Phe) was recorded against P. expansum, a plant pathogen responsible for causing post harvest decay of stored apples and oranges. To our knowledge, this is the first report on the isolation of these DKPs from Rhabditis EPN bacterial strain Bacillus sp.     

海洋弧菌(Vibrio sp.)LA-05发酵液粗提物的群体感应抑制活性及其发酵条件优化

【背景】海洋微生物代谢产物被认为是发掘新型天然活性物质的潜在来源,其中不可避免地包含新型群体感应抑制剂(quorum sensing inhibitors, QSIs)。响应面法是运用多元二次回归模型拟合各因素和响应值之间的函数关系,对各因素及其交互作用进行评价,可有效地从影响代谢产物发酵条件的各因素中预测出最佳条件。【目的】以紫色杆菌(Chromobacterium violaceum)ATCC 12472^T为指示菌株,研究海洋弧菌(Vibrio sp.)LA-05发酵液粗提物的群体感应抑制活性。在单因素试验基础上,应用响应面法对菌株发酵条件进行优化,以提高其发酵代谢产物中活性物质的产量。【方法】使用琼脂平板扩散法进行初筛后,通过分析发酵液粗提物对紫色杆菌生长曲线及紫色素产量的影响研究其群体感应抑制活性。在单因素试验基础上,采用Box-Behnken设计原则,以紫色素抑制率为响应值,通过响应面试验优化菌株发酵条件,从而提高其发酵液中活性物质的产量。【结果】在基本不影响紫色杆菌正常生长的浓度范围内,粗提物可不同程度地抑制紫色素的产生。模型预测结果表明,最优发酵条件...     

Purification of an antifungal compound,cyclo(l-Pro-d-Leu) for cereals produced by Bacillus cereus subsp. thuringiensis associated with entomopathogenic nematode

Mold spoilage is the main cause of substantial economic loss in cereals and might also cause public health problems due to the production of mycotoxins. The aim of this study was to separate and purify and to identify antifungal compounds of bacterium associated with novel entomopathogenic nematode and check the antifungal property of identified compound in particular food model systems. The antifungal compound was purified using silica gel column chromatography, TLC and HPLC and its structure was elucidated using NMR (¹H NMR, ¹³C NMR, ¹H–¹H COSY, ¹H–¹³C HMBC), HRMS and Marfey's method. Based on the spectral data, the active compounds were identified as diketopiperazine [cyclo(l-Pro-d-Leu)]. The antifungal activity of cyclo(l-Pro-d-Leu) was studied by MIC and paper disk assay against Aspergillus flavus MTCC 277 and Aspergillus niger MTCC 282 and best MIC value of 8 μg/ml was recorded against A. flavus. Cyclo(l-Pro-d-Leu) strongly inhibit mycelia growth of fungus and thereby affecting aflatoxin production. To investigate the potential application of the cyclo(l-Pro-d-Leu) and to eliminate fungal spoilage in food and feed, soybean and peanut were used as models. White mycelia and dark/pale green spores of A. flavus were observed in the control soybeans after 2-day incubation. However the fungal growth was not observed in soybeans treated with cyclo(l-Pro-d-Leu). Almost the same result was observed for peanuts treated with cyclo(l-Pro-d-Leu) for A. niger. The cyclo(l-Pro-d-Leu) was nontoxic to two normal human cell lines (FS normal fibroblast and L231 lung epithelial) up to 200 μg/ml. Thus the diketopiperazine derivative identified in the study may be a promising alternative to chemical preservatives as a potential biopreservative which prevent fungal growth and mycotoxin formation in food and feed.     

Characterisation of a thermostable and proteolysis resistant phytase from Penicillium polonicum MF82 associated with the marine sponge Phorbas sp.

Abstract

Phytases are widely used in human and animal nutrition, aquaculture, soil amendment, and in the production of lower myo-inositol phosphates for clinical purposes. Some of these applications, especially feed industry require robust enzymes. Since the marine environments are less studied compared to terrestrial environments, we evaluated the extracellular phytase activity of 110 marine derived filamentous fungal (MDFF) strains previously isolated from sponge and sediment samples of the Turkey. MDFF strains were qualitatively screened for their extracellular phytase activities and P. polonicum MF82 phytase was further characterized following partial purification. Optimum pH and temperature were determined as 5.5 and 60?°C respectively. A significant relative phytase activity was observed in the presence of urea and acetone. However, there was no phytase activity followed by the treatment with Triton X-100 and Tween 80. Characterization studies revealed that P. polonicum MF82 phytase has superior properties for industrial use including wide pH and temperature range for activity, high optimum activity temperature, high thermal and pH stability, resistance to many enzyme inhibitors including various heavy metals, denaturants, detergents, proteases and organic solvents. Phytase extracellularly produced by P. polonicum MF82 strain presents a good candidate for commercial applications. This study demonstrates that the MDFF strains are prolific sources for phytase and presents the first report about the production and characterization of the phytase from a marine-derived P. polonicum strain.     

Characterization and antifungal activity of the yellow pigment produced by a Bacillus sp. DBS4 isolated from the lichen Dirinaria agealita

This study emphasis the production of yellow pigment from endolichenic Bacillus sp. isolated from the lichen Dirinaria aegialita (Afzel. ex Ach.) B.J. Moore. Yellow pigment-producing twenty different strains were investigated. The hyperactive pigment-producing bacterial strain was identified as Bacillus gibsonii based on 99 % sequence similarity. Maximum bacterial pigment production appeared in Luria Bertani medium. Methanol extraction of the pigment and its partial purification using TLC was carried out. Furthermore, isolated pigments were characterized using UV-visible spectroscopy, FTIR spectroscopy, and GC-MS results related to the possibility of the carotenoid occurrence. The pigment also exhibited efficient antifungal activity against selected fungal pathogens of economic importance. Likewise, the pigment extract evaluated for the total antioxidant potential using Phosphomolybdenum and Ferric reducing antioxidant power assay and the results represented in Ascorbic Acid Equivalent (AAE)- 21.45 ± 1.212 mg/mL. The SC₅₀ of the pigment extract found to be 75.125 ± 0.18 µg/ml determined by the ABTS assay.     

Cyclo(d‐Tyr‐d‐Phe): a new antibacterial,anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode

A new microbial cyclic dipeptide (diketopiperazine), cyclo(d ‐Tyr‐d ‐Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d ‐Tyr‐d ‐Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d ‐Tyr‐d ‐Phe) recorded significant antitumor activity against A549 cells (IC₅₀ value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d ‐Tyr‐d ‐Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d ‐Tyr‐d ‐Phe). Flow cytometry analysis showed that the cyclo(d ‐Tyr‐d ‐Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase‐3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d ‐Tyr‐d ‐Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d ‐Tyr‐d ‐Phe) with synthetic cyclo(d ‐Tyr‐d ‐Phe) and cyclo(l ‐Tyr‐l ‐Phe). Natural and synthetic cyclo(d ‐Tyr‐d ‐Phe) recorded similar pattern of activity. Although synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded significant activity than natural and synthetic cyclo(d ‐Tyr‐d ‐Phe). The results of the present study reveals that cyclo(d ‐Tyr‐d ‐Phe) is more bioactive than cyclo(l ‐Tyr‐l ‐Phe). To the best of our knowledge, this is the first time that cyclo(d ‐Tyr‐d ‐Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d ‐Tyr‐d ‐Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.