Mycelial growth inhibition of plant pathogenic fungi by extracts of South African plant species

Eleven plant species were collected from the Blyde River Canyon area, Mpumalanga, South Africa. Methanolic crude extracts of the plant samples were concentrated to dryness, dissolved in sterile distilled water and bioassayed in the laboratory for their fungicidal potential against seven plant fungal pathogens, namely Botrytis cinerea, Fusarium oxysporum, Sclerotium rolfsii, Rhizoctonia solani, Verticillium dahliae, Botryosphaeria dothidea and Pythium ultimum, at equal concentrations. A modified agar dilution method was used to determine the inhibitory effect of the plant extracts on the mycelial radial growth of the plant pathogens. Antifungal activity was confirmed in all the plants, but not in all plant parts and also not against all the test organisms. The crude extract of Eucomis autumnalis performed best of all the plant extracts tested, as it showed significant antifungal activity against all seven of the plant pathogenic test organisms and compared favourably to the inhibition of the mycelial growth by a broad spectrum synthetic fungicide (carbendazim/ difenoconazole). The crude extract of Schrebera alata came out second best, in the sense that at least one plant part inhibited the mycelial radial growth of four of the seven test organisms by at least 50%.     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性.将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤,分离得到脂肽类抗生素的纯品.经IC50实验和抗菌谱测定,考察了脂肽类抗生素对多种植物病原菌的作用,确定了脂肽类抗生素的抗菌谱.深入研究表明,枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质,能够抑制灰霉病菌孢子的萌发和菌丝的生长.脂肽类抗生素和挥发性抑菌物质的协同作用,有助于提高枯草芽孢杆菌的生物防治效果.     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性。将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤, 分离得到脂肽类抗生素的纯品。经IC50实验和抗菌谱测定, 考察了脂肽类抗生素对多种植物病原菌的作用, 确定了脂肽类抗生素的抗菌谱。深入研究表明, 枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质, 能够抑制灰霉病菌孢子的萌发和菌丝的生长。脂肽类抗生素和挥发性抑菌物质的协同作用, 有助于提高枯草芽孢杆菌的生物防治效果。     

Biological activities from extracts of endophytic fungi isolated from Viguiera arenaria and Tithonia diversifolia

A total of 39 endophytic fungi have been isolated from Viguiera arenaria and Tithonia diversifolia, both collected in S?o Paulo State, Brazil. The isolates were identified based on their ribosomal DNA sequences. The ethyl acetate (EtOAc) extracts of all endophytic fungi were evaluated for their antimicrobial, antiparasitic and antitumoral activity. Antimicrobial screening was conducted using an agar diffusion assay against three pathogenic microorganisms: Staphylococcus aureus, Escherichia coli and Candida albicans. Antiparasitic activity was determined by enzymatic inhibition of gGAPDH of Trypanosoma cruzi and adenine phosphorybosiltransferase (APRT) of Leishmania tarentolae. Antitumoral activity was tested against human T leukemia cells by the Mosmann colorimetric method. All extracts showed activity in at least one assay: 79.5% of the extracts were cytotoxic against leukemia cells, 5.1% of the extracts were active against S. aureus, 25.6% against E. coli and 64.1% against Candida albicans. Only one extract showed promising results in the inhibition of parasitic enzymes gGAPDH (95.0%) and three were found to inhibit APRT activity. The cytotoxic extract produced by the strain VA1 (Glomerella cingulata) was fractionated and yielded nectriapyrone and tyrosol. Nectriapyrone showed relevant cytotoxic activity against both human T leukemia and melanoma tumor cell lines.     

A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans

BACKGROUND: Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development. RESULTS: We have generated and characterized deletion mutants for four genes supposed to function in a nematode Toll signaling pathway. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. On the other hand, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type. CONCLUSIONS: In C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter r?le, it functions in the context of a behavioral mechanism that keeps worms away from potential danger.     

In vitro antibacterial activity and physicochemical properties of a crude methanol extract of the larvae of the blow fly Lucilia cuprina

The emergence of multidrug‐resistant bacterial strains has prompted the reintroduction of maggot therapy in the treatment of chronic, infected wounds. Many previous studies have demonstrated the potent antibacterial activity of larval excretions/secretions of the blowfly Lucilia sericata (Meigen) (Diptera:Calliphoridae) against bacteria. However, the antibacterial activity of its sibling species, Lucilia cuprina (Wiedemann) (Diptera:Calliphoridae) against a wide range of pathogenic bacteria has never been determined. The aim of this study was to develop a new procedure to produce whole body extract of larvae of L. cuprina via methanol extraction as well as to demonstrate the in vitro antibacterial activity of this extract against seven selected wound pathogens (Staphylococcus aureus, methicillin‐resistant S. aureus, S. epidermidis, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli). The turbidimetric assay demonstrated that L. cuprina larval extract was significantly potent against all bacteria tested (P < 0.001). Additionally, colony‐forming unit (CFU), agar well diffusion and minimum inhibitory concentration assays have confirmed the apparent potency of larval extract against P. aeruginosa. The reconstituted larval extract was highly robust and thermally stable. These observations substantiated the feasibility of the methanol extraction method in the production of larval extract.     

Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens

Aims: A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus. Methods and Results: To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site‐directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application. Conclusions: We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested. Significance and Impact of the Study: We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.     

Increased responsiveness in feeding behaviour of Caenorhabditis elegans after experimental coevolution with its microparasite Bacillus thuringiensis

Immune responses, either constitutive or induced, are costly. An alternative defence strategy may be based on behavioural responses. For example, avoidance behaviour reduces contact with pathogens and thus the risk of infection as well as the requirement of immune system activation. Similarly, if pathogens are taken up orally, preferential feeding of pathogen-free food may be advantageous. Behavioural defences have been found in many animals, including the nematode Caenorhabditis elegans. We here tested nematodes from a laboratory based evolution experiment which had either coevolved with their microparasite Bacillus thuringiensis (BT) or evolved under control conditions. After 48 generations, coevolved populations were more sensitive to food conditions: in comparison with the controls, they reduced feeding activity in the presence of pathogenic BT strains while at the same time increasing it in the presence of non-pathogenic strains. We conclude that host-parasite coevolution can drive changes in the behavioural responsiveness to bacterial microbes, potentially leading to an increased defence against pathogens.     

博落回、虎杖和黄芩提取物对植物病原菌的抑制作用

研究了博落回(Macleaya cordata)、虎杖(Reynoutria japonica)和黄芩(Scutellaria baicalensis)提取物对三种植物病原细菌和六种病原真菌的抑制作用.结果显示:虎杖提取物对根癌土壤杆菌和番茄疮痂病菌表现出较强的抗细菌活性;博落回提取物表现出较强的抗真菌活性,对供试真菌的半抑制浓度(IC50)在0.04～0.76mg/mL之间.     

In vitro anti- biofilm and anti-bacterial activity of Sesbania grandiflora extract against Staphylococcus aureus

The main objective of this research is to investigate the anti-biofilm and anti-bacterial activity of Sesbania grandiflora (S. grandiflora) against Staphylococcus aureus. S. grandiflora extract were prepared and analyzed with UV –Vis spectroscopy, Fourier transform infrared spectroscopy, Dynamic light scattering. Biofilm forming pathogens were identified by congo-red assay. Quantification of Extracellular polymeric substance (EPS) particularly protein and carbohydrate were calculated. The efficacy of the herbal extract S. grandiflora and its inhibition against the pathogenic strain of S. aureus was also evaluated. The gradual decrease or disappearance of peaks reveals the reduction of protein and carbohydrate content in the EPS of S. aureus when treated with S. grandiflora. The antibacterial activity of S. grandiflora extract against the bacterial strain S. aureus showed that the extract were more active against the strain. To conclude, anti-biofilm and antibacterial efficacy of S. grandiflora plays a vital role over biofilm producing pathogens and act as a good source for controlling the microbial population.     

六种中药及其复方对鳗鲡致病性气单胞菌的体外抑制作用

针对病鳗内脏中分离的5 株致病性气单胞菌, 采用琼脂稀释法测定五倍子、石榴皮、大黄、虎杖、黄芩及黄连各味中药的最低抑菌浓度(MIC)和最低杀菌浓度(MBC); 再根据棋盘法设计15 种双联用药方和4 种三联用药方, 同样检测各组合配方的抑菌作用。实验结果表明, 6 味中药对养殖鳗鲡5 株致病性气单胞菌均有不同程度的抗菌效果, 其中五倍子的抑菌作用最强, 其次是大黄和石榴皮, 而黄连的抑菌效果最差; 15 种双联用药方较各味中药单用的抑菌活性绝大多数出现增强, 抑菌浓度至少减低39%, FICFIC≤0.5 表现显著增强抗菌活性的协同比例占23.3%; 4 种三联用药方对5 株致病性气单胞菌均具有显著的协同抑制效应, 复方中单味中药的抑菌浓度可以降低80%以上; 而双联用HC14 对4 株致病菌出现FIC≥2 的降低彼此抗菌活性的相互拮抗现象。由此说明合理运用不同中药的联用配伍, 不仅可提高单味中药的抗菌疗效, 而且大大减少了单一中药在实际养殖生产中的给药浓度, 降低药物在环境中的残留量, 防止残留药物造成环境污染, 并且降低用药成本, 提高水产养殖业的经济和社会效益。研究为中药复方防治细菌性鱼病提供科学理论参考。       

Antimicrobial activity of certain bacteria and fungi isolated from soil mixed with human saliva against pathogenic microbes causing dermatological diseases

Soil samples (collected from El-Madina El-Monawara, Kingdom Saudi Arabia) were mixed with human saliva, incubated in media suitable for bacterial and fungal growth and filtered. Eighteen bacterial and five fungal species were isolated and identified. The bacterial and fungal filtrates as well as the isolated species were evaluated for their antimicrobial activities against some pathogenic microbes causing dermatological diseases (Staphylococcus aureus, methicillin resistant S. aureus (MRSA) and Aspergillus niger). The bacterial filtrate showed significant antagonistic effect against S. aureus and methicillin resistant S. aureus (MRSA), whereas showed non inhibitory action on the pathogenic fungus. In contrast, the fungal filtrate antagonized the growth of the pathogenic fungus (A. niger) and did not produce any inhibitory effect on the two tested pathogenic bacteria. The isolated bacterial species showed different levels of antagonistic activities against the three tested microbes. Bacillus subtilis was described as potent isolate against the three pathogens, followed by Esherichia coli. However, Bacillus megaterium strongly inhibited the growth of the pathogenic bacteria only. On the other side, all the fungal filtrates of the isolated species, except Cochliobolus lanatus showed antagonistic activity against the pathogenic fungus (A. niger). The filtrate of Fusarium oxysporum and Emericella nidulans counteracted the growth of S. aureus, whereas, the growth of MRSA was inhibited only by the filtrate of E. nidulans. From the passage way of our respected prophet, how is never tells from him self, if any person complains from awound or ulcer, the messenger of Allah (prayers and peace be upon him) put his forefinger on the ground and lift it then he says: (In the Name of God, soil of our land, with the saliva of some of us, our sick person will get well after the permission of our God) Al-Bukhari. The meaning of this Hadith that the prophet takes his saliva on the forefinger then he put it on the soil and wipe on the wound place while saying the above Hadith that is shows the Prophet’s miracle, which is evidence of healing by using soil and saliva.     

链霉菌A01-chit33CT产纳他霉素和几丁质酶协同表达发酵条件优化

生物农药由于具有良好的生态效应和安全性,因此比化学农药更受到人们的青睐,生物农药的发展契合低碳、循环、清洁绿色经济发展理念。因此,寻求利于食品安全和环境保护,同时高效控制植物病害的新型生物农药成为时下及未来研究的热点。链霉菌以产生纳他霉素等抗生素起到生防作用。链霉菌株A01-chit33CT既可以产生纳他霉素又可以高表达几丁质酶活,生防效果大大增加。为确定链霉菌A01-chit33CT产纳他霉素和几丁质酶协同表达的发酵条件,初步探索了碳氮源和发酵条件对菌株产生纳他霉素和几丁质酶的影响。结果表明,葡萄糖促进纳他霉素的产生而抑制几丁质酶的表达,因此分两阶段添加葡萄糖和几丁质粉来达到二者协同表达。研究确定最佳发酵培养基为:葡萄糖40 g/L,几丁质粉10 g/L(发酵4 d添加),黄豆粉30 g/L,大豆蛋白胨10 g/L,CaCO35 g/L,MgSO4.7H2O 0.5 g/L,K2HPO40.5 g/L。最优发酵条件为:初始pH 6.0,温度28℃,转速180 r/min。在此条件下,链霉菌A01-chit33CT产纳他霉素达1.52 g/L,同时几丁质酶活达990 U/ml,二者比优化前的水平分别提高了1.95倍和2.27倍。     

Chemical and biological characterization of sclerosin, an antifungal lipopeptide

Pseudomonas sp. strain DF41 produces a lipopeptide, called sclerosin that inhibits the fungal pathogen Sclerotinia sclerotiorum . The aim of the current study was to deduce the chemical structure of this lipopeptide and further characterize its bioactivity. Mass spectrometry analysis determined the structure of sclerosin to be CH(3)-(CH(2))(6)-CH(OH)-CH(2)-CO-Dhb-Pro-Ala-Leu/Ile-Ala-Val-Val-Dhb-Thr-Val-Leu/Ile-Dhp-Ala-Ala-Ala-Val-Dhb-Dhb-Ala-Dab-Ser-Val-OH, similar to corpeptins A and B of the tolaasin group, differing by only 3 amino acids in the peptide chain. Subjecting sclerosin to various ring opening procedures revealed no new ions, suggesting that this molecule is linear. As such, sclerosin represents a new member of the tolaasin lipopeptide group. Incubation of S.?sclerotinia ascospores and sclerotia in the presence of sclerosin inhibited the germination of both cell types. Sclerosin also exhibited antimicrobial activity against Bacillus species. Conversely, this lipopeptide demonstrated no zoosporicidal activity against the oomycete pathogen Phytophthora infestans . Next, we assessed the effect of DF41 and a lipopeptide-deficient mutant on the growth and development of Caenorhabditis elegans larvae. We discovered that sclerosin did not protect DF41 from ingestion by and degradation in the C.?elegans digestive tract. However, another metabolite produced by this bacterium appeared to shorten the life-span of the nematode compared to C.?elegans growing on Escherichia coli OP50.     

3-hydroxypropionic acid as a nematicidal principle in endophytic fungi

3- Hydroxypropionic acid was isolated by bioactivity-guided fractionation of extracts obtained from submerged cultures of several endophytic fungi isolated from above-ground plant organs. This compound showed selective nematicidal activity against the plant-parasitic nematode Meloidogyne incognita with LD50 values of 12.5-15 microg/ml. Activity against the saprophytic Caenorhabditis elegans was fivefold lower. No antimicrobial, cytotoxic or phytotoxic effects were observed. Propionic acid and D- and L-lactic acids were not active against either nematode species. Based on morphological features and ITS, 18S and 28S rDNA analyses, the producing strains were identified as Phomopsis phaseoli isolated from the leaf of a tropical tree, and four strains of Melanconium betulinum isolated from twigs of Betula pendula and B. pubescens in Germany. This is the first report of 3-hydroxypropionic acid in fungi, and of the nematicidal activity of this metabolite.     

Nematicidal activity of 5-hydroxymethyl-2-furoic acid against plant-parasitic nematodes

A nematicide, 5-hydroxymethyl-2-furoic acid (1), was isolated from cultures of the fungus Aspergillus sp. and its structure was identified by spectroscopic analysis. Compound 1 showed effective nematicidal activities against the pine wood nematode Bursaphelenchus xylophilus and the free-living nematode Caenorhabditis elegans without inhibitory activity against plant growth, but 1 did not show any effective nematicidal activity against Pratylenchus penetrans.     

Exploring antifungal activities of acetone extract of selected Indian medicinal plants against human dermal fungal pathogens

A broad spectrum of medicinal plants was used as traditional remedies for various infectious diseases. Fungal infectious diseases have a significant impact on public health. Fungi cause more prevalent infections in immunocompromised individuals mainly patients undergoing transplantation related therapies, and malignant cancer treatments. The present study aimed to investigate the in vitro antifungal effects of the traditional medicinal plants used in India against the fungal pathogens associated with dermal infections. Indian medicinal plants (Acalypha indica, Lawsonia inermis Allium sativum and Citrus limon) extract (acetone/crude) were tested for their antifungal effects against five fungal species isolated from skin scrapings of fungal infected patients were identified as including Alternaria spp., Curvularia spp., Fusarium spp., Trichophyton spp. and Geotrichum spp. using well diffusion test and the broth micro dilution method. All plant extracts have shown to have antifungal efficacy against dermal pathogens. Particularly, Allium sativum extract revealed a strong antifungal effect against all fungal isolates with the minimum fungicidal concentration (MFC) of 50–100 μg/mL. Strong antifungal activity against Curvularia spp., Trichophyton spp., and Geotrichum spp. was also observed for the extracts of Acalypha indica, and Lawsonia inermis with MFCs of 50–800 μg/mL respectively. The extracts of Citrus limon showed an effective antifungal activity against most of the fungal strains tested with the MFCs of 50–800 μg/mL. Our research demonstrated the strong evidence of conventional plants extracts against clinical fungal pathogens with the most promising option of employing natural-drugs for the treatment of skin infections. Furthermore, in-depth analysis of identifying the compounds responsible for the antifungal activity that could offer alternatives way to develop new natural antifungal therapeutics for combating resistant recurrent infections.     

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.