Antifungal activity in vitro of Baccharis glutinosa and Ambrosia confertiflora extracts on Aspergillus flavus, Aspergillus parasiticus and Fusarium verticillioides

The aims of this study were to evaluate the antifungal properties of Baccharis glutinosa and Ambrosia confertiflora extracts against Aspergillus flavus, A. parasiticus and Fusarium verticillioides, and to isolate the group of compounds that are responsible for the antifungal activity. Samples of aerial parts from each plant were extracted with 70% methanol and sequentially partitioned with hexane, ethyl acetate, and n-butanol. The partitioned fractions were evaluated in their capacity to inhibit the radial growth of the three species of fungi. The active fraction was used for an assay-guided chromatography of antifungal extracts. The results showed that the extract from B. glutinosa partitioned in ethyl acetate (Bea) showed the highest antifungal activity against the three fungi. Bea completely inhibited the growth of F. verticillioides at 0.8 mg/ml, whereas the radial growth of A. flavus and A. parasiticus was inhibited 70% at 1.5 mg/ml. The purified antifungal fraction from Bea showed 72, 54, and 52% of antifungal activity, respectively.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.     

Molecular characterization of fungal endophytes from Calotropis procera plants in Taif region (Saudi Arabia) and their antifungal activities

Fungal endophytes were isolated from the leaves of Calotropis procera (Apocynaceae) collected from Taif region (Saudi Arabia). Thirty-three different taxa were recovered. The overall foliar colonization rate was 35.1%. A total of 161 isolates were obtained and identified into 33 distinct operational taxonomic units based on the sequencing of the internal transcribed spacer regions of the rRNA gene. The most prevalent fungi were Aspergillus flavus, Chaetomium globosum, Cochliobolus lunatus, Fusarium dimerum, F. oxysporum, and Penicillium chrysogenum. A total of 161 isolates were tested for antifungal activities against four plant pathogenic fungi (Alternaria alternata, Fusarium oxysporum, Botrytis cinerea, and Pythium ultimum), of which 33 isolates showed antifungal activity against at least one plant pathogenic fungi. Four isolates of Chaetomium globosum and three isolates of Myrothecium verrucaria showed the strongest antifungal activity. This study reported the occurrence of a much wider spectrum of fungi, when compared with previous work. Also, it confirmed the variation of different isolates from the same species in terms of antifungal activity.     

Natural Product‐Based Fungicides Discovery: Design,Synthesis and Antifungal Activities of Some Sarisan Analogs Containing 1,3,4‐Oxadiazole Moieties

A series of sarisan analogs containing 1,3,4‐oxadiazole moieties were synthesized by iodine‐mediated oxidative cyclization and screened in vitro for their antifungal activities at 50 μg/mL against five phytopathogenic fungi such as Valsa mali, Curvularia lunata, Alternaria alternate, Fusarium solani and Fusarium graminearum. 1,3,4‐Oxadiazole derivatives 7e , 7p , 7r , 7t and 7u exhibited potent and a broad spectrum of antifungal activities against at least three phytopathogenic fungi at the concentration of 50 μg/mL. Especially, compound 7r displayed more potent antifungal activities against five phytopathogenic fungi than the positive control hymexazol. The EC₅₀ of 7r against V. mali, C. lunata and A. alternate were 12.6, 14.5 and 17.0 μg/mL, respectively. Additionally, some interesting results of structure‐activity relationships (SARs) were also observed.     

In vitro kinetics and antifungal activity of various extracts of Terminalia chebula seeds against plant pathogenic fungi

The aim of the present study was to examine the efficacy of various seed extracts of Terminalia chebula as an antifungal potential against certain important plant pathogenic fungi. The organic extracts of methanol, ethyl acetate and chloroform at the used concentration of 1500 ppm/disc revealed remarkable antifungal effect as a fungal mycelial growth inhibitor against Fusarium oxysporum, Fusarium solani, Phytophthora capsici and Botrytis cinerea, in the range of 41.6–61.3%, along with MIC values ranging from 62.5 to 500 μg/ml. Also, the extracts had a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of B. cinerea. The results obtained from this study suggest that the natural products derived from Terminalia chebula could become an alternative to synthetic fungicides for controlling such important plant pathogenic fungi.     

Microbial conversion and in vitro and in vivo antifungal assessment of bioconverted docosahexaenoic acid (bDHA) used against agricultural plant pathogenic fungi

Microbial modification of polyunsaturated fatty acids can often lead to special changes in their structure and in biological potential. Therefore, the aim of this study was to develop potential antifungal agents through the microbial conversion of docosahexaenoic acid (DHA). Bioconverted oil extract of docosahexaenoic acid (bDHA), obtained from the microbial conversion of docosahexaenoic acid (DHA) by Pseudomonas aeruginosa PR3, was assessed for its in vitro and in vivo antifungal potential. Mycelial growth inhibition of test plant pathogens, such as Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum, was measured in vitro. bDHA (5 μl disc⁻¹) inhibited 55.30–65.90% fungal mycelium radial growth of all the tested plant pathogens. Minimum inhibitory concentrations (MICs) of bDHA against the tested plant pathogens were found in the range of 125–500 μg ml⁻¹. Also, bDHA had a strong detrimental effect on spore germination for all the tested plant pathogens. Further, three plant pathogenic fungi, namely C. capsici, F. oxysporum and P. capsici, were subjected to an in vivo antifungal screening. bDHA at higher concentrations revealed a promising antifungal effect in vivo as compared to the positive control oligochitosan. Furthermore, elaborative study of GC-MS analysis was conducted on bioconverted oil extract of DHA to identify the transformation products present in bDHA. The results of this study indicate that the oil extract of bDHA has potential value of industrial significance to control plant pathogenic fungi.     

Pr-1, a novel antifungal protein from pumpkin rinds

A novel antifungal protein, M_r = ca. 40 kDa, was isolated from pumpkin rind and designated Pr-1. When purified by anion exchange chromatography and HPLC, it inhibited growth of several fungi including Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani, as well as the yeast, Candida albicans, at 10–20 μM. It did not inhibit growth of Escherichia coli or Staphylococcus aureus even at 200 μM. Laser scanning microscopy of fungal cells exposed to rhodamine-labeled Pr-1 revealed that the protein accumulated and was localized on the cell surface. Uptake of the vital stain, SYTOX Green, was enhanced when fungal conidia were treated with Pr-1 suggesting that the protein has membrane permeabilization activity. Pr-1 was thermostable at 70°C and did not lyse human red blood cells at 128 μM suggesting that the protein may be useful as an antifungal agent with little, if any human cytotoxicity.     

黄花倒水莲内生真菌生物活性评价及HNLF-44菌株鉴定

为充分开发黄花倒水莲(Polygala fallax)的内生真菌资源，获得具有抗植物病原真菌、抗氧化活性的内生真菌，该文以黄花倒水莲内生真菌为研究对象，使用平板对峙法检测内生真菌对6种植物病原真菌的抑菌活性，测定内生真菌发酵液的DPPH清除自由基能力和总还原能力，评价内生真菌的抗氧化活性，并对具有强抑菌活性和抗氧化活性的菌株进行形态和ITS鉴定。结果表明：(1)黄花倒水莲内生真菌中有2株内生真菌对香蕉专化尖孢镰刀菌、柑橘树脂病菌、叶点霉菌、香蕉具条叶斑病菌、茄病镰刀菌、三七根腐病菌具有明显的抑菌活性，抑菌率在50.3%～91.4%之间，其中HNLF-5对柑橘树脂病菌的抑菌率为73.2%,HNLF-44对香蕉专化尖孢镰刀菌抑菌率为91.4%。(2)内生真菌发酵液具有良好的抗氧化活性，DPPH清除率均在80%以上，总还原能力吸光值范围为0.279 2～0.748 8。(3)HNLF-44菌株为链格孢属真菌。该研究表明，药用植物黄花倒水莲内生真菌具有较好的生物活性，为后续从黄花倒水莲内生真菌中挖掘潜在新型抑菌活性和抗氧化活性物质奠定了基础。     

Design,Synthesis, and Antifungal Activities of Novel 1,2,4‐Triazole Schiff Base Derivatives

With the aim to find new compounds with high antifungal activity, 21 4‐amino‐5‐substituted‐1,2,4‐triazole Schiff bases ( 2a  –  2g , 3a  –  3g , and 4a  –  4g ) were designed and synthesized. Their antifungal activities against Pythium solani, Gibberlla nicotiancola, Fusarium oxysporium f. sp. niveum, Gibberlla saubinetii, Alternaria iycopersici, Phytophthora capsici, Physalospora piricola, Cercospora arachidicola hori, and Fusarium oxysporium f. sp. cucumber were tested, parts of the compounds exhibited excellent antifungal activity. This research provides useful information for further study of antifungal agents.     

1‐Phenyl‐3‐pentanone,a Volatile Compound from the Edible Mushroom Mycoleptodonoides aitchisonii Active Against Some Phytopathogenic Fungi

Volatiles produced by mycelia of mushrooms with aromatic odour were investigated for their antifungal activity against plant‐pathogenic fungi. The results of the screening of 23 species of basidiomycetes revealed that volatile substances from mycelia of Mycoleptodonoides aitchisonii (TUFC10099), an edible mushroom, strongly inhibited the mycelial growth, spore germination and lesion formation on host leaves of some plant‐pathogenic fungi including Alternaria alternata, A. brassicicola, A. brassicae, Colletotrichum orbiculare and Corynespora cassiicola. The volatile compounds were isolated from the culture filtrate of M. aitchisonii, and 1‐phenyl‐3‐pentanone was identified as a major antifungal volatile. The compound had significantly inhibitory activity against plant‐pathogenic fungi at 35 ppm. This is the first report that the volatile compound produced by mycelia of M. aitchisonii has antifungal activity against plant‐pathogenic fungi.     

Characterisation of plant growth‐promoting rhizobacteria from rhizosphere soil of heat‐stressed and unstressed wheat and their use as bio‐inoculant

• High temperature induces several proteins in plants that enhance tolerance to high temperature shock. The fate of proteins synthesised in microbial cells or secreted into culture media by interacting microbes has not been fully elucidated. The present investigation aimed to characterise plant growth‐promoting rhizobacteria (PGPR) isolated from the rhizosphere of wheat genotypes (differing in tolerance to high temperature stress) and evaluate their performance as bioinoculant for use in wheat.
• Four bacterial strains, viz. Pseudomonas brassicacearum, Bacillus thuringiensis, Bacillus cereus strain W6 and Bacillus subtilis, were isolated from the rhizosphere of heat‐stressed and unstressed wheat genotypes. The wheat genotypes were exposed to high temperature stress at 45 °C for 10 days (3 h daily) at pre‐anthesis phase. Isolates were identified on the basis of morphology and biochemical characteristics, 16S rRNA gene sequencing and whole cell protein profiles. Results were further complemented by size exclusion chromatography (SEC) with fast protein liquid chromatography (FPLC) and SDS PAGE of 80% ammonium sulphate precipitates of the cell‐free supernatants.
• Isolates were positive for catalase, oxidases and antimicrobial activity . P. brassicacearum from the rhizosphere of the heat‐tolerant genotype was more efficient in phosphate solubilisation, bacteriocin production, antifungal and antibacterial activity against Helminthosporium sativum, Fusarium moniliforme and Klebsiella pneumonia, respectively. The inoculated seedlings had significantly higher root and shoot fresh weight, enhanced activity of antioxidant enzymes, proline and protein content. Total profiling of the culture with SDS‐PAGE indicated expression of new protein bands in 95 kDa in P. brassicacearum.
• Temperature‐induced changes in PGPR isolates are similar to those in the host plant. P. brassicacearum may be a good candidate for use in biofertiliser production for plants exposed to high temperature stress.

     

The effect of <Emphasis Type="Italic">Baccharis glutinosa</Emphasis> extract on the growth of mycotoxigenic fungi and fumonisin B<Subscript>1</Subscript> and aflatoxin B<Subscript>1</Subscript> production

The aim of this study was to evaluate the effect of Baccharis glutinosa isolated extract on the growth of Aspergillus flavus and Aspergillus parasiticus, and their aflatoxin B₁ production; and growth of Fusarium verticillioides, and their fumonisin B₁ production. The three fungi were exposed to an antifungal fraction, designated as fraction F6-1, isolated from B. glutinosa by methanolic extraction followed by silica gel chromatography. The growth of the fungi was evaluated in kinetics of radial extension growth, kinetics of spores germination, length and diameter of hyphae, spores diameter, as well as in aflatoxin B₁ and fumonisin B₁ production. Fraction F6-1 caused radial growth inhibition of the three fungi mainly F. verticillioides. Spores germination of A. flavus and A. parasiticus was delayed in the early stage of the incubation time, although they completely germinated at 27 h. In contrast, spore germination of F. verticillioides was inhibited 87.7% up to 96 h. The lengths and diameters of hyphae, and spore diameters of the three fungi, were significantly smaller in comparison with those of the controls, and several morphological alterations were observed. Concerning aflatoxin B₁ and fumonisin B₁, fraction F6-1 did not show any inhibition effect at the concentration used. Fraction F6-1 was able to significantly inhibit the development of the three fungi, mainly F. verticillioides. The strong inhibitory effect of F6-1 on hyphae and spores suggests that it interacted with the fungi cell walls, which caused severe deformities. Nevertheless, this fraction was unable in inhibiting mycotoxin production from the three fungi at the concentration tested.     

Rapid Identification of Four Fusarium spp. Complex by High-Resolution Melting Curve Analysis and their Antifungal Susceptibility Profiles

Fusarium species are globally distributed filamentous ascomycete fungi that are frequently reported as plant pathogens and opportunistic human pathogens, leading to yield loss of crops, mycotoxin contamination of food and feed products as well as damage to human and livestock. Human infections of Fusarium spp. are difficult to treat due to broad antifungal resistance by members of this genus. Their role as disease-causing agents in crops and humans suggests a need for antifungal resistance profiles as well as a simple, rapid, and cost effective identification method. Fusarium strains were isolated from food and clinical samples. High-resolution melting curve (HRM) analysis was performed using specific primers targeting internal transcribed spacer (ITS) region, followed with evaluation of specificity and sensitivity. The antifungal susceptibility of four Fusarium species was studied using the Sensititre YeastOne method. HRM analysis revealed reproducible, unimodal melting profiles specific to each of the four Fusarium strains, while no amplification of the negative controls. The minimum detection limits were 100–120 copies based on a 2 µl volume of template. Clear susceptibility differences were observed against antifungal agents by different Fusarium isolates, with amphotericin B and voriconazole displayed strongest antifungal effects to all the tested strains. We developed a simple, rapid, and low-cost qPCR-HRM method for identification of four Fusarium spp. (F. oxysporum, F. lateritium, F. fujikuroi, and F. solani). The antifungal susceptibility profiles supplied antifungal information of foodborne and clinical Fusarium spp. and provided guidance for clinical treatment of human infections.

     

Endophytic fungi diversity of aquatic/riparian plants and their antifungal activity <Emphasis Type="Italic">in vitro</Emphasis>

Two hundred and fourteen endophytic fungi were isolated from 500 segments of aquatic/riparian plants Ottelia acuminata, Myriophyllum verticillatum, Equisetum arvense, Cardamine multijuga, and Impatiens chinensis. They were identified to 31 taxa in which Cladosporium, Fusarium, and Geotrichum were the dominant genera. Among all isolates, 169 (79%) were anamorphic fungi, 1 (0.5%) was an teleomorphic ascomycete and 44 (21%) were sterile mycelia. There were significant differences in the colonization frequency of endophytes between the five plant species (X∼2=51.128, P<0.001, Chi-square test). The riparian plants harboured more endophytes than the submerged plants. The antifungal activity of these isolates against Fusarium solani and Phytophthora nicotianae in vitro were tested and 28 (13.1%) isolates showed antifungal activities with more than 30% growth inhibition rate against the two pathogens.     

Antifungal activity of phytochemicals from Eucalyptus hybrid leaves against some plant pathogenic and wood decay fungi

The in vitro fungitoxicity of the essential oil (EO), crude extracts, dried fractions and ursolic acid (UA) isolated from the abundantly available and underutilised leaves of Eucalyptus hybrid (Eucalyptus camaldulensis × Eucalyptus tereticornis) was assessed against three plant pathogenic fungi Aspergillus niger, Fusarium solani and Ganoderma lucidum causing pathogenesis in seeds, seedlings and trees, respectively, and Trametes versicolor and Pycnoporous sanguineus making wood to decay. The EO, methanol extract and UA demonstrated varied antifungal activity against all the fungi with minimum inhibitory concentrations ranging from 0.5 to 3.0%. The results of the study demonstrate a new chemical utilisation approach of the leaves of Eucalyptus hybrid towards the development of biofungicides in the management of above fungi of common occurrence in Indian forestry.     

Antimicrobial and antioxidant activities of methanol extracts of <Emphasis Type="Italic">Evax pygmaea</Emphasis> (Asteraceae) growing wild in Tunisia

Antimicrobial activity of methanolic extracts obtained from the aerial parts of Evax pygmaea was tested against five bacteria and two strains of phytopathogenic fungi using the agar diffusion and broth microdilution methods. Antioxidant properties were evaluated through the ability of the different fractions to scavenge the stable ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals. The TEAC (Trolox Equivalent Antioxidant Capacity) and IC₅₀ values of the fractions were calculated and compared. The experimental data indicated that all fractions exhibit moderate to appreciable antibacterial activities against all Gram-positive cocci and Gram-negative rods except Pseudomonas aeruginosa, but no antifungal activity was observed. Ethyl acetate and methanol fractions were found to cause significant free radical-scavenging effects in both assays. These results may suggest that E. pygmaea could be used as a natural preservative ingredient in the food and/or pharmaceutical industries.     

Effect of Cultivation Time on Production of Antifungal Metabolite(s) by Streptomyces hygroscopicus in Laboratory‐Scale Bioreactor

Biological control agents offer one of the best alternatives to reduce the use of pesticides . Fungi from the genera Alternaria, Colletotrichum and Fusarium are listed among the most important storage pathogens of apple fruits. During storage, transport and marketing, pathogenic fungi can cause significant losses of apple fruits. This investigation studied the potential of Streptomyces hygroscopicus as a biocontrol agent against pathogenic fungi obtained from apple fruit samples expressing rot symptoms. Production of antifungal metabolites by S. hygroscopicus was carried out in 3‐l bench‐scale bioreactor (Biostat^® Aplus, Sartorius AG, Germany) during 7 days. Fermentation was carried out at 27°C with aeration rate of 0.5 vvm and agitation rate of 200 rpm. The aim was to analyse bioprocess parameters of batch biofungicide production in medium containing glucose as a carbon source and to examine at which stage of bioprocess production of antifungal metabolite(s) against six phytopathogenic fungi occurs. In vitro antifungal activity of the produced metabolites against six fungi of the genera Colletotrichum, Fusarium and Alternaria grown on potato dextrose agar were determined every 24 h using wells technique. Antifungal activity of cell‐free culture filtrate and filtrate treated with high temperature were tested. The filtrate treated with high temperature did not show any antifungal activity suggesting that active components are thermo unstable. Stationary phase of growth occurred between the third and fourth day of cultivation when production of secondary metabolites begins. Obtained results showed that maximal antifungal activity is achieved on fifth and sixth day of S. hygroscopicus cultivation under defined conditions (inhibition zone diameter higher than 30 mm for all test fungi).     

Antifungal effects of volatile organic compounds from the endophytic fungus Cryptosporiopsis ericae Cc-HG-7 isolated from Coptis chinensis Franch

In this study, 136 strains of endophytic fungi were isolated from the Chinese traditional medicinal plant Coptis chinensis Franch. Of these, 129 strains were classified into 12 different genera according to morphological traits and internal transcribed spacer (ITS) gene sequence analyses. Their antifungal activities were assessed against the following fungi: Magnaporthe oryzae, Pythium graminicola, Cylindrocarpon destructans, Fusarium oxysporum, Cercospora zeae-maydis, Sclerotinia sclerotiorum, Setosphaeria turcica and Botrytis cinerea. Fourteen endophytic strains were active against at least one of the selected fungi. The most active strain Cc-HG-7 identified as Cryptosporiopsis ericae displayed inhibition rates of 81.42% and 72.00%, respectively, against S. sclerotiorum and S. turcica in dual culture technique. The volatile antifungal compounds were identified using headspace solid-phase microextraction, followed by gas chromatography mass spectrometry to investigate the potential biocontrol mechanisms of strain Cc-HG-7. The results suggested that the strain Cc-HG-7 could be a potential agent for the biological control of S. sclerotiorum and S. turcica.     

Isolation and biochemical characterization of a novel leguminous defense peptide with antifungal and antiproliferative potency

Leguminous plants have formed a popular subject of research owing to the abundance of proteins and peptides with important biological activities that they produce. The antifungal proteins and peptides have been purified from a number of leguminous species. However, research continues to discover novel antifungal plant-produced peptides and proteins are being needed, specially those novel ones with both antifungal activity and other significant bioactivities. The objective of this study was to isolate a novel peptide from Phaseolus limensis. A 6.8 kDa peptide designated Limyin, with both antifungal and antiproliferative activity, was isolated from the large lima bean (P. limensis) legumes. The isolation procedure consisted of extraction, precipitation, affinity chromatography on Affi-gel blue gel, ion chromatography on SP-Toyopearl, and gel filtration on Superdex 75. Its N-terminal sequence was determined to be KTCENLATYYRGPCF, showing high homology to defensin and defensin precursors from plants. It potently suppressed mycelial growth in Alternaria alternata, Fusarium solani, and Botrytis cinerea. Its antifungal activity was stable up to 80°C. It showed antiproliferative activity towards tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y. However, it had no effect on bacteria Staphylococcus aureus and Salmonella. The present findings make a significant addition of the research on leguminous plants.