Study on an antimicrobial protein produced by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> JSa-9 isolated from soil

Paenibacillus polymyxa strain JSa-9, a soil isolate showed in vitro inhibitory effects on several pathogens. A protein of about 71.9 kDa, isolated from a culture of JSa-9, exhibited broad-spectrum antibacterial and antifungal activities. The purification procedure consisted of ion-exchange chromatography on DEAE-Sephacel and Sephadex G-100 column chromatography. The purified protein was characterized to be a glycoprotein, which contained 53% of amino acids and 28% of carbohydrate. Its N-terminal sequence was determined as KCATIPVVIKHL and the amino acid composition showed no significant homology with any known antagonistic proteins from P. polymyxa. Because of the excellent antimicrobial activity, this protein may be a good prospect for food preservation and plant disease control.     

Characterization of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> inhibitory lipopeptide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> IB

Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae.     

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.

     

Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.     

The Antimicrobial Peptide Thanatin Reduces Fungal Infections in Arabidopsis

We explored the antifungal activity of thanatin, a 21 amino acid synthetic peptide from the hemipteran spined soldier bug Podisus maculiventris, against the mycotoxin‐producing plant pathogenic ascomycete Fusarium graminearum. In vitro germination assays showed complete inhibition of macroconidia germination and mycelia growth by >10 μm thanatin. Moreover, detached leaves of thanatin‐expressing Arabidopsis thaliana plants displayed enhanced resistance towards colonization with F. graminearum. Consistent with this, the plants showed also enhanced resistance of detached leaves to colonization with Botrytis cinerea. The results demonstrate a potential of thanatin for use in plant protection.     

Isolation and characterization of a proteinaceous antifungal compound from Lactobacillus plantarum YML007 and its application as a food preservative

Korean kimchi is known for its myriad of lactic acid bacteria (LAB) with diverse bioactive compounds. This study was undertaken to isolate an efficient antifungal LAB strain among the isolated kimchi LABs. One thousand and four hundred LABs isolated from different kimchi samples were initially screened against Aspergillus niger. The strain exhibiting the highest antifungal activity was identified as Lactobacillus plantarum YML007 by 16S rRNA sequencing and biochemical assays using API 50 CHL kit. Lact. plantarum YML007 was further screened against Aspergillus oryzae, Aspergillus flavus, Fusarium oxysporum and other pathogenic bacteria. The morphological changes during the inhibition were assessed by scanning electron microscopy. Preliminary studies on the antifungal compound demonstrated its proteinaceous nature with a molecular weight of 1256·617 Da, analysed by matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF). The biopreservative activity of Lact. plantarum YML007 was evaluated using dried soybeans. Spores of A. niger were observed in the negative control after 15 days of incubation. However, fungal growth was not observed in the soybeans treated with fivefold concentrated cell‐free supernatant of Lact. plantarum YML007. The broad activity of Lact. plantarum YML007 against various food spoilage moulds and bacteria suggests its scope as a food preservative.

Significance and Impact of the Study

After screening 1400 kimchi bacterial isolates, strain Lactobacillus plantarum YML007 was selected with strong antifungal activity against various foodborne pathogens. From the preliminary studies, it was found that the bioactive compound is a low molecular weight novel protein of 1256·617 Da. Biopreservative potential of Lact. plantarum YML007 was demonstrated on soybean grains, and the results point out YML007 as a potent biopreservative having broad antimicrobial activity against various foodborne pathogens.     

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.     

A pathogenesis related protein, AhPR10 from peanut: an insight of its mode of antifungal activity

A pathogenesis related protein (AhPR10) is identified from a clone of 6-day old Arachis hypogaea L. (peanut) cDNA library. The clone expressed as a ∼20 kDa protein in E. coli. Nucleotide sequence derived amino acid sequence of the coding region shows its homology with PR10 proteins having Betv1 domain and P loop motif. Recombinant AhPR10 has ribonuclease activity, and antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Mutant protein AhPR10-K54N where lys54 is mutated to asn54 loses its ribonuclease and antifungal activities. FITC labeled AhPR10 and AhPR10-K54N are internalized by hyphae of F. oxysporum and R. solani but the later protein does not inhibit the fungal growth. This suggests that the ribonuclease function of AhPR10 is essential for its antifungal activity. Energy and temperature dependent internalization of AhPR10 into sensitive fungal hyphae indicate that internalization of the protein occurs through active uptake.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .The nucleotide sequence of AhPR10 reported in this paper is submitted to NCBI Nucleotide Sequence Database under the Accession number AY726607.     

Studies on phytohemagglutinins I. Some properties of the lectins of horse gram seeds (Dolichos biflorus L.)

The anti-A₁ lectin of Dolichos biflorus L. seeds has been isolated from the active protein fraction obtained by 40–60% saturation by ammonium sulfate of a saline extract (1 : 5) followed by successive chromatography on DEAE-cellulose, Sephadex G-100 and DEAE-cellulose. The lectin has a molecular weight of 122 000 and contains 1.25% neutral sugar, 0.8% glucosamine and only one terminal amino acid, alanine. Neutral sugar components comprise mannose, glucose, fucose and xylose.On the other hand, affinity chromatography of the same active protein fraction on O-(N-acetyl-α-d-galactosaminy;) polyacrylamide yields with different electrophoretic mobilities and with an overall neutral sugar content of 3.7%. Electrophoresis on polyacrylamide gel reveals a diffused broad band of unequal intensity. On ultracentrifugation the lectin shows a molecular weight of 124 000 with a tendency to form a dimer. Lectin isolated by affinity techniques contains 0.17% Ca, 0.11% Zn, 0.07% Mn and 0.01% Mg; lectin obtained by conventional chormatographic procedure shows a somewhat decreased content of Zn and Ca.Digestion with pepsin and pronase of the lectin isolated by conventional procedures yields a mixture of fragments from which three glycopeptides designated as G1, G2A and G2B can be isolated by chromatography on Sephadex G-25 and preparative paper electrophoresis. G1 contains aspartic acid, threonine, proline, glycine, alanine, glucose and fucose and its calculated minimal molecular weight is approximately 2800; glycopeptide G2A contains aspartic acid, serine, glucosamine, mannose and fucose and has a calculated minimal molecular weight of approximately 1800.The purified lectin isolated by either of the two methods is active in concentration of 10 μg/ml against A₁-erythrocytes. Its erythroagglutinating activity is enhanced by Ca²⁺, Co²⁺, Ni²⁺ and Mg²⁺ and inhibiited by both N-acetyl-d-galactosamine and EDTA. The mixture of glycopeptides G2 isoalted by pronase digestion shows erythroagglutinating activity at the same concentration, but is nonspecific.     

Purification and partial characterization of a cellular retinol-binding protein from human liver

A protein with binding specificity for retinol was purified from human liver. [³H]Retinol was added to liver extracts and the [³H]retinol-binding protein isolated by conventional chromatographic techniques including ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephadex G-75 and G-50 and preparative isoelectric focusing. The yield was 10–15% in different preparations and the degree of purification was about 3000-fold. The purified protein had a molecular weight of about 15 000 as estimated from both gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulphate and was homogeneous in several electrophoretic systems. Isoelectric focusing of the purified protein gave a doublet band. Only one fluorescent band at pH 4.70 was seen if the protein solution was incubated with excess retinol prior to isoelectric focusing. The isolated protein did not react with antiserum to the retinol-binding protein of plasma. The amino acid composition and the amino terminal amino acid sequence for the first sixteen amino acids of the purified protein differed significantly from that of the plasma retinol-binding protein.     

Evaluation of antagonistic activities of Bacillus subtilis and Bacillus licheniformis against wood-staining fungi: In vitro and in vivo experiments

The antifungal activity of bacterial strains Bacillus subtilis EF 617317 and B. licheniformis EF 617325 was demonstrated against sapstaining fungal cultures Ophiostoma flexuosum, O. tetropii, O. polonicum, and O. ips in both in vitro and in vivo conditions. The crude active supernatant fractions of 7 days old B. subtilis and B. licheniformis cultures inhibited the growth of sapstaining fungi in laboratory experiments. Thermostability and pH stability of crude supernatants were determined by series of experiments. FT-IR analysis was performed to confirm the surface structural groups of lipoproteins present in the crude active supernatant. Partial purification of lipopeptides present in the crude supernatant was done by using Cellulose anion exchange chromatography and followed by Sephadex gel filtration chromatography. Partially purified compounds significantly inhibited the sapstaining fungal growth by in vitro analysis. The lipopeptides responsible for antifungal activity were identified by electrospray ionization mass spectrometry after partial purification by ion exchange and gel filtration chromatography. Four major ion peaks were identified as m/z 1023, 1038, 1060, and 1081 in B. licheniformis and 3 major ion peaks were identified as m/z 1036, 1058, and 1090 in B. subtilis. In conclusion, the partially purified lipopeptides may belong to surfactin and iturin family. In vivo analysis for antifungal activity of lipopeptides on wood was conducted in laboratory. In addition, the potential of extracts for fungal inhibition on surface and internal part of wood samples were analyzed by scanning electron microscopy.     

Identification of a biocontrol agent Bacillus vallismortis BV23 and assessment of effects of its metabolites on Fusarium graminearum causing corn stalk rot

Corn stalk rot, caused by Fusarium graminearum, is one of the most destructive diseases of maize in many regions of the world. A bacterial strain BV23 was isolated from corn rhizosphere that reduced corn stalk rot significantly in greenhouse studies in 2016 and 2017. BV23 was identified as Bacillus vallismortis, which showed antagonistic effects against a number of fungal pathogens, including F. graminearum, Rhizoctonia solani, Athelia rolfsii, and Thanatephorus cucumeris. BV23 had the greatest fungistatic effect on F. graminearum, inhibiting mycelial growth by 66.2%, conidial germination by 90.1%, and conidial production by 86.7%. The probable antifungal mechanism was assessed by examining the morphology and ultrastructure of F. graminearum hyphae. Treatment by BV23 culture supernatant resulted in coarser hyphae, induced cytoplasmic granulation, and increased cell membrane permeability of F. graminearum, causing cytoplasm leakage. These effects became increasingly obvious with increasing concentration (1%, 5% and 10%). Furthermore, the antifungal active substances were sensitive to heat.     

Brucin,an antibacterial peptide derived from fruit protein of Fructus Bruceae,Brucea javanica (L.) Merr

A novel antibacterial peptide specific to Streptococcus pyogenes was produced from dried fruit protein of Brucea javanica (L.) Merr. A mixture of active peptides from the fruit protein was produced in vitro by pepsin hydrolysis. The hydrolysate was purified by reverse‐phase HPLC, and antimicrobial peptides active against Gram‐negative and Gram‐positive bacteria were analysed using SDS‐PAGE and nanoLC‐MS/MS. Here, four possible peptides were obtained and chemically synthesized for comparative study of the growth inhibition of Strep. pyogenes. One chemically synthesized peptide with a molecular mass of 1168·31 Da, His‐Thr‐Leu‐Cys‐Met‐Asp‐Gly‐Gly‐Ala‐Thr‐Tyr, showed the most potent antibacterial activity against Strep. pyogenes. This 11‐amino acid peptide was named Brucin. Its bacterial inhibitory activity was 16‐fold and 12·5‐fold higher than penicillin G and chloramphenicol, respectively, with a MIC value of 20 μmol l^?1. The results suggest that Brucin, a potent antibiotic peptide, may be developed as an alternative drug for the treatment of the disease caused by Strep. pyogenes.

Significance and Impact of the Study

An antibacterial peptide, named Brucin with specificity for Streptococcus pyogenes, was produced in vitro from dried fruit protein of Brucea javanica (L.) Merr. by pepsin‐catalysed hydrolysis. Its inhibitory activity towards the Gram‐positive bacteria was higher than penicillin G and chloramphenicol. The result suggested that Brucin may be applied for the treatment of the disease caused by Strep. pyogenes^*.     

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.     

Biocontrol of Late Blight Disease (Phytophthora capsici) of Pepper and the Plant Growth Promotion by Paenibacillus ehimensis KWN38

For field application of a bacterial strain used to control Phythophthora capsici, we will need a biologically and economically efficient carrier medium. The known antagonist Paenibacillus ehimensisKWN38 was grown in a grass medium where it showed high antifungal and lytic enzyme activities. To demonstrate the potential of P. ehimensisKWN38 for biocontrol of late blight disease in pepper, pot trials were conducted by treating the 1‐month‐old plants with water (W), a selected grass medium (G3), G plus P. ehimensisKWN38 inoculation (G3P) or synthetic fungicide (F). The shoot dry weight in G3P was higher than that in W and F treatments at 15 days after zoospore infection (DZI). The root dry weight in G3P was also higher than that in W. The root mortality of G3 and W increased over 58 and 80% at 15 DZI, and some plants in those treatments wilted due to the failure of root physiology. The plants in G3P and F survived well because of their better root health conditions. Soil cellulase activity of G3P was consistently higher than that of W and F at earlier observation times (0, 2 and 6 DZI). The root β‐1,3‐glucanase activity of G3P promptly increased to maximum shortly after zoospore infection and reached the maximum value of 51.12 unit g^?1 of fresh weight at 2 DZI. All these results indicate that inoculation of P. ehimensisKWN38 to the root zone of potted pepper plants increases plant growth, root and soil enzyme activities and alleviates the root death caused by infection with P. capsici zoospores.     

Six New Pentacyclic Triterpenoids from the Fruit of Camptotheca acuminata

Six new pentacyclic triterpenoids were isolated from the fruit of Camptotheca acuminata. The chemical structures of the new compounds were elucidated by extensive spectroscopic analysis including HR‐ESI‐MS, IR, UV, 1D‐ and 2D‐NMR. Moreover, the antibacterial activities of compounds 1 , 2 , 4 , 5 , and 6 were evaluated against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Dickeya zeae. All these tested compounds showed moderate antibacterial activity against Bacillus subtilis and Dickeya zeae.     

Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens

Aims: To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product‐derived Bacillus amyloliquefaciens. Methods and Results: An unknown bacterial species cultured from the Yogu Farm? probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell‐free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. Conclusion: The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. Significance and Impact of the Study: This is the first report of intra‐species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin’s activity against bacterial vaginosis‐associated pathogens.     

A novel Geotrichum candidum lipase with some preference for the 2-position on a triglyceride molecule

Summary A new component of Geotrichum candidum lipase with a unique positional specificity was isolated from culture broth together with a known major component. The purification included DEAE-Sephadex A-50 ion exchange chromatography. Sephadex G-100 gel filtration, and Butyl Toyopearl 650S hydrophobic interaction chromatography. The newly isolated component, though only a minor one, cleaved the 2-positioned ester bond nearly twice as fast as the 1(3)-positioned ester bond of a triglyceride molecule. In contrast, the major component hydrolysed all the ester bonds indiscriminately, which is consistent with the widely accepted positional specificity of the lipase from G. candidum. Offprint requests to: A. Sugihara     

Tricalycoside,a New Cerebroside from Tricalysia coriacea (Rubiaceae)

A new cerebroside, named as tricalycoside ( 1 ), was isolated from the CH₂Cl₂/MeOH (1:1) extract of twigs and leaves of Tricalysia coriacea using repeated silica gel open column chromatography followed by preparative TLC and Sephadex LH‐20, together with six known compounds ( 2 – 7 ). The structure of the new compound was determined by analysis of 1D‐ and 2D‐NMR, MS data, chemical conversion, and by comparison of these data with those from the literature. Tricalycoside ( 1 ) possessed a weak antibacterial activity against Klebsiella pneumoniae (MIC = 75 μg/mL).     

Potential of a new strain of Bacillus subtilis CPA-8 to control the major postharvest diseases of fruit

Biocontrol potential of Bacillus subtilis strain CPA-8 was tested against the main postharvest diseases of orange, apple and stone fruit. Previously, CPA-8 growth was characterized and its antifungal activity in vitro determined against Botrytis cinerea, Monilinia laxa, M. fructicola, Penicillium digitatum, P. expansum, and P. italicum. In vivo activity against these pathogens was tested by treating fruits with cells, endospores or cell free supernatants. CPA-8 treatments cannot control decay caused by P. digitatum and P. italicum on oranges. The higher concentrations of CPA-8 studied were effective in controlling B. cinerea on apple, showing grey mold incidence from 70 to 12.5% in comparison with 100% in the control. However, in general, CPA-8 treatments were not effective in controlling P. expansum. The best results of CPA-8 treatments were obtained in stone fruit against M. laxa and M. fructicola where most treatments resulted in brown rot incidence of 0% compared with 70 and 90% in the control. Based on these results, cultures, cells and cell free supernatants at different concentrations were tested against M. laxa and M. fructicola on stone fruit. Most bacterial concentrations were effective in controlling M. laxa and M. fructicola as well as or better than Serenade® Max, in some treatments showing brown rot incidences of 0% in comparison with 100% of control. Bacterial populations of CPA-8 were maintained stable or increased up to 2-log inside wounds, showing the ability of the bacteria to colonize injured tissues. Experimental evidence suggests that B. subtilis CPA-8 has biocontrol potential for control of postharvest disease on several fruit types.