Novel bifunctional alkaline protease inhibitor: protease inhibitory activity as the biochemical basis of antifungal activity

An alkaline protease inhibitor (API) from a Streptomyces sp. NCIM 5127 was shown to possess antifungal activity against several phytopathogenic fungi besides its antiproteolytic (anti-feedent) activity [J. V. Vernekar et al. (1999) Biochem. Biophys. Res. Commun. 262, 702-707]. Based on the correlation between antiproteolytic and antifungal activities in several tests such as copurification, heat inactivation, chemical modification, and its binding interaction with the fungal protease, we demonstrate, for the first time, that the dual function of API is a consequence of its ability to inhibit the essential alkaline protease. The parallel enrichment of both the functions during purification together with the heat inactivation of API leading to the concomitant loss of the two activities suggested their presence on a single molecule. Chemical modification of API with NBS resulted in the complete loss of antiproteolytic and antifungal activities, with no gross change in conformation implying the involvement of a Trp residue in the active site of the inhibitor and the presence of a single active site for the two activities. Treatment of API with DTT abolished both the activities although the native structure of API remained virtually unaffected, indicating the catalytic role of the disulfide bonds. Inactivation of API either by active site modification or by conformational changes leads to the concurrent loss of both the antiproteolytic and antifungal activities. Experimental evidences presented here serve to implicate that the antifungal activity of API is a consequence of its protease inhibitory activity.     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.     

Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits

AIM: To study the antifungal activity and plant beneficial traits of a broad-spectrum antagonistic fluorescent pseudomonad strain, PUPa3. METHODS AND RESULTS: Strain PUPa3 was isolated from the rhizosphere soil of rice and identified as Pseudomonas aeruginosa on the basis of biochemical tests and by comparison of 16S rDNA sequences. This bacterium exhibits a broad-spectrum antifungal activity towards phytopathogenic fungi. The antifungal metabolite by PUPa3 was extracted, purified and characterized using nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Production of indole-3-acetic acid (IAA), siderophores, phosphatase and protease in PUPa3 was determined. Strain PUPa3 did not produce hydrogen cyanide, cellulase and pectinase. CONCLUSION: The antifungal metabolite produced by PUPa3 has been identified as phenazine-1-carboxamide (PCN) on the basis of NMR and MS data. Strain PUPa3 showed a broad-spectrum antifungal activity towards a range of phytopathogenic fungi. This bacterium also showed several plant growth-promoting traits but did not show the traits attributed to deleterious rhizobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the production of PCN as well as IAA for the first time by a saprophytic P. aeruginosa strain PUPa3. Because of the production of siderophore, growth hormone, protease and phosphatase and its innate fungicidal potential, this strain can be used as biofertilizer and antagonist against a range of phytopathogenic fungi that infect rice, groundnut, tobacco, chili, mango, sugarcane, tea, cotton and banana.     

Pn-AMPs,the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato,Lycopersicum esculentum

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.     

Antifungal Activity of Volatile Compounds Produced by an Edible Mushroom Hypsizygus marmoreus against Phytopathogenic Fungi

Volatile compounds with antifungal activity produced by edible mushrooms have potential as biological control agents to combat fungal diseases and reduce fungicide use in agriculture. Here we investigated the antifungal activity of volatile compounds produced by the edible mushroom Hypsizygus marmoreus (TUFC 11906) against eight phytopathogenic fungi. The results showed that volatile compounds from the mycelia and culture filtrates (CFs) of H. marmoreus had antifungal activity against some phytopathogenic fungi. Among them, the mycelial growth and conidial germination of Alternaria brassicicola were significantly inhibited by 60 and 100%, respectively. Moreover, the volatile compounds from CFs inhibited the lesion formation of A. brassicicola on detached cabbage leaves by 94%. The volatile compounds had higher antifungal activity against A. brassicicola than other fungi. With the removal of the volatile compounds from conidia of A. brassicicola, the conidia began to germinate, which indicates fungistatic activity of the compounds. The volatile compounds were isolated from the CFs of H. marmoreus, and the major volatile compound with antifungal activity was estimated to be 2‐methylpropanoic acid 2,2‐dimethyl‐1‐(2‐hydroxy‐1‐methylethyl)propyl ester. As the volatile compound produced by H. marmoreus is a product of an edible mushroom and has fungistatic activity against some phytopathogenic fungi, especially A. brassicicola, it may be possible to use the compounds as a novel safe agent for protecting crops in the field and during storage.     

Detection of phlD gene in some fluorescent pseudomonads isolated from Iran and its relative with antifungal activities

Fluorescent pseudomonads that produce antibiotic 2,4-diacetylphloroglocinol (2,4-DAPG) are important group of PGRP that inhibit a broad spectrum of plant pathogenic fungi. Studying on genetic diversity of 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads has been shown with special importance. The first step to investigate the genetic diversity of these bacteria is detecting of the genes required for the biosynthesis of this antibiotic. The objectives of the current study were detection of phlD gene within fluorescent pseudomonads by a PCR-based assay, and comparison of phenotypic and genotypic characteristics of fluorescent pseudomonads with proven biocontrol potential against some soil-borne phytopathogenic fungi. We used a collection of 47 fluorescent Pseudomonas spp. some with known biological control activity against Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var. parasitica, Pythium sp. and Fusarium sp. in vitro and the potential to produce known secondary metabolites such as, siderophore, HCN and protease. The results indicated that 66, 40.42, 63.82,48.94 and 27.65% of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively. Rhizoctonia solani recognized as the most vulnerable fungus. Among 47 strains, 76.59, 97.87 and 17% of strains produced protease, siderophore and HCN, respectively. We could detect phlD gene in strains P-5, P-32, P-47. Strain CHA0 was used as positive control for the detection this gene. Overall, there was no obvious link between the existence of phlD gene and inhibition of fungal growth or production of the antifungal metabolites in vitro. But in some strains such as CHA0 and P-5, we saw a link between the existence of phlD and antifungal activities. Studying on detection and diversity of phlD provides a fundamental knowledge for developing a rapid genetic screening system to identify a potential biocontrol strains.     

小桐子内生真菌及其抗真菌活性研究

从药用植物小桐子的根和茎中分离到内生真菌57株,经鉴定,它们分属于15个不同的分类单元。其中拟盘多毛孢为优势属,其次为拟茎点霉属和茎点霉属。用杨桃炭疽菌,香蕉疫霉和镰刀菌作指示菌对这些内生真菌的抗真菌活性进行检测,结果表明,有2株菌对杨桃炭疽菌具有抑菌活性。     

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。     

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18–10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.     

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides . A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.     

Evaluation of the antifungal activity of Rumex vesicarius L. and Ziziphus spina-christi (L) Desf. Aqueous extracts and assessment of the morphological changes induced to certain myco-phytopathogens

Many Plant extracts had proved a potential antifungal activity against a wide range of phytopathogenic fungi. The aim of this study was to evaluate the antifungal activity of the aqueous extracts of Rumex vesicarius L. and Ziziphus spina-christi (L) Desf. against some fungal species. The effect on growth inhibition, conidia germination, sporogenesis, morphological, and ultrastructural characterizations of fungal growth by scanning and transmission electron microscopes, have been investigated. Both plant extracts exhibited an antifungal activity against Fusarium, Helminthosporium, Alternaria, and Rhizoctonia species, besides, the sporogenesis of Alternaria and Fusarium species was suppressed. Both plants induced severe morphological changes in the hyphal shape and surface. We concluded that the aqueous extracts of these plants had strong antifungal activities. More investigations should be performed to evaluate the possible applications in agriculture and in vivo.     

A potent antifungal protein from Helianthus annuus flowersis a trypsin inhibitor

A 16-kDa protein was isolated from Helianthus annuus flowers by its ability to inhibit the germination of fungal spores. This protein, SAP16, displays an associated activity of trypsin inhibitor and was further purified to apparent homogeneity by affinity chromatography on trypsin-agarose. SAP16 causes the complete inhibition of Sclerotinia sclerotiorum ascospores germination at a concentration of 5 μg·mL^–1 (0.31 μM) and a clear reduction of mycelial growth at lower concentrations, indicating a strong antifungal potency against this natural pathogen of sunflower. Our data suggest that the antifungal ability of SAP16 would not be the result of the inhibition of a fungal protease. This study contributes to the characterization of the emerging family of antifungal proteins with an associated activity of trypsin inhibition and emphasizes their role in plant resistance against fungal attack.     

Herbal remedies for the management of seed-borne fungal pathogens by an edible plant Decalepis hamiltonii (Wight & Arn)

Abstract

Antifungal activity-guided assay of solvent extracts of Decalepis hamiltonii (Wight & Arn) (Asclepiadaceae) against important phytopathogenic fungi, known to cause diseases in sorghum, maize and paddy proved to be highly significant. Among the five solvent extracts tested, Petroleum ether extract showed highly significant antifungal activity. Phytochemical analysis revealed that the antifungal active principle is a phenolic compound. TLC separation of the phenolic fraction using chloroform as an eluting solvent revealed the presence of seven bands but the antifungal activity was observed only in band five with R_f value 0.77. The antifungal active compound is identified as 2-hydroxy-4-methoxybenzaldehyde based on Nuclear Magnetic Resonance (NMR) and mass spectral analysis. The Minimal inhibitory concentration (MIC) varied between 200 μg ml^?1 and 700 μg ml^?1 depending on the fungal species. Seed treatment of the active principle significantly increased seed germination and seed vigour with a corresponding decrease in seed mycoflora. The antifungal active compound was effective against all the 24 fungal species tested suggesting broad-spectrum antifungal activity. Comparative evaluation of the active principle with the synthetic fungicides revealed that the antifungal activity of the active principle obtained from the plant is better than that of synthetic fungicide. This plant being an edible one can be exploited in the management of seed-borne pathogenic fungi and the prevention of biodeterioration of grains and mycotoxin elaboration during storage.     

Antifungal Activity of a Bowman–Birk-type Trypsin Inhibitor from Wheat Kernel

A trypsin inhibitor from wheat kernel (WTI) was found to have a strong antifungal activity against a number of pathogenic fungi and to inhibit fungal trypsin-like activity. WTI inhibited in vitro spore germination and hyphal growth of pathogens, with protein concentration required for 50% growth inhibition (IC₅₀) values ranging from 111.7 to above 500 μg/ml. As observed by electron microscopy, WTI determined morphological alterations represented by hyphal growth inhibition and branching. One of the fungal species tested, Botrytis cinerea produced a trypsin-like protease, which was inhibited by the trypsin inhibitor. WTI, as well as other seed defence proteins, appear to be an important resistance factor in wheat kernels during rest and early germination when plants are particularly exposed to attack by potential soil-borne pathogens.     

A comparative study of transgenic canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) harboring either chimeric or native <Emphasis Type="Italic">Chit</Emphasis>42 genes against phytopathogenic fungi

Canola (Brassica napus L.), an agro-economically important crop in the world, is sensitive to many fungal pathogens. One strategy to combat fungal diseases is genetic engineering through transferring genes encoding the pathogenesis-related (PR) proteins such as chitinase which cause the chitin degradation of fungal cell wall. Chitinase Chit42 from Trichoderma atroviride (PTCC5220) plays an important role in biocontrol and has high antifungal activity against a wide range of phytopathogenic fungi. This enzyme lacks a chitin binding domain (ChBD) which is involved in binding activity to insoluble chitin. In the present study, we investigated the effect of chitin binding domain fused to Chit42 when compared with native Chit42. These genes were over-expressed under the CaMV35S promoter in B. napus, R line Hyola 308. Transformation of cotyledonary petioles was achieved by pBISM2 and pBIKE1 constructs containing chimeric and native Chit42 genes respectively, via Agrobacterium method. The insertion of transgenes in T₀ generation was verified through polymerase chain reaction (PCR) and Southern blot analysis. Antifungal activity of expressed chitinase in transgenic plants was also investigated by bioassays. The transgenic canola expressing chimeric chitinase showed stronger inhibition against phytopathogenic fungi that indicates the role of chitin binding domain.     

Isolation and characterization of a transposon mutant of Pseudomonas fluorescens AU63 deficient in antifungal activity against Pythium ultimum

Tn5 transposon mutagenesis via electroporation of Pseudomonas fluorescens AU63 was used to generate mutants deficient in antifungal activity against the phytopathogenic fungi Pythium ultimum and Thielaviopsis basicola. Mutant C-45 was obtained by an initial screen for the loss of antibacterial activity against Bacillus subtilis and a subsequent screen of mutants obtained for the loss of antifungal activity against pathogenic fungi. A single chromosomal insertion of Tn5 in the chromosome of Ps. fluorescens C-45 was confirmed by Southern blot hybridization. A metabolite responsible for the observed antibacterial and antifungal activities was identified using thin layer chromatography. The antimicrobial activities of the partially purified substance present in the parental strain and missing in the C-45 mutant were not affected by protease, high temperature, acid or alkali treatment. These results provide the basis for a structural analysis of this new antimicrobial substance and the genetic elucidation of its biosynthesis.     

A novel protease inhibitor in Bombyx mori is involved in defense against Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle using a plethora of hydrolytic enzymes including cuticle-degrading proteases and chitinases, which are important virulence factors. The insect integument and hemolymph contains a relatively high concentration of protease inhibitors, which are closely involved with defense against pathogenic microorganisms. To elucidate the molecular mechanism underlying resistance against entomopathogenic fungi and to identify a new molecular target for improving fungal resistance in the silkworm, Bombyx mori, we cloned and expressed a novel silkworm TIL-type protease inhibitor BmSPI38, which was very stable over a wide range of temperatures and pH values. An activity assay suggested that BmSPI38 potently inactivated the insecticidal cuticle-degrading enzyme (CDEP-1) produced by B. bassiana and subtilisin A produced by Bacillus licheniformis. The melanization of silkworm induced by CDEP-1 protease could also be blocked by BmSPI38. These results provided new insights into the molecular mechanisms whereby insect protease inhibitors provide resistance against entomopathogenic fungi, suggesting the possibility of using fungal biopesticides in sericulture.     

Cloning and heterologous expression of SS10, a subtilisin-like protease displaying antifungal activity from Trichoderma harzianum

Trichoderma harzianum parasitizes a large variety of phytopathogenic fungi. Trichoderma harzianum mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. A gene ( SS10 ) encoding a subtilisin-like protease was cloned from T. harzianum T88, a biocontrol agent effective against soil-borne fungal pathogens. The full-length cDNA was isolated by 5' and 3' rapid amplification of the cDNA ends. The coding region of the gene is 1302 bp long, encoding 433 amino acids of a predicted protein with a molecular mass of 45 kDa and a pI of 6.1. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) (EC 3.4.21.) and had a predicted active site made up of the catalytic residues Asp 187, His 218 and Ser 376. Northern experiments demonstrated that SS10 was induced in response to different fungal cell walls. Subtilisin-like protease gene SS10 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (17.8 U mL⁻¹) 60 h after induction with galactose. The optimal enzyme reaction temperature was 50 °C and the optimal pH was 8. The subtilisin-like protease exerted broad-spectrum antifungal activity against Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Cytospora chrysosperma .     

Synthesis and Antifungal Activity of Curcumol Derivatives

To discover novel and effective antifungal candidates, a series of new curcumol derivatives were designed, synthesized, and evaluated their antifungal activity against five phytopathogenic fungi by the mycelium growth rate method. Derivatives c4 , c22 and c23 exhibited excellent antifungal activity against Phomopsis sp. with EC₅₀ values of 3.06, 3.07, and 3.16 μM, respectively. Specifically, compound c4 exhibited the strongest antifungal activity against Phomopsis sp., which was 44 times that of pyrimethanil (EC₅₀=134.37 μM). The results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that compound c4 could cause cell senescence and death of Phomopsis sp. by changing the normal hyphal morphology and disrupting the normal metabolism of hyphal cells. Moreover, compound c4 showed excellent curative effect against Phomopsis sp. on kiwifruit. These findings confirmed that compound c4 has great potential as a potent antifungal agent.     

A peroxiredoxin Q homolog from gentians is involved in both resistance against fungal disease and oxidative stress

An antifungal protein (GtAFP1) showing antimicrobial activity against phytopathogenic fungi was purified from leaves of Gentiana triflora. The deduced amino acid sequence of the cDNA of the corresponding gene, GtAFP1, showed 94, 75, 72 and 63% amino acid identities with peroxiredoxin Q from Populus balsamifera x P. deltoides subsp. trichocarpa, Sedum lineare, Suaeda maritima and Arabidopsis thaliana, respectively. The GtAFP1 gene is suggested to be present in the genome in one to two copies and was expressed in the leaves, roots and stems. Expression of GtAFP1 was induced by treatment with salicylic acid, but not methyl jasmonate. Recombinant GtAFP1 protein showed not only antifungal activity but also thioredoxin-dependent peroxidase activity. Overexpression of GtAFP1 in tobacco plants improved tolerance not only against fungal diseases but also against oxidative stress. These results indicate that GtAFP1 might act as a disease and oxidative stress defensive gene in plants and could be useful for engineering stress-resistant plants.