Production of extracellular proteases by human pathogenic Trichoderma longibrachiatum strains

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, and N-Succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens.     

In vitro efficacy of ciprofloxacin alone and in combination with amoxycillin against Salmonella typhi isolates

Combined effect of ciprofloxacin (Ci) and amoxycillin (Ax) has been studied in vitro against 12 clinical isolates of S. typhi that showed Ci minimum inhibitory concentration (MIC) of > or =1 microg/ml. By agar dilution method, MIC values of Ax were 10-16 microg/ml for 11 isolates and 0.5 microg/ml for the remaining one isolate. The isolates, when treated with Ci and Ax in combination, showed fractional inhibitory concentration (FIC) of 0.004-0.256 microg/ml for Ci. FIC of Ax ranged from 6-10 microg/ml, except for a single isolate that showed Ax FIC of 0.25 microg/ml. Thus Ci was more efficacious in combination with Ax against S. typhi than Ci alone. The antibiotic combination exhibited an additive effect for all the isolates showing FIC index 0.504-0.832.     

茶树内生木霉种的鉴定及其在植物体内的定殖

采用分离自健康茶树叶片组织的一株长枝木霉Trichoderma longibrachiatum菌株CSN-18,研究茶树内生木霉的人工接种、再分离及其在茶树地上部组织中的内生性。该菌在PDA培养基上生长速度快,产孢量大。用CSN-18回接茶苗,接种后一个月可以从茶苗的茎和叶组织中再分离获得该真菌。接种后的茶苗没有观察到明显的病害表现;与对照相比,接种后6个月组培苗生长良好,叶色更绿;接种后1个月,实生苗生长正常。通过石蜡切片和苯胺蓝染色,在已接种的茶树组培苗的叶片内部组织中可观察到木霉的存在,从而证明了木霉能够在茶树地上部组织内定殖,是茶树的一种内生真菌。     

Etest for assessing the susceptibility of filamentous fungi

The purpose of this study was to evaluate the Etest as an in vitro antifungal susceptibility test method for different moulds originating from human samples and from the environment. A total of 50 isolates (1 Acremonium, 18 Aspergillus, 2 Cladosporium, 1 Epicoccum, 15 Penicillium, 2 Scopulariopsis and 11 Trichoderma strains) were tested by the Etest. Forty-six of the tested moulds (92%) were resistant to fluconazole with minimal inhibitory concentrations (MICs) > or = 256 microg ml(-1). There were strains resistant to ketoconazole among Aspergillus niger, A. ochraceus and Cladosporium spp. with MICs > 32 microg ml(-1). For fluconazole, no differences were observed using two different inocula, while for itraconazole, ketoconazole and amphotericin B, a 1 or less step 2-fold dilution difference in MIC was seen for the most of 10 selected strains. The MICs of fluconazole and amphotericin B obtained for Trichoderma strains by the Etest and the agar dilution method were also compared. MICs for fluconazole were in agreement, while MICs for amphotericin B were higher with 1 or 2 steps of 2-fold dilutions for most of Trichoderma strains in the case of the agar dilution method.     

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.     

Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization

To study the role of Trichoderma in sick building syndrome, it is essential to be able to accurately identify species. Forty-four strains of Trichoderma spp. isolated from Danish buildings damaged by water leaks were identified using ITS1 ribotyping and universally primed PCR, UP-PCR. Ribotyping allowed the assignment of the strains into three distinct groups. High similarity of UP-PCR banding profiles of the strains allowed species designation for almost all strains (43 out of 44) when compared with the UP-PCR banding profiles obtained from reference strains of T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum and T. viride. However, cross hybridization of UP-PCR products showed that the latter strain had high DNA homology to the ex-type strain of T. hamatum. The combined approach is a convenient way for reliable identification of Trichoderma strains.     

Isozyme analysis and relationships among three species in Malaysian Trichoderma isolates

Isozyme and protein electrophoresis data from mycelial extracts of 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia were investigated. The eight enzyme and a single protein pattern systems were analyzed. Three isozyme and total protein patterns were shown to be useful for the detection of three Trichoderma species. The isozyme and protein data were analyzed using the Nei and Li Dice similarity coefficient for pairwise comparison between individual isolates, species isolate group, and for generating a distance matrix. The UPGMA cluster analysis showed a higher degree of relationship between T. harzianum and T. aureoviride than to T. longibrachiatum. These results suggested that the T. harzianum isolates had high levels of genetic variation compared to the other isolates of Trichoderma species.     

Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline

Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 microg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 microg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.     

Cork taint of wines: role of the filamentous fungi isolated from cork in the formation of 2,4,6-trichloroanisole by o methylation of 2,4,6-trichlorophenol

Cork taint is a musty or moldy off-odor in wine mainly caused by 2,4,6-trichloroanisole (2,4,6-TCA). We examined the role of 14 fungal strains isolated from cork samples in the production of 2,4,6-TCA by O methylation of 2,4,6-trichlorophenol (2,4,6-TCP). The fungal strains isolated belong to the genera Penicillium (four isolates); Trichoderma (two isolates); and Acremonium, Chrysonilia, Cladosporium, Fusarium, Mortierella, Mucor, Paecilomyces, and Verticillium (one isolate each). Eleven of these strains could produce 2,4,6-TCA when they were grown directly on cork in the presence of 2,4,6-TCP. The highest levels of bioconversion were carried out by the Trichoderma and Fusarium strains. One strain of Trichoderma longibrachiatum could also efficiently produce 2,4,6-TCA in liquid medium. However, no detectable levels of 2,4,6-TCA production by this strain could be detected on cork when putative precursors other than 2,4,6-TCP, including several anisoles, dichlorophenols, trichlorophenols, or other highly chlorinated compounds, were tested. Time course expression studies with liquid cultures showed that the formation of 2,4,6-TCA was not affected by a high concentration of glucose (2% or 111 mM) or by ammonium salts at concentrations up to 60 mM. In T. longibrachiatum the O methylation of 2,4,6-TCP was catalyzed by a mycelium-associated S-adenosyl-L-methionine (SAM)-dependent methyltransferase that was strongly induced by 2,4,6-TCP. The reaction was inhibited by S-adenosyl-L-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings increase our understanding of the mechanism underlying the origin of 2,4,6-TCA on cork, which is poorly understood despite its great economic importance for the wine industry, and they could also help us improve our knowledge about the biodegradation and detoxification processes associated with chlorinated phenols.     

Identification of Trichoderma strains by image analysis of HPLC chromatograms

Forty-four Trichoderma strains from water-damaged building materials or indoor dust were classified with chromatographic image analysis on full chromatographic matrices obtained by high performance liquid chromatography with UV detection of culture extracts. The classes were compared with morphological identification and rDNA sequence data, and for each class all strains were of the same identity. With all three techniques each strain--except one--was identified as the same species. These strains belonged to Trichoderma atroviride (nine strains), Trichoderma viride (three strains), Trichoderma harzianum (10 strains), Trichoderma citrinoviride (12 strains), and Trichoderma longibrachiatum (nine strains). The odd strain was identified as Trichoderma hamatum by morphology and rDNA sequencing, but not by image analysis as no reference strains of this species were included. It is concluded that the secondary metabolite profile contains sufficient information for classification and species identification.     

ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum

The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu-Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Effectiveness of the antibiotics chloramphenicol and rifampin in the treatment of Streptococcus pneumoniae-induced meningitis and systemic infections

Streptococcus pneumoniae, a common pathogen in pediatric infections, has become resistant to penicillin and make these infections difficult to treat. Rifampin and chloramphenicol have been recommended as alternative therapies, since they are less costly and more accessible to communities with limited resources. However, their use may be restricted by the differing levels of resistance found in target populations. The objective was to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for chloramphenicol and rifampin in strains of S. pneumoniae. These strains were newly isolated from children under age 5 that had demonstrated systemic infections and meningitis. A subgroup of 107 isolates of S. pneumoniae was selected from 324 strains isolated during a period of 2 years (1994-1996). Among these isolates, 60 were penicillin-resistant and 47 were susceptible; 53 isolates were from children with meningitis. MIC and MBC for chloramphenicol and rifampicin were obtained by standard methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS). S. pneumoniae ATCC strain 49619 served as the control. An isolate was considered susceptible to chloramphenicol when MIC = 4 microg/ml and resistant when MIC = 8 microg/ml. A strain was considered susceptible to rifampin when MIC = 1 microg/ml and resistant when MIC = 4 microg/ml. MBC was determined by recording the lower concentration of the antibiotic that inhibited 99.9% of the initial inoculum. Chloramphenicol resistance was found in 21% of the 107 isolates. In the group susceptible to penicillin, 11% were resistant to chloramphenicol and in the group resistant to penicillin 28% was resistant to chloramphenicol as well. MBC was found > 4 microg/ml in 28% of the isolates susceptible to penicillin and in 60% of the resistant isolates. No isolates were found resistant to rifampin. However, 2 penicillin resistant isolates showed CBM > 1 microg/ml to rifampin, and one with CIM = 1 microg/ml had a MBC to rifampicin of 16 microg/ml. Meningitis isolates showed higher CIM and CBM than the group of total isolates. These data suggest that chloramphenicol is not recommended for invasive infections caused by S. pneumoniae in Colombia. Rifampin is a more effective therapy in combination with other antibiotics for treatment of this kind of infections. Further studies are necessary to clarify the significance of low levels of MBC to rifampin found in some strains, since this may affect the efficacy of therapies that include this antibiotic.     

Abietanes from Plectranthus grandidentatus and P. hereroensis against methicillin- and vancomycin-resistant bacteria

The antimicrobial activity of 10 natural abietanes isolated from Plectranthus grandidentatus and P. hereroensis acetonic extract was evaluated against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). The results revealed that the most active diterpenes were coleon U (1), 7alpha-acetoxy-6beta-hydroxyroyleanone (2) and horminone (3). Minimum inhibitory concentration (MIC) values ranging 0.98-15.63 microg/ml were obtained for MRSA clinical strains, and MIC values of 15.63 and 31.25 microg/ml were obtained for VRE clinical strains. Some structure-activity relationships are emphasized.     

In vitro water activity and pH dependence of mycelial growth and extracellular enzyme activities of Trichoderma strains with biocontrol potential

AIMS: Water activity (aw) and pH are probably the most important environmental parameters affecting the activities of mycoparasitic Trichoderma strains. Therefore it is important to collect information on the effects of these factors on mycelial growth and on the in vitro activities of extracellular enzymes involved in nutrient competition (e.g. beta-glucosidase, cellobiohydrolase and beta-xylosidase) and mycoparasitism (e.g. N-acetyl-beta-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) of Trichoderma strains with biocontrol potential. METHODS AND RESULTS: Water activity and pH dependence of the linear mycelial growth of five examined Trichoderma strains belonging to three different species groups was examined on yeast extract and soil extract media. Maximal growth rates were observed at aw 0.997 and pH 4.0 in the case of all strains. The activities of the examined extracellular enzymes at different aw and pH values were determined spectrophotometrically after incubation with chromogenic p-nitrophenyl and p-nitroaniline substrates. Maximal enzyme activities were measured at aw 0.950 for beta-glucosidase, trypsin-like protease and chymotrypsin-like protease, at 0.910 for cellobiohydrolase and at 0.993 for beta-xylosidase and N-acetyl-beta-glucosaminidase enzymes. Optimal pH values are suggested to be at 5.0 for beta-glucosidase, cellobiohydrolase and N-acetyl-beta-glucosaminidase, at 3.0 for beta-xylosidase, at 6.0 for trypsin-like protease and between 6.0 and 7.0 for chymotrypsin-like protease activities, respectively. CONCLUSIONS: Extracellular enzymes of the examined mycoparasitic Trichoderma strains are able to display activities under a wider range of aw and pH values than those allowing mycelial growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Data about the effects of aw and pH on mycelial growth and extracellular enzyme activities of Trichoderma reveal useful information about the applicability of biocontrol strains in agricultural soils with specific water and pH relations.     

Pathogenicity of Ochroconis gallopava isolated from hot springs in Japan and a review of published reports

Four strains of Ochroconis gallopava from 3 out of 15 Japanese hot springs were isolated. Colonies of the hot spring isolates were uniformly floccose and dark olive green on the surface and dark brown on their reverse side on potato dextrose agar (PDA) plates, however, they became felty, flat, and brownish-black, and produced a reddish-brown pigment after several times of subculture at room temperature. Shapes and sizes of conidia of the four strains were individual, while the D1/D2 domain of the large subunit ribosomal RNA gene sequences showed 99.7% identity in the GenBank database. The DNA pattern of the hot spring isolates amplified by species specific loop mediated isothermal amplification method were as the same pattern as that of a clinical isolate. The minimum inhibitory concentrations of antifungal agents to O. gallopava isolated from the hot springs were ranged from 0.5 to 1 μg/ml in amphotericin B, 1 to 16 μg/ml in flucytosine, 0.125 to 0.25 μg/ml in itraconazole, 1 to 4 μg/ml in miconazole, 16 to 64 μg/ml in flconazole and 0.03 to 0.5 μg/ml in micafungin. The isolates had fatal outcome in experimentally infected mice intravenously with severe invasiveness to brains and kidneys. These findings suggested that O. gallopava habitats in hot springs could be one of sources for infection.     

Improved technique on reversion of mycelial protoplast of Trichoderma reesei into mycelia

Important parameters in the regeneration of protoplasts are viability, the capacity to synthesize cell walls and the retention of properties of the parent cell. Mycelial protoplasts of Trichoderma longibrachiatum (Trichoderma reesei) have been regenerated. Factors influencing the regeneration of protoplasts of T. longibrachiatum QM 9414 were found to be, the nature of osmotic stabilizer, the concentration of osmotic stabilizer, pH, temperature, and the composition of regeneration medium. With glucose-mineral regeneration medium, the optimum conditions for protoplasts regeneration were 0.5 M KCl, pH 6.0 and temperature 30°C. With Czapek-Dox medium, the optimum conditions were 0.7 M mannitol, pH 6.0 and temperature 30°C. Maximum regeneration frequency of T. longibrachiatum protoplasts were obtained using glucose-mineral medium.     

Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea

Free-living nitrogen fixing bacteria were isolated from rhizosphere of seven different plant namely sesame, maize, wheat, soybean, lettuce, pepper and rice grown in Chungbuk Province, Korea. Five isolates with nitrogenase activity above 150nmol(-1) mg(-1) protein were identified based on, phenotypic and 16S rDNA sequences analysis. The strains were identified as Stenotrophomonas maltophilia (PM-1, PM-26), Bacillus fusiformis (PM-5, PM-24) and Pseudomonas fluorescens (PM-13), respectively. All the isolates produced indole-3-acetic acid (IAA), in the presence of tryptophan, ranging from 100.4 microg ml(-1) (PM-13) to 255 microg ml(-1) (PM-24). The isolate PM-24 (Bacillus fusiformis) exhibiting highest nitrogenase activity (3677.81 nmol h(-1) mg(-1) protein) and IAA production (255microg ml(-1)) has a promising potential for developing as a plant growth promoting rhizobacteria.     

Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinum L.)

Nine isolates of Trichoderma spp. were investigated for their ability to solubilize insoluble phosphate in Pikovskaya's broth and were compared with an efficient phosphate-solubilizing bacterium Bacillus megaterium subsp. phospaticum PB that was used as the reference strain. All 9 Trichoderma isolates were found to solubilize insoluble tricalcium phosphate to various extents. Trichoderma viride (TV 97) (9.03 microg x mL(-1)), Trichoderma virens (PDBCTVs 12) (9.0 microg x mL(-1)), and Trichoderma virens (PDBCTVs 13) (8.83 microg x mL(-1)) solubilized 70% of that solubilized by the reference strain Bacillus megaterium (12.43 microg x mL(-1)). Pot culture and field evaluations with Trichoderma harzianum (PDBCTH 10), Trichoderma viride (TV 97), and Trichoderma virens (PDBCTVs 12) using chickpea (Cicer arietinum L.) 'Annegeri-1' as the test plant and rock phosphate as the phosphorus source showed significantly increased P uptake in plants treated with Trichoderma harzianum (PDBCTH 10) followed by Trichoderma virens (PDBCTVs 12) and Trichoderma viride (TV 97). Inoculation of Trichoderma spp. also showed increased growth and yield parameters of chickpea compared with the uninoculated controls under both glasshouse and field conditions.