Microbiology of secondary infections in Buruli ulcer lesions; implications for therapeutic interventions.

BACKGROUND: Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans and is the second most common mycobacterial disease after tuberculosis in Ghana and Côte d'Ivoire. M. ulcerans produces mycolactone, an immunosuppressant macrolide toxin, responsible for the characteristic painless nature of the infection. Secondary infection of ulcers before, during and after treatment has been associated with delayed wound healing and resistance to streptomycin and rifampicin. However, not much is known of the bacteria causing these infections as well as antimicrobial drugs for treating the secondary microorganism. This study sought to identify secondary microbial infections in BU lesions and to determine their levels of antibiotic resistance due to the prolonged antibiotic therapy required for Buruli ulcer. RESULTS: Swabs from fifty-one suspected BU cases were sampled in the Amansie Central District from St. Peters Hospital (Jacobu) and through an active case surveillance. Forty of the samples were M. ulcerans (BU) positive. Secondary bacteria were identified in all sampled lesions (N = 51). The predominant bacteria identified in both BU and Non-BU groups were Staphylococci spp and Bacilli spp. The most diverse secondary bacteria were detected among BU patients who were not yet on antibiotic treatment. Fungal species identified were Candida spp, Penicillium spp and Trichodema spp. Selected secondary bacteria isolates were all susceptible to clarithromycin and amikacin among both BU and Non-BU patients. Majority, however, had high resistance to streptomycin. CONCLUSIONS: Microorganisms other than M. ulcerans colonize and proliferate on BU lesions. Secondary microorganisms of BU wounds were mainly Staphylococcus spp, Bacillus spp and Pseudomonas spp. These secondary microorganisms were less predominant in BU patients under treatment compared to those without treatment. The delay in healing that are experienced by some BU patients could be as a result of these bacteria and fungi colonizing and proliferating in BU lesions. Clarithromycin and amikacin are likely suitable drugs for clearance of secondary infection of Buruli ulcer.

Trichoderma from Brazilian garlic and onion crop soils and description of two new species: Trichoderma azevedoi and Trichoderma peberdyi.

Fifty four Trichoderma strains were isolated from soil samples collected from garlic and onion crops in eight different sites in Brazil and were identified using phylogenetic analysis based on combined ITS region, tef1-α, cal, act and rpb2 sequences. The genetic variability of the recovered Trichoderma species was analysed by AFLP and their phenotypic variability determined using MALDI-TOF. The strain clusters from both typing techniques coincided with the taxonomic determinations made from phylogenetic analysis. The phylogenetic analysis showed the occurrence of Trichoderma asperellum, Trichoderma asperelloides, Trichoderma afroharzianum, Trichoderma hamatum, Trichoderma lentiforme, Trichoderma koningiopsis, Trichoderma longibrachiatum and Trichoderma erinaceum, in the soil samples. We also identified and describe two new Trichoderma species, both in the harzianum clade of section Pachybasium, which we have named Trichoderma azevedoi sp. nov. and Trichoderma peberdyi sp. nov. The examined strains of both T. azevedoi (three strains) and T. peberdyi (12 strains) display significant genotypic and phenotypic variability, but form monophyletic clades with strong bootstrap and posterior probability support and are morphologically distinct from their respective most closely related species.

Extracts of Caesalpinia ferrea and Trichoderma sp. on the control of Colletotrichum sp. transmission in Sideroxylon obtusifolium seeds / Extratos de Caesalpinia ferrea e Trichoderma spp. no controle da tramissibilidade de Colletotrichum sp. em sementes de Sideroxylon obtusifolium (Sapotaceae)

ABSTRACT Recent research reports the importance of preserving plants in Brazilian semiarid regions, in this context, the scientific literature has reported different pharmacological studies from plant extracts with an antifungal potential, coming from forest species that can contribute as a control and management strategy in the transmission of phytopathogens. This study aimed to evaluate the effect of biotech treatments in controlling the transmission of Colletotrichum sp. in seeds of S. obtusifolium. In this study, 100 seeds were subjected to the following preventive treatments: fungicide Captan®, extract of Caesalpinia ferrea Mart. Ex. Tul., and biological control with Trichoderma spp. The biological control with Trichoderma spp. and the alternative control using C. ferrea extract provided a greater protection to seeds and seedlings of S. obtusifolium facing the transmissibility of Colletotrichum sp.The treatment based on plant extract is more efficient for this purpose only in large seeds and does not interfere on the germination percentage and speed. Therefore it is necessary to perform other studies with Trichoderma spp. and C. ferrea extract to test different doses of these products.

RESUMO Recentes pesquisas relatam a importância da preservação de plantas do semiárido brasileiro. Neste contexto, a literatura científica tem relatado diferentes estudos farmacológicos com extratos vegetais com potencial antifúngico proveniente de espécies florestais que podem contribuir como estratégia de controle e gerenciamento na transmissão de fitopatógenos. No presente estudo o objetivo foi avaliar o efeito de tratamentos biotecnológicos no controle da transmissibilidade de Colletotrichum sp. em sementes de S. obtusifolium. Neste estudo foram utilizadas 100 sementes submetidas aos seguintes tratamentos preventivos: fungicida Captan®, extrato de Caesalpinia ferrea Mart. Ex. Tul. e controle biológico com Trichoderma spp. O controle biológico com Trichoderma spp. e o alternativo com extrato de C. ferrea proporcionam maior proteção às sementes e plântulas S. obtusifolium quanto a transmissibilidade do Colletotrichum sp. O tratamento à base de extrato vegetal foi o mais eficiente para este fim, apenas em sementes de maior tamanho, por não interferir na porcentagem e velocidade de germinação. Portanto, faz-se necessário à realização de outros trabalhos com Trichoderma spp. e extrato de C. ferrea para testar doses diferentes desses produtos.

Antifungal activity of metabolites of the endophytic fungus Trichoderma brevicompactum from garlic.

The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8S- ITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4ß-acetoxy-12,13- epoxy-Δ(9)-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 µg mL(-1). Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 µg mL(-1). However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 µg mL(-1)). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.

Antifungal activity of metabolites of the endophytic fungus Trichoderma brevicompactum from garlic

The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8SITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4β-acetoxy-12,13-epoxy-Δ9-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 µgmL-1. Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 µgmL-1. However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 µgmL-1). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.

Combined inoculation of Pseudomonas fluorescens and Trichoderma harzianum for enhancing plant growth of vanilla (Vanilla planifolia).

This study was conducted to evaluate the plant growth promoting efficiency of combined inoculation of rhizobacteria on Vanilla plants. Based on the in vitro performance of indigenous Trichoderma spp. and Pseudomonas spp., four effective antagonists were selected and screened under greenhouse experiment for their growth enhancement potential. The maximum percentage of growth enhancement were observed in the combination of Trichoderma harzianum with Pseudomonas fluorescens treatment followed by Pseudomonas fluorescens, Trichoderma harzianum, Pseudomonas putida and Trichoderma virens, respectively in decreasing order. Combined inoculation of Trichoderma harzianum and Pseudomonas fluorescens registered the maximum length of vine (82.88 cm), highest number of leaves (26.67/plant), recorded the highest fresh weight of shoots (61.54 g plant(-1)), fresh weight of roots (4.46 g plant(-1)) and dry weight of shoot (4.56 g plant(-1)) where as the highest dry weight of roots (2.0806 g plant(-1)) were achieved with treatments of Pseudomonas fluorescens. Among the inoculated strains, combined inoculation of Trichoderma harzianum and Pseudomonas fluorescens recorded the maximum nitrogen uptake (61.28 mg plant(-1)) followed by the combined inoculation of Trichoderma harzianum (std) and Pseudomonas fluorescens (std) (55.03 mg plant(-1)) and the highest phosphorus uptake (38.80 mg plant(-1)) was recorded in dual inoculation of Trichoderma harzianum and Pseudomonas fluorescens.

[Identification and secondary metabolites of endophytic fungal strain PR35 from Paeonia delavayi].

OBJECTIVE: To identify the endophytic fungal strain PR35 separated from Paeonia delavayi and study chemical constituents of its secondary metabolites. METHOD: The fungal strain PR35 was identified by morphological observation and ITS rDNA sequence analysis. Various chromatographic methods were adopted to separate and purify its secondary metabolites, and their structures were identified by physiochemical properties and spectral data RESULT: The fungal strain PR35 was identified as Trichoderma longibrachiatum. Five compounds were separated from fermentation products of fungal strain PR35 and identified as 1-(2,6-dihydroxyphenyl)-3-hydroxybutan-1-one (1), 1-(2,6-dihydroxypheny) propan-1-one (2), 1-(2,4,6-trihydroxyphenyl) butan-1-one (3), 4-methoxy-1-naphthol (4), and cerevisterol (5). Among them, compounds 1-3 showed notable antifungal activities against Botrytis cinerea, Fusarium avenaceum and Hormodendrum compactum. CONCLUSION: The endophytic fungus T. longibrachiatum was separated from the plant P. delavayi for the first time. Five compounds were first separated from endophytic fungus of P. delavayi. Among them, compound 4 was separated from microbial fermentation products for the first time.

Efficacy of Trichoderma asperellum oil formulations on the control of cocoa black pod disease (Phytophthora megakarya).

The objective of this study was therefore to develop a formulation of conidia of T. asperellum with the aim of improving its efficacy. The formulations developed were oily dispersions. It was a combination of solvents consisting of groundnut oil or palm oil with structural agents and emulsifying-dispersing agents. Emulsification tests were carried out and the stability of the emulsions evaluated. The evaluation of the effect of co-formulants on the growth of conidia of T. asperellum was done by reading the optical densities of the formulated samples on multi-plates using a plate reader. The test on detached cocoa pods was done by treating the cocoa pods with selected formulations at 1.10(7) conidia/ml and inoculation of the treated cocoa pods was done 24 hours later with zoospores of P. megakarya at 1.10(5) zoospores/ml. The growth of necrosis on the fruits was measured daily. The screening of co-formulants and emulsification tests ended up with the selection of two formulations. The first composed of conidia of T. asperellum, groundnut oil, Tensiofix NTM and Tensiofix 869. The second differed from the first by utilisation of palm oil as the solvent. These formulations proved stable when diluted in water with 1% and 0.5% of sedimentation respectively after 24 hours. The viability test of the conidia indicated that the different formulations selected did not have a fungitoxic effect. The test on detached cocoa pods showed an improved efficacy of T. asperellum to control the disease. The growth rates of necrosis were 6.29 mm/day, 7.25 mm/day and 31.6 mm/day for treatment with formulation 1, pure conidia and control treated with water respectively.

Characterization of field isolates of Trichoderma antagonistic against Rhizoctonia solani.

The aim of the present study was to characterize sixteen isolates of Trichoderma originating from a field of sugar beet where disease patches caused by Rhizoctonia solani were observed. Use of both molecular and morphological characteristics gave consistent identification of the isolates. Production of water-soluble and volatile inhibitors, mycoparasitism and induced systemic resistance in plant host were investigated using in vitro and in vivo tests in both sterilized and natural soils. This functional approach revealed the intra-specific diversity as well as biocontrol potential of the different isolates. Different antagonistic mechanisms were evident for different strains. The most antagonistic strain, T30 was identified as Trichoderma gamsii. This is the first report of an efficient antagonistic strain of T. gamsii being able to reduce the disease in different conditions. The ability to produce water-soluble inhibitors or coil around the hyphae of the pathogen in vitro was not related to the disease reduction in vivo. Additionally, the strains collected from the high disease areas in the field were better antagonists. The antagonistic activity was not characteristic of a species but that of a population.

Seed treatment with Trichoderma harzianum alleviates biotic, abiotic, and physiological stresses in germinating seeds and seedlings.

Trichoderma spp. are endophytic plant symbionts that are widely used as seed treatments to control diseases and to enhance plant growth and yield. Although some recent work has been published on their abilities to alleviate abiotic stresses, specific knowledge of mechanisms, abilities to control multiple plant stress factors, their effects on seed and seedlings is lacking. We examined the effects of seed treatment with T. harzianum strain T22 on germination of seed exposed to biotic stress (seed and seedling disease caused by Pythium ultimum) and abiotic stresses (osmotic, salinity, chilling, or heat stress). We also evaluated the ability of the beneficial fungus to overcome physiological stress (poor seed quality induced by seed aging). If seed were not under any of the stresses noted above, T22 generally had little effect upon seedling performance. However, under stress, treated seed germinated consistently faster and more uniformly than untreated seeds whether the stress was osmotic, salt, or suboptimal temperatures. The consistent response to varying stresses suggests a common mechanism through which the plant-fungus association enhances tolerance to a wide range of abiotic stresses as well as biotic stress. A common factor that negatively affects plants under these stress conditions is accumulation of toxic reactive oxygen species (ROS), and we tested the hypothesis that T22 reduced damages resulting from accumulation of ROS in stressed plants. Treatment of seeds reduced accumulation of lipid peroxides in seedlings under osmotic stress or in aged seeds. In addition, we showed that the effect of exogenous application of an antioxidant, glutathione, or application of T22, resulted in a similar positive effect on seed germination under osmotic stress or in aged seed. This evidence supports the model that T. harzianum strain T22 increases seedling vigor and ameliorates stress by inducing physiological protection in plants against oxidative damage.

Development of a molecular approach to describe the composition of Trichoderma communities.

Trichoderma and its teleomorphic stage Hypocrea play a key role for ecosystem functioning in terrestrial habitats. However, little is known about the ecology of the fungus. In this study we developed a novel Trichoderma-specific primer pair for diversity analysis. Based on a broad range master alignment, specific Trichoderma primers (ITSTrF/ITSTrR) were designed that comprise an approximate 650bp fragment of the internal transcribed spacer region from all taxonomic clades of the genus Trichoderma. This amplicon is suitable for identification with TrichoKey and TrichoBLAST. Moreover, this primer system was successfully applied to study the Trichoderma communities in the rhizosphere of different potato genotypes grown at two field sites in Germany. Cloning and sequencing confirmed the specificity of the primer and revealed a site-dependent Trichoderma composition. Based on the new primer system a semi-nested approach was used to generate amplicons suitable for denaturing gradient gel electrophoresis (DGGE) analysis and applied to analyse Trichoderma communities in the rhizosphere of potatoes. High field heterogeneity of Trichoderma communities was revealed by both DGGE. Furthermore, qPCR showed significantly different Trichoderma copy numbers between the sites.

[Progress on the control of medicinal plants soil-borne disease by anti-microorganism].

Much success had been achieved in the following aspect: the filtration of anti-microorganism, the action mechanisms, the inhibitory action in the field and so on. Though the narrow object and the unstable effect really exist now, it still has a broad developing future for it's advantage in keeping ecological balance and in fitting the requirement of GAP.