Screening of marine resistant strain based on PKS and NRPS genes and the activity of its metabolites / 生物工程学报

Based on polyketide syntheses gene (PKS) and non-ribosomal peptide synthetases gene (NRPS), one strain with high anti-pathogenic activity was screened from 77 strains isolated from Arctic marine sediments and identified. By optimizing the composition of culture medium and fermentation conditions, the production of this strain's active metabolites was improved and the main metabolites were identified by HRMS, 1H NMR and 13C NMR. The antibacterial spectrum of the main metabolites and the effect of the metabolites on cucumber Fusarium wilt were also determined. The results showed that the strain was Bacillus velezensis and it showed growth promoting effect on plants. When the strain was cultured in 5 g/L maltose, 10 g/L tryptone, 10 g/L sodium chloride, at 30 ℃, 150 r/min for 60 h, the diameter of the inhibition zone increased from (16.23±0.42) to (24.42±0.57) mm. The metabolites of this strain mainly contain macrolide compound macrolactin A, which has antagonistic effect on a variety of pathogenic bacteria and fungi. Cucumber seedling experiments showed that the metabolites of this strain had a protective effect on cucumber Fusarium wilt, and showed a good potential for development and application as a biocontrol agent.

Isolation of Fusarium and identification of its toxins from tuberous root of Pseudostellaria heterophylla / 中国中药杂志

Fusarium is the major pathogen of root rot of Pseudostellaria heterophylla. This study aims to explain the possible distribution of Fusarium species and the contamination of its toxin-chemotypes in tuberous root of P. heterophylla. A total of 89 strains of fungi were isolated from the tuberous root of P. heterophylla. Among them, 29 strains were identified as Fusarium by ITS2 sequence, accounting for 32.5%. They were identified as five species of F. avenaceum, F. tricinctum, F. fujikuroi, F. oxysporum, and F. graminearum based on β-Tubulin and EF-1α genes. LC-MS/MS detected 18, 1, and 5 strains able to produce ZEN, DON, and T2, which accounted for 62.1%, 3.4%, and 17.2%, respectively. Strain JK3-3 can produce ZEN, DON, and T2, while strains BH1-4-1, BH6-5, and BH16-2 can produce ZEN and T2. PCR detected six key synthase genes of Tri1, Tri7, Tri8, Tri13, PKS14, and PKS13 in strain JK3-3, which synthesized three toxins of ZEN, DON, and T2. Four key synthase genes of Tri8, Tri13, PKS14, and PKS13 were detected in strains BH1-4-1, BH6-5, and BH16-2, which were responsible for the synthesis of ZEN and T2. The results showed that the key genes of toxin biosynthesis were highly correlated with the toxins produced by Fusarium, and the biosynthesis of toxin was strictly controlled by the genetic information of the strain. This study provides a data basis for the targeted prevention and control of exo-genous mycotoxins in P. heterophylla and a possibility for the development of PCR for rapid detection of toxin contamination.

Influence of iron and copper on the activity of laccases in Fusarium oxysporum f. sp. lycopersici

ABSTRACT Fusarium oxysporum f. sp. lycopersici is a phytopathogenic fungus that causes vascular wilt in tomato plants. In this work we analyze the influence of metal salts such as iron and copper sulphate, as well as that of bathophenanthrolinedisulfonic acid (iron chelator) and bathocuproinedisulfonic acid (copper chelator) on the activity of laccases in the intra (IF) and extracellular fractions (EF) of the wild-type and the non-pathogenic mutant strain (rho1::hyg) of F. oxysporum. The results show that laccase activity in the IF fraction of the wild and mutant strain increased with the addition of iron chelator (53.4 and 114.32%; respectively). With copper, it is observed that there is an inhibition of the activity with the addition of CuSO4 for the EF of the wild and mutant strain (reduction of 82 and 62.6%; respectively) and for the IF of the mutant strain (54.8%). With the copper chelator a less laccase activity in the IF of the mutant strain was observed (reduction of 53.9%). The results obtained suggest a different regulation of intracellular laccases in the mutant strain compared with the wild type in presence of CuSO4 and copper chelator which may be due to the mutation in the rho gene.

Diversidad genética de aislamientos de Fusarium graminearum sensu lato asociados con la fusariosis de la espiga en trigo de diversas localidades argentinas / Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina

La fusariosis de la espiga de trigo es una importante enfermedad para la región pampeana Argentina; Fusarium graminearum es el principal patógeno asociado. Se estudió el polimorfismo del ADN de un conjunto de aislamientos utilizando las técnicas de IGS-RFLP e ISSR. La técnica de IGS-RFLP produjo 41 bandas, 30 de ellas fueron polimórficas. El análisis de los ISSR mostró 87 bandas con 47 bandas polimórficas. La primera de estas metodologías fue más eficiente, ya que detectó mayor promedio polimórfico (59,91%) que la segunda (44,11%). Los valores promedio del contenido de información polimórfica (PIC) fueron 0,211 y 0,129, respectivamente. Se identificaron 20 haplotipos por IGS-RFLP, mientras que el análisis de los ISSR reveló 15 haplotipos. La agrupación de genotipos obtenida en ambos dendrogramas fue diferente. Los grupos genéticos obtenidos por la técnica de IGS-RFLP mostraron una asociación parcial con el origen geográfico. Este es el primer reporte que analiza la variabilidad genética en poblaciones de F. graminearum de trigo empleando marcadores IGS-RFLP e ISSR en Argentina

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers

Associação entre resiliência e qualidade de vida relacionada à saúde bucal em idosos / Association between resilience and quality of life related to oral health in the elderly

OBJETIVO: Avaliar a associação entre resiliência e qualidade de vida relacionada à saúde bucal, por meio de uma abordagem hierárquica baseada em um modelo teórico conceitual em uma coorte de idosos do Rio Grande do Sul. MÉTODOS: Foi conduzido um estudo transversal aninhado a um estudo de coorte, em 2008. Foram avaliados 498 idosos de Carlos Barbosa, Rio Grande do Sul. As medidas avaliadas foram sociodemográficas, comportamentos de saúde, qualidade de vida relacionada à saúde bucal, medida pelo Oral Health Impact Profile (OHIP-14), Escala de Resiliência e CPOD. A associação entre o potencial de resiliência e os impactos na percepção de saúde bucal relacionados à qualidade de vida foi verificada por meio de regressão binomial negativa. Razões das médias (RM) são apresentadas com seus intervalos de confiança de 95% (IC95%). RESULTADOS: Maiores médias do OHIP foram encontradas entre mulheres (6,7 ± 6,3; p = 0,011), moradores da zona rural (7,3 ± 6,7; p = 0,004) e solteiros (8,0 ± 6,3; p = 0,032). O modelo final da análise multivariada mostrou que ser morador da zona rural (RM = 1,32; IC95% 1,06 - 1,65) e casado (RM = 1,36; IC95% 1,07 - 1,72) foram variáveis independentemente associadas à qualidade de vida relacionada à saúde bucal. Não houve associação entre resiliência e qualidade de vida relacionada à saúde bucal. CONCLUSÃO: Os resultados sugerem que variáveis sociodemográficas estão associados à qualidade de vida relacionada à saúde bucal. A hipótese de que a resiliência pudesse exercer um papel importante no desfecho não foi confirmada. .

OBJECTIVE: To evaluate the association between psychological resilience and oral health related to quality of life through a hierarchical approach based on a conceptual theoretical model in a cohort of elderly residents in Rio Grande do Sul, Brazil. METHODS: We conducted a cross-sectional study nested in a cohort study in 2008. We evaluated 498 elderly residents in Carlos Barbosa, Rio Grande do Sul. The measures included sociodemographic questionnaire, health behavior, quality of life related to oral health (OHRQOL), measured by the Oral Health Impact Profile (OHIP-14), Resilience Scale, and DMFT. The association between resilience and potential impacts on perceptions of oral health-related quality of life was assessed through negative binomial regression. Mean ratios (MR) are presented with 95% confidence intervals (95%CI). RESULTS: Higher means of OHIP were found in women 6.7 ± 6.3; p = 0.011), in rural dwellers (7.3 ± 6.7; p = 0.004) and singles (8.0 ± 6.3; p = 0.032). The final model of multivariate analysis showed that being a rural dweller (MR = 1.32; 95%CI 1.06 - 1.65) and being married (MR = 1.36; 95%CI 1.07 - 1.72) were independently associated with OHRQOL. There was no association between resilience and OHRQOL. CONCLUSION: The results suggest that factors such as sociodemographic variables are associated with OHRQOL. The hypothesis that resilience might play a role in the outcome has not been confirmed. .

Influência de diferentes extratos aquosos de plantas medicinais no desenvolvimento de Colletotrichum gloeosporioides e de Fusarium moniliforme / Influence of different aqueous extracts of medicinal plants in the development of Colletotrichum gloeosporioides and Fusarium moniliforme

O objetivo deste trabalho foi avaliar a influência dos extratos aquosos das plantas medicinais alecrim, alho, cravo-da-índia, sálvia, capim-limão, orégano ou pimenta-do-reino no desenvolvimento in vitro de Colletotrichum gloeosporioides e de Fusarium moniliforme. Os extratos foram obtidos pela infusão de 60 g de cada planta medicinal em 200 mL de água fervente. Cada extrato aquoso foi fracionado em concentrações de 0, 5, 10 e 20% (p:v) e incorporado ao meio de cultivo BDA (batata-dextrose-ágar) antes da esterilização em autoclave. Posteriormente, um disco de 8 mm de diâmetro de micélio fúngico de cada patógeno foi transferido para o centro de placas de Petri. Após 24, 48 e 96 horas de incubação em câmara de crescimento a 22 ± 2 ºC e fotoperíodo de 12 horas avaliou-se o crescimento micelial de F. moniliforme e de C. gloesporioides. No último período de incubação, também se quantificou o número de conídios de cada fungo. Para o teste de germinação adicionou-se nas cavidades de placas de teste Elisa, uma alíquota de 40 µL de cada extrato nas concentrações de 0, 5, 10 e 20%, e outra alíquota, da suspensão de conídios de cada patógeno. Após 24 horas a 22 ± 2 ºC, no escuro, a germinação dos conídios foi paralisada com a adição de 20 µL de lactofenol; avaliou-se então a porcentagem de germinação de conídios. Os experimentos foram conduzidos no delineamento inteiramente casualizado em esquema fatorial 7 x 4 (extratos de plantas medicinais x concentrações) com quatro repetições. Para ambos os patógenos o extrato aquoso de alho e cravo-da-índia apresentaram maior ou total inibição do crescimento micelial, respectivamente, quando comparado com os demais extratos. Para C. gloeosporioides, o extrato de cravo-da-índia apresentou menor número de conídios em todas as concentrações testadas, e para o extrato de alho a 20%, também não foi observada a germinação de conídios. O extrato de alho foi eficiente em reduzir o número e a germinação dos conídios de F. moniliforme na concentração de 20%. Os extratos de alecrim, cravo-da-índia, orégano e pimenta-do-reino, nas maiores concentrações, tiveram efeito positivo na redução da produção de conídios deste mesmo fungo.

The objective of this study was to evaluate the influence of aqueous extracts of the medicinal plants rosemary, garlic, clove, sage, lemongrass, oregano and black pepper in the in vitro development of Colletotrichum gloeosporioides and Fusarium moniliforme. The extracts were obtained by infusing 60 g of each medicinal plant in 200 mL of boiling water. Each aqueous extract was fractionated in the concentrations of 0, 5, 10 and 20% (w:v) and incorporated into the PDA (potato dextrose agar) culture medium before sterilization by autoclaving. Later, an 8 mm diameter disc of each pathogen mycelium was transferred to the center of the Petri dishes. After 24, 48 and 96 hours of incubation in a growth chamber at 22 ± 2 ºC and a photoperiod of 12 hours, we evaluated the mycelial growth of F. moniliforme, and C. gloesporioides. In the last period of incubation, we quantified the production of conidia of each fungus. For the germination test, we added, into the wells of an ELISA test plates, a 40 µL aliquot of each extract at the concentrations of 0, 5, 10 and 20% and another aliquot of a suspension of conidia of each pathogen. After 24 hours at 22 ± 2 ºC in the dark, the germination of the fungi was stopped with the addition of 20 µL of lactophenol. Then, we evaluated the germination of conidia. The experiments followed a completely randomized 7 x 4 factorial design (medicinal plants x concentrations) with four replications. For both pathogens, the aqueous extract of garlic and clove showed a greater or total inhibition of the mycelial growth, when compared to the other extracts. For the C. gloeosporioides, the clove extract showed a lower number of conidia at all concentrations tested, and the garlic extract at 20% showed not conidial germination. The garlic extract was efficient to reduce the conidial number and germination of F. moniliforme at 20%. Extracts of rosemary, clove, oregano and black pepper, in the highest concentrations, had positive effect in reducing the production of spores of the same fungus.

Morphological and molecular characterization of Fusarium. solani and F. oxysporum associated with crown disease of oil palm

Crown disease (CD) is infecting oil palm in the early stages of the crop development. Previous studies showed that Fusarium species were commonly associated with CD. However, the identity of the species has not been resolved. This study was carried out to identify and characterize through morphological approaches and to determine the genetic diversity of the Fusarium species. 51 isolates (39%) of Fusarium solani and 40 isolates (31%) of Fusarium oxysporum were recovered from oil palm with typical CD symptoms collected from nine states in Malaysia, together with samples from Padang and Medan, Indonesia. Based on morphological characteristics, isolates in both Fusarium species were classified into two distinct morphotypes; Morphotypes I and II. Molecular characterization based on IGS-RFLP analysis produced 27 haplotypes among the F. solani isolates and 33 haplotypes for F. oxysporum isolates, which indicated high levels of intraspecific variations. From UPGMA cluster analysis, the isolates in both Fusarium species were divided into two main clusters with the percentage of similarity from 87% to 100% for F. solani, and 89% to 100% for F. oxysporum isolates, which was in accordance with the Morphotypes I and II. The results of the present study indicated that F. solani and F. oxysporum associated with CD of oil palm in Malaysia and Indonesia were highly variable.

Biochemical markers assisted screening of Fusarium wilt resistant Musa paradisiaca (L.) cv. Puttabale micropropagated clones.

An efficient protocol was standardized for screening of panama wilt resistant Musa paradisiaca cv. Puttabale clones, an endemic cultivar of Karnataka, India. The synergistic effect of 6-benzyleaminopurine (2 to 6 mg/L) and thidiazuron (0.1 to 0.5 mg/L) on MS medium provoked multiple shoot induction from the excised meristem. An average of 30.10 ± 5.95 shoots was produced per propagule at 4 mg/L 6-benzyleaminopurine and 0.3 mg/L thidiazuron concentrations. Elongation of shoots observed on 5 mg/L BAP augmented medium with a mean length of 8.38 ± 0.30 shoots per propagule. For screening of disease resistant clones, multiple shoot buds were mutated with 0.4% ethyl-methane-sulfonate and cultured on MS medium supplemented with Fusarium oxysporum f. sp. cubense (FOC) culture filtrate (5–15%). Two month old co-cultivated secondary hardened plants were used for screening of disease resistance against FOC by the determination of biochemical markers such as total phenol, phenylalanine ammonia lyase, oxidative enzymes like peroxidase, polyphenol oxidase, catalase and PR-proteins like chitinase, β-1-3 glucanase activities. The mutated clones of M. paradisiaca cv. Puttabale cultured on FOC culture filtrate showed significant increase in the levels of biochemical markers as an indicative of acquiring disease resistant characteristics to FOC wilt.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

Cloning of a novel xylanase gene from a newly isolated Fusarium sp. Q7-31 and its expression in Escherichia coli

A strain of Q7-31 was isolated from Qinghai-Tibet Plateau and was identified as Fusarium sp. based on its morphological characteristics and ITS rDNA gene sequence analysis. It has the highest capacity of degrading cell wall activity compared with other 11 strains. To do research on its xylanase activity of Fusarium sp. Q7-31 while the degrading the rice cell walls, the complete gene xyn8 that encodes endo-1, 4-¥â-xylanase secreted by Fusarium sp. Q7-31 was cloned and sequenced. The coding region of the gene is separated by two introns of 56bp and 55bp. It encodes 230 amino acid residues of a protein with a calculated molecular weight of 25.7 kDa. The animo acids sequence of xyn8 gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The nature peptide encodeing cDNA was subcloned into pGEX5x-1 expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expression fusion protein was identified by SDS-PAGE and Western blotting, a new specific band of about 52kDa was identified when induced by IPTG. Enzyme activity assay verified the recombinants proteins as a xylanase. A maxium activity of 2.34U/ mg, the xylanase had optimal activity at pH 6.0 and temperature 40¨¬C .

Detection of fumonisin producing Fusarium verticillioides in paddy (Oryza sativa L. ) using polymerase chain reaction (PCR) / Detecção de Fusarium verticillioides produtor de fumonisina em arroz empregando reação de polimerase em cadeia

The study reports the occurrence of fumonisin producing Fusarium verticillioides in 90 samples of stored paddy (Oryza sativa L.)collected from different geographical regions ofKarnataka, India. Fumonisin producing F. verticillioides was identified based on micromorphological characteristics and PCR using two sets of primers. One set of primers was F. verticillioides species specific, which selectively amplified the intergenic space region of rDNA. The other set of primers was specific to fumonisin producing F. verticillioides. Eight paddy samples were positive for F. verticillioides. Eleven isolates obtained from these samples were capable of producing fumonisin.

O estudo relata a ocorrência de Fusarium verticillioides produtor de fumonisina em 90 amostras de arroz armazenado, coletado de diferentes regiões geográficas de Karnataka, Índia. F. verticillioides produtor de fumonisina foi identificado baseado em características micromorfológicas e PCR empregando dois sets de primers. Um dos sets era F. verticillioides especie-específico, que amplificava seletivamente a região do espaço intergênico do rDNA. O outro set de primers era especifico para F. verticillioides produtor de fumonisina. Oito amostras de arroz foram positivas para F. verticillioides. Onze isolados obtidos dessas amostras foram produtores de fumonisina.

Mining microorganism EST databases in the quest for new proteins

Microorganisms with large genomes are commonly the subjects of single-round partial sequencing of cDNA, generating expressed sequence tags (ESTs). Usually there is a great distance between gene discovery by EST projects and submission of amino acid sequences to public databases. We analyzed the relationship between available ESTs and protein sequences and used the sequences available in the secondary database, clusters of orthologous groups (COG), to investigate ESTs from eight microorganisms of medical and/or economic relevance, selecting for candidate ESTs that may be further pursued for protein characterization. The organisms chosen were Paracoccidioides brasiliensis, Dictyostelium discoideum, Fusarium graminearum, Plasmodium yoelii, Magnaporthe grisea, Emericella nidulans, Chlamydomonas reinhardtii and Eimeria tenella, which have more than 10,000 ESTs available in dbEST. A total of 77,114 protein sequences from COG were used, corresponding to 3,201 distinct genes. At least 212 of these were capable of identifying candidate ESTs for further studies (E. tenella). This number was extended to over 700 candidate ESTs (C. reinhardtii, F. graminearum). Remarkably, even the organism that presents the highest number of ESTs corresponding to known proteins, P. yoelii, showed a considerable number of candidate ESTs for protein characterization (477). For some organisms, such as P. brasiliensis, M. grisea and F. graminearum, bioinformatics has allowed for automatic annotation of up to about 20 of the ESTs that did not correspond to proteins already characterized in the organism. In conclusion, 4093 ESTs from these eight organisms that are homologous to COG genes were selected as candidates for protein characterization

Production of mycotoxins by galactose oxidase producing "Fusarium" using different culture media

The original isolate of the galactose oxidase producing fungus "Dactylium dendroides, and other five galactose oxidase producing "Fusarium" isolates were cultivated in different media and conditions, in order to evaluate the production of 11 mycotoxins, wich are characteristic of the genus "Fusarium": moniliform, fisaric acid, deoxyvalenol, fusarenone-X, nivalenol, 3-acetyldeoxynivalenol, neosolaniol, zearakenol, zeralenone, acetyl T-2, and iso T-2. The toxicity of the culture extractes to "Artemia salina" larvae was tested