Infección grave de piel y partes blandas por Fusarium oxysporum asociado a la administración subcutánea de insulina / Severe skin and soft tissue infection due to Fusarium oxysporum associated with subcutaneous insulin administration

Resumen La diabetes mellitus (DM) es una enfermedad crónica muy prevalente. Dentro de los tratamientos para la DM se encuentra la insulina que es el agente antidiabético más potente, sin embargo, una proporción significativa de pacientes no logra alcanzar el objetivo de hemoglobina glicosilada (HbA1c). Los errores en la aplicación de insulina son un factor importante y corregible en muchos casos. Se presenta el caso de una paciente con DM, antecedentes de neuropatía diabética, enfermedad renal crónica estadio V en hemodiálisis, hipertensión arterial, estenosis aórtica con recambio por válvula protésica, y anticoagulada, con escasa adherencia a recomendaciones higiénico dietéticas. Debido a la mala técnica de aplicación de insulina y falta de higiene, desarrolló varias infecciones polimicrobianas de piel y partes blandas, con evolución tórpida de las úlceras y mala respuesta al tratamiento indicado. Durante su internación, de una úlcera se aisló Fusarium oxysporum. Es importante jerarquizar la relevancia de la educación diabetológica en pacientes insulinizados y el rol de los educadores en diabetes en el cuidado de los mismos. Por otro lado, destacar la importancia de la toma de cultivos mediante punción de partes blandas ante la aparición de signos locales de infección.

Abstract Diabetes mellitus (DM) is a very prevalent chronic disease. Among the treatments for DM, insulin is the most potent antidiabetic agent. However a significant proportion of patients fail to achieve Errors in the application of insulin are an important and correctable factor in many cases. We present the case of a patient with DM who, due to poor insulin application technique and hygiene, develops a skin and soft tissue infection with subsequent appearance of Fusarium oxysporum. It is important to emphasize the relevance of diabetes education in insulin ized patients and the role of diabetes educators in their care. On the other hand, it is important to emphasize the importance of taking cultures by soft tissue puncture in case of local signs of infection.

Screening of natural drug molecules against Fusarium oxysporum of ginseng root rot based on machine learning / 药学学报

italic>Fusarium oxysporum widely exists in farmland soil and is one of the main pathogenic fungi of root rot, which seriously affects the growth and development of plants and often causes serious losses of cash crops. In order to screen out natural compounds that inhibit the activity of Fusarium oxysporum more economically and efficiently, random forest, support vector machine and artificial neural network based on machine learning algorithms were constructed using the information of known inhibitory compounds in ChEMBL database in this study. And the antibacterial activity of the screened drugs was verified thereafter. The results showed that the prediction accuracy of the three models reached 77.58%, 83.03% and 81.21%, respectively. Based on the inhibition experiment, the best inhibition effect (MIC = 0.312 5 mg·mL-1) of ononin was verified. The virtual screening method proposed in this study provides ideas for the development and creation of new pesticides derived from natural products, and the screened ononin is expected to be a potential lead compound for the development of novel inhibitors of Fusarium oxysporum.

Analysis of Fusarium oxysporum infection on changes of Astragalus metabolism by metabolomic approach / 药学学报

italic>Astragalus is a commonly used Chinese medicinal material in traditional Chinese medicine (TCM), and with the increase of planting area in recent years, the damage of Astragalus root rot has worsened year by year, which seriously affecting its quality and yield. Fusarium oxysporum is one of the main pathogens causing root rot in astragalus. In this study, UPLC-Q-TOF-MS based metabolomic approach combined with multivariate statistical analysis were used to analyze the metabolite changes of Astragalus in response to F. oxysporum infection. The results showed that 62 metabolites in the Astragalus had significant changes after inoculation of F. oxysporum. Polar metabolites included 40 flavonoids, 8 saponins, 2 nucleosides, 1 vitamin, 1 organic acid, 1 amino acid; while lipid metabolites included 3 fatty acids, 1 diradylglycerols, 2 lysophosphatidylcholine, 1 lysophosphatidylglycerol, 1 phosphatidylinositol, 1 sterol lipid. Among these differential metabolites, the relative content of flavonoids, vitamin B2, tryptophan and salicylic acid were increased, while the relative content of saponins were decreased. Correlation analysis showed that the flavonoids were positively correlated with each other, and positively correlated with most lipids, but negatively correlated with most saponins. In addition, studies have shown that F. oxysporum infection is not an influencing factor for the generation of malonyl substitution of flavonoid. This study elucidates the effect of F. oxysporum infection on Astragalus from the perspective of plant metabolism, which provides a basis for exploring the interaction mechanism between the Astragalus and F. oxysporum and further promoting molecular breeding.

Effects of temperature and humidity on infection of Fusarium oxysporum in seedlings of Salvia miltiorrhiza / 中国中药杂志

Wilt disease is a major disease of cultivated Salvia miltiorrhiza, which is caused by Fusarium oxysporum. Since the infection process of F. oxysporum in plants is affected by environment factors, this study was conducted to reveal the relationship between disease severity and concentration of the pathogen in plants in the infection process of F. oxysporum in seedlings of S. miltiorrhiza by pot experiments and to reveal the effects of temperature and humidity on the infection process. The results showed that, after inoculation of S. miltiorrhiza seedlings with F. oxysporum, the pathogen in different parts was detected at different time, and it was first detected in substrates. With the continuous propagation of the pathogen(4-5 d), it gradually infected the roots and stems of the seedlings, and the plants had yellowing leaves and withering. The number of the pathogen reached the maximum in each part after 7-8 d, and then gradually decreased in the later stage of the disease. The concentration of the pathogen in substrates, roots and stems of S. miltiorrhiza showed a trend of decreasing after increasing with the aggravation of the disease and reached the maximum in the samples of moderate morbidity, while the concentration in the samples of severe morbidity decreased. In addition, the infection of F. oxysporum in seedlings of S. miltiorrhiza was affected by temperature and humidity. The suitable temperature was 25-30 ℃ and the suitable humidity was 80%-90%. This study could provide guidance for the experiments on pathogenicity of F. oxysporum, screening of biocontrol bacteria and controlling of wilt.

Identification and analysis of AP2/ERF gene family of Panax notoginseng and function of PnDREB84 gene / 药学学报

The AP2/ERF gene family is one of the largest transcription factor families in the plant kingdom, and plays an important role in response to biological and abiotic stresses, plant hormone responses, and plant growth and development. In this study, the AP2/ERF family of Panax notoginseng was identified by bioinformatics methods, and the physicochemical properties, structure, phylogenetic relationship, expression pattern and function of PnDREB4 gene of the family were analyzed. The results showed that 140 AP2/ERF family members were identified in P. notoginseng, which were divided into DREB, ERF, AP2, RAV and Sololit subgroups. The physicochemical properties and motifs of proteins were similar among the subgroups. There were 34 differentially expressed genes in the AP2/ERF family of Fusarium oxysporum infected P. notoginseng plants, and 19 genes were up-regulated. The expression level of PnDREB84 was up-regulated with the extension of Fusarium oxysporum infection time in the range of 0-96 h. The content of ABA and SA in P. notoginseng plants overexpressing PnDREB84 gene increased after 4 ℃ stress. The results showed that PnDREB84 gene plays a dual regulatory role in the process of biological stress and abiotic stress. PnDREB84 gene can be used as a potential molecular marker for the breeding of new varieties of P. notoginseng. The identification of AP2/ERF transcription factor and function analysis of PnDREB84 gene of P. notoginseng provided data support for the analysis of stress resistance mechanism of P. notoginseng and the breeding of new varieties.

Síntesis y actividad antifúngica in vitro frente a Fusarium oxysporum de amidas N-alquilsustituidas derivadas de 2-aminoácidos

Una serie de amidas N-alquilsustituidas 1-16 fueron sintetizadas a partir de malonato de dietilo y ésteres de alquilo derivados de los aminoácidos L-triptófano, L-alanina, L-fenilalanina y L-tirosina. Los métodos de síntesis empleados involucraron calentamiento por irradiación de microondas empleando tanto un ácido de Lewis (AlCl3) o 4-dimetilaminopiridina (DMAP) como catalizador y auxiliar nucleofílico, respectivamente. Los resultados sugieren que el uso de irradiación de microondas y de DMAP conlleva mejores rendimientos en un tiempo de reacción más corto. Para ilustrar las diferencias observadas, se presentan las propuestas mecanísticas de cada método de reacción para la formación de amidas N-alquilsustituidas. Finalmente, las amidas sintetizadas se evaluaron en condiciones in vitro frente a Fusarium oxysporum; mostraron actividad antifúngica a diferentes niveles (0,40 mM < IC50 < 29,1 mM), lo cual indicó que las variaciones de la actividad observada de este grupo de compuestos pueden deberse al efecto de la amida acíclica como bioisóstero no clásico de algunas fitoalexinas heterocíclicas.

N-alkyl substituted amides 1-16 were synthesized from diethyl malonate and alkyl esters derived from the amino acids L -tryptophan, L -alanine, L -phenylalanine, and L -tyrosine. In addition, a microwave-assisted protocol was employed using a Lewis acid (AlCl3) or dimethylaminopyridine (DMAP) as a catalyst and nucleophilic auxiliary, respectively, affording the desired compounds. The results suggest that DMAP-catalyzed reactions under microwave irradiation yield higher during short reaction times. Each reaction method's mechanistic proposals for forming N-alkyl-substituted amides are presented to illustrate the observed differences. The synthesized amides were evaluated under in vitro conditions against Fusarium oxysporum. The compounds exhibited antifungal activity at different levels (0.40 mM < IC50 < 29.1 mM). These results indicated that the observed activity variations of this compound group might be due to the effect of acyclic amide as a non-classical bioisostere of some heterocyclic phytoalexins.

Uma série de amidas N-alquil substituídas foram sintetizadas a partir de malonato de dietila e ésteres alquílicos derivados dos aminoácidos ʟ-triptofano, L -alanina, L-fenilalanina e L-tirosina. Os métodos de síntese empregados foram realizados usando aquecimento por irradiação de micro-ondas empregando um ácido de Lewis (AlCl3) ou dimetilaminopiridina (DMAP) como catalisador. Os resultados sugerem que a irradiação de micro-ondas usando DMAP leva a melhores rendimentos em um tempo de reação mais curto. Para ilustrar as diferenças observadas, são apresentadas as propostas mecanísticas de cada método de reação para a formação de amidas N-alquilsubstituídas. Finalmente, as amidas sintetizadas foram avaliadas in vitro contra Fusarium oxysporum, mostrando atividade antifúngica em diferentes níveis (0.40 mM < IC50 < 29.1 mM), o que indica que as variações observadas na atividade desse grupo de compostos podem ser devidas ao efeito de amida acíclica como um bioisóstero não clássico de algumas fitoalexinas heterocíclicas.

EFFECT OF DIFFERENT NITROGEN SOURCES ON THE GROWTH OF FUSARIUM OXYSPORUM F. SP. CORIANDRII CAUSING WILT OF CORIANDER

Nitrogen is one of the most important and essential nutrition required for the proper growth and development of pathogen. It is an important component required for protein synthesis and other vital functions. In the present study investigation has made on the effect of different nitrogen sources (sodium nitrate, ammonium nitrate, potassium nitrate and calcium nitrate) on the mycelial growth of Fusarium oxysporum f. sp. coriandrii causing wilt of Coriander. For this study wild sensitive and highly resistant isolate were selected. It was observed that, there was variation in the mycelial growth of the sensitive and resistant isolate on the different Nitrogen sources. However, Sodium nitrate was found to be the best source of nitrogen. Resistant isolate always showed highest growth as compared to the sensitive isolate. This research work helps to manage the growth of pathogen.

Extraction and analysis of apoplastic phenolic metabolites in carnation roots and stems (Dianthus caryo-phyllus L) / Extracción y análisis de metabolitos fenólicos apoplásticos en raíz y tallo de clavel (Dianthus caryophyllus L) / Extração e análise do meta-bólitos fenólicos apoplásticos na raiz e caule do craveiro (Dianthus caryophyllus L)

Abstract The present study outlines the conditioning of various parameters for the efficient removal of the apoplastic fraction of carnation enriched in polar compounds, mainly phenolics. Several studies can apply the described workflow to different plant species in the particular or global analysis of those metabolites in this peripheral extracellular space. Hence, using carnation (Dianthus caryophyllus L) roots and stems, we evaluated different infiltration solutions for removing apoplastic metabolites. The best outcome was obtained by using the buffer solution NaH2PO4-Na2HPO4 0.1 M pH 6.5/NaCl 50 mM, since the highest amount of apoplastic phenolic metabolites can be obtained with the slightest contamination of intracellular compounds. The metabolites were separated using HPLC-DAD-ESI-MS, affording chromatographic profiles with reasonable quality parameters based on resolution, selectivity, and number of theoretical plates. It was possible to identify eight differential compounds (one flavone and seven flavonols), whose core moieties consisted of (iso)pratol, kaempferide, (dihydro)kaempferol, quercetin, and myricetin-type flavonoids according to the test organ and the cultivar. We deduced that identified flavonoids are related to phytoanticipin-type metabolites in carnation, such as hydroxy-methoxyflavone, di-o-benzoylquercetin, and kaempferide disalicyloylrhamnoside, which are abundantly present in the resistant cultivar. The conditions described in this work are fundamental for delving into the role of apoplastic phenolic metabolites related to the defense mechanisms of this ornamental plant.

Resumen En el presente estudio se describe el acondicionamiento de algunos parámetros con fines de obtención eficiente de extractos apoplásticos enriquecidos en compuestos polares, principalmente fenólicos. Este flujo de trabajo descrito, incluso, puede ser aplicado a diferentes especies vegetales para ser empleado en el análisis particular o global de metabolitos en este espacio extracelular periférico. Para ello, usando raíces y tallos de clavel (Dianthus cariophyllus L), se evaluaron diferentes soluciones de infiltración para la extracción de los metabolitos apoplásticos. El mejor resultado se logró con la disolución amortiguadora NaH2PO4-Na2HPO4 0,1 M pH 6,5/NaCl 50 mM, porque se obtiene la mayor cantidad de metabolitos fenólicos apoplásticos, con la menor contaminación de compuestos intracelulares. Los metabolitos se separaron mediante HPLC-DAD-ESI-MS, obteniendo perfiles cromatográficos con parámetros de calidad razonables basados en resolución, selectividad y número de platos teóricos. Con estas condiciones, fue posible identificar ocho compuestos diferenciales (una flavona y siete flavonoles), cuyas estructuras básicas comprendían flavonoides del tipo (iso)pratol, kaempférido, (dihidro) kaempferol, quercetina y miricetina, según el órgano de prueba y la variedad. Los flavonoides identificados están relacionados con metabolitos de tipo fitoanticipina en el clavel, como hidroxi-metoxiflavona, di-o-benzoilquercetina y kaempférido disaliciloilrhamnósido, abundantemente presentes en la variedad resistente. Las condiciones descritas en este trabajo son fundamentales para profundizar en el papel de los metabolitos fenólicos apoplásticos relacionados con los mecanismos de defensa de esta planta ornamental.

Resumo O presente estudo descreve o condicionamento de alguns parâmetros para a obtenção eficiente de extratos apoplásticos enriquecidos em compostos polares, principalmente fenólicos. Este fluxo de trabalho descrito pode até mesmo ser aplicado a diferentes espécies de plantas para serem usadas na análise particular ou global de metabólitos neste espaço extracelular periférico. Para isso, utilizando raízes e caules de craveiro (Dianthus cariophyllus L), diferentes soluções de infiltração foram avaliadas para a extração de metabólitos apoplásticos. O melhor resultado foi obtido com a solução tampão NaH2PO4-Na2HPO40 0.1 M pH 6.5/NaCl 50 mM, pois a maior quantidade de metabólitos fenólicos apoplásticos é extraída. Os metabólitos foram separados por HPLC-DAD-ESI-MS, obtendo-se perfis cromatográficos com parâmetros de qualidade razoáveis baseados na resolução, seletividade e número de pratos teóricos. Nessas condições, foi possível identificar oito compostos diferenciais (uma flavona e sete flavonóis), cujas estruturas básicas compreendem flavonóides do tipo (iso) pratol, kaempferida, (dihidro)kaempferol, quercetina e miricetina, de acordo com o órgão e a variedade de craveiro utilizado. Os flavonóides identificados estão relacionados a metabólitos do tipo fitoanticipinas no craveiro, como hidroxi-metoxiflavona, di-O-benzoilquercetina e kaempférido disaliciloilramnósido, abundantemente ocorridos na cultivar resistente. As condições descritas neste trabalho são essenciais para se aprofundar no papel dos metabólitos fenólicos apoplásticos relacionados aos mecanismos de defesa dessa planta ornamental.

HPLC-DAD analysis, antifungal and antioxidant activity of Solanum dolichosepalum bitter extracts and fractions

Abstract Solanum dolichosepalum is a plant with anti-infective effects. It is a healing agent and has ethnopharmacological uses. In this study, the antifungal activity of extracts and fractions of this species on C. albicans and F. oxysporum was evaluated. The antioxidant activity was measured using the ABTS and DPPH methods, and by determining the total content of phenolic compounds. An HPLC-DAD qualitative analysis was carried out to identify phenolic compounds and alkaloids. Pearson's correlation coefficients were calculated. Inhibitory effects were found in all the extracts and fractions on the analyzed microorganisms. F. oxysporum was the microorganism most sensitive to the action of S. dolichosepalum extracts. All extracts and fractions showed antioxidant activity, with the acetone extract and the acetone fraction being those that generated the best results. The content of total phenolic compounds showed that acetone has a greater affinity with the phenolic compounds present in S. dolichosepalum. In this plant, p-Hydroxybenzoic, vanillic, ferulic, trans-cinnamic, caffeic, p-coumaric, and rosmarinic acids were found, as well as theobromine, quercetin, and luteolin. The content of total phenolic compounds was determined to be directly proportional to the inhibition of the ABTS and DPPH radicals, and the inhibition of the analyzed microorganisms. It was determined that the extracts and fractions obtained from S. dolichosepalum show antioxidant and antifungal activity.

Antagonismo de rizobacterias sobre hongos fitopatógenos, y su actividad microbiana con potencial biofertilizante, bioestimulante y biocontrolador / Antagonism of rhizobacteria on phytopathogenic fungi, and their microbial activity with biofertilizer, biostimulant and biocontroller potential

RESUMEN Las rizobacterias forman parte de la gran cantidad de microorganismos que actúan como agentes de biocontrol, produciendo metabolitos que inducen resistencia sistémica en las plantas que inhiben el crecimiento de patógenos. El objetivo de esta investigación fue evaluar la capacidad de diez rizobacterias de los géneros Rhizobium, Bradyrhizobium, Sinorhizobium, Ochrobactrum y Pseudomonas para producir ácido cianhídrico (HCN), sideróforos y ácido indol-acético (AIA), disolver fosfato, fijar nitrógeno e inhibir el crecimiento de fitopatógenos. Se realizaron todas las pruebas fisiológicas y bioquímicas correspondientes, así como la prueba de antagonismo in vitro contra los fitopatógenos Fusarium oxysporum, Colletotrichum gloeosporioides y Rhizoctonia solani. Cinco cepas produjeron una mayor cantidad de AIA en relación a las otras en presencia de triptófano, la cepa ES1 (Ochrobactrum sp.) produjo HCN, el 50 % de las cepas evaluadas liberaron sideróforos, el 60 % disolvió fósforo, y todas resultaron positivas para la fijación de nitrógeno. Nueve cepas inhibieron el crecimiento de F. oxysporum entre 40 % y 65 %, la cepa Alf (Pseudomonas fluorescens) inhibió además el crecimiento de C. gloeosporioides en un 22 %, y ninguna inhibió el crecimiento de R. solani. Los rizobios evaluados y la cepa de Pseudomonas fluorescens podrían ejercer efectos beneficiosos sobre las plantas a través de mecanismos directos e indirectos, o una combinación de ambos, lo que las convierte en una opción sostenible para la producción de cultivos.

ABSTRACT Rhizobacteria are part of the large number of microorganisms that act as biocontrol agents, producing metabolites that induce systemic resistance in plants and inhibit the growth of pathogens. The objective of this research was to evaluate the capacity of ten rhizobacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium, Ochrobactrum and Pseudomonas to produce hydrogen cyanide (HCN), siderophores and indole acetic acid (IAA), dissolve phosphate, fix nitrogen and inhibit the growth of phytopathogens. All the corresponding physiological and biochemical tests were carried out, in addition to an in vitro antagonism test against the phytopathogens Fusarium oxysporum, Colletotrichum gloeosporioides and Rhizoctonia solani. Five strains produced a greater amount of IAA with respect to the others in the presence of tryptophan, the strain ES1 (Ochrobactrum sp.) produced HCN, 50% of the evaluated strains released siderophores, 60% solubilized phosphorus and all were positive for nitrogen fixation. Nine strains inhibited the growth of F. oxysporum by 40% to 65%. The Alf strain (Pseudomonas fluorescens) inhibited the growth of C. gloeosporioides by 22% while none inhibited the growth of R. solani. The rhizobia tested and the Pseudomonas fluorescens strain may have favorable effects on plants through direct and indirect mechanisms, or a combination of both, making them a sustainable option for crop production.

Microbial synthesis of magnetite nanoparticles for arsenic removal

Apart from their vast applications, the magnetic property of magnetite nanoparticles make it as opt candidatefor the removal of arsenic from drinking water and other polluted water streams. Magnetite nanoparticleswere produced by microbial synthesis from Fusarium oxysporum with Ferric Chloride and magnetite ore assubstrates. The structure and morphology of magnetite nanoparticles were characterized by Fourier TransformInfrared (FTIR) Spectroscopy, UV-Vis spectroscopy, X-ray Diffraction (XRD), Scanning Electron Microscope(SEM), and their magnetic properties were characterized Vibrating Sample Magnetometer. The presence ofmagnetite nanoparticles was indicated by the XRD spectral pattern and their SEM micrograph showed in thenano ranged particles with an average diameter of 26.78 nm. Magnetic characteristic of magnetite nanoparticleswas indicated super paramagnetic properties with a saturation magnetic value of 90.01 emug−1. These magnetitenanoparticles were used to remove the arsenic in the water by simple magnetic adsorption process and theremoval efficiency was found to be 96%.

Antagonistic activity of volatile metabolites from Trichoderma asperellum / 生物工程学报

Trichoderma spp. is a kind of filamentous fungi with important biocontrol value. Twelve strains of Trichoderma spp. were isolated from the soils of different types of crops in Shaoxing, Zhejiang and Foshan, Guangdong. The antagonistic resistance to Fusarium oxysporum was compared by plate confrontation test. The further analysis of volatile secondary metabolites for two strains were carried out using HS-SPME-GC-MS analysis. The results showed that T. asperellum ZJSX5003 and GDFS1009 had fast growth ability, and the inhibition effects on F. oxysporum were 73% and 74% respectively. Six identical volatile metabolites were detected as follows 2-Methyl-1-propanol, 3-Methyl-1-butanol, 3-Methyl-3-buten-1-ol, Acetyl methyl carbinol, Butane-2,3-diol and 6-n-pentyl-2H-pyran-2-one (6-PAP). Among them, 6-PAP was validated to have a higher inhibitory effect on F. oxysporum in vitro. This study will provide basis for the development of biocontrol agents with metabolites of Trichoderma, such as 6-PAP.

Selección de genes de referencia para análisis transcripcionales en el modelo clavel (Dianthus Caryophyllus L.) - Fusarium oxysporum f. sp. Dianthi / Selection of reference genes for transcriptional analysis in the pathosystem carnation (Dianthus Caryophyllus L.) - Fusarium oxysporum f. sp. dianthi / Seleção de genes de referência para análise de transcrição no modelo cravo (Dianthus Caryophyllus L.) - Fusarium oxysporum f. sp. dianthi

Resumen En el presente trabajo se seleccionaron y validaron genes de referencia para estudios transcripcionales en el modelo clavel -Fusarium oxysporum f. sp. dianthi. Para ello, se seleccionaron genes asociados a procesos básicos celulares que han sido usados como genes de referencia en otros modelos planta-patógeno y se determinó el efecto de la inoculación del patógeno sobre su expresión. Se realizó un diseño de cebadores para los diferentes genes candidatos con el fin de verificar tanto su presencia en el genoma de claveles cultivados en Colombia, como su transcripción constitutiva en los diferentes tejidos por medio de la técnica de transcripción reversa y posterior reacción en cadena de la polimerasa (RT-PCR por sus siglas en ingles). Posteriormente, se evaluaron los niveles transcripcionales de los genes candidatos usando RT-qPCR en tallos y raíces de dos variedades con diferentes niveles de resistencia a la enfermedad, que fueron inoculados con este patógeno. La validación estadística realizada, usando ANOVA y los programas GeNorm y Normfinder, determinó que los genes codificantes para una histona H3 y el ARNr18S no presentan variación en sus niveles de expresión por efecto de la inoculación, permitiendo su uso como genes de referencia en estudios transcripcionales en esta interacción planta-patógeno.

Abstract In this research, reference genes were validated for transcriptional studies in the pathosystem carnation - Fusarium oxysporum f. sp. dianthi. Genes associated with basic cellular processes that have been used as reference genes in other plant-pathogen models were selected and the effect of the inoculation with the pathogen on their expression was determined. For this, it was necessary to design primers for each candidate genes to check their presence in the genome of carnations grown in Colombia and their constitutive transcription in different tissues of the plant using reverse transcription subsequent polymerase chain reaction (RT-PCR). The transcriptional levels of the candidate genes were evaluated using RT-qPCR in stems and roots inoculated with this pathogen for two cultivars with different levels of resistance to the disease. According to the statistical validation carried out using ANOVA and the GeNorm and Normfinder programs, the genes coding for a histone H3 and rRNA18S did not show any significant variation in their expression levels due to inoculation, so they can be used as reference genes in transcriptional studies in this plant-pathogen interaction.

Resumo Neste trabalho foram validados genes de referencia para estudos de transcrição no modelo cravo - Fusarium oxysporum f. sp. dianthi. Genes associados a processos básicos celulares, anteriormente usados como genes de referencia noutros modelos planta-patógeno, foram selecionados e se determinou o efeito, sobre a sua expressão, que apresentava a inoculação com o patógeno. Foi realizado um desenho de iniciadores para os diferentes genes candidatos para verificou a sua presença no genoma de cravos cultivados na Colômbia, e uma transcrição constitutiva em diferentes tecidos da planta com a técnica de transcrição reversa e posterior reação em cadeia da polimerase (RT-PCR). Em seguida, os níveis de transcrição dos genes candidatos foram avaliados por RT-qPCR em caules e raízes de duas variedades com diferentes níveis de resistência à doença, que tinham sido inoculados com o patógeno. A validação estatística realizada por ANOVA y com os programas GeNorm y Normfinder, permitiram determinar que os genes que codificam para uma histona H3 y para o ARNr 18S, não apresentam variação nos seus níveis de expressão por efeito da inoculação, sugerindo que podem ser usados como genes de referencia em estudos de transcrição nesta interação planta-patógeno.

Screening of plant growth promoting rhizobacteria for antifungal activity against Fusarium oxysporum

Aim: New species of Plant Growth Promoting Rhizobacteria (PGPR), with varying growth promoting and biocontrol ability are often being discovered. They facilitate plant growth either directly by secreting nutrients and hormones or indirectly by providing defence mechanism to the plant. The present study was undertaken to isolate PGPR from the rhizosphere of Solanum lycopersicum and Arachis hypogaea, and test their growth promoting ability and antifungal activity against Fusarium oxysporum. Methodology: PGPRs were isolated from the rhizosphere of S. lycopersicum and A. hypogaea by serial dilution of the rhizospheric soil and identified by 16s rDNA sequencing. The isolates were analysed for antifungal activity against F. oxysporum, indole 3-acetic acid (IAA) production and phosphate solubilisation. For the growth promotion assay, aseptically grown Vigna radiata seedlings were dipped separately in isolated bacterial suspension of PGPR (109 CFU ml-1) and planted in autoclaved soil. Plants were irrigated with 50% Hoagland solution for every 48 hr and maintained at 25 ± 2 °C with 16/8 hr of light and dark photoperiod. Growth promotion was examined in terms of differences in shoot length, root length, fresh weight and dry weight after 12 days of treatment. Results: Six isolates were found to have antifungal activity towards plant pathogen, F. oxysporum. Five isolates showed similarity to Pseudomonas aeruginosa (B7-1, B11-5, B3-1, Rh-1, Rh-2) and one to Pseudomonas putida (B53). All six strains were able to produce IAA, where B53 and B13-1 showed the highest production compared to other strains. P. putida B53 demonstrated the highest plant growth promotion activity by significantly (p<0.05) increasing the growth of V. radiata plants as evidenced by increase in shoot length, root length, fresh and dry weight. Interpretation: The results obtained from the present study supports that PGPRs like Pseudomonas sp. could serve as potential eco-friendly bio-fertilizer and bio- fungicide

Trichoderma species diversity in rhizosphere soils and potential antagonism with Fusarium oxysporum / Diversidade de espécies de trichoderma em solos de rizosfera e potencial antagonismo com fusarium oxysporum

Neste estudo foram isoladas seis espécies de trichoderma isoladas de solos rizosféricos de arrozais, bananeiras, dendezeiros, seringueiras, hortaliças e pastagens. As espécies são t. harzianum, t. viride, t. koningii, t. asperellum, and t. parareesei. O estudo morfológico como pigmentação, crescimento de colônias e estudos anatômicos como aparências de conidiação, tamanho de conídios, padrão de ramificação dos conidióforos, formas de phialides, ausência ou presença de clamidósporos foram realizados para identificar as espécies de trichoderma. As espécies de trichoderma harzianum foram abundantes no solo enquanto as de t. viren foram a segunda mais frequente no solo. Todas as espécies apresentaram atividade antagônica contra o fusarium oxysporum. Enquanto t. parareesei apresentou a maior atividade antagônica de 91,10% contra f.oxysporum, relatado como melhor agente antagonista para fitopatógeno.

In this study, six species of trichoderma isolated from rhizospheric soils of rice fields, banana trees, oil palm trees, rubber trees, vegetables and pastures were isolated. The species are t. harzianum, t. viride, t. koningii, t. asperellum, and t. I will stop. Morphological studies such as pigmentation, colony growth and anatomical studies such as appearance of conidia, size of conidia, branching pattern of conidiophores, forms of phialides, absence or presence of chlamydospores were performed to identify the species of trichoderma. The species of trichoderma harzianum were abundant in the soil while those of t. viren were the second most frequent in the soil. All species showed antagonistic activity against fusarium oxysporum. While t. parareesei presented the greatest antagonistic activity of 91.10% against f.oxysporum, reported as the best antagonistic agent for phytopathogen.

Isolation and Characterisation of Endophytic Strain Paenibacillus polymyxa SR19 from Urtica dioica and the Study of Their Effect against Fusarium oxysporum f. sp Tomato

In an attempt to obtain biological control agents for fusariose wilt, a total of 54 endophytic bacterial strains were isolated from the root of plants Urtica dioica and screened for in vitro antagonist activity against Fusarium oxysporum. Among the 54 bacterial isolates, 27 isolates exhibited more than 60% inhibition of mycelia growth of Fusarium oxysporum. The strain R19 which exhibited the most obvious antagonistic activity was selected for greenhouse studies. The SR19 had no pathogenicity and was identified as Paenibacillus polymyxa based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence. Growth chamber studies resulted in statistically significant increases in inoculating tomato seedling with the endophytic strain SR19 which in turn resulted in improving plant seedling stand by 32% and increasing fresh weights of root and fresh weight of aerial biomass of plants over the untreated pathogen control by 6.95 g and 7.96 g, respectively. Strain SR19 is a potential biological control agent that may contribute to the protection of tomato plants against fusariose wilt.

Isolation and Biochemical Characterisation of Endophytic Bacillus spp from Urtica dioica and Study of Their Antagonistic Effect against Phytopathogens

Endophytic bacteria have been isolated from the roots of Urtica dioica. A total of 54 endophytic bacteria were isolated from the underground parts using suitable surface sterilisation protocol. Three isolates R45a; R45b; R21a were tested for antagonism effect against Fusarium oxysporum, Colletotrichum gloeosporioides, Rhizoctonia solani, Phytophthora parasitica in dual culture method. Significant inhibitory effects on mecylial radial growth have been revealed with a percentage superior or equal to 75%. These strains were Gram-positive rods. Cultures on nutrient agar showed irregular, entirely cream coloured colonies that are strictly aerobic and capable of forming endospore. They belong probably to the genus of Bacillus spp.

Secondary metabolites from endophytic fungus Fusarium oxysporum isolated from Paeonia ostii / 中草药

Objective To study the secondary metabolites of endophytic fungus Fusarium oxysporum form Paeonia ostii. Methods The fermentation liquor of the fungal strain F. oxysporum were isolated and purified using various chromatographic methods. The structures of the compounds were identified by spectrum analysis. Results Twenty-three compounds were isolated from the fungal and their structures were identified as (2S)-proline (1), (22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol (2), cyclo-(S-Pro-S-Leu) (3), cyclo-(L-Ala-L-Pro) (4), cyclo-(Val-Pro) (5), cyclo-(R-Pro-S-Phe) (6), cyclo-(D-cis-Hyp-L-Phe) (7), cyclo-(trans-4-hydroxy- L-Pro-L-Phe) (8), cyclo-(Gla-Tyr) (9), cyclo-(trans-4-hydroxy-L-Pro-L-Leu) (10), L,L-cyclo-(Trp-Pro) (11), cyclo-(L-Leu-Gly) (12), N-(3-(1H-indol-3-yl)propyl)acetamide (13), indole-3-acetic acid (14), 2-piperidone (15), 2-pyrrolidinone (16), thymidine (17), (5S)-5-[(4-hydroxyphenyl)methyl]-2,4-imidazolidinedione (18), (3S,6S)-3-(butan-2-yl)-6-(1-hydroxyethyl) piperazine-2,5-dione (19), cyclo-(Ala-Leu) (20), cyclo-(S-Pro-S-Phe) (21), cyclo-(Tyr-Pro) (22), and cyclo-(L-Phe-L-Tyr) (23). Conclusion With the exception of compounds 2, 6, and 14, the other compounds are isolated from the fermentation liquor of the fungal strain F. oxysporum for the first time.

Antagonistic activity of Streptomyces thermocarboxydus to Fusarium oxysporum: The cause of leaf rot disease on Aloe Vera (Aloe barbadensis Mill.) in Bali, Indonesia

@#Aims: Aloe vera (Aloe barbadensis Miller) has been cultured in Bali, Indonesia since 2006. Its cultivation area is 170 haincluding 5 regencies i.e. Buleleng, Karangasem, Bangli, Badung and Gianyar. This study was conducted to isolate andidentify the Streptomyces sp. that potentially can be used to inhibit the growth of F. oxysporum and control the leaf rotdisease on aloe vera with and to study the structural responses of F. oxysporum to the treatment of Streptomycesculture filtrate.Methodology and results: Samples were collected from Serokadan, Kerobokan, and Saba, Bali, Indonesia. The isolateof Streptomyces sp. that resulted in the highest antifungal activity was further observed on its morphological andultrastructure characteristics using SEM and TEM. Identification was done by using 16S rRNA sequencing techniques. Agreenhouse experiment was conducted to evaluate the effectiveness of the filtrate of Streptomyces sp. to control the leafrot disease on aloe vera. The Streptomyces GYRRK was identified to be S. thermocarboxydus and the filtrate inhibitedthe growth of F. oxysporum by damaging cell wall and plasma membrane of macro conidia cell, micro conidia, andhypha. Treatment with the filtrate of S. thermocarboxydus with four sprays (one spray equal to 0.5 mL) over inoculatedleaves of aloe vera reduced the leaf rot disease by 68%.Conclusion, significance and impact of study: This result suggests that filtrate of S. thermocarboxydus potentiallycan be used as an alternative control agent against leaf rot disease on aloe vera in Bali.

Condiciones para el análisis electrofóretico de proteínas apoplásticas de tallos y raíces de clavel (Dianthus caryophyllus L.) para estudios proteómicos / Electrophoretic conditions for analysis of apoplastic proteins in stems and roots of carnation (Dianthus caryophyllus L.) for proteomic studies / Condições para a análise eletroforética de proteínas apoplásticas de caules e raízes de cravo (Dianthus caryophyllus L.) para estudos proteômicos

Resumen En el presente estudio se describe un flujo de trabajo que puede ser aplicado a diferentes especies vegetales, con el fin de obtener extractos apoplásticos que puedan ser usados para análisis proteómicos. Para ello, usando tallos y raíces de clavel, se evaluaron parámetros claves para la extracción de estas proteínas. Se determinó que para esta especie (Dianthus caryophyllus L.) se debe usar la solución amortiguadora fosfato de sodio 0,1 M pH 6,5, cloruro de sodio 50 mM y 0,1% β-mercaptoetanol para la infiltración; con tres tiempos de vacío de 20 s a 70 kPa y centrifugación a 1000 x g durante 20 min a 4 °C, seguido de precipitación y concentración de proteínas con el método Ácido tricloroacético/acetona. Bajo estas condiciones, se obtienen extractos que permiten análisis electroforéticos en 2D de proteínas de apoplasto, usando para el isolectroenfoque tiras con gradientes de pH 5-8 para raíz y pH 3-10 para tallo. Las condiciones descritas permitirán profundizar sobre el papel de las proteínas apoplásticas en diversidad de fenómenos biológicos que involucren esta especie vegetal.

Abstract In the present study a workflow, that can be applied to different plant species, to obtain a high quality apoplastic extract and for proteomic analysis is described. For that, using carnation roots and stems, some important parameters for the extraction of these proteins were evaluated. The best conditions for the infiltration were provided by using a 0.1 M sodium phosphate buffer pH 6.5, 50 mM sodium chloride and β -mercaptoethanol 0.1%, with 3 vacuum times at 70 kPa for 20 s and centrifuged at 4°C at 1,000 g for 20 min, and then a subsequent protein precipitation and concentration with the Trichloroacetic acid/acetone protocol. These conditions can be used to obtain a good quality extract for 2D electrophoretic analysis of apoplastic proteins. Strips with pH gradients between 5-8 and 3-10 were used to run isoelectric analysis for extracts obtained from apoplast in roots and stems, respectively. The conditions described can be used to perform studies focused on apoplastic proteins and its role in biological phenomena involving this plant species.

Resumo No presente estudo, é descrito um fluxo de trabalho que pode ser aplicado a espécies vegetais diferentes, para obter extratos apoplásticos que podem ser utilizados para análise proteômico. Para isto, utilizando os caules e as raízes do cravo, foram avaliados parâmetros considerados chave para a extração destas proteínas. Foi determinado que para esta espécie, deve ser utilizada uma solução tampão de fosfato de sódio 0,1 M pH 6,5 com cloreto de sódio 50 mM e 0,1% β-mercaptoetanol para a infiltração; com três aplicações de vácuo de 20 s a 70 kPa e centrifugação a 1000 x g por 20 minutos a 4°C, e subsequente precipitação e concentração de proteínas usando Ácido ricloroacético/acetona. Nestas condições se podem obter extratos que permitem a análise eletroforética 2D de proteínas do apoplasto utilizando para a focalização isoeléctrica tiras com gradientes de pH 5-8 para a raiz e de pH 3-10 para o caule. As condições descritas permitirão aprofundar sobre o papel das proteínas apoplásticas numa diversidade de fenômenos biológicos que envolvem esta espécie de planta.