Phenolic compounds and antifungal activity of ethyl acetate extract and methanolic extract from Capsicum chinense Jacq. ripe fruit / Compostos fenólicos e atividade antifúngica dos extratos acetato de etila e metanólico dos frutos maduros de Capsicum chinense Jacq

Food loss due to contamination caused by fungi has much impact on agriculture and leads to significant economic losses. Synthetic and natural fungicides have been used for avoiding losses of several food products due to fungal contamination. As a result, species of the genus Capsicum have been used for preserving food because of their chemical compounds with antifungal activity. Therefore, this study aimed at identifying some phenolic compounds found in both ethyl acetate extract (EAE) and methanolic extract (ME) from habanero pepper (C. chinense) ripe fruit by liquid chromatography tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS) and at evaluating their antifungal activities against fungi Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides. Extracts resulted from a sequential process of maceration. Antifungal activity was evaluated by the disk diffusion method (DDM) at the following doses of both diluted extracts: 25 µL, 50 µL, 100 µL and 200 µL. The chemical analysis showed that there were protocatechuic acid, gentisic acid, vanillic acid, kaempferol-3-O-robinobiosideo and naringenin in both extracts. EAE showed high inhibition of mycelial growth at both doses 100µL and 200µL against the three fungi while methanolic exhibited weak activity even at the highest dose under investigation. However, further in-depth studies are needed to reinforce their uses and practical applications to the agricultural field.

As perdas de alimentos por contaminação causada por fungos são de grande impacto negativo para a agricultura, gerando altos prejuízos econômicos. Para evitar as perdas de diversos produtos alimentícios pela contaminação fúngica são utilizados fungicidas sintéticos e naturais. As espécies do gênero Capsicum são usadas há muitos anos para auxiliar na conservação de alimentos por possuírem substâncias químicas com ação antifúngica entre outras. Neste contexto, o objetivo deste estudo foi identificar alguns compostos fenólicos por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (LC-ESI-MS/MS) presentes nos extratos acetato de etila (EAE) e metanólico (ME) dos frutos maduros da pimenta biquinho (C. chinense) e avaliar atividade antifúngica de EAE e ME contra os fungos Sclerotinia sclerotiorum, Rhizopus stolonifer e Colletotrichum gloeosporioides. Os extratos foram obtidos de forma sequencial, utilizando o procedimento de maceração. A atividade antifúngica foi avaliada seguindo a metodologia de difusão em disco, nas doses de 25 µL, 50 µL, 100 µL e 200 µL de cada extrato diluído. A análise química evidenciou a presença de ácido protocatequico, ácido gentisico, ácido vanílico, kaempferol-3-O-robinobiosídeo e naringenina em ambos os extratos. EAE revelou maior poder de inibição do crescimento micelial nas doses de 100µL e 200µL contra os três fungos testados, enquanto ME exibiu fraca atividade inclusive na maior dose investigada. Entretanto, estudos mais aprofundados ainda são necessários para consolidar seu uso e aplicação prática na área agronômica.

Phenolic compounds and antifungal activity of ethyl acetate extract and methanolic extract from Capsicum chinense Jacq. ripe fruit

Abstract Food loss due to contamination caused by fungi has much impact on agriculture and leads to significant economic losses. Synthetic and natural fungicides have been used for avoiding losses of several food products due to fungal contamination. As a result, species of the genus Capsicum have been used for preserving food because of their chemical compounds with antifungal activity. Therefore, this study aimed at identifying some phenolic compounds found in both ethyl acetate extract (EAE) and methanolic extract (ME) from habanero pepper (C. chinense) ripe fruit by liquid chromatography tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS) and at evaluating their antifungal activities against fungi Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides. Extracts resulted from a sequential process of maceration. Antifungal activity was evaluated by the disk diffusion method (DDM) at the following doses of both diluted extracts: 25 µL, 50 µL, 100 µL and 200 µL. The chemical analysis showed that there were protocatechuic acid, gentisic acid, vanillic acid, kaempferol-3-O-robinobiosideo and naringenin in both extracts. EAE showed high inhibition of mycelial growth at both doses 100µL and 200µL against the three fungi while methanolic exhibited weak activity even at the highest dose under investigation. However, further in-depth studies are needed to reinforce their uses and practical applications to the agricultural field.

Resumo As perdas de alimentos por contaminação causada por fungos são de grande impacto negativo para a agricultura, gerando altos prejuízos econômicos. Para evitar as perdas de diversos produtos alimentícios pela contaminação fúngica são utilizados fungicidas sintéticos e naturais. As espécies do gênero Capsicum são usadas há muitos anos para auxiliar na conservação de alimentos por possuírem substâncias químicas com ação antifúngica entre outras. Neste contexto, o objetivo deste estudo foi identificar alguns compostos fenólicos por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (LC-ESI-MS/MS) presentes nos extratos acetato de etila (EAE) e metanólico (ME) dos frutos maduros da pimenta biquinho (C. chinense) e avaliar atividade antifúngica de EAE e ME contra os fungos Sclerotinia sclerotiorum, Rhizopus stolonifer e Colletotrichum gloeosporioides. Os extratos foram obtidos de forma sequencial, utilizando o procedimento de maceração. A atividade antifúngica foi avaliada seguindo a metodologia de difusão em disco, nas doses de 25 µL, 50 µL, 100 µL e 200 µL de cada extrato diluído. A análise química evidenciou a presença de ácido protocatequico, ácido gentisico, ácido vanílico, kaempferol-3-O-robinobiosídeo e naringenina em ambos os extratos. EAE revelou maior poder de inibição do crescimento micelial nas doses de 100µL e 200µL contra os três fungos testados, enquanto ME exibiu fraca atividade inclusive na maior dose investigada. Entretanto, estudos mais aprofundados ainda são necessários para consolidar seu uso e aplicação prática na área agronômica.

Resistance to diseases of sour, sweet and wild passion fruit genotypes

The cultivation of passion fruit is important for Brazil, since the country is currently the largest producer and consumer of fruit in the world. However, the fields of passion fruit still face important problems due to the incidence and severity of diseases in the field. Thus, the present study aimed to assess resistance to bacterial and fungal diseases in 13 genotypes of sour, sweet and wild passion fruit, in field conditions in the Distrito Federal, Brazil. For this, a field experiment was installed in a randomized block design, with four replications and 13 treatments (genotypes). The characteristics of incidence, severity and degree of resistance for bacteriosis, septoriosis, scab and anthracnose diseases were evaluated in 5 fruits per plot of each genotype. Genetic parameters of the evaluated traits were also estimated. High heritability values and CVg/Cve ratio were observed for most of the evaluated characteristics. The genotypes presented mean values of incidence and severity of bacteriosis, septoriosis, scab and anthracnose different among them, and the one that presented the best results in the degree of resistance for all diseases was F1 (MAR20 # 24 x ECL7 P1 R4).

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.

Antimicrobial activities of endophytic fungi residing in Aloe vera against diabetic wound pathogens

Aims@#Endophytic fungi are organisms that colonize living plant tissues without causing significant symptoms of diseases. They are known as the repository of bioactive compounds. This study was aimed to isolate endophytic fungi associated with Aloe vera and to assess their antimicrobial activity against the diabetic wound pathogens that result to lower limb amputations. @*Methodology and results@#In this study, 84 endophytic fungi were isolated from A. vera by using 5% sodium hypochlorite and 70% ethanol as surface sterilants. On preliminary antimicrobial screening by agar plug assay, 12 (14.3%) isolates showed significant antagonistic activity on the test microorganisms. Two of the endophytes were identified as Aspergillus flavus and Colletotrichum gloeosporioides through morphological and molecular techniques. The fungal cultures fermented in shake flask were extracted subsequently with hexane, dichloromethane, ethyl acetate, and n-butanol. Ethyl acetate and dichloromethane extracts of A. flavus and C. gloeosporioides inhibited all test bacteria. The compounds in the extracts were seemed to be semi-polar in nature. On broth microdilution assay, minimal inhibitory and minimal lethality concentration of ethyl acetate extracts of the fungi were found in the range of 0.63−2.50 and 1.25−5.00 mg/mL, respectively. @*Conclusion, significance and impact of study@#This study reveals that A. vera harbours a wide diversity of endophytic fungi. A. flavus and C. gloeosporioides showing broad spectrum antimicrobial activity on wound pathogens may be potential sources of bioactive compounds. Further investigations should be conducted to isolate and identify the antimicrobial compound produced by these fungal isolates.

Use of phosphites in postharvest to control anthracnose of yellow passionfruit / Uso de fosfitos em pós-colheita para o controle da antracnose do maracujá-amarelo

Brazil is the largest producer of yellow passionfruit (Passiflora edulis f. flavicarpa) and one of its production problems is the anthracnose (Colletotrichum spp.). The use of fungicides on control of postharvest diseases is a method that protects the fruits during storage. However, precautions must be taken due to fungicide toxicity. The restriction to the use of fungicides in post-harvest led a demand for alternative methods of disease control, and, the phosphite application is one of these methods. Therefore, this work aimed to evaluate the effects of fruit immersion in phosphite on postharvest control of anthracnose. Two tests were developed in vitro to assess the effect on the fungus: phosphite Mg2 (40%P2O5+6%Mg), Zn (40%P2O5+10%Zn), Ca1 (30%P2O5+7%Ca) and K1 (40%P2O5+20%K2O). For the in vivo tests, passionfruit (Gigante Amarelo), were wounded and inoculated (50L; 106conidia mL-1). Two tests were done with: Cu (25%P2O5+5%Cu), 2.5mL L-1; Zn, 2.5mL L-1; K1, 2.5mL L-1; Mg1 (30%P2O5+4%Mg), 3mL L-1; Ca1, 3mL L-1; Ca2 (10%P2O5+6%Ca), 4mL L-1; K2 (40%P2O5+20%K2O), 1.5mL L-1; Mg2 (40%P2O5+6%Mg), 1.5mL L-1; K3 (20%P2O5+20%K2O) 1.75 mL L-1; K4 (30%P2O5+20%K2O), 1.75mL L-1. Other two tests with phosphites Mg2, Ca1, Zn and K1 were with CaCl2 (2%) was developed. In addition, phosphites were tested at 25, 50, 100 and 200% of the dose: K2 (100%; 1.5mL L-1) and Ca1 (100%; 3 mL L-1). The phosphites Mg2, Ca1, K1 and Zn in vitro have reduced mycelial growth and fungus conidia production. The phosphites K1, K2, Ca1 and Zn were the ones that most reduced the size of the anthracnose lesion. There were no differences among treatments, concerning the physico-chemical fruit properties analyzed (% fresh mass loss, total soluble solids, pH and titratable acidity).

O Brasil é o maior produtor mundial de maracujá-amarelo (Passiflora edulis f. flavicarpa) e um dos problemas para sua produção é a antracnose (Colletotrichum spp.). O uso de fungicidas no controle de doenças pós-colheita é um método que protege os frutos durante o armazenamento, mas, precauções adicionais devem ser tomadas quanto à sua toxidade, presença de resíduos e a provável seleção de fungos resistentes. A restrição ao uso de fungicidas na pós-colheita cresceu e levou à procura de alternativas de controle, e, entre tais está à aplicação de fosfitos. Diante disso, este trabalho objetivou avaliar os efeitos da imersão de frutos em soluções de fosfitos no controle da antracnose em pós-colheita. Dois testes in vitro foram feitospara avaliar o efeito de fosfitono fungo: Mg2 (40%P2O5+6%Mg), Zn (40%P2O5+10%Zn), Ca1 (30%P2O5+7%Ca) e K1 (40%P2O5+20%K2O). In vivo, frutos de maracujá (Gigante Amarelo), foram feridos e inoculados (50l; 106conídios mL-1). Dois testes foram feitos com: Cu (25%P2O5+5%Cu), 2,5mL L-1; Zn, 2,5mL L-1; K1, 2,5mL L-1; Mg1 (30%P2O5+4%Mg), 3mL L-1; Ca1, 3mL L-1; Ca2 (10%P2O5+6%Ca), 4mL L-1; K2 (40%P2O5+20%K2O), 1,5mL L-1; Mg2 (40%P2O5+6%Mg), 1,5mL L-1; K3 (20%P2O5+20%K2O), 1,75mL L-1; K4 (30%P2O5+20%K2O), 1,75mL L-1. Outros dois testes com fosfitos foram com CaCl2 (2%) e Mg2, Ca1, Zn e K1. Ainda, dois fosfitos foram testados a 25, 50, 100 e 200% da dose: K2 (100%; 1,5mL L-1) e Ca1 (100%; 3mL L-1). Os fosfitos Mg2, Ca1, K1 e Zn in vitro reduziram o crescimento micelial e a produção de conídios do fungo. Os fosfitos K1, K2, Ca1 e Zn foram que mais reduziram o diâmetro da lesão causada pelo patógeno. Ca1 e K1 com CaCl2 reduziram o tamanho das lesões. Não houve diferenças significativas entre os tratamentos, quanto as características físico-químicas analisadas (% perda de massa fresca, teor de sólidos solúveis totais, pH e acidez titulável) dos frutos.

Identification and Characterizations of Pathogenic Fungal Species Associated with Symptoms of Cassava Anthracnose in Ivory Coast

Cassava anthracnose is a plant disease that affects cassava stems, petioles and fruits. The aim of this study was to analyze the diversity of cassava anthracnose symptoms in Ivory Coast and then to identify and characterize the associated fungal genera. Surveys were carried out in all agricultural zones of the country from July to November, in 2014, 2015, 2016 and 2017. Infected samples consisting of stems cut with a small number of superficial cankers (0.3%), distorted stems (25.77%), and necrotic stems and petioles (65.18%) were collected. Also, withered and dried apical buds (8.76%) were harvested. Fungal pathogens derived from samples were Colletotrichum gloeosporioides (35.08%), Fusarium sp. (27.19%) and Botrytis sp. (19.73%) genera and undetermined strains (17.98%). Genera were characterized by morphological and microscopic characteristics. Parasitic pressure increased to 80 and 100% respectively for Botrytis sp. genus and Colletotrichum gloeosporioides and Fusarium sp genera. Fungal genera have caused lesions on stem and petioles in green house with diameters sizes 46, 71 and 72 mm respectively for genera Botrytis sp, Fusarium sp and Colletotrichum gloeosporioides. Aggressiveness index of Botrytis sp. genus was 3 and 4 respectively for Colletotrichum gloeosporioides and Fusarium sp. genera. The mycoflora of cassava aerial organs alteration, linked to the symptoms of anthracnose, is composed of genera of great economic importance and scientific interest.

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.

Alternative Control of Plant Pathogen Fungi Through Ethanolic Extracts of Avocado Seeds (Persea Americana Mill.)

ABSTRACT The aim of this study was to evaluate the antifungal action of ethanolic extracts of avocado seeds, by conducting two experiments in the laboratory of plant pathology of EPAMIG SUL/Lavras, MG, Brazil, in a completely randomized design with three replicates. The first consisted of the following treatments ('Breda' 3%, 'Breda' 2%, 'Margarida' 3%, 'Margarida' 2%, Control, and Ethanol) testing its inhibitory effect on two fungal species (Colletotrichum gloeosporioides and Monilinia fructicola) and the second consisted of different concentrations of extracts (0%, 0.25%, 0.50%, 0.75%, 1.0%, 1.5%, and 2.0%) on the fungus F fructicola. The evaluations were performed at three times (7, 14, and 21 days of incubation) by measurements of the mycelial diameter using a digital caliper. The data were submitted to analysis of variance and the averages were compared by Scott-Knott test at 5% probability. The results demonstrated the positive potential of the ethanolic extracts of avocado seed on the mycelial development of fungi M. fructicola and C. gloeosporioides during the evaluated days.

Effects of Crude Toxins from Colletotrichum gloeosporioides on Growth of Dendrobium officinale Kimura et Migo / 广州中医药大学学报

To explore the effects of crude toxins from Colletotrichum gloeosporioides(C. gloeosporioides)on the growth of Dendrobium officinale Kimura et Migo(D. officinale),and to provide early basis for thescreening and cultivation of the resistant variants of C. gloeosporioides. Methods Seedlings of D. officinal werecultivated in MS medium added with different concentrations of the crude toxins from C. gloeosporioides. Theeffects of the crude toxin on the growth of seedlings were observed, and the optimum resistance -selectionthreshold was preliminarily screened. Results In the concentration range of 5％ - 15％(volume fraction),crudetoxins from C. gloeosporioides increased the plant height,stem diameter,number of new bud,root number,and fresh mass of D. officinale,among which the effect of 5％(volume fraction)of crude toxins was the moststrongest. In the concentration range of 35％ - 40％(volume fraction),crude toxins suppressed the plant height,leaf number,number of new bud,root number,and the fresh mass of D. officinale. When cultured with 20％(volume fraction)crude toxins, D. officinale was blooming, and the flowers appeared variation phenomenon.Conclusion The crude toxins from C. gloeosporioides have biological activity and certain toxicity,which can beconsidered as a selection agent instead of pathogenin in vitro to screen the resistant variant of D. officinale,butthe optimum resistance-selection threshold still needs further research.

Metabonomics study of interaction mechanism between Dendrobium officinale and Colletotrichum gloeosporioides based on GC-MS / 中草药

Objective To investigate the metabolites related to the course of anthracnosis of Dendrobium officinale and its possible mechanism based on the technique of metabonomics, so as to provide theoretical support for the creation or breeding of disease resistant varieties of D. officinale. Methods The normal and infected by Colletotrichum gloeosporioides leaves of D. officinale were collected respectively, and to find out the differential metabolites through the sample pretreatment, GC-MS analysis, and bioinformatics analysis. Then the differential metabolites and analysis of metabolic pathways involved in the course of disease were carried on the preliminary discussion. Results The multidimensional statistical models of each analysis group were successfully established. The dispersion points of VIP > 1.0 were selected as the potential differential materials, combined with P < 0.05 in the analysis of single dimensional Statistics (test) as the standard to verify. A total of 84 differential metabolites screened from 305 identified metabolites were considered to be pathogenesis related metabolites. A total of 34 differential metabolites were found out to be involved in the metabolic pathways through the pathway enrichment analysis, and the obtained ZC-GB metabolic pathways were of significance. Conclusion Based on GC-MS technology, the metabolomics analysis of D. officinale samples (normal/infected) was carried out and the metabolites related to the course of anthracnosis of D. officinale were found out. It could lay the foundation for studying the disease of D. officinale and cultivating resistant varieties on molecular level.

Caracterización química y evaluación de la actividad antifúngica del aceite esencial foliar de Lippia alba contra Colletotrichum gloeosporioides

El objetivo de este estudio fue caracterizar y evaluar in vitro la eficiencia del aceite esencial foliar de Lippia alba contra Colletotrichum gloeosporioides. Se extrajo el aceite esencial a partir de las hojas mediante el método de hidrodestilación asistida por microondas (MWHD) y se identificaron sus metabolitos mediante una cromatografía de gases acoplada a espectrometría de masas (GC/MS). La evaluación in vitro se realizó empleando 4 concentraciones del aceite esencial (500, 1000, 3000 y 10000 ppm) diluidas en acetona; se utilizó un control positivo con Benomil 1 g/L, un control negativo con acetona y un testigo absoluto; la eficiencia del aceite se midió mediante el porcentaje de índice antifúngico (%I.A). El componente mayoritario en el aceite esencial fue el citral (34.62 a 40.03%) y que el mayor %I.A se encontró a la concentración de 10000 ppm (97.8%), muy similar a la del Benomil (100%). Lo anterior demostraría la eficacia del aceite esencial de L. alba para controlar a C. gloeosporioides, y su posible uso como fungicida biológico.

The aim of this study was to characterize and evaluate the in vitro efficiency of leaf essential oil of Lippia alba against Colletotrichum gloeosporioides. Essential oil from the leaves was extracted by the method of assisted microwave hydrodistillation (MWHD) and metabolites were identified by gas chromatography coupled to mass spectrometry (GC/ MS). The in vitro evaluation was performed using 4 concentrations of essential oil (500, 1000, 3000 and 10000 ppm) diluted in acetone; positive control with Benomyl 1 g /L, a negative control with acetone and absolute control was used; oil efficiency was measured by the percentage of antifungal index (% IA). It was found that the major component was in the essential oil citral (34.62 to 40.03%) and the highest %I.A was found to 10000 ppm concentration (97.8%) very similar to Benomyl (100%). This would demonstrate the efficacy of the L. alba essential oil to control C. gloeosporioides, and its possible use as a biological fungicide.

Development of a High-Resolution Multi-Locus Microsatellite Typing Method for Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is an economically important fungal pathogen causing substantial yield losses indifferent host plants. To understand the genetic diversity and molecular epidemiology of this fungus, we have developed a novel, high-resolution multi-locus microsatellite typing (MLMT) method. Bioinformatic analysis of C. gloeosporioides unannotated genome sequence yielded eight potential microsatellite loci, of which five, CG1 (GT)(n), CG2 (GT1)(n), CG3 (TC)(n), CG4 (CT)(n), and CG5 (CT1)(n) were selected for further study based on their universal amplification potential, reproducibility, and repeat number polymorphism. The selected microsatellites were used to analyze 31 strains of C. gloeosporioides isolated from 20 different host plants from India. All microsatellite loci were found to be polymorphic, and the approximate fragment sizes of microsatellite loci CG1, CG2, CG3, CG4, and CG5 were in ranges of 213–241, 197–227, 231–265, 209–275, and 132–188, respectively. Among the 31 isolates, 55 different genotypes were identified. The Simpson's index of diversity (D) values for the individual locus ranged from 0.79 to 0.92, with the D value of all combined five microsatellite loci being 0.99. Microsatellite data analysis revealed that isolates from Ocimum sanctum, Capsicum annuum (chili pepper), and Mangifera indica (mango) formed distinct clusters, therefore exhibited some level of correlation between certain genotypes and host. The developed MLMT method would be a powerful tool for studying the genetic diversity and any possible genotype-host correlation in C. gloeosporioides.

Production of secondary metabolite E2.2 from Phaleria macrocarpa endophytic fungus

Objective To isolate new endophytic fungus from Phaleria macrocarpa (P. macrocarpa) that is able to produce E2.2 compound. Methods Endophytic fungi were isolated from P. macrocarpa. Morphological and molecular identification was done to determine the species of the endophytic fungus. High performance liquid chromatography was used to determine the ability of this fungus to produce E2.2 compound and to quantify the total yield of E2.2 from fungal fermentation. Fermentation process was optimized by observing suitable medium, pH and length of fermentation process. Phloroglucinol and gallic acid addition were examined to determine the effect of each compound on E2.2 production. Results One endophytic fungus was successfully isolated from P. macrocarpa plant. Morphological and molecular identification showed that it was a Colletotrichum gloeosporioides which belonged to Glomerellaceae family. This fungus showed highest production of E2.2 when incubated in potato dextrose broth with initial pH value of the medium at 5, and was incubated for 15 days. Phloroglucinol was found to better enhance E2.2 production. Conclusions Colletotrichum gloeosporioides found in P. macrocarpa plant is promising as a potential alternative source of E2.2.

Production of secondary metabolite E2.2 from Phaleria macrocarpa endophytic fungus

Objective: To isolate new endophytic fungus from Phaleria macrocarpa (P. macrocarpa) that is able to produce E2.2 compound. Methods: Endophytic fungi were isolated from P. macrocarpa. Morphological and molecular identification was done to determine the species of the endophytic fungus. High performance liquid chromatography was used to determine the ability of this fungus to produce E2.2 compound and to quantify the total yield of E2.2 from fungal fermen-tation. Fermentation process was optimized by observing suitable medium, pH and length of fermentation process. Phloroglucinol and gallic acid addition were examined to determine the effect of each compound on E2.2 production. Results: One endophytic fungus was successfully isolated from P. macrocarpa plant. Morphological and molecular identification showed that it was a Colletotrichum gloeo-sporioides which belonged to Glomerellaceae family. This fungus showed highest pro-duction of E2.2 when incubated in potato dextrose broth with initial pH value of the medium at 5, and was incubated for 15 days. Phloroglucinol was found to better enhance E2.2 production. Conclusions: Colletotrichum gloeosporioides found in P. macrocarpa plant is prom-ising as a potential alternative source of E2.2.

Feohifomicosis causada por Colletotrichum gloeosporioides y Alternaria infectoria en un paciente trasplantado renal / Phaeohyphomycosis caused by Colletotrichum gloeosporioides and Alternaría infectoria in renal transplant recipient

Several species of black fungi have been reported as agents of subcutaneous phaeohyphomycosis. Although most of these fungi are considered opportunistic pathogens, they play an important role in phaeohyphomycosis, a disease considered an emergent mycosis among solid organ recipients. We report a case of phaeohyphomycosis caused by Alternaria infectoria of the left hand and the 4th finger of the right hand of a 68-year-old male who underwent a renal transplant 35 months before. The lesion was treated with surgical excision. One year later, the patient presented a new lesion on the 5th finger of the right hand, but this time caused by Colletotrichum gloeosporioides that was also removed surgically. Both lesions did not relapse after being removed. Antifungal susceptibility testing was performed against five antifungal drugs (amphotericin B, itraconazole, flucytosine, fluconazole and voriconazole). Alternaria infectoria was resistant to all five drugs and C. gloeosporioides was sensitive only to amphotericin B and voriconazole. We emphasize the need of histopathologic and microbiologic studies of new lesions of phaeohyphomycosis, since in this case the same patient was infected twice by two different fungi.

Diversas especies de hongos negros han sido descritos como agentes de feohifomicosis subcutáneas. A pesar que la gran mayoría de estos se consideran como oportunistas, juegan un papel importante en la feohifomicosis, enfermedad considerada una micosis emergente en pacientes trasplantados de órganos sólidos. Reportamos un caso de feohifomicosis subcutánea causada por Alternaria infectoria en el dorso de la mano izquierda y 4° falange de la mano derecha de un paciente de sexo masculino de 68 años de edad, que había sido sometido a un trasplante de riñón 35 meses antes. Se realizó escisión quirúrgica de la lesión. Después de un año, el paciente presentó una nueva lesión, esta vez en la 5° falange de la mano derecha causada por Colletotrichum gloeosporioides, lesión que también fue removida quirúrgicamente. El paciente no presentó nuevas lesiones después de las cirugías. Las pruebas de susceptibilidad antifúngica fueron realizados utilizando cinco antifúngicos (anfotericina B, itraconazol, 5-fluorcitosina, fluconazol y voriconazol). Alternaria infectoria presentó resistencia a los cinco antifíngicos y C. gloeosporioides fue susceptible solamente a anfotericina B y voriconazol. Enfatizamos la necesidad de realizar estudios histopatológicas y microbiológicos de nuevas lesiones de feohifomicosis, ya que el mismo paciente fue infectado por dos hongos diferentes.

Reação de progênies de maracujazeiro azedo à antracnose em condições de casa de vegetação / Reaction of passion fruit progenies to the antracnosis under greenhouse conditions

Neste trabalho objetivou-se avaliar e selecionar progênies de maracujazeiro- azedo com resistência à antracnose em condições de casa de vegetação. O experimento foi conduzido na estação experimental da biologia da Universidade de Brasília. Foram utilizadas sementes (propagação sexuada) coletadas no campo da fazenda Água Limpa. As progênies usadas nesse experimento foram: seleção MAR, 20#01, 20#05, 20#12, 20#15, 20#19, 20#21, 20#36, 20#40 e 20#2005, PL1, PL2, PL3, PL4, PL5, PL7, GA2, BRS Gigante Amarelo, MSC, BRS Sol do Cerrado, RC3, Roxo Australiano, AR01 e FB100. O delineamento experimental foi em blocos casualizados com parcela subdividida com 24 tratamentos (progênies), 4 repetições e 6 plantas úteis por repetição. A progênie MAR20#19 apresentou as menores incidências na folha, planta e severidade, 89,2%, 87,5% e 3,71, respectivamente. A progênie MAR20#19 comportou-se como moderadamente resistente e as outras progênies como altamente susceptíveis.

In this work, the objective was to evaluate and select progenies of passion fruit with resistance to anthracnose disease under conditions of a greenhouse. The experiment was conducted at the experimental station of biology of the University of Brasília. It was used seeds (sexual propagation) collected from the farm Água Limpa field water. The progenies used in this experiment were the selections MAR, 20#01, 20#05, 20#12, 20#15, 20#19, 20#21, 20#36, 20#40, 20#2005, PL1, PL2, PL3 , PL4, PL5, PL7, GA2, BRS Gigante Amarelo, MSCA, BRS Sol do Cerrado, RC3, Purple Australian, AR01 and FB100. The experimental design was randomized block with split plot with 24 treatments (progenies) 4 replicates and 6 plants per repetition. The progeny mar20#19 had the lowest incidences leaf, plant and severity, 89.2%, 87.5% and 3.71, respectively. The progeny mar20#19 was observed as moderately resistant and the other progenies as highly susceptible.

Reação em progênies de maracujá-azedo à antracnose em condições de campo / Passionfruit progenies reaction to anthracnosis in field

O presente trabalho objetivou avaliar a reação de progênies de maracujá-azedo à antracnose em condições de campo. Para tanto foi utilizado o delineamento em blocos casualizados com quatro repetições, em arranjo de parcela subdividida com 14 tratamentos e 8 plantas por parcela. Foram avaliadas, em quatro diferentes épocas (dezembro, janeiro, fevereiro e março), as progênies: MAR 20#36, MAR 20#09, MAR 20#03, MAR 20#23, MAR 20#46, GA2, AR 02, AR 01, FB 200, AP1, RC3, PCF-2, EC-RAM e FP 01. Utilizaram-se 10 frutos escolhidos ao acaso durante a colheita das 14 progênies, levando em conta a incidência e severidade da doença. O grau de resistência ao fungo Colletotrichum gloeosporioides foi avaliado utilizando uma escala de nota 1 a 4. Houve diferença significativa na avaliação da severidade e da incidência à antracnose entre as quatro épocas de avaliações. Foi apresentado nas épocas de Dezembro e Janeiro maior incidência da doença, diferindo significativamente das outras duas épocas. A maior severidade foi apresentada na época 2, com 1,29% da superfície dos frutos coberta com lesões, diferindo significativamente das outras três épocas de avaliação. Foi observada interação entre época e progênies quanto à severidade à antracnose apenas na época 2, com as progênies A09, MAR 20#23 e MAR 20#03. Na interação progênies dentro das épocas, FB 200 foi significativo na época 1, apresentando maior severidade. As progênies A09 e MAR 20#23 foram significativos na época 2. Na interação entre época dentro de progênie quanto à incidência, as progênies MAR 20#36, RC 3, MAR 20#23, AP 1 e MAR 20#46, diferiram significativamente tanto na época 1, quanto na época 2. Todas as progênies avaliadas foram consideradas moderadamente susceptíveis.

The purpose of this study was to evaluate the reaction of passionfruit progenies to anthracnosis in field. Randomized blocks with four repetitions, 14 treatments, eight plants per plot were managed in the field. The progenies MAR 20#36, MAR 20#09, MAR 20#03, MAR 20#23, MAR 20#46, GA2, AR 02, AR 01, FB 200, AP1, RC3, PCF-2, EC-RAM and FP 01 were evaluated in four different periods (December, January, February and March). Ten fruits per plot were randomly chosen during the harvest of the 14 genotypes, and the incidence and the severity of the diseases were considered. The resistance degree to the fungus Colletotrichum gloeosporioides was evaluated according to a note scale from 1 to 4. There was significant difference in the severity and incidence of anthracnosis among the four evaluation periods. The highest incidence of the disease occured in periods 1 and 2. The highest severity was in period 2, covering 1.29% of the surface of the wounded fruits. There was interaction between period and the progenies to anthracnosis only in period 2, embracing the progenies A09, MAR 20#23 and MAR 20#03. In the progenies interaction in the periods, FB 200 showed highest and significant severity in period 1. The progenies A09 and MAR 20#23 showed significance in period 2. The progenies MAR 20#36, RC 3, MAR 20#23, AP 1 and MAR 20#46 differed in incidence in periods 1 and 2. All progenies evaluated were considered partially susceptible.

Composição química e atividade in vitro de três óleos essenciais sobre Colletotrichum gloeosporioides do maracujazeiro / Chemical composition and in vitro activity of three essential oils on Colletotrichum gloeosporioides from passion fruit

Objetivou-se avaliar a ação de diferentes concentrações de óleos essenciais de três plantas medicinais sobre o crescimento micelial, a produção e a germinação de conídios de Colletotrichum gloeosporioides (Penz.). Utilizou-se os óleos essenciais de Lippia sidoides Cham., Cymbopogon citratus (DC) Stapf.) e Ocimum gratissimum L. em cinco concentrações (0 µL mL-1, 1 µL mL-1, 3 µL mL-1, 5 µL mL-1 e 7 µL mL-1) com 4 repetições no delineamento inteiramente casualizado, em esquema fatorial 5x3. Os óleos foram analisados por cromatografia gasosa acoplada com espectrometria de massas. Para todas as características verificou-se efeito significativo da interação "Óleo" versus "Concentração". O óleo essencial de C. citratus proporcionou a maior inibição do crescimento micelial em todas as concentrações testadas. Os outros óleos não se diferenciaram, exceto a partir da concentração de 5 µL mL-1, onde o óleo de L. sidoides foi superior ao óleo de O. gratissimum. Na produção e germinação de conídios, o óleo de C. citratus, foi superior aos óleos de L. sidoides e de O. gratissimum nas concentrações de 1 µL mL-1 e 3 µL mL-1, inibindo completamente a produção e germinação a partir da concentração de 3 µL mL-1. As concentrações influenciam em todas as variáveis, ajustando-se à equação do modelo linear. Observa-se o decréscimo nessas, à medida em que se aumentam as concentrações dos óleos testados. Os óleos essenciais testados possuem efeito fungitóxico e fungistático.

The objective of this study was to evaluate the action of different concentrations of essential oils of three medicinal plants on the mycelial growth, production and conidial germination of Colletotrichum gloeosporioides (Penz.). We used a completely randomized design, in a 5x3 factorial design, being five concentrations (0 µL mL-1, 1 µL mL-1, 3 µL mL-1, 5 µL mL-1 e 7 µL mL-1) x three oils from Lippia sidoides Cham., Cymbopogon citratus (DC) Stapf. and Ocimum gratissimum L., with four replications. The oils were analyzed by gas chromatography with mass spectrometry. There was a significant interaction of concentration versus species on all analyzed traits. The essential oil from C. citratus yielded the highest mycelial growth inhibition at all concentrations. The other oils were not differentiated, except at the concentration of 5µL mL-1, in which the oil of L. sidoides was more efficient that the oil of O. gratissimum. In conidial production and germination, the oil of C. citratus was superior to the oil of L. sidoides and O. gratissimum at the concentrations of 1 µL mL-1 and 3 µL mL-1, as it completely inhibited the production and germination of conidia at the concentrations of 3 µL mL-1 and above. The concentrations influenced all the variables, adjusting themselves to the linear model equation. We observed a decrease in the variables, as the concentrations of the oils tested are increased. The essential oils used have antifungal and fungicidal effect.

Antifungal activity using medicinal plant extracts against pathogens of coffee tree / Atividade antifúngica utilizando extratos de plantas medicinais contra fitopatógenos do cafeeiro

Generally, the medicinal plants have antifungal substances that can be used for the plant protection against phytopathogens. The objective of this study was to know the efficiency of aqueous extracts from medicinal plants against the major etiological agents of coffee tree. The aqueous extracts used were extracted from bulbs of Allium sativum, leaves of Vernonia polysphaera, Cymbopogon citratus, Cymbopogon nardus, Cordia verbenacea, Eucalyptus citriodora, Ricinus communis, Azadirachta indica, Piper hispidinervum and flower buds of Syzygium aromaticum. The etiological agents considered for this study were Cercospora coffeicola, Colletotrichum gloeosporioides, Fusarium oxysporum, Phoma tarda, Rhizoctonia solani and Hemileia vastatrix. The screening for harmful extracts was done based on mycelial growth and conidial germination inhibition. All experiments performed were in vitro conditions. The inhibition of mycelial growth was performed mixing the extracts with the PDA. This mixture was poured in Petri dishes. On the center of the dishes was added one PDA disc with mycelium. It was incubated in a chamber set to 25ºC. The evaluation was done daily by measuring the mycelial growth. The germination assessment was also performed with Petri dishes containing agar-water medium at 2%. These were incubated at 25ºC for 24 hours. After this period the interruption of germination was performed using lactoglycerol. The experiments were conducted in a completely randomized design. The most effective plant extracts against the micelial growth and conidial germination were V. polysphaera, S. aromaticum and A. sativum.

Geralmente, as plantas medicinais têm substâncias antifúngicas que podem ser utilizadas para a proteção das plantas contra fitopatógenos. O objetivo deste estudo foi conhecer a eficiência de extratos aquosos de plantas medicinais contra os principais agentes etiológicos do cafeeiro. Os extratos aquosos utilizados foram extraídos de bulbos de Allium sativum, folhas de Vernonia polysphaera, Cymbopogon citratus, Cymbopogon nardus, Cordia verbenacea, Eucalyptus citriodora, Ricinus communis, Azadirachta indica, Piper hispidinervum e botões florais de Syzygium aromaticum. Os agentes etiológicos considerados neste estudo foram Cercospora coffeicola, Colletotrichum gloeosporioides, Fusarium oxysporum, Phoma tarda, Rhizoctonia solani e Hemileia vastatrix. A triagem dos extratos foi realizada com base no crescimento micelial e na inibição da germinação de conídios. Todos os experimentos foram realizados em condições in vitro. A inibição do crescimento micelial foi realizada misturando-se os extratos com PDA. Esta mistura foi vertida em placas de Petri. No centro das placas foi adicionado um disco de PDA com micélio. Incubou-se em câmara programada para 25°C. A avaliação foi feita diariamente através da medição do crescimento micelial. O experimento sobre a germinação também foi realizado com placas com meio ágar-água a 2%. Estas foram incubadas durante 24 horas. Após este período, a interrupção da germinação foi realizada utilizando lactoglicerol. Os experimentos foram conduzidos em delineamento inteiramente casualizado. Os extratos de plantas mais eficazes contra o crescimento micelial e germinação de conídios foram V. polysphaera, S. aromaticum e A. sativum.