Distribución espacial de antracnosis (Colletotrichum gloeosporioides Penz) en aguacate en el Estado de México, México / Spatial distribution of anthracnose (Colletotrichum gloeosporioides Penz) in avocado in the State of Mexico, Mexico

Resumen El aguacate (Persea americana) es una especie cuyo cultivo es de gran importancia nutricional y económica para México; sin embargo, como cualquier otro cultivo, a menudo se ve afectado por plagas y enfermedades que limitan su comercialización a nivel mundial. El hongo fitopatógeno Colletotrichum gloeosporioides es el agente causal de la antracnosis en el aguacate y se manifiesta en las etapas tempranas del desarrollo del fruto, así como en poscosecha y durante el almacenamiento, en condiciones de alta humedad relativa (80%) y temperaturas desde los 20 ◦C. Las pérdidas económicas a causa de este hongo pueden ser de hasta el 20% de la producción. En el presente estudio se aplicaron métodos geoestadísticos para definir la distribución espacial de antracnosis en frutos de aguacate cultivar Hass en cuatro municipios del Estado de México, durante el periodo de enero a junio de 2017. La distribución de la antracnosis se ajustó a modelos gaussianos y exponenciales en la mayoría de los casos. Los mapas de infestación realizados mediante krigeado muestran más de un centro de agregación de la enfermedad. Este análisis permitió estimar la superficie infestada: se encontró una infestación de más del 50% en los primeros muestreos y de hasta un 98% en los muestreos de junio en todas las zonas estudiadas. © 2019 Publicado por Elsevier Espana, S.L.U. en nombre de Asociacion Argentina de Microbiologıa. Este es un art´ıculo Open Access bajo la licencia CC BY-NC-ND (http://creativecommons. org/licenses/by-nc-nd/4.0/).

Abstract Persea americana is a species of great nutritional and economic importance for Mexico, however, like any other agricultural crop, it is affected by pests and diseases that limit its worldwide commercialization. The phytopathogenic fungus Colletotrichum gloeosporioides is the causative agent of anthracnose in avocado and manifests itself in the early stages of fruit development as well as in post-harvest and storage, under conditions of high relative humidity (80%) and at temperatures from 20°C, causing losses economic up to 20% of production. Applying geostatistical methods the present study aims to define the spatial distribution of anthracnose in Hass avocado fruits in four municipalities of the State of Mexico during the period from January to June 2017. The results show that the distribution of anthracnose was adjusted to gaussian and exponential models in most, the infestation maps made through the kriging show more than one centerof aggregation of the disease, based on it the infested surface was estimated, finding an infestation of more than 50% in the first samples and up to 98% in the samplings belonging to the month of June in all the areas studied. © 2019 Published by Elsevier Espana, S.L.U. on behalf of Asociación Argentina de Microbiología. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/ licenses/by-nc-nd/4.0/).

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.

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.

Efeito fungitóxico do óleo essencial de aroeira da praia (Schinus terebinthifolius RADDI) sobre Colletotrichum gloeosporioides / Fungitoxic effect of essential oil from aroeira (Schinus terebinthifolius RADDI) on Colletotrichum gloeosporioides

Neste trabalho foi avaliado o efeito do óleo essencial do fruto de Schinus terebinthifolius sobre o crescimento micelial do fungo Colletotrichum gloeosporioides in vitro, e no desenvolvimento da antracnose no período de pós-colheita em mamões. As diferentes concentrações de óleo foram diluídas em Tween 80 a 8%. No experimento in vitro foram preparados meios de cultura BDA nas concentrações de 0,05; 0,10; 0,25 e 0,50% do óleo essencial. O controle negativo foi realizado apenas com meio BDA e o controle solvente com meio BDA e Tween 80 a 8%. A inibição do crescimento do fungo foi diretamente proporcional à quantidade do óleo e a maior inibição encontrada foi de 79,07% na concentração de óleo de 0,50%. No experimento in vivo os frutos do mamoeiro foram inoculados com o fungo em quatro tratamentos: com biofilme; com biofilme mais 0,50% do óleo; com fungicida Prochloraz e frutos controle. Embora o tratamento com óleo tenha sido eficiente contra o fungo, não foi indicado comercialmente, pois apresentou valores elevados de perda de massa fresca, de firmeza, e também sintomas de fitotoxidade. O óleo tem propriedade antifúngica contra C. gloeosporioides in vitro e in vivo, contudo, não é recomendado para o mamão em função da fitotoxidez.

This study evaluated the effect of essential oil from Schinus terebinthifolius fruit on the mycelial growth of the fungus Colletotrichum gloeosporioides in vitro and on the anthracnose development during the postharvest period of papaya fruits. The different oil concentrations were diluted in 8% Tween 80. For the in vitro experiment, PDA culture media were prepared at the concentrations of 0.05, 0.10, 0.25 and 0.50% essential oil. Negative control was prepared with PDA medium alone, while solvent control was prepared with PDA medium and 8% Tween 80. Fungal growth inhibition was directly proportional to the oil amount and the greatest inhibition was 79.07% at 0.50% oil concentration. For the in vivo experiment, papaya fruits were inoculated with the fungus in four treatments: with biofilm, with biofilm plus 0.50% oil, with the fungicide Prochloraz and control fruits. Although treatment with oil was efficient against the fungus, it was not commercially recommended since it presented high values of loss of fresh mass and firmness, as well as phytotoxicity symptoms. The oil has antifungal property against C. gloeosporioides both in vitro and in vivo; however, it is not recommended for papaya fruits due to its phytotoxicity.

Plant extracts for controlling the post-harvest anthracnose of banana fruit / Extratos de plantas para controle pos-colheita da antracnose da Banana

In banana, fruit rot is incited by Colletotrichum musae which has been the most serious post-harvest disease of immature and mature fruit. The usual control by fungicides prohibited in many countries reduces their commercial value. Therefore, two experiments were conducted to evaluate the antimicrobial activity of alternative products to the synthetic fungicides. First, berries naturally infected by anthracnose were immersed into Azadirachta indica and citric extracts at 2 and 4% (v/v) for 3 minutes and stored for 11 days under environmental conditions. Next, other berries were immersed into essential oil emulsions of Allium sativum, Copaifera langsdorfii, Cinnamomum zeylanicum and Eugenia caryophyllata at 5% for 3 minutes but stored for 11 days. Berries immersed into distilled water were used as control-treatments. The percentage of disease incidence observed in the control-treatment was similar to the ones observed in the extract of A. indica at 2%. The control-treatment showed disease severity of 75.13% and the percentage of disease control was 20.85%. Fruit immersed into distilled water presented less effectiveness than the ones immersed into citric extracts, which promoted the highest effectiveness. Citric extract at 4% was the most efficient treatment because the disease incidence was 19.44%, the disease severity was 9.34% and the disease control was 90.16%. Less severity and, consequently, more disease control were achieved by immersing the berries into the emulsion of essential oil of A. sativum, followed by treatments with C. langsdorfii, E. caryophyllata and C. zeylanicum.

Em pós-colheita, a podridão dos frutos causada por Colletotrichum musae é a doença mais importante da banana (Musa spp.), sendo presente em frutos verdes e maduros, tornando o produto pouco apresentável e inadequado à comercialização. Considerando-se os efeitos prejudiciais à saúde do tradicional método químico de controle e a proibição da utilização de fungicidas em muitos países, objetivou-se no presente trabalho avaliar bioprodutos com atividade antimicrobiana, considerados alternativos para o controle de antracnose em banana em pós-colheita. Os experimentos foram realizados com frutos de banana naturalmente infectados com Colletotrichum musae, submetidos à imersã, em extratos da planta Azadirachta indica e extratos cítricos (Ecolife), nas concentrações de 2 e 4% (v/v), permanecendo por 11 dias em condições ambientes. A emulsão composta de óleos essenciais das plantas Allium sativum, Copaifera langsdorfii, Cinnamomum zeylanicum e Eugenia caryophyllata também foi avaliada quanto a sua eficácia no controle do patógeno, permanecendo em condição ambiente por 11 dias. Frutos tratados com água destilada constituíram o tratamento controle. Os resultados obtidos demonstraram que frutos submetidos ao extrato aquoso de A. indica na concentração de 2% (88,89%) não diferiu do tratamento controle (100% de incidência da doença). O extrato cítrico a 4% promoveu percentuais de incidência, severidade e controle de 19,44; 9,34 e 90,16%, respectivamente, sendo o mais eficiente. Menor percentual de severidade e o maior percentual de controle da doença nos frutos foram proporcionados pela emulsão óleos essenciais de A. sativum, seguidos pelos tratamentos com C. langsdorfii, E. caryophyllata e C. zeylanicum.

Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot

Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host x pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.

Vegetative compatibility and heterokaryon formation between different isolates of Colletotrichum lindemuthianum by using the nit mutant system

Colletotrichum lindemuthianum, the causative agent of bean anthracnose, is one of the most common pathogens leading to expressive damage to plants beyond presenting noticeable variability. The knowledge on vegetative compatibility groups (VCGs) is of particular interest in asexual fungi as they subdivide the population in groups that can exchange genetic information via heterokaryosis and the parasexual cycle. Among the techniques used in studies about vegetative compatibility groups, the obtainment of nit mutants is apparent. This paper is aimed at obtaining heterokaryons between different isolates of C. lindemuthianum, grouping them in VCGs and evaluating their genetic variability by using the nit mutants system. Nit mutants were obtained from 20 single spore isolates. The mutants were phenotypically classified and paired for complementation and formation of heterokaryons so as to group them in VCGs. Seventeen mutants from the different phenotypic-rates were recovered: nit1, nit2, nit3 and nitM. At the same time, 10 mutants were selected for pairing and division of the anastomosis groups. Nine heterokaryons were obtained and the isolates were divided into 9 vegetative compatibility groups. In the combinations for the formation of anastomosis, 31 compatible combinations and 24 incompatible combinations were observed. It was concluded that the methodology used to select nit mutants in C. lindemuthianum made it possible to determine the vegetative compatibility groups and that such a technique was adequate to prove genetic variability.

Genetic relatedness of Brazilian Colletotrichum truncatum isolates assessed by vegetative compatibility groups and RAPD analysis

The genetic variation among nine soybean-originating isolates of Colletotrichum truncatum from different Brazilian states was studied. Nitrate non-utilizing (nit) mutants were obtained with potassium chlorate and used to characterize vegetative compatibility reactions, heterokaryosis and RAPD profile. Based on pairings of nit mutants from the different isolates, five vegetative complementation groups (VCG) were identified, and barriers to the formation of heterokaryons were observed among isolates derived from the same geographic area. No complementation was observed among any of the nit mutants recovered from the isolate A, which was designed heterokaryon-self-incompatible. Based on RAPD analysis, a polymorphism was detected among the wild isolate C and their nit1 and NitM mutants. RAPD amplification, with five different primers, also showed polymorphic profiles among Brazilian C. truncatum isolates. Dendrogram analysis resulted in a similarity degree ranging between 0.331 and 0.882 among isolates and identified three RAPD groups. Despite the lack of a correlation between the RAPD analysis and the vegetative compatibility grouping, results demonstrated the potential of VCG analysis to differentiate C. truncatum isolates genotypically similar when compared by RAPD.

Antimicrobial activity and potential use of monoterpenes as tropical fruits preservatives

Banana, papaya and pineapple are the most consumed tropical fruits in the world, being Brazil one of the main producers. Fungi Colletotrichum musae, Colletotrichum gloeosporioides and Fusarium subglutinans f.sp. ananas cause severe post harvest diseases and losses in fruits quality. The aim of this work was to evaluate the effectiveness of five monoterpenes to inhibit the mycelial growth and conidia germination of these three phytopathogens. The monoterpenes citral, citronellal, L-carvone, isopullegol and a-pinene were diluted in ethanol to final concentrations from 0.2 to 1 percent. All monoterpenes were found to inhibit the growth of the three studies fungi in a dose-dependent manner. Citral was the most effective of the oils tested and showed potent fungicidal activity at concentrations above 0.5 percent. Also, in vivo evaluation with these tropical fruits demonstrated the efficiency of citral to inhibit fungal growth. These results indicate the potential use of citral as a natural pesticide control of post-harvest fruit diseases.

Banana, mamão e abacaxi são as frutas tropicais mais consumidas no mundo, sendo o Brasil um dos principais produtores. Os fungos Colletotrichum musae, Colletotrichum gloeosporioides e Fusarium subglutinans f.sp ananas são os principais causadores de doenças e perdas em pós-colheita de frutas. A proposta deste estudo foi avaliar a eficácia de cinco monoterpenos em inibir o crescimento micelial e a germinação dos conídios destes três fitopatógenos. Os monoterpenos citral, citronelal, L-carvona, isopulegol e a-pineno foram diluídos em etanol à concentração final de 0,2 a 1 por cento. Todos os monoterpenos testados inibiram os três fungos estudados de maneira dose-dependente. Citral foi o mais efetivo dos óleos testados e apresentou uma potente atividade fungicida em concentrações acima de 0,5 por cento. Mais ainda, avaliação in vivo com estas frutas tropicais demonstrou a eficácia de citral como inibidor do crescimento fúngico. Estes resultados indicam o uso em potencial de citral como um pesticida natural no controle das doenças em pós-colheita de frutas tropicais.

Keratitis due to Colletotrichum dematium--a case report.

Colletotrichum dematium has been rarely reported from India before. The present case, a farmer, developed peripheral corneal ulcer five days following trauma with plant. At presentation his visual acuity was 6/60 (unaided) and 6/24P with pinhole. Slit lamp and fluorescent stain examination revealed paracentral corneal ulcer with irregular margins, stromal infiltration and multiple epithelial defects. Microbiological examination of corneal samples confirmed the initial diagnosis of fungal corneal ulcer and the fungus was identified as C.dematium. Patient was treated with topical natamycin and ciprofloxacin. Patient left against medical advice and was lost to follow up. This report emphasizes that Colletotrichum keratitis may not be rare. Early diagnosis may help in institution of specific therapy early in the disease.

Ensayos de inoculación in vitro de Colletotrichum gloeosporioides aislado de hojas asintomáticas de Citrus limon / In vitro inoculation assays of Colletotrichum gloeosporioidesisolated from symptomless leaves of Citrus limon

Los hongos endofíticos pueden colonizar la parte interna de los tejidos sin causar daños aparentes al hospedador. Colletotrichum gloeosporioides, agente causal de la antracnosis, Es la especie más frecuentemente aislada de plantas sanas de Citrus limon en Tucumán. El objetivo de este trabajo fue estudiar, mediante técnicas de inoculación in vitro, la capacidad de estos aislamientos de producir infecciones sintomáticas o asintomáticas en plantines de Citrange ‘Troyer’ (Poncirus trifoliata x Citrus sinensis) y C. Limon. Se determinó el poder germinativo de conidios de loa aislamientos Nº328 y 797 obtenidos de hojas asintomáticas, y el patogénico Nº. N1. Platines en tubos de ensayo y macetas fueron inoculados con una suspensión de 105 conidios/ml y mantenidos en cámara de crecimiento a 25-28ºC. Los conidios de todas las cepas alcanzaron el máximo de germinación (100 por ciento) entre 6 y 7 horas de la siembra. Los aislamientos Nº 328 y 797 indujeron síntomas de antracnosis (clorosis de las hojas, necrosis del tallo y hojas, y defoliación) únicamente en los platines en tubos de ensayo en macetas no desarrollaron la enfermedad. El aislamiento patogénico produjo síntomas de enfermedad en los platines en tubos y macetas. La frecuencia de infección asintomática de los platines en tubo de ensayo fue del 12,5 por ciento para las cepas Nº 328 y 797. Los síntomas de antracnosis producidos por estas 2 cepas en platines en tubos y no en macetas, indicarían que la falta de preservación de condiciones naturales, favorables al hongo, no resultan las apropiadas para determinar el tipo de simbiosis que ocurre en la naturaleza. Consideramos que la utilización de mutantes endofíticas y la realización de los ensayos en plantas de limón a campo, permitirían estudiar mejor el comportamiento de estos hongos en los tejidos de la planta.