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Abstracts 2017


PROGRAMA DE ABSTRACTS:
Presentaciones Orales
Carteles Científicos

XVIII Simposio Anual de Micología
22 de abril de 2017


Conferencia Magistral Dr. Carlos Chardón Palacios

Medicinal fungus and the course of treatment: The role of Ganoderma lucidum in breast cancerDra. Yaliz Loperena-Álvarez, Ph.D.
Pontificia Universidad Católica de Puerto Rico, Mayagüez

Natural products have been the sources of anticancer drugs since the beginning of treatments against disease. Approximately 36% of the new first-in-class small molecules medicine approved by US Food and Drug Administration are derived from natural products. Ganoderma lucidum is a basidiomycetus fungus of oriental origin, which as a long history of use for promoting health and longevity in China, Japan, and other Asian countries. The fungus belongs to the mushroom that grows on the lower trucks of deciduous trees. Ganoderma sp. have been used in multiple in vitro and in vivo models due to its anti-proliferative and growth inhibitory efficacy in cancer cells, especially breast cancer. Herein, this conference will emphasize in the progress of alternative and combinatorial therapeutics using G. lucidum extracts in breast cancer.

Presentaciones Orales

Differential sensitivity to QoI fungicide among isolates of Colletotrichum gloeosporiodes
 that cause anthracnose on yam
Bosques-Martínez, M.  and Feliciano-Rivera, M.
Department of Agro-Environmental Sciences, University of Puerto Rico, Mayagüez

Chemical control, based on Quinone outside Inhibitor (QoI-a.i. azoxystrobin) fungicides, has been essential for the management of foliar diseases including yam anthracnose. Colletotrichum gloeosporioides is the causal agent of yam anthracnose on cultivars of Dioscorea alata in Puerto Rico. Due to the excessive use of QoI fungicide and the lack of resistance management practices at farm level, control failures has been detected more frequently. Therefore, the objective of this research was to evaluate the sensitivity to azoxystrobin of two isolates of C. gloeosporioides. One of the isolates was collected from a farm exposed to QoI fungicide and the other isolate was collected from the germplasm of the Corozal Agriculture Experimental Station. Sensitivity to the product was determined through QoI fungicide amended selective media assays and in vitro colorimetric assays to evaluate mycelial growth and spore germination. Four concentrations of the fungicide with and without the alternative oxidase (AOX) inhibitor salicylhydroxamic acid (SHAM) were tested for sensitivity. Treatment effects were determined by ANOVA and treatment means separated by Fisher’s LSD using Infostat statistical software. In the assays evaluated both isolates showed significant differences among the doses tested, with the combination of azoxystrobin 10 µg/ml and SHAM 100 µg/ml being the most effective in reducing their growth. Results showed that C. gloesporioides collected from the farm exposed to QoI fungicide grew in the fungicide amended media in a manner similar to the non-amended treatment. However, the isolate that was not exposed to the fungicide grew in a dose-dependent manner indicating a high sensitivity response to the fungicide. In the absence of SHAM, the isolate collected from the farm exposed to QoI fungicide, had no significant response to the tested doses of azoxystrobin, indicating that the isolate was able to circumvent the effect of the fungicide by the activation of the AOX pathway. These results suggest that, in Puerto Rico, C. gloesporioides exposed to QoI fungicide has possibly developed a resistance to azoxystrobin.

Aceites esenciales, una alternativa segura para el tratamiento de dermatofitosis
Vázquez Alvarado, Y., Torres Caraballo, S., Pagán Medina, C., Morales Miranda, S., y Arroyo-González, Hna. N.
 Departamento de Biología, Pontificia Universidad Católica de Puerto Rico, Ponce

Los dermatofitos son hongos parásitos de la queratina, especialmente de las capas externas de uñas, piel y cabello, que dependen de esta proteína filamentosa para su propagación. Generalmente, estos hongos son tratados con medicamentos químicos que presentan para el humano un alto riesgo de daño hepático y un potencial carcinogénico. Estudios recientes han reportado que aceites esenciales, extraídos de plantas, exhiben actividad fungicida o fungistática además de un nivel muy bajo de toxicidad. El objetivo de esta investigación fue determinar el efecto de los aceites esenciales de Origanum vulgare (orégano), Cinnamomum zeylanicum (hoja de canela) y Eugenia caryophyllata (clavo) contra los dermatofitos Trichophyton rubrum,  Trichophyton mentagrophytes y Trichophyton tonsurans, que han tenido una incidencia en la zona sur de Puerto Rico de 45.2%, 25.8% y un 6.5%, respectivamente. La efectividad de los aceites esenciales se evaluó mediante la formación de halos de inhibición los cuales se midieron a los 7, 14 y 21 días. Los resultados mostraron que los aceites esenciales de orégano, hoja de canela y de clavo presentaron un efecto fungicida. Sin embargo, el fungicida de mayor efectividad fue el aceite esencial de orégano que presentó inhibición total de dermatofitos aun después de 21 días. En conclusión, estos resultados arrojan una promesa terapéutica inocua para combatir dermatofitos en humanos.

Botryosphaeriaceae as pathogens of tropical exotic fruits
 Soto Bauzó, A.1, Serrato Díaz, L. M. 1, Aviles Noriega, A. 2, Rivera Vargas, L. I. 3, Goenaga, R. 4, and Bayman Gupta, P. 1
1Department of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan
2East University, School of Science and Technology, Carolina
3Department of Crops and Agro-Environmental Sciences, University of Puerto Rico, Mayagüez
 4USDA-ARS, Tropical Agriculture Research Station, Mayagüez

Botryosphaeriaceae family fungi have been identified to cause dieback on tropical fruit crop trees. To identify the causative agent of dieback in rambutan and longan in Puerto Rico, a disease survey from 2008 to 2016 was conducted at seven orchards. Diseased tissue of rambutan, longan, mango, and mandarin showing different symptoms were surface-sterilized and plated on PDA. Forty-one isolates in the Botryosphaeriaceae family were isolated and identified using morphological and molecular characters. For morphology, four different media were used. Characters such as sporulation, colony color, and shape and size of conidia were used to identify species. The ITS1-5.8S-ITS2 of rDNA and partial sequences of β-tubulin and trans-elongation factor 1α (EF1-α) were amplified, sequenced and compared with sequences of Botrysophaeriaceae in GenBank. Pathogenicity tests were conducted on 129 rambutan and longan seedlings. Cross-inoculations were done using 10-day old mycelium disks of 5mm in diameter from pure cultures grown on PDA. Control seedlings were inoculated with PDA disks. Symptoms of dieback were evaluated at 8, 14, 30 and 60 days after inoculations (DAI).  Fourteen out of 41 isolates caused dieback in rambutan at 14 DAI. Five of these were collected from rambutan, four from longan, two from mango and three from mandarin. Ten isolates caused dieback in longan, of which nine were collected from rambutan, longan and mandarin (three each) and one from mango. At 60 DAI pycnidia were observed on the trunk of rambutan and longan trees. Five species, four Lasiodiplodia spp. and one Neofusicoccum sp., were identified to cause dieback disease in rambutan and longan. Cross-inoculations showed that pathogenicity is wide-spectrum, indicating that different planting alternatives should be considered for better crop management.

Estudio de la biodiversidad de hifomicetos acuáticos presentes en la espuma del río Usabón en Barranquitas
Arroyo-Berríos, N.L. 1, Negrón-Martínez, C. 2 y Lozada-Troche, C. 2
1Departamento de Ciencias Naturales, Universidad de Puerto Rico, Cayey
2Departamento de Biología, Universidad de Puerto Rico, Cayey

Los hifomicetos acuáticos, también conocidos como hongos ingoldianos, son un grupo de hongos microscópicos que se encuentran en hojas deciduas o cualquier material vegetal que cae en los ríos o arroyos. En los ecosistemas acuáticos, estos hongos juegan roles de gran importancia ya que son responsables de degradar la materia vegetal, facilitando así el reciclaje de nutrientes en niveles tróficos superiores. En el presente estudio, se realizó un censo de la biodiversidad de los hifomicetos acuáticos presentes en muestras de espuma obtenidas del río Usabón ubicado en Barranquitas P.R durante el periodo de agosto 2016 hasta noviembre 2016. Se colectaron muestras de espuma cada dos semanas las cuales fueron teñidas y observadas bajo un microscopio para realizar la identificación y cuantificación de los hongos ingoldianos presentes en las mismas. Cuarenta y cinco especies fueron identificadas en las muestras de espuma del río Usabón. Los resultados obtenidos indicaron que las especies dominantes fueron: Campylospora filicladia, Flagellospora curvula, Campylospora sp, Helicomyces colligatus, y H.torquatus. También se encontraron cerca de veinte especies ocasionales. Las variaciones en las frecuencias de los hongos en el río pudieron ser causadas por los cambios climatológicos, la disponibilidad de sustratos, la tendencia de prevalecer en las estaciones del año y su favorabilidad de subsistir.


Afiches de Investigaciones

Afiche 1
Caffeine degradation of Penicillium sp.
Galarza García, A. M., and Seda Miró, J.
 Biology Department, Pontifical Catholic University of Puerto Rico, Ponce

Caffeine degradation has been extensively studied in bacteria, such as Pseudomonas, and produce several non-toxic metabolites in the process. However, few fungal isolates have been reported to degrade theobromine, the non-toxic metabolite produced by bacteria in caffeine degradation. Due to toxic theophylline production in caffeine degradation by fungi, they have not been considered viable for the decaffeination process. As a result, few studies on caffeine and theobromine degradation by highly caffeine-tolerant fungal isolates have been explored. We aim to study caffeine and theobromine degradation from a fungal isolate (Pencillium sp.) that was initially isolated from a fresh apple product. Preliminary studies in our laboratory showed this fungal isolate to be highly tolerant to caffeine (25 mg/mL), which is an alkaloid commonly found in plants and is known for its role in plant defense. Our objective also includes detection of other metabolites produced from caffeine degradation. To perform our study, Penicillium sp. was cultivated in Potato Dextrose Agar (PDA) at 25°C for 5 days. Approximately 106 spores were then inoculated in a caffeine-sucrose (CS) medium containing 1 g/l of caffeine. Three biological replicates were performed including a positive and negative control. After 48 h of growth, 4 g/L of caffeine were added. Cultures were observed for 7 days and samples were taken every 12 hours. Caffeine degradation and metabolite production were analyzed using high performance liquid chromatography (HPLC). At present, results for caffeine degradation remain in progress and future efforts consist of analyzing theobromine degradation.

Afiche 2

Captura de micoflora viable en el interior del Edificio de Biología utilizando el Andersen Sampler
Noguera-Colón, A. J., Nazario-Ayala, V. M., Brull-Hayes, P.N. y Maldonado-Ramírez, S. L., PhD.
Departamento de Biología, Universidad de Puerto Rico, Mayagüez

Es conocido que los edificios y lugares públicos deben cumplir con ciertas reglas de limpieza e higiene para poder estar certificados por las agencias pertinentes dependiendo del uso y actividades en el edificio. En el caso del Edificio de Biología, donde se llevan a cabo investigaciones y hay una alta frecuencia de personas, no se pueden ignorar ninguno de los parámetros de higiene, incluyendo la calidad del aire. Recientemente se colectaron muestras de aire para determinar si los hongos presentes en el interior del Edificio de Biología de la Universidad de Puerto Rico, Recinto de Mayagüez, presentan alguna amenaza a la salud. Aquí se presentan los resultados de un muestreo realizado en enero 2017 exponiendo por 10 minutos platos de Malt Extract Agar suplementado con cloranfenicol como superficie de colección en un Andersen Sampler de dos etapas. Cada plato se incubó a 25±2°C por 96 horas y se anotó el crecimiento de colonias a las 48, 72 y 96 horas. Luego, se identificaron los diferentes morfotipos en cada plato y se transfirieron a cultivos puros.   Se observó una alta frecuencia de hongos históricamente clasificados como alérgenos, entre ellos Cladosporium sp. (16.6%) y Geotrichum sp. (1.49%).  También, se encontró Stachybotrys sp. (0.186%) en el aire y en una muestra con cinta adhesiva. Además, se encontraron hongos patógenos de plantas, como Rhizoctonia sp. (0.186%) y Nigrospora sp. (0.186%). Estos resultados son significativos, ya que hemos encontrados hongos patógenos para los humanos, los cuales ameritan ser eliminados del aire. Por lo tanto, muestreos posteriores demostrarán si se tomaron las medidas necesarias.

Afiche 3

Assessment of the abundance of fungi present in three canopy trimming experiment sites in the Luquillo Experimental Forest
Morales Pomalaza, B. L.1 and Cantrell Rodríguez, S. A.2
1Departamento de Biología, Universidad de Puerto Rico, Humacao
2Departamento de Biología, Universidad del Turabo, Gurabo

Natural phenomena like storms and hurricanes are known to cause disturbances in forests understory. These disturbances can have a direct effect on the different communities that inhabit the underbrush by altering key factors like soil moisture. Fungal communities are a very crucial part of biomass decomposition in forest understory ecosystems. In Puerto Rico, the development of a large-scale experiment called the Canopy Trimming Experiment (CTE), brought up data on how events like canopy trimming and debris deposition on the forest floor can affect the microbial communities in the soil, this included responses of fungal populations. The objective of this study was to assess the diversity of these populations in the different treatment sites of the CTE, to quantify and characterize it, and determine if there was any significant difference between treatments. Fungi were sampled from leaf litter and soil using various methods of isolation and fruiting bodies were collected and counted from three different blocks with two treatments, trim and control. DNA sequencing was performed with the purpose of creating sequencing data of the diversity of the fungal communities within the different CTE sites for future reference. It was found that there was no significant difference in the total number of fungi in leaf litter or the number of fruiting bodies between treatments. In contrast, total fungi in soil were found to be significantly more abundant in trim plots than in control plots. A total of thirty-three sampled fungi were sequenced and identified. Fungi in leaf litter appeared to not be affected by drying in the open canopy plots, even though the litter moisture has been shown to be reduced. Microfungi in soil was significantly more abundant in open canopy treatments, as hypothesized, because the moisture in the soil was apparently conserved while being reduced on litter, thus the difference in abundance between soils. Differences in abundance were found but in future studies differences in species composition between treatments would like to be explored.

Afiche 4

Diversity of Thraustochytrids associated with mangroves in Puerto Rico
Soto-López D., Rodríguez E., and Cafaro M.
Departamento de Biología, Universidad de Puerto Rico, Mayagüez

Omega-3 (ω-3) oil is known for its capability to reduce and treat heart related diseases. Nowadays the majority of our ω-3 oil is derived from fish, but some marine microorganisms can produce similar oils. Thraustochytrids are eukaryotic fungus-like protists known for their biotechnological potential for ω-3 oil and other novel compounds production via polyunsaturated fatty acids (PUFAs). This represents an alternative for the industry to mass produce high-purity oils, meaning that demand for PUFA-rich products is expected to increase. Thraustochytrids are decomposers usually found in tropical marine environments, typically mangroves, macroalgae and seagrasses. In Puerto Rico, Thraustochytrids diversity is unknown. Mangrove and seagrass leaf-litter samples were collected from Playa Rosada, Lajas, and Playa Buyé, Cabo Rojo to isolate strains by traditional culture methods in seawater media. Samples were processed in the lab under sterile conditions and placed in petri dishes with artificial or aged seawater marine media (0.1% yeast extract, 0.1% peptone, 0.5% glucose); incubated at 28C for 24-48 hours. Microbial growth was observed under a compound microscope to determine type of microorganism and individual strains were purified. Results include the isolation of various yeasts from both collecting sites and one Thraustochytrid from Lajas so far. These were morphologically identified and will be further characterized using molecular techniques. These findings will improve our understanding for Thraustochytrid’s diversity in Puerto Rico and explore its possibilities for PUFA production. Moreover, new opportunities for taxonomic and biotechnological research will be produced.

Afiche 5

Identification of Pseudocercospora griseola races in Puerto Rico
Navarro-Monserrat, E. D.,1 Serrato-Diaz, L.,1 Porch, T.,2 and Bayman, P.1
1Department of Biology, University of Puerto Rico - Rio Piedras, San Juan
2USDA-ARS, Tropical Agricultural Research Station, Mayagüez

Angular Leaf Spot (ALS), caused by the fungus Pseudocercospora griseola, is a frequent disease of the common bean (Phaseolus vulgaris) especially in tropical and subtropical regions. P.griseola and P.vulgaris have co-evolved through time and been classified of Mesoamerican or Andean groups. In Puerto Rico, isolates of P. griseola have been shown to be Mesoamerican; however, races have not yet been determined. This study focuses on the isolation and identification of races of P. griseola in Puerto Rico. Forty-five isolates were obtained from experimental research stations at UPR and USDA-ARS in Isabela and Juana Diaz. Races of P. griseola were identified by pathogenicity tests using a set of twelve differential bean cultivars defined by International Center for Tropical Agriculture (CIAT). P. griseola isolates were grown on V8 medium and incubated for 45 days. Conidia suspensions were used to inoculate 20-day old plants. Twenty days after inoculation symptoms were observed and the isolates were classified into races according to compatibility reactions. 21 isolates have been tested (9 from Juana Diaz and 12 from Isabela) and 12 races have been identified. These results suggest that the pathogen P.griseola may contain polymorphisms in its genome, possibly in virulence genes. This project can thus be used as a basis to determine candidate virulence genes and select for resistant bean varieties.

Afiche 6

Yeast associated with demosponges from Puerto Rico
Sánchez-Martínez, E.1, Rosado-Rodríguez, G.2 and  Maldonado-Ramírez, S.L.1
1Department of Biology, University of Puerto Rico, Mayagüez
2Department of Marine Sciences, University of Puerto Rico, Mayagüez

Sponges are known for their symbiotic associations with bacteria and to a lesser extent with fungi. It has been argued that the association between fungi and sponges is not truly symbiotic, and that fungi found are incidental. Nevertheless, vertically-transmitted endosymbiotic yeast has been observed by transmission electron microscopy in sponges of the genus Chondrilla.  The identity of the symbiont and its presence in other sponge species is still unknown. This work focused on studying the presence of yeasts in marine sponges commonly found in Puerto Rico. Samples were taken from colonies of Ircinia strobilina, Tedania ignis, and Chondrilla caribensis.  Dilution-plate method and high-throughput culturing by dilution-to-extinction were applied for the isolation of yeasts from sponge samples.  Plates were incubated for up to 60 days to allow the isolation of slow-growing colonies. A total of 36 yeast morphotypes were isolated and analyzed by sequencing the ITS region and D1/D2 domains of the large subunit ribosomal DNA. The brewer’s yeast, Saccharomyces cerevisiae, was isolated from all samples, comprising the first report of this organism as a common inhabitant in marine sponges in Puerto Rico. Other yeasts isolated include the halophilic black yeast, Hortaea werneckii, and an unidentified yeast belonging to the Sporidiobolales (Basidiomycota).

Afiche 7

Sensitivity of Colletotrichum alatae to a quinone outside inhibitor (QoI) fungicide
Escalera-García, I. A. and Feliciano-Rivera, M.
Department of Agro-environmental Sciences, University of Puerto Rico, Mayagüez

Yam (Dioscorea spp.) is a staple crop of economic importance in tropical and sub-tropical regions worldwide. Yams are very susceptible to diseases caused by a wide spectrum of pathogens including bacteria, nematodes, virus and fungi that significantly reduce yield and quality of the commercial product. Anthracnose disease, caused by Colletotrichum spp., is one of the most destructive diseases in yam (Dioscorea alatae) production in Puerto Rico. Chemical control, based on Quinone outside Inhibitor (QoI) fungicides, has been essential in the management of anthracnose. However, due to the excessive use of QoI fungicide control failures has been occurred in recent years. Hence, the purpose of this research was to measure the sensitivity of an isolate of Colletotrichum alatae collected from commercial fields to a QoI fungicide. Sensitivity to the product was determined using amended selective media assays, colorimetric assays, and in vitro spore germination assays with different fungicide concentrations (0.1, 1.0, 5.0 and 10.0 µg/ml label rate). Fungicide treatments were evaluated with and without the alternative oxidase inhibitor salicylhydroxamic acid (SHAM). Treatment effects were determined by ANOVA and treatment means separated by Fisher’s LSD using InfoStat Statistical Software. Results showed that C. alatae grew similar to the non-amended fungicide treatment at concentrations of 0.1, 1.0, 5.0 µg/ml. At the concentration of 10 µg/ml in combination with SHAM C. alatae showed a growth reduction. The same results were obtained in the colorimetric assays and in the spore germination assays. These results suggest that, in Puerto Rico, C. alatae have developed a resistance to QoI fungicides in both the mycelium and spore stage.

Afiche 8

Fungal diversity in green litter decomposition in a hurricane experiment
Velazquez Cruz, J1, Moreno, I.1, Barberena-Arias, M. F.1 and Cantrell, S. A.1
1Department of Biology, University of Turabo, Gurabo

Hurricanes generate disturbances in forests such as canopy opening, fallen trees and leaves which in turn alter physicochemical characteristics of the habitat, as well as, decomposer activity. Litter decomposition depends primarily on the interaction among climate, litter quality and biota; as a consequence any change in habitats will result in changes in these factors. Identifying the changes in the fungal community structure in soil and forest floor litter can help understand the factors that influence ecosystem recovery. This study is part of the Canopy Trimming Experiment 2 performed by the Luquillo LTER at El Verde Field Station. For this, three blocks (A, B and C) were selected, each with two plots of 20m x 20m, one plot for control and the other was subject to canopy opening and debris addition (Trim plus debris). This experimental design represents 3 blocks x 2 plots/block (1 trim+debris/ 1 control) x 3 subplots x 3 litterbag mesh sizes x 4 collecting times (3, 5, 12 and 24 weeks), for a total of 216 litterbags. A pool sample for each mesh size from the 3 subplots/per plot/time was obtained for DNA extraction. DNA was extracted using Power Soil DNA Isolation kit from MoBio. TRFLP technique was used to obtain profiles of fungal communities using the fungal ITS region. The diversity between samples was analyzed using NMDS and UPGMA Cluster analysis using the Bray-Curtis similarity index. Two way Permanova shows significant differences in fungal communities between blocks, control and trim plus debris, and mesh size. No significant differences were observed through time. In general, the diversity of fungal communities increase in the trim plus debris treatment. This increase is mainly due to the addition of detritus which increases soil moistures. Fungi are the main decomposers of detritus in the forest floor and the effect of natural disturbances such as a hurricane on leaf litter microbial communities will help us understand how litter decomposition might be affected. This one component of a larger study that is analyzing the interaction between arthropods, microorganisms, nutrient mineralization and disturbances.

Afiche 9
Tipos de hongos cultivables transportados por Erythrodiplax umbrata
(Insecta: Odonata: Libellulidae)
Ruiz-Arocho J., Santos-Flores, C. y Maldonado-Ramírez, S.
Departamento de Biología, Universidad de Puerto Rico, Mayagüez

El proceso de dispersión es una de las necesidades básicas que la mayoría de los seres vivos deben sobrellevar para la supervivencia óptima de su especie. En el reino Mycota, la dispersión puede verse influenciada no solo por factores abióticos, sino también por factores bióticos como dispersión en el tracto digestivo de animales y la dispersión en la superficie de los mismos. Un ejemplo de un animal con un alto grado de movimiento y en los cuales se han estudiado sus capacidades motoras, así como sus rangos de dispersión, son las libélulas (Insecta: Odonata). Por tal razón, en este experimento nos concentramos en conocer qué tipo de hongos cultivables pueden ser transportados y subsecuentemente dispersados por Erythrodiplax umbrata (Odonata: Libellulidae). Para cumplir con este objetivo, se colectaron nueve machos de la especie en una zona adyacente al Bosque de Miradero, Mayagüez PR. Se realizó una impresión individual de las áreas dorsales y ventrales de cada individuo en medio de cultivo de agar de harina de maíz (CMA) y los hongos encontrados fueron aislados para su identificación en medio de cultivo de agar de papa y dextrosa (PDA). Hasta el momento se han recuperado 66 morfotipos de los cuales se han logrado identificar hongos pertenecientes a los géneros Aspergillus, Spegazzinia, Alternaria; entre varias levaduras. A pesar de que una cantidad considerable de especímenes han sido clasificados como micelio carente de estructuras reproductivas y aún existen otros en proceso de esporulación, podemos inferir que, si cada morfotipo representa una especie distinta, la cantidad de especies que pueden ser transportadas por libélulas es considerablemente alta. Esto nos demuestra la importancia de las interacciones hongos-invertebrados y el rol que tienen estos insectos para con el movimiento y dispersión de hongos.

Afiche 10

Hongos fitopatógenos que afectan al cultivo de Hierbabuena (Mentha spicata L.) en Puerto Rico
Santiago Charneco, J. L., Simbaña Carrera, L.L., y Rivera Vargas, L. I.
Departamento de Ciencias Agroambientales, Universidad de Puerto Rico, Mayagüez

La Mentha spicata L. comúnmente conocida como hierbabuena, es una especie aromática. En Puerto Rico es valorada por sus cualidades aromáticas y su uso culinario. Esta planta aunque es producida bajo condiciones controladas en invernadero, es afectada por microorganismos patógenos que ocasionan pérdidas en su producción a pequeña escala. Los síntomas más comunes son: necrosis en hojas y tallos; y manchas cloróticas en hojas. Es por esto que el objetivo de este estudio fue identificar microorganismos fitopatógenos asociados al cultivo de hierbabuena. Se recolectaron plantas sintomáticas en Aguadilla crecidas en condiciones de invernadero, en Isabela para la venta en un centro comercial y en Mayagüez de una finca comercial. Los microorganismos se aislaron en medio de acidulado de agar de papa y dextrosa y se incubaron por 48 h a 28°C. Los hongos fueron caracterizados morfológicamente a través de microscopia de luz y de fases e identificados utilizando claves morfológicas. Se obtuvieron ocho aislados de hongos. Se identificaron los hongos fitopatógenos: Curvularia spp, Colletotrichum spp., Aspergillus spp. y aislados pertenecientes a la familia Botryosphaeriaceae. También se identificaron los hongos saprófitos: Paecilomyces spp. y Mucor sp. Además, se completaron los postulados de Koch en ramas sanas colocadas en cámaras húmedas. En un periodo de una semana, la mayoría de los aislados causaron clorosis en las hojas inoculadas y luego de dos semanas se observó necrosis intervenal y pudrición del tejido. Posteriormente se espera realizar pruebas de patogenicidad en tiestos y caracterizar molecularmente las especies de hongos patogénicas.

Afiche 11

Dynamics of microbial groups in response to simulated hurricane at
 El Yunque Rain Forest in Puerto Rico
González-Rosario, K. M.1,3, Cantrell, S. A.1, and Pérez-Jiménez, J.R.,1-3
1School of Natural Science and Technology
 2Interdisciplinary Research Institute
3Puerto Rico Institute for Microbial Ecology Research, Universidad del Turabo, Gurabo

Climate variability models predict increase in incidence and intensity of hurricanes. In Puerto Rico, hurricanes have impacted the Luquillo Rain Forest in many occasions resulting on canopy debris deposited in the forest floor.  Inhabiting microbes face input of complex plant biomass, increases in sunlight, and alterations in redox potential that impact their prevalence and contributions to biogeochemical cycles. A Canopy Trimming Experiment (CTE), that simulated the pass of a hurricane, has been done in the Tabonuco forest. Our goal is to determine temporal heterogeneity of two microbial groups (bacteria, fungi) in response to detritus deposition of simulated hurricane effect. Soil samples are being collected from plots, at different times. Two treatments are considered: with and without detritus deposition trimmed from the local canopy. Bacteria and fungi are being characterized independently by the molecular analyses of four distinctive genes (16S rDNA and ITS). Bacteria was homogeneous over time for the same plot suggesting microbial succession in which rare microbiota became more prevalent over time. In contrast, diversity increased over time where detritus was added. Bacterial and fungal communities exhibited spatial variation regardless the availability of plant debris. In contrast, microbial functional groups examined demonstrated greater heterogeneity in response to plant debris addition (treatments). In the future, characterization of specific taxa, qPCR and Next Generation Sequencing.

Afiche 12

Detecting antimycotic properties of gold and silver nanoparticles
against Aspergillus niger
Morales-Rodríguez, L.M. and Colón-Mena, Y.
Department of Natural Sciences, University of Puerto Rico, Aguadilla

Gold and silver nanoparticles (Au/Ag-NPs) have been shown to have inhibitory effects and antibacterial activity. Over the years, resistance to antibiotics and antimycotic agents by pathogenic mushrooms has become a major health problem. In this study, we evaluate the inhibitory effects of gold and silver nanoparticles against pathogenic fungus Aspergillus niger – causative agent of aspergillosis (lung disease) and otomycosis (fungal ear infections) in humans. To test the effects of metal nanoparticles (Me-NPs), we performed the Kirby-Bauer Disk Susceptibility Test on Mueller-Hinton agar plates. The plates (n=10) were inoculated with the test strain once the desired absorbance reading was achieved. First round, all standard paper disks (D=6mm) in five plates were impregnated with Au-NPs only. The second round of experiments followed the procedure previously described, but with Ag-NPs instead. Measurement of the inhibition halos was performed after 48 hours of incubation. Our results show that silver nanoparticles have a significantly higher inhibitory effect on the test strain -when compared to gold nanoparticles. Therefore, silver nanoparticles have a potent inhibitory effect against A. niger.

Afiche 13

Isolation and characterization of mycological bioprospects from the
 Arecibo Observatory Radio Telescope
Rivera-De Jesús, M. A. and Ríos-Velázquez, C.
Department of Biology, University of Puerto Rico, Mayagüez

The Arecibo Observatory is a single-dish radio telescope, once the largest in the world, situated within the vegetation of Arecibo, Puerto Rico that can send and receive powerful radio waves. Despite its unique environment, the radio telescope’s microbiological flora has seldom been studied, in particular its mycological flora. This study, in addition to describing the mycological flora of the area of the telescope, also focuses on exploring mycological bioprospects that could have or have acquired novel attributes with Biotechnological or Biomedical applications. The first stage involves the isolation of mycoflora present at the four radar support columns located at the periphery of the radar dish.  Samples were collected using sterile swabs, serially diluted, and dispersed on Potato Dextrose Agar (PDA) media. The samples were incubated at 25°C and the presence of colonies was monitored daily. The mycoprospects were isolated and analyzed macroscopic and microscopically. A total of 136 individuals have been isolated. The most mycological samples were collected on the northeastern and northwestern columns, with the northeastern column containing the highest amount of yeasts of all. Some of the genera found belong to Penicillium sp., Paecilomyces sp. and Aspergillus sp., in addition to a variety of yeasts that were also identified. Other macroscopic colony features indicate to more genera to be identified, demonstrating the mycological variety found in the area. Functionally, some of the mycological bioprospects isolated have shown antibiosis activity to other fungi and are to be further analyzed to discover potential antibiosis activity against bacteria.

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Ubiquity of Aspergillus spp. and other fungal Genera in green coffee beans
Rodríguez-Soto, M. M., Simbaña Carrera L, L. L., Dumas, J., and Rivera Vargas, L. I.
Department of Agro-Environmental Sciences, University of Puerto Rico, Mayagüez

Different species of Aspergillus produce diverse mycotoxins such as: aflatoxins and Ochratoxin A, known for their adverse effects on human health. These fungi colonize tissues, epiphytically or endophytically, of economic important crops including coffee, one of the most consumed drinks globally and a very important agricultural commodity in Puerto Rico. Studies have reported Aspergillus carbonarius as an important source of Ochratoxin A in coffee. Our study aimed to elucidate the occurrence of different fungi in green coffee beans, especially those characterized for their mycotoxin production, emphasizing Aspergillus spp. For this purpose, two-year-old cold preserved samples of green beans from six coffee cultivars (i.e. Frontón, Typica, Borbón, Catuaí, Catuaí honey and Limaní) harvested in Adjuntas, Puerto Rico were evaluated. To isolate fungi, green coffee samples were surface sterilized and placed on potato dextrose agar media amended with antibiotics. Fungal colonies were counted, purified and identified morphologically using light and phase microscopy. A total of 68 fungal colonies were isolated. Aspergillus spp. (44%) was the most ubiquitous endophytic fungal genera identified, followed by Chaetomium spp. (21%). Five different Aspergillus spp. morpho-groups were observed. Other fungal genera identified were: Penicillium spp.  (13%) and Rhizopus sp. (4%). Eighteen percent of the isolates did not produce reproductive structures and were not identified. Coffee cultivar Borbón showed the highest amount of fungal colonies (n=20) while cultivar Limaní showed the lowest (n=3). We demonstrate that a diverse group of fungal species colonize green coffee beans and survive cold storage conditions. These findings are important because of its known mycotoxins production and their implications in human health.

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Bioprospección y biocontrol de la Broca del Café con Beauveria bassiana
García Rodríguez, N. M., Mariño Cardenas, Y. A., y Bayman-Gupta, P.
Departamento de Biología, Universidad de Puerto Rico, Río Piedras

Reportada por primera vez en Puerto Rico en el 2007, la broca del café (Hypothenemus hampei) es el insecto plaga más dañino al café en Puerto Rico y el mundo. Actualmente, el manejo integrado de la broca suele usar el hongo entomopatógeno Beauveria bassiana (Bb) como control biológico. En Puerto Rico, este hongo es aplicado usando el producto con cepa comercial GHA (de Mycotrol® y BotaniGard®). Nuestro objetivo es evaluar el potencial uso de cepas locales de Bb y compararlas con la cepa comercial para el biocontrol de la broca del café. Cepas locales de Bb fueron colectadas de brocas parasitadas en cafetales de Puerto Rico. Se escogieron nueve de éstas y la cepa GHA, para utilizarse pruebas de virulencia in vitro aplicando una suspensión de 4 x 106 conidias/mL a brocas hembras en placas petri y documentando mortandad durante ocho días. Se transfirieron las brocas muertas a placas Petri con un papel filtro húmedo y se evaluó diariamente durante 10 días la presencia del micelio, para luego cuantificar las conidias producidas por broca. En el campo se comparó la persistencia de la cepa local más virulenta y la cepa GHA, donde se asperjaron árboles con cada una de éstas, cada dos semanas se contabilizaron los frutos con colonias esporuladas de Bb. Se observaron diferencias significativas en la virulencia de cepas locales y la cepa GHA, sin embargo, dos de las locales produjeron porcentajes de mortandad similares a los registrados por la cepa GHA. Además, una de éstas superó la cantidad de esporas producidas de la cepa GHA. En el campo, el tratamiento con la cepa local mostró mayor persistencia de Bb en comparación con el tratamiento con la cepa comercial. Estos resultados sugieren que las cepas locales de B. bassiana son un recurso genético de alto potencial para el biocontrol de la broca del café y otros insectos plagas.

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Isolation of Fusarium spp. inhibiting and Fumonisin B1 degrading microbes
from corn (Zea mays L.) and sorghum (Sorghum bicolor L.)
Rivera Hernández, O.1, Rivera-Vargas, L. I.1 and Kuldau, G.2
1Department of Agro-Environmental Sciences, University of Puerto Rico, Mayagüez
2Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University

Fumonisins are secondary metabolites produced by some species of the genus Fusarium. These are hazardous and prevalent mycotoxins in crops such as corn and sorghum. Fumonisins usually accumulate in grain affected by Fusarium Ear Rot caused by Fusarium verticillioides. The possibility of reducing harmful levels of these toxins by using fumonisin-degrading microbes seems promising. In the same way, using biological control to inhibit the growth of Fusarium species appears to be an efficient way of managing the disease. As a first step towards identifying possible biological controls, we isolated two bacteria from corn and sorghum with potential fungal inhibitory properties. Screenings of the samples as well as co-cultures were done in order to evaluate inhibiting organisms. Inhibition tests were conducted by co-culture of Fusarium spp. isolates M-8773 and M-8778, with potential inhibitory microbes on Waksman’s glucose agar. Lisogeny broth was used as a negative control. For microbes capable of degrading the mycotoxin, a suspension of corn and sorghum leaves was incubated in a mycotoxin enriched liquid mineral medium for four weeks. Medium changes were done between weeks to prevent organisms from taking carbon out of dead organisms. After incubation, four Gram-negative bacterial strains were isolated as potential degraders. Although this opens the possibility of finding degrading organisms in host plants for F. verticillioides is not certain that all the bacterial strains isolated are fumonisin decomposers. This can be explained through cooperative catabolism where some bacterial species degrade the products of other reactions that occurred first, obtaining carbon out of other compounds in the process. HPLC and PCR are contemplated to characterize true fumonisin degrading bacteria.

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Inventory of cultivable fungi from fresh casts of the
earthworm Yuisia olgae (Annelida, Benhamiidae): Preliminary results
Carrera-Martínez, R.,1,2, Rosado-Rodríguez, G.,1,3 and Maldonado-Ramírez, S.L.1
1Department of Biology, University of Puerto Rico, Mayagüez
2Warnell School of Forestry and Natural Resources, University of Georgia, Georgia
3Department of Marine Sciences, University of Puerto Rico, Mayagüez


Earthworm and fungi interaction have been the focus of many studies, as earthworms can disturb fungal diversity and species composition. However, few studies focused on the fungal communities associated with earthworms, including their fresh casts. Here, we propose a novel methodology to study fungal diversity associated with earthworm casts. Two specimens of the earthworm Yuisia olgae were collected together and maintained alive on a container with wet soil for about 12 hours. At the laboratory, the specimens were separated from the soil and externally cleaned with sterilized water until no remnants of soil particles were observed on the specimens. The earthworms were placed on two zip-lock bags previously sterilized with ethanol that contained a piece of sterilized paper towel soaked in sterilized water. One additional identical system was placed without earthworms to discard fungi that could be associated to the zip-lock bag system. The zip-lock bags were then incubated at room temperature in the dark for 24 hours. A sample of approximately 1mg of earthworm casts was extracted with a sterile swab. The serial dilution method was used to process the samples that were then cultivated on Potato Dextrose Agar at room temperature. The control system was sampled with a sterile swab and then cultivated directly on PDA. Fungal cultures from the earthworm surface were obtained with a sterile swab, which were then discarded as casts-associated morphotypes. Fungi were identified using morphological characters and DNA sequences of the ITS region. A total of 13 fungal species were isolated from the earthworm casts. Fusarium was the most abundant genus (frequency of 0.77), followed by Penincillium (0.13). Fusarium lichenicola, Thrichosporon sp., Clonostachys sp. and Aphanoascus sp. were isolated for the first time from earthworm casts. Additionally, these results contrasts with previous publications that suggests that earthworm feeding behavior is antagonistic to Fusarium.

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Bio-remediation of oil pollutants by fungi
González-Ferrer, S.M.1,  Hernández-Lamberty, M.1 , Peterson, K.1  and Cafaro, M.1
1Department of Biology, University of Puerto Rico, Mayagüez

Pollution in marine environments comes from certain components in the motor oil that indefinitely stay in these environments, especially in the sediments. Among the chemical components of diesel, motor oil, and other petroleum-based fuels are partially combusted polycyclic aromatic hydrocarbons (PAHs). These have stable ring structures difficult to degrade, hence harmful to the environment and even carcinogenic. Previous studies of lignin degradation by fungi have shown potential towards degrading PAHs due to the non-specific fungal secreted enzymes. Acknowledging this possibility, we designed an experiment to use fungi isolated from coastal mangroves to degrade waste motor oil, Purpureocillium lillacinum, Fusarium solani, Aspergillius niger, and Aspergillius caelatus. The experiment consisted of a base culture medium, containing aged sea water, NO3 solution, and used motor oil as the sole carbon source. Along with the motor oil, we tested other treatments consisting of lignin, cellulose, and a mixture of these on the fungal samples. The purpose was to use minimal sources of nutrients possible to fully measure fungal growth based on motor oil degradation. Our preliminary results indicate that all four fungi have the capacity to grow with motor oil as the only carbon source, as well as in cellulose, lignin, and the mixture of treatments. We found that a consortium consisting of all four fungal species was more efficient at using the waste motor oil as a carbon source than in the treatments containing single species isolates. We expect to analyze more fungal strains and other organisms to add to our synergistic consortium. We will also incorporate gas chromatography-mass spectrophotometry to measure actual degradation of the carbon compounds and characterize the enzymes that are responsible of the degradation process.

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Occurrence of Cryptococcus neoformans/gatti in dry forest areas
of the southwestern zone of Puerto Rico
Fumero Nazario, S., Velez Justiniano, Y., Rodriguez Golderos, G.M. and Ruiz Acevedo, A.
Department of Biology, University of Puerto Rico, Mayagüez

Cryptococcus neoformans/gatti are opportunistic and encapsulated yeasts that adversely affect primarily immunosuppressed patients. Although infections of C. neoformans/gatti are relatively rare in humans with a healthy immune system, its effects on wildlife and ecology are poorly understood. The yeasts live freely in the environment, mainly associated to pigeon droppings and to eucalyptus, or other tree detritus. Suspected carriers of C. neoformans/gatti were sampled to determine the presence of the yeast on succulent plant sources. Surface samples were collected during dry season from Pilosocereus royenii cacti at sites affected by an unknown insect caused disease at the southwest of Puerto Rico. The prospective C. neo/gatti isolates were preliminarily identified by melanin production on Niger Seed Agar and then confirmed by biochemical characterization.  Two out of thirteen samples showed yeast-like growth with non-capsulation. The presence of C. neo/gatti or other fungi helps to prove if the colonization process in lesions is initiated by an insect. In addition, the lesions in the cacti provide favorable environmental conditions for fungal community growth and development. Further studies to determine fungi-insect relationship are needed.

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Antibiotic potential of endophytic fungi associated with the Black Mangrove Avicennia germinans
in Cabo Rojo, Puerto Rico
Figueroa-Negrón, V.1 and Cafaro, M.J.1
1Department of Biology, University of Puerto Rico, Mayagüez

The search for new antimicrobial compounds has been of great interest in recent years. These compounds are naturally produced by microorganisms including endophytic fungi isolated from mangroves. This study focuses on endophytic fungi isolated from leaves and seeds of the black mangrove (Avicennia germinans) from Bahia Salinas, Cabo Rojo, Puerto Rico. We isolated 42 fungal endophytes from which nine identified strains were tested for antimicrobial secondary metabolite production. We selected fungal strains from Penicillium, Aspergillus flavus, Aspergillus clavatus, Stereum, Nigrospora, Purpureocillium, Engyodontium, Hortaea and Bionectria for the analysis. Four bacterial strains: Escherichia coli, Pseudomonas sp., Serratia sp. and Staphylococcus sp. and two yeast strains of Candida albicans and C. tropicalis were used to perform bioassays. Growth curves were prepared for yeast and bacteria. Fungal extracts were added after lag, exponential and stationary growth phase of each bacteria and yeast. Our results showed that Aspergillus flavus and Aspergillus clavatus extracts strongly decreased the growth of C. albicans and E. coli, while Nigrospora only affected the growth of E. coli. There was no significant change in the growth of C. tropicalis or the other bacterial strains. These results could open a way for future discoveries of new antimicrobial o antifungal compounds.

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Phytophthora spp. y especies de hongos asociadas a daños en la producción de
piña (Ananas comosus) en Puerto Rico
Veléz Negrón, Y. I., Simbaña Carrera, L. L. y Rivera Vargas, L. I.
Departamento de Ciencias Agroambientales, Universidad de Puerto Rico, Mayagüez

La piña (Ananas comosus) es un cultivo de importancia en Puerto Rico. En los últimos años la producción ha alcanzado 5,332 toneladas, valorándose en $5 millones para el periodo 2014-2015. Este cultivo se ve afectado por microorganismos como hongos y oomicetos que ocasionan graves pérdidas en la producción, incrementando la preocupación entre los productores. Phytophthora cinammomi, es uno de los patógenos más importantes que ocasiona una pudrición en la base del cogollo de la fruta, las hojas centrales presentan una coloración amarilla y las mismas pueden ser desprendidas con facilidad. Debido, a esta preocupación se esbozaron dos objetivos: determinar la presencia de Phytophthora spp. en zonas productoras de piña de la isla e identificar hongos asociados a otros daños observados en el campo. Las muestras de tejido vegetal enfermo fueron recolectadas en las localidades de Lajas, Guánica, Comerío y Santa Isabel. En el laboratorio se prepararon cámaras húmedas y material vegetal enfermo. A partir del micelio obtenido, se sembraron muestras directamente en medios sintéticos (i.e. PDA, V8 y PARPH). Las placas se incubaron de 48 horas a 28°C. Los microorganismos patógenos fueron identificados mediante microscopía de luz y de fases. Los géneros de microorganismos fueron identificados utilizando claves taxonómicas. Se han identificado los siguientes microorganismos: Phytophthora spp., Fusarium spp., Curvularia y tres aislados de hongos pertenecientes a la Familia Botryosphaeriaceae Hasta el momento el patrón de distribución sintomatológica propia de Phytophthora spp, está presente en todas las zonas muestreadas. Se estarán realizando pruebas de patogenicidad y caracterizándolos mediante análisis molecular con diferentes regiones genéticas (ITS del rDNA y el gen COI).

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