Relación in vitro entre cylindrocarpon cylindroides y neonectria fuckeliana asociados a cancros fustales de pinus radiata en Chile / In vitro relationship between cylindrocarpon cylindroides and neonectria fuckeliana associated with stem cankers on pinus radiata in Chile

Desde el punto de vista estrictamente micológico, se ha discutido por décadas la relación de Cylindrocarpon cylindroides como anamorfo de Neonectria fuckeliana, lo que entre otras consecuencias, ha incidido en la controversial ubicación taxonómica de este teleomorfo. Se añade a esto que en patología forestal se ha brindado poca atención a la presencia de C. cylindroides dentro del ciclo de la enfermedad causada por Neonectria fuckeliana en coníferas, razón por la cual los estudios específicos respecto de este anamorfo han sido también muy escasos. El presente trabajo tuvo como objetivo verificar la relación entre C. cylindroides y Neonectria fuckeliana, asociados a cancros en troncos de Pinus radiata en Chile, mediante el análisis de las características macro y micromorfológicas que ellos manifiestan en cultivos in vitro, además de análisis de tipo molecular. Se pudo constatar que ambos agentes dieron origen a colonias con idénticas características macro y micromorfológicas, destacando entre éstas la producción de abundantes conodióforos y microconidios del tipo Acremonium, el cual ya ha sido señalado en anteriores estudios como estado anamorfo de Neonectria fuckeliana. El que este mismo estado asexual del tipo Acremonium haya sido la manifestación in vitro tanto de C. cylindroides como de Neonectria fuckeliana, permitió confirmar la relación existente entre estos dos agentes en estudio, la que también fue ratificada mediante los análisis moleculares realizados.

From the point of view strictly mycological has been discussed for decades the relationship of Cylindrocarpon cylindroides as anamorph of Neonectria fuckeliana, which among other consequences, has influenced the controversial taxonomic status of this teleomorph. Add to this that in forest pathology has given little attention to the presence of C. cylindroides within the cycle of the disease caused by Neonectria fuckeliana on conifers, why specific studies regarding this anamorph have been very few. This study aimed to verify the relationship between C. cylindroides and Neonectria fuckeliana associated with cankers on trunks of Pinus radiata in Chile, by analyzing the macro and micromorphological characteristics they manifest in cultures in vitro, further analysis type molecular. It was found that both agents gave rise to colonies with identical macro and micromorphological characteristics, highlighting among them the production of abundant microconidia and conidiophores of Acremonium type, which has already been noted in previous studies as anamorph state of Neonectria fuckeliana. The asexual state that this same type of Acremonium has been the in vitro demons-tration of both C. cylindroides as Neonectria fuckeliana, allowed us to confirm the relationship between these two agents in the study, which was also ratified by the molecular analyzes performed.

Caracterización morfológica de Acremonium sp. asociado a Neonectria fuckeliana en Pinus radiata en Chile / Morphological characterization of Acremonium-like asociated with Neonectria fuckeliana in Pinus radiata in Chile

En este estudio se realizaron los primeros aislamientos del estado asexual del patógeno Neonectria fuckeliana asociado a cancros o ®revirado del pino¼ en plantaciones de Pinus radiata. Se caracterizaron morfológicamente las cepas del sinanamorfo semejante a Verticillium (Acremonium), obtenidas en cultivo in vitro a partir de peritecios. El material para los aislamientos consistió en trozos de corteza de P. radiata con presencia de peritecios, colectados en Toltén, región de La Araucanía, lugar donde se realizó el primer reporte de N. fuckeliana en Chile. Se utilizaron diez cepas del semejante a Acremonium para la caracterización morfológica, mediciones de estructuras fúngicas, ritmo de crecimiento in vitro y morfología de las colonias. Las colonias presentaron un micelio flocoso y ralo de bordes blanquecinos e irregulares, destacándose tres tipos de colonias, blancas, naranjo oscura y naranjo claro. Taxonómicamente, las cepas coinciden con las estructuras mencionadas en la literatura, caracterizándose por la presencia de glioconidios. Las fialides midieron entre 7 – 78,4 x 1,4 - 4,9 ìm. Los conidios, de formas ovoides y algunas bicelulares, midieron entre 4,2 - 8,4 x 2,6 - 3,5 ìm. El ritmo de crecimiento in vitro fue lento, completando su desarrollo a los 19 días con un promedio de 71 +/- 0,3 mm de diámetro, a una tasa de crecimiento diario de 3,8 mm. Los resultados obtenidos hacen necesario futuros estudios de carácter molecular para analizar la variabilidad genética poblacional que puede presentarse en Chile.

First Neonectria fuckeliana isolates in Chile. Strains of Acremonium obtained from in vitro peritecia culture were morphologically characterized. The samples were collected in Toltén, La Araucanía region, were Neonectria fuckeliana was first reported in Chile. The material used for the isolation came from pieces of Pinus radiata bark with peritecia. Ten Acremonium-like strains were used for characterization of fungal structures, in vitro growth and strains morphology. The colonies were a floccose mycelium and thin edges whitish and irregular, varying color highlighting three types of strains, white, dark orange and pale orange. Taxonomically, the strains match the structures referred in the literature, characterized by the presence of gliospores. The phialides dimensions ranged from 7 to 7.8 ìm long and 1.4 to 4.9 ìm wide. The conidio of ovoid shapes and some bicelular measured between 4.2 to 8.4 ìm in length and width 2.6 and 3.5 ìm. In vitro growth rates were slow, the complete development take19 days with a daily growth average of 71 +/-3 mm in diameter, at a rate of 3.8 mm. It is necessary future molecular studies to analyze the population genetic variation that may occur in Chile.

Viabilidade, confirmação taxonômica e detecção enzimática de espécies de Acremonium preservadas sob óleo mineral na Coleção de Culturas University Recife Mycology / Viability, taxonomic confirmation and enzymatic detection of Acremonium species preserved under mineral oil in the URM Culture Collection

Enzimas hidrolíticas secretadas por fungos têm um papel importante na patogenicidade das infecções. Objetivando avaliar a atividade enzimática foram testados 31 isolados de Acremonium mantidos na Coleção de Culturas University Recife Mycology. Fragmentos das culturas foram transferidos para caldo glicosado para reativação e posterior crescimento em meio ágar batata dextrose, para verificar viabilidade, pureza e confirmação taxonômica pela observação das características macroscópicas e microscópicas. Para detecção enzimática foram utilizados substratos de caseína do leite e gelatina para protease, amido para amilase e lecitina de soja para fosfolipase. Das 31 culturas, 26 (83,9 por cento) mantiveram-se viáveis e 24 (92,3 por cento) foram confirmadas taxonomicamente. Das 24 culturas, 12 (50 por cento) apresentaram atividade proteásica, duas (16,7 por cento) em caseína do leite, uma (8,3 por cento) em gelatina e nove (75 por cento) em ambos os substratos; 16 (66,7 por cento) degradaram amido. Nenhuma cultura apresentou atividade fosfolipásica. Conclui-se que espécies de Acremonium são capazes de produzir enzimas envolvidas na patogenicidade das infecções fúngicas.

Hydrolytic enzymes secreted by fungi play an important role in the pathogenesis of infection. With the aim of evaluating the enzymatic activity, 31 isolates of Acremonium stored in the University of Recife Mycology (URM) Culture Collection were tested. Culture fragments were transferred to glycoside broth for reactivation and further growth in potato dextrose agar medium in order to investigate viability and purity and to confirm the taxonomy through observing the macroscopic and microscopic characteristics. To detect enzymes, milk casein and gelatin were used as substrates for proteinase, starch for amylase and soy lecithin for phospholipase. Among the 31 cultures, 26 (83.9 percent) remained viable and 24 (92.3 percent) were confirmed taxonomically. Out of these 24 cultures, 12 (50 percent) presented proteinase activity, of which two (16.7 percent) were on milk casein, one (8.3 percent) on gelatin and nine (75 percent) on both substrates; 16 (66.7 percent) degraded starch. None of the cultures presented phospholipase activity. It was concluded that Acremonium species are able to produce enzymes that are involved in the pathogenicity of fungal infections.