Antiproliferative activity of biomass extract from Pseudomonas cedrina

Background: The study of plant-associated microorganisms is very important in the discovery and development of bioactive compounds. Pseudomonas is a diverse genus of Gammaproteobacteria comprising more than 60 species capable of establishing themselves in many habitats, which include leaves and stems of many plants. There are reports of metabolites with diverse biological activity obtained from bacteria of this genus, and some of the metabolites have shown cytotoxic activity against cancer cell lines. Because of the high incidence of cancer, research in recent years has focused on obtaining new sources of active compounds that exhibit interesting pharmacodynamic and pharmacokinetic properties that lead to the development of new therapeutic agents. Results: A bacterial strain was isolated from tumors located in the stem of Pinus patula, and it was identified as Pseudomonas cedrina. Extracts from biomass and broth of P. cedrina were obtained with chloroform:methanol (1:1). Only biomass extracts exhibited antiproliferative activity against human tumor cell lines of cervix (HeLa), lung (A-549), and breast (HBL-100). In addition, a biomass extract from P. cedrina was fractioned by silica gel column chromatography and two diketopiperazines were isolated: cyclo-(L-Prolyl-L-Valine) and cyclo-(L-Leucyl-L-Proline). Conclusions: This is the first report on the association of P. cedrina with the stems of P. patula in Mexico and the antiproliferative activity of extracts from this species of bacteria against human solid tumor cell lines.

Transferencia de nutrientes y crecimiento de Pinus greggii Engelm. inoculado con hongos comestibles ectomicorrícicos en dos sustratos / Nutrient transfer and growth of Pinus greggii Engelm. inoculated with edible ectomycorrhizal mushrooms in two substrates

La ectomicorriza es una simbiosis mutualista de enorme importancia en la producción de árboles de valor forestal. Uno de los criterios de selección de hongos ectomicorrícicos de gran interés es su comestibilidad, por la importancia económica, ecológica y cultural de los hongos comestibles ectomicorrícicos como un producto forestal no maderable. En este estudio se evaluó el efecto de la inoculación con los hongos comestibles ectomicorrícicos, Laccaria laccata, Laccaria bicolor y Hebeloma leucosarx, en el crecimiento y el contenido nutricional de Pinus greggii, crecido en un sustrato experimental, o en un sustrato comercial enriquecido con un fertilizante de liberación lenta. Dos años después de la siembra, se observaron diferencias en el crecimiento aéreo y radical y en el contenido de macro- y micronutrientes entre plantas inoculadas y no inoculadas, independientemente de la especie fúngica y del sustrato utilizado. Las plantas crecidas en el sustrato comercial tuvieron mayor crecimiento y contenido de nutrientes; sin embargo, sus porcentajes de colonización fueron menores que los de plantas crecidas en el sustrato experimental. Existieron diferencias en la transferencia de nutrientes a la parte aérea de las plantas inoculadas, al comparar entre las especies fúngicas implicadas. Se observó transferencia de Ca por L. laccata, de Na por L. bicolor y de Mn por H.leucosarx en el sustrato experimental. La selección de sustratos constituye un factor clave en la producción de plantas ectomicorrizadas y las tres especies de hongos comestibles ectomicorrícicos evaluados tienen un potencial relevante para la micorrización controlada de P. greggii.

An ectomycorrhiza is a mutualistic symbiosis of paramount importance in forestry and tree production. One of the selection criteria of ectomycorrhizal fungi that has currently gained importance is their edibility due to the economic, ecological and cultural relevance of edible ectomycorrhizal mushrooms as a non-timber forest product. The effect of the inoculation with three edible ectomycorrhizal mushrooms: Laccaria laccata, Laccaria bicolor y Hebeloma leucosarx, which are widely sold in Mexico, on the growth and nutrient contents of Pinus greggii grown in an experimental substrate and a commercial substrate enriched with a slow-release fertilizer, was evaluated. Two years after sowing, differences in terms of shoot and root biomass and macro and micronutrient contents between inoculated and non-inoculated plants, were recorded independently of the fungal species and the substrate. Despite the fact that plants grown in the commercial substrate had higher growth and nutrient contents, their ectomycorrhizal colonization percentages were smaller than those of the plants grown in the experimental substrate. The differences in the nutrient transfer to the inoculated plant shoots among the evaluated fungal species were recorded. Ca mobilization by L. laccata, Na by L. bicolor and Mn by H. leucosarx were observed in the plants growing in the experimental substrate. It has been demonstrated that the selection of substrates constitutes an important factor in the production of ectomycorrhizal plants and that the three evaluated species of edible ectomycorrhizal mushrooms have an enormous potential in the controlled mycorrhization of P. greggii.

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.

Detección del sinanamorfo Cylindrocarpon cylindroides var. Tenue sobre cancros causados por Neonectria fuckeliana en Pinus radiata / Detection of sinanamorph Cylindrocarpon cylindroides var. tenue on Neonectria fuckeliana cankers in Pinus radiata

El presente trabajo tuvo por finalidad reportar y analizar la presencia de estructuras fungosas (esporodoquios) sobre cancros causados por Neonectria fuckeliana en árboles de Pinus radiata en Chile. Las muestras fueron colectadas en las Regiones de La Araucanía y de Los Ríos y fueron analizadas en el laboratorio. Los resultados de los análisis morfológicos y taxonómicos permitieron concluir que los esporodoquios hallados correspondían al sinanamorfo de Neonectria fuckeliana (forma cylindrocarpon cylindroides var tenue), siendo el primer reporte en el país. Estos antecedentes contribuyen al conocimiento y comprensión del ciclo patológico y epidemiológico de Neonectria fuckeliana en plantaciones de Pinus radiata.

The present work was aimed at reporting and analyzing the presence of fungal structures (sporodochia) present on cankers caused by Neonectria fuckeliana in Pinus radiata trees in Chile. The samples were collected in the regions of La Araucanía and Los Ríos and analyzed in the laboratory. The results of morphological and taxonomic analysis led to the conclusion that thesporodochia found corresponded to sinanamorph of Neonectria fuckeliana (cylindrocarpon cylindroides var. tenue morph), the first report in the country. This work contribute to understanding and comprehension of the disease cycle and epidemiology of Neonectria fuckeliana on plantations of Pinus radiata.

Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in Portugal

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have beneficial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were collected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporocarps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effectiveness of the techniques used to amplify DNA polymorphic sequences, which enhances the ­characte­rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.

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.

Growth response of Pinus densiflora seedlings inoculated with three indigenous ectomycorrhizal fungi in combination

Pinus densiflora seedlings were inoculated with three indigenous ectomycorrhizal fungi (Cenococcum geophilum, Rhizopogon roseolus and Russula densifolia) in single-, two-, and three-species treatments. After 8 months, the colonization rates of each ectomycorrhizal species, seedling growth and the nutrition were assessed in each treatment. P. densiflora seedlings inoculated with different ECM species composition showed an increase in height and basal diameter and improved seedling root and shoot nutrition concentrations compared to control treatment. Generally, combined inoculation had a more positive influence on the seedlings than the single inoculation. The three-species inoculation presented the highest growth and basal diameter and concentration of most nutrients except potassium. In conclusion, the results provided strong evidence for benefits of combined inoculation with the indigenous ectomycorrhizal fungi on P. densiflora seedlings under controlled conditions.

Heavy metal toxicity on dehydrogenase activity on rhizospheric soil of ectomycorrhizal pine seedlings in field condition.

The present investigation was carried out to study the toxicity of heavy metals (Zn, Cd, Cu, Ni, Pb and Al) on the dehydrogenase activity of ectomycorrhizal (Suillus leutus, Scleroderma aurantium, Cenococcum graniforme and Boletus spp.) and non-mycorrhizal rhizospheric soil of pine seedlings. The treatment of heavy metals adversely affected the dehydrogenase activity. Inoculation of mixed ectomycorrhizal fungi harbored increased activity of dehydrogenase. It was observed that in absence of ectomycorrhizae, heavy metals drastically reduced the enzyme activity at higher concentration of metals.