Ecological impacts of Phlebiopsis gigantea biocontrol treatment against Heterobasidion spp. as revealed by fungal community profiling and population analyses

Wood decay fungi belonging to the species complex Heterobasidion annosum sensu lato are among the most common and economically important species causing root rot and stem decay in conifers of the northern temperate regions. New infections by these pathogens can be suppressed by tree stump treatments using chemical or biological control agents. In Finland, the corticiaceous fungus Phlebiopsis gigantea has been formulated into a commercial biocontrol agent called Rotstop (Verdera Ltd.). This thesis addresses the ecological impacts of Rotstop biocontrol treatment on the mycoflora of conifer stumps. Locally, fungal communities within Rotstop-treated and untreated stumps were analyzed using a novel method based on DGGE profiling of small subunit ribosomal DNA fragments amplified directly from wood samples. Population analyses for P. gigantea and H. annosum s.l. were conducted to evaluate possible risks associated with local and/or global distribution of the Rotstop strain. Based on molecular community profiling by DGGE, we detected a few individual wood-inhabiting fungal species (OTUs) that seemed to have suffered or benefited from the Rotstop biocontrol treatment. The DGGE analyses also revealed fungal diversity not retrieved by cultivation and some fungal sequence types untypical for decomposing conifer wood. However, statistical analysis of DGGE community profiles obtained from Rotstop-treated and untreated conifer stumps revealed that the Rotstop treatment had not caused a statistically significant reduction in the species diversity of wood-inhabiting fungi within our experimental forest plots. Locally, ISSR genotyping of cultured P. gigantea strains showed that the Rotstop biocontrol strain was capable of surviving up to six years within treated Norway spruce stumps, while in Scots pine stumps it was sooner replaced by successor fungal species. In addition, the spread of resident P. gigantea strains into Rotstop-treated forest stands seemed effective in preventing the formation of genetically monomorphic populations in the short run. On a global scale, we detected a considerable level of genetic differentiation between the interfertile European and North American populations of P. gigantea. These results strongly suggest that local biocontrol strains should be used in order to prevent global spread of P. gigantea and hybrid formation between geographically isolated populations. The population analysis for H. annosum s.l. revealed a collection of Chinese fungal strains that showed a high degree of laboratory fertility with three different allopatric H. annosum s.l. taxa. However, based on the molecular markers, the Chinese strains could be clearly affiliated with the H. parviporum taxonomical cluster, which thus appears to have a continuous distribution range from Europe through southern Siberia to northern China. Keywords: Rotstop, wood decay, DGGE, ISSR fingerprinting, ribosomal DNA

The Biocontrol Agent Phlebiopsis gigantea: Efficacy and Impacts on the Stump Bacterial Biota and Conifer tree Defences

Phlebiopsis gigantea has been for a long time known as a strong competitor against Heterobasidion annosum and intensively applied as a biological control agent on stump surfaces of Picea abies in Fennoscandia. However, the mechanism underlying its antagonistic activity is still unknown. A primary concern is the possible impact of P. gigantea treatment on resident non-target microbial biota of conifer stumps. Additional risk factor is the potential of P. gigantea to acquire a necrotrophic habit through adaptation to living wood tissues. This study focused on the differential screening of several P. gigantea isolates from diverse geographical sources as well as the use of breeding approach to enhance the biocontrol efficacy against H. annosum infection. The results showed a significant positive correlation between growth rate in wood and high biocontrol efficacy. Furthermore, with aid of breeding approach, several progeny strains were obtained that had better growth rate and control efficacy than parental isolates. To address the issue of the potential of P. gigantea to acquire necrotrophic capability, a combination of histochemical, molecular and transcript profiling (454 sequencing) were used to investigate the interactions between these two fungi and ten year old P. sylvestris seedlings. The results revealed that both P. gigantea and H. annosum provoked strong necrotic lesions, but after prolonged incubation, P. gigantea lesions shrank and ceased to expand further. Tree seedlings pre-treated with P. gigantea further restricted H. annosum-induced necrosis and had elevated transcript levels of genes important for lignification, cell death regulation and jasmonic acid signalling. These suggest that induced localized resistance is a contributory factor for the biocontrol efficacy of P.gigantea, and it has a comparatively limited necrotrophic capability than H. annosum. Finally, to investigate the potential impact of P. gigantea on the stump bacterial biota, 16S rDNA isolated from tissue samples from stumps of P. abies after 1-, 6- and 13-year post treatment was sequenced using bar-coded 454 Titanium pyrosequencing. Proteobacteria were found to be the most abundant at the initial stages of stump decay but were selectively replaced by Acidobacteria at advanced stages of the decay. Moreover, P. gigantea treatment significantly decreased the bacterial richness at initial decay stage in the stumps. Over time, the bacterial community in the stumps gradually recovered and the negative effects of P. gigantea was attenuated.

Producción de biomasa de nacedero (Trichanthera gigantea) en diferentes escenarios de sombra y frecuencias de cortes, en el Rancho Ebenezer, Niquinohomo, Masaya

Con el objeto de contribuir al uso y manejo de nuevas especies forrajeras como alternativas para la alimentación animal en las condiciones agroclimáticas del municipio de Niquinohomo, específicamente en el Rancho Agropecológico de Especies Menores Ebenezer (RAEME). Se realizó un trabajo investigativo donde se evaluó las características morfoestructurales de nacedero (Trichanthera gigantea), bajo tres escenarios de sombra (umbra, penumbra y a pleno sol) y cuatro frecuencias de corte (30, 60, 90 y 120 días) y su efecto sobre la producción de biomasa y contenido de proteína de nacedero. El estudio se ubicó en la comarca Hoja Chigüe #1 a una distancia de 4 Km al sur del municipio de Niquinohomo, Departamento de Masaya con una altitud de 400 msnm, con latitud de 11° 52′ y longitud de 86°03′ . El promedio de precipitación anual es de 1200 mm, con periodos lluviosos generalmente en los meses de Julio a Noviembre. Con una temperatura mínimas de 22° C y máximas de 38° C, variando estas durante todo el año. Los suelos son de textura franco arcilloso y una profundidad efectiva de 40 cm. Entre las características morfoestructurales, se evaluaron el grosor, altura, número de tallos y rebrotes, para lo cual se realizaron mediciones sobre cinco plantas (para cada condición), sembrados a una distancia de 1 m entre surco y entre planta, las cuales se seleccionaron al azar. Al iniciar el estudio se hizo un corte de uniformidad; después se realizó el corte para cada frecuencia en estudio, cuantificando la producción de forraje comestible (hoja/tallo tierno y la proporción entre ellas). Durante el ensayo no se hizo ninguna aplicación de fertilizante. Se encontró que la producción de biomasa fue mejor cuando los cortes se realizaron cada 120 días, siendo estas mayores cuando estaban en condiciones de sombra y penumbra (31 y 32 t ha-1 respectivamente). En cambio los contenidos de proteína fueron mayores cuando los corteseran menores de 60 días, los cuales fluctuaron entre 20 y 23%. Encontrándose una relación inversa entre la edad de corte y producción de biomasa con respecto al contenido de proteína.

The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose.

Circadian clocks are 24-h timing devices that phase cellular responses; coordinate growth, physiology, and metabolism; and anticipate the day-night cycle. Here we report sensitivity of the Arabidopsis thaliana circadian oscillator to sucrose, providing evidence that plant metabolism can regulate circadian function. We found that the Arabidopsis circadian system is particularly sensitive to sucrose in the dark. These data suggest that there is a feedback between the molecular components that comprise the circadian oscillator and plant metabolism, with the circadian clock both regulating and being regulated by metabolism. We used also simulations within a three-loop mathematical model of the Arabidopsis circadian oscillator to identify components of the circadian clock sensitive to sucrose. The mathematical studies identified GIGANTEA (GI) as being associated with sucrose sensing. Experimental validation of this prediction demonstrated that GI is required for the full response of the circadian clock to sucrose. We demonstrate that GI acts as part of the sucrose-signaling network and propose this role permits metabolic input into circadian timing in Arabidopsis.

Producción de biomasa de nacedero (Trichanthera gigantea) en diferentes escenarios de sombra y frecuencias de cortes, en el Rancho Ebenezer. Niquinohomo, Masaya

Con el objeto de contribuir al uso y manejo de nuevas especies forrajeras como alternativas para la alimentación animal, se realizó un estudio en el Rancho Agroecológico de Especies Menores Ebenezer (RAEME), ubicado a 4 km del Municipio de Niquinohomo, Departamento de Masaya. El cual está a una altitud de 384 msnm, con latitud norte de 11 °52'25. 1 8" y longitud oeste de 86°05'14.89", precipitación media anual es de 1,200 mm, con periodos lluviosos generalmente en los meses de Julio a Noviembre y con temperaturas mínimas de 22° e y máximas de 38° e, variando estas durante todo el afio. Los suelos son de textura franco arcilloso y una profundidad efectiva de 40 cm. Se utilizaron plantaciones de nacedero (Trichanthera gigantea), bajo tres escenarios de sombra ( 4 hrs sol o penumbra, 8 hrs sol o sombra y más de 8 hrs sol), los que fueron establecidos hacia tres años y tenían espaciamientos de 1 m entre surcos y 1 m entre plantas. Además se realizaron cuatro frecuencias de corte (30, 60, 90 y 120 días). Se evaluaron las características morfoestructurales de nacedero, como: grosor, altura y número de tallos y rebrotes. Así como la producción de biomasa forrajera y el contenido de proteína. Al iniciar el estudio se hizo un corte de uniformidad. Posteriormente se seleccionaron cinco plantas sobre las cuales se evaluaron las características morfoestructurales, la producción de biomasa y el contenido de proteína. Se encontró que los mejores resultados de la morfoestructura de nacedero se presentan bajo condiciones de sombra o 8 hrs sol, seguido de la penumbra o 4 hrs sol, así mismo que la producción de biomasa fue mejor cuando los cortes se realizaron cada 120 días, siendo estas mayores cuando estaban en condiciones de sombra y penumbra (3 l y 32 t ha•' respectivamente). En cambio los contenidos de proteína fueron mayores cuando los intervalos de cortes eran menores (60 días), los cuales fluctuaron entre 20 y 23%. Encontrándose una relación inversa entre la edad de corte y producción de biomasa con respecto al contenido de proteína. Se concluye que el nacedero requiere de sombra para su buen comportamiento morfoestructural y productivo.

A Comparison of the Range and Movements of Acmaea digitalis Eschscholtz, Acmaea scutum Eschscholtz, Acmaea limatula Carpenter and Lottia gigantea Sowerby

Ten limpets (4 Acmaea digitalis , 4 Acmaea scutum, 1 Acmaea limatula, and 1 Lottia gigantea) were marked and their movements observed over a thirteen day period. Recordings of positions were made on a map, and the path of each was drawn on the map from day to day. Acmaea digitalis showed the greatest range, mostly in a vertical direction, and moved usually at night during high tide. Acmaea scutum showed a more limited range in a horizontal direction, and moved both day and night during high tide. Acmaea limatula had a horizontal range similar to A. scutum,, but exhibited no movement during the day time. Lottia gigantea had the most restricted range of any limpet studied, and moved only at night during high tide. This is a student paper done for a University of California Berkeley Zoology class. Since UCB didn't have its own marine lab at the time, it rented space at Hopkins Marine Station where this work was done. Gene Haderlie went on to earn his Ph.D. from Berkeley and later became a Professor at the Naval Post Graduate School in Monterey. (PDF contains 23 pages)

A Comparison of the SNP Variation in the Calcification PMCA Gene of Lottia gigantea between a Santa Barbara Population and a More Acidic Monterey Bay Population

As the atmospheric levels of CO2 rise from human activity, the carbonic acid levels of the ocean increase, causing ocean acidification. This increase in acidity breaks down the calcified bodies that many marine organisms depend upon. Upwelling regions such as Monterey Bay in California have pH levels that are not expected to reach the open ocean for a few decades. This study reviews one of the common intertidal animals of the California coast, the Owl Limpet Lottia gigantea, and its genetic variation of the plasma membrane Ca2+ ATPase (PMCA) in relation to the acidity of its environment. The PMCA protein functions in the calcification process of many organisms. Specifically in limpets, this gene functions to form its protective shell. Single-nucleotide polymorphisms (SNPs) were found among five sections of the gene to determine variation between the acidic environment population in Monterey, California and the non-acidic environment population in Santa Barbara, California. While some variation was determined, the Monterey Bay and Santa Barbara Lottia gigantea populations are not significantly distinct at the PMCA gene. Sections B, C, and D were found to be linked. Only one location in Section B was found to have an amino acid change within an exon. Section A has the strongest connection to the sampling location. Monterey individuals were seen to be more genetically recognizable, while Santa Barbara individuals showed slightly more variation. Understanding the trends of ocean acidification, upwelling region activities, and population genetics will assist in determining how the ocean environment will behave in the future.

A set of polymorphic microsatellite loci for Vriesea gigantea and Alcantarea imperialis (Bromeliaceae) and cross-amplification in other bromeliad species

Fifteen polymorphic microsatellite markers were isolated and characterized in two species of Bromeliaceae: Vriesea gigantea and Alcantarea imperialis. The number of alleles observed for each locus ranged from three to 16. The loci will be used for studies of the genetic structure of natural populations, reproductive biology, and evolutionary relationships among and within these genera. A cross-amplification test in 22 taxa suggests that the markers will be useful for similar applications in numerous other bromeliad species.

Aristolactams and Alkamides of Aristolochia gigantea

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Description of an injury in a human caused by a false tocandira (Dinoponera gigantea, Perty, 1833) with a revision on folkloric, pharmacological and clinical aspects of the giant ants of the genera Paraponera and Dinoponera (sub-family Ponerinae)

A partir de um acidente causado pela picada de uma formiga falsa tocandira na mão de um pescador amador, os autores descrevem os achados clínicos locais observados, tais como edema, eritema e dor excruciante e a evolução do envenenamento, que cursou com fenômenos sistêmicos imediatos, como sudorese fria, náuseas, vômitos, mal estar, taquicardia e linfadenopatia axilar à esquerda. Após três horas, a dor intensa persistia e o paciente apresentou um episódio de hematoquesia, sem história anterior de enfermidades do trato digestivo, hematológicas ou vasculares. O uso de analgésicos (Tramal® 300 mg/dia), água quente e gelo não melhorou a dor, que arrefeceu em oito horas, tendo permanecido por cerca de 24 horas. São apresentados ainda os aspectos folclóricos, farmacológicos e clínicos relacionados às picadas de tocandiras.

8-benzylberbine and N-oxide alkaloids from Aristolochia gigantea

From the ethanol extract of Aristolochia gigantea Mart an 8-benzylberbine-type alkaloid and an N-oxide were isolated together with pinitol and sequoyitol. Structural assignments were based on analysis of spectral data, mainly by 2D NMR spectroscopic techniques. (C) 1997 Elsevier B.V. Ltd.

Aristolactamas e alcamidas isoladas de Aristolochia gigantea Mart

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)