PCR–DGGE method for in planta detection and identification of Phytophthora species

We have developed a method to detect multiple species of Phytophthora directly in infected plant tissues. The method is based on the polymerase chain reaction and uses Phytopththora‐specific primers and denaturing gradient gel electrophoresis (PCR–DGGE). The method distinguished most of the 16 Phytophthora species tested. Very closely related species might not, however, be identified using the method. The detection efficiency was high and successful in eight different plant tissues tested. The PCR–DGGE detection tool developed here will be a fast and inexpensive method suitable for pathogen surveys and research programmes.     

The North American P group of Heterobasidion annosum s.l. is widely distributed in Pinus pinea forests of the western coast of central Italy

The distribution of the North American P group of Heterobasidion annosum s.l., recently reported from a Pinus pinea forest in the surroundings of Rome, was studied using mating tests and DNA fingerprinting (mitochondrial DNA, random amplified microsatellite technique and two group‐specific markers). This fungus is present in several forests and small plantations along the Tyrrhenian coast of the Italian peninsula, within an area approximately 100 km long, extending from Fregene in the north to the National Park of Circeo in the south, and 27 km wide including the city of Rome. In pine forests of Castelporziano, Castel Fusano and Anzio, where US troops resided during the Second World War, the North American P group is more frequent than the European P group. The low number of mating alleles in the Italian population of the North American P group supports the hypothesis of its origin from a small number of introductions. The near 100% sexual compatibility between the North American and European P groups, together with inconsistencies in results obtained with different identification methods of these groups, suggests that hybridization between the North American and European P populations occurs occasionally.     

FDG‐PET IN HEALTHY AND EPILEPTIC LAGOTTO ROMAGNOLO DOGS AND CHANGES IN BRAIN GLUCOSE UPTAKE WITH AGE

Regional cerebral metabolism and blood flow can be measured noninvasively with positron emission tomography (PET). 2‐[¹⁸F]fluoro‐2‐deoxy‐D‐glucose (FDG) widely serves as a PET tracer in human patients with epilepsy to identify the seizure focus. The goal of this prospective study was to determine whether juvenile or adult dogs with focal‐onset epilepsy exhibit abnormal cerebral glucose uptake interictally and whether glucose uptake changes with age. We used FDG‐PET to examine six Lagotto Romagnolo dogs with juvenile epilepsy, two dogs with adult‐onset epilepsy, and five control dogs of the same breed at different ages. Three researchers unaware of dog clinical status visually analyzed co‐registered PET and magnetic resonance imaging (MRI) images. Results of the visual PET analyses were compared with electroencephalography (EEG) results. In semiquantitative analysis, relative standard uptake values (SUV) of regions of interest (ROI) drawn to different brain regions were compared between epileptic and control dogs. Visual analysis revealed areas of hypometabolism interictally in five out of six dogs with juvenile epilepsy in the occipital, temporal, and parietal cortex. Changes in EEG occurred in three of these dogs in the same areas where PET showed cortical hypometabolism. Visual analysis showed no abnormalities in cerebral glucose uptake in dogs with adult‐onset epilepsy. Semiquantitative analysis detected no differences between epileptic and control dogs. This result emphasizes the importance of visual analysis in FDG‐PET studies of epileptic dogs. A change in glucose uptake was also detected with age. Glucose uptake values increased between dog ages of 8 and 28 weeks and then remained constant.     

CEREBRAL GLUCOSE UTILIZATION MEASURED WITH HIGH RESOLUTION POSITRON EMISSION TOMOGRAPHY IN EPILEPTIC FINNISH SPITZ DOGS AND HEALTHY DOGS

In human epileptic patients, changes in cerebral glucose utilization can be detected 2‐deoxy‐2‐[¹⁸F] fluoro‐d ‐glucose positron emission tomography (FDG‐PET). The purpose of this prospective study was to determine whether epileptic dogs might show similar findings. Eleven Finnish Spitz dogs with focal idiopathic epilepsy and six healthy dogs were included. Dogs were examined using electroencephalography (EEG) and FDG‐PET, with epileptic dogs being evaluated during the interictal period. Visual and semi‐quantitative assessment methods of FDG‐PET were compared and contrasted with EEG findings. Three independent observers, unaware of dog clinical status, detected FDG‐PET uptake abnormalities in 9/11 epileptic (82%), and 4/8 healthy dogs (50%). Occipital cortex findings were significantly associated with epileptic status (P = 0.013). Epileptic dogs had significantly lower standardized uptake values (SUVs) in numerous cortical regions, the cerebellum, and the hippocampus compared to the control dogs. The lowest SUVs were found in the occipital lobe. White matter normalized and left‐right asymmetry index values for all pairs of homologous regions did not differ between groups. Visual evaluation of the EEGs was less sensitive (36%) than FDG‐PET. Both diagnostic tests were consensual and specific (100%) for occipital findings, but EEG had a lower sensitivity for detecting lateralized foci than FDG‐PET. Findings supported the use of FDG‐PET as a diagnostic test for dogs with suspected idiopathic epilepsy. Visual and semiquantitative analyses of FDG‐PET scans provided complementary information. Findings also supported the theory that epileptogenesis may occur in multiple brain regions in Finnish Spitz dogs with idiopathic epilepsy.     

Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase

Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.     

Adaptive optimization of crop production and nitrogen leaching abatement under yield uncertainty

The study develops a bio-economic crop management model that internalizes the environmental cost of nitrate pollution, accounts for stochastic weather, and includes an option for split fertilization. The integrated model is designed to indicate whether a producer can benefit from applying fertilizer several times during the growing season, in response to crop needs, rather than a single time, at sowing. The model is parameterized for the cultivation of spring malting barley (Hordeum vulgare L.) in Southern Finland. The costs of negative externalities from nitrogen leaching are internalized in the landowner’s decision problem through a pollution tax. The results indicate that without a pollution tax a single application of fertilizer gauged to meet the needs of the entire season is optimal. With a tax, the benefits of split application - applying varying amounts of fertilizer at selected stages of the growing season - increase significantly. In comparison to a single application of fertilizer at sowing, split fertilization improves yields, increases the total amount of fertilizer used, and reduces nitrogen leaching.     

Cronartium rust sporulation on hemiparasitic plants

Susceptibility of potential alternate host plants to pine stem rusts belonging to Cronartium spp. was artificially tested in Finland during 2012–2013. Forty‐three species representing 11 plant families were inoculated in the laboratory; 34 species (11 families) were inoculated in the greenhouse with aeciospores of Cronartium flaccidum or C. ribicola. Twenty‐one selected species (10 families) were also exposed to natural inoculum of C. flaccidum in the field in two severely affected Pinus sylvestris stands. After 5–8 weeks’ incubation, C. flaccidum sporulated on 17 species (nine families) in the laboratory, 17 species (eight families) in the greenhouse and seven species (five families) in the field. Cronartium ribicola sporulated on three species (three families) in the laboratory or greenhouse. All of the hemiparasitic plants that belong to Orobanchaceae were infected by C. flaccidum, and several species supported rust sporulation when exposed to natural inoculum. Susceptible species belonged to genera Veronica, Euphrasia, Castilleja, Pedicularis, Rhinanthus, Saxifraga, Loasa, Ribes, Tropaeolum, Swertia, Physalis, Nicotiana, Hyoscyamus, Paeonia, Apocynum, Impatiens, Vincetoxicum and Myrica.     

HPLC determination of extractable and unextractable proanthocyanidins in plant materials

This study developed a method for the determination of extractable and unextractable proanthocyanidins. Extractable proanthocyanidins were separated according to their degree of polymerization using normal phase HPLC. Unextractable proanthocyanidins were measured after acid-catalyzed depolymerization as flavan-3-ols (terminal units) and benzylthioethers (external units). Electrospray ionization mass spectrometry (ESI-MS) was used for the identification of proanthocyanidins in the samples. Hubaux-Vos detection limits were 0.01-0.15 ng/injection for extractable proanthocyanidins, with recovery rates from 69 to 91%. Detection limits for unextractable proanthocyanidin derivatives were 0.002-0.035 ng/injection with 80% recovery. The developed method was applied to the analysis of several fruit and berry samples. Results showed great variation in the proportion of unextractable proanthocyanidins in total proanthocyanidin content between samples, being highest in the green variety of table grape (63%) and lowest in the apple cultivar 'Valkeakuulas' (4.1%). The method reported herein is reliable and gives valuable information on the nature of proanthocyanidins in plant-derived foods.     

Hybridization between two biotypes of Gremmeniella abietina var. abietina in artificial pairings

Summary The ability of the European biotypes A and B of Gremmeniella abietina var. abietina to hybridize in artificial pairings was tested. Using three types of molecular markers progenies with parental markers mixed indicating that the two biotypes can hybridize were observed. However, the resulting progeny had a low rate of successful germination, grew poorly on artificial medium and one was most probably a partial diploid or heterokaryon. Thus, both biotypes are genetically distinct populations and should be described as separate species as soon as sufficient discriminating information is available.     

Phenolic acids in berries, fruits, and beverages

The contents of soluble and total phenolic acids were analyzed in samples of 29 berries and berry products, 24 fruits and fruit peels, and 12 beverages. Variation of phenolic acids in berries was also studied. Soluble phenolic acids were extracted with methanolic acetic acid, and a tentative quantification was performed by high-performance liquid chromatography (HPLC). The total phenolic acid content was determined by HPLC after alkaline and acid hydrolyses. The content of total phenolic acids as aglycones in the above samples varied from 0 (pear cider) to 103 mg/100 g fresh weight (rowanberry). Besides rowanberry, the best phenolic acid sources among berries were chokeberry (96 mg/100 g), blueberry (85 mg/100 g), sweet rowanberry (75 mg/100 g), and saskatoon berry (59 mg/100 g). Among fruits, the highest contents (28 mg/100 g) were determined in dark plum, cherry, and one apple variety (Valkea Kuulas). Coffee (97 mg/100 g) as well as green and black teas (30-36 mg/100 g) were the best sources among beverages. Caffeic acid dominated in all of these samples except in tea brews. Variation in the phenolic acid contents of the berries was either small or moderate.