DIGITAL IMAGE ANALYSIS OF CHROMATIN FIBRE PHENOTYPE AFTER ‘IN SITU’ DIGESTION WITH RESTRICTION ENDONUCLEASES

Restriction Endonucleases (REs) may recognize, cleave and remove DNA from fixed chromatin producing specific chromosome banding patterns. However, the modifications produced in the chromatin fibre are not easy to evaluate and compare. The aim of the present investigation was to visualize differences resulting in the texture of the chromatin fibre from metaphase chromosomes after each digestion using digital image analysis (DIA) facilities. To this purpose, metaphase chromosomes derived from a L-929 mouse cell line were digested with different REs (AluI,HpaII andHaeIII). Since light microscopy does not permit the observation of the chromatin fibre, DIA was performed on digitalized images of metaphase chromosomes under electron microscopy. The application of a LUT (Look Up Table) within the DIA software assigns a colour to each grey level of a digital image. The results obtained using a particular LUT, which permits the discrimination of specific chromatin fibre phenotypes resulting from each digestion, are reported and compared with those obtained under the light microscope.     

The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits

Numerous vegetable crops are susceptible to powdery mildew, but cucurbits are arguably the group most severely affected. Podosphaera fusca (synonym Podosphaera xanthii ) is the main causal agent of cucurbit powdery mildew and one of the most important limiting factors for cucurbit production worldwide. Although great efforts have been invested in disease control, by contrast, many basic aspects of the biology of P. fusca remain unknown.
Taxonomy: Podosphaera fusca (Fr.) Braun & Shishkoff. Kingdom Fungi; Phylum Ascomycota; Subdivision Pezizomycotina; Class Leotiomycetes; Order Erysiphales; Family Erysiphaceae; genus Podosphaera ; species fusca.
Identification: Superficial persistent mycelium. Conidia in chains, hyaline, ellipsoid to ovoid or doliform, about 24–40 × 15–22 µm, with cylindrical or cone-shaped fibrosin bodies, which often germinate from a lateral face and produce a broad, clavate germ tube and cylindrical foot-cells. Unbranched erect conidiophores. Cleistothecia globose, mostly 70–100 µm in diameter, dark brown/black. One ascus per cleistothecium with eight ascospores.
Host range: Angiosperm species that include several families, such as Asteracea, Cucurbitaceae, Lamiaceae, Scrophulariaceae, Solanaceae and Verbenaceae.
Disease symptoms: White colonies develop on leaf surfaces, petioles and stems. Under favourable environmental conditions, the colonies coalesce and the host tissue becomes chlorotic and usually senesces early.
Control: Chemical control and the use of resistant cultivars. Resistance has been documented in populations of P. fusca to some of the chemicals registered for control.     

Structure‐function analysis of npr1 alleles in Arabidopsis reveals a role for its paralogs in the perception of salicylic acid

Salicylic acid (SA) is necessary for plant defence against some pathogens, whereas NPR1 is necessary for SA perception. Plant defence can be induced to an extreme by several applications of benzothiadiazole (BTH), an analogue of SA. Thus, plants that do not perceive BTH grow unaffected, whereas wild‐type plants grow stunted. This feature allows us to screen for mutants in Arabidopsis thaliana that show insensitivity to BTH in a high‐throughput fashion. Most of the mutants are npr1 alleles, with similar phenotypes in plant weight and pathogen growth. The mutations are clustered in the carboxyl‐terminal part of the protein, and no obvious null alleles were recovered. These facts have prompted a search for knockouts in the NPR1 gene. Two of these KO alleles identified are null and have an intermediate phenotype. All the evidence presented lead us to propose a redundancy in SA perception, with the paralogs of NPR1 taking part in this signalling. We show that the mutations recovered in the screening genetically interact with the paralogs preventing their function in SA signalling.     

A rapid PCR method to discriminate between Tomato yellow leaf curl virus isolates

Tomato yellow leaf curl virus (TYLCV) was recently divided into two different species: Tomato yellow leaf curl virus‐Israel (TYLCV‐Is) and Tomato yellow leaf curl virus‐Sardinia (TYLCV‐Sar). There are no rapid methods by which TYLCV viruses may be assigned to either TYLCV‐Is or TYLCV‐Sar species. In the present work, using an extensive alignment of begomovirus sequences, TYLCV‐specific primers were designed and tested which allow the specific amplification of DNA fragments from any isolate of TYLCV. Also, a primer was designed and tested which allows the specific amplification of TYLCV‐Sar. Furthermore, a combination of these primers was selected to develop a duplex PCR method, which has the potential to detect either TYLCV‐Is or TYLCV‐Sar. The PCR methods were also highly effective with minimal sample preparation and allowed direct amplification of TYLCV from infected leaf extracts. This approach may be used in the laboratory as a tool for rapid, large‐scale diagnostics of TYLCV‐infected samples.     

Are predatory birds effective secondary seed dispersers?

We have studied the unusual phenomenon of secondary seed dispersal of Lycium intricatum seeds on a small oceanic Atlantic island (Alegranza, Canarian Archipelago) in which a small frugivorous lizard ( Gallotia atlantica ) and two different predatory birds participate, a shrike ( Lanius excubitor ) and a kestrel ( Falco tinnunculus ). Endemic lizards that are common prey of both bird species consume Lycium fruits. Lizard remains were significantly matched with the presence of Lycium fruits in the regurgitation pellets of the two predatory birds. Seeds were found in 7.3% of the lizard droppings, 31.0% of kestrel pellets and 55.7% of shrike regurgitations. The mean number of seeds per dropping or pellet was 4.8 ±4 in lizard, 20.2 ±34.5 in shrike and 6.7 ±8.1 in kestrel. The percentage of viable seeds showed significant differences among all four treatments, decreasing in the following direction: seeds collected directly from plants (98.0%), shrikes (88.0%), lizards (72.3%), and kestrels (31.7%). Seeds from Lycium fresh fruits and shrike pellets showed significantly higher germination rates than those from lizard droppings and kestrel pellets. While lizards and shrikes are effective seed dispersers, kestrel gut treatment decreases seed viability. Seed viability is always higher than seed germination in each of the four treatments. In this island environment, Lycium seeds are under an important random influence during the seed dispersal process. Secondary seed dispersal seems to acquire a relevant dimension in small and remote insular environments or isolated continental systems where interactions among the different elements involved are intense, all of them are abundant native residents, and they have been coexisting for a long time. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 75 , 345–352.     

Seasonal decline in provisioning effort and nestling mass of Blue Tits Cyanistes caeruleus: experimental support for the parent quality hypothesis

A common feature of many birds breeding in seasonal environments is that fitness‐related parameters such as nestling mass or survival decline as the breeding season progresses. Consequently, there is a tendency for early breeders to have better reproductive performance than individuals breeding later in the season. This variation could be caused by factors associated with the date of laying, such as changing environmental conditions (date hypothesis), or by differences in parental quality between early and late breeders (parent quality hypothesis). To evaluate the relative importance of both hypotheses, we manipulated hatch dates of Blue Tits Cyanistes caeruleus by exchanging clutches with different incubation stages and assessed the impact on nestling mass, nestling diet and provisioning rates. Mean nestling mass declined significantly over the season. This was the combined result of differences in parental quality, which dominated in the early part of the season, and the influence of hatching date (date effect per se), which prevailed later in the season. Nestling diet composition was apparently uninfluenced by the manipulation, suggesting that deteriorating food supplies are the primary reason for the seasonal variation in the nestling diet. Counter to the date hypothesis, delayed parents did not feed their young less than control pairs did, but in fact exhibited higher provisioning rates. Our results suggest that in this population, parental quality seems to constrain post‐hatching reproductive performance and such intrinsic limitations may help to explain why certain individuals breed later.     

Quaternary structure changes and kinetics of urease inactivation by L-usnic acid in relation to the regulation of nutrient transfer between lichen symbionts

Abstract L-usnic acid inactivates urease by the formation of high molecular weight aggregates which can reach a maximum of 700,000 under experimentation conditions previously described. The substrate, urea, itself provokes temporary aggregation states which may be either active or inactive; the latter being reversible. However, these inactive aggregates, of 820,000 molecular weight, are irreversibly stabilized by L-usnic acid. The active aggregates with molecular weights oscillating from 605,000 to 650,000, depending on whether they are formed in the presence or absence of inactivator, may combine with the substrate to form an apparently normal ES complex.