板栗疫病菌致病力分化的研究

中国板栗(Castanea mollissima Blume)对板栗疫病菌(Cryphonectria parasitica(Murr.)Barr)具有强的抗病力。但近十多年来,我国许多省份均发现有板栗疫病,据调查,广西有19个县市存在栗疫病,有的地方发病还相当严重。为了解释疫病发生的上述情况,并为生产和该病菌的更深入研究提供指导,作者在广西桂林、南宁、柳州、梧州、河池等地收集菌株,对疫病菌致病性的分化进行了研究,现将结果报道如下。 1 材料和方法 1.1 毒力参照菌株 法国已知弱毒株EPF为毒力参照株。 1.2 栗疫病菌的采集、分离和单孢纯化 1.2.1 标本采集:1988年12月～1989年6月,采自广西桂林、南宁、柳州、梧州、河池等地板栗病株。 1.2.2 菌株分离和纯化:将病枝或病树皮,用经灯焰灼烧过的刀片削去表皮,取坏死内皮约5×5mm^2,压于PDA上,28℃,光照培养,产孢后用微块法进行单孢纯化。 1.3     

Identification and characterisation of traditional chestnut varieties of southern Spain using morphological and simple sequence repeat (SSRs) markers

The maintenance of plant genetic resources requires the identification of places and agroforestry systems that support high levels of genetic diversity. The aim of this work was to clarify the number and names of chestnut ( Castanea sativa ) cultivars in the south of Spain. Accordingly, 100 grafted chestnut trees corresponding to 34 traditional names were evaluated using 10 qualitative morphological traits and seven simple sequence repeat (SSRs) markers. We identified 38 varieties, 12 in the Huelva region and 26 in Malaga, and synonymies and homonymies were discussed. This work demonstrated that the joint use of morphological traits and SSR markers is an efficient method to evaluate the agrobiodiversity of chestnut in this region. It was also stated that chestnut production in the south of Spain is a traditional system using varieties developed ' in situ '. This constitutes a genuine system of on-farm conservation, which is now threatened. Consequently, adopting strategies for its safeguarding are urgently recommended.     

The emission of corrosive vapours by wood. Sweet-chestnut (Castanea sativa) and wychelm (Ulmus glabrau) O-acetyl-4-O-methylglucuronoxylans extracted with dimethyl sulphoxide

1. O-Acetylated 4-O-methylglucuronoxylans were isolated from sweet chestnut and wych elm, either green or incubated at 48 degrees and 100% relative humidity for 36 weeks. 2. The chlorine-ethanolamine method of delignification resulted in a 50% loss of O-acetyl groups from green wych elm compared with an 18% loss from green sweet chestnut. 3. The acid-chlorite method gave an acceptable loss of O-acetyl groups in three cases, but incubated sweet chestnut showed a 44.6% loss. However, it is believed that this is due to the loss of simple O-acetylated xylose sugars resulting from glycosidic hydrolysis, rather than removal of O-acetyl groups by direct hydrolysis. Assuming that this occurs in a random manner, it is unlikely to have much structural significance. 4. Dimethyl sulphoxide extraction of chestnut holocellulose and elm holocellulose, green and incubated, yielded O-acetyl glucuronoxylans containing 10.2, 3.8, 13.1 and 7.7% O-acetyl groups respectively. 5. The location of these O-acetyl groups was determined by Bouveng's method in which phenyl isocyanate is used as a blocking group.     

Functional Organization of a Multimodular Bacterial Chemosensory Apparatus

Chemosensory systems (CSS) are complex regulatory pathways capable of perceiving external signals and translating them into different cellular behaviors such as motility and development. In the δ-proteobacterium Myxococcus xanthus, chemosensing allows groups of cells to orient themselves and aggregate into specialized multicellular biofilms termed fruiting bodies. M. xanthus contains eight predicted CSS and 21 chemoreceptors. In this work, we systematically deleted genes encoding components of each CSS and chemoreceptors and determined their effects on M. xanthus social behaviors. Then, to understand how the 21 chemoreceptors are distributed among the eight CSS, we examined their phylogenetic distribution, genomic organization and subcellular localization. We found that, in vivo, receptors belonging to the same phylogenetic group colocalize and interact with CSS components of the respective phylogenetic group. Finally, we identified a large chemosensory module formed by three interconnected CSS and multiple chemoreceptors and showed that complex behaviors such as cell group motility and biofilm formation require regulatory apparatus composed of multiple interconnected Che-like systems.     

A genetic linkage map of European chestnut (Castanea sativa Mill.) based on RAPD, ISSR and isozyme markers

A genetic linkage map of European chestnut (Castanea sativa Mill.) based on RAPD, ISSR and isozyme markers was constructed using the two-way pseudo-testcross strategy. A total of 96 individuals from a F₁ full-sib family was genotyped with 381 molecular markers (311 RAPDs, 65 ISSRs, 5 isozymes). Markers in testcross configuration, segregating 1:1, were used to establish two separate maternal and paternal maps including 187 and 148 markers, respectively. The markers identified 12 linkage groups based on the haploid number of chestnut. The female and male framework maps reached a total length of 720 and 721 cM (Kosambi), respectively, representing a 76% and 68% coverage of the overall genome. A total of 46 markers, found in intercross configuration, segregating 3:1 and 1:2:1, were used to identify homologous linkage groups between parental maps; out of 12 linkage groups 11 could be joined. RAPD and ISSR markers showed a good and comparable reliability, allowing for the first time the establishment of a saturated linkage map for European chestnut. These maps will be a starting point for studies on the structure, evolution and function of the chestnut genome. Identification of QTLs for adaptive traits in chestnut will be the primary target. Received: 3 July 2000 / Accepted: 16 October 2000     

Genetic structure of wild sweet chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill.) populations in northwest of Spain and their differences with other European stands

The genetic structure of 15 wild populations of Castanea sativa located in Galicia (northwest of Spain) were compared with another 10 Spanish, 2 Italian, and 2 Greek populations using 9 nuclear microsatellites. Additionally, 41 Castanea crenata, 19 Castanea mollissima and 54 accessions of varieties were used to detect the presence of Asiatic germplasm and grafted varieties in wild stands. Bayesian methods detected two main clusters in the European populations of sweet chestnut: a Northern Iberian Peninsula cluster included the northern and northwestern Spanish populations and a Mediterranean cluster contained central and southern Spanish, Italian and Greek populations. Further subdivisions were also detected. The Northern Iberian Peninsula cluster was divided into Cantabrian and Atlantic clusters and the Mediterranean cluster was divided into western and eastern Mediterranean clusters. Several populations were highly affected by domestication because they were classified as admixed among the four defined groups. Negative values for the fixation index were detected in northern and northwestern Spain showing an excess of heterozygotes that could be explained by the introduction of grafted varieties and its introgression with local populations or by natural selection in favor of heterozygotes produced by ink disease. The combined analysis with the Asiatic species also indicated their presence and possible introgression in the wild populations of northwestern Spain. The results of this study suggest that the current genetic structure of sweet chestnut can be explained by existence of several refugia during Pleistocene and also by human activity that changes the structure of the populations.     

Genetic characterisation of traditional chestnut varieties in Italy using microsatellites (simple sequence repeats) markers

European chestnut (Castanea sativa) is an important multipurpose tree that has been cultivated for wood and fruit in the Mediterranean basin since ancient times. Cultivation of traditional chestnut varieties has a long tradition in Italy, where cultivars have been selected over centuries as a function of the best nut traits. In this study, 94 grafted chestnuts corresponding to 26 representative cultivars from Italy were evaluated by seven simple sequence repeat (SSR) markers to establish whether they corresponded to varieties in the narrow sense. The results allowed 20 genotypes to be identified that corresponded to the same number of clones. In total, 52 alleles were identified, eight of which were exclusive. Cases of homonymies and synonymies were detected. Moreover, our results highlighted a considerable genetic uniformity among ‘Marrone-type’ cultivars and, on the contrary, a high genetic diversity among the evaluated cultivars demonstrating that this is a valuable germplasm and an important genetic resource to be preserved.     

Combining plant and animal traits to assess community functional responses to disturbance

There is a general consensus that functional traits are reliable indicators of adaptation of organisms to particular environmental characteristics. In this study we relate the combined distributions of species traits of plants and animals to disturbance regimes in chestnut forests of southern Switzerland affected by regular winter fires. We used co‐inertia analysis for combining the trait response of 471 invertebrate species (117 001 individuals) and 81 plant species at 23 sites with different fire and cutting histories. Trait response was assessed by calculating the variation in weighted mean traits averaged over the communities and by using mean traits in multivariate analyses. The analysis showed a strong association between plant and animal traits under fire constraints (Monte‐Carlo test, p=0.0045). Plants and animal distributions show parallel trends in responses to fire which selects traits relating to persistence (ability to survive), resilience (ability to recover) and mobility. Warmth‐demanding insects, herbivores, flying carnivores and pollinators were associated with recent fires, as were annual, ruderal and light‐demanding plant species with long flowering duration. Small arthropods feeding on dead wood and those with narrow habitat requirements were associated with low fire frequency and unburnt sites, as were competitive plants with large seeds favoring moist sites. The spatial association between plant and animal traits reflected adaptations that promote survival in the disturbance regime, while the disturbance acts as an environmental filter on the distribution and assemblage of the trait values within communities. This combined analysis of plant and invertebrate traits distributions illustrates how community and ecosystem responses can be monitored and the results generalized across localities and disturbance types. Analyses of traits that cross trophic levels provide powerful and promising tools for validating management procedures and controlling ecosystem functions.     

Taxonomy and ecology of <Emphasis Type="Italic">Castanea sativa</Emphasis> Mill. forests in Greece

Castanea sativa Mill. is an important species of the Balkan Peninsula with high ecological and economic value. This article contributes to a phytosociological synsystematic approach of the C. sativa plant communities in Greece that covers the southern part of the Balkan Peninsula and describes the ecological parameters involved in their distribution and taxonomy. Phytosociological data have been collected from 14 mountainous areas across continental Greece, which are considered representative of the main forest types of C. sativa in the area. The 14 mountains demonstrate a variety of environmental conditions. Five communities (associations) and seven sub-communities (sub-associations) have been identified and described using TWINSPAN, Corespondence Analysis and the Braun-Blanquet classification scheme. Castanea sativa (chestnut) forests of Greece can be distinguished as three broad groups, defined primarily by their geographical distribution. In northern and central Greece chestnut stands are characterized by their high floristic homogeneity, which is reflected in the identification of only one community per region. Chestnut forests in northern Greece, in particular, exhibit strong floristic similarities with those of the rest of the Balkans. In southern Greece, on the other hand, the southern distribution limit of chestnut in the Balkans, there is a greater floristic diversity between the sampled mountains which is reflected in the presence of three confirmed communities and two sub-associations. Several factors have been identified as important in determining the structure and floristic composition of chestnut forests in Greece. Whilst climate and the grazing regime are influential, the degree of silvicultural management appears to be the most important factor determining the floristic composition of chestnut forests and their long-term sustainability.     

A New Measure of Functional Evenness and Some of Its Properties

Functional evenness is increasingly considered an important facet of functional diversity that sheds light on the complex relationships between community assembly and ecosystem functioning. Nonetheless, in spite of its relevant role for ecosystem functioning, only a few measures of functional evenness have been proposed. In this paper we introduce a new measure of functional evenness that reflects the regularity in the distribution of species abundances, together with the evenness in their pairwise functional dissimilarities. To show how the proposed measure works, we focus on changes in functional evenness calculated from Grime’s classification of plant strategies as competitors (C), stress-tolerators (S) and ruderals (R) along a post-fire successional gradient in temperate chestnut forests of southern Switzerland.     

Spatial analysis of nuclear and mitochondrial RFLP genotypes in populations of the chestnut blight fungus, Cryphonectria parasitica

Spatial structure of both nuclear and mitochondrial RFLPs were studied in several populations of the chestnut blight fungus, Cryphonectria parasitica, using a variety of spatial autocorrelation tests designed to detect nonrandom patterns. Fungal individuals were sampled from cankers on infected chestnut trees, and the location of each tree was mapped. Single-locus nuclear RFLPs, nuclear fingerprints, and mitochondrial DNA haplotypes were determined for each individual. Individuals with the same DNA fingerprint genotypes occurred closer together than would be expected at random in four of the five plots, while mitochondrial DNA haplotypes were aggregated in all five plots. Genetic distances between individuals, expressed as one minus the proportion of shared restriction fragment size classes for fingerprints and mitochondrial haplotypes, were significantly correlated with Euclidean distances between individuals in four of the five populations, but these correlations were very weak (r < 0.18). The same DNA fingerprint and single-copy nuclear RFLP alleles occurred on the same trees or immediately neighbouring trees more often than would be expected at random. Most of the aggregation for all three genetic markers occurred among individuals within the same cluster of chestnut stems or on neighbouring trees. Lack of spatial autocorrelation in one population was probably due to sampling on a larger scale that was too coarse to detect any patterns. Significant aggregation of genotypes in C. parasitica is most likely caused by some degree of restricted dispersal within populations. The implications of restricted dispersal are discussed in relation to the breeding system and isolation by distance in populations of. C. parasitica.     

Pedigree estimation of the (sub) population contribution to the total gene diversity: the horse coat colour case

A method to quantify the contribution of subpopulations to genetic diversity in the whole population was assessed using pedigree information. The standardization of between- and within-subpopulation mean coancestries was developed to account for the different coat colour subpopulation sizes in the Spanish Purebred (SPB) horse population. The data included 166264 horses registered in the SPB Studbook. Animals born in the past 11 years (1996 to 2006) were selected as the 'reference population' and were grouped according to coat colour into eight subpopulations: grey (64 836 animals), bay (33 633), black (9414), chestnut (1243), buckskin (433), roan (107), isabella (57) and white (37). Contributions to the total genetic diversity were first assessed in the existing subpopulations and later compared with two scenarios with equal subpopulation size, one with the mean population size (13 710) and another with a low population size (100). Ancestor analysis revealed a very similar origin for the different groups, except for six ancestors that were only present in one of the groups likely to be responsible for the corresponding colour. The coancestry matrix showed a close genetic relationship between the bay and chestnut subpopulations. Before adjustment, Nei's minimum distance showed a lack of differentiation among subpopulations (particularly among the black, chestnut and bay subpopulations) except for isabella and white individuals, whereas after adjustment, white, roan and grey individuals appeared less differentiated. Standardization showed that balancing coat colours would contribute preserving the genetic diversity of the breed. The global genetic diversity increased by 12.5% when the subpopulations were size standardized, showing that a progressive increase in minority coats would be profitable for the genetic diversity of this breed. The methodology developed could be useful for the study of the genetic structure of subpopulations with unbalanced sizes and to predict their genetic importance in terms of their contribution to genetic variability.     

Predator complex of the horse chestnut leafminer Cameraria ohridella: identification and impact assessment

Abstract:  The control of Cameraria ohridella Deschka and Dimic (Lepidoptera, Gracillariidae) by natural enemies in Europe is poor. Thus, in the past 15 years epidemic population densities of the moth regularly caused a continuous pre-mature defoliation of horse chestnut trees, Aesculus hippocastanum L.. Whereas several studies investigated the parasitoid complex of the leafminer and revealed its inefficiency, only little is known about the predators of C. ohridella . The aim of this study was to identify the predator complex in European horse chestnut stands by visual observations and exposure experiments and to assess the impact of different predator guilds on C. ohridella populations by an exclusion experiment. Of all potential predators observed, only blue tits, Parus caeruleus L., great tits, Parus major L., marsh tits, Parus palustris L. and the southern oak bushcricket Meconema meridionale (Costa) were found to prey on the pre-imaginal leafminer stages. Bushcrickets have to our knowledge never before been recorded preying on leafminers; moreover M. meridionale showed a measurable negative impact on the C. ohridella population. However, birds seem to be of greater importance as predators of C. ohridella than arthropods. Predation rates of birds ranged from 2 to 4% of leafminer populations, which is of the same order of magnitude as published parasitism rates. We conclude that the augmentation of tit densities, for example via nesting boxes in chestnut trees, should be considered as a component of an integrated control strategy against the horse chestnut leafminer.     

Fluorescence quenching method for the determination of carbazochrome sodium sulfonate with aromatic amino acids

In Britton‐Robinson (BR) buffer medium (pH 3.3), carbazochrome sodium sulfonate (CSS) can react with some aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) to form a 1:1 complex by electrostatic attraction, aromatic stacking interaction and Van der Waals' force, resulting in fluorescence quenching of these amino acids. Maximum quenching wavelengths were located at 352 nm (CSS‐Trp system), 303 nm (CSS‐Tyr system) and 284 nm (CSS‐Phe system), respectively. The fluorescence quenching value (ΔF) was proportional to the concentration of CSS in a certain range. The fluorescence quenching method for the determination of CSS showed high sensitivity, with detection limits of 31.3 ng/mL (CSS‐Trp system), 44.6 ng/mL (CSS‐Tyr system) and 315.0 ng/mL (CSS‐Phe system), respectively. The optimum conditions of the reaction conditions and the effect of coexisting substances were investigated and results showed that the method had good selectivity. The method was successfully applied for the rapid determination of CSS in blood and urine samples. Based on the bimolecular quenching constant K_q, the effect of temperature and Stern‐Volmer plots, this study showed that quenching of fluorescence of amino acids by CSS was a static quenching process. Copyright © 2012 John Wiley & Sons, Ltd.     

Heterozygote advantage in the American chestnut, Castanea dentata (Fagaceae)

The American chestnut (Castanea dentata; Fagaceae) was a dominant canopy tree in the Appalachian Mountains of North America. Since the introduction of the chestnut blight fungus (Cryphonectria parasitica; Valsaceae) in America, the American chestnut has been reduced to a predominantly clonal, understory species. Our objective was to determine whether the ecological changes and absence of new recruits have influenced the population genetics of American chestnut. Leaf samples were collected from four populations in southwestern Virginia. Electrophoretic data from five polymorphic loci were used to determine the genetic diversity and population structure of the populations and subpopulations. Growth data and infection status were recorded for one of the populations to determine their relationship with heterozygosity. F statistics revealed a significant amount of differentiation among subpopulations and an excess of heterozygotes within subpopulations. Heterozygous individuals also had higher rates of vegetative growth. The superior performance and excess of heterozygotes suggests that selection favors heterozygous individuals. The prolonged absence of sexual reproduction in C. dentata has allowed subtle fitness differences to accumulate to the extent that they have had significant effects on the genetics of chestnut populations.     

Differential response among chestnut traditional varieties to the attack of Cydia splendana

Fruit production of Castanea sativa Miller (Fagaceae) in southern Spain is carried out in traditional orchards using a large number of local varieties. The main areas of production are located in the Natural Park of Sierra de Aracena y Picos de Aroche (Huelva) and the Genal river Valley (Malaga). These agroforestry systems have shown a high genetic diversity and represent an example of in situ conservation. In this region, chestnut mainly suffers from attacks by Cydia splendana (Hübner) (Lepidoptera: Tortricidae), whose larvae feed on the fruits, thereby reducing their quality and economic value. The aim of this study was to evaluate the differential response between the chestnut local varieties to the attack of C. splendana. For that purpose, trees from the most representative varieties were evaluated on various farms. In cases where it was necessary, the trees were identified with a set of eight microsatellite markers. The infestation rate of C. splendana (percentage of infested fruits of the total fruits sampled per tree) differed among varieties, indicating a high genetic component in this effect. The results obtained in this study open the possibility of using this differential response in the control of C. splendana.     

Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.     

Differentiation of isolates of Discula umbrinella (Teleomorph: Apiognomonia errabunda) from beech, chestnut, and oak using randomly amplified polymorphic DNA markers.

Genetic variation in 30 isolates of Discula umbrinella derived from beech, chestnut, and oak was assessed using randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphic markers. Polymerase chain reaction amplifications with 17 primers produced 134 different DNA fragments. Three RAPD fragments were subsequently used for Southern hybridization. By these techniques up to four different individuals could be detected in the same leaf. The presence of several individuals within a single leaf indicates a finely tuned balance between the endophyte and its host. Cluster analysis of all arbitrary primed amplified DNA fragments showed that the isolates could be placed into four groups corresponding to their host origin. The high percentage of private RAPD variants within groups is consistent with low gene flow.     

Differentiation of Alpine marmot populations traced by DNA fingerprinting.

As revealed by allozyme studies, the genetic variation of the Alpine marmot (Marmota m. marmota) has been reduced by a species-wide bottleneck at the end of the last glaciation. Therefore the more variable microsatellite loci were used as a genetic marker system to investigate variablility and differentiation of four autochthonous and four allochthonous populations founded by the release of small numbers of individuals during the last 150 years. The microsatellite loci detected by the DNA-probe (ATCC)₄ were found to be polymorphic in all populations, but the amount of variation was lower than in comparable mammalian species. In spite of founder effects the variation in the allochthonous populations was not significanlty reduced compared to the autochthonous populations. The autochthonous populations from Austria and from the eastern part of Switzerland were genetically similar, only the population from western Switzerland was clearly differentiated from the others. In the allochthonous populations similarities in the microsatellite patterns reveal genetic affinities to putative autochthonous source populations of the founder individuals.