Isolation and characterization of polymorphic microsatellites in European chestnut (Castanea sativa Mill.)

Thirteen polymorphic microsatellite loci were isolated from Castanea sativa (Mill.). Six contained dinucleotide repeats, six contained trinucleotide repeats, and one contained a compound microsatellite of a trinucleotide and a tetranucleotide repeat. The loci were characterized using C. sativa trees from three populations in the UK and the parents and six seedlings from a Turkish mapping population. The number of alleles revealed varied from two to 14 (mean = 5.15) per loci. Eight loci were found to be useful in the mapping family.     

Study of the temperature effect in three chestnut (Castanea sativa Mill.) cultivars' behaviour

The aim of this work was to analyse the effect of temperature in three chestnut cultivars, Aveleira, Judia and Longal. For this purpose, gas exchange, thylakoid membrane potential, photosynthetic pigment and lipid content data in July, September and October under different temperatures (31, 26 and 18 degrees C) were determined. With respect to gas exchanges, significant changes in photosynthesis rate of Aveleira were observed between July and September (7mumol CO2m(-2)s(-1)). In contrast, Judia and Longal showed a strong increase in this period, 6.1-8.5 and 4.9-6.7 micromol CO2m(-2)s(-1), for Judia and Longal, which represent an increase of about 15% and 43%, respectively. Similar patterns were detected in daylight photosynthesis measurements for Judia and Longal, in which an almost 60% decrease was observed, in contrast to 40% for Aveleira, from morning to midday, when temperatures increased from 27 to 34 degrees C. In addition to high photosynthetic rates in the hottest month, Aveleira was also the sunniest cultivar according its highest value on chlorophyll a/b ratio (3.65). Cultivars also presented maximal thylakoid membrane potential at different temperatures, with their values being 20.8, 17.8 and 17.2 degrees C for Aveleira, Longal and Judia, respectively. These results were also supported by thylakoid fatty acid composition which indicated that the unsaturation index of Aveleira (158) was the lowest in comparison with other two cultivars, 168 and 175, for Longal and Judia, respectively.     

Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.)

Here, we established a protocol for induction of somatic embryogenesis and plant regeneration from immature cotyledons of open-pollinated seeds of European chestnut (Castanea sativa Mill.) cultivars ‘Osmano?lu’ and ‘Sar?a?lama’. Basal media, Murashige and Skoog medium (MS), Driver and Kuniyuki Walnut medium (DKW), and Woody Plant Medium (WPM) supplemented with l-glutamine or casein hydrolysate, with or without silver nitrate, agar or gelrite, and various plant growth regulator (PGR) combinations were tested in initial cultures for induction of somatic embryos. The effects of initial cultures on the percentage of somatic embryos and average number of embryos per cotyledon explant, subcultured monthly, were determined at the end of 4 mo. Interactions were observed among the different treatments for ‘Osmano?lu’ cultivar, with the highest rates of somatic embryogenesis (4.7–9.7%) being obtained in MS, DKW, or WPM basal media supplemented with (1) 6-benzyladenine (BA; 1 mg/L)?+?kinetin (KIN; 2 mg/L)?+?indole-3-butyric acid (IBA; 0.01 mg/L); (2) BA (1 mg/L)?+?1-phenyl-3-(1,2,3-thiadiazol-5-yl; TDZ 0.1 mg/L)?+?IBA (0.01 mg/L), and (3) KIN (2 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L) PGR combinations plus l-glutamine or casein hydrolysate, with or without silver nitrate, and with either gelrite or agar. The highest percentages (12.0% and 11.2%) of somatic embryogenesis for ‘Sar?a?lama’ were obtained in DKW supplemented with PGR combinations of (1) BA (1 mg/L)?+?KIN (2 mg/L)?+?IBA (0.01 mg/L), (2) BA (1 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L), respectively. The average number of somatic embryos ranged between 0 and 0.65 per explant for ‘Osmano?lu’ and between 0 and 0.49 per ‘Sar?a?lama’ explant. For germination of somatic embryos, root, shoot, and plantlet regeneration, different treatments included desiccation, cold and gibberellic acid (GA₃), and BA alone or in combination with auxins (IBA or α-naphthaleneacetic acid, NAA; 0.1 mg/L). The highest rate of somatic embryos regeneration (27.5%) occurred using MS basal media with half-strength microelements containing 0.1 mg/L BA?+?0.1 mg/L NAA, after treatments of desiccation, or desiccation plus cold or GA₃ (3 mg/L).     

Effects of elevated CO2 on growth,photosynthesis and respiration of sweet chestnut (Castanea sativa Mill.)

Two year old sweet chestnut seedlings (Castanea sativa Mill) were grown in pots at ambient (350 µmol·mol^–1) and double (700 µmol·mol^–1) atmospheric CO₂ concentration in constantly ventilated greenhouses during entire growing seasons. CO₂ enrichment caused either no significant change or a decrease in shoot response, depending on yearly weather conditions. Similarly, leaf area was either reduced or unchanged under elevated CO₂. However, when grown under controlled conditions in a growth chamber, leaf area was enlarged with elevated CO₂.The CO₂ exchanges of whole plants were measured during the growing season. In elevated CO₂, net photosynthetic rate was maximum in May and then decreased, reaching the level of the control at the end of the season. End of night dark respiration of enriched plants was significantly lower than that of control plants; this difference decreased with time and became negligible in the fall. The original CO₂ level acted instantaneously on the respiration rate: a double concentration in CO₂ decreased the respiration of control plants and a reduced concentration enhanced the respiration of enriched plants. The carbon balance of a chestnut seedling may then be modified in elevated CO₂ by increased carbon inputs and decreased carbon outputs.     

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     

Reworking the idea of chestnut (Castanea sativa Mill.) cultivation in Roman times: New data from ancient Campania

Abstract

Charcoal analysis was carried out in two archaeological sites on the north slope of the Somma-Vesuvius volcano, not far from Naples. Both sites were inhabited between the 2nd century AD and AD 472, when a great Vesuvius eruption (so called Pollena eruption) buried them. In both sites, Castanea sativa wood was largely used for architectural structures as well as firewood. Ten ¹⁴C dates, spanning between the 1st and the 5th century AD, testify to a continuative use of this wood throughout the life of the sites. The comparison between archaeobotanical and pollen data from other sites in southern Italy suggests that chestnut woods were close to the sites studied and had a restricted spreading, possibly in a refugium area on Vesuvius' foothills. The match between archaeological charcoals and pollen data suggests that the Romans did not spread chestnut cultivation in the entire Campania region and that chestnut probably became a common timber in Campania starting from the 5th–6th century AD.     

Ectomycorrhizal community structure in a healthy and a Phytophthora-infected chestnut (Castanea sativa Mill.) stand in central Italy

Ink disease caused by Phytophthora cambivora is a major disease of sweet chestnut (Castanea sativa). In two C. sativa stands in central Italy, one (Montesanti) that is infected with P. cambivora and the trees showing symptoms of ink disease and another healthy stand (Puzzella), the ectomycorrhizal (ECM) community structure was investigated. On the roots of the surviving trees of the diseased stand, 29 different ECM species were determined compared to 23 in the healthy stand. Eleven ECM species were common to both stands; however, a number of species were unique to one of the stands. Cenococcum geophilum was dominant at both sites, but the percentage colonisation was much higher at Montesanti (40.8%) compared to Puzzella (27.2%). There was a switch in species from Russula vesca, Russula lepida and Russula azurea at Puzzella to Russula nigricans, R. lepida and Russula delica at Montesanti. Both R. vesca and R. azurea were found only at the Puzzella site. At the diseased site, the ECMs formed had a smaller root tip diameter, and the ECM at the healthy site had more abundant extramatrical hyphae.     

Carbon dioxide enrichment reduces shoot growth in sweet chestnut seedlings (Castanea sativa Mill.)*

Abstract. Two-year-old potted sweet chestnut seedlings were grown at 350 ppm CO₂ and 700 ppm, day and night in constantly ventilated tunnels during two full growing seasons, near Paris, France (48° N, 2° E). Enrichment with CO₂ caused an unusual shoot growth response. After the end of July, stem elongation ceased in 62% of the CO₂ enriched plants as compared with 37% in the control. The leaves of CO₂-enriched seedlings showed early senescence, indicated by premature yellowing and a decrease in chlorophyll content. This was associated with nutrient dilution brought about by the rapid growth of these trees. The increase in total dry weight of the CO₂-enriched seedlings was essentially the result of increase in the root dry weight (69%). Shoot weight decreased by 22% relative to the control. Total leaf area per enriched plant was 25% smaller than the control. This unusual pattern of growth and carbon allocation of the CO₂ treated Chestnut trees emphasizes the concept of a response specificity within trees to an increase of atmospheric CO₂.     

Upgrade of Castanea sativa (Mill.) genetic resources by sequencing of barcode markers

Journal of Genetics -     

In vitro conservation of chestnut (Castanea sativa) by slow growth

Slow growth storage has been achieved for Castanea sativa (cv. ‘Montemarano’) shoot cultures over a duration of 48 mo at a temperature of 8°C, where 82% of explants survived and were able to resume normal growth after transfer to standard culture conditions at 23°C. The evaluation of the chlorophyll content of leaves also showed no differences between material stored for 48 mo and control material subcultured at 23°C. With a storage temperature of 4°C, the survival of shoots was significantly lower at approximately 56% after 12 mo, and no plants recovered after 24-mo storage. The presence of 6-benzyladenine 0.44 μM in the culture medium proved to be necessary for the recovery of healthy shoots, while pre-treatments with different concentrations of abscisic acid did not significantly influence the survival of shoots following storage conditions. A low level of light during slow growth storage resulted in positive effects on the rate of shoot survival over the longest preservation periods.     

Bacillus subtilis an endophyte of chestnut (Castanea sativa) as antagonist against chestnut blight (Cryphonectria parasitica)

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos

These studies were designed to test if a binary vector containing the gfp, bar and oxalate oxidase genes could transform American chestnut somatic embryos; to see if a desiccation treatment during co-cultivation would affect the transformation frequency of different American chestnut somatic embryo clones; to explore the effects of more rapid desiccation; and to see if the antibiotics used to kill the Agrobacterium were interfering with the regeneration of the somatic embryos. Two days of gradual desiccation was found to significantly enhance transient GFP expression frequency. When this treatment was tested on six American chestnut clones, five were transformed and four of these remained embryogenic. Transformation was confirmed by Southern hybridization. Phenotypically normal transgenic shoots were regenerated and rooted. Vascular tissue specific expression of the oxalate oxidase gene was detected in one transgenic line. Carbenicillin, cefotaxime, and tricarcillin were found to not interfere with the regeneration of transformed embryos.     

The breakdown and decomposition of sweet chestnut (Castanea sativa mill.) and beech (Fagus sylvatica L.) leaf litter in two deciduous woodland soils

Summary Changes in the carbon, hydrogen, nitrogen and polyphenol content of chestnut and beech leaves were measured during the first year after fall.Chestnut leaves had an initial carbon, hydrogen and nitrogen content (by weight) of 48.71%, 5.56% and 0.77% respectively; beech had a similar carbon and hydrogen content (47.77% and 5.36%) but less nitrogen (0.56%).Both leaf litter types showed percentage increases in nitrogen content during the study period but only the beech showed absolute increases in nitrogen content of up to 66.7% of the initial weight present in the leaves. The percentage increases in the nitrogen content of chestnut litter were largely attributable to more rapid losses of non-nitrogenous leaf constituents while the weight of nitrogen present in the leaves remained relatively constant.The percentage carbon and hydrogen contents of the chestnut and beech litter showed changes of less than 1% throughout the year, indicating that carbohydrate losses were directly proportional to weight losses.Soil animals fed on chestnut leaves to a far greater extent than beech leaves; this difference did not appear to be directly attributable to differences between the nitrogen contents or C/N ratios of the two leaf litter species. The gross polyphenol contents of chestnut and beech litter showed an intraspecific inverse correlation with the feeding activities of soil animals on the leaves, but did not account for interspecific differences in leaf palatability since chestnut leaves were eaten when they contained higher polyphenol concentrations than beech leaves. However, there was an interspecific negative correlation between palatability and the presence of protocatechuic and gallic acids.     

Identification of QTLs affecting adaptive traits in Castanea sativa Mill

A QTL analysis for three different adaptive traits was performed in an F₁ progeny of Castanea sativa Mill. The female and male parents originated from two Turkish chestnut populations adapted to a drought and humid environment, respectively. QTLs for bud flush, growth and carbon isotope discrimination were detected over a 3‐year period. Bud set was also recorded in the last year of measurement. Thirty‐five individual QTLs were detected for phenology, 28 for growth and 17 for carbon isotope discrimination, most of them explaining a low to moderate proportion of the total phenotypic variance. QTLs were distributed throughout the whole genome. Temporally stable QTLs were identified for all the traits analysed, with phenology showing the higher proportion of stable QTLs. Interesting phenotypic correlations and co‐localizations among QTLs for different adaptive traits were observed, allowing the formulation of an hypothesis about the genetic adaptation of the female parent to drought.     

Plant species diversity changes in abandoned chestnut (Castanea sativa) groves in southern France

Over the last few decades, marked land use changes have taken place in many Mediterranean ecosystems. For example, many chestnut groves in France are now abandoned and have turned into 'natural' coppice stands while others are now clear-cut every 10 or 15 years for wood. Species composition and life form diversity of the plant communities have changed markedly both in the abandoned groves and in the periodically clear-cut coppice stands. What are the consequences of these changes on biodiversity at local and regional scales, and what are the implications for 'new forestry' management intended to conserve biodiversity at the same time as it optimizes productivity and profitability? To answer these questions we studied plant species diversity in the understorey strata along a successional gradient including cultivated grove; abandoned grove; young (15 years old), medium (40 years), and old (>55 years) coppice stands. The results showed that species richness decreased quickly but not steadily along this gradient, but life-form spectra data highlighted that the high levels of plant species biodiversity in cultivated groves were due primarily to the large number of therophytes (annual plants). At a shorter time scale, we also studied the consequences of experimental clear-cutting on plant species biodiversity. Direct measurements over four years showed that species richness and diversity in the understorey increased in the first two years after clear-cutting, but decreased during the 3rd and 4th years. This decrease corresponded closely with a steady increase in Leaf Area Index of the forest canopy. These results have been used to suggest some possible ways to manage the biodiversity in these ecosystems.     

Allozyme diversity in Chinese,Seguin and American chestnut (Castanea spp.)

Allozyme genetic variability in three chestnut (Castanea) species was investigated using 19 loci from ten enzyme systems. G-tests of heterogeneity of isozymic allele distribution showed significant differences between the three species at 15 of the 19 loci, and between the 13 C. mollissima populations at 13 of the 19 loci examined. C. mollissima was found to possess a significantly-higher value of mean gene heterozygosity (H=0.3050±0.0419), the percentage of polymorphic loci (P=84.21%) and the average number of alleles per locus (A=2.05), than any other species in the Castanea section Eucastanon. When the genetic variability of populations of C. mollissima from four regions in China was investigated, the population from the Changjiang river region showed a markedly higher mean gene heterozygosity (H=0.3480±0.0436) than populations from the other regions. Genetic relationships among the four regions were assessed by Nei's genetic identity I and standard genetic distance D. An approximately-identical distance between the population from the Changjiang river region and populations from the three other regions was observed, while populations from the latter regions showed almost the same genetic distance from each other. These data, when considered with information existing prior to this study, contribute to an understanding of the possible origin and progenitor of the chestnut species.     

Influence of mycorrhization on physiological parameters of micropropagated Castanea sativa Mill. plants

 Mycorrhizal micropropagated Castanea sativa plants were studied in terms of growth and physiological parameters following in vitro mycorrhization with Pisolithus tinctorius. Mycorrhization enhanced growth of micropropagated chestnut plants, increased their protein content and photosynthetic rates, decreased the respiratory rates and CO₂ compensation point. RuBisCO activity was not significantly different in mycorrhizal and control plants, although there was an increase in the amount of RuBisCO in the former. Mycorrhization increased plant biomass and improved plants physiological status, thus enhancing the acclimatization process. Accepted: 21 May 1997     

Fire-related features of wood anatomy in a sweet chestnut (Castanea sativa) coppice in southern Switzerland

Explorative wood anatomical analysis was conducted on a Castanea sativa stand in southern Switzerland, where a moderate-intensity surface fire burned in April 1997. Cross-sections were sampled at multiple heights from 20 scarred shoots, 20 apparently intact shoots, along with cores taken from 20 reference trees outside the fire area. Thin sections were prepared from uphill and downhill locations on the circumference for up to 5 years preceding and following the event year. The thin sections were visually observed in order to identify a response to the known fire event preserved in the wood anatomical structure. Anatomical features were observed at the uphill and downhill locations on both scarred and intact (unscarred) cross-sections, and they occurred in a subset of the observed samples. The features observed in both scarred and intact cross-sections were an apparent increase in vessel density and a decrease in lumen area of the second row of earlywood vessels, along with tyloses formation in the first row of earlywood vessels. Furthermore, the scarred region exhibited a zone of delayed cambial death following the fire, and the onset of woundwood was often initiated later in the season or the following year. Using the type of features and their location around the circumference, we inferred that the observed features may have formed in response to local heating of the cambium, and likely formed in response to canopy damage.