Interactive effect of CO2 enrichment and temperature on the photosynthesis of field-grown hinoki cypress (Chamaecyparis obtusa) branches

Two branches of a field-grown Chamaecy-paris obtusa tree were enclosed in chambers of an open gas exchange system for continuous CO₂ exchange measurements. One branch was subjected to ambient air (CO₂, 370 μmol mol^–1) and the other was subjected to CO₂-enriched air (800 μmol mol^–1). The CO₂ exchange rate of the branches, air temperature and photosynthetic photon flux density were recorded every 4 min by a computer during the two experimental periods of July 1994 to June 1995 (experiment 1) and April 1996 to August 1997 (experiment 2). The response of CO₂ gas exchange rate to light changed with the seasonal temperature. The highest saturated rate of net photosynthesis on a leaf area basis was observed in May and October in both CO₂ treatments when the mean daytime temperature was about 18–19°C. This temperature was almost equal to the yearly mean daytime temperature. Above and below this temperature, the saturated net photosynthesis rate decreased. The net photosynthesis rate was usually higher in the elevated CO₂ treatment. The ratio of monthly net photosynthesis rate in elevated CO₂ to that in ambient CO₂ was linearly related to the monthly mean daytime temperature. This ratio increased by 3.3% for each 1°C increase in the monthly mean daytime temperature; the highest ratio of 1.8 occurred in August. When the ratio was 1.0, the temperature was about 5–6°C, which was close to the mean daytime temperature of the coldest month. Elevated CO₂ increased per unit area net photosynthesis by 38.5% and 43.7% in experiments 1 and 2, respectively. Received: 29 March 1999 / Accepted: 22 October 1999     

Angle distribution of foliage in individual Chamaecyparis obtusa canopies and effect of angle on diffuse light penetration

 The vertical distribution of foliage angle and area of three Chamaecyparis obtusa trees was determined by the triangle method, which calculates foliage geometry using measured coordinates of the leaf ”corners”, in a 43-year-old plantation in central Japan. Vertical distribution patterns of leaf area were different depending on tree size, but the boundary heights, which divide the canopy into sunlit and shaded parts, were similar in the three sample trees. The value of the average foliage angle [I(Z)] at a given depth (Z) from the tip of the stem decreased continually from the upper to lower layers within the canopy. The vertical patterns of changes in I(Z) were different among the three trees, but could be expressed by the following allometric equation as a function of depth.

Identification of a chromosome-specific probe that maps within the Ph1 deletions in common and durum wheat

 The Ph1 (pairing homoeologous) gene is the major factor that determines the diploid-like chromosome behavior of polyploid wheat. This gene, which is located on the long arm of chromosome 5B (5BL), suppresses homoeologous pairing at meiosis while allowing exclusive homologous pairing. In an effort to tag the specific chromosomal region where this gene is located, we have previously microdissected chromosome arm 5BL from bread wheat and produced a plasmid library by random PCR amplification and cloning. In this work we isolated from this library a 5BL-specific probe, WPG90, and mapped it within the interstitial deleted chromosome fragments carrying Ph1 in common and durum wheat. A PCR assay of Ph1 based on WPG90 was developed that allows an easy identification of homozygous genotypes deficient for this gene. Received: 19 June 1996 / Accepted: 18 October 1996     

Conidioma production of the white root rot fungus in axenic culture under near-ultraviolet light radiation

 Conidiomata of the white root rot fungus were produced in axenic culture under near-ultraviolet light radiation. Pieces of sterilized Japanese pear twigs were placed on 7-day-old oatmeal agar culture in plates. The plates were further incubated for 5 days and then illuminated by near-ultraviolet light. Synnemata developed on the twigs within 5 weeks in 19 of 20 isolates tested, and conidia were observed in 12 of the 19 isolates. The synnemata and conidia produced were morphologically identical to those of Dematophora necatrix. Received: October 29, 2001 / Accepted: March 11, 2002     

A taxon-specific oligonucleotide probe for temperate zone soil isolates of Glomus mosseae

 The 5.8 S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus mosseae FL156 and UK118 were amplified by polymerase chain reaction (PCR) using ITS1 and ITS4 as primers. The amplification product from template DNA of UK118 was cloned and sequenced (569 bp); the amplified DNA from FL156 was sequenced directly (582 bp). There was a 95% sequence similarity between DNAs amplified from the two isolates; in contrast, major dissimilarities with partial sequences of seven other glomalean taxa were observed. Four oligonucleotide sequences unique to Glomus mosseae were identified as potential primers. Their specificity to Glomus mosseae was assessed by PCR amplification of genomic DNA from spores from 36 glomalean fungi: 13 isolates of Glomus mosseae, two Glomus monosporum, 10 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The Glomus mosseae isolates were from a broad range of temperate zone agricultural soils. Oligonucleotide pair GMOS1 : GMOS2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all Glomus mosseae isolates and two isolates of the closely related Glomus monosporum. This primer pair did not prime PCR when the template consisted of DNA from any of the other glomalean fungi or any of the nonmycorrhizal controls. In addition, a 24-mer oligonucleotide, designated GMOS5, hybridized with Glomus mosseae and Glomus monosporum DNA amplified by PCR using primer pairs ITS1 : ITS4 and GMOS1 : GMOS2. Colony-blot assays showed that GMOS5 hybridized to 100% and 97% of E. coli pUC19 clones of amplification products from Glomus mosseae FL156 and UK118 DNA templates, respectively, indicating that nearly all clones contained an homologous sequence. GMOS5 was used successfully to detect specifically Glomus mosseae in DNA extracted from colonized sudan grass (Sorghum sudanense L.) roots and amplified by PCR using the primer pair GMOS1 : GMOS2. The results confirm several previous indications that Glomus mosseae and Glomus monosporum are indistinguishable taxonomic entities. Accepted: 14 February 1998     

Molecular characterization of Pisolithus spp. isolates by rDNA PCR-RFLP

 Variation within ribosomal DNA (rDNA) genes of 19 isolates of Pisolithus from different geographic origins and hosts was examined by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis. The primers utilized amplify rDNA regions in a wide range of fungi. One amplified region includes the internal transcribed spacer (ITS), which has a low degree of conservation. The ITS amplification products (640–750 bp) were digested with a variety of restriction endonucleases. Cluster analysis based on the restriction fragments grouped the isolates into three distinct groups: group I contained isolates collected in the northern hemisphere, except Pt 1, group II contained those collected in Brazil and group III contained isolate Pt 1. Additional analysis of other rDNA regions, IGS, 17 S and 25 S rDNA, resulted in similar groups. The data suggest that the taxonomy and systematics of this ectomycorrhizal fungus should be revised. Accepted: 16 September 1998     

Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region

Using a fluorescence-based PCR-SSCP (single-strand conformation polymorphism), we verified imperfectibility in the paternal inheritance of chloroplast DNA (cpDNA) in Chamaecyparis obtusa (Cupressaceae) controlled crosses. An intraspecific sequence polymorphism of the intergenic spacer region between the trnD and trnY genes was utilized as a molecular marker. Of 361 progenies, in which the cpDNA haplotypes of their female and male parents were different, 352 (97.5%) possessed the same haplotypes as their male parents, and nine (2.5%) the same haplotypes as their female parents. The parentage of the nine progenies with female parental types was diagnosed using DNA fingerprinting based on fluorescence-based RAPD profiles. Their parentage showed convincing evidence of the low frequency of maternal inheritance. Moreover, heteroplasmy was observed in the open-pollinated seeds collected in a seed orchard. The confirmation of maternal plastid transmission in the full-sib families and the observation of heteroplasmy in seeds reveal that the paternal inheritance of cpDNA is not an exclusive phenomenon and that the mode of its inheritance is biparental in C. obtusa. Received: 15 April 2000 / Accepted: 13 July 2000     

Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS

 The primed in situ DNA labelling (PRINS) procedure was optimised for the rapid physical mapping of several types of repetitive DNA sequences on the mitotic chromosomes of Vicia faba, Pisum sativum and Secale cereale. A localization of the highly repeated FokI sequence on V. faba chromosomes was achieved after a 7-min total reaction time. In addition, we report a procedure for direct cycling-PRINS (C-PRINS), a variation of PRINS which involves a sequence of thermal cycles analogous to the polymerase chain reaction. Compared to PRINS, C-PRINS was more sensitive. Further work is needed to improve the sensitivity of the reaction to allow for the reliable detection of low-copy DNA sequences. Received: 17 September 1996 / Accepted: 18 October 1996     

Phylogenetic analysis of the internal transcribed spacer region of Japanese Lilium species

 The DNA from 16 Lilium species and one variety endemic to or naturalized in Japan were obtained and their internal transcribed spacer regions of nuclear ribosomal DNA (nrDNA) were amplified by PCR and sequenced by cycle sequencing. Phylogenetic analysis of the ITS sequences supported the validity of Comber’s classification system. It has also provided molecular evidence for the transfer of Lilium dauricum to sect. Sinomartagon. The phylogenetic relationships revealed by ITS DNA analysis were supported by previously published crossability data. The molecular phylogeny of Japanese Lilium species was discussed with reference to the putative migration routes of these species. Received: 30 June 1998 / Accepted: 19 October 1998     

Identification and partial purification of an oxidase from lignifying xylem of Leyland cypress (X Cupressocyparis leylandii)

 Oxidase activity was exclusively present in lignifying cells of developing xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl₂ extracts of crude cell walls and seems to be ionically associated with the cell walls. Oxidase activity was selected and concentrated using affinity chromatography on Concanavalin-A Sepharose which suggests that it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase band of apparent Mr 92 kD capable of oxidising N, N, N′, N′ - tetramethyl phenylene diamine/α-naphthol was identified after non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD oxidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition, the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised coniferyl alcohol to form insoluble material that yielded a Fourier transform infra-red spectrum similar to dehydrogenation polymers of coniferyl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but further studies are required to fully define this oxidase and its possible homology with other oxidases identified in the lignifying xylem of different species of trees. Received: 20 May 1997 / Accepted: 7 August 1997     

Genetic diversity of the ectomycorrhizal fungus Pisolithus tinctorius based on RAPD-PCR analysis

 Twenty Pisolithus tinctorius isolates from different geographic locations and different hosts were characterized by the random amplified polymorphic DNA technique. Thirteen arbitrary primers generated 87 DNA fragments, all of them polymorphic. These data were used to calculate genetic distances among the isolates. The pairwise genetic distances ranged from 1 to 100%, with an average of 58.7%. Cluster analysis based on the amplified fragments grouped the isolates according to their host and geographical origins. Group I contained isolates collected in Brazil and group II those collected in the Northern Hemisphere. In addition to the diversity seen at the molecular level, the isolates also showed host specificity. Greenhouse experiments demonstrated that isolates from the Northern Hemisphere colonized mainly Pinus whereas isolates from Brazil colonized only Eucalyptus. The molecular data suggest that the Pisolithus tinctorius isolates analyzed belong to two distinct groups. The data also suggest new guidelines for future investigations on the taxonomy and systematic of this important fungus species. Furthermore, these results support future experiments aimed at the selection and development of improved isolates of P. tinctorius. Accepted: 3 October 1997     

Effect of phosphate and the arbuscular mycorrhizal fungus Glomus intraradices on disease severity of root rot of peas (Pisum sativum) caused by Aphanomyces euteiches

 The effects of inorganic phosphate levels and the presence of arbuscular mycorrhiza on disease severity of Aphanomyces euteiches in pea roots were studied. Disease severity on roots and epicotyl as well as the oospore number within infected root tissue were correlated with the phosphorus (P) level in the growth medium. The arbuscular mycorrhizal fungus Glomus intraradices increased P uptake and the P concentration in the plant but reduced disease development in peas. Polyacrylamide gel electrophoresis followed by densitometry of glucose-6-phosphate dehydrogenase specific to A.euteiches was used to measure the activity of the pathogen in roots. The enzyme activity increased with disease severity and disease incidence, except in plants supplemented with P at the highest level, where a peak in activity was seen 12 days after inoculation with the pathogen, followed by a decrease in activity. The epicotyl of mycorrhizal plants showed a reduction in disease severity although this part of the plants was not mycorrhizal. Thus, an induced systemic factor may be responsible for increased resistance in mycorrhizal plants. Accepted: 21 August 1998     

Microsatellite markers isolated in olive ( Olea europaea L.) are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars

We have isolated and sequenced 52 microsatellites or simple sequence repeats (SSRs) from nearly 60 positive clones obtained from two ’Frantoio’ olive genomic libraries enriched in (AC/GT) and (AG/CT) repeats, respectively. The repeat-containing fragments obtained from genomic DNA restricted with Tsp509I were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs which gave correct re-amplification in the source genome were used to assay the polymorphism of 12 olive cultivars, namely four well-known cultivars (’Coratina’, ’Frantoio’, ’Leccino’, ’Pendolino’) and eight ancient cultivars grown locally near Lake Garda (’Casaliva’, ’Favarol’, ’Fort’, ’Grignan’, ’Less’, ’Raza’, ’Rossanel’, ’Trep’). The local cultivars were each re- presented by two to four long-lived individuals. The analysis was carried out using ³³P-labelled primers and 6% polyacrylamide sequencing gels. All except two microsatellites showed polymorphism, the number of alleles varying from 1 to 5. The average genetic diversity (H) was 0.55. The power of discrimination (PD) was 0.60. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individual plants of the same cultivar in four out of the eight local cultivars analysed. Several primer pairs (17%) amplified more than one locus. Received: 23 March 2001 / Accepted 17 May 2001     

Genetic homogeneity among isolates of Fusarium solani that cause soybean sudden death syndrome

  Fusarium solani f. sp. phaseoli is the etiological agent of soybean sudden death syndrome (SDS). This form species includes both members that cause SDS and those that do not. Despite the extensive use of SDS isolates in soybean plant breeding studies, no information regarding genetic relatedness of isolates is available. Sequencing of the D2 region of the large-subunit (28S) ribosomal DNA of 19 isolates of F. solani f. sp. phaseoli, both SDS and non-SDS isolates, resulted in identical sequences and thus indicated a very low level of genetic variation within the form species. Sequencing of the ITS region resulted in low-level intra-individual as well as intra-specific variation. Random amplified polymorphic DNA (RAPD) analysis was used for a genome-wide estimate of genetic variation and was able to resolve only two amplitypes of the SDS isolates. Thus, SDS isolates from throughout the U.S. comprise an almost clonal population with an extremely low level of genetic variation among individuals. Received: 22 November 1996 / Accepted: 4 April 1997     

Specifying the introgressed regions from H. argophyllus in cultivated sunflower (Helianthus annuus L.) to mark Phomopsis resistance genes

A method based upon targetting of intro-gressed markers in a Phomopsis-resistant line (R) of cultivated sunflower, issuing from a H. argophyllus cross was used to mark the Phomopsis resistance regions. Our study was based upon 203 families derived from a cross between an inbred line susceptible to Phomopsis (S1) and the introgressed resistant line (R). Families were checked for Phomopsis resistance level in a design with replicated plots and natural infection was re-inforced by pieces of contaminated stems. Thirty four primers were employed for RAPD analysis. Out of 102 polymorphic fragments between (S1) and H. argophyllus, seven were still present in (R) suggesting that they marked introgressions of H. argophyllus into (R). The plants were scored for the presence or absence of 19 fragments obtained from five primers, and the relationships between the presence/absence of fragments in plants and Phomopsis resistance/susceptiblity in the progenies was determined by using an analysis of variance. We found that at least two introgressed regions, as well as favourable factors from sunflower, contributed to the level of Phomopsis resistance in cultivated sunflower. Received: 28 June 1996 / Accepted: 5 July 1996     

Identification of DNA amplification fingerprinting (DAF) markers close to the symbiosis-ineffective sym31 mutation of pea (Pisum sativum L.)

 We demonstrate efficient genome mapping through a combination of bulked segregant analysis (BSA) with DNA amplification fingerprinting (DAF). Two sets of 64 octamer DAF primers, along with two PCR programs of low- and high-annealing temperatures (30°C and 55°C, respectively), appeared to be enough to locate molecular markers within 2–5 cM of a gene of interest. This approach allowed the rapid identification of four BSA markers linked to the pea (Pisum sativum L.) Sym31 gene, which is responsible for bacteroid and symbiosome differentiation. Three of these markers are shown to be tightly linked to the sym31 mutation. Two markers flanking the Sym31 gene, A21-310 and B1-277, cover a 4–5 cM interval of pea linkage group 3. Both markers were converted to sequence-characterized amplified regions (SCARs). The flanking markers may be potential tools for marker-assisted selection or for positional cloning of the Sym31 gene. Received: 2 July 1998 / Accepted: 8 October 1998     

X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11?Å resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.     

EST-library construction using spore RNA of the arbuscular mycorrhizal fungus Gigaspora rosea

RNA was extracted from activated spores of the arbuscular mycorrhizal fungus Gigaspora rosea. Double-stranded cDNA was synthesised, digested and cloned into the vector lambda-ZAP express. Of the 1,500 clones obtained, 1.5% carried inserts of the rRNA gene cluster. After excision, inserts from 50 randomly selected clones were sequenced. Database searches revealed that 62% of the clones had similarities to already known sequences. These mainly code for proteins involved in translation and protein processing, replication and the cell cycle and cell signal transduction. One fragment probably belonged to a metallothionein-encoding gene which may be involved in heavy-metal binding. The method presented is an easy and rapid way to obtain short fragments of coding regions for expressed sequence tag libraries. Accepted: 28 November 2000     

Presence and organization of an osmotic stress response domain in wild Triticeae species

Sixteen Triticeae species of the genera Aegilops L., Pseudoroegneria (Nevski) Löve, Taeniatherum Nevski and Thinopyrum Löve were investigated by PCR amplification for the presence of a wheat germin gene internal domain involved in osmotic stress resistance. In all of the species studied a single band of identical or very similar size was detected, After cloning and sequencing of these fragments, different degrees of homology were found with the original wheat domain, which suggested that in these species there are functional differences in the osmotic response involving the germin core.