Distance effect of co-occurring tree species on pine wilt disease incidence inPinus densiflora seedlings inoculated withBursaphelenchus xylophilus

Field-grownPinus densiflora seedlings were inoculated withBursaphelenchus xylophilus and the incidence of pine wilt disease was analyzed with respect to the spatial relationships between pine seedlings and adjacent seedlings of other tree species in a pure pine stand and three stands mixed withAlnus sieboldiana, Sarothamus scoparius or naturally associated species. The disease incidence was 60.9% in a 0–40 cm distance class from the nearestA. sieboldiana seedlings and then decreased with increasing minimum distance. The mean minimum distance between pine seedlings andA. sieboldiana was significantly shorter in diseased seedlings than in healthy ones. The highest disease incidence (53.9%) was found in a 0–40 cm distance class from the nearestS. scoparius, although the relationship with minimum distance was blurred by a high incidence in the 80-cm distance class. The mean minimum distance between pine seedlings was significantly short in diseased seedlings in a pure stand, whereas it was not so short between pine seedlings and other tree species in the two stands mixed withS. scoparius and the naturally associated species. The distance effect on disease incidence was noticeable inA. sieboldiana andP. densiflora at high density. This was not so clear inS. scoparius and was not found in the naturally associated species.     

Detection of Low Genetic Variation in a Critically Endangered Chinese Pine, Pinus squamata, Using RAPD and ISSR Markers

With only 32 individuals in the northeastern corner of Yunnan Province, China, Pinus squamata is one of the most endangered conifers in the world. Using two classes of molecular markers, RAPD and ISSR, its very low genetic variation was revealed. Shannon's index of phenotypic diversity (I) was 0.030, the mean effective number of alleles per locus (A_e) was 1.032, the percentage of polymorphic loci (P) was 6.45, and the expected heterozygosity (H_e) was 0.019 at the species level based on RAPD markers. The results of ISSR were consistent with those detected by RAPD but somewhat higher (I = 0.048, A_e = 1.042, P = 12.3, H_e = 0.029). The genetic variation of the subpopulation on the southwest-facing slope was much higher than that of the subpopulation on the northeast-facing slope, which may be attributed to the more diverse environment on the southwest-facing slope. The genetic differentiation between the two subpopulations was very low. The between-subpopulation variabilities, Φ_ST, calculated from RAPD and ISSR data were 0.011 and 0.024. Because of the lack of fossil records and geological historical data, it was difficult to explain the extremely low genetic diversity of the species. We postulate that this ancient pine might have experienced strong bottlenecks during its long evolutionary history, which caused the loss of genetic variation. Genetic drift and inbreeding in post-bottlenecked small populations may be the major forces that contribute to low genetic diversity. Human activities such as logging may have accelerated the loss of genetic diversity in P. squamata.     

大兴安岭山地樟子松径向生长对气候变暖的响应——以满归地区为例

基于树木年代学的理论和方法,建立了大兴安岭满归地区樟子松年轮宽度年表,分析了标准化年表与该区主要气候因子(温度和降水)之间的相关关系,揭示了气候因子对树木径向生长的影响.结果表明:当年4-8月的各月平均温度是研究区樟子松径向生长的主要限制因子;4-8月各月温度的不断升高对樟子松径向生长产生了不利影响.利用4-8月平均温度的变化模拟樟子松径向生长变异(1958 -2008年),发现随着区域气候暖干化趋势的加强,该区樟子松生长将呈现出衰退的特征.     

The Production of Transgenic Scots Pine (Pinus Sylvestris L.) via the Application of Transformed Pollen in Controlled Crossings

The study demonstrates the production of a transgenic Scots pine (Pinus sylvestris L.) seedling through the application of transformed pollen in controlled crossings. The pollen lots were transformed by particle bombardment, resulting in transient transformation frequencies varying from 15 to 49% of the germinated pollen grains, and bombarded pollen was used to pollinate megasporangiate strobili. Progeny was screened by histochemical, GUS assays, and selected seedlings were further analysed by PCR. PCR amplification revealed the presence of both the nptII and gusA genes in one seedling (23/237). Results were confirmed by Southern blot analysis. The morphology and growth of this transgenic seedling was normal. Although the transformation frequency of recovered plants was very low (1/14999), the present protocol suggests that production of transgenic Scots pine is possible without the use of any tissue culture methods or the involvement of marker genes, for selection of transformants.     

Drought as an Inciting Mortality Factor in Scots Pine Stands of the Valais, Switzerland

During the 20th century, high mortality rates of Scots pine (Pinus silvestris L.) have been observed over large areas in the Rhône valley (Valais, Switzerland) and in other dry valleys of the European Alps. In this study, we evaluated drought as a possible inciting factor of Scots pine decline in the Valais. Averaged tree-ring widths, standardized tree-ring series, and estimated annual mortality risks were related to a drought index. Correlations between drought indices and standardized tree-ring series from 11 sites showed a moderate association. Several drought years and drought periods could be detected since 1864 that coincided with decreased growth. Although single, extreme drought years had generally a short-term, reversible effect on tree growth, multi-year drought initiated prolonged growth decreases that increased a tree’s long-term risk of death. Tree death occurred generally several years or even decades after the drought. In conclusion, drought has a limiting effect on tree growth and acts as a bottleneck event in triggering Scots pine decline in the Valais.     

A partial genetic linkage map of slash pine (Pinus elliottii Engelm. var. elliottii) based on random amplified polymorphic DNAs

A set of 420 random, 10-base, oligonucleotide primers was screened for random amplified polymorphic DNA (RAPD) fragments within a sample of eight megagametophyte DNAs of a single slash pine (Pinus elliottii Engelm. var. elliottii) tree. The apparently repeatable RAPD fragments were further characterized within a sample of 68 megagametophytes from the same tree. Fragments segregating in a 11, present-to-absent, ratio were classified and mapped using multi-point linkage analysis. The analysis revealed 13 linkage groups of at least three loci, ranging in size from 28 to 68 cM, and nine linked pairs of loci. The 22 groups and pairs included 73 RAPD markers and covered a genetic map distance of approximately 782 cM. Genome size estimates, based on linkage data, ranged from 2880 to 3360 cM. Using a 30-cM map scale and including the 24 unlinked markers and the ends of the 13 linkage groups and nine linked pairs, the set of RAPD markers accounts for approximately 2160 cM or 64–75% of the genome. This extent of genomic coverage should allow for the efficient mapping of genes responsible for a reaction to the causal agent of fusiform rust disease, Cronartium quercuum (Berk.) Miyabe ex Shirai f. sp. fusiforme.     

Description of Meloidogyne pini n. sp., a Root-Knot Nematode Parasitic on Sand Pine (Pinus clausa), with Additional Notes on the Morphology of M. megatyla

Meloidogyne pini n. sp. is described from sand pine, Pinus clausa, in Georgia. The perineal pattern of the female has a large cuticular ridge surrounding a deeply recessed perivulval area. The lateral fields are marked by transverse striae. The female stylet is 14.6 μm long, and the knobs are small, rounded, and set off from the straight and narrow shaft. The excretory pore is near the level of the base of the stylet. The labial disc of the male is large, rounded, and fused with the crescent-shaped medial lips. The head region is smooth, the styler is 20.8 μm long, and the cone is more than twice as long as the shaft. The knobs are rounded and set off from the shaft. In the second-stage juvenile, the labial disc, medial lips, and lateral lips form one smooth, continuous, ovoid head cap. Mean juvenile length is 434 μm, stylet length is 12.8 μm, and tail length is 44.4 μm. M. pini n. sp. also parasitizes loblolly and slash pine. Additional morphological details of M. megatyla are presented.     

High-throughput genotyping and mapping of single nucleotide polymorphisms in loblolly pine (Pinus taeda L.)

The development and application of genomic tools to loblolly pine (Pinus taeda L.) offer promising insights into the organization and structure of conifer genomes. The application of a high-throughput genotyping assay across diverse forest tree species, however, is currently limited taxonomically. This is despite the ongoing development of genome-scale projects aiming at the construction of expressed sequence tag (EST) libraries and the resequencing of EST-derived unigenes for a diverse array of forest tree species. In this paper, we report on the application of Illumina’s high-throughput GoldenGate™ SNP genotyping assay to a loblolly pine mapping population. Single nucleotide polymorphisms (SNPs) were identified through resequencing of previously identified wood quality, drought tolerance, and disease resistance candidate genes prior to genotyping. From that effort, a 384 multiplexed SNP assay was developed for high-throughput genotyping. Approximately 67% of the 384 SNPs queried converted into high-quality genotypes for the 48 progeny samples. Of those 257 successfully genotyped SNPs, 70 were segregating within the mapping population. A total of 27 candidate genes were subsequently mapped onto the existing loblolly pine consensus map, which consists of 12 linkage groups spanning a total map distance of 1,227.6 cM. The ability of SNPs to be mapped to the same position as fragment-based markers previously developed within the same candidate genes, as well as the pivotal role that SNPs currently play in the dissection of complex phenotypic traits, illustrate the usefulness of high-throughput SNP genotyping technologies to the continued development of pine genomics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation of polymorphic microsatellite loci in Livistona chinensis (Jacq.) R.Br. ex Mart. var. boninensis Becc., an endemic palm species of the oceanic Bonin Islands, Japan

A set of microsatellites markers were developed for Livistona chinensis var. boninensis, an endemic palm tree of the Bonin Islands. We obtained 123 sequences containing unique microsatellites from an enriched library. Twelve loci were screened for their feasibility using 32 trees. They showed polymorphisms with two to nine alleles per locus. No significant deviation from Hardy–Weinberg equilibrium was observed for 11 loci. No genotypic disequilibrium was detected between any two of the loci. Total exclusionary powers for the first and the second parents were 0.978774 and 0.998987, respectively. These markers will allow us to investigate the gene flow within/among populations of the species.     

Evaluation of morphological and molecular variation in Plantago asiatica var. densiuscula, with special reference to the systematic treatment of Plantago asiatica var. yakusimensis

Morphological and molecular variations in Plantago asiatica L. var. densiuscula Pilg. were analyzed to evaluate the genetic basis for recognizing the dwarf variety P. asiatica var. yakusimensis (Masam.) Ohwi. Considerable variation in the leaf size of P. asiatica var. densiuscula was observed, and no morphological discontinuities were found between the dwarf types of P. asiatica var. densiuscula and P. asiatica var. yakusimensis. Morphological analysis of plants grown under standardized conditions revealed that both environmental plasticity and genetic differentiation contributed to the dwarfisms. Molecular phylogenetic analysis of rDNA internal transcribed spacer (ITS) regions and the SUC1 locus encoding a sucrose transporter revealed that P. asiatica var. yakusimensis was genetically unique although the differentiation level was low. From the above results, we concluded that P. asiatica var. yakusimensis should be reduced to a form of P. asiatica var. densiuscula. Furthermore, the geographic distribution of the SUC1 genotype suggested multiple origins of dwarves, and possible hypotheses for the origins of dwarves are discussed.     

Responses of the macroalga Gracilaria tenuistipitata var. liui (Rhodophyta) to iron stress

Chlorophyll (Chl), phycoerythrin (PE), total nitrogen (TN% dw) and Fein tissues were measured in Fe-deficient cultures of Gracilariatenuistipitata var. liui over a period of 60 days. ⁵⁵Fe uptakeand photosynthetic carbon fixation (NaH¹⁴CO³) werecompared in Fe-rich and Fe-deficient cultures and analyzed the effects ofFe-deficiency on the ultrastructure. The maximum carbon fixationdecreased significantly (p < 0.01) under Fe-deficiency. Thechlorophyll and phycoerythrin contents also declined with decreasing tissueiron content, falling, respectively, to 7.9 and 33.8% of their originallevel. Photosynthesis in Fe-deficient cells became light-saturated at lowerirradiance than the control. Total N in tissue decreased from 3.65 to2.49%. ⁵⁵Fe uptake rate for cultures grown on NO₃^-was measured following resuspension in either NH₄⁺ orNO₃^- as N source. Enhanced Fe uptake developedunder Fe stress, especially with cells resuspended in NH⁺₄-N medium. The Vmaxfor Fe uptake was higher with NH₄⁺ thanNO₃^- (62.8 versus 12.1 pmol mg dw^-1 h^-1). The requirement for N accelerates further Fe uptake. Ultrastructuralobservations of Fe-deficient cells showed reductions in chloroplast number,degeneration of lamellar organization, decrease in mitochondrial matrixdensity and variation in accumulation body number and morphology. During Fe-deficiency, the growth rate continued to decline and after 40days of iron deficiency, no further growth was detectable, and eventuallyiron deficiency resulted in chlorosis. The results suggest that the lowergrowth rate of Gracilaria tenuistipitata var. liui underFe-deficiency may result from largely from inhibition of photosynthesis andnitrogen utilization.     

RFLP mapping of resistance to the blackleg disease [causal agent,Leptosphaeria maculans (Desm.) Ces. et de Not.] in canola (Brassica napus L.)

We report the tagging of genes involved in blackleg resistance, present in the French cultivar Crésor of B. napus, with RFLP markers. A total of 218 cDNA probes were tested on the parental cultivars Crésor (resistant) and Westar (susceptible), and 141 polymorphic markers were used in a segregating population composed of 98 doubled-haploid lines (DH). A genetic map from this cross was constructed with 175 RFLP markers and allowed us to scan for specific chromosomal associations between response to blackleg infection and RFLP markers. Canola residues infested with virulent strains of Leptosphaeria maculans were used as inoculum and a suspension of pycnidiospores from cultures of L. maculans, including the highly virulent isolate Leroy, was sprayed to increase disease pressure. QTL mapping suggested that a single chromosomal region was responsible for resistance in each of the four environments tested. This QTL accounted for a high proportion of the variation of blackleg reaction in each of the assays. A second QTL, responsible for a small proportion of the variation of blackleg reaction, was present in one of four year-site assays. A Mendelian approach, using blackleg disease ratings for classifying DH lines as resistant or susceptible, also allowed us to map resistance in the region of the highly significant LOD scores observed in each environment by interval mapping. Results strongly support the presence of a single major gene, named LmFr ₁ controlling adult plant resistance to blackleg in spring oil-seed rape cultivar Crésor. Several RFLP markers were found associated with LmFr ₁.     

Induced chitinase activity in resistant wheat leaves inoculated with an incompatible race of Puccinia striiformis f. sp. tritici,the causal agent of yellow rust disease

Chitinase specific activity was measured spectrophotometrically in wheat leaf tissues during the compatible and incompatible interactions with Puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease. The wheat cultivar, Federation^* 4/Kavkaz, was inoculated with virulent (134E134A+) or avirulent (4EOA+) races of P. striiformis f. sp. tritici in the first leaf stage. The results showed that chitinase activity pattern was similar in both compatible and incompatible interactions up to 72 hrs after inoculation. However, the specific activity increased rapidly in the incompatible reaction thereafter. In susceptible reaction, chitinase activity gradually declined after 72 hrs post-inoculation reaching a level similar to that in the control plants two weeks after inoculation. Chitinase specific activity in resistance response was at least three times greater than that in the susceptible reaction two weeks following the inoculation. Electrophoresis of native polyacrylamide gel impregnated with 0.1% (w/v) glycol chitinas the substrate revealed the presence of eight chitinase isoforms with relative electrophoretic mobility (R_m) values ranging from 0.11 to 0.64 in the resolving gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation and characterization of microsatellite loci in the endangered tree Berchemiella wilsonii var. pubipetiolata and cross-species amplification in closely related taxa

Twenty-eight polymorphic microsatellite loci were developed and characterized for the endangered tree Berchemiella wilsonii var. pubipetiolata. The observed number of allele ranged from two to seven. The ranges of observed and expected heterozygosity were 0.039–1.000 and 0.038–0.816, respectively. In addition, this set of microsatellites produced robust cross-species amplification in other two endangered taxa: Berchemiella berchemiaefolia and Berchemiella wilsonii, suggesting these microsatellite markers should provide a useful tool for genetic and conservation studies of the globally endangered genus Berchemiella.     

Effectiveness of beta-exotoxin ofBacillus thuringiensis var.thuringiensis on the ability ofMeloidogyne sp. from brinjal (Solanum melongena L.) to survive

Beta-exotoxin produced byBacillus thuringiensis var.thuringiensis grown in the acid hydrolysates of wheat and rice brans caused 95% and 85% mortality respectively ofMeloidogyne sp. as against 72% of β-exotoxin produced on farm yard manure within 7 days. Acid hydrolysate of wheat or rice bran and solid farm yard manure proved to be the best media for growth ofB. thuringiensis var.thuringiensis.     

Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in wheat

Endophytic actinobacteria isolated from healthy cereal plants were assessed for their ability to control fungal root pathogens of cereal crops both in vitro and in planta. Thirty eight strains belonging to the genera Streptomyces, Microbispora, Micromonospora, and Nocardioidies were assayed for their ability to produce antifungal compounds in vitro against Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease in wheat, Rhizoctonia solani and Pythium spp. Spores of these strains were applied as coatings to wheat seed, with five replicates (25 plants), and assayed for the control of take-all disease in planta in steamed soil. The biocontrol activity of the 17 most active actinobacterial strains was tested further in a field soil naturally infested with take-all and Rhizoctonia. Sixty-four percent of this group of microorganisms exhibited antifungal activity in vitro, which is not unexpected as actinobacteria are recognized as prolific producers of bioactive secondary metabolites. Seventeen of the actinobacteria displayed statistically significant activity in planta against Ggt in the steamed soil bioassay. The active endophytes included a number of Streptomyces, as well as Microbispora and Nocardioides spp. and were also able to control the development of disease symptoms in treated plants exposed to Ggt and Rhizoctonia in the field soil. The results of this study indicate that endophytic actinobacteria may provide an advantage as biological control agents for use in the field, where others have failed, due to their ability to colonize the internal tissues of the host plant.     

Early postfire seed dispersal, seedling establishment and seedling mortality of Pinus coulteri (D. Don) in central coastal California, USA

Seedling recruitment in many highly serotinous populations of Pinus coulteri on California's central coast depends almost entirely on periodic, stand-replacing fire. Compared to serotinous pines of the Mediterranean Basin, little detailed information is available on the postfire demography of California closed-cone pines, including P. coulteri. In September 1996 a wildfire burned the 760-ha American Canyon Research Natural Area (RNA). Using aerial photography, we mapped burn severity of P. coulteri-chaparral woodlands and forests within the RNA. From May to September of 1997, we also quantified seedling establishment and mortality in relation to biophysical site characteristics including fire severity. Seventy-six percent of P. coulteri forests and woodlands experienced high-severity burns, 9% moderate-severity burns, and 15% low-severity or unburned. Of the 53 plots used for seedling counts, 70% were high-severity, 26% moderate-severity, and 4% low-severity. Seedling densities 13 months postfire were low (0.21 m^–2), but seedling mortality also was low (8.4%). Aerial seed bank size increased from north-facing to south-facing slopes and from high-severity to low-severity burns. Seedling recruitment was unrelated to burn severity and increased with the size of the canopy seed bank (cone density). Many seedlings established from rodent seed caches; 23% of the seedlings established in clumps from seeds cached by Dipodomys agilis, Chaetodipus californicus and Peromyscus maniculatus. Pinus coulteri seeds have low potential for dispersal by wind, but secondary dispersal by rodents moves seeds away from source trees and into neighboring chaparral. We discuss the potential importance of rodent seed caching to postfire demography of California and Mediterranean serotinous pines.     

Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

Fine structure of the flagellar apparatus of gametesin situ and motile zygotes of the green algaUlothrix flacca var.Roscoffensis (Ulotrichales) (Chlorophyta)

Summary This fine structural study ofUlothrix flacca (Dillw.) ThuretRoscoffensis variety (Berger-Perrot), a marineUlothrix, describes in detail the flagellar apparatus configuration of gametesin situ in the gametangia and in motile zygotes. The gametes's flagellar apparatus shows two basal bodies overlapping at their proximal end at a 30° angle, in an 11/5 o'clock configuration or with a counterclockwise absolute orientation. The basal bodies are interconnected by a non-striated band or capping plate. They are wrapped in their proximal part by an electron-dense sheath and obtured by a bilobed terminal cap. A cruciate microtubular root system having a 4-2-4-2 alternation pattern is present. A striated microtubule associated component (S.M.A.C.) or system I fibres accompany the two membered root R₂. The system II fibres or rhizoplasts along with striated bands associated to the microtubular roots, were not observed and are presumed to be absent.In the motile zygotes, the basal bodies are paired in a cruciate pattern. During the fusion process, two basal bodies, one of each pair, slide in a face to face position with a slight displacement into the 11/5 o'clock direction; the other two make a 30° counterclockwise rotation, thus making a 60° angle between the two basal bodies of each pair instead of 30° in the gamete.After comparison with the flagellar apparatus of other green alga gametes, it is concluded that the taxonomic affinities ofUlothrix flacca var.Roscoffensis, lie with theUlvophyceae sensuStewart andMattox 1978.Abbreviations CP capping plate - ER endoplasmic reticulum - G Golgi body - LG lipid globule - M mitochondria - MS presumed mating structure - N nucleus - R₂,R₄ microtubular roots - SH sheath - SMAC striated microtubule associated component - TC terminal cap - V vacuole - Ve vesicles in the anterior papilla - 1, 2, 1, 2 basal bodies numerotation