Genet Distribution of Ectomycorrhizal Fungus Suillus grevillei Populations in Two Larix kaempferi Stands over Two Years

Suillus grevillei in two Larix kaempferi stands was determined over two years by inter-simple sequence repeat (ISSR) polymorphism analysis using primers, (GTG)₅, (GCC)₅ and (GACA)₄. Thirty-five genets were identified from 67 sporocarps at the older stand (stand A in which the distribution of S. grevillei genet in 1997 was analyzed previously) in 1998, and 14 genets from 52 sporocarps at the younger stand (stand B) in 1997 and 1998. The characteristics of S. grevillei genets in stand A in 1998 were similar to those in 1997. A single genet was represented by 1.8 and 3.7 sporocarps on average in stands A and B, respectively. In stand A, 42 out of 61 genets, i.e., about 70% were represented by individual sporocarps compared to five out of 14 genets, i.e., about 35% in stand B. The largest and the average genet sizes was 6.8 m and less than 1 m in stand A, and 11 m and 2.3 m in stand B, respectively. A t-test showed the genet size in stand A to be significantly smaller than that in stand B. The above results indicate that the smaller genets of the S. grevillei population in stand A might be due to environmental conditions not genetic traits specific to this species. Observations over two years showed that although some genets formed sporocarps in both 1997 and 1998, many formed sporocarps only in one of the two years. Emerging positions of sporocarps in 1997 and 1998, which belonged to the same genet, were similar but not identical, about 2 m apart, suggesting mobility in the subterranean parts of ECM fungal genets. Received 10 April 2000/ Accepted in revised form 31 August 2000     

Analysis of genetic structure of a Suillus grevillei population in a Larix kaempferi stand by polymorphism of inter-simple sequence repeat (ISSR)

Clones of ectomycorrhizal fungi can colonize new areas through production of vegetative mycelium or spore dispersal, but the relative importance of these processes in nature is not known. In this study, sporocarps of an ectomycorrhizal fungus, Suillus grevillei , were mapped and sampled from a Larix kaempferi stand at the foot of Mt Fuji. DNA was extracted directly from each sporocarp, and DNA polymorphism was analysed by polymerase chain reaction (PCR) amplification of inter-simple sequence repeat (ISSR) regions primed by (GTG)₅, (GCC)₅ and (GACA)₄. Different sensitivities to detect polymorphism were found among the three primers, with (GACA)₄ showing the highest sensitivity. Forty seven sporocarps were analysed by the three ISSR primers and divided into 34 genets based on combination of PCR fingerprints. In the population 28 genets were represented by individual sporocarps. In most cases, sporocarps grown in aggregation (within a circle of 50 cm diameter) showed some different ISSR band patterns. These results suggest that genets of S. grevillei at the test site are relatively small. The genetic similarities between the 34 genets were also calculated and similarity groups were determined by the criterion that all similarity F values of genets within a group were not <80%. In general, the genets within a similarity group located close to each other. The results of multiple different but highly related genets in a small area suggest that the population of S. grevillei in this stand is not spread and maintained by clonal mycelium extension but is reproduced by spore dispersal.     

Comparative studies on the mycorrhization of Larix decidua and Picea abies by Suillus grevillei

Summary Mycorrhization of Picea abies has been achieved, for the first time, with six strains of Suillus grevillei by a new culture method, using activated charcoal paper and liquid medium as a substrate. Mycorrhization of P. abies and Larix decidua was compared, and the process was found to be significantly different in the two tree species. S. grevillei is not incompatible with P. abies, but it forms mycorrhizae more readily with L. decidua. Hyphal growth was clearly stimulated on the surface of roots of Larix but retarded on Picea. A well organized Hartig net was formed with both tree species, but wall protuberances were frequently observed on the outer cell walls of Picea cortex cells when the Hartig net was not fully developed. No conspicuous cell wall reactions occurred in Larix roots. Cell wall protuberances may be comparable to those in transfer cells and are interpreted as an alternative to Hartig net development. Anatomical differences between roots of Larix and Picea, and physiologically active substances such as recognition factors on the root surfaces, are discussed with respect to their responsibility for the different reactions of S. grevillei.     

Apoplastic Transport through the Fungal Sheath of Pinus sylvestris/Suillus bovinus Ectomycorrhizae

Roots of Pinus sylvestris L. were inoculated in vitro with the basidiomycete Suillus bovinus (Fr.) O. Kuntze. To investigate apoplastic transport in mycorrhizal and sterile roots of Pinus sylvestris, roots of intact plants were submerged for 20 h in 0.1% solutions of the fluorescent dye sulphorhodamine G (SR-G) or for 6 h in 1.5% solutions of lanthanum nitrate. Samples treated with the dye were cryofixed, freeze-dried or freeze-substituted and embedded into Spurr's medium, maintaining strictly anhydrous conditions to prevent movement of the water-soluble dye after cryofixation. Lanthanum-treated roots were fixed in glutaraldehyde, post-fixed in OsO₄, dehydrated in a graded acetone series and embedded in Spurr's resin. The apoplastic distribution of the two tracers were examined either using fluorescence optics (sulphorhodamine) or with the electron microscope (La³⁺). The yellow-green fluorescence of sulphorhodamine could be detected within the apoplast of the fungal sheath, the Hartig net and the host cortex, up to the endodermis. Electron-dense lanthanum deposits were located in the fungal sheath, the Hartig net and in the root cortex. Greater deposition was detected within the matrix material, in which the hyphae of the mantle are embedded. The apoplastic distribution of the two tracers within the plant root did not indicate any significant qualitative differences between sterile and mycorrhizal rootlets. In contrast to reports by other authors, we conclude that the fungal sheath does provide an apoplastic pathway for water and ions at least in Pinus sylvestris/Suillus bovinus mycorrhizae. However, the mobility of charged molecules, particularly cations, may be limited by the fungal matrix.     

Colonisation of Pinus halepensis roots by Pseudomonas fluorescens and interaction with the ectomycorrhizal fungus Suillus granulatus

Colonisation of Pinus halepensis roots by GFP-tagged Pseudomonas fluorescens Aur6 was monitored by epifluorescence microscopy and dilution plating. Aur6-GFP was able to colonise and proliferate on P. halepensis roots. Co-inoculation with the ectomycorrhizal fungus Suillus granulatus did not affect the bacterial colonisation pattern whereas it had an effect on bacterial density. Bacterial counts increased during the first 20 days of seedling growth, irrespective of seedlings being mycorrhizal or not. After 40 days, bacterial density significantly decreased and bacteria concentrated on the upper two-thirds of the pine root. The presence of S. granulatus significantly stimulated survival of bacteria in the root elongation zone where fungal colonisation was higher. The number of mycorrhizas formed by S. granulatus was not affected by co-inoculation with Aur6-GFP. Neither Aur6-GFP nor S. granulatus stimulated P. halepensis development when inoculated alone, but a synergistic effect was observed on seedling growth when bacteria and fungus were co-inoculated.     

过量铜、锌对外生菌根菌牛乳牛肝菌生物量、呼吸和糖酵解酶活性的影响

在过量铜或锌胁迫下,独立培养的外生菌根菌牛乳牛肝菌（Suillus     

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5′ gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.     

Ectomycorrhizae of Lactarius lignyotus on Norway spruce,characterized by anatomical and molecular tools

The ectomycorrhizae of Lactarius lignyotus on Norway spruce are comprehensively described by morphological and anatomical characteristics. Identification of ectomycorrhizae was performed by tracing mycelia to the fruitbodies and also by molecular tools, using polymerase chain reaction (PCR) amplification of the fungal DNA. The newly described ectomycorrhiza is compared to ectomycorrhiza of the related Lactarius picinus. The amplified DNA products of the two fungi and their ectomycorrhizae could be distinguished by characteristic fragments after digestion with Hinf1.Considered as part LV of the series Studies on ectomycorrhizae of the Institute for Systematic Botany, Munich; part LIV: Agerer et al. (1994)     

Defoliation effects on enzyme activities of the ectomycorrhizal fungus <Emphasis Type="Italic">Suillus granulatus</Emphasis> in a <Emphasis Type="Italic">Pinus contorta</Emphasis> (lodgepole pine) stand in Yellowstone National Park

Ectomycorrhizal (EM) basidiomycete fungi are obligate mutualists of pines and hardwoods that receive fixed C from the host tree. Though they often share most recent common ancestors with wood-rotting fungi, it is unclear to what extent EM fungi retain the ability to express enzymes that break down woody substrates. In this study, we tested the hypothesis that the dominant EM fungus in a pure pine system retains the ability to produce enzymes that break down woody substrates in a natural setting, and that this ability is inducible by reduction of host photosynthetic potential via partial defoliation. To achieve this, pines in replicate blocks were defoliated 50% by needle removal, and enzyme activities were measured in individual EM root tips that had been treated with antibiotics to prevent possible bacterial activity. Results indicate that the dominant EM fungal species (Suillus granulatus) expressed all enzymes tested (endocellulase D-glucosidase, laccase, manganese peroxidase, lignin peroxidase, phosphatase and protease), and that activities of these enzymes increased significantly (P < 0.001) in response to defoliation. Thus, this EM fungus (one of the more specialized mutualists of pine) has the potential to play a significant role in C, N and P cycling in this forested ecosystem. Therefore, many above-ground factors that reduce photosynthetic potential or divert fixed C from roots may have wide-reaching ecosystem effects.     

Population genetics and fine-scale genetic structure of Rhizopogon roseolus in the Tottori sand dune

We investigated the population genetics and fine-scale genetic structure of Rhizopogon roseolus. A total of 173 R. roseolus sporocarps were collected from two stands in the Tottori sand dune. We developed and applied five novel polymorphic microsatellite (SSR; simple sequence repeat) markers for sporocarp genotyping. In total, we identified 110 genets, most of which were small in size. Spatial autocorrelation analyses revealed a significantly positive genetic structure in short-distance classes. The inbreeding coefficient value was significant in both stands (F_IS = 0.18), while the F_ST value (F_ST = 0.020) indicated little genetic differentiation between the two populations. The majority of alleles were distributed in both stands with similar frequencies. These results suggest that short-distance spore dispersal plays a dominant role in generating new genets, and eventually increases the frequency of inbreeding in the Tottori sand dune, whereas rare gene flow between the two stands, possibly associated with spore dispersal by mycophagous animals, could reduce genetic differentiation.     

Microsatellite Analysis of Genetic Variation and Population Genetic Differentiation in Autotetraploid and Diploid Rice

Genetic diversity and population genetic structure of autotetraploid and diploid populations of rice collected from Chengdu Institute of Biology, Chinese Academy of Sciences, were studied based on 36 microsatellite loci. Among 50 varieties, a moderate to high level of genetic diversity was observed at the population level, with the number of alleles per locus (A _e) ranging from 2 to 6 (mean 3.028) and polymorphism information content ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (H _e) varied from 0.04 to 0.76 (mean 0.370) and Shannon’s index (I) from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed slightly higher levels of A _e, H _e, and I than the diploid populations. Rare alleles were observed at most of the simple sequence repeat loci in one or more of the 50 accessions, and a core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed genetic variability mainly among autotetraploid populations rather than diploid populations (F _st = 0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and an autotetraploid and its original diploid indica male sterile lines. Group II contained only the original IR accessions. Group III was more diverse than either Group II or Group IV, comprising both autotetraploid and diploid indica restoring lines. Group IV included a japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice were somewhat dissimilar, as variation exists that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some important agricultural characters that were not found in the original diploid rice varieties.     

Effects of monovalent cations on phosphate accumulation and storage of the ectomycorrhizal fungus Suillus bovinus

Comparative in vivo ³¹P-NMR studies of the fungus Suillus bovinus (L.: Fr.) O. Kuntze in pure culture have produced interesting new data. To investigate the response of phosphate metabolism to a change in external monovalent cations, samples were exposed to a Hoagland solution containing different monovalent cations Li⁺, Na⁺, K⁺, or Rb⁺ at 10 mM concentration. A method of nutrient cycling during analysis where the cation was changed and the phosphate kept constant allowed us to determine the kinetics of phosphate accumulation, storage and incorporation into polyphosphate following exposure to the range of test cations. Different external monovalent cations had different effects upon changes in the content of both phosphate and polyphosphate. Treatment with Li⁺, Na⁺, or Rb⁺ resulted in a change in phosphate accumulation to 60, 73, and 107% and in content of the intracellular mobile polyphosphate (polyP) to 119, 112, and 94%, respectively, compared with the control taken as 100%. The effect of each cation is related to its position in the periodic table. Reversing this process, i.e., exchanging with K⁺, returned phosphate metabolism to normal. Although, the increase in depolarization of the cell membrane should affect the internal pH, fungal metabolism using energy requiring mechanisms appeared necessary to maintain the intracellular pH. Thus, increasing contents of mobile polyP were the consequence of an increasing energy demand. On the other hand, the increasing depolarization of the cell membrane following the sequence Rb⁺ < K⁺ < Na⁺ < Li⁺ inhibited the net P_i accumulation. Furthermore, it is postulated that the P_i accumulation was also regulated by the intracellular content in polyP.     

长白落叶松人工林天然更新幼苗分布格局及其研究方法的比较

长白落叶松是东北东部地区主要造林树种之一．该文研究了55a生长白落叶松人工林天然更新幼苗的空间分布格局．测定了样地内（30m×18m）所有天然更新幼苗的坐标、地径和苗高．在幼苗坐标点图的基础上,进行了23个取样尺度（离散尺度）下的相邻格子法的空间格局分析：采用Poisson分布、负二项分布和正二项分布拟合幼苗的离散分布,方差均值比、Morisita指数和最近邻体法确定了空间分布格局,C-指数、Green指数和负二项参数K值度量了它们的聚集强度．利用更新幼苗的坐标点图,采用地统计学中自々半方差分析方法和空间点格局方法（采用Ripley二次分析法）研究了尺度连续变化条件下更新幼苗空间分布格局和聚集强度的变化并确定了其聚集规模,利用最近邻体法的计算方法分析了更新幼苗的自然定植特点．结果表明：1）更新幼苗所有离散尺度下（样方面积从1m^2到18m^2）均呈聚集分布．连续尺度下,在0—10．5m的半径区间内呈聚集分布,在10．5—12．3m的半径区间内呈随机分布．2）离散尺度下（样方面积从1m^2到18m^2）,聚集强度随尺度的增加而增加．连续尺度下,在0．10．5m的半径区间内,聚集强度随尺度的增加表现出先增加后降低的趋势,在半径（尺度）为4．2m处聚集强度最大．3）此次调查的样地范围内天然更新幼苗呈现聚集状态生长的平均最大聚集斑块（聚集规模）的半径为4．79m．4）天然更新幼苗之间的平均距离为0．30-0．42m,更新幼苗生长在与母树相距2．99—3．26m之外的空间．5）相邻格子法（传统的样方方法）和空间点格局分析法对格局的判别和格局强度的确定基本一致,但是空间点格局分析法能够反映出尺度连续变化条件下格局和格局强度的变化．它与最近邻体法和地统计学方法等以种群分布的坐标点图为数据源的     

Diversity changes in an intensively bred wheat germplasm during the 20th century

Plant breeding may lead to narrowing genetic diversity of cultivatedcrops, thereby affecting sustained selection gains in crop improvement. A totalof 47 microsatellite primer pairs (mapped to the 21 wheat genetic linkagegroups) were assessed in 75 Nordic spring wheat cultivars bred during the20^th century to determine the variation of genetic diversity in thisgermplasm throughout this period. The number of alleles ranged from one toseven, with an average of 3.6 alleles per microsatellite marker. A dendrogramresulting from analysis of the matrix of dissimilarities using the unweightedpair-group method with arithmetic average discriminated all cultivars andrevealed clusters of accessions released both from some geographical area inthe Nordic Region and the breeding era, i.e. before and after World War II. Geneticdiversity in this wheat material increased from 1900 to 1940 and again from1960 onwards. In between these two periods there was a loss of diversity, whichcould not be explained by changes in a single genome or in one or few chromosomesets. Effects of different selection within countries are revealed by cleardifferences in frequency of some microsatellite alleles. In adition somemicrosatellite alleles were lost during the first quarter of the century whileseveral new alleles were introduced in the Nordic spring wheat material duringthe second half of the century. These results suggest that genetic diversity inNordic spring wheat was enhanced by plant breeding in the first quarter of the20^th century and following a decrease during the second quarter wasincreased again by plant breeding.     

Cross transferability of SSR markers to endangered Cedrela species that grow in Argentinean subtropical forests,as a valuable tool for population genetic studies

Species of Cedrela with a high economic value from Northwest and Northeastern Argentina are severely exploited. This work evaluates whether 51 nuclear SSRs, developed to study phylogenetically close species in the Meliaceae family (Cedrela odorata, Cedrela fissilis, Swietenia humilis and Swietenia macrophylla), can be used to study C. fissilis, Cedrela balansae, Cedrela saltensis and Cedrela angustifolia. A 62.8% of the total of 194 SSRs/species combinations showed a successful, homologous and cross-species amplification. As expected, a great success in SSRs transferability among Cedrela species was observed. Twenty-one screened SSRs showed a successful amplification pattern in all target species and many of them were polymorphic (9, 13, 13 and 7 SSRs for C. fissilis, C. balansae, C. saltensis and C. angustifolia, respectively). The high number of evaluated SSRs from the Cedrela genus and Meliaceae family, allowed us to obtain a suitable set of validated markers that are highly variable and easily scored, and also identify those which were less sturdy. We were able to retain a useful set of markers for three of the target species, but not for C. angustifolia. This could be due to its greater phylogenetic and morphological distances to the other three species. The lack of SSRs developed for our target species, transforms the transferred SSRs reported here in a valuable tool to monitor the genetic consequences of forest overexploitation on Cedrela species.