Nitrogen-fixing Bacillus sp. associated with Douglas-fir tuberculate ectomycorrhizae

Nitrogenase activities, measured by acetylene reduction, were detected under microaerophilic field conditions in Douglas-fir tuberculate ectomycorrhizae. Tuberculate ectomycorrhizae consist of densely packed clusters of ectomycorrhizal rootlets enclosed in a supplementary fungal peridium-like layer. Nitrogenase activity was primarily in the external layer and was greatly enhanced with added sucrose. The bacterium isolated, a nitrogen-fixing, spore-forming Bacillus sp., is an aerobe but requires anaerobic conditions for nitrogenase activity. Respiration in the tuberculate complex by the fungus, roots, and associated mycorrhizosphere microbes probably contributes to maintaining a microaerophilic niche where nitrogen fixation can take place. Water extracts of peridium or mycorrhizal root tips enhanced nitrogenase activity of this associative Bacillus sp., thereby indicating a close nutritional relationship between this bacterium and the tuberculate mycorrhizae. Thiamine more significantly enhanced bacterial nitrogenase activity than biotin; no activity was detected with p-aminobenzoic acid. Even though the levels ofnitrogenase activities in the tubercles in situ were low, as measured by the present methods, they may indicate a significant contribution to the nitrogen dynamics of these nitrogen-limited Douglas-fir forests over a long-term period.     

不同林龄和密度马尾松人工林针叶和根系的生态化学计量特征

为了解不同林龄和密度马尾松人工林针叶和根系的养分变化特征,该文在广西南宁市横县镇龙林场选择了四种林龄(幼龄林、中龄林、成熟林和过熟林)和四种密度(低密度林、中低密度林、中高密度林和高密度林)马尾松林共八种林分,分析了马尾松针叶和根系的C、N、P含量和比值及其与土壤养分的关系。结果表明:(1)所有龄林与密度林的马尾松针叶N∶P比值均大于16,表明该地区马尾松明显受P限制,幼龄林更加明显。(2)马尾松针叶C含量随着林龄增长逐渐增大后下降,N与P含量呈微弱下降趋势,导致C∶N比值、C∶P比值和N∶P比值呈微弱上升趋势,但没达到显著水平;根系C含量、P含量和C∶N比值逐渐增大,N含量、C∶P比值和N∶P比值呈U字型且都在幼龄林最大;针叶和根系在成熟林阶段均具有较高的P含量和最高的C含量。(3)中密度林的马尾松针叶的C和N含量较高且P含量最高,C∶N比值较低且C∶P比值和N∶P比值最低;根系的C、N和P含量较高,而C∶N比值、C∶P比值和N∶P比值较低。(4)马尾松的根系养分尤其是P含量在不同龄林和不同密度林之间的变化比针叶更加剧烈,且其与土壤养分之间的相关性比针叶更强。综上结果表明,马尾松人工林受P限制,在低龄林加强P肥管理和选择合适的林分密度(中等密度)则有利于缓解马尾松受P限制的状态。     

七株外生菌根真菌与三种松苗菌根的形成能力

松树外生菌根菌资源丰富,但实际应用的种类不多。为筛选出与松苗形成菌根能力较强的菌种,采用播种接菌和芽苗截根移栽接菌两种方法,对7株外生菌根真菌与马尾松、湿地松和黑松3种松苗的菌根合成进行了研究。结果表明：形成的菌根以二叉分枝状为主,棒状菌根相对较少,多叉状菌根以马尾松较多。PC2形成的菌根表面菌丝厚且紧密,504、EG、Pt,形成的菌根表面菌丝紧密程度中等,而505、ZJ和HX形成的菌根其表面菌丝则比较稀疏;Pt1、Pt2、EG形成的菌根外延菌丝较长,而505、HX形成的菌根外延菌丝极短。截根接菌时的感染率和感染指数要高于播种接菌。504形成菌根的能力最强,在3种松苗上的菌根感染率都达100％,感染指数最高可达90,最低也达70;Pt2和EG与马尾松和黑松形成菌根的能力较强;而505和HX仅与马尾松形成菌根的能力较强;Pt1形成菌根的能力较差,在3种松苗上菌根感染率和感染指数都较低。在3种松苗中,马尾松的菌根化状况最好,其次为黑松,湿地松的菌根化状况较差。     

Diversity of ectomycorrhizal fungi naturally established on containerised Pinus seedlings in nursery conditions

The study examined the diversity of ectomycorrhizal fungi, naturally established on roots of containerised Pinus seedlings in a nursery, using PCR-RFLP and sequencing of the nuclear ribosomal internal transcribed spacer. Seventy-two samples, including ectomycorrhizae and fruit bodies, were examined. Molecular typing assigned the fungal symbionts to four ectomycorrhizal Boletales: Rhizopogon rubescens, Suillus bovinus, S. variegatus, and R. luteolus. R. rubescens was abundant (37.5%), while Suillus and R. luteolus species were moderately established (25-26%) and rare (2.8%), respectively. In addition, Rhizopogon species colonised P. nigra ssp. salzmannii seedlings, whereas Suillus species were identified on Pinus nigra ssp. nigra seedlings. The diversity and the ability of these naturally established symbionts under artificial nursery conditions were discussed. The molecular survey investigated here should contribute to successful monitoring of mycorrhizal application under both nursery and plantation conditions.     

Root surface phosphatase activities and uptake of 32P-labelled inositol phosphate in field-collected gray birch and red maple roots

 This study examined select, naturally-occurring tree mycorrhizae for differences related to efficiency of organic phosphorus hydrolysis in forest soils. We investigated the activity of several phosphatases and root respiration in field-collected ectomycorrhizae of American beech and gray birch and VAM of red maple. Root materials were collected in the early and late growing season from a common soil type. American beech occurred in a late-successional stand, whereas gray birch and red maple grew in a mid-successional stand. All of the root types examined had phosphatase activities with p-nitrophenyl phosphate, bis-p-nitrophenyl phosphate and phytic acid and thus the potential to mineralize monoester and diester forms of organic phosphorus. Rates of hydrolysis at pH 5.0 were greatest with p-nitrophenyl phosphate. Although enzyme activity varied with season and ectomycorrhizal morphotype, VAM roots of red maple consistently had the lowest enzyme activities on a length and dry weight basis. Comparison of ³²P uptake from inositol phosphate by gray birch and red maple roots suggested that phosphomonoesterase activity was linked to P uptake from this source. Differences between species in oxygen consumption rates were less pronounced than those observed for enzymatic activities, suggesting similar short-term energy demands by the root types examined. The quantitative differences observed between plants growing on a common soil potentially relate to differences in host demand or reflect differences in basic morphology and/or physiology of associated mycobionts. Further study is necessary to understand the importance of these enzymes in the functional ecology of mycorrhizal fungi. Accepted: 20 December 1996     

四种外生菌根真菌对油松幼苗的抗猝倒病和促生作用

为了筛选拮抗油松猝倒病菌及对油松苗木具有促生作用的外生菌根（ectomycorrhizal,ECM）真菌,通过对盆栽油松幼苗进行人工接种,测定了ECM真菌对油松根的侵染率、对猝倒病的防治效果以及对油松株高和地径的影响。结果表明,供试ECM真菌中,绒粘盖牛肝菌Suillus tomentosus、灰环粘盖牛肝菌Suillus laricinus、灰鹅膏菌Amaniata vaginata和血红铆钉菇Gomphidius viscidus可在盆栽条件下与油松合成菌根。灰鹅膏菌对油松猝倒病防治效果最好,为43%     

Ectomycorrhizae and ectomycorrhizal fungal fruit bodies in pine stands differentially damaged by pine wilt disease

We surveyed ectomycorrhizae, ectomycorrhizal fungal fruit bodies, and soil physical properties in one heavily damaged and two lightly damaged pine stands on Mt. Tsukuba, central Japan. The rate of ectomycorrhizal root tips was not different between heavily and lightly damaged pine stands. For ectomycorrhizae, Cenococcum geophilum had high relative abundance in the heavily damaged pine stand. The number of ectomycorrhizal fungal fruit bodies in the heavily damaged pine stand was much lower than that in the lightly damaged pine stands.     

不同林龄和密度对马尾松人工林凋落叶养分变化的影响

该文选择广西南宁市横县镇龙林场的4种林龄(幼龄林、中龄林、成熟林和过熟林)和4种密度(低密度林、中低密度林、中高密度林和高密度林)马尾松人工林共8种林分作为研究对象,分析了未破碎和破碎两个不同降解阶段的凋落叶C、N、P含量及其生态化学计量学特征。结果表明:(1)不同林龄中,凋落叶初始C、N含量在过熟林和成熟林中较高,P含量没有显著变化,且C∶N比值和C∶P比值从幼龄林到成熟林逐渐升高,说明较高林龄马尾松对N和P重吸收较低,而较低林龄马尾松对N和P重吸收较强,需要较大。(2)不同密度林中,随着林木密度的增加,凋落叶初始C含量逐渐升高,N含量无显著变化,P含量降低;高密度林凋落叶的初始C∶P比值和N∶P比值较高,说明高种植密度下马尾松可能对N和P养分的需求较大,P重吸收较强。(3)不同林龄和不同密度马尾松林的破碎凋落叶C含量、C∶N比值、C∶P比值和N∶P比值比未破碎凋落叶的低,N和P含量较高,说明凋落物在降解过程中出现N和P养分的富集现象。(4)中林龄和较高种植密度的马尾松破碎凋落叶与未破碎凋落物的C含量差值最大,C∶N比值和C∶P比值较低,说明这两种林分的凋落叶C的降解速率可能较大。上述结果说明,中龄林和中高、高密度林的马尾松可能对N和P养分的需求较大,重吸收效率较高,且凋落叶C的潜在分解速率较高,可能利于有机碳较快进入土壤中。     

The extraction and quantification of ergosterol from ectomycorrhizal fungi and roots

Recently, ergosterol analysis has been used to quantify viable fungal biomass in resynthesized ectomycorrhizae. An objective of our study was to quantify ergosterol in a range of ectomycorrhizal isolates under differing growth conditions. In addition, we tested the applicability of the method on field-collected roots of ectomycorrhizal and vesicular-arbuscular (VA) mycorrhizal plants. Quantification of sitosterol as a biomass indicator of plant roots was also undertaken. Ergosterol was not detected in roots of uninoculated Betula populifolia seedlings, and sitosterol was not detected in an ectomycorrhizal fungal isolate but was present in birch roots. Ergosterol was produced in all isolates examined, which represented the major orders of ectomycorrhizal fungi. The range of values obtained, from 3 to nearly 18 g ergosterol mg^-1 dry mass, agrees well with reported values for other mycorrhizal and decomposer fungi. Hyphal ergosterol was the same during growth on phytic acid and KH₂PO₄. Reduction of growth temperature from 25° C to 15° C had little effect on ergosterol content of cultures harvested at similar growth stages. Ergosterol and sitosterol were detected in field-collected ectomycorrhizae of B. populifolia and Pinus sylvestris and VA mycorrhizae of Acer rubrum and Plantago major. Both ergosterol content and ergosterol to sitosterol ratios were significantly lower in VA mycorrhizae than ectomycorrhizae. Calculations of viable fungal biomass associated with field-collected roots were in agreement with those reported by others using the method on resynthesized ectomycorrhizae. Estimates of total mass could be obtained for field-collected B. populifolia roots by a simultaneously using ergosterol to estimate fungal biomass and sitosterol to estimate root mass. Some potential applications and limitations of sterol quantification in studies of mycorrhizal physiology and ecology are discussed.     

Ectomycorrhizal specificity patterns in a mixed Pinus contorta and Picea engelmannii forest in Yellowstone National Park

We used molecular genetic methods to test two hypotheses, (i) that host plant specificity among ectomycorrhizal fungi would be common in a closed-canopy, mixed Pinus contorta-Picea engelmannii forest in Yellowstone National Park and (ii) that specificity would be more common in the early successional tree species, P. contorta, than in the invader, P. engelmannii. We identified 28 ectomycorrhizal fungal species collected from 27 soil cores. The proportion of P. engelmannii to P. contorta ectomycorrhizae was nearly equal (52 and 48%, respectively). Of the 28 fungal species, 18 composed greater than 95% of the fungal community. No species was associated exclusively with P. contorta, but four species, each found in only one core, and one species found in two cores were associated exclusively with P. engelmannii. These fungi composed less than 5% of the total ectomycorrhizae. Thus, neither hypothesis was supported, and hypothesized benefits of ectomycorrhizal specificity to both trees and fungi probably do not exist in this system.     

Root turnover and productivity of coniferous forests

Summary Fine roots and mycorrhizae have recently been shown to produce a major portion of the organic matter entering decomposition. Roots and mycorrhizae constitute 63 to 70% of total net primary production in Douglas-fir and Pacific silver fir stands. The importance of roots in primary production makes the method of root extraction from the soil important. Wet-sieving with small mesh screens is more effective than hand-sorting for fine roots and mycorrhizae. Screen size, the efficiency of recovery, the physiological status of the roots and coversion factors to derive biomass from the numbers of root tips should be stated. Published data is enhanced if the phenological status of the stand, its age, tree density, and soil texture are quoted.Given the large fluxes in fine root and mycorrhiza populations, single biomass estimates are not useful in studies of ecosystem structure and function. A better understanding needs accurate methods to distinguish live and dead roots, data on the production and turnover of large roots, and data on the transfer of nutrients accompanying the large input of roots to decomposition.     

The catalytic activity of nitrogenase in intact Azotobacter vinelandii cells

The influence of the growth conditions on the concentration of nitrogenase and on the nitrogenase activity, was studied in intact Azotobacter vinelandii cells. It was observed that whole cell nitrogenase activity could be enhanced in two ways. An increase of the growth rate of cells was accompanied by an increase in whole cell nitrogenase activity and by an increase in the concentration of nitrogenase in the cells. The molar ratio of Fe protein:MoFe protein was 1.47 +/- 0.17 and independent of the growth rate. Activity measurements in cell extracts showed that the catalytic activity of the nitrogenase proteins was independent of the growth rate of cells. The second way to increase whole cell nitrogenase activity was to expose cells to excess oxygen. Whole cells were exposed for 2.5 h to an enhanced oxygen-input rate. After this incubation nitrogenase activity was increased without an increase in protein concentration. It is calculated that the catalytic activity of the Fe protein in these cells was 6200 nmol C2H4 formed X min-1 X (mg Fe protein)-1. With these cells and with cells grown at a high growth rate, 50% of the whole cell activity is lost by preparing a cell-free extract. It will be demonstrated that this inactivation is partly caused by the activity measurements in vitro. When dithionite was replaced by flavodoxin as electron donor, a maximal catalytic activity of 4500 nmol C2H4 formed X min-1 X (mg Fe protein)-1 was measured in vitro for the Fe protein. The results are discussed in relation to the present model for nitrogenase catalysis.     

夏块菌与青刚栎形成外生菌根形态变化的研究

夏块菌（Tuber aestivum）是一种具有较高经济价值的菌根食用菌。对夏块菌与青刚栎（Cycloba-lanopsis glauca）在形成菌根过程中不同阶段的菌根形态变化进行了研究,结果显示：用夏块菌孢子液接种青刚栎苗后,第14天起开始形成淡乳色的外生菌根,外延菌丝刚毛状;第一至第二个月可形成黄褐色、褐色外生菌根,外延菌丝刚毛或羊毛状。外生菌根为单根,长1～4mm,直径150～250μm。菌套厚12～20μm,平坦或自菌根延伸出刚毛状菌丝,外延菌丝束黄绿色;哈替氏网菌丝直径1～1.5μm。菌根老化后变暗褐或萎缩。外延菌丝束呈黄绿色是夏块菌菌根区别于其它块菌菌根最重要的形态特征。     

贵州中部喀斯特山地不同植被生态系统细根生态特征及养分储量

为了解喀斯特生态系统退化过程中树木细根生物量和土壤养分的变化,选择贵州中部喀斯特山地乔木林、灌木林和灌草丛3种植被生态系统,比较分析不同深度(0～5 cm、5～10 cm和10～15 cm)土壤细根数量及其养分情况.结果表明:树木细根主要分布在0～10 cm土层,并随土层加深而减少.在0～10 cm土层中,乔木林、灌木林和灌草丛的活细根生物量分别占0～15 cm总细根生物量的42.78%、56.75%和53.38%,总活细根生物量的83.36%、86.91%和93.79%.不同植被下优势种植物细根生物量存在差异.0～5 cm土层乔木林活细根氮素和磷素储量均显著高于灌草丛和灌木林(P0.05),但灌木林和灌草丛间没有差异;5～10 cm土层乔木林活细根氮和磷储量显著高于灌草丛和灌木林(P0.05),灌木林下又显著高于灌草丛下(P0.05).0～10 cm土层的活细根生物量与植株地上部分生物量呈正相关,植物叶片氮、磷养分含量与细根比根长呈显著的负相关,说明细根的养分储量对地上生物量的建成和生态系统功能的发挥具有重要作用.     

Changes in diversity and storage function of ectomycorrhiza and soil organoprofile dynamics after introduction of beech into Scots pine forests

Diversity and storage function of mycorrhiza as well as soil organoprofile formation were investigated in a chronosequence of a pure Scots pine (Pinus sylvestris L.) stand, of Scots pine stands that were underplanted with beech (Fagus sylvatica L.) and in three pure beech stands of different age. Mycorrhiza diversity was higher in the pure beech stands compared to the pure pine stand. Beech and pine trees in the mixed stands had similar dominant mycorrhiza morphotypes. However, trees in two of the three pure beech stands were mycorrhized with other types. Mycorrhizal abundance and nutrient amounts of mycorrhizae associated with beech trees were higher in the mixed and in the pure beech stands compared to pine mycorrhizae indicating that nutrient uptake was higher in older beech than in older pine trees. Humus quality varied from pine to beech stands. Plant litter storage in the humus layer was highest in the youngest mixed stand and lowest in the oldest beech stand. Humus forms changed from moder grass-type in the pure Scots pine stand to mor-like moder and moder rich in fine humus with increasing age of beeches in the mixed stands. The older beech stands were characterised by oligomull and mull-like moder as the dominating humus forms. The ecologically favourable humus forms, i.e., nutrient rich humus forms in the older beech stands correlate well with the higher mycorrhizal diversity and abundance as well as the higher nutrient storage of their mycorrhizae in these stands. The results are also discussed with regard to the 'base-pump effect' of beech trees.     

Ectomycorrhizal mycelia reduce bacterial activity in a sandy soil

Abstract: Bacterial activity was studied in a growth system containing Pinus contorta seedlings inoculated with different mycorrhizal fungi. Nylon nets enabled separation of soil compartments with extramatrical mycorrhizal hyphae from soil compartments with roots and mycelium. In three separate experiments bacterial activity, estimated as thymidine incorporation, was reduced in soils with Paxillus involutus hyphae compared to controls without mycorrhizal hyphae. This effect was found irrespective of compartments with and without roots were compared. Laccaria bicolor only reduced the activity in one of these three experiments. Thelephora terrestris (tested in two experiments), Laccaria proxima, Suillus variegatus and Hebeloma crustuliniforme (one experiment), also reduced the thymidine and leucine incorporation rates of bacteria. The reduction for these fungi varied between 20% and 50% in all experiments. Numbers of viable bacteria appeared to be reduced by T. terrestris, L. proxima, S. variegatus and H. crustuliniforme in one experiment, while no effect was seen in the other experiments.     

Fossil ectomycorrhizae from the Middle Eocene

Fossil ectomycorrhizae were found recently among permineralized plant remains in the middle Eocene Princeton chert of British Columbia. The ectomycorrhizae are associated with roots of Pinus and have a Hartig net that extends to the endodermis, a pseudoparenchymatous mantle, and contiguous extramatrical hyphae that are simple-septate. The mycorrhizal rootlets lack root hairs and dichotomize repeatedly to form large, coralloid clusters. Reproductive structures are absent. Based on the morphological characteristics, and the identity of the host, the closely related basidiomycete genera Rhizopogon and Suillus are suggested as comparable extant mycorrhizal fungi. These exquisitely preserved specimens represent the first unequivocal occurrence of fossil ectomycorrhizae and demonstrate that such associations were well-established at least 50 million years ago.     

In vitro ectomycorrhizal formation in Picea glehnii seedlings

 Twenty isolates of ectomycorrhizal fungi – 3 from Picea glehnii, 12 from other coniferous trees, and 5 from decidous trees – were tested for the ability to form mycorrhizae with P. glehnii, using an in vitro synthesis technique. Macroscopically, mycorrhizal formation was observed 3 months after inoculation, when the lateral roots began to grow. Mycelial growth was observed in all inoculated treatments, generally around and along the roots. Six months after inoculation, seedlings were harvested and the mycorrhizae were observed microscopically. Fourteen of the 20 isolates formed ectomycorrhizae with a dense sheath and a deep Hartig net; 1 formed ectendomycorrhizae with a rudimentary mantle, a well-developed Hartig net and intracellular hyphae; 3 formed pseudomycorrhizae with a mantle but without the Hartig net; and only 2 of the fungi tested, Chalciporus pipeparatus 5/92 and Lyophyllum sp. 61/92, did not form mycorrhizae at all. P. glehnii was a good host species since it had low specificity to ectomycorrhizal fungi isolated from trees other than P. glehnii. Accepted: 6 May 1996     

Development of salix and populus mycorrhizae in metallic mine tailings

Summary Roots of two year old willow and poplar planted as cuttings in copper and iron mine tailings were stained intact and examined microscopically for mycorrhizal development. Incidence of mycorrhizae was related to tree vigor and to chemical and physical properties of the tailings. Results showed that no mycorrhizal development occurred on roots of these tree species in the copper tailings; inoculations with natural forest soil extract failed to initiate this symbiosis. Tops and roots of the willow and poplar were stunted and survival was poor. Roots in the iron mine tailings developed extensive ectomycorrhizae and trees showed good growth. Presence of mycorrhizae was confirmed and external morphology examined with the scanning electron microscope. Aseptic jack pine and naturally mycorrhizal white pine roots were used in microscopic comparisons with the hardwoods. re]19760220     

Mycorrhizal infection and ageing affect element localization in short roots of Norway spruce [Picea abies (L) Karst.]

Norway spruce [Picea abies (L.) Karst.] seedlings, nonmycorrhizal of mycorrhizal with Laccaria laccata or Paxillus involutus were grown in a quartz sand-nutrient solution system for 6 months and then treated with 5 M Pb for 4 days. Element contents of cortex cell wall of young, medium and old short roots were determined by X-ray microanalysis of longitudinal thin sections. The Pb content was influenced neither by age nor by the distance from the root tip (up to 1.7 mm) but was significantly lower in the P. involutus mycorrhizae than in the L. laccata mycorrhizae or in nonmycorrhizal short roots. In the P. involutus mycorrhizae, the P content of the cortex cell walls was twice as high in young mycorrhizae than in old mycorrhizae. In the nonmycorrhizal short roots and the L. laccata mycorrhizae, P content was influenced neither by age nor by distance from the root tip. The Ca and Fe contents of the cortex cell walls increased with age in the nonmycorrhizal short roots and the mycorrhizae. It is concluded that the element content of the cortex cell walls of short roots is strongly influenced by age, while the distance from the root tip seems to be of minor importance.