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.     

6种外生菌根真菌对西南桦幼苗的接种效应

【背景】西南桦是兼具内生、外生菌根的典型菌根营养型树种,菌根化育苗是其壮苗培育的有效措施。【目的】揭示外生菌根真菌对西南桦无性系幼苗生长和养分含量的影响,为其菌根化育苗筛选优良外生菌根真菌提供科学依据。【方法】以BY1、FB4、FB4+和A5等4个西南桦优良无性系为研究对象,选用土生空团菌(Cenococcumgeophilum)、松乳菇(Lactariusdeliciosus)、黄硬皮马勃(Scleroderma flavidum)、多根硬皮马勃(S. polyrhizum)、褐环乳牛肝菌(Suillus luteus)和红绒盖牛肝菌(Xerocomuschrysenteron)6个外生菌根真菌进行盆栽接种试验,分析接种处理间及无性系间苗高、地径、生物量以及养分含量差异。【结果】6个菌种均能与西南桦无性系幼苗形成外生菌根共生体,接种多根硬皮马勃与黄硬皮马勃显著促进了幼苗生长和养分吸收(P0.05),说明其与幼苗的亲和力明显优于其它菌种。尽管菌根侵染率在4个无性系之间无显著差异(P≥0.05),但各菌种对FB4、BY1幼苗生长的促进作用显著强于其它2个无性系。【结论】多根硬皮马勃和黄硬皮马勃可作为西南桦菌根化育苗的优选菌种。     

Forest reclamation of fly ash deposit: a field study on appraisal of mycorrhizal inoculation

An extensive field trial was established on a fly ash deposit (1) to evaluate whether the inoculation with arbuscular mycorrhizal fungi (AMF) and/or ectomycorrhizal fungi (EcMF) improves growth and survival of 13 planted tree species and (2) to trace the inoculated mycorrhizal fungi in tree roots after one growing season. Molecular methods were applied to characterize AMF and EcMF entering the studied system (inocula, native soil, and roots of nursery seedlings). Biometric parameters and mortality of the trees were recorded and the presence of AMF and EcMF in sampled trees was determined both microscopically and genetically. Mycorrhizal inoculation did not improve survival or growth of any tree species. Most AMF‐host and all EcMF‐host seedlings were highly precolonized already from the nursery. An abundant and diverse AMF community was also found in the field soil. The AMF inoculum taxa partially overlapped with AMF in the native soil and in the precolonized roots. After one season, the only two inoculum‐unique AMF taxa were detected in host species non‐precolonized or only partially precolonized from the nursery. The components of EcMF inoculum were not detected in any sampled tree. After the season, the ectomycorrhizal hosts maintained most of their original EcMF taxa gathered in nursery, some tree species were additionally colonized by EcMF probably originating from the soil. Our results show considerable self‐restoration potential of nature on the target site. Mycorrhizal inoculation thus did not bring any conclusive advantage to the planted trees and seems superfluous for reclamation practice on the fly ash deposit.     

Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza-forming Ceratobasidiaceae fungi

? We investigated the fungal symbionts and carbon nutrition of a Japanese forest photosynthetic orchid, Platanthera minor, whose ecology suggests a mixotrophic syndrome, that is, a mycorrhizal association with ectomycorrhiza (ECM)-forming fungi and partial exploitation of fungal carbon. ? We performed molecular identification of symbionts by PCR amplifications of the fungal ribosomal DNA on hyphal coils extracted from P. minor roots. We tested for a (13)C and (15)N enrichment characteristic of mixotrophic plants. We also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings. ? In phylogenetic analyses, most isolated fungi were close to ECM-forming Ceratobasidiaceae clades previously detected from a few fully heterotrophic orchids or environmental ectomycorrhiza surveys. The direct inoculation of fungal coils of these fungi resulted in ectomycorrhiza formation on P. densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P. minor, with fungal carbon contributing from 50% to 65%. ? This is the first evidence of photosynthetic orchids associated with ectomycorrhizal Ceratobasidiaceae taxa, confirming the evolution of mixotrophy in the Orchideae orchid tribe, and of ectomycorrhizal abilities in the Ceratobasidiaceae. Our new ectomycorrhiza formation technique may enhance the study of unculturable orchid mycorrhizal fungi.     

Kinetics of phosphate absorption by mycorrhizal and non-mycorrhizal Scots pine seedlings

Kinetics of net phosphate (P_i) uptake was measured on intact ectomycorrhizal and non‐mycorrhizal Pinus sylvestris seedlings using a semihydroponic cultivation method. The depletion of P_i in a nutrient solution was assessed over a 160–0.2 μM P_i gradient. Growth of the pine seedlings was P limited and measurements were performed 7 and 9 weeks after inoculation. Three ectomycorrhizal fungi were studied: Paxillus involutus, Suillus bovinus and Thelephoraterrestris. P_i uptake was extremely fast in plants colonised by P. involutus. The P_i concentration dropped below 0.2 μM within 4–5 h. In plants colonised with S. bovinus this occurred in 5–6 h and in plants associated with T. terrestris 8 h were needed to run through the whole concentration range. Non‐mycorrhizal plants of similar size and nutrient status decreased P_i to a concentration between 1 and 2 μM in 18 h. Data were curve fitted to a two‐phase Michaelis‐Menten equation. The apparent kinetic constants, K_m and V_max, for the high affinity P_i uptake system of the pine roots could be estimated accurately. V_max of this system was up to 7 times higher in pines associated with P. involutus than in non‐mycorrhizal seedlings. The intact extraradical mycelium greatly increased the absorption surface area of the roots (V_max). Non‐mycorrhizal plants had a K_m between 7.8 and 16.4 μM P_i. Plants mycorrhizal with P. involutus had K_m values between 2.4 and 7.2, plants colonised with S. bovinus had a K_m between 5.1 and 12.3, and seedlings associated with T. terrestris had a K_m from 4.6 to 10.1 μM P_i. All 3 ectomycorrhizal fungi had a strong impact on the P_i absorption capacity of the pine seedlings. The results also demonstrated that there is substantial heterogeneity in kinetic parameters among the different mycorrhizal root systems.     

Mycorrhiza formation and growth of Eucalyptus globulus seedlings inoculated with spores of various ectomycorrhizal fungi

 As many eucalypts in commercial plantations are poorly ectomycorrhizal there is a need to develop inoculation programs for forest nurseries. The use of fungal spores as inoculum is a viable proposition for low technology nurseries currently producing eucalypts for outplanting in developing countries. Forty-three collections of ectomycorrhizal fungi from southwestern Australia and two from China, representing 18 genera, were tested for their effectiveness as spore inoculum on Eucalyptus globulus Labill. seedlings. Seven-day-old seedlings were inoculated with 25 mg air-dry spores in a water suspension. Ectomycorrhizal development was assessed in soil cores 65 and 110 days after inoculation. By day 65, about 50% of the treatments had formed ectomycorrhizas. By day 110, inoculated seedlings were generally ectomycorrhizal, but in many cases the percentage of roots colonized was low (<10%). Species of Laccaria, Hydnangium, Descolea, Descomyces, Scleroderma and Pisolithus formed more ectomycorrhizas than the other fungi. Species of Russula, Boletus, Lactarius and Hysterangium did not form ectomycorrhizas. The dry weights of inoculated seedlings ranged from 90% to 225% of the uninoculated seedlings by day 110. Although plants with extensively colonized roots generally had increased seedling growth, the overall mycorrhizal colonization levels were poorly correlated to seedling growth. Species of Laccaria, Descolea, Scleroderma and Pisolithus are proposed as potential candidate fungi for nursery inoculation programs for eucalypts. Accepted: 7 May 1998     

Growth,root colonization and nutrient status of Helianthemum sessiliflorum Desf. inoculated with a desert truffle Terfezia boudieri Chatin

This study aims to investigate the effects of inoculation using Terfezia boudieri Chatin ascospores (ectomycorrhizal fungus) on growth, root colonization and nutrient status of Helianthemum sessiliflorum Desf. seedlings grown in pots on two-soil types (gypseous and sandy loam). Mycorrhizal seedlings had significantly increased their height and leaf number compared to non-mycorrhizal ones. Regardless of mycorrhizal inoculation treatments, the plants growing on gypseous soil showed higher growth as compared to sandy loam one. It appears that inoculation with T. boudieri changed root morphology, increasing branching of first-order lateral roots of H. sessiliflorum seedlings. The highest root mycorrhizal colonization was recorded in inoculated seedlings on sandy loam soil (89%) when compared to gypseous one (52%). N, P and K concentrations in mycorrhizal seedlings were significantly improved by fungal inoculation. It can be concluded that inoculation of H. sessiliflorum with T. boudieri increased growth attributes and improved plant nutritional status.     

Frost hardiness of mycorrhizal (Hebeloma sp.) and non-mycorrhizal Scots pine roots

The frost hardiness (FH) of mycorrhizal [ectomycorrhizal (ECM)] and non-mycorrhizal (NM) Scots pine (Pinus sylvestris) seedlings was studied to assess whether mycorrhizal symbiosis affected the roots’ tolerance of below-zero temperatures. ECM (Hebeloma sp.) and NM seedlings were cultivated in a growth chamber for 18 weeks. After 13 weeks’ growth in long-day and high-temperature (LDHT) conditions, a half of the ECM and NM seedlings were moved into a chamber with short-day and low-temperature (SDLT) conditions to cold acclimate. After exposures to a range of below-zero temperatures, the FH of the roots was assessed by means of the relative electrolyte leakage test. The FH was determined as the inflection point of the temperature-response curve. No significant difference was found between the FH of mycorrhizal and non-mycorrhizal roots in LDHT (?8.9 and ?9.8 °C) or SDLT (?7.5 and ?6.8 °C). The mycorrhizal treatment had no significant effect on the total dry mass, the allocation of dry mass among the roots and needles or nutrient accumulation. The mycorrhizal treatment with Hebeloma sp. did not affect the FH of Scots pine in this experimental setup. More information is needed on the extent to which mycorrhizas tolerate low temperatures, especially with different nutrient contents and different mycorrhiza fungi.     

Growth of mycorrhizal jack pine (Pinus banksiana) and white spruce (Picea glauca) seedlings planted in oil sands reclaimed areas

The effectiveness of ectomycorrhizal inoculation at the tree nursery seedling production stage on growth and survival was examined in jack pine (Pinus banksiana) and white spruce (Picea glauca) planted in oil sands reclamation sites. The seedlings were inoculated with Hebeloma crustuliniforme strain # UAMH 5247, Suillus tomentosus strain # UAMH 6252, and Laccaria bicolor strain # UAMH 8232, as individual pure cultures and in combinations. These treatments were demonstrated to improve salinity resistance and water uptake in conifer seedlings. The field responses of seedlings to ectomycorrhizal inoculation varied between plant species, inoculation treatments, and measured parameters. Seedling inoculation resulted in higher ectomycorrhizal colonization rates compared with non-inoculated control, which had also a relatively small proportion of roots colonized by the nursery contaminant fungi identified as Amphinema byssoides and Thelephora americana. Seedling inoculation had overall a greater effect on relative height growth rates, dry biomass, and stem volumes in jack pine compared with white spruce. However, when examined after two growing seasons, inoculated white spruce seedlings showed up to 75 % higher survival rates than non-inoculated controls. The persistence of inoculated fungi in roots of planted seedlings was examined at the end of the second growing season. Although the inoculation with H. crustuliniforme triggered growth responses, the fungus was not found in the roots of seedlings at the end of the second growing season suggesting a possibility that the observed growth-promoting effect of H. crustuliniforme may be transient. The results suggest that the inoculation of conifer seedlings with ectomycorrhizal fungi could potentially be carried out on a large scale in tree nurseries to benefit postplanting performance in oil sands reclamation sites. However, these practices should take into consideration the differences in responses between the different plant species and fungal strains.     

Elevated CO2 does not ameliorate effects of ozone on carbon allocation in Pinus halepensis and Betula pendula in symbiosis with Paxillus involutus

The effect of 700 μmol CO₂ mol⁻¹, 200 nmol ozone mol⁻¹ and a combination of the two on carbon allocation was examined in Pinus halepensis co-cultured with Betula pendula in symbiosis with the ectomycorrhizal fungus Paxillus involutus . The results show that under low nutrient and ozone levels, elevated CO₂ has no effect on the growth of B. pendula or P. halepensis seedlings nor on net carbon partitioning between plant parts. Elevated CO₂ did not enhance the growth of the fungus in symbiosis with the birch. On the other hand, ozone had a strong negative effect on the growth of the birch, which corresponded with the significantly reduced growth rates of the fungus. Exposure to elevated CO₂ did not ameliorate the negative effects of ozone on birch; in contrast, it acted as an additional stress factor. Neither ozone nor CO₂ had significant effects on biomass accumulation in the pine seedlings. Ozone stimulated the spread of mycorrhizal infection from the birch seedlings to neighbouring pines and had no statistically significant effects on phosphoenolpyruvate carboxylase (PEPC) or ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity in the pine needles or on PEPC activity in pine roots.     

菌根真菌与施肥对杜鹃苗养分吸收的影响

以2年生杜鹃(Rhododendron simsii)苗为研究对象,采用J1(杜鹃花类菌根真菌混合接菌)、J2(杜鹃花类菌根真菌和马尾松外生菌混合接菌)、J3(不接菌,对照)等3种接菌组合与不同氮磷钾施肥组合研究菌根真菌与氮磷钾肥对杜鹃苗生长及养分吸收的影响。结果表明,杜鹃幼苗的生长指标和氮磷钾含量总体表现为接菌处理优于不接菌处理,J2处理下施用尿素1.1 g·株-1、钙镁磷1 g·株-1、氯化钾0.7 g·株-1对杜鹃苗生长及氮磷钾含量积累的促进作用最为明显。数据表明,J2处理促进N向叶积累,促进K向茎叶积累,促进P向根茎积累;J1处理促进N和P向根茎积累,促进K向茎叶积累。     

The use of inositol hexaphosphate as a phosphorus source by mycorrhizal and non-mycorrhizal Scots Pine (Pinus sylvestris)

1. The external mycelia of the ectomycorrhizal fungi Thelephora terrestris and Suillus luteus , associated with Pinus sylvestris roots, exhibited a substantial extracellular acid phosphatase activity. The activity was positively correlated with the ergosterol concentration in the growth substratum and decreased with an increasing P nutrition.
2. The pioneer species T. terrestris grew best at a high P_i nutrition level whereas S. luteus , a 'late-stage' mycobiont, produced more active biomass at a low P_i nutrition level.
3. The phytase activity of the external mycelia could not be detected; at the root surface a phytase activity was observed. Mycorrhizas had significantly higher activities than uninfected roots.
4. The addition of a relatively high concentration of a soluble phytate to the growth substratum resulted in an increased relative growth rate (RGR) in both mycorrhizal and non-mycorrhizal plants. The influence of the mycorrhizal fungi on the use of the phytate-P was small, despite the phytase activity of the mycorrhizal feeder roots.
5. The addition of phytate fixed on a HPLC resin did not result in an increase of the RGR and P uptake neither in the non-mycorrhizal nor in the mycorrhizal Pines. The experiment did not support the hypothesis that phytate, which has a low solubility in soils, is a useful P source for ectomycorrhizal plants.     

Ericoid mycorrhizal fungi are common root associates of a Mediterranean ectomycorrhizal plant (Quercus ilex)

Mycorrhiza samples of neighbouring Quercus ilex and Erica arborea plants collected in a postcutting habitat were processed to see whether plants differing in mycorrhizal status harbour the same root endophytes. Three experiments were performed in parallel: (i) isolation, identification and molecular characterization of fungi from surface-sterilized roots of both plant species; (ii) re-inoculation of fungal isolates on axenic E. arborea and Q. ilex seedlings; (iii) direct inoculation of field-collected Q. ilex ectomycorrhizas onto E. arborea seedlings. About 70 and 150 fungal isolates were obtained from roots of Q. ilex and E. arborea, respectively. Among them, Oidiodendron species and five cultural morphotypes of sterile isolates formed typical ericoid mycorrhizas on E. arborea in vitro. Fungi with such mycorrhizal ability were derived from both host plants. Isolates belonging to one of these morphotypes (sd9) also exhibited an unusual pattern of colonization, with an additional extracellular hyphal net. Ericoid mycorrhizas were also readily obtained by direct inoculation of E. arborea seedlings with Q. ilex ectomycorrhizal tips. Polymerase chain-restriction fragment length polymorphism and random amplified polymorphic DNA analyses of the shared sterile morphotypes demonstrate, in the case of sd9, the occurrence of the same genet on the two host plants. These results indicate that ericoid mycorrhizal fungi associate with ectomycorrhizal roots, and the ecological significance of this finding is discussed.     

菌根接种对长苞铁杉幼苗生长的影响

采用天然长苞铁杉林富含菌根真菌的根际土壤,对长苞铁杉种子进行盆栽接种育苗试验,比较接种处理和未接种的长苞铁杉种子萌发率、菌根侵染率、幼苗生长、生物量分配及氮磷钾含量的差异。结果表明,外生菌根对长苞铁杉种子的萌发没有影响,对幼苗生长和氮磷钾养分的吸收有明显的促进作用,接种幼苗菌根感染率超过75%,其株高、根生物量、叶生物量和氮磷钾含量等均比对照显著增加,但对生物量在器官中的分配没有显著影响。     

Coinoculation efficacy of ectomycorrhizal fungi on Pinus patula seedlings in a nursery

 The coinoculation efficacy of the ectomycorrhizal fungi Laccaria laccata and Thelephora terrestris on the growth and mycorrhizal development of Pinus patula seedlings was studied and compared to individual inoculation of these fungi in a nursery. The total number of mycorrhizas was higher in seedlings inoculated with the combined inoculum than with the individual inocula. The colonization by T. terrestris was higher than L. laccata when the seedlings were inoculated with the two fungi simultaneously. Coinoculation significantly increased the height and dry weight of the seedlings compared with individual inoculation, both in steam-sterilized and unsterilized soil. Accepted: 12 May 1997     

Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell. seedlings

The development of ectomycorrhizas on inoculated eucalypt seedlings in commercial nurseries is often slow so that only a small percentage of roots are mycorrhizal at the time of outplanting. If mycorrhizal formation could be enhanced by co-inoculation with bacteria which promote rapid root colonisation by specific ectomycorrhizal fungi, as demonstrated by certain bacteria in the Douglas fir-Laccaria bicolor association, this would be of advantage to the eucalypt forest industry. Two bacterial isolates with a demonstrated Mycorrhization Helper Bacteria (MHB) effect on ectomycorrhiza formation between Pseudotsuga menziesii and Laccaria bicolor (S238), and seven Western Australian bacterial isolates from Laccaria fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaria spp. with Eucalyptus diversicolor seedlings. Mycorrhizal formation by L. fraterna (E710) as measured by percentage infected root tips, increased significantly (p < 0.05) by up to 296% in treatments coinoculated with MHB isolates from France (Pseudomonas fluorescens Bbc6 or Bacillus subtilis MB3), or indigenous isolates (Bacillus sp. Elf28 or a pseudomonad Elf29). In treatments coinoculated with L. laccata (E766) and the MHB isolate P. fluorescens (Bbc6) mycorrhizal development was significantly inhibited (p < 0.05). A significant Plant Growth Promoting Rhizobacteria (PGPR) effect was observed where the mean shoot d.w. of seedlings inoculated only with an unidentified bacterium (Elf21), was 49% greater than the mean of uninoculated controls (-fungus, -bacterium). Mean shoot d.w. of seedlings coinoculated with L. bicolor (S-238), which did not form ectomycorrhizas with E. diversicolor, and an unidentified bacterium (Slf14) or Bacillus sp. (Elf28) were significantly higher than uninoculated seedlings or seedlings inoculated with L. bicolor (S-238) alone. This is the first time that an MHB effect has been demonstrated in a eucalypt-ectomycorrhizal fungus association. These organisms have the potential to improve ectomycorrhizal development on eucalypts under nursery conditions and this is particularly important for fast growing eucalypt species where the retention time of seedlings in the nursery is of short duration (2–3 months).     

美花石斛菌根真菌接菌方式与接种效应初步研究

自然条件下,兰科菌根真菌的共生对于兰科植物种子萌芽和植株生长是必不可少的。为探讨有益共生真菌对兰科植物生长的促进作用,特别是在实验室环境下接菌方式的改变与接种效应直接的联系,本研究从野生美花石斛(Dendrobium loddigesii Rolfe.)新鲜营养根中分离、筛选出3种菌根真菌（M1、M2和M3）,采用单一接菌和混合接菌的接种方式,分析3个菌株及其不同接种方式对美花石斛生长的影响。研究得到优势菌株M1和M3,并证实混合接菌对美花石斛的生物量增长具有较好的正效应,两两混合接种方式M1-M2、M2-M3及3个混合接种方式M1-M2-M3均能较好的促进美花石斛生物量的积累。充分发挥混合接菌对兰科植物生长发育所产生的效能,提高生产效率,具有较强的现实意义。     

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     

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%