Effect of inoculation with putative plant growth-promoting rhizobacteria isolated from Pinus spp. on Pinus pinea growth, mycorrhization and rhizosphere microbial communities

Aims: In this study, 10 putative plant growth-promoting rhizobacteria (PGPR) were assayed for their ability to improve Pinus pinea growth and mycorrhization. Methods and Results: After an inoculation assay, except for two, all strains stimulated plant growth. All bacteria altered rhizosphere microbial communities as revealed by phospholipid fatty acid analysis; associating plant growth promotion with a decrease in biological diversity. Three strains were tested for their ability to enhance pine mycorrhization with wild fungi species. Only strain BB1 increased the total number of mycorrhizal root tips. Mycorrhizas present in the roots of each treatment were identified by ribosomal RNA sequencing and denaturing gradient gel electrophoresis analysis, detecting specificity between mycorrhizal species colonizing the roots and the inoculated PGPR. Conclusions: In conclusion, BB1 appears to be a good candidate to be developed into a biofertilizer directed to enhance pine growth and mycorrhization, which should result in a better establishment rate for plants used in reforestation. Significance and Impact of the Study: This study shows the potential of PGPR to improve fitness of forest tree specie. Moreover, the specificity between the bacteria inoculated and the mycorrhiza that the plant selects involve a potential biotechnological use in production of value-added fungi.     

Pinus halepensis,Pinus pinaster,Pinus pinea and Pinus sylvestris Essential Oils Chemotypes and Monoterpene Hydrocarbon Enantiomers,before and after Inoculation with the Pinewood Nematode Bursaphelenchus xylophilus

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, a serious threat to global forest populations of conifers, especially Pinus spp. A time‐course study of the essential oils (EOs) of 2‐year‐old Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris following inoculation with the PWN was performed. The constitutive and nematode inoculation induced EOs components were analyzed at both the wounding or inoculation areas and at the whole plant level. The enantiomeric ratio of optically active main EOs components was also evaluated. External symptoms of infection were observed only in P. pinaster and P. sylvestris 21 and 15 days after inoculation, respectively. The EO composition analysis of uninoculated and unwounded plants revealed the occurrence of chemotypes for P. pinaster, P. halepensis and P. sylvestris, whereas P. pinea showed a homogenous EO composition. When whole plants were evaluated for EO and monoterpene hydrocarbon enantiomeric chemical composition, no relevant qualitative and quantitative differences were found. Instead, EO analysis of inoculated and uninoculated wounded areas revealed an increase of sesquiterpenes and diterpenic compounds, especially in P. pinea and P. halepensis, comparatively to healthy whole plants EOs.     

威海天鹅湖大叶藻(Zostera marina)与日本鳗草(Zostera japonica)根际微生物群落结构及其驱动机制

[目的] 本文以威海天鹅湖大叶藻和日本鳗草根际沉积物为主要研究对象,探究不同生长时期的海草根际微生物群落结构多样性,并分析导致微生物群落结构差异的内在因素。[方法] 选取威海天鹅湖大叶藻和日本鳗草根际沉积物与非草区表层沉积物,采用高通量测序技术（Illumina MiSeq platform）解析海草不同生长时期下根际与非草区微生物群落特征。[结果] 微生物群落结构差异由海草生长时期以及海草是否定植共同驱动。在海草成熟期,丙酸菌属（Propionigenium）在大叶藻与日本鳗草根际有明显富集,其相对丰度分别为11.58%和14.26%;在海草幼苗期,脱硫球茎菌科（Desulfobulbaceae）在海草根际富集（大叶藻：2.299%,日本鳗草：4.092%）;在海草衰退期时,硫卵菌属（Sulfurovum）的相对丰度在根际较高（大叶藻：5.624%,日本鳗草：3.749%）。此外,海草生长时期对不同样品之间微生物群落结构差异的解释度最大（R²=0.20335,P=0.002）。PICRUSt2功能预测结果表明各功能基因在海草不同生长时期所呈现的趋势一致,但丰度上呈现出幼苗期 > 成熟期 > 衰退期的结果。[结论] 天鹅湖海草床沉积物微生物群落结构在海草不同生长时期呈现不同的多样性特征,具有明显的根际效应且不同种类海草的根际微生物群落无显著差异,不具有物种特异性。     

施磷对干旱胁迫下箭竹根际土壤养分及微生物群落的影响

以箭竹及其根际土壤作为研究对象,采用两因素随机区组实验,设置2种水分处理（正常浇水和干旱胁迫）和2种施磷量处理（施磷和不施磷）,探究施磷对干旱胁迫下箭竹根际土壤养分及微生物群落结构和多样性的影响。结果表明：（1）干旱胁迫显著降低了箭竹根际土壤中微生物量碳、可溶性有机氮和有效磷的含量,虽对箭竹根际土壤微生物群落的多样性无显著影响,但显著降低了箭竹根际土壤中总PLFA（phospholipid fatty acid contents）的含量和真菌、细菌、革兰氏阳性菌与革兰氏阴性菌的PLFA含量以及革兰氏阳性菌/革兰氏阴性菌的PLFA比值,显著改变了箭竹根际土壤微生物群落结构,结果显著降低了箭竹的生物量。（2）施磷显著增加了受旱箭竹根际土壤中微生物量碳和有效磷的含量,虽大体上对受旱箭竹根际土壤微生物群落的多样性无显著影响,但显著增加了受旱箭竹根际土壤中总PLFA和真菌PLFA的含量,并在一定程度上增加了细菌、革兰氏阳性菌、革兰氏阴性菌和放线菌的PLFA含量以及革兰氏阳性菌/革兰氏阴性菌和真菌/细菌的PLFA比值,也在一定程度上改善了受旱箭竹根际土壤微生物群落结构,从而改善受旱箭竹的生长。（3）主成分分析表明,干旱对箭竹根际土壤微生物群落结构的影响显著,而施磷的影响不明显。（4）相关分析发现,箭竹根际土壤微生物群落结构与箭竹根际土壤微生物量碳、可溶性有机氮及箭竹生物量呈显著正相关。综上,干旱降低了箭竹根际土壤养分含量和微生物生物量,改变了箭竹根际土壤微生物群落结构,抑制了箭竹的生长;施磷能增加受旱箭竹根际土壤养分含量和微生物生物量,改善受旱箭竹根际土壤微生物群落结构,进而改善受旱箭竹的生长。     

Effects of Microbial Community Diversity on the Survival of Pseudomonas aeruginosa in the Wheat Rhizosphere

Ecological theory suggests that microbial communities with greater microbial diversity would be less susceptible to invasion by potential opportunistic pathogens. We investigated whether the survival of the opportunistic pathogen Pseudomonas aeruginosa in the wheat rhizosphere would be affected by the presence of natural and constructed microbial communities of various diversity levels. Three levels of microbial community diversity were derived from wheat roots by a dilution/extinction approach. These wheat rhizosphere inocula, as well as a gnotobiotic microbial community consisting of seven culturable wheat rhizobacterial isolates, were introduced into the nutrient solution of hydroponically grown wheat plants on the day of planting. Phenotypic characterization of the culturable microbial communities on R2A medium, Shannon microbial diversity index, community-level physiological profiles, and terminal restriction fragment length polymorphisms were used to assess the varying microbial diversity levels. At day 7 the roots were invaded with P. aeruginosa and the number of P. aeruginosa colony forming units per root were measured at day 14. The average number of surviving P. aeruginosa cells was 3.52, 4.90, 7.18, 6.65 log₁₀ cfu/root in the high, medium, low, and gnotobiotic microbial community diversity level treatments, respectively. The invasibility of the rhizosphere communities by P. aeruginosa was inversely related to the level of diversity from the dilution extinction gradient. The gnotobiotic community did not confer protection against P. aeruginosa invasion. Although these data indicate that invasibility is inversely related to diversity, further study is needed to both reproduce these findings and define the specific mechanisms of the diversity effect.     

Effects of <Emphasis Type="Italic">Pinus pinea</Emphasis> litter on seed germination and seedling performance of three Mediterranean shrub species

Pinus pinea L. is a coniferous tree inhabiting the Mediterranean Basin, which appears widely distributed in the Iberian Peninsula, frequently associated to Mediterranean shrub species, such as Cistus salvifolius L., C. libanotis L. and Halimium halimifolium (L.) Willk. (Cistaceae). This study investigated the effects (chemical and physical) of P. pinea needles on the seed germination and early growth of these three shrub species. Laboratory experiments were carried out in order to assess an expected negative effect by using different treatments to seeds (physical layer and two aqueous extracts concentrations). Seed germination of all species was inhibited when treated with aqueous extracts and the degree of inhibition was higher when using the extract with the highest concentration, while no effect was observed with the physical layer treatment. Seedling performance was also sensitive to the different treatments, showing the most stressed values with the high concentration extract. The results from this study highlight the importance of allelopathy of P. pinea needles as a mechanism for controlling seed germination and seedling performance of some Mediterranean shrub species.     

Effect of Introduced Pseudomonas fluorescens Strains on Soil Nematode and Protozoan Populations in the Rhizosphere of Wheat and Pea

Abstract Previous studies have shown that inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 increased mineralization of organic nitrogen in the rhizosphere. In contrast, inoculation of the same strains onto wheat seeds reduced mineralization of N from organic residues incorporated into soil. In the present study, we report on a likely explanation of this phenomenon, which appears to be governed by the effect of plant-microbe interactions on bacterial-feeding nematodes and protozoa. In soil microcosm tests, inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 resulted in an increase in the number of nematodes and protozoa in the rhizosphere as compared to noninoculated controls. This trend was repeated using a model sand system into which the bacteriophagous nematode Caenorhabditis elegans was introduced. It was subsequently found that non-inoculated germinating pea seeds exerted a nematicidal effect on C. elegans, which was remedied by inoculation with either strain F113lacZY or F113G22. This suggests that nematicidal compounds released by the germinating pea seeds were metabolized by the microbial inoculants before they affected nematode populations in the spermosphere or rhizosphere of pea. In contrast, inoculation of wheat plants resulted in significantly lower nematode populations in the rhizosphere, whereas protozoan numbers were unaffected. No nematicidal effects of inoculated or noninoculated wheat seeds could be found, suggesting that microfaunal populations were affected at a later stage during plant growth. Because of their key roles in accelerating the turnover of microbially immobilized N and organic matter, plants that support a larger microfaunal population are likely to benefit from a higher availability of inorganic nitrogen. Therefore, an understanding of plant-microbe interactions and their effects on soil microfaunal populations is essential in order to assess the effects of microbial inocula on plant mineral nutrition. Received: 27 May 1999; Accepted: 15 July 1999; Online Publication: 17 December 1999     

Can the emergence of pine Diplodia shoot blight in France be explained by changes in pathogen pressure linked to climate change?

Sphaeropsis shoot blight, caused by Diplodia pinea and Diplodia scrobiculata, damage conifers throughout the world. In France, the first disease outbreaks were reported during the 1990s. The factors associated with the pathogen presence in stands and the relationship between pathogen and disease distributions were analysed in order to understand the Sphaeropsis emergence. Eighty‐two stands of Pinus nigra, Pinus sylvestris, Pinus pinaster and Pinus radiata were visited. Cones were collected on the ground to assess the pathogen frequency. Diplodia spp were isolated and determined by a species‐specific PCR test. The role of potential explaining factors of D. pinea prevalence on cones was analysed by logistic regression. D. pinea was the dominant species in visited stands. The main factors influencing the pathogen presence selected in the models were host species (the pathogen being less frequent on P. pinaster than on P. nigra and P. sylvestris cones), winter temperature and summer rain, which were both positively correlated with cone colonization. The climate became more favourable to D. pinea presence within the last 15 years compared with the previous 30‐year period. By contrast, future climatic changes over the next 40 years should have far less impact on the pathogen presence.     

Ecological Strategies of Soil Microbial Communities under Plants of Meadow Ecosystems

Dominant growth strategies of soil microbial communities of mown and unmown meadows were assessed with respect to the constants of saturation and maximal specific growth rate of microorganisms. The microbial community of mown-meadow soil was characterized by a greater biomass and activity due to prevalence of microorganisms with the r strategy, compared to the microbial community of unmown-meadow soil. In contrast to nonrhizosphere soil, rhizosphere soil was dominated by rapidly growing microorganisms with the r strategy. The dependence of the dominant ecological strategy of the rhizosphere microbial community on the vegetation stage of plants has been traced. Study of the effect of plant species on the growth strategies of rhizosphere microorganisms showed that the features of the K strategy are more pronounced in the following rhizosphere microbial communities of grasses at the same growth stage: r strategy–Bromopsis inermis L.–Poa pratensis L., P. compressa L.–Dactylis glomerata L.–Festuca pratensisL.–K strategy. In the absence of limitation by climatic factors, the growth strategies of rhizosphere microorganisms are determined by the competition between microorganisms and plants for nutrients.     

Combined Application of the Biological Product LS213 with Bacillus, Pseudomonas or Chryseobacterium for Growth Promotion and Biological Control of Soil-Borne Diseases in Pepper and Tomato

Recent works suggest that the combination of several PGPRs could be more effective than individual strains as a horticultural product. LS213 is a product formed by a combination of two PGPRs, Bacillus subtilis strain GB03 (a growth-promoting agent), B. amyloliquefaciens strain IN937a (an inducer of systemic resistance) and chitosan. The aim of this work is to establish if the combination of three PGPR, B. licheniformis CECT 5106, Pseudomonas fluorescens CECT 5398 and Chryseobacterium balustinum CECT 5399 with LS213 would have a synergistic effect on growth promotion and biocontrol on tomato and pepper against Fusarium wilt and Rhizoctonia damping off. When individual rhizobacterium and the LS213 were put together, the biometric parameters were higher than with individual rhizobacterium both in tomato and pepper, revealing a synergistic effect on growth promotion, being the most effective combination that of B. licheniformis and LS213. When P. fluorescens CECT 5398 was applied alone, it gave good results, which could be due to the production of siderophores by this strain. Biocontrol results also indicate that those treatments that combined LS213 and each of the bacteria (Treatments: T7 and T8) gave significantly higher percentages of healthy plants for both tomato (T7: 65%) and pepper (T7: 75% and T8: 70%) than the LS213 alone (45% of healthy plants for tomato and 60% for pepper) three weeks after pathogen attack. The effects in pepper were more marked than in tomato. The best treatment in biocontrol was the combination of P. fluorescens and LS213. In summary, the combination of microorganisms gives better results probably due to the different mechanisms used.     

Effects of 5-fluoro-2′-deoxyuridine on bacterial growth its use for DNA labelling

Experiments on the action of 5-fluoro-2′-deoxyuridine on growth ofEscherichia coli B, CECT 101;Pseudomonas fluorescens, CECT 318;Pseudomonas savastanoi, CECT 93;Micrococcus luteus, ATCC 4698;Bacillus cereus, CIP 52.58;Bacillus macerans, ClP 52.58 andBacillus subtilis, ATCC 6633, are described. The inhibition of growth is reversed by thymine plus uracil in all cases except inPseudomonas strains in which uracil alone is active, and in which no exogenous thymine is taken up, not even in the presece of 2′-deoxyguanosine. Growth conditions for improved labelling of bacterial DNA are discussed in the light of the results.     

秦岭典型林分夏秋两季根际与非根际土壤微生物群落结构

本试验主要以秦岭山脉锐齿栎(Quercus aliena var.acutidentata)、油松(Pinus tabuliformis)、华山松(Pinus armandii)、云杉(Picea asperata)4种典型林分为对象,利用BIOLOG微孔板法研究4种林分夏、秋两季根际与非根际土壤微生物群落代谢多样性。研究表明:(1)夏、秋季土壤根际与非根际的平均颜色变化率(AWCD)值截然不同,除秋季云杉外,其余处理均表现为非根际根际,且AWCD明显受到季节的影响。(2)夏秋两季4种林分根际与非根际土壤微生物功能多样性之间差异显著,其中锐齿栎林夏秋两季均表现为根际小于非根际,而其它3种针叶林则是夏季根际小于非根际,秋季根际大于非根际;锐齿栎林根际与非根际均为秋季低于夏季,其他3种针叶林则是非根际土秋季低于夏季,而根际土秋季高于夏季。(3)主成分分析显示各土壤微生物功能多样性具有显著差异,且4种林分夏秋两季根际与非根际主成分综合得分也有所不同,4种林分非根际土综合得分锐齿栎最高,其次是华山松和云杉,油松最低;根际土夏秋规律不同,夏季华山松和油松较高,云杉最低,秋季油松和云杉较高,锐齿栎最低,且综合得分与多样性指数达到显著或极显著的正相关。(4)冗余分析表明土壤理化性质的综合作用对土壤微生物群落功能多样性有显著影响。     

Changes in germination and glyoxylate and respiratory enzymes of Pinus pinea seeds under various abiotic stresses

Abstract

This study examined Pinus pinea seeds for their tolerance to osmotic potentials of ?0.30MPa (10% polyethylene glycol [PEG]), ?0.58MPa (18% PEG), ?0.80MPa (21% PEG), ?1.05MPa (24% PEG), pH values of 4, 5, 6, 7, 8, 9, 10, and different calcareous solutions (5, 10, 20 and 40% CaCO₃). The main enzymes of glyoxylate cycle and respiratory pathway were tested. Pinus pinea seeds under no stressful condition (Control) and 5% CaCO₃ reached 100% of germination. Higher concentrations of CaCO₃ (20, 40%) and lower pH (4–5) adversely affected seed germination percentage, glyoxylic and respiratory enzyme activities. PEG caused the most detrimental effects on Pinus seeds; increasing the osmotic potential the germination was completely inhibited. These results suggest that Pinus pinea is able to germinate in calcareous and alkaline soils rather than in soils with lower water availability and acidic conditions.     

Nitrogen effects on rhizosphere processes of range grasses from different successional seres

Nitrogen and rhizosphere microorganism effects on nitrogen and carbon dynamics of Sitanion hystrix (early successional species), Stipa comata and Poa secundu which are (mid-successional species), and Agropyron spicatum (late successional species) were evaluated in a growth chamber study. Rhizosphere inocula resulted in increased nitrogen in both root and shoot tissue, and also of water-extractable carbon in the rhizosphere. Plant species, rhizosphere inocula and nitrogen level showed a three-way significant interaction for total and plant-available nitrogen. Rhizosphere microbe presence resulted in higher plant-available nitrogen in the rhizosphere of S. hystrix and less with A. spicatum, suggesting nitrogen immobilization with the later successional grass. Higher nitrogen resulted in decreased active bacteria in the rhizosphere of all plants tested, and decreased fungal hyphal lengths in the rhizosphere of the later successional P. secunda and A. spicutum. Exudate carbon in the rhizosphere of the late successional species A. spicatum, was more recalcitrant, which also may contribute to nitrogen immobilization. These differential responses of early- and late-successional grasses may be important factors contributing to plant succession.     

Interactions between rhizosphere microorganisms under iron limitation

Bacillus polymyxa, Pseudomonas cepacia and Pseudomonas fluorescens are present in the rhizosphere of many crop plants. Little is known about microbial interactions in the rhizosphere. We investigated the type of interaction between these species under iron limitation. We found that, in mixed batch cultures, P. cepacia stimulates the growth of B. polymyxa and this stimulation can be observed also in low iron medium. Cell-free supernatants of cultures of P. fluorescens with various amounts of the siderophore pyoverdine also stimulate the growth of B. polymyxa. In this case we observed a positive correlation between pyoverdine concentration and growth stimulation. Purified pyoverdine also affects positively the growth of B. polymyxa.     

Risk assessment for engineered bacteria used in biocontrol of fungal disease in agricultural crops

A plant growth promoting rhizobacterium (PGPR)Pseudomonas fluorescens SBW25 (WT) protects a number of crop plant species from damping-off caused by Pythium ultimum. A genetically modified, phenazine-1-carboxylic acid (PCA) producing variant, 23.10, carries on its chromosome a single copy of phzABCDEFG, under the control of the P tac constitutive promoter. The genetically modified biological control agent (GM-BCA), 23.10, has improved biocontrol activity when compared to wild type SBW25, and can effectively suppress Pythium spp. present at up to 100 times normal field infestations. GM-BCA inocula establish high population densities which persist well in the phytosphere of several crop plants including pea, wheat and sugar beet, effectively suppressed infection and promoted increase in total plant biomass. It also has an improved spectrum of activity over other plant phytopathogens such as Fusarium spp. Gaeumannomyces graminis var. tritici, Phytophtora cinnamomi and Rhizoctonia solani. However in developing BCAs and in particular GMBCAs it is important to determine whether their use has any adverse effect in the environment. Any observed changes following inoculation with wild type BCA or GM BCA in microbial diversity (bacteria and fungi) were negligible when assessed by either quantitive selective plate count methods (CFU/g) or culture independent molecular assays (SSU rRNA based PCR-DGGE). Rhizosphere community diversity profiles (DGGE) in infected plants in the presence of inocula were highly similar to disease free systems. Histological assessment of the impact of inocula on established functional mycorrhizae associations were conducted on cores collected from an established field margin grassland pasture. No adverse impact on mycorrhizal colonization and root infection were recorded after addition of WT or GM-BCA bacterial inocula as a soil drench. This approach and the related culturable and culture independent methods have recorded only a minor, transient perturbation to microbial communities, but as far as we are aware this is the first direct demonstration that a functional, AFC producing GMM also has only a transient impact on mycorrhizal associations in established plant communities. In all instances studied the plant species, plant stage of development and disease, damping-off, had a greater impact on changes in rhizosphere diversity than the presence of an introduced GM bacterial inocula.     

马铃薯全生育期内根际微生物组变化规律

[目的]陆生植物根际环境与土壤中的微生物菌群关系密切,其根际微生物群落动态极可能直接影响着植物健康及养分高效利用。虽然根际益生菌已被证实可用于提高作物生产力,但由于缺乏对这些菌群组成动态变化规律的认识了解,它们的开发受到限制。研究马铃薯全生育期根际菌群的动态变化规律,探讨根际菌群变化与马铃薯发育时期的相关性,为针对马铃薯不同生长时期开发专用生物益生菌肥奠定理论基础。[方法]本研究着眼于马铃薯田间全生命周期微生物组动态变化,通过Illumina MiSeq高通量测序技术对不同时间点马铃薯根际细菌16S rRNA基因V3-V4区和真菌ITS区测序并对操作分类单位(OTU)进行聚类,分析样品间微生物群落的多样性特征,并通过机器学习的方法建立模型,将根际菌群与田间马铃薯发育时间相关联。[结果]根际菌群在马铃薯各个发育阶段随时间变化明显,营养生长阶段的微生物群落结构发生了显著变化,随着结薯期的开始逐渐稳定,直到块茎成熟后期根际菌群再次出现较大变化,且在不同施肥处理间呈现较大差异。进一步基于模型挖掘了与马铃薯发育时间相关联的22个特征细菌类群和16个特征真菌类群,其中苗期和结薯末期的特征类群分别为梭菌(Clostridium)和放线菌(Actinobacteria)。[结论]马铃薯的生长发育时期是影响根际微生物群落组成的主要因素,益生菌肥的添加主要影响马铃薯结薯末期的细菌微生物菌群结构。     

Growth rates of rhizosphere microorganisms depend on competitive abilities of plants and N supply

Abstract

Plant‐microbial interactions under N‐limiting conditions are governed by competitive abilities of plants for N. Our study aimed to examine how two plant species of strawberry, Fragaria vesca L. (native species) and Duchesnea indica (Andrews) Focke (an invasive plant in central Europe), growing in intra‐specific and inter‐specific competition alter the functions of rhizosphere microorganisms in dependence on N availability. By intra‐specific competition at low N level, a 2.4‐fold slower microbial‐specific growth rate was observed under D. indica characterized by smaller root biomass and lower N content in roots compared with F. vesca. By inter‐specific competition of both plants at low N level, microbial growth rates were similar to those for D. indica indicating that plants with stronger competitive abilities for N controls microbial community in the rhizosphere. Since a high N level smoothed the differences between plant species in root and microbial biomass as well as in microbial growth rates under both intra‐specific and inter‐specific competition, we conclude that competitive abilities of plant species were crucial for microbial growth in the rhizosphere only under N imitation.     

Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain

 Although Pinus pinea L. is an important forest species in the Mediterranean region, few reports exist on its ectomycorrhizal associates. Sixty isolates, obtained from fungal sporocarps collected in mixed forests of P. pinea in Catalonia (northeastern Spain), were tested for ectomycorrhiza formation on containerized P. pinea seedlings when applied as mycelial inoculum produced in peat-vermiculite. A total of 17 isolates, in 8 genera (Amanita, Hebeloma, Laccaria, Lactarius, Pisolithus, Rhizopogon, Scleroderma and Suillus), formed ectomycorrhizas and the percentages of mycorrhizal short roots varied among isolates and species from 13% to 89%. Some of these fungi are cited for the first time in association with P. pinea. The results indicate further fungal candidates for controlled inoculation of P. pinea seedlings in the nursery. Accepted: 29 October 1998