Genetic Diversity and Mating Type Distribution of Tuber melanosporum and Their Significance to Truffle Cultivation in Artificially Planted Truffieres in Australia

Tuber melanosporum is a truffle native to Europe and is cultivated in countries such as Australia for the gastronomic market, where production yields are often lower than expected. We assessed the genetic diversity of T. melanosporum with six microsatellite loci to assess the effect of genetic drift on truffle yield in Australia. Genetic diversity as assessed on 210 ascocarps revealed a higher allelic diversity compared to previous studies from Europe, suggesting a possible genetic expansion and/or multiple and diverse source populations for inoculum. The results also suggest that the single sequence repeat diversity of locus ME2 is adaptive and that, for example, the probability of replication errors is increased for this locus. Loss of genetic diversity in Australian populations is therefore not a likely factor in limiting ascocarp production. A survey of nursery seedlings and trees inoculated with T. melanosporum revealed that <70% of seedlings and host trees were colonized with T. melanosporum and that some trees had been contaminated by Tuber brumale, presumably during the inoculation process. Mating type (MAT1-1-1 and MAT1-2-1) analyses on seedling and four- to ten-year-old host trees found that 100% of seedlings but only approximately half of host trees had both mating types present. Furthermore, MAT1-1-1 was detected significantly more commonly than MAT1-2-1 in established trees, suggesting a competitive advantage for MAT1-1-1 strains. This study clearly shows that there are more factors involved in ascocarp production than just the presence of both mating types on host trees.     

Ecological indicators of Tuber aestivum habitats in temperate European beech forests

Although Tuber aestivum is widely distributed across Europe, little is known about its biology and ecology. We assess the vegetation composition of wild T. aestivum sites and use this information to characterise suitable habitats in Fagus sylvatica, Carpinus betulus and Ostrya carpinifolia (FCO) forests. Plant species composition at 16 naturally colonised T. aestivum sites in southwestern Germany and Switzerland was compared with that of 232 reference sites in Swiss FCO forest communities. The vegetation composition of truffle sites exhibits a high proportion of herb and shrub species that are indicators of a warm and dry climate, alkaline soil, and open tree canopy and thus typically occur in thermophilous FCO communities on calcareous bedrock. We conclude that T. aestivum is associated with a set of key species in addition to known host species. Based on their floristic composition, we identified FCO forest communities with high and low suitability for T. aestivum.     

Cultivation of Mediterranean species of Tuber (Tuberaceae) in British Columbia,Canada

Based on an assessment of soil and climatic conditions in British Columbia (BC), the Truffle Association of British Columbia (TABC) determined that the cultivation of Mediterranean Tuber melanosporum and Tuber aestivum might be possible in the warmer parts of the province. With the cooperation of independent truffle growers, TABC assessed the colonization of host tree roots collected from eight truffle orchards planted 2–7 years earlier using morphological and molecular criteria. Both Tuber species persisted on the roots of inoculated trees in six of the eight truffle orchards studied. The identity of Tuber ectomycorrhizas that had been characterized morphologically as differing from those of T. melanosporum and T. aestivum were determined using DNA sequence analysis to belong to three species of truffles native to the Pacific Northwest. One of those species, Tuber anniae, had been previously reported from BC, but the other two, Tuber menseri nom. prov. and Tuber beyerlei, are reported here from BC for the first time. Recently, production of three Périgord black truffles in one truffle orchard and one Burgundy truffle in another orchard demonstrates that these truffles are able to fruit in BC.     

Potential and limitations of Burgundy truffle cultivation

Burgundy truffles (Tuber aestivum syn. Tuber uncinatum) are the highly prized fruit bodies of subterranean fungi always occurring in ectomycorrhizal symbiosis with host plants. Successful cultivation can be achieved through artificial mycorrhization and outplanting of mostly oaks and hazel on suitable terrain. Here, we review ecological requirements, the influence of environmental factors, and the importance of molecular techniques for a successful cultivation of T. aestivum across Europe. The historical background and current knowledge of T. aestivum cultivation are discussed in light of its socioeconomic relevance.     

Using common mycorrhizal networks for controlled inoculation of Quercus spp. with Tuber melanosporum: the nurse plant method

The high cost and restricted availability of black truffle spore inoculum for controlled mycorrhiza formation of host trees produced for truffle orchards worldwide encourage the search for more efficient and sustainable inoculation methods that can be applied globally. In this study, we evaluated the potential of the nurse plant method for the controlled inoculation of Quercus cerris and Quercus robur with Tuber melanosporum by mycorrhizal networks in pot cultures. Pine bark compost, adjusted to pH?7.8 by liming, was used as substrate for all assays. Initially, Q. robur seedlings were inoculated with truffle spores and cultured for 12 months. After this period, the plants presenting 74 % mycorrhizal fine roots were transferred to larger containers. Nurse plants were used for two treatments of two different nursling species: five sterilized acorns or five 45-day-old, axenically grown Q. robur or Q. cerris seedlings, planted in containers around the nurse plant. After 6 months, colonized nursling plant root tips showed that mycorrhiza formation by T. melanosporum was higher than 45 % in the seedlings tested, with the most successful nursling combination being Q. cerris seedlings, reaching 81 % colonization. Bulk identification of T. melanosporum mycorrhizae was based on morphological and anatomical features and confirmed by sequencing of the internal transcribed spacer region of the ribosomal DNA of selected root tips. Our results show that the nurse plant method yields attractive rates of mycorrhiza formation by the Périgord black truffle and suggest that establishing and maintaining common mycorrhizal networks in pot cultures enables sustained use of the initial spore inoculum.     

New evidence for the symbiosis between Tuber aestivum and Picea abies

The Burgundy truffle (Tuber aestivum Vittad.), an ectomycorrhizal fungus living in association with host plants, is one of the most exclusive delicacies. The symbiosis with deciduous oak, beech, and hazel dominates our concept of truffle ecophysiology, whereas potential conifer hosts have rarely been reported. Here, we present morphological and molecular evidence of a wildlife T. aestivum symbiosis with Norway spruce (Picea abies Karst.) and an independent greenhouse inoculation experiment, to confirm our field observation in southwest Germany. A total of 27 out of 50 P. abies seedlings developed T. aestivum ectomycorrhizae with a mean mycorrhization rate of 19.6 %. These findings not only suggest P. abies to be a productive host species under suitable biogeographic conditions but also emphasize the broad ecological amplitude and great symbiotic range of T. aestivum. While challenging common knowledge, this study demonstrates a significant expansion of the species' cultivation potential to the central European regions, where P. abies forests occur on calcareous soils.     

Ectomycorrhizal fungal diversity in orchards of cultivated pecan (Carya illinoinensis; Juglandaceae)

Carya illinoinensis (pecan) belongs to the Juglandaceae (walnut family) and is a major economic nut crop in the southern USA. Although evidence suggests that some species in the Juglandaceae are ectomycorrhizal, investigations on their ectomycorrhizal fungal symbionts are quite limited. Here we assessed the ectomycorrhizal fungal diversity in cultivated orchards of C. illinoinensis. Five pecan orchards in southern Georgia, USA, were studied, three of which were known to fruit the native edible truffle species Tuber lyonii. We sequenced rDNA from single ectomycorrhizal root tips sampled from a total of 50 individual trees. Mycorrhizae were identified by ITS and LSU rDNA sequence-based methods. Forty-four distinct ectomycorrhizal taxa were detected. Sequestrate taxa including Tuber and Scleroderma were particularly abundant. The two most abundant sequence types belonged to T. lyonii (17%) and an undescribed Tuber species (~20%). Because of our interest in the ecology of T. lyonii, we also conducted greenhouse studies to determine whether this species would colonize and form ectomycorrhizae on roots of pecan, oak, or pine species endemic to the region. T. lyonii ectomycorrhizae were formed on pecan and oak seedlings, but not pine, when these were inoculated with spores. That oak and pecan seedling roots were receptive to truffle spores indicates that spore slurry inoculation could be a suitable method for commercial use and that, ecologically, T. lyonii may function as a pioneer ectomycorrhizal species for these hosts.     

Root-tip cutting and uniconazole treatment improve the colonization rate of Tuber indicum on Pinus armandii seedlings in the greenhouse

The Chinese black truffle Tuber indicum is commercially valuable. The main factors influencing the success or failure of a truffle crop include the mycorrhizal colonization rate and host plant quality. The effects of a plant growth regulator (uniconazole) and plant growth management technique (root-tip cutting) on T. indicum colonization rate and Pinus armandii seedling growth were assessed under greenhouse conditions. The results indicated that 10 mg l⁻¹ uniconazole or the combination of 5 mg l⁻¹ uniconazole and root-tip cutting constitutes an effective method for ectomycorrhizal synthesis based on an overall evaluation of colonization rate, plant biomass, plant height, root weight, stem circumference and antioxidant enzyme activities (SOD and POD) of P. armandii. The abundance of Proteobacteria in the rhizosphere of colonized seedlings might serve as an indicator of stable mycorrhizal colonization. This research inspires the potential application of uniconazole and root-tip cutting treatments for mycorrhizal synthesis and truffle cultivation.     

Identification and inoculation of fungal strains from Cedrus deodara rhizosphere involve in growth and alleviation of high nitrogen stress

Cedrus deodara is economically and ethnobotanically an important forest tree and is shown to be at decline in Northern areas of Pakistan in recent years mainly due to high concentration of Nitrogen in forests. Ectomycorrhizal (ECM) association forming fungi enables the forest trees to develop optimally by absorbing water from the rhizosphere through their absorptive hyphae and by making available the nutrients by mobilization of N and P from the organic substrates. This study was conducted to identify the ECM strains from C. deodara rhizosphere and to analyse the impact of high N load on the C. deodara seedlings to establish N critical load value for coniferous forests of Pakistan. Six new fungal strains were identified from the rhizosphere of C. deodara and were registered at GenBank (NCBI) as Emmia latemarginata strain ACE1, Aspergillus terreus strain ACE2, Purpureocillium lilacinum strain ACE3, Talaromyces pinophilus strain ACE4, A. fumigatus strain ACE5 and T. pinophilus strain ACE6 with accession numbers MH145426, MH145427, MH145428, MH145429, MH145430 and MH547115. Four out of six isolated strains were inoculated with seedlings of C. deodara singly and in consortium (CN) in combination with nitrogen load of 0 (C), 25 (T1), 50 (T2), 100 kg N ha⁻¹ yr⁻¹ (T3). Agronomic, physiological and gene expression studies for ExpansinA4 (EXPA4) and Cystatins (Cys) were made to analyse the impact of fungal strains in relation to high N stress. This study suggests a positive impact of T1 (25 kg N ha⁻¹ yr⁻¹) Nitrogen load and a negative impact of T3 (100 kg N ha⁻¹ yr⁻¹) on growth parameters and expression patterns of EXPA4 and Cys genes. Peroxidase (POX) activity decreased in the order ACE5 > ACE2 > C > ACE3 > ACE1 > CN. However, the results of Superoxide dismutase (SOD) showed decreasing trend in the order ACE5 > C > CN > ACE1 > ACE2 > ACE3. Strain ACE3 was shown to have a positive impact on the seedlings in terms of growth, physiology and expression of genes. Present study suggests that newly identified fungal strains showing positive impact on the growth and physiology of C. deodara could be used for the propagation of this economically important plant in Pakistan after pathogenicity test.     

Effects of Tabtoxinine-beta-Lactam on Nitrogen Metabolism in Avena sativa L. Roots

The effects of tabtoxinine-β-lactam (T-β-L) on nitrate uptake and glutamine synthetase (GS) and nitrate reductase (NR) activities in roots of Avena sativa seedlings were determined. Seven-day-old oat seedlings placed in a 10 mm KNO₃ and 0.5 mm T-β-L solution for 24 hours took up T-β-L and lost approximately 90% of their root GS activity. [³H]-T-β-L taken up by roots of seven-day-old oat seedlings was associated with GS immunoprecipitated from the extract of these roots. Total nitrate uptake and in vivo NR activity were decreased approximately 50% in the T-β-L treated roots. However, T-β-L uptake did not affect the induction phases of nitrate uptake or reduction, nor did it inhibit in vitro NR activity. Thus, the decrease in nitrate uptake and reduction is a secondary effect of T-β-L action. Roots of seven-day-old oat seedlings were inoculated with Pseudomonas syringae pv tabaci (Tox+) and the pathogen population in the rhizosphere was estimated by dilution plate count; 6 × 10¹³ bacteria were recovered after 3 days, as compared to the original inoculation with 7 × 10⁹ bacteria, indicating a significant growth of the pathogen in the rhizosphere. The bacteria recovered from the rhizosphere caused chlorosis in tobacco leaves and produced T-β-L in culture; 1 × 10¹⁴ bacteria were recovered from roots of seedlings inoculated with P. syringae pv tabaci (Tox−) using the same inoculation and assay procedure as for the pv tabaci (Tox+). Extracts of surface-sterilized roots previously inoculated with P. syringae pv tabaci (Tox+) did not produce viable bacterial cultures when plated out on a complete medium. Oat seedlings growing in sand culture and inoculated with P. syringae pv tabaci (Tox+) had developed chlorosis, and root GS activity had declined to less than 10% of controls after 3 days. Conversely, seedlings inoculated with P. syringae pv tabaci (Tox−) never developed chlorosis and maintained normal levels of GS activity. All oat plants inoculated with P. syringae pv tabaci (Tox+) died within 7 days after inoculation as compared to the plants inoculated with P. syringae pv tabaci (Tox−) which grew to maturity.     

Quantitative assessments of the host range and strain specificity of endophytic colonization by Klebsiella pneumoniae 342

Enteric bacteria, particularly Klebsiella, are common endophytes of plants. Endophytic colonization is important as these bacteria may be beneficial, either by providing fixed N or growth hormones to the host plant. In this work, we assessed the host range and strain specificity for endophytic colonization with Klebsiella pneumoniae 342 (Kp342) on five host plants. This strain was inoculated onto seedlings of Medicago sativa, Medicago truncatula, Arabidopsis thaliana, Triticum aestivum, and Oryza sativa. The type strain of K. pneumoniae, ATCC13883, was also inoculated on all of these hosts except M. truncatula. Both strains were labeled with GFP. Eight inoculum levels were used from 1 CFU to 10⁷ CFU per plant plus uninoculated controls. Six days after inoculation, the number of cells colonizing the rhizosphere and interior were determined. Inoculation with about one CFU of Kp342 was adequate to obtain high colonization levels on the rhizosphere and roots of all host plants. The type strain could colonize the interior of the host plant but the highest colonization levels were generally 100-fold lower than those obtained from Kp342 and those levels required at least 1000 cells in the inoculum. Thus, Kp342 was a more efficient colonizer of the plant apoplast. In addition, the monocots inoculated in this work were colonized endophytically in much higher numbers than were the dicots. Cells of Kp342 congregate at lateral root junctions suggesting the cells enter the plant through cracks created by lateral root extensions. The strain and host effects observed here suggest that endophytic colonization is an active process controlled by genetic determinants from both partners.     

Tuber aestivum association with non-host roots

Mycorrhizal fungi provide direct and functional interconnection of soil environment with their host plant roots. Colonization of non-host plants have occasionally been described, but its intensity and functional significance in complex plant communities remain generally unknown. Here, the abundance of ectomycorrhizal fungus Tuber aestivum was measured in the roots of host and non-host (non-ectomycorrhizal) plants in a naturally occurring T. aestivum colony using a quantitative PCR approach. The roots of non-host plant species found inside the brûlé area were extensively colonized by T. aestivum mycelium, although the levels were significantly lower than those found in host Carpinus betulus roots. However, fungal biomass concentration in the non-host roots was one to two orders of magnitude higher than that in the surrounding soil. This indicates existence of an important biotic interaction between T. aestivum mycelium and the non-host, mostly herbaceous plants. Roots, either host or non-host, thus probably constitute hot spots of T. aestivum activity in the soil ecosystem with as yet uncovered functional significance.     

A new duplex PCR assay for the rapid screening of mating-type idiomorphs of pathogenic Sporothrix species

Sporothrix schenckii and allied species are thermodimorphic fungi widely distributed in nature which causes human and animal sporotrichosis, the most common subcutaneous mycosis globally. Sporotrichosis is acquired after a traumatic inoculation of soil or plant material contaminated with Sporothrix propagules or through bites and scratches from diseased cats. In Ascomycota, the master regulators of sex are MAT genes that lie in a single mating-type locus, in Sporothrix these are determined by two nonhomologous alleles, MAT1-1 and MAT1-2. We assessed the whole-genome sequences of medically relevant Sporothrix to develop a single-tube duplex PCR assay to screen S. brasiliensis, S. schenckii, S. globosa, and S. luriei idiomorphs (MAT1-1 or MAT1-2) and understand the distribution and incidence of mating-type strains from natural populations. Using our duplex PCR assay, a 673 bp amplicon (α-box protein) was consistently amplified from all MAT1-1 isolates, while a 291 bp fragment was only amplified from the isolates harboring MAT1-2 (HMG box). Molecular evidence suggests heterothallism (self-sterility) as the unique mating strategy among the species evaluated. The mating-type identity of 93 isolates revealed a nearly equal distribution (1:1 ratio) of mating type alleles within species but deviating between different outbreak areas. Remarkably, for S. brasiliensis in Rio de Janeiro, we report an overwhelming occurrence of MAT1-2 (1:13 ratio; χ² = 10.286, P = 0.0013) opposing the high prevalence MAT1-1 in the Rio Grande do Sul (10:1 ratio; χ² = 7.364, P = 0.0067). Therefore, the population structure of Sporothrix species refers from paucity to regular cycles of sexual recombination in most of the studied regions. Our PCR-based mating-type diagnostic assay is proposed here as an important marker to track the geographical expansion during the long-lasting outbreak of cat-transmitted sporotrichosis driven by S. brasiliensis.     

Assessing ectomycorrhizal fungal spore banks of truffle producing soils with pecan seedling trap-plants

Background and Aims

Recently, the truffle species Tuber lyonii Butters was found to be dominant in ectomycorrhizal (EcM) fungal communities of cultivated pecan (Carya illinoinensis (Wangenh.) K. Koch). Many truffle fungi exhibit the trait of effectively colonizing plant roots via spores. We hypothesized that T. lyonii would be well represented in the spore bank of pecan orchard soils where it is found.

Methods

We used axenically-grown pecan seedlings as trap-plants to bait for EcM associates in soils collected beneath truffle-producing pecan trees. EcM fungi on seedlings were characterized through rDNA sequencing and were compared to EcM communities of adult trees in these orchards.

Results

Tuber lyonii mycorrhizas were well formed on seedlings inoculated with truffle spores, but were limited to just a few of the trap-plants grown in field soils. We compared EcM communities of adult pecan orchard trees to those on trap-plants and found distinct communities on each, with a high degree of similarity at the ordinal but not species level.

Conclusions

Although species of Pezizales are abundant in pecan EcM communities and as propagules in their soil spore banks, only a low level of T. lyonii was detected in soil spore banks beneath orchard trees naturally colonized by T. lyonii. Other factors including land-use history or orchard management may better explain this truffle species presence and abundance in pecan EcM communities.

     

Colonization of root tissues and protection against Fusarium wilt of rye (Secale cereale) by nonpathogenic rhizosphere strains of Fusarium culmorum

Colonization of rye (Secale cereale) tissues by nonpathogenic rhizosphere Fusarium culmorum isolates DEMFc2 and DEMFc5 and a pathogenic strain DEMFc37, and their effect on plant fresh weight were studied in pot experiments. Both rhizosphere isolates colonized the epidermis and the cortex but were not found in vessels, while the pathogen colonized all three layers of root cells. The numbers of pathogen CFU isolated from plant tissues were much higher than those of the rhizosphere isolates in spite of the same number of macroconidia used as inoculum (1 × 10⁵ g⁻¹ of soil). Inoculation of seedlings with DEMFc2 resulted in a 20% increase, with DEMFc5 in more than a 20% reduction, and with DEMFc37 in a 38% reduction of shoot fresh weight of 14-day-old plants. Pre-colonization of plants with (either of) the rhizosphere isolates and subsequent inoculation with the pathogen resulted in plant weights the same as those observed in plants inoculated with the rhizosphere strain alone. The disease severity index for shoots of plants pre-colonized with DEMFc2 was reduced from class 4 (86% diseased plants) observed for plants inoculated with the pathogen alone to class 2 (average of 8% diseased plants) when pre-treated with the rhizosphere strain. The CFU number of the pathogen isolated from the interior of roots of plants pre-colonized with the rhizosphere isolates was as low as 10% of the number isolated from plants inoculated with the pathogen alone. A study of in vitro interactions between the rhizosphere isolates and the pathogen suggests that changes in plant colonization by the pathogen and its effect on fresh weight of plants pre-colonized with the rhizosphere isolates were not connected with inhibition of its growth by a direct action of the rhizosphere isolates. The results suggest that strain DEMFc2 can be considered as a potential biocontrol agent.     

Biosafety and colonization of Burkholderia multivorans WS-FJ9 and its growth-promoting effects on poplars

Burkholderia cepacia complex (Bcc) is a group of bacteria with conflicting biological characteristics, which make them simultaneously beneficial and harmful to humans. They have been exploited for biocontrol, bioremediation, and plant growth promotion. However, their capacity as opportunistic bacteria that infect humans restricts their biotechnological applications. Therefore, the risks of using these bacteria should be assessed. In this study, Burkholderia multivorans WS-FJ9 originally isolated from pine rhizosphere, which was shown to be efficient in solubilizing phosphate, was evaluated with respect to its biosafety, colonization in poplar rhizosphere, and growth-promoting effects on poplar seedlings. Pathogenicity of B. multivorans WS-FJ9 on plants was determined experimentally using onion and tobacco as model plants. Onion bulb inoculated with B. multivorans WS-FJ9 showed slight hypersensitive responses around the inoculation points, but effects were not detectable based on the inner color and odor of the onion. Tobacco leaves inoculated with B. multivorans WS-FJ9 exhibited slightly water-soaked spots around the inoculation points, which did not expand or develop into lesions even with repeated incubation. Pathogenicity of the strain in alfalfa, which has been suggested as an alternative Bcc model for mice, was not detectable. Results from gene-specific polymerase chain reactions showed that the tested B. multivorans WS-FJ9 strain did not possess the BCESM and cblA virulence genes. Scanning electron microscopy revealed that the colonization of the WS-FJ9 strain reached 1.4?×?10⁴ colony forming units (cfu)?g^?1 rhizosphere soil on day 77 post-inoculation. The B. multivorans WS-FJ9 strain could colonize the rhizosphere as well as the root tissues and cells of poplars. Greenhouse evaluations in both sterilized and non-sterilized soils indicated that B. multivorans WS-FJ9 significantly promoted growth in height, root collar diameter, and plant biomass of inoculated poplar seedlings compared with controls. Phosphorus contents of roots and stems of treated seedlings were 0.57 and 0.55 mg g^?1 higher than those of the controls, respectively. Phosphorus content was lower in the rhizosphere soils by an average of 1.03 mg g^?1 compared with controls. The results demonstrated that B. multivorans WS-FJ9 is a nonpathogenic strain that could colonize the roots and significantly promote the growth of poplar seedlings.     

Morphological and physiological response of sour orange (Citrus aurantium L.) seedlings to the inoculation of taxonomically characterized bacterial endophytes

Entophytic bacteria (EBs) are very diverse and found in virtually all plant species studied. These natural EBs live insides the host plant and can be used to maximize crop and fruit yield by exploiting their potential. In this paper, EBs characterization from various citrus genotypes and their influence on the morphological and physiological functioning of sour orange (Citrus aurantium) seedlings are described. To assess the influence of 10 distinct EBs, three different techniques (injection, soil mix, and spray) were applied for single and mixed inoculation on sour orange (C. aurantium) seedlings. The selected strains were identified as firmicutes (Enterococcus faecalis, Bacillus safensis, Bacillus cereus, Bacillus megaterium, Brevibacillus borstelensis & Staphylococcus haemolyticus), and gamma Proteobacteria (Enterobacter hormachaei, Proteus mirabilis, Pseudomonas aeruginosa, & Pseudomonas sp.) by 16S rRNA gene sequencing. To investigate the influence of these EBs on host plant morphology, different parameters (morphometric) were recorded after five WOI (weeks of inoculation), including shoot/root length, shoot/root fresh and dry biomass, and biophysical analyses i.e., relative water content (RLWC). Physiological markers such as chlorophyll & carotenoid content, protein content, proline content, phenolics, and flavonoids were also analyzed to determine the influence of endophytes on sour orange seedlings. Five strains such as SM-34, SM-20, SM-36, SM-68, and SM-56 significantly improved the development and physiology of sour orange seedlings. Bacillus cereus and Pseudomonas aeruginosa produced the best outcomes in terms of plant growth. The relative quantification of bacterial inoculums was determined using real-time PCR. A rise in the number of bacterial cells in inoculated treatment suggests that bacterial strains survived and colonized successfully, and also shown their competitiveness with native bacterial community structure. As per the results of inoculation methods, soil mixing, and injection methods were determined to be effective for bacterial inoculation to plants but a variable trend was found for different parameters with test bacterial strains. After testing their impact on field conditions, these strains can be applied as fertilizers as an alternative to conventional chemical fertilizer, although in the context of mixed inoculation of bacterial strains, 5 M and 6 M performed best and enhanced plant growth-promoting activity.     

The cultivation of oak seedlings inoculated with Tuber aestivum Vittad. in the boreal region of Finland

Despite recent findings, truffles are rarely found in Finland. In 2006, we began to explore the cultivation potential of Tuber aestivum/uncinatum in Finland. In 2006–2008, roughly 1,200 Quercus robur seedlings and 200 Q. pubescens seedlings were planted in 20 orchards. We aimed to challenge the Southern European (France) tree provenances of oak seedlings in a boreal climate. Additional winter coverings made up of fabric or plastic and twigs prevented the seedlings’ mortality even when the air temperature was below ?30 °C during the second winter. The results showed that the top soil temperature at 15 cm depth has to be above ?5 °C to guarantee the survival of seedlings. Q. pubescens was more sensitive to low soil temperatures than Q. robur. Morphological and PCR analysis of root samples collected over 2007–2010 confirmed the presence of T. aestivum in all orchards despite unfavorable temperatures during the winter time. The first T. aestivum sporocarps were found under Q. robur in October 2012 in the orchards established in 2006 on old agricultural land, showing truffle cultivation to be successful in the boreal climate.