![[NF-2000 Database - AIR Program]](../images/Air.gif) |
AIR2-CT94-1149
Improvement of the Quality of Forest Seedlings and Mediterranean Reforestation Using Controlled Mycorrhizal Infection |
 |
Proposal No: | AIR2-CT94-1149 |
| Date Prepared: | November 1999 | |
| Source: | Final report May 1998 |
Objectives
The objective of the project was to use controlled mycorrhizal infection to produce forest seedlings that are best adapted to the conditions of the Mediterranean environment. To attain this objective, the quality of mycorrhizal plants produced in nurseries must be improved, by a rational choice of forest species and associated mycorrhizal fungi and by the optimisation of the techniques of production of the plants. The improvements achieved under nursery conditions was tested in situ in experimental reforestation trials. The objective of the project can be achieved through close collaboration between the various levels of the plantation chain: improvement, production and use of forest seedlings. Thus, the project called upon expertise of researchers from different disciplines to provide and expand the necessary theoretical basis. It also drew on the expertise of professionals in the chain of production and use of forest seedlings in order to ensure the technical and economical viability of the proposed innovations. These skills were exploited in the three main activities:
Activities
The screening of mycorrhizal strains adapted to the tree species studied and efficient in the soil conditions has led to the following results:
Isolation, in vitro culture and mycorrhizal capability of fungal strains Isolations were made from basidiomes of putative ectomycorrhizal fungi associated with Pinus pinea and Quercus suber in the Mediterranean area of northern Catalonia. A total of 112 isolates were obtained, most of them from species of the genus Boletus, Lactarius, Pisolithus and Rhizopogon. From a total of 61 isolates tested under non aseptic syntheses with P. pinea, 15 formed ectomycorrhizae with containerised seedlings. These isolates belonged to the genus Amanita, Hebeloma, Lactarius, Pisolithus, Rhizopogon, Scieroderma and Suillus. These results demonstrated, not only the symbiotic compatibility between these ectomycorrhizal isolates and Pinus pinea, but also their effectiveness when applied as vegetative inoculum produced in a peat-vermiculite carrier. Besides the 36 isolates already available several new strains belonging to 5 ectomycorrhizal fungi ( Suillus collinitus, S. mediterrraneensis, Rhizopogon sp., R. roseolus, Pisolithus tinctorius ) were isolated during 1997. As far as micropropagation of P. halepensis was concerned, in vitro-plants have been obtained and their multiplication have been optimised by testing different culture media and growth regulators. On the other hand, mycorrhiza formation was not observed in growth chamber conditions, probably due to the scarce root development of vitroplants.
Physiology of the mycelial growth of two mycorrhizal isolates on solid media The utilisation of phosphate and production of phytases by the mycelium of two strains (S. collinitus J3.15.24 and Pisolithus tinctorius F26) were studied on various solid media, including a mixture of peat-vermiculite saturated with nutrient solution (solid state fermentation). The mycelium of the two strains was able to produce phytases to release inorganic phosphorus from phytate. However, the production of biomass of S. collinitus J3.15.24 was greater on a potassium phosphate-phytate mixture than on phytate only, contrarily to P. tinctorius F 26. This strain was more adapted than S. collinitus J3.15.24 to the culture in solid state fermentation on vermiculite support.
Morphological and cellular characterisation of ectomycorrhizae formed by Suillus collinitus on pines and by Pisolithus tinctorius on pines and cork oaks Two Suillus collinitus strains isolated from Mediterranean and alpine zones were used to inoculate two pines (Pinus pinea and P. nigra var. corsicana ) typical of Mediterranean and alpine regions, respectively. The first events occurring, between the partners were analysed by using a morphological approach, giving particular attention to the interacting cell surfaces. The four combinations led to a comparable mycorrhizal infection degree and to a similar colonisation pattern. Hyphae mostly established the first contacts with the host surface causing the breakdown of a thin cuticle layer and developing under it. However, ultrastructural observations revealed that S. collinitus strain J3.15.24 from the Mediterranean region caused some limited defence reactions in the roots of P. nigra suggesting that the two fungal strains may have functional differences in their mycorrhizal capacities. Relevant differences were also found between two strains of Pisolithus tinctorius : F 28 resulted no effective mycorrhizal associations, while Pt 202 produced fully established mycorrhizae in the presence of Pinus pinea and Quercus suber roots.
Effects of mycorrhizal infection on the ionic exchanges at the root/soil interface Measurements in pH - stat system have indicated that, pH 4.8, mycorrhizal and non mycorrhizal Pinus pinaster exhibited OH- excretion, particularly for P. pinaster / Hebeloma cylindrosporum . At pH 6.5, H+ excretion was observed , especially for the associations with S. collinitus and Rhizopogon roseolus . At a given pH value, NO3- uptake was enhanced in mycorrhizal associations with S. collinitus and R. roseolus . The methodology using double barrelled H+ microelectrodes have been successfully applied to P. nigra ssp. laricio seedlings: Suillis collinitus J3.15.31 has greatly modified the evolution of pH in the apoplast of mycorrhizae compared to the non-mycorrhizal short roots, whereas S. collinitus J3.15.244 (more compatible with Mediterranean pines such as P. pinea and P. halepensis than with P. nigra ) did not change the behaviour of short roots associated with this isolate. Measurements of pH values, made directly on hyphae colonised soil or bare soil surface by a combined pH electrode fitted with a flat glass membrane, have indicated that for P. pinaster / R. roseolus cultivated on soil thin layer enriched or not by CaCO3, a strong acidification occurred at the level of mycorhizal roots. An acidification was also observed at the level of soil surface areas colonised by fungal hyphae, even in the presence of CaCO3. For P. pinaster / H. cylindrosporum, only a small decrease of pH was noted at the level of mycorrhizal roots.
Effects of mycorrhizal infection on the carbon balance of the host plant The effects of mycorrhizal infection with Hebeloma cylindrosporum D3.25.9 and Pisolithus tinctorius F 26 on plant biomass production of Pinus nigra ssp. laricio var. corsicana were enhanced by P fertilisation. However, the presence of the fungus did not increase the net photosynthesis rate nor the root respiration. The comparison of these results to those obtained with P. pinaster inoculated with the same fungal species has shown that R. rubescens has a beneficial effect on both host species whereas H. cylindrosporum was shown to strongly decrease the plant biomass, leading simultaneously to an increase of net photosynthesis and root respiration rates in P. pinaster. Carbon and nitrogen transfers between H. cylindrosporum and its host plant are probably regulated by the host plant itself.
Techniques of production of mycorrhizal inoculum and of controlled mycorrhizal infection in nurseries The infectivity of different types of inoculum and the application dosages needed to obtain a good mycorrhizal development in containerised Pinus pinea seedlings was investigated. Mycelium grown in peat-vermiculite was an effective inoculum source for Hebeloma crustiliniforme, Laccaria laccata and Pisolithus tinctorius. The rates of optima application ranged from 1 : 64 to 1 : 16 (inoculum substrate, v / v). Mycelium of Laccaria bicolor entrapped in alginate beads proved also to be an effective inoculum. Optimal application dosage has been established at 1 : 20 (inoculum substrate, v / v). On another hand, the low rate of spores needed to obtain a high proportion of ectomycorrhizal seedlings with Rhizopogon roseolus makes this fungal species an appropriate candidate to be implemented at industrial scale. In addition, the use P of mycelial slurries with or without agar was not effective to form ectomycorrhizae with P. halepensis even when this type of inoculum was viable in vitro. By contrast, acceptable levels of mycorrhizal infection have been obtained with mycelium grown in peat-vermiculite. Both fertilisation regime and substrate characteristics play an important role in the establishment and development of the ectomycorrhizal colonisation in nursery conditions. For P. pinea, the percentage of mycorrhizal seedlings and the percentage of ectomycorrhizal feeder roots were significantly higher in non fertilised plants, and these percentages decreased as the fertiliser concentration increased. Reducing the fertiliser concentration to a quarter of the recommended dosage promotes a good mycorrhizal colonisation without affecting significantly the plant development. At the same time, the use of peat-vermiculite substrate improved the ectomycorrhizal development in containerised seedlings compared to plants, produced in a peat-composted bark substrate. For P. halepensis, although the different fertilisation levels used have affected the plant growth, they seemed not to affect the development of mycorrhizal infection. Whatever the fertilisation regime, high degrees of mycorrhizal rates and mycorrhizal infection were obtained with Quercus suber / Pisolithus tinctorius (2 strains), whereas these two parameters depended on the fertilisation when Hebeloma sarcophyllum and Laccaria bicolor were used as mycorrhizal symbionts. In mycorrhizal P. halepensis and P. pinea containerised plants, an increase of the photosynthesis, a decrease of the N contents and an increase of the K contents in the stems were observed. By contrast, the mycorrhizal infection had very weak or no effects on the nutrient contents of P. nigra ssp. laticio var. corsicana and Q. suber plants in nursery. When P. halepensis plants were produced under low fertilisation regime, the mycorrhizal infection led to an increase of the N uptake by the plants under the nursery conditions of the south-eastern Spain.
Molecular characterisation of Suillus strains and Lactarius basidiomes The genome of 13 Suillus isolates, mostly originating from Mediterranean and alpine regions was screened by using a range of PCR-based techniques. In particular, microsatellite fingerprinting allowed separation of strains with different symbiotic capabilities (for example : Suillus collinitus strains J3.15.24 and J3.15.31), as well as strains originating from the same zone. The results suggest that a range of molecular probes used on the same isolates can provide both specific fingerprints for selected fungal strains and tools with which to investigate the physiological characteristics of ectomycorrhizal fungi. The genetic clustering of the different species of Lactarius sect. Dapetes has been refined. The species have fallen into main divergent groups : all the L. deliciosus genotypes (I) ; the four remaining species (L. deterrimus, L. salmonicolor, L. sanguifluus , L. semisanguifluus ) (II). In the group II, L. salmonicolor and L. deterrimus appeared clearly distant from each other and from L. sanguifluus and L. semisangfluus which have remained mixed. The assessment of the improvements previously achieved in nursery justifies the development of ecological studies in the laboratory and in experimental plantations
Ecological competence and competitivity of mycorrhizal strains in the soil conditions A minimum level of 40% mycorrhizal rate is required for survival and development of mycorrhiza after out-planting. Laccaria laccata is well adapted to acid soils whereas Hebeloma crustuliniforme fits better with neutral and alkaline soil. Suillus collinitus and Rhizopogon rubescens are both good candidates for dry location, the former being adapted to calcareous soils and the latter to acid soils. There is a soil-dependent discrepancy in the competitivity of two strains (D 45 and D 39) of L. deliciosus This reinforces preliminary observations made on the distribution of the ITS genotypes within natural populations of L. deliciosus: the A genotype of the strain D45 was shown to be the most frequent and exhibited the largest geographical distribution in the South of France.
Establishment and ecophysiological monitoring of the experimental reforestation plots The initial program of establishment of reforestation plots has been partially completed, especially in Catalonia and in Liguria, due to a partial supply. From the 11 mycorrhizal associations established by Participants L2 and L7 during 3 years in 15 experimental sites, it could be concluded that : (i) the effect of the site on the growth was strong for P. pinea ; (ii) Hebeloma crustuliniforme and Laccaria bicolor were efficient mycorrhizal fungi for P. nigra ssp. laricio var. corsicana and there was an interaction soil-symbionts ; (iii) Suillus collinitus, H. crustliliniforme and Pisolitlius tinctorius enhanced the growth of P. halepensis in a calcareous soil. Preliminary results from two sites reforested with mycorrhizal P. pinea in Catalonia indicated that plant survival was significantly affected by Rhizopogon sp. inoculation, after one year. At the same time, in some sites, plants inoculated with Rhizopogon roseolus showed a significant growth improvement compared to uninoculated plants. In general, a high mortality rate was noted after one year of plantation in Albacete province for uninoculated P. halepensis or P. halepensis inoculated with Pisolithus tinctorius. The dominant fungal genotype of ectomycorrhizae detected on short roots of P. pinea from an experimental plot including P. pinea / Suillus collinitus J3.15.2 plants was found to be closely related to Rhizopogon rubescens R 19.1, both in Spring and Autumn.
Conclusions
Important culture collections of mycorrhizal fungi associated with Pinus pinea, P. halepensis, P. sylvestris, P. nigra and Quercus suber are available in the laboratories of the participants concerned. The mycorrhizal capability of 15 isolates was assessed under non aseptic syntheses with P. pinea, but mycorrhizal formation was not observed in vitroplants of P. halepensis. Pisolithus tinctorius strain F26 was more adapted than Suillus collinitus strain J3.15.24 to the culture in solid state fermentation on vermiculite support. S. collinitus strain J3.15.24 caused some limited defence reactions in the roots of P. nigra. OH- and H+ excretions were detected in the rhizosphere of mycorrhizal and non-mycorrhizal P. pinaster. However, these excretions were the highest for mycorrhizal associations. Suillus collinitus J3.15.31 has greatly modified the evolution of pH in the apoplast of mycorrhizae compared to the non-mycorrhizal short roots of P. nigra ssp. laricio seedlings. Carbon and nitrogen transfers between Hebeloma cylindrosporum and the host plant (P. pinaster, P. nigra ssp. laricio .... ) were probably regulated by the host plant. Mycelium grown in peat-vermiculite was an effective inoculum source for Hebeloma crustuliniforme. Laccaria bicolor entrapped in alginate beads proved also to be an effective inoculum. However, the use of mycelial slurries was not effective to form ectomycorrhizae with P. halepensis. Both fertilisation regime and substrate characteristics play an important role in the establishment and development of the ectomycorrhizal colonisation in nursery conditions. Laccaria laccata, Hebeloma crustuliniforme, and Suillus collinitus and Rhizopogon rubescens are good candidates for acid, alkaline / neutral, and dry soils, respectively. There is a soil- dependent discrepancy in the competitivity of 2 strains of Lactarius deliciosus. Some mycorrhizal associations have shown good performances in soil conditions, and the follow-up of the experimental plots (survival rate and growth of plants, survival of the introduced strains) is in progress. A range of molecular probes used on the same isolates can provide both specific fingerprints for selected fungal strains and tools with which to investigate the physiological characteristics of ectomycorrhizal fungi.
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
- biomatnet@biomatnet.org
 |
 |
 |
News |
 |
Events |
|
|