A study of community ecology of ectomycorrhizal fungi assoiated with Abies firma Sieb. et Zucc.

(1) Seasonal occurrences and spatial distributions of fruiting bodies of ECM fungi

1. The species composition and the number of fruiting bodies of putative ECM fungi were surveyed for 3 years (1994-1996) in an Abies firma-dominated forest in central Japan. The survey was conducted in a 10 x 30 m plot for 67 times at about 10-day intervals.
   
2. A total of 39 species in 13 genera of ECM fungi were recorded, and an undescribed Russula species, Russula sp. 1, occurred most frequently both in each fruiting season and during the study period. The cumulative number of ECM fungal species increased from 29 through 34 to 39 over the three successive years.
   
3. Seasonal changes in numbers of ECM fungal species and fruiting bodies showed a bimodal pattern in each year, with one peak from late July to early August and the other larger peak from late September to early October. At the species level, the seasonal abundance of the fruiting bodies tended to show one striking peak for most of the occurred species.
   
4. A large number of fruiting bodies were observed on the forest floor of the west side of the plot, where A. firma were dominant. In contrast, fewer fruiting bodies occurred on the east side of the plot, occupied exclusively by the non-ECM trees, Chamaecyparis obtusa and Cryptomeria japonica. At the species level, R. ochroleuca, Russula sp. 1 and Strobilomyces confusus tended to produce their fruiting bodies on the forest floor directly under the crown of A. firma. On the other hand, individuals of Inocybe cincinnata, Gomphus floccosus and G. fujisanensis were independently aggregated in limited areas outside the crown of A. firma.
   
5. The four most frequent species of ECM fungal species, I. cincinnata, R. ochroleuca, Russula sp. 1 and S. confusus were selected for the analysis of interspecific spatial associations by means of the w index. The analysis was made combining the most dominant species, Russula sp. 1, and one of the other three species. Pairwise, Russula sp. 1 with I. cincinnata, with R. ochroleuca or with S. confusus showed an association that was exclusive, independent or overlapping, respectively.
   

1. Current-year or one-year-old seedlings of naturally grown A. firma (n=128) and soil block samples (10x10x10 cm; n=9) and soil core samples (3.2 cm in diameter x 10 cm; n=28) were taken from the plot for the microscopy of the root systems of A. firma seedlings and its mature trees. When the root tip was mycorrhizal sheathing with fungal mantles, it was morphologically characterized based on features of fungal partners.
   
2. In the stereoscopic microscopy, the root system of A. firma was found to be monopodial forms for the seedlings and monopodial pinnate or monopodial pyramidal forms for the mature trees. Some ECM types showed characteristic colors of fungal mantles: slightly purple (type 15), reddish brown (type 48) and blackish (types 35, 36 and 37), the most of the other types showed white-, cream- or brown-colored mycorrhizas.
   
3. In the light microscopy, ECM roots were examined with respect to the following fungal features: the plan view of the fungal mantle surfaces, the morphology and diameter of emanating hyphae, presence or absence of clamp connections, presence or absence of cystidia and their shapes. Consequently, forty-eight morphologically distinct ECM types were classified based on the macro- and microscopic features.
   
4. Twenty-five of the 48 ECM types were found in both seedlings and mature trees of A. firma root systems. Thirteen and 10 types were unique for the seedlings and the mature trees, respectively.
   

1. For the three most frequent fruiting species of ECM fungi, I. cincinnata, Russula sp. 1 and S. confusus, as a fruiting body at the plot, their fruiting bodies and ECM roots, which had been classified morphologically as described above (See (2)), involved in soil blocks (10x10x10 cm) from directly below the fruiting bodies were used for the extraction of fungal DNA. Following the DNA extraction, the internal transcribed spacer (ITS) region of ribosomal DNA was amplified, and the product of the amplification was digested with two enzymes, Alu I and Hinf I.
   
2. Diverse morphotypes ranging from 5 to 13 types, were occurred in each soil block. Types 41, 21 and 21 were dominated beneath the fruiting bodies of I. cincinnata, Russula sp. 1 and S. confusus, respectively.
   
3. Types 41, 21 and 16 were identical in both the length of the ITS region and the restriction fragment length polymorphism profiles of Alu I and Hinf I digests, to those of the fruiting bodies of I. cincinnata, Russula sp. 1 and S. confusus, respectively.
   
4. For fungal species of I. cincinnata and Russula sp. 1, their morphotypes were each dominated directly below ground of the fruiting bodies. On the other hand, type 16 formed by S. confusus was much less frequent even directly beneath the fruiting body.
   

1. The formation of ECM roots found on the root systems of current-year- and one-year-old A. firma seedlings, all of which were examined in ECM morphotypes as did in the section (2), were surveyed during 1995 to 1997. The spatial distribution of ECM roots of A. firma found on the seedlings and the mature trees were determined as well as that of the ECM morphotypes.
   
2. The formation of ECM roots on the seedlings was initiated just as same time as that of the lateral roots. Percentages of the ECM formation differed between 40 and 80 % increasing from summer to autumn for the current -year-old seedlings. For the one-year-old seedlings, the rate of ECM formations was more or less same levels, ca 70 %, during the sampling period. Each of the classified ECM types found on the seedlings was also tended to increase from summer to autumn.
   
3. ECM roots on the seedlings and on the mature trees of A. firma were classified into 37 and 23 morphotypes, respectively. Type 21 occurred most frequently in terms of both the number of ECM root tips and the frequency of occurrence per samples; seedlings or soil cores irrespective of the host ages. However, the most of ECM types were found less frequently and located in limited areas.
   
4. The ECM roots on the seedlings were evenly distributed all over the plot, whereas those of the mature trees were deviated and significantly decreased with the distance from the stem of the trees. Down to 10 cm in deep, the ECM roots of the mature trees were distributed more abundantly in the soil layer of 0-5 cm than that of 5-10 cm.
   
5. The occurrence position of type 21 found on the seedlings was tended to overlap with that of the mature trees involved in soil cores.
   

1. The occurrence of putative ECM fruiting bodies and the morphological and molecular examinations of ECM roots suggested that 53 ECM fungal species, at a minimum, had ECM associations with A. firma in the plot (300 m²). The complex environmental condition, where the plot was situated on the border of a man-made and a naturally regenerated forest, may contribute to the coexistence of diverse ECM fungi.
   
2. Both the occurrence season and location of ECM fruiting bodies differed between fungal species implying that the formation of the fruiting bodies can be partitioned among the species spatially and temporally. However, various types of ECM roots distributed locally under the soil. This would indicate that many species of ECM fungi were distributed coexistantly below ground.
   
3. For about 40 % of the classified ECM morphotypes, fungal partners were inferred to be Cenococcum geophilum, the genera Lactarius, Russula and Tuber, and unidentified Basidiomycetes, based on earlier references. Moreover, both the morphological and molecular examinations of ECM roots enabled us to specify the fungal partner of the roots unambiguously and, in fact, ECM roots formed by three fungal species: I. cincinnata, Russula sp. 1 and S. confusus were revealed at the first time in this study.
   
4. Russula sp. 1 was the most dominant ECM fungal species indicated by both fruiting bodies and ECM roots, suggesting that the formation of the fruiting body above ground can reflect well the abundance of the fungus below ground. Besides, type 21 formed by this fungus was the dominant ECM roots examined in both seedlings and mature trees of A. firma. It is thus that the possibility of interconnection between the plants by extraradical mycelium of the fungus was inferred.
   
5. For the occurrence of ECM fruiting bodies and of ECM morphotypes on A. firma seedlings and its mature trees, the rank-abundance relationships were well represented by the Geometric series: a few dominant and rare species together with a large number of other species which sequenced proportionally each other in the array of the rank. The shape of this pattern suggested that various ECM fungal species co-occurred in a certain area could be regulated by a structural rule for their existence. Adaptability to environmental conditions at the study plot and competitive ability for their food resources, e.g. root tips, among ECM fungi might be considered as factors for the establishment of the Geometric series.
   
6. Traits of community structure of ECM fungi and the significance of ECM symbiosis in forest ecosystems were overviewed and discussed on the basis of the results obtained with this study and earlier information so far.
   

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