Ryota Kataoka

Comparison of the bacterial communities established on the mycorrhizae formed on Pinus thunbergii root tips by eight species of fungi

The ectomycorrhizal (ECM) fungi associated with Pinus thunbergii seedlings grown on sand dune were identified by molecular method, and the diversity of bacteria associated with ECM and Extraradical mycelium were examined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rDNA. The mycorrhizal formation rate of 1-year old P. thunbergii seedlings was more than 95%. Cenococcum geophilum was the most dominant ECM fungus, followed by T01, RFLP-8, Russula spp., and Suillus sp. Bacterial community was most diverse with C. geophilum- and RFLP-8-mycorrhiza. Sequencing analysis showed that Burkholderia spp. and Bradyrhizobium spp. were on the surface of ECM short root of seven ECM. The fungi detected as extraradical mycelium using DGGE of 18S rDNA were Suillus bovinus and RFLP-8-mycorrhiza. Bacterial community on the extraradical mycelium was more diverse than those on ECM root tip. Burkholderia spp. and Bradyrhizobium spp. were found also on extraradical mycelium.

A new mycorrhizal helper bacterium, Ralstonia species, in the ectomycorrhizal symbiosis between Pinus thunbergii and Suillus granulatus

In a Robinia-pseudoacacia-dominated coastal forest in Tottori prefecture Japan, the growth and survival of Pinus thunbergii seedlings and the natural regeneration of P. thunbergii was disturbed by R. pseudoacacia. In order to improve the growth of P. thunbergii seedling in the Tottori sand dune, we tried to find a mycorrhiza helper bacteria (MHB) from P. thunbergii mycorrhizosphere in a Tottori sand dune. Two MHB, Ralstonia sp. and Bacillus subtilis, were selected from the nine bacterial species isolated from the mycorrhizosphere of P. thunbergii. The bacterial effect on the ectomycorrhizal fungus Suillus granulatus was investigated by confrontation assay and a microcosm experiment. The confrontation assay showed that Ralstonia sp. promoted the hyphal growth of S. granulatus. Moreover, the S. granulatus–P. thunbergii symbiosis was significantly stimulated by Ralstonia sp. and B. subtilis. Ralstonia sp. and B. subtilis were regarded as MHB associated with P. thunbergii. This is the first report of Ralstonia sp. as an MHB.

Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom Tricholoma matsutake

AbstactThe fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a ’shiro’. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.

Mycorrhizal Inoculants: Progress in Inoculant Production Technology

Of the seven types of mycorrhizae, the symbiotic association of plants with arbuscular mycorrhizae (AM) and ectomycorrhiza (ECM) is the most abundant and widespread. Mycorrhizal inoculant technology, especially of AM and ECM, appears to be a promising avenue for sustainable agriculture and forestry because of their extensive and productive association with plants. Production of mycorrhizal inocula is a complex procedure that requires commercial enterprises to develop the necessary biotechnological skill and ability to respond to legal, ethical, educational, and commercial requirements. At present, commercial mycorrhizal inocula are produced in pots, nursery plots, containers with different substrates and plants, and aeroponic systems, and by nutrient film technique, or in vitro. Different formulated products are now marketed, which creates the need for the establishment of standards for widely accepted quality control. Generally, preparation and formulation of mycorrhizal inocula are carried out by applying polymer materials with well-established characteristics and which are useful for agriculture and forestry. The most commonly used methods involve entrapment of fungal materials in natural polysaccharide gels, which includes immobilization of mycorrhizal root pieces, vesicles, and spores, in some cases coentrapped with other plant-beneficial microorganisms. Efforts should be devoted toward registration procedures of mycorrhizal inoculants to stimulate the development of mycorrhizal products industry. Biotechnology research and development in such activities must be encouraged, particularly with regard to interactions of mycorrhizal fungi with other rhizosphere microbes, and selection of new plant varieties with enhanced mycorrhizal traits to provide maximum benefits to agriculture and forestry.

Ectomycorrhizae and Their Importance in Forest Ecosystems

Ectomycorrhizal (ECM) associations involve the most diverse category of myocrrhizae. The diversity derives from the fungal partners; more than 5,000 species of fungi, mainly Basidiomycetes, with a limited number of Ascomycetes and Zygomycetes, make the relationship very diverse. On the contrary, however, relatively few families of plants such as Fagaceae, Pinaceae, Betulaceae, and Dipterocarpaceae are involved in the ECM associations. These plants, however, are distributed over wide areas of temperate and boreal forests, and are therefore economically important. ECM fungi make associations with plants by forming a sheath (mantle) around fine root tips with hyphae that grow inward between root cells of the cortex and make Hartig net, and emanate outward through the soil, increasing the surface area to absorb nutrients and water. Thus, the mycorrhizal fungi gain photosynthates and other essential substances from the plant and in return help the plant take up water and minerals. Pine wilt disease (PWD) is a globally serious forest disease, and also shows the importance of ectomycorrhizal relationships. Pine trees planted on a mountain slope were killed by PWD, but some trees survived at the top of the slope, where mycorrhizal associations developed far better than on lower slopes. ECM associations, beside fertilization, also increase the supply of water to the pines, and elevate host resistance against disease and parasites. Moreover, inoculation of pine seedlings with ECM fungi under laboratory conditions confirmed the increase in their resistance to PWD. Pine seedlings can tolerate the adverse effects of environmental stress such as acid mist when infected with ECM fungi. These fungi can also make a significant contribution to forest ecosystems by increasing biomass and creating a network among trees through which nutrients may transported. ECM fungi also improve the growth of host plants at the seedling stage. Many pioneer plants in wastelands are facilitated in their establishment by ECM. This association has been successfully applied to reforestation programs in tropical forests by inoculating mycorrhizae on to nursery seedlings.

Endophytic actinomycetes from Pinus thunbergii and their antifungal activity against Cylindrocladium sp.

The inside of Pinus thunbergii could be a reliable screening source for a useful agent in controlling plant disease. Isolation of endophytic actinomycetes from P. thunbergii and their potential as biocontrol agents against the plant pathogen Cylindrocladium sp. were investigated. Two endophytic actinomycetes, Streptomyces sp. and Microbispora sp., were isolated from surface-sterilised root tissues of P. thunbergii seedlings. The recovery test of these two endophytic actinomycetes from pine seedling showed that Streptomyces sp. was isolated from only roots, but Microbispora sp. was isolated from both roots and leaves. Thus, Microbispora sp. is able to move to leaves from roots. Moreover, we evaluated the potential of both strains as biocontrol agents against Cylindrocladium sp. Two weeks after inoculation of Cylindrocladium sp. alone, pine seedlings showed a 50% mortality rate. Co-inoculation of Cylindrocladium sp. and Microbispora sp. did not affect seedling mortality rate. However, inoculation with both Cylindrocladium sp. and Streptomyces sp. reduced seedling mortality to 12%. Streptomyces sp. could be a useful agent in controlling pine disease caused by Cylindrocladium sp. Thus, it seems that Streptomyces sp. may induce a local host defence reaction and Microbispora sp. systemically spreads to aerial parts through the transpiration stream.