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Interactions between trees and ectomycorrhizal (ECM) fungi are critical for the growth and survival of both partners. However, ECM symbiosis in endangered trees has hardly been explored, complicating conservation efforts. Here, we evaluated resident ECM roots and soil spore banks of ECM fungi from endangered Pinus amamiana forests on Yakushima and Tanegashima Islands, Kagoshima Prefecture, Japan. Soil samples were collected from remaining four forests in the two islands. The resident ECM roots in soil samples were subjected to molecular identification. Soil spore banks of ECM fungi were analyzed via bioassays using a range of host seedlings (P. amamiana, P. parviflora, P. densiflora and Castanopsis sieboldii) for 6–8 months. In all remaining P. amamiana forests, we discovered a new Rhizopogon species (Rhizopogon sp.1), the sequence of which has no match amoung numerous Rhizopogon sequences deposited in the international sequence database. Host identification of the resident ECM roots confirmed that Rhizopogon sp.1 was associated only with P. amamiana. Rhizopogon sp.1 was far more dominant in soil spore banks than in resident ECM roots, and its presence was confirmed in nearly all soil samples examined across the major remaining populations. While Rhizopogon sp.1 did not completely lose compatibility to other pine species, its infection rate in the bioassays was highest in the original host, P. amamiana, the performance of which was improved by the infection. These results indicate that Rhizopogon sp.1 is very likely to have a close ecological relationship with endangered P. amamiana, probably due to a long co-evolutionary period on isolated islands, and to play the key role in seedling establishment after disturbance. We may need to identify and utilize such key ECM fungi to conserve endangered trees practically.

We developed 13 microsatellite markers from a genomic library enriched for dinucleotide (CT) repeats in a dioecious evergreen tree, Myrica rubra . We screened loci from 32 adult trees. The number of alleles ranged from two to 14, and the expected heterozygosity ranged from 0.324 to 0.884. The total paternity exclusionary power where the mother was known equalled 0.99965. These loci are of clear value in studying mating system and parentage analysis of this species.

We examined the effectiveness of a new Bursaphelenchus xylophilus detection kit, based on loop-mediated isothermal amplification (LAMP), in old discs taken from the stem base of B. xylophilus-infested dead trees of Pinus armandii var. amamiana (PAAm) occurring in their natural habitats. LAMP products, representing a past B. xylophilus infection, were detected in two consecutive trials from 16 of 20 discs collected from PAAm trees that died between 2003 and 2006. Bursaphelenchus xylophilus were more frequently detected using LAMP in wood samples taken from sapwood than from heartwood. No significant differences in the detection of B. xylophilus using LAMP were observed in relation to the disc collection time (from 3 to 6 years before the analysis). Bursaphelenchus xylophilus were not detected using LAMP in four discs, although a B. xylophilus infection had been confirmed for the original PAAm trees at the time they were found dead. This may have resulted from the small amount of wood chips needed for the LAMP test or the reduced number and uneven distribution of the nematode in the old dead trees. The results indicate that the new B. xylophilus detection kit will be a very efficient tool for conducting retrospective analysis of PAAm mortality factors.

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