The needles of Japanese black pines with symptoms of disease were collected from the suburb around Shimane University, Matsue, Shimane Prefecture, Japan. We isolated endophytic and phytopathogenic fungi by two methods. The first, the surface sterilization method (Hata & Futai, 1995) , was used for isolation with minor modifi- cations. Needles were cut and dipped in 70% ethanol for 1 min, surface sterilized for 5 min in a solution of 10% sodium hypochlorite solution (Wako Pure Chemical Industries, Osaka, Japan), rinsed in sterilized distilled water twice, and then dried on sterilized filter paper. Surface-sterilized samples were placed on potato dextrose agar (PDA) plates containing chloramphenicol (20 μg/ml) and incubated at 26˚C ± 1˚C for 1 - 2 weeks. In another method, needles washed with tap water were put on wet filter paper in a plastic box and then incubated at 26˚C ± 1˚C in a growth chamber (LH-60FL3-DT, NK System, Osaka, Japan) for 1 to 3 weeks under a regime of 12 h of white light and 12 h of dark in order to form stroma of endophytic and phytopathogenic fungi on the needles. Spore masses formed on the needles were then picked up with a sterilized glass needle under a stereomicroscope (Goh, 1999; Choi et al., 1999) , placed on PDA plates containing chloramphenicol (20 μg/ml), and incubated at 26˚C ± 1˚C for 1-2 weeks.

The identity of the isolates was confirmed by sequencing of the rDNA ITS region. Fungal isolates were grown on PDA medium for 1 week at 26˚C ± 1˚C. Mycelia were scraped and harvested in 1.5-ml Eppendorf micro tubes. DNA extraction was carried out using a Nucleo Spin Plant II kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions, resuspended in 20 μl of TE buffer, and stored at −20˚C until use. The universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al., 1990) were used to amplify the ITS-5.8S-ITS regions between the 18S and 28S nuclear rDNA. PCR reactions were performed using a Thermal Cycler GeneAtlas (Astec, Fukuoka Japan). The reaction mixture (100 μl) contained about 20 ng of the fungal genomic DNA, 0.5 μM of each primer, 0.2 mM of each dNTP, 1× reaction buffer (10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl), and 2.5 U of Taq DNA polymerase (TaKaRa, Osaka, Japan). The amplification cycle consisted of an initial heat denaturation step at 94˚C for 2 min, followed by 25 cycles of 94˚C for 30 sec, 55˚C for 30 sec, and 72˚C for 30 sec, and a final extension at 72˚C for 10 min. The PCR products were electrophoresed in a 1% agarose gel in Tris-acetate-EDTA (TAE) buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.3), stained with ethidium bromide, destained in distilled water, and visualized under UV light (302 nm, UVP M-15V, UVP, Upland, CA). The PCR products were then excised and purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey- Nagel) following the manufacturer’s instructions. Sequencing reactions were performed using the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The DNA sequence analysis was performed on an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA). A computer analysis of the DNA sequence datawas performed using GENETYX^®- Mac (GENETYX, Tokyo, Japan). Comparisons between the DNA and the predicted aminoacid sequence as well as a phylogenetic analysis were carried out using the BLAST and CLUSTALW network programsat the DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp).

Sequences of each ITS region were aligned using CLUSTALW network programs at the DDBJ. Each specific primer pair within the ITS region was selected manually for species-specific detection. PCR reactions were performed by a Thermal Cycler GeneAtlas (Astec). The reaction mixture (20 μl) contained 10 ng of the fungal genomic DNA, 0.5 μM of each primer, 0.2 mM each dNTP, 1× reaction buffer (10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl), and 0.5 U of Taq DNA polymerase (TaKaRa). The amplification cycle consisted of an initial heat denaturation step at 94˚C for 2 min, followed by 25 cycles of 94˚C for 30 sec, 55˚C for 30 sec, and 72˚C for 30 sec; and a final extension at 72˚C for 10 min. PCR products were electrophoresed in a 1.5% agarose gel in TAE buffer, stained with ethidium bromide, destained in distilled water, and visualized under UV light (302 nm, UVP M-15V, UVP). An electrophoretogram was photographed using a gel documentation system (Print graph AE-6910FD, ATTO, Tokyo, Japan).

The needles of Japanese black pines with or without symptoms of disease were collected from the suburb around Shimane University, Matsue, Shimane Prefecture, Japan. A detached needle from the Japanese black pine was washed in distilled water in order to remove microbial adhesion on the needle surface. DNA extraction from the needle was carried out using the NucleoSpin Plant II kit (Macherey-Nagel), resuspended in 50 μl of TE buffer, and stored at −20˚C until use. The first PCR reactions were performed using primers ITS1 and ITS4 in order to amplify the ITS-5.8S-ITS rDNA fragment of the fungi as mentioned above. A second PCR reaction (nested PCR) was performed using each specific primer pair and 1 μl of one-twentieth (1:20) diluted first PCR reaction mixture as a template DNA as mentioned above.

We isolated 45 endophytic and phytopathogenic fungal candidates from P. thunbergii needles collected in Shimane prefecture, Japan (data not shown). The ITS regions of all of these fungi were sequenced. The frequent endophytic and phytopathogenic fungal candidates were Pestalotiopsis sp., L. conigenum, L. acicola, R. kalkhoffii, and S. pini-thunbergii. Thus, these five species were selected for further investigation (Table 1).

Sequences of ITS regions of L. conigenum, L. acicola, Pestalotiopsis neglecta, R. kalkhoffii, and S. pini-thun- bergii (Table 1) were aligned by ClustalW (Figure 1), and specific forward and reverse primer pairs of oligonucleotides were designed from a non-consensus sequence of the alignment (Figure 1 and Table 2). In silico, primer pair specificity was evaluated by searching the DDBJ database. The BLAST search with the sequences

of the primer pairs Lo1F/Lo1R, Le1F/Le1R, Pn1F/Pn1R, Rk1F/Rk1R, and Sp1F/Sp1R as a query showed the fungal sequences most similar to L. conigenum, L. acicola, Pestalotiopsis sp., R. kalkhoffii, and S. pini-thunbergii, respectively, suggesting that each primer pair was specific for detecting each phytopathgenic fungus by PCR amplification of the ITS-5.8S-ITS region (data not shown).

To confirm the specificity of the primer pairs, PCR was carried out using each primer pair and the genomic DNA of each phytopathogenic fungus as a template (Figure 2). No amplification was detected using the rbcLF/ rbcLR primer pair to target the RuBisCO large subunit gene of the Japanese black pine with each template of genomic DNA in all phytopathogenic fungi (Figures 2(a)-(h)), confirming that the rbcLF/rbcLR primer pair does not interact with the fungal genomic DNA used in this study. On the other hand, amplification was detected using the ITS1/ITS4 primer pair with each template of genomic DNA in all fungi (Figures 2(a)-(h)), indicating

that the ITS1/ITS4 primer pair could be used to generally amplify the ITS-5.8S-ITS rDNA of fungi as a universal primer. On the other hand, the use of each specific primer pair was successful for detecting the target species; only the primer pair Rk1F/Rk1R successfully amplified the target DNA in the predicted single band of 393 bp from R. Kalkhoffii (Figure 2(a), lane 3); only the primer pair Sp1F/Sp1R successfully amplified the target DNA in the predicted single band of 390 bp from S. pini-thunbergii (Figure 2(b), lane 4); only the primer pair Lo1F/Lo1R successfully amplified the target DNA in the predicted single band of 337 bp from L. conigenum (Figure 2(c), lane 5); only the primer pair Le1F/Le1R successfully amplified the target DNA in the predicted single band of 333 bp from L. acicola (Figure 2(d), lane 6); only the primer pair Pn1F/Pn1R successfully amplified the target DNAin the predicted single band of 431 bp from P. neglecta (Figure 2(e), lane 7); and furthermore, all amplification products were obtained using each primer pair with a DNA mixture of R. kalkhoffii, S. pini-thunbergii, L. conigenum, L. acicola, and P. neglecta (Figure 2(f), lanes 2 - 7). No amplification product was obtained using any of the primer pairs except ITS1/ITS4 as a fungal ITS universal primer with each template of genomic DNA from Cladosporium sp. (Figure 2(g)) and Diaporthe sp. (Figure 2(h)). These results suggested that each specific primer pair would be fit to detect each endophytic and phytopathogenic fungus even if several endophytic and phytopathogenic fungi were present in the same needle from a Japanese black pine.

We evaluated whether any endophytic or phytopathogenic fungi could be detected from a needle (Figure 3). Amplification products were obtained using the rbcLF/rbcLR primer pair to target the RuBisCO large subunit gene in sample A and sample B but not in sample C (Figure 3(b)). These results indicate that the RuBisCO large subunit gene would be detectable from the greening region of a needle, whereas it would not be detectable from

the withered region of a needle due to DNA degradation. In contrast, amplification products were obtained using ITS1/ITS4 to target the ITS-5.8S-ITS rDNA of fungi in samples A, B and C, indicating whether any fungi were present inside the needle. Furthermore, it was suggested that the fungal biomass of sample B and sample C would be greater than that of sample A because the amplification fragments of sample B and sample C were larger than that of sample A. Nested PCR revealed that S. pini-thunbergii and L. acicola were detected in all samples (A to C), whereas P. neglecta was only detected in sample B (Figure 3(c)). We further evaluated another needle (Figure 4). Amplification products were obtained using rbcLF/rbcLR to target the RuBisCO large subunit gene in sample D and sample E but not in sample F (Figure 4(b)). These results were almost the same except for sample D, in which no amplification products were observed. It was noted that S. pini-thunbergii and P. neglecta were detected in sample D even with no amplification products being observed (Figure 4(c)). On the other hand, R. kalkhoffii, S. pini-thunbergii, L. acicola, and P. neglecta were detected in sample E and sample F (Figure 4(c)), indicating that these fungi can exist inside the withered region of a needle.

We tried to evaluate the possibility that these fungi could be detected from needles preserved in a freezer for one week (Figure 5). As a result, amplification products were obtained using the rbcLF/rbcLR primer pair to target the RuBisCO large subunit gene in sample H and sample I, while amplification products were obtained using the ITS1/ITS4 primer pair to target the ITS-5.8S-ITS rDNA of fungi in samples G, H and I. Nested PCR revealed that R. kalkhoffii, S. pini-thunbergii, L. conigenum, and L. acicola were detected in sample G, whereas R. kalkhoffii, S. pini-thunbergii, L. acicola and P. neglecta were detected in sample H and sample I (Figure 5(c)).

Finally, we tried to evaluate the detection of the endophytic and phytopathogenic fungi from healthy needles without any lesions. It was clearly demonstrated that amplification products were obtained using the rbcLF/rbcLR primer pair to target the RuBisCO large subunit gene, whereas no amplification products were visually observed using the ITS1/ITS4 primer pair to target the ITS-5.8S-ITS rDNA of fungi in all samples (Figure 6(b)). Nested PCR revealed that there was no amplification product using five specific primer pairs in sample K and sample L, suggesting that R. kalkhoffii, S. pini-thunbergii, L. conigenum, L. acicola and P. neglecta were not present in the healthy needles in sample K and sample L (Figure 6(c)). Interestingly, nested PCR revealed that L. acicola and

P. neglecta were only detected in sample J, suggesting that L. acicola and P. neglecta were present as endophytic fungi in the healthy needle in sample J (Figure 6(c)).