1. Miscanthus and Reed, a large perennial wetland grass, are receiving considerable attention as potential bio-energy crops because of their ability to produce tremendous yields of biomass up to 45 Mg ha -1 yr -1. So far, barely yellow dwarf luteovirus and Pythium phragmitis have been reported to cause diseases on Miscanthus and reed, but bacterial disease was not reported in Miscanthus and reed. Thirteen Miscanthus and reed samples having necrotic lesions were collected from wet-lands located in Hampyeong, Jeju, Jeongseon, Jipyeong, Muan, and Yeoju in Korea. Totally 58 strains were isolated from the necrotic lesions of Miscanthus and reed. On the basis of partial or full 16S rDNA sequencing, 58 isolates were tentatively identified and turned out to belong to three taxonomical categories: Actinobacteria (18 strains), Firmicutes (13 strains), and Proteobacteria (27 strains). Among them, nine isolates caused necrotic symptoms on healthy Miscanthus, which were similar to the symptoms previously observed in the fields. Fig. 2. Naturally occurring necrotic lesions on Miscanthus (A) and reed (B) and the symptoms on Miscanthus generated by inoculation of the bacterial isolates obtained from the necrotic lesions (C). Fig. 3. Bacterial diversity in the necrotic lesions of Miscanthus (left) and reed (right). Culturable bacteria consist of three taxonomical groups Actinobacteria (18 strains, 32%), Firmicutes (13 strains, 23%), and Proteobacteria (27 strains, 45%). A B C D E F No.IsolateOriginGroupCloset relative speciesSequence similarity 1JSM-02MFPaenibacillus xylanexedens99.9% 2MWM-04M α Methylobacterium aminovorans99.9% 3MDM-01MγPantoea ananatis99.7% 4MDM-03MγPseudomonas lurida100% 5JSM-07MγPseudomonas oryzihabitans100% 6YLR-02RγPseudomonas rhodesiae99.9% 7LHR-02RγEnterobacter ludwigii99.9% 8JSM-04MAcCutobacterium flaccumfaciens100% 9JSM-06MAcPlantibacter flavus99.8% Ac, Actinobacteria; F, Firmicutes; α, α-Proteobacteria; γ, γ-Proteobacteria; M, Miscanthus; R, Reed 0.1 Pseudomonas rhodesiae CIP 104664 T (AF064459) Strain YLR-02 Pseudomonas lurida DSM 15835 T (AJ581999) Strain MDM-03 Pseudomonas grimontii CFML 97-514 T (AF268029) Pseudomonas tremae CFBP 6111 T (AJ492826) Strain MWM-03 Pseudomonas alcalophila AL15-21 T (AB030583) Pseudomonas flavescens B62 T (U01916) Strain JSM-07 Pseudomonas pachastrellae KMM 331 T (AB125367) Enhydrobacter aerosaccus LMG 21877 T (AJ550856) Pantoea vagans LMG 24199 T (EF688012) Pantoea ananas LMG 2665 T (Z96081) Strain MDM-01 Enterobacter gergoviae JCM 1234 T (AB004748) Enterobacter ludwigii EN-119 T (AJ853891) Strain LHR-02 Serratia nematodiphila DZ0503SBS1 T (NR044385) Alteromonas addita R10SW13 T (AY682202) Xanthomonas campestris LMG 568 T (X95917) Burkholderia cepacia ATCC 25416 T (M22518) Methylobacterium fujisawaense DSM 5686 T (AJ250801) Strain JSM-08 Methylobacterium radiotolerans JCM 2831 T (D32227) Strain HPLM-03 Methylobacterium salsuginis MP1 T (EF015477) Methylobacterium aminovorans CCM 4612 T (AJ851086) Methylobacterium extorquens JCM 2802 T (D32224) Strain MWM-04 Rhodobacter capsulatus ATCC 11166 T (D16428) Sphingomonas azotifigens NBRC 15496 T (AB217473) Curtobacterium flaccumfaciens LMG 3645 T (AJ312209) Strain JSM-04 Strain JSM-05 Strain YJM-09 Curtobacterium ammoniigenes NBRC 101786 T (AB266597) Strain YJM-06 Curtobacterium albidum DSM 20512 T (AM042692) Curtobacterium ginsengisoli DCY26 T (EF587758) Microbacterium testaceum DSM 20166 T (X77445) Microbacterium flavescens DSM 20643 T (Y17232) Plantibacter flavus DSM 14012 T (AJ310417) Strain JSM-06 Phycicoccus aerophilus 5516T-20 T (EF493847) Micrococcus luteus DSM 20030 T (AJ536198) Nocardioides simplex KCTC 9106 T (AF005009) Rhodococcus erythropolis MPU33 T (AB334770) Streptomyces griseus KCTC 9080 T (M76388) Paenibacillus xylanexedens B22a T (EU558281) Paenibacillus amylolyticus NRRL NRS-290 T (D85396) Strain JSM-02 Paenibacillus pabuli NCIMB 12781 T (X60630) Paenibacillus jamilae CECT 5266 T (AJ271157) Paenibacillus agaridevorans DSM 1355 T (AJ345023) Bacillus licheniformis DSM 13 T (X68416) Bacillus subtilis DSM 10 T (AJ276351) Staphylococcus epidermidis ATCC 14990 T (D83363) Lactobacillus delbrueckii subsp. delbrueckii ATCC 9649 T (AY050172) Flavobacterium aquatile ATCC 11947 T (M62797) γ-Proteobacteria α-Proteobacteria Actinobacteria Firmicutes 56 57 98 97 98 100 52 67 81 74 53 96 100 97 63 76 100 58 80 100 66 100 81 87 64 100 88 100 81 99 57 87 81 100 84 96 100 68 100 58 100 99 100 Healthy Miscanthus and Reed plants were artificially infected with the bacterial isolates obtained from naturally occurring necrotic lesions. The strains belonged to α- and γ- Proteobacteria caused necrotic lesions on the inoculation sites and the symptoms were similar to the symptoms observed previously in the fields. Bacteria were re-isolated from the necrotic lesions and were compared to original isolates on the basis of the cultural characteristics and 16S rDNA sequences. We found 9 strains have pathogenicity on Miscanthus and Reed. Among them, six strains were assigned to species of the genus Enterobacter, Pantoea, Pseudomonas, and Methylobacterium in Proteobacteria. Two strains were members of the genus Curtobacterium and Plantibacter in Actinobacteria and one strain was considered a species of the genus Paenibacillus in Firmicutes. These strains will provide important information on bacterial diseases of Miscanthus and reed valuable for biomass utilization. Discussion Identification of Bacterial Isolates from Miscanthus and Reed Necrotic Lesion and Reed Necrotic Lesion Dong Wan Lee 1, Jin-Sung Hong 2, Beom Seok Kim 1 and Jin-Won Kim 3 * 1 Division of Biotechnology, College of Life Science & Biotechnology, Korea University, Seoul 136-713, Korea 2 Institute of Natural Sciences, Division of Environmental & Life Sciences, Seoul Women’s University, Seoul 139-774, Korea 3 Department of Environmental Horticulture, University of Seoul, Seoul 130-743, Korea Bacterial diversity in necrotic lesions of Miscanthus and reed collected in the field Fig. 1. Procedures of pathogenicity test on Miscanthus based on the Koch’s postulates. (A)(B) (C) Inoculation of bacterial isolates Table 1. Bacterial isolates causing necrotic symptom on Miscanthus Phylogenetic tree of the bacterial isolates causing necrosis on Miscanthus This work was supported by a grant from the BioGreen 21 Program (No. PJ007446052012), Rural Development Administration, Republic of Korea. Numbers at the nodes indicate bootstrap values (>50%). Scale bar, 0.1 substitutions per nucleotide positions. AcknowledgementResultsMaterials and MethodsAbstract Test for Pathogenicity Isolation of bacteria