Massilia rhizosphaerae sp. nov., a rice-associated rhizobacterium with antibacterial activity against Ralstonia solanacearum.

A novel rhizobacterium, designated strain NEAU-GH312T, with antibacterial activity against Ralstonia solanacearum was isolated from rhizosphere soil of rice (Heilongjiang Province, PR China) and characterized with a polyphasic approach. Cells of strain NEAU-GH312T were Gram-stain-negative, aerobic, non-spore-forming, motile with peritrichous flagella and rod-shaped. Colonies were light orange, convex and semi-translucent on Reasoner's 2A (R2A) agar after 2 days of incubation at 28 °C. Growth was observed on R2A agar at 10-40 °C, pH 4.0-8.0 and with 0-5 % (w/v) NaCl. The respiratory quinone was ubiquinone Q-8. The major cellular fatty acids of strain NEAU-GH312T were C16 : 1 ω7c and/or C16 : 1 ω6c, C16 : 0 and C18 : 1 ω7c and/or C18 : 1 ω6c. The main polar lipids were phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Phylogenetic analyses confirmed the well-supported affiliation of strain NEAU-GH312T within the genus Massilia, close to the type strains of Massilia arvi THG-RS2OT (98.7 %), Massilia norwichensis NS9T (98.7 %) and Massilia kyonggiensis TSA1T (98.6 %). Strain NEAU-GH312T had a genome size of 6.68 Mb and an average DNA G+C content of 66.3 mol%. Based on the genotypic, phenotypic and chemotaxonomic data obtained in this study, strain NEAU-GH312T could be classified as representative of a novel species of the genus Massilia, for which the name Massilia rhizosphaerae sp. nov. is proposed, with strain NEAU-GH312T (=DSM 109722T=CCTCC AB 2019142T) as the type strain.

Massilia cellulosiltytica sp. nov., a novel cellulose-degrading bacterium isolated from rhizosphere soil of rice (Oryza sativa L.) and its whole genome analysis.

A bacterial strain, Gram-stain negative, rod-shaped, aerobic and cellulose-degrading, designated NEAU-DD11T, was isolated from rhizosphere soil of rice collected from Northeast Agricultural University in Harbin, Heilongjiang Province, North-east China. Base on 16S rRNA gene sequence analysis, strain NEAU-DD11T belongs to the genus Massilia and shared high sequence similarities with Massilia phosphatilytica 12-OD1T (98.46%) and Massilia putida 6NM-7 T (98.41%). Phylogenetic analysis based on the 16S rRNA gene and whole genome sequences indicated that strain NEAU-DD11T formed lineage related to M. phosphatilytica 12-OD1T and M. putida 6NM-7 T. The major fatty acids of the strain were C16:0, C17:0-cyclo and C16:1ω7c. The respiratory quinone was Q-8. The polar lipids profile of the strain showed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified polar lipid and an unidentified phospholipid. In addition, the digital DNA-DNA hybridization values between strain NEAU-DD11T and M. phosphatilytica 12-OD1T and M. putida 6NM-7 T were 45.4 and 35.6%, respectively, which are lower than the accepted threshold value of 70%. The DNA G + C content of strain NEAU-DD11T was 66.2%. The whole genome analysis showed the strain contained carbohydrate enzymes such as glycoside hydrolase and polysaccharide lyase, which enabled the strain to have the function of degrading cellulose. On the basis of the phenotypic, genotypic and chemotaxonomic characteristics, we conclude that strain NEAU-DD11T represents a novel species of the genus Massilia, for which the name Massilia cellulosiltytica sp. nov. is proposed. The type strain is NEAU-DD11T (= CCTCC AB 2019141 T = DSM 109721 T).

First Report of Leaf Spot Caused by Cladosporium tenuissimum on Panicle Hydrangea (Hydrangea paniculate) in China.

Panicle Hydrangea (Hydrangea paniculate) is an ornamental flowering plant native to China and Japan. In August 2019, leaf spot symptoms with about 30% disease incidence were observed on panicle hydrangea in two grower fields (about 0.1 ha in total) of Northeast Agriculture University, China (126.72°E, 45.74°N). Symptoms initially appeared on the lower and older leaves and showed small subcircular brown spots with dark-brown edges on both sides. As the disease progressed, the necrotic spots enlarged, became irregular, coalesced, and the infected leaf blighted in approximately 2 weeks. Panicle hydrangea leaf samples (n=15) from different plants that showed spot symptoms were collected and surface sterilized with 70% ethanol for 10 s, followed by 0.5% NaClO treatment for 4 min, and rinsed in sterile water 3 times. Thereafter, leaf samples were placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fifteen hyphal-tipped pure cultures were obtained. Colonies growing on PDA for 7 days were olive green to dark green, exhibited a velvet-like texture and sometimes were radially furrowed and wrinkled. Margins varied from white gray to dark green without prominent exudates. The back of the plate showed dark green to black. Conidiophores were up to 180 to 600 µm long, 2.8 to 4.5 µm wide (n=50), subcylindrical-filiform, straight, septate, and unbranched or rarely branched. Ramoconidia were 0 to 1 septate, cylindrical to clavate, smooth-walled, 8 to 22 µm long (n=50). Conidia were single-celled, lemon-shaped, smooth-walled and 2.0 to 5.0 µm (diameter) (n=50). To confirm the identity, three genomic DNA regions, internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (EF), and actin (ACT) of the representative isolate BAI-1 were amplified with primer pairs ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Bensch et al. 2012; Jo et al. 2018). DNA sequences of the isolate from ITS, EF, and ACT showed 99.81% (514/515 bp), 99.10% (219/221 bp), and 99.54% (216/217 bp) nucleotide identity with those of C. tenuissimum CBS 125995, respectively (GenBank accession nos. HM148197, HM148442, and HM148687). The sequences of isolate BAI-1 were deposited in GenBank (accession nos. MW045455, MW052465, and MW052466). To fulfill Koch's postulates, five healthy 2-year-old panicle hydrangea plants grown in pots were surface sterilized with 70% ethanol, washed twice with sterile distilled water, and sprayed with a conidial suspension of strain BAI-1 (adjusted to 1×106 conidia/ml using a hemocytometer), maintained in a greenhouse at 25°C and 85% relative humidity. Five plants sprayed with sterilized water served as controls. The inoculated plants showed leaf spot symptoms that were similar to those previously observed in the fields after 7 days, whereas control leaves remained healthy. The fungus was reisolated from symptomatic leaves and its identity was confirmed by morphological and molecular method. These experiments were repeated twice. So far, C. tenuissimum was reported to cause leaf spot of alfalfa (Han et al. 2019) and castor (Liu et al. 2019). To our knowledge, this is the first report of leaf spot disease in panicle hydrangea caused by C. tenuissimum in China. Leaf spot has a negative effect on the aesthetic value of panicle hydrangea, and this report will assist with monitoring distribution of the disease as well as developing management recommendations.

Rhodococcus oryzae sp. nov., a novel actinobacterium isolated from rhizosphere soil of rice (Oryza sativa L.).

A Gram-stain-positive, non-motile, creamy-white actinobacterium, which has an elementary branching rod-coccus life cycle was isolated from the rhizosphere soil of rice (Oryza sativa L.) collected from Northeast Agricultural University in Harbin, Heilongjiang province, north-east PR China, and its taxonomic status was examined by using a polyphasic approach. Results from the 16S rRNA gene sequence study showed that the isolate, designated strain NEAU-CX67T, belonged to the genus Rhodococcus and formed a cluster with Rhodococcus maanshanensis DSM 44675T, Rhodococcus kronopolitis NEAU-ML12T and Rhodococcus tukisamuensis JCM 11308T (98.3, 98.1 and 97.7% gene sequence similarity, respectively). The major fatty acids were C16 : 0, 10-methyl C18 : 0, C18 : 1 ω9c and C16 : 1 ω7c. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The major isoprenoid quinone was MK-8(H2). Whole-cell hydrolysates contained meso-diaminopimelic acid. Arabinose, galactose and ribose were detected as diagnostic sugars from whole-cell hydrolysates. Mycolic acids were detected. The genomic DNA G+C content of strain NEAU-CX67T was 64.6 mol%. Strain NEAU-CX67T exhibited low average nucleotide identity and digital DNA-DNA hybridization values with R. maanshanensis DSM 44675T (92.1 and 45.4 %) and R. tukisamuensis JCM 11308T (81.9 and 24.4 %). On the basis of results of phylogenetic, genotypic, physiological and chemotaxonomic analysis, strain NEAU-CX67T is considered to represent a novel species of the genus Rhodococcus for which the name Rhodococcus oryzae sp. nov. is proposed. The type strain is NEAU-CX67T (=DSM 107701T=CCTCC AB 2018233T).