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Mesorhizobium Qingshengii Sp. Nov., Isolated from Effective Nodules of Astragalus Sinicus

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International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2002–2007 DOI 10.1099/ijs.0.044362-0 Mesorhizobium qingshengii sp. nov., isolated from effective nodules of Astragalus sinicus Wen Tao Zheng,1 Ying Li, Jr,1 Rui Wang,1 Xin Hua Sui,1 Xiao Xia Zhang,2 Jun Jie Zhang,1 En Tao Wang1,3 and Wen Xin Chen1 Correspondence 1State Key Laboratory for Agro-Biotechnology, College of Biological Sciences, China Agricultural Xin Hua Sui University, Beijing, 100193, PR China [email protected] 2Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China 3Departamento de Microbiologı´a, Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico Nacional, 11340 Me´xico DF, Mexico In a study on the diversity of rhizobia isolated from root nodules of Astragalus sinicus, five strains showed identical 16S rRNA gene sequences. They were related most closely to the type strains of Mesorhizobium loti, Mesorhizobium shangrilense, Mesorhizobium ciceri and Mesorhizobium australicum, with sequence similarities of 99.6–99.8 %. A polyphasic approach, including 16S– 23S intergenic spacer (IGS) RFLP, comparative sequence analysis of 16S rRNA, atpD, glnII and recA genes, DNA–DNA hybridization and phenotypic tests, clustered the five isolates into a coherent group distinct from all recognized Mesorhizobium species. Except for strain CCBAU 33446, from which no symbiotic gene was detected, the four remaining strains shared identical nifH and nodC gene sequences and nodulated with Astragalus sinicus. In addition, these five strains showed similar but different fingerprints in IGS-RFLP and BOX-repeat-based PCR, indicating that they were not clones of the same strain. They were also distinguished from recognized Mesorhizobium species by several phenotypic features and fatty acid profiles. Based upon all the results, we suggest that the five strains represent a novel species for which the name Mesorhizobium qingshengii sp. nov. is proposed. The type strain is CCBAU 33460T (5CGMCC 1.12097T5LMG 26793T5HAMBI 3277T). The DNA G+C content of the type strain is 59.52 mol% (Tm). Astragalus sinicus Linn. (Chinese milk vetch), an herb- in turn improves the quality and flavour of rice (Lin & Gu, aceous legume originated in China, has been widely 1998). Although only Mesorhizobium huakuii has been cultured as a traditional green manure in wintry fallow described from the rhizobia of A. sinicus (Chen et al., 1991; paddy fields in southern China and Japan. It is also used as Murooka et al., 1993; Jarvis et al., 1997; Nuswantara et al., a soil fertilizer and in repairing the soil environment, which 1999), great diversity in the chromosomal genes has been revealed among the microsymbionts of this plant. In addition, the nodulation (nod) genes in the rhizobia of A. The GenBank/EMBL/DDBJ accession numbers reported herein are sinicus were found to be conserved (Murooka et al., 1993; as follows: JQ339788, JQ339776, JQ339785, JQ339775 and Guo et al., 1999; Zhang et al., 2000). JQ339793 for the 16S rRNA gene; JQ339818, JQ339806, JQ339815, JQ339805 and JQ339824 for the partial atpD gene; During a study of rhizobia nodulating A. sinicus in south-east JQ339851, JQ339839, JQ339848, JQ339838 and JQ339856 for the China (encompassing four provinces), 232 bacterial isolates partial glnII gene; and JQ339757, JQ339745, JQ339754, JQ339744 were isolated from root nodules collected in the fields by and JQ339762 for the partial recA gene for strains CCBAU 33460T, CCBAU 33430,CCBAU 33431,CCBAU 33446 and CCBAU 33455, standard methods (Vincent, 1970). All were classified within respectively. Plus JQ339911, JQ339899, JQ339908 and JQ339916 the genus Mesorhizobium based upon 16S rRNA gene for the partial nifH gene; and JQ339881, JQ339869, JQ339878 and sequence analysis (our unpublished data), with the methods JQ339886 for the partial nodC gene for strains CCBAU 33460T, described subsequently. Within these rhizobia, five isolates CCBAU 33430, CCBAU 33431 and CCBAU 33455, respectively. originating from Jiangxi province shared identical sequences Six supplementary figures and three supplementary tables are available of 16S rRNA genes and formed a unique lineage in a with the online version of this paper. subcluster together with the type strains of Mesorhizobium Abbreviations: Box-PCR, Box-repeat-based PCR; IGS, 16S–23S rRNA loti, Mesorhizobium shangrilense, Mesorhizobium ciceri and intergenic spacer; NJ, neighbour-joining; ML, maximum-likelihood. (Fig. 1). To clarify the taxonomic relationships of these five 2002 044362 G 2013 IUMS Printed in Great Britain Mesorhizobium qingshengii sp. nov. CCBAU 33446 (JQ339775) CCBAU 33455 (JQ339793) CCBAU 33460T (JQ339788) M. qingshengii sp. nov. CCBAU 33431 (JQ339785) CCBAU 33430 (JQ339776) 74/76 Model selected: GTR+I+G M. ciceri LMG 14989T (U07934) -lnL=2790.6831 67/80 T K=10 M. loti NZP 2213 (NR_025837) AIC= 5601.3662 68/80 M. shangrilense CCBAU 65327T (EU074203) Base frequencies: 73/72 M. australicum WSM2073T (AY601516) freqA =0.2444 freqC =0.2331 M. alhagi CCNWXJ 12-2T (EU169578) freqG =0.3129 100/100 M. camelthorni CCNWXJ 40-4T (EU169581) freqT =0.2095 Substitution model: T M. albiziae CCBAU 61158 (DQ100066) Rate matrix M. chacoense LMG 19008T (AJ278249) R(a) [A-C] =1.4942 R(b) [A-G] =1.5203 81/86 M. robiniae CCNWYC 115T (EU849582) R(c) [A-T] =3.0340 91/88 M. muleiense CCBAU 83963T (HQ316710) R(d) [C-G] =0.4775 65/79 M. temperatum SDW018T (AF508208) R(e) [C-T] =2.8207 68/89 R(f) [G-T] =1.0000 T M. mediterraneum LMG 17148 (AM181745) Among-site rate variation M. caraganae CCBAU 11299T (EF149003) Proportion of invariable sites T (I) = 0.8315 M. gobiense CCBAU 83330 (EF035064) Variable sites (G) M. metallidurans STM 2683T (AM930381) Gamma distribution shape 69/95 M. tianshanense CCBAU 3306 T (AF041447) parameter = 0.7122 M. tarimense CCBAU 83306T (EF035058) M. septentrionale SDW014T (AF508207) 81/95 M. silamurunense CCBAU 01550T (EU399698) M. plurifarium LMG 11892T (Y14158) M. amorphae ACCC 19665T (AF041442) M. opportunistum WSM2075T (AY601515) M. thiogangeticum SJTT (AJ864462) M. huakuii CCBAU 2609T (FJ491264) S. fredii USDA 205T (AY260149) 0.1 Fig. 1. Maximum-likelihood tree reconstructed from 16S rRNA gene sequences showing the phylogenetic relationships of the representative strains of Mesorhizobium qingshengii sp. nov. under the best-fit model shown. Bootstrap values of 65 % or more (ML/NJ) are provided at the nodes. The sequence of Sinorhizobium fredii USDA 205T was used as an outgroup. Bar, 10 % sequence divergence. strains, a polyphasic approach was performed in this Mesorhizobium huakuii, Mesorhizobium temperatum and study. All the bacterial strains used in the present study Mesorhizobium tianshanense. were cultured in TY medium (tryptone, 5.0 g; yeast Amplification of 16S rRNA, atpD, glnII and recA genes with extract, 3.0 g; CaCl , 0.6 g; pH 7.0–7.2) at 28 uC (except 2 primer sets P1/P6 (Tan et al., 1997), atpD255F/atpD782R, where indicated). recA41F/recA640R and glnII12F/glnII689R (Vinuesa et al., For 16S–23S intergenic spacer (IGS) RFLP, the IGS 2005), respectively, was performed with the protocols fragment was amplified with primer pair FGPS6/23S-38 originally described. Amplification of partial nifH and and the PCR protocol of Rasolomampianina et al. (2005). nodC genes was performed with primers nifH1F/nifH1R The genomic DNA extracted from each strain following the (Laguerre, et al., 2001) and nodC540/nodC1160 (Sarita method of Terefework et al. (2001) was used as template. et al., 2005), respectively. All the amplified fragments were The amplified genes were digested with HaeIII, HhaI and directly sequenced as described by Hurek et al. (1997). The MspI as specified by the manufacturer and the digested sequences were aligned with those of defined Mesorhizobium fragments were separated and visualized as described by species using the CLUSTAL W program in the MEGA 5.0 Terefework et al. (2001). The five strains showed five software package (Kumar et al., 2008). Unrooted trees were different patterns and were clustered as a group at 72 % constructed with the neighbour-joining (NJ) method similarity in the cluster analysis using the DICE coefficient (Saitou & Nei, 1987) and Jukes–Cantor distance (Jukes & and UPGMA method (Fig. S1 available in IJSEM Online). Cantor, 1969), respectively, and were bootstrapped using They were further grouped at 60.6 % similarity with the 1000 replications (Felsenstein, 1985). Maximum-likelihood type strains of Mesorhizobium caraganae, M. ciceri, M. loti, (ML) trees were also constructed using the PhyML 3.0 http://ijs.sgmjournals.org 2003 W. T. Zheng and others program (Guindon & Gascuel, 2003). The robustness of ML Nodulation and nitrogen-fixation abilities and host ranges topologies was inferred by non-parametric bootstrap tests are important features for symbiotic rhizobial species. In based on 100 pseudo-replicates of the data (Felsenstein, this study, cross nodulation tests were performed in 1985). The nucleotide substitution model was selected Leonard jars filled with vermiculite moistened with N-free by Akaike’s information criterion, as implemented in solution (Vincent, 1970). Except strain CCBAU 33446, the Modeltest 3.7 (Posada & Crandall, 1998). remaining four strains were able to nodulate with A. sinicus and occasionally with Astragalus adsurgens (four of the 10 In the phylogenetic analysis based on 16S rRNA gene plant replications nodulated) under laboratory conditions. sequences (1270 nt), the phylogenetic topologies were
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    Mohammad Saghir Khan l Almas Zaidi Javed Musarrat Editors Microbes for Legume Improvement SpringerWienNewYork Editors Dr. Mohammad Saghir Khan Dr. Almas Zaidi Aligarh Muslim University Aligarh Muslim University Fac. Agricultural Sciences Fac. Agricultural Sciences Dept. Agricultural Microbiology Dept. Agricultural Microbiology 202002 Aligarh 202002 Aligarh India India [email protected] [email protected] Prof. Dr. Javed Musarrat Aligarh Muslim University Fac. Agricultural Sciences Dept. Agricultural Microbiology 202002 Aligarh India [email protected] This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machines or similar means, and storage in data banks. Product Liability: The publisher can give no guarantee for all the information contained in this book. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. # 2010 Springer-Verlag/Wien Printed in Germany SpringerWienNewYork is a part of Springer Science+Business Media springer.at Typesetting: SPI, Pondicherry, India Printed on acid-free and chlorine-free bleached paper SPIN: 12711161 With 23 (partly coloured) Figures Library of Congress Control Number: 2010931546 ISBN 978-3-211-99752-9 e-ISBN 978-3-211-99753-6 DOI 10.1007/978-3-211-99753-6 SpringerWienNewYork Preface The farmer folks around the world are facing acute problems in providing plants with required nutrients due to inadequate supply of raw materials, poor storage quality, indiscriminate uses and unaffordable hike in the costs of synthetic chemical fertilizers.
  • Biserrula Pelecinus-Nodulating Mesorhizobium Sp

    Biserrula Pelecinus-Nodulating Mesorhizobium Sp

    Symbiotic Effectiveness, Phylogeny and Genetic Stability of Biserrula pelecinus-nodulating Mesorhizobium sp. isolated from Eritrea and Ethiopia Amanuel Asrat Bekuma A thesis submitted for the degree of Doctor of Philosophy Murdoch University, Perth Western Australia June 2017 ii Declaration I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. Amanuel Asrat Bekuma iii This thesis is dedicated to my family iv Abstract Biserrula pelecinus is a productive pasture legume with potential for replenishing soil fertility and providing quality livestock feed in Southern Australia. The experience with growing B. pelecinus in Australia suggests an opportunity to evaluate this legume in Ethiopia, due to its relevance to low-input farming systems such as those practiced in Ethiopia. However, the success of B. pelecinus is dependent upon using effective, competitive, and genetically stable inoculum strains of root nodule bacteria (mesorhizobia). Mesorhizobium strains isolated from the Mediterranean region were previously reported to be effective on B. pelecinus in Australian soils. Subsequently, it was discovered that these strains transferred genes required for symbiosis with B. pelecinus (contained on a “symbiosis island’ in the chromosome) to non-symbiotic soil bacteria. This transfer converted the recipient soil bacteria into symbionts that were less effective in N2-fixation than the original inoculant. This study investigated selection of effective, stable inoculum strains for use with B. pelecinus in Ethiopian soils. Genetically diverse and effective mesorhizobial strains of B. pelecinus were shown to be present in Ethiopian and Eritrean soils.