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Rhizobial Populations, Diversity, Nodulation, and Its Relevance to Legume Cropping Euan K

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Rhizobial Populations, Diversity, Nodulation, and Its Relevance to Legume Cropping Euan K Rhizobial populations, diversity, nodulation, and its relevance to legume cropping Euan K. James Marta Maluk Francesc Ferrando- Molina* Cathy Hawes Richard Quilliam* Pietro P.M. Iannetta diversity of rhizobia “rhizobia” can be a functional term to define bacteria able to fix nitrogen in symbiosis with legumes g a-rhizobia Rickettsia Nitrosovibrio b b-rhizobia a Bordetella Methylobacterium Burkholderia s.l. Bradyrhizobium Blastobacter Rhodopseudomonas Ralstonia solanacearum Paracoccus Ochrobactrum Alcaligenes Sinorhizobium Cupriavidus Rhizobium Agrobacterium Phyllobacterium Mesorhizobium Neisseria Azorhizobium Spirillum Devosia Azoarcus Xanthobacter Azospirillum outgroup d e 16S rRNA tree of proteobacteria (adapted from Moulin et al. 2001) Biological Nitrogen Fixation (BNF) + - 4+ N2 + 10H + 8e + 16ATP ® 2NH + H2 Root nodule on Sesbania rostrata cut open to reveal the Immunolocalisation of nitrogenase N2-fixing cells (*) Fe-protein in bacteroids (b) BNF “Green Manures” in action No fertiliser necessary, as you get your Nitrogen for free! Paddy field at IRRI, Manila Stem nodulated Sesbania rostrata Non-nodulated legumes are N- deficient Soybean ± inoculation Balruddery Farm, Angus, Scotland. July 2018. 1400 Comandor cleaned seed weights [kg per ha] 1200 1000 800 600 400 200 0 Control MF AMFonly RhizoLiq+A RhizLiqOnly Economical importance of BNF • Australia (soybean, peas, f. beans) • New Zealand (pasture legumes for dairy) • Brazil/Argentina (soybean, French bean) • USA (soybean, peas) • WortH billions of $ to tHe economies of tHese countries • Assisted by large research programmes into rHizobial inoculants and tHeir application (in partnership witH industry) Faba beans and peas • Faba beans (Vicia faba) and peas (Pisum sativum) are the UK’s main grain legumes, and are of great economic and agricultural importance as a source of protein for humans and animals (Prices: $300 & $350 per tonne, respectively). • Like many legumes they can have all their N- requirements supplied by forming symbioses with a common soil bacterium called Rhizobium. • Both are widely grown in temperate regions, such as East Scotland, often in rotation with non-legume crops, in which their capacity for BNF is utilised. • However, they are NOT inoculated with rhizobia and farmers simply rely on native rhizobial populations. How to reverse decline in pulse growing in UK • Domestically-grown f. beans suffer from massive competition from imported soybean. • F. beans suffer from “yield instability”, which makes them unreliable. • In part, this is linked to erratic BNF performance in the field and it can be corrected. BNF is adversely affected by: • Lack of suitable rhizobia in the soil leading to low nodulation and low BNF. • Inappropriate use of N-fertiliser (legumes will not fix N if fertiliser is present). • Inappropriate crop rotation/sequences. Experimental trials of Faba beans at JHI (Balruddery CSC) Centre for Sustainable Cropping (CSC) • At Balruddery Farm • Experimental research platform • Large scale and long-term • 6-course rotation • Split field design • Conventional (“C”) • Sustainable (“S” = compost only) • Crop yield and quality • Economics • Nutrient budgets • Soil structure • GHG emissions 12 • Above and below ground diversity Measuring BNF by Faba beans • BNF measured at early pod fill using tHe (delta) δ15N natural abundance technique. • Total N removed in tHe grains at final Harvest, and tHe N remaining in tHe field (shoots & roots) also estimated. • DNA extracted from soil and tested for presence of rHizobia • RHizobia isolated from nodules and genotyped. • Strains tested on peas, faba beans & lentils in greenHouse and field trials Fixed N at mid podfill (2012 - 2015) Estimated using 15N BNF kg ha-1 450 400 350 300 250 200 150 100 50 0 C S C S C S C S 2012 2013 2014 2015 N in crop residues after harvest (shoots + roots) N in residues kg ha-1 120 100 80 60 40 20 0 C S C S C S C S 2012 2013 2014 2015 Faba bean BNF in UK • Faba beans can fix >300 and peas >100 kg N ha-1 • After f. bean grain Harvest up to 90 kg N Ha-1 can be left in tHe soil for tHe use of tHe following (non-legume) crop. • WitH fertiliser at a price of $300 t-1 tHis BNF amounts to a considerable cost saving. • Dependent on tHe presence of good rHizobia in tHe soil. • THese are a source of potential elite inoculants for commercial exploitation. Rhizobial populations (qPCR) 16S rRNA & (qPCR)16SrRNA populations Rhizobial Rhizobium Rlv Rleg population g-1 soil dry weight Rlv population g-1 soil dry weight Farm management: Farm Conventional Year Year Year Integrated nodD nodD 97 I 99 81 III 150 isolates: 2 main genotypes of “Rlv” IV 93 99 II Rhizobium leguminosarum bv.viciae J-1 (GQ323717.1) Rhizobium leguminosarum bv.viciae Nvf1 (GQ323718.1) 82 54 76 JHI838 Phaseolus vulgaris Salamanca Spain 53 99 Rhizobium bangladeshense BLR175 T (JN649025.1) Lens culinaris Bangladesh Rhizobium lentis BLR9 (JN649014.1) Lens culinaris Bangladesh 66 Rhizobium leguminosarum bv. viciae CCBAU11080 (GQ323701.1) Liaoning Spring RLV-IV 96 JHI1249 Pisum sativum Orkney 69 JHI1084 Lathyrush sativus Washington USA Rhizobium leguminosarum bv. trifolii WSM2304 (DQ873524.1) 89 Rhizobium leguminosarum bv. trifolii (AJ306480) 99 Rhizobium leguminosarum bv. trifolii WSM1325 (DQ873523.1) Rhizobium sullae Hc 04N (KR732671.1) 99 Rhizobium phaseoli Ch24-10 (AHJU02000025.1) 94 Rhizobium etli CFN 42T (U80928.5) 97 Rhizobium gallicum R602T (CP006879.1) 63 Rhizobium tropici CIAT 899T (NC 020061.1) Azorhizobium caulinodans ORS571T (NC 009937) 0.1 Screening rhizobia Field Greenhouse Greenhouse screen of elite rhizobia (pea cv. Corus biomass increase) Dry biomass (g) 0-60d) 5x 12x Rhizobia isolate (code) Existing commercial strain 0. 0 1. 0 2. 0 3. 0 4. 0 Control “ Top 15”Top rhizobial strains on pea 3841 13 VF2 Dry weight above ground biomass (g) pea cv. Kareni (g)pea cv. ground biomass above Dry weight 362 24 387 E2-1B Lat.Lini2B -1 LegTech 42 LA -2A VF5 370 388 Rlv strains have different symbiotic preferences that may not be linked with their original hosts Stem dry weight (g) June 2019 Pea cv. Corus Faba beans cv. Vertigo 2.2 Lentils cv. Rosana 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 NC 3841 JHI24 JHI13 JHI42 JHI370 JHI362 JHI783 JHI387 JHI782 JHI974 JHI979 JHI388 JHI1266 JHI1253 JHI1093 JHI1236 JHI1238 Genomes • >50 Rlv strains sequenced by MicrobesNG (Univ. Birmingham) - ranging from high- to low-performing strains • Phylogenies constructed of: • Housekeeping genes (rrs, recA, atpD) • Nif genes (nifDH) • Nod genes (nodAD) nodAD nifDH rrs-recA-atpD JHI1418 Wiltshire UK 2017 JHI1418 Wiltshire UK 2017 JHI1592 Skye UK 2017 VF5 Yorkshire UK Rhizobium leguminosarum bv. viciae GB30 Poland Rhizobium leguminosarum bv. viciae GB30 Poland Rhizobium leguminosarum bv. viciae GB30 Poland JHI1592 Skye UK 2017 JHI1422 Wiltshire UK 2017 JHI1253 Orkney UK 2017 JHI1422 Wiltshire UK 2017 JHI1442 Orkney UK 2017 JHI985 Angus UK 2016 JHI1442 Orkney UK 2017 JHI1266 Orkney UK 2017 JHI788 Roxburghshire UK 2015 JHI1253 Orkney UK 2017 JHI1259 Orkney UK 2017 JHI787 Ethiopia 2015 JHI1259 Orkney UK 2017 VF5 Yorkshire UK 6 JHI388 CSC (C) 2013 rcr1045 1 rcr1045 7 JHI370 CSC (C) 2013 WSM1455 Greece 2002 8 JHI788 Roxburghshire UK 2015 0 JHI367 CSC (C) 2013 1 Group I JHI388 CSC (C) 2013 Group I WSM1455 Greece 2002 JHI362 CSC (I) 2013 JHI387 CSC (C) 2013 I JHI388 CSC (C) 2013 I JHI24 Angus UK 2011 JHI370 CSC (C) 2013 JHI387 CSC (C) 2013 JHI387 CSC (C) 2013 gsC JHI362 CSC (I) 2013 6 JHI370 CSC (C) 2013 JHI782 2015 gsC JHI42 Angus UK 2011 3 6 JHI367 CSC (C) 2013 8 rcr1045 6 JHI367 CSC (C) 2013 JHI362 CSC (I) 2013 6 JHI979 Angus UK 2016 JHI24 Angus UK 2011 9 JHI1418 Wiltshire UK 2017 JHI787 Ethiopia 2015 9 JHI979 Angus UK 2016 9 6 JHI42 Angus UK 2011 9 Rhizobium pisi DSM 30132 T 9 JHI1592 Skye UK 2017 5 JHI1259 Orkney UK 2017 7 JHI1093 Angus UK 2017 JHI985 Angus UK 2016 6 JHI1442 Orkney UK 2017 8 JHI1096 Angus UK 2017 JHI364 CSC (I) 2013 5 8 JHI364 CSC (I)2013 72 JHI985 Angus UK 2016 6 JHI953 CSC (I) 2016 9 9 2 JHI953 CSC (I)2016 Rhizobium leguminosarum bv. viciae UPM791 USA 9 8 VF2 Yorkshire UK 8 VF2 Yorkshire UK 8 JHI1253Rhizobium Orkney fabae UK 2017 CCBAU 33202 T Rlv-I China 7 JHI54 Angus UK 2011 JHI1266 Orkney UK 2017 8 JHI10 Angus UK 2011 1 JHI1093 Angus UK 2017 9 JHI1422 Wiltshire UK 2017 JHI13 Angus UK 2011 Group III 0 JHI1096 Angus UK 2017 8 8 JHI925 CSC (C)2016 6 1 JHI1438 Angus UK 2017 III 0 JHI1084 Washington State USA 2017 5 JHI1093 Angus UK 2017 10 9 0 Rhizobium leguminosarum bv. viciae USDA 2370 T Tunisia JHI24 Angus UK 2011 JHI1096 Angus UK 2017 9 0 0 9 Group III Rhizobium leguminosarum bv. trifolii SRDI943 Australia VF2 Yorkshire UK 59 Rhizobium pisi DSM 30132 T III 8 5 0 JHI787 Ethiopia 2015 8 Rhizobium leguminosarum bv. trifolii WSM1325 gsA JHI1266 Orkney UK 2017 1 6 gsA JHI42 Angus UK 2011 8 Rhizobium leguminosarum bv.trifolii CC275eAustralia 8 JHI953 CSC (I) 2016 0 6 7 JHI10 Angus UK 2011 9 JHI979 Angus UK 2016 0 4 JHI10 Angus UK 2011 3 99 JHI54 Angus UK 2011 JHI13 Angus UK 2011 9 JHI366 CSC (C) 2013 5 6 9 Rhizobium leguminosarum bv. trifolii CC278f USA JHI364 CSC (I) 2013 9 JHI1438 Angus UK 2017 gsD gsD 2 Rhizobium leguminosarum bv. viciae USDA 2370 T Tunisia 9 Rhizobium leguminosarum Norway 8 JHI1587 Cambridge UK 2017 9 9 Rhizobium laguerreae FB206 T Tunisia gsF 8 8 JHI54 Angus UK 2011 Group IV 2 JHI1415 Wiltshire UK 2017 1 Rhizobium leguminosarum bv. viciae CCBAU 11080 Rlv-IV China 5 Rhizobium bangladeshense BLR175 T Bangladesh IV JHI1600 Wiltshire UK 2017 Group II 9 Rhizobium binae BLR195 T Bangladesh Rhizobium leguminosarum bv.
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