Towards a Characterisation of the Wild Legume Bitter Vetch (Lathyrus Linifolius L

University of Dundee

Towards a characterisation of the wild legume bitter vetch (Lathyrus linifolius L. (Reichard) Bassler): heteromorphic seed germination, root nodule structure and N- fixing rhizobial symbionts Jacovo, E Dello; Valentine, T A; Maluk, M; Toorop, P; Lopez Del Egido, L; Frachon, N

Published in: Plant biology

DOI: 10.1111/plb.12902

Publication date: 2019

Document Version Peer reviewed version

Link to publication in Discovery Research Portal

Citation for published version (APA): Jacovo, E. D., Valentine, T. A., Maluk, M., Toorop, P., Lopez Del Egido, L., Frachon, N., Kenicer, G., Park, L., Goff, M., Ferro, V. A., Bonomi, C., James, E. K., & Iannetta, P. P. M. (2019). Towards a characterisation of the wild legume bitter vetch (Lathyrus linifolius L. (Reichard) Bassler): heteromorphic seed germination, root nodule structure and N-fixing rhizobial symbionts: heteromorphic seed germination, root nodule structure and N-fixing rhizobial symbionts. Plant biology, 21(3), 523-532. https://doi.org/10.1111/plb.12902

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Download date: 26. Sep. 2021 This article isprotected rights by All copyright. reserved. doi: lead todifferences version between this andthe ofPleasecite article Version Record. this as been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article has ac been † 8 7 6 5 4 Selsfield Road, Ardingly, West Sussex RH17 6TN, United Kingdom 3 2 1 N. Dello Jacovo, E. fixing (Reichard) Bässler): acharacterisation legume theTowards wild of bitter ( vetch Handling Editor: H.Pritchard Articletype : Research Paper DR. PIETRO PAOLOMICHELE IANNETTA (Orcid ID : 0000 LAURADR. LOPEZ DEL EGIDO(Orcid ID : 0000 TRACYDR. ANNEVALENTINE (Orcid ID 0000: These authorsThese contributed equally to this manuscript. Museo delle Scienze, Corso Lavoro del edella Scienza 3,38122, Trento Strathclyde University, 161 Cathedral Street, Glasgow, G4 0RE, Bitter Royal Botanic Garden, Seed Royal Botanic Gardens, Kew, The James Hutton Institute, University

Accepted 5 Article , Kenicer, G.

10.1111/plb.12902 Physiology - Vetch rhizobial rhizobial

of Pavia,of Via S.Epifanio

Ltd. 5 1,2,† , Park, L. , Syngenta Seeds B.V , symbionts. 11 The Cloisters, Wingate, CoDurham, 5PT, TS28 , Valentine, T.A.

heteromorphic 20AInverleith Row

cepted for publication and undergone forcepted and buthas not review publication fullpeer 5 , Goff, , Goff, M. Errol Road,

Comparative Plant and Fungal Biology Department,

1,† , 6

, Ferro, V.A. 14 , Maluk, M. . , 1600AA, – Invergowrie seed germination, root nodule structure root germination, nodule seed N and

27100 Pavia, Italy , - Edinburgh 0002 - 0002 1,†

7 Enkhuizen, , Bonomi, C. - 9064 - , Toorop 1660 ,

Dundee , - 0002 - EH3 5LR 2896) - 9131) , P.

, 3451 The

DD2 5DA, Scotland 8 3

, James,E.K. , Lopez Egido, del L. United Kingdom , Netherlands - United United Kingdom 4259)

Lathyrus linifolius Lathyrus United Kingdom ,

Italy

and Iannetta, P.P.M.

, Wakehurst Place,

United Kingdom 1,2,4

, Frachon,

L. L. - 1,*

This article isprotected rights by All copyright. reserved. Accepted ArticleAbstract *Corresponding a   

L. linifolius L. Brown were seeds largerand comprised a larger (69%). ofthe proportion seed batch %) Seed linifolius L. s and enzyme, nitrogenase) includ data. Ultrastructural Seed of synteny crops. However, withimportantpulse conservation, claimed therapeutic properties. key and species moth butterfly fixing linifolius L. Lathyrus Fax: Phone: e Address: Name: equeneces - mail: L. linifolius linifolius L. after scarification at differing optimal characterised

morphotypes

ed of species

+44 (0) 344 9285428 [email protected]

L. lini L. immunolabelling withnifH uthor .

root nodules yield

. A of coloniser lownutrient (N) novel croppingas model andnovel species. The latter genetic owing toits remain to be characterised. +44 (0)344 928 5429 DD2 5DA, Scotland, United Kingdom The James Hutton Institute, Errol Road, Invergowrie, Dundee, Pietro folius folius

details ed phylogenetically

had similar had similar

hetermorphic seeds of distinct colourhetermorphic seedsgreen classes: brown ofdistinct and Iannetta, P

and functionaland characterisation of . were characterised (Reichard)Bässler

: Endosymbiotic bacteria

appeared

. carbon and its T M.

us, the

, indeterminate instructureindeterminate using

- protein antibodies (recognisingfixing antibodies theNprotein :N ratios and weregerminableequally and :N ratios roots are known forroots are their known organoleptic

using physical, colourimetricchemical physical, andusing ( 16S species species tem bitter vetch

peratures r

soils soils RNA, RNA, regenerationattributesfunctional and were were has high

it isolated from theroot nodules ) nodA

supports biodiversitysuch as is a

( the N fixingroot nodules 16 and 20

potential for potential for

fabaceous nitrogen fabaceous nitrogen and ,

effective ( nodD o C, respectively C,

gene restoration capable of

( and ca. (N)

90 ) , . . This article This isprotected by copyright. All rights reserved. Accepted Liston,al., 1998;Kenicer et2005) varyyellow, incolour including violet, blueor white orange, red, purple, often assisted or by simple branched tendri The genus annual presentsboth andperennial witha species sprawling climbinghabit or considered primary ofdiversity tobethe andcentres secondary Regions and theMediterranean in around an basin speciesNorth inEurope, America, 160 species hemisphereare across whichwith52,30,78,24and thenorthern distributed Eurasia, tropical the and Africa Americas Miocenein the middle approximatelyM period, 14 Fabeae approximately thatevolved iscomposedof Mediterranean intheEastern 380species belongFabeae, of a tothefamily tribe subdivision Fabaceae, or The gen ArticleIntroduction Lathyrus linifolius Key words 

socio have in potentialapplication restoration The findingsand isolates have rhizobial potential leguminosarum fixing atmospheric us

Lathyrus : :

economically crop important

and , bitter vetch, seed germination,

L .

horticulture horticulture encompasses biovar N )

and accommodateand viciae . Asia,

using seeds. native thedata and resourcesAlso, rhizobial comparative . plants

South South America and East tropical Africa,respectively . The genus The s such as s such ls, and pollinator dependantls, andwhich flowers may referred togenericallyreferred as Peas’ ‘Sweet which d

legume and functional studies studies functional and

strains with with strains d inNorth andSouth Americaare y

Pisum a Lathyrus . Species of the tribe thenspread of to Species thetribe root nodules,root

application , Lens

high similarity is mad of the of

and Leguminosae Rhizobium Vicia for e upof approximately genus, respectively with ecological ecological

(Asmussen and (Asmussen .

related and to .

Rhizobium Rhizobium . The tribe . . This article isprotected rights by All copyright. reserved. recorded records indistribution onthe National Vegetation floristic Classification(NVC) Britain thoughis though andal., Rose 2014; O'Reilly, (20 linifolius Lathyrus interactions. etNilsson al.,2008) related of specialist tothebutterflies extinction ( lonicerae Phyllonorycter nigrescentella to semi horticultural species being cultiv sativus ( which include types domesticated characterised by Network,a mesophyte 2013), The latter (AsmussenListon,Lewis and 1998;Kenicer et al.,2005; etet al.,2005;Schaefer al.,2012) (BNFfixation can their food capacityand animal feedwithout toprovide Lathyrus Vicia faba

Acceptedoligophagous Article -

be productive on low nutrient soils asa nutrientsoils functionbe of onlow productive their capacity for biol 350 m) grasslands, with soils of lownutrientstatus soils grasslands, and4 350 m)with pHranging from - natural systems

it is it L.

includes ; (the Narrow property ( Schaefer et al., 2012) etSchaefer al., L.), ( pea absent

) ,

and butterflies andsuchasbutterflies moths, rare

many in is found in extends to extends

. their their Pisum sativum Pisum

lyfound the cold climatic extremesof the recordsarefoundNo regarding and inecologicaland interaction - BorderedFive species roots can sandy ofarid soils help the environments stabilise 2006) . ;

L extensivelyandgrazed in the The species isalsogeneticallyother species generaThe syntenouswith Grapholita jungiellaGrapholita .

which are agriculturalwhich andecological of due importance to . ated forated aesthetic linifolius Lathyrus linifolius Lathyrus .

L.)Indianknown cropsas and such lesser L. L. South EastSouth - Spot Burnet moth) Spot linifolius linifolius

(Reichard) Bässler Leucoptera lathyrifoliella form lathyrifoliella Leucoptera orobi

of England Leptidea sinapis Leptidea is present 59 in s reasons

N common pulse cropsas such faba bean ; speciesthis i orth Grapholita lunulana Grapholita

its involvement in involvement in its has value been its as for popularised non

an .

Europe - in I grazed semi and ts loss from such habitats from hasbeents loss such

(Grimeal., 2014) et - organic (N) nitrogen input

(

National Biodiversity for its % s , ,

important and is the woo of European territories, of European

aromatic flowers - natural low altitude low natural soil found throughoutfound ; and d whitebutterfly

- for its microorganism ogical N -

pea ( pea Zygaena to . It 7

is also is attraction

(Grime et Lathyrus Lathyrus ;

. . They In an ; .

This article This isprotected by copyright. All rights reserved. exp et al. and w T agreement to produce flavoured neutralspirit use Pratt 2012; Henderson1994; Johnston, and and Dickson, Step, linifolius flavour, a generalforas and/ortincture use latter sometim of beverages medicine torelieveexcessive flatulence,chest ailments famine periodthis the species was Lightfoot, 2012; Johnston, Moffat 1777; etal., 2014 historic speciesrecorded extensively have been Despite theabsence of ( not surprising that (1999)Rose 2009) in tables (JNCC, Grime e Acceptedherefore Article d ression of

, as a flouring agent, andasagent, were aflouring

hile (201 , al to offset the symptoms t al.(2014)

include liquor , records of the Scottish HighlandsrecordsScottish of the scientific evidencescientific itsimpact for

2 that theroot tubersfeelings were used topromote ofsatiation this species species this ) revealed that consumption ofbitter consumption vetch) revealedtubers that by significantlyaltered rats

. considered

Raw tubers werealso water the sliced withhot Raw and spirits, tubers infused orneutral hypothalamus genes metabolism. inregulating involved es afterroasting tomake flavoured beverages L . .

linifolius linifolius L also has the also - Quercus knots, liquory knots, L .

. linifolius linifolius

linifolius prized is scarceof arable crop incentres production of inebriation duetoexcess alcohol consumption

(W11)and . for fermented be tomake

in agricultural ecosystems, usesof ethnobotanical the Of theliterature sourcescited potential an indicator of ancient woodland its its - knots ,

but are from but predominantly root tubers whichof intimes wereforroot tubers usedsatiation .

in these regards (Beith, 1995; Cook, 1995;Hatfield,(Beith, 1995;Cook, 2004; Juniper communisJuniper Hence

for development and l iquorice iquorice

the many for names vernacular ;

Pennant, 2014; Vic ,

er and, as commonly aflavouring, most

, v which on occasions was distilled waswhich distilled onoccasions

is etch owing totheir liquorice as a scantresearch recent by Woods

dominated woodland (W19) dominated woodland 1899)

(Brenchley, 1920; here therapeutic and therapeutic seventeenth . .

, The tubers wereThe also tubers As such, it As

there isgeneral . kery, 1995)

L . linifolius such as

, century

is as a novel crop L

. - perhaps . the UK

like During . the the .

This article isprotected rights by All copyright. reserved. Analytical(Exeter Inc., USA) balance) oven driedfor 48h C andNcontent were measured for each seed morph Seed areathe repeated with “ respective ROI weretransferred onto each of the split images analysis each image was wasThis also used produceto region of i and s segment using a digital image dpi), (1200 and subsequent analysis using the places (ug) assessing the green brown or characterised heteromorphic as based onseed colours L morphometricSeed material and characterisation Materials and methods sequences from rhizobia isolated from related legumegenera rRNA) and and molecular optimise seed germination; 2) characterisation of 2004; vetchling, characterisation of the N the aspect also remains beto characterised with respect to germination their capacities L. linifolius seedstheir or reviewed reports dedicated theto species, and germinationthe of suppliers usefor restorationin andconservation projects, developed or as a novel crop Whether developed for study anacademic as model species, exploited .

Acceptedlinifolius Article eed measurements were acquired by using the - carbon fixing root nodule also

scanner ed and into tin containers combustionfor and processed with a L. aphaca

L. latifolius using ( symbiotic

seeds were purchased from has Adventurer® Explorer, Ohaus) the the Mean Gray Value

relative abundance of ea which describe : characterisation n i .

ntegrated density” included in the measurement ( heter These colour morphs were characterised using 300seeds seeds(100

itrogen Epson Expression 10000XL Pro the L. linifolius L. linifolius

at at 75

FIJI o or meadowor vetchling

( morphic seed

rhizobial symbionts nodA or s

split into

° SIOX SIOX ratio perennial peavine of C . , nodD

L. linifolius From rep each optimal method . we also report upon

seeds is importan of seeds,

plugin the . each component of a red

) genomes) s , as , as distinguished bydifferent their seed colours, and so this root nodule symbionts ch morphotype and ( Bitter Simple Interactive Object Ext also remains to bereported nterest (ROI) locating

test and from other , , licate

de Meyer al., et 2011) and s for seed germination : in: acomparativ -

seed seed treatments (temperature V hence ,

L. etch t by

) . 2 mg

nissolia However, there is a and saved N

Analyse particles colourimetric analysis using ahigh resolution

- there are no similar reports which characterise . , Ltd. fixing root ,

Three their Lathyrus which were categorised superficially either as were weighed ,

a commercial seed supplier or grassor vetchling, fresh weight – Fiji/

r using sequences of their core (16S .

as eplicates of 4seeds were weigh

green and e phylogenetic analysis with samethe

. for T species ( ImageJ software -

nodule nodule structure; and 3) “Integrated density” CE440™ iff format the particlethe analyses process was . each seed

. – , Therefore,

Our own observations show that

and

blue function on the binary image raction

(with Sartorius SE2 Ultra paucity via recorded L. pratensis there has

commercial native seed (RGB) . Elemental Analyser

and The ; .

(

Mutch Young, and

For the colorimetric Friedland et al., 2005) of scientificof peer herein

scarification) . images were

Image to scale . been been

T using a4 or per per he structure . is is productthe of we report on a

, Seeds .

meadow maximising s The only ,

replicate)

were taken the isolation

ed limited could be to decimal

- - and micro of of .

by ,

This article This isprotected by copyright. All rights reserved. when needed glasshouse allowed to drain from base the up, and produces even saturation throughout potsthe to just of sand medium comprised that 3:1 Sylvamix® Special (Melcourt, (domestic (Scotland, UK) wereSeeds treated using a standardised approach developed at Royalthe Botanic Garden Edinburgh Plant formation root harvesting growthfor nodule and moni were performed in control and treatment ( germinationAll trials were prepared in sterile containment the at James Hutton Institute, Dundee brown morph conditions theto seed embryo scarified manually using asharp scalpel blade, morpheach The germinated seeds and protrusion (1 After the initial imbibition ofset placed onto a tray within a sealed plastic bag (to avoid dehydration of the filter papers/seeds) in sterile disti diluted provideto 2.5 were surface sterilised by immersion with gentle shaking 3min for sodium in hypochlorite solution replicates To scarification Optimal germination tempe 56°27'23"N, 3°04'14"W 90 approximately Accepted Article investigate effect . tored abovetored as for optimal germination experiments.

mm diameter After sowing, horticultural replicates were placed into incuba

below the level of the ; ( , of seed scarification on germinationtotal rate and t lled water (SDW) and placed on two comprising at 0.01

o where they were subject to ambient conditions type - , 16 and 20 s

2 mm)2 until and were allowed nodulate to naturally. . optimal germinat

% , In April 2016 seeds were soaked

then stratified 2 mm (15 seeds(15

[v/v]), and placed . an

P The seeds were then germinated in dishesPetri as described abov etri dishes

Petri dishes LMS Cooled Incubator 600 (LMS without Ltd), light the % 12 seeds

. °

The pots wereThe then placed in an empty which container, was filled with water [v/v] active chlorine C time germination ceased

, the , the respective optimal germination temperatures phase ), andwithin), per internal rootingmedium taken for taken for .

replicate) in in an incubator ion ion for each colour morph each) rature seed heteromorphs of After addition of 7mL of

, plates were monitored daily, andgermination grit (2

were

onto the surface smallof pots

were used – fully randomised

tors at constant temperatures at either: 5, 10, 15,20 or a time frame six months of from January to 2016 June 50 6 .

mm) was to added cover (‘top F % or each

. . of seed

The The seeds were then washed three Data w with 5

disks 3MM of per

°C for 14dbefore transfer to the un overnight in watertap contai morph

the incision being made along axis the transverse morphotype s .

er to This allowThis e germinate

on tray

used to determine proportionthe of

otype o SDW theto petri .

www.melcourt.co.uk type Plants were not fed, but only watered 50

Whatmann

, the seeds of three replicates were was assessed using s a s

t and

for incubation and assessmentand s of a range of

. containing dry sieved, rooting (t

water to saturate the compost The pots wereThe then removed and 50 temperature combination ).

- dress’) thedress’) seeds adepthto

filter paper mm (90 radius) . -

dish Seed germination was for for the green and the temperature ning detergent was . )

times with

Germinat each dish was and scored as radicle ix ix replicates heated

e horticultural in darkin eed s , three ion tests

2 mL .

Each . 25 .

All Seeds

from ° C .

This article isprotected rights by All copyright. reserved. Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, US). µL SDW. removed using anaspirator and residual liquid was air dried. was washed with 200 70 µl 15 min (or volumes µL) (~655 of isopropanol (SigmaI9030). 0.1 10min.for : (25 24 : 1[v/v/v]; Sigma was P2069) added to each sample, vortexed and centrifuged 11 at (Sigma and P4850)) incubated at 37 Cl pH 7.5,1mM EDTA pH 8.0 g 1 for For DNA extraction, bacterial cells were harvested from cultures liquid by centrifugation at 11 characterisation. used prepareto 25 Beringer (1974) tryptone yeast (TY) broth (tryptone, 5g L selected and congo red mM yeast NaCl; extract, 0.5 g L 2 100EA) sterile deionized water % water and nodules Root nodules for bacterial isolation were harvested from plants juvenile Root molecular bacterial isolation and characterisations taken against the dos Reis et al. collected on slides, stained with UCT ultramicrotome light for and transmission electron microscopy 48for hat 60 nodules were then elongateif water gently removed fromrooting their medium andthe root systems carefully washed in running tap Root nodules were harvested from plantsjuvenile of around 5 ultrastructureRoot nodule 0 mL ofyeast mannitol agar (YMA; 54.89 mM mannitol; 2.87mM K

sodium

Accepted Article x volume (~17.5 µL) of 3

. using a JEOL 1400JEM TEM.

0 min Freshly harvested root nodules were gently sliced half in

were selected randomly and spread onto

Each DNA sample was assessed for its qu were ( d hypochlorite The aqueous phase of the upper layer (~175µL) was recovered, and was first mixed with - 0.025 g nitrogen ) ) and then fixed 20°C overnight) andpelleted by centrifugation at 11

. purified

Bacterial pellets were re , °C

(2010) then then .

Liquid cultures were in 100in

subject to anethanol % L ase surface sterilised by rinsing in . -

1 glycerol stocks for long . Single )

. . Sections for

Fe %

(Fisher Chemical S/5042/15) [ The Nodules were then crushed using sterile 90 mm sterile diameter triple vent

- LR White

( % [v/v]% ethanol and centrifuged 11 at in 2 in nifH dishes were incubated colonies

, 0.5 .5

- 0.5 from each of of from each two ) sodium acetate (pH5.2) 1

;

protein % glutaraldehyde% Fred and Fred Waksman, 1928;

TEM were treated by immunogold labelling using anantibody °

% . % w/v% sodium dodecyl sulphate and 10 µL proteinaseof K

C for 1h.

The nodules were then sectioned using aglass knife - from purified plates grown in a rotary shaker (150 suspended in 420 µLof freshly made lysis buffer ( toluidine blue and digital micrographs taken described as by - LR White LR White

according to - 1 ; yeast ; extract, 3g L - term

Then, 420 µLof phenol :

acrylic resin dilution series before DNA was precipitated by incubating at

storage and storage and extractto DNA for strain molecular 70 at 28 . ality andquantity using a

as as described by linifolius

v/v] % (James et al.

. was This later mixed °C for [v/v]

were used to inoculate 5 mL ofsterile for for Petri dishesPetri (

The The – Vincent 1970)

4 plants ethanol, followed by immersion 2.5 in 10 cm in

24 using asterile scalpel 1 ,

min, andrinsing three times with 000 x g 15min. for ; 6.05mM; CaCl p

, DNA pellet 000 x ellet pestles (SigmaZ359947 – , 48 h

. 2002) . James et James et al. (2011) rpm) 28 at

Light m Nodules were washed with tap 2

, as , as described abov HPO . g for 1 min. height Sterilin

chloroform isoamyl : alcohol S . Digital m ingle colonies were 4 with f icroscope sections were ; 0.81 ; n was .

ND with approximately 3x ° The plantswere 101VR20 C 2 - re

2H ilter sterilised - overnight

mM MgSO 1000

- Supernatant was The D suspended suspended 50in icrographs icrographs polymerisation

2 (longitudinally O; pH 6.8 .

The fixed 10 mM Tris on a ) containing) e - NA pellet 80 . ,

000 xg Three Three then

°C for , Leica 4 000 x - ; 1.71 ;

were ;

- This article This isprotected by copyright. All rights reserved. intercept and a linear regression t (1982)al. modellinear a as function of temperature according to the procedure analysis of the germination rate (1/ using the model type ‘ Boltzmann sigmo morph. Crawley (2012) germinationfinal percentage were analysed using ageneralised linear mo were compared 3.1.1 Results were analysed with Statistical analysis strains used inallphylogenetic in analyses are included measured in the number of substitutions the topology superior with log likelihood value distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting obtained automatically by 1000 bootstrap replicates and uniform among rates sites using the following parameters: Clustal Ω for alignment, complete deletion for gaps missing or data, Tamura Phylogenetic relationships were calculated numbers from MG546114 to MG546116. of S MG546091 geneAll sequences were deposited an ABI3730 DNA analyser. Purified products (Illustra™ ExoStar™ 1 extension at 72 comprised 95 amplification cycles ( primer 1.25 unit Go Supplementary bacterial isolates The primers and thermal profile he data for each equences

Accepted ArticleL .

; sativus ( Team, .

The The - For all primer co Nei

with

for the calculation species of cardinal temperatures were

(16S (16S rRNA)

g Taq model as 2015) ermin

°C 1.5 for min, each regression line

a Table Table 1 . °C for 1.5 between seed morphs

® G2 DNA polymerase (Promega

amplified from Germination rate was morphotype via comparison d al curveal . ation rate (Tamura Nei, and 1993)

Data obtained from their core

Supplementary N was fitted as to find T .

on , Each 50µLPCR reaction contained 1 µL DNA template, 1 mbinations initial denaturation was for 95°C for 2min, followed by the MG546092 to min applying Neighbor -

Linear that was fitted Microsoft excel and , was

the the was . and

(16S rRNA) and There was There also a final single extension cycle at 72 s DNA and extracted purified from isolates cultured from dry nodules

used used generateto annealing temp calculated - . separated into sub these least least t T in GenBankin with the accession numbers 50 b ) related) to temperature Table Table

through (base temperature for germination) used to calculate the optimal growth temperature for each

S have been deposited thein NCBI database with the accession MG546 the morphologicalthe characteri per - quares’, or

Step, GEStep, Healthcare Life Sciences) were sequenced using to using the 1

and as site the ) ) for each primer pair b -

Join and T analysis variance of (ANOVA) . 1/t

. 102 The trees were drawn to scale, with branch symbiotic RStudio (RStudio curve were

erature Accession numbers 50 gene ing

M7845), 0.2M7845), mM dNTP each and 0.4µM of each ; c - ( NLS with . M optimal and supra nodA

Both conducted in and BioNJ algorithms amatrix to of pairwise of germinatof aximum specific products to identify

. (

Initial tree(s) for heuristicthe search were shown in ) the t

. ) (

Two Two nodA T and . b Supplementary

using Excel

50 In the and T

L MG546 calculated approaches were adopted for the

and ikelihood methodikelihood based on the . ed

Version 1.1.383 Supplementary Each amplification cycle MEGA7 c

first

described s proportion against time for were identified as the nod ation - optimal temperature range 103 to

referenceand species case it was (GLM)del D

is 2010 as as the inflection point of ) genomes) given are in

and the CandN analysis linked the to fromMG546080 (Ku .

Table Table

Data obtained from the MG546

in in (Microsoft) mar al., et 2016)

Garcia °C Table Table and Rversion x root noduleroot , as described in

regressed using a

2 for 15 for reaction buffer, . 113

- 1 sub etHuidobro ), ), and

(

. x lengths

nodD min Briefly, - - ( optimal axis axis to fitted

) .

This article isprotected rights by All copyright. reserved. Fig percentage of modelling (GLM) showed that there is a statistically significant relat 2A) green 20 green (Fig Trials determining Optimal germination temperature scarification effectof and notdid discriminatory power tha may beargued that averagethe green red, and blue colour content their red, Seed colour analysis confirmed the that brownof seeds cannot be attributed weightthe of the seed greenwith seeds demonstrating significantly greater circularity andhigher SA:V ratio significantl The green seed averages for length, width, area, surface area (SA) andvolume (V) wereall average fresh weight of characteri brown’ (Fig visually by apparentdifferences theirin relative size and colour, eitheras ‘smaller green’ or ‘larger Lathyrus linifolius Morphological characterisation and Results made from polynomial the model andthe position of the maximum was prediction selected. polynomial regression calcula polynomial relationship between t this approach was fitted to the temperature response functions Optimal temperature was calculated for both morphs as the intercept of su temperature ranges and T

Accepted° Article . .

C, whereas for the brown ure performed with 2A At 25

morpho morphotype te

) show any differences between the two

2) showed that seed germination varied with seed incubation temperature . d by using the following

green and blue integrated colour densities and averag Similarly, asignificant s

ation y less tha °

. t C

50 1) , type

seed germination (

seed the optimal g data plotted . .

This was confirmed by weigh Green seeds represented 31

showed signific showed seed batches percentage germination dropped 40 to in Excel and n

, those values recorded for brown seeds morph

the individual n c

(maximum temperature for germination) integrated density germination values formula for maximumthe regression point against ermination temperature lies onthe maximum identified point in the o

morphotype types of total germination total of non of .

included The The second

P = R, using a relationship was found between : 50

c antly higher green seeds the the

onsidering

only was still significantly different indicating that the greater weight against temperature 0.8574; F= 0.2546; R² = five visible

to theirto two seed heteromorphs which could carbon

the constant G

approach involves . %

eneralized

The assessment of

t, trend was similar with an exception 5 at morph

% between y being

germination (72 colour morphs be can discriminated onthe basis of

colour discrimination andgermination = ax

higher for bothfor

the the : nitrogen temperatures o 2 slightly less

seeds in the types + bxc

70 . moisture content L

T - inear morph – scarified scarified seeds across a temperature series he maximum the of regression was . 80

B ratio

e relative pixel number (Table 0.08356; 0.08356;

the usethe of a a rown seeds were M % o s generalthe equation of the

over a temperature range from 5to percentage of (analysis types than that of ixed ), than), brownthe type (41 seed batch (Table 1 per rbon (C) to to the

ionship between temperature and

is is pixel may more have reliable Model .

A generalised linear y – .

b and supra = b/2a

for for this polynomial regression

and N -

, GLMM the the larger brown seeds 0.9467 be distinguished .

- germination and seed supra optimal In R prediction were also . S

second approach content pecifically, the

x + more elongated, - . ), ), optimal ) °

. After drying, C

with According to

ranges , where 1.7676619 ,

however,

% the the 1 ; ) . Fig .

the It .

; .

This article This isprotected by copyright. All rights reserved. lentis all the rhizobia isolated form complex. Comparative phylogenetic characterisation using ecuadorense leguminosarum sequence data showed that of n BLAST a perform phylogenetic the analysis, sequences from nodD Root nodule bacteria were isolated from the catalyses atmospheric di immunogold with labelled an antibody against infected cells in the BNF zone contained apparently functional bac appeared pink (due to leghaemoglobin content), indicati meristem; linifolius include majoritythe of repeat Pisum L CharacterisationNodulation 1/3 of in each case green andbrown seeds morph significantly improved seed germination by 10or almost 17 When incubated at estimates for brownthe for optimalthe temperature for greenthe fitting to temperatures types (P greenfor Assessments 16.3 According the to cardinal temperature model morph odulating genera were excluded from analysis further .

Acceptedlinifolius Article

and 20.3 , R.

- genes were , o o lacking Vicia types type

< 0.001; R - full data bangladeshense nodules show the gainst those held in NCBI the nucleotide data base

(P (P invasion

nodules were indeterminate , R. using the GLM (

, < and of estimated

° respectively

monophyletic C 0.001; 0.001;

the the and one isolate grouped with the binae

for greenfor and brown colour set gave estimates of optimal germination of Lathyrus sequenced (Fig . ,

the 2

seed germinationseed rate (1/t

biological N fixing, andsenescent = , R. R² = , 0.6063; y = temperature for optimumgermination rate cropped

at at achieving 50 morphotype - nitrogen gas biologically to useful N forms , R. 14.1 lentis

(Table 1 genera 0.7534 typical clade binae L. P = the

and 13.5 , R. linifolius legume

0.8574; F= 0.2546; R² = 12 potentially nodulating isolates tested, 11 segregated with R.

(Schaefer et al., 2012) ; 1A) discernto their identity potential as ’ arrangement which from base tip to comprise a continuum of -

) and y = bangladeshense 0.0002x (IRLC) . varied between 13.5 and20.3 °C.

G %

ermination rate (t

, and this nodule type is R. species species ( in 16orin 9d faster

0.0003x

C morphotype segregated with

laguerreae

for the green andbrown morph 2 of of L + 0.0054x 50 morph the .

the ; Fig; the Fethe linifolius Sprent al. et 2017) 2 optimal germination R.

sub + 0.0085x 16S r . , R. o etli

2B) - types

- (nifH) protein of the nitrogenase enzyme varied betw and - . family Faboideae ( zones (Fig

, aegyptiacum - Our phylogenetic analysis of . generaThese also belong to ve R. 50 root nodules and R 0.0045 by , respectively R. ), varied), significantly

NA a , respectively (Table of activeof BNF, and TEM showed rhizobial - pisi nodA

0.08356,

strain from fitting % (Altschul et al., 1990) pisi -

16.1 0.0158 , R for greenfor and brown seed n

typical of the ) , and alongside odA

gene sequence (Fig. showed 4) that gave . . een 14.1 for each seed batch

teroids (Fig

phaseoli 3A andB) polynomial . and

Light

and y and

), and), brown temperature

optimal germination =

L. 17.5 representing atime reduction - R. n Papilionaceae) which 0.7772632 their ly nodulating

sativus micrographs of mature , odD and 16.3

, R. respectively sophoriradicis

°C. Therefore . genetically syntenous

At harvest

models 16S rRNA, esperanzae 3C) which were compared using 1 R. between ;

L. s model shown) not -

leguminosarum

coloured seed coloured and non

were identified at °C, whereas

( , s x +1.7676619 16S rRNA grass pea the the to to average data carification

. rhizobia

scarified ‘

Polynomial the the nodules inverse were

, R. nodA , species -

estimates ) , which .

. and

To To L , R.

). ).

This article isprotected rights by All copyright. reserved. 3 with species such someas woodlands, wemay speculate that where it is found. Therefore, and since the species occurs mainly in improved though There are very published few peer orderin combined with seed treatment may improve also seed vigour andseedling survival feasibility Davy, 1997) frequently used Chemical (sulphuric acid) and mechanical based presence physicalof dormancy these in seeds Also, t grow its agreement respectively the morphotypes, respectively was more suitable theto data points available here ( werethan used here. Th real trend. Thus two data points approach al.,et 1982) data analyses to estimate germination and scarification treatment generally improve temperature influencenot analysis and also by the differences in their fresh and The distinction in germination through pre study is designed to can which implies Fabaceae The presence Discussion

Accepted Article , 1/T50 curve fit,

therefore - he increased germination

James et James et al. (2011) or moreor fold less soil N

th to to improve establishment was found to be limited with the o , including

, of of the lower during the warm season

since the temperature

) . I identified covers a range from 1 % .

germination traits , of an impermeable seed coat is a Whichever method is used

n the developmentthe mechanisms of and/or structures that

trigger seed and germination rate, t other

( treatme , the presentthe st at

this method more is appropriate when abroader range of temperatures are

two colour

least versus characterise these aspects and develop an effective strategy to improve

Lathyrus seed Vicia - 16.2 price n

- one e alternative method of fitting acurve to the 1/T t on t sowing techniques optimal temperature

) pre , reportedhas that doesit not appear to nodulate well, even in environments 0.87

. H imbibition and species species ( for for d side of the intersection

Lathyrus - udy only the latter was applied morphs (green and brown) is confirmed through the image colour

; owever germi met g the existencethe of . and (at 0.02

percentage r F s a een . or bothor hod - 4.1 . This L. linifolius reviewed reports on nodulation and BNF fixation by

ll 0.97 for the cardinal nation V. lutea

and 20.3 – , indicate

for possible application in large 1 (Basaran et al., 2012; Justice and Marks, 1943; Walmsley and the the tofit the data waslower in terms R of – ) 6.3

reflect 0.10 %) regardless to modelto the temperature xgermination data they morph

germination processes techniques, assuch seed

. (green morph and faster germination after scarification ptimal regression for ,

V. salvatica . d s

a o . cardinalThe temperature approach

may bemore N dependant soil than other related wild seed s C germination performance a preference for th

o the the adaptation of

scarification (James et al., 2011) hard seed coat for greenfor and types ) of of 16.1 , characteristic

which are not statistically sufficient to define the dry weight colour morph

natural - and 17.5

o fit linesfit and type

n order assessto the economic V. sativa appear

)

(without treatments) optimal brown

to 1 to usually . e

: green: and brown morphotypes, seed .

both morph

However

o The approach used in the present of the L species to C for .

priming . 3.5 Scarification may be also

. On the other hand, the nodules linifolius Two methodsTwo wereused allow the passage water of that to be

- seed morphs, respectively. This

scale productionscale – ) which) typically occupy soils

involves 20 many 50 g

(in terms final of r

een v , .3 the mostthe effective and

ersus t and rhizobia inoculation his heteromorphism germinate and o °

grasslands and/or – types to temperate climates C genera in

and 2

(brown morph

physical dormancy, (0.68 (0.68 temperature data

(Garcia comprising brown confirms the . and L. linifolius the family This formThis of

are in seed - seed seed develop 0.64 applied

Huidobro to forto o only type

using does does , .

) ,

This article This isprotected by copyright. All rights reserved. the seedof priming, bacteria (rhizobia) inoculation and the interactions between the treatments to select the interaction adopted here required consumer In summary, by and the ( with 2017) vast majoritythe are thein genus bacteria of the rhizobia specificity (Atkinson et al., Rö1994; lipochitooligosaccharide rhizobial particular Cummings2005; 2009; al., Fonseca et et al., 2012; Gehlot et al., 2013; Mutch and Young, 2004) The sequences of both association in the improvement of nodulating bacteria is production enhancement inte Shcherbakova (Cassan e have been found performance and yield production Rhizobia inoculation systems, controlled and environments. abundance approach Nevertheless,protein. actual quantification BNF of using techniques such as the legheam reported here were clearly effec Fig. 4;

AcceptedPisum, Vicia Article

large scale production most effective L raction nodA . nodA

. This also proved to be the case with linifolius Young et al. - o - on germination traits and how germination and production can be improved host recognitionhost (Fisher and Long, 1992; Van Rhijn and Vanderleyden, 1995). , the “canonical” nod genes t al.,t 2009; de Souza al., 2016; et Kumar et al., Schlindwein 2016; et al., 2008; host specificityhost ( globin s phylogenies show ahigh levelof consistency andconfirm s s sequences for and

of of the are L is is

. et al.,et 2017; Sorty et al., 2016) Pisum

, end uses to characteriseto morphology,seed germination traits and fundamental soil microbial isolates linifolius

), ), generaltheir structure and the fact that bacteroidstheir contained nitrogenase and , especially when species therefore

one wild legume species,

, Lathrus

(Unkovich al., 2008) et , 2006 can be included in this context Vicia for for functional applications nodA aligning . .

(Adu et al.

R In order to fulfil the requirements of large scale production knowledge is has the potential to bedeve ,

Sprent et al. 2017 of ) . fundamental for the application of this novel methodology crop in .

leguminosarum and This This segregation was even stronger with the presentthe study, the a and L is is reflected in the pplied . linifolius tive, as indicated by pink their internal colouration (due to Lathyrus with with those of nodD Rhizobium .

L P

. 2014 to ositive effect

hrig et al., 1994) linifolius nodA

(mostly) crop species were using technique .

) - Further investigation cross

specific specific rhizobia strains were used for the treatment “nod factors” (NF),which determines rhizobial host BC,

the )

biovar

. used remain to be carried out for the species natural in (Drouin et al., 1996; James al., et 2011; Villadas et al., .

This This becauseis they are involved in biosynthesisthe of

The The identification of specific R

germination and production F diversity nodulating their of bacteria - L ha . inoculation group span awide range species, of but urther investigation on the effectiveness of this

. . leguminosarum

s ve

linifolius here

characterisation of apparently effective

on viciae

been identified majo as loped as an alternative crop severalwith potential , whereas seed to assessto rhizobial isolate diversit s , including the sequenced type strain 3841 that demonstrate ling rhizobial isolates to improveto seed germination

growth and t

are he

b iovar nodD

now

that that the genetic

viciae needed to nodD gene, is involved in

good potential for is neededis later

r determinantr s pecies which all

(Fig sequence data, wi

development stages 15 N natural . consider the effect – .

5 rhizobia . . synteny

The The approach

segregated .

Nodulating s The L. linifolius

(Chen et al., of

shared use use in nodD

. th

all

- This article isprotected rights by All copyright. reserved. AcceptedBrenchley, W.E. (1920) Beringer, J.E. (1974) R factor transfer in Beith, M. (1995) Healing threads. Traditional medicines of the highlands and islands. Edinburgh: Basaran, U.,Mut, H., Asci, O.O., Ayan,I., Acar,Z. Germination (2012) pattern of naturally grown Atkinson, E.M., Palcic, M.M., Hindsgaul, O., Long, S.R. (1994). Biosynthesis of Asmussen, C.andListon, A. (1998) Chloroplast DNA ArticleAltschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, (1990) Basic D.J. local alignment search Adu, M.O.,Chatot, A., Wiesel, L., Bennett, M.J., Broadley, M.R.,White, P.J., Dupuy, (2014) L.X. A References (RESAS), division a of InstituteHutton are financially supported by the Rural & Environment Science & Analytical Services Agreement Number 607785 Actions) of Europeanthe Union's Seventh Framework Programme FP7/2007 The research leading to these results has received funding from People the Programme Curie (Marie Acknowledgements Microbiology, Polygon 294pp.ISBN, Production, Lathyrus Proceedings of the National Academy Sciences, of lipooligosaccharide Nod factors: NodA is requi Lathyrus (Fabaceae). American Journal Botany, of tool. Journal of Molecular Biology, Brassica rap scanner system high for

and 6

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species differentto methods an ,

www.nasstec.eu – 335. 74866199 - resolution quantificationresolution of variation in root growth dynamics of - 198.

Rhizobium le (9).

215 Longmans, Green and Company.

. The RoyalThe Botanic Gardens Edinburgh andthe James (3), (3), 403

guminosarum characters, phylogeny, andclassification of red for an N – 410. d seedbeds. International Journal of Plant 85 91

(18), 8418 (3), 387

- . Journal of General acyltransferase activity. – 387. - 8422.

- 2013/ under2013/ REA Grant

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This article This isprotected by copyright. All rights reserved. Accepteddos Reis, F.B., Simon,Jr., M.F.,Gross, E.,Boddey, R.M., Elliott, G.N., Neto, N.E., Loureiro Md De Meyer, S.E., Van Hoorde, K., Vekeman, B.,Braeckman, Willems, T., A.(2011) de Souza, E.M.,Bassani, V.L., Sperotto, R.A., Granada, C.E. (2016) Inoculation new of rh ArticleCummings, S.P.,Gyaneshwar, P., Vinuesa, P.,Farruggia, F.T., Andrews, Humphry, M., D., Elliott, Crawley, M.J. (2012 Cook, F.E.M. (1995) Chen, W.M., James, E.K., Chou, She J.H., F.,Cassan, Perrig, D., Sgroy, Masciarelli, V., O., Penna, C.,Luna, V. (2009) Phytologist, nitrogen fixation by Bontemps, C.,Goi, S.R., Young, Sprent, J.P., J.I., James,E.K. (2010) Nodulation and de Queiroz, L.P., Scotti, M.R., Chen, W.M., Noren, A., Rubio, M.C., deFaria, S.M., BiologySoil andBiochemistry, 43(12), 2384 rhizobiaof associated with indigenous legumes different in regions Flanders of (Belgium). sativa isolates improve nutrient uptake growth and of bean ( Microbiology, by Moreira, F.M., Sprent, J.I., Young, G.N., Nelson,A.,Orr, C., Pettitt, D., Shah, G.R., Santos, S.R., Krishnan, H.B., Odee, D., Mimosa pigra L.). European SoilJournal of Biology, germinationseed and early seedling growth in corn ( Az39 and Rhizobium (Agrobacterium)Rhizobium ). Journal of the Science of Foodand Agriculture, Bradyrhizobium japonicum ) 186 Economic botany data collection standard The R book , a newly discovered invasive plant in Taiwan. New Phytologist, 11 (4), 934 (10), 2510(10), Mimosa spp –

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strain IRBG74strain andother rhizobia. Environmental . thein Cerrado andCaatinga biomes Brazil. of u, S.Y., Yang, S.Z., Sprent, J.I. (2005) β

J.P., J.P., James, E.K. (2009) Nodulation of E109, inoculated singly in or combination, promote 45 , 28 - – 2396.

35.

Zea mays Phaseolus vulgaris

96 Royal Botanic Gardens (Kew). (10), 3446

L.) and soybean ( Azospirillum brasilense - 3453. ) ) and arugula ( - Sesbania Rhizobia from

Genetic diversity 168 Glycine max New izobial , 661

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Oxford University Press. No. 15. aintain mountain firewalls Sierrain Nevada National 15

- Felix, MartinezFelix, J.D., , 1343 Bl ackwell Scientific Publications, -

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(2), 92(2), - – Molina, E., Molina, Toro, N., 101.

International ., Cronin, A., Davis,

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Oxford, Vicia

, This article This isprotected by copyright. All rights reserved. Accepted Article leguminosarum amplification of nodD from community DNA revealsthe genetic diversity of Genome biology, genome of Rhizobium leguminosarum

in in soil. Environmental 7 (4), R34.(4),

Zézé, L. A.,Mutch,

has has recognizable core a M icrobiology, A., Young, J.P.W. (2001)

3 (6), 363(6), nd accessory components. - 370.

Direct Rhizobium nitrogen Carbon and (t Germination rate Germination (%) discrimination Colour (fresh Physical qualities Moisture content (%) Dry (mgweight seed Fresh weight (mg seed Proportion of batch (%) Parameter This article isprotected rights by All copyright. reserved. significance test. content(N) are expressed in proportional and absolute terms. presented for optimalthe g presentedis for shape), surface area (SA; major and minor axis, respectively (mm), circularity coefficient (where, 1 relative humidity. Physical qualities of each seed type presentedare as length and width across seed dry weight ( average data (± SE) on the basis of their relative: proportion of total seed complement (%), fresh and Table 50

Accepted, d) Article

seeds) 1 .

Characterisation of the heteromorphic seeds of

mg seed

scarified and non

- 1 - 1 ) and ) moisture content (%) of fresh weight after equilibration 5 at C:N Ratio Nitrogen (mg seed % Nitrogen Carbon (mg seed % Carbon Scarified seeds Not scarified seeds Scarified seeds Not scarified seeds Blue Green Red Blue Green Red SA V : ratio V (mm SA (mm Cirularity Area (mm Width (mm) Length (mm)

) -

1 )

ermination temperature for morpheach type. Seed carbon (C) and nitrogen

3 2 ) ), volume ), (V; 2

)

2 -

) scarified seeds (%).

Avg mm density Integrated

- 1 - )

)

2 mm

3 ) and ) SA V : ratio. Germination eachfor morph type 0.896 0.002 ± 5.14 ± 0.13 2.44 ± 0.04 2.66 ± 0.03 8.45 ± 2.35 14.87 0.49 ± 16.10 0.21 ± 2.8931 ± Green Seed heteromorph (colour type) 11.24 1.64 ± 0.61 ± 0.06 4.16 ± 0.50 6.70 ± 0.26 45.09 0.61 ± 15.8 ± 1.8 22.8 ± 1.8 91.6 ± 1.67 81.6 ± 7.4 4 40 ± 3 88 ± 113 ± 2 207 ± 20 453 ± 17 583 ± 14 1.62 ± 0.07 8.46 ± 0.34 13.13 0.03 ± Germination andgermination rate (t Lathyrus linifolius

P value

, denotes values for statistical 0.891 0.001 ± 5.33 ± 0.12 2.49 ± 0.04 2.72 ± 0.02 7.59 ± 2.51 18.28 1.12 ± 19.62 0.77 ± 2.8969 ± Brown 12.85 4.46 ± 0.77 ± 0.16 4.26 ± 1.10 8.24 ± 0.57 45.04 0.43 ± 9.37 ± 0.94 14.6 ± 0.69 86.7 ± 4.7 70.0 ± 5.8 33 ± 3 66 ± 2 97 ± 178 ± 20 352 ± 15 523 ± 12 1.53 ± 0.06 8.93 ± 13.17 0.03 ±

= perfect circle, 0 elongated L.. Results present 4

0.31

o C and 15 % 50 ) ) are 0.003 0.013 0.042 0.017 0.815 0.050 0.012 <0.001 P value 0.752 0.463 0.936 0.0717 0.948 <0.001 <0.001 0.040 0.017 0.018

This article This isprotected by copyright. All rights reserved. against theFe bacteroids cell; inan the section infected has (b) immunogold been withanantibody labelled amyloplasts/starch (s). Bargrains = 20µm; D). The are int infectedcells infected (*) cells inthe N view and, ofthe invasion(iz) meristemN (m), senescent =Bar 200µm; (s). zones throughfrom theinvasion(iz) zone and the meristem tothe N (m) micrographa showing inlongitudinal development matureall stages of profile of nodule Figure3. modelling; thesigmoidal andB),shows models. coloured morphs,respectively, seed lines fittedgeneralisedwhere: using A)shows linear andcurves dashed o lines show B) ( Figure 2. respectively. = mm. divisions Scale small bar shown here either basisto onthis the partitioned right discriminated onthebasis Figure 1. LegendsFigure ■ ) seed morph Accepted Article seed germinationseed(1/t rate Micrographs of Micrographs of The heteromorphic seedsThe heteromorphic of The responsetoseedThe incubation temperatu - (

o nifH) protein of protein nifH) types showing: - fixing zone; thesefixing zone; are binpanel labelled withbacteroids packed linifolius L. Lathyrus of their different aseithergreen. colours, brown or They are 50 erspersed withuninfected are interstitialfull of cells which

). Vertical bars of showSE themeansreplicates. Solid of3 f models fitted to describe data for the greenf modelsfor todescribe fitted data the nitrogen A ) the percentage oftotalnon percentage ) the B ) higher ma Lathyrus linifolius Lathyrus ase (arrows). Bar(arrows). ase = 1µm. D ) transmission electron micrograph) transmission of

- fixing zones (*). Bar = 100 µm; fixingBarµm; = (*). zones 100 nodule sections, showing:

gnification showing light micrograph a re ofnon - , orleft

L. ( - - bitter vetch bitter - hand sides of thehand image, sides of scarified germinated seeds and scarified, green and (○) scarified, brown

- fixing (*) andfixing (*) the ) can) be A -

) light and brown C ) rhizobial ) rhizobial - This article isprotected rights by All copyright. reserved. Accepted Article containingpositions and missing gaps dataeliminated. were are each code. strain T bootstrap replicates) and linifolius Lathyrus Figure 5 contai drawn scale,to with branch lengths measured thein number substitutions of end of each strain code. T >50 % basis of their probable species identity disce shown linifolius Figure 4.

, drawn toscale,withbranchdrawn in the number lengths ofsubstitutions measured

B) ning gaps andmissing were data eliminated.

are for representative isolates only (not duplicates), believed to bepotentially nodulating on the (1000 bootstrap replicates)

nodD root root nodule isolates for . Molecular phylogenetic analysis Molecular phylogeneticanalysis the using

gene he t he s root nodule isolates for

( are shown inthe tree.are strains''T'' The shownbyat are a type the end of 512 512 ree he t positions in thefinal in dataset).positions was ree

is is the rooted with rooted are shown in the rooted with nodA

gene (442 positions in the final dataset). The sequences using the rned by 16S rRNA BLAST results : Azorhizobium caulinodans Azorhizobium A Azorhizobium caulinodans ) 16S rRNA in (255 positions ) 16S thefinal dataset)

tree. The type strains are shown''T'' by a the at maximum likelihood (ML) method of M aximum aximum Only bootstrap>50 values %

L ikelihood (ML) method of ikelihood

ORS 571

ORS 571 per . Only bootstrap values

site. All positions T . The . The tree is T per . The tree

site. All Lathyrus (1000 s

;

This article isprotected rights by All copyright. reserved. Accepted Article