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CO~I~IISSION III: SY~lPOSIA

Transactions

15th World Congress of Soil Science

15 Bodcnkundlicher \Velt.kongress 15cmc Congrcs \ tond i:J.l de b Science du Sol 15:: Congrcso \ lund i:J. I deb Cic nti:J. del Suelo

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---------------Azospirillum brasilense:·Root Colonization of Weeds and Crop

Plants, Inter-Root Movement-arid Survival in Soils and Rhizospbere ·

Yoav Bashan ... , Gina Holguin·, Nieves Rodriguez', M. Esther Puente., and Ronald Ferrera-Cerrato•. ·Department of Microbiology, Division of Experimental Biology, The Center for Biological Research (CIB), P.O. Box 128, 23000 La Paz. B.C.S .. Mexico. "Seccion de Microbiologia, Centro de Edafologia. Colegio de Postgraduados, 56230 Chapingo, Mex., Mexico. · - ·. · _· · - ~ · · -

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Abstract A=ospirillum brasileuse was studied for'its root cotoniz.aiion of64 plant species, inter-root motility in wheat and soybean,. survival in 18 soil types from two countries and su~val in the rhizosphere. A. brasileuse was capable of colonizing all tested plant species. The root-to-root motility was monitored in agar, sand and light-textured soil The study used a motile wild-type (mot) strain and a motility deficient strain (mot') which was derived from the same wild type strain. The colonization level of inoculated roots was similar for both Strains. Mot• cells moved from inoculated roots (in agar, sand or light-textured soil) to non-inoculatei:l roots where they formed a band-type colonization composed of bacterial aggregates, 're&ardJes$ of the· plant species. The mot' strain did not move towards non-inoculated roots of either plant speaes--and usually stayed at the inoculation site and root tips. The primary factor gov~ng motility of mot•. Cells was the effect of attractants and repellents. In the rhizosphere, A. brasilense populations · maintained a high level for prolonged periods. Survival in soil varied. In Israeli soils. its population declined rapidly within a month. In Me:ucan soils., its population maintained a level equal to the original inoculation level or only slightly decreased. Soil factors affecting survival were the amounts of calcium, nitrogen and sand. We propose that: (i) A. brasilt:nse is not a plant-specific bacteria. (u) bacterial motility within the plant root system and between neighboring plants is a prerequisite in the root-bacteria recognition mechanism. It is an active process and a consequence of a non-specific bacterial chemotaxis, influenced by the balance between attractants and possible repellents cxtn.Jded by the root, but not directly dependent on nutrient deficiency, and (iii) survival_ in the soil in the absence of plants is influenced by the amounts of calcium, nitrogen and sand in ihe soil.

Introduction. The beneficial rhizosphere bacterium A.:ospirillllm has the potential to increase plant grov.-th and productivity ( 18,29). When A:ospirillum colonizes roots., the bacteria can be found an}•where in the root system of sevetal plant species (24), but have an une.xplained preference for the root-tip, the elongation zone and the root-hair zone (19,27,37). In the field, cells originating from soil surface inoculation using various types of inoculants can be found esnbedding the entire root system and at least as deep as 50 em. Funhermore. in plantless soil. A::ospirillum can migrate ~rizontally as far as 30 em from the original inoculation site towards growing plants (13,21). Thus, self-motility is an important asset for this bacteria. A.:ospirillum cells are chemotactic. both towards various chemoattractants in vitro (32,42,48) and possibly during their travel through the soil towards plants (7,14). They are aerotactic (3) and redox-tactic (30) as well. In soil, bacterial motility mainly depends on the presence of plants (13).

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Root rips ue ltl c:fficic:t:t v('l;tOr for pu.si~·e \'tnid truufc:r of~. Df"QJ/m.s.r: u cc:-::p l.S ~ en from the ii'I()(;U!~tion site ( 17). Ho\"~c:r,'dispcnion &nd coloniz:uion of A::DSpi,/lum, from lhe ioa<:'UUted seed to the c:nrire root system of lhe pl..:!.nc lfld to adjlCc::ll pl..:!.nu. h3s yef to be c:xpb.ined. In p!andc::u soil. the b.3ct:rU ue rapidly Uld Stron;ly absorbed into lhe d.ly ltld org:nic fnc:ion of the soil, lfld its motiiiry is ememdy ~tncted.. evet in watc:r ~tur.ued soil (15,16). After a few weelu. the bactc:rU.I popul..ltion diminished in some types of soils (!1.~5). CWnu of A:a.sptrillum specilicicy for CC'Uin <:ae:U species ue documc::1tcd (for ~ew see !!). ~t eo.id~ce ho"'~er. showed ocha'wisc. A..~pmllum en colonize scvc:r.U pb.nt species of clilfc:rent bot~cl fltl\ilies (9,18,:0.:~). Tbe full host t'ltl!:)e ofA;osptrillum 1-w not yet been de:ined..

The~ ~~r chu scud~ -..;~ .(i) ~~ duci~~ ;he ·plltlc hosi-t'ltlse of A. hras&t~.s.r:, (ii) to C:xplo"re the major medwusm(s) irrvo!~ in the coloni.r:uion of the: c:ntin: root srstem from seed inoc:uluion. 1Dd bow roots ofa.dj~cc::1t, non-inO<:\I.Uted pl&nu JI'C coloN-~ (iii) to uses.s the surviv:U oflhc: bacteriA

.' in the: pb.nt's rhiz.osphcre Wi in soil ~ (iv) to dc:tc:nnine soil f:lctors which ~r.:ly ;Urea soil survi~ · ofthe b.Jctc:::U.. · -· _-. • ·:.: ~. · ...... _, · :_~. · .. _... ·---:· --· · : ·.~-: ~ ·.~·-.:.-: .

.. -:.. 1\t:arc:ri:au and mcthol.b. ~ .

· Org:anisms :and ;rowth c:omJitions. The folio--in!; bJc:::rill str:jns were used in this srudy· {i) in motility studies, A::u.,pmllum bra.ni~·Ju.t Cd (ATCC 29710, hi~hly motile str.l.in, motl and a non-motile: spont~nc:ous mut~nt (mof) derived from the TnS mutlnt of str.sin Cd. Compared to its parental st~n. the strJin 2971 G-1 Ob is app:&renlly defective only in N:·fix.ltion and asgre-::.-1ting

· abilities: Other c.".:!J'3cleristia were idc::1ticl to the plln:-nlaJ Str.siru (Cd) u dc.aibcd in detail ciscwhc:re (2:!.25). The identic! znrisCI"'ic d-.u:~cterisrics of mot' a.Jlowcd ta to u.sc zncibodic:s t:iscd ag:1im: st~n Cd (mot) (unpublished cbi:&) for ELISA de1c:rmin.:uion (de:scibed l~c:r). The: mo( mut10t W'U isoiJted by. (a) ~IUti"S iu in:lbiliry tO S'\lr.1lTl1 on solid mCcfium surface (J 1 ), an<f (b) !isht mic:-oscopic prQ:IrJiions of ~c:c:ri:& from the los:uirhmic p~ of growth. or obt.:Uncd from very youns colonies (~4 h). The chosen mul:&nt ~s comple1e!y non-motile durin!; scv~ hours u an:&Jyz.c:d by an iiT'~SC JN.Iysis system. (ii) in root eoloni::uion scecning U\d in sur.i..,a.J srudic:s in soils. in :&ddition to srr:&in Cd ""C used J.lso A. hrast/<(11.f< S?-2~ 5 (:!). Whc::u ?l.:tnts (Tr111cum a .. sri•'ftm) c:v. iii.:.a.J (""•ntet ""he.:u) ltld soybon planes (Giyr:u~ me:) cv Pdl4. we-e used u :c:sr $p«ies in motility srudies SC'Cds were surflce-<iisinieaed with I~ :'bOO for S min. then thoroushly wuhcd with sterile de-ionized water Seeds "'CI'e imb1bcd for 5 h in ste:1le tzp ~tcr prior to tr.uufc:r :o rhc ~~ srowth c.~:unbcrs (IS X :o em: cont.:Uning two plltlts o.c."t.. one in c:~c.": comp:1nment), u dcsc:nbcd dscwhere in de1.:Ul ( 17). Altcm:ue l~cn (~pprox 0.5 c:n) of semi-solid 0.1 ~~ ~;.Jr UM1 rhe ori!)i!UJ soft lSJ.f (0 5~~) were used. The CoMruC:ion of the c.'umber was done: on ice to permit quick solidilic:~tion of the soft o:~slr !lyer on the cop of:hc: semi-solid !:tye". thc-c:by .:~void ins mixins bctw~ the lzycn. Our c:.'umbc:r \.lq,l modi lied from the: p~Jn of the: orisirW growth cl-.;unber by (i) re;lilcc:mc:m of the cc:t:trJI solid ;Jlnuion with l pmition tTUc!c of ~r:e :nc:taJ net wruc!'\ ~lowed the: b;ac:c:fiJ tO mo"e f:c:-::!~· ..nthm the two compJrlmc::'ltS of the: srowth .:~!:>cr .

but P~'C":''tcd :he roots from mec::ins. Uld (•i) siloS.$ '-C":''IIUltOn tubes to incn:.:.sc o.'~Sc::'! c!if::~on in the c:.'un-.ber ltld i)rc:vcnt lerotJ.."tiS of A. bf'C.J1h·tt.~

8Jc:cri;U s:r.sins we~ S"'"'n in nutn~t broth (D•i'c:o) prc:;::~red for p!.:~nt inoc.;IJtJon J.t a cor:c.c:~:r.uion of 10'" c:U/mJ l.S preo.-iousJ~ c!esc:~bd (6. !!) J.'1d inocu~Jted onto se:ds l.S .:!c:s...--:1be: eiSC\oWhc:re (17). In some: soilltld rtlizosphc::-c: s.ur.;~·al studi1:.1. .1 hishcr lc:-vd of!)ac:c:n3 1.7'hl0' and

1. .&.J -.:1 o• .:fu.'s so•l Ius llso ~ used -

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The inoc-.Jl.lted s.eec.!s ~C":"e pbced in the c.!umbc:r 1-2 cU)'1 be:ore the !'lOn-inocubted onc::s.. In most cxpcrimcr.u.. lon!>er inc.Jb~ion of incx:u.Wed scailinss c:re:uc:s lon!)C'f' roou comp:1ted to aon-<noa.:U.u:d s.eedlinss. It is :Usa known ~ the root tip md ldj:tcent short root-luit reo:;ion.. the pn:fctTc:d c:oloni..ction sites of A;,oJpmllum, grow fi.Sler ttwl the ~c::i:t c::.n move in scmi-51Jiid ~u and Wid (38). By su~c:rin~ planwion.. -= improved the odds of the baaC!U comple1ing the rnigr.uion from inocul..uc:d roots to non-inocub.ted roots. The cuin:: c::tperimc:nt luted. u most. 5 cbys. or unlil the roots rcc.'led t.be bottom of the srowth ciwnber. Older planu produced a mass of sccondJ.ty roou which complic:ued the llU.lysis. The pb.nted growth ch3mbcn were ~vc:d to an c:nvironmcr.L1lly con1.r0Ued. !use:- growtb d-.:1mber for t.be dur.uion of t.be c:"tpc:rimc:nt. Si.aUbt etpcrimc:nu I.Wns qu.1ru: Wid or tight-tc:"tn.u-cd 51Jil wc:re crried out in sim.illr, but Larsc:r. growth dwnbcn (20 X JO c::n) maae ofple:ugl.:t.u. ·. :-.•. - • Root coloni..ction srudic:s were done in plmts gro.....;ns in 500 ml bl.l.clc pl.utic pots cont:tining 2 ·soil cnixrun::· of pet: ~culitc:: sand (1: 1:1. v/v).. Crop plant seeds were disinfec1ed as dc:scnbcd lbove before inoc-.JUtion, but weed seds or ~roduc:ive Or:;Jns were inoculated .....;thout di.sinfc:".ion

·. bc:c::lwe of a Lld: of <Uu on these prop3gules' n::sponse to the disinfec:"..:lnt. The pl:tnts were grown for 30-;5 <Uys ~C" inoc-..:btion. Then, root popui.:won was mosu~ .lS described later. • . ~-: ·. > ... Survi~ of b.lc::eria in stc::ili.zed soils was done in a controUed growth ciulnbcr u .. J9:'C in ?~ sc.Jed !50 mJ pl.utic pots. The number of swvivins bac:eri:l was determined pcriodic::1Jly for. l~ <Uys ~the pbte count method on OAB N-fre:: ~c:dium (11). · .· · · · •·

. ·. Sand .and soil. rn motiliry studies. pure qu:LtU sa.nd wu used .u growth medium. To inae:ue tr.c: low w-.atcr tield..::1p:zciry of the sand(~/. vlv), very fine vcn:ticliite was incorpor.lted ituo the sand whic.1 inc:re.ued :he w:ltC'f' ticld-<::zplciry of the newly-formed :ni:c:::re tO .:o-1 •. The s;~nd wu stc:rilizcd in :tn ovc:n.:zt 180'"CforlOhpriortothe~pc:rimc:nu. ; . _·.. . . . : . . : . . . · ~ .. : . : .. Ia coloni:.:tion studies we ~ lisht-~cxturcd. s:&ndy-lo.un soil stenliz.r:d by ryndcli:::Won .....;th a wuer 5dd-ap:zciry of!.6~. (v/v), ors~c;; manc:r a :mtc:nt of I.J~. Utd cl:ay content of ·UY... . .... ; ~ ·, In srudies on SUM~ in the soil. ""C used the follo.....;ns 5oOiU: from lsnd: Tern R.os:s:a ·soils (R.':odoxdfs). Mc:!it~c.., brown forest 51Jils (F.:~plo~C":"olls); Re:ld.:in.1 soils of mowu.:Lins (Rcndolls). Brown bu.l.ltic soil$ (Xcrott.'letU}; Brown-red s.andy 5o0ils (H:aploxenJfs); Brown ~I.IYW soils • ~..sols (Chromoxc:rens); Alluvial soils (Xcrolluvc:nts); Brown Stej)pe soils (D.IcixC"Oris); R.c::-.6:-.a soils of vall~ (C:alciott.l.,ids); H.unnud:t 51Jils of mountlins (Gypsiorthids); Brown d.escrt sl.:det:al soils (Tonionhc:nu); Loess ~w soils {C.unbortl-~ds); Utd t..oc:s.sW sandy soils (Toni;:~cnts) ( J I). From Baja CJlifomiJ.. ~1c:Uc:o: El CJtriz.:U soils :tnd LDs P!:tr.es soils md from ~tlinb.nd ~k..Uco: Soil &om Cordoba in the sute of Ver.xc:·u.L :tnd two 51Jils.. ~ontecifio and TcquCSGui!Uhuac !Tom the SUle of~1c:Uco. ·

o~tc-<tion 2nd quantilic:uion or bacteria oa I"'O[S. [n motility studies. :n.Jjor ~c:crUJ c.oloniz::t.ion sitc:s on :he mot surf.1c:e wC'f'e visibly dc:eced (dnwn Jnd photogn;:hed) by the Tc:er.u::llium chlorice ('!IC}<c:ducing method of PauiGuin :tr.d Oobc:bncr (39) u modified by B:LS."ww and ~J.."lony ( 17). Light mic.""OSGOpic observ:~tions of i)ink ::ones sho ... •ed ftW.si,·e :asgre;:ue colo~rion whc:rc:u :-.on-pink zones sho...,C'd ne::.riy no aggresltc:s but only sinsJe c.e!ls in the root vicinicy (d.lu not sho'"'-rt. 21so in 17). B~ctcril ...,ere identified Jnd counted by the indireoc:t-EI..ISA method (J6) or combined .....;th the l.imucd E."lrichmc::t ~tetllod (::.J) whe!'l the number of bactcnl wu smaller :!-.:tn 10" c.~'~j (the !o"'er limit of our ELISA mc-.... .od) The lJt::r :ne:hod was .lmc::::dd .l.S foll.cW1· .lfter rr..a:k i!'lS :!:e ;:inl.. bar.c sites on :r---.spuent tl"lCi!'lS ;:l~C'f'. :!tc: roots "-cr: ~,pe!'lt:ally CJI inside the g:-o,..,!: .:.':~~e~ usin!:: J ~om: · rr.JCc: J;:;:ln.'U.s m.::cc: oi :l.lr:-:e·ste~ !i .:~ :o stamlc:ss-ssed ~o~

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spaced 10 mm apart on a plastic holder. Then. each root segment was picked up with flame-sterilized tweezers and transferred onto 1 ml OAB medium (11) in tissue culture plates. The rest of the Iimited-enriehment method was unchanged. · · _ Data for bacterial counts were collected from: (i) the last root segment containing the root tip and the elongation zone (as described by Levanony and Basban, (35)], (u) the root segment at a distance of 5-.6 em from the root tip, (w) the segment adjacent to the seed. (iv) the total of migrating bacteria in the non-inoculated roots, and (v) the total number of bacteria in inoculated root. The total number of bacteria per root was given in cases where the entire root was visibly covered with bacteria (a pink root). · .. ~ . __ .. - ·

The bacteria present in •artificial root• preparations made of sodium alginate beads (described later) were counted by dissolving the beads in 0.2 M potassium phosphate buffer (5), and were counted by conventional plate count method on OAB N-free medium ( ll ). Strict aseptic conditions were used in this study, although it is vinually impossible to obtain sterile assemblies, even with disinfected seeds. Nevertheless, the level of bacterial contamination "Was 'very low as verified by routine total bacterial counts of slightly sonicated roots (25 W, :t min) ·on nutrient agar plates. Fungal contamination was absent when evaluated on potato-deXtrose. agar plates and by stereoscopic microscopy of every colony that developed on the nutrient aga( plates. ~ :r:n·:: = • • ·•

In colo1'ization studie;i of weeds and crop plantS, bacteria were countas follows: The entire plant was removed from the pot and all loose ~sOil mixture• paiticles were shaken out. The roots were slightly rinsed in sterile de-ionized water, and· the bacteria were "counted either by indirect-ELISA or by the Limited Enrichment Method as descn"bed earlier. In soil survival studies, bacteria were counted by conventional plate count method on Nutrient Agar; ·r·~ '!..> . · ,_

• . - • . ••• -:. ·~·~( - . ~ • l~~~~~~ ~~~ft:?~ Chemoattractants and repellents. We used the chem~attractants glycine (10 mM), aspartic acid (10 mM), sodium malate (100 mM);-sOdiuin ... suCCinate (100 mM) {42,49), and the repellents p-nitrophenylglycerol (0.006%) or NaEDTA. '(O.l7%)(31), all of analytical grades. These chemicals were_ embedded in alginat~:beads or applied ~i-ectly to the roots as described later .

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Alginate bead chains as' .;~rtificial root" (AR). Chemicals were entrapped in solid, sterile, 3-4 mm diameter alginate beads, produced as described earlier (S). Using a hypodermic needle, the beads were aseptically and carefully threaded on a very fine nylon thread in strings of up to 15 em long. These Strings of beads, or •aruficial roots• (AR) simulated non-inoculated roots (beads with chemoattractams or repellents) and were embedded in the same agar compartments as plants. Chemotaxis of A. brasile11se towards sodium alginate was tested by the classic capillary test for bacteria in general (1) and the open channel chemotaXis system developed for A:ospirillum (3). It was found ~t alginate is not a chemoatt~c:t.a:nt for A. hraiilenM .

• · •• • - . .•• · "~ ... '1l" ... :"' · ·- -..:.-r: ... :. ' · .... :-. :,: Survival nue calculations related t~ s~il Parameters. This was calculated as follows: surviving cells in each soil were counted periodically at 7 day intervals: for up to 35-45 days as descn'bed above. This data was analyzed using Linear Regression Analysis (LRA) vs. time. This analysis produced a linear survival rate formula (y--ox+b) for each soil. Since the bacteria died with time in most soils, the linear regression coefficients (a's) had miws. values. These linear regression c:oefticients, from all soils, were analyzed again by LRA vs. a single soil paramet~ch time (such as nitrogen. pH etc.) which was measured separately in all soils.

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upcriment:ll o.J~ign .1no.J Sl:ltiSiic:~l .:1n:al~ i is A!l .:.' ;:c:'lmc:ms "'ere c:~r:-:C'\! .:>ut tn tn~ii.: .: c .: (one

gro""th ch.ltr.bcr or .1 pot .u l rc:~lic:ue) . .1nd c:.:c~ .... ,u rc;:e:~ted :.~ cimc:$ Sis:1iiiun,;c: is :;iH·n by StV~d..U'd E.'Tor (SE) of oc!\ column. Blctc:~l.l CQunts were tll..cn from chc: l\ef'll)C: of root se<tioru

&em 3 <fif!"c:rent s.eedlin!::-1- The whC.lt root system of SCC'dlin>S consi>.lcd of~ m:~in roots. the so~·bon

root systc::n oi youn:; sec:d.lin!,;.S c:onsisu:d of .1 sin~lc: root. l.inc:lt c:orrdJtioru were tc::.tetl for siylific::lnCc: lt 5'Y. conndc:ncc:.

Resulu A. bru.silcn.sc stnins Cl.l ~nd Sp-!.SS: M)()t colon~tion or w~s .:1nd crop pl:mu rrnm the ume botanic! f;~muic:s.. Root c:oloni.z.ltion by two stniru of A. bf'Wll.:"~ (Cd ll\d Sp-!.SS) in s.ce,jlin>S of6-' plant species bclon!;;in!;; to 19 diffe:-e:1t botll\iol flmilic:s re-.·alc:d thJt the blceri:l is c::p:ble of

c:clonizins ~ pLlnt species in sisnific:uu numbcn (T~le I). Different levds of c:oloni.z.ltion were d.ctcctcd between the f:milies.. bcin); hi!)hest in ~tl.I~CC.lC. F~:1coe Uld Sol.uucoe :1nd lowest in Brusiccoe .1.11d Api.1cac:. However, both stniru "'ere op.lh!e of coklni.zin~ weeds ll\d crop piV~u belonging to the =ne bounicl f:t.mi!y at simi!.u extent. In scnenl. smin Sp-!45 wu a slishtly better

r• coloniz:::r th:l.n stnin Cd (T~Ic: !). :. . . · - .·... ·

Movement of ..L hrusilcn.sc mot" .1nd ..L bi'TUilcn.sc mot' from inot:ubtrt.l M)()U to non-inocul:ucd rO<>U .or the ume pbnt species :mu be.rween differtnt species. In .1~..r tl":lyS. A. brczsil.ttm: mot'

misnted from inoculltc:d roots to the non-inoaJlated roots of the Jdjac:ent plll\t. irrespective of the pl.lnt species. Coloni:ction of the non-inocul.ltcd roots w.u banc!-like: i.e... most bacteria were

f ecncc:unted in c!dinc:d .::ones "'hile few "'ere c!et~ed in the rest of the root ( Fis I A. C). Roots tips

I. and sced·sites w~ l.lways coloni~etl by A. br:uile:uc: mot· (Fis I A.C). . . . fro. h A different p:mern wu de:eced m the A. brasilc:t4V: mot' mutant. &cen.:~ d1d not mov:: m t c:

iftoaJWed roots to the non-ii'IOC'.L!atcd roots. re-,;lrc!lc:s.s the species of the donor (inoculated} pl.lnt (Fig 1 E. G). The patte:n o( c.oloniz:uion in the: sc::d-inocul.lted whc:u· roots ~ ~.so di!Tere:~t. u

l. bac:c:ri.:~ were: conce:ltl":ltcd either ncr the inocul.:~ted seed or in the: root tip (Fis I E·H). In both plant

S{)«ie:s.. the tot~ levd of ec!oniution in the orisirully inocu13ted roots by the mot" and the mof sr~ru was hish .lnd simi!.u (Fis I B.D. F.H). · In ~ ll\d lisht·tC:."!. turcd soil. A. brastf.:,s< mot' moved bct...,een the roots of the ~e species or different species.. similu to its p.:~ttem in ·as:~r. No movement of A. brcstlm~ mot' from inoaJI:ucd to

non-illOC\;IJted roots was de:ec:ed. In m.1ny c::ues. the: inoc..Jiltc:d b.lc:eri.:~ rer:uincd .1t the 5Ced inocullt ion site ll\d did not misr.tte ... ~th the root tips. The b:~c:eril.l populJtion levds of roots in ~

or soil "'CfC: slmi!u to those in as~ (dJtJ not shown).

~1ovem~:nt of ..L brruilt:.r~u mot" aad tl bnuil~n.u mof from inocul:ueti roou towards

cht:mo:attncunu and ~pt:llenu .. When "mific:UJ root" (AR) c:ontajnins the c:.'lc:moJtt :-:lC.lnt

slycim: t'l:?l.lccd non-inocui.ltcd root Uld the: other complnment lu.d in<XUI;ued soybc:ln roots. A. bras,fmSI: mot" c:clls mi;nted tow.uds the: AR. The: s;~me wu :rue: when AR c:ont.Uning m.l.!.1tc: wu pl.1ced ..,;th inoc-..Jbted whet roots (dltJ not sho,.,-n). When the A. bras1l~tuc mor "'.u usc-d. no misr:uion tov..u::!s these .:~t:l":lc:t:lnts was obscr.e:! in SO)bc.:ln or :he m.:~.Lue-whot combinJtions (c!.lt.l not sho,.,n)

\\"hen the .ltt~C:ll\tS (,!;l~ci:~e.. maiJte. .uplrtrc Jcid lr.d suc:imc lcid) were placed on restric:ed .::ones

of t!:e · .-\r::fic.l.l root" (~'"S in Fis Z A·D). A. !Jru.stl.·m< mot' INSJ;~ted from the inoculltcd roots

alr::o~ to !he .lllr.lCUlt sites. Similu ~=Its wer:: obt.:Uned in t!:e combm:~t ioru 3fycine-soybo.1

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T:.&ble 1. Root coloniz:ttion of weeds and crop planu from the same botanical families by A. brasilen.s,e strains Cd and Sp-245. · . · . .. . . .

·. Weed/crop plant Botanical family cfu/g (d.wt) roots

Cd.

Annuals (winter weeds) .. · ;.:. . . . . .. ... . .

/saris alepplca sa>p. : : : <.··i: -"7:~::.:. ~·l.2f(;_7xl<Y Sinapsis ~oensis L. B~~~ ·: \?_·." S. Jto.8xl <Y .Erucaria myagroides (I...)Hal. : : ;::...:,: _..;;.·:: ,2.4±().5xl01

: · Brassica nigra (L.) Koch. ·' · ·:.::;;: :~~~:~·:J:;:-.~4.7,.ti).9xl_<Y

- ~o;o~: syri~~:~.) ~- ;. )/~:~·;;·~-~~~~-;{1~X7xlct Scolymus maculatus L. . . ;:-1·· .• · : · 3.8±().lxlct ChrisanJh_i!nu~_iegeium L. . . . · · ~ · 7.2±().6xl ct ChrisanthemumcoronariumL. : .· . :.-·:~ . :~-_: - 9.1±().6xl0~ ·Amhemis pS!tJdoctula Boiss. Astericeae ··. ·'.;-. · :~2.9±<>.Sxl ct Amhemis mtla11olepis Boiss. ;.:~ ... :..:.: ;-~.:.?"- _": ·6.3+0.8xlct 0 · · (L) DC · ·_,.:, ~;-!..'.:~~·r:.:·~s-+O ·lxl,.. . rmems miXla . . . : . . -· - · -:·- ~ . .:-r.,.: ,. J. _ . v Cichorium pumilum Jacq. · · · ·~· .rf~; ~:~'?:4=':'!-:~.3:s:!(>.3xl ct prr~ ttm1is (B_oiss)Bomm. { ;. :-1~}:t:.2,ti.s±l.2xl0''

7• ~Senecio Vf_llgaris L. - ·:;;.~~ · ·· · ..... r~'i~~';::·: -:-7_-:_;~2;4:!(>.Sxlct ~lltcio vemalisL. ~-· ·• · ·: ·:- ·-: ' --;; - - ~·· ··:· -~2.8~.7xiO'' .--Ammi vig,Qgd-(i..)Lam. Dm1cus aureus Desf.

.Ridolphia seget11m (L.)Moris.

Srel/aria media (L.)Vill. Silene gallica L.

4 . : .. .. "' -·

Beta Vulgaris L.

l.al-arera trim~stris L. Mal\-a nicaeensis All.

Urtica ure11S L. Urtica pi/ulifera L.

Phalaris paradoxa L. Phalaris brachystachys A\oe'i10 st~rilis L

Rammculus an-ensis L

... . -Apiaceae.

· ~

. - : ·· .-~.-~:~xi<Y 4.1±().4xlct S.7:!(>.9xlct -... ·

7.s±t.3xtct Caryophyllaceae . . 4.8±().6x I 0" . # ~.: ; · :· .~ · ... : ·~

Chenopodi~ -~-~ ·2..J±o.Sx10S Malvacea~ ·. _:_.:~. ~~~-~xiO'

.. · S.S±1.3xl~

Urticaceae

Poaceae

Ranunculaceae

18

2.8±(). 7~ l ()" . 5.8±l.hlct

6.2±().7xi0S 4.9±().Sxl0S 3.8±().9xl<1

6.3;!:1.4xl03

Sp-245

4·.6±Q.4xio' · 7.2±().6xl01

. 3.3±().7x19' ··6.6±Q.4xl0'

5.1±().3x.to• 4.7±().8xl04

1.4.:t().4x 1 OS 'l.l±().lxiO' ~.2±().7xl04

6. 7.:t().Sxl0" ': 82±().6xlct

S.6±Q.Sxlct : .. -~2±().8xl0S

2.6;t0.7x I ct 3.1±().7xlct

1.2±().3xlct 6. 7±1.4x I 04

8.8;!:1.6xl04

9.6;!:1.7xl0" 7.7±().6xl0"

2.4±().7xl0S

5.1±().4xl06

5.8±().3xl06

3.5~.6xl0" 7.5~.4xl0

4

7.5;tl.2xlct 7.3;!:1.4xiOS 4.7±().6xl0S

9.1±().7xlO'

(Continue Taulc 1.)

Weed/crop plant l3otanical family cfu/g (d.wt) roots

Lupinus hirsulus L. Medicogo ciliaris (L.)Krock. Fabaccae Vicia 1'11/gare L.

Papa1·~·r rlweas L. Papavcraceae

Cd.

4.4~0.4xl07

2.1±0.7xl07

8.9:!;0.8x I 06

Perennials (win let· weeds)

Gladiolu.\ sc:gt:lllm Gawl. lridaceae Cynara ,,yriaca Boiss. Compositae Geranium tuhcro.\'11111 L. Gcraniaccac Polygomm1 equisctifomw S.ei.S. Polygonaccae

6.8±0.7xl04

4.8±1.2x104

3.6:!;0.6x 1 o• 4.3±0.6xl04

Annuals (summer weeds)

Svlamnn 1'illo.\'lllll (L. )Lam. 2.8::!:0.6xl06

Datura ,\'/r{(IIIO//ill/11 L. Solanaceae 3.4::!:0.2xl06

S.:tana I'Crtictllala (L.)13.P. Poaccac 4.B:_0.4x 10~

Amaraulhus rclrojlc:xus L. Amaranthaccac 6.7:!;0.8xl0s A marantlws J.:rocci:ans L. 5. I ::!:0.6x I os

Chenopodium OJIII!ijfJiium Schrad. Chenopodiacc:~c :l.7'!_0.2xl04

l'c•·cnni:tl (summer wcccls)

Cynodou dectylon (L. )Pcrs. 5.3::!:0.4x I 01

Sorghum lwlepcnse (L. )Pcrs. Poaccac 6.3;:!:0.8xl0s

Comwhw/us OI'I'CIISis L. Convulvulaccae 5.5;:!:0.6x I 04

J~'cbalium claterium (L.)Rich. Cucurbitaccac 6.8::!: 1.2x I o•

Prosopis jorcota Eig Fabaceac 3.2;:!:0.5xl06

19

Sp-245

4.8:!;0.6x I 07

2.8±0.8x I 07

1.8:t0.5x107

6.1±0.5xl04

8.6;:!:0. 7x 1 o• 5.2±0.2x I 04

6.8;:!:0.8x I 04

7.7;:!:0.5xl04

4.7,:t0.5xl0b 5.3;:!:0. 7x I 06

6.2:!:0. 7x I os

6.9:t0.6xl0s 6.3,:tl.lx10~

5.7::!:0.7xl04

5.9;:!:0.8x 1 os 7.1:!;1.2x !Os

6.9;:!:1.3xl04

9.7;:!:1.4xl04

5.9::!:l.Ix 1 o~

(Continue Tnble 1.) ·------------·--------------------··------·--------·----............. _____ .,..,. _____________ .. __________ .., _____ .. ______ .,. __ Weed/crop pl:mt Botanicnl f:unily cfu/g (d wt) roots

Cd. Sp-245 -------· ........ ----------- ... ------........... .. -------...... ------......... -------------------------.. - ....... --------................ ---__. ______ _

\VItc;tl

l!atley Oat Sorghum Corn

.Tomat{) pepper Eggplant

Cucumber Melon Water melon

Canola

Cotton

carrot

Sugar beet

Soybean

l'oaccnc

Solanncc:Jc

Cucurhit;~ccae

Brassicaccae

1\piacl.':le

Crop plants

(,<J t CJ.Kx I()'

·1 .2_1_0.5x I o' 3.7:tO.JxiO' 5.6!0.5xl0' 2.1±0.5x I 07

3.6±0.6x I 0,.' 2.7;!:0.2xi0G

G 3.4:!_:l.lxl0

5.2:!_:0.4x I o• 3.4±0.5xl0• 68:t0.6xl01

7.6±0.4xl01

8.8.±0. 7x I O'

Chcnopodiaccae 6.5±1 .2.-.:10•

Fabaceac

:1 .2j_ () r,xJ(J1

(>.7± 1.2x I()~ 4.6:t0.6xl0s 8.8:t0.6xl05

2.8±0.2xl0'

4.6.±0.5x I 0'' 3. 1!0.8x l06

5. 1±0.6xl06

5.6.±0. 7x 1 o~ 4.2:t0.9x I o~ 9.8.±0.6x I 01

I. 1.±0.2x I o•

7.3± 1.3x I 06

7.2±0.6xlo•

5.3±0.6x I 07

I ----------------------------------------------------------------------------------------------------------------l Plant's nomenclature nccording 10 Cohen (26) and Zoh:~ry (50).

(Fig 2 !\), malate-soybean (Fig 2 11), malate-wheat (Fig ::! 0), or when combinations of three ' att ract:u·us (aspart: •t c, succinate and 111alatc) were u~ccl in tltc 1\R placet! wit h inoculated soybean root 1 (Fig 2 C).

When "artificial roots" loaded with the repellents p-nitrophenylglycerol or NaEOTA were placed in the opositc compartmetll of inoculated soybean roots, A. hmsi/1.'11.1'1! mot ' did not migrate towards the AR. Non-inoculntcd soybean roots, when loaded with the same repellents, also repeHed A. bmsilense mof along the root. except at the root tip zone (data not shown).

20

..

+ _, 0

:::ii

0

-100

1-100

-100

-200

inoc .

non-inoc.inoculo.lad

G

noo-inoc. inoc.

r-------------~ 7 B _,

c:: Cl.l

non-inoc. s E

0 Total mlgral. bactorla

·~ Tolal bacl.

ln lnoc. plo.nte E:a Root lip

I I 50-00mm

E.J Seed

non-inoc. -----;;.-

non-inoc.

D

F

j

4

2

3

tJO Cl.l ,, _, 0 0 ... 1-0 _, 0 0 J...

/

..... 0

0 z

b/J 0 ~

Fig 1. Movement of A. brasileme mot + and mor from inoculated roots to non-inoculated roots of different plant species in soft agar. A. bra~ilcnse mot • (A-D); A. brasilense mor (E-1-I). A and E- Movement from inoculated wheat roots to non-inoculated soybean roots. C and G - Movement from inoculated sovbean roots to non-inoculated wheat roots. B. D, F and 1-I - Number of bacteria on different zones of the root. Bars represent SE, and 0 mm represent seed site in the growth chamber. In Figs F and H, the 3 empty columns on the left, represent absence of bacteria from the respective root parLc; of inoculated roots. (Modified data after Appl. Environ. Microbiol.).

21

AR Natural 0

A

-100

non-inoc.

Soybean

succ.l acid

Soybean ,.q -200

non-inoc. soybean

~ 0 D E f±83 Colonized AR

OJD Non-colonized AR

<!)

Q

E3 Natural root

5 -100

non-inoc. whcnt inocula Le d

-200

Fig 2. Movement or A. brrrsilt.:nse mot" from inoculated roots towards chcrnoattractants located in restricted zones in AR (arrows). !Z"2J -hc:avily colonized root sections. nars represent SE, and 0 mm represent:; seed sitc::s in th~.: grClwth chamber. Drawn circles are only schematic and do not represent the actual si7.e of the heads. c;'vlodilicd clara after Appl. Environ. Microbiol.)

:!2

-0

0 z

T:1hlc 2. Surviv;ll (I~ days after inoculation) uf A. hru.,ilc!IISL' !>trains Cd and Sp-2~:-1 in the ~nil :wd rhizosphrrr of wh,·at (l\ lcx.) and tumato (lsr. :1nd BCS) plants in I H t:rJI('S or soils from divcr~r nrigins.

--- .... -........... -- ~ ............. ------------.. ---............ ---------------------... ------------ .. -----.. --........... -.. ---· ........... --- .. -........ ---------- -Soil name ami oritin

Terra Rossa (lsr.) Mediterranean brown

forest (lsr.) Rcndzina soils

or mot 1111ains (I sr.) Brown b<Jsahi~: (lsr.) Orown-rcd s<1ndy (lsr.) 13rown ;dluvinl

so il s-v~~rtisols (lsr.) Alluvi:d (lsr.) Rrown steppe ( lsr.) Rcnd;.ina soils

of valleys (lsr.) I lammada soils

of mountains (lsr.) m·own desert

skeletal (I sr.) Loess raw (Jsr.) Locssial s:n1dy (lsr.) Ycracru4 (1\kx.) Montecillo (Mex.) Tcquesqninahuac (Mcx.) Los Planes (BC:S) E1 Carrizal (BCSJ

I I

lnm:ulation level clit/ml

Rhizosphcre Soil

l:dO" 1:.;10''

4.2xl0"

1.77xl07 1.77xl07

Sut viv;li (cf'u/l'.J

Rhi:....osphcrc

4.2xl07

6.4x 107

2. 1xl07

R.4x10" 6 4x 10"

7.Sx 10'' 4.2x I 0' 2. 1xl0to

5.7xl07

4.4xl0~

3 8x I of· 6.6x 107

1.2x I 07

5.7x l0(' 5.9xl07

2.9x I 07

I .7x 107

I .Jx I (J7

Abbreviations: Isr.= Israel; Mex.= mainland Mexico; BCS = Oaja California Sur, Mexico. Means (n=1).

Soil

J.4 x 10'

3 3x I 0"

~Ux ! O'' 2 2x I 0~ 40

\ .1>..10' 2.3xl0' <>.6x I o=

4.4x l04

0

4 4.6xl01

4.5xl03

6.Sx l07

1.9xl09

2.1x10" 1. 1 X 10" 1.2x I o'·

Sur\'i\';11 of ,.1, hrasilcm>c in the rhi:r.osphr•·c nnd in soils rt·om clivcrsc origins. Survival of A. hmsd''"·"' strains Cd and Sp-245 in 18 diO"erent types of soils nnd in the rhiz.ospherc of plants growing in lhcsc soils was evaluated. Thirteen soils originated from Israel, 3 from mainland Mexico and 2 from the peninsula of Oaja California in Mexico. In general, A. hrasilcnse survived well in the rhizosphcrc or wheat and tomato plants regardless of soil type. However, in plantlcss soils, its survi,·ahility varied; numbers sharply declined in Israeli soils, slightly decreased in B~•ja California soils and were stable or even increased in mainland Mexico soils (Table 2).

23

Physic:tl :llld chemical factors :tiTrcting survival of A. brasilcnse iu soil. Ttn physical and chemical soil parameters (nitrogen, CaC01, org:mic matter, clay, silt, fine sand, rough sand, field capacity, pH and salinity) were analyzed by Linear Regression Analysis vs. the rate of A:o:.pirillum mort31ity (with time) in I 3 Israeli soils. Linear correlations were found betwt:en mon::.lity and the contem of C:1C01, nitrogen :~nd rough-sand. The higher these par:mteh.:rs were pre:>entetl in a given soil, the higher the monality rate was of the bacteria. A typical analysis is presentt:d in Fig 3, showing a signific:mt linear correlation between the amoum ofCaC01 and monality of A. hra.wle!IISI.f Cd.

-0.1

-0.2 ~-,~

• • • .. Q) -0.3 y=-0.154-0.00?x _, cd f-o

...- • r=-0.81 (!j

-0.4 :> • ·-:> h ::J

Cf)

-0.5

- 0 .6 • •

-0. j' --~L--,_J'-----'---_j ___ L_-.

0 10 20 30 40 SO GO

CaC~ (%) Fig. :> . Linl·ar regrc~sion aualysis between the surviv:tl rate of .-l. hmsiiL'IISl' Cd :tt1d the amouut of CaCO, present in 13 soil types !'rom l~rad. The regression cocllicicnt wns signilkant at 1':~0.05.

Discussiun. Root colonii'.:Jtion by Plant Growth-Promoting Rhizohacteria (PGJ>R) is an essential t\:quisi to.: wh~.:tt ~.: nh :111ci ng p l :~n t growth is the objective. Most bacl<:ria need to be in the plant vicinity in sullicicnt nnn1b~.:rs H> alli.:ct the pi:lm I i i'~.: eye!..: ( I K). Survival in soil in th~.: ahscm:c of host plams is also of crucinl importance !'or the inoculation indu!:try. On one hand, inoculated bacteria which can survive indelinitt:ly in the soil in sullicicnt numbers after the growing season and still be able to colonize the next crop, provide little incentive for funher development. Long-term survival would also cause concerns ahout environmental manipulation, especially in gen~.:tically-cngineered microorg:misms. On the other hand, a broad spectrum or host range is an obvious :l(lvantngc for any given beneficial bacterin, eliminating the need for developing many specilic crop-bacteria combinations and :1voiding confusion by growers, especially in less developed countries. A:o.~7nrillum,

24

a known P( ii'R. has some of these traits. Apparently its host r:mgc is wide and its survival in soil is limited in some soils ( 11\,4.'i ). The bacteria can increase the yield of many crop plants, not by the !'llflflt'C~ sion of ;my plant p;tthogens, but by direct ly aflccting plant growth through a y«.:l-lo-he-revcalcd mechanism(s) (I R)

This study provides fut thcr evidence about the broad host t ange and the non-spccili«.: nature of A:mpn·iflnm. Under controlled conditions. the bacteria colonit.cd the root systems ofCA plant spcci«.:s belonging to l'J differen t botanical families. It had no prcf~.:rcncc to crop plants or weeds. nor to annuals or fll'f'cnnials. This list strengthened previous studies showing Azmpirillnm's ubili ty to colonize dilli.:r«.:nt plant species wotldwidc either wild or c10p plants (I ~.40). It seems that A:ospwillmu is an optimal root colu111zcr. This !itct, as satisfactory as it might be lor the inoculation industty, should raise sotne points or caution. In an inoculated Iicld, the local weeds which arc alwnys present. might he enhanced too. Furt hermore, most studies on the host-mngc of Azospirillum were conducted under a limited scale (greenhouse, ascetic conditions and/or controlled conditions etc.). Therefore. before dra'~ ing dclinite conclusions, all these findings should be verified under field conditions as well. For practic;1l reasons, A:o.']Jirifl11111 cells arc usually incorporated into inoculant carriers composed of peat, vermiculite (29,46) or synthetic materials such as alginate (5) . These inoculants arc later mixed with the seeds before sowing in the field, or applied to the soil during seedling emergence (21 ). This common inoculation technology is usually carried out by standard agro-mcchanical sowing devices which arc inaccurate from a microbial Slilndpoint, i.e., unable to ensure that the bacteria will encounter the emerging root. Thus. bacterial movement from the inoculation site to the root site is essential if root coloniz:tt ion is to occur. This distance can range from a few microns to several centimeters, and bacterial movement must occur in an environment of fierce competition with other soil microfaun:~ which arc also seeking nutrients and root colonization sites on the growing seedlings (4). A:o.\piriflum and pseudomonad biocontrol agents have been known to colonize the entire root system of plants (20.211.33) This bacterial dispersion hns been partially allributed to passive transport by the root tip!' ( 17,33). I 10\":evcr, in contrast to some pseudomonades, A:mpirif/11111 docs not disperse by percolating water but rather, it adsorbs into the soil particles ( 14,15). Nevertheless, passive dispersion by W!lter, especially in semi-arid conditions which lack sunicicnt water, [where Azospirillmu showed its best performance (4:l}] can not explain the f.1ct that the entire root system is colonized by A:o.IJnri/lnm. Thercrorc. it is rea~onahle to assume that another enicicnt bacterial dispersion mechanism exi~ts. One aitn l>f this study was to reveal the majot component of this mechanism since dispersal lw•. b<:e11 identified ;r!. a crucial parameter in the deliberate ira<>culation of genetically-engineered beneficial bacteria (8,4 7 ). This study revealed that A. brasilc!IISC motility in the rhizosphcre is essential to colonization of the root system. All hough the non-motile mutant cells proliferated similar to the wild type, they failed to coloni:z.c neighboring roots, even though water percolation for passive transportation was always avnilnblc. Our results with A. hrasih•n,c give further support to studies which show that non-motile mutants of bcnclicial pscudt11110nade~ were impaired in their <1bility to move· towards seeds (44) or colonize roots (28) when compared to their wild type parent strains. Al though A;:o.vnri/111111 has previously shown chemotaxis towards specific compounds (34,42,49), it was not the case in this study. The bnctcria were attracted to non-specific chcmoaHractants commonly found in root exudates and to which many rhizobacteria might migrate. Therefore, it can be suggested that a non-spccilic chemotaxis to root exudates may be a preliminaty mechanism, operating over relatively long distances and before Other COIIlpOnents of the plant-bacteria rec5>gnition SyStCI11

25

I .J

(adh~o: ,i \'l' ulatt·rials, b .:t ins \! t ~ . ) t:tkl.' ovl.'r .t\1 tht:st: distallC\.'S, the pl:tnt root-ha~h-ria r~·,:ugnitilln llll.'l.:hani"--n ts indli:~tivl', as shO\\ n in this study hy bactct ia that migrated from root~ to "art ilicial rnP1',.

\\·c alsn su~gt·~t that thi!> dispt:rsion tllCl:hanism inctl!ases the survival of A:O.']Jinllum. --bJSfJinllum is in many ctst·s. tot;llly dl'pt:ndcnt on the prt•scnce of roots to survive becau!'e it sut vin.:~ p<lo!ly in SlHil~o: soils ( 1 :\,45). Tht: death of' the host plant will not diminish the bacterial population front the lield since thl! ha t: teria is capable of migrating 10 the neighboring plants, whether it is tht: same specit:s, a dil1~rc:nt spc<.:ics, or even 10 weeds. This non-specilic chemotactic trait of A:.ospm/111111,i~ app<lrt'nlly •.1" ,,,.,., II•:•" :o11y (,;,c;tl'r i: rl al ii :rei i• 111 1" 111r tr i~:11 t ~ Ot hcrwi!.C, it <.:an 11111 he cx plaim.:d wlty tl 11: bact ct ia ""!''all: ti ""' 1 ut~b c.:ontainin~ ~:sudatcs to other roots whid1 provide similar lllatct iab. Bacterial moveml.'nt fi·nm inocubt~·d roots which were still extruding nutrit.:nts to non i 1 1tr~ulat l.'d

roots. GHI lw ~·splainc.:d in at h:ast two ways (i) The h:u.:tcrial populatinn un tlK· rt.llll ~ t·ousulllc.:d more nutrit·nt" th:m th~.: nHlls c..:ould supply at a givt:n 1110111cnt. This crcmcd a temporary, tnllt il'tll·ddi~:icnt mic..: t tH'II\'irnllna:nl whid1 nwy have stinnll:ll l.'d migration towards al ternative nutri~.:tll :-.ources ( ii ) . . ·l:ost•iril/um ltlrtllt'd lll:linly :1g.grcg:tt\! t)')li.'S of' colonization, which n:striCtl!d I.'Cll lllt.)\'('lllt'nt due to the ptnduction or extensive librillar m:lll· rials (10,20.37) These aggregates r rovic.k an l'COingic..:al adv:unage over any single bacterial ..:<.:11 in the compl!tition for nutrients in thl! rhizl>sphert·. 'l'lll' rdill·t'. in order 10 survive. single <.:ells need to locate sites which lack aggregate coloniz:ition, so they migrate to non-inocul:ucd roots and coloni:te them, producing the new aggregates that wt'rc tlctccted in this swdy. Tht•rclllre, moving 10 an uncolonized root may be a way that single hactl.'ri:t l'an dl~~:tiw l y

C\llllllt'll.' with bactcri:tl agg.rt:gates fur nutrients. S;u-viv:ll ur A:ospll'il!twi in soil is inconclusive. On one hand. the bacteria survived poorly in ~lllllC

lsr:tdi soils ( 15.21 ) and excellent ly in l3razilian soils (2). One may assume that some of the chemical or physi.:al characteristics lll' the sui I may inlluence survival. The screening of I R soil typ..-s li·om I sr:t l'l and l\1csico rcvcakd that soil parantCtt.! rs arl! important to survival. The main limiting lirctnr li.>ulld w:~s the amount of calcium in the soil; secondary factors were nitrogen and sand c..:ontl' tll Thl.'se drcumstantiallindings nawrally require addition:~! experimental verification.

Conrlusious· we propose that (i) A. hrasi/,•nse is not a plant- specific ba<.:teria. (ii) bactt:t ial motility within the plant root systt·m and bctwl!cn neighboring plants is a prt:rcquisitc in the root ba~tt:ria

recognition mechanism. It is an active process and a consequence of a non-:-.pl't:ilic bacterial chemotaxi~. inllucnced by the halancc between attractants and possible! rc1wlktll~ t'Xtrudcd by the root , but not 41irl.'<.:tly <kpcndcnt on nu1rient dclicicncy, and (iii ) survival iu thl.' snil in thl' ahst·nt:\' or plants may be iullucnced by th.: amoum or calcium. nitrogen and sand in the soil.

Admuwh·dJ,:t·n•rnts. This study is dcdi<.:atcd to the memory of the late l'vlr. Avucr llashan liom hrad aud wa~ pa11ially supponcd hy Couo;cjo i'lat.:ional de Cicncia y Tccnologia (CONAC 'yT). l\11.'.xico \\'c thauk Mr Roy Jl(lwcrs lor constru<.: tive English corrections during vacation and to Drs. J. Dobcrcincr, EMBRAI'A. Orazil and M. Singh, GDF, Germany for donating A brasiknst: Sp-245 and 297 I 0- I 0 b, respectively.

2f.

1. Adler. J. 1973. t\ mcthocll(>r measuring chl·motaxis and usc of the method t(> detcnninc optimum cond itions li1r chelllolaxis hy t·:_,t ·I!,•J"ic!nu cn/1 .I ( icn Micr ohio I. 7•1 . 77-'> 1.

2 Baldani. V I.. D .• M.J\ . Alva11:1. de B .. .I I llaldani, :u1d J Dol,l:fl:im·r I 'JX(, h.tai,Ji:;luueut nl" inm:ulatcd /l ;mpirif/11111 SJlp. in till· I hii'.ll~ph<.: l C ollld in roots or lidd groW II Wlll;;l( :tlld sorghum. l'lant Soil 90. 35-46.

3. Barak . R .• I. Nur, Y. Okon. and Y. !Ienis. I 1)!!2 . J\erolact ic rc~ponse of A ;ovJirillnm hrusilcnsc . J Uactcriol. 152:643-6:19.

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