New genetic sources of resistance in the genus Phaseolus to individual and combined aluminium toxicity and progressive soil drying stresses

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1 New genetic sources of resistnce in the genus Phseolus to individul nd combined luminium toxicity nd progressive soil drying stresses Louis Butre, Idupulpti Ro, Philippe Lepoivre, José Polni, Césr Cjio, Jun Cusquer & Stephen Beebe Interntionl Journl of Plnt Breeding ISSN DOI / s

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3 DOI /s New genetic sources of resistnce in the genus Phseolus to individul nd combined luminium toxicity nd progressive soil drying stresses Louis Butre Idupulpti Ro Philippe Lepoivre José Polni Césr Cjio Jun Cusquer Stephen Beebe Received: 18 Februry 2011 / Accepted: 23 My 2011 Ó Springer Science+Business Medi B.V Abstrct Ben species nd genotypes show wide phenotypic vribility in reltion to luminium () resistnce nd progressive soil drying. The objective of this study ws to identify nd chrcterize sources of resistnce to toxicity nd progressive soil drying mong six genotypes of common ben (Phseolus vulgris), four of runner ben (P. coccineus), nd one of tepry ben (P. cutifolius), using hydroponic nd soil cylinder screening methods. One experiment on hydroponic screening of resistnce ws crried out using bsl nutrient solution with nd without 20 lm. Two experiments were crried out using two oxisols in 80 cm long soil cylinders with high (H) nd low (L) sturtion tretments. The three experiments showed n verge of % inhibition of root growth with H compred with L tretments. Differences in root development nd distribution were observed L. Butre I. Ro J. Polni C. Cjio J. Cusquer S. Beebe (&) Centro Interncionl de Agricultur Tropicl (CIAT), A. A. 6713, Cli, Colombi e-mil: s.beebe@cgir.org L. Butre P. Lepoivre Université de Liège (ULg), Gembloux Agro-Bio Tech, Unité de Phytopthologie, Pssge des Déportés, 2, 5030 Gembloux, Belgium L. Butre Institut des Sciences Agronomiques du Rwnd (ISAR), 47 Rue Député Kmunzizi, BP 5016 Kigli, Rwnd mong genotypes nd species. Two ccessions of P. coccineus (G Q, G Q) nd one Anden common ben genotype (ICA Quimby) were outstnding in root nd shoot growth in the H tretments. P. coccineus ccession (G Q) ws outstnding under combined stress of -toxic cid soil nd progressive soil drying. Accessions of P. coccineus my represent unique sources of resistnce for the improvement of common ben through interspecific crosses. Keywords Abiotic stress Acid soil uminium resistnce Root growth Screening methods Wter stress Abbrevitions H High luminium soil sturtion LA Lef re L Low luminium soil sturtion MRD Men root dimeter NRT Number of root tips RDW Root dry weight REGWQ Ryn Einot Gbriel Welsh Multiple Test R:S Root to shoot SDW Shoot dry weight SRL Specific root length TPRL48h Tp root length t 48 h of exposure to with nd without luminium in solution TPRL120h Tp root length t 120 h of exposure to with nd without luminium in solution

4 TRER TRL VRD29d VRD33d Introduction Tp root elongtion rte Totl root length Visul rooting depth t 29 dys Visul rooting depth t 33 dys Wter stress Well wtered Common ben (Phseolus vulgris L.) is one of the most importnt grin legumes for direct humn consumption in the world. The crop is minly produced on smll-scle frms in developing countries in Ltin Americ nd Afric where both biotic nd biotic stress fctors limit production. About 60% of the ben growing re is ffected by drought while bout 40% of the ben growing re is ffected by luminium () toxicity, resulting in yield reductions from 30 to 60% (Wortmnn et l. 1998; Thung nd Ro 1999). Abiotic stress resistnce is by its nture more complex physiologiclly, typiclly subject to lrge environmentl effects nd hs been less well studied thn biotic stress resistnce in common ben (Ro 2001). uminium is one of the most bundnt minerls in the soil, comprising pproximtely 7% of soil mss. At neutrl or wekly cidic ph, it exists in insoluble forms of lumino-silicte or oxide. However, in cidic soils, it is solubilised into phytotoxic form. Toxic levels dmge roots, restrict plnt size, nd lower yield in most crops (Villgrci et l. 2001). uminium toxicity results in inhibition of root elongtion (Mossor-Pietrszewsk 2001; Horst et l. 2010). The toxic effects of in soil cn be overcome by dding pproprite soil mendments such s lime (Pndey et l. 1994; Villgrci et l. 2001). However, lime ppliction usully must be repeted over severl yers nd is not ffordble to most smllholders in developing countries tht grow bens. Developing ben genotypes tolernt to cid soil conditions is n ecologiclly friendly, energyconserving, nd economicl solution for resourcepoor frmers in the tropics (Rngel et l. 2005). Genotypic differences in seed yield of common ben germplsm ccessions nd breeding lines hve been observed in field screening on -toxic cid soils tht were mended with or without lime (65% sturtion) (Ro 2001; Ro et l. 2004). These genotypic differences in seed yield could be relted to differences in resistnce to, nd cquisition nd utiliztion of nutrients for trnsport of photossimiltes to developing seeds. Significnt genotypic differences in resistnce in common ben were reported bsed on -inhibited root elongtion in nutrient solution (Foy 1988; Mssot et l. 1999; Rngel et l. 2005, 2007; Mnrique et l. 2006). Differentil genotypic response to stress contributes to identifiction of new sources of resistnce s well s improved understnding of mechnisms of resistnce in common ben (Rngel et l. 2005, 2007, 2009, 2010; Blir et l. 2009; López-Mrín et l. 2009). Resistnce to in common ben is ttributed to the relese of citrte by the root pex (Rngel et l. 2010) nd the expression of citrte trnsporter MATE (multidrug nd toxin extrusion fmily protein) gene is crucil for citrte exudtion (Etich et l. 2010). though the MATE gene expression ws prerequisite for citrte exudtion nd resistnce, genotypic difference in resistnce in common ben ws minly dependent on the cpcity to sustin the synthesis of citrte for mintining the cytosolic citrte pool tht enbles exudtion (Rngel et l. 2010; Etich et l. 2010). The initil -induced inhibition of root elongtion in both -resistnt (ICA Quimby) nd -sensitive (VAX 1) genotypes ws correlted with the expression of the ACCO (1-minocyclopropne-1- crboxylic cid oxidse) gene (Etich et l. 2010). Improving resistnce in plnts hs been limited by indequte screening methodologies (Villgrci et l. 2001). Screening for resistnce bsed on field dt would be strengthened by complimentry evlution of specific phenotypic nd physiologicl trits. Hydroponic systems hve been used with success in evlution of resistnce for mny crops nd re n ttrctive lterntive. They llow evlution of lrge number of genotypes quickly nd hve been used to identify prentl stock for soyben breeding (Binchi-Hll et l. 1998; Cmpbell nd Crter 1990; Crter nd Rufty 1993; Spehr 1994; Binchi-Hll et l. 2000; Silv et l. 2001). Common ben nd soyben both pertin to the tribe Phseolee, much more closely relted to ech other thn they re to Lotus, Medicgo, or other grin legumes such s lentils or chickpes (Choi et l. 2004). This opens the possibility of common genes nd/or

5 mechnisms between the species, nd suggests tht the ppliction of methods used in soyben might serve to discriminte genetic differences in common ben s well. Hydroponic systems for evlution of genetic mterils provide strict control of nutrient vilbility nd re widely used in genetic studies. To complement the hydroponic screening system, reserchers t CIAT developed greenhouse screening method using verticl soil cylinders with high (H) sturtion to quntify genotypic differences in root development nd distribution in -toxic soil conditions (CIAT 2008). Soil-bsed systems offer medium tht is more similr to field conditions. This methodology lso permits chrcteriztion of the root system, in terms of rooting depth nd brnching of fine roots in soil with known bulk density. Drought is mjor biotic stress in mny prts of the world (Johnsen et l. 1994). There is n urgent need for developing high yielding drought resistnt cultivrs tht use wter efficiently, reduce dependence on irrigtion wter nd ssocited production costs, increse nd stbilize yield in drought-prone environments, nd increse profit mrgins for producers (Muñoz-Pere et l. 2006). Selection for drought resistnce bsed on yield lone my not recover ll specific physiologicl ttributes s these might be expressed differentilly under distinct conditions (Subbro et l. 1995). Rooting pttern, especilly greter root length in lower soil strt, is n importnt drought resistnce mechnism for common ben (Sponchido et l. 1989). Drought resistnt ben genotypes could extend their roots to 1.2 m depth in drought environments, wheres the sensitive genotypes could not extend their roots beyond 0.8 m; nd these differences in rooting depths were reflected in overll shoot growth nd yield (White nd Cstillo 1988). Root systems show considerble rchitecturl vrition mong species, mong genotypes of given species, nd even with different prts of single root system (Lynch 1995). Wild reltives in mny legumes possess deep rooting cpbility tht could be trnsferred to cultivted legumes. A number of Phseolus species, such s P. cutifolius, P. retensis, nd P. coccineus, hve deep nd/or tuberous primry root ttributes (Singh nd White 1988). Understnding the genetic nd physiologicl mechnisms by which plnts cope with chnges in environmentl conditions is criticl for creting efficient strtegies to develop stress-resistnt cultivrs for sustinble production systems (Ro 2001). Abiotic stress fctors often co-occur in frmers fields. Roots tht re stunted by toxicity re inefficient in bsorbing both nutrients nd wter (Mossor-Pietrszewsk 2001). -resistnt plnts my be more drought tolernt nd require lower inputs of lime nd P fertilizer thn less resistnt genotypes (Little 1988). Yng et l. (2010) chrcterized the combined toxicity nd drought stress on root growth, with specil emphsis on /drought interction in the root pex of common ben. Using polyethylene glycol (PEG) to simulte osmotic stress (OS) or drought stress, they found tht OS enhnces resistnce by inhibiting ccumultion in the root pices of the -sensitive genotype (VAX 1). This llevition of toxicity ws found to be relted to the ltertion of cell wll porosity resulting from PEG-induced dehydrtion of the root poplst. Improving resistnce to two complex stresses such s toxicity nd wter stress () in common ben requires identifying new sources of resistnce mong P. vulgris ccessions nd in sister species including P. coccineus nd P. cutifolius. P. coccineus is plced within the secondry gene pool nd cn be crossed redily with common ben. P. cutifolius is considered to form prt of the tertiry gene pool, but cn lso be crossed to common ben using embryo rescue. The objective of this work ws to identify potentil prents bsed on phenotypic differences mong ben genotypes in root development nd root distribution under individul nd combined stress fctors of H toxicity nd progressive soil drying. Mterils nd methods Three greenhouse trils were conducted t CIAT hedqurters in Plmir (Lt N; Long W, titude 965 m) using hydroponic nd soil cylinder systems. For purposes of these studies, resistnce refers to the response of genotype to toxic in the hydroponic system, nd tolernce to -toxic cid soil conditions refers to tolernce to H sturtion in cid soil together with low vilbility of nutrients. A single hydroponic screening (Tril 1) employed low ionic strength nutrient solution to evlute root trits of seedlings grown with or without 20 lm in bsl nutrient solution (Rngel et l. 2005, 2007). One soil cylinder experiment (Tril 2) compred plnt response

6 in two oxisols with H nd low (L) sturtion. A second soil cylinder experiment (Tril 3) ws conducted to evlute plnt response to individul nd combined stress fctors of cid soil (H nd L sturtion) nd two levels of soil moisture (well wtered () nd wter stress () induced by progressive soil drying) in fctoril design. Plnt mterils Eleven ben genotypes were selected for the study from our previous work (CIAT 2005; S. Beebe, unpublished dt), including four runner ben (P. coccineus) ccessions (G Q, G Q, G Q nd G Q); six common ben genotypes including two lrge seeded bens of the Anden gene pool (ICA Quimby nd IJR, indeterminte Jmic red) nd four lines of the Mesomericn gene pool (VAX1, VAX3, VAX6, SER16); nd one tepry ben (P. cutifolius) ccession (G40159). The P. coccineus ccessions hd been identified in field screening of 155 entries of P. coccineus nd P. polynthus in n toxic field site in Sntnder of Quilicho, Colombi, bsed on shoot vigor (S. Beebe, unpublished results). P. cutifolius is drought resistnt desert species nd one of its ccessions, G40159 hd been identified s especilly drought resistnt (Ro et l. 2007). The VAX lines hd been selected for common bcteril blight resistnce in Sntnder of Quilicho during their development, nd VAX1 hd expressed good shoot vigor in toxic soils. These plnt mterils were evluted for their phenotypic differences under individul nd combined stresses of nd drought. For the hydroponic experiment, germinted seeds were trnsferred to smll pots contining sterile snd for root development nd were crefully removed from the snd fter 3 dys. Seedlings with uniform vigour nd tp root length were chosen for evlution in hydroponic system. For the two soil cylinder experiments, seeds were surfce sterilized with sodium hypochlorite (1% for 5 min) nd wshed with bundnt deionised wter. Seeds were germinted on filter pper for 2 3 dys before plnting in soil tubes. Evlution for resistnce using hydroponic system The hydroponic experiment (Tril 1) ws conducted during November nd December Plnts were grown in greenhouse with n verge temperture of 31.1/22.3 C (dy/night), reltive humidity of 48.0/67.3% (dy/night), nd with mximum photosynthetic ctive rdition of 1,100 lmol m -2 s -1 photon flux density t noon. Seedlings with uniform root length (5 7 cm) were selected for evlution with nutrient solution composed with 5 mm CCl 2, 0.5 mm KCl nd 8 lm H 3 BO 3 t ph 4.5, with or without 20 lm Cl 3 (Rngel et l. 2007; López- Mrín et l. 2009). Mononucler ( mono ) concentrtion ws mesured colorimetriclly using luminon or pyroctechol violet method ccording to Kerven et l. (1989). uminium tretment of 20 lm resulted in 16 ± 2 lm mono fter 24 h. Twenty liter plstic tnks were filled with 16 l of nutrient solution. Ech seedling ws plced in n individul comprtment in try floting on the solution, nd the nutrient solution ws permnently erted with compressor during the evlution. Acclimtion to low ph before pplying tretment ws mde by djusting the solution ph to 5.5 for 0 h, followed by ph 4.9 for 18 h nd lstly by ph 4.5 for 24 h (Rngel et l. 2007). Nutrient solutions were renewed every third dy. Plnts with the sme root length were distributed in pirs in ech tretment fter mesuring the length of tp root with ruler. The experimentl design ws rndomized complete block with five to ten replictions. Root morphologicl ttributes were evluted. Tp root length (TPRL) t 48 h (TPRL48h) nd t 120 h (TPRL120h) ws recorded. Tp root elongtion rte (TRER) ws determined t 48 h (TRER48h) nd fter 120 h (TRER120h) with nd without stress bsed on the initil mesurement of tp root length. TRER ws defined s the difference between the initil nd finl tp root length during the tretment period; nd -induced inhibition of TRER ws clculted ccording to Rngel et l. (2005): Inhibition of root elongtion ð% Þ ¼ ½ðTRER control TRER Þ=TRER control Š100 At hrvest, roots were seprted from the rest of the plnts, sved in plstic bgs nd refrigerted t 4 C while proceeding to nlyze imges using fltbed colour scnner, Epson Expression1680 Scnner. Differences in root morphologicl ttributes mong genotypes including totl root length (TRL), men root dimeter (MRD), nd number of root tips (NRT) were nlyzed using WinRhizo Ò softwre progrm.

7 Specific root length (SRL, root length per unit dry weight) ws clculted, nd the root dry weight (RDW) ws determined by drying roots t 65 C in n oven for 48 h. Evlution for individul nd combined stress in soil Two soil cylinder experiments were crried out, ech rrnged s three rndomized complete blocks. The first soil cylinder experiment (Tril 2) compred plnt response in Oxisols with L nd H sturtion nd ws conducted during June July 2007 in greenhouse in Plmir (CIAT/Colombi) with n verge temperture of 29.4/23.1 C (dy/night), reltive humidity of 57.2/79.4% (dy/night), nd mximum photosynthetic ctive rdition of 1,100 lmol m -2 s -1 photon flux density t noon. Stress of -toxic cid soil ws simulted using soils collected from Sntnder de Quilicho, Cuc Deprtment ( N lt., W long; 990 m ltitude), Colombi. Soil used in the stress tretment (H) ws chrcterized by ph of 4.11 nd 76% soil sturtion (0 10 cm) for top-soil (top 10 cm of the cylinder) nd 83% sturtion for subsoil (10 75 cm) with ph 4.14 (Tble 1). This tretment did not receive ny dditionl fertilizer ppliction to simulte H with low nutrient vilbility soil conditions tht re typicl of -toxic cid soils. Root nd shoot growth of ben genotypes under this tretment ws visully (bsed on symptoms) restricted by both -toxicity nd low vilbility of P. Soil used for L tretment ws chrcterized by ph of 4.45 nd 28% sturtion (0 10 cm) for topsoil nd 58% sturtion for subsoil (10 75 cm) with ph The soil cylinders for L tretment were pcked with Quilicho soil (described in Tble 1), previously fertilized with dequte mendments (g kg -1 soil) for top soil (0 10 cm): 3.69 N (ure), 5.30 P (triple superphosphte), 5.30 C (triple superphosphte), 4.08 K (KCl), 6.36 C (CCO 3 ), 6.36 Mg (MgCO 3 or dolomite lime), 0.49 S (elementl sulphur), 0.09 Zn (ZnCl 2 ), 0.11 CuCl 2 2H 2 O, 0.01 B (H 3 BO 3 ) nd 0.01 Mo (NMoO 4 2H 2 O); nd for subsoil (10 75 cm) N (ure), 21.2 P (triple superphosphte), C (triple superphosphte), K (KCl), C (CCO 3 ), Mg (MgCO 3 or dolomite lime), 1.97 S (elementl sulphur), 0.36 Zn (ZnCl 2 ), 0.46 CuCl 2 2H 2 O, 0.05 B (H 3 BO 3 ) nd 0.02 Mo (N- MoO 4 2H 2 O). This level of fertilizer ppliction ws designed to provide dequte supply of nutrients, nd it did not ffect sturtion nd ph of the mended soil. The polyethylene cylinders were inserted into PVC pipes nd were mintined t 80% field cpcity by weighing ech cylinder every 3 dys nd pplying wter to the soil t the top (Polní et l. 2009). Shoot nd root ttributes were evluted on plnts in soil tubes. Totl chlorophyll content (SPAD) ws mesured every week using SPAD-502 Chlorophyll meter (Minolt cmer Co., Ltd, Jpn). Visul rooting depth (VRD) ws determined t 29 dys fter plnting. At the time of hrvest (29 dys fter plnting), lef re (LA) ws determined by scnning leves of ech genotype using LI-3100 Are meter (LI-COR Biosciences). Shoot dry weight (SDW) ws mesured fter drying leves, stems nd pods in n oven t 70 C for 72 h. Ech soil cylinder ws sliced into six lyers representing different soil depths (0 5, 5 10, 10 20, 20 40, 40 60, cm), soil nd roots were collected, nd roots wshed nd clened to seprte living plnt roots from orgnic debris before Tble 1 Chemicl chrcteristics of two oxisols with H or L sturtion from Sntnder of Quilicho used for evluting cid soil tolernce sturtion Soil depth (cm) ph (cmol kg -1 soil) C (cmol kg -1 soil) Mg (cmol kg -1 soil) K (cmol kg -1 soil) st. (%) SOM (%) Avilble P (mg kg -1 ) High High Low Low SOM Soil orgnic mtter

8 scnning. Root length nd biomss distribution were determined for ech profile, but cutting of soil cylinders t different depths did not permit mesuring NRT. Differences in distribution of root length nd root biomss within soil cylinder mong genotypes were estimted by Gle nd Grigl (1987) symptotic eqution: Y ¼ 1 b d where Y is the frction of root length/biomss ccumulted from the soil surfce to depth d (cm), nd b is prmeter tht describes the shpe of the cumultive distribution with depth. Higher b vlues (closer to 1) indicte greter proportion of root length/biomss deeper in the soil profile. Lower b vlues (e.g. b = 0.920) imply greter proportion of root length/biomss nerer to the soil surfce. In second soil cylinder tril (Tril 3), individul nd combined stress of nd progressive soil drying were evluted in September 2008 under greenhouse conditions t n verge temperture of 30.7/23.3 C (dy/night), reltive humidity of 49.0/68.3% (dy/ night), nd t n verge photosynthetic photon flux density of 820 lmol m -2 s -1 during the dy. Plnts were grown in trnsprent tubes inserted in PVC pipes s previously described for screening with the sme soil type from Sntnder of Quilicho. The L sturtion tretment ws fertilized with dequte supply of nutrients s described bove for top soil nd subsoil. The experimentl design ws rndomized complete block with three repetitions nd with two levels of sturtion in soil (high nd low) nd two levels of wter supply ( nd due to progressive soil drying) in fctoril design. Cylinders of genotype-soil-moisture combintions were rndomized within ech block. Ech cylinder ws pcked with two types of soil (top-soil nd sub-soil); nd mintined t 80% of field cpcity by weighing every 3 dys (4,780 g for H sturtion tretment cylinders nd 4,910 g for the low sturtion). ws imposed to simulte progressive soil drying fter 10 dys of initil growth while for the tretment, wter ws pplied to the top of cylinders to mintin them t 80% of field cpcity (Polní et l. 2009). At hrvest, shoots nd roots of 33 dy-old plnts (23 dys without wter ppliction in the tretment) were seprted, nd LA mesured by scnning leves. Shoot biomss ws determined fter drying leves nd stems in n oven. Roots were processed in the sme wy s for previous soil cylinder tril (Tril 2) with individul stress tretment of -toxic cid soil lone nd the sme prmeters were determined. Sttisticl nlysis Anlysis of vrince ws performed by the ANOVA sttisticl procedure of SAS (SAS 9.1, ; SAS institute Inc.; SunOS 5.9 pltform). The mens were compred using Ryn Einot Gbriel Welsh (REGWQ) Multiple Test. This test controls the type I experiment-wise error rte. Mens for ech dependent vrible were grouped, nd mens with the sme letter re not significntly different (P \ 0.05 level). Differences between genotypes were nlyzed with the lest significnt difference (LSD). Correltion coefficients were clculted (PROC CORR) for ll pirs of genotypic mens cross ll replictions with ech tretment nd for ll three experiments. Regression nlysis ws crried with the PROC REG procedure. Results nd discussion The response of root ttributes (TRL, MRD, SRL, VRD) to stress ws used to ssess resistnce of bens to toxicity nd toxic cid soil. Vrition mong the 11 genotypes ws found in this study using both hydroponic (Tbles 2, 3) nd soil cylinder (Tbles 4, 5, 6, 7) screening systems. Reltionships between trits were considered bsed on expecttions of which combintions of trits would confer fvourble rection to toxicity. High vribility in root ttributes ws observed between genotypes except for TPRL t 48 h of tretment (TPRL48h) with 20 lm in hydroponic system. Since P. coccineus ccessions did not show ny difference in the hydroponic system between tretments with nd without in solution for 48 h, the exposure time ws extended to 120 h where significnt genotypic vrition (P \ 0.001) in resistnce ws observed. Evlution of resistnce in hydroponic system stress in hydroponic screening (Tril 1) ffected root growth chrcteristics of ll 11 genotypes tested. Vrition in response to stress (20 lm ) mong

9 Tble 2 Influence of -stress (with (20 lm ) nd without (0 lm )) on totl root length (TRL), tp root length (TPRL) t 48 h nd 120 h, root dry weight (RDW), men root dimeter (MRD), specific root length (SRL) nd number of root tips (NRT) of 11 ben genotypes from three Phseolus species grown in hydroponic system (Tril 1) Genotype TRL (m) TPRL48h (cm) TPRL120h (cm) RDW (g) MRD (mm) SRL (m g -1 ) NRT 20 lm 0 lm 20 lm 0 lm 20 lm 0 lm 20 lm 0 lm 20 lm 0 lm 20 lm 0 lm 20 lm 0 lm G Q G Q G Q G Q ICA Quimby b IJR b VAX 1 b SER 16 b VAX 3 b VAX 6 b G c Men LSD b P. coccineus P. vulgris P. cutifolius c

10 genotypes ws found for TRL, TPRL48h, TPRL120h, RDW, MRD, SRL nd NRT (Tble 2). Three P. coccineus ccessions (G Q, G Q, G Q) were highly resistnt to, wheres three Mesomericn common ben genotypes (VAX6, VAX3, SER16) were more sensitive. Genotype 9 tretment (Level of ) interction were found for SRL, NRT, MRD nd TRL (Tble 3). TRL ws highly correlted with TPRL48h (r = 0.77**), with TPRL120h (r = 0.92***), with RDW (r = 0.91***), nd with NRT (r = 0.96***). This suggests tht the tp root response to toxicity (TPRL5d) reflects the rection of the rest of the root system. In contrst López-Mrín et l. (2009) found tht while ben genotype G19833 showed higher TRER thn DOR364 in both control nd tretment solutions, differences in TRL between the two genotypes were less notble. Correltion ws lso found between TPRL48h nd NRT (r = 0.78**), nd between TPRL48h nd TPRL120h (r = 0.74**) (Tble 3). This ltter correltion ws perhps lower thn expected, considering tht it reflects mesurements of the sme trit on the sme plnts over 72 h period. Perhps different plnt responses were being expressed over this period, s noted by Rngel et l. (2007, 2010) in other trils. TRER cn be monitored esily nd is ssumed to be n indictor of resistnce. strongly ffected TRER with mens vrying from 0.15 to 1.0 mm h -1. TRER of three P. coccineus ccessions (G Q, G Q nd G Q) nd ICA Quimby (n Anden common ben) were high ([0.6 mm h -1 ) compred to other genotypes (Fig. 1A). The mjor toxicity symptom observed in plnts is inhibition of root elongtion rte clculted from the comprison of root elongtion rte with nd without toxic (Mrschner 1991; Ryn et l. 1993; Delhize nd Ryn 1995). TRER ws inhibited by %, with n verge of 42.7%. Three genotypes (VAX3, VAX1, G Q) were sensitive to with over 50%; six genotypes including SER16, IJR, G Tble 3 Correltion coefficients nd men squres for totl root length (TRL), tp root length (TPRL) t 48 h nd 120 h, root dry weight (RDW), men root dimeter (MRD), specific root length (SRL), nd number of root tips (NRT) of 11 ben genotypes grown under hydroponic system with two level of (20 lm nd 0 lm ) (Tril 1) Trits/source level/df TRL TPRL48h TPRL120h RDW MRD SRL NRT TRL TPRL48h (ns) ** 1 TPRL120h ** 0.74** *** 0.74** 1 RDW *** 0.13 (ns) 0.58 (ns) *** 0.59 (ns) 0.83** 1 MRD (ns) (ns) (ns) 0.61* (ns) (ns) 0.24 (ns) 0.57 (ns) 1 SRL (ns) 0.72* 0.26 (ns) (ns) -0.84*** (ns) 0.07 (ns) (ns) (ns) -0.87*** 1 NRT *** 0.43 (ns) 0.66* 0.86*** 0.16 (ns) 0.04 (ns) *** 0.78** 0.91*** 0.84*** 0.20 (ns) (ns) 1 Level of *** 4.54* 10.93*** *** *** 11894*** 5158*** Rep. ( level) (ns) 0.46 (ns) 0.68 (ns) (ns) (ns) (ns) (ns) Genotype *** 0.86* 2.33*** *** *** 5894*** 525.5*** Gen. 9 level * 0.1 (ns) 0.45 (ns) (ns) * 1713*** 73.77** Error ns Non significnt Sttisticl significnce t the * 0.5, ** 0.01, nd *** probbility levels, respectively

11 Tble 4 Influence of cid soil stress (H high luminium sturtion, L low luminium sturtion) on totl root length (TRL), men root dimeter (MRD), specific root length (SRL) nd VRD t 29 dys (VRD29d) for 29 dys-old plnts of 11 ben genotypes from three Phseolus species grown in soil cylinders under conditions (Tril 2) Genotype TRL (m) MRD (mm) VRD29d (cm) SRL (m g -1 ) H L H L H L H L G Q G Q G Q G Q I. Quimby b IJR b VAX 1 b SER 16 b VAX 3 b VAX 6 b G c Men LSD P. coccineus P. vulgris P. cutifolius 3Q, VAX6, ICA Quimby, nd G40159 were intermedite with inhibition between 37.5 nd 44.0%, wheres P. coccineus ccessions G Q nd G Q were resistnt with inhibition of 30.8 nd 21.3%, respectively (Fig. 1B), confirming the vribility observed previously between the two ben species (CIAT 2005), nd the level of resistnce reported in ICA Quimby (Rngel et l. 2007). Fine nd extensive roots tht continue to grow nd tht thicken less in the presence of toxic should be ble to explore toxic soil more efficiently (Eisenstt 1992; Villgrci et l. 2001). Genotypes grown in presence or bsence of for 120 h lso showed differentil response to for the increse of MRD, rnging from 8.1 to 20.5% (Fig. 1C). VAX6, G Q, G40159, nd ICA Quimby presented less increse of root dimeter (\9.43%). Five genotypes from different ben species nd gene pools (G Q, G Q, G40159, ICA Quimby nd VAX6) were outstnding for minimizing inhibition of TRER nd increse of root dimeter (Fig. 1). In contrst, sensitive line VAX1 presented inhibition of TRER nd increse of MRD of 60.3 nd 20.2%, respectively. Similr results were found before in evlution of resistnce mong 52 genotypes of common ben (CIAT 2005). Villgrci et l. (2001) reported tht number of bsl roots nd brnching from tp root were clerly reduced by in ll soyben genotypes. We found similr results for NRT (root brnching) nd for SRL which is ffected by number of fine brnches. As expected, SRL exhibited strong negtive correltion with verge root dimeter in the hydroponic system. However, the rnking of genotypes by SRL did not pper to correspond to rnking by other trits. Evlution for -toxic cid soil tolernce uminium-toxic cid soil in Tril 2 ffected ll root prmeters except for MRD (Tbles 4, 5). Averge vlues for L nd H tretments, respectively were: TRL, 59 nd 27.4 m; MRD, 0.30 nd 0.29 mm; VRD29d, 67.1 nd 56.7 cm; nd SRL, nd 78.7 m g -1. Genotypic differences were highly significnt for TRL, MRD, SRL nd VRD29d t 29 dys. Acid soil tolernt genotypes G Q, G Q nd G Q mintined good root structure in cid soil. Genotype 9 level interction ws significnt for SRL nd VRD29d t 29 dys. In

12 Tble 5 Correltion coefficients nd men squres for totl root length (TRL), men root dimeter (MRD), specific root length (SRL), VRD t 29 dys (VRD29d), lef re (LA), shoot dry weight (SDW) nd root:shoot rtio (R:S rtio) of 29 dys-old plnts of 11 ben genotypes evluted under conditions of high sturtion (H) nd low sturtion (L) in soil cylinders under conditions (Tril 2) Trits/source level/df TRL MRD SRL VRD29d LA SDW R:S rtio TRL L 1 H 1 MRD L 0.53 (ns) 1 H 0.58 (ns) 1 SRL L (ns) -0.79** 1 H 0.33 (ns) 0.27 (ns) 1 VRD29d L 0.65* 0.77** (ns) 1 H 0.78** 0.92*** 0.34 (ns) 1 LA L 0.15 (ns) 0.35 (ns) 0.02 (ns) 0.64* 1 H 0.52 (ns) 0.14 (ns) 0.22 (ns) 0.31 (ns) 1 SDW L 0.64* 0.57 (ns) (ns) 0.82** 0.69* 1 H 0.50 (ns) 0.38 (ns) 0.46 (ns) 0.47 (ns) 0.81** 1 R:S rtio L 0.47 (ns) 0.68* -0.79** 0.24 (ns) (ns) (ns) 1 H 0.81** 0.70* 0.05 (ns) 0.71* 0.15 (ns) 0.11 (ns) 1 Level of *** (ns) 12236*** 1774*** *** 19.58*** 0.34*** Rep. ( level) (ns) (ns) (ns) (ns) 2991 (ns) 0.19 (ns) (ns) Genotype *** 0.009*** 1342*** 3747*** 3235 (ns) 0.28* 0.06*** Gen. 9 level (ns) (ns) 986.2** 1815* 8193* 0.18 (ns) 0.012* Error ns Non significnt Sttisticl significnce t the * 0.5, ** 0.01, nd *** probbility levels, respectively H soil, TRL ws correlted with only two root trits, VRD29d t 29 dys (r = 0.78**) nd root to shoot rtio (R:S) rtio (r = 0.81**); wheres MRD ws highly correlted with VRD29d of 29 dy-old plnts (r = 0.92***) (Tble 5). This implies tht thicker roots were penetrting deeper into soil, nd reflects the fct tht P. coccineus presented high vlues of both trits. However, thicker roots did not necessrily represent less efficient use of biomss, s indicted by vlues of SRL. Wheres H soil induced serious reductions in SRL in common ben in Tril 2, three of the runner ben ccessions mintined their vlues of SRL. In Tril 3, G Q likewise showed miniml reduction in SRL in cid soil. In both soil trils runner ben presented thicker roots thn common ben under stress, nd yet runner ben presented SRL tht ws greter thn (in Tril 2) or comprble (in Tril 3) to common ben in stress, especilly compred to the Mesomericn genotypes SER 16, VAX 3 nd VAX 6. This implies tht the roots of runner ben re less dense thn roots of common ben. This my be n expression of erenchym development in runner ben, which would be very interesting trit to introgress to common ben. The tendency to thicker roots in P. coccineus ws more evident in the soil experiments thn in the hydroponic trils. Tolernce to combined stress in soil uminium levels 9 wter regimes in Tril 3 effects were lrge for TRL nd moderte for SRL. As expected, combined stress of -toxic soil nd ws the most inhibitory to TRL, followed by lone, nd then by lone, bsed on the tretment verges (Tble 6). However, s expected, -stress lone ws more inhibitory thn to SRL nd VRD33d. Genotypes differed for ll root trits considered (TRL, MRD, SRL, nd VRD33d) (Tble 7). Two sister lines of P. coccineus, G Q nd G Q, were the most tolernt to combined stress, presenting the highest vlues of

13 Tble 6 Influence of individul nd combined stress fctors of cid soil (H high luminium, L low luminium) nd wter stress ( well wtered, wter stress) on totl root length (TRL), men root dimeter (MRD), VRD t 33 dys (VRD33d) nd specific root length (SRL) of 33 dys-old plnts of 11 ben genotypes from three Phseolus species grown in soil cylinders (23 dys under ) (Tril 3) Genotype TRL (m) MRD (mm) VRD33d (cm) SRL (m g -1 ) L L H H L L H H L L H H L L H H G Q G Q G Q G Q I. Quimby b IJR b VAX 1 b SER 16 b VAX 3 b VAX 6 b G c Men LSD b P. coccineus P. vulgris P. cutifolius c

14 Tble 7 Correltion coefficients nd men squres for totl root length (TRL), men root dimeter (MRD), specific root length (SRL), VRD t 33 dys (VRD33d), lef re (LA), shoot dry weight (SDW), root:shoot rtio (R:S rtio) of 11 ben genotypes grown under combined stress of nd wter stress (23 dys under ) (Tril 3) Trits/source DF TRL MRD VRD33d SRL LA SDW R:S rtio TRL 1 MRD 0.43* 1 VRD33d 0.79*** 0.27 (ns) 1 SRL -0.2 (ns) -0.62*** (ns) 1 LA 0.62*** 0.56*** 0.58*** -0.39* 1 SDW 0.82*** 0.55*** 0.59*** -0.42* 0.62*** 1 R:S rtio 0.57*** 0.19 (ns) 0.59*** (ns) 0.32 (ns) 0.10 (ns) 1 Rep * 0.004** *** Wter * ** ** Rep 9 wter * * * *** 0.009** 598.0** *** *** 9.855** 0.804** Wter *** * *** 3.437* Rep 9 wter Genotype *** 0.005*** 313.9*** *** 13.55** 0.435* 0.355*** 9 genotype *** *** 128.4*** 24.11*** 0.819*** 0.088** Wter 9 genotype Wter 9 9 genotype * *** Error ns Non significnt, Wr wter regime Sttisticl significnce t the * 0.5, ** 0.01, nd *** probbility levels, respectively TRL nd mintining deeper root system (Tbles 6, 8). They were lso the two best under -stress lone but showed difficulties to develop deep rooting system under lone. Other reltively tolernt genotypes in combined stress were G Q nd ICA Quimby. Interctions of genotype 9 tretment were highly significnt for TRL, SRL, nd VRD (Tble 7). though combined stress of nd is generlly more dmging thn ech stress considered seprtely, in this study n unexpected interction between these two stress fctors ws observed. P. coccineus ccessions (G Q, G Q, nd G Q) nd to lesser extent ICA Quimby developed more roots, nd showed deeper rooting bsed on VRD33d with the combined nd thn with lone (Tble 6). TRL ws correlted with MRD (r = 0.43*), nd with root depth (r = 0.79***) in the combined stress tretment, wheres MRD ws negtively correlted with SRL (r =-0.62***) s expected (Tble 7). Root length nd dry weight distribution Results on genotypic differences in rooting strtegies estimted by the vlues of the extinction coefficient b for root length distribution in Trils 2 nd 3 showed tht the ccessions of P. coccineus hd greter proportion of root length t depth thn the other genotypes except for the Anden genotype IJR under H tretment in both s well s conditions (Tble 8). Similr effects were observed on b vlues for root biomss distribution (Tble 9). Root nd shoot ttributes Shoot trits were mesured only in the soil cylinder trils. If n improved root system with resistnce is to result in yield improvement, it is importnt tht it contribute to enhnced shoot development. Estimtion of shoot vigor of plnts grown in cid soil ws mde bsed on LA development nd SDW under individul (Tril 2) nd combined stress of nd (Tril 3).

15 A Tp root elongtion rte (mm h -1 ) B Induced inhibition of root elongtion (%) C incresed men root dimeter (%) b b b G Q G Q G Q ICA Quimby IJR G40159 G Q SER16 VAX3 VAX6 VAX1 b b VAX3 VAX1 G Q SER16 IJR G Q VAX6 ICA Quimby G40159 G Q G Q G Q VAX1 VAX3 IJR G Q G Q SER16 ICA Quimby Ben genotypes G40159 G Q VAX6 G Q ws the best genotype under -stress lone for combining n extensive root system, lrge leves nd vigorous seedlings. G Q ws found to be the best with similr potentil under combined stress of nd, but ws intermedite for lone for both trits, nd ws very poor for lone bc bc bc b bc bc Fig. 1 Root prmeters of 11 ben genotypes grown under hydroponic system under two levels of (20 lm nd 0 lm ) t ph 4.5 (Tril 1). A TRER in mm per hour under H (20 lm ); B -induced inhibition of TRER in per cent; C -induced increse of MRD in per cent. Brs represent mens ± SD, with 4 replictes. Different letters indicte differences between t P \ 0.05 (REGWQ test) b bc bc b bc bc b c c b c c b (Tble 10). Genotype 9 level interction ws significnt for LA nd R:S rtio nd highly significnt interction ws observed for genotype 9 ( nd wter regime) for LA nd R:S rtio nd SDW (Tble 7). Lrger LA development ws ccompnied by strong shoot biomss investment in stems, brnches nd petioles. P. vulgris genotypes SER16 nd VAX1, nd P. cutifolius G40159 expressed reltively higher vlues of LA nd shoot biomss under drought stress, but were strongly ffected by -toxic cid soil, nd were more sensitive in shoot prmeters under combined stress fctors of nd (Tble 10), indicting tht their cpcity to cquire nutrients nd wter for shoot growth ws reduced. The reltionships between shoot trits (LA nd SDW) nd TRL in stress lone (Tril 2) nd in combined stress of nd (Tril 3) in soil cylinder system were nlysed. Liner regression of LA on TRL in stress for the 11 genotypes showed positive reltionship (r = 0.81*) (Fig. 2A), nd similr reltionship ws observed between SDW nd TRL (r = 0.83*) (Fig. 2B) in stress. Reltionship between SDW nd TRL in combined stress of nd lso showed strong positive reltionship (r = 0.81*) (Fig. 2C). The ccessions of P. coccineus in most cses presented the highest vlues nd influenced the regression vlues hevily. The genotypes G Q, G Q nd G Q were outstnding under stress expressing deeper root system (Tble 4) nd mintining bove verge shoot development. l P. vulgris cultivrs nd the P. cutifolius ccession performed poorly in root development nd LA production with the exception of n Anden ben type IJR tht ws intermedite (Fig. 3A). Roots cn exert indirect control on lef growth which depends on the supply of cytokinins nd wter from roots (Lmbers et l. 1995). Results from short-term experiments indicted tht t low nutrient vilbility some species with high potentil root growth rte still grow fster thn those with low potentil root growth rte nd hve greter cpcity to cquire nutrients (Chpin 1980; Lmbers nd Poorter 1992; Ryser nd Lmbers 1995). Root trits cross experiments TRL ws nlysed t two levels of tretment both in hydroponic nd soil cylinder experiments. There ws strong liner reltionship between TRL per

16 Tble 8 Influence of individul nd combined stress fctors of cid soil (H high luminium, L low luminium) nd wter stress ( well wtered, wter stress) on root length distribution estimted by root length extinction coefficient b, tht describes the shpe of the cumultive root length distribution with depth, of 11 ben genotypes from three Phseolus species grown in soil cylinders (Tril 3) Genotype Individul stress Combined nd wter stress H L H L H L G Q G Q G Q G Q ICA Quimby b IJR b VAX 1 b SER 16 b VAX 3 b VAX 6 b G c Men LSD 0.05 NS 0.101* *** NS * * P. coccineus P. vulgris P. cutifolius plnt under 20 lm nd the control with 0 lm in hydroponic system (r = 0.94) (Fig. 3A). For soil cylinder system, the liner reltionship between TRL under L nd H sturtion ws lso strong (r = 0.84) (Fig. 3B). Four P. coccineus ccessions (G Q, G Q, G Q, nd G Q) showed greter vlues of TRL both with nd without in hydroponic system, nd in soil cylinder studies, three of these (G Q, G Q, nd G Q) mintined good root development with either L or H sturtion. The two screening methods were highly correlted (r = 0.85***) for TRL in H soil nd in nutrient solution with 20 lm (Fig. 3C), lthough correltion ws driven in lrge prt by the vlues of the P. coccineus ccessions. A significnt but lower correltion (r = 0.69*) ws found between TRL in L soil nd nutrient solution without in hydroponic system (Fig. 3D). Severl uthors hve exmined the reltionship of response with different screening methods with vrying results. Nrsimhmoorthy et l. (2007) compred three methods including hydroponics, soil, nd root stining for evlution of tolernce in Medicgo trunctul (Brrel Meic) germplsm nd found wek correltion, suggesting tht ech technique is distinct nd cnnot be substituted for ech other. A lrge discrepncy between hydroponics-bsed rtings of seedlings nd snd-culture-bsed rtings of soyben plnts ws found when tolernce ws expressed s percentge of controls, nd correltions between snd culture nd hydroponics-bsed results were found to be low (Villgrci et l. 2001). Horst nd Klotz (1990) compred 31 soyben genotypes using hydroponics nd soil systems nd detected positive though non-significnt reltionship (r = 0.79). In contrst, Cmpbell nd Crter (1990) demonstrted in their experiments good greement between tolernce rtings determined in solution culture nd pots with soil in the greenhouse when expressed s percentge of the control tretment. Recent reserch on penut indicted tht root chrcteristics of plnts grown in hydroponics were closely relted with those of plnts grown with soil in smll pot conditions (Girdthi et l. 2010). Hydroponic evlution identified the soyben cultivr Perry s sensitive even though it hd been found to

17 Tble 9 Influence of individul nd combined stress fctors of cid soil (H high luminium, L low luminium) nd wter stress ( well wtered, wter stress) on root biomss distribution estimted by root biomss extinction coefficient b, tht describes the shpe of the cumultive root biomss distribution with depth, of 11 ben genotypes from three Phseolus species grown in soil cylinders (Tril 3) Genotype Individul stress Combined nd wter stress H L H L H L G Q G Q G Q G Q ICA Quimby b IJR b VAX 1 b SER 16 b VAX 3 b VAX 6 b G c Men LSD 0.05 NS NS *** NS ** * P. coccineus P. vulgris P. cutifolius be tolernt in soil-bsed ssys with older plnts (Armiger et l. 1968; Foy et l. 1969, 1992; Devine et l. 1979; Spr et l. 1982; Horst nd Klotz 1990). VAX1 which ws found to be sensitive in hydroponic system ws found to be cid soil tolernt under field conditions becuse of its bundnt dventitious root system tht helped void toxicity. In our experience with the hydroponic system nd in -toxic cid soil, ech screening method permitted evlution of different spects of root behviour. For exmple, the hydroponic system enbled quntifiction of NRT (the most -sensitive prt of the root) which ws not possible with soil cylinders fter cutting the cylinders t different soil depths, wheres soil bsed screening reveled rooting bility to penetrte -toxic soil. In this sense the two methods were complementry. A strong reltionship ws observed between resistnce in hydroponics nd cid soil tolernce for some specific root trits. Significnt correltions were found between three root trits from hydroponic evlution (TRL, TPRL120h nd SRL) nd three other root trits from soil cylinder evlution (TRL, VRD nd MRD). Villgrci et l. (2001) concluded tht some genetic sources will lend themselves well to hydroponicsbsed screening while others my not. Of these trits, TRL nd VRD in soil re those tht we interpret s being the most relevnt mesures of root helth in n toxic environment. On the other hnd, no significnt correltion ws found between SRL in hydroponic nd soil cylinder experiments, nd genotypic rnking bsed on SRL in nutrient solution with 20 lm did not gree with the rnking in -toxic cid soil. While some trits express similrly in the two systems, others pprently do not. An unusul reltionship ws observed between SRL nd MRD. In the hydroponic system the correltion of SRL nd MRD ws highly negtive s expected: -0.84*** t 0 lm nd -0.87*** with 20 lm. With L stress in soil, the correltion of SRL nd MRD presented similr vlue: -0.79***. But in -toxic cid soil, the correltion ws slightly positive (0.27; NS). This difference reflects contrsting response of roots in soil s compred to the hydroponic system tht seems to be due to P. coccineus.

18 Tble 10 Lef re (LA), shoot dry weight (SDW) nd root to shoot rtio (R:S rtio) of 11 ben genotypes including 4 P. coccineus ccessions, 1 P. cutifolius nd 6 P. vulgris grown under individul nd combined stress of (H high luminium, L low luminium) nd wter stress conditions ( well wtered, wter stress) (Trils 2 nd 3) Genotypes Individul stress of (Tril 2) Individul nd combined stress of nd (Tril 3) LA (cm 2 ) SDW (g) R:S rtio LA (cm 2 ) SDW (g) R:S rtio H L H L H L L L H H L L H H L L H H G Q G Q G Q G Q I. Quimby b IJR b VAX 1 b SER 16 b VAX 3 b VAX 6 b G c Men LSD P. coccineus P. vulgris P. cutifolius b c