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Abenavoli Et Al-2016-Journal of Agronomy and Crop Science

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  DROUGHT STRESS Root Phenotyping For Drought Tolerance in Bean LandracesFrom Calabria (Italy) M. R. Abenavoli, M. Leone, F. Sunseri, M. Bacchi & A. Sorgon  a Dipartimento Agraria, Universit  a Mediterranea di Reggio Calabria, Reggio Calabria, Italy Keywords common bean; drought stress; landraces;root architecture; root morphology Correspondence A. Sorgon  aDipartimento AgrariaUniversit  a Mediterranea di Reggio CalabriaSalita MelissariI-89124 Reggio CalabriaItalyTel.: +390965324077Fax: +390965311092Email: asorgona@unirc.itAccepted January 23, 2015doi:10.1111/jac.12124 Abstract Common bean ( Phaseolus vulgaris ) is cultivated throughout Latin America andAfrica, and for the European community, in Italy and Spain, areas are mainly subjected to drought stress which is predict to worsen by regional climatic mod-els. The aims of this work were to identify the drought-tolerant and drought-sen-sitive bean landraces using drought tolerance and phenotypic plasticity indexesand to dissect the root morphological and 2D-architecture traits related todrought tolerance. Thirty-one landraces from diverse gene pools and areas of theCalabria region (South Italy), with different habits and morphological traits,were screened for drought tolerance in a hydroponic system. Root phenotypingwas conducted by image analysis. Drought tolerance screening identified twolandraces as drought tolerant and sensitive, respectively. Under drought stress,the drought-tolerant landrace exhibited several interesting root traits such as ahigher root length, surface area and, above all, the fineness of the whole rootsystems and, with emphasis, of the higher order roots. Drought stress inducedplastic root responses in both bean landraces but with contrasting patterns. Thedrought-tolerant landrace exhibited a dimorphic-rooted strategy, which could beincluded in future utility for bean breeding programmes in drought-proneenvironments. Introduction Common bean ( Phaseolus vulgaris ) is the most importantfood legume for the human diet providing proteins, vita-mins and minerals, especially in East and southern Africaand the Americas (Beebe 2012). In Europe, Italy and Spainare the main producer regions (FAOSTAT 2012).In developing countries, bean production is frequently subjected to drought (Thung and Rao 1999) that drastically reduces up to 60 % its yield (Beebe et al. 2011). Extensiveirrigation systems mitigate the negative impact of droughtstress in developed countries. However, the high consump-tion of irrigation water together with increasing frequency,intensity and duration of drought stress predicted by regio-nal climatic models in Europe (IPCC 2007) highlights theimportance to select high yielding drought-tolerant beancultivars. In bean, genetic improvement for drought toler-ance is based on worldwide germplasm genetic variability for adaptation to water deficiency (Mu ~ noz-Perea et al.2006, Beebe et al. 2008, Porch et al. 2009). In this context,Durango and Mesoamerica races belonging to MiddleAmerican gene pool provide high levels of drought toler-ance in common bean (Mu ~ noz-Perea et al. 2006, Beebeet al. 2008) as well as in the wild species,  P. acutifolius (Martinez-Rojo et al. 2007). However, a wide investigationin other wild species or landraces from different gene poolscould be useful to identify the morpho-physiological traitsrelated to drought tolerance. In particular, landraces adapted   to marginal areas could provide a valuable geneticsource for identifying drought-related traits for beanadaptation.Until now, the selection for drought tolerance in beanwas mainly based on yield and its components, phenotypicplasticity and morpho-physiological traits (Beebe et al.2013). Among the latter, root traits appeared to be morerelated to drought tolerance (Sponchiado et al. 1989,Mohamed et al. 2002, 2005) compared to the above-ground part, especially in bean (White and Castillo 1992).Nevertheless, root system has been less frequently considered as a source of drought tolerance traits in bean ©   2015 Blackwell Verlag GmbH,  202  (2016) 1–12  1 J Agro Crop Sci (2016) ISSN 0931-2250  breeding programmes (Beebe et al. 2013). Rooting depthand distribution are generally taken into account fordrought tolerance improvement (Sponchiado et al. 1989,Mohamed et al. 2002, 2005), although recently, an array of root traits or phenes have been also reported (Songsri et al.2008, Henry et al. 2012, Lynch and Brown 2012). Forexample, the root length ratio (RLR, root length per unit of the plant’s dry mass) is considered a better trait than rootlength for describing the plant’s potential for soil resourceacquisition under stress conditions (Ryser 1998) because itavoids the ‘allometric effects’ (Coleman et al. 1994) or the‘apparent plasticity’ (Weiner 2004). The RLR is constitutedby root mass ratio (RMR, root mass per unit of the plant’sdry mass), the allocation component, and root fineness(RF, root length per unit root volume) and tissue density (RTD, root dry mass per unit root volume), the structuralcomponents (Ryser 1998). Plants may produce longer rootseither by increasing biomass allocation or root finenessand/or reducing root tissue density, leaving biomass alloca-tion unchanged. Under drought stress condition, changesin the RTD were related to drought tolerance in sugar beet(Romano et al. 2013) as well as the RF was considered afunctional trait for drought-tolerant herbaceous tallgrassprairie species (Tucker et al. 2011) because it correlatedwith the root’s ability to take up water (Pem  an et al. 2006,Hern  andez et al. 2010, Rewald et al. 2011).The morphology of single root types has scarcely beenconsidered for the bean adaptation strategy to dry condi-tions. It is well known that different root types can be char-acterized by diverse anatomical, morphological andphysiological features (Waisel and Eshel 2002) which deter-mined different water uptake capacity among the root clas-ses (Rewald et al. 2011). Furthermore, the genetic variationin the growth rates of distinct root classes could be impor-tant for the plant’s adaptation to drought stress (Guo et al.2008, Hund et al. 2009). For example, different responsesamong root types in P uptake (Rubio et al. 2004), toleranceto combined P/drought stress (Ho et al. 2005) and rotresistance (Rom  an-Avil  es et al. 2004) have been observedin common bean.In this framework, the aims of this work were (i) theidentification of the drought-tolerant and drought-sensi-tive landraces in a bean collection from Calabria (SouthItaly) by specific drought indexes and (ii) the dissectionof root morphological traits mainly that related to the2D-architecture traits such as root orders, whorls andtips.Considering that the Mediterranean area is very suscepti-ble to the effects of climate changes especially in terms of frequency and magnitude of drought stress (IPCC 2007),the results of this study could allow to identify potentialdrought tolerant parents and root traits related to droughttolerance useful for the future bean breeding programmes. Materials and methods Screening for drought tolerance Plant material, growth condition and drought stress treat-ment  Thirty-one bean genotypes from different areas of Calabriaregion (South Italy) were selected based on variation forgene pools, site of srcin and habit (Mercati et al. 2013).Seeds were kindly provided by Agenzia Regionale Speri-mentazione e Servizi in Agricoltura (ARSSA  –   Calabria,Italy) (Table 1). A drought-tolerant American genotype, Table 1  Description, characteristics and code of the 31 Calabrianbean landraces, the drought-tolerant American genotype and thecommercial Italian variety used in this studyCode(ID#) Common NameSite ofsrcin 1 HabitusGenepool 2 1 Fagiolo uncino CS Climbing A5 Posa di montagna RC Climbing A12 Serpente CS Climbing A14 Suraca larga Unknown Climbing A17 A fava CS Climbing C18 Reniforme CS Climbing U24 Sbraca pasta RC Dwarf A25 Fasolu vasciu CS Dwarf M29 A cavolo Unknown Dwarf A31 Cannellino bianco CS Climbing A37 Fasolu quarantino CS Dwarf A40 Coc  o gialla CZ Dwarf A44 Fagiolo ciuncu 2008 CS Dwarf A45 Cannellino nano Italy Dwarf A49 Vovolacu CZ Climbing A50 Core di Ges  u CZ Climbing A51 Mangiatutto a granella CS Climbing A53 Sarrisa CZ Climbing A59 Selvaggia CZ Climbing U61 Vravalacu CZ Climbing A67 Povarella CZ Dwarf A70 Fagiolo cursuni dall’occhio CS Dwarf U78 Azzicca CS Climbing A83 Zicca o valana RC Climbing A85 Fag. bianco piccolo RC Climbing M87 Azzicca a caciumbalo CS Climbing A90 Cervineddu CS Climbing A91 Nicolisa CZ Climbing A92 Fagiolino RC Climbing M99 Favarula nera CS Climbing C100 Posa rossa di settembre RC Climbing A101 Monachella CZ Climbing ACO CO46348 USA Climbing A 1 Site of srcin: CS (Cosenza), CZ (Catanzaro), VV (Vibo Valentia), RC(Reggio Calabria). 2 Gene pool (Mercati et al. 2013): A, Andean; M, Middle American; C, P. coccineous ; U, Unknown. ©   2015 Blackwell Verlag GmbH,  202  (2016) 1–12 2 Abenavoli  et al.  CO46348 (Brick et al. 2008), kindly provided by Prof.Brick MA (Colorado State University, USA), and a com-mercial Italian variety (Cannellino nano) were used astesters.Seeds of bean genotypes were surface-sterilized for 2 minin 10 % NaOCl and germinated in the dark at 25  ° C, for2 d, in rolls paper soaked with 0.5 m M  CaSO 4  and thenplaced in growth chamber for 4 d under artificial light. Six days after seeding, six seedlings of uniform size of eachgenotype were transferred to the growing units (PVC tubes,4 cm diameter  9  50 cm height) each seedling in a separatetube. According to Fan et al. (2003), the aerated nutrientsolution (pH 6) contained 3 m M  KNO 3 , 2 m M  Ca(NO 3 ) 2 ,0.5 m M  MgSO 4 , 0.5 m M  (NH 4 ) 2 HPO 4 , 0.05 m M  Fe-EDTA,0.05 m M  KCl, 0.025 m M  H 3 BO 3 , 2  l M  MnSO 4 , 2  l M ZnSO 4 , 0.5  l M  CuSO 4  and 0.5  l M  (NH 4 ) 6 Mo 7 O 24 . Thenutrient solution was renewed every 2 days, and the pHwas daily adjusted to 6.0 by 0.1 N KOH. The growing unitswere then placed in growth chamber at 25/18  ° C and 70 %relative humidity and a 14/10 h light/dark cycle (PPFDabove shoot 300  l E m  2 s  1 ).The drought stress was simulated by the variation of theosmotic potential by adding the polyethylene glycol (PEG)in the nutrient solution. In particular, at phenologicalphase V3 (first true leaf), in drought stress plants (D),224 g l  1 of PEG 8000 (Sigma Aldrich P2139, St. Louis,MO, USA) was added to the nutrient solution to reach anosmotic potential of    0.6 MPa calculated using the equa-tion of Michel (1983). The final PEG concentration wasgradually achieved by the addition of 81.2 g l  1 PEG every 6 h. In control plants (C), the nutrient solution wasrenewed with the same above composition. Growth parameters and leaf morphology  At 0, 8, 24, 48, 56, 72, 144, 168 and 192 h from PEG treat-ment, the stem length (StL, cm) and leaf area (LA, cm 2 )were measured in each seedling. Stem elongation rate(StER, cm h  1 ) and leaf area expansion rate (LAER,cm 2 h  1 ) were calculated by the linear regression slopes of StL  vs  time and LA  vs  time.After 192 h of PEG treatment, the seedlings were har-vested and separated in leaves and stems, which wereplaced in an oven at 70  ° C for 2 days to determine the leaf (LDW, g) and stem dry weight (StDW, g), respectively. Theshoot dry weight (ShDW, g) was calculated by the sum of StDW and LDW. Finally, the leaf mass per area (LMA,g cm  2 ) was calculated by LDW/LA ratio (Mat  ıas et al.2012). Leaf relative water content  After 192 h of PEG treatment, one terminal leaflet of eachgenotype from C and D plants was collected and immedi-ately weighed (LFW, g), dipped in deionised water and leftin the dark for 48 h. Afterwards, they were again weighedto measure the leaf turgid weight (LTW, g) and then placedin an oven at 70  ° C for 48 h to determine the dry weight(LDW). The relative water content (RWC, %) of the leaf was calculated according to Gonz  alez and Gonz  alez (2003):RWC  ¼ ð LFW    LDW Þ = ð LTW    LDW Þ Phenotypic plasticity and drought stress indexes The ‘response coefficient’ (RC) as phenotypic plasticity index (Poorter and Nagel 2000) was calculated for eachgenotype with the following ratio:RC  ¼  V  C  = V  D where  V  C   and  V  D  represent the average values of traitsobtained under control and drought stress conditions. TheShDW, LDW, StER, LAER, RWC and LMA were the traitstook in account.The RC values equal to 1 indicated no response todrought stress, while RC < 1 and RC > 1 indicated the toler-ance and susceptibility to drought stress, respectively.The drought tolerance index (DTI) (Fernandez 1992,Ober et al. 2004) and the drought tolerance efficiency (DTE) (Fischer and Wood 1981) were calculated accordingto the following formulas:DTI  ¼ ð DM D = DM C  Þ = ð ADM D = ADM C  Þ DTE  ¼  DM D = DM C  where DM D  and DM C   are the dry weights of each genotype,and ADM D  and ADM C   are the average values of dry weights of shoot and leaves of all genotypes under droughtand control conditions, respectively. Root phenotyping  Plant material, growth condition and drought stress treat-ment  The #25 and #44 landraces, drought tolerant and droughtsensitive, respectively, were used in additional experimentson which growth condition and drought stress treatmentwere the same described in the screening for drought toler-ance.  Morphological root analysis After 192 h from PEG treatment, the seedlings of eachgenotype and drought stress treatment were harvested andseparated in shoots and roots. The root systems werestained with 0.1 % toluidine blue solution for 5 min andthen scanned at a resolution of 300 dpi (WinRhizo STD1600, Instruments R   egent Inc., Canada). WinRhizo Pro v.4.0 software package (Instruments R   egent Inc., CheminSainte-Foy, Qu  ebec, Canada) was used to measure the ©   2015 Blackwell Verlag GmbH,  202  (2016) 1–12  3 Root Phenotyping of Bean Landraces in Drought Condition  following parameters: length (L, cm), surface area (SA,cm 2 ) and volume (V, cm 3 ) of the basal (B), tap (T), basallateral (BL) and tap lateral (TL) roots. Further, the rootlength distribution among the following root classes diame-ter, as defined by Bohm (1979), was obtained: very fine(VF, 0  –  0.5 mm), fine (F, 0.5  –  1 mm) and large (L,  > 1mm).Images were used to count the number (N, n.) of the basalroots, the laterals of the basal and tap roots and the rootwhorls. Shoot (ShDW, g) and root (RDW, g) dry weightswere measured after oven-drying at 70  ° C for 48 h. Plantdry weight (PDW, g) was calculated by the sum of ShDWand RDW.Based on the above measurements, root length ratio(root length/whole plant dry weight, cm g  1 ), root massratio (root dry weight/whole plant dry weight, g g  1 ), rootfineness (root length/root volume, cm cm  3 ) and root tis-sue density (root dry weight/root volume, g cm  3 ) werecalculated. Statistical analysis A completely randomized design with three replicates forgenotype and treatment has been utilized. All data weretested for normality (Kolmogorov   –  Smirnoff test) andhomogeneity of variance (Levene median test) and, whererequired, the data were transformed.All parameters were analysed by two-way analysis of var-iance with the genotype and treatment level (control anddrought stress) as main factors. Subsequently, Tukey’s testwas used to compare the means of all parameters of eachgenotype and treatment level. For the RC values, the statis-tical significance was obtained by the probability level(P  <  0.05) of the Genotype  9  Treatment (G  9  T) interac-tion.Statistical analysis of the data was carried out using SPSSStatistics v. 15.0 (IBM Corp., Armonk, NY, USA) while thegraphics were prepared using SigmaPlot v. 8.0 (Jandel Sci-entific, San Rafael, CA, USA). Results Screening for drought tolerance The screening for drought tolerance among bean genotypeswas carried out by two different approaches: the ‘pheno-typic plasticity’ and the ‘tolerance indexes’.The first approach took into account the ‘response coef-ficient’ as phenotypic plasticity index (Poorter and Nagel2000) while  ANOVA  (genotype  9  environment interaction)represented a statistical evaluation of the genotype’s plastic-ity (Valladares et al. 2006) in response to drought stress.The phenotypic plasticity was calculated considering differ-ent drought-related traits such as growth parameters (shootand leaf dry weight, stem elongation and leaf areaexpansion rates), plant water status (leaf relative watercontent) and leaf morphology (leaf mass per area).The shoot dry weight significantly varied among thebean genotypes (0.122  –  1.078 g; P  <  0.001) and was signifi-cantly reduced by drought stress (P  <  0.001), (Table 2).G  9  T interaction of the ShDW was also statistically signif-icant (P  <  0.05), (Table 2), indicating a different response Table 2  Shoot and leaf dry weight and phenotypic plasticity (RC,response coefficient) of bean genotypes grown for 8 days with (D) orwithout 6 % PEG (C) in nutrient solutionGenotypes(ID#)Shoot dry weight (g) Leaf dry weight (g) C D RC C D RC  1 0.524 0.360 1.45 0.310 0.191 1.625 0.399  0.180  2.22 0.265  0.094  2.8112 0.297 0.307 0.97 0.176 0.192 0.9214 0.276 0.238 1.16 0.141 0.119 1.1917 0.705 0.630 1.12 0.368 0.311 1.1818 0.527 0.482 1.09 0.226 0.150 1.5124 0.287 0.232 1.24 0.176 0.137 1.2825 0.310 0.403 0.77 0.211 0.266 0.7929 0.448 0.282 1.59 0.306 0.182 1.6831 0.996  0.480  2.08 0.619  0.267  2.3237 0.376 0.330 1.14 0.251 0.220 1.1440 0.343 0.283 1.21 0.224 0.187 1.2044 0.404  0.186  2.18 0.232  0.075  3.1045 0.420 0.293 1.43 0.239 0.141 1.6949 0.571 0.438 1.30 0.349 0.240 1.4550 0.581  0.389  1.49 0.339 0.226 1.5051 0.326 0.312 1.04 0.196 0.187 1.0553 0.320 0.294 1.09 0.210 0.190 1.1059 0.592 0.463 1.28 0.369 0.264 1.4061 0.443  0.144  3.08 0.223 0.076 2.9667 0.446 0.391 1.14 0.276 0.234 1.1870 0.151 0.122 1.24 0.089 0.062 1.4378 0.350 0.303 1.16 0.220 0.192 1.1583 0.302 0.336 0.90 0.183 0.193 0.9585 0.491  0.278  1.77 0.294  0.139  2.1287 0.448 0.361 1.24 0.282 0.204 1.3890 0.670  0.340  1.97 0.451  0.219  2.0691 0.510 0.578 0.88 0.327 0.354 0.9292 0.369 0.324 1.14 0.241 0.207 1.1699 1.078  0.597  1.81 0.537  0.209  2.57100 0.635  0.440  1.44 0.395  0.236  1.68101 0.313 0.283 1.11 0.163 0.142 1.15CO 0.496 0.359 1.38 0.291 0.186 1.57Statistic 1 G 7.12*** G 4.20***T 38.42*** T 43.62***G  9  T 1.55* G  9  T 1.48*Bold: statistical difference between the mean of each bean germplasmat drought condition respect to the control plants (P  <  0.05, test ofTukey). 1 Statistical analysis:  ANOVA  two-way ( G , genotypes;  T  , treatment withPEG;  G  9  T  : genotypes  9  treatment interaction);  * 0.05  >  P  <  0.01; ** 0.01  >  P  <  0.001;  *** 0.001  >  P; NS not significant. ©   2015 Blackwell Verlag GmbH,  202  (2016) 1–12 4 Abenavoli  et al.
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