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Effects of shading on chlorophyll content, chlorophyll fluorescence and photosynthesis of subterranean clover

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Effects of shading on chlorophyll content, chlorophyll fluorescence and photosynthesis of subterranean clover
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  MISCELLANEOUS Effects of Shading on Chlorophyll Content, ChlorophyllFluorescence and Photosynthesis of Subterranean Clover R. P. Mauro, A. Occhipinti, A. M. G. Longo & G. Mauromicale Department of Agronomical, Agrochemical and Animal Production Sciences (DACPA), University of Catania, Catania, Italy Introduction Subterranean clovers represent a group of annual, pre-dominantly autogamous, self-reseeding pasture legumes,native to the Mediterranean Basin and temperate WesternEurope (Morley 1961). Among the three (possibly four)species making up the group,  Trifolium subterraneum  L.and  Trifolium brachycalycinum  Katzn. et Morley are themost widely distributed across the species’ centre of ori-gin (Pecetti and Piano 1998, Talamucci 2002). Their abil-ity to persist over many seasons, along with their rusticity and high nutritional feed value, has ensured their world-wide success as an introduced pasture species, and cur-rently they occupy some 53 Mha of improved pastureland (Talamucci 2002). Over the last decades, subterra-nean clovers have attracted renewed attention for covercropping in Mediterranean environments, as they rapidly achieve dense ground cover, show allelopathy againstweeds and generate long-lasting swards (Enache andIlnicki 1990, Ingels et al. 1998, Mauromicale et al. 2010).Over time, such swards would be expected to increase theorganic matter and nitrogen content of the soil, reducethe soil erosion and nitrogen leaching, release the previ-ously non-available soil phosphorus, encourage the gener-alist arthropod populations and heartworm communitiesor reduce pest and weeds pressure towards the cash crops(Bugg et al. 1990, Wyland et al. 1996, Campiglia 1999,Kamh et al. 1999, Mitchell et al. 1999, Hiltbrunner et al.2007, den Hollander et al. 2007, Campiglia et al. 2009,Pelosi et al. 2009). To provide most of the ecological ben-efits expected, cover crops must produce an adequateamount of biomass during their growth cycle, which, inturn, is the result of the species’ adaptation to the growthenvironment (Lu et al. 2000, Mauromicale et al. 2010).Modern Mediterranean orchards are characterized by high tree densities, so cover crops must be tolerant of much reduced light levels. Both inter and intra-specificvariation for the ability to thrive under conditions of low  Keywords cover cropping; photosynthesis; plant dryweight; shading;  Trifolium brachycalycinum ; T. subterraneum Correspondence G. MauromicaleDepartment of Agronomical, Agrochemicaland Animal Production Sciences (DACPA),University of Catania, Via Valdisavoia 5,95123 – Catania, ItalyTel.: +39 095 234 409Fax: +39 095 234 449Email: g.mauromicale@unict.itAccepted June 4, 2010doi:10.1111/j.1439-037X.2010.00436.x Abstract A field-experiment (2004/2005 and 2005/2006 seasons) was conducted in thecoastal plain of south-eastern Sicily (37  03 ¢ N, 15  18 ¢ E, 15 m a.s.l.), on a Cal-cixerollic Xerochrepts soil, aimed at quantifying the effect of shading on chlo-rophyll (Chl) content, Chl fluorescence, photosynthesis and growth of subterranean clover. Four levels of photosynthetically active radiation reduction(from 0 % to 90 %) were tested on  Trifolium brachycalycinum  cv. ‘Clare’ and Trifolium subterraneum  ecotype ‘Ragalna’. In both species shading progressively increased  F  v  / F  m , internal CO 2  concentration, diffusive leaf resistance and spe-cific leaf area (up to 8 %, 34 %, 18 % and 68 %, respectively), and decreasedChl content,  T  max  , photosynthetic rate and plant dry weight (up to 9 %, 24 %,79 % and 39 %, respectively). As plants aged, characteristic bell-shaped trendswere evident for photosynthetic parameters, with  F  v  / F  m  increasing up until theonset of flowering, and thereafter declining. This implies that  F  v  / F  m  may be auseful indicator of earliness in subterranean clover genotypes. The abovegrounddry biomass response to shading was both genotype- and season-dependent,but was predictable from the measurement of relative leaf Chl content. More-over, our results suggest that an improvement in the interaction betweenhost-rhizobium may represent a major potential breeding target for enhancingsubterranean clover tolerance to shading. J. Agronomy & Crop Science (2011) ISSN 0931-2250 ª  2009 Blackwell Verlag GmbH,  197  (2011) 57–66  57  irradiance have been widely documented (Baig et al.2005, Mu et al. 2010). Although some authors (Black 1957, Rossiter 1978, Balocchi and Phillips 1997) reportedthat subterranean clover growth is negatively affected by low irradiance, no breeding programmes had been under-taken to date towards enhancing its shading tolerance forcover cropping purposes.Over the last decades, developments in instrumentationand methodology have improved the precision of esti-mates of chlorophyll (Chl) content and fluorescence. As aresult, significant advances have been made in definingselection criteria in a multitude of field crops (Araus et al.1998, Earl and Tollenaar 1999, Lima et al. 1999, Zhanget al. 2000). Chl fluorescence is a quantitative and quali-tative indicator of light-dependent photosynthetic pro-cesses, and has been suggested as a screening method toimprove heat tolerance of some tropical fruit crops(Yamada et al. 1996), drought tolerance in durum wheat(Flagella et al. 1995) and heat tolerance in bean (Petkovaet al. 2007). The combined measurements of both relativeChl content and Chl fluorescence in the field have beenexploited to predict genotype adaptability, to describeplant ageing pattern in potato grown in Mediterraneanenvironment, and to elucidate the effects of the weed ho-loparasite  Phelipanche ramosa  (L.) Pomel (syn.:  Orobancheramosa  L.) infection on the photosynthetic machinery of greenhouse tomato plant (Mauromicale et al. 2006, 2008).The aim of this current research was to investigate theresponse of two subterranean clover entries to shading, by measuring their Chl content and fluorescence, photosyn-thetic rate and plant growth. We also set out to explorethe possibility of identifying physiological parameters,which could be employed as predictive tools in selectionprogrammes. Materials and Methods Site, climate and soil A field-experiment was conducted over two growing sea-sons (2004/2005 and 2005/2006) on the coastal plain of south-eastern Sicily (37º03 ¢ N, 15º18 ¢ E, 15 m a.s.l.), anarea where  Citrus  spp. orchards and table-grape vineyardsare common. The local climate is characterized by mildwet winters, and warm dry summers. Winter frosts arerare (two events over 30 years). Over the period 1959–1988, mean maximum day and minimum night tempera-tures during January were, respectively, 15.3 and 7.5   C,and the average annual rainfall was 447 mm. The moder-ately deep soil (   85 cm) is a Calcixerollic Xerochrepts(USDA, Soil Taxonomy), which at the beginning of theexperiment comprised 28 % clay, 25 % silt, 45 % sand,2 % organic matter, 1.8 &  total nitrogen, 68 mg kg ) 1 assimilable P 2 O 5  and 537 mg kg ) 1 exchangeable K 2 O. Thesoil pH was 8.4, its moisture capacity was 29 % of dry soil, and its wilting point was 11 %. All soil analyses wereperformed according to procedures approved by the Ital-ian Society of Soil Science. Experimental design, plant material and managementpractices The experiment was set out as a randomized split-plotdesign with four replications. The main treatments com-prised four imposed shading levels (removal of 0 %,40 %, 60 % and 90 % of sunlight, hereafter  S 0 ,  S 40 ,  S 60 and  S 90 ), with  T. brachycalycinum  cv. ‘Clare’ and  T. sub-terraneum  ecotype ‘Ragalna’ as sub-plots. ‘Ragalna’ seedwas obtained from the germplasm collection held at theDepartment of Agronomical, Agrochemical and AnimalProduction Sciences, University of Catania, the originalaccession having been collected from the Mount Etnaarea (Mauromicale et al. 1997). ‘Clare’ is a cultivarreleased in Australia in 1950 (Caporali and Campiglia2001). These entries were chosen because of their rapidgrowth, their similar height (  26 cm) but different leaf area (highest in ‘Clare’) and their ability to generatedense swards (Pardini et al. 1995, Mauromicale et al.1997, 2010). Shading was imposed by the erection of black polyethylene netting (‘Ombra 40’, ‘Ombra 60’ and‘Ombra super 90’, supplied by Carretta Tessitura Berg-amo, Italy) at   1.8 m above ground level. The mainplots were oriented in an E–W direction, and the nettingwas trained down to 0.3 m above ground level at eachend to avoid lateral irradiation and to minimize thedevelopment of microclimate variation within the plots.The effectiveness of the shading was tested on a monthly basis (three measurements per day inside and outsideeach shading structure) with a solarimetric bar (LicorLine Quantum LQA, One Meter Sensing Length; manu-factured by LI-Cor Inc., Lincoln, NE, USA). The sub-plotdimensions were 4  ·  4 m, while the main plots werespaced 3 m each other, to minimize reciprocal shadeinterference. The seedbed was shallow-tilled shortly before sowing by hand in September 2004 with 500 ger-minable seeds m ) 2 . After sowing, the beds were harrowedand uniformly rolled. Prior to seeding, 20 kg ha ) 1 N and50 kg ha ) 1 P 2 O 5  were incorporated into the soil. As theexperimental field hosted  Trifolium  spp. cultivations inthe previous seasons, no supplemental  Rhizobium  wasgiven. To promote crop establishment, the plots weresprinkler-irrigated to field capacity ( ) 0.03 MPa measuredwith tensiometers at 25 cm depth) on 28 September2004 and 22 September 2005. Otherwise, the only inter-ventions were occasional hand weeding throughout thetrial. Mauro  et al. 58  ª  2009 Blackwell Verlag GmbH,  197  (2011) 57–66  Meteorological measurements Air temperature (minimum, maximum and mean), rela-tive humidity (minimum, maximum and mean), soiltemperature at 20 cm (minimum, maximum and mean),wind direction and speed, global radiation, photosyntheti-cally active radiation (PAR), rainfall and evaporation wererecorded every half hour with a meteorological station(Mod. Multirecorder 2.40; ETG, Firenze, Italy) sited  15 m from the experimental field. Under the netting,mean maximum and minimum day temperatures wererecorded once a day, using conventional minimum andmaximum thermometers. Chlorophyll fluorescence and Chl content measurements Chlorophyll fluorescence parameters ( F  v  / F  m,  which repre-sents the ratio between variable and maximum fluores-cence, and  T  max  , the time at which fluorescence peaked)were recorded in the field, from non-detached leaves,with a portable fluorescence induction monitor (Fi m 1500; Alma Group Company, Hoddesdon, Herts, UK)over a period between 119 and 209 days after starter irri-gation (DAS) in the first season, and between 135 and210 DAS in the second season. The instrument’s clip wasfixed to 10 randomly chosen subclover plants growing inthe centre of each subplot. Measurements (saturationirradiance up to 3000  l mol m ) 2 s ) 1 ) were made between11:00 and 13:00 h on the adaxial side of the youngestfully expanded leaf, after a 20-min dark adaptation per-iod. Relative Chl content was measured using a portablechlorophyll meter (SPAD 502; Minolta Camera, Osaka,Japan). Before each set of measurements, the instrumentwas conveniently calibrated according to manufacturer’sinstructions. At each measurement date, 10 readings persubplot were taken between 146 and 209 DAS (the firstseason) and between 135 and 210 DAS (second season),from the adaxial side of the tallest expanded central leafletbelonging to the same plants used to measure Chl fluo-rescence; measurements avoided to place the Chl meterover central leaflets veins (Markwell et al. 1995). To mini-mize interactions with either plant water status and/ornatural irradiance level (Hoel and Solhaug 1998, Martı´nezand Guiamet 2004), SPAD measurements were made inthe morning, starting at 08:00 h. Photosynthetic rate, intercellular CO 2  concentration anddiffusive leaf resistance In the first season, the instantaneous leaf photosyntheticrate (Ph r ), intercellular CO 2  concentration ( C  i ) and diffu-sive leaf resistance ( R s ) were measured around 12:00 hinside a 250 cm 2 chamber in the closed circuit mode of aLicor Li-6200 Portable Photosynthesis system (LI-COR Inc.) over the period 119-209 DAS. Measurements weretaken from the youngest fully expanded leaf, generally onclear sunny days where PAR lays in the range 1500–1900  l mol photons m ) 2 s ) 1 . At each time point, dupli-cate measurements were taken from the same leaf of fiveof the plants per sub-plot previously used for Chl fluores-cence measurements. Plant dry weight and specific leaf area In both seasons, at 210 DAS, randomly sampled 0.25 m 2 patches were cut   1 cm above ground level, and plantswere counted. Subsamples of 50 plants were used to esti-mate the fresh weight and the leaf area (Area Measure-ment System; Delta-T Devices LTD, Burwell, Cambridge,UK), then they were oven-dried at 105   C for 72 h todetermine the dry weight. Specific leaf area was calculatedas the ratio between leaf area and dry weight. Statistical analysis Bartlett’s test was adopted to check for homoscedasticity,following which the data were subjected to a three-way  anova  for split-plots, applying a factorial combination of ‘shading level  ·  genotype  ·  measurement date’. Whenneeded, separated two-way   anova s were conducted ateach measurement date, as a factorial combination of ‘shading level  ·  genotype’. Aboveground dry biomass perplant and specific leaf area were subjected to a two-way  anova , considering the growing season as a randomeffect (Gomez and Gomez 1984). Means were separatedon the basis of Fisher’s protected LSD test (P  £  0.05). Foreach genotype tested, a correlation analysis was used todefine the relationship between plant dry weight and vari-ous physiological variables. Rainfall and temperature during the trial Total rainfall in the 2004–2005 season fell mostly betweenOctober and March (355 of 454 mm), as is typical for theMediterranean climate. The 2005–2006 season was ratherunusual, in that the rainfall amount was high (572 mm)and 81 % of it fell between October and January. Shadingprogressively reduced both the maximum and minimummean monthly temperatures by, respectively, 0.2–4.4   Cand 0.1–3.9   C, with the highest reduction affecting theperiod from June to August. All temperatures lay withinthe optimal range for subterranean clover growth, sinceacross the whole 2-year period, the mean maxima rangedfrom 22.9 to 26.7   C during emergence (October), andfrom 19.4 to 21.6   C during flowering (April), while inJanuary, the minima were above 5.8   C. Effects of Shading on Subterranean Clover ª  2009 Blackwell Verlag GmbH,  197  (2011) 57–66  59  Results Effect of shading on Chl content and Chl fluorescence The studied factors, namely shading level, genotype andmeasurement date, significantly affected Chl content,  F  v  / F  m  and  T  max  , in 2004–2005 season (Table 1). Indeed, inthe first season, Chl content decreased linearly as the levelof shading was increased, falling from 41.0 ( S 0 ) to 37.5( S 90 ) SPAD units (Table 2). In the second season, theshading depressed Chl content (39.4 SPAD units for  S 0 and 35.7 for  S 90 ) (Table 3) as well as reduced  T  max   (the S 90  level was 24 % less than the  S 0  level in the first seasonand 12 % lower in the second) (Tables 2 and 3). On theother hand,  F  v  / F  m  was positively correlated with thedegree of shading (the S 90  level was 8 % above that of   S 0 in the first season, and 7 % above in the second) Table 2  Chlorophyll (Chl) content, Chlfluorescence and photosynthetic variables ofsubterranean clover as affected by shadinglevel, genotype and measurement date(the first season)TreatmentChl content[SPAD units]  F  v  /  F  m  T  max  [ms]Ph r  l molCO 2  m ) 2 s ) 1 C  i  l molCO 2  mol ) 1 R s  s cm ) 1 Shading level S  0  41.0 a 0.780 c 415 a 11.3 a 189 d 2.2 b S  40  39.3 b 0.813 b 350 b 9.0 b 211 c 2.4 ab S  60  38.8 bc 0.821 ab 322 bc 7.4 c 228 b 2.6 a S  90  37.5 c 0.832 a 316 c 2.4 d 253 a 2.6 aTrend L** Q** Q** Q** L* Q**Genotype Trifoliumbrachycalycinum  ‘Clare’40.1 a 0.822 a 331 b 7.9 a 209 b 2.4 a Trifolium subterraneum  ‘Ragalna’38.2 b 0.801 b 370 a 7.2 b 234 a 2.4 aMeasurement date [DAS]119 – 0.794 c 427 a 9.3 a 205 c 3.2 a146 41.0 a 0.814 ab 402 a 9.1 a 206 c 1.9 b190 39.4 b 0.829 a 316 b 7.3 b 223 b 1.7 b209 37.0 c 0.808 bc 257 c 4.4 c 249 a 3.1 aTrend Q*** Q** L* Q* Q* Q*Different letters within columns and within factors indicate significantly different means accord-ing to Fisher’s protected LSD test (P  £  0.05). L, linear; Q, quadratic. *P  £  0.05; **P  £  0.01;***P  £  0.001. Relationship tested by regression analysis between shading level or measurementdate and responses of each variable. Table 1  F  -values of the main factors andtheir interaction on the observed variables,with the significance resulting from the  ANOVA VariableSource of variationShading (S) Genotype (G)Measurementdate/Season (M) (S)  ·  (G) (S)  ·  (M) (G)  ·  (M)2004–2005 seasonChl content 6.6*** 11.9*** 16.9*** NS NS NS F  v  /  F  m  16.6*** 7.1* 7.1*** NS NS NS T  max  16.8*** 11.2** 50.0*** NS NS NSPh r  190.0*** 7.2** 66.9*** NS NS NS C  i  84.7*** 75.0*** 28.3*** NS NS NS R s  2.8 * NS 52.1*** NS NS NS2005–2006 seasonChl content 6.8*** 6.6* 104.3*** NS NS NS F  v  /  F  m  26.3*** NS 11.9*** NS NS 4.4** T  max  5.8** NS 36.3*** NS NS NS2004–2006 seasonPlant dry weight 83*** NS – NS NS 7**Specific leaf area 15*** 23*** – NS NS NSNS: not significant; *, **, ***significant at P  £  0.05, 0.01 and 0.001, respectively.Mauro  et al. 60  ª  2009 Blackwell Verlag GmbH,  197  (2011) 57–66  (Tables 2 and 3). While Chl content and  T  max   progres-sively decreased with plant age,  F  v  / F  m  tended to increaseup until the onset of flowering, peaking at 190 DAS inthe first season and at 184 DAS in the second (Tables 2and 3). The performances of the two accessions wasmarkedly different in the first season when, averaged overthe other factors, ‘Clare’ had a significantly higher Chlcontent and  F  v  / F  m , but a lower  T  max   (Table 2). In thesecond season, ‘Ragalna’ showed a higher Chl contentthan ‘Clare’, but its  F  v  / F  m  time-course was different(Fig. 1). Effect of shading on photosynthetic rate (Ph r ), intercellu-lar  C  i  and diffusive leaf resistance ( R s ) Shading significantly affected Ph r  and  C  i , contributing alarge part of the overall variance for these traits; on theother hand, measurement date provided the major sourceof variation for  R s  (Table 1). As the level of shadingincreased from  S 0  to  S 90 , Ph r  fell by 79 % (from 11.3 to2.4  l mol CO 2  m ) 2 s ) 1 ), while  C  i  rose by 34 % (from 189to 253  l mol CO 2  mol ) 1 ) and  R s  by 18 % (from 2.2 to2.6 s cm ) 1 ) (Table 2). As the plants aged, Ph r  tended todecrease, falling by 9.3 (119 DAS) to 4.4  l molCO 2  m ) 2 s ) 1 (209 DAS), a trend which was opposite tothat experienced by   C  i  (Table 2).  R s  decreased progres-sively up to the onset of flowering, and thereafterincreased (Table 2). Irrespective of the other factors,‘Clare’ performed better than ‘Ragalna’, achieving both ahigher Ph r  (7.9 and 7.2  l mol CO 2  m ) 2 s ) 1 , respectively)and a lower  C  i  (209 and 234  l mol CO 2  mol ) 1 ) (Table 2). Effect of shading on plant dry weight and specificleaf area Plant dry weight and specific leaf area were both signifi-cantly affected by shading and season (Table 1). In thefirst season, the former decreased progressively in responseto shading from 1555 ( S 0 ) to 1390 ( S 60 ), and finally to949 mg DM plant ) 1 under the  S 90  level (Table 4). Theseshade treatment effects were even stronger in the secondseason, decreasing plant dry weight by 247 ( S 40 ), 266 ( S 60 )and 706 mg DM plant ) 1 ( S 90 ) (Table 4). As this result, inthe second season, plant dry weight under  S 90  was 4.6-fold Table 3  Chlorophyll (Chl) content and Chl fluorescence variables ofsubterranean clover as affected by shading level, genotype and mea-surement date (the second season)TreatmentChl content[SPAD units]  F  v  /  F  m  T  max  [ms]Shading level S  0  39.4 a 0.792 c 311 a S  40  38.9 ab 0.821 b 295 ab S  60  37.5 b 0.833 b 290 bc S  90  35.7 c 0.852 a 274 cTrend L*** L* L*Genotype Trifoliumbrachycalycinum  ‘Clare’37.0 b 0.825 a 287 a Trifolium subterraneum  ‘Ragalna’38.8 a 0.824 a 298 aMeasurement date [DAS]135 41.4 a 0.818 b 341 a158 42.4 a 0.833 a 301 b184 39.3 b 0.843 a 278 c210 28.4 c 0.804 b 250 dTrend Q** Q*** L*Different letters within columns and within factors indicate signifi-cantly different means according to Fisher’s protected LSD test(P  £  0.05). L, linear; Q, quadratic. *P  £  0.05; **P  £  0.01;***P  £  0.001. Relationship tested by regression analysis betweenshading level or measurement date and responses of each variable 0.750.800.850.90 Days after starter irrigation      F    v      /     F    m 2005–2006 Season TB TS 0.750.800.850.90 Days after starter irrigation      F    v      /     F    m 2004–2005 Season NSNSNS TB TS 119 190 209 146100135 158 184 210 100 (a)(b) Fig. 1  The  F  v  /  F  m  trend of the two subterranean clover genotypes dur-ing plant ageing, in 2004–2005 (a) and 2005–2006 (b) seasons. Verti-cal bars indicate the LSD values (P  £  0.05). T B ,  T. brachycalycinum ‘Clare’; T S ,  T. subterraneum  ‘Ragalna’; NS, not significant. Effects of Shading on Subterranean Clover ª  2009 Blackwell Verlag GmbH,  197  (2011) 57–66  61
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