new for review only · 2018. 6. 4. · for review only 1 red clover varieties with nitrogen fixing...

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Red Clover Varieties with Nitrogen Fixing Advantage During

the Early Stages of Seedling Development

Journal: Canadian Journal of Plant Science

Manuscript ID CJPS-2017-0071.R2

Manuscript Type: Article

Date Submitted by the Author: 01-Oct-2017

Complete List of Authors: Thilakarathna, Malinda ; University of Guelph, Plant Agriculture Papadopoulos, Yousef; Agriculture and Agri-Food Canada, Grimmett, Mark; Federal Government, Agriculture & Agri-Food Canada Fillmore, Sherry; Agriculture Agri-food Canada, Kentvile RDC Crouse, Matthew; Agriculture and Agri-Food Canada, Dalhousie University, Faculty of Agriculture, PO Box 550 100-5 Haley Institute

Prithiviraj, Balakrishnan ; Nova Scotia Agricultural College, Environmental Sciences

Keywords: root nodules, root exudates, red clover, genotypic variability, Nitrogen

https://mc.manuscriptcentral.com/cjps-pubs

Canadian Journal of Plant Science

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Red Clover Varieties with Nitrogen Fixing Advantage during the Early Stages

of Seedling Development

M.S. Thilakarathna1,2, Y.A. Papadopoulos3*, M. Grimmett4, S.A.E. Fillmore5, M. Crouse3, and B.

Prithiviraj6

1 Department of Biology, Dalhousie University, Halifax, NS, Canada B3H 4J1

2 Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada N1G 2W1

3 Agriculture & Agri-Food Canada, Faculty of Agriculture, Dalhousie University, Truro, NS,

Canada B2N 5E3

4 Agriculture & Agri-Food Canada, Charlottetown, PEI, Canada CIA 4N6

5 Agriculture & Agri-Food Canada, Kentville, NS, Canada B4N 1J5

6 Department of Plant Food and Environmental Sciences, Faculty of Agriculture, Dalhousie

University, Truro, NS, Canada B2N 5E3

3* Corresponding author: Yousef A. Papadopoulos

E-mail address: [email protected]

Telephone: +1 902-896-2452

Fax: +1 902-895-6734

Number of figures = 1

Number of tables = 4

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Abstract 1

Plant and environmental factors affect root nitrogen (N) exudation dynamics in legumes. To 2

better understand the genotypic variability and plant factors affecting root N release nodulation, 3

plant growth, tissue N content, and root N exudation of six (three diploid and three tetraploid) 4

red clover (Trifolium pratense L.) varieties were evaluated under controlled environmental 5

conditions during the first eight weeks of plant growth after rhizobia inoculation. Genotypic 6

differences were found for nodulation, plant dry weight, leaf area, root attributes (root length, 7

surface area, volume, and diameter), shoot and root N concentration, and N content. Genotypic 8

differences were found for root exudate N content in terms of NO3--N, NH4

+-N, and dissolved 9

organic N (DON). In general, root exudate inorganic N content was greater in tetraploid varieties 10

than in the diploids throughout the growth period. Root exudate DON content was greater than 11

the inorganic N content. The NO3--N content in root exudate was positively correlated with root 12

growth attributes and root N concentration while, NH4+-N content was positively correlated with 13

nodule number. Root exudate DON positively correlated with shoot N concentration and average 14

nodule dry weight. These results highlight the existence of genotypic differences among red 15

clover varieties for plant morphological factors affecting root N release during the early stages of 16

plant development. 17

18

Keywords: root nodules, root exudates, nitrogen, red clover, genotypic variability 19

20

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Introduction 24

Forage legumes are an important component of legume/grass pasture systems worldwide mainly 25

due to the role in providing significant nitrogen (N) input through the symbiotic nitrogen fixation 26

(SNF) and for enhancing the nutritional quality of the grazed stands. Estimates of the amount of 27

N fixed by forage legumes under legume/grass systems ranges from 13 to 682 kg N ha -1 yr-1 28

(Ledgard and Steele 1992). In total it is estimated that annually 50-70 Tg of N can be derived 29

from biological nitrogen fixation (BNF) in agricultural systems, where pasture and fodder 30

legumes contribute 12-25 Tg N annually (Herridge et al. 2008). Biologically fixed N is 31

incorporated into organic forms and is less prone to leaching and volatilization (Dixon and Kahn 32

2004). In agricultural systems, N credit from SNF can be beneficial under crop rotation and 33

intercropping/mixed cropping systems (Peoples et al. 1995), where the N losses are minimized 34

(Drinkwater et al. 1998). 35

Nitrogen fixed by legumes is released in to the soil mainly through root decomposition and 36

subsequent mineralization as well as N containing root exudates (Fustec et al. 2010). The 37

released N can be incorporated into the soil mineral N pool, taken up by plants, immobilized by 38

soil microbes, leached away from the soil system or lost through denitrification (Cameron et al. 39

2013; Thilakarathna et al. 2016a). Interestingly, part of the N fixed by legumes can be released 40

and taken up by neighboring non-legumes during their growth, which is referred to as N transfer 41

(Thilakarathna et al. 2016a). Nitrogen can be transferred underground from legumes to non-42

legumes through different mechanisms: decomposition of belowground legume tissues (Wichern 43

et al. 2008; Fustec et al. 2010), plant root exudates (Paynel et al. 2008; Fustec et al. 2010; 44

Lesuffleur and Cliquet 2010) and mycorrhizal-mediated N transfer (Høgh-Jensen 2006; He et al. 45

2009). Legume root exudates contain both low and high molecular weight N compounds (Badri 46

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and Vivanco 2009), which can transfer N to non-legumes (Paynel and Cliquet 2003; Paynel et al. 47

2008; Jalonen et al. 2009a, 2009b). 48

Among the different N-containing root exudates, ammonium and amino acids are the major 49

forms of N exuded by clover, with ammonium contributing the most (Paynel et al. 2001, 2008; 50

Lesuffleur and Cliquet 2010). Glycine and serine are the dominant forms of amino acids in 51

clover root exudates (Lesuffleur et al. 2007; Paynel et al. 2008), but many other amino acids 52

have also been reported (Paynel et al. 2008). Since plants take up N in both inorganic (NO3- and 53

NH4+) and organic (mainly as amino acids) forms (Näsholm et al. 2009; Richardson et al. 2009), 54

different forms of N compounds in the legume root exudates can act as potential N transfer 55

sources to non-legumes. 56

Plant and environmental factors affect root exudation of N compounds by legumes (Paynel 57

et al. 2008; Goergen et al. 2009; Jalonen et al. 2009b; Mahieu et al. 2009; van Kessel et al. 2009; 58

Thilakarathna et al. 2016a). Among different plant-associated factors, N fixation (Paynel et al. 59

2008), root N concentration (Jalonen et al. 2009a), and total plant N content (Mahieu et al. 2009) 60

were shown to stimulate N exudates. Furthermore, genotypic variability is one of the major 61

factors that govern N release by different legumes (Thilakarathna et al. 2016a). Variability 62

among different red clover varieties was observed for nodulation and plant growth characteristics 63

(Thilakarathna et al. 2012a), which may have resulted in variation on SNF and N transfer 64

(Thilakarathna et al. 2016b). Ploidy level was also shown to have an effect on nodulation and 65

SNF in legumes (Mergaert et al. 2006; Cannon et al. 2014). Higher ploidy level was associated 66

with larger cells in nodules, which can contain larger numbers of bacteroids and enhance SNF 67

(Mergaert et al. 2006). Our previous work found that ploidy level in red clover affected the 68

amount of N fixation and N transfer under field conditions (Thilakarathna et al. 2016b). Early in 69

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the growing season, N seems to be transferred from legumes to non-legumes as root exudates 70

rather than N derived from decomposing roots and nodule debris (Paynel and Cliquet 2003; 71

Gylfadóttir et al. 2007; Lesuffleur et al. 2013). Identifying genotypic variability among legume 72

varieties for N exudation and understanding the relationship between N exudation and plant 73

growth characteristics during the early growth stages of the legumes will help in development of 74

a management strategy to improve the N transfer from legumes to non-legumes, while 75

minimizing nitrate leaching into the ground water. 76

Red clover is a major legume crop used in temperate regions particularly with livestock 77

farming systems (Vega et al. 2015) and as a cover crop in rotations(Thilakarathna et al. 2015). 78

The objectives of the present study were: 1) to evaluate the genotypic variability among red 79

clover varieties for nodulation potential, plant growth traits, net N exudation in terms of NO3--N, 80

NH4+-N, and dissolved organic N during the early stages of seedling development and 2) to 81

identify the plant morphological factors (nodulation and root morphological parameters) 82

affecting net N exudation. 83

84

Materials and Methods 85

Plant Materials, Rhizobia Inoculation, and Growing Conditions 86

Six winter-hardy, double-cut type red clover varieties (see below) were selected for this study 87

based on: 1) superior productivity in Eastern Canada; 2) have strong nodulation profiles; 3) 88

genetic diversity (originated from unrelated populations) 4) ploidy (both diploid and tetraploid 89

types). Six red clover varieties including three diploid varieties: AC Christie, early flowering and 90

has no pubescence on the stems (Martin et al. 1999); Tapani, early flowering plants selected 91

from old stands in three Atlantic Provinces of Canada (Papadopoulos et al. 2008), CRS 15, new 92

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variety selected for high nitrogenase activity (Y.A. Papadopoulos, AAFC, personal 93

communication); and three tetraploid varieties: CRS 18, new variety selected for improved 94

seedling vigour (Y.A. Papadopoulos, AAFC, personal communication); CRS 39, new variety 95

selected for plant vigour (roots and top growth) and persistence (Y.A. Papadopoulos, AAFC, 96

personal communication); and Tempus, high yielding variety which is currently used as a 97

reference variety in registration trials Eastern Canada 98

(http://www.inspection.gc.ca/english/plaveg/variet/regvare.shtml). Seeds of the six varieties were 99

surface sterilized with 2% sodium hypochlorite for three minutes and washed with five changes 100

of sterile distilled water. Seeds were pre-germinated on wet sterile filter papers in the dark for 101

three days and three germinating seeds were transferred into plastic growth pouches (Mega 102

International, Minneapolis, MN, USA) containing deionized water. One week after germination, 103

the seedlings were thinned to one seedling per growth pouch and plants were inoculated with a 1 104

ml suspension of Rhizobium leguminosarum biovar trifolii (ATCC 14480) as in the method 105

described by Thilakarathna et al. 2012a). From the second week of plant growth plants were 106

supplied with quarter-strength Hoagland’s N-free nutrient solution 107

(http://www.caissonlabs.com/catalog.php), where the pH of the Hoagland’s solution was 108

adjusted to 5.8. Volume of the plant growing solution in each growth pouch was maintained at 109

approximately 25 ml during the trial. Plants were grown in a growth room with supplemental 110

lighting maintained with a photoperiod of 16 hours of daylight at 150 µmol m–2 s–1 and 8 hours 111

of dark (16 D: 8 N) at 23 ± 2 oC. 112

113

Collection of Root Exudates and Harvesting of Plants 114

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At 4, 6, and 8-weeks after inoculation with rhizobia the growing solution in each growth pouch 115

was collected, filtered through 0.45-µm micro filters and preserved at -20 ºC in 50 ml sterile 116

eppendorf tubes for detailed N analysis. After collection of the growing solution first and second 117

sampling, each growth pouch with red clover plant was immediately refilled with 25 ml of 118

quarter-strength N-free Hoagland’s nutrient solution. 119

Plants were harvested 8 weeks after rhizobia inoculation. The attributes determined were 120

days-to-nodule initiation, number of nodules at 4, 6, and 8 weeks, shoot dry weight (DW), root 121

DW, and average nodule DW at harvest. Leaves were scanned using Epson Expression 1000X 122

(Epson Canada Ltd., Markham, ON, Canada) and total leaf area was measured using the 123

WinFOLIA (Regents Instruments Inc., Québec City, QC, Canada) software system. A detailed 124

root morphological analysis was collected, including root volume, total root length, root surface 125

area, and average root diameter was measured using a WinRHIZO system (Regents Instruments 126

Inc., Québec City, QC, Canada). Shoot and root DW were measured after drying the plant in a 127

hot air oven at 65 ºC for 3 days. Dry plant samples were ground using a micro Wiley mill, 128

standard model 3 (Arthur H. Thomas Co., Philadelphia, USA), to pass through a 1-mm sieve. 129

130

Analysis of Plant Tissues and Root Exudates 131

Total N and carbon (C) content of the roots and shoots were analyzed by dry combustion at 1000 132

ᵒC followed by combustion gas stream analysis of N2 and CO2 using a Elementar Vario MAX 133

CN analyzer (Elementar Americas Inc., Mt. Laurel, NJ). The growing solutions collected from 134

the pouches were analyzed for NO3--N and NH4

+-N using flow injection analysis on a Lachat 135

QuikChem 8500 (Lachat Instruments, Loveland, CO) using Lachat methods 10-107-06-1-X and 136

10-107-04-1-A (Lachat Instruments, 2007; 2009). Standards and carrier were prepared from 137

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Hoagland's No 2 basal salt mix solution (http://www.caissonlabs.com/catalog.php). Total 138

dissolved N (TDN) was determined similarly following potassium persulfate digestion at 121 ᵒC 139

and subsequent NO3--N analysis with matrix matched standards on the above described flow 140

injection analyzer. 141

142

Statistical Analysis 143

The experimental design was a 6 × 6 Latin square design with each of the six red clover varieties 144

represented by 6 pouches (1plant/pouch) and repeated 10 times. Each attribute was analyzed 145

using the Latin square model with row, and column as random effects and red clover varieties as 146

the fixed effect within the ANOVA. The results were expressed at a significance level of P < 147

0.05. Orthogonal contrasts were used to assess differences between the red clover varieties based 148

on the different known varietal attributes such as ploidy. Using ANOVA, nodule number and 149

root exudates-N were analyzed across three time points (4, 6, and 8 weeks) with repeated 150

measurements expressed as the mean, linear, and quadratic coefficients across the growing 151

period. Principal component analysis (PCA) was used to explore the relationships between the 152

varieties and assess the relationship between the plant growth parameters measured and the 153

different types of N compounds exuded by red clover root systems during early growth. The 154

following variables were log10 transformed to meet assumptions of normality in the statistical 155

analysis; nodule number, number of days-to-nodule initiate, average nodule size, shoot and root 156

C and N concentrations, NH4+-N, NO3

--N and DON content of the growth medium containing 157

the root exudates. The backtransformed values are presented for reference. The statistical 158

analyses of data was conducted with the GenStat® (VSN International 2011) software. 159

160

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Results 161

Nodulation profiles 162

Mean nodule numbers were significantly different among the six red clover varieties during plant 163

growth (Table 1), where nodule number increased linearly during the growth period 164

(Supplementary Fig. 1). The diploid cultivar CRS 15 had the greatest mean nodule number 165

compared to the other diploid varieties. However, the three tetraploid varieties had a similar 166

number of nodules per plant (Table 1). Diploid varieties had greater specific nodulation than 167

tetraploid varieties. CRS 15 had the highest specific nodulation compared to all other selected 168

varieties. Days-to-nodule initiation varied slightly based on the red clover variety (Table 1). 169

Average nodule size was different among the red clover varieties, where CRS 15 formed smaller 170

nodules compared to the other varieties (Table 1). Tetraploid varieties formed larger nodules 171

(0.604 mg nodule-1) compared to the diploid varieties (0.352 mg nodule-1). CRS15 produced 172

smaller nodules than AC Christie and Tapani, but average nodule size was similar among 173

tetraploid varieties (Table 1). 174

(Please insert Table 1 here) 175

176

Dry Matter, Leaf Area and Root Morphology 177

Genotypic differences were found for shoot DW, root DW, total DW (shoot and root), and total 178

leaf area among the six red clover varieties as well as within ploidy levels (Table 2). Tempus had 179

the highest mean values for the above-mentioned four attributes, whereas CRS 15 had the lowest 180

among the six red clover varieties. Tetraploid varieties had higher shoot DW, root DW, total 181

DW, and leaf area compared to the diploid varieties (Table 2). Among the diploid varieties, CRS 182

15 had the lowest shoot DW, root DW, total plant DW and total leaf area, whereas Tempus had 183

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the highest mean values for these attributes among the tetraploids. The root growth indicators 184

assessed in this study (root length, surface area, volume and average diameter) varied among the 185

six red clover varieties (Table 2). Among the tetraploid varieties, Tempus had higher root surface 186

area, root volume and average root diameter. CRS 15 had smaller root volume and thin roots 187

compared to the other two diploid varieties. Tetraploid varieties had greater root length, root 188

surface area, root volume and root diameter (Table 2). 189


191

Plant Nitrogen and Carbon Profiles 192

Plant tissue N (%), C (%), N content, and C:N ratio were significantly different among the six 193

red clover varieties (Table 3). Among the diploid varieties, CRS 15 had lower shoot N%, tissue 194

N content (shoot, root and total), shoot and root C% (Table 3). Tempus had higher root C% and 195

tissue N content (shoot, root, and total) among the tetraploid varieties (Table 3). Tetraploid 196

varieties had greater shoot N%, root N%, shoot C%, root C%, shoot N content, root N content 197

and total plant N content (Table 3). Cultivar differences were also found for the plant C:N ratio 198

with CRS 15 having a greater C:N ratio than the other varieties (Table 3). Diploid varieties had 199

slightly higher C:N ratio compared to the tetraploid varieties. 200


202

Ammonium, Nitrate, and Dissolved Organic Nitrogen in Root Exudates 203

The consternation of NO3--N, NH4

+-N, and DON in root exudates were significantly different 204

among the red clover varieties during the 8-weeks of plant growth (Table 4). Among the diploid 205

varieties, root exudates of CRS 15 had the greatest NH4+-N consternation. However, the opposite 206

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trend was found for DON, where CRS 15 had less DON than AC Christie and Tapani. Root 207

exudates of Tempus had the greatest concentration of NH4+-N and NO3-N compared to CRS 18 208

and CRS 39. Generally, root exudates of the tetraploid varieties had greater NH4+-N, and NO3

--N 209

concentrations. During the 8-weeks of growth the NH4+-N concentration of the root exudates 210

increased linearly (Supplementary Fig. 2) whereas DON increased in a quadratic manner 211

(Supplementary Fig. 3). 212


214

Principal Component Analysis 215

The first two principal components explain 88% of the total variation for the 15 attributes 216

evaluated in this study (Fig. 1). This outlines differences and correlations among red clover 217

varieties. Component-1 distinguishes differences between the red clover varieties, where all the 218

tetraploid varieties were positive for score 1 while the diploids were negative. Differences 219

between varieties were driven by total plant N content and total plant DW, which were strongly 220

correlated (Fig. 1). Average root diameter, root volume, root N%, total leaf area, and root surface 221

area were also positively correlated with total plant N content and total plant DW (Fig. 1). For 222

component-2, differences between varieties were due to the contrast between the nodule number 223

and NH4+-N concentration in root exudates versus nodule size and DON in root exudates. CRS 224

15 had the greatest number of nodules but they were smaller in size than the other varieties. 225

Tempus, CRS 18, and Tapani had larger nodules but they were few in number. 226

(Please insert Fig. 1 here) 227

228

Discussion 229

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Beyond differences observed between the two ploidy levels there was significant variability for 230

the attributes measured within ploidy levels. Significant differences among the red clover 231

varieties for the nodulation profiles (nodule number, average nodule size, and days-to-nodule 232

initiation) highlight the genotypic variability among varieties for these attributes, which 233

corroborates with previous findings (Thilakarathna et al. 2012a). Furthermore, ploidy level 234

appears to contribute to the differences observed between the red clover varieties. The variety 235

CRS 15 had the most nodules, although their average dry weight was smaller than the other 236

varieties. Since the area infected by rhizobia is smaller with smaller nodules (King and Purcell 237

2001), the efficiency of N fixation is limited. On the other hand, nodule maintenance is energy 238

intensive (Foyer et al. 2005), and as having many nodules is costly for the plant in terms of 239

energy. These factors may have caused lower biomass production in CRS 15 compared to the 240

other varieties, which was also observed in our field studies (Thilakarathna et al. 2016b). 241

Generally, the tetraploid varieties formed larger nodules and had greater N fixation, which 242

indicates that large nodules are better for N fixation (Vikman and Vessey 1993). At higher 243

ploidy levels the cells of the nodules are larger creating space for more bacteroids, which 244

enhance the SNF (Mergaert et al. 2006). When available soil N is limited and/or for N is intense 245

early nodulation is important. Therefore, some of the tetraploid red clover varieties used in this 246

study may be better suited than the diploids under these growing conditions. 247

Genotypic variability among the six red clover varieties was also found for DM yield 248

(shoot, root, and total plant DW) and plant morphological characteristics including leaf area, root 249

length, root surface area, average root diameter, and root volume (Table 2), which is in 250

agreement with previous findings (Thilakarathna et al. 2012a, 2012b, 2016b). Tetraploid 251

varieties produced greater shoot and root biomass compared to the diploids, yielding an average 252

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of 58% higher total plant DM. Photosynthetic rate is positively related to whole plant leaf area 253

(Koyama and Kikuzawa 2009) and leaf N content (Reich et al. 1998). Therefore, the greater 254

yields of tetraploid varieties may be due to greater photosynthetic capacity (greater leaf area and 255

leaf N content) and higher efficiency of N fixation (large nodules). Because SNF is an energy 256

intensive process (Halbleib and Ludden 2000), photosynthetically-assimilated C needs to be 257

directed towards nodules to supply energy and for N assimilation. As the tetraploid red clover 258

varieties produced 40% more leaf area than the diploids, they are capable of supplying more C to 259

their nodules for higher N fixation. Furthermore, the extensive root systems in the tetraploids aid 260

in obtaining more macro and micronutrients from the nutrient medium for better plant growth 261

(Table 2). 262

Significant variety differences for tissue C and N concentrations, plant N content, and C:N 263

ratio correspond with the genotypic variability for the above attributes (Table 3). Compared to 264

the diploid varieties, tetraploid varieties had 17% greater shoot N concentration and 78% more 265

total plant N content at harvest, which also highlights their greater N fixation capacity. Since we 266

did not supply any external N for plant growth, total plant N equates with total fixed N. Although 267

CRS 15 had the most nodules, tissue N concentration and plant N content were less, which 268

confirms that having many nodules does not always result in higher N fixation, possibly due to 269

the high C cost for nodulation and nodule maintenance (Bourion et al. 2007). Our field results 270

also indicated that CRS 15 had the lowest N fixation among the six red clover varieties 271

(Thilakarathna et al. 2016b). 272

Generally, net exudation of inorganic N was greater in the tetraploid red clover varieties 273

compared to the diploids in terms of NH4+-N, and NO3

--N concentrations during their early 274

growth period (Table 4). However, in the longer term, under field conditions, genotypic 275

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variability for N transfer did not appear to be closely related to ploidy level (Thilakarathna et al. 276

2016b). Under field conditions, most of the N transferred from the red clover varieties to 277

bluegrass may have been derived from decomposing legume tissues belowground rather than 278

from N-containing root exudates. The net release of NH4+-N and NO3

--N by red clover varieties 279

during early growth was minor compared to DON. Total dissolved N is the combination of 280

dissolved inorganic N (NH4+-N and NO3

--N) and DON (amino acids, peptides, and proteins). In 281

general, 74% of total dissolved N in the red clover root exudates during the first 8 weeks of plant 282

growth was DON. Legumes tend to increase root exudation of N as plants mature (Jensen 1996; 283

Jalonen et al. 2009a). Similarly, over the eight weeks of growth in this experimentation net 284

exudation of NH4+-N and DON in the red clover varieties increased. It is also possible that most 285

of the released NH4+-N and NO3

--N were recaptured by the red clover plants, as dissolved 286

inorganic N is readily available for plant uptake. Plants are also able to recapture released amino 287

acids (Jones et al. 2005). Under liquid culture, there is a greater possibility for the plants to 288

reabsorb amino acids than from a soil medium (Badalucco and Nannipieri 2007) as the amino 289

acids are not bound to soil particulates nor immobilized by soil microbes. Therefore although 290

most of N released by the red clover plants in this study was dissolved organic N, measurements 291

of this N component is impacted by the ongoing efflux-influx mechanisms for N and does not 292

reflect efflux of N only. 293

Root exudates are one of the major mechanisms for transferring N to neighboring non-294

legumes (Paynel et al. 2008; Jalonen et al. 2009b), especially at early growth stages of legumes 295

(Lesuffleur et al. 2013). Plants uptake N mainly as dissolved inorganic N as well as DON 296

(Näsholm et al. 2009; Tegeder and Rentsch 2010). In general, AC Christie, CRS 39, and Tempus 297

are better candidates for root exudate mediated N transfer among the selected varieties based on 298

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their level of root exudate DON concentration. However, under soil conditions, DON can be 299

rapidly immobilized by soil microbes without plant uptake (Owen and Jones 2001; van Kessel et 300

al. 2009) due to their low C:N ratio (Uselman et al. 2000) and low diffusion coefficient in soil 301

(Jones et al. 2005), thus facilitating N transfer indirectly after microbial turnover (Jalonen et al. 302

2009b). 303

Nitrogen exudation is positively correlated with root N concentration (Jalonen et al. 2009a) 304

and total plant N content (Mahieu et al. 2009). In this study root exudate NH4+-N content was 305

positively correlated with nodule number, whereas NO3--N content was positively correlated 306

with root length and surface area. Despite having a smaller root structure, CRS 15 had a greater 307

NH4+-N content in root exudates, possibly due to its greater nodule numbers. However, exudate 308

DON content was positively correlated with average nodule dry weight and shoot N 309

concentration, which explain having lower DON in CRS 15 among the varieties. 310

In summary, we found significant genotypic variability among different red clover varieties 311

for nodulation, plant growth, plant N content, and root exudate N content. Root exudate NO3--N 312

was positively correlated with root growth attributes and root N concentration while, NH4+-N 313

positively correlated with nodule number. Dissolved organic N content of the root exudates 314

positively correlated with shoot N concentration and average nodule dry weight. A deeper 315

understanding of the genotypic variability among legume varieties for N exudation, and its 316

related attributes is required before criteria for efficient selection of varieties with improved N 317

transfer can be determined. Some of the genetic variability observed among the red clover 318

varieties for the parameters studied in this research can be attributed to differences in ploidy 319

level. As discussed, tetraploid varieties were generally superior to diploids with respect to the N 320

fixation parameters measured. However, the variability in magnitude of the N fixation attributes 321

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monitored in this work was great within ploidy levels. Therefore a singular conclusion that 322

tetraploid red clover varieties will have greater N fixation capacity is not possible at this time. 323

Future studies pursuing the development of molecular markers associated with the types of 324

N exudates and their magnitude in red clover varieties is warranted. This will aid in improving 325

our ability to monitor and understand the mechanisms of nitrogen recycling at the early stages of 326

stand establishment. Furthermore, this will assist red clover breeders to generate red clover 327

populations having the traits required to facilitate N transfer the establishments of companion 328

grasses in forage mixtures under sustainable production systems. 329

330

Acknowledgements 331

The technical support provided by Matthew Crouse and Jeff Franklin, the editorial assistance of 332

Christina McRae, EditWorks, and the many helpful comments provided by the internal review 333

process at the Atlantic Food and Horticulture Research Centre, are greatly appreciated. This 334

work was supported by an Agriculture and Agri-Food Canada research grant to Dr. Y.A. 335

Papadopoulos. 336

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References 346

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Badalucco, L., and Nannipieri, P. 2007. Nutrient transformations in the rhizosphere. in The 347 rhizosphere: biochemistry and organic substances at the soil–plant interface. 2nd ed (Eds R. 348 Pinton., Z. Varanini., P. Nannipieri) pp. 111–133. (Taylor & Francis: Boca Raton, FL). 349

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Tables 511

512

Table 1 Mean nodule number, specific nodulation at harvest, days-to-nodule initiation and average nodule dry weight of six red clover varieties. Data were log10 transformed to adhere and meet assumptions of normality in the analysis.

Red clover varieties Mean nodule number (# plant-1)

Specific nodulation (nodules g-1 root DWa)

Days-to-nodule initiation

Average nodule DW (mg nodule-1)

AC Christie 1.171 (14.8) 2.619 (416) 0.69 (4.9) -0.461 (0.346) Tapani 1.085 (12.2) 2.545 (351) 0.71 (5.1) -0.229 (0.590) CRS 15 1.224 (16.8) 2.860 (724) 0.69 (4.9) -0.670 (0.214) Tempus 1.166 (14.7) 2.265 (184) 0.66 (4.5) -0.229 (0.591) CRS 39 1.199 (15.8) 2.498 (315) 0.68 (4.8) -0.337 (0.460) CRS 18 1.088 (12.2) 2.361 (229) 0.69 (4.9) -0.091 (0.812) Mean by ploidy level

..Diploid 1.160 (14.5) 2.675 (473) 0.69 (4.9) -0.453 (0.352)

..Tetraploid 1.151 (14.2) 2.375 (237) 0.68 (4.7) -0.219 (0.604)

Grand mean 1.156 (14.3) 2.525 (335) 0.68 (4.8) -0.336 (0.461) SEMb 0.025 0.037 0.01 0.080 F-probability Ploidy nsc <0.001 0.013 <0.001 Cultivar (nd = 60) <0.001 <0.001 0.051 0.002

..C15 vs ACC, Tap 0.002 <0.001 ns 0.002

..Tem vs C39, C18 ns <0.001 0.014 ns

..C15 vs Tem 0.030 <0.001 ns 0.016

Note: Values in parentheses are de-transformed values. C15; CRS 15, ACC; AC Christie, Tap; Tapani, Tem; Tempus, C39; CRS 39 and C18; CRS 18. aDry weight bStandard error of mean cNot significantly different at p=0.05 level dNumber of replicates

513

514

515

516

517

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Table 2 Shoot plant dry weight, root dry weight, total dry weight (shoot and root), total leaf area, root length, root surface area, root volume and average root diameter of the six red clover varieties after 8 weeks of seedling growth.

Red clover varieties Shoot DWa

(mg)

Root DW (mg)

Total DW (mg)

Total leaf area (cm2)

Root length (cm)

Root surface area (cm2)

Root volume (cm3)

Average root diameter (mm)

AC Christie 88.8 59.6 148 19.5 408 40.5 0.335 0.317 Tapani 85.1 54.7 139 18.8 384 38.2 0.312 0.314 CRS 15 59.8 42.7 103 13.5 414 37.7 0.266 0.284 Tempus 141.4 107.1 248 26.8 460 60.0 0.628 0.412 CRS 39 105.7 74.0 180 22.5 516 55.6 0.494 0.343 CRS 18 107.1 82.7 190 22.7 449 52.5 0.518 0.386 Mean by ploidy

level ..Diploid 77.9 52.3 130 17.2 402 38.8 0.305 0.305 ..Tetraploid 118.1 87.9 206 24.0 475 56.0 0.546 0.381

Grand mean 98.0 70.1 168 20.6 438 47.4 0.426 0.343 SEMb 5.1 3.5 8 1.0 14 1.8 0.023 0.007 F-probability Ploidy <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Cultivar (nc = 60) <0.001 <0.001 <0.001 <0.001 0.005 0.050 <0.001 <0.001 ..C15 vs ACC, Tap <0.001 <0.001 <0.001 <0.001 nsd ns <0.001 0.041 ..Tem vs C39, C18 <0.001 <0.001 <0.001 <0.001 ns 0.009 <0.001 <0.001 ..C15 vs Tem <0.001 <0.001 <0.001 <0.001 ns 0.085 <0.001 <0.001

Note: All the measurements are per plant basis. C15; CRS 15, ACC; AC Christie, Tap; Tapani, Tem; Tempus, C39; CRS 39 and C18; CRS 18. aDry weight bStandard error of mean cNumber of replicates dNot significantly different at p=0.05 level

518

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Table 3 Nitrogen and carbon concentration (dry weight basis) of shoot and root, N content of the shoot, root and total plant (shoot + root) and C:N ratio of the six red clover varieties after 8 weeks of seedling growth. Data were log10 transformed to adhere and meet assumptions of normality in the analysis.

Red clover varieties Plant N (%) Plant C (%) N content (mg plant-1) C:N ratio Shoot Root Shoot Root Shoot Root Total

AC Christie 0.391 (2.46) 0.391 (2.46) 1.591 (39.0) 1.611 (40.8) 2.21 1.47 3.68 16.2 Tapani 0.375 (2.37) 0.403 (2.53) 1.589 (38.8) 1.614 (41.2) 2.06 1.40 3.44 16.4 CRS 15 0.294 (1.97) 0.383 (2.42) 1.580 (38.0) 1.605 (40.2) 1.26 1.05 2.31 17.9 Tempus 0.416 (2.61) 0.458 (2.87) 1.600 (39.8) 1.624 (42.0) 3.71 3.07 6.78 15.0 CRS 39 0.408 (2.56) 0.463 (2.90) 1.597 (39.5) 1.614 (41.1) 2.77 2.18 4.94 15.0 CRS 18 0.410 (2.57) 0.436 (2.73) 1.596 (39.5) 1.617 (41.4) 2.77 2.27 5.04 15.4 Mean by ploidy level

..Diploid 0.353 (2.26) 0.392 (2.47) 1.587 (38.6) 1.610 (40.7) 1.84 1.31 3.15 16.8

..Tetraploid 0.412 (2.58) 0.452 (2.83) 1.597 (39.6) 1.618 (41.5) 3.08 2.51 5.59 15.1 Grand mean 0.382 (2.41) 0.422 (2.64) 1.592 (39.1) 1.614 (41.1) 2.46 1.91 4.37 15.9 SEMa 0.016 0.008 0.002 0.002 0.14 0.10 0.22 0.16 F-probability Ploidy <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Cultivar (nb = 10) 0.001 0.090 0.007 <0.001 <0.001 <0.001 <0.001 <0.001

..C15 vs ACC, Tap <0.001 nsc <0.001 <0.001 <0.001 0.002 <0.001 <0.001

..Tem vs C39, C18 ns ns ns <0.001 <0.001 <0.001 <0.001 ns

..C15 vs Tem ns ns ns ns <0.001 <0.001 <0.001 <0.001

Note: Values in parentheses are de-transformed values. C15; CRS 15, ACC; AC Christie, Tap; Tapani, Tem; Tempus, C39; CRS 39 and C18; CRS 18. aStandard error of mean bNumber of replicates cNot significantly different at p=0.05 level

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Table 4 Mean NH4+-N, NO3--N, and dissolved organic N content in the root exudates containing growing solution of the six red clover varieties, collected at 4, 6 and 8 weeks of plant growth. Data were log10 transformed to adhere and meet assumptions of normality in the analysis.

Red clover varieties NH4+-N

(µg plant-1) NO3

--N

(µg plant-1) Dissolved organic Na (µg plant-1)

AC Christie -0.471 (0.338) -0.369 (0.428) 0.471 (2.96) Tapani -0.466 (0.342) -0.425 (0.376) 0.391 (2.46) CRS 15 -0.371 (0.426) -0.361 (0.436) 0.271 (1.87) Tempus -0.352 (0.444) -0.214 (0.610) 0.422 (2.64) CRS 39 -0.413 (0.386) -0.300 (0.502) 0.445 (2.79) CRS 18 -0.404 (0.394) -0.272 (0.535) 0.387 (2.44) Mean by ploidy level

..Diploid -0.436 (0.366) -0.385 (0.413) 0.378 (2.39)

..Tetraploid -0.390 (0.408) -0.262 (0.547) 0.018 (2.62)

Grand mean -0.413 (0.386) -0.323 (0.475) 0.398 (2.50) SEMb 0.013 0.021 0.030 F-probability Ploidy <0.001 <0.001 nsc Cultivar <0.001 0.012 0.001

..C15 vs ACC, Tap <0.001 ns <0.001

..Tem vs C39, C18 <0.001 0.007 ns

..C15 vs Tem ns ns ns Cultivar quadratic ns ns <0.001 Cultivar linear 0.001 0.004 0.004 Note: Values in parenthesis are de-transformed values. C15; CRS 15, ACC; AC Christie, Tap; Tapani, Tem; Tempus, C39; CRS 39 and C18; CRS 18. aDissolved organic N content was calculated as the difference from total dissolved N and NO3

- -N and NH4+ -N

bSEM; standard error of mean cNot significantly different at p=0.05 level

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Figure Caption

Fig. 1 Principal component analysis of nodulation (number and size), root attributes (length, surface area, volume, diameter), leaf area, total plant dry weight (DW), shoot and root N concentration, total plant N content, C:N ratio and mean N exuded (NH4

+-N, NO3--N, and DON)

during 8 weeks of growth in six red clover varieties.

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new for review only · 2018. 6. 4. · for review only 1 red clover varieties with nitrogen fixing...

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