Compatible rhizosphere competent microbial consortium add value to

Subscriber access provided by UNIV OF NEWCASTLE

Article

Compatible rhizosphere competent microbial consortium add value to nutritional quality in edible parts of chickpea Sudheer KUmar Yadav, Surendra Singh, Harikesh Bahadur Singh, and Birinchi Kumar Sarma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01326 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Compatible rhizosphere competent microbial consortium add value to nutritional quality

2

in edible parts of chickpea

3 4

Sudheer K. Yadav1, Surendra Singh1, Harikesh B. Singh2, Birinchi K. Sarma2*,

5

1

Department of Botany, Institute of Science, Banaras Hindu University, Varanasi-221005

6

2

Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras

7

Hindu University, Varanasi-221005

8 9

*Corresponding Author Email: [email protected]

10 11

Abstract

12

Chickpea is used as a high energy and protein source in human and livestock’s diets. Moreover,

13

chickpea straw can be used as alternative of forage in ruminant diets. The present study evaluates

14

the effect of beneficial microbial inoculation on enhancing the nutritional values in edible parts

15

of chickpea. Two rhizosphere competent compatible microbes (Pseudomonas fluorescens OKC

16

and Trichoderma asperellum T42) were selected and applied to seeds either individually or in

17

consortium before sowing. Chickpea seeds treated with the microbes showed enhanced plant

18

growth (88.93% shoot length at 60 DAS) and biomass accumulation (21.37% at 120 DAS).

19

Notably, the uptake of mineral nutrients viz. N (90.27, 91.45, 142.64%), P (14.13, 58.73,

20

56.84%), K (20.5, 9.23, 35.98%), Na (91.98, 101.66, 36.46%), Ca (16.61, 29.46, 16%) and

21

organic carbon (28.54, 17.09, 18.54%) was found in seed, foliage and pericarp of the chickpea

22

plants, respectively. Additionally, nutritional quality viz. total phenolic (59.7, 2.8, 17.25%),

23

protein (9.78, 18.53, 7.68%), carbohydrate content (26.22, 30.21, 26.63%), total flavonoid

24

content (3.11, 9.15, 7.81%) and reducing power (112.98, 75.42, 111.75%) was also found in

ACS Paragon Plus Environment


25

seed, foliage and pericarp of the chickpea plants. Most importantly the microbial consortium

26

treated plants showed maximum increase of nutrient accumulation and enhancement in

27

nutritional quality in all edible parts of chickpea. Nutritional partitioning in different edible parts

28

of chickpea was also evident in the microbial treatments compared to their uninoculated ones.

29

The results thus clearly demonstrated microbe-mediated enhancement in dietary value of the

30

edible parts of chickpea as seeds are consumed by human whereas pericarp and foliage (straw)

31

are used as alternative of forage and roughage in ruminant diets.

32

Keywords: Biofertilizers, Nutrient content, Nutritional value, Shikimic acid, Microbial

33

consortium, Phenolics

34

Introduction

35

The present world population of about 7.2 billion is expected to cross 9.6 billion by the end of

36

year 2050.1 In order to provide food to all by that time, the annual production of legumes needs a

37

significant jump. To achieve this onerous target and to meet the food requirement of the people,

38

the current agricultural practices in many developing countries have become heavily dependent

39

on the intensive use of chemical fertilizers and pesticides which led to severe problems to human

40

health and environment. Therefore, priority should be given to the use of microorganisms as

41

biofertilizers for food security and sustainable crop production as an ecofriendly approach.2 In

42

this context, recently attempts have been made more towards ‘nutrient rich high quality food’

43

production in sustainable agriculture to secure bio-safety.3 Hence the enhancement in nutritional

44

quality of food in their natural surroundings particularly in agricultural field conditions is best

45

adopted for the sustainable agriculture.4 The use of biofertilizers has been found to enhance the

46

plant growth, yield, nutrient content, activate plant defense and improved the signaling network.5

47

Recently, organic inputs have been recommended as an alternative of soil fertilization for


Page 2 of 32

Page 3 of 32


48

improvement of nutrient supply and maintenance of field management.6 In this context organic

49

farming is helpful in ensuring the food safety along with the maintenance of soil biodiversity.7

50

Furthermore, biofertilizers are having additional advantage due to their longer shelf life without

51

imparting any harmful effects to the ecosystem.8 Biofertilizers also maintain the soil fertility

52

through various processes such as N2-fixation, phosphate solubilization, mineralization, secretion

53

of plant growth regulating compounds, production of antibiotics and biodegradation of organic

54

matters.9

55

Chickpea (Cicer arietinum L.) is an important grain legume used all over the world as a

56

good source of minerals, proteins, vitamins, and regularly included in human diets. The prospect

57

of food legumes for the livestock food mainly depends on their nutrient contribution in diet.

58

Young and fresh chickpea leaves are also used as cooked green vegetable to provide plenty of

59

dietary nutrients in malnourished populations in certain parts of the world.10 Whereas, chickpea

60

is produced mostly for human consumption, it is however, also used as an alternative to supply

61

proteins and energy feed for livestocks. Chickpea straw is also used as fodder for ruminants.11

62

Chickpea straw is a primary and major by-product after harvesting and thrashing, and also

63

contributes equal or more than grain yield. It has higher nutritional quality and palatability than

64

cereals straw.12 Thus, it is evident that no part of chickpea is a waste as all parts are consumed at

65

different stages by human and livestock.

66

Rhizosphere competent microbes are used mostly as biofertilizers or biopesticides

67

successfully13. However, what impacts they may have on the nutritional quality of edible parts of

68

the introduced crops are not well known. Keeping this in view, we used two beneficial

69

rhizosphere competent microbes, viz., Trichoderma asperellum strain T42 (GenBank accession

70

JN128894) and Pseudomonas fluorescens strain OKC (GenBank accession JN128891) either



71

individually or in combination, and evaluated the possible impacts they could have on the

72

nutritional quality of edible parts of chickpea in the current study. We evaluated nutritional

73

values of chickpea seeds along with foliage and pericarp and compared the values with non-

74

microbe introduced plants. The experiment was conducted under natural field condition in order

75

to get an appropriate idea how the microbes influences the nutritional values of chickpea under

76

natural environmental conditions.

77

Materials and methods

78

Organisms

79

Pseudomonas fluorescens OKC (GenBank accession JN128891) was isolated from the

80

rhizosphere of okra (Abelmoschus esculentus) plant on King’s B agar medium, whereas

81

Trichoderma asperellum T42 (GenBank accession JN128894) was obtained from a pool of

82

Trichoderma isolates maintained in the Department of Mycology and Plant Pathology, Institute

83

of Agricultural Sciences, Banaras Hindu University and grown on Potato Dextrose Agar (PDA).

84

Both the strains were selected on the basis of their plant growth promotion activities as well as

85

compatibility between them.14 The Pseudomonas strain was identified by sequencing the 16s

86

rDNA region15, and the Trichoderma isolate was identified by sequencing the ITS region.16,17

87

Inoculum preparation, seed treatment and sowing

88

Seeds of chickpea (Cicer arietinum L. var. Avrodhi) were surface sterilized by using 1% sodium

89

hypochlorite for 3 min and further rinsed 10 times with sterilized distilled water. P. fluorescens

90

strain OKC was inoculated in King’s B broth and incubated at 28°C in incubator shaker at 100

91

rpm for 48h. Bacterial culture was collected in the form of pellet by centrifugation at 4°C

92

(10,000xg for 2 min), the resulting pellets were washed with sterilized distilled water thrice and

93

absorbance of the homogenous suspension was measured spectrophotometrically at 600 nm. The


Page 4 of 32

Page 5 of 32


94

optical density for CFU 108 ml-1 P. fluorescens OKC was determined as 0.393. Similarly, the

95

Trichoderma isolate T42 was inoculated on PDA and incubated at 28°C for 4 days. Spores of

96

T42 were harvested in sterilized distilled water, absorbance of the spore suspension was

97

measured at 600 nm and optical density for CFU 107 mL-1 of T42 was determined as 1.14. OKC

98

cell suspension and T42 spore suspension were mixed in equal volume for consortium treatment.

99

Both cell and spore suspensions were mixed with 1% carboxy methyl cellulose (CMC).18 The

100

treatments applied were as follows: OKC = P. fluorescens strain OKC, T42 = T. asperellum T42,

101

OKC + T42, Control = without any microbial treatment. For sowing, the field was divided into 4

102

square meter sized experimental plots. Row to row spacing was maintained at 30 cm whereas

103

plant to plant spacing was maintained at 15 cm. The four treatments were arranged in CRBD in

104

two blocks. Each treatment was replicated thrice. Prescribed agronomical practices were adopted

105

for chickpea cultivation in Uttar Pradesh of India.

106

Plant growth promoting traits

107

For recording the growth parameters, three chickpea plants were uprooted randomly from each

108

plot after 60 and 120 days after germination (DAG). To remove soil particles adhered to the

109

roots surface, the plants were thoroughly washed by placing them on a sieve (mesh size 1 mm)

110

under running tap water. The washed plants were initially air dried followed by oven-dried at 45-

111

50°C19 by placing them on two layers of blotting papers. Shoot length (SL), shoot fresh weight

112

(SFW), shoot dry weight (SDW), root length (RL), root fresh weight (RFW), root dry weight

113

(RDW) and total biomass (TB) were quantified after 60 days of sowing and data from the

114

replicated plots were pooled. Seed, foliage and pericarp yields were also recorded from all the

115

treatments. Seed, foliage and pericarp of 100 plants from replicated plots were used for dry

116

biomass measurement from each treatment.



117

Preparation of plant samples for analysis

118

Seed, pericarp and foliage were separated from 50 randomly selected plants from all replications,

119

mixed thoroughly and oven dried. Dried samples were then grounded and the ground powder

120

was used for further analysis.

121

Nutrient analysis

122

Approximately 200 mg of oven-dried fine powder of plant materials was dissolved in 5 ml of

123

concentrated sulphuric acid (AR grade). The flask was shaken in swirling motion and kept at

124

room temperature for 20 min. The content was boiled gently for 30 min and to that 1 ml of 4%

125

(v/v) perchloric acid (62%) was added. The content was further heated gently till the digest

126

became clear and kept at room temperature for cooling. Phosphorus (P) was extracted by nitric–

127

perchloric acid digestion and measured using the vanadomolybdophosphoric acid colorimetric

128

method.20 Potassium (K), sodium (Na) and calcium (Ca) was assayed using flame

129

spectrophotometer (Corning 400, UK)21 whereas total nitrogen (N) and total organic matter were

130

determined by using Kjeldahl method,22 and Walkley and Black23, respectively.

131

Total phenol content

132

Total phenol content (TPC) was determined following the method of Zheng and Shetty.24 Leaf

133

sample (0.1 g) was collected from the mixture of 50 randomly selected plants from all

134

replications and placed in 95% ethanol (5 ml) and kept at 0°C for 48 h. The samples were

135

homogenized individually and centrifuged at 13000 rpm for 10 min. To 1 ml of the supernatant,

136

1 ml of 95% ethanol, 5 ml of sterilized distilled water and 0.5 ml of 50% Folin-Ciocalteau regent

137

was added and the contents were mixed thoroughly. After 5 min, 1 ml of sodium carbonate (5%)

138

was added to the reaction mixture, the reaction mixture was allowed to stand for 1h and the

139

absorbance of the colour developed was recorded at 725 nm against a reagent blank. Standard


Page 6 of 32

Page 7 of 32


140

curves were prepared for each assay using various concentrations of gallic acid (GA; Sigma,

141

USA) in 95% ethanol. Absorbance values were converted to mg GA equivalents (GAE) g-1 FW.

142

Protein content

143

Protein content was estimated following the method of Lowry et al.25 using bovine serum

144

albumin as standard and expressed in terms of mg protein g−1 FW. 0.1g sample (collected

145

similarly as TPC) was extracted in 0.1M phosphate buffer (5ml, pH=7.0). The reagent A,

146

alkaline sodium carbonate (2%) was prepared in sodium hydroxide (0.1N) and reagent B, copper

147

sulphate (0.5%) in potassium sodium tartrate (1%). Further fresh alkaline copper solution was

148

prepared by adding 50ml of reagent A and 1ml of reagent B. The aliquot of 0.2ml sample extract

149

was made up to 1 ml by adding distilled water and alkaline copper solution was added (5ml). The

150

solution was mixed thoroughly and incubated at room temperature for 10 min. 0.5ml of diluted

151

folin-ciocalteau reagent (1:1) was added in the solution, mixed thoroughly and incubated at room

152

temperature for 30 min in dark. The blue color was developed and absorbance was taken at

153

660nm. The blank was prepared directly with 1ml of distilled water (without sample extract) and

154

followed the same procedure as done with extracted samples.

155

Total carbohydrate

156

Total carbohydrate was measured using anthrone method.26 Seed, foliage and pericarp samples

157

(0.1g each) were first hydrolyzed into simple sugars using dilute HCl (2.5N) in boiling water

158

bath for 3h. The solution was neutralized with solid Na2CO3 until the effervescence ceased and

159

the final volume was made up to 100ml. One ml aliquots was mixed thoroughly with 4ml

160

anthrone reagent and incubated in boiling water bath for 8 min. Absorbance of the dark green

161

color developed was recorded at 630nm upon cooling. Absorbance values were converted to mg

162

glucose g-1 dry weight (DW) using the standard curve of glucose.



163

Preparation of extracts from chickpea plant parts for determination of total flavonoid

164

content and reducing power

165

The extracts were prepared by dissolving 2.0 g powders of seeds, foliage and pericarp separately

166

from the oven dried and ground powders in 10 ml of methanol (50%) and incubated overnight at

167

room temperature. The extracts were filtered through sterilized Whatman No. 1 filter paper and

168

further fractionated with equal volume of ethyl acetate in separating funnel by shaking

169

vigorously. The ethyl acetate fractions were taken out separately, and the residue was

170

refractionated using the ethyl acetate. The pooled fractions were evaporated and dried samples

171

were dissolved in 2.0 ml of HPLC grade methanol and used for analysis.27

172

i.

Total flavonoid content (TFC)

173

TFC was quantified by method of Irshad et al.28 and expressed in quercetin equivalents of

174

standard curve. To 0.5 ml extract, 4ml of distilled water and 0.3ml of 50% NaNO2 solution were

175

added. After 5 min, 0.3 ml of 10% AlCl3 solution was added mixed thoroughly. Again after

176

another 5 min, 2 ml of 1 M NaOH was added and the volume was made up to 10 ml with 95%

177

ethanol. The solution was mixed thoroughly and absorbance was recorded at 510 nm. TFC was

178

expressed as mg quercetin equivalents g−1 FW.

179

ii.

Reducing power (RP)

180

The reducing power of the plant extracts was evaluated by modified ferric reducing antioxidant

181

power assay.29 To 500 µl aliquots of the extract, 1.0 ml methanol and 2.5 ml each of phosphate

182

buffer (pH 6.6) and 1% (w/v) potassium ferricyanide were added. The reaction mixture was

183

incubated at 50°C in a water bath for 20 min. The reaction was terminated by the addition of 2.5

184

ml TCA (10% w/v). Further above reaction mixture was diluted with an equal volume of

185

deionized water and 0.5 ml FeCl3 (0.1% w/v) was added. Absorbance was measured at 700 nm


Page 8 of 32

Page 9 of 32


186

against a reagent blank after 10 min. RP was expressed as ascorbic acid equivalent (1 ASE = 1

187

mM). The ASE/ml value is inversely proportional to reducing power.

188

High performance liquid chromatography (HPLC) analyses of seed, foliage and pericarp

189

Ground powders from different parts of chickpea were used for extraction of phenolic

190

compounds. One gram of the ground samples from seed, foliage and pericarp was extracted with

191

50% methanol (10 ml) separately. The solvent was removed under reduced pressure in rota-

192

evaporator (Eyela N–Nseries, Tokyo, Japan), the resulting residue was solubilized in HPLC

193

grade methanol, and specific phenolics were analyzed using quantitative HPLC. The HPLC

194

(Shimadzu LC-10A, Japan) was furnished with dual pump LC-10A binary system, UV detector

195

SPD-10A and Phenomenex (Torrance, USA) C18 column (4µm, 250×4.6 mm). The data were

196

integrated by Shimadzu LC solution series software. Compounds were separated with

197

acetonitrile and 1% acetic acid in a linear gradient program, starting with 18% acetonitrile,

198

changing to 32% in 15 min and finally to 50% in 40 min30. Solvent flow rate was maintained to

199

1.0 mL min-1. Results (µg g-1 DW) were calculated by comparing the peak areas (max 254 nm)

200

of the samples with those of standards (LC solution series software, Shimadzu, Japan).

201

Statistical analyses

202

Data from different experiments were recorded as mean ± standard deviation (SD) of at least five

203

replications (five repeated analyses in one experiment) and were subjected to analysis of

204

variance (ANOVA). The treatment mean values were compared by Duncan’s multiple range test

205

(DMRT) at p ≤ 0.05 significance level. The software used for analysis was SPSS version 16. The

206

Principal Component Analysis (PCA) was performed using R-program.

207

Results

208

Plant growth, biomass and yield



Page 10 of 32

209

Chickpea plants raised from seeds treated with the rhizosphere competent compatible microbes

210

either individually or in consortium showed increase in dry weight and yield of seed, foliage and

211

pericarp compared to the plants raised from untreated seeds (Fig. 1). Significant increase in dry

212

weight was observed in seed and pericarp (17.6 and 28.15%, respectively) in the plants raised

213

from seeds treated with the microbial consortium of P. fluorescens OKC and T. asperellum T42

214

compared to their untreated control counterparts. Dry weight of foliage was also increased over

215

the control but it was not significantly high. Although applications of the microbes OKC and T42

216

individually also resulted in increased biomass of different chickpea parts, but the increase in dry

217

weights in the consortium treated plants were higher. Similarly, there was significant increase in

218

root and shoot lengths in all microbial treatments but the consortium treated plants showed

219

maximum increase of shoot and root lengths (88.93 and 98.61%, respectively). Additionally, it

220

was also recorded that there was significant increase in, shoot fresh and dry weight, root fresh

221

and dry weight (RDW) as well as total biomass (88.62, 138.2, , 200, 71.75 and 123.63%,

222

respectively) in consortium treated plants compared to untreated control plants after 60 days of

223

sowing. Between the two microbes Trichoderma and Pseudomonas it was interesting to note that

224

the fungal strain T. asperellum T42 enhanced shoot biomass more compared to the bacterial

225

strain whereas the bacterial strain P. fluorescens OKC enhanced root biomass more compared to

226

the fungal strain.

227

Nutrient content

228

Nutrient contents (N, P, K, Na, Ca and total organic carbon) in seed, pericarp and foliage of

229

chickpea plants treated with the microbial consortium were significantly high compared to the

230

untreated control counterparts (except potassium in foliage). Interestingly, application of the

231

microbes individually also increased the nutrient contents in most of the cases but the difference


Page 11 of 32


232

was not significant in various occasion. The results thus show the significance of combining

233

compatible microbes over use of single microbes.

234

Total P content in seeds, pericarp and foliage of chickpea varied from 155.91 to 177.95,

235

43.87 to 68.81 and 91.51 to 145.26 mg g-1, respectively (Fig. 2A). Total P content in seed,

236

pericarp and foliage was 14.13, 56.84 and 58.73% higher, respectively, in the microbial

237

consortium treatment compared to the untreated control plants. However, the maximum P

238

content was found in seeds followed by foliage and pericarp. Similarly, N content also varied

239

from 2.88 to 5.48, 0.68 to 1.65 and 1.17 to 2.24% in chickpea seed, pericarp and foliage,

240

respectively, and maximum being recorded in the plants treated with the consortium of OKC and

241

T42 (Fig. 2B). N content was also recorded highest in the seeds followed by foliage and pericarp.

242

The K concentration also varied from 308.93 to 372.26, 658.7 to 895.73 and 477.16 to

243

521.2 ppm in seed, pericarp and foliage, respectively (Fig. 2C). Highest accumulation of K in

244

seeds, pericarp and foliage of the chickpea plants was also recorded in the consortium treatment

245

and the increase was 20.5, 35.98 and 9.23%, respectively, compared to the untreated control

246

plants. Unlike N and P contents, pericarp of the chickpea plants had maximum K concentration

247

followed by foliage and seed. However, the amount of K content in different parts varied

248

significantly. The K content in pericarp and seeds of chickpea plants treated with the microbial

249

consortium varied significantly but the difference in K content between pericarp and foliage did

250

not vary significantly. Similarly, richness in Na concentration was also highest in pericarp

251

followed by seed and foliage (Fig. 2D) similar to the K content. The concentration of Na ranged

252

from 9.86 to 18.93, 20.11 to 27.48 and 6.59 to 13.29 ppm in seed, pericarp and foliage,

253

respectively. However, unlike the K content, highest Na concentration was observed in OKC

254

treated plants compared to the microbial consortium treatment. Pericarp of chickpea plants



Page 12 of 32

255

contained 52.31% higher Na from OKC treated plants compared to their untreated control

256

counterparts. However, chickpea plants treated with the consortium of OKC and T42 had

257

maximum Na in seeds and foliage compared to Na concentration in all three parts in untreated

258

control counterparts. Chickpea plants treated with the consortium showed 91.98, 36.64 and

259

101.67% increase in Na concentration in seed, pericarp and foliage, respectively, to that of their

260

untreated control counterparts. The Ca concentration also showed a similar trend of

261

accumulation in different chickpea parts. Similar to K and Na, the pericarp of the chickpea plants

262

had maximum Ca concentration followed by foliage and seed. The Ca concentration ranged from

263

86.23 to 100.56, 505.6 to 586.5 and 151.86 to 196.6 ppm in seed, pericarp and foliage,

264

respectively, and maximum being in the plants treated with the microbial consortium (Fig. 2E).

265

The increase in Ca concentration in seed, pericarp and foliage in the consortium treated plants

266

was 16.61, 16 and 29.46%, respectively, compared to the untreated control counterparts.

267

The organic carbon content was also enhanced significantly in chickpea seed, foliage and

268

pericarp of the plants raised from seeds treated with the microbial consortium (Fig. 2F). The

269

organic carbon content ranged from 32.13 to 41.3, 32.77 to 38.37 and 27.18 to 32.22 mg g-1 in

270

seed, pericarp and foliage, respectively. A significant increase of 28.54, 17.09 and 18.54% in

271

organic carbon content was recorded in seeds, foliage and pericarp, respectively, of the chickpea

272

plants raised from seeds treated with the consortium compared to the untreated control.

273

Interestingly, no significant difference in organic carbon content was recorded in pericarp of the

274

chickpea plants raised from the seeds treated with P. fluorescens alone compared to the untreated

275

control.

276

Biochemical contents in chickpea


Page 13 of 32


277

Influence of the microbes on biochemical contents such as total phenolics, protein, carbohydrate

278

and flavonoids was also similar to their effect on host biomass enhancement and nutritional

279

contents. The microbial consortium treated plants showed significant increase in the biochemical

280

contents in different parts of chickpea compared to the untreated control plants and in several

281

instances over the individual microbial treatments.

282

Total phenolic content (TPC)

283

TPC was highest in the foliage of chickpea plants followed by pericarp and seed in all microbial

284

treatments compared to the untreated controls (Fig. 3A). TPC in foliage were nearly ten folds

285

higher compared to seeds and nearly 2 folds higher compared to pericarp. TPC ranged from 0.67

286

to 1.07, 4.23 to 4.96 and 7.15 to 12.36 mg g-1 in seed, pericarp and foliage, respectively. TPC

287

content did not vary significantly in most of the individual microbial treatments but the

288

variations were significant in the microbial consortium treatment. Increase in TPC in seed,

289

pericarp and foliage was 59.7, 17.25 and 2.8%, respectively, compared to their untreated control

290

counterparts.

291

Protein content

292

Protein content in seed, pericarp and foliage of chickpea plants treated with the microbial

293

consortium was higher compared to their untreated control plants (Fig. 3B). Protein content of

294

seed, pericarp and foliage of the chickpea plants treated with the consortium of OKC and T42

295

was 9.78, 7.68 and 18.53% higher, respectively, than their untreated control plants. The protein

296

content ranged from 659.7 to 724.3, 434.02 to 467.36 and 424.86 to 503.61 µg g-1 of dry weight

297

in seed, pericarp and foliage, respectively. Total protein content was highest in seed followed by

298

foliage and pericarp. The protein content in seed was higher than foliage and pericarp and its



Page 14 of 32

299

content was nearly equal in foliage and pericarp. However, statistically no significant difference

300

was recorded in the protein content of the pericarp of treated and untreated chickpea plants.

301

Total carbohydrate content

302

Total carbohydrate content varied from 56.67 to 71.53, 57.3 to 72.56 and 58.58 to 76.28 mg g-1

303

of dry weight in seed, pericarp and foliage, respectively (Fig. 3C). Chickpea plants treated with

304

the microbial consortium recorded maximum carbohydrate contents followed by the plants

305

treated with either Trichoderma or Pseudomonas alone. In contrast the untreated chickpea plants

306

had lowest carbohydrate content in their seed, pericarp and foliage compared to the microbial

307

treatments. Statistically significant increase in total carbohydrate content was recorded in the

308

consortium treated chickpea plant parts compared to the individual microbial treatments and

309

control plant parts.

310

Total flavonoid content (TFC)

311

Total flavonoid content in seed, pericarp and foliage of the chickpea plants varied from

312

0.998±0.001 to 1.029±0.0128, 1.139±0.003 to 1.22±0.003 and 1.257±0.016 to 1.371±0.014 mg

313

quercetin equivalents g−1 DW, respectively (Fig. 3D). Seeds, pericarp and foliage of the chickpea

314

plants treated with the microbial consortium of OKC and T42 showed 3.11, 7.81 and 9.15%

315

higher accumulation of flavonoid content compared to their untreated control counterparts. Total

316

flavonoid content was significantly high in all chickpea parts in the consortium treatment

317

compared to their untreated control counterparts. In contrast, no significant difference in

318

flavonoid content was observed in the chickpea parts of the individual microbial treatments of

319

either OKC or T42. Flavonoid content was highest in foliage followed by pericarp and least in

320

seed.

321

Reducing power (RP)


Page 15 of 32


322

Similar to total flavonoid contents the reducing power of seed, pericarp and foliage of chickpea

323

plants was high in all parts of the consortium plants compared to that of untreated control plants.

324

The percent increase in reducing power varied from 29.12 to 62.0, 27.92 to 59.11 and 33.7 to

325

59.11 in seed, pericarp and foliage, respectively (Fig. 4). Reducing power was significantly high

326

in all chickpea parts in the microbial consortium treatment compared to their individual

327

treatments and untreated control plants. Interestingly, the reducing power in all chickpea parts

328

treated with the microbial consortium was nearly equal.

329

HPLC analysis of phenolics and flavonoids

330

HPLC analyses revealed variations in the phenolic acid contents in different chickpea parts in

331

different treatments. Altogether 8 compounds viz., shikimic acid, and phenolic acids such as

332

gallic acid, t-chlorogenic acid, syringic acid, p-coumaric acid, ferulic acid, quercetin and

333

kaempferol were detected from the chickpea part extracts. Phenolic acid contents in foliage, seed

334

and pericarp were significantly high in microbial treatments particularly in the microbial

335

consortium treatment (except t-chlorogenic acid and quercetin in pericarp) compared to their

336

control counterparts (Table 1). Similarly, shikimic acid content was also significantly high in

337

both seed and foliage but the content is not-significantly high in pericarp. Individually, the

338

phenolic acids, viz., t-chlorogenic acid, syringic acid, and p-coumaric acid, and flavonoids

339

quercetin and kaempferol accumulated in higher contents in either pericarp or foliage in the

340

microbial consortium treatment whereas highest accumulation of the other two phenolics gallic

341

and ferulic acids was observed in individual treatment of OKC in pericarp. Among the three

342

different chickpea parts, phenolic acid accumulation was least in chickpea seeds. Nevertheless,

343

the phenolic acid contents in seeds were higher in all microbial treatments compared to the

344

untreated control counterparts.



Page 16 of 32

345

PCA analysis showed that total phosphorus and nitrogen contents are the major

346

components that clubbed with dry weight and protein content of the chickpea seeds in various

347

treatments where plants were raised from either individual Pseudomonas and Trichoderma

348

treated seeds or plants raised from the consortium treated seeds. Similarly, the mineral contents

349

of Na, Ca and K along with organic matter content were clubbed with the carbohydrate content

350

and reducing power of the pericarp of plants in various treatments that were raised from either

351

individual Pseudomonas and Trichoderma treated seeds or plants raised from the consortium

352

treated seeds. Similarly, the total phenolic content was clubbed with the total flavonoid content

353

in all treatments in foliage (Fig. 5).

354

Discussion

355

Chickpea is an important food component in large parts of the world31 but very little is known

356

about its potential health benefits compared to the other legumes. Few earlier studies showed that

357

chickpea consumption lower the serum total cholesterol levels32,33 and coronary heart disease

358

(CHD) risk34. Efforts are being made to increase nutritional value of food through breeding

359

programs, biotechnological interventions and dietary supplements to the food products.

360

Rhizosphere microbe-induced plant defense is well demonstrated, however, unifying studies

361

linking rhizosphere microbe-induced nutritional value in edible parts in crop plants are still

362

lacking. Therefore, in the present study, we compared the effects of two rhizosphere competent

363

and compatible microbes (P. fluorescens OKC and T. asperellum T42) in enhancing the

364

nutritional value in human and livestock edible parts of chickpea.

365

N, P and K are the three main inorganic mineral nutrients which are essentially required

366

for crop growth. They are also crucial constituents of several enzymes, hormones, amino acids

367

and genetic materials in plants that take part in various life functions.35-37 Rhizosphere competent


Page 17 of 32


368

microbes play a vital role in making these nutrients available for plant uptake and their use

369

efficiency by the host plants.13 We observed a direct correlation of increased dry weight and

370

protein content of seeds in microbial treatments with increase in total phosphorus and nitrogen

371

content. Similarly, we also observed that increase in minerals and organic matter also correlated

372

with carbohydrate content in chickpea in the microbial treatments. Additionally, the total

373

phenolic content was found directly correlated with total flavonoid content in foliage collected

374

from the plants raised from seeds treated with the microbes. These correlations could directly be

375

attributed with enhanced uptake of several important nutrients (N, P, K, Na, and Ca) by chickpea

376

in microbial treatments compared to their untreated control counterparts. Interestingly, chickpea

377

plants treated with the consortium of OKC and T42 showed maximum increase in nutrient

378

contents indicating the advantage of consortium treatment of compatible microbes over their

379

individual applications. Microbial consortium potentially mimics the natural environmental

380

conditions where soil microbes lives in community. Hence, in the present study enhanced

381

nutrient content in chickpea plant parts in the microbial consortium treatment could be attributed

382

to natural lifestyle with synergistic effects of the two microbes. Similar increase in N content is

383

also reported in chickpea plants treated with indigenous Mesorhizobium sp. and P. aeruginosa.38

384

Since, human and animals acquire most of the nutrients through food products, abundance of

385

nutrients in edible plant parts is therefore desirable as they affect our daily diets.

386

Further, significant increase in total phenolics, protein, flavonoid and carbohydrate

387

contents in consortium treated chickpea plant parts compared to untreated control plants is a true

388

indication of improved nutritional qualities in consortium treated plants. Since phenols are

389

important arsenal for plant defense against any invading pathogens and are closely associated

390

with free radical scavenging property, higher accumulation of phenolics in microbe treated



Page 18 of 32

391

chickpea plants is highly significant from health point of view. Similarly, polyphenols are also

392

involved in receptors and enzymes associated in the signal perception and transduction

393

pathways, and change the cellular redox conditions.39 Further, enhanced accumulation of

394

phenolics was also reported in Trigonella foenum-graecum when the plants were treated with

395

Bacillus lentimorbus.40 Individually shikimic acid is also important because it acts as a precursor

396

molecule for almost all phenolic compounds. It is interesting to note that shikimic acid

397

accumulation was more in pericarp and foliage compared to seeds in consortium treated seeds.

398

Higher shikimic acid accumulation indicates the additional advantage the host derives due to

399

enhanced phenylpropanoid activities leading to higher phenolics synthesis as these parts are

400

usually subjected to pathogen attacks. Similarly, a significant increase in quercetin and

401

kaempferol content in foliage and pericarp was also recorded in the chickpea plants raised from

402

the microbial consortium treated seeds. These flavonoids are known to act as antioxidants and

403

also enhance the antioxidant properties in foods41 by inhibiting the activity of alternative

404

oxidases.42 In diet, these flavonoids reduce lipid peroxidation and permeability of K due to

405

dysfunction of membrane in erythrocytes.43

406

The harmful effect of free radicals present in food and biological system are well known.

407

Free radicals may destroy food quality by converting them into poisonous food. Increased

408

reducing power stabilizes and stops free radical chain reactions by formation of stable products.

409

High RP may also increase with intracellular antioxidants, peptides of organism used to start

410

fermentation and their hydrogen-donating ability.27 Hence, increased RP in chickpea plant parts

411

in this study may be correlated with increased antioxidant levels such as total phenols and

412

flavonoids in chickpea plants raised from consortium treated seeds.


Page 19 of 32


413

Increasing use of cereals in livestock diet makes a competitive dispute with human feed

414

and nutrition whereas use of soybean in place of cereals is, however, a costly affair. Further,

415

livestock population in India is growing to meet the basic requirement of growing human

416

population, and at the same time demand for fodder supply has also increased. Thus, additional

417

resource such as chickpea straw has been accepted as feed for livestock. Therefore, livestock

418

feed with sustainably enhanced nutritional quality through use of rhizosphere competent

419

microbes will contribute significantly to the health of the livestock. Thus, use of proven

420

microbial consortium will not only increase the nutritional quality of the forage and roughage for

421

the livestock in countries like India but also reduce the demand and supply ratio.44 Therefore, use

422

of chickpea straw (both pericarp and foliage) is considered as one of the best alternatives to

423

overcome this problem due to their high nutritional value and harmless effect on egg production,

424

meat and milk quality.45

425

The current study thus advocates use of microbial consortia of compatible rhizosphere

426

microbes to improve nutrient content in plants, which also enhances yield. We demonstrated that

427

a useful microbial consortium can increase the nutritive value such as antioxidants of edible parts

428

of crop plants whose consumption could be a safe and effective way to battle against oxidative

429

stress in human and livestock.

430 431

Acknowledgements

432

SKY is grateful to Indian Council of Medical Research, New Delhi for financial assistance

433

[Grant No. 3/1/3/JRF-2012/HRD-66(80689)]. BKS is grateful to Indian Council of Agricultural

434

Sciences, New Delhi for financial assistance (Grant No. ICAR-NBAIM/AMAAS/2014-15/73).

435

References:



Page 20 of 32

436

(1) UNDESA. World population projected to reach 9.6 billion by 2050. URL

437

(http://www.un.org/en/development/desa/news/population/un-report-world-population-

438

projected-to-reach-9-6-billion-by-2050.html) (2013).

439

(2) Bhardwaj, D., Ansari, M.W., Sahoo, R.K., Tuteja, N. Biofertilizers function as key player

440

in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity.

441

Microb. Cell Fact., 2014, 13, 66.

442 443 444 445

(3) Raja, N. Biopesticides and biofertilizers: ecofriendly sources for sustainable agriculture. J. Biofertil. Biopestici., 2013, 1000e112:1000e112. (4) Patel, J.S., Singh, A., Singh, H.B., Sarma, B.K. Plant genotype, microbial recruitment and nutritional security. Front. Plant Sci., 2015, 6, 608.

446

(5) Patel, J.S., Sarma, B.K., Singh, H.B., Upadhyay, R.S., Kharwar, R.N., Ahmed M.

447

Pseudomonas fluorescens and Trichoderma asperellum enhance expression of Gα subunits

448

of the pea heterotrimeric G-protein during Erysiphe pisi infection. Front. Plant Sci., 2016,

449

6, 1206.

450

(6) Araujo, A.S.F., Santos, V.B., Monteiro, R.T.R. Responses of soil microbial biomass and

451

activity for practices of organic and conventional farming systems in Piauistate, Brazil.

452

Eur. J. Soil Biol., 2008, 44, 225–230.

453

(7) Megali, L., Glauser, G., Rasmann, S. Fertilization with beneficial microorganisms

454

decreases tomato defenses against insect pests. Agron. Sustain. Dev., 2013, 34, 649-656.

455

(8) Sahoo, R.K., Ansari, M.W., Pradhan, M., Dangar, T.K., Mohanty, S., Tuteja, N.

456

Phenotypic and molecular characterization of efficient native Azospirillum strains from rice

457

fields for crop improvement. Protoplasma, 2014, 251, 511-523.

458

(9) Sinha, R.K., Valani, D., Chauhan, K., Agarwal, S. Embarking on a second green revolution

459

for sustainable agriculture by vermiculture biotechnology using earthworms: reviving the

460

dreams of Sir Charles Darwin. Int. J. Agric. Health Saf., 2014, 1, 50–64.

461 462 463 464

(10) Ibrikci, H., Knewtson, S.J.B., Grusak, M.A. Chickpea leaves as a vegetable green for humans: evaluation of mineral composition. J. Sci. Food Agric., 2003, 83, 945–950. (11) Bampidis, V.A., Christodoulou, V. Chickpeas (Cicer arietinum L.) in animal nutrition: A review. Anim. Feed Sci. Tech., 2011, 168, 1-20.

465

(12) Maheri-Sis, N., Aghajanzadeh-Golshani, A., Cheraghi, H., Ebrahimnezhad, Y.,

466

Ghalehkandi, J.G., Asaadi-Dizaji, A. 2011. Dry matter degradation kinetics and


Page 21 of 32


467

metabolizable energy of chickpea (Cicer arietinum) straw in ruminants. Res. J. Biol. Sci.,

468

6, 635-638.

469

(13) Sarma, B.K., Yadav, S.K., Singh, S., Singh, H.B. Microbial consortium mediated plant

470

defense against phytopathogens: readdressing for enhancing efficacy. Soil. Biol. Biochem.,

471

2015, 87, 25–33.

472

(14) Jain, A., Singh, S., Sarma, B.K., Singh, H.B. Microbial consortium mediated

473

reprogramming of defense network in pea to enhance tolerance against Sclerotinia

474

sclerotiorum. J. Appl. Microbiol., 2012, 112, 537–550.

475

(15) Mishra, S., Nautiyal, C.S. Reducing the allelopathic effect of Parthenium hysterophorus

476

L. on wheat (Triticum aestivum L.) by Pseudomonas putida. Plant Growth Regul. 2012, 66,

477

155-165.

478

(16) Saxena, A., Raghuwanshi, R., Singh, H.B. Trichoderma species mediated differential

479

tolerance against biotic stress of phytopathogens in Cicer arietinum L. J. Basic Microbiol

480

2015, 55, 195-206.

481

(17) Patel, J.S., Kharwar, R.N., Singh, H.B., Upadhyay, R.S., Sarma, B.K. Trichoderma

482

asperellum (T42) and Pseudomonas fluorescens (OKC)-enhances resistance of pea against

483

Erysiphe pisi through enhanced ROS generation and lignifications. Front. Microbiol. 2017,

484

8, 306

485

(18) Yadav, S.K., Dave, A., Sarkar, A. Singh, H.B., Sarma, B.K. Co-inoculated biopriming

486

with Trichoderma, Pseudomonas and Rhizobium improves crop growth in Cicer arietinum

487

and Phaseolus vulgaris. Int. J. Agric. Environ. Biotechnol., 2013, 6, 255-259.

488 489 490 491 492 493 494 495

(19) Stuart, D.L., Wills, R.B. Effect of drying temperature on alkylamide and cichoric acid concentrations of Echinacea purpurea. J. Agric. Food Chem. 2003, 51, 1608-1610. (20) Jackson, N.E. Soil Chemical Analysis. Prentice Hall, Inc., Englewood Cliffs, NJ, 1973, p. 498. (21) Kemi Idowu, M., Adote Aduayi, E. Sodium-potassium interaction on growth, yield and quality of tomato in ultisol. J. Plant Interact. 2007, 2, 263-271. (22) Nelson, D.W., Sommers, L.E. Determination of total nitrogen in plant material. Agron. J., 1973, 65, 109–112.



Page 22 of 32

496

(23) Walkley, A., Black, I.A. An examination of the Degtjareff method for determining soil

497

organic matter, and a proposed modification of the chromic acid titration method. Soil Sci.,

498

1934, 37, 29-38.

499 500 501 502 503 504

(24) Zheng, Z., Shetty, K. Solid-state bioconversion of phenolics from cranberry pomace and role of Lentinus edodes β-glucosidase. J. Agric. Food Chem., 2000, 48, 895–900. (25) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. Protein measurement with the folin phenol reagent. J. Biol. Chem., 1951, 193, 265-275. (26) Hedge, J.E., Hofreiter, B.T., Whistler, R.L. Carbohydrate chemistry. Academic Press, New York (1962): 17.

505

(27) Jain, A., Singh, A., Chaudhary, A., Singh, S., Singh, H.B. Modulation of nutritional and

506

antioxidant potential of seeds and pericarp of pea pericarps treated with microbial

507

consortium. Food Res. Int., 2014, 64, 275–282.

508

(28) Irshad, Md., Zafarya, Md., Singh, M., Rizvi, M.M.A. Comparative analysis of the

509

antioxidant activity of Cassia fistula extracts. Int. J. Med. Chem. 2012, 2012 ID 157125.

510

(29) Singhal, M., Paul, A., Singh, H.P., Dubey, S.K., Gaur, K. Evaluation of reducing power

511

assay of chalcone semicarbazones. J. Chem. Pharm. Res., 2011, 3, 639-645.

512

(30) Singh, B.N., Singh, B.R., Singh, R.L., Prakash, D., Singh, D.P., Sarma, B.K., Upadhyay,

513

G., Singh, H.B. Polyphenolics from various extracts/fractions of red onion (Allium cepa)

514

peel with potent antioxidant and antimutagenic activities. Food Chem. Toxicol., 2009, 47,

515

1161-1167.

516

(31) Singh, A., Jain, A., Sarma, B.K., Upadhyay, R.S., Singh, H.B. Beneficial compatible

517

microbes enhance antioxidants in chickpea edible parts through synergistic interactions.

518

LWT-Food Sci. Technol. 2014, 56, 390-397.

519

(32) Ghorai, M., Mandal, S.C., Pal, M., Pal, S.P., Saha, B.P. A comparative study on

520

hypocholesterolaemic effect of allicin, whole germinated seeds of bengal gram and

521

guggulipid of gum gugglu. Phytother. Res., 2000, 14, 200-202.

522 523 524 525

(33) Mathur, K.S., Khan, M.A., Sharma, R.D. Hypocholesterolaemic effect of Bengal gram: a long-term study in man. Brit. Med. J., 1968, 1, 30-31. (34) Murty, C.M., Pittaway, J.K., Ball, M.J. Chickpea supplementation in an Australian diet affects food choice, satiety and bowel health. Appetite, 2010, 54, 282-288.


Page 23 of 32

526 527 528 529


(35) Maathuis, F.J.M. Physiological functions of mineral macronutrients. Curr. Opin. Plant Biol., 2009, 12, 250–258. (36) Krouk, G., Crawford, N.M., Coruzzi, G.M., Tsay, Y.F. Nitrate signaling: adaptation to fluctuating environments. Curr. Opin. Plant Biol., 2010, 13, 265-272.

530

(37) Chevalier, F., Rossignol, M. Proteomic analysis of Arabidopsis thaliana ecotypes with

531

contrasted root architecture in response to phosphate deficiency. J. Plant Physiol., 2011,

532

168, 1885-1890.

533

(38) Verma, J.P., Yadav, J., Tiwari, K.N., Kumar, A. Effect of indigenous Mesorhizobium

534

spp. and plant growth promoting rhizobacteria on yields and nutrients uptake of chickpea

535

(Cicer arietinum L.) under sustainable agriculture. Ecol. Eng., 2013, 51, 282-286.

536

(39) Halliwell, B., Rafter, J., Jenner, A. Health promotion by flavonoids, tocopherols,

537

tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not? Am. J. Clin.

538

Nutr., 2005, 81, 268S-276S.

539

(40) Nautiyal, C.S., Govindarajan, R., Lavania, M., Pushpangadan, P. Novel mechanism of

540

modulating natural antioxidants in functional foods: Involvement of plant growth

541

promoting rhizobacteria NRRL B-30488. J. Agric. Food Chem., 2008, 56, 4474–4481.

542 543 544 545

(41) Shahidi, F., Wanasundara, P.K. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr., 1992, 32, 67. (42) Shimoji, H., Yamasaki, H. Inhibitory effects of flavonoids on alternative respiration of plant mitochondria. Biol. Plantarum, 2005, 49, 117-119.

546

(43) Maridonneau-Parini, I., Braquet, P., Garay, R.P. Heterogenous effect of flavonoids on K+

547

loss and lipid peroxidation induced by oxygen-free radicals in human red cells. Pharm. Res.

548

Commun., 1986, 18, 61-73.

549 550

(44) Datta, D. Indian fodder management towards 2030: A case of vision or myopia. Int. J. Manage. Soc. Sci. Res., 2013, 2, 33-41.

551

(45) Vasta, V., Nudda, A., Cannas, A., Lanza, M., Priolo, A. Alternative feed resources and

552

their effects on the quality of meat and milk from small ruminants. Anim. Feed Sci. Tech.,

553

2008, 147, 223-246.

554 555 556



Page 24 of 32

557

Figures legends:

558

Fig. 1. Shoot fresh weight (SFW), Root fresh weight (RFW), Shoot dry weight (SDW), Root dry

559

weight (RDW) and Total biomass (TB) in g (A) and Shoot length (SL), Root length (RL) in cm

560

(B) of different treatments was recorded after 60 days. Dry weight of the seeds, foliage and

561

pericarp of chickpea raised from treatment with Pseudomonas and Trichoderma either singly or

562

in combination after 120 days (C). Data are means of three replicates. Vertical bars indicate the

563

standard deviations of the means. Different letters indicate the significant differences among

564

treatments within the results taken at the same time interval according to Duncan's multiple

565

range test at p ≤ 0.05

566

Fig. 2. Phosphorus (A), nitrogen (B), potassium (C), sodium (D), calcium (E) and organic matter

567

(F) content in the seeds, foliage and pericarp of chickpea raised from treatment with

568

Pseudomonas and Trichoderma either singly or in combination. Data are means of three

569

replicates. Vertical bars indicate the standard deviations of the means. Different letters indicate

570

the significant differences among treatments within the results taken at the same time interval

571

according to Duncan's multiple range test at p ≤ 0.05.

572

Fig. 3. Total phenolics (A), Protein (B), carbohydrate (C) and total flavonoid (D) content in the

573

seeds, foliage and pericarp of chickpea raised from treatment with Pseudomonas and

574

Trichoderma either singly or in combination. Data are means of three replicates. Vertical bars

575

indicate the standard deviations of the means. Different letters indicate the significant differences

576

among treatments within the results taken at the same time interval according to Duncan's

577

multiple range test at p ≤ 0.05.


Page 25 of 32


578

Fig. 4. Reducing power in the seeds, foliage and pericarp of chickpea raised from treatment with

579

Pseudomonas and Trichoderma either singly or in combination. Data are means of three

580

replicates. Vertical bars indicate the standard deviations of the means. Different letters indicate

581

the significant differences among treatments within the results taken at the same time interval

582

according to Duncan's multiple range test at p ≤ 0.05

583

Fig. 5. PCA ordination plot showing grouping of twelve treatments (shown by arrow) against

584

various enhanced nutrient contents (shown by triangles). Abbreviation: pericarp of control plants

585

(Cp), pericarp of Pseudomonas fluorescens OKC treated plants (Op), pericarp of Trichoderma

586

asperellum T42 treated plants (Tp), pericarp of consortium treated plants (OTp), foliage of

587

control plants (Cf), foliage of Pseudomonas fluorescens OKC treated plants (Of), foliage of

588

Trichoderma asperellum T42 treated plants (Tf), foliage of consortium treated plants (OTf),

589

seeds of control plants (Cs), seeds of Pseudomonas fluorescens OKC treated plants (Os), seeds

590

of Trichoderma asperellum T42 treated plants (Ts), seeds of consortium treated plants (OTs).

591

Total phosphate (TP), Nitrogen (N), Organic matter (OM), Sodium (Na), Potassium (K),

592

Calcium (Ca), Total dry weight (DW), Total phenolic content (TPC), Protein content (PC),

593

Carbohydrate content (C), Total flavonoid content (TFC) and Reducing power (RP).

594 595 596 597 598 599



Page 26 of 32

600

Table 1. Shikimic acid, phenolics and flavonoid contents in different parts of chickpea raised

601

from treatment with Pseudomonas and Trichoderma either singly or in combination†.

602

Pericarp (µg g−1) OKC T42 OKC + T42 Control a a b Shikimic acid 1767 ± 175.8 1640 ± 80.6 1564 ± 134 1508 ± 193b Gallic acid 52.2 ± 1.97a 43.2 ± 2.6ab 58.39 ± 10a 33.3 ± 13.8b a a a t-Chlorogenic acid 36.2 ± 0.07 35.44 ± 3.3 36.55 ± 1.3 34.32 ± 2a Ferulic acid 19.5 ± 1.21b 22.25 ± 1.5a 23.16± 4.1a 18.56 ± 1.8b bc b a Syringic acid 2.31 ± 1.11 3.09 ± 0.04 9.24 ± 0.2 1.03 ± 0.3c p-Coumaric acid 35.6 ± 1.91b 36.5 ± 2.6b 42.18 ± 2.6a 29.87 ± 2.1c a a a Quercetin 1.46 ± 0.07 1.94 ± 0.8 2.05± 0.1 1.40 ± 0.3a Kaempferol 17.94 ± 0.07a 12.09 ± 0.9b 18.94 ± 0.8a 11.56 ± 1.7b −1 Seed (µg g ) Shikimic acid 1740.7 ± 127.3b 1812.9 ± 219.5b 2045.4 ± 185.7a 1731.9 ± 301.7b Gallic acid 68.71 ± 2.8c 99.9 ± 14.1b 116.27 ± 4.8a 64.12 ± 9.2c t-Chlorogenic acid 35.46 ± 2.1b 30.49 ± 0.22c 46.26 ± 1.8a 29.74 ± 1.9c b c a Ferulic acid 17.91 ± 9.1 15.44 ± 6.18 23.59 ± 4.2 14.22 ± 7c Syringic acid 7.34 ± 0.2c 9.34 ± 0.8b 11.58 ± 0.02a 2.15 ± 0.72d bc bc a p-Coumaric acid 125.7 ± 3.6 127.02 ± 6.25 166.2 ± 51.3 119.71 ± 37c b b a 2.37 ± 0.48 4.78 ± 0.5 1.96 ± 1.5b Quercetin 2.5 ± 0.2 Kaempferol 10.76 ± 1.1a 7.04 ± 0.86b 10.88 ± 1.7a 6.87 ± 0.98b Foliage (µg g−1) b Shikimic acid 1385 ± 139.9 1752.5 ± 21.6a 1914.8 ± 9.4a 1363.1 ± 86.8b Gallic acid 98.1 ± 8.2b 103.67 ± 3.0a 109.62 ± 7.0a 94.01 ± 26b a b a 13.23 ± 0.6 55.62 ± 17.1 10.17 ± 1.7b Ferulic acid 43.12 ± 2.2 b b a Syringic acid 13.42 ± 1.1 13.19 ± 2.1 24.51 ± 1.8 11.51 ± 4b p-Coumaric acid 31.52 ± 3.4a 23.65 ± 3.3b 32.40 ± 14.3a 19.24 ± 1.4b b b a Quercetin 2.11 ± 0.2 2.81 ± 0.2 7.46 ± 1.4 1.62 ± 0.1b Kaempferol 103.41 ± 15.4c 131.19 ± 4.7b 151.25 ± 14.6a 97.84 ± 11.4d †Results are expressed as means of three replicates ± SD. Different superscript letters indicate

603

significant differences among treatments within the results taken at the same time interval

604

according to Duncan's multiple range test at p ≤ 0.05.

Phenolic acid

605


Page 27 of 32


Fig. 1



a

Page 28 of 32

A

B

a

a

c c a

a ab bc c

cc

a

bb

b

a

C

D ab bc

c a

b

a

c c c

a

a

E

a cc cc

F F

a c

d d

a

c

a

c c

Fig. 2


bc b

Page 29 of 32


a

10

dc Control

8

OKC

ab a ab b

T42 OKC+T42

4 2

c

ab b a

500

b aa

a

a

bb c

Control OKC

400

T42

300

OKC+T42

200 100 Pericarp

Foliage

a

90

a

a

bb

b cc

c

bb

c

60

Control

50

OKC

40

T42

30

OKC+T42

20 10 0 Pericarp

Seed

Seed

1.6

C Total Flavonoid Content (mg quercetin equivalents g−1 FW)

Seed

100 Carbohydrate (mg/g DW)

B

0 Pericarp

70

c

b

600

0

80

aa

700

b

12

6

800

A Protein (µg/g DW)

Total phenolic content (mM GAE/g of DW)

14

1.4

c bb

a c b bc

a

1.2

foliage

a babab

1

Control OKC

0.8

T42

0.6

OKC+T42 0.4 0.2 0

Foliage

D

Pericarp

Fig. 3


Seed

Foliage


Page 30 of 32

Reducing Power (% increase)

80 70

a

a

a

60 ab

50

b

40 30

b

b c

b

b c

bc

Control OKC T42 OKC+T42

20 10 0 Pericarp

Seed

Foliage

Fig. 4


Page 31 of 32


Fig. 5



Priming chickpea seeds with Pseudomonas and Trichoderma consortium enhances nutritional quality of edible parts 122x75mm (300 x 300 DPI)


Page 32 of 32

Compatible rhizosphere competent microbial consortium add value to

Recommend Documents