Metabolomes of Potato Root Exudates - American Chemical Society

Subscriber access provided by Northern Illinois University

Article

Metabolomes of Potato Root Exudates: Compounds That Stimulate Resting Spore Germination of the Soil-Borne Pathogen Spongospora subterranea Mark A. Balendres, David S Nichols, Robert S Tegg, and Calum R. Wilson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03904 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 19, 2016

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 34

Journal of Agricultural and Food Chemistry

1

Metabolomes of Potato Root Exudates: Compounds That

2

Stimulate Resting Spore Germination of the Soil-Borne

3

Pathogen Spongospora subterranea

4 5

Mark A. Balendres†, David S. Nichols‡, Robert S. Tegg†, and Calum R.

6

Wilson†*

7 8

†

9

Tasmania, 13 St. Johns Avenue, New Town, Tasmania 7008, Australia

New Town Research Laboratories, Tasmanian Institute of Agriculture, University of

10

‡

11

7001, Australia

Central Science Laboratory, University of Tasmania, Private Bag 74, Hobart, Tasmania

12 13

* Corresponding author (Tel: (+613)62266381; E-mail: [email protected])

14 15 16 17 18 19 20 21 22 23 24

1 ACS Paragon Plus Environment


Page 2 of 34

25


Page 3 of 34


Root exudation has importance in soil chemical ecology influencing

26

ABSTRACT:

27

rhizosphere microbiota. Prior studies reported root exudates from host and non-host plants

28

stimulated resting spore germination of Spongospora subterranea, the powdery scab pathogen

29

of potato, but the identity of stimulatory compounds was unknown. This study showed that

30

potato root exudates stimulated S. subterranea resting spore germination, releasing more

31

zoospores at an earlier time than the control. We detected 24 low-molecular weight organic

32

compounds within potato root exudates and identified specific amino acids, sugars, organic

33

acids and other compounds that were stimulatory to S. subterranea resting spore germination.

34

Given several stimulatory compounds are commonly found in exudates of diverse plant

35

species we support observations of non-host specific stimulation. We provide knowledge of S.

36

subterranea resting spore biology and chemical ecology that may be useful in formulating

37

new disease management strategies.

38 39

KEYWORDS: Powdery scab, root infection, Plasmodiophorid, HILIC UPLC-MS, zoospore

40

release, metabolomics

41 42 43 44 45 46 47 48 49 50



51

Page 4 of 34

INTRODUCTION

52

Spongospora subterranea (Wallr.) Lagerh. causes powdery scab and root disease in

53

potato.1 Spongospora diseases are major problems in potato production worldwide. The

54

pathogen downgrades the tuber fresh market-value, causes substantial economic losses in the

55

potato processing industry,2 and leads to failure of seed tuber certification and subsequent

56

losses in product value for seed potato producers.3 Primary zoospores initiate infection after

57

release from germinating resting spores that are formed into aggregates known as sporosori.4

58

Zoospores migrate to host roots, where they encyst and establish infections5 which develop

59

into plasmodia and may mature as zoosporangia or resting spores.6 Zoosporangia contain

60

secondary zoospores which are sources of secondary infection in tubers and roots after release

61

into the soil environment. When resting spores are released back into the soil they become the

62

new generation of primary inoculum. As such, the germination of S. subterranea resting

63

spores is the first crucial step in disease development. Despite its importance, knowledge of

64

the processes and the factors influencing resting spore germination is limited.7 Root exudates

65

play a critical role as stimulants of S. subterranea resting spore germination.4,8,9 Prior studies

66

report a lack of host specificity with exudates from both hosts and non-hosts capable of

67

stimulating germination. However, no attempts have been made to identify any stimulatory

68

compound(s) present in the root exudates.

69

An estimated 40-50% of the carbon fixed by plants is released as root exudates,

70

mostly as low molecular weight organic (LMWO) compounds, making root exudation a

71

significant carbon cost to the plant.10,11 Root exudates play important roles in rhizosphere

72

chemical ecology influencing interactions between the plant and other soil biota. They may

73

function as, but are not limited to, chemoattractants, inhibitors or stimulants of microbial

74

growth10 and may influence the colonization and activation of root-infecting pathogens.12,13

75

The response of soil microbes may vary depending on the root exudate’s organic composition.


Page 5 of 34


76

For instance, peanut root exudates differing in LMWO compound composition have varying

77

influence on spore germination of soil-borne peanut pathogens.14 The stimulatory effects of

78

potato root exudates to S. subterranea resting spore germination might also be explained by

79

their organic chemical composition.

80

Characterization of the root exudate metabolome can assist in gaining a better

81

understanding of plant-microbial interactions. The choice of techniques to be used in

82

metabolomic studies is important to achieve unbiased and successful characterization of a

83

metabolome. Metabolite analysis of aqueous samples, including root exudates, generally uses

84

gas or liquid chromatography which is usually coupled to mass spectrometry (MS). Gas-

85

chromatography is more suitable for volatile compounds, whilst liquid-chromatography (LC)

86

is commonly used for non-volatiles. Recently, new improvements have been made to LC

87

techniques, particularly for detecting polar, hydrophilic compounds. An improved hydrophilic

88

interaction ultra-performance liquid-chromatography mass spectrometry (HILIC UPLC-MS)

89

approach has been used to simultaneously identify more than 100 analytes from grapefruit

90

extracts in less than 30 min at detection levels as low as 5 ng/ml.15

91

The present study examined metabolomes of potato root exudates to gain insight into

92

their metabolite composition, and to determine if specific chemical constituents stimulate S.

93

subterranea resting spore germination. The bioassays were performed in a soil-less in vitro

94

system9 coupled with microscopy, and the targeted characterization of compounds in the root

95

exudates was achieved using a HILIC UPLC-MS methodology.15 This work advances the

96

understanding of the biology of S. subterranea resting spore germination and provides new

97

insights into the pathogen’s chemical ecology. Furthermore, the primary metabolic

98

composition of potato root exudates is outlined, expanding known metabolite constituents.

99

Potato genotypes used in this study have importance in potato breeding programs and in



Page 6 of 34

100

commercial production. Hence, the metabolite profile may compliment other “omics”

101

approaches for potato improvement.

102 103

MATERIALS AND METHODS

104

Potato Root Exudate Collections. Root exudates were collected from deionized water

105

extracts of tissue-cultured potato (Solanum tuberosum). Four potato cultivars that varied in

106

their known resistance to S. subterranea diseases were used. Cultivars ‘Agria’ and ‘Iwa’ are

107

highly susceptible to both tuber and root disease, whilst cvs. ‘Russet Burbank’ and ‘Gladiator’

108

have moderate to strong resistance to tuber infection and moderate resistance to root

109

infection1,16. Preparation and collection of potato root exudates were performed aseptically.

110

Potato tissue-cultured plantlets were grown in potato multiplication medium (MS salts and

111

vitamins, 30 g/L of sucrose, 40 mg/L of ascorbic acid, 500 mg/L of casein hydrolysate and 0.8%

112

agar, pH 5.8) under a 16 h photoperiod using white fluorescent lamps (65 µmol/m2/s) at

113

22 °C.17 After 2 weeks, plants were gently uprooted from the media, their roots were washed

114

with sterile deionized water, blotted dry on sterile tissue paper, then placed in a sterile

115

polycarbonate bottle containing 20 ml of fresh sterile deionized water (instrument

116

conductivity at 0.059 µS/cm). For exudate collection plants were incubated in the water

117

solution under the same light and temperature regime as before for varying incubation periods.

118

Firstly, to confirm the capability of potato root exudates to stimulate resting spore

119

germination and to assess possible influences of cultivar, a series of nine bioassays comparing

120

32 individual potato root exudates was conducted. Either two (‘Gladiator’ and ‘Agria’) or all

121

four cultivars were incubated in the water solution for 7 d prior to collection and testing. Then

122

for analysis of root exudate composition and association with stimulation of resting spore

123

germination a further 12 exudate solutions were collected. Exudates from all four cultivars

124

were collected following 2, 7 and 18 d incubation in the water solution. All exudate solutions


Page 7 of 34


125

were stored in sterile Falcon tubes at -20 °C, in the dark, until used. There were no signs of

126

microbial contamination in the root exudate solutions prior to use. Unless otherwise stated, all

127

chemicals used in this study were sourced from Sigma-Aldrich (St. Louis, MO).

128

Inoculum Preparation. The S. subterranea inoculum (resting spores) was prepared from

129

powdery scab-affected tubers, collected from Devonport, Tasmania, Australia (41.17 °S,

130

146.33 °E). Diseased tubers were washed with running tap water for 1-2 min, soaked in 2%

131

sodium hypochlorite (White King, Pental Products Ltd Pty, Melbourne, Australia) for 3 min,

132

quickly rinsed, and air-dried. Lesions were excised using a scalpel, dried for 4 d at 40 °C,

133

ground by mortar and pestle, and stored at 4 °C until used. The inoculum was approximately 1

134

y old when used. Inoculum contained approximately 6,900 sporosori/mg, as determined by

135

suspending 0.1 g of inoculum in 10 ml water and quantification using light microscopy.

136

Resting Spore Germination Assay. Presence of motile zoospores in the test solutions

137

indicated resting spore germination.9 Briefly, 1 mg of inoculum (6,900 sporosori) was

138

suspended either in 1.5 ml of root exudate, individual compound solutions (0.1 mg/ml), or in

139

deionized water (control) solutions, each in a 2 ml microcentrifuge tube covered with

140

aluminum foil. Tubes were incubated for 30 d at 15–18 °C. At 3-7 d intervals, 35 µl from

141

each treatment was subsampled. The number of motile zoospores in the subsample was

142

determined with a DM 2500 LED microscope (Leica Microsystem, Wetzlar, Germany) at

143

200X magnification, scanning in an inverted “S” manner within a 22 mm x 22 mm area of

144

each microscope-slide. S. subterranea zoospores were identified by morphology4 and motility

145

behavior.18 Assessments of each root exudate solution and 43 individual LMWO compounds

146

were repeated, respectively, seven and two times. Each assay consisted of three replications.

147

In the individual chemical bioassay, Hoagland’s solution and sterile deionized water were

148

added, respectively, as a stimulant and non-stimulant controls.



Page 8 of 34

149

Additional bioassays were performed to further validate S. subterranea zoospore

150

identity. The first used a modified tomato-bait test.19 A 100 µl subsample from each test and

151

control solution was transferred into a McCartney bottle containing 5 ml of non-sterile

152

deionized water and a healthy 2 week old tomato plant (cv. Grape). Tomato plants were left in

153

the solution for 24-48 h at 15-18 °C; to allow zoospores to encyst, and were then removed.

154

Roots were washed to remove any resting spores adhering to root surfaces. Each plant was

155

transferred to a new McCartney bottle containing 20 ml of non-sterile deionized water and

156

grown for an additional 2 weeks to allow the development of zoosporangia. This test was

157

repeated, using different zoospore solution sources. Sample roots were then cut, placed on

158

microscope slides, stained with 0.1% Trypan blue for at least 10 min, and zoosporangia20

159

were examined under a light microscope. In the second test, the number of zoospores

160

successfully attached to S. subterranea roots of cv. ‘Gladiator’ and cv. ‘Iwa’ host roots was

161

determined. Single, 2 cm long, roots were placed on microscope slides, which were flooded

162

with 70 µl of test solutions and incubated at room temperature for 10 min. Each test was

163

replicated three times. To ensure all roots received a similar exposure to zoospores, the test

164

was performed one root/cultivar/replicate at a time. The number of zoospores attached on the

165

roots were counted microscopically, at 200X magnification, by scanning the whole root.

166

Phytochemical Analyses. Detection of primary metabolites in potato root exudate

167

solutions was done using HILIC UPLC-MS.15 The UPLC assays were performed using a

168

Acquity UPLC H-class system (Waters, Milford, MA), and MS analyses using a Xevo triple

169

quadrupole MS system (Waters). HILIC separation was done on a 2.1 mm x 150 mm Acquity

170

1.7 µm BEH amide VanGuard column maintained at 60 °C and eluted with a two-step

171

gradient at 500 µL/min flow rate for 30 min. The mobile phases were composed of A

172

(acetonitrile/water, 95–5 (v/v), 0.1% formic acid. and 0.075% NH4OH) and B (acetonitrile–

173

water, 2–98 (v/v), 0.2% formic acid. and 0.1% NH4OH). LiChrosolv-grade acetonitrile was


Page 9 of 34


174

purchased from Merck (Darmstadt, Germany), whilst formic acid (>95%) and NH4OH were

175

purchased from Sigma-Aldrich. The gradient started with a 4 min isocratic step at 100%

176

mobile phase A, then rising to 28% mobile phase B over the next 21 min and finally to 60% B

177

over 5 min.15 The column was then equilibrated for 12 min in the initial conditions. Two

178

cycles of weak and strong solvent washing of the injection system were carried out between

179

injections. The injection volume was 10 µl and the column eluent was directed to the mass

180

spectrometer. Metabolite detection was achieved using selected ion monitoring and an

181

electrospray ionization source was applied operating in both positive and negative ion mode.

182

The parameters in the electrospray were set as follows; capillary voltage, −2.5 kV or 3 kV;

183

cone and desolvation temperatures, respectively, 150 and 400ºC with a desolvation gas flow

184

of 950 L/h and cone flow of 100 L/h. The cone voltage was optimized for each individual

185

analyte. The identity of metabolites was confirmed by detection of the expected [M+H]+ or

186

[M-H]- molecules at known retention indices15 by the analysis of standard chemicals, as both

187

pure standards and spiked into root exudates samples. The identity of amino acid metabolites

188

were also confirmed by tandem-MS multiple reaction monitoring experiments, with the

189

detection of known product molecules arising from selected precursor molecular species.15

190

Data and Statistical Analyses. Zoospore numbers were converted to counts per 100 µl

191

solution, prior to statistical analysis using SPSS statistical software, ver. 22, (IBM, Armonk

192

NY). An independent T-test and one-way analysis of variance (ANOVA) were performed for

193

data having, respectively, two and more than two treatments. Data containing two factors

194

were analyzed by two-way ANOVA. Multiple comparison of means was carried out using the

195

Fisher’s Least Significant Difference (LSD) test at 0.05 level of probability. HILIC UPLC-

196

MS analyses were done using MassLynx XS software.15 The limit of detection and limit of

197

quantitation of the analytes were determined at signal-to-noise ratios of 3 and 10 respectively.

198

A hierarchical cluster dendrogram was constructed using the average linkage (between group)



Page 10 of 34

199

method, with binary data measured using the squared Euclidean distance. A pattern (color)

200

map was constructed to represent the distribution (numbers) of the metabolite/compound

201

classes detected in various root exudates.

202 203

RESULTS

204

Spongospora subterranea Zoospore Identity. Motile biflagellate (of unequal length)

205

zoospores, of approximately 4-5 µm diameter, were observed in the majority of test potato

206

root exudates and occasionally in control (sterile deionized water) solution. The presence of

207

the two flagella on each zoospore were best observed at 400X magnification, but observation

208

of zoospore swimming patterns was best viewed at 200X magnification. Other uniflagellate

209

and biflagellate organisms were present, but their numbers were minimal and their movement

210

patterns distinct from those S. subterranea zoospore. Figure 1 shows that zoosporangia,

211

indicative of S. subterranea infection, developed when test solutions with S. subterranea

212

zoospores were exposed to tomato roots. Similarly zoospores were observed attached to

213

potato roots with a greater number of zoospores (P=0.008) attached to roots of the susceptible

214

cv. ‘Iwa’ (16.67 ± 2.60) than the resistant cv. ‘Gladiator’ (3.67 ± 0.33; Figure 1).

215

Stimulatory Effects of Potato Root Exudates to Resting Spore Germination. In

216

the first experiment across all nine assays, exposure of resting spores to 25 of the 32 potato

217

root exudates stimulated release of S. subterranea zoospores (Figure 2). Zoospores were

218

occasionally released in the control solution (four out of nine assays), but release was

219

substantially later (average first release was at 11.8 d) than in the potato root exudates (4.4 d).

220

Within individual assays statistically significant differences in zoospore numbers released

221

were found between potato cultivars but there was no consistent cultivar response between

222

assays (Figure 2).


Page 11 of 34


223

In the second experiment, the duration of incubation of potato roots prior to exudate

224

collection (2, 7, and 18 d) had no statistically significant effect (P=0.152) on stimulation of

225

resting spore germination and while cultivar showed a statistically significant effect

226

(P=0.050), there were again no obvious consistent trends (Figure 3). The interaction of

227

incubation period and cultivar had a clear statistically significant effect (P=0.033), with

228

differences between cultivar most evident in the 18 d incubation samples (Figure 3).

229

Metabolite Constituents of Various Potato Root Exudates. The HILIC UPLC-MS

230

analysis detected a total of 24 LMWO compounds from potato root exudates (Table 1). These

231

compounds ranged from 104-504 Da molecular weight, and included eight amino acids, one

232

sugar, three sugar alcohols, five organic acids and seven other organic compounds. Detection

233

of these compounds varied among root exudates. Asparagine, glutamic acid, glutamine,

234

proline, serine, pinitol, choline, trehalose and tyramine were detected in most of the potato

235

root exudates. However, some compounds were uniquely observed in a particular potato

236

cultivar root exudates or collection date. For instance, raffinose, dehydroascorbic and quinic

237

acid, and adenosine were, respectively, present only in ‘Iwa’, ‘Agria’, and ‘Gladiator’ root

238

exudates and histamine was only detected in the exudates incubated for 18 d. The hierarchical

239

cluster analysis revealed that potato root exudates could be divided at 80% similarity into

240

three groups based on the metabolite composition (Figure 4). Cluster 1 included all ‘Iwa’ root

241

exudates, cluster 2 was composed of mostly 2 and 7 d incubation and all ‘Russet Burbank’

242

root exudates, and cluster 3 were mostly 18 d incubation and ‘Gladiator’ root exudates.

243

Screening of Metabolites for Stimulants of Resting Spore Germination. Testing

244

individual compounds provided a more robust direct relationship of each compound to resting

245

spore germination stimulation (Figures 5 and 6).

246

subterranea resting spores was chemical-specific (Figure 5). By comparing capacity and

247

timing of zoospore release it was determined that five of the 18 compounds found in potato

Stimulation of germination of S.



Page 12 of 34

248

root exudates and seven of the 25 additional compounds tested were stimulant at 0.1 mg/ml,

249

resulting in zoospore release at least 8 d earlier than the water control (Figures 5 and 6). L-

250

Glutamine and tyramine had greater effects (P=0.045 and P=0.010, respectively) on resting

251

spore germination than the other compounds tested and the Hoagland’s stimulant control.

252

Mean accumulated zoospore numbers in other stimulant LMWO compounds, L-rhamnose,

253

cellobiose, L-aspartic acid, N-acetyl cysteine, piperazine, glucoronic acid, L-serine, succinate,

254

L-citrulline

255

control (P>0.05) but were deemed stimulant as zoospore release in the presence of these

256

compounds was at least 8 d earlier than in the deionized water control (Figure 6).

and citric acid, were not statistically different from the non-stimulant water

257 258

DISCUSSION

259

This study aimed at elucidating, at a metabolite level, the stimulatory effects of potato

260

root exudates on S. subterranea resting spore germination. LMWO compounds are the main

261

constituents of plant root exudates and are known stimuli of soil-borne plant pathogens.12,21 In

262

an initial series of in vitro bioassays we showed in most instances aqueous potato root

263

exudates stimulated S. subterranea resting spore germination, releasing zoospores earlier and

264

in greater numbers than the deionized water control. It was also clear that there was no

265

consistent cultivar effect, with variability between assays likely due to variations in plant

266

physiological condition. Using targeted HILIC UPLC-MS based metabolite profiling,15

267

covering major primary metabolites in a plant biological system, we identified 24 polar

268

LMWO compounds within potato root exudates. The majority of these compounds were

269

amino acids, which is in agreement with other plant species22,23 and from prior metabolomic

270

studies of potato tubers.24,25 The LMWO compounds, however, were not uniformly detected

271

across all potato root exudates, with more compounds detected in the extracts from the cvs

272

‘Agria’, ‘Gladiator’ and ‘Russet Burbank’ than in the ‘Iwa’ extracts. Hierarchical cluster


Page 13 of 34


273

analysis of the potato root exudate components separated exudates into three clusters

274

associated with cultivar and exudate incubation period. Similar variations have been observed

275

with both plant and environmental factors influencing release of LMWO compounds.26,27

276

Analysis of metabolites from tubers of different Solanum spp. indicated individual species

277

could be identified by their metabolic composition.24 Variability of root exudate composition

278

with plant physiological and environmental factors22 suggests these are less useful for

279

biotyping potato cultivars but with knowledge of their biosynthesis it may be possible to

280

investigate genetic and proteome changes in a plant grown under particular conditions. For

281

example, the HILIC UPLC-MS based metabolite profiling

282

extended to analyze metabolite change in S. subterranea-infected plants to gain further

283

insights into genes expressed by host plants during root infection.

15

used in this study could be

284

Pathogen germination or activation by host root exudates is a common phenomenon

285

among many soil-borne pathosystem, and this is particularly the case for environmentally-

286

resistant-spore producing pathogens.12 The stimulation of resting spore germination by potato

287

root exudates (Figure 2) corroborated the findings of earlier reports.8,9 While there could be

288

doubts that the zoospores identified, in this and other studies,4,9,18 may be those of other

289

flagellated contaminant organisms, additional testing showed that the detected zoospores

290

produced zoosporangia after host infection,1 and attached to potato roots in a differential

291

manner associated with known cultivar resistance28 as would be expected with S. subterranea.

292

Furthermore, the initial release of zoospores (4-5 d) coincides with the other reports which

293

have used in vitro approaches to examine S. subterranea resting spore germination.7 ,21

294

Root exudates from both host and non-host plants and their chemical constituents have

295

been suggested to play a part of the S. subterranea pre-infection activation.4,7, 9,19 Our study

296

supports these observations. In several other pathosystems individual host specific

297

compounds found within root exudates activate and chemotaxically attract the pathogen



Page 14 of 34

298

spores to a susceptible host plant to facilitate infection.10,12 Conversely, we have shown that a

299

diverse range of LMWO compounds are stimulatory toward S. subterranea resting spore

300

germination. Whilst we show these are produced by the potato host many of these compounds

301

are commonly found in root exudates from other plant species including non-hosts.22,23 Thus

302

we can conclude that resting spore germination is not host-specific. This is in agreement with

303

studies of the closely related Plasmodiophora brassicae.29-32 It is thus unsurprising that

304

production of stimulatory compounds was not associated with known potato cultivar

305

resistance with cultivar effects quite variable. Rather it is likely that production of stimulatory

306

LMWO compounds will be more strongly influenced by other factors such as the

307

physiological condition of the plant.27 It has been suggested that S. subterranea zoospores are

308

likely attracted to root exudates4 or specific chemical compounds within these exudates.7 This

309

has been well documented in other pathosystems33,34 and variation in the susceptibility of

310

plants can be associated with variation of chemo-attraction by the zoospores.35,36 We provide

311

preliminary data indicating zoospore attachment to potato host roots is affected by resistance

312

of the potato cultivar but to date no studies specifically examining chemotaxic attraction of S.

313

subterranea zoospores have been conducted.

314

Control of potato diseases caused by S. subterranea has been partly successful.37

315

Planting resistant cultivars has proven the most effective means for powdery scab control, but

316

cultivars lack effective strong resistance to root infection. The use of fungicides provides

317

partial efficiency, reducing disease impact,37,38 probably by reducing inoculum pressure or

318

delaying initial zoospore-host interaction. However, there have been general restrictions on

319

the use of synthetic chemicals for disease management due potential health and environmental

320

risks, and on crop toxicity.39 This has resulted in increased efforts to explore alternative

321

chemical and biological control methods. Several investigations used a variety of plant

322

extracts and their active chemical components for control of major vegetable and grain crop


Page 15 of 34


323

diseases.40 Others have used organic amendments41 and biological control agents to eradicate

324

or reduce inoculum levels and minimize disease outcomes,42 while some continuously test

325

other chemical substances.43 Few of these types of investigations have been applied to S.

326

subterranea diseases of potato.37 The present study identified several root exudate compounds

327

that stimulate S. subterranea resting spore germination and application in absence of a host

328

plant could assist in reducing soil inoculum levels by removing viable resting spores.7

329

We acknowledge that the tests were done in vitro, and that the root exudates were

330

collected from plants grown in sterile aqueous solutions. Compounds detected in this study

331

may differ from those exuded by plants grown under field conditions due to soil

332

environmental and biological factors. Nevertheless, studies using root exudates from in vitro

333

cultures provide clear indications of direct interactions of the pathogen with host root

334

exudates due to the absence of potential compounding factors which could affect pathogen

335

behavior.44 Moreover, the main objective of this study was to determine whether compounds

336

released by potato roots influence S. subterranea resting spore germination. Identifying

337

physiological and environmental factors that may influence potato root exudation, particularly

338

those influencing the release of chemical-stimulants in potato root exudates, is worthy of

339

further investigation.7

340

In conclusion, have demonstrated further support, and provide the first direct evidence,

341

to the previous findings that potato root exudates stimulate S. subterranea resting spore

342

germination.8,9 We also showed that these biologically active potato root exudates contain

343

polar LMWO compounds, and that specific compounds present within these exudates and

344

related compounds stimulate resting spore germination. This study offers new knowledge of

345

the LMWO chemical constituents in potato root exudates that directly influence resting spore

346

germination and thus, makes a contribution to knowledge of the pathogen’s biology and

347

chemical ecology. Active compounds are known from root exudates of other plants (including



Page 16 of 34

348

non-hosts) which supports the contention that resting spore stimulation is not host specific.

349

Our findings may also be of importance to other root-infecting pathogens of potato that may

350

be influenced by root exudation. From an integrated pest management perspective, the use of

351

non-pesticidal resting spore germination-stimulating chemical compounds to decrease the

352

levels of infective agents (zoospores) during cropping breaks, could provide a new method to

353

manage S. subterranea soil inoculum, and potentially decrease the subsequent disease

354

outcomes. 7

355 356

ACKNOWLEDGMENT

357

We thank Annabel Wilson (University of Tasmania; UTAS, Australia) for providing

358

tissue cultured potato plants, Sergey Shabala (UTAS) for certain chemicals, Ross Corkrey

359

(UTAS) for assistance in statistical analysis, and James Braselton (Ohio University, Athens,

360

OH) for technical advice and useful discussion.

361 362

SUPPORTING INFORMATION

363

The Supporting Information is available free of charge on the ACS Publication website.

364

a. Composition of the Hoagland’s solution used in this study.

365

b. List of potato root exudate metabolites, with selected ion monitoring, molecular

366

formulas, monoisotopic masses, precursor and product ions, ionization polarity,

367

retention time, cone and collision voltages.

368 369

FUNDING SOURCE

370

M. B. is supported by a Tasmanian Graduate Research Scholarship provided by the

371

University of Tasmania. This study was supported by Horticulture Innovation Australia

372

Limited: project number PT14002 (http://www.horticulture.com.au/) with funding from


Page 17 of 34


373

Simplot Australia Pty. Ltd., McCain Foods Australia Pty. Ltd., and matched funds from the

374

Australian Federal Government. The University of Tasmania provided in-kind support.

375 376

NOTE

377

The authors declare no competing financial interest.

378 379

REFERENCES

380

(1) Falloon, R. E.; Merz, U.; Butler, R. C.; Curtin, D.; Lister, R. A.; Thomas, S. M., Root

381

infection of potato by Spongospora subterranea: knowledge review and evidence for

382

decreased plant productivity. Plant Pathol. 2016, 65, 422-434.

383 384 385

(2) Wilson, C. R., Plant pathogens – the great thieves of vegetable value. Acta Hortic. 2016, in press. (3) Tegg, R. S.; Corkrey, R.; Wilson, C. R., A comparison of potato seed-tuber sampling

386

strategies using visual and DNA analyses to estimate incidence of major seed tuber-borne

387

pathogens. Eur. J. Plant Pathol. 2014, 139, 359-367.

388 389 390 391 392 393 394 395

(4) Kole, A. P., A contribution to the knowldge of Spongospora subterranea (Wallr.) Lagerh., cause of powdery scab of potatoes. Tijdschr. over Plantenziekten 1954, 60, 1-65. (5) Keskin, B.; Fuchs, W., Der infektionsvorgang bei Polymyxa betae. Arch. Mikrobiol. 1969, 68, 218-226. (6) Braselton, J. P., Ultrastructural karyology of Spongospora subterranea (Plasmodiophoromycetes). Can. J. Plant Pathol. 1992, 70, 1228-33. (7) Balendres, M. A.; Tegg, R. S.; Wilson, C. R., Key events in pathogenesis of spongospora diseases in potato: a review. Australas. Plant Pathol. 2016, 45, 229-240.

396

(8) Merz, U., Epidemiological aspects of powdery scab of potatoes caused by Spongospora

397

subterranea. In Proceedings of the 2nd Symposium of the International Working Group



398

on Plant Viruses with Fungal Vectors, Montreal, Canada, Hiruki, C., Ed., American

399

Society of Sugar Beet Technologists: Denver, CO, 1993; pp 104-106.

400

Page 18 of 34

(9) Fornier, N.; Powell, A. A.; Burgess, P. J. Factors affecting the release of primary

401

zoospores from cystosori of Spongospora subterranea assessed using monoclonal

402

antibody ELISA test, In Proceedings of the 3rd Symposium of the International Working

403

Group on Plant Viruses with Fungal Vectors, Dundee, Scotland, Sherwood, J. L.; Rush,

404

C. M., Eds. American Society of Sugar Beet Technologists: Denver, CO, 1996; pp 89-92.

405

(10) Bais, H. P.; Weir, T. L.; Perry, L. G.; Gilroy, S.; Vivanco, J. M., The role of root

406

exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant

407

Biol. 2006, 57, 233-266.

408 409 410 411 412 413 414

(11) Whipps, J. M., Carbon economy. In The Rhizosphere, Lynch, J. M., ed. John Wiley: Chichester, UK, 1990; p 59. (12) Schroth, M. N.; Hildebrand, D. C., Influence of plant exudates on root-infecting fungi. Annu. Rev. Phytopathol. 1964, 2, 101-132. (13) Nelson, E., Exudate molecules initiating fungal responses to seeds and roots. Plant Soil 1990, 129, 61-73. (14) Li, X.-G.; Zhang, T.-L.; Wang, X.-X.; Hua, K.; Zhao, L.; Han, Z.-M., The composition of

415

root exudates from two different resistant peanut cultivars and their effects on the growth

416

of soil-borne pathogen. Int. J. Biol. Sci. 2013, 9, 164-173.

417

(15) Gika, H. G.; Theodoridis, G. A.; Vrhovsek, U.; Mattivi, F., Quantitative profiling of polar

418

primary metabolites using hydrophilic interaction ultrahigh performance liquid

419

chromatography–tandem mass spectrometry. J. Chromatogr. A 2012, 1259, 121-127.

420 421

(16) Falloon, R. E.; Genet, R. A.; Wallace, A. R.; Butler, R. C., Susceptibility of potato (Solanum tuberosum) cultivars to powdery scab (caused by Spongospora subterranea f.


Page 19 of 34


422

sp. subterranea), and relationships between tuber and root infection. Australas. Plant

423

Pathol. 2003, 32, 377-385.

424

(17) Wilson, C. R.; Tegg, R. S.; Wilson, A. J.; Luckman, G. A.; Eyles, A.; Yuan, Z. Q.;

425

Hingston, L. H.; Conner, A. J., Stable and extreme resistance to common scab of potato

426

obtained through somatic cell selection. Phytopathology 2010, 100, 460-467.

427 428 429 430 431 432 433

(18) Merz, U., Microscopical observations of the primary zoospores of Spongospora subterranea f.sp. subterranea. Plant Pathol. 1997, 46, 670-674. (19) Merz, U., Infectivity, inoculum density and germination of Spongospora subterranea resting spores: a solution-culture test system. EPPO Bull. 1989, 19, 585-592. (20) Ledingham, G. A., Occurrence of zoosporangia in Spongospora subterranea, (Wallroth) Lagerheim. Nature 1935, 135, 394-394. (21) Nelson, E. B., Rhizosphere regulation of preinfection behavior of oomycete plant

434

pathogens. In Microbial Activity in the Rhizoshere, Mukerji, K. G.; Manoharachary, C.;

435

Singh, J., eds. Springer: Berlin, Germany, 2006; pp 311-343.

436

(22) Carvalhais, L. C.; Dennis, P. G.; Fedoseyenko, D.; Hajirezaei, M.-R.; Borriss, R.; von

437

Wirén, N., Root exudation of sugars, amino acids, and organic acids by maize as affected

438

by nitrogen, phosphorus, potassium, and iron deficiency. J. Plant Nutr. Soil Sci. 2011,

439

174, 3-11.

440

(23) Vančura, V.; Hovadík, A., Root exudates of plants. Plant Soil 1965, 22, 21-32.

441

(24) Dobson, G.; Shepherd, T.; Verrall, S. R.; Griffiths, W. D.; Ramsay, G.; McNicol, J. W.;

442

Davies, H. V.; Stewart, D., A metabolomics study of cultivated potato (Solanum

443

tuberosum) groups Andigena, Phureja, Stenotomum, and Tuberosum using gas

444

chromatography−mass spectrometry. J. Agric. Food Chem. 2010, 58, 1214-1223.



445

Page 20 of 34

(25) Roessner, U.; Wagner, C.; Kopka, J.; Trethewey, R. N.; Willmitzer, L., Simultaneous

446

analysis of metabolites in potato tuber by gas chromatography–mass spectrometry. Plant

447

J. 2000, 23, 131-142.

448 449 450

(26) Lynch, J. M.; Whipps, J. M., Substrate flow in the rhizosphere. Plant Soil 1990, 129, 110. (27) Neumann, G.; Romheld, V., The release of root exudates as affected by the plant

451

physiological status. In The rhizosphere: biochemistry and organic substances at the

452

soil–plant interface, 2nd edn.; Pinton, R.; Varanini, Z.; Nannipieri, P., eds. CRC Press:

453

Boca Raton, FL, 2007; pp 23-57.

454

(28) Hernandez Maldonado, M. L.; Falloon, R. E.; Butler, R. C.; Conner, A. J.; Bulman, S. R.,

455

Spongospora subterranea root infection assessed in two potato cultivars differing in

456

susceptibility to tuber powdery scab. Plant Pathol. 2013, 62, 1089-1096.

457

(29) Kowalski, K., Observations on the behaviour of resting spores of Plasmodiophora

458

brassicae in the presence of cruciferous and non‐cruciferous plant roots. Acta Hortic.

459

1996, 407, 419-422.

460

(30) Ohi, M.; Kitamura, T.; Hata, S., Stimulation by caffeic acid, coumalic acid, and corilagin

461

of the germination of resting spores of the clubroot pathogen Plasmodiophora brassicae.

462

Biosci. Biotechnol. Biochem. 2003, 67, 170-173.

463

(31) Suzuki, K.; Matsumiya, E.; Ueno, Y.; Mizutani, J., Some properties of germination-

464

stimulating factor from plants for resting spores of Plasmodiophora brassicae. Jpn. J.

465

Phytopathol. 1992, 58, 699-705.

466

(32) Mattey, M.; Dixon, G. R., Premature germination of resting spores as a means of

467

protecting Brassica crops from Plasmodiphora brassicae Wor., (clubroot). Crop Prot.

468

2015, 77, 27-30.


Page 21 of 34


469 470

(33) Bimpong, C. E.; Clerk, G. C., Motility and chemotaxis in zoospores of Phytophthora palmivora (Butl.) Butl. Annals Bot. 1970, 34, 617-624.

471

(34) Islam, M. T.; Tahara, S., Chemotaxis of fungal zoospores, with special reference to

472

Aphanomyces cochlioides. Biosci. Biotechnol. Biochem. 2001, 65, 1933-1948.

473 474 475 476 477 478 479

(35) Zentmeyer, G. A., Chemotaxis of zoospores for root exudates. Science 1961, 133, 15951596. (36) Deacon, J. W., Ecological implications of recognition events in the pre-infection stages of root pathogens. New Phytol. 1996, 133, 135-145. (37) Falloon, R. E., Control of powdery scab of potato: towards integrated disease management. Am. J. Pot. Res. 2008, 85, 253-260. (38) Thangavel, T.; Tegg, R. S.; Wilson, C. R., Monitoring Spongospora subterranea

480

development in potato roots reveals distinct infection patterns and enables efficient

481

assessment of disease control methods. PLoS ONE 2015, 10, e0137647.

482 483 484 485 486 487 488 489 490

(39) Aktar, M. W.; Sengupta, D.; Chowdhury, A., Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip. Toxicol. 2009, 2, 1-12. (40) Chitwood, D. J., Phytochemical based strategies for nematode control Annu. Rev. Phytopathol. 2002, 40, 221-249. (41) Gamliel, A.; Austerweil, M.; Kritzman, G., Non-chemical approach to soilborne pest management – organic amendments. Crop Prot. 2000, 19, 847-853. (42) Janisiewicz, W. J.; Korsten, L., Biological control of postharvest diseases of fruits Annu. Rev. Phytopathol. 2002, 40, 411-441. (43) Martin, F. N., Development of alternative strategies for management of soilborne

491

pathogens currently controlled with methyl bromide. Annu. Rev. Phytopathol. 2003, 41,

492

325-350.



493

Page 22 of 34

(44) Vranova, V.; Rejsek, K.; Skene, K. R.; Janous, D.; Formanek, P., Methods of collection

494

of plant root exudates in relation to plant metabolism and purpose: A review. J. Plant

495

Nutr. Soil Sci. 2013, 176, 175-199.

496 497


Page 23 of 34


498

FIGURE CAPTIONS

499 500

Figure 1. Photomicrographs of Spongospora subterranea plasmodia (Pa) and zoosporangia

501

(Za) in tomato roots, and zoospores (Zs) attached on potato root, which validates

502

zoospore identity.

503 504

Figure 2. Resting spore germination (zoospore release) of Spongospora subterranea as

505

influenced by deionized water (

506

(

507

population at different time intervals (line graph, left) and the accumulated

508

population at the end of incubation period (bar graph, right) are represented.

509

Arrows in black and blue indicate initial zoospore release, respectively, in root

510

exudates and deionized water (control). Vertical bars are standard errors (n=3).

511

Asterisks indicate treatments means, within same day, are statistically different

512

(p

Metabolomes of Potato Root Exudates - American Chemical Society

Recommend Documents