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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
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Journal of Agricultural and Food Chemistry
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Metabolomes of Potato Root Exudates: Compounds That
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Stimulate Resting Spore Germination of the Soil-Borne
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Pathogen Spongospora subterranea
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Mark A. Balendres†, David S. Nichols‡, Robert S. Tegg†, and Calum R.
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Wilson†*
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†
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Tasmania, 13 St. Johns Avenue, New Town, Tasmania 7008, Australia
New Town Research Laboratories, Tasmanian Institute of Agriculture, University of
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‡
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7001, Australia
Central Science Laboratory, University of Tasmania, Private Bag 74, Hobart, Tasmania
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* Corresponding author (Tel: (+613)62266381; E-mail:
[email protected])
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Root exudation has importance in soil chemical ecology influencing
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ABSTRACT:
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rhizosphere microbiota. Prior studies reported root exudates from host and non-host plants
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stimulated resting spore germination of Spongospora subterranea, the powdery scab pathogen
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of potato, but the identity of stimulatory compounds was unknown. This study showed that
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potato root exudates stimulated S. subterranea resting spore germination, releasing more
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zoospores at an earlier time than the control. We detected 24 low-molecular weight organic
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compounds within potato root exudates and identified specific amino acids, sugars, organic
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acids and other compounds that were stimulatory to S. subterranea resting spore germination.
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Given several stimulatory compounds are commonly found in exudates of diverse plant
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species we support observations of non-host specific stimulation. We provide knowledge of S.
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subterranea resting spore biology and chemical ecology that may be useful in formulating
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new disease management strategies.
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KEYWORDS: Powdery scab, root infection, Plasmodiophorid, HILIC UPLC-MS, zoospore
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release, metabolomics
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INTRODUCTION
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Spongospora subterranea (Wallr.) Lagerh. causes powdery scab and root disease in
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potato.1 Spongospora diseases are major problems in potato production worldwide. The
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pathogen downgrades the tuber fresh market-value, causes substantial economic losses in the
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potato processing industry,2 and leads to failure of seed tuber certification and subsequent
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losses in product value for seed potato producers.3 Primary zoospores initiate infection after
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release from germinating resting spores that are formed into aggregates known as sporosori.4
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Zoospores migrate to host roots, where they encyst and establish infections5 which develop
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into plasmodia and may mature as zoosporangia or resting spores.6 Zoosporangia contain
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secondary zoospores which are sources of secondary infection in tubers and roots after release
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into the soil environment. When resting spores are released back into the soil they become the
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new generation of primary inoculum. As such, the germination of S. subterranea resting
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spores is the first crucial step in disease development. Despite its importance, knowledge of
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the processes and the factors influencing resting spore germination is limited.7 Root exudates
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play a critical role as stimulants of S. subterranea resting spore germination.4,8,9 Prior studies
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report a lack of host specificity with exudates from both hosts and non-hosts capable of
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stimulating germination. However, no attempts have been made to identify any stimulatory
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compound(s) present in the root exudates.
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An estimated 40-50% of the carbon fixed by plants is released as root exudates,
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mostly as low molecular weight organic (LMWO) compounds, making root exudation a
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significant carbon cost to the plant.10,11 Root exudates play important roles in rhizosphere
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chemical ecology influencing interactions between the plant and other soil biota. They may
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function as, but are not limited to, chemoattractants, inhibitors or stimulants of microbial
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growth10 and may influence the colonization and activation of root-infecting pathogens.12,13
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The response of soil microbes may vary depending on the root exudate’s organic composition.
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For instance, peanut root exudates differing in LMWO compound composition have varying
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influence on spore germination of soil-borne peanut pathogens.14 The stimulatory effects of
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potato root exudates to S. subterranea resting spore germination might also be explained by
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their organic chemical composition.
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Characterization of the root exudate metabolome can assist in gaining a better
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understanding of plant-microbial interactions. The choice of techniques to be used in
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metabolomic studies is important to achieve unbiased and successful characterization of a
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metabolome. Metabolite analysis of aqueous samples, including root exudates, generally uses
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gas or liquid chromatography which is usually coupled to mass spectrometry (MS). Gas-
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chromatography is more suitable for volatile compounds, whilst liquid-chromatography (LC)
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is commonly used for non-volatiles. Recently, new improvements have been made to LC
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techniques, particularly for detecting polar, hydrophilic compounds. An improved hydrophilic
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interaction ultra-performance liquid-chromatography mass spectrometry (HILIC UPLC-MS)
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approach has been used to simultaneously identify more than 100 analytes from grapefruit
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extracts in less than 30 min at detection levels as low as 5 ng/ml.15
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The present study examined metabolomes of potato root exudates to gain insight into
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their metabolite composition, and to determine if specific chemical constituents stimulate S.
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subterranea resting spore germination. The bioassays were performed in a soil-less in vitro
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system9 coupled with microscopy, and the targeted characterization of compounds in the root
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exudates was achieved using a HILIC UPLC-MS methodology.15 This work advances the
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understanding of the biology of S. subterranea resting spore germination and provides new
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insights into the pathogen’s chemical ecology. Furthermore, the primary metabolic
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composition of potato root exudates is outlined, expanding known metabolite constituents.
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Potato genotypes used in this study have importance in potato breeding programs and in
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commercial production. Hence, the metabolite profile may compliment other “omics”
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approaches for potato improvement.
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MATERIALS AND METHODS
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Potato Root Exudate Collections. Root exudates were collected from deionized water
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extracts of tissue-cultured potato (Solanum tuberosum). Four potato cultivars that varied in
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their known resistance to S. subterranea diseases were used. Cultivars ‘Agria’ and ‘Iwa’ are
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highly susceptible to both tuber and root disease, whilst cvs. ‘Russet Burbank’ and ‘Gladiator’
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have moderate to strong resistance to tuber infection and moderate resistance to root
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infection1,16. Preparation and collection of potato root exudates were performed aseptically.
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Potato tissue-cultured plantlets were grown in potato multiplication medium (MS salts and
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vitamins, 30 g/L of sucrose, 40 mg/L of ascorbic acid, 500 mg/L of casein hydrolysate and 0.8%
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agar, pH 5.8) under a 16 h photoperiod using white fluorescent lamps (65 µmol/m2/s) at
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22 °C.17 After 2 weeks, plants were gently uprooted from the media, their roots were washed
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with sterile deionized water, blotted dry on sterile tissue paper, then placed in a sterile
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polycarbonate bottle containing 20 ml of fresh sterile deionized water (instrument
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conductivity at 0.059 µS/cm). For exudate collection plants were incubated in the water
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solution under the same light and temperature regime as before for varying incubation periods.
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Firstly, to confirm the capability of potato root exudates to stimulate resting spore
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germination and to assess possible influences of cultivar, a series of nine bioassays comparing
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32 individual potato root exudates was conducted. Either two (‘Gladiator’ and ‘Agria’) or all
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four cultivars were incubated in the water solution for 7 d prior to collection and testing. Then
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for analysis of root exudate composition and association with stimulation of resting spore
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germination a further 12 exudate solutions were collected. Exudates from all four cultivars
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were collected following 2, 7 and 18 d incubation in the water solution. All exudate solutions
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were stored in sterile Falcon tubes at -20 °C, in the dark, until used. There were no signs of
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microbial contamination in the root exudate solutions prior to use. Unless otherwise stated, all
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chemicals used in this study were sourced from Sigma-Aldrich (St. Louis, MO).
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Inoculum Preparation. The S. subterranea inoculum (resting spores) was prepared from
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powdery scab-affected tubers, collected from Devonport, Tasmania, Australia (41.17 °S,
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146.33 °E). Diseased tubers were washed with running tap water for 1-2 min, soaked in 2%
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sodium hypochlorite (White King, Pental Products Ltd Pty, Melbourne, Australia) for 3 min,
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quickly rinsed, and air-dried. Lesions were excised using a scalpel, dried for 4 d at 40 °C,
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ground by mortar and pestle, and stored at 4 °C until used. The inoculum was approximately 1
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y old when used. Inoculum contained approximately 6,900 sporosori/mg, as determined by
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suspending 0.1 g of inoculum in 10 ml water and quantification using light microscopy.
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Resting Spore Germination Assay. Presence of motile zoospores in the test solutions
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indicated resting spore germination.9 Briefly, 1 mg of inoculum (6,900 sporosori) was
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suspended either in 1.5 ml of root exudate, individual compound solutions (0.1 mg/ml), or in
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deionized water (control) solutions, each in a 2 ml microcentrifuge tube covered with
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aluminum foil. Tubes were incubated for 30 d at 15–18 °C. At 3-7 d intervals, 35 µl from
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each treatment was subsampled. The number of motile zoospores in the subsample was
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determined with a DM 2500 LED microscope (Leica Microsystem, Wetzlar, Germany) at
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200X magnification, scanning in an inverted “S” manner within a 22 mm x 22 mm area of
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each microscope-slide. S. subterranea zoospores were identified by morphology4 and motility
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behavior.18 Assessments of each root exudate solution and 43 individual LMWO compounds
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were repeated, respectively, seven and two times. Each assay consisted of three replications.
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In the individual chemical bioassay, Hoagland’s solution and sterile deionized water were
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added, respectively, as a stimulant and non-stimulant controls.
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Additional bioassays were performed to further validate S. subterranea zoospore
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identity. The first used a modified tomato-bait test.19 A 100 µl subsample from each test and
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control solution was transferred into a McCartney bottle containing 5 ml of non-sterile
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deionized water and a healthy 2 week old tomato plant (cv. Grape). Tomato plants were left in
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the solution for 24-48 h at 15-18 °C; to allow zoospores to encyst, and were then removed.
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Roots were washed to remove any resting spores adhering to root surfaces. Each plant was
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transferred to a new McCartney bottle containing 20 ml of non-sterile deionized water and
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grown for an additional 2 weeks to allow the development of zoosporangia. This test was
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repeated, using different zoospore solution sources. Sample roots were then cut, placed on
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microscope slides, stained with 0.1% Trypan blue for at least 10 min, and zoosporangia20
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were examined under a light microscope. In the second test, the number of zoospores
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successfully attached to S. subterranea roots of cv. ‘Gladiator’ and cv. ‘Iwa’ host roots was
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determined. Single, 2 cm long, roots were placed on microscope slides, which were flooded
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with 70 µl of test solutions and incubated at room temperature for 10 min. Each test was
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replicated three times. To ensure all roots received a similar exposure to zoospores, the test
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was performed one root/cultivar/replicate at a time. The number of zoospores attached on the
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roots were counted microscopically, at 200X magnification, by scanning the whole root.
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Phytochemical Analyses. Detection of primary metabolites in potato root exudate
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solutions was done using HILIC UPLC-MS.15 The UPLC assays were performed using a
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Acquity UPLC H-class system (Waters, Milford, MA), and MS analyses using a Xevo triple
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quadrupole MS system (Waters). HILIC separation was done on a 2.1 mm x 150 mm Acquity
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1.7 µm BEH amide VanGuard column maintained at 60 °C and eluted with a two-step
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gradient at 500 µL/min flow rate for 30 min. The mobile phases were composed of A
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(acetonitrile/water, 95–5 (v/v), 0.1% formic acid. and 0.075% NH4OH) and B (acetonitrile–
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water, 2–98 (v/v), 0.2% formic acid. and 0.1% NH4OH). LiChrosolv-grade acetonitrile was
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purchased from Merck (Darmstadt, Germany), whilst formic acid (>95%) and NH4OH were
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purchased from Sigma-Aldrich. The gradient started with a 4 min isocratic step at 100%
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mobile phase A, then rising to 28% mobile phase B over the next 21 min and finally to 60% B
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over 5 min.15 The column was then equilibrated for 12 min in the initial conditions. Two
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cycles of weak and strong solvent washing of the injection system were carried out between
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injections. The injection volume was 10 µl and the column eluent was directed to the mass
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spectrometer. Metabolite detection was achieved using selected ion monitoring and an
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electrospray ionization source was applied operating in both positive and negative ion mode.
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The parameters in the electrospray were set as follows; capillary voltage, −2.5 kV or 3 kV;
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cone and desolvation temperatures, respectively, 150 and 400ºC with a desolvation gas flow
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of 950 L/h and cone flow of 100 L/h. The cone voltage was optimized for each individual
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analyte. The identity of metabolites was confirmed by detection of the expected [M+H]+ or
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[M-H]- molecules at known retention indices15 by the analysis of standard chemicals, as both
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pure standards and spiked into root exudates samples. The identity of amino acid metabolites
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were also confirmed by tandem-MS multiple reaction monitoring experiments, with the
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detection of known product molecules arising from selected precursor molecular species.15
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Data and Statistical Analyses. Zoospore numbers were converted to counts per 100 µl
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solution, prior to statistical analysis using SPSS statistical software, ver. 22, (IBM, Armonk
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NY). An independent T-test and one-way analysis of variance (ANOVA) were performed for
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data having, respectively, two and more than two treatments. Data containing two factors
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were analyzed by two-way ANOVA. Multiple comparison of means was carried out using the
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Fisher’s Least Significant Difference (LSD) test at 0.05 level of probability. HILIC UPLC-
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MS analyses were done using MassLynx XS software.15 The limit of detection and limit of
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quantitation of the analytes were determined at signal-to-noise ratios of 3 and 10 respectively.
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A hierarchical cluster dendrogram was constructed using the average linkage (between group)
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method, with binary data measured using the squared Euclidean distance. A pattern (color)
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map was constructed to represent the distribution (numbers) of the metabolite/compound
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classes detected in various root exudates.
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RESULTS
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Spongospora subterranea Zoospore Identity. Motile biflagellate (of unequal length)
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zoospores, of approximately 4-5 µm diameter, were observed in the majority of test potato
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root exudates and occasionally in control (sterile deionized water) solution. The presence of
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the two flagella on each zoospore were best observed at 400X magnification, but observation
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of zoospore swimming patterns was best viewed at 200X magnification. Other uniflagellate
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and biflagellate organisms were present, but their numbers were minimal and their movement
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patterns distinct from those S. subterranea zoospore. Figure 1 shows that zoosporangia,
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indicative of S. subterranea infection, developed when test solutions with S. subterranea
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zoospores were exposed to tomato roots. Similarly zoospores were observed attached to
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potato roots with a greater number of zoospores (P=0.008) attached to roots of the susceptible
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cv. ‘Iwa’ (16.67 ± 2.60) than the resistant cv. ‘Gladiator’ (3.67 ± 0.33; Figure 1).
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Stimulatory Effects of Potato Root Exudates to Resting Spore Germination. In
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the first experiment across all nine assays, exposure of resting spores to 25 of the 32 potato
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root exudates stimulated release of S. subterranea zoospores (Figure 2). Zoospores were
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occasionally released in the control solution (four out of nine assays), but release was
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substantially later (average first release was at 11.8 d) than in the potato root exudates (4.4 d).
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Within individual assays statistically significant differences in zoospore numbers released
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were found between potato cultivars but there was no consistent cultivar response between
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assays (Figure 2).
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In the second experiment, the duration of incubation of potato roots prior to exudate
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collection (2, 7, and 18 d) had no statistically significant effect (P=0.152) on stimulation of
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resting spore germination and while cultivar showed a statistically significant effect
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(P=0.050), there were again no obvious consistent trends (Figure 3). The interaction of
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incubation period and cultivar had a clear statistically significant effect (P=0.033), with
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differences between cultivar most evident in the 18 d incubation samples (Figure 3).
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Metabolite Constituents of Various Potato Root Exudates. The HILIC UPLC-MS
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analysis detected a total of 24 LMWO compounds from potato root exudates (Table 1). These
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compounds ranged from 104-504 Da molecular weight, and included eight amino acids, one
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sugar, three sugar alcohols, five organic acids and seven other organic compounds. Detection
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of these compounds varied among root exudates. Asparagine, glutamic acid, glutamine,
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proline, serine, pinitol, choline, trehalose and tyramine were detected in most of the potato
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root exudates. However, some compounds were uniquely observed in a particular potato
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cultivar root exudates or collection date. For instance, raffinose, dehydroascorbic and quinic
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acid, and adenosine were, respectively, present only in ‘Iwa’, ‘Agria’, and ‘Gladiator’ root
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exudates and histamine was only detected in the exudates incubated for 18 d. The hierarchical
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cluster analysis revealed that potato root exudates could be divided at 80% similarity into
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three groups based on the metabolite composition (Figure 4). Cluster 1 included all ‘Iwa’ root
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exudates, cluster 2 was composed of mostly 2 and 7 d incubation and all ‘Russet Burbank’
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root exudates, and cluster 3 were mostly 18 d incubation and ‘Gladiator’ root exudates.
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Screening of Metabolites for Stimulants of Resting Spore Germination. Testing
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individual compounds provided a more robust direct relationship of each compound to resting
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spore germination stimulation (Figures 5 and 6).
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subterranea resting spores was chemical-specific (Figure 5). By comparing capacity and
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timing of zoospore release it was determined that five of the 18 compounds found in potato
Stimulation of germination of S.
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root exudates and seven of the 25 additional compounds tested were stimulant at 0.1 mg/ml,
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resulting in zoospore release at least 8 d earlier than the water control (Figures 5 and 6). L-
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Glutamine and tyramine had greater effects (P=0.045 and P=0.010, respectively) on resting
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spore germination than the other compounds tested and the Hoagland’s stimulant control.
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Mean accumulated zoospore numbers in other stimulant LMWO compounds, L-rhamnose,
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cellobiose, L-aspartic acid, N-acetyl cysteine, piperazine, glucoronic acid, L-serine, succinate,
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L-citrulline
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control (P>0.05) but were deemed stimulant as zoospore release in the presence of these
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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
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DISCUSSION
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This study aimed at elucidating, at a metabolite level, the stimulatory effects of potato
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root exudates on S. subterranea resting spore germination. LMWO compounds are the main
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constituents of plant root exudates and are known stimuli of soil-borne plant pathogens.12,21 In
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an initial series of in vitro bioassays we showed in most instances aqueous potato root
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exudates stimulated S. subterranea resting spore germination, releasing zoospores earlier and
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in greater numbers than the deionized water control. It was also clear that there was no
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consistent cultivar effect, with variability between assays likely due to variations in plant
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physiological condition. Using targeted HILIC UPLC-MS based metabolite profiling,15
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covering major primary metabolites in a plant biological system, we identified 24 polar
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LMWO compounds within potato root exudates. The majority of these compounds were
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amino acids, which is in agreement with other plant species22,23 and from prior metabolomic
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studies of potato tubers.24,25 The LMWO compounds, however, were not uniformly detected
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across all potato root exudates, with more compounds detected in the extracts from the cvs
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‘Agria’, ‘Gladiator’ and ‘Russet Burbank’ than in the ‘Iwa’ extracts. Hierarchical cluster
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analysis of the potato root exudate components separated exudates into three clusters
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associated with cultivar and exudate incubation period. Similar variations have been observed
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with both plant and environmental factors influencing release of LMWO compounds.26,27
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Analysis of metabolites from tubers of different Solanum spp. indicated individual species
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could be identified by their metabolic composition.24 Variability of root exudate composition
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with plant physiological and environmental factors22 suggests these are less useful for
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biotyping potato cultivars but with knowledge of their biosynthesis it may be possible to
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investigate genetic and proteome changes in a plant grown under particular conditions. For
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example, the HILIC UPLC-MS based metabolite profiling
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extended to analyze metabolite change in S. subterranea-infected plants to gain further
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insights into genes expressed by host plants during root infection.
15
used in this study could be
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Pathogen germination or activation by host root exudates is a common phenomenon
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among many soil-borne pathosystem, and this is particularly the case for environmentally-
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resistant-spore producing pathogens.12 The stimulation of resting spore germination by potato
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root exudates (Figure 2) corroborated the findings of earlier reports.8,9 While there could be
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doubts that the zoospores identified, in this and other studies,4,9,18 may be those of other
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flagellated contaminant organisms, additional testing showed that the detected zoospores
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produced zoosporangia after host infection,1 and attached to potato roots in a differential
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manner associated with known cultivar resistance28 as would be expected with S. subterranea.
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Furthermore, the initial release of zoospores (4-5 d) coincides with the other reports which
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have used in vitro approaches to examine S. subterranea resting spore germination.7 ,21
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Root exudates from both host and non-host plants and their chemical constituents have
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been suggested to play a part of the S. subterranea pre-infection activation.4,7, 9,19 Our study
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supports these observations. In several other pathosystems individual host specific
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compounds found within root exudates activate and chemotaxically attract the pathogen
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spores to a susceptible host plant to facilitate infection.10,12 Conversely, we have shown that a
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diverse range of LMWO compounds are stimulatory toward S. subterranea resting spore
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germination. Whilst we show these are produced by the potato host many of these compounds
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are commonly found in root exudates from other plant species including non-hosts.22,23 Thus
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we can conclude that resting spore germination is not host-specific. This is in agreement with
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studies of the closely related Plasmodiophora brassicae.29-32 It is thus unsurprising that
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production of stimulatory compounds was not associated with known potato cultivar
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resistance with cultivar effects quite variable. Rather it is likely that production of stimulatory
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LMWO compounds will be more strongly influenced by other factors such as the
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physiological condition of the plant.27 It has been suggested that S. subterranea zoospores are
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likely attracted to root exudates4 or specific chemical compounds within these exudates.7 This
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has been well documented in other pathosystems33,34 and variation in the susceptibility of
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plants can be associated with variation of chemo-attraction by the zoospores.35,36 We provide
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preliminary data indicating zoospore attachment to potato host roots is affected by resistance
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of the potato cultivar but to date no studies specifically examining chemotaxic attraction of S.
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subterranea zoospores have been conducted.
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Control of potato diseases caused by S. subterranea has been partly successful.37
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Planting resistant cultivars has proven the most effective means for powdery scab control, but
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cultivars lack effective strong resistance to root infection. The use of fungicides provides
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partial efficiency, reducing disease impact,37,38 probably by reducing inoculum pressure or
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delaying initial zoospore-host interaction. However, there have been general restrictions on
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the use of synthetic chemicals for disease management due potential health and environmental
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risks, and on crop toxicity.39 This has resulted in increased efforts to explore alternative
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chemical and biological control methods. Several investigations used a variety of plant
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extracts and their active chemical components for control of major vegetable and grain crop
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diseases.40 Others have used organic amendments41 and biological control agents to eradicate
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or reduce inoculum levels and minimize disease outcomes,42 while some continuously test
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other chemical substances.43 Few of these types of investigations have been applied to S.
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subterranea diseases of potato.37 The present study identified several root exudate compounds
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that stimulate S. subterranea resting spore germination and application in absence of a host
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plant could assist in reducing soil inoculum levels by removing viable resting spores.7
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We acknowledge that the tests were done in vitro, and that the root exudates were
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collected from plants grown in sterile aqueous solutions. Compounds detected in this study
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may differ from those exuded by plants grown under field conditions due to soil
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environmental and biological factors. Nevertheless, studies using root exudates from in vitro
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cultures provide clear indications of direct interactions of the pathogen with host root
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exudates due to the absence of potential compounding factors which could affect pathogen
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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
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germination and thus, makes a contribution to knowledge of the pathogen’s biology and
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chemical ecology. Active compounds are known from root exudates of other plants (including
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non-hosts) which supports the contention that resting spore stimulation is not host specific.
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Our findings may also be of importance to other root-infecting pathogens of potato that may
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be influenced by root exudation. From an integrated pest management perspective, the use of
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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
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We thank Annabel Wilson (University of Tasmania; UTAS, Australia) for providing
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tissue cultured potato plants, Sergey Shabala (UTAS) for certain chemicals, Ross Corkrey
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(UTAS) for assistance in statistical analysis, and James Braselton (Ohio University, Athens,
360
OH) for technical advice and useful discussion.
361 362
SUPPORTING INFORMATION
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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
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M. B. is supported by a Tasmanian Graduate Research Scholarship provided by the
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University of Tasmania. This study was supported by Horticulture Innovation Australia
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Limited: project number PT14002 (http://www.horticulture.com.au/) with funding from
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Simplot Australia Pty. Ltd., McCain Foods Australia Pty. Ltd., and matched funds from the
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Australian Federal Government. The University of Tasmania provided in-kind support.
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NOTE
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The authors declare no competing financial interest.
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FIGURE CAPTIONS
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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.
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Figure 2. Resting spore germination (zoospore release) of Spongospora subterranea as
505
influenced by deionized water (
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(
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population at different time intervals (line graph, left) and the accumulated
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population at the end of incubation period (bar graph, right) are represented.
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Arrows in black and blue indicate initial zoospore release, respectively, in root
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exudates and deionized water (control). Vertical bars are standard errors (n=3).
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Asterisks indicate treatments means, within same day, are statistically different
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(p