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Droplet Digital PCR Technology for Detection of Ilyonectria liriodendri from

Grapevine Environmental Samples

Article  in  Plant Disease · November 2019

DOI: 10.1094/PDIS-03-19-0529-RE

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1 Droplet Digital PCR Technology for Detection of Ilyonectria

2 liriodendri from Grapevine Environmental Samples

4 María del Pilar Martínez-Diz1,2, Marcos Andrés-Sodupe3, Mónica Berbegal4, Rebeca

5 Bujanda3, Emilia Díaz-Losada1 and David Gramaje3

7 1Estación de Viticultura y Enología de Galicia (AGACAL-EVEGA), Ponte San Clodio s/n

8 32428-Leiro-Ourense, Spain

9 2Universidade da Coruña, Facultade de Ciencias, Zapateira, 15071 A Coruña, Spain.

10 3Instituto de Ciencias de la Vid y del Vino (ICVV), Consejo Superior de Investigaciones

11 Científicas - Universidad de la Rioja - Gobierno de La Rioja, Ctra. de Burgos Km. 6, 26007

12 Logroño, Spain.

13 4Instituto Agroforestal Mediterráneo, Universitat Politècnica de València, Camino de Vera s/n,

14 46022 Valencia, Spain.

15

16 Corresponding Author: David Gramaje, Email: [Link]@[Link]

17

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18 Abstract

19 María del Pilar Martínez-Diz, Marcos Andrés-Sodupe, Mónica Berbegal, Rebeca Bujanda,

20 Emilia Díaz-Losada and David Gramaje. 2019. Droplet Digital PCR Technology for Detection of

21 Ilyonectria liriodendri from Grapevine Environmental Samples. XX:XX-XX.

22

23 Black-foot disease is one of the most important soilborne diseases affecting planting material in

24 grapevine nurseries and young vineyards. Accurate, early and specific detection and

25 quantification of black-foot disease causing fungi are essential to alert growers and nurseries to

26 the presence of the pathogens in soil, and to prevent the spread of these pathogens through

27 grapevines using certified pathogen-free planting material and development of resistance. We

28 comparatively assessed the accuracy, efficiency, and specificity of Droplet Digital PCR (ddPCR)

29 and real-time PCR (qPCR) techniques for the detection and quantification of Ilyonectria

30 liriodendri in bulk and rhizosphere soils, as well as grapevine endorhizosphere. Fungal

31 abundance was not affected by soil-plant fractions. Both techniques showed a high degree of

32 correlation across the samples assessed (R2=0.95) with ddPCR being more sensitive to lower

33 target concentrations. Roots of asymptomatic vines were found to be a microbial niche that is

34 inhabited by black-foot disease fungi.

35

36

37

38

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39 Soilborne pathogens can establish a parasitic relationship with their host plants in the

40 rhizosphere. To infect roots, pathogens have to compete with other microbial organisms of the

41 rhizosphere for available microsites and nutrients (Chapelle et al. 2016). To date, the complex

42 grapevine-soilborne pathogen interactions in the rhizosphere are not fully understood. Among

43 diseases caused by soilborne pathogens in grapevine, black-foot has received much attention in

44 recent decades from plant pathologists as it has been implicated in contributing to young

45 grapevine decline syndrome (Gramaje and Armengol 2011). Cylindrocarpon-like asexual

46 morphs belonging to the genera Campylocarpon, Cylindrocladiella, Dactylonectria, Ilyonectria,

47 Neonectria and Thelonectria have been associated with black-foot disease (Agustí-Brisach and

48 Armengol 2013; Carlucci et al. 2017; Lombard et al. 2014). The genus Ilyonectria represents one

49 of several newly established genera of fungi with Cylindrocarpon-like anamorphs (Chaverri et

50 al. 2011), with Ilyonectria liriodendri being one of the most prevalent causal agents of black-foot

51 disease (Agustí-Brisach and Armengol 2013).

52 Internal symptoms of black-foot diseased vines usually range from black, necrotic, sunken

53 lesions on roots to reddish brown discoloration in the base of the rootstock (Halleen et al. 2006).

54 Foliar symptoms associated with black-foot disease are practically indistinguishable from those

55 observed in Petri disease affected vines and include delayed bud break, chlorotic foliage with

56 necrotic margins, overall stunting, and wilting of leaves or entire shoots (Agustí-Brisach and

57 Armengol 2013). These symptoms may also resemble those associated with abiotic disorders

58 such as spring frost, winter damage, nutrient deficiency and/or water stress (Gramaje et al. 2018).

59 Black-foot disease is particularly important in grapevine nurseries and new plantations.

60 Cylindrocarpon-like asexual morphs produce conidia and some species also produce

61 chlamydospores in culture, which indicates that those propagules are likely to be produced on

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62 stem bases of infected vines and the diseased roots. The conidia are spread in soil water and the

63 chlamydospores can allow these pathogens to survive in the soil for extended periods of time

64 (Petit et al. 2011). Infection can occur through the small wounds made when roots break off

65 during the planting process, through the incomplete callusing of the lower trunk or through

66 wounds made in the grapevine propagation process, such as disbudding wounds, from which the

67 infection progresses downward to the base of the trunk (Halleen et al. 2006).

68 Traditionally, detection and identification of black-foot disease fungi in grapevine has been

69 performed by morphological approaches (Chaverri et al. 2011) or by multiplex PCR system

70 (Alaniz et al. 2009). Although reliable for a preliminary identification and classification, these

71 techniques are not practical to detect low levels of black-foot pathogens that anticipated during

72 early stages of infection. Recently, real-time PCR (qPCR) has become a useful technique for

73 increasing the sensitivity and specificity for detecting and quantifying Cylindrocarpon-like

74 asexual morphs (Agustí-Brisach et al. 2014; Langenhoven et al. 2018; Tewoldemehdin et al.

75 2011). The Digital PCR (ddPCR) has only recently been adapted to detect plant pathogens in

76 agricultural systems from biomedical disciplines where it showed to be more sensitive

77 technology compared with qPCR (Bahder et al. 2016, 2018; Dreo et al. 2014; Miotke et al. 2014;

78 Racki et al. 2014).

79 The objectives of this study were therefore: i) to design a ddPCR protocol that is capable to

80 detect and quantify I. liriodendri in soil and roots, ii) to evaluate the overall sensitivity of ddPCR

81 for detection of I. liriodendri compared with qPCR, and iii) to compare the abundance of I.

82 liriodendri in different habitats inside and outside of grapevine roots.

83

84 Materials and Methods

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85 Fungal Isolate selection and DNA serial dilutions. Ilyonectria liriodendri isolate BV-0596 was

86 obtained from the culture collection of the Instituto de Ciencias de la Vid y del Vino (ICVV)

87 (Spain). Fungal mycelium and conidia from pure cultures grown on potato dextrose agar for 2 to

88 3 weeks at 25ºC in the dark were scraped and homogenized in 2 ml tubes with 600 μl of P1

89 buffer of the kit E.Z.N.A. Plant Miniprep kit (Omega Bio-tek, Norcross, GA, USA) with 4 steel

90 beads of 2.38 mm and 2 of 3 mm diameter (Qiagen, Hilden, Germany) using a FastPrep-

91 24TM5G (MP Biomedicals, California, USA) at 5 m/s for 20 s twice. DNA integrity and quality

92 were assessed by gel electrophoresis visualizing the samples previously stained with RedSafe

93 (iNtRON Biotechnology, Lynnwood, WA, USA). DNA samples were quantified using the

94 Invitrogen Qubit 4 Fluorometer with Qubit dsDNA HS (High Sensitivity) Kit (Thermo Fisher

95 Scientific, Waltham, MA, USA). Serial dilutions ranging from 10,000 to 1 fg μl-1 of the DNA

96 were prepared for quantification purposes by ddPCR and qPCR. Three independent DNA

97 standard curves were obtained using separate pathogen DNA sources that were treated as

98 independent experiments.

99

100 TaqMan assay design and ddPCR parameters. Digital Droplet PCR (ddPCR) was performed

101 on a Bio-Rad QX200 system using a TaqMan assay. A probe was designed using the

102 PrimerQuest®Design Tool (Integrated DNA Technologies, Inc. Coralville, IA, USA) and labeled

103 at the 5’ end with Hetrachloro-6-carboxyfluorescin (HEX) and a double-quencher (internal ZEN

104 with 3’ Iowa Black FQ). The probe sequence is 5’-

105 /HEX/TCCGAGCGT/ZEN/CATTTCAACCCTCAA/3IABkFQ/-3’. Primers YT2F

106 (Tewoldemedhin et al. 2011) and Cyl-R (Dubrovsky and Fabritius 2007) were used in the

107 experiment. These primers amplify the main Cylindrocarpon-like asexual morphs associated

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108 with black-foot disease, in particular those belonging to the genera Dactylonectria, Ilyonectria,

109 Neonectria, and Thelonectria. Each reaction contained 1x Supermix for Probes (Bio-Rad

110 Laboratories, Hercules, CA, USA), 20 µM of each forward and reverse primer solution (final

111 concentration 750 nM for each primer), 10 µM of the probe and 2 µl of DNA template resulting

112 in a final volume of 20 µl. The PCR reactions were mixed, centrifuged briefly, and 20 μl

113 transferred into the sample well of a DG8TM cartridge (Bio-Rad). After adding 70 μl of

114 QX200TM droplet generation oil (Bio-Rad Laboratories) into the oil wells, the cartridge was

115 covered using a DG8TM gasket, and droplets generated using the QX200TM droplet generator

116 (Bio-Rad Laboratories). Droplets were carefully transferred into PCR plates using a multi-

117 channel pipette and the plate was sealed using PCR plate heat seal foil and the PX1TM PCR

118 plate sealer (Bio-Rad Laboratories). PCR was performed in a C1000 touch thermal cycler (Bio-

119 Rad Laboratories) using the following thermal cycling conditions: initial denaturation stage of

120 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 30 s and annealing

121 temperature (59°C) for 60 s and a final extension of 10 min at 98°C. PCR plates were transferred

122 into a QX200TM droplet reader (Bio-Rad Laboratories) and reads analyzed using QuantaSoftTM

123 software (Bio-Rad Laboratories). A thermal-gradient PCR experiment was conducted to

124 establish the optimal annealing temperature for the primers using DNA of I. liriodendri isolate

125 BV-0596 as a template.

126

127 Real-time PCR assay parameters. Real-time PCR assays were performed on a CFX384 real

128 time PCR system (Bio-Rad Laboratories) using the same primers and TaqMan probe as

129 described above. Each reaction contained 2 µl of DNA template, 5 µl of 1x Supermix for Probes

130 (Bio-Rad), containing 500 nM of probe and 750 nM of each primer. The reaction mix was

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131 adjusted to a final volume of 10 µl with sterile distilled water. Thermal cycling conditions were

132 as follows: 10 min of initial denaturation at 95°C, followed by 40 cycles of denaturation for 30 s

133 at 94°C and annealing at 62°C for 60 s. Both ddpCR and qPCR were performed at

134 BIODONOSTIA Health Research Institute (San Sebastián, Spain).

135

136 Environmental sample collection. Grapevine samples were collected at five young vineyards of

137 Tempranillo cultivar grafted onto 110 Richter rootstock maintained in La Rioja (Spain) located

138 between 2.2 to 14.9 km distance from each other (Supplementary Table S1). These vineyards

139 were under similar soil, climatic and management conditions. In each vineyard, three different

140 sample types were studied in June 2017 (flowering): bulk soil, soil surrounding roots

141 (rhizosphere) and roots (endorhizosphere). Four plants per vineyard were chosen to represent the

142 same aspect of the plant and position within the vineyard, and four samples were randomly

143 collected from each soil-plant fraction (bulk, rhizosphere and endorhizosphere). Sampled vines

144 did not show any symptom of disease or nutrient deficiency and root tissue did not have any rot

145 or necrosis that could be associated to black-foot or other diseases caused by soilborne

146 pathogens. A total of 60 samples were collected.

147 Bulk soil samples were collected with a sterile spade 1 m from each stem at depths of 40 to

148 50 cm. Homogenized dry soil was then passed through a 1-mm-pore size sieve and divided into

149 two subsamples, each one for I. liriodendri detection and quantification, and soil chemistry

150 analyses, respectively. Roots and rhizosphere soil samples were collected with a sterile spade

151 close to the stem at depths of 40 to 50 cm, where the root system was denser. All samples were

152 stored on dry ice in sterile bags at the time of sampling, and brought to the laboratory for further

153 processing within 24 h from the time of sampling. A total of 5 g of the sampled roots with

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154 rhizosphere soil particles attached were placed in sterile tubes containing 9 ml of physiological

155 solution (9 g/L NaCl). They were vortexed for 5 min to detach the soil particles and immediately

156 centrifuged at 1,503 g for 5 min. The supernatant was discarded and the remaining soil fraction

157 was used to represent the rhizosphere fraction. The roots devoid of soil particles were placed in a

158 new tube and surface sterilized according to Cherif et al. (2015).

159

160 DNA extraction. The bulk soil and rhizosphere DNA were extracted from 0.5 g sample using

161 the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany). For endorhizosphere DNA, before DNA

162 extraction, roots were sequentially washed in 70% ethanol and distilled water. Upon this

163 treatment, bark was carefully peeled out and the DNA was extracted from 0.5 g tissue using the

164 i-genomic Plant DNA Extraction Mini Kit (iNtRON Biotechnology, South Korea).

165

166 Standard curves determination. A standard curve was constructed with DNA dilution series of

167 I. liriodendri isolate BV-0596. Analyses were performed as previously described and the

168 standard curve was generated following the MIQE guidelines (Bustin et al. 2009). The estimated

169 number of target molecules per µl (ddPCR) and the quantification cycle (Cq) (qPCR) values

170 obtained for each specific isolate DNA dilution were plotted against the logarithm of the

171 concentration (fg µl-1) of each isolate DNA dilution.

172 Sensitivity of ddPCR and qPCR was assessed estimating the limit of detection (LOD) of both

173 techniques, using the resulting standards curves to determine the minimum DNA concentration

174 that can be detected in three consecutive assays. The amplification efficiency (E) and the

175 coefficient of determination (R2) of the standard curve were obtained using the specific software

176 of each system, QuantaSoftTM (Bio-Rad) for ddPCR and CFX Maestro (Bio-Rad) for qPCR.

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177 Signal threshold levels were set automatically by the instrument software and the LOD was

178 identified by the last dilution in which successful amplification of all DNA replicates occurred.

179

180 Quantification of black-foot pathogens in environmental samples. To compare both

181 quantification techniques in environmental samples, the 60 grapevine samples collected at 5

182 young vineyards in La Rioja were analyzed by both ddPCR and qPCR technologies. All samples,

183 along with a non-template control (NTC) reaction (water), two positive controls containing DNA

184 of a soil sample tested positive to I. liriodendri by ITS high-throughput amplicon sequencing,

185 and DNA extracted from a pure culture of I. liriodendri isolate BV-0596, were analyzed by

186 qPCR and ddPCR in triplicate. In addition, negative controls (serial dilutions of DNA from

187 grapevine and soil lacking target fungi) were used alone or spiked with the same amount of

188 target DNA in order to assess inhibition in the assays. The mean DNA concentration and the

189 standard deviation were determined from five replicates per dilution. For the qPCR results, copy

190 number was calculated with the following formula: (DNA amount (g) * 6.022 x 1023 (copy/mol)

191 / (DNA length (bp) * 660 (g/mol/bp)) (Lee et al. 2006; Lee et al. 2016), where DNA amount was

192 the concentration of DNA (g) and DNA length was the length of I. liriodendri BV-0596 genome,

193 60 Mbp (unpublished data). The efficiency of both ddPCR and qPCR technologies to quantify I.

194 liriodendri from environmental samples was compared. Values from the I. liriodendri DNA

195 concentration obtained with each technique were transformed by log (n/N ∗ 1000 + 1). Where n

196 was the DNA concentration detected on each sample and N was the total DNA concentration

197 detected. An analysis of correlation between both transformed datasets was performed in R

198 version 3.5 (R Core Team 2017) using the corrr package. DNA concentration values using both

199 quantification methods were calculated for each fraction and vineyard. Significance levels for

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200 mean values were determined by the Kruskal-Wallis one-way analysis of variance on ranks and

201 mean separation was conducted at P<0.05. The analysis was performed using R package

202 agricolae (Mendiburu 2015).

203

204 Results

205

206 Detection and quantification limit of genomic DNA of cultured I. liriodendri by ddPCR and

207 qPCR. The optimal annealing temperature for primers using pure culture I. liriodendri BV-0596

208 DNA in ddPCR was established at 59ºC. Both methods showed good linearity within the

209 quantification range with a high coefficient of determination (R2) of 0.9917 and 0.9893 and a

210 reaction efficiency of 0.83 and 0.97 for ddPCR and qPCR, respectively (Fig. 1). The minimum

211 target concentration detectable was the 5 fg μl-1 dilution for ddPCR and the 10 fg μl-1 dilution for

212 qPCR (Table 1), thus, the LOD was established at these concentrations for each technique. The

213 NTC showed no positive amplification.

214

215 Quantification of black-foot pathogens from environmental samples. DNA of I. liriodendri

216 was detected in all soil-plant fractions samples in the five vineyards assessed. Significant

217 differences in the abundance of I. liriodendri were detected among vineyards with both

218 techniques (P < 0.01). Concentrations ranged from 1.79 to 20.98 pg μl-1 in vineyard 1, 0.77 to

219 8.73 pg μl-1 in vineyard 2, 1.99 to 53.8 pg μl-1 in vineyard 3, 0.03 to 38.58 pg μl-1 in vineyard 4

220 and 0.34 to 29.43 pg μl-1 in vineyard 5 by qPCR (data not shown). In ddPCR, concentrations

221 ranged from 96 to 2,350 copies μl-1 in vineyard 1, 75 to 860 copies μl-1 in vineyard 2, 190 to

222 8,680 copies μl-1 in vineyard 3, 4.7 to 9,470 copies μl-1 in vineyard 4 and 40 to 1,920 copies μl-1

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223 in vineyard 5 (data not shown). Average number of copies in each vineyard per soil-plant

224 fraction obtained by ddPCR are shown in Table 2. In each vineyard, no significant differences in

225 the abundance of I. liriodendri were detected among soil-plant fractions with both techniques

226 (P>0.05). Overall, concentrations ranged from 0.5 to 38.68 pg μl-1 in bulk soil, 0.03 to 53.8 pg μl-

227 1 in rhizosphere and 0.78 to 107.73 pg μl-1 in roots fraction by qPCR. Average concentrations in

228 each soil-plant fraction were 10.07 pg μl-1 (bulk soil), 10.49 pg μl-1 (rhizosphere), and 11.53 fg

229 μl-1 (endorhizosphere). In ddPCR, concentrations ranged from 40 to 9,470 copies μl-1 in bulk soil,

230 4.7 to 5,270 copies μl-1 in rhizosphere and 75 to 8,680 copies μl-1 in roots fraction. Average

231 number of copies in each soil-plant fraction were 1,275 copies μl-1 (bulk soil), 1,028 copies μl-1

232 (rhizosphere), and 1,233 copies μl-1 (endorhizosphere). No PCR inhibition or positive droplets

233 noticed using negative controls by qPCR or ddPCR (Fig. 3), respectively. The correlation

234 analysis showed a high and positive significant correlation between I. liriodendri DNA

235 quantified using both the ddPCR and qPCR techniques (R2= 0.95) (Fig. 2).

236

237 Discussion

238

239 Digital PCR is the latest DNA quantification technology that can be broadly used in several

240 scientific fields (Cao et al. 2015; Hussain et al. 2016; Morisset et al. 2013; Palumbo et al. 2016;

241 Porcellato et al. 2016; Yang et al. 2014), including plant pathology (Bahder et al. 2018, 2019;

242 Voegel and Nelson 2018). The present study represents the first approach to assess the ddPCR as

243 a reliable tool to detect and quantify pathogenic fungi associated with grapevine trunk diseases.

244 In particular, black-foot disease is one of the main soilborne diseases affecting planting material

245 and young vineyards worldwide (Gramaje et al. 2018). DNA was not sheared prior to the

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246 experiments since our input DNA concentration was 10 ng/20 μL reaction. DNA shearing is

247 recommended for input DNA concentrations >66 ng/20 μL reaction using the Bio-Rad

248 QX100/200 system (Hindson et al. 2011). We found that both ddPCR and qPCR showed the

249 potential of being efficient techniques to detect and measure I. liriodendri DNA associated with

250 black-foot disease, with a strong correlation between them. These findings are in agreement with

251 those obtained by Kim et al. (2014), who found a high quantitative agreement between DNA

252 quantity measured with ddPCR and qPCR while examining population dynamics of bacteria in

253 soil.

254 Quantification of copy number from complex samples containing multiple target species may

255 be inaccurate. However, our attempt to design specific primers for black-foot disease genera

256 (Campylocarpon, Cylindrocladiella, Dactylonectria, Ilyonectria, Neonectria and Thelonectria)

257 from available gene sequences in the GenBank database (internal transcribed spacer region,

258 histone H3, translation elongation factor 1-alpha and β-tubulin genes) were unsuccessful, due to

259 lack of highly conserved gene regions among these closely related phylogenetic genera. In this

260 study, I. liriodendri BV-0596 genome size was used in the calculation of copy number across

261 samples and the DNA of this isolate was also used for the standard curve determination and to

262 establish the optimal annealing temperature for the primers. A limitation of this approach that

263 needs to be stated is that bias in the calculation of the copy number may be introduced due to

264 different genome sizes from the fungal species associated with black-foot disease: 58 Mbp in D.

265 macrodidyma isolate JAC15-245 (Malapi-Wight et al. 2015) and 64 Mbp in D. torresensis

266 isolate BV-0666 (Gramaje et al. 2019).

267 The ddPCR showed to be more sensitive as compared with qPCR in the detection and

268 quantification of this fungal pathogen at very low concentrations. Increased sensitivity of digital

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269 PCR over qPCR has been reported in other studies (Bahder et al. 2018; Cavé et al. 2016; Kim et

270 al. 2014; Porcellato et al. 2016) and similar sensitivity was highlighted by others (Blaya et al.

271 2016; Dreo et al. 2014) when comparing both techniques. Developing a robust ddPCR assay with

272 increased sensitivity of ddPCR over qPCR would be beneficial to researchers and diagnostic

273 laboratories by identifying early infections in grapevines and soil. Additional benefits were

274 reported in several recent studies for ddPCR such as it obviates the preparation of reference

275 DNA templates (Kim et al. 2014), the absolute quantitative target detection without the need of

276 standard curves construction (Yang et al. 2014) and that the inhibitory substances had a little

277 effect on DNA quantification using this technique (Hoshino and Inagaki 2012). These features

278 and the results obtained in the present study make ddPCR an attractive alternative for measuring

279 environmental samples allowing a better understanding and monitoring of fungal pathogens

280 associated with grapevine trunk diseases in the future.

281 The abundance of I. liriodendri was not affected by soil or plant as source of DNA. Recent

282 studies have shown that black-foot inoculum pressure in vineyard soils is frequently high (Reis et

283 al. 2013; Agustí-Brisach et al. 2014), even with the absence of grapevine (Cardoso et al. 2013;

284 Berlanas et al. 2017). Black-foot disease fungi can survive in soil for multiple years in the

285 absence of suitable host due to the production of chlamydospores after infected vines have been

286 removed (Petit et al. 2011). These pathogens have also been frequently associated with the roots

287 of herbaceous plants (Langenhoven et al. 2018) and weeds (Agustí-Brisach et al. 2011). It is also

288 important to note that Cylindrocarpon-like asexual morphs seem to be quite frequent in roots and

289 the endorhizosphere of diverse plants, not just only in grapevines. Several studies show that they

290 are indeed among the dominant fungi found in soils with strawberry (Xu et al. 2015) and forest

291 trees (Bonito et al. 2014), and in roots of apple in South Africa (Tewoldemedhin et al. 2011) and

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292 USA (Manici et al. 2018). This frequent and high occurrence of Cylindrocarpon-like asexual

293 morphs in different environments may explain the lack of specialization of these fungi to specific

294 plant associated ecological niches (rhizosphere and endorhizosphere).

295 Both the qPCR and ddPCR methods revealed that healthy grapevine plants harbor

296 Cylindrocarpon-like asexual morphs that are causal agents of black-foot disease, demonstrating

297 that these fungi can act as endophytic and/or latent pathogenic microorganisms in grapevine. In

298 the scientific literature, observations of black-foot disease fungi as endophytes colonizing

299 asymptomatic vines (Langenhoven et al. 2018; Berlanas et al. 2019) or other plant species

300 (Agustí-Brisach et al. 2011; Langenhoven et al. 2018) have been documented. Many of these

301 asymptomatic plants are cereals and brassicaceous crops, used in crop rotations in grapevine

302 nurseries (Langenhoven et al. 2018), and weeds, which may be present in field nurseries and

303 established vineyards along with cultivated crops (Agustí-Brisach et al. 2011; Langenhoven et al.

304 2018). The occurrence of black-foot disease pathogens in asymptomatic vines highlights the

305 urgent need to implement early, accurate and specific in planta detection and quantification of

306 these fungi to prevent the spread of black-foot disease in grapevine propagation material.

307

308 Literature Cited

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312 Evaluation of vineyard weeds as potential hosts of black-foot and Petri disease pathogens.

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314 Agustí-Brisach, C., Mostert, L., and Armengol, J. 2014. Detection and quantification of

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453

454 Acknowledgements

455 We thank Bodegas Bilbaínas and Bodegas Vivanco for providing the vineyards from which

456 samples were collected in this study. David Gramaje was supported by the DOC-INIA program

457 from the INIA, co-funded by the European Social Fund. María del Pilar Martínez-Diz was

458 supported by the FPI-INIA program from the INIA.

459

460 Figure captions

461

462 Fig. 1. Standard curves obtained using Ilyonectria liriodendri isolate BV-0596 DNA dilutions

463 ranging from 10,000 to 1 fg μl-1. The logarithm of the concentration of each isolate DNA dilution

464 was plotted against: a) Target DNA concentration (copy number µl-1), in order to construct the

465 ddPCR standard curve; b) Quantification cycle (Cq), in order to construct the qPCR standard

466 curve. Data points represent amplification results of five replicates. The reaction efficiency was

467 0.83% and 0.97% for ddPCR and qPCR analysis, respectively.

468

469 Fig. 2. The distribution of DNA concentration of I. liriodendri values is shown on the diagonal.

470 The bivariate scatter plot with a fitted line is displayed on the bottom of the diagonal and the

471 Spearman correlation value (P<0.05) is indicated on the top of the diagonal.

472

473 Fig. 3. Droplet digital PCR amplitude plot showing all accepted droplets with a clear distinction

474 between positives (green) and negatives (grey) in each soil-plant fraction (bulk soil, rhizosphere

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475 and roots), positive control (I. liriodendri isolate BV-0596 DNA) and non-template control

476 (NTC), confirming the assay optimization.

477

478 Tables

479 Table 1. Droplet digital PCR and Quantification Cycle (Cq) obtained in real-time PCR average

480 data for the serial dilutions of Ilyonectria liriodendri isolate BV-0596 DNA (n=5). Values

481 represent the mean±SE.

482

DNA concentration ddPCR qPCR

(fg μl-1) (copies μl-1) (Cq)
10,000 41.3 ± 0.98 26.0 ± 0.10
1,000 30.3 ± 0.32 29.3 ± 0.07
100 17.3 ± 0.36 32.6 ± 0.20
50 14.0 ± 0.84 33.9 ± 0.13
10 4.5 ± 0.45 36.3 ± 0.29
5 1.2 ± 0.16 N/A
1 N/A N/A

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484 Table 2. Droplet profile and digital PCR quantitation data from DNA extracts of the environmental samples from five vineyards (n=4). Values

485 represent the mean±SE from four replicates.

486

Bulk soil Rhizosphere soil Endorhizosphere

Accepted Accepted Accepted

(+) Droplets Copies μl-1 (+) Droplets Copies μl-1 (+) Droplets Copies μl-1
Droplets Droplets Droplets
Vineyard 1 17,400± 930 417 ± 143 293 ± 103 16,400 ± 1,420 1,250 ± 723 889 ± 495 16,200± 971 482 ± 75 370 ± 79
Vineyard 2 12,400± 1,570 194± 44.0 183 ±28 11,800 ± 1,480 442± 147 441 ± 147 11,500± 1,230 285 ± 123 321 ± 166
Vineyard 3 18,100± 606 2,910 ± 784 2,090 ± 610 18,500 ± 751 2,270 ± 1,210 1,820 ± 1,150 18,500± 670 3,410 ± 2,360 2,700± 2,000
Vineyard 4 16,600± 653 3,320 ± 1,820 3,190 ± 2,100 18,200 ± 282 1,830 ± 649 1,260 ± 453 16,700 ± 604 2,770± 1,000 2,300± 894
Vineyard 5 14,900± 1,160 833 ± 445 624 ± 298 16,000 ± 756 925 ± 561 722 ± 428 16,400± 755 640 ± 126 468 ± 96
(+) control1 19,200 ± 0 20 ±0 12 ±0 19,200 ± 0 20 ±0 12 ±0 19,200 ± 0 20 ±0 12 ±0
(+) control2 16,400± 0 411 ±0 299 ±0 16,400± 0 411 ±0 299 ±0 16,400± 0 411 ±0 299 ±0
(-) control3 16,100± 436 0 N/A 16,100± 436 0 N/A 16,100± 436 0 N/A
(-) control4 18,600±1,660 0 N/A 18,600±1,660 0 N/A 18,600±1,660 0 N/A
487
488 1DNA of a pure culture of I. liriodendri isolate BV-0596 (n=1)
489 2DNA of soil sample tested positive to I. liriodendri (n=1)
490 3DNA from grapevine (n=2)

491 4Water (n=2)

492

493

494

495

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496

497 Supplementary Table S1. Soil physicochemical properties and management practices of the five vineyards examined in this study. Values

498 represent the mean±SE.

499

500
Vineyard-1 Vineyard-2 Vineyard-3 Vineyard-4 Vineyard-5
Coordinates 42,583560º, 42,588604º, 42,539882º, 42,499781º, 42,593068º,
501 -2,853296º -2,868726º -2,766227º -2,781054º -2,851397º
Location Haro Haro Briones Briones Haro
Year of plantation 2013 2013 2014 2012 2013
502 Extension (ha) 0.93 8.32 3.00 5.11 1.96
Altitude (m) 478 482 501 497 499
Physicochemical properties
pH 8.1a±0.02 8.1±0.02 8.2 8.2 8.2±0.02
P mg/100g 3.39±0.25 2.6±0.2 2.4±0.3 3.1±0.3 3.2±0.2
K mg/100g 17.2±0.7 17.1±0.4 16.4±0.5 19.1±0.6 20.5±0.5
S mg/100g 4.3±0.5 3.9±0.4 4.1±0.3 4.1±0.4 4.2±0.4
Mg mg/100g 24.3±0.3 27.1±0.3 25.7±0.5 21.4±0.4 26.7±0.5
Mn mg/100g 3.5±0.8 2.9±0.7 2.8±0.7 2.8±0.7 2.8±0.8
Fe mg/100g 8.6±0.4 8.7±0.2 10.6±0.2 7.7±0.4 8.7±0.5
Ca mg/100g 3979.6±220.8 4346.9±120.4 4347.5±109.8 3731.3±176.4 3503.0±126.7
Na mg/100g 2.4±0.2 2.83±0.1 3.1±0.2 3.0±0.2 3.4±0.2
SOM% 0.95±0.03 1.05±0.2 1.05±0.03 0.95±0.05 1.07±0.08
Clay% 18.5±0.2 22.2±0.3 25.3±0.3 21.9±0.6 22.9±0.4
Sand% 39.9±0.3 33.8±0.4 34.3±0.5 35.1±0.6 34.3±0.9
Silt% 41.6±0.6 44.0±1.1 40.4±0.8 43.0±0.7 42.8±0.5
CO3Ca 14.15±0.02 15.67±0.23 13.9±0.1 14.5±0.4 14.9±0.6
CEC mekv/100g 8.9±0.3 10.4±0.1 11.2±0.2 9.6±0.2 9.1±0.3
EC mS/cm 0.14 0.16 0.16 0.15 0.16
Assim. Ca mekv/100g 15.1±0.2 17.4±0.2 16.5±0.2 15.5±0.3 14.8±0.4
Assim. Mg mekv/100g 1.57±0.05 1.81±0.11 1.75±0.12 1.70±0.15 1.63±0.21
Soil temperature (ºC) (July) 20.9 21.0 21.6 21.3 20.9
Soil management practices
Irrigation system Drip irrigation
Fertilization 2 applications per year
Pest management practices 5 spray treatments against powdery and downy mildew per year
Herbicide treatment Yes

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Figure 1

190x254mm (150 x 150 DPI)

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Figure 2

296x209mm (200 x 200 DPI)

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Figure 3

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