detection of black-foot disease pathogens in the grapevine nursery propagation process in spain

Detection of black-foot disease pathogens in the grapevinenursery propagation process in Spain

Carlos Agustí-Brisach & David Gramaje &

José García-Jiménez & Josep Armengol

Accepted: 25 April 2013 /Published online: 10 May 2013# KNPV 2013

Abstract Two commercial nurseries located inComunidad Valenciana region (central-eastern Spain)were sampled in 2010 to evaluate whether the grape-vine nursery propagation process could be a source ofblack-foot disease pathogens. Samples were takenfrom four sources of the propagation process: pre-grafting hydration tanks, scissors used for cuttingbuds, omega-cut grafting machines, and peat used forcallusing. DNA from these samples was extracted andmultiplex nested-PCR using primers specific for“Cylindrocarpon” pauciseptatum, Ilyonectria lirio-dendri and I. macrodidyma-complex (composed of I.alcacerensis, I. estremocensis, I. macrodidyma, I.novozelandica, I. torresensis, and two undescribedspecies) was used to identify the species present. Ilyo-nectria liriodendri and I. macrodidyma-complex weredetected in hydration tanks, scissors, graftingmachines and peat, I. macrodidyma-complex beingthe most frequent. Additionally, ten grafted cuttings

each from five grapevine scion/rootstock combina-tions were collected from each nursery immediatelyafter callusing, and again after one growing season in anursery field. Roots of these grafted cuttings andplants were sampled to isolate the fungal pathogens.Only I. torresensis was isolated after callusing, whileI. liriodendri, I. novozelandica and I. torresensis wereisolated after one growing season, showing the highestincidence at this latter sampling time. Moreover, DNAwas extracted from roots and analyzed as describedbefore. Ilyonectria liriodendri and I. macrodidyma-complex were also detected at both sampling times.The use of the multiplex nested-PCR technique im-proved the detection of I. liriodendri and I. macro-didyma-complex from grafted cuttings and plants inboth nurseries. This work shows that the grapevinenursery propagation process should be considered as apotential infection source for black-foot disease patho-gens, and confirms that infections caused by Ilyonec-tria spp. in grapevine planting material increasemarkedly after one growing season in nursery fields.

Keywords Black-foot . Inoculum sources . Plantingmaterial . Vitis vinifera

Introduction

Over the last decade, the importance of infected grape-vine propagation material as a major means of spreadof fungal trunk pathogens has been emphasized

Eur J Plant Pathol (2013) 137:103–112DOI 10.1007/s10658-013-0221-8

C. Agustí-Brisach : J. García-Jiménez : J. Armengol (*)Instituto Agroforestal Mediterráneo, UniversidadPolitécnica de Valencia,Camino de Vera s/n,46022 Valencia, Spaine-mail: [email protected]

D. GramajeDepartment of Crop Protection, Institute for SustainableAgriculture (IAS), Spanish National Research Council(CSIC), Alameda del Obispo s/n, APDO. 4084,14004 Córdoba, Spain

(Gramaje and Armengol 2011). Although these patho-gens usually manifest in older, mature grapevines astrunk diseases, some of them are frequently associatedwith nursery grapevines as symptomatic or asymptom-atic infections (Halleen et al. 2003; Fourie and Halleen2004).

Traditional grapevine propagation techniques userootstock or scion mother vines, from which dormantcuttings are taken for bench grafting, rooting, or fieldbudding. Some practices, such as hydration, cold stor-age conditions, and general standards of nursery hy-giene, can have a significant effect on the quality ofthe vines produced (Gramaje and Armengol 2011).When apparently healthy grafted nursery plants areexamined, a relevant number of them show blackdiscolourations and brown to dark streaks in longitu-dinal or transversal sections of the wood on stems,rootstocks or roots, from which Petri or black-footdisease pathogens, or Botryosphaeriaceae spp., canbe isolated (Halleen et al. 2003; Oliveira et al. 2004;Aroca et al. 2006; Halleen et al. 2006; Mostert et al.2006).

Regarding Petri disease pathogens, Phaeomoniellachlamydospora and species of the genus Phaeoacre-monium have been detected in grapevine propagationmaterial from nurseries in California (Dubrovski andFabritius 2007), Greece (Rumbos and Rumbou 2001),Italy (Serra et al. 2011), New Zealand (Whiteman et al.2003), Portugal (Oliveira et al. 2004), South Africa(Halleen et al. 2003; Retief et al. 2006) and Spain(Aroca et al. 2006; Giménez-Jaime et al. 2006). Dif-ferent stages of the grafted plant production processhave been identified as potential inoculum sources ofPetri disease pathogens. In Spain, Aroca et al. (2010)detected Pa. chlamydospora, Phaeoacremonium aleo-philum and Pm. parasiticum in hydration tanks,omega-cut grafting machines, scissors, and peat forcallusing during the grapevine propagation processby nested-PCR. These authors also isolated Pm. aleo-philum and Pm. parasiticum from scissors washingsand Pa. chlamydospora from peat samples using asemi-selective culture medium, demonstrating that vi-able propagules are present at different stages of thegrafted plant production process (Aroca et al. 2010).Recently, Gramaje et al. (2011) isolated Cadophoraluteo-olivacea and Ca. melinii from planting materialshowing black vascular streaking and decline symp-toms characteristic of Petri disease, and also detectedviable inoculum of Ca. luteo-olivacea from different

stages of the grapevine nursery process. Species ofBotryosphaeriaceae have also been detected on grape-vine propagation material in Greece (Rumbos andRumbou 2001), Italy (Spagnolo et al. 2011), Portugal(Oliveira et al. 2004), Spain (Aroca et al. 2006) andNew Zealand (Billones-Baaijens et al. 2013a, b).

Black-foot disease pathogens, included into thegenera Campylocarpon, “Cylindrocarpon” and Ilyo-nectria (Halleen et al. 2004, 2006; Schroers et al.2008; Chaverri et al. 2011; Cabral et al. 2012a, b),have also been frequently isolated from rootstockmother-plants, rooted rootstock cuttings, bench-graftand young grafted vines in California (Dubrovski andFabritius 2007), Greece (Rumbos and Rumbou 2001),Portugal (Oliveira et al. 2004) and South Africa(Halleen et al. 2003; Fourie and Halleen 2004). It iswell known that these pathogens are common in thesoil and it has been demonstrated that nursery fieldsites can harbour them, causing infection of graftedvines after some months of growth in nursery soils(Halleen et al. 2003, 2007; Chaverri et al. 2011). Infact, isolation studies conducted in South Africangrapevine nurseries by Halleen et al. (2003) demon-strated that soilborne inoculum of black-foot patho-gens infected grafted grapevines once planted innursery fields. Nevertheless, the presence of black-foot disease pathogens in grapevine nurseries in Spainhas not been explored.

Molecular techniques have played an importantrole in the identification and detection of black-footpathogens. In 2011, known species associated withblack-foot were transferred to the genus Ilyonectria(Chaverri et al. 2011). This change was a consequenceof the taxonomic revision of the genera Cylindrocar-pon and Neonectria¸ during which the five informalgroups of Cylindrocarpon were recognized as genera(i.e., Neonectria/Cylindrocarpon sensu stricto, Rugo-nectria, Thelonectria, Ilyonectria and the anamorphgenus Campylocarpon) (Chaverri et al. 2011). Al-though the causal agents of black-foot were transferredto Ilyonectria (Chaverri et al. 2011; Cabral et al.2012a), they are commonly referred to as “Cylindro-carpon” (e.g., “C.” liriodendri and “C.” macrodidy-mum) to distinguish them from true Cylindrocarponspecies belonging to the genus Neonectria (Chaverri etal. 2011). More recently, Cabral et al. (2012b) revealedthe existence of polymorphism in a wide collection ofisolates previously identified as “C.” macrodidymumby using the ribosomal RNA internal transcribed

104 Eur J Plant Pathol (2013) 137:103–112

spacer region, and the β-tubulin, histone H3 and trans-lation elongation factor 1-α gene regions. Consequent-ly, six new species of Ilyonectria (I. alcacerensis, I.estremocensis, I. novozelandica, I. torresensis, Ilyo-nectria sp. 1 and I. sp. 2) and I. macrodidyma sensustricto, which are morphologically rather similar, wererecognised into the I. macrodidyma-complex. To al-low rapid diagnoses of these species, Dubrovski andFabritius (2007) developed genus-specific PCR pri-mers (Cyl-F and Cyl-R) for the simultaneous detectionof “Cylindrocarpon” liriodendri and “C.” macrodidy-mum in grapevine nurseries. Alaniz et al. (2009)designed species-specific primer pairs (Mac1/MaPa2,Lir1/Lir2 and Pau1/MaPa2) which allow the quick andeasy detection of I. liriodendri, I. macrodidyma-com-plex and “C.” pauciseptatum and which have beenrecently used to detect inoculum sources of thesepathogens in a commercial grapevine nursery in Por-tugal (Cardoso et al. 2012).

The objective of this work was to evaluate which ofthe different stages in the propagation of grapevine inSpanish nurseries could be a source of black-footdisease pathogens. To this end, Ilyonectria spp. weredetected at different stages of the propagation processby multiplex nested-PCR and by isolation and geno-typing in the grapevine planting material before andafter the rooting phase in nursery fields.

Material and methods

Sampling of grapevine nurseries

Two commercial nurseries (A and B), located inComunidad Valenciana region (central-eastern Spain)were surveyed in 2010. Samples from nurseries weretaken arbitrarily at four stages of the propagation pro-cess: pre-grafting hydration tanks, washings from scis-sors used for cutting buds, washings from omega-cutgrafting machines, and peat from the callusing stage.Samples of tank water (approximately 100 ml each)were taken from the middle of the tank stirring slight-ly. Sampling was done in each hydration period whencuttings had been immersed for 2 to 3 days. A sampleof the tap water used to fill the tanks was also collect-ed. Scissors and omega-cut grafting machines werewashed with a sterile solution of 0.2 % Tween-20 atthe end of one working day, and 30 to 60 ml of everywashing was recovered in a sterile tube. Samples of

about 100 g of peat were taken after the callusing stagefor a batch of finished cuttings. One sample of peatwas also taken before use. In nursery A, the totalnumber of samples taken was 28 from hydration tanks,38 from scissors, 13 from omega-cut graftingmachines and five from peat after the callusing pro-cess, and in nursery B, the total number of samplestaken was 19 from hydration tanks, 23 from scissors,16 from omega-cut grafting machines and five frompeat after the callusing process.

In addition, 10 grafted cuttings from five bundles(200 grafted cuttings each) of different grapevinescion/rootstock combinations were collected per nurs-ery after callusing (50 grafted cuttings per nursery).The remaining grafted cuttings were immediatelyplanted in a nursery field in both nurseries. After onegrowing season, ten plants of the same five grapevinescion/rootstock combinations were also collected ineach nursery (50 plants per nursery).

Detection of Ilyonectria spp. and “C.” pauciseptatumfrom liquid samples

Liquid samples from hydration tanks, scissors andomega-cut grafting machines were centrifuged for15 min at 232 rcf; supernatant was discarded and theresulting liquid sample pellets were mixed. This liquidwas centrifuged again for 5 min at 930 rcf in order toobtain a sample pellet which was stored at −20 °C. TotalDNAwas extracted using the E.Z.N.A. Plant MiniprepKit (Omega Bio-tek, Doraville, USA). Before followingthe manufacturer’s instruction of the E.Z.N.A. PlantMiniprep Kit, pellets samples were shook using smallsteel balls in a vial filled with 600 μl of DNA extractionbuffer (P1) for 10 min at 50 Hz using TissueLyser LT(QIAGEN, Hilden, Germany). The homogenized sam-ples (600 μl) were then transferred to new vials and themanufacturer’s instructions were followed to completethe DNA extraction.

A nested-PCR combined with a multiplex PCR ap-proach was used for the detection of Ilyonectria spp. and“C.” pauciseptatum (Alaniz et al. 2009). The first PCRreaction was carried out with primers ITS1F/ITS4(Gardes and Bruns 1993) containing 1× PCR buffer,1.25 mMMgCl2, 80 μM of each dNTP, 0.2 μM of eachprimer, 0.7 U of Taq polymerase, and 1 μl of templateDNA (20 ng μl−1). The PCR reaction mix was adjustedto a final volume of 25 μl with water. The programconsisted of an initial step of 3 min at 94 °C, followed

Eur J Plant Pathol (2013) 137:103–112 105

by 35 cycles of denaturation at 94 °C for 30 s, annealingat 55 °C for 30 s, and elongation at 72 °C for 45 s. Afinal extension was performed at 72 °C for 10 min. ThePCR product was diluted with water 1:100 and 1 μl wasused as template DNA for the secondary PCR whichwas carried out with specific primers Lir1/Lir2, Mac1/-MaPa2 and Pau1/MaPa2 (Alaniz et al. 2009) to amplifyI. liriodendri, I. macrodidyma-complex and “C.” pauci-septatum, respectively. Each reaction contained 1× PCRbuffer, 2.0 mMMgCl2, 80 μMof each dNTP, 0.2 μMofeach primer, 0.7 U of Taq polymerase, and 1 μl oftemplate DNA. The PCR reaction mix was adjusted toa final volume of 25 μl with water. The cycling programconsisted of an initial step of 3 min at 94 °C, followed by30 cycles of denaturation at 94 °C for 30 s, annealing at54 °C for 30 s, and elongation at 72 °C for 45 s. A finalextension was performed at 72 °C for 10 min. Mul-tiplex nested-PCR product was visualized on 1.5 %agarose gels stained with ethidium bromide (Greenand Sambrook 2012).

Detection of Ilyonectria spp. and “C.” pauciseptatumfrom peat samples

For the direct extraction of DNA from peat samples,individual samples were lyophilized overnight and thencrushed using FRITSCH Variable Speed Rotor Mill-PULVERISETTE 14 (ROSH, Oberstein, Germany) un-til all the peat was ground to a fine powder. A total of250 mg of each air-dried peat sample was placed in1.5 ml vial with 700 μl warm (65 °C) extraction buffer(120 mMNaH2PO4, 2 % CTAB or SDS, 1.5 mMNaCl,pH 8.0, and 1 % w/v polyvinylpyrrolidone). Vials werethen shaken and incubated at 65 °C for 15min, followedby centrifugation at 1571 rcf for 10 min. Supernatants(400 μl) were then transferred to a new vial and ZR SoilMicrobe DNA MiniPrepTM (Zymo Research, Irvine,U.S.A.) was used for the further steps of DNA extrac-tion. Extracted DNAwas kept at −20 °C until its use inPCR amplifications. Multiplex nested-PCR reactions todetect Ilyonectria spp. and “C.” pauciseptatum werecarried out as described before.

Isolation and molecular detection of Ilyonectria spp.and “C.” pauciseptatum in grafted cuttings and plants

For the molecular detection of Ilyonectria spp. fromgrafted cuttings and plants, DNA extraction from 0.2 gof necrotic roots and multiplex nested-PCR reactions

were carried out as described for liquid and peatsamples.

In order to isolate Ilyonectria spp. and “C.” pauci-septatum from grafted cuttings and plants at each sam-pling time, root sections were cut from necrotic areas,washed under running tap water, surface-disinfested for1 min in a 1.5 % sodium hypochlorite solution, andwashed twice with sterile distilled water. Small rootpieces were then plated on potato dextrose agar (PDA,Biokar-Diagnostics, Zac de Ther, France) supplementedwith 0.5 g l−1 of streptomycin sulphate (Sigma-Aldrich,St. Louis, MO, USA). Plates were incubated for 10–15 days at 25 °C in the dark and all colonies weretransferred to PDA. Isolates were single-spored priorto morphological and molecular identification with theserial dilution method (Dhingra and Sinclair 1995).

Species of Ilyonectria were identified morphologi-cally by macroscopic characters including colony tex-ture, colour, and the shape of the growing margin onPDA. Conidia were observed and measured on Spezi-eller Nährstoffarmer Agar (SNA) with the addition of a1×1 cm piece of filter paper to the colony surface(Alaniz et al. 2007; Chaverri et al. 2011). For DNAextraction, fungal mycelium and conidia from pure cul-tures grown on PDA for 2 to 3 weeks at 25 °C in thedark were scraped and mechanically disrupted by grind-ing to a fine powder under liquid nitrogen using a mortarand pestle. Total DNAwas extracted using the E.Z.N.A.Plant Miniprep Kit following manufacturer’s instruc-tions. DNAwas visualized on 0.7% agarose gels stainedwith ethidium bromide and was stored at −20 °C. Theidentification of Ilyonectria spp. was confirmed by se-quencing part of the histone H3 gene using CYLH3Fand CYLH3R primers (Crous et al. 2004; Cabral et al.2012a, b). The sequences obtained were then blasted inGenBank.

Results

Detection of Ilyonectria spp. in liquid and peatsamples

Ilyonectria liriodendri and I. macrodidyma-complexwere detected in hydration tanks, scissors, omega-cutgrafting machines, or in peat for callusing, I. macro-didyma-complex being the most frequent (Table 1 andFig. 1). However, “C.” pauciseptatum was notdetected. In nursery A, I. liriodendri was detected in


five of 28 samples from hydration tanks and in two offive peat samples, while I. macrodidyma-complex wasdetected in 10 of 28 samples from hydration tanks,in six of 38 from scissors, and in all peat samples.Ilyonectria spp. were not detected from omega-cutgrafting machines. In nursery B, I. liriodendri wasonly detected in two of 19 samples from hydrationtanks, while I. macrodidyma-complex was detectedin five of 19 samples from hydration tanks, in sixof 23 samples from scissors, in four of 16 samplesfrom omega-cut grafting machines and in four offive peat samples. None of these pathogens weredetected in tap water and peat taken before plantprocessing.

Isolation and molecular detection of Ilyonectria spp.in grafted cuttings and plants

Results of the isolation and detection of Ilyonectria spp.from grafted cuttings and plants sampled after callusing

and after growth in nursery fields, respectively, areshown in Table 2. After callusing, only I. torre-sensis, was isolated from one grafted cutting innursery A, while no Ilyonectria spp. were isolatedin nursery B. However, the use of the multiplexnested-PCR technique allowed the detection of I.liriodendri and I. macrodidyma-complex in bothnurseries with higher frequency. In nursery A, I.liriodendri was detected from three of 50 graftedcuttings, belonging to two different scion/rootstockcombinations, and I. macrodidyma-complex wasdetected from 10 of 50 grafted cuttings belongingto three scion/rootstock combinations. In nurseryB, I. macrodidyma-complex was detected from fiveof 50 grafted cuttings belonging to three scion/-rootstock combinations.

After a growth season in nursery fields, I. lirioden-dri, I. novozelandica and I. torresensis were isolated inboth nurseries, I. novozelandica being the most fre-quent species. In nursery A, I. liriodendri was isolated

Table 1 Detection of Ilyonectria liriodendri and I. macrodidyma-complex in samples collected from nurseries at four differentpropagation stages

Stage of grapevine propagation process Nursery A Nursery B

I. liriodendria I. macrodidyma-complex I. liriodendri I. macrodidyma-complex

Hydration tanks 5/28b 10/28 2/19 5/19

Scissors 0/38 6/38 0/23 6/23

Omega-cut grafting machines 0/13 0/13 0/16 4/16

Peat for callusing 2/5 5/5 0/5 4/5

a Detection was performed by multiplex nested-PCRbNumber of samples from which I. liriodendri and I. macrodidyma-complex were identified/number of total samples evaluated

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 M

500 bp

Fig. 1 Identification of Ilyonectria liriodendri and/or I. macro-didyma-complex from liquid samples by nested multiplex poly-merase chain reaction (PCR). PCR amplification products withthree primer pairs: Lir1/Lir2, Mac1/MaPa2, and Pau1/MaPa2.Lanes 1 to 13: liquid samples from scissors sampled in nurseryA (Lanes 1 and 2) and in nursery B (Lanes 3 to 5), liquid samples

from hydration tanks sampled in nursery A (Lanes 6 to 8) and innursery B (Lanes 9 to 11), and liquid samples from omega-cutgrafting machines sampled in nursery B (Lanes 12 and 13); lanes14 to 16, positive control of I. liriodendri, I. macrodidyma-com-plex and “Cylindrocarpon” pauciseptatum, respectively; lane 17,negative control; lanes M, 100-bp DNA ladder


from six of 50 plants belonging to three scion/root-stock combinations, I. novozelandica was isolatedfrom seven of 50 plants belonging to four scion/root-stock combinations and I. torresensis was isolatedfrom four of 50 plants belonging to three scion/root-stock combinations. In nursery B, I. liriodendri wasisolated from seven of 50 plants belonging to fourscion/rootstock combinations, I. novozelandica wasisolated from eight of 50 plants analyzed belonging

to four scion/rootstock combinations and I. torresensiswas isolated from six of 50 plants analyzed belongingto three scion/rootstock combinations.

By making use of the multiplex nested-PCR, wedetected I. liriodendri and I. macrodidyma-complex inboth nurseries more frequently than by isolation. Innursery A, I. liriodendri was detected from six of 50plants, belonging to two different scion/rootstock com-binations, and I. macrodidyma-complex was detected

Table 2 Isolation and molecular detection of Ilyonectria spp. in grapevine nursery plants sampled after callusing stage and after onegrowing season in nursery fields

After callusing After growth in nursery fields

Nursery Scion/rootstock Isolationa Nested PCR Isolation Nested PCR

A Cabernet Sauvignon/110R

– – I. torresensis (2/10) I. macrodidyma-complex(3/10)

Cardenal/110R – I. macrodidyma-complex(1/10)c

I. liriodendri (1/10) I. macrodidyma-complex(4/10)

I. liriodendri (1/10) I. novozelandica (3/10)

Pardina/110R – I. macrodidyma-complex(2/10)

I. liriodendri (3/10) I. macrodidyma-complex(1/10)

I. liriodendri (2/10) I. novozelandica (1/10)

I. torresensis (1/10)

Mazuelo/110R I. torresensis(1/10)b

– I. novozelandica (2/10) I. liriodendri (1/10)

I. macrodidyma-complex(4/10)

Victoria/140Ru – I. macrodidyma-complex(7/10)

I. liriodendri (2/10) I. liriodendri (5/10)

I. novozelandica (1/10) I. macrodidyma-complex(6/10)I. torresensis (1/10)

B Tintorera/SO4 – I. macrodidyma-complex(1/10)


I. novozelandica (1/10) I. macrodidyma-complex(4/10)

Bobal/161.49 – I. macrodidyma-complex(1/10)

I. novozelandica (3/10) -

Airén/420 – – I. liriodendri (2/10) I. liriodendri (7/10)

I. torresensis (3/10) I. macrodidyma-complex(9/10)

Red Globe/140Ru – I. macrodidyma-complex(3/10)


I. novozelandica (2/10) I. macrodidyma-complex(7/10)

I. torresensis (2/10)

Airén/41B – – I. liriodendri (2/10) I. liriodendri (7/10)

I. novozelandica (2/10) I. macrodidyma-complex(9/10)I. torresensis (1/10)

a Identification was confirmed by sequencing part of the histone H3 gene (HIS) using CYLH3F and CYLH3R primersb Number of plants from which Ilyonectria spp. were isolated/number of total plants evaluatedc Number of plants from which Ilyonectria spp. were detected/number of total plants evaluated


from 18 of 50 plants belonging to all scion/rootstockcombinations analyzed. In nursery B, I. liriodendri wasdetected from 18 of 50 plants belonging to four differentscion/rootstock combinations, and I. macrodidyma-complex was detected from 29 of 50 plants belongingto four different scion/rootstock combinations. Co-infections caused by I. liriodendri and I. macrodi-dyma-complex were detected both immediately aftercallusing and after a growth season in nursery fields.

Discussion

Previous studies had detected Petri disease patho-gens such as Ca. luteo-olivacea, Pa. chlamydo-spora and Phaeoacremonium spp., as well asBotryosphaeriaceae spp. at different stages of thegrapevine nursery process or in propagation mate-rial (Aroca et al. 2010; Gramaje et al. 2011;Spagnolo et al. 2011). The present study demon-strates for the first time that inoculum of Ilyonec-tria spp. is also present at the different stages ofthe grapevine nursery propagation process. Wedetected I. liriodendri and I. macrodidyma-com-plex in hydration tanks, omega-cut graftingmachines, scissors and/or callusing peat from twodifferent nurseries by multiplex nested-PCR, I.macrodidyma-complex being the most frequent.These pathogens were not detected in tap waterand peat before use at their respective stages,which indicates that they were present in waterand peat after being in contact with infected plant-ing material. This is in concordance with theresults obtained by Aroca et al. (2010) and Gra-maje et al. (2011), in commercial Spanish grape-vine nurseries, who detected Ca. luteo-olivacea,Pa. chlamydospora, Pm. aleophilum and Pm. par-asiticum in hydration tanks, omega-cut graftingmachines, scissors and peat for callusing duringthe grapevine propagation process by multiplexnested-PCR or by isolating them on culture medi-um, but not from water and peat before use.

The high potential risk of mother vines as an inoc-ulum source of fungal grapevine trunk pathogens inthe vegetative propagation process has been demon-strated (Pascoe and Cottral 2000; Feliciano and Gubler2001; Edwards et al. 2003). Thus, water used in hy-dration tanks to soak cuttings could be contaminatedby field-acquired microorganisms on the bark of the

bud sections dispersing into the tank being also apotential inoculum source (Waite and May 2005;Aroca et al. 2010; Gramaje et al. 2011). Moreover,buds of hydrated canes are manually removed withscissors, and rootstock and scion cuttings aremechanically-grafted with an omega-cut grafting ma-chine causing wounds which make the propagationmaterial very susceptible to infection by fungal trunkpathogens (Gramaje and Armengol 2011). Accordingto this, our results suggest that infections caused byIlyonectria spp. can also occur during these stages ofthe nursery propagation process.

Moreover, in this work we confirmed that dur-ing the rooting phase in nursery fields the numberof plants infected with black-foot pathogensincreases markedly. By isolation on culture media,only one I. torresensis isolate was obtained fromone of the cuttings sampled immediately after cal-lusing. However, after one growing season in nurs-ery fields, I. liriodendri, I. novozelandica and I.torresensis were more frequently isolated fromrooted plants in both nurseries. These results arein agreement with those obtained by Halleen et al.(2003) who demonstrated that in South Africangrapevine nurseries less than 1 % of the callusedcuttings were infected with Ilyonectria spp. priorto planting in nursery soils for rooting, whereas50 % or more of the plants were infected by theend of the season. Similar results have beenobtained in Spain with Petri disease pathogenssuch as Pa. chlamydospora and Pm. aleophilum,which were more frequently isolated from grafted plantsafter plantation in nursery fields (Giménez-Jaime et al.2006).

Regarding the molecular detection of Ilyonectriaspp. on grafted cuttings and plants, a greater numberof positive samples were found in both nurseries, aswell as the two different sampling times. This could bedue to the fact that these pathogens may require ahigher level of host colonization to be detected byisolation on culture media (Aroca and Raposo 2007;Aroca et al. 2010) and to the high sensitivity of themultiplex nested-PCR technique (minimum of 100 pgof genomic DNA for “C.” pauciseptatum, 10 pg for I.liriodendri and 100 fg for I. macrodidyma-complex)(Alaniz et al. 2009). Furthermore, the multiplexnested-PCR technique allowed rapid identifica-tions. The analyses were performed in 6 h, whileculturing and subsequent fungal isolation took


between 10 and 20 days and misidentification wasnot ruled out. Thus, the multiplex nested-PCRtechnique will be useful for the detection of thesepathogens in a high throughput manner from manynurseries and planting material.

Taken together, these results demonstrate that thesoil in the nursery fields, where grafting plants areplanted once the grafting propagation process is over,is an important source of inoculum for Ilyonectria spp.During this period, the susceptible basal ends of mostof the nursery grafted cuttings are partly or even fullyexposed. The young callus roots also break during theplanting process, resulting in small wounds suscepti-ble to infection by Ilyonectria spp., which are commonand may be isolated as soil inhabitants, saprobes ondead plant material, root colonizers or pathogens, orweak pathogens of various herbaceous and woodyplants (Brayford 1993; Halleen et al. 2003, 2007).Recently, Ilyonectria spp. have been detected in natu-ral soils from grapevine nurseries in Spain by usingbait plants (Agustí-Brisach et al. 2013).

It is interesting to note that species belonging toI. macrodidyma-complex were the most frequentlydetected. These results agree with those obtainedby Alaniz et al. (2007), who reported the preva-lence of the I. macrodidyma-complex associatedwith black-foot disease in Spain. This fungal com-plex was also isolated frequently in Spain fromroots of weeds and bait plants grown in grapevinerootstock mother fields, nursery fields and commercialvineyards (Agustí-Brisach et al. 2011, 2013).

The use of propagation material infected with Ilyo-nectria spp. is contributing to the low percentagesurvival of vine plants observed in the last few yearsin Spain (Giménez-Jaime et al. 2006; Gramaje et al.2009), as well as the dissemination of these pathogens.In this context, a sanitation program is required toimprove the quality of grapevine planting material.Chemical control with fungicides (Fourie and Halleen2006; Rego et al. 2006; Halleen et al. 2007; Alaniz et al.2011) and biological methods such as the use of Tricho-derma (Fourie and Halleen 2006; Halleen et al. 2007),arbuscular mycorrhizae (Petit and Gubler 2006; Bleachet al. 2008) or soil biofumigation (Bleach et al. 2009)can be used as strategies to decrease the incidence andseverity of Ilyonectria spp. during the nursery propaga-tion process. Finally, standard hot water treatment pro-tocols at 50 °C for 30 min may be sufficient to controlIlyonectria spp. in grapevine propagation material

(Gramaje et al. 2010). Thus, an integrated managementprogram that includes biological, chemical, hot watertreatment and other control measures would ensure highphytosanitary quality plant production with low levelsof infection.

Acknowledgments This research was financially supportedby the Project RTA2010-00009-C03-03 (Instituto Nacional deInvestigación y Tecnología Agraria y Alimentaria INIA, Spain)and the European Regional Development Fund (ERDF), andperformed within the Programme VLC/Campus, MicroclusterIViSoCa (Innovation for a Sustainable Viticulture and Quality).We acknowledge V. Garrigues for technical assistance.

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