The endophytic mycota associated with Vitis vinifera in central Spain

The endophytic mycota associated with Vitis viniferain central Spain

Vicente González & María Luisa Tello

Received: 15 June 2010 /Accepted: 3 November 2010 /Published online: 17 November 2010# Kevin D. Hyde 2010

Abstract This study investigates the diversity of fungalendophytes associated with several varieties of grapevineswith differing modes of cultivation in the Madrid region(central Spain). Our goal is to screen for and identify newfungal biocontrol agents against vine diseases, especially thoseassociated with young plants produced in nurseries. A total of500 fungal strains representing 68 taxa from six locations wereisolated and characterised. Differences regarding cultivar andplant part processed were analysed in terms of composition andrelative abundance of species. Some of the more frequentlyisolated strains represented were Acremonium, Alternaria,Aureobasidium, Botryotinia, Cladosporium, Epicoccum,Fusarium, Gibberella, Nectria, Penicillium, Phoma andTrichoderma species. Botryosphaeria species and Phomopsisviticola were also frequently isolated and may be vinepathogens. Several species of Acremonium, Phoma(P. glomerata) and Chaetomium showed promising antago-nistic activity at the laboratory scale.

Keywords Antagonists . Fungal communities . Grapevine .

Petri disease . Species diversity

Introduction

Endophytic fungi inhabit plant organs colonizing internaltissues without causing visible disease symptoms (Petrini1991; Schulz and Boyle 2005; Zabalgogeazcoa 2008; Hydeand Soytong 2008). Endophytes have been found in all

plant species examined, being considered as ubiquitous inthe plant kingdom (Hyde and Soytong 2008; Oses et al.2008; Fröhlich et al. 2000; Sánchez et al. 2010; de Errasti etal. 2010; Tejesvi et al. 2010), and have even been foundassociated with lichens (Li et al. 2007) and sea grasses(Alva et al. 2002; Sakayaroj et al. 2010). The biodiversityof endophytes in a plant can be remarkable; in certainspecies, more than 100 endophytic taxa have been detected(Stone et al. 2004).

Many factors such as sampling site, tissue age, tissuespecificity or associated vegetation can influence the compo-sition of endophytic communities (Carroll and Carroll 1978;Fisher et al. 1986; Fisher and Petrini 1990; Johnson andWhitney 1989; Sieber et al. 1991a; Rodrigues 1994). Amongthese, geographical variation is recognised as one of the mostimportant. Numerous studies have reported that the abun-dance, diversity and species composition of endophytes canbe strongly influenced by the sampling locality (Bills andPolishook 1992; Arnold et al. 2001; Higgins et al. 2007).Taxa isolated from the same plant species tend to varyaccording to the location of each individual (Collado et al.1999, 2000). When comparing large geographic areas,endophytes have been shown to be more diverse in tropicalbiomes than in boreal or temperate regions (Arnold andLutzoni 2007). Endophyte diversity is also influenced bybioclimatic factors such as latitude, average temperature orannual rainfall, which are associated to some degree with thegeography (Arnold and Lutzoni 2007). The degree ofdisturbance suffered in a plant community can also affectthe composition of endophytic mycota (Gamboa andBayman 2001). Finally, age and phytosanitary status of thehost plant can also exert some effect on the composition ofendophytic mycota (Arnold et al. 2003).

Endophytic fungi show a remarkable chemical diversityof secondary metabolites and therefore possess an impor-

V. González (*) :M. L. TelloInstituto Madrileño de Investigación y Desarrollo Rural, Agrario yAlimentario (IMIDRA), Finca “El Encín”,Ctra. NII, Km 38,200,Alcalá de Henares, Madrid 28800, Spaine-mail: [email protected]

Fungal Diversity (2011) 47:29–42DOI 10.1007/s13225-010-0073-x

tant biotechnological potential (Tan and Zou 2001; Huanget al. 2009). Endophytes constitute a relatively underex-plored and attractive source of natural products suitable tobe exploited in medicine, agriculture or industry (Strobel2002; Strobel and Daisy 2003; Vicente et al. 2002;Polishook et al. 1993; Peláez et al. 1998, 2000; Dreyfuss1986; Brunner and Petrini 1992; Hensens et al. 1999; Aly etal. 2010; Lin et al. 2010).

Endophytes may also interact with their hosts, enhancingtheir growth and improving their resistance to environmen-tal stresses (Clay 1988; Wolock-Madej and Clay 1991;Knoch et al. 1993) or their capacity to resist attacks fromherbivores or other plant pathogenic fungi (Faeth and Fagan2002; Clay 1988, 1990; Leuchtmann et al. 2000). Severalstudies have shown that endophytic microorganisms play akey role in the host-pathogen interactions prior to thetriggering of the disease. Several mechanisms capable toprevent and/or restrict the development of plant pathogenshave been described for some endophytic species. Someendophytes can induce systemic resistance mechanisms andthe expression of defence genes against the attack of certainpathogens in their hosts (Arnold et al. 2003; Gwinn andGavin 1992), or may produce antibiotics that inhibit thegrowth of other fungi. Furthermore, endophytic speciescould eventually compete with plant pathogenic fungi forspace and nutrients.

Another important aspect of the host-pathogen infectionprocesses is that some phytopathogenic fungi could live asendophytes during part of their life cycle (Bisseger andSieber 1994; Franz et al. 1993; Sieber et al. 1991b). This isan important factor in plant-microbe interactions. Amongall fungal taxa able to penetrate and colonize plant tissuesin a given host, only a small proportion of these can causepathogenesis and disease. According to this view, one ofthe challenges of current phytopathology is the character-ization of the differences existing between the infectionprocesses carried out by endophytic vs. pathogenic micro-organisms. This could be applied to grapevine diseases,where the etiological agents of some of the most importantdiseases of the crop (i.e. excoriose, Petri disease, black foot,esca syndrome) are usually isolated living inside planttissues from both diseased and asymptomatic plants(Halleen et al. 2007; Mostert et al. 2000).

Several authors have investigated the diversity andecological role of endophytic communities in grapevine(e.g. Cardinali et al. 1994; Schweigkofler and Prillinger1999; Tiedemann et al. 1988; Casieri et al. 2009; Rodolfi etal. 2008). However, knowledge of the diversity, distributionor influence of these fungi in the development and/orprevention of certain fungal diseases is still incomplete.Mostert et al. (2000) investigated the type of behaviour ofPhomopsis viticola (the etiological agent of vine excoriose)on several grapevine tissues. Their results demonstrated that

some Phomopsis isolates behaved as primary pathogens,whereas others acted as true endophytes. Tiedemann et al.(1988) surveyed the endophytic diversity on grapevine,focusing mostly on vascular tissues of young plants.Recently, Halleen et al. (2007) tested the pathogenicity ofsome vascular endophytes from genus Phaeoacremonium,usually associated to vine plants whose pathogenic poten-tial remained still undefined.

There are no data on the endophytic fungal communitiesfrom Vitis vinifera of the Iberian Peninsula. Most studiesreported to date are confined to the characterization andepidemiology of pathogenic fungi associated to grapevineplants (Armengol et al. 2001a, b; Aroca et al. 2006;Giménez-Jaime et al. 2006; Sánchez-Torres et al. 2008),or describe the development of new techniques andmethods to prevent and control them (Gramaje et al.2009a, b). The present study is focused on the character-ization of species diversity of fungal endophytes associatedto several varieties and modes of cultivation of grapevinesexisting in the Madrid region of central Spain.

Materials and methods

Sampling and fungal isolation

Surveys were carried out in natural vineyards distributedalong the Madrid region during the period 2008–2009.Sampling sites were located into three vine-producing areasincluded within the P.D.O. (Protected Denomination ofOrigin) “Vinos de Madrid”, representing most of the vinevarieties included under such denomination (Table 1).Plants of different age (rooted cuttings, young plants,mature stands) and phytosanitary status (both symptomaticand asymptomatic) were sampled. Depending on the typeof vine variety, 3–14 plants were processed for fungalisolation. Samples of trunks / shoots and leaves were takenfrom each of the stands. Plant samples consisted either ofsmall leaf pieces (0.5×0.5 cm2), twig sections (1–1.5 cm inlength) or portions (0.7×0.7×0.3 cm3) from the inner partsof trunks. In addition, grapes and stalks from some of theplants sampled were also subjected to fungal isolation.

A surface sterilisation method (Schultz et al. 1993) wasused in order to suppress epiphytic fungi. Thus, plant partswere first washed (4–5 times) with sterile water, thenimmersed in 70% ethanol for 1 min, followed by 3%sodium hypochlorite for 15 min and lastly washed again 4–5 times with sterile distilled water. Sterilised plant materialwas placed in two synthetic culture media: water agar (WA)plates and potato dextrose agar (PDA) plates supplementedwith streptomycin sulphate (0.5 g/L) to prevent bacterialcontaminants. For each of the vine plants sampled, fourplates per medium (4 WA + 4 PDA), each containing 4–5

30 Fungal Diversity (2011) 47:29–42

fragments of the several plant parts assayed were incubatedat 25°C in the dark for 15–20 days. When fungal coloniesemerged from plant tissues into the agar, mycelial frag-ments were transferred to new PDA plates. These isolateswere maintained at 18°C with 12 h photoperiod.

Fungal identification

Whenever possible, the taxonomic identification of theendophytes isolated was based on morphological char-acters exhibited by these strains. To induce sporulationof sterile mycelia, strains were cultured on low-nutrientmedium (WA) containing sterilised fragments of V.vinifera leaves and supplemented with streptomycinsulphate (0.5 g/L). In the cases where sporulation failed,identification was attempted by sequencing of the internaltranscribed spacer (ITS) fragment of the rDNA region.PCR amplification of this region was performed asdescribed by Sánchez-Torres et al. (2008), and sequencedusing primers ITS4 and ITS5 (White et al. 1990).Sequence-based identification was achieved by searchingagainst the GenBank database using the FASTA algorithm(Pearson 1990). When the homology between a strainsequence and a sequence in GenBank was greater than97%, the match was accepted at the species level. Thegenus alone was accepted when similarity fell between 95and 97%. Strains with an ITS sequence showing adivergence greater than 5% with any entry at GenBankwere considered as unidentified. Although the degree ofsequence similarity can vary at both intra-genus or intra-species level depending on the type of fungal groupanalysed, arbitrary thresholds similar to those used in thepresent study have been previously used in otherendophyte-related studies to identify taxa (O’Brien et al.2005; Neubert et al. 2006; Sánchez et al. 2007, 2008).These parameters are consistent with those usuallyemployed to define species boundaries in many otherfungal taxa from different orders (Arenal et al. 2000).

Quantification of fungal diversity

To estimate the relationship between the number ofplants analysed and the number of taxa obtained, aspecies accumulation curve (Zak and Willig 2004)(Fig. 5) was performed using EstimateS software (Colwell2005) by means of random sampling without replacement ofthe fungal species data isolated for each plant according tothe methodology described in Colwell and Coddington(1994). To estimate the number of endophytic species thatare expected to be associated with V. vinifera plants, somenon-parametric, incidence-based estimators of species rich-ness (Marrugan 2004) such as Jacknife and Bootstrap werealso calculated. Independent species accumulation curvesincluding the non-parametric richness estimators alreadymentioned were also plotted for each of the severallocalities surveyed, to determine the quality of thesampling and the community structure separately(Fig. 6). In addition, Shannon’s index of diversity (H’)for the relative abundance of each of the taxa obtained(Zak and Willig 2004) was calculated.

Additional analyses were performed to determine theinfluence of several parameters on the abundance andcomposition fungal communities. Thus, the effect of bothhost (variety) and plant tissue processed on the compositionand distribution of the taxonomic groups isolated was alsoinvestigated.

Results

Isolation and fungal identification

This work presents findings of a 2-year survey aiming tocharacterise the endophytic mycota associated with severalvine cultivars in Madrid. The survey was carried out in sixvine-producing areas and a total of 42 field-sampled plants

Table 1 Geographical origin of the plants surveyed. Type of cultivar, sites and number of plants sampled per variety are indicated

Vine varieties Number of plants per sampling locality

“Tempranillo” 4 (Belmonte de Tajo); 4 (Valdilecha “Finca El Socorro”); 1 (Alcalá de Henares “Finca El Encín”) 9

“Moscatel Grano Menudo” 2 (Belmonte de Tajo); 2 (Valdilecha “Finca El Socorro”) 4

“Cabernet-Sauvignon” 2 (Valdilecha “Finca El Socorro”); 1 (Alcalá de Henares “Finca El Encín”) 3

“Malvar” 2 (Valdilecha “Finca El Socorro”); 1 (Alcalá de Henares “Finca El Encín”) 3

“Syrah” 2 (Valdilecha “Finca El Socorro”) 2

“Merlot” 2 (Valdilecha “Finca El Socorro”); 1 (Alcalá de Henares “Finca El Encín”) 3

“Garnacha” 3 (Campo Real); 6 (Villamanta); 7 (Cadalso de los Vidrios) 16

“Albillo” 1 (Alcalá de Henares “Finca El Encín”) 1

“Airén” 1 (Alcalá de Henares “Finca El Encín”) 1

Total Plants=42

Fungal Diversity (2011) 47:29–42 31

were processed. From these, a total of 585 endophyticfungal strains were obtained. Prior to taxonomic identifica-tion, a preliminary visual inspection was made in order toavoid the selection of identical strains arising from the sameplant stand. Among these, 488 isolates were identified onthe basis of morphological and/or molecular characters,while 97 strains were unidentified as sterile mycelia. Mostisolates emerged and were visible and ready for subcultur-ing in less than 6–7 days after the placement on cultureplates. PDA plates yielded more colonies per plantfragment (an average of 5 fungal colonies) than WA plates,with an average of 2 fungal colonies per fragment. About75% of the isolates produced spores (conidia or sexualspores) during the following 2 months. To induce sporula-tion of the remaining strains, cultures were transferred to alow-nutrient medium (WA) with the addition of V. viniferaleaves. Using this method, 42 additional isolates sporulatedand were identified by morphology. The remaining 78sterile isolates failed to produce reproductive structures andwere identified at the genus and species level by comparingtheir ITS sequences with those in GenBank using theFASTA search algorithm.

Species diversity of fungal endophytes

Sixty-eight operational taxonomic units (OTUs) represent-ing 51 fungal genera were recognised (Table 2). Amongthem, taxa belonging to the ascomycetes were by far themost abundant, represented either by sexual or asexualstages. Thus, 62 ascomycetous taxa (91.3%) were detected,whereas only two basidiomycetes (2.9%) and four zygo-mycetes (5.8%) were recovered. Thirty-four strains couldnot be identified using both microbiology or DNAsequence-based comparisons. However, FASTA searchesindicate the presence of 33 ascomycetous and one basidio-mycetous ITS sequences among these 34 strains not definedto species or genus level. Within the ascomycetes, the 62taxa isolated represented 18 orders, being the Xylariales,Sordariales, Pleosporales, Hypocreales, Eurotiales, Botryos-phaeriales and a collective group of incertae sedis, the mostrepresentative groups with more than five species each(Fig. 1) and representing 66% of the total ascomycetoustaxa. Among these highly represented orders, the Hypo-creales and the Pleosporales were by far the most abundant,with 13 and 10 OTUs respectively, representing 19.1% and14.7% of all the taxa identified in the study.

Our analyses revealed a low proportion of isolates ofvine phytopathogenic fungi. Some fungal taxa however,usually associated with different grapevine diseases wereisolated. These were mainly related with Petri disease(Phaeomoniella chlamydospora, Phaeoacremonium aleo-philum, P. inflatipes), Excoriose (Phomopsis viticola),Black Foot (Cylindrocarpon destructans) and Black Dead

Arm disease (Botryosphaeria spp.). Phytopathogens repre-sented a small proportion of the mycota characterised in ourstudy, and only Phomopsis viticola could be considered asfrequent, representing the 1.53% of all the strains charac-terized. Interestingly, we did not recover any wood decaybasidiomycetes associated with esca syndrome (no isolatesof Hymenochaetaceae or Stereaceae were found), eventhough some 40-year-old stands displaying typical symp-toms were sampled.

Some of the genera isolated were previously identified aspotential biocontrol agents, which makes them attractive forfurther investigation of their antimicrobial activities.Among these, we have isolated several strains belongingto genera Acremonium, Phoma (P. glomerata), Chaetomium(Ch. globosum), Epicoccum (E. nigrum), Fusarium (F.proliferatum) or Aureobasidium (A. pullulans) that haveshown promising activity in the laboratory (data notshown). Some of them have been reported to exhibitantagonistic activity against certain grapevine pathogens.

The relationships between the number of isolated strains(Fig. 2) or OTUs (Fig. 3) and sampling sites was alsoinvestigated. Our results showed that Belmonte de Tajo,Alcalá de Henares and Villamanta yielded the highestnumbers of isolates per sampling site, regarding bothnumber of strains (140, 89 and 62 respectively) (Fig. 2)and taxa (36, 27 and 28) (Fig. 3) isolated. However, whenconsidering the number of sampling campaigns in eachlocation, Campo Real is most diverse, since 67 isolatedstrains representing 22 OTUs were obtained after process-ing only three grapevine plants. Remarkably, Valdilechaappears to be the less diverse sampling site, where a total of67 strains representing 19 fungal species were obtained,after processing 14 vine plants. Interestingly, samples fromValdilecha come from an experimental farm, where thevineyards are fully monitored, and repetitive phytosanitarytreatments are routinely applied to these grapevine plots.

Most of the taxa obtained in this study could beconsidered as frequent species (Fig. 4), with 45 (66.1%)fungal species appearing in three or more plants (plurals),whilst a total of 13 (19,1%) singletons and 10 (14,7%)doubletons were isolated. This result suggests that themajority of the endophytic mycota of grapevine plantsanalyzed could be dominated by a group of relativelyconstant species, rather than by rare or occasional fungaltaxa.

Species accumulation curves were plotted to investigatethe relationship between the number of fungal taxa obtainedin the study and the sampling efforts (number of plantsanalysed) employed (Fig. 5). To calculate the speciesaccumulation curve (Sobs) we used the Mao-Tau estimatorthat compares species richness between two or moresampling sites with confidence intervals up to 95%. Weused also Jacknife and Bootstrap as richness estimators,


Table 2 Endophytic fungal isolates from leaves, twigs and berries of Vitis vinifera from six vine-producing zones of Madrid region

Sampling localitiesa

Fungal Taxab Belmonte de Tajo Campo Real Valdilecha Alcalá de Henares Cadalso de los Vidrios Villamanta Total isolatesc (488)

Is Pl Is Pl Is Pl Is Pl Is Pl Is Pl

Acremonium strictum T, L 6 2 0 0 0 0 6 3 0 0 1 1 13

Acremonium spp. T, L 10 6 7 7 2 2 12 3 4 4 2 1 37

Alternaria arborescens L 1 1 0 0 3 1 3 3 0 0 0 0 7

Alternaria alternata L 9 4 5 5 12 4 8 5 5 4 3 1 42

Alternaria sp. L 3 1 0 0 0 0 0 0 0 0 0 0 3

Alternaria tenuissima L 1 1 0 0 0 0 0 0 0 0 0 0 1

Arthrinium phaeospermum T, L 1 1 0 0 0 0 0 0 0 0 0 0 1

Aspergillus niger T 7 3 1 1 0 0 1 1 0 0 0 0 9

Aspergillus terreus L 2 1 0 0 0 0 0 0 0 0 0 0 2

Aspergillus sp. L 5 2 3 1 3 3 1 1 6 5 1 1 19

Aureobasidium pullulans T, L 2 1 5 2 0 0 3 1 2 2 5 2 17

Beauveria bassiana T 0 0 0 0 0 0 0 0 1 1 2 1 3

Botryosphaeria obtusa T 1 1 0 0 0 0 3 2 0 0 0 0 4

Botryosphaeria parva T 0 0 0 0 0 0 0 0 0 0 1 1 1

Botryosphaeria stevensii T 3 3 0 0 0 0 0 0 0 0 0 0 3

Botryotinia fuckeliana B 0 0 0 0 0 0 0 0 2 2 2 1 4

Botrytis cinerea B 0 0 0 0 7 4 0 0 0 0 0 0 7

Ceratobasidium cornigerum T 0 0 0 0 0 0 0 0 2 1 0 0 2

Cladosporium herbarum L 3 1 2 2 0 0 2 1 3 3 0 0 10

Chaetomium globosum T 0 0 0 0 0 0 0 0 2 2 0 0 2

Chaetomium sp. T 3 1 0 0 0 0 0 0 0 0 0 0 3

Colletotrichum sp. L 2 1 2 2 0 0 0 0 0 0 0 0 4

Cylindrocarpon destructans T 4 2 1 1 0 0 0 0 0 0 2 2 7

Cytospora chrysosperma T 0 0 0 0 0 0 0 0 0 0 1 1 1

Epicoccum nigrum L 6 4 11 5 4 4 6 2 3 3 4 3 34

Fusarium oxysporum L 0 0 0 0 6 3 7 3 0 0 3 1 16

Fusarium proliferatum T 12 5 0 0 0 0 0 0 0 0 0 0 12

Geotrichum sp. L 0 0 0 0 1 1 0 0 0 0 0 0 1

Giberella avenacea T 0 0 0 0 0 0 0 0 0 0 3 2 3

Gonatobotryum sp. T 2 2 0 0 1 1 0 0 0 0 0 0 3

Humicola sp. T 4 4 0 0 0 0 0 0 0 0 0 0 4

Hypoxylon serpens T 0 0 0 0 0 0 0 0 2 1 0 0 2

Libertella sp. L 2 1 3 3 0 0 1 1 0 0 1 1 7

Lecanicillium lecanii L 5 4 0 0 0 0 1 1 0 0 0 0 6

Leptosphaeria sp. L 0 0 0 0 0 0 1 1 0 0 0 0 1

Macrophomina phaseolina L 3 3 0 0 0 0 0 0 0 0 0 0 3

Mucor hiemalis L 0 0 0 0 0 0 4 4 0 0 0 0 4

Mucor racemosus L 0 0 0 0 0 0 0 0 0 0 2 2 2

Mucor spp. L 4 1 2 2 3 1 1 1 0 0 2 2 12

Nectria fuckeliana T 3 1 0 0 0 0 0 0 0 0 0 0 3

Nectria ramulariae T 0 0 0 0 0 0 1 1 0 0 0 0 1

Nigrospora oryzae T 0 0 0 0 3 3 0 0 0 0 0 0 3

Nodulisporium sp. L 4 4 0 0 2 1 0 0 0 0 0 0 6

Ophiostoma piceae T 0 0 0 0 0 0 0 0 0 0 2 1 2

Penicillium spp. L 10 4 4 2 6 5 3 3 13 6 5 2 41

Periconia igniaria L 0 0 0 0 3 3 0 0 1 1 2 1 6

Phaeoacremonium aleophilum T 3 1 0 0 1 1 1 1 0 0 0 0 5

Phaeoacremonium inflatipes T 0 0 3 3 0 0 0 0 2 1 2 2 7

Phaeomoniella chlamydospora T 0 0 0 0 0 0 1 1 0 0 0 0 1

Phialophora sp. L 0 0 0 0 0 0 0 0 0 0 3 1 3


because the distribution of fungal species in our study wasnot similar to those assumed by other estimators based inthe relationship between rare and abundant species, whichassume that communities are mostly composed by rarespecies (singletons or doubletons). In this study, most oftaxa obtained were represented by common species, usuallyfound more than 5–6 times in the survey (Fig. 4). Thus, a20.5% and a 38.2% of the total taxa were isolated morethan 10 and 5 times respectively.

The species accumulation curve reached asymptote (Fig. 5),indicating that sampling more plants would not yield manyadditional fungal taxa, and that the number of plants surveyedwas suitable to recover most of the species diversityharboured by grapevine plants. The species accumulationcurves were associated with two richness estimators (finalvalue of 72.88 for Jacknife and 72.86 for Bootstrap) (Fig. 5)showing that the expected diversity tends to be asymptoticand similar to the curve of observed species. Thus, all theaccumulation curves became asymptotic and very close to thespecies richness predicted by estimators, with the exception ofAlcalá de Henares and Campo Real samplings (Fig. 6a andb), where an increase of the sampled population couldeventually yield some new endophytic fungal taxa, speciallyin the last site that yielded a number of 22 identified OTUsafter analysing only three grapevine plants.

The value of Shannon’s index of diversity (H’) was 4.1when all the observed taxa were considered, and 3.17 and3.3 when calculated for subgroups of isolates obtained fromtrunks/twigs or leaves respectively.

Host effects

The influence of the type of grapevine cultivar on therichness (Fig. 7) and composition of endophytic mycotawas investigated. Since the sampling efforts were notequally distributed in the different survey sites, thedistribution of major taxonomic groups across the severalvarieties examined was compared. The seven main asco-mycetous orders mentioned above shared essentially thesame distribution pattern across the six cultivars morefrequently sampled (Fig. 8), with some minor differences.Thus, Hypocreales and Pleosporales were the most abun-dant group of fungi in all the cultivars examined, followedby the Eurotiales, that were especially abundant in somevarieties like Tempranillo and Merlot. However, someorders like the Sordariales and Botryosphaeriales were notrecorded in any of the varieties examined. Furthermore,Cabernet-Sauvignon plants displayed less variability thanthe rest of cultivars, where only species belonging to thePleosporales, Hypocreales and Eurotiales were isolated.

Table 2 (continued)

Sampling localitiesa

Fungal Taxab Belmonte de Tajo Campo Real Valdilecha Alcalá de Henares Cadalso de los Vidrios Villamanta Total isolatesc (488)

Is Pl Is Pl Is Pl Is Pl Is Pl Is Pl

Phoma glomerata T, L 0 0 1 1 0 0 0 0 3 2 1 1 5

Phoma sp. T 0 0 4 1 0 0 0 0 0 0 0 0 4

Phomopsis viticola T 1 1 0 0 3 1 4 1 1 1 0 0 9

Rhinocladiella atrovirens T 1 1 0 0 0 0 0 0 0 0 0 0 1

Rhizoctonia solani T 0 0 0 0 0 0 1 1 0 0 0 0 1

Rhizopus stolonifer L 2 2 1 1 0 0 1 1 1 1 9 3 14

Selenophoma sp. L 0 0 0 0 0 0 1 1 0 0 0 0 1

Slerotinia sclerotiorum T 2 2 0 0 0 0 0 0 0 0 0 0 2

Sordaria sp. T 0 0 0 0 0 0 4 2 0 0 0 0 4

Sporormiella intermedia T 0 0 2 2 0 0 0 0 0 0 0 0 2

Stemphylium sp. L 0 0 0 0 0 0 0 0 0 0 1 0 1

Torula sp. L 0 0 0 0 0 0 0 0 0 0 2 2 2

Trichoderma aureoviride T, L 3 1 4 4 1 1 0 0 2 1 1 1 11

Trichoderma harzianum T, L 4 1 2 2 5 2 2 2 4 3 3 3 20

Trichoderma sp. T, L 4 3 1 1 0 0 2 2 3 1 4 4 14

Truncatella angustata T 0 0 0 0 1 1 0 0 0 0 0 0 1

Ulocladium sp. L 2 2 2 2 0 0 0 0 0 0 0 0 4

Xylaria hypoxylon T 0 0 2 2 0 0 0 0 0 0 0 0 2

a Sampling localities; Is: number of isolates obtained of each fungal taxa; Pl: number of plants in which isolates were obtainedb Sampled plant tissues; T: twigs/branches, L: leaves, B: berriesc Total isolates: underlined numbers indicate fungal species with more than 10 strains isolated


As mentioned above, the vine varieties were mismatchedin terms of the number of plants analysed. However, whenGarnacha and Tempranillo (the two varieties with more plantssurveyed) were compared (Fig. 7), the species accumulationcurves were clearly different. Thus, the accumulation curvesof cultivar Garnacha (Fig. 7a) showed an asymptote for boththe observed species and species richness estimators. Thisindicates that the sampling efforts were enough to obtainfungal diversity harboured in this variety. In contrast,accumulation curves for the variety Tempranillo (Fig. 7b)exhibited a non-asymptotic profile for observed species, aswell as higher biodiversity rates expected when plottingrichness estimators, suggesting that sampling more plantstands might lead to obtaining new fungal taxa.

Tissue effects

Fungal diversity was shown to be different according to thetype of tissue analyzed, especially when the number of taxaobtained from each tissue sample was taken into account(Fig. 9a). Thus, mean numbers of taxa isolated per tissuesample from both leaves and branches were similar, whilethose from taxa obtained simultaneously from branches andleaves as well as those coming from berries were lowcompared with the former two plant parts, especially in thecase of those species obtained from both leaves andbranches. Moreover, when the mean number of strainsisolated from each tissue sample were analyzed (Fig. 9b),leaves gave the highest number of fungal isolates per tissuesample, whereas the number of colonies per tissue samplebelonging to the rest of plant parts were much lower.Specifically, 31 fungal taxa (110 isolates) were obtainedfrom trunks or twigs, while 27 OTUs (247 isolates) wereisolated from grapevine leaves and 8 OTUs (114 isolates)were obtained from both woody tissues and leaves. Twobotryosphariaceous taxa (3 isolates) were found in grapes.

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Results have shown that a greater percentage of fungal taxawere recovered from both woody tissues and leaves, andthe fact that ubiquitous species existing simultaneously inthese two plant parts were not much abundant. Finally,mean numbers of taxa isolated from berries suggest thatendophytic diversity associated to such tissues was domi-nated by a small number of taxa, regardless the number ofstrains obtained.

With regards to the distribution of isolates obtained fromeach type of tissue in each of the different cultivarsanalysed (Fig. 10), leaves again yielded the highest numberof fungal strains in all the varieties analysed, with theexception of cultivar Merlot. The percentage of strainsobtained from both branches and the combination branches/leaves was similar in four of the cultivars (i.e. Tempranillo,Garnacha, Airén and Moscatel Grano Menudo), while inthe other cultivars processed, endophytic isolates weredominated by strains obtained from leaves and the

combination branches/leaves, being less abundant the fungiisolated from branches. In general terms, our results suggestthat among the grapevine plants and varieties analysed, thenumber of fungal isolates obtained from the phyllospherewas significantly higher than those coming from lignifiedtissues (trunks / twigs).

Discussion

This study describes the composition of the endophytic fungalcommunities within the plant tissues of several cultivars of V.vinifera from the Madrid region (central Spain). In generalterms, the associated mycota is dominated by ascomycetesand asexual fungi, which represented about 91% of the totalnumber of OTUs obtained, which is in agreement withprevious reports (Cardinali et al. 1994; Casieri et al. 2009;Halleen et al. 2007; Tiedemann et al. 1988; Mostert et al.2000). These results are consistent with other studies dealingwith the endophytic fungal communities of woody tissues orshoots and leaves of different plant hosts (e.g. Bills 1996;Arnold 2007; Fröhlich et al. 2000; Rungjindamai et al.2008), which have found similar distribution patterns ofascomycetous fungi. The high proportion of Hypocreales,Pleosporales or Xylariales obtained suggests these groups asmain components of the endophytic mycota of many woodyplants. The low proportion of basidiomycetous and zygo-mycetous taxa found in our study is consistent with otherstudies (e.g. Bettucci and Saravay 1993; Sánchez et al. 2010;Chapela and Boddy 1988). Stone et al. (2004) pointed outthe low proportions usually reported in endophytic invento-ries could be due to a sampling bias favouring theoccurrence of sporulating and fast-growing species, general-ly belonging to asexual ascomycetes, rather than wood decaybasidiomycetes, although some 3 recent papers have sug-gested that basidiomycetes constitute an important compo-nent of certain endophytic communities (Rungjindamai et al.2008; Pinruan et al. 2010; Duong et al. 2006; Crozieret al. 2006). We did not recover any fungus from eitherthe Hymenochaetaceae (Fomitiporia) or the Stereaceae(Stereum), even though some plant stands had visualsymptoms of esca disease. Two basidiomycetous weredetected and belonged to the Ceratobasidiaceae, a well-known taxonomic complex of fast growing soil-borne fungi.

The species abundance of endophytic mycota of V.vinifera is mostly comprised of plural or frequent taxa(66%), rather than rare species (singleton and doubletonisolates). When considering rare or occasional species thoseappearing once (singles), the proportion of plural taxa was71%. This indicates that the endophytic mycota of grape-vines comprises a large proportion of characteristic species,repeatedly isolated, regardless of the plant part or cultivar.Such community structure differs to the endophytic mycota

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Fig. 4 Structure of fungal communities associated to V. vinifera plantssurveyed in the study. The graph shows isolate abundances for all theidentified OTUs in all the varieties sampled

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of other plant hosts (Sánchez et al. 2007, 2010; Pinnoi et al.2006; Kauhanen et al. 2006), where fungal communitieswere dominated by rare or occasional species. In this sense,Jacknife and Bootstrap methods employed do not assume aparticular species distribution, generating it instead from anumber of replicates in a random process. These results arein agreement with those obtained by Casieri et al. (2009), ina study of endophytic fungi associated with several Vitiscultivars in Switzerland. In this work, approximately 65%of total isolates identified by the authors represented pluraltaxa. The abundance of plural endophytic fungi fromgrapevine reported here is consistent with other endophytesurveys from tree or shrub species (Kowalski and Kehr1992; Sieber et al. 1991a; Widler and Müller 1984).

The H´ values obtained seem to indicate a similardiversity as that obtained for fungal endophytes in otherstudies (Zak and Willig 2004; Karamchand et al. 2009;Banerjee et al. 2009; Sánchez et al. 2010, etc.) and suggestthat V. vinifera may harbour a rich endophytic mycota.

Differences in terms of species diversity were observedwhen comparing the types of grapevine cultivars sampled.In spite of the heterogeneous nature of the samplings, whenthe two best sampled varieties (Garnacha and Tempranillo)were compared, different species accumulation curves wereobtained (Fig. 7). The observed and expected diversity werenot the same in the two varieties, indicating that not all ofthe varieties examined could harbour the same diversity

rates in terms of the number of fungal taxa obtained.Similar results have been reported previously in studies ofendophytic fungi of grapevine plants. Casieri et al. (2009)analysed the fungal communities of five grapevine cultivarsin Switzerland, and showed that the number and composi-tion of OTUs isolated differed between cultivars.

In general terms, we detected a low occurrence ofpathogenic fungal species associated with grapevines aswas found in previous studies (Casieri et al. 2009;Schweigkofler and Prillinger 1999; Tiedemann et al.1988), even though some reported causal agents of vinewood diseases were detected. Furthermore, Eutypa lata,the causal agent of eutypiosis was not found in any of thesamplings, in agreement with other studies (Mateo-Argomániz 1995; Muruamendiaraz et al. 2009). The lastauthors (Muruamendiaraz et al. 2009) considered thisdisease as restricted to northern areas of Spain, andusually occurring in vineyards with more than 300 mmof annual rainfall. An exception could be made concerningthe incidence of Phomopsis viticola, the causal agent ofexcoriose, which was isolated up to 9 times from at least 4cultivars. Our results agree with those obtained by Rodolfiet al. (2006), who reported high isolation rates for thisspecies in a study of Vitis endophytes from Italy. The highprevalence of this pathogen in endophyte surveys ofgrapevine would be consistent with the consideration ofthis species as an endophyte or as a latent pathogen

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a bFig. 6 Individual accumulationcurves plotted for two deviantlocalities sampled, showing therelationship between the numberof OTUs obtained and samplingefforts employed. Jacknife 1 andBootstrap species richness esti-mators are also plotted. a:Alcalá de Henares; b: CampoReal

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Fig. 7 Individual accumulationcurves plotted for two of thegrapevine cultivars sampled.Jacknife 1 and Bootstrap speciesrichness estimators are also plot-ted. a: Garnacha; b: Tempranillo


(Mostert et al. 2000). Other studies on the fungalcommunities of grapevines (Casieri et al. 2009) showeda low incidence of pathogenic species such as esca andPetri related fungi (including here Botryosphaeria spp.).

As pointed out in previous reports (Casieri et al. 2009;Schweigkofler and Prillinger 1999; Tiedemann et al. 1988),the number and composition of OTUs isolated from theseveral plant parts examined did not vary much betweenwoody tissues and leaves, with the exception of fruits,where a very low number of taxa were recovered fromgrapes. Low fungal colonisation rates of grapevine fruitsare not surprising, considering the shorter exposure tofungal inocula compared with the other tissues. Resultssuggest that all plant tissues contributed similarly to thecomposition of the endophytic mycota of the varietiessurveyed, although the highest rates of both strains and taxarecovered were observed in grapevine leaves. We have

found that 11,7% of the fungi were found in more than oneplant part, a slightly lower value than in other studies(Casieri et al. 2009), where 25% is reported. These resultssuggest the existence of independent infection points forthese taxa.

Some of the genera obtained in the study like Phoma (P.glomerata), Chaetomium (Ch. globosum), Acremoniumspp., Aureobasidium (A. pullulans) and Epicoccum (E.nigrum) need further comment. Those genera have beenrepeatedly reported to possess antifungal properties that areuseful against a number of plant diseases (El-Tarabily andSivasithamparam 2006; Schena et al. 1999; Sullivan andWhite 2000, etc.). They appear to be frequent componentsof almost every endophyte survey, regardless the type ofplant analysed. Other taxa present in our collection such asAlternaria alternata or Fusarium proliferatum have alsobeen identified as promising biocontrol agents against

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VINE VARIETIES

Xylariales

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Fig. 8 Distribution of majorascomicetous groups per someof the main vine varietiesprospected

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a bFig. 9 Distribution of isolatedfungi between the several plantparts analyzed; a: mean numbersof isolated species per tissuesample; b: mean numbers ofisolated strains per tissue sample


specific grapevine pathologies like grapevine downymildew caused by Plasmopara viticola (Musetti et al.2006; Falk et al. 1996; Bakshi et al. 2001), and were alsodetected in our study. Among all these fungal antagonists,A. pullulans, A. alternata, E. nigrum and F. proliferatumwere the most abundant taxa recovered in our study. Forinstance, Fusarium proliferatum, repeatedly isolated in ourstudy, has been employed to control grapevine downymildew caused by Plasmopara viticola (Bakshi et al. 2001;Falk et al. 1996). In these studies F. proliferatum isconsidered a mycoparasitic, cold-tolerant fungus, capableof controlling the development of P. viticola via secretion ofextracellular glucanolytic enzymes (Bakshi et al. 2001). Dueto the fact that we have isolated numerous strains (12) of thistaxon, a preliminary screening process will be performed todetect cold-tolerant isolates, prior to the in vitro antagonismassays.

Epicoccum nigrum represents another promising bio-control agent, currently being developed commerciallydue to its capability to produce secondary metabolites withantibiotic activity (Martini et al. 2009). Some authors haveprobed the biocontrol properties of E. nigrum againstpathogens such as Monilinia (Larena et al. 2005) as wellas other several grapevine pathogens like Plasmoparaviticola (Rodolfi et al. 2006; Kortekamp 1997) or Botrytiscinerea (Fowler et al. 1999). In our preliminary in vitroassays, some of the E. nigrum isolates were able to inhibitthe growth of several grapevine pathogens such as P.chlamydospora. Another fungus with potential for its useas microbial antagonist obtained in our study is Aureoba-sidium pullulans. This taxon is known to possess activityagainst a wide range of grapevine pathogens, includingpostharvest fungi (Schena et al. 1999, 2003). This black

yeast has a great potential for its use in biocontrol, due tothe numerous mechanisms that could be involved in theprotection processes reported (i.e. competition fornutrients and space, production of pectolytic enzymes,polysaccharides or antimicrobial metabolites) (Martini etal. 2009; El-Tarabily and Sivasithamparam 2006). Thisfungus has been frequently isolated (up to 19 strains) inour surveys, and preliminary biocontrol tests at laboratoryscale have demonstrated a high antagonist activity againstall the grapevine pathogens assayed up to date (F.mediterranea, S. hirsutum, P. chlamydospora, P. aleophi-lum and Phomopsis viticola). Further research with thisspecies is needed, aimed at the characterization anddevelopment of future biocontrol products. As mentioned,we have isolated other fungal genera that have beenreported to control grapevine pathogens, such as Alter-naria (A. alternata, A. tenuissima and A. arborescens).Taxa from this cosmopolitan group have been previouslycharacterized as endophytes of grapevine (Polizzotto et al.2008), and reported to inhibit etiological agents ofgrapevine diseases like Plasmopara viticola (Musetti etal. 2006) or Botrytis cinerea (Dugan et al. 2002).Furthermore, other “classical” fungal antagonists likethose from the genus Trichoderma were also isolated inour study with a total of 44 isolates, including T.aureoviride and T. harzianum. The latter is often utilisedcommercially to prevent and control fungal diseases(Monte 2001). Recent experiences on the biocontrol oftrunk diseases of grapevine have been carried out usingTrichoderma-based products (Di Marco et al. 2004; DiMarco and Osti 2007; Harvey and Hunt 2006, etc.).

In summary, we have found that endophytic communi-ties may constitute a source of new biocontrol fungal agents

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Fig. 10 Percentage of strainsisolated in each of the planttissues studied per vine variety


useful to control vine diseases, especially those associatedwith the decline of young grapevine plants produced innurseries.

Acknowledgements This study has been supported by the researchproject FP08-AL02 (IMIDRA-Comunidad de Madrid), “Caracterizaciónde la micoflora endofítica asociada a Vitis vinifera; su diversidad,distribución e implicación en la dinámica y etiología de las principalesenfermedades asociadas al cultivo”.

The authors also thanks Dr. Fernando Peláez (CNIO-ISCIII, Spain)and Dr. Gonzalo Platas (Fundación Medina Andalucia, Spain) theirhelpful criticisms to improve the manuscript.

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The endophytic mycota associated with Vitis vinifera in central Spain

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