Cohorts of arbuscular mycorrhizal fungi (AMF) in Vitisvinifera, a typical Mediterranean fruit cropemi4_160 594..604

Raffaella Balestrini,1† Franco Magurno,1†

Christopher Walker,2 Erica Lumini1 andValeria Bianciotto1*1Istituto Protezione Piante (IPP) del CNR c/oDipartimento di Biologia Vegetale dell’Università degliStudi di Torino, Viale Mattioli 25, 10125 Torino, Italy.2Royal Botanic Garden Edinburgh, 20A Inverleith Row,Edinburgh EH3 5LR, Scotland, UK.

Summary

In field conditions, grapevine roots normally are colo-nized by arbuscular mycorrhizal fungi (AMF).However, little is published, from either morphologi-cal or molecular studies, on the species compositionof these symbionts in production vineyards. The AMFbiodiversity of two Piedmont vineyards (at Neive andLessona), characterized by different soil features,was investigated by morphological and molecularanalyses. Several morphotypes were identified fromthe two vineyard soils. Community composition ofAMF, both in soil and root samples, was then analy-sed with molecular approach to amplify a portion(550 bp) of AM fungal SSU rDNA. Phylogenetic analy-ses show a different distribution of sequences fromthe two sites in the main glomeromycotan groups. Inthe Neive site, the Glomeraceae group A is the onlyone well represented whereas more groups werefound at Lessona. Among the more representativeoperational taxonomic units (OTUs), only one relatedto the Glomus irregulare phylotype was sharedbetween the two vineyard soils. The data obtained inthis work together with similar results in literature onthis important fruit crop reinforce the concept that thegeneral AMF assemblage structure and compositionin vineyards might be influenced more by soil typethan by host plant features (age, vegetative stages) ormanagement practices.

Introduction

Soil functions are essential for the biosphere, but the soilis perhaps the most difficult and poorly understood envi-ronmental matrix. It provides a wide range of ecosystemservices that underpin the sustainability of agriculture,forestry, horticulture, fruit production, and both semi-natural and natural terrestrial environments. The mainecological functions of soil include nutrient cycling,carbon storage and turnover, water retention, soil struc-turing, regulation of above ground diversity, bufferingand transformation of potentially harmful compoundsand elements (e.g. heavy metals and pesticides). Allthese functions are to some extent dependent on theactivity of living soil organisms (i.e. microbes, fauna andlive plant roots) and an adequate understanding of soilbiodiversity plays a key role in ensuring sustainable useof soil. Because soil functionality is highly dependent onmicroorganisms, measures of their activity, biomass anddiversity, including the presence and health of root sym-bionts, have often been proposed as indicators of soilquality. Progress made over the last decade has con-firmed that there is a wide range of soil microbialdiversity and activity that remains mostly unexplored.Recently, new strategies and more sophisticatedmethods have been developed to characterize soil biodi-versity, both in terms of variety and structure. More effortis needed to establish general patterns of variation inmicrobial diversity, mostly in relation to saprotrophic,pathogenic and symbiotic fungi. Among the most impor-tant soil organisms are the symbiotic fungi that formarbuscular mycorrhizae (AM; all in the phylum Glomero-mycota). They are thought to be the oldest group oforganisms living in symbiosis with land plants (Blackwell,2000; Redecker et al., 2000). AM fungi (AMF) colonizethe roots of most land plants, where they facilitatemineral nutrient uptake from the soil in exchange forplant-assimilated carbon (Smith and Read, 1997). AMFare obligate biotrophs. Although their spores can germi-nate in the absence of host plants, they depend on aliving photoautotrophic partner to complete their lifecycle and produce the next generation of spores. Thereis a need for basic information on the biology of thesefungi, at both species and cohort levels. Genomesequencing of representative AM fungi, such as Glomussp. (Martin et al., 2008; Stockinger et al., 2009), will

Received 14 December, 2009; accepted 12 February, 2010. *Forcorrespondence. E-mail v.bianciotto@ipp.cnr.it; Tel. (+39)0116502927; Fax (+39) 0116705962. †These authors equally contrib-uted to this work.

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perhaps provide new clues to strengthen our under-standing of the reproduction, biology and genetics inthese fungi.

In contrast, in the last decade, more information hasbeen gathered regarding the functional role of AM fungi inecosystems (van der Heijden, 2002). In particular, there isevidence that experimental (van der Heijden et al.1998a,b) and natural (Moora et al., 2004) AM fungal com-munities with different taxon composition may induce adifferent growth response in plants, and may thus influencethe structure and composition of vascular plant communi-ties (Klironomos, 2003; Munkvold et al., 2004). About 215glomeromycotan species have been so far described(Schübler et al., 2001), primarily on the basis of sporemorphology but increasingly supported by DNA sequenceinformation. The use of DNA sequencing methods and thenew metagenomics approaches (i.e. high-throughputmolecular diagnostic tools) has transformed our under-standing of the diversity ofAMs, in particular because it hasallowed the detection of AM fungi directly from natural andagricultural environmental samples (Lumini et al., 2010;Öpik et al., 2009). However, little is so far published onthe species composition of these symbionts in productionvineyards.

Vitis vinifera, cultivated for both fruit and beverage,represents one of the most economically important fruitcrop worldwide, with widespread cultivation and high com-mercial value (Vivier and Pretorius, 2002). Grapes (about67 Mt of grapes produced on 7 501 872 ha in 2007; http://faostat.fao.org) and their derivatives have a large andexpanding worldwide market. They can be grown at lati-tudes from 50°N to 40°S and up to 3000 m above sea level,with almost 98% of grape vineyards planted with V. viniferassp. sativa cultivars of Eurasian origin. A traditional icon ofthe Mediterranean diet, the grape has more recently beenextensively cultivated in the New World and its cultivation isnow moving to Asia. It has the potential to become a modelorganism for fruit trees, as mirrored by the two genomesequencing projects developed on grape (Jaillon et al.,2007; Velasco et al., 2007), and its genome was the firstfruit crop to be sequenced.

Vitis vinifera is cultivated as a perennial crop, tradition-ally (especially in Europe) non-irrigated and with a dif-fused distribution over a wide area of dry or semi-dryecosystems. Many viticulturists now aim to produce highquality wine, increasing profit from the land and reducingagronomic input through encouraging natural beneficialorganisms. Wine grape production is one such croppingsystem in which mineral fertilizer and cover crops arecombined to optimize soil nutrient content. In addition,maximizing yield is not the goal of wine grape production,so inputs from mineral fertilizers are relatively low com-pared with those of annual cropping systems. In relationto prospective use of AMF, this is an advantage over most

annual cropping systems in which negative effects of fer-tilization on AMF have been demonstrated (Treseder,2004). Vineyard soils support indigenous AMF (Dealet al., 1971; Possingham and Groot-Obbink, 1971; Mengeet al., 1983; Nappi et al., 1985; Cheng and Baumgartner,2004; Oehl et al., 2005; Lumini et al., 2010) and it is wellestablished that grapevines roots are colonized by AMF(Nappi et al., 1985; Schubert and Cravero, 1985; Karagi-annidis and Nikolaou, 1999; Schreiner and Mihara, 2009).AMF colonization of vines roots can result in increasedgrowth (Schubert et al., 1988; Biricolti et al., 1997; Linder-man and Davis, 2001), enhanced nutrient uptake(Schreiner, 2007) and improved drought tolerance(Nikolaou et al., 2003; Schreiner, 2003; Schreiner andLinderman, 2005; Schreiner et al., 2007).

Much of this work is from small scale experiments, butlittle is known about the species composition of AMFcolonizing grapevines in production vineyards. The mostcommon species reported from vineyard soils have beenGlomus intraradices (but see Stockinger et al., 2009 inrelation to nomenclatural confusion in this species),Glomus macrocarpum, Glomus mosseae and Paraglo-mus occultum (Menge et al., 1983; Nappi et al., 1985;Schubert and Cravero, 1985; Karagiannidis et al., 1997;Cheng and Baumgartner, 2004; Oehl et al., 2005). It is notclear which of these fungi found as spores in soil actuallycolonize roots of grapevines. Some might be associatedonly with other plants in the vineyard, such as cover cropsor weeds, that could play a role in maintaining AMFspecies diversity in the roots of grapevines. Molecularmethods can show which organisms are actually presentin roots of individual plants, but few such studies arepublished on the AM fungal composition in vineyards,which represent good candidates in studies devoted toverify the AMF communities in a land-use gradient fromnatural to conventionally managed ecosystems.

In this study we focused on the AM fungal biodiversityin two Mediterranean vineyards at Neive and Lessona inPiedmont, Italy (Fig. S1). We compared the AMF found inthe soil with those specifically in grapevine roots, as astep towards understanding their complexity. We soughtto discover if there are specific AMF-grapevine root asso-ciations and which, if any, environmental factors influencethe symbioses. While there are no doubts on the benefitsproduced by AM fungi, only with such comparisons willsufficient understanding be acquired to optimize theirapplication in a commercial context.

Results and discussion

Physical and chemical analyses of the soil

Results of physical and chemical analyses on soil areshown in Table S1. According to the United States Depart-

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ment of Agriculture classification both soils have a loamtexture. They had similar contents in N and P nutrients,but for pH and cationic exchange capacity (CEC) therewere strong differences between the two studied sites.Neive soil was quite alkaline (pH 8.28) whereas soil fromLessona was strongly acidic (pH 4.78). There is someevidence to show that pH in particular can affect the AMFspecies composition (Wang et al., 1993). No strong differ-ences in the physical properties (bulk density and aggre-gate stability) were found among the sampled areas, butsoil organic matter was lower in Neive than in Lessonasoils with the former having a lower mean C:N ratio. Thereis some work suggesting that sporulation of particularAMF can be stimulated increasing organic matter (Gryn-dler et al., 2004; Oehl et al., 2009), but there seems to beno study to assess if different fungi react differently in thisrespect.

AMF spores in soils

Extracted spores were identified morphologically, andspecies determined by reference to the literature (seehttp://www.amf-phylogeny.com) and from experience.Several morphotypes were identified and described(Fig. 1). In Lessona vineyard soil we detected members ofScutellospora (S. erythropus and S. dipurpurescens),Sclerocystis sinuosa (= Glomus sinuosum of someauthors), Paraglomus (probably P. occultum) and

Archeospora trappei. Extractions from the Neive vineyardsoil yielded a much more complex cohort of species. Incommon with the Lessona soil, both Paraglomus cf.occultum and S. sinuosa were present, but in addition,species from Glomus [G. mosseae, G. sp. (similar to G.mosseae, but with brownish spores), G. microaggrega-tum, G. aureum, G. cf. etunicatum, a ‘red brown laminate’species of Glomus, and a Glomus sp. similar to G. intrara-dices], and Archeospora trappei, along with Diversisporaspurca, and an undescribed species from Ambispora. It isnoteworthy that no members of Scutellospora were foundin the Neive soil. The lack of certainty indicated by ‘cf’ inspecies determination reflects the difficulties in identifyingspores from soil extractions. For example, only one spe-cimens of G. cf. etunicatum was found, and it wasdegraded. The group of Glomus spp. that contains G.intraradices can only be determined fully through molecu-lar means. The taxonomy of AM fungi is in fact continu-ously being revised and, i.e. a reference species formerlythought to be G. intraradices (DAOM 197198) has beennow reclassified as being more closely related to Glomusirregulare (Stockinger et al., 2009). Similarly, P. occultumis defined mainly through its molecular signature, andmorphological similar species are likely to be separableonly by molecular means. It is thus better to offer anelement of doubt when giving names to field-collectedspecimens of these organisms. The Glomeraceae cur-rently contains only a single genus, Glomus, but it is

Fig. 1. Spore morphotypes from Lessona and Neive Vineyard soil. Spores were extracted from the soil by a wet-sieving, sucrosecentrifugation technique developed from Jenkins (1964) for AMF spore extraction by Walker and colleagues (1982). They were then washedinto watch glasses with water and examined first under a dissecting microscope, and, after mounting on microscope slides in polyvinyl alcohollacto-glycerol (PVLG) (Omar et al., 1979), with or without the addition of Melzer’s reagent, through a compound microscope following themethods of Walker (1983). Voucher specimens (Walker, 1979) were retained in the herbarium of C. Walker, now included in the Herbarium ofthe Royal Botanic Garden Edinburgh (E). 1–8. Scutellospora erythropus. 1. Freshly extracted spores in water. Note the large variation inshape and colour. 2 & 3. Intact spores in PVLG showing the relatively small thin-walled bulbous base (top) and the prominent darkly colouredgermination shield (left). 4. Detail from spore crushed in PVLG with Melzer’s reagent, showing lack of reaction to this reagent. 5. Spore lightlycrushed in PVLG to reveal the ‘endospore’ with the prominent yellow-brown germination shield. 6 & 7. Detail of crushed spore showing thebulbous base and the colourless inner wall group and part of the germination shield. 8. Germination shield dissected from the spore, showingdetails of infolding around its periphery and germ tube initials (arrows). 9–16. Scutellospora dipurpurescens (possibly = S. calospora). 9.Freshly extracted spores in water. Note large shape variation and uniform colour. 10 & 11. Intact spores in PVLG showing prominent bulbousbase and shape variation. 12. Spore in PVLG-M showing the ‘amorphous’ plastic reaction of one of the inner wall components which hasreacted to become pale purplish pink. 13. Detail from the spore in 12, showing the wall groups (1–3). 14. Detail from crushed spore inPVLG-M showing two reacting wall components in the innermost wall group, used as a defining species characteristic. 15. Detail of thethick-walled, prominent bulbous base. 16. Lateral view showing the development of the germination shield (arrowed) by extrusion of theinnermost wall component. 17–19. Sclerocystis sinuosa (= Glomus sinuosum). 17. Slightly crushed entire sporocarp, showing sporessurrounding a central sterile plexus. 18. Individual spores can be seen at the edges of this specimen, crushed in PVLG. 19. Surface hyphaeon the sporocarp, showing their sinuous nature that gives the species its epithet. 20. Archaeospora trappei (or possibly Intraspora schenckii).Single, colourless spore detached from its sporiferous saccule. 21–22. Diversispora spurca. 21. Intact spores showing short subtending hypha(top, centre). 22. Lightly crushed spore in PVLG, showing the thick outer wall component and the flexible inner component. 23. Sporesextracted from 50 ml of soil beneath grapevines at Neive, showing the variety of glomeromycotan species present. 24. Glomus sp. in the G.mosseae group showing simple subtending hypha (narrow, parallel-sided). 25. Crushed spore in PVLG-M of Diversispora spurca showingslight yellow reaction to this reagent. 26. Spore corresponding to the description of Paraglomus occultum. 27. A collapsed dead spore of aglomeromycotan fungus in the G. intraradices group. 28. A degraded ‘red brown laminate’ spore of a Glomus sp. 29. Optical section of anacaulosporoid spore of Ambispora sp., showing distinctive spore contents. 30. Sinuous peridial hyphae of a sporocarp of Sclerocystis sinuosa(= G. sinuosum of some authors). 31 & 32. Ambispora sp. glomoid spore in PVLG-M (31) showing a pale yellow reaction, and acaulosporoidspore in PVLG (32). 33. A brownish funnel-shaped spore in the G. mosseae grouping. 34. Wall detail of a spore, represented by only a singlespecimen, of a Glomus sp. similar to G. etunicatum. 35. A pair of spores from a dead spore mass of G. aureum. 36. Two spores from a sporecluster of G. microaggregatum crushed in PVLG-M, showing no reaction to this reagent: only a single small cluster of moribund spores wasfound in this extraction.

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evident that this will need to be separated into differentgenera (Schübler et al., 2001; Redecker et al., 2007) andconsequently we have retained the older name for S.sinuosa rather than placing it within Glomus as do someauthors (Almeida and Schenck, 1990).

AMF cohort identification in soil and in root

The use of DNA sequencing methods and the newmetagenomics approaches has transformed our under-

standing of the diversity of AMs. There are (as at March2009) > 11000 glomeromycotan sequences assignedto about 950 distinct taxonomy identifiers in GenBank,only 96 of which are unambiguous species binomials(Helgason and Fitter, 2009). There are many sequencesdesignated ‘environmental’ or ‘uncultured’. Even allowingfor duplication, this suggests a high level of diversity notyet in culture. It is apparent that natural communitiesof AM fungi are biologically diverse, with large numbers oftaxa present, whether identified from spores or from DNA.

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This taxonomic diversity appears also to reflect functionaldiversity. Although large numbers of plant–fungus combi-nations occur naturally or can be created experimentally,not all fungus–plant combinations behave similarly. Inexperimental studies, there is usually one or more optimalcombination (at least as measured by plant performance),implying that the cost–benefit ratio differs with differentfungal–plant cohorts (Helgason and Fitter, 2009). Tounderstand factors influencing the structure of plantcommunities, more information is needed about thenatural distribution patterns and symbiotic associationsof AM fungi.

In our study, PCR was performed on three subsamplesof soil from each plant considered. Overall 1152 clonesfrom soil clone libraries were screened by PCR; out ofthese, 480 clones of each site were sequenced, for a totalof 960 sequences. The BLAST search in the GenBanksequence database revealed that 270 clones (56.25%) forLessona and 207 clones (43.13%) for Neive corre-sponded with AM fungi sequences showing a high degreeof similarity to sequences from taxa belonging to thephylum Glomeromycota (NCBI Gene Bank Accession No.GU353347–GU353956). No chimeric sequences weredetected in this study. In spite of the supposed AM primerspecificity, ‘contaminant’ sequences were detected,belonging to taxa from non-glomeromycotan fungi. Toimprove molecular species characterization and to allowspecies level community analyses in the field, a set ofnewly designed AMF-specific PCR primers, tested onspores isolated from soils and roots, was successfullydeveloped, but published well after the work here wascompleted (Krüger et al., 2009). Even if these primers hadbeen available, for this research it was desirable to usethe common NS31/AMmix primer set since a high numberof its targeted sequences are deposited in GeneBank. Inaddition, NS31/AM1 have been recently used in a pyrose-quencing study on AM fungal communities associated toroots in a boreonemoral forest, generating a high numberof sequences for this target (SSU rDNA; Öpik et al.,2009). Similarly, another set of new primers (the AML1/AML2 pair of Lee et al., 2008) were developed and testedon spores and roots. Nevertheless, we tested these

primers and discovered that their use did not substantiallymodify the AMF community detection in agricultural soils(unpublished data). We have for example observed thatthe genus Paraglomus was not detected, although itspresence was verified using specific primers GOCC56/GOCC427 (Millner et al., 2001). All together these obser-vations reinforce the difficulties of using AMF primers in amore complex matrix such as soil.

In our work, Glomeromycota sequences were thengrouped into operational taxonomic units (OTUs) corre-sponding to phylotypes with 98% similarity levels(OTU0.02). Considering each soil type, 33 OTUs fromLessona and 34 from Neive were generated. Both of therarefaction curves show a decreasing rate of accumula-tion of OTUs but did not reach the asymptote (Fig. S2A).This pattern indicates that our sequence sampling effortsobtained a large proportion of the diversity of soil AMFcommunities, at least for Lessona. This was supported bythe non-parametric richness estimators ACE and Chao1(Table 1). For Neive, in contrast, the number of observedOTUs is lower than the richness estimated by the ACEand Chao1 indices. The Shannon Wiener index values(H′Lessona = 2.62; H′Neive = 2.75; Table 1) were higher thanthose found in other highly exploited agrosystems andseem comparable to those of natural or semi-natural eco-systems (Öpik et al., 2006) as well as in Sardinia vine-yards (H′ = 2.50 and 2.75 for the tilled and no-tilled covercropped vineyards, respectively; Lumini et al., 2010).

Phylogenetic analyses showed a different distribution ofsequences from the two sites in the main glomeromyco-tan groups. Sequences of six families and species groupswithin the phylum were obtained for Lessona vineyard(Glomus group Aa, Glomus group Ab, Glomus group Ad,Glomus group B, Gigasporaceae and Diversisporaceae)(Fig. S3). The best represented were the Glomus groupAd, from which no species were recognised in the mor-phological survey, the Glomus group Ab and the Glomusgroup B. Also the family Gigasporaceae was well repre-sented, confirming the soil spore analysis. In the Neivevineyard, the sequences obtained mainly belong to theGlomus group A (Fig. S4). Some OTUs, comprising a highnumber of sequences, are positioned in two main clades:

Table 1. The ability of the two non-parametric richness estimators (ACE and Chao1), to predict the number OTUs, is compared with the numbersof observed OTUs (rarefaction index). OTUs from the soil and roots samples were generated at 98% level of similarity.

AMF community

Observed OTUsa Estimated OTUsa

O/Eb

Diversity indexa

Rarefaction ACE Chao1 H’

Neive soil 34 49.2 51 0.68 2.75Lessona soil 33 41.2 39.4 0.825 2.62Lessona roots 10 12.62 10.25 0.88 1.56

a. The values are calculated using the DOTUR software.b. O/E = No. OTUs observed divided by No. OTUs estimated by the two non-parametric richness estimators ACE and Chao1 (mean value).

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one related to G. sinuosum (= S. sinuosa) (group Ab) andthe other related to the phylotype ORVIN GLO 1E, previ-ously reported by Schreiner and Mihara (2009) in Oregonvineyards (Fig. S3). Few OTUs were found common tothe two considered vineyard soils, highlighting a very lowoverlap between the two AMF populations (Fig. 2A). Con-sidering the more representative OTUs, only one related

to G. irregulare (a generalist species) phylotype wasshared.

As expected, there is a poor overlap between the mor-phological and the molecular data. The lack of agreementbetween AMF spores and the molecular analysis in soilsis probably due to the fact that the primer couple used inthis work do not amplify genera such as Archeospora

Fig. 2. To verify the overlap between the soil AMF communities two sets of OTUs0.02 were generated, one with Lessona–Neive soil sequencespooled (A) and the second with those from Lessona soil and roots (B). Overlapping and exclusive OTUs were displayed by means ofrank-abundance diagrams (plotting the number of intra-OTU0.02 sequences as a function of the OTU rank). Singletons are not shown.

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and Paraglomus that were identified morphologically.However, in some cases we have found a correlationbetween spores and phylotypes such as in Neive for G.sinuosum. Interestingly, in Lessona site morphologicalanalysis revealed the presence of S. dipurpurescens,which it is suggested may be synonymous with Scutel-lospora calospora, which was identified by the molecularanalysis and BLAST searches. This result agrees withthose previously obtained both for spores in soils and inroots in Oregon vineyards as well as in Sardinia vineyardsoils (Lumini et al., 2010; Schreiner and Mihara, 2009).Although it has been demonstrated that the primers usedcan amplify Acaulospora species, the lack of sequencescorresponding to this genus in Lessona and Neive con-firms the results reported by Lumini and colleagues(2010), where the presence of Acaulospora sp. was foundonly in pasture and managed meadow and not associatedto Mediterranean vineyard soils. In addition, Schreinerand Mihara (2009) have suggested that fungi within thisfamily could be considered poor competitors (colonizers)in grape roots from vineyard samples. The fact thatmembers of the Acaulosporaceae have a low root and soilcolonization levels has been reported (Hart and Reader,2002; Hempel et al., 2007), suggesting that a differentcolonization strategy could be associated with differentglomeromycotan phylogenetic groups.

To compare the AMF communities outside and insidethe roots the same molecular approach was applied ongrape root collected in Lessona vineyard. A total of 192clones were sequenced and 133 sequences (69.27%),belonging to Glomeromycota, were used to generate atotal of 10 OTUs. Rarefaction curve, together with ACEand Chao1 indices, suggested that the number of theobserved OTUs is close to the estimate number, indicat-ing good sampling efficiency for AMF diversity of graperoots (Fig. S2B; Table 1). The 10 OTUs from roots wereall represented in soil (Figs 2B and 3), which, asexpected, maintain a higher AMF biodiversity than roots.By far the most abundant and diverse group in soiland roots samples was Glomus group A, in particularsequences belonging to Glomus group Ad and Glomusgroup Ab. Among them, sequences related to phylotypespreviously found in Pinot noir roots (Oregon vineyards)were identified: ORVIN GLO 3C, GLO3E, GLO2E.Glomus group B was only occasionally found in the rootsamples, while sequences belonging to Gigasporaceaehave been retrieved in the two compartments (soil androot). In particular, we have found sequences corre-sponding to S. dipurpurescens and related to the phylo-type ORVIN SCUT 1 identified as associated with graperoots in Oregon vineyards. The fact that AMF amplifiedfrom grape roots are clearly dominated by Glomus spp.

Fig. 3. Network by the SplitsTree4 V4.10 software (Hudson and Bryant, 2006) showing the phylogenetic relationships between the differentroots (10) and soil (33) OTUs of Lessona vineyard. The Kimura-2p method was used for the computation of distances with the referencessequences also included in the analyses. Green circles: OTUs from roots of grapevine. Red circles: OTUs from vineyard soil. White circles:glomeromycotan reference sequences from GeneBank. The dimension of dots is related to the number of sequences assigned for each OTU.

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is in agreement with the other paper published ongrape roots AMF communities in vineyards and withmany other AMF biodiversity studies in different ecosys-tems (Öpik et al., 2006; Alguacil et al., 2008; Schreinerand Mihara, 2009). In conclusion, the results obtained inthe Piedmont cover cropped vineyards, cultivated withthe same V. vinifera cv. Nebbiolo clone 308 (grafted on420A rootstock), suggest that the maintenance of adifferent AMF community seems to be affected by soilcharacteristics. For example, the presence of membersof the Diversisporales in Lessona is consistent with boththe ecology of this fungal group (thought to prefer sandysoils), and the edaphic features of this soil site. The rela-tionship between vineyard soils and AMF assemblagesfound in this work is not surprising since it has alreadyshown that the soil characteristics are an importantvariable influencing AMF communities (Helgason andFitter, 2009; Schreiner and Mihara, 2009). A recentpyrosequencing study on AMF communities in ‘Vermen-tino di Gallura’ Sardinia vineyards has shown that eachof the two considered vineyards has a peculiar AMFassemblage that seems to be related to the differentmanagement systems (tilled and no-tilled covercropped). However, a high number of common OTUswere shared between the two vineyards, which arecultivated close together on the same soil type. Theseresults demonstrate that when there is no differencein soil features, the composition of the AMF communitiesis similar and there is an overlapping in the two AMFassemblages (Lumini et al., 2010). All these data, includ-ing those obtained in this work, reinforce the concept thatthe general AMF assemblage structure and compositionin vineyards might be influenced primarily by soil typeand at less extent by host plant features (age, vegetativestages; Schreiner and Mihara, 2009) or managementpractices (Lumini et al., 2010). Interestingly, one of themost important difference between the two vineyard soilsis their pH (8.3 versus 4.8) that it has already reportedto be a likely driving force for structuring AM fungalcommunities (An et al., 2008; Helgason and Fitter, 2009for a review, Wang et al., 1985).

The results of this study, together with the few so farpublished on vineyard ecosystems, give us a chance toopen a window on reveal some of the factors responsiblefor the differences in AMF assemblages in this economi-cally important culture (http://faostat.fao.org). In future,the analyses of the AMF ‘gene bank’ sequences associ-ated with vineyards worldwide (South African, NewZealand, California, Argentina, etc.) could highlight on theAMF phylotypes most commonly found in vineyards. Theorganisms from which these phylotypes derive could thenbe species isolated and made available for research.Coming, as they would, from the low input viticulturesystems, the resultant cultures could then be tested as

candidates for application in reduced input cultivationsystems beyond those of V. vinifera.

Acknowledgements

The contributions to this work have been funded by theRegione Piemonte Tech4wine Project (CIPE 2006), ENDUREproject (European network of Excellence), IPP-CNR (AG-P02-006, Biodiversity project-Torino). C.W. was supported bya short mobility grant in the frame of the CNR InternationalExchange Program during his visit in Turin laboratories (May2008). We are grateful to Giulia Bughi Peruglia and RobertoOrrù for their help in molecular and morphological analyses.F.M. was supported by Tech4wine Project grant.

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Fig. S1. In this study we focused on the AM fungal biodiver-sity in two Mediterranean vineyards in the north-west and inthe south side of the Piedmont region of Italy. Lessona(45°33′17″E; 8°13′23″E; 260 m above sea level; A) andNeive (44°43′16″N; 8°4′59″E; 260 m above sea level; B) siteshave different pedoclimatic features but were stocked withthe same cultivar of Vitis vinifera (cv ‘Nebbiolo’ clone 308)grafted onto the same rootstock (420A). Four plants wererandomly chosen from each vineyard. From each plant, threesubsurface soil cores (5 cm Ø and 20 cm depth) were col-lected at 30 cm distance from stem in May 2008 to provide 24samples in total. DNA extraction from 0.5 g of soil sampleswas performed using a FastDNA Spin for Soil kit (Q-BIOgene, Heidelberg, Germany) according to a modified pro-tocol as reported by Luis and colleagues (2004). In theLessona vineyard, young root fragments from the sameplants were also collected. Two different root fragments(100 mg wet weight) from every plant were considered in theextraction step (DNeasy Plant Minikit; Quiagen, Hilden,Germany) and control PCR amplifications with V. vinifera-specific primers (GAPDH-f/GAPDH-r; Reid et al., 2006) wereperformed. To overcome the difficulties of mixed startingmaterial, species composition of AM fungi in soil and rootswas analysed by a nested PCR approach. The NS1/NS4universal primers (White et al., 1990) were used in the firstamplification followed by nested amplification with NS31/AMmix primers as described by Santos-Gonzalez and col-leagues (2007) to amplify a fragment of SSU rDNA (550 bp).Fig. S2. Rarefaction curves for the SSU rDNA sequencesfrom Lessona and Neive soils (2A) and from soil and roots ofLessona vineyard (2B) at similarity levels of 98%.Fig. S3. Phylogenetic tree of Lessona soil AMF communitiesbased on 550 bp of SSU rDNA sequences. Sequences werealigned by MUSCLE with those identified by the BLAST searchand with reference sequences of the major groups of Glom-eromycota. MEGA 4.0 software assessing Kimura-2p modelas distance method and 1000 replicates of non-parametricbootstrapping was used to construct a neighbour-joining (NJ)consensus tree. Bayesian phylogenetic inference analysiswas generated using MrBayes 3.1.1 (Ronquist and Huelsen-beck, 2003) with four simultaneous Markov chain MonteCarlo and over 500 000 generations for two runs. Contigsequences obtained for each OTU and 37 referencesequences obtained from GenBank have been used. Branchsupports were assigned as posterior probabilities on the con-sensus trees. The molecular evolutionary model for Bayesiananalyses was estimated with MrAIC.pl 1.4.3 (Nylander, 2004)

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resulting in GTR + I + G model. Contig sequences are indi-cates as LeS and classified in ascending order the LeS1being the most represented OTUs. Numbers in italics abovethe branches are bootstrap values from 1000 resamplingsdetermined for NJ analysis. Bold numbers above thebranches correspond to posterior probabilities derived fromBayesian analyses. The percentages of sequences clusteringwithin the main glomeromycotan groups are reported.Endogone pisiformis and Mortierella verticillata were used asoutgroups.Fig. S4. Phylogenetic tree of Neive soil AMF communitiesbased on 550 bp of SSU rDNA sequences. The consensustree derived from neighbour-joining (NJ) and Bayesian phy-logenetic inference analysis of contig sequences obtained foreach OTU and 37 references sequences obtained fromGenBank. Contig sequences are indicates as NeS and clas-sified in ascending order the NeS1 being most representedOTUs. Numbers in italics above the branches are bootstrapvalues from 1000 resamplings determined for neighbour-joining analysis based on Kimura 2-parameter genetic dis-tances. Bold numbers above the branches correspond to

posterior probabilities derived from Bayesian analyses. Thepercentages of sequences clustering within the main glom-eromycotan groups are reported. Endogone pisiformis andMortierella verticillata were used as outgroups.Table S1. Values of some soil properties in the two Piedmontvineyards.The Lessona site is a flat landscape in which grapevine is themain crop plant. This area is characterized by a temperateclimate with an annual rainfall average of approx. 1250 mmand an annual temperature average of 12–13°C. The soil wasclassified as an alfisol. In contrast, the Neive site is situatedin a typical hill landscape. The area is referred to as a xero-teric region with an annual rainfall average of 821 mm and anannual temperature average of 11°C. The soil was classifiedas an entisol.

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