Occurrence and richness of arbuscular mycorrizal fungi in vineyards with grapevine decline and dieback symptoms

Ocorrência e riqueza de fungos micorrízicos arbusculares em vinhedos com sintomas de declínio e morte da videira

Aelton dos Santos Bezerra Marcelo Betancur-Agudelo Edenilson Meyer Karl Kemmelmeier Sidney Luiz Stürmer Cláudio Roberto Fonsêca Sousa Soares Paulo Emilio Lovato About the authors

ABSTRACT:

This research identified arbuscular mycorrhizal fungi (AMF) in rhizosphere soil of grapevines with Grapevine Death and Decline symptoms (GDD) or asymptomatic healthy (H) plants, and characterized the relationship of AMF communities with soil chemical attributes. The AMF spore number ranged from 287 to 432 spores 50 cm-3 in soil with GDD plants, and from 357 to 464 spores 50 cm-3 in H plants, with no differences among vineyards or between GDD and H plants within each vineyard. We detected 42 species and 17 genera, and most taxa belonged to Acaulosporaceae or Glomeraceae. Claroideoglomus etunicatum, Funneliformis mosseae, and Archaeospora trappei were the most frequent species in all vineyards. Soil chemical attributes were not determinant for the occurrence of most fungal species; although, Entrophospora infrequens, Diversispora sp1 and Diversispora sp2 were associated with a vineyard having high soil copper. Vineyards harbor highly diverse AMF communities, which are determined by location.

Key words:
community structure; glomeromycota; soil factors; Vitis

RESUMO:

Este trabalho teve como objetivo identificar fungos micorrízicos arbusculares (FMA) em solo rizosférico de videiras com sintomas de declínio e morte da videira (D) e em plantas saudáveis (S), e caracterizar a relação das comunidades de FMA com atributos químicos do solo. O número de esporos de FMA variou de 287 a 432 esporos 50 cm-3 em solo em plantas D, e de 357 a 464 esporos 50 cm-3 em plantas S, sem diferenças entre vinhedos ou entre plantas D e S dentro de cada vinhedo. Detectamos 42 espécies e 17 gêneros, sendo que a maioria dos táxons pertencia a Acaulosporaceae ou Glomeraceae. Claroideoglomus etunicatum, Funneliformis mosseae e Archaeospora trappei foram as espécies mais frequentes em todos os vinhedos. Os atributos químicos do solo não foram determinantes para a ocorrência da maioria das espécies de fungos, embora Entrophospora infrequens, Diversispora sp1 e Diversispora sp2 estivessem associados a um vinhedo com alto teor de cobre do solo. Os vinhedos abrigam comunidades FMA altamente diversificadas, que são determinadas pela localização.

Palavras-chave:
estrutura da comunidade; glomeromycota; fatores do solo; Vitis

INTRODUCTION:

Expansion and renewal of vineyards are constrained by factors such as relief, land costs, and pests and diseases, which can affect the emergence of grapevine decline and dieback (GDD) (BASSO et al., 2017BASSO, M. F.; et al. Grapevine virus diseases: economic impact and current advances in viral prospection and management. Revista Brasileira de Fruticultura , v.39, n.1, p.1-22, 2017. Available from: <Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-29452017000104001 >. Accessed: Dec. 28, 2020. doi: 10.1590/0100-29452017411.
https://www.scielo.br/scielo.php?script=...
). Different pathogens may cause early GDD (VALENCIA et al., 2015VALENCIA, D.; et al. Dissemination of Botryosphaeriaceae conidia in vineyards in the semiarid Mediterranean climate of the Valparaíso Region of Chile. Phytopathologia Mediterranea . v.54, n.4, p.394-402, 2015. Available from: <Available from: https://oajournals.fupress.net/index.php/pm/article/view/5629 >. Accessed: Dec. 28, 2020. doi: 10.14601/Phytopathol_Mediterr-16055.
https://oajournals.fupress.net/index.php...
), which affect trunk, leaves, or fruits (BERTSCH et al., 2012BERTSCH, C.; et al. Grapevine trunk diseases: Complex and still poorly understood. Plant Pathology. v. 62, p.243-265, 2012. Available from: <Available from: https://bsppjournals.onlinelibrary.wiley.com/doi/full/10.1111/j.1365-3059.2012.02674.x >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1365-3059.2012.02674.x.
https://bsppjournals.onlinelibrary.wiley...
). Plants affected by GDD have low vigor, internerval leaf chlorosis, and weak, uneven branches (MENEZES-NETTO et al., 2016MENEZES-NETTO, A. C.; et al. Declínio e morte de videiras no estado de Santa Catarina: causas e alternativas de controle. Boletim Técnico. 175, 81p, 2016. Available from: <Available from: https://publicacoes.epagri.sc.gov.br/index.php/BT/article/view/422 >. Accessed: Dec. 28, 2020.
https://publicacoes.epagri.sc.gov.br/ind...
). The plant vegetative stage affects the symptoms since grapevines are more susceptible to onset of GDD at the beginning of the fruiting stage (AL-MAWAALI et al., 2013AL-MAWAALI, Q.; et al. Etiology, development and reaction of muskmelon to vine decline under arid conditions of Oman. Phytopathologia Mediterranea, v.52, n.03, p.457-465, 2013. Available from: <Available from: https://oajournals.fupress.net/index.php/pm/article/view/5534 >. Accessed: Dec. 28, 2020. doi: 10.14601/Phytopathol_Mediterr-11673.
https://oajournals.fupress.net/index.php...
). As a result, grapevines are deficient in potassium, magnesium, phosphorus, sulfur, and often have high leaf copper (TERRA et al., 2003TERRA, M. M.; et al. Avaliação do estado nutricional da videira ‘Itália’ na região de Jales, SP, usando o sistema integrado de diagnose e recomendação. Revista Brasileira de Fruticultura . v.25, n.2, p.309-314, 2003. Available from: <Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-29452003000200032 >. Accessed: Dec. 28, 2020. doi: 10.1590/S0100-29452003000200032.
https://www.scielo.br/scielo.php?script=...
). GDD results in low plant stand, and plants die before the investment in vineyard installation is recovered. Research on this subject in Brazil is scant and mostly focused on pathogenic fungi in the country’s southern region (GARRIDO et al., 2004GARRIDO, L., DA R.; et al. Fungi associated with grapevine showing decline and plant death in the state of Rio Grande do Sul, Southern Brazil. Fitopatologia Brasileira. v.29, n.3, p.322-324, 2004. Available from: <Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-41582004000300016 >. Accessed: Dec. 28, 2020. doi: 10.1590/S0100-41582004000300016.
https://www.scielo.br/scielo.php?script=...
; DAMBROS et al., 2016DAMBROS, R. N.; et al. Control of grapevine decline with the use of drains and ridges. Revista Brasileira de Fruticultura . v.38, n.2, p.1-7, 2016. Available from: <Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-29452016000200402&lng=en&tlng=en >. Accessed: Dec. 28, 2020. doi: 10.1590/0100-29452016448.
https://www.scielo.br/scielo.php?script=...
; MENEZES-NETTO et al., 2016). Among the procedures proposed to solve GDD problems, and the association between arbuscular mycorrhizal fungi (AMF) and grapevines is a possibility.

Mycorrhizas have a fundamental role in plant survival, growth, and development (SMITH & READ, 2008SMITH, S. E.; READ, D. J. Mycorrhiza l symbiosis. San Diego: Academic Press. 605 p. 3. ed. 2008. Available from: <Available from: https://www.sciencedirect.com/book/9780123705266/mycorrhizal-symbiosis >. Acessed: Dec. 28, 2020.
https://www.sciencedirect.com/book/97801...
). Arbuscular mycorrhizal fungi (AMF) are obligate symbiotrophs, associating with around 72% of plant species worldwide (BRUNDETT & TEDERSOO, 2018BRUNDETT, M. C.; TEDERSOO, L. Evolutionary history of mycorrhizal symbioses and global host plant diversity. New Phytologist. v.220, p.1108-1115, 2018. Available from: <Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14976 >. Accessed: Dec. 28, 2020. doi: 10.1111/nph.14976.
https://nph.onlinelibrary.wiley.com/doi/...
). AMF may reduce GDD occurrence and enhance plant growth (TROUVELOT et al., 2015TROUVELOT, S.; et al. Arbuscular mycorrhiza symbiosis in viticulture: a review. Agronomy for Sustainable Development. v.35, n.4, p.1449-1467, 2015. Available from: <Available from: https://www.researchgate.net/publication/282400867_Arbuscular_mycorrhiza_symbiosis_in_viticulture_a_review >. Accessed: Dec. 28, 2020. doi: 10.1007/s13593-015-0329-7.
https://www.researchgate.net/publication...
). GDD may also affect AMF root colonization, suggesting a complex relationship of these symbiotic fungi with other GDD causes (SCHREINER, 2003SCHREINER, R. P. Mycorrhiza l Colonization of Grapevine Rootstocks under Field Conditions. American Journal of Enology and Viticulture. v.54. p.143-149, 2003. Available from: <Available from: https://www.researchgate.net/publication/50993895_Mycorrhiza l_Colonization_of_Grapevine_Rootstocks_under_Field_Conditions >. Accessed: Apr. 12, 2021.
https://www.researchgate.net/publication...
; WASCHKIES et al., 1994WASCHKIES, C., et al. Relations between grapevine replant disease and root colonization of grapevine (Vitis sp.) by fluorescent pseudomonads and endomycorrhizal fungi. Plant Soil . v.162, p.219-227, 1994. Available from: <Available from: https://link.springer.com/article/10.1007%2FBF01347709#citeas >. Accessed: Apr. 11, 2021. doi: 10.1007/BF01347709.
https://link.springer.com/article/10.100...
). Mycorrhizas promote tolerance or resistance to pathogenic fungi through improved plant nutrition, compensation of root damage, competition for plant photosynthates or root colonization points, and activation of plant defense mechanisms (AZCON-AGUILAR & BAREA, 1996AZCON-AGUILAR, C.; BAREA, J. M. Arbuscular mycorrhizas and biological control of soil-borne plant pathogens - an overview of the mechanisms involved. Mycorrhiza. v.6, p.457-464, 1996. Available from: <Available from: https://link.springer.com/article/10.1007/s005720050147#citeas >. Accessed: Dec. 28, 2020. doi: 10.1007/s005720050147.
https://link.springer.com/article/10.100...
).

Measures to mitigate losses caused by GDD include use of resistant varieties or grafting. However, such practices are not enough to solve the problems. One possibility is inoculation with AMF isolated from vineyards, and a first step to develop this approach is to inventory the AMF communities associated with grapevines in field conditions (SILVEIRA, 2006). This research is a first step to identify AMF species occurring in areas with GDD to isolate them in single cultures for further testing under controlled conditions. We characterized AMF communities present in soils of grapevines with and without symptoms of GDD and investigate the effect of soil attributes on those AMF communities.

MATERIALS AND METHODS:

Field sampling

Soil samples were collected in four vineyards, with different ages and management histories, in the Rio do Peixe Valley, state of Santa Catarina, Brazil, a region responsible for most of the state grapevine production (BACK et al. 2013BACK, Á. J.; et al. Climate changes and grape production in Vale do Rio do Peixe, in the state of Santa Catarina. Revista Brasileira de Fruticultura, v.35, n.1, p.159-169, mar. 2013. Available from: <Available from: https://doi.org/10.1590/S0100-29452013000100019 >. Accessed: Jun. 14, 2021.
https://doi.org/10.1590/S0100-2945201300...
). Vineyards were located in the municipalities of Videira (V1 and V4), Pinheiro Preto (V2), and Tangará (V3) (Table 1). The region has two types of climate: humid mesothermal subtropical with hot summers (Cfa), and humid mesothermal subtropical with mild summers (Cfb), with annual rainfall ranging from 1.300 mm to 1.900 mm (PANDOLFO et al., 2002PANDOLFO, C.; et al. Atlas Climatológico do Estado de Santa Catarina. Florianópolis: Epagri. CD-ROM. 13 p.2002. Available from: <Available from: https://ciram.epagri.sc.gov.br/ciram_arquivos/atlasClimatologico/atlasClimatologico.pdf >. Accessed: Dec. 28, 2020.
https://ciram.epagri.sc.gov.br/ciram_arq...
).

Table 1
General information on four vineyards in the Rio do Peixe Valley, Santa Catarina,, Brazil.

All vineyards have conventional management system (not organic), using black oats as winter cover crops. The soil was sampled according to the Tropical Soil Biology and Fertility methodology (MOREIRA et al., 2008MOREIRA, F. M. S.; et al. A Handbook of tropical soil biology: sampling and characterization of below-ground biodiversity. 1ed. Earthscan., London, p.17-42, 2008. Available from: <Available from: https://www.researchgate.net/publication/259332766_A_Handbook_of_Tropical_Soil_Biology_Sampling_and_Characterization_of_Below-Ground_Biodiversity >. Accessed: Dec. 28, 2020.
https://www.researchgate.net/publication...
). In each vineyard, 14 samples were collected, seven around plants with GDD symptoms (GDD), and seven from asymptomatic, presumably healthy (H) plants. The symptoms of decline observed in plants were physiological decay, apparently caused by fungi, viruses, and other agents. At each point with an H or GDD plant, a central plant was georeferenced, and soil subsample (0-20 cm deep) was collected from six points around each plant: three sub-samples were collected at a 3.0-m distance from the central plant, and three other sub-samples collected 6.0 m away from the central plant. Those six sub-samples from each plant were pooled into a composite sample with approximately 1.0 kg of soil, kept at 4 °C until processing. In total, 56 samples were collected in the vineyards, 28 of healthy plants, and 28 of plants that presented GDD symptoms.

Soil fertility analysis

Soil fertility analyses followed the methods of EMBRAPA (1997EMBRAPA - Empresa Brasileira de Pesquisa Agropecuária. Centro Nacional de Pesquisa de Solos. Manual de métodos de análise de solo. Rio de Janeiro. Embrapa. 2 ed. 212p. 1977. Available from: <Available from: https://www.agencia.cnptia.embrapa.br/Repositorio/Manual+de+Metodos_000fzvhotqk02wx5ok0q43a0ram31wtr.pdf >. Accessed: Dec. 28, 2020.
https://www.agencia.cnptia.embrapa.br/Re...
). Active acidity was measured as pH in water 1:1 (v:v). Potassium (K), copper (Cu), and phosphorus (P) were extracted with Mehlich I solution and P was measured in UV-visible spectrometry in HCl 0.87 M and (NH4)6Mo7O24 4H20 solutions, and K was measured by flame photometry. Exchangeable aluminum (Al), calcium (Ca), and magnesium (Mg) were extracted with KCl 1 mol L-1 and quantified by atomic absorption spectrophotometry. Potential acidity (H++Al3+) was determined with calcium acetate buffered to pH 7.0 and determined volumetrically with NaOH. Resin-extracted phosphorus (Pr) was extracted with an anion exchange resin, colored with ammonium molybdate, and PC reducing solution (1-amino-2naphthol-4sulfonic acid, sodium sulfite, and sodium metabisulfite), and measured by UV-visible spectrometry (TEDESCO et al., 1995TEDESCO, M. J.; et al. Análises de solo, plantas e outros materiais. Boletim Técnico . Porto Alegre, Universidade Federal do Rio Grande do Sul. n.5, 174p, 1995. Available from: <https://www.bdpa.cnptia.embrapa.br/consulta/busca?b=ad&id=502314&biblioteca=vazio&busca=autoria:%22TEDESCO,%20M.J.%22&qFacets=autoria:%22TEDESCO,%20M.J.%22&sort=&paginacao=t&paginaAtual=2>. Accessed: Dec. 28, 2020.).

Morphological identification of AMF

A 50-cm³ volume of soil from each field sample was used to extract AMF spores by wet sieving (GERDEMANN & NICOLSON, 1963GERDEMANN, J. W.; NICOLSON, T. H. Spores of mycorrhizal endogone species extracted from soil by wet sieving and decanting. Transactions of the British Mycological Society. v.46, ed.2, p.235-244, 1963. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0007153663800790 >. Accessed: Dec. 28, 2020. doi: 10.1016/S0007-1536(63)80079-0.
https://www.sciencedirect.com/science/ar...
), followed by centrifugation in a 60% sucrose solution (JENKINS, 1964JENKINS, W. R. A rapid centrifugal floatation technique for separating nematodes from soil. Plant Disease Reporter. v.48, n.9, p.692-694, 1964. Available from: <Available from: https://www.cabdirect.org/cabdirect/abstract/19650801105 >. Accessed: Dec. 28, 2020.
https://www.cabdirect.org/cabdirect/abst...
). The supernatant was poured into stacked sieves of 180, 90, and 45 μm. Spores of each size class were placed on a microscope slide using polyvinyl alcohol-lactic acid-glycerol (PVLG) and PVLG + Melzer reagent as mounting media. Taxonomic identification of species was based on morphological descriptions available at the INVAM website (International Culture Collection of Arbuscular Mycorrhizal Fungi - http://invam.wvu.edu) and Blaszkowski (2012BLASZKOWSKI, J. Glomeromycota: W. Szafer Institute of Botany, Polish Academy of Sciencess, Kraków. 303 p. 2012.). We followed the classification proposed by REDECKER et al. (2013REDECKER, D.; et al. An evidence-based consensus for the classification of arbuscular mycorrhizal fungi (Glomeromycota). Mycorrhiza . v.23, n.7, p.515-531, 2013. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23558516/ >. Accessed: Dec. 28, 2020. doi: 10.1007 / s00572-013-0486-y.
https://pubmed.ncbi.nlm.nih.gov/23558516...
). AMF species identified in vineyards were registered in the National Genetic Resource Data Base (SisGEen - Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado), under number A9FA3BC.

Trap cultures

An aliquot of about 400g of each sample was used to establish trap cultures, according to MORTON et al. (1993MORTON, J. B.; BENTIVENGA, S. P. Germplasm in the international collection of Arbuscular and Vesicular arbuscular mycorrhizal fungi (INVAM) and procedures for culture development, documentation, and storage. Mycotaxon. v.48, n.1, p.491-528, 1993. Available from: <Available from: https://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=3811986 >. Accessed: Dec. 28, 2020. issn: 0093-4666.
https://pascal-francis.inist.fr/vibad/in...
). Each soil sample was placed in a 1.5 dm3 pot containing 40% soil collected in the field, 30% vermiculite, and 30% sterile sand, with Urochloa decumbens as the host plant. One month after seeding, plants were pruned to cause physiological disturbance and increase tillering. Plants were irrigated three times a week during the first two months, and after that establishment period, submitted to five-day periods without watering to stimulate AMF root colonization and sporulation. After five months, irrigation was suspended, and after another month, spores were extracted, mounted on slides, and identified as previously described.

Analysis of AMF species richness

Species richness was calculated as the number of AMF species identified from field samples and trap culture. The frequency of occurrence (F) was calculated by the equation F = (Ji/K)*100, where F is the frequency of species I, Ji is the number of samples in which the species was detected, and K is the total number of samples. Species frequency was classified as dominant (85% ≤ FO ≤ 100%), very common (50% ≤ FO < 85%), common (30% ≤ FO < 50%) and rare (FO < 30%) according to ZHANG et al. (2004ZHANG, Y.; et al. Survey of arbuscular mycorrhizal fungi in deforested and natural forest land in the subtropical region of Dujiangyan, southwest China. Plant and Soil. v.261, p.257-263, 2004. Available from: <Available from: https://link.springer.com/article/10.1023/B:PLSO.0000035572.15098.f6 >. Accessed: Dec. 28, 2020. doi: 10.1023/B:PLSO.0000035572.15098.f6.
https://link.springer.com/article/10.102...
).

Statistical analyses

Similarity among AMF communities from the four vineyards was estimated by the non-metric multidimensional scale (NMDS) based on Jaccard’s index, as described in CLARKE (1993CLARKE, K. R. Non-parametric multivariate analysis of changes in community structure. Australian Journal of Ecology. v.18, ed.1, p.117-143, 1993. Available from: <Available from: https://onlinelibrary.wiley.com/doi/10.1111/j.1442-9993.1993.tb00438.x >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1442-9993.1993.tb00438.x.
https://onlinelibrary.wiley.com/doi/10.1...
). The effects of the vineyard soil chemical attributes on AMF communities were analyzed by Canonical Redundancy Analysis - RDA (BORCARDT et al., 2011BORCARDT, D.;et al. Numerical Ecology with R., New York: Dordrecht London Heidelberg, p.234-240, 2011.). Those analyses were performed using the Vegan package in the Program R studio 3.1.4 (OKSANEN et al., 2013OKSANEN, J.; et al. Package ‘vegan’. R Packag. 261 p. 2013. Available from: <Available from: https://cran.r-project.org/web/packages/vegan/vegan.pdf >. Accessed: Dec. 28, 2020.
https://cran.r-project.org/web/packages/...
).

RESULTS:

Soil attributes varied among sites. Soil pH ranged from 5.1 to 6.5, and the V3 and V4 vineyards had values of 5.7 and 5.1 (Table 2), respectively, below the desirable value of 6.0 (Comissão de Química e Fertilidade do Solo, 2016COMISSÃO DE QUÍMICA E FERTILIDADE DO SOLO (CQFS-RS/SC). Manual de calagem e adubação para os estados do Rio Grande do Sul e Santa Catarina. Viçosa, Sociedade Brasileira de Ciência do Solo. 2016. 376p. Available from: <Available from: http://www.sbcs-nrs.org.br/docs/Manual_de_Calagem_e_Adubacao_para_os_Estados_do_RS_e_de_SC-2016.pdf >. Accessed: Jun. 16, 2021.
http://www.sbcs-nrs.org.br/docs/Manual_d...
). According to those regional criteria, exchangeable K was high or very high. Ca, Mg, Cu, organic matter, CEC, and Melich-extracted P were high for all areas, while resin-extracted P ranged from high to very high. The highest soil Cu concentration occurred in V3, the oldest vineyard.

Table 2
Soil chemical attributes in four vineyards in the Rio do Peixe Valley, Santa Catarina, Brazil.

Mean number of AMF spores ranged from 287 to 432 spores per 50 cm3 in soils with GDD plants and between 357 and 464 spores per 50 cm3 in asymptomatic plants (Figure 1). No differences in spore number were detected among vineyards nor between asymptomatic and GDD plants.

Figure 1
AMF spores 50 cm-3 of soil around grapevines with or without Grapevine Decline and Dieback (GDD) symptoms. V1 - V4: vineyard; GDD: plants with GDD symptoms; H: asymptomatic plants. Bars represent standard error of the mean.

Morphological characterization of AMF spores yielded 42 species and 17 genera belonging to Acaulosporaceae, Ambisporaceae, Archaeosporaceae, Claroideoglomeraceae, Diversisporaceae, Gigasporaceae, Glomeraceae, and Paraglomeraceae (Table 3). Entrophospora infrequens was detected but not allocated to any family, following REDECKER et al. (2013REDECKER, D.; et al. An evidence-based consensus for the classification of arbuscular mycorrhizal fungi (Glomeromycota). Mycorrhiza . v.23, n.7, p.515-531, 2013. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23558516/ >. Accessed: Dec. 28, 2020. doi: 10.1007 / s00572-013-0486-y.
https://pubmed.ncbi.nlm.nih.gov/23558516...
). The highest number of AMF species occurred V1 and V4 vineyards, with 31 and 30 species, while V2 and V3 had the lowest numbers, with 26 and 28 species. The families with the highest species richness (66% of all morphotypes recovered) were Glomeraceae and Acaulosporaceae, represented by 20 and 8 species. We detected 24 morphotypes in both trap cultures and field samples, and 15 morphotypes were reported only in field samples. Morphotypes detected exclusively in trap cultures were Glomus sp3 and Glomus sp4 from V4 and Sclerocystis sp1 from V1. The most frequent species were Claroideoglomus etunicatum, Funneliformis mosseae, and Archaeospora trappei, with global frequencies of 93, 89, and 84%, respectively. Those species were dominant or very common in soil from both GDD- and H-plants. Thirty-seven species (88% of the total richness) were shared by soils with GDD and H-plants (Figure 2). Species associated exclusively with H-plants were Acaulospora alpina, A. foveata, Rhizophagus fasciculatus, and Sclerocystis sp1, while the only exclusive species in GDD-plants was Oehlia diaphana (Table 3).

Figure 2
Venn diagram of the presence of AMF species in the soil around grapevines with Grapevine Decline and Dieback symptoms (GDD) or asymptomatic (H) in four vineyards (V1 to V4).

Table 3
Occurrence frequency and global frequency (F%) of arbuscular mycorrhizal fungus species in soil around grapevines with or without Grapevine Decline and Dieback (GDD) symptoms in four vineyards (V1 to V4).

The principal coordinate analysis (PCoA) indicated that vineyards separate AMF communities and that occurrence of AMF species within each vineyard did not differ between soils with H- and GDD-plants (Figure 3). The PERMANOVA demonstrated differences among vineyards (Table 4), confirming the PCoA findings.

Distance-based redundancy analysis (dbRDA) (Figure 4) showed that soil attributes were strongly correlated. Such was the case of organic matter with V4, K, and resin-extracted phosphorus (P.r) with V1 and V2, while Cu, pH, Ca, and Mg are related to V3 (Table 5). Part of the AMF species showed positive correlations with some soil attributes. Organic matter was positively related to the presence of Gigaspora margarita and Glomus microaggregatum, while Cu concentration had a positive correlation with Entrophospora infrequens, Diversispora sp2, and Diversispora sp3. The variables Mg, Ca, pH, and Cu showed a negative correlation with Gigaspora margarita and Glomus microaggregatum, and K and P.r had a negative correlation with Entrophospora infrequens, Diversispora sp2, and Diversispora sp3.

Figure 3
Principal coordinate analysis (PCoA) using a similarity coefficient of Jaccard for the presence-absence data of arbuscular mycorrhizal fungal species in the soil around grapevines with (GDD) or without (H) Grapevine Decline and Dieback (GDD) symptoms in four vineyards (V1 to V4).

Table 4
Permanova using Jaccard similarity coefficient for presence-absence data of AMF species in soil around grapevines with or without Grapevine Decline and Dieback (GDD) symptoms in four vineyards (V1 to V4).

Figure 4
Distance-based redundancy analysis (dbRDA) between soil chemical attributes and occurrence of arbuscular mycorrhizal fungi (AMF) species in the soil around grapevines with (D) or without (H) Grapevine Decline and Dieback (GDD) symptoms in four vineyards (V1 to V4).

Table 5
Eigenvalues explained variability and dbRDA analysis coordinates for chemical attributes of soil around grapevines with or without Grapevine Decline and Dieback (GDD) symptoms, in four vineyards (V1 to V4).

DISCUSSION:

This research is the first systematic survey of AMF communities in vineyards with plants showing symptoms of GDD and asymptomatic plants in southern Brazil. We based our inventory of AMF communities solely on morphological identification of spores from field samples and trap cultures, which may have limitations. Some studies showed that molecular approaches revealed different species composition in comparison to analyses of spores, roots or soil (HEMPEL et al., 2007HEMPEL, S.; et al. Differences in the species composition of arbuscular mycorrhizal fungi inspore, root and soil communities in a grassland ecosystem. Environ Microbiol. v.9, n.8, p.1930-1938, 2007. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/17635540/ >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1462-2920.2007.01309.x.
https://pubmed.ncbi.nlm.nih.gov/17635540...
). However, assessment of AMF community using spore morphology may reveal a higher number of species than molecular approaches (VIEIRA et al., 2018VIEIRA, C. K.; et al. Morphological and molecular diversity of arbuscular mycorrhizal fungi in revegetated iron-mining sites has the same magnitude of adjacent pristine ecosystems. Journal of Environmental Sciences. v.67, p.330-343, 2018. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S1001074217313529 >. Accessed: Dec. 28, 2020. doi: 10.1016/j.jes.2017.08.019.
https://www.sciencedirect.com/science/ar...
). Our results evidenced that AMF community composition between GDD and H were highly similar, and Claroideoglomus etunicatum, Funneliformis mosseae, and Archaeospora trappei were the most frequent fungi recovered in both types of vineyards. These results suggested grapevine physiological conditions do not determine AMF community composition and structure, which are more affected by the vineyard location.

AMF spore numbers were higher than in vineyards in the Brazilian Northeast (FREITAS et al., 2011FREITAS, N. D. O.; YANO-MELO, A. M. Soil biochemistry and microbial activity in vineyards under conventional and organic management at Northeast Brazil. Scientia Agricola. v.68, n.2, p.223-229, 2011. Available from: <Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-90162011000200013 >. Accessed: Dec. 28, 2020. doi: 10.1590/S0103-90162011000200013.
https://www.scielo.br/scielo.php?script=...
), in an arid climate, but they were lower than in Italian vineyards under Mediterranean climate (NAPPI et al., 1985NAPPI, P.; et al. Grapevine root system and VA mycorrhizae in some soils of Piedmont (Italy). Plant Soil. v.85, p.205-210, 1985. Available from: <Available from: https://link.springer.com/article/10.1007/BF02139624 >. Accessed: Dec. 28, 2020. doi: 10.1007/BF02139624.
https://link.springer.com/article/10.100...
). In V3-GDD plants, which had the lowest spore density, the value is 2.5 times higher than in organic and conventional vineyards in the same region, which had 115 and 45 spores 50 cm3 (BETTONI et al., 2016BETTONI, J. C.; et al. Colonização micorrízica de videiras cultivadas em sistemas orgânico e convencional no estado de Santa Catarina. Revista Agropecuária Catarinense. v.29, n.1, p.45-48, 2016. Available from: <Available from: https://publicacoes.epagri.sc.gov.br/index.php/RAC/article/view/310 >. Accessed: Dec. 28, 2020. issn: 6076.
https://publicacoes.epagri.sc.gov.br/ind...
). Several factors can impact AMF sporulation under field conditions, including moisture, soil factors, grapevine variety, and sampling season (JHA & SONGACHAN, 2020JHA, S. S.; L. S. SONGACHAN, L. S. Research on diversityand community composition of Arbuscular Mycorrhiza l Fungi species in India: A review. Plant Archives. v.20, n.2, p.4201-4226, 2020. <http://plantarchives.org/20-2/4201-4226%20(7134).pdf>. Accessed: Apr. 04, 2021.
http://plantarchives.org/20-2/4201-4226%...
). We sampled vineyards in Autumn, which has a large thermal amplitude and is linked to physiological and nutritional changes in grapevines that seem to favor AMF sporulation (RABATIN, 1979RABATIN, S. C. Seasonal and edaphic variation in vesicular-arbuscular mycorrhizal infection of grasses by Glomus tenuis. New Phytologist . v.83, ed.1, p.95-102, 1979. Available from: <Available from: https://nph.onlinelibrary.wiley.com/doi/abs/10.1111/j.1469-8137.1979.tb00730.x >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1469-8137.1979.tb00730.x.
https://nph.onlinelibrary.wiley.com/doi/...
). Although, spore production may differ with grapevine varieties (KARAGJANNIDIS et al., 1997KARAGJANNIDIS, N.; et al. Root colonization and spore population by VA-mycorrhizal fungi in four grapevine rootstocks. Vitis. v.36, n.2, p.57-60, 1997. Available from: <Available from: https://ojs.openagrar.de/index.php/VITIS/article/view/4856 >. Accessed: Dec. 28, 2020. doi: 10.5073/vitis.1997.36.57-60.
https://ojs.openagrar.de/index.php/VITIS...
), we ruled out this factor since two rootstocks (VR 043-43 and Paulsen-1103) were evaluated and we detected no differences between them. Soil P and organic matter were high in all vineyards (Table 2), which can affect root colonization and C allocation to spore production (FREITAS et al., 2011). Sporulation usually increases after phosphate application, an effect associated with P tolerance of some AMF species (SYLVIA & SCHENCK, 1983SYLVIA, D. M.; SCHENCK, N. C. Application of superphosphate to mycorrhizal plants sitimulates sporulation of phosphorus tolerant versicular arbuscular my corrhizal fungi. New Phytologist . v.95, p.655-661, 1983. Available from: <Available from: https://nph.onlinelibrary.wiley.com/doi/abs/10.1111/j.1469-8137.1983.tb03529.x >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1469-8137.1983.tb03529.x.
https://nph.onlinelibrary.wiley.com/doi/...
), and organic matter also promotes sporulation in vineyards soils (FREITAS et al., 2011).

Regardless of vineyard location and plant health, Claroideoglomus etunicatum, Funneliformis mosseae, and Archaeospora trappei were the most frequent species. We attributed the dominance of these species to their life strategies as they can be considered r-strategists. Claroideoglomus etunicatum has an extensive production of small spores in a short time (PINHEIRO et al., 2019PINHEIRO, E. M.; et al. Arbuscular mycorrhizal fungi in seedling formation of barbados cherry (Malpighia emarginata D.C). Revista Caatinga. v.32, n.2, p.370-380, 2019. Available from: <Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S1983-21252019000200370 >. Accessed: Dec. 28, 2020. doi: 10.1590/1983-21252019v32n210rc.
https://www.scielo.br/scielo.php?script=...
), Funneliformis mosseae rapidly colonizes ruderal habitats (SÝKOROVÁ et al., 2007SÝKOROVÁ, Z.; et al. The cultivation bias: different communities of arbuscular mycorrhizal fungi detected in roots from the field, from bait plants transplanted to the field, and from a greenhouse trap experiment. Mycorrhiza . v.18, p.1-14, 2007. Available from: <Available from: https://link.springer.com/article/10.1007/s00572-007-0147-0 >. Accessed: Dec. 28, 2020. doi: 10.1007/s00572-007-0147-0.
https://link.springer.com/article/10.100...
), while Archaeospora trappei seems to have a sporulation-based strategy to survive in extreme environments (SPAIN, 2003SPAIN, J. L. Emendation of Archaeospora and of its type species, Archaeospora trappei. Mycotaxon . v.87, p.109-112, 2003. Available from: <Available from: https://www.researchgate.net/publication/292229654_Emendation_of_Archaeospora_and_of_its_type_species_Archaeospora_trappei >. Accessed: Dec. 28, 2020.
https://www.researchgate.net/publication...
; OEHL & CHISTIAN, 2014OEHL, F.; CHRISTIAN, K. Multiple mycorrhization at the coldest place known for Angiosperm plant life. Alpine Botany. v.124, p.193-198, 2014. Available from: <Available from: https://link.springer.com/article/10.1007/s00035-014-0138-7#citeas >. Accessed: Dec. 28, 2020. doi: 10.1007/s00035-014-0138-7.
https://link.springer.com/article/10.100...
). These generalist species are usually ubiquitous and associate strongly with highly disturbed sites (OEHL et al., 2010OEHL, F.; et al. Soil type and land use intensity determine the composition of arbuscular mycorrhizal fungal communities. Soil Biology and Biochemistry. v.42, ed.5, p.724-738, 2010. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0038071710000234 >. Accessed: Dec. 28, 2020. doi: 10.1016/j.soilbio.2010.01.006.
https://www.sciencedirect.com/science/ar...
), and their life strategies include traits such as fast spore germination and mycelium extension (CANO & BAGO, 2005CANO, C.; BAGO, A. Competition and substrate colonization strategies of three polyxenically grown arbuscular mycorrhizal fungi. Mycologia. v.97, n.6, p.1201-1214, 2005. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/16722214/ >. Accessed: Dec. 28, 2020. doi: 10.3852/ mycologia.97.6.1201.
https://pubmed.ncbi.nlm.nih.gov/16722214...
). AMF species vary in carbon demand, which may define life strategies, interspecific variation, and occurrence of functional trade-offs (CHAGNON et al., 2013CHAGNON, P.; et al. A trait-based framework to understand life history of mycorrhizal fungi. Trends in Plant Science. v.18, ed.9, p.484-491, 2013. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S1360138513000885 >. Accessed: Dec. 28, 2020. doi: 10.1016/j.tplants.2013.05.001.
https://www.sciencedirect.com/science/ar...
; HART et al., 2001HART, M. M.; READER, R. J. Life-history strategies of arbuscular mycorrhizal fungi in relation to their successional dynamics. Mycologia . v.93, n.3, p.1186-1194, 2001. Available from: <Available from: https://www.jstor.org/stable/3761678?seq=1 >. Accessed: Dec. 28, 2020. doi: 10.2307/3761678.
https://www.jstor.org/stable/3761678?seq...
). Changes in growth rates in the microbial community by nutrient availability may reflect changes to a predominance by either r-type or K-type AMF (BLAGODATSKAYA et al., 2007BLAGODATSKAYA, E. V.; et al. Priming effects in Chernozem induced by glucose and N in relation to microbial growth strategies. Applied Soil Ecology. v.37, ed.1-2, p.95-105, 2007. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0929139307000704 >. Accessed: Dec. 28, 2020. doi: 10.1016/j.apsoil.2007.05.002.
https://www.sciencedirect.com/science/ar...
). Interestingly, F. mosseae and A. trappei were also detected in cohorts of AMF in grapevines from Italy (BALESTRINI et al., 2010BALESTRINI, R.; et al. Cohorts of arbuscular mycorrhizal fungi (AMF) in Vitis vinifera, a typical Mediterranean fruit crop. Environ. Microbiol. Rep. v.2, n.4, p.594-604, 2010. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23766230/ >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1758-2229.2010.00160.x.
https://pubmed.ncbi.nlm.nih.gov/23766230...
), suggesting some host preference for those two fungal species.

The AMF species richness reported in our study is higher than in previous studies in vineyards in USA (17 species) (SCHREINER & MIHARA, 2009SCHREINER, P. R.; MIHARA, K. K. The diversity of arbuscular mycorrhizal fungi amplified from grapevine roots (Vitis vinifera L.) in Oregon vineyards is seasonally stable and influenced by soil and vine age. Mycologia . v.101, n.5, p.599-611, 2009. Available from: <Available from: https://www.tandfonline.com/doi/abs/10.3852/08-169 >. Accessed: Dec. 28, 2020. doi: 10.3852 / 08-169.
https://www.tandfonline.com/doi/abs/10.3...
), and Italy (9 species) (BALESTRINI et al., 2010BALESTRINI, R.; et al. Cohorts of arbuscular mycorrhizal fungi (AMF) in Vitis vinifera, a typical Mediterranean fruit crop. Environ. Microbiol. Rep. v.2, n.4, p.594-604, 2010. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23766230/ >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1758-2229.2010.00160.x.
https://pubmed.ncbi.nlm.nih.gov/23766230...
). A possible explanation for this is that we sampled four different vineyards in distinct geographical locations and used trap cultures to further detect AMF species. Alternatively, high AMF species richness could result from the presence of black oats used as a cover crop in all vineyards. GDD- and H-plants shared most AMF species (88% of the total species richness), and species richness in both GDD- and H-plants were high, regardless of the vineyard. That suggested that GDD-plants do not affect the AMF species associated with them and that AMF species composition associated with vineyards is affected mainly by soil attributes (CHENG & BAUMGARTEN, 2004CHENG, X.; BAUMGARTNEN, K. Survey of arbuscular mycorrhizal fungal communities in Northern California vineyards and mycorrhizal colonization potential of grapevine nursery stock. HortScience: a publication of the American Society for Horticultural Science. v.39, n.7, p.1702-1706, 2004. Available from: <Available from: https://www.researchgate.net/publication/277766949_Survey_of_Arbuscular_Mycorrhiza l_Fungal_Communities_in_Northern_California_Vineyards_and_Mycorrhiza l_Colonization_Potential_of_Grapevine_Nursery_Stock >. Accessed: Dec. 28, 2020. doi: 10.21273/HORTSCI.39.7.1702.
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) and plant age (SCHREINER & MIHARA, 2009). The lack of differences between H- and GDD-plants may have occurred because plants were under the same management conditions in both situations. The dominance of Glomeraceae and Acaulosporaceae was expected, as previously reported in southern Brazil (ÁVILA et al., 2007ÁVILA, A. L.; et al. Occurrence of arbuscular mycorrhizal fungi in vineyards under diferent managements. Revista Brasieira de Agroecologia. v.2, n.1, p.641-644, 2007. Available from: <Available from: http://revistas.aba-agroecologia.org.br/index.php/rbagroecologia/article/view/6377 >. Accessed: Dec. 28, 2020. issn: 1980-9735.
http://revistas.aba-agroecologia.org.br/...
; SILVA et al., 2015SILVA, R. F.; et al. Influência do uso do solo na ocorrência e diversidade de FMAs em Latossolo no Sul do Brasil. Semina: Ciências Agrárias, Londrina. v.36, (3Supl1), p.1851-1862, 2015. Available from: <Available from: https://www.researchgate.net/publication/282070570_Influencia_do_uso_do_solo_na_ocorrencia_e_diversidade_de_FMAs_em_Latossolo_no_Sul_do_Brasil >. Accessed: Dec. 28, 2020. doi: 10.5433/1679-0359.2015v36n3Supl1p1851.
https://www.researchgate.net/publication...
) and the USA (SCHREINER & MIHARA, 2009). BOUFFAUD et al. (2016BOUFFAUD, M. L.; et al. Regional-scale analysis of arbuscular mycorrhizal fungi: the case of Burgundy vineyards. Oeno One. v.50, n.1, p.1-8, 2016. Available from: <Available from: https://oeno-one.eu/article/view/49 >. Accessed: Dec. 28, 2020. doi: 10.20870/oeno-one.2016.50.1.49.
https://oeno-one.eu/article/view/49...
) used molecular techniques and found high variability in AMF abundance and root colonization, and a correlation with plant management. As our research was restricted to morphological techniques, diversity may have been underestimated compared to molecular techniques. Species associated exclusively with H-plants were Acaulospora alpina, A. foveata, Rhizophagus fasciculatus, and Sclerocystis sp1, while the only species associated exclusively with GDD-plants was Oehlia diaphana (Table 3). Although, all five species were rare in both types of vineyards, their presence might indicate some selectivity for specific physiological conditions of grapevines or may be limited to a soil patch providing favorable conditions for a given AMF species. That indicates that they could be promising for future research in inoculation programs of grapevine or be tested in field conditions to verify their influence on plants with GDD symptoms.

The PCoA (Figure 3) and PERMANOVA analysis (Table 4) indicated that AMF communities are more distinct between vineyards than between H- and GDD-plants within the same vineyard. Similar results were detected in Italy, where vineyards harbored sequences from six species groups in one site, while those from other site harbored Glomus group A, and locations shared few OTUs (BALESTRINI et al., 2010BALESTRINI, R.; et al. Cohorts of arbuscular mycorrhizal fungi (AMF) in Vitis vinifera, a typical Mediterranean fruit crop. Environ. Microbiol. Rep. v.2, n.4, p.594-604, 2010. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23766230/ >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1758-2229.2010.00160.x.
https://pubmed.ncbi.nlm.nih.gov/23766230...
). Two types of natural filters interfered with an AMF community: the environmental filter, which selects species tolerant to environmental factors, and the host filter, which only allows colonization by compatible fungal partners (VÁLYI et al., 2016VÁLYI, K.; et al. Community assembly and coexistence in communities of arbuscular mycorrhizal fungi. The ISME Journal. v.10, p.2341-2351, 2016. Available from: <Available from: https://www.nature.com/articles/ismej201646 >. Accessed: Dec. 28, 2020. doi: 10.1038/ismej.2016.46.
https://www.nature.com/articles/ismej201...
). Considering that rootstocks (host filter) did not affect AMF communities, our data suggested that some environmental filters (e.g., cover crops) or historical processes (e.g., dispersion) might be acting to shape AMF communities in vineyards of Santa Catarina. Moreover, our results suggested that the physiological state of grapevine plants (GDD- or H-) has little or no influence on the soil AMF community composition.

No clear pattern appeared when the relationship between AMF species, soil attributes, and vineyards was analyzed. Soil P concentration in all four vineyards (Table 1) is high (Comissão de Química e Fertilidade do Solo, 2016), and this was particularly evident for V2, which has a history of phosphate fertilization, (Figure 4). V3 vineyard has the highest levels of Cu, due to its age and cultivation history. In the region, frequent phytosanitary treatments are applied to control fungal diseases, and many use copper-based products. Therefore, vineyards with a long history have high levels of soil Cu (Casali et al., 2008CASALI, C. A.; et al. Formas e dessorção de cobre em solos cultivados com videira na Serra Gaúcha do Rio Grande do Sul. Revista Brasileira de Ciência do Solo. v.32, n.4, p.1479-1487, 2008. Available from: <Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-06832008000400012&lng=en&nrm=iso >. Accessed: Dec. 28, 2020. doi: 10.1590/S0100-06832008000400012.
https://www.scielo.br/scielo.php?script=...
); although, the highest Cu values (65 mg kg-1 in V2) are not enough to cause toxicity to grapevines. Species associated with V2 are Entrophospora infrequens, Diversispora sp1 and Diversispora sp2., which suggested that they might have become adapted to high soil copper.

In conclusion, our results suggested that grapevines and, their associated cover crops, maintain a highly diverse AMF community, with high species richness and presence of most genera and families of Glomeromycota. Differences between vineyards were more related to geographical location than with rootstock or grapevine physiological state, i.e., GDD or healthy plants. Although, soil characteristics are an important determinant of AMF community assemblages (BALESTRINI et al. 2010BALESTRINI, R.; et al. Cohorts of arbuscular mycorrhizal fungi (AMF) in Vitis vinifera, a typical Mediterranean fruit crop. Environ. Microbiol. Rep. v.2, n.4, p.594-604, 2010. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23766230/ >. Accessed: Dec. 28, 2020. doi: 10.1111/j.1758-2229.2010.00160.x.
https://pubmed.ncbi.nlm.nih.gov/23766230...
; SCHREINER & MIHARA 2009SCHREINER, P. R.; MIHARA, K. K. The diversity of arbuscular mycorrhizal fungi amplified from grapevine roots (Vitis vinifera L.) in Oregon vineyards is seasonally stable and influenced by soil and vine age. Mycologia . v.101, n.5, p.599-611, 2009. Available from: <Available from: https://www.tandfonline.com/doi/abs/10.3852/08-169 >. Accessed: Dec. 28, 2020. doi: 10.3852 / 08-169.
https://www.tandfonline.com/doi/abs/10.3...
), our results showed a limited role of soil factors in shaping AMF communities in vineyards. An exception would be the strong association of E. infrequens, Diversispora sp1, and sp2 with high levels of Cu in V2. These species could be isolated in single culture for further research. Since the AMF community assembly was mainly affected by soil factors in each area, our research confirmed that AMF communities in vineyards are affected by characteristics of the plant’s host (e.g., rootstock, age) and management practices (e.g., cover crops, fertilization regime). The rich AMF community associated with grapevine represents a biotechnological potential for inoculation programs aiming to reduce fertilizer input and prevent GDD emergence.

CONCLUSION:

Arbuscular mycorrhizal fungal communities differ among vineyards, but not between soil under plants with grapevine decline and dieback symptoms (GDD) or asymptomatic. Soil chemical attributes were not determinant for occurrence of most AMF species, but copper is associated with the presence of Entrophospora infrequens, Diversispora sp1, and sp2. Claroideoglomus etunicatum, Funneliformis mosseae, and Archaeospora trappei were dominant and are promising for use in production of AMF inoculated grapevine plantlets.

ACKNOWLEDGMENTS

Empresa Brasileira de Pesquisa Agropecuária Uva e Vinho (Embrapa Uva and Vinho), Fundação de Amparo à Pesquisa do Estado de Santa Catarina (FAPESC), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001) funded this work. S.L.S, C.R.F.S.S., and P.E.L have CNPq Research Fellowship (Pq) Grants (Processes 307995/2019-4, 310124/2018-2, and 308334/2017-5, respectively).

REFERENCES

  • CR-2021-0011.R1

Publication Dates

  • Publication in this collection
    06 Sept 2021
  • Date of issue
    2022

History

  • Received
    07 Jan 2021
  • Accepted
    10 May 2021
  • Reviewed
    15 July 2021
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