Fungal community in chestnut orchards with different Hypholoma fasciculare aboveground abundance: potential implications for sustainable production Comunidade fúngica de soutos com diferentes abundâncias de Hypholoma fasciculare: potenciais implicações para a produção sustentável de castanheiro Francisca Reis1, Eric Pereira, Rui M. Tavares1, Paula Baptista e Teresa Lino-Neto1* 1 Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center (CBFP), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; 2 CIMO / School of Agriculture, Polytechnic Institute of Bragança, Campus Sta. Apolónia, 5300-253 Bragança, Portugal. (*e-mail: [email protected]) http://dx.doi.org/10.19084/RCA15153 Received/recebido: 2015.11.05 Received in revised form/recebido em versão revista: 2016.04.13 Accepted/aceite: 2016.04.20 ABSTRACT European chestnut (Castanea sativa Mill.) trees have a significant impact in the Portuguese economy, due to the production of chestnuts and wood, and other related activities, such as mushroom collection and hunt. Hypholoma fasciculare (Huds.) is a saprophytic fungus widely distributed in Trás-os-Montes (northeast of Portugal) chestnut groves that displays an in vitro strong antagonistic activity against ectomycorrhizal (EMC) fungi. In this study, the above- and belowground fungal diversity was evaluated in three chestnut orchards, containing distinct H. fasciculare sporocarps abundances in an attempt to better understand the potential of this fungus to attain an improved chestnut tree sustainable productivity. Aboveground analysis was performed based on macrofungi collection during fruiting seasons (spring and autumn) of two consecutive years. Belowground evaluation was based on the metabarcoding of chestnut orchards soil DNA, using the fungal barcode ITS1 and a high-throughput sequencing (454-sequencing) approach. Although all collected fruitbodies were identified as being produced by Basidiomycota fungi, a more diversified fungal community was revealed by the belowground approach. Both approaches have revealed a rich and abundant ECM community in all chestnut orchards. The correlation between the abundance of H. fasciculare fruitbodies and specific fungal guilds fruitbodies/reads suggests that this fungus may affect soil fungal community, mainly ECM and phytoparasites, as well as species composition of fungal communities. Although not conclusive, the results suggest that H. fasciculare presence could be critical for sustainable chestnut ecosystems. Keywords: Castanea sativa, chestnut orchard soils, ectomycorrhizal community, Hypholoma fasciculare, ITS metabarcoding. RESUMO O castanheiro europeu (Castanea sativa Mill.) tem um enorme impacto na economia Portuguesa, sobretudo devido à produção de castanha e de madeira, mas também de actividades relacionadas com a colheira de cogumelos e a caça. Hypholoma fasciculare (Huds.) é um fungo saprófita amplamente distribuído nos soutos da região de Trás-os-Montes (nordeste de Portugal) e que apresenta, em condições in vitro, uma forte actividade antagonista contra fungos ecto- micorrízicos. Neste estudo pretendeu-se avaliar a comunidade fúngica presente na superfície e no sub-solo de soutos contendo diferentes abundâncias de carpóforos de H. fasciculare, numa tentativa de compreender melhor a possibi- lidade deste fungo melhorar a produção sustentável do castanheiro. A diversidade fúngica à superfície do solo foi avaliada recorrendo à colheita de cogumelos durante o outono e a primavera, ao longo de dois anos consecutivos. A diversidade no sub-solo foi avaliada molecularmente pela extração do DNA do solo de soutos, amplificação da região ITS1 e sequenciação de nova geração (sequenciação 454). Apesar dos cogumelos colhidos serem todos produ- zidos por fungos Basidiomicetes, uma comunidade fúngica mais diversificada foi revelada pela abordagem molecular. No entanto, ambas as abordagens revelaram uma comunidade rica e abundante de fungos ECM em todos os soutos. A correlação efetuada entre a abundância de cogumelos de H. fasciculare e os grupos tróficos dos fungos presentes, avaliada por cogumelos/sequências, sugere que este fungo pode afetar a comunidade fúngica do solo, principalmente 124 Revista de Ciências Agrárias, 2017, 40(1): 124-132 os fungos ectomicorrízicos e fitoparasitas, assim como a composição em espécies da comunidade. Apesar de não serem conclusivos, os resultados sugerem que a presença de H. fasciculare poderá ser crítica para a sustentabilidade de ecos- sistemas de castanheiro. Palavras-chave: Castanea sativa, comunidade ectomicorrízica, Hypholoma fasciculare, identificação molecular por ITS, solos de soutos. INTRODUCTION assays (Pereira et al., 2011). However, despite the recognized role of H. fasciculare as an antagonist, Chestnut ecosystems are among the conservation there is still a limited understanding of its effect priorities in Europe (Habitat Directive 92/43/EEC, on soil microbial diversity and consequently on 1993). Chestnut tree (Castanea sativa Mill.) has a sustainable crop production. great economic importance, mainly due to the fruit value and high quality wood. Portugal is the third During the life cycle of many fungi, sexually largest producer in Europe of chestnut fruit (FAO, reproducing structures are visible to naked eye, 2013), where four different Protected Designations comprising the so-called aboveground compo- of Origin (PDO; Castanha da Terra Fria, Castanha dos nents of a fungal community. Different molec- Soutos da Lapa, Castanha da Padrela and Castanha de ular approaches based on the detection of fungal Marvão) were attributed. Taking into account the DNA in the soil have recently emerged and are economic and social value of this market, efforts included in the so-called belowground approaches have been made in order to preserve chestnut (Yoccoz, 2012). Differences in the accuracy of tradi- orchards ecosystems. The soil fungal diversity has tional and molecular approaches for describing been, not only important for mushroom collection fungal community became evident in the past activities, but also a major concern for chestnut years and are now known to bring complemen- tree sustainability. When evaluating the fungal tary information for ecological fungal research diversity and fruiting pattern associated with (Horton and Bruns, 2001; Baptista et al., 2015). In Portuguese chestnut orchards, the high abundance the present work, fruitbody surveys and soil ITS1 of a saprotrophic fungus – Hypholoma fasciculare – metabarcoding approaches are combined to study was reported in healthy and centenary chestnut the diversity of fungi in soils of three centenary orchards (Baptista et al., 2010). Recently, a deeper chestnut orchards displaying different abun- assessment of soil fungal diversity in Trás-os- dances of H. fasciculare. The presence of low/high Montes chestnut orchards, which combines both amount of this fungus was further correlated with traditional (fruit body collection) and molecular the natural abundance of different fungal guilds (next generation sequencing) approaches revealed in an attempt to better understand its effect on soil a high fungal diversity (Baptista et al., 2015). microbial diversity and consequently on chestnut Although the ectomycorrhizal (ECM) community grove sustainability. represented more than 50% of total fungal commu- nity, H. fasciculare was also detected by molecular methods (Baptista et al., 2015). There are evidences MATERIAL AND METHODS that this lignin-saprophytic cord-forming fungus presents a significant antagonistic action against The sampling region was located in the Natural different fungi that naturally occur in the soil of Park of Montesinho (Bragança, northeast of chestnut groves (Pereira et al., 2012). Taking advan- Portugal), where 100-year-old non-tilled chestnut tage from this high antagonistic activity, H. fascic- orchards were selected. Area of sampling is char- ulare was used in field trials for the biological acterized by having a sub-continental climate control of certain pathogens, such as Armillaria with long cold winters and short but hot and dry ostoyae (Romagn.) Herink. (Chapman et al., 2004). summers. Annual air temperature diverges among Although some H. fasciculare antagonistic interac- 10-14 0C (temperature ranging from 0 to 28 0C in tions could be indeed beneficial for chestnut tree January and July, respectively). Annual rainfall sustainability, others could be detrimental for average ranges from 1000 to 1200 mm, 90% of which ectomycorrhizal association, as revealed by in vitro occurring between October and April (Baptista, Reis et al., Fungal community related with Hypholoma fasciculare abundance 125 2007). Three chestnut orchards were selected (MID) and ITS2 primer sequence (White et al., 1990). (about 1.5 ha each), based on different amounts of PCR using FastStart Taq DNA Polymerase (Roche®) H. fasciculare fruitbodies observed in each orchard included the following thermal cycle conditions: from September 2002 to December 2005 (Baptista an initial denaturation at 94°C for 4 min, followed et al., 2010). The most abundant orchards (Ab and by 30 cycles of 94 °C for 30 sec, 50 °C for 45 sec Md) were located in the same region (Oleiros), and 72 °C for 45 sec and a final extension of 72 °C 500 m apart from each other (N 41º 51 W 6º 49; 899 for 10 min. Amplicons preparation for sequencing, m altitude). Ab orchard presented a higher abun- pyrosequencing on a Genome Sequencer GS FLX dance of H. fasciculare fruitbodies