Endophytic fungi associated with Araucaria angustifolia (Bertol.) Kuntze

Abstract

The diversity of endophytes and their ecological relationships with the endangered conifer Araucaria angustifolia (a critically endangered species) are unrevealed. This study aimed to characterize the diversity of endophytic fungi associated with A. angustifolia. To this end, we analyzed 90 fragments from five individuals collected from a mixed localized fragment in Guarapuava-PR, Brazil. The total DNA of 61 morphotypes was extracted and the Internal Transcribed Spacer (ITS) region was amplified and sequenced. The sequence analysis allowed the identification of 37 genera belonging to the phylum Ascomycota and the classes Eurotiomycetes, Dothideomycetes, and Sordariomycetes, divided into 11 orders and 13 families. Most of the isolated fungi belonged to the Sordariomycetes class (40%) and to the Xylaria genus (14%), while Eurotiomycetes was the minority class within the community. Our results reveal the high endophytic richness supporting the life cycle of A. angustifolia and reinforce the necessity for the conservation of this conifer, as many genetic resources can be lost owing to its irrational exploration.

Key words
Brazilian biome; diversity; endophytic; fungi

INTRODUCTION

The Araucaria forest is the main original forest type in southern Brazil, while it also occurs in some states in the southeastern region and northeastern Argentina (Carlucci et al. 2021). It is characterized by the presence of the conifer Araucaria angustifolia, commonly known as Paraná pine or Brazilian pine (Campanili & Schaffer 2010). Araucaria angustifolia is a gymnosperm that belongs to the order Pinales, class Pinopsida, and family Araucariaceae. Despite its apparent structural simplicity, the Araucaria forest has an enormous richness of species of the most varied taxa (Ribeiro et al. 2009).

Like many other Brazilian environments, the Araucaria forest suffers a sharp and continuous process of fragmentation. Originally, the Araucaria forest covered 20 million hectares in southern and southeastern Brazil (Campanili & Schaffer 2010). The intense anthropogenic pressure on the Araucaria forest reduced it to 12.6% of its original area by 2005 (Ribeiro et al. 2009), which applies to this day (Rezende et al. 2018, Carlucci et al. 2021). For this reason, A. angustifolia is considered a critically endangered species according to the International Union for Conservation of Nature (Thomas 2013).

It is important to study the Araucaria forest not only because this ecosystem has been reduced to small fragments, but also because it contains significant genetic resources that are still unknown to man (Solórzano-Filho & Kraus 1999). The extinction of A. angustifolia has resulted in serious consequences for the ecosystem, involving the loss of animal and plant diversity as well as microorganisms, many of which are still undescribed and possibly associated with this valuable plant species (Ribeiro & Cardoso 2012). For this reason, knowledge on the diversity of organisms associated with the Araucaria forest is necessary to stimulate the generation and dissemination of scientific and technological knowledge, in order to promote the conservation of its remnants and stimulate sustainable management.

Endophytic fungi are microorganisms that inhabit plant organs and tissues. Such fungi are distributed by colonizing intercellular spaces at some stage or throughout their life cycle, without causing apparent symptoms of disease or damage related to their presence in the host (Packiam & Dhakshinamoorthy 2021).

Endophytic species are of great interest to researchers because they can increase the adaptive value of the host species, help in their development, support them during adverse conditions, promote their stress tolerance, decrease the development of pathogens, and/or inhibit herbivory by producing metabolites (Jia et al. 2016, Alam et al. 2021, Packiam & Dhakshinamoorthy 2021, Jha et al. 2023).

The diversity of fungal endophytes is high, with more than 22 genera found among plant species (Hamzah et al. 2018, Segaran & Sathiavelu 2019). Knowledge on this group of organisms can elucidate the evolutionary history of plants and fungi (Kusari et al. 2014). Furthermore, as the diversity of endophytic fungi is influenced by plant species and environmental factors, the isolation of these fungi from previously unexplored plant communities may present great potential for the discovery of unique and undescribed secondary metabolites.

To the best of our knowledge, the fungal studies in A. angustifolia are focused on phytopathogenic fungi (Hodges & May 1972, Butin & Peredo 1986, Mendes et al. 2010), mycorrhizal fungi (Moreira et al. 2003, 2007, 2012, Silva et al. 2009, Zandavalli et al. 2008, Vilcatoma-Medina et al. 2018), and saprotrophic fungi (da Silva et al. 2021). However, to date, the diversity of endophytic fungi associated with A. angustifolia has not yet been assessed. The drastic reduction in the vegetation cover of the Araucaria forest indicates that it is an area of ​​extreme biological importance, highlighting its priority in research involving inventorying, conservation, and the rational exploitation of the remaining species. Therefore, we aimed to describe endophytic fungi associated with A. angustifolia.

MATERIALS AND METHODS

Samples and isolation of endophytic fungi

In order to isolate endophytic fungi, samples were collected from five A. angustifolia trees in Guará district, Guarapuava-PR (25°22’2.917”S 51°17’25.138”W). This area is a fragment of a mixed ombrophilous forest that covers approximately 300 ha. According to the Köppen-Geiger classification, the region is in the Cfb climate zone (mesothermal, humid throughout the year, and mild summer) (Maack 1981). The identification of A. angustifolia in site was possible because of the crown characteristics of the genus and this species.

In total, five A. angustifolia trees, approximately 10 m high and with no symptoms of disease, were chosen. From each individual, six branches (younger terminal part) were harvested by pruning shears and stored separately (for each tree) in plastic bags. Immediately after collection, the samples were taken to the laboratory for the isolation of endophytic fungi. Surface disinfection was performed for each sample. Initially, each of the six branches of each tree was cut into 10 cm pieces and the needles were removed. Then, the stems were washed in water and neutral detergent with a soft brush and rinsed in running water and distilled water. The branches were then placed in a laminar flow chamber and subjected to the methodology described by Sun et al. (2011).

After superficial disinfection, the branches were rinsed in sterile distilled water three times and placed on a sterile paper towel to dry. We distributed 100 µL of the water used for the last rinse in a Petri dish containing Sabouraud agar medium supplemented with chloramphenicol (0.05 g L^-1) and used it as the negative control. Three 0.5 cm fragments were obtained from each of the branches using tweezers and a sterile scalpel, totaling 90 fragments. These were later arranged equidistantly in Petri dishes with Sabouraud agar medium supplemented with chloramphenicol. The plates were incubated in an oven at 28 °C (±2) in the dark for up to 25 days. The fungi that developed from the plant tissue fragments were collected and placed on plates containing Sabouraud medium until pure colonies were obtained. The strains obtained were grouped into different morphotypes and stored at 4 °C, according to Castellani (1939). All fungi were registered in the Brazilian National System of Genetic Heritage Management and Associated Traditional Knowledge (SisGen) under the number AF5F498.

Molecular identification: DNA extraction, PCR amplification, and sequencing

Distinct fungal morphotypes were molecularly identified. Each strain was cultured in Czapeck medium supplemented with 1% dextrose with a pH of 5.6 and cultivated for 5 days at 28 °C (± 2). After cultivation, the fungal mycelia were filtered through a vacuum pump using sterile filter paper and washed with sterile ultrapure water. Immediately after filtration, the mycelia were macerated using a mortar and pestle with liquid nitrogen to obtain a very fine powder. The cetyltrimethylammonium bromide (CTAB) method proposed by Doyle & Doyle (1987) was used for DNA extraction.

After DNA extraction, amplification was performed using a thermocycler (Mastercycler®, Eppendorf, USA). The PCR amplification of the ITS1–5.8S–ITS2 DNA region was achieved in one fragment using ITS5 forward (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4 reverse (5’-TCCTCCGCTTATTGATATGC-3’) primers, as described by White et al. (1990). The PCR amplification mix of the ITS5-ITS4 comprised approximately 20 ng genomic DNA, 1× BiolaseTM buffer with 1.0 mM MgCl₂ (Bioline, London, UK), 10 µM of each primer (forward and reverse), 0.25 mM of each dNTP, and 1 unit of BiolaseTM DNA polymerase (Bioline). The reaction volume was adjusted with ddH₂O to a final volume of 20 μL. The amplification conditions were as follows: initial denaturation at 94 °C for 2 min; 30 cycles of 30 s at 95 °C (denaturation), 60 s at 50 °C (annealing), 60 s at 72 °C, and a final extension at 72 °C for 7 min.

The PCR products were purified using QIAquick PCR purification spin columns (Qiagen) and quantified using a NanoDrop 2000 spectrophotometer with a NanoDrop 2000/2000c (Thermo Fisher Scientific, Inc.).

The sequencing reactions were performed in 10 µL reactions using BigDye Terminator sequencing reagents and protocols (Applied Biosystems, Foster City, California, USA). The data were collected on an ABI-Prism 3500 automated sequencer (Applied Biosystems) by ACTGene Molecular Analyses at the Universidade Federal do Rio Grande do Sul. All sequences were deposited in GenBank.

BLAST (www.blast.ddbj.nig.ac.jp/) was used to verify the homology of the sequences obtained with those deposited in the GenBank NCBI database. Identification at the species level was performed when the sequences obtained presented, in relation to the homologous reference sequence, coverage greater than 80% and similarity greater than 97%. In addition, when the sequences obtained had homologies with more than 10 different species, the classification was performed only at the genus level (Raja et al. 2017).

Phylogenetic analysis

The phylogenetic tree was constructed using Bayesian analysis (MrBayes v.3.1.2). We used 37 sequences obtained from this study and included more than 25 sequences from the GenBank database. Outgroup taxa belonging to the species Neurospora crassa_AY681193 were used.

RESULTS

We obtained 102 filamentous fungi isolates from 90 fragments of A. angustifolia sampled. No growth of yeast colonies was observed. At the end of the morphological analysis, 61 morphologically distinct lineages were identified. Of these, one morphotype was selected for the sequencing of the ITS region of the rDNA gene.

Among the 61 morphotypes that were subjected to DNA extraction and amplification of the ITS sequence, 24 did not have their DNA amplified or did not have quality sequences. Therefore, further analyses were carried out with the 37 strains for which molecular identification was possible.

Some endophytes were identified at the genus level, whereas others were identified at the species level. Seventeen genera were observed: Annulohypoxylon, Aspergillus, Colletotrichum, Coniochaeta, Diaporthe, Fimetariella, Fusarium, Hypoxylon, Mycoleptodiscus, Muyocopron, Neofusicoccum, Neopestalotiopsis, Pestalotiopsis, Phyllosticta, Preussia, Trichoderma, and Xylaria. All genera belong to the phylum Ascomycota and to the classes Eurotiomycetes, Dothideomycetes, and Sordariomycetes, and are divided into 11 orders and 13 families (Table I). Figure 1 shows the diversity of the colony morphology (obverse and reverse) of the endophytic fungi that were identified. The results of the sequences demonstrate that the ITS region, considered a DNA barcode for fungi, was not effective in discriminating many of the strains under investigation at the species level.

Figure 1
Diversity of the colony morphology (obverse and reverse) of the endophytic fungi associated with A. angustifolia cultivated in Sabouraud agar at 25 °C, for 14 days.

Table I
Classification of endophytic fungi associated with A. angustifolia.

Endophytic fungi classification
Code	Class	Order	Family	Proposed species	GeneBank Acession
W12	Eurotiomycetes	Eurotiales	Aspergillaceae	Asper gillus fumigatus	MY996900
W23				Aspergillus fumigatus	MY996901
W44				Aspergillus flavus	MY996902
W16	Dothideomycetes	Pleosporales	Sporormiaceae	Preussia mínima	MY996903
W37		Botryosphaeriales	Botryosphaeriaceae	Neofusiococcum sp.	MY996904
W56				Phyllosticta capitalensis	MY996905
W411				Phyllosticta capitalensis	MY996906
W43				Phyllosticta capitalensis	MY996907
W19	Sordariomycetes	Coniochaetales	Coniochaetaceae	Coniochaeta velutina	MY996908
W26		Hypocreales	Hypocreaceae	Trichoderma koningiopsis	MY996909
W42			Nectriaceae	Fusarium sp.	MY996910
W48		Glomerellales	Glomerellaceae	Colletotrichum sp.	MY996911
W57		Muyocopronales	Muyocopronaceae	Mycoleptodiscus sp.	MY996912
W18				Muyocopron sp.	MY996913
W32		Diaporthales	Diaporthaceae	Diaporthe sp.	MY996914
W312				Diaporthe sp.	MY996915
W45				Diaporthe serafinie	MY996916
W41				Diaporthe sp.	MY996917
W28		Amphisphaeriales	Pestalotiopsidaceae	Neopestalotiopsis sp.	MY996918
W13				Pestalotiopsis sp.	MY996919
W24				Pestalotiopsis sp.	MY996920
W211				Neopestalotiopsis sp.	MY996921
W27		Xylariales	Hypoxylaceae	Annulohypoxylon stygium	MY996922
W11				Hypoxylon sp.	MY996923
W311				Hypoxylon sp.	MY996924
W315				Hypoxylon sp.	MY996925
W213			Xylariaceae	Xylaria sp.	MY996926
W114				Xylaria sp.	MY996927
W218				Xylaria sp.	MY996928
W313				Xylaria berteri	MY996929
W214				Xylaria sp.	MY996930
W310				Xylaria sp.	MY996931
W47				Xylaria sp.	MY996932
W15			Hypoxylaceae	Hypoxylon sp.	MY996933
W39			Xylariaceae	Xylaria berteri	MY996934
W111				Xylaria sp.	MY996935
W410		Sordariales	Lasiosphaeriaceae	Fimetariella rabenhorstii	MY996936

The phylogenetic tree analysis (Figure 2) revealed the presence of large clades, which correspond to the three fungal classes found (Sordariomycetes, Eurotiomycetes, and Dothideomycetes). Most of the isolated fungi belonged to the Sordariomycetes class (40%) and the Xylaria genus (14%). In contrast, Eurotiomycetes was recognized as a minority class within the community, with strains belonging to a single genus, including the Aspergillus flavus and Aspergillus fumigatus species. The class Dothideomycetes included representatives of two orders, three families, and three genera.

Figure 2
Phylogenetic tree of the ITS region nucleotide sequences of endophytic fungi associated with A. angustifolia. The tree was built with Bayesian analysis. The Bootstrap values are based on 1000 replicate runs, shown as percent. Neurospora crassa_AY681193 was used as the outgroup. The strains codes follows the name of fungal species.

DISCUSSION

This study presents, for the first time, the characterization of a community of cultivable endophytic fungi associated with individuals of A. angustifolia in southern Brazil. Compared to studies carried out with other gymnosperms and angiosperms, the number of species described in the present work is significant, considering the sampling effort of 90 fragments, from five individuals (Hormazabal & Piontelli 2009, Correia et al. 2018, Wang et al. 2019, Ferreira et al. 2020).

The endophytic species isolated were placed in 11 different orders, seven of which belonged to the Sordariomycetes class, with emphasis on the order Xylariales and genus Xylaria, which comprised the largest number of species. The Xylaria genus is one of the most diverse within the family Xylariaceae, with approximately 600 described species (Helaly et al. 2018). Xylaria species are typical examples of endophytes isolated from fragments of apparently healthy plants, and are predominant in these diversity studies (Vaz et al. 2014). In Brazil, these endophytes have also been reported to be abundant in other plants, such as Eucalyptus microcorys, Vochysia divergens, and Stryphnodendron adstringens (Lacerda et al. 2018, Noriler et al. 2018). According to Okane et al. (2012), fungi of the genus Xylaria are generalists and can colonize a wide variety of plants.

Fungi of the genera Diaporthe and Pestalotiopsis were also abundant, and both have already been reported as endophytic fungi in other studies (Banhos et al. 2014, Noriler et al. 2018); however, these fungi, along with others belonging to genera Neofusicoccum, Fusarium, Phyllosticta, and Colletotrichum, are considered pathogens of plants such as soybean, sunflower, pepper, bean, and coffee (Nguyen et al. 2009, Shetty et al. 2011, Lazarotto et al. 2014, Udayanga et al. 2015, Batista et al. 2019). Of these, Fusarium and Neofusicoccum have already been reported as pathogens of A. angustifolia (Auer & Grigoletti Junior 1997, Dalmas et al. 2013).

Endophytic relationships depend on the balance of antagonism between the fungus and its host. Throughout infection, mutualism, commensalism, and parasitism interactions can be established, and owing to this, fungi considered phytopathogenic, such as Fusarium and Colletotrichum, have already been isolated as endophytes, conferring benefits to host plants (Nandhini et al. 2018, Huang et al. 2020). In addition to the habit changes that occur because of balanced antagonism, genetic alterations that result in the conversion of parasites to non-parasites have already been observed (Freeman & Rodriguez 1993, Hacquard et al. 2016).

Many of the genera described herein have previously been reported as endophytes of other plants, including native Brazilian plants. Similarly, 10 genera described in the present study, including Xylaria, Diaporthe, and Pestalotiopsis were also associated with angiosperm species Vochysia divergens Pohl. and Stryphnodendron adstringens (Mart.) Coville belonging to the Brazilian Pantanal and Cerrado biomes, respectively (Noriler et al. 2018). Six genera associated with A. angustifolia were associated with Myrcia guianensis (Aubl.) DC. found in the Amazon (Banhos et al. 2014). A. fumigatus strains were previously isolated from the leaves of Erythrophleum fordii Oliver, a legume species of the Fabaceae family (Shi et al. 2015). Colletotrichum sp. is well recognized as an endophyte of a wide variety of plants distributed worldwide, including members of the Amaryllidaceae, Orchidaceae, Proteaceae, and Solanaceae families (Weir et al. 2012). Until now, there has been only one record of Fimetariella rabenhorstii in Brazil and is described as an endophytic of S. adstringens (Carvalho et al. 2012). These findings highlight that the genera of endophytic fungi found in the present study have the capacity to develop in a wide diversity of hosts.

Researchers have found it difficult to understand the evolutionary process of these microorganisms and their adaptation to the hosts (Gladieux 2018), as the diversity of endophytic fungi is influenced by the physiology of the host, environmental factors, and interactions with other organisms. Research shows that the community can vary in hosts from different places, is distinguished in different parts of the plant, and can be seasonally modified (Shi et al. 2016, Bowman & Arnold 2018). It is believed that endophytic fungi have host specificity, as observed in phytopathogenic fungal strains. However, it is difficult to understand this characteristic, which requires a larger number of plants to be sampled, in addition to the fact that specificity also occurs below the species level (Arnold 2007).

For the first time, we have described the apparent broad endophytic fungal diversity of A. angustifolia. The revealed community included 17 genera in the phylum Ascomycota. Among these, there was a high prevalence of Xylaria species. This study opens perspectives to bioprospecting as a strategy for conservation and sustainable use of A. angustifolia, a promising source of undiscovered and potentially useful genetic resources.

ACKNOWLEDGMENTS

This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001.

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Publication Dates

• Publication in this collection
  13 Sept 2024
• Date of issue
  2024

History

• Received
  08 Mar 2023
• Accepted
  06 May 2024