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Scientia Agricola

On-line version ISSN 1678-992X

Sci. agric. (Piracicaba, Braz.) vol.77 no.3 Piracicaba  2020  Epub Sep 05, 2019

http://dx.doi.org/10.1590/1678-992x-2018-0210 

Agricultural Microbiology

Endophytic fungi from Brachiaria grasses in Brazil and preliminary screening of Sclerotinia sclerotiorum antagonists

Danielly de Souza Gama1 
http://orcid.org/0000-0001-5812-9083

Ítalo Augusto Férrer Melo Santos1 
http://orcid.org/0000-0001-5433-3562

Lucas Magalhães de Abreu2 
http://orcid.org/0000-0003-1606-5709

Flávio Henrique Vasconcelos de Medeiros3 
http://orcid.org/0000-0003-0993-796X

Whasley Ferreira Duarte1 
http://orcid.org/0000-0002-1027-588X

Patrícia Gomes Cardoso*  1 
http://orcid.org/0000-0002-0797-2502

1Universidade Federal de Lavras – Depto. de Biologia, C.P. 3037 – 37200-000 – Lavras, MG – Brasil

2Universidade Federal de Viçosa – Depto. de Fitopatologia – Campus Universitário – 36570-977 – Viçosa, MG – Brasil

3Universidade Federal de Lavras – Depto. de Fitopatologia


ABSTRACT:

Fungal endophytes of Brachiaria, a nonhost of Sclerotinia sclerotiorum, may harbor species with antagonistic effects against this plant pathogen. The objective of this work was to investigate the diversity of endophytic fungi associated with different Brachiaria species and hybrids and evaluate their potential to inhibit the plant pathogen S. sclerotiorum. Stem samples from 39 Brachiaria spp. plants were collected in pasture fields and experimental areas of three states of Brazil resulting in 74 endophytes isolated. Twenty-eight species were identified by sequences of the Internal Transcribed Spacer (ITS) and 18S rDNA regions. Paraconiothyrium sp. was the most abundant endophyte, accounting for 24 % (14 isolates) of total, and it was isolated from B. ruziziensis, B. decumbens, B. humidicola, and B. brizantha. Phoma sorghina was the second most abundant taxon, followed by Sarocladium strictum, and Plenodomus sp. In vitro analyses showed that Paraconiothyrium sp., Sarocladium kiliense, Acremonium curvulum, Setophoma terrestris, Dissoconium sp., and Cladosporium flabelliforme exhibited antagonistic activity against S. sclerotiorum, with percentages of growth inhibition ranging from 25 to 60 (p < 0.05). Paraconiothyrium sp. BBXE1 (60 %), BBPB4.1 (60 %), BCMT4.1 (54 %), and S. kiliense (54 %) showed the highest values of Antagonism Percentages (AP). Therefore, fungi with inhibitory activity against S. sclerotiorum such as Paraconiothyrium sp. are naturally endophytic in Brachiaria grasses.

Keywords: Coniothyrium-like fungi; Urochloa; tropical forage grasses; antagonism; white mold

Introduction

Grass pastures are widely distributed in different regions of Brazil, and the success of their establishment depends on the use of robust forage species. Brachiaria (syn. Urochloa) species are among the most important forages for cattle feeding in the country, reflecting their adequate adaptation to different conditions (Valle et al., 2009).

The beneficial association of some agricultural grass species from temperate regions with vertically transmitted Clavicipitaceous fungal endophytes is a well stablished phenomenon (Saikkonen et al., 2006). This mutualism may protect plants against the growth of herbivore insects through the balance between antagonistic signaling pathways and increased availability of nutrients (Saikkonen et al., 2006, 2013). This type of symbiosis is not commonly observed in tropical grasses (Sánchez Márquez et al., 2012). Nevertheless, a large diversity of horizontally transmitted endophytic fungi in Poaceae species used for cattle feeding in tropical regions deserves further investigation (Rodrigues and Dias-Filho, 1996).

Grasses can be used for mulching or rotation with dicots to reduce disease inoculum, since they do not share the same range of pathogens (Gasparotto et al., 1982; Görgen et al., 2009, 2010). The white mold is one of these diseases, caused by Sclerotinia sclerotiorum. This pathogen has a wide host range, but it mainly affects dicotyledonous hosts, including important crops such as soybean, bean and cotton, causing water-soaked lesions that expand rapidly killing a large portion of the aerial plant tissues, which commonly become covered by the white mycelium of the pathogen (Bolton et al., 2006).

Management of white mold in soybean crop may include rotation/mulching with nonhost Poaceae and the combination with biological control, using for example Trichoderma harzianum (Görgen et al., 2009, 2010), a fungus reported as endophytic in Brachiaria grasses (Rodrigues and Dias-Filho, 1996; Kago et al., 2016). Thus, a plausible hypothesis is that fungal endophytes of Brachiaria, a nonhost of S. sclerotiorum (Görgen et al., 2009), may harbor species with antagonistic effects against this plant pathogen. Therefore, we investigated the diversity of fungal endophytes associated with several Brachiaria species in Brazil and evaluated in vitro antagonism of the isolates against S. sclerotiorum.

Materials and Methods

Sampling Brachiaria plants

Thirty-nine disease-free plants of different species and hybrids of Brachiaria were sampled: B. ruziziensis; B. decumbens cv. Basilisk; B. mutica cv. Angola; hybrid cv. Mulato I and cv. Mulato II; B. humidicola common, cv. Llanero and cv. Tupi; B. brizantha cv. Piatã, cv. Xaraés, and cv. Marandu, in which 11 were collected from pasture fields and 28 from experimental plots. The samples were collected during the rainy and dry seasons from Aug 2012 to Oct 2013 in the following locations: five at the Universidade Federal de Lavras (Lavras-MG, Brazil); seven at the Embrapa Amazônia Oriental (Belém-PA, Brazil); eight at the Embrapa Gado de Corte (Campo Grande-MS, Brazil); 19 at the Embrapa Gado de Leite (Juiz de Fora-MG, Brazil) (Figure 1). Plants in vegetative stage were harvested at 10 cm above the ground using pruning shears, placed in plastic bags and refrigerated for approximately 48 h until isolation (6 to 10 Apr). A cool box was used to transport samples to the laboratory.

Figure 1 Brazilian map showing the states and sites where plants were sampled. The map was created on the QGIS software version 2.18 using the Geographic Coordinate Reference System and Datum SIRGAS2000 from data of Instituto Brasileiro de Geografia e Estatística (IBGE). 

Isolation of endophytic fungi from Brachiaria plants

Defoliated stem samples were washed under tap water, cut into 10 cm fragments and placed in 50 mL sterile Falcon tubes. Surface disinfestation was done by successive washes with sterile distilled water (1 min), 96 % ethanol (2 min), sterile distilled water (1 min), 5 % sodium hypochlorite (2 min), and a final wash three times in sterile distilled water (1 min). Stem pieces were dried on sterile filter paper and cut into smaller pieces (0.5 cm) using a sterile scalpel. Fifteen stem pieces per plant sample were seeded onto Petri dishes containing PDA medium (potato infusion-dextrose-agar) amended with cefotaxime (0.25 g L−1) incubated at 25 °C. To assess the efficiency of disinfestation, aliquots of final rinse water of 100 µL were similarly seeded. Samples were examined daily and the endophytic fungi were individually transferred to new plates containing PDA/cefotaxime plates for purification. Isolates were stored with the Castellani's method (Castellani, 1939) at 4 °C in sterile tubes containing 1 mL of sterile distilled water.

ITS and 18S rDNA sequencing and molecular identification

The molecular identification of fungal isolates was performed using DNA sequences of ITS and 18S rDNA (White et al., 1990). Mycelia were scraped from colonies on PDA using a sterile toothpick and total DNA was extracted using the Microbial DNA Isolation Kit (MO BIO). Amplification reactions were performed in 30 µL reaction volumes containing 15 µL of kit, 12 µL of H2O, 10 pmol of each forward and reverse primer, and 1 ng of DNA. For ITS amplification, primers ITS1 (5‘-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5‘- TCCTCCGCTTATTGATATGC-3’) were used, and reaction conditions were: 95 °C for 2 min, followed by 35 cycles at 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 7 min. To amplify the 18S region, primers NS1 (5‘-GTAGTCATATGCTTGTCTC-3’) and NS6 (5‘-GCATCACAGACCTGTTATTGCCTC-3’) were used. Reactions were performed in a thermocycler at 94 °C for 1 min, followed by 35 cycles at 94 °C for 35 s, 55 °C for 50 s, and 72 °C for 2 min, with a final extension at 72 °C for 6 min.

The amplification products were purified and sent to Macrogen (Seoul, South Korea) for Sanger sequencing. The sequences were edited using the SeqAssem 07/2008 software and then compared with sequences of reference strains deposited in international culture collections available in the GenBank database (National Center for Biotechnology Information - NCBI) (https://www.ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool (BLAST) and identification was performed according to the identities found.

Antifungal activity of isolated endophytic fungi against Sclerotinia sclerotiorum

The evaluation of antifungal activity was adapted from Kelemu et al. (2001). The pathogenic strain UFLA44 of S. sclerotiorum used in this work was collected from common bean field cultivar Ouro Vermelho by Abreu and Souza (2015) in the municipally of Coimbra, state of Minas Gerais, Brazil. A small mycelial fragment of the endophyte was placed on one side of a Petri dish (9 cm diameter) containing PDA and incubated for 7 d at 25 °C. Next, 5 mm colony fragment of S. sclerotiorum UFLA44 obtained from sclerotia germination was placed on the opposite side the plate containing the endophyte (Figures 4A, B), where the phytopathogen was inoculated after the endophytic fungi because of its higher growth rate. The plates were incubated at 25 °C for additional 7 d and, at the end, the pathogen radial growth was the measured variable. This experiment was conducted in a completely randomized design with 225 experimental units: 74 endophytic fungi; phytopathogen alone, corresponding to the control (Figure 4C); with three replicates.

The Antagonism Percentage (AP) of j-esim replicate (n = 3) and i-esim treatment was calculated according to the equation: APij=(DMdm)/DMx100 , in which DM (cm) = diameter average of the S. sclerotiorum colony in the absence antagonist endophytic fungus and dm (cm) = diameter average of the S. sclerotiorum colony in dual culture with the antagonist endophytic fungus. Data were statistically evaluated by analysis of variance (ANOVA) and the means were compared using the Tukey test at 5 % probability level in SISVAR statistical software (version 5.6). The standard error of the mean was calculated and presented with the respective data.

Endophytes that exhibited antagonism against S. sclerotiorum were used in a second experiment to verify whether the observed inhibition resulted from the production of volatile or nonvolatile compounds by the endophytic fungi in the culture medium. The same methodology described in the first experiment was adopted, however, a bipartite Petri dish that prevented contact between the colonies was used.

Results and Discussion

We isolated 74 endophytic fungi from 39 stem samples of Brachiaria spp. collected in three states of Brazil (Figure 2). The 28 samples from experimental plots yielded 48 isolates, while 26 isolates were recovered from 11 samples collected in pasture fields, an overall recovery rate of two isolates per sample.

Figure 2 Endophytic fungi isolated from different species of Brachiaria for each sampling site in Brazil. 

Twenty-eight species of the endophytic fungi were identified by ITS and 18S rDNA sequences comparison, most belonging to Phylum Ascomycota (Figure 3A). Paraconiothyrium sp. was the most abundant endophyte (Figure 3B), accounting for 24 % (14 isolates) of total and isolated from all samples, except for one sample (hybrid cv. Mulato II and B. mutica). Phoma sorghina (11 isolates) was the second most abundant taxon, followed by Sarocladium strictum, and Plenodomus sp., both taxa represented by seven isolates. These four species accounted for approximately two-thirds of all isolates (68 %), but corresponded to 32 % of all species recovered.

Figure 3 Relative abundance at (A) phylum and (B) species level of endophytic fungi isolated from different species of Brachiaria

Paraconiothyrium, Phoma, and Plenodomus are Pleosporales genera belonging to different families of the Dothideomycetes class. Taxonomy of this group coniothyrium-like fungi is quite complex and subjected to periodical updates, mainly to accommodate the wealth of DNA sequence data and phylogenetic reassessments of ex-type and other reference strains preserved in culture collections (Verkley et al., 2014; Ariyawansa et al., 2015; Chen et al., 2015). On the other hand, the ITS marker is generally reliable for strain typing and identifications to genus level among this group of fungi (Verkley et al., 2014). Species of Phoma-like fungi are common endophytes of grasses, such as the temperate species Dactylis glomerata and Holcus lanatus (Sánchez Márquez et al., 2007, 2010). Two putative Phoma species were reported as endophytes of Brachiaria in a preliminary inventory conducted in Brazil (Rodrigues and Dias-Filho, 1996). P. sorghina, the second most abundant species and isolated from B. ruziziensis, B. decumbens, and B. humidicola, was also one of the most frequently isolated endophyte in perennial grasses Hyparrhenia hirta and Bothriochloa macra in Australia (White and Backhouse, 2007). Species of Paraconiothyrium were the dominant root endophytes of native grasses inhabiting semiarid grasslands in New Mexico (Khidir et al., 2010). We isolated endophytes from the lower part of plant stems and the root system of Brachiaria and the soil are possibly the source of the abundant Paraconiothyrium taxon, since this is a genus of common soil-borne fungi (Domsch et al., 2007).

Twelve isolates were identified as Sarocladium species, the third most common genus of Brachiaria endophytes. B. brizantha yielded Sarocladium sp., S. kiliense and S. strictum. From B. ruziziensis, S. spinificis and S. strictum were isolated. There is a high diversity of Sarocladium in grasses, mostly endophytes and some phytopathogens (e.g. S. oryzae). This diversity is shown by the description of two new species, Sarocladium spinificis, endophytic to coastal grass Spinifex littoreus in Taiwan, and S. brachiariae, recently described in B. brizantha China (Yeh and Kirschner, 2014; Liu et al., 2017). S. implicatum (formerly known as Acremonium implicatum) was identified as a seed-transmitted endophyte of Brachiaria species, where it may play a role in protecting plants against fungal pathogens, such as Drechslera spp., which causes leaf spots (Kelemu et al., 2001). Seedborne S. implicatum can colonize other plant parts after germination and provide fitness advantages to the host (Kago et al., 2016).

Two isolates of Meira sp. from B. ruziziensis were the sole representatives of Phylum Basidiomycota in this study. Species of this genus were described as acaropathogenic and can be found as endophyte of many plant species (Rush and Aime, 2013). Moreover, a recent study reported the isolation of Meira sp. as an endophyte of the temperate grass Ammophila arenaria (Sánchez Márquez et al., 2012).

In vitro analyses showed that the endophytic fungi Paraconiothyrium sp. (isolates BLMT2.1, BCMT4.1, BBPB4.1, and BBXE1), Sarocladium kiliense, Acremonium curvulum, Setophoma terrestris, Dissoconium sp. (isolate BMMT1.3) and Cladosporium flabelliforme exhibited antagonistic activity against S. sclerotiorum (p < 0.05), with percentages of growth inhibition ranging from 25 to 60 (Figure 4D). Paraconiothyrium sp. BBXE1, BBPB4.1, BCMT4.1, and S. kiliense showed the highest values of Antagonism Percentages.

Figure 4 (A) In vitro antagonism of Sarocladium kiliense (Sk) and (B) Paraconiothyrium sp. BBPB4.1 (P) in relation to Sclerotinia sclerotiorum (Ss) in dual cultures. (C) Growth of Ss after 7 days in the absence of antagonist. (D) Antagonism percentage values exhibited by selected endophytic fungi against Ss (p < 0.05). Means followed by same letter do not differ by the Tukey test at 5 % level (n = 3) and bars represent the standard error of the mean. Arrows in (A) and (B) show the inhibition zones. 

The growth inhibition of S. sclerotiorum is not likely caused by the synthesis of volatile molecules, because no inhibition was observed when these fungi were grown in bipartite plates. Coniothyrium minitans is a known mycoparasite capable of controlling plant diseases caused by fungal pathogens, including S. sclerotiorum (Whipps et al., 2008), reducing survival of sclerotia and production of apothecia (Zeng et al., 2012). Almeida et al. (2014) reported the isolation of graminin B, a compound with antibiotic activity obtained from fermentation of broths of species P. hawaiensis. Three isolates of Paraconiothyrium sp. showed the highest inhibition rates in our bioassays (BBXE1, BBPB4.1, and BCMT4.1), suggesting that other Paraconiothyrium species can be used in the biocontrol of white mold and that antibiosis is another mode of action of them.

One endophytic isolate of S. kiliense reduced the growth of S. sclerotiorum by more than a half through the excretion of metabolites in the medium and formation of inhibition halo (Figure 4A). The bioactivity of secondary metabolites produced by Sarocladium species is well documented in the case of S. oryzae, a producer of the phytotoxic helvolic acid and the antifungal compound cerulenin (Hittalmani et al., 2016). Secondary metabolites produced by an endophytic isolate of S. implicatum from Brachiaria inhibited the growth of Rhizoctonia solani, causal agent of foliar blight in Brachiaria, and Pyricularia oryzae, causal agent of rice blast (Kelemu et al., 2001). Considering that in vitro antagonism does not always predict efficacy, in vivo studies are needed to confirm these considerations since they were based only on in vitro test.

In the management of white mold caused by S. sclerotiorum, one of the recommendations is crop rotation of the host-plant with Brachiaria spp. This strategy can be potentialized by the application of Trichoderma harzianum over grass litter prior to the next sowing of the host plant, increasing eradication effects on the pathogen initial inoculum (Görgen et al., 2009). Although no Trichoderma sp. was recovered from Brachiaria sp., abundant Paraconiothyrium spp. may offer a reliable disease control. Trichoderma spp. has limited performance under cooler temperatures (Paula Júnior et al., 2012), the most favorable condition for S. sclerotiorum ascospore release and white mold epidemic outbreak (Bolton et al., 2006) as well as for C. minitans sclerotia parasitism (Whipps et al., 2008).

Once Brachiaria sp. harbors endophytic fungi, such as Paraconiothyrium sp. with inhibitory activity against S. sclerotiorum, rotation with Brachiaria sp. enriched with the antagonistic endophytic fungus may offer a package composed of two disease management tools by planting Brachiaria sp. This prospect should be explored in future in vivo studies with active application of endophytes to Brachiaria seeds or to the field. The bioactive compounds produced by these endophytic fungi also deserve further investigation, since they may be used in the control of plant diseases or other fields, such as medicine and industry.

Conclusions

Brachiaria spp. from different experimental plots and pasture fields in Brazil yielded 28 taxa of stem-associated endophytic fungi. Two-thirds of all isolates belong to four of the most common species, Paraconiothyrium sp., Phoma sorghina, Plenodomus sp., and Sarocladium strictum.

Four isolates of the most common endophytic fungus Paraconiothyrium sp. (BLMT2.1, BCMT4.1, BBPB4.1, and BBXE1) in addition to Sarocladium kiliense, Acremonium curvulum, Setophoma terrestris, Dissoconium sp. (isolate BMMT1.3), and Cladosporium flabelliforme exhibited in vitro antifungal activity against S. sclerotiorum. Tests with Brachiaria sp. enriched with the antagonistic endophytic fungus and the investigation of bioactive compounds produced should be explored in future studies.

Acknowledgements

To the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), for the scholarships granted to the first author; to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), for the financial support to this study (CAG-APQ-02100-13); to the researchers of the Universidade Federal de Lavras, Embrapa Gado de Corte, Embrapa Gado de Leite, and Embrapa Amazônia Oriental, for the providing the samples used in this work.

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Received: June 27, 2018; Accepted: October 29, 2018

*Corresponding author <patricia@ufla.br>

Edited by: Fernando Dini Andreote

Author's Contributions

Conceptualization: Gama, D.S.; Medeiros, F.H.V.; Cardoso, P.G. Data acquisition: Gama, D.S.; Cardoso, P.G. Data analysis: Gama, D.S.; Santos, Í.A.F.M. Design of methodology: Gama, D.S.; Medeiros, F.H.V.; Cardoso, P.G. Writing and editing: Gama, D.S.; Santos, Í.A.F.M.; Abreu, L.M.; Medeiros, F.H.V.; Duarte, W.F; Cardoso, P.G.

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