Cultivable root endophytic fungi associated with Acrocomia aculeata and its antagonistic activity against phytopathogenic oomycetes

Abstract

Macaw palm (Acrocomia aculeata Jacq.) is a palm, native to Brazilian territory that stands out due to the amount of oil produced with applications in the biodiesel industry, cosmetics, and food. Its commercial exploitation in Brazil, including phytosanitary management is based on concepts and practices of regenerative agriculture, which has the responsibility of sustainable cultivation by avoiding, for example, the use of chemical pesticides. Recently, root and stem rot disease were reported in macaw palm seedlings caused by Phytophthora palmivora. Managing this plant pathogen is complex, and the chemical control of this soil-borne oomycete is not viable, in addition to the negative impact on the environment. Many microorganisms are studied and used as biological control agents (BCAs) against pathogens, among them the community of endophytic fungi associated with plants. This is a sustainable biotechnological alternative for plant disease control. The community of cultivable endophytic fungi associated with healthy roots of macaw palm was explored using the extinction cultivation technique and a screening was carried out to select potential antagonists against oomycetes through the dual culture test. Specific gene regions from the best isolates were amplified for identification. A total of 250 isolates were obtained, and 46 were selected for in vitro tests against representatives of phytopathogenic oomycetes. After tests against Phytophthora heterospora, Phytophthora palmivora, Pythium aphanidermatum, and Pythium deliense, two isolates were selected as potential antagonists. The phylogenetic analysis of selected isolates showed that they belong to two different species: Talaromyces sayulitensis COAD 3605 and Epicoccum italicum COAD 3608. The percentage of inhibition of phytopathogenic oomycetes testedwas until 82% in the antagonism tests conducted. From the 46 isolates selected, only 2 were selected which showed great antagonistic activity towards all oomycetes tested. These fungi will be used in upcoming studies that aim to determine the effectiveness of endophytes in controlling diseases caused by oomycetes in the field.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-024-01482-z.

Introduction

Macaw palm (Acrocomia aculeata Jacq.) is a member of the Arecaceae, native to South and Central America, and can be found in almost the entire Brazilian territory [1]. Acrocomia aculeata stands out due to the amount of oil produced: between 4 and 6 ton of oil per hectare, depending on the density of crops, with applications in the biodiesel industry, cosmetics, and food [2]. This crop occurs mainly in areas with high solar radiation, and equally is adapted to sandy and clayey soils [3].

The commercial exploitation of macaw palm in Brazil is based on concepts and practices of regenerative agriculture, which has the responsibility for more sustainable cultivation. This is due to the largest macaw palm-producing company in the world, which aims to recover degraded Brazilian areas for planting this crop, has serious sustainability policies that are followed as a requirement for commercial exploitation [4]. This responsibility for the sustainable cultivation of macaw palm in Brazil comes from a global need to implement large-scale agriculture that is combined with environmental preservation [5]. Therefore, is necessary to sustainably manage diseases.

Recently, the etiology of the main disease found in a major macaw palm nursery in Brazil was described, the basal stem and root rot [6]. This disease affects nursery plants and can be transferred to the field when transplanting infected seedlings which leads to the death of young seedlings. The causal agent is the oomycete Phytophthora palmivora which was reported in commercial production areas. The Phytophthora genus is currently divided into 16 phylogenetic clades [7] and P. palmivora is into clade IV of this genus, in a sister position to P. heterospora. Both species are associated with rot disease [8].

Phytophthora palmivora is a plant pathogen of global concern, due to its broad host range and the economic losses it causes in the field. This oomycete can infect many plant species [9], with several reports in perennial crops such as Elaeis guineenses, Bactris gasipaes, Theobroma cacao, Caryca papaya and Artrocarpus heterophyllus [10–13]. The management of this soil-borne pathogen in commercial areas is complex, and the chemical control damages the environment, which is not consistent with the sustainability policies established for the cultivation of macaw palm in Brazil. In the case of oomycetes, copper products and other active ingredients such as ethylene bisdithiocarbamate manganese complex, mandipropamid, and zinc ethylene bisdithiocarbamate are recommended for chemical control but are classified as very harmful to the environment with toxic residues that are difficult to degrade in the soil [14]. In addition, chemical control is costly, due to the high investment and low efficiency of controlling soil-borne pathogens [15].

The use of biological control, although it is still expensive, has many advantages over chemical treatments. It is a sustainable alternative in this context because can reduce the pressure of these soil pathogens at a low cost, minimize negative environmental impact during phytosanitary management, and contribute to the reduction of the dependence on chemicals of agriculture [16]. Some microorganisms have been studied and used as biological control agents (BCAs) against plant pathogens, among them the community of endophytic fungi associated with plants has been showing great application potential worldwide [17–20]. Endophytic fungi colonize the internal tissues of plants, without causing apparent damage [21] and their antifungal activity is attributed to different mechanisms of action such as mycoparasitism, competition, and antibiosis that act to inhibit the growth of pathogenic organisms [22, 23]. A. aculeata-associated microbial community is still little explored with few on fungi associated with leaves and fruits, which leads to a lack of study of potential applications of their endophytic fungi in biological control for this crop [24, 25].

Considering the high productivity of oil in the macaw palm and their several uses in industry, its cultivation tied to the principles of regenerative agriculture, less information about the microbiota associated with the roots and their potential for application of the endophytic fungi for disease control, this study aims to (i) Explore the potential endophytic fungal associated with the roots of A. aculeata through the isolation of fungi, (ii) Identify effective biocontrol agents by assessing the in vitro antagonistic potential of these isolated endophytic fungi against P. palmivora and other oomycetes.

Materials and methods

Plant material and fungal isolation

Roots samples from three healthy 6-year-old Acrocomia aculeta plants were collected in Minas Gerais state, Brazil, in the cities of João Pinheiro (northwest region) in December 2021 and Viçosa (Zona da Mata region) in January 2022, at the S.Oleum company premises, amounting to three samples. For each sample, nine grams of root tissue were washed with tap water and submitted to the dilution-to-extinction technique [26, 27], with modifications. The material was surface disinfected by soaking in 70% ethanol (added two drops of tween each 100 mL) for 1 min and NaClO (2.5% activated chlorine) solution for 4 min and rinsed twice in sterilized saline solution (0,85% NaCl). Aliquots of 150 µl of sterilized saline solution were placed in solid YMC (10 g/L malt extract, 2 g/L yeast extract, 13 g/L agar) supplemented with streptomycin (50 mg/L) and tetracycline hydrochloride (50 mg/L) and incubated for 4 weeks, at 25 °C to verify the efficiency of the endophyte isolation technique. The plant material was subsequently crushed with saline solution (0,85% NaCl) in a blender 600 wats for 1 min and filtered in sieves to obtain fragments ranging from 106 to 212 μm in size. The fragments were centrifuged at 7000G for 5 min and resuspended in 20 ml of 0.1% carboxymethylcellulose (CMC) for obtaining the dilutions: 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Aliquots of 150 µl each dilution were placed in solid YMC and incubated for 4 weeks, at 25 °C. Daily monitoring of fungal growth was performed and the mycelia were transferred to potato dextrose agar (PDA, 39 g/L, KASVI). The different morphotypes of the isolated fungi were selected for the next steps.

Morphological characterization

A total of 250 isolates were obtained and 60 endophytic fungal representatives of each from morphotype were selected according to the characteristics of their colonies. These selected fungal isolates were grown on PDA and corn meal agar (CMA, 20 g/L), and kept at 20–25 °C for 2–3 weeks, for microscopic observation and colonies record. The microscopic preparations were mounted by placing the fungal structures on a drop of lactoglycerol solution in slides and covered with a coverslip. Photographs were taken with an Olympus BX53 microscope equipped with a digital camera Olympus Q- Color5™. The Olympus cellSens Dimension v. 1.9 software system was used for measuring the morphological characteristics of isolates. Isolates of species belonging to potential pathogenic genera were excluded during the morphological characterization for the next step, as recommended by Bettiol et al. [28].

Screening of endophytic fungi with antagonistic activity

This step was carried out to evaluate the ability of endophytic fungi to inhibit the growth of Phytophthora heterospora COAD 3414 (a highly aggressive isolate, used as a reference in the Laboratório de Micologia e Etiologia de Doenças Fúngicas de Plantas at Universidade Federal de Viçosa). The fungi with different morphotypes selected by the above-mentioned morphological characterizaiton were grown up on PDA for screening. An in vitro dual culture assay was run in which a 5-mm-diameter mycelial plug from 7-day-old of each endophytic fungi was placed at 1.5 cm to the edge of plastic Petri dishes (90 mm) containing PDA and a mycelial plug from 7-day-old of P. heterospora COAD 3414 was placed at 1.5 cm to the other edge of the dish. For the control treatment, only the mycelial plug containing P. heterospora was placed in the Petri dish. The Petri dishes were maintained at 25 °C until the pathogen grew fully on the plate. The growth inhibition (%) of the pathogen was determined by the formula: inhibition (%) = (R– r)/R * 100, where R is the radial growth of the pathogen in the control plate and r is its growth in the dishes with endophytes isolates [29]. The experiment was performed with 4 replicates in completely randomized designs.

The antagonistic activity of endophytic fungi was classified according to inhibition percentage. Isolates that showed the highest percentage of inhibition (above 30%), were selected for the identification and new in vitro antagonist activity tests for other oomycetes. Moreover, the cultures of selected isolates were stored in 2 ml microtubes containing sterile distilled water at ambient temperature, in 2 ml microtubes containing glycerol solution at -20 °C, and anhydrous silica gel at 5 °C [30, 31]. The isolates selected were also deposited in the culture collection “Coleção Octávio Almeida Drummond” (COAD) housed at Universidade Federal de Viçosa.

In vitro antagonistic test

The isolates that showed the highest percentage of inhibition for P. heterospora COAD 3414 were selected and used in new antagonism tests with the pathogenic oomycetes: P. palmivora (isolates COAD 3580 and COAD 3581) that cause root and basal bulb rot in A. aculeata, Pytium aphanidermatum COAD 3618, and Py. deliense COAD 3620. In vitro, dual culture assays were run with the selected isolates in the same way as above. The growth inhibition (%) of the pathogen was determined according to the formula in item 2.3. All experiments were repeated twice, each with 4 replicates in completely randomized designs. The antagonistic activity of endophytic fungi was classified according to inhibition percentage and analyzed using analysis of variance (ANOVA), followed by Tukey’s test; p-value ≤ 0.05.

The antagonistic activity through the antibiosis by volatile compounds for the isolates that showed the highest percentage of inhibition against P. palmivora COAD 3580 and COAD 3581, Py. aphanidermatum COAD 3618, and Py. deliense COAD 3620 was checked. In vitro, bioassays were realized in compartmentalized plastic Petri dishes containing PDA with endophytic fungi selected. A 5-mm-diameter mycelial plug from a 7-day-old of each endophytic fungi was inoculated to 2.25 cm on the edge of plastic Petri dishes containing PDA and after 7 days a mycelial plug from a 7-day-old of oomycetes was inoculated at 2.25 cm of the other edge of the dish compartment. For the control treatment, a mycelial plug containing mycelia of the oomycete was inoculated in the dish compartment. The dishes were stored at 25 °C until the pathogen grew fully on the control dish. The growth inhibition (%) of the pathogen by volatile compounds was determined by the formula: inhibition (%) = (R– r)/R * 100, as described in item 2.3. All experiments were repeated twice, each with 4 replicates in completely randomized designs. The data were subjected to the T student test.

DNA extraction, amplification, and sequencing

The selected isolates were grown on PDA with sterile cellophane paper, at 25 °C for 7 days. The mycelia were collected with a sterile toothpick and transferred to 2 mL microtubes containing 600 µl of Nuclei Lysis Solution of the Wizard Genomic DNA Purification kit (Promega), 100 mg polyvinylpyrrolidone (Sigma-Aldrich), and four steel beads (2.8 mm diameter). Next, the samples were mixed and crushed with the L-Beader 6 (Loccus Biotecnologia) for 60 s at 4,000 rpms. After maceration, the genomic DNA was extracted, according to Pinho et al. [32].

According to the morphological analysis and identification of the fungal genera, specific gene regions were selected for amplification. The internal transcribed spacer 1 and 2 regions and intervening 5.8 S rDNA region (ITS) and nuclear 28 S rDNA region (LSU) were amplified for isolates that did not produce any reproductive structures and therefore could not be identified to the genus level. The primers used for each region gene are described in Table 1. The PCR conditions for regions consisted of initial denaturation at 94 °C for 2 min; 34 cycles of denaturation at 94 °C for 30 s, primer annealing according to Table 1, primer extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min. The amplified fragments were purified and sequenced by Macrogen Inc., South Korea.

Table 1.

Primer sets in PCR assays for plant endophytic fungi

Target/Locus	Primer	Direction		Primer annealing temperature	Reference
Beta-tubulin (tub2)	T1	Foward		52 °C		[33]
	Tub4RD	Reverse				[34]
RNA polymerase II subunit 2 (RPB2)	RPB2-5F2	Foward		50 °C		[35]
	RPB2-7cR	Reverse				[36]
Large Subunit (LSU, 28 S) of the rRNA	LR6	Foward		52 °C		[37]
	ITS5	Reverse				[38]
Calmodulin (CaM)	CMD5	Foward		55 °C		[39]
	CMD6	Reverse
Beta-tubulin (tub2)	2ª	Foward		55 °C		[40]
	Bt2b	Reverse
RNA polymerase II subunit 2 (RPB2)	fRPB2-7cF	Foward		52 °C		[36]
	fRPB2-11aR	Reverse
Translation Elongation Factor 1- alpha (tef1-α)	EF1-983 F	Foward		56 °C		[41]
	EF1-2218R	Reverse
RNA polymerase II subunit 1 (RPB1)	RPB1af	Foward		51 °C		[42]
	RPB1cr	Reverse
Small Subunit (SSU, 18 S) of the rRNA	NS1	Foward		46 °C		[38]
	NS4	Reverse
Internal Transcribed Spacer (ITS)	ITS5	Foward		52 °C		[38]
	LR6	Reverse				[37]

Phylogenetic analysis

The contigs were prepared using the FinchTV software, and the new sequences were deposited in the GenBank. The sequences were compared against the GenBank database using the BLAST tool. Sequences of type cultures and reference isolates obtained from GenBank were included in the phylogenetic analyses to identify the isolates (Table S1, and S2). Nucleotide substitution patterns were determined using model test 2.1.7 [43]. The likelihood values were calculated, and the models were selected according to the Akaike Information Criterion (AIC).

The sequences were aligned using the MUSCLE ^® algorithm performed in the MEGA X software system [44]. Bayesian Inference (BI) phylogenetic tree was performed for the data sets of each isolate, using tools implemented in the CIPRES Science Gateway portal with the tool MrBayes v.3.2.7a [45]. The BI analysis was performed by the Monte Carlo method via Markov Chains (MCMC), and two independent analyses were performed simultaneously. Four MCMC chains were run in each analysis, which generated random trees up to 10,000,000 generations, with sampling every 1,000 generations. A total of 20,002 trees were sampled, and the first 2,500 trees (25%) of each analysis were discarded. The trees were visualized in the Fig.Tree software system and edited in graphics programs.

Results

Isolation, morphological characterization, and screening

A total of 250 endophytic fungi isolates were obtained from three samples of healthy root tissue from A. aculeata. According to the analysis of morphological characteristics of the colony and microscopic observations, 60 isolates were selected. The morphological characterization of selected fungi evidenced 13 Fusarium or Fusarium-like genera and 1 Pestalotiopsis-like genus that were excluded for subsequent analyses. Thus, the screening of potential antagonists with P. heterospora COAD 3414 was carried out with 46 endophytic fungi.

In the dual culture test, after the growth of P. heterospora COAD 3414 in the entire control plate, measurements were performed, and the inhibition percentage of each selected isolate was calculated (Table S3). According to the percentage of inhibition (above 30%), 11 isolates were selected for identification by phylogenetic analysis (Fig. 1) and for new in vitro antagonistic tests against other oomycetes (Table 2, Figure S1). Furthermore, these 11 isolates were deposited in the COAD collection.

Fig. 1.

Eleven isolates with percentage of inhibition between 30 and 100% against Phytophthora palmivora COAD 3414 selected for sequencing and in vitro tests with new oomycetes, cultivated in PDA and CMA. In order from left to right: COAD 3608, COAD 3603, COAD 3607, COAD 3606, COAD 3611, COAD 3612, COAD 3617, COAD 3609, COAD 3605, COAD 3604, COAD 3610. Scale bars = 1,8 cm. COAD: code corresponding to the collection of fungal cultures “Coleção Octávio Almeida Drummond”

Table 2.

Inhibition percentage of selected endophytes fungi against Phytophthora Heterospora COAD 3414 with inhibition above 30%

Endophytic fungi	Inhibition (%)
COAD 3606 COAD 3609 COAD 3608 COAD 3605 COAD 3603 COAD 3604 COAD 3610 COAD 3607 COAD 3617 COAD 3611 COAD 3612	70,27 64,86 62,16 38,74 37,84 36,94 35,13 33,33 30,94 30,63 30,63

COAD: code corresponding to the collection of fungal cultures Colecao Octavio Almeida Drummond

In vitro antagonistic test

The results of the dual culture tests carried out with the 11 selected isolates against P. palmivora showed high percentages of inhibition, as well as in the screening with P. heterospora COAD 3414 shown above (item 3.1). In the P. palmivora tests, COAD 3605, COAD 3608, COAD 3609, COAD 3607, and COAD 3606 differed significantly from the other endophytes (Tukey test; p-value ≤ 0.05), with the percentage of inhibition from 82.50% to 68 0.93% growth of P. palmivora in vitro. For the other isolates, the percentages of inhibition ranged from 44.28 to 22.14%, as shown in Table 3. For tests of endophytic fungi confronted with Pythium species, the inhibition rate varied in lower percentages, from 40.15% to values of null (Table 3). Despite lower values compared with the tests against P. palmivora, the treatments differed statistically from each other, with higher inhibition rates attributed to COAD 3608, COAD 3606, and COAD 3607 for Py. aphanidermatum, and COAD 3606, COAD 3609, COAD 3617, COAD 3607, COAD 3605, COAD 3608, and COAD 3612 for Py. deliense.

Table 3.

Inhibition percent inhibitionof selected endophytes fungi from screening against Phytophthora Palmivora COAD 3580, Phytophthora Palmivora COAD 3581, Pythium aphanidermatum COAD 3618, and Pythium Deliense COAD 3620

Endophytic fungi	P. palmivora COAD 3580 inhibition (%)	SD	P. palmivora COAD 3581 inhibition (%)	SD	Py. aphanidermatum COAD 3618 inhibition (%)	SD	Py. deliense COAD 3620 inhibition (%)	SD
COAD 3605 COAD 3608 COAD 3609 COAD 3607 COAD 3606 COAD 3611 COAD 3603 COAD 3617 COAD 3610 COAD3604 COAD 3612	82.50 a 73.93 a 73.21 a 72.86 a 68.93 a 40.36 b 31.43 b 31.43 b 27.86 b 23.93 b 23.93 b	15,33 8,34 3,64 3,95 4,61 7,08 16,99 5,74 14,90 4,19 9,83	80.00 a 78.21 a 77.50 a 69.28 a 71.07 a 39.64 bc 36.07 abc 44.28 b 22.14 d 29.28 cd 28.21 cd	10,20 3,22 1,46 3,32 1,13 3,00 3,10 5,24 9,48 4,13 7,24	6.09 bc 17.14 a 0.47 c 10.11 b 12.84 ab 0.10 c 0.10 c 3.01 c 0.10 c 1.85 c 1.17 c	2,11 6,38 0,98 2,38 1,11 0,19 0,29 3,75 0,19 2,14 0,86	35.61 a 35.23 a 40.15 a 39.01 a 40.15 a 0.00 b 0.00 b 38.64 a 0.00 b 0.00 b 33.84 a	8,79 0,76 4,71 3,98 6,61 0 0 0,87 0 0 0,71

COAD: code corresponding to the collection of fungal cultures “Coleção Octávio Almeida Drummond”. SD: Standard deviation. Different lowercase letters indicate significant differences between the percentage of inhibition of each isolate by Tukey’s test (p-value ≤ 0.05)

Fig. 2.

Dual culture tests with phytopathogenic oomycetes on the left side of the plate. Phytophthora heterospora COAD 3414 (a). Talaromyces sayulitensis COAD 3605 (b) and Epicoccum italicum COAD 3608 (c) against P. heterospora COAD 3414. Phytophthora palmivora COAD 3580 (d). T. sayulitensis COAD 3605 (e) and E. italicum COAD 3608 (f) against P. palmivora COAD 3580. P. palmivora COAD 3581 (g). T. sayulitensis COAD 3605 (h) and E. italicum COAD 3608 (i) against P. palmivora COAD 3581. Pythium aphanidermatum COAD 3618 (j). T. sayulitensis COAD 3605 (k) and E. italicum COAD 3608 (l) against P. aphanidermatum COAD 3618. Pythium deliense COAD 3620 (m). T. sayulitensis COAD 3605 (n) and E. italicum COAD 3608 (o) against Pythium deliense COAD 3620

The antagonism tests performed with two representative endophyte isolates (COAD 3605 and COAD 3608) against all phytopathogenic oomycetes tested are illustrated in Fig. 2.

In vitro, bioassays of volatile compounds were run with the isolates COAD 3605 and COAD 3608 (Table 4). The percent of in vitro inhibition caused by volatile compounds produced by the selected endophytic fungi ranged from 33.33 to 2.22%.

Table 4.

Inhibition percent of oomycetes by volatile compounds produced by best endophytic fungi

Endophytic fungi	P. palmivora COAD 3580 inhibition (%)	P. palmivora COAD 3581 inhibition (%)	Py. aphanidermatum COAD 3618 inhibition (%)	Py. deliense COAD 3620 inhibition (%)
COAD 3608 COAD 3605	24.16* (6,33) 3.33* (2,72)	33.33n.s. (4,36) 27.25n.s. (4,75)	21.11* (3,33) 6.66* (1,92)	12.22n.s. (3,56) 2.22n.s. (10,71)

COAD: code corresponding to the collection of fungal cultures “Coleção Octávio Almeida Drummond”. Asterisks indicate significant differences between the percentage of inhibition of each isolate by the student t-test (p ≤ 0.05). Standard deviation values ​​are indicated in parentheses

Phylogenetic analyses

According to the phylogenetic analyses using sequences from the selected regions for each isolate, the potential BCAs were identified into 11 different taxonomic groups, distributed within a phylum, three classes, five orders, nine families, and 11 genera, including a new genus (Table S4). All the 11 potential BCAs belong to the Ascomycota, and the most representative class was Sordariomycetes (seven isolates), followed by Dothideomycetes (three isolates) and finally the class Eurotiomycetes (one isolate). The most representative order was Hypocreales, followed by Pleosporales.

The single isolate within the class Eurotiomycetes (COAD 3605) corresponds to the genus Talaromyces sect. Talaromyces, forming a monophyletic clade of high support with the type T. sayulitensis species (Fig. 3). One of the isolates belonging to the Pleosporales order (COAD 3608) is Epicoccum italicum, in a monophyletic clade with high support for this species (Fig. 4). Phylogenetic tree inferred from Bayesian inference of concatenated alignments with 88 strains representing the genus Talaromyces sect. Talaromyces and 75 strains representing the genus Epicoccum are available in supplementary data (Figs S2 and S3). Information on the best models for the phylogenetic analyses of these two isolates is described in Table 5. Their morphological characteristics and microscopic observations of mycoparasitism are shown in Fig. 5 (T. sayulitensis COAD 3605) and Fig. 6 (E. italicum COAD 3608).

Fig. 3.

Phylogenetic tree inferred from Bayesian inference of a concatenated alignment with β-tubulin, CaM, and RPB2 sequence data of strains representing part of the genus Talaromyces sect. Talaromyces. Posterior probability values ​​above 0.7 are close to nodes. The tree is rooted to Talaromyces trachyspermus (CBS 373.48)

Fig. 4.

Phylogenetic tree inferred from Bayesian inference of a concatenated alignment with β-tubulin, RPB2, and LSU sequence data of strains representing part of the genus Epicoccum. Posterior probability values ​​above 0.7 are close to nodes. The tree is rooted to Didymella americana CBS 568.97 and D. americana CBS 185.85

Table 5.

Information about the datasets of each group in the phylogenetic analysis

	Talaromyces sayulitensis				Epicoccum italicum
Partition	NT	C	BM		NT	C	BM
β-TUB	88	500	HKY + I + G		73	339	GTR + I + G
CaM	88	559	SYM + I + G		-	-	-
RPB2	86	872	HKY + I + G		71	596	GTR + I + G
LSU	-	-	-		75	973	GTR + I + G

NT= number of taxa; C= characters; BM= best nucleotide substitution model

Fig. 5.

Talaromyces sayulitensis COAD 3605 and its mycoparasitism action. (a) Conidiophores. (b) Conidiophore. c-d. Conidiogenous cells and chain conidia. e. Conidia. f.T. sayulitensis above hyphae of P. palmivora (black arrow: P. palmivora COAD 3581 hyphae; white arrow: T. sayulitensis hyphae). a-c: Scale bars = 20 μm. d-f: Scale bars = 10 μm

Fig. 6.

Epicoccum italicum COAD 3608 and its mycoparasitism action. (a) Sporodochium. (b) Conidiogenous cells. (c) Dictyoconidia. e– f.E. italicum above hyphae of P. palmivora (black arrow: P. palmivora COAD 3581 hyphae; white arrow: E. italicum hyphae). d - h. Scale bars = 20 μm

Four genera belonging to the Hypocreales were identified by the phylogenetic analyses based on the ITS gene region and correspond to Nectriaceae: Ilyonectria COAD 3603, Gliocladiopsis COAD 3607, Mariannaea COAD 3609 and Cylindrocladiella COAD 3617. The isolate COAD 3612 is a single representative Niessiliaceae, belonging to the Niesslia. According to the analyses of the ITS and LSU gene regions, the isolate COAD 3611 corresponds to the genus Acrocalymma and forms a well-supported clade with the species A. vagum (data not shown).

Furthermore, the only isolate within the Chaetomiaceae family (COAD 3610) forms a well-supported sister clade with the genus Dichotomopilus. The phylogenetic analysis of this isolate, using the ITS region, indicates that COAD 3610 is a possible new species. The phylogenetic analysis of the isolated COAD 3603, carried out with the regions ITS, RPB1, SSU, and TEF1-α, showed that the species corresponds to the genus Pseudophialophora, representing a possible new species, and the isolate COAD 3604, which is a possible new genus of dark septate endophyte (DSE) belonging to the Latoruaceae family.

Discussion

Acrocomia aculeata is a native Brazilian species of great underexplored economic and scientific potential. This study accesses for the first time the endophytic fungal community associated with macaw palm and their potential for biocontrol of phytopathogens, especially the oomycetes causing the basal stem and root rot of macaw palm.

A total of 250 endophytic isolates were obtained from root samples from three A. aculeta healthy plants. Tropical trees harbor a great community of endophytic fungal species [46–48]; however, traditional isolation techniques may not elucidate all cultivable host-associated microbiota, due to the chosen technique and the cultivation and incubation conditions. The modified dilution-to-extinction culture method used in this work [26] may have guaranteed access to many roots’ endophytic isolates, resulting in more taxonomic information, phenotypic evaluations, and bioprospecting of isolates.

The number of cultivable isolates highlighted a great community of endophytic fungi associated with macaw palm root tissues that have not yet been explored. Studies on the microbial community associated with A. aculeta are scarce. As far as we know, it is restricted to the study of fermenting microorganisms in the fruits [49], and some on fungal isolation associated with palm leaves [24, 25]. In addition to being few, these studies have not yet explored the community of root fungi.

Information on the microbial community associated with other members of the Arecaceae family is more robust. Pinruan et al. [46] isolated 340 endophytic fungi from oil palm, with representatives of the phylum Ascomycota and Basidiomycota. Konta et al. [50] described a new genus and nine new fungal species associated with palm trees in Thailand. Despite the wealth of works on fungi associated with Arecaceae, much still needs to be explored regarding the potential applications of these fungi, mainly as biological control agents (BCAs). In our study, in addition to isolating endophytes associated with macaw palms, we also began to fill these gaps of possible BCAs associated with Arecaceae. Furthermore, all 11 isolates with antagonistic activity against tested oomycetes are from different genera and belong to eight distinct families, highlighting the taxonomic richness and application potential of different groups of root endophytes.

In the present study, the dual culture tests made it possible to verify the antagonistic activity of endophytic fungi through the mechanism of competition for space and nutrients on the Petri dish. This test is often used for the screening of potential antagonists [18, 51]. Despite the wide use of dual culture and its results leading to a good selection of isolates, this method can disfavor the competition of antagonists when the tested pathogens have a very fast growth rate [52]. This could explain the range of lower values for all isolates confronted with Py. aphanidermatum and Py. deliense compared to the results with P. heterospora and P. palmivora since Pythium is a fast-growing oomycete genus in PDA culture media.

Two isolates that showed high inhibition percentages for all tested oomycetes were identified: T. sayulitensis COAD 3605 and E. italicum COAD 3608. These isolates had high phylogenetic support in analyses, each one grouping into monophyletic clades of known species.

The isolate T. sayulitensis COAD 3605 which showed higher inhibition percentages for all tested oomycetes was reported for the first time in settled dust in Mexico [53] and already was found in solid oil shale and rhizospheric soil [54]. Savković et al. [55] checked the activity of cellulolytic and carbonate dissolution ability for T. sayulitensis indicating it is a species with potential for biodegradation. Matiz-Villamil et al. [56] already reported this species’ ability together with two species of Bacillus to accelerate the composting process of organic waste from pig farming (including pig carcasses). In addition, Lopez [57] verified the ability of T. sayulitenses to degrade rice straw for rice and tomato crops, increasing the growth of these plants. Despite the biotechnological possibilities, the species T. sayulitensis had not yet been studied as a candidate for BCA. Talaromyces sayulitensis is also a fast-growing species able to produce many spores in culture media, which is a desirable trait in the selection of BCAs [58].

The genus Talaromyces was initially described by Benjamin [59] and reclassified as the teleomorphic species of Penicillium. This genus is divided into seven sections and comprises species of various environments like soil, air, and plants [60]. Many species produce enzymes, compounds, and pigments. In addition, the ability of biocontrol and phosphate solubilization has already been reported for this genus [61–63]. For example, T. flavus can degrade the cell walls of Py. ultimum [61] and has antagonistic activities against P. capsica KACC, Fusarium oxysporum KACC, and Rhizoctonia solani AG2-1 [51].

The isolate E. italicum COAD 3608 also stood out among the endophytic fungi. That species was described by Chen et al. [64] and isolated for the first time from Acca sellowiana (Myrtaceae) in Italy. In this work, we are reporting E. italicum for the first time in Brazil. Epicoccum comprises species isolated from plants, soil, and air. Among the species of this genus, some stand out for their ability to control phytopathogens such as P. infestans and Fusarium ssp. Different biological control mechanisms are described for species of this genus, such as action antagonists, production of mycotoxins, and inducing resistance in the host plants [65].

There is already a vast literature demonstrating the ability of E. nigrum to control different phytopathogens, in addition to reports for Epicoccum layuense, Epicoccum dendrobii, Epicoccum mezzettii, and Epicoccum minitans. Also, recently, Rivera-Vega et al. [66] reported the ability of E. italicum to control soybean looper (Chrysodeixis includens) and nematode cysts (Heterodera glycines) for soybeans, indicating its potential for phytosanitary management. In the present study, high inhibition values were found for the isolate E. italicum COAD 3608, especially when confronted with P. palmivora COAD 3580 and COAD 3581 (73.93% and 78.21% respectively). Future in vivo tests with this species are important to verify its potential as BCA against oomycetes.

According to data from in vitro bioassays of antibiosis by volatile compounds, the isolates T. sayulitensis COAD 3605 and E. italicum COAD 3608 have a greater inhibition potential for P. palmivora when compared to Pythium species. Also, mycoparasitism may be another mechanism involved in the antagonism of these two endophytic fungi against oomycetes, according to the results of the dual culture test. One month after setting up the dual culture test, intense colonization of endophytic fungi under oomycetes in Petri dishes was observed, which leads us to suggest such a possibility.

During a study for the selection of BCAs, it is important to consider all characteristics of the isolate that restrict its registration and use. According to a review carried out by Köhl et al. [58], among the undesirable characteristics is the pathogenicity of the isolate in plants. Thus, isolates with pathogenicity reports and/or included in taxonomic groups with many pathogenicity reports should be carefully analyzed for possible selection as BCAs, mainly considering the species’ historic, as well as the effector genes, present in these isolates pathogenic and the phenomenon of horizontal transfer that can naturally occur between microorganisms. Considering this, despite good inhibition results, the isolates Ilyonectria sp. COAD 3609, Gliocladiopsis sp. COAD 3606, and Cylindrocladiella sp. COAD 3607, were not selected for in vivo tests.

These isolates are within Nectriaceae, which harbors many plant pathogenic taxa [67]. The genus Ilyonectria has 37 species described according to the Mycobank database [68], some reported as associated with rot diseases in different plants, including some members of Arecaceae. Aiello et al. [69], described Ilyonectria palmarum causing dry basal stem rot of different palm trees. It was recently described that I. robusta caused root rot and plant death in Aconitum carmichaelii in China [70], I. crassa and I. pseudodestructans caused potato tuber decay in North America [71], and different species of Ilyonectria were also associated with disease of black foot rot in Proteaceae in South African [72].

The genus Cylindrocladiella has 48 species according to Mycobank database [69] and some species are also reported associated with root rot and other plant diseases. Cylindrocladiella peruviana already have been reported as causing black foot disease on grapevine in China and stem and crown rot on avocados in Italy [73, 74]; Cylindrocladiella sp. is also associated with “black foot” of grapes in Crimea [75]; and C. pseudohawaiiensis associated to rot of fruits on almond tree in Brazil [76].

The genus Gliocladiopsis was introduced by Saksena [77] and currently comprises 21 species. Gliocladiopsis species have already been isolated from plants with rot symptoms [67], however, pathogenicity tests have not yet confirmed as causal agents of the rot. Lombard and Crous [78] highlighted that although this genus is associated with symptomatic plant tissues, its relevance as a plant pathogen still needs to be clarified. Therefore, due to the uncertainty that permeates Gliocladiopsis, applications of this genus in agriculture, especially in the control of causal agents of rot, require further investigations.

The identification of other isolates tested in this study (data not shown) was initially carried out at the genus level and showed some new taxa associated with A. aculeata, which is expected given the low number of studies on the microbiota of this palm tree. This work marks the beginning of access to an unexplored taxonomic richness of endophytic fungi in macaw palm and the new species and genera found in this first isolation of root endophytic fungi will be proposed soon, according to the nomenclatural rules.

Given the interest of this work in selecting possible BCAs associated with macaw palm against oomycetes, we chose to present the phylogenetic position only of the selected isolates for future in vivo tests against P. palmivora, Py. aphanidermatum, and Py. deliense. The isolates T. sayulitensis COAD 3605 and E. italicum COAD 3608 were chosen due to high inhibition rates in the dual culture test and based on desirable characteristics for biocontrol use. The selection of isolates was initially based on two mechanisms of antagonism, indicating that the mode of action of our BCA candidates can be through direct contact with the phytopathogen or through volatile compounds produced, expanding their application possibilities.

This is the beginning of the BCAs selection process, and the selected isolates still need to go through other levels of scrutiny (e.g. ecological tests, soil tests) before being defined as BCA, mainly because we still know little about endophyte host range or ecology, and its effect on disrupting the surrounding microbial community. Furthermore, it is known that the spectrum of action of a BCA is not always broad, that is, the same antagonistic strains may exhibit varying degrees of activity against different strains within the same pathogenic species. Therefore, screening with endophytic fungi isolated from A. aculeata with new phytopathogens is not discarded, in addition to new tests that elucidate other mechanisms of antagonistic action.

Conclusion

Macaw palm presents a great community of root endophytic fungi that are cultivable, including taxa unknown to science, and fungi with the potential to control oomycetes. Among the 250 fungi obtained, two were selected for future studies aimed at determining the effectiveness of endophytes for controlling diseases caused by oomycetes in the field. We may be on the right way to developing an effective biological control of root rot caused by Phytophthora palmivora and diseases caused by other oomycetes.

Electronic supplementary material

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Author contribution

Oliveira, J. A.: conceptualization, methodology, formal analysis, data curation, experimentation, processing of results, writing - original draft. Custódio, F. A.: data curation, writing - review. Pereira, O. L.: conceptualization, resources, writing - review, supervision, project administration, funding acquisition.

Funding

This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001; and Soleum Company.

Data availability The alignment datasets generated during the study are available from the corresponding author upon request. All other data generated or analyzed during this study are included in this published article, its supplementary files, and the Genbank/NCBI repository. Declarations Conflict of interest The authors declared no conflicts of interest concerning the research, authorship, and publication of this article.

Data availability

The alignment datasets generated during the study are available from the corresponding author upon request. All other data generated or analyzed during this study are included in this published article, its supplementary files, and the Genbank/NCBI repository.

Declarations

Conflict of interest

The authors declared no conflicts of interest concerning the research, authorship, and publication of this article.