Endophytic species of Induratia from coffee and carqueja plants from Brazil and its potential for the biological control of toxicogenic fungi on coffee beans by means of antimicrobial volatiles

Abstract

Several endophytic fungi have been reported to have produced bioactive metabolites. Some of them, including the Induratia species, have the capacity to emit volatile compounds with antimicrobial properties with broad spectrum against human and plant pathogens. The present study aimed to prospect the Induratia species producing volatile organic compounds (VOCs), in carqueja plants used in alternative medicine and coffee plants in Brazil. A total of 11 fungal isolates producing volatile metabolites were obtained by a parallel growth technique, using I. alba 620 as a reference strain. Phylogenetic relationships revealed the presence of at least three distinct species, I. coffeana, I. yucatanensis, and Induratia sp. SPME/GC/MS analyses of the VOCs in the headspace above the mycelium from Induratia species cultured for 10 days on PDA revealed the volatile profile emitted by I. coffeana CCF 572, I. coffeana COAD 2055, I. yucatanensis COAD 2062, and Induratia sp. COAD 2059. Volatile organic compounds produced by I. coffeana isolates presented antimicrobial activity against Aspergillus ochraceus, A. sclerotiorum, A. elegans, A. foetidus, A. flavus, A. tamari, A. tubingensis, A. sydowii, A. niger, A. caespitosus, A. versicolor, and A. expansum, sometimes by decreasing the growth rate or, mainly, by fully inhibiting colony growth. Fifty-eight percent of the target species died after 6 days of exposure to VOCs emitted by I. coffeana CCF 572. In addition, VOCs emitted by the same fungus inhibited the growth in A. ochraceus inoculated into coffee beans, which indicates that plants which have I. coffeana as an endophyte may be protected from attacks by this plant pathogen.

Introduction

Endophytic fungi are characterized by their ability to colonize plant tissues without causing any visible symptoms of disease for at least part of their life cycle [1, 2]. Their presence can promote the development of their host by producing a range of metabolites that may protect the host plant against different stress conditions, such as pathogenic invasions and drought [3, 4]. Several endophytic fungi have been reported to produce bioactive metabolites. However, little emphasis has been given to the production of volatile compounds with antimicrobial properties. Research involving the capacity of a fungus to emit antimicrobial volatile organic compounds (VOCs) has intensified since the discovery of Muscodor albus (current Induratia alba), an endophytic fungus isolated from Cinnamomum Zeylanicum, Breyne, which stands out for the production of antimicrobial volatiles with broad spectrum against human and plant pathogens [5, 6].

After the discovery of I. alba, other species of fungi have been linked with the production of antimicrobial VOCs. Most of these species have been isolated from healthy plant tissue, especially in tropical plants used in alternative medicine, such as Ananas ananassoides (Baker) L.B. Smith, Aegle marmelos (L.) Corr., Cinnamomum spp., and Myroxylon balsamum (L.) Harms [7–10]. The genus Induratia has the ability to produce VOCs that may inhibit the growth of some plant pathogens [11, 51]. Induratia coffeana obtained from Brazilian coffe plants produces VOCs which completely inhibits the growth of Botrytis cinereal [48] and presents fungicidal effect against A. ochraceus [49].

VOCs present in the mixture emitted by Induratia can vary between species, and the profile can affect the antimicrobial action spectrum of the volatiles emitted. Gas chromatography/mass spectrometry analyses of the mixture of VOCs produced by I. alba revealed the occurrence of at least 28 different compounds involving at least five classes of organic substances. The esters contribute a higher percentage to the mixture, followed by alcohols, acids, lipids, and ketones. The species produces 1-Butanol, 3-methyl-, acetate, and 1-Butanol, 3-methyl- in greater abundance [6]. Induratia vitigena already stands out in the production of naphthalene and its derivatives [6, 12].

VOCs of endophytic Induratia isolates, obtained from coffee plants, present antifungal, antibacterial, and anti-nematode activities. Induratia coffeana produces VOCs with antifungal activity against Botrytis cinerea and antibacterial activity against Staphylococcus aureus, Enterococcus faecalis and E. faecium. Moreover, Induratia coffeana produces volatile compounds with nematicidal activity against Meloidogyne incognita [48].

The versatility of endophytic fungi, such as the Induratia species, for the production of bioactive metabolites makes it a promising microbial control agent for use in the biological control of plant pathogens. Currently, few studies have been undertaken in Brazil to investigate the potential of endophytic fungi to emit VOCs with antimicrobial properties. The present study aimed to prospect the Induratia species producing antimicrobial VOCs, in carqueja plants and coffee plants in Brazil, to identify these VOCs and evaluate their antimicrobial activity against the Aspergillus species often associated with coffee beans.

Materials and methods

Microorganisms

The Aspergillus species used in this study were provided by the Coleção de Cultura de Microrganismos do Departamento de Ciências dos Alimentos—UFLA. Induratia alba CZ 620 was provided by Montana State University Mycological Collection.

Isolation and screening of endophytic fungi

Intact and asymptomatic leaves and branches from Baccharis trimera Less, Hyptis brevipes Poit, Ottonia anisum Sprengel, and Coffea arabica L plants were collected in the Atlantic Forest biome at the Parque Estadual da Serra do Brigadeiro forest reserve (20° 42′ 55″ S, 42° 26′ 51″ W), located in the Zona da Mata region, Minas Gerais state, Brazil. The plant tissue was gently washed with tap water, cut into 0.5 × 0.5-cm fragments, disinfested according to Zhang et al. (2010) [14] by immersion in 75% ethanol for 30 s, followed by immersion in 1% sodium hypochlorite, for 10 min, and finally washed three times in sterile distilled water. The isolation of endophytic fungi proceeded according to the parallel growth isolation technique, adapted from Worapong et al. (2001) [5] and Ezra et al. (2004) [13]. Both sides of a two-compartment plastic Petri dish were loaded with potato dextrose agar (PDA). A 5-mm-diameter micelial plug from a 10-day-old culture of I. alba CZ 620 producing antimicrobial volatile metabolites was inoculated on one side of the dish and grown for 10 days, at 20 ± 2 °C, in the absence of light. Plant fragments were then placed on the other side of the dish. The dish was sealed with polyvinil chloride (PVC) plastic film and incubated at 20 ± 2 °C, in the absence of light. Fungal hyphae emerging from the fragments were transferred to another Petri dish free from I. alba CZ 620 and incubated under the same previous conditions.

Screening for the production of VOCs with antimicrobial properties was performed by the ability of endophytic fungus to inhibit the growth of Rhizopus stolonifer (Ehrenb.) Vuill., Botrytis cinerea Pers, and Aspergillus ochraceus G. Wilh through the parallel growth technique previously mentioned. Both sides of a two-compartment plastic Petri dish were loaded with PDA. A 5-mm-diameter micelial plug from a 10-day-old culture of endophytic fungi supposedly producing antimicrobial volatile metabolites was inoculated on one side of the dish and grown for 10 days, at 20 ± 2 °C, in the absence of light. A 5-mm-diameter micelial plug from a 7-day-old culture of plant pathogenic fungus was then placed on the other side of the dish. The dish was sealed with pvc plastic film and incubated at 20 ± 2 °C, in the absence of light, for 48 h. The effect of the endophytic isolate on the growth of plant pathogen fungi was assessed by the presence or absence of mycelial growth in the inoculated dishes.

All isolates obtained in this work were deposited in the fungal culture collection “Coleção Octávio Almeida Drummond” (COAD) and “Coleção de Cultura Fúngica” (CCF), hosted at Universidade Federal de Viçosa.

Morphological characterization

The isolates were grown in potato dextrose agar (PDA, Sigma-Aldrich), 2% malt extract agar (MEA, Kasvi), and synthetic nutrient deficient agar (SNA), with and without dry and autoclaved plant tissue (corn stover, branches, and leaves of pine), at 25 °C, to induce the formation of reproductive structures. Micro-morphological characteristics were observed with a light microscope (Olympus BX53, Japan).

DNA extraction, PCR amplification, sequencing, and phylogenetic analysis

Genomic DNA was extracted from colonies originated from fragments of hyphae tip, grown in PDA, at 25 °C, with 12 daily hours of light, for 7 days. Fungal mycelium was scraped in the colony margins, and DNA extraction was carried out according to the protocol established by Pinho et. al (2012) [15], whose genomic DNA is extracted by Wizard® Genomic DNA Purification Kit (Promega Corporation, WI, U.S.A.) with adaptations.

Sequences of the internal transcribed spacer (its) and partial 28S (rRNA) regions of rDNA were amplified using primers ITS1 and ITS4, LR0R, and LR5, respectively [16, 17]. Polymerase chain reaction (PCR) products were purified and sequenced in Macrogen (South Korea), using the same primers employed for the amplification of the fragments. Nucleotide sequences were edited on the BioEdit 7.2.5 software system [18]. All sequences were verified manually, and ambiguous nucleotide positions were clarified using sequences of both DNA strands. The sequences were subjected to analysis on the Basic Local Alignment Search Tool (BLAST) on GenBank—National Center for Biotechnology Information (NCBI) and Unite databases. The sequences related for new taxa were downloaded from the Genbank (Table 1). The sequences with high similarity to each locus were taken along with representatives of each fungal species obtained in this study. The sequences obtained in this study and those from the GenBank—NCBI were aligned, using MUSCLE [19], and manually corrected on MEGA 7.0 software system [20]. Alignment regions with ambiguous sequences were excluded from the analysis, and gaps (insertions and deletions) were treated as missing data.

Table 1.

GenBank accession numbers of Induratia isolates included in this study. Type strains are indicated

Species	Strain	Host species/substrat	Origin	GenBank accession no. (ITS)	Reference
Induratia alba	620(T)	Cinnamomum zeylanicum	Honduras	AF324336	[5]
Induratia brasiliensis	LGMF1256(T)	Schinus terebinthifolius	Brazil	KY924494	[36]
Induratia camphorae	1639CCSTITD(T)	Cinnamomum camphora	India	KC481681	[37]
Induratia cinnamomi	CMU-Cib 461(T)	Cinnamomum bejolghota	Tailand	GQ848369	[8]
Induratia coffeana	COAD1842(T)	Coffea arabica	Brazil	KM514680	[27]
Induratia coffeana	CML4011	Coffea arabica	Brazil	MN658676	[48]
Induratia coffeana	CML4012	Coffea arabica	Brazil	MN658677	[48]
Induratia coffeana	CML4019	Coffea arabica	Brazil	MN658679	[48]
Induratia coffeana	COAD 2055	Baccharis trimera	Brazil	OP846018	This study
Induratia coffeana	ACJ06	Baccharis trimera	Brazil	OP846019	This study
Induratia coffeana	ACJ08	Baccharis trimera	Brazil	OP846020	This study
Induratia coffeana	CCF 572	Coffea arabica	Brazil	KM514681	This study
Induratia coffeana	COAD 2064	Coffea arabica	Brazil	KP826879	This study
Induratia crispans	B-23(T)	Ananas ananassoides	Bolivia	EU195297	[7]
Induratia darjeelingensis	1CCSTITD(T)	Cinnamomum camphora	India	JQ409997	[38]
Induratia equiseti	CMU-M2(T)	Equisetum debile	Thailand	JX089322	[4]
Induratia fengyangensis	ZJLQ024	Actinidia chinensis	China	HM034855	[14]
Induratia fengyangensis	ZJLQ023(T)	Actinidia chinensis	China	HM034856	[14]
Induratia ghoomensis	6 CCSTITD(T)	Cinnamomum camphora	India	KF537625	[39]
Induratia heveae	RTM5-IV3(T)	Hevea brasiliensis	Thailand	KF850713	[40]
Induratia indica	6(b)CCSTITD(T)	Cinnamomum camphora	India	KF537626	[39]
Induratia kashayum	16AMLWLS(T)	Aegle marmelos	India	KC481680	[4]
Induratia musae	CMU-MU3(T)	Musa acuminata	Thailand	JX089323	[4]
Induratia oryzae	CMU-WR2(T)	Oryza rufipogon	Thailand	JX089321	[4]
Induratia rosea	A3-5(T)	Grevillea pteridifolia	Australia	AH010859	[41]
Induratia sp.	COAD 2059	Coffea arabica	Brazil	OP846012	This study
Induratia sp.	CCF 573	Coffea arabica	Brazil	OP846016	This study
Species	Strain	Host species/substrat	Origin	GenBank accession no. (ITS)	Reference
Induratia sp.	CCF 574	Coffea arabica	Brazil	OP846014	This study
Induratia sp.	COAD 2063	Coffea arabica	Brazil	OP846015	This study
Induratia strobelii	6610CMSTITBR(T)	Cinnamomum zeylanicum	India	JQ409999	[10]
Induratia suthepensis	CMU462(T)	Cinnamomum bejolghota	Thailand	JN558830	[4]
Induratia suturae	SR-2011(T)	Prestonia trifida	USA	JF938595	[42]
Induratia thailandica	MFLUCC 17–2669(T)	Dead branch	Thailand	MK762707	[43]
Induratia thailandica	HKAS102323	Dead branch	Thailand	MK762708	[43]
Induratia tigerensis	2CCSTITD(T)	Cinnamomum camphora	India	JQ409998	[44]
Induratia vitigena	P15(T)	Paullinia paullinioides	Peru	AY100022	[12]
Induratia yucatanensis	B110(T)	Bursera sirnaruha	Mexican	FJ917287	[30]
Induratia yucatanensis	COAD 2062	Coffea arabica	Brazil	OP846013	This study
Induratia yucatanensis	COAD 2060	Coffea arabica	Brazil	OP846017	This study
Induratia yunnanensis	W-S-38(T)	Oplismenus undulatifolius	China	MG866046	[45]
Induratia ziziphi	MFLUCC 17–2662(T)	Dead branch	Thailand	MK762705	[43]
Induratia ziziphi	HKAS102300	Dead branch	Thailand	MK762706	[43]
Emarcea castanopsidicola	CBS 117,105(T)	Castanopsis diversifolia	Thailand	AY603496	[46]
Emarcea eucalyptigena	CBS139908(T)	Eucalyptus brassiana	Netherlands	KR476733	[47]

The phylogenetic analysis of Bayesian inference was performed on the CIPRES portal [21] using MrBayes v 3.2 [22], based on Markov Monte Carlo Chain (MCMC), with 10,000,000 generations, using the nucleotide substitution model informed by the Akaike information criterion (AIC) on MrModeltest software v.2.3 [23]. The trees were sampled every 1000 generations, and 25% of all trees obtained were burned. Posterior probabilities [24] were determined in the most consensus tree among the 7500 remaining trees. The generated tree was viewed in FigTree v. 1.4.3 [25] and edited with the use of graphics programs.

Activity of volatile metabolites produced by Induratia spp. against Aspergillus species

The antifungal activity of VOCs emitted by representative isolates of Induratia species was evaluated against Aspergillus sclerotiorum, A. caespitosus, A. elegans, A. expansum, A. flavus, A. foetidus, A. niger, A. sydowii, A. tamari, A. tubingensis, A. versicolor, and A. ochraceus [5, 13]. Both sides of a two-compartment plastic Petri dish were loaded with PDA. A 5-mm diameter micelial plug from a 10-day-old culture of the Induratia isolate was inoculated on one side of the dish and grown for 10 days, at 20 ± 2 °C, in the absence of light. A micelial plug from the margin of a 7-day-old culture of each Aspergillus sp. was then placed on the other side of the dish. The dish was sealed with pvc plastic film and incubated at 20 ± 2 °C, in the absence of light, for 6 days. Aspergillus sp. growing free from the Induratia isolate was used as the control. The inhibition percentage of Aspergillus sp. was measured by the colony growth rate, and its viability was investigated by the subculture of the pathogen on the tested dish into a non-treated PDA dish. The experiment was repeated twice, with five replicates.

Activity of volatile metabolites produced by I. coffeana CCF 572 against A. ochraceus inoculated on coffee beans

The activity of VOCs emitted by I. coffeana CCF 572 was tested against A. ochraceus associated with coffee beans. The bioassay was performed on plastic Petri dishes into two compartments. Both sides were loaded with PDA, and a 5-mm diameter micelial disc from a 10-day-old culture of I. coffeana CCF 572 was inoculated on one side of the dish and grown for 10 days, at 20 ± 2 °C, in the absence of light. Five coffee beans autoclaved three times at 120 °C; 1 kgf/cm² for 20 min were inoculated with A. ochraceus by immersion in a suspension of conidia (10⁵conidia. mL⁻¹) for 30 s were placed on the other side of the dish. The dish was sealed with pvc plastic film and incubated at 20 ± 2 °C, in the absence of light. Autoclaved coffee beans inoculated with A. ochraceus and placed on Petri dish, free from I. coffeana CCF 572 were used as the control 1, and autoclaved coffee beans placed on Petri dish, as the control 2. The effect of the treatment was evaluated by observation of the emergence of A. ochraceus conidiophores on coffee beans, using a stereoscopic microscope. The experiment was repeated twice, with five replicates (dishes).

VOCs identification

The VOCs produced by representative isolates of Induratia species were identified by solid-phase micro-extraction gas chromatography and mass spectroscopy (SPME-GC/MS), adapted from Strobel et al. (2001) [6]. The VOCs were extracted with a SPME syringe (SULPECO, USA), 50/30 µmdivinylbenzene/carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) on StableFlex fiber (SULPECO, USA) from the headspace above a 10-day-old culture of each fungal endophytic grown on PDA in a headspace vial of 20 mL. The SPME fiber was placed through a small hole drilled in the vial septum, and the adsorption was allowed to continue for 60 min with the fiber cooled by liquid nitrogen (CF-SPME) [26].

The analysis of VOCs was conducted with a Finnigan Trace DSQ GC/MS equipped with an ion trap mass spectrometer from Thermo Scientific (West Palm Beach, FL, USA); and a HP-5MScolumn (30 m × 0.25 mm × 0.25 μm) with a helium flow of 1.5 mL min⁻¹. The injector was operated in splitless mode for 10 min with an injector temperature of 250 °C. The oven temperature ramp started at 50 °C, increased to 70 °C, at a rate of 5 °C min⁻¹, maintained for 1 min, and then increased to 280 °C, at a rate of 10 °C min⁻¹, maintained for 2 min. The mass spectrometer was operated in electron ionization mode (EI), with energy of 70 eV. Data acquisition and data processing were performed by software systems. The VOCs produced by endophytic fungi were identified by comparing the obtained mass spectra with the National Institute of Standards and Technology (NIST) library and by comparing the calculated Kováts retention index with compounds under high quality match indicated by the NIST library. Comparative analyses were conducted on control vials containing only PDA. The compounds present in the control were removed from the data set obtained from the GC/MS of endophytic fungi.

Statistical analysis

The data obtained in this study were evaluated by one-way ANOVA, and the significance of the treatments was determined by Tukey’s HSD for multiple comparisons (P ≤ 0.05).

Results

Isolation and identification

A total of 11 fungal isolates producing volatile metabolites was obtained by a parallel growth technique using I. alba 620 as a reference strain. Eight isolates were obtained from the branches of coffee plants, and three, from B. trimera leaves (common name: carqueja).

The internal transcribed spacer regions 1 and 2 (including the 5.8S rRNA gene) (ITS) from endophytic isolates presented at least 99% similarity with Induratia sequences by BLAST in the GenBank and UNITE databases. Comparative studies of morphological characteristics indicate that, although belonging to the genus Induratia, neither conidia nor sporulation structures were observed under laboratory conditions.

Bayesian inference analysis of ITS-5.8S sequences derived from all Induratia-like isolates revealed phylogenetic relationships with the Induratia genus. In all the Induratia species described to date, the phylogenetic relationships revealed the presence of at least three distinct species, I. coffeana, I. yucatanensis, and Induratia sp. (Fig. 1).

Fig. 1.

Bayesian inference tree of Induratia using ITS-5.8S sequences of rDNA. The posterior probability values are indicated at the nodes. The Induratia isolates from this study are highlighted in bold. The analyses included 39 Induratia specimens and were rooted with Emarcea eucalyptigena and Emarcea castanopsidicola (Xylariaceae) for out-group. Bar = 0.03 substitutions per nucleotide position. The species in this study are highlighted in bold. Type strains of the species are indicated after the culture collection number (T)

Isolates COAD 2062 and COAD 2060 are phylogenetically close to I. yucatanensis and form a clade. Isolates COAD 2064 and CCF 572 comprise a group forming a clade with I. coffeana, together with the isolates COAD 2055, ACJO6, and ACJ08, obtained from carqueja leaves (Fig. 1). Among the isolates obtained from coffee plants, COAD 2059 and CCF 573 were phylogenetically related, similarly to CCF 574 and COAD 2063. These four isolates formed a clade not well resolved, and Induratia sp. COAD 2059, Induratia sp. CCF 573, Induratia sp. CCF 574, and Induratia COAD 2063 grouped with species of reference I. vitigena, I. suturae, I. equiseti, I. ziziphi, and I. thailandica (Fig. 1).

Activity of Induratia spp. volatiles against Aspergillus species

The VOCs emitted by different Induratiai solates obtained in this study were tested against the Aspergillus species, which are often associated with coffee beans. The volatile metabolites produced by CCF 572 and COAD 2055 isolates (both phylogenetically identified as I. coffeana) presented antifungal activity against all Aspergillus species tested, sometimes by decreasing the growth rate or fully inhibiting colony growth (Table 2). Aspergillus ochraceus, A. sclerotiorum, A. elegans, A. foetidus, A. flavus, A. tamari, A. tubingensis, A. sydowii, A. niger, A. caespitosus, and A. versicolor died after 6 days of exposure to VOCs emitted by I. coffeana CCF 572 isolated from coffee plants. VOCs emitted by I. yucatanensis COAD 2062 fully inhibited the growth of A. sclerotiorum, A. versicolor, and A. tamari, causing the demise of the latter. VOCs emitted by Induratia sp. COAD 2059 fully inhibited the growth of A. tamari, A. ochraceus, and A. niger and killed A. ocharaceus and A. niger.

Table 2.

Effect of the VOCs emitted by Induratia isolates on the growth of Aspergillus species

	Growth ratio after 6 days of exposure (% vs. control)
Aspergillus species	Induratia coffeana CCF 572	Induratia coffeana COAD 2055	Induratia yucatanensis COAD 2062	Induratia sp. COAD 2059
A. ochraceus	0 Dead	0 Dead	98.6 ± 1.3	0
A. sclerotiorum	0 Dead	0 Dead	0 Dead	91.1 ± 2.7
A. elegans	0	0	100 ± 4.2	100 ± 5.6
A. foetidus	0 Dead	0 Dead	97.7 ± 1.2	100 ± 1.6
A. flavus	0	0 Dead	99.3 ± 3.8	99.5 ± 2.9
A. tamari	0 Dead	0 Dead	0 Dead	0 Dead
A. tubingensis	0 Dead	0	99.0 ± 1.3	51.9 ± 4.2
A. sydowii	0 Dead	0	70.6 ± 0.9	41.7 ± 1.0
A. niger	0 Dead	17.6 ± 1.3	91.9 ± 2.9	0
A. caespitosus	0	0	79.8 ± 5.9	72.2 ± 3.1
A. versicolor	0	43.8 ± 3.7	0	100 ± 8.9
A. expansum	39.7 ± 1.1	64.5 ± 1.7	92.3 ± 2.0	97.3 ± 11.6

Growth ratio was calculated as the fraction of the value of colony diameter grown in the presence and in the absence of Induratia isolate and expressed as percentage

Tests were repeated three times and means ± standard deviation were calculated

Viability of each Aspergillus species was evaluated after 6 days in fresh PDA dishes (free of Induratia VOCs)

Volatile organic compounds emitted by I. coffeana CCF 572 also nullified A. ochraceus growth when inoculated into coffee beans. The inhibition of growth by VOCs in A. ochraceus inoculated into coffee beans was significant (P ≤ 0.01), and no growth in Aspergillus was observed in coffee beans treated with I. coffeana CCF 572 VOCs (Fig. 2).

Fig. 2.

Mycofumigation of A. ochraceus in coffee beans with I. coffeana CCF 572 VOCs. a(1–2) Control I, coffee beans without inoculation of A. ochraceus and non-fumigated. b(1–2) Control II, coffee beans inoculated with A. ochraceus. c(1–2), d(1–2) Coffee beans inoculated with A. ochraceus and mycofumigated with I. coffeana CCF 572

Profile of VOCS produced by Induratia species

The SPME/GC/MS analyses of the VOCs revealed the volatile profile emitted by I. coffeana CCF 572, I. coffeana COAD 2055, I. yucatanensis COAD 2062, and Induratia sp. COAD 2059 (Tables 3, 4, 5, and 6). The production of VOCs of the Induratia isolates was diversified. The highest number of volatile organic compounds was detected for the species Induratia yucatanensis COAD 2062. All isolates emitted the following compound (Cyclosativene). The highest peak area (%) observed was from the Cyclosativene compound for the isolates I. coffeana COAD 2062 (26.9) and I. coffeana ACJ01 (20.04), and 1H-Cyclopropa[a]naphthalene, decahydro-1,1,3a-trimethyl-7- methylene-, [1aS-(1aà,3aà,7aá,7bà)]- compound for the isolate Induratia sp. COAD 2059 (38.49).

Table 3.

GC/MS analysis of the volatile compounds emitted by Induratia coffeana CCF 572

Retention index (min)	Compound	Molecular formula	Peak area %	Kovats index	Calculated Kovats index
14.25	Pyrimidine, 2-chloro-4-ethyl-6-methyl-	C7H9ClN2	0.91	1174	1099
15.58	Cyclosativene	C15H24	9.18	1125	1150
16.05	Tricyclo[4.3.1.1(3,8)]undecane, 3-methoxy-	C12H20O	4.8	1273	1166
17.8	4-Amino-3,5-diethylpyridine	C9H14N2	7.8	1411	1422
18.73	Imidazo[5,1-f][1,2,4]triazine-2,7-diamine	C5H6N6	4.6	1570	1698
19.43	Dimethyl-[4-[2-(3-methylisoxazol-5-yl)vinyl]phenyl]amine	C14H16N2O	0.82	1812	1805
20.13	4(1H)-Pyrimidinone, 2,3-dihydro-1-methyl-6-(4-pyridinyl)-2-thioxo-	C10H9N3OS	1.6	1943	1829
20.82	2-Phenyl-6-chloro-benzofuran	C14H9ClO	1.51	1861	1852
22.77	1-Methyl-2-nitro-4-(1,2,2-trimethyl-cyclopentyl)-benzene	C15H21NO2	8.93	1951	1946

Table 4.

GC/MS analysis of the volatile compounds emitted by Induratia coffeana ACJ01

Retention Index (min)	Compound	Molecular formula	Peak area %	Estimated Kovats index	Calculated Kovats index
7.97	Phenylethyl alcohol	C8H10O	11.1	1136	989
14.02	1,2Bis[methyl(trimethylene)silyloxy]propane	C11H24O2Si2	12.37	1081	1096
15.58	Cyclosativene	C15H24	20.04	1125	1149
15.81	But-3-en-2-one, 4-(2-chloro-6-fluorophenyl)-	C10H8ClFO	4.4	1391	1158
15.94	1-Ethyl-12-oxatetracyclo[5.2.1.1(2,6).1(9,11)]dodecane	C13H20O	7.55	1391	1162
18.48	6,6,9a-Trimethyl-5,5a,6,7,8,9,9a,9b-octahydronaphtho[1,2-c]furan-1(3H)-one	C15H22O2	3.24	1807	1661
21.55	Cyclopropa[3,4]cyclohepta[1,2-a]naphthalene, 1,1a,1b,2,3,7b,8,9,10,10a-decahydro-5-methoxy-10-methylene-	C18H22O	4.32	1823	1875
22.77	2-(4a,8-Dimethyl-7-oxo-1,2,3,4,4a,7-hexahydronaphthalen-2-yl)-propionic acid	C15H20O3	14.46	1980	1946

Table 5.

GC/MS analysis of the volatile compounds emitted by Induratia yucatanensis COAD 2062

Retention index (min)	Compound	Molecular formula	Peak area %	Estimated Kovats index	Calculated Kovats index
12.63	Pyrazine, isopropenyl-	C7H8N2	1.22	988	1078
12.86	1,3,5,6,7-Pentamethylbicyclo[3.2.0]hepta-2,6-diene	C12H18	2.95	1121	1081
13.6	Cyclosativene	C15H24	13.24	1125	1091
13.82	Bicyclo[2.2.2]octa-2,5-diene, 1,2,3,6-tetramethyl-	C12H18	9.16	1168	1094
14.01	Cyclosativene	C15H24	2.93	1125	1097
15.59	Cyclosativene	C15H24	26.9	1125	1150
16.18	2,3,5-Trimethyl-6-ethylpyrazine	C9H14N2	0.42	1220	1171
16.39	1,2-Bis(diethylphosphino)ethane	C10H24P2	1.46	1269	1179
17.26	Tricyclo[4.4.0.0(2,7)]dec-8-ene-3-methanol, à,à,6,8-tetramethyl-, stereoisomer	C15H24O	6.07	1325	1257
17.8	2H-Pyrazol-3-ol, 5-furan-2-yl-	C7H6N2O2	4	1473	1422
18.32	1-Penten-3-one, 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-	C14H22O	0.48	1557	1637
21.14	(Z,Z)-3-Methyl-3H-cyclonona(def)biphenylene	C18H14	0.51	1882	1863
21.21	Benzo[c]2,7-naphthiridine-4,5(3H,6H)-dione	C12H8N2O2	0.38	1896	1865
22.21	9H-Xanthen-9-one, 4-methoxy-	C14H10O3	0.67	1963	1896
22.76	2-Cyanomethyl-2-methyl-8-methoxy-1,2,3,4-tetrahydroquinoline	C13H16N2O	5.94	1981	1945
22.87	(Z,Z)-3-Methyl-3H-cyclonona(def)biphenylene	C18H14	1.43	1882	1926

Table 6.

GC/MS analysis of the volatile compounds emitted by Induratia sp. COAD 2059

Retention index (min)	Compound	Molecular formula	Peak area %	Kovats index	Calculated Kovats index
12.03	Tricyclo[4.4.0.0(2,7)]dec-3-ene, 8-isopropyl-1,3-dimethyl-, (1S,2R,6R,7R,8S)-( +)-	C15H24	3.78	1300	1070
12.22	Cycloisolongifolene	C15H24	6.96	1197	1072
12.71	Cyclosativene	C15H24	0.65	1125	1079
12.88	Copaene	C15H24	2.53	1221	1082
13.51	Cyclosativene	C15H24	9.01	1125	1090
13.81	Cyclosativene	C15H24	9.67	1125	1094
14.02	12-Oxatetracyclo[4.3.1.1(2,5).1(4,10)]dodecane, 11-isopropylidene-	C14H20O	3.24	1272	1097
15.58	1H-Cyclopropa[a]naphthalene, decahydro-1,1,3a-trimethyl-7-methylene-, [1aS-(1aà,3aà,7aá,7bà)]-	C15H24	38.49	1398	1150
16.2	1- Dimethyl(pentafluorophenyl)silyloxycyclopentane	C13H15F5OSi	0.54	1293	1172
17.31	3,5-Dimethyl-1-dimethylisopropylsilyloxybenzene	C13H22OSi	0.69	1339	1268
21.54	4-Trimethylsilyl-9,9-dimethyl-9-silafluorene	C17H22S	1.46	1693	1875
22.76	2-Methyl-5-methoxy-3-(á-aminopropyl)-indole	C13H18N2O	3.62	1920	1945

Discussion

The genus Induratia has been isolated from healthy plant tissue in tropical regions throughout the world. Induratia species have already been described as isolates from different host plants. In this study, 11 Induratia isolates were obtained from coffee and carqueja plants in the Atlantic Forest biome in Brazil, by an isolation technique specific to the Induratia genus. These isolates were initially identified by comparative studies of morphological characteristics and the BLAST of ITS-5.8S sequences in the Genbank and UNITE databases. All sequences presented at least 99% identity with Induratia sequences. Through Bayesian inference analysis with the ITS-5.8S sequences of all Induratia species described to date, phylogenetic relationships revealed the presence of at least three distinct species, I. coffeana, I. yucatanensis, and Induratia sp. (Fig. 1).

Endophytic isolates of Induratia coffena and Induratia sp. obtained from coffee plants have already been reported in Brazil [48, 51]. In this study, I. coffeana was isolated from two different host plants (Coffea arabica and Bacchans trimera) and, similarly to other Induratia species, it presented no host specificity [13]. Induratiac coffeana was described by A. A. M. Gomes, D. B. Pinho, and O. L. Pereira [27] as endophytic in coffee plants and in the study referring to Induratia in Brazil featuring the Rubiaceae plant family. To date, reports on its occurrence are restricted to Brazil. Baccharis trimera (Asteraceae) is a South American plant used in traditional medicine for the treatment of rheumatism, hepatobiliary disorders, and diabetes [28]. Extracts of B. trimera have exhibited anti-inflammatory and analgesic properties [29]. To our knowledge, this is the first time that I. coffeana was isolated from B. trimera.

Induratia yucatanensis was first described as an endophyte from Bursera simaruba in Mexico, a plant native to tropical regions with medicinal properties [30]. However, in this study, I. yucatanensis COAD 2062 was isolated from coffee plants. Induratia yucatanensis was also isolated from coffee plants in studies carried out by Monteiro et al. (2017) [49], Monteiro et al. (2020) [44], Bastos et al. (2020) [50], and Mota et al. (2021) [51].

Four isolates obtained in this study (Induratia COAD 2059, Induratia sp. CCF 573, Induratia sp. CCF 574 and Induratia COAD 2063) could not be identified at the species level and were grouped in the same clade, together with I. equiseti, I. sutura, and I. vitigenus. Studies carried out based on the phylogeny of three loci, ITS, RPB2, and TUB2, also identified two Induratia isolates originated from Coffea arabica [48] as Induratia sp.

The description of new species of Induratia was obtained by phylogenetic species recognition coupled with the phenotype of the volatile profile emitted [4, 9]. However, a number of Induratia isolates that group in the same monophyletic clade with another species of Induratia have been proposed as a new species simply because of their different volatile profile [4]. Prudence and caution are necessary for a new species proposition based on differences in the volatile profile, since certain studies indicate that profiles of volatile metabolites may derive from various factors, including growing media and environment and enzyme activity for the biosynthesis of such metabolites [31].

The lack of more robust morphological characteristics for Induratia taxonomy coupled with the inconsistent pattern of volatile profile among isolates makes it difficult to identify the Induratia species. An alternative for better discriminating the Induratia species could be the use of other molecular markers to search for other DNA regions more informative to the phylogeny of this group, as observed with other fungi [32, 33].

The volatile profile analysis was performed for I. coffeana CCF 572, I. coffeana COAD 2055, I. yucatanensis COAD 2062, and Induratia sp. COAD 2059 (Tables 3, 4, 5, and 6). This is not the first time the volatile profile emitted by I. coffeana has been reported [48]. I. coffeana CCF 572 emitted at least nine volatiles (Pyrimidine 2-chloro-4-ethyl-6-methyl-, Cyclosativene, Tricyclo[4.3.1.1(3,8)] undecane- 3-methoxy-, 4-Amino-3,5-diethylpyridine, Imidazo [5,1-f][1,2,4]triazine-2,7-diamine, Dimethyl-[4-[2-(3-methylisoxazol-5-yl)vinyl]phenyl]amine, 4(1H)-Pyrimidinone, 2,3-dihydro-1-methyl-6-(4-pyridinyl)-2-thioxo-, 2-Phenyl-6-chloro-benzofuran, 1-Methyl-2-nitro-4-(1,2,2-trimethyl-cyclopentyl)-benzene) that could possibly be identified by comparisons with the NIST library and Kovats retention index (Table 3). Guimarães et al. (2021) [48] demonstrated that I. coffeana and Induratia sp. produced different VOCs. In this study, the isolate COAD 2055, which also belongs to I. coffeana, emitted a volatile profile, unlike I. coffeana CCF 572, and only one compound (Cyclosativene) was shared by these isolates (Table 4). Induratia coffeana CCF 572 and I. coffeana COAD 2055 were isolated from different host plants (from coffee and carqueja plants, respectively), and although they belong to the same species, the composition of their volatile profiles seems to vary between them. The difference in the mixture of VOCs emitted by isolates from the same species was also observed in I. alba isolates [13]. The characteristic mixture of volatile compounds produced by each Induratia species suggests adaptation to a unique ecological role in their respective ecosystems [30].

The profile of volatiles emitted by I. yucatanensis COAD 2062 was different from that of other I. yucatanensis isolates reported [30]. Pyrazine derivatives, sesquiterpenes, alcohols, and benzene derivatives were detected among 16 volatiles identified. Cyclosativene presented the highest percentage of peak per area in the mixture emitted by I. yucatanensis COAD 2062.

Induratia sp. COAD 2059 also produced a mixture of VOCs different from other Induratia reported, with at least 12 volatiles identified by comparison with the NIST library and Kovats retention index. 1H-Cyclopropa[a]naphthalene, decahydro-1,1,3a-trimethyl-7-methylene- [1aS-(1aα,3aα,7aβ,7bα)]- was the compound with the highest relative proportion (38.49%). The emission of a naphthalene derivative has also been observed in other Induratia species, especially I. vitigenus [12] species phylogenetically close to Induratia sp. COAD 2059.

The test of antifungal activity of VOCs emitted by selected Induratia isolates representative of each species obtained in this study was carried out against the Aspergillus species often associated with coffee beans and mycotoxin production [34]. Volatile organic compounds of all Induratia isolates tested somehow affected the growth of at least one of the Aspergillus species tested. However, I. coffeana isolates were the most effective in the inhibition and/or demise of the Aspergillus species tested, besides hindering the growth in A. ochraceus inoculated into coffee beans. The mixture of VOCs emitted by Induratia isolates presented antimicrobial activity against a wide range of microorganisms, including plant pathogen fungi, such as the Aspergillus species [35, 49]. The Volatile organic compounds of I. alba presented low inhibitory effects against fungi and bacteria, when tested separately, but when tested in a mixture, they acted synergistically and killed a broad range of fungi and bacteria [6].

The production of antimicrobial VOCs by endophytic fungi can act as a defense mechanism for host plants against the attack of plant pathogens, which favors the survival of the fungi that produce them, by preventing the colonization of their host plant tissue by other microorganisms competing for the same ecological niche [31]. Since the discovery of the antimicrobial activity of VOCs emitted by Induratia, they have been considered for use in agricultural, medical, and industrial applications [35]. The discovery of new Induratia isolates, especially in different ecological niches with high activity of competition and antagonism, is a promising source of biological control agents adapted to a particular environment that can be used on a specific site. Induratia isolates naturally present in coffee plants with antimicrobial activity against the Aspergillus species producing mycotoxins can protect their host plant from the attack by these plant pathogens. The total inhibition of growth in A. ochraceus in coffee beans by VOCs emitted by I. coffeana CCF 572 indicates that plants which have I. coffeana as an endophyte may be protected from attacks by this plant pathogen. Further studies should be undertaken to elucidate the activity mechanism of VOCs and establish methods of application of Induratia sp. as a biological control agent.

The discovery of new Induratia isolates, especially in different ecological niches with high level of competition and antagonism, is a promising source of biological control agents adapted to a particular environment that can be used on a specific site.

Funding

This work was financially supported by the Brazilian Coffee Research and Development Consortium (CBP&D/Café 10.18.20.047.00.00), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Declarations

Consent for publication

All the authors agree to publish the manuscript.

Competing interests

The authors declare no competing interests.