ArticlePDF Available

Fungal Endophytic Community Associated with Guarana (Paullinia cupana Var. Sorbilis): Diversity Driver by Genotypes in the Centre of Origin

Authors:

Abstract and Figures

Guarana plant is a native of the Amazon region. Due to its high amount of caffeine and tannins, the seed has medicinal and stimulating properties. The guarana industry has grown exponentially in recent years; however, little information is available about associated mycobiota, particularly endophytic fungi. The present study aimed to compare the distribution and diversity of endophytic fungi associated with the leaves and seeds of anthracnose-resistant and susceptible guarana plants produced in Maués and Manaus, Amazonas State, Brazil. A total of 7514 endophytic fungi were isolated on Potato Dextrose Agar, Sabouraud and Czapek media, and grouped into 77 morphological groups. Overall, fungal communities in guarana leaves and seeds were mainly composed by Colletotrichum and Fusarium genera, but also by Chondrostereum, Clonostachys, Curvularia, Hypomontagnella, Lentinus, Neopestalotiopsis, Nigrospora, Peroneutypa, Phyllosticta, Simplicillium and Tinctoporellus. Obtained results indicate that some members of Colletotrichum and Fusarium genera may have experienced dysbiosis during the guarana domestication process, suggesting that some individuals may behave as latent pathogens. The susceptible guarana genotype cultivated in Manaus presented higher fungal diversity. The relative abundance of taxa and diversity among samples suggests that communities are structured by genotype and geographic location. This is the first report of mycobiota in both guarana leaves and seeds.
Content may be subject to copyright.
Fungi
Journal of
Article
Fungal Endophytic Community Associated with
Guarana (Paullinia cupana Var. Sorbilis): Diversity
Driver by Genotypes in the Centre of Origin
Carla Santos 1, Blenda Naara Santos da Silva 2,3,
Ana Francisca Tibúrcia Amorim Ferreira e Ferreira 2,
Cledir Santos 3, * , Nelson Lima 1and Jânia Lília da Silva Bentes 2
1CEB-Centre of Biological Engineering, Micoteca da Universidade do Minho, University of Minho,
4710-057 Braga, Portugal; carla.santos@ceb.uminho.pt (C.S.); nelson@ie.uminho.pt (N.L.)
2Postgraduate Program in Tropical Agronomy, Federal University of Amazonas,
Manaus-AM 69067-005, Brazil; blenda.naara@gmail.com (B.N.S.d.S.);
ana.tiburcia@gmail.com (A.F.T.A.F.eF.); jlbentes@ufam.edu.br (J.L.d.S.B.)
3Department of Chemical Sciences and Natural Resources, BIOREN-UFRO, Universidad de La Frontera,
Temuco 4811-230, Chile
*Correspondence: cledir.santos@ufrontera.cl; Tel.: +56-452-596-726
Received: 30 June 2020; Accepted: 28 July 2020; Published: 31 July 2020


Abstract:
Guarana plant is a native of the Amazon region. Due to its high amount of caeine
and tannins, the seed has medicinal and stimulating properties. The guarana industry has grown
exponentially in recent years; however, little information is available about associated mycobiota,
particularly endophytic fungi. The present study aimed to compare the distribution and diversity
of endophytic fungi associated with the leaves and seeds of anthracnose-resistant and susceptible
guarana plants produced in Mau
é
s and Manaus, Amazonas State, Brazil. A total of 7514 endophytic
fungi were isolated on Potato Dextrose Agar, Sabouraud and Czapek media, and grouped into
77 morphological groups. Overall, fungal communities in guarana leaves and seeds were mainly
composed by Colletotrichum and Fusarium genera, but also by Chondrostereum,Clonostachys,Curvularia,
Hypomontagnella,Lentinus,Neopestalotiopsis,Nigrospora,Peroneutypa,Phyllosticta,Simplicillium and
Tinctoporellus. Obtained results indicate that some members of Colletotrichum and Fusarium genera
may have experienced dysbiosis during the guarana domestication process, suggesting that some
individuals may behave as latent pathogens. The susceptible guarana genotype cultivated in Manaus
presented higher fungal diversity. The relative abundance of taxa and diversity among samples
suggests that communities are structured by genotype and geographic location. This is the first report
of mycobiota in both guarana leaves and seeds.
Keywords: mycobiota; composition; diversity; genotypes; plant organs; geographical location
1. Introduction
The guarana plant (Paullinia cupana var. sorbilis Mart. Ducke) is an Amazonian species with
a center of origin in Mau
é
s city, of Amazon State-Brazil [
1
]. The seeds, the commercially exploited
plant part, are characterized by high amounts of caeine—about two to five times greater than the
content found in coee (Coea arabica L.), yerba mate (Illex paraguariensis A. St.-Hil.) and green tea
(Camellia sinensis L. Kuntze) [2].
The indigenous Sater
é
Mau
é
tribe associated the use of guarana seeds with strength, vitality and
disease prevention [
3
]. Its consumption results in changes in the nervous system [
4
], improvement of
physical performance [
5
], and increased cognitive response [
6
]. In addition, previous studies suggest it
J. Fungi 2020,6, 123; doi:10.3390/jof6030123 www.mdpi.com/journal/jof
J. Fungi 2020,6, 123 2 of 20
can have protective eects against neuropathologies [
7
9
], reduction in cardiovascular diseases [
10
],
weight loss in humans [
11
14
], and changes in intestinal microbiota [
13
]. Due to its antioxidative
action, products derived from guarana can replace synthetic food antioxidants and are used in the
manufacture of various cosmetics [
15
,
16
]. It also has antimutagenic, anticarcinogenic and antiallergenic
properties [
17
,
18
]. These stimulating, therapeutic and medicinal properties of guarana are related to its
chemical substances, such as tannins, xanthines, and especially caeine (1,3,7-trimetilxantina) [
19
].
The compounds, theobromine (3,7-dimethylxanthine), theophylline (1,3-dimethylxanthine) and tannins
represent 0.3%, 0.3% and 14% of the plant content, respectively [20,21].
The properties of guarana seeds make their characteristics highly requested by dierent industries.
Currently, Brazil is the main guarana producer in the world. Most of the production is consumed
in the domestic market by the carbonated beverages sector (45%), and remainder amount is used in
the manufacture of syrups, powders and pharma compounds in general [
22
]. The growing demand
for dierent healthy products among consumers, especially in the beverage sector, is expected to
increase the guarana market to USD 8.30 billion by 2021, representing an increase of 142% over the
value published in 2018, reflecting an increase of USD 5.86 billion [23].
Seeking to meet market demand, guarana industry has grown exponentially in Brazil. Currently,
guarana cultivation occupies 15 thousand hectares, distributed mostly in the Brazilian States of Bahia
(6500 ha) and Amazonas (8133 ha) [
24
]. Until 2016, the highest production per hectare was recorded in
Bahia. According to a previous study published by the Brazilian Institute of Geography and Statistics
(IBGE, Brazil), in the period from 2017 to 2018, a significant increase in the guarana production in
the State of Rond
ô
nia, Brazil, was observed. It reached 705.8 ton.ha
1
, leaving Amazonas in third
place in terms of productivity, with 501.5 ton.ha
1
, while Bahia State remained in the first place, with a
production of 862.1 ton.ha1[25].
In the Amazon, the tropical climate, characterized as hot and humid (annual average: 27.2
C
and 2101 mm [
26
]), favors the establishment of insects and pathogens that significantly aect plants.
The trips (Pseudophilothrips adisi Zur Strassen), anthracnose (Colletotrichum guaranicola Albuq.) and
oversprouting (Fusarium decemcellulare Brick) are the main factors related to the stagnation of guarana
production in the Amazon region [
27
30
]. Application of agrochemicals, cultural practices and insertion
of tolerant genotypes have been used to control these problems [28,31,32].
Recent studies have demonstrated the importance of microorganisms associated to plants as
biological source of new molecules and bioactive compounds. Such close relationships between
hosts and their associated communities of microorganisms (or microbiota) have led to the description
of the “holobiont” concept. This has been around since the early 20th century [
33
] but it is mostly
associated with the studies of Margulis, particularly [
34
]. More recently the term “hologenome” was
proposed—it corresponds to the entire metagenome of a holobiont, that is, the combined gene pool
of the host and its microbiota [
35
]. In this theory, the relationship between host and its microbiota
is a key aspect aecting the holobiont fitness to its environment [
35
]. Among the microorganisms
associated to plants, endophytic fungi stand out for their multiple interactions with the host and are a
good choice for exploitation of such kind of new molecules with biological activity against insects and
pathogens. During plant–fungus mutual interaction, endophytes decrease attacks by herbivore and
plant pathogens, favoring greater protection of the plant and production of vegetal biomass. In return,
plants provide essential nutrients to endophytic fungi, and produce hormones and amino acids that
modulate mycobiota by recruiting specific taxonomic groups [
36
,
37
]. However, not always do the
recruited endophyte groups result in positive eects for the host. Such microorganisms may antagonize
phytopathogens, facilitate disease or have neutral eect [
38
,
39
]. Positive, neutral or negative eects
depend on the environment and the dierent combinations between host and endophyte genotypes as
well as interactions with other organisms [40,41].
The understanding of the factors guiding the microbial community of guarana is extremely
important because it can elucidate how these microorganisms are structured in specific niches.
In addition, the symbiotic interactions between endophytic fungi and guarana have been little
J. Fungi 2020,6, 123 3 of 20
explored [
42
44
]. The aim of this study was to identify the taxonomic composition of endophytes
on leaves and seeds of guarana (Paullinia cupana var. sorbilis), and compare culturable fungi isolated
among dierent guarana genotypes and geographical locations of guarana production in the Amazon
region, Brazil.
2. Materials and Methods
2.1. Sampling
Healthy guarana leaves and fruits from susceptible (BRS Amazonas cultivar 300) and resistant
(BRS Mau
é
s cultivar 871) genotypes to both anthracnose and oversprouting diseases were collected
during November 2014 in the experimental fields of Embrapa Amaz
ô
nia Ocidental located in the
municipalities of Manaus (MAO, 2
56
0
33
00
S 59
56
0
07
00
W) and Mau
é
s (MBZ, 3
23
0
55
00
S 57
42
0
25
00
W),
in the state of Amazonas, Brazil. The collection was performed from 5 plants of each genotype in
both municipalities, totalizing 20 plants. The collected material was labelled according to the origin
and susceptibility with the following abbreviations: MAO 300 or MAO 871 (susceptible or resistant
genotypes from Manaus); and MBZ 300 or MBZ 871 (susceptible or resistant genotypes from Maués).
The labelled plant material was stored in paper bags, packed in ice and transported to the Laboratory
of Microbiology and Plant Pathology of the Federal University of Amazonas (UFAM) in Manaus
city, Brazil.
2.2. Fungal Endophyte Isolation
Endophytic fungi were isolated from leaves as described in [
45
]. Leaf fragments of 5 cm were
submitted to superficial disinfestation in ethanol (70%, 1 min), NaCl (2%, 1 min), ethanol (70%, 30 s),
followed by triple wash in sterilized distilled water. The edges of the disinfested fragments were
eliminated, obtaining samples of 0.5 cm
2
, deposited in Petri dishes with PDA (Kasvi, S
ã
o Jos
é
do
Pinhais, Paran
á
, Brazil), Sabouraud (Merck, Darmstadt, Germany) and Czapek (Difco
TM
, BD, Franklin
Lakes, NJ, USA) media supplemented with chloramphenicol 250 mg
·
L
1
(Amresco
®
, Solon, OH, USA).
For each culture medium, 100 fragments per plant were deposited, totaling 300 fragments per plant in
the three culture media.
For the isolation of endophytic fungi from seeds, 25 seeds from each origin (MAO 300, MAO 871,
MBZ 300 and MBZ 871) were used, totaling 100 seeds. The fruits were washed in running water
and the aryl was removed, followed by surface disinfestation in aqueous solution of ethanol (70%,
2 min), NaClO (3%, 5 min), ethanol (70%, 30 s), followed by rinse thrice in sterilized distilled water.
Five equidistant seeds were deposited in Petri’s dishes with PDA supplemented with chloramphenicol
(250 mg·L1).
The eectiveness of the disinfestation process was verified by deposition of 50
µ
L of the water
used in the last wash in the same culture media used for endophytic fungi isolation. Petri dishes
containing control, leaf fragments and seeds were kept at 28 C without photoperiod.
After 24–72 h, the first fragments of hyphae grown from leaves and seeds were transferred to
new Petri dishes containing PDA for isolation of fungal colonies. After fungal growth (7–10 days),
the macro-morphological characteristics were observed. For fungi visualization, slides were prepared
with lactophenol and cotton blue. Observation of reproductive fungal structures was carried out by
using a Carl Zeiss
®
(Oberkochen, Germany) light microscope and photographed with the AxioCAM
ERc 5s camera with a 40×objective.
Endophytic fungal colonies were subsequently quantified and grouped into morphotypes/OTU’s
(Operational Taxonomic Units) based on their cultural and reproductive structures
characteristics [4649]
.
In order to maintain genetic uniformity, monoconidial cultures were obtained from representatives of
each OTU [
50
]. All monoconidial isolates were preserved using the Castellani method and deposited
in the Chilean Culture Collection of Type Strains (WDCM 1111, http://ccct.ufro.cl/), hosted by the
Universidad de La Frontera (Temuco, Chile).
J. Fungi 2020,6, 123 4 of 20
2.3. DNA Extraction, Amplification and Sequencing
Genomic DNA was extracted from monoconidial cultures from representatives of each OUTs,
using the Wizard
®
Genomic DNA Purification Kit. DNA was quantified in 0.8% agarose gels using
50 ng lambda DNA (Promega, Madison, WI, USA) molecular weight marker, and the fragments were
visualized using the Loccus Biotechnology Molecular Imaging Transilluminator. The 260/280 ratio for
DNA quality and concentration was obtained using a Nanodrop
®
2000c spectrophotometer (Thermo
Fisher Scientific, Waltham, MA, USA), and the final concentration adjusted to 30 ng.µL1.
Internal transcribed spacer of ribosomal DNA (ITS) region was amplified using 0.2
µ
M of
ITS1/ITS4 primers [
51
], 1
×
VWR Taq DNA Polymerase Master Mix with 1 mM MgCl
2
(VWR, Radnor,
Pennsylvania, PA, USA), and approximately 50 ng of template DNA in a total 50
µ
L reaction volume.
PCR cycling conditions were: pre-denaturation at 95
C for 5 min; followed by 35 cycles of denaturation
at 95
C for 1 min, primer annealing at 56
C for 45 s, extension at 72
C for 90 s; and final extension at
72
C for 10 min, in a BioRad C-1000 thermocycler (BioRad, Hercules, CA, USA). Amplification success
was verified in 1% agarose gel and obtained amplicons were purified according to the NZYGelpure
kit (NZYtech, Lisbon, Portugal) protocol. Samples were sent for Sanger sequencing to Stab Vida
Lda (Madan Parque, Caparica, Portugal). Generated electropherograms were analyzed using 4Peaks
(by A. Griekspoor and Tom Groothuis, nucleobytes.com). Sequences were primarily analyzed using the
Blast algorithm from NCBI National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
Phylogenetic analysis was performed by multiple alignment of the obtained ITS sequences against
those of dierent species sequences retrieved from the NCBI database (Table S1). Alignment was
performed using the MUSCLE tool [
52
], implemented in MEGAX software (Institute of Molecular
Evolutionary Genetics, The Pennsylvania State University, USA [
53
,
54
]). Poorly aligned positions and
divergent regions were eliminated using the Gblocks v.0.91b online tool (Institut de Biologia Evolutiva
(CSIC-UPF), Barcelona, Spain, [
55
]). The most suitable substitution model was determined based on
the lowest Bayesian information criterion. A Maximum Likelihood (ML) tree—based on the Kimura
two-parameter [
56
] substitution model (K2), considering non-uniformity of evolutionary rates among
sites modelled using a discrete Gamma distribution (+G) with 5 rate categories, assuming that a certain
fraction of sites are evolutionary invariable (+I), and 1000 bootstrap replications [
57
]—was constructed
using MEGAX. All positions with less than 95% site coverage data were eliminated. The obtained tree
was edited in iTOL v.5.6 program (biobyte solutions GmbH, Heidelberg, Germany, [58]).
2.4. Fungal Diversity Analysis
The analyses of taxonomic composition were performed using the relative abundance matrix of
genera, later grouped in Operational Taxonomic Unit (OTUs), where each genus reflects one OTU.
Plots with genera taxonomic composition were constructed using Phyloseq R [59] and ggplot [60].
Analysis of endophytic fungi diversity was calculated by the program RStudio version 1.1.463
using the relative abundance of the taxa found in the samples. The alpha diversity, which analyzes the
diversity within each sample, was estimated by genotype (BRS Amazonas and BRS Mau
é
s) (q=0),
Shannon diversity (q=1), and Simpson diversity (q=2) in the range of geographic origin (Manaus
and Mau
é
s), using the Hill series, which takes into account the eective number of genera, package
iNEXT [61].
3. Results and Discussion
The analysis of diversity and taxonomic composition of the endophytic community of guarana
were based on the amount of fungi recovered from healthy guarana tissue samples in dierent culture
media, followed by the quantification and separation by morphotypes until identification through
molecular data. The diversity and composition of leaf and seed endophytes from two guarana cultivars,
both of which diered as to resistance/susceptibility to anthracnose and to oversprouting, considered
J. Fungi 2020,6, 123 5 of 20
the main diseases of this crop, were compared within a regional scale, which includes the municipalities
of Manaus and Maués Brazil, the latter being considered the center of origin of guarana.
As a result of the field collection, 7441 endophytes from 6000 guarana leaf fragments and 73 fungi
obtained from 100 seeds were isolated, distributed in two dierent cultivars and municipalities
according to Table 1. Leaf and seed fungi were grouped into 77 morphological groups. Such isolation
eort allows the capture of several diversity levels of the endophytic community associated with
guarana and it provides valuable resources for future studies, either in fungal taxonomy or
biotechnological applications.
Table 1.
Number of endophytic isolates from guarana plants (Paullinia cupana var. sorbilis), from the
cultivars susceptible BRS300 and resistant BRS871, collected in the municipalities of Manaus and Mau
é
s
AM, Brazil.
Leaves Seeds
Cultivar BRS300 BRS871 BRS300 BRS871 Total
Local Manaus 1533 1947 19 11 3510
Maués 2721 1240 23 20 4004
Total 4254 3187 42 31 7514
The amount of 77 morphotypes corresponded to 26 OTUs which were detected through molecular
sequencing. Initially, the morphotypes were separated according to cultural characteristics that justified
the groups’ distinction. However, dierent morphological groups were later identified within the same
genus. Similar results were observed in the study developed by Singh et al. [
62
], with the endophytic
fungi of Tectona grandis. The authors recovered 5089 isolates attributed to 45 distinct morphotypes,
identified based on the ITS region in just over 23 genera. Tan et al. [
63
] isolated 224 endophytic fungi
from various plant tissues of Dysosma versipellis, classified within 53 morphotypes and identified on
the basis of ITS in 29 dierent genera. Guo et al. [
64
] grouped an enormous amount of endophytes
of Leptocanna chinensis into 19 morphotypes. After sequencing the ITS region, the endophytes were
collected in only 3 genera: Diaporthe,Mycosphaerella and Xylaria. On the other hand, Wang et al. [
65
]
obtained a better approximation of the morphotype-taxon relation, the authors grouped the endophytic
fungi into 77 morphotypes, which were divided into 64 taxa based on the analysis of the ITS sequencing.
Most studies with cultivable fungi report similar results to ours, that is, higher number of morphotypes
and fewer genera. Nevertheless, it is important that the morphotypes are separated, as reported in
a review of traditional and molecular techniques used in studies of endophytic fungi diversity [
66
].
According to the authors, the arrangement within the dierent morphotypes does not reflect the actual
phylogeny of the taxa, but it is necessary because it assists in the separation and optimization of
molecular identification when one has a huge amount of individuals.
About 2486 fungi distributed in 16 morphotypes did not have their representatives identified
molecularly, among them members of Gilmaniela,Pithium,Phoma and Stemphillium. These representatives
needed the molecular analyses for eective identification, so they were demarcated as unidentified.
While morphology can be of great value for the dierentiation of some genera, some morphological
characteristics of fungi may cause confusion in people not specialized in a particular genus. For example,
conidiophore structure is a very helpful morphological characteristic to dierentiate between Aspergillus
and Penicillium. On the other hand, high levels of interspecific dierences in conidial dimensions,
septation and shape of aerial and sporodochial conidia in Neocosmospora hinder the morphological
dierentiation of this genus from Fusarium [
67
]. In addition, even renowned mycologists can commit
faults when performing fungal identification only on the basis of morphology, which is subject to
plasticity and changes caused by biotic and abiotic factors. Such endophytes lost the ability to grow in
synthetic culture medium after the storage period required to process all the 7514 isolates recovered,
approximately 3 to 4 months, leading to the diversity loss that these individuals could represent.
The preservation method used in this study was based on nutrient reduction, suitable for various fungi,
J. Fungi 2020,6, 123 6 of 20
as demonstrated in [
68
], which maintained 44 viable taxa preserved in distilled water for one year,
and in [
69
], who used the same method to maintain 151 basidiomycetes species for variable periods for
up to 7 years. However, preservation in distilled water can cause the death of the fungus due to the
absorption of water by osmosis [
70
]. Possibly, this happened with some of our isolates. For example,
members of Guignardia were initially quantified and separated into a morphotype G1, but none could be
retrieved for DNA extraction and sequencing. Only a fungal isolate could be recovered and sequenced
within morphotype G2, later identified as the teleomorph Phylosticta (CCCT 17.27, see Figure 1).
Many fungal isolates are lost annually because of the specificities necessary for the storage of certain
species, thus, in order to preserve individuals for future studies, endophytes with sequenced DNA
(Table S1) were deposited in the Colección Chilena de Cultivos Tipo (CCCT).
J. Fungi 2020, 6, x FOR PEER REVIEW 6 of 22
sequencing. Only a fungal isolate could be recovered and sequenced within morphotype G2, later
identified as the teleomorph Phylosticta (CCCT 17.27, see Figure 1). Many fungal isolates are lost
annually because of the specificities necessary for the storage of certain species, thus, in order to
preserve individuals for future studies, endophytes with sequenced DNA (Table S1) were deposited
in the Colección Chilena de Cultivos Tipo (CCCT).
Figure 1. Phylogenetic tree for ITS sequence data of the 136 strains isolated from guarana with other
species detailed in Table S1. Entorrhiza parvula TUB 021488
T
and E. citriformis PDD 70949
T
were used
as outgroup. The clade highlighted by red branches is composed by Basidiomycota species. The clade
highlighted by green branches is composed by Ascomycota species. Selected model: K2+G+I. The
percentage of trees in which the associated taxa clusters together in the bootstrap test (1,000 replicates)
is shown above the branches. The tree is drawn to scale with branch lengths measured in the number
of substitutions per site. All positions with less than 95% site coverage were eliminated. There were a
total of 282 positions in the final dataset.
The initial morphological identification allowed the separation of genera that produced sexual
structures, such as Aspergillus, Colletotrichum, Fusarium and Penicillium. However, a high number of
fungi was classified as Mycelia sterilia, among them the endophytes classified molecularly as
Chondrostereum, Diaporthe, Hypomontagnella, Lasiodiplodia, Lentinus, Muyocopron, Peroneutypa,
Figure 1.
Phylogenetic tree for ITS sequence data of the 136 strains isolated from guarana with other
species detailed in Table S1. Entorrhiza parvula TUB 021488
T
and E. citriformis PDD 70949
T
were
used as outgroup. The clade highlighted by red branches is composed by Basidiomycota species.
The clade highlighted by green branches is composed by Ascomycota species. Selected model: K2+G+I.
The percentage of trees in which the associated taxa clusters together in the bootstrap test (1000 replicates)
is shown above the branches. The tree is drawn to scale with branch lengths measured in the number
of substitutions per site. All positions with less than 95% site coverage were eliminated. There were a
total of 282 positions in the final dataset.
J. Fungi 2020,6, 123 7 of 20
The initial morphological identification allowed the separation of genera that produced sexual
structures, such as Aspergillus,Colletotrichum,Fusarium and Penicillium. However, a high number of fungi
was classified as Mycelia sterilia, among them the endophytes classified molecularly as Chondrostereum,
Diaporthe,Hypomontagnella, Lasiodiplodia,Lentinus, Muyocopron,Peroneutypa,Phomopsis,Phylosticta,
Polyporales and Tinctoporellus. Some isolates did not produce conidia, and others, such as members of
Nigrospora, lost that ability after the successive cultivation steps and under the analyzed conditions.
This inability to produce conidia under laboratory conditions indicates that growth was metabolically
unfavorable for the formation of reproductive structures [
66
]. Although conidiophores and conidia
represent commonly reported characteristics in the literature, their value can be limited, since more
than 50% of the total endophytic fungi usually does not sporulate on the used substrates [
71
,
72
].
Molecular biology analysis through amplification of the ITS region allowed the identification to
the genus level, especially of sterile fungi that could not be classified in any taxonomic setting
based on the morphology. The ITS region, known as the fungal barcode, is highly polymorphic,
easily amplified, with genetic information that allows intraspecific and interspecific distinction of
many members of the phyla Ascomycota and Basidiomycota, with reliable taxonomic classification at
the genus level for most fungi [
73
]. Based on the molecular data (Figure 1), it was possible to confirm
the identification of Aspergillus,Colletotrichum,Cladosporium,Curvularia,Fusarium,Neopestalotiopsis
and Penicillium, and other lesser common fungi in the laboratory routine, such as Chondrostereum,
Muyocopron,Peroneutypa and Tinctoporellus.
3.1. Culture Medium
Fungi isolated from guarana in dierent culture media resulted in the highest number of
endophytes in Czapek culture medium (2791 isolates), followed by PDA (2643) and Sabouraud
(2080). The number of genera obtained varied according to the culture medium used. The largest
number of unique OTUs, that is, genera exclusively found in a certain culture medium, was found
in PDA (Hypomontagnella,Muyocopron,Phyllosticta,Pseudopestalotiopsis and Talaromyces). The Czapek
and Sabouraud media provided only two unique OTUs obtained in these media (Peroneutypa and
Tinctoporellus). Three OTUs (Colletotrichum,Fusarium and Penicillium) were obtained from the four
samples (MAO 300, MAO 871, MBZ 300 and MBZ 871) in the three dierent culture media used.
The largest number of unique OTUs was isolated in PDA medium in the city of Mau
é
s (4), and in
the genotype BRS300 (5). The diversity parameters analyzed for the culture media (Figure 2a) show
that PDA has greater sample richness (q=0) when compared to Czapek and Sabouraud. However,
the parameters q=1 and 2 indicated that the three culture media did not diered in their capacity to
capture the diversity present in the guarana samples.
Our results show that isolation of endophytic fungi translated in the higher number of isolates in
Czapek and greater diversity in PDA medium. These results dier from those found in [
44
], where the
authors had better results regarding the incidence and diversity of guarana endophytes in Manioc
Dextrose Agar (MDA) when compared to PDA medium. The culture media reported in our study did
not diered in the diversity measures considered more reliable (q
1) [
74
]. However, PDA provided
five genera exclusively obtained in this culture medium, possibly due to the favorable conditions of
this medium in the development of most filamentous fungi [75].
J. Fungi 2020,6, 123 8 of 20
J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 22
Figure 2. Rarefaction and extrapolation curves of species alpha diversity (q = 0), Shannon diversity (q
= 1), and Simpson diversity (q = 2) for the following comparisons: (a) used culture media (PDA,
Czapek and Sabouraud); (b) plant tissue used for sampling (as no rarefaction was achieved for seeds,
the yellow arrow indicates the maximum q = 0 value estimated for seeds); (c) municipalities of Manaus
(MAO) and Maués (MBZ) where guarana samples were collected; and (d) susceptible (BRS300) and
resistant (BRS871) guarana genotype.
Our results show that isolation of endophytic fungi translated in the higher number of isolates
in Czapek and greater diversity in PDA medium. These results differ from those found in [44], where
the authors had better results regarding the incidence and diversity of guarana endophytes in Manioc
Dextrose Agar (MDA) when compared to PDA medium. The culture media reported in our study
did not differed in the diversity measures considered more reliable (q ≥ 1) [74]. However, PDA
provided five genera exclusively obtained in this culture medium, possibly due to the favorable
conditions of this medium in the development of most filamentous fungi [75].
3.2. Taxonomic Composition
The fungal community of guarana isolated here is mainly composed by members of Ascomycota,
phylum that also prevails in other vegetal species [76,77], with rare exceptions such as Hevea, where
most of the endophytes obtained in [78] are basidiomycetes. About 67% (5,005) of the endophytic
isolates obtained from guarana are inserted in the Ascomycotina, with members of the classes
Sordariomycetes (55%), Dothideomycetes (8%) and Eurotiomycetes (4%). Basidiomycotina group is only
represented by 0.3% (23) of the isolates, with members exclusively belonging to Agaricomycetes. The
families with the greatest relative abundance (RA) were Nectriaceae (17%), Glomerellaceae (16%),
Apiosporaceae (8%) and Diaporthaceae (6%). In total, 19 families and 25 genera were identified, as shown
in Figures 1 and 3.
In general, the taxonomic composition of guarana had two dominant OTUs, that is, with high
RA, Colletotrichum (16%) and Fusarium (15%). Another 12 OTUs had RA ranging from 1 to 7.9%, and
were considered as frequent, typical or common, making up half of the identified guarana isolates
(52.4%). The remaining 11 OTUs with AR < 1% were considered rare. The isolation of rare endophytic
fungi such as Cladosporium, Lentinus, Simplicillium, Pseudopestalotiopsis, Talaromyces and Tinctoporellus
BD A
SA B O U R A U D
0 2000 4000 0 2000 4000 0 200 0 4000
0
10
20
30
N um ber of individuals
Sp ecies diversity
interpolated extrapolated
0 1 2
M A O
M B Z
0 2000 4 000 600 0 8000 0 2000 4 000 600 0 8000
0
10
20
30
N um ber of individ uals
Sp ecies diversity
interpolated extrapolated
012
cultivar 300
cultivar 8 71
0 2500 5 000 750 0 0 25 00 5000 7 500
0
10
20
N um ber of ind ividuals
Sp ecies diversity
interpolated extrapolated
0 1 2
Leaf
0 2500 5 000 750 0 0 250 0 5000 7 500
0
10
20
N um ber of individuals
Sp ecies diversity
interpolated extrapolated
0 1 2
a) b)
c) d)
0 200 0 400 0 0 200 0 400 0 0 200 0 400 0
0
10
20
30
N u m ber of in dividua ls
S pe cies dive rsity
in te rpolated extrapo lated
0 1 2
PDA Czapek Sabouraud
Susceptible genotype (300) Resistant genotype (871)Maués (MBZ)Manaus (MAO)
Leaf
Species diversity
Number of individuals
Species diversity
Number of individuals
Species diversity
Number of individuals
Species diversity
Number of individuals
0 2000 400 0 0 2000 400 0 0 2000 400 0
0
10
20
30
N um be r of ind ividuals
S pecies diversity
interp olated extrapola te d
0 1 2
Figure 2.
Rarefaction and extrapolation curves of species alpha diversity (q=0), Shannon diversity
(q=1), and Simpson diversity (q=2) for the following comparisons: (
a
) used culture media (PDA,
Czapek and Sabouraud); (
b
) plant tissue used for sampling (as no rarefaction was achieved for seeds,
the yellow arrow indicates the maximum q =0 value estimated for seeds); (
c
) municipalities of Manaus
(MAO) and Mau
é
s (MBZ) where guarana samples were collected; and (
d
) susceptible (BRS300) and
resistant (BRS871) guarana genotype.
3.2. Taxonomic Composition
The fungal community of guarana isolated here is mainly composed by members of Ascomycota,
phylum that also prevails in other vegetal species [
76
,
77
], with rare exceptions such as Hevea, where most
of the endophytes obtained in [
78
] are basidiomycetes. About 67% (5005) of the endophytic isolates
obtained from guarana are inserted in the Ascomycotina, with members of the classes Sordariomycetes
(55%), Dothideomycetes (8%) and Eurotiomycetes (4%). Basidiomycotina group is only represented by
0.3% (23) of the isolates, with members exclusively belonging to Agaricomycetes. The families with the
greatest relative abundance (RA) were Nectriaceae (17%), Glomerellaceae (16%), Apiosporaceae (8%) and
Diaporthaceae (6%). In total, 19 families and 25 genera were identified, as shown in Figures 1and 3.
In general, the taxonomic composition of guarana had two dominant OTUs, that is, with high RA,
Colletotrichum (16%) and Fusarium (15%). Another 12 OTUs had RA ranging from 1 to 7.9%, and were
considered as frequent, typical or common, making up half of the identified guarana isolates (52.4%).
The remaining 11 OTUs with AR <1% were considered rare. The isolation of rare endophytic fungi
such as Cladosporium,Lentinus,Simplicillium,Pseudopestalotiopsis,Talaromyces and Tinctoporellus suggests
that the isolation and sampling procedures were appropriately employed. In general, the endophytes
isolated from guarana leaves and seeds represent little explored niches, and resulted in the first
report of 11 genera in endophytic communities of P. cupana:Clonostachys,Curvularia,Chondrostereum,
Hypomontagnella,Lentinus,Neopestalotiopsis,Nigrospora,Peroneutypa, Phyllosticta,Simplicillium and
Tinctoporellus. These genera, reported here for the first time in guarana, have already been cited in
studies on the biological control of pests and diseases in cultivated plants. For example, strong fungicidal
activity was evidenced by an extract produced from Lentinus crinitus, capable of inhibiting more than
J. Fungi 2020,6, 123 9 of 20
92% of the conidial spores of Fusarium sp. [
79
]. The fungus Clonostachys rosea is considered an eective
organism: entomopathogenic, mycoparasitic and nematophagus. Such capacities are associated
with, among other factors, the production of serine protease, an enzyme with important role during
biological control [
80
82
]. The strains C. rosea MpA/MpB and Bionectria sp. 6.21 reported in [
83
,
84
]
have antagonistic activity against phytopathogens through mycoparasitism and the production of
secondary metabolites that aid in the breakdown and degradation of the cell wall. Another mycoparasite
species, also found in guarana, is Simplicillium lanosoniveum S-599 which parasites fungi by secreting
proteases [85].
J. Fungi 2020, 6, x FOR PEER REVIEW 10 of 22
Figure 3. Genus level taxonomic composition of the fungal isolates obtained from guarana leaves and
seeds of susceptible (BRS300) and resistant (BRS871) plant genotypes from Manaus (MAO) and Maués
(MBZ), Amazonas, Brazil.
In the obtained leaves of MAO 300, five OTUs prevailed over the others, Diaporthe (10%),
Fusarium (9%), Guignardia (9%), Nigrospora (8%) and Colletotrichum (7%). In MBZ 300 only
Colletotrichum (25%) and Fusarium (29%), formed the group of predominant individuals. In the
genotype BRS871 from Maués the genera Nigrospora (30%), Fusarium (14%), Diaporthe (14%),
Clonostachys (10%) and Colletotrichum (8%) showed higher RA values. A smaller number of OTUs
prevailed in MAO 871, only Colletotrichum (17%) and Guignardia (8%). The foliar endophytic
community is composed of 20 OTUs, where 8 OTUs were found in both genotypes and
municipalities: Clonostachys, Colletotrichum, Curvularia, Diaporthe, Fusarium, Guignardia,
Neopestalotiopsis and Penicillium. The largest number of unique OTUs, that is, those found only in a
given sample, was found in MBZ 300 (4) and MAO 300 (2). In the MBZ 871 sample no unique OTU
was observed and only one unique OTU, Phyllosticta, was obtained from MAO 871.
The OTUs obtained from seeds varied according to plant location and genotype. The endophytes
from the MBZ 300 sample were mainly inserted in the genera Colletotrichum (22%), Clonostachys (26%),
Fusarium (13%), Talaromyces (13%), Diaporthe (9%) and Simplicillium (9%). In MAO 300, other groups
had high RA, Fusarium (47%), Clonostachys (21%), Aspergillus (11%) and Albonectria (11%). In cultivar
BRS871 the most abundant OTUs were Fusarium (15%) and Megasporoporia (15%) in MBZ 871, and
Cladosporium (55%) and Fusarium (27%) in MAO 871. The microbial seed community had a higher
number of unique OTUs in Maués, in samples MBZ 300 (2) and MBZ 871 (2). In Manaus, MAO 300
and MAO 871, one unique OTU was obtained in each sample, Aspergillus and Cladosporium,
respectively. Only one OTU (Fusarium) was isolated in all guarana genotypes and municipalities
studied. In the susceptible cultivar (BRS300), the highest total amount of OTUs, 8 and 6, were present
in the MBZ300 and MAO300 samples, respectively.
The mycobiota present in guarana plants is heterogeneous, varying in distribution and abundance
of genera according to plant genotypes and municipalities of sample collection. The present results
suggest that structuring of guarana fungal community (cultivable organisms) is directed both by the
genetics of the host plant as well as by the geographic location, especially in leaves. These results are in
line with previous studies of grapevines [99] and tomato [100] plants that have shown that different
0%
20%
40%
60%
80%
100%
MAO
300
MAO
871
MBZ
300
MBZ871
Lea
f
MAO
300
MAO
871
MBZ
300
MBZ871
Seed
Chondrostereum
Lentinus
Megasporoporia
Tinctoporellus
Curvularia
Muyocopron
Tal ar om yc es
Aspergillus
Penicillium
Cladosporium
Lasiodiplodia
Guignardia
Phyllosticta
Peroneutypa
Daldinia
Hypomontagnella
Pseudopestalotiopsis
Neopestalotiopsis
Nigrospora
Diaporthe
Colletotrichum
Simplicillium
Clonostachys
Albonectria
Fusarium
Unidentified
Leaf Seed
Genus
Relative abundance
Municipalities/Cultivar
Figure 3.
Genus level taxonomic composition of the fungal isolates obtained from guarana leaves and
seeds of susceptible (BRS300) and resistant (BRS871) plant genotypes from Manaus (MAO) and Mau
é
s
(MBZ), Amazonas, Brazil.
Most of the taxa associated with guarana were previously reported in the literature as having
beneficial ecological functions, like plant growth-promoting fungi (PGPF) which have the natural ability
to stimulate seedling vigor, seed germination rate, root morphogenesis and development, shoot growth,
yield, flowering, plant composition and photosynthetic eciency [
86
91
]. This ability can occur
through one or more mechanisms such as production of volatile organic compounds (VOC’s) and
phytohormones, antagonism to phytopathogens, amelioration of abiotic stresses and enhanced nutrient
availability [
92
]. For instance, Naraghi et al. [
93
] found that the endophyte Talaromyces flavus (TF-Po-V-50
and TF-Co-M-23) can promote the development and increase the biomass of cotton and potato plants
mediated by the seed treatment method; Hossain et al. [
94
] reported that Penicillium simplicissimum
GP17-2 could induce the host-plant defense system by the activation of multiple chemical signals.
The authors observed that Arabidopsis thaliana plants inoculated with GP17-2 presented a clear induced
systemic resistance (ISR) to Pseudomonas syringae pv. tomato DC3000. Other genera have also been
reported with the ability to increase plant growth such as Aspergillus, Cladosporium,Clonostachys,
Curvularia,Phomopsis and Talaromyces [
87
,
95
98
]. In the present study, it was possible to verify that the
same genera occur in the guarana endophytic community. One can hypothesize that these isolates
improve guarana plant growth through dierent mechanisms, but further studies are needed to confirm.
J. Fungi 2020,6, 123 10 of 20
3.3. Composition of the Endophytic Microbiota of Genotypes and Municipalities
The distribution of genera according to plant genotypes and collection municipalities is shown in
Figure 3.
In the obtained leaves of MAO 300, five OTUs prevailed over the others, Diaporthe (10%),
Fusarium (9%), Guignardia (9%), Nigrospora (8%) and Colletotrichum (7%). In MBZ 300 only Colletotrichum
(25%) and Fusarium (29%), formed the group of predominant individuals. In the genotype BRS871
from Mau
é
s the genera Nigrospora (30%), Fusarium (14%), Diaporthe (14%), Clonostachys (10%) and
Colletotrichum (8%) showed higher RA values. A smaller number of OTUs prevailed in MAO 871,
only Colletotrichum (17%) and Guignardia (8%). The foliar endophytic community is composed of
20 OTUs, where 8 OTUs were found in both genotypes and municipalities: Clonostachys,Colletotrichum,
Curvularia, Diaporthe,Fusarium,Guignardia,Neopestalotiopsis and Penicillium. The largest number of
unique OTUs, that is, those found only in a given sample, was found in MBZ 300 (4) and MAO
300 (2). In the MBZ 871 sample no unique OTU was observed and only one unique OTU, Phyllosticta,
was obtained from MAO 871.
The OTUs obtained from seeds varied according to plant location and genotype. The endophytes
from the MBZ 300 sample were mainly inserted in the genera Colletotrichum (22%), Clonostachys (26%),
Fusarium (13%), Talaromyces (13%), Diaporthe (9%) and Simplicillium (9%). In MAO 300, other groups had
high RA, Fusarium (47%), Clonostachys (21%), Aspergillus (11%) and Albonectria (11%). In cultivar BRS871
the most abundant OTUs were Fusarium (15%) and Megasporoporia (15%) in MBZ 871, and Cladosporium
(55%) and Fusarium (27%) in MAO 871. The microbial seed community had a higher number of unique
OTUs in Mau
é
s, in samples MBZ 300 (2) and MBZ 871 (2). In Manaus, MAO 300 and MAO 871,
one unique OTU was obtained in each sample, Aspergillus and Cladosporium, respectively. Only one
OTU (Fusarium) was isolated in all guarana genotypes and municipalities studied. In the susceptible
cultivar (BRS300), the highest total amount of OTUs, 8 and 6, were present in the MBZ300 and MAO300
samples, respectively.
The mycobiota present in guarana plants is heterogeneous, varying in distribution and abundance
of genera according to plant genotypes and municipalities of sample collection. The present results
suggest that structuring of guarana fungal community (cultivable organisms) is directed both by the
genetics of the host plant as well as by the geographic location, especially in leaves. These results are
in line with previous studies of grapevines [
99
] and tomato [
100
] plants that have shown that dierent
plant organs, genotypes of the same plant species and even sampling positions in the farmland can
harbor partially dierent microbiomes. Guarana had high dominance of Colletotrichum and Fusarium,
known as well established organisms, with dierent life style types [
98
]. Dierences in life style
depend on environmental conditions, fungal species, host and its maturity. Colletotrichum species life
styles, for example, can be broadly categorized as latent or quiescent, endophytic, hemibiotrophic and
necrotrophic [
101
103
]. Both genera have been extensively associated with the endophytic community
in dierent plants [
62
,
104
,
105
] but are also important pathogens of a wide range of hosts such as
pepper, soy, alfalfa and many other cultivable plants [
101
,
106
,
107
]. Several studies have shown that
pathogenic or parasitic fungi are found as endophytes [
108
112
]. In this situation, endophytes are
latent pathogens that infect the plant, and persist in a dormant phase without causing symptoms in
the host [
113
]. The symptoms and signs of the disease appear rapidly in response to physiological
changes of the plant, either by its stage of maturity or when subjected to nutritional and environmental
changes. Abiotic or biotic stress can trigger the pathogenic activity of endophytes when the host is
not able to limit fungal growth [
101
,
109
,
114
]. In leaves, both genera were observed with high RA
in Mau
é
s, origin of the dispersion of guarana, and in BRS300, cultivar susceptible to anthracnose
and to supersprouting. In seeds, members of Fusarium, were dominant in all samples, regardless of
genotype and locality. On the other hand, individuals from Colletotrichum were only obtained from
seeds of MBZ 300. This suggests that Fusarium endophytes possibly suer vertical transmission in
guarana, passing successively through generations and increasing their presence in the next generation
J. Fungi 2020,6, 123 11 of 20
of seedlings [
115
,
116
]. On the other hand, Colletotrichum endophytes are most probably acquired via
horizontal transmission, therefore being influenced by environment and geographical location.
Anthracnose and oversprouting are between the main diseases aecting guarana crops. The first
mainly aects aerial organs by severe tissue necrosis and the latter is characterized by malformed tissue
and organs in the nodes or branching points. Both diseases lead to guarana plant decline, aecting
plant growth and flowering, and to a reduction in crop productivity. Colletotrichum and Fusarium
endophytes are closely related with pathogens of anthracnose and oversprouting, and some members
may have undergone modifications related to the ecological pressure suered with the establishment
of guarana monocultures in Amazonas and favorable climatic conditions, such as humidity and
temperature extremes suitable for the multiplication of microorganisms. The period in which the
process of endophyte-pathogen modification occurred, that is, the product of the co-evolutionary
process, is an extremely short geological time, as demonstrated in [
117
]. The author suggested
a recent domestication of guarana populations and dispersion originating from Mau
é
s just over
600 years ago. The process of domestication, which relied in monoclonal cultures of guarana from
Mau
é
s and expansion throughout the Amazonas region [
19
], possibly caused “dysbiosis”, that is,
an imbalance in the microbial communities generating the transition of some members to a pathogenic
phase, a process already observed in Colletotrichum magna [
118
], Fusarium graminearum [
119
] and
Lasiodiplodia sp. [
120
]. These results suggest that Colletotrichum and Fusarium could represent potential
pathogens. However, the endophytic microbiota that makes guarana also includes other genera such
as Aspergillus,Clonostachys,Nigrospora,Phomopsis (Diaporthe) and Talaromyces, previously cited in the
literature as having a role in plant bioprotection. In this way, future studies could verify how these
endophytes influence the guarana plant in order to elucidate the types of interactions that occur,
positive, negative or neutral, and whether such interactions can be manipulated in favor of increased
production and protection of guarana.
3.4. Diversity Analysis
Several rarefaction and extrapolation curves of diversity measures comparisons are presented
in Figure 2. First we aimed to compare diversity values of dierent plant organs used for fungal
isolation. However, considering the very low number of individuals obtained from seeds (Table 1),
no rarefaction was achieved and diversity measures were not plotted. In Figure 2b, a reference to
the maximum q=0 rarefaction value obtained for seeds is shown. In comparison, it is possible to
see that leaves have greater richness (q=0), that is, a higher number of genera. Furthermore, Mau
é
s
(Figure 2c) and the susceptible guarana genotype BRS300 (Figure 2d) also have greater richness (q=0)
with no dierences observed in the other indexes studied (q=1, 2). It is interesting to note that the
rarefaction curves in Figure 2b–d were estimated by the combined analysis of leaf and seed isolates.
However, when analyzing them separately, the patterns of diversity change (Figure 4). The richness,
Shannon and Simpson diversity indexes analyzed by BoxPlots show that the municipality of Manaus
(MAO) and the susceptible genotype (BRS300) are the most diverse. In leaves, the greatest diversity
was observed in samples MAO 300 and MAO 871 while the seeds samples MAO 300 and MBZ 300 had
higher indices within the observed parameters. Variation in diversity estimates appears to be greater
in seed samples, which can be explained by the sensitivity of the Shannon index (q=1, Figure 4b)
and of the Simpson index (q=2, Figure 4c) to unique and abundant and only to abundant genera,
respectively. The lower number of analyzed seed isolates exacerbates the observed RA values for the
present OTUs, particularly in sample MAO 300, where two OTUs contain 68% of the RA.
J. Fungi 2020,6, 123 12 of 20
J. Fungi 2020, 6, x FOR PEER REVIEW 13 of 22
Figure 4.
Diversity indexes estimates based on fungal populations obtained from guarana leaves and
seeds of susceptible (BRS300) and resistant (BRS871) plant genotypes from Manaus (MAO) and Mau
é
s
(MBZ), Amazonas, Brazil. (a) species richness; (b) Shannon diversity; and (c) Simpson diversity.
J. Fungi 2020,6, 123 13 of 20
The study of the microbial community and its diversity can be influenced by the traditional
isolation method, since culture dependent methods are highly laborious and can hinder the isolation,
enumeration and maintenance of viable fungus species, as well as obtaining non-culturable and
biotrophic species [
62
,
121
]. Recently, diversity studies have been conducted using methods independent
of microorganism cultivation, such as Next Generation Sequencing (NGS) approaches. However,
these methods may produce errors and have been shown to overestimate the number of microorganisms
present in the samples [
66
,
121
124
]. A previous work comparing culture-based endophyte diversity
data with NGS data from the same host plant revealed that the culture-dependent method by itself has
the ability to reveal a real qualitative picture of fungal endophytes [
123
,
125
]. In addition, the traditional
culture method is the only way to isolate microorganisms for future studies in the laboratory in order
to explore the production of molecules that may be useful for various purposes.
In guarana, the diversity between geographical locations varied according to the source plant
material, possibly related to the more robust amount of fungi sampled in leaves, and genotype, with the
susceptible genotype having higher diversity indexes. This suggests that the microbial diversity of
guarana can be influenced by both genotype and geographical location. Our results are consistent with
previous studies, such as [
126
], where the authors demonstrated that even in the face of disturbances
such as the application of fungicides and presence of pests, the most determinant factors of the
endophytic community of Ageratina altissima were the locality and the cultivar. In another study that
analyzed the abundance, diversity, species composition and relative anity with the host of two
tree species, the community of endophytes diered according to the locality and host species [
127
].
Similar results were observed in the endophytic microbiota of Elymus mollis,Ammophila arenaria and
Ammophila breviligulata, diering from the soil microbial community, which was strictly influenced by
environmental factors, not by the cultivar or location [128].
In the present study, the susceptible genotype and, in general, the municipality of Manaus,
were shown to have greater diversity. Similar results were observed in the seeds and roots of guarana
studied in [
42
]. The authors related the richness of the susceptible genotype to the host vulnerability
to microbial infections. They also reported greater diversity in Manaus, correlated with the large
amount of inoculum that the planting received because it is located near an urban area, dierent from
the one found in Mau
é
s, located in a rural area. The genotypes covered in this study have some
similar characteristics. Both were originally selected from progenies located in the municipality of
Mau
é
s, with clonal propagation by rooting of cuttings, average annual seed yield (1.49 kg
·
plant
1
and 1.55 kg
·
plant
1
) and similar caeine contents (3.92% and 4.04%) [
129
,
130
]. However, they dier
in adapting to dierent conditions. Interestingly, the susceptible genotype observed in this study to
have greater endophyte diversity was reported in other studies to have better rooting of cuttings [
129
],
lower mortality rate [131] and higher yield of production (kg/plant) even with increased competition
for nutrients (plant/area) [
132
]. More recent works have shown that plant microbiota plays a key role
in host adaptation. The dynamic genetic change of the microorganisms provides the necessary time
for the host adaptation to the adverse conditions, improving plant adjustment and survival [
133
].
Considering the idea that the microbial community possibly exerts influence on the performance
and adaptation of the studied guarana plants, achieving a better understanding of how host genetic
variation and geographic location aects the microbial community should be pursued in future eorts
to incorporate biology into evolutionary ecology and agricultural science.
4. Conclusions
Guarana cultivable mycobiota is heterogeneous varying in distribution and abundance of genera
according to host plant genotype and geographic location. It is formed by 25 genera of endophytic
fungi, including the highly abundant Colletotrichum and Fusarium. Culture-dependent methods such as
the strategy adopted in this study give, by themselves, accurate qualitative pictures of fungal diversity
present in the host plant. Nevertheless, improvement of endophyte preservation and identification
techniques is necessary. In the present study, several isolates representing 16 morphotypes were left
J. Fungi 2020,6, 123 14 of 20
unidentified since they have lost their ability to grow in synthetic medium and could not be identified
based on molecular techniques, which lead to the potential diversity loss that these individuals
could represent.
The diversity was higher in the BRS300 susceptible cultivar and in the municipality of Manaus.
The main drivers of microbial community composition and diversity in guarana are plant genotype
and geographic location, as evidenced by the dierence of dominant endophytes in the sampling
units, presence of large number of OTUs found exclusively in certain samples, and diversity patterns.
This is in accordance with previous studies that have related susceptible genotype richness to host
vulnerability to microbial infections and the greater diversity in Manaus with large amount of inoculum
received by the planting.
Colletotrichum and Fusarium are known pathogens responsible, respectively, for anthracnose
and oversprouting diseases that compromise plant health and reduce crop productivity. In this
study, they were isolated as endophytes from healthy guarana tissue, possibly representing potential
pathogens. However, the endophytic community of guarana includes other fungal genera previously
associated with plant bioprotection.
Future studies focusing on how host genetic variation and geographic location aect the microbial
community present in guarana; what types of interactions between endophytes and with the host;
and whether such interactions can be manipulated to improve the fitness of the holobiont are of great
interest in order to increase guarana plant protection and production.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2309-608X/6/3/123/s1,
Table S1: Strains used for phylogenetic analysis. A list of GenBank ITS accession numbers for a set of selected
species and for the 136 guarana isolates amplified here is given.
Author Contributions:
Conceptualization, J.L.d.S.B., C.S. (Cledir Santos) and N.L.; methodology, C.S.
(Carla Santos), B.N.S.d.S. and A.F.T.A.F.eF.; investigation, C.S. (Carla Santos), B.N.S.d.S. and A.F.T.A.F.eF.;
resources, J.L.d.S.B., C.S. (Cledir Santos) and N.L.; data curation, C.S. (Carla Santos), C.S. (Cledir Santos) and N.L.;
writing—original draft preparation, B.N.S.d.S.; C.S. (Cledir Santos) and J.L.d.S.B. writing—review and editing, C.S.
(Carla Santos) and N.L.; supervision, C.S. (Cledir Santos), N.L. and J.L.d.S.B.; project administration, J.L.d.S.B.;
funding acquisition, J.L.d.S.B., C.S. (Cledir Santos) and N.L. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by the Coordenaç
ã
o de Aperfeiçoamento de Pessoal de N
í
vel Superior
(CAPES) project Pr
ó
-Amaz
ô
nia n
3287/13. C.S. (Carla Santos) and N.L. were supported by FCT under the scope
of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004)
funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional
Regional do Norte. C.S. (Cledir Santos) was supported by the Universidad de La Frontera (Temuco, Chile) with
partial funding from the Project PIA19-0001.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
References
1.
Atroch, A.L.; Nascimento Filho, F.J. Guarana—Paullinia cupana Kunth var. sorbilis (Mart.) Ducke. In Exotic
Fruits; Rodrigues, S., Silva, E.O., Brito, E.S., Rodrigues, S., Silva, E.O., Brito, E.S., Eds.; Academic Press:
Cambridge, MA, USA, 2018; pp. 225–236. [CrossRef]
2.
Babu, K.M.; Church, R.J.; Lewander, W. Energy drinks: The new eye-opener for adolescents. Clin. Pediatr.
Emerg. Med. 2008,9, 35–42. [CrossRef]
3. Lorenz, S.D.S. Sateré-Mawé: Os Filhos do Guaraná; Centro de Trabalho Indigenista: São Paulo, Brazil, 1992.
4.
Higgins, J.P.; Tuttle, T.D.; Higgins, C.L. Energy beverages: Content and safety. Mayo Clin. Proc.
2010
,85,
1033–1041. [CrossRef]
5.
Smith, N.; Atroch, A.L. Guaran
á
’s journey from regional tonic to aphrodisiac and global energy drink.
Evid. Based Complement. Alt. Med. eCAM 2010,7, 279–282. [CrossRef]
6.
Pomportes, L.; Davranche, K.; Brisswalter, I.; Hays, A.; Brisswalter, J.; Pomportes, L.; Davranche, K.;
Brisswalter, I.; Hays, A.; Brisswalter, J. Heart rate variability and cognitive function following a multi-vitamin
and mineral supplementation with added guarana (Paullinia cupana). Nutrients
2014
,7, 196–208. [CrossRef]
J. Fungi 2020,6, 123 15 of 20
7.
Bittencourt, L.d.S.; Zeid
á
n-Chuli
á
, F.; Yatsu, F.K.J.; Schnorr, C.E.; Moresco, K.S.; Kolling, E.A.; Gelain, D.P.;
Bassani, V.L.; Moreira, J.C.F. Guarana (Paullinia cupana Mart.) prevents
β
-amyloid aggregation, generation of
advanced glycation-end products (AGEs), and acrolein-induced cytotoxicity on human neuronal-like cells.
Phytother. Res. 2014,28, 1615–1624. [CrossRef]
8.
Boasqu
í
vis, P.F.; Silva, G.M.M.; Paiva, F.A.; Cavalcanti, R.M.; Nunez, C.V.; de Paula Oliveira, R. Guarana
(Paullinia cupana) extract protects Caenorhabditis elegans models for Alzheimer disease and Huntington disease
through activation of antioxidant and protein degradation pathways. Oxid. Med. Cell. Longev.
2018
,2018,
1–16. [CrossRef]
9.
De Oliveira, D.M.; Barreto, G.; Galeano, P.; Romero, J.I.; Holubiec, M.I.; Badorrey, M.S.; Capani, F.; Giraldez
Alvarez, L.D. Paullinia cupana Mart. var. sorbilis protects human dopaminergic neuroblastoma SH-SY5Y cell
line against rotenone-induced cytotoxicity. Hum. Exp. Toxicol. 2011,30, 1382–1391. [CrossRef]
10.
Ruchel, J.B.; Rezer, J.F.P.; Thorstenberg, M.L.; dos Santos, C.B.; Cabral, F.L.; Lopes, S.T.A.; da Silva, C.B.;
Machado, A.K.; da Cruz, I.B.M.; Schetinger, M.R.C.; et al. Hypercholesterolemia and ecto-enzymes of
purinergic system: Eects of Paullinia cupana.Phytother. Res. 2016,30, 49–57. [CrossRef]
11.
Bortolin, R.C.; Vargas, A.R.; Ramos, V.D.M.; Gasparotto, J.; Chaves, P.R.; Schnorr, C.E.; da Boit Martinello, K.;
Silveira, A.K.; Gomes, H.M.; Rabelo, T.K.; et al. Guarana supplementation attenuated obesity, insulin
resistance, and adipokines dysregulation induced by a standardized human western diet via brown adipose
tissue activation. Phytother. Res. 2019,33, 1394–1403. [CrossRef]
12.
Lima, N.D.S.; Teixeira, L.; Gambero, A.; Ribeiro, M.L. Guarana (Paullinia cupana) stimulates mitochondrial
biogenesis in mice fed high-fat diet. Nutrients 2018,10, 165. [CrossRef]
13.
Martel, J.; Ojcius, D.M.; Chang, C.-J.; Lin, C.-S.; Lu, C.-C.; Ko, Y.-F.; Tseng, S.-F.; Lai, H.-C.; Young, J.D.
Anti-obesogenic and antidiabetic eects of plants and mushrooms. Nat. Rev. Endocrinol.
2017
,13, 149–160.
[CrossRef] [PubMed]
14.
Santana,
Á
.L.; Macedo, G.A. Health and technological aspects of methylxanthines and polyphenols from
guarana: A review. J. Funct. Foods 2018,47, 457–468. [CrossRef]
15.
Basile, A.; Ferrara, L.; Pezzo, D.M.; Mele, G.; Sorbo, S.; Bassi, P.; Montesano, D. Antibacterial and antioxidant
activities of ethanol extract from Paullinia cupana Mart. J. Ethnopharmacol. 2005,102, 32–36. [CrossRef]
16.
Hamerski, L.; Vieira Somner, G.; Tamaio, N. Paullinia cupana Kunth (Sapindaceae): A review of its
ethnopharmacology, phytochemistry and pharmacology. J. Med. Plants Res. 2013,7, 2221–2229. [CrossRef]
17.
Avila-Sosa, R.; Montero-Rodr
í
guez, A.F.; Aguilar-Alonso, P.; Vera-L
ó
pez, O.; Lazcano-Hern
á
ndez, M.;
Morales-Medina, J.C.; Navarro-Cruz, A.R. Antioxidant properties of amazonian fruits: A mini review of
in vivo and in vitro studies. Oxid. Med. Cell. Longev. 2019,2019, 1–11. [CrossRef]
18.
Cadon
á
, F.C.; Rosa, J.L.; Schneider, T.; Cubillos-Rojas, M.; S
á
nchez-Tena, S.; Azzolin, V.F.; Assmann, C.E.;
Machado, A.K.; Ribeiro, E.E.; da Cruz, I.B.M. Guaran
á
, a highly caeinated food, presents
in vitro
antitumor
activity in colorectal and breast cancer cell lines by inhibiting AKT/mTOR/S6K and MAPKs pathways.
Nutr. Cancer 2017,69, 800–810. [CrossRef]
19.
Marques, L.L.M.; Ferreira, E.D.F.; Paula, D.M.N.; Klein, T.; Mello, D.J.C.P. Paullinia cupana: A multipurpose
plant—A review. Braz. J. Pharmacogn. 2018,29, 77–110. [CrossRef]
20.
Antonelli-Ushirobira, T.M.; Kaneshima, E.N.; Gabriel, M.; Audi, E.A.; Marques, L.C.; Mello, J.C.P. Acute
and subchronic toxicological evaluation of the semipurified extract of seeds of guaran
á
(Paullinia cupana) in
rodents. Food Chem. Toxicol. 2010,48, 1817–1820. [CrossRef]
21.
Marx, F. Analysis of guarana seeds II. Studies on the composition of the tannin fraction. Z. Lebensm.
Unters. Forsch. 1990,190, 429–431. [CrossRef]
22.
Pinto, C.E.D.L.; Atroch, A.L.; Fajardo, J.D.V.; Nascimento Filho, D.F.J. Seleç
ã
o de clones de guaranazeiro para
adaptabilidade e estabilidade no estado do Amazonas. Rev. Ciênc. Agrár. 2018,61, 1–7. [CrossRef]
23.
Market Data Forecast. Guarana Seed Extract Market by Form (Powder and Liquid) by Distribution (Health Stores,
Drug Stores, Online Retailing and Other Channels) by Application (Pharmaceuticals, Dietary Supplements, Cosmetics,
Food and Beverages, and Others), and by Region—Global Industry Analysis, Size, Share, Growth, Trends, And Forecast
To 2024; Custom Market Research Services: Albany, NY, USA, 2019; Volume 145.
24.
IBGE. Levantamento sistem
á
tico da produç
ã
o agr
í
cola. In Intituto Brasileiro de Geografia e Estatistica; IBGE:
Rio de Janeiro, Brazil, 2017.
25.
IBGE. Levantamento sistem
á
tico da produç
ã
o agr
í
cola: Pesquisa mensal de previs
ã
o e acompanhamento das
safras agrícolas no ano civil. In Intituto Brasileiro de Geografia e Estatistica; IBGE: Rio de Janeiro, Brazil, 2018.
J. Fungi 2020,6, 123 16 of 20
26.
Clima Mau
é
s: Temperatura, Tempo e Dados Climatol
ó
gicos Mau
é
s—Climate-Data.org. Available online:
https://pt.climate-data.org/america-do-sul/brasil/amazonas/maues-879673/(accessed on 27 May 2020).
27. Albuquerque, F.C. Antracnose do Guaraná; 1-37: Boletim Técnico; IAN: Belém, Brazil, 1960.
28. Araújo, J.C.A.; Pereira, J.C.R.; Gasparotto, L.; Arruda, D.M.R. O Complexo Superbrotamento do Guaranazeiro e
Seu Controle; Embrapa Amazônia Central: Manaus, Brazil, 2006.
29.
Gonçalves, J.R.C. Notas Sobre as Doenças e Pragas do Guaran
á
no Estado do Amazonas; IPEAN: Bel
é
m, Brazil, 1967.
30.
Tavares, A.M.; Atroch, A.L.; Nascimento Filho, D.F.J.; Pereira, J.C.R.; Ara
ú
jo, D.J.C.A.; Moares, L.A.C.;
Santos, L.P.; Garcia, M.V.B.; Arruda, D.M.R.; Sousa, N.R.; et al. Cultura do Guaranazeiro no Amazonas, 4th ed.;
Embrapa Amazônia Ocidental: Manaus, Brazil, 2005.
31.
Ara
ú
jo, J.C.A.; Pereira, J.C.R.; Gasparotto, L.; Arruda, D.M.R.; Moreira, A. Antracnose do Guaranazeiro e Seu
Controle; Embrapa Amazônia Ocidental: Manaus, Brazil, 2007; pp. 3–6.
32.
Ara
ú
jo, J.C.A.; Pereira, J.C.R.; Gasparotto, L.; Arruda, D.M.R.; Nascimento Filho, D.F.J.; Moreira, A. Avaliaç
ã
o
de fungicidas no controle da antracnose do guaranazeiro. In Proceedings of the Embrapa Amaz
ô
nia
Ocidental-Artigo em Anais de Congresso (ALICE), Manaus, Brazil, 31 October 2007.
33.
Baedke, J.; F
á
bregas-Tejeda, A.; Delgado, A.N. The holobiont concept before Margulis. J. Exp. Zoology Part B
Mol. Dev. Evol. 2020,334, 149–155. [CrossRef] [PubMed]
34.
Margulis, L.; Fester, R. Symbiosis as a Source of Evolutionary Innovation: Speciation and Morphogenesis; MIT Press:
Cambridge, MA, USA, 1991; p. 470.
35.
Zilber-Rosenberg, I.; Rosenberg, E. Role of microorganisms in the evolution of animals and plants:
The hologenome theory of evolution. FEMS Microbiol. Rev. 2008,32, 723–735. [CrossRef] [PubMed]
36.
Toju, H.; Guimar
ã
es, P.R.; Olesen, J.M.; Thompson, J.N. Assembly of complex plant-fungus networks.
Nat. Commun. 2014,5, 5273. [CrossRef] [PubMed]
37.
Vandenkoornhuyse, P.; Quaiser, A.; Duhamel, M.; Le Van, A.; Dufresne, A. The importance of the microbiome
of the plant holobiont. New Phytol. 2015,206, 1196–1206. [CrossRef]
38.
Busby, P.E.; Ridout, M.; Newcombe, G. Fungal endophytes: Modifiers of plant disease. Plant Mol. Biol.
2016
,
90, 645–655. [CrossRef]
39.
Raghavendra, A.K.H.; Newcombe, G. The contribution of foliar endophytes to quantitative resistance to
Melampsora rust. New Phytol. 2013,197, 909–918. [CrossRef]
40.
Ahlholm, J.U.; Helander, M.; Henriksson, J.; Metzler, M.; Saikkonen, K. Environmental conditions and host
genotype direct genetic diversity of Venturia ditricha, a fungal endophyte of birch trees. Evolution
2002
,56,
1566–1573. [CrossRef]
41.
Wäli, P.R.; Helander, M.; Nissinen, O.; Saikkonen, K. Susceptibility of endophyte-infected grasses to winter
pathogens (snow molds). Can. J. Bot. 2006,84, 1043–1051. [CrossRef]
42.
Azevedo Silva, F.; Liotti, R.G.; Boleti, A.P.D.A.; Reis,
É
.D.M.; Passos, M.B.S.; dos Santos, E.L.; Sampaio, O.M.;
Janu
á
rio, A.H.; Branco, C.L.B.; Silva, D.G.F.; et al. Diversity of cultivable fungal endophytes in Paullinia cupana
(Mart.) Ducke and bioactivity of their secondary metabolites. PLoS ONE 2018,13, e0195874. [CrossRef]
43.
Elias, L.M.; Fortkamp, D.; Sartori, S.B.; Ferreira, M.C.; Gomes, L.H.; Azevedo, J.L.; Montoya, V.Q.;
Rodrigues, A.; Ferreira, A.G.; Lira, S.P. The potential of compounds isolated from Xylaria spp. as antifungal
agents against anthracnose. Braz. J. Microbiol. 2018,49, 840–847. [CrossRef]
44.
Sia, D.E.; Marcon, J.; Luvizotto, D.; Quecine, M.; Tsui, S.; Pereira, J.; Pizzirani-Kleiner, A.; Azevedo, J.
Endophytic fungi from the Amazonian plant Paullinia cupana and from Olea europaea isolated using cassava
as an alternative starch media source. Springer Plus 2013,2, 579. [CrossRef]
45.
Souza, D.A.Q.L.; Souza, D.A.D.L.; Astolfi Filho, S.; Pinheiro, M.L.B.; Sarquis, M.I.D.M.; Pereira, J.O. Atividade
antimicrobiana de fungos endof
í
ticos isolados de plantas t
ó
xicas da Amaz
ô
nia: Palicourea longiflora (aubl.)
Rich e Strychnos cogens Bentham. Acta Amazon. 2004,34, 185–195. [CrossRef]
46.
Barnett, H.L.; Hunter, B.B. Illustrated Genera of Imperfect Fungi, 3rd ed.; Burgess Publishing Company:
Minneapolis, MI, USA, 1972; p. 241.
47.
Seifert, K.; Morgan-Jones, G.; Gams, W.; Kendrick, B. The Genera of Hyphomycetes; CBS-KNAW Fungal
Biodiversity Centre: Utrecht, The Netherlands, 2011; p. 997.
48.
Pitt, J.I. The Genus Penicillium and Its Teleomorphic States Eupenicillium and Talaromyces; Academic Press:
London, UK, 1979.
49.
Sutton, B.C. The Coelomycetes. Fungi Imperfecti with Pycnidia, Acervuli and Stromata; Commonwealth Mycol.
Inst.: Surrey, UK, 1980; Volume 696. [CrossRef]
J. Fungi 2020,6, 123 17 of 20
50.
Takashio, M. Single-spore and single-cell cultures of fungi. Two new methods particularly useful in the
isolation of fungal spores and cells. Ann. Microbiol. 1974,125A, 45–56.
51.
White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA
genes for phylogenetics. In PCR Protocls: A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H.,
Sninsky, J.J., White, T.J., Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Inoculum: London, UK,
1990; pp. 315–322.
52.
Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput.
Nucleic Acids Res. 2004,32, 1792–1797. [CrossRef] [PubMed]
53.
Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis
across Computing Platforms. Mol. Biol. Evol. 2018,35, 1547–1549. [CrossRef] [PubMed]
54.
Stecher, G.; Tamura, K.; Kumar, S. Molecular Evolutionary Genetics Analysis (MEGA) for macOS.
Mol. Biol. Evol. 2020,37, 1237–1239. [CrossRef]
55.
Castresana, J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis.
Mol. Biol. Evol. 2000,17, 540–552. [CrossRef]
56.
Kimura, M. A simple method for estimating evolutionary rates of base substitutions through comparative
studies of nucleotide sequences. J. Mol. Evol. 1980,16, 111–120. [CrossRef]
57.
Felsenstein, J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution
1985
,39, 783–791.
[CrossRef]
58.
Letunic, I.; Bork, P. Interactive tree of life (iTOL) v4: Recent updates and new developments. Nucleic Acids Res.
2019,47, W256–W259. [CrossRef]
59.
McMurdie, P.J.; Holmes, S. Phyloseq: An R package for reproducible interactive analysis and graphics of
microbiome census data. PLoS ONE 2013,8, e61217. [CrossRef]
60. Wickham, H. ggplot2: Elegant Graphics for Data Analysis; Springer: New York, NY, USA, 2009. [CrossRef]
61. Hsieh, T.C.; Ma, K.H.; Chao, A. iNEXT: An R package for rarefaction and extrapolation of species diversity
(Hill numbers). Methods Ecol. Evol. 2016,7, 1451–1456. [CrossRef]
62.
Singh, D.K.; Sharma, V.K.; Kumar, J.; Mishra, A.; Verma, S.K.; Sieber, T.N.; Kharwar, R.N. Diversity of
endophytic mycobiota of tropical tree Tectona grandis Linn.f.: Spatiotemporal and tissue type eects. Sci. Rep.
2017,7, 3745. [CrossRef] [PubMed]
63.
Tan, X.-M.; Zhou, Y.-Q.; Zhou, X.-L.; Xia, X.-H.; Wei, Y.; He, L.-L.; Tang, H.-Z.; Yu, L.-Y. Diversity and bioactive
potential of culturable fungal endophytes of Dysosma versipellis; a rare medicinal plant endemic to China.
Sci. Rep. 2018,8, 5929. [CrossRef] [PubMed]
64.
Guo, L.D.; Hyde, K.D.; Liew, E.C.Y. Identification of endophytic fungi from Livistona chinensis based on
morphology and rDNA sequences. New Phytol. 2000,147, 617–630. [CrossRef]
65.
Wang, Y.; Guo, L.D.; Hyde, K.D. Taxonomic placement of sterile morphotypes of endophytic fungi from
Pinus tabulaeformis (Pinaceae) in northeast China based on rDNA sequences. Fungal Divers.
2005
,20, 235–260.
66.
Sun, X.; Guo, L.D. Endophytic fungal diversity: Review of traditional and molecular techniques. Mycology
2012,3, 65–76. [CrossRef]
67.
Sandoval-Denis, M.; Crous, P.W. Removing chaos from confusion: Assigning names to common human and
animal pathogens in Neocosmospora.Persoonia 2018,41, 109–129. [CrossRef]
68.
Diogo, H.C.; Sarpieri, A.; Pires, M.C. Preservaç
ã
o de fungos em
á
gua destilada. Anais Bras. Dermatol.
2005
,
80, 591–594. [CrossRef]
69.
Burdsall, H.H.; Dorworth, E.B. Preserving cultures of wood-decaying Basidiomycotina using sterile distilled
water in cryovials. Mycologia 2007,86, 275. [CrossRef]
70. Okafor, N. Modern Industrial Microbiolohy and Biotechnology; Science Publishers: Enfield, NH, USA, 2007.
71.
Sarma, P.; Dkhar, M.S.; Kayang, H.; Kumar, M.; Dubey, N.K.; Raghuwanshi, R. Diversity of endophytic
fungi associated with the medicinally important aromatic plant Gaultheria fragrantissima.Stud. Fungi
2018
,3,
309–320. [CrossRef]
72.
Selim, K.A.; Waill, A.E.; Ahmed, M.T.; Ahmed, A.E.-B.; Tahany, M.A.-R.; Ahmed, I.E.-D.; Eman, F.A. Antiviral
and antioxidant potential of fungal endophytes of Egyptian medicinal plants. Fermentation
2018
,4, 49.
[CrossRef]
73.
Mahmoud, A.G.Y.; Zaher, E.H.F. Why nuclear ribosomal Internal Transcribed Spacer (ITS) has been selected
as the DNA barcode for fungi? Adv. Gen. Eng. 2015,4, 1–2. [CrossRef]
J. Fungi 2020,6, 123 18 of 20
74.
Chao, A.; Gotelli, N.J.; Hsieh, T.C.; Sander, E.L.; Ma, K.H.; Colwell, R.K.; Ellison, A.M. Rarefaction and
extrapolation with Hill numbers: A framework for sampling and estimation in species diversity studies.
Ecol. Monogr. 2014,84, 45–67. [CrossRef]
75. Alfenas, A.C.; Mafia, R.G. Métodos em Fitopatologia, 2nd ed.; Editora UFV: Viçosa, Brazil, 2016.
76. Carroll, G. Fungal endophytes in stems and leaves: From latent pathogen to mutualistic symbiont. Ecology
1988,69, 2–9. [CrossRef]
77.
Rajamanikyam, M.; Vadlapudi, V.; amanchy, R.; Upadhyayula, S.M.; Rajamanikyam, M.; Vadlapudi, V.;
amanchy, R.; Upadhyayula, S.M. Endophytic fungi as novel resources of natural therapeutics. Braz. Arch.
Biol. Technol. 2017,60. [CrossRef]
78.
Martin, R.; Gazis, R.; Skaltsas, D.; Chaverri, P.; Hibbett, D. Unexpected diversity of basidiomycetous
endophytes in sapwood and leaves of Hevea.Mycologia 2015,107, 284–297. [CrossRef]
79.
Figueiredo,
Á
.; Silva, A.C.e. Atividade
in vitro
de extratos de Pycnoporus sanguineus eLentinus crinitus sobre
o fitopatógeno Fusarium sp. Acta Amazon. 2014,44, 1–8. [CrossRef]
80.
Iqbal, M.; Dubey, M.; Gudmundsson, M.; Viketoft, M.; Jensen, D.F.; Karlsson, M. Comparative evolutionary
histories of fungal proteases reveal gene gains in the mycoparasitic and nematode-parasitic fungus
Clonostachys rosea.BMC Evol. Biol. 2018,18, 171. [CrossRef]
81.
Li, J.; Yang, J.; Huang, X.; Zhang, K.Q. Purification and characterization of an extracellular serine protease
from Clonostachys rosea and its potential as a pathogenic factor. Process Biochem.
2006
,41, 925–929. [CrossRef]
82.
Zou, C.G.; Xu, Y.F.; Liu, W.J.; Zhou, W.; Tao, N.; Tu, H.H.; Huang, X.W.; Yang, J.K.; Zhang, K.Q. Expression of
a serine protease gene prCIs up-regulated by oxidative stress in the fungus Clonostachys rosea: Implications
for fungal survival. PLoS ONE 2010,5, e13386. [CrossRef] [PubMed]
83.
Melo, D.I.S.; Valente, A.M.M.P.; Kavamura, V.N.; Vilela, E.S.D.; Faull, J.L. Mycoparasitic nature of Bionectria sp.
strain 6.21. J. Plant Protect. Res. 2014,54, 327–333. [CrossRef]
84.
Salamone, A.L.; Gundersen, B.; Inglis, D.A. Clonostachys rosea, a potential biological control agent for
Rhizoctonia solani AG-3 causing black scurf on potato. Biocontr. Sci. Technol. 2018,28, 895–900. [CrossRef]
85.
Vivas, J.M.S.; da Silveira, S.F.; dos Santos, P.H.D.; Carvalho, B.M.; Poltronieri, T.P.D.S.; Jorge, T.S.; Santos, J.S.;
Kurosawa, R.D.N.F.; de Moraes, R. Antagonism of fungi with biocontrol potential of papaya black spot
caused by Asperisporium caricae.Austr. J. Crop Sci. 2018,12, 827–833. [CrossRef]
86.
Hassan, M.M.; Daalla, H.M.; Modwi, H.I.; Osman, M.G.; Ahmed, I.I.; Gani, M.E.A.; El, A.; Babiker, G.E.
Eects of fungal strains on seeds germination of millet and Striga hermonthica.Univ. J. Agricult. Res.
2013
,2,
83–88. [CrossRef]
87.
Hung, R.; Lee Rutgers, S. Applications of Aspergillus in plant growth promotion. New Future Dev. Microbial
Biotechnol. Bioeng. 2016, 223–227. [CrossRef]
88.
Pereira, F.T.; Oliveira, D.J.B.; Muniz, P.H.P.C.; Peixoto, G.H.S.; Guimar
ã
es, R.R.; Carvalho, D.D.C.; Pereira, F.T.;
Oliveira, D.J.B.; Muniz, P.H.P.C.; Peixoto, G.H.S.; et al. Growth promotion and productivity of lettuce using
Trichoderma spp. commercial strains. Horticult. Bras. 2019,37, 69–74. [CrossRef]
89.
Waqas, M.; Khan, A.L.; Hamayun, M.; Shahzad, R.; Kang, S.-M.; Kim, J.-G.; Lee, I.-J. Endophytic fungi
promote plant growth and mitigate the adverse eects of stem rot: An example of Penicillium citrinum and
Aspergillus terreus.J. Plant Interact. 2015,10, 280–287. [CrossRef]
90.
Xia, C.; Li, N.; Zhang, X.; Feng, Y.; Christensen, M.J.; Nan, Z. An Epichloë endophyte improves photosynthetic
ability and dry matter production of its host Achnatherum inebrians infected by Blumeria graminis under
various soil water conditions. Fungal Ecol. 2016,22, 26–34. [CrossRef]
91.
Zavala-Gonzalez, E.A.; Rodr
í
guez-Cazorla, E.; Escudero, N.; Aranda-Martinez, A.; Mart
í
nez-Laborda, A.;
Ram
í
rez-Lepe, M.; Vera, A.; Lopez-Llorca, V.L. Arabidopsis thaliana root colonization by the nematophagous
fungus Pochonia chlamydosporia is modulated by jasmonate signaling and leads to accelerated flowering and
improved yield. New Phytol. 2017,213, 351–364. [CrossRef]
92.
Hardoim, P.R.; Van Overbeek, L.S.; Berg, G.; Pirttilä, A.M.; Compant, S.; Campisano, A.; Döring, M.;
Sessitsch, A. The hidden world within plants: Ecological and evolutionary considerations for defining
functioning of microbial endophytes. Microbiol. Mol. Biol. Rev. 2015,79, 293–320. [CrossRef] [PubMed]
93.
Naraghi, L.; Heydari, A.; Rezaee, S.; Razavi, M. Biocontrol agent Talaromyces flavus stimulates the growth of
cotton and potato. J. Plant Growth Regul. 2012,31, 471–477. [CrossRef]
J. Fungi 2020,6, 123 19 of 20
94.
Hossain, M.M.; Sultana, F.; Kubota, M.; Koyama, H.; Hyakumachi, M. The plant growth-promoting fungus
Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense
signals. Plant Cell Physiol. 2007,48, 1724–1736. [CrossRef]
95.
Hamayun, M.; Afzal Khan, S.; Ahmad, N.; Tang, D.-S.; Kang, S.-M.; Na, C.-I.; Sohn, E.-Y.; Hwang, Y.-H.;
Shin, D.-H.; Lee, B.-H.; et al. Cladosporium sphaerospermum as a new plant growth-promoting endophyte from
the roots of Glycine max (L.) Merr. World J. Microbiol. Biotechnol. 2009,25, 627–632. [CrossRef]
96.
Khalmuratova, I.; Kim, H.; Nam, Y.-J.; Oh, Y.; Jeong, M.-J.; Choi, H.-R.; You, Y.-H.; Choo, Y.-S.; Lee, I.-J.;
Shin, J.-H.; et al. Diversity and plant growth promoting capacity of endophytic fungi associated with
halophytic plants from the west coast of Korea. Mycobiology 2015,43, 373–383. [CrossRef] [PubMed]
97.
Priyadharsini, P.; Muthukumar, T. The root endophytic fungus Curvularia geniculata from Parthenium
hysterophorus roots improves plant growth through phosphate solubilization and phytohormone production.
Fungal Ecol. 2017,27, 69–77. [CrossRef]
98.
Chithra, S.; Jasim, B.; Mathew, J.; Radhakrishnan, E.K. Endophytic Phomopsis sp. colonization in Oryza sativa
was found to result in plant growth promotion and piperine production. Physiol. Plant.
2017
,160, 437–446.
[CrossRef]
99.
Berlanas, C.; Berbegal, M.; Elena, G.; Laidani, M.; Cibriain, J.F.; Sagües, A.; Gramaje, D. The fungal and
bacterial rhizosphere microbiome associated with grapevine rootstock genotypes in mature and young
vineyards. Front. Microbiol. 2019,10, 1142. [CrossRef]
100. Toju, H.; Okayasu, K.; Notaguchi, M. Leaf-associated microbiomes of grafted tomato plants. Sci. Rep. 2019,
9, 1787. [CrossRef] [PubMed]
101.
De Silva, D.D.; Crous, P.W.; Ades, P.K.; Hyde, K.D.; Taylor, P.W.J. Life styles of Colletotrichum species and
implications for plant biosecurity. Fungal Biol. Rev. 2017,31, 155–168. [CrossRef]
102.
Fesel, P.H.; Zuccaro, A. Dissecting endophytic lifestyle along the parasitism/mutualism continuum in
Arabidopsis.Curr. Opin. Microbiol. 2016,32, 103–112. [CrossRef] [PubMed]
103.
O’Connell, R.J.; Thon, M.R.; Hacquard, S.; Amyotte, S.G.; Kleemann, J.; Torres, M.F.; Damm, U.; Buiate, E.A.;
Epstein, L.; Alkan, N.; et al. Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by
genome and transcriptome analyses. Nat. Genet. 2012,44, 1060–1065. [CrossRef] [PubMed]
104.
Corr
ê
a, R.C.G.; Rhoden, S.A.; Mota, T.R.; Azevedo, J.L.; Pamphile, J.A.; de Souza, C.G.M.; Polizeli, M.D.L.T.D.M.;
Bracht, A.; Peralta, R.M. Endophytic fungi: Expanding the arsenal of industrial enzyme producers. J. Ind.
Microbiol. Biotechnol. 2014,41, 1467–1478. [CrossRef] [PubMed]
105.
Toghueo, R.M.K.; Zabalgogeazcoa, I.; V
á
zquez de Aldana, B.R.; Boyom, F.F. Enzymatic activity of endophytic
fungi from the medicinal plants Terminalia catappa,Terminalia mantaly and Cananga odorata.S. Afr. J. Bot.
2017
,
109, 146–153. [CrossRef]
106.
Eken, C.; Demirci, E. First report of Colletotrichum truncatum on alfalfa in Turkey. Plant Dis.
2007
,84, 100.
[CrossRef]
107.
Yang, H.C.; Haudenshield, J.S.; Hartman, G.L. First report of Colletotrichum chlorophyti causing soybean
anthracnose. Plant Dis. 2012,96, 1699. [CrossRef]
108.
G
ó
rzy´nska, K.; W˛egrzyn, E.; Sandecki, R.; Lembicz, M. Endophytic fungi and latent pathogens in the sedge
Carex secalina (Cyperaceae), a critically endangered species in Europe. Plant Protect. Sci.
2019
,55, 102–108.
[CrossRef]
109.
Photita, W.; Lumyong, S.; Lumyong, P.; McKenzie, E.H.C.; Hyde, K.D.; Photita, W.; Lumyong, S.; Lumyong, P.;
Hyde, M.E.H.C. Are some endophytes of Musa acuminata latent pathogens? Fungal Divers.
2004
,16, 131–140.
110.
Sessa, L.; Abreo, E.; Lupo, S. Diversity of fungal latent pathogens and true endophytes associated with fruit
trees in Uruguay. J. Phytopathol. 2018,166, 633–647. [CrossRef]
111.
Slippers, B.; Wingfield, M.J. Botryosphaeriaceae as endophytes and latent pathogens of woody plants:
Diversity, ecology and impact. Fungal Biol. Rev. 2007,21, 90–106. [CrossRef]
112.
Soumya, P.R.; Rukshana Begum, S.; Tamil Selvi, K.S. Endophytic fungi as latent pathogens in
Eichhornia crassipes (Mart.) Solms. Int. J. Adv. Sci. Res. Manag. 2018,3, 140–146.
113.
Petrini, O. Fungal endophytes of tree leaves. In Microbial Ecology of Leaves; Andrews, J.H., Hirano, S.S.,
Andrews, J.H., Hirano, S.S., Eds.; Springer: New York, NY, USA, 1991; pp. 179–197. [CrossRef]
114. Verhoe, K. Latent infections by fungi. Ann. Rev. Phytopathol. 1974,12, 99–110. [CrossRef]
115.
Shade, A.; Jacques, M.A.; Barret, M. Ecological patterns of seed microbiome diversity, transmission,
and assembly. Curr. Opin. Microbiol. 2017,37, 15–22. [CrossRef] [PubMed]
J. Fungi 2020,6, 123 20 of 20
116.
Shahzad, R.; Khan, A.L.; Bilal, S.; Asaf, S.; Lee, I.-J. What is there in seeds? Vertically transmitted endophytic
resources for sustainable improvement in plant growth. Front. Plant Sci. 2018,9, 24. [CrossRef] [PubMed]
117.
Sousa, N.R. Variabilidade Gen
é
tica e Estimativas de Par
â
metros Gen
é
ticos em Germoplasma de Guaranazeiro;
Embrapa Amazônia Ocidental: Maues, Brazil, 2004.
118.
Freeman, S.; Rodriguez, R.J. Genetic conversion of a fungal plant pathogen to a nonpathogenic, endophytic
mutualist. Science 1993. [CrossRef] [PubMed]
119.
Lofgren, L.A.; LeBlanc, N.R.; Certano, A.K.; Nachtigall, J.; LaBine, K.M.; Riddle, J.; Broz, K.; Dong, Y.;
Bethan, B.; Kafer, C.W.; et al. Fusarium graminearum: Pathogen or endophyte of North American grasses?
New Phytol. 2018. [CrossRef]
120.
Slippers, B.; Boissin, E.; Phillips, A.J.L.; Groenewald,J.Z.; Lombard, L.; Wingfield, M.J.; Postma, A.; Burgess, T.;
Crous, P.W. Phylogenetic lineages in the Botryosphaeriales: A systematic and evolutionary framework.
Stud. Mycol. 2013,76, 31–49. [CrossRef]
121.
Unterseher, M.; Gazis, R.; Chaverri, P.; Guarniz, C.F.G.; Tenorio, D.H.Z. Endophytic fungi from Peruvian
highland and lowland habitats form distinctive and host plant-specific assemblages. Biodivers. Conserv.
2013
,
22, 999–1016. [CrossRef]
122.
Ovaskainen, O.; Nokso-Koivisto, J.; Hottola, J.; Rajala, T.; Pennanen, T.; Ali-Kovero, H.; Miettinen, O.;
Oinonen, P.; Auvinen, P.; Paulin, L.; et al. Identifying wood-inhabiting fungi with 454 sequencing—What is
the probability that BLAST gives the correct species? Fungal Ecol. 2010,3, 274–283. [CrossRef]
123.
Tedersoo, L.; Nilsson, R.H.; Abarenkov, K.; Jairus, T.; Sadam, A.; Saar, I.; Bahram, M.; Bechem, E.; Chuyong, G.;
K
õ
ljalg, U. 454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results
but reveal substantial methodological biases. New Phytol. 2010,188, 291–301. [CrossRef]
124.
Unterseher, M.; Schnittler, M. Dilution-to-extinction cultivation of leaf-inhabiting endophytic fungi in beech
(Fagus sylvatica L.)—Dierent cultivation techniques influence fungal biodiversity assessment. Mycol. Res.
2009,113, 645–654. [CrossRef] [PubMed]
125.
Zhang, T.; Yao, Y.-F. Endophytic fungal communities associated with vascular plants in the high Arctic zone
are highly diverse and host-plant specific. PLoS ONE 2015,10, e0130051. [CrossRef] [PubMed]
126.
Christian, N.; Sullivan, C.; Visser, N.D.; Clay, K. Plant host and geographic location drive endophyte
community composition in the face of perturbation. Microbial Ecol.
2016
,72, 621–632. [CrossRef] [PubMed]
127.
Homan, M.T.; Arnold, A.E. Geographic locality and host identity shape fungal endophyte communities in
cupressaceous trees. Mycol Res. 2008,112, 331–344. [CrossRef]
128.
David, A.S.; Seabloom, E.W.; May, G. Plant host species and geographic distance aect the structure of
aboveground fungal symbiont communities, and environmental filtering aects belowground communities
in a coastal dune ecosystem. Microbial Ecol. 2016,71, 912–926. [CrossRef]
129.
De Arruda, M.R.; Cl
é
rio, J.; Pereira, R.; Moreira, A.; Geraldes Teixeira, W. Survival rate of guarana herbaceous
cuttings in dierent substrates. Cienc. Agrotecnol. 2007,31, 236–241.
130.
Garcia, T.B.; Nascimento Filho, D.F.J. O Cultivo do Guarana no Amazonas; Embrapa Amaz
ô
nia Ocidental:
Manaus, Brazil, 1999.
131.
Albertino, S.M.F.; Filho, F.J.D.N.; da Silva, J.F.; Atroch, A.L.; Galv
ã
o, A.K.D.L. Enraizamento de estacas de
cultivares de guaranazeiro com adubaç
ã
o de plantas matrizes. Pesqui. Agropecu. Bras.
2012
,47, 1449–1454.
[CrossRef]
132.
Pl
á
cido, C.G.; Moreira, A.; Moraes, L.A.C. Spacing and plant density in the yield components, nutritional
status, and soil fertility of guarana varieties grown in humid tropical Amazon. Commun. Soil Sci. Plant Anal.
2015,46, 1551–1565. [CrossRef]
133.
Rosenberg, E.; Zilber-Rosenberg, I. The hologenome concept of evolution after 10 years. Microbiome
2018
,
6, 78. [CrossRef]
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Chapter
In recent years the microbial associates of plants have been increasingly considered an essential tool for improving crop production while reducing the input of agrochemicals. With special reference to fungi, new achievements have shown that their application in crop management can go beyond the specific role that has been traditionally assigned to certain species and that strains combining aptitudes as plant growth promoters and antagonists of pests and pathogens can be used as multipurpose agents for improving plant fitness and crop yields. Species in the genus Talaromyces (Eurotiomycetes, Trichocomaceae) have been more and more widely reported for endophytic occurrence in investigations carried out in various agricultural and non-agricultural contexts. Our review of the available literature pointed out that 46 out of a total of over 170 species currently accepted in this genus have been found as endophytic associates of 281 plant species belonging to 108 families, with an evident raising trend that will likely lead to a notable increase of these figures shortly. Mutualistic interactions of these fungi with plants are analyzed in this paper regarding the ecological impact and applicative perspectives in sustainable agriculture.
Article
Plants of the genus Hevea present a great diversity of endophytic fungal species, which can provide bioactive compounds and enzymes for biotechnological use, and antagonist agents for plant disease biological control. The diversity of endophytic fungi associated with leaves of Hevea spp. clones in western Amazonia was explored using cultivation-based techniques, combined with the sequencing of the ITS rRNA-region. A total of 269 isolates were obtained, and phylogenetic analysis showed that they belong to 47 putative species, of which 24 species were unambiguous. The phylum Ascomycota was the most abundant (95.4%), with predominance of the genera Colletotrichum and Diaporthe, followed by the phylum Basidiomycota (4.6%), with abundance of the genera Trametes and Phanerochaete. Endophytic composition was influenced by the clones, with few species shared among them, and the greatest diversity was found in clone C44 (richness: 26, Shannon: 14,15, Simpson: 9.11). The potential for biocontrol and enzymatic production of endophytes has been investigated. In dual culture tests, 95% of the isolates showed inhibitory activity against C. gloeosporioides, and 84% against C. cassiicola. Efficient inhibition was obtained with isolates HEV158C and HEV255M (Cophinforma atrovirens and Polyporales sp. 2) for C. gloeosporioides, and HEV1A and HEV8B (Phanerochaete sp. 3 and Diaporthe sp. 4) for C. cassiicola. The endophytic isolates were positive for lipase (69.6%), amylase (67.6%), cellulase (33.3%), and protease (20.6%). The enzyme index ≥ 2 was found for amylase and lipase. The isolates obtained from rubber trees showed good antimicrobial and enzymatic potential, which can be tested in the future for use in the industry, and in the control of plant pathogens.
Article
Full-text available
In recent years, Lynn Margulis has been credited in various articles as the person who introduced the concept of holobiont into biology in the early 1990s. Today, the origin of evolutionary studies on holobionts is closely linked to her name. However, Margulis was not the first person to use this concept in its current context. That honor goes to the German theoretical biologist Adolf Meyer‐Abich, who introduced the holobiont concept nearly 50 years before her (in 1943). Although nearly completely forgotten today, in the 1940–60s he developed a comprehensive theory of evolutionary change through “holobiosis.” It had a surprisingly modern outlook, as it not only addressed tenets of today's evolutionary developmental biology (evo‐devo), like the origin of form and production of variation, but also anticipated key elements of Margulis' later endosymbiotic theory. As the holobiont concept has become an important guiding concept for organizing research, labeling conferences, and publishing articles on host‐microbiota collectives and hologenomes, the field should become aware of the independent origin of this concept in the context of holistic biology of the 1940s.
Article
Full-text available
The Molecular Evolutionary Genetics Analysis (MEGA) software enables comparative analysis of molecular sequences in phylogenetics and evolutionary medicine. Here, we introduce the macOS version of the MEGA software. This new version eliminates the need for virtualization and emulation programs previously required to use MEGA on Apple computers. MEGA for macOS utilizes memory and computing resources efficiently for conducting evolutionary analyses on Apple computers. It has a native Cocoa graphical user interface that is programmed to provide a consistent user experience across macOS, Windows, and Linux. MEGA for macOS is available from www.megasoftware.net free of charge.
Article
Full-text available
The Interactive Tree Of Life (https://itol.embl.de) is an online tool for the display, manipulation and annotation of phylogenetic and other trees. It is freely available and open to everyone. The current version introduces four new dataset types, together with numerous new features. Annotation options have been expanded and new control options added for many display elements. An interactive spreadsheet-like editor has been implemented, providing dataset creation and editing directly in the web interface. Font support has been rewritten with full support for UTF-8 character encoding throughout the user interface. Google Web Fonts are now fully supported in the tree text labels. iTOL v4 is the first tool which supports direct visualization of Qiime 2 trees and associated annotations. The user account system has been streamlined and expanded with new navigation options, and currently handles >700 000 trees from more than 40 000 individual users. Full batch access has been implemented allowing programmatic upload and export of trees and annotations.
Article
Full-text available
The microbiota colonizing the rhizosphere and the endorhizosphere contribute to plant growth, productivity, carbon sequestration and phytoremediation. Several studies suggested that different plants types and even genotypes of the same plant species harbor partially different microbiomes. Here, we characterize the rhizosphere bacterial and fungal microbiota across five grapevine rootstock genotypes cultivated in the same soil at two vineyards and sampling dates over two years by 16S rRNA gene and ITS high-throughput amplicon sequencing. In addition, we use quantitative PCR (qPCR) approach to measure the relative abundance and dynamic changes of fungal pathogens associated with black-foot disease. The objectives were to (1) unravel the effects of rootstock genotype on microbial communities in the rhizosphere of grapevine and (2) to compare the relative abundances of sequence reads and DNA amount of black-foot disease pathogens. Host genetic control of the microbiome was evident in the rhizosphere of the mature vineyard. Microbiome composition also shifted as year of sampling, and fungal diversity varied with sampling moments. Linear discriminant analysis identified specific bacterial (i.e., Bacillus) and fungal (i.e., Glomus) taxa associated with grapevine rootstocks. Host genotype did not predict any summary metrics of rhizosphere α- and β- diversity in the young vineyard. Regarding black-foot associated pathogens, a significant correlation between sequencing reads and qPCR was observed. In conclusion, grapevine rootstock genotypes in the mature vineyard were associated with different rhizosphere microbiomes. The latter could also have been affected by age of the vineyard, soil properties or field management practices. A more comprehensive study is needed to decipher the cause of the rootstock microbiome selection and the mechanisms by which grapevines are able to shape their associated microbial community. Understanding the vast diversity of bacteria and fungi in the rhizosphere and the interactions between microbiota and grapevine will facilitate the development of future strategies for grapevine protection.
Article
Full-text available
The aim of this study was to evaluate four strains of Trichoderma spp. (T. harzianum IBLF 006 WP, T. harzianum IBLF 006 SC, T. harzianum ESALQ 1306 and T. asperellum URM 5911) for seedling growth promotion in laboratory and head lettuce yield in field conditions. The experiment was carried out in a completely randomized design with four treatments (strains): IBLF 006 WP, IBLF 006 SC, ESALQ 1306 and URM 5911 and a non-inoculated (without Trichoderma) control. Each treatment consisted of 200 seeds, arranged in four replicates. Lettuce seeds cv. Astra were treated with 2 mL Trichoderma suspension (2.5 x 108 conidia mL-1 per each 100 g seeds) and submitted to growth assay in laboratory up to 7 days after sowing. For field experiment, we opened furrows, which were manually sprayed with 5 x 107 conidia mL-1. Afterwards, seedlings were transplanted (4 to 6 leaves of head lettuce cv. Mauren) and harvested 40 days later. Each treatment consisted of four replicates (1.2 x 1.2 m, 16 plants per plot) arranged in randomized blocks. In both experiments, a control without Trichoderma application was included, and we evaluated shoot length, root and total length, shoot, root and total fresh mass and shoot, root and total dry mass, shoot mass ratio, root mass ratio and shoot/root ratio. The germination (%) was evaluated by laboratory tests, whereas in field experiment, height, stem diameter, head diameter, number of leaves and yield were evaluated. The T. harzianum strain ESALQ 1306 provided the best head lettuce growth rate in laboratory test, which was confirmed in field experiment, in which the productivity (50.2 t ha-1) was superior when compared to the other strains (41.38 to 44.23 t ha-1) and the control (30.18 t ha-1).
Article
Full-text available
Brazil, Colombia, Ecuador, Peru, Bolivia, Venezuela, Suriname, Guyana, and French Guiana share an area of 7,295,710 km2 of the Amazon region. It is estimated that the Amazonian forest offers the greatest flora and fauna biodiversity on the planet and on its surface could cohabit 50% of the total existing living species; according to some botanists, it would contain about 16-20% of the species that exist today. This region has native fruit trees in which functional properties are reported as antioxidant and antiproliferative characteristics. Amazon plants offer a great therapeutic potential attributed to the content of bioactive phytochemicals. The aim of this mini review is to examine the state of the art of the main bioactive components of the most studied Amazonian plants. Among the main functional compounds reported were phenolic compounds, unsaturated fatty acids, carotenoids, phytosterols, and tocopherols, with flavonoids and carotenoids being the groups of greatest interest. The main beneficial effect reported has been the antioxidant effect, evaluated in most of the fruits investigated; other reported functional properties were antimicrobial, antimutagenic, antigenotoxic, analgesic, immunomodulatory, anticancer, bronchodilator, antiproliferative, and anti-inflammatory, including hypercholesterolemic effects, leishmanicidal activity, induction of apoptosis, protective action against diabetes, gastroprotective activity, and antidepressant effects.
Article
Full-text available
Bacteria and fungi form complex communities (microbiomes) in above- and below-ground organs of plants, contributing to hosts’ growth and survival in various ways. Recent studies have suggested that host plant genotypes control, at least partly, plant-associated microbiome compositions. However, we still have limited knowledge of how microbiome structures are determined in/on grafted crop plants, whose above-ground (scion) and below-ground (rootstock) genotypes are different with each other. By using eight varieties of grafted tomato plants, we examined how rootstock genotypes could determine the assembly of leaf endophytic microbes in field conditions. An Illumina sequencing analysis showed that both bacterial and fungal community structures did not significantly differ among tomato plants with different rootstock genotypes: rather, sampling positions in the farmland contributed to microbiome variation in a major way. Nonetheless, a further analysis targeting respective microbial taxa suggested that some bacteria and fungi could be preferentially associated with particular rootstock treatments. Specifically, a bacterium in the genus Deinococcus was found disproportionately from ungrafted tomato individuals. In addition, yeasts in the genus Hannaella occurred frequently on the tomato individuals whose rootstock genotype was “Ganbarune”. Overall, this study suggests to what extent leaf microbiome structures can be affected/unaffected by rootstock genotypes in grafted crop plants.
Article
Full-text available
Background The ascomycete fungus Clonostachys rosea (order Hypocreales) can control several important plant diseases caused by plant pathogenic fungi and nematodes. Subtilisin-like serine proteases are considered to play an important role in pathogenesis in entomopathogenic, mycoparasitic, and nematophagous fungi used for biological control. In this study, we analysed the evolutionary histories of protease gene families, and investigated sequence divergence and regulation of serine protease genes in C. rosea. Results Proteases of selected hypocrealean fungal species were classified into families based on the MEROPS peptidase database. The highest number of protease genes (590) was found in Fusarium solani, followed by C. rosea with 576 genes. Analysis of gene family evolution identified non-random changes in gene copy numbers in the five serine protease gene families S1A, S8A, S9X, S12 and S33. Four families, S1A, S8A, S9X, and S33, displayed gene gains in C. rosea. A gene-tree / species-tree reconciliation analysis of the S8A family revealed that the gene copy number increase in C. rosea was primarily associated with the S08.054 (proteinase K) subgroup. In addition, regulatory and predicted structural differences, including twelve sites evolving under positive selection, among eighteen C. rosea S8A serine protease paralog genes were also observed. The C. rosea S8A serine protease gene prs6 was induced during interaction with the plant pathogenic species F. graminearum. Conclusions Non-random increases in S8A, S9X and S33 serine protease gene numbers in the mycoparasitic species C. rosea, Trichoderma atroviride and T. virens suggests an involvement in fungal-fungal interactions. Regulatory and predicted structural differences between C. rosea S8A paralogs indicate that functional diversification is driving the observed increase in gene copy numbers. The induction of prs6 expression in C. rosea during confrontation with F. graminearum suggests an involvement of the corresponding protease in fungal-fungal interactions. The results pinpoint the importance of serine proteases for ecological niche adaptation in C. rosea, including a potential role in the mycoparasitic attack on fungal prey. Electronic supplementary material The online version of this article (10.1186/s12862-018-1291-1) contains supplementary material, which is available to authorized users.
Article
Prior to 1985, cultures at the Center for Forest Mycology Research were maintained on 1.5% malt extract agar test-tube slants. This system not only made it necessary to transfer the entire collection every year but also permitted genetic change because continual growth occurred. In 1985, the method of storing fungal cultures in sterile distilled water in cryovials was introduced. This study reports on the use of this method for long-term fungal storage. For varying periods up to 7 years, 151 miscellaneous species of wood-decaying Basidiomycotina were stored in sterile distilled water. Water storage has numerous advantages: culture viability or growth rate is not significantly influenced; isolates can be stored longer; genetic stability is greater; the method is quick, easy, and inexpensive, and requires less space.
Article
Obesity is a metabolic disorder associated with adverse health consequences that has increased worldwide at an epidemic rate. This has encouraged many people to utilize nonprescription herbal supplements for weight loss without knowledge of their safety or efficacy. However, mounting evidence has shown that some herbal supplements used for weight loss are associated with adverse effects. Guarana seed powder is a popular nonprescription dietary herb supplement marketed for weight loss, but no study has demonstrated its efficacy or safety when administered alone. Wistar rats were fed four different diets (low‐fat diet and Western diet with or without guarana supplementation) for 18 weeks. Metabolic parameters, gut microbiota changes, and toxicity were then characterized. Guarana seed powder supplementation prevented weight gain, insulin resistance, and adipokine dysregulation induced by Western diet compared with the control diet. Guarana induced brown adipose tissue expansion, mitochondrial biogenesis, uncoupling protein‐1 overexpression, AMPK activation, and minor changes in gut microbiota. Molecular docking suggested a direct activation of AMPK by four guarana compounds tested here. We propose that brown adipose tissue activation is one of the action mechanisms involved in guarana supplementation‐induced weight loss and that direct AMPK activation may underlie this mechanism. In summary, guarana is an attractive potential therapeutic agent to treat obesity.