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Describing the Unexplored Microorganisms Associated with

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Chapter 13
Describing the Unexplored
Microorganisms Associated with Guarana:
A Typical Tropical Plant

Bruna Durante Batista, Jaqueline Raquel de Almeida,

Thana Esashika Bezerra, João Lucio de Azevedo, and Maria Carolina Quecine

Abstract In Amazon rainforest, guarana (Paullinia cupana) is a typical tropical

plant with valuable pharmaceutical, economic, and social importance, especially for
Brazil, which is the only commercial producer of guarana. Despite its importance
and exclusive origins, knowledge regarding the microbial community associated
with guarana is incipient. The microorganisms associated with this plant were only
recently determined, and they were nearly unexplored until 2010 when two research
groups from São Paulo and Amazonas States joined in an unprecedented project to
explore the guarana microbiome. The main goal of the project was to find a biologi-
cal control for Colletotrichum, the causal agent of guarana anthracnose, which is an
important disease that severely affects the culture. Due to the huge diversity found
in the microbial community associated with guarana, the project has extended
beyond its original purpose, and other biotechnological applications have been
found for these new microorganisms. From the collection of fungi and bacteria
(endophytic and epiphytic, culturable and unculturable) that were obtained from
different ecological niches of guarana, several studies have been conducted aimed
at different applications, especially focusing on the control of plant pathogens, the
promotion of plant growth, and the discovery of new compounds.

Keywords Paullinia cupana • Amazonian rainforest • Biodiversity • Biological

control • Plant growth promotion • Mycovirus Endophytic • Epiphytic
• Rhizobacteria

B.D. Batista • J.R. de Almeida • J.L. de Azevedo • M.C. Quecine ( )

Department of Genetics, “Luiz de Queiroz” College of Agriculture, University of São Paulo,
11 Pádua Dias Av, P.O. BOX 83, 13400-970 Piracicaba, SP, Brazil
e-mail: mquecine@usp.br
T.E. Bezerra
University of Amazonas,
·
6200 General Rodrigo Otávio Av, Coroado I, 69067-005 Manaus, AM, Brazil

© Springer International Publishing AG 2017 293

J.L. de Azevedo, M.C. Quecine (eds.), Diversity and Benefits of Microorganisms
from the Tropics, DOI 10.1007/978-3-319-55804-2_13

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294 B.D. Batista et al.

13.1 Introduction

Approximately 200 species of guarana belong to the genus Paullinia in the family
Sapindaceae, including the species Paullinia cupana var. sorbilis, which is known
as “true guarana.” This plant occurs in the Amazonas, especially in Maués (Araujo
et al. 2002). Its production occurs in Amazonas and Bahia States and it is consumed
in pill form or diluted in water. Due to its pharmaceutical properties, guarana is also
a raw material for cosmetics; however, 70% of its production is destined for the soft
drink industry. More details regarding certain interesting guarana characteristics,
including production processes, markets, and seed chemical compositions have
been presented in a recent publication (Schimpl et al. 2013); however, only recently
has the guarana microbiome been accurately described.
A better understanding and manipulation of the plant microbiome has the poten-
tial to reduce the incidence of plant disease, increase agricultural production, reduce
chemical inputs, and reduce emission of greenhouse gases, resulting in more sus-
tainable agricultural practices. Despite this fact, the guarana microbiome is almost
unexplored and is an unknown reservoir for biotechnological application. The ben-
eficial plant-microbe associations in guarana have only recently been investigated.
In this chapter, both the pathogenic Colletotrichum and the beneficial plant microbes,
such as endophytes, epiphytes, and rhizosphere, will be described. We present
recent data that strongly suggest that microorganisms from guarana may improve
plant health and may act as growth promoters and agents of biocontrol for many
plant diseases. We also present new approaches for biological control, such as the
importance of mycovirus to the virulence of pathogenic fungi and the performance
of secondary metabolites produced by endophytes in controlling Colletotrichum
and other microorganisms during in vitro tests.

13.2 Pathogenic Microorganism: Colletotrichum, the Causal

Agent of Anthracnose in Guarana

The first studies related to plant-microbe interactions are frequently those involving
microorganisms that cause injury to the host, pathogens. In the same way, the initial
studies of interactions between microorganisms and guarana involved the causal agent
of anthracnose. Albuquerque (1960, 1961) was a pioneer in detecting the disease and
named the fungus Colletotrichum guaranicola, and described the lesion aspects in the
leaves and the general characteristics of the fungi, such as its germination, that indi-
cated the absence of a perfect phase. Subsequent publications (Duarte et al. 1995;
Veras et al. 1997) described other aspects, such as the fungal speed of germination,
conidia size, and morphophysiological variability under different carbon sources, and
detecting even hyphal anastomosis between strains, corroborating its high variability.
Menezes (2002) studied the biological and taxonomic aspects of Colletotrichum
and suggested that some strains are host-dependent, for instance, C. lindemuthianum
is typical in bean, C. musae in banana, whereas others such as C. gloeosporioides

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13 Describing the Unexplored Microorganisms Associated with Guarana… 295

and C. acutatum are pathogenic to a broad range of hosts. This author concluded that
morphological characteristics could not distinguish members of the Colletotrichum
genus. The genus is complex, and C. gloeosporioides is the most commonly encoun-
tered. However, the classification of C. guaranicola was based on the shape, conid-
ium size, and appressorium formation, which suggested that C. guaranicola belongs
inside the genus (Bentes and Barreto 2004).
Costa-Neto (2009) isolated fungi from P. cupana cultivated in the Federal University
of Amazonas (UFAM) experimental farm in Manaus and also in Maués. The author
found endophytic and pathogenic Colletotrichum strains. The pathogenic strains were
considered C. gloeosporioides, and its perfect phase was denominated Glomerella
cingulata. Interestingly, all of the pathogenic strains were obtained from Maués. Some
species, such as C. boninense, C. fragaria, C. acutatum, and some C. gloeosporioides
strains behaved as endophytes, demonstrating the difficulty for correct classification of
this pathogen. In a more recent study, Bentes and Costa-Neto (2011) obtained 20
strains of Colletotrichum from the municipalities of Manaus (Brazilian Agricultural
Research Corporation – EMBRAPA and Amazonas Federal University – UFAM) and
as well in the municipalities Presidente Figueiredo, Iranduba, and Maués all in
Amazonas State in Brazil. Using AFLP molecular markers, the authors concluded that
all strains constituted a single species but with intraspecific variability.
As mentioned, anthracnose is the main disease in P. cupana, affecting guarana
plantations, especially in the Amazonas State. Although P. cupana is native to
Amazon rainforest, the south of Bahia State has become the major Brazilian pro-
ducer of guarana. In Bahia, guarana is produced in small quantities, with a total area
of 6500 ha producing 400 kg/ha against a production of 174 kg/ha in Amazonas
State (Companhia Nacional do Abastecimento – CONAB 2016). Among the advan-
tages that Bahia has over Amazonas State are a good rainfall distribution, more
fertile soils, and, principally, a low incidence of diseases, such as anthracnose
(Guaraná 2004). All of these considerations led two groups, the UFAM and the
College of Agriculture “Luiz de Queiroz” from the University of São Paulo (USP),
to work together in a project that started in 2010. The aim of this project was to
study endophytic, epiphytic, and rhizospheric microorganisms associated with gua-
rana and to detect those that could serve as a control for anthracnose, promote plant
growth, or provide any other beneficial effects to the guarana and other cultures.
These studies were conducted with the financial support of Amazonas State Research
Foundation (FAPEAM) and São Paulo State Research Foundation (FAPESP), and
in the following sections, data obtained from these projects will be discussed.

13.3 Nonpathogenic Fungi and Bacteria Associated

with Guarana Plants

Due to the FAPESP/FAPEAM project in 2010, a satisfactory volume of information of

the guarana microbiome was obtained. Currently, there is a notable amount of data about
fungi and bacteria from leaves, stems and seeds, which are endophytic or epiphytic or

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296 B.D. Batista et al.

reside in the rhizosphere of this plant species. The search for a potential control agent of
anthracnose is still in progress. Additionally, the increased understanding of the guarana
microbial community that has offered new expectative and new microorganisms were
found with great potential for the promotion of plant growth, such as new candidates to
control diverse plant diseases. The following sections will describe the diversity of the
fungi and bacteria communities associated with different P. cupana organs and tissue
and their prospective uses in the agricultural, industrial, and pharmacological fields.

13.3.1 Fungi Associated with Guarana Plants

13.3.1.1 Endophytic Fungi in P. cupana Leaves

Guimarães (1998) isolated and identified endophytic fungi from guarana leaves
from three locations, two in the municipality of Manaus and one in Maués, which
indicated the presence of the genera Guignardia, Phomopsis, Xylaria, and
Colletotrichum and its perfect form Glomerella cingulata. The isolates of endo-
phytic Colletotrichum and the perfect form G. cingulate isolates were studied in
detail, and the genetic variability that was assessed using RAPD demonstrated high
variability among isolates that could indicate different species from the
Colletotrichum/Glomerella complex. As previously mentioned, Costa-Neto (2009)
also identified many species of Colletotrichum as endophytes from guarana. This
genus is commonly found as an endophyte in many plants as described by Azevedo
et al. (2002), Lu et al. (2004), and Pereira et al. (2007). In another study, Sia et al.
(2013) used two different culture media, Potato-Dextrose-Agar (BDA) and a new
MDA medium, consisting of Manihot Dextrose and agar, to isolate fungi from gua-
rana. Using the MDA medium, the authors were able to increase the number of
species isolated from P. cupana leaves. These authors also showed that MDA pre-
sented a greater richness and diversity of fungi. The genera Botrysphaeria,
Colletotrichum, Diaporthe, Phomopsis, Sordariomycetes, Xylaria, Aspergillus,
Bionectria, Cladosporium, Cochliobolus, Coniosporium, Guignardia, Pestalotiopsis,
Phanerochaete, and Pleosporales were described as endophytes in guarana with a
predominance of Colletotrichum, Phomopsis, Xylaria, and Guignardia.
The collection of microorganisms from guarana has been constantly increasing, and
in a recent study, Esashika-Bezerra (2015) isolated 522 fungi: 278 from leaves, 179
from stems, and 65 from the rhizosphere of guarana. Among the identified endophytes
were the genera Pestalotiopsis, Colletotrichum, Xylaria, Guignardia, Trichoderma,
Aspergillus, and Penicillium. Figure 13.1 shows the diversity of the isolates.
Esashika-Bezerra (2015) also evaluated the antagonistic activity of the fungal
isolates against C. gloeosporioides, and 55 isolates inhibited the growth of the
pathogen (Fig. 13.2). The five most promising isolates were classified as Penicillium
verruculosum, P. pinophilum, Geotrichum sp., Schizophyllum commune, and
Aspergillus flavus. Using in vivo tests against C. gloeosporioides, the pathogen was
inhibited, and none of these fungi had an effect on plant health. These results are

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13 Describing the Unexplored Microorganisms Associated with Guarana… 297

Fig. 13.1 Examples of the endophytic fungi isolated from the leaves, stems, and rhizosphere of P.
cupana (Esashika-Bezerra 2015)

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298 B.D. Batista et al.

Fig. 13.2 Antagonism assay for endophytic fungi against C. gloeosporioides 15 days after inocu-
lation in a petri dish containing PDA (Esashika-Bezerra 2015)

very significant, indicating the possibility of anthracnose control by microorganisms

originally isolated from P. cupana.
A similar study was conducted by Bonatelli et al. (2016) where 15 fungi were iso-
lated from guarana leaves showing typical anthracnose symptoms. The ITS1-5.8S-ITS2
sequencing was used to identify the fungi that were classified as Fusarium, Phomopsis,
Leptosphaeria, Pestalotiopsis, and Microdochium. The standard methodology to detect
the antagonistic activity was carried out, and two strains of Fusarium, one Pestalotiopsis

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13 Describing the Unexplored Microorganisms Associated with Guarana… 299

and one Microdochium showed significant inhibition of C. gloeosporioides. These

antagonistic fungi presented in vitro potential for the control of pathogens even for other
plants, which demonstrated the broad potential for biological control. These isolates
were also able to inhibit the growth of the pathogen in in vivo assays using detached
guarana leaves. These strains were then analyzed in vitro to produce chitinases, which
are enzymes known to affect pathogenic fungi (Collinge et al. 1993; Quecine et al.
2008) and to produce volatile compounds that could interfere with the growth of other
fungi (Strobel et al. 2001; Minerdi et al. 2009). One Fusarium strain produced chitinase,
and both strains of Fusarium and Pestalotiopsis produced volatile compounds. This
research may be improved with further evaluations of the control of anthracnose under
field conditions and the design of a fungi biocontrol formula for field application.

13.3.2 Endophytic Fungi and Bacteria from Guarana Seeds

Silva (2015) isolated endophytic fungi and bacteria from guarana seeds sampled in
Manaus and Maués. The endophytes were isolated after the seeds were treated to
remove epiphytes. The objective was to verify the presence and the amount of endo-
phytic fungi and bacteria in guarana seeds obtained from Amazonas State, which
has serious problems with anthracnose, and from the Bahia State, which has no
serious incidence of this disease. Interestingly, only a few fungi were obtained and
they were all from Bahia. The fungi belong to the genera Lasiodiplodia and
Fusarium. It is possible that the long transport time could have influenced the low
numbers of fungi isolated from the Amazonas State’s samples. It is interesting to
suggest that the low incidence of fungi from Amazonas State seeds could also be
due to the presence of an antimicrobial agent, as verified by Viswanatahan and
Samiyappan (2001), who demonstrated the presence of chitinases in guarana seeds
that could interfere with the growth of fungi and insects whose cell walls are com-
posed of chitin. Basile et al. (2005, 2013) also demonstrated the presence of antimi-
crobial compounds in P. cupana seeds capable of inhibiting fungi.
Using the same samples, Silva et al. (2016) isolated bacteria by grinding the seeds.
For bacteria, the number of colony-forming units (CFU) was similar for both loca-
tions (2.9 × 104 from Amazonas and 2.4 × 104 from Bahia). From Bahia, the predomi-
nant genera represented were Bacillus and Stenotrophomonas. From Amazonas,
Bacillus and Paenibacillus were predominant, whereas Ochrobactrum, Rhizobium,
and Microbacterium were found in lower numbers. To verify the presence of com-
pounds that could interfere with the development of the pathogen, the bacteria were
inoculated onto plates containing Colletotrichum, and the presence or absence of
zones of inhibition was noted. The pairwise test showed that 16 of the 102 evaluated
bacteria substantially reduced the growth of Colletotrichum. However, no in vivo
tests have yet been conducted to infer the pathogen control in greenhouse or field
conditions. The results seem to show that there is no correlation between the inci-
dence of anthracnose and endophytic microbiota in seeds. On seeds, there is probably
no factor from endophytic microbiota to explain the high incidence in Amazonas and

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300 B.D. Batista et al.

the low incidence in Bahia, and other factors such as climate, rainfall, soil fertility,
and cultivation practices may be involved as mentioned in other publications (Guaraná
2004). It is also important to mention that in Bahia, most of the guarana is planted as
seeds, whereas in Amazonas State’s clone cuttings are used. This could increase the
guarana genetic variability in Bahia and reduce it in Amazonas.

13.4 Bacteria in Guarana Leaves

13.4.1 Endophytic Bacteria

Endophytic bacteria have also been isolated from the leaves of P. cupana, and their
potential to increase the host fitness was investigated. As mentioned previously, the
principal limiting factor to produce guarana in Amazonas State is anthracnose, which
causes severe necrosis of young leaves and can affect all of the plant developmental
stages resulting in a reduction in fruit production of up to 88% (Araujo 2002). As
there is no effective control of this disease, the practices mostly used to minimize its
effects are the use of resistant clones, management, and chemical control. The fact
that plants of the same genotype can be found with and without the disease could be
related to the associated microbial community (endophytes and others), which makes
the study of affected and healthy plants interesting. Endophytes occupy the same
niches as pathogens, and biocontrol could occur by competition for the same space
(Lacava et al. 2004), synthesis of antimicrobials, enzymes, or siderophores (Araujo
et al. 2001, 2002), or induction of the plant resistance system (Gao et al. 2010). In
addition, culture-dependent methods, culture-independent methods, and genomic
libraries made from the endophytic bacterial community in the leaves can be used to
provide a more complete description of microbial resources in plants.
In an original study, endophytes were isolated from the leaves of guarana with and
without the symptoms of Colletotrichum, from Maués (AMBEV farm), Amazonas, in
June 2010 (Tsui 2012). Ninety-seven endophytes were collected from the leaves with
symptoms and 53 from asymptomatic leaves (apparently healthy). One hundred and
five of these isolates were identified using the partial sequence of the 16S rRNA gene,
and the most common genera found were Enterobacter, Stenotrophomonas,
Pseudomonas, Bacillus, Serratia, and Microbacterium. However, a predominance of
Stenotrophomonas was found in symptomatic leaves, and a predominance of
Enterobacter was found in asymptomatic leaves. Other genera in lower numbers were
also found, such as Staphylococcus, Dyella, Burkholderia, and Rhizobium in asymp-
tomatic leaves, and Curtobacterium, Cronobacter, Brevundimonas, Sphingobacterium,
Acinetobacter, Pantoea, and Erwinia in symptomatic leaves. A greater bacteria richness
was found in symptomatic leaves along with a greater diversity of bacteria. Antagonism
tests were conducted using the bacteria and Colletotrichum, and approximately 10% of
the bacteria showed some antagonistic effect against the fungus, with five bacteria from
symptomatic leaves and five from asymptomatic leaves selected and analyzed for chitin
production. One strain of Serratia marcescens from asymptomatic leaves demonstrated
great potential to inhibit Colletotrichum and high chitinolytic activity. A strain of

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13 Describing the Unexplored Microorganisms Associated with Guarana… 301

Erwinia sp. isolated from a symptomatic plant also strongly inhibited Colletotrichum.
This strain was selected to construct the mutant library using minitransposon Tn-10,
which can indicate if there are genes involved in fungus inhibition. These studies dem-
onstrate that endophytes can have a role in the reduction of the damage caused by
Colletotrichum in guarana and act as natural controllers or as a biological control of the
pathogen. However, there is a need for further studies.
In a detailed study, Bogas et al. (2015) used molecular processes to compare the
associations between endophytes in P. cupana leaves with and without anthracnose
symptoms. The 16S rDNA genes of the bacterial isolates were cloned. From the ten
generated libraries, 716 clones were estimated, and after the elimination of chimera
and chloroplast sequences, 263 clones from symptomatic plants and 272 from asymp-
tomatic plants were obtained. Regarding similarity, bacteria from symptomatic plants
presented 67 OTUs (Operational Taxonomic Units) and asymptomatic plants pre-
sented 61 OTUs with only 14.8% of the OTUs shared. The indices of richness (Chao1)
and diversity (Shannon-Weaver and Simpson) were similar in bacteria from plants
with and without symptoms. Proteobacteria (68.89%), Actinobacteria (24.07%),
Firmicutes (5.05%), Acidobacteria (1.28%), and Bacteroidetes (0.71%) were predom-
inant, and Bacteroidetes was only found in symptomatic plants. Alphaproteobacteria
(6.44%), Betaproteobacteria (38%), and Gammaproteobacteria (54.9%) were classi-
fied from the phylum Proteobacteria. A total of 34 different bacterial genera were
encountered (Fig. 13.3), principally Hydrogenophilus (25.3%), Pseudomonas
(16.6%), Propionibacterium (10.2%), Acinetobacter (6.7%), and Rubrobacter (5.4%).

Fig. 13.3 A phenetic analysis of the clones from the 16S rRNA gene libraries from the endophytic
bacterial community present in symptomatic and asymptomatic guarana leaves (Bogas et al. 2015)

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302 B.D. Batista et al.

According to Bogas et al. (2015), some genera were found exclusively or more
commonly in asymptomatic plants. Competition for space and nutrients or changing
an ecological niche can be suggested as causes for these results. It could also be
suggested that some pathogens could develop cell-wall degrading enzymes, which
increase the bacterial diversity in infected plants. Lytic enzymes could also act and
stimulate/induce resistance mechanisms. Some genera found in asymptomatic
plants could be involved in protection against diseases such as Pseudomonas,
Arsenophonus, Neisseria, and Haemophilus. Some members of the Pseudomonas
genus are used as biological control agents because they produce siderophores and
antibiotics and can induce systemic plant resistance (Ramesh et al. 2009; Krid et al.
2010). Neisseria and Haemophilus, although known as human pathogens, are
described as conferring benefits to plant hosts by promoting plant growth and con-
trol pathogens (Egamberdieva 2010).

13.4.2 Simultaneous Isolation of Epiphytic

and Endophytic Bacteria

Bonatelli (2012) also conducted a study involving the isolation of both epiphytic
and endophytic bacteria from guarana leaves. The author compared the bacteria
found in asymptomatic leaves with those from anthracnose symptomatic leaves.
The concentrations of epiphytic bacteria were 3 × 106 cfu/mL and 4 × 106 cfu/mL in
the asymptomatic and symptomatic leaves, respectively; for endophytic bacteria,
the concentrations were 3.1 × 106 cfu/mL and 5.1 × 106 cfu/mL in the asymptomatic
and symptomatic leaves, respectively. A total of 216 bacteria were identified by 16S
rDNA sequencing, representing 18 genera (Fig. 13.4).
The presence or absence of symptoms apparently influenced the bacterial com-
munity more than the condition of epiphytic or endophytic. The more abundant
genera appeared in all of the treatments, and the less abundant, in general, were
found in only one or more treatments. Burkholderia, Luteibacter, and Staphylococcus
genera occurred only in asymptomatic leaves, whereas Brevundimonas,
Microbacterium, Curtobacterium, and Sphingobacterium occurred only in symp-
tomatic leaves. Epiphytic Pantoea, Comamonas, Burkholderia, Luteibacter,
Microbacterium, Staphylococcus, and Sphingobacterium genera were found in
asymptomatic and symptomatic leaves.
Using an isolation method, a predominance of Proteobacteria phylum in asymp-
tomatic and symptomatic leaves was observed, and the presence of anthracnose was
the most important factor to modulate the structure of the bacterial community. In
symptomatic leaves, the most common genera were Pseudomonas, Stenotrophomonas,
and Pantoea regardless of whether they were evaluated by culture-dependent or
culture-independent methods. Culture-independent Acinetobacter, Acidobacter, and
Sphingobacteria were detected in symptomatic leaves, and Methylobacterium,
Beijerinckia, Bacillus, and a group of nonclassified Rhizobiales was detected in
asymptomatic leaves.

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13 Describing the Unexplored Microorganisms Associated with Guarana… 303

Fig. 13.4 Distribution of the isolated epiphytic and endophytic bacteria from the leaves of symp-
tomatic and asymptomatic leaves at the level of phylum (a); classes within the phylum
Proteobacteria (b) (Bonatelli 2012)

Epiphytic and endophytic isolated bacteria were tested in vitro for antagonistic
activity against Colletotrichum. The genera Bacillus, Stenotrophomonas, Pantoea,
Erwinia, Enterobacter, and Pseudomonas showed inhibition of the pathogen. To
identify the possible mechanisms involved in the inhibition of the pathogen, the
production of enzymes (cellulase, amylase, lipase, esterase, protease, polygalactu-
ronase, and pectin lyase) and siderophores by bacteria was evaluated. Interestingly,
it was possible to correlate the production of amylase, lipase, and polygalacturonase
with strains from symptomatic leaves. Concerning the antagonistic activity, 12.34%
and 8.33% of the endophytes from symptomatic and asymptomatic plants, respec-
tively, and 14.92% and 12.50% of epiphytes from symptomatic and asymptomatic

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304 B.D. Batista et al.

plants, respectively, showed inhibition of the fungus with no significant differences

between symptomatic and asymptomatic leaves. The best antagonistic genera were
Bacillus (seven strains), Stenotrophomonas, Pantoea, and Enterobacter (three
strains each); Pseudomonas and Acinetobacter (two strains each); and Serratia and
Comamonas (one strain each). The Bacillus strains were the best inhibitors of the
pathogen. Various strains produced enzymes and siderophores with no significant
differences between epiphytic and endophytic bacterial communities, whether from
symptomatic or asymptomatic plants. The presence of disease was the factor that
most influenced the cultivable bacterial community. Some authors have shown simi-
larity between epiphytic and endophytic communities, which suggests egress and
ingress phases (Newton et al. 2010). Other studies showed a greater diversity and
richness in the endophytic community (Santamaria and Bayman 2005; Kuklinsky-
Sobral 2003).
Bonatelli (2012) observed that in the noncultivated community, Proteobacteria
was the most common phylum (83.1% of the OTUs) followed by Acidobacteria
(7.87% of the OTUs), Bacteroidetes (3.22% of the OTUs), Actinobacteria (1.88% of
the OTUs), Firmicutes (1.69% of the OTUs), and Verrucomicrobia (1.31% of the
OTUs), with others achieving <1%. Table 13.1 shows a comparison between the
culture-dependent and culture-independent data.

13.5 Bacteria Isolated from the Rhizosphere

In another study conducted by Santos (2015), bacteria were isolated from the rhizo-
sphere of guarana (clone 800) from Maués (AMBEV farm) with and without
anthracnose symptoms. The bacteria community was accessed by isolation and a
culture-independent method using 16S rDNA pyrosequencing. The bacterial isola-
tion data suggested that the rhizosphere of healthy guarana showed greater richness
compared to that of guarana affected by Colletotrichum. In both cases, the
Bacteroidetes phylum was the most common, followed by Proteobacteria,
Acidobacteria, and then Actinobacteria. The most common genera in the symptom-
atic plants were Chryseobacterium, Flavobacterium, Pedobacter, and
Sphingobacterium, whereas in the asymptomatic plants the most common genera
were Rhizomicrobium, Acidicaldus, Sphingomonas, and Rhodoplanes. Some com-
mon genera to both symptomatic and asymptomatic plants were Pseudomonas,
Taibilla, Mucilaginibacter, Candidatus Koribacter, Granulicella, and Acidothermus.
Based on pyrosequencing, among the 614 OTUs identified (294 from symptomatic
and 427 from asymptomatic), 107 OTUs were common to the rhizosphere from
both asymptomatic and symptomatic plants with a similarity index of 97%.
According to the Chao and Ace indices, it was found that the diversity and richness
indices of the asymptomatic plant rhizosphere were higher than those indices of the
symptomatic plant rhizosphere. The results obtained by Santos (2015) indicate that
the pathogen, in this case the Colletotrichum sp., may modify the microbiota in the
rhizosphere around asymptomatic and symptomatic plants.

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13 Describing the Unexplored Microorganisms Associated with Guarana… 305

Table 13.1 Percentage of the genera accessed from culture-dependent and culture-independent
bacterial DNA (Bonatelli 2012)
Dependent Independent
Genera Symptomatic Asymptomatic Symptomatic Asymptomatic
Pseudomonas 19.9 1.4 17.7a 0.2
Erwinia 5.8 1.4 N.A. N.A.
Entereobacter 10.3 25.7 N.A. 0.1
Pantoea 17.3 1.4 14a 0
Serratia 3.2 7.1 10.9a 0.1
Acinetobacter 1.9 22.9 10.9a 0.1
Microbacterium 1.3 1.4 N.A. N.A.
Stenotrophomonas 20.5 2.9 1.3a 0
Brevundimonas 1.3 0 0.3 0.2
Sphingobacterium 1.3 0 3.5 1.1
Bacillus 12.8 25.7 0.2 0.4
Cronobacter 0.6 0 2.2a 0
Curtobacterium 0.6 1.4 1.0a 0.2
Comamonas 2.6 0 0.1 N.A.
Rhizobium 0.6 0.0 0.8a 0
Klebsiella 0.0 4.3 N.A. N.A.
Burkholderia 0.0 1.4 1.4 1.2
Dyella 0.0 1.4 0.2 0
Staphylococcus 0.0 1.4 0.1 0
N.A. not accessed, 0 accessed at a frequency below 0.01
a
Significant difference P > 0.05

Lima et al. (2016) also investigated the microorganisms from the P. cupana
rhizosphere but focused on the Actinobacteria Streptomyces hygroscopicus
ACTMS-9H. Based on a bioguided study, the methanolic biomass extract obtained
from submerged cultivation had the most potent antibacterial, antifungal, and cyto-
toxic activities. Using NMR, IR, and MS analyses, the authors found a fraction that
contained an alkaloid with antibacterial and antifungal activities (MIC values ≤1.9
and <3.9 μg/mL, respectively), concluding that the new compounds of S. hygro-
scopicus have potent antimicrobial, antifungal, and anticancer properties.

13.5.1 The Effect of Rhizosphere Microorganisms

on the Promotion of Plant Growth

Batista (2013) collected five rhizospheric soil samples from asymptomatic guarana
plants from Maués in November of 2010. After the isolation, approximately one
hundred of the bacteria were purified and identified by 16S rDNA gene sequencing.
Fourteen bacterial genera were found, and most of them belonged to the Proteobacteria
phylum followed by Firmicutes, Bacteroidetes, and Actinobacteria, with Burkholderia

jlazevedo@usp.br
306 B.D. Batista et al.

Fig. 13.5 The growth promotion in corn harvested 30 days after germination. On the left, the plant
inoculated with RZ2MS9 – Bacillus sp., and on the right, the control plant (noninoculated); on the
left, the plant inoculated with RZ2MS16 – Burkholderia ambifaria), and on the right, the control
plant (noninoculated) (Batista 2013)

and Bacillus as the most abundant genera. The author evaluated key plant growth-
promoting (PGP) traits in vitro (IAA and siderophore production as well as the abil-
ity to solubilize phosphorus and fix nitrogen) and the behavior of two multitrait PGP
strains, RZ2MS9 (Bacillus sp.) and RZ2MS16 (Burkholderia sp.) on corn and soy-
bean growth. Thirteen strains exhibited four PGP traits and mostly belonged to the
Burkholderia genus. Moreover, RZ2MS16 was genetically transformed to express
the heterologous green fluorescent protein (GFP) during in vivo monitoring. The
results in corn were highly positive (Fig. 13.5); Bacillus sp. (RZ2MS9) and B. ambi-
faria (RZ2MS16) increased the average heights of the treated corn plants by 39%
and 33%, respectively, compared to that of the control plants. Increases of 236 and
114% in the dry weight of the shoots and 248 and 136% in the dry weight of the roots
were observed relative to control plants for the RZ2MS9 and RZ2MS16 treatments,
respectively. RZ2MS9 and RZ2MS16 were also determined to promote guarana
growth; however, it is a perennial plant, while the preliminary assays have been
promising, and further research for longer periods of time should be conducted.
RZ2MS16:gfp was able to colonize plants’ roots and shoots, revealing an endo-
phytic behavior. These data suggest that PGPR from a typical tropical culture, such
as guarana, has the potential to be explored and used as bacterial inoculants within
a broad range of hosts (Tschoeke 2016).
Tschoeke (2016) also monitored the interaction between the strain of B. ambi-
faria (RZ2MS16) with the strains Bradyrhizobium diazoefficiens (SEMIA5080) and
Azospirillum brasilense (Ab-v5), which are both commercially used as inoculants in
soybean and corn, respectively. The synergistic effects of the interaction between
RZ2MS16 and the commercial inoculant were evaluated in a greenhouse experi-
ment. Strains RZ2MS16: pNKGFP, Ab-v5: pWM1013, and SEMIA5080: pWM1013

jlazevedo@usp.br
13 Describing the Unexplored Microorganisms Associated with Guarana… 307

promoted plant growth and colonized all of the evaluated niches in corn and soybean,
demonstrating the potential of the co-inoculation of bacteria from guarana and other
commercial inoculants.
Our aim was to better understand the molecular approaches involved in the promotion
of plant growth of the draft genome of B. ambifaria RZ2MS16. This bacterium isolated
from the guarana rhizosphere has been characterized in vitro regarding some features
related to plant growth promotion such as biological nitrogen fixation, indole acetic acid
production (IAA), and siderophore production. An in vivo assay with RZ2MS16 showed
that it was able to significantly promote corn, soybean, and sugarcane growth, showing
a broad host range. The genomic DNA was isolated using the DNeasy blood and tissue
kit (Qiagen, USA) and sequenced at the Center of Functional Genomics (University of
São Paulo, Campus “Luiz de Queiroz” – Brazil) using Illumina MiSeq.
The draft genome of RZ2MS16 is composed of 82 scaffolds with an average size of
98,730 bp, an N50 of 242,048 bp, and a GC content of 65.73%. The genome of
RZ2MS16 presents genes related to plant growth promotion, such as ferric sidero-
phores, and includes several genes involved with iron acquisition, transport, metabo-
lism, storage, and the uptake regulation protein (fur). The annotated genome also
revealed 21 genes involved with auxin biosynthesis. Enzymes related to
tryptophan-dependent and tryptophan-independent IAA biosynthetic pathways have
also been identified in this strain. Few genes were identified that were related to nitrogen
fixation, and the prediction of vnf nitrogenase suggests that the bacterium uses the vana-
dium nitrogenase system (Batista et al. 2016). These data may explain the plant growth
promotion ability of strain RZ2MS16. This genome information will facilitate formula-
tions for its practical application as a potential inoculant with a broad host range.

13.6 Mycovirus and Anthracnose Control

Many fungi contain virus particles, usually with double-stranded RNA (dsRNA)
genomes. In phytopathogenic fungi, these viruses have been studied because some
of them promote hypovirulence of the host fungus. Cases of hypovirulence have
also been observed in fungi that attack insects, such as the entomopathogenic fungi
(Dalzoto et al. 2006). In relation to pathogenic plant fungus, McCabe et al. (1999)
and Yu et al. (2010) highlighted the importance of hypovirulent fungus containing
mycovirus in the biological control of plant diseases. Pearson et al. (2009) also
discussed the possibility of transmission of viruses from hypovirulent strains to
virulent strains reducing the pathogenicity of the latter. Although mycoviruses are
common in some fungi, such as Fusarium (Kilic and Griffin 1998; Kwon et al.
2009), in the genus Colletotrichum, such common mycoviruses are not well
reported. Viral particles have been detected in Colletotrichum lindemuthianum
(Rawlinston et al. 1975) and in C. gloeosporioides (Dales et al. 1988). Recently,
Figueirêdo et al. (2012) reported the presence of mycovirus in pathogenic C. gloeo-
sporioides isolated from cashew, but in vivo hypovirulence tests were not per-
formed. Zhong et al. (2014) found that from 12 strains of Colletotrichum acutatum,

jlazevedo@usp.br
308 B.D. Batista et al.

a pepper pathogen, only one contained mycovirus, which was a gammapartitivirus.

Finally, Rosseto et al. (2016), who analyzed approximately 30 Colletotrichum
strains, found only one strain in the perfect form of the fungus (Glomerella cincu-
lata) containing a dsRNA virus. Esashika-Bezerra (2015), whose studies with fungi
isolated from guarana were cited previously, detected the presence of mycovirus in
a strain of the C. gloeosporioides complex by transmission electron microscopy.
The author inoculated the guarana strains of the fungus with the virus and patho-
genic strains without the virus and compared the symptoms after inoculation with
another joint inoculation of hypovirulent and pathogenic virus-free strains
(Fig. 13.6). The results were highly significant and showed that the hypovirulent
form and the mixture of the two fungi showed much less pronounced symptoms
than the virulent form separately inoculated on guarana leaves (Table 13.2).
These results indicate a new possibility for biological control of anthracnose in
guarana, although field trials are necessary to confirm the control. These data may
also indicate the transference of the mycovirus from one hypha to another by anas-
tomosis, as has been observed in Colletotrichum (Veras et al. 1997).

Fig. 13.6 Observation of a

mycovirus double-stranded
RNA virus genome
(dsRNA) in the filamentous
fungus Colletotrichum
gloeosporioides, isolate
CL3. The arrows show the
virus in fungal cell Bar
0.2 μm (Photo: Francisco
Tanaka – Esalq (2013)
(Esashika-Bezerra 2015))

Table 13.2 Symptoms of Pathogens Symptoms (cm)

in vivo inoculation of C.
gloeosporioides in young
leaves of P. cupana with and
without mycovirus
C. gloeosporioides (with mycovirus) 0.92 b
C. gloeosporioides (without 2.43 a
mycovirus)
C. gloeosporioides (with and 0.70 b
without mycovirus)
Control 0.00 c
Greenhouse at the Federal University of Amazonas –
UFAM. Manaus – AM (Esashika-Bezerra 2015)
The means followed by different letters show significant
differences (Tukey’s test at 5%)

jlazevedo@usp.br
13 Describing the Unexplored Microorganisms Associated with Guarana… 309

13.7 Secondary Metabolites of Bacteria and Fungi

from Endophytes of Guarana

The data obtained from bacteria and fungi from guarana permitted the detection of
many microorganisms that are important in growth promotion and in inhibition of
pathogens, such as Colletotrichum, the causal agent of anthracnose; some associations
between P. cupana and other beneficial microbes could lead to the establishment of
ecologically friendly cultivation of guarana and other plant species. The next step is to
detect which compounds are produced. For instance, a strain classified as Bacillus sub-
tilis has been described as a producer of more than 60 types of antibiotics (most with
antifungal action) and therefore could play a potential role in the control of plant dis-
ease. Bioassays and mass spectrometry analyses were performed, and in one fraction,
the members of the iturin family were detected and could be responsible for the control
of anthracnose caused by Colletotrichum and other pathogens in different cultures.
Sixteen endophytic fungi from guarana were analyzed in vitro, and some of them
showed inhibitory effects not only on Colletotrichum but also on C. acutatum from
pepper (Elias 2015). Aspergillus flavus produces a compound (asperfuran) that
inhibited the tested pathogenic fungi. Another product was also isolated from an
endophytic Xylaria sp. fungus, piliformic acid, which had highly inhibitory activity
toward C. gloeosporioides from guarana. Interestingly, Talaromyces aculeatus pro-
duced three compounds from the ester class, at least one with activity against C.
gloeosporioides. Based on these preliminary results, this is a promising new area of
research that could potentially yield novel bioactive compounds against pathogens
in guarana and other plants for future use in biological control programs.

13.8 Final Considerations: Application of Beneficial

Microorganisms in Guarana Cultivation

Microorganisms are increasingly considered essential for the survival of plants and ani-
mals. It is known that animals, including humans, contain microorganisms that live in
association with the host cells (fungi, bacteria, and viruses among others) and generally
produce beneficial effects. Similar observations have been made in plants that have a
wide variety of microorganisms coexisting with their hosts. These microorganisms
facilitate plant growth and can protect their hosts from adverse conditions such as stress.
Therefore, the general trend is to consider the plant-microorganism relationship as
holistic. Knowledge of the associated microbiota is essential in plant breeding programs
that focus not only on the economic value of the plant but also on its production in an
environmentally friendly and sustainable way. This chapter presents data, some very
promising, regarding the practical use of the microbiota from guarana. These results
were produced by the efforts of many researchers who continue to provide valuable data
on the microbial community and how it can be harnessed, which will permit cultivation
of guarana and other plants in more rational, efficient, disease-free, and pest-free ways.

jlazevedo@usp.br
310 B.D. Batista et al.

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