Bioresource Technology Reports 3 (2018) 22–26

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Bioresource Technology Reports

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Production of compounds by phytopathogenic fungi for biological control of T

aquatic macrophytes
Cristiano Daniel Moreiraa, Thamarys Scapinia, Siliandra Mullera, Jéssica Amroginskia,
Simone Golunskia, Leonardo Pandolfia, Leandro Galona, Rodrigo Josemar Seminoti Jacquesb,
Nariane de Andradeb, Caroline Borges Bevilacquab, Márcio Antônio Mazuttic, Gislaine Fongaroa,
⁎
Altemir J. Mossia, Helen Treichela,
a
Universidade Federal da Fronteira Sul — UFFS, Rodovia ERS 135 – km 72, n° 200, Erechim, RS, Brazil
b
Departamento de Solos/CCR - Universidade Federal de Santa Maria, Avenida Roraima, n° 1000, Cidade Universitária Bairro Camobi, Santa Maria – RS CEP 97105-900,
Brazil
c
Departamento de Engenharia Química, Universidade Federal de Santa Maria, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The intense urbanization and use of natural resources has been causing disturbances in the aquatic ecosystem,
Eicchonia crassipes such as the disorderly and dense increase of macrophytes, such as Eichhornia crassipes, Pistia stratiotes and
Pistia stratiotes Salvinia herzogii. In this context, we aim to evaluate the biological control of these plants by the production of
Salvinia herzogii phytopathogenic fungi compounds isolated from the foliar lesions of the plants collected. The microorganisms
Aspergillus flavus
were isolated and identified as Aspergillus flavus and Aspergillus miniscleroti. Afterwards, an experimental design
Aspergillus miniscleroti
Macrophytes biological control
was developed and the results showed that temperature and glucose concentration were determinant in biomass
production, being the largest biomass obtained 26.12 g/L and 45.95 g/L for A. flavus and A. miniscleroti, re-
spectively. The application of the compounds in the plants showed satisfactory results, presenting toxicity po-
tential in macrophytes, with a maximum deterioration of 88–94% for macrophytes E. crassipes.

1. Introduction Over the years, macrophytes management techniques have been

Macrophytes are aquatic plants that generally inhabit lentic ecosystems
and plays an essential role in natural processes, such as nutrient cycle and
metabolism (Bowden et al., 2017). These plants are relevant to the biolo-
gical balance of the ecosystem and have ample capacity of adaptability
(Kumar et al., 2017).
The occurrence of these plants in the environment is determined by
several factors, such as nutrient availability, water flow regime, plant
physiological demands, ability to tolerate local environmental conditions,
plant dispersal history and interactions with other species (Bowden et al.,
2017). However, although it is known that the presence of vegetation is one
of the key factor to distinguish a wetland from a pond or lagoon and also
vegetation assumes several benefits for aquatic environment such as water
quality amelioration, the intensity of human demand for natural resources
and disturbances in the aquatic ecosystem increase, turned aquatic plants in
a serious environmental disturbance, multiplying rapidly and densely
causing water loss from water bodies, decreasing the capacity of irrigation
channel flow, causing oxygen depletion and reducing phytoplankton
(Dodemaide et al., 2018; Haroon and Hussian, 2017).
studied to control the propagation and reduction of biomass, such as
mechanical removal and chemical control (Ding et al., 2006). Me-
chanical control is a method considered effective for the removal and
control of macrophytes in water resources, but the process results in a
large amount of humid biomass and causes another environmental
problem in the matter of safe disposal, because in most cases the bio-
mass, that could be used as an interesting source of biomass to produce
biogas through anaerobic digestion, for example, is discarded on land
or burned after drying, causing other environmental damage (Gusain
and Suthar, 2017). The chemical control of plants consists of the ap-
plication of chemicals capable of interfering in the development of
plants, such as the use of herbicides, but it is considered dangerous,
since contamination of the water with the chemicals may occur and
later the contaminated water course may be used for human and ani-
mals supply (Dall Armellina et al., 1999). As discussed, the approaches
of control have a failure rate for long-term sustainable suppression and
are sometimes economically unviable, thus new researches and in-
centives arise with the aim of developing biological control (Ding et al.,
2006).

⁎
Corresponding author.
E-mail address: helen.treichel@uffs.edu.br (H. Treichel).

https://doi.org/10.1016/j.biteb.2018.05.012
Received 7 May 2018; Received in revised form 30 May 2018; Accepted 31 May 2018
Available online 05 June 2018
2589-014X/ © 2018 Elsevier Ltd. All rights reserved.
C.D. Moreira et al.
Biological control consists of the application of natural interactions
that lead to relationships between species, seeking control from the
equilibrium between populations, offering a series of benefits, such as
greater target specificity and rapid degradation. In addition, products
developed for biological control have a variety of tools to be used alone
or in association with other control methods (Cordeau et al., 2016).
Biological control using pathogenic fungi for plants acts in a way that
significantly decreases plant productivity and growth, mainly through
the reduction of photosynthetic rates (Sutton et al., 2016). Phyto-
pathogenic fungi act on plants through the production of toxins that can
cause lesions and deficits in plant development (de Souza et al., 2017).
The species of macrophytes studied are very common in tropical and
subtropical climates, and are found in several parts of the world (Mary
Lissy and Madhu, 2010; Gupta et al., 2012). These plants are also
known and studied by the phytoremediation potential of contaminated
water, such as the studies of Mishra et al. (2008)), which demonstrated
the potential of the E. crassipes species in the treatment of wastewater
and the retention of heavy metals in treatment systems, demonstrating
relevant results of rapid and effective removal, becoming an alternative
to the treatment of secondary effluents or tertiary. The short flowering
times and rapid reproduction of these plants, permits application in
water remediation processes, decreasing the need to continuously add
of new plants, however, when in environments where it is not desirable,
the presence of the plant becomes a serious environmental problem
(Gupta et al., 2012). According to Mary Lissy and Madhu (2010)) only
ten plants of the species E. crassipes in only eight months could produce
a population of 655,330 individuals.
Considering the fungal potential for the biological control of weed
macrophytes, we aimed to develop biological control of weed macro-
phytes, P. stratiotes, E. crassipes and S. hergozii, through strains of phy-
topathogenic fungi capable of causing damage to the development of
plants, aiming the production of compounds through submerged fer-
mentation with posterior evaluation of the potential of foliar dete-
rioration of plants.
2. Material and methods
2.1. Isolation of microorganisms from the macrophytes
Macrophytes of species Eichhornia crassipes, Pistia stratiotes and
Salvinia herzogii were collected in the summer in different environ-
ments, such as dams, quarries and ponds, at coordinates S 27° 39.959′
W 52° 19.367′ e S 27° 37.630′ W 52° 18.630′; S 27° 37.785′ W 52°
17.374′; S 27° 42.915′ W 52° 16.899′, respectively. For collection pro-
cess plants were selected from the observation of foliar lesions, of which
pathogenic fungi were isolated. Aiming at fungi isolation, plants that
presented lesions were used. Samples of infected material were placed
in Petri dishes and maintained for subsequent isolation and growth of
microorganisms. The isolated plant material was multiplied in Petri
dishes containing Potato Dextrose Agar (PDA) and incubated at 35 °C
for a period of seven days. Posteriorly, successive replicates, depending
on fungus genus, were performed to obtain a single fungal isolate.
2.2. Fungal identification
Identification of the isolated microorganisms was performed by
extracting the DNA from the growth granule brackets in PDA culture
medium using the ZR Fungal/Bacterial DNA Mini Prep Kit (Zymo
Research). After the extraction of DNA, the complete area of the nrDNA
(ITS1-5.8S-ITS2) and part of the area of the gene β-Tubulin for fungal
were amplified with the primers ITS1 and ITS4e Bt2a e Bt2b, respec-
tively (White et al., 1990; Glass and Donaldson, 1995). The reactions of
amplification of the fragments objective were accomplished accord-
ingly (Campbell and Madenn, 1990). After the amplifications, the
electrophoresis was performed to verify the amplification in agarose gel
to 1.5 wt% and lid TBE 1X. The samples of DNA were red-faced with
23
Bioresource Technology Reports 3 (2018) 22–26
BlueGreen Loading Dye I® (LGC Biotecnology, Cotia, Brasil) and ob-
served in ultraviolet light. The products of PCR were purified with the
kit Gen Elute PCR clean-up Kit® (Sigma, Saint Louis, USA), following
the manufacturer's instructions. The sequencing of the samples was
performed in the sequencer ABI PRISM 3100 Genetic Analyzer (Applied
Biosystems).
The sequenced fragments were analyzed using the program Staden
Package 2.0.0b for the obtaining of the sequences consensus and then,
these sequences were deposited in GenBank and received their access
numbers (Staden et al., 2003).
For the identification of the isolated fragments, the sequence con-
sensus was compared with the others deposited in GenBank, and then
the ones that presented larger similarity using the tool BLASTn of
“National Center for Biotechnology Information databases” were
chosen. To select the species with larger verisimilitude, the correlation
head office was used using the software BioEdit (Hall, 1999), which
defines the correlation degree among two to two sequences of the
species.
2.3. Production of compounds from isolated fungi
Preliminary, tests were carried out on the production and applica-
tion of previously isolated fungi compounds in order to determine the
phytopathogenic potential for biological control of macrophytes.
To obtain the compound, a submerged fermentation process was
performed using previously isolated microorganisms which were
transferred to Erlenmeyers containing appropriately autoclaved fer-
mentative medium. The formulation of the medium was carried out in
the following proportions: 10 g/L glucose (C6H12O6), 7.5 g/L yeast ex-
tract, 10 g/L peptone, 2 g/L ammonium sulfate ((NH4)2SO4), 1 g/L
ferrous sulfate (FeSO4·7H2O) and 1 g/L manganese sulfate
(MnSO4H2O), incubated at 28 °C, 120 rpm for 72 h on an orbital shaker
(de Souza et al., 2015). After that, small scale applications were carried
out in plants.
2.4. Production of compounds by submerged fermentation
The optimization of the fermentation medium was carried out using
Experimental Design Methodology (Plackett-Burman) with 12 tests and
3 central points, where the variables investigated were glucose, peptone
and yeast extract, which varied in concentrations of 5 to 15 g/L. To
analyze the influence of the process parameters on biomass growth, the
effects of pH and temperature were investigated, evaluating the tem-
perature from 20 to 30 °C and the pH from 5 to 7.
2.5. Extraction and quantification of fungal biomass
After the fermentation process, the growth of the fungal biomass
was evaluated by the method of the dry mycelium weight. The cell mass
was separated from the liquid by filtration and kept in the greenhouse
for 48 h at 35 °C. After this period, the samples were weighed and the
filtrate (compound) was stored in plastic containers at a temperature of
approximately 6 °C until its application (de Souza et al., 2015).
2.6. Preliminary tests for the application of compound in the control of
macrophytes
The aquatic macrophytes were collected in the field and arranged in
boxes of water with a volume of 310 L during 8 days. Subsequently, the
plants were placed in an experimental area, where they were arranged
in buckets. The application of the extract was done manually with
spraying. After application, the plants were monitored for degradation
control based on the Horsfall-Barrett methodology (Campbell and
Madenn, 1990). This method makes it possible to evaluate the severity
of the lesion caused in the plants by a disease through a scale, presented
in Table 1.
C.D. Moreira et al. Bioresource Technology Reports 3 (2018) 22–26

Table 1 The exposure of the macrophytes to the fermentation medium was

Degree of severity attributed to leaf area of the injured plant.
Severity note Leaf area (injured plant%)
1 0
2 0–3
3 3–6
4 6–12
5 12–25
6 25–50
7 50–75
8 75–88
9 88–94
10 94–97
11 97–100
3. Results and discussion
The fungi isolated in PDA medium were identified as Aspergillus
minisclerotti and Aspergillus flavus. Using a comparative analysis by
BLASTn in NCBI, the consensus sequence showed 100% similarity and
the consensus sequence was deposited in GenBank under accession
number KX025102 and KX025103, respectively. These were subjected
to submerged fermentation in fermentation medium proposed by de
Souza et al. (2015)), where the production of fungal biomass was
27.5 g/L for A. minisclerotti and 24.3 g/L for A. flavus. Also, preliminary
results of application of the compound produced in the fermentation
presented promising results regarding the phytopathogenic effect in the
plants under test.
Aspergillus species are capable of producing a range of mycotoxins
(Kocube et al., 2013). Among the many Aspergillus species already
identified, fourteen (14) are responsible for the production of toxins,
among them the Flavi section, which includes the fungi A. flavus and A.
minisclerotti (Kocube et al., 2013). A. flavus is a filamentous fungus
considered a pathogen that causes allergic reactions and infections in
humans, animals and insects, such as aspergillosis (Yu et al., 2005).
Moreover, the fungus A. flavus cause damage to agricultural crops and
grains, to produce potent toxic and carcinogenic metabolites, such as
aflatoxins (Yu et al., 2005; Scully and Bidochka, 2009). This fungus is
able to survive in different environments, using various sources of or-
ganic nutrients such as plant leaves (Yu et al., 2005). A. miniscleroti
belongs to the same group as the fungus A. flavus, in addition it re-
sembles the production of aflatoxins B1 and B2, cyclopiazonic acid,
kojic acid and aspergylic acid (Pildain et al., 2008). In this case, the
leaves of macrophytes become an excellent environment for its devel-
opment.
performed after filtration process to separate the fungal biomass (only
the liquid medium was applied to the plants). Degradation results were
not effective in this assay and at the second moment, the application of
the compound was performed with the presence of fungal biomass,
presenting degradation results on the macrophytes species studied.
In relation to the decomposition and biochemical transformation of
the macrophytes using fungal extract, it is possible to emphasize a de-
gradative enzymatic action and still a fungal competition for the nu-
trients required by macrophytes, leading to the immobilization of ni-
trogen, through the conversion of inorganic nitrogen to fungal biomass
and consequent loss of nutritional quality of the plant and their de-
struction (Fell and Findlay, 1988). The absence of a degraded effect of
the macrophytes using only the filtered fermentation medium (without
fungicidal biomass) suggests that there is no action of toxic secondary
metabolites. A degrading action can be attributed to fungi as a bio-
control agent because of a competition for space and antibiosis, cor-
roborating experimental data (Figueiredo and Silva, 2014).
Table 2 shows the experimental matrix and the concentrations of
fungal biomass obtained in each trial. For the fungus A. flavus the
highest amount of biomass was found in Experiment 3 (26.12 g/L)
using a concentration of 15 g/L glucose, 5 g/L peptone and 10 g/L yeast
extract, temperature of 20 °C and pH 7. The fungus A. miniscleroti also
had the largest amount of biomass (45.95 g/L) in Experiment 3. When
evaluating the results, it is observed that the highest amounts of bio-
mass were obtained at higher glucose concentrations. This occurred due
to glucose being a readily assimilated carbon source which produces an
acceleration in fungi growth (Medina et al., 2008). In relation to pH, the
best results were found at neutral pH, a fact that is justified by Papa-
gianni, M. (Papagianni, 2004), that suggests that the greatest growth of
fungi occurs at pH close to neutrality.
Klaic et al. (2014)) analyzed the growth of Phoma sp. that showed
potential for the production of compounds, where the most promising
result for the composition of the medium consisted of glucose, peptone
and yeast extract at a concentration of 20 g/L, 20 g/L and 7.5 g/L re-
spectively, and pH 6, where a maximum concentration of fungal bio-
mass of 22 g/L was obtained. It was observed that with lower con-
centration of glucose and a considerable reduction in the concentration
of peptone, there was obtained a biomass concentration for A. flavus
and A. minisclerotti of 26.12 and 45.95 g/L, respectively, demonstrating
a significant increase when compared to the results obtained by (Klaic
et al., 2014) for Phona sp. where higher concentrations of nutrients
were used in the medium.
To assess the harmful effect of the compound on plants, the ap-
pearance of the macrophytes was visually assessed based on the

Table 2
Plackett-Burman experimental matrix used to evaluate the influence of process parameters and fermentation medium on the concentration of fungal biomass for
Aspergillus flavus and Aspergillus miniscleroti.
Assay T (°C) pH Glucose (g/L) Peptone (g/L) Yeast extract (g/L) Biomass (g/L)

A. flavus A. minisclerotti

1 30 (1) 5 (−1) 15 (1) 5 (−1) 5 (−1) 9.98 10.38

2 30 (1) 7 (1) 5 (−1) 15 (1) 5 (−1) 7.36 4.62
3 20 (−1) 7 (1) 15 (1) 5 (−1) 10 (1) 26.12 45.95
4 30 (1) 5 (−1) 15 (1) 15 (1) 5 (−1) 11.95 14.08
5 30 (1) 7 (1) 5 (−1) 15 (1) 10 (1) 9.70 6.64
6 30 (1) 7 (1) 15 (1) 5 (−1) 10 (1) 14.81 11.53
7 20 (−1) 7 (1) 15 (1) 15 (1) 5 (−1) 25.86 13.78
8 20 (−1) 5 (−1) 15 (1) 15 (1) 10 (1) 21.38 20.50
9 20 (−1) 5 (−1) 5 (−1) 15 (1) 10 (1) 10.07 8.35
10 30 (1) 5 (−1) 5 (−1) 5 (−1) 10 (1) 7.91 7.97
11 20 (−1) 7 (1) 5 (−1) 5 (−1) 5 (−1) 6.07 5.42
12 20 (−1) 5 (−1) 5 (−1) 5 (−1) 5 (−1) 14.03 10.63
13 25 (0) 6 (0) 10 (0) 10 (0) 7.5 (0) 10.62 11.27
14 25 (0) 6 (0) 10 (0) 10 (0) 7.5 (0) 10.79 10.09
15 25 (0) 6 (0) 10 (0) 10 (0) 7.5 (0) 10.99 10.28

24
C.D. Moreira et al.
Fig. 1. Preliminary test of application of the biocomposite in macrophytes of the species: (a) Salvinia hergozii; (b) Pistia stratiotes; (c) Eichhornia crassipes; (d) un-
treated plant species (control).
Horsfall-Barrett method. Observations were made at 5, 10, 15, 22 and
29 days after application of the compound in the plants. In Fig. 1, a
sequence of plant images is presented after application of the com-
pound.
For the species S. herzogii it was possible to observe that the appli-
cation of the compound caused lesions in the plant after the tenth
(10th) day, where it was observed a higher occurrence of necrosis on
the leaf surface, increasing gradually until the last day of observation.
Based on the evaluation of the methodology used, it was verified that
the severity score of the fungi lesions caused in the leaves of the mac-
rophyte S. herzogii was seven (7), when more than 50% and less than
75% of the plants presented lesions.
The species P. stratiotes suffered necrosis on the leaf surface after the
twenty-second (22nd) day of testing, and on twenty-ninth (29th) day it
presented a high index of lesions in all the leaves of the plant, revealing
rapid progression of degradation after the appearance of the first ne-
crotic areas. Based on the methodology, P. stratiotes presented a severity
score of six (6), when more than 25% and less than 50% of the plants
had lesions.
In the plant E. crassipes the appearance of lesions in the plants oc-
curred after the tenth (10th) day of application of the compound. This
species showed almost complete impairment of the leaf structure at the
end of the evaluation, showing that the compound can act effectively in
the biological control of this species. The evaluation based on the
methodology employed, the severity of lesions were scored in nine (9),
where 88% to 94% of the plants showed necrotic areas.
As the working performed by de Souza et al. (2015)), where they
verified that the effects caused by the action of the compound were
foliar lesions and in some cases the yellowing of the plants. It was ob-
served that the plants did not resist application of the compound, pre-
senting necrosis in a large part of the leaf tissue.
In the study of Souza et al. (de Souza et al., 2017), compounds were
produced from 39 phytopathogenic fungi isolated from weeds of the
rice crop in order to carry out the biological control of the plants. Of the
isolated fungal strains, 28 presented some phytopathogenic potential on
the herb. Using the same fermentative medium used in the preliminary
tests of our study, the maximum phytotoxicity observed by (de Souza
25
Bioresource Technology Reports 3 (2018) 22–26
et al., 2017) was 60% for the compound produced by the fungus
identified by Diaporthe sp. When compared to the results obtained in our
study, with the optimization of the fermentation medium, lower con-
centrations of salts were used and higher phytotoxicities were obtained
for the macrophytes S. hergozii (50–75%) and E. crassipes (88–94%),
showing effective potential on the biological control of macrophytes
using compounds produced from fungi A. flavus and A. miniscleroti.
4. Conclusion
The process of collecting, selecting and isolating microorganisms
from aquatic macrophytes of the species E. crassipes, P. stratiotes and S.
herzogii resulted in the isolation of pure cultures of the microorganisms
A. flavus and A. minisclerotti, which were used in the production of
compound. Thus, through an experimental design, the most promising
results for both microorganisms in relation to the biomass concentra-
tion were obtained when using 15 g/L glucose, 5 g/L peptone and 5 g/L
extract of yeast under conditions of pH 7 and temperature 20 °C. The
application of the compound in macrophytes showed promising results,
causing partial degradation in the three-species studied. Therefore, the
biological control through phytopathogenic fungi was positive and of
great relevance for the macrophytes control. Finally, if necessary, it is
believed that after the biocontrol, macrophytes biomass can be re-
moved through mechanical removal, but in a much lower total volume,
facilitating the handling.
Acknowledgments
The authors thank CNPq (306558/2014-9), CAPES (MCTI/FINEP/
CT-INFRA - PROINFRA - 02/2014) and FAPERGS (17/2551-0001 086-
8) for the financial support of this work.
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