Journal of Cleaner Production 234 (2019) 681e689

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Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

Aspergillus flavipes as a novel biostimulant for rooting-enhancement of

Eucalyptus

bora Zanoni do Prado a, Samara Louzada Oliveira a, Clarissa Hamaio Okino-Delgado a,
De
Susann Auer c, Jutta Ludwig-Müller c, Magali Ribeiro da Silva b,

lio Júnior da Costa Fernandes a, Caio Antonio Carbonari b,
Ce
William Fernando Zambuzzi a, Luciana Francisco Fleuri a, *
a ~o Paulo State University (UNESP), Institute of Biosciences, Botucatu, Brazil
Sa
b ~o Paulo State University (UNESP), School of Agriculture, Botucatu, Brazil
Sa
c €t Dresden, Institute of Botany, Dresden, Germany
Technische Universita

a r t i c l e i n f o a b s t r a c t

Article history: In recent years, forest breeding programs have increased Eucalyptus production for commercial pur-
Received 4 December 2018 poses; however, high-performing Eucalyptus clones have had problems with propagation, especially
Received in revised form when rooting. Nevertheless, studies have shown that inoculation of microorganisms producing indole-3-
18 June 2019
acetic acid (IAA) is an especially productive procedure to help these clones breed. In this study, we
Accepted 18 June 2019
Available online 21 June 2019
evaluated the production of IAA and analogues in 16 microbial strains. For the first time, a high IAA
production was described in the strain Aspergillus flavipes (ATCC® 16814™), and A. flavipes was shown by
Handling Editor: Prof. Jiri Jaromir Klemes LC-MS/MS to produce IAA through a tryptophan-dependent biosynthetic pathway. A. flavipes reached the
highest IAA production when cultivated under solid-state fermentation in an optimized medium
Keywords: composed of soybean bran, water and tryptophan. We mixed the fermentation products in solid form
Indole-3-acetic acid (SF) and liquid form (LF) with the substrate Carolina I® and then planted the cuttings of the hybrid
Solid-state fermentation Eucalyptus grandis x E. urophylla (clone IPB2). In fact, treatments with 40e120 mg kg
1 of SF increased
LC-MS/MS the adventitious rooting rate, root length and both root fresh and dry mass, while 120 mg kg
1 of LF
Vegetative propagation
increased root length and dry mass. Additionally, there was no toxicity on fibroblasts (NIH/3t3), and,
Cytotoxicity
therefore, the plant biostimulant was confirmed as a novel, non-toxic, and eco-friendly solution.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction trunk straightness, and wood quality characteristics (ArborGen,

2018). Despite the advantages in the field, the clone has diffi-
Clonal plantation of Eucalyptus based on vegetative propagation culties in rooting, demanding research to promote the adventitious
is successful mainly due to well-developed forest breeding pro- roots induction and development.
grams. However, vegetative propagation efficiency is prejudiced in The inoculation of some classes of microorganisms can assist in
some species due to specific endogenous and exogenous factors of overcoming this difficulty, stimulating adventitious root growth,
the mother plant, alterations of the root system architecture and increasing the absorption of nutrients and water, the host-plant
topophysis effects that influence the rooting potential (Peralta et al., biomass and the tolerance to stresses such as drought and dis-
2012). eases (Sukumar et al., 2013). When a substance can improve the
The clone IPB2 of the hybrid Eucalyptus grandis x Eucalyptus plant nutritional efficiency and abiotic stress and/or influence the
urophylla from ArborGen is classified as a clone SuperTree®. Such quality of the crop, regardless of its nutritional status, it is
clones are selected for a higher volume of wood, disease resistance, denominated as a plant biostimulant (du Jardin, 2015). The com-
mercial products are currently divided into five classes: microbial
inoculants, humic acids, fulvic acids, amino acids and seaweed
extracts (Calvo et al., 2014). The commercial biostimulants can
* Corresponding author.
contain one or more microorganisms and/or substances (du Jardin,
E-mail addresses: [email protected] (D. Zanoni do Prado), luciana.fleuri@
unesp.br (L.F. Fleuri). 2015).

https://doi.org/10.1016/j.jclepro.2019.06.211
0959-6526/© 2019 Elsevier Ltd. All rights reserved.
682 D. Zanoni do Prado et al. / Journal of Cleaner Production 234 (2019) 681e689

The global market of biological products for agriculture, which (1%, m/v) were inoculated with 100 mL of the microbial suspensions.
include biopesticides, biofertilizers, and biostimulants, may reach After 96 h of incubation at 30  C, for fungi, and 24 h of incuba-
US$ 10.05 billion until 2020, with an estimated annual increase of tion for bacteria at 33  C, 20 mL of Salkowski’s reagent were added
14.5%. The potential market growth is linked to the reduction of to the plates at room temperature. The reaction occurred for 30 min
chemical risks, better waste management and incentives from in the dark.
governmental agencies (Markets & Markets, 2016). The plates content were transferred to Falcon tubes and
Several studies have proven the effectiveness of microbial bio- centrifuged for 15 min at 6,000 rpm. Blanks were prepared by the
stimulants with the ability to provide additional phytohormones addition of Salkowki’s reagent in Petri plates with the culture
and/or their precursors (Wong et al., 2016). Phytohormones, such as media plus tryptophan (1%, m/v). The absorbance readings were
auxins, modulate the associations between plants and microor- compared to a standard curve of indole-3-acetic acid (Sigma
ganisms and coordinate cellular and metabolic responses associ- Aldrich®) and the results were expressed as mg of indole-3-acetic
ated with microbial growth in different plant tissues (Boivin et al., acid and analogues mL
1.
2016). The auxin indole-3-acetic acid (IAA) is the main regulator of
all aspects of plant development, acting on both basic cellular 2.2. Selection of the highest IAA producer for developing the
processes and macroscopic phenomena (Sauer et al., 2013), playing biostimulant
a central role in the formation of adventitious roots (Pacurar et al.,
2014). Beneficial effects on plants, as increases in shoot and root 2.2.1. Solid-state fermentation (SSF)
growth, lateral root and root hair numbers resulting from the The microorganism with the highest production of auxins in the
application of microorganisms of the genus Aspergillus, Trichodema, pre-test was evaluated for IAA and analogues production using
and Bacillus, producers of IAA, have been described in bean (Pha- different agro-industrial residues (cassava bagasse, wheat bran,
seolus vulgaris L.) (Hoyos-Carvajal et al., 2009), mung bean (Vigna soybean bran, distillers dried grains with solubles (DDGS) of maize
ratiata) (Hussein et al., 2016) and Arabidopsis thaliana (Salas-Marina and DDGS of sorghum) as substrates for SSF. The amino acid tryp-
et al., 2011). tophan (1%, m/m) was added to the substrates as an IAA biosyn-
Solid-state fermentation (SSF) is a microbial cultivation tech- thesis inductor. The SSF media was composed of 50% of substrate
nique that has been standing out in the development of industrial and 50% of distilled water (m/m). Erlenmeyer flasks (250 mL) with
bioprocesses, in the last two decades. SSF is advantageous due to the substrate (10 g) and water (10 mL) were sterilized for 20 min at
the lower energy requirement, higher product yield, lower waste- 121  C.
water production and lower risk of bacterial contamination. In The microbial suspensions (2 mL) were inoculated in the sub-
addition, it is considered an eco-friendly technique, mainly due to strate and incubated at 30  C for 120 h. Then, 50 mL of distilled
the use of solid agro-industrial wastes as substrate (Thomas et al., water was added and the content was filtered in two layers of
2013). cotton gauze.
Considering the adventitious rooting difficulties of Eucalyptus The Salkowski’s reagent (4 mL) was added to the filtration
clones and the IAA effects on rooting, this study aimed to produce a products (1 mL), the absorbance reading was performed in spec-
new plant biostimulant by SSF, evaluate the biostimulant effect on trophotometer at 535 nm and the results were expressed as mg of
the development of Eucalyptus grandis x Eucalyptus urophylla (clone indole-3-acetic acid and analogues mL
1.
IPB2) seedlings and test the product’s safety for fibroblasts.
2.2.2. Factorial planning
2. Materials and methods The SSF substrate with best production of auxinic compounds
was selected to be optimized by factorial planning. The pH was
2.1. Screening of microorganisms strains for IAA production measured both in the substrate and in the fermentation product.
The particle size (0.5, 1.0 and > 1.0 mm), the amount of water added
2.1.1. Microbial strains and inoculum preparation to the substrate (5, 10, 15 mL) and the tryptophan percentage (0.5,
The strains Aspergillus ustus (Bainer.) Thom & Church (IOC 4410), 1.0 and 1.5%, m/m) were selected as the independent variables,
Aspergillus niger van Tieghem (INCQS 40015), Aspergillus flavipes using the complete factorial planning of the 23 type with three
(ATCC® 16814™), Bacillus subtilis (CCGB 0030), Bacillus megaterium replications at the central point. Erlenmeyer flasks (250 mL) with
(CCGB 0146), Bacillus amyloliquefaciens (CCGB 0145), Trichoderma the substrate (10 g) and water (10 mL) were sterilized for 20 min at
atroviride (IOC 4503), Trichoderma koningii (INCQS 40331), Tricho- 121  C. The microbial suspensions (2 mL) were inoculated in the
derma harzianum Rifai (IOC 3844), were donated by Oswaldo Cruz substrate and incubated at 30  C for 120 h.
Foundation (FIOCRUZ). The strain Aspergillus niger 01 (CBMAI 2084)
was isolated by the Bioprocess Laboratory of Sa ~o Paulo State Uni- 2.2.3. Quantification of IAA by liquid chromatography and mass
versity, Botucatu, Brazil. The strains Bacillus subtilis (B, C, D, E, F, spectrometry (LC-MS/MS)
E27) were donated by Campinas State University (UNICAMP), The selected SSF conditions that gave the best preliminary re-
Brazil. sults were prepared with and without tryptophan in order to
The products from the fungal growth in potato dextrose agar confirm the IAA production by a metabolic pathway with trypto-
(PDA) slants for 96 h at 30  C and the bacterial growth in tryptic soy phan as a precursor. The extracts were centrifuged at 6,000 rpm for
agar (TSA) slants for 24 h at 33  C were suspended in 6 mL of ster- 10 min and filtered through a 0.22 mm membrane. IAA was quan-
ilized water. The suspension was adjusted to 107 spores mL
1. tified according to an adaptation of the method described by Prado
et al., 2019.
2.1.2. Quantification of IAA and analogues The samples (20 mL, pH 6.3) were injected in a LC-MS/MS system
The production of IAA and analogues by the microorganisms consisting of a High Efficiency Liquid Chromatograph, model Pro-
was determined by colorimetric measurement at 535 nm using eminence UFLC (Shimadzu®, Kyoto, Japan), equipped with two
Salkowski’s reagent (2% of FeCl3 in 35% of perchloric acid), ac- pumps LC-20AD, a self-injector SIL-20AC, a degasser DGU-20A5, a
cording to an adaptation of the methods described by Hoyos- controller system CBM-20A and an oven CTO-20AC., coupled to a
Carvajal et al. (2009) and Sarwar et al. (1992). Petri plates with mass spectrometer 3200 Q TRAP (Applied Biosystems®, Foster City,
PDA media, for fungi, and TSA media, for bacteria, with tryptophan USA).
 D. Zanoni do Prado et al. / Journal of Cleaner Production 234 (2019) 681e689 683

The LC separation was carried out by a Synergi 2.5 mm Fusion RP weighted in analytical balance for the dry mass determination.
100 Å chromatographic column (50  4.6 mm, Phenomenex) at
40  C. The samples were eluted with 1% of acetic acid diluted in 2.4. Product safety
milli-q water (v/v) (phase A) and 1% of acetic acid diluted in
methanol (v/v) (phase B) at the flow rate of 0.65 mL min
1. The 2.4.1. Cell viability
gradient program was as followed: 0e2 min, 45% phase B; 2e8 min, The biostimulant cytotoxicity in fibroblasts (NIH/t3t) was eval-
95% phase B; and 8e10 min, 45% phase B. The total run time was uated as described by Mosmann (1983). The cells were seeded 48 h
10 min, and the retention time of the compound in the chromato- before treatments in a 96-well plate at 5  104 cells mL
1. These
graphic column was 2.51 min. The electrospray ionization (ESI) was cells were selected because the mixing operation of the product to
operated in the negative ion mode. Mass parameters were Q1 mass the substrate requires contact of the operator’s hands with the
(174.19), Q3 mass (130.0), declustering potential (DP,
65 V), product.
entrance potential (EP,
10 V), collision energy (CE,
14 V), collision The microbial extracts were centrifuged at 6,000 rpm for 10 min
cell exit potential (CXP,
9 V). Analyst software (version 1.4.2) was and filtered through a 0.22 mm membrane. The cells were exposed
used to control sample acquisition and data analysis. to the biostimulant and to synthetic IAA (Sigma-Aldrich®, St Louis,
USA) at 0, 40, 80, 120 and 160 mg kg
1, the same concentrations of
2.3. Biostimulant effect in the hybrid Eucalyptus grandis x E. the greenhouse experiments. After the exposure time of 24 h, the
urophylla (clone IPB2) cell viability was subjected to MTT (3-(4,5-Dimethylthiazol-2-yl)-
2,5-Diphenyltetrazolium Bromide) (1 mg mL
1) treatment for 3 h.
2.3.1. Application forms Thereafter, the MTT solution was removed and 0.1 mL of DMSO
The aim of this experiment was to test if the SSF product is more (dimethyl sulphoxide) was added into each well for the solubili-
effective when applied in Eucalyptus in solid form (SF), with smaller zation of the dye. Then the absorbance readings were performed at
contact surface and longer contact time, or in liquid form (LF), with 570 nm using a microplate reader (SYNERGY-HTX multi-mode
wider contact surface and a shorter contact time. reader, Biotek, USA).
The crude/solid form (SF) was processed as described below to
obtain the liquid form (LF). The LF was prepared by adding distilled 2.5. Statistical analysis
water to the fermentative product in the proportion of 5:1 (5 mL of
water for each g of fermentation product), followed by filtration in The results were expressed as the mean values of three bio-
two layers of cotton gauze, the yield of the filtration process was logical replicates (mean ± standard deviation). Data were subjected
80%. Both preparations were mixed to the substrate Carolina I® to analysis of variance (ANOVA) and, when the F test was signifi-
(Sphagnum peat, rice husk, and vermiculite), both were adjusted to cant, means were compared by the Scott-Knott test (p  0.05). The
the doses 0, 40, 80, 120 and 160 mg kg
1 of IAA. Both forms of the Statistica software (version 10, Statsoft®) was used to perform the
fermentation products were placed in plastic tubes of 55 m3 and analysis.
moistened with nebulizers.
3. Results
2.3.2. Planting of cuttings
The experiment was conducted in the greenhouse of the com- 3.1. Production of IAA and analogues
pany Avam Flora, located in Aguas
de Santa Ba
rbara county, Sa
~o
Paulo State, Brazil (22 520 5000 S and 49 140 2000 W). The clone IPB2 All tested strains produced IAA and analogues in culture media
of the hybrid Eucalyptus grandis x E. urophylla from ArborGen was supplemented with tryptophan (1%, m/v). A high auxin derivative
selected for the experiment due to its large capacity of cellulose production was reported for the first time by the strain of Asper-
production and the low performance in greenhouse due to prob- gillus flavipes (ca 71 mg mL
1), while the productivity of the other
lems in adventitious rooting. strains varied from ca 3 (T. harzianum) to 23 mg mL
1 (B. subtilis E27)
Apical cuttings, from 3 to 6 cm length and from 1.5 to 2.5 mm (Fig. 1). Thus, the fungus A. flavipes was selected for the continua-
diameter were selected and planted in the prepared substrates. The tion of the study and cultivated under SSF.
experiments were implemented in a completely randomized The A. flavipes strain was cultivated in cassava bagasse, wheat
design with five treatments (four doses of IAA plus the control) and bran, soybean bran, maize DDGS and sorghum DDGS. There was no
three biological replicates (n ¼ 88). The cuttings were taken to the microbial growth in cassava bagasse and maize DDGS. The higher
greenhouse, where they remained for 30 days at 25-30  C and indolic compound production occurred using soybean bran as
relative humidity above 75% and then, were transferred to the substrate (183 mg mL
1), being increased about 2-fold in compari-
shade house (approximately 50% of full sun) for another 10 days, son to wheat bran (87 mg mL
1) and 5-fold in comparison to sor-
totalizing 40 days of the experiment. The water management was ghum DDGS (36 mg mL
1) (p  0.05) (Fig. 2). Thus, soybean bran
performed according to the need of irrigation, by observing the was selected for the optimization of IAA and analogues production
water saturation in the substrates. using factorial planning.

2.3.3. Assessment of rooting and development 3.2. IAA and analogues production using factorial planning
The survival percentage was evaluated in the transition from the
greenhouse to the shade house. At the end of the experiment, 15 In the factorial planning (R2 ¼ 0.86), the variables particle size
central seedlings were removed from each experimental unit to (mm), addition of water (mL) and tryptophan (%) and the interac-
perform the destructive evaluations. The plants were removed tion among all the variables, were not statistically significant for the
from the plastic tubes and washed in running water to remove the production of IAA and analogues using soybean bran as substrate.
substrate residues. Then, the seedlings were sectioned at the However, there was a great variation in the productivity of IAA and
interface between root and aerial part and determined, immedi- analogues due to the variation of the culture medium conditions.
ately after cutting, the rooting percentage, the mean lengths of the The lowest particle size (0.5 mm), highest water addition (15 mL)
shoots and roots, and the mean of fresh root mass. The roots were and lowest tryptophan addition (0.5%) induced the lowest IAA and
drained and then placed in a lyophilizer for 24 h at
60  C and analogues productivity (237 mg mL
1), while, under the same
684 D. Zanoni do Prado et al. / Journal of Cleaner Production 234 (2019) 681e689

(Fig. 3B), confirmed the results of the colorimetric analysis

(654 mg mL
1), about the high microbial production of IAA.
It was also verified that the production of IAA by A. flavipes oc-
curs using a tryptophan-dependent pathway. In the absence of
tryptophan, the production of IAA by A. flavipes reached
0.3 mg mL
1, while, adding 1.5% of tryptophan, the production was
increased to 515.5 mg mL
1 (Fig. 3A).

3.4. Rooting and development in response to the potential

biostimulant

The fermentation process decreased soybean bran pH from 6.5

to 6.3. The treatments with both SF and LF resulted in changes in
adventitious rooting and plant development (Figs. 4 and 5).
There was no significant difference between the treatments and
the control for both survival (Fig. 4A) and average shoot length
(Fig. 4C). There was an increase in the percentage of rooting by the
addition of 40 (89%), 80 (91%) and 120 mg kg
1 (82%) of SF in
comparison to the control (62%), while treatment with 160 mg kg
1
Fig. 1. Quantification of IAA (indole-3-acetic acid) and analogues (mg mL
1), produced
by the strains A. niger (01 and INCQS 40015), A. ustus (IOC 4410), A. flavipes (ATCC of IAA was statistically equal to the control (Fig. 4B). The same
16814), B. subtilis (CCGB 0030, B, C, D, E, F and E27), B. megaterium (CCGB 0146), pattern was followed in the variables average root length, roots
B. amyloliquefaciens (CCGB 0145), T. harzianum (IOC 3844) and T. atroviride (IOC 450). fresh and dry mass (Fig. 4D, E, and 4F). The treatments with 40
Mean followed by the same letter do not differ statistically according to the Scott Knott
(11.0 cm), 80 (10.9 cm), and 120 mg kg
1 (10.0 cm) were higher
test (p  0.05).
than the control (6.7 cm) and the application of 160 mg kg
1
(7.2 cm) of SF, for average root length (Fig. 4D). There was an in-
crease in root mass, fresh and dry, in treatments with 40 (19.1 and
1.7 mg, respectively), 80 (20.0 and 1.8 mg) and 120 (22.1 and
1.7 mg) mg kg
1 of IAA, in comparison to the control (14.0 and
1.3 mg) (Fig. 4EeF), while 160 mg kg
1 was similar to the control
(12.3 and 1.1 mg).
For the LF treatments, there was no significant difference for the
percentage of survival (Fig. 5A), rooting percentage (Fig. 5B) and
fresh root mass (Fig. 5E). The treatments with 80, 120 and
160 mg kg
1 of IAA (whose averages were, respectively, 9.5, 9.7 and
9.7 cm) did not alter the average length of the aerial part in relation
to the control (10.0 cm), while the treatment with 40 mg kg
1 of
IAA induced length reduction (8.5 cm) (Fig. 5C). The average root
length and the roots dry mass were only increased by the appli-
cation of 120 mg kg
1 of IAA (11.3 cm and 1.8 mg, respectively), in
comparison to the control (6.7 cm and 1.3 mg, respectively), while
the other treatments did not cause changes in those variables
(Fig. 5DeF).

3.5. Cell viability

Fig. 2. Quantification of IAA (indole-3-acetic acid) and analogues (mg mL
1) in extracts
from the solid-state fermentation of A. flavipes (ATCC® 16814™) in soybean bran,
wheat bran, and sorghum DDGS. The fibroblasts (NIH/3t3) viability differed in response to both
Means statistically compared by Scott Knott test (p  0.05). A. flavipes and synthetic IAA (Fig. 6AeB). The highest dose of syn-
thetic auxin (Fig. 6A) reduced the viability of NIH/3t3 in relation to
controls. The doses 120 and 160 mg kg
1 reduced NIH/3t3 viability
conditions of particle size and water and the highest tryptophan in comparison to 40 mg kg
1, while the application of 120 and
concentration (1.5%) was reported the highest production of IAA 160 mg kg
1 of A. flavipes IAA (Fig. 6B) increased NIH/3t3 viability in
and analogues (655 mg mL
1) (Table 1). relation to controls and other doses. Thus, for fibroblasts, the IAA
In addition, there was an increase by about ca 3.6-fold in the produced by A. flavipes, at the doses tested, can be considered non-
productivity of auxinic compounds in comparison to the initial toxic and advantageous in relation to the synthetic IAA, which was
conditions of soybean bran (183 mg mL
1) (Fig. 2). Thus, although shown as cytotoxic.
not statistically significant by factorial planning, the cultivation
conditions that resulted in higher IAA production were selected for 4. Discussion
the study continuation.
4.1. Production of IAA by Aspergillus flavipes
3.3. Quantification of indole-3-acetic acid by LC-MS/MS
Auxins can be analyzed by various methods such as qualitative
Three biological replicates were prepared (using the conditions (Hoyos-Carvajal et al., 2009), spectrophotometric (Sarwar et al.,
of the factorial planning that most increased the production of 1992), gas spectrometry (GC) (Barkawi et al., 2010), and high per-
auxin analogues) for the quantification of indole-3-acetic acid by formance liquid chromatography (HPLC) (Gupta et al., 2011; Lu
LC-MS/MS. The IAA productivity by A. flavipes (516 mg mL
1) et al., 2010) both coupled to mass spectrometry. However, the
 D. Zanoni do Prado et al. / Journal of Cleaner Production 234 (2019) 681e689 685

Table 1
Factorial planning for IAA and analogues (mg mL
1) optimization under solid-state fermentation, using A. flavipes and soybean bran and modifying the substrate conditions:
particle size (mm), addition of water (mL) and tryptophan (%).

Assay Particle size (mm) Water (mL) Tryptophan (%) IAA and analogues (mg mL
1)

1 0.5 5 0.5 317.77

2 >1.0 5 0.5 285.83
3 0.5 15 0.5 237.30
4 >1.0 15 0.5 307.84
5 0.5 5 1.5 365.52
6 >1.0 5 1.5 264.74
7 0.5 15 1.5 654.56
8 >1.0 15 1.5 384.56
9 1.0 10 1.0 450.84
10 1.0 10 1.0 383.86
11 1.0 10 1.0 359.09

100 mL
1) and Bacillus cereus (0.8 mg 100 mL
1) (Karadeniz et al.,
2006). Thus, in addition to unprecedented, the productivity of
A. flavipes under optimized conditions, in soybean bran
(515 mg mL
1), is higher than the productivity of other
microorganisms.
The auxin production of A. flavipes was exponentially increased
(from 0.3 to 515.5 mg mL
1) by tryptophan addition. IAA can be
synthesized using L-tryptophan as a precursor and other
tryptophan-independent pathways (Zhao, 2010). Although the
biosynthetic pathways of IAA production by microorganisms have
not yet been fully elucidated, it is described that tryptophan is used
as the main precursor for both bacteria and fungi, through in-
termediates such as indole-3-pyruvic acid (IPA), indole-3-
acetamide (IAN), and tryptamine (TAM) (Spaepen and
Vanderleyden, 2010). Thus, our study indicates that the produc-
tion of IAA by A. flavipes is most likely performed by a tryptophan-
dependent biosynthetic pathway.

4.2. Biostimulant effect in the hybrid Eucalyptus grandis x E.

urophylla (clone IPB2)

Both application forms (SF and LF) of the biostimulant

(A. flavipes cultivated in soybean bran) modified the growth and
development of the hybrid Eucalyptus grandis x E. urophylla (clone
IPB2). Thus the process and the product were patented and
deposited in the National Industrial Property Institute (INPI) under
the register BR 10 2018 007927 1. In the literature, there are many
reports about the effect of diverse categories of biostimulants, with
direct or indirect auxin effects, on plant growth and development
(Calvo et al., 2014).
Protein hydrolysates (PHs) are biostimulants composed of
peptides and amino acids. PHs have been associated to direct effects
in plants as increases in growth, yield, quality, and tolerance to
Fig. 3. Production of indole-3-acetic acid (IAA) by A. flavipes cultivated in soybean bran environmental and chemical soil stresses; and indirect effects as
(0.5 mm) supplemented with 15 mL of water, in the presence and absence of trypto- the stimulus of beneficial microorganisms growth, such as N2-
phan (1.5%) (A), and the chromatogram of the extract with tryptophan (MM ¼ 175,187)
fixing, P-solubilizing and IAA-producing bacteria (Colla et al., 2015).
and the secondary ions (MM ¼ 130.0 e 127.9) (B).
Seaweed extracts can also have biostimulant function. Extracts
from Laminaria and Ascophyllum nodosum, in general, stimulated
better the efficiency of the method, the higher are the costs to root growth, nutrition, esterase activity, and sugar content of maize
perform them. In our study, due to a high number of strains, we (Zea mays L.) plants. An elevate content of IAA (32.43 nM) was
firstly performed the quantification of IAA and analogues by a found in the most effective extract of A. nodosum for promoting root
spectrophotometric method in order to select the strain with the morphology traits (Ertani et al., 2018).
highest IAA production for tests under solid-state fermentation. The commercial formulation Humyk-Fer (Duclos International),
The selected strain, A. flavipes, was reported to produce IAA composed of 73% humic acid (HA), 27% fulvic acid (FA) and metal
under solid-state fermentation for the first time. Previously, the ions (Fe2þ, Kþ, Naþ), increased the growth of L. camara (Costa et al.,
production of IAA was reported by several microbial species, among 2008). In another study, IAA was detected in various extracts of HA
them, Bacillus subtilis (106.36 mg mL
1), Aspergillus ustus and all samples promoted root growth and proton pump activity in
(7.94 mg mL
1) (Salas-Marina et al., 2011), Aspergillus nidulans maize vesicles (Jindo et al., 2012).
(5 pmol mL
1) (Eckert et al., 1999), Bacillus megaterium (2.2 mg As reported in our study, some species of Aspergillus have also
686 D. Zanoni do Prado et al. / Journal of Cleaner Production 234 (2019) 681e689

Fig. 4. Survival (%) (A), rooting (%) (B), average shoot length (C), average root length (D), average root fresh mass (mg) (E) and average root dry mass (F), in response to treatments
with solid form (SF) of the biostimulant at 0, 40, 80, 120 and 160 mg kg
1 of IAA, 40 days after planting.
Mean followed by the same letter do not differ statistically according to the Scott Knott test (p  0.05).

been described in the literature as inoculants with biostimulant rooting. Woody pecan cuttings (Carya illinoinensis) were treated
function. The fungus Aspergillus ustus induced growth of shoots and with aqueous solutions of IAA and indole-3-butyric acid (IBA)
roots and increased the number of lateral roots and root hairs of (0.03%, 0.06%, and 0.09%) and naphthalene acetic acid (NAA) (0.06%,
Arabidopsis thaliana and Solanum tuberosum (Salas-Marina et al., 0.09%, and 0.12%), where the rhizogenesis occurred sooner with
2011), while the coinoculation of Aspergillus niger and Tricho- 0.09% of NAA (Zhao and Zhang, 2015). In Azadirachta indica A. Juss
derma harzianum, both producers of IAA, increased shoot length, (Neem), aqueous solutions of IBA, IAA, and NAA (100, 250, 500, 750,
root length and dry weight of shoot and root of chickpea (Cicer 1,000 and 1,500 mg L
1) were applied to cuttings and treatment
arietinum) (Yadav et al., 2011). with 500 mg L
1 of IBA induced a higher percentage of rooting
Indeed, there are growing evidences about the effectiveness of (Gehlot et al., 2015). Rooting and root system quality of black
diverse classes of biostimulant on plant growth and development. pepper (Piper nigrum cv. Bragantina) were improved when cuttings
In the future, biostimulant research should focus in the combina- were exposed to 4,000 mg kg
1 of IBA mixed with talcum powder
tion of biostimulant categories with complementary characteris- (Freire et al., 2017). In guava cuttings (Psidium guajava L.) CV. Safeda
tics, such as microbial inoculants with seaweed extracts or humic different auxins (IBA, IAA and, NAA 100 mg per 100 g talcum
substances (Calvo et al., 2014). powder) were applied. All auxins improved rooting, but cuttings
treated with IAA reached the highest survival percentage and
number of roots, 27.6 roots, while the treatments with IBA and NAA
4.2.1. Application forms resulted in 23 roots (Zamir et al., 2017).
Regarding the application forms, the SF from 40 to 120 mg kg
1 Although both aqueous and solid forms of IAA have shown
of IAA, increased rooting percentage, root length and fresh and dry promising results for cuttings rooting in several studies until now
root masses (Fig. 4); while LF increased root length and root dry there are no comparative discussions between the methods. For the
mass only in treatment with 120 mg kg
1 of IAA equivalents (Fig. 5). hybrid Eucalyptus grandis x E. urophylla (clone IPB2), the SF treat-
Natural and synthetic auxins have been applied to cuttings, both ment stood out in relation to the LF, exerting better effects and
in solution and in solid form, mixed with talc, aiming at inducing
 D. Zanoni do Prado et al. / Journal of Cleaner Production 234 (2019) 681e689 687

Fig. 5. Survival (%) (A), rooting (%) (B), average shoot length (C), average root length (D), average root fresh mass (mg) (E) and average root dry mass (F), in response to treatments
with liquid form (LF) of the biostimulant at 0, 40, 80, 120 and 160 mg kg
1 of IAA, 40 days after planting.
Mean followed by the same letter do not differ statistically according to the Scott Knott test (p  0.05).

Fig. 6. Viability of fibroblasts (NIH/3t3) under increasing doses (0, 40, 80, 120 and 160 mg kg
1) of synthetic indole-3-acetic acid (IAA) (Sigma®) (A) and produced by A. flavipes (B).
Means statistically compared by Scott Knott test (p  0.05).

requiring one-step less of processing. In addition, a carrier substrate 4.2.2. Inhibitory tendency
of the inoculant is important to provide a stable environment for There was an inhibitory tendency, on the majority of the
the inoculum and extend the product shelf-life (Malus
a et al., 2012). analyzed variables, due to the application of the highest dose of
Between the SF doses, we consider 40 mg kg
1 of IAA the most both SF and LF (160 mg kg
1 of IAA). At low concentrations, auxins
advantageous for rooting E. grandis x E. urophylla, for promoting stimulate growth and development processes in plants, but with
similar effects to 80 and 120 mg kg
1 at a lower cost of production. increasing concentration and activity in the tissues, the plant
688 D. Zanoni do Prado et al. / Journal of Cleaner Production 234 (2019) 681e689

growth is disturbed and the plant may be lethally affected product in the clone IPB2 of the hybrid Eucalyptus grandis x Euca-
(Grossmann, 2010). Therefore, our results suggest that the highest lyptus urophylla, increasing rooting. We consider the solid form (SF)
doses of SF and LF inhibited E. grandis x E. urophylla rooting and advantageous in comparison to the liquid form (LF), for showing
development. better-rooting results and one-step less of processing. The bio-
Synthetic auxin-like compounds are among the most successful stimulating effect of SF occurs in the range of 40e120 mg kg
1 of
herbicides used in agriculture, as they have greater stability in IAA, being recommended the lowest IAA dose due to the greater
plants than IAA, as well as systemic mobility and selective action preparation facility, less need of labor resources and lower pro-
(Grossmann, 2010). However, the most widely used auxinic her- duction costs.
bicide, 2,4-dichlorophenoxyacetic acid (2,4-D) generates waste, In addition to the positive effects on rooting, the product is non-
contaminating air, water, soil and food and the exposure to these toxic to fibroblasts (NIH/3t3), while the synthetic IAA showed
wastes can cause adverse effects on human health (Bukowska, toxicity in NIH/3t3 from the dose 120 mg kg
1. The biostimulant
2006), as genotoxicity (Madrigal-Bujaidar et al., 2001), neurotox- can also be considered environmentally sustainable and inexpen-
icity (Tayeb et al., 2010), hepatotoxicity (Bortolozzi et al., 2001) and sive, due to the use of agro-industrial wastes for its production.
renal toxicity (Uyanikgil et al., 2009). In contrast, microbial in-
oculants have been outstanding for solving environmental prob- Acknowledgments
lems (Calvo et al., 2014).
Due to the inhibitory effects of the highest dose on rooting, it is The authors gratefully acknowledge the financial assistance
possible that in addition to the biostimulating effect, the product from the Coordenaça ~o de Aperfeiçoamento de Pessoal de Nível
described in the patent BR 10 2018 007927 1, in high concentra- Superior - Brazil - Finance Codes 001 and 88881.133019/2016e01,
tions, could disturb the plant development, acting as herbicide. and the strains donation from Oswaldo Cruz Foundation (FIOCRUZ)
Thus, in order to verify this effect, future studies should test higher and Campinas State University (UNICAMP).
doses of the product and investigate the possible effects on envi-
ronmental toxicity and human health.
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