Chapter 5

Biodegradation of Paclobutrazol
A Plant Growth Regulator Used
in Irrigated Mango Orchard Soil
Fernanda Vaz, Ednaldo Santos-Filho, Suzyane Silva,
Silvany Arajo, Thatiana Stamford-Arnaud,
Andrea Bandeira, Ana Cristina Brasileiro-Vidal,
Newton Pereira Stamford,
Maria Aparecida Mouco and Ester Gouveia
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/60818

Abstract
Paclobutrazol

(PBZ),

[2RS,3RS]-1-[4-chlorophenyl]-4,4-dimethyl-2-(1H-1,2,4-

triazol-1-yl) pentan-3-ol, consists of a triazole ring and a benzene ring-chloro linked

to a carbon chain open. It is a plant growth regulator widely used in many crops in
order to produce fruit throughout the year by inhibiting gibberellin synthesis, a
hormone responsible for the vegetative plant growth. Actually, studies are showing
that paclobutrazol remains active in the soil for a long time, affecting the growth and
development of subsequent crops by reducing plant vigor. Biodegradation is an
effective and cheap process that can to degrade or transform contaminants to less toxic
or nontoxic. In this work, the biodegradation of paclobutrazol was studied using in
submersed culture and saturated and unsaturated soils. In these conditions, experi
ments with biostimulation and bioaugmentation were performed. In the experiments
carried out in submersed culture, with biostimulation by addition of glycerol, the PBZ
biodegradation was higher than that with PBZ as sole carbon source. The biodegra
dation of PBZ in unsaturated soils was more efficient when soil samples with a history
of application of PBZ were used. The highest number of applications of PBZ favored
biodegradation. The biodiversity of the microbiota in the soil favored the biodegra

2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.

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Biodegradation and Bioremediation of Polluted Systems - New Advances and Technologies

dation of PBZ aromatic rings. PBZ was not seen to be phytotoxic and the biodegraded
products increased the germination index.
Keywords: paclobutrazol, semi arid region of Brazyl, models, Pseudomonas

1. Introduction
Brazil has a great potential for fruit production, as it has area, climate, and enough water for
production throughout the year. The production of mango can be developed under different
climatic conditions, but it is commercially viable only within a well-defined range of temper
ature, rainfall, altitude, insulation, relative humidity, and winds. The fruit is native to tropical
climates, but it can be grown in subtropical regions of the planet. Mango (Mangifera indica L.)
plantations in the country occupy about 74,000 ha, generating a production of over 1.1 million
tons. Mango is produced in all regions of Brazil; however, the southeast and northeast regions
account for 94% of the total [1]. The area cultivated with mango in the northeast region has
increased by 20,000 ha in 10 years. In 2012, that amount alone was responsible for producing
more than 85% of the total exported by Brazil [2]. To foster exportation, however, it is necessary
to guarantee production whenever the market is receptive and to ensure that the quality of the
fruit corresponds to international food safety requirements [3].
The production of mango in Brazil can be divided into two different phases: the first one
characterized by extensive cultivation of local varieties with little or no use of technology; the
second one characterized by a high level of technology, such as irrigation, floral induction, and
improved varieties [4]. Mangos from the Brazilian semiarid region stand out in the national
scenario due to high yields and fruit quality, and also to the possibility of year-round produc
tion taking advantage of the climatic conditions as well as management techniques (irrigation,
pruning, and the use of growth regulators) for plant growth and blossom control [5].
The growth of the mango tree, as well as other tropical fruit trees, is not continuous but comes
in vegetative flushes of the terminal and axillary shoots of the branches, before the period of
dormancy. For the vegetative or floral growth to happen, two different processes occur in the
plant: the growth of the buds and the initiation of the sprouting. The bud starts to grow, which
includes the end of the dormancy and a quick development of the shoot. Along with the shoot
initiation, the induction happens, and it will define the vegetative type, floral or mixed [6].
Flowering in mango is a process that may occur during an extensive period (up to several
months) and can have its beginning altered, naturally or artificially, due to climatic
conditions, yield of the former harvest, or use of specific crop management techniques,
including plant growth regulators [7]. Most of the plant growth regulators inhibit the
gibberellin synthesis and can therefore be used for plant growth and flowering manage
ment [8]. Among the plant growth regulators used in fruit production, paclobutrazol (PBZ)
has shown efficiency in mango flowering management [9]. PBZ (Figure 1) must be applied
directly to the soil due to its low solubility, long residual activity, and lack of efficient foliar

Biodegradation of Paclobutrazol A Plant Growth Regulator Used in Irrigated Mango Orchard Soil
http://dx.doi.org/10.5772/60818

uptake [8]. The recommended doses range between 1.0 and 1.5 g, measured by tree crown
diameter, and dependent on the cultivar, climate, soil type, and plant nutrition. Paclobutra
zol is absorbed by the roots, conducted by the xylem to the leaves and buds, without
mobility by phloem [10]. It is persistent in the plant and soil, highly stable in the soil, and
its slow degradation lowers plant metabolism [8]. PBZ applied as a soil drench reduces
internode lengths and causes earlier and enhanced flowering in mango trees. These results
have been confirmed in different locations in the tropics [11].

Figure 1. Chemical structure of paclobutrazol.

Paclobutrazol doses applied, each year, are not always adequate because they do not take into
account the residue from previous applications. Paclobutrazol increases the compaction of
inflorescence in the Tommy Atkins mango proportionate to the applied dose [12]. High
dosage, which tends to reduce the panicle length of the treated plants (33% as compared to
control), results in the formation of very compact inflorescences, creating appropriate condi
tions for the incidence of diseases and pests as well as making phytosanitary control difficult
[9]. In addition to the phytosanitary problems, excessive doses of PBZ can inhibit vegetative
and floral growth longer than desirable, requiring more nitrate sprays to stimulate flowering.
The high cost of crop production, for all the reasons that have been mentioned, is only one of
the problems, as there is also the question of the accumulation of a chemical in the soil and
plant without knowing the consequences over the years, both for the production system and
the environment.
Soil application rather than foliar application of paclobutrazol has been found to be more
responsive in suppressing the vegetative growth and enhancing the reproductive growth in
mango trees [12, 13]. Studies have shown that paclobutrazol needs to be applied annually to
increase mango fruit yields [5]. However, the paclobutrazol treatments to the tree basins (soil

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under the canopy drip area within a radius of 1.5 m of the tree trunk) may result in its uptake
into the trees and thereby result in the persistence of its residues in the mango fruit and also
in the soil at the tree basin [13]. Such persistence of paclobutrazol residues in mango fruit may
lead to adverse effects on human health. The persistence of paclobutrazol residues in soil may
influence the soil microbial activity too. Soil microbial count of a mango orchard soil where
paclobutrazol was frequently applied has been shown to be reduced by up to 58% [14].
Soils are becoming polluted by pesticides because of the wide and, often indiscriminate,
use of these xenobiotic molecules in agricultural practice. In the soil, pesticides may be
involved in several stages, such as retention, transformation, and transport, the intensity of
which will affect the potential activity of agrochemicals [15]. Bioremediation is an effec
tive and cheap process that can degrade or transform contaminants to become less toxic or
nontoxic [1618]. Two processes have been found to increase the activity of microorgan
isms during bioremediation: biostimulation and bioaugmentation [19]. Biostimulation
involves the addition of nutrients and/or a terminal electron acceptor to increase the weak
activity of indigenous microbial populations by accelerating the decontamination rate since
the addition of one or more rate-limiting nutrients to the system improves the degrada
tion potential of the inhabiting microbial population [20, 21]. Bioaugmentation involves the
addition of external microbial strains (indigenous or exogenous) that have the ability to
degrade the target toxic molecules [22].
In this work, the biodegradation of paclobutrazol was studied using in submersed culture and
saturated and unsaturated soils. In these conditions, experiments with biostimulation and
bioaugmentation were performed.

2. Methodology
Soil samples were collected from irrigated mango orchards (M. indica L. cv. Tommy Atkins)
at the Bebedouro and Mandacaru Experimental Stations of the Brazilian Agricultural Corpo
ration (EMBRAPA Semirido), in the Municipalities of Petrolina (909 S, 4022 W), Pernam
buco state, and Juazeiro (924 S, 4024 W), Bahia state, both located in the So Francisco river
valley (northeast of Brazil). The two representative soils were a Yellow Ultisol (Bebedouro)
and a Vertisol (Mandacaru). These regions had been consecutively treated with PBZ, with an
average dose of 3.57 g of active ingredient per plant. The soil samples were collected 30 days
after the last application. An average of 1.5 kg of soil at depths of 15 and 30 cm was collected
from four points around eight plants. These samples were stored in a refrigerator for isolation
until the beginning of the experiments. Soil samples without a history of PBZ application were
also taken from the same farms.
The bacteria were isolated in a mineral medium [23], containing 0.25 g/L paclobutrazol (Cultar
25 SC, containing 25% of the active compound), which was used as the sole carbon source. 10
g of each soil sample (MSMandacaru without historical application of PBZ; MCManda
caru with historical; BSBebedouro without historical; BCBebedouro with historical) were
added to 100 mL medium in 500 mL flasks. These flasks were incubated at 30C in a rotatory

Biodegradation of Paclobutrazol A Plant Growth Regulator Used in Irrigated Mango Orchard Soil
http://dx.doi.org/10.5772/60818

shaker (200 rpm). Evidence for bacterium utilization of paclobutrazol was sought by streaking
turbid enrichment broths onto a mineral agar medium (15 g/L), containing paclobutrazol or
glucose as the sole carbon source, and then incubating these plates under the same enrichment
conditions. Pure cultures of paclobutrazol utilizing bacteria were obtained by streaking
distinct colonies present on the mineral agar medium plates onto Tryptone Soy Agar (TSA,
Oxoid). The isolates were identified by Gram staining test. The Gram-negative isolates were
streaked on three selective media for Pseudomonas: agar D4 [24]; agar Cetrimide (Merck) and
King [25]. Biodegradation experiments were accomplished in a mineral broth with PBZ (1
g/L) and glycerol (5 g/L) or glucose (10 g/L).
Biodegradation experiments in saturated soils (Yellow Ultisol and Vertisol) were conducted
in batch using paclobutrazol and paclobutrazol with added glycerol. The experiments were
performed under sterile (by Gamma radiation) conditions using the mixed culture of Pseudo
monas spp. Two concentrations of PBZ (10 and 25 mg/L) according to solubility in water (<26
mg/L) were used. The experiments were carried out in 60 mL flasks, where 5 g or soil was
added to a 25-mL volume of liquid. The microorganisms were added with about 107 cells/mL.
Control experiments were carried out only for the PBZ concentration of 25 mg/L without the
addition of microorganisms. These experiments were placed in a rotary shaker (200 rpm) at
30C for 35 days. Microbial concentration initial was 1.107 CFU/mL. This quantity corresponds
to the inoculum of microorganisms added to the experiment.
Biodegradation experiments were conducted with the collected soil samples with and without
history. Glycerol was added as the additional carbon source. To each 10 g of soil with (P-G:
PBZ and glycerol; P-NG: only PBZ) and without (NP-G: PBZ and glycerol; NP-NG: only PBZ)
PBZ application history, 30 g/g of PBZ was added from a solution prepared with a commercial
product (Cultar 25 SC). The experiments were carried out in 125 mL flasks at room tempera
ture, without stirring, for 63 days and in triplicate. Samples were withdrawn at 0, 7, 14, 21, 35,
48, and 66 days for the quantification of native microbial and residual PBZ. In experiments
with the addition of glycerol (P-G, NP-G), the concentration of this compound in the soil was
150 g/g. Microorganisms were not added to the soil.
A 24 factorial design to study the biodegradation of paclobutrazol was applied. A two-level
factorial design with 16 runs was employed to evaluate the individual and combined effects
of the four factors: glycerol, mineral medium, inoculum, and soil (Table 1). The levels of the
factorial design were glycerol (X1), with (+) and without addition (); mineral medium [28]
(X2), with (+) and without addition (); inoculum (X3), with (+) and without addition (); and
region of soil collection(X4), A (+) and B ().
Infrared spectra of the samples before (E1: 0 days) and after (E2: 70 days) the biodegradation
process using only PBZ or PBZ and glycerol in unsaturated soils were measured with FTIR
spectrophotometer (Vertex 70, Bruker). The analysis was done in IR region of 400 and 4000 cm1.
The determination of the phytotoxicity was carried out with samples of biodegradation
experiments (0 and 70 days), using only PBZ (4 g/g) or glycerol (2.4 mg/g) as additional carbon
source. Thirty seeds of Allium cepa (cv. Vale Ouro IPA-11) were germinated in individual Petri
dishes containing 20 mL for each treatment at room temperature for 72 h. Distilled water was

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Biodegradation and Bioremediation of Polluted Systems - New Advances and Technologies

Run

X1

X2

X3

X4

10

11

12

13

14

15

16

Table 1. Conditions of factorial design 24 experiments for the biodegradation of PBZ.

used as negative control, totalizing five treatments. After 24 h of treatment, seed germination
(%) and root length were measured per treatment, in order to determine seed germination
index (GI), as described in Equation (1):
GI ( % ) =

St Rt
* 100
Sc Rc

(1)

where St is the seed germination of treatment (%), Sc is the seed germination of negative control
(%), Rt is the root length of treatment (cm), and Rc is the root length of negative control (cm).

3. Results and discussion

3.1. Isolation of bacteria from soil resistant to paclobutrazol
A total of 37 strains were isolated from the soil samples, in which 89% were Gram-negative
bacteria. Eleven of these were identified as Pseudomonas (Figure 2). The growth curves of the
11 isolates of Pseudomonas showed exponential phase between 4 and 6 h. The maximum specific

Biodegradation of Paclobutrazol A Plant Growth Regulator Used in Irrigated Mango Orchard Soil
http://dx.doi.org/10.5772/60818

rates growth (max) were higher than 0.30 h1 (Table 2). These bacteria were selected by their
capacity to degrade diverse composites, as for example hydrocarbons [26], 2,4-dichlorophenol
[27], naphthalene [28], and organophosphates [29]. Pseudomonas also participates in metabolic
routes of compound degradation similar to paclobutrazol, as chlorobenzene [30] and atrazine
[31, 32]. Jackson et al. [33], in research with paclobutrazol biodegradation, isolated nine
Pseudomonas spp. with biodegradation capacity.

Figure 2. Bacteria isolated by enrichment of soil with paclobutrazol.

Pseudomonas spp.

max (h1)

BC8

0.36

MS9

0.45

BS19

0.49

BC20

0.78

BC21

0.96

MS23

0.65

MS26

0.59

MC27

0.68

BS31

0.71

BS32

0.68

BS33

0.68

Table 2. Growth maximum specific rates

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Biodegradation and Bioremediation of Polluted Systems - New Advances and Technologies

Two strains of Pseudomonas were identified as Pseudomonas aeruginosa; however, these were
not used in this work due their pathogenicity [34, 35]. Experiments with mixed cultures of the
soil samples MS, MC, BS, and BC were carried out using only PBZ as the carbon source to
evaluate PBZ biodegradation. TSA broth was inoculated with the cultures to activate them.
After 24 h, at 30C, bacteria was inoculated into 40 mL of the nutrient broth. After a period of
approximately 4 at 6 h of incubation, at 30C and 200 rpm, mixed cultures were prepared and
inoculated into 400 mL of mineral broth, as described by Ridgway et al. [23] and PBZ, 1 g/L.
Temperature and agitation conditions of this stage were similar to those for the inoculum.
Later, biodegradation experiments were accomplished in mineral broth with PBZ, 1 g/L and
glycerol, 5 g/L.
Biodegradation was for MS and BC mixed cultures that had reached the maximum in 20 days
of culture, with 47% (MS) and 43% (BC) of PBZ biodegradation. No relation was observed
between the PBZ biodegradation and the soil to have a history of application, probably due
the isolation to have been for enrichment. Since the results of the experiments with mixed
cultures MS and BC were similar, the culture MS was selected to continue with biodegradation
experiments.
The experiments carried out with glycerol as an additional carbon source grew and had PBZ
biodegradation higher than those with PBZ as sole carbon source. The maximum biodegra
dation reached about 75% in 10 days of culture. Jackson et al. [33], using Pseudomonas, obtained
a biodegradation of 79% in 39 days. On the other hand, Silva et al. [14] observed a 56%
biodegradation in 90 days, with mixed cultures of Bacillus, in an isolated soil sample with a
history of application. Table 3 presents PBZ biodegradation in relation to the time, found in
experiments using submersed culture. Lee et al. [28] observed that pyruvate can be used as an
additional carbon source to stimulate growth and aromatic hydrocarbons biodegradation for
Pseudomonas putida PG7.
Additional carbon

Microorganism

Biodegradation (%)

Time (days)

Pseudomonas

79

39

Jackson et al. [33]

Bacillus

56

90

Silva et al. [14]

Pseudomonas

47

20

Present work

Pseudomonas

75

10

Glycerol

Present work

Pseudomonas

Glucose

Present work

source

Reference

Table 3. Comparison of the PBZ biodegradation in submersed culture.

When glucose was used as an additional carbon source, the PBZ concentration remained
almost constant throughout the observation period (Table 3). PBZ biodegradation did not
occur probably due to glucose catabolic repression. The presence of a catabolic repressor, or
the presence of a carbon source that represses the expression of certain genes and operons
responsible for the utilization of alternative carbon sources, can result in a low concentration
inducing specific cometabolic routes [32].

Biodegradation of Paclobutrazol A Plant Growth Regulator Used in Irrigated Mango Orchard Soil
http://dx.doi.org/10.5772/60818

3.2. Paclobutrazol biodegradation in saturated soils

In the experiments with PBZ as the sole carbon source in saturated soils, there was no difference
in growth between the two PBZ concentrations used (10 and 25 mg/L). However, in experi
ments containing glycerol, a higher growth was observed when the concentration of PBZ 25
mg/L was used as a glycerol concentration of 50 mg/L. This can be attributed to an increased
amount of glycerol (125 mg/L) compared to experiments with the PBZ concentration of 10 mg/
L. The biodegradation of PBZ without an additional carbon source and 10 mg/L of PBZ was
approximately 43% after 14 days (Figure 3). However, with glycerol as an additional carbon
source, the biodegradation reached 70% in 28 days (Figure 4). The glycerol concentration
decreased rapidly and was completely consumed during 4 days, independently of the amount
utilized (Figure 5).

Figure 3. PBZ biodegradation. S1: yellow ultisol; S2: vertisol.

For the two soils and the two PBZ concentrations used (10 and 25 mg/L), there was a lag phase
(the period in which there is virtually no biodegradation) of approximately 2 days, when PBZ
was used as the sole carbon source. In all the experiments, biodegradation increased after a
certain period of time, approximately 28 to 14 days, with only PBZ and PBZ with glycerol,
respectively. The addition of a carbon source to the nutrient into the soil is believed to enhance
in situ bioremediation by stimulating the growth of microorganisms that are indigenous to the
subsurface and are capable of degrading contaminants [32].
3.3. Paclobutrazol biodegradation in unsaturated soils with and without a history of
application
Figure 6 shows the PBZ biodegradation kinetics in unsaturated soil without and with a history
of application, with and without the addition of glycerol. Experiments in P-G and P-NG soil
showed a sustained reduction after the 14th day and only around 1% of the PBZ remaining on

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Biodegradation and Bioremediation of Polluted Systems - New Advances and Technologies

Figure 4. PBZ biodegradation with glycerol as additional carbon source. S1: yellow ultisol; S2: vertisol.

Figure 5. Glycerol consumption. S1: yellow ultisol; S2: vertisol.

the 63th day. This ability of the native microbiota to degrade paclobutrazol was probably due
to the historical application. After repeated applications of some pesticides, the native
microorganisms in the soil can degrade these compounds as they become suited for agro
chemical use as a source of carbon for energy production and growth. Although there are some
other factors affecting the persistence of agrochemicals in the soil, such as temperature, pH of
the soil, chemical hydrolysis, and water content of the soil, microorganisms seem to play an
important role in the degradation of these compounds [34].

Biodegradation of Paclobutrazol A Plant Growth Regulator Used in Irrigated Mango Orchard Soil
http://dx.doi.org/10.5772/60818

Figure 6. PBZ residual over 63 days.

The PBZ residue in soil without application history of PBZ, containing glycerol (NP-G) or not
(NP-NG), was approximately 64% at 14 days. The lower biodegradation rate in NP-G and NPNG was due to the microbial not being adapted to PBZ since this soil had no history of
application.
The biodegradation in NP-G or NP-NG soils was clearly lower than that in P-G or P-NG soils.
Biodegradation was not significantly different up to 49 days for experiments in NP-G and NPNG soils. Similarly, in soil P-G and P-NG soils, biodegradation was not significantly different
up to 14 days. Maximum biodegradation occurred in soil with a PBZ application history within
63 days, regardless of the presence of glycerol. This was probably due to the low concentration
of glycerol added. Growth was similar; regardless of the addition of glycerol, comparing NPG with NP-NG soils and P-G- with P-NG soils. However, with respect to the soil with and
without history, there was higher growth to soil with history (P-G and P-NG).
The PBZ biodegradation kinetics modeling in the experiments without history (NP), but with
(G) and without (NG) glycerol (Figure 7) was similar and presented the highest fit following
a double first-order equation. PBZ was consumed at a rate k1 of 0.0894 and 0.1028 (Table 4),
respectively, in experiments with and without glycerol [35].
The kinetic modeling for the experiments with history (P) and with and without glycerol (G,
NG) differed greatly from experiments without history (NP) (Figure 8). Both followed a firstorder kinetics, where the PBZ was degraded at a constant k rate of 0.0573 and 0.0538 (Table
4), respectively.

95

regardless of the addition of glycerol, comparing NP-G with NP-NG soils and P-G- with P-NG soils. However, with respect to the
soil with and without history, there was higher growth to soil with history (P-G and P-NG).
The PBZ biodegradation kinetics modeling in the experiments without history (NP), but with (G) and without (NG) glycerol
(Figure 7) was similar and presented the highest fit following a double first-order equation. PBZ was consumed at a rate k1 of 0.0894
96 Biodegradation
and Bioremediation
of Polluted
- Newglycerol
Advances[35].
and Technologies
and 0.1028
(Table 4), respectively,
in experiments
withSystems
and without

120

A
100

PBZ (%)

80
60
40

Experimental
First Ordem

20

Double First Order

Logistic

0
0

14

28

42

56

70

56

70

Time (days)
120

B
100

PBZ (%)

80
60
40

Experimental
First Order

20

Double First Order

Logistic

0
0

14

28

42

Time (days)

Figure 7. Modeling kinetics of soil without history with and without glycerol. (A) Without glycerol; (B) with glycerol.
Figure 7. Modeling kinetics of soil without history with and without glycerol. (A) Without glycerol; (B) with glycerol.

Table 4. Constants and kinetic parameters of the biodegradation of paclobutrazol; NP-NG: soil without history and without glycerol;
NP-G: soil without
P-NG: soil with history
with history and with glycerol.
Soils history and with glycerol;
First order
Double and
first without
order glycerol; P-G: soilLogistics
C0 (%)
FirstKorder R
Double
first
order
Logistics
Soils
f
k1
k2
R
K
k
R
C0 (%)
K
R
f
k1
k
R
K
k
R
NP-NG
100
0.013
0.62
0.42
0.089
7.12 10112 0.89 5.76 101 5.84 102
0.88
2
0.88
NP-NG
100
0.013
0.62
0.42
0.089
7.12 11
1011 0.89 45.76 101 8 5.84 10
NP-G
100
0.009
0.51
0.32
0.103
2.69 10
0.81 5.00 10
6.09 10
0.60
8
0.60
NP-G
100
0.009
0.51
0.32
0.103
2.69 1011 0.81 5.00
104 7 6.09 10
4
P-NG
100
0.057
0.99
1.60
0.057
5.72 102
0.99
9.00

10
9.44

10
0.95
2
4
9.44 107
0.95
P-NG
100
0.057
0.99
1.60
0.057
5.722 10
0.99 9.00

10
P-G
100
0.054
0.98
1.00
0.054
5.35 10
0.98 3.77 105 3.72 5
108
0.93
3.72 108
0.93
P-G
100
0.054
0.98
1.00
0.054
5.35 102
0.98 3.77 10
Table 4. Constants and kinetic parameters of the biodegradation of paclobutrazol; NP-NG: soil without history and
without glycerol; NP-G: soil without history and with glycerol; P-NG: soil with history and without glycerol; P-G: soil
with history and with glycerol.

The kinetic modeling for the experiments with history (P) and with and without glycerol (G, NG) differed greatly from
experiments without history (NP) (Figure 8). Both followed a first-order kinetics, where the PBZ was degraded at a constant k rate of
0.0573 and 0.0538 (Table 4), respectively.

The kinetic modeling for the experiments with history (P) and with and without glycerol (G, NG) differed greatly from
experiments without history Biodegradation
(NP) (Figure 8).
followedaAfirst-order
kinetics,
where
thein PBZ
wasMango
degraded
at a constant
of Both
Paclobutrazol
Plant Growth
Regulator
Used
Irrigated
Orchard
Soil 97 k rate of
0.0573 and 0.0538 (Table 4), respectively.
http://dx.doi.org/10.5772/60818

120
Experimental

First Order

100

Double First Order

Logistic

PBZ (%)

80
60
40
20
0
0

14

28

42

56

70

Time (days)
120

Experimental
First Order

100

Double First Order

Logistic

PBZ (%)

80
60
40
20
0
0

14

28

42

56

70

Time (days)
Figure 8. Modeling kinetics of soil with history with and without glycerol. (A) Without glycerol; (B) with glycerol.

Vaz et al. [36] obtained excellent fits using double first-order kinetic and logistic models in
sterile soil and with addition of Pseudomonas spp., isolated from soil with no history. Mathe
matical models can help to identify high levels of toxic substances in soil or fruits of plants
treated with pesticides and indicate that such substances are able to be systematically moni
tored.
Paclobutrazol has been shown to be efficient in treating mango trees in semiarid conditions [9].
Because it needs to be applied directly into the soil, it is inconvenient since it remains and
affects future planting. Further, it is difficult to determine the dosage for each future use when
only empirical methods are used, as there may remain residue from the previous cycle of
application [8].
No quantification is done, nor is it always taken into consideration when deciding the dose.
Thus, the amount of paclobutrazol applied to the soil is not always appropriate, and risks of

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Biodegradation and Bioremediation of Polluted Systems - New Advances and Technologies

using doses above the recommended are great. The inflorescences on trees treated with high
doses are very compact [5], creating suitable conditions for the incidence of diseases and pests,
whose control is also hampered by the format of the panicles. Besides the phytosanitary
problems, excessive doses of PBZ can inhibit vegetative and floral sprouting longer than
desirable, requiring nitrate sprays to stimulate flowering. Thus, in addition to increasing the
cost of crop production, for all the reasons that have been mentioned, there is accumulation of
chemicals in the ground making the long-term consequences for the production system
unknown.
3.4. Paclobutrazol biodegradation in unsaturated soils with a history of applicationEffect
of bioaugmentation and biostimulation
PBZ biodegradation under the conditions of 24 factorial design is shown in Figure 9, where it
is possible to observe that biodegradation occurred under all conditions. Less biodegradation
was obtained in runs 1 and 9. In these runs, glycerol, the mineral medium, or inoculum had
not been added (control experiments). The biodegradation was possible probably due to action
of the native microbiota in the A and B soils since these soils had an application history [35].
Biodegradation was 79% and 60%, when only glycerol was added, in runs 2 and 10, respec
tively. However, biodegradation was about 85% when glycerol and the mineral medium were
added. The combination of bioaugmentation and biostimulation might be another promising
way to speed up the biodegradation of recalcitrant compounds. On the other hand, biodegra
dation reached 94% (runs 3 and 11) with the addition of the mineral medium only. The lack of
energy sources or electron acceptors or a lack of stimulation of the metabolic pathways
responsible for degradation can inhibit or delay the bioremediation [17, 35].
In runs 5 and 13, biodegradation reached only 38% and 29%, respectively. In these runs, only
the bacterial consortium was added. In runs with biostimulation and bioaugmentation
simultaneous (runs 6, 7, 8, 14, 15 and 16), a high level of biodegradation was achieved. Values
varied between 81% and 96%. Vaz et al. [37] studied the biodegradation of PBZ in two soils
under saturation conditions. A maximum value of 70% biodegradation within 28 days was
found in experiments where glycerol and the three strains of Pseudomonas spp. were used. In
the present study, higher values were found probably due to using soils with history of
application.
Glycerol (X1), mineral medium (X2), and inoculum (X3) were significant factors in the biode
gradation (Figure 10). The addition of glycerol, mineral medium, and inoculum increased the
biodegradation, regardless of soil used. However, higher biodegradation values (94% to 96%
biodegradation for the runs 11, 14, 15, and 16) were found in Soil B. In these runs, biostimulation
was applied by the addition of glycerol and/or the mineral medium. Among the main factors,
the most significant was the mineral medium followed by glycerol and inoculum. Both soils
(A and B) used in research were different with respect to the percentages of sand, silt, and clay.
The factorial design, however, did not differentiate significantly among these soil types.
In relation to the factors of interaction, only those factors involving the mineral medium (X2)
with glycerol (X1) or the inoculum (X3): X1X2 and X2X3, respectively, were significant. These

Biodegradation of Paclobutrazol A Plant Growth Regulator Used in Irrigated Mango Orchard Soil
http://dx.doi.org/10.5772/60818

4.0

110
100
90

3.0

80

2.5

70
60

2.0

50

1.5

40

1.0

30
20

0.5
0.0

Biodegradation (%)

Residual PBZ (g/g)

3.5

Biodegradation

Residual PBZ

10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Run
Figure 9. Biodegradation and PBZ residual for each run of factorial design.

Figure 10. Analysis of significance of independent factors presented as standardized Pareto charts of biodegradation.

effects were negative, indicating that the addition of the mineral medium with glycerol or the
inoculum did not favor the biodegradation of paclobutrazol. The addition of glycerol and
mineral medium was more significant (31.31) than the addition of the mineral medium and
inoculum (8.31).

99

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Biodegradation and Bioremediation of Polluted Systems - New Advances and Technologies

By applying multiple regression analysis on the experimental data, a polynomial model in

coded unit explains the role of each factor and its second-order interactions (Equation 2). The
negative and positive signs of regression coefficients indicate the antagonistic effect and
synergistic effect of each variable, respectively:
Y = 70.78 + 12.42X1 + 20.65X2 + 6.04X3 - 15.66X1X2 - 4.16X2 X3

(2)

where Y is biodegradation (%) and Xi is level factor in coded unit (+1 or 1), i = 14 for four
factors. Equation (2) demonstrates that glycerol (X1), the mineral medium (X2), and the
inoculum (X3) were responsible for the biodegradation observed. There are only two signifi
cant interaction effects (X1X2 and X2X3). This indicates the additional synergistic effect of these
factors. The analysis of variance (ANOVA) was applied to the experimental data and simulated
by the empirical model data. The F test was calculated as the ratio between the mean square
of regression and the residual mean square. The high value of F (137.63) test and the low p
value (0.012405) indicated the significance of the regression.
Figure 11 shows the Log UFCg1 observed in all runs of the factorial design 24 at zero and 40
days. The Log UFCg1 decreased in runs 1 and 9 due to depletion of nutrients in the soils since
only sterilized water was added (control runs). In runs 5, 6, 7, 8, 13, 14, 15, and 16, there was
also a decrease in the Log UFCg1 since in these runs bioaugmentation was performed and
probably the nutrients were not sufficient. On the other hand, in the runs with the addition of
glycerol and/or the mineral medium (2, 3, 4, 10, 11, and 12), there was an increase in Log
UFCg1, independent of the soil used.

Figure 11. Colony forming unity in each run of factorial design.

Biodegradation of Paclobutrazol A Plant Growth Regulator Used in Irrigated Mango Orchard Soil
http://dx.doi.org/10.5772/60818

101

3.5. Characterization of samples of biodegradation using FTIR spectroscopy

Figures 12 and 13 show an infrared analysis of PBZ dissolved in distilled water and only
methanol, respectively. The solvent used in the preparation of samples was methanol.
Comparing the spectra of PBZ and methanol, there is a band at 1650 cm1 only for spectra of
PBZ.
1.2
1.1
1.0

Absorbance

0.9
0.8
0.7

1651

0.6
0.5
0.4
0.3

PBZ

0.2
500

1000

1500

2000

2500

3000

3500

4000

4500

-1

Wave number (cm )

Figure 12. Fourier transformation infrared (FTIR) analysis of PBZ dissolved in distilled water.

Figure 12. Fourier transformation infrared (FTIR) analysis of PBZ dissolved in distilled water.
1.2

1.2
1.1

1.1
1.0

1.0
0.9

Absorbance

Absorbance

0.9
0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.4

0.3

MeOH
MeOH

0.3

0.2

0.2 500
500

1000

1000

1500

1500

2000

2000

2500

2500

3000

3000

3500

3500

4000

4000

4500

4500

-1

(cm-1 )

Wave
Wave number
number (cm )

Figure 13. Fourier

infrared
(FTIR)
Figure 13. Fourier transformation
infrared transformation
(FTIR) analysis of
methanol.

analysis of methanol.

Samples of soil E1 (only PBZ) and E2 (PBZ and glycerol) 0th day and 70th day of incubation are shown in Figures 14 and 15. A
comparison of FTIR spectra of samples after biodegradation (70th day) and before biodegradation (zero day) revealed the lack of the
band at 1650 cm1 corresponding to C=C and C=N stretching in the benzene and 1,2,4-triazole rings, respectively, which are
observed in the structure of paclobutrazol (Figure 1). Therefore, examination of this particular band confirmed the reduction of
paclobutrazol concentration in samples soils after 70 days of biodegradation.

MeOH
0.2
500

1000

1500

2000

2500

3000

3500

4000

4500

-1

102

Wave number (cm )

Biodegradation and Bioremediation of Polluted Systems - New Advances and Technologies

Figure 13. Fourier transformation infrared (FTIR) analysis of methanol.

Samples of soil E1 (only PBZ) and E2 (PBZ and glycerol) 0th day and 70th day of incubation
are shown in Figures 14 and 15. A comparison of FTIR spectra of samples after biodegradation
1
Samples of (70th
soil E1day)
(only
PBZ)
and biodegradation
E2 (PBZ and glycerol)
0th day
and 70th
day
of incubation
areatshown
in Figures
14 and 15
and
before
(zero day)
revealed
the
lack
of the band
1650 cm
omparison of FTIR
spectra
of
samples
after
biodegradation
(70th
day)
and
before
biodegradation
(zero
day)
revealed
the lack of
corresponding
to
C=C
and
C=N
stretching
in
the
benzene
and
1,2,4-triazole
rings,
respectively,
1
and at 1650 cm
corresponding
and C=Nofstretching
in the
benzene
and 1,2,4-triazole
rings,ofrespectively,
which
which
are observedtoinC=C
the structure
paclobutrazol
(Figure
1). Therefore,
examination
this
bserved in the structure of paclobutrazol (Figure 1). Therefore, examination of this particular band confirmed the reduction
particular band confirmed the reduction of paclobutrazol concentration in samples soils after
aclobutrazol concentration in samples soils after 70 days of biodegradation.
70 days of biodegradation.
1.2
1.1
1.0

Absorbance

0.9

1678

0.8
0.7
0.6
0.5
0.4

E1-0 days
E1-70 days

0.3
0.2
500

1000

1500

2000

2500

3000

3500

4000

4500

-1

Wave number (cm )

Figure 14. Fourier transformation infrared (FTIR) analysis of metabolites extracted with methanol before biodegradati
PBZ in experiments
without
(A)ofglycerol
as carbon
additional.
Figure 14. Fourieroftransformation
infrared (FTIR)
analysis
metabolites
extractedsource
with methanol
before biodegrada
tion of PBZ in experiments without (A) glycerol as carbon source additional.

Jackson et al. [33] observed 79% biodegradation after 39 days of incubation, possibly due
mineralization of the [14C]-label to CO2. Since the [14C]-label was located in the chlorobenzene
ring of paclobutrazol, the observed loss of labeled carbon indicated some degree of degrada
tion of this functional group by the Pseudomonas isolate. These authors concluded that PBZ is
at least partly degradable by bacteria (Pseudomonas and Alcaligenes) in pure culture, with the
chlorobenzene ring being catabolized but the 1,2,4-triazole ring was found to be resistant to
attack.
3.6. Phytotoxicity studies
Phytoxicity was evaluated based on the germination index, shown in Table 5. According
Paradelo et al. [38], phytoxicity between 50% and 80% is considered moderate, while above
80% is absent. These results indicated that the concentration of PBZ (4 g/g) used is not toxic
since index of germination was 83.1%. On the other hand, when was used in PBZ and glycerol,
this index decreased at approximately 3%. The metabolites produced during biodegradation

Figure 14. Fourier transformation infrared (FTIR) analysis of metabolites extracted with methanol before biodegradation
Biodegradation of Paclobutrazol A Plant Growth Regulator Used in Irrigated Mango Orchard Soil 103
of PBZ in experiments without (A) glycerol as carbon source additional.
http://dx.doi.org/10.5772/60818

1.2
1.1
1.0

Absorbance

0.9

1674

0.8
0.7
0.6
0.5
0.4

E2-0 days
E2-70 days

0.3
0.2
500

1000

1500

2000

2500

3000

3500

4000

4500

-1

Wave number (cm )

Figure 15.
15. Fourier
infrared
(FTIR)
analysis
of metabolites
extracted
with methanol
after theafter
biodegra
Figure
Fouriertransformation
transformation
infrared
(FTIR)
analysis
of metabolites
extracted
with methanol
the biodegradation
dation of PBZ in experiments
withinglycerol
as carbon
source
additional.
of PBZ
experiments
with
glycerol
as carbon source additional.

did not show phytotoxicity and increased index of germination, independently of the addition

Jackson
al. [33] observed
79%
biodegradation
days of without
incubation,
possibly of
due
mineralization of the [14C]-label to
of etglycerol.
However,
higher
increase after
was 39
observed
addition
glycerol.
14
CO2. Since the [ C]-label was located in the chlorobenzene ring of paclobutrazol, the observed loss of labeled carbon indicated some
Sample

Germination index (%)

Soil before biodegradation experiments (with PBZ 4 g/g)

83.1

Soil before biodegradation experiments (with PBZ 4 g/g and glycerol 2.4 mg/g)

80.7

Extracted metabolites after biodegradation experiments in the soil with PBZ

96.6

Extracted metabolites after biodegradation experiments in the soil with PBZ and glycerol

85.2

Table 5. Phytotoxicity studies of PBZ and metabolites produced after biodegradation (70 days) in experiments with
unsaturated soil with and without addition of glycerol.

4. Conclusions
The Pseudomonas isolated presents a great potential of paclobutrazol biodegradation. The
bacterial growth and the paclobutrazol biodegradation were higher in the experiments using
paclobutrazol and glycerol as carbon sources. These results indicate that glycerol can be
considered a carbon source that stimulates the growth and it does not inhibit the paclobutrazol
degradation by Pseudomonas spp.
The biodegradation of PBZ in unsaturated soils was more efficient when soil samples with a
history of application of PBZ were used. We concluded that this soil bacterium is better adapted

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Biodegradation and Bioremediation of Polluted Systems - New Advances and Technologies

for the degradation of the compound. Mathematical models can help to identify high levels of
toxic substances in soil treated with pesticides and indicate that such substances should be
systematically monitored.
Soils microorganisms were able to degrade PBZ (control experiments: 1 and 2 of the factorial
design), but only with a low increase in biodegradation (<25%). Simultaneous bioaugmenta
tion and biostimulation is not the best strategy. The highest number of applications of PBZ
favored biodegradation.
FTIR spectra indicate the biodegradation of PBZ aromatic rings. This probably happens
because of the biodiversity of the microbiotics in the soil. This is different from research
undertaken with a culture immersed in a mineral medium and a mixed Pseudonomas culture,
where only benzene chlorate has been degraded. Concentrations of 4 g/g of PBZ and 2.4 mg/
g of glycerol were not phytotoxic, and the biodegradation products increased the germination
index.

Acknowledgements
The authors acknowledge the financial support from Fundao de Amparo Cincia e
Tecnologia do Estado de Pernambuco (FACEPE) and Coordenao de Aperfeioamento de
Pessoal de Nvel Superior (CAPES).

Author details
Fernanda Vaz1, Ednaldo Santos-Filho1, Suzyane Silva1, Silvany Arajo2,
Thatiana Stamford-Arnaud1, Andrea Bandeira1, Ana Cristina Brasileiro-Vidal2,
Newton Pereira Stamford3, Maria Aparecida Mouco4 and Ester Gouveia1*
*Address all correspondence to: estergouveia@gmail.com
1 Department of Antibiotics, Federal University of Pernambuco, Recife-PE, Brazil
2 Department of Genetics, Federal University of Pernambuco, Recife-PE, Brazil
3 Department of Agronomy, Rural Federal University of Pernambuco, Recife-PE, Brazil
4 Brazilian Agricultural Research Corporation, Petrolina-PE, Brazil

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