Synthesis of thiodiazole copper microcapsules and release behavior of inhibiting R. solanacearum

Article abstract of DOI:10.1039/c3ra45744c

Microcapsules are one of the most useful devices to reduce the dosage of pesticides and prolong the duration of the active ingredient in target crops. In this study, thiadiazole copper (TDC) microcapsules were synthesized by in situ polymerization using p

Full text of DOI:10.1039/c3ra45744c

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Synthesis of thiodiazole copper microcapsules and
release behavior of inhibiting R. solanacearum†
Cite this: RSC Adv., 2014, 4, 4478
Chao Feng,* Chengsheng Zhang, Fanyu Kong and Jing Wang
Microcapsules are one of the most useful devices to reduce the dosage of pesticides and prolong the
duration of the active ingredient in target crops. In this study, thiadiazole copper (TDC) microcapsules
were synthesized by in situ polymerization using poly(urea-formaldehyde) and characterized by field
emission scanning electron microscopy, laser diffraction analysis, 3D optical microscopy,
thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy. The effects of pH and
temperature on the release of TDC were characterized by UV-visible absorption spectroscopy. The
relationship between the micromechanical behavior of the microcapsules and release of TDC was
studied by nanoindentation tests. The particle size distributions of the microcapsules (10–530 mm) were
ꢀ
controlled by different reaction parameters. The microcapsules were stable below 220 C, as determined
by TGA. The release kinetics indicated that the higher the temperature, the faster the release rate. The
TDC microcapsules were sensitive to pH, and the fastest release rate was observed at pH 4.0. The
maximum load, hardness, and Young's modulus under the same displacement conditions decreased
during the release. Water swelling was the major reason for TDC release from the microcapsules. The
pot experiments confirmed that the microcapsules exhibited long-term sustained release of TDC,
thereby protecting the tobacco from R. solanacearum.
Received 11th October 2013
Accepted 5th December 2013
DOI: 10.1039/c3ra45744c
www.rsc.org/advances
some of the key requirements of green chemistry to the
environment.
Tobacco bacterial wilt, caused by a soil-borne pathogen R. sol-
Introduction
16
Pesticide chemicals play an important role in the control of
pests and weeds diseases in agriculture and forestry; however, anacearum, is one of the most serious diseases affecting worldwide
17
environmental pollution, residual toxicity, and human diseases tobacco production. Moreover, thiodiazole copper (TDC), a new
18
are also caused by pesticides. Moreover, chemical fungicide effective for living bacterial disease, is widely used in
manufacturing involves large consumption of energy and China. TDC is safe for crops, shes, birds, honeybee, silkworms,
1,2
generates hazardous chemical waste.
dosage of pesticides and prolonged duration of active ingredi- environmental pollution. Therefore, TDC could be micro-
Therefore, reduced human beings, animals, and benecial insects, and does not cause
ents in target crops are of signicant interest.
encapsuled by polymers for control release applications.
One of the effective ways to solve these problems is to control
Moreover, nanoindentation is a suitable direct method to
obtain micromechanical properties of polymeric microcap-
3
the release of active ingredients from polymeric microcapsules,
4
,5–8
which is of signicant interest in drug delivery,
release,
fragrance sules, such as maximum load, hardness, and Young's modulus,
9
–11
12
13,14
19
food preservation, and self-healing materials.
compared to the indirect method (osmotic pressure method).
20
The utility of polymeric microcapsules for efficient cargo The Oliver–Pharr method has been widely used for the
storage stems from their ability to deliver chemicals (e.g., measurement of elastic modulus and hardness via nano-
15
pesticides) “just in time” to control the pests. Polymeric indentation in characterizing the mechanical behavior of
21–24
25
microcapsules protect the active ingredients from light and materials at small-scale.
Lee et al. introduced the nano-
other environmental factors. Moreover, polymeric microcap- indentation test to characterize the micromechanical behavior
sules effectively reduce chemical exposure, providing safety for of poly(melamine-formaldehyde) (PMF), as a polymeric shell
the operators. Therefore, polymeric microcapsules meet material. The PMF of self-healing microcapsules was shown to
behave as a viscoelastic plastic material. Furthermore, Wang
26
et al. introduced oxygen plasma treated carbon nanotubes to
Tobacco Integrated Pest Management of China Tobacco, Tobacco Research Institute of
Chinese Academy of Agricultural Sciences, Qingdao 266101, PR China. E-mail: poly(urea-formaldehyde) (PUF) microcapsules to enhance the
fengchao020511@163.com; Fax: +86 532 88701012; Tel: +86 532 88703236
elasticity of the microcapsule shells during the polymerization.
The improvement in micromechanical behavior of the micro-
capsule shells was conrmed by nanoindentation tests.
†
Electronic supplementary information (ESI) available: Additional UV-visible
absorption spectra and grade investigating method of tobacco disease are
provided. See DOI: 10.1039/c3ra45744c
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To the best of our knowledge, the synthesis of TDC micro-
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The size distributions of microcapsules were obtained using
capsules using PUF through in situ polymerization and the a Mastersizer 2000 Version 5.54, Malvern Instruments Ltd.
micromechanical behavior of microcapsule shells in the release Fourier transform infrared spectroscopy (FT-IR) tests were
process have not yet been reported. The objective of this research performed using a Thermo Scientic Nicolet iS10, Thermo
projectwastosynthesizeTDCmicrocapsulesthatcouldminimize Fisher Scientic Inc.
the initial release of the TDC, and instead provide a slow and
Nanoindentation tests were performed using an Agilent
sustained release to protect the tobacco from R. solanacearum. G200 NanoIndenter (Agilent Technologies, Inc., Chandler, AZ)
TDC was prepared via in situ polymerization, and the effects of pH with a three-sided pyramid diamond Berkovich indenter. The
ꢀ
and temperature on the release kinetics were studied. The indenter has a normal angle of 65.3 between the tip axis and
changes in the micromechanical behavior of TDC microcapsules the faces of the triangular pyramid.
was analyzed by nanoindentation tests. The trends in the
The release characterization of TDC microcapsules were
maximum load, hardness, and Young's modulus in the release examined by UV-visible transmittance spectra using a UV-3600
process were discussed. The disease index and control effect of Shimadzu.
different pesticide treatments to protect the tobacco from R. sol-
anacearum were studied by the pot experiments.
Microcapsule preparation
The microcapsules containing TDC were synthesized by in situ
polymerization of urea and formaldehyde using a modied
Materials and methods
Materials
27,28
literature process,
as shown in Fig. 1. TDC was completely
dissolved in SOAME, and then the mixture (15.0% w/v) was
slowly added to 35 mL of 1.5% sodium dodecyl benzenesulfo-
nate, urea, resorcinol, and ammonium chloride at room
temperature, and stirred for 30 min. One drop of ultra-
hydrophobic agent, either octane or hexadecane, was added to
the TDC mixture, as a costabilizer to decrease Ostwald
TDC and 20% TDC suspension concentrate (TDC SC) were
obtained from Zhejiang Longwan Chemicals Co., Ltd. Soybean
oil fatty acid methyl esters (SOAME) were obtained from West
Asia Chemical Corporation.
Tobacco seeds of Honghua Dajinyuan variety, which was
susceptible to tobacco bacterial wilt disease, were obtained
from Tobacco Research Institute of Chinese Academy of Agri-
cultural Sciences.
28
ripening and increase the hydrophobicity of the inner phase.
Then, formalin (37% formaldehyde) was added in the same
26
ratio as described previously by Wang et al. And, the pH was
The water used in all the experiments was produced by a
Millipore Milli-Q Plus 185 purication system and has a resis-
tivity level higher than 18.1 MU cm. Hydrochloric acid, soda lye,
and sodium sulde nonahydrate were obtained from Beijing
Chemical Works, CP. Sodium dodecyl benzene sulfonate, urea,
adjusted to 3.50 with sodium hydroxide. The temperature of
ꢀ
ꢀ
ꢁ
1
bath was slowly raised to 60 C at a rate of 1 C min . Aer 4 h
of continuous agitation, the heating and mechanical agitation
were stopped. Then, the reaction mixture with the polymerized
microcapsules were repeatedly washed with ethanol and water
to remove the impurities.
37 wt% formaldehyde solution, ammonium chloride, resor-
cinol, rhodamine B, and 1-octanol were obtained from Sigma
Aldrich. Rhodamine B was used to probe the shell. Fluorol
Green Golden 084 as a uorescence probe for the core was
obtained from Kunshan Haite Plastic Pigment Co., Ltd. All
chemicals were used as received.
Characterization
The morphology, physical properties, and stability of polymeric
microcapsules were characterized prior to embedding in the soil
for crop protection. The shell wall integrity and the morphology
of the microcapsules were observed by eld emission scanning
electron microscopy (FESEM) and laser diffraction analysis
(LDA). The FESEM images of the polymeric microcapsules were
acquired using a Hitachi S-4800 FESEM microscope. The optical
microscopy images of the polymeric microcapsules were recor-
ded using a VHX-1000, Keyence Co., Ltd.
The core–shell structure was obtained by a confocal laser
scanning microscope, Fluo ViewTM FV1000, Olympus Corp.
Fluorol Green Golden 084 was excited using a diode-pumped
solid-state laser with a wavelength of 488 nm; rhodamine B was
excited using a helium-neon laser with a wavelength of 543 nm.
The TGA data of the polymeric microcapsules were examined
using a TGA Q5000, TA Instrument.
Fig.
1 Flow chart of encapsulation method for preparing PUF
microcapsules containing TDC.
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Determination of core content of microcapsule
The core content of TDC microcapsule was determined by
29
extracting method with acetone as extracting solvent. The TDC
microcapsules were grinded, collected and washed with acetone
several times, then dried at room temperature. Knowing the
initial weight of microcapsules (M
shell of microcapsules (M ), the wall shell content (W
content (W ) of microcapsules were calculated as
i
), the weight of residual wall
r
s
) and core
c
M
r
W
s
¼
ꢂ 100%
(1)
(2)
M
i
W ¼ 1 ꢁ W
c
s
Greenhouse pot experiments
The pot experiments were conducted using a modied process
30
by Liu et al. in a greenhouse in Qingdao, Shan Dong province.
The surface sterilization were used to treat the tobacco seeds
with household bleach (2% NaOCl) for 6 min. Then, the seeds
were rinsed six times in Milli-Q water, and sown in a nursery
31
mixture of 3 : 3 : 4 of vermiculite, perlite, and turf. The
resulting medium was sterilized at 121 C for 2 h and placed
Fig. 2 FE-SEM images and size distributions of microcapsules under
different agitation rates. (A) and (B) under 500 rpm; (C) and (D) under
ꢀ
into a sterilized plastic tray. The plastic tray was placed over 1000 rpm; (E) and (F) under 1400 rpm.
ꢀ
distilled water and kept at 28–30 C and 60% relative humidity
for approximately 60 days. Appropriate fertilizers were applied
during the entire seedling stage.
As the agitation rate was increased from 500 to 1000 rpm, the
diameter of the microcapsules decreased to 50–400 mm, with a
mean diameter of 130 mm (Fig. 2D). Further, as the agitation rate
was increased from 1000 to 1400 rpm, the diameter of the
Infested soil inoculated by R. solanacearum
R. solanacearum was inoculated in a lysogeny broth medium microcapsules decreased to 15–105 mm containing well formed
ꢀ
and shaken on a rotary shaker for 24 h at 30 C and 150 rpm. shell walls, andwith a mean diameterof44 mm (Fig. 2F). As strong
The bacterial pellets were suspended in sterilized water and shear forces were generated under high agitation rate, large
35
mixed thoroughly with soils obtained from a eld located in droplets were broken up into small ones. Thus, the increase in
Fujian province. The soil properties are as follows: pH 6.50, the agitation rate resulted in smaller-diameter microcapsules.
ꢁ
1
ꢁ1
ꢁ1
1
4.11 g kg of total C, 2.02 g kg of total N, 1.24 g kg of total
The core contents of TDC microcapsules were 85.1, 78.7, and
P, 12.35 mg kg of available P, 10% of sand, 50% of silt, and 65.4 under 500 rpm, 1000 rpm, and 1400 rpm, respectively. The
0% of clay. The concentration of R. solanacearum was adjusted core contents of microcapsules prepared at lower agitation rate
ꢁ1
4
ꢁ
1
32
to 10 cfu g dry soil.
were higher than that of microcapsules prepared at higher
29
agitation rate. The weight fraction of core material in larger
microcapsule was relatively higher. Whereas that in smaller
microcapsule was relatively smaller.
Results and discussion
Microcapsule size
Confocal uorescence micrograph of TDC microcapsule was
shown in Fig. 3. The core and the shell were labeled with Fluorol
Green Golden 084 (green) and rhodamine B (red), respectively.
In the uorescence intensity cross-section prole (Fig. 3B), the
intensity of the rhodamine B (red) had two side peaks that
marked the shell. And, the intensity of the Fluorol Green Golden
The sizes of the microcapsules were analyzed by LDA. The sizes
of the microcapsules were controlled by the agitation rate in the
polymerization process.
33
The FE-SEM images of the TDC microcapsules under 500,
000, and 1400 rpm agitation rates are shown in Fig. 2A, C and
1
084 (green) concentrated in the middle of the yellow line that
E, respectively.
marked the core. Thus, confocal uorescence micrograph and
the uorescence intensity distribution demonstrated that the
microcapsules had a core–shell structure.
The TDC microcapsules obtained under 500 rpm agitation
rate had a diameter ranging from 85–530 mm, with a mean
diameter of 283 mm (Fig. 2B). In the polymerization, shear
forces generated by agitation could tear large oil droplets to
small ones. The equilibrium between interfacial tension of the
discrete oil droplets and shear forces depended on the agitation
Thermal stability of microcapsules
34
rate. Interfacial tension dominated at low agitation rate, and The thermal stability and overall quality of the TDC microcap-
dispersed droplets remained large.
sule were evaluated by TGA. The samples were examined using a
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ꢁ
1
peak at 1507.10 cm in the spectrum for TDC is attributed to
ꢁ1
cyano (C^N) groups. The peak at 1604.93 cm may be attrib-
uted to S–C]N groups (overlapped with NH peak) (Fig. 5a).
2
ꢁ
ꢁ1
Further, carboxylate (COO , 1455.79 cm ) and carbonyl (C^O,
746.39 cm ) peaks for SOAME were observed (Fig. 5b). These
ꢁ
1
1
characteristic peaks were also observed in the FT-IR spectra of
microcapsules, indicating the presence of TDC as the core
material in the synthesized microcapsules (Fig. 5c). Further-
ꢁ
1
more, peaks for urea group (CO–NH) at 1628.12 cm (over-
Fig. 3 (A) Confocal fluorescence micrograph of TDC microcapsule.
ꢁ1
lapped with C^O peak), amine group (N–H) at 1560.82 cm
,
(
B) Fluorescence intensity profile along the yellow line across the
ꢁ
1
29
and cyano group (C–N) at 1434 cm
for the shells were
microcapsule in (A).
observed (Fig. 5d). The characteristic peaks of shells are also
present in the microcapsules, as shown in Fig. 5c. Thus, the
comparison of FT-IR spectra conrmed that the synthesized
microcapsules contain TDC as the core material.
ꢁ
1
TGA Q5000 apparatus under a nitrogen ow (30 mL min ) at a
ꢀ
ꢁ1
ꢀ
heating rate of 10 C min from 25 to 550 C. The samples
(4–10 mg) were placed in a platinum crucible.
Fig. 4 shows a comparison of TGA curves between the TDC
microcapsules (with a stable shell wall) and TDC. All samples lost
mass in a single step. The onset points of mass loss of the TDC,
Characterization of TDC-release kinetics
Most of the prepared TDC microcapsules are circular in shape
and without any cracks on the surface (Fig. 6A). During the
immersion in soil, outer mechanical force could break the
shells of microcapsules, thereby forming microcracks on their
surfaces (Fig. 6B) and release the TDC from their core.
SOAME, TDC microcapsules, and microcapsule shells were 228,
ꢀ
1
65, 165, and 210 C, respectively. The SOAME and TDC micro-
capsules showed a similar trend in mass loss under the same
ꢀ
heating rate. The microcapsules were stable below 164
C
(
Fig. 4b). The TGA of the microcapsules, prepared by the opti-
Because environmental factors such as temperature
mized process (1400 rpm agitation rate) showed <5% weight loss
ꢀ
(
20–40 C) and soil acidity (pH 4.0–7.0) would affect the TDC
ꢀ
before 100 C (residual water). Then, a sharp weight loss
microcapsules in the actual soil application trials, these factors
were simulated for the release kinetics test.
The presence of TDC could not be directly analyzed by UV-
visible absorption spectra. Therefore, it was indirectly analyzed
ꢀ
occurred between 160 and 240 C, corresponding to TDC ther-
ꢀ
molysis (Fig. 4a) and loss of SOAME (boiling point 165 C)
(
Fig. 4d). Thus, the microcapsules were stable untill the release of
vaporized TDC. The TDC microcapsules were as stable as the PUF
microcapsules containing epoxy resins, as reported by Cosco
et al. The mass fraction of the TDC microcapsules and micro-
2 2
by adding 0.1 M sodium sulde nonahydrate (Na S$9H O) to
TDC, producing UV-visible active tizanidine (AMT). The UV-
visible absorption spectra of released AMT was recorded
between 270 and 350 nm using a UV-3600 Shimadzu with a slit
36
capsule shells were 10.6% and 7.9%, respectively, calculated by
26
Wang et al. The residue consisted of the reacted shell materials.
width of 1.0 nm, and 0.1 M Na
ence material.
2
S$9H
2
O was used as the refer-
This phenomenon indicates that the TDC from the microcap-
ꢀ
sules were completely lost in the heating process below 321 C.
The reaction between TDC and Na
2
S$9H O producing AMT
2
is shown in Fig. 7. AMT was used to indirectly measure the
concentrations of released TDC.
FT-IR spectra
The TDC microcapsules are light green in colour, as shown
in Fig. 8A, while, the hydrolysate copper sulde (CuS), produced
The FT-IR spectra of TDC, SOAME, microcapsules, and micro-
capsule shells are shown in Fig. 5. The strong transmittance
Fig. 4 Curves for mass loss of TDC (a), TDC microcapsules (b),
microcapsule shells (c), and SOAME (d) over the temperature range of Fig. 5 FT-IR spectra of TDC (a), SOAME (b), microcapsules (c), and
ꢀ
2
5 to 550 C.
microcapsule shells (d).
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Fig. 6 Optical microscopy images of TDC microcapsules (A) and
microcapsules with cracks on the surface of shell after outer mechanical
force treatment (B), recorded by depth-from-defocus method.
2 2
from the reaction between TDC and Na S$9H O, is black in
colour. Because the rough surfaces of the microcapsules
adsorbed the CuS, they became gray in colour, as shown in
Fig. 8B.
UV-visible spectra is a useful technique for determining the
37,38
absorption wavelength of the UV-active products.
The
released concentrations of AMT at different immersion times
were obtained from its UV-visible spectra. The increasing trend
of the absorption peak at 290.2 nm indicates that TDC contin-
ually releases into the water (Fig. S1†).
The common semiempirical exponential equation were used
to analyze the data obtained from TDC release experiments, and
to evaluate the inuence of pH and temperature on the release
3,12
kinetics as follows:
M
t
n
¼
Kt þ C
(3)
M
N
where, M
t
is the amount of TDC released at time t, M
N
is the
total amount of TDC in the microcapsules. K is the release
constant, C is a constant accounting for the initial burst effect,
Fig. 8 Optical microscopy images of prepared microcapsules (A), and
microcapsules released after treatment with 0.1 M Na S$9H O for 3
2 2
and n is the diffusional exponent describing the mechanism of days (B).
39
release depending on the geometry of the system. R is the
correlation coefficient that incorporates the matrix properties,
and t50 is the time taken to release 50% of TDC.
Using the data obtained for AMT, the corresponding TDC
The concentration gradient of a released active ingredient is release proles at various pH values and temperatures are
the major driving force for a Fickian diffusion system whose shown in Fig. 9A and B, respectively. The TDC release concen-
diffusion parameter is close to 0.50. While, the erosion of trations at different pH values show the same trend. The release
particles is the dominant driving force for a degradation concentrations increase rapidly until 90 h. Aer that, the release
controlled-release system whose diffusion parameter is close to concentrations increase slowly, and reaches a steady state
1.0. In the case of spheres, n value should be corrected to 0.43 concentration. However, the t50 values of different pH samples
and 0.85 corresponding to Fickian diffusion and degradation in Table 1 are 9.578, 5.494, 4.403, and 8.870 h for pH 4, 5, 6, and
40
controlled-release systems, respectively. As evidenced in 7, respectively, which are all below 10 h. The t50 values at pH 4
Table 1, the TDC release from the microcapsules was controlled
by Fickian diffusion.
The correlation coefficient (R) for all formulations is >0.97, Table 1 Parameters characterizing fitting of the model equation to the
TDC release data (P1, P2, P3, T1, T2, and T3 correspond to samples at
pH ¼ 4, pH ¼ 5, pH ¼ 6, pH ¼ 7, 20 C, 30 C, and 40 C, respectively)
and indicates an excellent correlation of TDC release prole
from the microcapsules, as shown by using eqn (3).
ꢀ
ꢀ
ꢀ
Sample
K
n
C
R
t50 (h)
P1
P2
P3
P4
T1
0.131
0.452
0.473
0.199
0.080
0.036
0.540
0.353
0.173
0.168
0.272
0.462
0.553
0.143
0.210
0.989
0.986
0.987
0.992
0.983
0.970
0.989
9.578
5.494
4.403
8.870
19.562
9.362
5.782
ꢁ0.106
ꢁ0.093
0.139
0.182
0.376
Fig. 7 Reaction between TDC and Na
2
S$9H
2
O forming tizanidine T2
T3
(
AMT), which is detected by UV-visible absorption spectra.
ꢁ0.195
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and 7 are larger than those of pH 5 and 6, indicating that the Nanoindentation test
microcapsules at pH 4 and 7 show long-term sustained release
The microcapsules need to be separated from one another for
nanoindentation test. Thus, the microcapsules were dispersed
in ethanol, and carefully separated on a smooth surface of glass
slide. Then, the back of the glass slide was strongly adhered to a
stainless steel sheet by hot melt adhesive. Moreover, the
continuous stiffness measurement (CSM) mode was choosen
of TDC. Thus, the TDC microcapsules are sensitive to pH, as
indicated by the release kinetic tests. However, the change in
pH did not signicantly change the value of n.
The shell made from PUF has a network structure at molec-
ular level. Thus, water could penetrate into the shell and swell the
19,41
shell of the microcapsule in the solution.
On different pH
ꢁ1
for nanoindentation test at a constant rate of 0.05 s up to 3
conditions, crosslinking reaction occurred on the shell of the
microcapsule. However, the effects of the changes in the pH on
the shell has not been clearly interpreted. For TDC microcap-
sules, water swelling was the major reason for active ingredient
release. The pH value inuenced the crosslinking reaction of the
shell, and had synergistic effect with water swelling.
ꢀ
mm depth. Tests were performed at 25 C. The holding time of
indentation was 10 s. The curve was measured three times with
the same sample on the distinct surface regions at each
indentation load. The hardness and Young's modulus values
were averaged over three indents. The microcapsule sizes are in
the range 15–105 mm with a mean diameter of ꢃ55 mm (Fig. 8A).
In the CSM technique, cycles of indentation, each of which
consists of continuous incremental loading and partial unloading,
In the temperature effect test, the t50 value for sample T1 is
signicantly larger than T2 and T3, indicating that the
microcapsules at lower temperature show long-term sustained
release of TDC. The t50 value changed from 19.562 to 5.782 h
42
are carried out until a nal desired depth is obtained. Fig. 10A
shows the typical loading–unloading curve of the TDC microcap-
sules at different immersion times. This behavior is similar to that
ꢀ
as the temperature was raised from 20 to 40 C. Higher
temperature could accelerate the rate of Fickian diffusion
process, resulting in higher release rate. Therefore, as expec-
ted, temperature is a key parameter affecting the release
process. The laboratory tests were conducted in water, and it is
expected that the release rate would be much slower in the
actual soil environment.
43
44
of viscoelastic-plastic polymer and polymer lms, respectively.
The “maximum load” could characterize the ability of
45
microcapsules to endure outer stress. The maximum load of
TDC microcapsules during the holding time was 29.192 mN
aer 30 min immersion, which is the largest load in the release
process. This result indicates that the micromechanical
behavior of the microcapsules in the initial immersion period is
nearly similar to that of prepared microcapsules.
Then, in the continuous immersion release process, the
maximum load of the microcapsules were 21.883, 9.734, 8.291,
and 4.829 mN, corresponding to 34, 90, 228, and 480 h
immersion, respectively. This nding proves that the immer-
sion process weakens the microcapsule's ability to endure outer
stress. These characteristics may correspond to the nature of
the rough surface of the microcapsule shell, which become
more viscoelastic aer absorbing the water. Thus, the shell of
microcapsules became more easily damaged under the inu-
ence of outer stress. The cracks increased in size and number on
the shell surfaces, release more core materials (TDC).
Hardness is dened as a measurement of the material's
resistance to local plastic deformation. Thus, the hardness, H,
was calculated from the data from the loading curve and was
dened as the maximum load, Pmax, divided by the contact
indentation area, A.
P
max
H ¼
(4)
A
Young's modulus (E) was calculated from the following
equation:
2
2
1
1 ꢁ v
1 ꢁ n
i
¼
þ
(5)
E
r
E
E
i
where, E is the reduced modulus, E and n are Young's modulus
r
i i
and Poisson's ratio for the specimen, and E and n are the same
46–48
parameters for the indenter.
Young's modulus-displacement and hardness-displacement
curves of the TDC microcapsules tested by using CSM at
Fig. 9 TDC release profiles from microcapsules at various pH values
(
A) and temperatures (B).
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different immersion times are shown in Fig. 10B and C. Both
the Young's modulus and hardness in the stable process show a
decreasing trend in the release process, similar to the maximum
load curves (Fig. 10A).
In the CSM test, the indenter became stable in the
displacement from 2000 to 3000 nm. The entire data for
Young's modulus and hardness are averaged in Fig. 10D. It
shows a clear decreasing trend vs. immersion time.
The hardness and Young's modulus are 0.048 and 1.468 GPa
aer 30 min immersion, respectively. Both of them sharply
decreased to 0.016 and 0.606 GPa aer 4 h immersion, respec-
ꢁ
4
tively. Further, they continuously decreased to 8.273 ꢂ 10 and
0.266 GPa aer 480 h immersion, respectively.
As the microcapsules swelling in water, the network struc-
ture shell became loose and weak. The change of the physical
structure led to the change of the micromechanical properties.
Therefore, the maximum load, hardness, and Young's
modulus exhibit similar decreasing trend during the TDC
release process. All of them decreased sharply until 90 h
immersion, then gradually decreased aer 90 h. This trend is
similar to the release kinetics of pH and temperature, as
shown in Fig. 9. Thus, the micromechanical behavior of the
TDC microcapsules was signicantly inuenced by immersion
in the release process.
Pot experiment
TDC molecule has two active groups owning fungicidal effect
(Fig. 7). One is thiazole group, which does not inhibit bacterial
growth outside of plants. However, it is highly effective inside
the plant. The active ingredient of TDC microcapsules taken
up by the plant causes severe damage to bacteria by thinning
49
the skin of bacteria leading to their death. The other is
copper ion that strongly inhibit both fungal and bacterial
+
+
growth. Aer exchanging with positive ions such as H and K
ions on the cell surface of disease-producing germs, copper
ions forms complex with the proteins of cell membrane
leading to their death. Moreover, copper ions can also pene-
trate into the cells of disease-producing germs and form
complex with enzymes, resulting in energy maladjustment
leading to their death. Under the combined action of these two
active groups, TDC can provide perfect fungicidal effect on
different germs.
In the pot experiment, the concentration of R. solanacearum
ꢁ
1
was adjusted to approximately 10 cfu g soil. The soils inocu-
lated without (for A treatment) and with (for B–D treatments)
pathogens were dened as health control and disease control,
respectively. The bacterial counts of R. solanacearum in the
various treatments are listed in Tables 2, which were deter-
mined at 3 days, 10 days, and 20 days aer the transplantation.
Four treatments in total were carried out. Each treatment had
three blocks (three replicates) which were randomly laid out,
and each block for each treatment had four pots. Thus, each
treatment had 12 pots. One tobacco seedling was transplanted
into each pot.
Fig. 10 Load-displacement (A), Young's modulus-displacement (B)
and hardness-displacement (C) curves of the TDC microcapsules
tested by using CSM at different immersion times: (a) 0.5 h, (b) 34 h, (c)
9
0 h, (d) 228 h, (e) 480 h, the average hardness and Young's modulus
of the microcapsules in the release process (D).
Aer about 60 days of incubation in nursery beds, the
tobacco seedlings were transplanted into soils. For C (TDC
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Table 2 Disease index and control effect of different treatments to R. solanacearum. The statistical method of disease index and control effect
are described in detail in the SI
3
days
10 days
20 days
Disease
index
Control
effect (%)
Disease
index
Control
effect (%)
Disease
index
Control
effect (%)
Treatments
A
Details
Soils without inoculation of R.
solanacearum
—
—
—
—
—
—
B
C
Soils inoculated with R. solanacearum
Soils treated with TDC SC and then
inoculated with R. solanacearum
Soils treated with TDC microcapsules
and then inoculated with R. solanacearum
1.8
0
—
100
12.7
4.4
—
65.4
30.1
12.7
—
57.8
D
0
100
2.0
84.2
7.5
75.1
SC) treatment, the tobacco seedlings were directly trans-
ꢁ
1
planted into the soil inoculated with 10 mL (0.05 g mL
active ingredient [ai]). For D (the TDC microcapsules were
synthesized inhouse) treatment, the tobacco plants were
transplanted into the soils that had been mixed with 0.5 g
of ai.
The four treatment samples did grow well for 3 days, as
shown in Fig. 11. However, one leaf of sample B became wilted,
indicating that the tobacco was infected with the inoculated R.
solanacearum aer 3 days. Further, sample B has did not inhibit
the propagation of R. solanacearum. The disease index of sample
B is 1.8, while the disease index and control effect of both
samples C and D are 0 and 100%, as listed in Table 2.
Moreover, some yellow areas were observed on the wilted leaf
of sample B, indicating that R. solanacearum could continuously
infect the plant until 10 days.
Meanwhile, no wilted leaves were observed in samples A and
D for 10 days. However, one leaf of sample C became wilted in
the same time, indicating that TDC SC could not effectively
control R. solanacearum, with a control effect of 65.4%.
At 20 days aer the transplantation, samples B and D
became completely wilted. The soils treated with pesticides (C
and D) had smaller counts of R. solanacearum than the control
soil. The disease incidence for samples C and D decreased to
5
7.8% and 75.1%, respectively, with the application of TDC.
Fig. 11 Effects of different treatments to R. solanacearum in the pot
experiment, (A): soils without inoculation of R. solanacearum, (B): soils
inoculated with R. solanacearum, (C): soils treated with TDC SC and
then inoculated with R. solanacearum, (D): soils treated with TDC
Moreover, the disease indexes for sample B, C, and D declined
from 30.1 to 12.7 and 7.5 aer 20 days.
In the pot experiments, water, existing in the soil, could
penetrate into the shells and swell the shells of the microcap- microcapsules and then inoculated with R. solanacearum.
sules. Due to the shell swelled, micro channels formed in the
shells, and the release process occurred.
Because of the slow and controlled release of TDC from the
microcapsules, the treated tobacco plants could be protected for
Conclusions
a long time. Therefore, the control effect of sample D is better TDC microcapsules were synthesized via in situ polymerization.
than that of sample C. It conrms that the microcapsules The size distributions of the microcapsules depend on the
effectively control the release of TDC in the pot experiment to agitation rates during the polymerization. The FT-IR spectra of
protect the tobacco from R. solanacearum.
the microcapsules and precursors conrmed that TDC was
Thus, the application of TDC microcapsules signicantly successfully encapsulated. The microcapsules were stable
ꢀ
suppressed the bacterial infection compared to TDC SC, both under 220 C, as measured by TGA. The release concentration of
at 10 and 20 days aer the transplantation. This effectiveness TDC during the release process was measured by UV-visible
was increased when the pesticide was applied with the rain absorption spectra. The release concentration of TDC signi-
water.
cantly increased until 90 h immersion, and then gradually
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4486 | RSC Adv., 2014, 4, 4478–4486
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