Microencapsulation of seed-coating tebuconazole and its effects on physiology and biochemistry of maize seedlings

Colloids and Surfaces B: Biointerfaces 114 (2014) 241–246

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Microencapsulation of seed-coating tebuconazole and its effects on physiology and biochemistry of maize seedlings Daibin Yang a , Na Wang a,b , Xiaojing Yan a , Jie Shi c , Min Zhang b , Zhenying Wang a , Huizhu Yuan a,∗ a State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Science, Beijing 100193, China b College of Agriculture, Sichuan Agricultural University, Wenjiang, 611130 Sichuan, China c Institute of Plant Protection, Hebei Academy of Agricultural and Forestry, Baoding, 071000 Hebei, China

a r t i c l e

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Article history: Received 27 March 2013 Received in revised form 7 October 2013 Accepted 9 October 2013 Available online 17 October 2013 Keywords: Microcapsule Tebuconazole Maize Plant growth regulating effect

a b s t r a c t Tebuconazole is a triazole systemic fungicide that is commonly used to treat fungal pathogens of crops, but at high doses can reduce seed germination. Seeds with microcapsulated tebuconazole were investigated to determine effects of this method on maize seedlings and the bioefficacy against maize head smut (Sphacelotheca reiliana). The ethyl cellulose (EC)-based microcapsules had encapsulation efficiency of 90.6%, and average size of 1.6 ␮m. A release kinetic study revealed that tebuconazole release from ECbased microcapsules fits the model (Mt /Mz = ktn + C). Glasshouse studies indicated that maize seedling emergence and growth were negatively affected in an exponential manner as predicted by model Y = A + B × e(−x/k) . However, microencapsulation could induce tebuconazole’s growth promoting effects by increasing emergence, shoot fresh weight, root fresh weight, carotenoid and chlorophyll content. Phytohormone analysis indicated the beneficial effects of microencapsulated tebuconazole were due to the sustained release of tebuconazole that appeared to influence the balance of phytohormones in maize seedlings. Contrary to conventional tebuconazole, microencapsulated seed-coated tebuconazole can lead to slightly increased gibberellins (GA) level and disappearance of abscisic acid (ABA) accumulation in maize. In addition, microcapsule formulation of tebuconazole was found to provide better protection against maize head smut when compared to conventional formulation. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Microencapsulation technology has received much attention in the past few years. If a plant is treated with microencapsulated functional agents, higher effectiveness and longer durability are expected [1,2]. In addition, microencapsulation may alleviate the detrimental responses of the plants to functional agents. In medicine, microencapsulation of Freund’s adjuvant within liposomes alleviated inflammation at the injection site [3]. In cell encapsulation, transplanted cells did not require immunosuppression because they were protected from immune rejection by an artificial, semipermeable membrane [4]. In plant protection, the microcapsule formulations of pesticides are superior to conventional formulations because they provide advantages in reducing degradation, decreasing dermal toxicity, reducing environmental pollution and providing an effective way to eliminate phytotoxicity [5].

∗ Corresponding author. Tel.: +86 10 62815941; fax: +86 10 62815941. E-mail address: [email protected] (H. Yuan). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.10.014

Triazole compounds are widely used as fungicides in plant protection because triazole fungicides provide a broad spectrum of fungicidal activity. Besides their fungicidal activity, triazole fungicides also have plant growth regulating properties [6]. But there is potential for this fungicide to be phytotoxic and retard plant growth when foliar applications or seed treatments deliver too much agent. Negative effects include low seed germination, suppressed seedling growth, especially reduction in shoot elongation [7,8]. The mechanism of the adverse regulatory effects appears to be that triazole fungicides shift the phytohormone balance in plant tissues and inhibit the biosynthesis of GAs, which leads to a transient raise in ABA content in plants [6,9]. However, the effects of triazole fungicides on plant growth are more complicated. In contrast to their growth retarding effects, triazole fungicides in some cases can promote plant growth. For example, Fletcher and Nath found triadimefon can prevent plant leaves from wilting and becoming senescent [10,11]. In carrot plants, hexaconazole and paclobutrazol increased plant fresh weight and dry weight [3,12]. Furthermore, the type of plant growth effect of triazole fungicides depends on plant stage of development. Plant regulatory effects of triadimefon were more pronounced when applied on seeds or

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young seedlings than on mature plants [4,13]. Triapenthenol and tebuconazole induced initial inhibition of stem extension and leaf expansion in oilseed rape (Brassica napus), but the plant recovered subsequently with compensatory growth [7]. Among these triazole fungicides, tebuconazole is one of the most commonly used fungicides in agriculture. Currently, it is an important fungicide for protecting maize seedlings from head smut (S. reiliana) when applied as a seed treatment. The known seed treatment product of tebuconazole is Raxil. As a triazole fungicide, tebuconazole also can be phytotoxic to maize seedlings, resulting in reduce seed emergence and deformed seedlings [14]. Therefore, it is a challenge for scientists to avoid negative effects and promote positive effects of tebuconazole. Microencapsulation is potentially an important tool that will allow scientists to accomplish this. The objective of this paper is to study the effects of tebuconazole on maize seeds and seedlings when it is delivered by microcapsules in a seed coating.

um PTFE filter. The amount of tebuconazole in the filtered solution was determined by reverse phase HPLC. 2.4. Greenhouse experiments Seeds of maize were treated with either Raxil (60 g AI L−1 ) or microcapsule formulation (100 g AI L−1 ). Both of the two formulations were applied at rates of 0.04, 0.1, 0.4, 1, 3 g AI kg−1 seed. All treatments were applied using a traditional method by stirring 100 g of seeds with fungicide formulation in 1 mL of water. The seeds of untreated control were stirred with 1 mL of tap water. The coated maize seeds were planted in peat substrate in glasshouse at 25 ◦ C. The emergence, plant height, shoot fresh weight, and root fresh weight of maize seedlings were measured 16 days after sowing (DAS) just when there was no new seedlings emerged. The seedlings of each treatment were uprooted randomly, washed and separated into shoots and roots, and then used for analysis of growth and biochemical constituents.

2. Materials and methods 2.5. Determination of photosynthetic pigments 2.1. Chemicals and maize seeds Technical tebuconazole (97.0%) was provided by Zhejiang Welldone Chemicals Co. Ltd (Zhejiang, China), and was used without further purification. EC (ethoxyl content 48%, viscosity 22 cps) was purchased from J&K Scientific Ltd. Commercial emulsifier 0203 (Polyoxyethylene castor oil + Calcium dodecylbenzene sulfonate, 1:1, w/w) was purchased from Jiangsu GPRO Group Co. (Jiangsu, China). Hydroxyl polyvinyl alcohol (17-88) was purchased from Beijing Organic Chemical Plant (Beijing, China). The flowable suspension of tebuconazole (Raxil, 60 g L−1 ) was the gift of Bayer CropScience AG. Seeds of maize (Nongda 108) were generously supplied by China Agricultural University (Beijing, China). 2.2. Preparation of polymeric microcapsules Microcapsules were prepared by the oil-in-water emulsion process. Briefly, 2.4 g of EC and 2.4 g of tebuconazole was dissolved in 20 mL of dichloromethane. A volume of 4.0 g Emulsifier 0203 was added to this organic solution as emulsifier. The organic solution was mixed with 200 mL of aqueous solution containing 2.0 g of hydroxypropyl cellulose. This mixture was sheared with Fluko FA25 high shear emulsifier at 20,000 rpm. Stirring with electric stirrer at room temperature was continued over night until the evaporation of dichloromethane was complete. The mixture was centrifuged at 12,000 rmp. The microcapsule formulation for seed treatment was prepared by redispersing the sediment in aqueous solution containing emulsifier 0203 (30 g L−1 ) and polyvinyl alcohol (17–88) (10 g L−1 ). The concentration of tebuconazole in this microcapsule formulation for seed treatment was 100 g L−1 . 2.3. Characterization of tebuconazole microcapsules Particle sizes of microcapsules were measured using a S-3400N scanning electron microscopy (SEM). At the same time, the particle sizes and their distribution of microcapsules were measured using a BT-9300H laser particle sizer (Dandong, China). The encapsulation efficiency of tebuconazole microcapsules was tested by the method of Asrar et al. [15]. The release study of tebuconazole from microcapsules was modified after the methods of Asrar et al. [15]. Briefly, 300 mg of microcapsule formulation was mixed with 500 mg of sodium benzoate (as preservative) and 150 mL of water. The mixture was then placed in a 500 mL glass bottle and agitated at room temperature by a magnetic stirrer. At intervals of day 0, 1, 3, 6, 12, 24, 48 after mixing, 1 mL of the mixture was removed and filtered through a 0.45

Chlorophyll and carotenoid were extracted and estimated by the method of Porra [16]. Carotenoid content was calculated by using the formula of Kirk and Allen [17]. 2.6. Determination of GA and ABA contents A volume of 0.2 g fresh sample tissues was extracted and homogenized in 2 mL of 80% methanol (containing 40 mg L−1 butylated hydroxytoluene as an antioxidant). The extract was incubated at 4 ◦ C for 48 h, and then centrifuged at 4000 rpm for 15 min at 4 ◦ C. The supernatant was passed through C18 Sep-Pak cartridges (Waters Corp., Millford, MA, USA), and the phytohormone fraction was eluted with 10 mL of methanol and then 10 mL of ether. The eluate was dried down by pure N2 at 20 ◦ C, and then the GA and ABA contents was assay by method of Wang et al. [18]. 2.7. Field experiments The experiments were carried out to assess the bioefficacy of two tebuconazole formulations: Raxil (60 g AI L−1 ) and microcapsule formulation (100 g AI L−1 ) in 2012 in Baoding, Hebei province, China. Coating maize seeds were conducted at the conventionally recommended rate of 0.12 g AI kg−1 seed by the method as mentioned above. Fields were seeded on May 22, and the following adjustments were adopted: distance in the row of 0.50 m; seed distance on the row of 0.30 m; sowing density of 60,000 seeds ha−1 ; plot area of 60 m2 . Artificial inoculation was applied by sowing each seed together with 100 g of diseased soil containing 0.1% S. reiliana inoculum. The inoculum was collected from the previous crop season. The insecticide treatments and the untreated control plots were arranged in a randomized block design, with three replications each. The maize head smut incidence was calculated as the percentage of S. reiliana infected ears per plot at crop maturity when disease symptoms were fully expressed. Field management was typical in this area, except for irrigation. Just before planting, the plot was irrigated to stimulate good germination and seedling establishment. Irrigation was then avoided during the early stages to enhance infection. 2.8. Statistical analysis Statistical analysis was conducted using PASW Statistics 18.0 (SPSS Inc, Chicago). Statistical analysis was performed using oneway analysis of variance (ANOVA) followed by Duncan’s multiple

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80 60 40 20 0 Fig. 1. SEM image of tebuconazole microcapsules containing tebuconazole.

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Cumulative release of tebuconazole (%)

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Dose (g AI kg seed) Raxil EC microcapsule

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Fig. 3. Emergence of maize seedlings after maize seeds were coated with different doses of Raxil and EC microcapsule formulations. The control treatment was set at zero. Error bars are standard errors. Bars marked with an asterisk (*) are statistically higher than control at P < 0.05.

40 35 30

After 48 days, 42.0% of tebuconazole was released. As sodium benzoate was added in the release system as preservative, the employed setup represents a situation that tebuconazole was gradually released from microcapsules into water without degradation. According to the theoretical analysis on diffusional release from microparticles, when the amount of release (Mt/ Mz ) is <60%, the empirical expression (1) below can be adopted to describe release kinetics[15,19] (Fig. 2)

25 20 15 10 5 0

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Time (days) Fig. 2. Cumulative release of tebuconazole from EC microcapsules. The curve was produced by fitting release data to the exponential model Mt/ Mz = ktn + C.

range test (DMRT) except analyzing the bioefficacy of tebuconazole. The values are mean ± SD for three samples in each group. P values ≤0.05 were considered as significant. 3. Results 3.1. Characterization of the microparticles containing tebuconazole Particle size and size distributions of the microcapsules were measured by laser diffraction. The size and shape of the microcapsules also were determined by SEM. The particle sizes measured by laser diffraction ranged from 0.5 to 5.0 ␮m with an average of 1.6 ␮m. The SEM characterization of the microcapsules containing tebuconazole shows the majority of the spherical particles range from 1 to 2 ␮m (Fig. 1). This size range agrees well with the size range measured by laser diffraction. The encapsulation efficiency was determined by analyzing the solution after washing the tebuconazole outside the microcapsules. The results indicated that the encapsulation efficiency was 90.6 ± 2.3%, which demonstrates that the solvent evaporation method was very efficient, with the majority of tebuconazole successfully encapsulated in microcapsules. 3.2. Release kinetics of microencapsulated tebuconazole in water Fig. 2 shows the release rate of tebuconazole in water from the EC-based microcapsules. As shown in Fig. 2, the cumulative release of tebuconazole increased gradually during the experiment.

Mt /Mz = kt n + C

(1)

where Mt/ Mz is the amount of tebuconazole released at time t, k is the constant that incorporates the matrix properties, the exponent n is a diffusion parameter, which is indicative of the transport mechanism, and C is a constant. In our study, the release data of tebuconazole from EC-based matrix also follows this empirical function. The values of k, n and C obtained from the initial 42% release of tebuconazole from EC-based matrix were 13.83, 0.22, and 9.37 with the correlation coefficient (r) = 0.9937. Here, the calculated n was close to the value of tebuconazole released from poly(styrene-co-maleic anhydride)-based microcapsules on addition of 30% poly(vinyl acetate) in wall materials, which provided high level of protection against wheat rust [15]. 3.3. Emergence of maize seedlings Seedling emergence is a key indicator to evaluate the safety of chemicals applied as seed coatings. As shown in Fig. 3, at rate of 0.04 g AI kg−1 seed, conventional tebuconazole (Raxil) did not affect emergence significantly. But the emergence of maize seedling was gradually reduced with increasing doses of tebuconazole. At rate of 3 g AI kg−1 seed, the emergence was only 4.0%. According to the results of statistical analysis, we can find the seedling emergence was negatively correlated with dose in the tested rate range when the emergence data were analyzed by applying the exponential model (2) Y = A + Be(−x/k)

(2)

where Y is the emergence, X is the dose of tebuconazole, and A, B, k are constant. The calculated values of A, B, k was 1.70, 90.31 and 0.84 with r = 0.9996. This result indicated the suppression of maize emergence induced by tebuconazole was exponentially dose dependent. However, the microencapsulation of tebuconazole can overcome this negative effect. The emergence of microcapsule

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Fresh weight of maize shoots and roots (g plant )

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Fig. 4. Fresh weight of maize seedling shoots and roots 16 DAS following maize seeds were coated with different doses of Raxil and EC microcapsule formulations. The control treatment was set at zero. Error bars are standard errors. Bars marked with an asterisk (*) are statistically higher than control at P < 0.05.

formulation treatments was above 95.0% at all rates, which was statistically greater than that of the control (Fig. 2). This result indicated that microencapsulation of tebuconazole could benefit seedling emergence.

3.4. Growth of maize seedlings Shoot height, shoot fresh weight and root fresh weight of seedlings were measured 16 DAS when the emergence of seedlings was completed. Reductions in shoot height, shoot fresh weight and root fresh weight of seedlings were observed under conventional tebuconazole treatments compared to untreated plants. Fig. 4 shows the fresh weight of seedling shoots and roots 16 DAS grown from tebuconazole coated seeds. As shown in Fig. 4, the fresh weight of maize seedling shoots and roots was gradually suppressed under conventional tebuconazole treatments as the rate of tebuconazole increased. Exponential model (2) also describes this dose dependent suppression with r > 0.96. In model (2), the value of k indicates the sensitivity of growth parameters to tebuconazole dose. In this study, value of k for shoot fresh weight (k = 0.59) was greater than root fresh weight (k = 0.26), which demonstrated that fresh weight of shoots was more susceptible to tebuconazole suppression than root fresh weight. At rate of 1.0 g AI kg−1 seed, shoot fresh weight was reduced by 49.6%, and root fresh weight by 33.7% when compared to control plants. Similarly, the shoot height data also agree with the exponential model (2) when r = 0.9108 and k = 0.94. Microencapsulation of tebuconazole can stimulate the growth of fresh weight of maize seedling shoots and roots. At all rates of tebuconazole under microcapsule treatments, fresh weight of seedling shoots and roots were greater than control plants (Fig. 4). At rate of 0.1 g AI kg−1 seed, microencapsulated tebuconazole increased fresh weight of shoots by 35.4% and of roots by 34.2%. At rate of 3 g AI kg−1 seed, fresh weight of shoots under microcapsule formulation treatment was numerically greater than control but this difference was not statistically significant. Under microencapsulation treatments, seedling shoot height recovered to the same height as the control plants.

chlorophyll content

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Dose (g AI kg seed) Raxil EC microcapsule Fig. 5. Carotenoid and chlorophyll contents in maize seedling leaves 16 DAS following maize seeds were coated with different doses of Raxil and EC microcapsule formulations. The control treatment was set at zero. Error bars are standard errors.

3.5. Effect of tebuconazole on photosynthetic pigment changes in maize seedling leaves As shown in Fig. 5, conventional tebuconazole treatment increased the carotenoid and chlorophyll contents in leaves at rate of 0.04 g AI kg−1 seed and 0.1 g AI kg−1 seed when compared to the control treatment. Relatively, more carotenoid was increased than chlorophyll. At rate of 0.04 g AI kg−1 seed, conventional tebuconazole treatment increased carotenoid content by 160.9%, but increased chlorophyll content only by 12.1% when compared to control. However, the carotenoid and chlorophyll content showed a downward trend in conventional tebuconazole treated maize seedlings with increasing tebuconazole doses (Fig. 5). At rate of 3 g AI kg−1 seed, the carotenoid content was not significantly different from control (P > 0.05), and the chlorophyll content was even significantly lower than control (P < 0.05). Microencapsulation altered the downward trend effect of tebuconazole on the carotenoid and chlorophyll contents in maize seedlings (Fig. 5). At all tested rates, tebuconazole increased the carotenoid and chlorophyll contents in maize seedlings to great level under microcapsule formulation treatments when compared to control treatment. At rate of 3 g AI kg−1 seed, the carotenoid content was 0.23 mg g−1 fresh weight, 2.4 times of control, and the chlorophyll content was 1.95 mg g−1 fresh weight, 1.3 times of control when tebuconazole was microencapsulated. 3.6. Effect of tebuconazole on phytohormonal changes in maize seedlings As shown in Fig. 6, the GA contents in leaves of seedling under conventional tebuconazole treatments were slightly lower than leaves from control plants. But the statistical tests showed that the difference in GA contents between conventional tebuconazole treatments and control was not statistically significant (P > 0.05). The GA contents in roots of maize seedlings under conventional tebuconazole treatments were almost at the same level as control and also were not statistically different from the control treatment (P > 0.05). For the microcapsule treatments, the GA levels in microcapsule treated seedling leaves were slightly greater than the control treatment and conventional tebuconazole treatments (Fig. 6). Microencapsulated tebuconazole had more profound influence on

-1

GA content in maize tissues(ng g fresh weight)

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leaves

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Disease incidence (%)

Bioefficacy (%)

4.0 ± 0.2a 2.2 ± 0.6b 20.7 ± 1.1c

80.3 ± 2.0a 89.6 ± 5.2b

Note: Values, in the same column, followed by different letters are statistically different at ˛ = 0.05. Student t-test was applied when comparing the bioefficacy of Raxil and microcapsule formulation.

roots

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Table 1 S. reiliana disease incidence and bioefficiency of tebuconazole against head smut in maize following seed treatment at rate of 0.12 g AI kg−1 seed.

Raxil Microcapsule formulation Control

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from 58.5 ng g−1 fresh weight to 117.4 ng g−1 fresh weight in roots. Therefore, following seed treatment, conventional tebuconazole led to gradually increased accumulation of ABA in maize seedlings as tebuconazole doses increased. Under microcapsule treatments, notably accumulation of ABA in maize seedlings was not observed, and the ABA contents were not significantly different from the control treatment (Fig. 7).

-1

Dose (g AI kg seed) Raxil EC microcapsule

120

leaves

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ABA content in maize tissues (ng g fresh weight)

Fig. 6. Effect of tebuconazole on the GA content in maize seedling leaves and roots16 DAS. The control treatment was set at zero. Error bars are standard errors. Bars marked with an asterisk (*) are statistically higher than control at P < 0.05.

60

3.7. Bioefficacy against maize head smut Bioefficacy of tebuconazole microcapsule formulation was evaluated in the field by assessing the incidence of head smut in maize. To ensure high disease incidence, each seed was inoculated by sowing seed together with S. reiliana chlamydospores. As shown in Table 1, there was 20.7% S. reiliana disease incidence in control plots, and tebuconazole provided a high level of protection against head smut in maize. Relative to Raxil, microcapsule formulation provided significantly better protection against head smut. This result indicates microcapsule formulation is a good alternative for commercialized tebuconazole formulations. 4. Discussion

30 roots

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Dose (g AI kg seed) Raxil

EC microcapsule

Fig. 7. Effect of tebuconazole on the ABA contents in maize seedling leaves and roots 16 DAS. The control treatment was set at zero. Error bars are standard errors.

GA content in roots than in leaves. At rate of 0.04 g AI kg−1 seed under microcapsule formulation treatment, the GA content in roots was increased by 65.3% relative to control, while it was only increased by 21.7% in leaves. In addition, the GA level in roots showed a downward trend with increasing doses of microencapsulated tebuconazole. At rate of 0.04 g AI kg−1 seed under microcapsule treatment, the GA content was 0.43 ng g−1 fresh weight, which was significantly higher than the control treatment (P < 0.05), while at rate of 3.0 g AI kg−1 seed, the GA content was 0.32 ng g−1 fresh weight, which was not significantly different from the control treatment (P > 0.05). In case of ABA level, conventional tebuconazole increased the ABA levels in leaves and roots of maize seedlings, and the ABA content showed an upward trend with increasing tebuconazole doses in leaves and roots (Fig. 7). In leaves, the ABA content increase from 77.2 ng g−1 fresh weight to 102.9 ng g−1 fresh weight, and

Our results show that maize seedling emergence is negatively correlated with the dose of tebuconazole as described by the exponential model (2). This result indicated that tebuconazole should be applied strictly at the recommended dose and the maize seeds should be coated uniformly by tebuconazole. Interestingly, this study shoes that EC-based microcapsule formulation of tebuconazole increases maize emergence. Theoretically, if the seed coating agents have plant growth promoting properties, the emergence of seedlings could be increased. In case of tebuconazole, it has the function of decreasing GA level and increasing ABA content. It was known that low GA level and high ABA is not helpful to seed germination and emergence, but helpful to seed dormancy [20,21]. After microencapsulation, the GA level increased slightly and ABA accumulation disappeared in the maize seedlings. This shifted phytohormone balance explained why microencapsulation of tebuconazole could lead to the increase in maize seedling emergence. As reported previously, suppression of plant growth by triazole fungicides was typical. For example, after treatment with increasing concentrations of tetraconazole, a number of morphological changes occurred in roots of maize seedlings: the root system was shorter, the primary and secondary roots were thickened, and there was a marked reduction in the rate of elongation [22]. In our study, regression analysis demonstrated shoot height, shoot fresh weight and root fresh weight were exponentially reduced with the increasing doses of tebuconazole, which agrees with the exponential model (2). On the contrary, microencapsulation of tebuconazole induced increase in seedling shoot fresh weight and root fresh weight under similar conditions. Simultaneously, the shoot height recovered to the same height as those of the control plants. These results indicated maize seedlings grew stronger than control seedlings when treated with microencapsulated tebuconazole.

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In addition to promoting seed emergence, microencapsulation of tebuconazole reduced the direct contact of tebuconazole with seeds and plants, which reduced phytotoxic effects. Normally, triazole compounds increase the carotenoid and chlorophyll content in plants. For example, hexaconazole and paclobutrazol increased the carotenoid and chlorophyll content in Daucus carota L [12,23]; Triadimefon and hexaconazole increased the carotenoid and chlorophyll content in Solenostemon rotundifolius [23]. Our results show that tebuconazole increased the carotenoid and chlorophyll content in maize seedlings at a relatively low dose and that overdose of tebuconazole was harmful to chlorophyll at early stage of seedlings. The downward trend in carotenoid and chlorophyll content in our study was contrary to the report of Lakshmanan et al. [24]. They found carotenoid and chlorophyll content showed a progressive increase in Plectranthus forskholii with increasing concentrations of hexaconazole. One of the reasons that led to this difference was that the young seedlings were more susceptible to chemical stress than mature plants and the investigations were conducted at different development stage of plants. Child et al. found that tebuconazole induced the initial inhibition of stem extension and leaf expansion of oilseed rape (B. napus), but there was a compensatory growth at the later development stage [7]. Montfort et al. found that initial suppression of wheat seedling height was induced 13 days after planting by triticonazole and triadimenol, but increased plant height was observed later [8]. In this study, the investigation was conducted just when the emergence was finished 16 DAS, whereas Lakshmanan et al. determined the carotenoid and chlorophyll contents in P. forskholii on the 165th day after planting [24]. Under treatment of microcapsule formulation, the notable increase in carotenoid and chlorophyll content can be explained that 90.6% of tebuconazole was encapsulated in the microparticles, and was gradually released into the soil. Therefore, the maize seeds and seedlings were exposed to relatively low doses of tebuconazole. The release profile of tebuconazole from microcapsules was in favor of increasing carotenoid and chlorophyll contents in maize seedlings since low dose of tebuconazole induced increase in carotenoid and chlorophyll content. It is well known that ABA and GA often play antagonistic roles in regulating seed dormancy, plant growth and development [25]. ABA can cause a significant inhibition of seed germination and suppression of growth and development, while GAs are required for germination and sustained seedling growth [26,27]. On the basis of shifted phytohormone balance, the alteration of growth effects of tebuconazole resulted from the changes in GA and ABA contents in seedlings. Triazole compounds are known inhibitors of GA biosynthesis [28,29]. Paclobutrazol, a triazole compound, downregulated the transcript level of GA metabolic genes during germination of maize seeds [30]. But in our study, tebuconazole did not significantly decrease the GA level in maize seedlings. Similarly, Grossmann et al. also found that triazole compound, BAS 111W, did not significantly change the level of GA in pumpkin seedlings at rate of 0.03–3.00 mg plant−1 and in 3-day-old oilseed rape seedlings at rate of 1–9 mg pot−1 under soil treatment [31]. However, microcapsule formulation treatment showed significantly increased GA level in leaves and roots. Particularly, the increased GA level showed a downward trend in roots. These results suggest that tebuconazole could increase the GA level when very low rate of tebuconazole is directly exposed to maize seeds or seedlings. Microencapsulation and controlled release of tebuconazole paved the way to offer sustained very low dose of tebuconazole over the time course. In case of ABA, our results were consistent with previous reports that compounds containing triazole structure can induce ABA accumulation in plants. For example, tetraconazole can induce modified levels of ABA and overexpression of an ABA regulated gene in maize [22]. It has been shown that the inhibiting effect of triazoles

is related to the increase in ABA content [32]. But obviously, microencapsulation of tebuconazole can induce disappearance of ABA accumulation in maize seedlings since ABA levels were almost at the same as those of control plants. This disappearance of ABA accumulation induced by microencapsulated tebuconazole is probably related to the lack of negative effects during plant emergence. Taken as a whole, microencapsulation and sustainable release of tebuconazole could shift the balance of phytohormones in maize seedlings in favor of stimulating emergence and growth of seedlings. In summary, microencapsulation is an effective way to overcome the detrimental effects of tebuconazole. Direct seed-coating with conventional tebuconazole reduced seed germination and inhibited seedling growth, whereas microencapsulation of tebuconazole eliminated these negative effects by inducing the plant growth promoting effects of tebuconazole on maize seedlings. Theoretically, the plant growth promoting effect of microencapsulated tebuconazole resulted from slightly increased GA level and disappearance of ABA accumulation in maize. Acknowledgements This work was funded by China Agriculture Research System (CARS-02) and the National Key Technology R&D Program of China (2012BAD19B01). The authors also gratefully acknowledge Dr. Hellmich, R.L, USDA-ARS, Corn Insects and Crop Genetics Research Unit, for comments and suggestions on this manuscript. References [1] K. Tsuji, J. Microencapsul. 18 (2001) 137–147. [2] S.R. Little, D.M. Lynn, Q. Ge, D.G. Anderson, S.V. Puram, J. Chen, H.N. Eisen, R. Langer, PNAS 101 (2004) 9534–9539. [3] S. Cohen, H. Bernstein, C. Hewes, M. Chow, R. Langer, PNAS 88 (1991) 10440–10444. [4] G. Orive, R.M. Hernandez, A.R. Gascon, R. Calafiore, T.M.S. Chang, P.D. Vos, G. Hortelano, D. Hunkeler, I. Lacik, A.M.J. Shapiro, J.L. Pedraz, Nat. Med. 9 (2003) 104–107. [5] B. Singh, D.K. Sharma, R. Kumar, A. Gupta, J. Hazard. Mater. 177 (2010) 290–299. [6] R.A. Fletcher, A. Gilley, T.D. Davis, N. Sankhla, Hortic. Rev. 24 (2000) 55–138. [7] R.D. Child, D.E. Evans, J. Allen, G.M. Arnold, Plant Growth Regul. 13 (1993) 203–212. [8] F. Montfort, B.L. Klepper, R.W. Smiley, Pestic. Sci. 46 (1996) 315–322. [9] K. Grossmann, Physiol. Plant. 78 (1990) 640–648. [10] R.A. Fletcher, V. Nath, Physiol. Plant. 62 (1984) 422–426. [11] N.K. Asare-Boamah, G. Hofstra, R.A. Flethcher, E.B. Dumbroff, Plant Cell Physiol. 27 (1986) 383–390. [12] R. Gopi, C.A. Jaleel, R. Sairam, G.M.A. Lakshmanan, M. Gomathinayagam, R. Panneerselvam, Colloids Surf., B 60 (2007) 180–186. [13] R.A. Fletcher, G. Hofstra, Plant Cell Physiol. 26 (1985) 775–780. [14] Y. Wang, D. Yang, H. Yuan, X. Yan, S. Qi, Nongyaoxue Xuebao 11 (2009) 59–64. [15] J. Asrar, Y. Ding, R.E. La Monica, L.C. Ness, J. Agric. Food Chem. 52 (2004) 4814–4820. [16] R. Porra, Photosynth. Res. 73 (2002) 149–156. [17] J.T.O. Kirk, R.L. Allen, Biochem. Biophys. Res. Commun. 21 (1965) 530–532. [18] Y. Wang, B. Li, M. Du, A.E. Eneji, B. Wang, L. Duan, Z. Li, X. Tian, J. Exp. Bot. 63 (2012) 5887–5901. [19] P.L. Ritger, N.A. Peppas, J. Controlled Release 5 (1987) 23–36. [20] A.R. Kermode, J. Plant Growth Regul. 24 (2005) 319–344. [21] B. Kucera, M.A. Cohn, G. Leubner-Metzger, Seed Sci. Res. 15 (2005) 281–307. [22] A. Ronchi, G. Farina, F. Gozzo, C. Tonelli, Plant Sci. 130 (1997) 51–62. [23] A. Kishorekumar, C.A. Jaleel, P. Manivannan, B. Sankar, R. Sridharan, R. Panneerselvam, Colloids Surf., B 60 (2007) 207–212. [24] G.M.A. Lakshmanan, C.A. Jaleel, M. Gomathinayagam, R. Panneerselvam, C.R. Biol. 330 (2007) 814–820. [25] F.A. Razem, K. Baron, R.D. Hill, Curr. Opin. Plant Biol. (2006) 454–459. ´ ´ [26] A. Badowiec, S. Swigo nska, E. Szypulska, S. Weidner, Acta Physiol. Plant. 34 (2012) 2359–2368. [27] K. Graeber, K. Nakabayashi, E. Miatton, G. Leubner-Metzger, W.J.J. Soppe, Plant Cell Environ. 35 (2012) 1769–1786. [28] R.S. Burden, T. Clark, P.J. Holloway, Pestic. Biochem. Physiol. 27 (1987) 289–300. [29] E. Nambara, T. Akazawa, P. McCourt, Plant Physiol. 97 (1991) 736–738. [30] J. Song, B. Guo, F. Song, H. Peng, Y. Yao, Y. Zhang, Q. Sun, Z. Ni, Gene 482 (2011) 34–42. [31] K. Grossmann, J. Kwiatkowski, C. Häuser, Physiol. Plant. 83 (1991) 544–550. [32] S.I. Chizhova, V.V. Pavlova, L.D. Prusakova, Russ. J. Plant Physiol. 52 (2005) 93–98.