Original Articles
炭酸カルシウム・ナノ粒子は作物の栄養状態を改善し耐虫性を高める
2015 年 40 巻 4 号 p. 208-213
詳細
2015 年 40 巻 4 号 p. 208-213
Nanoparticles and nanotechnology have been applied in agriculture, including for plant nutrition improvement, and pest control. Little research has been conducted on nano-calcium carbonate in plant protection and nutrition. In this study, we compared the effects of nano-calcium carbonate and colloidal calcium carbonate and found that nano-calcium carbonate treatments were better at increasing calcium content when sprayed on Tankan (Citrus tankan) leaves. Field tests on California red scale (Aonidiella aurantii) and Oriental fruit flies (Bactrocera dorsalis) both showed that nano-calcium yielded better control rates. Moreover, bioassays of Oriental fruit flies conducted using calcium carbonate particles yielded lower LC50 values for nano-calcium. Additionally, nano-calcium performed better in terms of protection efficacy against oviposition punctures from Oriental fruit flies on the Indian jujube. Nano-calcium yielded better overall results than colloidal calcium for fertilization, plant protection, and pest control.
Nanostructured materials typically consist of particles less than 100 nm in diameter. Due to their size, these materials have properties that are different from micrometric or larger-sized materials. These include differences in physical strength, chemical reactivity, and electrical conductivity (for details see Ref. 1)). The development of nanotechnology could play an important role in crop management.2) A variety of metal nanoparticles (aluminum (Al), silica (Si), and zinc (Zn)) and metal oxide-based polymers (Zinc oxide (ZnO) and titanium dioxide (TiO2)) are being developed for agricultural uses,2–4) and the application of technology at the nano level could play an important role in improving existing crop management techniques.2)
In recent years, researchers have studied the effects of nanostructured materials on plant germination and growth. For example, the penetration of nanostructured materials into seeds is thought to be responsible for the significantly higher germination rate and higher biomass production seen in certain plants.5) For pesticide applications, agricultural formulations of nanostructured materials may also possess useful nanopesticide formulation properties, such as the increased dispersion and wettability of agricultural formulations.4) The insertion of nano-scaled active ingredients into pesticides makes use of their specific properties to increase dispersion, wettability,4) and stability, and is designed to maximize the effectiveness of pesticides.6) For example, polyethylene glycol (PEG)-coated nanoparticles loaded with garlic essential oil have been shown to effectively control stored-product pests (Tribolium castaneum); this was demonstrated by Yang7) in a study that showed more than 80% control efficacy after five months.
Other researchers have used nanostructured materials as active ingredients of pesticides for crop protection.2) Nanostructured alumina shows insecticidal effects on two insect species (the rice weevil, Sitophilus oryzae, and the lesser grain borer, Rhyzopertha dominica).1) Nanosilica has also been used to control a range of agricultural insect pests.2,3) In addition, silver nanoparticles show insecticidal effects on the oleander aphid (Aphis nerii).8)
In this study, we compared the fertilization effects of nano-scale calcium carbonate (nano-Ca) and colloidal calcium carbonate (colloidal Ca) on Tankan (Citrus tankan Hayata) leaves. We also compared the control effectiveness of colloidal Ca and nano-Ca on infestations of the Oriental fruit fly (Bactrocera dorsalis) in fruits9) and on red scale insects (Aonidiella aurantii) that causes damage to Tankan plants. In addition, the effectiveness of colloidal Ca vs. nano-Ca for the protection of Indian jujubes (Zizyphus mauritiana) from damage by oviposition punctures from Oriental fruit fly females was also compared.
The calcium carbonate (CaCO3) nanoparticle (NP) suspension (nominal diameter 60 nm (20–100 nm)) was supplied by the Diamond Nano-biochem Co., Ltd. (Taichung, Taiwan). Water and nano-Ca were the main components, and the small particles of nano-Ca were suspended in distilled water. The concentration of the CaCO3 NP suspension (SC, pH 10) was 26% for the field test and 19.5% for the laboratory test. The bulk CaCO3 wettable powder (colloidal Ca 95%, Calufructus 95WP (particle size <10 µm)) was purchased from the Songzhou Chemical Industry Co. (Taichung, Taiwan) (Fig. 1). This formulation is also registered as a pesticide for the control of apple rust in Taiwan.
The pH value of colloidal Ca after 100-fold dilution in distilled water was between 7 and 8 and the pH value for nano-Ca after 100-fold dilution in distilled water was between 8 and 9, as tested using pH-Fix test strips (Macherrey-nagel, Germany).
2. Dilution preparation for fertilizers 2.1. Field testsTankan plants were separated into three groups: colloidal Ca (95%WP), nano-Ca suspension (26%SC), and tap water (CK), which was used as a control. The colloidal calcium was diluted 300 fold (w/v) in tap water and the nano-Ca was diluted 100 fold (v/v) before spraying. Each tree was sprayed with 3L of solution using a sprayer.
2.2. Laboratory tests 2.2.1. BioassaysBoth Ca fertilizers used were diluted using distilled water. The colloidal Ca was diluted into 6 different concentrations to a total volume of 500 mL: we first mixed 200 g of colloidal Ca (95%WP) with distilled water to get a total of 500 mL with a concentration of 38,000 mg/L; we then conducted serial dilutions twice for six different concentrations.
For the nano-Ca (19.5%SC) solution, we mixed 250 mL of nano-Ca with 250 mL of distilled water to get a concentration of 9,750 mg/L and then conducted serial dilutions twice for nine different concentrations.
2.2.2. Oviposition puncturesThe formulations used were (1) colloidal calcium (95%WP) and (2) nano-calcium suspensions (19.5%SC), with distilled water used as a control. The colloidal Ca was diluted to 4 different concentrations with a total volume of 500 mL, the dilution rates were 800- (0.625 g of colloidal Ca), 400- (1.25 g), 100- (5 g), and 50- (10 g) fold. The nano-Ca was diluted to 5 different concentrations with a total volume of 500 mL, the dilution rate being 800- (0.625 mL of nano-Ca), 400- (1.25 mL), 100- (5 mL), 50- (10 mL), and 20- (25 mL) fold.
3. Field testsTankan plants were grown at an elevation of approximately 900 m in the Heping District of Taichung City, Taiwan. The fertilizer and insect control experiments were conducted three months before harvest. This was after approximately 7 months of growth (plants were typically harvested after 10 to 11 months of growth). In the fertilizer experiment, a first application was conducted on December 18, 2011; the second application was on the January 17, 2012. In the insect control experiment, the first spraying took place on December 18, 2011, with a second spraying on December 25 of the same year.
Trees were separated into three groups and were sprayed with different treatments using a randomized block design. A total of three treatments were performed with three replications per experiment. Two calcium carbonate fertilizers were used, namely colloidal Ca (95%WP) and nano-Ca suspension (26%SC). A control group of tap water with no added calcium carbonate (CK) was also used.
3.1. Plant NutrientsThe Tankans were sprayed twice with a lapse of one month between sprayings. Samples were taken before the first treatment and two weeks after the second treatment. The samples were selected by cutting the fifth leaf of a branch from 8 different branch sections of each tree. Samples were dried at 70°C to a constant weight and were then ground to a powder. The powdered samples were collected and stored in airtight plastic bags until analysis. For each sample, two replicates (each of 0.3 g of dry leaves) were used to analyze the element content.
3.2. Analysis of element contentContent analysis for the elements nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) was conducted for the dried leaves. To test for N, the 0.3 g samples of dry leaves were digested in H2SO4 and then N levels were measured through distillation. Phosphorus was determined colorimetrically.10) K levels were measured using a flame emission photometer (Corning 410; Corning Science Products, Essex, UK). Magnesium content was measured using an atomic absorption spectrophotometer (GBC Avanta; Victoria, Australia). Calcium content was measured by atomic absorption spectrophotometer following digestion by two acids (nitric acid and perchloric acid11)).
3.3. Surveys of red scale insects on the TankansEach tree was treated using sprays either with or without calcium carbonate, and 9 replications were conducted for each experiment. Tankans were treated twice with a one-week period between sprayings. The numbers of live red scale insects (A. aurantii) were recorded for the first five leaves of a branch. A total of four branches (taken from different sections of each tree) were used to record the number of red scale insects in each tree. The numbers of live A. aurantii were recorded three times, specifically before the first treatment, one week after the first treatment, and one week after the second treatment. Excluding eggs, individuals from all stages were counted; from this, the rate of control of the insects was then determined. Data means were transformed to 
. The control rate (%)=(1-(treatment amount after treatment×CK amount before treatment)/(treatment amount before treatment×CK amount after treatment))×100.
Each tree was sprayed with treatments either with or without calcium carbonate, and 9 replications of the treatments were conducted. Ten fruits were randomly chosen from different sections of a tree. The number of fruits with visible damage from punctures (due to the action of Bactrocera dorsalis females) was recorded (1) before the first treatment, (2) one week after the first treatment, and (3) one week after the second treatment, after which the control rates were calculated as described above.
4. Laboratory tests 4.1. BioassaysThe strain of Oriental fruit fly used was originally collected in Taiwan in 1994, and has been maintained in a laboratory ever since. Specimens used in the bioassays were adult flies 7–14 days old. These were subjected to spray applications as described below or with distilled water as a control. Both calcium carbonate fertilizers used were diluted into 6–9 different concentrations. A total of 20 flies were placed in a caged area 11 cm in diameter, and 0.4 mL of the solution (of a specific concentration) was sprayed using a spray bottle (500 mL). Spraying was done four times in total (1.6 mL), and two replications were done for each concentration. All treated flies were maintained at a temperature of 24±2°C and in a 12 : 12 hr (L : D) photoperiod. The mortality rate was recorded for 72 hr, and then values for 50% lethal concentration (LC50) were calculated by log-probit analysis.12)
4.2. Oviposition puncturesFlies used in the cage experiments described were all provided with food (liquid food sucked in cotton wool (sugar : yeast : tap water, 4 : 1 : 5). To measure the oviposition effects, two formulations of calcium particles were tested on Indian jujubes (Z. mauritiana). The formulations were (1) colloidal Ca (95%WP) and (2) nano-Ca suspensions (19.5%), with distilled water as used as a control. The fertilizers were diluted into 4 or 5 different concentrations, and Indian jujubes were soaked in each concentration for 10 sec before being allowed to dry. For each dose used, three fruits were placed on plastic cylinders (2.5-cm diameter, 3.5-cm height) inside a net cage (25×25×25 cm3). For all experiments, the fruits were exposed to 10 mated females (8 to 10 days old) for 24 hr, and each treatment was replicated twice. The fruits were then removed for storage in sealed bags, and the number of punctures made by the B. dorsalis females was recorded after 72 hr.
5. Data analysisFor analyses of nutrients and pest damage in Tankan plants, the content of five elements and the damaged number of plants under different spray times (before spray, first spray, and second spray) using three different treatments were examined using ANOVA (F-test). Duncan’s multiple range tests were performed for multiple pairwise differences across treatments. The results were analyzed using R software.13) Any value of p<0.05 was considered statistically significant.
For oviposition puncture analysis, the number of punctures with each concentration at different treatments was compared with of the control treatment. Analyses of these single comparisons was evaluated using the Wilcoxon matched pairs test with Statistica 7.1 software.14)
The Ca concentrations for sprays on the Tankan plant were similar in the two treatments: the nano-Ca treatment was 2,600 mg/L and colloidal Ca was 3,170 mg/L. Concentrations of the five nutrient elements in the Tankans before application of the fertilizer are listed in Table 1. The Mg content was similar for all three treatments (including the water control treatment). However, two elements (N and K) were lower and P and Ca were higher in the control treatment than in the other two Ca treatments. After spraying twice, the increase of N and Mg was similar in all three treatments. The Ca content decreased in the control treatment but increased in the two Ca treatments. The nano-Ca treatment had 13 times the increase of Ca content over the colloidal Ca treatment. By contrast, the amount of K decreased in the two Ca treatments but increased in the control treatment. The K amounts after spraying were similar in the two calcium treatments.
| Treatment | Nutrients contents, g/kg | ||||
|---|---|---|---|---|---|
| N | P | K | Ca | Mg | |
| Before spray | |||||
| Control | 22.4±0.77a | 2.17±0.32a | 12.8±0.83a | 72.1±3.26a | 1.88±0.35a |
| Colloidal Ca | 23.3±1.52ab | 1.68±0.08b | 13.2±1.09ab | 67.6±12.9a | 1.95±0.14a |
| nano-Ca | 24.7±1.18b | 1.88±0.17b | 14.6±0.53b | 54.7±8.83b | 2.07±0.24a |
| After two sprays | |||||
| Control | 24.0±1.23a | 1.96±0.21a | 14.9±1.51a | 59.3±7.13a | 1.91±0.16a |
| Colloidal Ca | 25.8±0.86ab | 1.43±0.11b | 10.8±1.13b | 68.8±5.63ab | 2.20±0.22b |
| nano-Ca | 26.4±1.98b | 1.46±0.13b | 10.8±0.79b | 71.1±8.36b | 2.09±0.10a |
* Values are mean±SD. Each group was made up of three trees and the dry leaves from each group were separated into two portions for assay. Values with the different small notations a and b in each column indicate significant differences among the nutrients content measure of the three treatments as shown by Duncan’s multiple range tests (p<0.05).
The first sampling of leaves was undertaken during the flowering stage when the leaves gradually accumulate carbonates in preparation for fruit bearing. Unless the uptake rate of the nutrients was greater than the carbohydrate accumulation rate, the concentrations of nutrients in leaves decreased as compared with the leaves at flowering time.16) After application of Ca in Colloidal Ca and nano-Ca treatments, a greater Ca concentration was observed in the treated groups as compared to the control group (Table 1). The N concentration of leaves after two sprayings increased slightly, an effect that could be attributed to the N absorbed continuously during fruit growth. By contrast, the decrease in P and K concentrations after two sprayings was due to the lessened amount of P and K uptake as compared with the carbohydrate accumulation. After two sprayings, the P and K concentrations in the Ca-treatment groups were smaller than those in the control, which could be due to the greater increase in carbohydrate accumulation caused by the Ca treatment, resulting in a greater dilution effect.15)
The numbers of live red scales (A. aurantii) were similar with both the control and nano-calcium carbonate treatments, but were higher with the colloidal calcium treatment before spraying. After the first spraying, the density of red scales decreased in the nano-calcium carbonate, but the density of red scales from the other two treatments increased (Table 2). The control rate in the treatment of nano-Ca was 75% while the colloidal Ca was 50%. After the second round of sprayings, the nano-Ca treatment had the lowest density and the colloidal Ca treatment showed the second lowest density. The control rate in nano-calcium carbonate was approximately 75% while the colloidal Ca was approximately 66%.
| Treatment | Mean No. of scale insects/20 leaves (control rate, %) | ||
|---|---|---|---|
| Before sprays | After first spray | After second sprays | |
| Control | 8.00±6.60a | 24.0±7.68b | 23.4±9.84b |
| Colloidal Ca | 18.6±10.6b | 26.6±12.2b (52.3) | 18.7±13.0b (65.7) |
| nano-Ca | 7.22±4.32a | 5.33±3.39a (75.4) | 5.22±3.07a (75.3) |
* Values are mean±SD for 9 replications. Values with the different small superscript in each column were significantly different among three different treatments by Duncan’s multiple range tests at p<0.05. ** Values in parentheses indicate control rates. Control rate (%)=(1−(treatment amount after treatment×CK amount before treatment)/(treatment amount before treatment×CK amount after treatment))×100.
The fruit damage by Oriental fruit flies in Tankans was similar in all three treatments before spraying (Table 3). After the first spraying, the damage to fruit with nano Ca treatment was the lowest of all three treatments. However, this difference was not statistically significant (Duncan’s multiple range rests, p=0.054). The control rate in the nano-Ca treatment was 45.5% and the rate of control in the colloidal Ca treatment was 2.6%. After the second sprayings, the damage was lower in the nano-Ca treatment than in the other two treatments.
| Treatment | Mean No. of damage fruits/10 fruits (control rate, %) | ||
|---|---|---|---|
| Before sprays | After first spray | After second sprays | |
| Control | 0.67±1.00a | 2.44±1.33a | 3.00±1.22b |
| Colloidal Ca | 0.78±0.83a | 2.78±1.72a (2.6) | 3.00±1.41b (14.3) |
| nano-Ca | 0.67±0.71a | 1.33±1.22a (45.5) | 1.78±0.83a (40.7) |
* Values are mean±SD for 9 replications. Values with the different small superscript in each column were significantly different among three different treatments by Duncan’s multiple range tests at p<0.05. ** Values in parentheses indicate control rates. Control rate (%)=(1−(treatment amount after treatment×CK amount before treatment)/(treatment amount before treatment×CK amount after treatment))×100.
When flies recovered from the spraying treatment (about 2 min), they were able to walk. With colloidal Ca treatment, the flies would use their legs to clean their bodies for less than 30 sec and then resumed normal behavior. However, with the nano-Ca treatment, the flies quivered their proboscises and cleaned their bodies continually for 6 to 7 min. The mortalities of Oriental fruit flies against different concentrations of the two Ca particles are shown in Fig. 2 and the corresponding LC50 values are listed in Table 4. The value for colloidal Ca was 9,330 mg/L, whereas the value for nano-Ca was 6,530 mg/L but with little overlap of 95% fiducial limits (FL). When the adult flies were treated with more than 4,800 mg/L of Ca particles, the mortality caused by nano-Ca fertilizer were approximately 10% higher than that caused by colloidal Ca.
| Treatments | LC50 (95% FL) mg/L | Slope (±SE) | Chi-square | N |
|---|---|---|---|---|
| Colloidal Ca | 9,330 (6,400–13,900) | 1.82 (±0.22) | 4.56* | 240 |
| Nano-Ca | 6,530 (4,300–10,200) | 1.79 (±0.18) | 9.08* | 280 |
* Denotes significant difference by Chi-square test at the 5% level.
The results of the oviposition punctures in the Indian jujube by the Oriental fruit fly showed that nano-Ca had better protection efficacy against fruit fly oviposition (Table 5). Colloidal Ca significantly reduced the mean number of punctures on the fruits when diluted 100- or 400-folds, but no significant effect was found with less than 50- and 800-fold dilution. Nano-Ca had a significant effect on reducing the oviposition punctures at 20- to 800-fold dilution.
| Treatments | Dilution rate | Concentrations (mg/L) | Mean No. of punctures/fruit (Mean±SD)a |
|---|---|---|---|
| Control (water) | — | — | 10.00±3.35 |
| Colloidal Ca | 800 | 1,188 | 8.67±4.80 |
| 400 | 2,375 | 4.67±1.86* | |
| 100 | 9,500 | 5.17±2.93* | |
| 50 | 19,000 | 5.67±4.93 | |
| Nano-Ca | 800 | 244 | 2.83±3.43* |
| 400 | 488 | 4.50±3.15* | |
| 100 | 1,950 | 2.83±2.64* | |
| 50 | 3,900 | 1.50±1.22* | |
| 20 | 9,750 | 2.50±1.64* |
a Single comparisons (with control) were evaluated using Wilcoxon Matched Pairs Test (by Statistica 7.113)) and * denotes significant difference at the 5% level.
Calcium is a major essential plant elements. It can strengthen plants against diseases or increase crop yields. Calcium nutrition could reduce gray mold symptoms and the severity of postharvest Botrytis blight in rose flowers.16) The foliar application of calcium on Egyptian cotton can increase yields of seed cotton and lint and also improve fiber properties.17) After the field fertilizer experiment, the increased amount of calcium content of the nano-Ca treated leaves was 13 times that of leaves treated with colloidal calcium. This result revealed that nano-Ca was significantly more easily absorbed by Tankan leaves as compared to colloidal Ca. That the K content decreased while the Ca increased in both Ca treatments might be due to the antagonism between these two elements. Jakobsen18) found that Ca and Mg uptake decreased after excessive application of K fertilizer and could lead to Ca and Mg deficiency. In the same way, K uptake tends to decrease when Ca and Mg fertilizers are applied. The Ca content of the Tankan leaves increased significantly in our field tests. However, it should be noted that excessive spraying of Ca may induce potassium deficiency. The concentration and amount of Ca applied should be optimized to prevent such disadvantages.
Mineral particles could attach to insect cuticle surfaces and produce abrasions or block the spiracle and lead to biological and behavioral changes, including the reduction of feeding, moving, and oviposition.19) In this study, the control rate of nano-Ca was higher than that of colloidal Ca when used against A. aurantii on Tankan leaves and when used against Oriental fruit flies on Tankan fruit. These results show that the nano-Ca is the better repellency and control particle. Control rates after the first and second sprayings of nano-Ca were similar in both experiments, revealing that nano-Ca could bring plant protection efficacy with one spraying. The difference in the control rate between the colloidal Ca and nano-Ca treatments was higher in the fruit damage surveillance than in the red scale insect surveillance. This could be due to the fact that the rough and uneven surface of the fruit increased the difficulty of particle attachment and dispersal. The nano-Ca particles could still work well on this kind of surface but colloidal calcium could not.
The two Ca particles showed different protection efficacy on the oviposition behavior of Oriental fruit flies in the Indian jujube. Colloidal Ca reduced the puncture numbers significantly when used at 100 and 400 dilution rate while nano-Ca reduced the puncture numbers when used at from 20 to 800 folds dilution. The low efficacy of colloidal Ca in high concentration (low dilution rate) might be caused by low dispersibility. Although the puncture numbers of the colloidal calcium treatment could show apparent differences to the control group under suitable concentrations, it was still inferior to the nano-Ca treatment at every dilution rate. The concentration of colloidal calcium under same dilution rate was 4.87 folds to that of nano-Ca. This meant nano-Ca brought better efficacy under lower concentrations than colloidal Ca.
Insects can secrete a variety of cuticular lipids to form a watertight barrier and can avoid death from desiccation.3) When fly cuticles are covered with powder particles, the small particles may be absorbed into the cuticular lipids through physisorption, thereby causing death by desiccation.3) Due to better coverage and adherence from smaller particles, the Oriental fruit fly presented higher susceptibility to nano-Ca than to colloidal calcium in the susceptibility assay. The mortality caused by nano-Ca was higher than by colloidal calcium when concentrations reached 4,800 ppm or more. These results were similar to those of field tests.
The results of this study show that there are significant differences in the fertilizer effect of calcium and in the controlling efficacy of both Oriental fruit flies and red scale insects when treated with nano particles. Furthermore, nano-calcium carbonate could protect fruit from the oviposition of Oriental fruit flies.
As plant protection agents, calcium carbonate particles were relatively safe and friendly for human and environmental use as compared with chemical pesticides. Another advantage of calcium particles is the lower resistance possibility. Oriental fruit flies, as well as other tephritid flies, have shown resistance against various recently used insecticides including organophosphate, pyrethroids, and spinosad.20–23) Of the two Ca particles in our study, nano-Ca showed better performance in fertilization, plant protection, and pest control over colloidal Ca. The concentration in nano particles was only one tenth of that in the colloidal calcium. Thus, this inorganic nanomaterial may provide a cheap, reliable, and safe alternative for enhancing plant nutrition and crop protection.
The authors thank two anonymous reviewers for improving this manuscript and also thank Miss P.-F. Liu and Mrs. J.-J. Ku for assistance with data collection. This research was supported by the Diamond Nano-biochem Co., Ltd. (Taichung, Taiwan) under the grant SBIR100-19.
