Abstract［Objectives］ The effects of copperbased nutrient foliar fertilizer on photosynthetic characteristics, yield, accumulation and distribution of trace elements in various organs, disease prevention effect and soil enzyme activity were studied, so as to provide a theoretical basis for the application of foliar fertilizers in cotton production.
　　［Methods］ Through two years of field experiments, six treatments were set in total, namely spraying water (CK), traditional Bordeaux mixture (BDM), Kocide 2000 (KCD), copperbased nutrient foliar fertilizer (CF), ironcopperbased nutrient foliar fertilizer (CFFe), and zincboroncopperbased nutrient foliar fertilizer (CFZnB). Randomized block arrangement was adopted. Chlorophyll content in leaves was measured at each growth stage of the cotton. Photosynthetic characteristics of leaves were measured at the peak bolling stage. Plants were sampled at initial boll opening stage. The whole plant was divided into root, stem, leaf and cotton boll parts, in which the total copper, total zinc, total iron contents and accumulations were determined. Soil samples were collected from each plot, followed by the determination of soil enzyme activity. Disease index was investigated at bud, flowering and bollforming and boll opening stage.
　　［Results］ (1) Spraying CFFe, CFZnB, CF and KCD could significantly improve chlorophyll content of cotton leaves, and the CFFe treatment had the highest increase up to 13.30%, followed by the CFZnB treatment, which was 11.40% higher than the CK; and photosynthetic rate, stomata conductance and transpiration rate could be improved significantly, and the CFFe treatment showed the highest photosynthetic rate, which increased by 26.35% compared with the CK, followed by the CFZnB treatment, which increased by 17.96% compared with CK; and intercellular CO2 concentration was significantly reduced. (2) Spraying BDM, KCD, CF, CFFe and CFZnB can significantly increase total copper content and accumulation in various cotton organs (except the total copper content in the stem part of the CFZnB treatment; the CFZnB and CFFe treatments can significantly increase total zinc content and accumulation in various cotton organs; and spraying CFFe, CFZnB and CF can significantly increase total iron content and accumulation in various cotton organs (except the total iron content in the stem part of the CF treatment). (3) Spraying CFFe, CFZnB, CF, KCD and BDM greatly reduced the disease index at flowering and bollforming and boll opening stages. (4) The CFZnB and CFFe treatments had the highest soil urease activity, which was 7.14% higher than that of the CK, but the difference from the CK was not significant; the catalase activity of each treatment was significantly higher than that of the BDM treatment; and the sucrase activity of each treatment was significantly higher than that of the CK. (5) Spraying CFFe, CFZnB, CF and KCD significantly improved lint yield of cotton, and the CFZnB treatment showed the highest yield increase up to 12.34%, followed by the CFFe treatment, with an increase in the range of 8.77%-10.20%. 　　［Conclusions］ Copperbased nutrient foliar fertilizers have dual functions of disease control and prevention and plant nutrition and health care, and not only can significantly increase cotton yield, but also has certain disease prevention effect. It is recommended to use copperbased nutrient foliar fertilizers.
　　Key wordsCopperbased nutrient foliar fertilizer; Cotton; Photosynthetic characteristics; Yield; Disease index; Trace elements; Soil enzyme activity
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　　Received: June 2 2018Accepted: October 14, 2018
　　Qiang MA (1961-), male, P. R. China, senior agronomist, devoted to research about development and extension of new fertilizers.
　　*Corresponding author.
　　Copper is a micronutrient element necessary for plant growth, which is of great significance to normal physiological metabolism of crops［1］. As an ancient and new fungicide, copper has been playing an important role in practice［2］. Copper biocides represented by Bordeaux mixture have been used in orchards for a hundred years, and due to its wide antimicrobial spectrum, long duration, low possibility of causing drug resistance in pathogens, and low toxicity to humans and animals, they are still used in most parts of the world at a large quantity［3］. However, in the longterm use, traditional Bordeaux mixture also exhibits some shortcomings, such as tedious preparation method, inconvenient use, poor suspension performance, easy production of phytotoxicity, being alkaline, and inability to mix with most organic pesticides and leaving a large number of drug spots after sprayed on leaf and fruit surface, which not only influence plant photosynthesis, but also the appearance and quality of fruit［4-6］. Therefore, substitute products of Bordeaux liquid is developing at home and abroad.
　　Ludebao, Lujunling, Tonggaoshang, Lurutong and other products have been developed in China, but due to various reasons such as imperfect production technology, they had a short life on the market and were not widely applied at home and abroad. The products developed by foreign companies include Kocide produced by American Griffin Corporation, FunguranOH produced by Germany Uloli Agrochemical Co. Ltd., and Champion produced by American Yatelu Company. Among them, the development and promotion of Kocide is a major revolution in the production and use of Bordeaux liquid. So far, there are four generations of products, i.e., Kocide 101, 77% Kocide, Kocide 2000DF, and Kocide 3000, and the disease prevention and control effect of products is also gradually enhanced. A large number of studies have shown that the Kocide has a better disease prevention effect than that of similar products at home and abroad［7-8］. Although above copper preparations take a place in the Chinese market and have good antimicrobial effect, but most farmers cannot afford them due to their high price. At present, most Chinese peasants still use traditional Bordeaux mixture prepared in real time for use. 　　The development and production of biocides comparable to the US Kocide products, but low in price and in line with Chinas national conditions, is an important way to open up the Chinese copper biocide market, and is also an urgent need of farmers. Shandong Agricultural University has developed and produced a copperbased nutrient foliar fertilizer containing different trace elements, which has performed well in various crops and fruit trees［9-11］, and is an ideal substitute for domestic Bordeaux liquids. This product has a long attachment time on plant surface, which can enhance its antidisease effect and reduce leaching loss, thereby greatly reducing copper accumulation pollution in soil applied for many years, and can also reduce the spraying amount and spraying times. In use, water is added proportionally in the sprayer which is convenient to use, and its factoryscale production also greatly reduces the waste of resources, saves manpower and material resources, and can bring huge economic benefits［12］. This product can overcome the shortcomings of traditional Bordeaux mixture.
　　This study was conducted in Jingxian County, Hengshui City, Hebei Province, where the climate and soil conditions are suitable for cotton growth. Local farmers also have the habit of growing cotton, but many unfavorable factors restrict the development of cotton production, and pests and diseases are one of the restricting factors. Local cotton diseases mainly include blight, yellow wilt, stem blight, angular leaf spot, red rot, Cephalothecium roseum, soft rot, etc. In the past, cotton farmers used carbendazim, methyl thiophanate, and chlorothalonil to control these cotton diseases, but these agents have high cost and high toxicity to humans and animals. The application of copper biocides to control these diseases has the characteristics of low control cost, low toxicity to humans and animals, and low possibility of producing drug resistance in pathogens, and has achieved certain control effects. Using cotton copper biocides alternatively with other types of biocides to control cotton diseases will be more rational.
　　There have been some reports on the effects of copperbased nutrient foliar fertilizers on crops, but few studies have been conducted on their effects on cotton. In this study, two years of field experiments were conducted to study the effects of five foliar fertilizers on cotton growth and development, yield formation, photosynthetic pigment content, photosynthetic characteristics, accumulation and distribution of trace elements, disease prevention effect and soil enzyme activity, so as to provide a theoretical basis for the application of copperbased nutrient foliar fertilizer in cotton production. 　　Materials and Methods
　　Experimental location and materials
　　This experiment was carried out in Xiaoyang Village (37°32′N, 115°28′E), Liuzhimiao Town, Jing County, Hengshui City, Hebei Province from 2013 to 2014. The tested soil was calcaric ochriaquic cambosols in Chinese soil taxonomy. The soil was sandy loam, and the 0-20 cm layer contained organic matter 14.51 g/kg, total nitrogen 0.85 g/kg, nitrate nitrogen 20.30 mg/kg, nitrogen 14.08 mg/kg, available phosphorous 24.80 mg/kg, available potassium 98.80 mg/kg, available copper 1.30 mg/kg, available zinc 1.45 mg/kg, available iron 6.12 mg/kg and available boron 0.41 mg/kg, and had a pH value of 8.4. The tested cotton variety was Guoxinmian 3. The tested five kinds of foliar fertilizers were traditional Bordeaux mixture (BDM) (containing Cu 12.80%), American commercial copper preparation Kocide 2000 (KCD) (containing Cu 33.01% and Zn 0.03%), copperbased nutrient foliar fertilizer (CF) (containing Cu 32.89% and Zn 0.83%), ironcopperbased nutrient foliar fertilizer (CFFe) (containing Cu 3.289% and Fe 2.66%), and zincboroncopperbased nutrient foliar fertilizer (CFZnB) (containing Cu 32.89%, Zn 2.68% and B 2.88%), the latter three of which were developed and produced by National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, Shandong Agricultural University.
　　Experimental design
　　The experiment was carried out using a randomized block design with 6 treatments: ① spraying water (CK), ② traditional Bordeaux mixture (BDM), ③ Kocide 2000 (KCD), ④ copperbased nutrient foliar fertilizer (CF), ⑤ ironcopperbased nutrient foliar fertilizer (CFFe), and ⑥ zincboroncopperbased nutrient foliar fertilizer (CFZnB). Each treatment had three replicates, forming 18 plots in total. Each plot was 10 m long and 4 m wide, and had an area of 40 m2. The plant was planted in large and small rows, which had a spacing of 0.8 and 0.5 m, respectively, with an average row spacing of 0.65 m. The plant spacing was 0.32 m, the density was 48 077 plants/hm2, and there were 6 rows in a plot. The field was covered with plastic film, and before sowing, barnyard manure and diammonium phosphate were applied according to 14 m3/ hm2 and 250 kg/hm2, respectively. In 2013, cotton was planted on April 25, and seedlings emerged on May 3; and in 2014, cotton was planted on April 27, and seedlings emerged on May 4. From seedling stage, foliar fertilizers were sprayed on May 18-19 (seedling stage), June 2-3 (initial bud stage), June 18-19 (bud stage), July 2-3 (initial flowering stage), and July 18-19 (flowering and boll setting stage), for 5 times in total. 　　In order to ensure that the copper contents of the copper preparations were basically the same during each time of application, the traditional Bordeaux mixture was used in 390 times dilution, and KCD and the three kinds of copperbased nutrient foliar fertilizers developed by Shandong Agricultural University were used in 1 000 times dilution, so as to compare the properties of different copperbased and their functions and effects on crops. Based on the recommended dosage and recommended concentration of Kocide2000, the dilution times should be 1 000 times or more; CF, CFFe and CFZnB are basically the same as KCD because of the copper contents and active components basically the same as KCD, so all of them were used in 1 000 times dilution; and 390 times dilution of BDM is also a common concentration. The foliar fertilizers were applied to moisture both front and back surfaces of leaves without dropping off from them.
　　Sample collection time and method and determination time
　　On April 18, 2013 (before fertilization and sowing), 0-20 cm of ploughed soil was collected with a soil drill by Stype fivepoint method, as the basic soil sample, which was treated by steps of mixing thoroughly, air drying, pulverizing and sieving with a 2 mm sieve, for later testing in October. On July 26, 2014 (flowering and boll setting stage), 0-20 cm of ploughed soil was collected from each plot by Stype fivepoint method and mixed thoroughly, as the sample for testing of soil enzyme activity, which was airdried, pulverized and sieved with a 1 mm sieve, for testing in October. On September 4, 2014 (initial boll opening stage), plants were sampled. Each plot was divided into sampling region and yield calculating region. In the sampling region, samples were collected by diagonal line threepoint method. Three plants were taken from each plot into laboratory and divided into root, stem, leaf and boll parts, each of which was mixed thoroughly and treated by steps of deactivating at 105 ℃ for 30 min, dried at 70 ℃ to constant weight, grinding and sieving with a 2 mm sieve, for later testing in November and December.
　　Agricultural Biotechnology2019
　　Characteristics of each treatment and soil fertility evaluation
　　The traditional Bordeaux mixture was prepared in the same amount: copper sulfate∶quicklime=1∶1. Its active component is basic copper sulfate (［Cu(OH)2］3?CuSO4), which is a sky blue alkaline colloidal suspension. Due to the high solubility of copper sulfate, there are a large amount of free copper ions in Bordeaux mixture. Though copper ions are the main active antimicrobial component, excessive copper ions can cause damage to plants themselves, and are also toxic to environment. Kocide2000 is a thirdgeneration copperbased biocide developed by American Griffin Corporation in the mid1990s, which takes copper hydroxide as the main active component and has four characteristics. Firstly, it has higher free antimicrobial copper ions, and the dilution ratio is 1 000 times, which reduced its application rate. Secondly, it contains sixcarbon copper ions and thus has high activity. Thirdly, it can treat fungal and bacterial diseases, and would not cause production of drug resistance. At last, it is prepared in optimal polymeric granule form (DF), produces no dust and has the characteristic of easy subpackage and application, only copper and trace zinc have low nutritional function to plant, but have a stimulating effect on plant growth［7］. The effective component in these copperbased nutrient foliar fertilizers developed by Shandong Agricultural University containing different trace elements is basically the same as that of Kocide2000, i.e., copper hydroxide. These copperbased nutrient foliar fertilizers are added with effective microelements, iron, zinc and boron necessary for plants on the basis of the effective component ratio in traditional Bordeaux mixture, also provided with a carrier filler and various additives (such as dispersant, lubricant, adhesive, etc.), and finally concentrated to new suspensions, which have the advantages of strong adhesive force, high uniformity and greatlyreduced copper accumulation pollution. Due to the addition of effective trace elements required by plants, they can provide nutrients for growth and development of plants, and can play a dual role of supplementing micronutrients and controlling diseases［13］. 　　According to the soil fertility grading standard recommended by the second national soil survey, the test site of this study is located at moderate fertility level, and has moderate soil organic matter, total nitrogen, ammonium nitrogen and available potassium contents, and only nitrate nitrogen and available phosphorus contents are slightly higher than medium. The soil has a low effective boron content and belongs to the borondeficient soil, while cotton is a crop that is sensitive to boron and has a high boron content［14］. Although the soil available copper, available zinc and available iron contents are moderately or slightly high, the calcareous soil is alkaline at this test site, and trace elements such as copper, iron, zinc and boron are easily fixed by the soil, and exhibited a low plant absorption and utilization rate［15］.
　　Determination items and methods
　　The pH value of the soil solution was determined with a pH meter at the soilwater ratio of 1∶2.5. The ammonium nitrogen and nitrate nitrogen in the soil were extracted by 0.01 mol/L CaCl2 solution, and determined with a flow injection analyzer. Soil total nitrogen was determined by semimicro Kjeldahl method with a semiautomatic nitrogen analyzer. Soil available potassium was extracted with 1 mol/L ammonium acetate and determined by flame photometry. Soil available phosphorus was extracted with 0.5 mol/L NaHCO3 and determined by MoSb colorimetric method. Soil trace elements, copper, zinc and iron, were extracted by DTPA and determined with an atomic absorption spectrophotometer. Soil available boron was determined by curcumin colorimetry. Trace elements in plants were released by nitric acidperchloric acid combined digestion and then determined with an atomic absorption spectrophotometer［16］. Determination of soil enzyme activity: soil urease activity was determined by sodium phenolatesodium hypochlorite colorimetric method; catalase activity was determined by potassium permanganate titration; and sucrase activity was determined by 3,5dinitrosalicylic acid colorimetry［17］. Chlorophyll content was determined with an SPAD502 chlorophyll meter (based on the SPAD value) produced in Japan. The SPAD values of the functional leaves (four leaves of the main stem from top from seedling stage to initial boll opening stage, three leaves of the main stem from top at exuberant wadding stage) were measured at seedling stage (May 20), bud stage (June 20), flowering and bollforming stage (July 18), initial boll opening stage (September 2), and exuberant wadding stage (September 23). Leaf photosynthetic characteristics were measured by LI6400XT Portable Photosynthesis System. The photosynthetic characteristics of functional leaves (four leaves of the main stem from top) were determined on July 25, 2013 and July 26, 2014 at 9:00-11:00 am. 　　During cotton growth, the number of diseased leaves (buds, bolls), disease level and disease index were calculated. Disease index was calculated according to following equation: Disease index (%)=100×∑ (Number of diseased leaves (buds, bolls)×Representative value of each level)/(Total number of investigated leaves (buds, bolls)×Representative value of the highest level)［18］.
　　Data processing
　　The test data were statistically analyzed using DPS statistical software, and multiple comparisons were performed by the Duncan method.
　　Results and Analysis
　　Effects of different treatments on chlorophyll content in cotton leaves
　　Many studies have shown that the chlorophyll content of crop leaves has good correlation with the reading value of SPAD502 chlorophyll meter, which can be used for determination of crop chlorophyll［19］. It could be seen from Table 1 that spraying CFFe, CFZnB, CF and KCD could significantly improve chlorophyll content (SPAD value) of cotton leaves, and the test results in two years showed basically the same trend. The effects of different treatments on chlorophyll content in cotton leaves ranked as CFFe>CFZnB>CF>KCD>CK>BDM. At seedling stage, the differences of chlorophyll content in cotton leaves were between various treatments, and only the chlorophyll content of the CFFe treatment increased significantly compared with the CK. It increased by 8.36% and 7.25% in 2013 and 2014,, respectively. The differences between other treatments were not significant. At bud stage, the CFFe, CFZnB, CF and KCD treatments showed a leaf chlorophyll content significantly higher than the CK. At flowering and bollforming stage, the CFFe, CFZnB, CF and KCD treatments showed a chlorophyll content of leaves still significantly higher than that of the CK. At initial boll opening stage, the chlorophyll content of cotton leaves reached the peak, and those of the CFFe, CFZnB, CF and KCD treatments increased significantly compared with the CK. Among them, the CFFe treatment had the highest chlorophyll content, which increased by 13.30% and 10.53% compared with the CK in 2013 and 2014, respectively; and the CFZnB treatment had the second highest chlorophyll content, which increased by 11.40% and 8.21% compared with the CK in 2013 and 2014, respectively. At exuberant wadding stage, although the chlorophyll content of cotton leaves began to decrease, the CFFe, CFZnB, CF and KCD treatments still showed a value significantly higher than that of the CK, and only the increasing range slightly decreased. The chlorophyll content of the BDM treatment was the lowest at each growth stage, but the differences from CK were not significant. 　　Table 1SPAD value of cotton leaves under different treatments
　　YearTreatmentSeedling stage(May 20)Bud stage(June 20) Flowering and bollforming stage(July 18)Initial boll opening stage(Sept. 2)Exuberant wadding stage(Sept. 23)
　　2013CK31.12 b34.72 b38.64 b42.14 c41.33 b
　　BDM31.06 b33.81 b38.07 b40.65 c39.06 b
　　KCD32.27 ab36.96 a41.54 a45.05 b43.92 a
　　CF32.64 ab37.25 a42.33 a46.16 ab44.53 a
　　CFFe33.72 a38.33 a43.72 a47.70 a45.90 a
　　CFZnB32.85 ab37.86 a42.67 a46.94 ab45.18 a
　　2014CK34.50 b38.24 b42.33 c47.52 b45.14 b
　　BDM34.34 b38.05 b41.82 c47.24 b45.06 b
　　KCD35.43 ab40.26 ab45.28 b50.47 a48.15 a
　　CF35.83 ab40.92 a46.57 ab50.82 a48.92 a
　　CFFe37.04 a42.03 a48.02 a52.54 a50.56 a
　　CFZnB36.07 ab41.54 a47.03 ab51.40 a50.04 a
　　Means in the same vertical column with the same letter are not different significantly at the 5% level in the same year.
　　Effects of different treatments on photosynthetic characteristics of cotton leaves
　　Under normal conditions, increases of leaf transpiration rate and stomatal conductance are conducive to gas exchange, and could improve photosynthetic capacity of mesophyll cells, thereby improving leaf photosynthetic rate［20］. It could be seen from Table 2 that the CFFe, CFZnB, CF and KCD treatments could significantly increase photosynthetic rate, stomatal conductance and transpiration rate of cotton leaves, and the test results in the two years showed basically the same trend. The photosynthesis rates of the CFFe, CFZnB, CF and KCD treatments increased by 26.35%, 17.96%, 12.17% and 10.57%, respectively in 2013, compared with the CK. Among them, the photosynthesis rate of the CFFe treatment also increased significantly by 12.64% and 14.27%, respectively, compared with the CF and KCD treatments, while that of the BDM treatment was 1.52% lower than CK, without a significant difference. The photosynthesis rates of the CFFe, CFZnB, CF and KCD treatments increased by 25.97%, 17.74%, 12.03% and 10.45% respectively in 2014, compared with the CK. Among them, the photosynthesis rate of the CFFe treatment also increased significantly by 12.44% and 14.05% compared with the CF and KCD treatments, respectively, while that of the BDM treatment was 1.82% lower than the CK, without a significant difference from the CK. The stomatal conductance of the CFFe, CFZnB, CF, and KCD treatments increased significantly by 25.53%, 21.28%, 19.15%, and 14.89%, respectively in 2013, compared with the CK, and only the stomatal conductance of the BDM treatment was 2.13% lower than the CK, without a significant difference. The stomatal conductance of the CFFe, CFZnB, CF and KCD treatments increased significantly by 27.45%, 23.53%, 19.61%, and 15.69%, respectively in 2014, compared with the CK, and only the stomatal conductance of the BDM treatment was 1.96% lower than the CK, without a significant difference. The transpiration rates of the CFFe, CFZnB, CF and KCD treatments increased significantly by 22.59%, 19.53%, 15.16% and 10.64%, respectively in 2013, compared with the CK, but there was no significant difference between any two of them; and only the transpiration rate of the BDM treatment was 4.08% lower than the CK, without a significant difference. The transpiration rates of the CFFe, CFZnB, CF and KCD treatments significantly increased by 23.47%, 19.76%, 14.99% and 10.74%, respectively in 2014, but there was no significant difference between any two of them; and only the transpiration rate of the BDM treatment was 3.45% lower than the CK, without a significant difference. A high photosynthetic efficiency means that more light and raw materials are needed, and more CO2 is consumed. Therefore, intercellular CO2 concentration decreases with the photosynthetic efficiency increasing［21］. The intercellular CO2 concentrations of the CFFe, CFZnB, CF and KCD treatments decreased by 14.54%, 12.14%, 6.50% and 5.81%, respectively in 2013, compared with the CK, while the BDM treatment had an intercellular CO2 concentration 2.63% higher than the CK, without a significant difference. The intercellular CO2 concentrations of the CFFe, CFZnB, CF and KCD treatments decreased by 15.39%, 13.02%, 7.43% and 6.14%, respectively in 2014, compared with the CK, while the BDM treatment had an intercellular CO2 concentration 1.60% higher than the CK, without a significant difference. 　　Table 2Photosynthetic characteristics of cotton leaves at peak bolling stage under different treatments
　　YearTreatmentPhotosynthetic ratePn∥μmol/(m2?s)Stomatal conductanceGs∥mol/(m2?s)Intercellular CO2 concentrationCi∥μmol/molTranspiration rateTr∥mmol/(m2?s)
　　2013CK23.00 c0.47 c451.42 a6.86 b
　　BDM22.65 c0.46 c463.28 a6.58 b
　　KCD25.43 b0.54 b425.21 b7.59 a
　　CF25.80 b0.56 ab422.07 b7.90 a
　　CFFe29.06 a0.59 a385.78 c8.41 a
　　CFZnB27.13 ab0.57 ab396.60 c8.20 a
　　2014CK25.26 c0.51 c415.65 a7.54 b
　　BDM24.80 c0.50 c422.32 a7.28 b
　　KCD27.90 b0.59 b390.14 b8.35 a
　　CF28.30 b0.61 ab384.75 b8.67 a
　　CFFe31.82 a0.65 a351.67 c9.31 a
　　CFZnB29.74 ab0.63 ab361.53 c9.03 a
　　Means in the same vertical column with the same letter are not different significantly at the 5% level in the same year.
　　Effects of different treatments on the contents and accumulations of trace elements in cotton plants
　　Effects of different treatments on total copper contents and accumulations in different organs of cotton plants
　　The contents and accumulations of total copper, total zinc and total iron in various organs of cotton plants in different treatments (2014) are listed in Table 3. It could be seen that each treatment had significant effects on total copper contents and accumulations in various organs of cotton plants (the sampling date was September 4, at initial boll opening stage), and the total copper contents in various organs ranked as leaf>stem>cotton boll>root. Among different treatments, the BDM treatment had the highest total copper content, which increased significantly by 160.09%, 155.14%, 155.34% and 257.19% in the leaf, stem, boll and root, respectively, compared with the CK. The BDM treatment might cause excessive copper absorption in cotton, thereby inhibiting the growth of cotton［22］. The total copper content of the CFZnB treatment increased the smallest compared with CK, and the contents in the leaf, boll and root increased significantly by 19.14%, 40.33% and 46.67%, respectively, compared with the CK, while the difference in total copper content in the stem from the CK was not significant; and the remaining three treatments had the total copper content in each organ between the above two. The changing trend of total copper accumulation in various organs of cotton plants of different treatments was basically consistent with that of total copper content. 　　Effects of different treatments on total zinc contents and accumulations in different organs of cotton plants
　　It could be seen from Table 3 that cotton bolls had the highest total zinc content among the root, stem, leaf and boll parts, followed by leaves, and there was a small difference in total zinc content between the stem and root parts, both of which had a low content (the sampling date was September 4, at initial boll opening stage). Among the different treatments, the CFZnB treatment had the highest total zinc contents in various organs, and the total zinc contents in the boll, leaf, stem and root increased significantly by 39.93%, 22.24%, 36.00% and 49.59%, respectively, compared with the CK. The CFFe treatment had the second highest total zinc contents in various organs, which increased significantly by 17.05%-32.57% compared with the CK. The BDM treatment showed the lowest total zinc contents in various organs, which were lower than the CK by 7.99%-8.59%, without significant differences. The changing trend of total zinc accumulation in various organs of cotton plants of different treatments was basically consistent with that of total zinc content. The treatment applying zincboroncopperbased nutrient foliar fertilizer exhibited significantlyincreased zinc accumulation in cotton plants compared with the CK and other treatments (except the leaf, stem and root parts of the CFFe treatment), indicating that the application of zincboroncopperbased nutrient foliar fertilizer can effectively correct zinc deficiency symptoms in cotton［23］.
　　Table 3Total copper, zinc and iron contents and accumulations in different organs of cotton plant under different treatments in 2014
　　ElementTreatment
　　Content∥mg/kg
　　LeafStemRootCotton boll
　　Accumulation∥mg/plant
　　LeafStemRootCotton boll
　　Total copperCK6.74 d6.42 d2.85 e4.86 d0.25 e0.33 d0.03 d0.47 c
　　BDM17.53 a16.38 a10.18 a12.41 a0.64 a0.81 a0.12 a1.18 a
　　KCD10.18 b9.63 b7.02 b7.59 b0.40 b0.52 b0.09 b0.78 b
　　CF9.45 b9.10 b6.75 bc7.30 bc0.38 bc0.50 b0.08 b0.76 b
　　CFFe8.55 c8.22 c6.13 c6.96 c0.36 c0.48 b0.08 b0.74 b
　　CFZn8.03 c7.18 d4.18 d6.82 c0.33 d0.40 c0.05 c0.73 b
　　Total zincCK24.64 cd18.86 b18.39 cd25.07 cd0.91 c0.96 c0.21 c2.43 d
　　BDM22.67 d17.34 b16.81 d23.04 d0.82 d0.86 d0.19 d2.19 e
　　KCD27.62 b23.46 a19.54 c26.67 c1.09 b1.27 b0.24 b2.75 c
　　CF27.31 bc23.55 a19.38 c26.40 c1.08 b1.29 b0.24 b2.74 c 　　CFFe28.84 ab23.84 a24.38 b29.41 b1.22 a1.39 a0.32 a3.27 b
　　CFZn30.12 a25.65 a27.51 a35.08 a1.23 a1.44 a0.35 a3.75 a
　　Total ironCK295.06 de156.88 cd503.04 cd105.76 cd10.92 d8.00 d5.79 d10.25 d
　　BDM284.67 e151.31 d485.38 d101.16 d10.35 d7.59 d5.48 d9.72 d
　　KCD327.45 cd174.12 bc558.23 c116.45 c12.52 c9.54 c6.65 c11.76 c
　　CF339.13 c175.92 bc944.16 b205.29 b13.45 c9.93 c11.63 b21.32 b
　　CFFe587.75 a317.12 a1089.78 a239.08 a24.91 a18.53 a14.35 a26.55 a
　　CFZn409.09 b225.04 b982.23 ab211.89 b16.69 b12.66 b12.46 b22.65 b
　　Means in the same vertical column with the same letter are not different significantly at the 5% level in the same year.
　　Effects of different treatments on total iron contents and accumulations in different organs of cotton plants
　　It could be seen from Table 3 that the CFFe, CFZn and CF treatments could significantly improve total iron contents and accumulations in various organs of cotton plants (except the total iron content in the stem of the CF treatment, the sampling date was September 4, at initial boll opening stage). Among the four parts of cotton plants, the root had the highest total iron content, the total iron content of the boll was the lowest, and the total iron contents were in order of root>leaf>stem>boll. Among the different treatments, the CFFe treatment had the highest total iron contents in various organs, and the total iron contents in the root, leaf, stem and boll parts increased by 116.64%, 99.20%, 102.14% and 126.06%, respectively, compared with the CK. The CFZnB treatment had the second highest total iron contents in various organs, which also increased significantly by 38.65%-100.35% compared with the CK. The BDM treatment showed the lowest total iron contents in various organs, which increased by 3.51%-4.35% compared with the CK, without significant differences from the CK. The changing trend of total iron accumulations in various organs of cotton plants of different treatments was basically the same as that of total iron content, but the increasing range of total iron accumulation in each treatment was larger than that in the CK. It indicates that the application of ironcopperbased nutrient foliar fertilizer can make iron accumulate in cotton plants, thereby effectively correcting iron deficiency symptoms in cotton［23-24］.
　　Effect of different treatments on cotton disease control
　　The diseases investigated at bud stage included blight, Verticillium wilt, stem blight, etc. The diseases investigated at flowering and bollforming stage included blight, Verticillium wilt, red rot, pink rot, anthracnose, soft rot, stem blight, etc. The diseases investigated at boll opening stage included pink rot, red rot and epidemic disease of cotton bolls, and angular leaf spot and Verticillium wilt of plants. The disease index was calculated based on the survey results. The effects of different treatments on cotton diseases were different at different growth stages (Table 4). There were no significant differences at bud stage between different treatments in 2013 and 2014. The disease index of each treatment was significantly lower than that of the CK at flowering and bollforming stage and boll opening stage. The disease index was 21.69%-26.96% and 20.92%-26.94% lower than the CK at flowering and bollforming stage in 2013 and 2014, respectively. And the disease index was 23.38%-30.51% and 19.86%-25.94% lower than the CK at boll opening stage in 2013 and 2014, respectively. 　　Effect of different treatments on soil enzyme activity
　　Soil enzymes are an important constituent of soil. They are biocatalysts that produce specific biochemical reactions in soil, participate in various biochemical processes in soil and play an important role in soil development and soil fertility formation［25］. Copper preparations, that have not been absorbed by leaf surface, have a significant effect on soil enzyme activity. It could be seen from Table 5 that there were no significant differences in urease activity between different treatments (except the BDM treatment). The urease activity of the CFFe, CFZnB, CF and KCD treatments showed a nonsignificant increase compared with the CK, but the CFFe, CFZnB, CF, KCD and CK treatments were all significantly higher than the BDM treatment in urease activity. The CFFe and CFZnB treatments had the highest urease activity, which was 7.14% higher than the CK. The urease activity of the BDM treatment was significantly lower than that of the CK, which meant that it inhibited urease activity, while the CFFe, CFZnB, CF and KCD treatments slightly activated soil urease activity. There were no significant differences in soil catalase activity between the CFFe, CFZnB, CF and KCD treatments, which also had no significant differences from the CK, but the catalase activity in these five treatments was significantly higher than that in the BDM treatment, which inhibited soil catalase activity. The sucrase activity in various treatments significantly increased compared with the CK. Studies［26］ have shown that different soil enzymes are correlated with copper concentration to different degrees (sucrase>urease>catalase). At a low copper concentration, copper has an activating effect on the three kinds of enzymes, while at a high copper concentration, it has an inhibiting effect on the three enzymes［26］. In this study, the several applied copper preparations have a relatively low copper concentration, and the activating effect on sucrase was the highest at low copper concentrations as sucrase was most correlated with copper concentration; and the measurement of soil enzymes was performed in the period when cotton grew vigorously (flowering and bollforming stage), which was also the peak period of soil sucrase activity, the activating effect on which was also higher, while the peak period of soil catalase activity was before the flowering and bollforming stage［27］. Among the different treatments, the soil sucrase activity of the CFFe, CFZnB, CF and KCD treatments also significantly increased by 9.87%, 7.90%, 8.38% and 7.53%, respectively, compared with the BDM treatment. 　　Table 4Disease index of cotton under different treatments
　　Treatment
　　2013
　　Bud stageFlowering andbollforming stagesBoll opening stage
　　2014
　　Bud stageFlowering andbollforming stagesBoll opening stage
　　CK12.96 a24.67 a41.27 a13.85 a25.72a42.86 a
　　BDM12.64 a19.32 b31.21 b13.46 a20.34 b34.35 b
　　KCD12.55 a18.82 b30.45 b13.37 a19.65 b32.84 b
　　CF12.03 a 18.02 b28.68 b 13.21a18.79 b31.74 b
　　CFFe12.47 a 18.78 b29.36 b13.38 a19.53 b32.16 b
　　CFZnB11.83 a 18.19 b28.75 b13.28 a19.07 b31.83 b
　　Means in the same vertical column with the same letter are not different significantly at the 5% level in the same year.
　　Table 5Enzyme activity of soil under different treatments in 2014
　　Treatment Urease (NH3N, mg/g, 24 h)Catalase (0.1 mol/L KmnO4 ml/g)Sucrase (Gla, mg/g, 24 h)
　　CK0.28 a5.29 a37.36 c
　　BDM0.20 b5.00 b41.03 b
　　KCD0.29 a5.19 a44.12 a
　　CF0.29 a5.20 a44.47 a
　　CFFe0.30 a5.21 a45.08 a
　　CFZnB0.30 a5.19 a44.27 a
　　Means in the same vertical column with the same letter are not different significantly at the 5% level in the same year.
　　Effect of different treatments on cotton yield
　　Different treatments had significant effects on cotton yield and yield components (Table 6). The trends of test results in the two years were basically the same. The lint yields of the CFZnB, CFFe, CF and KCD treatments were significantly higher than that of the CK. In 2013, the lint yields of the CFZnB, CFFe, CF and KCD treatments increased significantly by 11.26%, 8.77%, 6.81% and 5.80%, respectively, compared with the CK. Among them, the CFZnB treatment had the highest lint yield, which also significantly increased by 5.16% compared with KCD treatment. In 2014, the lint yields of the CFZnB, CFFe, CF and KCD treatments were significantly higher than that of the CK by 12.34%, 10.20%, 7.80% and 5.96%, respectively. Among them, the CFZnB treatment had the highest lint yield, which also significantly increased by 6.02% compared with the KCD treatment. The BDM treatment had the lowest lint yield, which was 1.39% and 1.98% lower than the CK in 2013 and 2014, respectively, but the differences from the CK were not significant. According to the analysis of yield components, the main reason for the increase was that the CFFe, CFZnB, CF and KCD treatments promoted the increase of cotton boll number, and the numbers of cotton bolls in the CFFe, CFZnB and CF treatments were significantly higher than that in the CK, by 4.69%-7.28% and 5.75%-8.09% in 2013 and 2014, respectively; furthermore, the boll weight of the CFZnB treatment was significantly higher than that of the CK, 0.37 and 0.30 g (7.86% and 5.88%) higher than the CK, respectively, while there were no significant differences in boll weight between other treatments; the different treatments had no significant differences from the CK in lint percentage; and the prefrost lint yields in various treatments were significantly higher than that in the CK, 4.66%-6.31% and 4.85%-6.15% higher than the CK in 2013 and 2014, respectively, suggesting that various treatments could significantly promote early maturity of cotton, and spraying CFZnB, CFFe, CF and KCD not only could significantly improve cotton lint yield, but also could promote early maturity of cotton. 　　Table 6Cotton yield and its components under different treatments
　　YearTreatmentNumber of bollsBolls/hm2Boll weightgLint percentage%Prefrost lint yield%Seed cotton yieldkg/hm2Lint yieldkg/hm2
　　2013CK738 416 bc4.71 b37.26 a83.64 b2 991.03 c1 114.45 c
　　BDM731 431 c4.69 b37.25 a88.92 a2 950.15 c1 098.93 c
　　KCD766 872 ab4.81 ab37.34 a88.54 a3 157.80 b1 179.12 b
　　CF773 057 a4.85 ab37.35 a88.37 a3 186.93 ab1 190.32 ab
　　CFFe792 180 a4.86 ab37.38 a87.95 a3 242.85 ab1 212.18 ab
　　CFZnB774 850 a5.08 a37.40 a87.54 a3 315.49 a1 239.99 a
　　2014CK792 182 bc5.10 b37.21 a82.75 b3 494.81 c1 300.41 c
　　BDM782 675 c5.06 b37.21 a87.84 a3 425.70 c1 274.70 c
　　KCD828 505 ab5.16 ab37.26 a87.25 a3 697.95 b1 377.86 b
　　CF837 720 a5.19 ab37.31 a87.14 a3 757.44 ab1 401.90 ab
　　CFFe856 275 a5.20 ab37.40 a87.05 a3 831.82 ab1 433.10 ab
　　CFZnB839 609 a5.40 a37.43 a86.76 a3 902.87 a1 460.84 a
　　Means in the same vertical column with the same letter are not different significantly at the 5% level in the same year.
　　Discussion
　　Disease resistance in cotton
　　The commonality of all copper preparations is that the final antimicrobial effect is due to Cu ions released by effective components. The antimicrobial mechanism of the copper biocides has three explanations: firstly, copper ions enter the pathogen cells to coagulate or denature proteins; secondly, copper ions react withSH of pathogen cells, thereby destroying the action of enzymes; and thirdly, copper ions replace normal ions of pathogenic cell membrane［5］. Zinc can improve the disease resistance of oat, barley and winter rye. The results of this study showed that zinc has no obvious effect of improving disease resistance of cotton, and iron and boron have no antimicrobial effect. This is the same as the research results of Lu et al.［28］. Various treatments have different disease prevention effects at different growth stages of cotton. Their disease prevention effects were not obvious at bud stage of cotton growth, but were significant at the middle and late stages of cotton growth. This is consistent with the research results of Li［29］ and Xu［30］. The reason might be that at bud stage of cotton, the weather was windy, drought, not rainy, and the crop was not closed, resulting in good ventilation and light transmission, which were not conducive to the occurrence of diseases, while at flowering and bollforming stage and boll opening stage, the climate was hot and rainy, and the crop was closed, resulting in poor ventilation and light transmission, which were conducive to the occurrence of diseases; with the progress of plant development, the copper element in various treatments gradually accumulated in cotton plants, and could prevent and resist some fungal and bacterial diseases of cotton; and furthermore, the CFZnB, CFFe, CF, and KCD treatments could form a protective film on cotton leaves after spraying, which can also protect and control the disease of cotton Verticillium wilt［28］. Therefore, spraying CFZnB, CFFe, CF, KCD and BDM can improve the disease resistance of cotton. 　　Total copper content of cotton
　　The results of this experiment showed that copperbased foliar fertilizers had different effects on total copper contents in various organs of cotton. The total copper content of cotton was the highest in the BDM treatment and the lowest in the CFFe and CFZnB treatments. BDM itself has a low copper content (including Cu 12.80%), KCD contained Cu 33.01%, and CF, CFFe and CFZnB have a higher copper content (containing Cu 32.89%), so BDM was used in 390 times dilution, and KCD, CF , CFFe and CFZnB were used in 1 000 times dilution, which ensured that the copper contents of the copper foliar fertilizers spray at each time were basically the same. However, the amounts of copper absorbed by cotton were different, which was mainly because the form and structure of copper were different in different copperbased foliar fertilizers. The effective component of BDM is basic copper sulfate, and due to the high solubility of copper sulfate, there are a lot of free copper ions in Bordeaux mixture. Although copper ions are the main active component killing microbes, excessive copper ions can cause damage to the plants themselves, and the higher the concentration of free copper ions, the more copper ions plants absorb. The effective component of KCD, CF, CFFe and CFZnB is copper hydroxide, which has appropriate solubility, and not only has good disease prevention effect, but also is less possible to produce phytotoxicity. The microscopic crystal structure of copper hydroxide is fine needle crystal, and due to the crystallization characteristics and high efficiency of copper hydroxide, to achieve the same control effect, its concentration and dosage are significantly less than the Bordeaux mixture prepared by copper sulfate［6-8］. This is basically the same as the results of Wang et al.［31］, Song et al.［22］ and Xu et al.［32］.
　　Chlorophyll content and photosynthetic rate in cotton leaves
　　Copper, iron, zinc and boron are all essential micronutrients for plant growth and development. Copper is an element of various enzymes involved in the redox process in plants. It is present in the chloroplast plastocyanin and participates in electron transfer and photosynthetic phosphorylation of photosynthesis. Therefore, the addition of appropriate amount of copper can promote the formation of chlorophyll, thereby promoting photosynthesis and plant growth. However, excessive copper can poison plants and slow or stop plant growth［33］. However, excessive copper can cause phytotoxicity to plants, causing plants to grow slowly or stop growth［33］. Iron and zinc are essential nutrients for chlorophyll synthesis. Chlorophyll cannot be formed under irondeficiency, which leads to chlorosis［34］. Zincdeficient leaves suffer from chlorosis, small leaf and fasciation would occur at the tip of branches, which refers to as little leaf disease which can cause death of branches in severe case［35］. Although boron is not a constituent of plants, it has a good effect on the formation and stabilization of chlorophyll. When boron is deficient, new leaves whiten and old leaves yellow early. In this study, the soil had low available boron content and was borondeficient soil, while cotton is a crop that requires slightly more boron and is sensitive to boron［14］. Although the available copper, available zinc and available iron contents in the test soil were moderate or slightly high, trace elements such as copper, iron, zinc and boron can be easily fixed by soil as the test soil was alkaline calcareous soil, and exhibited a low absorption and utilization rate. Therefore, the effects of spraying foliar fertilizers containing trace elements of copper, iron, zinc and boron were obvious［15］. The results of this study showed that the BDM treatment had the lowest chlorophyll content, which might be because that the BDM treatment caused excessive absorption of copper by cotton and inhibited the increase of chlorophyll content; and the copper content in cotton plants of the KCD treatment cotton was not too high, because KCD promoted increase of chlorophyll due to its trace zinc content and structural features. The CF, CFFe and CFZnB treatments promoted the increase of chlorophyll content, because the three treatments contained iron, zinc and copper necessary for chlorophyll synthesis, or boron which has the effect of promoting chlorophyll formation and stabilization, plus their own characteristics. This is the same as the results of Wang et al.［31］ and Song et al.［22］. The CFFe treatment had the highest chlorophyll content and was superior to the CFZnB treatment, indicating that iron can better promote chlorophyll synthesis in leaves than zinc and boron, which is consistent with the results of Duan［23］ and Yin et al.［36］. Spraying CFFe, CFZnB, CF and KCD can significantly increase chlorophyll content in cotton leaves, and can also significantly increase photosynthetic rate of leaves, which is mainly because that chlorophyll content of leaves is positively correlated with photosynthetic rate［20］. Spraying CFFe, CFZnB, CF and KCD increased cotton yield by improving leaf chlorophyll content and increasing photosynthetic rate and photosynthetic capacity. The chlorophyll SPAD value of cotton leaves was higher at later growth stages than at middle growth stages, which had two reasons as below. Firstly, the rainfall was less before midJuly, the soil in the cotton field was relatively dry, and the chlorophyll content of the leaves increased at a lower speed; and from midJuly to September, there was more rainfall, the soil moisture was more suitable for cotton growth, and chlorophyll content of leaves increases faster. Secondly, chemical control was adopted in the field management, which meant to spray 1,1dimethylpiperidiniuchloride (DPC for short). It was sprayed at a low concentration for fewer times at middle growth stages, and at a high concentration at mid to late stages for more times, while some studies［37］ have shown that with the increase of the DPC amount, the chlorophyll content and SPAD value of cotton leaves increased［37］. 　　Lint yield of cotton
　　Yield is one of the important indicators to evaluate the growth of crops. The results of this study showed that the BDM treatment had the lowest lint yield, but the difference from the CK was not significant, which might be due to that the BDM treatment caused excessive absorption of copper by cotton and inhibited the increase of chlorophyll content as well as photosynthetic capacity, thereby inhibiting the growth and development of cotton and reducing production. However, at the same time, the BDM treatment can also kill pathogens and prevent disease, and improve the disease resistance of cotton. Combining the positive and negative effects, the BDM treatment only reduced yield slightly, which is basically the same as the results of Wang et al.［29］ and Song et al.［22］. The lint yields of the CFZnB, CFFe, CF and KCD treatments were significantly higher than that of the CK, specifically higher than the CK by 5.80%-11.26% and 5.96%-12.34% in 2013 and 2014, respectively. Among the various treatments, the CFZnB treatment had the highest lint yield, which was mainly due to the increase of boll number, and the CFZnB, CFFe and CF treatments all showed a boll number significantly higher than the CK, while the boll number of the KCD treatment was not significantly higher than that of the CK. The reason was mainly because that spraying CFZnB, CFFe, CF and KCD can significantly improve chlorophyll content in cotton leaves, and significantly increase photosynthetic rate and photosynthetic capacity of leaves, thereby promoting the growth of vegetative organs of cotton including the root, stem and leaf and the differentiation and development of reproductive organs and increasing the number of bolls, and these four treatments also improved the disease resistance of cotton, while there were no significant differences in boll number between the four treatments. The CFZnB treatment had the highest boll weight, which was significantly higher than that of the CK, which was mainly because that boron promoted the growth and development of reproductive organs, and promoted the growth and weight gain of cotton bolls［14］; and there were no significant differences in lint percentage between various treatments and the CK, indicating that the different copperbased foliar fertilizers had no significant effect on lint percentage, which is basically the same as the results of Lu et al.［28］ and Hai et al.［38］. From the perspective of the yield increasing effect on cotton lint, the CFZnB treatment was the best, followed by the CFFe treatment. 　　Conclusions
　　(1) Spraying CFFe, CFZnB, CF and KCD on leaves significantly improved the chlorophyll content and photosynthetic rate of cotton leaves and increased photosynthetic capacity. The CFFe treatment showed the largest increasing range, and its chlorophyll content and photosynthetic rate increased by 13.30% and 26.35%, respectively, compared with the CK, followed by the CFZnB treatment, which had the chlorophyll content and photosynthetic rate increased by 11.40% and 17.96%, respectively, compared with the CK.
　　(2) Spraying CFZnB, CFFe, CF and KCD can significantly improve cotton lint yield. The CFZnB treatment showed the highest yield increase up to 12.34%, followed by the CFFe treatment, with an increase in the range of 8.77%-10.20%.
　　(3) The copperbased nutrient foliar fertilizers developed by Shandong Agricultural University, as a type of foliar fertilizers, have dual functions of disease control and prevention and plant nutrition and health care, and not only can significantly increase cotton yield, but also has certain disease prevention effect. It is recommended to use zincboroncopperbased nutrient foliar fertilizers and ironcopperbased nutrient foliar fertilizers.
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