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HNUE JOURNAL OF SCIENCE Natural Sciences 2018, Volume 63, Issue 11, pp. 154-161 This paper is available online at http://stdb.hnue.edu.vn DOI: 10.18173/2354-1059.2018-0084 THE EFFECT OF USING BIOGRO FERTILIZER FOR REDUCING Cu AND Zn CONTENT IN Brassica integrifolia GROWN IN CONTAMINATED SOIL Tran Khanh Van, Do Thi Oanh, Nguyen Phuong Thao and Pham Thi Van Faculty of Biology, Hanoi National University of Education Abstract. The study on the effects of using BioGro fertilizer for reducing Cu and Zn content in Brassica integrifolia grown in contaminated soil which had total Cu and Zn concentration were 145.7 and 228.3 ppm, respectively was taken. The results showed that Biogro fertilizer had a positive effect to reduce Cu and Zn content in fresh biomass of vegetable. Moreover, the application of BioGro microbial fertilizer increased total chlorophyll content in leaves from 8.8 to 16.9 %, in comparison with the control. However, the vitamin C content decreased from 7.3 to 19.1% in treatments compared to control but not significantly different in treatments. With the dose of fertilizer application 1.5 times higher as recommended dose’s producer, some physiological parameters such as total chlorophyll content, conduced sugar content were significantly higher than other treatments. Especially, the Cu and Zn concentration in fresh leaves of B. integrifolia were within the range of limitation of heavy metals in vegetable by Vietnamese Ministry of Health. Therefore it would be recommended to apply BioGro fertilizer when grow B. integrifolia in Cu and Zn contaminated soil by Cu and Zn. Keywords: Brassica integrifolia, BioGro fertilizer, physiological parameter, contaminated by copper/zinc. 1. Introduction The environmental pollution near industrial zones and craft villages has been bad and it is getting worse and worse. In Vietnam, in recent years production of safe vegetables has increased thanks to the policies of promoting the development from the state agencies and enterprises [1, 2]. However, there is a lot of work to do to resolve the environmental pollution in general and soil contamination in particular. And environmental pollutions have big impacts on the quality of food, and heavy metal pollution in soil gives us an example, particularly, of its influence on plants as well as vegetables. The concern of the safety of food resources is what we put our priority in because it related to our daily life directly and constantly. The results showed that, during the period from 2005 to 2015, the safe vegetable area and production were variable but unstable [3]. However, in recent years production of safe vegetable has increased thanks to the policies of promoting the development from the state agencies and enterprises. The data in 2015 showed that the economic efficiency of the safe vegetable production in this locality is still low. We have initially surveyed the contents of some elements of heavy metal in soils for growing green vegetable in the suburbs of Hanoi (Bac Tu Liem District, Minh Khai Ward, Phuc Ly village) as Received October 29, 2018. Revised November 9, 2018. Accepted November 16, 2018. Contact Tran Khanh Van, e-mail address: vantk@hnue.edu.vn 154 The effect of using biogro fertilizer for reducing Cu and Zn content in Brassica integrifolia grown… well as the contents of such elements of heavy metals in some samples of vegetable such as lettuce, basella alba and mustard spinach. The survey results have shown that the total contents of heavy metals such as Cu and Zn in soil were 145.7 and 228.3 ppm, respectively which exceeded the allowable limits in National technical regulations (QCVN) [4] as well as Zn and Cu content in vegetables also higher than safety standards of FAO / WHO [5]. Thus, it is required to find a solution to grow safe green vegetable in such the situation that the soils have been contaminated. As a consequence, the usage of microbiological product seems to be one of the optimal solutions for improving this situation because of its safety to nature. And Basella integrifolia is one of the most popular vegetables in Vietnamese’ daily meal. Given such situation, we have carried out a study which we titled “The effect of using BioGro fertilizer for reducing Cu and Zn content in Brassica integrifolia grown on contaminated soil” aiming to contributing to resolving of the soil contamination issue. 2. Content 2.1. Materials and methodology 2.1.1. Materials Plant: Brassica integrifolia, is Providing by C.H Vietnam Co., Ltd. This plant is one of the most popular vegetables in Vietnam and it could be grown any time of year. Soil sampling site: at Phuc Ly commune, North Tu Liem District, Hanoi, Vietnam (N 21o03’52.9”, E 105o44’30.1’’) and surface soil was collected at the depth of 0- 20 cm. Table 1. Heavy metal concentration in soil at Phuc Ly Heavy metals in soils At Phuc Ly (ppm) Cu 145.7 50 Zn 228.3 200 A Vietnamese National Standard to agriculture soils (ppm) Factors of experiment Foliar application Fertigation We used 2 kinds of BioGro microorganism fertilizer: one is for foliar application and the other is to fertigation application. 155 Tran Khanh Van, Do Thi Oanh, Nguyen Phuong Thao and Pham Thi Van BioGro microorganism fertilizer provided by Center of Research and Application BioGro. We also determined the density of microorganism in these fertilizers as shown in Table 2 and we found that only Pseudomonas was approximately 100% as reported by producer. However, other microorganisms were counted just 20 - 40% as reported by producer. Table 2. Density of microorganisms in BioGro fertilizers BioGro fertilizer Microorganisms Average of density (CPU/ml) Rhodosporidium toruloides 86 x 105 Foliar application Rhodotorula mucilaginosa 85 x 105 Fertigation Candida tropicalis 66 x 105 Bacillus 6 x 102 Pseudomonas 10 x 107 Candida tropicalis 7 x 102 Table 3. Experimental treatments Treatment BioGro application method Dose CT 1 (control) Non application - CT 2 Foliar as recommended dose (RD) CT 3 Foliar × 1.5 RD CT 4 Fertigation RD CT 5 Fertigation × 1.5 RD CT 6 Foliar and Fertigation RD CT 7 Foliar and Fertigation × 1.5 RD Each treatment was repeated 4 times Time: From August to October 2018 Place: at the Experimental Gardens of Faculty of Biology, Hanoi National University of Education - Plastic pot size: 20 x 20 x 16 cm. - 5 kg soil taken from Phuc Ly per pot. Sowed 15 seeds of B. integrifolia per pot. Irrigation and after 15 days plants were removed to keep plant density to 4 plants/pot. Plants were cared for until harvest 40 days after planting. They were watered with “ditch water”. Collecting samples: after 40 days of sowing 156 The effect of using biogro fertilizer for reducing Cu and Zn content in Brassica integrifolia grown… 2.1.2. Parameters and monitoring methods Height of plant: Plants were measured from base of stem to tip of longest leaf, plus or minus 1 mm. Plant fresh weight: Plants were weighed on a balanced beam scale that had a 2.10-2g degree of precision. Total chlorophyll content in leaf: UV spectrophotometer Visible Model SPECORD 200 Plus with equation of Wettstein 1957 was used. Vitamin C content was determined by the iodine titration method. Nitrate content is determined by the colorimetric method. Total Cu, Zn content in soil: TCVN 6496, ISO11047:1995. Total Cu, Zn content in parts of the plant: AAS - Atomic Absorption Spectrophotometric NovAA 350 Analytik Jena. Destruction of plant sample involved use of wet ash method at 550oC. Density of microorganisms in BioGro fertilizer: direct measurement of microbial growth Data processing. The data were processed by statistical one–way ANOVA analysis (Turkey’s – b) with a significance level of α = 0.05. 2.2. Results and discussion 2.2.1. Effect of BioGro fertilizer to plant’s height The capability of a plant to grow in contaminated soils is important factor then the effect of BioGro fertilizer’s on the height of B. integrifolia grown in soils with different Cu and Zn concentrations is shown in Table 4. Table 4. The effect of BioGro fertilizer to plant’s height Plant’s height (cm) % compared to control Treatments CT1 (control) 12.45a ± 2.1 100 CT 2 13.80ba ± 1.5 111.6 CT 3 16.75cb ± 1.2 134.8 CT 4 17.50c ± 1.7 142.8 CT 5 21.65d ± 1.4 174.0 CT 6 21.50d ± 2.3 173.0 CT 7 21.75d ± 4.6 175.0 (Note: Values in the same column followed by the same letter are not significantly different at 0.05) The results in this Table indicated that the application of BioGro fertilizer as fertigation at rate of 1.5 times higher than recommended rate or the combination of both foliar and fertigation of BioGro significantly increased the height of plants. The plant’s height was higher at all treatments (except treatment CT 2) indicated that the application of Biogro fertilizer in contaminated Cu and Zn soil can reduce the toxicity of Cu and Zn, then stimulate plant’s growth. 2.2.2. Effect of BioGro fertilizer to fresh weight of B. Integrifolia Fresh weight is one of parameter to estimate the yield of plant as well as the photosynthetic efficiency whole plants. Therefore, fresh weight is importance parameter to evaluate the growth and development of plant. 157 Tran Khanh Van, Do Thi Oanh, Nguyen Phuong Thao and Pham Thi Van Figure 1. Effect of BioGro fertilizer to fresh weight of B. integrifolia The results in Figure 1 indicated that the fresh weight of B. integrifolia increased in treatments applied BioGro fertilizer as foliar and fertigation however no significant different was found between treatments. The possitive effect of BioGro fertilizer to the accumulate biomass in plant only found at application of fertilizer rate 1.5 RD (treatment CT7) compared to that in control. That might be the activities of microorganisms in fertilizer could enhanced the uptake of nutrition and helped metabolisms were stronger so the biomass of plant were increased [6, 7]. Therefore, in contaminated soil by Cu and Zn, it would apply micro fertilizer to stimulate growth of plant. The positive effect of micro-organic fertilizer to the productivity of cabbage was also found in the research of other author [8]. 2.2.3. Effect of BioGro fertilizer to total chlorophyll content in leaf of B. integrifolia These photosynthetic pigments have an important role in the formation of the products of photosynthesis. In the photosynthetic pigment, chlorophyll is the most important pigment. Total chlorophyll content has a decisive role to photosynthetic capacity of plants. Therefore, total chlorophyll content has an important role for the growth, development, yield formation and quality of products after harvest. Table 5. Total chlorophyll content in leaf of B. integrifolia Treatments CT 1 (control) CT 2 CT 3 CT 4 CT 5 CT 6 CT 7 Total chlorophyll (mg/g) 0.68a 0.74bc 0.75bc 0.72ab 0.77bc 0.75bc 0.79c % compared to control 100 108.8 110.5 106.5 114.1 110,4 116.9 (Note: Values in the same column followed by the same letter are not significantly different at 0.05) Total chlorophyll content varied from 0.68 – 0.79 mg/g fresh weight, the highest at treatment CT 7 and the lowest at treatment CT1 (control). Total chlorophyll content at treatments applied BioGro fertilizer were significantly higher than control but no different was found between treatment CT3, CT4, CT5, CT 6 and CT7. It is due to the microelement Cu affect chlorophyll biosynthesis and reliability of chlorophyll. However, when the concentration of Cu in soil was high (over the tolerance of plant), the effectiveness of microorganisms was reduced. 158 The effect of using biogro fertilizer for reducing Cu and Zn content in Brassica integrifolia grown… Microorganism fertilizer despite of strengthen the useful organisms that help fixed Cu in the rhizomes but the amount of fixed heavy metals might be limited. 2.2.4. Effect of BioGro fertilizer to vitamin C content One biochemical parameter to indicate the quality of vegetable is vitamin C content in leaf of B. integrifolia. Table 6. Vitamin C content in leaf of B. integrifolia Treatments Vitamin C content (mg %) % compared to control 0.41a ± 0,014 100 CT 1 (control) a CT 2 0.38 ± 0,010 92.7 CT 3 0.37a ± 0,015 89.3 CT 4 0.34a ± 0,014 83.4 CT 5 0.34a ± 0,015 81.9 a CT 6 0.35 ± 0,025 85.6 CT 7 0.34a ± 0,020 82.1 (Note: Values in the same column followed by the same letter are not significantly different at 0.05) The data in Table 6 indicated that the vitamin C content seemed decreasing in all BioGro fertilizer application treatments. However, there was no significantly different between vitamin C content in control and treatments. As we know that Cu is the essential trace element for plants, which is the main component of vitamin C, so Cu content is absorbed by the plant which affects the synthesis of vitamin C. Maybe Cu content in the leaf of BioGro application fertilizer was not as high as in the control then resulted in decreasing vitamin C content in all treatments. 2.2.5. Effect of BioGro fertilizer to nitrate content in leaf of B. integrifolia An importance nutritional quality factor of vegetables is low nitrate content. In this study, almost treatments gave the nitrate content were lower than the maximum limitation of Vietnamese Ministry of Health to B. integrifolia is 500 mg kg-1, except to control and treatment CT 2 in both winter and spring season. Through these data, we could say that the application of BioGro fertilizer as both foliar and fertigation had significant effect to reduce the nitrate content in B. integrifolia. Table 7. Nitrate content in leaf of B. integrifolia Treatments Nitrate content (mg/kg) % compared to control CT 1 (control) 533.1a ± 20.3 100 CT 2 520.7a ± 21.5 97.7 CT 3 408.2c ± 13.2 76.6 CT 4 495.5a ± 11.6 92.9 CT 5 470.5b ± 19.5 88.2 CT 6 445.5b ± 15.4 83.6 CT 7 380.5c ± 6.9 71.4 (Note: Values in the same column followed by the same letter are not significantly different at 0.05) 159 Tran Khanh Van, Do Thi Oanh, Nguyen Phuong Thao and Pham Thi Van 2.2.6. Effect of BioGro fertilizer on the accumulation of Cu and Zn in B. integrifolia According to Vietnamese Ministry of Health, if more than 30 and 40 mg kg-1 of Cu and Zn, respectively, is present in leafy vegetables, these vegetables are unsafe to eat. This study was carried out to with the goal of finding alternatives for safe vegetable production. The applied BioGro fertilizer to contaminated soil by Cu and Zn which aim to positively influence of the reduction of Cu and Zn accumulation in B. integrifolia leaves, and ensure safety for the users. To determine the ability to accumulate Cu and Zn in leaves, after 40 days of plantation, we conducted sampling and analysis of Cu content in fresh leaves. Figure 2. The accumulation of Cu in B. integrifolia In this study, Cu content in root was higher than leaf and stem and the average of Cu content of plant at treatment CT6, CT7 is clearly lower than that in other treatments (reduced from 30.7 to 37.6% in stem and 36.8 to 41.1% in leaf. At rate of 1.5 times higher to RD, the Cu content in leaf and stem decreased significantly. Therefore, the application of BioGro fertilizer by both foliar and fertigation may effect to reduce the Cu content in this plant and made this plant become safe to eat even it grown on Cu and Zn contaminated soil. Figure 3. The accumulation of Zn in B. integrifolia Similar pattern of Zn accumulation in B. integrifolia was found as Cu accumulation. The applications of BioGro fertilizer reduce 17.8 to 27% Zn accumulation in leaf compared to that in control. Fertigation of BioGro fertilizer also had a positive effect to Zn accumulation, reduced from 19.5 to 41.3% to control. Moreover, the application of both foliar and fertigation methods showed the lowest Zn content in leaf of this vegetable. That is due to microorganism fertilizer contain bacteria such as BHCM7-VK2 and DHCM20-AMF4. Activities of these useful bacteria helped improve soil and reduce toxicity when Cu content in the soil increases [6]. It is the accumulation of a large amount of Cu and Zn in the mycelium and spores of them should have reduced a significant amount of Cu in soil land resulting Cu and Zn concentrations accumulate in 160 The effect of using biogro fertilizer for reducing Cu and Zn content in Brassica integrifolia grown… the leaves were reduced. Besides, the accumulation of Cu and Zn in stems and roots system could also limit the accumulation of Cu and Zn onto leaves. Therefore, if the soil is contaminated by Cu and Zn, it could suggest applying BioGro fertilizer as fertigation or both foliar and fertigation to reduce the content of Cu and Zn in edible parts of vegetable so that such vegetable is safe to eat. 3. Conclusions The application of BioGro fertilizer in both foliar and fertigation had a positive effect to some physio-chemical parameters of B. integrifolia such as: Plant’s height, fresh weight increased significantly with the application of BioGro fertilizer in 1.5 times RD; Total chlorophyll content in experiment treatments were higher than those in control; Vitamin C content in leaf was not effected by Biogro fertilizer’s application in this study; The content of nitrate in leaves of experiment treatments were lower than the Ministry of Health’s maximum threshold (500 mgkg -1). Fertigation had more effect to reduce the Cu accumulation in plant in comparison to that foliar method. Using 1.5 times as recommended dose resulted in lower Cu content in leaves than RD. The Zn accumulation in plant was significantly lower in treatment by fertigation than that in foliar application thus safe to consumers. 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