Effects of Bacillus amyloliquefaciens Biofertilizer on Tea Yield and

Agricultural Science & Technology, 2014, 15(11): 1883-1887 Copyright 訫 2014, Information Institute of HAAS. All rights reserved

Molecular Biology and Tissue Culture

Effects of Bacillus amyloliquefaciens Biofertilizer on Tea Yield and Quality Zhihui BAI1*, Chenguang ZHOU1,2, Jianxi CAO3, Shengjun XU1, Shanghua WU1, Desheng LI2 1. Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; 2. College of Environment Science and Safety Engineering, Tianjin University of Technology, Tianjin 300384, China; 3. Tangshan Environmental Protection Bureau Fengrun Branch, Tangshan 064000, China

Abstract [Objective] This study was aimed to explore the effects of Bacillus amyloliquefaciens biofertilizer on tea yield and quality. [Method] The field plot experiment was conducted with the biofertilizer treatments and control to investigate 100-bud weight and main biochemical components. [Result] The treatments by Bacillus amyloliquefaciens biofertilizer, which was fermented using sweet potato starch wastewater (SPSW) as culture medium, improved 100-bud weight and tea quality significantly under the concentration of 0.8×108, 1.6×108 and 3.2×108 cfu/ml with the dose of 1 L/m2 for 4 times. At the optimum concentration of 1.6 ×108 cfu/ml, the biofertilizer treatment increased the 100-bud weight by 22.3% , water extracting materials by 21.9%, amino acids content by 8.83% , tea polyphenol content by 9.76% , and decreased theine content by 8.32% , respectively. Compared with the control, there was no significant difference between the SPSW treatment and the control. [Conclusion] The production of the B. amyloliquefaciens biofertilizer could consume SPSW, and the application of the biofertilizer could improved the tea yield and quality, which provided references for the development of ecological agriculture. Key words Bacillus amyloliquefaciens; Sweet potato starch wastewater; Tea; 100bud weight; Amino acid

ea is an important economic crop in China[1], and with the increase of population and the improvement of living standards, the consumption quantity and quality of tea is increasing. According to the data of the United Nations Food and Agriculture Organization (FAO), the tea consumption in increased from 537 800 t to 1 017 100 t in China from 2002 to 2009 [2-3]. Fertilization is one of the most important agricultural measures to increase tea yield, quality and sustainable development of tea garden [1, 4] . Currently, there are relatively many reports on tea fertilization, which are mainly focused on the application of chemical fertilizer, organic fertilizer and leguminous green manure [3, 5 -6], while there are few reports on the application of biofertilizer in tea gardens. In recent years, the tea garden environmental problems and ecological

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problems caused by the blind use and unreasonable fertilization mode have become increasingly prominent [ 7 -8] , however, with the characteristics of environmental protection and high efficient, biofertilizer has become the focus of research that special research on biofertilizer has been carried out in the developed countries like the USA, Japan [9-10]. China has also made some progress in the research and application of biofertilizer that various plant growth promoting bacteria (PGPB) have been isolated and applied in agriculture and forestry[11]. Bacillus amyloliquefaciens, a kind of PGPB, has very strong vitality and field planting ability [ 12 ] . The study of Kim et al. [13] showed that applying the fermentation broth of B. amyloliquefaciens had significant increasing effects on the germination and growth of watermelon. According to the existing lit-

Supported by the Key Research Program of the Chinese Academy of Sciences (KZZDEW-09-3, KZZD-EW-11-03); National Science and Technology Major Project (2014ZX07204-005). *Corresponding author. E-mail: [email protected] Received: September 28, 2014 Accepted: Novembr 1, 2014

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解淀粉芽孢杆菌微生物菌剂对茶叶产量和品质的影响白志辉 1*，周晨光 1，2，曹建喜 3，徐圣君 1，吴尚华 1，李德生 2 （1. 中国科学院生态环境研究中心，北京 100085 ；2. 天津理工大学环境科学与安全工程学院，天津 300384 ；3. 唐山市环境保护局丰润分局，唐山 064000 ）摘要 [目的] 考察解淀粉芽孢杆菌（Bacillus amyloliquefaciens ）微生物菌剂对茶叶产量和品质的影响。 [ 方法 ] 采用田间小区试验，设置对照组和不同菌剂使用浓度的处理组，测定各小区茶叶的百芽重和主要生化指标。 [结果] 以甘薯淀粉废水为培养基的解淀粉芽孢杆菌微生物菌剂，在 0.8×108、1.6×108、 3.2×108 cfu/ml 的使用浓度下，按 1 L/m2 的使用量，施用 4 次（约 16 个月）后，茶叶的百芽重和主要品质指标都有明显提高；其中，菌剂使用浓度为 1.6×108 cfu/ml 比较适宜，百芽重增加 22.3% ，茶叶中水浸出物提高 21.9% ，氨基酸提高 8.83% ，茶多酚增加 9.76% ，咖啡碱降低 6.36% ，而只将甘薯淀粉废水培养基稀释后直接使用，与不施肥的对照组无显著差异。 [ 结论] 解淀粉芽孢杆菌微生物菌剂的生产可以消纳甘薯淀粉废水，应用可以显著提高茶叶的产量和品质，为生态农业的发展提供了借鉴。关键词解淀粉芽孢杆菌；甘薯淀粉废水；茶叶；百芽重；氨基酸

基金项目中国科学院重点部署项目（KZZDEW-09-3 ，KZZD-EW-11-03 ）；国家科技重大专项（2014ZX07204-005 ）。作者简介白志辉（1971- ），男，河北内丘人，副研究员，博士，主要从事环境生物技术与微生物生态学研究，E-mail：[email protected]。 * 通讯作者。

收稿日期修回日期

2014-09-28 2014-11-01

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Agricultural Science & Technology

erature reports, B. amyloliquefaciens has an inhibitory effect on over 20 kinds of plant diseases and insect pests as well as fungal attack [14-19] . In other words, B. amyloliquefaciens has the double functions as biological pesticides and biological fertilizer. However, there is no report on the effect of B. amyloliquefaciens biofertilizer on tea yield and quality up to now . in this paper , the effects of B . amyloliquefaciens fertilizer on tea yield and quality was studied by applying the B. amyloliquefaciens biofertilizer, which was fermented using sweet potato starch wastewater (SPSW) to the tea garden, with the aim to provide certain scientific basis for the new development of ecological tea garden.

Culture of Bacillus amyloliquefaciens The B. amyloliquefaciens strains saved on the slant were inoculated into LB medium, and were made into liquid seed after shaking culture at 200 r/min for 36 h in the constant temperature shaking table of 32 ℃ . The liquid fermentation medium was made with the starch wastewater (also called sweet potato cell sap with COD of about 20 000 mg/L) from the sweet potato starch factory as the raw material through sterilizing for 20 min under 115 ℃ with pH adjusted to 7.0. And then 5% of seed liquid was inoculated into the liquid fermentation media, which were then continued with the shaking culture at 200 r/min in the constant temperature shaking table under different temperature. Samples were taken every 2 h, and plant count method was used to measure the living cell concentration in the fermentation liquid to plot the growth curve. Experimental design and fertilization treatments There were 5 treatments set in the test: treatment 1, the blank control (CK); treatment 2, sweet potato wastewater (FCK); treatment 3, 1% of B. amyloliquefaciens fermentation broth (0.8 × 108 cfu/ml) (H); treatment 4, 2% of B. amyloliquefaciens fermentation broth (1.6 ×108 (cfu/ml) (M); treatment 5, 4% of B. amyloliquefaciens fermentation broth (3.2 ×108 cfu / ml ) (L). Fertilization was applied in the way of furrow dressing. On December 15, 2010, the B. amyloliquefaciens fermentation broth (8 ×109 cfu/ml) was diluted 25 times, 50 times and 100 times with the SPSW, and then the diluted fermentation broths were applied to the H, M and L treatment field correspondingly with the application amount of 1 000 ml/m2, while treatment FCK applied with SPSW according to the application amount of 1 000 ml/m2, and no fertilizer to CK. B. amyloliquefaciens biofertilizer (fertilizer) was applied again on February 28, 2011, November 25, 2011 and February 28, 2012 with the same fertilization mode and fertilizing amount as the basic fertilizer. Measuring items and treatments On April 4, 2012, tea leaves were randomly picked at three sites in each

Materials and Methods Materials Generation situation of the study plot: "Fujian Baihaozao" is a quadrennial tea variety, which is planted in Hunan Xiangfeng Organic Tea Garden (113°19′E, 28°33′N) with an average elevation of 135 m where the landforms are mainly gentle slope hills. The area belongs to typical subtropical back humid monsoon climate with an annual average rainfall of 1 386 mm, average temperature of 16.5-34.5 ℃, and relative humidity of 65% -95% . The study plot with an area of about 1 000 m2 was a whole hilly flat. The experimental tea trees were planted in east-west direction with the row spacing of 100 cm, and the isolation belt was arranged between different treatments. All tea plants were under unified management, showing no obvious difference in growth, and regular fertilizing was not applied to the tea garden the year before the test. There were 3 repetitions for each treatment in random arrangement. SPSW: the SPSW (COD of about 20 000 mg/L) was produced by the Sweet Potato Processing Institute of Changsha Jinshu Food Co., Ltd. B. amyloliquefaciens: it was an A3 strain of B. amyloliquefaciens isolated from the rhizosphere soil of aquatic plant by our group, and it was preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 6676.

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treatment zone selected by using the 33 cm × 33 cm plank frame and the picking locations were marked. And then on April 9 and April 14, leaves were picked again at the marked places. At each picking, the bud density and 100-bud fresh weight were recorded. The quality components of the tea leaves were determined by measuring the new shoots of the buds with two leaves picked on April 4. The water extract of the tea samples were measure by using the boiling reflux extraction method (GB/T 83052002); tea polyphenols using iron tartrate colorimetric method (GB/T83132002); total amino acids using indene three ketone colorimetric method (GB/T8314-2002); theine determined by ultraviolet spectrophotometry (GB/T8312-2002). Quality index was detected through biochemical analysis after deactivating and fixing the fresh tea leaves in the oven and deducting water. Test data calculation and processing method The Excel 2010 and SPSS 20 were used for data analysis, and LSD multiple comparison method was to conduct the significant test.

Results and Analysis Culture of B. amyloliquefaciens The optimal culture temperature for B. amyloliquefaciens using SPSW as the raw material was preliminary explored, and the results were shown in Fig.1. The results showed that the cell concentration could reach 8 ×109 cfu/ml after culturing for 17 h at 32 ℃, which was consistent with the national standards of agricultural microbial fertilizer (GB20287-2006) (liquid inoculant ≥ 2×108 cfu/ml) requirements.

Fig.1 The growth curves of B. amyloliquefaciens at different culture temperatures

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Effects of Bacillus amyloliquefaciens fermentation broth on bud density As shown in Table 1, compared with the CK, treatment H and M showed significant increase in bud density at all three picking period. The bud density of treatment L showed significant increase from the treatment of those picked on April 4, and then with the density decreased, indicating that treatment L showed certain limitations in increase the bud density of tea. The probable reason might be that the concentration of the fermentation broth of treatment L was low, thus the lasting effect of probitics and nutrient substances became gradually insignificant. However, the mean values of the sampling points at different periods showed that all treatments increased the bud density of tea leaves significantly that compared with the CK, FCK increased by 4.6%, H by 36.3%, M by 42.1% and L by 21.6% , among which

treatment M showed the best effects with the largest increase. Effects of Bacillus amyloliquefaciens fermentation broth on 100bud weight The field trials (Table 2) showed that there was no significant change in 100-bud weight in treatment FCK, while the treatments applied with B . amyloliquefaciens fermentation broth showed significant increase in 100-bud weight, but there was no significant difference among the 3 treatments with different concentrations of B.amyloliquefaciens fermentation broth. Compared with the CK blank control, the application of B. amyloliquefaciens fermentation broth could increase the 100-bud weight by an average of 14.3% -22.3% . Treatment M showed the largest 100 -bud weight increase. Table 1 and 2 showed that treatment M also had the largest tea bud density, followed by treatment H, L, reflecting that B. amyloliquefaciens

Table 1 Effect of Bacillus amyloliquefaciens fermentation broth on bud density Treatment H

Bud density∥number/m2 April, 3rd

April, 9th

April 14th

Mean number/m2

Relative percentate∥%

570±32 a

363±36 ab

486±16 a

473±14 a

136

M

603±16 a

384±14 a

492±23 a

493±2 a

142

L

516±23 b

324±18 bc

426±10 b

422±9 b

122

FCK

435±14 c

294±23 cd

360±9 c

363±11 c

105

CK

393±29 c

276±10 d

372±10 c

347±10 c

100

Different letters among the treatments indicate significant differences at the level of P < 0.05. Table 2 Effect of Bacillus amyloliquefaciens on 100-bud weight Treatment H

100-bud weight//g April, 3

rd

April, 9

th

April 14

th

Mean weight∥g

Relative percentage∥%

23.96±1.38 a

22.01±0.98 a 22.74±2.69 a 22.90±1.25 a

121.7

M

24.10±1.54 a

23.47±0.51 a 21.46±2.56 a 23.01±1.39 a

122.3

L

23.61±0.28 a

15.59±1.08 b 24.38±0.36 a 21.52±0.39 a

114.3

FCK

20.75±0.95 b

11.65±1.42 c 21.37±1.74 a 17.92±0.83 b

CK

22.71±1.88 ab 10.34±0.67 c 23.42±0.34 a 18.82±0.31 b

95.2 100

Different letters among the treatments indicate significant differences at the level of P < 0.05. Table 3 Effect of Bacillus amyloliquefaciens on tea quality Treatment H

Water extract g/kg DW

Free amino acid g/kg DW

Tea polyphenol g/kg DW

Theine g/kg DW

565.1±15.8 b 27.63±0.03 a 259.2±1.68 b 41.66±0.33 d

Ratio of polyphenols and amino acids

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fermentation broth significantly increased the bud density and 100-bud weight and further improved tea yield. Effect of Bacillus amyloliquefaciens fermentation liquid on the quality of tea As shown in Table 3, compared with the CK, treatment FCK showed no significant changes in tea water extract and included biochemical components, thus there was no significant influence on tea quality. The tea amino acids and polyphenols content of the treatments applied with B. amyloliquefaciens fermentation broth showed great increase, all of which reached the significant level. Treatment M had the highest amino acid and tea polyphenols content, which increased by 8.82 and 9.76% respectively to the control CK. In addition, there were significant differences in tea polyphenols content among the groups treated with different concentrations of B. amyloliquefaciens fermentation broth, which also showed different degrees of decrease in theine content. Compared with the CK, the decrease of treatment M and H all reached significant level. Moreover, there were also significant differences in the theine contents among the treatments with different concentrations of B. amyloliquefaciens fermentation broth. It was probably related with the significant increase of amino acids and tea polyphenol of these treatmetns, since there always existed a trading off and taking turns relationships among the secondary products of the carbon and nitrogen metabolism of tea plant [6]. Table 3 showed that the water extracts of the treatments applied with B. amyloliquefaciens fermentation broth also increased significantly, which compared with the CK increased by 14.0% 21.9%; phenol ammonia ratio also increased by 2.18% -3.05% , but there was no significant difference between the treatments with different concentrations of B. amyloliquefaciens fermentation broth.

9.38±0.07 a

M

602.8±13.3 a 27.99±0.15 a 264.5±0.28 a 42.55±0.19 c

9.45±0.04 a

L

570.1±15.5 b 26.32±0.36 b 241.3±1.28 c 44.98±0.34 b

9.37±0.11 a

FCK

513.3±13.6 c 25.71±0.30 c 231.7±1.14 d 45.87±0.19 a

9.01±0.07 b

CK

494.4±14.6 c 25.72±0.41 c 241.1±1.85 c 45.44±0.19 ab

9.17±0.12 b

Different letters among the treatments indicate significant differences at the level of P < 0.05.

Conclusions and Discussion Bacillus amyloliquefaciens is a kind of plant growth promoting bacteria, belonging to the gram-positive bacteria. The study of LIU et al. [20] showed

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that after shooting for 5 d, the soil applied with B. amyloliquefaciens fermentation broth showed a 33.96% , 72.38% and 108.62% increase in the fresh weight, plant height and root length of maize compared with the control. SHEN et al. [21] inoculated the tobacco seedlings treated by B. amyloliquefaciens fermentation broth with TWV virus, and the results showed that the tobacco seedlings treated with B. amyloliquefaciens fermentation broth showed good growth trend, thick and green leaves, and the fresh weight, largest leaf length and width length of the treatment increased by 8.99% , 18.09% and 28.26% from the control. The test results indicated that B. amyloliquefaciens biofertilizer (fertilizer) could not only promote the growth of plant but also could inhibit the infection of pathogens. However, there was no report on the application of B. amyloliquefaciens fermentation broth to tea garden. In this study, SPSW was used to produce the B. amyloliquefaciens fermentation broth, which was then applied to the tea garden soil in the furrow application mode. The results showed that the B. amyloliquefaciens fermentation broth produced by SPSW reached the national agricultural standard that the microbial biomass reached up to 8 × 109 mlcfu/ml, and it was a new way for biofertilizer production with the advantages of reducing wastewater emission and low production cost. Compared with the treatments with no fertilizer application, the application of B. amyloliquefaciens biofertilizer could significantly improve tea yield and quality in increasing 100-bud weight by 14.3% -22.3% , water extract by 14.3%-21.94%, amino acid by 2.33%8.83% , tea polyphenols by 7.53% 9.76%, ratios of polyphenols to amino acid by 2.18%-3.06%, while reducing the theine by 6.36%-8.32%. It indicated that B. amyloliquefaciens could promote plant growth and improve quality. The mechanisms include the follows: first, some amino acids and lipid substances produced by the B. amyloliquefaciens could promote the growth and development of plants and the balance of soil mineral nutrients, such as the phytase generated from the metabolism [22], which could help to

convert the unabsorbable organic phosphorus in the soil into the absorbable phosphorus, improving phosphorus absorption efficiency of plants[23]. Second, B. amyloliquefaciens has inhibitory effect on various plant diseases and insect pests as well as tea fungus, which could reduce pests and diseases as well as adjust the microflora environment around the tea root system and balance soil nutrient elements, providing a suitable growth environment for tea plants, which is conducive to root development and nutrient absorption of tea plants [24]. In addition, many plant growth promoting bacteria have been verified to produce plant growth regulator in plant rhizosphere, such as IAA, CTK and other plant hormones, to promote plant growth and increase yield [25]. In this study, with furrow application as the fertilization mode, when the function range of B. amyloliquefaciens was certain, the growth -promoting effect improved with the increase of concentration, and the tea yield and quality were also increased. However, if the concentration was too high, the effects decreased. Therefore, it should control the application mode and concentration of biofertilizer in furrow application. In this study, using SPSW to ferment B. amyloliquefaciens to prepare the biofertilizer not only reduced pollutant emissions, but also made full use of the starch wastewater. In the meantime, the furrow application of B. amyloliquefaciens biofertilizer (fertilizer) could significantly improve the tea yield and quality. However, there were multiple ways of the microbial actions on plants, and furrow application also had the characteristics of large fertilization amount, long fertilization cycle and lasting fertilization effects. Therefore, it should optimize the application mode of leave surface fertilization with B. amyloliquefaciens as well as the combined application mode with chemical fertilizers. On the other hand, it also should carry on further study on the mechanism of the promoting effects of B. amyloliquefaciens on plant growth.

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Responsible proofreader: Xiaoyan WU Responsible editor: Na LI !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! (Continued from page 1864) type 2 isolates collected from some 2007, 48(8): 811-819. area of China with a PCR( 中国部分地 [14] LANG HW( 郎洪武 ), ZHANG GC( 张广 [9] CHEUNG AK. Transcriptional analysis 川 ), WU FQ ( 吴发权 ), et al. Detection 区猪圆环病毒 2 型的基因型分析 )[J]. of porcine circovirus type 2[J]. Virology, of serum antibody against postweanActa Veterinaria et Zootechnica Sinica 2003, 305(1): 168-180. ( 畜牧兽医学报 ), 2009, 40(9): 1358ing multisystemic wasting syndrome in [10] NAWAGITGUL P, MOROZOW I, 1362. pigs ( 断奶猪多系统衰竭综合征血清抗 BOLIN SR, et al. Open reading frame 体检测 )[J]. Chinese Journal of Veteri[18] XU N( 徐娜 ), LI BY( 李宝玉 ), LOU ZZ( 娄 2 of porcine circovirus type 2 encodes 忠子 ), et al. Advances in porcine cirnary Science and Technology ( 中国兽 a major capsid protein [J]. J Gen Virol, 医科技 ), 2000, 30(3): 3-5. covirus type II( 猪圆环病毒 II 型的研究 2000, 81(9): 2281-2287. 进展 )[J]. Biotechnology Bulletin ( 生物 [15] GE X, WANG F, GUO X, et al. Porcine [11] LIU J, CHEN I, KWANG J. Characteri技术通报 ), 2010, 6: 22-26. circovirus type 2 and its associated zation of a Previously Unidentified Vi[19] GRAU-ROMA L, CRISCI E, SIBILA M, diseases in China [J].Virus Res, 2012, ral Protein in Porcine Circovirus Type et al. A proposal on porcine circovirus 164(1-2): 100-106. 2-Infected Cells and Its Role in Virustype 2 (PCV2) genotype definition and [16] WANG XM( 王小敏 ), ZHANG WW( 张 Induced Apoptosis [J]. J Virol, 2005, 文文 ), HE KW ( 何孔旺 ), et al. Genotheir relation with postweaning multi79(13): 8262-8274. type analysis of porcine circovirus type systemic wasting syndrome (PMWS) [12] OLVERA A, CORTEY M, SEGALES J. 2 in eastern China from 2009 to 2010 occurrence [J]. Vet Microbiol,, 2008, Molecular evolution of porcine cir(2009-2010 年华东地区猪圆环病毒 2 128(1-2): 23-35. covirus type 2 genomes: phylogeny 型的基因型分析 ) [J]. Jiangsu Journal [20] HAMEL AL, LIN LL, SACHVIE C, et al. and clonality [J]. Virology, 2007, 357 of Agricultural Sciences ( 江苏农业学 PCR detection and characterization of (2):175-185. 报 ), 2011, 27(5): 1037-1042. type -2 porcine circovirus [J]. Can J [13] GAGNON CA, TREMBLAY D, TI[17] LI WJ ( 李文洁 ), LI WT ( 李文涛 ), YAN Vet Res, 2000, 64:44-525. JSSEN P, et al. The emergence of WD( 严伟东 ), et al. Survey on molecuporcine circovirus 2B genotype (PCVlar epidemiology of porcine circovirus 2B) in swine in Canada[J]. Can Vet J, Responsible editor: Xiaohui FAN

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