INTRODUCTION
HA are organic C-rich materials, which comprise the major fractions of humic substances (Canellas et al., Reference Canellas, Olivares, Okorokovha-Facanha and Facanha2002). They exhibit beneficial effects on reducing soil compaction and increasing nutrient availability (Nardi et al., Reference Nardi, Pizzeghello, Muscolo and Vianello2002). Many reports showed that HA enhanced seed germination and root, shoot and leaf growth in different plant species, such as cucumber, maize, pelargonium and wheat (Chen et al., Reference Chen, De Nobili and Aviad2004; Mora et al., Reference Mora, Bacaicoa, Zamarreno, Aguirre, Garnica, Fuentes and Garcia-Mina2010; Philippe et al. Reference Philippe, Boris and Marie2010). Treated plants showed a faster development and reached reproductive stage 3 to 5 days earlier than the control. However, the mechanism responsible for the function of HA is not yet clear. Some authors propose that humic substances promote plant growth by affecting hormone metabolism. HA had auxin activity on lateral root proliferation (Canellas et al., Reference Canellas, Olivares, Okorokovha-Facanha and Facanha2002; Zandonadi et al., Reference Zandonadi, Canellas and Facanha2007). Humic substances and its derivatives activated the auxin synthetic reporter (Dobbss et al., Reference Dobbss, Canellas, Olivares, Aguiar, Pereira, Azevedo, Spaccini, Piccolo and Facanha2010). The further research found that the beneficial effects of humic substances on shoot development could be directly associated with several active cytokinins and polyamines (Mora et al., Reference Mora, Baigorri, Bacaicoa, Zamarreño and García-Mina2012). On the other hand, Bidegain et al. (Reference Bidegain, Kaemmerer, Guiresse, Hafidi, Rey, Morard and Revel2000) found that humic substances from the chemically decomposed sawdust could increase total P uptake and yield in ryegrass by enhancing root development. Humic substances also could activate the root plasma membrane H+-ATPase activity and increase nitrate uptake rates in roots (Nardi et al., Reference Nardi, Pizzeghello, Muscolo and Vianello2002; Quaggiotti et al., Reference Quaggiotti, Ruperti, Pizzeghello, Francioso, Tugnoli and Nardi2004). In a low organic matter soil, there was a synergistic effect of HA and fertilizer on plant growth (Hartz and Bottoms, Reference Hartz and Bottoms2010). The above results suggested that humic substances can increase root uptake of mineral nutrients. It is possible that the combined application of HA and chemical fertilizer can accelerate the growth of plants. To investigate this hypothesis, the effects of HANPK chemical fertilizer were examined in field tobacco plants.
MATERIALS AND METHODS
Material incubation and experimental design
The experiments were performed in paddy soils of Binhe village, Guandu small town, Liuyang County, Hunan Province. The basic properties of paddy soil were 12.3 g kg−1 of organic matter, 130.5 mg kg−1 of alkali-hydrolyzable N, 33.6 mg kg−1 of readily available P, 105.2 mg kg−1 of readily available K, and pH 5.8. Four treatments were set as follows: the control; NPK; HA and HANPK. NPK indicated an application of 225 kg ha−1 of NPK (30-10-20) chemical fertilizer. HA indicated an application of 150 kg ha−1 of HA (Water solubility: 1000g kg−1, available N: 43.2 mg kg−1, available P2O5: 26.1 mg kg−1, available K2O: 87 mg kg−1, Produced by Shandong Hangao Biological Engineering Limited Company). HANPK indicated the mixture of 150 kg ha−1 of HA and 225 kg ha−1 of NPK. The control indicated no HA or NPK fertilizer application. 2000 kg ha−1 of organic manure was applied in each treatment as basal fertilizers. At the transplanting stage, 80% of HA, NPK or HANPK fertilizer was applied and 20% of HA, NPK or HANPK was applied at seedling stage (20 days after transplanting). Each treatment had four replicates. The particular application pattern was as follows: During transplanting, HA, NPK or HANPK was applied to a cultivation hole, and then a tobacco seedling was transplanted in the cultivation hole. After that, the seedling roots were covered with soil. There were 16,500 tobacco plants ha−1. The tobacco variety was K326, provided by China Tobacco Hunan Industrial Co. LTD, Changsha, China.
Measurement of root number, root length and root thickness
In tobacco fields, tobacco seedlings were grown for 45 days, and then harvested for root parameter measurements as shown in Tables 1 to 4. Tobacco roots were dug out, soaked in tap water and rinsed thoroughly with deionized water. Then, the numbers of the first- and second-order lateral roots were counted in water respectively. The length of tap root or lateral roots was measured with a ruler. When root length is less than 5 mm, the length or the number is not included in the calculation. Root diameter was measured at the middle point of root length manually and the data were classified into <2mm, 2–5mm and >5mm. After classification, root biomass was measured. In a hydroponic experiment, tobacco seedlings were grown for 16 days after germination. Roots with less than 5 mm-length or 0.5 mm-diameter are not included and counted. Average root length indicated the ratio of the total root length over the total root numbers. The data were shown in Table 6. Mature tobacco leaves were collected and used for analysis of leaf quality indices and leaf yield, please see the data in Tables 7 to 8.
Table 1. Effect of different treatments on the growth of tobacco seedlings.
Note: *Data indicated mean ± SE (n = 4). The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
Table 2. Effect of different treatments on lateral roots of tobacco seedlings.
Note: *Data indicated mean ± SE (n = 4). The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
Table 3. Effect of different treatments on root biomass under different soil layers.
Note: *Data indicated mean ± SE (n = 4). The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
Table 4. Effect of different treatments on root thickness of tobacco seedlings.
Note: *Data indicated mean ± SE (n = 4). The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
Measurement of root activity
Root activity of tobacco seedlings were measured by the method of α-naphthylamine oxidation. After 15-, 30-, 45- or 60-days transplanting, tobacco roots were dug out and washed in tap water and 55 mg L−1 of CaCl2 solution, and then submitted to a solution containing 50 mg L−1 of α-naphthylamine and pH 6.0 phosphate buffer for 10-min shaking slightly. After absorption, 2 mL of the solution was diluted with 10 mL of deionized water. Then, the diluted solution was reacted with 1 mL of sulfamic acid and 1 mL of sodium nitrite. After 25-min incubation, the solution was measured at 510 nm with a 754 spectrophotometer. Root activity can be calculated according to the oxidation of α-naphthylamine. For the details, please see Zhou (Reference Zhou1995).
Effect of TIBA on the growth of lateral roots
After germination, tobacco seedlings were grown in a nutrient solution (consisting of 240 mg L−1 CO(NH2)2, 505 mg L−1 KNO3, 118 mg L−1 Ca(NO3)2•4H2O, 136 mg L−1 KH2PO4, 246 mg L−1 MgSO4•7H2O, 22.5 mg L−1 EDTA-Fe, 0.125 mg L−1 CuSO4•5H2O, 2.8 mg L−1 H3BO3, 2.4 mg L−1 ZnSO4•7H2O, 2 mg L−1 MnSO4•H2O, 0.098 mg L−1 (NH4)2Mo7O4•4H2O) for 12 days, then submitted to nutrient solution containing 10 μmol L−1 2,3,5-triiodobenzoic acid (TIBA), a chemical inhibitor of polar auxin transport (Lomax et al., Reference Lomax, Muday and Rubery1995; Zhou et al., Reference Zhou, Yin and Xu2003) for 6 days. After treatment, the number and length of tobacco roots were counted using a ruler. The treatment solution was changed each week. TIBA was firstly dissolved in dimethyl sulfoxide (DMSO) as a stocking solution. When treatment was performed, TIBA was applied to the nutrient solution and its concentration was 10 μmol L−1. DMSO in the solution was less than 1 μmol L−1. The effect of DMSO on root growth was negligible in the experiment.
Measurement of chemical component of tobacco leaves
When tobacco leaves were mature, the 13th and 14th leaves from the top were harvested, baked and used for analysis of chemical components, including total sugar, reducing sugar, nicotine, total N (nitrogen) and K (potassium). After digestion with H2SO4-H2O2, N was measured by the Kjeldahl nitrogen determination method, K was determined by a flame spectrophotometry method. Total sugar, reducing sugar and water soluble sugar were measured by a copper reduction–titration method. Nicotine was determined by a Uv-vis spectrophotometry method. Shmuck value means the ratio of water soluble sugar and total protein. For more details, please see Bao (Reference Bao2000) and Wang (Reference Wang2003).
Statistical analysis
The experiments were performed in a completely randomized design and each treatment had four replicates. The experiment was replicated twice independently. The data were analysed statistically by Microsoft Office Excel 2000 and by Duncan's New Multiple range test, p < 0.05. Significant difference was analysed by using SAS software (SAS 8.1).
RESULTS
Effect of HANPK on the growth of tobacco roots
Many previous reports indicated that HA from various materials and compost could promote plant growth (Adani et al., Reference Adani, Genevini, Zaccheo and Zocchi1998; Bidegain et al., Reference Bidegain, Kaemmerer, Guiresse, Hafidi, Rey, Morard and Revel2000; Daur and Bakhashwain, Reference Daur and Bakhashwain2013; Mora et al., Reference Mora, Bacaicoa, Zamarreno, Aguirre, Garnica, Fuentes and Garcia-Mina2010; Mora et al., Reference Mora, Baigorri, Bacaicoa, Zamarreño and García-Mina2012). In this study, we examined the effect of HANPK on the growth of flue-cured tobacco roots in fields (Table 1). Results indicated that HANPK increased both root and shoot biomass of tobacco seedlings significantly. At 45 days after transplanting, shoot biomass with NPK, HA and HANPK treatment were 121.3%, 109% and 137.9% of that of the control. The corresponding values for root biomass were 117.9%, 106.3% and 129.7% respectively. However, HA, NPK or HANPK did not influence the ratio of root to shoot obviously.
To explore the mechanism of HANPK in enhancing tobacco growth, the number and average length of lateral roots were measured under different treatments. Results from Table 2 indicated that NPK, HA or HANPK did not influence the length of tap roots, and the number of the first-order lateral roots and their lengths significantly. Interestingly, HA or HANPK seemed to stimulate the growth of the second-order lateral roots. Compared with the control, the numbers of the second-order lateral roots under HA and HANPK were increased by 61.9% and 89.3% respectively. However, the average length of the second-order lateral roots did not vary greatly under different treatments (Table 2). Canellas et al. (Reference Canellas, Olivares, Okorokovha-Facanha and Facanha2002) also reported that treatment with HA could produce a hyper-induction of maize lateral roots. The proliferation of lateral roots induced an increase in both total radicular length and root surface area, which would benefit the uptake of water and nutrients.
In tobacco fields, the distribution of root biomass was also investigated in different soil layers using a root-digging method. The total soil profile was divided into three layers, i.e. 0–20 cm, 20–40 cm and 40–60 cm. Results indicated that most of root biomass was localized in 0–20 cm soil layer (Table 3). Root biomass in 0–20 cm soil layer was 72.9% to 81.6% of that in the total soil profile among different treatments. Compared with the control, HANPK increased root biomass by 51.1% in 0–20 cm soil layer and 34.9% in 0–60 cm soil layer respectively. A single application of NPK or HA influenced the distribution of root biomass in total soil layer slightly.
In addition, we measured the effect of HANPK on root thickness of tobacco seedlings (Table 4). Results indicated that root biomass with the diameter less than 2 mm under HA and HANPK was 43.7% and 64.8% higher than that of the control. NPK treatment did not enhance root biomass with less than 2 mm diameter in comparison to the control. The root biomass with 2–5 mm root diameter showed a similar varying pattern to the root biomass less than 2 mm root thickness among different treatments. No significant differences of root biomass with the diameter bigger than 5 mm were observed among different treatments. The results suggested that treatment with HA or HANPK contributed to the growth of more fine roots in comparison to the control or NPK treatment.
Effect of different treatments on the activity of tobacco roots
Except root biomass, we also measured the activity of tobacco roots at different transplanting days. Results from Table 5 indicated that HANPK stimulated the activity of tobacco roots obviously at early growth stages. Root activity with HANPK treatment was 128.4% and 143.2% of the control at 15 and 30 days after transplanting respectively. While treatment with NPK or HA influenced the activity of tobacco roots slightly in comparison to the control. At later growth stages (45 days or 60 days after transplanting), no significant differences of root activity were observed among different treatments (Table 5).
Table 5. Effect of different treatments on root activity of tobacco seedlings.
Note: *Data indicated mean ± SE (n = 4). The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
FW = fresh weight.
Inhibitory effect of TIBA on lateral root growth
HANPK stimulated the growth of lateral roots of flue-cured tobacco seedlings significantly (Table 2). To explore whether auxin was involved in or not, the effect of TIBA, an inhibitor of polar auxin transport on lateral root growth was investigated hydroponically as shown in Table 6. Results showed that the length of the first-order lateral roots and the number of the second-order lateral roots with HANPK treatment was increased by 32.3% and 58.6% in comparison to the control. But the addition of 10 μmol L−1 of TIBA to HANPK solution inhibited HANPK stimulating effect obviously (Table 6). Average length of the first- and the second-order lateral roots and number of the second-order lateral roots were reduced by 32.5%, 33% and 48.2%, respectively. Interestingly, TIBA did not influence the length of tap roots. Since the function of TIBA mainly blocked polar auxin transport, it was suggested that the stimulating effect of HANPK on lateral roots might be associated with indole-3-acetic acid (IAA) synthesis or transport (Table 6).
Table 6. Effect of TIBA and HANPK on lateral root growth of tobacco seedlings.
Note: *Data indicated mean ± SE (n = 4). The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
Effect of different treatments on tobacco leaf chemical components
Leaf chemical components are the major indices of tobacco leaf quality, which include total sugar, reducing sugar, nicotine, total nitrogen, potassium, etc. When these components were coordinated with each other, the quality of flue-cured tobacco leaves tended to be harmonious (Wang, Reference Wang2003; Xu et al., Reference Xu, Li and Xiao2008). Results from Table 7 indicated that different chemical components showed various varying patterns greatly. Total sugar and reducing sugar did not change greatly among different treatments although HANPK tended to increase their contents. HANPK treatment increased K uptake significantly. In comparison to the control, leaf K content with NPK, HA and HANPK treatment was elevated by 11.2%, 3.4% and 19.5%, respectively. NPK treatment increased nicotine content by 19.4%, while no differences of nicotine content were observed among HANPK, HA and the control. The results suggested that HA combined NPK compound fertilizer contributed to the elevation of the quality of tobacco leaves. Wang (Reference Wang2003) indicated that the quality of tobacco leaves increased with increasing sugar content. Low N content of tobacco leaves was better than that of high N content. The content of nicotine ranging from 1.5 to 3.5 was suitable for tobacco leaf quality. The data of Table 6 were within the scope of the above values.
Table 7. Effect of different treatments on the content of chemical components of flue-cured tobacco leaves.
Note: *Data indicated mean ± SE (n = 4). The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
Effect of different treatments on the main quality index ratio of flue-cured tobacco leaves
We also examined the main quality indices including shmuck value (water soluble sugar/total protein), nitrogen/nicotine, reducing sugar/nicotine and reducing sugar/total nitrogen as shown in Table 8. Shmuck value was not different among different treatments. Many reports indicated that it is suitable for highly qualified tobacco leaves when shmuck value approaches 3, and the ratio of reducing sugar to nicotine and the ratio of reducing sugar to nitrogen range from 6 to 10 (Wang, Reference Wang2003; Xu et al., Reference Xu, Li and Xiao2008). In this study, the above two indices ranged from 6.8 to 10.4, which were within the above scope. Noteworthy, HANPK increased the ratio of reducing sugar to nicotine by 13.6%. However, shmuck value and nitrogen/nicotine did not vary significantly (Table 8).
Table 8. Effect of different treatments on main quality indices of flue-cured tobacco leaves.
Note: *Data indicated mean ± SE (n = 4). The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
Effect of different treatments on the economic characteristic indices of flue-cured tobacco leaves
Tobacco yield, average price, output value and proportion of superior and medium leaves of flue-cured tobacco were the main economic characteristic indices, which reflected the quality and economic effect of tobacco leaves (Wang, Reference Wang2003). Results from Table 9 showed that HANPK significantly increased tobacco yield, proportion of superior and medium tobacco leaves, and output value in comparison to the control. The corresponding values were increased by 12.2%, 10.1% and 29.4%, respectively. The results suggested that HANPK treatment enhanced leaf yield and quality of flue-cured tobacco plants (Tables 8 and 9).
Table 9. Effect of different treatments on main economic indices of flue-cured tobacco.
Note: *Data indicated mean of four results. The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
Cost, benefits and output input ratio of tobacco leaves were also calculated under different treatments as shown in Table 10. Among four treatments, the input, output and benefit of HANPK were the highest, while those of the control were the lowest. NPK and HA treatment had similar inputs, but the output, benefit and output input ratio were different. HA treatment had a higher output, benefit and output input ratio than NPK treatment. The results suggested that the treatment of HA was better than NPK treatment. Results from Table 10 suggested that HANPK application was a great benefit to the production of tobacco leaves among these four treatments (Table 10).
Table 10. Costs, benefits and output input ratio of tobacco leaves under different treatments.
Note: *Data indicated mean of four results. The data with different letters in the same column indicated significant differences at p ≤ 0.05 (Duncan's test).
DISCUSSION
Many studies have shown that HA could not only enhance root, leaf and shoot growth, but also stimulate the seed germination of various crop species (Adani et al., Reference Adani, Genevini, Zaccheo and Zocchi1998; Bama and Selvakumari, Reference Bama and Selvakumari2009; Bidegain et al., Reference Bidegain, Kaemmerer, Guiresse, Hafidi, Rey, Morard and Revel2000; Canellas et al., Reference Canellas, Olivares, Okorokovha-Facanha and Facanha2002; Daur and Bakhashwain Reference Daur and Bakhashwain2013; Mora et al., Reference Mora, Bacaicoa, Zamarreno, Aguirre, Garnica, Fuentes and Garcia-Mina2010; Mora et al., Reference Mora, Baigorri, Bacaicoa, Zamarreño and García-Mina2012; Tahir et al., Reference Tahir, Khurshid, Khan, Abbasi and Kazmi2011). These positive effects are explained by the direct and indirect interaction of HA and roots at different physiological and metabolic processes. According to the previous results reported in research literatures (Fagbenro and Agboola, Reference Fagbenro and Agboola1999; Jindo et al., Reference Jindo, Martim, Navarro, Perez-alfocea, Hernandez, Garcia, Aguiar and Canellas2012), the positive effect of HA was first observed on plant growth such as the length or biomass of roots and shoots. Our results from Table 1 showed that the combined application of HANPK stimulated the growth of both roots and shoots of flue-cured tobacco plants significantly (Tables 1 and 2). Furthermore, HANPK increased K uptake by 19.5% (Table 7). The results suggested that the combined use of HA and NPK chemical fertilizer not only stimulated tobacco growth, but also enhanced the quality of flue-cured tobacco. In consistent with our results, Zhang et al. (Reference Zhang, Zhou, Zhao, Zhai, Wang, Alva and Paramasivam2013) performed an experiment to examine the effect of combination of chemical compound fertilizer and HA on yield and quality of apple. They found that the addition of HA to chemical compound fertilizers increased apple yield by 35%. In rice, a similar result was also observed that the application of HA combined with chemical fertilizer produced pronounced effects on root volume, root CEC and root length (Bama and Selvakumari, Reference Bama and Selvakumari2009). Cimrin and Yilmaz (Reference Cimrin and Yilmaz2005) indicated that a single application of HA to lettuce did not improve its yield, while the combined application of 120 kg ha−1 of phosphorus together with 300 kg ha−1 of HA increased the head weight of lettuce very much. The combination of HA and NPK chemical fertilizer might improve the availability of rhizosphere nutrients, and enhance the activities of microorganisms and enzymes, which thus stimulate plant growth (Du et al., Reference Du, Wang, Liu, Ma, Ma and Malhi2013; Nardi et al., Reference Nardi, Pizzeghello, Muscolo and Vianello2002).
Turgay et al. (Reference Turgay, Karaca, Unver and Tamer2011) indicated that the application of humic-fulvic substances alone showed different inclinations on selected soil characteristics in consecutive cropping seasons. Application of HA did not show significant effects on plant growth when soil organic carbon was high enough (Bernal et al., Reference Bernal, Alburquerque and Moral2009). Our results from Tables 1 and 9 supported the above view that a single application of HA did not influence tobacco growth and leaf yield significantly. This phenomenon might be associated with high content of active organic carbon in paddy soils. Since tobacco seedlings were planted in paddy soils in the system of tobacco–rice rotation, due to continuous straw returning, the contents of dissolved organic carbon and active organic carbon were high, which might retard the stimulating the effect of a single application of HA.
According to Pinton et al. (Reference Pinton, Cesco, Lacoletig, Astolfi and Varanini1999) and Schmidt et al. (Reference Schmidt, Santi, Pinton and Varanini2007), the beneficial effects of HA on plant nutrition have been attributed to the promotion of root development. Our results agreed with this observation that the combination of HANPK increased both dry weight and root numbers of tobacco plants significantly (Tables 1 and 2). Furthermore, HANPK increased the emergence of the second-order lateral roots greatly. The number of the second-order lateral roots was 189.3% of that of the control (Table 2). Similar results were also observed by other authors (Canellas et al., Reference Canellas, Olivares, Okorokovha-Facanha and Facanha2002; Hartwigsen and Evans, Reference Hartwigsen and Evans2000). In Arabidopsis, HA altered developmental programmes at an early stage of root cell differentiation, which resulted in an increased root absorptive surface area (Schmidt et al., Reference Schmidt, Santi, Pinton and Varanini2007). Modification of root structure might underline the beneficial effects of HANPK on early stages of tobacco growth. Unlu et al. (Reference Unlu, Unlu and Karakurt2010) found that foliar and soil HA fertilization could increase the accumulation of antioxidant compounds such as carotenoids and phenolic compounds in pepper fruit. Increased root activity might be associated with an increase of antioxidant enzyme activity in tobacco roots at under HANPK treatment (Table 5).
HA molecules have been reported to produce similar effects to IAA on root growth (Canellas et al., Reference Canellas, Olivares, Okorokovha-Facanha and Facanha2002; Schmidt et al., Reference Schmidt, Santi, Pinton and Varanini2007; Trevisan et al., Reference Trevisan, Pizzeghello, Ruperti, Francioso, Sassi, Palme, Quaggiotti and Nardi2010; Zandonadi et al., Reference Zandonadi, Canellas and Facanha2007). Interaction between plant hormones and humic substances might contribute to the effects of humic molecules on plant growth and development. Inhibition of IAA synthesis and transport would result in a negative effect on plant growth. Our hydroponic experiment supported the above hypothesis that the addition of TIBA, a polar auxin transport inhibitor, resulted in decreases of the number and average length of lateral roots (Table 6). The beneficial effects of HANPK on plant development suggest a possible involvement of auxin synthesis and transport. The phytohormone auxin was required to regulate the initiation and emergence phases of lateral root development (Casimiro et al., Reference Casimiro, Marchant, Bhalerao, Beeckman, Dhooge, Swarup, Graham, Inzé, Sandberg, Casero and Bennett2001). Jindo et al. (Reference Jindo, Martim, Navarro, Perez-alfocea, Hernandez, Garcia, Aguiar and Canellas2012) found that HA of different origins could increase the number of lateral roots by 22 to 111% in maize. HA from composted materials contain more carboxylic groups and more hydrophobic character, and thus produce morphological and biochemical effects on plant growth. In this study, HA from composted materials might exert a significant enhancing effect on lateral roots, and thus the number of lateral roots was increased by 58.6% in a hydroponic experiment (Table 6).
CONCLUSION
In conclusion, within the experimental conditions studied, we show that HANPK chemical fertilizer exhibits positive effects on tobacco growth and leaf quality. These results suggest that the combined use of HANPK fertilizer may generate various scientific and economic advantages.
Acknowledgments
The research work was supported by China Tobacco Hunan Industrial Limited Company Scientific Research Project (2011-YC-001) and the Guangdong Provincial Tobacco Monopoly Bureau Project of Science and Technology (201307).
