Inoculation of phosphate-solubilizing bacteria Bacillus thuringiensis B1 increases available phosphorus and growth of peanut in acidic soil

Abstract

Phosphate-solubilizing microorganisms play an important role in plant nutrition by enhancing phosphorus (P) availability to roots through converting the insoluble phosphates into soluble ions. We isolated phosphate-solubilizing bacteria (PSB) from acidic soil (Ultisols) in the field from the layer of 0–150 mm at a tea garden located at 28°38′26″ N and 116°24′27″ E. The capacity of bacterial isolates to solubilize mineral phosphate was tested on aluminum phosphate (AlPO₄) in liquid medium. Among these PSB, isolate B1 (identified as Bacillus thuringiensis) exhibited the maximum P-solubilizing ability and was particularly efficient at solubilizing AlPO₄ (up to 321 mg L⁻¹) in vitro. The isolate B1 was inoculated to an acidic soil to study its effect on phosphate solubilization and growth of peanuts (Arachis hypogeae). The Olsen-P in the tested soil increased from 14.7 to 23.4 mg kg⁻¹, with solubilization of 16.4 mg kg⁻¹ soil of Occluded-P after 14-day incubation. The inoculation by B1 significantly increased plant height (from 37.7 to 45.7 cm), number of branches (from 34.0 to 52.7 per plant), hundred-seed weight (from 42.1 to 46.9 g) and crude protein content (from 243.5 to 268.2 g kg⁻¹ dry weight). The phosphate-solubilizing B. thuringiensis strain B1 showed potential as a biological phosphorus fertilizer.

Keywords:

• Olsen-P
• phosphate-solubilizing bacteria
• peanut
• acidic soil

Introduction

Phosphorus (P) is one of the major essential macronutrients for plant growth and development. Most of soil P is in unavailable form and only about 1–2% of it may be taken up and incorporated into the aboveground plant parts (Juhi et al. 2011). Similarly, only a small proportion (about 10–20%) of applied P fertilizers can be used by plants, and the rest is rapidly converted into insoluble complexes in soil (Narsian & Patel 2000). A large percentage of P fertilizers applied to soils are converted to insoluble forms, thus increasing P fertilizer requirement (Podile & Kishore 2006). The frequent application of P fertilizers is not only costly but also depletes the nonrenewable phosphate-rock resources used in fertilizer manufacturing processes. In addition, many synthetic P fertilizers contain acids which tends to increase the acidity of the soil, which reduces the abundance and the diversity of beneficial organisms in soils, and which interferes with plant growth. Therefore, the attempts to improve P fertilizer efficiency and utilization of the plant-unavailable P in soils have long been a very important research topic.

The use of microbial fertilizers in agriculture has received more attention. Many soil microorganisms have the capacity to transform soil phosphate (Rodriguez & Fraga 1999). Inoculation of phosphate-solubilizing bacteria (PSB) improved solubilization of sparingly soluble phosphate compounds in soils, resulting in a higher crop yield (Whitelaw et al. 1997; Sundara et al. 2002). The PSB can solubilize inorganic phosphate (Zhu et al. 2011) through the processes of acidification, chelation, exchange reactions, and production of organic acids (Abd-Alla 1994; Singh & Reddy 2011).

Red clay soil (Ultisols in the American soil nomenclature) is widely distributed in tropical and subtropical climate zones and occupies 11.8% of the total land area in China. Rice production to support 22.5% of the population in the country (Lou et al. 2003) and the tea (Camellia sinensis) cultivation are usually on acidic, barren, and clay rich soils (volume-weight ~1.3 g cm−³, clay particle ≥40%, exchangeable aluminum 2–6 cmol(+) kg⁻¹, base saturation percentage ≈40%), which have a low concentration (<15 mg kg⁻¹) of soluble phosphate in soils causing P deficiency in crops (Lu 2000). The improvement of soil P is the most important issue in acidic soils because more than 80% of P in the soil were fixed by Fe and Al oxides and clay minerals (Xiang et al. 2003). Recently, the PSB strains have attracted the attention of agriculturists as soil inocula to improve plant growth and yield (Young 1994; Young et al. 1998; Goldstein et al. 1999; Fasim et al. 2002). Many PSB strains were reported in literatures, with some of them being able to almost substitute for synthetic P fertilizer in alkaline, calcareous, and acidic soils (Hao et al. 2006; Perez et al. 2007; Singh & Reddy 2011).

Little information is available on the isolation of PSB, increasing plant-available P and its benefit to plant growth in red soils (Ultisols). Hence, the objectives of this study were to (1) isolate bacteria from red soils with P-solubilizing capacity; (2) compare the PSB isolates for the capacity to solubilize aluminum phosphate (AlPO₄) in liquid medium; and (3) test the effect of the best PSB isolate on increasing plant-available P and facilitating growth of peanuts in red soils.

Materials and methods

Bacterial isolation

For isolating P-solubilizing bacteria, red clay soils (Ultisols) were sampled in the field from the layer of 0–150 mm at a tea garden located at 28°38′26″ North latitude and 116°24′27″ East longitude. The soils were collected from four non-P-amended plots and mixed into one sample. The sampled soil contains 7.48 g kg⁻¹ organic matter, 0.87 g kg⁻¹ total N, 0.1 g kg⁻¹ total P, 11.76 mg kg⁻¹ Olsen available P, and has a pH value at 5.76, which were assessed using methods according to Lu (2000) and Watanabe and Olsen (1965). The sample was homogenized in sterile MilliQ water and diluted by 10⁻⁴, 10⁻⁵, and 10⁻⁶. Aliquots of each dilution were spread on modified Pikovskaya’s (PVK) (Pikovskaya 1948) agar medium with pH adjusted to 7.0 (Nautiyal 1999). The modified PVK agar medium contained only AlPO₄ instead of Ca₃(PO₄)₂ (Lopez et al. 2011). Four isolates were obtained and purified using single colony streak plate method. Each isolate was separately cultured in liquid Luria-Bertani (LB) medium containing 10 g NaCl, 5 g yeast extract, and 10 g tryptone per liter with pH adjusted to 7.0. Bacterial isolates were freeze-stored with 30% (v/v) glycerol at –80°C. All growth media were sterilized prior to use by autoclaving for 30 min at 115°C.

Phosphate-solubilizing capacity in AlPO₄ liquid medium

Four replicates of each of four isolates (B1, B2, B3, and B4) were inoculated as 1-mL suspension (10⁸ CFU mL⁻¹) into 40 mL liquid PVK medium supplemented with 0.4 g AlPO₄ and were cultured in Erlenmeyer flask at 28°C for 6 days with continuous agitation (150 r min⁻¹). Three replicates of sterile water inoculated at the same dose were treated as blanks. The pH value of the medium was 5.2 before the experiment started. The flasks were sampled aseptically every 24 hours for the determination of acidity and soluble P. The acidity was assayed by reading on a pH meter, and the soil-available P was determined with molybdenum blue method, and the leach liquor was 0.5 mol L⁻¹ NaHCO₃ (Watanabe & Olsen 1965).

Each liquid medium of 20 mL of bacterial cultures were filtered through 0.2-m membrane filter. The filtrates were measured by High Performance Liquid Chromatography (HPLC) for organic acids, including oxalic acid, malic acid, tartaric acid, citric acid, succinic acid, and lactic acid. A filtrate of 20 μL was injected into HPLC (Model: Agilent HPLC1100, USA), which was equipped with Agilent Zorbax SB-Aq C₁₈ 250 × 4.6 mm (5 μm) column and detector DAD-G1315B. The mobile phase was 0.01 mol L⁻¹ (NH₄) H₂PO₄ with pH 2.5, flow rate 1 ml min⁻¹, and temperature 30°C. UV absorption was measured at 214 nm (Yi et al. 2008). The organic acids were identified by comparing their retention time with those of the standards which concentrations were described by Zhang et al. (2009).

Phosphate-solubilizing capacity in the soil incubation test

Based on the P-solubilizing capacity of PSBs in AlPO₄ liquid medium, strain B1 was selected for examining the effect of its inoculation on P solubilization in the acidic red clay soil (Ultisols). Samples of 100 g soil (Ultisols, pH 5.39, organic matter 6.43 g kg⁻¹, total N 0.87 g kg⁻¹, total P 0.15 g kg⁻¹ (Lu 2000); Olsen available P 14.8 mg kg⁻¹ (Watanabe & Olsen 1965); clay proportion ≥40%) were placed in individual Erlenmeyer flasks and were inoculated with strain B1 at 10 ml kg⁻¹ soil (with 10⁸ CFU mL⁻¹). Treated soils were incubated at 28°C and analyzed for soil pH and Olsen-available P after 1, 4, 7, 14, 21, and 28 days of incubation. The assay method was used (Jackson 1973). All treatments were repeated four times. Fractionation of inorganic soil P forms (Al-P, Fe-P, Occluded-P, and Ca-P) was measured after 1, 14, and 28 days of incubation (method according to Chang & Jackson 1957).

PSB population, soil pH, and peanut growth in pot experiment

To evaluate the P release and growth-promoting efficiency of the P-solubilizing bacterial strain B1, a pot experiment using the red clay soil (Ultisols, pH 5.32, total N 1.16 g kg⁻¹, total P 0.16 g kg⁻¹, organic matter 6.43 g kg⁻¹, Olsen-available P 7.6 mg kg⁻¹, and clay proportion ≥40%) planted with peanut was conducted at Nanjing Agricultural University (32°06′ N, 118°8′ E) in a polyvinyl chloride (PVC) covered greenhouse with 5 mW × 10 mL × 2.4 mH.

Four kilograms of nonsterile and sieved (1651 μm) red clay soil (Ultisols) were placed in a plastic pot (280 mm diameter × 300 mm height). Strain B1 was applied at 10 ml kg⁻¹ soil (with 10⁸ CFU mL⁻¹) for B1 treatment; sterile water was applied at 10 ml kg⁻¹ soil for control treatment.

Two peanut seeds were sown in each pot after the seeds were surface sterilized with sodium hypochlorite (1% v/v). Soil moisture was kept at 40% of field capacity. The pot experiment was arranged in completely randomized block design with four replicates for each of the two treatments, including a control treatment (blank) and a B1 inoculation.

The aboveground biomass of peanuts was harvested after plant height was recorded, and the roots were washed using slow-running water to remove soil particles and organic debris. After washing, the number of branches and nodules per root system and hundred-seed weight were recorded, and the seed crude protein content was measured (Lu 2000). The populations of PSB in the rhizosphere soil (Luo et al. 2010) were estimated at 15, 30, 60, 90, and 120 days after sowing, using a standard dilution plate counting technique and the modified PVK medium (see above for the composition).

Strain identification

Strain B1 was identified at the species level based on 16S rDNA genes (Yi et al. 2008) and GenBank sequence similarities (http://www.ncbi.nlm.nih.gov/).

Statistical analysis

Data were analyzed by SPSS 11.0 software. One-way analysis of variance (ANOVA) was done at the 0.05 significance level.

Results

Isolation of PSB strains

Four strains (B1, B2, B3, and B4) were found to form clear zones around their colonies on PVK agar plates. Among them, isolate B1 showed the maximum P-solubilizing activity as visualized by the size of the clear zone developed around the colony after 48-hour incubation.

P solubilization, pH change, and organic acid secretion of isolated PSBs with AlPO₄ liquid media

The liquid mediums by inoculating four PSBs showed significant increase in the solubilization of AlPO₄ (Figure 1). The levels of soluble P in culture filtrates increased significantly up to the fourth day, but decreased on fifth and sixth day in the B1 treatment. Although the other isolates were significantly different compared with control, the maximum P solubilization was recorded by B1 (321.12 mg L⁻¹). Small amount of phosphate was released during the process of sterilization. The concentration of soluble P in CK treatment increased from 13.14 mg L⁻¹ to 23.98 mg L⁻¹ during medium sterilization.

Figure 1. The effect of various PSB isolates on concentration of soluble P in Pikovskaya’s (PVK) liquid medium.

Note: CK – Sterile water added; B1 – PSB Isolate B1; B2 – PSB Isolate B2; B3 – PSB Isolate B3; B4 – PSB Isolate B4. The error bars are ± standard error, and the different letters denote the statistical difference among treatments on a particular sampling day, according to Duncan’s test (P ≤ 0.05). The tests were based on four replicates.

The growth of the isolates caused a significant increase in acidity. In about 6 days, the acidity of B1 treatment increased significantly from pH 5.0 to pH 3.3 (Figure 2). In contrast, the acidity in the control treatment did not significantly change (slightly increased from pH 5.0 to 4.7).

Figure 2. The effect of various PSB isolates on pH value of solutions of Pikovskaya’s (PVK) liquid medium.

Note: CK – Sterile water added; B1 – PSB Isolate B1; B2 – PSB Isolate B2; B3 – PSB Isolate B3; B4 – PSB Isolate B4. The tests were based on four replicates. The pH value of the medium was 5.2 for all the treatments before the experiment started.

The total quantity of organic acids excreted by the isolates was shown in Table 1. The isolates of B1 and B3 yielded higher concentrations of organic acids than that of B2 and B4. The isolate B1 showed a P-solubilizing ability by producing high concentration of organic acid at 516 mg L⁻¹ in the medium supernatant. There was a correlation between pH value and the concentration of organic acid when B1 was inoculated (R² = 0.55, P > 0.05). The blank control did not produce any organic acid in detectable amounts (Table 1), and the phosphate solubilization was the lowest.

Table 1. The quantity of organic acids excreted by the PSB isolates in Pikovskaya’s (PVK) liquid media.

	Quantity of organic acids (mg L^−1)
	Incubation time (days of culture)
PSB	0	1	2	3	4	5	6
CK	0	0a	0a	0a	0a	0a	0a
B1		516 ± 23d	272 ± 14d	287 ± 11d	51 ± 3b	51 ± 4b	40 ± 1b
B2		15 ± 1b	112 ± 19c	74 ± 9c	88 ± 1c	90 ± 6c	87 ± 2c
B3		90 ± 4c	771 ± 64e	485 ± 27e	386 ± 12d	433 ± 30d	401 ± 49d
B4		15 ± 1b	14 ± 1b	31 ± 2b	60 ± 5b	93 ± 6c	89 ± 1c

Note: CK – Sterile water added; B1 – PSB Isolate B1; B2 – PSB Isolate B2; B3 – PSB Isolate B3; B4 – PSB Isolate B4. Values are the mean ± standard error and different letters (a, b, c, d, and e) denote statistically difference among treatments on a particular sampling day according to Duncan’s test (P < 0.05). The tests were based on four replicates.

Effect of inoculation with B1 on available P, pH, and P transformation in soil incubation test

The variation trends of soil solubilized-P were in accordance with the liquid medium tested above: The soil soluble P climbed up and then declined after incubating 14 days and the solubilized-P in B1 treatment increased the most. The increment of soil Olsen-available P in B1 treatment rose to 23.4 mg kg⁻¹ after 14 days. The pH of the soil samples was gradually reduced in B1 treatment compared to control (Figure 3). The PSB B1 was able to solubilize almost 15.4 mg kg⁻¹ soil Occluded-P after incubating 14 days (Table 2). B1 could transform soil Occluded-P into the Olsen-available P for plant absorption.

Figure 3. The effect of PSB B1 on soil Olsen-P and pH value in the soil incubation test.

Note: CK – Sterile water added; B1 – PSB Isolate B1. The error bars are ± standard error. The tests were based on four replicates.

Table 2. Effect of inoculating PSB isolate B1 on forms of inorganic phosphorus in red clay soil (Ultisols) (mg kg⁻¹).

		The proportion of inorganic P (mg kg^−1)
		Incubation time (days of culture)
Inorganic phosphorus	Treatments	0	1	14	28
Al-P	CK	17.9	18.7 ± 0.3b	19.9 ± 1.0a	19.5 ± 0.8a
	B1		11.7 ± 1.0a	19.5 ± 1.0a	19.6 ± 0.1a
Fe-P	CK	4.7	4.8 ± 0.4a	5.4 ± 0.7a	5.8 ± 0.1b
	B1		4.4 ± 0.2a	4.8 ± 0.5a	0.7 ± 0.1a
Occluded-P	CK	112.3	114.7 ± 2.1b	117.6 ± 1.6b	123.6 ± 2.3b
	B1		108.6 ± 0.3a	102.2 ± 2.1a	103.7 ± 1.5a
Ca-P	CK	4.6	4.4 ± 0.1a	4.1 ± 0.1a	5.2 ± 0.4b
	B1		5.2 ± 0.1b	3.9 ± 0a	3.2 ± 0.1a

Note: CK – Sterile water added; B1 – PSB Isolate B1. Values are the mean ± standard error and different letters (a, b) denote statistical difference among treatments on a particular sampling day according to Duncan’s test (P < 0.05). The tests were based on four replicates.

Effect of inoculation with B1 on PSB population, soil pH, and peanut growth in pot experiment

B1-treated soil maintained higher numbers of PSB for at least 90 days, which indicated the presence of a facilitate effect on the growth and multiplication of B1, whereas the control failed to show any significant increase in their population (Table 3). The population density of PSB increased in the first 60 days and then decreased throughout the rest of the observation period in the B1 treatment. The pH of B1-treated soil was not very different from the control after 15 days, except for an unexpected temporary drop at 60 days (Figure 4). Although it was unable to differentiate native PSB from the introduced one, the introduced PSB B1 was a native PSB, but this went through the processes of purification and increment of population via manual cultivation in liquid medium. The soil test serves two purposes: (1) Test the effect of stimulating PSB in native soil during cropping; and (2) test the effect of overall P solubilization and the consequence of crop yields in native soil. Our results suggested that there were good positive impacts in both the cases.

Table 3. Concentration of PSB isolate B1 in peanut rhizosphere (in 10⁷ CFU g⁻¹ soil).

	Population density of PSB
	Incubation time (days of culture)
Treatment	0	15	30	60	90	120
CK	10.2 ± 1.1a	14.8 ± 2.3a	3.0 ± 0.3a	6.2 ± 0.7a	1.5 ± 0.2a	7.0 ± 1.2a
B1	10.2 ± 1.1a	30.4 ± 4.5b	16.7 ± 0.9b	96.3 ± 11.3b	41.2 ± 7.8b	11.8 ± 0.9b

Note: CFU – Colony Forming Unit; CK – Sterile water added; B1 – PSB Isolate B1. Values are the mean ± standard error and different letters (a, b) denote significant difference between treatments on each sampling day according to Duncan’s test (P < 0.05). The tests were based on four replicates.

Figure 4. The effect of PSB B1 on pH in soil of pots growing peanut.

Note: CK – Sterile water added; B1 – PSB Isolate B1. The error bars are ± standard error. The tests were based on four replicates.

The whole plant total P concentration increased from 9 g kg⁻¹ dry weight in control to 14 g kg⁻¹ dry weight in B1 treatment. Plant height and the number of branches per plant were significantly enhanced by the inoculation of B1 compared to the control. The maximum plant height of 45.7 cm was recorded in B1 treatment. There was a higher number of branches and nodules per plant in the B1 treatment. The plant hundred-seed weight was significantly higher with the inoculation of B1 (46.9 g per plant) than in the control (42.1 g per plant). The crude protein content of peanut seeds significantly increased from 243.5 g kg⁻¹ dry weight in control to 268.2 g kg⁻¹ dry weight in B1 treatment (Table 4).

Table 4. Effect of PSB isolate B1 on the growth parameters of peanut.

Treatment	Plant total P concentration (g kg^−1 dry weight)	Plant height (cm)	Branches (plant^−1)	Hundred seed weight (g)	Crude protein content (g kg^−1 dry weight)
CK	9.2 ± 0.1a	37.7 ± 1.8a	34.0 ± 2.0a	42.1 ± 1.5a	243.5 ± 0.3a
B1	14.4 ± 0.1b	45.7 ± 2.9b	52.7 ± 2.4b	46.9 ± 0.9b	268.2 ± 0.2b

Note: CK – Sterile water added; B1 – PSB Isolate B1. Values are the mean ± standard error and different letters (a, b) denoted statistically difference between treatments according to Duncan’s test (P < 0.05). The tests were based on four replicates.

Strain identification

In this study, only the identity of B1 was revealed, and the closest species of B1 strain was identified as Bacillus thuringiensis, and the similarity between their 16S rDNA genes was 99.99%. Thus, the isolate B1 was named as B. thuringiensis B1. We had applied for China patent for B1, the patent number is 201110095783.2. The NCBI accession number is S000979524.

Discussion

There are some differences among different P-solubilizing microbes in certain environments, which lead to significant difference of solubilizing functions under different conditions. In the present study, the P-solubilizing bacteria were isolated from the red clay soil (Ultisols) in a tea garden. The modified PVK medium was chosen for screening PSBs. Four PSBs had been isolated from the red clay soil in the tea garden. In the recent years, suggestions have been made for isolating PSB from the amended site for increasing efficiency. In this study, it was not targeted to isolate as much PSB as possible, nor targeted for solubilizing specific phosphate source, rather targeted for maximum survival rate of PSB in nonamended environment. The results showed that the solubilization efficiency of B1 was higher than other isolates after incubating it for 4 days; it then presented a decreasing tendency onward (Figure 1). A plausible reason for such an observation could be attributed to the availability of soluble form of phosphate, which has an inhibitory effect on further phosphate solubilization (Narsian et al. 1995). The negative effect of soluble P on microbial acid productivity might also be responsible for final soluble P concentration (Rohr et al. 1983). Also, the decrease in the concentration of organic acid was expected due to (1) typical short life cycle (32 to 48 hours) of Bacillus spp.; (2) population growth in constrained environment may decline after 28 to 90 hours of incubation (Renganathan et al. 2011; Li et al. 2012). Furthermore, organic acid may precipitate and chelate with cation in the soil (Illmer & Schinner 1992). All these may contribute to the limitations of biofertilizer application of PSB.

Some studies showed that phosphate-solubilizing microorganisms (PSM), including fungi and bacteria, have only a limited capacity to solubilize FePO₄ and AlPO₄ (Banik & dey 1983; Whitelaw et al. 1999). Those studies suggested that where poorly soluble Fe-P and Al-P occurred in high amounts, the microbial-mediated mobilization of P might be less effective. This might be one of the reasons to explain the low fertility of red clay soil. However, B1 was very efficient in solubilizing AlPO₄.

In each case, due to bacterial activity, lowering of pH coincided with increasing in the efficiency of phosphate-solubilizing activity. A maximum drop in pH was associated with a higher level of P solubilization, such as in B1, in which a large amount of P released from AlPO₄ (321.12 mg L⁻¹) was accompanied by a significant drop in pH. However, there was no significant correlation between pH and soluble P concentration (R ² = 0.32, P > 0.05). This result agreed with some previous research (Lin et al. 2001; Jorquera et al. 2008).

The red clay soil is very barren and clay proportion is ≥40%, and the adsorption ability of soil P is very strong. It can hardly provide enough P for the growth of plants. Generally speaking, it is hard to judge which form of inorganic P is effective (for plant growth within) in red clay soil. In calcareous soil, Al-P and Fe-P were often treated as effective P for plant growth; and the soil Occluded-P was acknowledged as useless P (Shen & Jiang 1992). If the PSB strains could solubilize the soil Occluded-P, they should secrete organic acids to solubilize the iron and aluminum coating which surrounded the soil Occluded-P (Gong et al. 2010). In the present study, B1 could solubilize the soil Occluded-P by secreting organic acid, thus leading to the increase of soil Olsen-available P. This consequence was agreed with by Shi and Wu (1998). Except Occluded-P, the soil Fe-P and Al-P also could be dissolved by organic acids (Jones & Darrah 1994; Dinkelaker et al. 1995; Iyamuremye et al. 1996; Luo et al. 1999).

The PSM strains have been studied for their phosphate-solubilizing activity and are often used as biofertilizers (Whitelaw et al. 1999; Singh & Reddy 2011). The hardly soluble phosphate can be changed by PSM into available phosphates for plant roots. As shown by Malboobi et al. (2009), the competitiveness of PSBs which they screened could increase biomass production and yield of potato in both greenhouse and field trials. In the present study, we found that B1 could increase plant biomass and yield. Researchers observed that coinoculation of PSB and rhizobia could increase both growth and yield of legumes and also improved the absorption of nitrogen and P in plants (Bashan & Holguin 1997; Rodriguez & Fraga 1999; Rosas et al. 2006). El-Komy (2005) demonstrated the beneficial influence of coinoculation of Azospirillum lipoferum and Bacillus megaterium for providing balanced nitrogen and P levels in wheat plants. Peanut (Arachis hypogaea L.) is an important cash and oil crop in low-hilly red soil region of South China and occupies 60–85% dry-farming area in the recent 20 years (Wang & Chen 2005). In this study, we found that the number of nodules per plant increased from 44.3 in the control treatment to 76.0 in the B1 treatment. It also revealed that nodule endophytes seemed to be an interesting group since the intimate link between B1 and the plant were thought to greatly improve the efficiency of the delivery of nutrients to the plant (Firáková et al. 2007; Hamdali et al. 2008).

Funding

This study was supported by the National Natural Science Foundation of China [grant number 41371263].

Additional information

Funding

Funding: This study was supported by the National Natural Science Foundation of China [grant number 41371263].