Introduction

Phosphorous is one of the seventeen essential nutrients for plant growth (Raghothama 1999) and the second most important macronutrient after nitrogen for crop production. The plant dry weight contains up to 0.5% phosphorus since it is required in huge amounts for nucleic acids, and it also involves in an array of other processes such as photosynthesis, or phospholipid functioning (Baleni and Negisho 2012). However, phosphorous is the least accessible macronutrient and hence often limiting in fertilizers used in agriculture. Its availability is not only low in soil but also in many agriculturally used fertilizers. Under acidic soil conditions, phosphate (P) forms scarcely soluble complexes with aluminum and iron and under alkaline soil conditions with calcium and magnesium (Baleni and Negisho 2012). Thus, for most of the soils on earth, accessibility of P limits crop production.

Rapeseed (B. napus L.) is one of the most important oil crops word-wide. It is estimated that the majority of the planting areas for Brassica napus in Asia are currently phosphorus deficient (Zhang et al. 2009; Yao et al. 2011). As a consequence, the P fertilizer application for B. napus planting is much higher than for the other crop species. Since excessive P fertilization entry into the soil pollutes the water and accelerates eutrophication (Yao et al. 2011), it is important to explore other ways to improve the access of Brassica napus to phosphorus.

Plants acquire P from the soil through direct uptake or indirectly via symbiotic mycorrhizal associations when they are formed (Lum and Hirsh 2003; Yadav et al. 2010; Gill et al. 2016). Mycorrhizal hyphae can penetrate the soil more efficiently than roots (Tinker et al. 1992; Tinker and Nye 2000; Lambers et al. 2008), and arbuscular mycorrhiza (AM) fungi also supports plant’s P uptake by hydrolyzing organic P compounds through acid phosphatases which they release into the soil (Baleni and Negisho 2012). It is estimated that the contribution of AM fungi to P uptake can increase from 49% under high P conditions to 77% under P limitation conditions (Thingstrup et al. 2000).

Piriformospora indica, an axenically cultivable root-colonizing endosymbiotic fungus, shares many features with AM fungi, it can colonize roots of a wide range of higher plants including those which cannot from symbioses with AM fungi and promotes nutrient uptake, disease resistance, stress tolerance and growth of their hosts (Gill et al. 2016; Unnikumar et al. 2013; Xu et al. 2017; Hosseini et al. 2017; Hussin et al. 2017; Zhang et al. 2018). These features open the possibility for many applications in the field by using the fungus as a biofertilizer, bioregulator, growth and yield stimulator as well as a biocontrol agent (Malla et al. 2004). Growth promotion induced by P. indica has been related to changes in the production and signaling of phytohormone, such as ethylene, auxin, gibberrelin or cytokinin (Vadassery et al. 2008; Camehl et al. 2010; Sirrenberg et al. 2007), however, the contribution of the fungus to the nutrient uptake into the host’s root is still not clear. Yadav et al. (2010) and Kumar et al. (2011) showed that the fungal P transporter PiPT participates in the promotion of maize growth by transferring P to the host under P-deprived condition. Shahollari et al. (2005) have shown that the uptake of radio-labelled P was improved by P. indica in Arabidopsis seedlings. In contrast, in barley and green gram, P. indica stimulated growth but not the overall amount of P in the hosts (Achatz et al. 2010; Ray and Valsalakumar 2010). The different observations can be caused by the different hosts, or different experimental conditions used for these studies. Here, we analyzed the role of P. indica for the P uptake in B. napus which was grown on media with different P sources.

The accessibility of P for the hosts in the soil depends on the available P form, the exudation of organic acids and/or protons and of phosphatase enzyme activities in the rhizosphere (Richardson and Simpson 2011). Ngwene et al. (2016) showed that P. indica solubilizes P from inorganic, but not organic P sources, and P solubilisation was not caused by enzymatic activities but rather decreasing pH in the medium. In contrast, Swetha and Padmavathi (2016) reported that a cell membrane preparation of the fungus has phosphatase activity in the presence of zinc phosphate. Also Malla et al. (2004) showed intracellular acid phosphatase activities of the fungus. That is, P. indica solubilizes P from inorganic P sources under in vitro conditions. However, it was not tested whether such an activity is symbiosis-specific or even stimulated in symbiotic interaction of P. indica with hosts. Therefore, we hypothesis that P. indica contributes to the P uptake of B. napus when different insoluble inorganic P forms were supplied, and then, we conducted the pot experiments with P. indica cultures to explore the ability of P. indica to release P from three different sources Ca3-P, Al-P or Fe-P into the symbiotic system of P. indica with B. napus. It is important to improve the utilization rate of phosphate fertilizer and reduce the environmental pollution due to the loss of phosphorus in rapeseed production.

Materials and methods

P. indica co-cultivation with B. napus and treatments with different insoluble P forms

The P. indica fungus was cultivated in a 250 ml Erlenmeyer flask with Aspergillus (ASP) medium. Cultures were incubated at 26 °C in the dark by shaking at 150 rpm. After 14 days, the liquid was removed by filtration and excess culture medium was carefully removed from the mycelia.

Seeds of B. napus (97,009 cultivars) were washed with distilled water for 30 min, then surface-sterilized with 75% ethanol for 1 min and 2% NaClO for 15 min, and finally rinsed 5 times with distilled water. The seeds were distributed evenly on a wet double-layer filter paper (sterilized at 121 °C), placed in a light incubator (GTOP-500Y, Beijing, China) at 28 °C in the dark for 2 days, and then cultured in the light for 1 day. Germinated seeds were inoculated by adding 3 ml of a spore suspension from P. indica. As control 3 ml sterilized spore suspension solution without the fungus was used. These seeds in plates were kept in a growth chamber at 25 °C in the light for 14 days. After 14 days, the root systems of the colonized seedlings were examined by staining with trypan blue and analyzed under a Leica microscope (DM5000B, Germany). The rate of colonization was calculated by the ratio of the length of infected root segments to the total length of checked root segments.Colonized and non-colonized (control) seedlings were transplanted in plastic pots (6.5 cm × 8 cm × 7 cm) (1 plant/pot) with 250 g sterilized sand, which was supplemented with different P sources: no P (control), tri-calcium phosphate (Ca3-P), iron phosphate (Fe-P) and aluminum phosphate (Al-P) at 1.0 g/kg P, respectively. For all 8 treatments, 16 replicates were performed. The pots were placed into a growth chamber at 25 °C with a 16 h-light and 8 h-dark photoperiod, and watered with 50 ml distilled water every second day and with 50 ml P-deficient Hoagland nutrient solution (Hothem et al. 2003) every 4th day. After 30 days, the aerial parts and roots were harvested separately.

Root scanning and P content analysis

After 30 days, the leaf areas of the second leaf from the top were determined for each plant by the plant image analyzer LA-S (Hangzhou, China). The roots were carefully washed with water and analyzed with the root scanner device and software WinRHIZO (Canada) for their total length, total surface area, total volume, average diameter and root tip number. After that, the shoot and root of the plants were dried at 65 °C in an oven for the determination of the dry weight. From these samples, the P content was determined with the SFA Segmented continuous flow analyzer (Alliance-Futura II, AMS, France).

Analysis of phosphatase activities in shoots and roots of B. napus and in the rhizospheric sand

Twelve seedlings per treatment removed from their pots and the sand attached to the root surface was collected and stored in sealed plastic bags at -20 °C. One-third of the sand was used for phosphatase activity assays, one-third for the determination of the organic acids, and the last part for P analysis. The assays were repeated 4 times. Phosphatase activities were determined spectrophotometrically using para-nitrophenol (pNPP) as substrate, according to Fornasier et al. (2011). Additionally, leaves and roots of Brassica napus seedlings were washed and immediately frozen in liquid nitrogen for RNA extraction.

RNA preparation and analysis of BnACP5 and BnPHt1;4 expression by RT-qPCR

Total RNA was extracted with TRIzol (Invitrogen) according to manufacturer’s instructions. One microgram of total RNA was subjected to first-strand cDNA synthesis using the Prime-Script RT reagent kit with gDNA Eraser (Takara). RT-qPCR was performed by the SYBR green method using the StepOne Plus Real-time PCR system (Applied Biosystems). Real-time quantitative reverse transcription PCR and the 2-∆∆Ct method (Livak and Schmittgen 2001) were used to calculate the cycle threshold value of each sample. Data are means ± SD (n = 4) of four replicates. RT-qPCR primers designed by Primer 5 are listed in Table 1.

Table 1 Primer sequences of P stress-responsive genes in B. napus probed in RT-PCR assays
Full size table

Analysis of organic acids and pH in the rhizosphere sands

The organic acids in the rhizospheric sand were analyzed by adding 10 ml of 5% 0.01 M KH2PO4 solutions (pH 2.73) to 3.0 g sands. The slurry was sonicated for 15 min and centrifuged at 4000 rpm for 10 min. The supernatant was then filtered through a 0.45 μm filtration membrane and 10 μl was subjected to Agilent 1200 Series HPLC (Agilent Technologies Inc., CA, USA) using a C18 column with 100% methanol for 5 min followed by 15 min 95% methanol and 5% 0.01 M KH2PO4, pH 2.73. The flow rate was maintained at 0.8 ml/min (Yin et al. 2015). Peaks were quantified using the following standards in 0.01 M KH2PO4, pH 2.73: oxalic (10.05 μg/ml), malic (102.3 μg/ml), citric (112.8 μg/ml), acetic (103.9 μg/ml) and tartaric (50.25 μg/ml) acids. Non-inoculated media was used as the control. The organic acid content was calculated by using the external standard peak area. The pH value was measured by a laboratory pH meter (FE28, Mettle Toledo, Swiss). The supernatant was adding 25 ml sterilized distilled water to 10 g sands. The pH of the sands before planting was 6.78.

Statistical analysis

The data obtained from four independent experiments were analyzed using SAS 9.2 and Microsoft Excel. All data were subjected to analyses of variance (ANOVA). Significant differences between treatments were analyzed by Duncan test. Differences were considered significant when p-values were below 0.05. Each value represents the mean of four independent experiments performed in triplicate.

Results

Growth of B. napus cultivated with or without P. indica and analysis of the root structure under different insoluble P forms

Microscopic inspection of B. napus roots inoculated with P. indica and stained with trypan blue showed that the fungus colonized successfully the root cortex (Fig. 1). The colonization rate with 80.2% was quite high, and we often found spores within the root cells (Fig. 1). P. indica stimulated positively the leaf areas and shoot dry weights of B. napus seedlings already when the seedlings were watered with media without any additional P source (Table 2, S1 Fig). The stimulatory effects of the fungus were even stronger when the seedlings were treated with media containing Ca3-P, Al-P or Fe-P. Also the root growth parameters were significantly greater for P. indica colonized plants, and the highest values for the total root lengths, surfaces, average diameters, total volumes and number of root tips were observed for seedlings exposed to Ca3-P solution (Table 3).

Fig. 1
figure 1

Colonization of B. napus roots by P. indica. After inoculation of the roots with a spore suspension of P. indica as described in the Methods part, co-cultivation of both symbionts was performed in soil for 14 days

Full size image
Table 2 Leaf area of B. napus plants grown with (+) or without (−) P. indica on different inorganic phosphate sources
Full size table
Table 3 The effect of P. indica on B. napus root parameters treated under different inorganic phosphate sources
Full size table

Effect of P. indica on the P concentration in shoot and root of B. napus and rhizosphere sand supplied with different P sources

The P concentration in shoots and roots of colonized and non-colonized B. napus by P. indica was grown on the soil supplemented with the different insoluble P sources (Fig. 2a, b). In all cases, the amounts of P in shoots and roots were higher in the presence of P. indica. The highest amount was measured for roots and shoots of plants exposed to Ca3-P and P. indica. Furthermore, the detectable P content in the rhizosphere sand samples was also higher when P. indica was present compared to samples from non-colonized plants, the values increased 1.4- and 2.6-fold on soil supplemented with Ca3-P and Fe-P, respectively, and there was no difference between P. indica colonized and non-colonized plants with Al-P (Fig. 2c).

Fig. 2
figure 2

Phosphorus concentration in shoot and root of P. indica colonized and uncolonized B. napus seedlings in pots watered with different insoluble phosphorus solutions (A: Shoot; B: Root; C: Rhizosphere soil). C: control; Ca3-P: tri-calcium phosphate; Al-P: aluminum phosphate; Fe-P: iron phosphate. Data are average values ±SD (n = 4) calculated from four independent experiments. Asterisks indicate significant differences between P. indica inoculated and non-inoculated seedlings (*p < 0.05)

Full size image

The effect of P. indica on phosphatase activities in shoots and roots of B. napus

The phosphatase activities in shoots and roots of B. napus with P. indica showed different trends depending on the application of Ca3-P, Al-P or Fe-P solutions (Fig. 3a, b). Phosphatase activity was only stimulated in shoots when P. indica colonized plants were grown on sand with Ca3-P or Fe-P solutions (Fig. 3a). However, in roots, the stimulatory effect of the fungus on the phosphatase activities was the highest for Al-P, followed by Ca3-P and Fe-P (Fig. 3b).

Fig. 3
figure 3

Acid phosphatase activities in shoot and root of P. indica colonized B. napus seedlings grown in rhizosphere soils watered with different insoluble phosphorus solutions (A: Shoot; B: Root). C: control; Ca3-P: tri-calcium phosphate; Al-P: aluminum phosphate; Fe-P: iron phosphate. Data are average values ±SD (n = 4) calculated from four independent experiments. Asterisks indicate significant differences between P. indica inoculated and non-inoculated seedlings (*p < 0.05)

Full size image

BnACP5 and BnPHt1;4 expression in shoots and roots of colonized and non-colonized B. napus on the different P sources

P. indica had different effects on the expression of the BnACP5 and BnPHt1;4 in shoots and roots of B. napus exposed to the different P sources (Fig. 4a, b). Expression of BnACP5 encoding a phosphatase was significantly increased by the fungus in roots exposed to Ca3-P, Al-P or Fe-P (Fig. 4a). However, in shoots, a stimulatory effect of the fungus could only be detected for plants exposed to Ca3-P (Fig. 4a).

Fig. 4
figure 4

Expression of BnACP5 and BnPHt1;4 in roots and leaves of B. napus grown in soil watered with different insoluble phosphorus solutions (A: BnACP5 gene; B: BnPHt1;4 gene; Left: Roots; Right: Leaves). C: control; Ca3-P: tri-calcium phosphate; Al-P: aluminum phosphate; Fe-P: iron phosphate. Data are average values ±SD (n = 4) calculated from four independent experiments. Asterisks indicate significant differences between P. indica inoculated and non-inoculated seedlings (*p < 0.05)

Full size image

The expression of BnPHt1;4 encoding a phosphate transporter of the PHT1 family in roots was also stimulated by P. indica under all treatments (Fig. 4b), in addition, only Ca3-P showed a stimulatory effect of the fungus in shoots. These results suggest that the effect of the fungus on the expression of BnACP5 and BnPHt1;4 is greater in roots than shoots.

Effect of P. indica on organic acids and pH value in the rhizosphere sands

HPLC analysis and pH value of the rhizosphere sands were performed to identify and quantify the organic acids produced after treatment of the plants with different P sources (Table 4). Oxalic, malic and citric acids were secreted into the rhizosphere sands after planting B. napus, while tartaric and acetic acids were not detected (Table 4). Overall, the organic acid production appeared to be a common event of occurrence when the P source applied to the soil in our data, however, the types and quantities of acid produced depended on the type of phosphate source. The citric acid was the major organic acid which was produced in response to Ca3-P or Al-P treatments whereas oxalic acid was mainly produced in response to Fe-P treatment. Furthermore, in the presence of P. indica, higher levels for these organic acids were found in the sand. It suggests that the fungus stimulated organic acid accumulation in the rhizosphere .

Table 4 Concentration of organic acids and pH in the rhizosphere sands with respect to different inorganic phosphate sources
Full size table
Fig. 5
figure 5

Mechanisms of solubilization of inorganic P planted B. napus seedlings colonized by P. indica in pots

Full size image

There was a significant decrease in the pH in the presence of P. indica (Table 4), with or without P addition, irrespective of the P source, compared to the cultures where P. indica was absent. The pH was reduced by 0.15, 0.2 and 0.33 unit for Ca3-P, Al-P and Fe-P, respectively. The result further proved that the organic acid was accumulated in the rhizosphere with different inorganic P source.

Discussion

At the pot experiments, the shoot and root growth of B. napus grown on soil supplemented with Ca3-P, Al-P or Fe-P as P source was clearly enhanced by P. indica, expect for average diameter supplemented with Ca3-P (Tables 2 and 3), and the amount of P in both organs was increased in the presence of the fungus. Furthermore, we demonstrate that the available P concentration in the rhizosphere sand was strongly stimulated by the fungus when Ca3-P or Fe-P was applied as additional P source (Fig. 2). These results suggest that P. indica stimulates the growth of the host by stimulating the P availability from the rhizosphere. The fungus releases P from P sources which are not accessible for the plant which is taken up by the roots directly or via a passage through the fungal hyphae. The performance of the fungus was mentioned in adult maize plants under P limitation conditions (Kumar et al. 2011). It was noticed that the root average diameter of B. napus seedlings with P. indica supplemented with Ca3-P was significantly decreased in our results. The possible reason was the changes in cytosolic Ca2+ concentration, which plays a role in affecting root architecture under low-P conditions (Niu et al. 2013). However, we also observed that the number of lateral root and root tip were increased significantly, which enabled the root to better explore the soluble phosphorus in the soil (Lynch and Brown 2001; Baleni and Negisho 2012; Niu et al. 2013). The contribution to the growth promoting effect of B. napus under field conditions needs to be further studied.

Phosphatase enzymes are believed to be important for P uptake. They have wide specificity in cleaving P ester bonds and hydrolysis of insoluble polyphosphates and organic phosphates. This has a strong influence on P uptake into roots and distribution within the cells (Swetha and Padmavathi 2016). In our data, the phosphatase activities in roots of B. napus were strongly stimulated by P. indica, when Ca3-P, Al-P or Fe-P was applied to the symbionts, and BnACP5 was higher expressed in roots. The phosphatase activities and BnACP5 expression in shoots showed different trends (Fig. 4a). These results indicate that the fungus mainly stimulated the phosphatases in roots of the host. The phosphatases from either plant or fungal origin convert insoluble P forms into soluble forms and make them accessible for the plants (cf. also Singh et al. 2000). However, in vitro culture, P. indica could solubilize P from Ca3-P and rock phosphate, but no relevant intra- or extracellular phosphatase activities were detected despite a stimulatory effect of the fungus on the expression of the relevant plant genes (Ngwene et al. 2016). Numerous reasons can explain this discrepancy, among which it is reasonable to assume that the fungus induces the enzymatic activities only under symbiotic conditions.

BnPht1;4 is a high-affinity P transporter of the PHT1 family, and plays a crucial role in the P starvation response. In this study, BnPHt1;4 expression was stimulated by P. indica in roots of Brassica napus with Ca3-P, Al-P or Fe-P, while in shoots a stimulatory effect was only detectable with Ca3-P (Fig. 4b). This suggests that P. indica has the potential to induce P transporter in B. napus roots under P deficiency condition there by facilitating B. napus to have better accessibility to soil phosphorus. This point was supported by the roots under P deficient conditions, and the gene may be regulated by both MYBCC and WRKY family transcription factors (Ren et al. 2014). Further studies need to show the transcription factors which are targeted by P. indica in the host under different inorganic P sources.

Organic anions such as citrate and malate are the major released root exudates, in response to P deficiency for mobilizing P for plant uptake (Dechassa and Schenk 2004). In our study, oxalic, malic and citric acids were accumulated in the rhizosphere sands when P. indica colonized B. napus plants were growing under Ca3-P or Al-P treatments, and oxalic acid was produced under Fe-P treatment (Table 4). The results were in accordance with Swetha and Padmavathi (2016), who also reported the production of oxalic acid and citric acid. However, tartaric, acetic, lactic and succinic acids were not found in our experiments. This could be caused by different experimental conditions or genotypes used (Corrales et al. 2007). However, organic acids in the culture medium were not detected probably because their amounts were too low (Ngwene et al. 2016). In our data, the malic and citric acid accumulation was decreased at Fe-P treatment, and the oxalic acid content was significantly higher than other treatments (Table 4). This resulted from different types of phosphorus sources (Vyas and Gulati 2009; Mardad et al. 2013). Moreover, the synthesis of organic acids is controlled by genes. One gene for a malate synthase and three genes for citrate synthases have been identified when P. indica was cultivated without the plant (Zuccaro et al. 2011). Therefore, the expression of these genes during the symbiotic interaction of P. indica with B. napus roots needs to be analyzed in detail.

Phosphorus solubilization is the combined effected of both drop in pH and organic acid production (Fankem et al. 2006). We also found that the pH value was decreased significantly when P. indica growth in the presence of Ca3-P, Al-P or Fe-P (Table 4). The most range of the reduced pH value was resulted from applying Fe-P and the minimum range was caused by Ca3-P. The probable reason is a buffering effect of the dissolved Ca3-P. Organic acids produced by the phosphate solubilizing microorganisms have been mainly involved in chelating the insoluble complexes of phosphate (Bagyaraj et al. 2000), therefore, the pH value in our experiment was above 6.0 in all treatments.

Notably, the P concentration in P. indica colonized B. napus shoots growing on soil supplemented with the different insoluble P sources was higher than in non-colonized plants. Since the alterations of the phosphatase activity and the expression of BnACP5 and BnPHt1;4 did show different trends in our experiments with the three different insoluble P sources, additional factors might contribute to the P transport from the roots to the shoots.

Phosphorus-solubilizing microorganisms and plants form a synergistic relationship in nature, presumably because it is beneficial for both partners. We proved that the pre-hypothesis was correct through our experiment, and secretion of organic acids and phosphatase enzymes might be crucial for such a scenario in the interaction studied here (cf. Malla et al. 2004; Singh et al. 2000). We propose that these processes participate in the promotion of plant growth and crop productivity. Solubilization of phosphate by P. indica interaction with Brassica napus occurs due to the combined effect of both phosphatase enzyme in roots and organic acid production in the rhizosphere soil. We have also presented a small model diagram of the mechanisms of solubilization of inorganic P planted B. napus seedlings colonized by P. indica in our experimental conditions (Fig 5).

Conclusion

The present study shows that the P levels in shoots and roots of P. indica colonized B. napus increases when the symbionts are growing under P limitation conditions with Ca3-P, Al-P or Fe-P as P sources. Especially under Ca3-P conditions, the expression of a high-affinity Pi transporter was remarkably stimulated in the roots, which indicates that the fungus mobilizes some P from inorganic sources for uptake, but higher expression of P transporter genes often demonstrates that P is still limiting. The symbionts respond to this situation by stimulating both phosphatase enzymes in the roots and the production of organic acid in the rhizosphere soil.