Xiaode Wang·Sainan Bian·Pengjie Chang·Ninghang Wang·Lingjuan Xuan·Mingru Zhang·Bin Dong·Chao Zhang·Jiasheng Wu·Yeqing Ying·Xiazhen Lin·Yamei Shen

Abstract Camptotheac acuminata Decne is a unique tree species in China with an important secondary metabolite,camptothecin (CPT), used in the treatment of cancer. Nitrogen (N) is an important element that affects plant growth and the accumulation of CPT. Reports on the effect of N on CPT synthesis from a genetic perspective are scarce. To explore the effects of different N sources and levels on CPT synthesis in C. acuminata, two-year-old seedlings were fertilized with different concentrations of pure ammonium sulphate,source of ammonium and potassium nitrate for nitrate Concentrations of 2.5, 5, 7.5, and 10 gpot? 1 and were used. The results showed that and treatments were best for growth and fresh weight of leaves. Compared with the other treatments, the CPT content, tryptophan synthase and tryptophan decarboxylase activities, and expression of the CaTSB and CaTDC1 genes under the 2.5 g and treatments peaked significantly at 30 days.However, the expression of CaTDC2 surpassed that of the other two genes at 60 days. Therefore, compared with source, the source was more beneficial for growth, and was better for CPT yield. Consequently, leaves of C. acuminata treated with 2.5 g could be harvested after 30 days to obtain maximum CPT content. CaTDC1 is more closely linked to CPT synthesis.The results of this study improved the production of CPT in C. acuminata via fertilization.

Keywords Ammonium sulfate·Camptotheca acuminata·Camptothecin·Gene expression·Potassium nitrate

Introduction

Camptotheca acuminata Decne is a native tree species in China, an important member of the Nyssaceae family, and has been exploited for many years for its medicinal properties (Hu et al. 2016). Camptothecin (CPT) is both a secondary metabolite and a pyrroloquinoline cytotoxic alkaloid that inhibits DNA topoisomerase (TOPO I) and kills cancer cells. It is a major component in some tissues of C. acuminata (Zhao et al. 2017). It was first isolated in 1966 and has received widespread attention due to its strong anti-tumour activity (Li et al. 2018). CPT anti-tumour drugs currently approved for clinics or clinical research are topotecan and irinotecan (Lin et al 2014). Increasing the different types of CPT-based products has been the focus of considerable research (Deepthi and Satheeshkumar 2016; Yang et al.2017).

Fertilization is an important way to increase CPT accumulation in leaves of C. acuminata (Feng et al. 2014; Yang et al. 2015). Nitrogen fertilizers promote plant growth and the accumulation of CPT (Kuang et al. 2016). Previous studies of C. acuminata have reported that N increased seedling growth and the accumulation of CPT (Pan et al. 2004). If C. acuminata plants suffer from low N levels, their survival could be influenced; additional CPT is produced to balance CPT metabolism and growth of C. acuminata (Sun and Yan 2008a). Pan et al. ( 2004) reported that, compared with,can enhance the formation of glutamate which can subsequently promote the synthesis of CPT; otherwise, the effects of different N forms on CPT levels are unknown.

Tryptophan synthase (TSB) and tryptophan decarboxylase (TDC) are key enzymes in the CPT biosynthesis pathway. Sun et al. ( 2008a) reported that TSB and TDC activities are intimately related to the synthesis of CPT, but the corresponding regulatory patterns of these two enzymes differ in response to treatment with different N levels. Regulation of the tryptophan synthesis pathway may respond simultaneously to the needs of protein synthesis and secondary metabolic processes in plants (Zhu et al. 2015). TDC can catalyse the decarboxylation of tryptophan to form tryptamine, which may be directly involved in the synthesis of other defensive substances such as N-ethyl serotonin (Sun and Yan 2008b).Several key enzymes, genes, and multiple regulatory sites are involved in the CPT pathway. Gene expression patterns may also help clarify the regulatory mechanism of CPT synthesis (Sadre et al. 2016).

Genes involved in the early steps of CPT biosynthesis have recently been identified (Fig. 1). CaTSB and CaTDC are genes that play important roles in the shikimic acid pathway (Sun et al. 2011; Góngora-Castillo et al. 2012; Hu et al.2016). The CaTSB gene is highly expressed during the early development of C. acuminata; in turn, this expression promotes high accumulation of CPT (Lu and Mcknight 1999).CaTDC is the gene involved in synthesizing tryptamine(Sharma et al. 2018), a precursor for the biosynthesis of both indoleacetic acid (Di et al. 2016) and terpenoid indole alkaloids (Alam et al. 2017). CaTDC is encoded by the CaTDC1 and CaTDC2 genes. CaTDC1 is regulated by the stage of developmental and is associated with CPT accumulation.CaTDC2 is involved in the pathogen invasion defence system. It is not expressed or, it is expressed in relatively low amounts, during the normal development of C. acuminata and can be induced under environmental stress (Liu et al.1999; Lorence and Nessler 2004). Larval defence strategies and synthesis mechanisms induced by environmental factors such as light (Liu et al. 2015), water (Ying et al. 2015), salicylic acid (Kai et al. 2014), and UV-B (Ultraviolet b-band)(Ruan et al. 2014) can affect the expression of genes that directly influence the accumulation of CPT. Exploring the mechanism of CPT metabolism in C. acuminata is beneficial for explaining the molecular mechanism of environmental factors that regulate CPT biosynthesis. However, no relevant studies have investigated the effects of different N forms and different N ratios on gene expression.

Fig. 1 Upstream pathway of the synthesis of the strictosidine backbone. TSB: b-subunit of tryptophan synthase; TDC:tryptophan decarboxylase;G10H: geraniol-10-hydroxylase;SCS: secologanin synthase;STR: strictosidine synthase;10-HGO: 10-hydroxy geraniol oxidoreductase

Therefore, to evaluate the different forms of N suitable for increasing CPT contents, C. acuminata seedlings were treated with two N sources at different concentrations. Morphological features, plant growth, CPT contents, and relevant enzymes and genes involved in CPT biosynthesis were analyzed. The information from this study will be valuable for facilitating our understanding of CPT biosynthesis and improving growth conditions for the commercial production of high CPT yields.

Materials and methods

Plant materials and growth conditions

Two-year-ol d C. acuminata seedlings from Jiujiang City,Jiangxi Province, China, were grown in a nursery of the Zhejiang Agriculture & Forestry University in April 2017. Each seedling was planted in a 30 cm × 40 cm pot with 10 kg soil.Over 85% of the root biomass was concentrated in the upper 10 cm of the soil containing media (peat:perlite:garden soil = 1:1:3 (v:v:v), pH = 6.8, 0.305 ms cm?1electrical conductivity (EC), approximately 86.6 mg kg?1hydrolyzable N, approximately 37.2 mg kg?1available phosphorus, and approximately 94.6 mg kg?1available potassium) (Allan 1935; Mc Lean and Watson 1985; Wu et al. 2016)). Healthy,uniform seedlings were selected and transported to a greenhouse, fertilized by two N sources, ammonium sulphate, and potassium nitrate (KNO3) , at different concentrations.

Four concentrations, 2.5, 5.0, 7.5 and 10.0 g pot?1(Kuang et al. 2016), were applied as two forms of N fertilizer, and converted to the corresponding analytically pure amount according to the corresponding N content. The controls were unfertilized. Three plants were treated in each concentration.To prevent fertilizer runoff, all plants were placed on trays.

Fertilization started on June 18 and ended on October 4. The plants were fertilized every 15 days (Sun and Yan 2008a). Each fertilizer treatment was dissolved in 200-mL water, and the controls had 200 mL of water only. Watering was outside the 10- cm radius of the stem to avoid damaging the roots. When ammonium (fertilizer was applied,7 μmol L?1of dicyanodiaminewere added to inhibit nitrification reactions. The experiment was conducted over five months and measurements made at the beginning and on the 30th, 60th, 90th, and 105th day.

Plant growth and leaf fresh weight

Heights of all the seedlings were measured on the first day and on the 30th, 60th, 90th, and 105th day. A mark was made on the stem base of each seedling (1-2 cm above the soil surface) before treatments. Heights were measured from the basal mark to the highest bud (to 0.1 cm). Stem diameter was measured with a digital vernier calliper (average 1 cm ± 0.02 cm per plant). Fresh leaves were harvested for biomass determination before and after treatment.

Extraction of CPT and analysis of CPT content

CPT was extr acted and analyzed according to Valletta et al.( 2010). The third and fourth leaves from the top were collected, ground and 100 mg and 50 mL of 60% methanol were mixed together for 30 min by sonication at room temperature. A 50- mL extract was filtered, vacuumed at 40 °C via a rotavapour apparatus, after which the methanolic extract was redissolved in HPLC-grade methanol (1 mL). A system that consisted of an HPLC pump (LC-20ATvp), a reversed phase column (Symmetry C18 4.6 × 250 nm) and a fluorescence detector (RF-10AXL) were used for CPT detection at 254 nm and 370 nm. The mobile phase consisted of acetonitrile:water (3:7 v/v), the flow rate was 1.0 mL min?1,and the measured wavelength 254 nm. The column temperature was 25 °C, and the injection volume 15 μL, based on the retention time and absorbance spectra of CPT reference solutions (10, 8, 5, 2, 1, and 0.5 mg mL?1). The CPT was identified and quantified.

TSB and TDC activity

The activit y of TSB was determined according to Last et al.( 1991). Frozen leaf samples were finely ground in liquid N at 4 °C. A 100-mg sample was ground to a paste with a pre-chilled mortar and pestle with 2 mL of 0.1 M potassium phosphate buffer (pH = 8.2), 600 mg of 100 pm glass beads, and 600 mg of polyvinylpolypyrrolidone (PVPP)(Sigma-Aldrich Shanghai China). The homogenates were sonicated for 60 s and cleared by centrifugation at 12,000 g for 15 min. The resultant supernatant fraction was used as an enzyme extract. Sixty micromoles of L-serine, 0.2 μmol indole, 80 μmol potassium phosphate (pH = 8.2), and 10 μg pyridoxal phosphate, together with 0.4 mL of plant extract were incubated under gentle agitation at 30 °C. The reaction was stopped by the addition of 0.5 mL methanol after 90 min and cleared by centrifugation at 7000 g for 5 min. The indole content of the supernatant fraction was measured at 270 nm by an HPLC system equipped with a detector (RF-10AXL fluorescence detector). The flow rate was 1 mL min?1,the isocratic mobile phase consisted of water: acetonitrile(50:50, v/v), and the column temperature was 25 °C. The measured amount of indole consumed in μmol h?1g?1indicated the enzyme activity.

Crude protein extract was obtained from approximately 1 g frozen leaf tissue in conjunction with 9 mL sodium phosphate buffer (pH = 7.8). Enzyme-linked immunosorbent assay (ELISA) kits were subsequently used to determine the enzymatic activity of TDC at 450 nm with the aid of a microplate spectrophotometer (Labsystems Multiskan MS 352, Finland). The activity of TDC was calculated based on the standard curve of the ELISA kit standard.

RNA extraction and quantitative real-time PCR(qRT-PCR)

The RNA of C. acuminata was extracted from 0.3 g frozen leaves with a RNA pure pot kit (Tiangen, Beijing, China).cDNA was synthesized using Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Takara, Dalian, China)and stored at ? 20 °C. The primers used were designed with Primer 5.0 software based on the C. acuminata transcriptome data available on the NCBI website (Table 1). Ten microliters of SYBR Premix Ex Taq? II (Takara, Dalian,China), 2 μL cDNA, 0.8 μL of each primer at 10 μM, and 6.4 μLconstituted each 20 μL reaction for qRT-PCR using a Light Cycler 480 II system (Roche, Basel, Switzerland). The qRT-PCR was carried out following the manufacturer’s protocol, and the thermal cycler conditions used were as follows: pre-denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 20 s. The actin gene was used as the control (Li et al. 2016).

Data analysis

Statistical analysis was performed with Excel 2007 and SPSS 20.0 software. Analysis of variance and multiple comparisons ( P < 0.05) were performed using the one-way ANOVA and Duncan methods. Drawings were constructed via Origin 9.0 software. The data in the graph were the means ± standard deviations.

Results

Morphological features and growth of C. acuminata

Over time, heights, stem diameters and total leaf biomass were significantly greater under theandtreatments than those of the controls ( P < 0.05) (Figs. 2, 3,4). Moreover, plant growth and total leaf biomass peaked by day105. Compared with other treatments, growth, stem diameters and total leaf biomass with 7.5 g pot?1of theandtreatments were significantly higher( P < 0.05). Compared with the controls at day 105, seedling heights were 22.7% and 66.9% greater, respectively, and stem diameters 40.5% and 58.1% greater, respectively. Total l eaf biomass in the 7.5 g pot?1andtreatments was 44.3% and 107.2% greater, respectively. Heights,stem growth, and total leaf biomass were better underconditions than those underconditions.

Different N sources and concentrations on leaf CPT content

The series of N sources and concentrations used significantly affected CPT contents (Fig. 5). Over time, the CPT content and yield sharply increased to the highest at 30 days and then decreased. Moreover, the CPT content in seedlings in t he 2.5 g pot?1ofandtreatments was significantly higher than seedlings in the other treatments( P < 0.05). After 30 days, compared with seedlings in the 5, 7.5 an d 10 g pot?1oftreatments, seedling CPT c ontent in the 2.5 g pot?1oftreatment increased by 21.7% , 55.6% and 86.7% , respectively, yields increased by 30.3% , 87.3% , and 150% , respectively. Compared with seedlings in the 5, 7.5 and 10 g pot?1treatments,seedling CPT content in the 2.5g pot?1treatment increased by 30.8% , 88.9% and 143% , respectively, and theyield by 15.2% , 43.7% and 67.3% , respectively. The results also indicate that CPT content was higher under thetreatments than under thetreatments (Fig. 5 A-B),but the CPT yield was higher under thetreatments than under thetreatments (Fig. 5 C-D).

Table 1 Primers sequence of target genes: the actin gene (used as a reference), tryptophansynthase ( CaTSB), tryptophandecarboxylase( CaTDC1) and tryptophan decarboxylase ( CaTDC2) genes

Fig. 2 Morphological changes in C. acuminata under different treatments of N. "NH" is ammonium N fertilizer and the figures indicate concentrations. "NO" is nitrate fertilizer and the figures are concentrations. CK = controls

Fig. 3 Effects of different concentrations of and on the growth. A Effects of on height of C. acuminata;B Effects of on height of C. acuminata; C Effects ofon stem diameter of C. acuminata; D Effects of on stem diameter of C. acuminata. CK = controls. Note: Data are means ± SDs. Letters indicate significant differences ( P ≤ 0.05) and the variance between different treatments

Fig. 4 Effects of different concentrations of and on leaf biomass (fresh weight). A Effects of on leaf biomass; B effects of on leaf biomass. Note: The data in the table are the means ± SDs. The different letters indicate significant differences ( P ≤ 0.05); CK = controls

Fig. 5 Effects of different concentrations of and on CPT content in C. acuminata. A Effects of on the CPT content; B effects of on the CPT content; C effects of on the total CPT yield; D Effects of on the total CPT yield. Note: Data are the means ± SDs. Different letters indicate significant differences ( P ≤ 0.05). CK = controls

The effect of N sources and concentrations on key enzyme activities

Fig. 6 Effects of different concentrations of and on TSB and TDC enzyme activity. A Effects of on activity of TSB; B effects of on activity of TSB; C effects of on activity of TDC; D Effects of on activity of TDC Note: Data are means ± SDs; letters indicate significant differences ( P ≤ 0.05)

TSB and TDC activities peaked by the 30th day, and then decreased after 60 days under both N sources (Fig. 6). The activities of the two enzymes in the 2.5 g pot?1of(Fig. 6 A, C) and(Fig. 6 B, D) by the 30th day were higher than in the other treatments. The TSB activity under both N sources and concentrations decreased after 60 days; enzyme activities in the treatments did not significantly differ from the controls, and there were no significant differences among the different concentrations ( P > 0.05). However, the TDC activity under both N sources and concentrations decreased after 60 days, and with theexception of seedlings in the 2.5 g pot?1withTDC activity was lower than that in the controls by the 105th day.

Gene expression that encode key enzymes in CPT biosynthesis under different nitrogen sources and concentrations

The expression patterns of genes involved in CPT biosynthesis were determined to understand the relationship between pigment accumulation and transcription levels related to CPT biosynthesis under different N sources and concentrations. In addition to the expression of CaTSB underconditions by 105 days (Fig. 7 B), the expression of CaTSB increased sharply after 30 days and then decreased from 60 to 105 days (Fig. 7 A-B). The expression of CaTSB with the 2.5 g pot?1treatment was significantly higher than in the other treatments at 30 and 60 days ( P < 0.05).

As time progressed, CaTDC1 expression under the two nitrogen sources and concentrations increased sharply by 30 days and then decreased. However, the expression of CaTDC1 in the treatments, including concentrations of 10.0 g pot?1, was always greater than in the controls(Fig. 7 C-D). The expression of CaTDC1 in the 2.5 g pot?1withand withwas the highest( P < 0.05), followed by treatment concentrations of 5.0,7.5 and 10.0 g pot?1in a decreasing order. The changes in CaTDC1 expression levels in response to treatment with 2.5 g pot?1correlated with CPT contents. Compared with those of CaTDC1, the expression levels of CaTDC2 were lower (Fig. 7 C-F). Nevertheless, expression levels of CaTDC2 in all the N treatments were greater than those in the controls, and peaked at 60 days in the 2.5 g pot?1treatment and at 90 days in the 2.5 g pot?1treatment (Fig. 7 E?F). Pearson correlation coefficients showed that under thetreatment,r = 0.971 ( P < 0.01), and under thetreatment,r = 0.925 ( P < 0.01) (Table 2).

Fig. 7 Effects of different concentrations of and on the expression levels of related genes ( CaTSB, CaTDC1,CaTDC2) in C. acuminata. A Effects of on the expression of CaTSB in C. acuminata; B effects of on the expression of CaTSB in C. acuminata; C effects of on the expression of CaTDC1 in C. acuminata; D Effects of on the expression of CaTDC1 in C. acuminata; E effects of on the expression of CaTDC2 in C. acuminata; F effects of on the expression of CaTDC2 in C. acuminata . Note: Data are means ± SDs. Letters indicate significant differences ( P ≤ 0.05)

Discussion

During growth, applications of N fertilizer can significantly promote overall plant performance and improve quality(Yadav et al. 2015). In this study, the addition ofandsignificantly increased heights, stem diameters and leaf fresh weight. The response towas better than the response toat the same concentration,indicating that, on one hand, two-year-old C. acuminata seedlings preferably take up; on the other hand,when N is continuously increased under, the accumulation ofions can have toxic effects and thus inhibit root growth (Roosta et al. 2018). Under highlevels, growth of seedlings is stagnant, because with excessiveaccumulation, soil pH was not conducive to the absorption of N by roots. Besides, excessive N was not conducive to the accumulation of biomass and disrupted the absorption and metabolism of N (Campbell 1988; Hu et al.2019).

CPT or camptothecin, as a natural secondary metabolite, may not be strictly controlled by the growth rhythm of C. acuminata but mainly affected by changes in the environment during the growing season (Yan et al. 2003).Previous studies have shown that N promotes the synthesisof CPT. The different N sources employed in this study resulted in significant differences in the accumulation of CPT. Camptothecin content under thetreatment was significantly greater than under thetreatment, but yields under thetreatment were significantly greater.is not conducive to C. acuminata growth. Freeions in plants are used to synthesize amino acids which are important compounds for the synthesis of proteins. Moreover, the accumulation ofions increases the synthesis of the tropane ring structure,and increased N fixation in leaves is better for metabolism and promotes the accumulation of camptothecin (Misra and Gupta 2006). Compared withis more beneficial for transferring N to roots to promote growth. In terms of the different concentrations of N, low concentrations (2.5 g pot?1) were more conducive to the synthesis of CPT and resulted in the highest synthesis, which is consistent with the findings of Toivonen et al. ( 1991) for Catharanthus roseus and for Festuca elata (Zhang et al. 2018). Nitrogen levels in carbon and N-rich secondary metabolites effect sugar and amino acid levels, which in turn affect CPT. Photosynthesis was inhibited to some extent (results not shown),and among the secondary metabolites, sugar and amino acid levels may be altered to promote CPT synthesis (Fritz et al. 2006).

Table 2 Pearson correlation coefficients between CPT contents and gene expression levels ( CaTSB, CaTDC1 and CaTDC2) in 2.5 g·pot ?1 of and

Table 2 Pearson correlation coefficients between CPT contents and gene expression levels ( CaTSB, CaTDC1 and CaTDC2) in 2.5 g·pot ?1 of and

Analysis Pearson correlation coefficients calculated using the paired t test to determine signification level: ** P < 0.01, * P < 0.05

Genes r P Treatment of NH 4 + -N CaTSB 0.883 0.047*CaTDC1 0.971 0.006**CaTDC2 0.118 0.85 Treatment of NO 3 ? -N CaTSB 0.86 0.061 CaTDC1 0.925 0.024**CaTDC2 ? 0.5 0.391

At present, the upstream portion of the CPT biosynthesis pathway is involved and includes the shikimate,the mevalonate, and the methyl erythritol pathways. Specifically, TSB and TDC are key enzymes involved in the CPT pathway which catalyzes the irreversible reaction of anthranilic acid to tryptophan. Moreover, the genes encoding enzymes related to secondary metabolism have been isolated and identified. High CaTSB and CaTDC1 expression is often equal to the CPT content (Lu and McKnight 1999).treatments had significant effects on CPT levels.is assimilated to glutamine underand glutamate is rapidly synthesized which may play an important role. The qRT-PCR analysis showed that CaTSB and CaTDC1 gene expression under thetreatment was significantly higher than under thetreatment. This is consistent with the results in Spinacia oleracea L. (Domínguez-Valdivia et al. 2008) and Chamaemelum nobile (L.) (Ková?ik and Klejdus 2014).

In addition, the regulation of the synthetic pathway of tryptophan may respond simultaneously to protein synthesis and secondary metabolic processes in plants.is highly amenable to N fixation, promotes the expression of CaTSB, and improves the synthesis of CPT. Under the treatment of, soil N was mainly used for root growth;the N content in the leaves decreased which led to the down regulation of CaTSB and CaTDC1 expression. The reduction of precursors (tryptophan and tryptamine) directly affected metabolism of the CPT synthesis pathway. Under low N stress, in order to maintain the balance between primary metabolism and secondary metabolism, the expression of synthetic genes increased and used to synthesize more precursors and promote the synthesis of CPT (Liu et al. 1999).Under highthe expression of CaTSB and CaTDC1 was down regulated. More N in plants is not used to form tryptamine precursors. Under hightreatment,may accumulate in tissues via diffusion,resulting inpoisoning which may inhibit the assimilation of N (Bensaddek et al. 2001), thus affecting the synthesis of CPT. Under hightreatment, soil pH may be affected which influences plant N metabolism and gene expression (Anderson et al. 2018). Studies have shown that the regulation of the tryptophan synthesis pathway may respond simultaneously to protein synthesis and secondary metabolic processes in plants. High expression of CaTSB may adapt to growth. Lower expression of CaTSB may be sufficient for CPT synthesis (Sun and Yan 2008a). This also explains the sudden increase of the expression of CaTSB on the 105th day but CPT had not changed accordingly. During the synthesis of CPT, TDC may be a rate-limiting factor and the activity of TDC may be affected by jasmonic acid(Sun et al. 2011). The results show that the expression of CaTDC1 did not correspond well with TDC enzyme activity.The precursor was converted to tryptamine but the content of CPT did not increase. This indicates that there may be other steps in the CPT synthesis pathway (Silvestrini et al. 2002).The expression of CaTDC2, which is a part of the induced defence system involved in pathogen invasion, was low under normal conditions. Upon induction, this gene may be partially expressed. Many factors affect CaTDC2 expression,including the accumulation ofions in the pots and the inhibition of nitrification. CaTDC2 participates in plant defensive systems and the results show that the expression of CaTDC2 was different under different N levels. There was no significant correlation between CaTDC2 expression and CPT synthesis (Table 2). CPT biosynthesis is a complex process and each pathway may affect CaTDC2 expression.

Conclusion

Different N sources are effective for growth and maximum CPT in C. acuminata.is a suitable N form for CPT biosynthesis; moreoverwas better for the expression of CaTSB and CaTDC1 which promoted CPT synthesis of C. acuminata seedlings. CaTDC1 is more closely linked to camptothecin synthesis. The synthetic pathway of CPT is complex; there may be multiple pathway regulations and multiple regulatory sites. The determination of more related enzyme activities and gene expression help to explain the regulatory relationships between nitrogen and CPT synthesis. In the production practice, low(2.5 g pot?1)may be used for fertilization, and after 30 days may be the best time to obtain maximum CPT. This study provides a good strategy for obtaining abundant camptothecin via suitable fertilization methods.