Adult-plant resistance of Panaxnotoginseng to nematodes and interspecific facilitation with pine trees

Root-knot nematode (RKN) is a soil-borne pathogen that severely damages Panaxnotoginseng. It has been recently shown that intercropping P.notoginseng in forests constitutes an innovative system that can promote P.notoginseng growth and suppress the spread of disease. However, there is little research on the interspecific interaction between pine trees and P.notoginseng. Here, 2 years of field experiments were designed to explore the effects of RKN infestation and P.notoginseng–pine tree intercropping on the growth and saponin synthesis of P.notoginseng. The results showed that RKN infestation could significantly reduce the root biomass, nutrient uptake and saponin accumulation of P.notoginseng seedlings. Interestingly, 2-year-old P.notoginseng plants grew normally (specifically, root biomass, saponin synthesis, nutrient uptake and root morphology were normal) under nematode infection, while the incidence of RKN disease was decreased compared with that of seedlings before transplantation, indicating that P.notoginseng has adult-plant resistance (APR) to RKN disease. Furthermore, P.notoginseng–pine tree intercropping could significantly reduce the incidence of RKN disease and increase the biomass, nutrient uptake and saponin synthesis of P.notoginseng under mild infection, and root architecture may play a key role. Above all, P.notoginseng showed APR to RKN disease, and P.notoginseng–pine tree intercropping represents a type of interspecific facilitation that can promote the growth and quality of P.notoginseng.


Introduction
Sanqi [Panax notoginseng (Burk.) F. H. Chen], a member of the Araliaceae family, is an important perennial herbaceous species with high medicinal and economic value Yang et al. 2019a). This species is distributed primarily in southwestern China and has been cultivated for more than 400 years in Yunnan Province Liu et al. 2019;Ye et al. 2019). P. notoginseng plants are often infected by many types of soil-borne pathogens, including fungi (Fusarium solani), bacteria (Pseudomonas spp.) and nematodes (Meloidogyne hapla), which results in yield and quality losses (Luo et al. 1999;Mao et al. 2013;Dong et al. 2013).
Root-knot nematode (RKN) is a plant-parasitic pest that can severely damage P. notoginseng, and it not only decreases the survival rates of seedlings but also suppresses growth and quality formation during subsequent growth stages (Hu et al.1998;Chen et al. 2002 (Yang et al. 2008(Yang et al. , 2019b, which infects mainly the lateral roots and fibres of P. notoginseng to form many galls, which disrupts normal physiological function of the roots (Dong et al. 2013;Borah et al. 2018;Yang et al. 2019b). Moreover, in the field, we found severe RKN infestation on P. notoginseng seedlings but only a mild infestation on the 2-or 3-year-old P. notoginseng. This phenomenon is similar to adult-plant resistance (APR) in other studies (Wang et al. 2005). For instance, resistance to leaf rust, stripe rust and powdery mildew in adult wheat (Pathan et al. 2006;Hiebert et al. 2010;Li et al. 2014); resistance to common rust in adult maize (Abedon and Tracy 1996); and age-related resistance to Pseudomonas syringae in Arabidopsis have been reported (Kus et al 2002). Hence, determining whether APR to RKN occurs in P. notoginseng is essential for seedling cultivation and subsequent growth of P. notoginseng. Agroforestry is an important intercropping system that has obvious advantages in improving land use efficiency, as this system effectively alleviates the prevalence of and damage by pests and diseases, ensuring yield stability (Trenbath 1999;Hong et al. 2017;Kimura et al. 2018;Zhang et al. 2019). The cultivation of P. notoginseng in forests constitutes an innovative intercropping system that has been developed and widely tested in southwestern China and can significantly increase social, economic and environmental benefits . Forest-medicinal plant intercropping systems can promote the growth and improve the quality of medicinal plants and can prevent pest and disease infestation Zhang et al 2020a). For example, a study showed that a mulberry-alfalfa intercropping system increased the relative abundance of beneficial fungi and promoted the growth of plants . Another study indicated that Tetrastigma hemsleyanum grown in accordance with a suitable cultivation pattern in a Phyllostachys edulis forest can reduce root rot disease and improve yield (Mao et al. 2018). Moreover, a previous study indicated that the environment under forests provides desirable shade, suitable temperature and moisture, adequate nutrients (soil rich in organic matter and microbes) and allelochemicals for P. notoginseng, which can promote growth . However, there is no information available about the direct interaction between pine trees and P. notoginseng in the field. Moreover, many previous studies have found that, through plant root interactions, intercropping can suppress soil-borne diseases and effectively control RKN diseases (Lv et al. 2018;Zhang et al. 2020b). For instance, marigold-sesamum or mulberry, angelica-marigold, tomato-marigold, tomato-garlic, tomato-mustard, and tomato-tobacco intercropping can reduce galls and numbers of second juveniles and effectively control RKN disease (Govindaiah et al. 1997;Xie et al. 2017;Selabat 2019). Therefore, it is very useful to determine the effects of P. notoginseng-pine tree agroforestry systems in forests on RKN damage.
In the present study, we attempted to use the "P. notoginseng-pine tree" model as a research object. First, we evaluated the effect of RKN infestation on the growth of P. notoginseng seedlings. Then, we studied the influence of seedling health (different RKN infestation levels) on the biomass, nutrient uptake, and saponin content of the 2-year-old P. notoginseng plants. Moreover, we explored the interactions between P. notoginseng and pine trees to determine the effects of the pine tree on the growth, quality and incidence of RKN of P. notoginseng.

Study site and experimental design
Two field experiments were carried out in Lancang County, Pu'er city, Yunnan Province, China (N22°48′18″, E99°46′56″; altitude of 1460 m above sea level), in 2018 and 2019. In January 2018, P. notoginseng seeds were sown in a 50 m × 40 m = 2000 m 2 plot with a nematode population of 21 s-stage juveniles (J2s)∕100 mL soil. Healthy seeds were surface sterilized with 1% sodium hypochlorite for 5 min and washed three times with sterile water, and the seeds were sown at a density of 5 cm × 5 cm (Wei et al. 2019). When the seeds germinated, a polyethylene net that allowed 10% light transmission was installed over the experimental plot to imitate the natural conditions for P. notoginseng growth (Wei et al. 2019). Normal field water management with no fertilizer was used in all the experiments (Wang et al. 2020). When the P. notoginseng seedlings were harvested, the effects of RKN infestation on the growth and quality of P. notoginseng seedlings were observed and evaluated.
In December 2018, the field experiment was conducted on new farmland with a nematode population of 17 J2s∕100 mL soil in accordance with a two-factor randomized block design with three replications. The first factor was the planting pattern-P. notoginseng-pine tree intercropping and P. notoginseng monoculture. The area of each replication (block) was 1.5 m × 20 m = 30 m 2 . In the intercropping system, pine trees were planted in the centre of 150 cmwide strips, and the spacing was 1 m per plant. The second factor was the P. notoginseng seedlings with different disease levels of RKN. The medium-sized P. notoginseng seedlings with different RKN infestation levels from experiment 1 were selected for field experiments in the following year. P. notoginseng was planted at the same inter-and intrarow spacing (15 and 12 cm) in both the monoculture and the intercropping systems. When P. notoginseng sprout, a polyethylene net was installed over the experimental plot, and water and fertilization were performed as described 1 3 previously (Wei et al. 2019;Wang et al. 2020). However, there was a severe drought in the experimental area in March to June 2019, which not only resulted in the decrease in soil water content in the topsoil, but also did not have enough water to irrigate P. notoginseng. At this time, P. notoginseng was in the stage of seedling sprout and aboveground growth. Drought seriously affected the normal seedling sprout of P. Notoginseng and even caused its death. At the end of the experiment, the incidence of P. notoginseng RKN disease, seedling survival rate, biomass, nutrient uptake, root morphology and saponin content were determined to evaluate the effects of P. notoginseng-pine tree intercropping and RKN infestation levels on the subsequent growth of P. notoginseng.

Sample collection and evaluation
In December 2018, the P. notoginseng seedlings were harvested at five uniformly distributed survey sites, and the soil was shaken off the roots. The severity of RKN of the root system was visually assessed for each plant by rating the percentage of roots with root tip galls, and it was based on the RKN rating chart of Bridge and Page, with slight modifications (Table 1) (Bridge and Page 1980;Win et al. 2015;Ulloa et al. 2016). Subsequently, the P. notoginseng seedlings of each RKN infestation level were separated into aboveground shoot and belowground root parts, and the roots were divided into large (fresh weight > 3 g), medium (fresh weight between 2~3 g), and small (fresh weight < 2 g) roots. Among them, the majority of samples used for the following year field experiments were medium-sized P. notoginseng plants, but there were not enough P. notoginseng seedlings at the disease levels 2-3. Therefore, the disease levels of 2-3 (11-50% of roots infected) were considered level 2 when planting in the second trial, and the disease level 4 was accordingly changed to 3 (> 50% of roots were infected).
In December 2019, the seedling survival rate was evaluated, and the 2-year-old P. notoginseng were collected at each plot. In the intercropping systems, we chose five uniformly distributed survey sites in each plot, and the area of each site was 1.5 m × 1 m = 1.5 m 2 , with one pine tree in the centre. The area collected in the monoculture systems was identical to that of the intercropping systems (Liu et al. 2020). All the samples were carefully washed with water; disease severity was assessed by examining the roots and was ranked between 0 and 3 according to previously described methods. The disease index was calculated as [∑ (the number of diseased plants in this index × Disease index)/(Total number of plants investigated × The highest disease index)] × 100 (Yang et al. 2014a).

Field crop research
At the end of experiments 1 and 2, the P. notoginseng plants were separated into aboveground shoots and belowground roots after the plant samples were harvested. In experiment 1, all the samples were divided into three different groups. In experiment 2, all the samples were divided into four different groups after the belowground roots were separated into taproots and fibrous roots, and three of the four groups were used for the same purposes as those in experiment 1. One group was used for determining dry biomass after the plant samples were immediately dried at 80 °C until a constant weight was reached (Wei et al. 2019). Another group was used to determine the nitrogen (N), phosphorus (P) and potassium (K) concentrations of the plant dry matter in accordance with previously described detection methods (Page et al.1982). The third group was used to determine the saponin content in the P. notoginseng taproot and fibrous roots by ultraperformance liquid chromatography (UPLC) detection methods as previously described (Yang et al. 2015). The fourth group was used to analyse the root morphology, as described below. The fibrous roots were scanned using an Epson Perfection V850 Pro scanner (Epson, Inc., Beijing, China), and the total root length, root diameter, root surface area, and root volume of the root samples at different RKN infestation levels were analysed by WinRHIZO™ software (Régent Instruments, Inc., Québec, Canada) (Wei et al. 2019;Liu et al. 2015).
The N, P and K concentrations of the dry matter in the P. notoginseng aboveground shoots and belowground roots were determined after digestion in a mixture with H 2 SO 4 and H 2 O 2 (Page et al. 1982). The N concentration in the plant dry matter was measured by the micro-Kjeldahl procedure (Kong et al. 2015;Prajjwal et al. 2017), the P concentration 1-10% of roots mildly infected with a few small knots 2 11-25% of roots infected, small knots but visible, main roots unaffected 3 26-50% of roots infected, Some larger knots visible, main roots unaffected 4 More than 50% of roots infected, larger knots predominant, knotting on some main roots, reduced root system was measured by the vanadomolybdate method (Yang et al. 2014b;Wu et al. 2017), and the K concentration was measured by flame photometry (Sedighe et al. 2018). Nutrient uptake was calculated as the product of nutrient concentration and biomass in the aboveground and belowground parts of P. notoginseng (Wei et al. 2019).

Analysis of saponin content
To determine the saponin accumulation of the P. notoginseng samples, five individual types of saponins, including R1, Rg1, Re, Rb1, and Rd, within the roots were identified and quantified by UPLC according to Yang's methods (2015), with slight modifications (Yang et al. 2015). Briefly, we weighed and transferred 0.2 g dried P. notoginseng root powder to a 50 mL tube and ultrasonicated it with 15 mL of methanol (MeOH): H 2 0 (75:25) at 40 °C for 40 min. The tube was then centrifuged for 10 min (12,000 × g, 4 °C). The supernatant was collected and stored at 4 °C until further determination. The samples were analysed using an Agilent UPLC system (1290 Infinity II) fitted with a diode array detector (DAD). Briefly, UPLC separations were performed using a Kinetex C18 column (100 × 4.6 mm, 2.6 μm). A multistep gradient was used for all separations, with an initial injection volume of 2 μL and a flow rate of 0.6 mL min −1 . The solvent was as follows: solvent A = ultrapure water; solvent B = acetonitrile (MeCN). The multistep gradient was as follows: 0-2 min, 20% (v/v) solution B; 2-7 min, 20-70% (v/v) solution B; 7-8 min, 70-85% (v/v) solution B; 8-10 min, 85% (v/v) solution B; and 10-10.5, min 85-20% (v/v) solution B. The column temperature was maintained at 40 °C. Chromatograms were recorded at 203 nm. The saponins (R1, Rg1, Re, Rb1, and Rd) in the samples were identified by comparing the results to those of authentic saponin standards. The saponin concentrations in samples were quantified using standard curves that showed the linear relationships between the peak areas and the concentrations.

Data analysis
The biomass, nutrient uptake and total saponin content of the P. notoginseng seedlings in different RKN infestation levels were analysed with SAS (SAS Institute Inc., USA) software, and the mean values (n = 3) were compared by the least significant difference (LSD) at the 5% level (Liu et al. 2020). The biomass, disease incidence, nutrient uptake, total saponin content and root morphology of the 2-yearold P. notoginseng with different RKN infestation levels in the intercropping and monoculture systems were analysed using SPSS version 18.0 software (SPSS, Inc. Chicago, IL, United States) (Wang et al. 2020), and the mean separation among treatments was compared by independent sample t tests (Zhang et al. 2020b). The difference in saponins content (R1, Rg1, Re, Rb1, and Rd) between the treatments was analysed using R software (version 2.15.3).

RKN infestation affects the biomass of P. notoginseng seedlings
The field data in 2018 showed that the RKN infestation could significantly affect the growth of P. notoginseng seedlings. Both the belowground dry biomass and the ratio of roots to shoots of P. notoginseng seedlings significantly decreased with increasing levels of RKN infestation (from 0 to 4 levels) in a dose-dependent manner ( Fig. 1; Table 2). In particular, the root growth of large-sized and mediumsized P. notoginseng seedlings was significantly inhibited when P. notoginseng plants were infected by RKN, but there were no significant effects on the small-sized P. notoginseng seedlings (Table 2).  Table 2 Effects of RKN infestation on the biomass of P. notoginseng seedlings All the data are presented as the mean ± standard error (SE) of three biological replicates. The data in the same column followed by different lowercase letters (a, b) showed significant differences among different RKN infestation levels at the 5% level by LSD.

Levels of RKN infestation
Aboveground dry biomass (g plant -1 ) Belowground dry biomass with different sizes (g plant -1 ) Ratio of roots to shoots (R/S) Total roots(g plant -1 ) Large-sized (g plant -1 ) Medium-sized (g plant -1 ) Small-sized (g plant -1 ) 0 0.13 ± 0.002b 0.64 ± 0.012a 1.08 ± 0.047a 0.66 ± 0.009a 0.33 ± 0.026a 4.76 ± 0.014a 1 0.14 ± 0.003ab 0.59 ± 0.007b 0.95 ± 0.028b 0.69 ± 0.012a 0.26 ± 0.028a 4.21 ± 0.127b 2 0.16 ± 0.005a 0.56 ± 0.003c 0.94 ± 0.009b 0.63 ± 0.015a 0.28 ± 0.018a 3.53 ± 0.115c 3 0.15 ± 0.011ab 0.59 ± 0.012b 0.93 ± 0.006b 0.64 ± 0.032a 0.32 ± 0.03a 4.03 ± 0.271b 4 0.14 ± 0.006ab 0.47 ± 0.006d 0.89 ± 0.015b 0.55 ± 0.023b 0.28 ± 0.009a 3.36 ± 0.124c Fig. 2 N, P, and K nutrient uptake of P. notoginseng seedlings among different RKN infestation levels. N (a), P (b), and K (c) uptake in the aboveground parts of each P. notoginseng plant. N (d), P (e), and K (f) uptake in the total roots of each P. notoginseng plant. N (g), P (h), and K (i) uptake in the large-sized, medium-sized and small-sized roots of each P. notoginseng plant. All the data are presented as the mean of three biological replicates, and the bars indicate the SEs. The different lowercase letters a, b above the error bars indicate significant differences among different RKN infestation levels for the same plant size at the 5% level by LSD. The different capital letters A, B denote significant differences among the different-sized P. notoginseng plants at the 5% level by LSD RKN infestation affects the nutrient uptake of P. notoginseng seedlings RKN infestation significantly affected the nutrient uptake (N, P, and K) of P. notoginseng seedlings ( Fig. 2; Fig. S1). RKN infestation increased the N and P uptake in the aboveground parts of P. notoginseng seedlings, and the highest N and P uptake occurred at disease level 2 (Fig. 2a, b); however, the aboveground nutrient concentration was not significantly different (Fig. S1a-c). Additionally, RKN infestation significantly increased the P uptake and the N and P concentration of total roots, whereas the N and K uptake and K concentration of total roots were not significantly different between diseased and healthy P. notoginseng (Fig. 2d-f; Fig. S1d-f). Further analysis revealed that RKN infestation could significantly suppress the N, P, and K uptake of largesized P. notoginseng, whereas mild infestation (levels 1-2) could increase the N, P and K uptake of medium-sized P. notoginseng (Fig. 2). Among them, the N, P, and K uptake in the large-sized roots at disease level 4 decreased by 9%, 14%, and 28% compared with that of healthy P. notoginseng (0 level) ( Fig. 2g-i).

RKN infestation affects the saponin accumulation of P. notoginseng seedlings
To investigate the effects of RKN infestation on saponin accumulation, the contents of the total saponins and five active saponins in P. notoginseng roots under different infection levels of RKN disease were measured. The data showed that RKN infestation could significantly reduce the content of total saponins (R1, Rg1, Re, Rb1, Rd) in the roots of each P. notoginseng plant but had no effect on saponin concentration ( Fig. 3a; Fig. S2). Among them, RKN infestation mainly reduced the content of total saponins in the roots of both the large-sized and the medium-sized P. notoginseng, but there were no significant differences in the roots of small-sized P. notoginseng across the different levels of RKN disease (Fig. 3a). A heatmap was constructed that illustrated that the concentrations of active saponins (R1, Rb1, and Rd) in the diseased roots of large-sized P. notoginseng and the contents of active saponins (Re, Rg1, and Rb1) in diseased roots of medium-sized P. notoginseng were significantly lower than those in healthy P. notoginseng roots (Fig. 3b, c), but there were no significant differences among the small-sized P. notoginseng (Fig. 3d). Furthermore, the total saponin and five active saponin contents and concentrations in the roots The different lowercase letters a, b above the error bars indicate significant differences among different disease levels within the same size at the 5% level by LSD. The different capital letters A, B denote significant differences among the different-sized P. notoginseng plants at the 5% level by LSD of large-sized P. notoginseng were significantly higher than those in the roots of both the medium-sized and the smallsized P. notoginseng, in accordance with a dose-dependent manner ( Fig. 3; Fig. S2).

Survival rate, disease incidence and disease index of 2-year-old P. notoginseng seedlings
As shown in Fig. 4, the seeding survival rate, disease incidence and disease index of 2-year-old P. notoginseng plants across different levels of RKN disease showed no difference between the intercropping and the monoculture systems (Fig. 4). However, the seeding survival rate of the 2-year-old P. notoginseng significantly decreased with increasing levels of RKN disease, while intercropping could significantly increase the survival rate of P. notoginseng seedlings with mild infection (1 level) (Fig. 4a).
With the exception of those at the 0 level, with respect to the P. notoginseng seedlings that were infected by RKN before planting, at one year after planting, the incidence of RKN disease of slightly infected P. notoginseng (1 level) decreased from 100 to 76% in the following year (Fig. 4b).
Moreover, the disease index of the 2-year-old P. notoginseng seedlings significantly increased with increasing level of RKN disease (Fig. 4c).

Fig. 4
Seeding survival rate, disease incidence and disease index of 2-year-old P. notoginseng in monoculture and intercropping systems. a Seeding survival rate of the 2-year-old P. notoginseng in monoculture and intercropping systems. b Disease incidence of RKN disease in the monoculture and intercropping systems. c Disease index of RKN disease in the monoculture and intercropping systems. The dif-ferent lowercase letters a, b above the error bars indicate significant differences between monoculture and intercropping. Significant differences are based on ANOVA tests (p < 0.05). The error bars indicate the standard errors of the means (n = 3). The different capital letters A, B denote significant differences among the P. notoginseng plants under different RKN infestation levels at the 5% level by LSD Table 3 The biomass of the 2-year-old P. notoginseng under different RKN infestation levels in the intercropping and monoculture systems All the data are presented as the mean ± SE of three biological replicates. The data in the same RKN infestation degrees and in the same column followed by different lowercase letters showed significant differences between the intercropping and the monoculture systems (Student's t test; p < 0.05; n = 3). The different capital letters (A, B) denote significant differences among the P. notoginseng plants under different RKN infestation levels at the 5% level by LSD.

Biomass of 2-year-old P. notoginseng with different RKN infestation levels in the intercropping and monoculture systems
Our data showed that the belowground biomass of the 2-year-old P. notoginseng regardless of total root, taproot and fibrous root biomass amounts per plant significantly increased in the P. notoginseng-pine tree intercropping systems compared with that of the 2-year-old P. notoginseng plants in the monoculture, but there were no effects on the aboveground biomass (Table 3). Further analysis showed that intercropping significantly increased the biomass of the taproot or fibrous roots of the 2-year-old P. notoginseng under mild infestation by RKN (1-2 levels). However, the promoting effects of intercropping were no longer detected in the 2-year-old P. notoginseng with higher infestation by RKN (3 levels), and there were no differences between the intercropping and the monoculture systems.

Nutrient uptake of 2-year-old P. notoginseng plants under different RKN infestation levels in the intercropping and monoculture systems
In the present study, the RKN infestation severity of P. notoginseng seedlings had little effect on the nutrient concentration and uptake of 2-year-old P. notoginseng ( Fig. 5;  Fig. S3). However, mild infection with nematodes (1 level) promoted N uptake in 2-year-old P. notoginseng, and it significantly increased the P concentration and uptake in the fibrous roots ( Fig. 5; Fig. S3). Further analysis showed that the N, P, and K nutrient uptake in the shoots of each P. notoginseng plant under the same RKN infestation degree was not significantly different between the plants in the The different lowercase letters a, b above the error bars indicate significant differences in the same RKN infestation degrees between the intercropping and the monoculture system (Student's t test; p < 0.05; n = 3). The different capital letters A, B denote significant differences among the P. notoginseng plants under different RKN infestation levels at the 5% level by LSD intercropping and the monoculture systems (Fig. 5a, d,  g). However, intercropping affected the P and K concentrations and the N, P, and K nutrient uptake of the roots of P. notoginseng plants ( Fig. 5; Fig. S3). Compared with monocropping, P. notoginseng-pine tree intercropping significantly increased the N and P uptake in the fibrous roots of each P. notoginseng plant, at the disease levels of 1 and 0, respectively (Fig. 5c, f). Additionally, the P uptake in the taproot of each P. notoginseng plant at the disease levels of 2 was significantly increased in the P. notoginseng-pine tree intercropping systems compared with the monoculture (Fig. 5e), whereas there were no effects on the N or K uptake (Fig. 5b, h).

Saponin accumulation in 2-year-old P. notoginseng with different RKN infestation levels in the intercropping and monoculture systems
To investigate the effects of P. notoginseng-pine tree intercropping on saponin accumulation, the contents of the total saponins and five active saponins in the fibrous roots and taproots of 2-year-old P. notoginseng with different RKN infestation levels under intercropping and monocropping were measured. Our results showed that the saponin concentrations of P. notoginseng in different disease grades were not significantly different from those of healthy P. notoginseng (Fig. S4), but mild infestation (1 level) could promote the synthesis of total saponins (Fig. 6). Moreover, the results indicated that intercropping affected the accumulation of total saponins (R1, Rg1, Re, Rb1, Rd) in the taproot of each P. notoginseng plant (Fig. 6a) but had no effect on the saponin concentration (Fig. S4). Intercropping could significantly increase the content of total saponins in the taproots at disease levels of 0 and 2 (Fig. 6a). Then, the heatmap showed that intercropping could significantly increase the contents of active saponins (R1, Rb1, Rd and Re, Rg1) in the taproot of plants at the disease level of both 0 and 2 (Fig. 6c). However, P. notoginseng-pine tree intercropping had no significant effect on the total saponin accumulation in the fibrous roots of each P. notoginseng plant (Fig. 6b). Notably, compared with monocropping, intercropping P. notoginseng with pine trees could significantly increase the content of Rd in the fibrous roots of each P. notoginseng plant at different RKN infestation levels (Fig. 6d).

Root morphology of 2-year-old P. notoginseng under different RKN infestation levels in the intercropping and monoculture systems
As shown in Fig. 7, intercropping could increase the root length, surface area and volume per plant (Fig. 7). Compared with monocropping, intercropping could significantly increase the root length and surface area per plant of the 2-year-old P. notoginseng under mild infestation (1 level), (Fig. 7a, b). However, the root volume per P. notoginseng plant was not significantly different between the intercropping and the monoculture systems (Fig. 7d). In addition, there were no significant differences in root diameter per plant among the P. notoginseng plants under different RKN infestation levels between the intercropping and monoculture systems (Fig. 7c).

RKN infestation decreased the growth and quality of P. notoginseng seedlings
RKN is one of the most economically damaging plant pathogens and causes stunted growth, resulting in quality and yield of in different crop species (Borah et al. 2018;Akram et al. 2019;Singh et al. 2019). In the present study, RKN infestation could significantly decrease the belowground biomass of P. notoginseng seedings, especially large-sized and medium-sized ones, and resulted in a lower root/shoot ratio of P. notoginseng (Table 2). In P. notoginseng production, farmers mainly use the taproots of P. notoginseng seedlings for transplanting, so these results indicated that RKN infection could affect the quality of P. notoginseng seedlings. Many previous studies reported that RKN infection causes great damage in different species, such as tomato, carrot, pepper, potato, and patchouli, resulting in poor growth and decreasing the quality and yield of the crops (Borah et al. 2018;Kepenekci et al. 2018;Singh et al. 2019). Upon further analysis, we found that RKN infestation The different lowercase letters a, b above the error bars indicate sig-nificant differences in the same RKN infestation degrees between the intercropping and the monoculture system (Student's t test; p < 0.05; n = 3). The different capital letters A, B denote significant differences among the P. notoginseng plants under different RKN infestation levels at the 5% level by LSD could significantly reduce the accumulation of saponins in the roots of P. notoginseng seedlings but showed no effect on saponin concentration ( Fig. 3; Fig. S2), indicating that the decrease in the synthesis of saponins was due to the reduction in biomass. Patchouli plant damage by RKNs mostly causes low oil yields and reduced essential oil quality (Pandey et al. 2009). Similarly, RKN causes a reduction in the yield, quality, and quantity of sweet potato tubers (Brito et al. 2020;Wendimu 2021). RKN infestation influences the nutrient uptake of plants (Khan et al. 2016(Khan et al. , 2018Tiwari et al. 2021). In the present study, RKN infestation had no effects on the nutrient uptake in the aboveground parts of P. notoginseng seedlings, but infestation could suppress the P and K uptake in the roots, especially in the roots of large-sized P. notoginseng seedlings (Fig. 2). Many previous studies have reported that the absorption capacity of the roots is generally impaired due to the formation of galls caused by Meloidogyne spp., which results in plants failing to acquire nutrients present in the soil, as evidenced by the 18%-28% lower N, P, and K contents of nematode-infected plants (Wilcox-Lee and Lorea 1987;Khan et al. 2016Khan et al. , 2018. Previous research also reported that infestation by parasitic nematodes causes a drastic reduction in herb and oil yields by influencing host nutrient contents and eventually affects the growth of plants (Melakeberhan et al. 1984;Tiwari et al. 2021). In conclusion, RKN infestation could significantly inhibit the belowground growth and nutrient and saponin accumulation, resulting in a decrease in the quality of P. notoginseng seedlings.

APR of P. notoginseng and planting within forests could effectively reduce nematode damage
In recent years, APR has been observed in many kinds of plants resistant to various diseases, and the disease index is a good indicator of the degree of APR in the field (Wang et al. 2005;Hao et al. 2007). In our research, with increasing levels of RKN disease of seedlings, the disease index of the 2-year-old P. notoginseng significantly increased, whereas the survival rate significantly decreased (Fig. 4a,  c), which may be closely related to the severe drought in the experimental area. Previous research also indicated that RKN infestation could reduce the resistance to environmental stresses of infested plants (Braun-Kiewnick et al. 2016;Kepenekci et al. 2018). Therefore, RKN infestation could reduce the adaptability of sanqi to extreme environmental conditions, resulting in a decrease in the survival rate of P. notoginseng. In addition, notably, the incidence of RKN disease of slightly infected P. notoginseng (1 level) decreased from 100 to 76% in the following year (Fig. 4b). These results indicated that P. notoginseng could better resist the damage of nematodes in the adult stage, which could also better explain the phenomenon that P. notoginseng seedlings were often severely damaged by nematodes, while the 2-or 3-year-old P. notoginseng were slightly damaged by nematodes in the field (Wang et al. 2021).
Planting diverse crop species can effectively control disease and reduce the damage caused by pests and diseases (Zhu et al. 2000;Ratnadass et al. 2012). In the present research, P. notoginseng-pine tree intercropping significantly increased the survival rate of P. notoginseng seedlings with mild infection (1 level) (Fig. 4a), indicating that the interaction between pine tree and P. notoginseng can increase resistance to nematodes and reduce their harmful effects. Many previous studies have also shown that cowpea or soybean intercropping with Telfairia occidentalis, pepper and Amaranthus can significantly reduce root galls and effectively suppress nematode infection (Agu 2008;Cookey et al. 2018). Therefore, the inhibition of RKN by pine trees may be an important reason why P. notoginseng RKN disease is rare in forests.

P. notoginseng-pine tree intercropping can improve the growth and quality of 2-year-old P. notoginseng
The infestation of nematodes mainly occurs in the lateral roots and root hairs of plants, preventing sufficient water and nutrient uptake by plants, resulting in poor growth, a decline in crop quality and yield and a reduction in resistance to other stresses (Devran et al. 2017;Khan et al. 2018;Kepenekci et al. 2018). In the present study, 2-year-old P. notoginseng seedlings infected by nematodes displayed no obvious inhibition in terms of biomass and saponin synthesis, and mild infection (1 level) could promote the growth and quality of P. notoginseng (Table 3; Fig. 6). These results showed that the 2-year-old P. notoginseng could maintain normal growth and quality formation under nematode infection and show a strong tolerance to RKN disease, it may be direct evidence to demonstrate that the P. notoginseng plants have APR to RKN disease. However, these results were inconsistent with those of previous studies showing that RKN mainly infects the roots of P. notoginseng, causes great damage to the normal physiological function of roots, and inhibits the growth of P. notoginseng (Dong et al. 2013;Yang et al. 2019b). The difference may be because the growth and quality of P. notoginseng in this study were evaluated only by individual plants and did not consider the seedling survival rate. Combining the effects of nematode damage on the seeding survival rate of the 2-year-old P. notoginseng mentioned previously with the results of the current study, we may conclude that the P. notoginseng plants have APR to RKN disease, which could ensure the normal growth and quality of P. notoginseng, but severe nematode infestation could affect the adaptability of P. notoginseng to adverse environments, resulting in high rates of sanqi death.
Intercropping can improve the yield and quality of plants by improving the rhizosphere soil ecological environment and soil quality Liu et al. 2021). In the present research, P. notoginseng-pine tree intercropping significantly increased the biomass of taproots and fibrous roots of 2-year-old P. notoginseng (Table 3). These results confirmed that pine trees are good neighbours of P. notoginseng. Then, some allelochemicals, such as α-terpineol and 2,3-butanediol, were from the volatile organic compounds (VOCs) and leachates of pine trees, which can reduce disease damage and alleviate negative plant-soil feedbacks of P. notoginseng, may play important roles (Ye et al. 2021;Li et al. 2022). At the same time, P. notoginseng-pine tree intercropping not only promoted root growth but also significantly increased the synthesis of saponins, with an especially significant increase in the contents of R1, Rb1 and Rd (Fig. 6). Similar results were reported by Liu et al. (2021), who found that Fallopia multiflora/Andrographis paniculata intercropping can improve the yield and quality of Fallopia multiflora . Gangadhar et al. (2018) also found a significantly higher oil yield and oil content of safflower in a safflower-linseed intercropping system in a suitable row proportion. The relay intercropping of Bt cotton in wheat could improve the growth and fibre quality of Bt cotton (Shah et al. 2019). Combining these results with the improvement of the survival rate of P. notoginseng in the intercropping system mentioned previously, we therefore concluded that P. notoginseng-pine tree intercropping promotes interspecific facilitation, which can improve the growth and quality of P. notoginseng. P. notoginseng-pine tree intercropping can promote the root growth of slightly infected P. notoginseng and increase nutrient uptake ability Plants can respond to surrounding environmental changes by optimizing root distribution and nutrient uptake capacity (Croft et al. 2012;Mou et al. 2013). In the present study, the nutrient uptake in the taproot of P. notoginseng first increased and then decreased with increasing levels of RKN disease but had no effect on the shoots (Fig. 5). These results suggested that the nutrient absorption capacity of the P. notoginseng root system significantly improved when the roots were slightly infested by nematodes. A previous study reported that inoculation of RKNs in bidi tobacco seedlings significantly increased K uptake in roots (Bairwa et al. 2012). In addition, the RKN lowest inoculum level had no effect on the N, P, or K content, while higher inoculum levels caused significant reductions in the N, P, and K contents of Lens culinaris (Hisamuddin and Azam 2010). The capacity of plant nutrient absorption mainly depends on root distribution and morphology. In this study, the root length, surface area and volume of P. notoginseng with mild infestation showed an increasing trend, which resulted in a significant decrease in the root surface area because of severe infestation (Fig. 7), indicating that mild infestation with nematodes could improve the nutrient absorption capacity of P. notoginseng by optimizing its root morphology. In previous studies, Ma et al. showed that nematode infestation increased seedling root length and root radius and enhanced root magnitude, altitude, and exterior path length (Ma et al. 2013(Ma et al. , 2014. These results can explain the differences in nutrient absorption of P. notoginseng with different nematode infestation levels. Nutrient uptake and root morphology of plants can greatly differ in intercropping systems (Raza et al. 2019;Duan et al. 2019). In our study, intercropping significantly increased the N, P and K uptake in the taproots of P. notoginseng but had no effects on the shoots (Fig. 5). At the same time, the root length and root surface area of intercropped P. notoginseng at the slightly infected were significantly increased (Fig. 7). These results indicated that intercropping improved the nutrient absorption capacity by promoting the root growth of P. notoginseng. Previous studies have shown that maize-faba bean and maize-chickpea intercropping significantly increased the root length and surface area of maize and ultimately promoted increased P uptake (Li et al. 2004;Zhang et al. 2012), whereas maize-alfalfa intercropping could significantly increase the P uptake and yield of alfalfa because of modifications to root morphology and distribution (Sun et al. 2019). Integrating our results of which intercropping significantly increased the survival rate of slightly infected P. notoginseng, as mentioned previously, we infer that the root optimization of P. notoginseng in this intercropping system may play a key role in P. notoginseng coping with extreme drought conditions, improving survival.

Conclusions
We used a P. notoginseng-pine tree intercropping system as a model to research RKN disease and the growth and quality of P. notoginseng under nematode infection. RKN infestation significantly reduced the growth and quality of P. notoginseng seedlings. 2-year-old P. notoginseng plants were highly tolerant to RKN disease, which corresponds to APR. Moreover, P. notoginseng-pine tree intercropping significantly reduced the incidence of RKN disease and significantly improved the growth and quality of slightly infected P. notoginseng, indicating that P. notoginseng-pine tree intercropping promotes interspecific facilitation. These "APR" or "P. notoginseng-pine tree intercropping" strategies can