Promoting Effects of Piriformospora indica on Plant Growth and Development of Tissue-Cultured Cerasus humilis Seedlings

Abstract

Piriformospora indica is a beneficial endophytic fungus that promotes plant growth and root development by colonizing plant roots. In order to investigate whether P. indica could promote the growth of tissue-cultured Cerasus humilis seedlings, in this study, we co-cultivated P. indica colony segments (P+) and P. indica spore suspensions (P++) in the rooting medium, and plant biomass as well as chlorophyll and root hormone contents of ‘3-19-3’ tissue-cultured C. humilis seedlings were determined under P+, P++, and CK (without fungus inoculation) treatments. The results showed that above-ground biomass and chlorophyll content of P+-and P++-treated tissue-cultured seedlings were significantly increased, and root peroxidase (POD), indole-3-acetic-acid (IAA) content, and root activities were significantly enhanced, while jasmonic acid (JA) and 1-aminocyclopropane-1-carboxylic acid (ACC) contents were reduced. Moreover, the growth-promoting effects of P++ treatment were found to be stronger than those of P+ treatment. Our results confirmed that P. indica was able to promote the growth of tissue-cultured C. humilis seedlings and effectively promoted root development by regulating hormone content. Therefore, the application of P. indica in the production of C. humilis is promising, especially in the cultivation of elite varieties.

1. Introduction

Cerasus humilis (Bge.) Sok, also known as ‘calcium fruit’ [1], is a shrubby fruit tree in the genus Prunus of the Rosaceae family and is known as an emerging third-generation fruit, along with blueberries and sea buckthorn [2]. Its fruits are brightly colored, moderately sweet and sour, with a strong aroma, and rich in a variety of vitamins, proteins, and minerals, which are beneficial to human health and have a high economic value [3]. At the same time, C. humilis fruits are rich in bioactive compounds such as flavonoids and carotenoids (β-carotene, lutein), which contribute significantly to its high antioxidant activity, so C. humilis fruits have great potential for health [4]. In addition, C. humilis is ecologically adaptable, able to grow in a wide range of unfavorable environments, and exhibits remarkable resistance to adversity [5]. Therefore, based on its strong ecological adaptability and multifunctional utilization value, C. humilis has drawn lots of attention from fruit growers and researchers. At present, in order to meet the needs of large-scale factory production of C. humilis seedlings, tissue culture technology has become an important means for their rapid propagation. This technology can not only effectively overcome the problems of long propagation periods and low propagation efficiency in the traditional propagation methods but also ensure the genetic stability and quality consistency of seedlings in large-scale planting for the purpose of fruit production [6]. However, during the growth and development of tissue-cultured C. humilis seedlings, issues such as weak shoot growth and poor root development persisted, resulting in low survival rates during transplanting [7]. Therefore, enhancing the growth quality and improving the physiological status of C. humilis seedlings have become key issues in the current propagation technology.

The interaction between plants and beneficial soil microorganisms, especially root endophytic fungi (such as arbuscular mycorrhizal fungi, AMF), is widely considered to be an important mechanism for promoting plant growth, increasing crop yield, and enhancing plant resistance to various environmental stresses [8]. Studies have shown that arbuscular mycorrhizal fungi can promote plant growth and improve plant tolerance to drought, salinity, disease, and other unfavorable environmental conditions by symbiosis with plant roots [9]. Among the arbuscular mycorrhizal fungi, Piriformospora indica from the Thar Desert of India is a strain with significant growth-promoting effects [10]. As a typical representative of AMF, P. indica not only has the functions of AMF to improve plant nutrient absorption, promote root development, and enhance stress resistance but also overcome the technical bottleneck of traditional AMF that is difficult to propagate in laboratory media [11]. In addition, the fungi have a wider range of plant host adaptability and are able to successfully colonize more than 200 plants [12]. According to reports, the colonization of P. indica occurs mainly in the roots of plants, especially in the cell differentiation zone [13].

A number of studies have shown that P. indica plays an important role in root development, plant growth, and survival rate of tissue-culture seedlings [13]. It has been shown that P. indica can effectively promote the number of roots of tissue-cultured banana seedlings and significantly shorten the rooting time [14]. Furthermore, Baishya et al. [15] found that P. indica significantly improves biomass productivity in Artemisia annua when co-cultured with Artemisia annua in vitro. At the same time, inoculation of tissue-cultured tobacco seedlings with P. indica was effective in reducing mortality rates [16]. Similarly, the application of P. indica in tissue-cultured gerbera seedlings also yielded positive results, including significantly increased survival rate and enhanced growth-related parameters and chlorophyll contents [17]. This phenomenon suggests that P. indica has important application values in not only promoting growth and root development but also improving the survival rate of tissue-cultured seedlings.

In our previous study [18], P. indica was utilized in C. humilis cutting propagation for the first time. By soaking the roots of C. humilis cuttings in the P. indica solution, the colonization rate was found to be as high as 90%. Further observations revealed that C. humilis cuttings inoculated with P. indica showed significant superiority over uninoculated seedlings in a number of growth indices. These results indicated that the symbiosis of P. indica and C. humilis plants significantly promoted their growth and development and had obvious growth-promoting effects. However, the application of this strain on tissue-culture propagation of C. humilis has not been extensively studied. Therefore, in the present study, the tissue-cultured seedlings of C. humilis variety ‘3-19-3’ were used as materials. In this study, we conducted P. indica inoculation in tissue-cultured C. humilis seedlings, and the growth parameters, photosynthetic pigment content, and hormone content were measured. The results of this study will contribute to optimizing the tissue-culture system in C. humilis and providing new strategies for C. humilis propagation and future cultivation.

2. Materials and Methods

2.1. Plant Materials and Fungal Preparation

In this study, tissue-cultured seedlings of Cerasus humilis variety ‘3-19-3’ obtained from the Cerasus humilis research group of Shanxi Agricultural University were used as experimental materials, and the Piriformospora indica strain was kindly provided by Dr. Cheng Chunzhen.

2.2. Explant Treatment

Two-thirds of the tender leaf branches at the top of the new shoots of seedlings of C. humilis variety ‘3-19-3’ were used for the experiment. These were rinsed with water for half an hour and then rinsed with distilled water twice. The clean branches were disinfected with 75% alcohol for 30 s, 2% sodium hypochlorite for two minutes, and then washed 3–4 times with sterile water. The tender leaf branches were dried on sterile filter paper and then cut to 1–2 cm long, and the stems with tender leaves were vertically inserted into MS + 1 mg L⁻¹ 6BA + 0.1 mgL⁻¹ IAA medium (Solarbio, Beijing, China). The C. humilis seedlings were cultured at 24 °C under 14 h of light (light intensity of 2000 Lx).

2.3. Fungus Preparation

A 0.5 cm piece of fungal plug was cut from the edge of potato dextrose agar (PDA) medium (Hopebiol, Qingdao, China) covered with P. indica mycelium, placed in the center of a new petri dish agar, and then cultured in a constant temperature incubator at 28 °C for one week to obtain enough mycelial growth/spore formation of P. indica for subsequent experiments. The treatment was repeated 10 times. Two P. indica inoculation treatments were set up. The first of the methods (P+) used a 0.5 cm mycelial plug taken from the growing edge of an actively growing culture of PDA. The second treatment, spore formation (P++), was performed by collecting chlamydospores according to the method described by Cheng et al. [14] and then adjusting the spore suspension to a final concentration of 2 × 10⁶ spores/mL for further use.

2.4. In-Vitro Inoculation

After three weeks of growth in the subculture medium (MS + 1 mg L⁻¹ 6BA + 0.1 mgL⁻¹ IAA), the tissue-cultured ‘3-19-3’ seedlings with 1–2 leaves were transferred to the rooting medium (1/2 MS + 1.2 mg L⁻¹ IAA) (Solarbio, Beijing, China) and cultured for two weeks under a 16 h photoperiod (light intensity of 2000 Lx), then seedlings with 3–4 leaves and about 3 cm height were selected for inoculation with P. indica.

This study uses three treatment methods. The P+ treatment consisted of two C. humilis tissue-culture seedlings, which were transferred to a new rooting medium, where a 0.5 cm P. indica fungal plug was placed onto the surface of the medium at 1 cm from the two seedlings. In the P++ treatment, 1 mL chlamydospore suspension (final concentration of 2 × 10⁶ spores ml⁻¹) was injected into the gap between the medium and the root of the tissue-culture seedlings. For the control group (CK), two tissue-culture seedlings were transferred to a tissue-culture bottle with the same rooting medium but without fungi. Each treatment was set up to include n = 20 replicates. The C. humilis seedlings were cultured for three weeks at 24 °C under 14 h of light (light intensity of 2000 Lx).

2.5. Piriformospora indica Colonization Detection

After two weeks, the colonization of tissue-cultured C. humilis by P. indica under P+ and P++ treatments was detected under the microscope using the Taipan blue staining method described in our previous study [18]. The colonization rate was determined as follows:

$$\mathit{colonization} \mathit{rate} \left(\%\right)=(\mathit{number} \mathit{of} \mathit{colonization} \mathit{root} \mathit{segments}/\mathit{total} \mathit{number} \mathit{of} \mathit{root} \mathit{segments}) \times 100$$

2.6. Determination of Plant Growth Parameters

After being co-cultured in tissue culture vessel for three weeks (control and P+/P++ treatments), C. humilis seedlings were then transferred to plant growth trays containing sterilized nutrient soil (peat/perlite = 1:1.5) and cultured in a growth chamber (Baidian Instrument and Equipment, Shanghai, China) at 23 °C, with a photoperiod of 16 h light/8 h dark and light intensity of 2000 Lx for two weeks. Weekly fertilization was conducted with ½-strength Hoagland’s solution.

On the first day and the 14th day after transplanting, the plant height, leaf number, total root length, root number, plant fresh weight, and plant dry weight of C. humilis seedlings were measured. Five seedlings were randomly selected from each treatment group and used as biological replicates.

2.7. Determination of Photosynthetic Pigments in Tissue-Cultured Cerasus humilis Seedling Leaves

After the tissue-cultured arising seedlings of C. humilis variety ‘3-19-3’ were grown in compost for two weeks, the photosynthetic pigments of the seedlings’ leaves were determined by the ethanol extraction method [19]. Five seedlings were randomly selected from each treatment group and repeated five times. Chlorophyll a, chlorophyll b, and carotenoids were determined by measuring absorbance at wavelengths of 665 nm, 649 nm, and 470 nm, respectively. The contents of chlorophyll a, chlorophyll b, and carotenoids were calculated according to the description of Yin et al [18].

2.8. Determination of Root Activity, POD Activity, and Hormone Content

After five weeks of successful establishment of the symbiotic relationship between the root system of C. humilis and P. indica, the colonized and non-colonized selected seedlings were collected, and the root activity was determined by the triphenyltetrazolium chloride (TTC) method [20]. Five seedlings were randomly selected from each treatment group and used as biological replicates.

In addition, the content of peroxidase (POD), indole-3-acetic-acid (IAA), jasmonic acid (JA), and 1-aminocyclopropane-1-carboxylic acid (ACC) in the roots of C. humilis seedlings were determined using the Plant140 enzyme-linked immunosorbent assay kit (Solarbio, Beijing, China) and the Multiskan SkyHigh 500C microplate reader (Thermo Fisher Scientific, Shanghai, China). Determination of each parameter was repeated 5 times.

2.9. Statistics Analysis

Statistical analysis was performed using SPSS 15.0 software, and the images were plotted using GraphPad Prism 10.0 software. The data were expressed as mean ± SDs of five biological replicates. A one-way ANOVA test was used for significance analysis in intra-group comparison.

3. Results

3.1. Piriformospora indica Colonization Detection Results

After two weeks of inoculation with P. indica, the roots of P+- and P++-inoculated seedlings were selected for observation under the 40-fold microscope, and it was found that clumps of spores were neatly arranged in the roots (Figure 1), which indicated that P. indica successfully colonized the roots of the tissue-cultured C. humilis seedlings. To further assess the colonization effect, the colonization rate was determined for both inoculation treatments. Thirty stained roots were randomly selected from P+ and P++ treatments for examination. The results showed that 23 segments (76.7%) were successfully colonized under P+ treatment, and 25 segments (83.3%) were successfully colonized under P++ treatment. These results indicate that both spore and colony segment inoculation can effectively infest the roots of tissue-cultured C. humilis seedlings with P. indica.

3.2. Effect of Piriformospora indica Colonization on Plant Phenotype of Tissue-Cultured Cerasus humilis Seedlings

On the first day of transplanting, it was observed that there were significant differences in the morphology of tissue-cultured C. humilis seedlings under different treatments. Compared with the control, C. humilis seedlings under P+ and P++ treatment groups showed significant increases in plant height, underground length, root number, leaf number, and plant fresh weight (Figure 2,

Table 1. Growth-related parameter changes in tissue-cultured seedlings after transplanting.

	1 d			14 d
	CK	P+	P++	CK	P+	P++
plant height (cm)	0.83 ± 0.10 c	1.89 ± 0.20 b	2.26 ± 0.13 a	1.85 ± 0.07 c	2.83 ± 0.10 b	3.29 ± 0.16 a
total root length (cm)	0.40 ± 0.10 b	0.86 ± 0.12 a	1.03 ± 0.11 a	0.74 ± 0.09 c	1.18 ± 0.09 b	1.64 ± 0.08 a
root number	2.00 ± 0.00 b	5.00 ± 1.00 a	5.00 ± 1.00 a	3.00 ± 1.00 b	4.00 ± 1.00 b	6.00 ± 1.00 a
leaf number	3.00 ± 2.00 c	8.00 ± 1.00 b	14.00 ± 1.00 a	9.00 ± 1.00 c	14.00 ± 2.00 b	17.00 ± 1.00 a
plant fresh weight (g)	0.12 ± 0.02 c	0.23 ± 0.03 b	0.28 ± 0.02 a	0.18 ± 0.02 c	0.27 ± 0.02 b	0.37 ± 0.02 a
plant dry weight (g)	0.02 ± 0.01 a	0.06 ± 0.01 a	0.08 ± 0.06 a	0.08 ± 0.01 c	0.14 ± 0.01 b	0.2 ± 0.02 a

Different lowercase letters (a, b, c) indicate significance less than 0.05 (p < 0.05). CK: plants without P. indica colonization; (P+): plants with fungi plug treatment; (P++): plants with spore treatment.

). However, the plant dry weight was the only parameter that did not show significant differences.

Two weeks after transplanting, we continued to observe the growth of plants in different treatment groups (Table 1). The results showed that except for the root number, the other parameters (plant height, underground length, leaf number, fresh weight, and dry weight) in the P+ and P++ groups were significantly higher than those in the control (p < 0.05). It is noteworthy that the P++ treatment group inoculated with P. indica chlamydospore suspension showed a more significant growth promotion effect than the P+ treatment group inoculated with P. indica colony segments. At the same time, it can be found from Figure 2 that the callus of the two treatment groups increased compared with the control group.

These results indicated that the inoculation of P. indica could promote the growth of C. humilis seedlings not only under tissue-culture conditions but also under growth chamber conditions. Moreover, the P++ treatment had a better growth-promoting effect than the P+ treatment.

3.3. Effects of Piriformospora indica Colonization on Root Activity of Tissue-Cultured Cerasus humilis Seedlings

To further evaluate the growth-promoting effects of P. indica colonization, the root activities of colonized and uncolonized C. humilis seedlings were determined by the TTC method. The results showed that the root activities of seedlings with successful colonization of P. indica (P+ and P++) increased by 13% and 35%, respectively, which were extremely significantly higher than those of the uncolonized seedlings (p < 0.01). Moreover, the root activity of C. humilis seedlings in the P++ group was also significantly higher than that of C. humilis seedlings in the P+ group (p < 0.01), showing a 19.6% increase (Figure 3).

3.4. Effects of Piriformospora indica Colonization on Photosynthetic Pigment Content in Leaves of Tissue-Cultured Cerasus humilis Seedlings

In order to understand the effect of P. indica colonization on photosynthetic pigment content in leaves of tissue-cultured C. humilis seedlings, photosynthetic pigment determination was conducted. The results showed that the photosynthetic pigment content of seedlings colonized with P. indica was significantly increased: the chlorophyll a contents of seedlings in the P+ and P++ groups were significantly increased by 19.1% and 25.1%, respectively, compared to the uncolonized seedlings, while the total chlorophyll contents also increased significantly by 17.7% and 35.9%, respectively. The contents of chlorophyll a and total chlorophyll in P++ were higher than those in the P+ group; however, the difference between the two groups was not significant (Figure 4).

In addition, the contents of chlorophyll b and carotenoids in the leaves of P+- and P++-treated C. humilis seedlings were 1.15-fold, 1.57-fold, 1.27-fold, and 1.94-fold of the control, respectively, and the differences were significant (p < 0.05). At the same time, differences in chlorophyll b and carotenoid contents between the P+ and P++ groups also reached a significant level (p < 0.05).

3.5. Effects of Piriformospora indica Colonization on POD Activity and Hormones in Roots of Tissue-Cultured Cerasus humilis Seedlings

The effects of P. indica colonization on root POD activity, IAA, JA, and ACC contents of tissue-cultured C. humilis seedlings were further studied (Figure 5).

The results showed that the root POD activities and IAA contents of P+ and P++ seedlings were significantly increased (p < 0.01). Specifically, the POD activities in P+- and P++-treated seedlings were 1.64-fold and 1.81-fold of the CK, respectively, while the IAA contents were 1.14-fold and 1.25-fold of the CK, respectively. Compared with the P+ treatment, the P++ treatment significantly increased the IAA content in the root system (p < 0.01); however, there was no significant difference in root POD content between the P+ and P++ treatments.

In addition, the JA and ACC contents in the roots of P+- and P++-treated seedlings were significantly lower than those of the CK (p < 0.05), accounting for 92 %, 79 %, 97 %, and 89 % of the CK, respectively. The differences in root JA and ACC contents between the P+ and P++ treatments reached a significant level (p < 0.05) and 15% and 9.0% reductions in root JA and ACC contents of the P++ group compared to the P+ group, respectively. The regulation of P. indica on root POD activity, IAA, JA, and ACC contents was similar to the results of our previous studies on C. humilis cutage seedlings, indicating that P. indica may be able to promote rooting of seedlings by increasing POD activity and IAA content and reducing JA and ACC contents at the same time.

4. Discussion

4.1. The Successful Colonization of Piriformospora indica Significantly Promoted the Growth of Tissue-Cultured Cerasus humilis Seedlings

P. indica has been shown to be able to colonize widely and establish symbiotic relationships with a variety of plants, covering a wide range of plant groups from monocotyledons to dicotyledons. However, the speed of root colonization and symbiotic function depend largely on the characteristics of host plants [21]. In this study, typical pear-shaped or oval spores were found in the cortical cells of C. humilis under the microscope. It is proven by this that P. indica could successfully colonize the roots of tissue-cultured seedlings of C. humilis variety ‘3-19-3’ and that multiple growth parameters of the aboveground parts of the seedlings showed significant improvement after inoculation with P. indica. This result is consistent with the growth-promoting effect of P. indica on other tissue-cultured seedlings, such as banana [14], strawberry [20], and Oncidium [22], in previous studies, further verifying its potential to promote plant growth.

It has been reported that P. indica promotes plant growth mainly by increasing root length and root number [23]. This is supported by the results of the present study, where we found that after P. indica successfully colonized the roots of C. humilis, the number and length of roots were significantly increased (p < 0.05). Similarly, P indica can significantly promote root development after establishing a symbiotic relationship with Bougainvillea tissue-cultured seedlings [24]. This result indicated that P. indica not only promoted plant root growth but also indirectly contributed to overall plant growth by enhancing root development.

4.2. After the Colonization of Piriformospora indica, the Root and POD Activity Increased, Which Was Beneficial to Root Growth and Development

It has been reported that P. indica can enhance the nutrient absorption capacity of host plants, thereby promoting plant growth and development [25]. The root activity level of plants directly determines the absorption efficiency of water and nutrients, so root activity plays a crucial role in the overall growth and development of plants [26]. The root activity of strawberry plants inoculated with P. indica was significantly up-regulated, indicating the positive effect of P. indica on plant roots [27]. Increasing root activity can not only enhance the ability of plants to absorb water and nutrients but also contribute to improving the adaptability of plants to maintain stable growth under complex and changing environmental conditions [28]. In the present study, the transplanted treatment group of seedlings showed significant improvement in root activity, indicating that colonization of P. indica improved root development of the tissue-cultured seedlings, which, in turn, impacted other morpho-physiological characteristics.

As a part of the plant antioxidant system, POD plays an important role in plant response to adverse environments [29], and some studies have shown that P. indica colonization can induce changes in POD activity in host plants [30]. The POD activity of peanuts increased significantly after inoculation with P. indica, which effectively reduced the damage of drought stress to peanuts [31]. In addition, POD is also widely considered an important marker and predictor of plant rooting performance [32]. Cheng et al., found that the colonization of longan roots by P. indica induced the up-regulation of POD activity, thus improving the growth environment of roots and promoting the rooting ability of plants [33]. In this study, the POD activity of roots was significantly increased after inoculation with P. indica, indicating that P. indica may improve the physiological state of roots by inducing POD activity, thus effectively enhancing the rooting ability of C. humilis seedlings.

Two inoculation approaches were used in these experiments and, as can be observed from the morphology of tissue-cultured seedlings (Figure 3), the treatment with spores showed a more significant effect (p < 0.05) in growth-promoting of C. humilis seedlings. This may be due to the fact that during the inoculation process, the spores can directly contact the roots of the seedlings, thus accelerating their colonization process in the roots, which, in turn, enhances the root activity and POD enzyme activity in the roots, ultimately showing a more obvious growth promotion effect [14].

4.3. The Colonization of Piriformospora indica Greatly Promoted the Accumulation of Photosynthetic Pigments in Tissue-Cultured Cerasus humilis Seedlings

Photosynthetic pigments are the key components of photosynthesis in plants [34]. Numerous studies have been conducted to show that P. indica can significantly increase the chlorophyll content of host plants, thereby promoting photosynthetic efficiency and overall plant growth performance. The interaction between the tissue-cultured aloe seedlings and P. indica can increase the plant biomass and chlorophyll content [35]. And the improvement of photosynthetic efficiency not only enhances plants’ growth vigor but also improves the stress resistance of plants. After the colonization of Dracocephalum kotschyi by P. indica, the photosynthetic pigment production of the inoculated group was significantly increased as a means of mitigating the adverse effects of cadmium stress in order to promote plant growth [36]. This study found that photosynthetic pigment production, including chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, were significantly increased in the leaves of C. humilis seedlings grown after inoculation with P. indica, which may be attributed to the fact that P. indica colonization improves nutrient uptake and transport in the C. humilis seedlings, and increases the synthesis of photosynthetic pigments, which, in turn, promotes C. humilis seedlings’ growth.

4.4. Piriformospora indica Promoted the Root Growth of Cerasus humilis Seedlings by Inducing IAA Biosynthesis and Inhibiting the Accumulation of JA and ACC

Piriformospora indica mediates plant growth mechanisms through a variety of pathways. One of the key factors is its interaction with plant hormones, such as auxin, jasmonic acid, and ethylene [37]. IAA, as the most important growth phytohormone, can significantly improve plant growth and development by regulating its content and distribution [38]. P. indica promotes plant growth by increasing IAA content, and previous studies have also shown that colonization of clover roots by P. indica promotes the transport of IAA to the above-ground parts, thereby accelerating plant growth and development [39]. In this study, it was also found that the IAA content of C. humilis seedlings inoculated with P. indica two weeks after transplanting was significantly higher than that of the control group.

In addition, it was also observed that the contents of JA and ACC in roots of C. humilis seedlings decreased after inoculation with P. indica, which may be because P. indica plays a regulatory role in the hormone levels and growth regulation of plants. Consistent with previous findings by Khabat et al., P. indica can inhibit the defense-related accumulation of jasmonic acid to promote plant growth [40]. In some cases, high concentrations of ethylene may have a negative effect on plant root growth [41]. Therefore, P. indica can further promote root development and overall plant growth by reducing ACC content and regulating plant hormone levels. Therefore, P. indica promoted plant growth by up-regulating IAA content and inhibiting JA and ACC content.

While this study found that P. indica could promote the growth of C. humilis tissue-cultured seedlings, it primarily focused on the physiological level. The underlying molecular mechanisms—such as potential growth regulatory genes like auxin signal-related growth regulator gene (GFR)—remain unexplored. Future studies incorporating transcriptomics and metabolomics would help elucidate these mechanisms, as demonstrated in similar articles [42,43]. In addition, the duration of the experiment was short (three weeks), and it may not be possible to capture potential effects such as seasonal adaptation.

5. Conclusions

Based on the results of this study, we constructed a preliminary model for the growth of tissue-cultured C. humilis seedlings after colonization with Piriformospora indica (Figure 6). Tissue-cultured seedlings in the P+ and P++ treatments showed significant growth improvement compared with non-colonized C. humilis seedlings, especially those inoculated with P. indica spore suspensions (P++), which showed superior growth-promoting effects. The colonization of P. indica promoted the accumulation of photosynthetic pigments in the leaves of C. humilis, enhanced the photosynthetic efficiency of the leaves, and provided sufficient energy for the plant to grow above ground. In terms of root development, the fungal colonization increased POD activity (an indicator of rooting capacity) and root activities, which helped improve the structure and function of the root system. In addition, P. indica colonization enhanced the rooting capacity of the root system by decreasing the content of JA and ACC in the root system while promoting the synthesis of IAA, which ultimately promoted the growth of C. humilis seedlings.