Comparative metabolomic profiling reveals molecular mechanisms underlying growth promotion and disease resistance in wheat conferred by Piriformospora indica in the field

ABSTRACT

Piriformospora indica, a plant root-colonizing basidiomycete fungus, exhibits strong growth-promoting activity in symbiosis with a broad range of plants. Here, we report the potential of P. indica to improve growth, yield, and disease resistance in wheat in the field. In the present study, P. indica successfully colonized wheat through chlamydospores and formed dense mycelial networks that covered roots. Plants subjected to the seed soaking (SS) treatment with P. indica chlamydospore suspensions enhanced tillering 2.28-fold compared to the non-inoculated wheat in the tillering stage. In addition, P. indica colonization promoted vegetative growth significantly during the three-leaf, tillering, and jointing stages. Moreover, the P. indica-SS-treatment enhanced wheat yield by 16.37 ± 1.63%, by increasing grains per ear and panicle weight and decreased damage to wheat shoot and root architecture markedly, with high field control effects against Fusarium pseudograminearum (81.59 ± 1.32%), Bipolaris sorokiniana (82.19 ± 1.59%), and Rhizoctonia cerealis (75.98 ± 1.36%). Most of the primary metabolites, such as amino acids, nucleotides, and lipids, involved in vegetative reproduction were increased in P. indica-SS-treatment plants, whereas secondary metabolites, such as terpenoids, polyketides, and alkaloids, decreased following P. indica inoculation. The up-regulated processes of protein, carbohydrate, and lipid metabolism indicated that P. indica colonization increased growth, yield, and disease resistance via the acceleration of plant primary metabolism. In conclusion, P. indica improved morphological, physiological, and metabolic substance levels, and further promoted its growth, yield, and disease resistance in wheat.

KEYWORDS:

• Piriformospora indica
• wheat
• growth-promoting
• disease resistance
• metabolomics

Introduction

Wheat is one of the major food crops for nearly a half of the global population.¹ However, wheat cultivation is adversely affected by various biotic and abiotic factors, so that further increase in wheat production has been impeded. Today, irrational application of fertilizers and fungicides leads to soil compaction, environmental pollution, and pesticide residues in soil and produce, among other environmental challenges.^2,3 In recent years, researchers have directed their attention to strategies for promoting wheat growth, adapting wheat production to environmental change, improving wheat yield and quality, and achieving sustainable agricultural development.^4,5

Among antagonistic fungi, symbiotic mycorrhizal fungi play important roles in soil nutrient acquisition and plant growth promotion, and can inhibit damage by aboveground and underground plant pathogens, thus improving plant performance and tolerance.^6–9 Piriformospora indica is a plant root-colonizing fungus belonging to the Basidiomycetes, based on nuclear DNA sequences from the D1/D2 region of the large ribosomal subunit. P. indica can replicate without any host plant.¹⁰ In addition, it has the capacity to grow under different substrates and its asexual stage forms chlamydospores. Following its discovery in the Indian Thar Desert in 1997,¹¹ P. indica has attracted considerable attention over the years owing to its growth and yield promotion abilities, besides its potential to confer systemic resistance against numerous of biotic and abiotic stress factors.^12–15 Furthermore, P. indica establishes beneficial interactions with numerous hosts, including monocotyledons, such as barley, wheat, rice, corn, and dicotyledons, such as Arabidopsis and tobacco.^16–18 In barley, root growth is strongly induced between 2 and 3 weeks following inoculation with P. indica, and further development of colonized roots is reflected by increased levels of expression of developmentally regulated genes.^18–20 In addition, the interaction of the endophytic fungus with Arabidopsis roots is accompanied by considerable nitrogen acquisition from the environment.¹¹ In interactions with Arabidopsis and tobacco, the fungus stimulates nitrate reduction, in contrast to the effect of arbuscular mycorrhizal fungi.²¹ In addition to the direct benefits of interaction between P. indica with plant host, application of autoclaved culture filtrates containing its exudates as well as cell-wall extracts have been demonstrated to promote plant growth strongly.²² Furthermore, P. indica has attracted considerable interest as a potential bioprotector agent.²³ P. indica is an excellent model of beneficial microbes since it primes plants for disease resistance against biotrophic and necrotrophic fungi,²⁴ oomycetes,²⁵ and viruses.²⁶ Recently, systemic resistance against a leaf pathogen, Blumeria graminis f. sp. hordei, was observed in Hordeum vulgare plants co-cultivated with P. indica.²⁷ Similarly, barley roots colonized by P. indica remained healthy when exposed to the Fusarium culmorum pathogen.¹⁸

The aim of the present study was to investigate and report the agronomic potential of P. indica fungus in wheat crop. Firstly, we demonstrate the wheat growth-promoting activity of the fungus in the field. In addition, we investigate the capacity of P. indica to resist wheat pathogens, including F. pseudograminearum, Bipolaris sorokiniana, and Rhizoctonia cerealis in the field. Finally, we perform root and leaf metabolomics analyses to reveal the potential underlying mechanisms via which P. indica regulates wheat growth and promotes yield.

Results

Piriformospora indica colonizes root cortical cells

P. indica colonization of the roots of wheat was identified by staining the cells with Trypan blue. Microscopic inspection of wheat co-cultured with P. indica after 7 d showed that the fungus colonized root cortical cells (Figure 1A). Dense mycelia were generated by germination of chlamydospores, which intertwined to form mycelial networks on the root surface (Figure 1B). This observation was further verified under Scanning Electron Microscopy (SEM) with root material grown in P. indica-inoculated substrate (Figure 1D-E).

Figure 1. Colonization pattern of P. indica in wheat roots.

Note: (A) The fungi form chlamydospores and germinate hypha. (B) Dense mycelia form mycelia networks on the root surface of wheat. (C) Roots of non-inoculated wheat were used as the control. Fungal structures were visualized following staining with 0.05% trypan blue. (D, E) Scanning Electron Microscopy (SEM) images of the colonization process of P. indica in wheat roots. Arrow indicated spores and circles indicated mycelia of P. indica. (F) SEM images of non-inoculated wheat, as the control.

Piriformospora indica promotes wheat growth

P. indica had a significant positive influence on the shoot and root architecture of wheat, which enhanced root and leaf vegetative growth (Table 1 and Figure 2). We selected potted wheat seedlings for use in the determination of the optimal concentrations of chlamydospore of P. indica in further field experiments. According to the results of preliminary experiments, the application of 1 × 10⁵/mL of P. indica spores had the optimal effects on wheat plant height and root length (Supplementary Table S1 and Figure S1). Subsequently, two classical methods of inoculation application, seed soaking (SS) and root irrigation (RI), were used to assess the effect of P. indica on biological characteristics of wheat in the field. Growth promotion was not obvious in the early three-leaf stage in the SS and RI treatments when compared to the control, whereas plant length in the SS treatment increased (1.17-fold) when compared to the non-inoculated wheat (Table 1). Additionally, the SS and RI plants showed significant increases in chlorophyll content, and plant fresh and dry biomass, when compared to those in plants without P. indica inoculation, in the tillering stage (Table 1). In particular, P. indica colonization promoted tillering by 2.28-fold in the SS treatment and 2.64-fold in RI treatment, when compared to that in the control. Furthermore, SS plants exhibited significantly higher plant length, root length, fresh and dry biomass in the jointing stage than in the control, and the fresh weight and dry weight increases were 2.17-fold and 2.27-fold, respectively (Table 1). The results indicate that P. indica promoted wheat growth in the field. In addition, at multiple growth stages, the SS treatment had greater effects on wheat than the RI treatment.

Figure 2. Effect of P. indica colonization on wheat at three-leaf, tillering, and jointing stages.

Note: The following treatments were set up: control (CK), P. indica inoculation via seed soaking treatment (SS), P. indica inoculation via root irrigation treatment (RI). Photos illustrate differences in vegetative growth and root architecture of wheat plants under P. indica colonization or alone at different stages.

Table 1. Effect of P. indica on biological characteristics of wheat.

Biological characteristics	Three-leaf stage			Tillering stage			Jointing stage
	CK	SS	RI	CK	SS	RI	CK	SS	RI
Plant height/cm	9.40 ± 0.15^a	11.00 ± 0.31^b	10.00 ± 0.31^a	21.13 ± 0.68^a	21.97 ± 1.16^a	22.06 ± 0.58^a	56.18 ± 2.56^a	66.85 ± 0.20^b	59.45 ± 6.44^a
Root length/cm	8.40 ± 0.31^a	7.43 ± 0.39^a	7.80 ± 0.40^a	10.27 ± 0.35^a	11.33 ± 0.69^b	12.50 ± 0.30^b	10.94 ± 1.00^a	13.85 ± 0.34^b	12.74 ± 0.12^b
SPAD	41.00 ± 0.81^a	39.00 ± 1.32^a	40.00 ± 0.42^a	36.07 ± 0.39^a	42.36 ± 1.29^b	40.62 ± 0.34^b	41.69 ± 1.07^a	50.01 ± 2.15^b	37.35 ± 0.53^a
Number of tillers per pot	n.d.	n.d.	n.d.	0.67 ± 0.27^a	1.93 ± 0.12^b	1.77 ± 0.33^b	n.d.	n.d.	n.d.
Shoot weight/g	2.1 ± 0.21^a	2.00 ± 0.14^a	2.10 ± 0.16^a	6.01 ± 0.32^a	9.85 ± 0.53^b	8.33 ± 0.48^b	28.69 ± 9.95^a	82.31 ± 3.13^b	28.55 ± 11.28^a
Root weight/g	0.20 ± 0.04^a	0.10 ± 0.03^a	0.10 ± 0.06^a	0.22 ± 0.05^a	0.44 ± 0.09^a	0.40 ± 0.07^a	1.22 ± 0.10^a	3.33 ± 0.39^b	1.84 ± 0.58^a
Total fresh weight/g	2.20 ± 0.18^a	2.1 ± 0.12^a	2.20 ± 0.17^a	6.25 ± 0.31^a	10.24 ± 0.59^b	8.79 ± 0.44^a	39.43 ± 9.95^a	85.64 ± 3.13^b	28.55 ± 11.28^a
Dry shoot weight/g	0.4 ± 0.03^a	0.4 ± 0.08^a	0.40 ± 0.03^a	1.39 ± 0.08^a	2.04 ± 0.14^b	2.01 ± 0.13^b	9.29 ± 2.67^a	21.00 ± 1.33^b	7.47 ± 2.67^a
Dry root weight/g	0.10 ± 0.03^a	0.10 ± 0.03^a	0.10 ± 0.01^a	0.08 ± 0.00^a	0.15 ± 0.02^b	0.15 ± 0.01^b	0.42 ± 0.05^a	1.05 ± 0.03^b	0.71 ± 0.12^b
Total dry weight/g	0.40 ± 0.03^a	0.50 ± 0.09^a	0.50 ± 0.04^a	1.46 ± 0.08^a	2.26 ± 0.17^b	2.15 ± 0.14^b	9.71 ± 2.70^a	22.06 ± 1.35^b	8.06 ± 2.97^a

Data in the table are means of nine points (with ten plants per point) from three plots. Different lowercase letters denote significant differences between P. indica inoculated and no-inoculated plants (P < 0.05). n.d., not detected.

Piriformospora indica enhances wheat yield

To assess whether P. indica colonization would increase grain yield, the wheat cultivar Zhongmai 100 was tested in an open-air field station in winter/spring. P. indica-SS-inoculated wheat grain yield increased by 16.37 ± 1.63% when compared with the control plant, with a high number of grains per ear and panicle weight (Table 2). Grain yield was relatively less affected in P. indica-RI-inoculated plants, with only a 1.67 ± 0.17% increase when compared with the non-inoculated wheat (Table 2). The results show that P. indica promoted wheat yield by increasing grains per ear and panicle weight.

Table 2. Effect of P. indica on yield characteristics of wheat.

Treat	Plant height/cm	Panicle length/cm	Grains per ear	Panicle weight/g	Yield (g/per 100 ears)	Growth rate (%)
CK	77.85 ± 2.26^a	8.00 ± 0.38^a	31.73 ± 4.24^b	1.90 ± 0.28^b	186.44 ± 20.33^b	-
SS	80.87 ± 1.85^a	8.00 ± 0.36^a	41.35 ± 6.50^a	2.34 ± 0.63^a	216.96 ± 23.50^a	16.37 ± 1.63
RI	80.13 ± 2.37^a	8.00 ± 0.42^a	37.59 ± 3.40^a	2.08 ± 0.31^ab	189.56 ± 17.55^b	1.67 ± 0.17

Data in the table are means of five points (with twenty plants per point) from each plot. Different lowercase letters denote significant differences between P. indica inoculated and no-inoculated plants (P <0.05).

Piriformospora indica induces disease resistance

Many studies have shown that P. indica could improve resistance to powdery mildew,^28,29 F. oxysporum, Verticillium dahlia, and Pepino Mosaic Virus.^30–32 We addressed the question of whether P. indica-inoculated wheat would also be more resistant to root fungal pathogen. Wheat was grown in soil containing the conidiophore or mycelium suspension of F. pseudograminearum, B. sorokiniana, and R. cerealis, and disease incidence was recorded. In the presence of P. indica, the deleterious effects of F. pseudograminearum, B. sorokiniana, and R. cerealis were significantly minimized. P. indica-SS-inoculated plants were more resistant to root disease than the RI treatment plants (Table 3). P. indica-SS-inoculated wheat exhibited enhanced control effects against F. pseudograminearum (81.59 ± 1.32%), B. sorokiniana (82.19 ± 1.59%), and R. cerealis (75.98 ± 1.36%). Moreover, P. indica-RI-treatment wheat showed improved control effects against F. pseudograminearum (64.55 ± 2.97%), B. sorokiniana (61.40 ± 0.75%), and R. cerealis (62.28 ± 1.98%). In addition, we observed that the P. indica-inoculated plants showed increasing plant length compared to the control plants and exhibited significantly fewer symptoms on wheat stems (Supplementary Figure S4). According to the results, P. indica exerts beneficial effects against the three major cereal pathogens that cause economic losses globally.

Table 3. Effect of P. indica on root infection by three fungal pathogens.

	Fusarium pseudograminearum		Bipolaris sorokiniana		Rhizoctonia cerealis
	Disease incidence (%)	Field control effect (%)	Disease incidence (%)	Field control effect (%)	Disease incidence (%)	Field control effect (%)
CK	30.52 ± 1.56^a	-	48.00 ± 2.60^a	-	42.50 ± 3.05^a	-
SS	5.62 ± 0.40^b	81.59 ± 1.32	8.55 ± 0.71^b	82.19 ± 1.59	10.21 ± 0.56^b	75.98 ± 1.36
RI	10.82 ± 0.89^c	64.55 ± 2.97	18.53 ± 0.31^c	61.40 ± 0.75	16.03 ± 0.79^c	62.28 ± 1.98

Data in the table are means of three replicates (with twenty plants per point) from each plot. Different lowercase letters in column Disease incidence denote significant differences between P. indica inoculated and no-inoculated plants (P <0.05). And the lowercase letters in column field control effect express significant differences between two treatments of P. indica inoculated.

Metabolic profiles of wheat tissue in response to Piriformosporaindica colonization

Although the beneficial effects of the root endophyte P. indica are widely recognized, the underlying mechanisms via which the positive effects are achieved are still unclear. To comprehensively explore the metabolic changes in different tissues in response to P. indica colonization, non-targeted metabolic analysis was performed on SS and RI-treatment roots and leaves in the jointing stage. As shown in Figure 3A, the metabolite profiling of SS treatment showed different changes in contrast to RI groups that included amino acids, fatty acids, terpenoids, polyketides, and other compounds. The number of up-regulated metabolites under the SS treatment was less than those in the RI treatment. A total of 930 metabolites were detected in wheat roots under the SS treatment. Among them, the levels of 127 metabolites increased and the levels of 803 metabolites decreased. Furthermore, 560 metabolites changed under the SS treatment in wheat leaf when compared with the control plant: the concentrations of 453 metabolites decreased and the concentrations of 107 metabolites increased. Under the RI treatment, the levels of 368 metabolites increased, whereas the levels of 192 metabolites decreased in roots, and the levels of 513 metabolites increased and the levels of 453 metabolites decreased in leaves.

Figure 3. Metabolic changes in wheat tissues under P. indica colonization in the jointing stage.

Note: (A) Differential metabolite accumulation in the roots and leaves of wheat under P. indica colonization. Blue columns represent up-regulated metabolites and orange columns represent down-regulated metabolites. (B) Heatmaps for wheat metabolite profiles under P. indica colonization. Positive mode Electrospray Ionization-Mass Spectrometry (ESI-MS) was used. The right column shows the compound ID from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Each group has three biological replicates, and each biological replicate comprises a pool of 10 plants.

To explore the trends of the different metabolites in wheat colonized by P. indica, significantly altered metabolites (P < 0.05) in the SS-root, SS-leaf, RI-root, and RI-leaf groups were selected for subsequent analysis and associated heatmaps plotted (Figure 3B, Supplementary Table S2). The metabolite profiles showed that P. indica colonization resulted in drastic changes in wheat root and leaf metabolites. We were particularly interested in metabolites that showed more than two-fold differences among SS-root, SS-leaf, RI-root, and RI-leaf groups. Amino acids (19), lipid (9), terpenoids and polyketides (18), and other secondary metabolites (24) are major groups of metabolites altered due to P. indica colonization. In SS-root, 24 metabolites were positively accumulated and 18 were decreased; in SS-leaf 21 compounds were positively accumulated and 41 were decreased due to P. indica infection. In addition, in RI-root, levels of 31 metabolites were increased, whereas only 13 were decreased; in RI-root, 32 metabolites showed increased accumulation and 21 decreased due to P. indica colonization. Metabolic profiles in SS samples showed different but largely negative effects on metabolite levels, while in the RI treatment, metabolite levels were largely increased. The concentrations of numerous metabolites involved in the metabolism of amino acids and lipids such as L-Allysine, N-(17-Hydroxylinolenoyl)-L-glutamine, and Icosenoic acid were increased in SS-root when compared with the control plant, including several terpenoids and polyketides, whereas other secondary metabolites such as Secologanin, L-Dopa, and Thebaine had much lower levels in SS-root. Conversely, in RI-root, most metabolites participated in lipid, terpenoid, and polyketide metabolism, and the levels of alkaloid-related metabolites increased under P. indica colonization when compared with the control. In addition, the levels of several secondary metabolites were much lower in SS-leaf and higher in RI-leaf than the level in the control plant. Notably, the concentrations of dopaquinone (C00822), N-Methylanthranilate (C03005), 3-methoxytyramine (C05587), and S-Adenosylmethionine (C00019), which belong to the anthocyanin and betalain biosynthesis pathways, were only increased in RI-leaf than in the other groups under P. indica infection.

To further explore the active physiological processes under P. indica colonization, 26 key expression pathways with the greatest degrees of change were determined (Figure 4). The metabolism of several amino acids, pyrimidine, lipids, and terpenoids increased following P. indica inoculation, whereas polyketide, flavone-related, and alkaloid metabolism were hindered under the SS treatment when compared with the control plants. However, in the RI treatment, primary metabolic processes, including amino acid, pyrimidine, and lipid metabolism, were decreased under the colonization of P. indica. Moreover, some pathways enriched in zeatin, anthocyanin, and alkaloid and other secondary metabolisms were increased following P. indica inoculation under RI treatment in contrast to the control.

Figure 4. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways analysis of altered metabolites under P. indica colonization.

Note: (A) Enrichment pathways of metabolites following P. indica inoculation under the seed soaking (SS) treatment. (B) Enrichment pathways of metabolites following P. indica inoculation under the root irrigation (RI) treatment. Bubble size represents the number of enrichment factors in the pathway. Bubble color change from purple to yellow indicates greater statistical significance.

Discussion

Global concerns over the overuse of synthetic pesticides have directed the attention of stakeholders and researchers to novel environmentally friendly fungicides or plant growth regulators. The application of useful antagonistic microorganisms, such as mycorrhizal fungi, rhizobacteria, and endophytic bacteria, has been considered as a promising alternative to globally available pesticides. P. indica is an excellent model of beneficial microorganisms because it can be genetically transformed and cultured under sterile conditions,³³ promote plant growth and yield,³⁴ enhance plant tolerance to abiotic stress,³⁵ and enable plants to resist biotrophic and necrotrophic fungi, oomycetes, and viruses.^24,25,30

In the present study, P. indica colonization of wheat root was identified using Trypan blue staining and SEM. Our results showed that the chlamydospores of P. indica germinated mycelia to assemble dense mycelial networks and packaged the wheat root (Figure 1). As a result of P. indica colonization, wheat vegetative growth was increased significantly, which were positively correlated with increases in total yield in field trials (Tables 1 and 2). P. indica-induced root growth and alterations in the root architecture have been observed in Chinese cabbage, Arabidopsis, maize, and rice.^36–39 In the present study, P. indica treatment increased wheat root length and plant height considerably, which in turn, promoted plant biomass (Table 1). Acceleration of root development enhances nutrient and water uptake, which subsequently promotes the growth of above ground plant parts. According to the results of previous studies, P. indica stimulates the expression of macronutrients transporter genes to improves the plant’s own uptake capabilities.^21,40,41 Moreover, P. indica can elevate the photosynthetic rate and growth phytohormones secretion, which helps in host’s growth and development.^39,42,43 We also observed that an increase in vegetative growth was positively correlated with an increase in total yield due to an increase in grains per ear and panicle weight following P. indica inoculation (Tables 1 and 2). In addition, the heading time of plants in the SS treatment was earlier than that in the non-colonized plants (data not shown). Achatz et al. (2010) reported that P. indica enhanced the grain yield of barley, which caused an increase in the number of ears per plant at early development stages. This indicates that the higher grain yield in P. indica-colonized plants not only results from the formation of more tillers but also from more rapid development, e.g., the observed earlier emergence of ears. Hence, P. indica colonization of wheat could promote plant growth and yield by enhancing plant capacity to absorb nutrients and promote the development of vegetative organs.

To explore the active physiological processes in wheat under colonization by P. indica, non-targeted metabolic analysis was performed on wheat roots and leaves following colonization by P. indica at the jointing stage. Different metabolic profiles were observed in different wheat tissues under different P. indica treatments. Primary metabolic processes, including amino acid and lipid metabolism, were up-regulated under the SS treatment when compared with the control plants. In addition, secondary metabolism processes were up-regulated following P. indica inoculation via the RI method in contrast to the P. indica non-inoculated wheat (Figure 3). Plant growth and yield are influenced by primary metabolism processes that synthesize protein, carbohydrate, and lipid-related substances.⁴⁴ In most of the host plants, it significantly stimulates growth by improving nutrient uptake into the hosts,^43,45,46 inducing early flowering,^47,48 and altering the accumulation of secondary metabolites.⁴⁹ Therefore, P. indica colonization promoted the growth and yield of wheat through inducing primary metabolism to enhance the synthesis of nutrient substances. Similarly, the accumulation of high amounts of primary metabolites increases osmotic pressure, which minimizes damage to cell structure and facilitates the maintenance of normal metabolic activities of plant cells.⁵⁰ Several antibiotic substances, such as penicillin and cephalosporin, were only increased in wheat root under P. indica colonization, which might induce antibacterial effects to protect plant from infection by pathogens (Supplementary Table S2). The pathways of cutin, suberine, and wax biosynthesis were up-regulated in leaves under the SS treatment in contrast to the control plant (Figure 4 and Supplementary Table S2). High cuticle production and stimulation of the expression of drought-responsive genes have been observed in P. indica-colonized Arabidopsis and Chinese cabbage under drought stress.^38,46 High cuticle production in P. indica-colonized plants can enhance wheat resistance to biological and abiotic stress factors by modulating stoma closure and regulating plant cell osmotic pressure, which promotes growth and disease resistance. In addition, some metabolites belonging to plant pigments biosynthesis (anthocyanin and betalain) displayed high accumulation in RI-leaf than in other groups under P. indica infection. These pigments protect plants from damage caused by UV and visible light, and act as antioxidants to reduce the production of ROS, which contributes to promoting the ability of resistance to biotic and abiotic stresses of plant.^51,52 Such metabolic results are also consistent with growth and yield promotion, via which P. indica accelerates nutrient acquisition and transition from the vegetative to the reproductive stage.

Two classical methods of pesticide application, seed soaking (SS) and root irrigation (RI), were used in the present study to evaluate the effect of P. indica on wheat growth, yield, and disease resistance. Overall, P. indica-SS-inoculated plants exhibited more significant growth, yield, and disease resistance enhancement than RI-treated plants. The SS treatment ensured interaction between wheat root tissues and P. indica, which facilitates P. indica colonization. However, the chlamydospores of P. indica might be more easily adsorbed by soil through RI treatment, which decreases the ratio of P. indica colonization. Moreover, environmental factors and farming practices might also affect the P. indica colonization efficiency, which reduces the promotion ability of fungus. To further exploit the growth promotion characteristics of P. indica, associated environmentally friendly seed soaking fungicides or plant growth regulators are under development.

Materials and methods

Fungal and plant material and cultivation

Piriformospora indica (CGMCC No.10325) was provided by Professor Karl-Heinz-Kogel at Justus-Liebig-University, Giessen, Germany. The wheat seeds (Zhongmai 100) were provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.

The field experiments were conducted on the experimental fields of the innovation facility of Tianjin Academy of Agricultural Sciences (39.43N, 116.96E), Tianjin, China, from October 2021 to June 2022. Warm temperate subhumid monsoon weather prevails in the area, with an average precipitation of 23 mm during the experiments. Supplementary Figure S2 shows the monthly averages of temperatures and precipitation. The soil of the experimental fields belongs to loam (pH 7.1–8.0). 600 kg/ha complex fertilizer (18% N, 22% P₂O₅ and 5% K₂O) was spread at the sowing stage of wheat growth. Water deficiency was supplemented when needed. For field experiments, experimental plots were 2 m² (= 2 × 1 m) and 110 grains were sowing per 1 m².

P. indica was cultured on potato-sucrose agar (PSA) plates (200 g/L potato, 20 g/L sucrose, and 15 g/L agar) at 25°C. To prepare chlamydospore suspensions of P. indica, after culture on PSA plates for 14 d, 0.02% tween-80 was added to the plates and the plates washed using spreader. Subsequently, the suspension was fully mixed using a vortex oscillator for 1 min and sterilized water was added to adjust the chlamydospore concentration to 1 × 10⁵/mL. For field assessment, 5000 of wheat seeds were washed with distilled water, then soaked into 75% ethyl alcohol for 5 min and divided into three parts, including the control (CK), soaking seed (SS), and root-irrigation (RI) groups. In the SS group, the seeds were soaked in chlamydospore suspensions of P. indica (1 × 10⁵/mL) for 30 min. In the RI group, the suspension of P. indica (1 × 10⁵/mL) was uniformly irrigated in a seed furrow before sowing, which 1 m of seed furrow irrigated with 100 mL suspension. Similar to treatment RI, 100 mL of suspension without fungi was added to the treatment CK. The three groups of wheat seeds were sown at the same time, with three replicates for each treatment. The schematic diagram of the field experimental area is shown in Figure S3.

Colonization observation of P. indica

The colonization status of P. indica was observed under a microscope by Trypan blue staining as described in a previous work.¹⁸ The sterilized wheat seeds were immersed in double-distilled water at 25°C for 1 week. Subsequently, the wheat seedlings were co-cultured with a chlamydospore suspension of P. indica (1 × 10⁵/mL) for another 1 week. The roots of co-cultured seedlings were washed thoroughly and cut into 1-cm segments. They were immersed in 10% KOH overnight and then washed with water three times. The root samples were dipped in 1% HCl for 5 min and then washed with water three times, again. They were then stained with 0.05% trypan blue for 4 min and then washed with water at six times. The samples were mounted on glass sides and then observed and recorded under a Nikon ECLIPSE NI-U research microscope (Nikon, Tokyo, Japan).

For SEM observation⁵³, the roots of co-cultured seedlings were cut into small pieces and washed three times using double-distilled water, and then immersed in 2.5% glutaraldehyde for 30 min. After washing several times in double-distilled water, the samples were dehydrated in a stepwise manner in 50%, 70%, 80%, 90%, and 100% ethyl alcohol, for 15 min at each step. The samples were dried at 4°C overnight and coated with ETD 2000C ion coater (Beijing Elaborate Technology Development Ltd., Beijing, China) and observed under a ZEISS MERLIN Compact Scanning Electron Microscope (Zeiss, Jena, Germany).

Vegetative growth and total yield assessment

To assess vegetative growth, three sampling locations in each plot, and 10 plants in each location were selected randomly to determine wheat plant growth at the three-leaf, tillering and jointing stages. The five-point sampling method, with 20 plants at each point and in each experimental plot, was used to calculate the average yield.

Disease resistance assessment

In vitro tests were conducted to investigate the antagonistic activity of P. indica against F. pseudograminearum, B. sorokiniana, and R. cerealis. Three groups of wheat containing CK, SS, and RI, were cultured in experimental plots for 1 week before being inoculated with pathogens. For F. pseudograminearum and B. sorokiniana, the concentrations of conidiophore were adjusted to 1 × 10⁵/mL. For R. cerealis, the mycelium and medium were broken up using tissue homogenizer. Each seed furrow in the experimental plots was irrigated with 100 mL conidiophore or mycelium suspension. After 3 weeks, disease incidence was calculated to assess the disease resistance capacity of P. indica.

Metabonomic analysis

Similar amounts of leaves and roots from 10 plants (one leaf per individual) were pooled at the jointing stage for each treatment as biological replicates, and three such replicates were used in metabolomic analyses. Leaf samples from each group were cut into small pieces and extracted from a 1-mL mixture of methanol and water (1:1, v/v). After fully grinding, the extracted metabolites (5 μL) were loaded on a Waters ACQUITY UPLC BEH C18 column (5 μm, 150 × 4.6 mm; Waters Crop., Milford, MA, USA) and then analyzed using a Waters UPLC I-Class/Xevo G2 ×S-Quadrupole Time-of-flight system (Waters Corp., Milford, MA, USA) at 4°C with a flow rate of 0.3 mL/min under an isocratic elution with water (0.1% formic acid) and acetonitrile (0.1% formic acid). Mass spectrometry detection was performed under the following conditions: capillary voltage: 0.5 kV; cone voltage: 35 V; extractor voltage: 4.0 V; source temperature: 11°C; desolvation temperature: 550°C. The detection was carried out in the positive ESI-MS mode. Metabolic data were collected and analyzed using MassLynx NT 4.1 software with QuanLynx program (Waters Corp). The relative concentrations (g/DW) of different metabolites in leaf and root tissues in each biological replicate were formatted as comma separated values (.csv). Significantly regulated metabolites in leaf and root between two-group means (P. indica colonization over control) were determined based on fold change and false discovery rate (Log2FC > 2 or Log2FC < 0.5 and P < 0.05). A pathway analysis was performed to better elucidate the function of the altered (significant at P < 0.05) metabolites in clusterProfile package,⁵⁴ via the KEGG pathway database (http://www.genome.ad.jp/kegg/pathway.html).

Statistical analysis

In this study, all data are presented as the mean ± SE of three biological replicates. The significance of differences was performed using IBM SPSS Statistics version 21.0 (IBM Corporation, Armonk, NY, USA) and analyzed using a one-way analysis of variance (ANOVA) with Duncan’s multiple range test at a 5% significance level.

Author contributions

X-RY, MY, and LL designed the experiment. Y-JL and M-YB performed the experiments. S-SQ, G-SL, and QW analyzed the data. Y-JL wrote the manuscript. X-RY, MY, and LL revised the manuscript.

Acknowledgments

The authors would like to thank Professor Karl-Heinz-Kogel at Justus-Liebig-University, Giessen, Germany, for the gift of the Piriformospora indica. The authors gratefully acknowledge associate researcher Rui Chen at the Institute of Germplasm Resources and Biotechnology, Tianjin Academy of Agricultural Sciences, for technical assistance in metabolic analysis.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592324.2023.2213934.

Additional information

Funding

The author gratefully acknowledges support from Key Research and Development Projects of Hebei Province (Grant No. 19226505D) and National Natural Science Foundation of China (Grant No. 31801948) as well as Natural Science Foundation of Hebei Province (Grant No. C2021202005) for financial supports.