Introduction

Cremastra appendiculata (D. Don) Makino (C. appendiculata) is a valuable herbal with a medicinal application history of more than a thousand years (Liu et al. 2021). It has become an endangered species and a scarce traditional Chinese medicine in Orchidaceae in China and is listed as a national secondary protected plant. Many scholars have studied the chemical constituents and pharmacological effects of C. appendiculata and found that it has antitumor (Zhang et al. 2021), hypotension (Ikeda et al. 2005), immune regulation (Zhao et al. 2017) and anti-inflammatory (Hur et al. 2010) effects. In recent years, the price of C. appendiculata has skyrocketed due to its scare resource. The main reasons are the low seed germination rate, slow growth rate under natural conditions and easy invasion by pathogens during artificial cultivation, resulting as a high mortality. Therefore, we attempted the rapid tissue propagation of C. appendiculata tubers. However, it was supering that a large number of plant tissues were contaminated by microorganisms despite a strict aseptic operation throughout the whole process. Based on this, we found that a large number of endophytes colonized C. appendiculata tubers.

Plant endophytes are microorganisms living in healthy plant organs but do not cause disease symptoms, and endophytic lifestyles play crucial roles in plant development, growth, fitness and diversification (Hardoim et al. 2015). They have the same or similar anabolic pathway as the host plants, which can promote plant growth and reproduction to a certain extent (Wani et al. 2015). Beneficial microbes also exert pressure on the survival of plant pathogens, thereby reducing their pathogenic effects on plants (such as the production of antibiotics and extracellular hydrolases) (Afzal et al. 2019).

Currently, the control of plant diseases and insect pests and the promotion of crop growth mostly rely on pesticides, chemical fertilizers and other chemical agents, most of which are artificially synthesized and have a slow degradation rate (Wang et al. 2018). Long-term and large-scale use of these agents has led to pesticide residues and increased the number of resistant weeds. In the process of killing bacteria, these agents also threaten beneficial microorganisms, cause water and soil pollution and even affect the ecological balance (Wang et al. 2020a). On the other hand, these agents induce pathogens to become drug-resistant and aggravate plant diseases, which are difficult to cure. To solve this problem, biocontrol preparations have attracted attentions, and endophytes are of the greatest interest. Biological control refers to the means of controlling plant diseases by living organisms such as animals, plants and microorganisms or their secondary metabolites (Zhang et al. 2020). It has the characteristics of low toxicity, safety and high efficiency, which meet the needs of environmental protection and sustainable agricultural development, and is an important strategy as an alternative to chemical control. Thus, it has gradually become an ideal method of disease prevention and control (Tarek et al. 2020; Cook 1993).

At present, many biocontrol isolates, such as Bacillus, Pseudomonas, Streptomyces sp. and Trichoderma sp. have been studied, developed and successfully commercialized (Wang et al. 2020b; Maes et al. 2020; Vurukonda et al. 2018; Vos et al. 2015). Among the plant growth-promoting rhizobacterias (PGPRs), strains from Bacillus and Pseudomonas are the most studied and exploited. The 7 strains isolated from barley roots by Yang (2019) had antagonistic activity against Fusarium oxysporum and effectively inhibited the occurrence of watermelon Fusarium wilt. Wang et al. (2020a) found that Pseudomonas aeruginosa CQ-40 was able to control tomato grey mould and promote tomato seed germination, seedling growth and fruit preservation. P. fluorescens is an inhibitory agent in the soil, which stifles the viability of F. oxysporum (Jain et al. 2021). However, due to the impact of cost and the stability of antibacterial activity, whether biocontrol fungi can be isolated from C. appendiculata tubers remains to be further studied.

C. appendiculata is often vulnerable to pathogen in artificial cultivation stage and is easy to has leaf spot, anthracnose, grey mould, leaf blight and other diseases (Wu et al. 2020). Anthracnose is caused by Colletotrichum sp., which mainly damages the leaves of plants and even lead to the death of C. appendiculata (Zhu 2006). Cercospora sp., Phyllosticta citricarpa, Tolypocladium cylindrosporum and other phytopatogenic fungi can cause leaf spot, which frequently occurs in the middle and lower parts of the leaf, and cause the death of leaf segment above the disease spot (Qin et al. 2017). The occurrence of these diseases will directly affect the survival of C. appendiculata. In summary, it is important to evaluate the antagonistic effect of C. appendiculata endophytes on pathogens and their abilities to promote plant seed germination and plant growth. Hence, we focused on beneficial microbial strains from C. appendiculata tubers with plant growth-promoting activity and antagonistic activity. We isolated and screened endophytic fungi from C. appendiculata tubers that can promote plant growth and inhibit pathogens to provide and develop potential biocontrol strains to control plant fungal diseases and promote crop growth. At the same time, the plant utilization rate of rare orchids was improved, and a good theoretical guidance and technical basis for the development of a new biocontrol agent instead of chemical pesticides were provided.

Materials and methods

Separation, purification and screening of endophytic fungi

C. appendiculata were obtained from Mianyang City, Sichuan Province in China on October 21, 2020, which were identified by Prof. Zhishan Ding. A voucher sample (No. Ding 0005) was kept in the laboratory of School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University. Thirty-five healthy C. appendiculata tubers were selected and added to 2% sodium hypochlorite (v/v) for 15 min, 75% alcohol for 30 s and then sterile water for three washes, followed by cutting into pieces. The tubers were placed on potato dextrose agar (PDA) (Hangzhou Microbial Reagent Co., Ltd., Zhejiang, China) plates. After incubation at 28 °C (Ren et al. 2021) for 10 days, the mycelium at the tip of the fungal colonies was selected and transplanted to new PDA plates to separate the cultures (Fig. S1). This was repeated several times, after which purified colonies were obtained.

Antagonic effect of fungal endophytes on phytopatogenic fungi

Three pathogens that cause anthracnose and leaf spot in various plants including Colletotrichum fructicola, Alternaria alternata, and Alternaria longipes were provided by Dr. Fangmei Zhou (Zhejiang Chinese Medicine University, China). The endophytic fungi from C. appendiculata tubers and pathogens were incubated in 20 mL of solidified PDA plates at 28 °C. A 7-day-old fungal pathogen mycelial disc (5 mm) was placed in the centre of 9-cm Petri dish PDA plates. The endophytes from C. appendiculata tubers were inoculated 15 mm from the central pathogen block. The PDA plates inoculated with pathogens alone were served as the control. After 7 days for incubation at 28 °C, the colony diameters of fungal pathogens were measured and compared to the control plate.

The fungistatic rate was calculated using the equation:

$$\mathrm{the\ fungistatic\ rate}\ \left(\%\right)=\left[\left({D}_{C}-{D}_{E}\right)/{D}_{C} \right]\times 100$$

where DC represents the colony diameter of three pathogens on a control plate and DE indicates the colony diameter of three pathogens on a plate that co-culture with endophytic fungi from C. appendiculata tubers (Bilański and Kowalski 2022). This experiment was repeated three times.

Identification of the fungi

Morphological identification: Four strains with fungistatic activity were inoculated on PDA medium plates and placed at 28 °C for 5 days to allow the colony area to cover 2/3 of the plate. Colony characteristics, such as morphology, size, colour and shape, were observed and recorded.

Molecular identification: Three genes, ITS, 18S rRNA and 28S rRNA were amplified for identification to identify the endophytes (Liu et al. 2015). The PCR system was composed of template DNA (1.0 µL), 1 × TSE101 mix (45 µL), upstream primer (2.0 μL) and downstream primer (2.0 μL). The thermocycling conditions were 1 cycle of 2 min at 98 °C, 35 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 10 s, extension at 72 °C for 10 s and followed by 72 °C for 5 min. The PCR products were then recovered from the gel, and the recovered products were sent to Beijing Qingke Biotechnology Co., Ltd. (Beijing, China) for sequencing analysis. The sequencing results were compared by fungal sequences in NCBI GenBank using BLAST software (http://www.ncbi.nlm.nih.gov/). The phylogenetic tree was constructed with MEGA version 7.0. The nucleotide sequences generated in this study have been deposited in the GenBank database under the accession numbers shown in Table 2.

Determination of the antifungal activity of the culture filtrates of endophytic fungi

DJL-6, DJL-9, DJL-10 and DJL-15 were inoculated in 100 mL of potato dextrose broth (PDB) medium in 250-mL conical flasks in shake culture (160 rpm) at 28 °C for 5 days. The culture filtrates (CFs) were acquired by centrifuging the culture at 12000 g for 10 min and then filtering through 0.22 μm MS® PES syringe filters. Then, the CFs were added to PDA at a proportion of 1:4 (v/v) with a final CF concentration of 20%. Sterile water was added to PDA as control (Jin et al. 2021). Mycelial discs of C. fructicola, A. alternata and A. longipes were inoculated on the CF-amended and control PDA. The colony diameters of three pathogens were measured after culturing for 7 days at 28 °C.

The growth inhibition rate was calculated by the following equation:

$$\mathrm{the\ inhibition\ rate}\ \left(\%\right)=\left[\left({D}_{C}-{D}_{CF}\right)/{D}_{C} \right]\times 100$$

where DCF represents the colony diameter in the treatment amending with the CFs and DC represents the colony diameter in the control PDA.

Determination of the effects of endophytic fungi on plant growth and disease resistance

The ability of the endophytic fungi to secrete extracellular enzymes was determined. Four isolates were inoculated on carboxymethylcellulose sodium (CMC) medium, chitinase medium and skim milk agar medium (Brzezinska and Jankiewicz 2012; Lin et al. 2019; Muniroh et al. 2019), cultured at 28 °C for 5 days and then observed for a transparent zone. The secretion of cellulase, chitinase and protease was evaluated and measured by the presence of the transparent zone. The production of β-1,3-glucanase was determined by dinitrosalicylate (DNS) (Ueki et al. 2019). Glucose was used as a standard. Four isolated cultures were inserted into PDB and oscillated for 5 days at 160 rpm and 28 °C. Then, the cultures were centrifuged at 12,000 g for 10 min. The supernatant was used as the crude extract of the enzyme. Then, 1 mL dextran substrate (100 mg laminarin in 100 mL 0.2 mol/L pH 5 sodium acetate-acetic acid buffer) and 1 mL of crude enzyme were mixed, and the mixture was incubated at 40 °C for 30 min. DNS solution (2 mL) was added to stop the reaction at 100 °C, and the absorbance was measured at 540 nm (model Epoch2, BioTek, America). The production of β-1,3-glucanase was calculated according to the glucose standard curve.

For the determination of nitrogen fixation ability, four fungi were inoculated on Ashby nitrogen-free medium (Jing et al. 2020), cultured at 28 °C for 5 days and observed for whether the strains grew in the medium; the experiment was repeated three times.

For determination of the ability to dissolve phosphate and potassium, phosphate solubilization and potassium dissolution tests were conducted using Pikovskaya agar (PKO) (Castro et al. 2018) and potassium fungi screening agar plate (Kushwaha et al. 2020) assays. Endophytic fungi were cultured at 28 °C for 5 days. The PKO plate was observed to determine whether there was a clear circle and whether the colony on the potassium fungi screening agar plate grew towards potassium to determine whether the strain had the ability to dissolve phosphorus and potassium.

To test for the ability to produce siderophores, a chrome azurol S (CAS) assay was used to detect the production of siderophores (Jamali et al. 2020). The strains were inoculated on the medium and then placed at 28 °C for 5 days, and the presence of an orange halo around the colony was observed. The experiment was repeated three times.

Quantitative estimation of IAA production was carried out using the Salkowski reagent (1 mL 0.5 M FeCl3, 50 mL 35% HClO4) (Gang et al. 2019). To determine the amount of IAA produced by the four strains, a standard curve was prepared using a series of IAA dilutions at 0, 5, 10, 15, 20, and 25 mg/L. Then, 1 mL centrifuged fungal fluid was added to 1 mL Salkowski reagent and incubated for 30 min in darkness. The colour densities of the mixtures were read using a microplate reader at 530 nm absorbance. The experiment was repeated three times.

Effects of endophytic fungi on seeds germination and seedlings growth

Pot experiments were conducted to examine the germination promotion effect of endophytic fungi on C. appendiculata seeds. Endophytic fungi were cultured in 250-mL conical flasks containing 150 mL PDB on an orbital shaker at 160 rpm and 28 °C for 5 days. The mycelium was filtered and washed with sterile water three times, mixed with 500 g sterile soil and put into a 20 cm × 15 cm × 11 cm pot dish. The soil used for the pot experiment was collected from a greenhouse at Zhejiang Chinese Medical University, Hangzhou, China. The soil was steam-sterilized twice at 121 °C for 1.5 h continuously. C. appendiculata seeds were surface-disinfected and sown in a mixture of mycelium and sterile soil at the same density, after which a 5 cm × 4 cm area was selected from each group to determine the quantity sown. The treatments with only sterile soil (without endophytic fungi) were used as controls.

After various treatments were applied, pot experiments were carried out at 23–25 °C and 65–85% relative humidity with 13 pots (each strain was subjected to three replicates). The germination rate of C. appendiculata seeds in the potted soil of each group was calculated and analysed in the same 5 cm × 4 cm area at 20 days, 40 days, 60 days and 80 days after inoculation with the endophytic fungi. This experiment was replicated three times.

Same-size grain soybean seeds were selected and added to 2% sodium hypochlorite (v/v) for 15 min and then 75% alcohol for 30 s, followed by thorough washing three times in sterile water. Endophytic fungi were cultured in 250-mL conical flasks containing 150 mL PDB on an orbital shaker at 160 rpm and 28 °C for 7 days. The cultures were centrifuged at 8000 r/mim for 20 min, washed and resuspended in sterile water. The number of spores in suspension of endophytic fungi was counted by hemocytometer (Yang 2019). Then the spore suspension of four isolates was diluted in the order of 0 times, 10 times, 102 times, 103 times and 104 times. Eighteen soybean seeds were soaked in each dilution of the gradient for 3 h, while sterile water was used as a negative control. The seeds were placed in sterile petri dishes containing moist filter gauze at 25 °C in the dark. The soybean seedlings were harvested after 6 days of the endophytic fungi inoculation treatment, the germination rate of soybean seeds in each group was calculated, and the radicle length, fresh weight and dry weight were determined. The optimum concentrations of the spore suspension of the 4 strains were selected. The average values of these parameters were calculated and replicated three times.

Statistical analysis

All the above screening tests adopted a completely random design (CRD) with 3 replicates per experiment to maintain the consistency of the experiment. The results were subjected to analysis of variance (ANOVA), while means were assessed using least significant difference (LSD) at P < 0.05. Statistical analyses were performed with R software (GraphPad Prism 8.2).

Results

Screening of endophytic fungi with fungistatic activity

A total of twenty-one fungi were isolated and purified from C. appendiculata tubers. Dual culture tests were used to evaluate the inhibition of plant pathogens of the isolates. Table 1 shows that only 4 endophytes of C. appendiculata had inhibitory effects on three kinds of pathogens. DJL-9 showed the strongest inhibition activity, and the fungistatic effects on C. fructicola, A. alternata and A. longipes were 72.15 ± 1.07%, 63.42 ± 2.38% and 68.21 ± 1.27%, respectively. DJL-6, DJL-10 and DJL-15 could also inhibit the growth of the three pathogens to a certain extent, but the effect was not as obvious as that of DJL-9. The most frequent type of interaction between pathogens vs. endophyte was from physical contact of mycelia (Fig. 1a2-a4, b2, b4, c1, c2, c4), while a1, b1, b3 and c3 showed an inhibition zone between pathogens and endophyte with the width of the inhibition zone less than 2 mm (Fig. 1a1, b1, b3, c3).

Table 1 Antagonistic activity of our collected fungal isolates from C. appendiculata tubers on 3 pathogenic fungal isolates
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Fig. 1
figure 1

Inhibition of growth of pathogens by selected endophytic fungi strains. a CK C. fructicola. b CK A. alternata. c CK A. longipes. a1, b1, c1 Inhibition of three pathogens growth with DJL-6. a2, b2, c2 Inhibition of three pathogens growth with DJL-9. a3, b3, c3 Inhibition of three pathogens growth with DJL-10. a4, b4, c4 Inhibition of three pathogens growth with DJL-15. Notes: “CK” means without any endophytic fungi isolated from C. appendiculata tubers. In the picture, endophytes are on the left side of the medium and pathogens are on the right

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Identification of endophytic fungi

Morphology assessments were conducted. DJL-6 colonies were yellow, flat and opaque, with rough edges and irregular shapes. The DJL-9 colonies were white and transparent, and the hyphae radiated outwards with irregular shapes on PDA medium plates. The DJL-10 colonies were round, dry and convex, with a black centre and a white edge that was opaque. DJL-15 colonies were brown, dry and convex, and the hyphae are flocculent, opaque and irregular in shape on PDA medium (Fig. 2).

Fig. 2
figure 2

Morphological characteristics of endophytic fungi on PDA medium culture. a Colony morphology of DJL-6. b Colony morphology of DJL-9. c Colony morphology of DJL-10. d Colony morphology of DJL-15

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For molecular identification, the sequences of four endophytic fungi were obtained by sequencing (showed in supplementary information 2) and compared with the GenBank database and reference strains. Here, the use of 18S rRNA and 28S rRNA gene sequence analysis allowed a phylogenetic differentiation down to the levels of families or genera, and ITS sequence further identified 4 endophytes to species. The endophytes were assigned as particular species only if minimum threshold similarity was ≥ 99% compared to the most closely related strain (Yuan et al. 2010). The phylogenetic tree generated using the phylogenetic analysis of sequences in the GenBank database and reference strains is presented in Fig. 3. DJL-6 was identified as Colletotrichum gloeosporioides, DJL-9 as Trichoderma tomentosum, DJL-10 as Colletotrichum godetiae, and DJL-15 as Talaromyces amestolkiae. The maximum identities for each isolate exceeded 99% with an E-value of 0 and the gene sequences of 4 strains were submitted to the GenBank database, and the accession numbers were obtained (Table 2).

Fig. 3
figure 3

rDNA ITS, 18S and 28S rRNA sequence phylogenetic tree of four endophytic fungi isolated from C. appendiculata tubers. The numbers at branch points were the significant bootstrap values (expressed as percentages based on 1000 replicates). The scale bar represents 0.05 substitutions per nucleotide position

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Table 2 Identification of fungus isolates obtained from C. appendiculata tubers by rDNA ITS, 18S rRNA and 28S rRNA gene sequence analysis
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Inhibition of pathogens by the CFs of endophytic fungi

The 4 endophytic fungi could produce antifungal substances inhibitory to C. fructicola, A. alternata and A. longipes. Figure 4 shows that the diameter of pathogens in the control group was significantly larger than that in the culture medium with CFs. Among them, the CFs of DJL-15 showed the strongest inhibition activity, and the inhibition rate against C. fructicola, A. alternata and A. longipes were 38.89 ± 1.81%, 52.73 ± 3.26% and 62.07 ± 2.45%, respectively. The CFs of DJL-6, DJL-9 and DJL-10 can also inhibit the growth of pathogens.

Fig. 4
figure 4

Inhibition of growth of pathogens by the culture filtrates of the endophytic fungi. a CK C. fructicola. b CK A. alternata. c CK A. longipes. a1, b1, c1 Inhibition of three pathogens growth with the culture filtrates of DJL-6. a2, b2, c2 Inhibition of three pathogens growth with the culture filtrates of DJL-9. a3, b3, c3 Inhibition of three pathogens growth with the culture filtrates of DJL-10. a4, b4, c4 Inhibition of three pathogens growth with the culture filtrates of DJL-15. Notes: “CK” means the pathogens grow without any culture filtrates of endophytic fungi

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Characteristics of endophytic fungi related to growth promotion and disease resistance

All strains tested were able to produce IAA in PDB medium with and without L-tryptophan, and the production of IAA was significantly increased in the medium with L-tryptophan, as shown in Table 3. In PDB medium supplemented with L-tryptophan, the highest amount of IAA (7.60 ± 0.32 mg/L) was produced by DJL-15, followed by DJL-6 and DJL-10, while DJL-9 produced the lowest amount of IAA (3.17 ± 0.16 mg/L). In the PDB medium with no L-tryptophan supplement, DJL-15 produced the highest amount of IAA (2.85 ± 0.14 mg/L), while DJL-9 produced the lowest amount of IAA (1.58 ± 0.11 mg/L).

Table 3 Characteristics of endophytic fungi related to growth promotion and disease resistance
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The results showed that all the four endophytic fungi could produce β-1,3-glucanase, and DJL-10 had the highest value (12.76 ± 0.18 mg/L). DJL-6 and DJL-10 could produce chitinase, and DJL-10 also showed cellulase activity.

Four isolates were tested for phosphate solubilizing activity in PKO media plates, and a clear transparent circle appeared around the colony, showing that the ability to solubilize phosphate was positively exhibited by the four endophytic fungi, except for DJL-9. All the strains showed proteolytic activity, as tested by the ability to produce a transparent zone in a skim milk medium plate assay. All the strains were able to fix nitrogen and resolve potassium, as indicated by the strains growing in Ashby nitrogen-free medium and on potassium fungi screening agar plates. However, none of the fungal strains were positive for iron siderophore complexes.

Endophytic fungi promoted seed germination and seedling growth

The endophytic fungi were assessed for their ability to promote germination of C. appendiculata seeds. In the pot experiment under greenhouse conditions, the growth of C. appendiculata seeds was slow without inoculation of endophytic fungi, and less than 5% of the seeds showed germination trend after 80 days. After inoculation with the endophytic fungi, all the isolates were able to significantly increase the germination rate of C. appendiculata seeds, and the seeds of all experimental groups showed a favourable growth trend. Among them, the mycelia of DJL-9 promoted seed germination of C. appendiculata most obviously. In the sterile soil mixed with DJL-9 mycelia, the germination rate was 15.06 ± 0.28% (Fig. 5).

Fig. 5
figure 5

Effects of endophytic fungi from C. appendiculata tubers on germination of C. appendiculata seeds. Results are means ± SE, statistically significant differences between treatments, p < 0.05

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The endophytic fungi were also assessed for their ability to promote the germination and growth of soybean. The optimal spore suspension concentrations of DJL-6, DJL-9, DJL-10 and DJL-15 for promoting soybean seed germination and growth were, respectively, 1.0 × 107, 1.0 × 108, 3.5 × 108 and 2.0 × 109 cfu/mL (Fig. 6). Compared to noninoculated soybean seeds, those inoculated with endophytic fungi showed a significantly enhanced germination rate (Table 4). These four endophytic fungi significantly increased the length of the soybean radicle. All the isolates, except for DJL-9, significantly increased dry weight compared to uninoculated soybean, and only DJL-6 and DJL-15 significantly increased fresh weight of soybeans. DJL-6 had the most obvious growth-promoting effect on soybean seeds; the radicle length of soybean reached 18.83 ± 1.18 cm, and the fresh weight and dry weight increased most significantly, at 0.956 ± 0.11 g and 0.168 ± 0.02 g, respectively.

Fig. 6
figure 6

Effects of endophytic fungi spore suspension with different dilution ratios on soybean seedling morphology. a DJL-6. b DJL-9. c DJL-10. d DJL-15. Sterile water control (1), spore suspension (2), tenfold diluted spore suspension (3), 102-fold diluted spore suspension (4), 103-fold diluted spore suspension (5), 104-fold diluted spore suspension (6)

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Table 4 Effect of spore suspension of endophytic fungi on morphological indexes of soybean seeds
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Discussion

In the present study, four strains with antagonistic activity against a variety of plant pathogens were isolated from C. appendiculata tubers. In vitro and in vivo experiments showed that the strain was beneficial to plants in a variety of ways, including inhibiting pathogens and promoting seed germination and plant growth.

Results have shown that biocontrol strains have disease resistance and growth-promoting effects on several vegetables, fruits and Chinese herbal medicines (Olanrewaju et al. 2017). C. fructicola, A. alternata and A. longipes are common plant pathogens in agricultural production, and they cause anthracnose, leaf spot and leaf blight diseases on various plants, such as carrots (Vintal et al. 2002), potatoes (Shoaib et al. 2014), apples (Li et al. 2019) and peaches (Usman et al. 2021). C. appendiculata grows in humid conditions and is susceptible to fungal infection. Infection of these three pathogens causes anthracnose, leaf spot disease and even the death of the plant, which poses a great threat to the survival of C. appendiculata (Jain et al. 2021). If endophytic fungi of C. appendiculata tubers can inhibit the growth of these three pathogens, it can be potentially developed into biological agents and applied in agricultural production.

Endophytes can act as biocontrol agents to directly or indirectly antagonize pathogens through various ways, including antibiosis, mycoparasitism, growth enhancement (endophytes promote plant growth by microbial hormones and reducing the effects of the disease) and competition (Thambugala et al. 2020). In this experiment, 4 strains isolated from C. appendiculata tubers differently demonstrated the effects of promoting plant growth and resisting pathogens. In the dual culture test, they mainly showed in vitro antagonistic activity against plant pathogens through physical contact of mycelia, and they can inhibit the growth of pathogens by competing for living space and nutrients in the medium. Meanwhile, their CFs also inhibited the growth of three plant pathogens, which shows the four endophytic fungi can produce fungistatic metabolites. They all promoted the germination of C. appendiculata seeds and the germination and growth of soybean seeds. These indicate the biocontrol potential of the four endophytic fungi.

In our experiment, we used three genes to identify four endophytes, which were 18S rRNA, 28S rRNA and ITS. Both the ITS region and 18S rRNA genes are broadly applied in molecular studies of fungi (Liu et al. 2015). Research showed that the ITS region could reveal higher richness, diversity and more dynamic than 18S rRNA. It could be concluded that ITS region is more rapid and precise for fungal community analysis than 18S rRNA (Weber et al. 2009). Also, the 28S rRNA gene beyond the D1-D2 region is useful for designing broad-range fungal PCR assays with a good ability to distinguish between species (Khot et al. 2009). Trichoderma plays an effective role in inhibiting pathogens and promoting plant growth. Around 90% of fungal biocontrol agents against pathogenic microorganisms belong to different strains of Trichoderma (Sood et al. 2020). Khruengsai et al. (2021) found that the volatile compounds produced by two T. afroharzianum strains inhibited the growth of F. oxysporum and F. proliferatum and significantly reduced the post-harvest decay of chili fruits. Appropriate doses of T. asperellum spore can increase the germination and radical length of vegetable crops, including tomato, brinjal, chilli, okra, ridge gourd and guar (Singh et al. 2016). In this study, DJL-9 was identified as T. tomentosum, which has a strong inhibitory effect on pathogens and promotion effects on plant growth. However, at present, there are relatively few studies on T. tomentosum and few reports on its biocontrol effect. Further studies on T. tomentosum will be carried out in the future.

In a study by Prieto et al. (2021), T. amestolkiae showed strong hydrolytic activity. Some β-glycosidases produced by T. amestolkiae catalyse the transfer of sugar molecules to specific glycosides through glycosylation and degrade plant biomass by secreting complex extracellular enzyme mixtures, which play an important role in destroying the cell structure of plant pathogens. In line with this result, in this study, strain DJL-15 produced a variety of hydrolytic enzymes and showed different antifungal activities against three plant pathogens in vitro.

DJL-6 and DJL-10 were identified as C. gloeosporioides and C. godetiae. In the existing literature reports, Colletotrichum is considered as the pathogenic fungus, which has a wide host range, often damaging trees, vegetables, medicinal plants and the roots, stems, leaves and flowers of field crops, fruits and their seedlings. It causes plant withering, fruit rot and seedling death without overt symptoms, and even results in severe economic losses (Cannon et al. 2012; Boufleur et al. 2021). However, in this study, we found that Colletotrichum (DJL-6 and DJL-10) can play a biocontrol role. Jin et al. (2017) also found that the Fusarium strain YCEF005 could promote the growth of tobacco seedlings, which significantly increased the number of leaves, fresh weight and dry weight of roots and shoots. A strain of C. gloeosporioides isolated from Justicia gendarussa is a novel endogenetic Taxol-producing fungus (Chen et al. 2020). These results indicate that some pathogenic fungi can appear as endophytes under specific conditions and produce compounds similar to the host. This conclusion needs further study and discussion.

The fungal cell walls are mainly composed of chitin and β-1,3-glucan (Fesel and Zuccaro 2016). Studies have found that some PGPR secrete chitinase, cellulase, protease and other hydrolases to decompose peptidoglycan from the pathogenic bacterial cell wall and extracellular virulence factors to achieve disease resistance (Abdelshafy et al. 2020). In our experiment, all four isolates had proteolytic activity and β-1,3-glucanase production capacity, and DJL-10 could also produce chitinase and cellulase. These isolated strains exhibited different antifungal activities.

In addition, endophytes can directly promote plant growth by improving the utilization rate of phosphorus and potassium, nitrogen fixation and production of IAA and other plant growth hormones (Lata et al. 2018). The capacity of IAA production, phosphate solubilization capacity, potassium solubilization capacity, nitrogen fixation capacity and production capacity of siderophores have been intensively studied. Most microorganisms isolated from plant roots can secrete IAA (Patten and Glick 1996). IAA is a plant growth regulator that promotes root growth and the absorption of certain nutrients. In the present study, the 4 endophytic fungi isolated from C. appendiculata tubers all had the ability to produce IAA, and the IAA yields of the 4 isolates were significantly increased after the addition of L-tryptophan. Regarding phosphate solubilization, phosphorus-containing inorganic salts were able to promote root formation, growth, seedling development and flower bloom, enabling an earlier mature of fruits and seeds and improving the quality and yield of the crops (Matos et al. 2017). Strain DJL-9 in the present study lacked the ability to solubilize inorganic phosphate, while the other three strains had strong phosphate solubilizing ability. Potassium ions can improve the activity of many enzymes involved in photosynthesis, thus enhancing plant photosynthesis and increasing plant resistance to stress (Liu et al. 2019). All the strains selected in our study showed potassium solubilization activity. Nitrogen fixation is the process by which organisms reduce nitrogen in the atmosphere to ammonia. Nitrogen is a synthetic element of plant proteins, nucleic acids, chlorophyll and many enzymes that can promote photosynthesis and other biological activities, thus promoting plant growth and development (Lindstrom and Mousavi 2020). All the strains in the present study could play a great role in nitrogen fixation. Therefore, a variety of mechanisms suggest that the strains in this study can promote plant growth and control diseases. Many biocontrol fungi, such as T. asperellum and Penicillium chrysogenum, can produce iron siderophore complexes (Qi and Zhao 2013; Chowdappa et al. 2020). However, none of the strains in this study exhibited iron siderophore complex production capacity.

Orchids have a low seed germination rate and slow plant growth in the natural environment and are susceptible to fungal infection, which leads to their scarce quantity. In current studies, natural control is a method for disease management. Tissue culture and the rapid propagation of C. appendiculata have been successfully studied (Mao et al. 2007), but there are few reports on improving the seed germination rate. In this study, by mixing soil with the mycelium, we found that compared with the blank control group, the mycelium of endophytic fungi could significantly improve the germination rate and growth rate of C. appendiculata seeds and soybean seeds. These results indicated that four endophytic strains of C. appendiculata tubers had growth-promoting effect on plants and are expected to be used in the cultivation of C. appendiculata, alleviating its resources shortage and generating social and economic benefits. In addition, the fungistatic activity of the four endophytic strains was significant, which lays a practical and theoretical foundation for the development of new biological pesticides, reduces the use of chemical fertilizers, pesticides and the possibility of environmental pollution and contributes to the cause of environmental protection.