Symbiotic seed germination and seedling growth of mycorrhizal fungi in Paphiopedilum hirsutissimun (Lindl.Ex Hook.) Stein from China

ABSTRACT

Similar to other orchid species, Paphiopedilum hirsutissimum (Lindl.ex Hook.) Stein, relies on nutrients provided by mycorrhizal fungus for seed germination and seedling development in the wild owing to a lack of endosperm in its seeds. Therefore, obtaining suitable and specialized fungi to enhance seed germination, seedling formation, and further development is considered a powerful tool for orchid seedling propagation, reintroduction, and species conservation. In this study, we investigated the diversity, abundance, and frequency of endophytic fungal strains in the root organs of P. hirsutissimum. One family and five genera of the fungi were isolated and identified through rDNA-ITS sequencing. The ability of isolated fungi to germinate in vitro from the seeds of this species was evaluated, and the development of P. hirsutissimum protocorm has been described. The findings showed that the treatments inoculated with endophytic fungal DYXY033 may successfully support the advanced developmental stage of seedlings up to stage 5. In addition, scanning electron microscopy (SEM) revealed that the mycelium of this strain began to invade from either end of the seeds up to the embryo, extending rapidly from the inside to the outside. Its lengthening resulted in the bursting of the seed coat to form protocorms, which developed into seedlings. The results showed that DYXY033 has a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions and is a representative mycorrhizal fungus with ecological value for the species. In summary, this strain may particularly be significant for the protection of P. hirsutissimum species that are endangered in China. In the long run, it may also contribute to global efforts in reintroducing orchid species and in realizing in situ restorations of threatened orchid populations.

KEYWORDS:

• Paphiopedilum hirsutissimum
• mycorrhizal fungi
• symbiotic germination
• seedling development
• reintroduction

1. Introduction

Under natural conditions, orchid seeds are difficult to germinate because of their extremely small, immature embryos that contain virtually no food reserves, the tough and impermeable layers of the seed coat, and the accumulation of inhibitory substances, among others^1–3. Therefore, nearly all orchid species share a symbiotic relationship with mycorrhizal fungi that allows seed germination, protocorm formation, and seedling development⁴. Owing to the lack of developed endosperm, orchids rely on compatible mycorrhizal fungi for supplying carbon and energy sources, and other nutrients until they reach autotrophy levels^5,6. A majority of fungi that encourage seed germination are orchid mycorrhizal fungus (OMF), which form pelotons inside the cells of adult roots or protocorms and seedlings⁷. Some orchid species that are mycoheterotrophic during seed germination, and seedling development maintain distinct association(s) with members of a restricted group of fungal partners throughout their lifetime, whereas others switch symbionts while transitioning from the protocorm to the seedling adult stage or in plants response to various environmental stresses^8–13. Different mycorrhizal fungi play various roles in seed germination, protocorm formation, and the subsequent seedling development stages of orchids¹⁴. Although some endophytic fungi promote seed germination, they become incompatible as symbionts during the growth of seedlings^15–17. Some studies have reported that orchids form mycorrhizal symbionts with Rhizoctonia-like fungi, including mycorrhizal fungi belonging to the families Ceratobasidiaceae, Sebacinaceae, and Tulasnellaceae^18,19. However, many orchid seeds do not germinate or develop without their compatible fungus symbiont because the mycorrhizal specificity associated with an orchid in situ seed symbiotic germination is often limiting²⁰. According to reports, some fungal isolates can promote orchid seed germination and seedling growth under controlled laboratory conditions (i.e., in vitro), and the consequently exhibit potential specificity^20–22. Therefore, it is essential to isolate, identify and screen beneficial specificity mycorrhizal fungi in orchids when seed germination, reintroduction and in situ restoration is used for species conservation⁴.

Paphiopedilum hirsutissimum (Lindl.ex Hook.) Stein is a rare epiphytic or terrestrial orchid restricted to Southwest and Southern China, Northeast India, Laos, Myanmar, Northern Thailand, and Northern Vietnam²³. Owing to its unique flower shapes, gorgeous colors, and long flowering cycle. This species flowers from April to May and has high ornamental and cultivation value. However, wild populations and individuals of P. hirsutissimum have considerably reduced in number owing to illegal harvesting, habitat destruction, anthropogenic effects. Therefore, P. hirsutissimum was classified as a second-class protected plant in China and was included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora²⁴. In recent years, the distribution fragmentation of wild P. hirsutissimum in Guizhou, China has intensified. This species has become prone to extinction, which calls for immediate attention and focused conservation measures^16,20,25. Symbiotic is considered a successful approach for in vitro seed germination and development of the genus Paphiopedilum , which will ultimately help in the propagation and conservation of endangered orchid species^14;26;27.Therefore, studies to comprehend its biology of seed germination and development as well as strategies to enable its establishment in vitro would be of great significance toward the conservation of populations of this species.

Most studies have reported that symbiotic orchid seed germination has become an efficient orchid propagation method²⁸. Therefore, mycorrhizal symbiotic seed germination is considered to be a key process in orchid seedling reproduction, reintroduction, and species conservation. Obtaining compatible and specific OMF for enhancing seed germination, protocorm development, and seedling growth is critical. This work was generally aimed at (a) isolating, identifying, and screening the mycorrhizal fungi exhibiting compatibility and specificity of symbiotic seed germination and seedling development of P. hirsutissimum; (b) evaluating the effects of fungal isolates on seed germination and seedling development of P. hirsutissimum, and (c) observe the different developmental growth stages of symbiotic seed germination. The specific mycorrhizal fungi obtained from this study will be used ex situ and in situ for the conservation of P. hirsutissimum. They could also be used for artificial propagation and conservation of orchids and provide fundamental guidance for the symbiotic techniques for P. hirsutissimum.

2. Materials and methods

2.1 Plant material

All root samples were collected from the native populations of Wangmo County and cultivated at the Orchid Conservation Center of the Guizhou Academy of Forestry (Guizhou Province of South Western, China). The mature capsules of P. hirsutissimum that had not undergone dehiscence were collected from the Orchid Conservation Center after 200 days following cross-pollination by hand (Cultivated populations)(Figures. 1a,b).

Figure 1. a: Flowering of P. hirsutissimum (Lindl.), b: Capsules; Longitudinal and transverse profiles of the roots of P. hirsutissimum. Arrows represent pelotons (c, d).

2.2 Isolation, purification, and conservation of endophytic fungi from P. hirsutissimum

Mycorrhizal fungi were isolated using an improved technique as used by Zhu et al.²⁹. First, fresh 6 P. hirsutissimum root samples were placed in a petri dish filled with sterile distilled water, and root hairs and velamen were gently scraped off using a scalpel on a super-clean worktable. Subsequently, after microscopic examination, brownish zones containing pelotons were chosen, cut off the root segments with pelotons, and rinsed 3 times with sterile distilled water. Then, the root segments were disinfected with 70% ethanol for 30 s, 0.1% mercuric chloride for 5 min, and finally washed with sterile distilled water 5 times. The root segments are cut into 2 cm long segments and then gently scraped segments with a scalpel and tweezers to release individual pelotons from the exodermis to the endodermis, which were then placed in a 60 mm diameter petri dish containing sterile water, and incubated in the dark at 24°C. Second, microscopically examined after 24 h of incubation, the pelotons in sterile distilled water were observed under an optical microscope. The germinated pelotons were found in the dark field of the optical microscope and transferred to 1 cm² potato dextrose agar medium (PDA: composition—200 mL of extract from 200 g boiled potatoes, 18 g dextrose, 12 g agar, and 800 mL deionized water) using a 1 mL Eppendorf micropipette, and incubated at 24°C. The pelotons emerging hyphae are observed under an optical microscope, cut off from 1 cm² PDA medium, and then transferred to fresh PDA plates for purification, and incubated at 24°C. After repeating this purification step 4–5 times, pure cultures were obtained and stored at 4°C.

2.3 Morphological identification of endophytic fungi of P. hirsutissimum

2.3.1 Morphological characteristics of colonies and mycelia

On PDA plates, the purified fungal strains were plated and incubated at 24°C. The colony morphological characteristics of each strain were observed and recorded on PDA medium daily, including colony morphology and mycelium cell structure.

All the purified fungal strains were inoculated in the middle of the PDA plates, then a sterile coverslip was placed into the medium at a 45-degree angle using forceps. When the hyphae grew to about 2/5 of the cover glass, the cover glass was taken out, and the structural characteristics of the hyphae are observed under an optical microscope³⁰.

2.3.2 Ultrastructure of dolipore septum

1. Sampling and fixing: discarded the culture medium and added it to the electron microscope fixative to fix for 2-4 h at 4°C. The cells were centrifuged at low speed to the bottom of the tube, showing cell masses the size of mung bean, wrapped in 1% agarose, and rinsed three times for 15 min each with 0.1 M phosphate buffer (pH 7.4).

2. Postfixing: 1% osmic acid, 0.1 M phosphate buffer (pH 7.4) was fixed at 20°C for 2 h. Rinsed three times for 15 min each with 0.1 M phosphoric acid buffer (pH 7.4).

3. Dehydration: the concentration gradient was followed by 50%–70%–80%–90%–95%–100%–100% alcohol–100%, acetone–100% acetone dehydration for 1 time, each time 15 min.

4. Infiltration: place tissue in an infiltration solution containing a 1:1 acetone: Epon812 ratio and let it soak for 2-4 h or overnight. The tissue was then embedded under pure Epon812 and allowed to soak for 5-8 h.

5. Embedding: embedded tissue with pure embedding agent Epon812 and polymerized for 48 h in the drying oven at 60°C.

6. Slicing: sliced 60–80 nm ultra-thin slices with an ultra-thin slicer.

7. Staining: fixed the slices on the copper screen, stained with uranyl acetate and lead citrate, each stained for 15 min, and the slices were dried at room temperature overnight.

8. Observation: the ultrastructure was observed with a transmission electron microscope.

2.4 Molecular identification of endophytic fungi of P. hirsutissimum

Three hundred microliters of fungus liquid were transferred into a 1.5 mL centrifuge tube and mixed with 8 µL of lysozyme before being enzymolysed at room temperature for 30 min. A total of 300 µL of digestion solution was added, homogenized, 4 µL RNase A was added, homogenized, and incubated at 55°C for 10 min. After that, 4 µL Pro-teinase K was added and incubated at 55°C for 30 min. The supernatant was transferred into a GenClean column sleeved in a 2 mL collection tube after being centrifuged at 12,000 rpm for 5 min. The GenClean column was removed after 1 min of centrifugation at 8,000 rpm, and the waste liquid in the collection tube was discarded. The GenClean column was placed back into the collection tube, 500 µL of wash solution was added, and the tube was centrifuged at 8,000 rpm for 1 min. The GenClean column was removed, and the waste in the collection tube was discarded once more. The GenClean column was removed, and the waste in the collection tube was discarded once more. The GenClean column was placed back into the collection tube and centrifuged at 12,000 rpm for 1 min to remove the residual wash solution. Put the GenClean column into a new clean 1.5 mL centrifuge tube, add 50 µL elution buffer to the center of the GenClean column, and bake at 37°C for 2 min. Centrifuged at 12,000 rpm for 1 min.

The primers for internal transcribed spacer (ITS) sequencing of the fungi were ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The polymerase chain reaction was performed in a 30-μL reaction mixture solution containing 1 μL of dNTP, 1 μL of the enzyme,1 μL each of the downstream and upstream primers, 21 μL of double-distilled water, and 3 μL of 10×Taq Buffer (Shanghai Sangon Biotech Co., Ltd). The amplification reaction comprised were as follows: initial denaturation at 95°C for 10 min, followed by 35 cycles of 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min by 35 cycles, and a final extension for at 72°C 5 min. The fragments were then purified and sequenced by an external service provider (Shanghai Sangon Biotech Co., Ltd, China). The sequences were used to perform a BLAST (Basic Local Alignment Search Tool) search against the NCBI (National Center for Biotechnology Information) GenBank nucleotide database to compare the sequences with the most similarity to identify each fungal isolates^27,30.

2.5 Symbiotic germination

The mycelium of each fungal isolates was inoculated onto 30 mL of PDA medium (PDA: 200 mL of extract from 200 g boiled potatoes, 18 g dextrose, 10 g agar, and 800 mL deionized water) into a 200 mL volume of tissue-culture bottles. This was repeated six times per treatment and the treatment without fungal inoculum served as controls (six replicate bottles). All treatments were placed in biochemical incubator in the dark at 24°C. After 2 weeks of incubation, it will be used for symbiotic seed germination tests.

Symbiotic seed germination trials were performed using mature P. hirsutissimum fruit capsules to assess the effect of the fungal isolates on embryo growth and differentiation. First, 1 mature and grow well fruit capsule was surface sterilized using 70% ethanol for 1 min, followed by 0.1% mercuric chloride for 12 min, and rinsed five times with sterile distilled water. Then, the fruit capsules were cut with a sterile scalpel and forceps, gently shaken, and the seeds were evenly sown on fungus-PDA medium. Approximately 200 seeds were sown in each tissue culture bottle¹⁴.

The incubation temperature was 22–24°C, light intensity was 1200 lux, and the light-dark ratio was 18 h:6 h. After 2 weeks of inoculation and culture, bottles were examined and recorded for germination and development regularly (e.g., weekly and monthly) during dark incubation. As reported in Stewart et al.³¹ seed germination and seedling developmental growth stages of P. hirsutissimum are as follows: stage 0, no germination; stage 1, rupture of the testa by enlarging embryo; stage 2, formation protocorm by expanding embryo to spherical; stage 3, the appearance of the protomeristem; stage 4, the emergence of the leaf; and stage 5, elongation of leaf to form a seedling.

In subsequent analyses, the average of the six replicates of tissue-culture bottle cultures was calculated. The germination rate: number of germinating seeds/total number of inoculated seeds × 100%.

2.6 Morphological observations of the seed symbiotic germination stages

For the study of the morphological features of seeds, protocorms, and seedlings at different developmental stages, the samples were examined after co-culture². The specimens were photographed under an inverted microscope (Olympus CKX53) equipped with a digital camera (Nikon D610). Furthermore, the samples were examined via scanning electron microscopy (SEM; Hitachi SU8100). The samples were washed thrice with 0.1 M PB (pH 7.4) and then transferred into 1% OsO₄ in 0.1 M PB (pH 7.4) for 1–2 h at room temperature. Next, the tissue blocks were washed thrice in 0.1 M PB (pH 7.4), for 15 min each time. Then, the blocks were dehydrated in a gradient series of ethanol (30%, 50%, 70%, 80%, 90% and 95%) and two changes of 100% ethanol for 15 min each and then in isoamyl acetate for 15 min. After drying the samples with Critical Point Dryer, they were attached to metallic stubs using carbon stickers and sputter-coated with gold for 30 s. Finally, the samples were observed and imaged by SEM.

2.7 Effects of mycorrhizal fungi on the growth of P. hirsutissimum seedlings

The test seedlings were obtained from the asymbiotic seeds germination of P. hirsutissimum. The tested strains and seedlings were symbiotically co-cultured on 6 replicate plates of the PDA medium¹⁴. This step was repeated 10 times for each treatment, and the treatment without fungal inoculum served as control (10 replicate bottles). First, the PDA media were inoculated with a 1 × 1 cm² block of strains into each tissue culture bottle and then cultured in the dark at 28°C for 7 days, after which the test seedlings were inoculated into tissue culture bottles. Finally, all cultures were sealed and incubated at 24°C–26°C/1200 lux under LED tissue culture lamps for a 12-h photoperiod. After 12 weeks of inoculation, the fresh weight, leaf length, and root length were measured for each seedling.

2.8 Phylogenetic analyses

All sequences of the fungal strains were subjected to BLAST in the NCBI BLAST similarity search tool to determine their closest match from the GenBank database. The phylogenetic trees were constructed by the neighbor – joining (NJ) method with the MEGA version 6.0 software with reference to the available DNA data obtained during this study.

2.9 Statistical analysis

The experimental data were analyzed by statistical analysis software SPSS (Statistical Package for the Social Sciences v.21.0). The Student-Newman-Keuls multiple (S-N-K) comparison in the single-factor analysis of variance method was used to determine whether there were significant differences between the different experimental treatments at the p ≤ 0.05.

3. Results

3.1 Morphological and molecular identification of endophytic fungi

Fungal pelotons could be observed in nearly all root sections of P. hirsutissimum used in this study (Figures. 1c,d). Observation under an optical microscope showed that the pelotons were unevenly distributed in the root cortex cells of orchids. This finding is consistent with the findings of previous studies^14,32. In total, 70 endophytic fungal strains were obtained from the mycorrhizal tissues of P. hirsutissimum roots with a variable diversity. All the obtained strains belonged to Ascomycota and Basidiomycota. Among them, the greatest diversity of endophytic fungi was found in Tulasnellaceae sp. Xylaria sp., and Fusarium sp., with a frequency of isolation of 57.58%, 27.14%, and 7.15%, respectively. This was followed by Fusarium sp., Nemania abortive, and Acremonium alternatum, which exhibited a frequency of isolation of 5, 4, and 1 colony, respectively (Tables 1,2).

Table 1. Occurrence and frequency of the fungal isolates.

Fungal Isolate	Number of Strains	Isolation Frequency(%)
DYXY023	3	4.29
DYXY0003	1	1.43
DYXY004	7	10.00
DYXY001	5	7.14
DYXYY2	3	4.29
DYXYXY1	4	5.71
DAXY0016C	8	11.43
DYXY013C	11	15.71
DYXY002	5	7.14
DYSH004	3	4.29
DYXY111	2	2.86
DYXY112	2	2.86
DYXY033	16	21.86

Table 2. Identification of ITS sequences of fungal isolates from P. hirsutissimum roots, based on the closest match in the GenBank database.

Fungal Isolate	Assigned identity based on most similar GenBank accession	Identity (%)	Query Coverage (%)	Total Score	GenBank Accession Number
DYXY023	Tulasnella sp.	99.65	92	1033	MH348614.1
DYXY0003	Acremonium alternatum	98.96	99	1031	KT192193.1
DYXY004	Xylaria venosula	99.15	98	1059	EF026149.1
DYXY001	X. apiculata	98.81	99	1048	KP133330.1
DYXYY2	Fusarium redolens	99.64	100	1026	MT182983.1
DYXYXY1	Nemania abortiva	99.82	98	1018	MN844448.1
DAXY0016C	Epulorhiza sp.	98.76	99	1110	EF393626.1
DYXY013C	Tulasnellaceae sp.	96.41	100	1051	MZ129307.1
DYXY002	X. arbuscula	99.83	99	1085	GU300090.1
DYSH004	Tulasnellaceae sp.	95.53	94	926	MZ129307.1
DYXY111	Xylaria sp.	99.63	96	976	MT601883.1
DYXY112	F. oxysporum	99.41	97	922	MT997880.1
DYXY033	Tulasnella sp.	99.36	100	1133	GQ241830.1

Orchids form mycorrhizal symbioses with Rhizoctonia-like fungi, including members of the Tulasnellaceae Serendipitaceae and Ceratobasidiaceae. Based on morphological characteristics and phylogenetic analysis of the sequences of rDNA internal transcribed spacer (ITS), 41 fungal strains were assigned to the family Tulasnellaceae (Figure 2).

Figure 2. Neighbor-joining phylogenetic tree analysis of the ITS sequences of mycorrhizal fungi isolated from the P. hirsutissimum roots. Bootstrap values (%) out of 1000 resamplings are represented at each node.

To provide a reference for the morphological classification and identification of Rhizoctonia-like fungi from the roots of orchids, only the Rhizoctonia-like fungi that displayed unique cultural characteristics from among mycorrhizal fungal isolates were given codes and subjected to further micromorphological characterization in this study.

DYSH004 displayed dolipore septum with imperforate and curved parenthesomes composed of a central electrontransparent layer and two electron-dense layers surrounding it (Figure 3c). Hyphae grew close to the medium, and they exhibited irregular edges, dendritic branching, uniform thickness, and a light brown color. In the later period, the aerial hyphae of the colonies turned white (Figure 3a). Moniliform cells were elliptical and cylindrical shaped, with a size of 6.39–12.03 × 12.88–20.78 μm in diameter, hyphae of 3.06–5.27 μm in diameter, and a cell length of 43.67–90.51 μm (Figure 3b).

Figure 3. Colony cultured on a PDA medium of isolate DYSH004(a), the morphology of monilioid cells(b,bar = 95 μm), the ultrastructure of dolipore septum (c); colony cultured on a PDA medium of isolate DYXY013C(d), the morphology of monilioid cells(e,bar = 70 μm), the ultrastructure of dolipore septum (f); colony cultured on a PDA medium of isolate DYXY023(g), the morphology of monilioid cells(h, bar = 70 μm), the ultrastructure of dolipore septum (i); colony cultured on a PDA medium of isolate DYXY033(j), the morphology of monilioid cells(k, bar = 70 μm), the ultrastructure of dolipore septum (l); colony cultured on a PDA medium of isolate DAXY0016C(m), the morphology of monilioid cells(n,bar = 75 μm), the ultrastructure of dolipore septum (o).

Figure 4. Continued.

Figure 4. Symbiotic seed germination and seedling developmental stages of P. hirsutissimum inoculated with mycorrhizal fungus DYXY033 cultured on PDA medium. Stage 1, rupture of the testa by enlarging embryo (arrow) (a-1, a-2); stage 2, protocorm formation by expanding embryo (arrow) (b-1, b-2); stage 3, the appearance of the protomeristem (arrow) (c-1,c-2); stage 4, the emergence of the leaf (arrow) (d-1, d-2); stage 5, elongation of the leaf to form a seedling (arrow) (e-1, e-2, f-1, f-2). Seeds’ germination and development on the wall of the bottle covered with mycelium (arrow) (g). Seedling with “cilium” rhizoids (arrow) (h). Reticulated fungal hyphae (arrow) (i). Rooting on the bottle wall (arrow) (j). P. hirsutissimum seedlings after 270 days of inoculation (k). Th seedlings are planted in the greenhouse (l).

DYXY013C exhibited dolipore septum with imperforate parenthesomes, and curved, composed of a central electrontransparent layer and two electron-dense layers surrounding the central layer (Figure 3f). Hyphae grew close to the medium without aerial hyphae and displayed smooth-surfaced, creamy-yellow, and even edges. In the later period, colonies turned light brown with irregular and waxy edges (Figure 3d). Subglobose and branched monilioid cell chains, measuring 10.64–13.51 × 12.28–18.57, constituted approximately 5–6 monilioid cells. Hyphae were 2.37–4.97 μm in diameter and cell length was 23.57–75.97 μm (Figure 3e).

DYXY023 exhibited dolipore septum with imperforate parenthesomes, and curved, composed of a central electrontransparent layer and two electron-dense layers surrounding the central layer (Figure 3i). In the early stage, colonies were creamy-yellow, hyphae grew close to the medium, with even edges, and aerial mycelia failed to thrive. In the later period, colonies turned light-brown with velvet edges, and exhibited irregular and aerial mycelia (Figure 3g). Monilioid cells were ellipsoidal or globose, with branched monilioid cell chains; they had an average size of 10.64–14.97 × 15.76–26.57 and were composed of approximately 4–5 monilioid cells. Hyphae were 2.97–6.38 μm in diameter and cell length was 47.31–176.87 μm (Figure 3h).

DYXY033 displayed dolipore septum with imperforate parenthesomes, and curved, composed of a central electrontransparent layer and two electron-dense layers surrounding the central layer (Figure 3l). The observed colonies were pale beige, waxy, irregular, with serrated edges, and without aerial hyphae in their early stage. Later, colonies turned brown and had small spherical bulges (Figure 3j). Monilioid cells were oval and displayed branched monilioid cell chains, with an average size of 10.56–13.72 × 20.81–27.35; they were composed of approximately 10–12 monilioid cells. Hyphae were 2.97–6.38 μm in diameter and cell length was 47.31–136.87 μm (Figure 3k).

DAXY0016C exhibited dolipore septum with imperforate parenthesomes, and curved, composed of a central electrontransparent layer and two electron-dense layers surrounding the central layer (Figure 3o). The observed colonies were pale beige in their early stage, hyphae grew close to the medium, and aerial mycelia did not flourish. Later, colonies became light-brown, velvet, and displayed even edges, with uniform thickness (Figure 3m). Hyphae were 2.52–5.31 μm in diameter and had a cell length of 35.79–101.31 μm. Moniliform cells were globose- to subglobose-shaped, branched monilioid cell chains, measuring 10.32–13.46 × 12.13–21.58 μm, which clustered to develop compact mass clusters of monilioid cell chains that formed tufts (Figure 3n).

3.2 In vitro symbiotic seed germination and seedling development

In this study, the effects of endophytic fungi on seed germination and protocorm development of P. hirsutissimum were monitored and evaluated after sowing them for 5 months (Table 3). Seed germination and seedling development growth stages of symbiotically cultured P. hirsutissimum (adapted from Stewart and Zettler³¹, as described above). The results showed that the germination rate index of P. hirsutissimum seeds in treatments inoculated with endophytic fungi DYXY013C, DYSH004, DYXY033, and CK was significantly higher than that in other treatments (stages 1,2). The developmental rate index of seedlings in treatments inoculated with endophytic fungi DYXY013C, DYSH004, and DYXY033 was significantly higher than that in other treatments (Stage 3). The treatments inoculated with endophytic fungal DYXY033 supported the growth of seedlings to an advanced developmental stage of up to stage 5. In addition, P. hirsutissimum seedlings inoculated with mycorrhizal fungal DYXY033 substantially increased the developmental rate index of seedlings at stages 1 to 3 in comparison with those inoculated with mycorrhizal fungi DYXY013C and DYSH004. The development of seedlings in treatments inoculated with endophytic fungi DYXY013C and DYSH004 was arrested at stage 3. The germination and development of each stage seedlings inoculated with dominant mycorrhizal fungal DYXY033 are shown in Figures 4a-i.

Table 3. Effect of PDA medium and endophytic fungi on germination and development of P. hirsutissimum seeds.

Fungal Isolate	Germination and growth stage (germination and growth rate)
	Stage 1%)	Stage 2%)	Stage 3%)	Stage 4%)	Stage 5%)
DYXY0003	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
DYXY004	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
DYXY001	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
DYXYY2	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
DYXYXY1	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
DYXY013C	62.67 ± 4.73d	31.33 ± 4.16c	26 ± 4.58b	0.00 ± 0.00a	0.00 ± 0.00a
DYXY002	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
DYSH004	52.67 ± 3.79c	34.33 ± 2.52c	24.67 ± 1.53b	0.00 ± 0.00a	0.00 ± 0.00a
DYXY111	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
DYXY112	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
DYXY033	78.33 ± 3.21e	73.33 ± 3.51d	63.33 ± 4.16c	57.00 ± 3.00b	56.67 ± 3.01b
CK	28 ± 2.65b	11.33 ± 1.15b	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a

Normal sterilized P. hirsutissimum seeds were used as control (CK).

Figure 4. Continued.

3.3 Seed germination and seedling development co-cultured with the fungus DYXY033

Based on the abovementioned results, the changes in the morphological characteristics during seed germination and subsequent protocorm development in symbiotic germination were carefully observed under SEM. The mature seeds of P. hirsutissimum had a tough seed coat (ck, Figure 5a). After 10 days of inoculation, the hyphae and monilioid cells began to wind around and wrap the seed (Figure 5b), begin to invade from either end of the seeds (Figure 5c), and enlarge the embryo by absorbing water, while retaining the seed coat around the embryo (Figures. 5b,d). After 20 days, the hyphae and monilioid cells had wrapped around the seed coat (Figure 5e), invaded the embryo, extended rapidly from the inside to the outside, and lengthened up enough to burst the seed coat (Figure 5f). After 30 days, the embryo continued to absorb water and swell, eventually causing the rupture of the seed coat and exposing the embryo partially, followed by developed into elliptical-shaped protocorms (Figures. 5g,h). After 40 days, the epidermal cells at the base of the protocorm began to bulge and form rhizoids (Figure 5i). Subsequently, the embryo developed into a protocorm, and the embryo polarity was established by the development of a protomeristem at the apex, with the appearance of the dorsal crest, cilium rhizoids, and shoot apical meristem (Figures. 5k,l). After 50 days, the leaf primordia emerged (Figure 5m). After 60 days, it began to differentiate into the first leaf (Figure 5n).

Figure 5. SEM images of the mycorrhizal fungus DYXY033 co-cultured with P. hirsutissimum seed and seedling development over 0–60 days. A mature seed of P. hirsutissimum (a). Hyphae and monilioid cells wound and wrapped around the seed (b). Hyphae and monilioid cells show invasion from the base of the seed (arrow) (c). Enlarged embryo after absorbing water (d). Hyphae and monilioid cells wrapped around the seed coat (arrow) (e). Hyphae and monilioid cells breaking through the seed coat (arrow) (f). Embryo continued to enlarge and the seed coat broke open, exposing the embryo partially (g). Clusters of moniliform cell chains colonized on either end of the embryo (arrow) (h). Epidermal cells at the base of the protocorm bulging to form rhizoids (arrow) (i). Hyphae invading epidermal cells of the embryo (arrow) (j). The appearance of the dorsal crest, shoot apical meristem, and cilium rhizoids (arrow) (k, l). Emergence of the leaf primordia (m). Differentiation into the first leaf (arrow) (n).

Figure 5. Continued.

3.4 Effects of different mycorrhizal fungi on the seedling growth of P. hirsutissimum

We selected five fungal isolates for inoculation with P. hirsutissimum seedlings in order to determine the beneficial mycorrhizal fungus. After 90 days of culturing, DAXY0016C, DYSH004, DYXY013C, DYXY023, and DYXY033 showed significantly increased seedling fresh weight when compared with CK (p < 0.05). However, when compared with DAXY0016C, DYSH004, DYXY013C, DYXY023, and CK, DYXY033 co-culture resulted in significantly increased seedling fresh weight and leaf length (p < 0.05). DYXY033 significantly promoted the root numbers when compared with DAXY0016C and CK (p < 0.05). Furthermore, only DYXY033 significantly promoted the root length when compared with CK (p < 0.05). These results indicated that the five fungal isolates could promote seedling growth and development, although the best one was demonstrated by isolate DYXY033 (Figures. 6a-d).

Figure 6. Effects of mycorrhizal fungi on the growth of P. hirsutissimum seedlings 90 days after inoculation. Co-culture with the PDA media on seedling growth of P. hirsutissimum. Bars indicate the mean change in the plant fresh weight (a), root length (b), leaf length (c), and root number (d). The mean values with different letters are significantly different.

4. Discussion

In the life cycle of an orchid, symbiotic mycorrhizal fungus plays an important role. As a result, it has been suggested that employing OMF to support orchid recovery initiatives is a useful strategy for orchid conservation³³. However, mycorrhizal fungi with in situ seed symbiotic germination are usually so restrictive that prerequisite for improving seed germination and seedling development is the isolation, identification, and acquisition of effective OMF^20,34,35. In this study, 70 fungal isolates were isolated from P. hirsutissimum roots. All the isolated strains belonged to Ascomycota and Basidiomycota. According to reports, OMF mainly belonged to Basidiomycota, Ascomycetes, and Mucoromycota, among which Basidiomycota was the most common and the earliest discovered OMF^27,36–38. The most common genera of OMF are Epulorhiza, Ceratorhiza, Ceratobasidium, Sistotrema, Tulasnella, Russula, Mycena, Tricharina, Peziza, and Flavodon^27;39–42;⁴³. Therefore, the results of endophytic fungi isolated from P. hirsutissimum were consistent with those of previous studies. Among them, endophytic fungi of P. hirsutissimum with morphological and molecular characteristics showed that endo-phytic fungi DYXY023, DAXY0016C, DYXY013C, DYSH004, and DYXY033 belonged to the typical orchid mycorrhizal family Tulasnellaceae, which were the dominant fungal endophytes. These fungi are associated with a wide variety of terrestrial and epiphytic orchids and speed up the germination of seeds as well as the development rate of protocorm and seedlings of orchids⁴. Previous studies have revealed that many mycorrhizal fungi isolated from species of Paphiopedilum were members of Tulasnella and Epulorhiza^{14,27,30,44,45}, which is consistent with the results obtained in our study. Based on the comparison of ITS sequences, endophytic fungi DYXY0003, DYXY004, DYXY001, DYXYY2, DYXYXY1, DYXY002, DYXY111, and DYXY112 were identified as A. alternatum, Xylaria venosula, X. apiculate, Fusarium redolens, N. abortive, X. arbuscula, Xylaria sp., and F. oxysporum, respectively. Among them, studies have reported that Xylaria and Fusarium are the dominant endophytic fungi often found in association with orchids. Species of Xylaria and Fusarium were ubiquitous in the roots of Dendrobium nobile and D. chrysanthum⁴⁶, and Xylaria has reportedly been found in the green orchid Anoectochilus setáceus⁴⁷. Cypripedium reginae, C.parviflorum, and Platanthera grandiflora are three green orchids whose seed germination has previously been reported in the literature to be caused by Fusarium^48,49. As far as is known, the endophytic fungus found in orchids of the species N. abortive or A. alternatum has never been reported in the literature. Our results showed a high diversity of endophytic fungi associated with P. hirsutissimum, including mycorrhizal and nonmycorrhizal fungal endophytes, which is consistent with the results of previous studies^6,37,50.

The results of symbiotic seed germination in this study showed that mycorrhizal fungi DYXY013C, DYSH004, and DYXY033 could promote the germination of P. hirsutissimum. In addition, DYXY033 could promote the seedling formation and rooting to stage 5, whereas DYXY013C and DYSH004 could promote the appearance of the protomeristem only to stage 3. Further research is required because it is unclear which aspect prevented the protocorm of P. hirsutissimum from developing later. Therefore, more experiments are needed to clarify the role of other fungal isolates in affecting the establishment of seedlings. Regarding the other endophytic fungi tested, the seeds were parasitized and could not promote germination and protocorm formation caused by the excessive growth of the strains in the PDA medium. According to the report, the type and composition of the medium was the factor affecting the seed germination and seedling growth of Paphiopedilum. In subsequent studies, periodic transfers of the seeds and protocorms to a new coculture medium and altering the concentration and composition of the medium were sufficient to resolve this problem^4,6,51,52. Although Xylaria and Fusarium species were the predominant endophytic fungi of P. hirsutissimum, their role as mycorrhizal fungi has not been illustrated and their ecological function remains unclear⁴⁶. It is difficult to designate the ecological roles of these endo-phytic fungi because their impacts on the seed may not reflect ecological competency in the adult plant⁴.

During the process of orchid symbiotic germination, the existence of an inner seed coat makes it difficult for the seed embryo to absorb water and expand, making it an important obstacle in seed germination³. According to the literature, the lignocellulose contents (especially of lignin) in Cremastra appendiculata seeds sharply decreased after symbiosis with Coprinellus disseminatus². The present results revealed that the strain DYXY033 had a positive effect on the seed germination and seedling development of P. hirsutissimum. Moreover, according to the results of SEM (Figures. 5f,g), DYXY033 was symbiotic with the seeds and invaded the embryo, followed by lengthening to grow and bursts through the seed coat. This event successfully promoted the germination and development of P. hirsutissimum, which may be attributed to its ability to decompose wood and provide the necessary nutrients and carbohydrates for seedlings’ growth². The results demonstrated that the inner seed coat was one of the main obstacle to seed germination. However, its specific symbiotic mechanism remains unknown and its function and molecule mechanism warrants further research.

Some OMFs are believed to have potential specificity because they can successfully promote orchid seed germination, protocorm development, and seedling establishment in vitro conditions^20,22,53. In this study, we used symbiotic seed germination experiments to verified the potential specificity of mycorrhizal fungus DYXY033. The results indicate that fungus DYXY033 with potential specificity could promote seed germination, seedling formation and rooting of P. hirsutissimum.Moreover, fungal DYXY033 mycelium grew vigorously on simple PDA medium and the tissue culture bottles were entirely covered along the wall of the tissue culture bottle, after culturing for 2 weeks. According to Figure 4g-j, the seeds of P. hirsutissimum germinated, grew, and rooting on the wall of the bottles covered with mycelium and without coming into contact with the PDA medium. It was concluded that the mycelium of fungal DYXY033 was compatible and supported seed germination without requiring PDA medium as nutritional support. In other words, the PDA medium promoted the mycelium growth of fungal DYXY033, and the mycelium promoted seed germination, seedling development, and rooting of P. hirsutissimum. Furthermore, DYXY033 supported the growth, development, and survival of P. hirsutissimum seedlings after transplanting in greenhouse conditions (Figure 4l). Results showed that DYXY033 forms a good symbiotic system with P. hirsutissimum seed and seedlings. This method that symbiotic (co-culture with fungus DYXY033) seed germination and seedling development methods require a relatively simple culture medium formula, which is cost-effective and time-saving advantages^14,28. Therefore, this this can be used as an alternative method for the artificial propagation of P. hirsutissimum.

Ecological specificity of mycorrhizal fungi has been proved to be valuable for the conservation of orchids, contributing to future orchid production, reintroduction in both in situ and ex situ conservation efforts, and under in vitro experimental conditions²⁰. In this study, we obtained an endophytic mycorrhizal fungal DYXY033 with compatibility and specificity from the roots of P. hirsutissimum and this could be potential for mass seedling propagation in rare orchid conservation initiatives. In future research, it should focus on testing the ecological specificity of strain DYXY033. When considering direct sowing for reintroduction and conservation, strain DYXY033 carrying ecological specificity should be tested in the wild. However, due to the high complexity of in situ symbiotic seed germination in orchid, it faces great challenges^16,20. Therefore, to succeed in P. hirsutissimum population in situ restoration and reintroduction programs, innovative and comprehensive measures are needed to design efficient, reliable, and practical programs, such as sowing time, the density of the fungal patch, optimal fungus or fungal combination, refining media composition, and seed bag design, etc.⁵⁴

Acknowledgments

We would like to acknowledge the research funding provided by the Science and Technology Department of Guizhou Province, China.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

The datasets used or analyzed in the current study are available from the corresponding author upon reasonable request.

Additional information

Funding

The work was supported by the  National key wildlife protection subsidy project: The Capacity Improvement Project of State Key Laboratory of Southwest Karst Mountain Biodiversity Conservation of National Forestry and Grassland Administration [2019-000411-05-01-001641]; Scientific Search Projects of Guizhou Forestry Bureau [2019] 02;Scientific Search Projects of Guizhou Forestry Bureau [2022] 36 .