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; reintroduction; seedling development; symbiotic germination.

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).

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.

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).

Figure 4.

Continued.

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.

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.