Specific in situ visualization of the pathogenic endophytic fungus Aciculosporium take, the cause of witches' broom in bamboo

Abstract

The endophytic fungus Aciculosporium take (Ascomycota; Clavicipitaceae) causes continuous shoot growth in bamboo. The colonized shoot eventually results in witches' broom formation but maintains normal leaf arrangement and branching pattern. To analyze the mechanism of well-regulated symptom development, the location of the fungal endophytic hyphae in host tissues was visualized. A colorimetric in situ hybridization technique using a species-specific oligonucleotide probe targeting the 18S rRNA of A. take was used. In situ hybridization was performed on tissue sections of diseased shoots with or without external signs of fungal colonization. Specific signals were detected in intercellular spaces of the bamboo tissues. Most signals were detected in the shoot apical meristem and the leaf primordia. In addition, fewer signals were detected in the lateral buds, juvenile leaves, and stems. These results indicate that A. take grows endophytically, particularly in the shoot apical meristem of the host. The location of A. take hyphae suggests that the mechanism of symptom development can be explained by the action of exogenous fungal auxin, which continuously induces primordium initiation within the host.

FIG. 1.

Bamboo shoot colonized by the endophytic fungus Aciculosporium take. (a to c) Schematic of witches’ broom symptom development on a bamboo species of Phyllostachys. (a) The normal shoot ceased to grow after three to five leaves developed. (b) An A. take-colonized shoot grew out with extraordinarily small leaves but maintained normal leaf arrangement. The shoot continued to grow with successive thin phytomers. (c) After stromata (arrows) were formed at the shoot apex, the lateral buds began to grow but maintained a normal branching pattern. This sequence repeats. (d to f) Bamboo (P. pubescens) shoot colonized by A. take. (d) Early symptom. Diseased shoots (DS) have leaves that are much smaller than normal leaves (NL). Bar, 1 cm. (e) Developed symptom shows typical witches’ broom appearance. Bar, 1 cm. (f) Ascostroma (AS) is formed on a conidiostroma (CS) at the apex of the diseased shoot. The conidiostroma is surrounded by a sheath. Bar, 1 mm.

FIG. 2.

Dot blot hybridization to evaluate the specificity of oligonucleotide probe Aci65 for genomic DNA. The specific oligonucleotide probe Aci65 targeting A. take 18S rRNA showed a positive signal only on A. take genomic DNA. Universal oligonucleotide probe R898, targeting fungal 18S rRNA, showed positive signals on all genomic DNAs of A. take and several other related clavicipitaceous fungi.

FIG. 3.

Species-specific visualization of Aciculosporium take by ISH of bamboo shoot tissue. (a) ISH with digoxigenin-labeled oligonucleotide probes on serial cross sections of bamboo bud tissue bearing an A. take ascostroma. Hybridization signals are shown in blue-purple. The A. take-specific probe (Aci65) showed positive signals in fungal structures, including pseudoparenchyma (pp), perithecia (pt), and asci. Furthermore, the signals were shown in intercellular spaces of juvenile leaf (jl) and stem (st). No signal was shown in sheath (sh) and epidermal cells of the stem. The positive control probe (R898) is identical to probe Aci65. The negative control probe (non-Aci65, the complementary sequence of probe Aci65) and no-probe control showed no reaction. Several microphotographs were combined into one large image. Bars, 1 mm. (b to i) ISH with oligonucleotide probe Aci65 on tissues of bamboo shoot colonized by A. take with no external signs of fungal colonization. Positive signals are shown in blue-purple. Magnified views of the boxed regions are shown in c, d, e, g, and i. (b) Longitudinal section of a whole shoot. Several microphotographs were combined into one large image. Bar, 1 mm. (c) Hyphae were observed in the tissue of the fourth lateral bud. Bar, 100 μm. (d) Hyphae were observed in the tissue of the second lateral bud and stem. Bar, 100 μm. (e) Many hyphae were observed in the tissues of the shoot apical meristem (sam) and leaf primordia (lp). Intercellular spaces of the meristem were especially filled with hyphae. Bar, 100 μm. (f) Cross sections of internode stem (st) and sheaths (sh). No signal was detected in sheaths. Bar, 500 μm. (g) Hyphae (arrows) were detected in intercellular spaces of stem. Bar, 100 μm. (h) Cross sections of juvenile leaf. Several hyphae (arrows) were detected in intercellular spaces. Bar, 500 μm. (i) A hypha (arrow) was observed in the intercellular space of the vascular bundle. Bar, 100 μm.