An efficient grafting method for phytoplasma transmission in Catharanthus roseus

Abstract

Background: Phytoplasmas are parasitic plant pathogens that reside intracellularly within the sieve tube cells. Phytoplasmas induce various symptoms, including floral virescence, phyllody, leaf yellowing, and witches'-broom. Currently, it is challenging to culture phytoplasma in vitro. In the laboratory, phytoplasmas are generally maintained in alternative host plants, such as Catharanthus roseus. Grafting is used to transmit phytoplasmas among the alternative hosts. During the experiment, scions from infected plants are grafted onto healthy plants using a side grafting method. However, the practice has certain limitations, including its inability to be applied to small plants and its irregular disease incidence.

Results: Here, we demonstrate a new approach, penetration grafting, to overcome the limitations of side grafting. This grafting method allows phytoplasma to be efficiently and uniformly transmitted into the inoculated plants. No significant difference was observed in phytoplasma accumulation between both grafting techniques. However, penetration grafting allows rapid symptom development, saving waiting time and reducing space usage.

Conclusions: This study provides a reliable and stable method for experiments that require grafting transmission.

Keywords: Catharanthus roseus; Grafting; Phytoplasma symptom; Phytoplasma transmission.

Fig. 1

Diagram of grafting technique (A) The disease scions from phytoplasma-infected plants were cut into a ‘V’ shape and inserted into the slanting cut made on the healthy stock plants of periwinkle. The red arrowhead indicates the grafting site of the plant. (B) A box cutter is used to make a slit on the stem of the rootstock, and the infected scion cut into a 'V' shape is then inserted into the slit. (C) comparison of the completed grafting

Fig. 2

Floral and leaf symptoms of periwinkle leaf yellowing (PLY) phytoplasma-infected periwinkle plants (A) Flowers of healthy periwinkle; (B to D) flowers of PLY phytoplasma-infected periwinkle plants; (E) leaves of healthy periwinkle; (F and G) leaves or shoots of PLY phytoplasma-infected periwinkle plants. Flowers that exhibit discoloration or partial yellowing of leaves were defined as the S1 symptom stage (B and F). Flowers showing partial virescence are at the S2 stage (C), while flowers that have completed virescence or shoot proliferation are at the S3 stage (D and G)

Fig. 3

(A) Changes in the pathogenesis of PLY phytoplasma-infected periwinkle plants with different grafting methods. (B) Days required from S1 to S3. Each sample was determined from at least 6 biological replications. The symptom stages are defined according to Fig. 2; that are S0 (no visible symptom), S1 (flower discoloration or partial leaf yellowing), S2 (partial floral virescence), and S3 (completed floral virescence or shoot proliferation). The error bar indicates the SE of each set of replicates; * indicates p value < 0.05, and *** indicates p value < 0.001. dpi = days post-inoculation

Fig. 4

(A) Standard curve of real-time PCR Ct values of periwinkle ubiquitin gene. (B) Standard curve of real-time PCR Ct values of PLY phytoplasma SecY  gene. (C) The mean number of phytoplasma genome copies was measured in the apical leaves at the S3 stage (flowers that have completed virescence or plants showing shoot proliferation symptoms). Each group was determined from at least 6 biological replications. The error bar indicates the SE of each set of replicates