Peanut witches' broom (PnWB) phytoplasma-mediated leafy flower symptoms and abnormal vascular bundles development

Abstract

The peanut witches' broom (PnWB) phytoplasma causes virescence symptoms such as phyllody (leafy flower) in infected peanuts. However, the obligate nature of phytoplasma limits the study of host-pathogen interactions, and the detailed anatomy of PnWB-infected plants has yet to be reported. Here, we demonstrate that 4',6'-diamidino-2-phenylindole (DAPI) staining can be used to track PnWB infection. The DAPI-stained phytoplasma cells were observed in phloem/internal phloem tissues, and changes in vascular bundle morphology, including increasing pith rays and thinner cell walls in the xylem, were found. We also discerned the cell types comprising PnWB in infected sieve tube members. These results suggest that the presence of PnWB in phloem tissue facilitates the transmission of phytoplasma via sap-feeding insect vectors. In addition, PnWB in sieve tube members and changes in vascular bundle morphology might strongly promote the ability of phytoplasmas to assimilate nutrients. These data will help further an understanding of the obligate life cycle and host-pathogen interactions of phytoplasma.

Keywords: 4′,6′-diamidino-2-phenylindole staining; broom phytoplasma; internal phloem; leafy flower; peanut witches'; vascular bundle.

Figure 1.

Peanut witches' broom (PnWB)-infected peanut plants were collected from peanut fields of Taiwan.(A) The geographic locations of PnWB isolates in Taiwan are indicated by red asterisks. PH refers to the Penghu Island; CH refers to the Changhua area; YL refers to the Yunlin area. Bar, 100 km. (B) Witches' broom symptoms of PnWB-infected peanuts collected from the Yunlin and Changhua areas. PnWB CH refers to PnWB-infected peanuts collected from Changhua. PnWB YL refers to PnWB-infected peanuts collected from Yunlin. Bar, 1 cm. (C) Healthy (left panel) and PnWB-infected (right panel) peanut flowers. ba: banner; w: wings; ke: keel; ov: ovary. Bar, 0.2 cm.

Figure 2.

Peanut witches' broom (PnWB) infection alters vascular bundle development.(A) Transverse sections of healthy (left panel) and PnWB-infected (right panel) C. roseus stems stained with toluidine blue O. Green arrowheads indicate pith rays. The enlarged photograph shows the xylem (xy). Bars, 0.2 mm. (B) Transverse section of healthy (left panel) and PnWB-infected (right panel) C. roseus stems stained with 4′,6′-diamidino-2-phenylindole (DAPI). White arrowheads indicate PnWB-infected cells. Bars, 0.05 mm. (C) Cross-section of PnWB-infected stem tissue (upper panel) stained with DAPI. Red arrowheads indicate PnWB-infected cells. Cross-section of healthy stem tissue (lower panel), which was used as a negative control. Pith (pi), pith ray (pr), xylem (xy), cambium zone (cz), phloem (ph), phloem fiber (pf), and cortex (co) are indicated by green arrows. Bar, 100 µm.

Figure 3.

Microscopic observation of peanut witches' broom (PnWB)-infected stem. (A) Detection of PnWB-infected cells by confocal microscopy. The stem sections were stained with 4′,6′-diamidino-2-phenylindole (DAPI). Red arrowheads indicate infected sieve tube members. Bar, 25 µm. (B) Ultrathin sections of sieve tube members from healthy (panel i) and PnWB-infected (panel ii) Catharanthus roseus plants. Red arrowheads indicate PnWB phytoplasma cells. The pleomorphic morphology of the PnWB cell is shown. DNA fibrils (DF) (panel iii), and ribosomes (R) and cell membrane (M) of PnWB (panel iv). CW, cell wall of the host cell. SB, smaller bodies. Bars, 1 µm.