Seed Transmission of Beet Curly Top Virus and Beet Curly Top Iran Virus in a Local Cultivar of Petunia in Iran

Abstract

Beet curly top virus (BCTV) and beet curly top Iran virus (BCTIV) are known as the causal agents of curly top disease in beet and several other dicotyledonous plants in Iran. These viruses are transmitted by Circulifer species, and until now, there has been no confirmed report of their seed transmission. A percentage (38.2-78.0%) of the seedlings developed from the seeds of a petunia local cultivar under insect-free conditions showed stunting, interveinal chlorosis, leaf curling, and vein swelling symptoms, and were infected by BCTV when tested by PCR. Presence of BCTV in seed extracts of petunia local cultivar was confirmed by PCR and IC-PCR, followed by sequencing. Agroinoculation of curly top free petunia plants with a BCTV infectious clone resulted in BCTV infection of plants and their developed seeds. These results show the seed infection and transmission of BCTV in a local cultivar of petunia. Similar experiments performed with BCTIV showed that this virus is also seed transmissible in the same cultivar of petunia, although with a lower rate (8.8-18.5%). Seed transmission of curly top viruses may have significant implications in the epidemiology of these viruses.

Keywords: beet curly top viruses; geminiviruses; petunia; seed transmission; transmission rate.

Figure 1

Detection of beet curly top virus (BCTV-Svr) and beet curly top Iran virus (BCTIV) in infected petunia plants. (a) Interveinal chlorosis, vein swelling and severe leaf curling in a BCTV-Svr infected petunia plant developed from the seed of the local cultivar; (b) non-symptomatic curly top free plant of local cultivar; (c,d) agarose gel electrophoresis analysis of PCR products of BCTV-Svr and BCTIV amplified from total DNA extracts of naturally infected petunia plants by specific primer pairs as indicated below each panel; (e) agarose gel electrophoresis analysis of PCR products of BCTV-Svr amplified from total DNA of seed extracts of petunia local cultivar by specific primer pairs as indicated below the panel. C⁺ = Positive control: cloned DNA of BCTIV (pBin-1.4-BCTIV in D) and BCTV-Svr (pBin-1.7 BCTV-Svr in E); HP = healthy (curly top free) plant. M = Marker (DNA ladder mix, Fermentas, Waltham, MA, USA).

Figure 2

Vertical transmission of BCTV-Svr and BCTIV through three generations of naturally infected petunia plants of local cultivar. (a) Schematic representation of an experiment on vertical transmission of BCTV-Svr and BCTIV through three generations (G1, G2 and G3). Healthy plants are shown with green leaves and those from symptomatic infected plants are shown in yellow color; (b) agarose gel electrophoresis analysis of PCR products of BCTV-Svr and BCTIV amplified from total DNA extracts from each plant in each generation using specific BCTV-Svr358^V/877^C or BCTIV-474^v/1155^c primer pair. C⁺ = Positive control: cloned DNA of BCTV-Svr (pBin-1.7 BCTV-Svr) or of BCTIV (pBin-1.4-BCTIV). HP = healthy (curly top free) plant. C⁻ = Negative control: distilled water. M = Marker (DNA ladder mix, Fermentas).

Figure 3

Agarose gel electrophoresis analysis of immunocapture PCR products using a BCTV antibody and specific BCTV-Svr358^V/877^C primer pair. Lane 1, extract of leaf tissue of healthy plants of local cultivar as negative control; lanes 2–6, extracts of leaf tissue of naturally infected plants of local cultivar; lanes 7 and 8, extracts of seeds of naturally infected plants of local cultivar; lanes 9 and 10 extracts of seeds of BCTV-Svr-agroinoculated plants of PCSP cultivar, lanes 11 and 12 extract of seeds developed from healthy plants of PCSP cultivar. M = Marker (DNA ladder mix, Fermentas).

Figure 4

Detection of BCTV-Svr in agroinfected petunia plants and their developed seeds. (a) Severe curly top symptoms in a BCTV-Svr-agroinoculated plant of local cultivar; (b) curly top free (healthy) plants of local cultivar; (c) a BCTV-Svr-agroinoculated plant of PCSP cultivar showing interveinal chlorosis, leaf curling and vein swelling; (d) healthy plants of PCSP cultivar; (e,f) agarose gel electrophoresis analysis of IC-PCR products using a BCTV antibody and the BCTV-Svr358^V/877^C primer pair (Table 1); (e) lanes 1 and 2, extracts of leaf tissue of healthy plants of PCSP cultivars as negative controls; lanes 3 and 4, extracts of leaf tissue of BCTV-Svr-agroinoculated plants of local and PCSP cultivars, respectively; (f) lanes 1–3, extracts of seeds of healthy plants of local cultivar as negative controls; lanes 4 and 5 extracts of seeds of BCTV-Svr-agroinoculated plants of local cultivar; lane 6, extract of seeds of BCTV-Svr-agroinoculated plants of PCSP cultivar. M = Marker (DNA ladder mix, Fermentas).

Figure 5

(a) Linearized diagram of BCTV-Svr genome. Arrows indicate coding sequences and purple dotted box indicates the region used for preparing probe for PCR ELISA (DIG labeling) and BCTV-Svr358^V/877^C primer pair; (b) Southern blot hybridization of total DNA extracted from: a healthy PCSP plant as negative control (lane 1), an agroinoculated PCSP plant as positive control (lane 2), an infected plant developed from seed of local cultivar (lane 3), seeds of healthy PCSP used as negative control (lane 4), seeds of agroinoculated PCSP cultivar (lane 5) and seeds of naturally infected local petunia cultivar (lane 6). Loaded DNA of each sample stained with ethidium bromide is shown at the bottom of the blot panel. sc (BCTV-Svr supercoil double-strand DNA, which is shown by the arrows); ss (BCTV-Svr single-strand DNA).