نوع مقاله : مقالات پژوهشی
1 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، ایران
2 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه فردوسی مشهد
عنوان مقاله [English]
Beet curly top disease is one of the most important sugar beet viral diseases. Numbers of viruses in the family Geminiviridae, including Beet Curly Top Iran Virus (BCTIrV), Beet Curly Top Virus (BCTV), and Turnip Curly Top Virus (TCTV) have generated curly top symptoms in sugar beet. BCTIrV belongs to the genus Becurtovirus, but BCTV and TCTV put in Curtovirus and Turncurtovirus genera, respectively. BCTIrV is known as the most prevalent causal agent of curly top disease in Iran. It has circular single-stranded DNA genomes with 2.8–3.2 kb nucleotides length. The genome encapsidate in quasiicosahedral twinned particle with 22 nm diameters. BCTIrV is transmitted by Circulifer Haematoceps leafhopper naturally. Non-uniform transmission and time-consuming process of reproduction of a virus-free population of the leafhopper make experimental transmission of BCTIrV troublous. Generation of an infectious clone is another strategy for efficient inoculation of BCTIrV to host without dependency on its natural insect vector. The aim of this study is comparison of pathogenicity of two infectious clones of BCTIrV (1.1 and 1.4 mer constructs) in several hosts and optimization of criteria for efficient reproduction of the vector for transmission of the virus.
Material and Method
The complete genome of BCTIrV was cloned in pBlueScript II SK (+) previously. Plasmid extraction from the bacterial cells was carried out using plasmid extraction procedure described by Kotchoni et. al. (24). The 1.4 and 1.1 BCTIrV infectious clones were made by joining 1029 and 308 kb fragments respectively to a unit length of BCTIrV genome. The infectious clones were agroinoculated to seedling s of sugar beet. DNA extraction from newly grown leaves of agroinoculated plants was performed using CTAB. Polymerase chain reaction (PCR) using specific primer pair for BCTIrV coat protein gene (CP) was carried out to identify infectivity of the constructs. To optimize the reproduction of Circulifer hematoceps in greenhouse conditions, Beta vulgaris, Solanum lycopersicum, Capsicum annuum, Nicotiana glutinosa, Sesamum indicum and Amaranthus retroflexus plants were used as hosts. To determine rate of reproduction of C. Hematoceps in greenhouse conditions, firstly optimum temperature for the leafhopper reproduction was determined, then C. Hematoceps population was counted at optimum temperature in 21, 45, and 60 days after release. In transmission test, an adult C. Hematoceps was used to transmit BCTIrV to sugar beet seedling.
Sequencing data and RFLP pattern using EcoRI/XhoI restriction endonuclease were confirmed integrity of the 1.4 and 1.1 constructs. Four weeks after inoculation, BCTIrV was identified in newly grown leaves of sugar beet seedlings using PCR. Eight weeks after inoculation yellowing and leaf curling symptoms were generated on infected plants. Circulifer hematoceps was successfully reproduced on B. vulgaris, S. indicum and A. retroflexus in greenhouse. Moreover, S. lycopersicum, C. annuum, N. glutinosa were not suitable hosts for the leafhopper reproduction. The maximum, minimum, and optimum of daily temperature required for C. hematoceps reproduction was 34°C, 18°C and 29°C, respectively in a period of 45 days after the leafhopper release. Also, the best fitted host for C. hematoceps multiplication was A. retroflexus, however, BCTIrV was not infective in this plant. BCTIrV was successfully transmitted from pG-BCTIrV 1.4 and pG-BCTIrV 1.1 agroinoculated B. vulgaris plants to healthy sugar beet using C. hematoceps. Fifth weeks after the leafhopper feeding systemic symptoms of the virus were developed.
The curly top is a destructive disease of sugar beet which is a threat to sugar beet production. Previously, several BCTIrV infectious clones with different lengths were made by other researchers. The pG-BCTIrV 1.4 is similar to constructs of previous studies. In this study, pG-BCTIrV 1.1 that is the smallest infectious construct of BCTIrV with a length of 3153 nucleotides, is successfully constructed for the first time. The 1.1 mer infectious clone will provide a facility for induction of point mutation in the BCTIrV genome to identify the role of genetic elements in virus pathogenicity via reverse genetic approach. The results indicated no significant difference in infection efficiency, symptoms severity, and vector transmission rate between pG-BCTIrV 1.4 and pG-BCTIrV 1.1 constructs. Forth weeks after inoculation symptoms were observed in B. vulgaris plants that vector transmission by pG-BCTIrV 1.4 and pG-BCTIrV 1.1, but Taheri et. al. (2012) represented symptoms appearance at two weeks post inoculation. This discrepancy may cause by differences in host susceptibility, virulence of the virus, or environmental conditions. The results revealed that, the preferential host for C. haematoceps multiplication was A. retroflexus in greenhouse conditions. The population of C. haematoceps increased up to 11.1-fold on this plant in 45 days. In the current study, infectivity of BCTIrV in A. retroflexus not identified either using agroinoculation or vector transmission, but Jahanbin et. al. (2015) represented BCTIrV infection in 20 % of agroinoculated A. retroflexus plants.
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