Abstract
Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germplasm. In this study, we develop two quick, sensitive, and reliable protocols for real-time quantitative PCR (qPCR) of Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV) using two sets of TaqMan probes and primers targeting the reverse transcriptase/ribonuclease H (RT/RNase H) region. The two assays had a detection limit of 100 copies of plasmid DNA and were 100 times more sensitive than conventional PCR. High specificity of the two assays was observed with respect to SCBIMV and SCBMOV. A total of 176 sugarcane leaf tissue samples from Fujian and Yunnan provinces were collected and analyzed in parallel by conventional PCR, SCBIMV-qPCR, and SCBMOV-qPCR. The SCBIMV-qPCR and SCBMOV-qPCR assays indicated that 50% (88/176) and 47% (83/176) samples tested positive, respectively, whereas only 29% (51/176) tested positive with conventional PCR with the primer pairs SCBV-F and SCBV-R. We demonstrate for the first time that SCBIMV and SCBMOV occur in China and reveal coinfection of both Badnavirus species in 29% (51/176) of tested leaf samples. Our findings supply sensitive and reliable qPCR assays for the detection and quantitation of SCBV in sugarcane quarantine programs.
Highlights
Sugarcane bacilliform virus (SCBV, genus Badnavirus, family Caulimoviridae) was reported for the first time in 1985 in Cuba [1]
Various variable nucleotides were found in the two sets of primers and probes of Sugarcane bacilliform IM virus (SCBIMV)-QLD and Sugarcane bacilliform MO virus (SCBMOV)-MOR by comparison of reverse transcriptase (RT)/ribonuclease H (RNase H) sequences with one another and with the other ten SCBV genotypes
The primers and probes sequences of SCBIMVqPCR and SCBMOV-quantitative polymerase chain reaction (PCR) (qPCR) were specific with respect to individual SCBV isolate, SCBIMV-QLD or SCBMOV-MOR
Summary
Sugarcane bacilliform virus (SCBV, genus Badnavirus, family Caulimoviridae) was reported for the first time in 1985 in Cuba [1]. SCBV can infect sugarcane (Saccharum spp.), Sorghum halepense, Brachiaria sp., Panicum maximum, and Rottboellia exaltata in a semipersistent manner via the insect vectors pink sugarcane mealy bug (Saccharicoccus sacchari) and gray sugarcane mealy bug (Dysmicoccus boninsis). It can mechanically infect rice (Oryza sativa), banana (Musa sp.), and sorghum (Sorghum vulgare L.) using partially purified extracts or by Agrobacteriummediated inoculation, but not by use of cutting implements or machinery [4,5,6]. Leaf freckle disease is caused by SCBV with symptoms of mottling, chlorosis, and stunted growth, with foliar symptoms varying across different host Saccharum and viral strains [5,6,7]
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