Molecular Diagnosis Of Malvastrum Leaf Curl And Tomato Yellow Leaf Curl Diseases In Fujian And Zhejiang Provinces, China

Geminiviruses (Family Geminiviridae) are a group of plant pathogenic viruses characterized by their unique twinned icosahedra particles, which encapsidate a circular single stranded DNA genome. They cause destructive diseases in many crops and are emerging as a serious threat to crop production worldwide. Recently, many distinct geminiviruses belonging to the genus begomovirus, which are transmitted exclusively by the whitefly vector, Bemisia tabaci, have been isolated from both weed and crop plant species in China. Majority of these distinct begomoviruses were isolated from South China.Accurate and reliable diagnosis of geminivirus plant diseases is an important prerequisite in the development of disease control strategies. This however, cannot be achieved by observing symptoms expression in host plants due to symptom similarities among geminiviruses and the occurrence of mixed infections involving two or more virus species. Molecular diagnosis has emerged as an accurate and reliable tool for the identification of specific viral pathogens involved in such infections. In this study, we carried out molecular diagnosis of Malvastrum leaf curl and Tomato yellow leaf curl diseases in Fujian and Zhejiang provinces of South East China.Leaf curling and vein banding disease symptoms were observed in Malvastrum coromandelianum, a perennial weed plant species found growing wild in fields within Fujian Province. Based on the symptoms expression, a begomovirus was suspected to be the cause agent and a molecular diagnosis was carried out to identify and characterize the specific viral pathogen responsible. Six leaf samples (FJ1, FJ2, FJ3, FJ4, FJ5 and FJ6) were collected from M. coromandelianum plants showing leaf curl and vein banding symptoms and total DNA was extracted from symptomatic leave tissues using the CTAB method. Partial viral DNA fragments, approximately 500bp long, were amplified from all the six samples by PCR using the degenerate primer pair PA/PB, specific for monopartite begomoviruses. The partial fragments were cloned and sequenced. Comparison of nucleotide sequence identity between the six isolates and other reported begomovirus DNA sequences showed that they are isolates of the same begomovirus, sharing 98-100% sequence identity among them and 90-93% sequence identity with the previously reported Malvastrum leaf curl Guangdong virus (MLCuGdV). This indicates that the six plant samples were infected by isolates of MLCuGdV. Isolate FJ3 was randomly selected for cloning and sequencing the complete viral genomic DNA. The complete viral genomic DNA of isolate FJ3 was found to be 2765 nucleotides long with all the characteristic features of a begomovirus. FJ3 DNA shares the highest (92.9%) nucleotide sequence identity with the previously described GD6 isolate of MLCuGdV (MLCuGdV-[GD6]) from Guangdong Province. This confirms that FJ3 is an isolate of MLCuGdV, proposed to be designated as MLCuGdV-[FJ3]. Consistent with previous reports, the Fujian isolates were not found associated with a putative satellite begomovirus DNAβmolecule either by PCR or southern blot hybridization analysis. In phylogenetic analysis, FJ3 DNA shows low affinity to other reported Malvastrum-infecting begomoviruses. Instead, it shows high affinity and clusters together with Papaya-infecting begomoviruses isolated from the same agro-ecological zone of South China. Further comparisons of amino acid sequences of polypeptides predicted to be encoded by various ORFs indicate that FJ3 is a chimeric molecule that has arisen by interspecific recombination among Papaya leaf curl China virus or Papaya leaf curl Guangdong virus and other unidentified ancestors.Tomato yellow leaf curl virus (TYLCV) and Tomato leaf curl virus (ToLCV) cause two of the most devastating viral diseases of cultivated tomato in tropical and subtropical regions of the world, and are spreading to new areas from their epicenters in the Middle East and the Mediterranean basin. TYLCV has not been reported in Mainland China prior to 2006, and begomoviruses has not been reported Zhejiang Province prior to 2006. However, in the autumn-winter cropping season of 2006, yellow leaf curl and stunting symptoms, characteristic of a begomovirus infection were reported in field grown tomato within Zhejiang Province. To determine the specific viral pathogens responsible, a molecular diagnostic survey was carried out in Zhejiang Province. Leaf samples were collected from tomato plants expressing yellow leaf curl and stunting symptoms in different districts of Zhejiang Province. Total DNA was extracted from the leaf tissues using the CTAB method and partial viral DNA fragments (Approx. 500 bp long) were amplified by PCR with the primer pair PA/PB, cloned and sequenced. Alignment of the 500 bp sequences showed that the 28 samples could be divided into two types. Type 1 isolates share high sequence identity (98-99%) with the previously reported TYLCV isolates, while Type 2 isolates share high sequence identity (97-98%) with ToLCTWV isolates. The complete DNA sequences of three TYLCV and two ToLCTWV randomly selected isolates share high (98-99%) sequence identity with their respective strains. The putative satellite DNAβmolecule was not detected in any of the samples either by PCR or Southern blot hybridization analysis.To complete Koch's postulates and resolve the etiology of ToLCTWV, infectious clones carrying a 1.4 mer hemidimer of ToLCTWV isolate ZJ16 DNA fragment were constructed and used for agro-inoculation of plants. The clone was infectious in Nicotiana benthamiana and tomato seedlings, initiating mild leaf yellowing and blistering symptoms at 14 days post inoculation (dpi) and symptoms are being monitored.