Serological and molecular diagnosis and management of the yellowing disease complex of sweet orange

Gazipur

The yellowing disease complex followed by decline is a severe constrained to citrus cultivation in Bangladesh. Yellowing symptoms including vein corking, chlorosis which are often confusing with the micronutrient deficiency. Identification and the genetic variability among causal agent/s and its insects vector, epidemiology, field diagnostics and management will be the major focus of the project.

An extensive survey program will be conducted at affected regions to know the existing disease incidence. Immuno-nucleobased diagnosis (DAC-ELISA; PCR and/or RT-PCR) and indexing using pathogenicity test will be carried out to diagnosis for the yellowing disease complex of citrus in Bangladesh. For field diagnosis using immunoassay we will generate in house antibody against Bangladeshi isolates. The insect vector/s will be identified by sequencing of COI. For management of the disease, cross protection and vector control will be carried out.

Yellowing disease status was assessed at seven districts in different citrus species. A total of 780 samples were collected from different locations in different citrus species and characterized through considering phenotypic, molecular diagnosis and biological indexing. The characteristics symptoms induced by CTV are yellowing, stem pitting and vein clearing also observed in Citrus reticulata, Citrus sinensis, Citrus aurantifolia. Citrus assamensis; Citrus macroptera ; Citrus limon confirmed by DAC-ELISA followed by RT-PCR. Among the 780 samples tested by DAC-ELISA, 176 samples found positive against in-house antibody raised against CTV. The molecular characterization based on the partial CP gene sequencing and genotype specific RT-PCR of the Bangladeshi CTV isolates revealed that closely related to the Resistance Breaking (RB) genotypes. Symptomatic 190 samples of Citrus Sinensis tested by PCR and found positive 87 samples. The biological indexing of CTV in Rangpur lime and HLB in Rangpur lime; Trifoliate orange and Pamelo has been confirmed by grafting. Our immune-nuclobased diagnosis revealed that HLB in more frequent in sweet orange than CTV. The Asian citrus psyllid (ACP) has been identified by amplification of species specific primer and insect transmission test of HLB in trifoliate orange. Our pathogenicity test (Graft transmission and insect transmission test) suggested that natural mild genotypes of CTV might be a suitable option for cross protection against virulent strain of CTV as well as HLB.

We developed sensitive protocol for diagnosis of CTV and CLas and their insect vector that are responsible for yellowing disease complex of sweet orange in Bangladesh. For field diagnosis using immunoassay later flow for large number of samples an in-house polyclonal antibody production is in progress. For management of yellowing disease complex of sweet orange, the searching of resistant or tolerant root stock, mild CTV strain and vector control are in progress.

By proper implementation of the project a suitable disease management technology will be developed and the yield of sweet orange will be increased and the socio-economic condition of the farmers will be uplifted.

After completion of the project, the citrus researchers (academician) will get the information for further advanced research for host-pathogens-vector interactions for development of yellowing disease resistance citrus variety and highly effective environmental and eco-friendly disease management technology that will reduce enormous yield loss of this sweet orange. Major objectives of this project to develop ImmunoStrip for field diagnosis and cross protection (either natural mild strain or genetic manipulation of the virulent strain) a kind of immunization against insect transmissible diseases which might be translatable strategies to other crops and diseases.