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Title: Development of a qPCR technique to screen for resistance to Asiatic citrus canker

Author
item McCollum, Thomas
item Stange, Richard
item Albrecht, Ute
item Bowman, Kim
item Niedz, Randall
item Stover, Eddie

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2010
Publication Date: 3/1/2011
Citation: Mccollum, G., Stange, R., Albrecht, U., Bowman, K., Niedz, R., Stover, E. 2011. Development of a qPCR technique to screen for resistance to Asiatic citrus canker. Acta Horticulturae. 892:173-182.

Interpretive Summary: Asiatic citrus canker is a bacterial disease threatening sustainability of the Florida citrus industry. We are working to develop citrus cultivars resistant to citrus canker. Methods for screening large numbers of plants to identify resistant individuals as well as tools to help us understand the interaction between the canker pathogen (Xanthomonas citri) and its citrus host are essential components in the process of developing resistant varieties. We have evaluated whole-plant spray inoculation (WPSI) with the bacterium that causes citrus canker as a method of screening for resistance. WPSI requires minimal effort and numerous hybrids can be evaluated concurrently. Our initial WPSI trial included 68 genotypes of citrus, citrus hybrids and citrus relatives; a wide range of disease expression was observed. We are currently using WPSI to screen numerous citrus trees in our program to identify canker resistant individuals. Although WPSI is an effective screeening method, it requires inoculation of whole plants and takes at least one month after inoculation to obtain results. We have conducted studies to determine if detached shoots can be used rather than whole plants, a method that shows promise for screening without exposing source plants to pathogens. We have developed a method that uses polymerase chain reaction (PCR) for quantification of the canker-causing bacterium in plant tissue; results obtained with PCR are well correlated with those obtained using the more laborious and time consuming plate count method; additionally the PCR method is not affected by the presence of non-target bacteria. PCR evaluations have revealed several aspects regarding the interaction of the bacterium with its citrus host. For example, growth of the bacterium is more rapid in leaves of grapefruit, the commercial citrus type most susceptible to citrus canker, than in kmquat, a citrus type highly resistant to canker. We are continuing to utilize these methods in our efforts to develop citrus varieties resistant to canker.

Technical Abstract: Asiatic citrus canker (Acc) (causal organism Xanthomonas citri subspc. citri (Xcc) is threatening sustainability of the Florida citrus industry. Resistant cultivars, whether developed through conventional breeding or genetic transformation, will be he best solution for dealint with Acc. In Florida, prior to 2005, regulatory constraints made experiments with Xcc very difficult to conduct. Greenhouse experiments with Xcc are now permitted, expanding opportunities for resistance screening. We evaluated whole-plant spray inoculation (WPSI) with Xcc for resistance screening of citrus germplasm. WPSI requires minimal effort and numerous hybrids can be evaluated concurrently. Our initial WPSI trial included 68 genotypes of citrus, citrus hybrids and citrus relatives; a wide range of disease expression was observed. We are currently using WPSI to screen transgenic citrus to determine if non-tissue-specific expression of antimicrobial peptides provides resistance to Xcc. Although WPSI is an effective screening method, it requires inoculation of whole plants and takes at least one month after inoculation to obtain results. Detached shoot inoculation methods show promise for screening without exposing source plants to pathogens. We have developed an RT-PCR method for quantification of Xcc in plant tissue and results are highly correlated with plate count data; RT-PCR is much more efficient than plate counting and is not affected by the presence of non-target bacteria. RT-PCR evaluations have revealed several aspects of the citrus/Xcc pathosystem. For example Xcc growth is more rapid in leaves of grapefruit than in kumquat, suggesting that kumquat resistance to Xcc may occur prior to hypersensitivity. A disadvantage of the RT-PCR method is that dead cells cannot be distinguished from live cells. We are currently evaluating the use of ethidium monoazide as a means for overcoming this limitation.