This project focuses on identifying viruses that infect aphids and asian citrus psyllids (ACPs), and assessing their potential to be developed into virus induced gene silencing (VIGS) vectors whose role is to inhibit the spread of pathogens transmitted by these insects. Aphids and ACPs are vectors of Citrus tristeza virus (CTV) and Candidactus Liberibacter species (the bacterium associated with Huanglongbing [HLB] disease), respectively. The recent arrival of the ACP in Southern California has many far-reaching implications for the future of the California citrus industry, and it behooves us to develop a variety of different measures to prevent its further spread. However, the chemical control of ACPs and aphids is challenging due to their rapid reproductive rate and/or clonal reproduction, their ability to infest different plant hosts, and, in the case of ACPs, their confinement in urban areas. Instead of focusing on controlling the insects, our short-term goal is to take advantage of state-of-the art sequencing and bioinformatics tools, as well as our knowledge in virology and insect manipulation to identify viruses of aphids and ACPs. We will next evaluate their potential to be developed into VIGS vectors by testing their biological activity and determining how well they can be maintained in the insect population. Our long term goal (NOT within the scope of this project) is to use VIGS to disrupt the association between the insects and pathogens, thereby impeding the transmission of the latter. Viruses of plant infesting insects have been identified over the last 10 years; therefore this is not a new concept. However, not all viruses are ideal candidates for development into VIGS vectors. Our goal is to identify those that have simple to work with genetic compositions, and are prevalent but not lethal to the insects so they can be easily manipulated and stably maintained in the insect population. So far, very little has been done to identify such viruses in the insect pests of citrus.
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Identification and characterization of unknown citrus pathogens are essential for developing
protocols to detect and exclude pathogens from citrus germplasm and commercial groves. However, the etiology of many
citrus diseases including more than half of the 25 graft transmissible citrus diseases listed in APS’s “Compendium of Citrus
Diseases”, is unknown. We have recently reported new methods for rapid virus/viroids discovery by deep sequencing and
assembly of total small RNAs from an infected plant or insect (Wu et al., Proc Natl Acad Sci 107:1606). Use of this
approach in the past year has allowed us to discover a new viroid in grapevine and two viroid candidates in citrus that share
no significant sequence homology with known viroids. We propose to process all of the citrus disease isolates available in
CCPP’s disease collection in Riverside as well as samples from citrus diseases or abnormalities where viral pathogens were
originally hypothesized but have not been conclusively demonstrated or ruled out as potential causal agents. We will obtain
complete genome sequences and develop diagnostic tools for, important citrus viruses and viroids identified for disease
survey and CCPP operations. Our method requires no prior enrichment for virus/viroid and thus overcomes major
limitations of current methods.
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