Richard F. Lee

Citrus tristeza virus and its aphid vector Toxoptera citricida: threats to citrus production in the Caribbean and Central and North America.

The CTV-BrCA complex represents a real threat to citrus production in the countries of the Caribbean Basin and Central and North America. The promptness in recognizing the situation by scientists, government officials, and citrus growers of this geographical area will pay dividends by delaying the occurrence of CTV epidemics. Immediate strategies should include preventing any further introduction of any severe CTV isolate into the region, and preventing any further dissemination of the virus via infected budwood. Continued education is essential to make prevention work as well and as long as possible. Eradication and suppression should be considered where the number of infected trees is small and they are restricted to well-defined locations. Largescale suppression should be guided by analysis of cost-benefit ratios and accurate survey information. Long-range movement of plant materials infested with the BrCA should be carefully avoided. Field evaluation of alternate rootstocks is essential in all areas where sour orange is threatened by CTV. Intermediate strategies include deployment of MSCP as other options fail, especially in the context of an integrated pest management scheme (61). Long-range strategies include development of immune scion varieties through genetic engineering and breeding. Several areas that need additional research have been identified by scientists and citrus growers at the various international workshops held in Costa Rica in 1991 and in Venezuela in 1992 (27,29). These areas are summarized as follows: 1) development of rapid methods to differentiate among mild, DI, and SP strains of CTV; 2) development of virus resistance in commercially desirable cultivars by either biotechnology methods, including somatic hybridization, production of transgenic plants, and genetic engineering approaches, or conventional breeding to transfer the CTV immunity present in some citrus relatives into acceptable cultivars; 3) gathering of data on distribution and spread of CTV, as affected by strains of CTV, vector type, and dynamics, hosts, and location effects; 4) developing a better understanding of virus-aphid relationships to determine how CTV is affected by aphid species, virus strain, and hosts; 5) developing biological control methods for the BrCA as part of an integrated pest management system to reduce spread of CTV; and 6) developing improved methods of MSCP

Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes

Abstract. Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3′ end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Graft-transmissible diseases of citrus

Tristeza, meaning sadness in Spanish or Portuguese, was the name first given to a decline of trees on sour orange rootstock in South America. Tristeza was later shown to be caused by Citrus tristeza virus (CTV), a member of the closterovirus group within the Closteroviridae. Seedling yellows disease is a synonym used for tristeza. CTV is probably the most destructive citrus virus in the world. There have been documented losses of about 30 million trees on sour orange rootstock in Brazil and Argentina in the 1940s and 1950s, of 6.6 million in Venezuela in the 1980, and an estimated 10 million trees in Florida and other Caribbean Basin countries, all these losses were due to the introduction of Toxoptera citricida, commonly called the brown citrus aphid (BrCA), which is the most efficient vector of CTV (Rocha-Pena et al. 1995). The BrCA has been increasing its geographical area throughout the Caribbean Basin since 1989 and was first found in Florida in November 1995 (Halbert, 1998). Even without the BrCA, CTV has killed about 10 million trees Spain (Cambra et al. 1988) and millions more which are not well documented in Israel, Florida, California and in other areas (Bar-Joseph et al. 1989).

Comparative sequence analysis of the coat proteins of biologically distinct citrus tristeza closterovirus isolates

The genome of citrus tristeza closterovirus (CTV) consists of a 20 kb single-stranded RNA encapsidated in a 2000 nm long, flexuous particle. Double-stranded (replicative form) RNAs purified from CTV-infected tissue were used to prepare complementary DNA libraries that involved initial first-strand cDNA synthesis followed by selective amplification of the coat protein gene. CTV-specific antisera were used to select clones expressing the coat protein. The coat protein genes of seven Florida and four exotic isolates that differ in their biological properties were cloned and sequenced. The gene is 669 base pairs long and encodes a 223 amino acid protein. There was a greater than 80% homology at both nucleotide and amino acid levels among all the isolates examined. However, comparisons showed that each isolate was found to have several unique amino acid residues. Several blocks of amino acid residues were conserved among all the isolates. A cluster dendrogram showed greater similarities among groups of mild and severe Florida isolates that differed significantly from those of the geographically distinct, exotic CTV isolates.

Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease

Citrus tristeza virus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region.

Transgenic resistance to Citrus tristeza virus in grapefruit.

Grapefruit (Citrus paradisi) transgenic plants transformed with a variety of constructs derived from the Citrus tristeza virus (CTV) genome were tested for their resistance to the virus. Most transgenic lines were susceptible (27 lines), a few were partially resistant (6 lines) and only one line, transformed with the 3′ end of CTV was resistant. Transgene expression levels and siRNA accumulation were determined to identify whether the resistance observed was RNA-mediated. The responses were varied. At least one resistant plant from a partially resistant line showed no steady-state transgene mRNA, siRNA accumulation and no viral RNA, implicating posttranscriptional gene silencing (PTGS) as the mechanism of resistance. The most resistant line showed no transgene mRNA accumulation and promoter methylation of cytosines in all contexts, the hallmark of RNA-directed DNA methylation and transcriptional gene silencing (TGS). The variety of responses, even among clonally propagated plants, is unexplained but is not unique to citrus. The genetics of CTV, host response or other factors may be responsible for this variability.

Effects of Insecticides on Behavior of Adult Bactericera cockerelli (Hemiptera: Triozidae) and Transmission of Candidatus Liberibacter psyllaurous

ABSTRACT The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera:Triozidae), is a serious pest of potatoes (Solanum tuberosum L.) that can cause yield loss by direct feeding on crop plants and by vectoring a bacterial pathogen, Candidatus Liberibacer psyllaurous. Current pest management practices rely on the use of insecticides to control the potato psyllid to lower disease incidences and increase yields. Although many studies have focused on the mortality that insecticides can cause on potato psyllid populations, little is known regarding the behavioral responses of the potato psyllid to insecticides or whether insecticides can decrease pathogen transmission. Thus, the objectives of this study were to determine the effects of insecticides on adult potato psyllid behaviors, the residual effects of insecticides on potato psyllid behaviors over time, and effects of these insecticides on Ca. L. psyllaurous transmission. Insecticides tested included imidacloprid, kaolin particle film, horticultural spray oil, abamectin, and pymetrozine. All insecticides significantly reduced probing durations and increased the amount of time adult psyllids spent off the leaflets, suggesting that these chemicals may be deterrents to feeding as well as repellents. Nonfeeding behaviors such as tasting, resting, and cleaning showed variable relationships with the different insecticide treatments over time. The insecticides imidacloprid and abamectin significantly lowered transmission of Ca. L. psyllaurous compared with untreated controls. The implications of our results for the selection of insecticides useful for an integrated pest management program for potato psyllid control are discussed.

Huanglongbing: An overview of a complex pathosystem ravaging the world's citrus

Citrus huanglongbing (HLB) has become a major disease and limiting factor of production in citrus areas that have become infected. The destruction to the affected citrus industries has resulted in a tremendous increase to support research that in return has resulted in significant information on both applied and basic knowledge concerning this important disease to the global citrus industry. Recent research indicates the relationship between citrus and the causal agent of HLB is shaped by multiple elements, in which host defense responses may also play an important role. This review is intended to provide an overview of the importance of HLB to a wider audience of plant biologists. Recent advances on host-pathogen interactions, population genetics and vectoring of the causal agent are discussed.

Distribution and Characterization of Citrus tristeza virus in South Florida Following Establishment of Toxoptera citricida

The incidence of Citrus tristeza virus (CTV) was found to increase significantly in southern Florida within 2 years after the establishment of its most efficient vector, Toxoptera citricida (Kirkaldy). Increased incidence of both mild and severe strains was documented, with the incidence of severe strains increasing more than mild strains. Molecular probes capable of differentiating mild, quick decline and various types of stem-pitting strains demonstrated that trees often were infected with more than one strain of CTV, with trees containing up to five different strains. Some CTV strains detected in the southeast urban corridor of Florida and in commercial groves in southwest Florida were found to react with probes specific for stem-pitting strains known from elsewhere in the world. The implications of the presence of these CTV strains in Florida and their possible presence in citrus budwood scion trees are discussed.

Serological techniques for detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the most economically important virus disease of citrus. In the last ten years, remarkable progress has been achieved in the development and improvement of new serological methods for CTV detection so that serology has become a dependable tool for many research, extension and regulatory purposes worldwide. CTV-specific polyclonal antisera and monoclonal antibodies have been developed in different research laboratories and used extensively in a wide range of different studies. This review describes the diverse serological methods developed for CTV detection and analyzes the advantages, disadvantages, relative sensitivity, applications, and present status of each method.