Cecile J. Robertson

Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus

ABSTRACT Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.

A plant virus evolved by acquiring multiple nonconserved genes to extend its host range

Viruses have evolved as combinations of genes whose products interact with cellular components to produce progeny virus throughout the plants. Some viral genes, particularly those that are involved in replication and assembly, tend to be relatively conserved, whereas other genes that have evolved for interactions with the specific host for movement and to counter host–defense systems tend to be less conserved. Closteroviridae encode 1–5 nonconserved ORFs. Citrus tristeza virus (CTV), a Closterovirus, possesses nonconserved p33, p18, and p13 genes that are expendable for systemic infection of the two laboratory hosts, Citrus macrophylla and Mexican lime. In this study, we show that the extended host range of CTV requires these nonconserved genes. The p33 gene was required to systemically infect sour orange and lemon trees, whereas either the p33 or the p18 gene was sufficient for systemic infection of grapefruit trees and the p33 or the p13 gene was sufficient for systemic infection of calamondin plants. Thus, these three genes are required for systemic infection of the full host range of CTV, but different genes were specific for different hosts. Remarkably, either of two genes was sufficient for infection of some citrus hybrids. These findings suggest that CTV acquired multiple nonconserved genes (p33, p18, and p13) and, as a result, gained the ability to interact with multiple hosts, thus extending its host range during the course of evolution. These results greatly extend the complexity of known virus–plant interactions.

Asymptomatic spread of huanglongbing and implications for disease control

Significance Huanglongbing (HLB) is a vector-transmitted bacterial infection of citrus trees that poses a major threat to the citrus industry in Florida, Texas, and California. Current control strategies that focus on the vector, the Asian citrus psyllid Diaphorina citri, are usually initiated when the trees become symptomatic, anywhere from 10 mo to several years after initial infection. We show, experimentally, that newly infected young leaves can become infectious within 10–15 d after receiving an inoculum of bacteria from an adult psyllid. We then show by microsimulation of the asymptomatic spread of HLB through a grove under different invasion scenarios and control strategies that reduction of up to 75% of adult psyllids and nymphs can enhance citrus production. Huanglongbing (HLB) is a bacterial infection of citrus trees transmitted by the Asian citrus psyllid Diaphorina citri. Mitigation of HLB has focused on spraying of insecticides to reduce the psyllid population and removal of trees when they first show symptoms of the disease. These interventions have been only marginally effective, because symptoms of HLB do not appear on leaves for months to years after initial infection. Limited knowledge about disease spread during the asymptomatic phase is exemplified by the heretofore unknown length of time from initial infection of newly developing cluster of young leaves, called flush, by adult psyllids until the flush become infectious. We present experimental evidence showing that young flush become infectious within 15 d after receiving an inoculum of Candidatus Liberibacter asiaticus (bacteria). Using this critical fact, we specify a microsimulation model of asymptomatic disease spread and intensity in a grove of citrus trees. We apply a range of psyllid introduction scenarios to show that entire groves can become infected with up to 12,000 psyllids per tree in less than 1 y, before most of the trees show any symptoms. We also show that intervention strategies that reduce the psyllid population by 75% during the flushing periods can delay infection of a full grove, and thereby reduce the amount of insecticide used throughout a year. This result implies that psyllid surveillance and control, using a variety of recently available technologies, should be used from the initial detection of invasion and throughout the asymptomatic period.

Differential tropism in roots and shoots infected by Citrus tristeza virus

Virus tropism is a result of interactions between virus, host and vector species, and determines the fate of an infection. In this study, we examined the infection process of Citrus tristeza virus (CTV) in susceptible and resistant species, and found that the tropism of CTV is not simply phloem limited, but tissue specific. In resistant species, virus infection was not prevented, but mostly restricted to the roots. This phenomenon was also observed after partial replacement of genes of one CTV strain from another, despite both parental strains being capable of systemic infection. Finally, the roots remained susceptible in the absence of viral gene products needed for systemic infection of shoots. Our results suggest that all phloem cells within a plant are not equally susceptible and that changes in host or virus may produce a novel tropism: restriction by the host to a location where further virus spread is prevented.

A novel method forCitruspropagation: Seed grafting

Summary We have developed a novel technique for grafting citrus seeds onto citrus rootstock plants that resulted in successful graft-take with normal vascular connections between the emerging seedling stem tissues and the rootstock plant. The method was found to be suitable for producing grafted plants from seeds of six cultivars and hybrids of Citrus and the citrus relative Murraya paniculata, using four common Citrus rootstocks. Plants produced by this method developed normally and were established in the field more rapidly than those produced by the common practice of grafting the rootstocks with budwood derived from seedlings prepared from seed in soil-based media. Seed grafting is expected to find a range of uses in breeding programmes; for example, by reducing the time required for the evaluation of hybrid seedlings, in cases where the female parent is mono-embryonic, for testing for vertical transmission of pathogens, and for screening for pathogen resistance among hybrid and mutagenised seed sources.