Citrus Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The causal agent of HLB in Florida is thought to be 'Candidatus Liberibacter asiaticus'. In this work, we examined the responses of 30 different genotypes of citrus to Florida isolates of 'Ca. L. asiaticus' under controlled conditions in the greenhouse or growth room. Although 'Ca. L. asiaticus' was able to multiply in all of the plants, a wide range of responses was observed among different hosts. Based on the symptoms developed and the ability of plants to continue growth, the different genotypes were grouped into four categories: sensitive, which exhibited severe chlorosis on leaves, greatly reduced growth, and eventual death; moderately tolerant, which exhibited some scattered distinct symptoms but little or no growth reduction and no plant death; tolerant, which exhibited very minimal symptoms; and genotypes, which exhibited variable reactions. Interestingly, although 'Ca. L. asiaticus' was unevenly distributed within each particular plant, comparison of titers of the bacterium in different citrus genotypes revealed that most accumulated similar levels of 'Ca. L. asiaticus', demonstrating that there is no strict correlation between bacterial titer and severity of disease. Incubation of infected plants in the growth room with continuous light greatly affected symptoms production by reducing the time before distinctive symptoms developed and significantly increasing severity of chlorosis of leaves of all citrus genotypes. These results provide additional evidence of the correlation between disruption of phloem translocation of carbohydrates during HLB infection and the appearance of chlorotic symptoms in leaves of infected trees. We also examined interaction between 'Ca. L. asiaticus' and Citrus tristeza virus, which usually occurs in trees that become infected with HLB, and found no synergistic effect of the two pathogens. We trust that observations reported here will provide reagents for further examination of the 'Ca. L. asiaticus'-citrus interaction to advance the understanding of how 'Ca. L. asiaticus' causes disease and to develop methods or trees to overcome the disease.

Citrus greening (Huanglongbing [HLB]) is one of the most destructive diseases of citrus worldwide. The causal agent of HLB in Florida is thought to be 'Candidatus Liberibacter asiaticus'. Understanding of the early events in HLB infection is critical for the development of effective measures to control the disease. In this work, we conducted cytopathological studies by following the development of the disease in citrus trees graft inoculated with 'Ca. L. asiaticus'-containing material under greenhouse conditions to examine the correlation between ultrastructural changes and symptom production, with the main objective of characterizing the early events of infection. Based on our observations, one of the first degenerative changes induced upon invasion of the pathogen appears to be swelling of middle lamella between cell walls surrounding sieve elements. This anatomical aberration was often observed in samples from newly growing flushes in inoculated sweet orange and grapefruit trees at the early "presymptomatic" stage of HLB infection. Development of symptoms and their progression correlated with an increasing degree of microscopic aberrations. Remarkably, the ability to observe the bacterium in the infected tissue also correlated with the degree of the disease progression. Large numbers of bacterial cells were found in phloem sieve tubes in tissue samples from presymptomatic young flushes. In contrast, we did not observe the bacteria in highly symptomatic leaf samples, suggesting a possibility that, at more advanced stages of the disease, a major proportion of 'Ca. L. asiaticus' is present in a nonviable state. We trust that observations reported here advance our understanding of how 'Ca. L. asiaticus' causes disease. Furthermore, they may be an important aid in answering a question: when and where within an infected tree the tissue serves as a better inoculum source for acquisition and transmission of the bacterium by its psyllid vector.

Superinfection exclusion, a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or a closely related virus, has been described for various viruses, including important pathogens of humans, animals, and plants. The phenomenon was initially used to test the relatedness of plant viruses. Subsequently, purposeful infection with a mild isolate has been implemented as a protective measure against virus isolates that cause severe disease. In the medical and veterinary fields, superinfection exclusion was found to interfere with repeated applications of virus-based vaccines to individuals with persistent infections and with the introduction of multicomponent vaccines. In spite of its significance, our understanding of this phenomenon is surprisingly incomplete. Recently, it was demonstrated that superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus, occurs only between isolates of the same strain, but not between isolates of different strains of the virus. In this study, I show that superinfection exclusion by CTV requires production of a specific viral protein, the p33 protein. Lack of the functional p33 protein completely eliminated the ability of the virus to exclude superinfection by the same or a closely related virus. Remarkably, the protein appeared to function only in a homology-dependent manner. A cognate protein from a heterologous strain failed to confer the exclusion, suggesting the existence of precise interactions of the p33 protein with other factors involved in this complex phenomenon. Superinfection exclusion or homologous interference is defined as the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus. The phenomenon has been described for various virus-host systems, including viruses that cause serious diseases in humans, animals, and plants (2, 9, 10, 11, 14, 19-21, 23, 28, 29, 31, 35, 37, 38, 55, 61-64, 74-77). From an evolutionary standpoint, superinfection exclusion can be a powerful strategy that determines the genetic structure of the virus population. Superinfection exclusion protects the virus from a related competing secondary virus targeting the cell that has been successfully infected by the primary virus. Besides elimination of competition for host resources, the phenomenon could function as a means to maintain the stability of viral sequences because it prevents replication of two or more viral genomes in the same cell, thus reducing the likelihood of the recombination or reassortment of viral genes, with the latter event of particular importance for the evolution of segmented viruses (18,27). From a practical standpoint, superinfection exclusion can have both positive and negative attributes. Referred to as cross-protection, this phenomenon has been implemented as an agricultural practice in which purposeful infection with a mild isolate was used as a protective measure against isolates of the virus that cause severe disease (reviewed in...

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