Graft Transmission and Cultivar Reaction of Citrus to ‘Candidatus Liberibacter americanus’

‘Candidatus Liberibacter’ is a genus of gram-negative bacteria composed of three species–‘Ca. Liberibacter asiaticus’, ‘Ca. Liberibacter africanus’ and ‘Ca. Liberibacter americanus’ (5,13). They are transmitted in the field by psyllids (2,9,17), colonize the phloem of the infected plant (7), and cause an important citrus disease named huanglongbing (HLB). The disease is also known as greening in South Africa, mottle leaf in the Philippines, dieback in India, and vein phloem degeneration in Indonesia (1). While the behavior of the first two species is well known and has been studied in several countries, the behavior of ‘Ca. Liberibacter americanus’ has received little attention since it was first described in 2004 from HLB-affected trees in Brazilian citrus orchards (13). ‘Ca. Liberibacter asiaticus’ has also been found in Brazil (3). Independent of the Liberibacter species, HLB management has been achieved through the elimination of affected trees to reduce inoculum, chemical control of vector populations, and the use of healthy young trees for planting. The adoption of these practices is expensive because of the need to have all trees surveyed by welltrained inspectors and the cost of psyllid control (14). To improve HLB management, a better understanding of the complex interaction that exists among host, pathogen, and vector is necessary. One obstacle for this understanding is that no one has been able to isolate and culture the pathogen on artificial media (1). Therefore, all research that involves pathogen inoculation and transmission require the use of infected plants as direct sources of inoculum. Liberibacter can be transmitted to citrus plants with the insect vector (Diaphorina citri or Trioza erytreae), dodder (Cuscuta sp.), or grafting (2,5,9,17; Hsiang, 1956, cited by Bové [1]). The choice of method will depend on the research purpose. Psyllid transmission is the natural process that occurs in the field, but its experimental use is complicated because of the low efficiency of pathogen transmission and the need to use a large number of insects. Dodder and shoot grafting are artificial, but because of the high efficiency of pathogen transmission, these methods have been used to investigate some aspects of the disease including host reaction to pathogen infection, host range, and pathogen aggressiveness. Since the first report of the presence of HLB in Brazil and the finding that a new species of Liberibacter was involved, several studies using graft inoculation have been carried out in our greenhouses. Here we present the results of three experiments: i) to determine the best affected twig tissue as a source of inoculum; ii) to evaluate the disease reaction in the main commercial Brazilian citrus cultivars and species used for fruit production or rootstocks; and iii) to determine the efficiency of pathogen propagation from individual buds through the usual process of producing young nursery trees for commercial planting.