Quantitative Distribution of Candidatus Liberibacter asiaticus in Citrus Plants and Fruits Infected by Citrus Huanglongbing

Authors: Li, Wenbin; LEVY, LAURENE - USDA APHIS BELTSVILLE MD; Hartung, John

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/29/2008
Publication Date: 2/1/2009
Citation: Li, W.N., Levy, L., Hartung, J.S. 2009. Quantitative Distribution of Candidatus Liberibacter asiaticus in Citrus Plants and Fruits Infected by Citrus Huanglongbing. Phytopathology. 99:139-144.

Interpretive Summary: Citrus greening, also known as huanglongbing, is the most important disease affecting citrus in the world today. The disease is lethal, the pathogen is spread rapidly by an insect vector, there is no effective treatment for diseased trees, and the disease has recently become established in both Brazil and Florida where 90% of the world’s orange juice is produced. The bacterium that causes the disease has not been cultured, and as a result very little is known about the important details of the biology of the pathogen and host interaction. We previously developed a sensitive and quantitative assay that can detect the pathogen in infected plant tissue. In this study we used our new quantitative test to determine the amounts of bacteria present in infected tissues of six citrus varieties. Our results show, for the first time, that the pathogen is completely systemic in infected trees, reaching substantial populations in the roots, bark, branches, leaves and fruit. The amount of bacteria present in different samples however can vary by as much as 10,000-fold within tissues of individual trees. Our results will assist in the development of more effective and efficient sampling methods to use in conjunction with our assay in disease surveys. The results also provide important baseline information about the levels of the pathogen present within infected trees, and will therefore be of interest to both regulatory and research scientists involved in the fight against citrus greening disease.

Technical Abstract: Citrus huanglongbing (HLB), or greening disease, is caused by any of three non culturable Gram-negative bacteria belonging to Candidatus Liberibacter spp. The pathogens are transmitted by citrus psyllids to all commercial varieties of citrus. The diseases are lethal and have recently become widespread in both São Paulo, Brazil and Florida, USA, the locations of the largest citrus industries in the world. Asiatic HLB, the form of the disease found in Florida, is caused by Ca. Liberibacter asiaticus (Ca. Las) and is the subject of this report. The non culturable nature of the pathogen has hampered research and little is known about the distribution of the pathogen in infected trees. In this study, we have used a quantitative PCR assay to systematically quantify the distribution of the pathogen in all tissues of six species of citrus either identified in the course of disease survey efforts in Florida or propagated in a greenhouse in Beltsville, MD. Populations of Ca. Las in leaf midribs, leaf blades and bark samples varied by a factor of 1000 among samples prepared from the six species tested and by a factor of 100 between two sweet orange trees tested. In naturally infected trees, above ground portions of the tree averaged 1010 Ca. Las genomes per g of tissue. Similar levels of Ca. Las were observed in some, but not all root samples from the same plants. In samples taken from greenhouse inoculated trees, population levels of the pathogen varied systematically from 104 genomes/g at the graft inoculation site to 1010 genomes/g in some leaf petioles. Root samples from these trees also contained Ca. Las at 107 genomes/g. Ca. Las was also readily detected and quantified in symptomatic fruit tissues. The highest levels of the pathogen in fruit tissues were found in the locular membranes and septa (108 genomes/g), with 100 fold lower levels of the pathogen in the meso and pericarp of such fruit. Our results demonstrate both the ubiquitous presence of Ca. Las in symptomatic citrus trees as well as great variation between individual trees and among samples of different tissues from the same trees. Our methods will be useful in both the management and scientific study of citrus Huanglongbing or greening disease.