2012 Annual Report
1a.Objectives (from AD-416):
Determine the role of seed-transmitted Las bacteria in HLB development, and the difference between seed transmitted Las and HLB-causing Las populations.
1b.Approach (from AD-416):
Quantitation and typing seed transmitted Las population and revealing variations of Las genome by sequencing analysis.
This is related to inhouse project objectives 1a: Characterize the etiology, molecular biology and genetics of ‘Candidatus Liberibacter asiaticus (Las),’ the bacterium associated with citrus huanglongbing (HLB) and 2a: Develop improved detection methods for GRSV, Las and Xanthomonads on citrus and strawberry.
High percentage of seedlings carried Candidatus Liberibacter asiaticus (Las) bacteria at extreme low titer, and the bacterial titer did not reach the same level as those of naturally huanglongbing (HLB)-infected plants even after more than four years incubation in the seedlings.
We have evaluated hundreds of seedlings from different citrus plants, including typical and atypical HLB-affected sweet orange, sour orange, grapefruit, Pomelo and trifoliate during 2007-2011. Various phenotypes, such as stunting and yellow shoot (>50%), blotchy mottle and vein corky (<1%) on the leaves were observed in the seedlings. However, most, if not all seedlings returned to normal growth after rich fertilization. Two types of seedlings were subjected to analysis: one type was those from source plants with atypical HLB symptom, and the other type was from source plants with typical HLB symptom. Due to the low titer of bacterium in seed transmitted seedlings, an effort for detection of extreme low titer of Las has been developed and carried out by using one set of primers LJ900fr targeting two putative effector genes (hyvI/hyvII) for ultra sensitive quantitative polymerase chain reaction (qPCR) using SYBR® Green, this detection method has been published recently. Using this method, high percentage of seedlings from HLB-infected trees were tested carry extreme low titer of Las bacterium, and after four years, Las-positive seedlings still not developed typical HLB disease. The presence of Las bacterium in phloem cell from Las positive seed transmitted sweet orange plant was indicated under TEM (transmission electron microscope). The results suggest that the seed-transmitted Las bacterial population is weak or nonpathogenic and may different from the HLB-causing population.
Low percentage of the Asian citrus psyllids could acquire Las bacterium from “the Las-positive seedlings” but the seed-transmitted Las remained very low titer in the psyllids.
Las-free adult psyllids were reared on the Las-positive seedlings inside screen cage. New generation of the adult psyllids were collected for PCR assay. A total of 241 psyllids from trifolia and sweet orange were tested for the presence of Las. Only 10.3% percent were considered positive with Ct value less than 31 by qPCR using LJ900fr primer, which were estimated 1000 fold less than those of the psyllids from HLB-infected tree. If these psyllids can transmit the seed-transmitted Las into citrus plants and cause HLB disease remain to be further investigated.
The populations of Las bacteria in seedlings were not the same composition as those naturally HLB-infected citrus plants. However, the trials to get the sequence of Las population were unsuccessful due to extremely low titer of bacteria.
Using the new genome information based on the prophage region, we were able to develop PCR protocols to differentiate the populations of Las bacteria in naturally HLB-infected plants in Florida. Currently there were at least six different populations associated with bacterial titer, different citrus varieties or symptoms. However, only one or combinations of two of these populations were detected from seed transmitted seedlings by nest PCR using primers specific for different populations. It is important to note that there were two populations which were present in HLB-infected plants and seedlings but not in the Las-infected psyllids. All these results imply that seed transmitted Las bacteria do not have all components of the Las populations that cause typical HLB disease and multiply well in the plants. Several trials were performed using large amounts of leaf tissue from seed transmitted plants to concentrate the bacteria to get the sequence of the Las population, but these attempts were unsuccessful due to extremely low titers of bacteria. We are currently studying the relationship between population and symptom as well as titer and the role of the seed-transmitted Las by grafting the seedlings with different Las populations together.