Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 910 - Grapefruit | 1040 - Molecular biology | 50% |
| 212 - Pathogens and Nematodes Affecting Plants | 920 - Orange | 1060 - Biology (whole systems) | 50% |
We have identified seed coats of several citrus varieties as having high levels of 'Ca. Liberibacter asiaticus' DNA and in immature seeds and use microscopy to demonstrate viability of bacteria. We will determine if there are optimal stages of development of the seed for extraction of bacteria by sampling populations of seeds collected from fruit at various stages of development. We will test various mechanical methods to purify intact cells from the seed coats.
This research relates to inhouse project objective: 1a. Characterize the etiology, molecular biology and genetics of �Candidatus Liberibacter asiaticus (Las),� the bacterium associated with citrus huanglongbing (HLB) . We have done initial studies on purifying Liberibacter cells from psyllid nymphs and adult psyllids. Our initial attempts to purify Liberibacter cells from nymphs were not productive. Based on quantitive polymerase chain reaction (qPCR) data, the calculated population of Liberibacter cells in a representative portion of the nymphs was much lower than for an equivalent number of adults. Fractionation of Percoll gradients loaded with extracts from nymphs and analysis of the Liberibacter content of the fractions indicated lower than desired recovery of Liberibacter cells from nymphs. Our initial results using adult psyllids were more promising. A sampling of the adults showed a much higher level of Liberibacter than in the nymphs. The Liberibacter numbers from Percoll gradient fractions indicated that Liberibacter cells purified from adult psyllids band much higher up in the gradient than when extractions are made from plant tissue. Fluorescence In Situ Hybridization (FISH) microscopy performed on these fractions indicate that Liberibacter cells are embedded in a matrix which likely is remnant insect tissue. This suggests that the current buffer extraction does not effectively dislodge Liberibacter cells from insect membranes. We will continue working with adult psyllids and will modify the extraction buffers to include different non-ionic detergents as a means of dislodging bacteria from insect tissues and increasing the recovery of "free" bacterial cells. If the detergents act as desired, we expect to see a greater banding of Liberibacter cells further down in the Percoll gradient, indicating an increased number of "free" bacterial cells.