1a.Objectives (from AD-416)
1) Attempt transmission from a pure culture of the bacterium isolated from infected orange plants by using micro-injection methodology to artificially infect citrus psyllids, and by using membrane feeding of citrus psyllids on an artificial HLB medium. . 2)Complete Koch's postulates using artificially loaded psyllids.
1b.Approach (from AD-416)
A pure culture of the suspected HLB bacterium will be micro-injected into the abdomen of healthy adult citrus psyllids. Psyllids will be fed on healthy sweet orange seedlings for 14 days, assayed by PCR for the presence of the bacterium, and plants will be observed for 4-6 months. Concurrently, healthy adult psyllids will be placed on stretched parafilm membrane sacks containing an artificial rearing medium and the HLB bacterium. Psyllids will be allowed to feed for 48 hours, placed on sweet orange seedlings for 14 days, assayed by PCR for the presence of the bacterium and plants will be observed for 4-6 months. Symptoms will be recorded, plants assayed by PCR, the bacteria will be isolated, DNA extracted, and sequenced to determine if the same HLB bacterium is present.
The objective of this work is to attempt to complete Koch’s postulates (proof of pathogenicity) of a pure culture of a suspect causal bacterium of Huanglongbing disease. A pure culture of the suspect bacterium will be introduced into the Asian citrus psyllid using micro-injection and/or by feeding the psyllid on membrane sachets containing cultured bacteria. Initial studies indicate the psyllid can inject the bacterium into stretched parafilm membrane sachets and retrieve it from membrane sachets. Procedures for micro-injection are currently being developed. Monitoring was accomplished through e-mail, internet and phone calls.