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Citrus Greening Bacterium Is Now Available in Culture — So What’s Next?

Citrus Industry

By Nabil Killiny
A dynamic research collaboration between several labs at Washington State University (Gang, Beyenal and Omsland labs), University of Arizona (Brown lab) and University of Florida (Killiny lab) recently reported an important step in the long-sought culture of the bacterium [Candidatus Liberibacter asiaticus (CLas)] associated with huanglongbing (HLB). The work, published in the journal Biofilm, describes a new approach (a membrane biofilm reactor) developed for growing the bacterial culture from diseased extracts of Hamlin sweet orange leaves and stems. It also outlines a modified formula of the medium used in the biofilm reactor for growing the bacteria. These modifications included adding vitamins and other essential nutrients, such as salts and trace minerals. So far, the lab-grown bacterial biofilm culture has been sustained and sub-cultured for more than two years in the labs.

Scientists worldwide have thus far been unable to cultivate the bacterium (CLas) associated with citrus greening disease in a pure or sustained culture. Attempts in the United States included co-culturing with Actinobacterium and other organisms, using citrus vein extract and a growth factor, and adding citrus juice to the growth medium. Recently, a Japanese group successfully co-cultured the Ishi-1 strain of CLas with Comamonadaceae, FlavobacteriaceaeMicrobacteriaceae and Pseudomonadaceae in a modified medium. These attempts only resulted in short-term viability and limited growth in vitro. These failed attempts at culturing provided unique insights into the world of CLas.

In the current host-free biofilm culture, obtaining the genome of CLas and determining the chemical composition of psyllid haemolymph (blood) and citrus phloem sap (the two sites where the bacterium multiplies) helped identify key nutrients required for growing the bacterium in a continuous biofilm culture. However, the biofilm contains other bacteria which are believed to support CLas growth.

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