M. J. Davis, Huiqin Chen, and Maria Peacock. University of Florida, IFAS Citrus Research and Education Center

Transcription

1 M. J. Davis, Huiqin Chen, and Maria Peacock University of Florida, IFAS Citrus Research and Education Center

2 aflèche, D., and Bové, J. M Mycoplasmas dans les agrumes atteints de "greening", de "stubborn" ou de maladies similaires. Fruits d'outre-mer, 25(6): hosh, S. K., Raychaudhuri, S. P., Varma, A., and Nariani, T. K Isolation and culture of mycoplasma associated with greening disease. Current Science 40: arnett, H. M Isolation of the greening organism. Citrus and Subtrop. Fruit J. 611:4-6. avis, M. J., Mondal, S. N., Chen, H. Q., Rogers, M. E., and Brlansky, R.H Cocultivation of Candidatus Liberibacter asiaticus with actinobacteria from citrus with huanglongbing. Plant Dis. 92: echler, A., Schuenzel, E. L., Cooke, P., Donnua, S., Thaveechai, N., P k E S A L S h d W L D V D ds h d

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4 Sequences producing significant alignments: Score (Bits) gi gb U RBU11014 Rickettsia bellii 16S rrna gene, 2789 gi gb L RIRRGDF Rickettsia bellii 16S ribosomal RN 2763 gi gb U RPU42084 Rickettsia PAR 16S ribosomal RNA 2759 gi gb AY Rickettsia endosymbiont of Tetrany gi gb CP Rickettsia felis URRWXCal2, complete 2718 gi gb AE Rickettsia conorii str. Malish 7, gi gb U RRU11021 Rickettsia rickettsii 16S rrna ge 2686 gi gb L RIRRGDE Rickettsia sp. 16S ribosomal RNA ( 2680 gi dbj AB Rickettsia sp. PAR-OY gene for gi gb U U Rickettsia honei 16S ribosomal RNA gi gb L RIRRGDI Rickettsia massiliae 16S ribosomal 2676 gi gb L RIRRGDM Rickettsia massiliae (strain Mt gi gb AF Rickettsia heilongjiangii 16S ribo gi gb AF Rickettsia sp. RpA4 strain RpA4 16S gi gb AF Rickettsia sp. Chad 16S ribosomal RNA 2670 gi gb U RAU17644 Rickettsia australis 16S ribosoma 2670 gi gb L RIRRGDO Rickettsia rhipicephali (strain gi gb U RCU12460 Rickettsia conorii ITT S rr 2666

5 Unidentified bacterium isolated from babaco or mountain papaya (hybrid of Carica stipulata and C. pubescens). It is an alpha proteobacterium based on its 16S rrna gene sequence. Not the papaya bunchy top rickettsia 16S rrna sequences differ PBT bacterium did not grow on media developed for the Babaco bacterium

6 Score E Sequences producing significant alignments: (Bits) gi gb AY Candidatus Liberibacter americanus gi gb AY Candidatus Liberibacter sp. LSg gi gb AY Uncultured bacterium clone M gi gb U Afipia genosp. 6 16S ribosomal R 1417 gi gb AF Uncultured bacterium clone RB7C gi emb AJ Methylocapsa acidiphila gi gb DQ Candidatus Liberibacter asiaticus gi gb DQ Candidatus Liberibacter asiaticus gi dbj AB Liberibacter sp. gene for 16S rrna, p 1348 gi gb DQ Candidatus Liberibacter asiaticus gi gb AY Candidatus Liberibacter asiaticus gi gb L Liberibacter africanus 16S ribo 1330 gi gb AY Candidatus Liberibacter asiaticus gi dbj AB Liberibacter endosymbiont of Diap gi gb L Liberibacter asiaticus 16S ribo 1300 gi gb AY Sinorhizobium sp. ORS S rib

7 The Babaco bacterium appears to be the most closely related species to the Liberibacter spp. that can be grown in axenic culture at the present time. It follows that the Babaco bacterium might be used in a model system for the development of media and procedures for culturing Liberibacter spp.

8 Brain Heart Infusion ACES KOH α-keto Glutaric Acid Mannitol Ferric Pyrophosplate Arabinose Folic Acid DS2 Insect Cell Culture Medium Fetal Bovine Serum per liter 12 g 10 g 3.76 g 2 g 5 g 0.25 mg 1 g 100 mg 250 ml 150 ml

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10 >98% identity of 16S rrna gene with that of Propionibacterium acnes Aerotolerant anaerobe Detection by P. acnes-specific PCR Healthy and diseased citrus - positive Infectious and non-infectious psyllids - positive

11 Liberibacter was growing in association with an actinobacterium. Single colony isolations retained mixed culture at a high frequency. Suggested that both bacteria benefit from the presence of the other. Growth of the actinobacterium overshadowed that of Liberibacter. PCR detection of Liberibacter was often weak and inconsistent

12 Alternative Approaches to Co- Culturing Liberibacter Co-culture with Acanthamoeba polyphaga Attempts were not successful Co-culture with insect cells Attempts using a Drosophia melanogaster S2 cell line have not been successful Attempts using Diaphorina citri cell lines are in progress Co-culture with organs of Diaphorina citri Attempts using alimentery canal expants are underway

13 Biofilm of bacteria on external surface of Diaphoria citri midgut.

14 Fluorescent stained bacteria washed from Diaphorina citri alimentary canal

15 DAPI stained alimentary canal FISH stained alimentary canal Flourescent in situ Hybridization (FISH) probe based on sequence of the OI1 16s rrna primer labeled with CY3

16 FISH stained Las cells washed from alimentary canal surface

17 Las biofilm on exterior surface of psyllid midgut

18 Las biofilm on exterior surface of psyllid midgut

19 Alimentary canal explants are incubated on media designed to support growth of Ca. L. asiaticus Growth of Ca. L. asiaticus is monitored microscopically Goal is to wean Ca. L. asiaticus from explant and grow it axenically in improved media

20 Provides a high inoculum dose of Ca. L. asiaticus Evidence of growth can be monitored microscopically and media modified accordingly

21 Ca. L. asiaticus found in approximately only 20-30% of psyllids Original concentration of Ca. L. asiaticus is imposable to predict Non-target microorganisms are often present and overrun culture

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