European Journal of Plant Pathology

, Volume 146, Issue 2, pp 349–368 | Cite as

First occurrence of Diaphorina citri in East Africa, characterization of the Ca. Liberibacter species causing huanglongbing (HLB) in Tanzania, and potential further spread of D. citri and HLB in Africa and Europe

  • Mpoki M. ShimwelaEmail author
  • Hossein A. Narouei-Khandan
  • Susan E. Halbert
  • Manjunath L. Keremane
  • Gerald V. Minsavage
  • Sujan Timilsina
  • Deogracious Protas Massawe
  • Jeffrey B. Jones
  • Ariena H. C. van BruggenEmail author


Citrus surveys were conducted at high (>700 m), medium (300-600 m) and low (<200 m) altitudes in Tanzania in 2014/15. Adults and nymphs of Trioza erytreae (del Guercio) were abundant in the highlands and less abundant at medium altitudes. Unexpectedly, adults and nymphs of Diaphorina citri Kuwayama, the Asian citrus psyllid, were found and collected at medium altitudes, around Morogoro. No psyllids were observed at low altitudes. Severe huanglongbing symptoms and tree decline were evident at high altitudes, while mild and few symptoms were observed at intermediate and low altitudes, respectively. DNA was extracted from leaf and psyllid samples and subjected to conventional PCR (cPCR) with seven different primer sets and RT qPCR with two primer sets. cPCR bands were sequenced and subjected to phylogenetic analysis. Candidatus Liberibacter africanus (Laf) was detected in highland leaf and T. erytreae samples from high and medium altitudes by all methods. Sequences from leaves and psyllids were similar to those from South Africa. Candidatus Liberibacter asiaticus (Las) was detected by qPCR in medium altitude leaf samples, but cross-reaction with Laf was likely because presence of Las was not confirmed by cPCR and sequencing. Neither Laf nor Las were detected in D. citri samples. This is the first reported occurrence of D. citri in Africa. Predictions were made of the potential distribution of D. citri and Las in Africa and along the Mediterranean coast using the correlative models MAXENT and Multi-Model Framework. Additional surveys at medium and low altitudes and quarantine measures are recommended.


Asian citrus psyllid Citrus greening Emerging pathogen Invasive species Species distribution models Vector 



This work was made possible by the generous support of the American people through the United States Agency for International Development (USAID)-funded Innovative Agricultural Research Initiative project (iAGRI) (Award No. CA-621-A-00-11-00009-00). The opinions expressed herein are those of the author(s) and do not necessarily reflect the views of the U.S. Agency for International Development or the United States Government. We are grateful to the Esther B. O’Keeffe Foundation for contributing to this research. We thank Prof. Dr. T. J. Msogoya, Head of Department of Crop Science and Production at Sokoine University of Agriculture (SUA) for allowing us to use molecular laboratory facilities. We also thank Dr. Fen Beed, Dr. George Mahuku and Haruni Murith at the International Institute of Tropical Agriculture (IITA) in Dar es Salaam for their support. We thank Dr. Kevin A. Williams, Florida Department of Agriculture and Consumer Services, FDACS/DPI, for examining the parasites recovered from Diaphorina citri nymphs. We thank Debra Jones, FDACS/DPI, and Prof. Dr. Gerhard Pietersen, University of Pretoria, South Africa, for positive controls for Las and Laf, respectively. We thank Dr. John S. Hernandez-Nopsa and Dr. Marina Ascunce, University of Florida, for reviewing the manuscript. This is Entomology Contribution No. 1290, Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Bureau of Entomology, Nematology, and Plant Pathology.

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  1. Aubert, B. (1987). Trioza erytreae del Guercio and Diaphorina citri Kuwayama (Homoptera: Psylloidea), the two vectors of citrus greening disease: Biological aspects and possible control strategies. Fruits, 42, 149–162.Google Scholar
  2. Bassanezi, R. B., Belasque Jr., J., & Montesino, L. H. (2013a). Frequency of symptomatic trees removal in small citrus blocks on citrus huanglongbing epidemics. Crop Protection, 52, 72–77.CrossRefGoogle Scholar
  3. Bassanezi, R. B., Montesino, L. H., Gimenes-Fernandes, N., Yamamoto, P. T., Gottwald, T. R., Amorim, L., & Bergamin Filho, A. (2013b). Efficacy of area-wide inoculum reduction and vector control on temporal progress of huanglongbing in young sweet orange plantings. Plant Disease., 97, 789–796.CrossRefGoogle Scholar
  4. Belasque Jr., J., Bassanezi, R. B., Yamamoto, P. T., Ayres, A. J., Tachibana, A., Violante, A. R., Tank Jr., A., Di Giorgi, F., Tersi, E. E. A., Menezes, G. M., Dragone, J., Jank Jr., R. H., & Bové, J. M. (2010). Lessons from huanglongbing management in São Paulo State, Brazil. Journal of Plant Pathology, 92, 285–302.Google Scholar
  5. Bertolini, E., Felipe, R. T. A., Sauer, A. V., Lopes, S. A., Arilla, A., Vidal, E., Mourão Filho, F. A. A., Nunes, W. M. C., Bové, J. M., López, M. M., & Cambra, M. (2014). Tissue-print and squash real-time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors. Plant Pathology, 63, 1149–1158.CrossRefGoogle Scholar
  6. Boava, L. P., Sagawa, C. H. D., Cristofani-Yaly, M., & Machado, M. A. (2015). Incidence of ‘Candidatus Liberibacter asiaticus’-infected plants among citrandarins as rootstock and scion under field conditions. Phytopathology, 105, 518–524.CrossRefPubMedGoogle Scholar
  7. Bové, J. M. (2006). Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology, 88, 7–37.Google Scholar
  8. Bové, J. M. (2014). Heat-tolerant Asian HLB meets heat-sensitive African HLB in the Arabian Peninsula! Why? Journal of Citrus Pathology, 1(1) [accessed on 12/09/15].
  9. Chiyaka, C., Singer, B. H., Halbert, S. E., Morris, J. G. & van Bruggen, A. H. C. (2012). Modeling huanglongbing transmission within a citrus tree. Proceedings of the National Academy of Sciences, 109, 12213–12218.Google Scholar
  10. Coy, M. R., Hoffmann, M., Kingdom Gibbard, H. N., Kuhns, E. H., Pelz-Stelinski, K. S., & Stelinski, L. L. (2014). Nested-quantitative PCR approach with improved sensitivity for the detection of low titer levels of Candidatus Liberibacter asiaticus in the Asian citrus psyllid, Diaphorina citri Kuwayama. Journal of Microbiological Methods, 102, 15–22.CrossRefPubMedGoogle Scholar
  11. da Graça, J. V., Douhan, G. W., Halbert, S. E., Keremane, M. L., Lee, R. F., Vidalakis, G., & Zhao, H. (2015). Huanglongbing: an overview of a complex pathosystem ravaging the world’s citrus. Journal of Integrative Plant Biology . doi: 10.1111/jipb.12437. PubMedGoogle Scholar
  12. Evers, G., & Grisoni, M. (1991). Present situation of the citrus greening disease in Tanzania and proposal for control strategies. Fruits, 46(2), 171–177.Google Scholar
  13. Farnsworth, D., Grogan, K. A., van Bruggen, A. H. C., & Moss, C. B. (2014). The potential economic cost and response to greening in Florida citrus. Choices, 29, 1–6.Google Scholar
  14. Gutierrez, A. P., & Ponti, L. (2013). Prospective analysis of the geographic distribution and relative abundance of Asian citrus psyllid (Hemiptera: Liviidae) and citrus greening disease in North America and the Mediterranean basin. Florida Entomologist, 96, 1375–1391.CrossRefGoogle Scholar
  15. Halbert, S. E. & Manjunath, K. L. (2004). Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: A literature review and assessment of risk in Florida. Florida Entomologist, 87, 330–353. CrossRefGoogle Scholar
  16. Halbert, S.E., Manjunath, K. L., Ramadugu, C., & Lee, R.F. (2012). Incidence of huanglongbing-associated ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri (Hemiptera: Psyllidae) collected from plants for sale in Florida. Florida Entomologist, 95, 617–624. CrossRefGoogle Scholar
  17. Hocquellet, A., Toorawa, P., Bové, J. M., & Garnier, M. (1999). Detection and identification of the two `Candidatus Liberobacter sp. ' associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the beta operon. Molecular and Cellular Probes, 13, 373–379.CrossRefPubMedGoogle Scholar
  18. Hollis, D. (1984). Afrotropical jumping plant lice of the family Triozidae (Homoptera: Psylloidea). Bulletin of the British Museum (Natural History), 49, 1–102.Google Scholar
  19. Hollis, D. (1987). A new citrus-feeding psyllid from the Comoro Islands, with a review of the Diaphorina anoena species group (Homoptera). Systematic Entomology, 12, 47–61.CrossRefGoogle Scholar
  20. Ichinose, K., Miyazi, K., Matsuhira, K., Yasuda, K., Sadoyama, Y., Tuan, D. H., & Bang, D. V. (2010). Unreliable pesticide control of the vector psyllid Diaphorina citri (Hemiptera: Psyllidae) for the reduction of microorganism disease transmission. Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 45, 466–472.CrossRefGoogle Scholar
  21. Inoue, H., Ohnishi, J., Ito, T., Tomimura, K., Miyata, S., Iwanami, T., & Ashihara, W. (2009). Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage. Annals of Applied Biology, 155, 29–36.CrossRefGoogle Scholar
  22. Jagoueix, S., Bové, J. M., & Garnier, M. (1996). PCR detection of the two ‘Candidatus’ liberobacter species associated with greening disease of citrus. Molecular and Cellular Probes, 10, 43–50.CrossRefPubMedGoogle Scholar
  23. Keremane, M. L., Ramadugu, C., Rodriguez, E., Kubota, R., Shibata, S., Hall, D. G., Roose, M. L., Jenkins, D., & Lee, R. F. (2015). A rapid field detection system for citrus huanglongbing assiciated ‘Candidatus Liberibacter asiaticus’ from the psyllid vector, Diaphorina citri Kuwayama and its implications in disease management. Crop Protection, 68, 41–48.CrossRefGoogle Scholar
  24. Kogenaru, S., Yan, Q., Riera, N., Roper, M. C., Deng, X., Ebert, T. A., Rogers, M., Irey, M. E., Pietersen, G., Rush, C. M., & Wang, N. (2014). Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real-time PCR. BMC Microbiology, 14, 39. [accessed 12/11/2015]
  25. Lallemand, J., Fos, A., & Bové, J. M. (1986). Transmission de la bacteria associé à la forme africaine de la maladie du “greening” par le psylle asiatique Diaphorina citri Kuwayama. Fruits, 41, 341–343.Google Scholar
  26. Lee, J. A., Halbert, S. E., Dawson, W. O., Robertson, C. J., Keesling E., & Singer, B. H. (2015). Asymptomatic spread of huanglongbing and implications for disease control. Proceedings of the National Academy of Sciences, 112, 7605–7610, doi: 10.1073/pnas.1508253112.Google Scholar
  27. Li, W., Levy, L., & Hartung, J. S. (2006). Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. Journal of Microbiological Methods, 66, 104–115.CrossRefPubMedGoogle Scholar
  28. Lin, H., Coletta-Filho, H. D., Han, C. S., Lou, B., Civerolo, E. L., Machado, M. A., & Gupta, G. (2013). Draft genome sequence of “Candidatus Liberibacter americanus” bacterium associated with citrus huanglongbing in Brazil. Genome Announcements, 1(3), e00275–e00213. doi: 10.1128/genomeA.00275-13 .accessed on 12/10/2015CrossRefPubMedPubMedCentralGoogle Scholar
  29. Liu, Y. H., & Tsai, J. H. (2000). Effects of temperature on biology and life table parameters of the Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae). Annals of Applied Biology, 137, 201–206.CrossRefGoogle Scholar
  30. Lopes, S. A., Frare, G. F., Bertolini, E., Cambra, M., Fernandes, N. G., Ayres, A. J., Marin, D. R., & Bové, J. M. (2009). Liberibacters associated with citrus huanglongbing in Brazil: ‘Candidatus Liberibacter asiaticus’ is heat tolerant, ‘Ca. L. americanus’ is heat sensitive. Plant Disease, 93, 257–262.CrossRefGoogle Scholar
  31. Magomere, T.O., Obukosia, S. D., Mutitu, E., Ngichabe, C., Olubayo, F., & Shibairo, S. (2009). Molecular characterization of Candidatus Liberibacter species/strains causing huanglongbing disease of citrus in Kenya. Electronic Journal of Biotechnology, 12, 1–14. CrossRefGoogle Scholar
  32. Manjunath, K. L., Halbert, S. E., Ramadugu, C., Webb, S. E., & Lee, R. F. (2008). Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida. Phytopathology, 98, 387–396.CrossRefPubMedGoogle Scholar
  33. Monzó, C., Urbaneja, A., & Tena, A. (2015). Los psílidos Diaphorina citri y Trioza erytreae como vectores de la enfermedad de cítricos huanglongbing (HLB): reciente detección de T. erytreae en la Península Ibérica. Boletín SEEA, 1, 29–37.Google Scholar
  34. Narouei-Khandan, H. A., Halbert, S. E., Worner, S. P., & VanBruggen, A. H. C. (2015). Global climate suitability of citrus huanglongbing and its vector, the Asian citrus psyllid, using two correlative species distribution modeling approaches, with emphasis on the USA. European Journal of Plant Pathology. doi: 10.1007/s10658-015-0804-7.Google Scholar
  35. Nelson, W. R., Munyanez, J. E., MsCue, K. F., & Bové, J. M. (2013). The pangaean origin of “Candidatus Liberibacterspecies. Journal of Plant Pathology, 95, 455–461.Google Scholar
  36. Orce, I. G., Sendin, L. N., Marano, M. R., Vojnov, A. A., Castagnaro, A. P., & Filippone, M. P. (2015). Novel set of real-time PCR primers for simultaneous detection of Liberibacter species associated with citrus huanglongbing. Scientia Agricola, 72, 252–259.CrossRefGoogle Scholar
  37. Patt, J. M., & Sétamou, M. (2010). Responses of the Asian citrus psyllid to volatiles emitted by the flushing shoots of its Rutaceous host plants. Environmental Entomology, 39, 618–624.CrossRefPubMedGoogle Scholar
  38. Pérez-Otero, R., Mansilla, J. P., & del Estal, P. (2015). Detección de la psila Africana de los cítricos, Trioza erytreae (del Guercio, 1918) (Hemiptera: Psylloidea: Triozidae), en la Península Ibérica. Arquivos Entomoloxicos Galegos, 13, 119–122.Google Scholar
  39. Posada, D. (2008). jModel test: phylogenetic model averaging. Molecular Biology and Evolution, 25, 1253–1256.CrossRefPubMedGoogle Scholar
  40. Razi, M. F., Keremane, M. L., Ramadugu, C., Roose, M., Khan, I. A., & Lee, R. F. (2014). Detection of citrus huanglongbing-associated ‘Candidatus Liberibacter asiaticus’ in citrus and Diaphorina citri in Pakistan, seasonal variability, and implications for disease management. Phytopathology, 104, 257–268.CrossRefPubMedGoogle Scholar
  41. Saponari, M., De Bac, G., Breithaupt, J., Loconsole, G., Yokomi, R. K., & Catalano, L. (2010). First report of ‘Candidatus Liberibacter asiaticus’ associated with huanglongbing in sweet Orange in Ethiopia. Plant Disease, 94, 482.CrossRefGoogle Scholar
  42. Shen, W., Cevallos-Cevallos, J. M., Nunes da Rocha, U., Stansly, P. A., Roberts, P. D., & van Bruggen, A. H. C. (2013a). Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with huanglongbing. European Journal of Plant Pathology, 137, 727–742.CrossRefGoogle Scholar
  43. Shen, W., Halbert, S. E., Dickstein, E., Manjunath, K. L., Shimwela, M. M., & van Bruggen, A. H. C. (2013b). Occurrence and in-grove distribution of citrus huanglongbing in north Central Florida. Journal of Plant Pathology, 95, 361–371.Google Scholar
  44. Sisterson, M. S., & Stenger, D. C. (2013). Roguing with replacement in perennial crops: conditions for successful disease management. Phytopathology, 103, 117–128.CrossRefPubMedGoogle Scholar
  45. Stover, E., & McCollum, G. (2011). Incidence and severity of huanglongbing and Candidatus Liberibacter asiaticus titer among field-infected citrus cultivars. Hortscience, 46, 1344–1348.Google Scholar
  46. Swai, I. S. (1988). Citrus diseases in Tanzania. Tropical horticulture XII, Acta Horticulturae, 218, 329-332.Google Scholar
  47. Swai, I. S., Evers, G., Gumpf, D. J., & Femi Lana, A. (1992). Occurrence of citrus greening disease in Tanzania. Plant Disease, 76, 1185.CrossRefGoogle Scholar
  48. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: molecular Evolutionary Genetics analysis Version 6.0. Molecular Biology and Evolution, 30, 2725–2729.CrossRefPubMedPubMedCentralGoogle Scholar
  49. Teixeira, D. C., Saillard, C., Couture, C., Martins, E. C., Wulff, N. A., Eveillard-Jagoueix, S., Yamamoto, P. T., Ayres, A. J., & Bové, J. M. (2008). Distribution and quantification of Candidatus Liberibacter americanus, agent of huanglongbnig disease of citrus in Sao Paulo State, Brasil, in leaves of an affected sweet orange tree as determined by PCR. Molecular and Cellular Probes, 22, 139–150.CrossRefPubMedGoogle Scholar
  50. Tiwari, S., Mann, R. S., Rogers, M. E., & Stelinski, L. L. (2011). Insecticide resistance in field populations of Asian citrus psyllid in Florida. Pest Management Science, 67, 1258–1268.CrossRefPubMedGoogle Scholar
  51. Toorawa, P. (1998). La maladie du huanglongbing (greening) des agrumes a L’Ile Maurice. In Detection de “Candidatus liberobacter [sic] asiaticum” et “Candidatus liberobacter [sic] africanum: dans les agrumes et les insects vecteurs (186p). Doctoral Thesis: L’University de Bordeaux.Google Scholar
  52. Yang, C. K., & Li, F. S. (1984). Nine new species and a new genus of psyllids from Yunnan. Entomotaxonomia, 6, 4251–4266.Google Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2016

Authors and Affiliations

  • Mpoki M. Shimwela
    • 1
    Email author
  • Hossein A. Narouei-Khandan
    • 1
  • Susan E. Halbert
    • 2
  • Manjunath L. Keremane
    • 3
  • Gerald V. Minsavage
    • 1
  • Sujan Timilsina
    • 1
  • Deogracious Protas Massawe
    • 4
  • Jeffrey B. Jones
    • 1
  • Ariena H. C. van Bruggen
    • 1
    Email author
  1. 1.Department of Plant Pathology and Emerging Pathogens InstituteUniversity of FloridaGainesvilleUSA
  2. 2.Florida Department of Agriculture and Consumer Services, Division of Plant IndustryGainesvilleUSA
  3. 3.United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository for Citrus and DatesRiversideUSA
  4. 4.Sokoine University of AgricultureMorogoroTanzania

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