LIBRO-CONGRESO-CITRUS

S12O01
New insights into the citrus Huanglongbing complex and potential solutions to this devastating
disease
Duan Y.P., Zhou L.J., Zhang M.Q., Vahling-Armstrong C., Hao G.X., Zou H.S., and Hoffman M.
USDA-ARS-USHRL, USA. Yongping.Duan @ ars.usda.gov
Citrus Huanglongbing (HLB) is a century-old and emerging disease that impedes citrus production worldwide.
Candidatus Liberibacter asiaticus (Las) is the globally prevalent species of HLB bacteria. Here we describe
molecular characterizations of Las, and our newly-developed control methods. From a genomic standpoint,
we revealed that Las has a significantly reduced genome and unique features adapted to its intracellular
lifestyle. For such a small genome, a large fraction, about 1/15, originates from prophages/phages. There are
at least two prophages in the Florida isolates. Frequent recombination and reassortment of these prophages/
phages may contribute to the evolving diversity and plasticity of the bacteria. There are at least 8 different
types of Las populations that exist in different hosts, different geographical origins, and in a single infection.
Furthermore, the different Las populations may be responsible for the titer variations, such as the extremely
low-titer seen with seed-transmitted Las bacteria from citrus and of infected Murraya paniculata. Using
functional genomics, we have revealed several aspects of Las biology that may contribute to its pathogenicity
including a high affinity zinc uptake system (ZnuABC), which allows Las to compete with its host for the limited
zinc supply and an ATP translocase that facilitates parasitization of the host’s energy supply. In addition,
Las encodes two novel autotransporter proteins that target mitochondria and may modulate host energy
biosyntheses. Although no flagellum has yet been observed, Las encodes a functional flagellin that triggers the
citrus basal defense response. In spite of the difficulty with HLB control, our newly-developed thermotherapy
and chemotherapy methods eliminated or suppressed Las bacteria in the- infected plants, providing potential
solutions for this devastating disease.
S12O02
Tissue-print and squash real-time PCR for direct detection of Candidatus Liberibacter spp. in
citrus plants and insect vectors
Bertolini E. 1 , Lopes S. 2 , Felipe R.T.A. 3 , Teresani G. 1 , Mourão F.A.A. 3 , Colomer M. 4 , Bové J.M. 5 , López M.M. 1 , and
Cambra M. 1
1 Instituto Valenciano de Investigaciones Agrarias (IVIA), Protección Vegetal y Biotecnología, Moncada, Valencia, Spain; 2 Fundo de
Defesa da Citricultura (FUNDECITRUS), Plant Pathology, Araraquara, São Paulo, Brazil; 3 Universidade de São Paulo (USP), Produção
Vegetal, Piracicaba, São Paulo, Brazil; 4 Plant Print Diagnostics S.L. (PPD), Technical Department, Guadassuar, Valencia, Spain; and
5 Institut Nationale de la Recherche Agronomique (INRA) and Université Victor Ségalen, Bordeaux, France. mcambra@ivia.es
Huanglongbing (HLB), the most severe disease of citrus, is seriously threatening the industry worldwide. The
accurate detection of the Candidatus Liberibacter species associated with HLB, is essential for control and
management of the disease. Real-time PCR is a very appropriate technique for sensitive and specific detection,
but the need for nucleic acids purification, greatly limits the number of samples that can be processed and
highly increases the time as well as the cost of the analyses. Direct real-time PCR detection of Ca. Liberibacter,
overcoming nucleic acids purification, is successfully achieved by systems of sample preparation such as
tissue-print, spot or squash methods on membranes. New, universal Ca. Liberibacter primers were designed,
patented and used with TaqMan probe for accurate detection of plant and psyllid-vector targets immobilized
on nylon or paper membranes. A complete kit based on this technology was successfully validated in an
international ring-test and diagnostic parameters were estimated. HLB targets were amplified from 35% of
1,359 squashed Diaphorina citri individuals having fed on HLB infected plants in Brazil. All samples (1,013)
collected from citrus trees during extensive surveys in Canary Islands, Spain, where Trioza erytreae is widely
present, tested negative; the bacterium was not detected in citrus or in psyllid species. The developed method
demonstrated high efficiency in detecting the HLB bacterial agents in routine analyses of the many samples
required for prevention and efficient management of the disease.
XII INTERNATIONAL CITRUS CONGRESS 2012 - 193
S12