LIBRO-CONGRESO-CITRUS

S12
have not been completely fulfilled. Pigeon pea witches’-broom phytoplasma and Aster yellow phytoplasma
were detected from citrus samples with characteristic HLB symptoms from Brazil and China, respectively.
These two phytoplasmas might also be associated with HLB. In order to elucidate the “Ca. Liberibacter” spp.
and the phytoplasma association with HLB in the P. R. China, 472 samples were collected from Guangdong,
Guangxi, Yunnan, Guizhou, Sichuan, Chongqing, Jiangxi, Fujian, and Zhejiang Provinces from 2008 to 2011. The
symptoms included leaf chlorosis, blotchy mottle, midrib yellowing, zinc nutritional deficiency, iron nutritional
deficiency and fruit colour inversion. Using primer set OI1/OI2c, PCR products digested with Xba were used
for identifying “Ca. Liberibacter asiaticus” and “Ca. Liberibacter africanus”. Primer set GB1/GB3 was used for
the detection of “Ca. Liberibacter americanus”. Nested PCR with phytoplasma-universal primer sets P1/P7
with fU5/rU3 and R16mF2/R16R1 with R16mF2n/R16R2 were used for the detection of phytoplasma. Of the
472 samples, 261 were positive for “Ca. Liberibacter asiaticus”. Samples with symptoms of blotchy mottle,
zinc nutritional deficiency and fruit colour inversion had higher detection rate than those one with other
symptoms. However, all samples were negative for “Ca. Liberibacter africanus”, “Ca. Liberibacter americanus”
and phytoplasma. Our results suggested that “Ca. Liberibacter asiaticus” was highly associated with HLB in
China, and phytoplasmas might not be closely related with the etiology of HLB.
S12P04
Rapid on site detection of the HLB/Citrus Greening causal agent “Candidatus Liberibacter
asiaticus” by AmplifyRP, a novel rapid isothermal nucleic acid amplification platform.
Russel P. 1 , Amato M. A. 2 , and Bohannon R. 1
1 Agdia Inc., United States; and 2 Agdia Biofords, France. marcos.amato@biofords.com
Huanglongbing (HLB), also called Citrus Greening, has become the most serious threat to the citrus industry
worldwide. Although the disease first emerged in the southeastern part of Asia, it is now well established all
along the American continent, including Brazil, Florida and California which are the world most important citrus
production areas. In the Asian and American continents the disease is associated with the insect-vectored,
obligate intracellular bacterium “Candidatus Liberibacter asiaticus”. The bacterium resides in the host phloem
cells and it is transmitted by the Asian Citrus Psyllid, Diaphorina citri. Early pathogen detection is the key success
factor to protect free areas from pathogen introduction. Unfortunately, the disease has a long incubation period,
during which pathogen concentration is very low and non-systemically distributed. This makes diagnostic, disease
control and eradication programs extremely difficult. Early detection is not possible by using traditional serological
methods. Agdia has recently developed a new rapid molecular detection technique, AmplifyRP, allowing PCRlevel
sensitivity detection within minutes, in the field. AmplifyRP uses a Recombinase-polymerase methodology
for DNA amplification at a single temperature. In contrast to conventional or real-time PCR, AmplifyRP has no
DNA purification requirements, requires no thermocycling, and results can be read using small and user-friendly
devices. A portable florescence reader or a lateral flow device (similar to Agdia’s ImmunoStrip®) can be used to
visualize results in as little as 30 minutes, compared to several hours for conventional PCR. AmplifyRP eliminates
the need for expensive PCR equipment, a large number of chemicals reagents, and the need for technically
trained staff. Given its characteristics, AmplifyRP is the ideal tool to monitor HLB progression by early detection
of “Ca. Liberibacter asiaticus” in citrus trees or in vector insects.
S12P05
Diversity of “Candidatus Liberibacter asiaticus”, “Candidatus Liberibacter africanus” and
“Candidatus Liberibacter americanus” based on 23S/5S rDNA sequences
Liao H. 1 , Bai X. 2 , Li Y. 3 , Chen C. 4 , Yang L. 5 , Xu N. 6 , Huang H. 6 , and Wang X. 6
1 Horticulture Research Institute of Guangxi Academy of Agricultural Sciences (HRIGAAS), Citrus Research Group, China; 2 Department
of Agriculture of Guangxi (DAG), Citrus Research, China; 3 Guangxi Crop Genetic Improvement and Biotechnology Lab (GCGIBL),
Citrus Research, China; 4 Guangxi Citrus Research Institute (GCRI), Citrus Breeding, China; 5 State Key Laboratory of Subtropical
Agro-Bioresources Conservation and Utilization (SKLSACU), Citrus Research, China; and 6 Horticulture Research Institute of Guangxi
Academy of Agricultural Sciences (HRIGAAS), Citrus Research Group, China. liaohuihong2001@gmail.com
Primers based on the 23S/5S rDNA conserved sequences of “Candidatus Liberibacter asiaticus” (Las),
“Candidatus Liberibacter africanus” (Laf) and “Candidatus Liberibacter americanus” (Lam) were designed to
200 - VALENCIA CONFERENCE CENTER, 18th-23rd NOVEMBER 2012