Lower reproductive rates of Asian citrus psyllid (Hemiptera: Psyllidae) on 'Tahiti' acid lime than on 'Valencia' sweet orange

ABSTRACT The 'Tahiti' acid lime and orange trees are hosts of 'Candidatus Liberibacter asiaticus' (CLas), the pathogen associated with the severe Asian form of huanglongbing (HLB), the most devasting citrus disease. They are also hosts of the vector of CLas, the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama. Relatively small numbers of lime trees occur in gardens and small orchards near large commercial 'Valencia' sweet orange orchards in Brazil. Applications of insecticides to suppress populations of ACP on the lime trees are usually nil or less frequent than in the orange orchards. Abundance of the psyllid on lime trees may therefore increase the risk of CLas spreading to the orchards. Because the abundance of the psyllid is influenced by the suitability of the trees as hosts, we compared reproductive potential of the insect on the two hosts in a controlled environment chamber (CEC) and in a greenhouse (GH). Daily temperature and relative humidity averaged 22ºC and 60% inside the CEC and 24°C and 70% inside the GH. Two pairs of adult male and female psyllids were caged for 3 days on new shoots and the fecundity and durations of development and survival of eggs and nymphs evaluated. Overall, acid 'Tahiti' was 3.5 times less suitable to ACP than 'Valencia'. Fecundity and survival of nymphs were 27% and 59% lower, and the life cycle 34% longer on 'Tahiti' than on 'Valencia'. Potential impacts of the results on CLas spread and HLB control are discussed.

Identification of Citrus medica var. sarcodactylis Infected with Citrus Huanglongbing / 中国实验方剂学杂志

Objective::To analyze and compare different samples in many aspects to identify Citrus medica var. sarcodactylis infected with Huanglongbing(HLB) timely and accurately, in order to prevent and control the disease in time. Method::HLB was identified through character analysis, reverse transcription-polymerase chain reaction(RT-PCR), enzyme digestion reaction and Real-time PCR. Result::In terms of characters, there were typically variegated yellow leaves and relatively small fruit, even with deformity but without " red nose fruit" among C. medica var. sarcodactylis infected with HLB. All of these can be used as the basis for the preliminary identification of HLB in the fields. According to the RT-PCR test results and enzyme digestion reaction, when the primer was OI1/OI2c, there was specific band of 1 160 bp, which could be cut into 520 bp and 640 bp by Xba I enzyme. These results were consistent with the characters of other citrus plants infected with HLB. According to the Real-time PCR detection results, C. medica var. sarcodactylis infected with HLB had amplification curves and dissolved peaks, with the melting temperature was 82 ℃ and Ct between 24.6 to 28.2, while the normal plants were not amplified. Conclusion::Character analysis can be used to roughly distinguish HLB in the fields, but with a certain subjectivity. RT-PCR or Real-time PCR can be used to identify C. medica var.sarcodactylis infected with HLB in a timely and accurate manner, and qPCR detection is more sensitive and quantitative. Through the combination of character analysis and molecular identification, C. medica var.sarcodactylis infected with HLB can be determined more timely and accurately.

Occurrence and distribution of Diaphorina citri (Hemiptera: Liviidae) and Tamarixia radiata (Hymenoptera: Eulophidae) in Pará state, Brazil / Ocorrência e distribuição de Diaphorina citri (Hemiptera: Liviidae) e Tamarixia radiata (Hymenoptera: Eulophidae) no estado do Pará

This study presents and discusses the results of a survey for the presence of Diaphorina citri Kuwayama and its natural enemies in Citrus spp. citrus and Murraya sp. myrtle plants in Pará State, Brazil. From 2013 to 2017, observations were made in five mesoregions of the State, and where D. citri was present, branches were examined to quantify eggs, nymphs and mummies with parasitoid emergence holes and to obtain parasitoids. D. citri adults were used to detected the bacterium through nested polymerase chain reaction (NESTED-PCR). Psyllids were found in citrus and myrtle plants. A total of 583 parasitoid specimens of Tamarixia radiata (Waterston) were obtained. The bacterial detection test was negative for Candidatus Liberibacter spp. Psyllids and parasitoids were present in the Lower Amazon, Metropolitan area, Northeastern Pará, Southeastern Pará and Southwestern Pará mesoregions. The presence of D. citri in citrus, with was previously absent in this host, alters the risk situation for Huanglongbing (HLB) in the State. These results will aid in developing preventive or containment measures through phytosanitary protection.(AU)

No presente estudo, são apresentados e discutidos os resultados de levantamento para a presença de Diaphorina citri Kuwayama e inimigos naturais em plantas de citros Citrus spp. e murta Murraya sp. no estado do Pará. No período de 2013 a 2017 foram realizadas observações em cinco mesorregiões do estado. Na presença de D. citri, ramos foram coletados com a quantificação de ovos, ninfas, múmias com orifício de emergência de parasitoide e obtenção de parasitoides. Adultos de D. citri foram analisados para a detecção da bactéria por meio de nested polymerase chain reaction (NESTED-PCR). O psilídeo foi constatado em citros e murta. Foram obtidos 583 exemplares do parasitoide Tamarixia radiata (Waterston). O teste de detecção da bactéria foi negativo, indicando ausência de Candidatus Liberibacter spp. O psilídeo e o parasitoide estão presentes nas mesorregiões do Baixo Amazonas, Metropolitana, Nordeste Paraense, Sudeste Paraense e Sudoeste Paraense. A presença de D. citri em citros, até então ausente nesse hospedeiro, altera a situação de risco do Huanglongbing (HLB) para o Estado. Esses resultados auxiliarão na formulação de medidas preventivas ou de contenção pela defesa fitossanitária.(AU)

Identification and Molecular Characterization of Candidatus Liberibacter asiaticus (Citrus Huanglongbing Disease Pathogenic Agent) and Its Control by Plant Extracts in Bangladesh

Aims: To identify and characterize Citrus Huanglongbing disease causing pathogen Candidatus Liberibacter asiaticus and to evaluate its biological control using medicinal plant extracts. Study Design: The study was designed based on standard laboratory protocol. Place and Duration of Study: Professor Joarder DNA and Chromosome Research Lab, Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh between June 2015 and September 2016. Methodology: Causal pathogen of Citrus Huanglongbing disease was isolated from infected leaves. Different types of biochemical and morphological characterizations of Candidatus Liberibacter asiaticus were done. 16S rDNA primers (27F and 1391R) were used to amplify genomic DNA of Candidatus Liberibacter asiaticus. Sequencing of 16S rDNA sequence of Candidatus Liberibacter asiaticus were performed. Sensitivity pattern of Candidatus Liberibacter asiaticus against several standard antibiotics were done. Antimicrobial activity test was observed using two solvents extracts of four medicinal plants by disc diffusion method in vitro condition. Results: Candidatus Liberibacter asiaticus showed positive and negative response to different biochemical test. Candidatus Liberibacter asiaticus showed gram negative in gram staining test. Candidatus Liberibacter asiaticus showed highest 35±0.5 mm and lowest 8±0.2 mm zone of inhibition against amoxycillin and kanamycin respectively. Approximately 1300bp band was found in PCR amplification and phylogenetic tree analysis of 16S rRNA gene sequence of Candidatus Liberibacter asiaticus showed 75% similarity with Candidatus Liberibacter asiaticus strain 374.15. Candidatus Liberibacter asiaticus showed highest 16±0.5 mm diameter of zone of inhibition at 60 µg/ml concentration for ethanol extract of Cuscuta reflexa. Conclusion: This study will be helpful for proper identification, characterization and control of Citrus Huanglongbing disease causing pathogen Candidatus Liberibacter asiaticus in an eco-friendly way.

Métodos para la detección presuntiva de Huanglongbing (HLB) en cítricos / Methods for presumtive detection of Huanglongbing (HLB) in Citrus

RESUMEN Huanglongbing (HLB) es considerada mundialmente como la enfermedad más amenazadora para la citricultura, y ha impactado principalmente en Asia, Sudáfrica y Brasil. A la fecha, no se cuenta con un tratamiento efectivo para la detección de esta enfermedad, que pueda ayudar a disminuir su propagación y la consecuente eliminación de árboles infectados. El método más confiable hasta ahora, para la identificación del HLB, es la prueba de reacción en cadena de la polimerasa (qrt-PCR), la cual es costosa y tardada. Esta es la primera revisión de los diferentes métodos existentes o en desarrollo para la detección e identificación del HLB; clasificados en métodos de análisis y reconocimiento de patrones en imágenes, espectrofotométricos, cromatográficos y moleculares a través del insecto vector. Algunos de estos métodos representan alternativas innovadoras con diferente nivel de eficiencia en tiempo, costo y confiabilidad en la detección de árboles enfermos de HLB o en el manejo de la enfermedad, con relación a los tradicionalmente utilizados.

ABSTRACT Huanglongbing (HLB) is considered worldwide as the most threatening disease for citrus and has impacted mainly in Asia, South Africa and Brazil. Until now, no effective treatment for the detection of this disease (is available, which can help diminish its spread and the consequent removal of infected trees). The most reliable identification HLB test method to date is the polyme-rase chain reaction (qrt-PCR), which is costly and time-consuming. This is the first review of the different existing or developing methods for HLB detection and identification, classified into analysis and patternrecognition in images, spectrophotometric, chromatographic and molecular through the insect vector. Some of these methods represent innovative alternatives with different levels of efficiency in time, cost and reliability in detecting diseased trees or in managing the disease, compared to those traditionally used.

Detection of Huanglongbing Pathogen in Citrus medica L. var. sarcodactylis (Noot.) Swingle by Polymerase Chain Reaction Analysis / 广州中医药大学学报

[Objective] To establish an effective way for the detection of Huanglongbing pathogen in Citrus medica L. var. sarcodactylis (Noot. ) Swingle by polymerase chain reaction (PCR) analysis, and to provide evidence for the early diagnosis of the culture of pathogen-free plantlets and for the prevention and control of diseases. [ Methods ] The incidence of Huanglongbing disease was investigated in the main producing area of Citrus medica L. var. sarcodactylis (Noot.) Swingle. By using the special primers designed by the DNA sequence of Citrus Huanglongbing pathogen, PCR amplification of DNA from the samples was conducted. [Results] Symptoms similar to the Citrus Huanglongbing disease occurred in Citrus medica L. var. sarcodactylis (Noot.) Swingle growing in Zhaoqing, Guangdong. The results of PCR analysis showed that a special DNA fragment of 400bp had been detected in the affected plants of Citrus medica L. var. sarcodactylis (Noot. ) Swingle, but it was not found in the healthy plants, indicating that the symptoms were caused by the Huanglongbing pathogen. [ Conclusion ] PCR analysis is an effective way for the detection of Huanglongbing pathogen in Citrus medica L. var. sarcodactylis ( Noot. ) Swingle.