Huanglongbing

Transmission of plant pathogens through insect vectors is a complex biological process involving interactions between the host plants, insects, and pathogens. Simultaneous impact of the insect damage and pathogenic bacteria in infected host plants induce volatiles that modify not only the behavior of its insect vector but also of their natural enemies, such as parasitoid wasps. Therefore, it is essential to understand how insects such as the predator ladybird beetle responds to volatiles emitted from a host plant and how the disease transmission alters the interactions between predators, vector, pathogens, and plants. In this study, we investigated the response of Propylaea japonica to volatiles from citrus plants damaged by Diaphorina citri and Candidatus Liberibacter asiaticus through olfactometer bioassays. Synthetic chemical blends were also used to determine the active compounds in the plant volatile. The results showed that volatiles emitted by healthy plants attracted more P. japonica than other treatments, due to the presence of high quantities of D-limonene and beta-ocimene, and the lack of methyl salicylate. When using synthetic chemicals in the olfactory tests, we found that D-limonene attracted P. japonica while methyl salicylate repelled the predator. However, beta-ocimene attracted the insects at lower concentrations but repelled them at higher concentrations. These results indicate that P. japonica could not efficiently search for its host by using volatile cues emitted from psyllids-and Las bacteria-infected citrus plants.

Huanglongbing (HLB), is the most devastating citrus disease in the world. Two of the three known HLB-associated (Candidatus Liberibacter spp.) species are present in the Americas. Among them, 'Ca. L. asiaticus' (CLas) was detected in Venezuela in 2017, although early characteristics HLB symptoms such as yellow shoots were first noticed in 2016. In this study, plant material from suspect citrus and from knowing HLB hosts, were collected in 17 municipalities in the states of Aragua, Carabobo, Yaracuy and Portuguesa (Venezuela) between August and November 2017, to determine CLas relative incidence and host range. Samples were then evaluated by PCR, using primers targeting the β-operon locus of ribosomal proteins. Out of 113 HLB-symptomatic citrus plants, 87 (76.9 %) were positive for CLas. Citrofortunella microcarpa, Swinglea glutinosa, and Murraya paniculata known host plants of the vector, the Asian citrus psyllid Diaphorina citri Kuwayama, were also found infected in all regions analyzed. Further spread of the pathogen is expected, given the nationwide distribution and abundance of the vector. Given the HLB-infection rates found in this study, additional surveys in eastern and western states are recommended to evaluate the spread and severity of the disease which will greatly assist the design of efficient management strategies based on research data. El huanglongbing de los cítricos en Venezuela: distribución parcial e incidencia relativa de Candidatus liberibacter asiaticus en la región centro-norte RESUMEN El "Huanglongbing" (HLB) es actualmente la enfermedad más devastadora de los cítricos. Dos de las tres especies de bacterias que se asocian al HLB (Candidatus Liberibacter spp.), han sido encontradas en las Américas. En Venezuela, la especie 'Ca. L asiaticus' (CLas) fue detectada en 2017, aunque los síntomas tempranos característicos asociados a la enfermedad, tales como brotes amarillos fueron notificados por productores en varias regiones del país desde el año 2016. En este estudio, se colectó material vegetal proveniente de árboles de cítricos, así como de hospederos conocidos del HLB, en 17 municipios de los estados Aragua, Carabobo, Yaracuy y Portuguesa (Venezuela) entre Agosto y Noviembre de 2017, con el objetivo de determinar la incidencia relativa y el rango de hospederos de CLas en la región central del país. Las muestras fueron evaluadas mediante PCR, utilizando cebadores para amplificar la región del locus de las proteínas ribosomales en el operón-β de la bacteria. De un total de 113 plantas de cítricos sintomáticas, 87 (76.9 %) resultaron positivas para CLas. Adicionalmente, se encontraron plantas de Citrofortunella microcarpa, Murraya paniculata y Swinglea glutinosa (hospederas del vector del HLB, el psílido asiático de los cítricos, Diaphorina citri Kuwayama) infectadas con CLas en todos los estados incluidos en el estudio. Dado el porcentaje de infección que revela esta investigación, se recomienda realizar muestreos en los estados orientales y occidentales, a fin de conocer la diseminación e incidencia de la enfermedad en Venezuela y formular estrategias eficientes de manejo y control basadas en evidencia científica. Palabras clave: enverdecimiento de los cítricos, detección, distribución, HLB, PCR, Venezuela.

The Asian citrus psyllid, Diaphorina citri, is a major pest of citrus and vector of citrus greening (huanglongbing) in Asian. In our field-collected psyllid samples, we discovered that Fuzhou (China) and Faisalabad (Pakistan), populations harbored an obligate primary endosymbiont Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.), and a secondary endosymbiont, Wolbachia surface proteins (WSP) which are intracellular endosymbionts residing in the bacteriomes. Responses of these symbionts to different temperatures were examined and their host survival assessed. Diagnostic PCR assays showed that the endosymbionts infection rates were not significantly reduced in both D. citri populations after 24 h exposure to cold or heat treatments. Although quantitative PCR assays showed significant reduction of WSP relative densities at 40°C for 24 h, a substantial decrease occurred as the exposure duration increased beyond 3 days. Under the same temperature regimes, Ca. Carsonella ruddii density was initially less affected during the first exposure day, but rapidly reduced at 3–5 days compared to WSP. However, the mortality of the psyllids increased rapidly as exposure time to heat treatment increased. The responses of the two symbionts to unfavorable temperature regimes highlight the complex host-symbionts interactions between D. citri and its associated endosymbionts.

In 2017, branches were collected
from Citrus sp. trees showing mild to severe yellowing on the
shoots, lopsided and greening fruits and tree decline in various
regions in Venezuela. To verify the presence of CLas, total DNA
was extracted from single leaf midribs and subjected to PCR
amplification with primers sets OI1/OI2c targeting the 16S
rDNA locus (Jagoueix et al. 1994) and A2/J5 targeting the
beta-operon locus of ribosomal proteins (Hocquellet et al.
1999). A total of 87 out of 104 samples (83.7%) tested positive
with both primer sets. The OI1/OI2c primers generated an
amplicon of approximately 1160 bp and digestion by XbaI
yielded two DNA fragments of approximately 640 bp and
520 bp, suggesting the presence of CLas (Jagoueix et al. 1994).
The A2/J5 primer set generated a ~703-bp amplicon, suggesting
‘Candidatus L. africanus’ was not present.