Citrus huanglongbing: validation of Real-Time PCR (qPCR) for the detection of Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus in Colombia

Abstract

Citrus huanglongbing (HLB) is the most destructive citrus disease. Two of the three known HLB-associated CandidatusLiberibacter species were recently found to be present in the Americas. In this study, eggs, nymphs and adults of Diaphorinacitri Kuwayama (Hemiptera: Liviidae) and suspect citrus plant materials were collected in 25 municipalities in the departments of Cundinamarca, Santander, Valle del Cauca, Meta and Quindio (Colombia). The detection sensitivity, specificity and assay performance of the 16S rDNA-based real-time PCR (qPCR) were validated for the field survey of the disease in Colombia. The validation confirmed the reliability and robustness of the real-time PCR method for the detection of HLB bacteria in host citrus plant tissues and the vector D. citri. The diagnosis was performed for Candidatus Liberibacter asiaticus (Ca. L. asiaticus) and for Candidatus Liberibacter americanus (Ca. L. americanus) on 168 citrus plant material samples and 239 insect samples. Neither Ca. L. asiaticus nor Ca. L. americanus were detected in the host plants or insects vector, confirming the absence of the disease in the citrus-producing areas of Colombia.

Dna Barcoding of Anemone Obtusiloba Var. Potentiloides: A Threatened and Endemic Plant From Western Himalayan Region of Pakistan

“…The real-time qPCR method targets had limited identification of one to ten copies per gene and low copy genes. The identification of low titer levels can be variable when the target sample is very dilute that caused inability to get reproducible results (Angel et al, 2015). Therefore, the targeted gene amplification by PCR of a recently revealed highly repetitive conserve region of Ca.…”

Screening nested-PCR primer for ‘Candidatus Liberibacter asiaticus’ associated with citrus Huanglongbing and application in Hunan, China

“…Currently, many methods based on PCR have been used to detect HLB infection, such as conventional PCR [3, 11, 12, 38], SSR[13], immune capture-PCR[17]. LAMP[16,39], PCR- RFLPs[28,40], droplet digital PCR[15], TaqMan qPCR[25], qPCR[11,12,41], qRT-PCR[4,23], and nested PCR[19]. Although these methods have worked well in symptomatic samples, they were subject to false negatives because HLB- associated Las may have a low titer and be unevenly distributed in citrus trees and even among cells within discrete tissues, yielding both positive and negative samples from various tissue samples originating from the same citrus tree [12, 42].…”

Potential functions of the shared bacterial taxa in the citrus leaf midribs determine the symptoms of Huanglongbing

“…The detection methods commonly used for HLB include PCR and qPCR, which are well-established for their accuracy and widespread use in HLB detection ( Ángel et al., 2014 ). However, there is currently no consensus on the threshold for distinguishing between HLB-positive and -negative samples.…”

“…The barcode and primers were removed, followed by merging, filtering, and decontamination of the pairedend sequences using QIIME2. Taxonomic classification was performed using the SILVA database with a similarity threshold of 97% (Almeida et al, 2018). The resulting high-quality data was then analyzed using R version 4.0.2, utilizing packages such as 'barplot' and 'vegan' for creating stacked histograms.…”