Detection of Huanglongbing (Citrus Greening) Disease by Nucleic Acid Spot Hybridization

Abstract

Polymerase chain reaction (PCR) amplifi cation with primers specifi c to the rDNA region successfully amplifi ed the 1160-bp DNA fragment from a Huanglongbing (HLB)-infected sweet orange sample with mottling symptoms leaves, but not from healthy sweet orange plants. The PCR product of 1160-bp was used as probe labeled with biotin for detection of the HLB pathogen in the nucleic acid spot hybridization (NASH) test. It was found that the HLB pathogen could be detected up to 1:100 dilution in HLB-infected tissue. Total DNA extracted from HLB-infected tissue was diluted 2-fold as 900 ng in TE buffer and spotted on a nitrocellulose membrane. Strong signals were observed up to 225 ng of DNA dilution, whereas a moderate signal was recorded at 112 ng. No hybridization signal was observed in the healthy samples, while strong signals were observed in the positive control.

LC-MS/MS Method for the Determination and Quantitation of Penicillin G and Its Metabolites in Citrus Fruits Affected by Huanglongbing

“…To prepare 800 μL of the individual calibration standards, 100 μL of the penicillin G-d 5 internal standard intermediate standard (40 ng/mL) was added to each of six different autosampler vials. Then to each vial was added 100 μL of each intermediate standard (2,5,20,100,200, 300 ng/mL). This was followed by the addition of 600 μL of ammonium acetate buffer.…”

“…The extracted matrix samples were evaporated in 50 mL centrifuge tubes and labeled 1−6. To each sample of dried matrix was added 100 μL of the correspondingly labeled mixed intermediate standard (2,5,20,100,200, 300 ng/mL) along with 700 μL of ammonium acetate buffer.…”

“…The fortified samples were made by spiking 2 g of the different citrus matrices (lemon, orange, and grapefruit) with 100 μL of four intermediate mixed standard solutions (2,5,20, and 200 ng/mL) and 100 μL of the internal standard intermediate solution (40 ng/mL). Overall, four spikes were made for each matrix to reach final concentrations of 0.1, 0.25, 1, and 10 ng/g of penicillin G, penillic acid, and penilloic acid in each spike.…”

Identification of the Virulence Factors of Candidatus Liberibacter asiaticus via Heterologous Expression in Nicotiana benthamiana using Tobacco Mosaic Virus

“…Because HLB is a major threat to the citrus industry [1], numerous research studies have been conducted, including studies on the identification of the causal agent [3,61], the cellular structure change of the infected trees [27,62], confirmation of the causal agent from putative-infected trees [5,9,10,12,63], and detection of the putative-infected trees [6,8,13,14]. In spite of the studies that were done to understand the pathogenesis of Ca Las [30,32,33,34,64] and the gene regulation of Ca Las [65], the virulence mechanism is still largely unknown, because of the unavailability of the mutant library.…”

“…The Asian form ( Candidatus Liberibacter asiaticus, Ca Las) is transmitted either by Asian citrus psyllid, Diaphorina citri , or by grafting. The recent research on HLB focused mainly on either the detection HLB by different methods [5,6,7,8,9,10,11,12,13,14,15,16,17] or citrus responses to HLB at the transcriptional level, translational level, and nutritional-status level [18,19,20,21,22,23,24,25,26,27,28,29]. Efforts were also made to understand the virulence mechanisms of Ca Las.…”