Crop Research

The pathogen Candidatus Liberibacter asiaticus (CLas) is the cause of Huanglongbing (HLB) or citrus greening (CG) disease, which is one of the devastating diseases causing a significant crop reduction in citrus crops (Family: Rutaceae). It is a systemic pathogen which lives in citrus crop phloem tissues. Detection of Ca. L. asiaticus (CLas) casuing citrus Huanglongbing (HLB) disease was facilitated by taking samples from three different citrus orchards located in Tirupattur, Dharmapuri, Krishnagiri district. Midrib injection method by using syringe needle or microneedles used has early transmission of Ca. L. asiaticus (CLas) in phloem tissue of acid lime leaves. Total genomic DNA is extracted from inoculation of citrus leaves using a modified CTAB method to identify DNA from both healthy and infected samples. A 100 mg leaf midrib sample was crushed in liquid nitrogen, homogenized in 500 µl CTAB, incubated for 45 minutes at 60°C in a water bath, centrifuged for 5 minutes at 12000 rpm, and the supernatant was then transferred to a fresh tube, to which 5 µl of RNase-A was added, and incubated for 20 minutes at 37°C. For phase separation, an equal volume of chloroform/isoamyl alcohol (24:1) was added, vortexed for five minutes, and centrifuged for two minutes at 12000 rpm. The upper phase was then transferred to a new tube, in which 500µl of chilling isopropanol was added, kept at -20°C for 15 minutes, centrifuged at 12000 rpm, and the pallet was cleaned twice with 70% ethanol. The specific primers OI1/OI2c and A2/J5 had been used in a polymerase chain reaction (PCR) for detection, and they showed a specific band of 1160 bp and 703 bp, respectively. These were amplified from diseased leaves whereas no product from healthy citrus plant.

Citrus greening or Huanglongbing (HLB) is a rapidly spreading disease caused by Candidatus Liberibacter species, which inhabit the phloem of citrus plants. It is primarily transmitted by psyllid insects. Managing HLB is challenging due to the bacterium's phloem-restricted nature, making diagnosis and treatment with antimicrobials difficult. Here in the present study, the bacterial consensus sequences were used for the primer design with In silico approaches and validated by PCR. The study was carried out in the Malwa region of Madhya Pradesh, India, which is known for citrus cultivations and remains affected by the HLB disease. Phylogenetic analysis of 100 Candidatus Liberibacter 16S rRNA sequences from NCBI was conducted using MEGA 6, with Malwa-specific refinement via MAFFT alignment. Eight previously deposited sequences formed the basis for the study. A consensus sequence was derived and used to design region-specific primers with GeneFisher. PCR validation was carried out with standard thermal cycling and gel electrophoresis. Phylogenetic analysis of 100 Candidatus Liberibacter 16S rRNA sequences from NCBI was conducted using Phylogenetic analysis showed high genetic variation among Liberibacter strains, supporting the need for region-specific detection. Eight sequences from the Malwa region were aligned using BioEdit, confirming conserved primer sites. Consensus primers were designed using GeneFisher and optimized for 16S rRNA targets. PCR validation identified 22 positives out of 100 samples, with primer set 2 detecting all target species effectively. The study concludes that primer set 2 enables rapid and accurate detection of HLB-causing bacteria in the Malwa region.