Molecular diagnosis conrol and identification of tolerance in citrus against greening (Huanglongbing)

Abstract

Citrus greening (Huanglongbing) is the most destructive disease of citrus. Control of this disease is necessary for the survival of citrus industry. Husain and Nath (1927) described severe damage caused by populations of Diaphorina citri at Sargodha from 1915 to 1920 without any mention the greening disease. Candidatus Liberibacter, a gram negative bacterium is the causal organism of the disease. The natural vector of the disease is citrus psylla. There are two species of this vector Diaphorina citri Kuwayama (Hemiptera: Sternorryncha: Liviidae) and Trioza erytreae (del Guercio) (Hemiptera: Sternorrynca: Triozidae) reported in case of citrus greening. The disease can also be transmitted through budding/grafting. The proposed study was aimed to identify the tolerance in citrus germplasm and to test the effect of antibiotic and heat treatment for cleaning the huanglongbing infected budwood. Conventional as well as quantitative real time polymerase chain reaction method was used for the detection of Candidatus Liberibacter. Monitoring of natural vector of the disease Diaphorina citri kuwayama in the field was also done for identification of tolerance against huanglongbing in the germplasm. Symptom appearance as a result of inoculation of the citrus germplasm was recorded. Huanglongbing was successfully detected in the budwood used for all the experiments; 1160bp and 703bp bands were observed by using OI1/OI2c and A2/J5 primers, respectively. Βeta operon region of huanglongbing pathogen was amplified from greening affected sweet orange (Citrus sinensis) from Pakistan using primer set A2/J5 and sequenced. Alignment results indicated 99% sequence homology with more than 90 blast hits in the NCBI Genebank. Budwood from those source plants was used in all experiments of the present study. Clausena Harmandiana, Glycosmis Pentaphylla, Poncirus trifoliate and Bitter Sweet orange showed HLB negative results by qPCR indicating them tolerant for huanglongbing. Screening was also done on the basis of Asian citrus psyllid (Diaphorina citri kuwayama) presence on citrus germplasm. On 13 accessions of the total germplasm no Asian citrus psyllid was found in the field. Out of the total, 36.5% accessions showed huanglongbing symptoms. Conventional polymerase chain reaction showed 28.37% of the total germplasm positive for huanglongbing, whereas real time quantitative polymerase chain reaction confirmed presence of Candidatus Liberibacter asiaticus in 94.60% of the accessions. Negative results were obtained by using tetracycline. Application of 1000ppm tetracycline and 75ppm penicillin gave results. In case of temperature treatment, 68°C and combination of tetracycline 1000ppm + 50°C temperature showed results.