Evaluation of susceptibility of ten Citrus Genotypes to ‘Candidatus Liberibacter asiaticus’ the causal agent of citrus greening.

Citrus greening (HLB) is one of the most damaging citrus diseases worldwide. As well, the three most important causal agents of this disease in the world include "Candidatus (Ca.) Liberibacter asiaticus ("CLas"), Ca. Liberibacter africanus ("CLaf"), and Ca. Liberibacter americanus ("CLam"). Accordingly, these were reported from Asian-American, African, and American countries, respectively. Of note, "CLas" species is found as the most important causal agent of this disease in all countries in the world, including Iran. It seems that the most effective way to reduce HLB is finding those cultivars that are resistant or tolerant to HLB. Accordingly, in the present study, an attempt was made to evaluate the reaction of a number of common citrus cultivars to this bacterium.

In the present research, ten commercial rootstocks and cultivars of the common citrus in the south of Iran, including Mexican lime, sour orange, mandarin orange, Kinnow mandarin, Orlando tangelo mandarin, local orange, Valencia orange ,sweet lemon, grapefruit, and Lisbon lemons were prepared as seedlings. After checking their health, they were inoculated with the citrus greening agent and then kept in an insect-free greenhouse at night at 25°C and during the day at 30°C.By passing two months from the inoculation, the pathogen was detected by performing specialized tests in inoculated seedlings. Thereafter, three two-year-old seedlings from each one of the above-mentioned rootstocks and cultivars were grafted with two infected buds. Two months after the inoculation, normal and nested PCR tests were performed on the vein tissue extract of the infected seedlings, once every two months, for a 12-month duration. Finally, both sensitivity and relative resistance of cultivars were evaluated based on the average number of days until the detection of the first Ca. L. asiaticus by PCR and also the incubation period of the disease, in each seedling.

The causal agent of this disease ("CLas") was detected at the earliest time(in Valencia oranges and Orlando tangelo mandarin after 180 and 200 days, respectively) after the inoculation with infected graft, compared to other cultivars and rootstocks. Among the studied species, the "CLas" was detected in both Mexican lime and Lisbon lemon with the longest delay (by passing 330 and 300 days from the inoculation, respectively). According to the results, "CLas" was detected in sour orange and sweet lemon 290 and 280 days after the inoculation with infected graft, respectively. Based on the average number of days until the detection of the first Ca. L. asiaticus by PCR and also the incubation period of the disease, in each seedling, Mexican lime and Lisbon lemon were found as the most tolerant species and Valencia oranges and Orlando tangelo mandarin were found as the most susceptible ones to HLB. Moreover, sour orange and sweet lemon seedlings were moderately susceptible ones; and grapefruit, local orange, local, and Kinnow mandarin were moderately tolerant to HLB.

Due to the reason that this bacterium does not grow in conventional medium, in this study, it was not possible to multiply it in artificial medium and then contaminate the plant with it. Therefore, performing the inoculation through grafting the seedlings with HLB infected buds was considered as the most appropriate method for seedlings inoculation with the pathogen. The faster the pathogen can settle and multiply and the symptoms appear in the host, the greater the susceptibility of the host to the disease. Early pathogen’s detection and symptoms’ observation in some hosts such as Valencia oranges and Orlando tangelo mandarin, indicate better bacterial proliferation in both cultivars. Notably, no symptoms of the disease were observed in Mexican lime and Lisbon lemons after 15 months, and one year after the inoculation in sour orange and sweet lemon plants. There is evidence that all known citrus cultivars are affected to this pathogen with different degrees of susceptibility. Based on the results of this study and the results of previous researchers in the world, it seems that researchers should look for the sources of resistance factors to HLB in more species and cultivars. Correspondingly, these sources can be used in the following two ways: used as a rootstock or scion, and used in the efforts of breeders to produce a resistant cultivar.