Graft transmission efficiencies and multiplication of 'Candidatus Liberibacter americanus' and 'ca. Liberibacter asiaticus' in citrus plants.

In Brazil 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus' cause huanglongbing (also known as greening), the most destructive citrus disease. A shift in pathogen prevalence was observed over time, with a disproportional increase in 'Ca. L. asiaticus' occurrence. Graft transmission experiments were used for a comparative study of both species using budsticks from symptomatic branches of field-affected trees as inoculum. The plants were inoculated with 'Ca. L. asiaticus' or 'Ca. L. americanus' alone, or simultaneously with both species. Symptom manifestation and conventional and quantitative real-time polymerase chain reaction were used for plant evaluations. 'Ca. L. americanus' was detected mainly in symptomatic plants and 'Ca. L. asiaticus' was detected in symptomatic plants as well as in infected plants prior to symptom manifestation. Transmission percentages varied from 54.7 to 88.0% for 'Ca. L. asiaticus' and 10.0 to 45.2% for 'Ca. L. americanus' in two experiments. In co-inoculated plants, 12.9% contained 'Ca. L. americanus' only, 40.3% contained 'Ca. L. asiaticus' only, and 19.3% contained both species. Average bacterial titers for 'Ca. L. asiaticus' and 'Ca. L. americanus', in log cells per gram of leaf midrib, were 6.42 and 4.87 for the experimental plants and 6.67 and 5.74 for the field trees used as the source of inoculum. The higher bacterial populations of the 'Ca. L. asiaticus'-infected plants provided an explanation for the disproportional increase in field prevalence of this species over time, based on the greater likelihood for pathogen transmission by the insect vector.

Liberibacters Associated with Citrus Huanglongbing in Brazil: 'Candidatus Liberibacter asiaticus' Is Heat Tolerant, 'Ca. L. americanus' Is Heat Sensitive.

In São Paulo State, Brazil, 'Candidatus Liberibacter americanus' and 'Candidatus Liberibacter asiaticus' are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with 'Ca. L. asiaticus' or 'Ca. L. americanus' was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any 'Ca. L. americanus'-infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, 'Ca. L. asiaticus'-infected trees had high titers at 32 and 35°C, but not at 38°C, while 'Ca. L. americanus'-infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of 'Ca. L. asiaticus' is not yet affected at 35°C, while a temperature of 32°C is detrimental to 'Ca. L. americanus'. Thus, 'Ca. L. americanus' is less heat tolerant than 'Ca. L. asiaticus'. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

Graft Transmission and Cultivar Reaction of Citrus to 'Candidatus Liberibacter americanus'.

Little is known about 'Candidatus Liberibacter americanus', a causal agent of huanglongbing or greening disease in Brazil, or its interaction with citrus trees. Greenhouse experiments were conducted with the objective of determining conditions favorable for transmission from field affected trees to young potted plants, to evaluate the reaction of multiple citrus species to the disease, and to determine the efficiency of pathogen propagation from individual buds. Single buds or bark pieces of various sizes or bark plus wood (budstick) that were removed from symptomatic or asymptomatic branches were used as sources of inoculum. Transmission success was evaluated through polymerase chain reaction analysis of total DNA extracted from leaf samples. Beginning at 4 to 5 months after inoculation, infected plants manifested leaf mottling and symptoms similar to those of iron, manganese, and zinc deficiencies. Blotchy mottled leaves appeared only on sweet oranges and Murcott tangor. Pathogen transmission was higher for these citrus cultivars and species (31.2 to 65.2%) than for limes, mandarins, or Swingle citrumelo (2.0 to 25.0%). Deformed small fruits with brownish columellae also developed on sweet oranges. Only buds and budsticks served as sources of inoculum and the larger the piece of tissue, the higher the transmission efficiency. Experiments initiated during the winter showed lower rates of graft tissue survival but relatively higher percentages of pathogen transmission.