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.

An Experimental Inoculation System to Study Citrus-Xylella fastidiosa Interactions.

Difficulties in reproducing the citrus variegated chlorosis (CVC) disease symptoms in experimental plants have delayed implementation of studies to better understand the essential aspects of this important disease. In an extensive study, cultivars of sweet orange (Citrus sinensis) were inoculated with Xylella fastidiosa using procedures that included root immersion, and stem absorption, pricking, or infiltration of the inoculum into plants of different ages. Inoculum consisted of 5-day-old cultures or cell suspensions of CVC strain 9a5c diluted in phosphate-buffered saline. Inoculated plants and controls were grown, or transferred just after inoculation, to 5-liter pots or 72-cell foam trays. Approximately 4, 5, 9, and 12 months after inoculation, leaves were collected and processed for polymerase chain reaction analysis or X. fastidiosa isolation on BCYE agar medium. Root immersion and stem inoculation of 4- and 6-month-old plants resulted in low percentages of symptomatic (0 to 7%) and plants positive by isolation (0 to 9%). Pinpricked or injected stems of 1-month-old seedlings resulted in high percentages of plants symptomatic (29 and 90% in Pera Rio, 75, 59, and 83% in Valencia, and 77% in Natal) or positive by isolation (26 and 93% in Pera Rio, 98, 96, and 83% in Valencia, and 77% in Natal). In foam trays, the seedlings grew less, the incubation period was shorter, and disease severity was higher than in pots. This system allows testing of higher numbers of plants in a reduced space with a more precise reproduction of the experimental conditions.

Weeds as Alternative Hosts of the Citrus, Coffee, and Plum Strains of Xylella fastidiosa in Brazil.

In Brazil, Xylella fastidiosa is present in citrus (Citrus sinensis), coffee (Coffea arabica), and plum (Prunus sp.) crops, causing the citrus variegated chlorosis (CVC), coffee leaf scorch (CLS), and plum leaf scald (PLS). Also present in these crops and infesting weeds, which ultimately could serve as sources of inoculum for the cultivated trees, are diverse populations of xylem-feeding leafhopper vectors. In order to assess host range of X. fastidiosa among weeds and to better understand their role in epidemics, field surveys, mechanical inoculations, and insect transmission tests were conducted. Polymerase chain reaction (PCR) and culture plating were used to detect the pathogen from plant tissues. X. fastidiosa was detected in 10 out of 23 species of the weed plants sampled in two citrus groves affected by CVC. None of the weed plants showed external symptoms. In the greenhouse, the average percentages of infection on plants mechanically inoculated with the CVC, CLS, and PLS strains of X. fastidiosa were, respectively, 25, 10, 0 in Medicago sativa; 70, 45, 20 in Echinochloa crus-galli; 45, 30, 0 in Brachiaria decumbens; 72, 70, 40 in Brachiaria plantaginea; 13, 10, 0 in Digitaria horizontalis; 31, 30, 0 in Solanum americanum; and 17, 0, 0 in Bidens pilosa. Symptoms were observed only in S. americanum and citrus and only when inoculated with the CVC strain. In insect transmission tests, the grass leafhopper Ferrariana trivittata was first caged on citrus plants showing CVC symptoms and then on healthy citrus and on the four most common weeds. No plants tested positive by PCR or culture, or showed symptoms for at least 4 months after inoculation. The amount of X. fastidiosa cells that may accumulate in weeds inoculated by leafhoppers is probably under insect acquisition thresholds, a factor that would limit their importance to the CVC epidemics, as studies on spatial distribution of diseased citrus trees over time indicate.

Experimental electron density of urea-phosphoric acid (1/1) at 100 K.

The deformation electron density of the urea-phosphoric acid adduct has been studied from 100 K X-ray and neutron diffraction experiments. Data were interpreted according to the Hirshfeld model. The long hydrogen bonds show characteristics of electrostatic interaction. Deformation density maps on the short hydrogen bond shows hydrogen more strongly bonded to urea than to phosphoric acid, and peak maxima at almost midway between the two O-H bonds.