Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Huanglongbing (HLB) has been a serious threat to the citrus industry worldwide. After its first report in São Paulo State, the main citrus production area in Brazil, the disease spread to the States of Minas Gerais, Paraná and Mato Grosso do Sul. Attempts to cure plants or remiss disease symptoms and damages have been evaluated and showed to be inefficient and nonviable. The development of resistant or tolerant varieties to the bacteria or its insect vector, the Asian citrus psyllid Diaphorina citri, is still a long term challenge. Earlier HLB management has been based on preventive measures such as planting of healthy nursery trees, elimination of diseased trees, and vector control. Supported by both research data and citrus grower experiences, HLB management in São Paulo and Triângulo/Sudoeste Mineiro citrus belt has been improved from measures individually applied only into the orchards to regional disease management, including differentiated psyllid control in the orchards based on tree location and shoot flushing, area-wide coordinated control of psyllids, and removal of inoculum sources in noncommercial properties in the vicinity of commercial orchards. In addition, the negative impact of HLB on orchard production and longevity has been reduced with wide adoption of better cultural practices such as high-density planting, irrigation, and adequate nutrition. Unlike in other countries where HLB reached epidemic levels, the management of HLB in São Paulo and Triângulo/Sudoeste Mineiro citrus belt has been considered a success case and has ensured the maintenance of citrus production and competitiveness of the Brazilian citrus industry while new, more durable, and sustainable measures are not yet available.

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC-and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

Genetically distinct strains of the plant bacterium Xylella fastidiosa (Xf) are responsible for a variety of plant diseases, accounting for severe economic damage throughout the world. Using as a reference the genome of Xf 9a5c strain, associated with citrus variegated chlorosis (CVC), we developed a microarray-based comparison involving 12 Xf isolates, providing a thorough assessment of the variation in genomic composition across the group. Our results demonstrate that Xf displays one of the largest flexible gene pools characterized to date, with several horizontally acquired elements, such as prophages, plasmids, and genomic islands (GIs), which contribute up to 18% of the final genome. Transcriptome analysis of bacteria grown under different conditions shows that most of these elements are transcriptionally active, and their expression can be influenced in a coordinated manner by environmental stimuli. Finally, evaluation of the genetic composition of these laterally transferred elements identified differences that may help to explain the adaptability of Xf strains to infect such a wide range of plant species

The biology and behaviour of the psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae), the major insect vector of bacteria associated with huanglongbing, have been extensively studied with respect to host preferences, thermal requirements, and responses to visual and chemical volatile stimuli. However, development of the psyllid in relation to the ontogeny of immature citrus flush growth has not been clearly defined or illustrated. Such information is important for determining the timing and frequency of measures used to minimize populations of the psyllid in orchards and spread of HLB. Our objective was to study how flush ontogeny influences the biotic potential of the psyllid. We divided citrus flush growth into six stages within four developmental phases: emergence (V1), development (V2 and V3), maturation (V4 and V5), and dormancy (V6). Diaphorina citri oviposition and nymph development were assessed on all flush stages in a temperature controlled room, and in a screen-house in which ambient temperatures varied. Our results show that biotic potential of Diaphorina citri is not a matter of the size or the age of the flushes (days after budbreak), but the developmental stage within its ontogeny. Females laid eggs on flush V1 to V5 only, with the time needed to commence oviposition increasing with the increasing in flush age. Stages V1, V2 and V3 were most suitable for oviposition, nymph survival and development, and adult emergence, which showed evidence of protandry. Flush shoots at emerging and developmental phases should be the focus of any chemical or biological control strategy to reduce the biotic potential of D. citri, to protect citrus tree from Liberibacter infection and to minimize HLB dissemination.

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.

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.

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, 'Candidatus Liberibacter asiaticus', 'Candidatus Liberibacter africanus' and 'Candidatus Liberibacter americanus', is essential for the preventive control of the disease. Real-time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of 'Ca. Liberibacter' species was achieved by direct tissue-printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real-time PCR. This simplified method was validated and could detect HLB-liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB-infected trees. The use of direct assays as template showed good agreement with use of purified DNA (j = 0Á76 AE 0Á052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB-infected trees with or without symptoms. A commercial ready-made kit based on this technology showed 96% accuracy in intra-laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.