Insight into resistance to 'Candidatus Liberibacter asiaticus,' associated with Huanglongbing, in Oceanian citrus genotypes.

Huanglongbing (HLB), the most destructive citrus disease, is associated with unculturable, phloem-limited Candidatus Liberibacter species, mainly Ca. L. asiaticus (Las). Las is transmitted naturally by the insect Diaphorina citri. In a previous study, we determined that the Oceanian citrus relatives Eremocitrus glauca, Microcitrus warburgiana, Microcitrus papuana, and Microcitrus australis and three hybrids among them and Citrus were full-resistant to Las. After 2 years of evaluations, leaves of those seven genotypes remained Las-free even with their susceptible rootstock being infected. However, Las was detected in their stem bark above the scion-rootstock graft union. Aiming to gain an understanding of the full-resistance phenotype, new experiments were carried out with the challenge-inoculated Oceanian citrus genotypes through which we evaluated: (1) Las acquisition by D. citri fed onto them; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top-grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back-pruning. Overall, results showed that insects that fed on plants from the Oceanian citrus genotypes, their canopies, new flushes, and leaves from rooted cuttings evaluated remained quantitative real-time polymerase chain reaction (qPCR)-negative. Moreover, their budwood pieces were unable to infect sweet orange through grafting. Furthermore, sweet orange control leaves resulted infected when insects fed onto them and graft-receptor susceptible plants. Genomic and morphological analysis of the Oceanian genotypes corroborated that E. glauca and M. warburgiana are pure species while our M. australis accession is an M. australis × M. inodora hybrid and M. papuana is probably a M. papuana × M. warburgiana hybrid. E. glauca × C. sinensis hybrid was found coming from a cross between E. glauca and mandarin or tangor. Eremocitrus × Microcitrus hybrid is a complex admixture of M. australasica, M. australis, and E. glauca while the last hybrid is an M. australasica × M. australis admixture. Confirmation of consistent full resistance in these genotypes with proper validation of their genomic parentages is essential to map properly genomic regions for breeding programs aimed to generate new Citrus-like cultivars yielding immunity to HLB.

Modeling 'Candidatus Liberibacter asiaticus' Movement Within Citrus Plants.

The phloem-limited 'Candidatus Liberibacter asiaticus' (Las) causes huanglongbing, a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in the greenhouse, and the impacts of temperature on plant colonization in growth-chamber experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by quantitative PCR (qPCR) or symptoms at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, maintained at from 8 to 20°C, from 18 to 30°C, or from 24 to 38°C daily range, and assessed by qPCR of samples taken from noninoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to analysis of variance and binomial generalized linear model and logistic regression analyses. Probabilities of detecting Las in greenhouse plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day-1 average speed of movement. In growth chambers, the temperature impacted plant colonization by Las, new shoot emission, and symptom expression. After a 7-month exposure time, Las was absent in all new shoots in the cooler environment (average three per plant), and present in 70% at the milder environment (six shoots, severe symptoms) and 25% in the warmer environment (eight shoots, no visible symptoms). Temperature of 25.7°C was the optimum condition for plant colonization. This explains the higher impact and incidence of huanglongbing disease during the winter months or regions of milder climates in Brazil.

Evidence That 'Candidatus Liberibacter asiaticus' Moves Predominantly Toward New Tissue Growth in Citrus Plants.

'Candidatus Liberibacter asiaticus' (Las) is an unculturable, phloem-limited, insect-transmitted bacterium associated with the Asiatic form of huanglongbing (HLB), the most destructive citrus disease. In Asia and the Americas, it is transmitted by the Asian citrus psyllid (Diaphorina citri Kuwavama). Despite considerable research, little is known about the processes involved in plant infection and colonization by Las. This study was conducted to determine whether the basal portion (below girdling) of the plant is an important route for Las to move laterally from a point of inoculation on a branch to pathogen-free branches elsewhere in the canopy, and to quantify the influence of actively growing tissues on vertical upward (acropetally) or downward (basipetally) movement of Las. Nongirdled and fully or partially girdled stems of potted plants of 'Pera' sweet orange, graft-inoculated above or below girdling, were sampled in distinct regions and assessed by qPCR, 6 months postinoculation. Las invaded all regions of partially and nongirdled plants but remained restricted to the inoculated regions of fully girdled plants, evidence that in planta bacterium movement is limited to the phloem. In fully girdled plants, starch accumulated above the girdling site, probably because of changes in flow of phloem sap. To study the influence of actively growing tissues, inoculated 'Valencia' sweet orange plants were kept intact or were top- or root-pruned to force production of new tissues, and sampled at 15-day intervals. Las migrated rapidly and most predominantly toward newly developing root and leaf tissues. The rapid and predominant movement of Las to newly developed shoots and roots would explain failures of canopy heat treatments and pruning to cure HLB-affected trees, and reinforces the need to protect rapidly growing new shoots from feeding by D. citri in order to minimize transmission and spread of the pathogen by the vector within and between orchards.

Resistance to 'Candidatus Liberibacter asiaticus,' the Huanglongbing Associated Bacterium, in Sexually and/or Graft-Compatible Citrus Relatives.

Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated 'Candidatus Liberibacter asiaticus' (Las) becomes strategic to warrant crop sustainability, but no resistant Citrus genotypes exist. Some Citrus relatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible with Citrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible with Citrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately these Citrus relatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Most Citrinae species examined were either susceptible or partially resistant to Las. However, Eremocitrus glauca and Papua/New Guinea Microcitrus species as well as their hybrids and those with Citrus arose here for the first time as full-resistant, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs or attempting using them directly as possible new Las-resistant Citrus rootstocks or interstocks.

Differential Responses of Tobacco to the Citrus Variegated Chlorosis and Coffee Stem Atrophy Strains of Xylella fastidiosa.

Xylella fastidiosa comprises a diverse group of xylem-limited, insect-transmitted bacterial pathogens. In Brazil, the citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) diseases are caused by X. fastidiosa subspecies pauca transmitted by common insect vectors. No simple protocol allowing strain discrimination exists, making epidemiological studies, which are important for devising control measures, difficult to undertake. Here, we show that both strains can easily be distinguished based on the pattern of leaf symptoms that they induce on pin prick-inoculated tobacco seedlings, namely small orange lesions and large necrotic lesions induced by the CVC and CSA strains, respectively. These differential responses allowed us to investigate whether mixed strain infections would occur in citrus or coffee trees in the field. Seedlings were individually inoculated with X. fastidiosa colonies recovered from citrus or coffee plants from various locations at three different times. No mixed infections were detected. In two experiments, the citrus and coffee strains infected only their original hosts as well as tobacco. The usefulness of this tobacco bioassay as a tool to study X. fastidiosa spread was demonstrated. It provided evidence that, over the years, the CVC and CSA pathogens have remained limited to their original hosts, despite crop proximity and the presence of sharpshooter vectors that favor transmission of the bacteria to and between both host species.

Improvement in the Excised Citrus Leaf Assay to Investigate Inoculation of 'Candidatus Liberibacter asiaticus' by the Asian Citrus Psyllid Diaphorina citri.

Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Brazil, is associated mainly with the bacterium 'Candidatus Liberibacter asiaticus' transmitted by the psyllid Diaphorina citri. The aim of this study was to improve our understanding of the 'Ca. L. asiaticus' infection process by exposing excised, fully expanded, immature citrus leaves in 50-ml Falcon tubes to one, four, or eight adults from a 'Ca. L. asiaticus'-exposed colony for 1-, 3-, 7-, or 15-day periods for access to inoculation (IAP). The leaves were incubated at 26°C for 1, 3, 7, 15, and 21 days (incubation period [IP]). Infection frequencies and 'Ca. L. asiaticus' titers were assessed by quantitative polymerase chain reaction (qPCR). 'Ca. L. asiaticus' infection was a function of leaf age, number of insects, IAP, and IP. In general, higher infection rates were observed on younger leaves inoculated with higher numbers of insects and after longer IAP and IP. The immature excised leaf method allowed determination of 3 to 7 days as the range of time required by 'Ca. L. asiaticus' to reach qPCR detectable levels. Even though leaf survival could be prolonged by the maintenance of a branch segment at the base of the leaf petiole, leaf degradation, visible after about 15 days IP, did not allow observation of the entire infection process which, in the intact plant, culminates with the appearance of the blotch mottling symptom on leaf blades.

Seasonal Variation of 'Candidatus Liberibacter asiaticus' Titers in New Shoots of Citrus in Distinct Environments.

The major citrus area of Brazil occupies near 450,000 ha between the Triângulo Mineiro (TM) region of Minas Gerais State and the south of São Paulo State (SPS). Significant climatic variation occurs between regions which could affect huanglongbing (HLB) progress, which is lower in TM. To investigate this possibility, young sweet orange shoots were sampled periodically over 2 years to determine 'Candidatus Liberibacter asiaticus' titers in naturally infected trees in orchards in Analândia, central SPS, and Frutal and Comendador Gomes, within TM. Data-loggers recorded local temperature and relative humidity hourly. In the laboratory, five 'Ca. L. asiaticus'-free Diaphorina citri adults were placed on each sampled shoot for 48 h to feed and acquire the pathogen. Shoots and insects were individually analyzed by quantitative polymerase chain reaction to determine 'Ca. L. asiaticus' titers. The incidence of 'Ca. L. asiaticus'-positive shoots, 'Ca. L. asiaticus' titers, and acquisition rates were lower for shoots from Comendador Gomes than those from Frutal or Analândia. Stronger association was observed between 'Ca. L. asiaticus' titers and the number of hours below 15°C (h < 15°C) or above 30°C (h > 30°C), and cumulative rainfall registered during the 15 days prior to sampling of shoots on each occasion. 'Ca. L. asiaticus' titers associated positively with h < 15°C and rainfall and negatively with h > 30°C. The slower spread and lower incidence of HLB in TM may be related to lower incidences of 'Ca. L. asiaticus'-positive young shoots and lower titers of 'Ca. L. asiaticus' in the same shoots as a consequence of the warmer and drier conditions.