Murraya paniculata and Swinglea glutinosa as Short-Term Transient Hosts of 'Candidatus Liberibacter asiaticus' and Implications for the Spread of Huanglongbing.

Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) Diaphorina citri, the principal vector of 'Candidatus Liberibacter asiaticus' (Las). Las is the pathogen associated with huanglongbing (HLB), the Asian form of which is the most devastating disease of Citrus species and cultivars (Rutaceae: Aurantioideae). M. paniculata is a common ornamental and S. glutinosa is grown as an ornamental, a citrus rootstock, and a hedgerow fence plant. Because of the uncertain status of these plants as reservoirs of Las, a series of cross-inoculation bioassays were carried out in different environments, using infected Valencia sweet orange (Citrus × aurantium) infected shoot tops as a source of inoculum and D. citri nymphs and adults reared on M. paniculata and S. glutinosa to inoculate pathogen-free Valencia orange plantlets. In contrast to sweet orange, Las was more unevenly distributed and reached much lower titers in M. paniculata and S. glutinosa. Infections in M. paniculata and S. glutinosa were also transient. Very few insects that successfully acquired Las from M. paniculata and S. glutinosa were able to transmit the pathogen to healthy citrus. Transmission rates were low from M. paniculata (1.0%) and S. glutinosa (2.0%) and occurred only in a controlled environment highly favorable to Las and ACP using 10-day-old adults that completed their life cycle on Las-positive plants. Our study showed that in HLB-endemic areas, M. paniculata and S. glutinosa can be deemed as epidemiologically dead-end hosts for Las and are not important alternative hosts of the pathogen for transmission to citrus. However, under a combination of conditions highly favorable to Las infection and transmission and in the absence of effective quarantine procedures, these plants could eventually serve as carriers of Las to regions currently free from HLB.

Impacts of huanglongbing on fruit yield and quality and on flushing dynamics of Sicilian lemon trees.

Introduction: The severe Asian form of huanglongbing (HLB), a vascular disease associated with the phloem-limited bacterium 'Candidatus Liberibacter asiaticus', is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. Disease impacts are known for sweet oranges and acid limes but not lemons. Methods: In a five-year study (2017-2021) we compared yield and fruit quality between naturally-infected and healthy 5-yr-old trees of Sicilian lemon 'Femminello', and shoot phenology on both lemon and 'Valencia' orange, both grafted onto 'Swingle' citrumelo, grown in southeastern São Paulo State, Brazil. HLB severity (percentage of tree canopy area with HLB symptoms) was assessed every 3-4 months, fruit yield and quality in May (2017 to 2019) or June/July (2020-2021), and vegetative and reproductive shoots fortnightly on 50-cm-long branches. The development of ACP on one-year-old seedlings of five lemon varieties, 'Tahiti' acid lime, 'Valencia' orange, and orange jasmine was evaluated. Results: Symptoms increased from 11% in 2017 to 64% in 2021, and a monomolecular model estimated 10 years for symptoms to occupy >90% of the tree canopy. On average, production of trees with symptom on 20%, 50% or 80% of the canopy respectively dropped by 18%, 38%, and 53% compared to healthy trees. Fruits of symptomatic branches of lemons were 4.22% lighter and the number of dropped fruits did not correlate with symptom severity. Flushing on symptomatic branches started earlier by 15 to 55 days as compared to the healthy branches of lemon and orange. On diseased trees, vegetative and reproductive shoots respectively increased by 24.5% and 17.5% on lemon and by 67.2% and 70.6% on sweet orange, but fruit set was reduced by 12.9% and 19.7% on lemon and orange trees, respectively. ACP reproduced similarly on all tested plants. Discussion: The fast symptom progress, significant yield reduction, and earlier flushing on diseased trees, providing conditions highly favorable for the pathogen to spread, reinforce the need of prompt diseased tree removal and frequent ACP preventive control to manage HLB in lemons as in any other citrus crop.

Early Population Dynamics of "Candidatus Liberibacter asiaticus" in Susceptible and Resistant Genotypes After Inoculation With Infected Diaphorina citri Feeding on Young Shoots.

Huanglongbing is a highly destructive citrus disease associated with "Candidatus Liberibacter asiaticus" (Las), a phloem-limited and non-culturable bacterium, naturally transmitted by the psyllid Diaphorina citri. Although diverse approaches have been used to understand the molecular mechanisms involved in the pathogen-host interaction, such approaches have focused on already infected and/or symptomatic plants, missing early events in the initial days post-inoculation. This study aimed to identify the time course of Las multiplication and whole-plant colonization immediately following inoculation by infected psyllids feeding for 2 days. Thus, the experimental approach was to track Las titers after psyllid inoculation in new shoots (NS) of Citrus × sinensis (susceptible), Murraya paniculata (partially resistant), and Bergera koenigii (fully resistant). Soon after psyllid removal, Las titers dropped until the 10-12th days in all three species. Following this, Las titers increased exponentially only in C. × sinensis and M. paniculata, indicating active bacterial multiplication. In C. × sinensis, Las reached a stationary phase at ∼5 log Las cells/g of tissue from the 40th day onward, while in M. paniculata, Las increased at a lower rate of up to ∼3 log Las cells/g of tissue between the 40th and 60th days, decreasing gradually thereafter and becoming undetectable from the 160th day onward. In B. koenigii, Las titers decreased from the start and remained undetectable. In C. × sinensis, an average of 2.6 log of Las cells/g of tissue was necessary for Las to move out of 50% of the NS in 23.6 days and to colonize the rest of the plant, causing a successful infection. Conversely, the probability of Las moving out of the NS remained below 50% in M. paniculata and zero in B. koenigii. To our knowledge, this is the first study on Las dynamics and whole-plant colonization during the earliest stages of infection. Identification of critical time-points for either successful multiplication or Las resistance may help to elucidate initial events of Las-host interactions that may be missed due to longer sampling intervals and at later stages of infection.

Optimization of sampling and monitoring of vegetative flushing in citrus orchards.

Citrus trees produce flushes throughout the year, but there are no criteria established for a precise shoot monitoring in orchards under tropical climate. Methods for quantification of flush dynamics would be useful for horticultural and pest management studies because different insect vectors feed and reproduce on flushes. We estimated the minimum number and distribution of trees for sampling and determined the flushing pattern over time in 'Valencia Late' orange trees grafted onto 'Swingle' citrumelo rootstock. Shoots within a square frame (0.25 m2) on two sides of the canopy were counted and classified by their phenological stage. The minimum number of samples was estimated using the mean number of shoots and area under the flush shoot dynamics (AUFSD). The temporal and spatial distribution analysis was performed by Taylor's power law and by multiple correspondence analysis (MCA). Additionally, a shoot maturity index (SMI) based on visual qualitative assessment of flushes is proposed. Considering the mean number of shoots, it was necessary to sample two sides of 16 trees to reach a relative sampling error (Er) of 25%, whereas by the AUFSD, only five trees were necessary to reach an Er of 10%. Flushes were predominantly randomly distributed over time and space. Testing eight transects, sampled trees should be distributed throughout the block, avoiding sampling concentration in a certain area. MCA showed that the west side and the upper sampling positions of trees were more likely to be associated with younger shoots. AUFSD and the evaluation of both sides of the canopy yielded a smaller number of trees to be assessed. The SMI was a reliable metric to estimate the shoot phenology of orange trees, and correlated well (R2 > 70%) with the mean number of shoots within the square frame. Therefore, SMI has the potential to make shoot monitoring in the field more practical.

Lower reproductive rates of Asian citrus psyllid (Hemiptera: Psyllidae) on 'Tahiti' acid lime than on 'Valencia' sweet orange

ABSTRACT The 'Tahiti' acid lime and orange trees are hosts of 'Candidatus Liberibacter asiaticus' (CLas), the pathogen associated with the severe Asian form of huanglongbing (HLB), the most devasting citrus disease. They are also hosts of the vector of CLas, the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama. Relatively small numbers of lime trees occur in gardens and small orchards near large commercial 'Valencia' sweet orange orchards in Brazil. Applications of insecticides to suppress populations of ACP on the lime trees are usually nil or less frequent than in the orange orchards. Abundance of the psyllid on lime trees may therefore increase the risk of CLas spreading to the orchards. Because the abundance of the psyllid is influenced by the suitability of the trees as hosts, we compared reproductive potential of the insect on the two hosts in a controlled environment chamber (CEC) and in a greenhouse (GH). Daily temperature and relative humidity averaged 22ºC and 60% inside the CEC and 24°C and 70% inside the GH. Two pairs of adult male and female psyllids were caged for 3 days on new shoots and the fecundity and durations of development and survival of eggs and nymphs evaluated. Overall, acid 'Tahiti' was 3.5 times less suitable to ACP than 'Valencia'. Fecundity and survival of nymphs were 27% and 59% lower, and the life cycle 34% longer on 'Tahiti' than on 'Valencia'. Potential impacts of the results on CLas spread and HLB control are discussed.

Citrus flush shoot ontogeny modulates biotic potential of Diaphorina citri.

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.

Flexible regression model for predicting the dissemination of Candidatus Liberibacter asiaticus under variable climatic conditions.

Greening, or Huanglongbing (HLB), poses a severe threat to global citrus cultivation, affecting various citrus species and compromising fruit production. Primarily transmitted by psyllids during phloem feeding, the bacterium Candidatus Liberibacter induces detrimental symptoms, including leaf yellowing and reduced fruit quality. Given the limitations of conventional control strategies, the search for innovative approaches, such as resistant genotypes and early diagnostic methods, becomes essential for the sustainability of citrus cultivation. The development of predictive models, such as the one proposed in this study, is essential as it enables the estimation of the bacterium's concentration and the vulnerability of healthy plants to infection, which will be instrumental in determining the risk of HLB. This study proposes a prediction model utilizing environmental factors, including temperature, humidity, and precipitation, which play a decisive role in greening epidemiology, influencing the complex interaction among the pathogen, vector, and host plant. In the proposed modeling, it addresses non-linear relationships through cubic smoothing splines applications and tackles imbalanced categorical predictor variables, requiring the use of a random-effects regression model, incorporating a random intercept to account for variability across different groups and mitigate the risk of biased predictions. The model's ability to predict HLB incidence under varying climatic conditions provides a significant contribution to disease management, offering a strategic tool for early intervention and potentially reducing the spread of HLB. Using climatological and environmental data, the research aims to develop a predictive model, assessing the influence of these variables on the spread of Candidatus Liberibacter asiaticus, essential for effective disease management. The proposed flexible model demonstrates robust predictions for both training and test data, identifying climatological and environmental predictors influencing the dissemination of Candidatus Liberibacter asiaticus, the vascular bacterium associated with Huanglongbing (HLB) or greening.