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Citrus Huanglongbing (HLB, also known as "citrus greening"), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), a deadly citrus disease which has significantly downsized the entire industry worldwide. The intractable and incurable disease has brought the citriculture an enormous loss of productivity. With no resistant varieties available, failure of chemical treatments despite repeated applications, and hazardous consequences to environmental health, have led to large-scale research to find a sustainable cure. Inside plants, the key determinants of health and safety, live the endophytic microbes. Endophytes possess unrivaled plant benefiting properties. The progression of HLB is known to cause disturbance in endophytic bacterial communities. Given the importance of the plant endophytic microbiome in disease progression, the notion of engineering microbiomes through indigenous endophytes is attracting scientific attention which is considered revolutionary as it precludes the incompatibility concerns associated with the use of alien (microbes from other plant species) endophytes. In this review, we briefly discuss the transformation of the plant-pathogen-environment to the plant-pathogen-microbial system in a disease triangle. We also argue the employment of indigenous endophytes isolated from a healthy state to engineer the diseased citrus endophytic microbiomes that can provide sustainable solution for vascular pathogens. We evaluated the plethora of microbiomes responses to the re-introduction of endophytes which leads to disease resistance in the citrus host. The idea is not merely confined to citrus-HLB, but it is globally applicable for tailoring a customized cure for general plant-pathogen systems particularly for the diseases caused by the vascular system-restricted pathogens. [ABSTRACT FROM AUTHOR]

Citrus greening or Huanglongbing (HLB) has caused sweet orange (Citrus sinensis) yield in Florida to decrease by 55% since the disease was first discovered in 2005. As a consequence, the profitability and sustainability of citrus (Citrus sp.) production in Florida have been jeopardized, as evidenced by the 62% reduction in the number of citrus growers statewide. Because there is still no effective treatment or management strategy to cure the disease, it is crucial to optimize grove practices and management. The use of improved rootstocks could increase the tolerance of citrus scions to biotic and abiotic stresses, thereby allowing growers to cope better with the impact of HLB in the field. We used yield data collected from commercial trials over the course of multiple seasons to assess the side-by-side performance of various commercially available rootstocks developed by the two major breeding programs in Florida in HLB-endemic field conditions. We found that some of the rootstocks attained not only statistically significant differences in yield relative to the control but also meaningful differences in revenue. Those estimates provide evidence regarding the effect of rootstock during the first few seasons after planting. Our findings are useful to improve growers’ decision-making processes regarding rootstock selection for new groves.

Citrus huanglongbing (HLB) has been recognized for a century yet control and management remain elusive despite over 90 years of intensive research. The bacterial pathogen is an insect endosymbiont that was most likely inadvertently introduced into citrus where it found a compatible environment for growth in citrus phloem cells and therefore jumped from the animal to plant kingdom. Because the genus citrus did not coevolve with the bacteria it has no resistance and little tolerance to it and the resulting vascular disease is severe. The winged insect vector of the bacteria, the Asian citrus psyllid (ACP), is an exotic introduced species in its own right, prolific, and difficult to control even on a regional spatial scale. The resulting disease has a long latent period prior to symptom expression and a challenging cryptic period during which detection by convention PCR and other methods can be elusive. The result is an unusually rapid increase and spread of the resulting disease. This article offers some nonconventional perspectives to examine this unusual and devastating pathosystem to stimulate though toward improved control/mitigation.

BackgroundHuanglongbing (HLB) disease is still the greatest threat to citriculture worldwide. Although there is not any resistance source in the Citrus germplasm, a certain level of moderated tolerance is present. A large-scale analysis of proteomic responses of Citrus may help: 1) clarifying physiological and molecular effects of disease progression, 2) validating previous data at transcriptomic level, and 3) identifying biomarkers for development of early diagnostics, short-term therapeutics and long-term genetic resistance.ResultsIn this work we have conducted a proteomic analysis of mature leaves of two Citrus genotypes with well-known differing tolerances to HLB: Navel orange (highly susceptible) and Volkameriana (moderately tolerant). Pathway enrichment analysis showed that amino acid degradation processes occurred to a larger degree in the Navel orange. No clear differences between the two genotypes were observed for primary metabolic pathways. The most important finding was that four glutathione-S-transferases were upregulated in Volkameriana and not in Navel orange. These proteins are involved in radical ion detoxification.ConclusionsUpregulation of proteins involved in radical ion detoxification should be considered as an important mechanism of increased tolerance to HLB.

Essential nutrients for citrus [‘Bingo’ (Citrus reticulata, Blanco)] production are important for different functions, including photosynthesis, resistance to disease, and productivity. During the past 15 to 20 years, citrus production in Florida has significantly declined as a result of the devastating citrus greening disease also called huanglongbing (HLB). Therefore, a greenhouse study was conducted for 2 years, starting in 2018, at the University of Florida/Institute of Food and Agricultural Sciences Citrus Research and Education Center in Florida to evaluate the effect of varying rates of iron on the growth and development of 2-year-old HLB-affected ‘Bingo’ (Citrus reticulata, Blanco) trees on Kuharske citrange rootstock. Four treatments were used in a randomized complete block (HLB status) design with seven single tree replicates for each treatment. The treatments applied were 0.0 (control), 5.6 (standard fertilization, lx), 11.2 (2x), and 22.4 (4x) kg⋅ha−1 iron on HLB-affected and healthy (non-HLB) citrus trees. Data including trunk diameter, tree height, and leaf samples were collected, processed, and analyzed at 3-month intervals for 2 years. At the end of the second year, trees were destructively sampled and processed as above-ground and below-ground biomass. Tree heights were different among iron rates of HLB-affected trees (P < 0.001); however, they were similar for non-HLB trees for both years. Higher average trunk diameters (P < 0.001) were observed for HLB-affected trees that received the 2x rate compared with the 1x rate and the control. In 2019, non-HLB trees showed 13% to 40% higher iron concentrations in leaves than HLB-affected trees. However, leaf iron concentrations were comparable for HLB-affected and non-HLB trees in 2020. Above-ground biomass for HLB-affected trees had between 33% and 44% more biomass (P < 0.01) than below-ground biomass for the corresponding iron fertilization. Iron accumulation correlated positively with all studied nutrients in the above-ground parts for both HLB-affected and non-HLB trees. A 95% confidence interval at which total biomass was nearly maximum corresponded to an iron rate of 9.6 to 11.8 kg⋅ha−1, which was close to the 2x rate. Therefore, soil iron application using the aforementioned rates may be appropriate for better growth and development of young HLB-affected trees.

Comparison of the metabolic changes prior to symptom development upon infection with Candidatus Liberibacter asiaticus (CLas), the bacterium associated with citrus greening disease, between citrus hosts with different tolerances is lacking. The objective of this study was to compare the early response of Lisbon lemon (Citrus limon) and Washington navel orange (Citrus sinensis [L.] Osbeck), two citrus species commercially important to California, to CLas through graft inoculation. Here, we compare the transcriptome, proteome, and metabolome response, using RNA sequencing, liquid chromatography tandem mass spectrometry, and 1H nuclear magnetic resonance, respectively, from our two recently published studies examining the response of the lemon and navel oranges separately, and introduce new micronutrient data from inductively coupled plasma mass spectrometry analysis, focusing on lemons at 10 and 14 weeks postgrafting (wpg), and navels at 8 and 18 wpg, prior to symptom development. Several micronutrients accumulated in presymptomatic infected lemons compared with controls, whereas little change was observed in the navels. Photosynthesis proteins were substantially altered by CLas infection in navels, with fewer changes observed in lemons. The metabolome differed between control and infected navels throughout infection, although differences between control and infected lemons were identified only after symptom expression. Taken together, these findings highlight differences in response to CLas between two varieties with differing tolerances.[Figure: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

[Display omitted] • The toxicity of 17 pesticidal proteins was tested against D. citri adults. • Of these Tpp78Aa1 was toxic to D. citri adults with an LC 50 of 204 µg/mL. • CLas infection did not impact Tpp78Aa1 toxicity to D. citri. The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri , but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC 50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC 50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri -active proteins may provide valuable tools for suppression of D. citri populations. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a vascular disease of Citrus caused by three species of the α-proteobacteria “Candidatus Liberibacter”, with “Candidatus Liberibacter asiaticus” (CLas) being the most widespread and the one causing significant economic losses in citrus-producing regions worldwide. However, Persian lime (Citrus latifolia Tanaka) has shown tolerance to the disease. To understand the molecular mechanisms of this tolerance, transcriptomic analysis of HLB was performed using asymptomatic and symptomatic leaves. RNA-Seq analysis revealed 652 differentially expressed genes (DEGs) in response to CLas infection, of which 457 were upregulated and 195 were downregulated. KEGG analysis revealed that after CLas infection, some DEGs were present in the plant–pathogen interaction and in the starch and sucrose metabolism pathways. DEGs present in the plant–pathogen interaction pathway suggests that tolerance against HLB in Persian lime could be mediated, at least partly, by the ClRSP2 and ClHSP90 genes. Previous reports documented that RSP2 and HSP90 showed low expression in susceptible citrus genotypes. Regarding the starch and sucrose metabolism pathways, some genes were identified as being related to the imbalance of starch accumulation. On the other hand, eight biotic stress-related genes were selected for further RT-qPCR analysis to validate our results. RT-qPCR results confirmed that symptomatic HLB leaves had high relative expression levels of the ClPR1, ClNFP, ClDR27, and ClSRK genes, whereas the ClHSL1, ClRPP13, ClPDR1, and ClNAC genes were expressed at lower levels than those from HLB asymptomatic leaves. Taken together, the present transcriptomic analysis contributes to the understanding of the CLas-Persian lime interaction in its natural environment and may set the basis for developing strategies for the integrated management of this important Citrus disease through the identification of blanks for genetic improvement.

Diaphorina citri Kuwayama (D. citri) is an insect vector of phloem-limited ‘Candidatus Liberibacter asiatus’ (CLas), the presumed pathogen of citrus Huanglongbing (HLB). Recently, our lab has preliminarily found it acquired and transmitted Citrus tristeza virus (CTV), which was previously suggested to be vectored by species of aphids. However, the influences of one of the pathogens on the acquisition and transmission efficiency of the other pathogen remain unknown. In this study, CLas and CTV acquisition and transmission by D. citri at different development stages under field and laboratory conditions were determined. CTV could be detected from the nymphs, adults, and honeydew of D. citri but not from the eggs and exuviates of them. CLas in plants might inhibit CTV acquisition by D. citri as lower CTV–positive rates and CTV titers were detected in D. citri collected from HLB-affected trees compared to those from CLas–free trees. D. citri were more likely to obtain CTV than CLas from host plants co-infected with the two pathogens. Intriguingly, CTV in D. citri facilitated the acquisition and transmission of CLas, but CLas carried by D. citri had no significant effect on the transmission of CTV by the same vector. Molecular detection and microscopy methods confirmed the enrichment of CTV in the midgut after a 72-h acquisition access period. Collectively, these results raise essential scientific questions for further research on the molecular mechanism of pathogen transmission by D. citri and provide new ideas for the comprehensive prevention and control of HLB and CTV.

Huanglongbing (HLB) is one of the most severe citrus diseases in the world, causing huge economic losses. However, efficient methods of protecting citrus from HLB have not yet been developed. microRNA (miRNA)-mediated regulation of gene expression is a useful tool to control plant diseases, but the miRNAs involved in regulating resistance to HLB have not yet been identified. In this study, we found that miR171b positively regulated resistance to HLB in citrus. Upon infection with HLB bacteria, the bacteria were detected in the second month in the control plants. However, in the miR171b-overexpressing transgenic citrus plants, the bacteria could not be detected until the 24th month. RNA-seq data indicated that multiple pathways, such as photosynthesis, plant–pathogen interaction, the MAPK signaling pathway, etc., might be involved in improving the resistance to HLB in miR171b-overexpressing plants compared with the control. Finally, we determined that miR171b could target SCARECROW-like (SCL) genes to downregulate their expression, which then led to promoted resistance to HLB stress. Collectively, our results demonstrate that miR171b plays a positive regulatory role in resistance to citrus HLB, and provides a new insight into the role of miRNAs in the adaptation of citrus to HLB stress.

Huanglongbing (HLB, or citrus greening) has been one of the devastating diseases in citrus production. The infection of HLB can seriously affect the photosynthetic capacity and growth of citrus plants. Thus, early, accurate, and rapid in-situ diagnosis of HLB can be highly critical to reducing the economic losses for the citrus industry. This study aims to develop an in-situ diagnosis of citrus HLB disease for the photosynthetic response of citrus leaves using sun-induced chlorophyll fluorescence. Four groups were labelled on the citrus trees with the asymptomatic HLB (aHLB), symptomatic HLB (sHLB), and macular (with symptoms similar to HLB) infection, as well as the healthy trees in the orchard. A Li-6800 portable photosynthetic system was utilized to measure the photosynthetic parameters (net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), transpiration rate (Tr), stomatal conductance (Gs)) of citrus leaf samples. The photosynthetic CO2 response curve (A-Ci) was obtained under the different carbon dioxide concentrations, further to calculate the maximum leaf carboxylation rate (Vcmax) and the maximum electron transfer rate (Jmax). Then, the upward and downward sun-induced chlorophyll fluorescence (SIF) and reflectance spectra of leaf samples were collected using an analytical spectral devices (ASD) spectrometer combined with a FluoWat clip. Finally, the photosynthetic pigments content (chlorophyll a, chlorophyll b, and carotenoids) of the leave samples were measured using the spectrophotometry in the laboratory, and the true infection status of the leaves was confirmed using the real-time quantitative polymerase chain reaction (qPCR). A systematic investigation was made on the photosynthetic parameters and the pigments content of the leave samples. An optimal combination of wavebands was selected using the competitive adaptive reweighted sampling (CARS) algorithm and reflectance spectra. The upward (Up) and downward (Dw) SIF yield indices (Up687, Up741, Dw687, Dw741, Up687/741, Dw687/741) were constructed using the peak position (687 nm and 741 nm) of the SIF spectra. Furthermore, the classification models of HLB were established to combine with the K-nearest neighbor (KNN) algorithm, according to the optimal wavebands of reflectance spectra and SIF yield indices. The results showed that the infection of HLB pathogen led to a significant decrease in the photosynthesis of citrus leaves even at the asymptomatic stage, indicating an excellent performance of SIF signals in the early diagnosis of HLB. The diagnostic accuracies of KNN models with the leaf reflectance using the optimal wavebands were 72.7% and 75.6% for aHLB and sHLB leaves, and 82.2% and 64.1% for healthy and macular leaves, respectively. By contrast, the diagnostic accuracies of KNN models using the Up687/741 (ratio of upward SIF yield at 687 nm to 741 nm) SIF yield index for aHLB and sHLB were 84.8% and 91.1%, and that of healthy leaves and macular leaves were 88.9% and 82.1%, respectively. Consequently, the KNN models with the leaf SIF spectra presented a higher potential in the early diagnosis of HLB than those with leaf reflectance spectra. These findings can provide a strong reference for the early, rapid, and in-situ diagnosis of citrus HLB in the incubation period. [ABSTRACT FROM AUTHOR]

Citrus greening (HLB) is an incurable bacterial disease severely affecting most citrus production regions. Evaluating the economic feasibility of control practices is challenging due to the complex intertemporal interactions among the pathogen, the vector, and the host. We propose a stochastic evaluation framework to systematically analyze the long-term economic performance of a broad range of management strategies. Different control approaches are evaluated in a hypothetical application in Texas. Results highlight the detrimental effects of the disease and the importance of developing cost-effective control options. A substantial loss in value is expected regardless of the intervention actions implemented. [ABSTRACT FROM AUTHOR]

Microorganisms have dynamic and complex interactions with their hosts. Diverse microbial communities residing near, on, and within the plants, called phytobiome, are an essential part of plant health and productivity. Exploiting citrus-associated microbiomes represents a scientific approach toward sustained and environment-friendly module of citrus production, though periodically exposed to several threats, with Huanglongbing (HLB) predominantly being most influential. Exploring the composition and function of the citrus microbiome, and possible microbial redesigning under HLB disease pressure has sparked renewed interest in recent times. A concise account of various achievements in understanding the citrus-associated microbiome, in various niche environments viz., rhizosphere, phyllosphere, endosphere, and core microbiota alongside their functional attributes has been thoroughly reviewed and presented. Efforts were also made to analyze the actual role of the citrus microbiome in soil fertility and resilience, interaction with and suppression of invading pathogens along with native microbial communities and their consequences thereupon. Despite the desired potential of the citrus microbiota to counter different pathogenic diseases, utilizing the citrus microbiome for beneficial applications at the field level is yet to be translated as a commercial product. We anticipate that advancement in multiomics technologies, high-throughput sequencing and culturing, genome editing tools, artificial intelligence, and microbial consortia will provide some exciting avenues for citrus microbiome research and microbial manipulation to improve the health and productivity of citrus plants. [ABSTRACT FROM AUTHOR]

Objective: 'Candidatus Liberibacter asiaticus' (CLas) is associated with the devastating citrus 'greening' disease. All attempts to achieve axenic growth and complete Koch's postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic 'Ca. Liberibacter' spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes. Results: Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of 'essential' Lcr genes to CLas. [ABSTRACT FROM AUTHOR]

Citrus Huanglongbing (HLB) is the most devastating disease of citrus caused by the Gram-negative phloem-limited bacterium " Candidatus Liberibacter asiaticus" (CLas). It can be transmitted by the Asian citrus psyllid " Diaphorina citri ," by grafting, and by the holoparasitic dodder. In this study, the non-natural host periwinkle (Catharanthus roseus) was infected via dodder (Cuscuta campestris) from CLas-infected citrus plants, and the asymptomatic leaves (AS) were subjected to transcriptomic and small-RNA profiling. The results were analyzed together with a transcriptome dataset from the NCBI repository that included leaves for which symptoms had just occurred (S) and yellowing leaves (Y). There were 3,675 differentially expressed genes (DEGs) identified in AS, and 6,390 more DEGs in S and further 2109 DEGs in Y. These DEGs were commonly enriched in photosystem, chloroplast, membrane, oxidation-reduction process, metal/zinc ion binding on GO. A total of 14,974 DEGs and 336 DE miRNAs (30 conserved and 301 novel) were identified. Through weighted gene co-expression network and nested network analyses, two critical nested miRNA–mRNA regulatory networks were identified with four conserved miRNAs. The primary miR164-NAC1 network is potentially involved in plant defense responses against CLas from the early infection stage to symptom development. The secondary network revealed the regulation of secondary metabolism and nutrient homeostasis through miR828-MYB94/miR1134-HSF4 and miR827-ATG8 regulatory networks, respectively. The findings discovered new potential mechanisms in periwinkle–CLas interactions, and its confirmation can be done in citrus–CLas system later on. The advantages of periwinkle plants in facilitating the quick establishment and greater multiplication of CLas, and shortening latency for disease symptom development make it a great surrogate for further studies, which could expedite our understanding of CLas pathogenesis. [ABSTRACT FROM AUTHOR]

Improving nutrient uptake and tree health play an important role in managing Huanglongbing (HLB)-affected citrus trees in Florida. A greenhouse experiment was conducted to evaluate the effect of increasing rates of manganese (Mn) on growth and development of sweet orange [Citrus sinensis (L.) Osbeck] trees at the University of Florida's Institute of Food and Agricultural Sciences (UF/IFAS) Citrus Research and Education Center in Lake Alfred, FL. Half the trees were graft-inoculated with the HLB pathogen and the remainder were used as the HLB-free (non HLB) control trees. Four rates of Mn (0.0 kg·ha-1 Mn (Control), 5.6 kg·ha-1 Mn (1x--standard rate), 11.2 kg·ha-1 Mn (2x--standard rate), and 22.4 kg·ha-1 Mn (4x--standard rate) were split applied quarterly to both sets of the trees in a completely randomized design. There were seven single tree replicates for each treatment. Response variables measured were trunk diameter, tree height, leaf Mn concentration, plus above- and belowground biomass. The accumulated Mn in leaf tissues significantly increased trunk diameter but did not affect tree height for both HLB-affected and non-HLB trees, the 2x rate had the maximum value for trunk diameter relative to the 4x rate. This study established a positive correlation between soil available Mn with Fe and Cu, but negative correlation with B and Zn. A strong correlation of -0.76, -0.69, and 0.65 was observed between soil Mn and B, Zn, and Cu, respectively, as compared with 0.49 with Mn and Fe. Among HLB-affected trees, the 2x rate gave the most belowground dry matter, which was 3% greater than the control and 5% greater than 4x. Aboveground dry matter had at least 30% more biomass than belowground matter among all treatments within HLB-affected trees. For small and medium roots, Mn accumulation increased with Mn application until 2x rate and decreased thereafter for HLB-affected trees. The results from our study showed an Mn rate of 8.9-11.5 kg·ha-1 Mn, as the optimum Mn level for young 'Valencia' HLB-affected trees in Florida. [ABSTRACT FROM AUTHOR]

Simple Summary: Citrus cultivation is affected in many parts of the world because of a devastating disease, huanglongbing (HLB) or citrus greening. In Florida, nearly all commercial citrus is compromised due to HLB, and the disease has spread to other citrus-growing regions of the United States, California and Texas. In California, testing Asian citrus psyllids (ACPs) for the HLB pathogen has been an essential part of integrated pest management. ACP and HLB surveys are essential for disease management in areas where HLB is not widespread. We developed improved ACP traps that can be deployed in the field along with the standard yellow sticky traps. The reusable traps were designed with Rhinoceros computer software and a 3D printer. These traps can be deployed for several months and provide a dynamic sampling mechanism for an improved disease survey strategy. In the present study, ACPs from the 3D-printed traps are collected in a preservative and appear suitable for HLB testing. The evaluation of traps in Florida and California under laboratory, greenhouse, and field conditions indicates that the 3D-printed traps can capture ACPs with about the same efficiency as the sticky traps. They are easy to handle and provide an important field tool for HLB management. Citrus huanglongbing (HLB) is a severe problem for citrus cultivation. The disease management programs benefit from improved field tools suitable for surveying the ACP vector (Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae)) and the associated pathogen. In the present study, we utilize three-dimensional (3D) printers and design tools to develop traps that can capture and preserve ACPs. Three novel, 3D-printed traps were designed and evaluated: stem trap, and cylinder traps 1 and 2. The traps and yellow sticky cards were deployed weekly for 8 months in 2 non-commercial citrus groves in Florida; in California, the traps were evaluated for 12 months in field cages and 4 citrus groves. The stem traps captured lower numbers of ACPs at all experimental sites compared to the cylinder traps. Capture rates in the cylinder traps were comparable to the sticky trap, making the device a viable tool for monitoring field ACPs. The two main advantages of using the reusable 3D traps over standard methods of ACP and HLB surveys include dynamic sampling that can be conducted year-round and the capture of ACPs that can be preserved and tested. Improved trapping may facilitate quick management decisions and mitigate HLB. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is an important plant hormone, which can control seed and bud dormancy, organ size control, senescence and death, and participate in both biological and abiotic stress, inhibit plant growth, and participate in plant disease resistance. In order to determine the content of ABA in plants quickly and accurately, a new type of ABA immunosensor was developed. To improve the detection performance of the sensor, the detection performance of the sensor was increased by modifying GR-COOH and SA on the electrode surface. The concentration of GR-COOH, SA, and ABA-Antibody were optimized, the optimal conditions for the three materials were 1.5 mg/ml, 1.25 mg/ml and 0.5 mg/ml. The immunosensor was constructed based on the electrode impedance changes (△Z ) due to the binding reaction of ABA antibody and antigen . It was found that the sensor showed linear relationship with ABA in the response range of 10 pmol/L~1 μmol/L, R2 was 0.99927, and the detection limit was about 10 pmol/L. The sensor also had good selectivity and stability. Using the electrochemical immunosensor, the content of ABA in navel orange leaf that have been successfully inoculated with citrus Huanglongbing by PCR was determined, and healthy plants were used as control. The test results showed that the impedance changes(△Z ) of healthy leaves and diseased leaves were 72 and 823, respectively, which indicated that the level of ABA in the infected plants increased significantly. The sensor provides a tool for the detection of plant hormone levels under disease stress. The results showed that the content of ABA increased in the leaves of navel orange infected by citrus Huanglongbing, which indicated that ABA played an important role in plant disease resistance. Furthermore, the changes of gene expression of key enzymes CitZEP in ABA synthesis pathway were studied, The results showed that the expression of CitZEP increased in plants infected with Huanglongbing disease, and the results were consistent with the detection results of the sensor, which indicated that the sensor had good practicability. [ABSTRACT FROM AUTHOR]

The "citrus greening disease" or huanglongbing (HLB) is caused by the Gram-negative bacteria Candidatus Liberibacter spp. One vector of HLB, the African citrus psyllid Trioza erytreae (del Guercio, 1918), was recorded for the first time in 2014, in mainland Spain, and since then, it has spread throughout the Iberian Peninsula. Spiders could be natural enemies of T. erytreae. In this work, we assessed the potential of a model spider species, selected according to the most abundant guild in the field, as a natural enemy of T. erytreae. Theridiidae was the most successful family, capturing adults of T. erytreae in the field. Kochiura aulica (Theridiidae) was selected as a model for functional response assays in the laboratory. The effect of web-building plasticity on the functional response of K. aulica was investigated using two types of arena. The spiders developed tubular-like or branch-like webs according to the spatial constraints. The web architecture significantly shifted the trapping efficiency of K. aulica. Functional responses for tubular and branch webs were both Type-II, but more psyllid prey were captured and killed in the tubular webs than in branch webs. In addition, significantly more psyllids survived in branch-like webs. Our results suggest that web architecture is a key factor driving the number of captured adults of the psyllid. Space web builders could be successful natural enemies of T. erytreae, although further research is needed to clarify the influence of web-building plasticity on the functional response and trapping efficiency of spiders. [ABSTRACT FROM AUTHOR]

Heat treatment has been applied in previous studies to treat diseased plants and trees affected by heat-sensitive pathogens. Huanglongbing (HLB) is a heat-sensitive pathogen and the optimal temperature–time for treating HLB-affected citrus trees was estimated to be 54 °C for 60 to 120 s from indoor experimental studies. However, utilizing this method in orchards is difficult due to technical difficulties to effectively apply heat. Recently, a mobile thermotherapy system (MTS) was developed to in-field treat HLB-affected trees. This mobile device includes a canopy cover that covers the diseased tree and a system to supply steam under the cover to treat the tree. It was proven that the temperature inside the canopy cover can reach the desired one (i.e., 54 °C) to kill bacteria. However, for HLB, the heat should penetrate the tree’s phloem where the bacteria live. Therefore, measuring the heat penetration inside the tree is very critical to evaluate the performance of the MTS. In this study, a heat transfer model was developed to simulate the heat penetration inside the tree and predict the temperature in the phloem of the diseased tree during the in-field heat treatment. The simulation results were compared with in-field experimental measurements. The heat transfer model was developed by a comparative analysis of the experimental data using the ANSYS software. Results showed that the temperature in the phloem was 10–40% lower than the temperature near the surface of the bark. Simulation results were consistent with experimental results, with an average relative error of less than 5%.

Abstract Background The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect pest species responsible for damages of citrus fruit quality and tree health. This insect is a vector of bacteria ‘Candidatus Liberibacter’ spp. a putative causal agent of citrus greening disease known as Huanglongbing (HLB), considered one of the most destructive diseases of citrus orchards worldwide. Disease management is mainly based on vector control using pesticides which can affect natural enemies that play an important role in pest control. The entomopathogenic fungi (EPF) Beauveria bassiana (2067 and 2121) and Metarhizium anisopliae (2411) were achieved by applying a suspension of 1 × 107 spores against D. citri nymphs and artificially infected the citrus seedlings under controlled and semi-field conditions. Also, the activity of these EPFs on the coccinellid and lacewing predators was evaluated. The effects of the main chemicals used in citrus plantations on the selected fungi for possible combined use was also investigated. Results The obtained results showed that under controlled conditions, the percentage of insect mortality produced by EPF varied between 82.8 and 85.9%. Under semi-field conditions, the infection and mortality of D. citri caused by the M. anisopliae 2411 strain was significantly higher than that of B. bassiana 2067, 78.9 and 51%, respectively. Non-significant effect of EPF on the natural enemies of D. citri was observed, suggesting that EPF and predators had compatible effects. Conclusion This study clearly demonstrated that EPF were able to kill D. citri and can be explored as a promissory biocontrol candidate. Simultaneous use in an integrated pest management program could be possible by applying EPF combined with pesticides.

Huanglongbing (HLB), which is mainly associated with “Candidatus Liberibacter asiaticus” (CLas), is currently threatening citrus production around the world. This destructive century-old disease results in callose deposition in phloem plasmodesmata and starch overaccumulation in leaf chloroplasts. Heat treatment with high humidity has proven to be effective in eliminating the CLas titers in potted citrus trees. This study explored the curative mechanisms and impact of heat treatment (from 32 to 48 °C with 75% ± 5% relative humidity, 14 h in light and 10 h in dark for four days) on mandarin trees (Citrus reticulata Blanco) that were severely infected by CLas using Solexa/Illumina's digital gene expression (DGE) profiling. A total of 838 differentially expressed genes (DEGs) were identified, of which 510 were downregulated. Protein-protein interaction analysis of the DEGs indicated that the thermotherapy of HLB trees downregulated the circadian clock related genes in chloroplasts and upregulated the RNA-editing process in the mitochondria, characterized by 141 genes encoding pentatricopeptide repeat-containing proteins (PPR) being all upregulated. Furthermore, the expression of phenylpropanoid and flavonoid biosynthesis genes, and auxin-induced genes were suppressed. The defense response pathway of the thermotherapy treated diseased trees was apparently disrupted, as indicated by the downregulation of LRR receptor-like serine/threonine-protein kinase FLS2 gene. This study improves our understanding of the curative mechanisms of heat treatment on HLB trees and CLas biological regulation. Importantly, this provides new insights on the involvement of RNA modification by PPR genes and changes in the circadian clock after the heat stress treatment of CLas-infected citrus.

Huanglongbing (HLB) is an incurable disease that affects citrus trees. To better understand the transmission of HLB, the mathematical model is developed to investigate the transmission dynamics of the disease between Asian citrus psyllid (ACP) and citrus trees. Through rigorous mathematical derivations, we derive the expression of the basic reproduction number (R0) of HLB. The findings show that the disease-free equilibrium is locally asymptotically stable if R0 1 the system is uniformly persistent. By applying the global sensitivity analysis of R0, we can obtain some parameters that have the greatest influence on the HLB transmission dynamics. Additionally, the optimal control theory is used to explore the corresponding optimal control problem of the HLB model. Numerical simulations are conducted to reinforce the analytical results. These theoretical and numerical results provide useful insights for understanding the transmission dynamics of HLB and may help policy makers to develop intervention strategies for the disease.

Citrus Huanglongbing (HLB, also known as “citrus greening”), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus.

Microorganisms have dynamic and complex interactions with their hosts. Diverse microbial communities residing near, on, and within the plants, called phytobiome, are an essential part of plant health and productivity. Exploiting citrus-associated microbiomes represents a scientific approach toward sustained and environment-friendly module of citrus production, though periodically exposed to several threats, with Huanglongbing (HLB) predominantly being most influential. Exploring the composition and function of the citrus microbiome, and possible microbial redesigning under HLB disease pressure has sparked renewed interest in recent times. A concise account of various achievements in understanding the citrus-associated microbiome, in various niche environments viz., rhizosphere, phyllosphere, endosphere, and core microbiota alongside their functional attributes has been thoroughly reviewed and presented. Efforts were also made to analyze the actual role of the citrus microbiome in soil fertility and resilience, interaction with and suppression of invading pathogens along with native microbial communities and their consequences thereupon. Despite the desired potential of the citrus microbiota to counter different pathogenic diseases, utilizing the citrus microbiome for beneficial applications at the field level is yet to be translated as a commercial product. We anticipate that advancement in multiomics technologies, high-throughput sequencing and culturing, genome editing tools, artificial intelligence, and microbial consortia will provide some exciting avenues for citrus microbiome research and microbial manipulation to improve the health and productivity of citrus plants.

Citrus Huanglongbing (HLB) is the most devastating disease of citrus caused by the Gram-negative phloem-limited bacterium “Candidatus Liberibacter asiaticus” (CLas). It can be transmitted by the Asian citrus psyllid “Diaphorina citri,” by grafting, and by the holoparasitic dodder. In this study, the non-natural host periwinkle (Catharanthus roseus) was infected via dodder (Cuscuta campestris) from CLas-infected citrus plants, and the asymptomatic leaves (AS) were subjected to transcriptomic and small-RNA profiling. The results were analyzed together with a transcriptome dataset from the NCBI repository that included leaves for which symptoms had just occurred (S) and yellowing leaves (Y). There were 3,675 differentially expressed genes (DEGs) identified in AS, and 6,390 more DEGs in S and further 2109 DEGs in Y. These DEGs were commonly enriched in photosystem, chloroplast, membrane, oxidation-reduction process, metal/zinc ion binding on GO. A total of 14,974 DEGs and 336 DE miRNAs (30 conserved and 301 novel) were identified. Through weighted gene co-expression network and nested network analyses, two critical nested miRNA–mRNA regulatory networks were identified with four conserved miRNAs. The primary miR164-NAC1 network is potentially involved in plant defense responses against CLas from the early infection stage to symptom development. The secondary network revealed the regulation of secondary metabolism and nutrient homeostasis through miR828-MYB94/miR1134-HSF4 and miR827-ATG8 regulatory networks, respectively. The findings discovered new potential mechanisms in periwinkle–CLas interactions, and its confirmation can be done in citrus–CLas system later on. The advantages of periwinkle plants in facilitating the quick establishment and greater multiplication of CLas, and shortening latency for disease symptom development make it a great surrogate for further studies, which could expedite our understanding of CLas pathogenesis.

Abstract Although insecticides are essential for the effective pest management, the development of sustainable formulations remains a challenge in modern agriculture. This work focuses on the encapsulation of thiamethoxam in polymeric micelles, a nanodelivery system. The nanomicelles, prepared by thin-film hydration method, presented 94.5 ± 10.26% of encapsulation efficiency, average size of 85.14 ± 2.23 nm, zeta potential of −5.3 mV, and acceptable homogenous distribution. Water solubility of thiamethoxam nanomicelles showed an increase by 41.7% in relation of thiamethoxam compound. Toxicity assessment was applied to Raphidocelis subcapitata Korshikov and Artemia salina Linnaeus organisms. The EC50 values obtained for microalgae of polymeric nanomicelles > polymeric nanomicelles loaded thiamethoxam > commercial pesticide. EC50 values obtained for Artemia salina in all treatments could not be compared due to lack of sensitivity shown by this organism to the adopted treatments. Nanoformulation effectiveness was evaluated against D. citri caged in citrus plants grown under greenhouse conditions, in comparison to that of standard formulations. Results have suggested that thiamethoxam nanomicelles might be effective in controlling D. citri around dose 2-fold lower than commercial formulations. Highlights Nanomicelles are prepared based on Pluronic ® F127 and Pluronic® P123 Thiamethoxam is effectively encapsulated in micelle nanostructures Thiamethoxam nanoinsecticide is tested against Diaphorina citri Kuwayana Nanomicelles are prepared based on Pluronic ® F127 and Pluronic® P123Thiamethoxam is effectively encapsulated in micelle nanostructuresThiamethoxam nanoinsecticide is tested against Diaphorina citri Kuwayana [ABSTRACT FROM AUTHOR]

Huanglongbing is caused by Candidatus Liberibacter asiaticus (C Las) and transmitted by Diaphorina citri. D. citri harbors various insect-specific viruses, including the Diaphorina citri flavi-like virus (DcFLV). The distribution and biological role of DcFLV in its host and the relationship with C Las are unknown. DcFLV was found in various organs of D. citri , including the midgut and salivary glands, where it co-localized with C Las. C Las-infected nymphs had the highest DcFLV titers compared to the infected adults and C Las-free adults and nymphs. DcFLV was vertically transmitted to offspring from female D. citri and was temporarily detected in Citrus macrophylla and grapefruit leaves from greenhouse and field. The incidences of DcFLV and C Las were positively correlated in field-collected D. citri samples, suggesting that DcFLV might be associated with C Las in the vector. These results provide new insights on the interactions between DcFLV, the D. citri , and C Las. • Diaphorina citri flavi-like virus (DcFLV) spreads systemically and transmitted vertically in D. citri. • DcFLV and Candidatus Liberibacter asiaticus (CLas) accumulate in D. citri midguts and salivary glands. • DcFLV and CLas co-occur in field collected psyllids. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a serious threat to the U.S. citrus industry because it spreads huanglongbing (HLB), a bacterial and incurable citrus disease. The nymphal parasitoid, Tamarixia radiata Waterston (Hymenoptera: Eulophidae), has been extensively released for biological control of D. citri in South Texas since 2010 but provides insufficient control. An 'attract-and-kill' (AK) device was evaluated for its compatibility with T. radiata for suppression of D. citri on dooryard citrus. The AK device is visually attractive to D. citri adults because it is the same color as young citrus flush and kills individuals on contact with the toxicant beta-cyfluthrin. This study evaluated 1) lethality of AK devices to T. radiata adults under lab conditions; 2) efficacy of AK devices for year-round psyllid suppression on individual dooryard lemon trees; 3) discovery and parasitism of D. citri colonies by T. radiata on lemon trees with or without AK devices. Contact with AK devices for 5 s or more was lethal to adult parasitoids. Deployment of 20 AK devices per tree provided significant year-round suppression of D. citri on infested lemon trees and reduced mean attack intensity (cumulative psyllid-days) of adults by 66% and nymphs by 82%. Discovery and parasitism rates of D. citri colonies by T. radiata were similar on control trees and trees protected by AK devices. An AK device that targets adult psyllids could be used to effectively complement biological control of D. citri by T. radiata in residential landscapes. Graphical Abstract [ABSTRACT FROM AUTHOR]

Citrus producers in many countries are facing serious problems with the emergence of citrus huanglongbing (HLB). This study was conducted to 1) detect the HLB pathogen complex in Upper Awash citrus production areas; 2) detect the presence of insect vectors of the pathogen and 3) understand the relationship between huanglongbing and nutritional profile of citrus. Leaf samples were collected from symptomatic orange and mandarin trees. Insect were collected using sticky trap and stem-Tap sampling techniques. Total DNA was isolated from leaves. Real time PCR was used to screen for the presence of 'Candidatus Liberibacter' species while conventional PCR was done to detect particular species. Field history was recorded for nutrient deficiency and amendments, as well as symptoms associated with deficiency. Leaf tissue analysis was conducted to quantify the level of different elements in HLB infected and none infected samples. All citrus species and varieties were found to be vulnerable to HLB infection. The result was verified by real time PCR that indicated the presence of 'Ca. Liberibacter' species. All conventional PCR tests indicated the presence of 'Ca. Liberibacter asiaticus'. No other species were detected in the samples. The pathogen was primarily transmitted by pruning materials and Diaphorina citri. The pathosystem was associated with nutrient deficiency such as decrease in zinc in which the management has shown to aggravate the disease pressure. The study provided insights on the distribution, insect vector and nutritional profiles of HLB infection on citrus trees. Future studies should focus on other marker data to determine the nature of origin and variability of this important citrus pathogen in Ethiopia. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a highly destructive disease that decreases the yield and quality of Citrus medica L. var. sarcodactylis Swingle (C. medica var. sarcodactylis) and poses a great threat to the development of the global citrus industry. To explore the influence of HLB infection on C. medica var. sarcodactylis, levels of photosynthetic pigments, malondialdehyde (MDA), and carbohydrates, as well as antioxidant enzyme activities, were measured. The results show that HLB infection decreased photosynthetic pigment content, increased MDA content and antioxidant enzyme activities, and caused various changes in carbohydrate levels in stem, fruit, and leaf tissues. Transcriptomic analysis of C. medica var. sarcodactylis was also used to identify key genes related to the carbohydrate metabolic synthesis pathway in C. medica var. sarcodactylis. The C. medica var. sarcodactylis ADP-glucose pyrophosphorylase1 (CmAGP1), CmAGP2, C. medica var. sarcodactylis Granule-bound starch synthase (CmGBSS), C. medica var. sarcodactylis Sucrose synthases1 (CmSUS1), CmSUS2, C. medica var. sarcodactylis Sucrose phosphate synthase (CmSPS), C. medica var. sarcodactylis alkaline/neutral invertase1 (CmNi1), CmNi2, CmNi3 and CmNi4 were successfully cloned and identified, and differential expression analysis showed that HLB infection significantly upregulated these genes in stems and leaves. In conclusion, HLB infection causes cellular damage, a reduction in photosynthetic capacity, decreased pathogen resistance, and severe disorders in carbohydrate metabolism in C. medica var. sarcodactylis. [ABSTRACT FROM AUTHOR]

‘Candidatus Liberibacter asiaticus’ (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host. [ABSTRACT FROM AUTHOR]

‘Candidatus Liberibacter asiaticus’ is associated with the devastating citrus disease Huanglongbing (HLB). It is transmitted by grafting infected material to healthy plants and by the feeding of the Asian citrus psyllid (Diaphorina citri). Previously, we demonstrated that a metabolomics approach using proton-nuclear magnetic resonance spectroscopy discriminates healthy from diseased plants via grafting. This work assessed the capability of this technology in discriminating healthy and diseased plants when the bacterium is vectored by psyllids. One-year-old greenhouse-grown ‘Lisbon’ lemon trees were exposed to either carrier psyllids (exposed, n = 10), or psyllids that were free of ‘Candidatus Liberibacter asiaticus’ (control, n = 6). Leaf metabolites were tracked for 1 year and disease diagnosis was made using quantitative PCR. Overall, 31 water-soluble metabolites were quantified in leaves, including four sugars and 12 amino acids. Analysis via nonmetric multidimensional scaling and principal component analysis revealed significant differences between the leaf metabolome of control versus infected trees beginning at 8 weeks postexposure, including alterations in glucose and quinic acid concentrations. These findings provide a longitudinal overview of the metabolic effects of HLB during the early phases of disease, and confirm previous experimental work demonstrating that infection elicits changes in the leaf metabolome that enables discrimination between healthy and infected plants. Here we demonstrate that the mode of inoculation (i.e., graft versus psyllid) results in a similar pathology. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), or citrus greening disease, has significantly decreased citrus production all over the world. The disease management currently depends on the efficient application and adequate distribution of insecticides to reduce the density of the disease vector, the Asian citrus psyllid. Here, we use a novel fluorescent-based method to evaluate insecticide distribution in an HLB-infected citrus grove in Florida. Specifically, we evaluated six different locations within citrus trees, the top and bottom sides of leaves, the effect of application approach (tractor versus airplane), and different application rates. We found that despite the insecticide distribution being highly variable among the different locations within a tree, the top of the leaves received an average increase of 21 times more than the bottom of the leaves. Application by tractor also resulted in a 4- to 87-fold increase in insecticide coverage compared with aerial application, depending on the location in the tree and side of the leaf. When taken to context with the type of insecticide that is applied (systemic vs. contact), these results can be used to optimize a pest management strategy to effectively target psyllids and other pests while minimizing the time and money spent on insecticide application and reducing risk to the environment. [ABSTRACT FROM AUTHOR]

‘Candidatus Liberibacter asiaticus’, the putative causal agent of citrus greening, is not available in pure culture yet. In addition to trees of citrus and citrus relatives, ‘Ca. L. asiaticus’ can grow in Madagascar periwinkle (Catharanthus roseus). Using gas chromatography-mass spectrometry, we compared the phloem sap composition in sweet orange ‘Valencia’ (Citrus sinensis) and periwinkle plants after the infection with ‘Ca. L. asiaticus’. Interestingly, in contrast to our previous studies of total leaf metabolites, we found that, compared with uninfected phloem sap, the organic acids implicated in the tricarboxylic acid cycle (TCA) cycle including citrate, isocitrate, succinate, fumarate, and malate were reduced significantly in the infected phloem saps of both species. As a result of the reduction of organic acids content, the pH of infected phloem saps was increased. We hypothesize that the bacterial growth induces the mitochondrial TCA cycle in parenchyma cells to produce more of these compounds to be used as a bacterial carbon source. Once these compounds reach a low level in the phloem sap, the bacterium may send a signal, yet to be identified, to initiate a feedback loop to further induce the TCA cycle. Phloem blockage might be another reason behind the reduced translocation of TCA cycle intermediates within the phloem. The net result, localized availability of organic acids, likely benefits bacterial growth and may explain the unequal distribution of ‘Ca. L. asiaticus’ within infected trees. These findings may help in designing media for the pure culturing of ‘Ca. L. asiaticus’. [ABSTRACT FROM AUTHOR]

Citrus greening, or huanglongbing (HLB), currently is the most destructive disease of citrus. HLB disease is putatively caused by the phloem-restricted α-proteobacterium ‘Candidatus Liberibacter asiaticus’. This bacterium is transmitted primarily by the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). Most animal pathogens are considered pathogenic to their insect vectors, whereas the relationships between plant pathogens and their insect vectors are variable. Lately, the relationship of ‘Ca. L. asiaticus’ with its insect vector, D. citri, has been well investigated at the molecular, biochemical, and biological levels in many studies. Herein, the findings concerning this relationship are discussed and molecular features of the acquisition of ‘Ca. L. asiaticus’ from the plant host and its growth and circulation within D. citri, as well as its transmission to plants, are presented. In addition, the effects of ‘Ca. L. asiaticus’ on the energy metabolism (respiration, tricarboxylic acid cycle, and adenosine triphosphate production), metabolic pathways, immune system, endosymbionts, and detoxification enzymes of D. citri are discussed together with other impacts such as shorter lifespan, altered feeding behavior, and higher fecundity. Overall, although ‘Ca. L. asiaticus’ has significant negative effects on its insect vector, it increases its vector fitness, indicating that it develops a mutualistic relationship with its vector. This review will help in understanding the specific interactions between ‘Ca. L. asiaticus’ and its psyllid vector in order to design innovative management strategies. [ABSTRACT FROM AUTHOR]

Background Huanglongbing, a devastating disease of citrus, is caused by the obligate, intracellular bacterium "Candidatus Liberibacter asiaticus" (C Las). C Las is transmitted by Diaphorina citri , the Asian citrus psyllid. Development of transmission-blocking strategies to manage huanglongbing relies on knowledge of C Las and D. citri interactions at the molecular level. Prior transcriptome analyses of D. citri point to changes in psyllid biology due to C Las infection but have been hampered by incomplete versions of the D. citri genome, proper host plant controls, and/or a lack of a uniform data analysis approach. In this work, we present lessons learned from a quantitative transcriptome analysis of excised heads, salivary glands, midguts, and bacteriomes from C Las-positive and C Las-negative D. citri using the chromosomal length D. citri genome assembly. Results Each organ had a unique transcriptome profile and response to C Las infection. Though most psyllids were infected with the bacterium, C Las-derived transcripts were not detected in all organs. By analyzing the midgut dataset using both the Diaci_v1.1 and v3.0 D. citri genomes, we showed that improved genome assembly led to significant and quantifiable differences in RNA-sequencing data interpretation. Conclusions Our results support the hypothesis that future transcriptome studies on circulative, vector-borne pathogens should be conducted at the tissue-specific level using complete, chromosomal-length genome assemblies for the most accurate understanding of pathogen-induced changes in vector gene expression. [ABSTRACT FROM AUTHOR]

Background: Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CaLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. Results: A one-time (7 days) inoculation access period with CaLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response after a long period of infection without herbivory (270 and 360 days). In contrast, while repeated (monthly) 'pulses' of 7 day feeding injury by psyllids stimulated immunity in CaLas-infected citrus by increasing SA in leaves initially (up to 120 days), long-term (270 and 360 days) repeated herbivory caused SA to decrease coincident with upregulation of genes associated with SA metabolism (BMST and DMR6). Similarly, transcriptional responses and metabolite (SA and its analytes) accumulation in citrus leaves exposed to a continuously reproducing population of D. citri exhibited a transitory upregulation of genes associated with SA signaling at 120 days and a posterior downregulation after long-term psyllid (adults and nymphs) feeding (270 and 360 days). Conclusions: Herbivory played an important role in regulation of SA accumulation in mature leaves of C. sinensis, whether or not those trees were coincidentally infected with CaLas. Our results indicate that prevention of feeding injury inflicted by D. citri from the tritrophic interaction may allow citrus plants to better cope with the consequences of CaLas infection, highlighting the importance of vector suppression as a component of managing this cosmopolitan disease. [ABSTRACT FROM AUTHOR]

Citrus production has been declining in Florida since 2004, mainly because of Huanglongbing (HLB), a deadly disease that is affecting an increasingly large proportion of worldwide citrus production. Nutrients such as nitrogen, calcium, and magnesium play an important role in defense mechanisms and could extend the productive life of HLB-affected citrus trees, but interactions among these nutrients remain unclear. The main objective of this study was to determine the response of HLB-affected trees to N, Ca, and Mg applications, using Valencia trees (Citrus sinensis) on Swingle citrumelo (C. paradisi Macf. × P. trifoliata) rootstock established on a Candler fine sand. Applications of N at 168, 224 (current recommended rate), and 280 kg N ha−1 were the main plots. Split-plots consisted of a grower standard treatment receiving only basal Ca (51 kg ha−1) and Mg (56 kg ha−1); supplemental Ca only (total Ca inputs: 96 kg ha−1); supplemental Mg only (total Mg inputs: 101 kg ha−1); and supplemental Ca (total Ca inputs: 73.5 kg ha−1) and Mg (total Mg inputs: 78.5 kg ha−1). Leaf N, Mg and Ca concentrations, soil extractable Ca, Mg, NO3−-N and NH4+-N, and soil pH and base saturation were measured five times during an 18-month period. Different N rates did not affect leaf nutrient and soil extractable concentrations, and the application of Ca did not increase soil Ca extractable concentration relative to the grower standard, although it had weak effects on leaf Ca. In contrast, Mg was effective at increasing both leaf and soil Mg concentrations. Overall, our results suggest a greater impact of Mg fertilization on soil and nutrient concentrations than N or Ca fertilization, and limited evidence for strong interactions among these nutrients. [ABSTRACT FROM AUTHOR]

Rootstocks modulate several characteristics of citrus trees, including vegetative growth, fruit yield and quality, and resistance or tolerance to pests, diseases, soil drought, and salinity, among other factors. There is a shortage of scion and rootstock cultivars among the combinations planted in Brazil. "Ponkan" mandarin and "Murcott" tangor grafted on "Rangpur" lime comprise the majority of the commercial mandarin orchards in Brazil. This low genetic diversity of citrus orchards can favor pest and disease outbreaks. This study aimed to evaluate the agronomic performance, Huanglongbing (HLB) tolerance, and fruit quality of "Emperor" mandarin on five different rootstocks for nine cropping seasons under the subtropical soil-climate conditions of the North region of the state of Paraná, Brazil. The experimental design was a randomized block, with six replications, two trees per block, and five rootstocks, including "Rangpur" lime, "Cleopatra," and "Sunki" mandarins, "Swingle" citrumelo, and "Fepagro C-13" citrange. The evaluations included tree growth, yield performance, fruit quality, and HLB disease incidence. "Emperor" mandarin trees grafted on "Rangpur" lime and "Swingle" citrumelo had early fruiting and high yield efficiency. "Rangpur" lime also induced the lowest tree growth, but low fruit quality. Trees on "Swingle" citrumelo and "Fepagro C-13" citrange showed low scion and rootstock affinity and produced fruits with high total soluble solids (TSS), with a lower number of seeds for those from trees on "Fepagro C-13" citrange. "Cleopatra" and "Sunki" mandarins induced higher juice content, while fruits from trees on "Cleopatra" also had higher TSS/titratable acidity (TA) ratio. "Emperor" mandarin trees were susceptible to HLB regardless of the rootstocks. Overall, "Cleopatra" and "Sunki" mandarins, "Swingle" citrumelo, and "Fepagro C-13" are more suitable rootstocks for "Emperor" mandarin under Brazilian subtropical conditions than "Rangpur" lime. [ABSTRACT FROM AUTHOR]

Citrus are economically important fruit crops to which infectious diseases like citrus canker caused by Xanthomonas citri subs. citri, citrus variegated chlorosis caused by Xylella fastidiosa, "huanglongbing" associated with the presence of Candidatusliberibacter species, anthracnose caused by Colletotrichum gloeosporioides and citrus black spot caused by Phyllosticta citricarpa, impose significant losses. Control measures involve chemical treatment of orchards but often, eradication of infected plants is unavoidable. To circumvent the environmental impacts of pesticides and the socio-economic impacts of eradication, innovative antimicrobial approaches like photodynamic inactivation are being tested. There is evidence of the susceptibility of Xanthomonas citri subs. citri and C. gloeosporioides to photodynamic damage. However, the realistic assessment of perspectives for widespread application of photodynamic inactivation in the control of citrus diseases, necessarily implies that other microorganisms are also considered. This review intends to provide a critical summary of the current state of research on photodynamic inactivation of citrus pathogens and to identify some of the current limitations to the widespread use of photodynamic treatments in citrus crops. [ABSTRACT FROM AUTHOR]

BACKGROUND: Processed kaolin is a promising tactic that can be utilized for managing Diaphorina citri Kuwayama infestation. However, the frequency of kaolin application to protect citrus trees during flushing remains undetermined. The objective of this study was to ascertain the frequency of kaolin application (2%) required to reduce D. citri infestation and dispersal in flushing citrus orchards and to measure the spectral reflectance of kaolin‐sprayed leaves. RESULTS: Trees sprayed with kaolin at 7‐ and 14‐day intervals showed an 80% psyllid reduction compared to untreated trees. In addition, there was lower psyllid dispersal in the kaolin‐sprayed plots. Spectral measurements (wavelengths between 320 and 700 nm) showed an increased reflectance of 35.4% (adaxial leaf side) and 21.9% (abaxial leaf side) in mature leaves, and 9% (adaxial leaf side) and 2.2% (abaxial leaf side) in kaolin‐sprayed immature leaves compared to untreated trees. CONCLUSION: Application of kaolin (2%) at 7‐ and 14‐day intervals can be an important and sustainable tool for reducing D. citri infestation and dispersal in flushing citrus orchards. The reduced D. citri population in plots with both kaolin application intervals may be related to the visual effect (whitish aspect) and increased light reflectance in citrus trees, which are probably the main mechanisms by which kaolin interferes on D. citri host finding behavior. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

An active miniature inverted-repeat transposable element (MITE), MCLas-A, was previously identified from ‘Candidatus Liberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease). To explore the recent transposition status of MCLas-A, 389 ‘Ca. L. asiaticus’ strains collected from nine regions in China were amplified using a specific primer set and three representative ‘Ca. L. asiaticus’ strains were analyzed by next-generation sequencing (NGS) approach. PCR and genomic analysis showed that the entire MCLas-A was only present in 1.80% (7/389) and the jumping-out type of the MITE was predominant (81.23%) in samples tested, suggesting high frequency transposition occurred in ‘Ca. L. asiaticus’ strains recently collected from China. Biological roles of transposition of the active MITE remain to be determined.

Six new hybrid rootstocks, ‘US-1279’, ‘US-1281’, ‘US-1282’, ‘US-1283’, ‘US-1284’, and ‘US-1516’, were released from the U.S. Department of Agriculture (USDA) citrus breeding program to provide improved tree tolerance to huanglongbing (HLB), the most destructive disease facing the citrus industry in the United States and many other parts of the world. Five of these new rootstocks were released based on field performance in trials with the rootstocks propagated by stem cuttings, rather than the traditional propagation using nucellar seedlings. In this study, we evaluated the fruit, seed, and seedling characteristics of these new rootstocks, along with four other USDA rootstocks of commercial importance. The study included a determination of the percentage of true-to-type and off-type seedlings by both plant morphology and simple sequence repeat (SSR) markers. All 10 rootstocks produced an acceptable number of seeds and good seedling emergence from those seeds. The rootstocks ‘Swingle’, ‘US-802’, ‘US-812’, ‘US-1283’, ‘US-1284’, and ‘US-1516’ had a high percentage of true-to-type seedlings and correspondingly good potential to be propagated by seeds. However, no true-to-type plants were observed among seedlings from the rootstocks ‘US-1279’, ‘US-1281’, and ‘US-1282’, indicating that economical seed propagation will be impossible for these cultivars. The 10 SSR marker sets used in this study were observed to easily differentiate the 10 rootstocks studied, and readily distinguished true-to-type and off-type seedlings among progeny from all 10 rootstock clones. This study presents information of significant value for commercial nurseries involved in propagation of citrus rootstocks, and those involved in citrus rootstock breeding and development around the world. We propose the use of these 10 SSR marker sets as readily applicable for accurate identification of most citrus rootstock cultivars and their true-to-type seedlings.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is one of the most destructive pests of citrus and the primary vector of Candidatus Liberibacter spp., which cause citrus Huanglongbing (HLB). Microbial insecticides based on entomopathogenic fungi are safer alternatives to chemical pesticides. They can mitigate the spread of HLB by suppressing D. citri populations in citrus groves. Purpureocillium lilacinum strain ZJPL08, previously isolated from D. citri cadavers, is highly pathogenic to D. citri. We determined the virulence of strain ZJPL08 conidial suspensions and the efficacy of a wettable powder formulation. The LC50 of strain ZJPL08 against D. citri at 7 d post-inoculation was 2.11 × 104 conidia/mL, and the LT50 of a 1.0 × 108 conidia/mL suspension was 3.14 d in the laboratory. Under the same conditions, the LC50 of the wettable powder was 1.39 × 104 conidia/mL, and the LT50 was 2.64 d. The corrected mortality caused by the 1500-times diluted solutions of strain ZJPL08 wettable powder in D. citri reached 86.67% at 7 d post-treatment and 100% at 14 d post-treatment under field conditions. Our study revealed that strain ZJPL08 infected D. citri by hyphae penetrating directly through the cuticle and into the body cavity and tissues. These findings show that P. lilacinum strain ZJPL08 has potential for the effective control of D. citri.