Liberibacter Asiaticus Research Articles

The Effect of Host Plant Species on the Detoxifying Enzymes of the Asian Citrus Psyllid,Diaphorina citri(Hemiptera: Liviidae)

Summary Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a phytophagous insect and the vector of the bacterium ‘Candidatus' Liberibacter asiaticus. This is the likely causal pathogen of huanglongbing, which results in decline and possible death of citrus trees. It has been shown that host plants can affect the detoxification enzyme profile of arthropods. Here, we examined the effect of rearing D. citri on various host plant species with respect to activity of general esterases (ESTs), glutathione S-transferases (GST), and cytochrome P450 monooxygenases (P450s). These enzymes were selected because they are known to metabolize a wide diversity of insecticides and are known to directly contribute to resistance in D. citri. We reared D. citri on Citrus sinensis L., Bergera koenigii L. and Murraya paniculata (L.) Jacq. (Sapindales: Rutaceae). Following 12 generations of rearing, the activities of EST, GST, and P450 enzymes were compared between the colonies raised on the different host plants. The GST activi...

Effects of Cold-Acclimation, Pathogen Infection, and Varying Temperatures on Insecticide Susceptibility, Feeding, and Detoxifying Enzyme Levels inDiaphorina citri(Hemiptera: Liviidae)

Abstract Infection of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), with ‘Candidatus' Liberibacter asiaticus (Las), the causal pathogen of citrus greening disease or huanglongbing, increases psyllid susceptibility to insecticides. Diaphorina citri populations in citrus occur year-round in tropical and sub-tropical habitats, and thus insecticide applications for managing this plant disease vector occur over a wide temperature range (10–40 °C). During the winter season, D. citri is occasionally exposed to periods of freezing temperatures, when temperatures fall below -6.5 °C. In this investigation, we compared insecticide susceptibility of uninfected and Las-infected D. citri at various temperatures (20–37 °C). Cold-acclimated (6 ± 1 °C) D. citri adults were less susceptible to neonicotinoid insecticides as compared with non-acclimated controls, but this trend was not observed for other insecticides tested. A positive correlation between temperature and percentage mortality caused b...

Incidence of 'Candidatus Liberibacter asiaticus'-Infected Plants Among Citrandarins as Rootstock and Scion Under Field Conditions.

Huanglongbing (HLB), caused by the bacterium 'Candidatus Liberibacter' spp., is currently one of the most serious diseases of citrus plants and has caused substantial economic losses. Thus far, there is no source of genetic resistance to HLB in the genus Citrus or its relatives. However, several studies have reported Poncirus trifoliata and some of its hybrids to be more tolerant to the disease. The main objective of this study was to report differences in the incidence of 'Ca. L. asiaticus' infection in citrandarin plants, hybrids from Sunki mandarin (Citrus sunki (Hayata) hort. ex Tanaka), and trifoliate orange Rubidoux (P. trifoliata (L.) Raf.)), after conducting an extensive survey under field conditions. These hybrid plants were established for approximately 7 years in an area with a high incidence of 'Ca. L. asiaticus'-infected plants. We selected two experimental areas (area A and area B), located approximately 10 m apart. Area A consists of Pera sweet orange (C. sinensis (L.) Osb.) grafted onto 56 different citrandarin rootstocks. Area B consists of citrandarin scions grafted onto Rangpur lime (C. limonia Osb.) rootstock. Bacteria in the leaves and roots were detected using real-time quantitative polymerase chain reaction. The incidence of 'Ca. L. asiaticus'-infected plants was 92% in area A and 14% in area B. Because infected plants occurred in both areas, we examined whether the P. trifoliata hybrid rootstock influenced HLB development and also determined the distribution of 'Ca. L. asiaticus' in Citrus tree tissues. Although this survey does not present evidence regarding the resistance of P. trifoliata and its hybrids in relation to bacteria or psyllids, future investigation, mainly using the most promising hybrids for response to 'Ca. L. asiaticus', will help us to understand the probable mechanism of defense or identifying compounds in P. trifoliata and its hybrids that are very important as strategy to combat HLB. Details of these results are presented and discussed in this article.

Infection Density Dynamics of the Citrus Greening Bacterium "Candidatus Liberibacter asiaticus" in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants.

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, "Candidatus Liberibacter asiaticus," and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of "Ca. Liberibacter asiaticus" in field populations of D. citri with experiments using field-collected insects to address how "Ca. Liberibacter asiaticus" infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from "Ca. Liberibacter asiaticus"-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were "Ca. Liberibacter asiaticus" positive. The infections were systemic across head-thorax and abdomen, ranging from 10(3) to 10(7) bacteria per insect. In spring, the infection densities were low in March, at ∼ 10(3) bacteria per insect, increasing up to 10(6) to 10(7) bacteria per insect in April and May, and decreasing to 10(5) to 10(6) bacteria per insect in late May, whereas the infection densities were constantly ∼ 10(6) to 10(7) bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with "Ca. Liberibacter asiaticus" infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected "Ca. Liberibacter asiaticus"-infected insects suggested that (i) "Ca. Liberibacter asiaticus"-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼ 10(6) bacteria per insect) of "Ca. Liberibacter asiaticus" density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits "Ca. Liberibacter asiaticus" to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.

Nutrient Management Options for Florida Citrus: A Review of NPK Application and Analytical Methods

Citrus production in Florida is ranked first in the United States. Success of the citrus industry in the state relies heavily on sound water and nutrient management practices. Recently, citrus production has been declining due to the escalating prevalence of the citrus greening (Liberibacter asiaticus) and canker (Xanthomonas axonopodis) diseases. One option being explored is the manipulation of nutrient management scenarios to increase and enhance tree productivity. The paper presents a review of the management, analytical and application methods of three major nutrients Nitrogen (N), Phosphorus (P), and Potassium (K) on Florida's sandy soils with low organic matter (OM) and high leaching potential due to heavy annual rains (∼1200 mm). The NPK management options for Florida citrus are compared with those of other citrus producing regions around the world. Also, the critical tissue and soil nutrient concentrations for optimal and high citrus production are discussed. The review paper should provide important nutrient management guidelines to citrus growers in Florida and other regions with similar climatic and soil conditions.

Single chain antibodies against ‘Ca. Liberibacter asiaticus’

Author(s): Yuan, Q.; Jordan, R.; Brlansky, R.H.; Minenkova, O.; Hartung, J.S. | Abstract: Antibodies are widely used as microbiological reagents, but antibodies that recognize ‘Ca. Liberibacter asiaticus’ are generally lacking.n We have developed and applied immunization and affinity screening methods to create a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19.n The antibody population is enriched for antibodies that bind antigens of ‘Ca. Liberibacter asiaticus’ and Diaphorina citri.n The primary library has more than 107 unique antibodies and the genes that encode them.n We have screened this library of antibodies for antibodies that bind to specifically chosen proteins that are present on the surface of ‘Ca. Liberibacter asiaticus’.n These proteins were used as ‘bait’ for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule expressing protein KpsF; a protein component of the type IV pilus (CapF); and two flagellar proteins FlhA and FlgI.n These scFvs have been used in ELISA and dot blot assays against purified protein antigens and ‘Ca. Liberibacter asiaticus’ infected plant extracts.n We also have isolated scFv that bind to surface exposed portions of the TolC proteins and of a protein called InvA.n These proteins may have critical roles in pathogenicity.n Thus far, screening of these scFvs is more efficient when using phage bound, rather than soluble scFvs.n We have demonstrated a technology to produce antibodies and select at will and against any protein target encoded by ‘Ca. Liberibacter asiaticus’.n Future applications will include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.

Visualization of ‘Candidatus Liberibacter asiaticus’ Cells in Citrus Seed Coats with Fluorescence In Situ Hybridization and Transmission Electron Microscopy

‘Candidatus Liberibacter asiaticus’ is bacterium implicated as the causal agent of the economically damaging disease of citrus called huanglongbing (HLB).  The bacterium is spread by movement of infected citrus propagation material and by the Asian citrus psyllid, Diaphorina citri Kuwayama.  Seed transmission is a possible additional route of dissemination for the pathogen.  Some published studies on seed transmission found abundant bacterial DNA in seed coats but no infections of germinated seedlings.  No direct observations of bacteria in seed coats were made in these studies.  In vascular bundles from citrus seed coats, we confirmed the presence of bacterial cells with transmission electron microscopy (TEM) and Fluorescence In Situ Hybridization (FISH).  The physical measurements and the morphology of individual bacterial cells revealed by TEM and FISH were consistent with those described in the literature for ‘Ca. Liberibacter asiaticus’.  No bacterial cells were observed in seed coats from non-infected trees.  A library of clones of prokaryote 16S rRNA gene sequences amplified from a non-infected tree contained no ‘Ca. Liberibacter asiaticus’ sequences, whereas 95% of the sequences in a similar library prepared from an infected tree were ‘Ca. Liberibacter asiaticus’, providing additional data that the bacterial cells observed by TEM and FISH in seed coats from infected trees were ‘Ca. Liberibacter asiaticus’.

Preliminary Evaluation of the Single-Tree, Huanglongbing Find in California

Huanglongbing (HLB, citrus greening) associated with ‘Candidatus Liberibacter’ species is a widespread devastating citrus disease not previously reported in California (CA). In March 2012, ‘C. Liberibacter asiaticus’ (CLas) was detected from an Asian citrus psyllid (ACP, Diaphorina citri) sample from Los Angeles, CA at the Jerry Dimitman Laboratory of the Citrus Research Board. Subsequent citrus plant surveys within a 400m area of the CLas-positive ACP sample performed by the California Department of Food and Agriculture identified an infected multi-grafted citrus tree at a residence in Los Angeles, CA. The CLas-positive tree was removed and nucleic acids from different plant tissues (i.e. roots, trunk, stems, and leaves) were extracted and distributed to several federal, state, and university laboratories nationwide for preliminary evaluation.  Labs attempted to identify the species and graft types of the infected citrus, study the genetic characteristics and genome diversity of the detected bacterium, as well as test for other graft-transmissible citrus pathogens (GTCP). Preliminary data suggested that one type of lemon was the original rootstock that received over 20 citrus grafts. CLas DNA population analysis suggested a possible single Asian origin. Preliminary tests indicated the possible presence of other GTCP. Further evaluations on the CA CLas-positive find are ongoing.

Whole Genome PCR Sequencing Strategy for ‘Candidatus’ Liberibacter asiaticus: Analyzing Sequence Diversity Among U.S. Isolates

Author(s): Shatters, Jr., R. G.; Morgan, J. K.; Jara-Cavieres, A.; Stover, E.; Powell, C. A.; Duan, Y.-P.; Moore, G. | Abstract: The inability to culture the ‘Candidatus’ Liberibacter asiaticus (CLas) bacterium has greatly hindered research on the etiology of the citrus disease Huanglongbing which is associated with this bacterium.n This is especially true with respect to possible links between strain/isolate diversity and disease symptom variations and development.n Past genetic marker research indicates that there is considerable CLas isolate diversity even within Florida (a location that has only recognized the presence of the disease since 2005); however, no effort has been made to correlate this diversity with symptom differences.n To advance our understanding of CLas geographic spread and strain/isolate diversity, we have developed a whole-genome PCR amplification strategy that can be used in conjunction with next generation genome sequencing to rapidly obtain near whole genome sequence for specific isolates.n This method was used to generate genome sequence data (~93% of the total genome with an average ~300x coverage) from numerous isolates within Florida that are known to be different with respect to previously characterized genetic markers, and also to compare genomic sequence with isolates that may induce different symptoms within citrus.n Results will be presented that show the diversity among Florida CLas isolates and will also be contextualized within the diversity observed sequence variation among global isolates.

Screening of citrus and its close relatives for tolerance to huanglongbing

Huanglongbing (HLB), a devastating disease of citrus, has become a serious problem for the citrus industries in Brazil and Florida, and both the disease and its psyllid vector, Diaphorina citri continue to spread to other citrus growing regions. Host resistance or tolerance to the pathogen would be extremely valuable to the citrus industry. A field trial was established in Fort Pierce, Florida where HLB has become endemic to assess the HLB tolerance level of different cultivars of citrus and citrus relatives. Over 800 seedlings representing over 100 accessions (8 replications of each) belonging to 18 genera of the subfamily Aurantioideae and family Rutaceae were evaluated over a period of four years. Leaf samples were collected at 6 month intervals during the spring and fall seasons and tested for the presence of HLB associated bacterium, Candidatus Liberibacter asiaticus (LAS) by real time PCR. While most accessions were found to be susceptible to HLB, the bacterium (LAS) was not detectable in about 20 accessions for up to four years of analysis. These include many trifoliates and trifoliate hybrids, some species of Bergera, Casimiroa, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Naringi, and Zanthoxylum. Information on varietal tolerance of citrus and its relatives to HLB is very important for management of the disease.  While most accessions in Citrus were susceptible, partial resistance was observed in some clonal populations of Citrus latipes. The probable basis of resistance is being investigated.

Translating Anatomical Structures and Functional Genomics of Candidatus Liberibacter asiaticus and solanacearum Into Circulative, Propagative Vector-Mediated Transmission Processes

Ca. Liberibacter asiaticus is the putative fastidious bacterial causal agent of citrus greening disease, also known as Huanglongbing (HLB), translated from Chinese as yellow dragon disease. The HLB bacterial pathogen is indigenous to Asia but has been introduced and dispersed to citrus throughout the Americas. A related bacterium that is indigenous to the Americas causes damage to potato (zebra chip) and tomato (vein-greening) and other solanaceous hosts. The causal agents are propagative and circulative in the psyllid vector, Diaphorina citri (Kuwayama) and Bactericera cockerelli (Sulc.), the Asian citrus and potato (or tomato) psyllid, respectively. The specific psyllid proteins that are indirectly or directly involved in the circulative, propagative transmission pathway are not known. However, if proteins were known that function at key points in the pathway e.g. post-ingestion, infection, biofilm formation, nutrition, circulation, and/or acquisition were known, such knowledge could be exploited to knock out their expression and abate pathogen transmission. To this end a combined approach involving functional genomics and anatomical localization of the bacterium is being implemented.  Results indicate that Ca. Liberibacter establishes biofilms on the outer surfaces of the alimentary canal and salivary glands of the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama and the potato psyllid (PP) Bactericera cockerelli Sulc. In silico transcript profiling of infected and uninfected ACP and PP identified a number of mis-expressed, unique transcripts (unitrans). Functional predictions (gene ontology associations) implicate certain of these unitrans in Ca. Liberibacter infection of the psyllid host and/or in psyllid-mediated Ca. Liberibacter transmission processes.

Nutritional Analysis of Flowers from ‘Valencia’ Orange Trees Infected with Huanglongbing

Author(s): Saccini, V. A.V.; Dos Santos, D. M.M.; Medina, C. L.; Machado, R. S.; Cruz, F. J.R. | Abstract: In the mid2000s, Brazil has reported the disease citrus greening or huanglongbing (HLB), considered one of the most destructive diseases in plants by its complexity. As an agent associated with HLB, we have bacteria: Candidatus Liberibacter americanus, Liberibacter asiaticus , Ca. Liberibacter africanus. Symptoms of greening may be masked by other symptoms generated from some diseases, besides such symptoms are similar to those caused by various mineral deficiencies. The objective of this study was to verify if the HLB affects the accumulation of nutrients in citrus flowers. The experiment was in Valencia oranges trees (C. sinensis) on Rangpur lime (C. limonia) with 12 years of age. Treatments consisted of: 1) symptomatic branches of flowers (PCR+), 2) asymptomatic branches of flowers (PCR+) and 3) flowers of healthy plants (PCR-). The levels of macronutrients and micronutrients were measuredn in December of 2011. The results showed lower levels of N, P, K, Ca, Mg, B, Fe, Mn in the flowers of diseased plants (asymptomatic and symptomatic) compared with the levels of certain nutrients in healthy plants. The reduction in nutrient concentration mobile (N, P and K) as not mobile in the phloem (Ca and B) shows that the absorption and distribution of nutrients were reduced in young tissues and justifies the diversity of symptoms found. The nutrients are involved in the activation / inactivation of enzymes related to metabolism and biosynthesis of plant hormone, auxin, gibberellins and cytokinins, which regulate the abortion of plant organs.

High incidence of preharvest colonization of huanglongbing-symptomatic citrus sinensis fruit by Lasiodiplodia theobromae (Diplodia natalensis) and exacerbation of postharvest fruit decay by that fungus.

Huanglongbing (HLB), presumably caused by the bacterium "Candidatus Liberibacter asiaticus," is a devastating citrus disease associated with excessive preharvest fruit drop. Lasiodiplodia theobromae (diplodia) is the causal organism of citrus stem end rot (SER). The pathogen infects citrus fruit under the calyx abscission zone (AZ-C) and is associated with cell wall hydrolytic enzymes similar to plant enzymes involved in abscission. By means of DNA sequencing, diplodia was found in "Ca. Liberibacter asiaticus"-positive juice from HLB-symptomatic fruit (S) but not in "Ca. Liberibacter asiaticus"-negative juice. Therefore, the incidence of diplodia in fruit tissues, the impact on HLB-related postharvest decay, and the implications for HLB-related preharvest fruit drop were investigated in Hamlin and Valencia oranges. Quantitative PCR results (qPCR) revealed a significantly (P < 0.001) greater incidence of diplodia in the AZ-C of HLB-symptomatic (S; "Ca. Liberibacter asiaticus" threshold cycle [CT] of <30) than in the AZ-C of in asymptomatic (AS; "Ca. Liberibacter asiaticus" CT of ≥30) fruit. In agreement with the qPCR results, 2 weeks after exposure to ethylene, the incidences of SER in S fruit were 66.7% (Hamlin) and 58.7% (Valencia), whereas for AS fruit the decay rates were 6.7% (Hamlin) and 5.3% (Valencia). Diplodia colonization of S fruit AZ-C was observed by scanning electron microscopy and confirmed by PCR test and morphology of conidia in isolates from the AZ-C after surface sterilization. Diplodia CT values were negatively correlated with ethylene production (R = -0.838 for Hamlin; R = -0.858 for Valencia) in S fruit, and positively correlated with fruit detachment force (R = 0.855 for Hamlin; R = 0.850 for Valencia), suggesting that diplodia colonization in AZ-C may exacerbate HLB-associated preharvest fruit drop.

"Candidatus Liberibacter asiaticus" prophage late genes may limit host range and culturability.

"Candidatus Liberibacter asiaticus" is an uncultured alphaproteobacterium that systemically colonizes its insect host both inter- and intracellularly and also causes a severe, crop-destroying disease of citrus called huanglongbing, or citrus "greening." In planta, "Ca. Liberibacter asiaticus" is also systemic but phloem limited. "Ca. Liberibacter asiaticus" strain UF506 carries two predicted prophages, SC1 and SC2. Bacteriophage particles have been observed in experimentally "Ca. Liberibacter asiaticus"-infected periwinkle but not in any other host. Comparative gene expression analysis of predicted SC1 late genes showed a much higher level of late gene expression, including holin transcripts (SC1_gp110), in "Ca. Liberibacter asiaticus"-infected periwinkle relative to "Ca. Liberibacter asiaticus"-infected citrus. To functionally characterize predicted holin and endolysin activity, SC1_gp110 and two predicted endolysins, one within SC1 (SC1_gp035) and another well outside the predicted prophage region (CLIBASIA_04790), were cloned and expressed in Escherichia coli. Both SC1 genes inhibited bacterial growth consistent with holin and endolysin function. The holin (SC1_gp110) promoter region was fused with a uidA reporter on pUFR071, a wide bacterial host range (repW) replicon, and used to transform Liberibacter crescens strain BT-1 by electroporation. BT-1 is the only liberibacter strain cultured to date and was used as a proxy for "Ca. Liberibacter asiaticus." pUFR071 was >95% stable without selection in BT-1 for over 20 generations. The reporter construct exhibited strong constitutive glucuronidase (GUS) activity in culture-grown BT-1 cells. However, GUS reporter activity in BT-1 was suppressed in a dose-dependent manner by crude aqueous extracts from psyllids. Taken together with plant expression data, these observations indicate that "Ca. Liberibacter asiaticus" prophage activation may limit "Ca. Liberibacter asiaticus" host range and culturability.

Identification of Genes Expressed Differentially in Grapefruit Infected with Candidatus Liberibacter asiaticus in the Late Stage of Disease

Citrus greening or Huanglongbing (HLB), a destructive disease of citrus worldwide, was reported from south of Iran in 2007. The molecular basis of compatibility and disease development in this system is poorly understood. We have carried out a cDNA-AFLP analysis to analyse gene expression of grapefruit infected by Candidatus Leiberibacter asiaticus in the late infection stage. We have applied a cDNA-AFLP approach on grafted infected grapefruit trees at the representing symptoms stage in susceptible host. Selective amplifications with 10 primer combinations allowed the visualization of approximately 24 transcript-derived fragments (TDFs) in the leaves of graft-inoculated trees, which were differentially expressed. We sequenced 14 fragments, which were identified as grapefruit transcripts after homology searching, whereas 12 were not homologous to sequences in NCBI databases. Many grapefruit genes spanning almost all functional categories were upregulated during infection, especially genes involved in ATP synthesize, cytochrome P450 synthesize, isoflavone 2'-hydroxylase, zeaxanthin epoxidase, cellulose synthase, DNA repair protein, aconitate hydratase 2 and citrus tristeza virus resistance gene. This study provides the first global catalogue of grapefruit genes expressed during inoculation, together with their functional annotations. This will help to elucidate the molecular basis of the resistance process and identify genes and chemicals that could help to inhibit the pathogen.

Liberibacter crescens gen. nov., sp. nov., the first cultured member of the genus Liberibacter.

The Gram-stain-negative, rod-shaped bacterial isolate BT-1(T) is the closest relative to the genus 'Candidatus Liberibacter' cultured to date. BT-1(T) was recovered from the phloem sap of a defoliating mountain papaya in Puerto Rico. The BT-1(T) 16S rRNA gene sequence showed that strain BT-1(T) is most closely related to members of the genus 'Ca. Liberibacter' sharing 94.7% 16S rRNA gene sequence similarity with 'Ca. Liberibacter americanus' and 'Ca. Liberibacter asiaticus'. Additionally, average nucleotide identity, 16S rRNA gene sequences and conserved protein sequences supported inclusion of the previously described species of the genus 'Ca. Liberibacter' in a genus with BT-1(T). The prominent fatty acids of isolate BT-1(T) were C18 : 1ω7c (77.2%), C16 : 0 OH (4.8%), C18 : 0 (4.4%) and C16 : 0 (3.5%). Both physiological and genomic characteristics support the creation of the genus Liberibacter, as well as the novel species Liberibacter crescens gen. nov., sp. nov. with type strain BT-1(T) ( = ATCC BAA-2481(T) = DSM 26877(T)).

Economic Potential of Producing Tahiti Limes in Southern Florida in the Presence of Citrus Canker and Citrus Greening

This article assesses the profitability of a hypothetical 5-acre tahiti lime (Citrus latifolia) orchard in southern Florida in the presence of citrus canker (Xanthomonas axonopodis pv. citri) and citrus greening [Candidatus Liberibacter asiaticus (LAS)]. To account for the uncertainty associated with the presence of these diseases, a stochastic budgeting technique was employed in the analysis, incorporating stochastic prices and yields based on discussions with industry experts and researchers. The analysis focused on three possible types of management strategies currently practiced by citrus (Citrus sp.) growers in Florida: 1) production without any specific control activities for citrus canker and citrus greening, 2) canker and greening management without removal or replacement of infected/suspicious trees, and 3) canker and greening management with removal and replacement of infected trees. The analysis was carried out for a 20-year time horizon and average net return per acre and rate of return on investment were considered. The results suggest that despite the presence of disease, it would be profitable to produce tahiti limes in southern Florida. This is because the tahiti lime offers some resistance to both citrus greening and canker and will produce even if minimal attention is paid to controlling the diseases. Of the three management strategies investigated, strategy 2 offers the best prospect in terms of high net returns and highest probability of achieving or surpassing the desired rates of return on investment of 12% per annum. The key finding from the study is that the production of tahiti limes in southern Florida can be profitable if steps are taken to manage the diseases, but contrary to popular view, it might be better to wait until the trees become fully unproductive before disposing of them.

Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with Huanglongbing

Intensive insecticide and nutrient management have been attempted worldwide to reduce citrus huanglongbing (HLB) symptom development and yield loss. However, effects of insecticide and nutrient applications on HLB have been poorly understood. Leaf nutrients, jasmonic and salicylic acid contents, cycle threshold (Ct) values of Ca. Liberibacter asiaticus (Las), and community structure of endophytic α-proteobacteria were evaluated after insecticide treatment, ‘nutrition’ treatment (including systemic resistance inducing agents), or both in comparison with a control in a two-factor field experiment in 2008–2012. Leaf N, Mn, Zn and B significantly increased whilst Cu decreased after nutrient applications. Salicylic acid significantly increased in old leaves treated with insecticides, nutrients or both, and in young leaves treated with nutrients only. The jasmonic acid concentration was highest after the nutrition treatment in both old and young leaves. Ct values of Las and leaf area and weight significantly increased after long-term nutrient applications in 2011 and/or 2012. Redundancy analysis of the endophytic α-proteobacteria community structure indicated that the communities were mainly separated according to nutrient applications, which were positively associated with Ct values of Las and Ca, Mn, Zn, B, Mg, and Fe contents in leaf samples collected in 2012. Thus, effects of insecticides on HLB were significant in the early 2-year period whilst nutrients had significant effects on Las content and leaf size and weight after at least 3 years of application.

Development of Primers and Probes for Genus and Species Specific Detection of 'Candidatus Liberibacter Species' by Real-Time PCR.

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different 'Candidatus Liberibacter species', 'Ca. Liberibacter asiaticus', 'Ca. Liberibacter americanus', and 'Ca. Liberibacter africanus', which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of 'Ca. Liberibacter' at the genus level, providing increased convenience. Recent molecular analyses of 'Ca. Liberibacter species' and other bacteria suggest that the rpoB gene (encoding the β-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus 'Ca. Liberibacter species', enabling detection of 'Ca. Liberibacter' at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected 'Ca. Liberibacter solanacearum', a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

Association of ‘Candidatus Liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing‐affected trees prior to appearance of foliar symptoms

Huanglongbing (HLB) is a systemic disease of citrus caused by phloem‐limited bacteria ‘Candidatus Liberibacter’ spp. with ‘Ca. Liberibacter asiaticus’ (Las) the most widespread. Phloem‐limited bacteria such as liberibacters and phytoplasmas are emerging as major pathogens of woody and herbaceous plants. Little is known about their systemic movement within a plant and the disease process in these tissues. Las movement after initial infection was monitored in leaves and roots of greenhouse trees. Root density, storage starch content, and vascular system anatomy in relation to Las presence in field and greenhouse trees, both with and without symptoms, showed the importance of root infection in disease development. Las preferentially colonized roots before leaves, where it multiplied and quickly invaded leaves when new foliar flush became a sink tissue for phloem flow. This led to the discovery that roots were damaged by root infection prior to development of visible foliar symptoms and was not associated with carbohydrate starvation caused by phloem‐plugging as previously hypothesized. The role of root infection in systemic insect‐vectored bacterial pathogens has been underestimated. These findings demonstrate the significance of early root infection to tree health and suggest a model for phloem‐limited bacterial movement from the initial insect feeding site to the roots where it replicates, damages the host root system, and then spreads to the rest of the canopy during subsequent leaf flushes. This model provides a framework for testing movement of phloem‐limited bacteria to gain greater understanding of how these pathogens cause disease and spread.