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.     

Up‐regulation of PR1 and less disruption of hormone and sucrose metabolism in roots is associated with lower susceptibility to ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las), is a devastating disease of citrus trees in Florida. Previous work showed that the rootstock cultivar Cleopatra mandarin (Citrus reticulata) has a higher population of Las in roots than Swingle citrumelo (C. paradisi × Poncirus trifoliata). Las reduced fibrous root biomass and sucrose content in Cleopatra mandarin more than in Swingle citrumelo. To understand the mechanisms for susceptibility to Las infection, sucrose and hormone metabolism status were evaluated in Cleopatra mandarin and Swingle citrumelo. In fibrous roots of Cleopatra mandarin, higher expression of genes related to sucrose cleavage was consistent with lower sucrose content compared to noninoculated seedlings at 5 weeks post‐root trimming (wpt). In fibrous roots of Swingle citrumelo, both sucrose content and gene expression related to sucrose cleavage were less disrupted by Las infection compared to Cleopatra mandarin at 5 wpt. Genes associated with salicylic acid (SA), ethylene (ET) and abscisic acid (ABA) synthesis, and ABA signalling, phospholipases D (PLD), and phospholipase A2 (PLA2) were activated by Las infection at 5 wpt in Cleopatra mandarin. Expression of downstream effectors of SA, i.e. NPR1, WRKY70 and PR1, did not change in Cleopatra mandarin, suggesting inhibition of the response to SA by the elevation of ABA, ET and PLD. In contrast, the up‐regulation of PR1, lower response of sucrose metabolism genes and down‐regulation of biosynthesis of phytohormones indicates that Swingle citrumelo activates a more effective defence against this biotrophic pathogen than Cleopatra mandarin.     

Effects of pymetrozine, an antifeedant of Hemiptera, on Asian citrus psyllid, Diaphorina citri, feeding behavior, survival and transmission of Candidatus Liberibacter asiaticus

BACKGROUND: Vector‐borne plant disease management can be enhanced by deployment of antifeedants in addition to the use of broad‐spectrum neurotoxic insecticides. The effects of pymetrozine on Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, feeding behaviour, survival and transmission of Candidatus Liberibacter asiaticus (Las), the presumed causal pathogen of huanglongbing, were investigated. RESULTS: Pymetrozine applied at 52 and 104 µg mL^?1 to citrus plants [Swingle citrumelo (X Citroncirus webberi Ingram and Moore)] modified the feeding behavior of ACP and increased the amount of time spent performing non‐penetration behaviors while decreasing the time spent performing ingestion behaviors compared with the controls 1 day after treatment. However, the antifeedant effect of pymetrozine subsided 5 days after application. Pymetrozine reduced the survival of both adults and nymphs on treated plants compared with the control. However, it had a greater impact on survival of nymphs than on survival of adults. Pymetrozine applied at 52 and 104 µg mL^?1 on Las‐infected ‘Valencia’ sweet orange plants [Citrus sinensis L. (Osbeck)] reduced acquisition (12 and 21% respectively) and transmission (11 and 18% respectively) of Las by feeding ACP adults compared with the controls; however, these reductions were not statistically significant. CONCLUSIONS: Pymetrozine exhibited moderate antifeedant effects by modifying the feeding behavior of ACP adults with short residual activity. The impact of pymetrozine on survival of nymphs was greater than on adults at the higher concentrations tested. Pymetrozine also reduced the acquisition and transmission of Las by feeding ACP adults up to 21 and 18%, respectively, compared with untreated controls. Copyright © 2010 Society of Chemical Industry     

Frequency and occurrence of the carrot pathogen ‘Candidatus Liberibacter solanacearum’ haplotype C in Finland

Occurrence of ‘Candidatus Liberibacter solanacearum’ (CLso) was studied in field‐grown carrots (Daucus carota) in different regions of Finland. In addition, the frequency of CLso in carrots and in field populations of its vector, the carrot psyllid (Trioza apicalis), was studied in southwestern Finland. CLso was detected in six of the seven regions where the main carrot cultivation areas are located. The highest disease incidence was found in southwestern Finland, in the area where this carrot pathogen was originally found. In the Tavastia Proper and Southwest Finland regions, CLso was detected in 26 out of 30 randomly chosen fields inspected in 2013 and 2014, and in a third of those fields more than 10% of plants showed symptoms. Of those carrots showing both psyllid feeding‐associated leaf curling and CLso infection‐associated leaf discolouration symptoms, 77% were CLso positive in the PCR test. Some symptomless carrots from the affected fields also tested positive. Of the carrot psyllid individuals collected from the same area, 60% were CLso positive. Elsewhere, disease incidence was variable in South Ostrobothnia in western Finland and low but established in South Savonia in eastern Finland. CLso was not detected in the North Ostrobothnia region. Sequencing of the amplified DNA fragments confirmed that the bacteria in the carrot samples from different areas within Finland all represented CLso haplotype C. The frequent occurrence and wide distribution of this pathogen, transmitted by a psyllid that does not migrate over long distances, suggest that it is persistent in Finland.     

Contrasting canopy and fibrous root damage on Swingle citrumelo caused by ‘Candidatus Liberibacter asiaticus’ and Phytophthora nicotianae

Huanglongbing (HLB), associated with the phloem‐limited bacterium ‘Candidatus Liberibacter asiaticus’ (Las), is devastating trees in citrus orchards of Florida. Additionally, Phytophthora nicotianae, omnipresent in citrus soils, causes root rot that reduces water and nutrient uptake by fibrous roots. To investigate fibrous root damage and replacement and canopy size in relation to infection of fibrous roots by Las and P. nicotianae, rootstock seedlings of Swingle citrumelo (Citrus paradisi × Poncirus trifoliata) were inoculated with Las or P. nicotianae in two greenhouse pot trials. Phytophthora nicotianae caused root damage within 5 weeks post‐inoculation, which led to greater reduction of canopy size than for Las‐infected seedlings by the end of the experiment. Las increased accumulation of fibrous root biomass at 5 weeks post‐root trimming (wpt) in the 2014 trial and at 11 wpt in the 2015 trial. New root length was not consistently increased by Las. Reduced total leaf area of symptomless Las‐infected seedlings compared to noninoculated controls might be due to the combined effect of altered carbohydrate allocation between shoots and roots and altered leaf morphology.     

Detection by PCR of Candidatus Liberibacter asiaticus, the bacterium causing citrus huanglongbing in vector psyllids: application to the study of vector–pathogen relationships

Citrus huanglongbing (HLB), previously called greening, is a serious citrus disease in Asia, eastern and southern Africa. It is caused by Candidatus Liberibacter asiaticus (Las), a phloem-limited, nonculturable bacterium transmitted by the Asian citrus psyllid ( Diaphorina citri ) in Asia. A PCR-based assay was developed for monitoring Las in vector psyllids using a rapid DNA extraction from psyllid bodies and PCR amplification. The entire procedure for Las detection in psyllids can be completed within 5 h. Using this method, Las can be accurately detected in psyllid adults as well as nymphs in different instar stages. The assay is sensitive enough for Las detection in single-psyllid extract from adult, fifth, fourth and third instars. In a transovarial transmission experiment, Las was not detected in eggs or in offspring produced by Las-carrying psyllid females. In a retention test, the Las-carrying psyllids remained Las-positive for 12 weeks after they were moved to common jasmine orange, a Las-immune plant. From these experimental results it was concluded that Las persists in the Asian citrus psyllid vector, but is not transovarially transmitted by the vector. These data help in understanding epidemiological characteristics of Las and psyllids in citrus HLB.     

Effect of Candidatus Liberibacter asiaticus infection on susceptibility of Asian citrus psyllid, Diaphorina citri, to selected insecticides

BACKGROUND: In the present investigation, the effect of Candidatus Liberibacter asiaticus (Las), a bacterium considered to be responsible for causing huanglongbing (HLB) disease in citrus, on the physiology of its vector, the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama, was determined. Specifically, the effects of Las infection on the susceptibility of ACP to selected insecticides were determined. Furthermore, total protein content and general esterase activity were quantified in Las‐infected and uninfected ACP to gain insight into the possible mechanism(s) responsible for altered susceptibility to insecticides owing to Las infection. RESULTS: LC₅₀ values were significantly lower in Las‐infected than in uninfected ACP adults for chlorpyrifos and spinetoram. Furthermore, there was a general trend towards lower LC₅₀ values for three other insecticides for Las‐infected ACP; however, the differences were not statistically significant. Total protein content (µg mL^?1) was significantly lower in Las‐infected (23.5 ± 1.3 in head + thorax; 27.7 ± 1.9 in abdomen) than in uninfected (29.7 ± 2.1 in head + thorax; 35.0 ± 2.3 in abdomen) ACP. Likewise, general esterase enzyme activity (nmol min^?1 mg^?1 protein) was significantly lower in Las‐infected (111.6 ± 4.5 in head + thorax; 109.5 ± 3.7 in abdomen) than in uninfected (135.9 ± 7.5 in head + thorax; 206.1 ± 23.7 in abdomen) ACP. CONCLUSION: Susceptibility of ACP to selected insecticides from five major chemistries was greater in Las‐infected than in uninfected ACP. The lower total protein content and reduced general esterase activity in Las‐infected than in uninfected ACP may partly explain the observed higher insecticide susceptibility of Las‐infected ACP. Copyright © 2010 Society of Chemical Industry     

Different symptoms in carrots caused by male and female carrot psyllid feeding and infection by ‘Candidatus Liberibacter solanacearum’

Carrot psyllid Trioza apicalis was recently found to carry the plant pathogenic bacterium ‘Candidatus Liberibacter solanacearum’ (CLs). To confirm the transmission of bacteria by the psyllids and to dissect the symptoms caused in carrot plants by psyllid feeding and CLs infection, a greenhouse experiment with single psyllids feeding on separate plants was performed. A positive correlation was found between the amount of CLs bacteria in the psyllids and in the corresponding plants exposed to feeding, indicating CLs transmission. The female psyllid feeding caused more severe damage than male feeding, and resulted in a substantial decrease in the root weight. Female psyllid feeding also significantly reduced the carrot leaf weight and increased the number of curled leaves. The number of curled leaves was also increased by the nymphs when their number exceeded 10 per plant. A high titre of CLs bacteria significantly reduced root weight, while not affecting the weight or number of the leaves. However, the amount of CLs correlated with the number of leaves showing discolouration symptoms. Microscopy of infected carrot plants revealed that the phloem tubes throughout the whole plant, from leaf veins to the root tip, were colonized by bacteria. The bacterial cells appeared to be long and thin flexible rods with tapering ends and a transversally undulated surface. Microscopy also revealed collapsed phloem cells in the infected carrots. Damage in the phloem vessels is likely to reduce the sucrose transport from source leaves to the root, explaining the observed leaf discolouration and reduction in root weight.     

Influence of temperature on infection and establishment of ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in citrus plants

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘Candidatus Liberibacter asiaticus’ and ‘Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘Ca. L. asiaticus’, which has taken over from ‘Ca. L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘Ca. L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘Ca. L. asiaticus’ was distributed throughout the inoculated plants, with mean C_t values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘Ca. L. asiaticus’ reached the highest titres in mature leaves (mean C_t value = 26·7) of citrus plants maintained at 22/27°C. ‘Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean C_t value in mature leaves = 33·6).     

Liberibacters associated with orange jasmine in Brazil: incidence in urban areas and relatedness to citrus liberibacters

Two surveys (2005/2006 and 2009) were conducted in the state of São Paulo, Brazil, to investigate the incidence of ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’, two liberibacters associated with citrus huanglongbing (HLB) disease and both transmitted by Diaphorina citri, in orange jasmine (Murraya exotica), a widespread ornamental tree in cities and villages. The graft‐transmissibility of the two species, and their DNA relatedness to citrus‐associated liberibacters, were also investigated. Quantitative PCR was applied to PCR‐positive orange jasmine and HLB‐positive citrus growing in backyards and orchards to assess their inoculum source potentials. Liberibacters were detected in 91 of 786 sampled orange jasmine plants in 10 of 76 sampled locations. PCR‐positive trees exhibited yellow shoots and/or dieback symptoms indistinguishable from those on PCR‐negative trees. ‘Candidatus Liberibacter americanus’ was more common in 2005/2006 (96·6%) and ‘Ca. L. asiaticus’ in 2009 (84·8%). rplJ nucleotide sequences were identical within all populations of either species. Graft transmission succeeded only in homologous host combinations, including ‘Ca. L. americanus’ (2/10) from/to orange jasmine and ‘Ca. L. americanus’ (5/18) and ‘Ca. L. asiaticus’ (5/9) from/to citrus. Symptoms were mild and developed less rapidly in orange jasmine than in citrus, probably as a result of lower liberibacter multiplication rates. Respective titres of ‘Ca. L. americanus’ and ‘Ca. L. asiaticus’ in orange jasmine averaged 4·3 and 3·0 log cells g^?1 tissue, compared with 5·5 and 7·3 in citrus. The results indicate that orange jasmine does not favour liberibacter multiplication as much as citrus. However, its importance in HLB epidemics should not be underestimated as it is a preferred host of D. citri and is not under any strict tree‐eradication programme or measures for insect control.     

Economic justification for quarantine status – the case study of ‘Candidatus Liberibacter solanacearum’ in the European Union

International agreements on plant health and trade require that regulating a pest should be justified by economic impact assessment. Economic impact assessments are usually qualitative, weakening the objective and transparency of the regulation decision. This study assessed the potential economic impacts of the invasion of the plant pathogenic bacterium ‘Candidatus Liberibacter solanacearum’ into the European Union in order to economically justify a decision on its quarantine status. Direct economic impacts resulting from yield loss in potato and tomato were computed using partial budgeting at a regional scale, while total economic impacts on the potato and tomato markets were computed using partial equilibrium modelling at the EU scale. Annual direct impacts at the most likely infestation level were estimated at €222 m for the whole EU. Uncertainty analysis showed a distribution of foreseeable annual impacts with a 5th percentile of €192 m, and a 95th percentile of €512 m. Increased market prices of potato and tomato resulting from reduced supply were found to increase profits for non‐infested producers and to compensate in part for the production losses of infested producers, with consumers paying for this mitigation of impacts on producers. The expected negative impact on societal welfare at the most likely infestation level is less than the estimated direct impacts, viz. €114 m/year. The potential economic impacts of ‘Ca. L. solanacearum’ in the European Union are demonstrably of major importance. Therefore, a decision to categorize this organism as a quarantine pest is supported.     

Effect of pyriproxyfen,a juvenile hormone mimic,on egg hatch,nymph development,adult emergence and reproduction of the Asian citrus psyllid,Diaphorina citri Kuwayama

BACKGROUND: The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is a vector of bacteria presumably responsible for huanglongbing (HLB) disease in citrus. In this laboratory study, an investigation was made of the activity of pyriproxyfen, a juvenile hormone mimic, on ACP eggs, nymphs and adults to evaluate its potential as a biorational insecticide for inclusion in an integrated pest management (IPM) program for ACP. RESULTS: Irrespective of egg age, timing or method of treatment, a significantly lower percentage of eggs (5–29%) hatched after exposure to 64 and 128 µg mL^?1 of pyriproxyfen. Only 0–36% of early instars (first, second and third) and 25–74% of late instars (fourth and fifth) survived to adults following exposure to 16, 32 and 64 µg mL^?1 of pyriproxyfen. However, 15–20% of adults that emerged following treatment as late instars exhibited morphological abnormalities. Furthermore, pyriproxyfen adversely affected reproduction (fecundity and fertility) of adults that emerged from treated fifth instars or that were treated topically with 0.04 µg as adults. CONCLUSIONS: Application of pyriproxyfen at 64 µg mL^?1 resulted in greater inhibition of egg hatch and suppression of adult emergence compared with lower rates. Pyriproxyfen also markedly reduced female fecundity and egg viability for adults that were exposed either as fifth instars or as newly emerged adults. The ovicidal, larvicidal and reproductive effects against ACP suggest that pyriproxyfen is suitable for integration into an IPM program for ACP. Copyright © 2009 Society of Chemical Industry     

Tissue‐print and squash real‐time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR. This simplified method was validated and could detect HLB‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.     

Molecular characterization,phylogenetic comparison and serological relationship of the Imp protein of several ‘Candidatus Phytoplasma aurantifolia’ strains

The immunodominant membrane protein Imp of several phytoplasmas within the ‘Candidatus Phytoplasma aurantifolia’ (16Sr‐II) group was investigated. Eighteen isolates from Iran (11), East Asia (5), Africa (1) and Australia (1) clustered into three phylogenetic subgroups (A, B and C) based on the 16S rDNA and imp genes, regardless of geographic origin. The imp gene sequences were variable, with more non‐synonymous than synonymous mutations (68 vs 20, respectively), even though many of the non‐synonymous ones (75%) produced conservative amino acid replacements. Eight codon sites on the extracellular region of the protein were under positive selection, with most of them (75%) coding for non‐conservative amino acid substitutions. Full‐length (21 kDa) and truncated (16 kDa) Imp proteins of two economically important Iranian phytoplasmas [lime witches’ broom (LWB) and alfalfa witches’ broom (AlWB‐F)] were expressed as His‐tagged recombinant proteins in Escherichia coli. An antiserum raised against full‐length recombinant LWB Imp reacted in western blots with membrane proteins extracted from LWB‐infected periwinkle and lime, indicating that Imp (19 kDa) is expressed in infected plants and is a membrane‐associated protein. The same polyclonal antibody also detected native Imp in proteins from periwinkles infected by phytoplasmas closely related to LWB (subgroup C) only, confirming phylogenetic clustering based on 16S rDNA and imp genes. Imp proteins of LWB and AlWB‐F isolates were also recognized by an antiserum raised against an enriched preparation of AlWB‐F phytoplasma cells, demonstrating the antigenic properties of this protein.