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.     

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts,rootstocks and the Asian citrus psyllid

The use of proper management strategies for citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las) and transmitted by Asian citrus psyllid (ACP) (Diaphorina citri), is a priority issue. HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP populations. Here, dynamic populations of Las in different citrus hosts and each instar of ACP were studied, together with the seasonal growth and distribution of Las in different tissues, using conventional and TaqMan real‐time PCR. Different levels of susceptibility/tolerance to HLB were seen, resulting in different degrees of symptom severity and growth effects on hosts or rootstocks. Troyer citrange, Swingle citrumelo and wood apple were highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis of Las, mature and old leaves contained high concentrations in cool temperatures in autumn and spring. Las was detected earlier through psyllid transmission than through graft inoculation, and the amounts of Las (AOL) varied in different hosts. Thus, different AOL (10⁴–10⁷ copy numbers μL^?1) and Las‐carrying percentages (LCP; 40–53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP of psyllids were not affected by increasing the acquisition‐access time. The present study has significant implications for disease ecology. The combination of early detection, use of suitable rootstocks and constraint of psyllid populations could achieve better management of HLB disease.     

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.     

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.     

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.     

Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds

A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.     

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.     

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.     

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.     

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).     

A yellows disease system with differing principal host plants for the obligatory pathogen and its vector

A stolbur‐type phytoplasma is the putative pathogen of grapevine yellows disease that causes economic damage to vineyards in most growing areas around the world. The pathogen is known to be transmitted to vines by two planthoppers, Hyalesthes obsoletus and Reptalus panzer; the latter is found in Europe but has not yet been observed in Israel. The establishment of a vector–pathogen–plant relationship requires that the pathogen and the vector meet on a shared host plant. This does not happen in the ecosystem examined here, where two different principal host plants for the obligate pathogen and its vector exist: the pathogen is established on vines, while its vector, H. obsoletus, develops on Vitex agnus‐castus. The present study verified that: (i) the vector cannot complete its life cycle on vines; (ii) V. agnus‐castus does not grow in the immediate vicinity of vines, and does not harbour the pathogen; and (iii) the pathogen is not vertically transmitted from mother to offspring. Moreover, in a thorough search of plants in vine growing areas, no other plants were found that host both the vector and the pathogen. However, it was found that the planthopper can acquire the phytoplasma from infected vines. Nonetheless, this does not prove the ability of the planthopper to further transmit the pathogen to vines and does not explain the presence of the vector on the non‐preferred vines. Thus, the enigma of the pathogen–vector–host triangle in this system remains unresolved.     

Tomato can be a latent carrier of ‘Candidatus Liberibacter solanacearum’, the causal agent of potato zebra chip disease

‘Candidatus Liberibacter solanacearum’ was recently described as the causal agent of potato zebra chip disease. This pathogen occurs in North America, New Zealand, and Northern Europe on various crops, and may spread to other potato growing regions. Observation on ‘Ca. L. solanacearum’‐infected tomato and potato plants propagated in growth chambers over 5 years indicated that tomato plants (cvs Moneymaker and Roma) can be a latent carrier of ‘Ca. L. solanacearum’. Tomato plants graft‐inoculated with scions from latently infected tomato plants remained symptomless, but tested positive in a species specific PCR assay. ‘Ca. L. solanacearum’ was consistently detected in the top, middle and bottom portion of the symptomless tomato plants, including stem, petiole, midrib, vein, flowers and fruits. In tomato fruits, ‘Ca. L. solanacearum’ was evenly distributed in the tissues at the peduncle and style ends, as well as in the pericarp, and columella placenta tissues. This is the first report that ‘Ca. L. solanacearum’ is present in a plant reproductive organ. In contrast, potato plants (cvs. Jemseg, Atlantic, Shepody, Frontier Russet, Russet Burbank, Red Pontiac, and Russet Norkotah) grafted with scions from the same latently infected tomato plants resulted in typical symptoms of purple top, leaf scorch, and other disease symptoms in plants and brown discoloration in the vascular ring and medullary rays in tubers.     

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.     

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.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.