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.     

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.     

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.     

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.     

A reappraisal of the current status of <Emphasis Type="Italic">Tilletia indica</Emphasis> as an important quarantine pest for Europe

Tilletia indica, the fungus responsible for Karnal bunt disease of wheat, is currently recognised as a quarantine pest by both the EU and EPPO. The evidence that has been used to justify this status is reviewed and found to be in need of reappraisal. Yield losses caused by the pathogen are insignificant and disease levels are rarely high enough to cause serious quality problems. The sole reason for its designation as an important quarantine pest would seem to lie in the serious implications for trade should the pathogen be detected in an exporting country. Since there is strong evidence to suggest that T. indica may not even establish in Europe, it would seem logical to reclassify the pathogen as the cause of a minor disease that is likely to have little quarantine significance for Europe. However, since most wheat-growing countries have strict quarantine regulations aimed at preventing the introduction of T. indica, this reclassification is unlikely to occur unless plant health authorities around the world can agree on a new status of reduced importance for T. indica that suits its actual potential as a pest. David Jones is a retired employee of the Central Science Laboratory (CSL). Statements and opinions expressed in this paper are those of the author and are not the views of the Central Science Laboratory.     

Seasonal dynamics of ground cover in Cirsium arvense – a basis for estimating grazing losses and economic impacts

Analysis of the economics of managing Cirsium arvense in grazed pastures worldwide has been hampered by a lack of data on the within‐year seasonal dynamics of the weed and its impact on the yield of palatable herbage. To redress this, the seasonal pattern in within‐patch percentage ground cover of the weed was determined from measurements on 39 dairy and 66 sheep and/or beef cattle farms in New Zealand during 2009–2010 and 2010–2011. This pattern was then scaled using farmer estimates of peak whole‐farm cover to derive mean monthly covers for dairy, beef, sheep/beef, sheep and deer farms. These monthly covers corresponded, respectively, to mean annual percentage covers of 2.7, 1.7, 3.0, 5.9 and 2.9% and to mean annual percentage losses in pasture growth (palatable herbage yield) of 3.6, 2.3, 4.0, 7.8 and 3.9%. The latter, in combination with 2011–2012 farm statistics, revealed that C. arvense caused a national loss in pastoral farm gross revenue in New Zealand in 2011–2012 of $685 million ($446 m dairy, $233 m sheep/beef, $6 m deer). Beyond the scope of this paper, the monthly covers and their corresponding monthly losses in pasture growth provide a basis for modelling the economic impacts of C. arvense and its management at a farm scale. More generally, the analytical method that we have developed is appropriate for evaluating the economic impact of any weed in a grazed pasture, particularly those exhibiting pronounced seasonal patterns in occupancy, such as annuals and deciduous perennials.