Introduction

‘Candidatus Liberibacter’ species are a highly variable group of non-culturable fastidious gram-negative bacteria that colonize the phloem of their host plants. The bacterium accumulates in sieve elements. This leads to collapsed conductive pathways, interrupts assimilate transport, and results in general cell death (Haapalainen, 2014, Miles et al., 2010). The ‘Ca. Liberibacter’ species have different temperature optima and varying degrees of tolerance to high temperatures (Munyaneza et al., 2012).

Plant diseases with major economic impact are associated with ‘Ca. Liberibacter’ and psyllids, their reported vectors. The best known example is huanglongbing disease in rutaceous plants caused by the three species ‘Ca. Liberibacter asiaticus’, ‘Ca. L. americanus’, and ‘Ca. L. africanus’. These are transmitted by the psyllids Diaphorina citri and Trioza erytreae. Huanglongbing disease caused damage worth $1 billion per year in the period 2015–2020 in the U.S. alone (Li et al., 2020). Another species of ‘Ca. Liberibacter’ is ‘Ca. Liberibacter solanacearum’ (Lso). Lso has a wide range of host plants, especially in the families of solanaceous and apiaceous crops and causes zebra chip disease in potatoes. This disease has been reported to cause yield losses and additional costs of $11 million per year in the Pacific Northwest of the U.S. alone for plant protection measures to control its vector Bactericera cockerelli (Levy et al., 2020; Sandanayaka et al., 2013).

Until now, several haplotypes of Lso have been described. The haplotypes A, B, C, D, and E are known to infect cultivated crops of major economic importance. Haplotypes A and B are transmitted by Bactericera cockerelli to solanaceous plants in the Americas and New Zealand (only haplotype A in NZ). In Germany and northern Europe, Trioza apicalis is the known vector transmitting haplotype C to carrot (Daucus carota) plants (Munyaneza et al., 2015; Nissinen et al., 2014; Ragnarsson et al., 2016). Haplotypes D and E have been found in apiaceous plants in several countries in the EU and the Mediterranean Basin. The main vector for these haplotypes is Bactericera trigonica; additionally, Bactericera tremblanyi was found in Morocco, but is not a vector of the bacterium (Antolinez et al., 2017). Haplotypes D and E were also found in carrot plants in Tunisia (Othmen et al., 2019) but the vector is still unidentified. In Israel, haplotype D is transmitted by the psyllid Bactericera trigonica to carrot plants in this region (Mawassi et al., 2018).

Both the diseases huanglongbing and zebra chip as well as their related vectors are not present in Europe. In contrast, the psyllid T. apicalis, as one vector of Lso that infests carrot plants is present in Europe.

In addition to vector-bound transmission of Lso by psyllids such as T. apicalis, transfer from Lso-infected seeds to seedlings has been considered as another pathway of transmission in carrots. Lso was detected in seedlings grown from infected carrot seed lots (Bertolini et al., 2015) but such transmission could not be observed in other studies (Mawassi et al., 2018; Nissinen et al., 2021; Oishi et al., 2017) or contrasting results were found (Loiseau et al., 2017).

In this study, we focus on Lso infection of carrot plants as transmitted by T. apicalis. Damage to carrot plants caused by T. apicalis is described as curled foliage and reduced growth, especially of the root, and proliferation of lateral roots (Markkula et al., 1976; Nehlin et al., 1994; Rygg, 1977). Since Lso was not known at that time, no distinction was made whether the damage was caused by the psyllid T. apicalis or due to infection with Lso. Following the first study of Lso with T. apicalis and carrot plants by Munyaneza et al. (2010), the symptoms associated with Lso infections in carrot plants were studied. Typical symptoms associated with Lso infection in carrot plants are reported to be leaf curling, yellow and purple discolouration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots (Alfaro-Fernández et al., 2012, Haapalainen, 2014; Munyaneza et al., 2015), which are in general the same as for T. apicalis (see above). The EPPO Bulletin (2020) names these symptoms “yellows decline”. However, there is no official name for any disease caused by Lso in carrot plants. Moreover, the symptoms associated with Lso are rather unspecific and other biotic stresses can induce similar symptoms on carrot plants. For example, Carrot Red Leaf Virus, also causes discolouration of the foliage (Yoshida, 2020), aster yellows phytoplasma belonging to the genus ‘Candidatus Phytoplasma’ causes chlorosis, stunting, twisting and proliferation of plant stems, and the development of adventitious roots (Frost et al., 2013) and Spiroplasma citri causes yellow-purple discoloration and also stunting of shoots and roots as well as formation of secondary roots (Lee et al., 2006). Therefore, it is difficult to clearly attribute symptoms on carrot plants and carrot root weight losses to T. apicalis and/or Lso infections.

The amount of Lso infection in carrot plants and psyllids can vary between different areas, even within the same country (Haapalainen et al., 2017). The Lso infection rate of carrot plants in Norway varied from 33 to 100% and in T. apicalis individuals from 21 to 56% (Munyaneza et al., 2014). In Finnish carrot samples, Lso was found in 6% of the asymptomatic samples (Munyaneza et al., 2011). Munyaneza et al. (2015) reported that 50% of the symptomatic carrot plants in Germany were tested Lso-positive, whereas 24% of T. apicalis psyllids were tested positive.

So far, relevant carrot yield losses in Europe caused by Lso and T. apicalis have been mainly reported from Scandinavian countries. For example, in Finland, Lso has been described as a cause for significant carrot weight loss in carrot plants by Nissinen et al. (2021). In Germany, Lso was detected in carrot plants and T. apicalis for the first time in 2014 (Munyaneza et al., 2015). However, for Germany there are no reports of significant yield losses in carrots due to Lso and no comparable studies are available.

In this study, we focus on the major area Lower Saxony for organically grown carrot. In this region, organic carrot plant production has increased and the focus is on carrot root weight and not marketability as fresh vegetables. Organically grown carrot plants are a major cash crop for farmers in this region. Thus, pests and pathogens affecting carrot plant weight are of high economic interest to the involved parties.

The aim of this on-farm field study was to: (i) analyse the loss of carrot root weight under field conditions due to Lso infection, (ii) analyse the relation between Lso infection in T. apicalis and infection rates in carrot plants at harvest (and in one year at three time points during the growing season). (iii) Compare symptoms associated with Lso infection of carrot plants described earlier and symptoms observed in this study.

Materials and methods

Field study sites, plant material, insect material

The study was conducted on carrot fields of organically managed farms in a carrot growing region in Lower Saxony, Germany, from 2018 to 2021. In total, eight different fields were investigated: four fields (field 1, 3, 5, and 7) from Farm A with carrot variety Romance and Bolero F1 and Naturland certification; four fields (field 2, 4, 6, and 8) from Farm B with carrot variety Solvita and Demeter certification (see Table 1). All fields were irrigated by farmers as required. The fields were divided into 20 cells of approximately the same size (5 × 4 cells along the longer/shorter side of the field). Section S1 in the Supplementary Material provides detailed information on the carrot fields including sketches of the field shape, cells, and positions of the traps described below.

Table 1 Dates of sowing and sampling as well as number of carrot samples with and without discoloured foliage in the years 2018 to 2021
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The fields were monitored for the occurrence of T. apicalis with two Rebell orange sticky traps (Andermatt, Grossdietwil, Switzerland) per field from May to July. The traps were placed at the edge and 20 m inside the field of the western field site. Traps were changed and screened for insects once per week. Psyllids were identified morphologically using a binocular, subsequently removed from the sticky traps, and stored in 70% Ethanol at room temperature until analysis by PCR (OEPP/EPPO, 2020). Additional T. apicalis were collected on the fields using net sweeping.

Carrot plants were sampled from the eight fields for visual assessment and testing for Lso. ISPM Standard 31 was followed to determine the minimum number of samples per field for a 95% confidence level of detection and 5% level of detection which resulted in 59 carrot plants per field. Four carrot plants were collected per cell, with the aim of sampling two carrot plants with discolouration and two carrot plants without discolouration (random sampling within each group). In cases this was not feasible, e.g. only carrot plants with/without foliage discolouration present, more samples with/without discolorations were collected to obtain a total of four samples per cell independently of foliage discolouration. The samples were collected in September from all fields.

Additional carrot samples from field 3 and field 4 were collected in August and October 2019 using the same sampling schema. The sampling dates and number of carrot plants per field and year are shown in Table 1.

Each sampled carrot plant was assessed for visible features previously described in the literature: discoloured and curled foliage, proliferation of secondary roots. The weight of the whole carrot plant was determined for carrot samples from 2019. In the years 2020 and 2021, the root weight and foliage weight were recorded separately, to assess a possible yield loss of carrot roots. Section S2.2 from the Supplemental Material provides pictures of the symptoms described before as well as pictures of carrot plants that were considered as symptomless.

Temperature and precipitation data from 2018 to 2021 were obtained from the weather station Uelzen for the fields of farm A and from weather station Wendisch-Evern for the fields of farm B (Table S4 in the Supplemental Material).

Lso detection in carrot roots by PCR

The presence of Lso in carrot root samples was detected by PCR. Total DNA was extracted from the middle of the carrot root using a CTAB buffer extraction method modified after Munyaneza et al. (2010). Five hundred milligrams of carrot tissue were homogenized with a hammer and three to five ml of CTAB extraction buffer (2% w/v CTAB; 20 mM EDTA.Na2.2H2O; 1.4 M sodium chloride, 100 mM tris ultrapure) were added after the homogenization step. Five hundred µl of the homogenate were incubated at 65 °C and 1200 rpm (Thermomixer comfort, Eppendorf) for 60 min. After incubation, 300 µl of chloroform was added. Samples were vortexed, followed by another incubation step at 22 °C and 300 rpm for 5 min. The samples were inverted thoroughly and centrifuged at 4 °C and 10,410 × g for 15 min (Eppendorf Centrifuge 5415 R). Three hundred µl of the aqueous phase were transferred to a new tube and 210 µl of ice-cold Isopropanol (70%) were added and centrifuged at 4 °C and 10,410 × g for 15 min. After discarding the supernatant, the DNA was washed with 500 µl of ice-cold Ethanol (80%), followed by vortexing and centrifuged again at 4 °C and 10,410 × g for 15 min. The DNA pellet was dried at 45 °C and suspended in 50 µl of sterile nuclease-free water.

The presence of Lso was tested with three primer pairs (Table S5 in the Supplemental Material) following the OEPP/EPPO (2020) protocol. The PCR analyses were performed in a total volume of 25 µl using 2 µl DNA template, 12.5 µl of the MyFi Mastermix (Maeridian Bioscience), 0.5/1.25/1 µl forward and reverse primer (0.2/0.5/0.4 µM), and PCR grade water to fill up to 25 µl for the fragments 16S, 16-23S, and 50S respectively (Table S5 in the Supplemental Material). The initial denaturation step lasted 120/30/30 s at 94/98/94 °C. The amplification in 35/35/40 cycles with denaturation for 30/10/30 s at 94/98/94 °C was followed by annealing for 30/20/30 s at 62/55/53 °C and elongation for 60/30/30 s at 72/72/72 °C followed by the final extension for 10/7/7 min at 72 °C. The amplified fragments were visualized by agarose gel electrophoresis using the fluorescent dye HDGreen DNA-Dye (Intas Science Imaging Instruments GmbH, Germany).

To confirm the presence of Lso haplotype C in carrot samples, a selection of Lso-positive samples was subjected to sequencing of the three fragments 16S, 16S-23S, and 50S according to OEPP/EPPO (2020). The PCR products were purified using MSB Spin PCRapace kit (Invitek Molecular, Germany) according to manufacturer’s instructions. Sequencing was performed by LGC Biosearch Technologies (Berlin, Germany). The haplotype of each sample was determined following OEPP/EPPO (2020) using a custom software package “haplotype-lso” (v.0.4.4), that is available at https://github.com/holtgrewe/haplotype-lso.

Lso detection in T. apicalis by PCR

Total DNA was extracted from T. apicalis individuals using the Blood and Tissue Kit (Qiagen GmbH, Germany) following the manufacturer’s recommendations. Insect DNA was tested for the presence of Lso by PCR following the protocol of Munyaneza et al. (2010), using the primer pair OA2/OI2c (Table S5 in the Supplemental Material). OA2 is a Lso specific primer (Liefting et al., 2009). The PCR was performed in a total volume of 25 µl with 2 µl DNA template, 12.5 µl MyFi Mix, 0.5 µl of each primer (10 µM), and 9.5 µl PCR grade water. The initial denaturation of 3 min at 94 °C was followed by the amplification in 39 cycles with denaturation for 20 s at 94 °C, annealing for 20 s at 62 °C, and elongation for 60 s at 72 °C, followed by the final extension for 5 min at 72 °C.

Assessment of Lso DNA degradation in psyllids

The potential degradation of the bacterium in T. apicalis insects by decay processes due to the time on the traps was investigated in a subsequent laboratory experiment. A sticky trap with ten T. apicalis individuals from our laboratory population was incubated in a climate chamber for one week at 23 °C during daytime (16 h) and 17 °C during night-time (8 h). Another ten T. apicalis individuals were analysed directly. The presence of Lso was analysed as described in Section "Lso detection in T. apicalis by PCR".

Data analysis

Statistical analysis was performed using R version 4.2.1. Section S3 in the Supplemental Material provides a detailed transcript of the performed statistical tests. A significance level of 5% was used.

To summarize, we first considered results within each field and per discoloured/non-discoloured plants. We subsequently pooled samples within each field regardless of their foliage being discoloured or not. Tests were performed using Student’s t-test (which assumes normal distribution) and the non-parametric Wilcoxon rank sum test (to account for data not appearing normally distributed such as carrot root weight on field 8). To account for false positives due to multiple testing, e.g., when testing for each field, Bonferroni correction was applied.

Results

Influence of Lso infection on carrot weight

Because of the targeted sampling strategy, the difference in carrot plant weight for each field was first considered separately for the discoloured and non-discoloured plants. First, the plant/root weight was tested for differences between Lso-positive and negative plants. No significant difference could be detected except for field 8 for discoloured plants (Student's t-Test, P-value = 0.00386 and 0.0382 after Bonferroni correction). However, this is based on only four Lso-positive samples vs. 31 Lso-negative ones. Further, the plant weight distribution of the Lso-negative samples does not appear to be normally distributed. Thus, a non-parametric Wilcoxon rank sum test was performed which gives an uncorrected P-value of 0.09. The details on this statistical analysis, where discoloured and non-discoloured plants are considered separately, can be found in Sections S3.4 and S3.5 in the Supplemental Material.

The targeted sampling strategy that we used may introduce certain biases when pooling plants with and without discolouration. Pooling all plants from a field increases the power of statistical tests and accepting this bias is possible when the results after pooling are considered carefully. In the following, we consider the results after pooling. The results described above relate to not-pooled data.

Figure 1 shows the root (1A)/total plant (1B) weight of the samples for Lso-positive and negative samples for each field which was tested for significant difference using Student’s t-Test. In the following, we limit the description to root weight only. Only field 8 showed a significant difference between Lso-positive/negative samples (P-value 0.00064 before and 0.00384 after Bonferroni correction). When dropping the assumption of normality and using a Wilcoxon rank sum test, the corrected P-value is 0.274. Sections S3.6 and S3.7 in the Supplemental Material contain the details of the statistical analysis.

Fig. 1
figure 1

Influence of ‘Candidatus Liberibacter solanacearum’ (Lso) infection on carrot root (A) and plant (B) weight. Figure 1A shows the root weight of Lso infected (positive) and non-infected (negative) carrot plant samples on fields 5–8 in the years 2020 and 2021. Figure 1B shows the total plant weight of the Lso positive and negative carrot plant samples of fields 3–8 in the years 2019 to 2021. The figures show boxplots overlaid with the individual values as point clouds. The upper and lower borders of the box indicate the interquartile range (IQR) while the tick bars indicate the medians while the whiskers indicate median ± 1.5*IQR

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Supplementary Table S3 shows the results of the three PCR tests done for each carrot sample. Using three PCR tests as recommended in the EPPO Bulletin (2020) led to more precise Lso detection as for some Lso positive samples, single PCRs showed a negative result. Additionally, we note that all Lso-positive carrot plants sequenced were infected with Lso haplotype C (data not shown), matching previous reports of Lso haplotypes in carrots.

To illustrate the variability of growth and root development of Lso-positive plants, the two samples 116 and 141 from field 3 were selected (Fig. 2). They were tested Lso-positive but displayed diverse development of the root and a great weight range. Sample 141 (Fig. 2A) had red and yellow discolouration of the foliage and stunted root growth but no lateral roots. In contrast, sample 116 (Fig. 2B) showed only marginal yellow discolouration, no stunted root growth, and no lateral roots. The carrot root weight also differed greatly. Sample 141 had a total weight of 32.6 g while sample 116 weighed 16 times that with a total weight of 536.6 g. In comparison, the mean weight of Lso free carrot root samples of field 3 was 256 g.

Fig. 2
figure 2

‘Candidatus Liberibacter solanacearum’ positive carrot samples number 141 (A) and number 116 (B), that were collected from field 3 in September 2019

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No formal, direct assessment of carrot yield in terms of total harvested carrot weight per hectare was performed in this study. However, the participating farmers did not report any economically relevant loss of carrot root weight.

Lso infection rate and T. apicalis numbers

The Lso infection rate of carrot roots showed a wide variation between fields and years ranging from 2.5% to 80% (Fig. 3). In 2020 and 2021 (Fields 5–8), the rate of Lso infected plants was much lower (2% to 20%) than in the previous two years on Fields 1–4 (53% to 80%). This difference is also significant as the P-value is 0.0294 by Wilcoxon rank sum test (cf. Section S3.7 in the Supplementary Material).

Fig. 3
figure 3

‘Candidatus Liberibacter solanacearum’ (Lso) infection rate of carrot roots in relation to numbers of Trioza apicalis. The bars show the proportion of Lso-positive plants per field and year. The numbers above the bars indicate the total number of T. apicalis on two sticky traps per field in the three month period from May to July on the respective field

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There was also a great variation in the numbers of T. apicalis per field (Fig. 3). Field 5 (2020) had the highest number of T. apicalis with 169 individuals trapped in total from May to July but did not have the highest Lso infection rate. In field 3 (2019) we found 80% of Lso infected plants but only 36 T. apicalis individuals were caught. No significant correlation was found between the number of T. apicalis and percentage of Lso-positive carrots per field (Spearman P-value: 0.93, Pearson P-value: 0.87, cf. Section S3.8 in the Supplementary Material).

Beside the Lso infection rate in carrot plants, we also analysed the Lso infection rate of T. apicalis (Table 2). In addition to this, Table 3 shows the number of field-collected T. apicalis in the years 2018 to 2021 which were analysed using PCR to detect Lso. The number of Lso-positive T. apicalis varied between 0 and 46% (Table 2). The Lso infection rates of T. apicalis were low considering the high infection rates and decreased from 2019 to 2021. In total, only 21 out of 184 individuals were Lso-positive.

Table 2 Results of the Trioza apicalis PCR tests with Lso specific primers. Trioza apicalis were field-collected with both sticky traps and net sweeping from 2018 to 2021
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Table 3 Numbers of total carrot plant samples, as well as count of Candiatus Liberibacter solanacearum (Lso)-positive and negative carrot samples, and Lso infection rates at the three sampling dates in 2019 on fields 3 and 4
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Assessment of Lso DNA degradation in psyllids

All psyllids of the directly analysed group and 90% of the incubated psyllids were Lso-positive. We therefore assume that no relevant decay processes occur on sticky traps either in the climatic chamber nor in the field.

Lso infection rate and symptoms of carrot foliage

In our field study sites, carrot foliage showed yellow and red and to a lesser extent, brown and violet discolouration (Fig. 4 and Table S1 in the Supplemental Material). Yellow was the most prevalent foliage discolouration in 2018, 2020, and 2021, while red foliage discolouration was found in 2019. In this study, curled foliage was not found on any carrot sample from 2018 to 2021. Lateral root formation was observed in 6%, 22%, 9%, and 16% of carrot samples in 2018, 2019, 2020, and 2021 respectively. Detailed data can be found in Supplementary Table S1.

Fig. 4
figure 4

Carrot plants with ‘Candidatus Liberibacter solanacearum’ (Lso) infection and discolouration of foliage. The bars show the proportion of plants with/without foliage discolouration per field and the proportion of Lso-positive tested samples with the respective discolouration per field in the years 2018 to 2021. The numbers in the bars indicate the number of carrot samples with the respective colour of foliage

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Most Lso-positive samples had discoloured foliage. However, not all samples with discolouration were Lso-positive. Especially on field 7, the number of samples with and without discolouration (43 vs. 38 samples) were nearly equal, but only 19.8% of the carrot plants were tested Lso-positive (Table 1 and Table S1 in the Supplemental Material). On fields 1, 5, and 6, more than 50% of Lso-negative plants had discolouration.

We tested for correlation between foliage discolouration and Lso infection for all plants with Fisher’s exact test which was found to be significant with a P-value of 4 × 10–10. When testing for correlation within the individual fields, we found a significant correlation only for 2 of the 8 fields (field 2 and 4, P-value of 0.00007 for field 2, 0.001 for field 4). The details of this statistical analysis are shown in Section S3.4 of the Supplemental Material including tables of all P-values.

Interestingly, a decreasing trend in the Lso infection rate of fields 3 and 4 was observed during the carrot growing season (Table 3). In field 3, the infection rate in August was 85% and decreased to 80% in September and 52% in October, although carrot plants of all three sampling dates showed various foliage discolouration. This decrease was more pronounced in field 3 than in field 4. Field 4 showed a decrease in the infection rate from 65% in August and 52% in September to 44% in October.

Weather data

The average temperature from May to September 2018 was 18 °C, and therefore higher compared to an average temperature of 14–16 °C in 2019, 2020 and to 2021 (Table S4 in the Supplementary Material).

Discussion

Regarding the influence of Lso infection on root weight, we found the following. The degree of Lso infection in the carrot plants was high in the years 2018 and 2019. However, the farmers did not report any loss of yield even for fields with higher infection rates of 50%-80%. While we did not perform a direct assessment of yield, we measured the carrot root and total plant weight of collected carrot samples. We performed a thorough statistical analysis for an association of carrot weight and the presence of Lso but found no statistically significant association. We do not consider the only exception of field 8 as convincing as it is based on four Lso-positive samples out of 80 and overall variation in carrot root weight was very high on this field (from less than 100 g to above 600 g). This corresponds to an infection rate of only 5% of the sampled carrot plants, the second lowest in the whole study. Further, no economically relevant carrot yield loss was reported by the farmer.

To the best of our knowledge, a relation between Lso infection and a reduction of carrot weight under field conditions was only discussed by Nissinen et al. (2021). These authors observed the highest carrot root weight loss in the dry and warm summer of 2016 and could only show a correlation between Lso titre and root weight loss in this year. We note that Nissinen et al. (2021) found a correlation between Lso titre and root weight loss while we measured presence of Lso only. It remains unclear why T. apicalis and Lso would cause a relevant carrot weight loss only under certain environmental conditions.

One possible explanation could be different temperature conditions at various growing regions. One factor influencing ‘Ca. Liberibacter’ species population dynamics is temperature. The optimum temperature for Lso multiplication is between 27 °C and 32 °C (Munyaneza et al., 2012). Lso infection in potatoes can lead to large yield losses when temperatures are in this range (Soliman et al., 2013). On the other hand, propagation of Lso slows down at temperatures below 17 °C and above 32 °C (Munyaneza et al., 2012). Nissinen et al. (2021) assume a lower temperature range for haplotype C in carrots when compared to haplotype A and B in potatoes. They measured higher relative bacterial titres in carrot plants when temperatures were higher than 15 °C. This could be a possible explanation for the higher infection rates in 2018 on surveyed fields in Germany. As described in Section "Weather data" (cf. Table S4 in the Supplementary Material), the average temperature from May to September was higher in 2018 than in 2019 to 2021. We could not confirm a carrot root weight reduction under the more moderate temperate climatic conditions in Germany. Remarkably, this was the case in organically grown carrot plants in Germany where no insect pest management for T. apicalis was applied in the studied fields. In contrast, the vector T. apicalis was chemically controlled in the study by Nissinen et al. (2021).

Also, there are several differences between this study and the one by Nissinen et al. (2021). While no irrigation was performed in the Finnish study, the farmers in this study irrigated their fields as needed. Nissinen et al. suggest irrigation for improving plant growth to avoid the vulnerable plant stages to coincide with high psyllid pressure. Further, in several years both the psyllid pressure and Lso infection rate were higher in the study by Nissinen et al. (2021) than in this study. Also, the growing season in Scandinavian countries is much shorter than in Germany which may also affect the overall impact of psyllid pressure and Lso infection.

The Lso infection rate in carrot plants from the sampled fields varied greatly and from year to year. These variations are comparable to those found in Finland (Nissinen et al., 2021) and Norway (Munyaneza et al., 2014).

We found a significant correlation between the previously described phenotype of foliage discoloration and the presence of Lso. This agrees with Nissinen et al. (2014) who found an increasing number of discoloured leaves with an increasing titre of Lso in infected carrot plants. However, we also found Lso infected plants without visible symptoms, which is in accordance with Munyaneza et al. (2011), who they analysed carrot samples from Finland and found Lso in 5.5%, 31.3% and 80% of asymptomatic plants samples, plants with curled leaves and plants with curled leaves and discolouration respectively. In Norway, Munyaneza et al. (2014) found Lso in 15.4% and 33.3 to 100% of asymptomatic and symptomatic carrot plant samples, respectively. We emphasize that the phenotypes observed in this study can also be caused by other pathogens. Red discoloration of the foliage can also be caused by phytoplasmas and viruses e.g., Carrot Red Leaf Virus (Yoshida, 2020) and yellow discoloration by various fungal diseases or lack of nutrients. Therefore, discoloration of the carrot foliage is not a good predictor for a ‘Ca. L. solanacearum’ infection. Proliferation of secondary roots and leaf curling are also reported to be ‘Ca. L. solanacearum’ symptoms in carrots. We found that only a small proportion of infected plants developed lateral roots. This symptom is not a robust feature of a ‘Ca. L. solanacearum’ haplotype C in carrots, as Haapalainen et al. (2017) also suggested. Proliferation of secondary roots can also be caused by soil borne nematodes (Pedroche et al., 2009). Leaf curling can also be induced by sucking insects that feed on the carrot plant. In addition to T. apicalis, other sucking insects are colonising carrot plants such as the aphid Cavariella aegopodii. These aphids are also known to be responsible for similar leaf symptoms by transmission of Carrot mosaic potyvirus (OEP/EPPO, 2001). Since the visible symptoms associated with Lso infection in carrot plants are unspecific, future studies should employ a broader screening for additional pathogens such as viruses and phytoplasmas.

When considering the infection rates as determined at different time points, we found that the Lso infection rate of the investigated carrot fields decreases during the growing season (Table 3). This is in line with the findings of Sumner-Kalkun et al. (2020) who also found decreasing numbers of positive carrot plant samples after more than ten weeks of the study period. So far, there is no explanation for this effect in the literature. Moreover, Nissinen et al. (2021) describe a rising Lso titre over time. As we did not measure titre, no direct comparison with the report by Nissinen et al. (2021) is possible. However, there are potential explanations for the decreasing infection rates. One possible explanation is potential inhibition of PCR in the more mature roots because of higher content of phenolic compounds or polysaccharides, such as reported by Malvick and Grunden (2005). Another potential explanation could be that the distribution of the Lso bacteria within the plant changes. For example, Wang et al. (2021) reported different Lso titres over time after infection where the highest concentration changed from the root in earlier stages to the foliage in later stages. A related possible explanation is that the overall ratio of bacteria to plant material grows smaller over time because of the plant growing and the bacteria concentration falls below the detection limit.

Our study showed a great variation of the Lso infection rate in T. apicalis in different years and fields. A reason for this might be a great variation in the transmission efficiency of Lso haplotype C from T. apicalis to carrot plants. There are similar results in the literature for other Lso haplotypes and their psyllids vectors. In B. cockerelli, Munyaneza et al. (2007) pointed out that only six out of nine B. cockerelli populations transmitted Lso to potatoes resulting in the zebra chip disease. Hung et al. (2004) found that the proportion of ‘Ca. L. asiaticus’ carrying psyllids varied from 55 to 75% in different populations.

We could not observe any correlation between the number of T. apicalis and the proportion of Lso-positive tested carrot plants. This is different from the observations from Finland (Nissinen et al., 2021). Moreover, we observed high Lso infection rates of carrot plants, despite low numbers of the vector T. apicalis. So far, T. apicalis is the only known vector for Lso haplotype C in carrot plants. However, there might be further transmission pathways of Lso to carrot plants, such as other psyllids or further insect vectors. Pitino et al. (2014) found that adult mealybugs (Ferrisia virgata) were able to transmit ‘Ca. L. asiaticus’ to periwinkle and citrus leaves. In our study, we found numerous insects of the Heteropteran on the sticky traps, as well as different aphid species (data not shown), and many of them are known as vectors for, e.g., viruses. To understand the epidemiology of Lso infection in carrot, it would be very useful to conduct further studies to reveal potential additional vectors which could also transmit the bacterium to carrot plants.

Regarding our methodology, using a random rather than a targeted sampling scheme in future studies would allow for a more unbiased estimates of infection rates. This would also allow to assess the suitability of discolouration as a predictor for Lso infection. Regarding the sampling scheme, we recommend a two-step approach for future studies where first the overall infection rate is roughly estimated based on random sampling of few carrot plants. In the case of higher infection rates, random sampling can continue. Otherwise, targeted sampling should be considered, depending on the objective of the study.

In contrast to our original hypothesis, we could find no evidence that either Lso or T. apicalis pose an economic risk to organically grown carrots in Lower Saxony, Germany. We could confirm the presence of Lso haplotype C in both carrot plants with high infection rates and the psyllid. However, no economically relevant carrot yield losses due to Lso infection have been reported by the farmers participating in this study. This is in line with lack of evidence for such losses in Germany. Even more, we could not find evidence for reduction of carrot root or total plant weight because of Lso infections. Apparently, carrot plants infected with Lso can develop without economically relevant reduction of weight. Overall, our results indicate that the role of Lso as causal agent in the occurrence of carrot root weight loss is of minor relevance in organically managed carrot fields in Lower Saxony, Germany. We remark that while zebra chip disease and huanglongbing disease cause economic damages in the millions, no such reports of economic damages in these dimensions exist for Lso haplotype C in carrot plants.

Economically relevant carrot root weight loss might still be caused by a combination of infection with Lso and further biotic or abiotic influences, e.g., drought stress or infections with funguses, viruses or phytoplasma. In a subsequent study, samples could be analysed further for such pathogens. During our study, T. apicalis was present only in very small numbers on carrot fields and most of the T. apicalis captured in this study tested negatively for Lso and were considered as not infected. Nevertheless, we consider monitoring of the situation as necessary.