Risk assessment of ‘Candidatus Liberibacter solanacearum’ transmission by the psyllids Bactericera trigonica and B. tremblayi from Apiaceae crops to potato

Candidatus Liberibacter solanacearum (Lso) is bacterium transmitted by psyllids to Solanaceae and Apiaceae plants. So far, Lso is found in Europe affecting Apiaceae. In the Mediterranean region, Bactericera trigonica is the only known vector of Lso, but the leek-onion psyllid Bactericera tremblayi is another widespread psyllid and potential vector of Lso. Commonly, carrot, leek and potato are cultivated in the same zones and it is uncertain if these psyllid species are able to transmit Lso to potato plants. Here, we assessed the transmission of Lso by B. trigonica and B. tremblayi to potato plants. B. trigonica showed preference to ingest from the phloem, settle and oviposit on carrot and celery but not on potato. This was correlated with high Lso transmission rates to both carrot (80%) and celery (70%) but very low to potato (≤3%). B. tremblayi preferred leek over carrot and potato, the latter being the less preferred host. B. tremblayi readily ingested from the phloem of infected carrots but failed to transmit Lso from carrot to carrot. Our study shows that the risk of Lso transmission from Apiaceae to potato by B. trigonica is very low, and that B. tremblayi is not a likely vector of Lso.


Results
Probing behaviour of Bactericera trigonica and Bactericera tremblayi on different hosts. Stylet penetration process of B. trigonica and B. tremblayi in different host plants was monitored using the electrical penetration graph technique (EPG). The probing duration per insect (PDI) showed no differences between carrot and celery but was shorter when B. trigonica probed on potato than on carrot or celery, (F = 7.17, df = 2, 47, P = 0.002) ( Table 1). In contrast to the PDI, the time from the first probe to the first phloem salivation (E1) was longer for insects probing on potato than for those on carrot or celery (F = 8.10, df = 2, 47 P = 0.001) ( Table 1). Bactericera trigonica spent more time in non-probing activities (NP) on potato compared with celery or carrot (F = 5.71, df = 2, 47, P = 0.006). The duration of the stylet pathway (C) and the time to the first probe from the start of the EPG were not significantly different when B. trigonica probed on carrot, potato or celery (Duration of C: F = 0.97, df = 2, 47, P = 0.385; Time to the first probe from the start of EPG: H = 2.88, df = 2, P = 0.236) ( Table 1). On the other hand, B. trigonica probed in similar proportions from the xylem tissues (G waveform) of celery and potato but probed more from xylem of carrot compared to potato. (Number of G: H = 12.09, df = 2, P = 0.002). Additionally, B. trigonica failed to reach the phloem tissues of potato plants because no phloem salivation (E1) or ingestion (E2) waveforms were detected. By contrast, E1 and E2 waveforms were commonly detected when B. trigonica probed carrot or celery (Table 1).
For B. tremblayi, the time to the first probe from the start of the EPG was different between leek and potato but not between leek and carrot or when comparing carrot and potato (H = 10.38, df = 2, P = 0.006) ( Table 1). The duration of stylet pathway (C) was similar in all plants tested (F = 2.00, df = 2, 47, P = 0.140). The duration of non-probing (NP) and the time from the first probe to the first phloem salivation (E1) were significantly shorter in leek than those in carrot or potato and no differences were observed between carrot and potato (Duration of NP: H = 23.85, df = 2, P = 0.000; Time from first probe to first E1: H = 17.87, df = 2, P = 0.000) ( Table 1). Probing duration per insect (PDI) was higher in leek than that in carrot or potato and no differences were detected between carrot and potato (F = 9.71, df = 2, 47, P = 0.000) ( Table 1). Data showed that EPG variables related to xylem activities were similar among all species of plants tested (Number of G: H = 1.81, df = 2, P = 0.403; Duration of G: F = 0.30, df = 2, 47 P = 0.739) (Table 1). Moreover, B. tremblayi reached the phloem tissues of all plants tested but showed a clear preference to probe longer and to spend more time in phloem-associated waveforms on leek than on carrot or potato (Number of E1: H = 21.27, df = 2, P = 0.000; Duration of E1: H = 28.94, df = 2, P = 0.000; Duration of E2: H = 30.50, df = 2, P = 0.000) (Table 1). Additionally, although not statistically significant, B. tremblayi reached the phloem salivation phase (E1) more frequently on carrot than on potato (Table 1). Furthermore, B. tremblayi ingested phloem sap (E2) from carrots but failed to ingest phloem sap from potatoes ( Table 1). infected plants/30 receptor plants with at least one infected psyllid per plant). Therefore, although the results showed clearly that B. tremblayi acquired the bacterium, the pathogen was not transmitted to carrots by this species of psyllid.

Settling preference, oviposition and Lso transmission on different hosts. Bactericera trigonica.
Settling preferences and oviposition on carrot, celery and potato as well as Lso transmission by B. trigonica were evaluated in non-choice and dual-choice assays. Adult B. trigonica did not show a preference between the Apiaceae plants tested in the non-choice and dual-choice assays. In both assays, the settling and oviposition preference of B. trigonica was similar for carrot and celery ( Fig. 1a,b,e,f). However, settling and oviposition were significantly reduced on potato compared with carrot or celery in both the non-choice and dual-choice assays ( Fig. 1a,b,c,d,g,h). The percentage of infested plants was also higher for carrot or celery than that for potato, but when comparing carrot and celery in both non-choice and dual-choice assays, no differences in the percentage of infested plants were observed (Supplementary Table S1).
The transmission of Lso to the different plants tested in both non-choice and dual-choice assays was different for B. trigonica (Fig. 2). In non-choice tests, B. trigonica did not transmit Lso to potatoes (Fig. 2a). By contrast, B. trigonica efficiently transmitted Lso to carrot and celery, which showed similar percentages of transmission (Fig. 2a). In potato-carrot and potato-celery treatments in the dual-choice assay, Lso transmission was always significantly higher to carrot or celery than to potato (Fig. 2c,d). However, in the carrot-celery treatment, Lso transmission was not significantly different between the two plant hosts (Fig. 2b).
Bactericera tremblayi. Settling and oviposition of B. tremblayi on leek, carrot and potato were also evaluated in non-choice and dual-choice assays. Settling preference of B. tremblayi was significantly different among all host plants tested in the non-choice and dual-choice assays (Fig. 3). Our data indicated that B. tremblayi preferred to settle and oviposit on leek compared with carrot or potato in both types of assay ( Fig. 3a,b,c,d,g,h). Additionally, B. tremblayi preferred to settle and oviposit on carrot compared with potato ( Fig. 3a,b,e,f). Compared with potato, a higher percentage of leek or carrot were infested, although no differences were detected between carrot and leek (Supplementary Table S2).

Discussion
Lso is a persistently transmitted pathogen that has a close relationship with the psyllid vectors 15,17 . For successful transmission, the stylet of Lso infected psyllids must reach and inoculate infected saliva into the phloem tissues of a susceptible host plant 5,18,22 . Because of this, the study of psyllid host preferences and stylet penetration process is fundamental for a complete understanding of the epidemiology of Lso in different crops. In this work, we showed  In this study, B. trigonica clearly preferred to settle and oviposit on Apiaceae compared with potato plants. Settling and oviposition of B. trigonica on potato was very low, even when other plant species were not available (non-choice assay). The EPG data were consistent with this marked preference pattern for Apiaceae by B. trigonica and demonstrated that B. trigonica engaged in sustained feeding from the phloem of carrot and celery but failed to reach and feed from the phloem of potato plants. Since psyllids use the phloem sap as the primary source of sugars and amino-acids 6 , feeding from phloem tissues is fundamental for the reproduction and the ability of a psyllid species in order to colonize a given host. Therefore, this information suggests that B. trigonica cannot colonize potato because it could not continuously feed from the phloem tissues of potato plants.
On the other hand, phloem feeding is an absolute prerequisite for transmission, thus only colonizing vectors capable of sustained phloem feeding can efficiently spread phloem-restricted pathogens 26,27 . Actually, inoculation success is directly correlated with the duration of the salivation phase performed by the vector just before phloem ingestion 5,28 . Our results support these assumptions because high Lso transmission rates were detected in plants on which B. trigonica settled for long periods and salivated and ingested from the phloem. However, null or very low Lso transmission was obtained for potato on which B. trigonica showed poor settling, low oviposition and neither salivation nor phloem ingestion. Interestingly, despite no phloem related activities were observed by EPGs in an 8 h period, a very low percentage of potato plants tested positive for Lso in the dual-choice assays. This result suggest that eventually a few individuals of B. trigonica may have reached the phloem of potato in the extended 72 h period used in our dual-choice assays.
According to the results of this study, the risk of Lso transmission mediated by B. trigonica from Apiaceae to potato would be very low. Although highly infected carrot and celery crops commonly overlap in growing zones with potato, our data suggest that B. trigonica may land on but would not settle or feed from the phloem of potato. This result is consistent with few B. trigonica captured in potato crops growing near to carrot crops 24 . With these conditions, the primary transmission of Lso mediated by B. trigonica would be very unlikely. Moreover, if primary transmission does occur, secondary dispersion from the infected potato to other potatoes is likely be very low because B. trigonica cannot feed in a sustained way from the phloem tissues and therefore cannot colonize potato.
Generally, the results of this work were consistent with and complements well the work performed by Munyaneza et al. 22 . They showed that the risk of Lso transmission from potato to carrot is negligible because the potato psyllid B. cockerelli is not able to efficiently localize and feed from the phloem tissues of carrot plants. In our study, the risk of movement of Lso mediated by B. trigonica from carrots to potato was very low for a similar reason. However, assessing the risk of cross transmission from carrot to potato in Europe is complex because more than one vector could be involved in the transmission of this pathogen. For example, the role of T. apicalis in cross transmission from carrot to other economically important crops in northern Europe remains unknown and requires further investigation.  Additionally, other psyllid species catalogued as potential vectors of Lso should receive special attention, e.g., B. tremblayi or B. nigricornis 24 . Our data showed that B. tremblayi, preferred to settle, oviposit and feed on leek but also settled and fed from phloem on carrots. This result was unexpected because the attempts to rear B. tremblayi on carrots were unsuccessful, and to our knowledge, the life cycle of this psyllid cannot be completed on carrot 7 . However, for psyllids, adults and nymphs may display different host plant specificity, and adults of some psyllid species may feed temporarily from plant species unsuitable for nymphal development 8 . Based on this scenario, our results suggest that in the absence of a plant suitable for reproduction, adults of B. tremblayi might use carrot as a temporary food plant. The EPG results support such use because B. tremblayi also ingested phloem sap from carrots. Although feeding from phloem was correlated with the capacity of this species to acquire Lso, we also showed that this psyllid species did not transmit the bacterium to carrot. Lack of transmission could be explained by different factors. For example, it has been reported that when Lso is acquired, the bacteria must pass through the insect midgut epithelium to infect the haemolymph and then enter the salivary glands 15,29 . It is uncertain if Lso is able to complete circulation in the body of B. tremblayi since this process has not been studied in this psyllid species. Finally, in this Lso-psyllid interaction, it is possible that longer latency periods than normal are required for the replication of Lso in the psyllid body, which provides another explanation for the absence of transmission. However, we did not test for latency periods in B. tremblayi, and the only reported latency period for Lso is in B. cockerelli 29 ; therefore, additional research is recommended to fully understand the Lso infection process in this psyllid species.
In conclusion, our results suggest that the host plant preferences of B. trigonica strongly influences the host range of Lso in the Mediterranean region. As a consequence, the risk of Lso transmission from carrot to potato mediated by B. trigonica is negligible, but tests of the transmission ability of other psyllid species that feed on potato might identify other risks. Additionally, we concluded that the hypothesis was not supported that B. tremblayi is a competent vector of Lso. . Colonies were tested for Lso by real-time PCR; the percentage of infection of the B. trigonica colony was 97%, whereas B. tremblayi was Lso-free. Dr. Jaime Cubero from the National Institute for Agronomic Research (INIA), Madrid, Spain, kindly identified the Lso haplotype in our B. trigonica colonies as haplotype E, according to the procedure described by Nelson et al. 12 . For the assays, 5-7-d-old, adult psyllids were collected from the psyllid colonies with a handmade vacuum aspirator one hour after the beginning of the experiments. Groups of insects used in the preference and transmission assays contained similar proportions of males and females. EPG analysis of psyllid probing behaviour. The probing behaviour of B. trigonica and B. tremblayi was monitored on different plants (carrot, celery and potato for B. trigonica and carrot, leek and potato for B. tremblayi) using the electrical penetration graph (EPG) technique. A gold wire electrode (2 cm length, 20 μ m diameter) was attached to the insect pronotum following the procedure described by Antolínez et al. 23 . A second electrode (copper, 10 cm length, 2 mm diameter) was inserted into the soil of the plant container. The psyllids were starved for 1 h during acclimatization between the time of wiring and the beginning of EPG recording. Then, the psyllids were placed on the abaxial surface of a fully expanded leaf and were allowed to probe and feed on the test plant for 8 h. The EPG recordings were obtained using an eight channel DC EPG system (type Giga-8;

Methods
Wageningen University, The Netherlands). The signal was digitized using a DI-710 board (Dataq ® Instruments, Akron, OH, USA), and the digitized data were loaded onto a PC and analysed with Stylet+ software (EPG Systems, Wageningen, The Netherlands). A minimum number of 15 replicates were obtained for each plant and psyllid combination. EPG waveforms previously described for psyllids were identified according to Pearson et al. 4 as follow: non-probing (NP), intercellular stylet pathway (C), salivation into phloem sieve elements (E1), phloem sap uptake from the sieve elements (E2) and active intake of xylem sap from xylem vessels (G). The following EPG parameters were calculated to describe pathway activity and phloem or xylem activity according to Backus et al. 30 : probing duration per insect, PDI, is the amount of time an average insect has the stylet inserted; number of waveform events per insect, NWEI, is the sum of the number of events of a particular waveform divided by the total number of insects in each treatment; total waveform duration (min) per insect, WDI, is the sum of durations of each event of a particular waveform divided by the total number of insects in each treatment; and waveform Scientific RepoRts | 7:45534 | DOI: 10.1038/srep45534 duration per event by insect, WDEi, is the average duration of events of a given waveform by an insect in a cohort. The variable time from the first probe to the first E1 was calculated according to Sarria et al. 31 .

Lso detection by real-time PCR.
To detect Lso in plants, plant DNA was purified following the CTAB (cetyltrimethyl ammonium bromide) protocol 32 . To detect Lso in psyllids, psyllid DNA was obtained following the squash protocol 33 . Then, Lso was detected by real-time PCR in plant and psyllids using the primers, TaqMan probe and procedure described by Bertolini et al. 16 .

Transmission of Lso by Bactericera tremblayi.
Adult psyllids of B. tremblayi were tested for both the acquisition of Lso from carrots and the inoculation of carrots with Lso. Groups of psyllids were exposed to Lso-infected carrot plants for an acquisition access period (AAP) of 72 h. Then, the psyllids were removed from carrots and transferred to leek plants for 15 d (latency period of Lso). After the 15 d, 400 psyllids were collected from the leek plants and transferred to 100 healthy carrot plants, each contained in a transparent, plastic cylindrical cage (4 psyllids/plant). The psyllids had access to the entire plant for an inoculation access period (IAP) of 24 h. Later, the psyllids were removed and tested individually using real-time PCR. Plants exposed to groups of insects that tested negative for Lso by real-time PCR (70 plants) were discarded from the analysis. Plants exposed to groups of insects that tested positive for Lso (30 plants) were sprayed with 1 g L −1 of Confidor ® (Bayer, Kansas City, MO, USA) on days zero and 10 and were maintained under greenhouse conditions for eight weeks to test for Lso by visual inspection of symptoms and by real-time PCR.
Settling preference, oviposition and Lso transmission on different hosts. Non-choice assays. The assays used a set of three cages with each cage containing one treatment. The following three treatments were evaluated: (T1) 36 carrot plants, (T2) 36 celery plants and (T3) 36 potato plants. Each treatment was replicated three times, and the replicates were rearranged to minimize location effects. Each cage contained potted plants arranged in a square (six rows and six columns, with 12.5 cm between plants). The cages were 1 × 1 × 1 m and covered with an aphid-proof mesh net. On a flight platform similar to that described by Fereres et al. 34 , two hundred Lso-infected individuals of B. trigonica were released. The flight platform was place 0.5 m above the test plants inside each cage. Insects were released at solar noon in greenhouse conditions similar to those described for insect rearing. All cages were rotated 180° daily to avoid orientation bias. The percentage of psyllids settled per test plant was determined, and the eggs per plant were counted 72 h after psyllid release. Settling preference was calculated as the percentage of insects settled on plants per cage. The percentage of plants infested by at least one insect was also calculated. Then, test plants were sprayed with 1 g L −1 of Confidor ® (Bayer, Kansas City, MO, USA) and re-sprayed 10 d later to avoid further Lso transmission. Plants were maintained in a separate glasshouse under greenhouse conditions. Eight weeks after the experiment was completed, the percentage of plants infected with Lso was evaluated by visual inspection of symptoms and by real-time PCR as described above.
In a separate assay, the percentage of infested plants, settling and oviposition of B. tremblayi were also evaluated using a similar procedure to that described for B. trigonica. In these assays, the following treatments were evaluated: (T1) 36 leek plants, (T2) 36 carrot plants, and (T3) 36 celery plants. Because of the lack of transmission shown by B. tremblayi in the previous transmission experiments (see results section), the individuals of B. tremblayi used in this assay and in the dual-choice assay were Lso-free. Thus, Lso transmission was not evaluated for B. tremblayi. This assay was also repeated three times, and the parameters evaluated were identical to those described for B. trigonica.
Dual-choice assays. For these assays, we used a similar procedure to that described for the non-choice assays. However, each experimental arena contained a combination of two different plant species (18 plants of each species were alternately arranged in a square 6 × 6 layout, with 12.5 cm between plants) (Supplementary Fig. S1). To assess the preference of B. trigonica, the assay included three treatments of the following plant combinations, with each in a separate cage: (T1) carrot-celery, (T2) celery-potato and (T3) potato-carrot. Additionally, a separate assay was performed to assess the host plant preference of B. tremblayi. This assay included the following treatments: (T1) carrot-leek, (T2) carrot-potato, and (T3) leek-potato. The parameters that were evaluated and the methodology used in this assay were identical to those described for the non-choice assays.
Statistical analyses. All behavioural variables were tested for normality using the Shapiro-Wilk W-test and were transformed when required by either sqrt(x + 1) or ln(x + 1). Comparisons among EPG treatments were performed using one-way ANOVA for Gaussian variables or with Kruskal-Wallis tests when normality was not achieved. For the preference assays, the percentage of insects settled per cage, the percentage of plants infected with Lso and the percentage of infested plants were transformed by arcsin x when required to reduce heteroscedasticity and achieve normality. Following transformation, values were rechecked for normality using Shapiro-Wilk W-tests, and then the means for each treatment were compared using one-way ANOVA for the non-choice tests. For the dual-choice tests, pairwise comparisons between combinations of plants in each treatment were performed with a Student's t-test or with a Mann-Whitney U-test when normality was not achieved. Data were analysed using the SPSS 21 statistical software package (IBM Corp).