All Citrus trees belong to the family Rutaceae, subfamily Aurantioideae and are native to the tropical and subtropical regions of Southeast Asia, northeast India, Yunnan Province in southwest China, northern Myanmar, the Indochinese peninsula and the Malaysian archipelago (Zhong and Nicolosi, 2020). It is the world’s largest fruit crop, cultivated in more than 140 countries in the world [143.755, 6 million tons in 2020, (FAO, 2021)]. The main threat of this tree crop is a disease called Huanglongbing (HLB) or citrus greening, which has provoked the decline of many citrus industries that have faced the disease (Gottwald, 2010). The disease was first reported in China in 1919 but the causal agents were only discovered in 1970 by electron microscopy (EM) (Bové, 2006). It is caused by three main species of uncultivable, phloem-restricted, Gram-negative bacteria of the genus Liberibacter. Three different bacteria species pathogenic to Citrus inducing HLB had been described so far: the Asian species named Candidatus Liberibacter asiaticus (CLas), the African species Ca. Liberibacter africanus (CLaf) and the American species Ca. Liberibacter americanus (CLam) (Bové, 2006). The former species is described as the most aggressive species among all three causing more severe symptoms of HLB, and being able to grow in a wide range of temperatures, even above 30°C (Bové, 2006). Symptoms affect the entire citrus plant, from roots to leaves, even altering the chemical characteristics and sensory attributes of the fruit, and eventually killing the plant (Bové, 2006; Baldwin et al., 2010; Dala-Paula et al., 2019).

HLB disease is transmitted by grafting and mainly by two species of phloem-sap feeding psyllids (Bové, 2006). One species originating from Asia, the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae) is the vector of CLas (Martinez and Wallace, 1967) and CLam (Teixeira et al., 2005a; Texeira et al., 2005b). The second species originating from Africa, the African citrus psyllid (AfCP), Trioza erytreae (Del Guercio, 1918) (Hemiptera: Triozidae) (McClean and Oberholzer, 1965) is the vector of CLaf (Bové, 2006). Both psyllid species are very sensitive to changes in temperature and relative humidity. The AfCP, develops under cool and moist climate, the optimum temperature for its preimaginal development is between 17°C and 25°C, and its juvenile stages can be completed in 17 and 43 days at 25 and 14°C, respectively (Catling, 1973). The ACP has a higher heat tolerance and its development from egg to adult can be completed in 17 days with optimal population growth potential at 25-28°C (Hall, 2008).

Huanglongbing disease affects several countries in Asia, sub-Saharan Africa, the islands of the Indian Ocean and the Americas (except for Bolivia, Chile, Perú, and Uruguay), but the Mediterranean Basin and Australia are still free from it (Ferrarezi et al., 2020). However, T. erytreae has recently been described in southern Europe in Portugal and Spain and is extending its range in both countries eastwards reaching the Basque region just at the border of southern France and southwestwards, reaching the Algarve region in southern of Portugal (Cocuzza et al., 2017; EPPO, 2021; Benhadi-Marín et al., 2022). Most studies on CLas transmission have been conducted with D. citri, the species widely present in Asia and America, while those on CLaf have exclusively used T. erytreae as a vector as the pathogen and the vector were previously restricted to the African continent. However, in recent years CLas has been introduced into Africa where both psyllid vector species are present and the epidemiological implications of such introduction are unknown (Ajene et al., 2020b). As one of CLas’s potential vectors is already present in southern Europe, the high heat tolerance, strong symptoms induction leading to the death of trees, high aggressiveness, and wide distribution of CLas, make this disease the main threat to this major citrus-growing area. However, the transmission ability of CLas by T. erytreae has never been fully proven, as i) when the experiment was carried out (in the 1970s), it was not possible to discriminate HLB species, and ii) it was tested on very few plants (Massonie et al., 1976). Recently, CLas was reported from field-collected adults of T. erytreae feeding on HLB infected citrus trees in Ethiopia and Uganda (Ajene et al., 2020b), but no transmission assay was conducted to prove its transmission capacity. Thus, there is no information on the risk of spread of CLas in a geographical area where T. erytreae is present. To clarify the transmission capacity of CLas by T. erytreae, we compared the transmission capacities of the two psyllids D. citri and T. erytreae for CLas in laboratory experiments. These experiments were conducted in La Réunion, an island in the southwest part of the Indian Ocean where the two-psyllid species are present, and the two HLB species CLas and CLaf have been reported (Catling, 1972; Garnier et al., 1996; Lu et al., 2021; Wang et al., 2021).

All field collected leaves of both mandarin cultivars, V1 and V8, were CLas positive. All plants or excised leaves where insects from the two species tested CLas negative were always found CLas negative (negative controls).

For AAP leaves, the positivity threshold was 29.27. A high significant difference on Cqs between the two CLas-infected cultivars was found (Deviance test, P = 1.41 ^-08) with significantly lower predicted averaged Cq for V1 than V8. There was no significant effect of temperature (Deviance test, P = 0.60) nor between the two psyllid species tested, T. erytreae and D. citri (Test of deviance, P = 0.22) ( Figure 2 ; Table 1 ) on the Cq values.

For IAP1 excised leaves, the positivity threshold was 32.86. A significant difference between psyllid species (Deviance test, P = 0.02) and between cultivars on Cq values was found (Deviance test, P = 0.03) ( Figure 3A ; Table 1 ). In IAP1, 22.22% (n=27) of the excised leaves were tested CLas positive by D. citri, and 9.43% (n=53) by T. erytreae after the 7-day IAP. When looking at the initial CLas-infected sources used for acquisition (AAP) (V1 and V8 cultivars), all IAP1 positive excised leaves (for both psyllid species) were derived from the V1 cultivar ( Figure 3A ; Table 1 ).

For IAP2 excised leaves, the Cq threshold was 33.12. No significant effect of temperature, initial CLas-infected source cultivar, or psyllid species was observed on the number of positive psyllids (Deviance test, P > 0.09). We observed 34.62% (n=26) of the excised leaves that were CLas positive when inoculated by D. citri, and 33.33% (n=45) when inoculated by T. erytreae after the last 7-day IAP on observed data ( Figure 3B ; Table 1 ). When predicted values were calculated by emmeans (D. citri predicted values= 23% [1-89%, 95% CI]; T. erytreae predicted values=4% [0-51%, 95% CI]), strong discrepancies were observed with the observed values, most probably due to too small samples. When looking at the initial CLas-infected source plants leaves used for acquisition (AAP) (both V1, V8), 66.67% (n=9) and 90% (n=10) IAP2 positive excised leaves were derived from V1 cultivar for D. citri and T. erytreae, respectively ( Figure 3B ; Table 1 ).

For IAP3 seedlings, the positivity threshold was 33.28. No significant effect of temperature, initial CLas-infected source cultivar, or psyllid species was observed (Deviance tests, P > 0.51). In total, the predicted transmission rates on 3-month old plants, using one insect by plant, were 7% [1-26%, 95% CI] for D. citri and 9% [3-29%, 95% CI] for T. erytreae ( Figure 4 ; Table 1 ) with averaged Cq from 17 to 22 ( Table 1 ). No more positive plants were observed after three more months (i.e., 6-month old plants).

The CLas infection status and bacterial titer for all adult psyllids that were used in the experiment (except the ones that were lost) were assessed after the IAP 3 ( Figure 5 ). The positivity threshold for this experiment was 31.66. Significant differences were found in the proportion of CLas positive psyllid adults between the two species (Deviance test, P = 0.003) with 78% [63-88%, 95% CI] of D. citri adults and 92% [80-97%, 95% CI] of T. erytreae adults that were CLas positive. There was an effect of the initial CLas-infected source cultivars used for acquisition (Deviance test, P = 1.28e^-5). No temperature effect was noticed in the proportion of positive psyllid adults. However, when taking into account Cq values, significant interactions were found between the initial cultivars AAP cultivars and psyllid species (Test of deviance, P= 0.025) as well as for temperature ranges and psyllid species (Test of deviance; P=0.04) ( Figure 5 ; Supplementary Data Figure 1 ). These significant interactions were mostly due to T. erytreae samples that had much higher Cq values when cultivar V8 was used as a CLas-infected source for the AAP at the lowest temperature range ( Supplementary Data Figure 1 ). Higher differences were found for T. erytreae, with lower averaged Cq values for V1 (20.18 [19.07-21.30, 95% CI] and of 18.57 [17.32-19.83, 95% CI] at 23°C and 26°C, respectively) than for V8 (24.24 [22.79-25.68, 95% CI] and of 20.89 [19.23-22.55, 95% CI] at 23°C and 26°C, respectively). For D. citri, higher Cq values were observed on average compared to T. erytreae, but with fewer differences between V1 (25.37 [24.23-26.51, 95% CI] and 26.14 [24.87-27.42, 95% CI] at 23°C and 26°C, respectively) and V8 (27.15 [24.88-29.41, 95% CI] and 26.44 [24.73-28.15, 95% CI] at 23°C and 26°C, respectively) were observed ( Supplementary Data Figure 1 ).

Our results demonstrated the ability of T. erytreae to acquire and transmit CLas equally well as D. citri, with no effect of the initial bacterial inoculum or temperature ranges tested.

When testing the bacterial titer of different mandarin cultivars as CLas-infected source plants for the initial inoculum (AAP) we were able to choose two cultivars (V1 and V8) that were found to have significantly different bacterial titers, both showing strong symptoms in the field. The highest bacterial titer was observed for V1, a cultivar known to be HLB susceptible (Koizumi et al., 1993). These differences in bacterial titers between cultivars had already been shown in the field and could reflect the capacity of the V8 cultivar to diminish bacterial multiplication (Alves et al., 2021). Another hypothesis to explain those observed variations could be that a different variant or strain of CLas had infected V1 and V8 cultivars, even in trees next to each other in a same orchard. Genetic studies of the CLas species revealed that even within a given region, it might comprise several different variants (Bastianel et al., 2005; Teixeira et al., 2008).

To our knowledge, this is the first time that plant-to-plant transmission experiments using CLas-infected plants and T. erytreae as the vector species have been performed. Both excised leaves and seedlings were used for both acquisition and inoculation tests. Our results clearly show the efficacy of T. erytreae to acquire and transmit efficiently the bacteria (i.e., in a similar way as D. citri its primary vector) highlighting the threat of having this vector invading the commercial citrus growing areas of southern Europe (Cocuzza et al., 2017; Benhadi-Marín et al., 2022). More importantly, our results highlight that the most aggressive and destructive C. Liberibacter species (CLas) can be spread efficiently by T. erytreae in regions where D. citri has not yet been established. Our findings have important epidemiological implications as CLas in addition to CLaf can be efficiently spread in regions where T. erytreae is present. This stresses the importance of putting in place extreme precautions to prevent the introduction of CLas-infected material in countries where T. erytreae is already established such as Spain or Portugal that have a very important citrus industry.

The initial AAP inoculum bacterial titer was shown to be important since most of the effective transmissions to the final IAP by the two-psyllid species on seedlings came from the V1 cultivar with the highest initial bacterial titer. A 14-day AAP using nymphs should have ensured a better acquisition rate for insects (Inoue et al., 2009; Ammar et al., 2016), which was verified here with CLas-positive rates obtained for D. citri and T. erytreae of 67 and 83%, respectively. Nonetheless, despite such a positive number of CLas-positive insects, only 7% and 9% of transmissions were recorded on IAP3 seedlings for single insect transmission of T. erytreae and D. citri, respectively. Similar results were observed with single insect transmission of D. citri (with transmission rates of 6.7% after 31 days post AAP) showing intermittent and random transmission over series of IAP (Canale et al., 2017). To be noticed, recent results obtained for D. citri with CLas on citrus seedlings with young shoots gave much higher transmission rates (over 56% of transmission), however this could be possibly explained by the experimental design, slightly different (regarding to AAP and IAP times) and the citrus cultivar used as the recipient of CLas (Lopes and Cifuentes-Arenas, 2021). Indeed, Volkameriana cultivar was considered as a moderately tolerant cultivar to CLas compared to sensitive sweet oranges, but still with comparable high bacterial titers (Folimonova et al., 2009). Furthermore, different bacterial strains or psyllid populations between both studies could also play a role in these discrepancies, as in La Réunion the CLas strain is slightly different from others (Lu et al., 2021) such as the ACP psyllid populations which were found genetically different from the new invasive populations in Kenya (Wang et al., 2021) as being present on the island for over 50 years (Bové, 2006).

T. erytreae had significantly higher bacterial titers than D. citri ( Supplementary Data Figure 1 ), but with no higher transmission rates on seedlings in IAP3, or on excised leaves in IAP1 and IAP2, compared to D. citri. Trioza erytreae had been shown to acquire (1 hour AAP was enough) and efficiently transmit CLaf within less than 7 days post AAP, quicker than D. citri with CLas (van Vuuren and van der Merwe, 1992; Ammar et al., 2013; Ammar et al., 2016). So, these lesser transmission rates observed for T. erytreae with CLas (compared to CLaf) might reflect a lesser adaptation of T. erytreae to CLas, as CLas never co-evolved due to their non-overlapping geographical distribution until recently (case of Ethiopia and Kenya where both CLas and T. erytreae are together (Ajene et al., 2020a; Ajene et al., 2020b)). This geographical isolation also contributes to explaining why all transmission tests with CLas have been conducted exclusively with D. citri, and all the CLaf transmission tests have been conducted with T. erytreae. Our results showing the almost equal transmission ability of CLas by T. erytreae need however to be further refined and further experiments should be conducted to better determine the latency period, the persistence or the intermittence of transmission over the life span of T. erytreae.

According to our results T. erytreae has the capacity of acquisition of CLas bacteria at its larval stage and ability to transmit it during its feeding at the adult stage to a CLas-free plant. This implies that CLas, known to be transmitted in a propagative-circulative manner by D. citri (Inoue et al., 2009; Ammar et al., 2016; Canale et al., 2017; Ammar et al., 2018), might also have the same capacity to multiply and circulate up to the salivary glands of T. erytreae, a psyllid of a different area of origin and from another family. Nonetheless, further experiments are needed to determine the pathways of passage and multiplication of the bacteria in the insect body and to be able to compare them with the system of D. citri and CLas.

No significant effect was observed between both temperature regimes tested (17-23°C/20-26°C) on transmission rates of both psyllid species on excised leaves or seedlings. Similar results were observed in the USA on CLas-infected plants incubated at 20°C, 27°C, and 32°C where similar bacterial titers and symptom induction were obtained (Folimonova et al., 2009). Those tested temperature ranges are also compatible with the ones where T. erytreae is already present in Southern Europe (Cocuzza et al., 2017; Benhadi-Marín et al., 2022). This area is then at high risk if the bacteria is introduced, consequences would be tremendously important knowing that this region, still HLB-free, is the 5^th worldwide citrus producer, after China, the USA, Brazil, and India (FAO, 2021).Furthermore, a recent study has proven also the risk of the potential introduction of this vector in high citrus growing areas of Mexico that would be most suitable for this vector than D. citri with CLas already present (Espinosa-Zaragoza et al., 2021). Preventive measures and an action plans were proposed in previous studies to reduce the risk of introduction and/or establishment of CLas and CLaf in the Mediterranean area (Durán-Vila et al., 2014; Aragón et al., 2022). Thus, in the light of the results of our study and the recent increase in the range of T. erytreae, these proposed measures should be taken into account even more. In conclusion, this study clearly demonstrated the ability of T. erytreae to acquire and transmit CLas. The presence of T. erytreae in the southern Europe is of great concern given its ability to disseminate the most aggressive variant of HLB (CLas). Thus, extreme precautions to prevent any entry of CLas into Europe should be adopted.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Conceptualization, BR, HD; Data collection, BR, MG, PT, FM, SR; Data analysis and interpretation, PT, FC, BR, HD; Writing—original draft, HD; Writing, review and editing, BR, HD, AF, FC; Funding acquisition, BR, HD, AF. All authors contributed to the article and approved the submitted version.