The Teide broom, Spartocytisus supranubius (L.f.) Christ ex G. Kunkel, is a species endemic to the Canary Islands. It is the dominant legume and the most characteristic species of the summit shrubland vegetation of the Tenerife high mountain ecosystem. In the past, intensive grazing and its use as forage (Pérez de Paz et al., 1986; Rodríguez-Delgado and Elena-Roselló, 2006) were the main threats to this highly palatable legume, from which it was protected after the establishment of Teide National Park (N.P.) in 1954 (del Arco-Aguilar and Rodríguez-Delgado, 2018). However, these were far from being its only problem, as in this ecosystem it also has to cope with several other biotic and abiotic stresses. In the decade 1980–1990, the favorable trend due to N.P. protection reversed and S. supranubius populations started to recede (Cubas et al., 2018; Ibarrola-Ulzurrun et al., 2019; Bello-Rodríguez et al., 2020).

In recent decades, regeneration of S. supranubius populations has been compromised by introduced herbivores, mainly rabbits (Cubas et al., 2018). Although present in the N.P. since their introduction in the sixteenth century, larger populations of rabbits have been favored by the milder climatic conditions in the last few decades (Bello-Rodríguez et al., 2020). On the other hand, these broom populations thrive between 2,000 and 3,100 m a.s.l. (Kyncl et al., 2006), where conditions are highly demanding for plants. The Mediterranean bioclimate is diverse throughout the Teide N.P. varying from dry Supramediterranean in the southern part to dry-subhumid Supramediterranean in the north, and semiarid or arid Oromediterranean at the summit of Teide peak (del Arco-Aguilar et al., 2006; del Arco-Aguilar and Rodríguez-Delgado, 2018). Broom plants have to face intense sunlight exposure and a high daily and annual temperature variation with means ranging from below 0°C in winter to over 25°C in summer. The annual precipitation is 330–450 mm, mostly occurring in winter (when some snow and frost episodes are possible) and the summer is extremely dry, with a drought period from May to September (del Arco-Aguilar et al., 2006). Morphological adaptations of S. supranubius include deep roots, small ephemeral deciduous leaves and multibranched photosynthetic stems, which branch almost from the base (Supplementary Figure 1). These features mold this species well to its natural environment (Wheeler and Dickson, 1990), where specimens up to 4 m high and 10 m diameter can be found (Olano et al., 2017). In addition to the harsh climatic conditions of the alpine ecosystem, prolonged drought in recent decades and an overall temperature increase due to global warming are causing a progressive deterioration of S. supranubius adult individuals (González-Rodríguez et al., 2017; Olano et al., 2017; Martín-Esquivel et al., 2020).

Nitrogen (N) availability can be another stress factor for broom plants in soils of Teide N.P. As a legume, S. supranubius can establish a symbiosis with N-fixing rhizobia. This is fundamental, since broom plants grow on poorly developed soils, where N content is very low and limits vegetation establishment (Arbelo et al., 2009; Díaz et al., 2011; Rodríguez et al., 2014). Therefore, biological nitrogen fixation (BNF) seems to be the key input of this element into the soil of this ecosystem (Wheeler and Dickson, 1990; Pulido-Suárez et al., 2021), highlighting the need for the maintenance of the Teide broom population. Thus, given the ecological importance of this legume in the N cycle, its decline could compromise the maintenance of the ecosystem and affect its total productivity.

Symbiotically-efficient-rhizobia inoculants have been widely and successfully applied in legume crop agriculture for about a century. Moreover, in the last decades the use of rhizobia inoculants has also been tested with other, different purposes. Chaer et al. (2011) describe several examples of successful inoculation of leguminous trees in the reclamation of degraded areas, resulting in soil recovery and the restoration of the ecosystem biodiversity. In addition, several studies have characterized the rhizobia nodulating different threatened legume species and have identified efficient N-fixers (Donate-Correa et al., 2007; Lorite et al., 2010; Fonseca et al., 2012; Martínez-Hidalgo et al., 2016; Safronova et al., 2017). Even so, rhizobial inoculation is not a common practice in natural ecosystems for legume species conservation and only rarely have they been used for this purpose. In recent years, Navarro et al. (2014) carried out a good example, applying rhizobial inoculation in a field trial to conserve the endangered legume Lupinus mariae-josephae. They achieved a successful plant reproductive cycle dependent on seedling inoculation with effective bradyrhizobia, reporting higher seed production and yield (Navarro et al., 2014).

In a previous study we described that strains of genus Bradyrhizobium are the main microsymbionts of S. supranubius (Pulido-Suárez et al., 2021) and showed, coinciding with Wheeler and Dickson (1990), the positive influence of this symbiosis in the ecosystem, since the soils around this legume have higher N content. However, the symbiotic effectiveness of the S. supranubius bradyrhizobia has not yet been evaluated nor how these endosymbiotic bacteria promote growth of the broom plants in their natural habitat. Hence, in the present study our aim was to evaluate the effect of rhizobial inoculation on S. supranubius when growing under the environmental conditions of its natural habitat of Teide N.P. We hypothesized that inoculation of broom plants with selected rhizobial strains before transplanting them on the field would produce more robust plants, better prepared to cope with the local external stresses.

Spartocytisus supranubius, the dominant species in the Teide N.P. is currently experiencing a decrease in population (Cubas et al., 2018; Martín-Esquivel et al., 2020). In order to slow down this decline, periodic transplanting of juvenile individuals is a frequent strategy in Teide N.P. About 10,000 broom plants are produced each year, a fraction of which is planted in the wild for ecological restoration purposes. The usual protocol followed by the environmental managers at the Teide N.P. includes a 2–3 years acclimatization period outside the greenhouses before transplanting them at the field. However, high mortality rates among transplanted individuals is observed, especially in the southern area of the park, where it is two or three times higher than in the northern area. Despite the relevance of N-fixation associated with this legume has already been evidenced (Wheeler and Dickson, 1990; Pulido-Suárez et al., 2021), the inoculation with rhizobia has never been incorporated into the legume species conservation strategies. In the present study we have assessed the response of S. supranubius to the rhizobial inoculation at the three transplanting stages followed by technicians at the Teide NP: growing seedlings at the greenhouses, during the acclimatization period, and in a field trial.

Growing legumes under greenhouse conditions is a very common practice to assess the symbiotic efficiency of the rhizobial inoculation. However, it is worth mentioning that here we evaluate the response of S. supranubius to the rhizobial inoculation in greenhouses located in Teide N.P., at 2050 m a.s.l., therefore in conditions similar to its natural habitat in the high mountain ecosystem of Tenerife. Although under greenhouse conditions plants are protected from some stresses (such as solar irradiation and winds), inside this greenhouse temperatures are similar to the outside and frozen plants are not rare on the coldest days of the year. In addition, the substrate used in the N.P. to grow seedlings in the greenhouse stage is not sterile, containing local soils and fertilized peat, so the presence of native rhizobia and some N input is possible. Despite of that, our results showed that broom seedlings responded positively to the inoculation with selected rhizobia, enhancing the natural nodulation and promoting plant growth, pointing to the effectiveness of these “extra” nodules compared to the more weakly nodulated control plants. The results for those inoculated with strains SSUT18 and SSUT109 were particularly positive as broom seedlings with these two strains doubled the biomass (FW and DW) of control plants and showed more branches, a key factor for this plant since photosynthesis mainly occurs in its photosynthetic stems (Kyncl et al., 2006). However, strain SSUT74 did not perform with the expected effectiveness, neither in the greenhouse nor in the field trial experiment. An important difference was that the substrate used to grow plants in the previous screening experiment was sterile vermiculite, while the experiments of this study were performed in non-sterile soils. The results obtained with SSUT74 inoculated plants might reflect poor root colonization and/or its inability to compete with other less effective, more competitive native strains in the soils, which could have occupied the nodules instead. In fact, an ERIC-PCR fingerprint from nodules recovered in the summer sampling (Supplementary Figure 8) seem to support this. Whereas, ERIC profiles confirmed nodule occupancy for SSUT18 and SSUT109, for SSUT74 it was observed that at least some nodules contained a different strain. This result was unexpected, because this strain had been isolated from local soils, so other unknown factors could be involved.

In addition to promoting growth, broom-seedling inoculation also had positive effects on AWC (Table 1) and plant survival (Supplementary Table 2). AWC in inoculated seedlings was statistically higher than controls. Interestingly, this effect seems unrelated with effectiveness, as this positive response to inoculation included strain SSUT74, which could be classified as symbiotically inefficient. Many studies have shown the sensitivity of the rhizobium-legume symbiosis to water deficit (Zahran, 1999; Marino et al., 2007; Marinković et al., 2019). However, few studies deal with the benefits of rhizobial inoculation of legumes under drought stress. An interesting example is the study by Agele et al. (2017), who observed positive effects in four legume species under several water stress levels (statistically significant for the less severe watering regime). Also, a significant delay in drought-induced leaf senescence was found in nodulated Medicago truncatula in comparison to non-nodulated plants (Staudinger et al., 2016). In turn, Defez et al. (2019), showed that overproduction of rhizobial indole-acetic acid inside root nodules positively affected drought stress response in nodulated alfalfa plants. Our preliminary results point to a positive effect of rhizobial inoculation on water content, at least during the early development stages of broom plants, and it will require further studies to confirm this tendency and investigate the mechanisms behind it.

Seedling mortality (above 60%) in the park greenhouse was considerably high during the first months. At this early stage, seedlings benefited from rhizobial inoculation, showing a survival rate about 20% higher when inoculated with strains SSUT18 and SSUT109. These results were similar to those by Navarro et al. (2014) who reported survival rates 25% higher for the rhizobia-inoculated pre-germinated seeds of Lupinus mariae-josephae, compared to uninoculated controls, after six months in the field. It has been shown that the first months of growth are critical for plant survival in alpine ecosystems (Forbis, 2003). Therefore, by promoting growth and decreasing mortality in this delicate early stage of seedling development, rhizobial inoculation can contribute to the success of the early steps of replanting campaigns.

Field experiments to study the effect of rhizobial inoculation on legumes are often complicated or difficult. Many failures occur because many adverse environmental conditions influence symbiosis under field conditions (Bordeleau and Prévost, 1994; Zahran, 1999) and/or due to a lack of competitiveness of the introduced rhizobia inoculants are easily supplanted by more competitive, less effective native rhizobia (diCenzo et al., 2019; Irisarri et al., 2019).

Seasonal variations are of great relevance to plant growth, especially in alpine ecosystems. Our first sampling in the field was in July. Although for other ecosystems the summer could already be an unfavorable time, in the alpine ecosystem of Teide N.P. spring comes late and early summer is still beneficial to plants in the park, so this sampling can be considered representative of a favorable period. The second sampling was 4 months later, in mid-November (fall). Early fall in the park is usually unfavorable after the extremely dry summers. However, the first rains had already fallen before the November sampling, which made this sampling more positive than originally expected.

The results obtained for the field experiment for both seasons were good, showing a significant benefit of inoculation for most parameters. The lack of statistical significance in some of the studied parameters could be explained by the natural high morphological variability of S. supranubius.

Plants inoculated with the SSUT18 strain had significantly higher number and weight of nodules, greater sizes (biovolume) and biomass. This strain also had the highest N content per plant that, being not statistically significant in our experimental conditions, it still contributed to more N per planted hectare than control plants, a fact that deserves to be taken into consideration. Six-months transplanted juvenile-brooms inoculated with this strain produced 0.2 g N per plant. This means that in the small field trial experiment we performed with 20 plant/treatment, SSUT18 would have produced 4 g N. Total nitrogen contents in soils in the majority of the areas of the park are below 3 g.kg-1 and mainly contained as organic matter (Arbelo et al., 2009). Some calculations using the same transplanting experimental design of one plant every three meters would give N values for one planted hectare after 6 months in the field of 135 g·ha-1 for the control plants (spontaneously nodulated by rhizobia present in the local soils), but of 180 g·ha-1 for those plant inoculated with strain SSUT18, which is about 25% more N in a planted ha. Therefore, taking this into account, even this moderate N content detected in this field trial still could be important for the ecosystem maintenance in these N-poor soils.

Destructive methods, albeit providing accurate measurements of biomass, obviously have the disadvantage of eliminating the samples (Catchpole and Wheelert, 1985), which in case of threatened species is a serious drawback. Therefore, for S. supranubius in the first July sampling, together with the destructive method we also performed a non-destructive approach and calculated the apparent biovolume. Following the description of Gough (2010), who describes young S. supranubius as cone shaped, the biovolume was first estimated using the cone volume formula. However, when establishing a correlation between the biovolume and biomass (SDW), the regression coefficient was low. It has been pointed out that the apparent biovolume is sometimes difficult to estimate accurately given the spatial arrangements of organs (such as branches), and because it is a combination of plant tissue and void (Pottier and Jabot, 2017). Consequently, to obtain a more accurate biovolume, we considered individual Teide broom branches as cylinders (Figure 1C). The regression coefficient obtained using a cylindric approach is considered a good fit for an allometric equation, and is similar to other results in previous studies (Pottier and Jabot, 2017). This good correlation indicates that a non-destructive estimation of biovolume is a simple method that could be used as a tool to indirectly determine plant growth when plant destruction is not possible, desired or permitted, thus monitoring the effect of inoculation on growth several times during longer periods.

Height was also greater in inoculated broom plants in the two samplings (especially in July). This could be advantageous for the plants' establishment, since plant height has been associated with competitive vigor (Cornelissen et al., 2003). Furthermore, although more speculative, faster growth of inoculated plants could also have another positive effect, indirectly contributing to reduce the negative impact of rabbits. Cubas et al. (2018) considered that individuals 70–100 cm high are tall enough to escape rabbit grazing. Kyncl et al. (2006) established a lower escape height from rabbit herbivory, at 55 cm. Our inoculated brooms reached about 50 cm height, and hence by introducing rhizobial inoculation in the greenhouse and during the acclimatization stage, it is expected that in a shorter period of time the inoculated broom plants can reach this key rabbit grazing-resistant threshold, thus ensuring survival outside the exclosure plots. Furthermore, SSUT18- and SSUT109-inoculated broom plants had on average 10 branches more than uninoculated controls, a finding that deserves to be highlighted because the number of branches is important for this species for two reasons. One, most of the photosynthesis is carried out by its stems, due to its small, deciduous leaves (Wheeler and Dickson, 1990). Two, the broom also presents clonal growth, that is, its branches have the capacity to root and form new individuals (Kyncl et al., 2006). This strategy has proven to be an important propagation method for this species (Kyncl et al., 2006).

We could not however evaluate survival in the field trial experiment, since all planted individuals survived. In order to explain this apparent 100% survival rate, it must be taken into account that the field experiment started with a set of 30 juvenile broom plants (2 years old), which were inoculated twice several months before transplanting them into the field. For transplantation, we selected only 20 healthy-looking individuals per treatment, with well-established nodulation. In fact, of the original 30 plants, some did die during acclimatization (Supplementary Table 2). However, survival was still much higher (above 90%) than scored in seedlings.

In the field trial, the effect of the rhizobia on plant water content was not clear. Contrasting with results for inoculated seedlings, only slightly higher, statistically non-significant AWC values were registered (Supplementary Table 1). Whether the fact that inoculated plants were already more than 2 years old when inoculated or some environmental conditions in the study area might have had an influence is not clear. Different sensitivity to water deficit has been reported among rhizobial strains (Mahler and Wollum, 1980; Fuhrmann et al., 1986; Busse and Bottomley, 1989) and so, isolation of water-stress adapted rhizobia strains may well be possible. The results for water content of inoculated seedlings encourage us to further study the effect of inoculation with selected bradyrhizobial strains, so as to alleviate drought stress in broom plants. Given the long drought episodes in the N.P. (Olano et al., 2017, del Arco-Aguilar and Rodríguez-Delgado, 2018), it would be extraordinarily desirable to demonstrate positive effects of rhizobial inoculation.

Inoculation with Bradyrhizobium strains SSUT18 and SSUT109 significantly enhanced biomass and biovolume and improved survival of S. supranubius seedlings and juvenile individuals. These were tested after growing in their natural ecosystem in the Teide N.P., under greenhouse and field conditions. By producing larger, more robust plants in a shorter time compared to uninoculated broom plants, bradyrhizobia inoculation reduces the greenhouse period and juvenile plants better cope with and survive stresses in field trials. Therefore, inoculation with selected rhizobia is a successful strategy to be integrated into conservation campaigns for threatened legume species. It could become a key tool in replanting and reforestation campaigns, aiding the maintenance of this fundamental key species in the delicate high mountain ecosystem of Tenerife.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, MT210556; https://www.ncbi.nlm.nih.gov/genbank/, MW439206; https://www.ncbi.nlm.nih.gov/genbank/, MW439207; https://www.ncbi.nlm.nih.gov/genbank/, MT321501; https://www.ncbi.nlm.nih.gov/genbank/, MW439203.

ML-B conceived the research and funding acquisition. ML-B and ÁG-R designed methodology. ML-B, LP-S, MA-A, and JM-E planned and set up the greenhouse and the field experiments. ÁG-R and LP-S took measurements of plant physiological parameters. ML-B, MA-A, and LP-S collected and took measures of the plant samples. LP-S and ML-B analyzed the data. JC and LP-S performed non-destructive methods and edited figures. ML-B and LP-S wrote the manuscript. All authors revised and made significant contributions to the write-up and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.