Aiming to improve our understanding of the functions that phyllosphere microbial communities might play in plants growing in extreme arid environments, we applied a high resolution sampling scheme to studying the phyllosphere microbial communities of two desert keystone trees (Acacia raddiana and Acacia tortilis). We investigated both the endophytic and epiphytic bacterial communities to understand the: (i) intra- and inter-individual spatial variation of the microbial community within a tree (the variation within the same tree caused by different sides of the canopy, and the variation between neighboring trees of the same species sampled at the same time and site) (ii) host species variation (variation of the microbial community caused by the host (tree) species (i.e., Acacia raddiana compared with neighboring Acacia tortilis), (iii) temporal variation of the microbial community within the same tree species and canopy side (samples collected from the same trees but at different seasons).
Our results demonstrate that the epiphytic bacterial communities were more sensitive to changes in the external environmental conditions, compared with the endophytic bacterial communities that were more stable between different environmental conditions (e.g., seasons) but varied among host tree species. Surprisingly, up to 60% of the total bacterial communities (the combined endophytic and epiphytic microbiome populations) were unclassified below family level, highlighting the uniqueness of the microbiome associated with acacia trees in the arid environment in the Arava.
The epiphytic bacterial diversity was found to be significantly higher than the endophytic bacterial community (Table 2). In terms of the overall observed number of OTU’s, the epiphytic bacterial community was shown to have double the diversity compared to its endophytic bacterial community counterpart. Similar findings at early and late leaves development in Origanum vulgare also found the total number of colony-forming units (CFU) of endophytic communities (1.8 ± 0.1) was less than half of the CFU of epiphytic bacterial communities (5.0 ± 0.2) . However, our results contradict previous work on microbiomes associated with Arabidopsis thaliana ) that showed epiphytic bacterial diversity indexes were lower than those measured for the associated endophytic bacterial communities . A recent study on the epiphytic and endophytic fungal diversity in leaves of olive trees growing in Mediterranean environments, showed that the epiphytic fungal communities had higher diversity indices compared to the endophytic diversity estimates . The fact that our epiphytic OTU diversity was higher than the endophytic diversity is particularly surprising, considering previous publication indicated that as the conditions inside the plant might be more favorable compared to the hostile conditions outside . This might explain the different findings comparing epiphytic and endophytic bacterial abundance and diversity in other studies, but in our case, both A. raddiana and A. tortilis had a lower epiphytic bacterial diversity compared to epiphytic bacterial diversity throughout the sampling month, including the hot and harsh conditions of the desert summer (Table S5). This discrepancy finding in our results and previously document findings could be unique to desert plants. Plants grown in desert environments are subjected to continuous stress conditions including increased salt concentration in endophytic compartments , decreasing stomatal conductance and increased concentration of abscisic acid and many other metabolites and enzymes . These plant responses were shown to affect plants-microbiome colonization [32, 38, 39]. Moreover, our results showed that the endophytic and epiphytic bacterial communities were significantly different from each other (Fig. 2A). In fact, endophytic but not epiphytic bacteria communities, differed between the two acacia species (Fig. 3A, 3B, 4) – specific to the host (acacia tree). This potentially indicates that endophytic bacteria were horizontally transmitted and that they might be more affected by genotypic factors rather than abiotic factors [4, 5, 21].
Similar to other findings indicating the changes in bacterial communities in phyllosphere following different environmental and biotic factors [38, 39], our results show seasonality to be the major driver of community composition in epiphytic bacteria (Fig. 4A and Fig. 6), including a specific abiotic parameters such as; humidity, temperature, precipitation and VPD (Fig. 7A and B). While these results highlight the significant effect of temperature on both epiphytic and endophytic bacterial communities, the effect of microclimate (different canopy sides) on the epiphytic bacterial diversity (Table 3) and community composition (Figs. 5 and 6) showed no significant variation for the different canopy sides for both species. This could be explained by the difference between monthly temperature, humidity and precipitation hinders back these effects of canopy side variation.
We also showed that the bacterial community compositions found in this study, differ from other epiphytic or endophytic microbiome found in tropical, subtropical and temperate regions, which are mostly dominated by high abundance of Alphaproteobacteria, Bacteroidetes and Acidobacteria [1, 40, 41]. In our study, the major differences between epiphytic and endophytic bacterial communities were due to the differential abundance of four major unclassified OTU’s belonging the bacterial families of Bacillaceae (Firmicutes phylum) and Comamonadaceae (Betaproteobacteria phylum) for the endophyte of A. raddiana and A. tortilis, respectively (Fig. 8). Other unclassified OTU’s belonging to the bacterial families of Geodematophilaceae and Micrococcaceae (both belonging to Actinobacteria phylum) were found in the epiphyte bacterial communities (Fig. 8). These bacterial families were also found in other studies investigating extreme conditions that investigated the metagenomic signatures of Tamarix phyllosphere [10, 42, 43] and other desert shrubs , highlighting the importance and the relationship of these found bacterial communities in desert plants adaptation to arid environment . However, the exact link between these different bacterial groups and their functional diversity is still to be investigated, such studies could shed the light of specific metabolites and enzymes that these adaptive bacterial groups exhibit in such an environment and at different stress conditions. Learning from the long coevolved plants-microbiome form naturally occurring plants in harsh conditions is invital under the current rate of climate change and the urgent need for new innovative solutions that can be learned from these interactions for more adaptive arid land agriculture.