Our research on two common Nigerian medicinal plants, Euphorbia lateriflora and Ficus thonningii, showed that they are densely colonized by different phyllosphere bacteria.
Fungal and bacterial marker genes were abundantly detected through qPCR in relation to archaea. Bacteria and fungi are the most abundant groups inhabiting the phyllosphere, and several reports have confirmed this with high marker gene abundance using quantitative PCR, amplicon and metagenome sequencing approaches [1, 62, 63]. It has been argued that the abundance of archaea is frequently underestimated, and the full potential of the archaeaome is poorly understood due to limitations in current assays [64–66]. However, archaea have been reported in high abundances in the rhizosphere [63, 66, 67]. The phyllosphere is probably less suited for archaeal proliferation.
The high abundance determined by estimation of colony forming units of bacteria per gram (CFU/g) of leaves suggests that the leaves of E. lateriflora and F. thonningii in Ibadan are heavily colonized. Phyllosphere bacterial populations typically range from 101 to 108 CFU/g depending on the plant species and on its environmental, climatic and geographical habitat [5, 53, 68–72]. The numbers recorded in this study are in the upper range, similar to those obtained from sampling of roots and rhizosphere environment [5, 73]. This may be attributable to the plants’ intrinsic properties and differences in environmental conditions from other phyllosphere studies, as the vast majority of studies originated outside Nigeria and West Africa. There are relatively less fluctuations in temperature and humidity in tropical Nigeria, which may favour the proliferation of bacteria in the phyllosphere, resulting in large populations.
Overall, Pseudomonadota was the most abundant phylum in all replicates of the two plants. Pseudomonadota is frequently the most dominant phylum in several phyllosphere studies [1, 74]. We observed that the dominance of Pseudomonadota was highly influenced by Alphaproteobacteria (75 to 90% of total ASVs depending on the sample), which was due to an overabundance of the Sphingomonas genus. Sphingomonas is a major genus in the phyllosphere of many plants and colonizes a wider variety of plants when compared to the more commonly detected Pseudomonas genus [75–79]. Sphingomonads are well adapted to withstand harsh conditions and competitive life in the phyllosphere [75, 80, 81], where they are associated with antagonism, plant protection from disease [76, 82, 83] and geochemical cycling.
To visualize the composition of other taxa in the microbiome more clearly, we removed all ASVs mapping to Sphingomonas spp. and repeated the analyses. The most abundant class shifted to Gammaproteobacteria, followed by Bacilli, Alphaproteobacteria and Actinomycetota. There were visible differences in the abundance between the two plants. For instance, Actinomycetia, Alphaproteobacteria and Bacilli were more abundant in E. lateriflora, while Gammaproteobacteria were more abundant in F. thonningii. However, the observed differences were not statistically significant. The differences in composition and abundance between the plants were more evident at lower taxonomic levels, where Oxalobacteraceae was exclusively detected in F. thonningii. Overall, the compositions of the microbiome of both plants were similar, which may be partly ascribed to similar environmental and climatic conditions, as the plants were collected in close locations. It has been established that environmental conditions and geographical location have a profound influence on phyllosphere microbiome composition [84]. The host plant is also considered a significant influential factor of microbiome composition and abundance and is argued to be the most significant single factor from studies that have observed similar composition from identical plants in different locations [85, 86]. However, several studies have also reported similarities in different plants within the same location and differences between identical plants in different locations [84, 87]. It is evident that an interplay of factors influences the composition of the phyllosphere microbiome, and none of these can be singly ascribed to a single factor [88]. However, the combination of location and host plant seems to be significantly involved in microbiome composition [4]. Nevertheless, there are remarkable overlaps between the phyllosphere microbiome composition of diverse plants irrespective of geographical location, plant genetics, and other factors [74, 84].
The most dominant fungal classes, Dothideomycetes and Sordariomycetes, are commonly detected in high numbers in the phyllosphere [74, 84, 88]. Differences in fungal composition between the plants were more visible compared to bacteria, and this was statistically significant. Similar to bacterial composition, the plants shared some ASVs, notably Cladosporiaceae, Corynesporascaceae and Mycosphaerellaceae. The commonly detected fungal families, Cladosporiaceae, Didymellaceae and Nectriaceae, were also found in our study. We observed less fungal diversity than bacterial diversity, which is consistent with other reports of phyllosphere epiphytes [74, 88].
The taxonomic composition of bacterial isolates via culture was specific and unique for each plant species. We note that 71 (56%) and 38 (40%) of the isolates characterized from E. laterifora and F. thoningii, respectively, were Pseudomonadota belonging to the species Escherichia coli. Phenotypic examination demonstrated that 38 out of 41 of these isolates from E. lateriflora produced both acid and gas from lactose at 44°C, strongly suggesting that their origin is recent feacal contamination from warm-blooded animals. All but one of the E. coli isolates from F. thonningii did not show these properties, suggesting that they are likely of environmental origin, preferably called type 2 Escherichia sp., and may indeed be stable members of the phyllosphere. This unique ecological succession on the leaves presumably indicates that height is a factor to consider in the type of organisms that colonize the phyllosphere microbiome, as F. thonningii is a tree, while E. lateriflora is a shrub. At least some mammalian-associated E. coli lineages are known to be associated with diminished phyllosphere abundance [89], which may account for the relatively smaller number of non-E. coli isolates in this study compared with other culture-based phyllosphere studies. Dominance of Enterobacteriaceae other than Pantoea sp. in the phyllosphere, although relatively unpopular, has been reported. Enterobacteriaceae, including antimicrobial resistant Shigella and Enterobacter, were found to dominate the phyllosphere or arugula (Eruca sativa), a commonly consumed vegetable in Graz, Austria, with resultant implications for the heath of those who consume it raw [90]. A study of banana phyllospheres in Uganda similarly reported an overdominance of enteric bacteria in the banana pseudostems [91]. The overdominance of Enterobacteriaceae may be linked to the ubiquitous use of organic manure in agriculture, and as the average annual temperature is approximately 26°C and 27°C in Uganda and Nigeria, respectively, enteric bacteria will be able to thrive for extended periods. Additionally, birds perching on trees may produce droppings that may introduce enteric organisms into phyllospheres.
All non-E. coli isolates belonged to three phyla, Pseudomonadota, Actinomycetota and Bacillota, which are common on plants [67, 92, 93]. The majority of isolates from E. lateriflora belonged to the families Comamonadaceae (Pseudomonadota), Enterobacteriaceae (Pseudomonadota) and Pseudomonadaceae (Pseudomonadota), and those from F. thonningii belonged to the families Enterobacteriaceae (Pseudomonadota) and Bacillaceae (Bacillota). Other less dominant families include Brevibacteriaceae (Actinomycetota), Micrococcaceae (Actinomycetota), Xanthomonadaceae (Pseudomonadota), Microbacteriaceae (Actinomycetota), Sphingomonadaceae (Pseudomonadota) Staphylococcaceae (Bacillota) and Methylobacteriaceae (Pseudomonadota). Thirteen and nineteen genera were represented on E. lateriflora and F. thonningii, respectively, while 29 isolates, designated ‘unknown’, could not be classified into any genera based on the cut offs as set by Kim et al., (2014). Fourteen (48.3%) of these isolates were Burkholderiales, and work to characterize and report these potential novel species is ongoing. Isolates belonging to all three phyla, Pseudomonadota, Actinomycetota and Bacillota, were detected in both E. lateriflora and F. thonningii. However, the composition of the phyllosphere at lower classification levels is unique to each plant. The Bacillota genera Bacillus, Lysinibacillus and Staphylococcus were found in both plants but in higher proportions in F. thonningii. Similarly, Pseudomonas was detected in higher proportions in E. lateriflora. Important plant-associated taxa, including Pantoea and Xanthomonas, were found only in F. thonningii. Actinomycetales, represented only by Brevibacterium, were isolated exclusively in E. lateriflora, while Micrococcales were isolated exclusively in F. thonningii. Overall, E. lateriflora and F. thonningii shared seven bacterial genera and differed by four and nine unique genera, respectively. Several factors, including host plant intrinsic characteristics, plant location, environmental factors and microbiome interactions, influence phyllosphere microbiome composition [94–96]. Of these factors, the host plant is the most significant, as evident from the similarity in the phyllosphere microbiome of identical plants across different locations, and may account for the differences observed in the present study [4, 97–99]. Some of the isolates identified are dynamic in their interactions with plants. Pseudomonas oryzihabitans, for instance, could be pathogenic, causing stem and leaf rot in Chinese muskmelon [100] and rice panicle blight and grain discolouration [101]. Despite being a plant pathogen, P. oryzihabitans can also be beneficial and has been shown to promote plant growth and prevent colonization by pathogenic fungi and nematodes [102–104]. Other plant pathogens identified include Pantoea allii, Xanthomonas perforans and Xanthomonas euvesicatoria [105–107].
There was a contrast in the abundance of taxa by culture and culture-independent methods, and this is usually the case [63]. However, there were overlaps in the dominant organisms detected by both methods, including Sphingomonadaceae, Enterobacteriaceae, Bacillaceae, Pseudomonadaceae and Xanthomonadaceae.
The majority of the genera identified in this study have been reported to be found in other plants. [68, 69, 108–110]. However, at the species level, only a few species have been reported on plants. Pseudomonas taiwanensis, for instance, was first classified after isolating it from soil [111], while Pantoea allii was first classified after isolation from onion plants [105]. Actinomycetota, represented in this study by Microbacterium, Brevibacteria and Micrococcus, has been reported [112, 113] to produce secondary metabolites with diverse activities, including antimicrobial activity and promotion of plant growth. Similarly, the genera Pseudomonas and Pantoea consist of members exhibiting antagonistic properties [13, 68] and are commonly known to coexist with plants in a symbiotic relationship. The occurrence of these genera on E. lateriflora and F. thonningii analysed in this study could contribute positively to the antimicrobial activity of the plant. Indeed, Egamberdieva et al., (2017) reported a correlation between the proportion of antagonistic bacteria and the antimicrobial properties of two plants that were discordant for medicinal activity. The medicinal plant employed in the above study (Hypericum perforatum) housed a significant population of antagonistic bacteria compared to its nonmedicinal counterpart, Ziziphora capitata. As has been reported previously [32, 114], hexane, ethyl-acetate and methanol extracts of E. lateriflora and F. thonningii show activity against type cultures of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Citrobacter freundii, as they did in this study. Phyllosphere isolates obtained from both plants showed wider zones of inhibition to the extracts of both plants, whereas only a handful were susceptible to ciprofloxacin and/or chlorocresol. In the same vein, phyllosphere isolates were typically inhibited by the plant extracts, as shown by the MICs, and not by chlorocresol. MIC values were lower for the E. lateriflora extract than for F. thonningii across board. However, one Xanthonomas perforans, one Micrococcus luteus and two strains of Microbacterium testaceum all isolated from F. thonningii had higher MICs to the E. lateriflora extract.
Overall, the data do not support our hypothesis that phyllosphere bacteria on leaves are resistant to the antimicrobial secondary metabolites extracted from plants. While it is possible that the chemical nature of antimicrobial principles is altered during extract preparation, it is more probable that secondary metabolites produced by healthy plants are embedded within the tissues of the plants, and as such, bacteria leaving on the surfaces of the plants may not have direct contact with such metabolites unless the plant is injured. Our findings also point to the possibility that antimicrobial resistance to active plant metabolites may be uncommon in nature, pointing to the potential of active principles to yield therapeutics less likely to be compromised by antimicrobial resistance. As only two plants were evaluated in this study, the generalizability of our findings is presently unknown and should be the subject of future research.