This study revealed that the bacterial OTU richness of the three bamboo species in autumn was significantly higher than that in spring. The Phyllosphere of F. ferax had a greater diversity of bacterial OTUs than that of A. spanostachya and Y. lineolate. The dominant phyllosphere bacteria of the three bamboo species are Proteobacteria, Acidobacteria, Bacteroides and Actinomycetes. In a warm and humid climate, the diversity and richness of phyllosphere bacteria in the three bamboo species in spring were significantly higher than in autumn. The overall importance of seasonality to the structure and composition of the phyllosphere microbial community has been confirmed by many studies (Thompson et al., 1993; Copeland et al., 2015). Proteobacteria, Acidobacteria, Bacteroides and Actinomycetes are often detected in a variety of forests, indicating that these organisms have a wide ecological range and an ability to adapt to many environments (Isabelle et al., 2016; Feng et al., 2019). In this study, the relative abundance of the Proteobacteria in the three bamboo species were all above 60%, and the differences between the bamboo species were not significant, indicating that Proteobacteria played a dominant role in the phyllosphere microbial community. Its changing in turn may impact health of the staple food bamboo foraged by giant panda around Xiaoxiangling mountains. In spring, the relative abundance (15.29%) of Acidobacteria in F. ferax was significantly lower than that of A. spanostachya (21.16%) and Y. lineolate (25.67%). Actinomycetes are gram-positive bacteria that can decompose cellulose and lignin (Taibi et al., 2012).
The bacterial diversity and abundance of all three bamboo species in autumn were significantly higher than that in spring, which was similar to the result of Zheng and colleagues (2011), who found that the number of phyllosphere microbial community of Pinus tabulaeformis varied significantly between different seasons, with the largest diversity and abundance in autumn, followed by summer and the least in spring. The higher temperature and humidity in summer and autumn contributed to the higher diversity and richness than that in spring, while the higher altitude and longer low temperature in winter lead to the lower diversity and abundance (Isabelle et al, 2016). Airborne microorganisms can settle directly onto the leaf layer and their diversity and density may vary with time (daily and seasonal patterns)and other environmental events (Liu et al., 2020). In addition, agricultural practices such as harvesting and planting also have an impact on the movement of airborne microorganisms via disturbances to the leaf surface, precipitation or rain splash, and soil pollution (Redford et al., 2009 ).
The Mantel test found that elevation, distance from water, tree diameter at breast height, mean height of bamboo, mean base diameter of bamboo, tree height, shrub coverage and the number of shrubs, all significantly affected phyllosphere microbial community (Table S5). Jackson and colleagues (2006) found that the changes in the phyllosphere bacterial community in resurgent ferns were related to rainfall and humidity. Similarly, Laforest and colleagues (2016) found that the host species, habitat and climate (average summer temperature and precipitation) drove the phyllosphere bacterial community structure in temperate trees. In this study, elevation had the strongest relationship with phyllosphere microbial community. Elevation was significantly and positively correlated to many bacteria phyla abundance, such as Proteobacteria (Fig. 5). However, with an increase in elevation, both the Shannon and Sobs indices declined (Fig. S6). Higher elevations generally have fewer and more widely distributed water sources, and lower temperatures can limit the fluidity of microbial cell membranes and proteins, which are not conducive to microbial reproduction and growth (Zhang et al., 2014).
Changes to the phyllosphere microbial community could impact the degradation and absorption of plant nutrients and the metabolism of enzymes (Fazal et al., 2021). In this study, we used PICRUSt to predict the function of phyllosphere bacteria of three bamboo species foraged by giant pandas. Our data shows that the gene function spectrum of phyllosphere bacteria of F. ferax was significantly different from the other two bamboo species. The relative abundance of gene types in the membrane transport secretion system, signal transduction and oxidative phosphorylation metabolism in the third level of the KEGG pathway for F. ferax were lower than for the other two bamboo species (Fig. 6). This indicated that the low-altitude F. ferax may need less energy and protein to maintain the physiological activities of some phyllosphere bacteria. While the high-altitude A. spanostachya and Y. lineolate may require more material and energy to adapt to the colder, lower oxygen environment. It is worth noting that the relative abundance of gene types in transporters and ABC transporters were the most expressed pathways in membrane transport at level 2. Hamana and colleagues (2012) revealed that ABC transporters can protect animals from the barrier of toxic substances. In addition, genes related to replication and repair may help reduce damage to biomolecules and may help bamboo adapt to high altitude environments. However, our results are only based on predicted metagenomics, and do not represent the actual function of leaf-peripheral bacteria.
Food microbes can affect the gut microbes of animals (Kohl and Dearing 2014;Kohl et al. 2016). Lei and colleagues (2020) found significant associations of certain bacteria and fungi between bamboo and the gut of giant panda. The diversity of bamboo bacteria was also positively correlated with that of gut bacteria in giant panda. Giant pandas prefer to consume bamboo that grows naturally at high altitudes, probably because the total number of endophytic bacteria in high altitudes tends to be lower (Helander et al., 2013). There's a lot of work needed to fully understand the relationship between the food microbes and gut microbes of pandas. To explore the relevance of these genes in the environmental adaptability of giant pandas and bamboo, further research is needed to directly sequence the metagenomics of the phyllosphere microbial community and determine whether there are specific enzymes related to digestion in giant pandas.