Successional changes in bacterial phyllosphere communities are plant-host species dependent

Abstract

Phyllosphere microbial communities are increasingly experiencing intense pulse disturbance events such as drought. It is currently unknown how phyllosphere communities respond to such disturbances and if they are able to recover. We explored the stability of phyllosphere communities over time, in response to drought stress, and under recovery from drought on temperate forage grasses. Compositional or functional changes were observed during the disturbance period and whether communities returned to non-stressed levels following recovery. Here, we found that phyllosphere community composition shifts as a result of simulated drought but does not fully recover after irrigation is resumed and that the degree of community response to drought is host species dependent. However, while community composition had changed, we found a high level of functional stability (resistance) over time and in the water deficit treatment. Ecological modeling enabled us to understand community assembly processes over a growing season and to determine if they were disrupted during a disturbance event. Phyllosphere community succession was characterized by a strong level of ecological drift, but drought disturbance resulted in variable selection, or, in other words, communities were diverging due to differences in selective pressures. This successional divergence of communities with drought was unique for each host species. Understanding phyllosphere responses to environmental stresses is important as climate change-induced stresses are expected to reduce crop productivity and phyllosphere functioning.

Importance: Leaf surface microbiomes have the potential to influence agricultural and ecosystem productivity. We assessed their stability by determining composition, functional resistance, and resilience. Resistance is the degree to which communities remain unchanged as a result of disturbance, and resilience is the ability of a community to recover to pre-disturbance conditions. By understanding the mechanisms of community assembly and how they relate to the resistance and resilience of microbial communities under common environmental stresses such as drought, we can better understand how communities will adapt to a changing environment and how we can promote healthy agricultural microbiomes. In this study, phyllosphere compositional stability was highly related to plant host species phylogeny and, to a lesser extent, known stress tolerances. Phyllosphere community assembly and stability are a result of complex interactions of ecological processes that are differentially imposed by host species.

Keywords: drought; microbial community assembly processes; pasture grass; phyllosphere; plant-microbe relationships; resilience.

Fig 1

Plant hosts showed few measured changes during the 10-week drought period despite a significant decrease in soil moisture in each of the three host species (A). Plant measurements taken throughout the experimental period include (B) leaf relative water content, (C) relative chlorophyll content, and (D) electrolyte leakage. (E) Proline content was measured (dry leaf mass) at the end of the drought period and the end of the recovery period. (F) Root mass was measured at three different depths at the end of the experimental period before rewatering occurred. Significant differences between treatments on an individual species are represented by asterisks, where *P < 0.05, **P < 0.001, and ***P < 0.001. Significant differences between host species are represented by lowercase letters. Letters that are the same represent no significant difference between host species.

Fig 2

Phyllosphere bacterial community diversity was measured for each host species and treatment over the experimental period using (A) observed species richness and (B) evenness. Community evenness on tall fescue hosts was the only alpha diversity metric to show a significant response to drought (P = 0.001). Differences in diversity levels between host species are represented by the lower-case letters at the top of each plot. Significant differences are represented by different letters, and no difference is represented by the same letter. Trends over time were determined using linear models. R² values are represented at the bottom of the plots for significant trends. N.S., not significant.

Fig 3

Relative abundances of phyllosphere bacterial classes were significantly different between the plant host treatments and had different responses to drought. Communities from tall fescue and ryegrass hosts were found to be more similar to each other than they were to orchardgrass using hierarchical clustering using Euclidean distance and average linkage. The red triangle separates the drought period from the recovery period. Legend column A represents classes from orchardgrass hosts that were significantly different from ryegrass. Column B represents classes from orchardgrass hosts that were significantly different from tall fescue. Column C represents classes that were different between ryegrass and tall fescue. Black asterisks indicate differences in both control and drought treatments, green asterisks indicate differences between control only, and yellow asterisks indicate differences between drought treatments. Significance levels are assigned as P > 0.05 (not significant); *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001.

Fig 4

Phyllosphere bacterial communities from each host species became more distinct over time and were significantly impacted by drought stress. Non-metric multidimensional scaling (NMDS) ordination was plotted using Bray-Curtis distances for (A) the beginning of the experimental period (week 1), (B) early drought period (week 3), (C) late drought period (week 9), and (D) end of the recovery period (week 13). (E) PERMANOVAs were conducted for each corresponding period to understand community stability by determining when communities under disturbance showed signs of change and if they were able to recover from the disturbance event. Significance levels are assigned as P > 0.05 (not significant); *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001.

Fig 5

Phyllosphere community assembly processes were different on each of the different host species, but all three hosts had high levels of undominated processes. Boxplots show the β-nearest taxon index (βNTI) values (A, C, and E) and RC_BC values (B, D, and F) comparing communities from the irrigated control treatment to communities from the drought treatment for each sampling week. Dashed red lines represent the significance cutoffs for βNTI (|βNTI| > 2) and RC_BC (|RC_BC| > 0.95). RC_BC values that fall between the dashed red lines represent undominated processes.

Fig 6

Treatment effects on phyllosphere community assembly processes for each of the host species. While undominated processes were prevalent on each host species, the defined processes varied. The percentage of contribution was calculated by dividing the number of significant pairwise comparisons for each process by the total number of pairwise comparisons.

Fig 7

The effects of host species and treatment on phyllosphere bacterial biomass (cell numbers) over time. The total number of bacterial cells per leaf material from each host species was calculated by washing bacterial cells off the surface of leaves, direct counts using epifluorescence microscopy, and comparing to leaf areas using ImageJ. Each watering treatment is represented by five biological replicates for each host species on each sampling day. After the first sampling day, orchardgrass and tall fescue showed stable bacterial biomass, but ryegrass had more day-to-day variation.

Fig 8

Rates of nitrogen fixation (A) and abundances of the nifH genes (B) were significantly different between host species but were not affected by the drought treatment. Nitrogen fixation rates were measured using stable isotope tracing, exposing the bacterial communities to ¹⁵N₂. The abundance of nifH genes was measured using qPCR and standardized to the number of copies per gram of leaf material. Significant differences between host species, determined by linear models with Tukey’s Honest Significant Difference post hoc analysis, are represented by lowercase letters in the upper left corner of each plot. Different letters indicate statistical differences between hosts.