(Table 1. A, B, C, D.)
Insect attack affects plant performance in the following generation
In order to understand how plant biomass accumulation was affected in response to the observed shift in the microbiome upon insect attack, a second generation of plants was grown in the absence of herbivore insects reusing the soil of the first generation. There was a clear reduction in the biomass of all crops grown in the substrate that previously experienced insect attack when compared with the plants sown in the control substrate (Figure 5). The most significant reduction in biomass was observed in beans (78.49%) and red beet (70.58%). In addition, we observed a less significant reduction in the biomass of sweet corn (35.84%), whereas tomato plants did not show a significant difference in biomass production. Sweet corn, beans, Arabidopsis,and red beet were found to be statistically significant; ANOVA one-way (p<0.05) followed by the T-Student (Bonferroni) (Figure 5).
Interestingly, comparison of biomass changes between generation one and two across beans and Arabidopsis plants showed that the plant biomass of the second generation grown under the microbiome shifted soil (Figure 5) was more dramatically reduced than the reduction observed from the actual insect feeding during the first generation (Figure 1).
Discussion
Recent rhizosphere microbiome studies have shown that insect infestation reshapes the overall microbiome structure in single crops (Kong et al, 2016; Li-Li et al., 2018). However, there is a limited understanding of the rhizosphere microbiome modulation across a variety of plant crops sampled at the same time, and the potential recruitment of plan beneficial microbial taxa in response to insect attack.
The present study evaluates the magnitude of the legacy effects of aboveground insect damage in rhizosphere microbiomes across five plant species. Our results showed that plant species shifted the microbial community composition in response to the herbivorous insect attack, presumably for promoting the recruitment of several plant-beneficial bacterial groups. PGPR bacteria taxa such as Azospirillum ,Burkholderia , and Arthrobacter increased significantly after insect attack across plant species. Our finding agrees with Kong et al. (2016) which demonstrates that whitefly infestation in aboveground organs leads to the recruitment of specific bacterial groups (e.g. Pseudomonas ssp .) conferring beneficial traits to pepper plants.
Our study showed a core rhizobiome that was consistently responsive in changes of relative abundance between control and insect attack treatments and across plant species; these include the phylaActinobacteria, Verrucomicrobia, Bacteroides, Firmicutes andAcidobacteria . These phyla significantly shifted in response to insect attack for most crops. At the genus level, every plant species held a specific microbiome comprised by unique bacteria (Supplementary table 8). Nine out of the 49 bacterial genera shared by conditioned soils across plant species became significantly more abundant. These genera include Azospirillum , Achromobacter ,Arthrobacter , Hydrogenophaga, and Burkholderia . In addition, every crop species showed at least one overabundant beneficial genus compared to the others. This observation may suggest that plant species select a specific group of microbes to exert a similar function due to difference in each plant species’ root exudate-derived metabolome profile (Hubbard et al, 2019). Most of the shared microbial species that significantly shifted are known to be beneficial for the plant. For instance, Azospirillum and Burkholderia are two well-known free-living nitrogen-fixing bacteria, as well as taxa associated with soil disease suppression (Jing and Qingye, 2012; Mendes et al., 2011). Members of the Achromobacter genus are known to be endophytes and root plant growth promoters (Bertrand et al, 2000; Jha and Kumar, 2009), and Arthrobacter induces nutrient solubilization and growth promotion (Banerjee et al, 2010; Velazquez-Becerra et al, 2011). These taxa are also implicated in the induction of plant immunity. It is established that PGPR bacteria have an IRS-eliciting effect in certain plant species (Shouan et al., 2001; Zehnder et al., 2000). For instance,Burkholderia inoculation incremented the accumulation of resistance-related enzymes (chitinase and β-1,3-glucanase), and carbohydrate and lipid-based molecular patterns related to defense-differential gene expression in corn and wheat (Elya et al., 2010; Madala et al., 2012).
It has been suggested that changes induced by aboveground herbivory in a present plant season can affect the performance of plants in subsequent growth seasons. This effect is known as the ecological soil legacy (Kardol et al, 2007; Van de Voorde et al, 2011; Wurst and Ohgushi, 2015). Soil legacy effects on plants are also linked to soil biota (Bezemer et al, 2013). For instance, Kostenco et al. (2012) demonstrated that feeding by aboveground insect herbivory on ragwort (Jacobaea vulgaris ) induced changes in the composition of soil fungi. In our second-generation study, we observed a significant decrease in biomass accumulation from plants grown under the disturbed soil (soil from plants grown under insect herbivory) compared with the control (fresh soil). This finding suggests that even though a subset of beneficial microbes was significantly promoted by the plant in response to aboveground damage, the cost of induced systemic resistance may outweigh the potential benefits of the recruited bacterial taxa in the first generation. This tradeoff is known as the ‘costs of resistance,’ and implies plant fitness reduction in response to herbivory (Bergelson and Purrington, 1996; Strauss et al, 2002;). We hypothesized that the observed rise in beneficial members of bacteria communities can accumulate in the soil until they are capable of exerting a significant impact on plant fitness. It is worth noting that the observed bacteria may also be acting belowground by signaling plant hormone systems, which is not necessarily translated in an immediate gain in biomass in the next generation. This supposition warrants further testing.
In summary, aboveground herbivory impacts rhizosphere microbial communities across plant species. Plants modulate PGPRs (plant-growth promoting rhizobacteria), increasing their abundance underTricoplusia ni attack. This increase in abundance of beneficial bacteria taxa is not reflected in biomass growth after the following generation of herbivory damage.
ACKNOWLEDGMENTS
We thank members of Professor Vivanco’s group for helping us with the discussion and providing valuable comments. We also thank Dr. Alison K. Hamm (USDA- ARS Soil Management and Sugar Beet Research Unit, Fort Collins, CO, USA) for technical assistance. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant 2014/50275-9, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant 482737/2012-3 to MCSF, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001, a USDA-Cooperative Agreement, and the Colorado State University Agricultural Experiment Station. MLM was the recipient of the FAPESP/DR fellowship 2016/18001-1 and FAPESP/BEPE fellowship 2017/05465-2. MCSF is also a research fellow of CNPq.
CONFLICT OF INTEREST
We have no declaration of conflict of interest.