Biochar-Enhanced Resistance to Botrytis cinerea in Strawberry Fruits (But Not Leaves) Is Associated With Changes in the Rhizosphere Microbiome

Abstract

Biochar has been reported to play a positive role in disease suppression against airborne pathogens in plants. The mechanisms behind this positive trait are not well-understood. In this study, we hypothesized that the attraction of plant growth-promoting rhizobacteria (PGPR) or fungi (PGPF) underlies the mechanism of biochar in plant protection. The attraction of PGPR and PGPF may either activate the innate immune system of plants or help the plants with nutrient uptake. We studied the effect of biochar in peat substrate (PS) on the susceptibility of strawberry, both on leaves and fruits, against the airborne fungal pathogen Botrytis cinerea. Biochar had a positive impact on the resistance of strawberry fruits but not the plant leaves. On leaves, the infection was more severe compared with plants without biochar in the PS. The different effects on fruits and plant leaves may indicate a trade-off between plant parts. Future studies should focus on monitoring gene expression and metabolites of strawberry fruits to investigate this potential trade-off effect. A change in the microbial community in the rhizosphere was also observed, with increased fungal diversity and higher abundances of amplicon sequence variants classified into Granulicella, Mucilaginibacter, and Byssochlamys surrounding the plant root, where the latter two were reported as biocontrol agents. The change in the microbial community was not correlated with a change in nutrient uptake by the plant in either the leaves or the fruits. A decrease in the defense gene expression in the leaves was observed. In conclusion, the decreased infection of B. cinerea in strawberry fruits mediated by the addition of biochar in the PS is most likely regulated by the changes in the microbial community.

Keywords: RNA sequencing; biochar; metabarcoding; microbiome; plant defense; strawberry.

FIGURE 1

Scoring Botrytis cinerea infection on strawberry plant leaves and fruits. (A) Strawberry plant leaves were inoculated at 8 weeks of plant growth. In total, three fully expanded leaves per plant were inoculated and per condition [peat substrate (PS)] or biochar-amended PS (PS + BC)] and 12 plants were inoculated. The disease was scored with a value of 0 (no infection) to 4 (100% infected leaf) at 7, 9, and 12 DAI. The score abundances over all plants and leaves are visualized. (B) Strawberry fruits (n = 6 per time point per tray) were inoculated with B. cinerea after picking. Fruits were discarded once infection symptoms were observed, and the AUDPC was calculated based on this measurement and was visualized per treatment. The AUDPC value per tray (n = 5) is represented in a boxplot. In total, this was repeated four times, indicated by the values 1–4. Statistical significances are indicated with asterisk (**p < 0.01; ***p < 0.001).

FIGURE 2

Effect of the addition of biochar on the bacterial (the V3–V4 fragment of the 16S rRNA gene) and fungal (ITS2 gene) community of the strawberry rhizosphere over time. (A) The principal coordinate analysis plot showing that the bacterial and fungal community shift over time (symbols are getting bigger when going further in time during the experiment). Only minor effects of the addition of biochar can be observed on the bacterial community (left), while for the fungal community (right), an effect of the addition of biochar can be identified. (B) Bacterial genera are consistently increased by the addition of biochar in the first 2–3 weeks of the experiment. (C) The fungal genus Byssochlamys is consistently increased in the strawberry rhizosphere due to the addition of biochar. For all plots, samples taken from PS are indicated in light gray, while biochar-amended PS is indicated in black. Statistical significances are indicated with an asterisk (*p < 0.05).

FIGURE 3

Chemical characterization of the PS with plants, strawberry fresh leaves, and strawberry fruits over 11 weeks of plant growth. (A) Plant-available nitrogen (N) (NO₃-N and NH₄-N), phosphorous (P) concentrations of either of the PS mixtures [without or with 2 g of biochar per liter added (PS + CH)] in which plants were grown (left), and NO₃-N, NH₄-N, and SO₄ concentrations of either PS mixtures in which no plants were grown (right). Dots represent the mean value with the SD as error bars (n = 3). If no values occur, the measurement was below the detection limit. (B) Total N and P contents (in mg/pot) measured in plant leaves per plant on weeks 3, 6, and 9 of the experiment. Mean values are represented as lines, and dots represent the values of the biological replicates (n = 3). (C) Concentrations of N (percentage on DM) and P (in g/kg DM) of strawberry fruits are represented (n = 5). Replicates are considered as all fruits picked from all plants grown in either PS or PS + BC at one specific time point (at 5, 6, 7, 8, and 9 weeks of plant growth). Statistical significances are indicated with an asterisk (*p < 0.05).

FIGURE 4

Gene expression in strawberry leaves. (A) Gene ontology (GO) enrichment analysis (biological process) of genes differentially expressed in PS vs. PS + BC + I comparison. The size of the boxes corresponds with the significance of the GO terms. (B) Relative gene expression values (by reverse transcription quantitative PCR) of six defense genes in strawberry for PS and biochar treatments (PS + BC), inoculated with B. cinerea (I) or mock-inoculated. Expression values are expressed in log2 fold changes (mean ± SE). The PS treatment is used as the control treatment; for this treatment, the expression value is set to 0. Statistically significant differences are indicated with different letters.