Pochonia chlamydosporia synergistically supports systemic plant defense response in Phacelia tanacetifolia against Meloidogyne hapla

Abstract

The network of antagonistic, neutral, and synergistic interactions between (micro)organisms has moved into the focus of current research, since in agriculture, this knowledge can help to develop efficient biocontrol strategies. Applying the nematophagous fungus Pochonia chlamydosporia as biocontrol agent to manage the root-knot nematode Meloidogyne hapla is a highly promising strategy. To gain new insight into the systemic response of plants to a plant-parasitic nematode and a nematophagous fungus, Phacelia was inoculated with M. hapla and/or P. chlamydosporia and subjected to transcriptome and metabolome analysis of leaves. While the metabolome proved quite stable except for the early time point of 48 h, comparison of the single P. chlamydosporia with the combined treatment revealed even larger effects after 6 d compared to 48 h, aligning with the later root infestation by P. chlamydosporia compared to M. hapla. Simultaneous exposure to both microorganisms showed a stronger overlap with the single M. hapla treatment than P. chlamydosporia. Changes of transcripts and metabolites were higher in the combined treatment compared to the individual inoculations. The results support the conclusion that P. chlamydosporia induces plant defense in a distinct and beneficial manner if combined with M. hapla although plant defense is partly suppressed by the endophytic growth. The results tentatively suggested that the application of P. chlamydosporia as a biocontrol agent against M. hapla can be more effective by supporting these tritrophic interactions with specific additives, such as phytohormones or amino acids in the formulation.

Keywords: biological control; biotic stress; plant defense; plant-parasitic nematodes; synergistic effects; systemic plant response.

Figure 1

Quantification of Pochonia chlamydosporia and Meloidogyne hapla in substrate, extent of P. chlamydosporia root colonization and amount of M. hapla eggs in Phacelia tanacetifolia 28 d after inoculation with M. hapla (MH), P. chlamydosporia (PC) and their combination (MH+PC). (A) Colony forming units of P. chlamydosporia per g substrate 28 d after inoculation (mean ± SE, n=12). (B) Percentage of intersects where hyphae were present relative to the total number of intersects counted for each treatment 28 d after inoculation (mean ± SD, n=12). (C) Total number of M. hapla in substrate for each treatment 28 d after inoculation (mean ± SE, n=12). (D) Number of M. hapla eggs per g root dry weight for each treatment 28 d after inoculation (mean ± SE, n=12). C, control; MH, inoculated with M. hapla; PC, inoculated with P. chlamydosporia; MH+PC, inoculated with M. hapla and P. chlamydosporia. Different letters indicate significance of difference (Kruskal–Wallis test with Dunn’s post hoc test, p < 0.05).

Figure 2

Transcriptome analysis of Phacelia tanacetifolia for the systemic effects after inoculation with Meloidogyne hapla (MH), Pochonia chlamydosporia (PC) and a combination of both (MH+PC). Total leaf RNA was subjected to RNAseq. Principal component analysis (PCA) (A) 48 h or (C) 6 d after inoculation displays the clustering of the three replicates for each treatment. Specificity and overlap of differentially expressed genes (DEGs) in P. tanacetifolia (B) 48 h and (D) 6 d after inoculation are shown in Venn diagrams (FDR < 0.05, upregulated: log2FC > 1 or downregulated log2FC < -1, n = 3). To globally investigate the additive effect of the combined inoculation (hypothesis 2), upregulated transcripts of MH+PC (FDR < 0.05, log2FC > 1) were compared to single inoculated treatments (Kruskal–Wallis test with Dunn’s post hoc test, p < 0.001), (E) 48 h (341) or (F) 6 d (390) after inoculation and downregulated transcripts of MH+PC (FDR < 0.05, log2FC < -1) were compared to single inoculated treatments (G) 48 h (515) or (H) 6 d (514) after inoculation.

Figure 3

Analysis of functionally annotated transcripts of Phacelia tanacetifolia for the systemic effects after inoculation with Meloidogyne hapla (MH), Pochonia chlamydosporia (PC) and a combination of both (MH+PC). Leaf RNA was subjected to RNAseq. Specificity and overlap of differentially expressed genes (DEGs) in P. tanacetifolia (A) 48 h and (B) 6 d after inoculation are shown in Venn diagrams (FDR < 0.05, upregulated: log2FC > 1 or downregulated log2FC < -1, n = 3).

Figure 4

Heatmap of stress markers in Phacelia tanacetifolia in response to Meloidogyne hapla (MH), Pochonia chlamydosporia (PC) and a combination of both (MH+PC). Heat maps with log2-fold changes of transcripts that were less regulated in MH+PC compared to MH (hypothesis 1: MH > 1 and MH+PC < MH or MH < -1 and MH+PC > MH) for one or both harvesting time points ( Supplementary Table S3 ). KEGG pathways: phenylpropanoid biosynthesis (ath00940), glutathione metabolism (ath00480), ascorbate and aldarate metabolism (ath00053), and peroxisomes (ath04146).

Figure 5

Heatmap of transcripts related to plant defense signaling in Phacelia tanacetifolia in response to Meloidogyne hapla (MH), Pochonia chlamydosporia (PC) and a combination of both (MH+PC). Heat maps with DEGs (FDR < 0.05) for plants inoculated with M. hapla (MH) or P. chlamydosporia (PC) or with a combination of both organisms (MH+PC) in comparison to uninoculated control plants. Heatmaps show log2-fold changes for treatments compared to the control for harvest 48 h and 6 d after inoculation fitting the parameters of hypothesis 2. KEGG pathways: Plant hormone signal transduction (ath04075), α-Linolenic acid metabolism (ath00592), MAPK signaling pathway (ath04016), Plant-pathogen interaction (ath04626).

Figure 6

Relative amounts of selected Phacelia tanacetifolia metabolites from central sugar metabolism (A–C), citric acid cycle (D–F), phytosterols (G, H) and myo-inositol (I, J), amino acids (K–N) and shikimate (O) in response to Meloidogyne hapla (MH), Pochonia chlamydosporia (PC) and a combination of both (MH+PC). Box plots show the peak areas of characteristic compound mass/charge ratio of the metabolites normalized to ribitol (217 mz-1) and leaf dry weight. The treatments with M. hapla (MH), with P. chlamydosporia (PC) or with a combination of both organisms (MH+PC) were compared with the uninoculated control plants (48 h after inoculation = C1, MH1, PC1, MH+PC1; 6 d after inoculation = C2, MH2, PC2, MH+PC2; 28 d after inoculation = C3, MH3, PC3, MH+PC3). The horizontal line in the boxes indicates the median and x the mean (n=6). Values with no letter in common are significantly different (Kruskal–Wallis test with Dunn’s Test for multiple comparisons with Benjamini and Hochberg (1995) correction as post-hoc test, p < 0.05). n.s. indicates no significant differences between the treatments for the respective harvesting time point.