Colony morphology and sclerotial biomass of P. umbellatus under exogenous different concentrations of oxalic acid
After cultivation for 30 d, the sclerotia with red asterisks* in the No OA group (Fig. 1A) and blue asterisks in the Low OA group (Fig. 1B) entered the SD stage, and interwoven mycelia with round, oval or irregular sclerotia distributed on the edge of the petri dishes, as depicted in Fig. 1A,B. From the concentrations of exogenous oxalic acid below 1 mg/mL, it was observed that oxalic acid at 0.05 mg/mL served as the most suitable concentration in P. umbellatus sclerotial formation (Fig. 1B), compared to that of the control group (Fig. 1A). P. umbellatus sclerotial biomass in the Low OA group increased significantly (P < 0.05). In addition, the minimum concentration of oxalic acid that completely inhibited P. umbellatus sclerotial development was 1.10 mg/mL (Fig. 1C, Table 1); thus, no sclerotia formed in the High OA group. With the increase of oxalic acid concentrations at certain ranges, P. umbellatus sclerotia was promoted or inhibited.
Colony morphology of P. umbellatus under exogenous different concentrations of oxalic acid. (A), (B) and (C) represent the colony morphological characteristics of P. umbellatus mycelia in the No OA, Low OA and High OA groups after cultivation for 30 d respectively. Red or blue asterisks represent round, oval or irregular sclerotia in the No OA and Low OA groups.
Transcriptome sequencing and de novo assembly and sequence annotation
To obtain the P. umbellatus mycelial transcriptomic expression profiles following treatment with oxalic acid, two libraries from a low-concentration oxalic acid (Low OA) group and a high-concentration oxalic acid (High OA) group and one library from a no-oxalic-acid (No OA) control group were constructed. Illumina sequencing data of P. umbellatus could be obtained in the NCBI BioProject and the Sequence Read Archive (SRA) database with the ID PRJNA669949. In total, 284,190,976 raw reads were generated (Supplementary Table 1), and 276,265,498 clean reads remained after the adaptor sequences, ambiguous nucleotides and low-quality sequences were removed. The assembly of clean reads included 22,523 unigenes in the range of 201–19,256 bp, and the numbers of assembled unigenes with different interval lengths are shown (Supplementary Fig. 1A).
In total, 22,523 nonredundant unigenes were subjected to similarity analysis according to the 7 public databases. Among these unigenes, 12,864 (57.11%) had the highest similarity matches in the NT database followed by 11,236 (49.88%) unigenes in the SwissProt database. Specifically, 10,914, 10,645, 10,571, 6934 and 4063 unigenes were functionally annotated in the other five databases (NR, GO, PFAM, KOG and KO), accounting for 48.45%, 47.26%, 46.93%, 30.78% and 18.03%, respectively, as shown in Supplementary Table 2.
According to GO, 10,645 unigenes were grouped into three major functional ontologies, including biological process, cellular component and molecular function (Supplementary Fig. 1B).
Identification of DEGs in the mycelia of P. umbellatus treated with different concentrations of oxalic acid
Due to the analysis of the DEGs, RPKM values were calculated, and the Pearson’s correlation coefficients (R2) were high between the three biological replicates in the same group, ranging from 0.902 to 1. The R2 of the samples between the different groups ranged from 0.705 to 0.816 (Supplementary Fig. 1C). The DEGs that met the criteria (adjusted P value < 0.05 and |log2 fold change|> 1) between the Low OA and the No OA group, the High OA and the No OA group were analysed, and the DEGs with higher expression levels in the Low OA and High OA groups were classified as ‘upregulated’, while those with lower expression levels in both groups were classified as ‘downregulated’ (Supplementary Fig. 1D). There were 725 DEGs between the High OA and the control group, and among them, 299 were upregulated and 426 were downregulated. There were 459 DEGs between the Low OA and the control group and among them, 231 were upregulated and 228 were downregulated (Supplementary Fig. 1D). According to the Venn diagram (Supplementary Fig. 1E), there were 231 common DEGs in both of the compared groups, and there were 59 DEGs upregulated and 47 downregulated in both cases. There were 44 DEGs upregulated in High OA compared with No OA but downregulated in Low OA compared with No OA. There were 81 DEGs upregulated in Low OA compared with No OA but downregulated in High OA compared with No OA.
DEGs related to oxidative stress, calcium signaling and energy metabolism
In previous studies, it was demonstrated that P. umbellatus sclerotial formation was closely associated with oxidative stress and that calcium channel blockers and calmodulin inhibitors inhibited P. umblelatus sclerotial development; therefore, we were strongly interested in the DEGs related to oxidative stress and the calcium signaling pathway5,8. In comparison to that of the control (No OA) group, DEGs encoding such oxidases as NADPH oxidase, alcohol oxidase, cytochrome c oxidase (subunits 1, 2 and 3), NADH dehydrogenase subunit 1, NADH dehydrogenase subunit 4, glycerol 2-dehydrogenase were upregulated in the Low OA group but were downregulated in the High OA group. Meanwhile, DEGs encoding such reductases as aldo/keto reductase and nitrite reductase were downregulated in the Low OA group but upregulated in the High OA group. Also, specific DEGs, such as cytochrome c reductase, ribonucleotide reductase alpha subunit and quinone reductase, were all upregulated, while such enzymes as galactose oxidase, sorbitol dehydrogenase, succinate dehydrogenase and alanine dehydrogenase, were all downregulated in the High OA group in comparison to that of the No OA group. Similarly, specific DEGs in the Low OA group, such as NAD-dependent glutamate dehydrogenase, glutamate dehydrogenase and NAD-aldehyde dehydrogenase, were all upregulated in the Low OA group compared to the control group (Supplementary Table 3). The same DEGs, c10001_g1, c9468_g4, c43_g1, c9143_g1, c3657_g1, c524_g1 and c5285_g1, were upregulated in the Low OA group but downregulated in the High OA or downregulated in the Low OA group while upregulated in the High OA group (Supplementary Table 3).
The DEGs related to calcium signaling (i.e. calmodulin / calcium motif containing genes) such as c2043_g1 c8739_g2 and c3257_g1 were all upregulated in the Low OA group but downregulated in the High OA group (Supplementary Table 3).
In comparison to that of the control group, c4703_g1 was upregulated in the Low OA group but downregulated in the High OA group. In addition, c7209_g1 and c6742_g2, were significantly highly expressed in the Low OA group, while c9487_g1, c9511_g1 and c9756_g1 were all downregulated in the High OA group, in comparison to the control group (Supplementary Table 3). The unigene sequences of the DEGs were listed in Supplementary Table 4.
ROS content and the fluorescence intensity of ROS in P. umbellatus mycelia were affected by different concentrations of oxalic acid
In the No OA group, the ROS content of P. umbellatus mycelia had accumulated to a certain extent after cultivation for 30 days as shown in Fig. 2A, while in the Low OA group, the ROS level was greater than that of the control group (Fig. 2B); however, in the High OA group, the ROS content was considerably lower than that of the control group (Fig. 2C). Compared with the control group, the fluorescence intensity of ROS increased in the Low OA group but decreased significantly in the High OA group (P < 0.05, Fig. 2D).
ROS content and fluorescence intensity of ROS in P. umbellatus mycelia affected by different concentrations of oxalic acid. After cultivation for 30 d, the ROS content of P. umbellatus mycelia in the No OA (A), Low OA (B) and High OA groups (C) is shown. Images are representative of three independent experiments (n = 30). Scale bar, 10 μm. The mean grey scale value of ROS in different groups of No OA, Low OA and High OA from three independent experiments is shown, with *standing for P < 0.05, compared to the control (No OA) group (D).
Validation of the DEGs using qRT-PCR
Six unigenes were selected and examined at the transcriptional level by qRT-PCR, to validate the reliability of the RNA-Seq data (Fig. 3). All the selected unigenes showed that the trend of the expression patterns were consistent with that of the transcriptomic data, which indicated the validity of the RNA-seq results.
Validation of the DEGs using qRT-PCR. Fold changes of the expression of the selected DEGs including c3657_g1, c9143_g1, c9468_g4, c10001_g1, c4703_g1 and c43_g1 between Low OA or High OA and No OA by qRT-PCR and RNA-seq were represented.
The net Ca2+ and H2O2 fluxes during P. umbellatus growth were affected by different concentrations of oxalic acid using NMT
The negative values represented the influx of Ca2+ and H2O2 (Fig. 4A,B) and the positive values represented the efflux of H2O2 (Fig. 4B). After cultivation for 30 d, Ca2+ influx was measured in the mycelia of P. umbellatus over a period of time in different groups, and the net Ca2+ uptake was observed to increase in the Low OA group but decreased in the High OA group significantly (P < 0.05) in comparison to that of the No OA group (Fig. 4A). The H2O2 in the High OA group presented slight efflux, with the net fluxes ranging from 4.05 to 9.77 pmol cm−2 s−1 (Fig. 4B); however, H2O2 presented influx in the No OA group and the Low OA group (Fig. 4B), and there were significant differences between the Low OA and High OA groups, compared to that of the control group (P < 0.05). The significant positive correlationship with Pearson’s correlation coefficient (r = 0.991, P < 0.01) between the flux measurement of Ca2+ and H2O2 was determined by correlationship analysis using SPSS, and the scatter plot is presented in Supplementary Fig. 1F.
Real-time Ca2+ and H2O2 flux measurements of P. umbellatus mycelia affected by different concentrations of oxalic acid using NMT. (A) Real-time Ca2+ influxes detection. (B) Real-time H2O2 fluxes measurement. The values of the net Ca2+ and H2O2 fluxes are the means ± SD (n = 30), with * representing for P < 0.05.
