Mycelium growth and sporulation of Tk1 strain in different media
The mycelium weight of T. koningiopsis Tk1 differed between different media. The mycelium weights of the Tk1 strain grown for 7 d in Fries3, PD, and PS media were 1.73, 1.29, and 0.94 g, respectively (Figure 1A). After 7 d, the spore pair values in the Fries3, PD, and PS media were 8.6, 8.4, and 8.3, respectively (Figure 1B).
Filtrate antifungal bioassay
The axenic fermentation extracts of T. koningiopsis Tk1 from different media were obtained. Both the extract and media were added to the PDA medium. The results showed that the media did not significantly impact the mycelial growth of F. oxysporum. Moreover, the axenic fermentation extract in the three media differed. A total of 3 mL axenic fermentation extract was added to 7 mL PDA for each test. Both axenic fermentation extracts from Fries3 and PS media showed better inhibition of pathogenic F. oxysporum. After 3 d, the pathogen colony diameters in plates with extract added from Fries3, PS, and PD media were 23.5 mm (inhibition rate of 22.95%), 20.25 mm (inhibition rate of 33.61%), and 30.00 mm (inhibition rate of 1.64%), respectively; the pathogen colony diameter in PDA was 30.50 mm. After 5 d, the colony diameter in plates with extract added from Fries3, PS, and PD media were 45.00 mm (inhibition rate of 33.58%), 44.50 mm (inhibition rate of 34.32%), and 58.75 mm (inhibition rate of 13.28%), respectively; the pathogen colony diameter in CK was 67.75 mm (Figure 2).
Sequencing assembly
Averages of 58,150,778, 61,910,742, and 48,690,148 raw reads were obtained from sequencing the Fries3, PD, and PS cDNA libraries of T. koningiopsis Tk1, respectively. After trimming adaptor sequences and eliminating low-quality reads, 56,134,944, 59,723,474, and 46,770,465 clean reads were obtained from Fries, PD, and PS samples, respectively (Supplementary Table S1). After combined assembly, the datasets resulted in 14,208 unigenes with a mean length of 2124 base pairs (bp), N50 of 4001 bp, and N90 of 803 bp (Supplementary Table S2).
Functional annotation of the assembled unigenes
Annotation was conducted using the BLASTx and BLASTn programs with an E-value cut-off of 10−5. A total of 12,201 (85.87%) unigenes was annotated by at least one of the databases: 10,796 (75.98%) unigenes were annotated by the NCBI-NR database, 3025 (21.29%) unigenes by the KO database, 11,158 (78.53%) unigenes by the NCBI-Nt database, 8614 (60.62%) by Swiss-Prot, 8748 (61.57%) unigenes by the PFAM database, 8748 (61.57%) unigenes by the GO database, and 3817 (26.86%) by the KOG database (Figure 3A). BLASTx homology search in the NCBI-NR database showed that T. koningiopsis transcriptomes had the best blast match to Trichoderma sequences, primarily to T. gamsii (43%) and T. atroviride (19.7%) (Figure 3B). All unigenes in the T. koningiopsis transcriptomes were subjected to GO analysis for the three categories molecular function, biological process (BP), and cellular component (CC) (Figure 3C). In the molecular function (MF) GO category, the most abundant transcripts were linked to binding and catalytic activity. In the BP category, the most enriched term was cellular process, with 5532 unigenes. The metabolic process and single-organism process terms were also abundant. In the CC category, cell and cell parts were the most enriched terms.
Analysis of DEGs
The square of the Pearson correlation coefficient (R2) was used to measure the correlation between samples. The R2 values for Fries3, PD, and PS samples were 0.959, 0.964, and 0.917, respectively (Supplementary Figure S1).
DEGs were normalized using the reads per kilobase of transcript per million mapped reads method. Compared with Fries3, PD showed 2618 DEGs, of which 1416 were upregulated and 1202 were downregulated. Compared with PS, PD showed 2293 DEGs, of which 1120 were upregulated and 1173 were downregulated. Compared with Fries, PS showed 876 DEGs, of which 382 were upregulated and 494 were downregulated (Figure 4A). Comparison of the DEGs from the Fries3 vs PD group and from the PS vs PD group revealed 1520 common DEGs; 1098 DEGs belonged only to the Fries3 vs PD group and 773 DEGs belonged only to the PS vs PD group. Comparison of the DEGs from the Fries3 vs PD group and from the Fries3 vs PS group revealed 557 common DEGs; 2043 DEGs belonged only to the Fries3 vs PD group and 301 DEGs belonged only to the Fries3 vs PS group. Comparison of the DEGs from the Fries3 vs PS group and PS vs PD group revealed 405 common DEGs; 471 DEGs belonged only to the Fries3 vs PS group and 1888 DEGs belonged only to the PS vs PD group. Comparison of DEGs in all three groups revealed 208 DEGs common between the three groups (Figure 4B). The heatmap showed that each group had some highly expressed genes, whereas the expression profiles of Fries3 and PS were similar (Figure 4C).
GO analysis of DEGs
The unigenes were compared with the GO database by BLAST, and the DEGs were analyzed for GO enrichment. The numbers of DEGs in the Fries3 vs PD group enriched for BP, MF, and CC were 735, 1011, and 0, respectively. The DEGs enriched in BP were mainly concentrated in three subcategories including oxidation-reduction process (383, 10.51%), transmembrane transport (249, 13.34%), and monocarboxylic acid metabolic process (103, 5.52%). The DEGs enriched in MF were mainly concentrated in 6 subcategories including oxidoreductase activity (394, 21.1%); coenzyme binding, cofactor binding (182, 9.75%); flavin adenine dinucleotide binding (74, 3.96%); oxidoreductase activity, acting on CH-OH group of donors (89, 4.77%); and oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen (66, 3.54%) (Figure 5A). The number of DEGs in the Fries3 vs and PS group enriched for BP, MF, and CC were 158, 762, and 0, respectively. DEGs enriched in BP were mainly concentrated in the subcategories of oxidation-reduction process (158, 25.44%). DEGs enriched in MF were mainly concentrated in four subcategories including oxidoreductase activity (167, 26.89%), coenzyme binding (90, 14.49%), cofactor binding (93, 14.98%), and catalytic activity (412, 66.34%) (Figure 5B). The numbers of DEGs in the PS vs PD group enriched for BP, MF, and CC were 236, 681, and 0, respectively. DEGs enriched in BP were mainly concentrated in the subcategories of transmembrane transport (236, 14.07%). DEGs enriched in MF were mainly concentrated 3 subcategories including oxidoreductase activity (338, 20.16%), coenzyme binding (162, 9.66%), and cofactor binding (181, 10.79%) (Figure 5C).
KEGG analysis of DEGs
The DEGs were searched in the KEGG database to identify physiological processes in which they might participate. The DEGs in the Fries3 vs PD group were enriched in metabolic pathways such as beta-alanine metabolism, amino sugar and nucleotide sugar metabolism, propanoate metabolism, tyrosine metabolism, sphingolipid metabolism, tryptophan metabolism, glycerolipid metabolism, starch and sucrose metabolism, glycerophospholipid metabolism, arachidonic acid metabolism, and phenylalanine metabolism. They were also enriched in biosynthesis pathways such as various types of N-glycan biosynthesis and fatty acid biosynthesis (Figure 6A). DEGs in the Fries3 vs PS group were enriched in several pathways such as amino sugar and nucleotide sugar metabolism, sulfur metabolism, propanoate metabolism, glycolysis/gluconeogenesis, methane metabolism, fatty acid degradation, pentose and glucuronate interconversions, nicotinate and nicotinamide metabolism, beta-alanine metabolism, starch and sucrose metabolism, glycerophospholipid metabolism, regulation of autophagy, peroxisome, phagosome, pentose phosphate pathway, glutathione metabolism, glyoxylate and dicarboxylate metabolism, cyanoamino acid metabolism, folate biosynthesis, and homologous recombination (Figure 6B). DEGs in the PS vs PD group were enriched in many pathways associated with ribosomes, beta-alanine metabolism, peroxisomes, amino sugar and nucleotide sugar metabolism, sphingolipid metabolism, propanoate metabolism, various types of N-glycan biosynthesis, ubiquitin-mediated proteolysis, valine, leucine and isoleucine degradation, regulation of autophagy, glycerophospholipid metabolism, glutathione metabolism, N-glycan biosynthesis, steroid biosynthesis, MAPK signaling pathway – yeast, vitamin B6 metabolism, one carbon pool by folate, thiamine metabolism, sulfur relay system, and phenylalanine metabolism (Figure 6C). Pathways common between the top 20 DEGs’ of the 3 groups were beta-alanine metabolism, peroxisome, amino sugar and nucleotide sugar metabolism, propanoate metabolism, and glycerophospholipid metabolism.
CAZyme analysis of DEGs
By comparing the protein sequences using the CAZymes analysis toolkit software package, 352 CAZyme families with 325 genes were annotated. Among them, there were 39 (11%) carbohydrate binding modules (CBM), 61 (17%) carbohydrate esterase (CE), 140 (40%) glycoside hydrolases (GH), 71 (20%) glycosyl transferases (GT), and 41 (12%) auxiliary activities (AA) (Figure 7A). Within CBM, CE, GH, GT, and AA, there were 18, 12, 49, 28, and 8 gene families, respectively (Supplementary Table S3).
The numbers of CAZymes in DEGs of the Fries3 vs PD group, PS vs PD group, and Fries3 vs PS group were 134, 97, and 46, respectively. Within the 134 CAZymes in DEGs of the Fries3 vs PD group, there were 22 AAs (13 upregulated and 9 downregulated), 18 CBMs (12 upregulated and 6 downregulated), 15 CEs (10 upregulated and 5 downregulated), 61 GHs (33 upregulated and 27 downregulated), and 18 GTs (7 upregulated and 11 downregulated). Interestingly, 10 were not expressed in the PD group but were highly expressed in the Fries3 group (Figure 7B). Within the 97 CAZymes in DEGs of the PS vs PD group, there were 13 AAs (10 up and 3 down), 11 CBMs (7 up and 4 down), 11 CEs (5 up and 6 down), 48 GHs (25 up and 23 down), and 14 GTs (6 up and 8 down). Interestingly, 6 were not expressed in the PS group but were highly expressed in the PD group (Figure 7C). Within the 46 CAZymes in DEGs of the Fries3 vs PS group, there were 10 AAs (3 up and 7 down), 8 CBMs (4 up and 4 down), 5 CEs (2 up and 3 down), 22 GHs (9 up and 13 down), and 1 GT (up) (Figure 7D).