Identification of the Ilex macrocarpa anthracnose pathogen and the antifungal potential of the cell-free supernatant of Bacillus velezensis against Colletotrichum fioriniae

Abstract

Introduction: Anthracnose is a significant fungal disease that affects tree growth and development, with Colletotrichum spp. exhibiting host non-specificity and targeting various organs, making disease control challenging.

Methods: This study aimed to identify the pathogenic species causing anthracnose in Ilex macrocarpa in Nanchong, Sichuan Province, and screen effective fungicides, particularly biological ones. The pathogen was identified as Colletotrichum fioriniae through morphological observation, pathogenicity assays, and molecular biological methods. Three biological and five chemical fungicides were evaluated for their effects on the mycelial growth and spore germination rate of the pathogen.

Results: The results indicated that prochloraz was the most effective chemical fungicide, while the cell-free supernatant (CFS) of Bacillus velezensis had the most significant inhibitory effect among the biological fungicides. Transcriptome analysis revealed that the CFS of B. velezensis significantly reduced the expression of genes associated with ribosomes, genetic information processing, membrane lipid metabolism, and sphingolipid biosynthesis in C. fioriniae. Additionally, the glutathione pathway's expression of various genes, including key genes such as GST, GFA, Grx, TRR, and POD, was induced. Furthermore, the expression of 17 MFS transporters and 9 ABC transporters was increased. Autophagy-related ATGs were also affected by the B. velezensis CFS.

Discussion: These findings suggest that the B. velezensis CFS may inhibit C. fioriniae through interference with ribosomes, genetic information processing, cell membrane metabolism, and energy metabolism. These results provide potential target genes for the B. velezensis CFS and insights into the antifungal mechanism by which B. velezensis inhibits C. fioriniae.

Keywords: Bacillus velezensis; Colletotrichum fioriniae; Ilex macrocarpa; anthracnose; biocontrol.

Figure 1

Isolation of fungi and pathogenicity test. (A) Brown sunken necrotic leaf spots causing by Colletotrichum fioriniae on Ilex macrocarpa in the field; (B,C) Colonial morphology of XLHR17 on PDA; (D,E) Mycelium and conidiospore of XLHR17; (F,G) Symptoms on leaves 7 days after inoculated by XLHR17 and sterile distilled water.

Figure 2

Phylogenetic tree generated by maximum likelihood analysis based on concatenated sequences of the ITS, HIS3, CHS-1, ACT, TUB2, and GAPDH. The dataset contained 31 ingroup taxa, and the tree is rooted with C. orchidophilum. The ex-type (exepitype) strains are marked with a *. RA × ML bootstrap values equal to or greater than 70% are shown at the nodes. The isolate used in present study, XLHR17, is highlighted in green.

Figure 3

Inhibitory effect of thiophanate-methyl on C. fioriniae. (A) The appearance of colony size and spore number of C. fioriniae inhibited by thiophanate-methyl; (B) Colony diameter; (C) Inhibition rate. p < 0.05 was marked with a *. Similarly hereinafter.

Figure 4

Inhibitory effect of prochloraz on C. fioriniae. (A) The appearance of colony size and spore number of C. fioriniae inhibited by t prochloraz; (B) Colony diameter; (C) Inhibition rate.

Figure 5

Inhibitory effect of imazalil on C. fioriniae. (A) The appearance of colony size and spore number of C. fioriniae inhibited by imazalil; (B) Colony diameter; (C) Inhibition rate.

Figure 6

Inhibitory effect of paclobutrazol on C. fioriniae. (A) The appearance of colony size and spore number of C. fioriniae inhibited by paclobutrazol; (B) Colony diameter; (C) Inhibition rate.

Figure 7

Inhibitory effect of carbendazim on C. fioriniae. (A) The appearance of colony size and spore number of C. fioriniae inhibited by carbendazim; (B) Colony diameter; (C) Inhibition rate.

Figure 8

Inhibitory effect of B. velezensis CFS on C. fioriniae. (A) The appearance of colony size and spore number of C. fioriniae inhibited by B. velezensis CFS; (B) Colony diameter; (C) Inhibition rate.

Figure 9

Inhibitory effect of B. licheniformis CFS on C. fioriniae. (A). The appearance of colony size and spore number of C. fioriniae inhibited by B. licheniformis CFS; (B) Colony diameter; (C) Inhibition rate.

Figure 10

Inhibitory effect of B. subtilis CFS on C. fioriniae. (A) The appearance of colony size and spore number of C. fioriniae inhibited by B. subtilis CFS; (B) Colony diameter; (C) Inhibition rate.

Figure 11

Enriched GO terms and KEGG pathways of DEGs after B. velezensis CFS treatment. GO enrichment analysis of upregulated genes (A) and downregulated genes (B). KEGG enrichment analysis of upregulated genes (C) and downregulated genes (D). The size of the dots denotes the number of enriched genes, and the color intensity denotes the significant level.

Figure 12

(A) Heat map of DEGs that involved in Steroid biosynthesis. ERG1, squalene epoxidase; ERG7, lanosterol synthase; CYP5, cytochrome b5; ERG24, C-14 sterol reductase; ERG25, C-4 sterol methyloxidase; ERG26, C-4 sterol decarboxylase; ERG27, C-3 sterol Keto reducatse; ERG6, C-24 Sterol methyl-transferase; ERG2, C-8 Sterol isomerase; ERG 3, C-5 Sterol desaturase; ERG 5:C-22 Sterol desaturase; ERG 4, C-24 sterol reductase. The heat map was generated by log (fold change) using Graphpad prism software: red, increase; green, decrease. (B) Heat map of DEGs that may be involved in sugar metabolism. GK, Glucokinase; PGI, Phosphoglucose isomerase; HK, Hexokinase; tpsB, trehalose 6-phosphate synthase; AMY, Alpha-amylase A; gLaA, Glucoamylase; CTS, Chitosanase; nagA, 6-Phosphate-N-acetylglucosamine deacetylation; nagB, 6-phosphate glucosamine deaminase; PFK, phosphofructokinase. The heat map was generated by log (fold change) using Graphpad prism software: red, increase; green, decrease. (C) Heat map of DEGs that may be involved in sugar metabolism. GDH, Glutamate dehydrogenase; argJ, Amino-acid acetyltransferase; GSS, glutathione synthase; GSH, glutathione; GR, glutathione reductase; PGD:6-phosphogluconate dehydrogenase; GPX, glutathione peroxidase; GTT, Glutathione transporter; GSSG, glutathione disulfide; GST, Glutathione S-transferase; TRR, Thioredoxin reductase; GRX, Glutaredoxin; SOD, Superoxide dismutase; CAT, Catalase. The expression image was generated using Graphpad prism software: red, increase; green, decrease.