Additional file 1 of From phyllosphere to insect cuticles: silkworms gather antifungal bacteria from mulberry leaves to battle fungal parasite attacks
Creators: name: Zhao, Pengfei
name: Hong, Song
name: Li, Yuekun
name: Chen, Haimin
name: Gao, Hanchun
name: Wang, Chengshu
Publisher: name: figshare
Year: 2024
Subjects: subject: Microbiology
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Screening of Msp1 deletion in Ma. sciuri by colony PCR (a) and verification by sequencing of PCR products (b). The putative mutant 3 (mut3) was not a successful gene deletion mutant. PAM, protospacer adjacent motif within the used CRISPR RNAs (crRNAs). Figure S2. Silkworm cuticular bacterial CFU counting and estimation of bacterial OTU diversity. a, b Comparison of the cuticular bacterial CFUs formed on the marine agar (a) and GYC medium (b) among the different ages of silkworm larvae. Two-tailed Student’s t-test was conducted between samples: *, P &lt; 0.05; ***, P &lt; 0.001; ****, P &lt; 0.0001. c, d Comparison of the Shannon (c) and Simpson (d) diversity indices among the ectomicrobiotas of the different ages of silkworm larvae. One-way ANOVA analysis was conducted to compare difference between samples: the column labelled with different capital letters, P &lt; 0.01; different lower letters, P &lt; 0.05. Ten independent replicates (three insects per replicate) were included for each sample. Figure S3. Screening and evaluation of bacteria isolated from silkworm surfaces for antifungal activity. a, b Inhibition or non-inhibition of M. robertsii (a, for 12 h) and B. bassiana (b, for 16 h) spore germination by different bacteria isolated from silkworm cuticles. Fungal spores (5 × 106 conidia/ml) were germinated in LB with the addition of bacterial cells each at 0.01 OD600. c, d The ethyl acetate extracts of Ma. sciuri have no effect on inhibiting M. robertsii (c) and B. bassiana (d) spore germination. Ma. sciuri was inoculated in LB for 24 h and the cultures were centrifuged, and both the supernatant and bacterial cells were extracted and used for inhibition assays. Figure S4. Inhibition of fungal spore germination and growth by Ma. sciuri. a Microscopic images showing the inhibition or non-inhibition of Metarhizium and Beauveria spore germination by different bacteria. Fungal spores (5 × 106 conidia/ml) were co-incubated with bacterial cells (each at a final value of OD600 = 0.01) in LB broth for 12 h (M. robertsii) or 16 h (B. bassiana) prior to imaging. Spore germination in LB was used as a mock control. Ungerminated fungal spores are arrowed. Bar, 5 μm. b Confrontation test of bacterial inhibition or non-inhibition of fungal growth for different times. Fungi were inoculated with 1 μl of spore suspensions (1 × 106 conidia/ml each). The bacterial strips were prepared by soaking the filter-paper strips in bacterial cells (OD600 = 5) for 30 sec before inoculation. The strips without bacterial cells were used as mock controls. DPI, day post inoculation. Figure S5. PCR verification of the presence of different bacteria. a Verification of the specific PCR primers for detecting Ma. sciuri (Ma. sc). Different isolates of Ma. sciuri are as shown in Table S1. The bacterial species Glutamicibacter mishrai (G. mi), Enterobacter asburiae (E. as), and Agrobacterium larrymoorei (A. la) were included as negative controls. b Verification of the presence of Ma. sciuri on mulberry leaves and leaf-fed silkworms. c Verification of the absence of Ma. sciuri in Silkmate fodder and fodder-fed silkworms. L4S, slough of the 4th instar larvae. d PCR verification of the symbiotic presence of Ma. sciuri in the 5th instar silkworms fed with the bacterium-free fodder. The insects were fed with mulberry leaves till the end of the 4th instar. e Images showing the silkworms conventionally reared (CR) with mulberry leaf and axenically reared with sterile Silkmate fodder. f, g Verification of the obtained axenic silkworm larvae by PCR of bacterial 16S rDNA (f) and plating of insect homogenates for bacterial colony formation (g). The CR silkworms were used as a positive control. Figure S6. Pretreatment of silkworm diets using the Ma. sciuri cells has no negative effect on insect development. a, b Feeding of the 5th instar larvae with the mulberry leaves soaked in Ma. sciuri cells (from 0.01 – 5 OD600 in sterile BPS buffer) has no obvious negative effect on the pupa (a) and cocoon (b) weight of silkworms. c, d Feeding of the 5th instar larvae with the artificial Silkmate fodder added with different amount of Ma. sciuri cells has no obvious negative effect on the pupa (c) and cocoon (d) weight of silkworms. One-way ANOVA analysis was conducted: ns, not significant. Table S1. Primers used in this study. Table S2. Isolation of cuticular bacteria from silkworm larvae. Table S3. Mass spectrometry detection of culture filtrate proteins secreted by Ma. sciuri.