Potato dextrose agar (pda)

The mangrove rhizosphere soil samples from one K. obovata tree and one A. ilicifolius tree were collected by the 1–2 mm soil tightly adhered to the 10–20 cm underground roots from East Harbour National Nature Reserve (Hainan, China) in April 13, 2018. The 5 g rhizosphere soil of each sample was put into a conical flask containing 100 ml sterile water and cultured by shock for 1 h. The soil suspension was diluted 100 times, and then 0.2 ml of the mixed suspension was added to the Potato Dextrose Agar (PDA media, purchased from HOPEBIO, China). The single colonies were toke from coated plate and then purified the single colony again. After purified, the isolates were inoculated in PDA media and incubated in a 28°C constant temperature incubator for 2–5 day until the mycelium growth was observed and the colony characteristics were documented. In this way, three fungal species were isolated from the rhizosphere soil of Kandelia obovata, and four fungal species were isolated from the rhizosphere soil of A. ilicifolius. The strains were then deposited at China National GeneBank (Qingdao), BGI-Qingdao, China.

All commercially available strains used in this study were purchased from China General Microbiological Culture Collection Center (CGMCC), including L. monocytogenes (CGMCC NO. 1.10753), E. faecium (CGMCC NO. 1.131), E. faecalis (CGMCC NO. 1.2135), S. aureus (CGMCC NO. 1.879), S. epidermidis (CGMCC NO. 1.4260), B. subtilis (CGMCC NO. 1.3358), A. baumanii (CGMCC NO. 1.6769), P. aeruginosa (CGMCC NO. 1.2421), P. stutzeri (CGMCC NO. 1.1803), P. fluorescens (CGMCC NO. 1.3202), E. coli (CGMCC NO. 1.2389), S. fiexneri (CGMCC NO. 1.1868), F. oxysporum (CGMCC NO. 3.6785), F. solani (CGMCC NO. 3.5840), F. graminearum (CGMCC NO. 3.3490), A. niger (CGMCC NO. 3.316), A. fumigatus (CGMCC NO. 3.5835) and A. ochraceus (CGMCC NO. 3.5830). Bacterial strains grew at 37 °C in nutrient broth (OXOID, Hampshire, UK) and fungal strains were cultured in potato dextrose agar (Hope Bio, Qingdao, China) at 28 °C, respectively.

Beauveria bassiana strains (BB Fafu-13 and BB Fafu-16) and I. fumosorosea (IF Fafu-1) used in this study were obtained from the fungal culture bank of Fujian Agriculture and Forestry University, Fuzhou, Fujian, China PR. The fungi were originally isolated from mycosed D. citri adults collected from a private citrus orchard in Rixi, a satellite town of Fuzhou, Fujian province, South Eastern part of China (26°21´8.88´´N, 119°16´9.52´´E). The fungal strains were sub-cultured on potato dextrose agar (PDA, Qingdao Hope Bio-Technology Co., Ltd. Qingdao, China), and stored at 25 °C in an incubator. All isolates (BB Fafu-13, BB Fafu-16 and IF Fafu-1) were identified morphologically using the taxonomy keys of Humber [38 ,39 ], and by using sequence data from the ITS region of nuclear rRNA (GenBank accession numbers MG844430 for BB Fafu-13, MG844431 for BB Fafu-16, and MG837716 for IF Fafu-1). The strains were selected for the current study based on the source and the findings of our previous studies [25 (link),40 (link)].

Fungi and bacteria associated with rhizosphere soils (within a 1 mm vicinity of the roots) were isolated using the traditional serial dilution technique [20 (link),51 (link)]. Afterward, 1 g of each rhizosphere soil sample was added to 99 mL of ddH₂O, and a 10-fold serial dilution was made from the suspension (10⁻³ to 10⁻⁶). Dilutions of 10⁻⁴ and 10⁻⁵ were used to isolate fungi, and 10⁻⁵ and 10⁻⁶ were used to isolate bacteria. One hundred microliters of each diluted suspension was spread evenly onto potato dextrose agar (PDA, Hopebio, Qingdao), malt extract agar (MEA, Huankai Microbiol, Guangzhou) and Czapek-Dox media (CDM, Huankai Microbiol, Guangzhou) (to isolate the fungi) or onto tryptic soy agar (TSA, Hopebio, Qingdao) (to isolate the bacteria). We added 0.02 g L⁻¹ tetracycline hydrochloride and 0.05 g L⁻¹ streptomycin sulfate to the PDA to eliminate bacterial contamination. The PDA plates were incubated at 28 °C in darkness for 3 days, and the TSA plates were incubated for 1–2 days until colonies appeared. The colonies were removed and transferred to fresh PDA or TSA plates for purification.

The F. graminearum strain W3008 was kindly provided by the College of Plant Science and Technology of Huazhong Agricultural University, China [59 (link)]. The strain was routinely cultured at 25 °C on potato dextrose agar (PDA, Hopebio, Qingdao, China) plates and was preserved in 20% disinfected glycerol at −80 °C for long-term storage [60 (link)]. Thymol (HPLC grade standard, purity > 98%, B21153, Shanghai yuanye Bio-Technology Co., Ltd. Shanghai, China) was dissolved in acetone into a 100 mg/mL stock solution, protected from light, and stored at 4 °C.

A. alternata was cultured in potato dextrose agar (PDA, Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) at 28 °C for two weeks. Afterward, the plates were flooded with sterile distilled water and then gently rubbed with a sterile glass spreading rod to release spores. Spore suspensions were filtered through four layers of sterile cheesecloth to remove mycelial fragments, and the spore concentration was adjusted to 1 × 10⁶ spores mL⁻¹ with the aid of a hemocytometer prior to use.