Strains and crpksgene
C rosea 67-1 was originally isolated from a vegetable plantation in Hainan Province, China. S. sclerotiorum was isolated from soybean affected by Sclerotinia white mold in Heilongjiang Province (Zhang et al. 2004). All strains are preserved in the Biocontrol of Soilborne Diseases Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.
The polyketide synthases encoding gene crpks was isolated from the genome of C. rosea 67-1 and its sequence was deposited in GenBank under accession number KY701733.
Gene cloning and bioinformatics analysis
Full-length crpks was cloned from the genome of C. rosea 67-1 using the PSneiF and PSneiR primer pair (Table 1) with 25 μL reaction system contained 2 μL DNA template, 1 μL PSneiF and PSneiR primer pairs, 12.5 μL PCR mix and 8.5 μL ddH2O. The PCR program was conducted as follows: 94°C for 3 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 3 min, with a final extension at 72°C for 10 min.
The molecular weight and isoelectric point of the Crpks protein were calculated using the ExPASy program. The hydrophobicity profile, signal peptide and transmembrane regions prediction were investigated using the ProtScale program, Signal 4.1 procedure and TMPRED software, respectively (Hofmann and Stoffel 1993; Gasteiger et al. 2005; Petersen et al. 2011). The functional domains of Crpks were detected using antiSMASH fungal version (Blin et al. 2019). Sequences that were closely similar with Crpks were identified by NCBI Blast searches and used for construct phylogenetic tree. Phylogenetic tree of Crpks was conducted by using MEGA version 5.1 with the neighbor-joining method under Kimura two-parameter model and bootstrap values were based on 1000 replications (Saitou and Nei 1987).
Gene knockout and validation
The pKH-KO plasmid, containing a trpc promoter, a hygromycin B resistance gene, and two uracil-specific excision reagent cloning sites, was used for construction of the crpks knockout vector as previously described (Sun et al. 2018), with the PUF/PUR and PDF/PDR primer pairs used for amplification of the upstream and downstream regions (Table 1). Knockout vector was verified using electrophoresis and DNA sequencing.
Transformation of vectors into C. rosea was conducted as previous described with minor revised (Sun et al. 2017). Fresh C. rosea spores were incubated on potato dextrose (PD) broth and cultivated at 26 °C on a rotary shaker with the speed of 180 rpm for 12 h. Then mycelium of C. rosea were collected by a 125-μm sterile sieve, and then washed several times with sterile distilled water and 0.7 mol/L NaCl. The cell wall of C. rosea mycelium was hydrolyzed with 40 mg/mL snail enzymes for 3 h on a rotary shaker with the speed of 100 rpm at 28 °C. The protoplasts were filtered with a sterile microfiber filter and then collected by centrifugation at 1500×g for 10 min. Finally the protoplasts were kept in STC buffer (50 mL 1 mol/L Tris-HCl with pH value of 8.0, 200 g sucrose, 5.55 g of CaCl2 in 1 L distilled water) with the concentration of 107 protoplasts/mL.
Knockout vector was gently added into protoplasts suspension of C. rosea and placed on ice for 20 min. Then added PTC solution (10 mL 1 mol/L Tris-HCl with pH value of 8.0, 20 mL 2.5 mol/ L CaCl2, and 400 g polyethylene glycol 4000 in 1L distilled water) and keep room temperature for 20 min. The mixture were then incubated into TB3 (200 g sucrose, 3 g yeast extract and 3 g casein acid hydro-lysate in 1 L distilled water) and cultured on a rotary shaker with the speed of 100 rpm at 26 °C for 16 h. Then the mixture was centrifuged at 2500×g for 10 min and the precipitation was suspended in STC buffer. Finally the suspension was mixed into TB3 plate with 300 μg mL-1 of hygromycin B and cultivated at 26 °C for 3-7 days. Mutants obtained from TB3 plates were transferred into potato dextrose agar (PDA) plates containing 300 μg/ml hygromycin B for screening, and then cultured for three generations to verify genetic stability.
Genomic DNA of mutants was extracted using a Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Ltd, Hangzhou, China). PCR amplification was performed to validate positive mutants using the PSneiF and PSneiR primer pair for which it was not possible to amplify the crpks open reading frame (ORF), FPSF and FPSR primer pair for which it can amplify the fragments beyond upstream and downstream regions (-3893 bp) were considered to positive deletion mutants designated as ∆crpks.
Fresh spores of the wild-type strain (WT) and the positive mutant with the equal concentration were inculcated and cultured on PDA plates at 26 °C for 1 week. The morphology of colonies and mycelia were observed, and the diameter of colonies was measured using Vernier calipers. Fresh spores of the WT and the positive mutant with the equal concentration were inoculated into PD broth and cultivated at 26 °C for 36 h on a rotary shaker with the speed of 180 rmp. Then 2% fermentation broth was transferred into chlamydospores production medium as previously described (Sun et al. 2018). Chlamydospores were collected after 48 and 72 h and counted under a microscope. The experiment was conducted with three replicates.
Mycoparasitism S. sclerotiorum sclerotia
S. sclerotiorum sclerotia were collected as previously described (Sun et al. 2015c). Sclerotia were surface-sterilized with 1% NaClO for 3 min, and immersed in C. rosea spore suspensions (107 spores/ml) for 10 min. Then sclerotia were placed on sterile filter paper for moist cultured in a Petri dish at 26°C for 7 days. The severity of Sclerotia parasitism was investigated on a 4-grade scale: 0 = no C. rosea was detected on the surface of sclerotia; 1 = rare hyphae of C. rosea was observed on sclerotia; 2 = sclerotia were covered with hyphae of C. rosea but keep firm; 3 = sclerotia were covered with hyphae of C. rosea and exhibit softening. The experiment was conducted with three replicates.
Biocontrol efficacy against soybean Sclerotinia white mold
Field trials of biocontrol efficacy were performed in soybean plants at the 9-compound leaves stage of development. Leaves were sprayed with C. rosea spore suspensions (5×106 spores/ml) and then with an equal volume of S. sclerotiorum mycelial suspension after 2 h. Carbendazim and sterile distilled water were used as positive and negative controls, respectively. The disease index of soybean Sclerotinia white mold was investigated after 7 days using a previously defined 9-grade scoring system: 0 = no symptoms; 1 = less than 5%; 3 = 5–10%; 5 = 11–25%; 7 = 26–50%; 9 = over 50%. The experiment was conducted with three replicates.
The statistical software SAS 9.1.3 (SAS Institute Inc., Cary, NC, USA) was used for analysis of variance. Duncan’s multiple range test was used to compare the means of biocontrol efficacy of C. rosea against soybean Sclerotinia white mold. t-tests were used to compare the means of other treatment groups. P < 0.05 was considered to indicate statistical significance.