Sclerotinia sclerotiorum Agglutinin Modulates Sclerotial Development, Pathogenicity and Response to Abiotic and Biotic Stresses in Different Manners

Sclerotinia sclerotiorum is an important plant pathogenic fungus of many crops. Our previous study identified the S. sclerotiorum agglutinin (SSA) that can be partially degraded by the serine protease CmSp1 from the mycoparasite Coniothyrium minitans. However, the biological functions of SSA in the pathogenicity of S. sclerotiorum and in its response to infection by C. minitans, as well as to environmental stresses, remain unknown. In this study, SSA disruption and complementary mutants were generated for characterization of its biological functions. Both the wild-type (WT) of S. sclerotiorum and the mutants were compared for growth and sclerotial formation on potato dextrose agar (PDA) and autoclaved carrot slices (ACS), for pathogenicity on oilseed rape, as well as for susceptibility to chemical stresses (NaCl, KCl, CaCl2, sorbitol, mannitol, sucrose, sodium dodecyl sulfate, H2O2) and to the mycoparasitism of C. minitans. The disruption mutants (ΔSSA-175, ΔSSA-178, ΔSSA-225) did not differ from the WT and the complementary mutant ΔSSA-178C in mycelial growth. However, compared to the WT and ΔSSA-178C, the disruption mutants formed immature sclerotia on PDA, and produced less but larger sclerotia on ACS; they became less sensitive to the eight investigated chemical stresses, but more aggressive in infecting leaves of oilseed rape, and more susceptible to mycoparasitism by C. minitans. These results suggest that SSA positively regulates sclerotial development and resistance to C. minitans mycoparasitism, but negatively regulates pathogenicity and resistance to chemical stresses.


Introduction
Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan plant pathogenic fungus with a wide range of host plants, including oilseed rape (Brassica napus L.), soybean (Glycine max Merr.) and sunflower (Helianthus annuus L.), causing huge economic losses for production of these crops [1,2]. It produces sclerotia, which can survive for a long time in soil or in plant debris [3]. Under humid and low temperature conditions, the sclerotia germinate to form apothecia, where ascospores are produced and discharged into the air, and finally spread onto plant tissues to initiate infection [4,5]. As a necrotrophic pathogen, the ascospores of S. sclerotiorum usually rely on extracellular nutrients (e.g., senescent flower petals, plant exudates from wounds) to germinate, and consequently, the germ tubes grow to form mycelia and infection cushions (e.g., appressoria-like structures) to cause infection on plant leaves, stems, pods, fruits and seeds [6,7].
Fungi have the capacity to adapt to various environmental stresses, such as oxidative and osmotic stresses in their life cycles. The cell wall of fungi can maintain cell morphology and protect cells from damage by biotic and abiotic stresses. Fungi sense environmental stresses through the cell wall integrity (CWI) signaling system, transmit the signals to the complementary mutant ∆CmSp1C [34]. The cultural media used in this study included potato dextrose agar (PDA) made of fresh potato, and autoclaved carrot slices (ASC) made of fresh carrot tubers (100 g in each 250-mL glass flask). PDA was used to incubate C. minitans and S. sclerotiorum, and ASC was used to incubate S. sclerotiorum alone for the production of sclerotia.

Extraction of DNA/RNA and cDNA Synthesis
The mycelium grown for 2 d on cellophane film overlays on PDA was collected. Genomic DNA (gDNA) was extracted from the mycelial samples of each fungal strain or mutant using the CTAB method [35]. The total RNA was extracted also from the mycelia of each strain or mutant using Trizol ® reagents (Invitrogen, Carlsbad, CA, USA). The RNA was reverse-transcripted into cDNA with the reagents in the PrimeScript RT Reagent Kit with a gDNA Eraser (TaKaRa, Dalian, China), using the protocol recommended by the manufacturer; the resulting cDNA was used for detecting the expression of SSA with the primer pair SSAf/SSAr (Table S1).

Disruption of SSA
For the disruption of SSA, the full length of SSA and its flanking sequences was downloaded from NCBI (https://www.ncbi.nlm.nih.gov/ (GenBank Acc. No. CP017814.1). The upstream and downstream DNA sequences of that gene were PCR-amplified using the primer pairs SSA-HyF/R and SSA-ygF/R, respectively, with the gDNA of WT of S. sclerotiorum as the template (Table S1). The resulting DNA sequences were separately ligated to the hygromycin gene vector pUCH18 [36], the inserted DNA fragments were PCRamplified using the primers SSA-HyF/Hy-R and gR-F/SSA-ygR, using the recombinant plasmid as the template (Table S1), and the resulting DNA amplicons were sequenced for validation of insertion accuracy. Then, the amplified fragments were transformed into the protoplasts of WT of S. sclerotiorum using polyethylene glycol (PEG) to replace the SSA gene with the hygromycin gene (Hyg) [33]. The protoplasts were plated on the protoplast regeneration medium TB3 [37], and the emerging fungal colonies were individually picked out and incubated on PDA amended with hygromycin B (50 µg/mL) for PCR identification. Disruption of SSA in six mutants (∆SSA-138, ∆SSA-175, ∆SSA-178, ∆SSA-179, ∆SSA-181, ∆SSA-225) was confirmed using Southern blotting. The gDNA from these mutants as well as the WT was digested with Bgl II; the DNA fragments were separated via agarose gel electrophoresis, transferred to a piece of nylon membrane film, and detected using a Biotin-labeled P1 probe with the procedure recommended by the manufacturer (GE Healthcare, Amersham Biosciences, Buckinghamshire, UK).

Complementation of SSA
For in situ complementation of the SSA-deletion mutant ∆SSA-178, the SSA upstream and downstream fragments in S. sclerotiorum WT were PCR-amplified using primers SSA-UpF/R and SSA-DownF/R (Table S1), respectively, and separately ligated to the neomycin vector pGNW containing the neomycin resistance gene (Supplementary Figure S1). The inserted DNA fragments were PCR-amplified using primers SSA-UpF/NeoR and NeoF/SSA-DownR (Table S1), using the recombinant plasmid DNA as the template; the resulting amplicons were then sequenced for validation of sequence accuracy. They were used to transform the protoplasts of ∆SSA-178 with the aid of PEG, the resulting protoplasts were plated on TB3 media for regeneration at 20 • C, and the fungal colonies were individually picked out and transferred to PDA amended with neomycin (50 µg/mL). They were identi-fied via PCR, and expression of SSA was detected via RT-PCR using the specific primer pairs listed in Table S1.

Determination of Mycelial Growth Rates and Sclerotial Formation
The WT, the SSA disruption mutants (∆SSA-175, ∆SSA-178, ∆SSA-225), and the complementary mutant ∆SSA-178C of S. sclerotiorum were separately inoculated on PDA in Petri dishes (9 cm in diameter) and on autoclaved carrot slices in 250-mL flasks. There were five dishes and five flasks for the WT, each mutant, and the complementary mutant. The PDA cultures were incubated at 20 • C for 24 and 48 h for observation of the colony diameter in each dish, and for 10 d for observation of sclerotial formation on each culture. The colony diameter data were used to calculate radial growth rates, expressed as mm per day (mm/d). The flask cultures were incubated at 20 • C for 20 days and the sclerotia in each flask were harvested by washing in water before being counted and weighed after air drying. For observation of sclerotial structure, sclerotinia obtained from ASC cultures were fixed and sliced, processed using the procedures described by Zhou and colleagues (2022) [37], and observed under a light microscope at 200× magnification.

Assay for Response to Chemical Stresses
The WT and the mutants (∆SSA-175, ∆SSA-178, ∆SSA-225, ∆SSA-178C) of S. sclerotiorum were separately inoculated on PDA alone (control) and on PDA amended with NaCl (0.5 mol/L), KCl (0.5 mol/L), CaCl 2 (0.5 mol/L), sorbitol (1 mol/L), mannitol (1 mol/L), sucrose (1 mol/L), sodium dodecyl sulfate (SDS, 0.1 mg/mL), or H 2 O 2 (3, 5, 10 mmol/L), with five dishes (replicates) for the WT or each mutant in each medium. The cultures were incubated at 20 • C in dark for 24 and 48 h, the diameter of the colony in each dish was measured, and the data of the two measurements for the WT or each mutant in each medium was used to calculate the mycelial growth rate (GR). The reduced growth rate (RGR) was calculated as follows: RGR (%) = 1 − GRT/GRC × 100, where GRT and GRC represent mycelial growth rates of the WT/mutants in the presence and absence of an investigated chemical, respectively.

Pathogenicity Test
Seeds of Brassica napus 'Zhongshuang No. 9' were sown in plastic pots filled with the potting mix (Zhengjiang Peilei Organic Manure Manufacturing Co., Ltd., Zhengjiang, China). The trays were maintained in a growth room (20 • C, 16 h light/8 h dark) for 10 d and watered as required. The seedlings in the pots were thinned to leave one in each pot, and were further incubated for 50 d. Young, fully expanded leaves were detached from the plants and placed in five rows on moisturized paper towels in a plastic tray (52 × 33 × 7 cm, length × width × height), with five leaves in each row. Mycelial agar plugs (5 mm in diameter) were removed from 1-day-old PDA cultures of the WT or each mutant and individually inoculated on the leaves, in a row, with mycelia on the agar plugs facing the leaves, one agar plug per leaf. The tray was covered individually with transparent plastic films and placed in the growth chamber (20 • C) for two days. The diameter of the leaf lesion around each agar plug was measured. The test was repeated four times.

Dual Culturing
Dual cultures were performed to determine the efficacy of the C. minitans WT (Chy-1), the disruption mutant ∆CmSp1, and the complementary mutant ∆CmSp1C of C. minitans in the mycoparasitic colonization of the colonies of WT, ∆SSA-178, and ∆SSA-178C, using the procedures described by Zeng and colleagues [33]. Briefly, the dual cultures were established via inoculation of C. minitans first in Petri dishes (9 cm in diameter) each containing 20 mL PDA amended with bromophenol blue (0.001%, w/v), 1 cm from the rim of the dishes. The cultures were incubated at 20 • C for 4 d; then, S. sclerotiorum was inoculated in these C. minitans cultures at a 7 cm distance from the inoculation point of C. minitans. There were seven to eight cultures (replicates) for each combination of C. minitans and S. sclerotiorum strains or mutants. The dual cultures were further incubated at 20 • C for 12 d. Areas colonized by S. sclerotiorum (yellow color) and C. minitans (blue color) in each dual culture were observed, and the size of the blue-colored area was recorded to indicate the mycoparasitic efficacy of C. minitans against S. sclerotiorum.

Data Analysis
A univariate procedure in SAS 8.1 software (SAS Institute, Cary, NC, USA) was used to analyze the data on sclerotial number and weight per flask in autoclaved carrot slices, and the data on relative growth rates on PDA alone or PDA plus the stress chemicals, as well as the size values of the C. minitans-colonized areas in the dual cultures. The means of each parameter for WT and each mutant in single cultures, as well as for Chy-1 + WT and each of the other combinations (Chy-1 + ∆SSA-178, Chy-1 + ∆SSA-178C, ∆CmSp1 + WT, ∆CmSp1 + ∆SSA-178), were compared using Student's t test at α = 0.05 or 0.01.

Identity of SSA
The SSA gene in S. sclerotiorum 1980 (sscle_01g001830) has an open reading frame (ORF) that is 652 bp long, with three introns and four exons; it encodes a protein with 153 aa, containing the RichB_lectin_2 domain from aa 45 to aa 135 (91 aa long). It was 100% identical to the SSA in S. sclerotiorum S1954 (GenBank Acc. No. ABE97202.1) (Figure 1), suggesting that the protein encoded by sscle_01g001830 is the agglutinin SSA (ABE97202.1), and sscle_01g001830 was herein designated as SSA.
using the procedures described by Zeng and colleagues [33]. Briefly, the dual cultures were established via inoculation of C. minitans first in Petri dishes (9 cm in diameter) each containing 20 mL PDA amended with bromophenol blue (0.001%, w/v), 1 cm from the rim of the dishes. The cultures were incubated at 20 °C for 4 d; then, S. sclerotiorum was inoculated in these C. minitans cultures at a 7 cm distance from the inoculation point of C. minitans. There were seven to eight cultures (replicates) for each combination of C. minitans and S. sclerotiorum strains or mutants. The dual cultures were further incubated at 20 °C for 12 d. Areas colonized by S. sclerotiorum (yellow color) and C. minitans (blue color) in each dual culture were observed, and the size of the blue-colored area was recorded to indicate the mycoparasitic efficacy of C. minitans against S. sclerotiorum.

Data Analysis
A univariate procedure in SAS 8.1 software (SAS Institute, Cary, NC, USA) was used to analyze the data on sclerotial number and weight per flask in autoclaved carrot slices, and the data on relative growth rates on PDA alone or PDA plus the stress chemicals, as well as the size values of the C. minitans-colonized areas in the dual cultures. The means of each parameter for WT and each mutant in single cultures, as well as for Chy-1 + WT and each of the other combinations (Chy-1 + ΔSSA-178, Chy-1 + ΔSSA-178C, ΔCmSp1 + WT, ΔCmSp1 + ΔSSA -178), were compared using Student's t test at α = 0.05 or 0.01.

Identity of SSA
The SSA gene in S. sclerotiorum 1980 (sscle_01g001830) has an open reading frame (ORF) that is 652 bp long, with three introns and four exons; it encodes a protein with 153 aa, containing the RichB_lectin_2 domain from aa 45 to aa 135 (91 aa long). It was 100% identical to the SSA in S. sclerotiorum S1954 (GenBank Acc. No. ABE97202.1) (Figure 1), suggesting that the protein encoded by sscle_01g001830 is the agglutinin SSA (ABE97202.1), and sscle_01g001830 was herein designated as SSA. . ★ and ▲ represent amino acids for the carbohydrate-binding and dimer assembly, respectively. Arrows indicate β-strands (β1 to β12).

Disruption and Complementation of SSA
The SSA gene in WT was replaced by the hygromycin gene (Hyg) (Figure 2A), and a total of 700 transformants showing the trait of hygromycin resistance were obtained. They were identified via PCR detection of the hygromycin resistance gene (Hyg) using the primer pair HYG-F/HYG-R (Table S1, Figure 2B), as well as the up and downstream regions of SSA using the primer pairs SSA-UpF/SSA-UpR and SSA-DownF/SSA-DownR, respectively. Six mutants (ΔSSA-138, ΔSSA-175, ΔSSA-178, ΔSSA-179, ΔSSA-181, ΔSSA-225) were finally obtained. They were further verified via PCR detection of the SSA ORF using the primer pair SSAF/SSAR (Table S1). The result showed that only the WT was detected to have the SSA ORF, whereas the six mutants did not show any positive detection of the SSA ORF ( Figure 2B).
Southern blotting with the probe P1 ( Figure 2A) indicated that the WT produced a single hybridization band of ~2.5 kb in size, whereas four of the six mutants (ΔSSA-175, and represent amino acids for the carbohydrate-binding and dimer assembly, respectively. Arrows indicate β-strands (β1 to β12).

Disruption and Complementation of SSA
The SSA gene in WT was replaced by the hygromycin gene (Hyg) (Figure 2A), and a total of 700 transformants showing the trait of hygromycin resistance were obtained. They were identified via PCR detection of the hygromycin resistance gene (Hyg) using the primer pair HYG-F/HYG-R (Table S1, Figure 2B), as well as the up and downstream regions of SSA using the primer pairs SSA-UpF/SSA-UpR and SSA-DownF/SSA-DownR, respectively. Six mutants (∆SSA-138, ∆SSA-175, ∆SSA-178, ∆SSA-179, ∆SSA-181, ∆SSA-225) were finally obtained. They were further verified via PCR detection of the SSA ORF using the primer pair SSAF/SSAR (Table S1). The result showed that only the WT was detected to have the SSA ORF, whereas the six mutants did not show any positive detection of the SSA ORF ( Figure 2B).
The disruption mutant ΔSSA-178 was transformed with the full-length ORF of SSA to complement SSA deficiency in that mutant. A mutant (ΔSSA-178C) showing neomycin resistance was obtained, and SSA was positively detected via PCR in ΔSSA-178C ( Figure  2D). Expression of SSA was detected using RT-PCR in WT as well as in ΔSSA-175, ΔSSA-178, ΔSSA-225, and ΔSSA-178C. The result showed that while WT and ΔSSA-178C had an expression of SSA, the remaining three disruption mutants (ΔSSA-175, ΔSSA-178, ΔSSA-225) had no detectable expression of SSA ( Figure 2E,F).     Figure 3A,B), and no significant differences were detected between WT and the mutants (p > 0.05). After incubation for 10 d, WT and ∆SSA-178C formed black mature sclerotia on the colonies and at the rim of the Petri dishes ( Figure 3A). However, the disruption mutants ∆SSA-175, ∆SSA-178, and ∆SSA-225 formed immature sclerotia with water drops on the sclerotial surface or sclerotial primordia in the colony center ( Figure 3B). After incubation for 15 d, some of sclerotia in the cultures of the three disruption mutants became black, indicating that maturation of the sclerotia in the cultures of the mutants was delayed, compared to those in the cultures of WT and the complementary mutant ( Figure 3B). mm/d ( Figure 3A,B), and no significant differences were detected between WT and the mutants (p > 0.05). After incubation for 10 d, WT and ΔSSA-178C formed black mature sclerotia on the colonies and at the rim of the Petri dishes ( Figure 3A). However, the disruption mutants ΔSSA-175, ΔSSA-178, and ΔSSA-225 formed immature sclerotia with water drops on the sclerotial surface or sclerotial primordia in the colony center ( Figure 3B). After incubation for 15 d, some of sclerotia in the cultures of the three disruption mutants became black, indicating that maturation of the sclerotia in the cultures of the mutants was delayed, compared to those in the cultures of WT and the complementary mutant ( Figure 3B). On autoclaved carrot slices (20 °C, 20 d), the WT and the complementary mutant ΔSSA-178C formed 103 and 89 sclerotia per flask, respectively, with the average sclerotial weight at 3.4 and 3.6 g per flask, respectively ( Figure 4A,C,D). The disruption mutants ΔSSA-175, ΔSSA-178, and ΔSSA-225 formed significantly (p < 0.01) fewer but larger sclerotia, with the average yield ranging from 60 to 68 sclerotia per flask, and the average sclerotial weight ranging from 4.7 to 5.3 g per flask ( Figure 4A,C,D). Interestingly, the cortex layer (outside) of the sclerotia formed by the WT and the complementary mutant was significantly thicker than that of the disruption mutants (ΔSSA-175, ΔSSA-178 and ΔSSA-225) ( Figure 4B,E). These results suggest that SSA plays an important role in the sclerotial development of S. sclerotiorum. On autoclaved carrot slices (20 • C, 20 d), the WT and the complementary mutant ∆SSA-178C formed 103 and 89 sclerotia per flask, respectively, with the average sclerotial weight at 3.4 and 3.6 g per flask, respectively ( Figure 4A,C,D). The disruption mutants ∆SSA-175, ∆SSA-178, and ∆SSA-225 formed significantly (p < 0.01) fewer but larger sclerotia, with the average yield ranging from 60 to 68 sclerotia per flask, and the average sclerotial weight ranging from 4.7 to 5.3 g per flask ( Figure 4A,C,D). Interestingly, the cortex layer (outside) of the sclerotia formed by the WT and the complementary mutant was significantly thicker than that of the disruption mutants (∆SSA-175, ∆SSA-178 and ∆SSA-225) ( Figure 4B,E). These results suggest that SSA plays an important role in the sclerotial development of S. sclerotiorum.

Effect of SSA Disruption on the Response of S. sclerotiorum to Chemical Stresses
The WT, the disruption mutants ΔSSA-175, ΔSSA-178, and ΔSSA-225, and the complementary mutant ΔSSA-178C grew rapidly on PDA alone at rates ranging from 22.1 to 22.3 mm/d. However, in the presence of the stress chemicals, the growth rates of the WT and the mutants were reduced to 2.2-17.0 mm/d ( Figure 5A). The results also showed that the stress chemicals had different effects on the WT, the complementary mutant, and the disruption mutants regarding the extent of growth rate reduction. On PDA amended with NaCl, sorbitol, mannitol, KCl, sucrose, CaCl2 and SDS, the average growth rates were reduced by 33%, 48%, 49%, 50%, 55%, 81%, and 90% (compared to their growth rates on PDA alone), respectively, for WT and ΔSSA-178C, whereas the growth rates were reduced by 24%, 39%, 32%, 43%, 43%, 77% and 82%, respectively, for ΔSSA-175, ΔSSA-178 and ΔSSA-225 ( Figure 5B). Statistical analysis showed that in response to each stress chemical, the disruption mutants had significantly lower (p < 0.05 or 0.01) percentages of growth rate reduction than those for WT and ΔSSA-178C ( Figure 5B), indicating that the SSA disruption mutants were less sensitive than the WT and the complementary mutant to the investigated chemical stresses.

Effect of SSA Disruption on the Response of S. sclerotiorum to Chemical Stresses
The WT, the disruption mutants ∆SSA-175, ∆SSA-178, and ∆SSA-225, and the complementary mutant ∆SSA-178C grew rapidly on PDA alone at rates ranging from 22.1 to 22.3 mm/d. However, in the presence of the stress chemicals, the growth rates of the WT and the mutants were reduced to 2.2-17.0 mm/d ( Figure 5A). The results also showed that the stress chemicals had different effects on the WT, the complementary mutant, and the disruption mutants regarding the extent of growth rate reduction. On PDA amended with NaCl, sorbitol, mannitol, KCl, sucrose, CaCl 2 and SDS, the average growth rates were reduced by 33%, 48%, 49%, 50%, 55%, 81%, and 90% (compared to their growth rates on PDA alone), respectively, for WT and ∆SSA-178C, whereas the growth rates were reduced by 24%, 39%, 32%, 43%, 43%, 77% and 82%, respectively, for ∆SSA-175, ∆SSA-178 and ∆SSA-225 ( Figure 5B). Statistical analysis showed that in response to each stress chemical, the disruption mutants had significantly lower (p < 0.05 or 0.01) percentages of growth rate reduction than those for WT and ∆SSA-178C ( Figure 5B), indicating that the SSA disruption mutants were less sensitive than the WT and the complementary mutant to the investigated chemical stresses.

Effect of SSA Disruption on the Response of S. sclerotiorum to H 2 O 2 Stresses
The WT, ∆SSA-175, ∆SSA-178, ∆SSA-225, and ∆SSA-178C grew rapidly on PDA alone at rates of about 22 mm/d; they colonized the entire dish after incubation for 48 h ( Figure 6A). In the presence of H 2 O 2 (3, 5, 10 mmol/L), however, growth of these strains was inhibited to 11.2 to 19.6 mm/d; as a result, the dishes were partially colonized by these strains (Figure 6A). In cultures with H 2 O 2 at 3 mmol/L, the growth rates of these five strains were reduced by~12% without significant difference (p > 0.05) between each mutant and WT in the average percentage of growth rate reduction ( Figure 6B). In cultures with H 2 O 2 at 5 and 10 mmol/L, the growth rates of WT and ∆SSA-178C reduced by 27% and 48%, respectively; the values were higher than those (19% and 36%) for the three disruption mutants, respectively ( Figure 6B). Statistical analysis indicated that under each concentration of H 2 O 2 , ∆SSA-178C did not significantly (p > 0.05) differ from WT in the value of growth rate reduction; however, the disruption mutants significantly differed from the WT in the value of growth rate reduction, indicating that the SSA disruption mutants were more resistant to H 2 O 2 than the WT and the complementary mutant.

Effect of SSA Disruption on the Response of S. sclerotiorum to H2O2 Stresses
The WT, ΔSSA-175, ΔSSA-178, ΔSSA-225, and ΔSSA-178C grew rapidly on PDA alone at rates of about 22 mm/d; they colonized the entire dish after incubation for 48 h ( Figure 6A). In the presence of H2O2 (3, 5, 10 mmol/L), however, growth of these strains was inhibited to 11.2 to 19.6 mm/d; as a result, the dishes were partially colonized by these strains ( Figure 6A). In cultures with H2O2 at 3 mmol/L, the growth rates of these five strains were reduced by ~12% without significant difference (p > 0.05) between each mutant and WT in the average percentage of growth rate reduction ( Figure 6B). In cultures with H2O2 at 5 and 10 mmol/L, the growth rates of WT and ΔSSA-178C reduced by 27% and 48%, respectively; the values were higher than those (19% and 36%) for the three disruption mutants, respectively ( Figure 6B). Statistical analysis indicated that under each concentration of H2O2, ΔSSA-178C did not significantly (p > 0.05) differ from WT in the value of growth rate reduction; however, the disruption mutants significantly differed from the WT in the value of growth rate reduction, indicating that the SSA disruption mutants were more resistant to H2O2 than the WT and the complementary mutant.

Effect of SSA Disruption on the Pathogenicity of S. sclerotiorum
In humid conditions (20 • C, 48 h), WT, ∆SSA-175, ∆SSA-178, ∆SSA-225, and ∆SSA-178C infected leaves of oilseed rape and formed necrotic lesions around the inoculation plugs ( Figure 7A). The WT and the complementary mutant formed lesions with average diameters of 22.2 and 22.9 mm, respectively; these measurements were significantly (p < 0.05 or 0.01) smaller than those of the lesions formed by the disruption mutants, which had average diameters ranging from 26.2 to 30.5 mm ( Figure 7B). This comparison suggests that SSA negatively regulates pathogenicity of S. sclerotiorum. J. Fungi 2023, 9, x FOR PEER REVIEW 11 of 16

Effect of SSA Disruption on the Pathogenicity of S. sclerotiorum
In humid conditions (20 °C, 48 h), WT, ΔSSA-175, ΔSSA-178, ΔSSA-225, and ΔSSA-178C infected leaves of oilseed rape and formed necrotic lesions around the inoculation plugs ( Figure 7A). The WT and the complementary mutant formed lesions with average diameters of 22.2 and 22.9 mm, respectively; these measurements were significantly (p < 0.05 or 0.01) smaller than those of the lesions formed by the disruption mutants, which had average diameters ranging from 26.2 to 30.5 mm ( Figure 7B). This comparison suggests that SSA negatively regulates pathogenicity of S. sclerotiorum.

Effect of SSA Disruption on the Resistance of S. sclerotiorum to Mycoparasitism by C. minitans
On PDA amended with the pH indicator bromophenol blue, dual cultures of S. sclerotiorum and C. minitans showed two contrasting colors (e.g., yellow and blue) which indicate colonization by S. sclerotiorum (e.g., production of oxalic acid) and C. minitans (e.g., degradation of oxalic acid), respectively, and the change of the color from yellow to blue in the dual cultures reflects the invasion of the S. sclerotiorum colonies by C. minitans [33]. The size of the blue area (BA) in the dual cultures varied not only with C. minitans WT Chy-1, ΔCmSp1, and ΔCmSp1C, but also with WT, ΔSSA-178, and ΔSSA-178C ( Figure 8A). The six types of dual cultures had the average BA size which accounted for 30% to 38% of the total size of the Petri dishes ( Figure 8B); the dual culture Chy-1 + ΔSSA-178 had the largest BA size (38%), whereas the dual culture ΔCmSp1 + WT had the smallest BA size (30%). This comparison suggests that the WT of C. minitans Chy-1 is more aggressive than ΔCmSp1 in invasion of the colonies of WT; the colonies of the disruption mutant ΔSSA-178 of S. sclerotiorum are less resistant than those of WT and ΔSSA-178C to invasion by C. minitans. Therefore, SSA positively affects the resistance of the colonies of S. sclerotiorum to mycoparasitism by C. minitans.

Effect of SSA Disruption on the Resistance of S. sclerotiorum to Mycoparasitism by C. minitans
On PDA amended with the pH indicator bromophenol blue, dual cultures of S. sclerotiorum and C. minitans showed two contrasting colors (e.g., yellow and blue) which indicate colonization by S. sclerotiorum (e.g., production of oxalic acid) and C. minitans (e.g., degradation of oxalic acid), respectively, and the change of the color from yellow to blue in the dual cultures reflects the invasion of the S. sclerotiorum colonies by C. minitans [33]. The size of the blue area (BA) in the dual cultures varied not only with C. minitans WT Chy-1, ∆CmSp1, and ∆CmSp1C, but also with WT, ∆SSA-178, and ∆SSA-178C ( Figure 8A). The six types of dual cultures had the average BA size which accounted for 30% to 38% of the total size of the Petri dishes ( Figure 8B); the dual culture Chy-1 + ∆SSA-178 had the largest BA size (38%), whereas the dual culture ∆CmSp1 + WT had the smallest BA size (30%). This comparison suggests that the WT of C. minitans Chy-1 is more aggressive than ∆CmSp1 in invasion of the colonies of WT; the colonies of the disruption mutant ∆SSA-178 of S. sclerotiorum are less resistant than those of WT and ∆SSA-178C to invasion by C. minitans. Therefore, SSA positively affects the resistance of the colonies of S. sclerotiorum to mycoparasitism by C. minitans.

Discussion
This study revealed that SSA in S. sclerotiorum 1980 is an agglutinin with 100% identity to SSA in S. sclerotiorum S1954 [21]. SSA in S. sclerotiorum was successfully disrupted, alongside three disruption mutants (ΔSSA-175, ΔSSA-178, ΔSSA-225). Moreover, one of the disruption mutants, namely ΔSSA-178, was complemented with the SSA gene, and the complementary mutant ΔSSA-178C was then generated. These mutants as well as the WT are essential research materials for evaluating the biological functions of SSA.
Agglutinins are glycoproteins; they are synthesized early in fungal growth and exported outside, accumulating on the hyphal cell wall where they are activated to perform certain biological functions [17]. Agglutinins can specifically recognize carbohydrates, thereby playing a cell-to-cell adhesion role, thus modulating cell differentiation, cell recognition, and interaction [16,38,39]. In the present study, we found that disruption of note the yellow color area colonized by S. sclerotiorum due to production of oxalic acid and the blue color area colonized by C. minitans due to degradation of oxalic acid. (B) Histogram showing the percentages of the C. minitans-colonized area. * and ** represent significant differences between the WT and an investigated mutant at p < 0.05 and p < 0.01, respectively, according to Student's t test.

Discussion
This study revealed that SSA in S. sclerotiorum 1980 is an agglutinin with 100% identity to SSA in S. sclerotiorum S1954 [21]. SSA in S. sclerotiorum was successfully disrupted, alongside three disruption mutants (∆SSA-175, ∆SSA-178, ∆SSA-225). Moreover, one of the disruption mutants, namely ∆SSA-178, was complemented with the SSA gene, and the complementary mutant ∆SSA-178C was then generated. These mutants as well as the WT are essential research materials for evaluating the biological functions of SSA.
Agglutinins are glycoproteins; they are synthesized early in fungal growth and exported outside, accumulating on the hyphal cell wall where they are activated to perform certain biological functions [17]. Agglutinins can specifically recognize carbohydrates, thereby playing a cell-to-cell adhesion role, thus modulating cell differentiation, cell recognition, and interaction [16,38,39]. In the present study, we found that disruption of SSA did not affect the mycelial growth of S. sclerotiorum on PDA; however, it affected sclerotial formation (through development) on PDA (through delayed sclerotial maturity) and on autoclaved carrot slices (it reduced sclerotial number, but increased sclerotial size and weight). It is well recognized that fungal sclerotia are a texturally hard, multicellular and nutrient-rich structure that can survive for a long time in adverse environments. Sclerotial development is a complicated process usually consisting of at least three stages, namely initiation, development, and maturation [40]. At the sclerotial initiation and development stages, fungal hyphal cells usually aggregate, and cell-to-cell recognition may become involved to form sclerotial primordia. This study observed that SSA disruption mutants were delayed for sclerotial maturity in the PDA cultures ( Figure 3A), and formed less but larger sclerotia than the WT and the complementary mutant in the carrot cultures ( Figure 4). These results suggest that SSA may become involved in sclerotial formation. On the other hand, the SSA disruption mutants were not completely blocked (suppressed) during sclerotial formation, implying that besides SSA, other agglutinins or signalling pathways may be involved in the sclerotial formation of S. sclerotiorum. We used the amino acid sequence of SSA to search other homologs in the genome of S. sclerotiorum 1980, and two homologs, namely XP001584968.1 and XP001587104.1, were identified ( Figure S2). This result implies that physical contact signaling may participate in the sclerotial development or maturation in S. sclerotiorum.
Previous studies have shown that S. sclerotiorum is a typical necrotrophic plant pathogen [41] as it owns multiple plant-attacking weapons such as cell wall-degrading enzymes (CWDEs) and oxalic acid (OA) [42]. This study found that the WT, SSA disruption mutants, and the complementary mutant caused necrotic lesions on leaves of oilseed rape ( Figure 7A). This result suggests that disruption of SSA did not completely eliminate the pathogenicity of S. sclerotiorum. The disruption mutants might retain the capability to produce CWDEs, as indicated by maceration of leaf tissues ( Figure 7A), and to produce OA, as shown by the presence of the yellow color in PDA cultures amended with bromophenol blue ( Figure 8A). However, compared to the WT and the complementary mutant, the SSA disruption mutants produced significantly larger (p < 0.05 or 0.01) leaf lesions on the leaves of oilseed rape. This result suggests that the disruption of SSA can enhance the aggressiveness of S. sclerotiorum in infecting leaves of oilseed rape. There are two possible reasons for this result: one is enhanced production of the pathogenesis-related chemical elements such as CWDEs, and the other one is reduced sensitivity to chemical, osmotic, and oxidative stresses from the leaves, as the disruption mutants became less sensitive to NaCl, KCl, CaCl 2 , sorbitol, mannitol, sucrose, SDS, and H 2 O 2 .
C. minitans is an obligate mycoparasite of S. sclerotiorum; it can attack both hyphae and sclerotia, resulting in sclerotial collapse and hyphal lysis, respectively [43,44]. Previous studies have shown that the mechanisms involved in mycoparasitism include production of extracellular enzymes, including chitinase, glucanases, and proteases [29,45], and elimination of OA toxicity via the degradation of OA [33]. Dual cultural assays have shown that invasion of the colonies of S. sclerotiorum by C. minitans causes the succession of S. sclerotiorum with C. minitans through the mycoparasitic interaction of C. minitans on S. sclerotiorum [46]. This study observed that C. minitans invaded the colonies of S. sclerotiorum with and without SSA in the dual cultures of the two fungi ( Figure 8A). This result suggests that SSA is probably not the key agglutinin for determining the mycoparasitic specificity of C. minitans. Interestingly, the colonies of the SSA disruption mutants showed more susceptibility than those of the WT and the SSA complementary mutant to the C. minitans invasion, implying that SSA may positively modulate resistance to the mycoparasitism of C. minitans.
In summary, this study obtained three SSA disruption mutants (∆SSA-175, ∆SSA-178, ∆SSA-225) and the complementary mutant ∆SSA-178C. On PDA, the disruption mutants did not differ from WT and ∆SSA-178C in their growth rate, but they did affect sclerotial development. On autoclaved carrot slices, they formed fewer but larger sclerotia than WT and ∆SSA-178C. The disruption mutants became less sensitive to seven chemical stresses and H 2 O 2 stresses; however, they became more aggressive in infecting leaves of oilseed rape, and more susceptible to mycoparasitism by C. minitans, compared to WT and ∆SSA-178C. Therefore, SSA positively regulates sclerotial development and resistance to mycoparasitism by C. minitans, and negatively regulates pathogenicity and responses to abiotic environmental stresses.