The Involvement of the Laccase Gene Cglac13 in Mycelial Growth, Germ Tube Development, and the Pathogenicity of Colletotrichum gloeosporioides from Mangoes

Colletotrichum gloeosporioides is one of the most serious diseases that causes damage to mangoes. Laccase, a copper-containing polyphenol oxidase, has been reported in many species with different functions and activities, and fungal laccase could be closely related to mycelial growth, melanin and appressorium formation, pathogenicity, and so on. Therefore, what is the relationship between laccase and pathogenicity? Do laccase genes have different functions? In this experiment, the knockout mutant and complementary strain of Cglac13 were obtained through polyethylene glycol (PEG)-mediated protoplast transformation, which then determined the related phenotypes. The results showed that the knockout of Cglac13 significantly increased the germ tube formation, and the formation rates of appressoria significantly decreased, delaying the mycelial growth and lignin degradation and, ultimately, leading to a significant reduction in the pathogenicity in mango fruit. Furthermore, we observed that Cglac13 was involved in regulating the formation of germ tubes and appressoria, mycelial growth, lignin degradation, and pathogenicity of C. gloeosporioides. This study is the first to report that the function of laccase is related to the formation of germ tubes, and this provides new insights into the pathogenesis of laccase in C. gloeosporioides.


Introduction
The phytopathogen C. gloeosporioides is widespread and has infected more than 470 plants [1], such as pear (Pyrus spp.) [2], apple (Malus domestica) [3], and strawberry (Fragaria × Ananassa) [4]. It also infects many tropical fruits, such as mango (Mangifera indica) [5], banana (Musa spp.) [6], and papaya (Carica papaya) [7], as well as some tropical cash crops, such as rubber (Hevea brasiliensis) [8] and cassava (Manihot esculenta) [9]. Moreover, it results in the occurrence of anthracnose. C. gloeosporioides has infected the young fruit of plants with symptoms, such as blackening, irregular shapes, and slightly sunken spots on the fruit. With the gradual increase in the disease spots, the fruit eventually rots [10]. In general, C. gloeosporioides is a latent infection of the cuticle or epidermal cells in immature fruits, and it is not revealed until the fruit is ripe [11,12]. Mango anthracnose is one of the significant diseases of both the mango growth and post-harvest periods.
It is also the most ruinous of all mango diseases and is common in mango-producing areas in the world [10]. When the fruit is planted in high humidity, the incidence of mango anthracnose can be close to 100%, which significantly reduces the economic benefits in various industries and causes significant harm [10,13,14].
In the vast majority of cases, two methods of infection have been identified for the phytopathogenic fungi: one involves mechanical force, and the other is via the secretion of secondary metabolites, such as various cell-wall hydrolases, to promote infection [15]. C gloeosporioides forms germ tubes through the germination of conidia attached to the host surface. Then, the extension top of the germ tubes expands to produce appressoria, and the melanocytes become swollen and form infection nails, invading the host [16]. The germ tube, as one of the infectious structures differentiated during the process of C. gloeosporioides infection [17], usually plays an important role.
Moreover, in Candida albicans, the formation of germ tubes is a sign of the transformation of colonized strains to pathogenic strains, and this can also improve their invasiveness and adhesion in the tissues [18]. In the black-spot disease Dendrobium, the germ tube of its pathogen invades the dendrobium and secretes toxins and enzymes, destroying the tissues, producing a large number of mycelia, and diffusing to the tissues around the stomata to form a mycelium, which then causes tissue disintegration [19]. Beauveria brongniartii penetrates the body wall of the poplar trunk-elephant larva (Cryptorhynchus lapathi) in the form of a germ tube or mycelium, and then it produces pathogenic effects on the larva [20]. Therefore, it follows that germ tubes play a role in a variety of pathogens [17][18][19][20].
Laccase, a copper-containing polyphenol oxidase [21], exists in the genome as a gene family and has been reported in several species [22]. At present, fungal laccase has been the most widely studied, and it has a variety of functions related to melanin formation, appressoria penetration, pathogenicity, growth rate, etc. Its role in different pathogenic plant fungi, and even within the same pathogen, has varied [23,24]. Lignin is one of the main components of lignocellulose [25]. It enhances the rigidity and hydrophobicity of plant cell walls [26] and is an important barrier against pests and diseases [27]. Genes such as PAL (phenylalanine ammonia lyase), 4CL (4-coumaric acid-coenzyme ligase), CAD (cinnamon alcohol deoxidase), and COMT (caffeic acid-O-methyltransferase) play important regulatory roles in the lignin-synthesis pathway [28].
As a recognized lignin-degrading enzyme, laccase plays an important role in the pathogenesis process [29]. It destroys the lignin structure of plant cell walls, thus, opening the first line of defense for pathogen infection [29]. It also oxidizes phenolic and non-phenolic substances produced by the host during the pathogenesis process and promotes the colonization and expansion of fungi in the host tissues [29]. To date, many studies have reported that laccase is related to the pathogenicity of many plant pathogens. In Cryphonectria parasitica, laccase was related to its pathogenicity and the synthesis of melanin [30,31].
In Colletotrichum tuberculosis, loss pathogenicity occurred after knocking out the laccase gene LAC2 [32]. In Setosphaeria turcica, melanin synthesis was hindered after StLAC1 mutation, and the pathogenicity was significantly lower than that in the wild-type [33]. StLAC2 participated in the regulation of lignin degradation and affected the infection process of S. turcica [34,35]. In previous studies, we found that laccase participated in the growth regulation of mycelia, which were continuously produced by the germ tubes, forming filamentous or tubular mycelia [36].
Nikita et al. found that 11,050 genes in C. gloeosporioides were differentially expressed in the process of germ tube formation by whole transcriptome analysis, which encoded cell-wall-degrading enzymes, germination, mycelial growth, host-fungus interaction, and virulence [37]. Among them, pectin lyase, glycosyl hydrolase family 76, and glucosemethanol-choline (GMC) oxidoreductase genes were highly up-regulated during this process [37]. Li et al. confirmed that the expression of the pectin lyase gene was related to laccase in C. gloeosporioides [38].
As a member of the glycosyl hydrolase family, laccase could slightly inhibit the production of cellulase, according to Hernández et al. [39]. Aryl alcohol oxidase (AAO) belongs to the GMC oxidoreductase superfamily [40]. The products released from its oxidation of benzyl alcohol interacted with laccase to degrade linocellulose and also inhibited the re-polymerization of laccase oxidation products [40,41]. Therefore, we speculated that laccase was indeed related to germ tubes.
Based on this conjecture, we chose the laccase gene family in C. gloeosporioides as the research object in order to observe the effect of laccase gene deletion on germ tube formation and reveal the role of the laccase gene in C. gloeosporioides. This could provide a basis for analyzing the pathogenic mechanism of C. gloeosporioides.

Strains and Culture Conditions
The wild-type strain A2 (No. CATAS-A2) used in this study was provided by Laboratory 405, Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences. It was obtained from the Baodao Xincun Plant Base, Danzhou City, Hainan Province, in May 2006 [36].
The strains involved in this experiment were cultured in darkness at 28 • C in PDA (potato 200 g, glucose 20 g, agar 16 g, and adding water to 1 L); PDA containing 0.04% guaiacol (used to observe extracellular laccase enzyme activity); 0.1% H 2 O 2 (used to analyze the antioxidant capacity of strains), 0.4 M CaCl 2 , 1 M NaCl, and 1 M KCl (used to analyze the effects of hypertonic stress on strains) [42]; and 30.0 g/L saccharose, 15.8 g/L glucose, and 30.0 g/L soluble starch, which were substituted for sucrose in Czapeck (CZA, NaNO 3 3 g, K 2 HPO 4 1 g, MgSO 4 0.5 g, KCl 0.5 g, FeSO 4 0.01 g, sucrose 30 g, and added water to 1 L, used to determine the utilization of carbon and nitrogen sources by strains) by equal weight. Similarly, 3.5 g/L L-glutamine, 1.9 g/L ammonium nitrate, and 3.1 g/L ammonium sulfate were substituted for NaNO 3 in CZA by equal mass. In addition, the colony diameters were measured and statistically analyzed.

Extraction of Plasmids, DNA, RNA, and Expression Levels
The centrifugation of the Escherichia coli strain DH5α solution cultured the knockout and complementary vector in LB medium (tryptone 10 g, yeast extract 5 g, NaCl 10 g, add water to 1 L, and adjust pH of the medium to 7.0) and extracted the plasmid according to the instructions of the Plasmid Mini Kit I (OMEGA, Norcross, GA, USA). We scraped the mycelium on the PDA plate and placed it into a 2 mL round-bottom centrifuge tube (after placing three steel balls into the centrifuge tube in advance).
Then, we placed it into a grinder to grind and extract DNA according to the steps in the instructions of the Fungal gDNA Kit (OMAGA, Norcross, GA, USA). Next, we placed the treated blades in a prepared mortar and ground the material into a fine powder with liquid nitrogen. We then extracted the total RNA, according to the RNAprep pure plant total RNA extraction kit (centrifugal column type) (TIANGEN, Beijing, China) and observed the imaging with agarose gel electrophoresis. We also used TIANScript cDNA First-Stand Kit (TIANGEN, Beijing, China) to reverse transcribe the cDNA.
For the leaves used in the expression measurement, we first selected the light-green mango leaves that were consistent in the variety (Tainung No. 1) and health status, soaked them in 1% NaClO solution for 5 min, rinsed them with sterile water three times, and then placed them on a sterile operation platform to dry. We gently pricked the leaves with sterilized needles, and then added 3.14 × 10 7 conidium/mL conidia suspension and moisturizing culture to the wound under dark conditions.
We observed the infection site at 0, 6, 12, 24, 36, 48, and 72 h. Then, we quickly froze the sample in liquid nitrogen and stored it in a refrigerator at −80 • C until use. Each treatment was repeated three times. Using 18s rDNA as the internal reference gene, the relative expressions of Cglac13 during different time periods of infection were analyzed with primer Cglac13-qPCR-F/R by quantitative real-time PCR.

Primer Design
According to the obtained nucleotide sequence of the Cglac13 gene, we designed the primers in Primer 5.0 software. Cglac13-qPCR-F/R was used for qRT-PCR to analyze the expression of Cglac13, 5Cglac13-MHF/MHR, 3Cglac13-MHF/MHR, and hygB-F/R for knockout vector construction; 5Cglac13-CF/CR, 3Cglac13-CF/CR, and Bar-F/R (F4/R4) for complementary vector construction; and Cglac13H-F/R (F1/R1), H850/H852 (F2/R2), Cglac13 BAR-F/R (F3/R3), and Bar-F/R (F4/R4) for detection of the knockout and complementary mutants (Table 1). Table 1. The primers used in this study. All primers were designed by Primer 5.0 software, and the complete sequence of each primer is shown in Text S1. Among them, the F1/R1 primer pair and the F3/R3 primer pair were designed based on the nucleotide sequence of the knockout vector and the complementary vector, which were specific primers. The former contained the full length of hygB, while the latter contained the full length of the two segments of Cglac13 and Bar, and both had upstream and downstream homologous arms. The F2/R2 primer pair and the F4/R4 primer pair were used to detect whether the hygB and Bar gene fragments had been inserted into the mutant.

Identification of Cglac13
We submitted the amino acid sequence of Cglac13 to the National Biotechnology Information Center (NCBI) of the United States (https://blast.ncbi.nlm.nih.gov/, accessed on 10 November 2022), and then we used the protein basic local alignment search tool (BLASTP) to analyze the sequence. According to the similarity with the sequence of Cglac13, 30 amino acid sequences were screened.
The 30 amino acid sequences were derived from Xylaria multiplex Furthermore, we compared Cglac13 with other amino acid sequences containing the 30 amino acid sequences screened from NCBI and the sequences from published studies in this gene family, and then we made 1000 bootstrap replicates. The phylogenetic tree was constructed using the neighbor-joining method in MEGA 11.0, and we provide the details by website (https://itol.embl.de/, accessed on 14 November 2022).
We analyzed the DNA and cDNA sequences of Cglac13 using DNAssist 1.0 software; then, we uploaded the amino acid sequence of Cglac13 to the SWISS-MODEL online website (https://swissmodel.expasy.org/, accessed on 25 November 2022), which used a homologous modeling method to predict the three-dimensional structure of the protein.
Following a similar protocol, we submitted the sequence of Cglac13 to the CD-Search tool in NCBI to predict the conserved domain (https://www.ncbi.nlm.nih.gov/, accessed on 29 November 2022). In addition, we constructed a flowchart for the identification of Cglac13 as shown in Figure S2.

Construction of Plasmids and Acquisition of the Knockout Mutant and Complementary Strain
The resistance gene hygromycin (hygB) used in the experiment was derived from the vector pGH14, which was modified based on plasmid pCT74 [43]. The resistance gene glyphosate (Bar) was derived from the vector pCAMBIA3300. According to the nucleotide sequence of Cglac13, specific primers were designed to amplify the 5' fragments and 3' fragments. We used the DNA of a wild-type strain as a template, amplified the 5' fragments (445) and 3' fragments (458), and then designed primers based on the sequences of the pGH14 plasmid and amplified hygB fragments (1412). Next, we used the In Fusion cloning technique to construct the knockout plasmid [44].
For the complementary construct, a fragment consisting of 5' fragments (2140), the Bar gene (552), and 3' fragments (260) was sequenced using the In Fusion cloning technique to construct the complementary plasmid. All plasmids were propagated in the Escherichia coli strain DH5α. In this experiment, the correct knockout and complement vectors were used to obtain knockout mutant and complementary strains through PEG-mediated proto-plast transformation. The former was transferred into the wild-type, while the latter was transferred into the mutant and screened in the corresponding resistance plate.

Conidia Germination, Germ Tube Elongation, and Appressorium Formation
For the experiment with conidia, germ tubes, and appressoria, we used the wild-type, mutant ∆Cglac13H and the complementary strain C-∆Cglac13H in 100 mL potato dextrose broth (PDB) and cultured by shaking a conical flask for 3 days at 28 • C, at 180 rpm in the dark. We observed the quantity of conidia on the hemocytometer, and then we collected the conidia, diluted them with sterilized water, and adjusted them to 2 × 10 6 conidium/mL. The conidia suspension of each strain was placed on a glass slide, and we observed the changes in the conidia at 0, 2, 4, 6, 12, 24, and 36 h with a DM2500 Leica microscope (Leica, Wetzlar, Germany). We used cellSens Dimension 1.11 Software to calculate the conidia germination rate and appressoria formation rate. In addition, we observed the germ tubes of wild-type and mutants at 12 h, statistically analyzed the number and proportion of germ tubes in these two strains and imaged the results.

Pathogenicity Assays
In the pathogenicity observation, we selected fruits (firmness: 7 N·cm −2 ; color: L: 90.69; a: 4.83; b: 33.31; and TSS: 12.8%) that were consistent in the variety (Tainung No. 1) and health status, with a smooth surface, physiological maturity, and no damage. The specific disinfection method was the same as in Section 2.2. We used it to observe and measure the changes in pathogenicity after the Cglac13 knockout. The treatment method was similar to that of the leaves, with the exception of inoculating each strain cake on the wound. We inoculated the wild-type, mutant ∆Cglac13H, and complementary strain C-∆Cglac13H with the same cultivation time on the mango fruit, which were treated according to two groups, wounded and unwounded.
After inoculation, the fruits were stored at 28 • C, RH ≥ 95% for 3 days, and then we observed and measured the disease spots. We cut the infected position of the mango peel on the third day. We used the Lignin Content Detection Kit (Solarbio, Beijing, China) and QuantStudio 6 Flex (Applied Biosystems, Waltham, MA, USA) to determine the lignin content and the expression of genes related to lignin biosynthesis of the wild-type, mutant ∆Cglac13H, and complementary strain C-∆Cglac13H.

Statistical Analysis
All the data were processed in IBM SPSS Statistics 26, and we used the One-Way ANOVA test for analysis. We assumed equal variance by the selected Duncan's method (p < 0.01). In this study, uppercase letters indicate an extremely significant difference (p < 0.01), and lowercase letters indicate a significant difference (p < 0.05).

Identification and Phylogenetic Analysis of Cglac13
The phylogenetic analysis results indicated that the Cglac13 protein and Colletotrichum spp. bilirubin oxidase protein KAI8207922.1 (a copper-containing polyphenol oxidase) were clustered on the same branch. Cglac13 is a homologous gene of the laccase genes LAC1, Cglac3, Cglac5, Cglac6, Cglac7, and Cglac10, which was studied at an earlier stage ( Figure 1A). DNAssist showed that the total length of Cglac13 was 1883 bp, the length of CDS was 1725 bp, and there were four exons and three introns in Cglac13 ( Figure 1B). Cglac13, with multiple Cu binding sites and a CuRO-3-BOD conserved domain, belonged to the cupredoxin superfamily ( Figure 1C,D). compared to the observations at 0 h, the relative expression levels of Cglac13 were from 8.66 to 19.07 of the study period. In summary, the Cglac13 gene had a positive response to the infection of mango by C. gloeosporioides.  These results indicated that Cglac13 is a copper-containing polyphenol oxidase. The expression levels of Cglac13 in the mango leaves at different time points of C. gloeosporioides infection were analyzed by qRT-PCR ( Figure 1E,F). The results showed that Cglac13 remained highly expressed throughout the infection process of C. gloeosporioides. As compared to the observations at 0 h, the relative expression levels of Cglac13 were from 8.66 to 19.07 of the study period. In summary, the Cglac13 gene had a positive response to the infection of mango by C. gloeosporioides.

Validation of Gene Knockout Mutant and Complementary Strain
The knockout and complement vectors of Cglac13 were constructed by homologous recombination (Figure 2A), and the knockout and complement transformants were obtained by polyethylene glycol (PEG)-mediated protoplast transformation. We used two primer pairs, F1/R1 and F2/R2, for the PCR amplification in order to detect the knockout mutant of the transformants. The F1/R1 primer pair amplified specific 2199 bp bands for the knockout mutant and knockout vector pCglac13H, which were lower than the wild-type bands, indicating that the Cglac13 gene had been successfully replaced by hygB ( Figure 2B).
At the same time, a specific bright band at 610 bp in the knockout mutant and the knockout vector pCglac13H was found in the amplified transformants of the F2/R2 primer pair, indicating that the hygB gene was successfully transferred to the transformants. Similarly, the PCR amplification of primer pairs F3/R3 and F4/R4 was used to detect the complementary transformants ( Figure 2B), and the complete electrophoretogram of these four pairs of primers is shown in Figure S1. In summary, the knockout mutant and complementary strain of Cglac13 were successfully obtained, and they were named ∆Cglac13H and C-∆Cglac13H, respectively.
The inoculated wild-type, the knockout mutant ∆Cglac13H, and the complementary strain C-∆Cglac13H were added to the culture media under different conditions to observe the mycelial growth in each strain ( Figure 2C,D). In the PDA plate culture containing guaiacol, there was an obvious red circle in the wild-type and complementary colonies but not in the knockout mutant ( Figure 2C). The results showed that, in the PDA media containing NaCl and KCl and in the CZA media containing ammonium nitrate and ammonium sulfate, the colony diameters of the knockout mutant, wild-type, and complementary strain had the largest differences, as compared to those in other conditions, and the colony diameters decreased by 51.81%, 42.93%, 46.03%, and 52.29%, respectively ( Figure 2C,D).
In the PDA medium containing 0.1% H 2 O 2 , the colony diameter of the mutant significantly increased ( Figure 2C,D). In addition, in the basic PDA medium, the PDA medium containing CaCl 2 and the other CZA media containing carbon and nitrogen sources, the colony diameter of mutant also decreased significantly compared to the wild-type.

Cglac13 Involves Conidial Germination and Appressoria Formation
We collected the conidia of the wild-type, knockout mutant ∆Cglac13H, and complementary strain C-∆Cglac13H. The conidia of the knockout mutant ∆Cglac13H germinated normally, produced germ tubes, and formed melanized appressoria normally. The germ tubes of the mutant were thinner and longer than those in the wild-type ( Figure 3A). The number and percentage of germ tubes in the mutant ∆Cglac13H and the wild-type were observed and counted at 12 h ( Figure 3B,C). Knockout mutant ∆Cglac13H and wild-type conidia germinated one, two, or three germ tubes.

Cglac13 Is Required for Pathogenicity
Concerning the plant pathogenic fungus, the pathogenicities of wild-type, mutant ∆Cglac13H, and complementary strain C-∆Cglac13H on the physiological maturity of the As compared to the wild-type, the percentage of germination from two to three germ tubes was significantly higher in the knockout mutant ( Figure 3C). The percentages of the knockout mutant and wild-type that germinated two germ tubes were 59.44% and 34.33%, respectively ( Figure 3C). During the process of germination, we observed that the germination rate of ∆Cglac13H was significantly higher than those of the wild-type and complementary strain ( Figure 3D). We observed the formation of appressoria under a microscope ( Figure 3E).
We found that appressoria began to form in the wild-type and complementary strain after 4 h, and the melanized, mature appressoria appeared 6 h afterwards. The appressorium formation rate in the wild-type reached 18.17%, while the germ tube of the knockout mutant continued to expand at this time, and few appressoria were formed. After 24 h, the appressorium formation rate of the mutant reached 20.13%. Then, the appressoria of the mutant gradually blackened and matured at 36 h. At this moment, the appressorium formation rate of the wild-type reached approximately 87.20%. The knockout of the Cglac13 gene delayed the appressorium formation of C. gloeosporioides and significantly reduced the formation rate ( Figure 3A,E). The results showed that Cglac13 regulated the conidia germination, the germ tube development, and appressorium formation in C. gloeosporioides.

Cglac13 Is Required for Pathogenicity
Concerning the plant pathogenic fungus, the pathogenicities of wild-type, mutant ∆Cglac13H, and complementary strain C-∆Cglac13H on the physiological maturity of the mango fruits were studied ( Figure 4A). The wild-type and complementary strain inoculated with stab wounds had significantly more damage to the pericarp tissue, while the disease spots inoculated with the knockout mutant were significantly smaller ( Figure 4B). In the puncture inoculation, the disease spot diameters of the knockout mutant were significantly lower than those in the wild-type ( Figure 4C). There were no significant differences in the diameters of the disease spot between the complementary strain and wild-type.
Without the puncture inoculation, the disease spot diameters of the knockout mutant were significantly smaller than those in the wild-type and complementary strain ( Figure 4D). As a recognized lignin-degrading enzyme, laccase was used to study the lignin content and the expression levels of the lignin-synthesis pathway and related genes in the mango peels inoculated on the third day. The lignin content of the knockout mutant was higher than in the wild-type and complementary strain with extremely significant differences ( Figure 4E). At the same time, the relative expression levels of the related genes in the mutant were also significantly higher than in the wild-type and complementary strain ( Figure 4F). The results suggest that Cglac13 participated in the degradation of lignin and significantly affected the pathogenicity of C. gloeosporioides with mangoes. shown in the illustration, the left side is the stab wound treatment, and the right side is the non-stab wound treatment. The circle shows the inoculated strains, which are wild-type, mutant, and complementary strain from top to bottom. (B) Virulence assays on mango fruits of the wild-type, mutant ∆Cglac13H, and complementary strain C-∆Cglac13H. The picture shows the disease spots on the 3rd, 4th, 5th and 6th days. (C) Statistical analysis of the spot diameter in wild-type, mutant ∆Cglac13H, and complementary strain C-∆Cglac13H, with stab inoculation (cm) (p < 0.01). (D) Statistical analysis of the spot diameter of wild-type, mutant ΔCglac13H, and complementary strain C-∆Cglac13H, without stab inoculation (cm) (p < 0.01). (E) The percentage content of lignin in mango peels inoculated in wild-type, mutant ∆Cglac13H, and complementary strain C-∆Cglac13H, on the third day (p < 0.01). (F) The relative expression of genes related to lignin synthesis in mango peel inoculated in the wildtype, mutant, and complementary strains, on the third day (p < 0.01). In this experiment, MiActin (JF737036.1) [45] was used as the reference gene, and the fruit peel inoculated in the wild-type was the control. We calculated the relative expression levels of MiPAL [46], Mi4CL (XM_044609362.1) [47], MiCAD (XM_044654805.1) [47], and MiCOMT (XM_044626972.1) on fruit peels in the (F) The relative expression of genes related to lignin synthesis in mango peel inoculated in the wildtype, mutant, and complementary strains, on the third day (p < 0.01). In this experiment, MiActin (JF737036.1) [45] was used as the reference gene, and the fruit peel inoculated in the wild-type was the control. We calculated the relative expression levels of MiPAL [46], Mi4CL (XM_044609362.1) [47], MiCAD (XM_044654805.1) [47], and MiCOMT (XM_044626972.1) on fruit peels in the inoculated mutant and complementary strain. (A, B, and C in the figure indicate extremely significant differences, while a, b, and c indicate significant differences.)

Discussion
Previous studies showed that the vegetative growth of hyphae is the prerequisite for producing conidia and infecting host cells in Magnaporthe oryzae [48]. The results of this experiment showed that the colony diameter of the knockout mutant ∆Cglac13H was significantly different from that in the wild-type in different media. With the exception of the significant increase in the colony diameter on the guaiacol and H 2 O 2 plates, the others were significantly decreased.
Among them, the difference in the colony diameter was the most obvious on the plate with KCl, NaCl, ammonium nitrate, and ammonium sulfate as substrates, indicating that, when compared to other hypertonic conditions, the tolerance of mutant ∆Cglac13H to KCl and NaCl was significantly lower, and the utilization rate of the nitrogen sources, such as ammonium nitrate and ammonium sulfate, was significantly reduced. The research results of Cañero et al. are consistent with the results of this study, where the laccase gene lcc3 of Fusarium oxysporum was involved in regulating the nutrient growth, carbon source metabolism, and oxidative stress [49]. In addition, the knockout of Shlac in Scleromitrula shiraiana had similar results [50].
Fungal laccase participated in regulating the morphogenesis, growth, and development of pathogens [29]. Compared to our previous research, the mycelial growth results of LAC1 [36] and Cglac13 on PDA are consistent. In the relevant stress experiments, LAC1 [36] and Cglac10 are similar to Cglac13. In addition, in the experiment on carbon and nitrogen source utilization, the results of LAC1 [36], Cglac10, and Cglac13 are consistent. Therefore, we conclude that Cglac13 is involved in the mycelial growth, the stress response, and the utilization of carbon and nitrogen sources in C. gloeosporioides.
Plant pathogen fungi, such as M. oryzae and C. gloeosporioides, could produce turgor through the appressoria, form a penetration peg, and invade the host, thus, causing plant disease [15,51]. We found that, compared to the wild-type, the appressorium formation rate and pathogenicity of the mutant ∆Cglac13H significantly decreased, which was consistent with the results of LAC1 and Cglac10 [36,52]. In addition, the knockout of the CgHOS2 [53], CgHSF1 [8], CgGa1 [54], CgCPS1 [55], CgOPT2 [56], and CgEnd3 [57] genes in C. gloeosporioides had the same results. This illustrates that Cglac13 significantly affected the appressorium formation and pathogenicity in C. gloeosporioides.
A germ tube is one of the infection structures of pathogenic plant fungi. In Metarhizium anisopliae, the virulent pr1 gene regulates appressorium development but does not regulate conidia germination and germ tube elongation [58,59]. In Verticillium dahliae, the knockout CPMO-1 and CPMO-2 genes reduced the number of microsclerotia and produced more germ tubes [60]. In C. gloeosporioides, the CgHOS2 [53] and PniiA-Cghsf1 [8] genes regulated the lengths of the germ tubes and the morphology of appressoria [8,53].
In an experiment on CgHSF1, Xuesheng Gao et al. found that the mutant PniiA-Cghsf1 weakened melanin synthesis by affecting the transcription of melanin-related genes and the formation of germ tubes and appressoria, thereby reducing the pathogenicity [8]. In contrast to these studies, the number of germ tubes after the deletion of Cglac13 was statistically analyzed in this experiment. We calculated the proportions of the wild-type, mutant ∆Cglac13H, and complementary strain C-∆Cglac13H producing one, two, or three germ tubes at 12 h.
We found that the Cglac13 gene regulated both the number and length of the germ tubes ( Figure 5), and, when compared to the knockout mutant, the wild-type showed decreases in these values by 42.24% and 63.10%, respectively. The results showed that Cglac13, as a member of the laccase gene family, was involved in the regulation of germ tube development.
Inappropriate temperature and relative humidity conditions can lead to the failure of conidia germination and delay the formation of appressoria. In this experiment, we speculated that the decrease in pathogenicity in the knockout mutant would be related to their formation of germ tubes, as this would promote the elongation of germ tubes and result in a decrease in the ability to form adherents and a delay in their formation time as well as a reduction in the success rate of the pathogen infection and, thus, the pathogenicity. The percentage refers to the proportion of one, two, or three germ tubes of conidium germination of the wild-type and Cglac13 gene knockout compared to the total number of germinated tubes. For the pathogenicity part, we used the type with the largest number of germination tubes in the wild-type and Cglac13 gene knockout conidia as examples. The number of conidium germinations with one germ tube in the wild-type was the largest, and the number of conidium germinations with two germ tubes in the Cglac13 gene knockout was the largest. "**" indicates an extremely significant difference compared to the wild-type (p < 0.01).

Conclusions
In this study, knockout mutant and complementary strains of Cglac13 were obtained through PEG-mediated protoplast transformation. Our results showed that Cglac13 was involved in regulating the formation of germ tubes and appressoria, mycelial growth, lignin degradation, and pathogenicity in C. gloeosporioides. For the first time, we proved that the laccase gene was related to the formation of germ tubes, and we provided new insights into the pathogenesis of laccase from C. gloeosporioides. In addition, we speculated that laccase could also be related to the formation of germ tubes in filamentous fungi other than C. gloeosporioides.
Author Contributions: Conceptualization, H.Z. and J.P.; methodology, M.Z., C.X., and H.Z.; validation, Q.T. and L.D.; formal analysis, C.X. and X.L.; investigation, X.L.; resources, H.Z.; data curation, C.X. and X.L.; writing-original draft preparation, H.Z. and M.Z.; writing-review and editing, Figure 5. Schematic model for the process variation of C. gloeosporioides infection in mango fruit caused by a Cglac13 gene knockout. The percentage refers to the proportion of one, two, or three germ tubes of conidium germination of the wild-type and Cglac13 gene knockout compared to the total number of germinated tubes. For the pathogenicity part, we used the type with the largest number of germination tubes in the wild-type and Cglac13 gene knockout conidia as examples. The number of conidium germinations with one germ tube in the wild-type was the largest, and the number of conidium germinations with two germ tubes in the Cglac13 gene knockout was the largest. "**" indicates an extremely significant difference compared to the wild-type (p < 0.01).
The infection of the pathogen in the host is affected by many factors, among which temperature and relative humidity are important environmental factors [61]. Inappropriate temperature and relative humidity conditions can lead to the failure of conidia germination and delay the formation of appressoria. In this experiment, we speculated that the decrease in pathogenicity in the knockout mutant would be related to their formation of germ tubes, as this would promote the elongation of germ tubes and result in a decrease in the ability to form adherents and a delay in their formation time as well as a reduction in the success rate of the pathogen infection and, thus, the pathogenicity.

Conclusions
In this study, knockout mutant and complementary strains of Cglac13 were obtained through PEG-mediated protoplast transformation. Our results showed that Cglac13 was involved in regulating the formation of germ tubes and appressoria, mycelial growth, lignin degradation, and pathogenicity in C. gloeosporioides. For the first time, we proved that the laccase gene was related to the formation of germ tubes, and we provided new insights into the pathogenesis of laccase from C. gloeosporioides. In addition, we speculated that laccase could also be related to the formation of germ tubes in filamentous fungi other than C. gloeosporioides.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.