Biological Characteristics of Verticillium dahliae MAT1-1 and MAT1-2 Strains

Verticillium dahliae is a soil-borne plant pathogenic fungus that causes Verticillium wilt on hundreds of dicotyledonous plant species. V. dahliae is considered an asexually (clonal) reproducing fungus, although both mating type idiomorphs (MAT1-1 and MAT1-2) are present, and is heterothallic. Most of the available information on V. dahliae strains, including their biology, pathology, and genomics comes from studies on isolates with the MAT1-2 idiomorph, and thus little information is available on the MAT1-1 V. dahliae strains in the literature. We therefore evaluated the growth responses of MAT1-1 and MAT1-2 V. dahliae strains to various stimuli. Growth rates and melanin production in response to increased temperature, alkaline pH, light, and H2O2 stress were higher in the MAT1-2 strains than in the MAT1-1 strains. In addition, the MAT1-2 strains showed an enhanced ability to degrade complex polysaccharides, especially starch, pectin, and cellulose. Furthermore, several MAT1-2 strains from both potato and sunflower showed increased virulence on their original hosts, relative to their MAT1-1 counterparts. Thus, compared to MAT1-1 strains, MAT1-2 strains derive their potentially greater fitness from an increased capacity to adapt to their environment and exhibit higher virulence. These competitive advantages might explain the current abundance of MAT1-2 strains relative to MAT1-1 strains in the agricultural and sylvicultural ecosystems, and this study provides the baseline information on the two mating idiomorphs to study sexual reproduction in V. dahliae under natural and laboratory conditions.


Introduction
Sexual reproduction, a ubiquitous feature of the eukaryotic kingdom, can accelerate the adaptation to continuously changing ecological niches. It also allows the repair of random epigenetic or conventional genetic damage by recombination with homologous chromosomes [1,2]. However, approximately 20% of the fungal species reproduce only by asexual means, with no recognized sexual cycle [3,4]; and in Ascomycotina, nearly 40% of the taxa were deemed asexual [5]. Such strictly asexual organisms are thought to be less flexible than sexual organisms, relying solely on random mutations to adapt to changing environments; therefore, they are often considered evolutionary dead ends, may ensue. Therefore, a comparative analysis of the basic growth and pathogenic characteristics of strains carrying the two mating-type idiomorphs will provide important data for the genetic analysis of the two idiomorphs and to evaluate the possibility of sexual reproduction in V. dahliae.

Characteristics of V. dahliae MAT1-1 Strain P48 and MAT1-2 Strain P50
To obtain comparative information on the MAT1-1 and MAT1-2 strains, V. dahliae strain P48 with MAT1-1 and P50 with MAT1-2 isolated from potato in Inner Mongolia, China, were selected. The upper sides of the P48 and P50 cultures displayed white dense mycelia on the PDA medium that progressively darkened on the undersurface of the cultures as the melanized microsclerotia were produced, at about 7 days after incubation ( Figure 1A). During this interval, strain P50 produced more melanin than P48 ( Figure 1A). The conidia from both strains were hyaline, elongated in clusters on phialides borne in whorls on branched aerial hyphae ( Figure 1A), typical characteristics of V. dahliae [46].
A number of PCR primers have been designed to amplify loci or markers associated with race (D or ND phenotype) and mating type idiomorphs [35,[46][47][48]. The PCR assays indicated that strain P48 had the MAT1-1 marker and P50 the MAT1-2 marker, confirming that P48 and P50 represented strains with MAT1-1 and MAT1-2 idiomorphs, respectively ( Figure 1B). Strain P50 was also characterized as the ND pathotype and belonged to race 2, while strain P48 contained the race 2 marker, and it neither carried the D nor the ND marker, suggesting that both P48 and P50 were race 2 strains and that P50 likely is not able to cause the defoliating phenotype on hosts such as olive, cotton, and okra. The well-characterized, highly virulent defoliating strain Vd991 represents the D pathotype, belongs to race 2, and carries the MAT1-2 marker. It was used as a control in these experiments.
To detect the sequence conservation and variability of the two mating-type master regulatory genes in strains P48 and P50, the coding sequences of the MAT1-1-1 and MAT1-2-1 genes from these two strains were aligned with the corresponding genes from eight other sexually reproducing fungi, two of which were homothallic (Fusarium graminearum, Aspergillus nidulans) and six were heterothallic (Magnaporthe grisea, Aspergillus fumigatus, Neurospora crassa, Saccharomyces cerevisiae, Penicillium marneffei), along with those of the V. dahliae strain VdLs. 17, which has been used as the reference genome in many studies [43]. The results showed that both P48 and P50 had the conserved α or HMG domains similar to other sexual fungi, and most amino acids in the α or HMG domains were also highly conserved ( Figure 1C).

Growth of MAT1-1 and MAT1-2 Strains under Different Culture Conditions
The growth characteristics of the MAT1-1 strain P48 were compared with those of the MAT1-2 strain P50 in response to a range of temperatures, pH treatments, light/dark regimes, carbon sources, and stress conditions. Furthermore, the growth of five additional isolates of MAT1-1 (S2, S11, S109, P51, and P56) and MAT1-2 (S1, S12, S23, P52, and P90) strains from sunflower (S) and potato (P) was also determined to ensure that the results obtained with P48 and P50 were not unique to these strains. Detailed information on these strains is provided in Table S1.

Temperature
V. dahliae has an optimal temperature range of 22−27 • C, and limited growth occurs above 32 • C [49]. The MAT1-1 strain P48 produced a predominantly white mycelium, but the MAT1-2 strain P50 produced less mycelium, displayed faster colony growth, and accumulated higher amounts of melanin following incubation for 15 days at 25 • C (Figure 2A).
V. dahliae has an optimal temperature range of 22−27 °C, and limited growth above 32 °C [49]. The MAT1-1 strain P48 produced a predominantly white mycelium the MAT1-2 strain P50 produced less mycelium, displayed faster colony growth, a cumulated higher amounts of melanin following incubation for 15 days at 25 °C (F 2A).
The growth rates of both P50 and P48 were significantly lower at 28 °C and relative to 25 °C (Figure 2A). However, even at 28 °C, the colony diameter of the M strain P50 was significantly larger than that of the MAT1-1 strain P48 (Figure 2A,B) °C, the growth of both strains was restricted and was not significantly different (F 2A,B). These results suggest that temperatures <28 °C had fewer influences on the M 2 strain P50 than on the MAT1-1 strain P48, but once the temperature exceeded 30 ° growth of both strains was nearly arrested.  The growth rates of both P50 and P48 were significantly lower at 28 • C and 30 • C relative to 25 • C ( Figure 2A). However, even at 28 • C, the colony diameter of the MAT1-2 strain P50 was significantly larger than that of the MAT1-1 strain P48 (Figure 2A,B). At 30 • C, the growth of both strains was restricted and was not significantly different (Figure 2A,B). These results suggest that temperatures <28 • C had fewer influences on the MAT1-2 strain P50 than on the MAT1-1 strain P48, but once the temperature exceeded 30 • C, the growth of both strains was nearly arrested.
Additional MAT1-1 and MAT1-2 V. dahliae strains, under the optimum temperature (25 • C), showed no detectable differences in their growth rates for up to 15 days incubation, except that the MAT1-1 strains mainly developed white mycelia with less melanin accumulation than the MAT1-2 strains ( Figure 2C and Figure S1). At 28 • C and 30 • C, the diameters of the MAT1-2 strains were significantly higher than those of the MAT1-1 strains ( Figure 2C and Figure S1). Higher temperatures therefore restrict growth and melanin production in both MAT1-1 and MAT1-2 strains, but these effects were more pronounced in the MAT1-1 strains than in the MAT1-2 strains.

pH
Verticillium wilt of cotton normally occurs in near-neutral to alkaline soils at a pH in the range 6-9. At pH 5.5 or below, the growth, microsclerotia production, and survival of V. dahliae are generally inhibited [49]. Under laboratory conditions, the optimum pH for V. dahliae on the PDA medium was approximately 6.5. To determine the differences in the growth of the two idiomorphs, three PDA plates at pH 7.0, 8.0, and 9.0 each were incubated at 25 • C. Strain P50 produced more melanin under these pH values than at pH 6.5 (Figures 2A and 3A). Melanin accumulation increased with increasing pH, with the maximum melanin enrichment occurring at pH 9.0 ( Figure 3A). However, the melanin production by the MAT1-1 strain P48 showed no obvious change with the pH ( Figure 3A). Increasing pH did not alter the growth rates of either strain, but at each pH tested, P50 exhibited a higher colony growth than strain P48 ( Figure 3A,B). Alkaline pH stimulated the accumulation of melanin by the two mating-type strains, but had no quantifiable influence on colony growth rates.  (A) Growth phenotype of P48 and P50 at pH 7, pH 8, and pH 9 after culturing for 15 days. (B) Colony diameters of P48 and P50 at pH 7, pH 8, and pH 9 after culturing for 15 days. (C) Colony diameter of MAT1-1 and MAT1-2 strain populations at pH 7, pH 8, and pH 9 after culturing for 15 days. P and S indicate strains isolated from potato and sunflower, respectively. Asterisks * and ** indicate significant differences; p < 0.05 and p < 0.01, respectively, according to an unpaired Student's t-test.

Light/Dark
The growth of strains P48 and P50 was evaluated on a PDA medium at 25 °C under dark and light conditions. Under the dark conditions examined, both the P48 and P50 strains produced melanin, although the growth rates and melanin accumulation in strain P50 were higher relative to those of P48 ( Figure 4A). However, under the light conditions Growth phenotype of P48 and P50 at pH 7, pH 8, and pH 9 after culturing for 15 days. (B) Colony diameters of P48 and P50 at pH 7, pH 8, and pH 9 after culturing for 15 days. (C) Colony diameter of MAT1-1 and MAT1-2 strain populations at pH 7, pH 8, and pH 9 after culturing for 15 days. P and S indicate strains isolated from potato and sunflower, respectively. Asterisks * and ** indicate significant differences; p < 0.05 and p < 0.01, respectively, according to an unpaired Student's t-test. The growth rates of the five additional MAT1-1 strains were not altered by the pH, but melanin accumulation was reduced at pH 8 and 9, regardless of whether they were isolated from potato or sunflower. In contrast, MAT1-2 strains from potato and sunflower displayed higher growth rates at higher pH, and melanin production was relatively unaffected compared to the MAT1-1 strains ( Figure 3C and Figure S2). Overall, relative to the MAT1-1 strains, MAT1-2 strains not only grew faster but also accumulated more melanin under near-neutral and alkaline pH.

Light/Dark
The growth of strains P48 and P50 was evaluated on a PDA medium at 25 • C under dark and light conditions. Under the dark conditions examined, both the P48 and P50 strains produced melanin, although the growth rates and melanin accumulation in strain P50 were higher relative to those of P48 ( Figure 4A). However, under the light conditions examined, neither P48 nor P50 produced visible melanin, with both forming only white mycelia ( Figure 4A). The colony diameter of P48 was smaller under light conditions than under dark conditions, but the colony diameters of the P50 strain showed no obvious differences between light and dark conditions ( Figure 4B). Under the light or dark conditions, the growth rates of P50 were higher than those of P48 ( Figure 4B). These results suggested that light interfered with the melanin production in both strains and limited the rate of growth of the MAT1-1 strain P48 but did not influence the growth rate of the MAT1-2 strain P50.  Asterisks * and ** indicate significant differences; p < 0.05 and p < 0.01, respectively, according to an unpaired Student's t-test.
The influence of light was further examined on the growth of five additional strains from MAT1-1 and MAT1-2. Under the light conditions tested, there was no visible melanin production in the MAT1-1 strains, with the exception of S2 ( Figure S3). Similarly, melanin accumulation in the MAT1-2 strains was also minimal under light, with only strains S23 and P52 producing small amounts of melanin ( Figure S3). The growth rate of MAT1-2 The influence of light was further examined on the growth of five additional strains from MAT1-1 and MAT1-2. Under the light conditions tested, there was no visible melanin production in the MAT1-1 strains, with the exception of S2 ( Figure S3). Similarly, melanin accumulation in the MAT1-2 strains was also minimal under light, with only strains S23 and P52 producing small amounts of melanin ( Figure S3). The growth rate of MAT1-2 strains whether under dark or light conditions was higher relative to that of the MAT1-1 strains; and MAT1-2 strains produced more melanin under the dark conditions than the MAT1-1 strains ( Figure 4C and Figure S3). These results indicate that light restricted melanin production in both mating-types but had relatively less influence on the growth rate of MAT1-2 strains versus MAT1-1 strains.

Carbon Source
Verticillium species are capable of metabolizing a wide range of carbon sources, including glucose, mannose, rhamnose, sucrose, xylose, and cellobiose [50][51][52]. To examine the complex nutrient utilization capacity of the two MAT strains, their growth rates and phenotypes were evaluated on media containing different carbon sources, including the relatively simple disaccharide sucrose, but also complex polysaccharides like starch, pectin, and cellulose. The growth rates of the MAT1-2 strain P50 were higher compared to that of the MAT1-1 strain P48 on each of the media containing different carbon sources, and P50 also produced more melanin ( Figure 5A,B). The higher growth of strain P50 suggested that the MAT1-2 strain has an increased capacity to degrade complex carbon sources, especially the complex polysaccharides pectin, cellulose, and starch. The MAT1-1 strain P48 produced mainly white mycelia on starch-, sucrose-and pectin-containing media with no apparent melanin production ( Figure 5A). Melanin production was also significantly enriched in the MAT1-2 strain P50 on starch, sucrose, and pectin media, in addition to producing the typical white mycelia ( Figure 5A). The growth of both mating type strains was limited on the medium containing cellulose as a carbon source, and there was little visible melanin production on this medium ( Figure 5A). Both strains utilized pectin, but their ability to utilize cellulose was relatively weak compared with other complex polysaccharides examined.
Based on these results, three complex carbon sources were further evaluated on the larger panel of MAT1-1 and MAT1-2 strains. The MAT1-2 strains produced more melanin and less white mycelia than the MAT1-1 strains on the starch medium ( Figure S4). When pectin served as the carbon source, both MAT1-1 and MAT1-2 strains produced melanin and mycelia ( Figure 5C and Figure S4). When cellulose was the carbon source, similar to P48 and P50, the growth of other MAT1-1 and MAT1-2 strains was limited, with scant mycelia, small colony diameters, and little melanin accumulation ( Figure S4). The colony diameters of all MAT1-2 strains were larger relative to those of the MAT1-1 strains on media containing the three selected carbon sources ( Figure 5B and Figure S4). Since pectin and cellulose are some of the chief constituent complex polysaccharides in plants, MAT1-2 strains may have an enhanced capability, compared to MAT1-1 strains, to degrade plant cell wall components during infection and colonization processes.

Stress Tolerance (Osmotic Stress, Oxidative Stress, Cell Wall Integrity Stress)
To evaluate the stress tolerance of the two mating type strains, their growth on media that imposed osmotic stress (sorbitol-containing medium at three concentrations: 1, 1.5, and 2 mol/L), oxidative stress (H 2 O 2 -containing medium at three concentrations: 0.9, 1.2, and 1.8 mmol/L), and cell wall integrity stress (Congo red-containing medium at three concentrations: 100, 200, and 300 µg/mL) was evaluated.
significantly enriched in the MAT1-2 strain P50 on starch, sucrose, and pectin media addition to producing the typical white mycelia ( Figure 5A). The growth of both ma type strains was limited on the medium containing cellulose as a carbon source, and th was little visible melanin production on this medium ( Figure 5A). Both strains utili pectin, but their ability to utilize cellulose was relatively weak compared with other c plex polysaccharides examined. Based on these results, three complex carbon sources were further evaluated on larger panel of MAT1-1 and MAT1-2 strains. The MAT1-2 strains produced more mela and less white mycelia than the MAT1-1 strains on the starch medium ( Figure S4). W pectin served as the carbon source, both MAT1-1 and MAT1-2 strains produced mela and mycelia (Figures 5C and S4). When cellulose was the carbon source, similar to and P50, the growth of other MAT1-1 and MAT1-2 strains was limited, with scant myc small colony diameters, and little melanin accumulation ( Figure S4). The colony diame of all MAT1-2 strains were larger relative to those of the MAT1-1 strains on media c taining the three selected carbon sources (Figures 5B and S4). Since pectin and cellu are some of the chief constituent complex polysaccharides in plants, MAT1-2 strains m have an enhanced capability, compared to MAT1-1 strains, to degrade plant cell wall c ponents during infection and colonization processes. Under osmotic stress, the growth rate of both P48 and P50 decreased as the sorbitol concentration increased, and there was no apparent melanin production under sorbitol stress. While there were no differences in the growth rates between the two strains under sorbitol concentrations of 1 and 1.5 mol/L, the growth rate of the P50 strain was higher relative to that of strain P48 at 2 mol/L sorbitol ( Figure S5A,B). Similarly, under cell wall integrity stress, the growth rates of both P48 and P50 decreased as the Congo red concentration increased ( Figure S5C,D). Strain P50 produced more melanin at 100 and 200 µg/L Congo red than P48 ( Figure S5C,D). Under the cell wall stress conditions tested, there were also no apparent differences in growth rate between the two strains ( Figure S5D). The growth rates of both strains decreased as the oxidative stress from H 2 O 2 concentration increased. However, there was almost no growth of strain P48 at the concentration of 1.8 mmol/L H 2 O 2 ( Figure 6A,B). Unlike the other stress conditions examined, the growth rate of the MAT1-2 strain P50 was higher than that of the MAT1-1 strain P48 in response to H 2 O 2 , indicating that the MAT1-2 strain P50 had a greater resistance to oxidative stress than the MAT1-1 strain P48.
The growth rates of both strains decreased as the oxidative stress from H2O2 conc increased. However, there was almost no growth of strain P48 at the concentra mmol/L H2O2 ( Figure 6A,B). Unlike the other stress conditions examined, the gr of the MAT1-2 strain P50 was higher than that of the MAT1-1 strain P48 in re H2O2, indicating that the MAT1-2 strain P50 had a greater resistance to oxida than the MAT1-1 strain P48.  (0.9, 1.2, 1.8 mmol/L) at 15 days of growth. P and S indicate strains isolated from potato and sunflower, respectively. Asterisks ** and *** indicate significant differences; p < 0.01 and p < 0.001, respectively, according to an unpaired Student's t-test.
Colony diameters of the additional MAT1-1 strains evaluated were significantly smaller in size as the H 2 O 2 concentration increased. At 1.8 mmol/L H 2 O 2 none of the MAT1-1 strains grew except P56, which produced very small colonies ( Figure S6). The growth rates of the MAT1-2 strains were also slower as the H 2 O 2 concentrations increased, the only difference being that the colony diameters were larger relative to those observed in the MAT1-1 strains

Pathogenicity of MAT1-1 and MAT1-2 Strains on Their Original Hosts
The virulence of the MAT1-1 and MAT1-2 strains was evaluated on potato or sunflower. Most of the MAT1-2 strains from potato were more virulent than the MAT1-1 strains on potato, except for strain P57 (Figure 7). However, the overall virulence of MAT1-2 and MAT1-1 strains from sunflower was not significantly different on sunflower (Figure 7).

Pathogenicity of MAT1-1 and MAT1-2 Strains on Their Original Hosts
The virulence of the MAT1-1 and MAT1-2 strains was evaluated on potato or sunflower. Most of the MAT1-2 strains from potato were more virulent than the MAT1-1 strains on potato, except for strain P57 (Figure 7). However, the overall virulence of MAT1-2 and MAT1-1 strains from sunflower was not significantly different on sunflower ( Figure  7). S11, S109, S31, and S29; MAT1-2 strains: S1, S12, S47, S23, and S38) three weeks after the inoculation of sunflower. Columns with different letters represent a statistical significance of p < 0.05 according to Duncan's new multiple range test.

Discussion
We compared the growth and the virulence of V. dahliae strains of opposite mating types derived from sunflower and potato. The MAT1-2 strains exhibited higher growth rates and melanin production under high temperatures, alkaline pH, light, and oxidative stress (Figures 2-4 and 6). Additionally, MAT1-2 strains showed an enhanced ability to S11, S109, S31, and S29; MAT1-2 strains: S1, S12, S47, S23, and S38) three weeks after the inoculation of sunflower. Columns with different letters represent a statistical significance of p < 0.05 according to Duncan's new multiple range test.

Discussion
We compared the growth and the virulence of V. dahliae strains of opposite mating types derived from sunflower and potato. The MAT1-2 strains exhibited higher growth rates and melanin production under high temperatures, alkaline pH, light, and oxidative stress (Figures 2-4 and 6). Additionally, MAT1-2 strains showed an enhanced ability to degrade complex polysaccharides, especially starch, pectin, and cellulose ( Figure 5). Correspondingly, some MAT1-2 strains from potato also exhibited increased virulence relative to the MAT1-1 strains on potato. However, the difference in virulence between the two mating types were not apparent on sunflower (Figure 7). This research documents the overall higher adaptability of the MAT1-2 strains, that confers them greater fitness. This may help explain the major imbalance in the frequencies of the two mating idiomorphs in the agricultural and sylvicultural ecosystems, and provides information for future inquiries into the potential sexual cycle of V. dahliae under experimental conditions. Temperature, as the single most important environmental factor, not only governs the germination of V. dahliae microsclerotia and disease development but also influences the geographic distribution of V. nonalfalfae and V. dahliae [49,53]. V. dahliae is mainly distributed in subtropical and temperate areas and has an optimal temperature in the range 22-27 • C, but limited growth occurs above 32 • C. Devaux and Sackston [54] demonstrated that almost no microsclerotia formed at temperatures over 30 • C. Abundant microsclerotia formed between 18 and 30 • C within 2−5 days and after 30 days at 5 • C, and no microsclerotia formed at 32 • C [55]. Colony growth and melanin production were altered as temperatures rose from 25 • C to 30 • C. At 30 • C, especially, the colony growth rates decreased and there was almost no melanin accumulation (Figure 2A,B). Similarly, the larger panel of MAT1-1 and MAT1-2 strains showed no growth rate differences at 25 • C. The growth rate of the MAT1-2 strains was higher relative to those of the MAT1-1 strains as the temperature increased ( Figure 2C), and also accumulated more melanin up to 30 • C ( Figure S1). High temperature affected MAT1-2 strains less than MAT1-1 strains, indicating a stronger temperature tolerance of V. dahliae MAT1-2 strains. Melanin production in V. dahliae is also linked with protection against high temperature stress [56].
The maintenance of a constant pH was the single most important factor in maintaining optimal growth rates of V. dahliae, and in cotton production, the disease normally occurs in soils that are neutral to alkaline (pH 6-9). Under acidic pH (5.5 or lower), the mycelial growth, microsclerotia production, and survival are all reduced [49]. Increases in the pH slightly reduced growth and melanin synthesis in the MAT1-1 strain P48 ( Figure 3A). In contrast, growth was not influenced significantly with increasing pH, but melanin production increased in the MAT1-2 strain P50 ( Figure 3A). Overall, the optimum pH for MAT1-1 strains was approximately 7.0, but the MAT1-2 strains could grow and produce melanin under a broad pH range.
For a soil-borne and plant xylem-invading fungus like V. dahliae, a dark environment may represent an optimal condition for V. dahliae growth and proliferation. Some of the earliest research findings on melanin production in V. albo-atrum (reclassified as V. alfalfae and V. nonalfalfae by Inderbitzin et al. [46]) suggest that its resting mycelium formed sooner in total darkness than in daylight [57]. Caroselli et al. [58] also claimed that "green light" inhibited growth and that more microsclerotia were formed in "red light" and darkness. Conidial production by V. dahliae was greatly enhanced, but microsclerotia production was completely inhibited by blue light [59,60]. Some previous studies indicated that melanin occurs as electron-dense granules in the outer cell wall of microsclerotia, and melanin deposition is typically coupled with later stages of microsclerotial formation in V. dahliae [61,62]. Under light conditions, except for a few strains, there was almost no melanin production for all strains as compared to incubation in the dark ( Figure 4A and Figure S3). Under light conditions, reduced melanin synthesis decreases the survival of V. dahliae, as these strains are highly sensitive to UV radiation [56]. This also corresponds to previous research showing that unmelanized conidia are more prone to rapid desiccation and damage by UV radiation [58,63].
Verticillium spp. are capable of metabolizing a wide range of carbon sources, including glucose, fructose, arabinose, galactose, mannose, cellobiose, ribose, and xylose, as well as complex polysaccharides such as starch, pectin, and cellulose [52]. V. dahliae encodes a wide range of secreted pectinolytic and cellulolytic enzymes that may initiate infection and symptom development [43,64]. Thus, the capacity of V. dahliae to utilize different carbon sources is crucial for the infection of various host plants and the proliferation in host cells, including in the xylem, which consists of complex polysaccharides such as pectin, cellulose, and lignified cellulose [65]. The growth rates and melanin production of the MAT1-2 strain P50 were higher than those of the MAT1-1 strain P48 when sucrose, starch, cellulose, and pectin were used as carbon ( Figure 5 and Figure S4). The cellulose utilization by both strains, however, was lower compared with the other three carbon sources. This was consistent with the growth of other V. dahliae strains on a cellulose-containing medium [66]. Although the growth rates of MAT1-2 strains were higher than in the MAT1-1 strains on media containing different polysaccharides, the melanin production in the MAT1-2 strains in general was not limited, unlike in strain P48 ( Figure 5C and Figure S4). Nonetheless, the MAT1-2 strains generally had a higher polysaccharide utilization ability relative to the MAT1-1 strains.
Congo red, sorbitol, and H 2 O 2 have been widely used to detect fungal cell wall integrity and resistance to high concentrations of osmotic regulators and oxidative stress, respectively. Congo red interacts with various polysaccharides and exhibits a particularly high affinity with chitin and cellulose, which disturbs fungal cell wall morphogenesis [67][68][69]. The osmotic regulator sorbitol at high concentrations causes osmotic stress [70,71]. In response to pathogen infection, plants tend to produce more H 2 O 2 to inhibit pathogen proliferation, and the reactive oxygen scavenging capacity has been viewed as an important evaluation index to assess pathogen infection ability and pathogenicity [56]. Under the Congo redand sorbitol-induced stress, the growth rates and melanin production of both MAT1-1 strain P48 and MAT1-2 strain P50 were impaired, except for the higher melanin production at relatively low Congo red concentrations (100 and 200 µg/mL) by strain P50 ( Figure  S5). These results suggest that the sensitivities of the two strains to Congo red and sorbitol were similar. However, MAT1-1 and MAT1-2 strains responded differently to H 2 O 2 stress, suggesting differing reactive oxygen scavenging abilities in the two mating-type populations.
In conclusion, under a higher temperature, alkaline pH, response to light, and oxidative stress, the MAT1-2 strains exhibited higher growth rates and melanin production relative to those of the MAT1-1 strains. In addition, the MAT1-2 strains also had a higher complex polysaccharide degradation ability, suggesting that the MAT1-2 strains had wider environmental adaptability, that gave them greater fitness and survivability. These advantages may also confer the MAT1-2 strains higher virulence on select host plants. As demonstrated in this study, there was higher virulence of the MAT1-2 strains from potato when assayed on potato but such differences were not apparent with the two mating-type strains from sunflower when assayed for virulence on sunflower. Nonetheless, relative to the MAT1-1 strains, MAT1-2 strains exhibit an increased ability for environmental and potential host adaptation. The results of this research support the hypothesis that MAT1-2 strains are more adaptable, and therefore far more abundant in nature than the MAT1-1 strains. This research also provides the baseline information on the two mating idiomorphs to pursue studies of a potential sexual cycle in V. dahliae under different experimental conditions.

Growth of V. dahliae Strains under Various Environmental Factors
To determine the growth rates and colony morphology of V. dahliae MAT1-1 and MAT1-2 strains under different temperatures, pH changes, carbon sources, light and dark conditions, and conditions causing osmotic and cell wall stress, conidial suspensions of every strain were prepared at 5 × 10 6 conidia/mL, and 2 µL was transferred to the center of Petri plates containing different media.
For temperature sensitivity assays, MAT1-1 and MAT1-2 strains were incubated on a potato dextrose agar (PDA) medium at 25 • C, 28 • C, and 30 • C, respectively. Three plates for each temperature were incubated in each experiment, and the experiment was repeated three times. The pH of the PDA medium was adjusted to 7.0, 8.0, and 9.0 using 1 M HCl or NaOH to determine the pH sensitivity of all isolates at 25 • C. Growth under light and dark conditions was tested on a PDA medium, with each strain incubated at 25 • C. For the carbon source utilization assays, the strains were cultured at 25 • C in basic C'zapek medium agar plates that included sucrose (30.0 g/L), starch (17.0 g/L), pectin (10.0 g/L), or cellulose (10.0 g/L) as the carbon source, respectively [72]. Again, three plates at each pH and isolate were incubated per experiment, and the experiment was repeated three times.
PDA plates containing 0.9, 1.2, and 1.8 mmol/L 30% H 2 O 2 were used to test all MAT1-1 and MAT1-2 isolates to oxidative stress. For osmotic stress experiments, strains P48 and P50 were inoculated and cultured on a complete medium (CM, Yeast extract 6.0 g/L, Casein acids hydrolysate 6.0 g/L, sucrose 10.0 g/L) at 25 • C supplemented with 1, 1.5, and 2 mol/L sorbitol, respectively. For cell wall stress assays, strains P48 and P50 were incubated on CM plates that were supplemented with 100, 200, and 300 µg/mL Congo red (Sigma-Aldrich) at 25 • C.
The growth of all strains under different culture conditions were evaluated after 15 days of incubation by measuring the colony diameters. Each strain was cultured on five plates for each of the treatments, and the experiments were repeated two times. Unpaired Student's t-tests were performed to determine the statistical significance among strains at p ≤ 0.05, p ≤ 0.01, or p ≤ 0.005.

DNA Extraction and Genotype Identification Assays of V. dahliae Strains
High conidial suspensions (1 × 10 8 conidia/mL) of V. dahliae strains P48, P50, and Vd991 were placed on a PDA medium and incubated at 25 • C, and the mycelium was collected after 7 days. The total genomic DNA was extracted for the identification of mating types, races, and D and ND types. Genomic DNA of each isolate was extracted using a FastPure Plant DNA Isolation Mini Kit (Vazyme, Nanjing, China) following the manufacturer's instructions and was stored at −20 • C for polymerase chain reaction (PCR) assays.
For the genotype identification assays, D/ND, race1/2, and MAT1-1/MAT1-2 were determined by PCR with previously developed primers [35,73,74] (Table S2). All PCR assays in this study were performed in 20 µL reaction volumes using 2×Taq Mester Mix (Dye Plus) P112-AA (Vazyme, Nan Jing, China). PCR was performed under the following conditions: an initial 94 • C denaturation step for 10 min, followed by 30 cycles at 94 • C for 30 s, annealing at 58 • C for 30 s, extension at 72 • C for 1 min, and a final extension of 10 min at 72 • C. The PCR products were detected by 1% agarose (Sigma-Aldrich, St. Louis, MO, USA) gel dyed with GelStain (TransGen, Beijing, China) and electrophoresis for 20 min at 120 V in 1×TAE buffer. Then the image was obtained with the Bio-Rad's ChemiDoc XRS system.

Gene Cloning and Bioinformatics Analysis
The mycelia of strains P48 and P50 were collected 7 days after culture at 25 • C on a PDA medium. The total RNA of P48 or P50 was extracted using the Total RNA Miniprep Kit (Axygen, MA, USA), and first-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). The targeted genes (MAT1-1-1 and MAT1-2-1) were amplified from the prepared cDNA using the primers listed in Table S2.

Virulence of MAT1-1 and MAT1-2 Strains
Ten MAT1-1 (P48, P51, P56, P89, P91, S2, S11, S109, S31, S29) and MAT1-2 (P50, P52, P57, P88, P90, S1, S12, S23, S47, S38) V. dahliae strains were selected for virulence assays. Assays were performed as described previously by Xiao et al. [75], with some modifications. All strains were cultured on a PDA medium for 7 days, transferred to a wheat bran medium, and incubated at 25 • C for 15 days until the medium was covered with mycelium. Conidia were harvested with distilled water and filtered with Miracloth (Solarbio, Beijing, China), and a 1 × 10 7 conidia/mL inoculum suspension was used in the virulence assays. Sunflower (cv. He 15) seeds were sown with 5 seeds in each pot, and potatoes (cv. LD 5009) were planted with two tubers in each pot, with each tuber containing at least 2-3 bud eyes. The pots were incubated at 28 • C in the greenhouse with alternating 16 h/8 h light-dark periods. Four-week-old sunflower and potato seedlings were inoculated by flooding 200 mL conidial suspension as described by Alkher et al. and Xiao et al. [75,76]. The plants were examined for symptoms weekly, and the quantification of disease parameters was performed at three weeks post-inoculation following the methods of Xiao et al. and Alkher et al. [75,76]. Five pots of sunflowers and ten pots of potatoes were used for each V. dahliae strain, and the experiment was repeated three times. The quantification of disease parameters was done as follows: Potato Verticillium wilt was rated on a 0 to 5 scale, where 0 = healthy plants with no foliar chlorosis and necrosis; 1 = visible leaf chlorosis with less than 1% necrosis; 2 = up to 40% chlorosis and 1-20% necrosis; 3 = up to 65% chlorosis and 21-35% necrosis; 4 = 100% chlorosis, 36-70% necrosis; and 5 = 100% chlorosis, 71-100% necrosis.
The disease indices for the potato and sunflower virulence assays were calculated according to the formula: =100 × ∑ (number of diseased leaves in each scale rating × representative value in each rating)/(total number of leaves examined × maximum rating value).
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22137148/s1, Figure S1: Growth phenotype of MAT1-1 and MAT1-2 strain populations at 25 • C, 28 • C, and 30 • C on PDA medium after culturing for 15 days. Figure S2: Growth phenotype of MAT1-1 and MAT1-2 strain populations on PDA after culturing for 15 days in medium adjusted to pH 7, pH 8, and pH 9. Figure S3: Growth phenotype of MAT1-1 and MAT1-2 strain populations under light and dark conditions for 15 days. Figure S4: Growth phenotype of MAT1-1 and MAT1-2 strain populations after culturing for 15 days in media types containing different carbon sources. Figure S5: Growth of strains P48 and P50 under two stress tolerance conditions. Figure S6: Growth of MAT1-1 and MAT1-2 strain populations under conditions of oxidative stress. Table S1: Information on all MAT1-1 and MAT1-2 strain populations. Table S2: Primers information used in this study