which produces a blue-green pigment known as xylindein, *Scytalidium cuboideum* (Sacc. & Ellis) Sigler & Kang, which produces a red pigment known as draconin red (Figure 1) that exists as both orange and red crystals, and *Scytalidium ganodermophthorum* Kang, Sigler, Y.W. Lee & S.H. Yun, which produces an unnamed yellow pigment. Further studies showed that polyester absorbed more pigment than other synthetic fabrics and that these colors were stable over time [17,18]. Hinsch [19] later confirmed that *S. cuboideum* and *C. aeruginosa* could be used to dye textiles with no mordant and attributed this to the carbon-carbon interaction of the main quinone structures.

<figure><img src="image_110.png" /><figcaption>**Figure 1.** The structure of dramada, the orange and red crystal pigment from *Scytalidium cuboideum*.</figcaption></figure>

Despite the success of early testing, the fungal pigments mentioned above never gained commercial traction. This is likely due to the need for the pigments to be carried in DCM, which is a potential human carcinogen and a known greenhouse gas [20–22]. Other solvents were tested for their ability to extract the pigments, and while tetrahydrofuran (THF), acetonitrile (ACN), and acetone were all moderately capable of extraction, all three interacted with the pigments and caused color change [23]. Due to these issues, an entirely new type of solvent was explored: natural oils. These oils proved to be highly successful at carrying the fungal pigments, though not at extracting them, with raw linseed oil allowing for the highest pigment stability of those tested [24]. However, of the tested pigments, only the red pigment from *S. cuboideum* was found to effectively color fabric, although it showed variation in coloration based on the material to which it was applied [25].

The purpose of study was to compare the dyeing capability, heat stability (in different washing temperatures), and pH stability (washing in different detergents), of three soft-rot spalting fungi, carried in raw linseed oil, on various fabrics. While it is already known that the pigments from these fungi are reasonable textile dyes when carried in DCM [17,18], this carrier is not practical for commercial scale use due to toxicity issues. An alternative carrier proposed is raw linseed oil, which is known to carry the pigments at high concentrations but has never been tested in textile applications. Using the oil carrier would result in a dyeing process with fewer environmental issues than the DCM process. Analysis via scanning electron microscopy (SEM) will further the understanding of the interaction between the fungal pigments and the fabrics. The research will assess if the oil changes the structure of the pigments, how the pigments bind to textiles, and if the reaction of the pigments changes due to temperature and pH changes based upon the oil carrier.

This work prepares the fungal pigments to fully enter into the commercial dye industry by assessing the viability of a more eco-friendly carrier and removing the largest hurdle currently facing the soft-rot fungal pigments, their DCM requirement for application.
## 2. Materials and Methods

All testing methodology followed either accepted the American Society of Testing Material (ASTM) standards (such as for fabric testing) or were based on testing used in previous work with these spalting pigments on textiles (see citations below).

### 2.1. Fungal Growth and Pigment Extraction

Cultures of *S. cuboideum* (UAMH 4802, isolated from oak lumber, location unknown) were used to inoculate malt agar plates amended with white rotted wood chips, following the procedure outlined in Robinson et al. [23]. Plates were allowed to grow for 2–3 weeks before they were dried for 24 h in a fume hood, extracted into DCM, and standardized to CIE $L*a*b*$ values of $L = 82.32, a = 26.84,$ and $b = 13.19$, as described in Robinson et al. [23]. After the standardization, raw linseed oil (Sunnyside) was mixed with the pigment carried in DCM and placed on a stir plate for 48 h in order to evaporate the solvent and leave the pigment suspended in the oil, following the methodology by Palomino Agurto et al. [25]. This pigmented oil was used for further testing.

### 2.2. Fabrics Tested

Unfinished 100% fabrics of polyester, nylon, cotton, and wool were used (see Table S1). Per each type of fabric, two different densities were used: low-density and high-density. To establish a difference between low-density and high-density, a minimum difference of 40 yarns per inch in the count number was established. Low-density nylon was discarded because its finishing process did not allow dying by the pigmented oil.

### 2.3. Mechanical Testing

Mechanical testing was done to evaluate the impact of the oil-pigments on tensile and tear strength of the fabrics, with samples for mechanical testing prepared according to the American Society of Testing Material (ASTM 2013). Submersion dying was carried out using borosilicate glass beakers (250 mL) (brand VWR, Randor, PA, USA), filled with 100 mL of pigmented oil from *S. cuboideum*. Samples from tested fabrics were separately submerged in the pigment solution in the uncovered beakers for 1 h. Once the time of exposure was achieved, samples were removed from the borosilicate beakers and placed over a metal mesh to dry for 48 h.

#### 2.3.1. Tear Strength Test

The tear strength test was performed according to the ASTM D1424 using a falling-pendulum digital Elmendorf-type (produced by SDL ATLAS, Rock Hills, SC, USA) to measure the force required to propagate a single-rip tear. This test was performed on the samples of the four fabrics in filling directions. Twelve repetitions were performed on both dyed and undyed control samples (treated with only raw linseed oil), making a total of 36 samples per fiber fabric and 144 samples in total.

Estimated least squares means tests with Tukey adjustment was performed using SAS 9.8 (SAS Institute, Cary, NC, USA) to compare the effect of treatment and fabric type on stress (MPa) or force (N). The least squares means for main effects were reported if the main effect was statistically significant and not part of any statistically significant interaction or when the main effect was statistically significant and the effect of that factor was always in the same direction for the interacting variable(s). Selected samples were later evaluated using SEM analysis (below) to assess the behavior of pigment crystals inside the fabric structures.

#### 2.3.2. Tensile Strength Test

The breaking (tensile strength) test was performed according to the ASTM D5034-09 (2013), using the Universal Testing Machine Instron Model 5582 (produced by Instron Company, Norwood, MA, USA). Samples were loaded into the tool and then exposed to tensile forces until breaking occurred.
The test was repeated for 30 samples for each fabric/density type, with 10 controls, 10 samples dyed with only raw linseed oil, and 10 samples dyed with pigmented oil. After the tensile strength test, affected samples were analyzed using SEM to evaluate any physical change in the attachment of the pigment to the fibers.

Estimated least squares means tests with Tukey adjustment were performed using SAS 9.8 (SAS Institute, Cary) to compare the effect of treatment and fabric type on f (N), and least squares means for main effects were reported if the main effect was statistically significant and not part of any statistically significant interaction or when the main effect was statistically significant and the effect of that factor was always in the same direction for the interacting variable(s). Selected samples were later evaluated using SEM analysis (below) to assess the behavior of pigment crystals inside the fabric structures.

## 2.4. Analysis of Colorfastness

### 2.4.1. Color Variation Across Fabrics

Difference in pigment uptake and coloration were compared between fabrics. Fabric samples were cut into 5.08 × 5.08 cm, and dripping was used to apply fungal pigment. This consisted of applying pigment suspended in raw linseed oil onto the surface of each fabric sample using a disposable pipette. Fifteen drops of the oil-solubilized pigments were applied onto each fabric sample using a 1 mL pipette (brand Gilson, Lewis Center, OH, USA), with drops weighing an average 0.02443 g.

Control samples were compared to samples colored using the dripping methodology, and color analysis was performed on the samples using a Konica Minolta Chroma Meter CR-5 colorimeter, using the CIE $L*a*b*$ color space, with the ΔE calculation 2000. A one-way ANOVA was performed to test for the effect of fabric type on the response of color variation (ΔE) using SAS 9.8 (SAS Institute, Cary).

### 2.4.2. Laundry Test

Colorfastness to laundering with and without bleach was performed on the fabric samples dyed with the dripping method. The washing test was performed according to the AATCC method 61-2013 (Colorfastness to Laundering: Accelerated Using a Launder-O-Meter) in a Launderometer model LEF (produced by SDL ATLAS). Twelve 5.08 × 5.08 cm samples of each tested fabric type were prepared using dripping methodology (above), with six replicated exposed to bleach in testing and six without exposure. Samples were placed in a canister containing 50 stainless steel balls and AATCC standard detergent solution (with or without bleach). The sealed canisters were placed in the launder-O-meter with hot water (49 °C) for 45 min. When the process finished, samples inside the canister were rinsed and air-dried for 48 h. The color was measured in the Konica Minolta Chroma Meter CR-5, utilizing the CIE $L*a*b*$ color space. A general linear model was used to analyze the interactions between the independent variables of laundry, fabric type, heat, and time of heating with a four-way ANOVA and Tukey-Kramer test ($p < 0.05$) to determine significant differences between groups. One sample for each fabric/treatment was randomly selected and was taken to be analyzed with SEM (below) to visualize any change from the control samples. The response of prepared pigmented fabrics to heating was then compared, with samples previously subjected to heat testing.

### 2.4.3. Heat Testing

Testing samples were prepared following methods described for the laundry test (using the same number of samples), then heated to one of three common dryer temperatures (low = 50 °C, medium = 65 °C, and high = 80 °C) in a forced air oven to simulate a home dryer. Initial color readings were taken after samples dried using a Konica Minolta Chroma Meter CR-5 colorimeter using the CIE $L*a*b*$ color space, using the ΔE calculation 2000. Color change was then again measured after 30 and 60 min exposure to heat, and samples were removed with tweezers and transported in baggies to prevent dirtying. Once the color was measured, two samples were randomly selected from each
fabric/treatment and analyzed via SEM (below) to visualize any change that occurred due to the heat. A repeated measurement ANOVA with a mixed model was performed to determine the difference in ΔE and any interactions between the independent variables fabric, time, heat, and treatment, and a Tukey Kramer test was performed to classify possible interactions.

### 2.4.4. Qualitative Analysis: SEM

The purpose of SEM was to visualize the physical interaction between the pigment when carried in raw linseed oil and the fabrics and how this varied between treatment groups and the control. All tested samples (noted above) were dried for 48 h at room temperature, then were mounted on aluminum studs 5 mm in diameter using carbon adhesive tape. Samples were coated following Vega Gutierrez (2016), with gold-palladium in a Cressington Sputter Coater 108 Auto (Cressington Scientific Instruments, Inc, Cranberry Twp, PA, USA) for 35 s to allow the samples to generate a coating that decreased the electron charging and increased the contrast on the sample images. Samples were analyzed with a FEI QUANTA 600F environmental SEM (FEI Co., Hillsboro, OR, USA). The electron spot used was 2 Å and 2 kV (high voltage).

## 3. Results

### 3.1. Influence of Fabric Type on Coloration of Samples

The color intensity of dyed fabrics was found to vary significantly (df = 619, F = 150.15, *p* < 0.001) depending on the type of fabric, with Tukey groupings showing that low-density cotton had the most pigmentation (ΔE = 35.58 ± 1.73), followed by high-density cotton (33.21 ± 3.54), and low-density polyester (27.61 ± 4.22). Nylon, high-density polyester, high-density wool, and low-density wool were not found to differ significantly and showed lower coloration overall.

### 3.2. Mechanical Testing

#### 3.2.1. Tensile Strength

No significant difference was seen in tensile strength between untreated fabric samples, fabric samples treated with linseed oil alone, and samples treated with pigmented oil (df = 2, F = 1.75, *p* = 0.1806). The fabric type was found to significantly affect the sample tensile strength (df = 6, F = 114.55, *p* < 0.0001), though no interaction was found between the interaction of fabric type and treatment, which would have indicated differences in strength changes for one or more of the fabrics due to the pigment (df = 12, F = 0.25, *p* = 0.9944).

SEM analysis showed differences in fiber morphology after tensile testing between tested fabric varieties. High-density cotton showed flattened or curvy twisted fibers close to the break area, with hook-like ends at the ends of fibers (Figure S1). Low density cotton also showed curvy, twisted fibers close to break point, with split fiber ends (Figure S2). Nylon showed flattening at the area of breakage and wide separation of fibers, with oil accumulation visible on their surface (Figure S3). High-density polyester showed uneven broken fiber ends and minor flattening of broken ends (Figure S4). Low-density polyester in contrast showed an irregular twisted surface and wavy fibers in the failure area, with oil accumulation evident (Figure S5). High-density wool showed flattened and elongated fibers with a sharp angle on failure sites, showcasing the separation of the cuticle from the fiber core (Figure S6). Low-density wool showed similar characteristics to high-density wool, with clear damage from the test (Figure S7).

#### 3.2.2. Tear Strength

The tearing strength test showed a significant interaction between fabric type and treatment (df = 12, F = 9.76, *p* < 0.0001), with low density cotton showing a significant difference between untreated samples and samples treated with pigmented or non-pigmented linseed oil. Control samples
tore at a significantly lower strength of 912.58 N compared to pigmented oil samples. Tukey groupings showed that the low-density wool with oil had the highest tear strength performance (5068.36 N), while high-density cotton treated with oil had the lowest performance (649.67 N) in this test.

High-density cotton showed clear flattened, broken ends with accumulated oil at the ends (Figure S8), while low-density cotton showed twisted sharp ends and wrinkled fibril bundles (Figure S9). High-density wool showed an even shape in the failure area with no loss of cuticle integrity, though a flattened core was present (Figure S10). Low-density wool showed cuticle loss and irregular surfaces, with breaking areas showing visible structural damage (Figure S11). Nylon showed un-flattened fibers with occasional bracket-like structures with accumulation of oil close to the break area on fiber ends, though most sections did not show this oil clumping (Figure S12). High-density polyester showed angled edges and flattened fibers, with oil accumulation and waviness seen in broken fiber ends (Figure S13). These was also seen in low-density polyester, which also showed twisting in the fibers (Figure S14).

### 3.3. *Laundry and Heat Testing*

Color change in response to laundry and heat processes was found to vary in response to testing conditions. The four-way ANOVA interaction between fabric type, laundry type (bleach/no bleach), temperature, and time was not found to be significant, nor were any three-way interactions. Significant two-way interactions included those between heating time and temperature (df = 4, f = 9.07, *p* = 0.0001), laundry type and temperature (df = 4, f = 7.66, *p* = 0.0005), heating time and temperature (df = 2, f = 9.07, *p* = 0.001), fabric type and temperature (df = 9, f = 1.88, *p* = 0.0349), and fabric type and laundry treatment (df = 8, f = 24.67, *p* < 0.0001). Overall, these significant interactions suggested that fabrics responded differently to pigmentation, with bleach leading to discoloration and increased exposure to heat (either over time or at higher temperatures) leading to more intense color. Drying temperature seemed especially important, as it was present in all significant interactions, although its effects were mediated by other variables.

In general, the addition of bleach resulted in less color difference between pigmented samples and controls, although for low-density cotton, the addition of bleach resulted in a significantly higher ΔE (more color change). Polyester and nylon showed significant degradation of the pigments after laundry treatment, both with and without bleaching, as can be seen in Figure 1. However, in this case, the ΔE value does not tell the whole story. ΔE measures the total change in color, and, while in most cases in this study this change reflects the intensity of the red coloration, when bleach was applied on a number of fabrics, the hue of the pigmentation changed. In wool of both densities, the color changed from red to blue, and in cotton, the color changed from red to dark purple (Figure 2). Overall, bleach caused a more visible color change on natural fabrics over synthetic fibers. Synthetic fabrics followed a different pattern, presenting less color change after washing and drying.

In addition to the changes in color, as measured by ΔE, and the variation of this effect upon different fabrics, bleach seemed to have a profound effect on the applied pigment in oil, as was qualitatively seen in SEM. While fabrics did not appear degraded in SEM, pigments on samples showed evidence of breakdown. Figure 1 shows pigments looking different in the bleached sample compared to the unbleached sample on the same fabrics at the same drying temperature and time. The analysis of SEM images identified the loss of the pigment layer produced by the interaction of the fabric with the oil. As can be seen in Figure 3, the degradation of oil layers occurred regardless of whether bleach was used during laundry tests. When bleach was used, it severely altered the dye color. All cases showed a strong contrast in comparison with the initial coloration and/or a dramatic loss of the dyed area.
<figure><img src="image_111.png" /><figcaption>**Figure 2.** Differences in color and saturation between various fibers treated with 15 drops of dramada-pigmented oil across laundry without bleach, laundry with bleach, and the control (unwashed samples).</figcaption></figure>
<figure><img src="image_112.png" /><figcaption>**Figure 3.** SEM images of a bleach/no bleach sample at the same temperature, showing degradation. (a) Low density polyester (control 15 drops); (b) low density polyester laundry without bleach; (c) low density polyester laundry with bleach.</figcaption></figure>

No dramada crystals were seen in cotton fabrics, with high density cotton showing a smooth covering of oil with no presence of crystals, which became porous after laundry treatment without bleach, and, in the presence of bleach, no oil was found on the samples (Figure S15). Low density cotton showed a less smooth surface of applied pigment with fissures on the outer layer, likely due to retention in the most superficial layer (Figure S16b). After washing with and without bleach, low density cotton showed retention of a thick layer of pigmented oil with an irregular porous shape (Figure S16). Nylon also showed no crystals in the pigmented oil layer, with an irregular surface that was actually smoother after laundry without bleach. In contrast, laundry with bleach resulted in stripping of much of the oil layer remaining only in spaces between yarns (Figure S17).

High density wool also did not form visible crystals within the pigment layer, which formed a thick, even layer of oil that remained after laundry without bleach. However, laundry with bleach showed visible interstices and a porous layer (Figure S18). The low-density wool allowed for a higher degree of infiltration of the oil into the lower layer of the fabric than the high-density wool. Low density wool also showed a thick pigment layer covering the fibers, though interactions with inner layers of the wood fibers made it difficult to distinguish if a granular surface seen where pigment was directly applied was due to pigment/wool interaction or not. After laundry without bleach, the oil absorbance of the cotton became higher than wool or polyester, as seen through the interstices in the oil layer (indicating a dryer oil coat) (Figure S19c), with samples exposed to bleach also showing a reduction in oil and flaking along a porous surface.

In contrast to other fabrics, polyester showed high concentrations of dramada crystals in below oil layers. High-density polyester showed a somewhat more even oil distribution, with a smoother surface seen after laundry with bleach and a lack of visible coating after laundry with bleach (Figure 4). Low density polyester also showed a reduced oil layer after laundry, along with several facture points. Oil also seemed to be degraded by detergent and bleach, forming cube-like structures between fibers and an overall porous surface (Figure 5). Low density polyester also showed a reduced oil layer after laundry, along with several facture points (Figure S20).
<figure><img src="image_113.png" /><figcaption>**Figure 4.** SEM images of high-density polyester samples: (a) control; (b) sample treated with 15 drops of pigmented oil, showing presence of dramada crystals; (c) pigmented sample after laundry treatment with no bleach, showing a somewhat smoothed surface; (d) pigmented sample after laundry treatment with bleach showing no visible oil layer.</figcaption></figure>

<figure><img src="image_114.png" /><figcaption>**Figure 5.** Distinctive features seen in polyester. (a) High-density polyester sample with a dramada crystal; (b) SEM image showing low-density polyester sample after the laundry test with bleach.</figcaption></figure>
### 3.4. Heat Testing

Significant variations of ΔE were seen between pigment treated fabrics exposed to drying conditions at either 50, 65, or 80 °C conditions for either 30 or 60 min. The four-way interaction between all these variables was not statistically significant (df = 12, F = 1.47, *p* = 0.1366), though two three-way interactions were found to be statistically significant. Fabric type and heating were found to influence color change after heating (df = 2, F = 7.41, *p* = 0.0008), with all fabrics having higher ΔE value after the heat treatment. In addition, the interaction between time, temperature, and treatment was significant (df = 12, F = 3.21, *p* = 0.0003), with an increase in ΔE after the heat process; a difference that can be seen in Figure 6. Tukey groups showed that high-density cotton and low-density polyester showed the highest (ΔE), while nylon showed the lowest (ΔE).

<figure><img src="image_115.png" /><figcaption>**Figure 6.** Fabrics dyed with pigmented oil before and after the heat treatment (30 min, 50 °C).</figcaption></figure>

## 4. Discussion

The red pigment produced by *Scytalidium cuboideum*, carried in oil, was found to successfully dye a variety of fabric types without impact on tested mechanical properties, though wide variation in response to colorfastness treatments was seen. The color of the oil pigment varied based on fabric. These differences could be seen in the presence and morphology of the pigmented oil layer under SEM.
The presence of the pigment in oil layer did not affect material strength or tensile strength. As natural dyes have historically been associated with weakening of fabric and decreases in tear strength due their need to use mordants for effective dying [26–28], this is a significant benefit for use of spalting fungal pigments.

Pigmented low density cotton showed a significant difference in tear strength in comparison to untreated samples, but no significance was seen between pigmented oil and non-pigmented oil. This suggests that any differences might be due to the absorption of oil, which could have influenced slippage, leading to tearing. It is also possible that cellulose functional groups may have interacted with pigmented oil and formed a stronger bond; however, this would require further investigation. The fact that significance was only seen in this fabric type further suggests that this effect was not due to the pigment presence and instead suggests that factors, such as fabric thickness and diffusion, may influence dye absorption. Additionally, SEM showed that the pigmented oil did not stay in the internal structures of the high-density cotton but it did stay in the internal structures of low-density cotton, which had more space to absorb it. Both tensile strength and tear strength testing results mirrored those found by Hinsch [19], who reported no significant differences in strength values after application of *S. cuboideum* pigment in the DCM carrier.

While pigment presence was not seen to influence mechanical testing, clear differences in coloration of applied pigment were seen between fabric varieties with both cotton varieties and low-density polyester, showing significantly higher coloration than other fabric types (p < 0.001). It is likely that structural differences between fabric types are responsible for these differences in coloration. The more complex shapes created by natural fibers presented a different surface, leading to different interactions with the pigmented oil. A deep view with SEM showed that polyester fibers seemed to have the dye attached mostly in the outer areas of the sample. Polyester fabrics are normally hydrophobic materials; therefore, their affinity for oil-based compounds was supposed to be higher than that of cotton and wool (which are normally lipophobic materials). Previous research [1,19] did not show any relationship between this feature of the fabrics and their ability to be dyed; however, both studies found that polyester had a general affinity for natural dyes. This relationship still needs to be explored with other types of polyester and fabric blends.

While differences were seen between fabric varieties, density differences did not result in consistent patterns of pigment behavior. While it was speculated that the crystal structure contained in pigment from *S. cuboideum* could be entrapped differentially in fabrics of varying densities, SEM investigations showed that fabric density was also not related to crystal presence, with pigmented oil staying in the fibers without forming crystals. In most cases, a thin layer of oil formed on the surface and a marked accumulation was found in the intersections of each yarn. More crystals may have formed with time, as crystals from *S. cuboideum* have been shown to appear in oil after 60 days, potentially due to oxidation processes [29].

The natural fibers retained coloration better than the synthetics, with wool fabrics, in particular, showing stable color after laundering. This has been seen with other fungal dyes (although not from spalting fungi), specifically with *Talaromyces australis* (C.M. Visagie, N. Yilmaz & J.C. Frisvad) and *Penicillium murcianum* (C. Ramírez & A.T. Martínez) [30]. In most cases, laundry with detergent alone was sufficient to remove the oil layer from the applied pigment. It is likely that the detergent used in the laundry test affected the triester groups of the linseed oil through a complex formation with available -OH⁻ groups. However, complete removal was not seen on wool, and cotton samples though the coloration persisted, suggesting that the oil may influence pigment fixation, creating a mordant-like effect that has been described in other studies [31].

The pigmented oil layer degraded on exposure to bleach, likely through a saponification reaction. The strong alkalinity of bleach is also likely responsible for the color change seen in laundry samples. The pigment from *S. cuboideum* is known to turn blue under basic conditions, instead of its normal red coloration [32,33]. The mechanism behind this color change is under investigation but has not yet been described. It is possible that the variation in response to bleach seen may have been due to
physical protection of pigment-functional groups pigment by the oil layer or by pigment binding to fabric surfaces.

While bonding mechanisms of fungal pigment attachment to different fabrics were not tested, these likely impacted the differential responses seen in colorfastness. It is possible that hydroxy groups in cotton, and potentially amino acids in wool, may form hydrogen bonds with the methoxy and hydroxy groups present in the fungal pigment dramada. For the polyester fabric, it is likely that the aromatic π-electron system of the phthalic unit may be interacting with the π-electron system of dramada, providing a strong bond between the fabric and the pigmented oil. This interaction has already been reported with an anthraquinone present in natural dyes [1]. It is also interesting that, in previous research studies, polyester fabrics were successfully dyed with *S. cuboideum* when using DCM as a carrier [18] and later with raw linseed oil [25]. However, there is a need to further investigate which interaction causes the bonding between the fabric and the dye.

Heating resulted in changes of fabric color, as it was present in all statistically significant interactions identified, with all fabrics brightening except nylon, which may have been due to low initial pigment uptake. Under SEM, a superficial layer could be seen on the dyed areas after heating. This layer was also seen in samples subjected to laundry testing and it appeared to be made of oil: it presented as a thin, compacted, smooth surface, and, in some cases, it showed interstices or pores. This layer was best seen in high-density wool and low-density cotton, which showed the best performance, as measured by ΔE after the heat test. The layer may have been formed due to evaporation, oxidation, or polymerization of the oil under the effect of the temperatures. The polymerization process of linseed oil is well known [34] and this process may have allowed for molecular stacking of the pigment into its crystal formation. Using high temperatures to accelerate the evaporation of the raw linseed oil has been previously reported in the preparation of oilcloths as a way to reduce drying time [35], although no oil layer, such as the one found in this research, has ever been reported. It is also notable that previous research has shown that temperatures higher than 70 °C have been shown to degrade the red color of dramada [29], although this effect was not seen during this research. It is possible that the oil provided some protection for the pigment.

While this research showed that a range of fabrics could be effectively dyed using fungal pigment from *Scytalidium cuboideum*, this method of dying presented colorfastness challenges, especially for synthetic fibers. In addition, the texture of completed cloth may be an obstacle, as the oil does not polymerize to a dry layer quickly, making samples oily. However, treatment of cotton with linseed oil to create a waterproof layer, known as an oilcloth, was commonly done in the past [36] and may be a sustainable waterproofing option. Testing other cellulosic fibers, such as bamboo, rayon, or hemp, should also be investigated, as should the water repellence of the pigmented oil layer. In addition, new technology would likely need to be developed to allow for industrial processing of quantities of this pigmented oil. The economics of this product would require further research. In addition, natural oils have been researched for protection of exterior wood in service [37,38], which may be an additional use.

The challenges seen using pigment from *S. cuboideum* in linseed oil may limit the ability of this technology to be adopted in the market. However, the use of spalting fungal pigments like that from *S. cuboideum* still have great potential for use in the textile industry, especially as they can be used without the need for mordants [18]. Other natural pigments, such as those from plant sources, have relied on mordants for their color stability [39–42], and the lack of mordants needed for fungal pigments represents a significant advantage. While it was hoped that linseed oil would provide a less toxic alternative to DCM, future work into identifying new solvents appropriate for use with dramada would be helpful, especially for use with synthetic fabrics. Recent work using pigment from *S. cuboideum* in inkjet printers may be a particularly effective method of textile dying with fungal pigments [43].
**5. Conclusions**

Red pigment from *Scytalidium cuboideum* carried in linseed oil was found to effectively dye a variety of fabric varieties, with natural fibers showing better coloration and colorfastness overall. Mechanical properties were found to be essentially unaffected by the application of pigment. Colorfastness was shown to vary among fabrics, with application of bleach associated with a color shift from red to blue of the applied pigment. Heating was found to result in the production of a bright red fabric, which may have potential as a sustainable waterproofing treatment.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2309-608X/6/2/53/s1, Figure S1: SEM images of high-density cotton exposed to tensile strength test; Figure S2: SEM images of low-density cotton exposed to tensile strength test; Figure S3: SEM images of nylon exposed to tensile strength test; Figure S4: SEM images of high-density polyester exposed to tensile strength test; Figure S5: SEM images of low-density polyester exposed to tensile strength test; Figure S6: SEM images of high-density wool exposed to tensile strength test; Figure S7: SEM images of low-density wool exposed to tensile strength test; Figure S8: SEM images of high-density cotton for tear strength test; Figure S9: SEM images of low-density cotton for tear strength test; Figure S10: SEM images of high-density wool for tear strength test; Figure S11: SEM images of low-density wool for tear strength test; Figure S12: SEM images of nylon for tear strength test; Figure S13: SEM images of high-density polyester for tear strength test; Figure S14: SEM images of low-density polyester for tear strength test; Figure S15: SEM images of high-density cotton samples from laundry test; Figure S16: SEM images of low-density cotton samples from laundry test; Figure S17: SEM images of nylon samples from laundry test; Figure S18: SEM images of high-density wool samples from laundry test; Figure S19: SEM images of low-density wool samples from laundry test; Figure S20: SEM images of low-density polyester samples.

**Author Contributions:** Conceptualization, S.C.R. and H.-L.C.; methodology, S.C.R. and H.-L.C.; validation, S.C.R. and H.-L.C.; formal analysis, M.E.P.A., S.M.V.G., and H.-L.C.; investigation, M.E.P.A.; resources, S.C.R. and H.-L.C.; data curation, M.E.P.A. and S.M.V.G.; writing—original draft preparation, M.E.P.A. and S.V.M.G.; writing—review and editing, R.C.V.C., S.M.V.G., H.-L.C., and S.C.R.; supervision, S.C.R. and H.-L.C.; project administration, S.C.R. and H.-L.C.; funding acquisition, S.C.R. and H.-L.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Walmart Foundation Manufacturing Fund.

**Acknowledgments:** Authors gratefully acknowledge Oregon State University Electron Microscopy Facilities, Peter Eschbach, and Teresa Sawyer and would like to thank Ariel Muldoon for help with statistical analysis.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Review

# Use of the Versatility of Fungal Metabolism to Meet Modern Demands for Healthy Aging, Functional Foods, and Sustainability

Jacqueline A. Takahashi <fnref n="1" />,* (D), Bianca V. R. Barbosa <fnref n="1" />, Bruna de A. Martins <fnref n="1" />(D), Christiano P. Guirlanda <fnref n="2" /> and Marília A. F. Moura <fnref n="2" />(D)

Received: 31 August 2020; Accepted: 27 September 2020; Published: 15 October 2020

<figure><img src="image_118.png" /></figure>

check for
updates

**Abstract:** Aging-associated, non-transmissible chronic diseases (NTCD) such as cancer, dyslipidemia, and neurodegenerative disorders have been challenged through several strategies including the consumption of healthy foods and the development of new drugs for existing diseases. Consumer health consciousness is guiding market trends toward the development of additives and nutraceutical products of natural origin. Fungi produce several metabolites with bioactivity against NTCD as well as pigments, dyes, antioxidants, polysaccharides, and enzymes that can be explored as substitutes for synthetic food additives. Research in this area has increased the yields of metabolites for industrial applications through improving fermentation conditions, application of metabolic engineering techniques, and fungal genetic manipulation. Several modern hyphenated techniques have impressively increased the rate of research in this area, enabling the analysis of a large number of species and fermentative conditions. This review thus focuses on summarizing the nutritional, pharmacological, and economic importance of fungi and their metabolites resulting from applications in the aforementioned areas, examples of modern techniques for optimizing the production of fungi and their metabolites, and methodologies for the identification and analysis of these compounds.

**Keywords:** fungi; secondary metabolites; metabolomics; NTCD; additives; functional foods; nutraceuticals; sustainability; healthy aging

## 1. Health and Modern Food Demands

Health concerns have always existed among humans. Although some conditions and diseases cannot be avoided yet, the manifestation of several non-transmissible chronic diseases (NTCD) with high prevalence in patients over 60 years of age such as diabetes, cardiovascular, and neurodegenerative diseases can be delayed by adhering to a healthy lifestyle, which among other factors, is directly correlated to eating habits. The physiological effects associated with the consumption of certain foods are thus becoming very popular. Several types of diets and foods such as the fat-free diet, low-carb diet [1], Mediterranean diet [2], and soy-based diet [3] have been adopted in the quest for healthy aging. Several effects of NTCD have also been postponed through calorie restriction diets in animal models [4]. However, prolonged caloric restriction in humans generates undesirable effects; thus, alternative ways of preventing NTCD have been sought through the development of drugs, foods, and/or nutraceuticals that have both health-promoting and anti-aging effects, without causing adverse effects [5]. There is an

<footnote n="1">¹ Department of Chemistry, Exact Sciences Institute, Universidade Federal de Minas Gerais, Pres. Antônio Carlos Avenue, 6627, Pampulha, Belo Horizonte 31270-901, MG, Brazil; biancavrb99@gmail.com (B.V.R.B.); bruna.almeidamartins@gmail.com (B.d.A.M.)</footnote>

<footnote n="2">² Department of Food Science, Faculty of Pharmacy, Universidade Federal de Minas Gerais, Pres. Antônio Carlos Avenue, 6627, Pampulha, Belo Horizonte 31270-901, MG, Brazil; cpguirlanda@gmail.com (C.P.G.); mourafmari@gmail.com (M.A.F.M.)</footnote>

<footnote>* Correspondence: jat@qui.ufmg.br</footnote>
increasing trend to combine the use of nutraceuticals with pharmacotherapy, even among individuals with non-aging-related diseases.

As oxidative stress is among the metabolic factors and pathways most related to cell aging, the consumption of nutraceuticals and functional foods with antioxidant activity has increased. Antioxidants can benefit the human body by directly or indirectly neutralizing reactive oxygen species (ROS), modulating metabolic pathways and gene expression, and activating mechanisms of cellular stress and autophagy that delay aging through pathways unrelated to ROS [5]. The nutraceutical market has already reached market values up to USD 117 billion [6]. Nutraceuticals can be classified into several categories based on the level of innovation and area of application (Table 1) [7,8]. Another trend associated with health improvement and NTCD prevention is that of consuming natural foods or foods containing natural, rather than synthetic, additives such as natural flavoring agents, acidulants, and colorants. Several people also prefer vegetarian and vegan diets, which involve restrictions in food and additives of animal origin to different extents; such diets are mainly motivated by the environmental impacts of livestock farming and animal welfare and have prompted studies on the possible effects of these diets on health [9] and increased the market demand for new vegetarian and vegan food products.

Filamentous fungi are capable of responding to different demands for the development of functional foods, nutraceuticals, and bioactive substances that can be used as medicines or in the food industry, either through the use of their biomass or the metabolites produced by them. A comprehensive review of the various aspects of fungal biotechnology and industrial applications was recently published by Meyer et al. [10].

The consumption of fungi as food mainly involves the consumption of mushrooms (Ascomycota and Basidiomycota phyla), which are enjoyed worldwide, sometimes as delicatessen or gourmet products. However, the role of fungi in human and animal health extends much further than the recognized health benefits of mushrooms [11]. Many fungal species are commercially available as supplements or nutraceuticals, and the fungal metabolites produced by these organisms including many non-Basidiomycota species as functional foods have multiple pharmacological activities. Examples of fungal species used as functional foods include many species of microscopic filamentous fungi that are easily cultivated under ex situ scalable conditions such as some well-known Penicillium, Aspergillus, and Fusarium species. However, lesser known species such as Ashbya gossypii also play an important role in the production of food additives such as riboflavin (vitamin B2) (1) (Figure 1) [12]. Other important fungal products associated with health improvement are enzymes such as β-galactosidase, which hydrolyzes lactose from dairy foods, is produced by filamentous fungi such as Trichoderma sp., and is helpful for lactose-intolerant individuals [13].

<table><caption>**Table 1.** Examples of food additives and nutraceuticals from fungi.</caption><thead><tr><th>Category</th><th>Active Component</th><th>Bioactivity</th><th>Fungal Source</th><th>References</th></tr></thead><tbody><tr><td>Nutrient</td><td>Vitamin C (55)</td><td>Antioxidant</td><td>Dictyophora indusiata</td><td>[14]</td></tr><tr><td rowspan="2">Secondary Metabolites</td><td>Resveratrol</td><td>Antioxidant</td><td>Pleurotusostreatus</td><td>[15]</td></tr><tr><td>Agmatine (28)</td><td>Neurological benefits</td><td>Aspergillus oryzae</td><td>[16]</td></tr><tr><td>ω-6</td><td>γ-linolenic acid</td><td>Anti-inflammatory</td><td>Mucor circinelloides</td><td>[17]</td></tr><tr><td>Polyunsaturated fatty acid</td><td>Arachidonic acid</td><td>Development of the nervous central system and enhancement of immune response</td><td>Mortierella alpina</td><td>[18]</td></tr><tr><td>Probiotic</td><td>Whole cell</td><td>Increase of beneficial bacteria population in gastrointestinal tract</td><td>Saccharomyces boulardii</td><td>[19]</td></tr><tr><td rowspan="2">Nutraceutical Enzymes</td><td>Fibrino(geno)lytic enzymes</td><td>Antithrombotic</td><td>Penicillium sp.</td><td>[20]</td></tr><tr><td>Lipase (Lipopan F)</td><td>Decrease glycemic response</td><td>Rhizopus microsporus</td><td>[21]</td></tr><tr><td>Fortified Nutraceuticals</td><td>Folate in fermented maize-based porridge</td><td></td><td>Rhizopus oryzae</td><td>[22]</td></tr><tr><td></td><td></td><td></td><td>Saccharomyces cerevisiae</td><td>[23]</td></tr></tbody></table>

This review discusses the usefulness of metabolites produced by fungi for food and pharmaceutical purposes closely linked to health improvement and the prevention of NTCD, respectively, as lead compounds, additives, nutraceuticals, supplements, and functional ingredients. The health benefits of fungal metabolites are highlighted. Cutting-edge tools for yield improvement and thus the scaling up
of fungal metabolite production as well as the means for ensuring a successful circular economy in this area are also discussed.

<figure><img src="image_119.png" /><figcaption>**Figure 1.** Chemical structures of some fungal-derived food additives (1–4, 22, 51–55).</figcaption></figure>

## 2. Natural Food Additives from Fungi

Prompted by the growing evidence for the association between natural compound intake and health, there is a demand for the replacement of synthetic food additives with natural products. Fungal metabolites feature many properties that have been explored for such replacement. This section presents the potential of fungal metabolites as food additives, focusing on their versatility as coloring agents, producing species, and recent achievements and challenges in this area.

Fungi provide several food additives and technology adjuvants such as organic acids, colorants, and fatty acids including some ω-3 and ω-6 class fatty acids, which are essential for human metabolism. Citric acid (2) and fumaric acid (3) (Figure 1), metabolites of *Aspergillus niger* and *Rhizopus oryzae*, respectively, are outstanding food additives that are industrially produced. Citric acid (2) and gluconic acid (4) (Figure 1) are fungal products with the highest commercial-scale production [24]. Citric acid (2) has a number of applications as an antioxidant, preservative, acidulant, pH control agent, and flavor regulation agent, with market numbers predicted to reach USD 3.6 billion in 2020 [24].

The harmful effects of synthetic food colorants on human health such as attention-deficit/hyperactivity disorder, asthma and allergies, cancer, and neurological disorders have accelerated the search for natural substitutes. The use of the yellow coloring compounds tartrazine, quinoline, and sunset as well as the red coloring compound amaranth [25] has been regulated and supervised by the World Health Organization. In addition, sustainability issues have contributed to the decreased acceptability of non-biodegradable synthetic colorants that are difficult to remove from effluents, causing toxic effects on plants, bacteria, algae, fishes, and crustaceans [26]. Fungal pigments and dyes have emerged as alternatives to these synthetic food additives [27].

Food dyes are coloring materials soluble in food substrates, while food pigments are insoluble in food and need to be carried by vehicles that bind to the food instead [28] (ACS, 2020). Food colorants are pigments or dyes approved for use as food additives [29] (FDA, 2017). A wide spectrum of natural colors can be obtained from metabolites of fungi of different genera such as *Eurotium*, *Fusarium*,
*Monascus*, and *Penicillium*, isolated from marine and terrestrial environments and extracted by techniques considered environmentally friendly including those employing ultrasound, pressurized liquid, microwaves, and pulsed electric field [30]. The fungal metabolites have a wide color range, which can be represented by carotenoids such as lycopene (red) (5), β-carotene (yellow-orange) (6), and astaxanthin (pink-red) (7); azafilones such as monascorubrin (orange) (8) and ankaflavin (yellow) (9); and the quinine derivatives alizarin (purple-red) (10) and bikaverin (red) (11) (Figure 2). Arpink Red (12) (Figure 2), an anthraquinone produced by *Penicillium oxalicum*, has been approved by the Codex Alimentarius for use as a food colorant in meat products, dairy, confectionery, ice cream, and alcoholic and non-alcoholic beverages [31,32]. Naftoquinone hydrosoluble metabolites (purple color) were produced by a soil-originated strain of *Fusarium oxysporum* using a simple culture medium containing glucose, ammonium sulfate, and salts [33]. Crystalline neoechinulin A (13) (ivory color), neoechinulin B (14), and cladosporin (15) (all having a yellow color) (Figure 2) are described as metabolites of *Eurotium amstelodami*, *Eurotium herbariorum*, and *Eurotium rubrum* isolated from outdoor and indoor samples in Canada and grown in medium containing sucrose, yeast extract, and salts [34].

The filamentous fungus *Monascus purpureus* is one of the first species that was used to produce natural colorants. It is traditionally consumed in Asia in fermented red rice, which is important in Chinese cuisine, and used in folk medicine as a regulator of digestive and circulatory functions [35,36]. Some species of the genus *Monascus* produce the secondary metabolite monacolin K (16) (Figure 2), which consists of a lactone with a free hydroxyl-acid moiety. Aside from its coloring property, monacolin K (16) has a high antioxidant activity and is marketed as a hypocholesterolemic drug known as lovastatin. The pigments of yellow, orange, and red color produced by the *Monascus* genus are considered safe for human consumption [37].

The development of mutant strains of *Monascus* has enabled an increase in available pigments including monashin (17) (Figure 2), which is obtained through the mutation of the enzyme polyketide synthase and has an antioxidant activity [35]. The monascin (18) (Figure 2) (yellow) produced by *M. purpureus* after activation by the transcription factors DAF-16/FOXO increased the production of superoxide dismutase and thermal shock protein HSP16.2, improving survival in a worm model [38]. Monascin (18) also reduced non-alcoholic fatty liver disease and increased AMPK levels and γ 1α co-activator of the receptor activated by peroxisome proliferator in mice [39]. In another study, a new azaphylone, monapurpureusone (19), and a new brownish natural product, monapurpureusin (20) (Figure 2), were obtained from a mutant strain of *M. purpureus* cultivated in fermented rice extract. Both compounds presented superoxide radical scavenging activity (EC₅₀ = 176.2 and 271.2 µM, respectively), with monapurpureusone (19) superior to the control gallic acid (237.1 µM), and anti-inflammatory activity (IC₅₀ = 27.5 and 24.9 µM, respectively), with both compounds superior to the control quercetin (35.9 µM) [36].

Red and orange pigments were produced by a strain of *Talaromyces albobiverticillius* isolated from a marine environment at pH 6.5, and their colors were shown to be dependent on the fermentation period (198.6 and 229.0 h, respectively) [40]. Optimal conditions for the production of pigments were also described for *F. oxysporum* (red color, rate C:N = 9, blue LED light, and absence of co-culture) and *Aspergillus chevalieri* (yellow color, rate C:N = 20, glucose as the carbon source, UV and red light, and co-cultivation with *Kluyveromyces marxianus*) [41]. The production of natural food colorants such as melanins, azaphilones, flavins, phenazines, and quinines by filamentous fungi has also been reported for Basidiomycetes [28].

The replacement of synthetic colorants with fungal pigments and dyes enables the production of safer and healthier foods. Compared with plant sources of natural pigments, fungal sources are more economically attractive, considering the relative ease of yield maximization by manipulating fungal fermentative parameters instead of relying on seasonal factors, as may occur in the production of pigments and dyes of plant origin.
<figure><img src="image_120.png" /><figcaption>**Figure 2.** Chemical structures of some colored fungal secondary metabolites (5–20).</figcaption></figure>
Nevertheless, some challenges need to be considered for regulating new food-related colorants such as the possibility of interactions with the food matrix, which causes undesirable sensorial changes, loss of color stability, and contamination by toxic substances such as mycotoxins. Some alternatives proposed for these problems are based on the controlled release of the colorant using microencapsulation as well as the use of nanoformulations to eliminate undesirable aromas and flavors [30]. Spray-drying microencapsulation was successfully used in broth fermented by three species producing yellow dyes, *Aspergillus keveii*, *Penicillium flavigenum*, and *Epicoccum nigrum*. Encapsulation with three adjuvants (maltodextrin, modified starch, and gumarabic) provided pigment retention above 70% [41]. With respect to problems linked to the production of mycotoxins such as citrinin (21) (Figure 3) produced by *Monascus*, strategies vary from changes in cultivation and fermentation conditions to disruption of genes encoding the production of mycotoxins in question to create non-mycotoxin-producing mutant strains [30].

<figure><img src="image_121.png" /><figcaption>**Figure 3.** Chemical structures of some mycotoxins (21, 29) and classes of mycotoxins (30, 31) frequently produced by fungal species.</figcaption></figure>

The use of fungal metabolites as natural colorants contributes greatly to the development of healthier foods. Nonetheless, several other additives of fungal origin such as substances related to taste and pH (acidulants, flavorings, and sweeteners), texture (thickeners and emulsifiers), and increased shelf life (antioxidants and preservatives) have been studied. Some examples can be found in Table 2. The application of some of these additives including exopolysaccharides is not restricted to the food industry. Fungal exopolysaccharides produced by species such as *Phellinus linteus*, *Ganoderma lucidum*, *Fusarium* sp., *Pleurotus* spp., *Inonotus obliquus*, and *Aureobasidium pullulans* have been employed to aid moisture retention in confectionery, increase the viscosity and crystallization of sugar, and as stabilizers, emulsifiers, and thickening agents [42,43]. These compounds are also of great interest to textiles, food, cosmetics, and pharmaceutical industries and are also important in agriculture as preservatives, bioherbicides, and microbicides.
<table><caption>**Table 2.** Examples of non-coloring natural additives from fungal origin.</caption><thead><tr><th>Function</th><th>Compound Name and Structure</th><th>Fungus</th><th>Main Uses</th><th>References</th></tr></thead><tbody><tr><td>Acidulant</td><td>Malic acid<br><img src="malic_acid.png" alt="Malic acid structure"></td><td>A. niger</td><td>Candies, soft drinks, baked goods</td><td>[44]</td></tr><tr><td>Antioxidant</td><td>Gallic acid<br><img src="gallic_acid.png" alt="Gallic acid structure"></td><td>A. niger</td><td>Any food susceptible to oxidation</td><td>[45]</td></tr><tr><td>Thickener agent</td><td>Galactomannans and β-1,3-glucans<br><img src="galactomannans.png" alt="Galactomannans structure"></td><td>Aspergillus terreus</td><td>Sauces, soups, puddings, fillings</td><td>[46]</td></tr><tr><td>Emulsifier</td><td>Sorbitol<br><img src="sorbitol.png" alt="Sorbitol structure"></td><td>Neopetalotopsis sp.</td><td>Baked goods and desserts</td><td>[47]</td></tr><tr><td>Flavoring agent</td><td>Dihydro-β-ionone<br><img src="dihydro-ionone.png" alt="Dihydro-ionone structure"></td><td>Volvariellavolvacea</td><td>Beverages, ice creams, candies</td><td>[48]</td></tr><tr><td>Preservative</td><td>Natamycin<br><img src="natamycin.png" alt="Natamycin structure"></td><td>Streptomyces gilvosporeus</td><td>Fungicide used as biopreservative in dairy and meat products</td><td>[49]</td></tr><tr><td>Sweetener</td><td>Mogroside V<br><img src="mogroside_V.png" alt="Mogroside V structure"></td><td>Diaporthe angelicae and Fusarium solani</td><td>Juices, soft drinks, cereals, confectionary, baked goods</td><td>[50]</td></tr></tbody></table>

Metabolic engineering of microorganisms has been successfully employed for producing 42 out of the 316 food additives from numerous species of fungi and bacteria currently approved by the European Union [44]. Using this technique, the production of glutamic acid, a metabolite capable of providing “umami” flavor to food, though fermentation with *Corynebacterium glutamicum* was optimized. The production of malic acid (22) (Figure 1), an acidulant used in food and beverages, was increased after overexpression of the genes encoding its precursors in the species *S. cerevisiae*, *Aspergillus flavus*, *Aspergillus oryzae*, and *A. niger* [51,52]. Metabolic engineering can also be used to introduce heterologous routes for enzyme production in microorganisms (e.g., to produce enzymes of plant origin) [51]. Changes in the molecular structures and colors of microbial pigments were made using the engineering system CRISPR-Cas9, which cleaves specific microbial DNA sites and introduces changes in one or more target genes [30]. For instance, *Escherichia coli* was used for the production of β-carotene (6) through genomic editing to introduce insertions, deletions, and substitutions in regions of the lacZ, galK, and ldhA genes using the CRISPR-Cas9 system [53].

A yeast species, *Yarrowia lipolytica*, was engineered by introducing genes for the production of β-ketolase and β-hydroxylase of seaweed and bacteria origin, increasing the yield of astaxanthin
(7) (285 ± 19 mg/L; 47% of total carotenoids) [54]. This was a great achievement, given the lower productivity of astaxanthin (7) by yeasts like *Xanthophyllomyces dendrorhous* (10.2 mg/L) [55] and microalgae like *Haematococcus pluvialis* (84.8 mg/L) [56] and *Chlorella zofingiensis* (12.5 mg/L) [57]. Astaxanthin (7) has a high market value (USD 2500–7000/kg), 5–20 times higher than that of β-carotene (6) produced by the microalga *Dunaliella salina* (USD 300–500/kg) [54,58].

Overall, tools such as genetic manipulation based on transcriptomic analysis, induction of mutations, cloning, and insertion of heterologous plasmids into species with well-known genomes have enabled the production of large quantities of metabolites by previously non-producing species [59]. The elucidation and manipulation of the different stages in the transcription and secretion of amylases, xylanases, and cellulases in filamentous fungi has enabled their overexpression [60]; these enzymes are widely used in bakery products to improve the quality of dough through hydrolysis of long-chain carbohydrates and non-starch polysaccharides (cellulose and arabinoxylans).

It was hypothesized that enzymes produced by *A. oryzae* and *A. niger* could cleave the anti-nutritional factors present in flour, in addition to hydrolyzing proteins and carbohydrates into smaller molecules and thus making them more accessible for digestion and releasing phenolic compounds from the matrix. However, the fungi used the amino acids present in the flour for their own metabolism, which negatively affected the protein quality of the final product [61]. A new fermented food product was thus developed using stale bread as a substrate for the fungus *Neurospora intermedia*, which converted 65% of the starch into 21% of protein, in addition to supplementing the final product with minerals and vitamins; although there was a reduction in the amounts of proline, glutamic acid, and phenylalanine, the overall amino acid composition was improved [62].

### 3. Benefits, Research, and Industrial Applications of Fungal Metabolites

The scope of the pharmacological activity of fungal metabolites seems to be as endless as the structural diversity. However, several issues in this area must be addressed such as the extent of in vivo effects. The wide range of beneficial biological effects of fungal metabolites can be related to the prevention and treatment of NTCD, and some such effects have identified several potential compounds for developing new drugs.

Terrein (23) (Figure 4), for instance, is a secondary metabolite biosynthesized in high concentrations (537.26 ± 23.42 g/kg crude extract) by *A. terreus* [63]. The anti-inflammatory and antioxidant properties of terrein (23) were reported in in vitro studies [64]. These important medicinal properties and the high initial yield of this metabolite allow the large-scale production and technological development of the *A. terreus* crude extract for the prevention of some age-related NTCD.

Compounds presenting cytotoxicity against tumor cells form one of the most important classes of fungal metabolites. The world incidence of cancers is high, with cancer affecting one in five people at some point in life. The number of cancer cases registered in 2018 was 18.1 million people, a number that could double in 2040 [65]. Cancer is a multifactorial disease that affects people regardless of age, gender, or origin. As an NTCD, cancer is worrying in several ways, since working-age people can be affected, treatment is costly and long, mortality rates are high, and individuals often have health issues even after cure.

Many reports on fungal metabolites with cytotoxic activity show the vast arsenal of molecules that fungi provide to combat cancer. Table 3 provides examples of antitumor metabolites produced by fungi. Species from different genera or secondary metabolites produced by them sometimes present inhibitory power more pronounced than or relatively close to that presented by standard compounds. This was observed for hypocriol A (24) and F (25) (Figure 4), isolated from the strain *Hypocrea* sp. [66], and for sesquiterpene strichocaranes E (26) and F (27) (Figure 4), isolated from the entomopathogenic fungus *Isaria fumosorosea* [67], in comparison to the standard cisplatin. Many human tumor cell lines can be inhibited by fungal metabolites such as breast (MDA and MCF-7) [67], cervical and lung [66], colorectal [63], gastric and liver [68], and pancreatic [69] cell lines. The majority of compounds
presented in Figure 4 are terpenes with different degrees of hydroxylation as well as linear and cyclic nitrogen-bearing compounds with free NH groups.

<figure><img src="image_122.png" /><figcaption>Figure 4. Cont.</figcaption></figure>
<figure><img src="image_123.png" /><figcaption>**Figure 4.** Chemical structures of metabolites (23–28, 32–41, 43–50) and class of bioactive compounds (42) produced by fungal species.</figcaption></figure>
<table><caption>**Table 3.** Cytotoxic activity of some fungal secondary metabolites against several human tumor cell lines.</caption><thead><tr><th rowspan="2">Metabolite</th><th rowspan="2">Fungal Origin</th><th rowspan="2">Yield</th><th colspan="4">Cytotoxic Activity</th><th rowspan="2">Reference</th></tr><tr><th>Human Tumor Cell</th><th>IC50</th><th>Control</th><th>IC50</th></tr></thead><tbody><tr><td rowspan="6">7-Desmethyl-6-methylbostrycoidin (32)</td><td rowspan="6">F. solani</td><td rowspan="6">7 mg/4.3 g extract</td><td>Breast (MDA MB 231)</td><td>0.73 µM</td><td rowspan="6">Doxorubicin</td><td>0.07 µM</td><td rowspan="6">[69]</td></tr><tr><td>Pancreatic (MIA PaCa2)</td><td>0.64 µM</td><td>0.04 µM</td></tr><tr><td>Cervical (HeLa)</td><td>0.71 µM</td><td>0.05 µM</td></tr><tr><td>Non small-cell lung (NCI H1975)</td><td>0.34 µM</td><td>0.03 µM</td></tr><tr><td>Lung fibroblast (WI38)</td><td>6.42 µM</td><td>0.35 µM</td></tr><tr><td>Breast (MDA MB 231)</td><td>1.51 µM</td><td>0.07 µM</td></tr><tr><td rowspan="6">7-Desmethylscorpinone (33)</td><td rowspan="6">F. solani</td><td rowspan="6">5 mg/4.3 g extract</td><td>Pancreatic (MIA PaCa2)</td><td>0.98 µM</td><td rowspan="6">Doxorubicin</td><td>0.04 µM</td><td rowspan="6">[69]</td></tr><tr><td>Cervical (HeLa)</td><td>0.96 µM</td><td>0.05 µM</td></tr><tr><td>Non small-cell lung (NCI H1975)</td><td>0.61 µM</td><td>0.03 µM</td></tr><tr><td>Lung fibroblast (WI38)</td><td>5.84 µM</td><td>0.35 µM</td></tr><tr><td>Ergosterol (43)</td><td>Penicillium chrysogenum</td><td>ni</td><td>Breast (MCF-7)</td><td>0.10 mM</td><td>ni</td><td>ni</td></tr><tr><td rowspan="2">Ergosterol peroxide (44)</td><td rowspan="2">V. volvacea</td><td rowspan="2">500 mg/725 g extract</td><td>Prostate (PC-3M)</td><td>27.98 ± 0.97 µM</td><td>5-Fluorouracil</td><td>64.35 µM</td><td rowspan="2">[68]</td></tr><tr><td>Prostate (PC-3M)</td><td>23.15 ± 1.54 µM</td><td>5-Fluorouracil</td><td>64.35 µM</td></tr><tr><td>3β, 5α, 9α, 14 α-Tetrahydroxy-ergosta-7,22-dien-6-one (45)</td><td>V. volvacea</td><td>15 mg/725 g extract</td><td>Liver (HepG2)</td><td>20.72 ± 0.76 µM</td><td>5-Fluorouracil</td><td>54.74 µM</td><td rowspan="2">[68]</td></tr><tr><td rowspan="2">3β, 5α, 9α-Trihydroxy-ergosta-7,22-dien-6-one (46)</td><td rowspan="2">V. volvacea</td><td rowspan="2">12.5 mg/725 g extract</td><td>Gastric (SGC-7901)</td><td>12.03 ± 0.77 µM</td><td rowspan="2">5-Fluorouracil</td><td>75.05 µM</td></tr><tr><td>Liver (HepG2)</td><td>5.90 ± 0.44 µM</td><td>54.74 µM</td></tr><tr><td rowspan="6">Hypocriol A (24)</td><td rowspan="6">Hypocrea sp.</td><td rowspan="6">50.3 mg/63.2 g extract</td><td>Colorectal (HCT116)</td><td>18.6 ± 0.7 µM</td><td rowspan="6">Cisplatin</td><td>18.8 ± 1.9 µM</td><td rowspan="6">[66]</td></tr><tr><td>Cervical (HeLa)</td><td>7.7 ± 0.4 µM</td><td>14.7 ± 0.8 µM</td></tr><tr><td>Lung (A549)</td><td>25.3 ± 2.5 µM</td><td>13.8 ± 1.2 µM</td></tr><tr><td>Breast (MCF-7)</td><td>19.7 ± 0.4 µM</td><td>17.6 ± 2.4 µM</td></tr><tr><td>Colorectal (HCT116)</td><td>2.7 ± 0.6 µM</td><td>18.8 ± 1.9 µM</td></tr><tr><td>Cervical (HeLa)</td><td>4.6 ± 0.1 µM</td><td>14.7 ± 0.8 µM</td></tr><tr><td rowspan="4">Hypocriol F (25)</td><td rowspan="4">Hypocrea sp.</td><td rowspan="4">11.2 mg/63.2 g extract</td><td>Lung (A549)</td><td>15.3 ± 1.6 µM</td><td rowspan="4">Cisplatin</td><td>13.8 ± 1.2 µM</td><td rowspan="4">[66]</td></tr><tr><td>Breast (MCF-7)</td><td>23.6 ± 1.3 µM</td><td>17.6 ± 2.4 µM</td></tr><tr><td>Colorectal (HCT-116)</td><td>12.13 µM</td><td>0.11 µM</td></tr><tr><td>Hepatocellular (HepG2)</td><td>22.53 µM</td><td>0.85 µM</td></tr><tr><td rowspan="8">Trichocarane E (26)</td><td rowspan="8">I. fumosorosea</td><td rowspan="8">30 mg/200 g extract</td><td>Breast (MDA)</td><td>0.13 µg/mL</td><td rowspan="8">Cisplatin</td><td>2.90 µg/mL</td><td rowspan="8">[67]</td></tr><tr><td>Breast (MCF-7)</td><td>2.46 µg/mL</td><td>1.14 µg/mL</td></tr><tr><td>Ovary (SKOV-3)</td><td>1.01 µg/mL</td><td>3.80 µg/mL</td></tr><tr><td>Cervical (Hela)</td><td>2.32 µg/mL</td><td>2.24 µg/mL</td></tr><tr><td>Lung (A549)</td><td>1.40 µg/mL</td><td>2.13 µg/mL</td></tr><tr><td>Liver (HepG2)</td><td>1.87 µg/mL</td><td>0.62 µg/mL</td></tr><tr><td>Breast (MDA)</td><td>0.89 µg/mL</td><td>2.90 µg/mL</td></tr><tr><td>Breast (MCF-7)</td><td>4.38 µg/mL</td><td>1.14 µg/mL</td></tr><tr><td rowspan="4">Trichocarane F (27)</td><td rowspan="4">I. fumosorosea</td><td rowspan="4">41 mg/200 g extract</td><td>Ovary (SKOV-3)</td><td>1.46 µg/mL</td><td rowspan="4">Cisplatin</td><td>3.80 µg/mL</td><td rowspan="4">[67]</td></tr><tr><td>Cervical (Hela)</td><td>4.57 µg/mL</td><td>2.24 µg/mL</td></tr><tr><td>Lung (A549)</td><td>1.66 µg/mL</td><td>2.13 µg/mL</td></tr><tr><td>Liver (HepG2)</td><td>3.66 µg/mL</td><td>0.62 µg/mL</td></tr></tbody></table>

Note: ni = not informed.
Despite the huge number of fungal bioactive metabolites, there is controversy regarding the consumption or development of new drugs from metabolites biosynthesized by toxin-producing fungi. Terrein (23) and agmatine (28) (Figure 4) are examples of biologically active metabolites produced by *Aspergillus*, a genus often associated with the production of mycotoxins [71,72]. However, species from *Aspergillus* and *Fusarium* genera, known sources of toxins such as zearalenone (29), trichothecenes (30), and fumonisins (31) (Figure 3), have shown a biotechnological potential beyond the production of mycotoxins. For example, azaanthraquinone derivatives, 7-desmethyl-6-methylbostrycoidin (32), and 7-desmethylscorpinone (33) (Figure 4), isolated from *F. solani* cultures, showed significant activity against tumor cell lines (Table 3) [69]. The mycoprotein "Quorn", a popular fungal food for human consumption, is produced using mycelia of *Fusarium* sp. [72]. Therefore, disregarding the potential of species from mycotoxin-producing genera is unnecessary in the research and industrial development of food products and medicines.

Cancer and neurodegenerative diseases are pathologies targeted by antioxidant therapies, not only for treatment but also for prevention, as proven by pre-clinical, clinical, and epidemiological studies [73]. Although compounds and foods with antioxidant activity have been targeted by many studies [5,74], some brief considerations are worthwhile. Despite encouraging data from several screenings pointing to a very significant number of fungal products with antioxidant activity, it is important to note that most of the experiments in these studies were not applied to tissues and organ systems, and the pharmacokinetic aspects of the absorption of these substances by the human organism were not evaluated [74]. Thus, the straightforward extrapolation of the results of these screenings to in vivo human applications is not possible [75].

Conversely, the in vitro antioxidant profile of a natural product can be improved. This was observed in a study in which three strains of *M. circinelloides* (CBS 277.49, WJ11, and CBS 108.16) were grown for different time periods (three, five, and seven days) in different culture media (standard Kendrick medium and Ratledge and modified Kendrick and Ratledge media (MKR), with nitrogen deficiency) and evaluated for phenolic compounds and antioxidant capacity. The total phenolics (TPC) and flavonoids (TFC) were improved for CBS 277.49, while CBS 108.16 produced a higher amount of condensed tannins (TCT). The ethanol extract obtained from CBS 277.49 (five days of growth in MKR medium) presented the best results regarding neutralization of the2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS⁺) radical, copper reducing power, and ferric reducing power. The latter had a higher ferric reducing power than those of the standards BHT and α-tocopherol [76].

Despite the relevance of cancer as an NTCD, several other diseases linked to aging limit a healthy life as people age. Accumulated dietary deficiencies throughout life and loss of immunity increase mortality rates associated with non-fatal diseases such as infections. Chronic inflammation is another worrisome condition that increases the risk of developing various pathologies such as hypertension, diabetes, and neurodegenerative diseases [77]. Although early diagnosis and measures to avoid aging-related pathologies and their consequences are essential, drugs for existing diseases continue to be required. Table 4 presents the structures of fungal metabolites associated with the treatment or control of infections, inflammatory and neuroinflammatory conditions, and Alzheimer's disease and examples and extents of the biological activities of these compounds.
<table><caption>**Table 4.** Examples of non-cytotoxic biologically active fungal secondary metabolites.</caption><thead><tr><th rowspan="2">Fungal Species</th><th rowspan="2">Bioactive Compound</th><th colspan="3">Bioactivity</th><th rowspan="2">Health Benefit</th><th rowspan="2">References</th></tr><tr><th>Target of Inhibitory Activity</th><th>Value</th><th>Control</th></tr></thead><tbody><tr><td>Cladosporium sphaerospermum</td><td>Cladosin L</td><td>Staphylococcus aureus</td><td>MIC = 25–50 µM</td><td>ni</td><td>Antibacterial</td><td>[78]</td></tr><tr><td>Fusarium chlamydosporum</td><td>Chlamydosterol A</td><td>5-lipoxygenase (5-LOX)</td><td>IC<sub>50</sub> = 3.06 µM</td><td>Indomethacin IC<sub>50</sub> = 1.13 µM</td><td>Anti-inflammatory</td><td>[79]</td></tr><tr><td>Hypoxylon sp.</td><td>Hypoxylon xanthone A</td><td>LPS-induced NO production</td><td>>70% at 1 µM</td><td>Minocycline >60% at 1 µM</td><td>Anti-neuroinflammatory</td><td>[80]</td></tr><tr><td>Rhizopycnis vagum</td><td>Rhizovagine A</td><td>Acetylcholinesterase enzyme</td><td>IC<sub>50</sub> = 43.1 µM</td><td>Tacrine hydrochloride IC<sub>50</sub> = 6.1 µM</td><td>Treatment for Alzheimer's disease</td><td>[81]</td></tr><tr><td>Saccharicola sp.</td><td>Speciosin U</td><td>Acetylcholinesterase (huAChE-ICER)</td><td>IC<sub>50</sub> = 0.037 ± 0.01 mg.mL<sup>-1</sup></td><td>Galantamine IC<sub>50</sub> = 0.076 ± 0.01 mg.mL<sup>-1</sup></td><td>Treatment for Alzheimer's disease</td><td>[82]</td></tr><tr><td>Trichoderma sp.</td><td>Coniothyrinone A</td><td>Vibrio anguillarum</td><td>MIC = 1.56 µM</td><td>Ciprofloxacin MIC = 0.625 µM</td><td>Antibacterial</td><td>[83]</td></tr></tbody></table>

ni = not informed.
The use of epigenetics for the production of new secondary metabolites through the activation of biosynthetic routes involving transcriptional genes previously silenced is an approach to increase and diversify the production of bioactive secondary metabolites by fungi. The use of a mutant strain of *Fusarium graminearum* lacking H3K27 methyltransferase after removal of the secondary metabolism repressor *kmt6* gene was reported as being advantageous for obtaining 22 metabolites, three of which had not been previously identified to be produced by this species: N-ethyl anthranilic acid (34), N-phenetylacetamide (35), and N-acetyltryptamine (36) (Figure 4). The production of the double mutant *kmt6fus 1*, with elimination of the production of compounds of the class fusarins, enabled the discovery of two new sesquiterpenes, tricinolone (37) and tricinolonic acid (38) (Figure 4) [84].

## 4. Mushrooms as Functional Foods for Preventing Aging-Related Non-Transmissible Chronic Diseases (NTCD)

The intake of edible mushrooms as functional foods associated with health improvement has been widely described [85]. Some metabolites obtained from mushrooms have been introduced into the market as they have antitumor properties or immunostimulants such as the polysaccharide lentinan produced by the edible mushrooms *Lentinula edodes* (Shiitake) and *G. lucidum*. *G. lucidum* latter is consumed in traditional East Asian medicine as bitter tea (hot aqueous extraction) and can also be obtained as a dry powder. The pharmacological properties of several mushroom metabolites have been demonstrated such as those of polysaccharides produced by *G. lucidum*, which were able to increase the survival of worms by activating the transcription factors DAF-16/FOXO [86].

*Hirsutella sinensis* produces polysaccharides with prebiotic properties related to insulin resistance and diabetes control [87,88]. Spermidine (39) (Figure 4) produced by several species [89], proved capable of prolonging the life of mice by reducing histone acetyltransferase EP300 [90], while epidemiological evidence indicates that spermidine (39) intake may also contribute to the reduction of human mortality [91]. Considerable amounts of the metabolite lovastatin (40) (Figure 4), a drug registered as low-density lipoprotein (LDL) cholesterol lowering, were detected in the widely consumed mushrooms *Agaricus bisporus* (30.79 mg/100 g dry weight), *Cantharellus cibarius* (67.89 mg/100 g dry weight), *Imleri abadia* (6.21 mg/100 g dry weight), and *L. edodes* (0.95 mg/100 g dry weight) [92].

However, the most prominent example of a mushroom with nutraceutical properties may be *Cordyceps militaris*, a rare and naturally occurring entomopathogenic medicinal mushroom in the Himalayan Mountains, Tibet, Nepal, and India. Studies have reported that consuming *C. militaris* extracts significantly increases glucose metabolism, thereby decreasing the glucose level in the blood. In addition, consumption of this mushroom provides protection against diabetic nephropathy [93]. *C. militaris* produces polysaccharides in the fructification body that are active toward α-glucosidase [94] and have immunomodulatory activity; this suggests their incorporation into functional foods and dietary supplements [95]. Anti-adipogenic activity was reported for a fermentation mix containing strawberry, silkworm pupae, and *C. militaris* [96]. Several nutraceutical products containing *Cordyceps* are available in the global market. Among the benefits claimed by their manufacturers are the promotion of mental health and benefits to the vascular system ("Cordyceps active"), cognition support ("Mushroom Plus"), anticancer and antioxidant activity ("Bhutan Cordyceps Tea"), strengthening of the cardiovascular system ("MRM Cordyceps CS-4 Strain"), and immune system support ("MycoNutri Cordyceps Organic") [97]. Cordycepin (41) (Figure 4) is the main metabolite produced by *C. militaris* and is very effective in reducing the accumulation of LDL, total cholesterol, triglycerides, and hyperlipidemia caused by high-fat diets [98]. Cordycepin (41) has several pharmacological properties such as anti-inflammatory, immunomodulatory, antioxidant, anti-aging, anticancer, antiviral, cardio, and hepatoprotective properties, among others [97]. This range of activities results in health effects that may help in postponing aging-linked NTCD. *C. militaris* also has high nutritional value and contains proteins, phenolic compounds, steroids, and lectins [99].

Another mushroom species with high functional and nutraceutical potential of high market value is *Agaricus subrufescens* (synonymy *Agaricus blazei* and *Agaricus brasiliensis*) [100]. It is commercialized
in several countries such as Brazil (brand name "Sun mushroom"), China (Ji Song Rong), and Japan (Himematsutake) [101]. Sun mushroom contains polyphenols and polysaccharides and is known to decrease oxidative stress and prevent NTCD; it is indicated to have antioxidant, antitumor, anti-inflammatory, and immunomodulatory properties [102].

Another class of bioactive compounds of increasing prominence found in edible mushrooms includes antcins (42) (Figure 4), steroids that contain an ergostane-type skeleton and are produced by *Antrodia* species such as *Antrodia cinnamomea* and *Antrodia salmomea*. Studies suggest that these compounds are promising agents in the treatment of cancer, inflammation, diabetes, and diseases resulting from oxidative stress, among others. The aforementioned species have been historically used in communities of Taiwan for treating various diseases such as diarrhea, abdominal pain, hypertension, dermatological irritation, and intoxication by food, alcohol, and drugs [103]. A study tracking 36,499 middle-aged and elderly Japanese men over an average of 13.2 years found a positive relationship between regular mushroom consumption and decreased incidence of prostate cancer [104]. Table 5 summarizes the classes of compounds and health benefits from some mushroom species cited in this section.

<table><caption>**Table 5.** Mushroom-originated compounds and health benefits related to non-transmissible chronic diseases (NTCD).</caption><thead><tr><th>Mushroom Species</th><th>Popular Name</th><th>Origin</th><th>Related Compounds</th><th>Health Benefits</th><th>References</th></tr></thead><tbody><tr><td>G. lucidum</td><td>Reishi</td><td>China and Eastern Asia</td><td>Polysaccharides</td><td>Antioxidant activity related to DAF-16/FOXO activation</td><td>[85]</td></tr><tr><td>H. sinensis</td><td>Caterpillar</td><td>Tibet</td><td>Polysaccharides</td><td>Prebiotic properties related to insulin resistance and diabetes control</td><td>[87,88]</td></tr><tr><td>L. edodes</td><td>Shitake</td><td>Eastern Asia</td><td>Spermidine (39)</td><td>Reduction on age-dependent memory impairment</td><td>[91]</td></tr><tr><td>A. bisporus</td><td>Champignon</td><td></td><td></td><td></td><td>[94]</td></tr><tr><td>C. cibarius</td><td>Chanterelle</td><td></td><td></td><td></td><td>[92]</td></tr><tr><td>I. badia</td><td>Bay bolete</td><td>Eastern Europe</td><td>Lovastatin (40)</td><td>LDL-cholesterol lowering</td><td>[92]</td></tr><tr><td>L. edodes</td><td>Shitake</td><td></td><td></td><td></td><td>[92]</td></tr><tr><td rowspan="2">C. militaris</td><td rowspan="2">Caterpillar</td><td rowspan="2">China, Tibet</td><td>Polysaccharides</td><td>Immunomodulation improvement</td><td>[94,95]</td></tr><tr><td>Cordycepin (41)</td><td>Total and LDL-cholesterol lowering, reduction of hyperlipidemia caused by high-fat diets</td><td>[96]</td></tr><tr><td>A. subrufescens</td><td>Sun Mushroom</td><td>Eastern North America</td><td>Polyphenols, polysaccharides</td><td>Decrease of oxidative stress, preventing diseases</td><td>[100-102]</td></tr><tr><td>A. cinnamomea</td><td>Niu-Chang-Chih</td><td>Taiwan</td><td>Antcins (42)</td><td>like cancer and inflammation</td><td>[103]</td></tr></tbody></table>

## 5. Toward a Sustainable Production of Fungal Metabolites

As the demand for preventive medicines, nutraceuticals, new drugs, food additives, and other health-related products of natural origin grows, the need for scaling up is also increasing. For bioactive metabolites of plant origin, efforts to increase production may be slow because production sometimes dependent on the seasonality of plants and a long plant growth period. Thus, efforts to increase the production of bioactive compounds have been directed toward microorganism-based options, such as metabolic engineering. Modifications in the culturing of fungi have been successful in the yield improvement of biomass and bioactive compounds. Moreover, special consideration is being given to endophytic fungi, especially those able to produce metabolites biosynthesized by their host plants. Table 6 shows some interesting examples of bioactive compounds produced by endophytic fungi after optimization of fermentation conditions. The good outcomes in this area were exemplified by Torres-Mendoza et al. [105] who reported, from 2001 to 2019, 224 patents related to metabolites from endophytic fungi applied to agricultural, biotechnology, pharmaceutical, and food industries, most of which used species from the *Aspergillus*, *Fusarium*, *Trichoderma*, *Penicillium*, and *Phomopsis* genera.
<table><caption>**Table 6.** Bioactive compounds produced by endophytic fungi under different fermentation conditions.</caption><thead><tr><th>Fungal Species</th><th>Host Plant</th><th>Target Compound</th><th>Health Benefit</th><th>Methodology</th><th>Target Parameters</th><th>Reference</th></tr></thead><tbody><tr><td>A. terreus</td><td>Coconut tree</td><td>L-Asparaginase</td><td>Treatment of acute lymphocytic leukemia</td><td>Factorial experimental design. Increase of scale(5-l bioreactor system).</td><td>pH, temperature, inoculum concentration.</td><td>[106]</td></tr><tr><td>F. solani</td><td>Chonemorpha fragrans</td><td>Camptothecin</td><td>Anticancer</td><td>Box-Behnken design using one factor at a time method.</td><td>Carbon and nitrogen sources, ethanol concentration, pH, temperature, stirring speed, incubation period, precursors and elicitors.</td><td>[107]</td></tr><tr><td>Penicillium bilaiae</td><td>Phoenix dactylifera</td><td>Acidic protease</td><td>Increasing in food digestibility</td><td>Response surface methodology. Plackett-Burman design. Box-Behnken design.</td><td>Temperature, initial pH, carbon and nitrogen sources, metal ions, detergents and enzyme inhibitors.</td><td>[108]</td></tr><tr><td>P. ostreatus</td><td>ni</td><td rowspan="2">Lovastatin (40)</td><td rowspan="2">Anti-hypercholesterolemic</td><td>Response surface methodology.</td><td>Nutrients, particle size of the solid substrate, temperature, incubation time.</td><td rowspan="2">[109]</td></tr><tr><td>Meyerozymaguilliermondii</td><td>leaves of Hibiscus rosa-sinensis</td><td>One parameter at time approach.</td><td>Nutrients, pH, inoculum size, temperature, addition of metallic ions, modulators, precursors.</td></tr><tr><td>A. niger</td><td>ni</td><td>Urease</td><td>Diuretic</td><td>Response surface methodology.</td><td>Strains, incubation time, temperature, pH, biomass, inoculum size, nitrogen content and moisture.</td><td>[111]</td></tr><tr><td>Spissomycesendophytica</td><td>Balanophorfungosa</td><td>Melanin</td><td>Radioprotective, thermoregulator, antitumor, and antiviral</td><td>One parameter at time approach.</td><td>Inhibitors, culture medium, temperature, pH.</td><td>[112]</td></tr><tr><td>E. nigrum</td><td>Taxus baccata</td><td>Taxol</td><td>Anticancer</td><td>One parameter at a time approach. Mutant strains.</td><td>Culture medium, stirring speed, temperature, incubation period, pH, medium volume, inoculum age, inoculum size, carbon source, nitrogen source, phosphorus source, gamma radiation dose.</td><td>[113]</td></tr><tr><td>Alternaria brassicae</td><td>Huperzia serrata</td><td>Huperzine A</td><td>Acetylcholinesterase inhibitor</td><td>Multifactorial statistical approaches. Plackett-Burman. Central composite designs.</td><td>Culture medium composition, medium volume, inoculum age, inoculum size, incubation period, ethanol addition, pH, temperature.</td><td>[114]</td></tr><tr><td>F. oxysporum</td><td>Dioscoreazingiberensis</td><td>Diosgenin</td><td>Anti-cancer, anti-thrombic, anti-diabetic, cardioprotective, osteoarthritis protective activity</td><td>One parameter at time approach.</td><td>Culture medium, antibiotics, temperature.</td><td>[115]</td></tr><tr><td>E. nigrum</td><td>Terminalia arjuna</td><td>Digoxin</td><td>Regulating the heart rhythm and strengthening heart diffusion</td><td>One parameter at time approach.</td><td>Culture medium, temperature, elicitors, incubation time, pH, medium volume, inoculum age, inoculum size, gamma irradiation mutagenesis.</td><td>[113]</td></tr><tr><td>Penicillium mallochii</td><td>A beech tree bark from Balikesir, Turkey</td><td>Orange-red pigment</td><td>Decreasing in allergic responses to synthetic pigments</td><td>One parameter at time approach.</td><td>Culture medium, pH, temperature.</td><td>[116]</td></tr><tr><td>Tausonia pullulans</td><td>Vinca minor</td><td>Vincamine</td><td>Improvement of cerebrovascular and cognitive disorders and reduction the effects of certain types of stroke</td><td>Protoplasts optimization.</td><td>Incubation time, temperature, modulators, protoplast inactivation method (heat, ultraviolet, microwave, sodium nitrite, and diethyl sulfate).</td><td>[117]</td></tr></tbody></table>

ni = not informed.
Yield enhancement in microbial production is one of the most challenging issues, but good progress has been reported. The production of fungal metabolites can also be optimized by co-cultivation with other fungal or bacterial species and variation of chemical parameters, such as composition of the culture medium, and physical parameters, such as temperature, pH, stirring speed, intensity and color of light, and oxygenation. The metabolic modulation resulting from these approaches depends on the fungal species; thus different fungi have been explored to produce fungal metabolites with new industrial applications around the world [38].

Biosynthesis of the natural polyamide agmatine (28) by *A. oryzae*, a fungus generally recognized as safe, was described during the fermentative process of sake production (Japanese rice wine) [118]. Optimization of the initial yield of agmatine (28) produced by *A. oryzae* in the presence of *S. cerevisiae* in a solid state (3.5 mM agmatine) was achieved by varying some fermentative parameters. At pH 5.3, the production of agmatine (28) increased to 6.3 mM. An increase of over 100% in the initial productivity was obtained by adding L-lactic (pH 3.0, 8.2 mM agmatine), succinic acid (pH 3.5, 8.7 mM agmatine), and citric acid (2) (pH 3.2, 8.3 mM agmatine) in the fermentation medium. Therapeutic evidences indicate that agmatine (28) is a promising lead compound against several NTCD that affect the central nervous system, such as Alzheimer's disease [119]. In 2019 only, the number of people affected by dementia was estimated at 50 million, with the worrying forecast of this number to be trebled by 2050 [120]. Therefore, new molecules for the treatment of Alzheimer's disease and other types of dementia are highly desired. The pharmacological properties and yield of agmatine (28) in fermentation by *A. oryzae* suggest the possibility of its incorporation into nutraceuticals [119]. In excess, agmatine (28) may present toxicity and can enhance the toxic action of other biogenic amines, such as histamine and tyramine, produced by the decarboxylation of amino acids by food-fermenting microorganisms. Therefore, determining a proper intake for agmatine (28) is necessary to mitigate risks and allows for the numerous aforementioned health benefits [16].

The preference for faster, less expensive, and green approaches has sped up research on bioactive fungal metabolites. Some promising analytical tools, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS), have been useful for addressing some bottleneck problems in this area, such as fungal identification. Genetic and morphological methods, although very efficient, require expertise, time, and resources that may be saved by the use of analytical instrumentation [121]. Such improvements start during screening steps, in the initial laboratory prospection of relevant species and promising fermentative conditions, and continue to the production steps, with the use of alternative substrates for fungal growth, such as agro industrial residues, towards a circular economy. Thus, extracts intended for screening are prepared on a reduced scale, using lower amounts of reagents, such as solvents, during extraction. In addition, modern analytical tools coupled with hyphenated techniques have been recently used for the analysis of a greater number of extracts, without the need for prior isolation of fungal metabolites from extracts. Biochemometrically derived fingerprints obtained by gas chromatography, high-performance liquid chromatography, MS, and nuclear magnetic resonance spectroscopy (NMR), together with statistical analysis also enable the establishment of straightforward associations between metabolite content and biological activities detected for a large number of crude extracts [70,122].

Although MS is the most widely used technique for this kind of analysis [123], NMR, in particular ¹H NMR metabolomic analysis, has gained ground by enabling the nondestructive identification of molecules in complex mixtures originating from various biological materials, such as plants and fungi extracts, foods, biological fluids, and tissues [124,125]. NMR is comparable to MS in criteria, such as efficiency, speed, reproducibility, and ease of sample preparation; however, it has advantages in terms of sample recovery and isotope detection. It is also independent of ionization potential and does not require complex internal standards [126]. Bi-dimensional biochemometric NMR evaluation of the crude extract of a marine-derived strain of *P. chrysogenum* enabled the attribution of the anti-proliferative activity of the extract against breast cancer cell lines to ergosterol (43) (Figure 4; Table 3), a structural metabolite common in fungi [70]. In addition, other metabolites of the ergostane
class, ergosterol peroxide (44), 3β, 5α, 9α, 14 α-tetrahydroxy-ergosta-7,22-dien-6-one (45), and 3β, 5α, 9α-trihydroxy-ergosta-7,22-dien-6-one (46) (Figure 4), were detected as metabolites of the edible mushroom *V. volvacea* and proved to be active against human cancer cell lines (Table 3) [68].

An interesting application of NMR-based metabolomics for the comparison and origin identification of edible mushrooms was reported. Multivariate analyses, such as PCA, were used to compare the chemical profiles of species, enabling the differentiation of *Kuehneromyces mutabilis* and *Hypholom acapnoides*, and the direct identification of 17 secondary metabolites in their extracts. Statistically significant differences were observed in the $^1$H NMR data in the variation of composition based on the collection site and restrictedness of metabolites in *K. mutabilis*. Upon comparing *H. capnoides* and *K. mutabilis*, a higher diversity of sugars was observed in *H. capnoides*, while *K. mutabilis* presented significantly higher amounts of some organic compounds, such as fumaric acid (3), showing the versatility of the $^1$H NMR technique for comparing fungi in terms of metabolites [127].

Other sophisticated approaches combining UPLC-ESI-TOF/MS, differential off-line LC-NMR, and quantitative $^1$H NMR (qHNMR) analysis were used to identify D-Phe-L-Val-D-Val-L-Tyr (47), D-Phe-L-Val-D-Val-L-Phe (48), and cis-bis(methylthio)silvatin (49) (Figure 4) in the aerobic fermentation of *Penicillium roqueforti* with and without L-Tryptophan enrichment. Another metabolite, roquefortine C (50) (Figure 4), was identified as an antimicrobial agent against *Bacillus subtilis* and *E. coli* [126]. $^1$H NMR-based metabolomics, applied in a study on extracts of 11 species of edible mushrooms, identified dry *Pleurotus geesteranum* and *Hericium erinaceus* and fresh *Pleurotus sapidus* as the most prominent species in terms of metabolite production. In addition, the use of $^1$H NMR led to the detection of more than 100 different metabolites of interest to the food industry including trehalose (51), mannitol (52), and glucose (53) (Figure 1). The analysis also showed high levels of carbohydrates and proteins, in addition to considerable amounts of vitamins A (54) and C (55) (Figure 1) and amino acids as nutritional compounds [14]. Therefore, NMR-based metabolomics has been successful in selecting fungi with outstanding potential for further technological development. Metabolomic tools have also been applied for the evaluation of organoleptic properties and interference of external factors with food quality [128] and nutrient quantification such as in the determination of the nutritional contents of 11 *Capsicum annuum* cultivars in terms of ascorbic acid (vitamin C) (55) [129]. In the near future, once there is sufficient data in this area, these useful metabolomic tools could be applied in several meta-studies such as for the determination of vitamin C (55) content in edible mushrooms. One study [130] reported vitamin C contents between 31.16 ± 0.93 (*Calvatia gigantea*) and 108.11 ± 3.22 (*Lepis tagilva*) mg/kg dry matter. In another study [14], the vitamin C contents ranged from 0.5 to 111.4 mg/100g dry weight. The highest levels of vitamin C were produced by the species *D. indusiata* and *A. subrufescens* at 111.4 and 69.7 mg/100 g dry weight, respectively [14], values close to the daily levels of vitamin C (55) recommended in some countries (75 to 110 mg) [131].

Metabolomic approaches have also been applied to the quantification of aflatoxins in industrialized baby food [132], to determine adulteration and its effect on the safety of nutraceuticals [133], and for differentiating extracts of various biomaterials such as crops [134] and fungi [14].

While metabolomic tools provide speed for research, technological development for future industrial applications of fungal metabolites must consider that the modern economy is increasingly challenged to transform traditional processes into sustainable production chains, minimizing waste, and reusing biomass for applications in the food and drug industries, even for well-established processes such as citric acid (2) production. The global market value of citric acid (2) was estimated to increase to USD 3.6 billion in 2020 and maintain an annual growth of 5% until 2025. Despite the successful industrial experience for citric acid (2) production, research to increase sustainability in bio-refineries is ongoing, opening new possibilities such as clustering-related fermentation production including olive oil and wine to reach models for circular bioeconomy [135].

Fortunately, the production of nutraceuticals and functional foods from biomass, extracts, and fungal metabolites greatly addresses the challenge of sustainability improvement, allowing the incorporation of low-cost, underutilized, and abundant materials into the industrial fermentation
process. Many agro-industrial residues available all over the world are sources of peptides, prebiotic dietary fibers, and hydrolyzed or smaller organic molecules such as phenolics, carotenoids, and tocopherols, among other classes. These features make them good materials for fungal cultivation as well as interesting substrates for the production of bioactive components from macromolecules [59,136]. For example, fermentation of pomegranate bark residue by *A. niger* resulted in the production of citric acid (2) with reduced production costs, while adding value to a residue usually directed for disposal [137]. High yields (7.8 to 14.4 g/L) of dry biomass, with protein contents ranging from 33.8 to 50.8%, and ethanol were recovered from the fermentation of effluents from the wheat starch industry by the fungi *A. oryzae* and *R. oryzae*. This kind of process contributes to the development of a circular economy, because the large volume and high chemical demand of oxygen in effluents from the starch industry represent an environmental burden and an additional cost as they create a need for effluents to be treated before being discarded [138]. The filamentous fungi *Actinomucor elegans* and *Umbelopsis isabelin* were simultaneously used for the enrichment of white grape-producing bagasse, a residue with little nutritional attractiveness. The fungi were able to increase the γ-linolenic acid and carotenoid contents, improving the nutritional content of bagasse, and thus creating the possibility of reintroducing bagasse into the production chain as a low-cost functional food for humans [139].

## 6. Conclusions

Fungi and their metabolites have important industrial applications in high-value-added products and have potential for the development of nutraceuticals that can contribute to the prevention of NTCD and improve health, especially in terms of human aging. In addition, fungi are suitable in the production of natural food additives such as colorants and stabilizers that have lower health risks than synthetic food additives, and bioactive metabolites for pharmacological use such as enzymes, statins, and antitumor agents. Fungal antioxidants have applications in both food preservation and the combat of oxidative stress in the human body, with positive outcomes for several diseases such as cancer. The use of metabolic engineering techniques has facilitated the overcoming of some obstacles to explore the pharmacological potential of fungi, even those producing toxic substances such as some species of the genera *Monascus*, *Aspergillus*, and *Fusarium*. Modern approaches have been successfully utilized to evaluate the interference of additives derived from fungi with the organoleptic properties and quality of food. Strategies currently available for scaling up metabolite production include direct genetic alteration with tools such as CRISPR-Cas9 and gene recombination. Research on the use of agro-industrial byproducts for sustainable fungal fermentation has shed light on its remarkable economic importance to the production of natural additives, food, drugs, and nutraceuticals. Further in vivo antioxidant activity studies of fungal metabolites are still scarce; however, new insights are required to expand the use of metabolites from filamentous fungi to improve human health in the 21st century.

**Author Contributions:** All authors participated in the layout of this review. J.A.T., B.V.R.B., B.d.A.M., C.P.G., and M.A.F.M. identified the literature for review and drafted the manuscript. In addition, J.A.T. conceived and critically revised the manuscript, B.V.R.B. and B.d.A.M. provided the tables and figures, and C.P.G. and M.A.F.M. formatted the document. All authors have read and approved the final version of the manuscript.

**Funding:** The authors gratefully acknowledge financial support from the Brazilian Agencies Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG PPM-00255-18); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grants 304922/2018-8, 141601/2018-3, 142517/2018-6, 121994/2019-8); the National Institute of Science and Technology–INCT BioNat/CNPq (Grant 465637/2014-0); and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)–Finance Code 001.

**Acknowledgments:** We thank Michel Almeida for kindly preparing the graphical abstract illustration.

**Conflicts of Interest:** The authors declare no conflict of interest.
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Review

# Selenium Biofortification of Crop Food by Beneficial Microorganisms

Yuanming Ye, Jingwang Qu, Yao Pu, Shen Rao, Feng Xu and Chu Wu

College of Horticulture & Gardening, Yangtze University, Jingzhou 434025, China; y.m.ye2020@gmail.com (Y.Y.); qujw@yangtzeu.edu.cn (J.Q.); puyao456@163.com (Y.P.); raoshen1989@163.com (S.R.); xufeng198@126.com (F.X.)

* Correspondence: wuchu08@yangtzeu.edu.cn; Tel.: +86-716-806-6262

Received: 30 March 2020; Accepted: 26 April 2020; Published: 3 May 2020

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**Abstract:** Selenium (Se) is essential for human health, however, Se is deficient in soil in many places all around the world, resulting in human diseases, such as notorious Keshan disease and Keshin-Beck disease. Therefore, Se biofortification is a popular approach to improve Se uptake and maintain human health. Beneficial microorganisms, including mycorrhizal and root endophytic fungi, dark septate fungi, and plant growth-promoting rhizobacteria (PGPRs), show multiple functions, especially increased plant nutrition uptake, growth and yield, and resistance to abiotic stresses. Such functions can be used for Se biofortification and increased growth and yield under drought and salt stress. The present review summarizes the use of mycorrhizal fungi and PGPRs in Se biofortification, aiming to improving their practical use.

**Keywords:** selenium; biofortification; transporters; mycorrhizal fungi; plant growth-promoting rhizobacteria (PGPRs)

## 1. Introduction

At present, it is widely accepted that selenium (Se) possesses multiple physiological functions in various biological systems as an integral part of a range of proteins containing Se. Therefore Se is important for human health. However, Se distribution in the earth's crust is greatly uneven, ranging from 0.005 mg·kg⁻¹ in Finland to 8000 mg·kg⁻¹ in Tuva-Russia [1]. Se deficiency has been reported in many places all around the world including China, North America, New Zealand, Australia, Sweden, and Finland [2–5]. Some notorious diseases are directly related to Se deficiency, such as Keshan disease and Keshin-Beck disease, two endemic diseases related to Se deficiency. Keshan disease was first prevalent at large scale in 1935 in Keshan county, Heilongjiang province, China. Keshan disease generally occurs in children and women of childbearing age and its symptoms are related to impairment of cardiac function, cardiac enlargement, and arrhythmia [6]. Although the main factor was not determined for the disease in etiology, it was closely related to Se because it was found that there was an obvious Se deficiency in local soil, and Se supplementation could partly control the disease. An investigation analyzed some physiological parameters, including blood Se level, glutathione peroxidase-1 (GPx-1) activity, and variance at codon 198 in GPx-1 gene, and found that the main risk factors for the disease were low GPx-1 activity, Keshan disease family history, and living in an endemic area [7], suggesting that Keshan disease is closely related to low GPx-1 activity. Kaschin-Beck disease is an osteoarthropathy, which manifests as severe dysarthrosis of joints, shortened fingers and toes, and in severe cases dwarfism. In China, the disease is prevalent in the Tibetan Plateau [8,9]. An investigation carried out by Zhang et al. [8] showed that the levels of environmental Se were very low, and Kaschin-Beck disease in the Tibetan Plateau was much severe with decreasing environmental Se under the Se-deficient condition, suggesting the relationship between Kaschin-Beck disease and Se deficiency in the Tibetan Plateau. In addition, Se is related to other human diseases and health, such as
cancer [10–13], muscle disease [14], and healthy aging and longevity [15–17]. Therefore, it is essential to maintain Se homeostasis in human body [18,19]. It was estimated that Se intake of >900 µg·day⁻¹ is harmful, and intake of <30 µg·day⁻¹ is not enough [20]. Some data have shown that over 800million people all around the world might suffer from Se deficiency [21–26]. Therefore, sufficient dietary Se uptake is important for human health.

Acquired Se is converted into some proteins that contain at least one of the two amino acids (i.e., selenocysteine (SeCys) and selenomethionine (SeMet)) as a key component (i.e., selenoproteins). Human health and diseases are related to selenoproteins, and selenocysteine is regarded as the 21st proteinogenic amino acid. The human genome encodes about 30 selenoproteins. In the article written by Reeves and Hoffmann [27], they described functions of selenoproteins in detail. Among the selenoproteins in human, glutathione peroxidases (GPx5) seem to be more important, because they include eight proteins (GPx1–GPx8) having antioxidant properties with multidimensional roles in living cells, ranging from H₂O₂ homeostasis to regulation of apoptosis [28]. Therefore, enough Se uptake is essential for functional maintenance of these selenoproteins. Since Se is deficient in many places all around the world, Se fortification in food is necessary. In view of high toxicity of selenite and selenate, Se biofortification is relatively bio-safe. Organic Seleno-compounds act as potential therapeutic and chemo-preventive agents that function as antioxidants, enzyme modulators, antitumor, antimicrobials, antihypertensive agents, antivirals, and cytokine inducers [29]. Organic seleno-compounds are provided with crop food [30–35], vegetables [36–42], fruits [43–45], and even nuts [46–48]. Therefore, how to increase concentrations of organic seleno-compounds in these plants is of significance for improvement of dietary Se acquisition by human being.

Se biofortification may be carried out by multiple ways, such as application of Se fertilizers on leaves [49–53] and in soil [52,54,55]. Se-enriched organic fertilizers are also applied. For example, Bañuelos et al. [56] used Se-enriched *Stanleya pinnata* to cultivate Se-enriched broccoli and carrots, and found that more than 90% of organic Se was converted to inorganic selenate and selenite. Se foliar application seems to be most effective way to fortify Se uptake in most arable crops [52,57]. However, a contrary result was observed by Lyons et al. [58]. They found foliar application was less efficient than application to soil at planting (at application rates of 40 and 120 g·ha⁻¹, respectively) in Australian trials. The agronomic application of Se fertilizers are more expensive and short-term solutions, especially in large-scale fields. Relatively, agronomic Se biofortification with beneficial microorganisms (BMOs) is a more inexpensive and long-term solution, especially in poor places and Se-rich places, such as Enshi, Hubei province, China [59] and Pineridge Natural Area, a seleniferous site west of Fort Collins, CO, USA [60].

In the present article, we focus on the roles of beneficial microorganisms in Se biofortification and our aim is to improve use of beneficial microorganisms in practice.

## 2. Improvement of Se Biofortification by BMOs

Symbiosis of plants with BMOs is helpful for plant growth and to increase in micronutrition uptake and resistance to abiotic and biotic stresses. Based on the characteristics of BMOs, BMOs can be used for Se biofortification. BMOs, including mycorrhizal fungi (endos- and ectomycorrhizal fungi), root endophytic fungi (REFs), and PGPRs, are popular in biofilmed biofertilizers. Arbuscular mycorrhizal fungi (AMFs) are preferential to colonize in roots of angiosperms, and ectomycorrhizal fungi are popular in gymnosperms. Most REFs possess a wide range of plant hosts.

### 2.1. Arbuscular Mycorrhizal Fungi

Arbuscular mycorrhizal fungi are used for Se biofortification because of their ability to enhance nutrition uptake of their host plants (Table 1). Functions of mycorrhizal fungi have been the primary focus of research, especially those involved in phosphate uptake. The genomes of these fungi encode some high-affinity inorganic phosphate transporters and some of them have been isolated and identified [61–65]. On the other hand, *in planta*, some symbiosis-specific phosphate transporters
can be induced by symbiosis with mycorrhizal fungi [61,66–72]. Thus, the interaction between plants and mycorrhizal fungi strengthens phosphate uptake and transportation to host plants [73–77]. Similarly, there are some sulfate transporters encoded by genomes of mycorrhizal fungi, such as sulfate transporters GBC38160.1 and GBC25943.1 and sulfate permeases PKY50973.1 in arbuscular mycorrhizal fungus *Rhizophagus irregularis*, sulfate transporters EDR02618.1 and EDR02177.1, and sulfate permeases EDR11271.1 and EDR00466.1 in ectomycorrhizal fungus *Laccaria bicolor*. Since Se and sulfur (S) belong to the same element family (VI-A), the chemical properties of Se are very similar to S. Se is absorbed as selenate or selenite, which is metabolized via the sulfur assimilation pathway in plants, leading to biosynthesis of SeCys, SeMet, and other Se isologs of various S metabolites [78–82]. Se can be transported by sulfur transporters to host plants, just like phosphate transported between mycorrhizal fungi and their host plants, such as the high-affinity sulfate permease [83] and the high-affinity sulfate transporters Sultr1:1 and Sultr1:2 [84–86]. The two sulfate transporters are proton-sulfate symporters, such that for every molecule of selenate entry into root cells, three protons are taken up. Sulfate transporters function in Se accumulation in food crops. Wheat genotype 'Puelche' is the most Se-tolerant and has the greatest Se accumulation among the three wheat genotypes studied (i.e., 'Puelche', 'Tinto', and 'Kumpa'), such that its Se accumulation was related to the strongest transcript level of the sulfate transporter TaeSultr4.1 in roots [87]. In addition, other transporters also take part in Se transport, such as silicon transporters in rice [88] and tomato [89], phosphate/orthophosphate transporters in wheat [90], rice [91–93], tomato [89], and yeast (*Saccharomyces cerevisiae*) [94,95], and monocarboxylates transporters in yeast (*S. cerevisiae*) [96]. Thus, it is reasonable to explain the experimental results that plant availability of selenate and selenite was influenced by the competing ions phosphate and sulfate [97,98]. Competition between phosphate and Se uptake led to decrease in Se accumulation translocation coefficients, and Se concentrations in wheat roots, stems, leaves, and spikes when phosphate fertilizers were applied to selenite fertilized soil [99]. However, a different case occurred. An investigation was carried out on sulfate and selenate uptake in *Astragalus* species (two Se hyperaccumulators *A. racemosus* and *A. bisulacatus* and two closely related non-accumulators *A. glycyphyllos* and *A. drummondii*), and results showed that sulfur deficiency increased Se accumulation, and increased Se supply increased sulfate accumulation in both root and shoot tissues [100]. In certain *Astragalus* species, the high expression of sulfate transporters led to enhanced ability of Se uptake and translocation, and therefore contributed to the Se hyperaccumulation trait. At present, except for sulfate and phosphate transporters, it is not clear whether other transporters have their homologous proteins in mycorrhizal fungi. If these homologous proteins occur in mycorrhizal fungi, they could mediate Se transport to host plants. On the other hand, decreases in sulfate bioavailability and mycorrhizal symbiosis enhanced expression of sulfate transporters, resulting in increase in ability to absorb sulfate and consequent uptake of Se [101–104]. Similarly, Se deficiency also enhances expression of sulfate transporters, resulting in an increase in Se uptake, and mycorrhizal symbiosis also enhances Se uptake.

<table><caption>**Table 1.** Arbuscular mycorrhizal fungi (AMFs) and root endophytic fungi (REFs) often used for Se biofortification.</caption><thead><tr><th>Microbes</th><th>Microbial Types</th><th>Host Plants</th><th>References</th></tr></thead><tbody><tr><td><i>Funneliformis mosseae</i></td><td>AMF</td><td><i>Triticum aestivum</i>, <i>Lactuca sativa</i>, <i>Asparagus officinalis</i>,</td><td>[105–108]</td></tr><tr><td><i>Glomus claroideum</i></td><td>AMF</td><td><i>Triticum aestivum</i></td><td>[109]</td></tr><tr><td><i>Glomus fasciculatum</i></td><td>AMF</td><td><i>Allium sativum</i></td><td>[110]</td></tr><tr><td><i>Glomus irtraradices</i></td><td>AMF</td><td><i>Allium sativum</i></td><td>[55]</td></tr><tr><td><i>Glomus mosseae</i></td><td>AMF</td><td><i>Lolium perenne</i>, <i>Allium sativum</i>, <i>Medicago sativa</i>, <i>Glycine max</i>, <i>Zea mays</i></td><td>[110–112]</td></tr><tr><td><i>Glomus versiform</i></td><td>AMF</td><td><i>Triticum aestivum</i></td><td>[105]</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Microbes</th><th>Microbial Types</th><th>Host Plants</th><th>References</th></tr></thead><tbody><tr><td>Rhizophagus intraradices</td><td>AMF</td><td>Lactuca sativa, Asparagus officinalis, Lactuca sativa, Allium cepa</td><td>[106–108,113]</td></tr><tr><td>Alternaria seleniiphila</td><td>REF</td><td>Stanleya pinnata</td><td>[114]</td></tr><tr><td>Alternaria astragali</td><td>REF</td><td>Astragalus bisulcatus</td><td>[114]</td></tr><tr><td>Aspergillus leporis</td><td>REF</td><td>Stanleya pinnata</td><td>[114]</td></tr><tr><td>Fusarium acuminatum</td><td>REF</td><td>Astragalus racemosus</td><td>[114]</td></tr><tr><td>Trichoderma harzianum</td><td>REF</td><td>Allium cepa</td><td>[106]</td></tr></tbody></table>

Some evidence supports the role of mycorrhizal fungi in enhancing Se uptake in plants. Wheat seedlings were inoculated with *Glomus versiform* or *Funneliformis mosseae* in hydroponic culture medium for eight weeks, the two arbuscular mycorrhizal fungi significantly increased selenate and selenite uptake by wheat root, but they did not show effect on uptake of SeMet [105]. Meanwhile, compared to non-mycorrhizal roots, mycorrhizal roots showed significantly higher $V_{max}$ for selenate and selenite uptake (179.6 vs. 55.93 nmol·g⁻¹DW·h⁻¹ for selenate and 1688.0 vs. 860.3 nmol·g⁻¹DW·h⁻¹ for selenite). Higher Se accumulation was carried out through up-regulating the expression of three genes encoding sulfate transporters, i.e., TaSultr1:1, TaSultr1:3, and TaSultr2:1, in the mycorrhizal roots, especially TaSultr1:1. In mycorrhizal roots with G. *versiform* and F. *mosseae*, the relative expressions of TaSultr1:1 gene was significantly up-regulated by 2.18-fold and 2.12-fold, respectively. Garlic (*Allium sativum* L.) is an important condimental species. This species is popular all around the world because of its diallyl disulfide, a component of garlic, which can inhibit proliferation of various cancer cells (e.g., colon, lung, and skin cancer cells) and WEHI-3 leukemia cells [115–117]. Garlic is used for Se biofortification with mycorrhizal fungi. A survey of applying selenate fertilizer and mycorrhizal fungus *Glomus intraradices* to soil was conducted, and the results showed mycorrhizal addition increased the Se uptake of garlic by 10-fold to 15 µg·g⁻¹DW, and fertilization with selenate and amendment of mycorrhizal fungi strongly increased the Se concentrations in garlic to around 1% [55]. Further analyses showed that the amendment of soil with the mycorrhizal fungus and/or selenate increased selenate concentrations in garlic, but did not affect distribution of detected Se species in garlic. In Se-contaminated soil, mycorrhizal fungi inoculation increased Se accumulation of plants. Alfalfa, maize, and soybean seedlings were cultivated in the soil contaminated with different levels of Se, and results showed that mycorrhizal fungi inoculation decreased Se accumulation in roots and shoots of all the plants at low Se levels (0 or 2 mg·kg⁻¹), but increased Se accumulation in alfalfa shoots and maize roots and shoots at Se level of 20 mg·kg⁻¹ [112]. Contrary results were observed on ryegrass (*Lolium perenne* cv. 'Barclay') [111]. Their results showed that Se concentrations in roots of ryegrass were not affected by mycorrhizal inoculation with the AMF G. *mosseae*, but mycorrhizal inoculation significantly reduced Se concentrations in shoots [111], further decreasing Se uptake in whole plants. Lettuce (*Lactuca sativa* L.) is one of the most consumed leaf vegetables in some places around the world because of its good properties, such as high levels of antioxidants (such as carotenoids, polyphenols, ascorbate, α-tocopherol) and dietary fiber [118–120], thus it is suitable for Se biofortification to enhance dietary Se consumption. When two lettuce cultivars 'Batavia Rubia Munguia' (BRM) and 'Maravilla de Verano' (MV) were treated with Se compounds (selenite, organic Se compounds SeU and SeCH₃) and AMFs (a mixture of Rhizophagus intraradices and Funneliformis mosseae), their growths were continuously improved by AMFs, except for BRM under treatment of SeCH₃ [106]. The positive effect of AMFs on plant biomass was different among lettuce cultivars and forms of seleno-compounds, and BRM lettuce plants showed the highest mycorrhizal efficiency index (MEI) under treatment of SeU, MV lettuce plants with the highest MEI under SeCH₃, suggesting that the two lettuce cultivars possessed preference for different seleno-compounds when they were inoculated with AMFs. Meanwhile, AMFs inoculation significantly affected mineral accumulation in
the leaves of BRM lettuce. In general, mycorrhizal inoculation significantly increased levels of macro and micronutrients, but significantly reduced Se levels in leaves of BRM lettuce. Significant interaction occurred about Se levels in shoots of BRM lettuce between seleno-compounds and AMFs inoculation. Similar status occurred on MV lettuce. Under treatment of selenite, AMFs inoculation reduced Se concentrations in leaves of MV lettuce. In contrast, under treatment of organic seleno-compound SeCH3, MV lettuce never accumulated detectable levels of Se in leaves, regardless of whether they were inoculated with AMFs nor. Treatment of organic seleno-compound SeU slightly increased Se concentrations in leaves of MV lettuce without AMFs inoculation [106]. Other research showed similar results [108]. All the results suggest that combination of seleno-compounds and AMFs inoculation does not increase Se levels in lettuce leaves, although it increases levels of some macro- and micronutrients and antioxidants. Therefore, some AMFs are not suitable for Se biofortification in lettuce. Of course, other AMFs should be chosen to examine their role in Se biofortification in lettuce under treatment of seleno-compounds. At present, it is not clear whether lettuce symbioses with some ectomycorrhizal fungi. Thus, more research is necessary for Se biofortification in lettuce.

Consversa et al. [107] investigated the effect of Se fern application and AMFs (*Rhizophagus intraradices* and *Funneliformis mosseae*) inoculation on Se biofortification for two years, such that Se fern application was carried out on green asparagus (*Asparagus officinalis* L.). Their experimental results showed that Se levels in non-mycorrhizal *A. officinaliscv.* 'Grande' plants increased in trial A1 as exogenous selenate levels increased. Under selenate treatment of 75 and 125 g·ha⁻¹, Se concentrations in spears increased 4.7 and 6.4-fold on a dry weight basis compared to control, respectively. Similar results occurred in trial B1. In trial B1, Se concentrations in spears were significantly affected by the interaction between Se amendment and AMFs inoculation. In spears of plants without Se amendment, Se levels were similar in mycorrhizal and non-mycorrhizal plants. All the results suggest a combination of Se amendment and mycorrhizal fungi greatly improve Se biofortification in *A. officinalis* and the combination should be recommended in field by large scale. However, contrary results have also been observed. When the AMF *Glomus mosseae* was used for inoculation with alfalfa (*Medicago sativa* L. cv. 'Chuangxin'), maize (*Zea mays* cv. 'ND108'), and soybean (*Glycine max* cv. 'Zhonghuang No. 17'), mycorrhizal inoculation significantly decreased Se concentrations in roots with the highest reduction for alfalfa (50-70%), while it was less than 40% for maize and soybean, Se concentrations in shoots decreased by 7-38% for mycorrhizal treatment, and the difference caused by inoculation influence was insignificant among the plant species [112]. When Se was added at the levels of 0 and 2 mg·kg⁻¹, the total Se accumulation in roots and shoots of all the three plant species were lower in mycorrhizal than in non-mycorrhizal treatment, while the opposite pattern was observed in roots of maize and shoots of alfalfa and maize when Se was applied at 20 mg·kg⁻¹ [112]. These results show negative effects on Se accumulation in these plant species when low levels of exogenous Se were added.

In addition, some ectomycorrhizal fungi can accumulate Se in their fruit bodies [121-123], suggesting their ability to acquire Se. Some of these ectomycorrhizal fungi are edible, thus they are used for biofortification of Se in fruit bodies. Few investigations on the role of REFs in Se biofortification have been carried out (Table 1). In general, REFs, especially members of the genus *Trichoderma*, can colonize roots of some host plants, thus they can be widely used for Se biofortification. At present, there are not reports on roles of dark septate fungi in Se biofortification of food crops.

Taken together, mycorrhizal inoculation might increase Se accumulation in some crop species, leading to Se biofortification of crops. For some crop species, more investigations are needed, especially for interactions between mycorrhizal fungi and crop species. For the abovementioned negative effects of G. mosseae on Se accumulation in alfalfa, maize, and soybean, more mycorrhizal fungi and root endophytic fungi should be used to investigation.

## 2.2. Se Biofortification by PGPRs

Plant growth-promoting rhizobacteria (PGPRs) are popular in improving nutrition uptake, plant growth, and resistance to abiotic stresses [124-127]. Some of them possess the ability to solubilize
phosphate in soil. Such ability could be used for Se biofortification (Table 2), because in some soil agrotypes, such as volcanic Andisols in southern Chile, Se bioavailability is very low. On the one hand, Se can form stable complexes with clays and/or can be strongly absorbed onto oxy-hydroxides of aluminum, iron, or manganese, and remain low in terms of bioavailability to plants [128–130]. On the other hand, oxyanions of Se, i.e., selenite and selenate, are bioavailable to plants. When selenate and selenite are supplied to soil, they are rapidly reduced to insoluble forms (e.g., Se-metal ion complex), leading to their low bioavailability (less than 10% only). The Se fertilizers that are not acquired by plant roots readily after application are not bioavailable to plants in the next season or the next year [131]. Thus, Se re-solubility in soil is very important. Although there are no report concernsregarding Se-solubilizing PGPRs at present, some seleno-bacteria have been studied [30,35,109,132,133]. Trivedi et al. [35] isolated and identified some endophytic seleno-bacteria from the various tissues of *Ricinus communis* plants and molecular identification analyses showed that they were *Paraburkholderia megapolitana*, *Alcaligenes faecalis*, and *Stenotrophomonas maltophilia*. Among the three bacteria, *P. megapolitana* was most effective in improving the growth of *Glycine max* plants under drought and enhancing Se biofortification which was 7.4-fold higher compared to control. The synergistic effect on Se biofortification and increased drought tolerance is important for plants grown in arid and semi-arid places with Se deficiency. A great number of people all around the world are dependent on wheat as their main component of diet, thus it is important to fortify Se in wheat grains [30]. Many studies have been carried out on Se biofortification in wheat. Durán et al. [109] evaluated the effects of Se acquisition by wheat plants through the co-inoculation of native seleno-bacteria strains *Stenotrophomonas* sp. B19, *Enterobacter* sp. B16, *Bacillus* sp. R12, and *Pseudomonas* sp. R8, both individually and in mixture, as a seleno-nanosphere source with AMF *Glomus claroideum*. They found that Se concentrations in plant tissues in inoculated plants were significantly higher than those of un-inoculated controls.Meantime, regardless of presence of AMF *G. claroideum*, Se concentrations in grains of wheat plants inoculated with *Enterobacter* sp. B16 were higher than those of plants inoculated with the rest of the microbial strains. In addition, PGPRs showed their synergistic role in improving Se concentrations with AMFs. When plants were inoculated with the seleno-bacteria strains and *G. claroideum*, Se concentrations in grains were 23.5% higher than those in non-mycorrhizal plants. The synergisms might be related to the relationship between seleno-bacteria strains and AMFs, because the seleno-bacteria could acquire more nutrition from the hyphae of their neighboring AMFs or ectomycorrhizal fungi [134–137]. Moreover, Durán et al. [132] isolated two Se-tolerant endophytic bacteria *Acinetobacter* sp. E6.2 and *Bacillus* sp. E5. They studied production of seleno-compounds (SeMet and seleno-methyl-selenocysteins (MeSeCys)) by the two bacteria, but they did not study the effects of the two bacteria on Se biofortification. Co-application of Se fertilizers and seleno-bacteria sometimes leads to changes in bacterial population. When Se-tolerant bacteria and Se amendment were supplied to wheat in Andisols, Se amendment stimulated population growth of two bacterial groups (*Paenibacillaceae* and *Brucellaceae*), but inhibited other bacterial groups (Clostridia, *Burkholderiales*, *Chitinophagaceae*, and *Oxalobacteraceae*) [133]. Meanwhile, Se concentrations in roots and leaves of wheat plants inoculated with Se-tolerant bacterial strains *Pseudomonas* sp. R8 and *Stenotrophomonas* sp. B19 were significantly higher than those of the un-inoculated controls. Higher Se biofortification is related to the Se tolerance of the two bacteria, because higher Se concentrations in roots and leaves were also observed when wheat plants inoculated with *Stenotrophomonas* sp. B19 were grown at concentrations of 5 and 10 mM of selenite, compared to those grown at 2 mM [133]. The results suggested that Se in seleno-bacteria could be transferred into their host plants. Effects of other Se-tolerant bacteria on Se biofortification were also investigated. When wheat plants were inoculated with two Se-tolerant bacterial strains *Bacillus cereus* YAP6 and *Bacillus licheniformis* YAP7, Se concentrations in the stems of the Se-treated wheat plants were increased up to 375%, and Se concentrations in kernels increased up to 154% of those in un-inoculated Se-treated wheat plants [34]. Meanwhile, the *Bacillus* strains can produce auxin, leading to increased number of leaves and greater biomass and shoot length [34]. When wheat plants were inoculated with *Bacillus pichinotyi* in the presence of selenate, they posed significantly higher biomass, shoot length, and spike length compared
to un-inoculated plants [33]. Meanwhile wheat plants inoculated with *B. pichinotyi* had significantly higher Se concentrations in wheat kernels (167%) and stems (252%), compared to un-inoculated plants. Overall, greater biomass means higher Se biofortification, which is important for crops cultivated by large scale in field. Rhizobia not only fixes nitrogen, but also helps Se accumulation. Data from Alford et al. [138] showed rhizobia significantly increased shoot biomass and Se accumulation in shoots of the Se-hyperaccumulator *Astragalus bisulcatus* and the nonhyper accumulator *A. drummondii*. The dual roles of rhizobia are of significance for organic Se production.

<table><caption>**Table 2.** Plant growth-promoting rhizobacteria (PGPRs) often used for Se biofortification.</caption><thead><tr><td>Microbes</td><td>Host Plants</td><td>References</td></tr></thead><tbody><tr><td>Acinetobacteria sp. E6.2</td><td>-</td><td>[132]</td></tr><tr><td>Acinetobacter sp.</td><td>Triticum aestivum</td><td>[139]</td></tr><tr><td>Alcaligenes faecalis</td><td>Ricinus communis, Glycine max</td><td>[35]</td></tr><tr><td>Anabaena sp.</td><td>Triticum aestivum</td><td>[30,140]</td></tr><tr><td>Bacillus amyloliquefaciens</td><td>Arabidopsis thaliana</td><td>[141]</td></tr><tr><td>Bacillus axarquiens</td><td>Triticum aestivum</td><td>[139]</td></tr><tr><td>Bacillus cereus</td><td>Triticum aestivum</td><td>[34]</td></tr><tr><td>Bacillus licheniformis</td><td>Triticum aestivum</td><td>[34]</td></tr><tr><td>Bacillus mycoides</td><td>Brassica juncea</td><td>[142]</td></tr><tr><td>Bacillus pichinotyi</td><td>Triticum aestivum</td><td>[33]</td></tr><tr><td>Bacillus sp. E5</td><td>-</td><td>[132]</td></tr><tr><td>Bacillus sp. E6.1</td><td>Triticum aestivum</td><td>[139]</td></tr><tr><td>Bacillus sp. R12</td><td>Triticum aestivum</td><td>[109]</td></tr><tr><td>Bacillus subtilis</td><td>Allium cepa</td><td>[113]</td></tr><tr><td>Calothrix sp.</td><td>Triticum aestivum</td><td>[30,140]</td></tr><tr><td>Enterobacter ludwigii</td><td>Triticum aestivum</td><td>[139]</td></tr><tr><td>Enterobacter sp. B16</td><td>Triticum aestivum</td><td>[109]</td></tr><tr><td>Klebsiella oxytoca</td><td>Triticum aestivum</td><td>[139]</td></tr><tr><td>Paraburkholderia megapolitana</td><td>Ricinus communis, Glycine max</td><td>[35]</td></tr><tr><td>Providencia sp.</td><td>Triticum aestivum</td><td>[30,140]</td></tr><tr><td>Pseudomonas sp. R8</td><td>Triticum aestivum</td><td>[109,133]</td></tr><tr><td>Rhizobium sp.</td><td>Astragalus bisulcatus, A. drummondii</td><td>[138]</td></tr><tr><td>Rhizosphere bacteria</td><td>Scirpus robustus, Polypogon monspeliensis</td><td>[143]</td></tr><tr><td>Se-tolerant bacteria</td><td>Brassica juncea</td><td>[144]</td></tr><tr><td>Stenotrophomonas maltophilia</td><td>Ricinus communis, Glycine max, Brassica juncea</td><td>[35,142]</td></tr><tr><td>Stenotrophomonas sp. B19</td><td>Triticum aestivum</td><td>[109,133]</td></tr></tbody></table>

Interestingly, volatile organic compounds (VOCs) released by PGPRs improve Se biofortification of plants. VOCs from *Bacillus amyloliquefaciens* BF06 significantly increased photosynthesis and growth of *Arabidopsis* plants and these VOCs led to an obvious increase in expressions of some genes encoding sulfate transporters and Se concentrations in plants [141]. VOCs released by *B. amyloliquefaciens* could not increase Se biofortification of *Arabidopsis* *Sultr1:2* mutants. All the results suggested sulfate transporters with high expression mediate Se uptake, as shown above. Meanwhile, the results indicate an unknown mechanism that PGPRs improves Se biofortification. The question is inevitable, how do the VOCs improve expression of sulfate transporters?
Taken together, Se amendment could improve population growth of some Se-tolerant bacteria; if these bacteria show synergistic effect on Se biofortification, they could be mixed in some biofilmed biofertilizers specific to certain crops and vegetables, thus, their combinative amendment along with Se fertilizers become a Se biofortification tool in sustainable agriculture [52,145].

### 3. Concluding Remarks and Perspectives

Since Se is essential for human health, Se biofortification must be carried out in Se-deficient places by various ways on food crops, vegetables, fruits, and nuts. Foliar and soil fertilization are effective for enhancing Se accumulation in crops. However, the two ways are expensive for large-scaled food crops, especially in poor places. Moreover, the effect of the two ways is short-term and they easily cause area source pollution. BMOs improve Se uptake and accumulation in food crops. Therefore, combination of BMOs and soil fertilization is a good approach to Se biofortification of crop food. At present, for Se biofortification by BMOs, there remain some questions to resolve. The first relates to the synergisms among these beneficial microorganisms. Biofilmed biofertilizers often include many BMOs. As they can compete for nutrition from their common host plants, some of them are possibly antagonistic. Therefore, before they are mixed in biofilmed biofertilizers, the synergism should be examined in detail. The second relates to Se biofortification and phytoremediation. Phytoremediation of Se is popular in Se-rich places. The plants harvested in phytoremediation could be used as the organic source of Se. However, attention must be paid to the fact that there are possibly other heavy metals in the harvested plants. In addition, transgenic plant technology has been used for phytoremediation and Se biofortification. Since some people are very sensitive to genetically modification of crop plants, application of transgenic plants for Se biofortification should be careful. The third relates to the use of Se hyperaccumulators. Some Se hyperaccumulators, such as *Stanleya pinnata* and *Astragalus bisulcatus* and *Cardamine enshiensis*, should be paid more attentions, especially *C. enshiensis*, because it is edible and can be directly used in food. The fourth relates to the use of Se nanoparticles. Relatively, Se nanoparticles are less toxic and more eco-friendly for both humans and the environment. More researches are necessary for use of Se nanoparticles, especially for the production of Se nanoparticles using plants and fungi. The fifth relates to increased plant resistance to abiotic stresses. Exogenous Se compounds and seleno-bacteria synergistically improve plant resistance to abiotic stresses, thus the synergism should be well used for plant resistance to abiotic stresses, especially drought and salt stress. Finally, the sixth relates to functions of root endophytic fungi and dark septate fungi. The two types of fungi possess ecological functions similar to mycorrhizal fungi, and they often colonize many plant species. However, very little attention has been paid to them.

**Author Contributions:** C.W. and F.X. planned the whole article and outline, and provided some references. C.W. and J.Q. revised the second manuscript. Y.Y. wrote the rough manuscript; Y.P. and S.R. reviewed the rough manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China (grant numbers 31870378); Fujian University Key Laboratory for Plant-Microbe Interaction (grant number PMI2018KF2).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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