Endophytic Fungi from the Four Staple Crops and Their Secondary Metabolites

Endophytic fungi are present in every plant, and crops are no exception. There are more than 50,000 edible plant species on the planet, but only 15 crops provide 90 percent of the global energy intake, and “the big four”—wheat, rice, maize and potato—are staples for about 5 billion people. Not only do the four staple crops contribute to global food security, but the endophytic fungi within their plant tissues are complex ecosystems that have been under scrutiny. This review presents an outline of the endophytic fungi and their secondary metabolites in four staple crops: wheat, rice, maize and potato. A total of 292 endophytic fungi were identified from the four major crops, with wheat having the highest number of 157 endophytic fungi. Potato endophytic fungi had the highest number of secondary metabolites, totaling 204 compounds, compared with only 23 secondary metabolites from the other three crops containing endophytic fungi. Some of the compounds are those with specific structural and pharmacological activities, which may be beneficial to agrochemistry and medicinal chemistry.


Introduction
There are more than 50,000 edible plant species on the planet, but only a few hundred contribute meaningfully to our diet [1].In fact, just 15 crops provide 90 percent of the global energy intake and "the big four"-wheat, rice, maize and potato-are staples for about 5 billion people [2,3].The most productive staple crop in the world is maize, which yielded 1.16 billion tons in 2022 alone, followed by wheat, rice and potatoes at 808, 776 and 375 million tons, respectively [4].Such reliable, widespread crops are the basis of food systems and human subsistence.
Endophytic fungi are defined as fungi that spend the whole or part of their life cycle colonizing inter-and/or intracellularly inside the tissues of the host plants, typically including non-vascular plants, ferns and allies, conifers and angiosperms [5,6].Considering crop-microbe interactions, endophytic fungi are also extremely important for agricultural sustainability.The presence of these endophytic fungi provides benefits to host plants, including enhanced resistance to herbivores and pests, increased competitiveness and improved tolerance to abiotic stresses such as the occurrence of heavy metals and high salinity, and may affect the yield and quality of the crop [6].In a recent study, twelve strains of isolated endophytic fungi from the tissues of Cotoneaster multiflorus were shown to promote plant growth and performance, and enhance the fitness of their host plants' resistance to biotic and abiotic stresses [7].Grasses are colonized by Acremonium endophytes, which are known to protect their hosts from insect attacks, nematodes and plant diseases [6].In addition, endophytes have been recognized as important sources of a variety of new biologically active secondary metabolites potentially useful for human medicine, with anticancer, antimicrobial and other activities, and also could be potential sources of novel natural products with industrial and agrochemical potential [8][9][10].
It is estimated that there are over one million fungal endophytes existing in nature [11].Up to now, just over 0.1 million fungi associated with higher organisms have been described [12].Thus, the global diversity of most endophytic fungi is unknown, and still a large number of species and ecosystems need to be explored.This review brings insight into the diversity and bioactive potential of these endophytic fungi in four staple crops, which will contribute to their effective utilization.

Wheat
Wheat (Triticum aestivum ssp.Aestivum) is a high-yielding crop that first originated in the Fertile Crescent region of the Middle East about 10,000 years ago and began to spread around the world [13].Before 7000 B.C., Mesopotamia began to grow wheat and barley and raise goats [14].Wheat was later utilized as a burial item for the Egyptian King Tutankhamun in 1325 B.C. [15].It was not until the 16th century that the Spanish brought wheat to the New World [16].Today, wheat is the staple food for more than 35% of the world's population and is one of the major commodity grains, accounting for a large share of the international food trade [17].

Rice
Rice (Oryza sativa L.) is a cereal crop that produces edible grain seeds.The seeds can be eaten in their natural form, but rice may be more productive and reliable as its main crop.Rice is native to China, and spread westward through India to Europe and eastward to Korea and Japan [18].Rice was first cultivated in China in 6000 B.C. until 2800 B.C., when it became one of the five grains grown in China [19].After that, until 330 B.C., Alexander the Great brought back rice wine from India.Later, in 1519-1522, the Magellanic fleet saw rice in the Malay Archipelago [20,21].In 1690, "Carolina Gold Rice" became an important industry in the New World [22].Over the past 70 years, increases in rice yields have resulted from increases in grain yields per hectare; from 1950 to 1980, increases in yields were due to the introduction of dwarfing genes; and from 1980 to the present, increases in yields have been attributed to the development of high-yielding varieties, including hybrids, and to improvements in crop management practices, such as nitrogen fertilization [23][24][25].Today, rice is the staple food for half of the world's population.Rice is an important commodity, both in rice-growing regions and around the world.

Maize
Maize (Zea mays L.) is known as the "Queen of Cereals" and is one of the most extensively planted cereal crops in the world, ranking first in terms of production [26].Beginning in 5000 B.C., maize was cultivated in Mesoamerica [27].In 1492, Columbus discovered that the native peoples of the New World were growing corn and introduced it to the Old World, where it spread rapidly over the next two centuries [28,29].In order to increase the yield of corn, it has been cross-pollinated with remarkable results [30].Later, technological advances were achieved to extract corn oil from maize, which is appreciated for its high energy content, and there are indications that corn oil has uses for boosting biocontrol agent fungal efficiency [31].In the 1960s, high-fructose corn syrup (HFCS) was developed for use in the manufacture of food and beverages.By the mid-1980s, it had completely replaced sucrose in most beverages in the United States and was consumed as a safe and reliable sweetener [32].Approximately two-thirds of today's maize is utilized largely for animal feed, but maize may also be used as a prime extract for motor fuel ethanol, opening up a new avenue for the development of sustainable biofuels [33].

Potato
Potatoes (Solanum tuberosum L.) are an essential staple crop, ranking second only to wheat, rice and maize.At the moment, 1.3 billion people worldwide use potatoes, and this type of root "food" is becoming increasingly popular in developing nations; nevertheless, potato whole flour preservation time can be up to 15 years, and some governments have even recognized potato as a strategic reserve food.Archaeological evidence suggests that the earliest humans to encounter and potentially consume the potato lived on the western coast of South America around 13,000 years ago during the Last Ice Age.During the Last Ice Age, when glaciers receded from the Altiplano, prototypes of farmed potatoes arose in the mountains of modern-day Peru and Bolivia [34][35][36].The oldest potato samples date back some 8000 years, and the domestication of the potato probably took place around Lake Titicaca, on the present-day border between Peru and Bolivia, and then gradually spread to the surrounding area [34,37].The first step in domesticating potatoes is to select bitter-free tubers.Following that, the selection of non-bitter potato varieties and their cultivation and dissemination led to the potato's domestication, allowing it to spread far over the world.China is by far the largest potato producer, accounting for 32% of the global planted area and 27% of global potato production [38].

Endophytic Fungi
Endophytic fungi (EFs) are a fascinating host-associated fungal community that colonizes the intercellular or intracellular regions of host tissues, benefiting their hosts while obtaining an advantage [39].EFs play an important role in agricultural growth and development by controlling pests and diseases, producing growth regulators and boosting crop adaptation to harsh circumstances [40,41].

Wheat Endophytic Fungi
The present research identified fungi of the genera Alternaria, Acremonium, Cladosporium, Chaetomium, Epicoccum, Fusarium and Phoma, which have been described as wheat endophytes in the literature [42][43][44][45][46][47][48].The species Aureobasidium pullulans were identified in almost all the studied wheat cultivars [42].Table 1 shows the comparison between the reviewed wheat endophytic fungi and the plant organs from which they originate.The vast majority of the wheat endophytic fungi reviewed belong to the genera Alternaria, Bipolaris, Cladosporium, Epicoccum, Fusarium, Penicillium and Sarocladium.Only nine endophytic fungi-Alternaria sp., Cladosporium sp., Fusarium oxysporum, Fusarium sp., Penicillium chrysogenum, Penicillium olsonii, Penicillium sp., Sarocladium sp. and Sarocladium strictumcan be isolated from the roots, leaves, stems and fruits of the plant.In addition, the root endophytic fungi were the most diverse.Some representatives of the Trichoderma genus, isolated from wheat, can reduce the severity of diseases caused by P. triticis, Mycosphaerella graminicola, and B. sorokiniana and A. alternata in wheat [42].Furthermore, the genera Alternaria and Cladosporium are capable of spreading vertically through the seed and thus infecting wheat [49].

Rice Endophytic Fungi
The vast majority of reported rice endophytic fungi belong to the genera Acremonium, Aspergillus, Chaetomium, Cladosporium, Fusarium, Penicillium, Sarocladium, Talaromyces and Trichoderma (Table 2).The dominant fungal genera Penicillium and Aspergillus coexisted in the stems and roots (Table 2) [50].Aspergillus flavus, Eupenicillium javanicum, Microsphaeropsis arundinis, Penicillium rubens, Talaromyces pinophilus and Trichoderma zelobreve were simultaneously detected in the roots, shoots and stems of the rice plants (Table 2).Only two strains of the endophytic fungi Acremonium sp. and Arthrobotrys sp. were able to coexist in the roots, leaves, stems and fruits of the plant, and Aspergillus ustus and Sarocladium oryzae DX-THL3 colonized just the leaves (Table 2).Furthermore, practically all rice endophytic fungi were discovered from the roots (Table 2).Several studies have shown that rice endophytic fungi promote rice growth; for example, Cladosporium sphaerospermum produces gibberellins (GA7 and GA4), which increase rice biomass [51,52].In addition, Talaromyces pinophilus, Aspergillus flavus and Trichoderma sp. were able to increase several plant growth parameters such as plant height, the number of tillers, total chlorophyll, photosynthesis rate, etc., as well as the accumulation of phytochemicals (total phenolic compounds and anthocyanins) and antioxidant capacity in rice seeds [53].

Potato Endophytic Fungi
There have been less studies on potato endophytic fungi than on wheat, rice and maize endophytic fungi.Almost all endophytic fungi originated in potato roots, while no endophytic fungi originated in potato fruits.Aspergillus carneus, Bipolaris eleusines, Boeremia exigua, Cephalotrichum gorgonifer, Chaetomium subaffine, Trichothecium crotocinigenum, Xylaria curta E10 and Xylaria cf.curta are all endophytic fungi originating from potato roots, leaves and stems (Table 4).Among other things, it was shown that Boeremia exigua showed good inhibitory activities on the growth of Phytophthora infestans, which causes late blight to the potato plant as well as to other plants [58].Chaetomium globosum is currently isolated only from potato stems (Table 4).In addition, Colletotrichum coccodes and Cylindrocarpon destructans are common endophytic fungi in potato roots and both are weak plant pathogens (Table 4) [59].

Terpenoid Compounds
Terpenoids are a type of natural product made up of basic "C5" units known as isoprene.Thus, terpenoids are classified according to the number of isoprene units, with sesquiterpenoids (C15), diterpenoids (C20), sesterterpenoids (C25) and others [99]  in rice leaves infected with the pathogen, and both of them inhibited the germ tube elongation of both phytopathogenic fungi, Pyricularia oryzae and Bipolaris oryzae, and their effective concentrations were lower than those found in the conidial germination assay; the IC50 values for germ tube elongation were more than 10 times smaller than those for conidial germination [112].Moreover, 143, 146 and 147 had relatively strong activities on the elongation of P. oryzae with IC50 values of 6.6, 7.8 and 8.1 µM, respectively, and 144 and 145 had relatively strong activities on the elongation of B. oryzae with IC50 values of 9.8 µM [112].In addition, a series of diterpenoids (148-156) (Figure 4) have also been identified from rice blast leaves infected with P. oryzae [113][114][115].

Diterpenoids
Two diterpenoids (116-117) (Figure 4) were isolated from the coffee medium of the maize endophyte fungus Fusarium sp.Among them, 117 showed significant antifungal activity against the phytopathogen A. alternata from Coffee arabica with an MIC value of 2 µg/mL [98].A casbane-type diterpene (118) (Figure 4) was obtained from UV-irradiated rice leaves, which inhibited the spore germination (IC 50 30 ppm) and germ tube growth (IC 50 10 ppm) of the rice blast fungus [106].Eight diterpenoids (119-126) (Figure 4) were obtained from rice husks, of which 122-123 and 125-126 exhibited moderate α-glucosidase inhibitory activity with IC 50 values ranging from 21.33 to 81.39 µg/mL, respectively [107].One diterpenoid, oryzalactone (127), also containing a lactone ring, was obtained from rice leaves, which inhibited conidial germination and germ tube elongation, suggesting that they have antifungal activity against P. oryzae [108].Ten diterpenes and diterpene derivatives (128-137) (Figure 4) were isolated from UV-irradiated rice leaves.Compounds 128 and 130 exhibited weak antifungal activity and may be biosynthetic intermediates of the rice phytotoxins momilactones and oryzalexin S, respectively [109].Compounds 132 and 133 showed the same and relatively high inhibitory activity against the mycelial growth of M. oryzae [109].The treatment of suspension-cultured rice cells with mycelial extracts of the potato pathogenic fungus Phytophthora infestans produced large amounts of diterpenoids (138-142).The ED 50 values of these compounds (138-142) in preventing the spore germination of the rice pathogenic fungus Magnaporthe grisea were 6, 20, 4, 7 and 25 µg/mL, respectively, and indicated that the hydroxyl group at the C-1 position was the main functional moiety for the high antifungal activity of these compounds [110,111].The benzene ring-containing diterpenoids abietoryzins A-E (143-147) (Figure 4) were detected in rice leaves infected with the pathogen, and both of them inhibited the germ tube elongation of both phytopathogenic fungi, Pyricularia oryzae and Bipolaris oryzae, and their effective concentrations were lower than those found in the conidial germination assay; the IC 50 values for germ tube elongation were more than 10 times smaller than those for conidial germination [112].Moreover, 143, 146 and 147 had relatively strong activities on the elongation of P. oryzae with IC 50 values of 6.6, 7.8 and 8.1 µM, respectively, and 144 and 145 had relatively strong activities on the elongation of B. oryzae with IC 50 values of 9.8 µM [112].In addition, a series of diterpenoids (148-156) (Figure 4) have also been identified from rice blast leaves infected with P. oryzae [113][114][115].

Alkaloid Compounds
Alkaloids were originally defined as alkaline substances extracted from a plant with a biological activity, and later extended to nitrogen-containing compounds derived from secondary or specialized metabolism [116].

Cytochalasins
Fourteen pyrrolidine/perhydroanthracene (5/6/6/6 tetracyclic skeleton)-fused ring systems of cytochalasins (177-190) (Figure 7) were isolated from the potato endophytic fungus X. curta E10.Compounds 177 and 181 exhibited potent inhibitory activity against the MCF-7 cell line with IC50 values of 2.03 and 0.85 µM, and compound 179 exhibited moderate inhibitory activity with an IC50 value of 13.9 µM, and it was hypothesized that the hydroxyl group at the C-7 position and the acetylation of OH-21 might be the key factors for the cytotoxic activity [84,85].Compounds 191-195 (Figure 7) were similarly isolated from the potato endophyte fungus Xylaria cf.curta, in which compound 191 showed a significant and selective inhibitory effect on LPS-induced B lymphocyte cell proliferation (IC50 value of 2.42 µM), which was 29 times more than against T-cell [117].In view of the structure/activity relationship, polar substitutions at positions 5 and 6 may be important for the inhibitory effects on B-cell proliferation.The oxygen substitution at position 7 could be a key pharmacophore for immunosuppressive activities, and the compounds with 7β-OH or 7-oxo have better activity than that of compounds with 7α-OH.6) and compounds 164-166 were isolated from the potato endophytic fungus B. eleusines, and compound 161 exhibited weak antifungal activity against E. floccosum, inhibiting it by 65.06% at 100 µM [111].Two steroidal compounds (167-168) (Figure 6) were isolated from the wheat pathogenic fungus M. majus 99049 [96].Compound 167 was active against HeLa cells with an IC 50 value of 51.9 µg/mL.In addition, 167 also showed weak anti-mycobacterium tuberculosis (MTB) with an MIC value at 80 µg/mL.And 167 showed moderate anti-methicillin-resistant Staphylococcus aureus (MRSA) activity with an MIC value of 25 µg/mL, while 168 showed weak anti-MRSA activity (MIC = 100 µg/mL) [96].Eight steroidal compounds (169-176) (Figure 6) were isolated from the endophytic fungus Sarocladium oryzae DX-THL3 from the leaf of Dongxiang wild rice (Oryza rufipogon Griff.), and compounds 169-170 and 172-175 exhibited antibacterial activity against S. aureus with MIC values of 64, 4, 8, 1, 4 and 16 µg/mL, 173 also showed antibacterial activity against Bacillus subtilis with an MIC value of 64 µg/mL, compounds 170, 173 and 175 showed some potent antibacterial activity against E. coli with MIC 64 µg/mL, and all of these compounds were inactive against Xanthomonas oryzae pv.oryzicola at 128 µg/mL [73].

Alkaloid Compounds
Alkaloids were originally defined as alkaline substances extracted from a plant with a biological activity, and later extended to nitrogen-containing compounds derived from secondary or specialized metabolism [116].

Alkaloid Compounds
Alkaloids were originally defined as alkaline substances extracted from a plant with a biological activity, and later extended to nitrogen-containing compounds derived from secondary or specialized metabolism [116].

Cytochalasins
Fourteen pyrrolidine/perhydroanthracene (5/6/6/6 tetracyclic skeleton)-fused ring systems of cytochalasins (177-190) (Figure 7) were isolated from the potato endophytic fungus X. curta E10.Compounds 177 and 181 exhibited potent inhibitory activity against the MCF-7 cell line with IC 50 values of 2.03 and 0.85 µM, and compound 179 exhibited moderate inhibitory activity with an IC 50 value of 13.9 µM, and it was hypothesized that the hydroxyl group at the C-7 position and the acetylation of OH-21 might be the key factors for the cytotoxic activity [84,85].Compounds 191-195 (Figure 7) were similarly isolated from the potato endophyte fungus Xylaria cf.curta, in which compound 191 showed a significant and selective inhibitory effect on LPS-induced B lymphocyte cell proliferation (IC 50 value of 2.42 µM), which was 29 times more than against T-cell [117].In view of the structure/activity relationship, polar substitutions at positions 5 and 6 may be important for the inhibitory effects on B-cell proliferation.The oxygen substitution at position 7 could be a key pharmacophore for immunosuppressive activities, and the compounds with 7β-OH or 7-oxo have better activity than that of compounds with 7α-OH.Moreover, the substitution of 21-OAc could increase the immunosuppressive activity as well [117].Cytochalasins D1 and C1 (196,197), which possess a unique eleven-membered macrocycle with an oxygen bridge, were also obtained from this fungus, and they showed moderate cytotoxicity against the HL-60 cell line with IC 50 values of 12.7 and 22.3 µM, respectively [118].An unprecedented 6/7/5/6/6/6 fused ring system with two chlorine substitutions, xylarichalasin A (198), was also obtained from this fungus, which showed significant cytotoxicity against the SMMC-7721 and MCF-7 cell lines (IC 50 values of 8.6, 6.3 µM), superior to the positive drug cisplatin [86].Three cytochalasins (curtachalasins C-E, 199-201) with an unprecedented bicyclic [3.3.1]lactam core structure and thirteen 19,20epoxycytochalasans (201-214) were isolated from extracts of the liquid fermentation of X. cf.curta [87,119].Compound 199 showed significant resistance reversal activity against fluconazole-resistant C. albicans, compound 208 showed significant specific cytotoxicity against the HL-60 cell line with an IC 50 value of 1.11 µM, and compounds 202 and 213 were moderately cytotoxic against the cancer cell line HL-60 (IC 50 values of 13.31 and 10.04 µM, respectively) [87,119].Eight cytochalasins boerechalasins A-G (215-221) and compound 222 were isolated from the potato endophytic fungus B. exigua, and compound 220 exhibited moderate cytotoxicity against the MCF-7 cell line, with an IC 50 value of 22.8 µM [120].

Other Alkaloids
Three alkaloid derivatives (223-225) (Figure 8) were isolated from the wheat endophytic fungus M. majus 99049, and they showed weak inhibitory activity against the HUH-7 human hepatoma cell with IC 50 values at 80 µg/mL [96].The compound caffeine (226) was isolated from the maize endophytic fungus Fusarium sp. and it showed significant antifungal activities against the plant pathogen A. alternata with MICs of 1 µg/mL [98].Pipecolisporin (227), a new pipecolic acid containing hexapeptide, was isolated from cultures of the wheat endophytic fungus Nigrospora oryzae.The compound (227) displayed remarkable antiparasitic activity against Plasmodium Falciparum and Trypanosoma cruzi, with an IC 50 value comparable to that of benznidazole, currently used in the treatment of Chagas disease, and no toxicity against a panel of five human carcinoma cell lines [121].
fluconazole-resistant C. albicans, compound 208 showed significant specific cytotoxicity against the HL-60 cell line with an IC50 value of 1.11 µM, and compounds 202 and 213 were moderately cytotoxic against the cancer cell line HL-60 (IC50 values of 13.31 and 10.04 µM, respectively) [87,119].Eight cytochalasins boerechalasins A-G (215-221) and compound 222 were isolated from the potato endophytic fungus B. exigua, and compound 220 exhibited moderate cytotoxicity against the MCF-7 cell line, with an IC50 value of 22.8 µM [120].Three alkaloid derivatives (223-225) (Figure 8) were isolated from the wheat endophytic fungus M. majus 99049, and they showed weak inhibitory activity against the HUH-7 human hepatoma cell with IC50 values at 80 µg/mL [96].The compound caffeine (226) was isolated from the maize endophytic fungus Fusarium sp. and it showed significant antifungal activities against the plant pathogen A. alternata with MICs of 1 µg/mL [98].Pipecolisporin (227), a new pipecolic acid containing hexapeptide, was isolated from cultures of the wheat endophytic fungus Nigrospora oryzae.The compound (227) displayed remarkable antiparasitic activity against Plasmodium Falciparum and Trypanosoma cruzi, with an IC50 value comparable to that of benznidazole, currently used in the treatment of Chagas disease, and no toxicity against a panel of five human carcinoma cell lines [121].

Conclusions
This review evaluates the endophytic fungi and secondary metabolites of four major crops: wheat, rice, maize and potato.Wheat had the highest number of endophytic fungi, followed by maize, rice and potato.Although potato endophytic fungi were the least abundant, they had the highest number of metabolites.In addition, 12 secondary metabolites were derived from wheat endophytic fungi, 8 compounds from rice endophytic fungi and only 3 compounds from maize endophytic fungi.Secondary metabolites derived from potato endophytic fungi play important roles in antimicrobial, plant growth regulation and human drug discovery.Although the chemical composition of endophytic fungi has been the focus of natural product research, only a small fraction of the chemical composition of endophytic fungi from the other three staple food crops (wheat, rice and maize) has been explored.Therefore, the diversity of endophytic fungi related to the four staple foods as well as their chemical composition still needs to be explored.

Conclusions
This review evaluates the endophytic fungi and secondary metabolites of four major crops: wheat, rice, maize and potato.Wheat had the highest number of endophytic fungi, followed by maize, rice and potato.Although potato endophytic fungi were the least abundant, they had the highest number of metabolites.In addition, 12 secondary metabolites were derived from wheat endophytic fungi, 8 compounds from rice endophytic fungi and only 3 compounds from maize endophytic fungi.Secondary metabolites derived from potato endophytic fungi play important roles in antimicrobial, plant growth regulation and human drug discovery.Although the chemical composition of endophytic fungi has been the focus of natural product research, only a small fraction of the chemical composition of endophytic fungi from the other three staple food crops (wheat, rice and maize) has been explored.Therefore, the diversity of endophytic fungi related to the four staple foods as well as their chemical composition still needs to be explored.

Figure 2 .
Figure 2. Chemical structures of other ketones.

Figure 8 .
Figure 8.Chemical structures of other alkaloids.

Author
Contributions: Y.F. and B.S. conceptualized the review.Y.F.wrote the review with the help of B.S. B.S. reviewed the text and structured the review.All authors have read and agreed to the

Figure 8 .
Figure 8.Chemical structures of other alkaloids.

Table 1 .
Endophytic fungi isolated from wheat plants.
√Present in plant organs.

Table 2 .
Endophytic fungi isolated from rice plants.

Table 4 .
Endophytic fungi isolated from potato plants.