Patents on Endophytic Fungi for Agriculture and Bio- and Phytoremediation Applications

Plant endophytic fungi spend all or part of their lives inside host tissues without causing disease symptoms. They can colonize the plant to protect against predators, pathogens and abiotic stresses generated by drought, salinity, high concentrations of heavy metals, UV radiation and temperature fluctuations. They can also promote plant growth through the biosynthesis of phytohormones and nutrient acquisition. In recent years, the study of endophytic fungi for biological control of plant diseases and pests has been intensified to try to reduce the ecological and public health impacts due the use of chemicals and the emergence of fungicide resistance. In this review, we examine 185 patents related to endophytic fungi (from January 1988 to December 2019) and discuss their applicability for abiotic stress tolerance and growth promotion of plants, as agents for biocontrol of herbivores and plant pathogens and bio- and phytoremediation applications.


Introduction
An endophytic fungus is any organism inhabiting plant organs that, at certain point in its lifetime, can colonize tissues without causing apparent harm [1]. Endophytic fungi have been a proven source of secondary metabolites with potential uses as anticancer, antibiotics, antivirals, anti-inflammatories, antioxidants, neuroprotective agents, insecticides and antifungals, and have multiple applications in biotechnological developments in pharmaceutical, agriculture, cosmetic, food industry and environmental processes [2]. In the last decades, studies of endophytic fungi have resulted in a number of patents linked to the production of biologically active secondary metabolites and in biotransformation processes [3].
Moreover, interaction between fungi and their hosts drives changes in the host metabolism, altering the response to environmental stress and predator attack. Additionally, this interaction leads to the production of secondary metabolites by both the fungi and the host, which further enhance the capability to respond to the environment [4][5][6][7].
The use of endophytic fungi for environmental applications such as growth promotion, relief of abiotic stress, biocontrol of pest and plant pathogens and bio/phytoremediation has gained important attention in recent years due to the concern about global climate change and contamination in soils and natural sources that increases stress in crops, limiting and reducing the production [8][9][10][11]. Furthermore, basic and applied research has been conducted to develop processes, methodologies and technologies that resulted in a considerable number of patents with new proposals to overcome some of these

Abiotic Stress Tolerance and Growth Promotion of Plants
The principal abiotic stress factors in plants include drought, salinity, high heavy metal concentrations, UV radiation and temperature fluctuations [12]. Abiotic stress affects the cellular pathways of plants, resulting in negative changes to their physiology and morphology [12]. Endophytic fungi have been shown to help their host plant to overcome abiotic stress and promote plant growth through the biosynthesis of phytohormones (indole-3-acetic-acid, gibberellins, cytokinins, ethylene, acetoin, 2, 3-butanediol) and nutrient absorption and uptake [12][13][14].

Biocontrol of Herbivores and Plant Pathogens
Crop plant diseases represent a major threat in agriculture [101]. The number of chemicals that can be effectively used to control pathogens has been reduced due to the emergence of fungicide resistance along with an increased awareness of the negative associated ecological and public health impacts [101]. Due to these problems, study of the biological control of plant diseases with endophytes has intensified in recent years [101]. Endophytes have been shown to protect their hosts against diseases, reducing infection levels and inhibiting the growth of pathogens [102,103]. The proposed mechanisms used by endophytes are the production of antimicrobial and structural compounds, niche competition, and the induction of plant immunity [104].
Endophytic fungi of different genera such as Beauveria, Cladosporium, Metarhizium, Muscodor, Trichoderma, and others have also been described in patents to control pests or different plant diseases (Table 3).

Bio-and Phytoremediation
Bioremediation is a process that uses microorganisms, plants or enzymes to detoxify contamination in natural sources. In phytoremediation, plants and their own metabolic system can extract toxic chemicals from water, soil and air. This chemicals or contaminants include metals and metalloid pollutants, carcinogenic agents, industrial organic waste material, inorganic pesticides and herbicides, chlorinated products, excess nutrients and radionuclides [10,11,192].
Endophytic fungi have the capability to degrade small and large organic compounds by enzymatic reactions, decompose environmental contaminants, and improve the soil microenvironment [193]. They can also increase the ability of host plants to remove contaminants from soil, water, sediment, and air [194], and to modulate morphological and physiological functions in the host plant improving its resistance to metals and providing different detoxification routes such as extracellular scavenging and complexation, compartmentalization and volatilization [14,195]. Figure 1 shows different bioremediation techniques involving endophytic fungi.

Bio-and Phytoremediation
Bioremediation is a process that uses microorganisms, plants or enzymes to detoxify contamination in natural sources. In phytoremediation, plants and their own metabolic system can extract toxic chemicals from water, soil and air. This chemicals or contaminants include metals and metalloid pollutants, carcinogenic agents, industrial organic waste material, inorganic pesticides and herbicides, chlorinated products, excess nutrients and radionuclides [10,11,192].
Endophytic fungi have the capability to degrade small and large organic compounds by enzymatic reactions, decompose environmental contaminants, and improve the soil microenvironment [193]. They can also increase the ability of host plants to remove contaminants from soil, water, sediment, and air [194], and to modulate morphological and physiological functions in the host plant improving its resistance to metals and providing different detoxification routes such as extracellular scavenging and complexation, compartmentalization and volatilization [14,195]. Figure 1 shows different bioremediation techniques involving endophytic fungi. Some patents describe the use of endophytic fungi for bioremediation and phytoremediation ( Table 4). Strains of the genus Fusarium have been reported to induce phytoremediation in heavy metal-contaminated soil [196], repair uranium-polluted water bodies [197], and decontaminate and decompose human and animal waste [198]. Additionally, the endophytic fungi Y2R14 and RWDL4-1 can be used to treat wastewater polluted by cadmium [199]. Heavy metals such as mercury, cadmium, arsenic, chromium, and lead are toxic at low concentrations. They can be accumulated in the ecosystem inside living organisms and are capable of entering the food chain [200]. The functions of several organs of the human body can be affected by heavy metals, and some of these substances can cause cancer by long-term exposure [200]. Uranium is a radioactive substance and is also harmful for the environment and human beings [197]. The use of microorganisms to repair large areas of farmland pollution can reduce costs, the use of large amounts of chemicals, and secondary pollution [196].
Species of Phomopsis and Xylaria have been reported to degrade the herbicide MCPA (2-methyl-4-chlorophenoxyacetic acid) in water and soil [201,202]. Additionally, several genera of fungi can be used to produce high-laccase content for soil bioremediation [203].

Patents that Claim Multiple Applications
A small number of patents comprised more than one possible application (Table 5); this is the case of the applications for Neotyphodium uncinatum to induce insect resistant and drought tolerance in plants [204]; Phoma sp. can improve salt stress resistance, promote the growth and increase biomass Some patents describe the use of endophytic fungi for bioremediation and phytoremediation ( Table 4). Strains of the genus Fusarium have been reported to induce phytoremediation in heavy metal-contaminated soil [196], repair uranium-polluted water bodies [197], and decontaminate and decompose human and animal waste [198]. Additionally, the endophytic fungi Y2R14 and RWDL4-1 can be used to treat wastewater polluted by cadmium [199]. Heavy metals such as mercury, cadmium, arsenic, chromium, and lead are toxic at low concentrations. They can be accumulated in the ecosystem inside living organisms and are capable of entering the food chain [200]. The functions of several organs of the human body can be affected by heavy metals, and some of these substances can cause cancer by long-term exposure [200]. Uranium is a radioactive substance and is also harmful for the environment and human beings [197]. The use of microorganisms to repair large areas of farmland pollution can reduce costs, the use of large amounts of chemicals, and secondary pollution [196].
Species of Phomopsis and Xylaria have been reported to degrade the herbicide MCPA (2-methyl-4-chlorophenoxyacetic acid) in water and soil [201,202]. Additionally, several genera of fungi can be used to produce high-laccase content for soil bioremediation [203].

Patents that Claim Multiple Applications
A small number of patents comprised more than one possible application (Table 5); this is the case of the applications for Neotyphodium uncinatum to induce insect resistant and drought tolerance in plants [204]; Phoma sp. can improve salt stress resistance, promote the growth and increase biomass in crop plants such as wheat and rice [205]; Clonostachys rosea promotes plant growth, stress resistance and reduces dependency on chemical pesticides [206,207]; Fusarium sp. stimulates plant growth and reduces heavy metal absorption in tobacco [208], and Rhizoctonia sp. fosters plant growth and stress resistance in Anoectochilus roxburghii [22]. Not disclosed wild soybean Treatment of wastewater polluted by the heavy metal cadmium [199] 1 Some patents just provided a common name for the host organism. 2 A list of the group of fungi is in Table S1.

WO2000062600A1
Neotyphodium uncinatum meadow fescue Import desired traits: include no adverse effects on herbivore, insect resistance, drought tolerance and improved persistence in the plants. [204] CN104293681A Phoma sp. Not disclosed Improving salt stress resistance in rice and wheat. Promotion of growth in rice seedling, delaying salt damage of wheat in saline and alkaline land. Increasing biomass accumulation in wheat. [205] US20160007613A1 Clonostachys rosea Not disclosed Promotion of plant vigor, health, growth, yield, and resistance to competitive stress. [206] WO2007107000A1 Clonostachys rosea Not disclosed Enhanced plant vigor, health, growth, yield, reducing environmental stress and reduction of dependency on chemical pesticides for pest control. [207] CN103849572A Fusarium sp. Not disclosed Promoting plant growth and reduction of heavy metal absorption in tobacco. [208] CN101953261A Rhizoctonia sp.

Anoectochilus roxburghii
Growth of A. roxburghii, improved the reproductive rate, survival rate and stress resistance. [22]

WO2019115582A1
Group of several fungi 2 Hordeum murinum Increased yield and biomass in cereal crops, and promotes biotic and abiotic stress resistance in cereal crops Group of several fungi 2 Hordeum murinum subsp. murinum Improving dry shoot weight, mean dry grain weight and suppression of seed-borne infection in a cereal crop. [35] We found two patents, whose applications implicated the use a plural number of fungi (genus/species); one of them claims the capability to increase biomass and promote biotic and abiotic stress resistance in cereal crops [37], the other claims to improve dry shoot weight, mean dry grain weight and suppression of seed-borne in cereal crops [35].

Discussion
In the present review, we highlight a wide number of endophytic fungi that have been patented for developing processes, methodologies, or new techniques in applications that include but are not restricted to (a) alternatives to overcome biotic and abiotic stress and to reduce the use of chemicals associated with environmental toxicity in agricultural practices, (b) the degradation of harmful compounds, and (c) improvement in the ability of plants to remove contaminants from soil, water, and air. Abiotic stress tolerance and growth promotion of plants, and biocontrol of herbivores and plant pathogens, were the most patentable applications of endophytic fungi with 88 and 90 patents, respectively; concerning bio-and phytoremediation, 7 patents were recorded for the period 1988-2019 ( Figure 2). The most representative genera of these applications belong to Alternaria, Aspergillus, Chaetomium, Fusarium, Penicillium and Muscodor.

Discussion
In the present review, we highlight a wide number of endophytic fungi that have been patented for developing processes, methodologies, or new techniques in applications that include but are not restricted to (a) alternatives to overcome biotic and abiotic stress and to reduce the use of chemicals associated with environmental toxicity in agricultural practices, (b) the degradation of harmful compounds, and (c) improvement in the ability of plants to remove contaminants from soil, water, and air. Abiotic stress tolerance and growth promotion of plants, and biocontrol of herbivores and plant pathogens, were the most patentable applications of endophytic fungi with 88 and 90 patents, respectively; concerning bio-and phytoremediation, 7 patents were recorded for the period 1988-2019 ( Figure 2). The most representative genera of these applications belong to Alternaria, Aspergillus, Chaetomium, Fusarium, Penicillium and Muscodor. Studies of endophytic fungi ecology have allowed the understanding of the multiple interactions they develop with their host, other endophytes, as well with herbivores and pathogens that put the host under abiotic stress. Nonetheless, it is evident that one individual or group of endophytes can be used for mitigation stresses from different origins. Due to the concerns about global climate change and its implications in food security, there are an increased interest to develop applications for the use of endophytic fungi in abiotic stress tolerance and growth promotion of important food crops [209], as well as the use for biocontrol of herbivores and plant pathogens. This increment can be noted since 2011 as shown in Figure 3. The loss of growing areas due to contamination and the recovery of spaces contaminated by heavy metals, organic and inorganic compounds will lead the focus of research on endophytic fungi for bio-and phytoremediation applications.
Considering the abundance of endophytic fungi under study, the development of patentable applications like those reviewed here, and other applications still unexplored like fungal pigments [210], has become a prominent research area for this class of microorganisms. Studies of endophytic fungi ecology have allowed the understanding of the multiple interactions they develop with their host, other endophytes, as well with herbivores and pathogens that put the host under abiotic stress. Nonetheless, it is evident that one individual or group of endophytes can be used for mitigation stresses from different origins. Due to the concerns about global climate change and its implications in food security, there are an increased interest to develop applications for the use of endophytic fungi in abiotic stress tolerance and growth promotion of important food crops [209], as well as the use for biocontrol of herbivores and plant pathogens. This increment can be noted since 2011 as shown in Figure 3. The loss of growing areas due to contamination and the recovery of spaces contaminated by heavy metals, organic and inorganic compounds will lead the focus of research on endophytic fungi for bio-and phytoremediation applications.

Future Perspectives
The use of endophytic fungi to improve the nutrients absorption in plants can change the optimum usage of organic and inorganic fertilizers [211]. The capability of endophytic fungi to increase biotic and abiotic stress tolerance in plant hosts is an unexplored area for agricultural purposes; the control of pests and diseases under climate change conditions [211]; studies in fungal species related to develop resistance to changes in their environment could lead their application in food production in limited resources areas and as an important alternative for crop production for human sustainability. Many endophytes are now often recognized as symbionts with unique and intimate interactions with the plant host [10]. The genetic engineering of fungi is an easier process than in plants. The genetic modification of endophytic fungi with useful genes could contribute, with new traits, to the inoculation of plants [212].
The use of endophytic fungi on remediation of contaminated ecosystems is an interesting prospect for further studies. Fungi that could increase the capacity of CO2 absorption by plants, degradation and biotransformation of waste, enhance food production without altering its quality or those that provided drought resistance/nutrient absorption capability to plant species related to human or animal feeding could be areas of significance to develop new applications and patents. The investigations applied in these fields are forwarded by the advance in the techniques used for the characterization of endophytic fungi and also by the technological advances in analytical techniques for carrying out studies of chemical processes at the cellular level.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: List of patens grounded in the use of several endophytic fungi to develop applications.   Considering the abundance of endophytic fungi under study, the development of patentable applications like those reviewed here, and other applications still unexplored like fungal pigments [210], has become a prominent research area for this class of microorganisms.

Future Perspectives
The use of endophytic fungi to improve the nutrients absorption in plants can change the optimum usage of organic and inorganic fertilizers [211]. The capability of endophytic fungi to increase biotic and abiotic stress tolerance in plant hosts is an unexplored area for agricultural purposes; the control of pests and diseases under climate change conditions [211]; studies in fungal species related to develop resistance to changes in their environment could lead their application in food production in limited resources areas and as an important alternative for crop production for human sustainability. Many endophytes are now often recognized as symbionts with unique and intimate interactions with the plant host [10]. The genetic engineering of fungi is an easier process than in plants. The genetic modification of endophytic fungi with useful genes could contribute, with new traits, to the inoculation of plants [212].
The use of endophytic fungi on remediation of contaminated ecosystems is an interesting prospect for further studies. Fungi that could increase the capacity of CO 2 absorption by plants, degradation and biotransformation of waste, enhance food production without altering its quality or those that provided drought resistance/nutrient absorption capability to plant species related to human or animal feeding could be areas of significance to develop new applications and patents. The investigations applied in these fields are forwarded by the advance in the techniques used for the characterization of endophytic fungi and also by the technological advances in analytical techniques for carrying out studies of chemical processes at the cellular level.