ABSTRACT
Plant endophytes are microorganisms that live inside plant tissues and establish a symbiotic relationship with their hosts. This relationship confers various benefits to plants, such as disease resistance, stress tolerance, and nutrient uptake efficiency. Therefore, plant endophytes are considered as potential "biostimulants" that can enhance plant performance and productivity. In this article, we review the characteristics of plant endophytes and their roles and applications in agriculture. How plant endophytes can act as microbial pesticides, microbial fertilizers, and stress regulators, and how they can produce growth hormones and other beneficial substances will be reviewed in this study. We also compare plant endophytes with chemical pesticides and fertilizers, and highlight their advantages and challenges. It has been demonstrated that plant endophytes have broad application prospects and can be used in crop production, which can help improve production efficiency, reduce dependence on chemical fertilizers and pesticides, and meet the expectations of sustainable agriculture. We suggest that future research should focus on exploring combinations of different endophytes or metabolites to achieve more comprehensive and effective plant growth promotion and protection effect.
Keywords: Endophyte, Microbial Pesticides, Microbial Fertilizer, Biostimulants
INTRODUCTION: PLANT ENDOPHYTES
Since the beginning of plants on Earth, millions of years of evolution have enabled them to develop a variety of mechanisms to cope with biotic and abiotic stresses. In particular, the symbiotic relationship established with microorganisms can obviously enhance the plant's ability to cope with stress and promote its growth and development, thus bringing about mutual survival advantages. Plant endophytes refer to microorganisms that grow inside plant sieve tubes or tissues. They spend all or part of their life cycles in plant tissues and form a symbiotic relationship with plants without causing any harm to the host plant (Bacon, White, & Stone, 2000). Endophytes can be mainly divided into two categories: bacteria and fungi. They can survive in the roots, stems, leaves, flowers and other parts of plants. The diversity of the species is high and widely present in various plants. Usually as a single plant, there are many types of endosymbiotic bacteria in the body, including several genera (Rosenblueth & Martinez-Romero, 2006). Some endophytes can systematically activate the plant's immune system and protect plants from diseases (Ho et al., 2015); some can obtain nutrients from the soil and transport them into the plant (Liu et al., 2017) and promote plant growth and development
(Hung et al., 2023; Hwang et al., 2021; Yaish, Al-Lawati, Jana, Vishwas Patankar, & Glick, 2016). There are also endophytes bacteria that can provide protection against abiotic stresses such as drought (Rolli et al., 2015), low temperature (Su et al., 2015), and high salinity (Hwang et al., 2022). They can even prevent herbivores from ingesting the plant (Bush, Wilkinson, & Schardl, 1997) and prevent the invasion of other competing plants (Saikkonen et al., 2013). Due to the characteristics of the above-mentioned endophytes, plant endophytes and their derivatives have great application potential. They are a promising agricultural technology that can provide effective and environmentally friendly sustainable solutions, which can not only improve agricultural production, but can also improve food quality and safety. In particular, improving agricultural productivity is an important task for countries around the world to cope with the world's population which is increasing to 9.1 billion in 2050. However, the reduction of global arable land due to urban expansion, climate change and poor land management is a major obstacle to this task. In the field of gene editing, in addition to technology such as genetically modified crops as breeding methods, biostimulants based on endophytes are expected to become another useful alternative in agricultural technology without necessarily changing its basic genetic structure.
Application of plant endophytes in microbial pesticides
Microbial pesticides are agricultural chemicals derived from microorganisms (such as bacteria, fungi, viruses, etc.) that can control plant diseases, pests, weeds, etc. by directly suppressing or killing them, or by interfering with their life cycles. Microbial pesticides are more environmentally friendly and less harmful to non-target organisms than chemical pesticides, and they also have some advantages in preventing resistance. Some examples of microbial pesticides are: Bacillus thuringiensis (or Bt), which produces a toxin called "crystal protein" that selectively targets and kills specific insect pests, such as Lepidoptera, Coleoptera, and Diptera, while having little impact on most other organisms, including humans, other mammals, and birds. Trichoderma fungi, which can induce plant disease resistance by competing with, producing antibiotics against, and inhibiting the growth of other plant pathogenic fungi, and can be used as microbial fungicides to prevent and control diseases such as powdery mildew, root rot (Rhizoctonia solani). Pseudomonas fluorescens competes with weeds in the soil for water, nutrients, and space, and reduces the resources available to weeds, making it difficult for them to grow and develop. Some Pseudomonas fluorescens strains are able to produce some secondary metabolites that can selectively hinder root formation, root growth and tiller germination of specific weeds (Kennedy, 2016). That is the reason why it can be used as a microbial herbicide.
Plant endophytes play an important role in assisting plants in resisting plant diseases. Endophytes usually form a symbiosis with plant roots and occupy the space in the plant roots and rhizosphere. This occupancy itself can hinder the invasion of pathogens, thereby reducing the chance of pathogens coming into contact with the plant. Competition between endophytes and pathogens can be termed competitive exclusion and helps maintain plant health. Some endophytes are capable of producing antibiotics that have inhibitory or killing effects against plant pathogens. The production of this antibiotic helps reduce the number of pathogens in the soil and reduces the risk of disease near plant roots. Burkholderia seminalis 869T2 (Re-classified from Burkholderia cenocepacia 869T2) is an endophytic bacterium that exists in the root tissue of vetiver grass (Chrysopogon zizanioides). It was isolated and studied because of its ability to inhibit the banana yellow leaf disease pathogen Fusarium oxysporum f. sp. cubense tropical race 4. The study found that this strain has the ability to produce the broad-acting fungal antibiotic pyrrolnitrin, and its genome contains multiple genes related to pyrrolnitrin production, including prnA, prnB, prnC, and prnD. In field trials, it was found that 7 months after inoculation with the 869T2 endophytic strain, in fields severely infected with Foc TR4, the incidence of yellow leaf disease on banana seedlings was only 3.4%, while the incidence in the control treatment was 24.5% (Ho et al., 2015). Endophytes can also assist plants in responding to pathogens by regulating the balance of plant hormones, such as ethylene and jasmonic acid, allowing them to respond to diseases more effectively. The 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was found in the genome of the 869T2 endophytic strain, which indicates that 869T2 has the ability to regulate the ethylene concentration in the host plant and assist the plant's response to pathogen invasion (Ho et al., 2015). The endophyte Bacillus vallismortis BS07 can also regulate the jasmonic acid pathway through secondary metabolites: cyclic dipeptides to make host plants resistant to Pseudomonas infection
(Noh et al., 2017). The above-mentioned Pseudomonas fluorescens, which can suppress weeds, is actually a plant endophyte (Lally et al., 2017).
The above-mentioned studies have confirmed that plant endophytes have great potential in plant disease control. Plant endophytes are usually based on naturally occurring microorganisms that form symbiotic relationships with plants. Compared with chemical pesticides, the application of plant endophytes is more eco-friendly and helps reduce negative impacts on the environment. Many endophytes are highly specific for specific plant pathogens and can target specific diseases while having less impact on non-target plants and organisms. Plant endophytes form a symbiotic relationship with plants and can promote plant growth, development and nutrient absorption. This helps improve the overall health of the plant, making it more resistant to disease. Using plant endophytes as a means of biological control can reduce reliance on chemical pesticides. Compared with chemical pesticides, plant endophytes are much more eco-friendly and can help reduce negative impacts on the environment, such as pesticide residues, ecological damage, and agricultural product quality. Therefore, plant endophytes are in line with the principles of sustainable agriculture and can help achieve a more environmentally friendly and ecologically friendly agricultural practice.
Application of plant endophytes in microbial fertilizers
Microbial fertilizer is a kind of fertilizer with microorganisms as the main active ingredient, usually composed of a series of microorganisms that are beneficial to plant growth. These microorganisms include bacteria, fungi, actinomycetes, etc., which provide various functions beneficial to plant growth, promote plant growth and improve soil fertility. Common microbial fertilizers include: Nitrogen-fixing bacteria (such as Rhizobium spp., Azotobacter spp., etc.) are a type of microorganisms that can convert nitrogen contained in the atmosphere into a form that can be absorbed by plants. These microorganisms can form a symbiotic relationship with plants, promote nitrogen absorption by plants, and improve plants' nitrogen use efficiency. Phosphorus is an important element for plant growth, but it usually exists in the soil in inorganic form and is not easily absorbed by plants. Phosphorus-dissolving bacteria and phosphorus-dissolving fungi (such as Bacillus spp., Pseudomonas spp., Mycorrhizal fungi, etc.) can convert fixed phosphorus in the soil into plant-absorbable forms and promote plant absorption of phosphorus. There is a type of microorganisms that produce some metabolites that promote plant growth, such as growth hormone (Auxin), steroid hormone (Gibberellin, GA), etc. They usually promote plant growth, increase plant stress resistance, and increase plant yield. Representative microorganisms include Bacillus spp., Pseudomonas spp., etc.
Plant endophytes can help plants to obtain and transport nutrients from the soil into plants and promote plant growth and development, giving them the potential to develop as microbial fertilizers. The cell structure called "hyphae" in plant endophytic fungi is the bridge between them and the plant roots. Mycelium forms extensive networks in the soil, extending its delicate structure into areas beyond the reach of plant roots. This increases the plant's root surface area, allowing it to absorb water and nutrients from the soil more efficiently. In addition to providing additional root surface area, it also maximizes material exchange between plants and mycelium by forming a symbiotic structure with plant roots. Plants provide carbohydrates and other organic matter to endophytes, while endophytes provide mineral nutrients such as phosphorus, nitrogen, calcium, etc. that are difficult for plants to obtain directly from the soil. This is extremely important for plants, because some minerals exist in inorganic or organic forms that are difficult for plants to absorb directly, and mycelium can help transform these forms, making them easier for plants to absorb and utilize. Among them, phosphorus transport is an area that plant endophytes are particularly good at. Phosphorus is an important element in plant growth and development, but it usually exists in organic form in the soil and is difficult to be directly absorbed by plants. Plant endophytes convert organic phosphorus in the soil into soluble phosphate through their hyphae, making it easier for plants to absorb. The cooperation of this symbiotic system allows plants to more efficiently acquire phosphorus in limited soil, thereby promoting their growth and development. Our previous study also found that plant endophytes can promote the expression of genes related to plant root hair development (such as: FLA6, XTH13), and enable the host plant to have better root development, which can also indirectly make it easier for plants to obtain nutrients from the soil and moisture. Burkholderia seminalis 869T2 has the ability to secrete IAA (2.76 ± 1.07 μg/mL) in the presence of L-tryptophan at a concentration of 500 mg/L. In field trials, it was found that in addition to helping bananas resist yellow leaf disease pathogens, it also has a significant effect on promoting plant growth. Banana plants inoculated with the 869T2 strain will have significantly higher plant heights and wider plant circumferences (Ho et al., 2015). Strain 869T2 has been confirmed to be endosymbiotic with a variety of different leafy vegetables, including Arabidopsis, green cabbage, Chinese cabbage, lettuce, amaranth, etc., and promote the growth of these leafy vegetables. The study also pointed out that the 869T2 strain has the ability to produce IAA, produce siderophores, and dissolve phosphate, which shows that this endophyte may promote plant growth through plant growth hormones and help plants absorb nutrients. In addition, the study also found that the 869T2 strain can cause peppers and okra to bloom earlier and have better fruit quality
(Hwang et al., 2021). There are also studies on applying the 869T2 strain to the field cultivation of head lettuce and cabbage. The 869T2 endophytic strain can enable cabbage to maintain good growth status in higher temperature seasons, and can also reduce the accumulation of nitrate (NO3−) and sulfate (SO42−) in lettuce. It has confirmed that the 869T2 endophytic strain can improve the harvest quality of leafy vegetables, especially by increasing fresh weight, dry weight and total soluble sugar accumulation, and can save 50% of fertilizer costs per hectare (Hung et al., 2023).
The application of plant endophytes to microbial fertilizers has multiple advantages and also has many exciting prospects. Endophytes promote the absorption of nutrients by plants, rather than directly providing more nutrient sources like traditional fertilizers. This allows the application of endophyte biofertilizers to improve plants' utilization efficiency of organic and inorganic nutrients in the soil, thereby reducing the use of organic and inorganic nutrients in the soil. The reduced demand for chemical fertilizers helps slow down the pollution of soil and water bodies. This further helps maintain soil ecological balance and reduce the spread of pathogens in the soil. This has a positive impact on soil health and ecosystem stability. In line with the principles of sustainable agriculture, this has positive implications for long-term agricultural sustainability and can improve agricultural production efficiency. Future studies may explore combinations of different endophytes to achieve more comprehensive and effective plant promotion and protection. The synergistic effect of different endophytes may bring multiple beneficial effects.
Application of plant endophytes in biostimulants
In recent years, the term “biostimulants” has appeared. Due to their characteristics, it is difficult to classify them as biopesticides or biofertilizers. The current definition of biostimulants is: including some active substances and/or microorganisms, applied to plants or around plant roots, these substances and/or microorganisms can stimulate the natural physiological responses of plants to enhance or benefit plant root growth, indirectly promote the improvement of plant nutrient absorption efficiency and improve the tolerance to abiotic stress and the quality of crops (du Jardin, 2015). The ingredients of biostimulants mainly come from natural organic substances, such as algae, humic acid, amino acids, plant extracts, etc. These ingredients are usually obtained by hydrolysis, extraction, or fermentation and are used in a purified form. Biostimulants contain a variety of ingredients that are beneficial to plants, including growth hormones, enzymes, antioxidants, microbial metabolites, etc. These ingredients can have a combined positive effect on plant growth and development. The organic matter in biostimulants helps improve soil structure and promote microbial activity, thereby increasing soil fertility and ecosystem stability. In fact, the above-mentioned microbial fertilizers do not directly provide excess nutrients like traditional fertilizers, but indirectly promote plant growth, and their characteristics are more like biostimulants.
Plant endophytes help plants resist abiotic stress, which is also an ability with great application potential. Especially due to climate change, plant growth environments around the world are facing more and more abiotic stresses such as high temperature, drought, and high salt. Plant endophytes can interact with the plant's immune system and activate the plant's defense mechanism. This includes initiating the plant's immune response and inducing the production of disease-resistant proteins and antioxidant substances to improve the plant's resistance to abiotic stress. It can also affect the synthesis and balance of plant hormones, especially ethylene, jasmonic acid, kinetin, etc. These hormones play an important role in plant defense responses, helping plants cope with abiotic stress. In addition, it improves the efficiency of plant absorption of nutrients, especially some microorganisms that are beneficial to plants such as Azotobacter and phosphorus-dissolving bacteria. By increasing the plant's nutritional status, the plant is more likely to cope with abiotic stress. Even some endophytes can produce antioxidant substances to reduce the degree of oxidative stress suffered by plants. This has a positive impact on resistance to abiotic stresses such as high temperature, drought, etc. Bacillus megaterium BP-R2 was isolated from the surface-sterilized root tissue of the halophyte Bolboschoenus planiculmis. The strain itself is highly tolerant to NaCl and produces IAA. The BP-R2 endophytic strain can be inoculated into Arabidopsis and Chinese cabbage, and allows the host plants to achieve better growth status under high-salt and drought stresses. Under salt and drought stress, accumulation of hydrogen peroxide (H2O2) content, electrolyte leakage (EL) and malondialdehyde (MDA) concentration was lower in BP-R2-inoculated plants
(Hwang et al., 2022).
The secondary metabolites of plant endophytes also have the potential to be used in the development of biostimulants. The 869T2 strain produces a compound: Pyrroloquinoline quinone (PQQ), an enzyme cofactor and antioxidant utilized by such as lactate, glucose, alcohol, and methanol dehydrogenases. The rhizosphere bacterium Pseudomonas fluorescens B16, which promotes plant growth, plays a vital role in stimulating plant growth through PQQ, which is a plant growth promoting factor. Application of PQQ can promote the increase in height, flower number, fruit number and total fruit weight of hydroponic tomatoes (Solanum lycopersicum). It can also increase the fresh weight of cucumber (Cucumis sativus) seedlings. The study also found that PQQ scavenged reactive oxygen species and hydrogen peroxide in injured cucumber leaves, suggesting that PQQ acts as an antioxidant in plants (Choi et al., 2007). In addition, PQQ is an important enzymatic cofactor for PQQ-dependent glucose dehydrogenase (GDH), which produces gluconic acid to promote phosphate dissolution and promote plant growth (Crespo, Boiardi, & Luna, 2011). On the other hand, the BP-R2 strain produces a compound designated as cyclodipeptides (CDPs): Cyclo(L-Ala-Gly). This type of small molecule compound has auxin-like activity and regulates plant growth through cross-species signaling (Ortiz-Castro et al., 2011). Cyclic dipeptides can also induce plant immunity and can defend against plant infection by pathogenic bacteria by activating the host plant's own jasmonic acid pathway
(Noh et al., 2017). However, the molecular mechanism of how the CDPs function on plant remained to be elucidated in the future.
CONCLUSION
Plant endophytes are microorganisms that live inside plant tissues and establish a symbiotic relationship with their hosts. They have great potential for agriculture and ecosystem management, as they can help achieve the goals of sustainable agriculture development. These goals include reducing the need for chemical fertilizers and pesticides, improving crop stress resistance and soil texture, and achieving environmentally friendly agriculture. Endophytes can also help plants cope better with extreme weather conditions caused by climate change, such as heat, drought, and floods, and maintain stable agricultural production. Moreover, those endophytes can help improve soil ecosystems, promote the activity of beneficial microorganisms, and improve soil health, which have positive effects on the restoration of ecosystems and the increase of biodiversity. Endophytes can also promote the absorption of nutrients by plants and help improve the nutritional value of crops, making them more suitable as food and feed. Furthermore, endophytes can inhibit the growth of pathogens and pests, and play a key role in reducing the use of agricultural chemicals and improving crop yields and quality, which can help reduce potential harm to human health and the environment. Additionally, endophytes can be incorporated into new agricultural production systems, such as smart farming, vertical farming, urban farming, and soilless cultivation, which can help increase crop yield and quality while conserving resources and reducing dependence on land. Therefore, emphasizing the importance and application of plant endophytes can help promote environmental education and improve farmers and the public's awareness of and protection of soil ecosystems.
ACKNOWLEDGMENTS
The funding for these studies were provided by the Ministry of Agriculture, Executive Yuan, Taiwan (110AS-1.6.1-BQ-B3; 111AS-1.6.1-ST-a7), the Ministry of Education of Taiwan (the Higher Education Sprout Project), and National Science and Technology Council, Taiwan (MOST107-2321-B-005-009-; MOST108-2321-B-005-004-; MOST109-2321-B-005-025-; MOST110-2321-B-005-008 -) to CC Huang.
REFERENCES
Bacon, C., White, J., & Stone, J. (2000). An overview of endophytic microbes: endophytism defined. Microbial endophytes, 1.
Bush, L. P., Wilkinson, H. H., & Schardl, C. L. (1997). Bioprotective alkaloids of grass-fungal endophyte symbioses. Plant Physiology, 114(1), 1.
Choi, O., Kim, J., Kim, J.-G., Jeong, Y., Moon, J. S., Park, C. S., & Hwang, I. (2007). Pyrroloquinoline Quinone Is a Plant Growth Promotion Factor Produced by Pseudomonas fluorescens B16. Plant Physiology, 146(2), 323-324. doi:10.1104/pp.107.112748
Crespo, J. M., Boiardi, J. L., & Luna, M. F. (2011). Mineral phosphate solubilization activity of Gluconacetobacter diazotrophicus under P-limitation and plant root environment. Agricultural Sciences, 2.
du Jardin, P. (2015). Plant biostimulants: Definition, concept, main categories and regulation. Scientia Horticulturae, 196, 3-14. doi:https://doi.org/10.1016/j.scienta.2015.09.021
Ho, Y.-N., Chiang, H.-M., Chao, C.-P., Su, C.-C., Hsu, H.-F., Guo, C.-t., . . . Huang, C.-C. (2015). In planta biocontrol of soilborne Fusarium wilt of banana through a plant endophytic bacterium, Burkholderia cenocepacia 869T2. Plant and Soil, 387(1), 295-306. doi:10.1007/s11104-014-2297-0
Hung, S.-H. W., Huang, T.-C., Lai, Y.-C., Wu, I.-C., Liu, C.-H., Huarng, Y.-F., Huang, C.-C. (2023). Endophytic Biostimulants for Smart Agriculture: Burkholderia seminalis 869T2 Benefits Heading Leafy Vegetables In-Field Management in Taiwan. Agronomy, 13(4), 967. Retrieved from https://www.mdpi.com/2073-4395/13/4/967
Hwang, H. H., Chien, P. R., Huang, F. C., Hung, S. H., Kuo, C. H., Deng, W. L., . . . Huang, C. C. (2021). A Plant Endophytic Bacterium, Burkholderia seminalis Strain 869T2, Promotes Plant Growth in Arabidopsis, Pak Choi, Chinese Amaranth, Lettuces, and Other Vegetables. Microorganisms, 9(8). doi:10.3390/microorganisms9081703
Hwang, H. H., Chien, P. R., Huang, F. C., Yeh, P. H., Hung, S. W., Deng, W. L., & Huang, C. C. (2022). A Plant Endophytic Bacterium Priestia megaterium StrainBP-R2 Isolated from the Halophyte Bolboschoenus planiculmis Enhances Plant Growth under Salt and Drought Stresses. Microorganisms, 10(10). doi:10.3390/microorganisms10102047
Kennedy, A. C. (2016). Pseudomonas fluorescens strains selectively suppress annual bluegrass (Poa annua L.). Biological Control, 103, 210-217. doi:https://doi.org/10.1016/j.biocontrol.2016.09.012
Lally, R. D., Galbally, P., Moreira, A. S., Spink, J., Ryan, D., Germaine, K. J., & Dowling, D. N. (2017). Application of endophytic Pseudomonas fluorescens and a bacterial consortium to Brassica napus can increase plant height and biomass under greenhouse and field conditions. Frontiers in Plant Science, 8, 2193.
Liu, H., Carvalhais, L. C., Crawford, M., Singh, E., Dennis, P. G., Pieterse, C. M., & Schenk, P. M. (2017). Inner plant values: diversity, colonization and benefits from endophytic bacteria. Frontiers in microbiology, 8, 2552.
Noh, S. W., Seo, R., Park, J. K., Manir, M. M., Park, K., Sang, M. K., . . . Jung, H. W. (2017). Cyclic Dipeptides from Bacillus vallismortis BS07 Require Key Components of Plant Immunity to Induce Disease Resistance in Arabidopsis against Pseudomonas Infection. Plant Pathology Journal, 33(4), 402-409. doi:10.5423/Ppj.0a.11.2016.0255
Ortiz-Castro, R., Díaz-Pérez, C., Martínez-Trujillo, M., del Río, R. E., Campos-García, J., & López-Bucio, J. (2011). Transkingdom signaling based on bacterial cyclodipeptides with auxin activity in plants. Proceedings of the National Academy of Sciences, 108(17), 7253-7258. doi:doi:10.1073/pnas.1006740108
Rolli, E., Marasco, R., Vigani, G., Ettoumi, B., Mapelli, F., Deangelis, M. L., . . . Daffonchio, D. (2015). Improved plant resistance to drought is promoted by the root-associated microbiome as a water stress-dependent trait. Environmental Microbiology, 17(2), 316-331. doi:https://doi.org/10.1111/1462-2920.12439
Rosenblueth, M., & Martinez-Romero, E. (2006). Bacterial endophytes and their interactions with hosts. Molecular Plant-Microbe Interactions, 19(8), 827-837. doi:10.1094/Mpmi-19-0827
Saikkonen, K., Ruokolainen, K., Huitu, O., Gundel, P. E., Piltti, T., Hamilton, C. E., & Helander, M. (2013). Fungal endophytes help prevent weed invasions. Agriculture, Ecosystems & Environment, 165, 1-5.
Su, F., Jacquard, C., Villaume, S., Michel, J., Rabenoelina, F., Clement, C., . . . Vaillant-Gaveau, N. (2015). Burkholderia phytofirmans PsJN reduces impact of freezing temperatures on photosynthesis in Arabidopsis thaliana. Frontiers in Plant Science, 6, 810. doi:10.3389/fpls.2015.00810
Yaish, M. W., Al-Lawati, A., Jana, G. A., Vishwas Patankar, H., & Glick, B. R. (2016). Impact of soil salinity on the structure of the bacterial endophytic community identified from the roots of caliph medic (Medicago truncatula). Plos One, 11(7), e0159007.
Application of Plant Endophytes in Sustainable Agriculture
ABSTRACT
Plant endophytes are microorganisms that live inside plant tissues and establish a symbiotic relationship with their hosts. This relationship confers various benefits to plants, such as disease resistance, stress tolerance, and nutrient uptake efficiency. Therefore, plant endophytes are considered as potential "biostimulants" that can enhance plant performance and productivity. In this article, we review the characteristics of plant endophytes and their roles and applications in agriculture. How plant endophytes can act as microbial pesticides, microbial fertilizers, and stress regulators, and how they can produce growth hormones and other beneficial substances will be reviewed in this study. We also compare plant endophytes with chemical pesticides and fertilizers, and highlight their advantages and challenges. It has been demonstrated that plant endophytes have broad application prospects and can be used in crop production, which can help improve production efficiency, reduce dependence on chemical fertilizers and pesticides, and meet the expectations of sustainable agriculture. We suggest that future research should focus on exploring combinations of different endophytes or metabolites to achieve more comprehensive and effective plant growth promotion and protection effect.
Keywords: Endophyte, Microbial Pesticides, Microbial Fertilizer, Biostimulants
INTRODUCTION: PLANT ENDOPHYTES
Since the beginning of plants on Earth, millions of years of evolution have enabled them to develop a variety of mechanisms to cope with biotic and abiotic stresses. In particular, the symbiotic relationship established with microorganisms can obviously enhance the plant's ability to cope with stress and promote its growth and development, thus bringing about mutual survival advantages. Plant endophytes refer to microorganisms that grow inside plant sieve tubes or tissues. They spend all or part of their life cycles in plant tissues and form a symbiotic relationship with plants without causing any harm to the host plant (Bacon, White, & Stone, 2000). Endophytes can be mainly divided into two categories: bacteria and fungi. They can survive in the roots, stems, leaves, flowers and other parts of plants. The diversity of the species is high and widely present in various plants. Usually as a single plant, there are many types of endosymbiotic bacteria in the body, including several genera (Rosenblueth & Martinez-Romero, 2006). Some endophytes can systematically activate the plant's immune system and protect plants from diseases (Ho et al., 2015); some can obtain nutrients from the soil and transport them into the plant (Liu et al., 2017) and promote plant growth and development
(Hung et al., 2023; Hwang et al., 2021; Yaish, Al-Lawati, Jana, Vishwas Patankar, & Glick, 2016). There are also endophytes bacteria that can provide protection against abiotic stresses such as drought (Rolli et al., 2015), low temperature (Su et al., 2015), and high salinity (Hwang et al., 2022). They can even prevent herbivores from ingesting the plant (Bush, Wilkinson, & Schardl, 1997) and prevent the invasion of other competing plants (Saikkonen et al., 2013). Due to the characteristics of the above-mentioned endophytes, plant endophytes and their derivatives have great application potential. They are a promising agricultural technology that can provide effective and environmentally friendly sustainable solutions, which can not only improve agricultural production, but can also improve food quality and safety. In particular, improving agricultural productivity is an important task for countries around the world to cope with the world's population which is increasing to 9.1 billion in 2050. However, the reduction of global arable land due to urban expansion, climate change and poor land management is a major obstacle to this task. In the field of gene editing, in addition to technology such as genetically modified crops as breeding methods, biostimulants based on endophytes are expected to become another useful alternative in agricultural technology without necessarily changing its basic genetic structure.
Application of plant endophytes in microbial pesticides
Microbial pesticides are agricultural chemicals derived from microorganisms (such as bacteria, fungi, viruses, etc.) that can control plant diseases, pests, weeds, etc. by directly suppressing or killing them, or by interfering with their life cycles. Microbial pesticides are more environmentally friendly and less harmful to non-target organisms than chemical pesticides, and they also have some advantages in preventing resistance. Some examples of microbial pesticides are: Bacillus thuringiensis (or Bt), which produces a toxin called "crystal protein" that selectively targets and kills specific insect pests, such as Lepidoptera, Coleoptera, and Diptera, while having little impact on most other organisms, including humans, other mammals, and birds. Trichoderma fungi, which can induce plant disease resistance by competing with, producing antibiotics against, and inhibiting the growth of other plant pathogenic fungi, and can be used as microbial fungicides to prevent and control diseases such as powdery mildew, root rot (Rhizoctonia solani). Pseudomonas fluorescens competes with weeds in the soil for water, nutrients, and space, and reduces the resources available to weeds, making it difficult for them to grow and develop. Some Pseudomonas fluorescens strains are able to produce some secondary metabolites that can selectively hinder root formation, root growth and tiller germination of specific weeds (Kennedy, 2016). That is the reason why it can be used as a microbial herbicide.
Plant endophytes play an important role in assisting plants in resisting plant diseases. Endophytes usually form a symbiosis with plant roots and occupy the space in the plant roots and rhizosphere. This occupancy itself can hinder the invasion of pathogens, thereby reducing the chance of pathogens coming into contact with the plant. Competition between endophytes and pathogens can be termed competitive exclusion and helps maintain plant health. Some endophytes are capable of producing antibiotics that have inhibitory or killing effects against plant pathogens. The production of this antibiotic helps reduce the number of pathogens in the soil and reduces the risk of disease near plant roots. Burkholderia seminalis 869T2 (Re-classified from Burkholderia cenocepacia 869T2) is an endophytic bacterium that exists in the root tissue of vetiver grass (Chrysopogon zizanioides). It was isolated and studied because of its ability to inhibit the banana yellow leaf disease pathogen Fusarium oxysporum f. sp. cubense tropical race 4. The study found that this strain has the ability to produce the broad-acting fungal antibiotic pyrrolnitrin, and its genome contains multiple genes related to pyrrolnitrin production, including prnA, prnB, prnC, and prnD. In field trials, it was found that 7 months after inoculation with the 869T2 endophytic strain, in fields severely infected with Foc TR4, the incidence of yellow leaf disease on banana seedlings was only 3.4%, while the incidence in the control treatment was 24.5% (Ho et al., 2015). Endophytes can also assist plants in responding to pathogens by regulating the balance of plant hormones, such as ethylene and jasmonic acid, allowing them to respond to diseases more effectively. The 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was found in the genome of the 869T2 endophytic strain, which indicates that 869T2 has the ability to regulate the ethylene concentration in the host plant and assist the plant's response to pathogen invasion (Ho et al., 2015). The endophyte Bacillus vallismortis BS07 can also regulate the jasmonic acid pathway through secondary metabolites: cyclic dipeptides to make host plants resistant to Pseudomonas infection
(Noh et al., 2017). The above-mentioned Pseudomonas fluorescens, which can suppress weeds, is actually a plant endophyte (Lally et al., 2017).
The above-mentioned studies have confirmed that plant endophytes have great potential in plant disease control. Plant endophytes are usually based on naturally occurring microorganisms that form symbiotic relationships with plants. Compared with chemical pesticides, the application of plant endophytes is more eco-friendly and helps reduce negative impacts on the environment. Many endophytes are highly specific for specific plant pathogens and can target specific diseases while having less impact on non-target plants and organisms. Plant endophytes form a symbiotic relationship with plants and can promote plant growth, development and nutrient absorption. This helps improve the overall health of the plant, making it more resistant to disease. Using plant endophytes as a means of biological control can reduce reliance on chemical pesticides. Compared with chemical pesticides, plant endophytes are much more eco-friendly and can help reduce negative impacts on the environment, such as pesticide residues, ecological damage, and agricultural product quality. Therefore, plant endophytes are in line with the principles of sustainable agriculture and can help achieve a more environmentally friendly and ecologically friendly agricultural practice.
Application of plant endophytes in microbial fertilizers
Microbial fertilizer is a kind of fertilizer with microorganisms as the main active ingredient, usually composed of a series of microorganisms that are beneficial to plant growth. These microorganisms include bacteria, fungi, actinomycetes, etc., which provide various functions beneficial to plant growth, promote plant growth and improve soil fertility. Common microbial fertilizers include: Nitrogen-fixing bacteria (such as Rhizobium spp., Azotobacter spp., etc.) are a type of microorganisms that can convert nitrogen contained in the atmosphere into a form that can be absorbed by plants. These microorganisms can form a symbiotic relationship with plants, promote nitrogen absorption by plants, and improve plants' nitrogen use efficiency. Phosphorus is an important element for plant growth, but it usually exists in the soil in inorganic form and is not easily absorbed by plants. Phosphorus-dissolving bacteria and phosphorus-dissolving fungi (such as Bacillus spp., Pseudomonas spp., Mycorrhizal fungi, etc.) can convert fixed phosphorus in the soil into plant-absorbable forms and promote plant absorption of phosphorus. There is a type of microorganisms that produce some metabolites that promote plant growth, such as growth hormone (Auxin), steroid hormone (Gibberellin, GA), etc. They usually promote plant growth, increase plant stress resistance, and increase plant yield. Representative microorganisms include Bacillus spp., Pseudomonas spp., etc.
Plant endophytes can help plants to obtain and transport nutrients from the soil into plants and promote plant growth and development, giving them the potential to develop as microbial fertilizers. The cell structure called "hyphae" in plant endophytic fungi is the bridge between them and the plant roots. Mycelium forms extensive networks in the soil, extending its delicate structure into areas beyond the reach of plant roots. This increases the plant's root surface area, allowing it to absorb water and nutrients from the soil more efficiently. In addition to providing additional root surface area, it also maximizes material exchange between plants and mycelium by forming a symbiotic structure with plant roots. Plants provide carbohydrates and other organic matter to endophytes, while endophytes provide mineral nutrients such as phosphorus, nitrogen, calcium, etc. that are difficult for plants to obtain directly from the soil. This is extremely important for plants, because some minerals exist in inorganic or organic forms that are difficult for plants to absorb directly, and mycelium can help transform these forms, making them easier for plants to absorb and utilize. Among them, phosphorus transport is an area that plant endophytes are particularly good at. Phosphorus is an important element in plant growth and development, but it usually exists in organic form in the soil and is difficult to be directly absorbed by plants. Plant endophytes convert organic phosphorus in the soil into soluble phosphate through their hyphae, making it easier for plants to absorb. The cooperation of this symbiotic system allows plants to more efficiently acquire phosphorus in limited soil, thereby promoting their growth and development. Our previous study also found that plant endophytes can promote the expression of genes related to plant root hair development (such as: FLA6, XTH13), and enable the host plant to have better root development, which can also indirectly make it easier for plants to obtain nutrients from the soil and moisture. Burkholderia seminalis 869T2 has the ability to secrete IAA (2.76 ± 1.07 μg/mL) in the presence of L-tryptophan at a concentration of 500 mg/L. In field trials, it was found that in addition to helping bananas resist yellow leaf disease pathogens, it also has a significant effect on promoting plant growth. Banana plants inoculated with the 869T2 strain will have significantly higher plant heights and wider plant circumferences (Ho et al., 2015). Strain 869T2 has been confirmed to be endosymbiotic with a variety of different leafy vegetables, including Arabidopsis, green cabbage, Chinese cabbage, lettuce, amaranth, etc., and promote the growth of these leafy vegetables. The study also pointed out that the 869T2 strain has the ability to produce IAA, produce siderophores, and dissolve phosphate, which shows that this endophyte may promote plant growth through plant growth hormones and help plants absorb nutrients. In addition, the study also found that the 869T2 strain can cause peppers and okra to bloom earlier and have better fruit quality
(Hwang et al., 2021). There are also studies on applying the 869T2 strain to the field cultivation of head lettuce and cabbage. The 869T2 endophytic strain can enable cabbage to maintain good growth status in higher temperature seasons, and can also reduce the accumulation of nitrate (NO3−) and sulfate (SO42−) in lettuce. It has confirmed that the 869T2 endophytic strain can improve the harvest quality of leafy vegetables, especially by increasing fresh weight, dry weight and total soluble sugar accumulation, and can save 50% of fertilizer costs per hectare (Hung et al., 2023).
The application of plant endophytes to microbial fertilizers has multiple advantages and also has many exciting prospects. Endophytes promote the absorption of nutrients by plants, rather than directly providing more nutrient sources like traditional fertilizers. This allows the application of endophyte biofertilizers to improve plants' utilization efficiency of organic and inorganic nutrients in the soil, thereby reducing the use of organic and inorganic nutrients in the soil. The reduced demand for chemical fertilizers helps slow down the pollution of soil and water bodies. This further helps maintain soil ecological balance and reduce the spread of pathogens in the soil. This has a positive impact on soil health and ecosystem stability. In line with the principles of sustainable agriculture, this has positive implications for long-term agricultural sustainability and can improve agricultural production efficiency. Future studies may explore combinations of different endophytes to achieve more comprehensive and effective plant promotion and protection. The synergistic effect of different endophytes may bring multiple beneficial effects.
Application of plant endophytes in biostimulants
In recent years, the term “biostimulants” has appeared. Due to their characteristics, it is difficult to classify them as biopesticides or biofertilizers. The current definition of biostimulants is: including some active substances and/or microorganisms, applied to plants or around plant roots, these substances and/or microorganisms can stimulate the natural physiological responses of plants to enhance or benefit plant root growth, indirectly promote the improvement of plant nutrient absorption efficiency and improve the tolerance to abiotic stress and the quality of crops (du Jardin, 2015). The ingredients of biostimulants mainly come from natural organic substances, such as algae, humic acid, amino acids, plant extracts, etc. These ingredients are usually obtained by hydrolysis, extraction, or fermentation and are used in a purified form. Biostimulants contain a variety of ingredients that are beneficial to plants, including growth hormones, enzymes, antioxidants, microbial metabolites, etc. These ingredients can have a combined positive effect on plant growth and development. The organic matter in biostimulants helps improve soil structure and promote microbial activity, thereby increasing soil fertility and ecosystem stability. In fact, the above-mentioned microbial fertilizers do not directly provide excess nutrients like traditional fertilizers, but indirectly promote plant growth, and their characteristics are more like biostimulants.
Plant endophytes help plants resist abiotic stress, which is also an ability with great application potential. Especially due to climate change, plant growth environments around the world are facing more and more abiotic stresses such as high temperature, drought, and high salt. Plant endophytes can interact with the plant's immune system and activate the plant's defense mechanism. This includes initiating the plant's immune response and inducing the production of disease-resistant proteins and antioxidant substances to improve the plant's resistance to abiotic stress. It can also affect the synthesis and balance of plant hormones, especially ethylene, jasmonic acid, kinetin, etc. These hormones play an important role in plant defense responses, helping plants cope with abiotic stress. In addition, it improves the efficiency of plant absorption of nutrients, especially some microorganisms that are beneficial to plants such as Azotobacter and phosphorus-dissolving bacteria. By increasing the plant's nutritional status, the plant is more likely to cope with abiotic stress. Even some endophytes can produce antioxidant substances to reduce the degree of oxidative stress suffered by plants. This has a positive impact on resistance to abiotic stresses such as high temperature, drought, etc. Bacillus megaterium BP-R2 was isolated from the surface-sterilized root tissue of the halophyte Bolboschoenus planiculmis. The strain itself is highly tolerant to NaCl and produces IAA. The BP-R2 endophytic strain can be inoculated into Arabidopsis and Chinese cabbage, and allows the host plants to achieve better growth status under high-salt and drought stresses. Under salt and drought stress, accumulation of hydrogen peroxide (H2O2) content, electrolyte leakage (EL) and malondialdehyde (MDA) concentration was lower in BP-R2-inoculated plants
(Hwang et al., 2022).
The secondary metabolites of plant endophytes also have the potential to be used in the development of biostimulants. The 869T2 strain produces a compound: Pyrroloquinoline quinone (PQQ), an enzyme cofactor and antioxidant utilized by such as lactate, glucose, alcohol, and methanol dehydrogenases. The rhizosphere bacterium Pseudomonas fluorescens B16, which promotes plant growth, plays a vital role in stimulating plant growth through PQQ, which is a plant growth promoting factor. Application of PQQ can promote the increase in height, flower number, fruit number and total fruit weight of hydroponic tomatoes (Solanum lycopersicum). It can also increase the fresh weight of cucumber (Cucumis sativus) seedlings. The study also found that PQQ scavenged reactive oxygen species and hydrogen peroxide in injured cucumber leaves, suggesting that PQQ acts as an antioxidant in plants (Choi et al., 2007). In addition, PQQ is an important enzymatic cofactor for PQQ-dependent glucose dehydrogenase (GDH), which produces gluconic acid to promote phosphate dissolution and promote plant growth (Crespo, Boiardi, & Luna, 2011). On the other hand, the BP-R2 strain produces a compound designated as cyclodipeptides (CDPs): Cyclo(L-Ala-Gly). This type of small molecule compound has auxin-like activity and regulates plant growth through cross-species signaling (Ortiz-Castro et al., 2011). Cyclic dipeptides can also induce plant immunity and can defend against plant infection by pathogenic bacteria by activating the host plant's own jasmonic acid pathway
(Noh et al., 2017). However, the molecular mechanism of how the CDPs function on plant remained to be elucidated in the future.
CONCLUSION
Plant endophytes are microorganisms that live inside plant tissues and establish a symbiotic relationship with their hosts. They have great potential for agriculture and ecosystem management, as they can help achieve the goals of sustainable agriculture development. These goals include reducing the need for chemical fertilizers and pesticides, improving crop stress resistance and soil texture, and achieving environmentally friendly agriculture. Endophytes can also help plants cope better with extreme weather conditions caused by climate change, such as heat, drought, and floods, and maintain stable agricultural production. Moreover, those endophytes can help improve soil ecosystems, promote the activity of beneficial microorganisms, and improve soil health, which have positive effects on the restoration of ecosystems and the increase of biodiversity. Endophytes can also promote the absorption of nutrients by plants and help improve the nutritional value of crops, making them more suitable as food and feed. Furthermore, endophytes can inhibit the growth of pathogens and pests, and play a key role in reducing the use of agricultural chemicals and improving crop yields and quality, which can help reduce potential harm to human health and the environment. Additionally, endophytes can be incorporated into new agricultural production systems, such as smart farming, vertical farming, urban farming, and soilless cultivation, which can help increase crop yield and quality while conserving resources and reducing dependence on land. Therefore, emphasizing the importance and application of plant endophytes can help promote environmental education and improve farmers and the public's awareness of and protection of soil ecosystems.
ACKNOWLEDGMENTS
The funding for these studies were provided by the Ministry of Agriculture, Executive Yuan, Taiwan (110AS-1.6.1-BQ-B3; 111AS-1.6.1-ST-a7), the Ministry of Education of Taiwan (the Higher Education Sprout Project), and National Science and Technology Council, Taiwan (MOST107-2321-B-005-009-; MOST108-2321-B-005-004-; MOST109-2321-B-005-025-; MOST110-2321-B-005-008 -) to CC Huang.
REFERENCES
Bacon, C., White, J., & Stone, J. (2000). An overview of endophytic microbes: endophytism defined. Microbial endophytes, 1.
Bush, L. P., Wilkinson, H. H., & Schardl, C. L. (1997). Bioprotective alkaloids of grass-fungal endophyte symbioses. Plant Physiology, 114(1), 1.
Choi, O., Kim, J., Kim, J.-G., Jeong, Y., Moon, J. S., Park, C. S., & Hwang, I. (2007). Pyrroloquinoline Quinone Is a Plant Growth Promotion Factor Produced by Pseudomonas fluorescens B16. Plant Physiology, 146(2), 323-324. doi:10.1104/pp.107.112748
Crespo, J. M., Boiardi, J. L., & Luna, M. F. (2011). Mineral phosphate solubilization activity of Gluconacetobacter diazotrophicus under P-limitation and plant root environment. Agricultural Sciences, 2.
du Jardin, P. (2015). Plant biostimulants: Definition, concept, main categories and regulation. Scientia Horticulturae, 196, 3-14. doi:https://doi.org/10.1016/j.scienta.2015.09.021
Ho, Y.-N., Chiang, H.-M., Chao, C.-P., Su, C.-C., Hsu, H.-F., Guo, C.-t., . . . Huang, C.-C. (2015). In planta biocontrol of soilborne Fusarium wilt of banana through a plant endophytic bacterium, Burkholderia cenocepacia 869T2. Plant and Soil, 387(1), 295-306. doi:10.1007/s11104-014-2297-0
Hung, S.-H. W., Huang, T.-C., Lai, Y.-C., Wu, I.-C., Liu, C.-H., Huarng, Y.-F., Huang, C.-C. (2023). Endophytic Biostimulants for Smart Agriculture: Burkholderia seminalis 869T2 Benefits Heading Leafy Vegetables In-Field Management in Taiwan. Agronomy, 13(4), 967. Retrieved from https://www.mdpi.com/2073-4395/13/4/967
Hwang, H. H., Chien, P. R., Huang, F. C., Hung, S. H., Kuo, C. H., Deng, W. L., . . . Huang, C. C. (2021). A Plant Endophytic Bacterium, Burkholderia seminalis Strain 869T2, Promotes Plant Growth in Arabidopsis, Pak Choi, Chinese Amaranth, Lettuces, and Other Vegetables. Microorganisms, 9(8). doi:10.3390/microorganisms9081703
Hwang, H. H., Chien, P. R., Huang, F. C., Yeh, P. H., Hung, S. W., Deng, W. L., & Huang, C. C. (2022). A Plant Endophytic Bacterium Priestia megaterium StrainBP-R2 Isolated from the Halophyte Bolboschoenus planiculmis Enhances Plant Growth under Salt and Drought Stresses. Microorganisms, 10(10). doi:10.3390/microorganisms10102047
Kennedy, A. C. (2016). Pseudomonas fluorescens strains selectively suppress annual bluegrass (Poa annua L.). Biological Control, 103, 210-217. doi:https://doi.org/10.1016/j.biocontrol.2016.09.012
Lally, R. D., Galbally, P., Moreira, A. S., Spink, J., Ryan, D., Germaine, K. J., & Dowling, D. N. (2017). Application of endophytic Pseudomonas fluorescens and a bacterial consortium to Brassica napus can increase plant height and biomass under greenhouse and field conditions. Frontiers in Plant Science, 8, 2193.
Liu, H., Carvalhais, L. C., Crawford, M., Singh, E., Dennis, P. G., Pieterse, C. M., & Schenk, P. M. (2017). Inner plant values: diversity, colonization and benefits from endophytic bacteria. Frontiers in microbiology, 8, 2552.
Noh, S. W., Seo, R., Park, J. K., Manir, M. M., Park, K., Sang, M. K., . . . Jung, H. W. (2017). Cyclic Dipeptides from Bacillus vallismortis BS07 Require Key Components of Plant Immunity to Induce Disease Resistance in Arabidopsis against Pseudomonas Infection. Plant Pathology Journal, 33(4), 402-409. doi:10.5423/Ppj.0a.11.2016.0255
Ortiz-Castro, R., Díaz-Pérez, C., Martínez-Trujillo, M., del Río, R. E., Campos-García, J., & López-Bucio, J. (2011). Transkingdom signaling based on bacterial cyclodipeptides with auxin activity in plants. Proceedings of the National Academy of Sciences, 108(17), 7253-7258. doi:doi:10.1073/pnas.1006740108
Rolli, E., Marasco, R., Vigani, G., Ettoumi, B., Mapelli, F., Deangelis, M. L., . . . Daffonchio, D. (2015). Improved plant resistance to drought is promoted by the root-associated microbiome as a water stress-dependent trait. Environmental Microbiology, 17(2), 316-331. doi:https://doi.org/10.1111/1462-2920.12439
Rosenblueth, M., & Martinez-Romero, E. (2006). Bacterial endophytes and their interactions with hosts. Molecular Plant-Microbe Interactions, 19(8), 827-837. doi:10.1094/Mpmi-19-0827
Saikkonen, K., Ruokolainen, K., Huitu, O., Gundel, P. E., Piltti, T., Hamilton, C. E., & Helander, M. (2013). Fungal endophytes help prevent weed invasions. Agriculture, Ecosystems & Environment, 165, 1-5.
Su, F., Jacquard, C., Villaume, S., Michel, J., Rabenoelina, F., Clement, C., . . . Vaillant-Gaveau, N. (2015). Burkholderia phytofirmans PsJN reduces impact of freezing temperatures on photosynthesis in Arabidopsis thaliana. Frontiers in Plant Science, 6, 810. doi:10.3389/fpls.2015.00810
Yaish, M. W., Al-Lawati, A., Jana, G. A., Vishwas Patankar, H., & Glick, B. R. (2016). Impact of soil salinity on the structure of the bacterial endophytic community identified from the roots of caliph medic (Medicago truncatula). Plos One, 11(7), e0159007.