THE HIDDEN WORLD WITHIN PLANTS

This is a transcription of the article “Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes” from Pablo R. Hardoim et al.

Before writing I would like to share with all of you this amazing video that can endorse the endophytes for food security and food insurance for the present and for the future.

All plants are inhabited internally by diverse microbial communities comprising bacterial, archaeal, fungal, and protistic taxa. These microorganisms showing endophytic lifestyles play crucial roles in plant development, growth, fitness, and diversification. The increasing awareness of and information on endophytes provide insight into the complexity of the plant microbiome. The nature of plant-endophyte interactions ranges from mutualism to pathogenicity. This depends on a set of abiotic and biotic factors, including the genotypes of plants and microbes, environmental conditions, and the dynamic network of interactions within the plant biome.

The author made a complete review considering the concept of endophytism, involving the latest insights into evolution, plant ecosystem functioning, and multipartite interactions.

The German botanist Heinrich Friedrich Link was the first to describe endophytes, in 1809. At that time, they were termed “Entophytae” and were described as a distinct group of partly parasitic fungi living in plants. Since then, many definitions have evolved; for a long time, they mostly addressed pathogens or parasitic organisms, primarily fungi. Only Béchamp described so-called microzymas in plants, referring to microorganisms. Generally, in the 19th century, the belief was that healthy or normally growing plants are sterile and thus free of microorganisms (postululated by Pasteur. Nevertheless, Galippe reported the occurrence of bacteria and fungi in the interior of vegetable plants and postulated that these microorganisms derive from the soil environment and migrate into the plant, where they might play a beneficial role for the host plant. Other studies in the late 19th century and the beginning of the 20th century confirmed the occurrence of beneficial microorganisms within plants. Nevertheless, contrasting views on the existence of plant-beneficial endophytes prevailed at that time. Nowadays, it is a well-established fact that plants are hosts for many types of microbial endophytes, including bacteria, fungi, archaea, and unicellular eukaryotes, such as algae and amoebae.

More recently, in 1991, Orlando Petrini defined endophytes as “all organisms inhabiting plant organs that at some time in their life cycle can colonize internal plant tissues without causing apparent harm to their host”. Since then, many definitions have been formulated, essentially all pertaining to microorganisms which for all or part of their life cycle invade tissues of living plants without causing disease. Although this endophyte definition has been the basis of many studies and might be a pragmatic approach to distinguish between endophytes and pathogens, it has some drawbacks and raises some questions.

Plant-microbe symbioses:  Different groups of bacteria and fungi interact with higher plants. Genetic links between the association of plants with arbuscular mycorrhizal fungi (AMF) and root nodule symbioses have been found, suggesting that at least segments of bacterial and fungal endophytic populations coevolved with each other and with their host. Mutualistic interactions leading to adaptive benefits for both partners occasionally evolved to even more complex forms, in which more than two partners were involved.

Plant-fungus symbioses: Are known to have occurred during early colonization of land by terrestrial plants. The fungal group Glomeromycota has for a long time been the prime candidate for interaction with the first terrestrial plants, in the Ordovician era, but members of the Mucoromycotina are also speculated to have had symbiotic interactions with the first terrestrial plants. The association between AMF and plants evolved as a symbiosis, facilitating the adaption of plants to the terrestrial environment. The oldest known fossils representing terrestrial fungi with properties similar to those of AMF were collected from dolomite rocks in Wisconsin and are estimated to be 460 million years old, originating from the Ordovician period. It was therefore assumed that terrestrial AMF already existed at the time when bryophyte-like, “lower” plants covered the land. All other plant-AMF interaction types, such as ectomycorrhiza and orchid and ericoid mycorrhiza, appeared later and are considered to be derived from the first interactions between AMF and the first terrestrial plants.

Evolution of Plant-Bacterium Symbioses:  The best-described plant-bacterium interaction is the one between leguminous plants and rhizobia. The interactions of nitrogen-fixing bacteria belonging to the genera Azorhizobium, Bradyrhizobium, Ensifer, Mesorhizobium, Rhizobium, and Sinorhizobium are capable of inducing differentiation in root nodule structure, as demonstrated in Fabaceae and Parasponia plants. Typical symptoms in roots of leguminous plants infected by rhizobia are curling of root hairs and the appearance of infection threads and, finally, nodule primordia in the inner root layers—these are all processes mediated by signal exchange between plants and rhizobia. In primordium cells, the bacteria become surrounded by the plant membrane, and together, the bacteria and plant structure form the symbiosome, in which atmospheric nitrogen is fixed and transferred in exchange for carbohydrates. Symbiosomes have a structure similar to that of mycorrhizal arbuscules, which are also surrounded by a plant membrane. It is interesting that a number of legume-nodulating rhizobial strains form endophytic associations with monocotyledonous plants, such as rice, maize, and sugarcane, and dicotyledonous plants, such as sweet potato. Although nodule primordia were not observed, rhizobial nifH transcripts were found inside roots of rice and sugarcane plants. The contribution of rhizobium-assimilated nitrogen to the total nitrogen pool in nonleguminous plants is still a matter of debate.

Lyfestyles of Endophytes:  Microorganisms can be strictly bound to plants and complete a major part or even their entire life cycle inside plants. Microorganisms requiring plant tissues to complete their life cycle are classified as “obligate.” Well-documented examples of obligate endophytes are found among mycorrhizal fungi and members of the fungal genera Balansia, Epichlo?, and Neotyphodium, from the family Clavicipitaceae (Ascomycota). On the other extreme are “opportunistic” endophytes that mainly thrive outside plant tissues (epiphytes) and sporadically enter the plant endosphere (101). Among these are rhizosphere-competent colonizers, such as bacteria of the genera Pseudomonas and Azospirillum and fungi of the genera Hypocrea and Trichoderma. It is interesting that endophytes, which are transmitted vertically via seeds, are often recovered as epiphytes, suggesting that various endophytes might also colonize surrounding host plant environments. Between these two extremes is an intermediate group, which comprises the vast majority of endophytic microorganisms, the so-called “facultative” endophytes. Whether facultative endophytes use the plant as a vector for dissemination or are actively selected by the host is still a matter of debate. However, facultative endophytes consume nutrients provided by plants, which would in fact reduce the ecological fitness of the host plant. This point is therefore often used as an argument that the so-called facultative endophytes must be mutualists in plants, even if the details of the interaction are unclear.

Colonization Behavior of Fungal Endophytes:  Successful colonization by endophytes depends on many variables, including plant tissue type, plant genotype, the microbial taxon and strain type, and biotic and abiotic environmental conditions. Different colonization strategies have been described for clavicipitaceous and nonclavicipitaceous endophytes. Species of the Clavicipitaceae, including Balansia spp., Epichlo? spp., and Claviceps spp., establish symbioses almost exclusively with grass, rush, and sledge hosts, in which they may colonize the entire host plant systemically. They proliferate in the shoot meristem, colonizing intercellular spaces of the newly forming shoots, and can be transmitted vertically via seeds. Some Neotyphodium and Epichlo? species may also be transmitted horizontally via leaf fragments falling on the soil. At the stage of inflorescence development, the mycelium of Neotyphodium can also colonize ovules and be present during infructescence development in the scutellum and the embryo, as demonstrated for Lolium perenne. When the inflorescence of the grass host develops, Epichlo? can also grow over the developing inflorescence and form stromata, which can be differentiated sexually with the help of Botanophila flies.

Colonization Behavior of Bacterial Endophytes: Many bacterial endophytes originate from the rhizosphere environment, which attracts microorganisms due to the presence of root exudates and rhizodeposits. Mercado-Blanco and Prieto suggested that the entry of bacterial endophytes into roots occurs via colonization of root hairs. To a certain extent, stem and leaf surfaces also produce exudates that attract microorganisms. However, UV light, the lack of nutrients, and desiccation generally reduce colonization of plant surfaces, and only adapted bacteria can survive and enter the plant via stomata, wounds, and hydathodes. Endophytes may also penetrate plants through flowers and fruits via colonization of the anthosphere and carposphere. Depending on the strain, various colonization routes have been described, and specific interactions have been suggested (133, 134). Several of these routes involve passive or active mechanisms enabling bacteria to migrate from the rhizoplane to the cortical cell layer, where the plant endodermis represents a barrier for further colonization (130, 135). For bacteria that can penetrate the endodermis, the xylem vascular system is the main transport route for systemic colonization of internal plant compartments (134), whereas others colonize intercellular spaces locally. Bacteria have been shown to colonize xylem vessels, and the sizes of the holes of the perforation plates between xylem elements are sufficiently large to allow bacterial passage (130, 134, 136–138). However, vertical spread of bacteria through plants may take several weeks (139), and it is unclear why bacterial endophytes progress so slowly in the vascular system. Bacteria might even migrate to reproductive organs of Angiospermae plants and have been detected in the inner tissues of flowers (epidermis and ovary), fruits (pulp), and seeds (tegument) of grapevines (18) and in pumpkin flowers (140), as well as in the pollen of pine, a Gymnospermae plant (141). Suitable niches for colonization by bacterial endophytes have been described for different plant taxonomic groups, including Bryophytes, Pteridophytes, Gymnospermae, and Angiospermae (17, 130, 142) (Fig. 1). Overall, it is not known whether endophytes need to reach a specific organ or tissue for optimal performance of the functions which have been identified for endophytes.

Functions of Endophytes: Some endophytes have no apparent effects on plant performance but live on the metabolites produced by the host. These are termed commensal endophytes, whereas other endophytes confer beneficial effects to the plant, such as protection against invading pathogens and (arthropod) herbivores, either via antibiosis or via induced resistance, and plant growth promotion. A third group includes latent pathogens. Generally, endophytes can have neutral or detrimental effects to the host plant under normal growth conditions, whereas they can be beneficial under more extreme conditions or during different stages of the plant life cycle. For example, the fungus Fusarium verticillioides has a dual role both as a pathogen and as a beneficial endophyte in maize. The balance between these two states is dependent on the host genotype, but also on locally occurring abiotic stress factors that reduce host fitness, resulting in distortion of the delicate balance and in the occurrence of disease symptoms in the plant and production of mycotoxins by the fungus. However, beneficial effects have also been demonstrated, e.g., strains of the endophytic fungus F. verticillioides suppress the growth of another pathogenic fungus, Ustilago maydis, protecting their host against disease.

Plant Growth Promotion and Protection against Biotic and Abiotic Stresse

ISR and production of antibiotic secondary metabolites

Researchers suggested in 1988 that endophytes play a role in the defense systems of trees. Because life cycles of endophytes are considered to be much shorter than the life cycle of their host, they may evolve faster in their host, resulting in higher selection of antagonistic forms that contribute to resistance against short-living pathogens and herbivores. Later, in 1991, Carroll suggested that endophyte-mediated induced resistance occurs in Douglas fir trees. Endophytes may induce plant defense reactions, so-called induced systemic resistance (ISR), leading to a higher tolerance of pathogens. There is increasing evidence that at an initial stage, interactions between beneficial microorganisms and plants trigger an immune response in plants similar to that against pathogens but that, later on, mutualists escape host defense responses and are able to successfully colonize plants. Bacterial strains of the genera Pseudomonas and Bacillus can be considered the most common groups inducing ISR, although ISR induction is not exclusive to these groups. Bacterial factors responsible for ISR induction were identified to include flagella, antibiotics, N-acylhomoserine lactones, salicylic acid, jasmonic acid, siderophores, volatiles (e.g., acetoin), and lipopolysaccharides. The shoot endophyte Methylobacterium sp. strain IMBG290 was shown to induce resistance against the pathogen Pectobacterium atrosepticum in potato, in an inoculum-density-dependent manner. The observed resistance was accompanied by changes in the structure of the innate endophytic community. Endophytic community changes were shown to correlate with disease resistance, indicating that the endophytic community as a whole, or just fractions thereof, can play a role in disease suppression. In contrast to bacterial endophytes, fungal endophytes have less frequently been reported to be involved in protection of their hosts via ISR.

Production of additional secondary metabolites: Secondary metabolites are biologically active compounds that are an important source of anticancer, antioxidant, antidiabetic, immunosuppressive, antifungal, anti-oomycete, antibacterial, insecticidal, nematicidal, and antiviral agents. In addition, endophytes produce secondary metabolites that are involved in mechanisms of signaling, defense, and genetic regulation of the establishment of symbiosis. Besides the production of secondary metabolite compounds, endophytes are also able to influence the secondary metabolism of their plant host. This was demonstrated in strawberry plants inoculated with a Methylobacterium species strain, in which the inoculant strain influenced the biosynthesis of flavor compounds, such as furanones, in the host plants. Recently, bacterial endophytes, along with bacterial methanol dehydrogenase transcripts, were localized in the vascular tissues of strawberry receptacles and in the cells of achenes, the locations where the furanone biosynthesis gene is expressed in the plant. Similarly, biosynthesis and accumulation of phenolic acids, flavan-3-ols, and oligomeric proanthocyanidins in bilberry (Vaccinium myrtillus L.) plants were enhanced upon interaction with a fungal endophyte, a Paraphaeosphaeria sp. strain.

Iron homeostasis

Protection against biotic and abiotic stresses

Plant growth stimulation

Nitrogen fixation

In summary, various mechanisms in endophytes can explain the profound effects that endophytes have on their plant hosts. A recent report indicates that endophyte infection can also affect the gender selection of the host plant (237), which suggests that many new properties remain to be identified among endophytes.

Deciphering the behavior of Endophytes by Comparative Genomic Analysis: A comparative genomic and metabolic network study revealed major differences between pathogenic (n = 36) and mutualistic (n = 28) symbionts of plants in their metabolic capabilities and cellular processes. Genes involved in biosynthetic processes and functions were enriched and more diverse among plant mutualists, while genes involved in degradation and host invasion were predominantly detected among phytopathogens. Pathogens seem to require more compounds from the plant cell wall, whereas plant mutualists metabolize more plant-stress-related compounds, thus potentially helping in stress amelioration. The study revealed the presence of secretion systems in pathogen genomes, probably needed to invade the host plants, while genomic loci encoding nitrogen fixation proteins and ribulose bisphosphate carboxylase/oxygenase (RubisCO) proteins were more exclusive to mutualistic bacteria. Bacteria carrying relatively large genomes are often able to successfully colonize a wide range of unrelated plant hosts, as well as soils, whereas strains with smaller genomes seem to have a smaller host range

Multitrophic Interactions: The plant biome comprises the plant and multiple fungal and bacterial players, including both pathogens and mutualists, and is characterized by a dense network of multitrophic interactions, which are still poorly understood. Particularly in the case of tight interactions between the plant host and endophytes, signaling and recognition processes are highly important, inducing molecular, physiological, and morphological changes. However, plant-associated microorganisms may also influence plant pathways and phenotypes more generally. Quambusch et al. reported distinct endophytic communities for easy- and difficult-to-propagate cherry genotypes, indicating the need for a specific microbiome, or at least specific microbiome components, for plant growth in general. The interaction of endophytes with their plant host may also affect its relationship with other microbes.

Interactions between Endophytes and Pathogens/Pests: Endophytes may increase the defense against herbivores, including insects that transmit pathogens. Deterrence of herbivores is known to be mediated via in planta production of biologically active alkaloids in grasses by endophytes, which can reduce arthropod feeding and, consequently, damage to the host. However, in relation to wild grasses, Faeth and Saari reported that herbivore abundance and species richness may be even greater on endophyte-infected plants with high alkaloid contents than on endophyte-free plants; they argued that herbivores may develop detoxification pathways. Endophyte infection in grasses has also been tested for reducing aphid-transmitted virus infections . Endophyte infection and alkaloid production resulted in reduced aphid feeding, as expected, but no effect on virus titers could be observed. Nevertheless, the impact of virus infection on the host was reduced in endophyte-infected plants, indicating that the endophyte induced a host response, which was probably responsible for this effect. The interactions between plants, endophytes, aphids, and viruses were also influenced by the host and endophyte genotypes as well as by abiotic factors, such as temperature.

Interactions between Endophytes and Other Symbionts: In addition to the interactions between endophyte communities and phytopathogens, endophytes interact with other symbiotic microorganisms. Foliar fungal endophyte species composition was reported to be altered by AMF colonization. Wearn et al. suggested that there is competition or antagonism between AMF and root endophytes, as they found negative correlations between mycorrhizal colonization and the presence of endophytes in roots of herbaceous grassland species. Some studies suggest that AMF colonization of grasses may be affected by the production of alkaloids or other allelopathic compounds by fungal endophytes. However, different AMF species or strains may behave/interact differently with plants and endophytes. For grasses, Larimer et al. reported that Glomus mossae enhanced endophyte growth through increased tiller production, and in return, G. mossae showed higher colonization levels. On the other hand, colonization by another AMF species, Glomus claroideum, declined in endophyte-infected plants. In Pinus sylvestris, the interaction between a bacterial endophyte and an ectomycorrhizal fungus was shown to be species dependent, as endophytic Methylobacterium extorquens enhanced the growth of pine seedlings with one fungal species but decreased the growth when coinoculated with another ectomycorrhizal fungus.

In conclusion, the plant biome is characterized by multiple and complex interactions between the plant, the associated microbiota, i.e., endophytes with different functions, including pathogens, and the environment. The plant phenotype not only is determined by the response of the plant to the environment but also is regulated by the associated microbiota, the response of the microbiota to the environment, and the complex interactions between individual members.

CONCLUDING REMARKS

Technological developments, especially with respect to “-omics” technologies, will revolutionize our concepts on endosphere microbiomes. At present, we are better able to distinguish between properties specific to phytopathogens, endophytes, and other microorganisms from soil and plant habitats. This will allow us to better understand mutualists and pathogens, because from an ecological perspective, the boundaries between both groups are not always clear. Furthermore, microbial groups previously thought to be distinctive of other environments, such as human pathogens in warm-blooded animals, have been demonstrated to thrive in plants. Genomics will teach us how microbial groups from other environments adapt to plant environments and will reveal the minimal genetic requirements for successful penetration and internal colonization of plants. Novel technologies will also allow us to investigate multiple interactions between microbial groups associated with plants and the plant host itself. Nowadays, we have a better capacity to analyze impacts of invading microorganisms on the whole endophytic community composition and functioning, and vice versa. We can also better explain the resilience of plants upon invasion by potentially deleterious microorganisms by the functioning and complexity of the endophytic communities. We must learn more, however, about the still unknown roles of endophytes, particularly the so-called commensal endophytes. This group, which causes no apparent effects on plant performance but lives on the metabolites produced by the host, is presumably the most dominant functional group among endophytes by quantity. We expect to find hidden functions within this group and to learn more about the complexity of microbial interactions within plants, including the consequences for the host plant. We also need to learn more about the interactions between endophytes and plants as well as the mechanisms employed by all partners. It will be highly relevant to elucidate the physiological conditions present in endophytes and plants during colonization, as it can be expected that an endophyte will have different characteristics inside the plant compared to growth in the soil or in the lab. Similarly, research is needed to better understand under which conditions and by which mechanisms microorganisms exhibit harmful, beneficial, or neutral effects on plant performance. By implementing new technologies and multidisciplinary approaches, our understanding of endophyte biology and ecology will consistently evolve further, leading to a better knowledge of the plant holobiome.