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Applied Microbiology Assignment

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ENDOPHYTES AND THEIR SIGNIFICANCE

SUBMITTED BY: Aditya Kanwal 2012BT09 M.Tech 1st Year MNNIT, Allahabad

CONTENTS 1. Introduction 2. Identification 3. Transmission and Relationship 4. Mechanism of Endophytic Growth 5. Bacterial Endophytes 6. Fungal Endophytes 7. Benefits and Uses 8. A Case Study 9. References

INTRODUCTION
As the word suggests, Endos meaning inside and phyton meaning plant, an endophyte is an endosymbiont that colonizes internal plant tissues. However, this term is also used for endosymbiotic fungi in algae. It can be a fungi or a bacterium and may or may not be beneficial to the plant. Though most of the endophytic relationships are mutualistic, some of them may even be parasitic in nature. A common characteristic for all fungal and bacterial endophytes is that they are symptomless, atleast for a short period of time, and mostly the effected plants are not easy to differentiate from the normal ones. Endophytes are usually present in the inter-cellular spaces but can be present in the intra-cellularly. The endophytes need amino acids, simple sugars, and a variety of vitamins to survive and acquire them from the apoplasm of the host. Endophytes have been found in almost all the species of plants and some algae. Certain endophytes share unique relationships with their host and colonize only that specific host, like Neotyphodium australiense that colonizes only specific austrailian population of grass. On the other hand, there are certain endophytes like Piriformospora indica that have been seen to effect almost all plant species. These endophytes have been seen to help plants in many ways by providing improving nutritional uptake in root, producing toxins to fend off the predators, improving biotic and abiotic stress tolerance etc. For humans, they have been studied for their use in medicine by identification of various primary and secondary metabolites released by them(eg. Taxol), improving plant yield, in generating biotic and abiotic resistance and in phytoremediation. The most frequently isolated endophytes are the fungi. It turns out that the vast majority of plants have not been studied for their endophytes. Thus, enormous opportunities exist for the recovery of novel fungal forms, taxa, and biotypes. Hawksworth and Rossman estimated there may be as many as 1 million different fungal species, yet only about 100,000 have been described. As more evidence accumulates, estimates keep rising as to the actual number of fungal species. It seems obvious that endophytes are a rich and reliable source of genetic diversity and novel, undescribed species. Finally, in our experience, novel microbes usually have associated with them, novel natural products. This fact alone helps eliminate the problems of dereplication in compound discovery.[5]

IDENTIFICATION
Endophytes can colonize multiple species of plants or can be host specific. Endophytic species are very diverse; it is thought that only a small minority of all existing endophytes have been characterized. A single leaf of a plant can harbor many different species of endophytes, both bacterial and fungal[3]. Endophytes can be identified in several ways, usually through amplifying and sequencing a small piece of DNA. Some endophytes can be cultured from a small piece of their host plant in an appropriate growth medium. Not all endophytes present can be cultured in this way because amplification of ground up plant tissue using fungal or bacterial specific primers has revealed the existence many cryptic species. Grass leaf endophytes can be seen as coiled tubes of hyphae under the microscope at 400X following clearing of the leaf sheaths in ethanol and staining with aniline blue[3]. Autofluorescent protein (AFP) methods are now a key tool or studying processes such as microbeplant interactions and biofilm formation. These techniques have been utilized to detect and enumerate microorganisms in situ on plant surfaces and in planta. One of these AFP strategies uses a marker system, which encodes the green fluorescent protein (GFP). Workers have developed GFP cassettes for chromosomal integration and expression of gfp in a variety of bacteria. Bacterial cells with chromosomal integration of gfp can be identified by epifluorescence microscopy or confocal laser scanning microscopy[4]. For characterization, microbial communities inhabiting stems, roots and tubers of various varieties of plants can be analysed by 16S rRNA gene-based techniques such as terminal restriction fragment length polymorphism analysis, denaturing gradient gel electrophoresis as well as 16S rRNA gene cloning and sequencing[4]. Rationale for Plant Selection[5]: It is important to understand the methods and rationale used to provide the best opportunities to isolate novel endophytic microorganisms. Thus, since the number of plant species in the world is so great, creative and imaginative strategies must be used to quickly narrow the search for endophytes displaying bioactivity. A specific rationale for the collection of each plant for endophyte isolation and natural product discovery is used. Several reasonable hypotheses govern this plant selection strategy and these are as follows: 1. Plants from unique environmental settings, especially those with an unusual biology, and possessing novel strategies for survival are seriously considered for study.

2. Plants that have an ethnobotanical history (use by indigenous peoples) that are related to the specific uses or applications of interest are selected for study. These plants are chosen either by direct contact with local peoples or via local literature. Ultimately, it may be learned that the healing powers of the botanical source, in fact, may have nothing to do with the natural products of the plant, but of the endophyte (inhabiting the plant). 3. Plants that are endemic, having an unusual longevity, or that have occupied a certain ancient land mass, such as Gondwanaland, are also more likely to lodge endophytes with active natural products than other plants. 4. Plants growing in areas of great biodiversity also have the prospect of hosting endophytes with great biodiversity. A real example for application of these strategies is: An aquatic plant, Rhyncholacis penicillata, was collected from a river system in Southwest Venezuela where the harsh aquatic environment subjected the plant to constant beating by virtue of rushing waters, debris, and tumbling rocks and pebbles. This created many portals through which common phytopathogenic oomycetes could enter the plant. Still, the plant population appeared to be healthy, possibly due to protection from an endophytic product. This was the environmental biological clue used to pick this plant for a comprehensive study of its endophytes. Eventually, a potent antifungal strain of Serratia marcescens, was recovered from R. penicillata and was shown to produce oocydin A, a novel antioomycetous compound . It is conceivable that the production of oocydin A by S. marcescens is directly related to the endophytes relationship with its higher plant host. Currently, oocydin A is being considered for agriculture use to control the ever threatening presence of oomyceteous fungi such as Pythium and Phytophthora[5].

TRANSMISSION AND RELATIONSHIP


Endophytes may be transmitted either vertically (directly from parent to offspring) or horizontally (from individual to unrelated individual)[3]. Vertically transmitted fungal endophytes are asexual and transmit via fungal hyphae penetrating the hosts seeds. Bacteria can also be transmitted to seeds through parent host. In this, seeds become the common diaspore for plant and the endophyte. This is mostly mutualistic and the plant mainly gains from this relationship as their reproductive fitness is intimately tied. On the other hand, horizontally transmitted endophytes are sexual and transmit via spores that can be spread by wind or insect vectors and in case of bacteria via bacterial infection. In this, sometimes, the host is rendered sterile by the endophyte and thus horizontal transfer remains the only option. Since they spread in a similar way to pathogens, horizontally transmitted endophytes are often closely related to parasitic behaviour. Fossil evidence suggests that the plant-endophyte relationship is as old as 400 million years i.e. when the plants first begin to colonize land. This is what makes the plantendophyte relationship so intimate. Both plants and the endophytes gain from this mutualistic relationship.

What Endophytes gain: Nutrition: The endophytes get amino acids, vitamins, simple sugars and various other nutrients directly from the apoplast (free diffusional space outside the plasma membrane) of the plant tissues. Protection: They are protected form desiccation, surface feeding insects, competition from other microbes, etc.

Transmission: A vast number of endophytes, mainly fungal, are transmitted vertically i.e asexually from host to it's seeds directly, thus maintaining a continuity. This way, the endophytes doest have to spent its energy in struggling for survival and for passing on its genes. Bacterial endophytes on the other hand can also be transmitted via insects. What Plants gain: Growth facilitation: Certain mycorhizzae called endomycorrhizae are known to fix nitrogen. They fulfill the nitrogen requirements of their host plants. Nitrogen fixation takes place in the absence of oxygen, low nitrogen and controlled phoshorus levels in these fungi. Also, phosphorus availability is increased via solubilization of various kinds of phosphates like calcium phosphate and iron phosphate with the help of various enzymes and chemicals released by the endomycorrhizae. Various other mineral ions like iron and calcium are also absorbed and utilized in the similar way. Another property of certain endophytes is the production of plant growth factors like Indole 3-acetic acid (an auxin) that is directly involved in the regulation of plant growth. The improved nutrient uptake can also be credited to the fact that endophytes modify the internal environment of the tissues they inhabit physiologically. Protection against Biotic stresses: Studies of fungal infection consistently reveal that plants with endophytes are less likely to suffer substantial damage, and herbivores feeding on infected plants are less productive. There are multiple modes through which endophytic fungi reduce insect and herbivore damage by releasing a variety of chemical mediators. These modes include reduced apetite, reduced development rate, reduced growth and reduced survival. Also, the endophytes alter the nutrient content due to the inherently nutrientexchange based economy of the plant-endophyte association. It has been seen that the infection by fungi directly alters the chemical composition of plants, with corresponding impacts on their herbivores. Endophytes frequently increase apoplastic carbohydrate concentration, altering the C:N ratio of leaves and making them a less efficient source of protein. Apart from this, enzymes like esterases, lipases, proteinases and cellulases, various antibiotics, antivirals, antifungals and antimicrobials (for eg. phenolic derivatives) are released extracellularly to prevent invasion of other microbes.

Improvement of abiotic stress tolerance: Endophytes have been known to degrade various kinds of xenobiotics to much simpler and comparatively less hazardous chemical compounds. These xenobiotics usually interfere with plant growth and its performance in natural environment. If degraded, there would be no abiotic stress on the plant and they can grow normally. Also, the products of degradation can be utilized by plants in various metabolic pathways. Endophyte infected plants are seen to be more tolerant to drought like conditions. By improving nutritional uptake, endophytes are directly making plants more fit and healthy and less prone to abiotic stresses and drought like conditions. Fitness and competitive ability: Fungal endophytes which provide defensive services to their host plants may exert selective pressures favouring association through enhanced fitness relative to uninfected hosts. The fungus Neotyphodium spp. infects grasses and increases fitness under conditions with high levels of interspecific copmpetition It does this through a combination of benefits including anti-herbivore defenses and growth promoting factors. Influence of Endophytes on other organisms: On Mammals: Endophytes, especially Fungi, produce alkaloids like ergot and indolediterpene that are toxic to mammalian herbivores. Endophytes like Neotyphodium sp. of grass release chemical substances that can create a variety of neurological symptoms in farm animals like spasms and sleepiness. Various other symptoms can be produced like fescue foot in which the cattle that graze on Neotyphoidium infected tall fescue grass (Lolium arundinaceum) can develop a condition known as "fescue foot" in which blood is reduced to extremities over a long period of time which eventually leads to the tail or hooves rotting off. On Insects: Alkaloids like lolines and peramines have been seen to be inhibitory for insects. They can cause various symptoms like reduction of apetite or even death. On other microbes: Infection from other microbes is inhibited by allelopathic effects (i.e. by biochemical substances released by the endophyte).

MECHANISM OF ENDOPHYTIC GROWTH


The inner space of plants represents an ecological niche where numerous species of fungal and bacterial endophytes live. Almost all known plant species associate with endophytes. Some endophytes infect the plant parts locally, being restricted to a small tissue area, for example Endomychorrihae in roots. On the other hand, some infect the host plant systematically as in Neotyphodium that infects almost all the plant parts (in grass). Although bacteria are prokaryotes and fungi are eukaryotes, they share many attributes of their associations with plant hosts, e.g. both colonise root tissues interand intra-cellularly, and often systemically. They do, however, differ somewhat in their modes of colonisation.[1] Endophytic infection can occur in one of the two ways: In embryo stage i.e. from current host to its seeds. This route of infection is employed exclusively via fungal endophytes that form a network of hyphae around the seeds and then infect them. This kind of relationship is more intimate. At some later stage of plant growth through tissue openings like stomata or through wounds caused by some physical damage, enzymes released by endophytes or via vectors like insects. Both bacterial and fungal endophytes can cause infection in this way. When an endophyte is infecting a plant through wound, its initial source of nutrition is host exudates, dead cortex cells and plant debris. At later stage, i.e. steady state, the nutritional sources usually comes from apoplastic and symplastic fluids. Bacteria primarily colonise intercellularly and rarely have been found to colonize intracellularly. They are frequently found in the vascular tissues (Xylem and Phloem) of host plants, which is advantageous for distribution. On the other hand, fungal colonisation can be both inter- and intra-cellular, the hyphae often forming intracellular coils. They rarely colonize vascular tissues. Formation of specialized structures are also seen sometimes, especially in the case of bacterial endophytes where node like structures are formed.

BACTERIAL ENDOPHYTES
Being an endophyte means protection from the external environment for the bacteria. These bacteria generally colonize the intercellular and ever intracellular spaces, and they have been isolated from almost all plant compartments. Endophytic bacteria have been isolated from both monocotyledonous and dicotyledonous plants, ranging from woody tree species, such as oak and pear, to herbaceous crop plants such as sugar beet and maize[4]. Many different endophytic species can colonize a plant at once. The colonization can be in different parts of the plant like roots, stem, leaves, etc or many different specie can colonize the same region(like leaves) at once. This has been proven by various kinds of molecular analysis and culture techniques. A vast amount of bacterial endophytes have been discovered and characterized till now. They belong to a variety of Families. Examples include Acinetobacter, Bacillus, Burkholderia, Herbaspirillium, Methylobacterium, Nocardioides, Paracoccus Paenibacillus, Pseudomonas, Serratia, Staphylococcus, Taxomyces etc.

FIGURE: Image of colonized endophytic Enterobacter sp.(By Taghavi S & Nykypanchuk D[10])

Various plant species have been studied for the presence of bacterial endophytes. An example for the study of Bacterial endophytes in a plant cultivar is presented below.

FIGURE: These are the bacterial endophytes extracted from various bean cultivars and phylogenetic relationship between them. Bar represents their localization(Lopez et al, 2010 [6])

FUNGAL ENDOPHYTES

All plants in natural ecosystems appear to be symbiotic with fungal endophytes. This highly diverse group of fungi has formed a deep impact on the host plant communities by forming a very intimate relationship in some cases. Despite more than 100 year of research resulting in thousands of journal articles, the ecological significance of these fungi remains poorly characterized. Historically, two endophytic groups (clavicipitaceous (C) and nonclavicipitaceous (NC)) have been discriminated based on phylogeny and life history traits[7]. Based on symbiosis, nonclavicipitaeceous fungi can futher be divided into three subtypes:

FIGURE: Fungal classification based on symbiotic criteria(Rodriguez et al, 2009 [7]) As in the case of bacterial endophytes, a vast amount of fungal endophytes have been discovered and characterized till now. For example Acremonium, Balansia, Cladosporium, Ceratopycnidium, Epichloe, Idriella, Neotyphodium, Piriformospora, Stagnospora, etc. FIGURE: Slender tubes of Neotyphodium coendophialum in the intercellular spaces of a grass.(By Dr. Nick Hill, UGA [9] )

Various plant species have been studied for the presence of fungal endophytes. An example for the study of fungal endophytes in a grass Bouteloua gracilis and Sporobolus cryptandrus is presented below.

FIGURE: Molecular phylogeny of the most common fungal endophytes found in grasses Bouteloua gracilis and Sporobolus cryptandrus. Codes represent sequences obtained preceded by the host and followed by the year. (Khidir et al, 2010 [8]).

BENEFITS AND USES:


As discussed previously, endophytes play many roles in their plant hosts. Owing to their properties, they have been studied and utilized in various ways by humans. Major uses can be classified under four headings as: For Medicinal puposes: Endophytes release a wide variety of secondary metabolites beneficial to us in one or the other way. They have been utilized for the production of: Antibiotics and Antimicrobials: Ecomycin, Kakadumycin, Pyrrocidines, Munumbicins, Altersolanol are amongst the variety of antimicrobial agents produced by various endophytic bacteria and fungi. Anticancer compounds: Taxol is an anticancer compound produced by almost all the plants in the Taxus genus and endophytes inside these trees. It has been used by humans for treating patients with lung, ovarian and breast cancer. Antivirals: Endophytes, mainly bacteria, also release certain antiviral compounds like Cytonic Acids A and B. Antifungals: Endophytes also release a variety of antifungal agents, targeted maily for parasitic fungi so as to avoid competition related to food and space. Antifungals like Fusaricidin A-D, Oocidin A and many other have been harvested from various endophytes for this purpose. Antimalarial:

Coronamycin, has been seen to possess antimalarial and antifungal properties. It is released by an endophytic bacterium of Streptomyces sp. and produced in a vine found in the forests of amazon. A variety of Antioxidants and Immunosuppresants have also been found to be produced.

In Plant yield and growth: Via modification of plant vascular system, nutrient solubilization, phytostimulation, etc. Endophytes, both bacterial and fungal, have been seen to increase the organic and inorganic nutrient digestibility and uptake in various cases depending on the hostendophyte relationship in each case, Degradation of complex organic molecules leads to the release of various simple organic and inorganic molecules that are metabolized by the plants. Some endophytic relationships also play dual role in which the endophyte degrades the Xenobiotic compound to simpler organic molecules which can then be incorporated into various metabolic pathways by the host plant. In Plant protection: By the release of antimicrobial compounds, tolerance to biotic and abiotic stresses, by competing for space and nutrition and thus preventing growth of other parasitic microbes, etc. Plant-microorganism symbiotic association based on mutual exploitation has more opportunity to survive in environment. There are increasing numbers of studies focusing on fungal endophytes because they benefit plant growth and protection. Plants inoculated with fungal endophytes usually display promotion in growth and resistance to diseases caused by pathogens. Fungal endophyte mediated plant resistances to pathogens have been well studied in agricultural crops, grass systems and forest trees.[11] In Phytoremediation: For biotransformation of Xenobiotics (like Phenol, Chlorophenols, BTEX, Toulene etc.) to much simpler and comparatively harmless compounds. A promising field to exploit plant-endophyte partnerships is the remediation of contaminated soils and (ground) water. Many plant growth promoting endophytes can assist their host plant to overcome contaminant-induced stress responses, thus providing improved plant growth. For phytoremediation of toxic metals, endophytes possessing a metal-resistance/sequestration system can lower metal phytotoxicity and affect metal translocation to the above-ground plant parts. Furthermore, endophytes that can degrade organic contaminants and deal with, or even better, improve extraction of the metals offer promising ways to improve phytoremediation of mixed pollution.[12]

A CASE STUDY[13]
Neotyphodium:
Neotyphodium is a genus conatining species of endophytic fungi. These endophytes are asexual, seed-borne symbionts of cool-season grasses, and grow intercellularly throughout the aerial tissues of their hosts, including shoot apical meristems, leaf sheaths and blades, inflorescences, seeds and embryos. For eg: Neotyphodium austrailiense, endosymbiont of Echinopogon ovatus, a grass endemic to Austrailia and New Zealand. Life Cycle: All Neotyphodium species infect new grass plants solely by growing into the seeds of their grass hosts, and infecting the growing seedling with the help of their hyphae. This is known as vertical transmission and is repeated in each flowering season. It has been proposed that vertically transmitted symbionts evolve to be mutualists since their reproductive fitness is intimately tied to that of their hosts. In fact, some positive effects of Neotyphodium on their host plants include increased growth, drought tolerance, and herbivore and pathogen resistance. Resistance against herbivores has been attributed to alkaloids produced by the symbiotic Neotyphodium. Although grass-Neotyphodium symbioses have been widely recognized to be mutualistic in many wild and cultivated grasses, the interactions can be highly variable and sometimes antagonistic, especially under nutrient-poor conditions in the soil. 5. Relationship and Coevolution: The Neotyphodium display a number of central features that suggest a very strong and ancient association with their grass hosts. The symbiosis appears to have existed already during the early grass evolution that has spawned today's grasses. This is suggested by phylogenetic studies indicating intimate relationship between Neotyphodium species and the grass hosts they inhabit. Growth of the fungal symbiont is very tightly regulated within its grass host, indicated by a largely unbranched mycelial morphology and remarkable synchrony of grass leaf and hyphal extension of the fungus which occurs via intercalary elongation of the endophyte's hyphae, a process so far not found in any other fungal species, indicating specialized adaptation of the fungus to the dynamic growth environment inside its host.

Bioactive Compounds: Many Neotyphodium endophytes produce a diverse range of natural product compounds with biological activities against a broad range of herbivores. 1) Ergoline alkaloids are characterized by a ring system derived from 4-prenyl tryptophan. Among the most abundant ergot alkaloids in symbiotic grasses is ergovaline, comprising an ergoline moiety attached to a bicyclic tripeptide containing the amino acids L-proline, L-alanine, and L-valine. These have activity against certain mammalian herbivores and some parasitic insects. 2) Another group of alkaloids are the indole-diterpenoids, such as lolitrem B, which are produced from the activity of several enzymes, including prenyltransferases and various monooxygenases. These have been seen to possess biological activity against mammalian herbivores, and also activity against some insects. 3) Peramine is a pyrrolopyrazine alkaloid thought to be biosynthesized from the guanidinium-group-containing amino acid L-arginine, and pyrrolidine-5-carboxylate, a precursor of L-proline, and is an insect-feeding deterrent. 4) The loline alkaloids are 1-aminopyrrolizidines with an oxygen atom linking bridgehead carbons 2 and 7, and are biosynthesized from the amino acids L-proline and L-homoserine. The lolines have insecticidal and insect-deterrent activities comparable to nicotine. N-formylloline, an insecticidal alkaloid produced in several Neotyphodiumgrass symbiota. Loline accumulation is strongly induced in young growing tissues or by damage to the plant-fungus symbiotum.

Peramine N-formylloline Ergovaline

REFERENCES
[1] Schulz B & Boyle C. " What are Endophytes?"(2006), Microbial Root Endophytes, Vol. 9, Soil Biology. [2] Bacon, Charles W, James F., Microbial Endophytes. (2000). [3] http://en.wikipedia.org/wiki/Endophyte(as on 09-2012 ) [4] Ryan R P, Germaine K, Franks A, Ryan D J & Dowling D N."Bacterial endophytes: recent developments and applications"(2008) , FEMS Microbiol Lett. 278, 19 [5] Strobel G & Daisy B."Bioprospecting for Microbial Endophytes and their natural products"(2003), Microbiol. Mol. Bio. Rev., Vol. 67 (4), 491-502 [6] Lopez A L, Rogel M A, Orrilo E O, Romero J M & Romero E M.Phaseolus vulgaris seed -borne endophytic community with novel bacterial species such as Rhizobium endophyticum sp. Nov..(2010) Systematic and Applied Microbiology, Vol. 33 (6), 322-327 [7] Rodriguez R J, White J F, Arnold A E & Redman R S., Fungal endophytes: diversity and functional roles.(2009). New Phytologist. Tansely Review. [8] Khidir H H, Eudy D M, Alfaro A P, Herrera J, Natvig D O & Sinsabaugh R L.A general suite of fungal endophytes dominate the roots of two dominant grasses in the semiarid grasslands(2010), Journal of Arid Environments, Vol. 74 (1), 35-42. [9] Hancock D W, Hicks R, Kichler J M & Smith R C, Georgia Forages: Grass Species, CAES Publications. Article on University of Georgia Webportal. [10] Martin F, Poplar growth promoting Endophytic Bacterium(2010) Article on MycorWeb Fungal Genomics.

[11] Gao F K, Dai C C & Liu X Z,Mechanisms of fungal endophytes in plant protection against pathogens(2010), African Journal of Microbiology Research Vol. 4(13), 1346-1351. [12] Nele W, Daniel V L, Safiyh T & Jaco V, Phytoremediation: plant-endophyte partnerships take the challenge(2009). [13] Excerpt from en.wikipedia.org/wiki/ Neotyphodium as on 10/10/2012.