Review article

Endophytic fungi, characteristics and their potential for genetic manipulation

Aghafakhr Mirlohi1, Mohammad Reza Sabzalian1 and Mojtaba Khayyam Nekouei2

1Department of Plant Breeding, Isfahan University of Technology, Isfahan, 2Agriculture Biotechnology Institute of Central Region of I.R. Iran.

Abstract scientists and biotechnologist to use them as a surro- Unique benign endophytes from Ascomycets have gate target of transformation. wide distribution among grass species. The symbiotic Keywords: Endophyte, Molecular marker, Neotypho- fungi enhance plant characters including performance, dium, Transformation insect and mammalian deterrence, nematode resist- ance and tolerance to drought, salt and other biotic Table of contents: and abiotic stresses. Endophytes from genus Introduction Neotyphodium (Acremonium) are of the major focus than their ancestors, and Epichloe species, because Endophyte, as Bio-Control Agents the formers have lost their sexual reproduction. Genetic diversity of endophytes based on molecular Therefore they should be genetically stable, and most Markers importantly, they cannot disassociate from host tis- Transformation of endophytes by marker genes sues, and are transferred vertically. They are mater- Gene knocking out and elimination of undesirable traits nally inherited and are therefore attractive for genetic Future prospects transformation without the concern about gene escape. Some marker genes have been successfully Concluding remarks transferred to endophyte Neotyphodium coenophialum and Neotyphodium lolii existing in Festuca arundi- nacea Schreb. and Lolium perenne L., respectively. INTRODUCTION Furthermore, gene silencing has been proved to be feasible for eliminating traits, which are economically The well-known biological agents of plant protection harmful. Methods of direct DNA uptake using polyeth- ylene glycol (PEG) and electroporation have been in recent years are endophytic fungal symbionts, found to be useful in transformation of these fungi. which predominantly are found in wild species of Transgenic fungi can be reinserted into the host with- cool-season grasses (Schardl and Phillips, 1997; Miles out need to tissue culture. The endophytic genes et al., 1998). Fungal endophytes, defined as fungi that responsible for a specific trait can be isolated and spend their life cycle within the aerial parts of host transferred to grass species or other microorganisms plant, form non-pathogenic fungi that systemically for direct exploitation of secondary metabolites and endophytic enzymes. Considering advancements in occupy intercellular open spaces (Bacon and De this filed, endophytes can open new horizons faced to Battista, 1991 and Schardl, 1996) and have key roles for ecological success of host plants (Selosseet al., 2004). The well-known grass endophytes are classified in the tribe Balansieae of the family Clavicipitaceae

Correspondence to: Aghafakhr Mirlohi, PhD (Ascomycets). In the grass family Poaceae, fungal Tel: +98 311-3913450, Fax: +98 311-3912254 endophytes have been found in Poeae tribe in the gen- E-mail: [email protected] era Festuca,Lolium , andPoa (Bacon, 1995), Bromeae

IRANIAN JOURNAL of BIOTECHNOLOGY, Vol. 2, No. 2, April 2004 75 tribe in the genusBromus (White Jr, 1987), Stipeaephytes grown in culture except of Lolines that merely tribe in the genusstipa (White Jr and Morgan –Jones, have been detected in host plants. Apparently, plant is 1987; Bruehlet al., 1994; Kaiseret al ., 1996),a bio-enhancer for production of lolines, however, Meliceae tribe (White Jr, 1987), Aveneae tribe (WhitePorter (1994) poses an idea that lolines may be an Jr, 1994), and Triticeae tribe. In Triticeae, theendophyte-induced fungal production of plant. All four class- endophytes have been found inElymus (White Jr, es have anti-insect activities but peramine is the most 1987),Hordeum (Wilson et al., 1991b) and wildpotent alkaloid in this respect. Lolitrem B is associat- species ofTriticum (Marshal et al., 1999). The mosted with the disorder ryegrass staggers commonly widely known and demonstrated endophytes fromobserved in sheep grazing perennial ryegrass and genus Neotyphodium are N. coenophialum (Morgan- ergovaline can cause maladies among cattle grazed on Jones and Gams) Glenn, Bacon and Hanlin,N. lolii infected tall fescue called fescue toxicosis. Ergovaline (Latch, Chiristensen, and Samuels) Glenn, Bacon andcan reduce lactation and fertility, cause miscarriage Hanlin andN. uncinatum (Gams, Petrini and Schmidt) and stillborn offspring (Schardl and Phillips, 1997). Glenn, Bacon and Hanlin that infect tall fescue The approaches for mitigating or eliminating toxi- (Festuca arundinacea Schreb.) perennial ryegrasscosis are: 1) production and replanting with endo- (Lolium perenne L.) and meadow fescue F.( pratensis phyte–free cultivars, 2) alleviating toxic symptoms by Huds.), respectively. vaccination against ergot alkaloids with It also seems that endophytes discovered inMetoclopramide, Domperidone and Prazosin, 3) Hordeum and Triticum should be from genus selecting endophyte genotypes with low toxic alkaloid Neotyphodium. Meanwhile endophytes have been accumulation, and 4) genetic engineering of the endo- found in some Iranian species such asBromus phytes (Schardl and Phillips, 1997; Thompson and tomentellus and Melica persica, beside species ofGarner, 1994). Because of the natural role of Festuca ( Khayyam Nekoueiet al., 2000; KhayyamNeotyphodium in biological protection, the approach Nekouei et al., 2001; Mohammadi and Mirlohi, 2003)of removing the endophyte is neither rational nor prac- and Lolium that possibly belong Neotyphodiumto tical (Tsaiet al., 1992). Other ways are available but (Mirlohi and Sabzalian, 2002) (Table 1). Interactiongenetic engineering involves extra advantages. between Neotyphodium endophytes and cool-seasonCloning of genes involved in the ergot alkaloid biosyn- grasses culminates a defensive mutualism in which thetic pathway and their eventual manipulation simul- endophytes produce a range of alkaloids or stimulatetaneously via gene knockout or anti-sense strategies, the host grass to synthesize alkaloids and other sec-would provide a means for identifying roles for ergot ondary metabolites that protect macrosymbiont (host)alkaloids and genetic analysis of biosynthetic path- from mammalian, insect, and nematode herbivoresways (Panaccioneet al., 2001). Finally this technique (Malinowski and Belesky, 2000). Thus the ecological-provides conditions for introducing exotic encoding ly diverse grasses and their successful adaptations maygenes that produce insecticide delta toxins, proteinase be due to this association (Baconet al., 1997). Theyinhibitors etc. have no sexual reproduction and even sometimes do not produce asexual spores. For this reason,Endophytes as bio-control agents: The most impor- Neotyphodium endophytes are dependent on the hosttant effects of endophytic fungi have been observed for their life and instead they produce protective alka-against wild life, particularly insects feeding on green loids to support host species. Four alkaloid classestissues of the plant. The first of this, was Argentine have so far been associated with the fungal endo-stem weevil Listronotus( bonariensis), when endo- phytes; the ergot alkaloids (e.g., lysergic acidphyte–free and plants were heavily damaged by stem wee- ergovaline), the indole diterpenes (e.g., paxillinevil larvae, and but damage on endophyte-infected plants lolitrem B), a pyrrolopyrazine (peramine) and wasthe sat-negligible (Rowan and Latch, 1994). Later, sod urated aminopyrrolizidines (loline, norloline,web wormsN- (Crambus spp.), billbugsSphenophorus ( acetylloline, and N-formylloline, which are all calledparvulus), fall armywormsSpodoptera ( frugiperda) “Lolines”). Other compounds such as ergosterol, har- and hairy chinch bugs (Blissus leucopterus hirtus) man, norharman and halostachin and plant growth reg- were found to be affected by endophytes and now this ulators have been reported in some endophytelist has been fully prolonged (Clementet al ., 1994; researches, but their role and mechanism is under Sabzalianet al., 2003; Sabzalianet al., 2004). investigation (Porter, 1994). Three of the four Evidencemen- suggesting protection against root- feeding tioned alkaloid classes have been produced by endo-insects are rare but authors have shown that roots of

76 Mirlohi et al. Table 1- List of plant species that have been so far identified to be harboring endophyte in Iran (data not published ).

*. Species and regions have been arranged based on priority of identification. endophyte–free tall fescue plants can be severelytion of tall fescue seeds highly infected with endo- attacked by insects and this is not the case in endo-phyte, presence ofRizoctonia zea can be easily phyte infected plants (Fig. 1), probably due to ignored,alkaloid as if it is not present. In other hand, resistance accumulation in rhizospher (data not published). Fourto fungal pathogens has not been crucial. For instance, classes of fungal metabolites are responsible for insectauthors have not found significant difference between resistance in endophyte infectedFestuca and Lolium endophyte–free and endophyte infected meadow fes- species. These are indole diterpenes, ergot alkaloids,cue and tall fescue attacked byBlumeria graminis peramine, and lolines (Rowan and Latch, 1994).(data not published). Endophytes can indirectly dimin- Indole diterpenes include lolitrems, lolitriol, and ishpax- viral diseases by host insect deterrence.et West al. illine that are basically neurotoxin and tremorgenic (1993)in have shown fewer incidence of barley yellow mice and sheep but might have anti-insect activity. Outdwarf virus (BYDV) on tall fescue infected with of the ergot alkaloids, ergovaline is the most activeNeotyphodium coenophialum possibly through reduc- against stem weevil and also cause heat stress in cattle.tion in insect population. Others are less important in this respect. Loline alka- Effect of endophytic fungi on endoparasitic nema- loids and peramine have the most potent anti-insecttodes has been taken the most attention after insects. properties. They can also contribute to make someMeloidogyne marylandi, a root knot nematode can be other known and unknown benefits. Presumably loline alkaloids were shown to reduce germination rate of competitor weed seeds (Malinowski and Belesky, 2000). It seems that mechanisms of resistance to insects through endophyte infection consist both antixenosis and antibiosis via alkaloid production. Resistance to insects is so important that may serve as bio-indicator for presence of endophyte. Wilsonet al . (1991a) in a bioassay with Russian wheat aphidDiuraphis ( noxia) reported significantly fewer aphids on perennial rye- grass plants containing endophyte as compared to those lacking endophyte. Beside endophyte influence on insects, there are several reports of endophyte activity against fungal diseases, viral pathogens and nematode herbivores.Figure 1. Influence of endophyte on root colonization by mealy bugs. Endophyte-infected plant of genotype 83 is free from white Gwinn and Gavin (1992) have shown that in germina-clonies of mealybugPhenococcus solani (left).

IRANIAN JOURNAL of BIOTECHNOLOGY, Vol. 2, No. 2, April 2004 77 deterred when endophyte exists in above ground tis-in Neotyphodium lolii and Neotyphodium sues of perennial ryegrass (Ballet al., 1997). There coenophialumare and thus can distinguish these two similar results about Pratylenchus scribneri species but does not produce any band from the minus (Kimmons et al., 1990). Mechanisms of resistanceDNA to control. Specific primers have also been devel- nematodes have yet to be approved, but could be dueoped to detect endophytes in plants. This technique to some alkaloids or indirectly by maintaining generalcan be used for detecting endophytes in seed lots and health. to measure percentage of endophyte infection (Doss Having known of endophyte effects on biologicaland Welty, 1995 and etDoss al., 1998). control, they may serve as genetic sources of insectTredway et al. (1999) used amplified fragment resistance and tolerance to biotic stresses. length polymorphism (AFLP) analysis to estimate phylogenetic relationships amongEpichloe and Genetic diversity of endophytes based on molecular Neotyphodium fungi and observed that clades of markers: Advances in molecular biology including Epichloe festucae correspond to the host species from those for molecular hybridization, cloning, restrictionwhich they have been isolated. This may be due to endonuclease digestions, protein profiling and nucleiccoevolution in grass–endophyte combinations. Schardl acid sequencing have provided many new tools for theand Siegel (1993) posed that if co-evolution is faithful, investigation of phylogenetic relationship particularlythe branching order of the endophytes on the phyloge- amongst microorganisms like endophytes. Dependingnetic tree should correspond to the branching order of on the information required, the most appropriate tech-the host from which they were obtained, but the result niques can be used. Lauchtmann and Clay (1990) showedused discrepancy in this theory. Most of this dis- isozyme variation for grouping of endophyte crepancyfrom pertains toEpichloe typhina biotypes that Epichloe and asexual derivatesNeotyphodium ( ). The can cross-inoculate several grass species. electrophoretic profiles did not reveal much difference Microsatellites or simple sequence repeats (SSR), between sexually and asexually reproducing endo-have been identified in endophytic fungi that the core phytes, but endophytes from different populationssequence and of these repeats consists of (CA)n, (GA)n, different host species showed distinct enzymatic band-(CAA)n, (GAA)n, or (ATC)n. Primers targeted to ing patterns. Multibanded electromorph was observed flanking sequences of these microsatellites have been that might be due to multiple copies of the gene withconstructed that can be used for identification of endo- different allozymes via heterokaryosis and/or phytesaneu- both in culture and in plants (Moonet al ., 1998 ploidy (Lauchtmann and Clay, 1990). Such conclusionand Van Zijll de Jonget al., 2003). Presence of multi- has been resulted in a hypothesis that some endophytesple bands in some samples has confirmed the hybrid have been evolved from hybridization between twonature of the endophytes. Meanwhile the presence of separate species (Tsaiet al., 1994). They realizedmore that than one band may be due to coexistence most tall fescue endophytes have two or three distinctbetween two or more endophytes as shown (Fig. 2) for copies ofβ -tubulin tub-2( ) gene, whileEpichloe iso- amplification with ITS (Internal Transcriped Spacers) late and meadow fescue endophyteNeotyphodium ( primers (Khayyam Nekouei, 2001). These primers can uncinatum) had only onetub-2 gene. It is theorizedbe attached to the conserved sequences ofrrn locus that endophytes when coexist in a single host can beand amplify the internal transcribed spacers of this vegetatively fused and produce a new species afterregion. subsequent events such as inter-chromosomal inter- Recently, a genomics program has been focused on change and deletion of extra chromosomes. EST (Expressed Sequence Tagged) ofNeotyphodium Christensen et al. (1993) also used isozyme analysisendophytes. to The aim of this program is to discover the discriminate endophytic isolates and found taxonomicgenes contributing in host colonization, nutrient sup- grouping peculiar to any of three grass species,F. ply to the endophytic fungus, biosynthesis of active arundinacea ,F. pratensis and L. perenne. secondary metabolites and physiological mechanisms Beside biochemical markers, PCR–based markers leading to enhanced plant vigor and tolerances to have been used to distinguish and detect endophyticstresses (Spangenberget al., 2001; Van Zijll deet Jong fungi of grasses. Arbitary and specific primers haveal., 2002). More information may help about been designed to detect endophyte purely or withingrass/endophyte association to use this system to host–endophyte DNA mixture (Ganjali et al., 2004).increase plant performance and improve grass toler- Liu et al. (1995) showed that a decamer primerance to biotic and abiotic stresses (Spangenberget al., (5´–CCGAGGTGAG-3´) amplifies various fragments 2001).

78 Mirlohi et al. another transformation experiment. They used green fluorescent protein gene gfp() to expressed it in Neotyphodium lolii, as an endophyte Loliumof perenne. They also used PEG solution as proposed by Murray et al. (1992), but the efficiency of transforma- tion was lower than Murray’s report. Expression gfpof could be visualized by epifluoresence microscope. With transfer of this gene there was no need to add substrate, which is required for β-glucuronidase (GUS) activity (Mikkelsenet al., 2001). The transformation works of Murray and co-work- Figure 2. PCR amplification product obtained by using ITS1 anders provided the idea of utilization of these microor- ITS4 primers from tall fescue genotypes containing more than one endophyte (lane 1 and lane 3) (Khayyam Nekouei, 2001).ganisms as a surrogate carrier for exotic genes instead of plant genomes. The importance of this system basi- cally relates to inability of genes inserted into endo- Transformation of endophytes by marker genes: An phytic genome to flow among plants due to cross–pol- important advancement in biotechnological applica- lination. This is due to maternal inheritance of endo- tions of endophytic fungi is the advent of transforma-phyte and inhibited gene escaping. tion systems that can deliver the novel genes into these organisms (Schardl, 1994). Methods have been devel-Gene knocking out and elimination of undesirable oped to introduce foreign genes into hyphae of endo-traits: Widespread application of endophytic fungi in phytes. These techniques have been linked with inocu-forages and agronomic crops depends on elimination lation of endophyte-free plants by transgenic ofendo- alkaloids contributing to mammalian toxicity. phytes. Such inoculation technique was firstly report-Among the approaches suggested for remedial treat- ed by of Latch and Christensen (1985). They mentplaced of livestock toxicity, genetic alteration of the fun- endophyte mycelium into the coleoptile tissue of ster-gus plays an important role in elimination or reduction ile seedling growing on water agar (Latch andof toxic factors (Tsaiet al., 1992). Two strategies have Christensen, 1985; Johnson-Cicaleseet al ., 2000 andbeen suggested for this purpose. The first one relies on Bouton et al., 2002). Other technologies consistdisruption of of effective genes involved in biosynthetic infection of somatic embryos (Kearneyet al ., 1991)pathway (Panaccioneet al., 2001 and Wang et al., and inoculation of callus culture (Johnsonet al., 1986).2004). In another strategy, scientists are looking for The first method has been widely used mainly due microorganismsto (bacterial or fungal) that can symbiot- its independent nature to callus formation. ically metabolize toxic agents (Panaccioneet al ., Murray et al. (1992) conducted the first experiment2001). In the first strategy, it was used a gene fragment to produce transformed endophyte. They utilizednamely CP. 605 fromClaviceps purpurea and found a Neotyphodium sp. strain 187 BB that could not pro-similar peptide synthetase geneNeotyphodium in sp., duce neurotoxin lolitrem B in perennial ryegrass. Theyendophytic fungi ofLolium perenne. The hph gene obtained protoplasts of fungus by means of Novozymwas inserted into this fragment and used for transfor- 234 application and used a buffer containing a 40%mation ofNeotyphodium sp. Knockout construct was polyethylene glycol (PEG) 4000 solution and addedadded to protoplasts as had been described by Murray plasmid vectors which individually carried et al. (1992). After transformation, fungus was used hygromycin resistance hph( ) andβ -glucuronidase for inoculation of perennial ryegrass and transfor- (uidA) genes. Transformed hyphae were successfullymants, established compatible association with grass transferred to host plant as described by Latch andhost (Panaccioneet al., 2001). In this experiment,lpsA Christensen (1985).et Tsai al. (1992) alsohph used gene that is essential for ergopeptine biosynthesis in gene for transformation of Neotyphodium Neotyphodium was disrupted. Extraction of surrogate coenophialum. After protoplast production, they usedtransformed plant and detection of ergovaline by fluo- gene pulser in an electroporation system to deliverrescence HPLC showed that knockout containing sym- marker gene into competent protoplast. They conclud-biota could not produce ergovaline (Panaccioneet al., ed that transformation of endophyte does not affect2001) and some other ergot alkaloids (Panaccioneet host compatibility compared with non-transformed al., 2003). type. Mikkelsenet al., (2001) in Denmark, performedFuture Technologies by using gene replacement

IRANIAN JOURNAL of BIOTECHNOLOGY, Vol. 2, No. 2, April 2004 79 and gene disruption techniques or expression of anti-ties should expedite reaching to this goal. The use of sense RNA will be beneficial for alleviating disadvan-endophytes in agriculture, industry and medicine will tageous traits (but ecologically advantageous) and helpbe extensive and they will have an especial place in breeders use this association in agronomically impor-future biological research. tant crops. Concluding remarks: In summary, molecular tech- Future Prospects: With respect to grass species, nology with genetically modifying endophytic fungi endophytes can provide resistance to mammalian,offers approach to express exotic genes in plant host. insects and nematode herbivores as well as droughtUse of this technique removes difficulties in regenera- tolerance and improved competition and persistence.tion of genetically modified plants particularly in In the past, the selectable options for remediationrecalcitrant of species and concerns about gene flow into animal toxicities were treatment with vaccines, selec-wild species. Methods available for manipulating grass tion of plant and endophyte genotypes, which wereendophytes provide an efficient technique for exploit- low in toxic alkaloids, or necessarily eradicationing of novel traits in indigenous plant species. Also it is endophyte. Nowadays, genetic manipulation of endo-conceivable to isolate the endophytic genes responsi- phyte can produce infected grass cultivars with stress ble for desirable traits and transfer them into plant resistance and insect deterrence, without affecting ani-genome. However, reaching to this goal we need to mal life (Porter, 1994). Endophytes may improveprovide our knowledge of biochemical and physiolog- unique sources of natural plant growth regulators, ical basis of endophyte-host interactions based on insecticides, herbicides and pharmaceutical useful present biodiversity of endophytes. Unfortunately, this compounds (Strobel, 2003). Production of anti-tumor,knowledge is still superficial and neither biochemical anti-cancer agents such as the leucinostatins from pathways nor endophyte biodiversity are well under- some Acremonium endophytes has been reported stood. Toxic effect of endophyte on mammalian con- (Strobelet al., 1997). Also ergot alkaloids with medic-sumption is another problem that solutions for elimi- inal applications and several unknown antibiotics havenation of this unwanted trait have been suggested. been found (Porter, 1994; and Mantle, 1987),Overall, but promising future for wide use of endophyhes unfortunately, progress in this respect is very slowin and different branches of science is predicted. research may reveal an endless treasure trove of medicinal agents. Acknowledgment Endophytes can also have industrial applications Thanks are given to the head of Fozveh research station of (Stone et al., 2000). For example they can produceIsfahan Research Center of Natural Resources and Animal enzymes such as xylanase βand- gluconase (Sotuet Science for supporting the germplasm collection. The al., 2002; Moyet al ., 2002) probably useful inauthors food are also grateful to the following people for providing and animal feed industries. Furthermore, somegermplasm: Mr. M. Jarrah, Isfahan Park Organization, Isfahan; Mr. M. Modarres, Isfahan Pakan Bazr Seed Co., Acremonium species have genes encoding phenol oxi-Isfahan; Mr. S. Azadi, Isfahan Research Center of Natural dase that can be used for production of enzymaticResources, Isfahan and R. Mohammadi, Isfahan University bleach (Gouka et al., 2001). of Technology. Endophytes transformed with exogenous DNA can be used as surrogates for transgenic grass plants. TheyReferences may encode insecticide delta toxins fromBacillus thuringiensis strains or proteinase inhibitors (Schardl,Bacon CW. (1995) Toxic endophyte-infected tall fescue and 1994). Endophytes transformed with fungicide and range grasses-historic perspectives.J Animal Sci. 73: 861-870. herbicide resistance genes will have ecologicallyBacon CW and De Battista J. (1991) Endophytic fungi of advantages for host plant and themselves. It may begrasses. In:Hand Book of Applied Mycology, Vol. 1. possible to isolate some of genes responsible for insectSoil and Plants, D. K. Avora, ed. (Marcel Dekker, New or nematode resistance from endophyte and introduce York), PP. 231-256. them into plant genome. These genes may be alteredBacon so CW, Richardson MD and White Jr JF. (1997) that to be more effective or have broader range for pro- Modification and uses of endophyte-enhanced turf tection. grasses: a role for molecular technology.Crop Sci. 37: Because of their importance for human, preserva- 1415- 1425. tion of these microorganisms is worth considering.Ball OJP, Bernard, EC and Gwinn, KD. (1997) Effect of selected Neotyphodium lolii isolates on root knot nem- Making scientists informed about endophyte proper-

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