REGULAR ARTICLE

Alleviation of salinity stress in plants by endophyticplant-fungal symbiosis: Current knowledge, perspectivesand future directions

Sneha Gupta & Martino Schillaci & Robert Walker &Penelope M. C. Smith & Michelle Watt & Ute Roessner

Received: 11 February 2020 /Accepted: 22 June 2020# The Author(s) 2020

Abstract Salinization of soil with sodium chloride ionsinhibits plant functions, causing reduction of yield ofcrops. Salt tolerant microorganisms have been studied toenhance crop growth under salinity. This review de-scribes the performance of endophytic fungi applied tocrops as a supplement to plant genetics or soil manage-ment to alleviate salt stress in crops. This is achieved viainducing systemic resistance, increasing the levels ofbeneficial metabolites, activating antioxidant systemsto scavenge ROS, and modulating plant growth phyto-hormones. Colonization by endophytic fungi improvesnutrient uptake and maintains ionic homeostasis bymodulating ion accumulation, thereby restricting thetransport of Na+ to leaves and ensuring a low cytosolicNa+:K+ ratio in plants. Participating endophytic fungienhance transcripts of genes encoding the high AffinityPotassium Transporter 1 (HKT1) and the inward-rectifying K+ channels KAT1 and KAT2, which playkey roles in regulating Na+ and K+ homeostasis.Endophytic-induced interplay of strigolactones playregulatory roles in salt tolerance by interacting with

phytohormones. Future research requires further atten-tion on the biochemical, molecular and genetic mecha-nisms crucial for salt stress resistance requires furtherattention for future research. Furthermore, to designstrategies for sustained plant health with endophyticfungi, a new wave of exploration of plant-endophyteresponses to combinations of stresses is mandatory.

Keywords Endophytic fungi . Biochemical changes .

Ionic homeostasis . Osmoregulation . Hormones .

Salinity . Roots . Soil . Inoculants .Microorganisms

Soil salinity affects agriculture globally

The beginning of the 21st century has been marked byglobal scarcity of water resources, increased environ-mental pollution and salinization of soil and fresh water.Two major threats for agricultural sustainability areincreased human population and reduction in arableland available for crop cultivation (Shahbaz andAshraf 2013). Several environmental stresses such ashigh winds, extreme temperatures, drought, salinity andflood have impacted on the production and cultivationof agricultural crops. Among these, soil salinity is one ofthe most significant environmental stresses resulting inmajor reductions in cultivatable land area, and decreasedcrop productivity and quality. It is estimated that 50% ofall arable land will be impacted by salinity by 2050(Shrivastava and Kumar 2015) and that globally, soilsalinity results in more than US$12 billion in annuallosses due to reduced crop productivity (Jägermeyr and

https://doi.org/10.1007/s11104-020-04618-w

Responsible Editor: Boris Rewald

S. Gupta (*) :M. Schillaci :R. Walker :M. Watt :U. RoessnerSchool of BioSciences, University of Melbourne, Parkville, VIC,Australiae-mail: snehagupta14@gmail.com

P. M. C. SmithCentre for AgriBiosciences, Department of Animal, Plant and SoilSciences, School of Life Sciences, La Trobe University,Bundoora, VIC, Australia

/ Published online: 9 July 2020

Plant Soil (2021) 461:219–244

http://crossmark.crossref.org/dialog/?doi=10.1007/s11104-020-04618-w&domain=pdf

Frieler 2018). Salinity is recognized as the main threat toenvironmental resources in several countries, affectingalmost 1 billion ha worldwide, which represents about7% of the earth’s continental area (Shrivastava andKumar 2015). Consequently, it is important to under-stand the crop responses to this major soil and plantstress to minimize economic loss and improve foodsecurity.

Soil is defined as being saline when the electricalconductivity (EC) of the saturation extract (ECe) in theroot zone exceeds 4 dSm− 1 at 25oC and has an ex-changeable sodium of 15% (w/v). Salinization also in-cludes excessive accumulation of ions such as calcium(Ca2+), magnesium (Mg2+), sodium (Na+), sulphates(SO4

2−), and chlorides (Cl−) in the soil, inhibiting plantgrowth and cellular functions. The most abundant ion inmost salt-affected soils is Na+ and hence the exchangephase is dominated by Na+. A secondary process oftenassociated with saline soils is alkalinisation, creating acondition known as sodicity. This results in the degra-dation of soil physical properties and porosity, leadingto reduced water and air flow and increased soil hard-ness and crusting.

Apart from affecting soil physical properties, highsoil salinity directly and adversely affects plants- bothnative vegetation and introduced crops, severely affect-ing seed germination, root growth, and the physiologicalfunctions of crops (Oster and Jayawardane 1998). It hasbeen estimated that worldwide 20% of total cultivatedand 33% of irrigated agricultural land is affected by highsalinity. This is mainly due to the toxicity of the salt ionsdirectly on the plant cells but also through generalosmotic effects of the soil around the roots of the plant.High osmotic potentials at the soil-root interface reducethe ability of the plant to absorb water from the soil(Machado and Serralheiro 2017).

Native plants have evolved mechanisms to toleratelow rainfall and high salinity over hundreds of thou-sands of years (Steffen et al. 2009). However, in the past200 years, human activities have intensely disrupted thenatural hydrological balance in many regions of theglobe. This has resulted in significant consequencesfor the distribution of salt in all landscapes leading tosevere degradation of both natural and agricultural en-vironments. It is predicted that the total area of landaffected by salinity will increase drastically over thenext few decades if effective solutions are not imple-mented. These solutions would involve significantchanges to our present systems of management

including research and development of strategies toimprove salt tolerance in crops and improve mecha-nisms to mitigate its consequences (Rengasamy 2002,2006).

Effects of salt stress on above-groundand below-ground organs of plants

Plants have two major systems, the above-ground or-gans (shoots) and below-ground organs (roots). Eachsystem has morphological, physiological and anatomi-cal differences that affect plant performance differently(Gregory 2007). However, while these two systemsgrow and function as a separate site for the uptake ofnutrients and other resources, they are coupled, and theirfunctions need to form an integrated system. The above-ground system is highly dependent on the developmentof below-ground organs and without a sufficiently de-veloped root system, the above-ground system cannotfully mature (de Willigen and van Noordwijk 1987).

Salinity limits vegetative and reproductive develop-ment by inducing physiological dysfunctions, and thishas profound implications on different harvested organssuch as leaf, stem, root, shoot, fruit or grain. The com-plex phenomenon of tolerance and response to salt stressinvolves dynamic changes in growth, physiology, met-abolic pathways and gene expressions (Atkinson andUrwin 2012; Munns and Tester 2008). Strategies usedto mitigate against salt stress include proline accumula-tion within cells (Matysik et al. 2002), modulation ofhormones and accumulation of glycine betaine andpolyols (Gupta and Huang 2014). They also involvegeneration of nitric oxide (NO) and compounds to com-bat formation of reactive oxygen species (ROS). NOdirectly or indirectly triggers expression of severalredox-regulated genes. NO also reacts with lipid radicalsthus preventing lipid oxidation, exerting a protectiveeffect by scavenging superoxide radicals and formationof peroxynitrite that can be neutralised by other cellularprocesses. NO also helps in the activation of manyantioxidant enzymes including catalase (CAT), ascor-bate or thiol-dependent peroxidases (APX), glutathionereductases (GR) and superoxide dismutase (SOD).

The effect of salinity on leaf growth, biomass pro-duction and grain yield on several crops are well docu-mented (Hasanuzzaman et al. 2013; Munns et al. 2011;Munns and Tester 2008; Sun et al. 2014). The extent towhich plants are damaged by salinity depends on several

220 Plant Soil (2021) 461:219–244

factors including species, genotype, plant growth phase,ionic strength, duration of salinity exposure, composi-tion of salinizing solution and, most importantly, whichplant organ is exposed (Robin et al. 2016).

Munns (2005) hypothesized that salinity damage inplants occurs in two temporal phases. The first phase ofgrowth reduction occurs rapidly after exposure and isdue to an osmotic effect, while the second phase, whichis a slower process, is due to the accumulation of saltions, mainly in older leaves. Early symptoms of thesecond phase of growth reduction include damage toold leaves and a reduced photosynthetic capacity(Munns et al. 2006). At the plant organ level, shootshave been demonstrated to be more sensitive to salinitythan roots (Munns and Tester 2008). However, roots areexposed to salinity stress before leaves and can respondrapidly through changes in elongation (Rahnama et al.2011) and function (Shelden et al. 2016). The roots arecrucial for a myriad of physiological processes includ-ing water and nutrient uptake, preventing toxic sub-stances from reaching photosynthetic tissue, signal ex-change with shoots, anchoring of plants, and providingmechanical support to the above-ground organs.

The root-soil interface/ The rhizosphere

Roots and their growing substrate are intrinsically con-nected, and they mutually influence each other at allstages of plant life (Gregory 2006). The interface be-tween roots and the soil is a complex and often ill-defined zone. Compounds are released from roots intothe surrounding soil matrix resulting in changes to itschemical and physical properties. The narrow zone ofsoil that surrounds and is influenced by plant roots isknown as the rhizosphere. The term rhizosphere wasfirst defined over a century ago by Hiltner (1904) andrecently, redefined by Pinton et al. (2007) as the mostdynamic interface on earth that includes soil influencedby the root, along with the root tissues. The rhizosphereis home to a vast number of microorganisms (Morganet al. 2005; Pinton et al. 2007), and consists of threedistinct zones: (a) the endorhizosphere, which includespart of the cortex and endodermis in which microbesoccupy the apoplastic space; (b) the rhizoplane, which isthe medial zone immediately next to the root consistingof the root surface and mucilages; and (c) theectorhizosphere, which extends from the rhizoplaneout into the bulk soil (Lynch 1990).

The root system architecture is greatly influenced bysoil conditions (Rich andWatt 2013), including nutrientgradients and concentrations of nitrate and phosphorus(Ho et al. 2005; Paterson et al. 2006). Roots also affectthe surrounding nutrient composition by the release oforganic compounds that play a vital role in mineralizingnutrients. The compounds released from the roots intothe surrounding soil are generally part of rhizodeposits(Jones et al. 2009), which include a range of substancesfrom sloughed-off root cells and tissues, mucilages,volatiles, and soluble lysates and exudates from dam-aged and intact cells (Curl and Truelove 1986; Dakoraand Phillips 2002; Watt 2009). Abiotic factors influencethe root system (Bekkara et al. 1998; Brimecombe et al.2000; Groleau-Renaud et al. 1998; Watt and Evans1999) with roots responding by secreting a differentcombination of compounds to protect against negativeeffects and encourage positive microbial interactions(Badri and Vivanco 2009). These secreted compoundsusually induce an interactive metabolic cross-talk in-volving diverse biosynthetic networks and pathways.

Root exudates include both secretions (includingmucilage) that are actively released from the root anddiffusates which are passively released because of os-motic differences between soil solution and the rootcells (McNear 2013). Inorganic root exudates includeions, water, ubiquitous H+ and electrons. Although theconcentration of inorganic compounds make up far lessof the root exudate composition compared to organiccompounds but their role is still significant (Khorassani2008; Uren 2000). Organic compounds can be classifiedinto high molecular weight compounds, such as com-plex molecules including polysaccharides secreted byroot cap cells and epidermal cells at the apical zone, andlow molecular weight compounds that include arabi-nose, fructose, glucose, amino acids, organic acids,plant hormones and phenolic compounds (Bertin et al.2003). Due to the richness of inorganic and organiccompounds in rhizodeposits, the rhizosphere is hometo specialised microbes that are able to utilise thesecompounds as an energy source.

Several recent and comprehensive reviews have beenwritten covering the diversity and activity of microor-ganisms at within roots and in the rhizosphere, as well asthe functions and effects of microorganisms in nutrientturnover and supply to the plant (Garcia et al. 2016;Jacoby et al. 2017; Smith and Smith 2011; Udvardi andPoole 2013). In the following section, the use of micro-organisms as one of the key approaches used to alleviate

221Plant Soil (2021) 461:219–244

abiotic stresses, with the focus on using fungi as a majorbeneficial microbe will be discussed.

Alleviating salt stress by association with endophyticfungi

Diverse metabolic and genetic strategies used by plant-associated microbes can reduce the impact of salt stressand other abiotic stresses arising from extreme environ-mental conditions (Gopalakrishnan et al. 2015; Singh2014). Induced Systemic Tolerance (IST) is the termused to describe microbe-mediated induction of abioticstress responses (Meena et al. 2017). In these beneficialsituations, rhizosphere microorganisms not only per-ceive and respond to signal molecules secreted by plantroots, they also release diverse signalling molecules thatinfluence plants, resulting in increased biotic and abioticstress resistance or tolerance, as well as root develop-ment and plant growth (Zhang et al. 2017a). Microbialinteractions with plants induce several local and system-ic responses that improve the metabolic capacity ofplants to respond to salt stress (Nguyen et al. 2016).This microorganisms-based plant biotechnology hasproven to be more efficient in many cases than plantbreeding and genetic modification approaches (Smith2014).

Beneficial effects due to plant root interactionswith endophytic fungi

In recent years the ability of mycorrhizal fungi to inducetolerance against salt stress in crops has been documented(Gangwar and Singh 2018) (Fig. 1). In a mycorrhizalassociation, the fungus colonizes the host plant’s roottissues, either intracellularly as in arbuscular mycorrhizalfungi (AMF), or forms extracellular exchangemechanismsoutside of the root cells, as in ectomycorrhizal fungi. Thus,mycorrhiza fungi can be categorised as endo- inside planttissue, or ecto- associated with the external rhizosphere ornot penetrating root cells. For the purpose of clarity, thisreview will only focus on endomycorrhizal (termed asendophytic for this review) fungi.

Penetration and colonisation of plant roots appears tobe essential for some endophytic fungal strains that arereported to promote plant growth and provide protectionagainst pathogens. For example, some species belong-ing to the genus Trichoderma can colonize local sites(Metcalf and Wilson 2001) on roots, mediated by

hydrophobins- (Viterbo et al. 2004) and expansin-likeproteins (Brotman et al. 2008) present in the outermostcell wall layer that coats the fungal cell surface. Otherrhizosphere-competent Trichoderma spp. colonize en-tire root surfaces for long periods of time (Harman 2000;Thrane et al. 1997) or penetrate the epidermis and thecortex (Yedidia et al. 1999). Once hyphae penetrateroots, a series of fungal bioactive compounds can beproduced inducing plant biochemical mechanisms(Harman 2006). The callose-enriched wall appositionsin the root cell limit the growth of the Trichoderma spp.to a small area (epidermis and cortex), preventing theentry of Trichoderma spp. into the vascular stele(Hermosa et al. 2012; Yedidia et al. 1999). Arbuscularmycorrhiza fungi (AMF) are another group of endo-phytic fungi. Their hyphae penetrate plant cells, produc-ing structures that are either balloon-like (vesicles) ordichotomously branching invaginations (arbuscules) asa means of nutrient exchange. The fungal hyphae do notin fact penetrate the protoplast (i.e. the interior of thecell), but invaginate the cell membrane. Dark septateendophytic (DSE) fungi are also root endophytes, char-acterized by intense dark pigmentation and the forma-tion of septate and melanized hyphae and occasionallymicrosclerotia (Knapp et al. 2015; Yuan et al. 2016).They can be found in plant cortical cells inter- andintracellularly and are present in several environments(Li et al. 2019; Santos et al. 2017). In contrast to the vastinformation on AMF, information on the role of DSEfungi in the ecosystem is limited.

Colonization of several crops with endophytic fungihas been reported to induce systemic resistance to path-ogens, mitigate stress by increasing the levels of protec-tive metabolites and osmoprotectants, activate antioxi-dant systems to prevent damage caused by ROS, de-creasing salt induced root respiration and modulate thephytohormone profile tominimize salt effects on growthof plants (Ghaffari et al. 2016; Jogawat et al. 2013; Liet al. 2017; Nia et al. 2012; Rewald et al. 2015; Zhanget al. 2019a). These effects are in coordinated to im-prove plant growth and resilience to salinity stress.These ameliorative effects can be evaluated in terms ofimproved plant growth exhibited by endophyte colo-nized (ENC) plants in comparison to non-endophytic(NENC) colonized plants.

Salinity triggers a decrease in stomatal conductance,thus decreasing the CO2:O2 ratio and increasing photo-respiration (Kangasjärvi et al. 2012). This causes anincrease in stomatal resistance to transpiration and an

222 Plant Soil (2021) 461:219–244

increase in the rate of tissue respiration. Under theseconditions, photosynthetic capacity is limited, and theplant uses its own photo-assimilates, resulting in de-creased growth. Rewald et al. (2015) showed that inNENC Ulmus glabra seedlings there was a significantincrease in fine root respiration under salt stress ascompared to their ENC counterparts. This suggestedthat colonization by endophytic fungi can prevent amajor increase of root respiration under moderate NaClstress, enabling trees to deploy more assimilated C forgrowth and, theoretically, improve defence mechanismsagainst other stress factors occurring in urbanenvironments.

Endophytic fungi are effective against several rootdiseases (Azcón-Aguilar and Barea 1997; Borowicz2001) and impart stress tolerance to plants (Duc et al.2018; Evelin et al. 2019; Yasmeen et al. 2019),but canalso enhance susceptibility to biotrophic leaf pathogens(Gernns et al. 2001; Waller et al. 2005). These endo-phytes have been frequently reported to not only protectagainst plant pathogens and pests but also impart strongtolerance against several abiotic stresses in crops(Gangwar and Singh 2018).

In the past decade, significant progress has been madeto understand several mechanisms of salt toleranceimparted by endophytic fungi. In the following sections,

Fig. 1 Potential beneficial effects of root colonisation of plants byendophytic, symbiotic fungi in saline soil conditions, summarisedfrom the literature. Salinity results in reduced root biomass due tosalt-induced inhibition of cell division and affect the total biomassyield (1) (left). Plant colonized with endophytic fungi improvesbiomass accumulation by modifying root architecture and in-creased nutrient absorption (1a) (right). Salt accumulation createscompetition for nutrient uptake and transport. This results inimbalance of the ionic composition of plant, affecting plant’sphysiological traits (2) (left). Endophytic fungi improve expres-sion of genes and upregulate several cation transporters, resultingin improved nutrient uptake and maintenance of ionic homeostasis(2a) (right). Increase of salt in soil lowers soil water potential

resulting in cellular dehydration (3) (left). Endophytic fungi negatethis effect by mediating accumulation of osmolytes consequentlyimproving plant’s water status (3a) (right). Increasing salinitycauses oxidative stress due to imbalance in reactive oxygen spe-cies generation and quenching activities of antioxidants (4) (left).Endophytic fungi improve the antioxidant systems of plants re-ducing oxidative stress under salt stress (4a) (right). Salt stresshinders photosynthesis by reducing uptake of magnesium anddecreasing chlorophyll concentration which eventually reducescarbon dioxide supply to RuBisCo (5) (left). Endophytes have apositive effect on photosynthesis under salt stress (5a) (right). Seetext for relevant references and further details

223Plant Soil (2021) 461:219–244

current understanding of biochemical and physiologicalchanges that occur in salt stressed plants inoculated withendophytic fungi will be covered. This will include ad-vances made recently toward better understanding of themechanisms that contribute to salt stress alleviation inENC plants. Finally, gaps in our understanding of themechanisms will be identified and research challenges tobe met in future studies will be discussed.

Mechanisms of salt tolerance in ENC plants

Increase in total biomass

Total biomass is usually evaluated as an indicator of theplant’s ability to tolerate salinity. Several studies havehighlighted that endophytic fungi impart salinity tolerancein host plants by virtue of higher biomass as compared toNENC plants. Endophytic fungus colonization has beendemonstrated to increase biomass in Zea mays L. (Rhoet al. 2018), soybean (Hamayun et al. 2017), Vochysiadivergens Pohl (Farias et al. 2019), Solanum lycopersicum(Azad and Kaminskyj 2016), Brassica juncea (Ahmadet al. 2015), Oryza sativa L. (Saddique et al. 2018) and,Triticum aestivum L. (Zhang et al. 2019b).

The total biomass can also be assessed by measuringplant relative growth rate (plant weight increment perplant weight unit). This includes measurement of the netassimilation rate (NAR) (the increase in plant weight perleaf area unit), the leaf area ratio (LAR) and root relativegrowth rate (RGRplant). Balliu et al. (2015) investigatedthe effects of commercially available AMF inoculant(Glomus sp. mixture) on growth and nutrient acquisitionin tomato (Solanum lycopersicum L.) plants grown inmedia with different levels of salinity. Salinity stressimmediately and significantly reduced the LAR, NARand RGRplant in NENC as compared to ENC plants.Similarly, Sallaku et al. (2019) showed that AMF alle-viates the salinity stress in cucumber plants by extendingtheir root length and root surface area and even morethrough enhancing their photosynthetic rate (NAR) ascompared to NENC plants.

Alteration of root architecture

Root branching and root system architecture play asignificant role in determining the composition of exu-dates (Badri and Vivanco 2009). Changes in the rootsystem architecture for regulating salt acquisition and

translocation are crucial for enhancing plant resistanceto salt stress (Jung and McCouch 2013). Barley plantsexperienced a decline in primary root growth undersaline conditions due to salt-induced inhibition of celldivision and elongation of root epidermal cells, whilesimultaneously stimulating lateral root development(Rahnama et al. 2011). Endophytic fungi can modulatethe plant’s ability to modify root architecture (Salope-Sondi et al. 2015; Vahabi et al. 2016). Yun et al. (2018)observed that the length and volume of roots weregreater in ENC than in NENC maize plants under salineconditions and similar observations have been reportedin Hordeum vulgare (Waller et al. 2005) and Oryzasativa L. (Kord et al. 2019). Improved root systemsenable the plant to utilize water and minerals fromnon-saline areas until exploitation of areas affected bysalt cannot be avoided (Jogawat et al. 2013). Thoughfew studies have shown the ability of endophytic fungito alter root architecture under saline conditions forbeneficial purposes, much remains to be investigatedon endophytic fungi influenced root architecture forbetter water and nutrient uptake in saline conditions.

Osmoregulation

Upon exposure to saline environments, plants undergo areduction in water absorbing capacity from the soil,disrupting cell water relations and inhibiting cell expan-sion. In order to negate these effects, plants employosmoregulation as a mechanism to tolerate salt stress(Munns and Tester 2008). This is achieved by accumu-lation of osmolytes in the form of proline, glycine beta-ine, sugars, organic acids, polyamines and amino acidscontributing to osmotic adjustment (Hasegawa et al.2000). These osmolytes, often termed as compatiblesolutes, are organic compounds of lowmolecular weightthat are water soluble and non-toxic at high concentra-tions (Chen and Murata 2011).

Under salt stress, ENC plants have been shown topossess higher osmotic potential than NENC plants(Contreras-Cornejo et al. 2014) due to accumulation ofosmolytes (Ahmad et al. 2015; Song et al. 2015) (Fig.2). Osmolytes are also involved in quenching reactiveoxygen species (ROS), maintaining membrane integri-ty, and stabilizing enzymes. Osmolytes are also de-scribed as osmoprotectants (Azad and Kaminskyj2016; Li et al. 2017). Endophytic symbiosis can influ-ence the concentration and profile of polyamines andorganic acids in plants (Chen et al. 2019; Zhao et al.

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2014). Polyamines help retain ion homeostasis in plantcells by enhancing the uptake of nutrients and water(Pang et al. 2007). Organic acids may increase theavailability of nitrogen, phosphorus and potassium (N,P and K) in soil (Samolski et al. 2012). The role ofspecific osmolytes in improving salt tolerance is ENCplants are discussed below.

Proline

Proline is one of the most common osmoprotectants thataccumulates in plants during salt stress, thereby

ameliorating the negative effects of salinity. Prolinehas been observed to protect cell walls under osmoticstress, protect protein integrity and to increase enzymat-ic activity by acting as a molecular chaperone. Prolinealso has a role in scavenging ROS and shows singletoxygen quenching ability (Kaur and Asthir 2015). De-spite these benefits, there are conflicting reports on therole of endophytic fungi in proline accumulation in saltstressed plants. Several studies reported increases inproline contents in ENC plants compared to NENCplants, while others have reported lower proline contentsin ENC plants (Table 1). Higher proline content in ENC

Fig. 2 Salinity stress induced osmotic stress tolerance mecha-nisms in plants. Increase in salt in soil lowers the soil waterpotential of plant cells. This reduces water uptake by plants andconsequently causes cellular dehydration (1) (left). To combat thisissue, plants accumulate osmolytes, such as proline, sugars andpolyamines in higher concentration. Osmolyte accumulation re-sults in lowering of cellular water potential and maintains afavourable gradient for water uptake from soil to roots. Endophytic

fungi alleviate osmotic stress by influencing the expression ofspecific genes, P5CS, pyroline-5-carboxylate synthase (1a) (right),involved in the biosynthesis of the osmolyte proline, activation ofstarch degrading enzyme, glucan-water dikinase (1b) (right) andforming tripartite symbiosis with roots and rhizobia (1c) (right) toelevate the accumulation of sugars and by increasing the biosyn-thesis of polyamines such as spermidine and spermine (1D) (right).See text for relevant references and further details

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Tab

le1

Examples

ofstudieson

effectsof

saltstress

andendophyticfungio

nosmoticregulatio

nin

plants

S.N

o.Saltlevel

(mM

NaC

l)Plant

Fungus

Parametersassessed

Effectsof

References

Salin

ityEndophytic

fungio

nsalt

stressed

plants

10,100

Zeamays

Yarrow

ialip

olytica

Shootp

rolin

econtent,total

flavonoid,totalp

henolics,

phytohormoneanalysis

Increased

Controlledtheproductio

nof

proline

Janetal.(2019)

20,100,

200,300

Hordeum

vulgare

Epichloë

brom

icola

Free,solubleconjugated

and

insolubleboundform

sof

polyam

ine(prolin

e),

putrescine,sperm

idineand

sperminecontent

Increasedproline

Proline,Sp

ermidine,total

spermine-

increasedunder

higher

stress

conditions,

Putrescine,freeform

ofspermine-

significantly

decreasedathigher

salt

treatm

ents

Chenetal.(2019)

30,50

Solanum

lycopersicum

Pirifo

rmospora

indica

Shootp

rolin

econtent

Highlyincreased

Significantly

reduced

Abdelazizetal.(2019)

440,100,

175,250

Medicago

truncatula

Pirifo

rmospora

indica

Shootp

rolin

econtent

Contin

ually

enhanced

inlin

ewith

theincreased

saltconcentration

Significantly

increasedthan

un-colonized

plants

Lietal.(2017)

50,100,200

Brassicajuncea

Trichoderm

aharzianum

Oilandprolinecontent,

pigm

ents,enzym

aticassay

Increasedwith

maxim

umaccumulationof

59.12%

at200mM

NaC

l

Furtherincrease

to70.37%

(Ahm

adetal.2015)

60,150

Triticumaestivum

Trichoderm

alongibrachiatu-

m

Water

contentinleaves

and

roots,chlorophyllcontent,

shootp

rolin

econtent

Increased

Highestincrease

win

plants

pretreated

with

fungus

under150mM

NaC

lstress

Zhang

etal.(2016)

70,70,

150,240

Oryza

sativa

Fiveisolates

ofTrichoderm

asp.

Leafwater

content,

chlorophyllcontent,prolin

econtent,mem

brane

stability,lipid

peroxidatio

nandexpression

ofstress

relatedgenes

Increased

Furtherincreased

Raw

atetal.(2016)

80,100,200,

300,400,

500

Triticumaestivum

Pirifo

rmospora

indica

Totalbiom

ass,photosynthetic

pigm

ents,com

patib

lesolutes

Increased

Furtherincreased

Zarea

etal.(2012)

226 Plant Soil (2021) 461:219–244

plants has been attributed to – (i) favouring a decline inionic influx inside cellular masses thus helping plants tomaintain their osmotic balance; (ii) increasing the ex-pression of the gene encoding Pyrroline-5-carboxylatesynthase (P5CS) enzyme which is involved in prolinebiosynthesis; and (iii) increasing activity of the P5CSenzyme (Rawat et al. 2016). Besides its role as anosmolyte proline can act as a stress marker. In ENCtomato plants, proline accumulation was reduced whenthe toxic effects of salinity were reduced followingcolonization of an endophytic fungus, Piriformosporaindica (Abdelaziz et al. 2019).

Sugars

In salt stressed plants, the accumulation of total solublesugars, such as glucose, sucrose, dextrins and maltose,serves as an osmoprotection as they can stabilize the cellmembrane and protoplast. These sugars also protectwater soluble enzymes from high intracellular concen-trations of inorganic ions (Liang et al. 2018). The syn-thesis of soluble sugars from starch and sucrose in plantsis upregulated by the activities of sucrose anabolizingenzymes such as α- and β-amylase, which convertstarch into dextrins and maltose, respectively (Preiss2018). Sucrose phosphate synthase and sucrose syn-thase catalyse the synthesis of sucrose, while β-fructofuranosidase catalyses the breakdown of sucroseto glucose and fructose (Peng et al. 2016). In plantsgrown under saline conditions, sucrose undergoes de-composition in order to meet the requirements for glu-cose (Munns and Tester 2008).

There have been reports that show the role of endo-phytic fungi in enhancing accumulation of solublesugars in salt stressed plants (Qi and Zhao 2013; UmaShaanker 2014; Zhang et al. 2019b). These sugars act aschemoattractant signals to soil rhizobia (el ZaharHaichar et al. 2014). These chemoattractants can directmovement to microorganisms in response to chemicalgradients- a behaviour known as chemotaxis. This che-motactic response of microorganisms to root exudatesplay key role in initiating communication between plantroots and microbes. Yang et al. (2015) reported that thecolonization by Phomopsis liquidambari could stimu-late sugar secretion from the rhizodeposition ofsloughed off cells and root debris of rice, thereby pro-viding carbon to the endophytic fungi. Another study ofP. liquidambari on peanut showed increased solublesugar contents in leaves. This was due to the ability of

the fungus to form tripartite symbiotic associations withpeanut roots and rhizobia. This tripartite associationsignificantly enhanced peanut nodulation (Zhang et al.2017b). Here, sucrose derived from photosynthesis wastransported to bacterial inoculated root nodules and washydrolysed by sucrose synthase into UDP-glucose andfructose. This was due to the allocation of more carbonby the endophyte toward peanut and rhizobia symbiontsby increased soluble sugar content, leading to moreactive nodule carbon metabolism in ENC plants.

Furthermore, Sherameti et al. (2005) also suggestedthat one of the major starch-degrading enzymes, glucan-water dikinase, activated by the fungus in colonizedroots, is responsible for the increase in soluble sugarsin ENC plants. Similar results were obtained byGhabooli (2014) with Piriformospora indica increasingthe level of soluble sugars, including glucose, fructose,and sucrose, in inoculated plants under salt stressconditions. Recently, Zhang et al. (2019a) demonstratedthat T. harzianum improved salt tolerance of cucumberseedlings by enhancing accumulation of sugars. Thisresults in adjustment of the osmotic potential for cellularwater retention and turgor maintenance, thereby mini-mizing the adverse effects of salt stress by balancing thesolute potential (Bai et al. 2019).

Organic acids

Other important osmolytes in plants are organic acidssuch as citric acid and malic acid. They are found inplant vacuoles and the regulation of their metabolismplays a crucial role in providing tolerance to salt stress(Guo et al. 2010). Fungal endophytes have been report-ed to induce the release of organic compounds by theroots (Yang et al. 2015; Zhang et al. 2014), thusinfluencing the concentrations and profile of organicacids in plants. One of the major plant nutritional disor-ders associated with increased salinity in soil is iron (Fe)deficiency. Endophytes can enhance Fe acquisition bytheir host through their ability to secrete organic acidswhich chelate and solubilise iron in the soil (Chen et al.1998; Khan et al. 2006). A study by Zhao et al. (2014)demonstrated that the release of organic acids fromendophytes, resulted in ferric solubilization to formorganic ferric salts that can be assimilated directly byplants under saline conditions. It has also been shownthat ENC plants have better nutrient uptake capacity anddistribution within plant tissues due to modulation of theroot architecture and nutrient availability in the soil.

227Plant Soil (2021) 461:219–244

These benefits are imparted by increases in organicacids produced by ENC plants (Samolski et al. 2012;Zhao et al. 2014). Limited research has been done onunderstanding the mechanisms underlying the changesin organic acids in ENC plants, thus this topic calls forfurther investigation.

Polyamines

Polyamines (PA) are low molecular weight nitrogenousaliphatic molecules that participate in physiological pro-cesses such as activation of antioxidant systems, cellgrowth and development, and in cellular osmoregula-tion in plants under salt stress (Singh et al. 2018). PAalso regulate ion channels, either by direct binding or viaPA-induced signalling molecules (ROS and NO). PAsalso regulate the activity of ion channels indirectly bymembrane depolarization. The hyperpolarization-activated Ca2+ influx and the NO-induced release ofintracellular Ca2+ result in a higher cytoplasmic Ca2+

concentration, which is a major component in generalstress responses such as stomatal movements (Wani2018; Williams 1997). They are either present in free,soluble conjugated (covalently conjugated with smallmolecules such as phenolic acids) or insoluble (boundwith macromolecules such as proteins, DNA and RNA)forms. These compatible solutes accumulate under saltstress and include putrescine (Put, diamine), spermidine(Spd, triamine) and spermine (Spm, tetramine)(Minocha et al. 2014; Todorova et al. 2013).

Differences in PA (Put, Spd, Spm) responses undersalt-stress have been reported in several species (Singhet al. 2018) and it remains unclear which polyamineplays the major role in imparting salt tolerance. Chenet al. (2019) demonstrated that the putrescine contentwas significantly reduced in ENC plants compared toNENC plants in high stress conditions whereasspermidine and spermine content showed the oppositepattern. It was suggested that salinity stress toleranceinduced by endophytic fungus Epichloë bromicola cor-related with enhanced conversion of putrescine tospermidine and spermine. The fungus also convertedthe free forms and soluble conjugated forms of poly-amines to insoluble bound forms of polyamines.

Modulation of the polyamine pool to help toleratesalt stress by arbuscular mycorrhizal fungi (AMF) iswell explored (Evelin et al. 2009). However, researchon polyamine metabolism during the interactions be-tween endophytic fungi and plants under salt stress is

underrepresented and many questions remain unan-swered. For example, most plant polyamine researchrelates to changes in free polyamines, and where poly-amine conjugates have been measured, substantialchanges have been detected. The precise role of poly-amines, free or conjugated, in ENC plants remains un-clear. Further investigations, focusing on understandingendophyte-facilitated modulation of polyamines, in-cluding the intracellular localization of free polyaminesand conjugates associatedwith salt tolerance in plants, isneeded. Already some of the key genes involved in thebiosynthetic pathways have been cloned making it pos-sible to manipulate polyamine metabolism using molec-ular genetic approaches (Malmberg et al. 1998). Hence,genetic manipulation of polyamine levels in ENC plantsmay allow valuable insights into the role of these com-pounds especially in studies of plant tolerance to saltstress.

Nutrient acquisition and ionic homeostasis

High salt (Na+ and Cl−) in the soil disturbs nutrientavailability by imposing competition during uptake,translocation or distribution within the plant. This maysuppress nutrient associated activities resulting in unde-sired ratios of Na+:K+, Na+:Ca2+, and Ca2+:Mg2+

(Munns et al. 2011). This in turn results in imbalanceamong ionic composition of the plant subsequentlyaffecting plants physiological traits (Hasegawa et al.2000; Munns et al. 2006). However, endophytic symbi-osis has been shown to improve assimilation of nutrientsand assist in maintenance of ionic homeostasis in hostplants grown in saline conditions (Table 2).

Although the effects of AM fungi on plant nutrientacquisition are commonly discussed based on the dif-ferences of nutrient concentration in plant tissues, therelative uptake rate of nutrient elements (RUR) hasrecently been suggested as a better tool to distinguishthe differences among treatments over a short period, asthe nutrient concentration could be largely influencedby the dilution effect of fast growth in young plants.Balliu et al. (2015) found that RUR values of ENCtomato plants grown in both non-saline and moderatesaline conditions were higher than in non-inoculatedseedlings. Similarly, another study showed the en-hancement effect of AMF inoculation on the nutrientuptake capacity of cucumber seedlings after salt stress(Sallaku et al. 2019).

228 Plant Soil (2021) 461:219–244

Tab

le2

Examples

ofstudieson

theeffectsof

salinity

andendophyticfungio

nnutrient

concentrationandionicratio

sin

plants

S.

No.

Saltlevel(m

MNaC

l)Plant

Fungus

Parametersassessed

Effectsof

References

Salin

ityEndophytic

fungio

nsalt

stressed

plants

10,140

Cucum

issativus

Phomaglom

erata

LWL2

and

Penicilliumsp.

LWL3

Na+,K

+,C

a2+,M

gcontent

Significantincreases

inNa+

and

decreasesin

K+,M

g2+and

Ca2

+levels

Significantly

higher

levelsof

K+,M

g2+andCa2

+ions,

particularly

incase

ofPenicillium

sp.and

P.g

lomerataandinhibitthe

uptake

ofNa+

Waqas

etal.(2012)

20,100,200

Zeamays

Pirifo

rmospora

indica

Na+,K

+content

IncreasedNa+

inrootsand

shoots,K

+in

shootsand

decreasedK+in

roots

Significantd

ecreased

levelsof

Na+

andK+in

rootsand

increase

inshoots

Yun

etal.(2018)

30,75,100

Arabidopsis

thaliana

Pirifo

rmospora

indica

Transcriptlevelsof

several

genesknow

nto

encode

proteins

involved

inNa+

and

K+homeostasisandthe

abiotic

stress

markergene

relativ

eto

DesiccationA

(RD29a)

Increased-

expression

ofthe

stress

markergene,R

D29a,

expression

levelo

fAtHKT1,

K+contentinrootsand

shoots,D

ecreased

Na+

contentinrootsandshoots

Decreased-expression

ofthe

stress

markergene,R

D29a,

furtherdecrease

inNa+

content,Fu

rtherincreased

expression

levelo

fAtHKT1,

andK+content

Abdelazizetal.

(2017)

40,100

Arabidopsis

thaliana

Trichoderm

avirens

andTrichoderm

aatroviride

Na+

content

Decreased

Na+

contentinroots

FurtherdecreasedNA+content

inroots

Contreras-Cornejo

etal.(2014)

50,150,300,450,

600

Hordeum

vulgare

Epichloe

Na+,C

,P,N

,K+content,C:N,

C:P,N

a+:K

+,N

:Pratio

sIncreasedNa+,N

,PandK+

contents,ionicratios,no

significanteffectonCcontent

FurtherincreasedN,P

andK+

contents,N

:Pratios;

Decreased

C:N,C

:P,N

a+:K

+

ratio

s

Song

etal.(2015)

60,100,300

Hordeum

vulgare

Pirifo

rmospora

indica

Na+,K

+,C

a2+,ionicratio

sIncreasedNa+,C

a2+,D

ecreased

K+:Na+,C

a2+:Na+

ratio

sIncreasedK+,K

+:Na+,

Ca2

+:Na+,D

ecreased

Na+

content

Ghabooli(2014)

70,50,100,150

Loliu

marundinaceum

Neotyphodium

coenophialum

Na+,K

+,C

a2+,and

Mg2

+

contentinleaves,rootsand

sheath

Atlow

ersaltconcentration-

inleaves,decreased

K+,sim

ilar

Na+,C

a2+unaffected;in

sheath,decreased

K+,similar

Na+andCa2

+

Ath

ighersaltconcentration-

inleaves,decreased

Na+,sim

ilar

K+,M

g2+unaffected

Atlow

ersaltconcentration-

inleaves,increased

K+,sim

ilar

Na+,C

a2+unaffected;in

sheath,increased

K+,similar

Na+andCa2

+

Ath

ighersaltconcentration-

inleaves,increased

Na+,sim

ilar

K+,

Mg2

+increasedatall

concentrations

Yin

etal.(2014)

229Plant Soil (2021) 461:219–244

Phosphorus

Phosphorus (P) and nitrogen (N) are two of the mostimportant and essential elements for plant growth withcrucial roles in cell function and metabolism (Uchida2000). Increased salt in soil occludes P to plants due toits precipitation with other cations (de Aguilar et al.1979), thereby creating soil-induced P deficiency inplants. This affects the normal growth of the plant andcauses older leaves to die prematurely (Niu et al. 2012).Increased P acquisition in ENC plants under salineconditions is attributed to (i) increased availability ofphosphates in soil due to the conversion of insolublephosphates into soluble forms through the process ofacidification, chelation and exchange reactions; (ii) abil-ity of endophytic fungi to absorb P at lower thresholdsowing to the expression of a high affinity Pi transporter,PiPT, and (iii) ability of endophytic fungi to interactwith diverse rhizobacteria which have inorganicphosphate-solubilizing capabilities by virtue of produc-tion of a variety of organic acids and acid phosphatases(Johri et al. 2015; Meena et al. 2010; Ngwene et al.2016; Singh et al. 2009; Srividya et al. 2009; Swethaand Padmavathi 2016). This effective P uptake in ENCplants aids in transporting absorbed phosphorus toleaves, prompting plant growth; increasing absorptionof nutrients and biomass accumulation (Wu et al. 2019),consequently alleviating the adverse effects of salinity.

Nitrogen

Nitrogen plays a crucial role in cell function and metab-olism (Chokshi et al. 2017). Plants absorb N as nitrate(NO3

−), ammonium (NH4+) ions, and also as organic

compounds such as amino acids and peptides (Rentschet al. 2007; Tegeder and Rentsch 2010) but absorption iscompromised by salinity due to N immobilisation. Ni-trate reductase (NR, E.C.1.6.6.1) catalyses reduction ofNO3

− to NO2− and its activity is nitrate-inducible. The

NR activity is the limiting step in the conversion ofNO3

− to amino acids (Campbell 1999). Nitrate reductaseactivity in leaves is largely dependent on nitrate fluxfrom roots (Ferrario-Méry et al. 1998) and is severelyaffected by osmotic stress induced by NaCl (Baki et al.2000). A number of reports have shown that endophyticfungal colonization assists in N uptake under stressconditions (Khan et al. 2011a; Sherameti et al. 2005;Song et al. 2015). Recruitment of N in endophyticinteractions differs from mycorrhizal interactions in

which the fungus preferentially recruits ammoniumrather than nitrate from the soil (Boukcim and Plassard2003; Gage 2004). Song et al. (2015) showed that inENC plants, N content increased in both the shoots androots with increasing salt concentrations. The funguswas suggested to be involved in the cell’s antioxidantand ROS-scavenging enzymes where N is an essentialcomponent (Cabot et al. 2014; Khan et al. 2014). An-other study by Sherameti et al. (2005) showed a signif-icant increase in growth of ENC plants that was pro-posed to be associated with a stimulation of the NADH-dependent NR, the key enzyme of nitrate assimilation inplants.

Na+:K+ ratio

Increased levels of Na+ in cells impairs important bio-chemical mechanisms required for plant growth andsurvival. Sodium accumulation alters cellular Na+:K+

ratios thereby reducing the availability of K+ that isrequired for activity of various enzymes and for theregulation of osmotic pressure and stomatal closure.Increased Na+ also competes with K+, disrupting cellu-lar metabolism in roots and leaf tissues (Abdelaziz et al.2017). This eventually increases the Na+:K+ ratios in thecytosol, and subsequently disrupts enzyme activity, pro-tein synthesis, turgor maintenance, photosynthesis andstomatal movement (Evelin et al. 2019).

High Na+:K+ ratios in plants indicate higher levels ofstress. Hence, plants must maintain low levels of Na+ tobe able to resist the deleterious effects of salinity. ENCplants consistently have lower Na+:K+ ratios thanNENC plants under saline conditions. Reza Sabzalianand Mirlohi (2010) demonstrated that the toxic effect ofNa+ was mitigated in grasses inoculated with endophyt-ic fungi by increasing K+ concentration and thus main-taining the Na+:K+ ratio in plants. Similar results werefound by Song et al. (2015) and Alikhani et al. (2013)where endophytic fungi modulated ion accumulation incolonized barley plants by decreasing the foliar Na+:K+

ratio. Restricting the transport of Na+ to leaves andensuring a low cytosolic Na+:K+ ratio are importantways plants can increase their tolerance to high saltlevels (Berthomieu et al. 2003; Cuin et al. 2003). In-crease of K+ concentration is also related to mechanismsthat control turgor pressure (Beckett and Hoddinott1997). Song et al. (2015) also showed that the lowerNa+:K+ ratios observed in ENC plants decreased thelevel of toxic ions and osmotic influence on plants under

230 Plant Soil (2021) 461:219–244

salt stress. Another study on barley plants inoculatedwith endophytic fungi showed a decreased Na+:K+ ratiocompared to uninoculated plants, indicating that thisratio is a reliable indicator of the severity of salt stress,or for screening plant genotypes for high Na+ tolerance(Ghabooli 2014) (Table 2).

Plants control Na+ homeostasis through a variety ofmembrane proteins, antiporters, nonspecific cationchannels, Na+ and K+ transporters, vacuolar ATPasesand aquaporins, and the plasma membrane (PM)(Grabov 2007). Recently, Abdelaziz et al. (2017) pos-tulated a molecular basis of establishing a balanced ionhomeostasis of Na+:K+ ratio in ENC plants. InoculatedArabidopsis plants had enhanced transcript levels of thegenes encoding the high Affinity Potassium Transporter1 (HKT1) and the inward-rectifying K+ channels KAT1and KAT2, which play key roles in regulating Na+ andK+ homeostasis. Subsequently, lower Na+:K+ ratioswere confirmed in the Arabidopsis line gl1-HKT:AtHKT1;1 that expresses an additionalAtHKT1;1 copy driven by the native promoter. Thisstudy demonstrated that endophytic colonization pro-motes plant growth under saline conditions by modulat-ing the expression level of the major Na+ and K+ ionchannels, which helps in the establishment of a balancedion homeostasis of Na+ and K+ under salt stress condi-tions (Abdelaziz et al. 2017).

Oxidative stress

Salt stress (osmotic and ionic stress) also interferes withproper cellular functions of plants due to enhancedproduction of ROS, which can lead to oxidative damagein several cellular components such as lipids, proteinsand DNA (Gupta and Huang 2014). ROS consist of agroup of chemically reactive oxygen molecules such ashydroxyl radical (OH-), H2O2, O2

− and O2− and areproduced as a result of interrupted pathways in plantmetabolism that cause transfer of high energy electronsto molecular oxygen (Gill and Tuteja 2010). Broad hostrange endophytic fungi can confer effective tolerance toROS under abiotic stress conditions such as salinity(Mastouri et al. 2010; Rodriguez et al. 2008). (Redmanet al. 2011); Singh et al. (2011) reported that exposure tohigh salt conditions caused ROS accumulation in toma-to, rice, panic grass, and dunegrass without endophyticassociations, whereas the ENC plants had decreasedROS accumulation through various pathways (Fig. 3).

Plants have two ways to counteract the adverse con-sequences of ROS, mainly enzymatic and non-enzymatic antioxidative systems. The enzymatic systemincludes catalase (CAT), ascorbate peroxidase (APX),superoxide dismutase (SOD), glutathione reductase(GR), dehydroascorbate reductases (DHAR) andmonodehydroascorbate reductases (MDHAR). Thenon-enzymatic antioxidant system consists of ascorbicacid (AsA), glutathione (GSH), carotenoids andosmoprotectants that play roles in quenching toxic by-products of ROS.

Baltruschat et al. (2008) reported increased activityof CAT, APX, GR and DHAR in the root tissues ofbarley under saline conditions. Increased activity ofDHAR was seen in P. indica colonized barley leadingto detoxification of ROS and an enhanced ratio ofascorbic acid to neutralize oxygen free radicals (Foryerand Noctor 2000). Azad and Kaminskyj (2016) usedH2O2 localization as a proxy to assess accumulation ofROS and showed that ENC plants had lower H2O2levels in their leaves following NaCl-stress, confirmingthe role of endophytes to reduce stress-induced ROSgeneration.

Also, Zhang et al. (2016) reported that ENC plantshad higher SOD, peroxidase (POD) and CAT activitysuggesting that the coordination of POD and CAT ac-tivity along with SOD activity played a central protec-tive role in the O2

− and H2O2 scavenging process inENC plants (Ahmad et al. 2015). Increased activity wasa result of increased expression of the genes encodingthe enzymes (Zhang et al. 2016). Under saline condi-tions, endophytic colonization also increases the con-centrations of non-enzymatic antioxidant moleculessuch as AsA, GSH and carotenoids in plants as shownby several studies (Jan et al. 2019; Jogawat et al. 2013;Prasad et al. 2013; Waller et al. 2005).

Salinity increases the level of lipid peroxidation(Hernández 2019; Yu et al. 2020) which results inhigher membrane permeability and loss of ions fromthe cells (Gupta and Huang 2014). NaCl treatment ofENC plants resulted in higher rates of lipid peroxidationin salt-sensitive plants than in salt-tolerant plants sug-gesting that the rate of lipid peroxidation can be used asan indicator to measure how effectively ENC plantscope with salt stress (Baltruschat et al. 2008). Anotherstudy showed that ENC plants contained higher ascor-bate concentrations in roots compared with NENCplants, while the ratio of ascorbate to dehydroascorbatewas not significantly altered and CAT, APX, GR,

231Plant Soil (2021) 461:219–244

DHAR and MDHAR activities were increased. Thesechanges were consistent with the decrease of leaf lipidperoxidation observed in these experiments (Walleret al. 2005). Similar results were shown by Mastouriet al. (2010) and Zhang et al. (2001) where ENC plantshad significantly reduced accumulation of lipid perox-ides than cont rol p lan ts under sa l t s t ress .Malondialdehyde (MDA), a product of lipid peroxida-tion, is generally regarded as an indicator of free radicaldamage to cell membranes caused by oxidative stress.

Zhang et al. (2016) reported that salt stressed ENCplants had a 15% reduction in MDA compared to saltstressed NENC plants. Table 3 lists some of the studiesreporting changes in lipid compositions due to endo-phytic symbiosis in salt stressed plants.

Photosynthesis

Salt stress hinders photosynthesis resulting in an enor-mous loss in crop productivity (Sudhir and Murthy

Fig. 3 Oxidative stress tolerance mechanisms in salt stressedplants. Increase in salinity causes oxidative stress in plants due toredox imbalance between ROS (reactive oxygen species) andantioxidants. This results in molecular and cellular damage andmembrane peroxidation eventually disturbing the normal function-ing of the cell. Several antioxidant enzymes are induced to combatsalt stress including catalyse (CAT), ascorbate peroxidase (APX),superoxide dismutase (SOD), peroxidase (POX), glutathione

reductase (GR), dehydroascorbate reductase (DHAR) andmonodehydroascorbate reductase (MDHAR). Ascorbate (AsA),glutathione (GSH) and carotenoids are non-enzymatic antioxi-dants that are produced to counteract the adverse consequencesof salt stress. In endophyte colonized (ENC) plants, the tolerancemechanism in reinforced by activating an efficient antioxidantsystem that abates the oxidative damage caused due to salt stress

232 Plant Soil (2021) 461:219–244

Tab

le3

Examples

ofstudieson

theeffectsof

salin

ityandendophyticfungio

nlip

idperoxidatio

nin

plants

S.N

o.Saltlevel

(mM

NaC

l)Plant

(Fam

ily)

Fungus

Param

etersassessed

Effectsof

References

Salin

ityEndophytic

fungio

nsalt

stressed

plants

10,100,

200,300

Oryza

sativa

Pirifo

rmospora

indica

Totalsolubleproteins,lipid

peroxidatio

n(m

easuredthiobarbitu

ricacid

reactiv

esubstances

inshootsandroots),free

prolinecontent,andenzyme

antio

xidants(catalase(CAT:

EC1.11.1.6),glutathionereductase(GR:

EC1.6.4.2),superoxide

dism

utase(SO

D:E

C1.15.1.1),

ascorbateperoxidase(A

PX:E

C1.11.1.11))activ

ity

Increasedlip

idperoxidatio

n,SO

D,A

PX,C

AT,G

R;

Decreased

totalsoluble

proteins,freeproline

content

Increasedtotalsoluble

proteins,prolin

e,further

increase

inSOD,A

PX,

CAT,G

R;D

ecreaseinlip

idperoxidatio

n

Bagherietal.

(2013)

20,100

Glycine

max

Fusarium

verticillioides

Lipid

peroxidatio

n,antio

xidant

enzyme

analysis,gibberellins,A

BA,salicylic

acid

IncreasedABA,S

A,lipid

peroxidatio

n;Decreased

CAT,S

OD,P

OD,S

A,

ABA

IncreasedCAT,S

OD,P

OD;

Decreased

lipid

peroxidatio

n,ABA,S

Asignificantly

Radhakrishnan

etal.(2013)

30,100

Glycine

max

Fusarium

verticillioides

andHum

icola

sp

Protein

content,catalase

activ

ity,A

BA

content,SA

content,lip

idperoxidatio

n(m

easuredin

term

sof

malondialdehyde-M

DAcontent)

IncreasedABA,S

A,lipid

peroxidatio

n;Decreased

CAT,S

OD,P

OD,S

A,

ABA,lipid

peroxidatio

n

Significant

three-fold

reductionin

MDAlevel,

ABA,S

A;Increased

CAT,

SOD,P

OD

Radhakrishnan

etal.(2015)

40,100,

175,250

Medicago

truncatula

Pirifo

rmospora

indica

Proline,MDA,S

odium

ion,

antio

xidant

enzymes

IncreasedMDA,N

A+in

shoots,slig

htincrease

inprolinecontent;Decreased

POD,S

OD,C

AT

Highestincreasedin

proline

contentw

ithincrease

inPOD,S

OD,C

AT;

decreasedMDA,N

A+in

shoots

Lietal.(2017)

50,200

Cucum

issativus

Trichoderm

aharzianum

Antioxidant

enzymes,K

+content,

K+/Na+

ratio

,Na+

content,ethylene

levels,M

DAlevelsas

ameasure

oflip

idperoxidatio

n

IncreasedMDAlevels,N

a+

content,ethylene

levels;

Decreased

antio

xidant

enzymes,K

+content,

K+/Na+

ratio

Improved

activ

ities

ofantio

xidant

enzymes,

increasedK+content,

K+/Na+

ratio

;decreased

Na+

content,ethylene

levels,M

DAlevels

Zhang

etal.

(2019a)

233Plant Soil (2021) 461:219–244

2004). Salt stress has been shown to degrade D1 and D2proteins of the photosystem II reaction centre. Theseproteins play crucial roles in protein phosphorylationcoupled with the flow of electrons (Jansen et al. 1996).Salt stress also results in decreased photosynthetic pig-ments by reducing the activity of enzymes that synthe-size them. Osmotic shock resulting from salt stress leadsto reduced leaf area and decrease in stomatal and meso-phyll conductance (Chaves et al. 2009). This limits CO2availability and assimilation which consequently affectsRuBisCO (Seemann and Critchley 1985). DecreasingCO2 assimilation also increases the risk of the accumu-lation of electrons in thylakoid membranes and predis-poses the photosynthetic apparatus to increased energydissipation. Thus, to dissipate this energy, photosystemII loses excess electrons causing injury to photosynthet-ic tissues and affecting the net photosynthetic rate(Redondo-Gómez et al. 2010).

Plants can protect the photosystems from light in-duced inhibition and damage in several ways such asminimizing harvesting of light and dispersion of excessenergy by non-photochemical quenching (NPQ) (LimaNeto et al. 2015). An increase in NPQ can limit quantumyield (Baker 2008) but ENC plants are reported to havelower NPQ, therefore symbiosis enhances photosynthet-ic efficiency by proficient conversion of harvested lightinto chemical energy and minimizing NPQ (Pehlivanet al. 2017). Endophytic fungi are also known to rein-force these mechanisms and reduce the negative effectsof salinity on plant photosynthetic capacity (Jogawatet al. 2013; Molina-Montenegro et al. 2018). Table 4lists some of the studies in the last decade on effect ofsalinity and endophytes on photosynthesis in plants.Endophytic symbiosis combats the negative effects ofsalt stress on photosynthesis in several ways. ENCplants have shown improved water status resulting inlarger leaf area and higher stomatal conductance andeventually better assimilation of carbon dioxide (Zareaet al. 2012).

Magnesium (Mg) is one of the essential macronutri-ents for plant growth and is involved in numerousphysiological and biochemical processes such as photo-synthesis, enzyme activation and synthesis of nucleicacids ad proteins (Chen et al. 2018). It is the central atomof the tetrapyrrole ring of chlorophyll a and bmolecules,which are the major pigments for photosynthetic lightabsorption (Wilkinson et al. 1990). Salt reduces uptakeof Mg2+ thus also reducing the concentration of chloro-phyll in leaves (Sudhir and Murthy 2004). ENC plants

maintain higher chlorophyll concentration by improvingthe uptake ofMg2+ (Jogawat et al. 2013; Yin et al. 2014)and this leads to maintenance of plastid integrity andenhanced photosynthetic efficiency (Johnson et al.2014).

Another way in which endophytes induce defencesystems in plants under saline conditions is by upregu-lating the ascorbate-glutathione (ASH-GSH) cycle; forexample Kumar et al. (2012) described that during saltstress, the endophytic fungus P. indicamaintains a highantioxidative environment by defence system priming,especially the ascorbate–glutathione (ASH–GSH) cycleleading to maintenance of plastid integrity and thereforeenhanced photosynthetic efficiency in colonised plantsduring abiotic stress (Johnson et al. 2014). ENC plantsalso confer the benefit of maintaining the integrity ofphotosystem II by repairing salt-induced degradation ofD1/D2/Cytb 559 complex by the accumulation of gly-cine betaine in ENC plants (Rivero et al. 2014). Glycinebetaine is also known to stabilise PSII pigment-proteincomplexes and protect the activities of RuBisCO andrubisco activase enzymes responsible for fixing CO2 inAM fungi (Talaat and Shawky 2014).

Hormonal regulation

Induction of phytohormones is also one of the strategiesplants use to mitigate abiotic stresses that ultimatelyenhance plant growth and productivity in stressful envi-ronments (Ryu and Cho 2015). Phytohormones, oftenregarded as plant growth regulators, are compounds thatare derived from plant biosynthetic pathways actingeither locally or via transport to other sites within theplant to mediate growth, development and nutrient allo-cation (Peleg and Blumwald 2011). These includeabscisic acid (ABA), gibberellins (GA), ethylene(ETHY), cytokinins (CKs), brassinosteroids (BRs) andauxins, particularly indole acetic acid (IAA). To initiatesuitable plant responses to environmental stimuli, thereis interplay between these hormones to modulate bio-chemical and physiological processes (Saeed et al.2017).

It is known that some strains of endophytic fungi canproduce plant hormones, especially gibberellins (GAs),to help the plant to tolerate or avoid abiotic stress(Contreras-Cornejo et al. 2009; Khan et al. 2011b; Wal-ler et al. 2005). Hamayun et al. (2010) reported thatinoculation with the endophytic fungi Phoma herbarumshowed increased plant biomass and elevated

234 Plant Soil (2021) 461:219–244

Tab

le4

Examples

ofstudieson

theeffectsof

salin

ityandendophyticfungio

nphotosynthesisin

plants

S.No.

Saltlevel(m

MNaC

l)Plant

Fungus

Param

etersassessed

Effectsof

References

Salin

ityEndophytic

fungionsalt

stressed

plants

10,200,300

Oryza

sativa

Pirifo

rmospora

indica

Photosyntheticpigm

ent

content[chlorophyll

(Chl)a,Chl

b]

Decreased

Increased

Jogawatetal.

(2013)

20,50,150

Lactucasativa(lettu

cevar.Rom

aine)

andSolanum

lycopersicum

(tomato

var.Moneymaker)

Colobanthus

quitensis

(AFE001)

and

Descham

psia

antarctica(AFE002)

The

netp

hotosynthesis

rate(A

),and

transpirationrate

(EC),water

use

efficiency

(WUE)for

photosynthesisas

the

ratio

between

photosyntheticrate

andtranspiration

(A/EC)

Decreased

Amax,

WUE

Significantly

increased

Amax,W

UE

Molina-Montenegro

etal.(2018)

30,100,200,300,400,

500

Triticumaestivum

Pirifo

rmospora

indica

andAzospirillum

spp.

Photosynthetic

pigm

ents(Chl

a,b,

ab)

Decreased

Significantly

increased

with

inoculationof

both

organism

s

Zarea

etal.(2012)

40,300,500

Solanumlycopersicum

Fusariumculmorum

Photosystem

II(PsII)

efficiency

Decreased

Increased

Azadand

Kam

inskyj

(2016)

235Plant Soil (2021) 461:219–244

production of active GAs including GA1, GA3, GA4,and GA7 in salt-stressed soybean. Similar results wereshown by Waqas et al. (2012), where salt-stressed cu-cumber plants inoculated with Penicillium sp. had largershoot growth and plant biomass that was attributed tothe secretion of bioactive GAs. A study on salt-stressedcucumber plants inoculated with Trichodermaasperellum Q1 alleviated the suppression effects of saltstress by altering the phytohormone levels (IAA, GAand ABA) and the phosphate solubilization ability (Leiand Zhang 2015). Three bioactive GAs, i.e. GA4, GA9and GA12 were more abundant in ENC plants grownunder salt stress compared to NENC plants (Khan et al.2011c), and this mitigated the adverse effects of salinityand improved growth.

Endophytic symbiosis under saline conditions has apositive influence on the endogenous concentration ofauxins (Contreras-Cornejo et al. 2009). Contreras-Cornejo et al. (2014) evaluated the expression of theauxin-responsive marker gene DR5:uidA which wasupregulated in ENC plants compared to their counter-parts under saline conditions speculating that, by pro-viding auxins, Trichoderma spp. could restore auxinhomeostasis and, consequently growth and develop-ment could be normalized when grown under salt stress.

Perspectives and future directions

Evolution has led to complex interactions between awide diversity of microorganisms and plants; many ofthem resulting in the establishment of a symbiotic rela-tionship between them (Hassani et al. 2018). Theseinteractions beneficially impact plant survival, biodiver-sity, fitness and ecosystem function (Bai et al. 2018;Rosier et al. 2016; Sasse et al. 2018). Growing evidenceindicates that endophytic associations can also be im-portant for plant fitness, development of the immunesystem, tolerance to abiotic stresses, nutrient acquisitionand disease suppression (Hiruma et al. 2016; Khan et al.2015; Khare et al. 2018; Soliman et al. 2015; Terhonenet al. 2016; Zuccaro et al. 2014). This review highlightssome of the numerous mechanisms by which endophyt-ic symbiosis promotes salt tolerance in plants. However,there are several challenges and issues that future re-search should address for comprehensive understandingof these mechanisms. It is well established how osmoticadjustment in plants under salt stress via enhanced ac-cumulation of osmolytes is achieved using endophyticsymbiosis. However, the biochemical, molecular and

genetic mechanisms are largely unexplored. Therefore,there is a need to understand these phenomena by in-vestigating genes encoding enzymes used for the syn-thesis of molecules that are crucial for salt stress resis-tance. Therefore, dedicated research into unravelling themolecular basis of osmolyte accumulation in ENCplants will broaden our understanding of the mecha-nisms involved.

In recent years, new compounds, such as polyamines,and strigolactones have been implicated in improvingplant tolerance to salt stress (Fahad et al. 2015).Strigolactones (SL) play regulatory roles to combatabiotic stress, including salinity, and in order to be fullyeffective, they need to modulate and interact with otherphytohormones, especially auxin and ABA. SLs arealso involved in several aspects of plant development;suppression of secondary branches in shoots, regulationof leaf senescence, stimulation of internode length andinduction of endophytic symbiosis (de Saint Germainet al. 2013; Lopez-Raez et al. 2017; Yamada et al.2014). This group of sesquiterpene lactones is responsi-ble for hyphal branching and successful colonisationwithin roots by producing 5-deoxy-strigol, followed bythe formation of a pre-penetration apparatus (Genreet al. 2005). Recently, SL secreted by roots ofArabidopsis thaliana was found to act as a signal mol-ecule for colonization of endophytic Mucor sp.(Rozpądek et al. 2018). Studies on auxin and ABAinvolvement with endophytes under salt stress has beenexplored, but further research is required to investigatethe role of strigolactones secreted by ENC plants inameliorating salt stress.

The root is the primary location in plants that sensessalt stress. The PM constitutes the interface between acell and its surroundings and plays an important role incell wall biosynthesis, ion transport, endocytosis, sens-ing of environmental stimuli, and cellular signal trans-duction (Mansour et al. 2015). PM lipids and proteins insalt tolerant plants are protected from oxidative attackthrough enhanced antioxidant systems, a mechanismthat minimizes lipid and protein oxidation whileretaining PM integrity (Mansour 2013). Though lipidperoxidation has been elucidated in ENC plants undersalt stress, lipid metabolism in the PM in root tissues isyet to be investigated. Hence future research that eval-uates how endophytic symbiosis influences thesechanges under saline conditions is warranted.

Limited studies are available to understand the role ofendophytic fungi in modifying the photosynthetic

236 Plant Soil (2021) 461:219–244

capacity of plants to alleviate the negative effects ofsalinity as described in previous sections. Salt stresshas been shown to degrade proteins of the PSII reactioncentre. These proteins play fundamental roles in phos-phorylation of proteins (Jansen et al. 1996). Studies inthe past have focused on understanding how AMFsymbiosis acts to maintain the integrity of PSII showingthe upregulation of the genes encoding these proteinsunder salt stress (Chen et al. 2017). However, researchon maintenance of these proteins by endophytic fungiunder salt stress is a field to explore.

Metabolomics is increasingly being utilized for gen-erating deep insights into abiotic stress responses. Sev-eral studies have focused on exploring and discoveringcompounds that stimulate ENC plant growth by allevi-ating stress using various technologies (Chetia et al.2019; Kusari and Spiteller 2012; Mazlan et al. 2019;Tawfike et al. 2018). However, molecular signallingmechanisms employed by endophytic fungi under salineconditions are yet to be explored. The high-throughputmass spectrometric profiling of cellular metabolites ofplant-associated endophytes under the influence of saltstress could help to reveal the level of interference by thestressor in overall cellular homeostasis. Thus, future‘omics-based research is required to generate compre-hensive information on specific plant-endophytic fungi-salt stress systems to resolve facts behind precise mech-anisms of stress tolerance in crop plants.

Although this review covers mechanisms and strate-gies employed by plants under salt stress, in nature plantsoften face multiple biotic and abiotic stresses instead of asingle stress. These combinations of stresses exert morecomplex effects on plant fitness which eventually resultsin potential differences from the responses elicited undersingle stresses. (Bai et al. 2018) demonstrated that tomatodeveloped integrated responses via genetic componentsand cross-talk of signalling pathways under combinedsalinity and pathogen stresses. This shows that plantsmust have evolved to mitigate a combination of stresses.Addressing specific questions related to multiple stressessuch as how beneficial microorganisms and pathogens orcombined abiotic stresses interact would facilitate thedesign of strategies for sustained plant health under di-verse environmental stresses.

In conclusion, directing future research on endophyt-ic symbiosis under salinity in order to understand theabove-mentioned challenges will help improve ourknowledge and understanding of the mechanisms ofendophyte facilitated salinity tolerance in host plants.

Acknowledgements SG thanks the University ofMelbourne forproviding the Melbourne Research Scholarship for financialassistance.

Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format,as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, andindicate if changes were made. The images or other third partymaterial in this article are included in the article's Creative Com-mons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article's Creative Com-mons licence and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy ofthis licence, visit http://creativecommons.org/licenses/by/4.0/.

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