Critical Reviews in Biotechnology

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Endophytic fungi: resource for gibberellins and

crop abiotic stress resistance

Abdul Latif Khan, Javid Hussain, Ahmed Al-Harrasi, Ahmed Al-Rawahi & In-
Jung Lee

To cite this article: Abdul Latif Khan, Javid Hussain, Ahmed Al-Harrasi, Ahmed Al-Rawahi & In-
Jung Lee (2015) Endophytic fungi: resource for gibberellins and crop abiotic stress resistance,
Critical Reviews in Biotechnology, 35:1, 62-74, DOI: 10.3109/07388551.2013.800018

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Published online: 28 Aug 2013.

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ISSN: 0738-8551 (print), 1549-7801 (electronic)

Crit Rev Biotechnol, 2015; 35(1): 62–74

! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2013.800018

REVIEW ARTICLE

Endophytic fungi: resource for gibberellins and crop abiotic stress

resistance
Abdul Latif Khan1,2, Javid Hussain1, Ahmed Al-Harrasi1, Ahmed Al-Rawahi1, and In-Jung Lee2
1
Department of Biological Science & Chemistry, College of Art and Science, University of Nizwa, Sultanate of Oman, and 2School of Applied
Biosciences, Kyungpook National University, Daegu Republic of Korea

Abstract Keywords
The beneficial effects of endophytes on plant growth are important for agricultural ecosystems Abiotic stresses, crop growth and physiology,
because they reduce the need for fertilizers and decrease soil and water pollution while endophytic fungi, gibberellins
compensating for environmental perturbations. Endophytic fungi are a novel source of
bioactive secondary metabolites; moreover, recently they have been found to produce History
physiologically active gibberellins as well. The symbiosis of gibberellins producing endophytic
fungi with crops can be a promising strategy to overcome the adverse effects of abiotic Received 9 January 2013
stresses. The association of such endophytes has not only increased plant biomass but also Revised 31 March 2013
ameliorated plant-growth during extreme environmental conditions. Endophytic fungi repre- Accepted 18 April 2013
sent a trove of unexplored biodiversity and a frequently overlooked component of crop Published online 27 August 2013
ecology. The present review describes the role of gibberellins producing endophytic fungi,
suggests putative mechanisms involved in plant endophyte stress interactions and discusses
future prospects in this field.

Endophytic fungi: the unexplored trove broad varieties of floral resources and collected from various
ecosystems like hot deserts (Hoffman & Arnold, 2008),
‘‘Endophyte’’ means ‘‘within the plant’’. Microorganisms
tundra (Higgins et al., 2007), mangroves (Gilbert et al., 2002)
(bacteria/fungi) living inside plant tissues (leaves, stems,
and temperate forests (Arnold et al., 2007). Hawksworth
roots, and fruits) without causing any symptoms of disease in
(1991) estimated a global diversity of 1.5 million fungal
the host are classified as endophytes (Saikkonen et al., 2004;
species, suggesting a ratio of six species per vascular plant
Schulz & Boyle, 2005). This definition excludes mycorrhizal
species. In the past century, many of the 0.1 million fungi that
fungi but does not imply that endophytic fungi are not
have been described were those associated with various
cultivable on artificial media (Gallo et al., 2008). The
higher organisms as either parasites or saprophytes on dead/
paleomycological evidence of endophytic symbioses with
dying biological materials (Gallo et al., 2008; Krings et al.,
plants has been estimated to be approximately 400 million
2007; Schulz & Boyle, 2005; Schulz et al., 1999). Thus, this
years old thus, placing it in the same geological period as
raises the question, where are the remaining 0.9 million
mycorrhizal symbioses (Krings et al., 2007; Rodriguez &
fungi? Recently, Hibbett et al. (2011) and Hawksworth (2011)
Redman, 2008). On the basis of evolutionary relatedness,
suggested that this diversity could be more than 8.7 million
taxonomy, plant hosts and ecological niche, endophytes are
species. However, the global diversity of these endophytic
divided into two major groups: (i) calvicipitaceous or class I
fungi is limitless and still a large number of species and
endophytes which inhabit some grasses and (ii) non-
ecosystems need to be explored. A significant amount of work
calvicipitaceous or class II endophytes which are associated
has also been carried out on endophytes derived from forest
in the asymptomatic tissues of non-vascular plants, ferns and
ecosystems (Arnold et al., 2007). Despite the great import-
allies, conifers and angiosperms (Harman, 2011; Rodriguez
ance of agriculture systems to our daily lives and food
et al., 2009).
production, research related to crops is still frequently
In last three decades of the 20th century, most studies on
overlooked. Research has been carried out on the isolation
endophytic fungi have been conducted on the population and
and characterization of plant growth-promoting bacteria and
taxonomy involving three plant families, the Coniferaceae,
fungi and to some extent on phyllospheric endophytes from
Ericaceae and Poaceae. Endophytes have been isolated from
grass and forest ecosystems; however, root endophytes
associated with crops are not well known. The present
review focuses on the endophytic fungi recovered from crop
Address for correspondence: In-Jung Lee, Crop Physiology Lab, School plants and their role in improving crop productivity under
of Applied Biosciences, Kyungpook National University Daegu, 702-
701, South Korea. Tel: 82 53 950 5708. Fax: 82 53 958 6880. E-mail: abiotic stresses (such as salinity, drought, temperature and
ijlee@knu.ac.kr heavy metal pollution) while producing gibberellins.
DOI: 10.3109/07388551.2013.800018
Endophytic potential to produce gibberellins
Endophytic fungi are an important and novel resource of
naturally bioactive compounds having potential applications
in agriculture, medicine and the food industry. A significant
number of interesting molecules have been produced by
endophytes, including flavonoids, peptides, alkaloids, ster-
oids, phenolics, terpenoids, lignans, and volatile organic
compounds with many of them biologically active (Kusari
et al., 2012). In fact, a previous study by Schulz & Boyle
(2005) showed that about 51% of biologically active metab-
olites originate from endophytes compared to only 38% of
novel substances originating from other soil microflora. In the
past two decades, many novel bioactive compounds with
antimicrobial, insecticidal, cytotoxic, and anticancer proper-
ties have been successfully isolated and characterized from
endophytic fungi.
During the long period of co-evolution, a friendly
relationship has been established between each endophyte
and its host plant. Some endophytes have the ability to
produce the same kind of bioactive compounds as those
originating from their host plants (Bérdy, 2005; Kusari et al.,
2012). One of the most interesting examples is the production
of the world’s first billion dollar anticancer drug, paclitexel
(Taxol) from Pestalotiopsis microspora (Strobel et al., 1996).
The endophyte colonizes the Himalayan yew tree without
causing apparent injury to the host plant. This potential has
set the stage for increasing interest in fungal endophytes
(Gallo et al., 2008; Kusari et al., 2012). However, looking at
the metabolomic potential of endophytic fungi, the isolation
and quantification of plant hormones have been frequently
overlooked. Plant hormones are known as signaling molecules
or chemical messengers which can play an important role in
plant growth and development (Hedden et al., 2002). These
also control the responses required for changing environmen-
tal conditions in which they trigger specific biochemical,
physiological and morphological responses. From the last
decade or so, it has been known that endophytes have the
capability to produce selected plant hormones, especially
gibberellins.
Gibberellins (GAs) are ubiquitous plant hormones that
elicit various metabolic functions required for the growth of a
plant like seed germination, stem elongation, sex expression,
flowering, formation of fruits and senescence (Bomke &
Tudzynski, 2009). GAs were initially isolated from the
pathogenic fungus (Fusarium fujikuroi: a teleomorph of
Gibberella fujikuroi)) of rice which causes the disease
known as ‘‘bakanae or foolish seedling’’ (Sawada, 1917).
The fungal attacked plants had significantly higher shoot
elongation but empty grains. After World War II, the first
three gibberellins GA1, GA2 and GA3 were isolated
(Takahashi et al., 1955), which stimulated international
research related to improved plant growth and development.
Up until now, more than 136 GAs have been identified
(Bomke et al., 2008; Hedden et al., 2002) which are named
based on their discovery (http://www.plant-hormones.info/
gibberellin_nomenclature.htm).
In plants, an immense amount of work has been carried out
to understand the role and biosynthesis pathways of
Gas; however; in microorganisms, the work has been
Gibberellins producing endophytes stress tolerance 63
steadily progressing. Bomke et al. (2008) reported that GAs
in fungi are synthesized from acetyl-CoA via the mevalonic
acid (MVA pathway) and are then converted to farnesyl
diphosphate (FDP; Figure 1). At the beginning of the GAs
pathway, consequent intermediates like geranylgeranyl
diphosphate (GGDP), ent-copalyl diphosphate (CDP), ent-
kaurene and ent-kaurenoic acid lead to the production of
GA12-aldehyde which is identical in higher plants and G.
fujikuroi (Bomke & Tudzynski, 2009; Bomke et al., 2008).
Following conversion to GA12-aldehyde, the pathway in
plants and fungi differs. In plants, GA12-aldehyde is converted
to GA12 and then is oxidized either to form GA9 or GA53,
which later on is converted to GA20. However, in plants, the
final step for the formation of bioactive GAs is the
3b-hydroxylation of GA9 and GA53 to give GA4 and GA1,
respectively (Bomke & Tudzynski, 2009). In contrast to
plants, GA12-aldehyde from fungi, such as G. fujikuroi, is
3b-hydroxylated to GA14-aldehyde and then oxidized to form
GA14. The subsequent conversion of GA14 to GA4 is
comparable to the production of GA9 and GA20 in plants
(Figure 1). Desaturation of GA4 results in the formation of
GA7 and then GA3 which is the main product in G. fujikuroi.
GA1 is formed as a minor product by 13-hydroxylation of
GA4 and is not converted to GA3 (Bomke & Tudzynski,
2009). During the exploration of GAs from endophytic fungi,
interestingly, GA5 has been also detected which is a precursor
of bioactive GA3 (Khan et al., 2008). Very few studies (mostly
endophytes; Table 1) have shown the presence of GA5 in their
fungal cultures (Khan et al., 2008). Probably, this might be an
interesting point of chemotaxonomic difference between
endophytes and other fungi because it has not been found in
Fusarium and Sphaceloma.
Gibberella fujikuroi and their anamorphs and telemorphs
have been studied for the presence of GAs gene clusters
(Figure 1); however, few of them have been reported to
produce GAs (see review Bomke & Tudzynski, 2009).
Similarly, another plant pathogen Sphaceloma manihoticola
is capable of producing considerable amounts of GA4 in its
cultures (Rademacher, 1994). In comparison to the seven gene
clusters of G. fujikuroi, S. manihoticola has only five GAs
biosynthesis genes (Bomke et al., 2008). Another strain of the
fungus, Phaeosphaeria sp. L487 produces GA1, GA4 and GA9
while it also has GAs biosynthesis related genes (see review
Kawaide, 2006). Other fungal species such as Aspergillus
niger (Bilkay et al., 2010), Neurospora crassa (Kawanabe
et al., 1983), Sporisorium reilianum (Matheussen et al., 1991)
and Agaricus bisporus (Pegg, 1973) have been identified to
either produce GAs during their growth or contain GAs
biosynthesis gene clusters. Most of these GAs producing
fungi belong to Ascomycetes compared to Basidiomycetes
suggesting an active GAs synthesis ability of the former.
In the case of Basidiomycetes, many of the species are
reported to have endophytisim while their secondary metab-
olite potential is also known to a lesser extent but regarding
phytohormones very little is known about them. These are
Piriformospora indica (Verma et al., 1998) and
Scolecobasidium tshawytschae (Hamayun et al., 2009d).
The latter has been known to produce GAs while the former
has been recently found to produce indole-3-acetic acid and
indole-3-lactate (Hilbert et al., 2012). However, the
64 A. L. Khan et al. Crit Rev Biotechnol, 2015; 35(1): 62–74

FDP GGDP CDP ent-kaurene ent-kaurenoic acid

CH2

H
H CH2 H CH2
HO OH
H CHO H CHO H
COOH COOH COOH
GA14-Aldehyde GA12-Aldehyde ent-7α-hydroxy
kaurenoic acid

H CH2
H CH2
H COOH
HO COOH
H COOH CO2
COOH GA12
GA14
O
CO2
OC H CH2

O H COOH
GA9

OC H CH2
O OH
HO
H COOH
GA4 OC H CH2

O H COOH
GA20
OC O
O OH H CH2
HO
H COOH OC H CH2
OC H CH2
GA7 HO
HO H COOH
H COOH GA1
GA4

O OH OH
O

OC H CH2 OC H CH2
HO HO
H H COOH
COOH
GA3 GA1
F. fujikuroi
Figure 1. Biosynthesis pathways of gibberellins in fungi. The various types of GAs are produced by F. fujikuroi and Phaeosphaeria sp. with the help of
a wide array of enzymes/genes. Both the fungi differ in their final product, that is, it is GA3 from F. fujikuroi in most cases while it can be GA1 as noted
in the GAs biosynthesis of Phaeosphaeria sp. The same pathway is comparable to endophytes as well. FDP – farnesyl diphosphate; GGDP –
geranylgeranyl diphosphate; CDP – ent-copalyl diphosphate.

production potential of gibberellins is not yet known and
requires further studies in this respect. Even in the case of
Ascomycetes, little research has been conducted on the GA
producing root endophytic fungi. Until now, 21 different
endophytic fungal species have been identified for their
potential to produce GAs in axenic cultures (Table 1). These
endophytes were isolated from the rhizospheric continuum of
the endophyte plant environment.
All the isolated endophytes have the potential to produce
GA1, GA3, GA4, GA7, GA9 and GA12 which suggest that
Table 1. Gibberellins producing endophytic fungi isolated from various crop/plants.

Host plants Endophyte Gibberellins (GAs in ng/ml) References

DOI: 10.3109/07388551.2013.800018

Glycine max L. Aspergillus fumigatus sp. LH02 GA4(8.38), GA9(2.16), GA12(1.56) Khan et al., 2011a
Cladosporium sphaerospermum GA1(0.24), GA3(8.9), GA4(2.58), GA7(1.37), GA5(1.2), Hamayun et al., 2009a
GA15 (1.1), GA19 (2.1), GA24 (1.8)
Phoma herbarum GA1 (0.11), GA3(2.91), GA4 (3.21), GA7 (1.4), GA9 (0.05), Hamayun et al., 2009b
GA12 (0.23), GA15 (0.42), GA19 (0.53), GA20 (0.06)
Chrysosporium pseudomerdarium GA1 (0.24), GA3 (8.5), GA4 (2.58), GA9 (1.39), GA15 (1.2), GA19 (1.4), GA20 (2.1) Hamayun et al., 2009c
Penicillium minioluteum LHL09 GA4 (12.84), GA7(48.91) Khan et al., 2011c
Scolecobasidium tshawytschae GA1 (0.3), GA3 (17.84), GA4 (18.58), GA7 (8.95), GA15 (0.45), GA24 (1.07) Hamayun et al., 2009d
Monochoria vaginalis Aspergillus sp. and Penicillium sp. GA3 (2.8), GA4 (2.6), GA7 (6.68), GA9 (1.61), GA24 (0.18) Ahmad et al., 2010
Cucumis sativus Cladosporium sp. MH-6; GA1 (0.81), GA3 (4.34), GA4 (9.31), GA9 (0.74), Hamayun et al., 2010a
Phoma sp. GAH7 GA15 (0.97), GA19 (1.67), GA20 (0.46)
Phoma glomerata LWL2, Penicillium sp. LWL3 GA1 (8.720), GA3 (2.420), GA4 (0.220), GA7 (4.2) Waqas et al., 2012
Paecilomyces formosus LHL10; GA1 (3.3), GA3 (1.1), GA4 (18.2), GA8 (37.2), GA9 (5.5), Khan et al., 2012a
Exophiala sp.LHL08 GA12 (1.4), GA20 (2.2), GA24 (13.6) Khan et al., 2011b
GA1 (3.546), GA3(3.98), GA4(121.50), GA5(1.50), GA7(133.47),
GA9(2.12), GA12(27.81), GA20(4.12),
Elymus mollis Gliomastix murorum KACC43902 GA1(0.32), GA3(5.76), GA4 (0.82),GA7(0.1), GA5(0.59), GA20(0.25), GA24(2.03) Khan et al., 2009a
Chrysanthemum coronarium Penicillium sp. MH7 GA1(1.37), GA3(5.88), GA4(8.62), GA7(2.05), GA9(0.83), GA12(0.44), Hamayun et al., 2010b
GA15(0.74), GA19(1.16), GA20(0.98)
Capsicum annuum Chaetomium globosum LK4 GA1(0.67), GA4(21.8), GA9(0.51), GA12(13.4), GA20(1.11) Khan at al., 2012b
Ixeris repenes Penicillium citrinum GA1(1.95), GA3(3.83), GA4(6.03), GA7(2.35), GA5(0.365), GA9(0.65), GA12(0.11), Khan et al., 2008
KACC43900 GA15(0.72), GA19(0.67), GA20 (0.30) GA24 (1.40)
Carex kobomugi Ohwi Arthrinium phaeospermum KACC43901 GA1(0.5), GA3(8.8), GA4(4.7), GA7(2.2), GA5 (0.4), GA9(0.6), GA12(0.4) Khan et al., 2009b
GA15(0.4), GA19(0.9), GA24(1.8)
Sesamum indicum Penicillium commune GA1(71.69), GA3(252.4), GA4(612.0), GA7(259.0), GA9(202.69) Choi et al., 2005
KNU5379
Calystegia soldanella Cadophora malorum GA1(1.213), GA3(1.292), GA4(3.6), GA7 (1.328), GA9 (0.796), GA12 (0.417), You et al., 2012
GA20(0.302), GA24(1.351), GA34(0.076), GA53(0.051)

The GAs were analyzed through GC-MS SIM analysis using full-scan mode (the first trial) and three major ions of the supplemented with [2H2] GA internal standards and the fungal GAs were monitored
simultaneously. The retention time was determined using hydrocarbon standards to calculate the Kovats retention index value and GA quantification was based on the peak area ratios of the non-deuterated
(extracted) GAs to the deuterated GAs. These data were calculated in nano-grams per milliliter of endophytic fungal.
Gibberellins producing endophytes stress tolerance
65
66 A. L. Khan et al.
these endophytes might have GAs biosynthesis pathways
similar to plants. However, it was noted that the GAs quantity
detected in the culture filtrates was very minute. The average
GA1 quantity ranged between 0.1 to 78.0 ng/ml, GA3 from 1.0
to 250.0 ng/ml, GA4 from 1.5 to 625.0 ng/ml, GA7 from 0.1 to
260.0 ng/ml, GA9 from 0.1 to 200.0 ng/ml and GA12 from 0.1
to 25.0 ng/ml. For other inactive GAs, the quantity recovered
from the cultures of each endophyte ranged from 0.1 to
5.0 ng/ml. The only bioactive GAs, which are used for plant
growth and development, are GA3 and GA4. The utilization of
bioactive GAs at endogenous and exogenous levels can have
an essential function in plant growth and yield, and tolerating
abiotic stimuli (Khan et al., 2012ab). Among endophytes
(Table 1), Paecilomyces formosus LHL10, Exophiala
sp.LHL08, Penicillium commune and Aspergillus fumigatus
LH02 have a significantly higher capacity to synthesize GA4
ranging from 3 to 600 mg/L (Choi et al., 2005; Khan et al.,
2011a, b, 2012a).
Although these quantities are less than that of industrial
level extraction from Gibberella fujikuroi (Lale & Gadre,
2010; Rodrigues et al., 2012), it still needs further assessment
in different substrates and growth media to assess the
quantification potential of GAs. GA3 is a high value
industrially important biochemical selling at USD 27–36/g
(Rodrigues et al., 2012) in the international market. As the
purity and potency increases, the price increases. Therefore,
its exogenous use at present is limited to premium crops only.
Most of the strains isolated in the above studies are known
plant pathogens; however, due to the switch over to the
endophytic mode of life, they have ameliorative symbiotic
effects as observed when these were applied to the host plants.
Most of the gibberellins producing endophytes in Table 1
were isolated and screened to differentiate GAs producing
strains. For this purpose, mutant rice variety waito-c was
used. Waito-c rice is a GAs biosynthesis mutant line with a
passive dy gene which synthesizes bioactive GAs through the
C13-hydroxylation pathway (Khan et al., 2012a) and has a
dwarf phenotype. During screening experiments, waito-c rice
seeds were treated with uniconazol to further suppress the
GAs biosynthesis pathway (Ikada et al., 2001). Using waito-c
rice can help in detecting the bioactive metabolite potential of
culture filtrates (Hamayun et al., 2010ab; Khan et al., 2011a,
2011b, 2011c). Besides, mutant and normal varieties of rice
like Dongjin-byeo (normal phenotype and active GAs
biosynthesis pathway) were also used to compare the effects
of endophytes. There is still a need to study the effects of such
endophytes on various crop varieties (tomato, maize, pea,
etc.; ga1, ga4, and ga5; na, lh-2, and ls-1) which have
suppressive GAs biosynthesis. The microbial synthesis of
plant growth regulators (e.g. GAs) can be a crucial element in
crop productivity (Hassan, 2002). However, little or no
information is available on the role of these bioactive strains
in stressful environments, especially drought, temperature and
salinity.
Some of the GAs like GA5, GA15, GA53, GA24, and GA19
were detected in axenic endophytic cultures (Table 1). These
different GAs were found in the cultures of Cadophora
malorum, Penicillium sp., Paecilomyces formosus and Phoma
sp. suggesting a unique adoptability and potential similarity to
host-plants. GAs biosynthesis in F. fujikuroi and
Crit Rev Biotechnol, 2015; 35(1): 62–74
S. manihoticola, however, was different from Phaeosphaeria
sp. L487, in which the key intermediates (such as GA14 and
GA3) were not detected (Bomeke et al., 2008). This suggests
there is a plant-like GAs biosynthesis pathway in these
endophytes. A similar difference can be seen with the
endophytes in Table 1. The authors did not report the
presence of GA14, which is the main GA in fungal GAs
biosynthesis and is formed after the conversion of GA12
(Bomeke et al., 2008). The presence of GA5, GA15, GA53,
GA24, and GA19 suggests that because these endophytes live
inside plants, they may have adopted similar characteristics as
observed for Pestalotiopsis microspora (Strobel et al., 1996).
However, such confirmation needs further studies at the
transcriptomics level because various related markers are
available.
Two way traffic: endophytes and crops
Studies have focused on the isolation of endophytes from
asymptomatic plant tissues indicating that individual species
express mutualistism, commensalism, or parasitism lifestyles
when re-inoculated back into the original host species.
Endophytes are already growing in plants either from the
seed development stage through the parental stage of the plant
(Faeth, 2002) or switching their lifestyle to endophyte via
rhizosphere into plants (Figure 2; Rodriguez et al., 2009). In
response to fungal contact through MYC factor development,
the plant increases calcium oscillation and activates defense-
related genes which differentiate the interaction as either
parasitic or mutualistic (Bonfante & Genre, 2010; Harman,
2011; Hause & Fester, 2005; Oldroyd et al., 2009; Ronald &
Shirasu, 2012).
In plants, the intercellular spaces consist of a series of
nonliving components (fluid inorganic ions such as potassium,
calcium, sulphur, phosphorus, and chlorine) of the plant
forming apoplasm and symplasm. The intercellular spaces are
located in the cortical tissue of the root and in the parenchyma
tissue of the stems and leaves (Bonfante & Genre, 2010;
Kuldau & Bacon, 2008). Although the mechanism for this
nutrient transport is still poorly understood, there is evidence
that fungal endophytes possess some hydrolytic enzymes and
metabolites, which can be used in nutrient acquisition (Kuldau
& Bacon, 2008; Oldroyd et al., 2009). The endophytic germ
tube approaches a root causing the abandonment of apical
dominance, forming an aspersorium, which appears to induce
the movement of the plant nucleus towards the contact site
(Figures 2 and 3). The cytoskeletal elements and the
endoplasmic reticulum forms the pre-penetration apparatus
along the axis of the nuclear movement (Bonfante & Genre,
2010). The structure is entered with an infection hypha, from
which colonization of the root cortex begins (Figure 2). Initial
infestation is accompanied by a balanced induction of plant
defense genes (Oldroyd et al., 2009). When the fungus finally
reaches the inner cortex, it penetrates the cell wall and builds
up a hyphal/yeast like structure. The fungus continues to
spread further in the root and also colonizes the surrounding
tissues and soil (Kogel et al., 2006).
Fungal endophytes benefit from inhabiting the plant’s
interior because it is a protected niche, in which there is
relatively little competition from other microorganisms, and a
DOI: 10.3109/07388551.2013.800018 Gibberellins producing endophytes stress tolerance 67

Figure 2. Microscopic analysis showed the active association and habitation of an endophyte (P. resedanum) recovered from pepper roots. The
Capsicum annuum plants inoculated with the endophyte (a) and after three weeks of symbiotic association, the roots showed the presence of the
endophyte (b). Pepper plants were inoculated with P. resedanum fungal spores (105 CFU). After 6 h of inoculation, the spores were attached to the
secondary roots of the pepper plants Bar ¼ 100 mm (c). After one week of endophytic-fungal association, P. resedanum was observed in the endodermal
cells (d) Bar ¼ 200 mm; and pericyle regions of the root Bar ¼ 20 mm (e). The endophytic freeze-dried cells were found in the meta-xylem of the roots
(f-g). Bar ¼ 25 mm; x ¼ 1.2 k and x ¼ 15.0 k. cc – cortex cells; ec – endophytic cells.

reliable source of nutrients. In a mutualistic mode of living
with plants, endophytic fungi increase plant fitness by
dissuading herbivory and pathogenic attacks while facilitating
plant growth through the uptake of essential nutrients,
efficient use of water and counteracting environmental cues
(Schulz & Boyle, 2005). During association, host plants
provide endophytic fungi with a protective sanctuary, access
to nutrients, and dissemination to the next generation, e.g.,
members of Clavicipitaceous and Dikarya (Hyde & Soytong,
2008). Symbiosis of endophytic fungi offer advantages to host
plants in the transport and assimilation of essential biochem-
icals such as nitrogen, phosphorous, etc. necessary for plant
growth and development (Figure 3; Harrison, 2005; Khan
et al., 2012a, b; Kogel et al., 2006; Waller et al., 2005) and
such functions can help the plant to counteract stressful
events.
Endophytes re-program stress responses
Despite extensive research in plant stress responses (Tuteja &
Sopory, 2008), two questions remain unanswered: (i) what are
the mechanisms by which plants adapt to abiotic stress; (ii)
why is there not a greater diversity and ecological distribution
of stress-tolerant plants. The surge in investigating mutualistic
endophytic fungi will help plants to grow well and to tolerate
the extreme environmental conditions (Schulz & Boyle,
2005). An ever-increasing human population and a demand
for agriculture productivity have further enhanced efforts that
emphasize research on such unique aspects. Endophytic fungi
have been known to play an essential role in plant growth,
especially grasses; however, few reports have elucidated their
symbiosis with crops. Recently, the ecological roles of some
endophytes have begun to be elucidated (Arnold et al., 2007;
Harman, 2011; Redman et al., 2001; Waller et al., 2005). Only
a few studies have shown that endophytes confer stress
tolerance to host species and have a significant role in the
survival of at least some crop plants in high-stress environ-
ments (Rodriguez et al., 2004; Supplementary Information I).
Piriformospora indica, a basidiomycete, recovered from
the rhizosphere of Prosopis juliflora and Zizyphus nummu-
laria in the Thar Desert (India), has been known to promote
the growth of various plants/crops (Oelmüller et al., 2009;
68 A. L. Khan et al. Crit Rev Biotechnol, 2015; 35(1): 62–74

Figure 3. Environmental continuum and endophytic interaction with a plant during abiotic stress conditions. The rhizosphere continuum provides a
sanctuary to the microbe in transportation, reproduction and accessibility to nutrients via the plant roots. Once the switch over is successful and
mutualism is established, it lasts for generations throughout the plant’s life. The plant provides a safe haven to the endophyte while facilitating it with
food. In return, it extends diverse benefits to the host plant ranging from an influx of nutrients to regulating the essential biochemicals after exposure to
abiotic stresses. Thus, the effects on the environmental continuum are ameliorated by promoting the metabolism of the phyllosphere involving the
rhizosphere. The ’þ’, ’-’ and ’-/þ’ in the phyllosphere show the increase, decrease and altered activities of various processes during plant development,
respectively, under stress conditions and endophytic association.

Verma et al., 1998). This fungus colonizes plant roots and
increases the biomass of both monocot and eudicot plants
(Verma et al., 1998). In barely, it improved seed yield,
germination and ripening while it enhanced abiotic stress
tolerance in Arabidopsis (Baltruschat et al., 2008; Oelmüller
et al., 2009). Furthermore, the mutualistic association of
P. indica with barley protected it from the adverse effects of
salinity stress (Waller et al., 2005). Since P. indica was
isolated from desert/drought environments, it was suggested
that it might confer drought tolerance to inoculated-plants.
P. indica when inoculated into Chinese cabbage successfully
colonized the plants while its continued association promoted
root/shoot growth and lateral root formation (Druege et al.,
2007; Schäfer et al., 2007). The endophyte extended tolerance
to the inoculated plants under polyethylene glycol induced
drought stress by enhancing antioxidant activities, photosyn-
thetic efficiency and stability to the thylakoid membrane
(Figure 3). The transcriptomic expressions of drought related
genes were significantly up-regulated in the P. indica
colonized plants. This association was further strengthened
by the activation of Ca2þ signaling and related proteins
(Schäfer et al., 2007). P. indica role in heat, salinity and metal
stress has not been fully explored and requires further studies
at transcriptomics levels to understand the mechanism
involved therein. P. indica was associated with barley under
higher salinity stress (Baltruschat et al., 2008). The non-
inoculated barley plants had significant adverse effects on
plant growth because the plants had higher lipid peroxidation
while P. indica infected plants had overcome the negative
effects induced by salinity (Baltruschat et al., 2008; Druege
et al., 2007). Another endophyte, Sebacina vermifera, which
is closely related to P. indica, induces ethylene production in
Nicotiana attenuata. When in symbiosis with barley exposed
to salinity and heat stress, it activates ethylene biosynthesis
and antioxidant enzymes to confer stress tolerance (Barazani
et al., 2007). In contrast to arbuscular mycorrhizal fungi,
P. indica can be easily grown on synthetic media allowing for
large scale propagation and possible use in agriculture
production (Oelmüller et al., 2009; Waller et al., 2005).
Similarly, Khan et al. (2011a, b, c, d) isolated Aspergillus
fumigatus sp. LH02, Penicillium minioluteum LHL09,
Metarhizium anisopliae LHL07, and P. funiculosum LHL06
DOI: 10.3109/07388551.2013.800018
from the roots of Glycine max L. and their association with
the host plant resulted in significantly higher plant biomass
(shoot length, fresh/dry weights) and improved growth under
moderate and high salinity stress. The non-infected plants had
visible symptoms of leaf necrosis and stunted growth and
photosynthesis rates compared to the inoculated ones
(Supplementary information 1). Although these isolated
endophytes are latent plant pathogens and can cause severe
plant diseases, due to their switch over to the endophytic
mode of life in the present case, their effects were ameliora-
tive to the host plants. Additionally, the endophytic stains
were also active in producing GAs in their growth medium;
therefore, in symbiosis with these fungi, the authors observed
growth promotion results. A similar role was reported for
Neotyphodium that secreted alkaloids presumed to have an
active role in enhancing the resistance of the host against
abiotic stresses when habituating Festuca arundinacea
(Schardle et al., 2004).
Besides salinity stress, various endophytes have been
recently reported to confer drought, heat and metal stress
tolerance to cucumber, pepper and tomato plants. Endophyte
Chaetomium globosum LK4 recovered from Capsicum
annuum plants, which grow in drought stressed environments,
promoted shoot length and plant biomass growth as well as an
increase in the photosynthesis rate (Khan et al., 2012b).
Phoma glomerata LWL2, Penicillium sp. LWL3, Exophiala
sp. LHL08 and Paecilomyces formosus LHL10 were isolated
from the roots of cucumber plants growing in agricultural
fields. LHL08 and LHL10 symbiosis helped cucumber plants
overcome the negative effects of salinity (60 and 120 mM),
drought (polyethylene glycol-15%) and temperature (low 5 C
and high 40 C) stresses (Khan et al., 2012a, b, c, d; Waqas
et al., 2012). The endophyte-infected plants had a higher plant
biomass, chlorophyll content and leaf area during stress
conditions while the non-infected counter-part had reduced
plant growth. The association resulted in a reduced level of
electrolytic leakage inside the plant tissues upon exposure to
seven days of salinity, heat and drought stress. Additionally,
the endophytic fungal association also retrieved larger amount
of water from sources inaccessible to the non-infected plants
under stress conditions (Figure 2).
Redman et al. (2001) demonstrated the growth promoting
effects of plant pathogenic fungi Colletotrichum species.
The Colletotrichum magna (path-1), C. orbiculare (683),
C. gloeosporioides (95-41A), C. musae (927), and C. magna
(L2.5) colonized asymptomatically with various crop plants
and established nonpathogenic symbioses with the plants.
Besides a growth stimulatory response, the inoculation
resulted in drought tolerance in the plants. Adopting similar
switchovers, Fusarium culmorum (FcRed1), Curvularia
protuberata (Cp4666D) and (CpYNP5C) infection resulted
in higher biomass and drought and salinity stress tolerance in
rice plants (Redman et al., 2011).
Recently, endophytes have been shown to also have a role
in the bioremediation of heavy metals (Li et al., 2012). Some
of the endophytic fungal strains have been found to ameliorate
the adverse effects of heavy metal stress on crop plants. In this
regard, endophyte Mucore sp. CBRF59 was recovered from
rapeseed plants which were growing in metal contaminated
soils. The endophyte was found to possess the capability to
Gibberellins producing endophytes stress tolerance 69
extend metal tolerance to crop plants (Deng et al., 2011).
Endophytic fungus Microsphaeropsis sp. LSE10 has been
shown to be involved in the biosorption of cadmium from
modified hyper accumulator Solanum nigrum L. (Xiao et al.,
2010).
The role of endophytic fungi in metal tolerance has been
recently elucidated by Li et al. (2012); however, this study
focused more on bacterial strains than fungal ones. Few
endophytes like Microsphaeropsis, Mucor, Phoma,
Alternaria, Peyronellaea, Steganosporium, and Aspergillus
have been known to grow well in a polluted medium and
protect plants from the adverse effects of metal stress (Li
et al., 2012). Very little is known about endophytes such as
Penicillium and its role in host-plant resistant to metal stress.
Some strains of Penicillium janthinellum and P. simplicissi-
mum grow well in a high copper (Cu) medium (Iskandar et al.,
2011) suggesting that they bear a higher biosorption capacity
against Cu. Previous studies showed that some strains of
Penicillium can extend tolerance to host plants against metal
toxicity. For example, the strain of P. janthinellum and
P. simplicissimum reduced the aluminum and zinc toxicity
because it produced citric acid (Zhang et al., 2002). Similarly,
an isolate of Penicillium sp. removed cadmium (Cd) during its
incubation period (Massaccesi et al., 2002). The findings of
these studies suggest that the strains/species of Penicillium
can mitigate Cd and other metal-related toxicity, which can be
attributed to their potential to produce bioactive metabolites
or enzymes (Massaccesi et al., 2002; Zhang et al., 2002).
Previously, it was noted that P. funiculosum produces
bioactive gibberellins which could contribute to the ability
of the fungus to convert toxic metal into stable complexes. A
similar effect of stabilizing the negative effects of sodium
chloride toxicity was also observed when this endophyte was
inoculated into soybean plants (Khan et al., 2011d). Such
beneficial effects could be also attributed to the reduced level
of metal inside the roots and shoots of soybean plants in
symbiosis with the endophyte.
This potential of fungal endophytes could be attributed to
two major features. First, because endophytes are isolated
from host-plants growing in normal or stressed environments,
there is a greater chance that they might extend similar
benefits because it is offered earlier to the host from which it
was isolated. A similar kind of behavior was observed with
Piriformospora indica, which was recovered from rhizosphere
of Prosopis juliflora in the Thar Desert (Verma et al., 1998).
Second, the adverse effects of stress can also be reduced if the
endophyte is potentially synthesizing bioactive metabolites.
Such metabolites can benefit the host while providing an
additional arsenal to the host to combat the negative effects of
the stress. Such capabilities were previously noted by
Schardle et al. (2004) and Redman et al. (2011). Khan et al.
(2011a,b,c,d; 2012a,b) also found that the production of
gibberellins by Penicillium funiculosum LHL06 and
Exophiala sp. LHL08 extended greater benefits to the host
cucumber and soybean plants during salinity and heat stress.
All these studies suggest that mutualistic endophytes can
significantly increase plant growth and yield during changing
environments (as shown in Figure 3). Influencing the root
physiology of soybean plants while delineating the ameliora-
tive effects on shoot growth is an ideal strategy to achieve the
70 A. L. Khan et al.
dual uses of increased crop productivity as well as reduced
toxic effects from copper pollution.
Endogenous oxidative stress and phytohormonal
regulations
Oxidative stress modulation
The antioxidant enzymes produced in plants include reduced
glutathione (GSH), superoxide dismutase (SOD), ascorbic
peroxidase (APX), catalase (CAT), peroxidase (POD), poly-
phenol oxidase (PPO), and so on. Among antioxidant
enzymes, peroxidase and catalase are supposed to play a
central role in abiotic stresses; however, their mechanisms are
still controversial. The activities of these antioxidants vary
greatly among various plant and fungal species as well as in
different environmental conditions during mutualistic symbi-
osis. There are reports which suggest that the activities of
such enzymes increase during the initial symbiosis so that
mutualism can develop; however, other findings suggest that
due to mutualistic symbiosis and the active role of endophytes
in counteracting stress, their activities are less activated
during stress (Figure 3). The latter part also suggests that
these endophytes might help plants avoid stress while reduced
stress means less regulation of the antioxidant apparatus
shown by Khan et al. (2012a, b). Polyphenol oxidase activity
was either not activated or reduced during association with
endophytic fungi under stress conditions (Khan et al., 2012a).
Catalase activity, on the other hand, is involved in the
elimination of H2O2 from mitochondria and microbodies and
can regulate plant stress responses. Studies by Hamilton &
Bauerle (2012), Khan et al. (2012b) and Redman et al. (2011)
have shown that catalase becomes higher in endophyte-treated
plants under heat stress indicating the regulation of H2O2 at a
cellular level. Similarly, the peroxidase activity may be
important in scavenging or utilizing the H2O2 generated in
oxidative stress. During heat stress, a significantly higher
peroxidase activity in endophyte-treated plants was observed
compared to the endophyte-free control indicating that plants
reduced the H2O2 concentration inside the cells. Heat stress
reduced the peroxidase activity in cucumber plants and this
reduction was mitigated by inoculation with endophytic fungi
(Khan et al., 2012c). Antioxidant enzymes activities normally
increase with salinity (Mittler et al., 2004). Endophytic fungi
have both increased peroxidase and catalase activities in
cucumber plants during heat stress (Khan et al., 2012a,b).
When exposed to salinity stress, endophyte P. indica symbi-
osis increased the tolerance of salt-sensitive barley (Hordeum
vulgare). The ascorbate concentrations were significantly
higher in P. indica-colonized plants compared to the non-
inoculated control plants (Baltruschat et al., 2008). Other
antioxidant enzymes like catalase, ascorbate peroxidase,
glutathione reductase, dehydroascorbate reductase and mono-
dehydroascorbate reductase were significantly increased in
endophyte-treated plants under salinity stress (Rodriguez
et al., 2008b; Waller et al., 2005).
Recently, it has been shown that endophytic fungi have a
broad host range and can confer effective tolerance to ROS
under abiotic stress conditions such as salinity (Rodriguez
et al., 2008a). The mechanism responsible for endophyte-
mediated regulation of the plant antioxidant enzyme system is
Crit Rev Biotechnol, 2015; 35(1): 62–74
still poorly understood. However, antioxidant scavengers can
enhance membrane stability against ROS attack while
malondialdehyde (MDA) content can be used to assess
abiotic stress induced injury to the functional membranes of
plants (Mittler et al., 2004). It has been shown that peroxides
of polyunsaturated fatty acids generate MDA, and in many
stresses, MDA is the abundant breakdown product of
aldehydic lipid (Baltruschat et al., 2008). It has been
estimated that more than 75% of the measured MDA is
derived from triunsaturated (trienoic) fatty acids such as
a-linoleic acid (18:3; Davey et al. 2005). Previous results
showed a decline in lipid peroxidation, reduced glutathione
content and superoxide anion activity in plants having
endophytic fungal association under heat stress (Baltruschat
et al., 2008; Khan et al., 2012c, d; Rodriguez, et al., 2004). It
suggests a decrease in membrane injury and lower oxidative
stress in fungus-treated plants than in fungi-free plants (White
& Torres, 2010; Figure 3).
Lipids are the major components of biological membranes
conferring permeability and fluidity to membranes and can
bind enzymes (Davey et al., 2005). Lipid desaturation could
be an important component of plant tolerance in response to
salt stress (Baltruschat et al., 2008). However, the effects are
more devastating on cellular membranes and their function
during drought and heat stress. In Arabidopsis, grown under
high temperature, the total lipid content in membranes
decreased to about one-half and the ratio of unsaturated to
saturated fatty acids decreased to one-third compared to the
levels at normal temperatures (Baltruschat et al., 2008).
Changes in the composition of membrane lipids are
associated with solute leakage. For example, higher electro-
lytic leakage occurs with an increase in the linolenic acid (a
fatty acid particularly prone to oxidation) content and a
decrease in linoleic acid content. However, it was observed
that low leaf electrolytic leakage results in lower linolenic and
higher linoleic acid contents (Khan et al., 2012c, d). Similar
results have been observed in other crop species (Falcone
et al., 2004; Upchurch, 2008); however, in relation to
endophytes, there is still a lack of understanding on the
physiological responses to stress. In the case of salinity stress,
oleic acid is generally reduced in the roots of plants.
Inoculation with P. indica under salinity caused a significant
reduction in oleic acid in barley leaves (Baltruschat et al.,
2008). Exophiala sp. LHL08, P. formosus LHL10 and
P. indica induced changes in the fatty acid composition
after salinity and heat stress. Such effects on the fatty acid
composition of host plants may indicate a symbiotic adaptive
strategy mediated by the endophyte to cope with stress
(Baltruschat et al., 2008). Ameliorative effects might depend
on the following: (i) the host physiology from which the
endophyte was isolated from and (ii) change in its ecological
lifestyle. It is believed that the ameliorative effects of
P. indica on other crop plants are comparable to the effects
of the host growing in its original habitat – the arid Thar
Desert (Baltruschat et al., 2008; White & Torres, 2010).
According to Khan et al. (2012a, b, c), endophytes
associated plants had to activate the antioxidant apparatus to
a lesser degree due to the reduced level of stress. When the
level/intensity of stress convened to the endophyte-inoculated
plants is lower; the degree of activation of the antioxidant
DOI: 10.3109/07388551.2013.800018
apparatus to tackle abiotic stress is also lower. However, in
other findings by Waller et al. (2005) and Hamilton & Bauerle
(2012), different scenarios were observed. Endophytes have
the potential to produce antioxidants which can help in
establishing symbiosis and support plant growth during
extreme environmental conditions (Hamilton et al., 2010).
This too depends on the endophyte’s potential to produce
various types and amounts of antioxidants; however, their role
in plant growth and development during stress has not yet
been fully explored. Additionally, the type of symbiosis
(mutualistic) with antioxidant producing endophytes will
ultimately have different physiological effects (Hamilton
et al., 2010).
Abscisic acid
Abscisic acid (ABA) is pivotal in plant growth because it
promotes stomatal closure to minimize water loss and then
mediates stress damage through activation of many stress-
responsive genes, which collectively increases the plant’s
stress tolerance (Mauch-Mani & Mauch, 2005). An enhanced
ABA level causes an increase in cytosolic Ca2þ concentration
and the subsequent activation of plasma membrane-localized
anion channels (Yamaguchi-Shinozaki & Shinozaki, 2006).
Increased ABA also causes an increase in H2O2 production,
which serves as a signaling intermediate to promote stomatal
closure (Tuteja & Sopory, 2008). Conversely, recent findings
suggest that during endophytic association under abiotic
stress, ABA synthesis is down-regulated (Khan et al., 2011a,
b, c, d; 2012a, 2011b; Waqas et al., 2012). The ABA
responses were almost similar in all stress conditions of
salinity, drought, heat and heavy metal, although the
quantities of ABA synthesis were different because the
intensities of the various stresses were different (Khan et al.,
2011a, b, c, d). This suggests that during stress, symbiosis of
endophytic fungi shows an ABA-independent response. Even
with the symbiosis of mycorrhizal fungi, the effect may
fluctuate among different classes of microorganisms and plant
species because some earlier reports have also found lower
ABA levels during such interactions (Mauch-Mani & Mauch,
2005). It could be postulated that endophyte producing GAs
as an external source in addition to the plant’s own capability
to synthesize GAs can result in a hyper-sensitive response of
endophytic symbiosis with plants under stress conditions
(Tuteja & Sopory, 2008). The same low level of ABA was also
observed when crop plants were exogenously treated with
GA3, a known crop growth regulator during stress (Gangwar
et al., 2011; Hamayun et al., 2010c). Higher ABA syntheses
during stress is also correlated with the inhibition of leaf
expansion and shoot development in different species;
however; endophyte treated plants had a significantly higher
leaf area, photosynthesis and shoot development compared to
the non-inoculated control plants (Khan et al., 2011a, b, c, d;
Redman et al., 2011; Waqas et al., 2012).
Salicylic acid
Salicylic acid (SA) causes Systemic Acquired Resistance
(SAR) and/or Induce Systemic Resistance (ISR; Hayat et al.,
2010). SAR is directly-dependent on the SA biosynthesis
pathway with the up-regulation of genes that encode
Gibberellins producing endophytes stress tolerance 71
pathogenesis related PR proteins (PR1, PR3, PR5, etc.).
Additionally, endogenous SA was shown to potentiate the
generation of reactive oxygen species (ROS) in photosyn-
thetic tissues of Arabidopsis thaliana during salt and osmotic
stresses thus, participating in the development of stress
symptoms. Endogenous SA regulates ROS-induced oxidant
production which can lead to cell death during a hypersen-
sitive response thus, acting as a signal for the development of
SAR. Under a mutualistic relationship, SA production initi-
ates ISR (Pozo & Azcon-Aguilar, 2007). This results in
enhanced SA levels which not only cause ISR but also
mediates the adverse effects of abiotic stresses.
The endophyte associated with crop plants resulted in a
higher level of endogenous SA during exposure to various
stresses like heat, salinity and water deficiency. This suggests
that the endophyte might induce ISR during stress to avoid
greater losses to the plant biomass. Previously, some studies
have shown that the initiation of a mutualistic relationship
between plant and fungi results in ISR and improves
plant performance against biotic and abiotic stresses
(Vicente & Plasencia, 2011). Endophyte Penicillium spp.
are known as potential inducers of ISR in various plants.
This is in agreement with a recent report (Waller et al., 2005)
in which systemic disease protection of barley (Hordeum
vulgare) infected with the endophytic-fungus Piriformospora
indica was not accompanied by increased levels of PR genes
while the same was true for the Penicillium resadenum LK6
under different abiotic stress conditions (unpublished results).
Thus, during endophytic fungal responses, the initiation of
ISR is a key physiological function under stress which needs
further elucidation at the ’omic’ levels. Increased SA
synthesis, however, also counteracts the abiotic stress induced
oxidative burst (Lee & Park, 2010; Waller et al., 2005). Our
scientific understanding of the molecular mechanisms by
which endophytes influence the outcome of plant abiotic
stress tolerance is still marginal.
Jasmonic acid
Jasmonic acid (JA) is a plant signaling molecule that responds
to defend the plant from biotic stress damages (Kunkel &
Brooks, 2002). It is proficient against microbial infection,
herbivory and elicitor treatments (Ren & Dai, 2012).
Previously, Miché et al. (2006) reported on the interaction
of a mutualistic endophyte of rice and other grass species,
Azoarcus sp. strain BH72, with rice roots while applying
exogenous JA. The results showed that the external addition
of JA inhibited the successful colonization of the endophyte
which suggests that in a compatible interaction with endo-
phytes, JA-inducible stress or defense responses are appar-
ently not important. The JA content, however, was
significantly increased when Atractylodes lancea plantlets
were associated with endophyte Gilmaniella sp. AL12 (Ren &
Dai, 2012). Exogenous application of JA suppressed JA and
volatile oil production; however, upon endophyte treatment,
only volatile production was induced. This suggests that JA
acts as a downstream signaling molecule under oxidant-
mediated volatile oil accumulation and endophytic fungal
association while SA is involved in a complementary
interaction (Ren & Dai, 2012). In another study, fungal
72 A. L. Khan et al.
endophytes Phoma glomerata LWL2 and Penicillium sp.
LWL3 improved the growth of cucumber plants during
salinity and drought stress while the endogenous JA content
increased with endophyte inoculation and drought stress
compared to salinity and control treatments (Waqas et al.,
2012). Khan et al. (2011a) showed that the endophyte
Aspergillus fumigatus sp. LH02 can reprogram soybean
plant growth during sodium chloride induced salinity stress
while the endogenous JA levels increased compared to non-
inoculated control plants. These different studies suggest an
altered mechanism of JA responses during endophytic fungal
symbiosis which is either dependent on the expression of
related stress hormones or by the environmental continuum
(Figure 3). Such cross-talk among multitudes of signaling and
responses exists which need to be elucidated in plant cells and
in their signal transduction networks (Miché et al., 2006).
Gibberellins
Very little is known about the status of endogenous gibber-
ellins in response to environmental stresses, particularly under
endophytic interaction. GAs synthesis inside plants is antag-
onistic in relation to ABA biosynthesis during abiotic stress
(Hedden et al., 2002). Physiologically active GAs (GA3 and
GA4) are activated to improve plant growth. As mentioned
earlier, exogenous application of GAs will not only enhance
plant growth but will also increase plant tolerance to stress.
This is due to the increase in the synthesis of endogenous GAs
which promotes shoot growth by cellular elongation and
overall plant development (Gangwar et al., 2011; Hamayun
et al., 2010c; Tuna et al., 2008). This was observed when
cucumber plants treated with salinity and drought conditions
had significantly higher bioactive GAs contents when
associated with endophytes (Khan et al., 2012a, c, d).
Interestingly, ABA has recently been shown to negatively
regulate SA-mediated defenses by down-regulating SA bio-
synthesis (Horvath et al., 2007). This is consistent with the
well-known ABA/GAs antagonistic regulation of many
aspects of plant development. Thus, it seems clear that
ABA and GAs are able to control plant immune responses by
modulating the levels of SA. Thus, up until now, research
findings have suggested that endophytes stimulate further
phytohormonal signaling to help crop plants counteract stress.
The increase in endogenous GAs, abnormally lower ABA,
significantly higher SA and altered/or inactive JA biosynthe-
sis might be responsible for extending tolerance to stress crop
plants. However, these postulates need further studies to
elucidate the interaction and physiology during such diverse
environmental conditions.
Future perspectives
In grasses, endophytes have been known to confer stress
tolerance; however, being a source of food security for
humans, little is known about crops and their endophytic
fungal wealth. This area needs to be explored to fully identify
the capacity of endophytes to not only improve plant biomass
and yield but also to confer stress resistant to broader varieties
of crop plants. Most of the studies on endophyte stress
resistance have been undertaken in controlled environments
while extensive field level trails have been ignored.
Crit Rev Biotechnol, 2015; 35(1): 62–74
Additionally, extremophilic endophytic fungi exploration
has been overlooked. Other than P. indica, there is no other
example of an active extremophilic endophyte. Endophytes
are famous for the production of bioactive metabolites but
less is known about their potential to secret phytohormones
although a few species have recently been shown to do this.
Research in this area is growing and nascent reports are
available on such potential; however, the synthesis of other
hormones like SA, auxins, ABA, cytokinin, ethylene,
jasmonic acid, etc. are still unexplored. Endophytes may
also be assessed for their ability to produce polyamines
(putrescine, spermidine and spermine), sugar, organic acid,
amino acid, antioxidants, enzymes, etc. because these func-
tional biochemicals play an essential role in plant
stress physiology. Most GAs have been extracted, purified
and quantified with chromatographic and spectroscopic
techniques while little attention given to the detailed eluci-
dation using nuclear magnetic resonance techniques to
confirm the structures. Besides quantification of GAs in
endophytic cultures, there is a dire need to elucidate and
confirm the biosynthetic makeup of GAs from these GAs
producing endophytes as was conducted in the case of G.
fujikuroi, S. manihoticola and Phaeosphaeria sp. L487
(Figure 1). Molecular markers and mutants are now available
for GAs gene cluster analysis to confirm biosynthesis
pathways.
The inter- or intra-connecting mechanisms involved in
extending stress tolerance to host plants, especially in crops,
still needs to be fully elucidated. Now, with the abundance of
various crop mutants, understanding crop plant stress physi-
ology, signaling of plant hormones, bioactive secondary
metabolites and genomics expressions during endophyte-plant
interaction will be an important step towards higher crop
production. The interactions of ‘‘omics’’ are still poorly
understood regarding endophyte-plant-symbiosis. The
Arabidopsis signal transduction mutants are available and
reverse genetics could be used to accelerate the disclosure of
the molecular basis of symbiosis and its beneficial effects on
the host. Despite this perspective, differences in signaling
pathways relevant to agronomically important traits exist
between Arabidopsis and cereals, which justify a strong
emphasis on future cereal research. Studies have been
performed mostly on drought and salinity stress tolerance
by endophytic fungi, while temperature and heavy metal
stresses are less studied with crop plants. The potential of
endophytic fungi in bioremediation, biosensors and biocata-
lyst development needs comprehensive assessment and
further exploration.
Acknowledgement
We would like to thank the financial supporters (Eco-
Innovation Project, Korean Government’s R & D program
on Environmental Technology and Development, Republic of
Korea) of this research.
Declaration of Interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
DOI: 10.3109/07388551.2013.800018
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