Endophytic Fungus Aspergillus japonicus Mediates...

Research ArticleEndophytic Fungus Aspergillus japonicus Mediates Host PlantGrowth under Normal and Heat Stress Conditions

Ismail1 Muhammad Hamayun 1 Anwar Hussain 1 Amjad Iqbal 2

Sumera Afzal Khan3 and In-Jung Lee 4

1Department of Botany Abdul Wali Khan University Mardan 23200 Pakistan2Department of Agriculture Abdul Wali Khan University Mardan 23200 Pakistan3Centre of Biotechnology and Microbiology University of Peshawar Pakistan4School of Applied Biosciences Kyungpook National University Daegu 41566 Republic of Korea

Correspondence should be addressed to Muhammad Hamayun hamayunawkumedupkAmjad Iqbal amjadiqbal147yahoocom and In-Jung Lee ijleeknuackr

Received 22 June 2018 Revised 7 October 2018 Accepted 24 October 2018 Published 6 December 2018

Academic Editor Shoji Mano

Copyright copy 2018 Ismail et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We have isolated an endophytic fungus with heat stress alleviation potential from wild plant Euphorbia indica L The phylogeneticanalysis and 18S rDNA sequence homology revealed that the designated isolate was Aspergillus japonicus EuR-26 Analysis of Ajaponicus culture filtrate displayed higher concentrations of salicylic acid (SA) indoleacetic acid (IAA) flavonoids and phenolicsFurthermore A japonicus association with soybean and sunflower had improved plant biomass and other growth features underhigh temperature stress (40∘C) in comparison to endophyte-free plants In fact endophytic association mitigated heat stress bynegotiating the activities of abscisic acid catalase and ascorbic acid oxidase in both soybean and sunflowerThe nutritional quality(phenolic flavonoids soluble sugars proteins and lipids) of the A japonicus-associated seedlings has also improved under heatstress in comparison to endophyte-free plants From the results it is concluded that A japonicus can modulate host plants growthunder heat stress and can be used as thermal stress alleviator in arid and semiarid regions of the globe (where mean summertemperature exceeds 40∘C) to sustain agriculture

1 Introduction

Temperature stress is becoming more persistent especiallyin arid semiarid and tropical zones of the globe causinggreat extortions to food crops Rising in the total meanglobal temperature also results in saline conditions throughmoisture evaporation from the soil Plants are vulnerableto various stresses because of their sessile nature In suchabnormal state reactive oxygen species (ROS) are generatedin high concentrations which cause apoptosis or prematurecell death when exposed for a longer time Different ROSspecies including singlet oxygen (1O

2) superoxide (O

2

minus1)hydroxyl radical (OH) and hydrogen peroxide (H

2O2) con-

taining free electrons are leaked in chloroplast and mito-chondria from electron transport chain [1] ROS are trappedby biological membranes where they are detoxified by localantioxidants except H

2O2[2] But all plants contain a specific

enzymatic antioxidant system in the form of superoxidedismutase (SOD) catalase (CAT) ascorbic acid oxidase(AAO) and peroxidase (POD) which neutralize ROS beforethey come into action to damage the cell components [3]CAT and AAO specifically target ROS by lowering theirenergy or disturbing their oxidizing chain reactions [4]CAT is known to have multigene family responsible forencoding vital plant proteins These vital proteins havesignificance in localization regulation and expression ofstress receptive genes in cells facing different environmentalstresses [5] Similarly AAO helps in controlling the levelof membrane bound antioxidant 120572-tocopherol as well asreorganization of cell wall during different environmentalstresses [6]Moreover plants also have the potential tomodu-late biochemical and physiological modifications dependingon variable capacity to identify stimuli and conduct signals[7]

HindawiBioMed Research InternationalVolume 2018 Article ID 7696831 11 pageshttpsdoiorg10115520187696831

2 BioMed Research International

Phytohormones like jasmonic acid (JA) abscisic acid(ABA) and salicylic acid (SA) serve as a signaling compoundduring stress [8] Different phytohormones respond differ-ently during biotic and abiotic stresses for example ABAregulates stomatal closure plant growth and developmentunder stressful conditions [9] SA controls plant growthdevelopment flower induction stomatal response ethylenesynthesis and respiration [10] Similarly JA is known tohave a role in the biosynthesis of defensive secondarymetabolites and proteins [11] JA also helps in modulation ofmany physiological phenomena like resistance to insects andpathogen pollen development senescence and root growth[12] Endophytic fungi that reside inside plant tissues withoutcausing any visible symptoms are one of the best bio agentsthat help in restoration of plant growth under stress [13]Theyalso have a role in enhancement of host growth increasingof nutrient absorption reduction in disease severity andimproving of host resistance against environmental stresses[13] Vital secondary metabolites like hormones (GAs IAASA and JA) flavonoids phenolics and proline are alsosecreted by endophytic fungi in their culture filtrate and prob-ably in their host tissues [14] Plants lacking in endophyticfungi are more susceptible to drought salinity heat andpathogenic stresses hence overall yield is highly reduced insuch abnormal situations as compared to endophyte-hostedplants [15] The present study was an attempt to explore suchendophytic fungi that have the potential to improve host plantgrowth and yield as well as enhance their immunity underthermal stress

2 Materials and Methods

21 Plant Sample Collection and Endophyte Isolation A wildplant Euphorbia indica L was collected from the desertarea of District Noshera Khyber Pakhtunkhwa Pakistan andprocessed for the isolation of endophytic fungi using Khanet al [16] procedure Fungal isolates were purified on potatodextrose agar (PDA) medium and kept in refrigerator at4∘C For longer storage PDA slants were made [17] Forthe collection and analysis of fungal secondary metabolitesendophyte isolates were grown in 50 ml of Czapek mediumfor 7 days in a shaking incubator set at 120 rpm at 28∘C

22 Screening Bioassay of fungal Filtrate on Rice SeedlingsScreening bioassay of fungal filtrate was carried out on riceseedlings by applying 100120583l of fungal filtrate on the tip ofrice seedlings (Fakhr-e-Malakand variety provided by theAgricultural Biotechnology Institute National AgriculturalResearch Center Islamabad Pakistan) at the two leavesstage grown in 08 water-agar medium in growth chamber(daynight cycle 14 h 28∘C plusmn 03 10 h 25∘C plusmn 03 relativehumidity 70) for 7 days Root-shoot length and fresh anddry weights were analyzed after one week of filtrate treatmentand compared with Czapek and distilled water treated plants

23 Fungal Identification For molecular identification ofendophytic fungi Khan et al [16] method was appliedInternal transcribed region of 18S rRNA was amplified byusing primers ITS1 (51015840-TCC GTA GGT GAA CCT GCG

G-31015840) and ITS4 (51015840-TCC TCC GCT TAT TGA TAT GC-31015840) The sequence obtained was then imperiled to BLASTn1for sequence homology approximation Neighbor joining(NJ) tree was applied for the phylogenetic analysis using theMEGA 7 software

24 Inoculation of Aspergillus japonicus EuR-26 to Soybeanand Sunflower Endophytic fungus A japonicus was grownin 250 ml conical flask containing 50 ml Czapek broth andkept in shaking incubator set at 120 rpm for 7 days at 28∘CPellet and supernatant was separated using filter paper Onemilligram of fungal biomass per 100 g of sterilized sandwas inoculated to pots containing 9 seeds of soybean andsunflower each and shifted to growth chambers set at 25∘Cand 40∘C for two weeks while supernatant was used forthe determination of secondary metabolites Half strengthHoagland solution (10 ml) was applied to the pots at 48 hintervals Growth parameters were evaluated after 2 weeksof incubation in growth chamber [18] The experimentswere performed in triplicate (each replicate consisted of 9seedlings)

25 IAA and SAAnalysis in the Culture Filtrate of A japonicusFor the determination of IAA in the filtrate of A japonicusBenizri et al [19] protocol was used Two ml Salikowskireagent was mixed well with 1 ml of pure fungal filtrate andthen kept in the dark for half an hour at 25∘C OD wastaken at 540 nm with the help of PerkinElmer Lambda 25spectrophotometer Different concentrations (10 20 30 4060 80 and 100 120583gml) of the IAA (Sigma Aldrich) were usedto construct the standard curve

For the determination of SA the method of Warrier etal [20] was followed Exactly 299 ml freshly prepared FeCl

3

(01) solution was mixed with 100 120583l of fungal filtrate Afterthe appearance of violet color ODwas observed at 540 nm Astandard curve was plotted by taking different concentrations(100 200 300 400119908 and 500 120583gml) of pure SA (SigmaAldrich)

26 ABA Analysis in Sunflower and Soybean For the analysisof ABA contents in soybean and sunflower the protocol ofYoon et al [21] was followed Fresh leaves of soybean and sun-flower (05 g each) were ground in liquid nitrogen Amixtureof isopropanol (15 ml) and glacial acetic acid (285 ml) wasadded to the grounded leaves The mixture was filtered anddehydrated by means of a rotary evaporator Diazomethanewas then added to the mixture and analyzed by GCndashMS SIM(6890N network GC system equipped with 5973 networkmass selective detector Agilent Technologies Palo Alto CAUSA) The Lab-Base (Thermo Quset Manchester UK) datasystem software was used to observe responses to ions withmz values of 162 and 190 for Me-ABA and 166 and 194 forMe-[2H6]-ABA ABA ([2H6]-ABA) from Sigma Aldrich wasused as internal standard

27 Analysis of Antioxidants in Soybean and SunflowerSeedlings Luck [22] protocol was followed in the deter-mination of CAT concentration in soybean and sunflowerseedlings Fresh leaves (2 g) of soybean and sunflower were

BioMed Research International 3

Table 1 Effect of A japonicus filtrate on the growth of rice seedlings

Growth attributes Control (DW) Control (Czk) A japonicusShoot Length (cm) 126 plusmn 025a 138 plusmn 034ab 1567 plusmn 052b

Root Length (cm) 63 plusmn 038a 66 plusmn 018ab 81 plusmn 035b

Dry Weight Shoot (g) 00034 plusmn 0001a 00049 plusmn 0003a 00086 plusmn 0004b

Dry Weight Root (g) 00147 plusmn 0001a 00155 plusmn 0004a 00182 plusmn 0004b

Chlorophyll (SPAD) 2073 plusmn 15a 227 plusmn 056b 259 plusmn 018c

DW= distilled water Czk = Czapek broth medium Data are means of 3 replicates (each replicate consisted of 9 seedlings) with standard error For each set oftreatment the different letter indicates significant differences at p (lt 005) as estimated by Tukey HSD test

crushed in phosphate buffer (10 ml) and centrifuged forfive minutes at 10000 rpm H

2O2-phosphate buffer (3 ml)

was then added to the supernatant (40 120583l) and the OD wasobserved at 240 nm H

2O2-free phosphate buffer solution

was used as blank The enzyme unit was calculated as theamount of enzyme required to decrease the absorbance at 240nm by 005 Oberbacher and Vines [23] protocol was used tomeasure the concentration of AAO in soybean and sunflowerseedlings Fresh leaves (01 g) of soybean and sunflower wereground in phosphate buffer (2 ml) and centrifuged for fiveminutes at 3000 rpm About 3 ml of the substrate solution(88 mg of ascorbic acid in 300 ml phosphate buffer pH 56)was mixed with 100 120583l of supernatant and the OD was notedafter every 30 seconds till 5 minutes at 265 nm

28 Analysis of Phenolics Proline and Flavonoids in theCulture Filtrate of A japonicus Sunflower and SoybeanCai et al [24] procedure was employed for the analysis ofphenolics in soybean sunflower and fungal filtrate Differentconcentrations (100 200 300 500 600 700 and 900mgml)of gallic acid (Sigma Aldrich) were used to construct astandard curve For the determination of proline the methodof Bates et al [25] was followed but after somemodificationsA standard curve was plotted against different concentrations(2 4 6 8 and 10 120583gml) of standard proline (Sigma Aldrich)and the OD was taken at 520 nm For the analysis of totalflavonoids the method of Mervat et al [26] was used A stan-dard curve was constructed by using different concentrationsof pure quercetin (15 30 60 120 240 and 480 120583gml SigmaAldrich) and the OD were taken at 415 nm

29 Analysis of Total Proteins Lipids and Soluble SugarsLowry et al [27] method was employed for the analysis oftotal proteins in the seedlings of soybean and sunflowerA standard curve was drawn using different concentrations(20 40 60 80 and 100 120583gml) of BSA (Sigma Aldrich) andthe OD was observed at 650 nm For the determination oftotal lipids we used the method of Van Hand et al [28]with some modifications A standard curve was constructedusing different concentrations of canola oil (10 40 70 100130 and 160 120583gml) and the OD was noticed at 490 nmFor the analysis of total soluble sugars in the soybean andsunflower seedlings the method of Mohammadkhani andHeidari [29] was used A standard curve was constructed byusing different concentrations (20 40 60 80 and 100 120583gml)of glucose (Sigma Aldrich) and the OD was measured at485 nm

210 Statistical Analysis All the experiments were performedin triplicate ANOVA (one-way analysis of variance) was usedfor the analysis of data and means were compared by TukeyHSD test at p lt 005 using SPSS-20 (SPSS Inch Chicago ILUSA) for windows

3 Results

31 Endophytes Isolation and Their Preliminary Screening onRice Seedlings We isolated 14 different endophytic fungi(Figure S1) from the wild plant of Euphorbia indica Land initially screened on rice seedlings for their growthpromoting or inhibiting activities (Figure S2) Endophyteswere first isolated on Hagem minimal medium and thenpurified on PDA on the basis of morphological differencesAfter screening on rice seedlings at the two leaves stagegrowth parameters were recorded after 1 week of filtratetreatment (Table 1) EuR-26 isolate was found to be growthpromoter and chosen for molecular identification

32 Molecular Identification of Fungal Isolate EuR-26 Freshmycelium was used for the extraction of genomic DNAwhile applying ITS1 (51015840-TCC GTA GGT GAA CCT GCGG-31015840) and ITS4 (51015840-TCC TCC GCT TAT TGA TAT GC-31015840) Sequence of ITS region of related fungi was comparedwith the nucleotide sequence of our fungal isolate EuR-26 with the help of the BLAST search program Sequenceof 18S rDNA exhibited maximum similarity (92) with AJaponicas The phylogenetic consensus tree was built from 14(13 reference and 1 clone) by neighbor joining (NJ) methodusing MEGA 7 software (Figure 1) Our isolate EuR-26 madea clad with A japonicus supported by 92 bootstrap valuein the consensus tree Combining results of phylogeneticanalysis and sequence homology suggested that the strainEuR-26 was A japonicus

33 Analysis of SecondaryMetabolites in theCulture Filtrates ofA japonicus Culture filtrate ofA japonicuswas analyzed forthe presence of important secondary metabolites includingIAA SA flavonoids and phenolics Different concentra-tions of secondary metabolites were IAA (1919 120583gml) SA(6311120583gml) flavonoids (556 120583gml) and phenolic (44mgml) (Figure 2)

34 Growth Features of A japonicus-Associated Soybeanand Sunflower Both A japonicus-associated soybean andsunflower at 25∘C and 40∘C showed significant increase in


0

0

0

92

2

22

12

4

3

15

JF7704351 Aspergillus japonicus

EuR-26

DQ1962051 Aspergillus japonicus

KC1288151 Aspergillus japonicus

KX5758521 Aspergillus japonicus

KU7290791 Aspergillus fijiensis

JF4394601 Aspergillus aculeatus

JX5014121 Aspergillus aculeatus

MG6825031 Aspergillus violaceofuscus

FR7338051 Aspergillus violaceofuscus

FJ0377551 Aspergillus niger

KT7269191 Aspergillus niger

JQ3165201 Aspergillus uvarum

NR 1353301 Aspergillus uvarum

63

Figure 1 Phylogenetic consensus tree using neighbor joining (NJ)method for the identification of isolate EuR-26 using 14 taxa 13 referencesand 1 cloneThe isolate was identified as Aspergillus japonicus as having 92 bootstrap value

IAA (gml) SA (gml) Flavonoids (gml) Phenolics (mgml)

CF of A japonicus

0

20

40

60

80

Con

cent

ratio

n

Figure 2 Analysis of secondary metabolites secreted by A japonicus (EuR-26) in their culture filtrate (CF) grown for 7 days in Czapekmedium in shaking incubator set at 120 rpm at 28∘CThe bars labeled with different letters are significantly different at p lt 005 as estimatedby Tukey HSD test The bars represent SE of a triplicate data (each replicate consisted of 9 seedlings)

various growth parameters including chlorophyll contentshoot length shoot fresh and dry weights as compared toendophyte-free plants At room temperature (25∘C) soybean(Table 2) and sunflower (Table 3) seedlings have morechlorophyll root-shoot length and dry weight than underheat stress condition (40∘C)

35 Modulation in A japonicus-Associated Soybean andSunflowerrsquos Endogenous Flavonoids Phenols and ProlineImportant secondary metabolites like flavonoids pheno-lics and prolines were analyzed in soybean and sunflowerseedlings inoculated with A japonicus at 25∘C and 40∘Cin growth chamber An increase has been noted in Ajaponicus-associated (4113 120583gg) and control (3796 120583gg)soybean seedlings at 40∘C as compared at 25∘C (3153 120583ggin experimental and 2091120583gg in control seedlings) while

endophyte-aligned seedlings have more flavonoids contentas compared to the endophyte-free seedlings Similarlysunflower seedlings aligned with A japonicus have moreflavonoids (4104 120583gg at 25∘C and 3443 120583gg at 40∘C) thancontrol plants (3618 120583gg at 25∘C and 2464 120583gg at 40∘C)at both temperatures while a decrease has been noted atthermal stress as compared to normal condition (Figure 3(a))We also found a slight reduction in the phenolics and prolineconcentration inA japonicus-aligned soybean and sunflowerseedlings as compared to control plants At 25∘C endophyte-aligned soybean has 28 mgg and endophyte-free seedlingshave 313mgg of phenolicswhile at 40∘C endophyte-alignedsoybean has 589 mgg and endophyte-free seedlings have 78mgg of phenolics Sunflower seedlings inoculated with Ajaponicus at 25∘C have 138 mgg and control plants have 201mgg of total phenolics while at 40∘C endophyte-associated


Table 2 Effect of A japonicus on the growth features of soybean

Growth attributes 25∘C 40∘CControl A japonicus Control A japonicus

Chlorophyll (SPAD) 326 plusmn 021a 374 plusmn 04d 29 plusmn 246a 332 plusmn 007c

Shoot Length (cm) 387 plusmn 176b 483 plusmn 12e 26 plusmn 093a 355 plusmn 126bc

Root Length (cm) 163 plusmn 39abc 133 plusmn 233ab 10 plusmn 06ab 12 plusmn 26abcd

Dry Weight Shoot (g) 014 plusmn 001a 0147 plusmn 001ab 008 plusmn 00003ab 009 plusmn 00001ab

Dry Weight Root (g) 008 plusmn 0002ab 010 plusmn 001bc 001 plusmn 00001ab 0062 plusmn 0001e

Data aremeans of three replicates (each replicate consisted of 9 seedlings) with standard errors For each set of treatment the different letter indicates significantdifferences at p (lt 005) as estimated by Tukey HSD test

Table 3 Effect of A Japonicas on the growth features of sunflower


Chlorophyll (SPAD) 39 plusmn 129a 433 plusmn 06ab 384 plusmn 195a 399 plusmn 312a

Shoot Length (cm) 237 plusmn 088a 252 plusmn 043ab 205 plusmn 045a 21 plusmn 153ab

Root Length (cm) 9 plusmn 058a 116 plusmn 087abcd 6 plusmn 058b 6 plusmn 025b

Dry Weight Shoot (g) 0085 plusmn 0009ab 0088 plusmn 0007ab 004 plusmn 00006b 0037 plusmn 00002a

Dry Weight Root (g) 0024 plusmn 0001a 0028 plusmn 0002a 0014 plusmn 00003b 002 plusmn 00002c


sunflower has 353 mgg and endophyte-free seedlings have434 mgg of phenolics (Figure 3(b)) A significant reductionwas found in the total proline concentration in soybean (771120583gg at 25∘C and 1446 120583gg at 40∘C) and sunflower (129120583gg at 25∘C and 168 120583gg at 40∘C) seedlings aligned with Ajaponicus as compared to control soybean (115 120583gg at 25∘Cand 164 120583gg at 40∘C) and sunflower (309 120583gg at 25∘C and373 120583gg at 40∘C) seedlings (Figure 3(c))

36 Reduction in the Concentration of CAT and AAO Asignificant reduction was detected in the concentration ofCAT andAAO in soybean and sunflower seedlings associatedwith A japonicus as compared to control plants Endophyte-aligned soybean has 046 enzyme unitg tissue and sunflowerhad 025 enzyme unitg tissue CAT at 25∘C as comparedto endophyte-free soybean 07 enzyme unitg tissue andsunflower 043 enzyme unitg tissue At 40∘C the soybeanseedlings had 095 enzyme unitg tissue and sunflower had06 units of enzyme unitg tissue as compared to controlsoybean 114 enzyme unitg tissue and sunflower 098 enzymeunitg tissue (Figure 4(a)) A similar decrease was found inthe amount of AAO in soybean and sunflower aligned withA japonicus at 25∘C and 40∘C as compared to endophyte-freesoybean and sunflower (Figure 4(b))

37 Reduction in Endogenous ABA Content in Soybeanand Sunflower A significant reduction was found in theconcentration of ABA in soybean and sunflower seedlingsassociated with A japonicus as compared to control seedlingsat 40∘C Endophyte-associated soybean has 2556 ngg andendophyte-free seedling had 4642 ngg at 25∘C At 40∘Cthe experimental soybean had 11378 ngg and controlseedling has 26246 ngg of ABA concentrations Simi-larly Endophyte-associated sunflower has 2605 ngg and

endophyte-free seedling has 2731 ngg at 25∘Cwhile at 40∘Cexperimental sunflower has 6032 ngg and control seedlinghas 6578 ngg of ABA concentrations (Figure 5)

38 Enhancement in the Nutritive Value of Soybean andSunflower Nutritive values of soybean and sunflower weredetermined by quantifying total sugar protein and lipidcontents A japonicus-associated soybean has 2028 120583ggwhile control plants have 19639 120583gg of soluble sugars at25∘C and at 40∘C experimental soybean seedlings had 16903120583gg while control seedlings have 13375 120583gg total sugarcontent A similar increase was observed in the total sugarconcentrations of sunflower seedlings inoculated with anendophytic fungus as compared to endophyte-free seedlingsunder heat stress (Figure 6(a)) A significant increase wasdetected in the total protein contents of soybean (30283 120583ggat 25∘C and at 2408 120583gg 40∘C) and sunflower (18915 120583ggat 25∘C and at 22729 120583gg 40∘C) aligned with A japonicus ascompared control seedlings of soybean (21367 120583gg at 25∘Cand at 16304 120583gg 40∘C) and sunflower (20413 120583gg at 25∘Cand at 12584 120583gg 40∘C) (Figure 6(b)) An enhancement inthe total lipids was found in A japonicus-inoculated soybeanand sunflower as compared to A japonicus-free soybean andsunflower at 25∘C and 40∘C (Figure 6(c))

4 Discussion

Endophytic fungi are known to have symbiotic associationwith plants in natural ecosystem [30] The endophytes areresponsible for synthesizing a large number of vital bioactivesecondary metabolites including phenolics flavonoids andalkaloids that have importance in the fields of medicinefood and agriculture [31] Endophytes have a defensiverole for their host plant in providing resistance against


A

CE F

DEF

B

D

Control EuR-6 Control EuR-6 Control EuR-6 Control EuR-6

Soybean Sunflower

Flavonoids

25∘C 40

∘C 25∘C 40

∘C

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cent

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gg)

(a)

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Soybean Sunflower

Phenolics

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(b)

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Soybean Sunflower

Proline

25∘C 40

∘C 25∘C 40

∘C

048

121620

Con

cent

ratio

n (

gg)

(c)

Figure 3 Effect ofA japonicus on the concentration of (a) flavonoid (b) phenolics and (c) proline in soybean and sunflower seedlings grownat 25∘C and 40∘C For each set of treatment the different letter indicates significant differences at p (lt 005) as estimated by Tukey HSD testThe bars represent SE of a triplicate data (each replicate consisted of 9 seedlings)

abiotic and biotic stresses as well as improving overallgrowth and yield [32] We isolated endophytic fungi fromwild plant Euphorbia indica L found in xeric conditionand screened them for their growth promoting potentialunder thermal stress Fungal filtrate was initially screenedon rice seedlings (Fakhr-e-Malakand variety provided bythe Agricultural Biotechnology Institute National Agricul-tural Research Center Islamabad Pakistan) for their growthpromoting or inhibiting activities The rice was selectedbecause their response is very easy and quick to growthhormones like GAs and IAA secreted by endophytic fungi[33] Culture filtrate of A japonicus significantly enhancedrice growth features and suggested that this endophyte hasthe potential for plant growth promotion which was later

on confirmed by detecting IAA in their culture filtrate Plantgrowth promoting potential of endophytic fungi and bacteriawas previously reported by Mei and Flinn [34] Endophyticfungi improve plant growth and development by secretingbioactive secondary metabolites and sharing of mutualisticgenes that can work better for host performance [32] Pro-longed exposure of plants to different stresses like droughtsalinity and heat results in growth inhibition accelerationof apoptosis and premature cell death [35] Present studyon the association of A japonicus with roots of soybeanand sunflower caused a significant increase in chlorophyllcontent root-shoot length and biomass of host plants Theincrease in chlorophyll contents in endophyte-associatedplants might be associated with increased photosynthetic


CAB

DD

ABA

D

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Soybean Sunflower

CAT

00

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tsg

tissu

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Soybean Sunflower

AAO

00

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tsg

tissu

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25∘C 40

∘C 25∘C 40

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(b)

Figure 4 Effect of A japonicus on the concentration of (a) CAT and (b) AAO in soybean and sunflower seedlings grown at 25∘C and 40∘COne enzyme unit was calculated as the amount of enzyme required to decrease the absorbance at 240 nm for CAT and 265 nm for AAO by005 units For each set of treatment the different letter indicates significant differences at p (lt 005) as estimated by TukeyHSD testThe barsrepresent SE of triplicate data (each replicate consisted of 9 seedlings)

BA

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Soybean Sunflower

ABA

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gg)

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∘C 25∘C 40

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Figure 5 GC MS quantification of endogenous ABA concentration in soybean and sunflower grown at 25∘C and 40∘C with and without Ajaponicus For each set of treatment the different letter indicates significant differences at p (lt 005) as estimated by Tukey HSD testThe barsrepresent SE of a triplicate data (each replicate consisted of 9 seedlings)


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Figure 6 Analysis of (a) soluble sugars (b) proteins and (c) lipids of soybean and sunflower seedlings grown at 25∘C and 40∘C with- andwithout-endophytic fungal strain A japonicus For each set of treatment the different letter indicates significant differences at p (lt 005) asestimated by Tukey HSD test The bars represent SE of a triplicate data (each replicate consisted of 9 seedlings)

rate Sun et al [36] reported similar observations that theendophyte Piriformospora indica successfully inhibited thedrought initiated losses in chlorophyll contents by maintain-ing the photosynthetic rate Similarly plant endophytes havebeen noticed to promote plant growth and biomass underabiotic stress conditions Penicillium resedanum Curvulariaprotuberate Alternaria sp and Trichoderma harzianum areone of the best examples that have controlled the shoot androot lengths and biomass production of the host plants underabiotic stress [37ndash39] The host plant growth promotion bythe endophytes under abiotic stress can be attributed to

the IAA and GA production [40ndash42] This means that Ajaponicus as an endophyte helped the host plants (soybeanand sunflower) to grow normal under heat stress ie 40∘CAlso it is possible that the IAA producing A japonicusmightalso be helpful in alleviating biotic and abiotic stresses in hostplant species Our results strongly supported and confirmedthe findings of Mei and Flinn [34] who reported that IAAproducing fungal and bacterial endophytes can improverice growth under drought salinity and high temperaturestress Besides the plant growth promotion A japonicas havealso improved the nutritional quality of both soybean and


sunflower under stress condition TheA japonicas associatedsoybean and sunflower seedlings have higher amounts ofsoluble sugars total proteins and total lipids as comparedto the nonassociated seedlings of both species when grownunder 40∘C Similar observations have been noted by Cliftonet al [43] who said that the alleviation in nutritional qualityof soybean was due to fungi M brunneum Certainly theendophytes might help the host plants to absorb optimumamounts of nutrients from rhizosphere to fulfill its nutrientrequirements even under stress conditions

Signaling by phytohormones especially ABA is one of theimportant defense strategies by higher plants under abioticstresses ABA significantly increases during abiotic stresseslike high temperature in rice seedlings [44] Heat stresscause upregulation of genes responsible for ABA biosyn-thesis while it downregulates the genes involved in ABAcatabolism [45] We found low levels of ABA in A japonicus-aligned soybean and sunflower exposed to thermal stress ascompared to control plants indicating that endophytic fungihave the capacity to lower the effect of heat stress for hostplants

Low concentration of ROS is required for better signalinggrowth and development while their high concentration isinjurious to plant tissues leading to premature cell deathPlants display a remarkable collection of enzymatic andnonenzymatic antioxidant defense systems that neutralizeROS toxicity in stressful conditions [46] CAT is known tobe involved directly while AAO as indirectly in ROS detoxi-fication High concentration of antioxidants including CATand AAO determines stress severity and susceptibility inplants [47] A significant decrease was found in the activitiesof CAT and AAO in A japonicus-associated soybean andsunflower under thermal stress as compared to endophyte-free plants CAT and AAO have a role in degrading excessof H2O2formed in microbodies and mitochondria of plants

under stress as well as regulate stress responses in plantsOur results confirmed the work of Waqas et al [10] whoobserved that endophytes-hosted plants are more resistant toenvironmental stresses than endophyte-free plants

Plants accumulate high content of proline in response todifferent environmental stresses as an osmolyte a bufferingagent for cellular redox and a scavenger of free radicalspotential [48] It also plays a role in alleviating cytoplasmicacidosis and keeping proper proportion of NADP+NADPHnecessary for harmonious plant-cellular-metabolism [49]and proteins [50] After the harsh stressful condition hoardedproline quickly disintegrates releasing high concentration ofpowerful reducing agents which speed up ATP synthesis andrenovation of stress-induced damage [51] Some abiotic stressresponsive genes that have proline responsive elements likePRE ACTCAT in their promoter regions are also expressedby proline accumulated under stress [52] High concentra-tion of proline in our controlled soybean and sunfloweras compared to A japonicus-associated plants confirmedthat proline accumulation enhances under abiotic stressesincluding high temperature [53] while endophytic fungi helpin reducing the stress severity for the host plant

Phenolics are natural compounds synthesized in responseof changing environmental conditions and essential for plants

in defensivemechanisms [54]Different phenolic compoundsare accumulated by higher plants as defense tools againstdifferent biotic and abiotic stresses [55] Accumulation ofphenolics by plants during stressful condition is one ofthe common indicators of plants Thus our results thattemperature stress enhances phenolics in soybean and sun-flower strongly support the research work of Lattanzio etal [56] Moreover decrease in the amount of phenolics inA japonicus-associated soybean and sunflower suggests thatendophytic fungi have a role in alleviating high temperaturestress in host plants On the contrary the amounts offlavonoids increased in A japonicus-associated soybean andsunflower seedlings in comparison to the A japonicus-freeseedlings From the results we can argue that A japonicascan ameliorate the production of flavonoids in both speciesto improve its health protective effect on human beingsThe results of the present study are in accordance with theobservations of Yang et al [57] who observed high totalflavonoids in fungal inoculated wine grapes

5 Conclusion

The current study revealed that endophyte-associated plantshave promoted the growth of soybean and sunflowerseedlings under normal as well as high temperature stressas compared to endophyte-free plants Moreover the inter-action of endophytic fungi with economically vital cropscan significantly improve their nutritive quality and quantityHence the uses of endophytic fungi not only promote growthof the plants under normal condition but also alleviatethermal stress in crop plants Therefore the application ofA japonicus to crop plants is suggested in the future forsustainable agriculture

Data Availability

The authors confirm that the whole data is presented in themanuscript and supplementary files

Ethical Approval

Our study does not involve any human animal or endangeredspecies

Consent

No consentapproval at the national or international level orappropriate permissions andor licenses for the study wasrequired

Conflicts of Interest

The authors declare that there are no conflicts of interest offinancial or nonfinancial nature related to this manuscript

Authorsrsquo Contributions

All the authors have equally contributed in this manuscript


Acknowledgments

This research was supported by the Basic Science ResearchProgram through the National Research Foundationof Korea (NRF) funded by the Ministry of Education(2017R1D1A1B04035601)

Supplementary Materials

Supplementary 1 Figure S1 colonies of endophytic fungigrown on Hagem minimal medium and purified on PDAmedia plates isolated from Euphorbia indica L Differentfungal colonies (14) were isolated from the host plant 9 fromroots and 5 from stem ie EuR-1 EuR-2 EuR-3 EuR-4 EuR-5 EuR-6 EuR-8 EuR-23 EuR-26 EuS-1 EuS-2 EuS-3 EuS-5and EuS-14 (EuR represents Euphorbia indica L root whileEuS represents Euphorbia indica L stem) Supplementary2 Figure S2 screening bioassay of fungal culture filtrates(100120583l) isolated from Euphorbia indica L on rice seedlingsat 2 leaves stage grown in 08 water-agar medium for 2weeks at 25∘C Reading taken after 1 week of culture filtrateapplication 14 sets of pots (3 pots in each set) are shown Eachset has 3 treatments includingCzapek control (right) distilledwater control (left) and endophyte cultural filtrate (middle)(Supplementary Materials)

References

[1] M Ashraf and P J C Harris ldquoPotential biochemical indicatorsof salinity tolerance in plantsrdquo Journal of Plant Sciences vol 166no 1 pp 3ndash16 2004

[2] L J Quan B Zhang W W Shi and H Y Li ldquoHydrogenperoxide in plants a versatile molecule of the reactive oxygenspecies networkrdquo Journal of Integrative Plant Biology vol 50 no1 pp 2ndash18 2008

[3] E H Alici and G Arabaci ldquoDetermination of SOD POD PPOand cat enzyme activities in Rumex obtusifolius Lrdquo AnnualResearch amp Review in Biology vol 11 no 3 pp 1ndash7 2016

[4] A Mhamdi G Queval S Chaouch S Vanderauwera F VanBreusegem and G Noctor ldquoCatalase function in plants a focuson Arabidopsis mutants as stress-mimic modelsrdquo Journal ofExperimental Botany vol 61 no 15 pp 4197ndash4220 2010

[5] Y Su J Guo H Ling et al ldquoIsolation of a novel peroxisomalcatalase gene from sugarcane which is responsive to biotic andabiotic stressesrdquoPLoSONE vol 9 no 1 Article ID e84426 2014

[6] P L Conklin ldquoRecent advances in the role and biosynthesis ofascorbic acid in plantsrdquo Plant Cell amp Environment vol 24 no4 pp 383ndash394 2001

[7] H Shao Z Liang andM Shao ldquoChanges of some anti-oxidativeenzymes under soil water deficits among 10 wheat genotypes attillering stagerdquo Journal of the Science of Food and Agriculturevol 86 2005

[8] K Shinozaki and K Yamaguchi-Shinozaki ldquoGene networksinvolved in drought stress response and tolerancerdquo Journal ofExperimental Botany vol 58 no 2 pp 221ndash227 2007

[9] A Wasilewska F Vlad C Sirichandra et al ldquoAn update onabscisic acid signaling in plants and more sdot sdot sdot rdquoMolecular Plantvol 1 no 2 pp 198ndash217 2008

[10] M Waqas A L Khan M Kamran et al ldquoEndophytic fungiproduce gibberellins and indoleacetic acid and promotes host-plant growth during stressrdquoMolecules vol 17 no 9 pp 10754ndash10773 2012

[11] V Balbi and A Devoto ldquoJasmonate signalling network inArabidopsis thaliana Crucial regulatory nodes and new physi-ological scenariosrdquo New Phytologist vol 177 no 2 pp 301ndash3182008

[12] O Lorenzo J M Chico J J Sanchez-Serrano and R SolanoldquoJASMONATE-INSENSITIVE1 encodes a MYC transcriptionfactor essential to discriminate between different jasmonate-regulated defense responses in arabidopsisrdquo The Plant Cell vol16 no 7 pp 1938ndash1950 2004

[13] R J Rodriguez C J Woodward and R S Redman ldquoFungalinfluence on plant tolerance to stressrdquo Biocomplexity of Plant-Fungal Interactions pp 155ndash163 2012

[14] X Sun L-D Guo and K D Hyde ldquoCommunity compositionof endophytic fungi in Acer truncatum and their role indecompositionrdquo Fungal Diversity vol 47 pp 85ndash95 2011

[15] K Saikkonen S Saari andM Helander ldquoDefensive mutualismbetween plants and endophytic fungirdquo Fungal Diversity vol 41pp 101ndash113 2010

[16] S A Khan M Hamayun H Yoon et al ldquoPlant growthpromotion and Penicillium citrinumrdquo BMCMicrobiology vol 82008

[17] S A Khan M Hamayun H-Y Kim et al ldquoA new strainof Arthrinium phaeospermum isolated from Carex kobomugiOhwi is capable of gibberellin productionrdquo Biotechnology Let-ters vol 31 no 2 pp 283ndash287 2009

[18] N Misra and U N Dwivedi ldquoGenotypic difference in salinitytolerance of green gram cultivarsrdquo Journal of Plant Sciences vol166 no 5 pp 1135ndash1142 2004

[19] E Benizri A Courtade C Picard and A Guckert ldquoRoleof maize root exudates in the production of auxins by Pseu-domonas fluorescensM31rdquo Soil Biology amp Biochemistry vol 30no 10-11 pp 1481ndash1484 1998

[20] R Warrier M Paul and M Vineetha ldquoEstimation of salicylicacid in Eucalyptus leaves using spectrophotometric methodsrdquoGenetics and Plant Physiology vol 3 no 1-2 pp 90ndash97 2013

[21] J Y Yoon M Hamayun S-K Lee and I-J Lee ldquoMethyljasmonate alleviated salinity stress in soybeanrdquo Journal of CropScience and Biotechnology vol 12 no 2 pp 63ndash68 2009

[22] H LuckMethods in Enzymatic Analysis Academic Press NewYork NY USA 1974

[23] M F Oberbacher and H M Vines ldquoSpectrophotometric assayof ascorbic acid oxidaserdquo Nature vol 197 no 4873 pp 1203-1204 1963

[24] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medicinalplants associated with anticancerrdquo Life Sciences vol 74 no 17pp 2157ndash2184 2004

[25] L S Bates R P Waldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[26] M M M El Far and H A A Taie ldquoAntioxidant activitiestotal anthocyanins phenolics and flavonoids contents of somesweetpotato genotypes under stress of different concentrationsof sucrose and sorbitolrdquo Australian Journal of Basic and AppliedSciences vol 3 no 4 pp 3609ndash3616 2009

[27] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951


[28] E Van Handel ldquoRapid determination of glycogen and sugarsin mosquitoesrdquo Journal of the American Mosquito ControlAssociation vol 1 no 3 pp 299ndash301 1985

[29] N Mohammadkhani and R Heidari ldquoDrought-induced accu-mulation of soluble sugars and proline in two maize varietiesrdquoWorld Applied Sciences Journal vol 3 no 3 pp 448ndash453 2008

[30] J Zhao L Zhou JWang et al ldquoEndophytic fungi for producingbioactive compounds originally from their host plantsrdquoCurrentResearch Technology and Education Topics in Applied Microbi-ology and Microbial Biotechnology vol 1 pp 567ndash576 2010

[31] C Liu T Liu F Yuan and Y Gu ldquoIsolating endophyticfungi from evergreen plants and determining their antifungalactivitiesrdquo African Journal of Microbiology Research vol 4 no21 pp 2243ndash2248 2010

[32] R J Rodriguez J F White Jr A E Arnold and R SRedman ldquoFungal endophytes diversity and functional rolesrdquoNew Phytologist vol 182 no 2 pp 314ndash330 2009

[33] M Hamayun A Hussain S A Khan et al ldquoKinetin modulatesphysio-hormonal attributes and isoflavone contents of soybeangrown under salinity stressrdquo Frontiers in Plant Science vol 6 p377 2015

[34] C Mei and B S Flinn ldquoThe use of beneficial microbial endo-phytes for plant biomass and stress tolerance improvementrdquoRecent Patents on Biotechnology vol 4 no 1 pp 81ndash95 2010

[35] M Iqbal andMAshraf ldquoGibberellic acidmediated induction ofsalt tolerance in wheat plants Growth ionic partitioning pho-tosynthesis yield and hormonal homeostasisrdquo Environmentaland Experimental Botany vol 86 pp 76ndash85 2013

[36] C Sun J M Johnson D Cai I Sherameti R Oelmuller andB Lou ldquoPiriformospora indica confers drought tolerance inChinese cabbage leaves by stimulating antioxidant enzymes theexpression of drought-related genes and the plastid-localizedCAS proteinrdquo Journal of Plant Physiology vol 167 no 12 pp1009ndash1017 2010

[37] A L Khan M Waqas and I-J Lee ldquoResilience of Penicilliumresedanum LK6 and exogenous gibberellin in improving Cap-sicum annuum growth under abiotic stressesrdquo Journal of PlantResearch vol 128 no 2 pp 259ndash268 2015

[38] R S Redman Y O Kim C J Woodward et al ldquoIncreasedfitness of rice plants to abiotic stress via habitat adaptedsymbiosis a strategy for mitigating impacts of climate changerdquoPLoS ONE vol 6 no 7 Article ID e14823 2011

[39] K Azad and S Kaminskyj ldquoA fungal endophyte strategy formitigating the effect of salt and drought stress on plant growthrdquoSymbiosis vol 68 no 1ndash3 pp 73ndash78 2016

[40] A Hussain S T Shah H Rahman M Irshad and A IqballdquoEffect of IAA on in vitro growth and colonization of Nostocin plant rootsrdquo Frontiers in Plant Science vol 6 2015

[41] M Hamayun A Hussain S A Khan et al ldquoGibberellins pro-ducing endophytic fungus Porostereum spadiceum AGH786rescues growth of salt affected soybeanrdquo Frontiers in Microbi-ology vol 8 2017

[42] L Bilal S Asaf M Hamayun et al ldquoPlant growth promot-ing endophytic fungi Asprgillus fumigatus TS1 and Fusariumproliferatum BRL1 produce gibberellins and regulates plantendogenous hormonesrdquo Symbiosis pp 1ndash11 2018

[43] E H Clifton S T Jaronski B S Coates E W Hodgson and AJ Gassmann ldquoEffects of endophytic entomopathogenic fungi onsoybean aphid and identification of Metarhizium isolates fromagricultural fieldsrdquoPLoSONE vol 13 no 3 Article ID e01948152018

[44] A S Raghavendra V K Gonugunta A Christmann and EGrill ldquoABA perception and signallingrdquo Trends in Plant Sciencevol 15 no 7 pp 395ndash401 2010

[45] S Toh A Imamura A Watanabe et al ldquoHigh temperature-induced abscisic acid biosynthesis and its role in the inhibitionof gibberellin action inArabidopsis seedsrdquoPlant Physiology vol146 no 3 pp 1368ndash1385 2008

[46] G K Agrawal S Tamogami H Iwahashi V P Agrawal and RRakwal ldquoTransient regulation of jasmonic acid-inducible riceMAP kinase gene (OsBWMK1) by diverse biotic and abioticstressesrdquo Plant Physiology and Biochemistry vol 41 no 4 pp355ndash361 2003

[47] F Waller B Achatz H Baltruschat et al ldquoThe endophyticfungus Piriformospora indica reprograms barley to salt-stresstolerance disease resistance and higher yieldrdquo Proceedings ofthe National Acadamyof Sciences of the United States of Americavol 102 no 38 pp 13386ndash13391 2005

[48] P B Kavi Kishor S Sangam R N Amrutha et al ldquoRegulationof proline biosynthesis degradation uptake and transport inhigher plants Its implications in plant growth and abiotic stresstolerancerdquoCurrent Science vol 88 no 3 pp 424ndash438 2005

[49] P D Hare and W A Cress ldquoMetabolic implications of stress-induced proline accumulation in plantsrdquo Plant Growth Regula-tion vol 21 no 2 pp 79ndash102 1997

[50] V Srinivas and D Balasubramanian ldquoProline is a protein-compatible hydrotroperdquo Langmuir vol 11 no 7 pp 2830ndash28331995

[51] P D Hare W A Cress and J Van Staden ldquoDissecting theroles of osmolyte accumulation during stressrdquo Plant Cell ampEnvironment vol 21 no 6 pp 535ndash553 1998

[52] V Chinnusamy A Jagendorf and J-K Zhu ldquoUnderstandingand improving salt tolerance in plantsrdquo Crop Science vol 45no 2 pp 437ndash448 2005

[53] W Claussen ldquoProline as a measure of stress in tomato plantsrdquoJournal of Plant Sciences vol 168 no 1 pp 241ndash248 2005

[54] S Caretto V Linsalata G Colella G Mita and V LattanzioldquoCarbon fluxes between primary metabolism and phenolicpathway in plant tissues under stressrdquo International Journal ofMolecular Sciences vol 16 no 11 pp 26378ndash26394 2015

[55] R Nakabayashi and K Saito ldquoIntegrated metabolomics forabiotic stress responses in plantsrdquo Current Opinion in PlantBiology vol 24 pp 10ndash16 2015

[56] V Lattanzio A Cardinali C Ruta et al ldquoRelationship ofsecondarymetabolism to growth in oregano (OriganumvulgareL) shoot cultures under nutritional stressrdquo Environmental andExperimental Botany vol 65 no 1 pp 54ndash62 2009

[57] M Yang M Ma M Yuan et al ldquoFungal endophytes as ametabolic fine-tuning regulator for wine graperdquo PLoS ONE vol11 no 9 Article ID e0163186 2016

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


Phytohormones like jasmonic acid (JA) abscisic acid(ABA) and salicylic acid (SA) serve as a signaling compoundduring stress [8] Different phytohormones respond differ-ently during biotic and abiotic stresses for example ABAregulates stomatal closure plant growth and developmentunder stressful conditions [9] SA controls plant growthdevelopment flower induction stomatal response ethylenesynthesis and respiration [10] Similarly JA is known tohave a role in the biosynthesis of defensive secondarymetabolites and proteins [11] JA also helps in modulation ofmany physiological phenomena like resistance to insects andpathogen pollen development senescence and root growth[12] Endophytic fungi that reside inside plant tissues withoutcausing any visible symptoms are one of the best bio agentsthat help in restoration of plant growth under stress [13]Theyalso have a role in enhancement of host growth increasingof nutrient absorption reduction in disease severity andimproving of host resistance against environmental stresses[13] Vital secondary metabolites like hormones (GAs IAASA and JA) flavonoids phenolics and proline are alsosecreted by endophytic fungi in their culture filtrate and prob-ably in their host tissues [14] Plants lacking in endophyticfungi are more susceptible to drought salinity heat andpathogenic stresses hence overall yield is highly reduced insuch abnormal situations as compared to endophyte-hostedplants [15] The present study was an attempt to explore suchendophytic fungi that have the potential to improve host plantgrowth and yield as well as enhance their immunity underthermal stress

2 Materials and Methods

21 Plant Sample Collection and Endophyte Isolation A wildplant Euphorbia indica L was collected from the desertarea of District Noshera Khyber Pakhtunkhwa Pakistan andprocessed for the isolation of endophytic fungi using Khanet al [16] procedure Fungal isolates were purified on potatodextrose agar (PDA) medium and kept in refrigerator at4∘C For longer storage PDA slants were made [17] Forthe collection and analysis of fungal secondary metabolitesendophyte isolates were grown in 50 ml of Czapek mediumfor 7 days in a shaking incubator set at 120 rpm at 28∘C

22 Screening Bioassay of fungal Filtrate on Rice SeedlingsScreening bioassay of fungal filtrate was carried out on riceseedlings by applying 100120583l of fungal filtrate on the tip ofrice seedlings (Fakhr-e-Malakand variety provided by theAgricultural Biotechnology Institute National AgriculturalResearch Center Islamabad Pakistan) at the two leavesstage grown in 08 water-agar medium in growth chamber(daynight cycle 14 h 28∘C plusmn 03 10 h 25∘C plusmn 03 relativehumidity 70) for 7 days Root-shoot length and fresh anddry weights were analyzed after one week of filtrate treatmentand compared with Czapek and distilled water treated plants

23 Fungal Identification For molecular identification ofendophytic fungi Khan et al [16] method was appliedInternal transcribed region of 18S rRNA was amplified byusing primers ITS1 (51015840-TCC GTA GGT GAA CCT GCG

G-31015840) and ITS4 (51015840-TCC TCC GCT TAT TGA TAT GC-31015840) The sequence obtained was then imperiled to BLASTn1for sequence homology approximation Neighbor joining(NJ) tree was applied for the phylogenetic analysis using theMEGA 7 software

24 Inoculation of Aspergillus japonicus EuR-26 to Soybeanand Sunflower Endophytic fungus A japonicus was grownin 250 ml conical flask containing 50 ml Czapek broth andkept in shaking incubator set at 120 rpm for 7 days at 28∘CPellet and supernatant was separated using filter paper Onemilligram of fungal biomass per 100 g of sterilized sandwas inoculated to pots containing 9 seeds of soybean andsunflower each and shifted to growth chambers set at 25∘Cand 40∘C for two weeks while supernatant was used forthe determination of secondary metabolites Half strengthHoagland solution (10 ml) was applied to the pots at 48 hintervals Growth parameters were evaluated after 2 weeksof incubation in growth chamber [18] The experimentswere performed in triplicate (each replicate consisted of 9seedlings)

25 IAA and SAAnalysis in the Culture Filtrate of A japonicusFor the determination of IAA in the filtrate of A japonicusBenizri et al [19] protocol was used Two ml Salikowskireagent was mixed well with 1 ml of pure fungal filtrate andthen kept in the dark for half an hour at 25∘C OD wastaken at 540 nm with the help of PerkinElmer Lambda 25spectrophotometer Different concentrations (10 20 30 4060 80 and 100 120583gml) of the IAA (Sigma Aldrich) were usedto construct the standard curve

For the determination of SA the method of Warrier etal [20] was followed Exactly 299 ml freshly prepared FeCl

3

(01) solution was mixed with 100 120583l of fungal filtrate Afterthe appearance of violet color ODwas observed at 540 nm Astandard curve was plotted by taking different concentrations(100 200 300 400119908 and 500 120583gml) of pure SA (SigmaAldrich)

26 ABA Analysis in Sunflower and Soybean For the analysisof ABA contents in soybean and sunflower the protocol ofYoon et al [21] was followed Fresh leaves of soybean and sun-flower (05 g each) were ground in liquid nitrogen Amixtureof isopropanol (15 ml) and glacial acetic acid (285 ml) wasadded to the grounded leaves The mixture was filtered anddehydrated by means of a rotary evaporator Diazomethanewas then added to the mixture and analyzed by GCndashMS SIM(6890N network GC system equipped with 5973 networkmass selective detector Agilent Technologies Palo Alto CAUSA) The Lab-Base (Thermo Quset Manchester UK) datasystem software was used to observe responses to ions withmz values of 162 and 190 for Me-ABA and 166 and 194 forMe-[2H6]-ABA ABA ([2H6]-ABA) from Sigma Aldrich wasused as internal standard

27 Analysis of Antioxidants in Soybean and SunflowerSeedlings Luck [22] protocol was followed in the deter-mination of CAT concentration in soybean and sunflowerseedlings Fresh leaves (2 g) of soybean and sunflower were

















3 Results






0

0

0

92

2

22

12

4

3

15


EuR-26













63



CF of A japonicus

0

20

40

60

80

Con

cent

ratio

n



























4 Discussion



A

CE F

DEF

B

D


Soybean Sunflower

Flavonoids

25∘C 40

∘C 25∘C 40

∘C

0

10

20

30

40

50

Con

cent

ratio

n (

gg)

(a)

D C

HG

B A

F E


Soybean Sunflower

Phenolics

25∘C 40

∘C 25∘C 40

∘C

0

5

10

15

Con

cent

ratio

n (m

gg)

(b)

FE

HG

C A D B


Soybean Sunflower

Proline

25∘C 40

∘C 25∘C 40

∘C

048

121620

Con

cent

ratio

n (

gg)

(c)





CAB

DD

ABA

D

BC


Soybean Sunflower

CAT

00

04

08

12

16

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(a)

CDBC

DBCD

BA

E

D


Soybean Sunflower

AAO

00

10

20

30

40

50

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(b)


BA

F

E

A A

D C


Soybean Sunflower

ABA

0

50

100

150

200

250

300

Con

cent

ratio

n (n

gg)

25∘C 40

∘C 25∘C 40

∘C



E E

CD

C

F

AB



25∘C 40

∘C 25∘C 40

∘C

0

50

100

150

200

250

Con

cent

ratio

n (

gg)

(a)

ABC

C

AB

BCABC AB

A

BC


Soybean Sunflower

Total protein

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(b)

E

F

CDE

AB

C

ABBC


Soybean Sunflower

Total Lipids

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(c)












5 Conclusion


Data Availability


Ethical Approval


Consent







Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


















3 Results






0

0

0

92

2

22

12

4

3

15


EuR-26













63



CF of A japonicus

0

20

40

60

80

Con

cent

ratio

n



























4 Discussion



A

CE F

DEF

B

D


Soybean Sunflower

Flavonoids

25∘C 40

∘C 25∘C 40

∘C

0

10

20

30

40

50

Con

cent

ratio

n (

gg)

(a)

D C

HG

B A

F E


Soybean Sunflower

Phenolics

25∘C 40

∘C 25∘C 40

∘C

0

5

10

15

Con

cent

ratio

n (m

gg)

(b)

FE

HG

C A D B


Soybean Sunflower

Proline

25∘C 40

∘C 25∘C 40

∘C

048

121620

Con

cent

ratio

n (

gg)

(c)





CAB

DD

ABA

D

BC


Soybean Sunflower

CAT

00

04

08

12

16

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(a)

CDBC

DBCD

BA

E

D


Soybean Sunflower

AAO

00

10

20

30

40

50

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(b)


BA

F

E

A A

D C


Soybean Sunflower

ABA

0

50

100

150

200

250

300

Con

cent

ratio

n (n

gg)

25∘C 40

∘C 25∘C 40

∘C



E E

CD

C

F

AB



25∘C 40

∘C 25∘C 40

∘C

0

50

100

150

200

250

Con

cent

ratio

n (

gg)

(a)

ABC

C

AB

BCABC AB

A

BC


Soybean Sunflower

Total protein

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(b)

E

F

CDE

AB

C

ABBC


Soybean Sunflower

Total Lipids

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(c)












5 Conclusion


Data Availability


Ethical Approval


Consent







Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0

0

0

92

2

22

12

4

3

15


EuR-26













63



CF of A japonicus

0

20

40

60

80

Con

cent

ratio

n



























4 Discussion



A

CE F

DEF

B

D


Soybean Sunflower

Flavonoids

25∘C 40

∘C 25∘C 40

∘C

0

10

20

30

40

50

Con

cent

ratio

n (

gg)

(a)

D C

HG

B A

F E


Soybean Sunflower

Phenolics

25∘C 40

∘C 25∘C 40

∘C

0

5

10

15

Con

cent

ratio

n (m

gg)

(b)

FE

HG

C A D B


Soybean Sunflower

Proline

25∘C 40

∘C 25∘C 40

∘C

048

121620

Con

cent

ratio

n (

gg)

(c)





CAB

DD

ABA

D

BC


Soybean Sunflower

CAT

00

04

08

12

16

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(a)

CDBC

DBCD

BA

E

D


Soybean Sunflower

AAO

00

10

20

30

40

50

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(b)


BA

F

E

A A

D C


Soybean Sunflower

ABA

0

50

100

150

200

250

300

Con

cent

ratio

n (n

gg)

25∘C 40

∘C 25∘C 40

∘C



E E

CD

C

F

AB



25∘C 40

∘C 25∘C 40

∘C

0

50

100

150

200

250

Con

cent

ratio

n (

gg)

(a)

ABC

C

AB

BCABC AB

A

BC


Soybean Sunflower

Total protein

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(b)

E

F

CDE

AB

C

ABBC


Soybean Sunflower

Total Lipids

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(c)












5 Conclusion


Data Availability


Ethical Approval


Consent







Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018
























4 Discussion



A

CE F

DEF

B

D


Soybean Sunflower

Flavonoids

25∘C 40

∘C 25∘C 40

∘C

0

10

20

30

40

50

Con

cent

ratio

n (

gg)

(a)

D C

HG

B A

F E


Soybean Sunflower

Phenolics

25∘C 40

∘C 25∘C 40

∘C

0

5

10

15

Con

cent

ratio

n (m

gg)

(b)

FE

HG

C A D B


Soybean Sunflower

Proline

25∘C 40

∘C 25∘C 40

∘C

048

121620

Con

cent

ratio

n (

gg)

(c)





CAB

DD

ABA

D

BC


Soybean Sunflower

CAT

00

04

08

12

16

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(a)

CDBC

DBCD

BA

E

D


Soybean Sunflower

AAO

00

10

20

30

40

50

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(b)


BA

F

E

A A

D C


Soybean Sunflower

ABA

0

50

100

150

200

250

300

Con

cent

ratio

n (n

gg)

25∘C 40

∘C 25∘C 40

∘C



E E

CD

C

F

AB



25∘C 40

∘C 25∘C 40

∘C

0

50

100

150

200

250

Con

cent

ratio

n (

gg)

(a)

ABC

C

AB

BCABC AB

A

BC


Soybean Sunflower

Total protein

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(b)

E

F

CDE

AB

C

ABBC


Soybean Sunflower

Total Lipids

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(c)












5 Conclusion


Data Availability


Ethical Approval


Consent







Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



A

CE F

DEF

B

D


Soybean Sunflower

Flavonoids

25∘C 40

∘C 25∘C 40

∘C

0

10

20

30

40

50

Con

cent

ratio

n (

gg)

(a)

D C

HG

B A

F E


Soybean Sunflower

Phenolics

25∘C 40

∘C 25∘C 40

∘C

0

5

10

15

Con

cent

ratio

n (m

gg)

(b)

FE

HG

C A D B


Soybean Sunflower

Proline

25∘C 40

∘C 25∘C 40

∘C

048

121620

Con

cent

ratio

n (

gg)

(c)





CAB

DD

ABA

D

BC


Soybean Sunflower

CAT

00

04

08

12

16

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(a)

CDBC

DBCD

BA

E

D


Soybean Sunflower

AAO

00

10

20

30

40

50

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(b)


BA

F

E

A A

D C


Soybean Sunflower

ABA

0

50

100

150

200

250

300

Con

cent

ratio

n (n

gg)

25∘C 40

∘C 25∘C 40

∘C



E E

CD

C

F

AB



25∘C 40

∘C 25∘C 40

∘C

0

50

100

150

200

250

Con

cent

ratio

n (

gg)

(a)

ABC

C

AB

BCABC AB

A

BC


Soybean Sunflower

Total protein

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(b)

E

F

CDE

AB

C

ABBC


Soybean Sunflower

Total Lipids

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(c)












5 Conclusion


Data Availability


Ethical Approval


Consent







Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



CAB

DD

ABA

D

BC


Soybean Sunflower

CAT

00

04

08

12

16

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(a)

CDBC

DBCD

BA

E

D


Soybean Sunflower

AAO

00

10

20

30

40

50

Uni

tsg

tissu

e

25∘C 40

∘C 25∘C 40

∘C

(b)


BA

F

E

A A

D C


Soybean Sunflower

ABA

0

50

100

150

200

250

300

Con

cent

ratio

n (n

gg)

25∘C 40

∘C 25∘C 40

∘C



E E

CD

C

F

AB



25∘C 40

∘C 25∘C 40

∘C

0

50

100

150

200

250

Con

cent

ratio

n (

gg)

(a)

ABC

C

AB

BCABC AB

A

BC


Soybean Sunflower

Total protein

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(b)

E

F

CDE

AB

C

ABBC


Soybean Sunflower

Total Lipids

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(c)












5 Conclusion


Data Availability


Ethical Approval


Consent







Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



E E

CD

C

F

AB



25∘C 40

∘C 25∘C 40

∘C

0

50

100

150

200

250

Con

cent

ratio

n (

gg)

(a)

ABC

C

AB

BCABC AB

A

BC


Soybean Sunflower

Total protein

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(b)

E

F

CDE

AB

C

ABBC


Soybean Sunflower

Total Lipids

25∘C 40

∘C 25∘C 40

∘C

0

100

200

300

400

Con

cent

ratio

n (

gg)

(c)












5 Conclusion


Data Availability


Ethical Approval


Consent







Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018










5 Conclusion


Data Availability


Ethical Approval


Consent







Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Acknowledgments




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Endophytic Fungus Aspergillus japonicus Mediates...

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Transcript of Endophytic Fungus Aspergillus japonicus Mediates...