assessment of plant growth promoting and abiotic stress...

Research ArticleAssessment of Plant Growth Promoting and Abiotic StressTolerance Properties of Wheat Endophytic Fungi

Farhana Alam Ripa Wei-dong Cao Shuai Tong and Jian-guang Sun

Key Laboratory of Microbial Resources Ministry of AgricultureInstitute of Agricultural Resources and Regional PlanningChinese Academy of Agricultural Sciences Beijing 100081 China

Correspondence should be addressed to Jian-guang Sun sunjianguangcaascn

Received 6 December 2018 Revised 22 February 2019 Accepted 6 March 2019 Published 27 March 2019

Academic Editor Atanas Atanassov

Copyright copy 2019 Farhana Alam Ripa et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aims of the present work were to isolate and characterize fungal endophytic communities associated with healthy wheat(Triticum aestivum L) plants collected from the North China Segregated endophytes were screened for their PGP traits abioticstresses (heavy metals salinity drought and temperature) and antibiotic sensitivity A total of 16 endophytic fungi were isolatedusing the culture-dependent approach from different tissue parts of wheat plants Based upon their internal transcribed spacer(ITS) rDNA gene sequencing 15 out of 16 isolates were selected for further analysis In the contemporary investigation a numberof the tested endophytes exhibited fairly good 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) (003plusmn0011 to 143plusmn001120583mol 120572-KB mgminus1 protein hrminus1) indole acetic acid (IAA) (1125plusmn004 to3612plusmn0004120583gmlminus1) and phosphate solubilizing index(PSI) (208plusmn003to516plusmn036) activities More than 30 isolates gave positive result for siderophore and ammonia tests whereasall exhibited catalase activity but only 2 (582PDA1 and 582PDA11) produced hydrogen cyanide Trichoderma strains showed saltheavy metals and drought tolerance at high levels and also exhibited resistance to all the tested antibiotics Strain 582PDA4 wasfound to be the most temperature (55∘C) tolerant isolateThe findings of this study indicated that the microbial endophytes isolatedfrom wheat plants possessing a crucial function to improve plant growth could be utilized as biofertilizers or bioagents to establisha sustainable crop production system

1 Introduction

Globally wheat is considered as one of the major cereal cropsAccording to Food and Agricultural Organization (FAO) ofthe United Nations its demand will be amplified up to 746million tons by 2020 [1] This raise in production desiresto be accomplished in spite of the budding challenges tomodern agriculture as well as precincts in the applicationof pesticides [2] concerns about the accessibility and envi-ronmental impact of fertilizer inputs [3] and the potentialharmful impacts of climate alteration on wheat yields anddisease spectrum [4] Cultivation of high yielding varietiesof crops rigorous cropping system and unevenness useof chemical fertilizers are the core factors which developnutrient discrepancy in soil squat yield shrinkage of soilfertility and stumpy quality of food Hence it turns to asevere problem to develop sustainable tactics for mitigationof unfavorable effect of intensive practices used by peasants

[5] The question of primary production sustainability isheightened more for wheat Agricultural scientists aroundthe planet are working round the clock to look for noveloptions to enhance agricultural productivity sustainabilitybut it undoubtedly represents an immense challenge forthem The use of beneficial microbial symbionts of plantswith the objective of improving agricultural productivityis one of the most important sustainable practices [6]Concerning to reduce the harmful effects of the conven-tional methods of agriculture innovative schemes based onmicrobial inoculation are currently gaining more attentionPlants and microorganisms form a symbiotic alliance withreimbursement for both cohorts Additionally plant-microbesymbiosis influences plant growth and health which effi-ciently ameliorates agricultural traits and improve soil qualityand nutrient cycling [7ndash9]

Normally a number of microbes are found to acquirenutrients for their continued existence through interaction

HindawiBioMed Research InternationalVolume 2019 Article ID 6105865 12 pageshttpsdoiorg10115520196105865

2 BioMed Research International

with plants which might be impartial harmful (parasitism)or beneficial (mutualism or symbiosis) to the host [10 11]Microbes that inhabit within the plant tissues devoid of doingsubstantive damage or acquiring remuneration other thansecuring their residency are considered as lsquoendophytesrsquo Plantshave been blessed by nature with diversified population ofendophytic microorganisms including beneficial bacteriafungi and actinomycetes They spend their entire or partof the life cycle living inside the plant causing no visualsymptoms of disease [12] Organization of such useful plantcoupled microbes is continuously gaining attention amongscientific community and at the view point of industries dueto their aptitude to advance plant quality and growth [13 14]

Species of fungi that reside within living plant tissuewithout causing symptoms of disease in their host are knownas fungal endophytes [15] They are the major members ofendophytic population that dwell entirely within plant tissuesand may be allied with roots stems andor leaves Everyplant harbor at least one or more endophytic fungi in theuniverse [16 17] In recent years they have been extensivelystudied in diverse geographic and climatic regions and werefound to be ubiquitous inside plant tissues and rich in speciesdiversity [18ndash21] Researchers found their precious roles innutrients supply environment acclimatization biotic andabiotic stresses protection growth promotion and enhancingcommunity biodiversity of host plants [22ndash26] They canalso act as warden against predators [27] and contestantsof microbial pathogens [28] Previous reports showed thatmany grass species indicated vegetative growth improvementin the presence of their fungal symbionts that have beenprimarily credited to enlarge plant fitness [29 30] Notwith-standing late examinations have demonstrated that plantgrowth promotion might be attributed to the discharge ofplant growth advancing secondary metabolites (gibberellinsauxins cytokinin) by the endophytic fungi in the rhizosphere[29] Literature survey additionally demonstrated that PlantGrowth Promoting Fungi (PGPF) maintain plant growththrough the generation of a number of significant enzymeslike ACCD urease catalase etc phosphate solubilizationsiderophore and IAA formation and antagonism to phy-topathogens and take a crucial part in plant growth [30ndash33] Earlier reports showed that antibiotic resistant PGPendophytes could be a good source of biocontrol agents [3435]

Although formerly a number of researches were con-ducted on wheat endophytic fungi [36ndash38] but till nowno report has been found on their PGP traits along withtheir resistance pattern to abiotic stresses and antibioticsTherefore we designed this study to evaluate the plant growthpromoting traits along with abiotic stress tolerance andantibiotics resistance properties of wheat endophytic fungiwhich would be a potential source of biofertilizers in asustainable organic crop production system in the foreseeablefuture

2 Material and Methods

21 Plant Sampling and Isolation of Endophytic FungusWheat plant samples were collected from Henan (Jinshui

District-34∘ 461015840 225910158401015840 N 113∘ 431015840 96210158401015840 E altitude 100meters) Shandong (Dezhou-37∘261015840N 116∘161015840E altitude 50meters) and Hebei (Anxin county-38∘551015840N 115∘561015840E altitude80 meters) provinces of Northern China on 20th April 2017and processed in the laboratory within 24 h They werechecked carefully for any disease symptoms or superficialdamage and were washed thoroughly in running tap waterto eradicate the superficial dirt of plant parts In the wakeof washing the samples were separated into leaves stemsand roots Endophytic fungi were isolated from the soundand asymptomatic roots leaves and stems of the exper-imented plant samples based on the published protocols[38 39] Competence of the surface sterilization strategy wasconfirmed by engrave technique [14] The sterilized plantparts were placed aseptically on potato dextrose agar (PDA)medium and incubated at room temperature for 5-7 d Thefungi appeared from the edges of the inoculated parts wereisolated and identified and pure cultures were maintained onpotato dextrose agar slants

22 Molecular Identification and Phylogenetic Analysis ofthe Isolated Endophytic Fungus Molecular identification wascarried out on the basis of fungal internal transcribed spacer(ITS) rDNA rejoins amplification and sequence analysesGenomic DNA was extracted with Tiangen Fungus DNA kit(Biotech Beijing Co Ltd) according to the manufacturerrsquosinstructions Amplification of fungal ITS region was carriedout with ITS1 and ITS4 as forward and reverse primerrespectively [40] The PCR products were checked for theexpected size on 1 agarose gel and were sequenced bySangon sequence service provider (Sangon Inc BeijingChina) The nucleotide sequences were compared againstnucleotide databases using the NCBI BLASTn program toidentify the closest known taxa The ITS-rDNA gene alongwith their closest homology sequences were aligned usingmultiple sequence alignment program CLUSTALW algo-rithm implemented inMEGA 7 software with default param-eters [41] Phylogenetic tree was constructed with neighbor-joining method byMEGA7 program As a statistical supportbootstrap replications (1000) were used for the nodes in thephylogenetic tree Based on molecular identification report15 fungi were chosen for further study To take scanningelectron micrographs of wheat fungi they were dehydratedin a graded mounting ethanolic series critical point-dried(CO2) coated with a thin layer of gold and observed

by means of a scanning electron microscope (Hitachi S-3400N) Some SEM images of wheat fungi are given inFigure 1

23 Evaluation of Some Important PGPProperties All isolateswere screened for awide range of vital PGPproperties ACCDpotentiality of the fungal isolates were checked by both qual-itative and quantitative ways using Dworkin and Foster (DF)minimal salts media [42] supplemented with 3 mM ACC asthe sole nitrogen source [43ndash45] Final ACCD activity wasexpressed in nanomol 120572-ketobutyrate (120572-KB) mgminus1 proteinhminus1 IAA production ability was tested using Salkowskirsquosreagent as delineated by Acuna et al [46] Siderophore man-ufacturing capability of the selected endophytes was studied

BioMed Research International 3

Mycelium Spore

(a)

Mycelium

Spore

(b)

Mycelium

Spore

(c)

Mycelium

Spore

(d)

Spore

Mycelium

(e)

Spore

Mycelium

(f)

Mycelium

Spore

(g)

Spore

Mycelium

(h)

Spore

Mycelium

(i)

Spore

Mycelium

(j)

Spore Mycelium

(k)

Spore

Mycelium

(l)

Figure 1 Electromicrographs of wheat fungi in 11 days old PDA plates ((a) T aureoviride (b) T harzianum (c) F proliferatum (d) Pjanthinellum (e) A flavus (f) A tenuissima (g) T funiculosus (h) P aurantiogriseum (i) A stellatus (j) C cladosporioides (k) A alternate(l) F equiseti)


by universal Chrome Azurol S (CAS) agar plate method [47]Phosphate solubilization property of all fungal isolates wastested by bromophenol blue (001-0001 mg literminus1) added toPikovskayarsquos agar media [48] Ammonia (NH

3) urease and

catalase production aptitude were studied as in previouslydescribed methods [49] Cyanogenesis (hydrogen cyanide-HCNproduction) property of the fungal isolates was checkedwith picric acid soaked filter paper containing PDA plates[50]

24 Abiotic Stress Tolerance A number of abiotic stresstolerance tests (salt heavy metals drought and temperature)were conducted on different levels with fresh cultures of theisolated endophytic fungiThe intrinsic salinity resistance testcultures were checked by observing their growth on PDAmedia amended with different concentration (25-10 wv)of sodium chloride (NaCl) at 28∘C for 5 d Heavy metaltolerance stress was assayed by growing the fungi on freshlyprepared PDAplates amended with a variety of soluble heavymetal salts (nickel-Ni lead-Pb copper-Cu cadmium-Cd andcobalt-Co) in different concentrations ranging from 50 to300 120583g mlminus1 at 28∘C for 5 d [43] Tolerance to drought stresswas evaluated by Leo Daniel et al method [50] with 10-40polyethylene glycol (PEG 6000 Da) amended PDA platesTemperature resistance was assessed by incubating fungi atdiverse temperature regime (namely 5 15 25 35 45 and55∘C) for 5 d [51]

25 Antibiotic Sensitivity Test Antibiotic resistance is con-sidered as one of the parameters to search for efficientbiological control agents [52] and previous literature showedthat this property can initiate plant growth to a certainextent [34 35 53] Here we have evaluated antibioticsensitivity of the isolated strains against nystatin (10 120583g)ketoconazole (50 120583g) and itraconazole (30120583g) soaked discs (6mm diameter) by Kirby Bauer disc-diffusion assay Depend-ing on the inhibition zone organisms were grouped asresistant or sensitive according to the published literatures[54 55] Each experiment was repeated thrice for eachfungus

26 Statistical Analysis Microsoft Excel 2013 was used toaccomplish statistical analysis The phylogenetic tree wasconstructed withMEGA7 software All the experiments werecarried out in triplicate Means and standard deviations wereestimated and applied

3 Results

In the contemporary study we have assayed a number ofPGP traits of the isolated fungi which might play a crucialrole on plant growth in both direct and indirect manners (allresults are given in Table 2) In ACCD screening 11 fungipassed qualitative test and were chosen for quantitative assayamong them 9 endophytic fungal strains exhibited negligibleto moderate enzyme activity (003plusmn0011 to 143plusmn001120583mol 120572-KB mgminus1 protein hrminus1) Nine fungi gave positive responsefor IAA with isolate 582PDA4 being the top producer

(21125plusmn0009120583gmlminus1) which proved them to be plant growthpromoters Siderophore producing aptitude was found foronly 3 (581PDA1 582PDA6 and 582PDA7) isolates in vari-able extents as evidenced by the formation of orange haloaround the colony in CAS plate Among the 15 fungi 11strains were marked to be phosphate solubilizers showingapparent halo zones around their colonies on Pikovskayarsquosagar medium in variable phosphate solubilizing index (PSI)scale (208plusmn003 to 516plusmn036)HCN test was positive for only2 isolates while NH

3was produced by around 34 of the

tested microbes For urease and catalase tests we have foundthat none of the isolates possess urease enzyme but all of themexhibited catalase activity

The abiotic stress tolerance capabilities of the isolateswerechecked by inspection of their growth in different levels ofsalt heavy metals drought and temperature Utmost growthtolerance was revealed by 582PDA6 and 582PDA7 under saltstress condition whereas almost all the fungi were able togrow at 5 of salt (except 582PDA5 and 582PDA11) whichproved them to be supportive for saline prone agriculturalareas (Table 3) Again the fungal endophytes exhibited aspeckled level of resistance to the tested heavy metals Strains582PDA6 and 582PDA7 were found to be resistant against allthe tested concentrations of heavy metals while other isolateswere resistant to different concentrations of the metals indifferent levels (Table 4) This study revealed that all theselected wheat endophytic fungi are able to resist drought invariable range Profuse sporulation was observed in presenceof 10-20 PEG concentrations for all strains whereas at 35PEG concentration sporulation was completely abolished forall strains Highest drought tolerance potency was observedby strain 582PDA6 (Table 5) Here we also studied the effectof different range of temperature on the tested fungal isolatesand found the optimum growth temperature of maximumstrains is 25∘C The least and utmost temperature toleratedby the isolates were recorded as 5∘C (581PDA1) and 55∘C(582PDA4) respectively (Table 6) So we may say that thesemicrobes can help to tolerate temperature stress to certainextent

Antibiotic resistance pattern of all the experimentedstrains varied fromantibiotic to antibiotic Four fungal strains(581PDA3 581PDA4 582PDA6 and 582PDA7) were foundto be resistant against all three antibiotics tested while threefungi 581PDA2 581PDA5 and 581PDA7 were sensitive to allthree antibiotics (Table 7)

4 Discussion

The need to increase food production and exhaustion ofwheat genetic resources has increased interest in alternativeapproaches for wheat improvement including the use ofendophytes Previous studies of wheat endophytes showedthat all wheat cultivars contain a relatively wide range ofendophytes predominantly fungi [56 57] By inspiring withthe previous research current investigation was designedto isolate and characterize endophytic fungi associated withwheat as well as screening their potentiality to support plantgrowth through polyphasic approach Sixteen endophytic


582PDA9 Fusarium proliferatum 100 (MF4716681)

581PDA2 Fusarium oxyporum 100 (FJ3608991)

581PDA4 Fusarium incarnatum 100 (KU2047601)

582PDA11 Fusarium equiseti 100 (MF1667651)

582PDA6 Trichoderma aureoviride 100 (HQ5969361)

582PDA7 Trichoderma harzianum 100 (KX3430871)

581PDA5 Alternaria alternata 100 (KY0265921)

582PDA1 Cladosporium cladosporioides 100 (MF3725801)

582PDA4 Talaromyces funiculosus 100 (AB8939411)

582PDA13 Aspergillus stellatus 100 (KU8666651)

581PDA3 Penicillium aurantiogriseum 100 (GU5662341)

582PDA8 Penicillium janthinellum 100 (KY4273601)

582PDA5 Aspergillus flavus 100 (MF3198931)

581PDA1 Aspergillus niger 100(KY7025761)

581PDA7 Alternaria tenuissima 100 (MF4351451)

100

100

100

94

100

68

100

7870

69

99

0050

Figure 2 Phylogenetic tree constructedwith ITS-rDNA sequences of fungal endophytic isolates obtained from tissue sections of wheat usingneighbor-joining method Evolutionary analyses were conducted in MEGA7

fungi were isolated from leaf stem and root of differentwheat plant samples and were identified using their ITS-rDNA sequences and based on their sequencing result wehave chosen 15 isolates for our work (Table 1) The sequencesof close relatives were obtained fromGen Bank to reconstructthe phylogenetic tree (Figure 2) After identification allisolates were screened for PGP and stress tolerance propertiesand antibiotic sensitivity The majority of the isolated fungusexhibited a number of relevant growth promoting param-eters including different enzyme activity (ACCD ureaseand catalase) inorganic phosphate solubilization IAA HCNsiderophore andNH

3production (Table 2)They also showed

a variable level of resistance against different abiotic stressesand tested antibiotics

Scientists have proved that ACCD-containing PGPFs cansuccessfully protect against growth inhibition by floodingelevated salt drought and presence of fungal and bacterialpathogens nematode damage and existence of high levels ofmetals and organic contaminants as well as low temperaturestress In the contemporary investigation among the 15isolated fungi 11 were able to show negligible ACCD activity

(003plusmn0011 to 143plusmn001120583mol 120572-KB mgminus1 protein hrminus1) Pre-vious document showed that a squat level of ACCD activitysuch as or more than 002120583mol 120572-KB mgminus1 protein hrminus1 isadequate for a microbe to elevate plant growth as a PGPE[30] IAA plays a role in cell growth slows down the growthof side shoots promotes abscission forms xylem and phloemtissue and also affects the growth and elongation of roots[57] Among the tested fungi 9 were able to produce IAAwithisolate 582PDA4 being the top producer (Table 2) Based onabove grounds we may state that for the investigated strainsIAA production executes a crucial role in the regulation ofthis PGP characteristic Siderophore producing endophytesare beneficial for plants because they inhibit phytopathogensby shrinking the accessibility of iron to pathogens andthus hamper their growth inside the plants and indirectlyaccelerate the plant growth We have checked siderophoreproducing ability of the isolated fungi by universal CAS agartechnique and found 3 (581PDA1 582PDA6 and 582PDA7)isolates having siderophore production ability in the formof orange halo around the colonies (Table 2) Optimisticgrowth response has been documented in diverse crop plants


Table 1 List of unique isolates from wheat plant and identification of the most closely related species using the ITS sequence to perform annrnt BLAST search at the National Center for Biotechnology Information

Strain Homologousmicroorganism ( Identity) Accession no

581PDA1 Aspergillus niger 100 KY7025761581PDA2 Fusarium oxyporum 100 FJ3608991

581PDA3 Penicilliumaurantiogriseum 100 GU5662341

581PDA4 Fusarium incarnatum 100 KU2047601581PDA5 Alternaria alternata 100 KY0265921581PDA7 Alternaria tenuissima 100 MF4351451

582PDA1 Cladosporiumcladosporioides 100 MF3725801

582PDA4 Talaromyces funiculosus 100 AB8939411582PDA5 Aspergillus flavus 100 MF3198931582PDA6 Trichoderma aureoviride 100 HQ5969361582PDA7 Trichoderma harzianum 100 KX3430871582PDA8 Penicillium janthinellum 100 KY4273601582PDA9 Fusarium proliferatum 100 MF4716681582PDA11 Fusarium equiseti 100 MF1667651582PDA13 Aspergillus stellatus 100 KU8666651

Table 2 PGP traits of isolated endophytic fungi

Strain 120572 KB 120583mol mgminus1 protein hrminus1 IAA 120583gmlminus1 Siderophore production (PSI) NH3production HCN Urease Catalase

581PDA1 099plusmn0005 - + 264plusmn034 ++ - - ++581PDA2 - 165plusmn0005 - - - - - +581PDA3 054plusmn0015 - - 264plusmn034 - - - +581PDA4 - - - 313plusmn037 - - - +581PDA5 061plusmn002 - - 211plusmn017 - - - +581PDA7 073plusmn0015 262plusmn014 - 208plusmn003 - - - +582PDA1 099plusmn001 - - - - + - +582PDA4 - 3612plusmn0004 - 229plusmn019 + - - +582PDA5 057plusmn0005 400plusmn0003 - - +++ - - ++582PDA6 141plusmn0005 1125plusmn004 ++ 409plusmn022 ++ - - ++582PDA7 143plusmn001 212plusmn005 ++ 249plusmn016 - - - +582PDA8 064plusmn001 550plusmn0007 - 516plusmn036 - - - +582PDA9 003plusmn0011 1375plusmn0018 - 218plusmn013 + - - +582PDA11 - 21125plusmn0009 - 249plusmn017 - + - +582PDA13 089plusmn001 - - 217plusmn002 + - - +- = negative + = poor growth ++ = moderate growth +++ = excellent growth Values represent mean of triplicate readings plusmnSD

inoculated with phosphate solubilizing endophytes [58] Ourfindings showed that 12 out of 15 tested endophytic fungigave PSI ranging from 211plusmn017 to 516 plusmn036 (Table 2)HCN and NH

3have indirect effect on growth promotion of

plants HCN is volatile in nature and competent to revealantifungal action whereas NH

3can assist to assure the

nitrogen requirement of the host plant and in large amountsuppresses the colonization of plants by pathogens [59]HCN test was positive for only 2 (582PDA1 and 582PDA11)isolates whileNH

3was produced by around 34of the tested

microbes (Table 2) For urease and catalase tests we have

observed that none of the isolates possess urease enzymebut all of them exhibited positive response for catalaseenzyme (Table 2) Catalase enzyme leads a foremost taskin organism protection against toxic free radicals that areproduced predominantly beneath environmental mechani-cal and chemical stresses and could promote plant growthvia an indirect way [60] In the current investigation allfungi gave positive response for catalase enzyme hencewe can say they indirectly enhance plant growth Thesefindings are in agreement with those published previously[61]


Table 3 Salt tolerance property of the endophytic fungi

Strain 25 5 75 10581PDA1 + + + -581PDA2 + + + -581PDA3 + + + -581PDA4 + + + -581PDA5 + + + -581PDA7 + + + -582PDA1 + + - -582PDA4 + + - -582PDA5 + - - -582PDA6 + + + +582PDA7 + + + +582PDA8 + + + -582PDA9 + + + -582PDA11 + - - -582PDA13 + + - -Here + = growth - = no growth

It is well known that abiotic stress leads to a series ofmorphological physiological biochemical and molecularchanges that adversely affect plant growth and productivity[62]Therefore selection screening and application of stresstolerant PGPF for better farming would considerably facil-itate the farming community by overcoming such extremeclimate changes Additionally such microbial application isalso acknowledged to conquer the fatal effect of chemicalfertilizers and pesticides Therefore with growth promotingactivities screening we have also taken initiative to drag outand be acquainted with promising wheat endophytic fungiwith abiotic stress tolerance and antibiotic sensitivity forbetter plant growth promotion

Salt tolerant microbes are a prospective bioresource forsaline prone areas On the other hand previous researchshowed if these endophytes also possess plant growth pro-moting traits they would be ideal for use in sustainableagriculture [63] Out of the 15 fungal isolates of wheat plants1333 exhibited tolerance to high salt concentration (10NaCl) Heavy metal contamination in the environment hasturned into a severe issue because they are not degradablelike organic pollutants and accumulate in different parts ofthe food chain which is a threat to plants and animal healthIn this perspective previous research gave information ofdiverse endophytes having ability to trim down the stressposed on plants by the presence of heavy metals amplifythe accessibility of metal for plant uptake and promote plantgrowth [64 65] In our current study we have also founda number of wheat endophytic fungi exhibiting resistancetowards the tested heavy metal salts (Ni Cu Cd Co andPb) in a variable range along with their PGP properties Cropplant-associated microbes having good drought toleranceproperty are recently getting increased attention By influenc-ing plant morphology development and physiological andbiochemical responses to stress fungal endophytes can pro-voke mechanisms of drought escaping drought lenience anddrought recovery in their hosts [66 67] In our investigation

we have observed that all the selected wheat endophytic fungiare able to resist drought in variable range A number ofendophytes have been studied that help plants to cope upwithtemperature stress and also encourage growth promotionof diverse crops at different climates [68] We have studiedeffect of different temperature on all the tested fungal isolatesand found the optimum growth temperature of maximumstrains is 25∘C The least and utmost temperature toleratedby the isolates were recorded as 5∘C (581PDA1) and 55∘C(582PDA4) respectively So it is tempting to conclude thatthese microbes can help to tolerate temperature stress tocertain extent

Uses of different types of materials such as heavy metalsalong with antibiotics in plants generate a selective pressurein the environment that consequently leads to themutation inorganism which will help them to survive and multiply [69]Previous research showed that antibiotic resistance propertyof endophytes can accelerate plant growth [34 35 53]With this deliberation the antibiotic resistance among PGPFwas checked and we noticed their resistance pattern variedfrom antibiotic to antibiotic It has also been reported thatunder environmental conditions of metal stress metal andantibiotic resistant microorganisms will adapt faster by thespread of R-factors than by mutation and natural selectionThe discrepancy in the resistance to many tested antibioticsprobably due to the variation in growth conditions andexposure of PGP microbes to stress conditions or toxic stuffsas well as existence or nonexistence of resistance mechanismsthat could be encoded either by chromosome andor R-plasmid [68 69]

5 Conclusion

Thepresent research revealed thatwheat plant is an ecologicalniche for different putative fungal endophytes The plantgrowth promoting ability of these microbes may be dueto their capability to secret elevated amounts of various


Table4Growth

ofthetestedendo

phytes

indifferent

concentrations

ofheavymetalsa

mendedagar

plates

Strain

Nickel

Copper

Cadm

ium

Cobalt

Lead

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

581PDA1

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

581PDA2

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA3

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA4

++

++

--

++

++

+-

++

++

--

++

++

--

++

++

+-

581PDA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA7

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

582P

DA1

++

++

+-

++

++

++

++

++

--

++

++

+-

++

++

++

582P

DA4

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA6

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA7

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA8

++

++

--

++

++

--

++

++

--

++

++

--

++

++

+-

582P

DA9

++

++

++

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA11

++

++

--

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA13

++

++

--

++

++

++

++

++

+-

++

++

+-

++

++

++

Here+deno

tesg

rowth-

deno

tesn

ogrow

th


Table 5 Drought resistance property of the isolated fungi at different concentrations of PEG

Strain 10 20 30 35 40581PDA1 3491 2672 1804 452 -581PDA2 7333 6367 5467 70 -581PDA3 6656 4930 1341 - -581PDA4 5978 4888 469 - -581PDA5 7158 5451 3997 763 -581PDA7 6847 4248 2356 1449 -582PDA1 2947 898 - - -582PDA4 4064 1839 672 - -582PDA5 5261 3560 78 - -582PDA6 8415 6979 5459 3568 -582PDA7 7804 6759 5563 4656 -582PDA8 3957 1196 2278 - -582PDA9 3981 3344 1673 - -582PDA11 3779 1245 519 - -582PDA13 3723 1213 323 - -Here - denotes no growth

Table 6 Growth of the endophytic fungi at different temperatures

Strain 5∘C 15∘C 25∘C 35∘C 45∘C 50∘C 55∘C581PDA1 + ++ +++ +++ + + ndash581PDA2 - + +++ ++ + ndash ndash581PDA3 ndash + +++ ++ + ndash ndash581PDA4 ndash + +++ ++ + ndash ndash581PDA5 - ++ +++ ++ + + ndash581PDA7 - ++ +++ ++ + + ndash582PDA1 + ++ +++ +++ ndash ndash ndash582PDA4 - ++ +++ ++ + + +582PDA5 - ++ +++ ++ + + ndash582PDA6 ndash ++ +++ ++ + ndash ndash582PDA7 ndash ++ +++ ++ + ndash ndash582PDA8 ndash ++ +++ ++ + ndash ndash582PDA9 ndash ++ +++ ++ + ndash ndash582PDA11 ndash ++ +++ ++ ++ + ndash582PDA13 ndash ++ +++ ++ ++ + ndashHere - = no growth + = poor growth ++=moderate growth +++= excellentgrowth

favorable growth promoting metabolites and therefore assisttheir host plants to survive beneath stress condition Thefindings of this study motivate us to advance investigationon the selected fungal endophytes in order to develop astrapping Bioagent with spacious applicability to multifieldand hereafter emerge as a thriving bioinoculum leading onthe way to organic food crops for a better tomorrow byplummeting the extreme uses of chemicals

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Table 7 Antibiotic sensitivity of endophytic fungi of T aestivum

Strain Nystatin Ketoconazole Itraconazole581PDA1 + ndash ndash581PDA2 - ndash ndash581PDA3 + + +581PDA4 + + +581PDA5 - ndash ndash581PDA7 - ndash ndash582PDA1 - + +582PDA4 +++ ndash ndash582PDA5 + ndash ndash582PDA6 + + +582PDA7 + + +582PDA8 - + +582PDA9 + ndash ndash582PDA11 - + +582PDA13 - ++ ++Here - = sensitive to antibiotic + = poorly resistant ++ = moderatelyresistant +++ = highly resistant

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 31870003) and carried out inKey Laboratory of Microbial Resources Ministry of Agricul-tureInstitute of Agricultural Resources and Regional Plan-ning Chinese Academy of Agricultural Sciences BeijingChina


References

[1] OECDFAO OECD-FAO Agricultural Outlook 2011-2020OECD Publishing and FAO 2011

[2] J M Barroso ldquoRegulations commission implementing regula-tion (EC) no 11072009 of the European parliament and of thecouncil as regards the list of approved active substancesrdquoOfficialJournal of the European Union p 153 2011

[3] J E Olesen M Trnka K C Kersebaum et al ldquoImpacts andadaptation of European crop production systems to climatechangerdquo European Journal of Agronomy vol 34 no 2 pp 96ndash112 2011

[4] R M Rees J A Baddeley A Bhogal et al ldquoNitrous oxidemitigation in UK agriculturerdquo Soil Science amp Plant Nutritionvol 59 no 1 pp 3ndash15 2013

[5] E Elkoca M Turan and M F Donmez ldquoEffects of singledual and triple inoculations with bacillus subtilis bacillusmegaterium and rhizobium leguminosarum bv phaseoli onnodulation nutrient uptake yield and yield parameters ofcommon bean (phaseolus vulgaris l cv rsquoElkoca-05rsquo)rdquo Journalof Plant Nutrition vol 33 no 14 pp 2104ndash2119 2010

[6] A D Freitas C L Vieira C E Santos N P Stamford andM dLyra ldquoCaracterizacao de rizobios isolados de Jacatupe cultivadoem solo salino no Estado de Pernanbuco Brasilrdquo Bragantia vol66 no 3 pp 497ndash504 2007

[7] A L Khan M Waqas A R Khan et al ldquoFungal endo-phyte Penicillium janthinellum LK5 improves growth of ABA-deficient tomato under salinityrdquo World Journal of Microbiologyand Biotechnology vol 29 no 11 pp 2133ndash2144 2013

[8] C Karthik M Oves R Thangabalu R Sharma S B San-thosh and P Indra Arulselvi ldquoCellulosimicrobium funkei-like enhances the growth of Phaseolus vulgaris by modulatingoxidative damage under Chromium(VI) toxicityrdquo Journal ofAdvanced Research vol 7 no 6 pp 839ndash850 2016

[9] A Puri K P Padda and C P Chanway ldquoSeedling growthpromotion and nitrogen fixation by a bacterial endophytePaenibacillus polymyxa P2b-2R and its GFP derivative in cornin a long-term trialrdquo Symbiosis vol 69 no 2 pp 123ndash129 2016

[10] H Shen W Ye L Hong et al ldquoProgress in parasitic plantbiology Host selection and nutrient transferrdquo 13e Journal ofPlant Biology vol 8 no 2 pp 175ndash185 2006

[11] P H Thrall M E Hochberg J J Burdon and J D BeverldquoCoevolution of symbiotic mutualists and parasites in a com-munity contextrdquo Trends in Ecology amp Evolution vol 22 no 3pp 120ndash126 2007

[12] J Hallmann A Quadt-Hallmann W F Mahaffee and J WKloepper ldquoBacterial endophytes in agricultural cropsrdquo Cana-dian Journal of Microbiology vol 43 no 10 pp 895ndash914 1997

[13] C G Carroll ldquoFungal mutualismrdquo in Fungal Mutualism C GCarroll and D TWicklow Eds pp 254ndash327 Dekker NY USA1992

[14] B Schulz A Rommert U Dammann H Aust and D StrackldquoThe endophyte-host interaction a balanced antagonismrdquoMycological Research vol 103 no 10 pp 1275ndash1283 1999

[15] 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

[16] E S Gaylord R W Preszler and W J Boecklen ldquoInteractionsbetween host plants endophytic fungi and a phytophagousinsect in an oak (Quercus grisea x Q gambelii) hybrid zonerdquoOecologia vol 105 no 3 pp 336ndash342 1996

[17] O Petrini T N Sieber L Toti and O Viret ldquoEcologymetabolite production and substrate utilization in endophyticfungirdquo Natural Toxins vol 1 no 3 pp 185ndash196 1992

[18] S R Ghimire N D Charlton J D Bell Y L Krishnamurthyand K D Craven ldquoBiodiversity of fungal endophyte commu-nities inhabiting switchgrass (Panicum virgatum L) growingin the native tallgrass prairie of northern Oklahomardquo FungalDiversity vol 47 pp 19ndash27 2011

[19] H-Y Li M Shen Z-P Zhou T Li Y-L Wei and L-BLin ldquoDiversity and cold adaptation of endophytic fungi fromfive dominant plant species collected from the Baima SnowMountain Southwest Chinardquo Fungal Diversity vol 54 pp 79ndash86 2012

[20] F N Rivera-Orduna R A Suarez-Sanchez Z R Flores-Bustamante J N Gracida-Rodriguez and L B Flores-CoteraldquoDiversity of endophytic fungi ofTaxus globosa (Mexican yew)rdquoFungal Diversity vol 47 pp 65ndash74 2011

[21] A Tanwar and A Aggarwal ldquoMultifaceted potential of bioinoc-ulants on red bell pepper (F1 hybrid IndamMamatha) produc-tionrdquo Journal of Plant Interactions vol 9 no 1 pp 82ndash91 2014

[22] G Berg ldquoPlant-microbe interactions promoting plant growthand health perspectives for controlled use of microorganismsin agriculturerdquoApplied Microbiology and Biotechnology vol 84no 1 pp 11ndash18 2009

[23] S K Gond V C Verma A Mishra A Kumar and R NKharwar ldquoRole of fungal endophytes in plant protectionrdquoin Management of Fungal Plant Pathogens A Arya and AE Perello Eds pp 183ndash197 CAB International WallingfordLondon 2010

[24] R N Kharwar V C Verma VC G Strobel and D Ezra ldquoTheendophytic fungal complex ofCatharanthusroseus (L) G DonrdquoCurrent Science vol 95 pp 228ndash233 2008

[25] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[26] R Pandey A K Mishra S Tiwari H N Singh and A KalraldquoEnhanced tolerance of Mentha arvensis against Meloidogyneincognita (Kofoid and White) Chitwood through mutualisticendophytes and PGPRsrdquo Journal of Plant Interactions vol 6 no4 pp 247ndash253 2011

[27] M R Seigel and L P Bush ldquoToxin production in grassen-dophyte associationsrdquo in 13e Mycota G C Carroll and PTudzynski Eds pp 185ndash207 Springer-Verlag Heidelberg 1997

[28] S Scannerini A M Fusconi and Mucciarelli ldquoThe effect ofendophytic fungi on host plant morphogenesisrdquo in CellularOrigin and Life in Extreme Habitats J Seckbach Ed pp 427ndash447 Kluwer Academic Publishers DordrechtTheNetherlands2001

[29] M Hamayun S A Khan A L Khan et al ldquoGrowth promotionof cucumber by pure cultures of gibberellin-producing Phomasp GAH7rdquo World Journal of Microbiology and Biotechnologyvol 26 no 5 pp 889ndash894 2010

[30] B R Glick ldquoBacteria with ACC deaminase can promote plantgrowth and help to feed the worldrdquo Microbiological Researchvol 169 no 1 pp 30ndash39 2014

[31] J W Kloepper R M Zablotowicz E M Tipping and RLifshitz ldquoPlant growth promotion mediated by bacterial rhizo-sphere colonizersrdquo in 13e Rhizosphere and Plant Growth D LKeister and P B Cregan Eds pp 315ndash326 Kluwer AcademicPublishers Dordrecht The Netherlands 1991


[32] R Malla R Prasad P H Giang U Pokharel R Oelmuellerand A Varma ldquoCharacteristic features of symbiotic fungusPiriformospora indicardquo Endocytobiosis and Cell Research vol 15pp 579ndash600 2004

[33] S A Wakelin R A Warren P R Harvey and M H RyderldquoPhosphate solubilization by Penicillium spp closely associatedwith wheat rootsrdquo Biology amp Fertility of Soils vol 40 no 1 pp36ndash43 2004

[34] F Yasmin R Othman K Sijam and M S Saad ldquoCharac-terization of beneficial properties of plant growth-promotingrhizobacteria isolated from sweet potato rhizosphererdquo AfricanJournal ofMicrobiology Research vol 3 no 11 pp 815ndash821 2009

[35] R Rangeshwaran J Raj and P Sreerama Kumar ldquoResistanceand susceptibility pattern of chickpea (Cicer arietillum L)endophytic bacteria to antibioticsrdquo Journal of Biological Controlvol 22 no 2 pp 393ndash403 2008

[36] M Hubbard J J Germida and V Vujanovic ldquoFungal endo-phytes enhance wheat heat and drought tolerance in terms ofgrain yield and second-generation seed viabilityrdquo Journal ofApplied Microbiology vol 116 no 1 pp 109ndash122 2014

[37] T Sieber T K Riesen E Muller and P M Fried ldquoEndophyticfungi in four winter wheat cultivars (Triticum aestivum L) dif-fering in resistance against stagonospora nodorum (berk) castamp germ =septoria nodorum (berk)rdquo Journal of Phytopathologyvol 122 no 4 pp 289ndash306 1988

[38] R N Kharwar A Mishra S K Gond A Stierle and D StierleldquoAnticancer compounds derived from fungal endophytes theirimportance and future challengesrdquoNatural Product Reports vol28 no 7 pp 1208ndash1228 2011

[39] G Strobel BDaisy U Castillo and JHarper ldquoNatural productsfrom endophytic microorganismsrdquo Journal of Natural Productsvol 67 no 2 pp 257ndash268 2004

[40] T J White T Bruns and S Lee ldquoAmplification and directsequencing of fungal ribosomal RNA genes for phylogeneticsrdquoin PCR Protocols A Guide to Methods and Applications M AInnis D H Gelfand J J Sninsky and T J Whte Eds pp 315ndash322 Academic Press Inc NY USA 1990

[41] S Kumar G Stecher and K Tamura ldquoMEGA7 molecularevolutionary genetics analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[42] MDworkin and JW Foster ldquoExperiments with somemicroor-ganisms which utilize ethane and hydrogenrdquo Journal of Bacteri-ology vol 75 pp 592ndash601 1958

[43] M M Bradford ldquoRapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[44] M Honma and T Shimomura ldquoMetabolism of 1-aminocyclo-propane-1-carboxylic acidrdquo Agricultural and Biological Chem-istry vol 42 no 10 pp 1825ndash1831 1978

[45] D M Penrose and B R Glick ldquoMethods for isolating and char-acterizingACCdeaminase-containing plant growth-promotingrhizobacteriardquo Physiologia Plantarum vol 118 no 1 pp 10ndash152003

[46] J J Acuna M A Jorquera O A Martınez et al ldquoIndole aceticacid and phytase activity produced by rhizosphere bacilli asaffected by pH and metalsrdquo Soil Science amp Plant Nutrition vol11 no 3 pp 1ndash12 2011

[47] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[48] N C SNautiyal SMehta andP Pushpangadan ldquoCompositionfor qualitative screening of phosphate solubilizing microorgan-isms and a qualitative method for screening microorganismsrdquoUnited States Patent Patent No 6638730 B2 2003

[49] V J Szilagyi-Zecchin A C IkedaMHungria et al ldquoIdentifica-tion and characterization of endophytic bacteria from corn (Zeamays L) roots with biotechnological potential in agriculturerdquoAMB Express vol 4 no 1 pp 1ndash9 2014

[50] A E Leo Daniel G S Praveen Kumar A S K Desai andMir Hassan ldquoIn vitro characterization of Trichoderma viridefor abiotic stress tolerance and field evaluation against rootrot disease in Vigna mungo Lrdquo Journal of Biofertilizers ampBiopesticides vol 2 no 111 2011

[51] M Jida and F Assefa ldquoPhenotypic and plant growth promotingcharacteristics of leguminosarum viciae from lentil growingareas of Ethiopiardquo African Journal of Microbiology Research vol5 pp 4133ndash4142 2011

[52] N Bhagya S S M Sheik K R Sharma and ChandrashekarldquoIsolation of endophytic colletotrichum gloeosporioides penzfrom salacia chinensis and its antifungal sensitivityrdquo Journal ofPhytological Research vol 36 pp 20ndash22 2011

[53] S Siddiqui Z A Siddiqui and I Ahmad ldquoEvaluation of fluo-rescent Pseudomonads and Bacillus isolates for the biocontrolof a wilt disease complex of pigeon peardquo World Journal ofMicrobiology andBiotechnology vol 21 no 5 pp 729ndash732 2005

[54] V Kumar A Kumar K D Pandey and B K Roy ldquoIsolationand characterization of bacterial endophytes from the roots ofCassia tora Lrdquo Annals of Microbiology vol 65 no 3 pp 1391ndash1399 2015

[55] M Comby S Lacoste F Baillieul C Profizi and J DupontldquoSpatial and temporal variation of cultivable communities ofco-occurring endophytes and pathogens in wheatrdquo Frontiers inMicrobiology vol 7 2016

[56] S Larran A Perello M R Simon and V Moreno ldquoTheendophytic fungi from wheat (Triticum aestivum L)rdquo WorldJournal of Microbiology and Biotechnology vol 23 no 4 pp565ndash572 2007

[57] J Vacheron G Desbrosses M L Bouffaud et al ldquoPlantgrowth promoting rhizobacteria and root system functioningrdquoFrontiers in Plant Science vol 4 p 356 2013

[58] N Oteino R D Lally S Kiwanuka et al ldquoPlant growthpromotion induced by phosphate solubilizing endophytic Pseu-domonas isolatesrdquo Frontiers inMicrobiology vol 6 pp 1ndash9 2015

[59] F N Mbai E N Magiri V N Matiru J Nganga J and VC S Nyambati ldquoIsolation and characterization of bacterialroot endophytes with potential to enhance plant growth fromKenyan Basmati ricerdquo American International Journal of Con-temporary Research vol 3 no 4 pp 25ndash40 2013

[60] A Kumar A Kumar S Devi S Patil C Payal and S NegildquoIsolation screening and characterization of bacteria fromrhizospheric soils for different plant growth promotion (PGP)activities an in vitro studyrdquo Recent Research in Science andTechnology vol 4 p 1 2012

[61] F Wang X Cui Y Sun and C-H Dong ldquoEthylene signalingand regulation in plant growth and stress responsesrdquo Plant CellReports vol 32 no 7 pp 1099ndash1109 2013

[62] R Hayat R Khalid M Ehsan I Ahmed A Yokotaand and SAli ldquoMolecular characterization of soil bacteria for improvingcrop yield in Pakistanrdquo Pakistan Journal of Botany vol 45 pp1045ndash1055 2013

[63] S Aishwarya N Nagam T Vijaya and R V Netala ldquoScreeningand identification of heavy metal-tolerant endophytic fungi


Lasiodiplodia theobromae from Boswellia ovalifoliolata anendemic plant of tirumala hillsrdquo Asian Journal of Pharmaceu-tical and Clinical Research vol 10 no 3 pp 488ndash491 2017

[64] H He Z Ye D Yang et al ldquoCharacterization of endophyticRahnella sp JN6 from Polygonum pubescens and its potentialin promoting growth and Cd Pb Zn uptake by Brassica napusrdquoChemosphere vol 90 no 6 pp 1960ndash1965 2013

[65] E Ngumbi and J Kloepper ldquoBacterial-mediated drought toler-ance Current and future prospectsrdquo Applied Soil Ecology vol105 pp 109ndash125 2016

[66] S S K P Vurukonda S Vardharajula M Shrivastava and ASkZ ldquoEnhancement of drought stress tolerance in crops by plantgrowth promoting rhizobacteriardquoMicrobiological Research vol184 pp 13ndash24 2016

[67] Y Bashan and G Holguin ldquoProposal for the division ofplant growth-promoting rhizobacteria into two classifica-tions biocontrol-PGPB (plant growth-promoting bacteria) andPGPBrdquo Soil Biology amp Biochemistry vol 30 no 8-9 pp 1225ndash1228 1998

[68] P A Wani and O I Irene ldquoScreening of microbes for theirmetal antibiotic resistance and plant growth promoting activ-ityrdquo Current Research in Bacteriology vol 7 no 1 pp 22ndash312014

[69] U Thacker R Parikh Y Shouche and D Madamwar ldquoReduc-tion of chromateby cell-free extract ofBrucella sp isolated fromCr(VI) contaminated sitesrdquo Bioresource Technology vol 98 no8 pp 1541ndash1547 2007

Hindawiwwwhindawicom

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Anatomy Research International

PeptidesInternational Journal of



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GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

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BioMed Research International

Cell BiologyInternational Journal of



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ArchaeaHindawiwwwhindawicom Volume 2018


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MicrobiologyHindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom


with plants which might be impartial harmful (parasitism)or beneficial (mutualism or symbiosis) to the host [10 11]Microbes that inhabit within the plant tissues devoid of doingsubstantive damage or acquiring remuneration other thansecuring their residency are considered as lsquoendophytesrsquo Plantshave been blessed by nature with diversified population ofendophytic microorganisms including beneficial bacteriafungi and actinomycetes They spend their entire or partof the life cycle living inside the plant causing no visualsymptoms of disease [12] Organization of such useful plantcoupled microbes is continuously gaining attention amongscientific community and at the view point of industries dueto their aptitude to advance plant quality and growth [13 14]

Species of fungi that reside within living plant tissuewithout causing symptoms of disease in their host are knownas fungal endophytes [15] They are the major members ofendophytic population that dwell entirely within plant tissuesand may be allied with roots stems andor leaves Everyplant harbor at least one or more endophytic fungi in theuniverse [16 17] In recent years they have been extensivelystudied in diverse geographic and climatic regions and werefound to be ubiquitous inside plant tissues and rich in speciesdiversity [18ndash21] Researchers found their precious roles innutrients supply environment acclimatization biotic andabiotic stresses protection growth promotion and enhancingcommunity biodiversity of host plants [22ndash26] They canalso act as warden against predators [27] and contestantsof microbial pathogens [28] Previous reports showed thatmany grass species indicated vegetative growth improvementin the presence of their fungal symbionts that have beenprimarily credited to enlarge plant fitness [29 30] Notwith-standing late examinations have demonstrated that plantgrowth promotion might be attributed to the discharge ofplant growth advancing secondary metabolites (gibberellinsauxins cytokinin) by the endophytic fungi in the rhizosphere[29] Literature survey additionally demonstrated that PlantGrowth Promoting Fungi (PGPF) maintain plant growththrough the generation of a number of significant enzymeslike ACCD urease catalase etc phosphate solubilizationsiderophore and IAA formation and antagonism to phy-topathogens and take a crucial part in plant growth [30ndash33] Earlier reports showed that antibiotic resistant PGPendophytes could be a good source of biocontrol agents [3435]

Although formerly a number of researches were con-ducted on wheat endophytic fungi [36ndash38] but till nowno report has been found on their PGP traits along withtheir resistance pattern to abiotic stresses and antibioticsTherefore we designed this study to evaluate the plant growthpromoting traits along with abiotic stress tolerance andantibiotics resistance properties of wheat endophytic fungiwhich would be a potential source of biofertilizers in asustainable organic crop production system in the foreseeablefuture

2 Material and Methods

21 Plant Sampling and Isolation of Endophytic FungusWheat plant samples were collected from Henan (Jinshui

District-34∘ 461015840 225910158401015840 N 113∘ 431015840 96210158401015840 E altitude 100meters) Shandong (Dezhou-37∘261015840N 116∘161015840E altitude 50meters) and Hebei (Anxin county-38∘551015840N 115∘561015840E altitude80 meters) provinces of Northern China on 20th April 2017and processed in the laboratory within 24 h They werechecked carefully for any disease symptoms or superficialdamage and were washed thoroughly in running tap waterto eradicate the superficial dirt of plant parts In the wakeof washing the samples were separated into leaves stemsand roots Endophytic fungi were isolated from the soundand asymptomatic roots leaves and stems of the exper-imented plant samples based on the published protocols[38 39] Competence of the surface sterilization strategy wasconfirmed by engrave technique [14] The sterilized plantparts were placed aseptically on potato dextrose agar (PDA)medium and incubated at room temperature for 5-7 d Thefungi appeared from the edges of the inoculated parts wereisolated and identified and pure cultures were maintained onpotato dextrose agar slants

22 Molecular Identification and Phylogenetic Analysis ofthe Isolated Endophytic Fungus Molecular identification wascarried out on the basis of fungal internal transcribed spacer(ITS) rDNA rejoins amplification and sequence analysesGenomic DNA was extracted with Tiangen Fungus DNA kit(Biotech Beijing Co Ltd) according to the manufacturerrsquosinstructions Amplification of fungal ITS region was carriedout with ITS1 and ITS4 as forward and reverse primerrespectively [40] The PCR products were checked for theexpected size on 1 agarose gel and were sequenced bySangon sequence service provider (Sangon Inc BeijingChina) The nucleotide sequences were compared againstnucleotide databases using the NCBI BLASTn program toidentify the closest known taxa The ITS-rDNA gene alongwith their closest homology sequences were aligned usingmultiple sequence alignment program CLUSTALW algo-rithm implemented inMEGA 7 software with default param-eters [41] Phylogenetic tree was constructed with neighbor-joining method byMEGA7 program As a statistical supportbootstrap replications (1000) were used for the nodes in thephylogenetic tree Based on molecular identification report15 fungi were chosen for further study To take scanningelectron micrographs of wheat fungi they were dehydratedin a graded mounting ethanolic series critical point-dried(CO2) coated with a thin layer of gold and observed

by means of a scanning electron microscope (Hitachi S-3400N) Some SEM images of wheat fungi are given inFigure 1

23 Evaluation of Some Important PGPProperties All isolateswere screened for awide range of vital PGPproperties ACCDpotentiality of the fungal isolates were checked by both qual-itative and quantitative ways using Dworkin and Foster (DF)minimal salts media [42] supplemented with 3 mM ACC asthe sole nitrogen source [43ndash45] Final ACCD activity wasexpressed in nanomol 120572-ketobutyrate (120572-KB) mgminus1 proteinhminus1 IAA production ability was tested using Salkowskirsquosreagent as delineated by Acuna et al [46] Siderophore man-ufacturing capability of the selected endophytes was studied


Mycelium Spore

(a)

Mycelium

Spore

(b)

Mycelium

Spore

(c)

Mycelium

Spore

(d)

Spore

Mycelium

(e)

Spore

Mycelium

(f)

Mycelium

Spore

(g)

Spore

Mycelium

(h)

Spore

Mycelium

(i)

Spore

Mycelium

(j)

Spore Mycelium

(k)

Spore

Mycelium

(l)




3) urease and





3 Results







4 Discussion


















100

100

100

94

100

68

100

7870

69

99

0050

































5 Conclusion



Table4Growth

ofthetestedendo

phytes

indifferent

concentrations

ofheavymetalsa

mendedagar

plates

Strain

Nickel

Copper

Cadm

ium

Cobalt

Lead

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

581PDA1

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

581PDA2

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA3

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA4

++

++

--

++

++

+-

++

++

--

++

++

--

++

++

+-

581PDA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA7

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

582P

DA1

++

++

+-

++

++

++

++

++

--

++

++

+-

++

++

++

582P

DA4

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA6

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA7

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA8

++

++

--

++

++

--

++

++

--

++

++

--

++

++

+-

582P

DA9

++

++

++

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA11

++

++

--

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA13

++

++

--

++

++

++

++

++

+-

++

++

+-

++

++

++

Here+deno

tesg

rowth-

deno

tesn

ogrow

th







Data Availability






Acknowledgments



References











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Mycelium Spore

(a)

Mycelium

Spore

(b)

Mycelium

Spore

(c)

Mycelium

Spore

(d)

Spore

Mycelium

(e)

Spore

Mycelium

(f)

Mycelium

Spore

(g)

Spore

Mycelium

(h)

Spore

Mycelium

(i)

Spore

Mycelium

(j)

Spore Mycelium

(k)

Spore

Mycelium

(l)




3) urease and





3 Results







4 Discussion


















100

100

100

94

100

68

100

7870

69

99

0050

































5 Conclusion



Table4Growth

ofthetestedendo

phytes

indifferent

concentrations

ofheavymetalsa

mendedagar

plates

Strain

Nickel

Copper

Cadm

ium

Cobalt

Lead

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

581PDA1

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

581PDA2

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA3

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA4

++

++

--

++

++

+-

++

++

--

++

++

--

++

++

+-

581PDA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA7

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

582P

DA1

++

++

+-

++

++

++

++

++

--

++

++

+-

++

++

++

582P

DA4

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA6

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA7

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA8

++

++

--

++

++

--

++

++

--

++

++

--

++

++

+-

582P

DA9

++

++

++

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA11

++

++

--

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA13

++

++

--

++

++

++

++

++

+-

++

++

+-

++

++

++

Here+deno

tesg

rowth-

deno

tesn

ogrow

th







Data Availability






Acknowledgments



References











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




3) urease and





3 Results







4 Discussion


















100

100

100

94

100

68

100

7870

69

99

0050

































5 Conclusion



Table4Growth

ofthetestedendo

phytes

indifferent

concentrations

ofheavymetalsa

mendedagar

plates

Strain

Nickel

Copper

Cadm

ium

Cobalt

Lead

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

581PDA1

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

581PDA2

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA3

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA4

++

++

--

++

++

+-

++

++

--

++

++

--

++

++

+-

581PDA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA7

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

582P

DA1

++

++

+-

++

++

++

++

++

--

++

++

+-

++

++

++

582P

DA4

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA6

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA7

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA8

++

++

--

++

++

--

++

++

--

++

++

--

++

++

+-

582P

DA9

++

++

++

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA11

++

++

--

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA13

++

++

--

++

++

++

++

++

+-

++

++

+-

++

++

++

Here+deno

tesg

rowth-

deno

tesn

ogrow

th







Data Availability






Acknowledgments



References











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


















100

100

100

94

100

68

100

7870

69

99

0050

































5 Conclusion



Table4Growth

ofthetestedendo

phytes

indifferent

concentrations

ofheavymetalsa

mendedagar

plates

Strain

Nickel

Copper

Cadm

ium

Cobalt

Lead

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

581PDA1

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

581PDA2

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA3

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA4

++

++

--

++

++

+-

++

++

--

++

++

--

++

++

+-

581PDA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA7

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

582P

DA1

++

++

+-

++

++

++

++

++

--

++

++

+-

++

++

++

582P

DA4

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA6

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA7

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA8

++

++

--

++

++

--

++

++

--

++

++

--

++

++

+-

582P

DA9

++

++

++

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA11

++

++

--

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA13

++

++

--

++

++

++

++

++

+-

++

++

+-

++

++

++

Here+deno

tesg

rowth-

deno

tesn

ogrow

th







Data Availability






Acknowledgments



References











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




























5 Conclusion



Table4Growth

ofthetestedendo

phytes

indifferent

concentrations

ofheavymetalsa

mendedagar

plates

Strain

Nickel

Copper

Cadm

ium

Cobalt

Lead

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

581PDA1

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

581PDA2

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA3

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA4

++

++

--

++

++

+-

++

++

--

++

++

--

++

++

+-

581PDA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA7

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

582P

DA1

++

++

+-

++

++

++

++

++

--

++

++

+-

++

++

++

582P

DA4

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA6

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA7

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA8

++

++

--

++

++

--

++

++

--

++

++

--

++

++

+-

582P

DA9

++

++

++

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA11

++

++

--

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA13

++

++

--

++

++

++

++

++

+-

++

++

+-

++

++

++

Here+deno

tesg

rowth-

deno

tesn

ogrow

th







Data Availability






Acknowledgments



References











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table4Growth

ofthetestedendo

phytes

indifferent

concentrations

ofheavymetalsa

mendedagar

plates

Strain

Nickel

Copper

Cadm

ium

Cobalt

Lead

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

50100

150

200

250

300

581PDA1

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

581PDA2

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA3

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA4

++

++

--

++

++

+-

++

++

--

++

++

--

++

++

+-

581PDA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

581PDA7

++

++

--

++

++

++

++

++

--

++

++

--

++

++

++

582P

DA1

++

++

+-

++

++

++

++

++

--

++

++

+-

++

++

++

582P

DA4

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA5

++

++

--

++

++

--

++

++

--

++

++

--

++

++

--

582P

DA6

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA7

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

582P

DA8

++

++

--

++

++

--

++

++

--

++

++

--

++

++

+-

582P

DA9

++

++

++

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA11

++

++

--

++

++

+-

++

++

+-

++

++

+-

++

++

+-

582P

DA13

++

++

--

++

++

++

++

++

+-

++

++

+-

++

++

++

Here+deno

tesg

rowth-

deno

tesn

ogrow

th







Data Availability






Acknowledgments



References











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








Data Availability






Acknowledgments



References











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



References











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


assessment of plant growth promoting and abiotic stress...

Documents