Plant Growth Promoting fungi PGPF - Copy.ppt

Evaluation the ability of PGPF to Induce Plant Systemic Resistant ( ISR )

Detection of phytohormone biosynthesis e.g. IAA and GAs by indigenous fungi isolated from the rhizosphere to elect promising strains for further study and application.
Investigation for the ability of these rhizosphere fungi to produce of Aminocyclopropane Carboxylic acid Deaminase (ACCD)

7) Gene Expression analysis of (SidD) genes which involved in siderophores- production for the selected plant growth-promoting fungi under heavy metal stress
Tohamy Anwar Tohamy

1. Studies on Rhizosphere Fungi for Plant Growth
Promotion and Stress Mitigation
هداهجا نم ةففخملاو تابنلاومنل ةزفحملا ريفسوزيرلا تايرطف يلع تاسارد
Tohamy Anwar Tohamy Mohamed
B.Sc. (Microbiology 2012)
Under Supervision
of
Dr. Khalid A. Hussein
Assistant Prof. of Microbiology
Botany & Microbiology Department,
Faculty of Science, Assiut University
Prof. Dr. Saad S. M. El-Maraghy
Professor of Microbiology
Botany & Microbiology Department,
Faculty of Science, Assiut University
2021

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Outlines
Introduction
Aims
Materials and Methods
Results
Discussion
Conclusion

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Introduction
Rhizosphere area

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Fungi Strategies for Plant Growth Promotion(PGP)
P-Solublization
Phytohormones
(e.g. Indol acetic acid & GAs )
(Biocontrol)
Via induced system resistance (ISR)
e.g. Chitinase, Glucanase and PR-1)
Siderophores Production
1-aminocyclopropane-1-carboxylic acid
(ACC) deaminase (ACCD)
and HM effect on expression of ACCD
Biofilm production
Introduction

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Introduction
Stress Mitigation

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Biocontrol via induced system resistance (ISR)
(Chitinase , Glucanase and pathogenesis related protein-1 (PR-1) as plant defense genes )
Introduction
PRR plant recognition receptor
PAMP Pathogen ----PTI Pathogen Triggered Immunity---هثحتسم هعانم--ISR------PR expression
Pathogen molecules-----ETI Effector Triggered Immunity --- --هبستكم هعانمSAR----HR

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Phytohormones (Indol acetic acid & GAs )
Introduction

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P-Solublization
Introduction

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Siderophores(Greek for iron carrier) is a low
molecular weight (500-1000 Daltons), high affinity ferric
iron-chelating compound secreted by organisms.
(Wittenwiler 2007).
Siderophores
Introduction

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1-aminocyclopropane-1-carboxylic
Deaminase (ACCD)
Introduction

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Extracellular Polymeric Matrix
Biofilm formation
Introduction

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Outlines
Introduction
Aims
Materials and Methods
Results
Discussion
Conclusion

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1)CollectionandcharacterizationofRhizospherefungalstrainspriortoplant
growth-promotingtraitsinvestigation.
2)Evaluationofthesiderophoresproductionabilitybydifferentfungalstrains
Isolatedfromtherhizosphere
3)Investigationfortheinorganicphosphate-solubilizationabilitybyPlantGrowth
PromotingFungi(PGPF)candidates
Aimsofthework

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Aimsofthework
4)EvaluationtheabilityofPGPFtoInducePlantSystemicResistant(ISR)
5)Detectionofphytohormonebiosynthesise.g.IAAandGAsbyindigenousfungi
isolatedfromtherhizospheretoelectpromisingstrainsforfurtherstudyand
application.
6)Investigationfortheabilityoftheserhizospherefungitoproduceof
AminocyclopropaneCarboxylicacidDeaminase(ACCD)
7)GeneExpressionanalysisof(SidD)geneswhichinvolvedinsiderophores-
productionfortheselectedplantgrowth-promotingfungiunderheavymetalstress

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8)GeneExpressionof(ACCD)genewhichinvolvedin(ACC)deaminase-
productionfortheselectedplantgrowth-promotingfungi(PGPF).
9)Investigationfortheeffectofthepotentfungalstrainson“GreenhousePlant
Growth”inordertoimprovethequalityandproductivityofanimportantcrop.
10)Evaluationofthepotentialityofcandidatefungalstrainstoenhance/improve
planttoleranceagainstsalinitystressandtoacceleratetheearlyseed
germinationofkeyplantcrops.
Aimsofthework

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Introduction
Aims
Materials and Methods
Results
Discussion
Conclusion
Outlines

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Materials and methods
Wheatgrains(TriticumaestivumL.)wereprovidedby
(AgriculturalResearchCenter,Egypt).
Morethan170isolateswerecollectedfrom
therhizosphereofwheat(T.aestivumL)Assiut,Egypt.
1. Isolation and identification

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Materials and methods
Identification of Strains
(Moubasher 1993).
( Domsh 1980).
C
B
D
A
1. Isolation and identification

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Potent isolates for mol. identification
The universal primers ITS1: 5’ TCC GTA GGT GAA CCT GCG G 3’ and
ITS4: 5’-TCCTCCGCTTATTGATATGC -3’ were used for fungal amplification
(Herlemann et al. 2011).
GenBank database using the BLAST search (NCBI).
The online tool PhyML (www.phylogeny.fr),
visualization of the tree was achieved using
TreeDyn.
Materials and methods
Identification of Strains
1. Isolation and identification

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Materialsandmethods
ChromeazurolS(CAS)agarplateassaywasused.
CAS-agarhalfplatetechniquewasconducted
accordingto(SchwynandNeilands1987).
Thesiderophoresunitswerecalculatedas
[(Ar-As)/Ar]100=%siderophoresunits;
2.Siderophoresbiosynthesisassay

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where Ar is the absorbance value of the reference,
and As is the absorbance value of the sample. The
investigation was conducted in three replicates. The
average of the three replicates was used for all
analyses.
Materialsandmethods
Siderophoresbiosynthesisassay
2.Siderophoresbiosynthesisassay

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Materials and methods
Phosphatesolublizationassay
SolubilizationoftheprecipitatedCa
3(PO
4)
2
onPikovskaya’sagarmedium(Pikovskaya1948).
Phosphate solubilization was estimated quantitatively
by inoculating 10 mL of Pikovskaya’s broth )Pingale and
Virkar, 2013; Yasmin andBano, 2011).
3.Phosphatesolublizationassay

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PreparationofBarleygraininoculums(BGI)
accordingto(Hossainetal.,2008)
potentisolates
Aspergillusflavus,A.niger,Pencilliumchrysogenum,P.
citrinum,andTrichodermakoningiopsis
Rhizocotinasolanipathogenwasisolatedfromrootrot
infected(PhaseolusvulgarisL.)
Materials and methodsA
B C
D E
F G
4.ISRExperimentFORpotentisolates

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Cultivation of T. aestivumin soil
Autoclaved soil + T. aestivum grains
Cultivated at RT
11-h day and 13-h night cycle.
Materials and methods
Induction Treatments with BGI
Autoclaved soil + Powdered BGI (0.5% w/w) five PGPF
Chemicalinductiontreatment
syntheticISRinducerbenzothiadiazole(0.3mMBTH)A
B C
D E
F G
4.ISRExperimentFORpotentisolates

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Materials and methodsA
B C
D E
F G
ChallengeInoculationanddiseaseassessment
-After3weeksinasoilsystem.
PlantswereinoculatedbyBGIofRhizocotinasolani
pathogenaccordingto(Hossainetal.,2008).
-After7daysafterthepathogenapplication,disease
severitywasmeasuredaccordingto(Ryuetal.,2003).
-Gene expression analysis of PR-1.2, Chi-1, and Glu-2
4.ISRExperimentFORpotentisolates

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Materials and methods
RT-PCRanalysis
RNAExtraction
RNAwasisolatedbythephenol/chloroformextraction
accordingtothemethoddescribedbyKayetal.(1987).
cDNAsynthesis
Forthegeneexpression,complementaryDNA
(cDNA)wassynthesized
QRT-PCRanalysisforRNAs
TherelativeexpressiondataoftotalRNAwas
calculatedaccordingtothe2
−ΔΔCT
procedureand
presentedasfoldchange(LivakandSchmittgen,2001).

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Materials and methods
Indole-3-acetic acid production
•By using Czapek-Dox broth media
supplemented with tryptophan.
•A standard curve of IAA concentration was
designed to calculate the equivalent IAA
concentration produced by each fungal
strain in the bioassay media. (Brick 1991).
5.DetectionofIAAandGasbiosynthesise.g.

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Materials and methods
5.DetectionofIAAandGasbiosynthesise.g.
Gibberellins’ production
•Gibberellic acid was produced on synthetic
Czapek-Dox broth (Difco) media. According to
(Cho et al., 1979)
•Gibberellic acid in the ethyl acetate phase
wasmeasured by UV spectrophotometer 254
nm.
•at The amount of gibberellic acid was calculated
from the standard curve (Bruckner 1991).

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Materials and methods
ACC deaminase activity was measured by the determination of α-
ketobutyrate Minimal media containing ACC as a sole nitrogen
source (Bhagat 2013 ) (Bhagat 2014).
1-aminocyclopropane-1-carboxylicacid(ACC)
deaminaseproducingplantgrowthpromoting
fungihydrolyzeACCintoammoniaandα-
ketobutyratereducestresslevelsofethyleneand
consequentlydecreasingtheseverityofstress.
Fungus
6.EstimationofACCdeaminaseactivity(ACCD)

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Materials and methods
7. Detection of gene Expression of (SidD) genes which involved in siderophores-production
for the selected plant growth-promoting Yeast under heavy metal stress
Eightyeast strains were select from
35yeast isolates, namely YEAST-1,
-2, -5, -6, -16, -17, -30, and YEAST-
34, were subjected to siderophores,
IAA, and ACCD production and
phosphate solubilization activity.
strains were identified18S rRNA
genes sequence
Yeast isolation and identification

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•CAS-agar plates with different concentrations of
CdCl
2and Pb(NO
3)
2(0, 200, 400, 600,800 and 1000 μM) inoculated with
yeast strains and incubated at 27 ᴼC for 4 days.
Inculcation and Cultivation
Materials and methods
7. Detection of gene Expression of (SidD) genes which involved in siderophores-production
for the selected plant growth-promoting Yeast under heavy metal stress
2 potent
Yarrowia lipolytica
Trichosporon ovoides
showed significantly
higher resistance to
heavy metals

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Materials and methods
Inculcation and Cultivation •Color development or orang halos were detected.
7. Detection of gene Expression of (SidD) genes which involved in siderophores-production
for the selected plant growth-promoting Yeast under heavy metal stress

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Materials and methods
sidD-forward (5-ACGCAACCGACTGGTTGTT -3) and
sidD-reverse (5-ATTCGTGCGAGACTCGGAT -3)
according to (Pandey 2014) .
RNA Extraction, cDNA synthesis and Quantitative real-time PCR
7. Detection of gene Expression of (SidD) genes for the selected plant growth-promoting
Yeast under heavy metal stress

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Materials and methods
8. Detection of gene Expression of (ACCD) genes which involved in(ACC) deaminase
production for the selected plant growth-promoting Yeast
ACC deaminase activity was measured
by the determination of α-ketobutyrate
Minimal media containing ACC, (1,3 and
5) mMas a sole nitrogen source (Bhagat
2013 and (Bhagat 2014).
Inculcation and Cultivation

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RNA Extraction, cDNA synthesis and Quantitative real-time PCR
•ACCD (forward) 5-CGGGAGGAAGCCGTATTACA -3
•ACCD (Reverse) 5-CGACCCTGTCACAGCACAAA -3 (Viterbo et al 2010)
Materials and methods
8. Detection of gene Expression of (ACCD) genes which involved in s1-
aminocyclopropane-1-carboxylic acid (ACC) deaminase production for the selected plant
growth-promoting Yeast

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Materials and methods
9. Investigation for the effect of the potent fungal strains on “Greenhouse Plant
Growth” in order to improve the quality and productivity of an important crop.

37. Seeds of
crops
Surface
sterilization
10 Seeds on
filter paper
Introduction of
saline solution
Soaking in
0, 50, 100, 200 mM NaCl
25℃incubation at
dark condition
Growth chamber
4 days growing
Examination and recording
results every 24 h
Germination experiment
Materials and methods
10. Evaluation of the potentiality of Yeast strains to enhance plant tolerance against salinity
stress and to accelerate the early seed germination of key plant crops.

38. Seeds of
crops
Surface
sterilization
Inoculation
of seeds
Soaking for 2 h
at 200 rpm shaking
Imbibing yeast
into seeds
Soaking in
0, 50, 100, 200 mM NaCl
Growth chamber
4 days
growing
Record number of seeds
germinated every after 24 h
25℃incubation at
dark condition
Germination experiment
Yeasts vs. Salinity stress
Materials and methods

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Materials and methods
11.Yeast biofilm formation with crystal violet (CV) staining test
(Di Domenico et al. 2016).
Polystyrene coated 96-well sterile plate
BHI only as
control
Constant count of Yeast sus.
37ºc for 48 h
Wash ( D.W)Dry for 30 min
Stain attached biofilm
forming Yeast ( CV stain )
Wash ( D.W)
Dissolving the dye that incorporated
with the cells forming biofilm using ethanol acetone

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Introduction
Aims
Materials and Methods
Results
Discussion
Conclusions
Outlines

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41Fungi ID Species
IAA Production
µg ml
-1
1 Aspergillus niger Tiegh.,1867 298.7 ± 26.33
2 Penicillium islandicum, sopp,1912 224.2 ± 36.87
3 Penicillium chrysogenum ,thom,1920 252.1 ± 72.41
4 Penicillium chrysogenum ,thom,1920 578 ± 362.1
5 Penicillium chrysogenum ,thom,1920 626.9 ± 549.7
6 Penicillium chrysogenum ,thom,1920 338.7 ± 129
7 Penicillium chrysogenum ,thom,1920 291.7 ± 3.292
8 Penicillium chrysogenum ,thom,1920 886.6 ± 341.7
9 Penicillium chrysogenum ,thom,1920 581.2 ± 482.5
10 Penicillium chrysogenum ,thom,1920 281.9 ± 100.1
11 Penicillium chrysogenum ,thom,1920 216.7 ± 56.62
12 Penicillium chrysogenum ,thom,1920 567.3 ± 429.9
13 Penicillium chrysogenum ,thom,1920 1094 ± 67.81
14 Aspergillus niger Tiegh.,1867 585.9 ± 447
15 Penicillium chrysogenum ,thom,1920 249.8 ± 106
16 Penicillium chrysogenum ,thom,1920 286.6 ± 30.1
17 Aspergillus niger Tiegh.,1867 452.3 ± 613.6
18 Penicillium chrysogenum ,thom,1920 230.7 ± 287
19 Aspergillus fumigatus 286.6 ± 308.1
20 Penicillium chrysogenum ,thom,1920 187.9 ± 134.3
21 Aspergillus fumigatus 359.7 ± 375.9
22 Penicillium chrysogenum ,thom,1920 375.9 ± 420
23 Cladosporium cladosporioides 564.5 ± 693.2
24 Penicillium chrysogenum ,thom,1920 20.77 ± 8.5
25 Penicillium chrysogenum ,thom,1920 634.8 ± 433.2
26 Pencillium islandicum 333.1 ± 158
27 Penicillium oxalicum 488.1 ± 96.77
28 Cladosporium cladosporioides 310.8 ± 32.92
29 Acremonium strictum 641.3 ± 119.8
30 Penicillium chrysogenum ,thom,1920 774.4 ± 44.77
31 Penicillium corylophilum 750.7 ± 490.4
32 Cladosporium cladosporioides 159 ± 202.8
33 Penicillium islandicum 707.4 ± 248.8
34 Cladosporium cladosporioides 1103 ± 50.69
35 Cladosporium cladosporioides 609.2 ± 460.2
36 Penicillium chrysogenum ,thom,1920 235.4 ± 93.48
37 Cladosporium cladosporioides 635.7 ± 828.2
38 Penicillium chrysogenum ,thom,1920 484.4 ± 631.3
39 Aspergillus niger Tiegh.,1867 301.5 ± 371.3
40 Cladosporium cladosporioides 258.6 ± 331.8
41 Penicillium oxalicum 465.8 ± 634
42 Cladosporium cladosporioides 579.8 ± 696.5
43 Cladosporium cladosporioides 194.4 ± 163.3
44 Cladosporium cladosporioides 178.1 ± 199.5
45 Cladosporium cladosporioides 654.3 ± 847.9 Fungi ID Species
IAA Production
µg ml
-1
46 Cladosporium cladosporioides 514.7 ± 632
47 Cladosporium cladosporioides 291.7 ± 386.4
48 Cladosporium cladosporioides 275.9 ± 344.3
49 Cladosporium cladosporioides 407.6 ± 150.1
50 Penicillium chrysogenum 458.8 ± 306.8
51 Cladosporium cladosporioides 547.7 ± 199.5
52 Penicillium chrysogenum ,thom,1920 632.9 ± 580.6
53 Aspergillus niger Tiegh.,1867 296.8 ± 276.5
54 Cladosporium cladosporioides 432.3 ± 524.7
55 Cladosporium cladosporioides 368.5 ± 356.8
56 Penicillium citrinum 140.9 ± 167.9
57 Penicillium islandicum 303.3 ± 38.18
58 Penicillium islandicum 935.9 ± 282.4
59 Aspergillus niger Tiegh.,1867 852.2 ± 71.7
60 Cladosporium cladosporioides 616.6 ± 653.7
61 Penicillium citrinum 31.94 ± 13.8
62 Cladosporium cladosporioides 38.93 ± 30.28
63 Cladosporium herbarum 50.56 ± 49.37
64 Aspergillus niger Tiegh.,1867 19.84 ± 3.29
65 Fusarium oxysporum 755.8 ± 672.8
66 Cladosporium cladosporioides 710.2 ± 650.4
67 Penicillium chrysogenum ,thom,1920 256.3 ± 78.34
68 Penicillium chrysogenum ,thom,1920 581.2 ± 357.5
69 Penicillium chrysogenum ,thom,1920 1110 ± 133
70 Cladosporium cladosporioides 571.9 ± 458.8
71 Penicillium chrysogenum ,thom,1920 372.2 ± 117.2
72 Aspergillus falvus 816.3 ± 441.1
73 Penicillium islandicum 1080 ± 273.9
74 Penicillium chrysogenum ,thom,1920 812.6 ± 535.9
75 Yeast 418.8 ± 1221.1
76 Yeast 301 ± 25.67
77 Penicillium chrysogenum ,thom,1920 936.9 ± 440.4
78 Aspergillus niger Tiegh.,1867 817.7 ± 204.7
79 Penicillium chrysogenum ,thom,1920 762.3 ± 413.4
80 Cladosporium herbarum 266.1 ± 355.5
81 Cladosporium herbarum 575.2 ± 435.8
82 Aspergillus niger Tiegh.,1867 846.6 ± 311.4
83 Penicillium chrysogenum ,thom,1920 482.1 ± 280.4
84 Penicillium chrysogenum ,thom,1920 680.9 ± 536.5
85 Penicillium chrysogenum ,thom,1920 669.7 ± 780.1
86 Cladosporium cladosporioides 496.5 ± 614.2
87 Fusarium solani 308.5 ± 361.4
88 Penicillium chrysogenum ,thom,1920 276.8 ± 306.1
89 Aspergillus niger Tiegh.,1867 302.4 ± 1.3
90 Cladosporium funiculosum 686.4 ± 15.14 Fungi ID Species
IAA Production
µg ml
-1
91 Aspergillus falvus 736.2 ± 67.15
92 Fusarium oxysporum 1073 ± 42.13
93 Penicillium chrysogenum ,thom,1920 78.03 ± 32.92
94 Penicillium chrysogenum ,thom,1920 48.24 ± 18.43
95 Yeast 69.65 ± 65.83
96 Yeast 31.48 ± 13.17
97 Aspergillus niger Tiegh.,1867 1092 ± 197.5
98 Penicillium chrysogenum ,thom,1920 1436 ± 376.6
99 Penicillium chrysogenum ,thom,1920 284.7 ± 38.18
100 Penicillium chrysogenum ,thom,1920 303.3 ± 35.55
101 Cladosporium cladosporioides 1192 ± 15.8
102 Cladosporium cladosporioides 928 ± 44.77
103 Cladosporium cladosporioides 453.7 ± 1.975
104 Cladosporium cladosporioides 521.2 ± 23.7
105 Penicillium chrysogenum ,thom,1920 638 ± 524.7
106 Alternaria alternata 1179 ± 782.1
107 Penicillium chrysogenum ,thom,1920 478.8 ± 285.1
108 Aspergillus niger Tiegh.,1867 681.8 ± 535.2
109 Aspergillus niger Tiegh.,1867 666.9 ± 776.2
110 As.flavus 524.9 ± 654.4
111 Pencillium citrincum 259.1 ± 291.6
112 Penicillium chrysogenum ,thom,1920 322 ± 370
113 Penicillium chrysogenum ,thom,1920 615.7 ± 476
114 Penicillium chrysogenum ,thom,1920 1182 ± 736.7
115 Penicillium chrysogenum ,thom,1920 462.1 ± 289
116 Penicillium chrysogenum ,thom,1920 632.9 ± 501.6
117 Penicillium islandicum 619.9 ± 793.9
118 Penicillium islandicum 537 ± 597.8
119 Penicillium islandicum 272.1 ± 254.8
120 Penicillium islandicum 294.5 ± 344.3
121 Cladosporium cladosporioides 292.6 ± 19.09
122 Penicillium islandicum 664.6 ± 5.267
123 Cladosporium cladosporioides 703.7 ± 52.67
124 Cladosporium cladosporioides 1004 ± 23.04
125 Penicillium islandicum 59.87 ± 1.975
126 Penicillium islandicum 113.9 ± 0.658
127 Cladosporium cladosporioides 101.8 ± 13.82
128 Penicillium islandicum 37.53 ± 19.09
129 Cladosporium cladosporioides 90.37 ± 9.8
130 Cladosporium cladosporioides 84.34 ± 15.23
131 Penicillium islandicum 90.48 ± 9.9
132 Penicillium islandicum 87.36 ± 10.9
133 Cladosporium cladosporioides 84.52 ± 17.24
134 Aspergillus flavus 89.23 ± 9.02
135 Aspergillus flavus 89.43 ± 9.34 Fungi ID Species
IAA Production
µg ml
-1
136 Penicillium islandicum 87.15 ± 12.45
137 Aspergillus flavus 89 ± 10.14
138 Aspergillus flavus 89.48 ± 9.3
139 Aspergillus flavus 90.92 ± 6.9
140 Aspergillus flavus 88.3 ± 11.25
141 aspergillus flavus 145.061± 23
kh1 Aspergillus ustus 137.613 ±34
kh2 Trichoderma harzianum 106.89 ± 11
kh3 Trichoderma harzianum 179.508 ± 25
kh4 penicillium chrysogenum ,thom,1920 109.683 ± 13.2
kh5 fusarium solani 126.441 ±45
kh6 Trichoderma harzianum 117.131 ± 56
kh7 Trichoderma harzianum 105.028 ± 49
kh8 penicillium chrysogenum ,thom,1920 82.684± 35
kh9 fusarium solani 109.683± 65
kh10 Trichoderma harzianum 129.234± 37
kh11 penicillium chrysogenum ,thom,1920 114.338 ± 76
kh12 fusarium solani 96.649 ± 18
kh13 Trichoderma harzianum 110.614 ± 15
kh14 Trichoderma harzianum 128.303 ± 19
kh15 Trichoderma harzianum 97.58 ± 65
kh16 penicillium chrysogenum ,thom,1920 136.682 ± 85
kh17 fusarium solani 141.337 ± 98
kh18 Trichoderma harzianum 105.959 ± 95
kh19 Trichoderma harzianum 122.717 ± 5.25
kh20 fusarium oxysporum 10.997 ± 14.5
kh21 penicillium chrysogenum ,thom,1920 123.648 ± 18.5
kh22 fusarium solani 110.614 ± 25
kh23 Trichoderma harzianum 127.372 ± 15.9
kh24 Trichoderma harzianum 107.821 ± 11
kh25 As.ustus 139.475 ± 15.7
kh26 As.ustus 126.441 ± 14
kh27 Trichoderma harzianum 139.475 ± 102
kh28 As.ustus 108.752 ± 96
kh29 As.ustus 118.993 ± 56
kh30 cladosporium cladosporioides 118.993 ± 34
Results
Table .1. Isolated strains

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Siderophores production
Fig.1. Quantitative spectrophotometric assay for siderophore
production by fungal species isolated from wheat (T. aestivum)
rhizosphere.
Results

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Fig. 2 . Plate showing growth of isolate which indicates
ACC utilization using ACCD aminase producing yeast.
Fig.4. The quantitative detection of
IAA
Fig.3. Example for solubilization of
precipitated Tricalcium-phosphate
Ca
3(PO
4)
2by Trichoderma
koningiopsisshould be measured
according to halo width (mm).
ACCD and IAA production and Phosphate-solubilization activity
Results
control

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Table. 2. Gene expression levels for the studied plant defensive genes
between wheat affected with PGPF, chemical inducer, and control groups.Treatment PR-1.2 Chi-1 Glu-2
Control 1 ± 0.00d 1 ± 0.00d 1 ± 0.00d
Aspergillus flavus 27.90 ± 5.70a 1.79 ± 0.79d 1.48 ± 0.10d
Aspergillus niger 34.80 ± 6.60a 3.02 ± 0.78ab 2.39 ± 0.73b
Penicillium chrysogenum 4.65 ± 4.32d 2.388 ± 1.28cd 2.60 ± 0.54b
Pencillium citrinum 8.36 ± 5.95c 2.164 ± 0.19d 2.25 ± 0.89bc
Trichoderma koningiopsis19.56 ± 6.03b 4.827 ± 2.40a 24.84 ± 1.40a
BTH 7.41 ± 4.71c 3.143 ± 1.14b 1.92 ± 0.49c
Role of plant-growth promoting fungi (PGPF) in defensive genes
expression of Triticum aestivum against wilt disease
Results

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45Treatment
Co ntr o l
A. flavus A. niger
P . chr yso genum
P . citr inum
T . ko ningio p sis
BT H
Gene expression levels
0
10
20
30
40
50
PR-1.2
Chit-1
Glu-2
Disease severity
Results
Fig.5. Disease severity against Rhizoctonia solani R43 in Triticum aestivumL.
treated with PGPF strains and BTH.Expression of the plant-specific defensive
Role of plant-growth promoting fungi (PGPF) in defensive genes
expression of Triticum aestivum against wilt disease

46. 6/20/2024
46A
B C
D E
F G
Fig .6. disease severity
in the untreated control was significantly higher than
all treatments.
Plants that received PGPF inoculation and BTH
treatment showed less severe leaves lesions than
the untreated plant.
A) untreated control; B) Aspergillus flavus;
C) A. niger; D) Pencillium chrysogenum;
E) P. citrinum; F) Trichoderma koningiopsis;
G)synthetic SAR inducer (BTH), separately.
Results
Role of plant-growth promoting fungi (PGPF) in defensive genes
expression of Triticum aestivum against wilt disease

47. 6/20/2024
47
Fig.7.Phylogenetictreedesignedbyneighbor-closinganalysisof18SrRNAgenesequencetoshow
thepositionandsimilaritypercentageoftheisolatedPGPFstrains.AspergillusflavusND41,A.niger
A7,Penicilliumchrysogenumzy32,P.citrinumFIBSR4,andTrichodermakoningiopsisT-440.The
lengthsoftheuprightlinesarearbitrary;thelengthsofthetransversallinesareproportionaltogenetic
distances.Bootstrapfrequenciesaregivenforamultipledatasetof100trials.
Results
Phylogenetic tree designed by neighbor-closing analysis of 18S rRNA gene sequence of
potent isolates

48. 6/20/2024
48
Fig. 8. 18s rRNA gene sequence similarity for the selected root-associated
fungal strains.
Results
18s rRNA gene sequence similarity for the selected root-associated fungal strains.

49. 6/20/2024
49
Salinity effect on Triticum aestivumSeeds germination
Fig .9. Examples of yeast treatment effect on Triticum aestivum
seeds germination
A –Control B -Yarrowia lipolytica(1)
C -Saccharomyces cerevisiae (34) D -Candida subhashii(17)
Results

50. 6/20/2024
50
Results
Salinity effect on Triticum aestivumSeeds germination
Table.3. Effect of yeast treatment on germination seeds of Triticum
aestivum: under salinity stress:
Root lengthsNaCl2 Mm Control Yarrowia lipolytica (1)Candida diddensiae (2) Trichosporon gamsii (5) Trichosporon ovoides (6)
0 1.773 ± 0.21 a 1.933 ± 0.35 a 1.99 ± 0.31 a 2.1633 ± 0.35 ab 1.49 ± 0.13 a
50 1.073 ± 0.11 a 1.233 ± 0.15 a 1.09 ± 0.1 a 1.633 ± 0.15 a 1.9 ± 0.1 a
100 1.563 ± 0.15 a 1.367 ± 0.3 a 0.8 ± 0.23 a 1.4 ± 0 a 1.733 ± 0.11 a
200 1.31 ± 0.15 a 1.533 ± 0.15 a 0.967 ± 0.1 a 2.44 ± 0.37 b 1.167 ± 0.45 a
NaCl2 Mm Control Yarrowia lipolytica (16)Candida subhashii (17)Saccharomyces cerevisiae (30) Saccharomyces cerevisiae (34)
0 1.773 ± 0.21 a 1.96 ± 0 a 1.133 ± 0.14 a 1.793 ± 0.32 a 1.99 ± 0.21 a
50 1.073 ± 0.11 a 1.6 ± 0 a 1.23 ± 0.15 a 1.633 ± 0.12 a 1.35 ± 0.21 a
100 1.563 ± 0.15 a 1.5 ± 0.26 a 1.1 ± 0 a 1.7 ± 0.2 a 1.333 ± 0.35 a
200 1.31 ± 0.15 a 1.1 ± 0.65 a 1.2 ± 0.12 a 2.223 ± 0.25 ab 0.888 ± 0.168 a

51. 6/20/2024
51
Results
Salinity effect on Triticum aestivumSeeds germination
Table.4. Effect of yeast treatment on germination seeds of Triticum
aestivumunder salinity stress:
shoot lengthsNaCl2 Mm Control Yarrowia lipolytica (1)Candida diddensiae (2) Trichosporon gamsii (5) Trichosporon ovoides (6)
0 1.8 ± 0.11 a 3± 0.32 ab 6.7 ± 0.64 c 3.663 ± 0.55 ab 4 ± 0.7 b
50 2.09 ± 0.136 a 3.33 ± 0.35 ab 6.9 ± 0.7 c 3.553 ± 0.15 ab 3.9 ± 0.661 b
100 2.12 ± 0.131 a 3.167 ± 0.53 ab 6.8 ± 0.7 c 3.76 ± 0.6 ab 4.733 ± 0.166 b
200 2.23 ± 0.15 a 3.555 ± 0.55 ab 6.067 ± 0.92 c 3.433 ± 0.55 ba 4.2.167 ± 0.15 b
NaCl2 Mm Control Yarrowia lipolytica (16)Candida subhashii (17)Saccharomyces cerevisiae (30) Saccharomyces cerevisiae (34)
0 1.8 ± 0.11 a 5.8 ± 0.5 bc 3.533 ± 0.15 ab 3.9 ± 0.1 ab 3.867 ± 0.23 ba
50 2.09 ± 0.136 a 5.5 ± 0 bc 3.553 ± 0.17 ab 3.733 ± 0.78 ab 3.5 ± 0.13 ab
100 2.12 ± 0.131 a 5.5 ± 0.27 bc 3.55 ± 0 ab 3.87 ± 0.2 ab 3.333 ± 0.89 ab
200 2.23 ± 0.15 a 5.333 ± 0.77 bc 3.2 ± 0.55 ab 3.133 ± 0.85 ab 3.9233 ± 0.068 ab

52. 6/20/2024
52
Effect of yeast treatment on Triticum aestivumgrowth in greenhouse
Fig.10. Effect of yeast treatment on plant growth of Triticum aestivum: Shoot and
Root lengths in compare with control.
Results
Yarrowia lipolytica (1)
Candida diddensiae (
2
)
Trichosporon gamsii (5) Trichosporon ovoides (
6
)
Yarrowia lipolytica (
16
)
Candida subhashii (17)
Saccharomyces cerevisiae (
30
)
Saccharomyces cerevisiae (
34
)
control

53. 6/20/2024
53
Table.5. Effect of yeast treatment on plant growth of Triticum aestivum:
shoot lengths in compare with control
Results
Effect of yeast treatment on Triticum aestivumgrowth in greenhouseControl Yarrowia lipolytica (1)Candida diddensiae (2) Trichosporon gamsii (5) Trichosporon ovoides (6)
1.99 ± 0.11 a 3± 0.32 ab 6.7 ± 0.64 c 3.663 ± 0.55 ab 4 ± 0.7 b
2.32 ± 0.136 a 3.33 ± 0.35 ab 6.9 ± 0.7 c 3.553 ± 0.15 ab 3.9 ± 0.661 b
2.13 ± 0.131 a 3.167 ± 0.53 ab 6.8 ± 0.7 c 3.76 ± 0.6 ab 4.733 ± 0.166 b
2.37 ± 0.15 a 3.555 ± 0.55 ab 6.067 ± 0.92 c 3.433 ± 0.55 ab 4.2.167 ± 0.15 b
Control Yarrowia lipolytica (16)Candida subhashii (17) Saccharomyces cerevisiae (30) Saccharomyces cerevisiae (34)
1.99 ± 0.11 a 5.8 ± 0.5 bc 3.533 ± 0.15 ab 3.9 ± 0.1 b 3.867 ± 0.23 b
2.32 ± 0.136 a 5.5 ± 0 bc 3.553 ± 0.17 ab 3.733 ± 0.78 b 3.5 ± 0.13 ab
2.13 ± 0.131 a 5.5 ± 0.27 bc 3.55 ± 0 ab 3.87 ± 0.2 b 3.333 ± 0.89 ab
2.37 ± 0.15 a 5.333 ± 0.77 bc 3.2 ± 0.55 ab 3.133 ± 0.85 ab 3.9233 ± 0.068 b

54. 6/20/2024
54
Results
Effect of yeast treatment on Triticum aestivumgrowth in greenhouse
Table.6.EffectofyeasttreatmentonplantgrowthofTriticumaestivum:
RootlengthsincomparewithcontrolControl Yarrowia lipolytica (1)Candida diddensiae (2) Trichosporon gamsii (5) Trichosporon ovoides (6)
2.773 ± 0.21 a 3.933 ± 0.35 ab 6.99 ± 0.31 c 3.1633 ± 0.35 ab 3.49 ± 0.13 ab
2.073 ± 0.11 a 3.233 ± 0.15 ab 6.09 ± 0.1 c 3.633 ± 0.15 ab 3.9 ± 0.1 b
2.563 ± 0.15 a 3.367 ± 0.3 ab 6.8 ± 0.23 c 3.4 ± 0 ab 3.733 ± 0.11 b
2.31 ± 0.15 a 3.533 ± 0.15 ab 6.967 ± 0.1 c 3.44 ± 0.37 ab 4.167 ± 0.45 b
Control Yarrowia lipolytica (16)Candida subhashii (17) Saccharomyces cerevisiae (30) Saccharomyces cerevisiae (34)
2.773 ± 0.21 a 5.96 ± 0 bc 3.133 ± 0.14 ab 3.793 ± 0.32 b 3.99 ± 0.21 b
2.073 ± 0.11 a 5.6 ± 0 bc 3.23 ± 0.15 ab 3.633 ± 0.12 ab 4.35 ± 0.21 b
2.563 ± 0.15 a 5.5 ± 0.26 bc 3.1 ± 0 ab 3.7 ± 0.2 b 4.333 ± 0.35 b
2.31 ± 0.15 a 5.1 ± 0.65 bc 3.2 ± 0.12 ab 4.223 ± 0.25 b 3.888 ± 0.168 b

55. 6/20/2024
55
Biofilm producer yeast strains
Table.7. Biofilm producer yeast strains
High producer isolates a,
Moderate producers ab
Weak producers b
Results
Yarrowia
lipolytica (1)
Yarrowia lipolytica (
16
)
Saccharomyces cerevisiae (30)
Control
Fig.11. CV assay for
Biofilm producer yeast strainsYarrowia lipolytica YEAST-1 2.134 ± 0.15 a
Candida diddensiae YEAST-2 1.943 ± 0.036 a
Trichosporon gamsii YEAST-5 1.571 ± 0.055 a
Trichosporon ovoides YEAST-6 1.75 ± 0.023 a
Yarrowia lipolytica YEAST-16 1.347 ± 0.019 ab
Candida subhashii YEAST-17 1.765 ± 0.005 a
Saccharomyces cerevisiae YEAST-30 0.437 ± 0.017 b
Saccharomyces cerevisiae YEAST-34 1.233 ± 0.055 ab

56. 6/20/2024
5661
3416
ControlYeast-34Yeast-1Yeast-5Yeast-30Yeast-16Yeast-17Yeast-2Yeast-6
Fig.12.Plantgrowth-promotingactivitiesoftheisolatedrhizosphereyeasts;A)IAA
productionvariationamongthedifferentheavymetal-resistantstrains;B)thephosphate
solubilizationonPikovskaya’sagarmedium;C)theACCDactivityonminimalmedia-
containingACCasasolenitrogensourcebythedifferentisolatedyeaststrains.
Results
Potent isolated yeast

57. 6/20/2024
57Yeast strains
Siderphores %
(Unites)
IAA
(µg ml
-1
)
Phosphat sol.
(mg ml
-1
)
ACCD
(µmol ml
-1
)
Yarrowia lipolytica 57.11 ± 2.36bc51.53 ± 2.5c 1.03 ± 0.07cd39.36 ± 0.42b
Candida diddensiae 62.38 ± 0.87b 30.87 ± 1.8d 1.26 ± 0.07c22.44 ± 1.21d
Trichosporon gamsii 63.04 ± 2.48b 82.63 ± 4.3a 1.32 ± 0.08c29.37 ± 1.41c
Trichosporon ovoides 67.4 ± 0.33a 43.33 ± 3.0c 2.98 ± 0.09a35.45 ± 1.60b
Yarrowia lipolytica 74.85 ± 0.78a 11.09 ± 2.7e 0.93 ± 0.09d52.36 ± 0.72a
Candida subhashii 55.83 ± 3.371c23.09 ± 1.9de2.25 ± 0.04bc21.55 ± 0.96d
Saccharomyces cerevisiae 8.16 ± 3.96e 65.26 ± 2.9b 2.54 ± 0.01b20.49 ± 0.83d
Saccharomyces cerevisiae 24.7 ± 2.376d72.03 ± 7.6ab 2.38 ± 0.05b29.23 ± 1.40c
Table.8. Plant growth promoting activities of the selected heavy metal-
resistant yeast strains.
Results
Potent isolated yeast

58. 6/20/2024
58Cd
2+
Concentration (µM)
Control200 400 600 800 1000
Gene expression of
SidD
gene
in
Y. lipolytica

0
1
2
3
4
5
SidD gene Pb
2+
Concentration (µM)
Control200 400 600 800 1000
Gene expression of
SidD
gene
in
Y. lipolytica


0
1
2
3
4
5
SidD gene Cd
2+
Concentration (µM)
Control200 400 600 800 1000
Gene expression of
SidD
gene
in
T. ovoides


0
1
2
3
4
5
6
7
SidD gene Pb
2+
Concentration (µM)
Control200 400 600 800 1000
Gene expression of
SidD
gene
in
T. ovoides

0.0
0.2
0.4
0.6
0.8
1.0
SidD gene A B
C D
Fig.13.Effectofheavymetalsstressonthegeneexpressionofsiderophoresbyyeast
strains.(A)SidDgeneexpressioninY.lipolyticaunderdifferentlevelsofCd
2+
ions.(B)SidD
geneexpressioninY.lipolyticaunderdifferentconcentrationsofPb
2+
ions.(C)SidDgene
expressioninT.ovoidesunderdifferentlevelsofCd
2+
ions.(D)SidDgeneexpressioninT.
ovoidesunderdifferentconcentrationsofPb
2+
ions.
Results
Expression of SidDGene under Heavy Metal Stress in Plant
Growth-Promoting Yeasts

59. 6/20/2024
59Strains
Cd
2+
MIC
(µM)
Pb
2+
MIC
(µM)
Max identity (%) Strain of closest identity Identification
YEAST-1 2000 1500 100% Yarrowia lipolytica F45 Yarrowia lipolytica
YEAST-2 1750 1750 98% Candida diddensiae MITS575 Candida diddensiae
YEAST-5 1500 1750 100% Trichosporon gamsii CBS8245 Trichosporon gamsii
YEAST-6 1750 1750 100% Trichosporon ovoides IFM 63839Trichosporon ovoides
YEAST-16 1750 2000 99% Yarrowia lipolytica JCM 2320 Yarrowia lipolytica
YEAST-17 1500 1500 100% Candida subhashii UAMH 10744 Candida subhashii
YEAST-30 1750 1500 100% Saccharomyces cerevisiae FI25-1FSaccharomyces cerevisiae
YEAST-34 1750 1500 99% Saccharomyces cerevisiae MIBA781 Saccharomyces cerevisiae
Table.9. Heavy metal-resistant yeasts isolated from rhizosphere
soils and their identification.
Results
MIC for potent isolated yeasts

60. 6/20/2024
60A
Candida diddensiae MITS575
Candida subhashii UAMH 10744
Saccharomyces cerevisiae MIBA781
Saccharomyces cerevisiae FI25-1F
Trichosporon gamsii CBS8245
Trichosporon ovoides IFM 63839
Yarrowia lipolytica F45
Yarrowia lipolytica JCM 2320
Fig.14.Phylogeneticanalysisoftheisolatedheavymetal-resistantyeaststrains;
A)Thephylogenictreeshowingthelengthsoftheuprightlinesarearbitrary;the
lengthsofthetransversallinesareproportionaltogeneticdistances.Bootstrap
frequenciesaregivenforamultipledatasetof100trials;
Results
Heavy metal-resistant yeasts isolated

61. 6/20/2024
61B
Candida diddensiae MITS575
Candida subhashii UAMH 10744
Saccharomyces cerevisiae MIBA781
Saccharomyces cerevisiae FI25-1F
Trichosporon gamsii CBS8245
Trichosporon ovoides IFM 63839
Yarrowia lipolytica F45
Yarrowia lipolytica JCM 2320
B)18s rRNA gene sequence similarity for the isolated heavy metal-
resistant yeast strains.
Results
Heavy metal-resistant yeasts isolated

62. 6/20/2024
62
Expression of ACCDGene in Plant Growth-Promoting Yeasts PGPY
Fig.15.AnalysisoftheACCDmRNAsusingQuantitativeReal-TimePCR.ACCDmRNA
induction by 1, 3 and 5 mM ACC.after a 24-h incubation. Values were normalized against
noninduced control .
The results are relative mRNA expression (mean standard error) of three replicate
cultures.
Results

63. 6/20/2024
63
Introduction
Aims
Materials and Methods
Results
Discussion
Conclusion
Outlines

64. 6/20/2024
64
Discussion
Beneficial microbes are those microbiomes of plant roots able to improve
plant health by facilitating minerals uptake e.g. solubilization the inorganic
phosphate or production of siderophores.
Inducedsystemicresistance(ISR)isanimportantmechanismbywhich
plantgrowth-promotingbacteriaandfungimaintainthewholeplantbodyfor
improvinggrowthandenhancingitsdefenseagainstabroadrangeof
pathogens.
OurresultsshowedahighaccumulationofPR-1.2,Chi-1,andGlu-2inthe
wheatplantsinoculatedwithPGPFcomparingtothosetreatedwithBTH.

65. 6/20/2024
65
Discussion
JA-mediatedresponsesareinducedwhenaplantiswoundedbyinsect
herbivoryorduringpathogenlesionformation;theyarealsoinvolvedinISR
(Waltersetal.,2013).
Kimetal.(2015)analyzedgeneexpressioninpathogenesis-related(PR),they
foundthat,thevolatilesofBacillussp.JSincreasedtheregulationofPR-2and
acidicPR-3.
BarleygraininoculumofPGPFstrainsinducesystemicresistanceinTriticum
aestivumL(Khatrietal.,2017).

66. 6/20/2024
66
ManystudieshavedemonstratedthatSAplaysacriticalroleintheplant
defenseagainstpathogens.
SAlevelincreasesinbothinfectedandintactleavesinresponsetopathogen
penetration,theexogenousapplicationofSAcanactivatethepathogenesis-
related(PR)genesandlaunchSAR(Mathysetal.,2012).
TheobtainedresultsindicatedthatPR-1.2,Chi-1,andGlu-2genesareinvolved
inplantdefenseresponsepathwaysofwheatplantsafterRhizoctoniasolani
attack.
Discussion

67. 6/20/2024
67
Dimkpaetal.(2009)observedthatCd,Cu,andNistimulatedsiderophores
productionbyStreptomycessp.strains.
heavymetalsotherthanironinduceproductionofsiderophoressuggeststhat
thesechelatorsmightplayaroleinmicrobialtoxicmetaltolerance(Husseinand
Joo,2017)
Discussion

68. 6/20/2024
68
Yuatal.(2017)revealedthatBacillusjunceacouldproduceanexcessof
siderophoreandmightbepossiblyusedtoincreasethephytoextractionof
heavymetalsfromsoil.
Khannaetal.(2019)detectedthatmetalresistantPGPRundermetaltoxicity
improvedphotosyntheticpigments
enhancedexpressionofmetaltransportergenes.
Discussion

69. 6/20/2024
69Cd
2+
Concentration (µM)
Control200 400 600 800 1000
Gene expression of
SidD
gene
in
Y. lipolytica

0
1
2
3
4
5
SidD gene Pb
2+
Concentration (µM)
Control200 400 600 800 1000
Gene expression of
SidD
gene
in
Y. lipolytica


0
1
2
3
4
5
SidD gene Cd
2+
Concentration (µM)
Control200 400 600 800 1000
Gene expression of
SidD
gene
in
T. ovoides


0
1
2
3
4
5
6
7
SidD gene Pb
2+
Concentration (µM)
Control200 400 600 800 1000
Gene expression of
SidD
gene
in
T. ovoides

0.0
0.2
0.4
0.6
0.8
1.0
SidD gene A B
C D
Our results
Discussion

70. 6/20/2024
70
Brotmanetal.,(2013)indicatedthatFungiwhichexpressACCDcanmitigate
thedifferentstressesthataffectplantcropsanddevelopment
OurresultsdemonstratedACCdeaminase-producingmicrobesmay
improveplantgrowthbydecliningthedetrimentaleffectofstressexertedby
ethylene.
OurresultsindicatedoverexpressionofACCDgene
Discussion

71. 6/20/2024
71
Introduction
Aims
Materials and Methods
Results
Discussion
Conclusion
Outlines

72. 6/20/2024
72
Data demonstrated that disease severity(lesions size) was significantly
decreased in PGPF treated wheat
Conclusion
PGPF are economically and environmentally beneficial for plant growth
promotion.
PGPF induced plant systemic immunity ISRand evaluatedexpression of
pathogenesis-related proteins of wheat plants including PR-1.2, Chi-1, and,
Glu-2 after infection by Rhizoctoniasolani, the causal agent of wheat wilt
disease.
.
PGPF gives novel and different modes of action for plant protection
strategies to improve cropping systems.
Salinity stress mitigation –HM stress mitigation
Biofilm forming protection

73. 6/20/2024
73
Metal-resistantmicroorganismsabletoproducesiderophoreshave
recentlybeensuggestedtoimprovegrowthanddecreasetheheavymetals
accumulationwithinplants.
Inthisstudy,eightyeastisolateswereobtainedfromTriticumsativum
rhizospheresoilaffiliatingwithYarrowia,Candida,Trichosporon,and
Saccharomyces
Showedrelativelyhighheavymetaltolerance,wereanalyzedfor
siderophores,IAA,andACCDproduction,andphosphatesolubilization.
Y.lipolyticaF45andY.lipolyticaJCM2320tolerated2mMofCdandPb.For
theremainingyeaststrains
Conclusions

74. 6/20/2024
74
SidDgeneisthemostlikelyresponsibleforthebiosynthesisofsiderophoresin
fungi.
Thepresenceof600–800μMheavymetal,butunderironlimitation,induced
thesidDgeneexpressionduringthegrowthofT.ovoidesIFM63839andY.
lipolyticaJCM2320strains.Thedatacurrentlyavailablementionthat
siderophoresareproducedbyyeastsnotsolelytoscavengeforiron.
Thisstudyconsideredtheconceptthatsiderophoresplayasignificantrolein
protectingtherhizobialmicrobesagainstheavymetalstoxicityanddiscusses
thepotentialcontributioninplantgrowthpromotion.
Conclusions

75. 6/20/2024
75
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76. 6/20/2024
76
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77. Thank you
6/20/2024
77
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78

79. 6/20/2024
79
Expression of SidDGene under Heavy Metal Stress in Plant
Growth-Promoting Yeasts
•Atotalof35rhizosphereyeastisolateswereselectedat
randombasedondifferencesinthemorphologicalfeaturesof
thecoloniesand
•testedforheavymetalMICusingCdCl
2andPb(NO
3)
2
•Thestrongestheavymetalresistantstrainsweresubjectedto
siderophores,IAA,andACCDproductionandphosphate
solubilizationactivity.
•Eightoftheyeaststrains,namelyYEAST-1,YEAST-2,YEAST-
5,YEAST-6,YEAST-16,YEAST-17,YEAST-30,andYEAST-
34,showedsignificantlyhigherresistancetoheavymetals
whencomparedwiththeotherstrainsinvestigatedinthe
presentstudy.
•Therhizosphereheavymetal-resistantstrainswereidentified
andanalyzedbasedontheir18SrRNAgenessequence
Results

80. 6/20/2024
80
Discussion
The result showed that PGPF are Beneficial microbes
able to
Improve plant health Facilitating minerals uptake
Solubilizationthe inorganic phosphate Production of siderophores, IAA and GAs
Induced systemic resistance (ISR) ACCD Production

81. 6/20/2024
81
Induced systemic resistance (ISR)
is an important mechanism
Plant Growth-Promoting Fungi (PGPF ) maintainthe whole plant body for
improving growth and enhancing its defense against a broad range of pathogens.
Discussion
Increasedexpression of
chitinase gene (PR-3) and β-1, 3-glucanase (PR-
2) may contribute to
higher resistance against fungal pathogens.
shows a
distinctive difference observed in the expression
of wilt disease symptomsbetween untreated
and PGPF-treated
wheat plants

82. 6/20/2024
82
Therefore,theysuggestedthatmicro-organismspossessgrowth-promotingtraits
thatcanreducemetaltoxicityinplantsKhannaetal.(2019).
Itisstillunclearhowheavymetalsotherthanironionsstimulatesiderophores
production(HusseinandJoo,2019).
Discussion

83. 6/20/2024
83
Discussion
BGIactivates plant immunity
(ISR) (SAR)
for control of Rhizoctonia solani R43
fungal lesions and disease severity are reduced in compare toBTH
The results showed a high expression of PR-1.2, Chi-1, and Glu-2 in the wheat
plants inoculated with PGPF comparing to those treated with BTH and untreated
one

84. 6/20/2024
84
Discussion
The result showed that siderophore-producing microorganisms
have been also considered important for enhancing metal tolerance
in plants
eight yeast strains showed heavy metal-resistance and plant-growth promotion
properties.
Two strains, namely YEAST-6 and YEAST-16 showed high siderophoreproduction
and heavy metal-resistance, were investigated for
sidDgene expression
under different levels of Cd and Pbtoxicity stress. SidD gene expression
was induced by yeasts growing under iron-limiting conditions and excess of other
heavy metal.
Expression of sidDgene increases in the presence of 600–800 μMheavy metal but
under iron limitation.

85. 6/20/2024
85
Discussion
Expression of ACCDGene under nitrogen deficiency Stress in Plant
Growth-Promoting Yeasts PGPY
The data obtained show that ACC deaminase is highly expressed in
34 saccharomyces cerevisiaewith 511.4 ±209 fold.
The results showed that ACCD enzyme not only be related to plant growth
promotion ability, but May also play role in microorganism’s development
ACC (mM) 1.0 3.0 5.0
noninduced control 1.13 ±0.032 0.8 ±0.021 0.9231 ±0.023
1 Yarrowialipolytica 0.4235 ±0.04 3.226 ±0.017 113.1 ±1.39
2 Candida diddensiae 4.782 ±0.049 115.8 ±1.29 149 ±5
5 Trichosporon gamsii 23.37 ±0.059 3.429 ±0.034 4.554 ±0.023
6 trichosporonovoides 5.064 ±0.013 3.853 ±0.016 7.433 ±0.1
16 Yarrowialipolytica 21.69 ±0.25 19.22 ±0.2 14.31 ±0.3
17 Candida subhashii 28 ±0.6 21.96 ±0.25 6.947 ±0.08
30 saccharomyces cerevisiae 20.25 ±0.39 0.4987 ±0.01 0.5959 ±0.0013
34 saccharomyces cerevisiae 49.63 ±0.589 2.668 ±0.11 511.4 ±209

86. 6/20/2024
86
Discussion
The result showed that the Eight isolates enhance seed germination of
triticum aestivum L.under salinity stress showed significant difference for
in compare to control.
2Candida diddensiaehave significant effect on shoot protection against
salinity lead to increase shoot length to 6.7 ±0.64 mm in zero salinity and
its activities still stable in high salt concentrations.
Salinity Stress
2Candida diddensiaetreated bot showed the most affected treatment in
both shoot and root 6.7 ±0.64, 6.99 ±0.31 mm respectively.
“Greenhouse Plant Growth”

87. 6/20/2024
87
ACC (mM) 1.0 3.0 5.0
noninducedcontrol 1.13 ±0.032 0.8 ±0.021 0.9231 ±0.023
1 Yarrowialipolytica 0.4235 ±0.04 3.226 ±0.017 113.1 ±1.39
2 Candida diddensiae 4.782 ±0.049 115.8 ±1.29 149 ±5
5 Trichosporongamsii 23.37 ±0.059 3.429 ±0.034 4.554 ±0.023
6 trichosporonovoides 5.064 ±0.013 3.853 ±0.016 7.433 ±0.1
16 Yarrowialipolytica 21.69 ±0.25 19.22 ±0.2 14.31 ±0.3
17 Candida subhashii 28 ±0.6 21.96 ±0.25 6.947 ±0.08
30 saccharomyces cerevisiae 20.25 ±0.39 0.4987 ±0.01 0.5959 ±0.0013
34 saccharomyces cerevisiae 49.63 ±0.589 2.668 ±0.11 511.4 ±209
Expression of ACCDGene under nitrogen deficiency Stress in
Plant Growth-Promoting Yeasts PGPY
Results

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Materials and methods
Determination of minimum
inhibitory concentrations
(MICs)
7. Detection of gene Expression of (SidD) genes which involved in siderophores-production
for the selected plant growth-promoting Yeast under heavy metal stress
lowest concentration of metal that
inhibit visible growth of the isolate.

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ACCD production
Fig. 2 . Plate showing growth of isolate which indicates ACC utilization
using ACCD aminase producing yeast.
Results

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Phosphate-solubilization activity
Results
Fig.3. Example for solubilization of precipitated Tricalcium-phosphate Ca
3
(PO
4)
2by Trichoderma koningiopsisshould be measured according to halo
width (mm).

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IAA production
Fig.4. The quantitative detection of IAA
Results
control