Plant defense responses against Radopholus   similis in East African Highland bananas   (EAHB-AAA) inoculated with endophy...
Banana production• 3rd most important fruit crop in the world•Types grown: cooking, brewing and dessert• Uganda has the hi...
Major banana production constraintsBiotic factors• Pests such as banana weevil and plant parasitic  nematodes (most import...
Radopholus similis damage
Cosmopolites sordidus damage
Current control strategies for banana weevil            and the nematode  • Clean planting materials (tissue culture plant...
EndophytesEndophytes are all organisms that, at some  time in their life cycle, live symptomlessly  within plant tissues  ...
Endophyte research at IITA-Uganda• A collection endophytic isolates with biocontrol  potential (From 1995)• In vitro and i...
Continued• Induced resistance determined as the most likely  mode of action against R. similis (Athman 2006, PhD  Thesis, ...
Induced resistance• Refers to the activation of plant defense responses  by biotic or abiotic elicitors• Resistance is exp...
Gaps• Development of markers to trace endophytes within  plant• Need to determine biochemical changes in banana  following...
Overview of PhD Research
Development of marked isolates for plant          colonization studies Accepted for publication in Biocontrol 2009 (DOI 10...
Colony morphology of a benomyl-resistant   mutant and wild-type isolate on PDA   V5w2                Wild-type isolate    ...
Root colonization by benomyl-resistant                        mutant isolates of Emb2.4o and V5w2                      100...
Root colonization by chlorate-resistant                       mutant isolates of Emb2.4o and V5w2                      100...
Study of biochemical changes induced    in banana following endophyte inoculation and nematode challenge
A. Expression analysis of putative banana       defense genes using qRT-PCRPublished in Molecular Plant Pathology 2007; 71...
Experimental design•   Banana cvs susceptible (Nabusa) and tolerant    (Kayinja) to R. similisTreatments1. Plants of both ...
Results• Susceptible cv: genes not up-regulated after  endophyte inoculation or nematode challenge• Tolerant cv: up-regula...
B. cDNA-AFLPs for identification of genesup-regulated in banana following endophyte                colonization Why cDNA-A...
Summary of treatments and sampling timeTreatments           Zero hr   2 DPI    7 DPI      30 DPINabusa control            ...
Root &rhizome          RNA                 mRNA & cDNA     Selective                 extraction          synthesis       a...
Results• 50 differentially expressed fragments cloned and  assigned putative identities• Fragments of interest included  ...
Expression analysis of up-regulated genes            using qRT-PCR• Expression studied in susceptible and tolerant  banana...
Nabusa                                                                            Nabusa                   18.00          ...
C. Activities of phenylpropanoid pathwayenzymes following endophyte inoculation and              R. similis challenge • PA...
PAL activity (Comparisons by cultivar and time point) Cultivar Treatment            7 DPI 7 DPNC   30 DPNC   60 DPNC Nabus...
POX activity (Comparisons by cultivar and time point)Cultivar   Treatment              7 DPI 7 DPNC   30 DPNC   60 DPNCNab...
PPO activity (Comparisons by cultivar and time point)Cultivar   Treatment              7 DPI 7 DPNC   30 DPNC   60 DPNCNab...
R. similis population densities in roots of               challenged plantsCultivar   Treatment                   7 DPNC  ...
Dual inoculation of isolates Emb2.4o                 and V5w2Published in Biocontrol Science and Technology 2009; 19:639-655
Effect of dual inoculations on plant    colonization, growth and pest controlInoculation treatmentsa. V5w2 (chlorate mutan...
Root and Rhizome colonization 4 weeks after                    inoculation                 80                             ...
Effect of Dual inoculation on pest control• Reduced nematode densities in plants inoculated with V5w2  alone and in those ...
Nematode densities in roots 12 weeks post challenge Inoculation treatment           Total nematodes (x100)/100g root      ...
Weevil damage to the rhizome 12 weeks after                              challenge               90                a      ...
Effect of Dual inoculation on plant growth• Dually inoculated plants challenged with R. similis  showed increased height a...
Conclusions• Chemically marked F. oxysporum endophytes are  stable mutants that can be effectively used to  determine actu...
Continued • Other than direct up-regulation, F. oxysporum   endophytes prime defense genes for up-regulation   following R...
Acknowledgements  BMZ for Funding
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Plant defense responses against Radopholus similis in East African Highland bananas(EAHB-AAA) inoculated with endophytic non-pathogenic Fusarium oxysporum

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Banana production,Study of biochemical changes induced in banana following endophyte inoculation and nematode challenge

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Plant defense responses against Radopholus similis in East African Highland bananas(EAHB-AAA) inoculated with endophytic non-pathogenic Fusarium oxysporum

  1. 1. Plant defense responses against Radopholus similis in East African Highland bananas (EAHB-AAA) inoculated with endophytic non-pathogenic Fusarium oxysporum Pamela Paparu Supervisors: Prof Altus Viljeon, Dr Thomas Dubois and Dr Daniel Coyne International Institute of Tropical Agriculture FABI, University of Pretoria
  2. 2. Banana production• 3rd most important fruit crop in the world•Types grown: cooking, brewing and dessert• Uganda has the highest consumption, with an estimated250 kg per capita consumption annually• World exports projected to reach15 million tons by 2010
  3. 3. Major banana production constraintsBiotic factors• Pests such as banana weevil and plant parasitic nematodes (most importantly Radopholus similis)• Diseases such as Black sigatoka, Fusarium wilt, Banana streak virus, banana bunchy top virus and Banana bacterial wiltAbiotic factors• Loss of soil fertility due to poor soil management• Socio-economic factors (land shortage, migrations)
  4. 4. Radopholus similis damage
  5. 5. Cosmopolites sordidus damage
  6. 6. Current control strategies for banana weevil and the nematode • Clean planting materials (tissue culture plants and hot water-treated suckers) • Chemical control • Cultural practices e.g Field sanitation and trapping of adult weevils • Host plant resistance • Biological control of weevils using Beauveria bassiana and that of R. similis using Fusarium oxysporum endophytes is being investigated
  7. 7. EndophytesEndophytes are all organisms that, at some time in their life cycle, live symptomlessly within plant tissues pseudostem base inner roots corm outer corm
  8. 8. Endophyte research at IITA-Uganda• A collection endophytic isolates with biocontrol potential (From 1995)• In vitro and in vivo efficacy tests against target pests• Development of inoculation methods that result in high colonization percentages (Paparu et al.2006-Journal of Crop Improvement 16: 81-95)• Root colonization patterns have also been determined (Paparu et al. 2006-Annals of Applied Biology 149:1-8)
  9. 9. Continued• Induced resistance determined as the most likely mode of action against R. similis (Athman 2006, PhD Thesis, University of Pretoria) Split-root experiments A= Inducer half: Endophyte-inoculated B= Respondent half: Nematode challenged Nematode numbers were reduced in respondent half, compared to A B control• On-farm testing of F. oxysporum endophytes fornematode control is on-going
  10. 10. Induced resistance• Refers to the activation of plant defense responses by biotic or abiotic elicitors• Resistance is expressed locally or systemicallyTypes of induced resistance• Systemic acquired resistance (SAR)- activated following exposure to pathogens or wounding; signal molecule involved in its pathway is Salicylic acid• Induced systemic resistance (ISR)- resistance induced in plants following root colonization by rhizobacteria; signal molecule is Jasmonic acid
  11. 11. Gaps• Development of markers to trace endophytes within plant• Need to determine biochemical changes in banana following resistance induction• Effect of dual inoculation of isolates on plant colonization, growth and control of target pests
  12. 12. Overview of PhD Research
  13. 13. Development of marked isolates for plant colonization studies Accepted for publication in Biocontrol 2009 (DOI 10.1007/s10526-009-9221-1)• Generated benomyl and chlorate resistant mutants• Mutants were tested for growth on PDA and plant colonization abilities, in comparison with wild-type isolates
  14. 14. Colony morphology of a benomyl-resistant mutant and wild-type isolate on PDA V5w2 Wild-type isolate Mutant isolate
  15. 15. Root colonization by benomyl-resistant mutant isolates of Emb2.4o and V5w2 100 Emb2.4o V5w2 90 80% root colonization 70 60 50 40 30 20 10 0 1 2 3 8 1 4 6 8 w2 o .4 BR BR BR BR BR BR BR BR V5 b2 Em
  16. 16. Root colonization by chlorate-resistant mutant isolates of Emb2.4o and V5w2 100 90 80 Emb2.4o V5w2% root colonization 70 60 50 40 30 20 10 0 2 3 4 6 2 4 9 12 w2 o .4 HR HR HR HR HR HR HR V5 b2 HR C C C C C C C Em C
  17. 17. Study of biochemical changes induced in banana following endophyte inoculation and nematode challenge
  18. 18. A. Expression analysis of putative banana defense genes using qRT-PCRPublished in Molecular Plant Pathology 2007; 71: 149-157 • Genes screened included PR-1, Catalase, Endochitinase, Peroxidase, PAL and Lectin • These genes were up-regulated following colonization of tolerant banana cvs by Fusarium oxysporum f.sp. cubense (Foc) (Van den Berg 2006, University of Pretoria; Forsthy 2006, University of Queen’s Land)
  19. 19. Experimental design• Banana cvs susceptible (Nabusa) and tolerant (Kayinja) to R. similisTreatments1. Plants of both cvs not inoculated (control)2. Plants of both cvs inoculated with isolate V5w2 and sampled at 2 and 33 days after inoculation3. Endophyte-inoculated plants of both cvs challenged with R. similis and sampled 3 days after challenge4. Control plants of both cvs challenged with nematodes and sampled 3 days after challenge
  20. 20. Results• Susceptible cv: genes not up-regulated after endophyte inoculation or nematode challenge• Tolerant cv: up-regulation of Catalase and PR-1 at zero hr, 33 DPI and 3 DPNC
  21. 21. B. cDNA-AFLPs for identification of genesup-regulated in banana following endophyte colonization Why cDNA-AFLPs? • Allows detection of changes in gene expression between samples • Requires no prior sequence information
  22. 22. Summary of treatments and sampling timeTreatments Zero hr 2 DPI 7 DPI 30 DPINabusa control +Km-5 control +Nabusa + V5w2 + + +Nabusa + Emb2.4o + + +Km-5 + V5w2 + + +Km-5 + Emb2.4o + + + DPI = Days Post Inoculation + = Sampling time
  23. 23. Root &rhizome RNA mRNA & cDNA Selective extraction synthesis amplification AFLP gelsampling Polymorph ic bands sequenced Zero hr V5w2 Emb2.40
  24. 24. Results• 50 differentially expressed fragments cloned and assigned putative identities• Fragments of interest included  Lipoxygenase (associated with ISR and involved in phenylpropanoid pathway)  Glycolate oxidase (involved in oxidative burst)  Coronatine insensitive 1(signal molecule essential for jasmonate responses)  Β-1,3-glucan synthase and cellulose synthase (cell wall appositions)  ABC transporter (implicated in transport of defense molecules)
  25. 25. Expression analysis of up-regulated genes using qRT-PCR• Expression studied in susceptible and tolerant banana cvs following endophyte inoculation and nematode challenge• Genes included ABC transporter, COI 1, β-1,3- glucan synthase and lipoxygenase
  26. 26. Nabusa Nabusa 18.00 0.80 Kayinja Kayinja 16.00 0.70 14.00 0.60Expression ratio Expression ratio 12.00 0.50 10.00 0.40 8.00 0.30 6.00 4.00 0.20 2.00 0.10 0.00 0.00 ZeroHr E2DPI E33DPI ERS CRS ZeroHr E2DPI E33DPI ERS CRS ABC-transporter β-1,3-glucan synthase 4.50 Nabusa Nabusa 0.10 4.00 Kayinja 0.09 Kayinja 3.50 0.08Expression ratio Expression ratio 3.00 0.07 0.06 2.50 0.05 2.00 0.04 1.50 0.03 1.00 0.02 0.50 0.01 0.00 0.00 ZeroHr E2DPI E33DPI ERS CRS ZeroHr E2DPI E33DPI ERS CRS Coronatine Insensitive 1 Lipoxygenase
  27. 27. C. Activities of phenylpropanoid pathwayenzymes following endophyte inoculation and R. similis challenge • PAL, POX and PPO are major enzymes in the phenylpropanoid pathway (leading to phenolic and lignin synthesis) • Root protein extracts were assayed for PAL, POX and PPO activity following endophyte inoculation and nematode challenge • Roots were also assessed for nematode population density Paper submitted to Nematology Journal
  28. 28. PAL activity (Comparisons by cultivar and time point) Cultivar Treatment 7 DPI 7 DPNC 30 DPNC 60 DPNC Nabusa Control Nabusa Control + R.similis Nabusa V5w2 Nabusa V5w2 + R.similis Km-5 Control Km-5 Control + R.similis Km-5 V5w2 Km-5 V5w2 + R.similis
  29. 29. POX activity (Comparisons by cultivar and time point)Cultivar Treatment 7 DPI 7 DPNC 30 DPNC 60 DPNCNabusa ControlNabusa Control + R. similisNabusa V5w2Nabusa V5w2 + R. similis Km-5 Control Km-5 Control + R. similis Km-5 V5w2 Km-5 V5w2 + R. similis
  30. 30. PPO activity (Comparisons by cultivar and time point)Cultivar Treatment 7 DPI 7 DPNC 30 DPNC 60 DPNCNabusa ControlNabusa Control + R. similisNabusa V5w2Nabusa V5w2 + R.similis Km-5 Control Km-5 Control + R. similis Km-5 V5w2 Km-5 V5w2 + R.similis
  31. 31. R. similis population densities in roots of challenged plantsCultivar Treatment 7 DPNC 30 DPNC 60 DPNCNabusa Control + R. similis 0 83.3 a 6913.9 aNabusa V5w2 + R. similis 83.3 a 0 3915.1 b Km-5 Control + R. similis 249.9 a 0 499.8 c Km-5 V5w2 + R. similis 83.3 a 83.3 a 249.9 c Means within a column followed by different letters are significantly different (P ≤ 0.005 Turkey’s)
  32. 32. Dual inoculation of isolates Emb2.4o and V5w2Published in Biocontrol Science and Technology 2009; 19:639-655
  33. 33. Effect of dual inoculations on plant colonization, growth and pest controlInoculation treatmentsa. V5w2 (chlorate mutant)b. Emb2.4o (benomyl mutant)c. V5w2 + Emb2.4od. Control
  34. 34. Root and Rhizome colonization 4 weeks after inoculation 80 Saprophytic strains 70 60 Roots V5w2% Colonization 50 Emb2.4o 40 30 Rhizome 20 10 0 E m ol E m ol w2 w2 2 2 o o .4 .4 5w 5w r r t t V5 V5 on on b2 b2 +V +V C C o o .4 .4 b2 b2 Em Em
  35. 35. Effect of Dual inoculation on pest control• Reduced nematode densities in plants inoculated with V5w2 alone and in those inoculated dually• Reduced weevil damage to the rhizome
  36. 36. Nematode densities in roots 12 weeks post challenge Inoculation treatment Total nematodes (x100)/100g root Rep 1 Rep 2 Control 217.7 a 217.3 a Emb2.4o 249.4 a 223.5 a V5w2 55.9 b 218.1 a Emb2.4o + V5w2 79.8 b 122.2 b Means within a column followed by different letters are significantly different (P ≤ 0.005 Turkey’s)
  37. 37. Weevil damage to the rhizome 12 weeks after challenge 90 a a Emb2.4o 80 ab a ab a a V5w2 ab ab Dual 70 b Control b b 60 Damage (%) 50 40 30 20 10 0 Rhizome periphery Inner rhizome Outer rhizomeFor each plant organ, bars followed by different letters are significantlydifferent at P ≤ 0.05 (Tukey’s)
  38. 38. Effect of Dual inoculation on plant growth• Dually inoculated plants challenged with R. similis showed increased height and girth in one replicate• Effect on growth neutral in replicate 2
  39. 39. Conclusions• Chemically marked F. oxysporum endophytes are stable mutants that can be effectively used to determine actual plant colonization percentages• Defense pathways activated following banana root colonization by pathogenic and non-pathogenic F. oxysporum are dissimilar• There is significant evidence for the involvement of JA-induced defense pathway in endophyte- mediated defense responses against R. similis
  40. 40. Continued • Other than direct up-regulation, F. oxysporum endophytes prime defense genes for up-regulation following R. similis challenge • Dual inoculation of isolates Emb2.4o and V5w2 increases plant colonization and reduces R. similis populations in the screenhouse • The effect of dual inoculation of isolates Emb2.4o and V5w2 on plant growth and weevil damage needs further investigations
  41. 41. Acknowledgements BMZ for Funding

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