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Modern Biocontrol
Agents and
Biofertilizers
Prof. Mark Laing
Plant Pathology, UKZN
& Plant Health Products (Pty) Ltd
Infected Ant
The Future is Here, for BCAs
1. Fewer pesticides available
2. A wider range of BCAs
3. Better formulations, shelf-life, simplicity
4. Biofertilizers:
• N fixing – nodules and free-living
• Mobilization of P and micronutrients
• Buffering abiotic stress:
• Tolerance of heat, cold, drought, waterlogging, soil
toxins, toxic metals
5. Soluble silicon - a powerful tool to add
Beneficial Microbes for Agriculture
– available of coming soon !
1. Biocontrol Agents to control
• Diseases, pre- and post-harvest - most
• Pests - most
• Nematodes – coming now
Added benefit
– Growth stimulation esp. seed and roots
2. Biofertilizers to enhance fertilization
• N fixation – Rhizobium, Azotobacter, etc.
• P mobilization
• Micronutrient mobilization – Mo, Bo, Zn, Fe
What growers need: Registered, commercial BCA
products: effective, affordable, stable, simple
Field Crop Soil Problems
• Seed death, damping off and uneven stand
(sub-lethal infection)
• Root diseases of adult plants
– Cause - Pythium, Rhizoctonia, Fusarium spp., Ggt
(take-all)
– Solution - seed treat with Trichoderma (e.g., Eco-T
from PHP)
• It stimulates early germination
• It colonizes the emerging root
• It stimulates more lateral root formation
• It kills incoming pathogens and nematodes
• It degrades allelochemicals in the soil
• It buffers versus abiotic stress factors
Biocontrol of Rhizoctonia on
cucumber seedlings
Trichoderma coils,
penetrates, kills.
Enzymes digest cell
walls and contents.
Feeding like a spider on
a fly.
D
T
R
B
TP
R
C
T
C
R
A
0
20
40
60
80
100
120Untreated
Cruizeronly
Eco-T
onlyCruizer+
Eco-T
TrichoPlus
only
Cruizer+
TrichoPlus
avg.rootweight
Effects of treatments on root wet weight
A Maize Trial with 2 Trichodermas & Cruiser
Mechanisms of Biocontrol
1. Hyper-parasitism (eats fungus)
2. Antibiosis (chemicals that attack pathogens)
3. Competitive displacement (eats
nutrients first)
4. Triggering resistance in host (SAR)
5. Binds iron needed by pathogen
(releases binding agents, siderophores)
Kenyan Lisianthus with Fusarium oxysporum
root rot
No treatment
Trichoderma applied weekly
Survival(%)
0
20
40
60
80
100
Control
Rhizoc
Tdr+
Rhizoc
Tst+
Rhizoc
Treatment
Survival(%)
TotalDryWeight
0
2
4
6
8
10
Control
Rhizoc
Tdr+
Rhizoc
Tst+
Rhizoc
Treatment
DryWeight(g)
Biocontrol of Rhizoctonia on
cucumber seedlings
Foliar Diseases – 2 examples
• Rust biocontrol– fungus Sphaerellopsis filum
attacks pustules and stops carryover – isolate, screen,
register. Studies on biology, epidemiology, efficacy over
seasons, economics of its use, culture conditions.
• Powdery mildew biocontrol - Ampelomyces
quisqualis (AQ). AQ10 was commercialized previously.
PHP has worked out its production but it needs
registration trials.
A rust pustule colonized by S. filum
S. filum – black spore
capsules in the old rust
pustule
A new rust pustule
Infection of wheat rust spore by S. filum
(Plachecka 2005)
17
Rust
Spore
Rust Spore
Ampelomyces quisqualis attacking grape powdery
mildew, U. necator
18
A pycnidium of A. quisqualis
developed inside a spore of pm,
showing the powdery mildew colony
on a grape leaf in the background.
Photos - D. Gadoury
Spores of A. quisqualis emerging
from a pycnidium of A. quisqualis,
formed in place of a pm conidium
Post-Harvest vs Moulds and Insects
• Eco-77 – colonize flowers of wheat
and maize to stop Aspergillus &
Fusarium (source of mycotoxins)
• Yeasts: e.g. YieldPlus, B13
• Control of weevils & moths (USDA
too)
– Beauveria bassiana
– Bacillus thurungiensis
Untreated
Control
Yeast B13
Rice Weevil (Sitophilus oryzae) infected
with Beauvaria bassiana
Rice Weevil (Sitophilus oryzae) infected
with Beauvaria bassiana
Insect Pests
• Aphids – Conidiobolus ?
• Caterpillars – Viruses, Btk, Bb
• Bollworm - viruses
• Red spider – Beauveria bassiana
• Thrips – Bb
• Beetles, weevils – Bb, EPNs
• Whitegrubs – B. brognartia, Bb, Bt
Beauveria bassiana parasitizing
army worm larvae
Beauveria-infected
white grub adult
Beauveria-infected
black vine weevil
Dead red spider mites after Eco-Bb x K silicate
on brinjal (Gatarahiya & Laing, 2008)
0
20
40
60
80
100
120
0 1 2
Beauveria rate (g l-1)
%deadmites
(adults+juveniles)
No Si
With Si
Bollworm on Wheat Infected by Virus
Beetle grub filled with EPN’s
Pictures taken from the University of Florida extension service web-site
http://edis.ifas.ufl.edu/IN468
Entomopathogenic nematode entering an insect larva
EPN
Larva
Nematode (Eelworm) Biocontrol
• Trichoderma (Eco-T) is suppressive
• Nematode trapping fungi – Clonostachys
rosae eats nematode larvae
• Bacillus thurungiensis strains kill if eaten
• Bayer – Votivo = Bacillus firmus
• Products that eat nematode eggs – to kill
all eggs in top 300mm of soil, requires
2000 kg / ha vs recommended 2kg / ha
• Rotate with a Brassica crop (e.g., canola,
cabbage) – mustard gases released
• Nematodes in sheep and goats !
Nematode attacked by a nematode-
trapping fungus
1. Eliminates sub-lethal pathogens
2. Plant hormones released
3. Mineralization – P, Mo dissolved
4. Detoxification – allellochemicals
and metals
5. Environmental buffering
Growth Stimulation by Trichoderma:
Modes of action
No
Treatment
Trichoderma
Nutrient uptake can be enhanced by
alterations to root structure
Untreated
1.8 area units
Eco-T seed treatment
3.2 area units
Eco-T treated maize root
numerous root hairs
Untreated maize root
Few root hairs
Nutrient uptake is enhanced by increased
number of root hairs
Root and shoot lengths of perenial
ryegrass (69 days)
0
50
100
150
200
250
Untreated
Treated
Untreated
Treated
Feb planting March planting
Length(mm)
Shoot
Root
Abiotic Stress: some Trichoderma
strains and beneficial soil bacteria buffer plants
from extreme environmental conditions:
• Waterlogging
• Drought
• pH extremes
• Nutritional stress
• Pathogen / Environment interaction
Eco-T® buffers vs water-logging
Effects of soil moisture in lettuce (inoculated with Pythium)
0
100
200
300
400
500
600
700
800Yield(g)wetweight
uninoculated control
inoculated control
biocontrol
Treatments
optimum irrigationhalf double
0
20
40
60
80
Meanwetweight(g)
control(u) control(i) biocontrol
Treatments
6C
12C
18C
24C
30C
Effect of water temperature on biological interactions
in hydroponic lettuce (inoculated with Pythium)
40
60
80
100
120
140
160
Meantotalwetweights(g)
control(u)
control(i)
biocontrol
Treatments
pH4
pH5
pH6
pH7
Effect of pH on disease severity and biocontrol activity
Formulations and Applications
• Crucial to activity – eg Bb
• Application must maximize BCA activity
– e.g., Eco-T on seed vs drenching
• Needs to be simple, accurate,
affordable to attract farmers
• Must not conflict with farmer operations
• New approach for field crops – Eco-T in
John Deere talc+graphite lubricant for
planter bins
Biofertilizers
• Symbiotic N fixation – only legumes
– Rhizobium nodules
– Promiscuous strains under development for other legumes
– Room storage at 200C
• Free Living N fixation – for all crops
– Azotobacter, Azospirillum sp.
– Burkholderia sp., Klebsiella
• P access, dissolved from clay, rock phosphate
– Bacillus spp.
– Trichoderma
– Mycorrhizae
• Micronutrient access (Mo, Bo, Zn, Cu, Fe)
– Trichoderma
– Mycorrhizae
Soybean –
Rhizobium nodules
(Strain WB74)
Red nodule =
fixing N
Green nodule =
parasite !
Effect of microbial treatments on Soybean yield
0
500
1000
1500
2000
2500
untreated
Eco-T
1x
Eco-T
2x
Rhizobium
Eco-T
+
R
hizobium
Treatments
Yield(g)
Series1
Rhizobium vs waterlogging stress in wattle
• N fixation for all the
other crops
• Free-living diazotrophic
bacteria to fix nitrogen in the
root zone
• How much NPK fertilizer can
be replaced by the best
Azotobacter strains ?
• To date: 65% replacement of
normal NPK fertilizer without
affecting normal plant
growth, using best strains
• P solubilization by other
bacteria
Figure Increased rooting in treated plants (left)
Soluble Silicon in Plant Health
• Silicon deficient soils - >70% of Africa short of soluble Si
• No deficiency symptoms but both physical and
biochemical role in some plants (Si uptake is active)
• Grasses deposit Si as phytoliths into leaves; cane cuts
• Action vs Pests – accelerated resistance
• Action vs Diseases – accelerated SAR (immune system)
• Improves orderly cellulose deposition
• Stress tolerance
– Cold, frost
– Heat
– Drought
– Al, Mn, Cd
• Synergistic with biocontrol agents
Typical Opal Phytoliths (maize & grass)
Liang, Sun, Zhu, Christie 2006
• Silicon effectively mitigates abiotic
stresses:
– Manganese, aluminum and heavy metal
toxicities
– Salinity
– Drought
– Chilling and freezing stresses
• Mechanisms poorly understood
Abiotic Stress Buffering by Si
Cold Stress:
bananas after 40C x 4 days
The Future of BCAs
1. Fewer pesticides available
2. A wider range of BCAs
3. Better formulations, shelf-life, simplicity
4. Biofertilizers:
• N fixing – nodules and free-living
• Mobilization of P and micronutrients
• Buffering abiotic stress:
• Tolerance of heat, cold, drought, waterlogging, soil
toxins, toxic metals
5. Soluble silicon - a powerful tool to add
Potential Problems with BCA & BFZ
• Foreign strains, not well adapted to SA soils
• Many strains need to be screened to find one really
powerful strain – requires time, money and research
facilities
• Good BCA but poor formulation = ineffective
• Spores counts too low – 105 /g instead of 107 /g
• Contamination by other fungi
• It is not a chemical, but a living organism. Back seat
of a car for 4 hours will kill it.
• Relatively short-lived except spore-formers (Bacillus),
6-12 months
56

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@No tll bca and biofertilizers 2012 09 04 a

  • 1. Modern Biocontrol Agents and Biofertilizers Prof. Mark Laing Plant Pathology, UKZN & Plant Health Products (Pty) Ltd
  • 3. The Future is Here, for BCAs 1. Fewer pesticides available 2. A wider range of BCAs 3. Better formulations, shelf-life, simplicity 4. Biofertilizers: • N fixing – nodules and free-living • Mobilization of P and micronutrients • Buffering abiotic stress: • Tolerance of heat, cold, drought, waterlogging, soil toxins, toxic metals 5. Soluble silicon - a powerful tool to add
  • 4. Beneficial Microbes for Agriculture – available of coming soon ! 1. Biocontrol Agents to control • Diseases, pre- and post-harvest - most • Pests - most • Nematodes – coming now Added benefit – Growth stimulation esp. seed and roots 2. Biofertilizers to enhance fertilization • N fixation – Rhizobium, Azotobacter, etc. • P mobilization • Micronutrient mobilization – Mo, Bo, Zn, Fe
  • 5. What growers need: Registered, commercial BCA products: effective, affordable, stable, simple
  • 6. Field Crop Soil Problems • Seed death, damping off and uneven stand (sub-lethal infection) • Root diseases of adult plants – Cause - Pythium, Rhizoctonia, Fusarium spp., Ggt (take-all) – Solution - seed treat with Trichoderma (e.g., Eco-T from PHP) • It stimulates early germination • It colonizes the emerging root • It stimulates more lateral root formation • It kills incoming pathogens and nematodes • It degrades allelochemicals in the soil • It buffers versus abiotic stress factors
  • 7. Biocontrol of Rhizoctonia on cucumber seedlings
  • 8. Trichoderma coils, penetrates, kills. Enzymes digest cell walls and contents. Feeding like a spider on a fly. D T R B TP R C T C R A
  • 10. Mechanisms of Biocontrol 1. Hyper-parasitism (eats fungus) 2. Antibiosis (chemicals that attack pathogens) 3. Competitive displacement (eats nutrients first) 4. Triggering resistance in host (SAR) 5. Binds iron needed by pathogen (releases binding agents, siderophores)
  • 11. Kenyan Lisianthus with Fusarium oxysporum root rot No treatment Trichoderma applied weekly
  • 13.
  • 14.
  • 15. Foliar Diseases – 2 examples • Rust biocontrol– fungus Sphaerellopsis filum attacks pustules and stops carryover – isolate, screen, register. Studies on biology, epidemiology, efficacy over seasons, economics of its use, culture conditions. • Powdery mildew biocontrol - Ampelomyces quisqualis (AQ). AQ10 was commercialized previously. PHP has worked out its production but it needs registration trials.
  • 16. A rust pustule colonized by S. filum S. filum – black spore capsules in the old rust pustule A new rust pustule
  • 17. Infection of wheat rust spore by S. filum (Plachecka 2005) 17 Rust Spore Rust Spore
  • 18. Ampelomyces quisqualis attacking grape powdery mildew, U. necator 18 A pycnidium of A. quisqualis developed inside a spore of pm, showing the powdery mildew colony on a grape leaf in the background. Photos - D. Gadoury Spores of A. quisqualis emerging from a pycnidium of A. quisqualis, formed in place of a pm conidium
  • 19. Post-Harvest vs Moulds and Insects • Eco-77 – colonize flowers of wheat and maize to stop Aspergillus & Fusarium (source of mycotoxins) • Yeasts: e.g. YieldPlus, B13 • Control of weevils & moths (USDA too) – Beauveria bassiana – Bacillus thurungiensis
  • 21. Rice Weevil (Sitophilus oryzae) infected with Beauvaria bassiana Rice Weevil (Sitophilus oryzae) infected with Beauvaria bassiana
  • 22. Insect Pests • Aphids – Conidiobolus ? • Caterpillars – Viruses, Btk, Bb • Bollworm - viruses • Red spider – Beauveria bassiana • Thrips – Bb • Beetles, weevils – Bb, EPNs • Whitegrubs – B. brognartia, Bb, Bt
  • 25. Dead red spider mites after Eco-Bb x K silicate on brinjal (Gatarahiya & Laing, 2008) 0 20 40 60 80 100 120 0 1 2 Beauveria rate (g l-1) %deadmites (adults+juveniles) No Si With Si
  • 26. Bollworm on Wheat Infected by Virus
  • 27. Beetle grub filled with EPN’s Pictures taken from the University of Florida extension service web-site http://edis.ifas.ufl.edu/IN468
  • 28. Entomopathogenic nematode entering an insect larva EPN Larva
  • 29. Nematode (Eelworm) Biocontrol • Trichoderma (Eco-T) is suppressive • Nematode trapping fungi – Clonostachys rosae eats nematode larvae • Bacillus thurungiensis strains kill if eaten • Bayer – Votivo = Bacillus firmus • Products that eat nematode eggs – to kill all eggs in top 300mm of soil, requires 2000 kg / ha vs recommended 2kg / ha • Rotate with a Brassica crop (e.g., canola, cabbage) – mustard gases released • Nematodes in sheep and goats !
  • 30. Nematode attacked by a nematode- trapping fungus
  • 31. 1. Eliminates sub-lethal pathogens 2. Plant hormones released 3. Mineralization – P, Mo dissolved 4. Detoxification – allellochemicals and metals 5. Environmental buffering Growth Stimulation by Trichoderma: Modes of action
  • 33. Nutrient uptake can be enhanced by alterations to root structure Untreated 1.8 area units Eco-T seed treatment 3.2 area units
  • 34. Eco-T treated maize root numerous root hairs Untreated maize root Few root hairs Nutrient uptake is enhanced by increased number of root hairs
  • 35.
  • 36.
  • 37. Root and shoot lengths of perenial ryegrass (69 days) 0 50 100 150 200 250 Untreated Treated Untreated Treated Feb planting March planting Length(mm) Shoot Root
  • 38. Abiotic Stress: some Trichoderma strains and beneficial soil bacteria buffer plants from extreme environmental conditions: • Waterlogging • Drought • pH extremes • Nutritional stress • Pathogen / Environment interaction
  • 39. Eco-T® buffers vs water-logging
  • 40. Effects of soil moisture in lettuce (inoculated with Pythium) 0 100 200 300 400 500 600 700 800Yield(g)wetweight uninoculated control inoculated control biocontrol Treatments optimum irrigationhalf double
  • 41. 0 20 40 60 80 Meanwetweight(g) control(u) control(i) biocontrol Treatments 6C 12C 18C 24C 30C Effect of water temperature on biological interactions in hydroponic lettuce (inoculated with Pythium)
  • 43. Formulations and Applications • Crucial to activity – eg Bb • Application must maximize BCA activity – e.g., Eco-T on seed vs drenching • Needs to be simple, accurate, affordable to attract farmers • Must not conflict with farmer operations • New approach for field crops – Eco-T in John Deere talc+graphite lubricant for planter bins
  • 44. Biofertilizers • Symbiotic N fixation – only legumes – Rhizobium nodules – Promiscuous strains under development for other legumes – Room storage at 200C • Free Living N fixation – for all crops – Azotobacter, Azospirillum sp. – Burkholderia sp., Klebsiella • P access, dissolved from clay, rock phosphate – Bacillus spp. – Trichoderma – Mycorrhizae • Micronutrient access (Mo, Bo, Zn, Cu, Fe) – Trichoderma – Mycorrhizae
  • 45. Soybean – Rhizobium nodules (Strain WB74) Red nodule = fixing N Green nodule = parasite !
  • 46. Effect of microbial treatments on Soybean yield 0 500 1000 1500 2000 2500 untreated Eco-T 1x Eco-T 2x Rhizobium Eco-T + R hizobium Treatments Yield(g) Series1
  • 47. Rhizobium vs waterlogging stress in wattle
  • 48. • N fixation for all the other crops • Free-living diazotrophic bacteria to fix nitrogen in the root zone • How much NPK fertilizer can be replaced by the best Azotobacter strains ? • To date: 65% replacement of normal NPK fertilizer without affecting normal plant growth, using best strains • P solubilization by other bacteria
  • 49. Figure Increased rooting in treated plants (left)
  • 50. Soluble Silicon in Plant Health • Silicon deficient soils - >70% of Africa short of soluble Si • No deficiency symptoms but both physical and biochemical role in some plants (Si uptake is active) • Grasses deposit Si as phytoliths into leaves; cane cuts • Action vs Pests – accelerated resistance • Action vs Diseases – accelerated SAR (immune system) • Improves orderly cellulose deposition • Stress tolerance – Cold, frost – Heat – Drought – Al, Mn, Cd • Synergistic with biocontrol agents
  • 51. Typical Opal Phytoliths (maize & grass)
  • 52. Liang, Sun, Zhu, Christie 2006 • Silicon effectively mitigates abiotic stresses: – Manganese, aluminum and heavy metal toxicities – Salinity – Drought – Chilling and freezing stresses • Mechanisms poorly understood Abiotic Stress Buffering by Si
  • 54. The Future of BCAs 1. Fewer pesticides available 2. A wider range of BCAs 3. Better formulations, shelf-life, simplicity 4. Biofertilizers: • N fixing – nodules and free-living • Mobilization of P and micronutrients • Buffering abiotic stress: • Tolerance of heat, cold, drought, waterlogging, soil toxins, toxic metals 5. Soluble silicon - a powerful tool to add
  • 55. Potential Problems with BCA & BFZ • Foreign strains, not well adapted to SA soils • Many strains need to be screened to find one really powerful strain – requires time, money and research facilities • Good BCA but poor formulation = ineffective • Spores counts too low – 105 /g instead of 107 /g • Contamination by other fungi • It is not a chemical, but a living organism. Back seat of a car for 4 hours will kill it. • Relatively short-lived except spore-formers (Bacillus), 6-12 months
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