Download Beneficial and Pathogenic Microbes in Agriculture

Sustainable Palm Oil Research Unit (SPOR)
Sustainability Challenges in
Oil Palm Mono-cultivation:
Are Microbes the Solution?
Khim-Phin,Chong
Sustainable Palm Oil Research Unit (SPOR)
Universiti Malaysia Sabah
Content of Talk:
Oil Palm Success and Challenges: In Brief
Fundamental of Microbial in Agriculture
Case Studies:
-Roles of Microbes in Oil Palm Improvement:
-Roles of Microbes in Enhancing Oil Palm Disease Resistant
-Microbial Diversity in Oil Palm Soil
Conclusions
Oil Palm
World Palm Oil production increase from 13 to 28% from
1990 to 2011, with increase of exports from 36 to 57%
Two key exporting countries: Malaysia (47%) and
Indonesia (46%). Account 93% of world palm oil exports
Malaysia: 5 Million Ha of Oil Palm. Contributing over 11%
of global supply of edible oils and fats with 0.1% of the
total global agricultural land area
High Land Productivity Oil Palm vs Other Oil
Seeds:
11x more than soyabean
10x more than sunflower
7x more than rapeseed
In 2011, Malaysia’s Export revenue of oil palm
products reached a record high of RM80.4
billion, an increase of 34.5% against RM59.8
billion achieved in 2010.
Palm Oil Industry:
At Crossroad or Under
Real Threat?
Workers
SocioEnvironment
Human
Capital
Productivity
Cost of Production
Soil Environments
• Surface and
subsurface soils are
typically nutrientpoor environments
for microbes
• Rhizosphere is
enriched in nutrients
as a result of nearby
plant activities
Rhizosphere
• Rhizoplane
– soil in direct contact
with plant root
• Endophytes
– microbes attached to
root surface
Increasing organic C
Decreasing moisture
Organic material in rhizosphere
• Exudates
– low molecular weight compounds
released from plant cells in a nonmetabolic manner (leakage)
• Secretions
– compounds metabolically released
from plant cells
• Lysates
– compounds released from moribund
cells during autolysis
• Plant mucilage
– plant polysacchrides
• Some bacteria and blue green
algae are able to fix nitrogen
from the atmosphere to enrich
soil with nitrogen and increase
its fertility.
• These microbes are commonly
called biological nitrogen
fixers.
• Rhizobium bacteria is involved in the
fixation of nitrogen in leguminous
plants (pulses). Rhizobium lives in
the root nodules of leguminous
plants, such as beans and peas, with
which it has a symbiotic relationship.
• Sometimes nitrogen gets fixed
through the action of lightning.
• Our atmosphere has 78% nitrogen gas. Nitrogen is one of the
essential constituents of all living organisms as part of
proteins, chlorophyll, nucleic acids and vitamins. The
atmospheric nitrogen cannot be taken directly by plants and
animals.
• Certain bacteria and blue green algae present in the soil fix
nitrogen from the atmosphere and convert into compounds of
nitrogen.
• Once nitrogen is converted into these usable compounds, it can
be utilized by plants from the soil through their root system.
• Nitrogen is then used for the synthesis of plant proteins and
other compounds. Animals feeding on plants get these proteins
and other nitrogen compounds.
• When plants and animals die, bacteria and fungi present in the
soil convert the nitrogenous wastes into nitrogenous compounds
to be used by plants again.
• Certain other bacteria convert some part of them to nitrogen gas
which goes back into the atmosphere. As a result, the
percentage of nitrogen in the atmosphere remains more or less
constant.
Beneficial root-microbe interactions
• Atmosphere contains 1015 tons N2 gas
– Biological nitrogen fixation
– Minimum of 70 million tons N fixed/year
Sources of Fixed Nitrogen
Biological
Lightning
Fertilizer
25%
10%
65%
Vegetative Growth
Improvement-Nursery
(Johor Estate)
• Combinations of Bacillus spp,
Aspergillus spp & Pseudomonas
spp
Feb 2010 : Seed sowing
May 2010 : Transplanting to main
nursery
June 2010 : 1st Application
Oct 2010 : 2nd Application
Dec 2010 : Field transplanting
Microbes treated
Untreated
Courtesy of Agrinos
Results show the averages for each parameters measure for
microorganisms treated oil palm seedlings compared to control
(Kalimantan Nursery, Indonesia)
Observation (Avg)
Treated
Control
Frond Length (cm)
133.8
125.6
Leaflet Length (cm)
41.8
36.8
Number of Total Fronds
21.2
20.4
Girth (cm)
37.2
30.4
Root Length (cm)
122.8
111.4
Seedling Height (cm)
154.8
140.2
Courtesy of Agrinos
Yield-Immature Plantings
(Tawau Estate)
• Nitrogen based fertilizer
reduction:
1st Yr Planting-0%
2nd Yr Planting-15%
Subsequent Yr-25%
• Yield Improvement (%)
1st Yr Harvesting-80%
2nd Yr Harvesting-120%
3rd Yr Harvesting-49%
4th Yr Harvesting-17%
5th Yr Harvesting-11%
3.57% bigger girth
10% > LAI
Courtesy of Agrinos
Soil Analysis
Block
Depth(cm)
Opt
pH
Exchg. K
Available P
Organic C
(p.p.p.m)
(%)
Total
Available
Organic N
(%)
4.55.5
0.30
400
20.00
2.50
0.25
TreatBlk 1
0-15
5.6
0.38
187
20.30
0.77
0.13
TreatBlk 1
15-35
5.8
0.17
199
11.80
0.99
0.18
ContBlk 2
0-15
5.5
0.66
178
6.90
0.66
0.12
ContBlk 2
15-35
4.7
0.65
137
5.10
0.37
0.09
Courtesy of Agrinos
Yield-Mature Plantings
(Sandakan Estate)
• 1st application of
microbes: 10 years age
• Yield Improvement (%)
1st Yr treatment-12%
2nd Yr treatment-15%
3rd Yr treatment-25%
4th Yr treatment-28%
Years of treatment
*Salinity and sandy problem
Courtesy of Agrinos
Percentage of Ganoderma infection after two months based on ergosterol
(fungal sterol) content and growth on GSM for oil palm seedlings pre-treated
with various combination of microbes
Microbes
Ergosterol
(Average)
Infection
percentage
GSM
percentage
Bacillus spp. and Trichoderma
spp.
0.19 µg/mL-1
30 %
30%
Lactobacillus, Nattobacillus and
Yeasts
0.19 µg/mL-1
60 %
60 %
Bacillus spp, Aspergillus spp. and
Pseudomonas spp.
0.23 µg/mL-1
100 %
100 %
Control (Infect without microbes)
1.52 µg/mL-1
100%
100%
Control (Healthy)
0 µg/mL-1
0%
0%
Percentage of Ganoderma infection after four months based on ergosterol
(fungal sterol) content and growth on GSM for oil palm seedlings pre-treated
with various combination of microbes
Microbes
Ergosterol
(Average)
Infection
percentage
GSM
percentage
Bacillus spp. and Trichoderma
spp.
0.22 µg/mL-1
60 %
60%
Lactobacillus, Nattobacillus and
Yeasts
0.34 µg/mL-1
50 %
50%
Bacillus spp, Aspergillus spp. and
Pseudomonas spp.
0.22 µg/mL-1
60 %
60 %
Control (Infect without microbes)
2.35 µg/mL-1
100%
100%
Control (Healthy)
0 µg/mL-1
0%
0%
A
Ergosterol
A
B
C
Ergosterol
HPLC Chromatograms of ergosterol, peak was detected at
RT 7-8
min. (A) Ergosterol standard (B) Healthy palm (C) Infected palm.
Ergosterol peaks are arrowed.
B
Oil palm roots cultured on Ganoderma Selective
Media (GSM) after 5 days of incubation.
A: Infected oil palm roots, indicated by
Ganoderma growth. B: Uninfected oil palm roots.
Ganoderma-inoculated oil palm seedling roots treated with
different combination of microbes. A: Bacillus sp and
Trichoderma sp. B: Bacillus sp., Aspergillus sp. and
Pseudomonas sp. C: Lactobacillus, Nattobacillus and Yeasts ,
D: Control. Bar= 3 cm
Blue line: control
Green line: Microbes treated
2nd App
1st App
Oil palm estate soil inoculated with Ganoderma boninense and treated with
combinations of Bacillus spp, Aspergillus spp and Pseudomonas spp
Courtesy of Agrinos & UPM
2nd App
Hyphal extension of G. boninense in sterile and non sterile soil and frond debris (FD).
Colonised wheat grains were used as inoculum source. a: Mycelial grown in FD after 4
days. b: Extension in FD after 10 days. c: Growth in soil after 4 days. d: Hyphal extension
in soil after 10 days
Cooper, R. (2011) in Sustainable Agriculture: An Insight into Ganoderma
2nd App
Assessment on field application is currently on going. Data
collection due mid Nov 2012
Courtesy of Martin Kong, One Good Earth
Population density of microorganisms in all soil samples collected from oil palm plantation of Sapi Nangoh,
Sandakan.
Sample
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
S17
S18
S19
S20
Growth medium
NA(Bacteria)
3.2 x 103
5.1 x 103
7.0 x 102
7.2 x 104
3.8 x 103
3.0 x 106
4.0 x 103
2.4 x 107
1.6 x 103
3.6 x 103
7.0 x 102
5.0 x 106
1.2 x 106
4.6 x 103
6.7 x 105
1.7 x 103
5.7 x 103
3.2 x 103
3.2 x 103
PDA (Fungi)
5.0 x 102
1.0 x 102
3.0 x 102
3.0 x 102
9.0 x 102
1.0 x 102
1.0 x 102
1.0 x 102
1.0 x 102
MEA (Yeast)
2.8 x 102
1.3 x 104
1.5 x 104
4.7 x 104
7.4 x 103
5.3 x 103
1.5 x 103
4.8 x 103
1.1 x 103
2.0 x 106
3.6 x 106
4.2 x 104
1.9 x 103
7.9 x 103
6.4 x 104
7.2 x 104
6.3 x 102
4.7 x 104
3.1 x 103
4.7 x 103
Cont…..
Sample
S21
S22
S23
S24
S25
S26
S27
S28
S29
S30
S31
S32
S33
S34
S35
NA(Bacteria)
2.0 x 103
2.0 x 105
1.3 x 103
3.2 x 103
2.2 x 104
3.1 x 104
3.3 x 104
3.1 x 104
1.9 x 104
3.6 x 103
1.6 x 103
7.3 x 103
5.6 x 104
1.5 x 103
1.2 x 103
Growth medium
PDA (Fungi)
1.0 x 102
3.0 x 102
1.0 x 102
3.0 x 102
1.0 x 102
1.0 x 102
8.0 x 102
2.0 x 102
-
MEA (Yeast)
1.8 x 103
9.6 x 103
1.6 x 103
1.1 x 103
8.0 x 102
1.6 x 103
7.6 x 103
2.4 x 103
4.8 x 103
2.0 x 103
2.5 x 103
2.7 x 103
4.7 x 103
4.0 x 102
4.9 x 103
Identification using Biolog technique
Microrganisms
Bacteria
Genus
Species
Bacillus
B. thuringiensis
B. pumilus
B. humi
B. lichenformis
B. albus
B. pseudomycoides
B. amyloliquefaciens
A. woluwensis
A. globiformis
K. sedentarius
R. picketii
C. boris
C. mycetoides
B. epidermis
R. fascians
R. minuta
R. graminis
R. pustula
G. selenospora
F. neoformansbacillisporus
F. fuzhouensis
S. johnsonii
B. albus
C. albidus
P. solitumwestling
P. neoechinulatum
C. herbarum
F. tricinctum
A. kiliense
Arthobacter
Actinomycetes
Kytococous
Ralstonia
Corynebacterium
Yeast
Brevibacterium
Rhodotococcus
Rhodotorula
Fungi
Guilliermondella
Filobasidiella
Fellomyces
Sporidiobolus
Bulleromyces
Cryptococcus
Penicilium
Cladosporium
Fusarium
Acremonium
Microbes Identification based on Biolog technique
GEN III microplate (left) showed purple colour -change pattern due to the carbon utilization by the bacteria after 24
hr of incubation. Based on the colour changes, bacteria was identified as Bacillus pumilus (squared in red) with the
probability of 1.0 and 0.764 similarity.
Bacterial culture (left) isolated from soil sample which identified as B.pumilus based
on Biolog identification technique. Observation of B. pumilus (right) under light
microscope using 40x magnification power. Bar=100µm
Oil Palm
Bacterial population: 102 to 107
cfu/g of soil
Forest
Bacterial populations: 105 to 108
cfu/g of soil
No growth was observed from soil
sample of S6 on NA.
Fungal population: only 102cfu/g of
soil.
Fungal population: 105 to 106 cfu/g
of soil
No growth was observed on PDA
from soil of S3, S4, S7, S8, S13,
S14, S15, S16, S18, S19, S27, S28,
S29, S30, S31, S34 and S35.
All soil samples (S1-S35) showed
the presence of yeasts
Yeasts population: 102 to 106 cfu/g
of soil
Yeasts population: 103 cfu/g of soil
Conclusion
• Soil microbial ecosystem is complex but combinations of
microbes have the ability to enhance Oil Palm nutrient
uptake, increase availability of nutrient to Oil Palm and
suppress pathogens attack especially Ganoderma
• Ganoderma is weak competitor with many other
microorganisms. We are the one who provides suitable
soil condition for them to become the champion!
• Is extensive use of agrochemicals lead to depletion of
microbial population in oil palm soil? Need further
investigation
• More funding is required to accelerate research related to
potential indigenous microbes for the
betterment/enrichment of agriculture/plantation soil for
the future.
One Good Earth (M) Sdn Bhd
All my Postgraduates & Co-researchers