Studies on the suppression of cacao diseases with Ecuadorian endophytic Bacillus spp. Rachel L. Melnick PhD Candidate, Dept. Plant Pathology The Pennsylvania State University January 23, 2008
Dec 14, 2015
Studies on the suppression of cacao diseases with Ecuadorian endophytic Bacillus spp.
Rachel L. MelnickPhD Candidate, Dept. Plant PathologyThe Pennsylvania State UniversityJanuary 23, 2008
Hypothesis Endophytic Bacillus spp. from cacao
may provide sustainable control of cacao diseases, through long-term colonization and activation of plant defense mechanisms Susceptible
tissue, no bacteria
Tissue with bacteria
Resistant tissue, no bacteria
Initial PhD research•Collected endophytes from elite cacao
trees at INIAP plantation
•Screened endophytes for biocontrol potential
•Four endophytic Bacillus spp. could be potential biocontrol agents ▫There initially were 70 isolates
INIAP Experiment•Four clonal cacao
genotypes EET19 -S A2162 - MR A2634 - MR CCN51 – S/T IMC67 - Seedling
Five bacterial treatments• Silwet control• Bacillus A20• Bacillus ET• Bacillus CT• Bacillus CR
Application •Initial application May 2007 (start of
dry season•Sprayed Log 8.0 CFU/mL with Silwet
L-77•Plants maintained in greenhouse for 3
weeks
TimelineMay 2007
Dec 2007
Initial Application
Dry S
easo
n
Phytosanitation and reapplication
Rain
y Seaso
n
Mar 2008
Reapplication
Jun 2007 Planted in the field
May 2008
Phytosanitation and reapplication
Dry S
easo
nDec 2008 Phytosanitation and
reapplication
Measure endophytic colonization•Removed standard area of leaf tissue•Surface sterilized•Triturated and plated
Dry season results (2007)•Bacteria survived endophytically in
foliage throughout the dry season▫Initial concentration log 6.8 CFU/cm2 ▫Concentration after 6 months ~ log 4.8
CFU/cm2
▫Populations stabilized at ~ log 4.8 ± 0.3 Applied Bacillus were 1000 fold higher than
total culturable background (control leaves)
Dry season results (2007)•Low disease pressure•Measured several variables
▫Swelling▫No. infected stems
Dry season results – Bacteria
Bacterial treatment
Control A20 CT ET CR
Dis
ea
se In
cid
en
ce (
% o
f dis
ea
sed
pla
nts
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Dry season results - Genotype
Cacao genotype
EET19 CCN51 A2634 A2126 IMC67
Dis
ease
inci
denc
e (%
of
dise
ased
pla
nts)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Significant genotype/bacteria combo•Combining biocontrol with resistant
genotype is better for reducing disease
Genotype and bacterial treatment
% o
f d
ise
ase
d p
lan
ts
0.0
0.2
0.4
0.6
0.8
Days after bacterial application
0 20 40 60 80 100 120
Dis
ea
se
se
ve
rity
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5A20 CT Control CR ET
Rainy season results 2008
Phytosanitation and reapplication
Reapplication
Rainy Season Results
Bacterial Treatments
A20 CT Control CR ET
AU
DP
C (
Dis
ea
se
Se
ve
rity
/tim
e)
0
20
40
60
80
100
120
140A
AB
A A
B
Conclusions•Successful year round disease
reduction by Bacillus pumilis ET
•Three applications a year are enough to reduce disease
•Combining bacteria with resistance in the plant probably increase disease suppression
Current and future research•Verification study at Nestlé Farm
▫Cultivars: EET19, EET95, SIL1▫Treatments: Control, CT, ET
•Mode of action▫Induced resistance▫Microbial ecology▫Antibiosis
•Biological Control of Frosty Pod▫Started this week
Nestlé experiment•Four genotypes with varying
susceptibility•Endophytes: CT & ET•Established May 2008, rated Sept
2008Cultivar Treatment % Living
Disease Severity
EET19 Control 70 0EET95 Control 80 0.22SIL1 Control 50 0EET19 ET 70 0EET95 ET 90 0SIL1 ET 10 *EET19 CT 100 0EET95 CT 80 0.25SIL1 CT 50 0
Induced resistance•Determine if endophytes affect gene
expression of plant defense genes
•Seedlings grown in growth chambers in double magenta boxes
•Quantitative Real Time PCR (Q-PCR)▫Collaboration with USDA-ARS SPCL
•Potential for microarray in the future
Microbial ecology•Determine if beneficial endophyte replace
neutral endophytes▫More beneficial, more disease suppression
•Automated Ribosomal Intergenic Spacer Analysis (ARISA)
•Measure species diversity and abundance in community
Peak h
eig
ht
(flu
ore
scen
ce
un
its)
Antibiosis•Conducted plate assays•Will be screening Bacillus spp. for the
presence of antimicrobial genes and gene products
•Collaboration with Dr. Dan Roberts USDA-ARS SASL
Biological control of frosty pod•Treatments: Control, A20, ET, CR, CT•400 pods (80 reps) sprayed at INIAP on
nacional▫Hand pollinated pods
•400 pods (80 reps) at Rio Lindo on CCN51▫Open pollinated pods
Overall conclusions•Biological control shows potential to
reduce witches’ broom of cacao
•Combining IPM strategies is best for managing cacao diseases
•Bacillus spp. impacts cacao gene expression
Acknowledgements◦ Penn State
Adviser: Paul Backman Mark Guiltinan Megan Hayden Seila Maximova Sharon Pishak Anissa Poleatewich Ann Young
• USDA-ARS SPCL▫ Bryan Bailey▫ Mary Strem
• INIAP• Carmen Suárez• Karina Solis• Danilo Vera
Support: USAID
IPM-CRSP SANREM-CRSP
USDA-ARS SPCL Penn State University
Dept of Plant Pathology College of Agricultural
Sciences Tag-a-long (Office of
International Programs) Competitive Grant
◦ Nestlé Martín AlvarezCABI Jayne Crozier