Studies on the suppression of cacao diseases with Ecuadorian endophytic Bacillus spp.

Rachel L. MelnickPhD Candidate, Dept. Plant PathologyThe Pennsylvania State UniversityJanuary 23, 2008

Previous research•Endophytic Bacillus sp. from tomato

colonized cacao foliage and reduced disease

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

Field Trial

Research•Have conducted 2o months of field trials

for biological control of witches’ broom of cacao

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

Results

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

Plant Disease Management Reports:

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 & Future

Research

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

Samples of data…•Some graph here

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