Dec. 4

- 6, 2013 

## DuPont Pioneer SAP Comments:

Corn Rootworm Resistance

Monitoring

Clinton D. Pilcher, Ph.D.

DuPont Pioneer2 

# Challenges

Science Solutions

Deliver 

# Collaboration

Growers, Industry

Academics, Regulators3 

# Corn Rootworm (CRW) Management

and Monitoring at Pioneer

Proactive Management

Unexpected Damage (UXD) Process

Sub

- lethal Seedling Assay (SSA )

• Dr. Analiza Alves4 

# Managing and Monitoring

TimeResistance Allele Frequency

0.1 0.5 1.0 

### • Best Management Practices (BMPs)

• Management Based on “Field History”

• Integrated Refuge & Pyramids

• External Education & Outreach • UXD Process 

## Proactive Reactive5 Rootworm

& Wind

Damage

Aug 28, 2001

Photo: Alex Woodall

DuPont Pioneer

Kamrar

, IA 

### 1999

– Soybeans



## - 23 bu /a 

## - 32 bu /a 

# Know the Field History for BMPs6 

# UXD Process and Characterization

Year 0 Year 1 Year 2

**Grower Call**

**Field Evaluation**

**Adult Collection** Thresholds Met = Characterization

**Farm**

**- Wide BMPs**  **Standardized Assays (SSA & GH)**

Thresholds Met = Characterization

**Adult Collection**  **Research**

**Stakeholder Communication Assays** 

**Confirmation**7 

# Regulation Should Encourage:

Uniformity in UXD process

Technical collaboration/coordination

among stakeholders

Practical solutions for

growers and registrants

Sound science and

knowledge sharingDec. 4

- 6, 2013 

## DuPont Pioneer SAP Comments:

Corn Rootworm Resistance

Monitoring

Analiza Alves, Ph.D.

DuPont Pioneer9 

# Diet bioassay

• Sanitation

• Low sensitivity to toxins

• Discriminating concentrations are impractical



# Other approaches

• On

- plant assays may offer

increased sensitivity

• Sub

- lethal Seedling Assay (SSA) 

# Challenges of Rootworm Resistance

Monitoring & Detection10 

# 59122 Effect is More Chronic Than Acute

Susceptible larvae extracted after 17 days of exposure



### Initial feeding does not result in

direct mortality 

## Isoline 5912211 

# Pioneer Sub

- lethal Seedling Assay (SSA)

Larval Development DistributionProportion of Population



## 59122

Isoline

Output: Mean instar ratio = Mean instar Bt

Mean instar non

- Bt Instar

Source: Nowatzki, et al. 200812 

# Initial Observations on CRW Field

Populations SSA Trials

Variability of SSA results from field CRW populations

higher than expected

Potential loss of insects to pupation

Need for better separation between resistant and control

laboratory population



# SSA Further Optimized13 

# Optimization Results

– Larval RecoveryRecovery Rate (%)



## – Exposure Length ( o C Degree Days) 

## +

Shorter exposure increases insect recovery Isoline, 500 eggs

Isoline, 1000 eggs Treatments

59122, 500 eggs

59122, 1000 eggs



### Initial

SSA 56% increase

in larval

recovery14 

# Optimization Results

– Instar Means

Instar mean

Shorter exposure increases treatment separation 

## – Exposure Length ( o C Degree Days) 

## +15 

# Improvements

– Data Analysis

Mean Instar Ratio

Mean instar Bt

Mean instar non

- Bt



## Percent 3 rd 

## Instar Ratio

Percent 3 rd 

## instar Bt

Percent 3 rd 

## instar non

- Bt16 0.15 0.95 

## Percent 3 rd 

## Instar Ratio

RR lab colony 

## Mean Instar Ratio

Increased separation between lab resistant and field

monitoring populations 

# Improvements

– Data Analysis

% %

0.687 0.98717 

# Assay Characterization for CRW

Resistance Assessments

On

- plant assays to assess resistance against

59122 (Cry34/35) and Pioneer experimental

event (mCry3A)

• SSA

• Greenhouse efficacy trial18 

# SSA: % Larval Return

mCry3A

- RR mCry3A

- SS 59122

- RR 59122

-

SS

Data suggest mortality is indicative of

susceptibility for mCry3A* Larval recovery on 59122 (Cry34/35)

treatment is not informative 59122 mCry3A * Isoline Event: 59122 mCry3A * Isoline 59122 mCry3A * Isoline 59122 mCry3A * Isoline

* Pioneer experimental event19 

# SSA: Mean Instar Ratio

mCry3A

- RR mCry3A

- SS 59122

- RR 59122

-

SS

Instar assessment aligns with resistance development 59122 mCry3A * 59122 mCry3A * 59122 mCry3A * 59122 mCry3A * Event:Diff. betweentrts 

## –

**+**

* Pioneer experimental event20 

# High NIS May Not Mean Resistance

Infest Rate Low High

Know how a trait responds in an assay! Isoline

Single

CRW

eventsMedian Root Rating(0-3 Node Injury Score) 

# Greenhouse Root Efficacy Bioassay21 

# Summary Remarks

EPA has recommended on

- plant assays for CRW

resistance monitoring

On

- plant assays have the capability of detecting

CRW resistance

Result interpretation is event specific

Assay characterization and

validation are necessary