The Challenges and Rewards of Combining

Applied and Basic Research in the Context of

International Research

Robert L. Gilbertson

Department of Plant Pathology

University of California-Davis

Plant Pathology: Endless Opportunities

• A ‘job’ that I enjoy

• New challenges and opportunities every day

• One of these opportunities has been

international research

• This story involves ‘forks in the road’

and the question of ‘breath vs. depth’

• It is also a story of how people

shape your career in so many ways

Formative Years and

My Introduction to Plant Pathology

• Always fascinated by nature

• Looking under logs and stones and

exploring tide pools; catching frogs, toads,

and snakes; and a love for animals

• Biology was my favorite class

• B. S. in Wildlife Biology at UMass-Amherst

• Veterinary School/M. S. in Parasitology?

• Summer job with Dr. William Manning

• M. S. in plant pathology with Dr. Manning

on Fusarium crown stem and root rot of asparagus

• Interest in Entomology because of a little fly

Extension technician and landscaper:

Solidified my passion for plant pathology

• Extension Technician in Entomology

with Dr. Dave Ferro

• Also worked as a landscaper in Earthcare

• Gordon Fletcher-Howell

-An appreciation for nature

-Attention to detail

-Strong work ethic

-Great sense of humor

• Taught me about plants and exposed me to

plant pathology in the real world

• A decision to pursue

a Ph.D. in plant pathology

Colorado State University (CSU)

• Ph.D. in the Department of Botany and Plant

Pathology under the direction of

Dr. Earl G. Ruppel, a USDA Plant Pathologist

• Worked on a project on the influence

of cropping systems on soil borne fungi

• ‘I need it yesterday’

• Attention to detail in writing;

his goal was to get a manuscript back

without a single edit!

• He made me a far better writer

• However, on the tenth iteration of their theses,

my students may not be so happy with Dr. R!

Colorado State University-Bill Brown

• An interaction with Dr. William (Bill) Brown, Professor

and Extension IPM Coordinator

• Energetic, upbeat and a passion for plant pathology

• An opportunity to work on stalk rot of corn

• Became the subject of my Ph.D.

and Bill was my co-advisor

• Had to listen to listen to me

‘get on my soapbox’

and complain about the CSU curriculum

• One of his passions was international agriculture

and this would have a lasting impact…

Teaching-CSU

• Students get much of their teaching experience

being Teaching Assistants (TAs)

• As a Ph.D. student at CSU, I was a TA for

Dr. Ralph‘Tex’ Baker in Intro Plant Path

• I will never for forget our first (and last)

meeting to plan the laboratory section of the

class

• He gave me an outline, told me where the

prepared slides were and said: ‘Good luck’

• Time to put my practical knowledge to work!

• Luckily, I found Ervin H. Barnes

UW Madison: Breath or Depth?

• Completed my Ph.D. at CSU in 1985

• Interviewed for a faculty position at UC Berkeley

• I took a postdoctoral position

at UW-Madison working on

diseases of common bean in Brazil

• Under the direction of Drs. Donald

‘Doc’ Hagedorn and Douglas P. Maxwell

• Part of the USAID-funded Bean/Cowpea

Collaborative Research Support

Program (CRSP)

• Involved going to Brazil

UW Madison-Don ‘Doc’ Hagedorn

• Dr. Ruppel was a student of Dr. Don ‘Doc’ Hagedorn

• A world expert in diseases of common beans

and peas

• A breeder/pathologist who generated

many resistant varieties

• A yearly ritual: plant the W7 root rot plot

• A new professor was going to solve root rot with

with a biocontrol agent! Doc gave her a spot in

the W7 root rot plot…

• Doc said he knew it was time to retire

‘when he was hiring his student’s student!’

Initially worked on common bacterial blight (CBB) diseased caused

by Xanthomonas campestris pv. phaseoli

and X. fuscans subsp. fuscans

• An important bacterial disease of common

bean in Brazil

• New methods of screening for resistance

and investigating the ecology of the bacteria

• Identified non-pathogenic xanthomonads

associated with bean debris

• Developed molecular tools (DNA probes,

RFLPs etc.) to investigate applied questions

• One day Dr. Doug Maxwell called me to his

office...

Winds of change at the Bean Cowpea CRSP

“Bob they are cancelling the Bean/Cowpea CRSP activities in

Brazil, but want us to work on bean golden mosaic virus. Bob, do

you want to work on bean golden mosaic virus?”

(In other words, do you want to keep your job?)

OK, what is bean golden mosaic (BGM)?

• A viral disease threatening bean production

throughout Latin America

• Symptoms: stunting, distorted growth and

striking yellow mosaic

• Transmitted by the sweet potato whitefly,

Bemisia tabaci

• Caused by Bean golden mosaic virus

(BGMV), a member of a new type of plant

virus with twinned (geminate) virions and

a circular single-stranded DNA genome

• I am thinking I should have paid more

attention in Virology class with Dr. Agrios!

A story about two viruses that cause the same disease

• Dr. Robert Goodman characterized BGMV-PR,

a New World bipartite begomovirus (DNA-A and DNA-B)

• Differences between BGMV from the Caribbean

and Brazil

• Worked with BGMV-PR in Florida with

Dr. Ernie Hiebert and then went to EMBRAPA

Rice and Beans Research Station in Goiania to

work on BGMV-BZ

• Confirmed BGMV-BZ was different than BGMV-PR

• Time to get to the molecular level...

• Not a worthy effort? Bob Goodman said ‘Why do

you want to do that? We have already done it’

Visit Brazil to work on BGMV

Living in Goiania I’m liking it here in Brazil!

Bring on the churrasco and the Antartica Hanging out with the locals

A story about two viruses

that cause the same disease

• Geminivirus DNA detected in the extract

• Time to bring on a ‘heavyweight’ to help

with the cloning: Dr. Paul Ahlquist

• ‘Shotgun’ cloned fragments of the BGMV-BZ

genome were ~80% identical to BGMV-PR!

• BGMV-BZ was a different virus!

• Good thing we did not listen to

Bob Goodman!

• Now to fulfill Koch’s postulates

A story about two viruses that cause the same disease

• Sequences of the full-length DNA-A and DNA-B clones of

BGMV-BZ confirmed it was genetically distinct from

Caribbean and Mexican isolates

• However, establishing infectivity was proving difficult

• All the methods we tried were unsuccessful

• We were running out of ideas… (and Dr. Maxwell’s patience)

A fortunate encounter with Agracetus

• Company using a gene gun driven by an electric

discharge to introduce DNA into soybeans

• ‘You have the DNA and we

have the gun-let’s have a blast’

• Could geminivirus DNA be introduced

into beans with the particle gun?

• Why not? Nothing to lose…

• Worked with Dr. David Russell

• Primary leaves didn’t work, maybe radicles could be

the ticket?

• Don’t forget the controls!

Golden beans: Two viruses do cause

the same disease

• ‘A watched pot never boils’

• Yellow vein symptoms appeared in

primary leaves of beans bombarded

with BGMV BZ A+B…could it be?

• No question when beautiful golden

mosaic symptoms appeared in trifoliolate leaves!

• Koch’s postulates fulfilled and a tool for genetic studies!

• Now a commonly used method to introduce

viral (and other) DNAs into plants

• Agroinoculation is an alternative

Parallel or local evolution of crop-infecting begomoviruses

Invasive polyphagous

whiteflies

(B. tabaci biotype B)

feed on non-agricultural

plants infected with

indigenous begomoviruses

Viruliferous whiteflies

move to crop plants and

introduce indigenous

begomoviruses

Mutation/

recombination

of indigenous viruses

leads to emergence of

new crop-infecting

begomoviruses, which

typically causes severe

disease symptoms

in this new host

Arrival of a new M.S. student from Costa Rica

• Dr. Maxwell assigns me to help

train Ms. Maria R. Rojas

• She is smart and a fast learner

• She is also very attractive and

has a great sense of humor

• I’m in trouble…

• I propose and we get married…

• She moves to UC Davis, earns

her Ph.D. in Plant Biology and

becomes one the world experts in

microinjection!

A dream come true: A faculty position!

• After many applications (Dr. Ruppel was getting tired

of writing letters of recommendation), I interviewed for

faculty positions at Purdue and UC Davis

• Two job offers…what to do?

• The UC Davis position is a

seed pathologist-virologist; an opportunity

to combine breath and depth!

• More opportunities for research and funding

• Off to California!

Pseudorecombination and

recombination leads to the

emergence of a new

virulent DNA virus

• Passage of a weakly virulent

pseudorecombinant bipartite

begomovirus (TA+BB) leads to increased

virulence and increased DNA-B levels

• Correlated with the exchange of the

origin of replication-the BB acquired the

TA origin-via intermolecular

recombination!

• No wonder that there are now

322 species of begomoviruses-the

most of any type of plant virus!

ToMoV-A BDMV-B

ToMoV-A BDMV-B

+

+

Passage

through

N. benthamiana

• Infectious, but plants had

attenuated symptoms and

reduced levels of DNA-B

• Severe symptoms and

equal levels of DNA-B

• Intermolecular recombination

in the common region

Pseudorecombination and recombination leads to the

evolution of a more virulent bipartite begomovirus

• An approach for disease management that

combines multiple strategies based on

knowledge of pathogen biology

• Goal is efficient management with

minimal inputs of pesticide; economically

and environmentally friendly

• Three basic steps:

1. Correct pathogen ID

2. Understanding pathogen biology/

disease epidemiology

3. Development and evaluation of an

integrated management strategy

Integrated Pest Management (IPM)

The introduction of the exotic

Tomato yellow leaf curl virus

into the Dominican Republic:

An opportunity to apply

molecular tools and IPM

• Strange disease symptoms appeared in

tomatoes in 1992

• Associated with high whitefly populations

• Within 2 years, the disease was threatening

the entire processing tomato industry

• Symptoms looked like those of tomato yellow

leaf curl, a disease not known to occur in the

New World

Identification of

Tomato Yellow Leaf Curl Virus

in the Dominican Republic

pTY-DR1

• Overlapping PCR primers

based on TYLCV sequence

amplified a 2.8 kb DNA

fragment

• Restriction map was identical

to that of TYLCV

• The complete sequence of

the cloned fragment was

97% identical to TYLCV!

Nucleic Acid Squash Blot

Hybridization

*

**

*

**

*

**

*

**

• Squash tissue samples onto

nylon membranes

• Prepare membranes

(i.e., denature DNA)

• Prepare and denature probe

(cloned geminivirus DNA)

• Hybridization/wash

• Expose to X-ray film

-Positive sample: black spot

-Negative sample: no signal

TYLCV in the DR: Basic

Research Yields a Practical

Solution

• TYLCV had spread to the major

processing tomato

growing locations within 1-2 yrs

• Tomato was the primary host

• A three-month whitefly

host-free period

was proposed as a strategy

to break the disease cycle

• A driving force behind this effort

was Ing. Miguel Sanchez

PCR Detection of TYLCV in Whiteflies: An Approach to

Validate the Benefit of the Whitefly Host-free Period

Mehta et al., 1994

Add 100 μl STE

buffer and grind

from DRto CA

Whiteflies

in EtOH

Remove EtOH

and wash with

STE buffer

Whiteflies collected

from plant crops

and/or weeds

in the DR

Spin 10’at

13,000 rpm

Use 20 μl of

supernatant

for PCR

Whiteflies

in EtOH

PCR detection of TYLCV in whiteflies

collected from tomatoes during

the growing season

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Host free period is a key component of the IPM strategy

• The host-free period was implemented

along with a number of other practices

• Evidence that the host-free period was

effective:

-4-8 week delay in the appearance of

TYLCV symptoms

-Dramatic drop in detection of

TYLCV in whiteflies

• This IPM approach allowed for the

recovery of the processing tomato

industry in the DO

PCR detection of TYLCV in whiteflies

collected from various plants during the host-free period

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Detection of TYLCV in whiteflies

IPM for TYLCV in the Dominican Republic

• Three month whitefly host-free period

• Varietal selection

-early: early maturing hybrids

-late: TYLCV-resistant varieties

• Management of whitefly populations

• Extensive sanitation after harvest

• Monitoring TYLCV in whiteflies/

plants

• Tomato yields for exceeded pre-TYLCV levels

Déjà vu all over again:

Introduction of another invasive virus into the DO,

Tomato chlorotic spot virus (TCSV)

• Unusual disease symptoms appear in

processing tomatoes in the DO in 2012

• Included stunting and distorted growth,

chlorosis, mosaic/mottle and necrosis of

leaves and necrotic areas on fruit

• Shown to be caused by this thrips-vectored

tospovirus from South America

New methods for sample preparation

facilitate international research

• Allows application of PCR/RT-PCR to samples

from diverse geographical locations at a central

laboratory

• Absorption strips (AgDia)

• FTA cards (Whatman)

Samples applied to

absorption strips

in Mali, West Africa

Samples applied to

FTA cards in the DO

Tomato Production in West Africa is Threatened

By Viral Disease Epidemics

Tomato Is a Very Important Cash Vegetable Crop in WA

Objectives of USAID

IPM-Collaborative Research

Support Project (CRSP) and

Application of Biotechnology

Support Program II (ABSP II)

1. Identify virus(es) involved

2. Develop an understanding of the

biology of the viruses

3. Develop an IPM strategy

Flow chart showing

the steps involved in

the detection and

characterization of

whitefly-transmitted

geminiviruses

(begomoviruses)

infecting tomato and

other crops in

West Africa

Tomato diseases in West Africa are caused

a complex of locally evolved begomoviruses

• Seven new begomovirus species and three betasatellites

characterized

• Koch’s postulates fulfilled with full-length

infectious clones

• Host range limited to solanaceous crops

• Agroinoculation used to identify resistant varieties

• IPM strategy involving regional sanitation,

host-free period and resistant/tolerant hybrid

varieties was devised

Test plant

2nd

3rd

ToLCMLV

TYLCMLV

ToYLCRV

Implementation of the IPM strategy

• The Baguineda irrigated rice-vegetable

perimeter in Mali was selected as a test site

• Tomato production severely impacted by

begomovirus diseases

• Meetings were conducted with chiefs of the

local villages to present the IPM strategy

(the AIDS disease of the tomato plant)

• The rainy season months (June-August)

were selected for the host-free period

• In 2005, the IPM program was implemented

Application of a voluntary host-free

period for management of

tomato begomoviruses in West Africa

• Sanitation program

• Tomato and pepper-free period (June-August)

• Seeds of early maturing hybrid tomatoes

were donated by seed companies and

distributed to selected farmers

• Monitoring program of the part of the Ph.D.

of Dr. Moussa Noussourou

• Reduced virus incidence and whitefly

populations and impressive yields!

Application of an IPM strategy

for management of

begomoviruses in West Africa

• The IPM strategy has allowed for the return of

tomato production to Baguineda

• Yields were greater than pre-virus levels

• Evidence of sustainability

• Farmers have embraced the program and

want to purchase hybrid seed

• Host-free periods were planned for other

tomato-growing areas in the Mali

• Then came the coup d'état in Mali...

Why participate in international research?

• Address important disease problems in countries

with serious food security issues

• Establish collaborations with colleagues in foreign

countries

• Exciting new research opportunities

• Broaden your horizons by learning about the

culture, history and language of a new country

and seeing exotic disease and pests problems

• New opportunities for research funding

• Serve as ambassadors for UC Davis and

the United States

Teaching-What goes around comes around

• As a teacher at UC Davis I have not followed

the ‘Tex Baker model’

• My goals has been to give students ‘their money’s worth’

• Providing fresh high quality specimens, sharing

my experiences and participating in labs

• Over the years, I learned where to find disease

specimens in Davis

• The thrill of finding a great specimen!

• A family effort: Lifting my daughter over a fence to

get ‘beautiful’ cucurbit virus symptoms-

Teaching-Put your money where your mouth is

• ‘Breath and depth’: an ‘opportunity’ to

teach classes with amazing colleagues

• Intro Plant Path with Rick Bostock

• Plant Bacteriology with Bruce Kirkpatrick

• The integrative Plant Virus Vector Interactions

with Bill Lucas and Diane Ullman

• Advanced Plant Pathology Lab

• A plant pathology module in an undergrad IPM

class

• Diseases of Field, Vegetable and Fruit and

Nut Crops with multiple faculty colleagues

What has worked for me?

• A passion for plant pathology

• Being curious and observant

• Self-motivation, strong work ethic

and a drive to succeed;

but with integrity and respect for all

• Why can I do something,

not why can’t I do something!

• Willingness to sacrifice

• Incredible colleagues, students and

post-docs

• APS: an organization that brings us together

and keeps the field alive and relevant

• A life-long interest and passion for

international agriculture