Post on 29-Aug-2018
transcript
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
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• 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