The Challenges and Rewards of Combining Applied and Basic Research in the Context of International Research

Transcription

1 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

2 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

3 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

4 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

5 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!

6 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

7 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

8 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

9 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!

10 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...

11 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?)

12 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!

13 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

14 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

15 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

16 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)

17 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!

18 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

19 Parallel or local evolution of crop-infecting begomoviruses Invasive polyphagous whiteflies (B. tabaci biotype B) feed on non-agricultural plants infected with 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 Viruliferous whiteflies move to crop plants and introduce indigenous begomoviruses

20 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!

21 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!

22 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!

23 Pseudorecombination and recombination leads to the evolution of a more virulent bipartite begomovirus ToMoV-A + BDMV-B Infectious, but plants had attenuated symptoms and reduced levels of DNA-B Passage through N. benthamiana Severe symptoms and equal levels of DNA-B ToMoV-A + BDMV-B Intermolecular recombination in the common region

24 Integrated Pest Management (IPM) 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

25 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

26 Identification of Tomato Yellow Leaf Curl Virus in the Dominican Republic 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! pty-dr1

27 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

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29 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

30 PCR Detection of TYLCV in Whiteflies: An Approach to Validate the Benefit of the Whitefly Host-free Period to CA from DR Whiteflies collected from plant crops and/or weeds in the DR Whiteflies in EtOH Whiteflies in EtOH Remove EtOH and wash with STE buffer Add 100 μl STE buffer and grind Mehta et al., 1994 Use 20 μl of supernatant for PCR Spin 10 at 13,000 rpm

31 - control - control PCR detection of TYLCV in whiteflies collected from tomatoes during the growing season Northwest South Northwest South controls + controls

32 Pumpkin Pumpkin Wild cucumber Eggplant Pumpkin Cotton Wild cucumber Pumpkin Wild cucumber Melon Melon Tomato Wild cucumber Watermelon Melon Pepper Wild cucumber Eggplant Wild cucumber 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 PCR detection of TYLCV in whiteflies collected from various plants during the host-free period Pumpkin Pumpkin Wild cucumber Pumpkin Cotton Pumpkin Wild cucumber Melon Melon Tomato Wild cucumber Watermelon Melon Pepper Wild cucumber Northwest South Northwest South This IPM approach allowed for the recovery of the processing tomato industry in the DO Detection of TYLCV in whiteflies

33 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

34 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

35 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 FTA cards in the DO Samples applied to absorption strips in Mali, West Africa

36 Tomato Production in West Africa is Threatened By Viral Disease Epidemics

37 Tomato Is a Very Important Cash Vegetable Crop in WA

38 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

39 Flow chart showing the steps involved in the detection and characterization of whitefly-transmitted geminiviruses (begomoviruses) infecting tomato and other crops in West Africa

40 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 ToLCMLV TYLCMLV ToYLCRV 3rd 2nd Test plant

41 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

42 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!

43 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...

44 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

45 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-

46 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

47 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