IPM CRSP Trip Reports

IPM CRSP Trip Reports Country(s) Visited: United States of America (USA) Dates of Travel: June 14 to 29, 2014 for Dr. Mutaqin June 22 to 25, 2014 for Dr. Schnabel Travelers Names and Affiliations: Dr. Kikin Hamzah Mutaqin (participant) Department of Plant Protection Bogor Agricultural University, Bogor, Indonesia. Dr. Guido Schnabel, Clemson University, School of Agricultural, Forest, and Environmental Sciences, Clemson SC 29634. Rationale and Purpose of Trip: Following a 2013 visit to key citrus areas in Indonesia, plant pathologists Jim Adaskaveg, Guido Schnabel, and Helga Foerster from the University of California, Riverside and Clemson University recommended that a Clean Plant Network must be implemented to provide growers with pathogen-tested planting stock. There are still many areas of production with little to no signs of serious diseases but that could all change dramatically if Tristesa or HLB or bacterial canker infested planting stock were to be brought in and cultivated. Clean plant networks were established in the US and many other countries and are viewed as an essential part of Integrated Pest Management. The purpose of this trip was to provide training for Dr. Kikin Mutaqin that would enable him to start a Clean Citrus Plant Network in Indonesia. Dr. G. Schnabel attended part of this workshop to be able to assist with the implementation in the future. Sites Visited: Citrus Clonal Protection Program (CCPP), University of California Riverside, CA Department of Plant Pathology and Microbiology, University of California Riverside, CA Department of Botany and Plant Sciences, University of California Riverside, CA Rubidoux Quarantine Facility-CCPP, Rubidoux, CA

National Clonal Germplasm Repository for Citrus and Dates, USDA-ARS Pacific West Area, Riverside, CA Citrus Clonal Protection Program (CCPP) Lindcove Foundation, Exeter CA Lindcove Research & Extension Center, Exeter CA Willits & Newcomb Inc., Arvin CA B&Z Nursery, Porterville CA Description of Activities/Observations: This was an intense, well organized, and excellent training that covered comprehensive aspects of how to generate and maintain pathogen-tested citrus planting material for nurseries that then can be distributed to farmers. During the training it became clear that providing citrus growers with pathogen-tested citrus material (rootstocks and scion) is going to be critical for sustained citrus production in Indonesia. Our long term goal is to exclude devastating citrus diseases such as viruses, phytoplasmas and bacteria from trees that growers plant for citrus production. To the best of our knowledge such a system is not yet in place in Indonesia, thus systemic spread of sick planting stock from nurseries to growers is a major concern. The most important pathogens threatening the Indonesian citrus industry are huanglongbing/hlb also called citrus greening (Liberibacter asiaticum), Tristeza Virus, and bacterial canker (Xanthomonas axonopodis pv. citri). The establishment of a clean plant program should be done in small steps to avoid mistakes, gain experience, and gain grower confidence. Action plan to get a Clean Citrus Plant Program initiated (start small, expand with experience) Find out how farmers currently obtain trees, what cultivar/rootstocks are they using (if any); visit citrus research station in East Java (in Malang county); talk to big government-supported nursery in West Java (in Garut county) to find out what the existing programs provide. Find out most popular varieties. Allocate/build two greenhouses (A and B) for this project. They need to have solid walls, high sanitation standards, big enough entrance space to store stuff. Allocate/hire a skilled and reliable greenhouse technician Learn to grow citrus trees in pots in greenhouse B, keep the other one (A) clean for clean plants

Order pathogen-tested indicator plants and learn to grow them in greenhouse A; Every two years retest clean citrus varieties with indicator plants and a technician for this project. Greenhouses should have solid walls and double door entrance with and via PCR or ELISA. Obtain most popular varieties (pathogen-free) from Australia, or China or California. Grow them in greenhouse A. Obtain budwood from these plants to supply nursery. Every two years test them for pathogens. Inoculated indicator plants should be moved to greenhouse B. Train a key nursery how to grow trees from seeds and how to graft scions on rootstocks. Make sure nurseries uses clean rootstocks and provide nursery with pathogen-tested buds of popular cultivar for propagation (Commercial nursery should graft onto rootstocks and sell trees). Team up with Horticulture person you trust. He is in charge of training nurseries to produce trees and to help with grower demonstration plot. He should not interfere with your responsibility of greenhouse activities. Demonstration trials: conduct demonstration trials in key citrus growing areas (Pak Pak Bharat, Garut, Malang) on farmer ground. Compare grower standard (air layering?) vs clean plants from your program. Timeline Timeline (6 month intervals) Activities 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 survey growers, nursery, research station x greenhouse and technician allocation x x year 1 year 2 year 3 year 4 year 5 year 6 year 7 year 8 year 9 year 10 learn to grow trees in pots x x x x popular varieties x x x x x x x x x x x x x order and maintain indicator plants x x x x x x x x x x x x conduct indexing (greenhouse and lab) x x x x x x produce clean trees on rootstocks x x x x x x x x x x x x demonstation trials x x x x x x x x x x x provide nursery consistently with clean buds x x x x x x x x x x x Potential pitfalls. Pathogen-tested local variety may not be available from elsewhere. In this case California offered to help clean up these varieties in Riverside (6 months) OR/AND Dr.

Mutaqin will clean it up in his lab. If the research station in East Java has data on variety performance, that may help decide what variety to order. Activities by topic. Dr. Mutaqin learned about the history and importance of citrus industry in USA, especially California, and chronological development of the Citrus Clonal Protection Program. He learned about (i) the US laws/rules and regulations related to citrus production and transportation at the country and state level, (ii) how to generate germplasm that is free of relevant pathogens using thermotheraphy and shoot tip grafting techniques, (iii) principles and practical aspects of propagating citrus planting material using graftting technique (unification of budwood or scion to rootstocks), and (iv) the requirements of establishment of a successful pathogen-tested citrus planting material program. Activities by date. June 14, 2014 Saturday Departure from Jakarta, arrived in Ontario International Airport. Dr. G. Vidalakis picked me up from the airport to Riverside. A short discussion made about general aspect of citrus production and its problem in a country. June 15, 2014 Sunday Visit University of California Riverside campus for early orientation. June 16, 2014 - Monday In the morning, participant visited greenhouse located at UC Riverside campus, where citrus plants infected with pathogens maintained and related experiments conducted (Dr. G. Vidalakis) First thing done for participant when arrived in campus was to fix schedule for participant and instructors for entire period of training. Participant visited CCPP laboratorium to learn pathogen detection/indexing, nucleic acid extraction system, Polymerase chain reaction (PCR) system, including quantitative realtime PCR (qrt-pcr) and other lab techniques. (Dr. S. Kamenidou) The instructor gave presentation and led discussion about comphrehensive aspects of citrus production, including: History of Citrus Clonal Protection Program (CCPP), The history of Washington navel orange (Figure 1), regulation system of citrus in the USA

and state of California, overview of CCPP responsibility and function, other related institutions. Principles and design of greenhouse or screenhouse and other facilities required for citrus propagation and clean up program. Explanation and demostration about transportation of citrus planting material (budwoods) under federal regulation. (Dr. G. Vidalakis) In the afternoon participant did activities at CCPP lab to learn about principle and demostration of nucleic acid extraction from citrus plant parts using automated extraction machine at CCPP lab (Figure 2). (Dr. S. Kamenidou) June 17, 2014 - Tuesday In the morning, participant visited university field experimental field & station for citrus near UC Riverside. It as also shown the oldest building yet historical in campus, i.e. Citrus Experiment Station building, from which University of California Riverside started (Mr. G. Greer) Morning activity continued, participant visited CCPP Citrus Quarantine facilities located at Rubidoux, CA to learn about administrative and database for citrus testing, overview newly built greenhouses designed to meet insect/vector proof requirement. It was also demonstration and practice for participant of shoot tip grafting (micrografting) technique to free citrus planting materials from pathogens (Figure 3). The instructor also explained facilities for liberating citrus planting material from pathogens using heat treatment (thermotheraphy) (Mr. G. Greer) In the afternoon, participant addressed some questions about many aspect during discussion with the instructor (Dr. G. Vidalakis). June 18, 2014 - Wednesday In the morning participant visited Citrus Variety Collection located next to UC Riverside campus. The scientist responsible for the collection take participant a tour to introduce history of citrus in California, citrus biological species and varieties and its characteristics (Citrus includes pumello, papeda, mandarin and many varieties resulted from crossing among them, such as sweet orange, sour orange, lime, lemon, grapefruit etc), other citrusrelated species such as jessamine orange. The scientist also gave participant to taste many different varieties of citrus fruits (Figure 4). (Dr. Tracy Kahn)

In the afternoon, participant attended lab activities at CCPP lab at UC Riverside mainly to learn about total nucleic acid (DNA or RNA) extraction technique from citrus plant part and the use of Quantitative Real time PCR (qrt-pcr) using SYBR system for citrus indexing and pathogen detection and diagnosis. The instructor showed the participant the equipment and entire process how to do the extraction and the PCR (Mrs. Nina Tan) June 19, 2014 - Thursday In the morning, participant visited USDA ARS Pacific West Area-National Clonal Germplasma Repository for Citrus & Dates located in Riverside. Dr. R. Krueger explained the function and activities held in the station. He showed participant greenhouse and its citrus plants, cryopreservation technique and experiment for long storage of citrus germplasm. (Dr. R. Krueger) Another scientist, Dr. Richard F. Lee, had discussion with the participant and explained about citrus huanglongbing disease, citrus biological and molecular phylogenetic, citrus indexing both based on biological assay (indicator plants) and lab assay (ELISA, molecular methods), organizing and establishing citrus germplasm database. (Dr. R. F. Lee) In the station, another scientist demosntrated a very simple, time efficient yet sophisticated technique to detect HLB pathogen from its insect vector (asian citrus psyllids, Diaphorina citri) using Loop mediated isothermal amplification (LAMP), a single tube technique for the amplification of DNA. (Dr. M. Keremane) In the afternoon, participant back to the CCPP lab to come with Mrs. Nina Tan to see result of qrt-pcr and analysis from computer. June 20, 2014 - Friday In the morning participant did activity in Rubidoux quarantine facility to leanr and practice about citrus indexing using grafting budwood or bark chip to rootstock. To do disease indexing of citrus plant, it is important to know about disease symptom, especially which are caused by graft transmissible pathogens, for example viroid, tristeza virus, psorosis, and stubborn spiroplasma (Figure 5). In additon participant did practice to use grafting technique to produce citrus rootstock planting material (Figure 6).(Mr. Greg Greer)

In the afternoon, participant did activity with lab members at greenhouse located in UCR campus to do slash inoculation of citrus pathogen to indicator plants. (Mrs. Nina Tan, Tyler and Brandon). Slash inoculation is an inoculation technique by using blade slahsing to make wounds at plant stem for entry of pathogen inoculum applied as suspension (Figure 7). (Mrs. Nina Tan) At the end of the day, participant came with Ms. Tavia to do qrt-pcr with Taqman system and show the result on the computer (Figure 8). (Ms Tavia) June 21, 2014 - Saturday In the morning participant visited to post office located at Riverside to send packages for participant s former PhD advisors in Oklahoma State University, Stillwater USA and made contact with them. Participant visited Riverside downtown In the afternoon participant attended a gathering/party in the instructor s house. All CCPP lab member were there (Dr. G. Vidalakis) June 22, 2014 - Sunday In the morning Participant met Drs. Guido Schnabel (just arrived from Clemson University, SC) and Dr. James Adaskaveg and Dr. Helga Foster (UC Riveside faculty members). We did campus touring, including visited Dr. Adaskaveg & Dr. Foster s plant mycology lab at Fawcett Hall, UC Riverside. We also went to the mountain peak next to the campus to see campus ciew from above. The UCR people also showed avocado plantation on the way down from the peak to campus (Dr. J. Adaskaveg and Dr. H. Foster) In the afternoon the participant and Dr. G. Schnabel had discussion to overview activities done during first week of training and to talk about future program and implementation June 23, 2014 - Monday In the morning, participant, Dr. Schnabel and Dr. Vidalakis had discussion about the citrus program In the afternoon, participant and Dr. Schnabel met Dr. Mikeal Roose, a UCR professor for citrus breeder & genetics, to talk about aspects of citrus cultivation, propagation, breeding, molecular identification and also agricultural system and extension (Dr. M. Roose)

In the afternoon, attended how to do qrt-pcr using Taqman system conducted by a doctoral student at CCPP lab (Ms. Nina Tan). June 24, 2014 - Tuesday In the morning, participant had discussion with Dr. Schnabel to talk about future plan of pathogen-tested citrus planting material program to be implemented in participant home country (Indonesia). The discussion resulted in a draft of the action plan and timeline (Dr. G. Schnabel) The draft was showed to Dr. Vidalakis for correction and advise (Dr. G. Vidalakis) In the afternoon, participant and Dr. Schnabel met with Dr. Helga Foster and her four students who work at her Mycology Lab at UC Riverside. Each of us talked about our position and own activities. Dr. Foster and each of students also talke about their research project, including almond, olive and citrus disease and epidemiology. June 25, 2014 - Wednesday In the morning participant travelled by car from Riverside to Central Valley (San Joaquin Valley) for about 3 hours with Greg Greer (Greg Greer). First place visited in Central Vallley was Willits & Newcombe, Inc citrus nursery company located in Arvin CA. They showed conventional yet modern greenhouses with positive pressure system to keep insect or vector away from the house. The greenhouse used for rootstock citrus plant propagation. In addition the have new system greenhouse called airstream inflatable greenhouse made from a huge ballon (size 0.5 acre) with positive pressure created by two giant fan inside. The Airstream Greenhouse provides an ideally ventilated, insect protected environment (Figure 9). (Mr. G. Greer, Mr. Gary) At the same place I met with Dr. Rock Christiano, Staff Research Associate, Manager/ Lindcove Research & Extension Center University of California, who would be the instructor for the rest of the training. (Dr. R. Christiano) At the end of the day, participant travelled from Arvin to Visalia, where the hotel for participant located (Dr. R. Christiano) June 26, 2014 - Thursday In the morning, participant visited CCPP Lindcove fundamental and protected block with to learn about: Positive pressurized greenhouse design and system, planting medium for citrus seedling or rootstock in pot, watering/irrigation and fertilizing system for citrus in

greenhouse, solarization technique to recycle citrus plant wastes as well as to remove possible soil pathogenic microorganisms, how citrus germplasm and planting material maintained in foundation and protected foundation block, laboratory citrus quality testing facility, collecting and extraction seed from fruit for propagation, collecting, storage and delivering budwood (Figure 10 and 11). (Dr. R. Christiano) Participant visited B & Z Incl. a citrus nursery company located in Porterville, CA which has also eleven 0.5 acre airstreaming greenhouses for propagation of citrus planting materials. (Dr. R. Christiano) In the afternoon, participant back to Lindcove to visit University of California Agricultural and Natural Resources, Lindcove Research & Extension Center. A Scientist brought participant to experimental field to see citrus variety collection and field performance and characteristics of citrus varieties, irradiated citrus mutant plant (breeding program through irradiation), other commodities (avocado and olive) (Figure 13). (Mr. Kurt Schmidt) Participant also visited a laboratory where a citrus fruit grading machine for reasearch and testing purpose. The machine equipped with conveyor and operated with computer has ability to sort and grade citrus fuitr based on size and shape, texture/color perfection in huge capacity. (Mr. Kurt Schmidt) June 27, 2014 - Friday Participant visited Visalia downtown in the morning. The rest of the day was for wrap up and writing trip report June 28, 2014 - Saturday Participant travelled from Visalia to Fresno by car for about 1 hour, then departed from Fresno International airport to Indonesia June 29, 2014 - Sunday Arrived in Jakarta in the midnight

List of Contacts Made: Name Dr. Georgios Vidalakis Dr. Sofia Kamenidou Mrs. ina Tan Title/Organization Director/Citrus Clonal Protection Program, UC Riverside Administration manager/citrus Clonal Protection Program, UC Riverside Doctoral student/dept. Of Plant Pathology UC Riverside Contact Info (address, phone, email) (951) 827-3763 2401 Boyce Hall georgios.vidalakis@ucr.edu sofia.kamenidou@ucr.edu Boyce Hall, UCR Stan016@ucr.edu Ms. Tavia Lab Manager Boyce Hall, UCR Tyler Undergraduate Student Boyce Hall, UCR Brandon Part time employee Boyce Hall, UCR Greg Greer Dr. Tracy L. Kahn Dr. Mikeal L. Roose Robert R. Krueger Dr. Richard F. Lee Dr. Manjunath Keremane Dr. James E. Adaskaveg Technician/Rubidoux Quarantine Facility Principal Museum Scientist/ Citrus Variety Colllection - Dept. of Botany and Plant Sciences UC Riverside Professor of Genetics, Citrus & Asparagus Breeding/ Dept. of Botany and Plant Sciences UC Riverside Horticulturist & Curator/ USDA ARS Pacific West Area-National Clonal Germplasma Repository for Citrus & Dates Research Leader, Research Plant Pathologist/ USDA ARS Pacific West Area- National Clonal Germplasma Repository for Citrus & Dates Plant Pathologist/ USDA ARS Pacific West Area- National Clonal Germplasma Repository for Citrus & Dates Professor/Dept. Plant Pathology, UC Riverside Rubidoux Quarantine Facility, Riverside 951-827-7360 900 University Ave. 4170 Batchelor Hall, Riverside CA 92521-0124 Tracy.kahn@ucr.edu 951-827-4317 900 University Ave 4121 Batchelor Hall, Riverside CA 92521-0124 Mikeal.roose@ucr.edu 951-827-4399 100 Martin Luther King Blvd Riverside, CA 92507-5437 Robert.krueger@ars.usda.gov, Robert.krueger@ucr.edu 951-827-4399 100 Martin Luther King Blvd Riverside, CA 92507-5437 Richard.Lee@ars.usda.gov 951-827-4399 100 Martin Luther King Blvd Riverside, CA 92507-5437 Manjunath.keremane@ars.usda.gov 900 University Ave. 242 Fawcett Lab. Riverside, CA 92521

Dr. Helga Foster Dr. Rock Christiano Mrs. Jackie Maxwell Professor/Dept. Plant Pathology, UC Riverside Staff Research Associate, Manager/ Lindcove Research & Extension Center University of California CCPP Lindcove Foundation President/Willits & Newcomb Inc. Jim.adaskaveg@ucr.edu 900 University Ave. 242 Fawcett Lab. Riverside, CA 92521 Helgaf@ucr.edu (559) 429-9718 22963 Carson Avenue Exeter, CA 93221 Rock.christiano@ucr.edu 661-327-9345 PO Box 428 Arvin, CA 93203 jackiem@wncitrus.com Mr. Gary Employer/B & Z Inc. 1857 S. Newcombe Porterville, CA 93257 (559) 781-7438 Mr. Kurt Schmidt Dr. Guido Schnabel Principal Superintendent of Agriculture/University of California Agricultural and Natural Resources, Lindcove Research & Extension Center Professor & Extension Plant Pathologist/School of Agricultural, Forest & Environmental Sciences (559) 592-2408 Ext 153 22963 Carson Avenue, Exeter, CA 93221 krschmidt@ucdavis.edu 864-656-6705 105 Collings St. 220 BRC, Clemson SC 29634 schnabe@clemson.edu

Appendix (Figures): Figure 1. Washington navel orange first parent tree in USA/California brought from Bahia (Brazil) in 1870 which is still alive until now in Riverside Figure 2. Nucleic acid (DNA or RNA) total extraction from citrus plant part: sample preparation (left), sample grinding machine (center), MagmaxTM nucleic acid extraction automated machine (right)

Figure 3. Shoot tip grafting (micrografting) to produce pathogen-free citrus: preparation of rootstock seedling grown on MS medium (above row), preparation and unification of shoot tip onto rootstock seedling done aseptically in laminar air floe cabinet (center row), micrografted citrus plant in tube grown in incubator at 27 C, followed acclimatization in growth chamber (below row) Figure 4. UC Riverside citrus variety collection (left), Dr Tracy Kahn explain characteristic of a citrus variety (right)

a b c d Figure 5. Citrus graft-transmissible diseases: Tristeza (a), Psorosis (b), Viroid (c), Tatter leaf (d), Fatal yellow (e), stubborn (f) e f a b c Figure 6. Grafting to unifiy a scion with rootstock: making cut on rootstock and preparing a scion (a), a scion intergrated onto rootstock (b, c)

Figure 7. Slash inoculation Figure 8. quantitative real time polymerase chain reaction (qrt-pcr) for indexing or detecting citrus from pathogen: sample/template and reaction preparation (left), running reaction in comterized RT PCR machine (center), setting qrt-pcr program (right)

Figure 9. Positive pressurized greenhouses at Willits & Newcomb Inc. citrus nursery company in Arvin CA: inside regular greenhouses (above) Inside airstreaming greenhouse and its giant fan to create positive pressure (below) Figure 10. Lindcove citrus foundation and protected block facilities

Figure 11. Laboratorium facility in Lindcove for citrus quality assesment (left), citrus variety seeds for propagation of rootstock (right) Figure 12. Airstreaming greenhouses at B & Z Inc citrus nursery in Porterville, CA Figure 13. Lindcove experimental field: citrus (left), avocado (center), olive (right)