New Huanglongbing Detection in Riverside

By John Krist

Chief Executive Officer of the Farm Bureau of Ventura County. Contact him at john@farmbureauvc.com.

Nearly 600 scientists, government agency representatives and members of the agricultural community gathered in Riverside last month for the sixth International Research Conference on HLB. This was the first iteration of the IRCHLB to take place in California, but like its predecessors in Florida, the conference drew a global sampling of smart people trying to figure out an answer to the most pressing question facing the worldwide citrus industry: How do we stop Huanglongbing and the Asian citrus psyllid from destroying it?

As was the case at previous conferences, no definitive answer to that question was provided by any of the hundreds of research presentations and posters. Work continues on every imaginable mechanism for disrupting the lethal vector-host-pathogen complex: breeding HLB-resistant or HLB–tolerant citrus rootstocks and scions; disrupting the ability of ACP to reproduce, feed or acquire the disease-causing bacteria; treatments to cure or reduce symptoms of infection; genetically or biologically based methods for killing ACP more efficiently. The “solution,” however, remains as elusive as it was during all previous biennial conferences.

But there have been important advances. This year, researchers presented results from their use of a tool that did not exist as recently as seven years ago. And it offers the prospect of rapid development of a suite of interventions — HLB-tolerant trees, crippled psyllids, perhaps even a lethal agent that attacks the bacteria themselves — that could change the course of the epidemic.

The new approach is being made possible by a gene-editing technology known as CRISPR. The term (pronounced “crisper”) is an acronym for “Clustered Regularly Interspaced Palindromic Repeats,” and it refers to odd DNA fragments discovered 20 years ago by scientists examining the genome of various families of microbes.

The short, repeated fragments of DNA were distinctive and appeared unrelated to the remainder of the microbial genome. Additional investigation revealed similar structures in the genomes of microbes from vastly different families of life, suggesting that they were not random but performed some function useful to microbial survival.

Research over the next decade revealed that CRISPRs were a feature of bacterial immune systems. Bacteria attacked by viruses (known as bacteriophages, or just phages) evolved a way to duplicate small snippets of the invader's DNA, insert it into their own genome, and pass that modified genetic code along to subsequent generations of bacteria. If attacked again by a phage containing that same snippet of DNA, the bacterial DNA recognizes it and triggers a targeted enzyme response that severs the viral genome at precisely that point, disabling the virus.

These discoveries led eventually to a technique by which researchers can pretty much treat the genome of any organism as a set of Lego blocks, its pieces plugged in, removed or replaced virtually at will. Using tools developed in the lab, but replicating the function of the CRISPR complex in microbes, they can dispatch custom-made genetic packets into living organisms, where they can remove, silence, activate or replace specific genes and their functions. 

Since scientists first reported six years ago that this adaptive microbial immune system could be repurposed into a simple and reliable technique for gene editing, thousands of researchers have been exploring its application in a wide variety of fields. One of them is the quest for a solution to HLB. And at this year's conference, several teams reported progress toward using the CRSPR system to modify the genome of all three elements of the pest-host-disease triad: ACP, the HLB bacteria, and citrus trees. 

Perhaps the most remarkable of these reports carried an inscrutable title: “BAPC-assisted-CRISPR-Cas9 Delivery into Nymphs and Adults for Heritable Gene Editing (Hemiptera).” 

Hidden within that bland language was a dramatic achievement: Two scientists — Wayne Hunter at the U.S. Department of Agriculture's Fort Pierce lab in Florida, and John Tomich of Kansas State University — injected a special variant of the CRISPR package, designed to knock out two genes in the ACP genome, into a female adult psyllid. Her offspring inherited a CRISPR-modified genome in which those two genes — one governing physiological development, and the other eye color — had been deleted. 

As nymphs, the genetically modified ACP took much longer to develop and had lower survival rates. As adults, the survivors had strange white eyes and malformed wings, and their lifespan was one-third of normal.

Those would be useful achievements on their own. But the experiment also demonstrated a powerful tool for determining the functions of other ACP genes by disabling them and observing the results on psyllid morphology, physiology or behavior. This could potentially lead to one of the Holy Grails of ACP-HLB research — a psyllid that is incapable of transmitting the disease-causing bacterium. 

Driving that genetic trait throughout the ACP population would halt the epidemic in its tracks. Growers could greatly reduce, if not eliminate, their reliance on pesticides as a means of slowing disease spread by suppressing the vector population — currently the only viable strategy. 

There were several other presentations about research to use CRISPR or other techniques to genetically modify the citrus genome to improve HLB tolerance — a much more rapid way of generating potential new rootstocks than the years-long process required to screen varieties and rear new trees — and to disable or kill the bacteria themselves.

The speed with which this field of knowledge is evolving, and the remarkable power of the CRISPR technology, left many of us who heard these presentations hopeful. After years of tantalizing research “breakthroughs” that have failed to yield meaningful and deployable interventions, the fight against ACP and HLB may soon be waged on our terms – not the bug's. 

Until transgenic psyllids move from the lab to the field, however, we're stuck with our current approach to disease and vector management: Suppress ACP populations with chemicals, identify and remove infected trees as quickly as possible, and replant using disease-free nursery stock. But there was exciting news on this front as well. 

From the beginning, the effort to identify and remove HLB-positive trees has been hampered by the long lag time between initial infection and confirmation using the classic DNA testing that serves as the regulatory gold standard. Because the test looks specifically for sequences of DNA that are unique to the bacteria in samples of ACP or plant tissue, it can provide incontrovertible and direct evidence of infection. From a legal standpoint, this is key when regulators act to force removal of positive trees. 

The problem, however, is one of sampling. A mature citrus tree may have 200,000 leaves, yet in the early months and even years of infection, the bacteria are not distributed uniformly through the canopy. Typical samples submitted for DNA analysis may consist of as few as 12 leaves per tree. Chances of picking the right leaves are minuscule when the bacterial population is small, and perhaps isolated only in a particular branch. The odds are stacked against the sampling effort from the start. 

This is why a major branch of the global research effort is devoted to developing early detection technologies — methods of confirming probable HLB infection based on changes in tree physiology that come about within days or weeks of infection. Being able to identify and remove infected trees before significant numbers of ACP have had the opportunity to feed on them and acquire the bacteria would greatly improve our ability to influence the course of the epidemic.

Several good candidates have been identified and validated, but they require intensive laboratory processing, and capacity limitations have stymied their wide use. But the most promising of these technologies doesn't require a lab at all.

As reported here after the 2017 IRCHLB, a team of dogs trained according to protocols developed by researchers at USDA and North Carolina State University have demonstrated the ability to correctly identify HLB-positive trees as little as two weeks after infection. During field trials, their accuracy rate has been up to 98 percent.

From the outset of this project, which has been funded through a federal grant, lead researcher Tim Gottwald of the USDA theorized that the dogs were identifying a change in the suite of volatile organic compounds emitted by trees. This is known to occur almost immediately upon infection, as the plant mounts a physiological defense against the invading bacteria. Sick trees don't smell like healthy trees, and Gottwald's theory has been that dogs can tell the difference.

At this year's conference Gottwald declared that he'd been wrong. In more recent experiments, his team injected the HLB bacterium into plants that are utterly unlike citrus — periwinkle, tobacco, dodder. Although the dogs had been trained on citrus, they correctly identified the inoculated non-citrus plants even though they smelled nothing like oranges or lemons. He then took it one step further, and ran the dogs past cages containing ACP, some of which had been infected in the lab. The dogs were able to correctly identify infected psyllids as well. And bugs don't smell anything like plants.

To confirm his new hypothesis, he exposed the dogs to an array of bacterial cultures, some of which included the bacteria known to cause HLB. The dogs were able to identify those, too. Gottwald now believes that when the dogs identify citrus tree as infected with HLB, it's because they are picking up the actual scent of the bacteria within it. 

If that's the case, it would provide a powerful argument for immediate and mandatory removal of suspect trees very early in the disease process: The dogs would providing direct evidence of infection. From a regulatory standpoint, it should be no different from a positive DNA test result.

The current risk-based HLB survey being conducted throughout the state — consisting of systematic collection of plant tissue and psyllid samples for DNA testing — has been heavily weighted toward urban areas, which is how the exploding epidemic in Los Angeles, Orange and Riverside counties was discovered. The current tally of HLB-positive trees removed there now exceeds 1,200, all of them in urban yards. 

Less attention has been paid, however, to rigorous surveying of commercial groves. This places an imperative on arranging for trained dogs and handlers, now based in Florida,  to begin their work here. Nothing is more important than finding HLB in commercial groves while it is early enough for tree removal to stop or slow the spread of the epidemic. The CRISPR research has opened the door to an entirely new array of potential weapons against HLB, but the citrus industry needs to survive long enough to use them.

Photo:

HLB deformed fruit

News from the Ventura County ACP-HLB Task Force

Winter 2019 area-wide treatment cycle has begun

This is a reminder that the Winter 2019 area-wide management (AWM) treatment window opened Jan.7. You are encouraged to file pesticide use reports (PURs) electronically through CalAgPermits, Agrian, or another system that allows treatments reports to be rapidly filed and recognized. Treatment reminders will be emailed (mailed for those without an email record) about 2-3 weeks prior to the treatment window. In some instances, our contact list may only have contact information for your farm manager, pest control advisor, or pest control operators. If you are not receiving emails, please contact one of your grower liaisons (see contact information below) to receive information about ACP, HLB and your citrus.

Website redesigned

The Citrus Pest & Disease Program (CPDPP) launched a redesign of its website to provide members of California's citrus industry easier access to the key maps, regulatory updates and events they need to stay informed on the fight against HLB in California. The website is at www.citrusinsider.org.

HLB confirmations continue to increase

At least 1,024 residential trees had been confirmed as infected with HLB as of January 2019. No HLB-positive trees have been found in commercial groves. The HLB quarantine boundaries and the latest tally of HLB confirmations, updated weekly, is available online at https://citrusinsider.org/maps-and-quarantines/.

Report neglected and abandoned citrus

Help prevent neglected and abandoned citrus from serving as a breeding ground for ACP and the spread of HLB by reporting its location County Agricultural Commissioner's office at (805) 388-4222. If your citrus is not worth the resources required to protect it from ACP and HLB, it may be a good time to consider removing the trees. Tree-removal assistance is offered to small growers through the California Citrus Mutual and Bayer's ACT NOW program. For more information contact Joel Reyes at  jreyes@cacitrusmutual.com or (559) 592-3790.

Calendar

UCR Citrus Day (Jan. 29)

This year's UC Riverside Citrus Day will be on Tuesday, Jan. 29. Agenda and registration information can be found by clicking here.

International Research Conference on HLB/Citrus Virologist Conference (March 10-15)

The joint International HLB and Citrus Virologist conference will be at the Riverside Convention Center in Riverside, CA. Registration and more information can be found here.

Citrus Pest and Disease Prevention Committee meeting (March 12)

The CPDPC is charged with advising the state on management of the Citrus Pest and Disease Prevention Program. The CPDPC and subcommittee meetings are open to the public, and options for participation include in person or by webinar and conference call. Click here to view and register for upcoming committee and subcommittee meetings. Attendance is free. 

esources

University of California ACP area-wide materials list and ACP monitoring protocols

Movement of bulk citrus materials list

Ventura County AWM maps and schedule

Citrus Pest and Disease Prevention Program

Ventura County ACP-HLB Task Force mailing list signup

Contact your grower liaisons if you have additional questions: 

Sandra Zwaal

szwaal2@gmail.com

(949) 636-7089

Cressida Silvers

cressidasb@gmail.com

(805) 284-3310

The best way to delay arrival of HLB in our area and minimize its impact is to keep ACP suppressed down to the lowest level possible.  By treating in coordination with neighbors in an areawide approach, grower ACP treatments can have a greater impact on ACP populations than treating independently and out of sync with neighbors. Best Management Practices, such as making sure all equipment arriving and leaving your grove is free of citrus stems and leaves, can also greatly reduce the risk of HLB-positive psyllids entering your grove.

CITRUS REMOVAL PROGRAM: Citrus trees that are neglected or abandoned may harbor ACP and HLB, increasing risk to other citrus in the area. Abandoned and neglected trees may be reported to Cressida Silvers at 805-284-3310,  or the county Ag Commissioner's office. The Citrus Matters ACT NOW program may be able to assist in citrus removal. For more information contact Joel Reyes at  jreyes@cacitrusmutual.com or (559) 592-3790. 

Asian Citrus Psyllid / ACP

There have been no ACP detections in San Luis Obispo County since our last update.

Huanglongbing / HLB

The most recent map and totals for HLB detections are posted at the website https://citrusinsider.org/maps/. As of November 16, the total number of trees that have tested positive for the HLB bacterium is 948, still all in LA, Orange, and Riverside Counties. All HLB detections have been on residential properties and the infected trees have been or are being removed. No HLB has been found in commercial groves to date.

Clarification on Field Cleaning Requirements for Movement of Bulk Citrus

To clarify the approved mitigation measures for bulk citrus fruit movement, the California Department of Food and Agriculture (CDFA) has updated the Asian Citrus Psyllid (ACP)-Free Declaration form. The current options that allow growers to meet the ACP-free standard when shipping fruit to a different ACP regional quarantine zone are the “spray and harvest,” “field cleaning with machine” and “wet wash” methods. Field cleaning must be done by machine, not by hand.

To read the full article, click here: https://citrusinsider.org/2018/11/clarification-on-field-cleaning-requirements-for-movement-of-bulk-citrus/

Upcoming CPDPC Meetings

• Joint Science and Technology Subcommittee and Regulatory Task Force meeting Thur., December 6 at 1:30 pm in Sacramento. Agenda attached, including link to join by webinar/phone.
• CPDPC Operations Subcommittee meets Wed., December 12 at 9 am in Visalia. Agenda attached with link to join by webinar/phone.
• The next meeting of the CPDPC Full Committee will be January 9 in Visalia. Agenda is pending. 
• All meeting agendas and eventually the minutes are posted at https://www.cdfa.ca.gov/citruscommittee/ . All meetings are free and open to the public, and accessible via webinar.  

Additional Resources

• General information on the ACP/HLB program, including quarantine information: https://citrusinsider.org/
• Biology of ACP and HLB, detection maps and recommendations for monitoring, eradication and management : http://ucanr.edu/sites/acp/
• Summaries of the latest research to combat HLB: http://ucanr.edu/sites/scienceforcitrushealth/

And Now it's in Marin County

SACRAMENTO — Marin County has been placed under quarantine for the Asian citrus psyllid (ACP) following the detection of one ACP in the City of Novato. The entire county is included in the quarantine zone.

The ACP is an invasive species of concern because it can carry the disease huanglongbing (HLB), also known as citrus greening.  All citrus and closely related species, such as curry leaf trees, are susceptible hosts for both the insect and disease.  There is no cure once the tree becomes infected. A diseased tree will decline in health and produce bitter, misshaped fruit until it dies.  In California, HLB has been detected at residential properties in Los Angeles, Orange and Riverside counties. This plant disease does not affect human health.

Residents with backyard citrus trees in the quarantine area are asked not to transport or send citrus fruit or leaves, potted citrus trees, or curry leaves from the quarantine area. For commercial citrus, the quarantine prohibits the movement of citrus and curry leaf tree nursery stock, including all plant parts except fruit, out of the quarantine area. The quarantine also requires that all commercial citrus fruit be cleaned of leaves and stems prior to moving out of the quarantine area.  An exception may be made for nursery stock and budwood grown in USDA-approved structures that are designed to keep ACP and other insects out.

ACP quarantines are in place in Alameda, Contra Costa, Fresno, Kern, Kings, Madera, Merced, Monterey, Placer, San Benito, San Joaquin, San Luis Obispo, San Mateo, Santa Clara, Solano, Stanislaus, Tulare, Yolo, Imperial, Los Angeles, Orange, Riverside, San Bernardino, San Diego, Santa Barbara, and Ventura counties, as well as Marin.

Residents in the area who think they may have seen ACP or symptoms of HLB on their trees are urged to call CDFA's Pest Hotline at 1-800-491-1899 or a local agricultural commissioner's office  For more information on the ACP and HLB, please visit: www.cdfa.ca.gov/go/acp. Residents are also asked to follow these steps:

• Inspect trees for the Asian citrus psyllid and Huanglongbing monthly, and whenever watering, spraying, pruning or tending trees. Psyllids are most noticeable when new leaves are growing on the tips of the branches.
• As part of your tree care, visit your local nursery or garden center to get advice on products that can help protect your citrus tree.
• Do not move citrus plants, foliage or fruit into or out of your area, and especially across state or international borders. This could unknowingly contribute to spread of the pest and disease.
• When planting a new citrus tree, be sure to get your tree from a reputable, licensed nursery in your local area.
• When grafting citrus trees, only use registered budwood that comes with source documentation, such as the budwood offered through the Citrus Clonal Protection Program.
• Be sure to dry out citrus tree clippings or double bag them before removing the plant material from the property.

–California Department of Food and Agriculture

VISALIA – Last week's California Citrus Conference marked a major milestone for growers, and it wasn't just the 50th anniversary of the Visalia-based Citrus Research Board (CRB). It was a resounding revelation that new research may cure the greatest threat to the citrus industry in the next few years.

Michelle Heck, a molecular biologist with the USDA Agricultural Research Service, discusses her research to breed a new citrus pest that does not transmit the fatal tree disease known as huanglongbing. Photo by Michael Alvarez.

Michelle Heck, PhD, told the crowd of citrus growers at the Wyndham Hotel on Oct. 10 that her team might only need that much time to inbreed a generation of Asian citrus psyllids that are incapable of transmitting the deadly tree disease known as huanglongbing (HLB). The disease has already destroyed China's citrus industry, decimated Florida and Texas growing regions and is currently killing the citrus industry in Brazil.

One grower commented, “China's been dealing with this for 100 years and Brazil for 14 years. We've had this for four to five years in California and we are already knocking on the door of nailing it. That's impressive!”

Heck, a molecular biologist with the USDA Agricultural Research Service, was the first to lead a team of scientists to study the proteins involved in the interaction of the pest, plant and pathogen. One of those proteins creates a blue color in the blood of some psyllids. Her research revealed that psyllids containing the blue protein are far less efficient at transmitting HLB to the plant than others. She then bred those psyllids and took their progency and raised them on orange jasmine hedges, better known as Murraya, a plant the psyllids are attracted but is HLB resistant. The combination of the pest and plant reduced transmission of HLB to healthy citrus leaves from 32% to 2.9%.

Heck said the next steps are to continue breeding the pests that are poor transmitters of the disease to create a line of psyllids that do not transmit HLB at all. She said it would take another two years to breed an “optimized line” of the psyllid but once that was complete, that line could begin mass breeding for release.

“By sheer numbers, we can tip the scales [in the fight against HLB],” she said, “but it's unknown if these lines will out compete other psyllids [in the field].”

One grower asked if the non-transmitting line of the pest would be considered a genetically modified organism, or GMO, a distinction that could hurt fruit grown in groves with the new pest. Heck said all of the psyllids would be bred natuarally, so there is no genetic alteration of the insect itself.

“This is something the anti-GMO groups should feel good about,” Heck said.

Best Case Scenario

Victoria Hornbaker, Statewide Citrus Program Manager for the California Department of Food and Agriculture (CDFA), called the current HLB situation in California a best case scenario. She said the Citrus Pest and Disease Prevention Program's (CPDPP) No. 1 priority is to quickly detect and remove diseased trees. Shortly after the discovery of the first HLB tree in 2012, California's myriad of citrus agencies worked together to quickly implement measures to control movement of fruit and nursery stock, monitor and suppress the ACP population, and begin working on ways to detect the disease and possibly cure it.

“Instead of all commercial groves being covered by a quarantine, we said we're going to quarantine the whole state,” Hornbaker said.

By limiting the movement of citrus in and out of different quarantine zones, there is less likelihood of transporting trees from an infected area to an uninfected area. If any infected trees are discovered, they are removed, destroyed and replaced with a healthy tree. There are many early detection techniques (EDTs) being studied throughout the country, including looking for patterns in leaves, chemicals produced by trees in response to HLB, and studying molecules of the bacteria causing the disease. A recent analysis of these EDTs showed that most are about 95% effective in identifying an infected tree, and that losing 5% of healthy trees is an acceptable loss compared to devastation caused by the disease spreading unchecked.

While early detection methods of ACP are still being perfected, the fight to control the spread of the psyllid is not. After research identified the microscopic parasitic wasp radiate terminaxia as the natural enemy of the psyllid, they began working to mass produce and release them. To date, more than 11 million wasps have bee released in citrus growing regions since 2013, the closest being in Kern County.

Reproduced from Sun Gazette:

http://www.thesungazette.com/article/news/2018/10/17/pests-that-spread-citrus-disease-are-key-to-cure/