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

Critical Leaf Nutrient Thresholds to Diagnose Deficiencies in HLB Trees

By Arnold Schumann, Laura Waldo, Tripti Vashisth, Alan Wright and Kelly Morgan

Huanglongbing (HLB) disease severely impacts the nutrient status of citrus trees, particularly by stunting the feeder roots and causing measurable deficiencies of nutrients in the roots and canopies. Visible symptoms of nutrient deficiencies on citrus foliage are characteristic but not diagnostic of HLB disease.

Recent HLB research efforts have focused on manipulating nutrient deficiencies with fertilizer applications to mitigate HLB symptoms and help keep affected trees alive and productive. Efficient diagnosis of nutrient imbalances in HLB-affected trees (both deficiencies and excesses) is necessary to make fertilizer remedies cost-effective and productive. Published thresholds defining deficient, low, optimal, high and excess ranges of nutrient concentrations in citrus leaves were derived long before HLB spread in Florida. Therefore, we conducted a series of survey studies to verify and improve those existing thresholds in HLB-endemic Florida citrus groves.

Traditionally, nutrient thresholds were obtained empirically from long-term replicated fertilization trials conducted decades ago, most notably where “missing nutrient” experiments would, over many years, cause those nutrients to become deficient in trees not receiving certain fertilizers. By comparing treatment plots to fully fertilized control plots, the fruit yield reduction and other symptoms caused by the nutrient deficiency could be quantified.

Critical threshold concentrations (CTCs) derived for each nutrient from those studies were then published and used as thresholds to diagnose leaf tissue samples collected from commercial groves. In the current study, we used a sample survey method and the Diagnosis and Recommendation Integrated System (DRIS) instead of “missing nutrient” experiments in order to save considerable time.

STUDY SETUP
Leaf tissue samples were collected quarterly from 2016 to 2018 in three citrus-growing regions of Florida (11 locations from the Central Ridge, five from the east coast Indian River and six from the southwest Flatwoods). Soil samples were collected annually, and tree canopy measurements, leaf size, starch content, quantitative polymerase chain reaction (qPCR) assay and other measurements were conducted periodically for the same sites. In this article, we will focus only on the leaf tissue nutrients of sweet orange trees and assume that most of the sampled trees were HLB-positive, based on the qPCR analyses.

ABOUT DRIS
In an April 2009 Citrus Industry article, DRIS was introduced as a promising method for interpreting nutrient levels in plant tissues. See “Potential use of DRIS for leaf nutrient diagnosis in Florida citrus” (www.crec.ifas.ufl.edu/extension/trade_journals/trade_journals2009.shtml).

The DRIS method produces an index for each plant nutrient calculated from a series of leaf concentration ratios of that nutrient with all other measured nutrients, relative to a set of nutrient norms (standards) that were previously calculated from a high-yielding grove. We obtained the DRIS norms from leaf samples collected in the summer of 2003, from a 22-year-old block of Hamlin orange trees on Cleopatra mandarin rootstock near Fort Meade, Florida, spaced 20 by 25 feet and with an average fruit yield over four years of 770 boxes per acre. The leaf sampling for DRIS norm development coincided with a peak yield of 970 boxes per acre in the 2003–04 season, prior to Hurricane Charlie and the appearance of HLB in Florida.

DETERMINING DEFICIENCIES
The application of DRIS to diagnose nutrient deficiencies in leaf tissue from HLB-affected trees is best done in conjunction with the CTCs. The CTCs are the thresholds for each nutrient that determine whether a given tree or grove would be responsive to fertilizer supplying that nutrient. Traditionally the CTCs occupy a spot on the plant response curve for each nutrient concentration of about 90 percent maximum yield or growth.

When multiple deficiencies occur, as is often the case with HLB-affected trees, the DRIS indices provide information about the relative severity and ranking of the deficiencies, which the CTCs do not accomplish. Dealing with multiple nutrient deficiencies efficiently is important because of the biological stoichiometry that determines the ideal proportions of nutrients in a plant, just as, for example, a water molecule consists of exactly two atoms of hydrogen and one of oxygen.

Liebig's law of the minimum applied to plant growth, and often visualized as a leaking barrel, (Figure 1) demonstrates that it is the most limiting nutrient that limits growth and yield, regardless of the amounts or types of other nutrients available. Another analogy is that a gasoline engine will not run faster if only the gasoline flow is increased without proportionately increasing the air (oxygen) supply, such as if the choke control is active. The engine may flood and stall if too much gasoline is supplied without increasing the most limiting ingredient for combustion at that time (oxygen).

For example, consider the results in Table 1 of leaf tissue analysis from an HLB-affected tree.

DRIS indices have a theoretical optimum at zero, with more negative values indicating possible nutrient deficiency, and increasing positive values indicating possible nutrient excess. In the Table 1 example, Mg<Mn

Figure 1. Scatter plot of leaf manganese Diagnosis and Recommendation Integrated System (DRIS) indices versus manganese concentrations in HLB-affected citrus. The breakpoint between red and green regression lines indicates the location of the critical threshold concentration (CTC = 22.1 mg/kg).

CALCULATING CTCs
To examine the validity of currently available CTCs for nutrient diagnosis in HLB-endemic Florida citrus, we used scatter diagrams of the DRIS indices for each nutrient plotted against the nutrient concentrations. This technique has been used before in other crops to determine CTCs from nutrient survey data, including loblolly pine, cotton and signalgrass. Figure 2 shows that the scatter plot for manganese in our survey of HLB-affected groves follows two data trends of different slopes, with the breakpoint indicating the CTC for that nutrient on the x-axis. We used segmented linear regression to determine the correct breakpoint.

The process was repeated for each nutrient in order to determine the preliminary CTCs listed in Table 2. The optimal nutrient ranges in Table 2 were estimated from the intercept of the regression line with zero on the y-axis (DRIS). By comparing our new CTCs with published CTCs, it appears that deficiency diagnoses for copper, boron, potassium, manganese and zinc could be underestimated by currently available CTCs when used for HLB-affected trees.

The optimal nutrient concentrations calculated from this study are mostly in agreement with ranges previously published by the University of Florida Institute of Food and Agricultural Sciences (UF/IFAS), except that boron and potassium optimal levels were estimated slightly higher in the survey than those previously published. These results are preliminary, and in this study, CTCs could only be calculated for those nutrients where deficiencies occurred in the survey data. We plan to augment this survey with more leaf nutrition data to better define and expand CTC estimates to be used for diagnosing HLB-affected citrus for all nutrients.

Please contact us if you have leaf nutrient data from your groves that you are willing to share in this study, or if you would like to use the DRIS program which will be published online to a UF/IFAS web page.

SUMMARY
In this article we provided updates on research efforts to improve the nutrition of HLB-affected Florida citrus by revising the diagnostic CTC of nutrients in leaf tissue. We also revisited the DRIS method for improving diagnosis of multiple nutrient deficiencies that are common in HLB-affected groves, including potassium, magnesium, manganese, zinc, iron, boron and copper. Additional leaf nutrient data will be obtained for an expanded survey, to be analyzed and corroborated with results of recently completed micronutrient fertilization experiments.

Acknowledgements: We thank the grower cooperators who offered their groves for our nutrient surveys, including Gapway Groves, Ben Hill Griffin Groves, OrangeCo, Brent Shirard, Michael Monroe and English Brothers. We are grateful to the Citrus Research and Development Foundation for financial support of this project.

Arnold Schumann (schumaw@ufl.edu) and Laura Waldo are soil scientists, and Tripti Vashisth is a horticulturist, all at the UF/IFAS Citrus Research and Education Center in Lake Alfred. Alan Wright is a soil scientist at the UF/IFAS Indian River Research and Education Center in Fort Pierce. Kelly Morgan is a soil scientist and center director at the UF/IFAS Southwest Florida Research and Education Center in Immokalee.

Table 1.  Leaf tissue analysis of HLB-affected orange trees

Figure 1. Manganese concentration of leaf tissue

Table 2. Critical nutrient concentration thresholds

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