- Author: Ben Faber
Texas A&M AgriLife researchers have made a discovery that will help combat fastidious pathogens, which cost U.S. agriculture alone billions of dollars annually.
For the past few years, Kranthi Mandadi, Ph.D., a Texas A&M AgriLife Research scientist and associate professor in Texas A&M's Department of Plant Pathology and Microbiology, along with his colleagues, has been working on developing new biological technologies to fight fastidious or “unculturable” pathogens. Mandadi and members of his team are based at the Texas A&M AgriLife Research and Extension Center in Weslaco.
The results of their work, “Plant hairy roots enable high throughput identification of new antimicrobials against Candidatus Liberibacter spp.” were recently published in Nature Communications.
Fastidious plant diseases and their costs
Fastidious plant pathogens infect citrus, tomatoes, potatoes, grapes, peppers and other crops grown throughout Texas. Often transmitted by insect vectors, these disease agents cause billions of dollars of damage each year. The U.S. citrus industry alone would save $3 billion per year through control of just one of these diseases — citrus greening. Additionally, the fastidious pathogen that causes Pierce's Disease in grapes is the No.1 threat to the $1 billion wine industry in Texas.
“Currently, invasive fastidious pathogens are causing several major outbreaks in row crops, specialty crops and citrus, with immense costs to Texas and the U.S.,” said Juan Landivar, Ph.D., director of the AgriLife center at Weslaco, which has been involved in efforts to combat fastidious plant pathogens for many years.
Landivar said an expanded effort against fastidious plant diseases would protect the health of crops, environments, economies and people across the country.
A way to grow “unculturable” bacteria
Some plant pathogens can be grown as pure cultures in the laboratory in the presence of artificial nutrient solutions. Being able to culture disease agents in the lab facilitates their study by providing researchers with a reliable supply of experimental material. However, an estimated 99% of bacteria in the world are fastidious, or unable to grow outside their native environment.
“The greatest obstacle to understanding and controlling fastidious pathogens was the inability to cultivate them in a laboratory setting and to screen for lots of potential therapies,” said Leland “Sandy” Pierson, Ph.D., professor and head of Texas A&M's Department of Plant Pathology and Microbiology. “But Dr. Mandadi and his team have developed a breakthrough method as an alternative means to propagate fastidious bacteria. These bacteria are believed to be responsible for Huanglongbing, also known as citrus greening disease, and other insect-vectored diseases such as potato zebra chip and tomato vein greening disease.”
The breakthrough came in the form of the “hairy root” system. This technology utilizes the pathogen-infected host tissues to produce so-called hairy roots that can serve as biological vessels for the propagation of these pathogens in the laboratory.
“Classical microbiological techniques developed early in the 19th century cultured animal and mammalian viruses in host cells, tissues and embryonated eggs,” Mandadi said. “In a similar manner, we hypothesized that plant hairy roots could be suitable for propagating fastidious pathogens. And indeed, hairy roots supported the accumulation and growth of fastidious plant bacteria.”
Microbial hairy roots appear similar to normal root tissues that develop from the plant and mimic a bacterium's natural environment, he said. This allows the growth of the fastidious pathogens in controlled laboratory conditions.
Expedited screening for antimicrobial treatments
While microbial hairy root cultures are not traditional “pure” test tube cultures, they allow on-demand access to the fastidious bacterium in the laboratory. This enables the expedited screening of diverse antimicrobials like chemical inhibitors, immune modulators as well as gene/CRISPR-based therapies.
Other advantages are that hairy root cultures are easy to produce in the laboratory and can be maintained for several months to a year in laboratory growth chambers. Depending on the pathogen and the efficacy of screening, it is also at least four times faster than conventional screening methods, according to Sonia Irigoyen, Ph.D., and Manikandan Ramasamy, Ph.D., both AgriLife Research scientists and co-authors of the study.
In addition, the hairy root bioassays are scalable, so they can be used to pre-screen from a few to several hundred potential therapies simultaneously in a high-throughput manner. The microbial hairy root system can also be used to obtain mechanistic insights into antimicrobial function.
“Use of this technique has already led to the discovery of six new antimicrobial peptides with proven efficacy in plant materials,” Mandadi said. “These antimicrobials, either singly or in combination, could be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.”
Collaborators in the fight
“Typically, the type of breakthrough Dr. Mandadi and his team came up with is unusual for a university system off-campus center, as such centers usually have limited personnel and resources,” Landivar said. “Fortunately, the support we have received from the Texas A&M University System and other funding agencies and collaborators has helped make it possible for the Weslaco center to perform this world-class-level research.”
Besides a team of researchers at the Weslaco center, Mandadi collaborates with scientists at Texas A&M University, Texas A&M University Kingsville-Citrus Center, University of Florida, University of California System, and industry stakeholders including Citrus Research and Development Foundation, Texas Citrus Pest and Disease Management Corporation, Bayer and other entities.
Southern Gardens Citrus, a subsidiary of U.S. Sugar in Florida, has partnered with Texas A&M to commercialize the hairy root system as well as new therapies for application in the field.
Landivar also said funding from the U.S. Department of Agriculture National Institute of Food and Agriculture's Emergency Citrus Disease Research and Extension program, NIFA ECDRE, and support from the Foundation for Food and Agricultural Research and AgriLife Research's Insect-Vectored Disease Grant are making it possible to facilitate development of innovative technologies and discovery of therapies to combat diseases caused by fastidious bacteria.
To expand on his research, Mandadi recently partnered on a new project with Citrus Research and Development Foundation, Bayer, Southern Gardens Citrus, University of Florida and University of California-Davis. That project is funded by the NIFA ECDRE program. The overall goal is to bring together academics, growers and agrochemical industry to discover, develop and commercialize therapies for citrus greening disease.
Mandadi said use of the hairy root system has already been instrumental in finding several potential new treatments for citrus greening and potato zebra chip, as described in the Nature Communications article.
“We hope this technology can be further expanded to find even more therapies against current and emerging fastidious pathogens and, ultimately, with the support of industry, deploy them as field-ready products,” he said.
/h3>/h3>/h3>/h3>- Author: Ben Faber
A recent Ventura County ACP-HLB Task Force sponsored webinar was held, the topics and speakers listed below The PDF's from the speakers are available online
Webinar Agenda 8-13-20
9:30 a.m. |
Welcome, and update on status of HLB in California: Leslie Leavens, chair, Ventura County ACP-HLB Task Force. |
9:45 a.m. |
Update on area-wide participation rates and CDFA buffer treatments: Sandra Zwaal, Ventura County Grower Liaison |
10 a.m. |
Final report on 2017-2020 ACP surveying project in Ventura County, and overview of Phase Two research: Beth Grafton-Cardwell, IPM Specialist and Research Entomologist, University of California-Riverside, and Director of Lindcove Research and Extension Center (retired); and Monique Rivera, Assistant Extension Specialist, Department of Entomology, UC Riverside. |
10:40 a.m. |
Final report on deployment of detection canines to scout Ventura County commercial groves for evidence of early HLB infection: John Krist, CEO, Farm Bureau of Ventura County. |
11 a.m. |
Implications of canine detection data for HLB management in Ventura County commercial citrus: Neil McRoberts, western regional director, National Plant Diagnostic Network, and professor of plant pathology, UC Davis. |
11:30 a.m. |
Adjourn |
For more information, contact us by email at taskforce@farmbureauvc.com.
- Author: Ben Faber
Last week, UCR issued a news release entitled UCR Discovers First Effective Treatment for Citrus-destroying Disease, which shares the news of a licensing agreement being reached with Invaio Sciences. This is extremely exciting news about potentially promising research that could significantly change the future of the citrus industry, both here and in all citrus growing area in the world. Now it will be important to complete studies on the effectiveness of this therapy in greenhouse and field studies.
As yet, there are no published scientific papers describing the methodology by which they discovered it, or the field or lab trials through which they determined its efficacy. As has been the case so often with ACP-HLB, there have been promising strategies that on further inspection fail to meet the criteria of a “cure”.
If it really works in a commercial field setting, it would be a great complement to the canine surveys, since it would be much easier to clear infection from a tree when it is still in the early stages and has not yet become systemic.
The Citrus Research Board has a wait-and-see approach - Glad that something is in the works to be commercialized, but waiting for more test results. This might be a real breakthrough. Read the CRB's response to the recent news:
- Author: Ben Faber
A Solution for the devastating bacteria causing citrus huanglongbing?
UC Riverside scientists have found the first substance capable of controlling Citrus Greening Disease, which has devastated citrus farms in Florida and also threatens California.
Oranges afflicted with Citrus Greening Disease. (UCR)
The new treatment effectively kills the bacterium causing the disease with a naturally occurring molecule found in wild citrus relatives. This molecule, an antimicrobial peptide, offers numerous advantages over the antibiotics currently used to treat the disease.
UCR geneticist Hailing Jin, who discovered the cure after a five-year search, explained that unlike antibiotic sprays, the peptide is stable even when used outdoors in high heat, easy to manufacture, and safe for humans.
“This peptide is found in the fruit of greening-tolerant Australian finger limes, which has been consumed for hundreds of years,” Jin said. “It is much safer to use this natural plant product on agricultural crops than other synthetic chemicals.”
Currently, some growers in Florida are spraying antibiotics and pesticides in an attempt to save trees from the CLas bacterium that causes citrus greening, also known as Huanglongbing or HLB.
The Asian citrus psyllid, pictured here, spreads the bacterium that causes Citrus Greening Disease. (Mike Lewis/UCR)
“Most antibiotics are temperature sensitive, so their effects are largely reduced when applied in the hot weather,” Jin said. “By contrast, this peptide is stable even when used in 130-degree heat.”
Jin found the peptide by examining plants such as the Australian finger lime known to possess natural tolerance for the bacteria that causes Citrus Greening Disease, and she isolated the genes that contribute to this innate immunity. One of these genes produces the peptide, which she then tested over the course of two years. Improvement was soon visible.
“You can see the bacteria drastically reduced, and the leaves appear healthy again only a few months after treatment,” Jin said.
Because the peptide only needs to be reapplied a few times per year, it is highly cost effective for growers. This peptide can also be developed into a vaccine-like solution to protect young healthy plants from infection, as it is able to induce the plant's innate immunity to the bacteria.
Jin's peptide can be applied by injection or foliage spray, and it moves systemically through plants and remains stable, which makes the effect of the treatment stronger.
The treatment will be further enhanced with proprietary injection technology made by Invaio Sciences. UC Riverside has entered into an exclusive, worldwide license agreement with Invaio, ensuring this new treatment goes exactly where it's needed in plants.
“Invaio is enthusiastic to partner with UC Riverside and advance this innovative technology for combating the disease known as Citrus Greening or Huanglongbing,” said Invaio Chief Science Officer Gerardo Ramos. “The prospect of addressing this previously incurable and devastating crop disease, helping agricultural communities and improving the environmental impact of production is exciting and rewarding,” he said. “This is crop protection in harmony with nature.”
The need for an HLB cure is a global problem, but hits especially close to home as California produces 80 percent of all the fresh citrus in the United States, said Brian Suh, director of technology commercialization in UCR's Office of Technology Partnerships, which helps bring university technology to market for the benefit of society through licenses, partnerships, and startup companies.
“This license to Invaio opens up the opportunity for a product to get to market faster,” Suh said. “Cutting edge research from UCR, like the peptide identified by Dr. Jin, has a tremendous amount of commercial potential and can transform the trajectory of real-world problems with these innovative solutions.”
UV Riverside news release:
https://news.ucr.edu/articles/2020/07/07/uc-riverside-discovers-first-effective-treatment-citrus-destroying-disease
/span>/h4>/h4>- Author: Ben Faber
An Automated Delivery System for Therapeutic Materials to Treat HLB Infected Citrus
Ozgur Batuman1 and Louise Ferguson2
¹Southwest Florida Research and Education Center, University of Florida, Immokalee, FL; 2UC Davis, Department of Plant Sciences, University of California Davis, Davis CA
Why is this research needed?
In 2005, a disease called Huanglongbing (HLB, citrus greening, was identified in Florida's commercial citrus groves. The disease is caused by a bacterium that affects all citrus cultivars by disrupting the flow of nutrients from the source of production, to the site of use, causing tree decline. HLB weakens the root system, increases early fruit and leaf drop, lowers tree productivity and fruit quality and ultimately kills the tree. The disease has spread to all the major production regions in Florida. Economic losses have exceeded more than $4 billion dollars. Currently, more than 95% of Florida's trees are infected. There is currently no cure for the disease.
Efforts to control HLB have been unsuccessful as the bacterium cannot be cultured, literally grown, in a petri dish, and once in the plant it proliferates within the citrus phloem. Phloem is the system that transports sugars from their site of production, the leaves, to plant parts that use sugars, the roots or flowers.Phloem transport is generally downward but can be upward as well.
Once the HLB bacterium is in a tree's phloem it has the potential to infect the entire tree. It is exceedingly difficult to introduce any control agent into the phloem with the conventional control methods of foliar spraying or soil drenching.
Thus far, no treatment preventing HLB infection, or controlling the bacterium once within the tree, has been developed. Potential chemicals are being investigated, but in order to test them, direct or indirect phloem delivery, where the bacterium proliferates, is needed. Therefore, an effective method of delivering an effective volume of theraputics into the phloem is needed to evaluate potential treatments.
What is the focus of this project?
Our project focuses on developing a method of delivering therapeutic liquid materials, bactericides, microbial metabolites, RNAi, or biologicals, into the citrus vascular tissues, both the xylem which conducts water and nutrients upward from the roots and the phloem, which conducts sugars and other metabolic products downward from the leaves. We are investigating diffusion, trunk punctures with a surrounding liquid reservoir for passive uptake and infusion, low pressure active injections. We are focusing on these methods as foliar sprays and root drenches have not been successful phloem delivery methods.
Who will be doing the research?
The project is led by plant pathologist Dr. Ozgur Batuman with colleagues at the Southwest Florida Research and Education Center (SWFREC) at University of Florida in Immokalee. This four-year project will also study the citrus vascular system with a multidisciplinary research team including UF Plant Pathologists Drs. Nabil Killiny and Amit Levy at Lake Alfred, SWFREC UF Plant Physiologist Ute Albrecht, Citrus Horticulturist Fernando Alferez, Precision Ag. Engineer Yiannis Ampatzidis, Agricultural and Natural Resources Economist Tara Wade, University of California-Davis Extension Specialist Louise Ferguson and Texas A&M-Kingsville Citrus Center Plant Pathologist Veronica Ancona as well as number of graduate students, postdocs, and Florida, Texas and California citrus industry members.
How will this research be done?
Our earlier research involving comparisons of delivery methods including foliar sprays, soil drenching and trunk injection determined Needle-Assisted Trunk Infusion (NATI) was the best potential delivery method (Figure.1). In initial experiments, using NATI, 1 ml of rhodamine (1%) dye was injected into the trunks of one-year-old citrus seedlings. A visible red color, indicative of rhodamine uptake and movement, was detected in the upper-most leaves within 30-60 min and an increase in color intensity was observed within 24 hours. Similar results were observed in two-year-old grafted Valencia plants within 48 hours. If the NATI delivery method can be automated, large numbers of trees could be treated quickly. Once the delivery method has been developed, implementation will be tested with potential treatments developed within other research projects.
Our proposed automated delivery would consist of a robotic arm with several modules at the end of the arm, installed on an ATV or tractor. One module with needles would grip and puncture the trunk, a second module would wrap a reservoir around the trunk below the punctures and third module would fill the reservoir. (Figure 2). Hopefully, a robotic arm plus automated system will be inexpensive enough for growers to purchase and simple enough to use.
Another approach is disease prevention; application pf prophylactic chemicals that prevent infection. In this scenario our system would be used treat healthy young trees with bactericides or boost their immune system. When infected by the ACP the bacterium would either be killed or suppressed, perhaps below the level that harms tree growth and productivity. This option is analogous to the vaccinations that prevent diseases in humans and animals.
What are the greatest challenges and opportunities.
The greatest challenge is successful phloem delivery. The greatest opportunity is that, if successful, we will have developed a method that will allow much more precise deliver of theraputics to citrus trees. For example, if an effective phloem delivery method is developed, it could be used to control insects that feed on citrus plant parts. Or, it could be used to deliver growth regulators, perhaps nutrients and carbohydrates, to roots and fruits to increase growth, development and fruit quality; much like an intravenous injection functions in an animal.
Among the questions we hope to investigate are:
- When, what kind of, and what amount of therapeutics can be applied by NATI?
- At what frequency?
- What type of citrus tree: cultivar, age, infected, healthy is the best for treatment by NATI?
- Can we kill the bacterium? How and when to assess a change in bacteria titer after treatment?
- When will become available and be economically feasible for growers?
Figure 1. Distribution of rhodamine (red dye; 1%) applied by NATI in various tissues (left) of grafted and non-grafted young citrus plants grown in the greenhouse (right). Photos taken 2 weeks after the treatments. Treatments and tissues observed are indicated. Yo = year-old.
Figure 2. Projected automated delivery system (ADS); an ATV with extendable arm with NATI and the cover placement systems on the arm guided onto the tree trunk (upper panel), and closeup of NATI and cover placement system (panel below).
Acknowledgement
The United States Department of Agriculture National Institute of Food and Agriculture (USDA-NIFA) Grant # 2019-70016-29096.
For more information, please visit this project's dedicated website:
https://swfrec.ifas.ufl.edu/programs/citrus-path/automated-delivery/