The infection, citrus yellow vein disease, was discovered 64 years ago in Riverside and has never been seen elsewhere in the world. Decades later, UC Riverside researchers have finally unraveled the associated pathogen's genetic codes — a significant step toward harnessing its unique properties.
A paper describing this work was published recently in the journal Frontiers in Microbiology. It opens the door to testing whether this apparently benign infection could be used as a vehicle to transport antibacterial and antiviral agents into citrus plants' vascular systems, where infections usually take place.
Citrus crops face a highly uncertain future due to Huanglongbing, also known as citrus greening disease. In the past decade, the disease has caused a 72% decline in oranges used for juice, and a 21% decrease in the American fresh citrus fruit market. Growers in other parts of the world are similarly affected, and it continues to spread unabated.
Though there are promising treatments and disease-resistant hybrids being developed for Huanglongbing, none are yet commercially available.
Cells use RNA to convert the information stored in DNA into proteins that carry out different functions. Yellow vein disease is associated with small, independently mobile RNA, also called iRNA, which spreads through a plant's vascular system. This spreading mechanism could be a new way to send treatments for Huanglongbing or other diseases into plants.
The story of this promising research starts in 1957 with Lewis Weathers, a UC Riverside plant pathology professor.
“He found four limequat trees with beautiful, bright veins on their leaves, almost fluorescent yellow,” said Georgios Vidalakis, a plant pathology professor at UCR, UC-ANR cooperative extension specialist, and principal investigator on the new paper.
“That color was recognized as a disease, and samples of it were deposited at the Citrus Clonal Protection Program disease bank where it was waiting for us to study decades later,” Vidalakis said.
Based on Weathers' experiments, it did not appear as though the disease is carried by any animal or other microorganism, Vidalakis said.
“We think it was introduced into the limequats in a single event, and once those plants were destroyed, it never spread to other citrus in California.”
Researchers have learned, however, how the disease spread between cells in infected plants. The iRNA disguises itself with plant proteins that lets it pass through cellular connective tissue. This ability to travel inside citrus trees may allow the iRNA to send protective molecules into noncitrus plants as well, including grapes, olives, and cacao.
Yellow vein disease iRNA is also surprisingly small, even for a microscopic organism. Plant viruses typically have four to 10 genes on an average, each with at least one function. The iRNA of this disease has only one functional gene.
“The iRNA is amazing because it's able to manipulate plant cells to help it replicate, despite having only one functional gene,” explained Kiran Gadhave, a UCR microbiologist, corresponding author of the paper, and lead researcher of the iRNA project. “In addition to its potential therapeutic value, it's just a scientific curiosity. This is as small as it gets.”
Though they believe the pathogen to be benign, the research team is doing additional testing to make sure it won't affect fruit quality or quantity, tree height, or any other markers of health. Symptoms in greenhouse-grown trees were mild. Now this is being tested in a field trial in the living laboratory at the UCR Agricultural Experiment Station AgOps.
One day, iRNA could be used alongside gene editing technologies like CRISPR, in which cells are trained to recognize and destroy nucleic acids from invading plant pathogens. There are challenges with both methods that need to be overcome first, like managing unintended effects, costs, and efficient delivery.
“The common limitations of both approaches can be overcome by matching their strengths, the same way we integrate different disease management solutions,” Gadhave said.
Progression of citrus yellow vein disease, starting from healthy (left) to infected (right). (Gerardo Uribe/UCR)
Citrus greening, also called Huanglongbing (HLB), is devastating the citrus industry. Florida alone has experienced a 50 to 75 percent reduction in citrus production. There are no resistant varieties of citrus available and limited disease control measures.
Some scientists think it is possible that orange juice could one day become as expensive and rare as caviar. In an effort to prevent this, three plant pathologists at the University of California-Berkeley and United States Department of Agriculture conducted research into ways to boost citrus immunity and protect the valuable fruit against citrus greening.
Because the bacteria that causes citrus greening cannot be grown in a lab, scientists have to find novel ways to conduct experiments. The University of California-Berkeley/USDA team looked at many different strains of the bacteria that cause citrus greening to see if they could identify peptides (a compound of two or more amino acids) that would trigger immune responses.
"This was a long list, so we narrowed it down by selecting small peptides that were a bit different in their peptide sequence, which might imply that the bacterium had made those sequence changes so that they wouldn't be recognized by the plant immune system," explained Jennifer D. Lewis, group leader of the research team. "Then we further narrowed that list to peptides from strains that caused disease in citrus."
Through this research, they showed that two peptides could trigger immune responses in multiple plant species, including citrus. These peptides may play a role in preventing or reducing yield loss from citrus greening.
According to Lewis, "We thought it was particularly interesting that some of the peptides predicted to elicit a response, could actually trigger immune responses in multiple plant species. This suggests that the immune response to these peptides is conserved across species."
RIVERSIDE, Calif. (http://www.ucr.edu) -- Researchers at the University of California, Riverside have made an important step in understanding the molecular mechanism of huanglongbing (HLB), a destructive disease that is a serious threat to the citrus industry worldwide.
HLB, also known as citrus greening disease, has devastated groves in Asia, South America, and the southern U.S., costing the Florida citrus industry billions of dollars since 2005. Since 2012, the disease has been spreading in California's residential areas, prompting serious concerns about the state's commercial citrus groves.
HLB is associated with a species of bacteria called Candidatus Liberibacter asiaticus (CLas), which is transmitted by a tiny insect called the Asian citrus psyllid (ACP). Infected trees show leaf mottling, deformed and discolored fruits, and premature fruit drop. There is no cure for the disease and once a tree is infected it typically dies within three to five years.
An important step to developing HLB-resistant citrus varieties is to better understand how the bacterium infects trees and causes disease.
"Citrus trees, like all plants, have complex immune systems to prevent pathogenic infection, so the question is 'how does the CLas pathogen evade that immunity so it can cause disease?' said Wenbo Ma, a professor of plant pathology in UCR's College of Natural & Agricultural Sciences.
In a paper published Monday in Nature Communications, a team led by Ma reported a significant breakthrough in understanding the disease mechanism of HLB. They discovered that the bacterium secretes a protein--called Sec-delivered effector 1 (SDE1)--that helps infect plants. SDE1 works by attacking specific proteases--called papain-like cysteine proteases (PLCPs)--that could otherwise help the citrus trees resist infection.
"In the diseased trees we studied, the protein levels of some PLCPs were increased, presumably attempting to combat the bacterial infection," Ma said. "However, the bacterium fights back, by inhibiting the enzymatic activity of PLCPs through SDE1."
Ma said since scientists cannot grow CLas in the laboratory, the team used a surrogate system comprising the model plant Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae that was genetically engineered to produce SDE1. Using this system, they show that SDE1 promotes bacterial infection. This study is among the first to describe the molecular tactics employed by CLas to colonize citrus plants.
"This study represents an important step towards better understanding the HLB disease mechanism, which will help us develop novel approaches to control this unstoppable disease," Ma said.
The team is now investigating the molecular details of how SDE1 suppresses citrus PLCPs with the aim to use the CRISPR gene editing system to modify the proteases to become resistant to the inhibitory effects of SDE1.
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The title of the paper is 'An effector from the Huanglongbing-associated pathogen targets citrus proteases.' In addition to Ma, authors from UC Riverside are Kelley Clark, Simon Schwizer, Eva Hawara, Deborah Pagliaccia, Liping Zeng, Jinxia Shi, Pengcheng Wang and Yinsheng Wang. Other authors include Jessica Yvette Franco, Thomas Liebrand and Gitta Coaker from UC Davis; Zhiqian Pang and Nian Wang from the University of Florida; Fatta B. Gurung and Veronica Ancona from Texas A&M University; and Renier van der Hoorn from the University of Oxford.
Funding was from the United States Department of Agriculture (USDA) National Institute of Food and Agriculture.
IMAGE: An orange tree that is infected with huanglongbing. Leaf symptoms begin on one or a few branches and slowly spread to the rest of the tree. view more
Extension Specialist in Biological Control, Department of Entomology, University of California Riverside, CA 92521mark.hoddle@ucr.edu
Well, what a dramatic title, and it is certainly an unabashed rip off from Winston Churchill too! However, many growers in California view the threat posed to the long term viability and profitability of citrus by Asian citrus psyllid (ACP) and huanglongbing (HLB) as a war! ACP is a small sap sucking insect that is native to the Indian subcontinent and it has emerged as a global threat to citrus because it vectors a bacterium (Candidatus Liberibacter asiaticus [CLas]) that causes a lethal and incurable citrus disease, huanglongbing (sometimes referred to as citrus greening). Trees displaying HLB go into decline and some varieties may die in as little as 5-8 years post-infection. Symptoms may include chlorotic leaves (Fig. 2) and premature fruit drop. Fruit that are retained become misshapen as they develop, ripen irregularly (hence the common name citrus greening), and have a bitter taste. Florida's “war” with ACP and HLB started around 1998 when the psyllid was first found, then in 2005 the first citrus trees with HLB symptoms were discovered. Some economic estimates of the ACP-CLas epidemic in Florida suggest that more than 8,000 jobs have been lost, production has declined by 23%, and revenues have dropped by 16% .
ACP was first detected in southern California in 2008, and in 2012, the first citrus trees with HLB symptoms were found in Hacienda Heights in Los Angeles County. Subsequently, HLB has been confirmed in San Gabriel, also in LA County. Detections of ACP in the San Joaquin Valley (SJV) are increasing, especially around Bakersfield, and these finds make the largest citrus production areas in California highly vulnerable to CLas. One response to the ACP invasion has been to go after the vector in urban residential areas where ACP enjoys a relatively good life on backyard citrus trees that are treated infrequently with pesticides. The intention of this urban-residential-ACP control program was to knock back psyllid numbers to low levels with the goal of reducing the rate of spread of ACP and CLas through urban areas and into commercial citrus production zones. Vector reduction have been attempted in two different ways; spraying insecticides and biological control. The California Department of Food and Agriculture aggressively pursued residential spraying of citrus during the initial stages of the ACP campaign in southern California, but this quickly became unsustainable due to cost and the speed at which ACP was spreading through the urban landscape. Spraying is still used in areas were ACP populations are small and localized, such as those being found in parts of the SJV.
The second approach has been to run a classical biological control program targeting ACP. Classical biological control introduces natural enemies from the home range of the pest into the invaded area with the goal of establishing these beneficial agents so that they permanently suppress pest populations to less damaging levels. This approach necessitated searches for ACP natural enemies in the native range of the pest, a process referred to as foreign exploration. Because ACP has a huge putative native range, foreign exploration was conducted in Punjab Pakistan (Fig. 3), an area with ~70% climate match with the major citrus production areas in the SJV. A good climate match, in theory, should result in natural enemies that are pre-adapted to California's hot dry summers and cool damp winters. From September 2010 to April 2013, six expeditions searching for ACP natural enemies were conducted in Pakistan. The University of Agriculture Faisalabad (UAF) was the home base for this project. Collaboration with UAF Faculty was excellent, and the Vice Chancellor of UAF, Dr. Iqrar Khan, is a UC Riverside graduate in plant pathology, and Mike Roose was Iqrar's major professor!
Two species of parasitoid, Tamarixia radiata and Diaphorencyrtus aligarhensis, were found attacking ACP in Pakistan. Both species were returned to the Insectary and Quarantine Facility at UC Riverside. Mandatory host range and host specificity tests were conducted over a 2-3 year period and results indicated that both species likely posed little environmental risk to California and it was concluded that both species offered significant benefits because of their ability to parasitize and feed on ACP nymphs. Both of these processes kill ACP nymphs. Consequently, USDA-APHIS issued release permits and CDFA took over the mass rearing of these parasitoids. As of June 2016, more than 3 million Tamarixia and 170,000 Diaphorencyrtus have been released in southern California.
Initial results of the ACP biocontrol program are promising. Tamarixia appears to have established widely in southern California, and in combination with other species of natural enemies, especially generalist predators like lacewing and syrphid fly larvae, significant reductions in ACP populations have been documented. Diaphorencyrtus lags behind Tamarixia because it was the second parasitoid out of the quarantine pipeline, but multiple recoveries have been made at about 60% of sites where this species has been released. However, the impact and rate of spread of Diaphorencyrtus and whether it can compete successfully with Tamarixia is unknown and the subject of Citrus Research Board (CRB) and USDA-MAC sponsored research.
Although the major biocontrol efforts have centered on ACP in residential areas in southern California, the focus of the biocontrol program, especially with respect to Tamarixia, is beginning to face northwards, and battle lines are being drawn around and through Bakersfield and Tulare. The redirection of effort has been the subject of intense discussion at recent CRB BioControl Taskforce meetings. The emerging consensus is that movement of biocontrol agents into urban areas north of the Tehachapi mountains is needed. It is highly likely that in late 2016 or early 2017 Pakistani mercenaries will be released for the first time into the SJV to hunt down ACP.Asian citrus psyllid, Diaphorina citri, is an invasive pest in California. Immature psyllids, the nymphs (A) acquire the HLB-causing bacteria when feeding on infected plants. (B) Adult psyllids can carry bacteria between trees and inoculate healthy plants when feeding (Photos by Mike Lewis, Center for Invasive Species Research, UC Riverside)
University of California Cooperative Extension Ventura County 669 County Square Drive, Suite 100 Ventura, CA 93003 Phone: 805.645.1451 Fax: 805.645.1474
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