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Combating Citrus Greening Disease

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ARS combats citrus greening disease (also known as Huanglongbing, or HLB) through disease detection, prevention, and mitigation research. Citrus greening represents the greatest threat to the $3.35 billion U.S. citrus industry. It is caused by a bacterial pathogen, Candidatus Liberibacter asiaticus (CLas), which is spread by the Asian citrus psyllid. Since the psyllid’s discovery in Florida in 1998, the industry has lost 60 percent of acreage and closed about 80 percent of juice plants and packinghouses. The disease has spread to Texas, California, Georgia, Arizona, and Louisiana. The following advancements in FY 2019 highlight ARS’s ongoing citrus greening response efforts. Hyperlinked accomplishment titles point to active parent research projects.

Canine detection of citrus greening in California to mitigate an impending statewide epidemic. HLB epidemics continue to spread worldwide and devastate the citrus industry. The key to mitigating HLB is early detection and rapid response. ARS researchers in Fort Pierce, Florida, have trained 20 dogs to detect HLB shortly after a tree has been infected (i.e., within 2 to 4 weeks after infection). The prior gold standard used to detect HLB, polymerase chain reaction (PCR), requires months to perform. The dogs can also detect infection with 99 percent accuracy, whereas the accuracy of PCR detection is approximately 30 to 35 percent, because even though the assay is good, it is difficult for humans to select infected tissue from a tree with some 200,000 leaves. In 2018–2019, California growers, the California Department Food and Agriculture (CDFA), industry leaders, and regulatory personnel concluded that the canines were more than 92 percent accurate in detecting infection even under highly variable and inhospitable conditions. The canines are being integrated and deployed by CDFA to detect asymptomatic incipient infections of HLB for early response (tree removal). Simulations indicate how the impending epidemic is greatly mitigated by incorporating canine detection teams in early detection practices.

New citrus trees for U.S. growers. Huanglongbing (HLB) disease has been devastating to the Florida citrus industry and severely threatens citrus production in other parts of the United States. The use of tolerant rootstocks and scions can be effective in ameliorating disease effects. ARS researchers in Ft. Pierce, Florida, have released three HLB-tolerant citrus rootstocks that produced sweet orange trees with improved health, fruit yield, and fruit quality over multiple years in an HLB-endemic environment. ARS researchers have also released the first citrus scion cultivar, called ‘US SunDragon’, that has good fruit quality and tolerance to HLB disease. ‘US SunDragon’ is being widely tested in Florida as a breeding parent, for niche fruit, and for home-owner plantings. Initial tests of juice quality show promise for inclusion of ‘US SunDragon’ in orange juice blends. The new tolerant rootstocks and scions will allow continued profitable production of citrus in the presence of HLB.

New treatment for citrus greening. Many bacterial pathogens in plants are difficult to target because they are protected by biofilms. A set of novel morpholino chemicals—small pieces of DNA that are antisense (i.e., oriented in the opposite direction of normal DNA)—were discovered by ARS scientists in Fort Pierce, Florida, to move through the biofilm containing the bacterium that causes citrus greening disease, thus killing the bacteria. Now patented by ARS, this strategy, which also worked in potato against Zebra chip disease, represents a new means to protect fruit, nut trees, and vegetables from numerous important plant diseases.

Improved protocol for detecting citrus greening infections. Detection of citrus HLB pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) infection triggers regulatory action and affects management decisions by citrus growers, so early detection is a critical component of HLB management. Many diagnostic methods and protocols have been developed, including official protocols used by the Animal and Plant Health Inspection Service (APHIS). However, detection of CLas in citrus trees is challenging in the absence of HLB symptoms because samples collected from CLas-infected trees often test negative for infection when tree is actually infected. This is known as a false negative. Research conducted by ARS scientists in Fort Pierce, Florida, determined a hierarchical sampling strategy that, at the tree level, reduces the probability of false negative diagnoses of CLas. The hierarchical sampling scheme is based on where CLas is most likely to be found in a tree. Results demonstrated that this new sampling method can detect CLas infections within 24 hours after infection occurs. The results also have been shared with APHIS to improve official protocols for CLas infection detection and monitoring in citrus groves.

A new sensitive method to detect low concentration samples of CLas. Clas, which is vectored by the Asian citrus psyllid, is the bacterium associated with the devastating citrus disease huanglongbing (HLB). Eradication of Clas inoculum sources is the first line of defense against the spread of HLB but erratic distribution and low titer of the pathogen in citrus limits efficacy of diagnosis using real-time polymerase chain reaction (qPCR) to detect the targeted Clas 16S rRNA gene. ARS researchers in Parlier, California, along with a scientist in the Central California Tristeza Eradication Agency, developed duplex real-time PCR and droplet digital PCR (ddPCR) tests to definitively detect low-titer Clas samples using two different genetic targets (16S and RNR) in the Clas chromosome. Sensitivity of the duplex assay was shown to detect down to two DNA copies per typical PCR reaction volume. Using low-titer (marginally positive) samples of Clas in leaf tissue and ACP, detection of Clas was greater with the RNR than the 16S gene target. Therefore, the duplex ddPCR described above can be used to confirm questionable results based on the regulatory standard 16S target.

Citrus greening strains in southern California have different origins. ‘Candidatus Liberibacter asiaticus’ (CLas) is a bacterium associated with huanglongbing (HLB) that was first detected in Florida in 2005 and in California in 2012. HLB has devastated citrus throughout Florida and has now been detected in more than 1,494 infected citrus trees in urban southern California. The only way to control HLB is to remove infected trees. Repeated insecticide application to kill the Asian citrus psyllid (ACP), the insect that serves as a vector for HLB, is neither economically, environmentally, nor biologically sustainable. ARS researchers in Parlier, California, along with scientists from the California Department of Food and Agriculture, the Animal and Plant Health Inspection Service, and South China Agricultural University, examined genomic diversity among 10 Clas strains from southern California by next-generation sequencing and detected prophages in all Clas strains. The California strains formed four prophage groups associated with different collection sites and were more closely related to strains in Asia rather than Florida, which indicated that the Clas pathway of entry was from independent introductions at multiple times. This information is important to growers in formulating HLB management strategies for different pathogen strains and for regulatory agencies in prioritizing and optimizing pathogen interdiction surveys.