Citrus greening disease



Citrus greening disease or yellow dragon disease (calque of abbr. HLB) is a disease of citrus caused by a vector-transmitted pathogen. The causative agents are motile bacteria, Liberibacter spp. The disease is transmitted by the Asian citrus psyllid, Diaphorina citri, and the African citrus psyllid, Trioza erytreae, also known as the two-spotted citrus psyllid. It has no known cure. It has also been shown to be graft-transmissible.

Three different types of HLB are currently known: the heat-tolerant Asian form, and the heat-sensitive African and American forms. The disease was first described in 1929 and first reported in South China in 1943. The African variation was first reported in 1947 in South Africa, where it is still widespread. Eventually, it affected the United States, reaching Florida in 2005. Within three years, it had spread to the majority of citrus farms. The rapid increase in this disease has threatened the citrus industry not only in Florida, but the entire US. As of 2009, 33 countries have reported HLB infection in their citrus crop.

Symptoms
HLB is distinguished by the common symptoms of yellowing of the veins and adjacent tissues (hence the "yellow dragon" name given by observing Chaozhou farmers as early as the 1870s ); followed by splotchy mottling of the entire leaf, premature defoliation, dieback of twigs, decay of feeder rootlets and lateral roots, and decline in vigor, ultimately followed by the death of the entire plant. Affected trees have stunted growth, bear multiple off-season flowers (most of which fall off), and produce small, irregularly shaped fruit with a thick, pale peel that remains green at the bottom and tastes very bitter. Common symptoms can often be mistaken for nutrient deficiencies; however, the distinguishing factor between nutrient deficiencies is the pattern of symmetry. Nutrient deficiencies tend to be symmetrical along the leaf vein margin, while HLB has an asymmetrical yellowing around the vein. The most noticeable symptom of HLB is greening and stunting of the fruit, especially after ripening.

Transmission
HLB was originally thought to be a viral disease, but was later discovered to be caused by bacteria, carried by insect vectors. HLB infection can arise in various climates and is often associated with different species of psyllid insects. For example, citrus crops in Africa become infected under cool conditions as the bacteria are transmitted by the African citrus psyllid Trioza erytreae, an invasive insect that favors cool and moist conditions for optimal activity. Citrus crops in Asia, however, are often infected under warm conditions as the bacteria are transmitted by the Asian citrus psyllid Diaphorina citri.

The young larval stage is the most suitable for acquisition of ca. L. asiaticus by the Asian citrus psyllid Diaphorina citri, and some cultivars show greater efficiency in transmitting the disease to the vector than others. Temperature also shows a great influence in the parasite-host relationship between the bacteria and the insect vector, affecting how it is acquired and transmitted by the insects.

The causative agents are fastidious phloem-restricted, Gram-negative bacteria in the gracilicutes clade. The Asian form, ca. L. asiaticus is heat tolerant. This means the greening symptoms can develop at temperatures up to 35 °C. The African form, ca. L. africanus, and American form, ca. L. americanus, are heat sensitive, thus symptoms only develop when the temperature is in the range 20–25 °C. Although T. erytreae is the natural vector of African citrus greening and D. citri is the natural vector of American and Asian citrus greening, either psyllid can in fact transmit either of the greening agents under experimental conditions.

Distribution
Distribution of the Asian citrus psyllid that is a vector of the citrus greening disease, is primarily in tropical and subtropical Asia. It has been reported in all citrus-growing regions in Asia except mainland Japan. The disease has affected crops in China, India, Sri Lanka, Malaysia, Indonesia, Myanmar, the Philippines, Pakistan, Thailand, the Ryukyu Islands, Nepal, Saudi Arabia, and Afghanistan. Areas outside Asia have also reported the disease: Réunion, Mauritius, Brazil, Paraguay, and Florida in the U.S. since 2005, and in several municipalities in Mexico since 2009    On March 30, 2012, citrus greening disease was confirmed in a single citrus tree in Hacienda Heights, Los Angeles County, California. The first report of HLB in Texas occurred on January 13, 2012, from a Valencia sweet orange tree in a commercial orchard in San Juan, Texas. Prospects are bleak for the ubiquitous backyard citrus orchards of California as residential growers are unlikely to consistently use the pesticides which provide effective control in commercial orchards.

The distribution of the African citrus psyllid includes Africa, Madeira, Saudi Arabia, Portugal, and Yemen. This species is sensitive to high temperatures and will not develop at temperatures greater than 25 °C. It is also a vector of the African strain of huanglongbing (Candidatus Liberibacter africanus), which is also sensitive to heat. This strain of HLB is reported to occur in Africa, (Burundi, Cameroon, Central African Republic, Comoros, Ethiopia, Kenya, Madagascar, Malawi, Mauritius, Reunion, Rwanda, South Africa, St. Helena (unconfirmed), Swaziland, Tanzania, Zimbabwe), Saudi Arabia, and Yemen. The disease was not reported in the EU as of 2004

History
The effort to culture Candidatus Liberibacter asiaticus (CLas), a vasculature-restricted citrus pathogen associated with citrus greening disease, has been a significant challenge in plant pathology. Despite several attempts, CLas has not been successfully established in pure culture, remaining elusive compared to other fastidious bacteria associated with citrus diseases. Over the past decade, transient cultures of CLas have been reported, marking incremental progress toward its sustained cultivation. Notably, cocultivation with Propionibacterium acnes facilitated CLas growth in vitro, highlighting the potential for mutualistic bacterial relationships in supporting CLas's nutritional and chemical needs. Further experiments achieved viable CLas cultures for weeks using commercial grapefruit juice and biofilms, though these did not constitute independent cultivation. Attempts to culture CLas on Liber A medium suggested pathogenicity in citrus, yet this claim was met with skepticism and has not been reproducible. Recent efforts involving coculture with phloem-associated microbiota indicated possible growth through DNA quantification, but direct viability measures were lacking. The research underscores the complexity of CLas's nutritional and environmental requirements, suggesting that association with specific microflora is essential for its growth. Despite these advances, the cultivation of CLas remains a challenging frontier, necessitating further studies for reproducible and sustainable culture methods.

Limitations
Unlike many bacteria, CLas demonstrates a specialized metabolic reliance on a diverse range of nutrients directly sourced from its host environments. Remarkably, CLas bypasses traditional glycolytic pathways for energy production, likely due to the absence of key glycolytic enzymes, and instead leans on alternative metabolic routes, including a dependence on the tricarboxylic acid (TCA) cycle for processing organic acids—a crucial carbon source found abundantly within the phloem sap of its plant hosts The bacterium's nutrient acquisition strategies extend to the assimilation of both proteinogenic and nonproteinogenic amino acids, as well as choline, through specialized transport systems, despite its inability to synthesize all amino acids de novo. This nuanced interaction with its environment highlights an evolved adaptation to extract and utilize host-derived nutrients, even in the face of its reduced genomic capacity. Furthermore, CLas’s exploitation of host nucleotides and cofactors through specific transport proteins underlines its sophisticated survival strategies within the host's microenvironments.

Environmental conditions also significantly impact CLas's culturability. The bacterium favors slightly acidic conditions, thriving in the naturally low pH found within citrus phloem sap and in vitro media such as commercial grapefruit juice. Additionally, CLas exhibits limited aerobic respiration capabilities, a result of lacking a terminal oxidase complex, and shows a preference for microaerophilic conditions that reflect the oxygen levels of its natural phloem habitat. These specific requirements complicate efforts to culture CLas outside its host, as the replication of these conditions in vitro is challenging. Genetic factors further constrain the cultivation of CLas. The bacterium’s genome has undergone significant reduction, losing pathways and enzymes crucial for independent growth. For example, the absence of the glyoxalase pathway, necessary for mitigating methylglyoxal toxicity, and the lack of biosynthetic pathways for essential lipids, necessitate the supplementation of culture media with specific nutrients or cofactors. Moreover, the presence of bacteriophage genes within CLas adds complexity, potentially affecting its pathogenicity and culturability. The combination of these unique growth requirements and genetic limitations underscores the inherent challenges in cultivating CLas, necessitating innovative culturing approaches to overcome these hurdles and advance our understanding of citrus greening disease. A report by Zheng et al, (2024) entitle: Towards the completion of Koch's postulates for the citrus Huanglongbing bacterium, Candidatus Liberibacter asiaticus. in Horticulture Research 11(3):1-7, describes establishing CLas cultures. This opens the door to a wide range of research experiments on effective CLas microbicides that were previously not possible.

Model system
Liberibacter crescens has emerged as a valuable model system for studying CLas. Unlike CLas, L. crescens can be cultured axenically and does not require a plant or insect host, offering insights into the genus's early evolutionary stages. This system has been instrumental in functional genomic studies of CLas genes, allowing researchers to explore CLas physiology and pathogenicity mechanisms in a controlled environment. Moreover, the ability of L. crescens to form biofilms under modified culture conditions provides a unique platform for studying biofilm formation, a critical aspect of CLas's lifecycle in insect vectors.

Despite the advances with L. crescens and the theoretical potential of cell culture infection models and bacterial cocultures, the axenic culturing of CLas remains a significant challenge. The development of CLas transformation protocols, which has been successful with L. crescens but not yet with CLas, is considered a critical step forward. This approach, whether through direct transformation or leveraging L. crescens as a surrogate, holds promise for unlocking the complexities of CLas biology and ultimately devising effective strategies to combat citrus greening disease.

Control
Some cultural practices can be effective in managing this disease. Cultural methods include antibacterial management, sanitation, removal of infected plants, frequent scouting, and most importantly, crisis declaration. Tracking the disease will help prevent further infection in other affected areas and help mitigate more local infections if detected early enough. The Asian citrus psyllid has alternative hosts that may attract psyllids to citrus plants in the vicinity such as Murraya paniculata, Severinia buxifolia, and other plants in the family Rutaceae.

No cure for citrus greening disease is known, and efforts to control it have been slow because infected citrus plants are difficult to maintain, regenerate, and study. Ongoing challenges associated with mitigating disease at the field-scale include seasonality of the phytopathogen (Liberibacter spp.) and associated disease symptoms, limitations for therapeutics to contact the phytopathogen in planta, adverse impacts of broad-spectrum treatments on plant-beneficial microbiota, and potential implications on public and ecosystem health.

No naturally immune citrus cultivars have been identified; however, creating genetically modified citrus may be a possible solution, but questions of its acceptability to consumers exist. A researcher at Texas AgriLife Research reported in 2012 that incorporating two genes from spinach into citrus trees improved resistance to citrus greening disease in greenhouse trials. Field tests by Southern Gardens Citrus of oranges with the spinach genes in Florida are ongoing.

A resistant variety of mandarin orange called 'Bingo' has been bred at the University of Florida. Other varieties can have a partial tolerance to the disease.

Antibiotics
Researchers at the Agricultural Research Service of the United States Department of Agriculture have used lemon trees infected with citrus greening disease to infect periwinkle plants in an effort to study the disease. Periwinkle plants are easily infected with the disease and respond well when experimentally treated with antibiotics. Researchers are testing the effect of penicillin G sodium and biocide 2,2-dibromo-3-nitrilopropionamide as potential treatments for infected citrus plants based on the positive results that were observed when applied to infected periwinkle. In June 2014, the USDA allocated an additional US$31.5 million to expand research combating citrus greening disease.

Certain antibiotics, specifically streptomycin and oxytetracycline, may be effective in the fight against citrus greening disease and have been used in the United States but have been banned in Brazil and the European Union. In 2016, the EPA allowed use of streptomycin and oxytetracycline on orchards with citrus fruits like grapefruits, oranges and tangerines in Florida on an emergency basis, this approval was expanded and broadened to other states for oxytetracycline in December 2018. Further expansion of medically important antibiotics is proposed by the EPA but opposed by the FDA and CDC, primarily as antibiotic resistance can be expected to develop and affect human health.

Peptides
University of California scientists have discovered a peptide that prevented and treated citrus greening disease in greenhouse trials;, it is being tested in field trials. The university entered into an exclusive license with Invaio to develop an enhanced injectable version of the product.

Antisense oligonucleotides
Researchers from the Agricultural Research Service, USDA at the U.S. Horticultural Research Laboratory in Fort Pierce, FL along with University of Florida Collaborators have developed two types of antisense oilgos (FANA and Morpholino's), and demonstrated efficient delivery into citrus trees, potato and tomato plants. The oligonucleotide FANA (2′-deoxy-2′-fluoro-arabinonucleotide) RNA-targeting technology into citrus trees and potato plants for management of bacterial pathogens and arthropod pests. The FANA ASO technology is a single nucleotide strand of 20–24 nt in length that incorporates 2′F- chemically modifications of nucleotides, along with a phosphorothioate backbone and modified flanking nucleotides, in their structure called “gapmers,” produced by AUM LifeTech., Inc. These unique modified structures of FANA “triggers” enables gymnotic activity that self-delivers into cells, moving systemically in treated plants and insects, with significant suppression of their RNA targets. Reported is the FANA suppression of two plant-infecting bacterium Candidatus Liberibacter asiaticus, CLas (in citrus trees), and C. Liberibacter solanacearum, CLso (in potato and tomato) ]

Morpholino's: These researchers demonstrated that PPMOs moved systemically in plants, fruit trees, and grapevines, such that they could suppress CLas in infected citrus trees and the psyllid vectors. Furthermore, the PPMOs designed to endosymbiotic bacteria of the psyllid vectors, can reduce psyllid populations by targeting and suppressing the insects endosymbionts, the bacteria which are essential for psyllid survival. Morpholino's must be covalently linked with a charged molecule or peptide, such as a cell-penetrating peptide, CPP, to enter bacteria (both Gram-positive or Gram-negative). After the MOs are conjugated to a carrier, they are referred to as PPMO, CPPMO, PMO, or similar. For more protocols, references at GeneTools, LLC, website (http://www.gene-ools.com/morpholino_antisense_oligos). Short PPMOs of 14–18nt are used to target bacteria, using a sequence, referred to as the external guide sequence, EGS, that when complexed with the target RNA modifies the shape to resemble a tRNA precursor which renders the target RNA susceptible to cleavage by ribonuclease P (RNase-P).

Cover crops
Some success has been reported using a cover crop strategy. The citrus trees were not free of the disease bacteria, yet a healthy soil environment allowed them to produce fruit and remain profitable.