Citrus Greening Disease (Huanglonbing, HLB):
Introduction: Citrus greening disease or Huanglonbing, HLB, is a bacterial plant disease that is spread form one citure tree to the next by a small insect called the Asian citrus pysllid. The bacteria phloem-limited Liberibacter asiaticus, is a gram negative bacteria that traels throughout the tree’s system and begins to estroy the fine roots of the tree (and thus the tree’s vascular system), making the tree unable to maintin its tree canopy. As the disease progresses, the tree’s leaves begin to yello on one side more than the other and becomes as smaller, the tree produces small and lopsided fruit, and the fruit prematurely drops from the tree. If left untreated, the tree eventually dies. (Schweighofer, US 9,894,899).
Huanglongbing (HLB), also known as citrus geening, yellow shoot, or yellow gragon, is a bacterial disease affectivng citrus plants. In Florida, HLB is a major contributor to the decline in orange production. Roy (US 2019/0059392)
HLB or citrus greening disease was first reported in southern China in 1919, but it has been suggested to have originated in Africa. The diasese is now found in about 40 different Asian, African, North and South American countreis and has recently become a serious threat in Florida, California, Louisiana, Texas and Brazil, all of which are major citrus production locations. (Rouseff, US 2010/0074972)
Huanglongbing (“yellow dragon” in Chinese) is a disease of citrus that has taken on great importance in Mexico. It is also known by the initials HLB and the English word Greening or Ex-Greening. The disease is very destructive, because it causes total losses in citrus product. HLB symptoms include but are not restricted to leaf yellowing, reduced production of fruits and practically, the absence of seeds. (Xoconostle Cazares, US 2016/0186201).
Definitions:
—Biopesticides: is defined by the US Environmental Protection Agency (EPA) to be pesticides derived form natural materials and categorizes them as either biochemical pesticides, containing substances that control pests by nontoxic mechanisms, microbial pesticides, consisting of microorganisms that typically produce bioactive natural products (BNPs), or plant-incorporated protectants with activity produced by plants because of added genetic material. (Yang, US 2023/0309565)
–Phloem: is the vascular tissue in plants that conducts sugars and other metabolic products downward form the leaves. The HLB bacteria metabolizes the sugar that would otherwise be transported through the phloem to other parts of the plant, leading to stunted growth and eyllowing of plant parts. Roy (US 2019/0059392)
Bacteria associated with HLB :
Citrus greening disease is caused by the phloem-limited fastidious prokaryotic alpha-proteobacterium Candidatus Liberibacter spp., Ca. africanus, and Ca. L. americanus. Rouseff, US 2010/0074972)
It has been determined that HLB is casued by a group of bacteria that form a coherent clade, generally called Candidatus Liberibacter, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus, and Candidatus Liberibacter americanus. (Xoconostle Cazares, US 2016/0186201)
The HLB bacgteria a phloem-limited fastidious prokaryotic alpha-proteobacterium Candidatus Liberibacter spp. (CLas or often termed Las), most notably Ca. L. asiaticus, Ca. L. africanus, and Ca. L. americanus. Ca. L. asiaticus, and its verctor Diaphorina citri, also known as Asian Citrus Psyllid (ACP) are the major cause of HLB disease in North America. (Roy (US 2019/0059392))
In 2004, when HLB was seen for the first tiem in the Americas and more precisely Brazil, two liberibacter species were identified: (i) a new species, Ca. L. americanus (Lam), infecting most of the affected trees, and (ii) the known Asian liberibacteri, Las, present in a minority of trees. All three citrus liberibacters are uncultured and phloem-limited. That is, these bacteria live in plants exclusively within living plant phloem cells. Las is the most widely distributed by far. (Gabriel, US 2016/0278376)
—Candidatus Liberibacter asiaticus:
—Candidatus Liberibacter psyllarous:
—Phytoplasma:
Vectors:
Two pysllids, Diaphorina citri Kuwayama and Trioza erytreaei, are known to vector HLB. Rouseff, US 2010/0074972)
One of the vectors of the Candidatus Liberibacter spp bacteria cuasing HLB is Diaphorina citir Kuwayama (Hemiptera: Psyllidae); this insect has an embryonic period ranging from 9.7 days at 15 degrees C to 3.5 days at 28 degrees C; the eggs are placed at the end of the tender shoots of the plant, on and between young folded leaves, appearing frequently in large numbers in the same twig. (Xoconostle Cazares, US 2016/0186201)
Citrus Suseptible:
Among the citrus that are susceptible to HLB infection, the more severe symptoms occur in the Mexican lime, orange (Citurs sinensis), Mandarin orange (Citurs reticulata), and grapefruits (Citrus reticulataxCiturs paradisi) while less severe symptoms are present in limes (Citrus limon) and tangerines (Citrus paradisi), Citrus limonia, Citrus Limetioides. (Xoconostle Cazares, US 2016/0186201)
Treatment:
Most treatments involve a combination of spraying an insecticide like carbaryl, imidacloprid and aldicarb to kill the asisn citures psylli and micronutrients such as manganese on boron directly on the elaves. Potassium salicylate can also be appllied. However, studies have not shown that enhanced foliar nutrition does anything to counteract the disease. As soon as the treatment is stopped, the symptoms return. Additionally, the insecticides can find their way into consumer products like orange juice and pose a risk to consumer health. (Schweighofer, US 9,894,899)
—Antibiotics:
Other treatments require injecting an antibiotic like tetracycline and penicillin into the trees. Sometimes the tetracycline is used alone or followed by the penicillin. Other times the tetracylcine is combined with soluble copper or zinc sulfate. Treatment usually occurs two or more times at regular intervals and comprises trunk integrity. Injection requires dirlling a hole inot the tree about half the depth of the tree’s trunk diametr, installing an injection screen into the hold, and coupling a supply hose to the screw. After treatment the hold is sealed. Drilling might be a good way to get antibiotics and insecticides into the phloem and xylem of a tree. However, it is labor intensive, wakens the trunk and leaves the trunk vulnerable for antoehr bacteria, fungus, or insect to enter the trunk. (Schweighofer, US 9,894,899)
To control HLB the use of tetracycline has been reported, which interferes with protein synthesis in bacteria or of penicillin, which inhibits the synthesis of peptidoglicane, a structural component of the bacterial cell wall. Although the result was marked reduction in symptoms, the use of bacteriostatic tetracylcine was discarded because of the phytotocity produced thereby; besides, the use of antibiotics in plants and animals for human ocnsumpton is not recommended. (Xoconostle Cazares, US 2016/0186201)
—RNAi:
Hunter (US 2017/0211082) discloses that a dsRNA generated form D. citri trehalase gene is effective in reducing fitness and/or survival of D. citri. Thus, genetically altered plants expressing the dsRNA and plants to which dsRNA solutions are applied increase D. citri mortality and reduce D. citri infestation. With reduced D. citri population, the spread of microorganisms for which D. citri is a vector is reduced. Such microorganisms include, but are not limtied to C. Liberibacter speceis, including: CLas, CLam and CLaf.
El-Mohtar (US 2025/3034991) discloses that high levels of NPR1 protein is correlated with HLB tolerance or resistance. Citrus trees genetically modfied to overexpress Arabidopsis NPR1 can create tolerance to HLB. Howeer, these trees are genetically modifed (GM) and thus have to go through a long approval process. Thus a pnael of negative immune regulators were knocked down to thereby increase citrus NPR1 protein levels. The method invovles introducing into a citrus plant a viral vector engineered to produce an RNA intefering (RNAi) inducer targeting a negative immune regulator CsNPR3.
—Grafting with HLB tolerant/resistant cirus:
Grafting is a technique which includes joining of tissues from two or more different plants to form one composite plant. A vascular connection between the two plant segments is formed at the site of the graft union such that transport of water and/or nutrients occurs between the joined plant segments to enable growth of the composite plant. An upper part of a first plant, for example a scion, can be grafted onto a lower part of a second plant, for example a rootstock, to form the composite plant. Raveh (US 2024/0365720)
Raveh (US 2024/0365720) discloses a a method of producing an HLB tolerant citurs which includes grafting an HLB tolerane or resistant citrus interstock on an HLB susceptible or toelrant citurs rootstock.
—Extracts:
–-Canberry composition: Roy (US 2019/0059392) discloses a method of treating or inhibiitng infection of a plant by contacting the plant with an amount of a cranberry compoisition effective ot inhibit the plant pathogen.
–Oak Leaf extract: Cano Mogrovejo (US 2021/0186031) disclsoes compositions and methods for suppression in HLB in a subject having HLB (or otherwise for treatment of symptoms of HLB, in a subject in need thereof. The compositions inclue oak leaf extract which is applied to and around citrus plants having or suspected of having HLB.
–Control Psyllid infestation:
—-RNAi:
Interfering RNA (iRNA) reers to a double stranded RNA molecule that mediates RNA inteference (RNAi). At elast one strand of the duplex or double stranded region of a siRNA is substantially homologous to a target RNA molecule. The strand complementary to a target RNA molecules is the “antisense strand”, the strand homolocougs to the target RNA molecule is the “sense strand” and is also complementary to the RNAi antisense strand. RNAi may also contain additional sequences such as linking sequences, or loos and other folded structures. Brown (US 2024/0043864)
Brown (US 2024/0043864) discloses vectors taht include sequences for controlling psyllid infestation of plants by RNA interference (RNAi) and transgenic plants transformed with these vectors. In some embodimetns, the dsRNA includes a sense region with at least 90% sequence identity to a sequence that inclues at least 15 consecutive nucleotides and an antisense region that includes a second sequence complementary to the sense region.