Open access peer-reviewed chapter

Devious Phloem Intruder Candidatus Liberibacter Species Causing Huanglongbing: History, Symptoms, Mechanism, and Current Strategies

Written By

Palaniyandi Karuppaiya, Junyuan Huang and Muqing Zhang

Submitted: 08 April 2022 Reviewed: 28 April 2022 Published: 15 June 2022

DOI: 10.5772/intechopen.105089

From the Edited Volume

Current and Emerging Challenges in the Diseases of Trees

Edited by Cristiano Bellé

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Abstract

Huanglongbing (HLB) or greening is a devastating phloem-intruding bacterial disease that generates various symptoms in leaves and fruits, threatening the global citrus industry. Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus, and Candidatus Liberibacter americanus are the causative agents of HLB in citrus-producing regions around many countries, and these proteobacteria are being vectorized by Diaphorina citri and Triozaerytreae. The lack of HLB-resistant citrus cultivars, the rapid spread of disease, and the fastidious nature of HLB-proteobacteria have made it difficult to mitigate HLB in the citrus field. There are numerous reports on the control of HLB disease using thermotherapy, chemotherapy, plant defense activators, brassinosteroids, and nanoemulsions. However, there is no evidence of such applicability of the methods mentioned above to complete the elimination or suppression of the pathogen to control HLB disease. We aim to provide an overall picture of HLB disease, its distribution, causal organism, pathogenic mechanism, and current and future strategies for combat against citrus Huanglongbing disease. This review may prompt the researchers toward an integrated and environmentally sustainable methodology for the mitigation/elimination of HLB pathogens.

Keywords

  • citrus
  • Huanglongbing
  • Candidatus Liberibacter
  • control strategy
  • psyllid

1. Introduction

Citrus fruits are the most predominantly produced fruits worldwide. The citrus species, Rutaceae family, is one of the major fruit crops in the world, which has provided an immune-enhancing source of vitamin C, nutrients, and medicinal value since ancient times [1]. Citrus crops are cultivated in more than 135 countries worldwide [2]. Worldwide citrus production is estimated at over 124.3 million tons annually [3]. Cultivated commercial citrus plants, consisting of rootstock and scion varieties, have a significant impact on scion growth, fruit quality, yield, and tolerance to biotic and abiotic stresses [4, 5]. Therefore, the selection of rootstock may make a significant contribution to the success or failure of the planting process [2]. However, various biotic and abiotic stresses impede citrus production worldwide, among which Huanglongbing is one of the significant pernicious diseases devastating the citriculture industry in the last few decades. Citriculture industries in Asia, Africa, and America have suffered massive economic losses due to the devastating Huanglongbing (HLB) malady [6].

Citrus HLB (Yellow dragon disease or citrus greening) is one of the highly ruinous diseases in citrus species caused by proteobacteria Candidatus Liberibacter species. The casual organisms of HLB have not been successfully cultured on axenic culture to date, and the prevalence of the HLB pathogen in citrus plants was evaluated using a diagnostic polymerase chain reaction (PCR) technique. Diaphorina citri Kuwayama (Asian citrus psyllid (ACP)) and Triozaerytreae (African citrus psyllid (AfCP)) transmit HLB disease from one citrus plant to another and also feed on many other species of the Rutaceae family [7]. The ACP resides in warm and humid zones and is most prevalent in Asia, the Indian subcontinent, Saudi Arabia, Reunion, and Mauritius. Now, ACP has also spread to South and Central America, such as Brazil, USA, and Mexico [8]. AfCP thrives in cold weather and is sensitive to the sweltering climate. AfCP resides in Africa, Cameroon, South Africa, Yemen, Madagascar, and Madeira Island [9]. HLB was first identified as a significant issue of unknown disease in citrus by farmers in southern China at the end of the nineteenth century [10]. HLB was first known a century ago as Citrus “Dieback” in India and “Yellow Dragon shoot Disease” in China, with a clear impact on citrus production in many countries, followed by South Africa, the Philippines, Indonesia, Thailand, Brazil, and the United States [11].

Citrus are susceptible to HLB, that is, nearly all commercial citrus and some citrus relatives. Poncirus trifoliate citrus, some P. trifoliate hybrids, and a few lemon varieties are considered more HLB tolerant [12]. The most efficient and sustainable strategy against citrus HLB is breeding resistant citrus cultivars. However, conventional citrus breeding is a long-term process that takes about 20 years to develop a new variety. Further, breeding efficiency is affected by gametophytic cross-incompatibility, heterozygosis, pollen-ovule sterility, apomixis, seedlessness, graft incompatibilities, polyembryony, and unstable characteristics [13]. Genetically engineered resistant citrus varieties are yet to be available for commercial cultivation due to the lack of acceptance of GMOs from farmers and consumers. It will, therefore, take many years to develop a promising resistant cultivar against HLB [14].

Many strategies to combat HLB were initiated, such as thermotherapy, antibiotics, plant defense initiators, pesticide, vector control management, chemotherapy, nanotechnology, and a transgenic approach [15, 16]. Beta-lactams, tetracyclines, and silver nanoparticles have obtained better results against HLB malady [17, 18]; however, the emergence of antibiotic resistance to microorganisms and indirect effects on human health and the environment is a significant and increasing risk that certainly restricts the use of antibiotics at the field level [18]. However, no effective strategies to eliminate or repress the HLB pathogens have been identified. This review attempts to provide an overall picture of HLB disease, distribution, casual organism and its pathogenic mechanism, and vector control management, and post the current and possible strategies to mitigate/combat HLB malady in the field.

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2. Incidence, distribution, symptomology, and detection of citrus Huanglongbing

2.1 Incidence of Huanglongbing

HLB (also known as Yellow Dragon/shoot disease) was first identified as an unknown disease in citrus trees by citrus farmers in Guangdong Province, China, at the end of the nineteenth century [19], but studies suggest that HLB most likely originated in Taiwan in 1870 where it was known as Likubin (“Drooping disease”) [20, 21]. Later, the HLB spread to other parts of China; by 1935, it had become a severe disease of citrus species [21]. Like HLB, dieback was first described in the central parts of India in the middle of the eighteenth century [22]. At that time, it might have been limited, but HLB was recorded in Assam in the eighteenth century and, by 1912, was a devastating disease in Bombay, India. However, the Citrus tristeza virus might cause this disease. Raychaudhuri [23] exhibited that dieback was the same as HLB. African greening disease was first identified in a sweet orange orchard in parts of South Africa in 1929 [24]. Outside of China, HLB was known as the “Greening” disease in South Africa, where extensive research was conducted in the 1950s. In Indonesia, the HLB disease was first noticed in the 1940s and is called the “citrus phloem degeneration” disease [25]. Reinking, in 1919, first described this disease in English as yellowing and leaf mottle of citrus noticed in China. According to International nomenclature rules, the name “Huanglongbing” was considered the official name by citrus pathologists at the 13th conference of the International Organization of Citrus Virologists in China [26]. “Huanglong” means yellowing of the shoot, as well as the yellow dragon (the symptom appears almost like a yellow dragon over the infected trees) and “bing,” which means disease in Chinese [10]. Since the discovery of HLB, it has been named differently worldwide [27]. HLB was known outside under the name “citrus dieback” in India [23], “mottle leaf disease” in the Philippines [28], “vein phloem degeneration disease” in Indonesia [25], “yellow branch,” “blotchy mottle,” or “greening” disease in South Africa [29].

2.2 Geographical distribution

Globally, HLB has been considered one of the significant threats to citrus commercial and sustainable production. HLB was confirmed in citrus-producing regions of various countries, such as Nepal, Bangladesh, Thailand, Pakistan, Japan, Vietnam, Cambodia, Laos, Malaysia, Central African Republic, Comoros, Ethiopia, Kong Hong, Kenya, Madagascar, Malawi, Mauritius, Saudi Arabia, Reunion, Rwanda, Yemen, Zimbabwe, Somalia, Tanzania, Swaziland, and various region of United States of America including California, Florida [7, 27]. HLB has been reported in 24 countries and territories in East, South, Southwest Asia, East, and South Africa. Since then, it has been widely spread in other Asian, American, and African countries [27].

2.3 Symptomology

HLB symptoms are more evident in cold weather conditions than in hot seasons [30]. It is difficult to specify the period between when the citrus tree is affected by HLB and the onset of disease symptoms. It will exhibit in different parts of the plants or only in infected sectors when it eventually manifests symptoms. It is, therefore, difficult to diagnose and control at the early stage of HLB disease [31]. The HLB-infected tree exhibits symptoms in various parts of the plant depending on the stage of infection. If infection occurs soon after propagation, the entire tree gets affected and turns yellow all over the canopy, which leads to a decline irrevocably. Both the symptoms and the causative organisms were restricted to the infected sector in the event of later infection [27]. Only the infected sector will exhibit symptoms in the case of citrus trees affected by HLB, while the remaining parts will show normal growth and good-quality fruits. The symptoms observed on the HLB-affected tree include a heavy drop in the leaf and out-of-season flushing and blooming. Chronically, HLB-affected trees displayed stunting growth, twig dieback, sparse yellow foliage, or severe fruit drop [24]. The initial stage of HLB is vein yellowing [32], and the secondary level includes (infected leaves) small, upright with various chlorotic patterns similar to that caused by nutrient deficiency, such as zinc, sulfur, iron, boron, manganese, and calcium [33, 34]. In severe cases, the leaves were utterly void of chlorophyll, except for rounded green spots located on the leaves at random places [24]. The most accurate diagnostic symptom for HLB is that the infected fruits are small, lopsided, and taste bitter and salty. HLB-affected trees with premature shedding of green fruit drops while remaining on the tree, in which fruits with yellow halo-like lesions were staying green on the shaded side, hence the name “greening” [7, 34]. Root systems are developed in severely infected trees that exhibit poorly formed roots with few fibrous roots due to undernourishment [24] and repression of new root growth and rootlets decay [10].

HLB disease is challenging to diagnose based on symptoms, particularly during the early stages of the disease. Numerous symptoms of HLB might occur, and citrus trees are often caused by other diseases or nutrient deficiencies that may lead to similar symptoms [11, 30, 35]. Symptoms could be aggravated by other pathogens being coinfected. Several reports from Asian countries postulated that HLB-affected citrus trees are commonly coinfected with the Citrus tristeza virus (CTV) [7]. Interestingly, some CTV isolates protect trees against HLB infection [36]. Blotchy mottle leaf is a principal diagnosis of HLB that could be misinterpreted with other diseases, such as stubborn citrus disease caused by Spiroplasma citri, a severe infection of CTV phytophthora root rot, zinc deficiency, and waterlogging. Furthermore, it can also be confused with symptoms of leaf-related stress and mineral deficiency [37]. Early stages of citrus blight are also associated with the symptoms of zinc deficiency [38]. For these confusing symptoms of the disease, an unequivocal diagnosis technique is needed for HLB disease.

2.4 Method of HLB detection

Early identification and isolation of Canditatus Liberibacter species-infected trees are effective management approaches used to limit the spread of HLB from invading HLB disease-free citrus orchards in local and international trade [39]. Visual examination is one of the most commonly employed approaches for detecting citrus HLB disease. Traditionally, early detection of HLB disease relied primarily on various symptoms in the field, such as blotchy mottle leaf, yellow shoot, aborted seed, and lopsided fruit with green color remaining at the stylar end [40]. Nevertheless, this approach is highly affected by subjective interpretation, diagnostic errors can be higher than 30%, and other biotic and abiotic stress-related problems may worsen diagnosis. HLB symptoms might be confused with diseases such as Citrus Tristeza Closterovirus, Phytophthora infection, citrus blight, and specific nutrient deficiencies [41]. Thus, the availability of advanced technologies that enable early and rapid detection of HLB pathogens is crucial [42]. Currently used methods for the diagnosis and confirmation of HLB disease include serology, enzymatic assay, enzyme-linked immunosorbent assays (ELISA), transmission electron microscopy, DNA probes, conventional polymerase chain reaction (PCR), quantitative PCR (qPCR), Fourier transform infrared spectroscopy (FTIR), and mid-infrared spectroscopy. Pereira et al. [43] developed a method for early diagnosis using X-ray fluorescence. The laser-induced breakdown spectroscopy (LIBS) combined with chemometric strategies is used to predict the condition of orchard plants infected with Canditatus Liberibacter species successfully. However, these methods did not provide early diagnosis except for the LIBS method. Recently, Tran et al. [44] reported a sensitive and selective label-free biosensor that combines the physical and chemical advantages of carbon nanomaterials such as single-walled carbon nanotubes (SWNTs) in a field-effect transistor (FET)/chemiresistor architecture with selective antibodies against Sec-delivered effector 1 (SDE1), a secreted protein biomarker, for the detection of HLB. Detailed HLB detection techniques have recently been reviewed [42, 45].

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3. Causal agents of citrus Huanglongbing

The bacterium associated with citrus HLB was Candidatus Liberibacter species, which belongs to the alpha-proteobacteria determined by the 16 s ribosomal DNA sequences and the operon [9]. Proteobacteria associated with HLB disease in citrus are successively referred to as Candidatus Liberibacter asiaticus (Las) found in the majority of HLB-affected countries, Candidatus Liberibacter africanus (Laf) limited to African countries, and Candidatus Liberibacter americanus (Lam) limited to America [15]. How Candidatus Liberibacter bacterium established its association with citrus species remains unclear.

Scientific classification of Candidatus Liberibacter.

Kingdom: Bacteria.

Phylum: Proteobacteria.

Class: Alphaproteobacteria.

Order: Rhizobiales.

Family: Rhizobiaceae.

Genus: Candidatus Liberibacter.

3.1 In vitro culture of Candidatus Liberibacter species associated with HLB

The isolation of Candidatus Liberibacter species, causing HLB in an artificial culture medium, was a primary target for many researchers. Davis et al. [46] attempted to isolated Candidatus Liberibacter asiaticus in a culture medium from young angular green shoots from HLB-affected trees. A growth film appeared on the bottom of the tube containing broth AD medium. After single-colony isolation, Las and the actinobacteria closely related to Propionibacterium acnes remained together. Thus, Las was not isolated in axenic culture. Moreover, actinobacteria are prevalent residents of citrus and psyllids, whether Las is present. Sechler et al. [47] successfully cultivated a single colony of all three Candidatus Liberibacter species from HLB-affected leaf midveins and petiole sap in a new medium designated Liber A. The isolated cells were ovoid to rod-shaped, 0.3 to 0.4 by 0.5 to 2.0 μm, often with fimbriae-like appendages. They isolated two Las and one Lam strains from non-inoculated tissues of inoculated trees and seedlings 9 and 2 months later.

3.2 The pathogenic mechanism of Candidatus Liberibacter

Candidatus Liberibacter species are gram-negative, phloem-restricted bacterium associated with the pernicious disease of citrus HLB. Although Candidatus Liberibacters have been cultivated in artificial media, traditional molecular and genetic analyses have been difficult to perform owing to declining viability in culture [46, 48]. This difficulty has significantly limited efforts to comprehend the mechanisms of Liberibacter virulence. To date, most insights into the mechanisms of Liberibacter pathogenesis have been acquired through genomic analyses of Liberibacter sequences, host plant transcriptomic, proteomic, and metabolomic data associated with Liberibacter infection, and studies involving surrogates such as Sinorhizobium and E. coli, and expression in planta [15]. Evidence suggests that Liberibacters species associated with HLB live solely within the phloem tissues of host citrus plants [15]. Las bacterium resides inside a sieve tube and companion cells [47, 49]. The relatively consistent symptomology among various symptom-expressing hosts is one of the hallmarks of diseases caused by Liberibacter species [15].

3.2.1 Liberibacter secretion system and effector protein

The secretome of a pathogenic bacterium represents an array of molecules that play offensive roles during colonization, among which effectors are an important class of proteins capable of suppressing defense and/or manipulating host physiology [50, 51]. Interestingly, Las contain type I secretion systems (T1SSs) and a complete general secretory pathway (Sec), but lack other secretion systems (T2SS and T3SS) [15, 52], which play a significant role in extracellular pathogenic attacks on plant and animal host [16].

Liberibacter genome analyses found a complete T1SSs system in Las and Laf, but not in Lam [15]. Genes encode for serralysin and hemolysin; a T1SSs effector protein has been identified in Las and Laf genomes [53]. Serralysin is a metalloprotease secreted by gram-negative bacteria to inactivate peptides and antimicrobial proteins produced by the host plant. Las bacterium might use serralysin to degrade antimicrobial proteins in the host as its defense mechanism. This degraded protein is used for growth and metabolism by the Las bacterium as a carbon and nitrogen nutrient [16]. On the other hand, the hemolysin gene has been identified in all sequenced Liberibacters, which play an essential role in bacteria survival in the host plant. Las-produced hemolysin triggers ion leakage and water molecules from the host cell that lead to host cell apoptosis [16, 54].

The Secretary pathway (Sec) or Sec-translocon facilitates these effector proteins’ transports outside the cytoplasm membrane vital for bacterial viability. The Sec machinery also secretes essential virulence factors in some plant-pathogenic bacteria [15]. Candidatus Liberibacter species have a general secretory pathway, which may lead to the secretion of effector proteins [55]. Since Candidatus Liberibacter species are phloem-resided bacteria, there is an inference that the bacteria secrete effector proteins directly into the cytoplasm of the host cells and modulate their physiology [56]. The effector protein CLIBASIA_05315 was located in transgenic citrus chloroplasts, resulting in leaf chlorosis and plant growth retardation [57]. Several research groups are currently focusing on identifying and characterizing the effector proteins of Candidatus Liberibacter species, and it is expected that we will have an improved view of this pathogenic mechanism of bacteria in a few years.

3.2.2 Lipopolysaccharides

Lipopolysaccharides (LPS), also known as endotoxin, are critical components derived from the outer membranes of gram-negative bacteria consisting of lipid A, an oligosaccharide core, and an O-antigen. LPSs are involved in outer membrane functions that are crucial for bacterial growth, survival against antimicrobial chemicals, and virulence, particularly within a host-parasite interaction. Lipid A is highly conserved, then the oligosaccharide core and O-antigen [15, 16]. LPSs are classical activators of defense responses in plants during plant-pathogen interaction [58]. Las bacteria use gene encoding active salicylate hydroxylase (SahA) to degrade salicylic acid (SA) and suppress plant defense mechanism. Intriguingly, the SahA gene is highly expressed in planta, while it is not expressed in psyllid vectors [56]. Las impedes SA-mediated defense responses in the phloem using its SA hydroxylase and maintains significant bacterial titer in citrus HLB disease progression over several years before the tree irrevocably declines. It is yet to be determined whether LPSs of Liberibacter cause callose accumulation in the phloem.

3.2.3 Flagella

The bacterial flagellum organelle, an intricate multiprotein essential for its rotational propulsion, promotes host colonization through adherence and induces plant immune modulation [15]. Las flagella have been reported to trigger host plant defense in planta as a pathogen-associated molecular pattern (PAMP) [59]. Microscopic studies found that flagella have not been observed in the Candidatus Liberibacter species that reside in the phloem in HLB-infected samples [11]. Despite the small size of the genome, genes associated with flagella biosynthesis have been identified in the sequenced Liberibacter genome [15, 16, 51, 52]. The genes fliF, flgI, and flgD expressed in flagellar assembly and the motB gene associated with the motor function were overexpressed in planta.

The flbT, an essential flagellin regulatory protein that acts as a regulatory checkpoint for flagellin gene expression, is found in the Las bacterial genome, whereas it is not in the Lam genome. The absence of flbT in the Lam genome results in no PAMP activation in planta [60]. Conversely, flgL, flgK, and fliE were overexpressed in psyllid [61].

3.2.4 Prophages

Several pathogenic bacteria harbor prophages or phage remnants integrated into their genome, encoding lysogenic genes that are proven or suspected virulence factors [59]. Las- and Lam-sequenced genome contains two potential prophages, Type 1 represents prophage SC1, and Type 2 represents prophage SC2. SC1 involved in the lytic cycle of forming phage particles. SC2 was implicated in the lysogenic conversion of Las pathogenesis [60, 62]. Type 3 prophage (P-JXGC-3) was identified in Las samples collected from Southern China. This prophage carries another bacterial defense system, such as a restriction-modification system (RM system) [63]. This RM system is fortified with endonucleases, which cleaves invading DNA that protects host DNA by altering specific sequences [64]. Type 1 and Type 2 prophages were not detected in the Las strain from Southern China. It is not clear whether these strains contain prophages or have unknown prophages. There are no comprehensive studies to describe the Las prophage repertoire [65]. Among strains observed in an extensive survey of Las isolates in China, it was typical for Las to have a single prophage, with Guangdong isolates harboring mainly the type 2 prophage, whereas isolates from Yunnan are dominated by the type 1 prophage [65]. The Las strain genome from Japan does not contain prophages [56]. Among the Las whole-genome sequences recently reported from different geographic areas around the globe, eight Las genomes contain extensive prophage sequences [63]. A survey of prophage prevalence in southern China revealed active prophage-phage interactions in the Las bacterial strains [63]. The exact function of the RM system has yet to be experimentally determined in Type 3 prophages. However, the lack of a prophage in many Las strains does not relate to the lack of HLB symptoms because Ishi-1 and the Guangdong isolates, which do not contain any prophages, induce similar HLB symptoms as isolates containing prophages [54, 65]. Overall, this evidence suggests that prophages contribute to bacterial virulence but are not required for Las pathogenicity.

3.3 Phloem dysfunction of HLB-affected citrus

Las bacteria reside within phloem and colonize sieve tubes [15, 16, 66]. Phloem dysfunction is a primary modification due to hyperactive differentiation of vascular cambium and hypertrophy of parenchyma cells surrounding the necrotic phloem pocket that may determine the development of HLB symptoms [32, 67]. HLB-associated Liberibacter secretes virulence factor and Sec-dependent effectors (SDEs) into phloem that stimulates HLB symptoms by interfering with either phloem or companion cell protein and genes of the host [15]. The secreted SDEs and virulence factors may interact with plastids, mitochondria, vacuole, and endoplasmic reticulum in the host phloem and target host genes and proteins to promote pathogen growth and disease development and suppress host immune responses [15]. Eventually, it leads to phloem malfunction in the host plant due to the Liberibacter virulence factors and SDE effects on sieve tubes and companion cells, which provide protein and transcripts to the sieve elements. Necrotic phloem was found in the HLB-infected plants due to starch (Figure 1) and callose deposition [32]. Callose accumulation was observed in sieve plates of Las-infected citrus [67]. Phloem dysfunction is generally associated with phloem sieve elements plugged with extensive deposition of callose and phloem protein 2 [67, 68], followed by phloem cell wall distortion and sieve element collapse [69]. Subsequently, photoassimilate transport was significantly blocked due to necrotic phloem [15, 16, 66, 68], which might result in substantial quantities of starch particles in almost all living cells of aerial parts, including phloem parenchyma and the sieve tube elements [32, 70]. The excessive accumulation of starch and zinc deficiency in chloroplast disrupts the thylakoid resulting in nonuniform loss of chlorophyll that triggers noticeable blotchy mottle appearance in the HLB-infected leaves [40, 70, 71]. The anatomical transverse section of HLB-infected leaf midrib exhibited phloem collapse with cell wall distortion and thickening in Valencia sweet orange and SB siblings [72]. In addition, hyperactive vascular cambium regenerates new phloem in the HLB-infected trees, consisting of assemblies of sieve elements, companion cells, and phloem parenchyma cells, but lacks phloemic fibers [72].

Figure 1.

SEM micrographs of transverse section of healthy and HLB-infected citrus petiole. A and B. Healthy plant; C and D. HLB-infected plants.

In addition to anatomical changes, several metabolic imbalances and genetic reprogramming are noticed in HLB-affected plants [57, 66]. Salicylic acid and downstream signaling play a key role in provoking plant defense mechanisms against biotrophic pathogens [73, 74]. Wang and Trivedi postulated that a protein with potential salicylate hydroxylase activity might convert salicylic acid into catechol [75]. Salicylic acid pathway depression was observed in HLB-susceptible citrus plants [76]. Based on the Candidatus Liberibacter and plant interactions mechanism literature, we suggest the pathogenic mechanism of Candidatus Liberibacter species associated with citrus HLB in the following model (Figure 2).

Figure 2.

Illustration of Candidatus Liberibacter virulence mechanisms in the plant.Candidatus Liberibacter species associated with HLB (red circles) live in phloem elements. Phloem is mainly liable for the distribution of the carbohydrate from the source to the sink. Nutrients are transmitted to the phloem either through the apoplastic pathway or the symplastic pathway. Candidatus Liberibacter species may secrete effector protein (sec-dependent effectors) and virulence factors (orange and blue circle, red triangles) into phloem sieve elements and companion cells to interfere with host target (genes and protein) that can cause cell necrosis, cell death, and phloem malfunction. Effectors or virulence factors may interfere with phloem organelles, such as mitochondria, plastids, or endoplasmic reticulum, to trigger cellular responses. Some effectors (SDEs) may directly or indirectly affect the expression of target genes. In addition, Candidatus Liberibacter species may trigger plant immune responses through pathogen-associated molecular patterns leading to cell death and callose accumulation, resulting in inhibition of phloem transportation. The presence of Candidatus Liberibacter species and its metabolic activity may interfere with the function of the phloem by interrupting the osmatic gradients and integrity of phloem transportation. Abbreviations: PMPs—Pathogen-associated molecular patterns; RFO—Raffinose family oligosaccharide.

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4. Transmission of citrus HLB

The graft transmitted HLB was due to a viral disease [77]. Soon afterward, similar opinions were put forward in South Africa, strengthened by the results of grafting trials showing that greening was inconsistently transmitted to healthy plants. Lin [21] confirmed that HLB was transmitted through grafting in China, thus establishing the causative agent as a pathogen. McClean and Oberholzer [78] confirmed the graft transmissibility of African greening in 1965. The pathogen is not easily transmitted to progeny trees propagated by buds from infected trees, possibly due to sieve tube necrosis and uneven pathogen distribution, but more transmission occurs if stem pieces are used. No infection could be obtained when material from apparently healthy sectors of diseased trees was used. In 1964, natural spread by exposing seedlings to insects in a HLB-affected orchard developed yellowing symptoms similar to greening [79].

Two insect vectors are responsible for the rapid transmission of citrus HLB from Las-infected citrus to healthy citrus species, Asian citrus psyllid D. citri in Asia and America, and the African citrus psyllid, Triozaerytreae in Africa. The acquisition feeding period is 30 min or longer, and the pathogen remains latent for 3–20 days. The inoculation feeding period is 1 hour or more [80].

Asian citrus psyllid is widespread around the world and found in hot and humid conditions and lower-lying areas in China, India, Myanmar, Taiwan, Philippine Islands, Malaysia, Indonesia, Sri Lanka, Pakistan, Thailand, Nepal, Ryukyu Islands (Japan), Afghanistan, Saudi Arabia, Reunion, and Mauritius [81]. Asian citrus psyllid firstly evolved in India [82], then spread in South America in the 1940s, invading Brazil, Argentina, and Venezuela, and then invaded the West Indies (Guadeloupe), Abaco Island, Grand Bahama Island, Cayman Islands, and the USA in the 1990s. In 2001, ACP was found in the Dominican Republic, Cuba, Puerto Rico, and Texas [83, 84]. Asian citrus psyllid has been reported more recently in many new Americas, including Mexico, Costa Rico, Belize, Honduras, and the states of Alabama, Arizona, California, Georgia, Louisiana, Mississippi, and South Carolina, USA [85].

African citrus psyllid (AfCP) thrives in cool and moist temperatures, at higher areas about 100 to 500 m above sea level. And it is sensitive to excessive heat and exists in Africa from the islands of the Indian Ocean through east and central Africa to South Africa, Saudi Arabia, Yemen, the northwestern region of the Iberian Peninsula, Cameroon, Kenya, Ethiopia, Zimbabwe, Tanzania, Malawi, Galicia, northern Portugal, Swaziland, Madagascar, Rwanda/Burundi, and Reunion [86]. Psyllid populations in Africa, Saudi Arabia, and Yemen might be able to adapt and settle under a wide range of environmental conditions, such as equatorial, arid, and warm temperate climates with varying temperatures and rainfall [86].

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5. Current strategies to combat citrus HLB

5.1 Vector control

5.1.1 Biocontrol of vector

Biocontrol uses natural enemies by import, augmentation, and conservation to control the population density of disease pathogens or pests in agriculture [87]. Asian citrus psyllid (Diaphorinacitri) was controlled using a practical method of import (from Asia) and free of Tamarixia radiata in Florida citrus groves [88]. Natural enemies, Diaphorencyrtus aligarhensis and Tamarixia radiata, were imported from Taiwan and Vietnam to Florida citrus orchard and released as a biocontrol agent for D. citri [89]. T. radiata became more widely established parasitoid wasps than its counterpart D. aligarhensis. In urban and suburban regions, the release of T. radiata could considerably benefit commercial citrus growers by reducing latent psyllid populations and preventing the further spread of HLB disease [87]. T. radiata has been firmly established in commercial citrus-producing countries, including Réunion Island [90], the Philippines [91], Indonesia [92], Guadeloupe [93], and the USA [94], where it is spread throughout the state [88]. It appeared inadvertently in Brazil, Venezuela, Mexico [95], Puerto Rico [83], and Texas [31].

In addition to wasps, many insects native to Florida are recognized as D. citri predators, including several ladybeetle and spiders [81]. Cyclonedas anguinea, Harmonia axyridis Pallas (Coccinellid beetles), and Olla v-nigrum Mulsant were the leading killer of nymphal psyllids. In addition to Ceraeochrysa, Hibanavelox (becker), spider, and Chrysoperla rufilabris Burmeister (lacewings), Tamarixia radiata (parasitoid) also contributed to the additional mortality in Florida citrus groves. Coccinellid beetles are considered one of the most important natural enemies of D. citri populations in central Florida. Besides this, intraguild predation (IGP) causes more than 95% of immature T. radiate mortality [96]. Van den Berg et al. [97] noted that the spiders are the critical predators of T. erytreae, followed by chrysopids, coccinellids, syrphids, hemerobiids, Hemiptera, and predatory mites in citrus groves under the control of an integrated management program. Adults and larvae of O. v-nigrum (Mulsant) preying on premature Asian citrus psyllid were noticed in citrus groves throughout Florida [98].

A range of fungi species was reported to infect Asian citrus Psyllid, particularly under humid conditions [81], including entomopathogenic fungi, Isaria fumosorosea, Lecanicillium lecanii, Beauveria bassiana, Capnodium citri, Cladosporium sp. nr. Oxysporum, Metarhizium anisopliae, and Hirsutella citriformis that were used against HLB vectors as biopesticides [99, 100, 101]. Isaria fumosorosea is reported to have great potential to control different insect pests [102]. H. citriformis conidia with synnemata produced in vitro and in vivo were subjected to adult D. citri exhibits an increased mortality rate [100, 103]. In a 2-year field investigation in citrus groves in Florida, adult D. citri (ACP) was killed by H. citriformis following the rainy season [104]. In laboratory conditions, the fungal strains of Isaria fumosorosea (ESALQ-1296) and Beauveria bassiana (ESALQ-PL63) accounted for 77.8 and 78.4% of adult D. citri mortality, respectively, while in semifield conditions, adult D. citri mortality rate was as high as 83.5% with ESALQ-PL63 and 80.6% with ESALQ-1296. During 1 year, the monthly use of these two fungal strains in commercial citrus groves exhibited adult D. citri mortality ranging from 96.1% in December 2011 to 57.8% in October 2012. In addition, this study found that the mortality rate increased under high humidity conditions [99]. Isariajavanica and Acrostalagmus aphidum were also identified as biopesticides against D. citri in China [105]. The use of fungal species, such as Metarhizium anisopliae, Cordyceps bassiana, and Isaria fumosorosea, was shown to decrease larger populations of nymphs than adults of D. citri in the Persian lime groves [106].

5.1.2 RNA interference for vector control

RNA interference, a process in which a double-stranded RNA exerts a silencing effect on the complementary mRNA, has become a powerful tool in entomology. Advantages, such as ease of use, specific targeting, and lack of environmental persistence, make RNAi techniques highly attractive for crop protection against many insect pests [107]. The main challenges in using RNAi-based pest control methods are compelling target gene selection and reliable delivery of dsRNA. The over-expression of dsRNAs in transgenic plants has induced RNAi in targeted insects [108, 109]. The transgenic approach in citrus, however, is slow and difficult. Hajeri et al. [110] targeted D. citri endogenous Awd (abnormal wing development disc) gene for silencing by using a CTV-RNAi vector, resulting in impaired wings in D. citri that would potentially limit the ability to fly and successful transmission of CLas bacteria between citrus trees in the field. In addition, decreased Awd gene in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. Taning et al. [111] postulated that a small dose of dsRNA (dsAK, dsSOD) administered through in Planta system (iPS) bioassay was sufficient to trigger the RNAi mechanism, causing significant suppression of the targeted transcript and increased mortality in ACP.

5.1.3 Horticultural mineral oil for vector control

Petroleum-based horticultural mineral oils (HMO) are a vital constituent of integrated management programs for many pathogens and several phytophagous arthropods pathogens that affect the productivity of fruits, vegetables, and ornamentals in the commercial cultivation field as well as greenhouse conditions [112]. Since the 1980s, HMOs have been employed to control mites and scales in China [113]. HMO controls citrus leaf miner, citrus rust mite, citrus red mite, red scale, chaff scale, spiny whitefly, and Asian citrus Psyllid in citrus [114, 115]. By lowering the number of HMOs used in treatment to 0.25 ± 0.5% and maximizing the number of sprays during each season, a significant level of pest control was achieved without the threat of phytotoxicity. The combined treatment with oils and Isaria fumosorosea showed that the survival rate of adult psyllids was lower than that of oils used alone [116]. Kumar et al. [117] postulated that the combined treatment of entomopathogenic Isaria fumosoroseaand HMOs dramatically reduced D. citri populations, where the maximum mean survival for D. citri was 12.5 ± 0.7 days. Similarly, Tansey et al. [118] revealed that mixes of insecticide and HMO application considerably decreased the populations of nymph and adult D. citri in Valencia orange groves in Florida. Conversely, Qureshi et al. [119] disclosed that HMO alone did not control D. Citri populations because the mean suppression of nymph and adults for more than 3 weeks was only 36 and 50%, respectively.

5.2 Las bacterial control

5.2.1 Antibiotics

Antibiotics are crucial for controlling bacterial diseases in fruit-bearing trees, vegetables, and ornamentals. Although antibiotics can be detected on plant surfaces using delicate analytical chemistry techniques for up to a month after application, their ability to inhibit bacterial growth is lost within a week [120]. In-plant disease control, nearly 40 antibiotics were screened; only streptomycin and tetracycline were used extensively in fruit trees [121]. The only commercially applied treatment for HLB was tetracycline, which is bacteriostatic rather than bactericidal, in Reunion Island’s orchards [122, 123]. Tetracycline was the only approved antibiotic injection in trees injected directly into the trunks of HLB-affected citrus trees in China, Indonesia, India, Taiwan, and South Africa during the 1970s [36, 117, 124]. Although the symptoms of HLB were considerably decreased, this antibiotic trunk injection method was not in practice owing to its phytotoxicity and labor costs. The use of penicillin-carbendazim antibiotics in citrus trees showed significant control of HLB disease. The antibiotic disadvantage is a reduction in the fruit size owing to phytotoxicity and the residues of the antibiotics in citrus fruits [125]. The development of therapeutic compounds and bactericidal agents to control devastating HLB could provide an additional solution for an effective integrated disease management program. However, other than selective antibiotics, nonselective bactericide is recommended for general use in most crops, particularly citrus [126]. The combination treatment of streptomycin with penicillin efficiently eliminated or repressed the Las bacterium compared with the separate administration of either antibiotic [126]. The treatment of penicillin combined with streptomycin also significantly reduced the bacterial titer of Las in greenhouse citrus plants. Kasugamycin and Oxytetracycline combination therapy via trunk injection significantly reduced HLB bacterial titer in the field. However, the combination of kasugamycin and streptomycin was not effective against the bacterium of Las [127]. Penicillin with oxytetracycline combination therapy has been more effective in controlling citrus pathogens [128] but may require annual treatment [20]. Among the 31 tested antibiotics, some were effective at reducing and eliminating Las bacterial titers in inoculated rootstock and the treated scions of citrus plants, such as ampicillin, carbenicillin, penicillin, cefalexin, rifampicin, and sulfadimethoxine [20]. Oxytetracycline has therefore been suggested to be used more frequently in combination treatment [129, 130] with penicillin or kasugamycin against HLB to control the progression of bacterial resistance and maximize the antibiotic efficacy against HLB pathogenic bacteria [131]. The Environmental Protection Agency (EPA) of the USA allows citrus growers to spray streptomycin and oxytetracycline as routine treatments in the citrus field several times per year [132]. Oxytetracycline (1 g/L) was delivered to leaves of HLB-infected trees through the foliar application, and oxytetracycline was found in all leaves, although at reduced levels than in the directly applied leaves [132]. However, the phytotoxicity of tetracycline should be considered [20]. Antibiotics tested to combat HLB malady are tabulated inTable 1.

S.NoAntibioticsWorking concentration (mg/L)EffectivenessPhytotoxicity
1Actidione25HighHighest
2Validoxylamine A100PartlyLess
3Zhongshengmycin100PartlyLess
4Amikacin sulfate100NoneNIL
5Gentamicin sulfate100NoneNIL
6Hygromycin B150PartlyNIL
7Kanamycin sulfate100PartlyNone
8Kasugamycin hydrochloride100NoneNIL
9Neomycin hydrate trisulfate50NoneNIL
10Spectinomycin dihydrochloride pentadrate20PartlyNone
11Streptomycin sulfate100NoneNIL
12Tobramycin20NoneNIL
13Ampicillin sodium100HighLess
14Carbenicillin disodium100HighLess
15Penicillin G potassium100HighLess
16Cefalexin100HighLess
17Vancomycin hydrochloride40NoneNIL
18Lincomycin hydrocloride100NoneNIL
19Cycloserine50PartlyNIL
20Rifamycin sodium50PartlyLess
21Rifampicin50HighLess
22Rifaximin50PartlyLess
23Colistinmethane sulfonate sodium20NoneNIL
24Polymixin B sulfate300NoneNIL
25Cinoxacin300NoneNIL
26Ciprofloxacin hydrochloride300PartlyNIL
27Sulfadimethoxine sodium100PartlyModerate
28Sulfamethoxazole100PartlyModerate
29Sulfathiazole sodium100PartlyModerate
30Chloramphenicol30PartlyLess
31Oxytetracycline hydrochloride100HighHighest

Table 1.

Antibiotics effectiveness against CLas bacterium and phytotoxicity.

5.2.2 Thermotherapy

Heat treatment or thermotherapy of planting material is a century-old disease control method that has proven effective against various pathogenic microorganisms. Thermotherapy, simple in principle, can eliminate the conserved pathogens depending on temperature/time regime and can cause mild injuries to the host during the treatment. Heat is mainly generated by water, vapor, or air [133]. The main advantage of thermotherapy treatment is that it is more environmentally friendly than harmful agrochemicals. Thermotherapy has proven to be an effective strategy against HLB that helps to enhance the vigor of citrus trees and promotes new root growth and development. The efficacy of thermotherapy against HLB pathogens depends on the temperature and citrus varieties [134]. Therapy could recuperate HLB-affected citrus plants by eliminating or suppressing Las bacterial titers at temperatures above 40°C [6, 134]. Candidatus Liberibacter asiaticus is a heat-tolerant phloem-limited bacteria that can withstand a temperature of about 35°C, while Candidatus Liberibacter americanus is heat-sensitive [135]. Thermotherapy could eliminate HLB pathogens from valuable horticultural trees associated with shoot tip grafting [136].

Lin opined on eliminating yellow shoot disease with water-saturated hot air treatment of graft wood 48–58°C with no loss of tissue viability [137]. In India, the thermotherapy of budwood at 47°C for 2 hours of diminished disease incidence, and more prolonged treatment eradicated the pathogen [138]. Heat treatment at temperatures around 38–40°C for 3 or 4 weeks killed HLB pathogens in young infected plants or citrus seedlings grafted with infected tissues [138, 139]. In South Africa, HLB-infected budwoods were treated with hot water baths at 51°C for 1 hour, 49°C for 2 hours, and 47°C for 4 hours, eliminating HLB pathogens with some loss of viability at higher temperatures [140]. In HLB-affected trees topped with polyethylene fiberglass sheets for 2 to 5 months, the number of diseased fruits decreased. However, this technique is not feasible for extensive use in citrus groves [27]. The HLB-affected citrus seedlings were continuously exposed to 40 to 42°C heat therapy for 7 to 10 days, significantly reducing titer or eliminating Las bacteria. This treatment can be helpful to combat HLB-affected plants in greenhouse and nursery settings [134]. Ehsani et al. [141] also postulated a decrease in HLB symptoms in groves of citrus trees after heat treatment. The combined thermo- and chemotherapy of sulfathiazole sodium or sulfadimethoxine sodium was more effective at 45°C than in thermotherapy alone, chemotherapy alone, or a combination of thermotherapy at 40°C and chemotherapy [142]. The temperature treatment at 45°C for 8 h per day for a week and a combination of ampicillin sodium, actidione, and validoxylamine A as a bark paint on grapefruits plant significantly reduced Las titer [143]. Two-year-old graft HLB-affected citrus reticulate treated with thermotherapy at 45°C and 48°C showed diminished HLB symptoms and Las titers 8 weeks after treatment in the greenhouse condition [144]. Commercial and residential citrus trees covered with portable plastic enclosures exposed to elevated temperatures through solarization showed vigorous growth in 3–6 weeks after treatment. Although commercial citrus trees showed Las after heat treatment, many trees generated extensive flushes and grew strongly for 2 to 3 years after therapy [145]. Inner bark heat treatment with 60°C–0.03 MPa-30s in 9-year-old citrus plants exhibited significantly reduced Las bacterial titer with vigorous plant growth from all treated HLB-affected trees [146]. Abdulridha et al. [147] reported that HLB-affected trees with canopy cover were treated with combined hot water and steam therapy at 55°C for 90 seconds. The temperature distribution inside the canopy cover was not uniform; the canopy temperatures were more significant than the trunk temperatures. The mobile thermotherapy treatment needs to be improved to increase the temperatures around the tree trunk to nearly the same temperature as a canopy. Vincent et al. [132] postulated that heat treatment from 43 to 54°C for no longer than 45 s showed adverse effects on citrus tree growth.

HLB is a systemic disease. Efficient elimination of Las bacteria from the entire citrus tree, including roots, is vital to managing the disease. The current thermotherapy challenge is that although adequately elevated temperatures can reach the above-ground areas of the plant, killing temperatures are unlikely to be attained at the roots where the temperature is mitigated by the soil [148]. Therefore, heat treatment is unlikely to reduce the populations of HLB pathogens in the roots, which then acts as a site for canopy reinfection during flushes. The efficacy of heat treatment in eliminating Las bacterial populations in underground roots must be enhanced to become a feasible part of integrated citrus HLB management [15]. To overcome this barrier, Hoffman et al. [134] suggest that heat treatment, coupled with chemotherapy in HLB-affected plants, can lead to a potential future strategy for controlling citrus HLB.

5.2.3 Plant defense activators to combat HLB

Trunk injection is an alternative target-precise technique for efficiently delivering plant protective chemicals in tree fruit crops. It harnesses the rapid transportation ability of the xylem that enables therapeutic compounds’ translocation and subsequent distribution into the canopy where plant protection is needed [149]. There has been limited research on the trunk injection of antibiotics and plant defense activators for better disease control. Several recent field studies have demonstrated the utility of trunk injection of bactericides and plant defense activators in disease management [150].

Treatments with β-aminobutyric acid (BABA), 2,1,3-benzothiadiazole (BTH), 2,6-dichloroisonicotinic acid (INA), ascorbic acid (AA), and the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DDG) plant defense inducers individually or in combination found effective in suppressing Las bacterial population in plants and sustaining fruit production to a certain extent. Treatment with BABA and BTH was the most effective in reducing the Las population in plant tissues compared with other plant defense inducers [151]. Hu and Wang proved that trunk injection of oxytetracycline in HLB-affected trees exhibited long-lasting suppression of Las populations. It also prevented the tree decline by promoting new growth without the disease [152]. Trunk injections of salicylic acid, potassium phosphate, acibenzolar-S-methyl, and oxalic acid in the HLB-affected tree significantly suppressed the Las titer and HLB disease progress [150].

Brassinosteroids (BRs) are a class of steroid hormones that regulate gene expression, growth, and developmental processes in response to biotic and abiotic stress [153]. The plant defense mechanism of brassinosteroids was mediated by leucine-rich repeat receptor kinase (LRR-RK) BAK1, which serves as a coreceptor for both microbe-associated molecular patterns (MAMPs) and steroid hormone [154], which binds to BRs and FLS2 eliciting microbe-induced immunity. BR treatment showed increasing disease resistance against many pathogens [6]. Canales et al. [155] postulated that applying epibrassinolide as a foliar spray in HLB-infected plants improved immunity against Candidatus Liberibacter asiaticus in greenhouse and field citrus plants. Candidatus Liberibacter asiaticus titer was markedly reduced in epibrassinolide-treated plants due to the enhanced defense gene expression in the citrus leaves. However, the molecular mechanism of BRs in plant responses under normal and environmentally challenging conditions has remained unclear [155].

5.3 Nanoemulsions to deliver chemicals against Las bacteria

HLB is caused by Las proteobacteria that reside in the phloem of infected citrus trees. It is, therefore, challenging to deliver effective compounds into the phloem through a foliar spray. The presence of wax, cutin, and pectin in plant cuticles prevents the effective bactericidal compounds from entering the phloem through a foliar spraying method. The use of chemical adjuvant enhanced the foliar uptake of agrochemicals [156, 157]. However, foliar spray treatment, including the combination of antibiotic PS and adjuvants in dimethyl sulfoxide and Silwet L-77, did not significantly impact the HLB-affected citrus trees [128]. Therefore, there is a need for candidate adjuvants, which can potentially increase the permeability of citrus cuticles to deliver antimicrobial compounds into citrus phloem.

Nanoemulsions or submicron emulsions are colloidal dispersion systems with average droplets size ranging from 50 to 1000 nm that has extensively studied for delivering chemical compounds. Nanoemulsions were pondered as thermodynamically and kinetically stable isotropic dispersions, composed of two immiscible liquids such as water and oil, stabilized by an interfacial film composed of an appropriate surfactant and co-surfactant to form a single-phase [158]. However, the approach efficacy relies on nanoemulsions droplet characteristics, such as low surface tension, tiny size, ample surface area, and low interface tension [159]. Our research group postulated that water in oil nanoemulsions containing ampicillin coupled with adjuvant Brij 35 was used as a foliar spray to enhance the permeability through the citrus cuticle into the phloem and more efficiently eliminated Las bacteria in HLB-affected citrus in planta [160]. Ampicillin showed the lowest phytotoxicity to citrus trees infected with Las bacteria [20]. However, the US Environmental Protection Agency (EPA) has not approved the commercial use of ampicillin in crops due to the development of resistant bacterial strains [160]. In another study, oil in water nanoemulsions was formulated using a spontaneous emulsification method, where five different antimicrobial compounds alone combined with Cremophor EL (viscous oil), acetone, and Span 80/Tween 80, which formed tiny droplets, were effectively applied to the bark for efficiently control HLB [161].

Silver nanoparticles (AgNPs) are one of the most investigated and used in agricultural science to enhance the yield and sustainable development of the crop. This has long been reported to have significant antibacterial, antifungal, antiviral, and pesticide effects. AgNPs are used as foliar sprays to prevent the development of rot, mold, fungi, and other plant pathogens [162]. Stephano-Hornedo et al. [18] evaluated the commercially available AgNPs to directly eradicate Candidatus Liberibacter asiaticus (CLas), responsible for HLB in the citrus field. The 93 HLB-infected citrus trees administered foliar and trunk injections of silver nanoparticles showed a remarkable reduction of 80–90% in bacterial titer by both methods than control. Compared with other effective treatments involving b-lactam antibiotics, the effectiveness of AgNPs is 3- to 60-fold higher when administered by foliar spray and 75- to 750-fold higher when injected via tree trunk. Thus, the silver nanoparticles could be a sustainable method for mitigating citrus HLB. However, AgNPs toxicity to a citrus tree and the environment needs to be warranted before its commercial use.

5.4 Transgenic approach to combat HLB

Globally, insect pests are responsible for significant crop losses through direct harm and transmission of plant diseases [163]. The best long-term alternative strategy for managing citrus HLB is to develop disease-resistant cultivars in commercial citrus production. Due to the lack of resistant cultivars, developing HLB-resistant plants by conventional citrus breeding is difficult. Resistance occurs in citrus relatives, such as kumquat, where its genetic background influences the quality and yield of the fruit [164]. In addition, conventional citrus breeding is labor- and time-consuming, and very costly as citrus species are polygenic, extremely heterozygous plants with a long juvenile phase. The genetic transformation approach is an essential strategy that would aid in incorporating disease-resistant genes into citrus cultivars to combat the HLB disease. The progression of citrus breeding through genetic transformation is still early, indicating a lack of molecular pathogenesis understanding of innate disease resistance in citrus [165].

Systemic acquired resistance (SAR), a natural plant defense response mechanism, has been well characterized in Arabidopsis thaliana. SAR entails signal molecule salicylic acid (SA) to activate defense mechanisms. In response to SA, the non-expression of pathogenesis-related gene 1 (NPR1) is translocated to the nucleus, where it triggers the expression of pathogenic related (PR) genes by interacting with TGA transcription factors, thereby provoking SAR [166, 167]. Arabidopsis mutants contain deficiencies in the NPR1 gene showing decreased PR gene expression induced by SA and SAR, leading to increased susceptibility to pathogens [167, 168]. Conversely, overexpression of the NPR1 gene in Arabidopsis increased the disease resistance to bacteria and oomycete pathogens. Interestingly, the over-expression of AtNPR1gene in most plant species does not provoke noticeable adverse effects on plant growth and development [169]. Thus, NPR1 is a target gene for the genetic transformation of nonspecific resistance in crop plants.

Dutt et al. [170] postulated that the overexpression of the AtNPR1 gene in Hamlin and Valencia orange cultivars resulted in trees with normal phenotypes, and exhibited increased resistance to HLB. Transgenic trees showed reduced disease severity, and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field. The phloem-expressed NPR1 gene was equally effective in increasing disease resistance by triggering several indigenous gene expressions involving plant defense mechanisms of signaling pathways. In addition to triggering resistance to HLB, the observed SAR response could protect citrus trees from other major fungal and bacterial diseases, such as black spots and citrus canker [170].

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6. Conclusions

HLB is one of the century-old diseases in the history of citrus pathology. The global spread of HLB disease causes economic loss in most citrus-producing countries. The causal agent of HLB, Candidatus Liberibacter, impedes understanding its pathogenic mechanism, fastidious nature, and unculturable in artificial conditions. Future research will focus on the isolation and pure culture of these proteobacteria. The rootstock and scion of some tolerant varieties have been noted and used by citrus growers in citriculture. These tolerant varieties have the potential to suppress the progression of HLB in Las-infected trees. The HLB management program recommends the intense psyllid control and removal of Las-infected trees in citrus groves. Citrus farmers focus on maintaining productivity in the HLB-affected trees by using plant defense activators, micronutrients, and fertilizers and paying more attention to water irrigation systems. Besides, thermotherapy is still an efficient methodology for eliminating and suppressing the causal agents in citrus scions and rootstock. Antibiotics alone or combined with other bactericides have also shown to be effective against citrus HLB. However, antibiotics need to evaluate their phytotoxicity before their commercial use. The combination of thermotherapy and antibiotics, plant defense activators, and thermotherapy provides controlled HLB management efficiency. The formation of nanoemulsion in water in oil (W/O) and/or oil in water (O/W) could offer a practical methodology for the targeted delivery of antimicrobial compounds to the phloem of citrus by foliar spraying method to control citrus HLB. In addition, transgenic orange cultivars over-expressing the AtNPR1 gene exhibited enhanced resistance to HLB. Transcriptome analysis between susceptible, tolerant, and resistant citrus varieties provides new insights into HLB tolerance by revealing defense-related genes, biological pathway signaling, hormones, transporters, carbohydrate metabolism, phloem-related genes, and secondary metabolism. In addition, some potential targets have also been identified, such as DMR6-like and NPR1-like genes for future HLB-tolerant citrus breeding [171]. Epibrassinolide as a foliar spray in HLB-affected plants improved the immunity against Candidatus Liberibacter asiaticus in the greenhouse and citrus field. However, further studies on the impact of eBL and nanoemulsion loaded with antibiotics in HLB-affected citrus plants are warranted to understand the complexity of citrus pathophysiology and fruit productivity. Researchers have investigated many control strategies to combat Candidatus Liberibacter species, but no effective management strategies have been developed. More studies are needed to investigate a sustainable and environmentally friendly strategy to control citrus HLB in the form of an antimicrobial agent in citrus groves. Meanwhile, biotechnological approaches such as transgenic, gene editing, and host-induced gene silencing may provide an unprecedented opportunity for long-term HLB management tools.

Based on the extensive prevention strategy experiments in the citriculture field by Chinese farmers, it has been shown that the control of HLB disease can be carried out in the three-pronged approach.

  1. HLB-free seedlings. Selection of HLB-free citrus saplings, rootstocks, and scions. Furthermore, infected root stocks/scions might be cured through thermotherapy.

  2. Removal of infected plants. Identification of HLB-affected plants by utilizing a suitable pathogen detection system and removing infected trees or infected sectors.

  3. Suppress the psyllid. Control of psyllids to reduce the spread of HLB pathogens in the field and biological control of vectors might be desirable methods to control the vector populations rapidly and cost-effectively.

Nanotechnology-driven farming is still early, but it is an exciting and challenging field of research to be developed in the future, especially if the proper emphasis is placed on understanding the fundamental interactions between nanoscale materials and crop plants [172]. Future nanotechnology will enable the development of biosensors for early diagnosis of disease, new methods for suppression of disease pathogens in field and greenhouse conditions, and new molecular tools for understanding pathogenic mechanisms in pathogens and plants [173]. Nanotechnological investigations in phytopathology have increased dramatically over the last decade. Nanomaterials can be engineered as biosensors to diagnose plant diseases and as a means of delivery of genetic material, probes, and agrochemicals. Nanotechnology has been incorporated into disease management strategies, diagnostic tools, and molecular tools. Nanotechnologies could provide an alternative treatment to citrus farmers to be integrated into their existing HLB management programs in the citrus groves.

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Acknowledgments

This work was funded by the Science and Technology Major Project of Guangxi (Gui Ke AA18118046).

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Conflict of interest

The authors declare no conflict of interest.

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Written By

Palaniyandi Karuppaiya, Junyuan Huang and Muqing Zhang

Submitted: 08 April 2022 Reviewed: 28 April 2022 Published: 15 June 2022