Recent progress in the understanding of Citrus Huanglongbing: from the perspective of pathogen and citrus host

Abstract

Citrus Huanglongbing (HLB) is a devastating disease spread by citrus psyllid, causing severe losses to the global citrus industry. The transmission of HLB is mainly influenced by both the pathogen and the citrus psyllid. The unculturable nature of the HLB bacteria (Candidatus Liberibacter asiaticus, CLas) and the susceptibility of all commercial citrus varieties made it extremely difficult to study the mechanisms of resistance and susceptibility. In recent years, new progress has been made in understanding the virulence factors of CLas as well as the defense strategies of citrus host against the attack of CLas. This paper reviews the recent advances in the pathogenic mechanisms of CLas, the screening of agents targeting the CLas, including antimicrobial peptides, metabolites and chemicals, the citrus host defense response to CLas, and strategies to enhance citrus defense. Future challenges that need to be addressed are also discussed.

Introduction

Citrus is one of the most important horticultural crops in the world, grown in more than 114 countries and regions around the globe (Talon and Gmitter 2008; Deng 2022). Citrus huanglongbing (HLB) is mainly associated with three species of phloem-limited bacteria, ‘Candidatus Liberibacter asiaticus’ (CLas), ‘Ca. L. africanus’ (CLaf) and ‘Ca. L. americanus’ (CLam), which are mainly spread by citrus psyllid (Diaphorina citri Kuwayama, D. citri) (Hu et al. 2021). In 1919, researchers firstly discovered HLB symptoms in Chaoshan, China, but it did not get enough attention at that time (Bové 2006). Currently, HLB is present in Africa, Asia, America (USA, Mexico, Brazil, Cuba) and Oceania, posing a continuous threat to citrus cultivation and causing a substantial economic impact on citrus-growing regions worldwide (Mendonça et al. 2017; Milne et al. 2018; Zhou 2020).

All commercial citrus varieties are susceptible to HLB, severely limiting the development of the citrus industry. Typical symptoms in infected citrus include leaves showing uniform or mottled yellowing, greening similar to deficiency symptoms, with veins protrusion in severe cases (Bové 2006; Liu et al. 2015), abnormal fruit color, small fruits, and sour fruit flavor, stunted roots and reduced fibrous roots, yellowing shoots, shrinking canopy and ultimately tree death (Batool et al. 2007; Rosales and Burns 2011; Graham et al. 2013).

The inability to continuously culture CLas in vitro has significantly impeded the study of its pathogenic mechanisms. CLas can infect all commercial citrus varieties, so the exploration and utilization of citrus disease-resistant resources and the creation of resistant varieties are crucial for the long-term development of the citrus industry. This paper reviews recent advances in the mechanisms of interaction between CLas-secreted virulence factors and target genes in citrus, the screening and development of drugs targeting CLas, the citrus host defense response to CLas and strategies to resist CLas infection. We also discussed future challenges that need to be addressed for HLB control.

Pathogenesis of CLas

The process of CLas invasion into citrus is shown in Fig. 1. CLas can colonize citrus tissues including seed coats, anthers, filaments, pollen grains, stigma, ovary, receptacle, roots and phloem (Hilf 2011; Wang et al. 2022a). CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores and can change their morphology to move through the sieve pore (Achor et al. 2020). The parasitic and pathogenic mechanisms of CLas leading to HLB symptoms can be summarized as following aspects. Firstly, phloem blockage hinders nutrient transport. The phloem is the conduit for nutrient transport in plants and a favorable site for pathogen colonization. After invading citrus, CLas binds to the plasma membrane at the sieve pores of the phloem and induces the expression of CALLOSE SYNTHASE 7 and PHLOEM PROTEIN 2 genes, leading to the deposition of callose, which blocks the sieve pores and thickening of the phloem cells, obstructing the transport of plant nutrients and ultimately causing HLB symptoms (Kim et al. 2009; Fan et al. 2012; Achor et al. 2020; Lewis et al. 2022). Secondly, nutrient consumption disrupts the host’s metabolic balance. CLas parasitism in citrus is heavily dependent on host nutrients. CLas potentially utilizes the ABC ATP-binding cassette transporter to acquire nutrients from the host for its proliferation, which extensive consumption depletes the host’s nutrient resources. Additionally, metabolic changes in the phloem caused by the pathogen alter the osmotic pressure, damaging phloem function and transport activity, which results in HLB symptoms (Duan et al. 2009; Wang and Trivedi 2013; Achor et al. 2020). Thirdly, CLas interferes with the host's immune responses. CLas infection triggers a chronic immune response in citrus, inducing the up-regulation of NADPH oxidase-encoding genes RBOHB, RBOHD and RBOHF, and the reduction of antioxidant enzymes contributing to the production of reactive oxygen species (ROS) (Ma et al. 2022). The accumulation of ROS in the phloem leads to the death of sieve elements and companion cells, impairing nutrients transport capacity. The gene SahA (CLIBASIA_00255), which encodes salicylate hydroxylase in CLas, is significantly up-regulated during infection, leading to the degradation salicylic acid (SA) produced by the host in response to the pathogen, thereby suppressing the plant’s immune responses (Li et al. 2017). Lastly, CLas secretes virulence factors to promote plant susceptibility. CLas induces symptoms of HLB through the secretion of effector proteins or other virulence factors that interact with targets in the plant (Jones and Dangl 2006), and research in this area has progressed relatively rapidly in recent years.

Fig. 1.

The process of CLas invasion into citrus and strategies to control CLas. After CLas infects citrus, the expression of transcriptional activators in CLas are elevated, which promotes the secretion of various virulence factors (SDEs, ncSecPs, etc.) through the secretion system to interact with targets in citrus (Mackey et al. 2014; Pagliai et al. 2015). The virulence factors can inhibit multiple signaling pathways in the plant, reduce plant defense responses and promote susceptibility to CLas, which ultimately leads to the development of HLB symptoms (Hu et al. 2021). Developing antimicrobial peptides or chemicals (SAMP, G6P3510, bortezomib, etc.) targeting the key transcriptional activators in CLas, the essential components of the secretion system, the critical virulence factors and targets of effectors in citrus is a potential approach to control HLB (Barnett et al. 2019; Huang et al. 2021; Wang et al. 2024)

Virulence factors secreted by CLas and their interaction with citrus host

CLas can secrete proteins via the type I secretion system (T1SS), Sec-dependent secretion system, or other methods to interact with and infect the host. With the deepening of research, more virulence factors secreted by CLas are being identified (Table 1). These factors promote CLas infection and colonization by influencing plant growth and developmental processes and interfering with defense responses. Recently, sec-delivered effectors (SDEs) have been extensively studied, some of which the interaction mechanisms with targets in citrus have been revealed. For example, SDE1 encoded by CLIBASIA_05315 inhibits the activity of the citrus defense papain-like cysteine proteases (PLCPs). Overexpression of SDE1 in Duncan grapefruit increases sensitivity to CLas and the HLB symptoms appear earlier. Furthermore, SDE1 can down-regulate the host protein DEAD-box RNA helicase (DDX3) and promote yellowing symptoms (Clark et al. 2018, 2020; Zhou et al. 2020). SDE3 (CLIBASIA_00420), a conserved effector in bacteria, promotes the proliferation of Pseudomonas syringae pv. tomato DC3000 and Phytophthora capsici when overexpressed in Arabidopsis. In citrus, SDE3 interacts with CsGAPC1 protein, a negative regulator of autophagy, leading to the degradation of the autophagy protein CsATG8 and suppression of immune responses. Overexpression of SDE3 in citrus enhances CLas colonization (Shi et al. 2023a). Overexpression of SDE4405 (CLIBASIA_04405) in citrus activates CsATG8-mediated autophagy and significantly down-regulates immune-related genes, such as CsPR1, CsWRKY22 and CsTGA. Interestingly, knocking out the GAPC gene in Arabidopsis and Nicotiana benthamiana, and overexpression of the CsATG8 gene in N. benthamiana, can enhance disease resistance, but this has not been validated in citrus (Zhang et al. 2020; Shi et al. 2023b). SDE15 acts as an inhibitor of programmed cell death (PCD) induced by Xanthomonas citri ssp. citri strain A^w (Brunings and Gabriel 2003), which associates with citrus CsACD2 protein to suppress citrus defense and facilitate CLas infection. Overexpression of CsACD2 has a similar effect as SDE15 while silencing CsACD2 induces autoimmunity and cell death (Yao and Greenberg 2006; Pang et al. 2020). These target genes of SDEs are potential targets for molecular design breeding for disease resistance.

Table 1.

Subcellular localization and function of the virulence factors of CLas proposed in this paper

Virulence factors	Subcellular localization	Function	Reference
SDE1	chloroplast vesicles	Inhibit PLCP and interact with DDX3	Clark et al. 2018; Clark et al. 2020; Zhou et al. 2020
SDE3	cell nucleus, cytoplasm	Degradation of cellular autophagy proteins and inhibition of immune responses	Shi et al. 2023a
SDE15	free distribution in cells	Inhibit plant immunity response	Ying et al. 2019; Pa Pang et al. 2020
CLIBASIA_03875	cell nucleus, cytoplasm, cytomembrane	Inhibit plant immunity response	Zhang et al. 2019
SDE4405	plasma membrane	Promote cellular autophagy and down-regulate expression of immune-related genes	Zhang et al. 2020; Shi et al. 2023b
SDE115	cell nucleus, cytoplasm	Down-regulate immune-related genes to reduce the levels of SA and JA	Du et al. 2022a, b
SDE460	cell nucleus	Interferes with host hormone signaling, MAPK signaling pathway	Liu et al. 2019; Wang et al. 2023
SDE410, SDE4435, SDE4955	cell nucleus, cytoplasm, cytomembrane	Down-regulate immune-related gene expression and enhance peroxidase activity	Li et al. 2024a, b
SECP8	cell nucleus, cytoplasm, cytomembrane	Inhibit plant PCD and down-regulate defense gene expression	Shen et al. 2022
CLIBASIA_04425	cytoplasm, cytomembrane	Inhibit the expression of SA pathway-related genes	Zhang et al. 2023
SC2_gp095, CLIBASIA_RS00445, CLIBASIA_RS00940	—	Inhibit of Rboh-mediated H2O2 signaling and oxylipins accumulation	Jain et al. 2015; Jain et al. 2018; Jain et al. 2019
AGH17470	cell nucleus, cytoplasm	Up-regulates defense gene expression and increases SA content	Du et al. 2022a, b
AGH17488	cytoplasm, cytomembrane	Promote CsAPX6 activity and reduce ROS accumulation	Du et al. 2023
CLIBASIA_03915	cytoplasm	Cause phloem necrosis in the senescent leaves of N. benthamiana	Li et al. 2020
CLIBASIA_04250	cell nucleus, cytoplasm
CLIBASIA_00470, CLIBASIA_04025	cytoplasm	Inhibite plant growth	Ying et al. 2019
CLIBASIA_05150	golgi	Cause cell death	Hao et al. 2019
CLIBASIA_04065C	mitochondrion
LasP235	cell nucleus	Cause leaf chlorosis and plant growth retardation	Hao et al. 2019

"-"means unknown

There are also some SDEs that can inhibit plant defense responses, but their targets in citrus are still unknown. For instance, CLIBASIA_03875 is the first PCD suppressor identified from CLas, which can suppress the hypersensitive response (HR) and H₂O₂ accumulation by regulating CNGCs, BI-1 and WRKY33 in N. benthamiana (Zhang et al. 2019). SDE115 (CLIBASIA_05115) plays a crucial role during the early stage of CLas infection in citrus, significantly down-regulating PR and WRKY gene expression and reducing salicylic acid (SA) and jasmonic acid (JA) levels, exacerbating HLB symptoms (Du et al. 2022b). The involvement of SDE460 in CLas infestation of citrus host is dependent on temperature changes. At 25℃, SDE460 inhibits immune responses and promotes CLas infestation by affecting several pathways such as hormone signaling and MAPK signaling pathway. At 32℃, SDE460 was prevented from entering the plant cell nucleus, and its pathogenicity was reduced (Wang et al. 2023; Li et al. 2024a, b). SDE4310, SDE4435 and SDE4955, when overexpressed in Arabidopsis, suppress the expression of PR2, PR5 and ICS1. These effectors enhance the stability of peroxidases CAT2, CAT3, PXG2 and PrxQ, and interact with GAPA to modulate ROS bursts and cell death, thereby regulating host defenses (Li et al. 2024a, b). SECP8 (CLIBASIA_05330) is an inhibitor of PCD, and the SAR-related genes CsPR1, CsPR2 and FRK1 were significantly down-regulated in SECP8 overexpressed citrus inoculated with CLas, which produced significant dwarfing symptoms and yellowing phenotypes. Overexpression of SECP8 can also increase susceptibility to citrus canker. Interestingly, expression of SECP8 in N. benthamiana suppressed the necrosis and dwarfing phenotypes caused by SDE1, but the relationship between SECP8 and SDE1 remain unknown (Shen et al. 2022). CLIBASIA_04425 can activate the expression of CsPP2, CsAPS1 and CsGBSS1 in citrus while suppress the expression of SA pathway-related genes, such as CsEDS1, CsNDR1 and CsNPR1 (Zhang et al. 2023). Expressed in N. benthamiana by tobacco mosaic virus (TMV), CLIBASIA_00470 and CLIBASIA_04025 can cause symptom of stunting, CLIBASIA_05150 and CLIBASIA_04065C (C-terminal part of CLIBASIA_04065) induced cell death (Ying et al. 2019). Expression of CLIBASIA_03915 and CLIBASIA_04250 in N. benthamiana by TMV caused phloem necrosis in senescent leaves (Li et al. 2020).

The bacterial cytoplasm delivers proteins called non-classical secreted proteins (ncSecPs), which can also inhibit plant defense responses and promote pathogen infestation. Researchers have identified 27 ncSecPs from the CLas genome, of which 10 are localized to the cytoplasm, cell membrane and inclusion bodies. These ncSecPs can inhibit the plant’s HR and H₂O₂ accumulation, promoting CLas infection like the previously mentioned PCD inhibitors (Du et al. 2021). Overexpression of LasP235 in Carrizo citrange showed leaf chlorosis and plant growth retardation (Hao et al. 2019). SC2_gp095, CLIBASIA_RS00940 and CLIBASIA_RS00445 are peroxidases that can inhibit Rboh-mediated H₂O₂ signaling. CLIBASIA_RS00445 can also inhibit the accumulation of oxylipins induced by tert-butyl hydroperoxide, thereby suppressing the HR response in plants (Jain et al. 2015, 2018, 2019). AGH17470 is the first ncSecP identified from the prophage region of the CLas genome that can induce HR. When expressed in citrus, it significantly up-regulates the expression of SA-mediated signaling genes such as CsNPR1, CsTGA and CsPR1, and increases SA levels (Du et al. 2022a). Similarly, AGH17488, encoded by the phage region of the CLas genome, enhances the activity of the host ascorbate oxidase APX6 and increases the localization of CsAPX6 in the membrane, thereby inhibiting plant immunity by reducing ROS accumulation. Transient expression of AGH17488 and CsAPX6 in citrus can increase susceptibility to citrus canker (Du et al. 2023). Additionally, it was found that CLas could promote the expression of DcitSGP1 and DcitSGP3 proteins in D. citri, which enhances the feeding behavior of D. citri on citrus and mediates the suppression of the JA pathway in citrus, thereby inhibiting defense responses (Liu et al. 2024). The virulence factors secreted by CLas are primarily localized in the nucleus, cytoplasm and cell membrane. During different stages of CLas infection in the host, these virulence factors exert various functions. They interact with targets in the host to suppress the expression of plant immunity-related genes, influence the levels of ROS, SA and JA, affect plant PCD response, and mediate PTI and ETI responses. This facilitates the long-term growth and colonization of CLas in the host.

Citrus host defense response to CLas

Elucidating the mechanism of disease resistance and identification of disease-resistant genes from HLB-resistant/tolerant resources can promote breeding for HLB-resistant new cultivars. Currently, based on the analyses of some HLB-tolerant resources, several resistance mechanisms have been elucidated (Fig. 2). Firstly, some HLB-tolerant resources probably accelerate growth and development to compensate for damage. Generally, CLas cause severe damage to the phloem after infecting citrus, such as phloem blockage and callose deposition. While in two HLB-tolerant varieties, ‘Bearss’ lemon and ‘LB8-9’ Sugar Belle mandarin, their phloem had better regeneration capacity, which might be the reason for their HLB-tolerance (Deng et al. 2019). Secondly, citrus may activate the immune response to CLas. After infecting the host, CLas suppress the host’s defense response to promote its proliferation. Immune response-related proteins, including Cys-rich secretory proteins, pathogenesis-related proteins, glutathione-S-transferases, catalase and thioredoxin, were significantly up-regulated in the resistant variety Australian finger lime (Citrus australasica) compared with the susceptible sweet orange after CLas infestation. These proteins enhance the host redox response to CLas infestation resulting in excess ROS and alleviating HLB symptoms (Weber et al. 2022). In Persian lime (Citrus latifolia), genes such as ClPR1, ClNFP, ClDRL27 and ClSPK are up-regulated upon HLB infection, suggesting their potential role in combating CLas (Estrella-Maldonado et al. 2023). Thirdly, the accumulation of defense-related metabolites may enhance disease resistance. The metabolites in different citrus varieties infested with CLas varied significantly (Rao et al. 2019). In disease-resistant varieties, the levels of certain sugars (raffinose, fructose, glucose and inulin) are significantly lower than in susceptible varieties. While the levels of some amino acids, flavonoids, terpenoids and unknown compounds, are higher in disease-resistant varieties, which may contribute to their disease resistance (Albrecht et al. 2016). Significantly different accumulations of cyanidin 3-rutinoside, hesperidin and gardenoside have been observed in the fruit pith of three varieties with diversified tolerance to HLB (Li et al. 2024a, b). Determination of volatile metabolites in 14 citrus varieties revealed that compounds with inhibitory effects, such as aldehydes and monoterpenes (linalool, laurinene, d-limonene, etc.), were higher in disease-resistant varieties, and high accumulations of limonene, γ-terpinene and β-myrcene were detected in the rinds of disease-resistant citrus hybrids, which may be involved in inhibiting the CLas (Hijaz et al. 2016; Huang et al. 2017). After infection by CLas, ‘Shatian’ pomelo exhibited a significantly higher release of terpenoids, including β-caryophyllene, β-ocimene, and others, accompanied by the upregulation of terpene synthesis genes CgTPS1 and CgTPS2 (Wen et al. 2023). In addition, stable antimicrobial peptide (SAMP), identified from citrus disease-tolerant materials, has the dual function of directly inhibiting CLas growth and inducing plant immune responses (Huang et al. 2021).

Fig. 2.

Citrus host defense response to CLas and strategies to enhance citrus resistance. Citrus can enhance disease resistance through accelerating growth and development, enhancing autoimmune response and accumulation of defense related metabolites (Deng et al. 2019; Rao et al. 2019; Weber et al. 2022; Estrella-Maldonado et al. 2023). For the HLB management, some phytohormones (melatonin, salicylic acid, etc.) can be used to activate the plant immune system (Lu et al. 2016; Hu et al. 2018; Nehela and Killiny 2020; Shahzad et al. 2024). Exogenous application or enhancing endogenous synthesis of antimicrobial metabolites (luteolin, quercetin, apigenin, etc.) can help plants defend against CLas (Zuo et al. 2019; Yang et al. 2021a, b). In addition, exploring the resistant mechanisms of HLB-resistant citrus resources, identifying the disease-susceptible gene or pathogen-target genes, and breeding disease-resistant cultivars by genetic manipulation and other technologies is a basic solution to HLB

In conclusion, the significant resistance to CLas observed in these HLB-tolerant varieties is the result of a multifaceted set of factors. By leveraging the mechanisms outlined above, we can further refine strategies for controlling HLB. Firstly, existing HLB-tolerant varieties can be utilized in cross-breeding programs to create disease-resistant populations, identifying disease-tolerant and susceptible genes and expand genetic resource pool (Deng et al. 2019). Secondly, immune-related genes activated by CLas in tolerant varieties should be fully explored, validated and integrated with other genetic resources to support molecular breeding efforts (Estrella-Maldonado et al. 2023). Lastly, differential metabolites, such as secondary metabolites, hormones and antimicrobial peptides, between susceptible and resistant varieties should be further studied to aid in the development of anti-CLas agents (Huang et al. 2021; Rao et al. 2019). Additionally, microbial control may be also an important strategy for HLB management in the future.

Development of antimicrobial metabolites

The treatment of HLB by trunk injection of antibiotics is less efficient and has ecological safety risks. The problem of antibiotic residues and whether they make CLas resistant to drugs are still unknown. Many metabolites of citrus have bacteriostatic activity. It is of great significance for the resistance breeding and green control of HLB to increase the content of antibacterial metabolites in citrus or to develop new agents using these metabolites (Rao et al. 2019). Analysis of secondary metabolites in citrus leaves showed that the contents of flavonoids and phenolics were higher in disease-tolerant citrus varieties, and these two types of metabolites have high antioxidant capacity, which may be associated with citrus tolerance to CLas (Hijaz et al. 2020). Flavonoids, coumarins and lignins all belong to the plant phenylpropanoid pathway metabolites, and many of these compounds possess significant antimicrobial effects (Mathesius 2018). Flavonoids, characterized by a C6-C3-C6 skeleton, are polyphenolic compounds with diverse biological activities, including antioxidant and anti-inflammatory properties. The antibacterial mechanisms of flavonoids involve damaging the cytoplasmic membrane, inhibiting nucleic acid synthesis and disrupting energy metabolism (Cushnie and Lamb 2011). Specifically, 8-prenylnaringenin and 8-prenylflavanone glabranine significantly inhibit Staphylococcus aureus biofilms (Manner et al. 2013), while kaempferol exhibits inhibitory activity against both S. aureus and Escherichia coli (Teffo et al. 2010). Other compounds such as quercetin, sinapic acid, naringin and apigenin act as effective antagonists in cell signaling pathways (Vikram et al. 2010). For CLas, several flavone and flavonol compounds, luteolin, quercetin and apigenin, were reported to selectively inhibit the endoribonuclease activity of RNA ribonuclease YbeY in CLas, indicating a potential application of these metabolites in inhibition of CLas proliferation (Zuo et al. 2019). Methylation of hydroxyl groups in flavonoid structures reduces their antimicrobial activity, whereas hydrophobic substituents enhance this activity. Beyond direct antibacterial effects, flavonoids can reduce the vitality of infected plant cells, protecting host cells from bacterial toxicity and minimizing PCD responses (Paolillo et al. 2011). Hydroxycoumarins disrupts bacterial cell membranes by targeting the lipopolysaccharide biosynthesis pathway, leading to bacterial death (Yang et al. 2021a, b). Furthermore, ruscogenin inhibits transcriptional regulator XpsR in Ralstonia solanacearum, thereby suppressing extracellular polysaccharide synthesis and exerting antibacterial effects (Yang et al. 2021a, b).

Crops with high levels of volatile compound are more resilient to stressful environments (Meena et al. 2017). Expressing citrus terpene synthase gene CsTPS21 in N. benthamiana led to the synthesis of β-ocimene, which effectively repelled psyllid (Bin et al. 2023). Plants release sesquiterpene compounds like DMNT and TMTT, which can repel harmful insects or attract natural enemies to defend against insect attacks. Furthermore, expression of two cytochrome P450 genes in citrus, CsCYP82L1 and CsCYP82L2, promoted the release of DMNT and TMTT, enhancing the avoidance response against psyllid (Sun et al. 2024). The volatile (Z)-3-hexenol and (Z)-3-hexenol produced by tobacco leaves can attract the bite of psyllid, while L-nicotine in tobacco can resist insects (Zheng et al. 2023). Methyl mercaptan, DMS and DMDS released by guava can activate the expression of defense genes such as PAL and PR1 in citrus and increase the content of polyphenols, lauric acid and methyl salicylate to enhance resistance to psyllid. Therefore, planting N. benthamiana and guava in citrus zones is beneficial to the control of HLB (Ling et al. 2022). It has great potential to identify bacteriostatic metabolites and develop new agents to replace antibiotics for the control of HLB (Fig. 2).

Development of antimicrobial peptides

Antimicrobial peptides (AMPs) are a class of widely studied antimicrobial compounds found across various organisms (Patel and Akhtar 2017). AMPs play a role by disrupting membrane permeability and promoting oxidative stress (Brogden 2005; Yun and Lee 2016). For instance, Lipid Transfer Protein 1 (LTP1), a cationic, multifaceted protein belonging to the pathogenesis-related protein (PR14) family, can not only bind to jasmonic acid and lipids involved in plant immunity, but also disrupt pathogen cell membrane integrity, making it a potential effective antimicrobial agent (Rode et al. 2024). In recent years, the development of AMPs targeting CLas holds promise for controlling HLB (Table 2). As CLas resides in the phloem and most plant-derived AMPs cannot be transported to the phloem, researchers respectively fused two human antimicrobial peptides, lysozyme and β-defensin 2, with a phloem-restricted protein (CmPP16), and expressed them in citrus. The citrus plants expressing the two AMPs showed attenuated HLB symptoms, enhanced photosynthetic capacity and reduced CLas titer. The use of a phloem-specific promoter to express the AMP cecropin B gene in the phloem has also enhanced citrus tolerance to CLas (Zou et al. 2016; Guerra-Lupián et al. 2018). Using citrus hairy roots to culture CLas and screen for inhibitory compounds, two AMPs and four chemical compounds (chloroxylenol, cinoxacin, duartin and cyclopentolate hydrochloride) with inhibitory effects on CLas proliferation comparable to tetracycline were identified (Irigoyen et al. 2020). A stable antimicrobial peptide (SAMP), screened from HLB-tolerant Microcitrus australiasica, is thermally stable and can move through the citrus vascular system. When applied it to treat CLas-infected citrus plants, SAMP can not only reduce CLas titer and disease symptoms in HLB-positive trees but also induce innate immunity to prevent and inhibit infections (Huang et al. 2021). Nodule-specific cysteine-rich peptides (NCRs) identified from Mediterranean legumes can reduce CLas proliferation in citrus leaves and decrease CLas acquisition by the vector psyllid (Higgins et al. 2024). Researchers have also identified a peptide with a high affinity to BamA, the outer membrane protein of CLas. By fusing AMPs with that peptide, specifically targeting antimicrobial peptides (STAMPs) which can inhibit BamA and subsequently inhibit the proliferation of CLas have been designed (Mallawarachchi et al. 2024). Targeting TolC, a key protein in the CLas T1SS efflux pump system, three AMPs (plantaricin JLA-9, darobactin and urechistachykinin II) have been identified (Wang et al. 2022b). Based on a dual-specificity serine/tyrosine phosphatase (STP) in CLas, which localized in the host cytoplasm and cell membrane and mediates host PCD response, researchers identified two competitive inhibitors of STP, G6P3510 and G6P6373, which can effectively inhibit CLas proliferation. Peroxidases play crucial roles in the proliferation of pathogenic bacteria. Designing ligands targeting peroxiredoxin in CLas can inhibit its proliferation (Gupta et al. 2021; Wang et al. 2024).

Table 2.

Mechanism of antimicrobial peptides and chemicals proposed in this paper

AMPs/Chemicals	Function and mechanism	Reference
LTP1	Promote plant immunity and disrupts pathogen cell membranes	Rode et al. 2024
Lysozyme, β-defensin 2	Inhibit CLas proliferation and promotes plant photosynthesis	Guerra-Lupián et al. 2018
cecropin B	Inhibit CLas proliferation	Zou et al. 2016
SAMP	Induce the expression of plant defense genes that acts on bacterial cell membranes	Huang et al. 2021
NCRs	Inhibit CLas proliferation and CLas uptake by D. citri	Higgins et al. 2024
STAMPs	Target the CLas outer membrane protein BamA	Mallawarachchi et al. 2024
Plantaricin JLA-9, Darobactin, Urechistachykinin II	Target T1SS efflux pump key protein TolC in CLas	Wang et al. 2022b
G6P3510, G6P6373	Competitive inhibitors of STP in CLas	Wang et al. 2024
Tolfenamic acid	Target the RNA polymerase binding protein PrbP in CLas	Gardner et al. 2016; Pan et al. 2017
Benzbromarone, 4’-Demethylepipodophyllotoxin	Target the CLas transcriptional regulator LdtR in CLas	Mackey et al. 2014; Pagliai et al. 2015; Barnett et al. 2019
Bortezomib, ChemDiv C549-0604, ChemDiv D244-0326	Inhibit the CLas transcriptional regulator VisNR in CLas	Barnett et al. 2019
Rosiglitazone	Inhibit the CLas transcriptional regulator RpoH in CLas	Barnett et al. 2019

Development of antimicrobial chemicals

In addition to antimicrobial peptides, some special chemicals can also inhibit the growth of CLas in citrus (Table 2). Tolfenamic acid can interact with the RNA polymerase binding protein PrbP in CLas and inhibit its proliferation (Duan et al. 2009). Benzbromarone can inhibit a transcription activator LdtR, which plays a crucial role in CLas response to osmotic stress, by binding to the amino acid sites within the Benz1 pocket of LdtR (Mackey et al. 2014; Pagliai et al. 2015). Though a high-throughput screening system, five compounds were identified to have inhibitory effect on the activity of CLas transcription activators LdtR, RpoH and VisNR (Barnett et al. 2019). However, inhibiting the activity of CLas transcription factors alone does not fully suppress the proliferation of the pathogen. Further research is needed to identify critical pathogenic factors and develop highly effective and precisely targeted antimicrobial peptides or chemicals (Fig. 1).

Strengthen the citrus immune system

The plant immune system is an important defensive barrier against pathogen infestation. Inducing the expression of immune-related genes in citrus can enhance resistance before CLas infestation, which is a proactive strategy for HLB control (Fig. 2). Several phytohormones and secondary metabolites can activate the immune system of the plant. Brassinosteroids, which are hormones involved in various physiological processes and associated with plant resistance, can increase resistance to various pathogens. Spraying 24-epibrassinolide (eBL) in greenhouses and fields can induce the up-regulation of citrus defense-related genes such as SOD, PAL and AOS, significantly reducing CLas titer (Lu et al. 2016). Exogenous application of melatonin can increase the levels of salicylic acid and other hormones and reduce CLas titer in citrus (Nehela and Killiny 2020). Treatment of sweet orange with gibberellin acid mitigated plant oxidative stress, enhanced defense responses and promoted carbohydrate transport in the plant to mitigate the effects caused by CLas (Shahzad et al. 2024). Exogenous application of MeSA, a natural community immune response signal, can effectively induce defense gene expression and mitigate HLB symptoms (Cheng et al. 2023). The application of γ-aminobutyric can induce the up-regulation of multiple hormone-related genes and the accumulation of amino acids, enhancing antioxidant defense response to HLB (Nehela and Killiny 2023). Trunk injection of salicylic acid and acibenzolar-S-methyl significantly induced the expression of PR1 and PR2, while injection of potassium phosphate and oxalic acid significantly induced the expression of PR2 and PR15, leading to systemic acquired resistance in citrus (Hu et al. 2018). Some researchers have also suggested that HLB is a citrus autoimmune disease and that CLas infestation of citrus induces a systemic chronic immune response and produces symptoms of HLB (Ma et al. 2022). Inducing systemic acquired resistance to combat CLas is a relatively slow process compared to the immediate effects of antibiotic injection. In addition, many mineral elements such as magnesium, calcium, iron and manganese tended to decrease in HLB infected citrus, and nutrition supplementation of HLB infected trees may also inhibit HLB development (Dong et al. 2021). Soil and foliar application of calcium, magnesium and boron fertilizers can improve the structure of soil microbial communities and enrich probiotics in the inter-root zone (Zhou et al. 2021).

Role of microorganisms in the control of HLB

Biological control offers a promising alternative strategy for managing HLB. Plant diseases often induce shifts in plant-associated microbial communities, and endophytes can modulate plant defenses through ethylene, jasmonic, or salicylic acid pathways (Fadiji and Babalola 2020). Root-associated endophytes, in particular, may contribute to plant defense against pathogens. For instance, disease-resistant tomato cultivars exhibit greater root endophytic bacterial diversity and a higher presence of antagonistic organisms (Upreti and Thomas 2015). Similarly, Endophytic Bacteria-39, isolated from citrus, has been shown to reduce the incidence of citrus canker (Rabbee et al. 2022). In the case of HLB, the disease disrupts the rhizosphere-to-rhizoplane enrichment process, reducing the abundance of rhizoplane-enriched genera and impairing plant host-microbiome interactions (Zhang, et al. 2017). Studies comparing microbial diversity in leaves from 24 citrus varieties found that asymptomatic citrus exhibited higher total numbers of endophytes compared to symptomatic plants. Bacillus and Curtobacterium were dominant in symptomatic citrus, and higher densities of Bacillus were associated with lower HLB severity, suggesting that Bacillus subtilis may play a role in enhancing citrus resistance to HLB (Munir, et al. 2020). Additionally, inoculation of Burkholderia strains isolated from the healthy citrus root-associated microbiome could trigger the expression of genes involved in induced systemic resistance in HLB-infected plants (Zhang, et al. 2017). Endophytes, due to their ability to colonize citrus tissues more effectively than other bacteria, represent promising candidates for biological control (Blaustein et al. 2018). In the future, more targeted screening of microbial colonies in citrus roots and leaves is worth exploring to identify potential biocontrol agents against CLas.

Conclusion and Perspectives

HLB is a major threat to the world’s citrus industry. The occurrence and spread of HLB primarily involve three factors: the pathogen, the vector D. citri and the citrus host (Aidoo et al. 2023). Understanding these factors is crucial for effective disease management. Currently, research on virulence factors secreted by pathogenic bacteria and their pathogenic mechanisms has been progressing rapidly, but it remains to be demonstrated which of these virulence factors are most critical for disease causation, and this is also related to the research and development of drugs targeting the virulence factors (Hu et al. 2021). Another efficient solution to HLB is to explore the resistance resources, analyze the mechanism of disease resistance, and breed for resistant cultivars, which requires further long-term screening and evaluation of disease resistant citrus resources.

Overall, a comprehensive strategy for HLB control must address the pathogen, the vector and the host. In terms of pathogenic bacteria and the host, future research should focus on several key areas. Firstly, explore the in vitro culture method of CLas and analyze the pathogenic mechanism. The main virulence factors of CLas remain to be discovered, which is the basis for screening and developing environmentally friendly drugs for CLas. Secondly, evaluation and application of disease-resistant citrus resources is important for the identification of disease resistance genes and breeding for resistance to HLB. Genetic manipulation of the targets of pathogenic effectors by gene editing technology is also a potential strategy to improve the HLB resistance of currently planted citrus varieties. Thirdly, the research and development of bacteriostatic metabolites and AMPs may be an effective way to reduce current losses caused by rapid outbreaks of HLB. In addition, biological control using citrus-associated endophytes presents another avenue for disease management. In the near future, alternative solutions will be proposed to increase the content of bacteriostatic metabolites in citrus or develop green pesticides as substitutes for antibiotics.

Author contribution

Kun Yang prepared the initial manuscript with the help of Bin Hu, Wang Zhang and Tao Yuan. Yuantao Xu conceptualized and revised the manuscript. All authors have approved the final version of the manuscript for publication.

Funding

This study was funded by grants from the National Natural Science Foundation of China (31925034, U23A20198), Key Research and Development Program of Hubei (2022BBA155) and National Postdoctoral Program for Innovative Talents (BX20200146).

Data availability

Not applicable.

Declarations

Institutional review board

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Informed consent

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Conflict of interest

The authors declare that they have no conflict of interest.