A host‐derived chimeric peptide protects citrus against Huanglongbing without threatening the native microbial community of the phyllosphere

Abstract

Introduction

The application of host‐derived antibacterial peptides has been highlighted as a potential efficacious and safe tool for the treatment of Huanglongbing (HLB), the most devastating disease of citrus. However, pathogenic bacteria such as HLB‐causing Candidatus liberibacter asiaticus (CLas) often develop resistance against the host antibacterial peptides. We showed that chimeras containing two different host antibacterial peptides not only retain antibacterial activity but also overcome bacterial resistance and enhance plant defence responses. Also, chimeric peptides can have an off‐target impact on the structure and function of plant‐associated microbiomes. However, there is a lack of understanding of the impact of the chimeric peptide therapy on the microbial structure in the citrus phyllosphere while reducing the CLas titre. Here, we aim to evaluate the efficacy of a chimeric peptide (UGK17) to reduce CLas titre, inducing plant defence response and impacting the microbiome associated with the citrus phyllosphere.

Material and Method

Leaf samples were collected from orange and grapefruit trees in Texas and identified as old and young leaves according to their maturity. We collected three different types of leaves based on their infection and symptoms: healthy, symptomatic (infected with typical symptoms), and asymptomatic (infected without symptoms). In planta assay was performed by dipping the leaves in the 0, 5 and 25 μM of UGK17 solutions for 48 h. The quantifications of CLas titre and pathogenesis‐related (PR) gene expression were done by quantitative polymerase chain reaction (qPCR) and reverse transcription‐qPCR, respectively. Amplicon sequencing was done to evaluate the impact of UGK17 on individual bacterial community structures. In addition, we performed an ex planta assay to assess the effect of UGK17 on the growth of bacterial isolates including Liberibacter crescens instead of unculturable CLas predominant in the phyllosphere.

Result

The UGK17 treatment reduced the CLas titre in both asymptomatic and symptomatic citrus leaves, regardless of the age of the leaves. The UGK17 application augmented the PR gene expression. In ex planta assay, the growth of L. crescens along with four other strains belonging to the family Rhizobiaceae, was significantly inhibited by the UGK17 while the growth of 74 strains were unaffected. Additionally, there was no statistically significant changes in the microbial community structure with UGK17 treatment.

Conclusion

Our results suggest that the chimeric peptide therapy is a promising solution to combat HLB by targeting mainly Gram‐negative pathogens and enhancing the plant immune responses without impairing the indigenous microbial community in the citrus phyllosphere.

INTRODUCTION

Citrus greening or Huanglongbing (HLB), caused by phloem limited and insect (Asian citrus psyllid [ACP]) vectored bacteria, Candidatus liberibacter spp. (Candidatus Liberibacter asiaticus [CLas] in Florida) is the most destructive disease of citrus (Blaustein et al., 2018; Wang & Trivedi, 2013). HLB damage to citrus includes weakened root systems, discoloured leaves, premature fruit drop, and bitter juice (Wang & Trivedi, 2013; Wang, Pierson, et al., 2017). While the disease, or its vector, has impacted all citrus-growing regions of the United States, it has had significant impacts on commercial citrus-growing regions of Florida and Texas. Moreover, the pathogen is poised to expand from residential areas of California to threaten commercial production. In Florida, the harvest for the state's signature plunged to 45.1 million boxes in 2022; an 69% drop from 149.8 million boxes in 2005, the year when HLB was first detected in the state (USDA, 2023). Florida has lost over $8.0 billion in revenue, 149,338 citrus acres, and over 8000 jobs to HLB (USDA, 2023). If yields continue to decline and trees keep dying, the US citrus industry will lose relevance resulting in serious economic impact over time.

To date, neither a cure nor an economically viable strategy for the management of diseased trees is available to the industry. Producing resistant cultivars through conventional breeding is difficult due to the lack of commercially available resistant rootstock/scion combinations (Dutt et al., 2015). Other strategies used, or in development, for HLB treatment and prevention include: (i) blocking transmission by ACP using insecticides (Boina et al., 2010; Rehberg et al., 2022; Sétamou et al., 2010; Srinivasan et al., 2008) and RNA interference targeted to ACP genes involved in transmission (Britt et al., 2020); (ii) reducing the level of CLas and maintaining plant growth by thermal, nutritional, and chemical therapy (Hoffman et al., 2013; Li et al., 2016, 2019); (iii) killing CLas by antibiotics (Hu & Wang, 2016; Zhang et al., 2011); (iv) improving soil biodiversity and harnessing beneficial bacteria (Bazany et al., 2022; Stokes et al., 2023; Zhang et al., 2017); and (v) enhancement of the HLB tolerance by transgenic technology (Dutt et al., 2015; Hao et al., 2016; Stover et al., 2013). These disease management strategies have achieved varying degrees of success (Li et al., 2020). However, citrus growers need other and more efficacious, safe, and cost-effective tools for HLB therapy.

The current lack of methods to effectively combat HLB makes the discovery of innovative therapeutic measures imperative for the survival of the citrus industry. In this regard, host-therapy based on the natural defence genes that constitute pathogenesis-related (PR) or defence peptides/proteins has emerged as a potential tool for the treatment and prevention of HLB (Basu et al., 2022; Huang et al., 2021; Weber et al., 2022). The defence peptides or proteins can kill the bacteria cell directly by forming pores in the cell membrane, interacting with nucleic acids, or inhibiting protein synthesis and activity of enzymes (Dandekar et al., 2012; Montesinos, 2007, 2023; Zhang et al., 2021). PR proteins or peptides can function as signalling molecules that modulate the action of some components of the plant immunity-signalling pathways, suggesting that some of these peptides may play a fundamental role as components of feedback loops that regulate the duration and intensity of a plant defence response (Allaker, 2008; Basu et al., 2022; Campos et al., 2018). Recently, Huang et al. (2021) identified novel peptides from HLB-tolerant citrus cultivars and reported the enhancement of plant immunity in the peptide-injected trees. Stover et al. (2013) reported the bactericidal activity of synthetic peptides on Agrobacterium tumefaciens and Sinorhizobium meliloti which are phylogenetically related to CLas. Previous research has shown that transgenic citrus-expressing peptides reduced both CLas load and HLB symptoms (Soares et al., 2020). While transgenic citrus will offer a complementary strategy that will provide a more sustained solution, this will require significant time for field efficacy studies and regulatory processes before being handed over to the growers (Graham et al., 2020; Su et al., 2023). The combination of two different peptides resulting in a chimera has been showing increase of the therapeutic potential compared to individual peptides (Basu et al., 2022; Gupta & Stover, 2020). For example, Basu et al. (2022) designed chimeric helix-turn-helix (HTH) peptides by joining two different helical amphipathic peptide segments involved in the plant's innate immunity. These chimeras not only show bactericidal activity on infected plants but also augment a plant's innate immune system during infection. Although host-derived peptides are efficient antibacterial agents and are suitable alternatives to antibiotics in citrus production, there are concerns about their unwanted consequences on environmental sustainability and human health.

Plant-associated microbiomes play key roles in plant nutrition, health, and resistance to various biotic and abiotic stresses (Trivedi et al., 2022). Host-derived peptides can directly impact the selected microbial groups acting as toxins and killing the microbes. In addition, the induction of plant defence responses by host-derived peptides can indirectly influence the structure and function of the plant-associated microbiome (Hacquard et al., 2017; Trivedi et al., 2020). The microbiome plays a central role in plant and soil health (Trivedi et al., 2022), so microbial dysbiosis can lead to decreased plant performance. On the other hand, if peptides are selective against the pathogen, their application can restore the microbiome that can benefit plant growth. HLB is known to have significant impacts on both the structure and functions of plant-associated microbiome (Hu et al., 2022; Li et al., 2021; Liu et al., 2023; Trivedi et al., 2016). The HLB-mediated changes in the plant-associated microbiome is postulated to have a detrimental influence in overall citrus health (Trivedi et al., 2012). Little is known about the consequences of host-derived peptide application on the plant-associated microbiome, and the investigation of unintended effects is rarely considered (Lei et al., 2021; Weinhold et al., 2018). A detailed understanding on the molecular and ecological aspects of interactions between host-derived peptides with plant/insect microbiomes and the impact of these interactions on the environmental sustainability of production systems is required to make science-based decisions about the environmental impact of peptide application in the natural environment. However, to the best of our knowledge, no detailed studies have evaluated the nontarget effect impact of the application of host-derived antimicrobials on plant-associated microbiomes.

The application of host-based therapy shows clear benefits for HLB management and are the only few silver lining for the citrus industry on the verge of destruction due to HLB (Basu et al., 2022; Wang, 2021). Although the application of peptides provides a robust HLB management tool, there are concerns about the off-target effects of the host-derived antimicrobial peptides on the citrus microbiome. Here, we conducted experiments to evaluate: (a) The efficacy of a newly developed host-derived chimeric peptide (UGK17) in reducing CLas titre in the leaves of two citrus varieties showing different symptoms; (b) the impact of peptide application on the augmentation of PR genes involved in plant immune response; and (c) the impact of the peptide application on the structure of phyllosphere microbiome. Overall, our results will provide information to promote host-mediated antimicrobial therapy as a promising solution for protecting the citrus industry against HLB without having off-target impacts on citrus-associated microbiome.

MATERIALS AND METHODS

Sample collection

We collected leaf samples from mature Valencia sweet orange (Citrus sinesis) and Rio Red grapefruit (Citrus paradisi) trees grafted on sour orange rootstock. These trees are in Texas A&M University-Kingsville's South Research Farm in Weslaco, TX. From each variety, three types of samples were collected: (a) healthy (no symptoms on the entire tree); (b) symptomatic (showing asymmetric leaf mottling); and (c) asymptomatic (samples collected from the same branch as infected but with no symptoms). Samples were collected from 15 trees from each variety. Also, the collected samples were divided into old and young leaves according to their maturity. Four leaves were taken randomly for each UGK17 treatment from the collected samples. Samples were shipped to the lab at Colorado State University overnight in blue ice.

Experimental setup

Details on the design and synthesis of the chimeric peptide UGK17 used in this study is provided in Basu et al. (2022). Briefly, UGK17 is a combination of two segments belonging to proteins associated with plant innate immunity. The UGK17 was custom synthesised after performing molecular modelling to construct a library of peptide chimeras by joining two antibacterial α/β segments from citrus proteins. The selection process focused on identifying energetically stable chimeras with a high predicted antibacterial activity. Figure 1 illustrates the experimental setup in this study. A previous study (Basu et al., 2022) has shown that UGK17 is effective in CLas clearing at 25 μM UGK17 treatment. Here, in addition to 25 μM concentrations, we examined the effectiveness of UGK17 treatment in reducing CLas titre at a lower concentration (5 μM). The peptide was diluted in water to make 5 and 25 μM concentrations. Leaf samples belonging to two varieties and different symptoms were placed in individual Petri dishes. The Petri dishes with two leaves were filled with 25 mL of the UGK17 solutions with 5 or 25 μM concentrations or water (control treatment). The Petri dishes were left in room temperature in the dark for 48 h till they absorbed 5–6 mL of the respective solution. Four biological replicates from each treatment were collected, and immediately frozen in liquid nitrogen, and stored at −80°C for DNA/RNA extraction.

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DNA/RNA extraction

The frozen samples were grinded using mortar pastel. DNA and RNA were extracted using DNeasy Plant Pro and RNeasy PowerPlant kits (Qiagen), respectively. The extracted DNA/RNA was quantified using NanoDrop 2000 (Thermo Fisher Scientific) and stored at −80°C for further use.

Quantification of CLas

All quantitative polymerase chain reaction (qPCR) assays were performed in a 96-well plate using an ABI Prism 7500 Sequence detection system (Applied Biosystems). Primer set CQULA04F-CQULAP10P-CQULA04R was used to target the β-operon region of CLas, and qPCR reactions were performed according to the conditions described in Trivedi et al. (2009). Each individual sample was replicated four times on a 96-well plate and the whole reaction was repeated twice to verify the consistency of the method. Results were analysed using ABI Prism software. Raw data were analysed using the default settings (threshold = 0.2) of the software.

PR gene expression

To determine PR gene expression, contamination of genomic DNA was removed by RNA treatment with a Turbo-DNA free kit (Ambion). (Reverse transcription-qPCR) RT-qPCR assays were conducted to determine the expression of three PR genes (PR-1, PR-2, and PR-5), using an Applied Biosystems 7500 Fast real-time PCR system, with the QuantiTect SYBR green RT-PCR kit (Qiagen). The PCR conditions were 30 min of reverse transcription at 50°C, followed by 15 min of predenaturation at 95°C and 40 cycles of 15 s of denaturation at 94°C, 30 s of annealing at 55°C, and 30 s of extension at 72°C. The housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase-C was used as an endogenous control. Melting curve analysis was conducted to verify the specificity of the RT-qPCR products. The products were run on a 2% agarose gel to confirm the presence of only a single band. Two technical replicates and three biological replicates were used for each gene. The relative fold change in target gene expression was calculated using the formula $${2}^{ \mbox{-} \unicode{x02206}\unicode{x02206}{C}_{{\rm{t}}}}$$ (Livak & Schmittgen, 2001).

Ex planta bacterial growth assays

The ex planta bacterial growth assay was done to investigate the impact of the chimeric peptide application on the growth of individual bacteria from a culture collection maintained in the CSU lab. We selected 81 bacterial isolates that represent dominant microbial groups present in the plant phyllosphere (Trivedi et al., 2020, 2011). We also included Candidatus liberibacter crescens strain BT-1 (BAA-2481^TM), which represents the closest culturable relative of CLas. A precultivated bacterial culture dilution (25 μL corresponding to approximately 1 × 10⁵–2 × 10⁵ bacterial cells (OD₆₀₀ = 0.001]) was mixed with 75 μL of Luria-Bertani broth (ATCC medium 2870 for L. crescens) with or without 25 μM of the UGK17. The bacterial culture was grown in a rotating shaker (200 rpm) at 28°C for 48 h. The cultivation of bacterial strains was replicated three times. The optical density (OD) was measured at 600 nm in 96-well flat-bottom plates (Thermo Fisher Scientific) using the plate reader (Infinite M Nano⁺; Tecan).

The phylogenetic tree was constructed based on the 16S sequences by using Clustal Omega (Madeira et al., 2022). The heat map was generated based on the z-score of the percentage change of the OD₆₀₀. The visualisation of the phylogenetic tree and a heat map was done by ITOL. The significant differences between the cell culture with and without the UGK17 for each strain were determined by Student's t-test at p < 0.05.

Microbiome analysis

The diversity and community structure of bacteria and fungi was determined by amplicon sequencing using an Illumina MiSeq platform. We used the primer sets 515F/806R (Caporaso et al., 2012) and ITS1F/ITS2R (Caporaso et al., 2012) to amplify a portion of the bacterial 16S ribosomal RNA (rRNA) gene and fungal ITS1 region, respectively. The 0.5 μM of mitochondrial peptide nucleic acid and plastid peptide nucleic acid clamps (PNA Bio) per reaction were added to block the 16S rRNA amplification of host mitochondria and chloroplast, respectively. The amplicons were sequenced by an Illumina Miseq platform at the Colorado State University Next Generation Sequencing facility. Bioinformatics processing was performed using a combination of USEARCH (Edgar, 2010) and UNOISE3 (Edgar, 2016). Operational taxonomic units (OTU) tables based on 97% sequence similarity were generated using the USEARCH pipeline. Adapters and primers were removed using cutadapt (Martin, 2011). Then samples were demultiplexed. Paired-end reads were merged, and quality was assessed with fastQC. The merged pairs were discarded if they contained ambiguous nucleotides, had a low (Q < 20) quality score, or were short in length (<100 bp). The representative set database was created using the UCLUST and UPARSE algorithms (Edgar, 2013). Unique sequences were clustered into unique OTUs using DADA2 and DeNoised using uNoise3 as described (Xiong et al., 2021). OTU tables were generated by mapping reads to the representative set database. OTUs were counted at the sample level. Taxonomic identification of bacteria and fungi was obtained against the Silva (Quast et al., 2012) and UNITE database (Nilsson et al., 2019), respectively.

Samples were evaluated separately for bacterial (16S) and fungal (ITS) communities. Samples were rarified to the lowest occupancy of 14,000 and 6800 reads for 16S rRNA and ITS, respectively. We used the R package ‘mctools’ to analyse microbial community structure (Leff, 2017). To examine beta diversity, Bray–Curtis dissimilarity distances were calculated then ordinated in nonmetric multidimensional scaling (NMDS). Permutational multivariate analysis of variance (PERMANOVA) models were generated to determine significant differences between disease infection, age of the leaves, symptoms, and the UGK17 treatment. To examine alpha diversity, Shannon diversity indexes were calculated and evaluated through general linear models. Tukey honestly significant difference tests were used to determine the influence of the above variables on alpha-diversity. Using information from the in vitro experiment, we performed random forest (RF) analysis to identify keystone microbes that are affected by the UGK17 treatments (Trivedi et al., 2017). A significance level of p < 0.05 was used for the impact of the UGK17 application on the structure and diversity of the citrus phyllosphere microbiome as compared to the control.

RESULTS AND DISCUSSION

The host-derived chimeric peptides reduces the CLas titre in the citrus leaves

In this study, C_t value was used to estimate the quantity of the CLas titre in the samples (Grosdidier et al., 2017). Our results demonstrated that the application of chimeric peptide UGK17 significantly increased the C_t value, which can be interpreted as a smaller CLas titre (Figure 2). The C_t values of the healthy citrus leaves was higher than 35 regardless of the peptide treatment, indicating the extremely small number of CLas. We found that the UKG17 application decreased pathogen titre in both the citrus varieties with equal effect. Also, we observed that the UGK17 is equally effective in reducing the CLas titre at both the concentrations used in the study. As expected, asymptomatic leaves have less CLas titre compared to symptomatic leaves. However, both 5 and 25 μM of UGK17 treatment increased the C_t value from the range of 24–35. The increase of C_t value over five can be interpreted as the decrease of colony forming unit ∼100, which is assumed that UGK17 decreased the CLas titre effectively even with low dosage (Grosdidier et al., 2017). Also, the C_t values from asymptomatic leaves treated with the UGK17 reached 35 indicating the protection of citrus leaves against CLas before the manifestation of symptoms. There are several reports on the design and application of host-derived peptides to provide protection against HLB (Basu et al., 2022; Huang et al., 2021; Stover et al., 2013). The chimeric peptide used in this study has previously been reported to decrease CLas titre in grapefruit and showed nontoxicity to human and host cells (Basu et al., 2022; Gupta & Stover, 2020). The effectiveness of peptides in suppressing pathogens depends on various factors including disease severity, scion, and age of leaves (Leekha et al., 2011; Moretta et al., 2021). Here we show that the chimeric peptide can efficiently reduce the CLas titre in the citrus leaves and the bactericidal activity might not be affected by host, maturity of leaves, and severity of symptoms. The reduction in CLas titre is comparable to previous reports using the similar (Basu et al., 2022) or other peptides (Huang et al., 2021). Our results suggest that the chimeric peptide can be one of the potential strategies to manage the HLB by reducing the CLas titre effectively in low dosages.

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The host-derived chimeric peptide enhance the plant's innate immunity-related gene expression

Plants recognise pathogens and protect themselves by plant innate immunity composed of pathogen-associated molecular pattern-triggered immunity (PTI) resulting in the activation of the mitogen-activated protein kinase (MAPK), MAPK kinase, and MAPK kinase kinase (Nishad et al., 2020). While pathogens may suppress PTI by using the effector, effector-triggered immunity activated by Nod-like receptor can enhance the disease resistance (Yuan et al., 2021). Salicylic acid (SA), jasmonic acid, and ethylene are also important signalling hormones as a part of plant innate immunity (Nishad et al., 2020). The activation of plant immune response results to the production of PR proteins which have antimicrobial activity (Ali et al., 2018). As PR proteins are involved in the systemic acquired resistance, they protect plants from reinfection (Durrant & Dong, 2004). It has been proposed that the host antimicrobial peptides may possess the intrinsic ability to directly induce the production of the PR proteins (Jain & Khurana, 2018). We did not test this hypothesis due to the unavailability of the bacteria-free citrus leaves. Here, we monitored the accumulation of PR gene transcripts in the asymptomatic and symptomatic leaves of grapefruit and orange in the UGK17 application treatments and compared the expression with the controls to evaluate the augmentation of plant immune response (Figure 3). As 5 and 25 μM of UGK17 treatment showed same amount of reduction on CLas quantity in Figure 1, we considered the expression from the samples treated with 5 μM of UGK17. The results shown are for the young leaves as we were not able to extract high-quality RNA from the old leaves for both the citrus varieties. For the expression of PR genes, we tested the expression of the PR-1, PR-2, and PR-5 genes, considered markers of plant-immune response (Fu & Dong, 2013; Li et al., 2017; Molinari et al., 2014). The expression of all the PR genes increased with the UGK17 application (at a concentration of 5 μM) for both citrus varieties. We observed higher expression levels of PR genes in symptomatic as compared to the asymptomatic samples for both the citrus varieties. Our result indicates the peptide-mediated increases in the plant immune response as a mechanism to reduce CLas titre. CLas produces an active salicylate hydroxylase and the degradation of SA has been reported to decrease the expression of defence-related genes in citrus (Li et al., 2017). Application of exogenous plant defence inducers reduced HLB severity in field trials (Li et al., 2016). Our results suggest that the chimeric peptide is a dual-functional peptide that can reduce CLas titre and suppress disease symptoms in HLB-positive trees and augments plant's systemic defence responses against new infection.

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Host-derived chimeric peptide has a selectively negative impact on the growth of few bacterial isolates

The HTH peptides prefer to attach to Gram-negative bacterial membranes, particularly pathogens such as CLas (Gupta & Stover, 2020). The bacterial growth assay was done to estimate the specificity of the chimeric peptide to target CLas. In this assay, Liberibacter crescens is used as the closest cultured relative of CLas. Most of the strains were not affected by the UGK17 treatment. The OD₆₀₀ of the L. crescens decreased 58.7% under the UGK17 treatment compared to the control (Figure 4). Four isolates belonging to the family Rhizobiaceae including S. meliloti, Pararhizobium sp., Neorhizobium sp., and Rhizobium acidisoli showed significant decrease in growth with the UGK17 treatment compared to control. However, not all the members of family Rhizobiaceae were impacted by the UGK17 treatments. Interestingly, Gram-positive bacterial isolates such as Streptomyces venezuelae and Arthrobacter pascens showed a significant increase in the growth with the UGK17 treatment compared to the control. Overall, our results suggest that the chimeric peptide will have a direct impact on the growth of pathogen and few related species without impacting the overall bacterial isolates present in the phyllosphere.

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Host-derived chimeric peptide has minimal impact on the phyllosphere microbiome in citrus

The NMDS analysis showed clear separation (PERMANOVA p = 0.001) between healthy and infected (both symptomatic and asymptomatic) phyllosphere-associated bacterial and fungal communities for both the tested varieties with different leaf ages (Figure 5). Thus, our results are per the previous studies that have reported the significant impact of HLB infection and disease severity on leaf microbiome (Srivastava et al., 2022; Wang, Stelinski, et al., 2017; Xu et al., 2018). This is because that HLB causes the imbalanced carbohydrate partitioning and deficiencies of micronutrients such as Fe, Mn, and Zn (Blaustein et al., 2017). As the disease symptom severity is correlated with the CLas titre, the competitive relation between the CLas and native microbiota can affect the microbial community in phyllosphere (Blaustein et al., 2017). The clustering of the UGK17-treated samples at both the concentrations (5 and 25 μM of the UGK17) with the water-treated samples indicated that the UGK17 application has a nonsignificant impact on the bacterial and fungal community associated with the phyllosphere of old and young leaves of citrus and orange (Figure 5). The alpha diversity shown as the Shannon index for both bacterial and fungal community was observed to be similar between both the young and old grapefruit and orange leaves (Figure 6). For most of the cases our results did not show significant (p > 0.05) differences between control and peptide treatments. However, old symptomatic grapefruit leaves had shown a significant decrease in the Shannon index for bacterial and fungal community under the peptide treatment compared to the nontreated leaves. Relative abundance of CLas has been negatively correlated with alpha (Blaustein et al., 2017) therefore decrease in the CLas titre due to the UGK17 treatments can cause increase in microbial diversity in some instances. Machine learning-based RF analysis showed that only three bacterial species responded to the peptide application (CLas, Clostridium sensu stricto and Clostridium aerotolerans). The most prominent was CLas (mean decrease accuracy = 0.31). The other impacted bacterial species only constituted less than 0.5% of the relative abundance of the entire bacterial communities. For fungal communities, we did not observe any indicators for the UGK17 treatments. Our observations indicate that the host-derived chimeric peptides have minimal impact on phyllospheric microbial community structure despite the significant shifts of microbial structure by HLB infection. As the chimeric peptide is also a product of the plant's innate immune responses, the induction of plant defence response will have minimal impact on the native microbiota (Trivedi et al., 2020). Consequently, the chimeric peptide derived from the host might target the CLas without impacting on microbial structure in the citrus phyllosphere.

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CONCLUSION

In this study, the chimeric peptide derived from plants had shown effective reduction of CLas regardless of the scion, age of the leaves, and disease symptoms. Also, the augmentation of PR gene expression with the peptide treatment showed that the peptide can decrease the pathogen titre by enhancing the plant immune response. Moreover, the chimeric peptide has minimal impact on the microbial community structure in phyllosphere with the promotion of the plant-beneficial microbial growth while it specifically targets the pathogen. These findings provide the possibility of the chimeric peptide as a promising solution to cure the infected citrus trees and/or protect the healthy trees against the HLB in the field conditions. Further, the field study with different pathogens or practices for the effective uptake will provide the potential of the host-derived chimeric peptide on large-scale agricultural industry for sustainable agriculture.

AUTHOR CONTRIBUTIONS

Jeongyun Choi: Data collection, formal analysis; investigation; visualization; methodology; writing-original draft; writing–review and editing. Supratim Basu: Data collection, investigation; writing-review and editing. Abigail Thompson: Data collection, investigation; writing–review and editing. Kristen Otto: Data collection, investigation; writing–review and editing. Elena V. Sineva: Data collection, investigation; writing–review and editing. Madhurababu Kunta: Conceptualization; investigation; resources; methodology; funding acquisition; writing–review and editing. Goutam Gupta: Conceptualization; investigation; resources; methodology; supervision; funding acquisition; writing–review and editing. Pankaj Trivedi: Conceptualization; investigation; resources; methodology; supervision; funding acquisition; writing-original draft; writing–review and editing.

ACKNOWLEDGEMENTS

The authors thank Mr. Zachary Tiernan (Colorado State University) for help with lab work. This work was supported by a NIFA Grant (Grant No. 2020-70029-33199; ‘Providing practical solutions for Huanglongbing treatment and prevention’).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The raw sequence data related to this study has been submitted in the NCBI Sequence Read Archive under SRA accessions number PRJNA1011376.

ETHICS STATEMENT

The authors confirm that they have followed the ethical policies of the journal.

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