Huanglongbing Research Articles

Detection of citrus greening disease and field efficacy of anti-pathogen chemicals against the disease in mandarin (Citrus reticulata Blanco.) in Gulmi, Nepal

Citrus cultivation in Nepal faces a persistent decline due to the widespread prevalence of Huanglongbing (HLB), also known as Citrus Greening Disease, across citrus-growing regions. This has resulted in significant economic losses for farmers, prompting them to actively seek preventive and remedial measures. In a study conducted at Resunga Municipality and Dhurkot Rural Municipality, Gulmi, aimed at addressing this decline, 51 orchards were assessed for HLB using the starch iodine test. Concurrently, the efficacy of anti-pathogen chemicals in managing citrus greening disease in mandarin (Citrus reticulata Blanco) was evaluated. An experiment was designed, incorporating eight treatments in a Randomized Complete Block Design (RCBD) with three replications. Each replication included eight treatments designated as follows: T1: Neem oil 5ml/l + Lentinan 2ml/l, T2: Neem Oil 5ml/l + B. amyloliquefacians 5ml/l, T3: Neem oil 5ml/l + Pseudomonas 5ml/l, T4: Imidacloprid 0.02% + Copper Oxychloride 0.025%, T5: Imidacloprid 0.02% + Streptocycline 250ppm, T6: B. thuringiensis 2ml/l + Pseudomonas 5ml/l, T7: B. thuringiensis 2ml/l + B. amyloliquefacians 5ml/l, T8: control. Results revealed that among the orchards tested, 18 were positive for HLB, representing 35.2% of the sample size. Notably, plots treated with Neem oil + B. amyloliquefacians and B. thuringiensis + B. amyloliquefacians exhibited the most significant reduction in disease severity compared to the control. Based on these findings, foliar application of Neem oil at a concentration of 5ml/l along with B. amyloliquefacians at 5ml/l, and B. thuringiensis at 2ml/l along with B. amyloliquefacians at 5ml/l at monthly intervals showed promising results in reducing the severity of citrus greening.

Insecticide Efficacy of Green Synthesis Silver Nanoparticles on Diaphorina citri Kuwayama (Hemiptera: Liviidae).

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of Liberibacter asiaticus Jagoueix et al. and Liberibacter americanus Teixeira et al., causal agents of the critical yellow dragon disease or Huanglongbing (HLB), which affects citrus production worldwide. Recently, green synthetic nanoparticles have emerged as a potential alternative to control of agricultural insect pests. The insecticide effect of silver nanoparticles (AgNPs) on 2nd instar nymphs of D. citri under laboratory and greenhouse conditions was evaluated. Mortality was recorded 24, 48, and 72 h after application on D. citri nymphs under both laboratory and greenhouse conditions. The laboratory results showed that AgNPs caused 97.84 and 100% mortality at 32 and 64 ppm, respectively, 72 h after treatment. In the greenhouse, AgNPs caused 78.69 and 80.14% mortality using 64 and 128 ppm 72 h after application. This research is the first to evaluate the green synthesis AgNPs on D. citri and are a promising strategy to control the pest.

An examination of trends in U.S. orange and grapefruit juice consumption

AbstractThe average U.S. consumer has been drinking less orange and less grapefruit juice over time. Per capita consumption is down more than 50% since the mid‐2000s. In this study, we propose and estimate a count data model. The number of times an adult consumes 100% orange or 100% grapefruit juice over a 2‐day period is modeled as a function of income, product prices, and some characteristics of the consumer such as level of education, being on a diet, and taking nutrient supplements that proxy for health concerns and nutrition knowledge. Data used in the study were collected between January 2005 and March 2020 on over 30,000 adult consumers. Tests for overdispersion favor a negative binomial model instead of the more restrictive Poison regression model. Estimation results confirm that rising juice product prices and concern over product healthfulness have been contributing to long run trends in orange and grapefruit juice consumption. Prices for these citrus juices have increased in recent years due in part to the citrus greening disease and hazardous weather in Florida. Trends in orange and grapefruit juice consumption can impact consumer diet quality, fruit growers, and agribusiness firms responsible for transforming raw fruit into juice and juice products. [EconLit Citations: D12, Q13].

Candidatus Liberibacter asiaticus influences the emergence of the Asian citrus psyllid Diaphorina citri by regulating key cuticular proteins.

The Asian citrus psyllid, Diaphorina citri, is the primary vector of the HLB pathogen, Candidatus Liberibacter asiaticus (CLas). The acquisition of CLas shortens the developmental period of nymphs, accelerating the emergence into adulthood and thereby facilitating the spread of CLas. Cuticular proteins (CPs) are involved in insect emergence. In this study, we investigated the molecular mechanisms underlying CLas-promoted emergence in D.citri via CP mediation. Here, a total of 159 CP genes were first identified in the D.citri genome. Chromosomal location analysis revealed an uneven distribution of these CP genes across the 13 D.citri chromosomes. Proteomic analysis identified 54 differentially expressed CPs during D.citri emergence, with 14 CPs exhibiting significant differential expression after CLas acquisition. Five key genes, Dc18aa-1, Dc18aa-2, DcCPR-24, DcCPR-38 and DcCPR-58, were screened from the proteome and CLas acquisition. The silencing of these 5 genes through a modified feeding method significantly reduced the emergence rate and caused various abnormal phenotypes, indicating the crucial role that these genes play in D.citri emergence. This study provides a comprehensive overview of the role of CPs in D.citri and reveals that CLas can influence the emergence process of D.citri by regulating the expression of CPs. These key CPs may serve as potential targets for future research on controlling huanglongbing (HLB) transmission.

Citrus-mediated gene silencing of cytochrome P450 suppresses insecticide resistance and increases mortality in Diaphorina citri.

Asian citrus psyllid, Diaphorina citri, is a hemipteran that vectors the causal pathogen of citrus greening disease, or huanglongbing (HLB). HLB is a tree killing disease that has severely limited citrus production globally. Unfortunately, there is no cure for this disease, and mitigation depends on multiple insecticide applications to reduce vector populations. Silencing of cytochrome P450 expression associated with detoxification enzymes by RNA interference is known to increase susceptibility of D. citri to insecticides. However, dsRNA was previously introduced into psyllids by topical applications. The possible application of this technology for pest management will require effective field delivery of the dsRNA. Therefore, we evaluated a virus vector (Citrus tristeza virus; 'mild strain' T36) to deliver gene silencing directly to this sap-sucking insect via plant phloem. Citrus macrophylla plants inoculated with CTV expressing a truncated consensus sequence of CYP450 (CTV-tCYP450) constantly produced small interfering RNA in the plant phloem that targeted five cytochrome p540 (CYP450) genes in D. citri. Insecticide susceptible D. citri reared on citrus infected with CTV-tCYP450 were subsequently more susceptible to imidacloprid, fenpropathrin, carbaryl, and chlorpyrifos than those reared on citrus infected with wildtype CTV or non-infected negative controls. Additionally, nymph survival and adult lifespan were significantly reduced when psyllids were reared on CTV-tCYP450 citrus plants compared with controls. Interestingly, similar results were obtained after one and two generations of rearing. Finally, field-collected psyllids from areas with known broad-spectrum insecticide resistance were rendered more susceptible to imidacloprid and fenpropathrin after feeding on CTV-tCYP450 citrus trees as compared with those reared on controls. The integration of citrus-mediated RNA inference targeting psyllid detoxification enzymes could function as a resistance management tool and reduce insecticide input in an integrated pest management program for HLB. © 2024 Society of Chemical Industry.

Fruit Size as an Indicator of Fruit Drop in Huanglongbing-affected ‘Valencia’

Increased preharvest fruit drop rates and reduced fruit sizes are common symptoms associated with Huanglongbing (HLB) in sweet orange. Small fruit may be more likely to drop during preharvest fruit drop. The objective of the current study was to determine whether fruit size could be used as an indicator of the preharvest fruit drop likelihood. Nearly 1400 fruit were surveyed over the following three timepoints across two years: 11 Mar 2022, 15 Apr 2022, and 22 Mar 2023. Each fruit was evaluated to determine the equator and peduncle diameter, fruit detachment force (FDF), type of detachment (mechanically broken or abscission), and lopsidedness. The total soluble solids content was determined for a subset of fruit. The FDF was consistently lower in abscising fruit than in mechanically broken fruit, suggesting that the physiological process of abscission had begun in these fruit and that they were more likely to drop. The fruit diameter was significantly smaller in abscising fruit than in mechanically broken fruit on 11 Mar 2022 and 22 Mar 2023, but not on 15 Apr 2022. Similarly, the fruit diameter and FDF were also significantly and positively correlated, but this relationship was weaker at time points late in the season (closer to harvest). These findings suggest that small fruit are indeed more likely to drop early in the preharvest period. The abscising fruit had total soluble solids contents similar to those of the mechanically broken fruit. Therefore, we hypothesized that the smaller fruit ceased growing and are able to respond to abscission signals earlier than the larger fruit. Therefore, any strategies to mitigate fruit drop, such as the use of plant growth regulators, should be applied early in the season when the fruit are still actively growing. When growth ceases, the fruit are vulnerable to drop.

Citrus rootstock breeding in an HLB-endemic environment with an industry in crisis

The arrival (2005) and rapid establishment in Florida of the most devastating disease known to citriculture, namely Huanglongbing or Citrus Greening Disease, has caused dramatic changes to the long-term citrus rootstock breeding program at the University of Florida’s Citrus Research and Education Center (UF-CREC). Since all commercial scion/rootstock combinations have proven to be susceptible to the disease, there is an urgent need to quickly develop improved rootstocks with good horticultural attributes that can mitigate the disease in trees grafted with commercial scions. This report describes our efforts to achieve this goal. Our approach is to rapidly develop and screen a large population of genetically-diverse hybrid rootstock candidates. Multiple available sources of potential rootstock germplasm are being exploited in our efforts. We have identified several good rootstock candidates that are showing improved HLB tolerance. These are now being propagated either by seed or micropropagation as needed for advanced multi-site trials with multiple scions. This research offers potential for the ultimate solution to HLB, leading to sustainable and profitable citriculture in HLB-endemic areas

“Extreme hybrids” from the Australian citrus rootstock breeding program

Citrus is one of the most genetically diverse fruit trees used by humans, and yet few rootstock breeders have ventured beyond a narrow range of parents. For the last 25 years we have explored wider graft and sexual compatibility within the citrus subfamily (Aurantioideae) than any previous attempts, and have identified new genera and species for rootstock breeding. Whilst the vast majority of this work has met with failure, it has identified factors such as sexual incompatibility, late-acting lethality and poor field adaptation as the reasons why many citrus relatives are not currently amenable to rootstock breeding. A major breakthrough early in the program was the discovery that Oceania citrus species are extremely sensitive to Citrus tristeza virus (CTV) when used as rootstocks, and the realisation that resistance must be introgressed if we are to ever discover useful traits masked by this disease sensitivity. Bridging hybrids were required to transfer this virus resistance and a newly discovered species (Citrus wakonai) was employed to speed-up the process. After three generations of crossing, our “extreme hybrids” with Oceania parentage now show commercial performance equivalent to industry standards. By providing citrus growers with a range of high-performance rootstocks from extremely complex genetic backgrounds we can increase the biological diversity of orchards without compromising production. Our hope is that these “extreme hybrids” may help in the battle against Huanglongbing (HLB) disease.

Enhancing Plant Disease Recognition on Mobile Devices: A Hybrid Approach with CNNs, Rule-Based Systems, and Machine Learning

The food industry has significantly boosted the agricultural economy of India, historically known as the largest producing nation with a strong agricultural identity. The primary crops include grains, fruits (such as potatoes, oranges, tomatoes), sugarcane, and specially cultivated grains and cotton. Maharashtra, a state in India, has experienced substantial economic growth, particularly attributed to citrus and cotton industries. This growth has generated employment opportunities and holds significant potential for the state's economic advancement. To sustain the prosperity of these industries, the government is actively addressing concerns related to disease control, Labor costs, and global market dynamics. In recent times, citrus canker, citrus greening, and black spots on cotton have emerged as severe threats to citrus crops in Maharashtra. Farmers are troubled by the costs associated with tree loss, scouting efforts, and the use of chemicals to control diseases. An automated detection system could play a crucial role in preventing and minimizing these losses, thereby safeguarding the industries, farmers, and the overall economy. This research focuses on developing disease detection through pattern recognition methods for various crops. The image detection process has some main categories which are image acquisition, image processing, and pattern recognition. Image preprocessing is used to process the image in a clear and clean format. Pattern recognition methods are then utilized to classify samples into different crop conditions. To assess classification approaches, results will be compared across different crops for disease detection. The goal is to demonstrate a classification accuracy surpassing existing models, reaching the highest. The research primarily aims to showcase the feasibility of disease detection based on visible symptoms on fruits or leaves. Data collection and initial knowledge acquisition are planned through offline and online approaches. The overall motive is to have accurate detection of the fruits and leaves thereafter using those results for diseases detection. Keywords: Machine Learning Algorithms, Plant disease detection, Image Recognition, Rule-Based mechanism, CNN, Detection technologies, Color Grading

Comparative Transcriptome and sRNAome Analysis Suggest Coordinated Citrus Immune Responses against Huanglongbing Disease.

Citrus Huanglongbing (HLB), caused by the phloem-inhibiting bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease, intimidating citrus production worldwide. Although commercially cultivated citrus cultivars are vulnerable to CLas infection, HLB-tolerant attributes have, however, been observed in certain citrus varieties, suggesting a possible pathway for identifying innate defense regulators that mitigate HLB. By adopting transcriptome and small RNAome analysis, the current study compares the responses of HLB-tolerant lemon (Citrus limon L.) with HLB-susceptible Shatangju mandarin (Citrus reticulata Blanco cv. Shatangju) against CLas infection. Transcriptome analysis revealed significant differences in gene expression between lemon and Shatangju. A total of 1751 and 3076 significantly differentially expressed genes were identified in Shatangju and lemon, respectively. Specifically, CLas infected lemon tissues demonstrated higher expressions of genes involved in antioxidant enzyme activity, protein phosphorylation, carbohydrate, cell wall, and lipid metabolism than Shatangju. Wet-lab experiments further validated these findings, demonstrating increased antioxidant enzyme activity in lemon: APX (35%), SOD (30%), and CAT (64%) than Shatangju. Conversely, Shatangju plants exhibited higher levels of oxidative stress markers like H2O2 (44.5%) and MDA content (65.2%), alongside pronounced ion leakage (11.85%), than lemon. Moreover, microscopic investigations revealed that CLas infected Shatangju phloem exhibits significantly more starch and callose accumulation than lemon. Furthermore, comparative sRNA profiles revealed the potential defensive regulators for HLB tolerance. In Shatangju, increased expression of csi-miR166 suppresses the expression of disease-resistant proteins, leading to inadequate defense against CLas. Conversely, reduced expression of csi-miR166 in lemon plants enables them to combat HLB by activating disease-resistance proteins. The above findings indicate that when infected with CLas, lemon exhibits stronger antioxidative activity and higher expression of disease-resistant genes, contributing to its enhanced tolerance to HLB. In contrast, Shatangju shows lower antioxidative activity, reduced expression of disease-resistant genes, significant ion leakage, and extensive callose deposition, possibly related to damage to plant cell structure and blockage of phloem sieve tubes, thereby promoting the development of HLB symptoms.

A Combinatorial Single-Molecule Real-Time and Illumina Sequencing Analysis of Postembryonic Gene Expression in the Asian Citrus Psyllid Diaphorina citri.

Huanglongbing (HLB) is a systemic plant disease caused by 'Candidatus Liberibacter asiaticus (CLas)' and transmitted by Diaphorina citri. D. citri acquires the CLas bacteria in the nymph stage and transmits it in the adult stage, indicating that molting from the nymph to adult stages is crucial for HLB transmission. However, the available D. citri reference genomes are incomplete, and gene function studies have been limited to date. In the current research, PacBio single-molecule real-time (SMRT) and Illumina sequencing were performed to investigate the transcriptome of D. citri nymphs and adults. In total, 10,641 full-length, non-redundant transcripts (FLNRTs), 594 alternative splicing (AS) events, 4522 simple sequence repeats (SSRs), 1086 long-coding RNAs (lncRNAs), 281 transcription factors (TFs), and 4459 APA sites were identified. Furthermore, 3746 differentially expressed genes (DEGs) between nymphs and adults were identified, among which 30 DEGs involved in the Hippo signaling pathway were found. Reverse transcription-quantitative PCR (RT-qPCR) further validated the expression levels of 12 DEGs and showed a positive correlation with transcriptome data. Finally, the spatiotemporal expression pattern of genes involved in the Hippo signaling pathway exhibited high expression in the D. citri testis, ovary, and egg. Silencing of the D. citri transcriptional co-activator (DcYki) gene significantly increased D. citri mortality and decreased the cumulative molting. Our results provide useful information and a reliable data resource for gene function research of D. citri.

Induction of Somatic Embryogenesis of Pontianak Siamese Orange Cotyledon Cultures on Murashige Skoog Media with the Addition of 2,4-D and Kinetin

Conventional citrus propagation has weaknesses, one of which is susceptible to CVPD(Citrus Vein Phloem Degeneration) and other diseases which are the main cause of the decline in population and production of pontianak siam orange plants in West Kalimantan. One alternative to in vitro propagation of citrus seedlings is the induction of somatic embryogenesis. The purpose of the study was to obtain the best combination of 2,4-D and kinetin concentrations for callus induction and somatic embryo induction from Pontianak siam orange cotyledon explants, observing the developmental phase (stage) of embryos formed. The implementation of the study used a factorial completely randomized design (CRD) consisting of 2 factors. The first factor is the concentration of 2,4-D consisting of 4 concentration levels, namely 0; 0.5; 1; 1.5; 2 mg/L. The second factor is kinetin concentration consisting of 4 concentration levels, namely 0; 0.5; 1; 1.5; 2 mg/L.The observation parameters include the time of callus emergence (hst), the percentage of explants forming callus (%), the texture and color of the callus, observing the stage of the embryo found. The results showed that 72% of treatments were able to induce callus from pontianak siam orange cotyledons with an average callus appearance at 7 hst. The callus formed is compact and crumbly with varied callus colors, namely white, yellowish, greenish and brownish. The best treatment that produces embryogenic callus is the combination of D0.5K0.5; D1K1; D0.5K1.5; D1.5K1.5 and D2K2 with the characteristics of crumbly callus and yellowish and brownish colors. The results of microscopic observations found that the somatic embryo phase formed was the pro-embryo phase.

Chromosome-Scale, De Novo, Phased Genome Assemblies of Three Australian Limes: Citrus australasica, C. inodora, and C. glauca.

Huanglongbing (HLB) is a severe citrus disease worldwide. Wild Australian limes like Citrus australasica, C. inodora, and C. glauca possess beneficial HLB resistance traits. Individual trees of the three taxa were extensively used in a breeding program for over a decade to introgress resistance traits into commercial-quality citrus germplasm. We generated high-quality, phased, de novo genome assemblies of the three Australian limes using PacBio long-read sequencing. The genome assembly sizes of the primary and alternate haplotypes were determined for C. australasica (337 Mb/335 Mb), C. inodora (304 Mb/299 Mb), and C. glauca (376 Mb/379 Mb). The nine chromosome-scale scaffolds included 86-91% of the genome sequences generated. The integrity and completeness of the assembled genomes were estimated to be at 97.2-98.8%. Gene annotation studies identified 25,461 genes in C. australasica, 27,665 in C. inodora, and 30,067 in C. glauca. Genes belonging to 118 orthogroups were specific to Australian lime genomes compared to other citrus genomes analyzed. Significantly fewer canonical resistance (R) genes were found in C. inodora and C. glauca (319 and 449, respectively) compared to C. australasica (576), C. clementina (579), and C. sinensis (651). Similar patterns were observed for other gene families associated with potential HLB resistance, including Phloem protein 2 (PP2) and Callose synthase (CalS) genes predicted in the Australian lime genomes. The genomic information on Australian limes developed in the present study will help understand the genetic basis of HLB resistance.

The Use and Impact of Antibiotics in Plant Agriculture: A Review.

Growers have depended on the specificity and efficacy of streptomycin and oxytetracycline as a part of their plant disease arsenal since the middle of the 20th century. With climate change intensifying plant bacterial epidemics, the established success of these antibiotics remains threatened. Our strong reliance on certain antibiotics for devastating diseases eventually gave way to resistance development. Although antibiotics in plant agriculture equal to less than 0.5% of overall antibiotic use in the United States, it is still imperative for humans to continue to monitor usage, environmental residues, and resistance in bacterial populations. This review provides an overview of the history and use, resistance and mitigation, regulation, environmental impact, and economics of antibiotics in plant agriculture. Bacterial issues, such as the ongoing Huanglongbing (citrus greening) epidemic in Florida citrus production, may need antibiotics for adequate control. Therefore, preserving the efficacy of our current antibiotics by utilizing more targeted application methods, such as trunk injection, should be a major focus. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Enterobacteriaceae as a Key Indicator of Huanglongbing Infection in Diaphorina citri.

Extensive microbial interactions occur within insect hosts. However, the interactions between the Huanglongbing (HLB) pathogen and endosymbiotic bacteria within the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) in wild populations remain elusive. Thus, this study aimed to detect the infection rates of HLB in the ACP across five localities in China, with a widespread prevalence in Ruijin (RJ, 58%), Huidong (HD, 28%), and Lingui (LG, 15%) populations. Next, microbial communities of RJ and LG populations collected from citrus were analyzed via 16S rRNA amplicon sequencing. The results revealed a markedly higher microbial diversity in the RJ population compared to the LG population. Moreover, the PCoA analysis identified significant differences in microbial communities between the two populations. Considering that the inter-population differences of Bray-Curtis dissimilarity in the RJ population exceeded those between populations, separate analyses were performed. Our findings indicated an increased abundance of Enterobacteriaceae in individuals infected with HLB in both populations. Random forest analysis also identified Enterobacteriaceae as a crucial indicator of HLB infection. Furthermore, the phylogenetic analysis suggested a potential regulatory role of ASV4017 in Enterobacteriaceae for ACP, suggesting its possible attractant activity. This research contributes to expanding the understanding of microbial communities associated with HLB infection, holding significant implications for HLB prevention and treatment.

Economic and Social Impact of Huanglongbing on the Mexico Citrus Industry: A Review and Future Perspectives

The citrus industry is of great importance in Mexico, with an estimated production value of USD 2.4 billion and the potential to generate up to 28 million employees per year. Approximately 69,000 Mexican families depend on this sector. However, it is under serious threat from the disease known as Huanglongbing (HLB). The rapid spread of this disease has caused significant economic losses, impacted the livelihoods of citrus growers, and led to substantial job losses. Currently, HLB is not under control in Mexico, and as the management strategies applied have proven to be ineffective, yields have not been recovered to pre-HLB levels, and production costs have significantly increased. Therefore, it remains the most destructive citrus disease. This review paper describes the current situation of the disease, its economic and social impact, and the measures adopted for its management in Mexico. Future perspectives highlight advances in research based on new biotechnological tools as an eco-friendly management alternative. Practical suggestions to address HLB are also mentioned in our analysis.

Dynamics of 'Candidatus Liberibacter asiaticus' Growth, Concentrations of Reactive Oxygen Species, and Ion Leakage in Huanglongbing-Positive Sweet Orange.

Citrus Huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves-late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

The beta pore-forming bacterial pesticidal protein Tpp78Aa1 is toxic to the Asian citrus psyllid vector of the citrus greening bacterium

The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri, but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC50 of approximately 204µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri-active proteins may provide valuable tools for suppression of D. citri populations.

Reduction of Wolbachia in Diaphorina citri (Hemiptera: Liviidae) increases phytopathogen acquisition and decreases fitness.

Wolbachia pipientis is a maternally inherited intracellular bacterium that infects a wide range of arthropods. Wolbachia can have a significant impact on host biology and development, often due to its effects on reproduction. We investigated Wolbachia-mediated effects in the Asian citrus psyllid, Diaphorina citri Kuwayama, which transmits Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease. Diaphorina citri are naturally infected with Wolbachia; therefore, investigating Wolbachia-mediated effects on D. citri fitness and CLas transmission required artificial reduction of this endosymbiont with the application of doxycycline. Doxycycline treatment of psyllids reduced Wolbachia infection by approximately 60% in both male and female D. citri. Psyllids treated with doxycycline exhibited higher CLas acquisition in both adults and nymphs as compared with negative controls. In addition, doxycycline-treated psyllids exhibited decreased fitness as measured by reduced egg and nymph production as well as adult emergence as compared with control lines without the doxycycline treatment. Our results indicate that Wolbachia benefits D. citri by improving fitness and potentially competes with CLas by interfering with phytopathogen acquisition. Targeted manipulation of endosymbionts in this phytopathogen vector may yield disease management tools.

Climate change mitigation and seasonal infestation patterns of citrus psyllid Diaphorina citri: implications for managing huanglongbing (HLB) disease in tangerine citrus

The citrus psyllid, Diaphorina citri, is a recognized vector of the devastating Huanglongbing (HLB) disease in citrus plants, posing a significant threat to global citrus production. As climate change continues to influence agroecological conditions, comprehending the population dynamics of this vector is imperative for effective disease management. This study delved into the relationship between climate variations and D. citri infestation patterns in tangerine citrus orchards from January to December 2018. Leveraging a dataset of 12 monthly observation sets, we comprehensively analyzed the abundance of the citrus psyllid population. Our investigation unveiled a clear association between climate variability and the incidence of citrus psyllid infestations and HLB occurrences. Importantly, our findings revealed that D. citri infestations were confined to the budding phase. Contrary to expectations, rainfall and temperature did not correlate positively with the development of the D. citri population or the prevalence of HLB disease in tangerine citrus. These results suggest other factors significantly affect HLB spread, impacting disease management. Further research is needed to identify and understand these factors, providing valuable insights for mitigating climate change’s impact on citrus cultivation.