Candidatus Liberibacter Americanus Research Articles

Molecular identification and characterization of “Candidatus Liberibacter spp.” from major citrus growing region of Malwa, Madhya Pradesh, India

One of the most damaging diseases impacting yearly yield and productivity is huanglongbing, often known as greening of citrus fruits and plants. Huanglongbing is caused by the Candidatus Liberibacter species, of which three primary species are known worldwide: Candidatus Liberibacter africanus (CLaf), Candidatus Liberibacter americanus (CLam), and Candidatus Liberibacter asiaticus (CLas). In the current study, Candidatus Liberibacter infection and the occurrence of huanglongbing disease were examined in the Malwa region of Madhya Pradesh, which is home to the Nagpur Mandarin cultivar. A total of 100 samples were gathered for the current study from 14 villages in the chosen area, primarily Mandsaur, Neemuch, Ratlam, Ujjain, Dewas, Indore, Shajapur, Rajgarh, Dhar, and Jhabua. Here, out of 100 samples, 22 were determined to be positive, while the remaining 100 were negative despite having modest physiological and morphological characteristics that resembled symptoms of HLB. Sequencing results from 08 samples in the current investigation indicate that Candidatus Liberibacter asiaticus (05), Candidatus Liberibacter americanus (02), and Candidatus Liberibacter africanus (01) are all positive. In the study, eight 16S rRNA genes sequences targeting OI1/OI2c were submitted to GenBank with for Candidatus Liberibacter africanus (1) accession no: OQ725636.1 for Candidatus Liberibacter americanus (2) Accession: OQ725635.1 and OQ725634.1 and Candidatus Liberibacter asiaticus (5) for Accession: OQ725633.1, OQ725632.1, OQ725631.1, OQ725630.1 and OQ725629.1. Candidatus Liberibacter species isolated and sequenced in the current study had a greater degree of mutation (41 point mutations), according to single nucleotide polymorphism analysis. Present study provides occurrence and prevalence of Candidatus Liberibacter asiaticus as key causative agent for huanglongbing in the Nagpur Mandarin cultivar. Higher prevalence and occurrence of Candidatus Liberibacter species also affect other agriculture crops and hence a trend and pattern in genomic profile was investigated via SNP analysis that provides details of mutation. Genomic profile of Candidatus Liberibacter species is highly recommended for the development of antibacterial to tackle Candidatus Liberibacter species driven agriculture economical losses.

Gene silencing of cathepsins B and L using CTV-based, plant-mediated RNAi interferes with ovarial development in Asian citrus psyllid (ACP), Diaphorina citri.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D. citri females as a result of knockdown of DCcathB and DCcathL genes mediated by CTV dsRNA. These results showed that Psyllids fed on plants treated with the CTV-dsRNA exhibited downregulation of the Vg gene, one of the most important genes associated with embryogenic and female development, which was associated with dsRNA-mediated silencing of the two cathepsin genes. Based on our findings, a CTV-based strategy for delivering RNAi via plants that targets DCcathB and DCcathL genes may represent a suitable avenue for development of dsRNA-based tools to manage D. citri that limits the spread of HLB.

Trapping and Repellent Techniques for Huanglongbing Management in Citrus Orchards: Innovative Strategies to Combat Vector-Mediated Disease Transmission

The global increase in human population has heightened the demand for citrus fruits and their derivatives, necessitating effective management strategies to mitigate the impact of huanglongbing (HLB) disease. Caused by gram-negative bacteria such as ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’, and ‘Candidatus Liberibacter americanus’, HLB is primarily transmitted by the insect vector Diaphorina citri. While traditional vector suppression methods using insecticides have proven ineffective, innovative approaches are required to control disease spread. This study aims to address the persistent nature of the vector and its ability to transmit HLB throughout its life cycle by emphasizing trapping and repellent techniques. By implementing early detection methods, utilizing disease-resistant citrus varieties, employing disease-free seeds, enhancing nutritional regimes, and reducing vector populations, promising results have been achieved in reducing vector populations and limiting HLB inoculum. The implications of this research underscore the importance of adopting trapping and repellent strategies, alongside the use of disease-free citrus plants, to effectively manage HLB and safeguard citrus plantations worldwide.

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

Differentiation of ‘Candidatus Liberibacter asiaticus’ in Saudi Arabia based on tandem repeat variability in genomic locus

Citrus greening, or huanglongbing, is a destructive disease threatening many citrus worldwide, and drastically altering the global dynamics of the citrus industry. The disease is caused by one of several unculturable bacterial species belonging to ‘Candidatus Liberibacter’. The recent availability of complete genome sequences of ‘Candidatus Liberibacter asiaticus’ (CLas) has facilitated comprehensive assessments of genomic variability using a range of approaches, including short tandem repeat analysis. The objective of this study was to evaluate the genetic diversity of CLas populations in Saudi Arabia based on tandem repeat number (TRN) within the CLIBASIA_01645 locus, predicted to encode the bacteriophage C1 repressor protein. Results indicated that the genotype richness of the Saudi Arabian CLas isolates was conserved by 27% based on the TRN locus. Four different genotypes TRN2, TRN3, TRN4, and TRN5 were identified. However, the TRN2 and TRN5 were the most dominant genotypes. All four of the TRN genotypes were associated with CLas-positive mandarin (Citrus reticulata) or sweet orange (C. sinensis) citrus trees. The diversity (H = 0.69) and evenness (H'=0.914) were overall relatively high, with the northern region of Saudi Arabia harboring the highest diversity (0.7) and evenness score (0.9–1.0). Phylogenetic analysis of the CLas-bacteriophage C1 repressor protein of the Saudi Arabian isolates indicated CLas was more closely related to ‘Candidatus Liberibacter africanus’ than to ‘Candidatus Liberibacter americanus’.

Climate-induced range shifts of invasive species (Diaphorina citri Kuwayama).

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a destructive, invasive species that poses a serious threat to the citrus industry wherever it occurs. The psyllid vectors the phloem-limited bacteria 'Candidatus Liberibacter americanus' and 'Ca. L. asiaticus', causal agents of the incurable citrus greening disease or huanglongbing (HLB). It is essential to understand which regions and areas are suitable for colonization by ACP to formulate appropriate policy and preventive measures. Considering its biology and ecology, we used a machine learning algorithm based on the MaxEnt (Maximum Entropy) principle, to predict the potential global distribution of ACP using bioclimatic variables and elevation. The model predictions are consistent with the known distribution of ACP and also highlight the potential occurrence outside its current ecological range, that is, primarily in Africa, Asia and the Americas. The most important abiotic variables driving the global distribution of ACP were annual mean temperature, seasonality of temperature and annual precipitation. Our findings highlight the need for international collaboration in slowing the spread of invasive pests like D. citri. © 2022 Society of Chemical Industry.

Probing the Application of OmpA-Derived Peptides to Disrupt the Acquisition of 'Candidatus Liberibacter asiaticus' by Diaphorina citri.

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria 'Candidatus Liberibacter asiaticus' (CLas) and 'Candidatus Liberibacter americanus' (CLam) are associated with HLB in Brazil but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid [ACP]) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel, more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison with CLas, suggesting a possible role in host interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides, aiming to evaluate acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5, and Pep6 in artificial diet significantly reduced the acquisition of CLas, whereas increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas, and sweet orange plants stably absorbed and maintained this peptide for as long as 3 months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

Prevalent Transmission of 'Candidatus Liberibacter asiaticus' over 'Ca. Liberibacter americanus' in a Long-Term Controlled Environment.

In Brazil, citrus huanglongbing (HLB) is associated with 'Candidatus Liberibacter americanus' (CLam) and 'Ca. Liberibacter asiaticus' (CLas). However, there are few studies about HLB epidemiology when both Liberibacter spp. and its insect vector, the Asian citrus psyllid (ACP, Diaphorina citri), are present. The objective of this work was to compare the transmission of HLB by ACP when both CLam and CLas are present as primary inoculum. Two experiments were performed under screenhouse conditions from April 2008 to January 2012 (experiment 1) and from February 2011 to December 2015 (experiment 2). The experiments were carried out with sweet orange plants infected with CLam or CLas as inoculum source surrounded by sweet orange healthy plants. One hundred Liberibacter-free adult psyllids were monthly confined to the source of inoculum plants for 7 days with subsequent free movement inside the screenhouse. Fortnightly, nymphs and adults of psyllids were monitored. Psyllid and leaf samples were collected periodically for Liberibacter detection by PCR or quantitative PCR. CLas was detected more frequently than CLam in both psyllid and leaf samples. No mixed infections were detected in the psyllids. A clear prevalence of CLas over CLam was observed in both experiments. The final HLB incidences were 16.7 and 14.5% of Liberibacter-positive test plants, and CLas was detected in 92.3 and 93.1% of these infected plants. Mixed infection was observed only in 3.8% of infected test plants in experiment 1. These results endorse the shift in the prevalence of CLam to CLas observed in citrus orchards of São Paulo, Brazil.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

Understanding Citrus Greening Disease and Its Possible Management Strategies in Nepal

Huanglongbing (HLB), also known as citrus greening, is a devastating disease of citrus that has decimated several citrus orchards throughout the world. The disease is associated with three species of unculturable and phloem-limited bacteriae, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. The most common species of bacteria found in Nepal is Candidatus Liberibacter asiaticus which is transmitted by an insect vector, Asian citrus psyllid (Diaphorina citri). This disease has been detected in several economically important citrus production areas of Nepal, which resulted in heavy yield loss. No cure for the disease has been discovered yet and it is essential to practice proper management strategies to maintain citrus health and sustain citrus production under HLB pressure. Several disease management approaches such as pathogen-free nursery establishment, use of disease tolerant rootstock cultivars, proper irrigation and nutrient supply, removal of HLB affected trees, and control of psyllid with frequent insecticide application are widely practiced throughout the world. This review article highlights the characteristics of the citrus greening disease and its insect vector and gives insights into their management techniques. Several technologically advanced options available to minimize the HLB infection might not be feasible currently in Nepal due to economic and topographic constraints. This article also aims to bring into focus the cost-effective methods that growers in Nepal can practice to mitigate the impact of HLB disease in their citrus orchards. Int. J. Appl. Sci. Biotechnol. Vol 9(4): 227-238.

PM 7/121 (2) ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’.1This Standard should be used in conjunction with PM 7/76 (5) Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2014–09. Revised in 2021–04.

Introduction to Diagnostics Standards

Introduction to Diagnostics Standards

Comparison of the performance of the main real-time and conventional PCR detection tests for ‘Candidatus Liberibacter’ spp., plant pathogenic bacteria causing the Huanglongbing disease in Citrus spp.

Huanglongbing disease affects the Rutaceae family and is associated with three phloem-limited bacterium species: Candidatus Liberibacter asiaticus, africanus and americanus. These species are considered quarantine pathogens in the world, and pose major risks for citrus production and industry. Due to the low titer and the uneven distribution of the bacteria within its host plant, conventional PCR detection protocols can lead to false negative results, especially for early detection. Herein, three real-time PCR diagnostic methods recommended by the EPPO and FAO for asiaticus and africanus species detection were evaluated for their performance and compared with a conventional duplex PCR. Assessments were done as part of an international cooperative project under the EUPHRESCO guidance. Intra-laboratory assessment of the analytical specificity and analytical sensitivity was performed on 33 target or non-target DNA samples and seven target DNA samples were used to determine the sensitivity. Thereafter, repeatability, reproducibility, and concordance odds ratio were assessed on 20 target or non-target DNA samples through a collaborative test performance study organized among eight international laboratories. Results showed that the Li protocol proved to be the best method for asiaticus and africanus species detection, along with the conventional duplex PCR; whereas the Morgan protocol showed high performance only for asiaticus species. Interlaboratory reproducibility was high, suggesting that these real-time PCR methods can be readily transferred to diagnostic laboratories.

The Scary Disease in Citrus: Greening

Turunçgil yeşillenme hastalığı (Huanglongbing-HLB, Greening Disease) turunçgil ağaçlarında ekonomik kayıplara neden olan bakteriyel bir hastalıktır. Hastalığı oluşturan patojenin bilimsel ismi Candidatus liberibacter’dir. HLB ye sebep olan şu ana kadar üç farklı Candidatus tür’ü saptanmıştır. Bunlardan Candidatus liberibacter asiaticus (CLas), dünya genelinde HLB ile ilişkili en yaygın Liberibacter türüdür. Diğer iki tür olan, Candidatus liberibacter americanus (CLam) ve Candidatus liberibacter africanus (CLaf) ise daha az yoğunlukta ve ekolojilere özel olarak yaygınlık gösterirler. Hastalık Hindistan’da 1929 yılında “turunçgillerde geriye ölüm” olarak tanımlanmıştır. Ancak bu hastalık 1890’lı yıllarda Çin de ‘yellow shoot diseas, sarı sürgün hastalığı’ olarak çok iyi bilinmekte olması nedeniyle çoğu bilim insanı hastalığın ilk ortaya çıktığı yeri Çin olarak kabul etmektedir. Turunçgil yeşillenme hastalığına karşı bugüne kadar yüzde yüz etkili herhangi bir mücadele yöntemi bulunamamıştır. Bu da, patojenin ne kadar tehlikeli ve önemli olduğunu ortaya koymaktadır. Hastalık, turunçgil üretim alanlarında veya ülkeler arasında vektör böceklerle, (Afrika Turunçgil Psillidi olan Trioza erytreae Del Guercio, diğer bir Psillid türü olan Asya Psillidi Diaphorina citri Kuwayama) veya enfekteli aşı gözleri ile taşınmaktadır. HLB, özellikle portakal, mandarin, tangelo ve greyfurt fidan veya ağaçlarında yaşa bakmaksızın etkilidir. Enfekteli ağaçlarda ortaya çıkan tipik hastalık belirtileri çinko eksikliği, bazı abiyotik stres faktörlerinin belirtileri veya diğer bazı turunçgil hastalıklarının oluşturduğu belirtiler ile karıştırılabilmektedir.

Long-term, sustained feeding by Asian citrus psyllid disrupts salicylic acid homeostasis in sweet orange

BackgroundPhloem-feeding insects are known to modulate the salicylic acid (SA) signaling pathway in various plant-insect interaction models. Diaphorina citri is a phloem feeding vector of the deadly phytopathogens, Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, and the interactions of D. citri with its host that may modulate plant defenses are not well understood. The objectives of this study were to investigate the molecular mechanisms involved in transcriptional regulation of SA modification and activation of defense-associated responses in sweet orange (Citrus sinensis) exposed to various durations (7-, 14- and 150- days) of continuous feeding by D. citri.ResultsWe quantified expression of genes involved in SA pathway activation and subsequent modification, as well as, associated SA metabolites (SA methyl ester, 2,3-DHBA, and SA 2-O-β-D-glucoside). NPR1 and PR-1 expression was upregulated in plants exposed to continuous feeding by D. citri for 14 days. Expression of BSMT-like, MES1-like and DMR6-like oxygenase, as well as, accumulation of their respective SA metabolites (SA methyl ester, 2,3-DHBA) was significantly higher in plants exposed to continuous feeding by D. citri for 150 days than in those without D. citri infestation. Concomitantly, expression of UGT74F2-like was significantly downregulated and its metabolite, SA 2-β-D-glucoside, was highly accumulated in trees exposed to 150 d of feeding compared to control trees without D. citri.ConclusionsD. citri herbivory differentially regulated transcription and SA-metabolite accumulation in citrus leaves, depending on duration of insect feeding. Our results suggest that prolonged and uninterrupted exposure (150 d) of citrus to D. citri feeding suppressed plant immunity and inhibited growth, which may highlight the importance of vector suppression as part of huanglongbing (HLB) management in citrus.

Host-free biofilm culture of “Candidatus Liberibacter asiaticus,” the bacterium associated with Huanglongbing

Inability to culture the phloem-restricted alpha-proteobacterium “Candidatus Liberibacter asiaticus” (“Ca. L. asiaticus”) or the closely related species (“Candidatus Liberibacter americanus” and “Candidatus Liberibacter africanus”) that are associated with Huanglongbing (HLB) hampers the development of effective long-term control strategies for this devastating disease. Here we report successful establishment and long-term maintenance of host-free “Ca. L. asiaticus” cultures, with the bacterium growing within cultured biofilms derived from infected citrus tissue. The biofilms were grown in a newly designed growth medium under specific conditions. The initial biofilm-based culture has been successfully maintained for over two years and has undergone over a dozen subcultures. Multiple independent cultures have been established and maintained in a biofilm reactor system, opening the door to the development of pure culture of “Ca. L. asiaticus” and the use of genetics-based methods to understand and mitigate the spread of HLB.

Screening nested-PCR primer for 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing and application in Hunan, China.

Citrus Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. Sensitive and accurate assays are vital for efficient prevention of the spread of HLB-associated “Candidatus Liberibacter spp”. “Candidatus Liberibacter spp” that infect Citrus includes “Candidatus Liberibacter asiaticus” (Las), “Candidatus Liberibacter africanus” (Laf) and “Candidatus Liberibacter americanus” (Lam). Of them, Las is the most widespread species. In this study, a set of nested PCR primer pairs were screened to diagnose Las, and the nested PCR method greatly enhanced the sensitivity to detect Las up to 10 times and 100 times compared to qPCR and conventional PCR, respectively. Totally, 1112 samples from 5 different citrus cultivars in 39 different counties and cities were assayed by nested PCR. The results show that 384 samples were HLB-infected; the highest positive detection rate was 79.7% from the lopsided fruit samples, and the lowest positive detection rate was 16.3% from the apical dieback samples. The results indicate that the designed nested PCR primer pairs can detect Las from different symptomatic tissues, different citrus cultivars and different geographic regions. The set of nested PCR primers designed in the present study will provide a very useful supplementation to the current approaches for Las detection.

Molecular Characterization and Detection of 16SrIII Group Phytoplasma Associated with Huanglongbing Symptoms.

When huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' was infecting most of the trees while 'Ca. L. asiaticus' was present in a minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus-growing regions in Brazil. A phytoplasma from the 16SrIX group was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter spp. In this report, HLB samples testing negative for 'Ca. L. asiaticus', 'Ca. L. americanus', and 16SrIX phytoplasma were infected with 16SrIII phytoplasmas. Coinfection with 'Ca. L. asiaticus' and 16SrIII was also found. The 16S ribosomal RNA (rRNA) gene sequences from 22 samples were obtained and sequenced, confirming that the 16SrIII group phytoplasma is associated with HLB symptoms in SP and MG States. Ten single-nucleotide polymorphisms (SNPs) were found in the 1,427-bp 16S rRNA gene sequences from 16SrIII phytoplasmas from citrus, whereas none was detected in 16S rRNA gene sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group-specific primers, sequenced from a subset of nine samples, and assembled into three groups based on eight SNPs. SNPs in 16S rRNA gene and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB-associated microbiomes.

Identification and Distribution of Haplotypes of Diaphorina citri (Hemiptera: Liviidae) in Jamaica and the Caribbean.

Diaphorina citri Kuwayama (Hemiptera: Psyllidae), also known as the Asian citrus psyllid, is a vector of the citrus huanglongbing (HLB) disease. D. citri transmits all three known strains of the HLB pathogen: Candidatus Liberibacter africanus, Candidatus Liberibacter americanus, and Candidatus Liberibacter asiaticus. The study involved 92 psyllids representing the Caribbean Basin and reference samples representing countries within Asia, North America, and South America. This study was aimed at characterizing D. citri on a molecular level in order to determine the haplotype diversity and uniqueness within Jamaica and the Caribbean Basin. D. citri-specific primers were used to amplify an 821 bp gene fragment from the mitochondrial cytochrome c oxidase subunit I gene (mtCOI). The statistical parsimony program, TCS, was used to determine the 12 haplotypes found within the Caribbean, with haplotypes H2 and H7 being the most prominent. The H2 haplotype was found to belong to the South Western Asia group originating from India. H2 represented 54% of the sequenced samples and dominated the Greater Antilles, 22% were grouped as H7, dominating the Lesser Antilles, while the remaining 24% of the sequences were grouped in the remaining 10 haplotypes and were variants seen within the Greater and Lesser Antilles.

Physiologic, Anatomic, and Gene Expression Changes in Citrus sunki, Poncirus trifoliata, and Their Hybrids After 'Candidatus Liberibacter asiaticus' Infection.

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely 'Candidatus Liberibacter asiaticus' (Las), 'Candidatus Liberibacter africanus', and 'Candidatus Liberibacter americanus'. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.