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Reactive oxygen species (ROS) are pivotal in signal transduction processes in plant–pathogen interactions. The ROS signaling pathways involved in Candidatus Liberibacter asiaticus (CLas) and Xanthomonas citri subspecies citri (Xcc) infections in Citrus sinensis (sweet orange) are unclear. In this study, we comprehensively identified ROS metabolism-associated genes, including 9 NADPH oxidase (RBOH), 14 superoxide dismutase (SOD), 1 catalase (CAT), 9 peroxiredoxin (PrxR), 5 ascorbate peroxidase (APX), 4 glutathione peroxidase (GPX), 3 monodehydroascorbate reductase (MDAR), 2 dehydroascorbate reductase (DHAR), 2 glutathione reductase (GR), 24 thioredoxin (Trx), and 18 glutaredoxin (GLR) genes in C. sinensis. An analysis revealed variable gene structures but conserved motifs and domains in ROS subfamilies. A comparative synteny analysis with Arabidopsis thaliana and Vitis vinifera indicated evolutionary conservation of most ROS metabolism-associated genes, with some originating from gene duplication events post-species divergence in C. sinensis. Expression profiling revealed five up-regulated genes and four down-regulated genes during both CLas and Xcc infections. Promoter analysis revealed numerous stress-responsive elements in the promoter of ROS metabolism-associated genes. Protein–protein interaction network analysis highlighted the involvement of ROS metabolism in various biological processes. A comparison of ROS metabolism-associated genes between C. sinensis and Poncirus trifoliata indicated multiple gene gain and loss events within ROS subfamilies of C. sinensis. This study enhances our understanding of ROS metabolism in C. sinensis and sheds light on citrus–pathogen interactions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Huanglongbing (HLB) is the most devastating citrus disease across Africa, America and Asia. It is associated with three species of bacteria belonging to the genus 'Candidatus Liberibacter', transmitted by the psyllid vector Trioza erytreae, among others. This insect has spread throughout part of the Iberian Peninsula, threatening citrus orchards with the possible arrival and transmission of HLB. Currently, there are no curative methods of control for the disease, so finding plant material less suitable for psyllids can reduce the risk of spreading. We have evaluated how the citrus combination, rootstocks (Flying dragon, 'Cleopatra' mandarin, Carrizo citrange, Forner-Alcaide no.5, Forner-Alcaide no.517 and Citrus macrophylla) and scion cultivars ('Star Ruby', 'Clemenules', 'Navelina', 'Valencia Late', 'Fino 49' and 'Ortanique') determine the biology of T. erytreae. Our results show that T. erytreae behaves differently depending on the combination, and is the least suitable for its survival with 'Ortanique' on Forner-Alcaide no. 517 and 'Valencia Late' on Carrizo citrange. The psyllid preferred 'Fino 49' grafted onto Carrizo citrange for egg laying rather than Flying Dragon. Trioza erytreae (Del Guercio, 1918) (Hemiptera: Triozidae) is a citrus pest which produces gall symptoms on leaves and transmits bacteria associated with the citrus disease Huanglongbing, 'Candidatus Liberibacter' spp. In the present work, the biology and behaviour of T. erytreae were studied in different rootstock–cultivar combinations. Six rootstocks were used, Flying dragon (FD), 'Cleopatra' mandarin (CL), Carrizo citrange (CC), Forner-Alcaide no.5 (FA5), Forner-Alcaide no.517 (FA517) and Citrus macrophylla (CM), and six scion cultivars: 'Star Ruby', 'Clemenules', 'Navelina', 'Valencia Late', 'Fino 49' and 'Ortanique'. Survival and oviposition were evaluated in a no-choice trial, and preference in a choice trial, all of them under greenhouse conditions. Trioza erytreae did not show a clear settle preference for any citrus combination. However, it was able to lay more eggs in 'Fino 49' grafted on CC than on FD. In terms of survival, 'Ortanique' grafted onto FA5 was more suitable than when grafted onto FA517, and in the case of 'Valencia Late', when it was grafted onto CM rather than CC. Our results showed that T. erytreae behave differently depending on the citrus combination. [ABSTRACT FROM AUTHOR]

Citri Reticulatae Pericarpium, especially the pericarp of Citrus reticulata Blanco cv. Chachiensis (PCRC), is an important edible and medicinal ingredient for health and pharmacological properties. Citrus Huanglongbing, a devastating disease that currently threatens the citrus industry worldwide, is caused by a phloem-limited alpha-proteobacterium, "Candidatus Liberibacter asiaticus" (CLas). The industry of cultivar Chachiensis has been suffering from HLB. Although HLB affected the quality of citrus fruit, whether the quality of PCRC was affected by HLB remains unclear. In this study, we compared the metabolite profiles between HLB-affected and healthy PCRC from three sources: fresh, 6-month-old, and 9-year-old PCRC, through the untargeted LC–MS method. Compared to healthy controls, various types of bioactive compounds, mainly flavonoids, terpenoids, alkaloids, coumarins, polysaccharides, and phenolic acids, accumulated in HLB-affected PCRC, especially in the HLB-affected 9-year PCRC. In particular, isorhamnetin, isoliquiritigenin, luteolin 7-O-beta-D-glucoside, limonin, geniposide, pyrimidodiazepine, scoparone, chitobiose, m-coumaric acid, malonate, and pantothenic acid, which contributed to the pharmacological activity and health care effects of PCRC, were highly accumulated in HLB-affected 9-year-old PCRC compared to the healthy control. Multibioassay analyses revealed that HLB-affected 9-year-old PCRC had a higher content of total flavonoids and total polyphenols and exhibited similar antioxidant capacity as compared to healthy controls. The results of this study provided detailed information on the quality of HLB-affected PCRC. [ABSTRACT FROM AUTHOR]

Citrus is affected by many diseases, and hence, the movement of citrus propagative materials is highly regulated in the USA. Currently used regulatory pathogen detection methods include biological and laboratory-based technologies, which are time-consuming, expensive, and have many limitations. There is an urgent need to develop alternate, rapid, economical, and reliable testing methods for safe germplasm exchange. Citrus huanglongbing (HLB) has devastated citrus industries leading to an increased need for germplasm exchanges between citrus growing regions for evaluating many potentially valuable hybrids for both HLB resistance and multilocational performance. In the present study, Next-Generation Sequencing (NGS) methods were used to sequence the transcriptomes of 21 test samples, including 15 well-characterized pathogen-positive plants. A workflow was designed in the CLC Genomics Workbench software, v 21.0.5 for bioinformatics analysis of the sequence data for the detection of pathogens. NGS was rapid and found to be a valuable technique for the detection of viral and bacterial pathogens, and for the discovery of new citrus viruses, complementary to the existing array of biological and laboratory assays. Using NGS methods, we detected beet western yellows virus, a newly reported citrus virus, and a variant of the citrus yellow vein-associated virus associated with the "fatal yellows" disease. [ABSTRACT FROM AUTHOR]

Candidatus Liberibacter asiaticus (CLas) is associated with Citrus Huanglongbing (HLB), a devastating disease in the US. Previously, we conducted a two-year-long monthly HLB survey by quantitative real-time PCR using root DNA fractions prepared from 112 field grapefruit trees grafted on sour orange rootstock. Approximately 10% of the trees remained CLas-free during the entire survey period. This study conducted 16S metagenomics using the time-series root DNA fractions, monthly prepared during twenty-four consecutive months, followed by microbial co-occurrence network analysis to investigate the microbial factors contributing to the CLas-free phenotype of the aforementioned trees. Based on the HLB status and the time when the trees were first diagnosed as CLas-positive during the survey, the samples were divided into four groups, Stage H (healthy), Stage I (early), II (mid), and III (late) samples. The 16S metagenomics data using Silva 16S database v132 revealed that HLB compromised the diversity of rhizosphere microbiota. At the phylum level, Actinobacteria and Proteobacteria were the predominant bacterial phyla, comprising >93% of total bacterial phyla, irrespective of HLB status. In addition, a temporal change in the rhizosphere microbe population was observed during a two-year-long survey, from which we confirmed that some bacterial families differently responded to HLB disease status. The clustering of the bacterial co-occurrence network data revealed the presence of a subnetwork composed of Streptomycetaceae and bacterial families with plant growth-promoting activity in Stage H and III samples. These data implicated that the Streptomycetaceae subnetwork may act as a functional unit against HLB. [ABSTRACT FROM AUTHOR]

Due to human activities and changes in land use, the spatiotemporal pattern of citrus has undergone significant changes after the outbreak of Huanglongbing (HLB). We selected time-series Sentinel-2 images to delineate citrus orchard areas following the onset of HLB. This was conducted to extract citrus orchards in Xunwu County between 2017 and 2022. The spatial and temporal patterns and their influencing factors were investigated by spatial analysis. Results show (1) a notable decline in total citrus orchard area by 216.70 km2, primarily witnessed in orchards without insect-proof screens (IPS), shifting towards cropland, bush, and IPS areas. Contrastingly, citrus orchards with IPS exhibited a modest increase from 7.82 km2 to 111.39 km2, predominantly converting from areas lacking IPS, cropland, and bare land. (2) Spatial distribution patterns revealing a "cold in the south and hot in the north" trend. Orchards without IPS are concentrated in central and northern regions, while those with IPS are clustered predominantly in the north, with a recent shift towards the northeast. (3) Landscape analysis indicating a trend of fragmentation of citrus orchards, while a gradual dispersion of orchards without IPS and those with IPS showcased enhanced concentration and aggregation. (4) Orchards with IPS predominantly occupy regions characterized by an elevation ranging between 300 m and 400 m, primarily in the southeast, southwest, and southern directions. These areas exhibit slopes averaging between 10° and 15°, with surface temperatures ranging from 18 °C to 26 °C. Additionally, these orchards tend to be situated in proximity to impervious surfaces and roads. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB; also known as citrus greening) is the most destructive bacterial disease of citrus worldwide with no known sustainable cure yet. Herein, we used non-targeted metabolomics and transcriptomics to prove that γ-aminobutyric acid (GABA) accumulation might influence the homeostasis of several metabolic pathways, as well as antioxidant defense machinery, and their metabolism-related genes. Overall, 41 metabolites were detected in 'Valencia' sweet orange (Citrus sinensis) leaf extract including 19 proteinogenic amino acids (PAA), 10 organic acids, 5 fatty acids, and 9 other amines (four phenolic amines and three non-PAA). Exogenous GABA application increased most PAA in healthy (except L-threonine, L-glutamine, L-glutamic acid, and L-methionine) and 'Candidatus L. asiaticus'-infected citrus plants (with no exception). Moreover, GABA accumulation significantly induced L-tryptophan, L-phenylalanine, and α-linolenic acid, the main precursors of auxins, salicylic acid (SA), and jasmonic acid (JA), respectively. Furthermore, GABA supplementation upregulated most, if not all, of amino acids, phenolic amines, phytohormone metabolism-related, and GABA shunt-associated genes in both healthy and 'Ca. L. asiaticus'-infected leaves. Moreover, although 'Ca. L. asiaticus' induced the accumulation of H2O2 and O2•− and generated strong oxidative stress in infected leaves, GABA possibly stimulates the activation of a multilayered antioxidative system to neutralize the deleterious effect of reactive oxygen species (ROS) and maintain redox status within infected leaves. This complex system comprises two major components: (i) the enzymatic antioxidant defense machinery (six POXs, four SODs, and CAT) that serves as the front line in antioxidant defenses, and (ii) the non-enzymatic antioxidant defense machinery (phenolic acids and phenolic amines) that works as a second defense line against 'Ca. L. asiaticus'-induced ROS in citrus infected leaves. Collectively, our findings suggest that GABA might be a promising alternative eco-friendly strategy that helps citrus trees battle HLB particularly, and other diseases in general. [ABSTRACT FROM AUTHOR]

Ageratum conyzoides (A. conyzoides) is commonly found or intentionally planted in citrus orchards due to its ability to provide habitat and breeding grounds for the natural enemies of citrus pests. This study aims to expand from a switching Huanglongbing model by incorporating the effects of A. conyzoides, vector preferences for settling, and pesticide application intervals on disease transmission. Additionally, we establish the basic reproduction number R 0 and its calculation for a general switching compartmental epidemic model. Theoretical findings demonstrate that the basic reproduction number serves as a threshold parameter to characterize the dynamics of the models: if R 0 < 1 , the disease will disappear, whereas if R 0 > 1 , it will spread. Numerical results indicate that the recruitment rate of A. conyzoides not only affects the spread speed of Huanglongbing but also leads to paradoxical effects. Specifically, in cases of high infection rates, a low recruitment rate of A. conyzoides can result in a decrease, rather than an increase, in the basic reproduction number. Conversely, a high recruitment rate can accelerate the spread of Huanglongbing. Furthermore, we show how different vector bias and pesticide spraying periods affect the basic reproduction number. [ABSTRACT FROM AUTHOR]

The devastating citrus disease, Huanglongbing (HLB), is associated with 'Candidatus Liberibacter sp.' and transmitted by citrus psyllids. Unfortunately, HLB has no known sustainable cure yet. Herein, we proposed γ-aminobutyric acid (GABA) as a potential eco-friendly therapeutic solution to HLB. Herein, we used GC/MS-based targeted metabolomics combined with gene expression to investigate the role of GABA in citrus response against HLB and to better understand its relationship(s) with different phytohormones. GABA supplementation via root drench boosts the accumulation of endogenous GABA in the leaves of both healthy and 'Ca. L. asiaticus'-infected trees. GABA accumulation benefits the activation of a multi-layered defensive system via modulating the phytohormone levels and regulating the expression of their biosynthesis genes and some pathogenesis-related proteins (PRs) in both healthy and 'Ca. L. asiaticus'-infected plants. Moreover, our findings showed that GABA application stimulates auxin biosynthesis in 'Ca. L. asiaticus'-infected plants via the activation of the indole-3-pyruvate (I3PA) pathway, not via the tryptamine (TAM)-dependent pathway, to enhance the growth of HLB-affected trees. Likewise, GABA accumulation was associated with the upregulation of SA biosynthesis genes, particularly the PAL-dependent route, resulting in higher SA levels that activated CsPR1, CsPR2, CsPR5, and CsWRKY70, which are prominent to activation of the SA-mediated pathway. Additionally, higher GABA levels were correlated with an enhanced JA profile and linked with both CsPR3 and CsPR4, which activates the JA-mediated pathway. Collectively, our findings suggest that exogenous GABA application might be a promising alternative and eco-friendly strategy that helps citrus trees battle HLB. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), caused by the Candidatus Liberibacter spp., is the most devastating disease in the citrus industry. HLB significantly affects and alters the microbial community structure or potential function of the microbial community of leaves and roots. However, it is unknown how the microbial community structure of the pericarp with different pigments is affected by Candidatus Liberibacter asiaticus (CLas). This study identified the enriched taxa of the microbial community in the citrus pericarp with normal or abnormal pigment and determine the effects of HLB on the pericarp microbial community using 16S rRNA-seq. The alpha and beta diversity and composition of microbial communities were significantly different between normal and abnormal pigment pericarp tissues of ripe fruits infected by CLas. Firmicutes, Actinobacteriota, Bacteroidota, Acidobacteriota, and Desulfobacterota dominated the pericarp microbiota composition in WDYFs (whole dark yellow fruits) samples. The relative abundance of most genera in WDYFs was higher than 1%, such as Burkholderia, and Pelomonas. However, with the exception of the HLB pathogen, the relative abundance of most genera in the abnormal-colored pericarp samples was less than 1%. CLas decreased the relative abundance of pericarp taxonomic. The predicted function of microbial was more plentiful and functional properties in the WDYF sample, such as translation, ribosomal structure and biogenesis, amino acid transport and metabolism, energy production and conversion, and some other clusters of orthologous groups (COG) except for cell motility. The results of this study offer novel insights into understanding the composition of microbial communities of the CLas-affected citrus pericarps and contribute to the development of biological control strategies for citrus against Huanglongbing. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a vascular disease of Citrus caused by three species of the α-proteobacteria "Candidatus Liberibacter", with "Candidatus Liberibacter asiaticus" (CLas) being the most widespread and the one causing significant economic losses in citrus-producing regions worldwide. However, Persian lime (Citrus latifolia Tanaka) has shown tolerance to the disease. To understand the molecular mechanisms of this tolerance, transcriptomic analysis of HLB was performed using asymptomatic and symptomatic leaves. RNA-Seq analysis revealed 652 differentially expressed genes (DEGs) in response to CLas infection, of which 457 were upregulated and 195 were downregulated. KEGG analysis revealed that after CLas infection, some DEGs were present in the plant–pathogen interaction and in the starch and sucrose metabolism pathways. DEGs present in the plant–pathogen interaction pathway suggests that tolerance against HLB in Persian lime could be mediated, at least partly, by the ClRSP2 and ClHSP90 genes. Previous reports documented that RSP2 and HSP90 showed low expression in susceptible citrus genotypes. Regarding the starch and sucrose metabolism pathways, some genes were identified as being related to the imbalance of starch accumulation. On the other hand, eight biotic stress-related genes were selected for further RT-qPCR analysis to validate our results. RT-qPCR results confirmed that symptomatic HLB leaves had high relative expression levels of the ClPR1, ClNFP, ClDR27, and ClSRK genes, whereas the ClHSL1, ClRPP13, ClPDR1, and ClNAC genes were expressed at lower levels than those from HLB asymptomatic leaves. Taken together, the present transcriptomic analysis contributes to the understanding of the CLas-Persian lime interaction in its natural environment and may set the basis for developing strategies for the integrated management of this important Citrus disease through the identification of blanks for genetic improvement. [ABSTRACT FROM AUTHOR]

Diaphorina citri Kuwayama (D. citri) is an insect vector of phloem-limited 'Candidatus Liberibacter asiatus' (CLas), the presumed pathogen of citrus Huanglongbing (HLB). Recently, our lab has preliminarily found it acquired and transmitted Citrus tristeza virus (CTV), which was previously suggested to be vectored by species of aphids. However, the influences of one of the pathogens on the acquisition and transmission efficiency of the other pathogen remain unknown. In this study, CLas and CTV acquisition and transmission by D. citri at different development stages under field and laboratory conditions were determined. CTV could be detected from the nymphs, adults, and honeydew of D. citri but not from the eggs and exuviates of them. CLas in plants might inhibit CTV acquisition by D. citri as lower CTV–positive rates and CTV titers were detected in D. citri collected from HLB-affected trees compared to those from CLas–free trees. D. citri were more likely to obtain CTV than CLas from host plants co-infected with the two pathogens. Intriguingly, CTV in D. citri facilitated the acquisition and transmission of CLas, but CLas carried by D. citri had no significant effect on the transmission of CTV by the same vector. Molecular detection and microscopy methods confirmed the enrichment of CTV in the midgut after a 72-h acquisition access period. Collectively, these results raise essential scientific questions for further research on the molecular mechanism of pathogen transmission by D. citri and provide new ideas for the comprehensive prevention and control of HLB and CTV. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is one of the most severe citrus diseases in the world, causing huge economic losses. However, efficient methods of protecting citrus from HLB have not yet been developed. microRNA (miRNA)-mediated regulation of gene expression is a useful tool to control plant diseases, but the miRNAs involved in regulating resistance to HLB have not yet been identified. In this study, we found that miR171b positively regulated resistance to HLB in citrus. Upon infection with HLB bacteria, the bacteria were detected in the second month in the control plants. However, in the miR171b-overexpressing transgenic citrus plants, the bacteria could not be detected until the 24th month. RNA-seq data indicated that multiple pathways, such as photosynthesis, plant–pathogen interaction, the MAPK signaling pathway, etc., might be involved in improving the resistance to HLB in miR171b-overexpressing plants compared with the control. Finally, we determined that miR171b could target SCARECROW-like (SCL) genes to downregulate their expression, which then led to promoted resistance to HLB stress. Collectively, our results demonstrate that miR171b plays a positive regulatory role in resistance to citrus HLB, and provides a new insight into the role of miRNAs in the adaptation of citrus to HLB stress. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a highly contagious and devastating citrus disease that causes huge economic losses to the citrus industry. Because it cannot be cured, timely detection of the HLB infection status of plants and removal of diseased trees are effective ways to reduce losses. However, complex HLB symptoms, such as single HLB-symptomatic or zinc deficiency + HLB-positive, cannot be identified by a single reflection imaging method at present. In this study, a vision system with an integrated reflection–transmission image acquisition module, human–computer interaction module, and power supply module was developed for rapid HLB detection in the field. In reflection imaging mode, 660 nm polarized light was used as the illumination source to enhance the contrast of the HLB symptoms in the images based on the differences in the absorption of narrow-band light by the components within the leaves. In transmission imaging mode, polarization images were obtained in four directions, and the polarization angle images were calculated using the Stokes vector to detect the optical activity of starch. A step-by-step classification model with four steps was used for the identification of six classes of samples (healthy, HLB-symptomatic, zinc deficiency, zinc deficiency + HLB-positive, magnesium deficiency, and boron deficiency). The results showed that the model had an accuracy of 96.92% for the full category of samples and 98.08% for the identification of multiple types of HLB (HLB-symptomatic and zinc deficiency + HLB-positive). In addition, the classification model had good recognition of zinc deficiency and zinc deficiency + HLB-positive samples, at 92.86%. [ABSTRACT FROM AUTHOR]

In Florida, almost all citrus trees are affected with Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas). We characterized various parameters of HLB-affected sweet orange trees in response to yield-improving nutritional treatment, including canopy volume, canopy density and CLas Ct values, and found that the treatment improved yield and maintained canopy density for over three years, whereas untreated HLB-affected trees declined in canopy density. The nutritional treatment did not affect CLas titer or the tree canopy volume suggesting that canopy density is a better indicator of fruit yield. To further validate the importance of canopy density, we evaluated three independent orchards (different in tree age or variety) to identify the specific traits that are correlated with fruit yields. We found that canopy density and fruit detachment force (FDF), were positively correlated with fruit yields in independent trials. Canopy density accurately distinguished between mild and severe trees in three field trials. High and low producing HLB trees had the same Ct values. Ct values did not always agree with CLas number in the phloem, as visualized by transmission electron microscopy. Our work identifies canopy density as an efficient trait to predict yields of HLB-affected trees and suggests canopy health is more relevant for yields than the CLas population. [ABSTRACT FROM AUTHOR]

Citrus are one of the most relevant fruit crops in the Mediterranean basin, which is the second-highest citrus-growing region in the world. However, these crops are mainly grown under Mediterranean semi-arid conditions, characterized by long periods of drought and torrential rain. In this work, we have assessed the response of three promising HLB-tolerant citrus rootstocks (Orange-14, UFR-1, and B11R3T27) to the application of four water stress conditions (Control, Mild water stress, Drought, and Flooding), comparing them with Carrizo citrange. Aerial plant symptoms were recorded during the experimental period, whereas plant water parameters, including stomatal conductance, leaf water potential, and relative water content, were obtained at the end of the assay. For all assessed rootstocks and variables, drought treatment was the most limiting factor, with Carrizo citrange being the most suitable rootstock under this condition. Flooding was the second restrictive treatment, in which UFR-1 was the least affected rootstock. Mid-water stress with 50% water requirements did not differ from the Control treatment, which can help save water resources in semi-arid regions. This information can be helpful for the citrus industry to increase the efficiency of citrus crops subject to water stress in semi-arid regions. [ABSTRACT FROM AUTHOR]

"Candidatus Liberibacter asiaticus" (CLas) is a phloem-restricted α-proteobacterium that is associated with citrus huanglongbing (HLB), which is the most destructive disease that affects all varieties of citrus. Although midrib is usually used as a material for CLas detection, we recently found that the bacterium was enriched in fruits, especially in the fruit pith. However, no study has revealed the molecular basis of these two parts in responding to CLas infection. Therefore, we performed transcriptome and UHPLC–MS-based targeted and untargeted metabolomics analyses in order to organize the essential genes and metabolites that are involved. Transcriptome and metabolome characterized 4834 differentially expressed genes (DEGs) and 383 differentially accumulated metabolites (DAMs) between the two materials, wherein 179 DEGs and 44 DAMs were affected by HLB in both of the tissues, involving the pathways of phenylpropanoid biosynthesis, phytohormone signaling transduction, starch and sucrose metabolism, and photosynthesis. Notably, we discovered that the gene expression that is related to beta-glucosidase and endoglucanase was up-regulated in fruits. In addition, defense-related gene expression and metabolite accumulation were significantly down-regulated in infected fruits. Taken together, the decreased amount of jasmonic acid, coupled with the reduced accumulation of phenylpropanoid and the increased proliferation of indole-3-acetic acid, salicylic acid, and abscisic acid, compared to leaf midribs, may contribute largely to the enrichment of CLas in fruit piths, resulting in disorders of photosynthesis and starch and sucrose metabolism. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is an important citrus disease associated with the phloem-limited, uncultured bacterium 'Candidatus Liberibacter asiaticus'(CLas). Effective treatments against CLas have to be validated in the field, however, methods for the field assessment of treatment effectiveness are time-consuming, in part because DNA-based assays, including quantitative PCR (qPCR), cannot differentiate between live and dead bacterial DNA. The aim of this study was to develop a method for rapid the evaluation of HLB therapies in field experiments. To this aim, a DNA extraction method from citrus leaf tissues with propidum monoazide (PMA), a dye that binds covalently to dsDNA making it unavailable for amplification in subsequent qPCR reactions, was optimized. The results indicated that the efficacy of PMA-qPCR was highly dependent on the primer set used. Primers targeting the 16S region of CLas showed a clear distinction between qPCR from PMA-treated and non-treated samples, while the RNR and LJ900 primers did not show significant differences between the DNA extraction methods. The PMA-qPCR viability analysis of CLas from citrus cuttings treated with different ampicillin (Amp) concentrations showed that all concentrations reduced CLas titers significantly starting 4 days after the initial treatment, unlike the water treatment, which did not show any change. This method was used for assessing the antibacterial activity of Amp, Streptomycin, Oxytetracycline (OTC), and a water control in field tests. The PMA-qPCR results indicated that Amp and OTC displayed significant antibacterial activity against CLas by 8 days post-injection, which was not detected in the non-PMA qPCR analysis. This method could allow the rapid validation of treatments against CLas in field experiments and facilitate the implementation of effective management strategies against HLB. [ABSTRACT FROM AUTHOR]

Background: Huanglongbing (HLB) is currently one of the most devasting diseases in citrus plants worldwide. Resistance against its causal agent, Candidatus Liberibacter asiaticus (CLas), in commercial Citrus species remains a challenge, even though they show differences in CLas multiplication. Methods: A total of 14 citrandarins and their parents (Sunki mandarin and Poncirus trifoliata cv. Rubidoux) were top-grafted onto the canopy of potted 'Valencia' sweet orange plants with high CLas titers. The grafted genotypes were tested for CLas infection and physiological effects of the disease (starch accumulation and callose deposition) for 12 months. Results: All tested genotypes were infected by CLas during the time frame of the experiment. However, a decrease in the infection rate ranging from 50% to 80% for the hybrids H109, H126, H157, and H222 was observed 360 days from the top-grafting. CLas was undetected by real-time PCR in H106 at the end of the experiment, which had low levels of starch and callose deposition. Conclusions: CLas infected all of the tested citrandarins, but a decrease in the infection rate over time was detected for some specific genotypes, which led to less starch accumulation and callose deposition. [ABSTRACT FROM AUTHOR]

Simple Summary: The severe Asian form of huanglongbing is the most serious disease of citrus in Malaysia. It is caused by a phloem-limited bacterium 'Candidatus Liberibacter asiaticus' and transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama. Minimising the devastating impacts of the disease requires effective integrated pest management (IPM) programs to limit detrimental environmental and health consequences of over-reliance on synthetic pesticides. Key components of such programs include grower awareness, planting of pathogen-free trees, monitoring psyllid and disease incidence, removal of diseased trees, and judicious use of insecticides, mineral oils, and natural enemies to suppress incidence of the psyllid. Significant progress has been made towards implementing the programs. The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, transmits 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium associated with the severe Asian form of huanglongbing (HLB), and the most destructive disease of citrus. The pathogen and the psyllid, both of South Asian origin, are now widespread in citrus regions of Asia and the Americas. There is no cure for the disease. Application of synthetic pesticides, in some instances more frequently than fortnightly, to minimise incidence of ACP in citrus orchards, has not prevented inevitable impacts of the disease in regions of Asia where CLas is present. Despite the inevitable spread of the disease, significant progress has been made in Sarawak since the mid-1990s towards effectively implementing integrated pest management (IPM) programs for stemming the impact of the disease and detrimental consequences of over-reliance on synthetic pesticides. Growers are encouraged to plant pathogen-free trees, remove diseased trees, monitor incidence of the psyllid, and to use pesticides judiciously to reduce their detrimental impacts on natural enemies. Knowledge has been enhanced through research on seasonal incidence of the psyllid, use of mineral oils, development of protocols and iodine–starch test kits for detecting infected trees, PCR for confirming the presence of CLas in symptomatic leaves, methods for monitoring incidence the psyllid, and training extension staff and growers. However, major impediments to increasing the average longevity of trees beyond <5 years in poorly managed orchards, based on marcotting (air layering), and >12 years in well-managed orchards, based on pathogen-free trees, still need to be addressed. These include grower knowledge, marcotting, aggressive marketing of synthetic pesticides, high prices of mineral oils, spray application procedures, and better reliance on natural enemies of the psyllid. [ABSTRACT FROM AUTHOR]

This system was not well characterized in I D. citri. i Our comprehensive in silico and bioinformatic analyzes using protein-protein BLAST of I Drosophila melanogaster i as a model against I D. citri i in the NCBI and I Diaphorina citri i OGS v2.0 protein databases revealed seven Toll pathway-related genes in I D. citri. In addition, the presence of a death domain in the Tube protein of I D. citri i and I D. melanogaster i does not require their association with a protein kinase family protein, as death domains are observed in many proteins lacking catalytic function or a protein kinase domain, and this result agrees with findings by Towb et al. [[78]]. The BLASTp search of I D. melanogaster i produced significant similarities to the embryonic polarity protein Dorsal-like isoform X1 (XP 017305228.1), embryonic polarity protein Dorsal-like isoform X2 (XP 026675878.1) and embryonic polarity protein Dorsal-like isoform X3 (XP 017305229.1). In our protein-protein BLAST search of the Dif/dI protein sequence from I D. melanogaster i against the I D. citri i , both genes hit the same protein of embryonic polarity protein Dorsal-like. The protein sequences from Pelle gene were identified using protein-protein BLAST (BLASTp) using Pelle gene (Pll, AAA28750.1) from D. melanogaster, as a query sequence against D. citri and other insects available in GeneBank, the National Center for Biotechnology Information website (NCBI,, 15 April 2021). [Extracted from the article]

Huanglongbing (HLB) is an obstinate disease in the citrus industry. No resistant citrus resources were currently available, but various degrees of Huanglongbing tolerance exist in different germplasm. Citrus junos is emerging as one of the popular rootstocks widely used in the citrus production. However, its responses to the HLB causal agent, Candidatus Liberibacter asiaticus (CLas), were still elusive. In the current study, we investigated the physiological, anatomical, and metabolomic responses of a C. junos rootstock 'Pujiang Xiangcheng' by a controlled CLas grafting inoculation. The summer flushes and roots were impaired at 15 weeks after inoculation, although typical leaf symptomatic phenotypes were not obvious. The chlorophyll pigments and the photosynthetic rate were compromised. The phloem sieve tubes were still working, despite the fact that the callose was deposited and the starch granules were accumulated in the phloem cells. A wide, targeted metabolomic analysis was carried out to explore the systematic alterations of the metabolites at this early stage of infection in the leaves and root system. The differentially accumulated metabolites in the CLas-affected leaves and roots compared with the mock-inoculation control tissues revealed that distinct responses were obvious. Besides the commonly observed alteration of sugar and amino acids, the active break down of starch in the roots was discovered. The different types of fatty acids were altered in the two tissues, with a more pronounced content decline in the roots. Our results not only provided fundamental knowledge about the response of the C. junos rootstock to the HLB disease, but also presented new insights into the host–pathogen interaction in the early stages. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), which is caused by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus,' is an economically important disease of citrus in many regions of the world. Due to the significant damage caused by the HLB disease in recent years, the use of antibiotics was recommended for the therapy of this destructive disease. Products with active ingredients oxytetracycline and streptomycin have been approved for the control of the HLB via foliar application. However, previous work raised questions about the efficacy of foliar delivery of antibiotics in the field. In this study, we examined the effects of a variety of adjuvants on the uptake of oxytetracycline and streptomycin using the foliar application. We also compared the efficiency of foliar application of oxytetracycline and streptomycin with trunk injection. The 'Ca. L. asiaticus' titers in citrus plants were measured using quantitative PCR, and the levels of antibiotics were determined using the ELISA assay. Our results include extremely low levels of oxytetracycline and streptomycin in leaves that were covered during foliar application, indicating that neither streptomycin nor oxytetracycline was successfully systemically delivered by foliar application even after being mixed with adjuvants. Likewise, the 'Ca. L. asiaticus' titer0 was not affected by any of the foliar applications. High levels of streptomycin were detected in leaves that were exposed to direct foliar application, indicating that streptomycin was adsorbed or bound to citrus leaves. On the other hand, the trunk injection of oxytetracycline resulted in high levels of this antibiotic in leaves and significantly reduced the level of 'Ca. L. asiaticus' titer in citrus trees. Unfortunately, the trunk injection of streptomycin resulted in low levels of streptomycin in citrus leaves and did not affect the 'Ca. L. asiaticus' titer, indicating that streptomycin was either bound in the xylem of citrus trees or it was not applied in sufficient quantity required for the inhibition of 'Ca. L. asiaticus.' Taken together, our current results demonstrated that foliar application of oxytetracycline and streptomycin did not effectively deliver antibiotics in citrus despite using adjuvants. Our results also suggested that oxytetracycline could be more effective against the HLB pathogen than streptomycin, which is possibly due to differences between the two in systemic movement in citrus trees. [ABSTRACT FROM AUTHOR]

'Candidatus Liberibacter asiaticus' (CLas) is a bacterium that causes Huanglongbing, also known as citrus greening, in citrus plants. 'Candidatus Liberibacter solanacearum' (Lso) is a close relative of CLas and in the US it infects solanaceous crops, causing zebra chip disease in potato. Previously, we have identified the Lso hypothetical protein effector 1 (Lso-HPE1). This protein uses a signal peptide for secretion; disrupts programmed cell death; and interacts with tomato RAD23c, d, and e proteins, but not with RAD23a. In this study, we evaluated whether CLIBASIA_00460, the CLas homolog of Lso-HPE1 interacted with citrus RAD23 proteins and disrupted their programmed cell death. Based on the yeast two-hybrid assay results, CLIBASIA_00460 interacted with citrus RAD23c and RAD23d, but not with citrus RAD23b. These results were confirmed using bimolecular fluorescence complementation assays, which showed that these interactions occurred in cell puncta, but not in the nucleus or cytoplasm. Additionally, CLIBASIA_00460 was able to disrupt the PrfD1416V-induced hypersensitive response. Therefore, based on the similar interactions between Lso-HPE1 and CLIBASIA_00460 with the host RAD23 proteins and their ability to inhibit cell death in plants, we propose that these effectors may have similar functions during plant infection. [ABSTRACT FROM AUTHOR]

The citrus industry has been threatened by Huanglongbing (HLB) for over a century. Here, an HLB-induced Arabidopsis RPM1-interacting protein 4 (RIN4) homologous gene was cloned from Citrus clementina, and its characteristics and function were analyzed to determine its role during citrus–Candidatus Liberibacter asiaticus (CLas) interactions. Quantitative real-time PCR showed that RIN4 was expressed in roots, stems, leaves and flowers, with the greatest expression level in leaves. Its expression was suppressed by gibberellic acid, indole-3-acetic acid, salicylic acid and jasmonic acid treatments, but was induced by abscisic acid and salt treatments, as well as wounding. The transient expression of a RIN4-GFP showed that RIN4 was localized in the cell membrane. RIN4-overexpressing transgenic C. maxima cv. 'Shatianyou' plants were obtained, and some transgenic plants showed greater sensitivity to CLas infection and earlier HLB symptoms appearance than non-transgenic controls. Results obtained in this study indicated that the upregulated expression of RIN4 in HLB diseased citrus may aid CLas infection. [ABSTRACT FROM AUTHOR]

Simple Summary: Citrus cultivation is affected in many parts of the world because of a devastating disease, huanglongbing (HLB) or citrus greening. In Florida, nearly all commercial citrus is compromised due to HLB, and the disease has spread to other citrus-growing regions of the United States, California and Texas. In California, testing Asian citrus psyllids (ACPs) for the HLB pathogen has been an essential part of integrated pest management. ACP and HLB surveys are essential for disease management in areas where HLB is not widespread. We developed improved ACP traps that can be deployed in the field along with the standard yellow sticky traps. The reusable traps were designed with Rhinoceros computer software and a 3D printer. These traps can be deployed for several months and provide a dynamic sampling mechanism for an improved disease survey strategy. In the present study, ACPs from the 3D-printed traps are collected in a preservative and appear suitable for HLB testing. The evaluation of traps in Florida and California under laboratory, greenhouse, and field conditions indicates that the 3D-printed traps can capture ACPs with about the same efficiency as the sticky traps. They are easy to handle and provide an important field tool for HLB management. Citrus huanglongbing (HLB) is a severe problem for citrus cultivation. The disease management programs benefit from improved field tools suitable for surveying the ACP vector (Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae)) and the associated pathogen. In the present study, we utilize three-dimensional (3D) printers and design tools to develop traps that can capture and preserve ACPs. Three novel, 3D-printed traps were designed and evaluated: stem trap, and cylinder traps 1 and 2. The traps and yellow sticky cards were deployed weekly for 8 months in 2 non-commercial citrus groves in Florida; in California, the traps were evaluated for 12 months in field cages and 4 citrus groves. The stem traps captured lower numbers of ACPs at all experimental sites compared to the cylinder traps. Capture rates in the cylinder traps were comparable to the sticky trap, making the device a viable tool for monitoring field ACPs. The two main advantages of using the reusable 3D traps over standard methods of ACP and HLB surveys include dynamic sampling that can be conducted year-round and the capture of ACPs that can be preserved and tested. Improved trapping may facilitate quick management decisions and mitigate HLB. [ABSTRACT FROM AUTHOR]

The "citrus greening disease" or huanglongbing (HLB) is caused by the Gram-negative bacteria Candidatus Liberibacter spp. One vector of HLB, the African citrus psyllid Trioza erytreae (del Guercio, 1918), was recorded for the first time in 2014, in mainland Spain, and since then, it has spread throughout the Iberian Peninsula. Spiders could be natural enemies of T. erytreae. In this work, we assessed the potential of a model spider species, selected according to the most abundant guild in the field, as a natural enemy of T. erytreae. Theridiidae was the most successful family, capturing adults of T. erytreae in the field. Kochiura aulica (Theridiidae) was selected as a model for functional response assays in the laboratory. The effect of web-building plasticity on the functional response of K. aulica was investigated using two types of arena. The spiders developed tubular-like or branch-like webs according to the spatial constraints. The web architecture significantly shifted the trapping efficiency of K. aulica. Functional responses for tubular and branch webs were both Type-II, but more psyllid prey were captured and killed in the tubular webs than in branch webs. In addition, significantly more psyllids survived in branch-like webs. Our results suggest that web architecture is a key factor driving the number of captured adults of the psyllid. Space web builders could be successful natural enemies of T. erytreae, although further research is needed to clarify the influence of web-building plasticity on the functional response and trapping efficiency of spiders. [ABSTRACT FROM AUTHOR]

Citrus species, including sweet oranges, grapefruits, pomelos, and lemons, are the most widely cultivated trees and consumed fruits worldwide. In citrus orchard management, the control of Huanglongbing (HLB) disease and its insect vector Diaphorina citri (Asian citrus psyllid, ACP) represents a major global challenge. Consumers have been increasingly pushing the citrus production chain toward a more sustainable system, including stringent measures to prevent the use of chemical pesticides. In recent years, biotechnological advances have offered safe and environmentally friendly alternatives for crop production. Technologies such as RNA interference (RNAi)-mediated gene silencing have emerged as innovative tools for agricultural pest management. Here, we provide an overview of RNAi as a promising approach for ACP control and discuss the associated challenges. Despite the availability of specific silencing sequences aimed at a target gene of the insect pest, the uptake of double-stranded RNA is limited in hemipteran insects. In this context, improved delivery methods, stability maintenance, and RNAi response are considered the factors contributing to the increased effectiveness of exogenous RNAi against hemipteran pests. These approaches can serve as potential tools for efficient ACP control. [ABSTRACT FROM AUTHOR]

The plant rhizosphere microbiome is known to play a vital role in plant health by competing with pathogens or inducing plant resistance. This study aims to investigate rhizosphere microorganisms responsive to a devastating citrus disease caused by ‘Candidatus Liberibacter asiaticus’ (CLas) infection, by using 16S rRNA sequencing and metagenome technologies. The results show that 30 rhizosphere and 14 root bacterial genera were significantly affected by CLas infection, including 9 plant resistance-associated bacterial genera. Among these, Amycolatopsis, Sphingopyxis, Chryseobacterium, Flavobacterium, Ralstonia, Stenotrophomonas, Duganella, and Streptacidiphilus were considerably enriched in CLas-infected roots, while Rhizobium was significantly decreased. Metagenome analysis revealed that the abundance of genes involved in carbohydrate metabolism, such as glycolysis, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, was significantly reduced in the CLas-infected citrus rhizosphere microbial community. Likewise, the abundance of genes involved in phosphoinositide signaling and phosphoinositide metabolism, which play important roles in energy metabolism (such as carbohydrate metabolism and lipid metabolism), was also decreased in the CLas-infected samples. Taken together, our results indicate that CLas infection could affect the resistance potential and energy metabolism of the citrus rhizosphere microbial community, which may help us to understand the rhizosphere responses to plant disease and thus facilitate the development and application of antagonistic microorganism products in citrus industry. [ABSTRACT FROM AUTHOR]

Heat treatment has been applied in previous studies to treat diseased plants and trees affected by heat-sensitive pathogens. Huanglongbing (HLB) is a heat-sensitive pathogen and the optimal temperature-time for treating HLB-affected citrus trees was estimated to be 54 °C for 60 to 120 s from indoor experimental studies. However, utilizing this method in orchards is difficult due to technical difficulties to effectively apply heat. Recently, a mobile thermotherapy system (MTS) was developed to in-field treat HLB-affected trees. This mobile device includes a canopy cover that covers the diseased tree and a system to supply steam under the cover to treat the tree. It was proven that the temperature inside the canopy cover can reach the desired one (i.e., 54 °C) to kill bacteria. However, for HLB, the heat should penetrate the tree's phloem where the bacteria live. Therefore, measuring the heat penetration inside the tree is very critical to evaluate the performance of the MTS. In this study, a heat transfer model was developed to simulate the heat penetration inside the tree and predict the temperature in the phloem of the diseased tree during the in-field heat treatment. The simulation results were compared with in-field experimental measurements. The heat transfer model was developed by a comparative analysis of the experimental data using the ANSYS software. Results showed that the temperature in the phloem was 10-40% lower than the temperature near the surface of the bark. Simulation results were consistent with experimental results, with an average relative error of less than 5%. [ABSTRACT FROM AUTHOR]

Sweet oranges in Puerto Rico and other citrus-producing areas in the world have been greatly affected by the Huanglongbing disease (HLB). Historically, most of Puerto Rico's citrus production has been located from 0-600 m above sea level, where fruit acquire a desirable color and flavor due to climatic conditions. However, higher populations of the disease vector Diaphorina citri Kuwayama have been reported at these elevations in Puerto Rico. Although only 6.4% of the land area is located above 600 m, it is composed mainly of environmentally sensitive or non-arable land where Inceptisols are the dominant soil order. For that reason, 'Marr's Early' and 'Pera' sweet orange (Citrus sinensis) scions grafted on 'Carrizo', 'HRS 802', and 'HRS 812' rootstocks were planted in Alonso clay series Inceptisol (Oxic Humudepts) at 649 m in 2010. Tree growth parameters (height, diameter, canopy volume) and yield efficiency were measured. Fruit quality was determined from juice content (%), total soluble solids [Brix], and pH. Leaf tissue analyses showed an optimum range for Ca, Mg, Na, P, B, Cu, and Zn, an indicator of tree health. A few were high (i.e., N and P) or in excess (i.e., Fe), but no clear connection to specific scions or rootstocks could be established. Tree height, tree diameter, fruit production, and juice content were higher in both sweet oranges grafted on 'HRS 802' compared with those on 'HRS 812' and 'Carrizo'. Therefore, 'HRS 802' rootstock can be recommended to local farmers growing sweet oranges in Alonso series soil. [ABSTRACT FROM AUTHOR]

The Florida citrus industry is going through major changes and considerable replanting. Many growers are planting varieties, especially rootstocks, that are potentially productive under Huanglongbing (HLB; Candidatus Liberibacter asiaticus) prevalent conditions. However, the high demand for new plant material has put tremendous pressure on citrus nurseries and has created a bottleneck in production. Often it can take more than one year to produce field-transplant ready plants in nurseries; therefore, there is a critical need to accelerate plant production. This three-part study was conducted with the following objectives: (1) determine citrus nursery trends; (2) evaluate use of compost for rootstock germination; and, (3) evaluate use of compost for rootstock seedling growth. According to the nursery survey, rootstock seed germination and seedling growth were the most time-consuming, taking 6-8 months. Moreover, it was apparent from the survey results that 44% of the citrus nurseries were experimenting with potting mixes to achieve adequate plant growth and quality. Our greenhouse study demonstrated successful use of yard waste compost in place of peat moss in a potting mix. With use of 37% to 50% of compost in the potting mix, the overall germination rate and mean emergence time were improved to 70% in less than four weeks for US-897 rootstock as compared to no compost in the potting mix. In addition, 37% to 50% compost resulted in higher biomass accumulation in seedlings. When seedlings of rootstocks C-32 and Flying Dragon were grown with 37% to 50% compost, the growth rate and, therefore, percentage of successful budding were significantly increased as compared to no compost. In addition, substrate analysis indicated that a high compost potting media was rich in mineral nutrients, hence the use of fertilizer in nurseries could be minimized. Altogether, use of compost in place of peat moss seems promising and could accelerate germination and growth of rootstocks, reducing the production time as well as cost. [ABSTRACT FROM AUTHOR]

The chemical composition of volatile oils from 22 genotypes of Citrus and related genera was poorly differentiated, but chemometric techniques have clarified the relationships between the 22 genotypes, and allowed us to understand their resistance to D. citri. The most convincing similarities include the synthesis of (Z)- -ocimene and (E)-caryophyllene for all 11 genotypes of group A. Genotypes of group B are not uniformly characterized by essential oil compounds. When stimulated with odor sources of 22 genotypes in a Y-tube olfactometer D. citri preferentially entered the arm containing the volatile oils of Murraya paniculata, confirming orange jasmine as its best host. C. reticulata x C. sinensis was the least preferred genotype, and is characterized by the presence of phytol, (Z)- -ocimene, and -elemene, which were not found in the most preferred genotype. We speculate that these three compounds may act as a repellent, making these oils less attractive to D. citri. [ABSTRACT FROM AUTHOR]

In an earlier study, an electronic tongue system (e-tongue) has been used to differentiate between orange juice made from healthy fruit and from fruit affected by the citrus greening or Huanglongbing (HLB) disease. This study investigated the reaction of an e-tongue system to the main chemicals in orange juice that impact flavor and health benefits and are also impacted by HLB. Orange juice was spiked with sucrose (0.2-5.0 g/100 mL), citric acid (0.1%-3.0% g/100 mL) and potassium chloride (0.1-3.0 g/100 mL) as well as the secondary metabolites nomilin (1-30 μg/mL), limonin (1-30 μg/mL), limonin glucoside (30-200 μg/mL), hesperidin (30-400 μg/mL) and hesperetin (30-400 μg/mL). Performance of Alpha MOS sensor sets #1 (pharmaceutical) and #5 (food) were compared for the same samples, with sensor set #1 generally giving better separation than sensor set #5 for sucrose, sensor set #5 giving better separation for nomilin and limonin, both sets being efficient at separating citric acid, potassium chloride, hesperitin and limonin glucoside, and neither set discriminating hesperidin efficiently. Orange juice made from fruit over the harvest season and from fruit harvested from healthy or HLB-affected trees were separated by harvest maturity, disease state and disease severity. [ABSTRACT FROM AUTHOR]

Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae) was first released in California for biological control of Diaphorina citri (Hemiptera: Liviidae) in December 2014. The establishment and parasitism rates of D. aligarhensis, along with those of another introduced species, Tamarixia radiata (Hymenoptera: Eulophidae), first released in 2011, were assessed at 15 D. aligarhensis release and 24 no-release control sites over the period 2016–2018. Study sites with citrus trees that were infested with D. citri eggs, nymphs, and adults, were located in residential areas in southern California that spanned three different climatic zones: coastal, intermediate, and desert interior sites. Parasitism rates of D. aligarhensis were low, averaging 0.62% compared to 21.2% for T. radiata which had spread naturally and established widely through the study area approximately one year earlier. Recoveries of D. aligarhensis at release sites were made eight times in 2016 and 2017. Conversely, T. radiata was recovered consistently at 34 of the 39 sites surveyed. Analyses indicated that parasitism of D. citri nymphs by T. radiata exhibited delayed density-dependence with a 12-month lag associated with reductions of D. citri densities by 50%. Irrespective of the climatic zone, the highest frequency of parasitized D. citri nymphs for T. radiata was recorded during peak periods of citrus flush growth from March through June and October through November each year. The findings reported here suggest that it is unlikely D. aligarhensis has established in California and that competition from T. radiata may, in part, have contributed to establishment failure. Consequently, biological control efforts targeting D. citri in California should focus on T. radiata. [ABSTRACT FROM AUTHOR]

Citrus greening or huanglongbing (HLB) is commonly known as yellow dragon disease and affects citrus production worldwide. Therefore, it has a significant impact on and deleterious effects in the agro-industrial sector. Significant efforts have been made to combat this disease and mitigate its destructive impact on citrus production, but still, there is no effective biocompatible treatment available to control HLB disorder. This study is considered the first biocompatible approach to evaluate the potential of phytogenic selenium nanoparticles (SeNPs) to improve the health of HLB-infected 'Kinnow' mandarin plants. Polymerase chain reactions (PCRs) with specific primers were used to detect HLB disease in 'Kinnow' mandarin plants, and PCR products were sequenced to identify Candidatus Liberibacter asiaticus (CLas), and accession numbers for CLas1 and CLas2, MZ851933 and MZ851934, respectively, were obtained. SeNPs were synthesized by using Allium sativum L. clove extract as a reducing, capping, and stabilizing agent and various techniques such as UV-visible spectrophotometry, energy dispersive X-rays, scanning electron microscopy (SEM), Fourier transformed infrared spectroscopy (FTIR), and X-ray diffraction analysis (XRD) were used to confirm the biogenesis of SeNPs. Different concentrations of SeNPs (25, 50, 75, and 100 mg L−1) were exogenously applied to HLB-infected 'Kinnow' mandarin plants and obtained spectacular results. The obtained results from the current study proved that 75 mg L−1 of SeNPs was most effective to improve the chlorophyll, carotenoids, relative water content (RWC), membrane stability index (MSI), total soluble sugar (TSS), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), total flavonoid content (TFC), and total phenolic content (TPC) and significant decrease was observed in hydrogen peroxide (H2O2), malondialdehyde (MDA), and proline (PRO) contents of HLB-infected 'Kinnow' mandarin plants as compared to untreated diseased citrus plants. In conclusion, these results allow us to synthesize the SeNPs formulation as a promising management strategy to treat the HLB disease in citrus plants. [ABSTRACT FROM AUTHOR]

Citrus relatives are a relevant source of valuable traits for use in citrus breeding, including resistance to diseases such as Huanglongbing (HLB). Resistant rootstocks may impact tree responses to HLB. This requires graft compatibility, which has been poorly investigated within the Aurantioideae. In this study, the biometric characteristics and the anatomy of the graft union of 86 scion/rootstock combinations were assessed. This comprised 18 genotypes/species and 8 genera from Citrinae, Balsamocitrinae, and Clauseninae subtribes sensu Swingle and Reece. Most graft combinations were found to be noncompatible. Phylogenetic proximity did not ensure successful grafting as, for example, Orange jasmine autografts failed, whereas some intergeneric grafts were successful (>60% of graft-take). Plant scion height was directly related to graft-take, but the correlation between the scion and rootstock stem diameters was not a reliable indicator of graft compatibility. Rangpur/Tabog, Tabog/Rangpur, Wampee/Rangpur, Wampee/Pomeroy, Wampee/Swingle, Pomeroy/Wampee, and Swingle/Wampee were the most compatible intergeneric graft combinations. Graft-take success for this was at similar levels to those of sweet orange grafted on common citrus rootstocks. The position as a scion or rootstock in the combination affected the performance and was specific to the genotypes tested. The lack of differentiation between xylem-derived calli and the accumulation of phenolic compounds at the graft union were clear anatomical and biochemical markers, respectively, of incompatibility for most Aurantioideae combinations. In the field, within a set of the ten most promising combinations, Hamlin/Rangpur (control) was the only one that became infected by 'Candidatus Liberibacter asiaticus'. This was first observed 12 months after planting. Overall, the assessment of biometric traits and anatomy of the graft union allowed Aurantioideae genotypes to be divided into four clusters, with respect to their graft compatibility, as follows: fully compatible with high graft-take and plant growth; potentially compatible with high graft-take but lower plant growth; partially incompatible with lower graft-take and poor plant growth; and fully incompatible with a complete absence of graft-take. [ABSTRACT FROM AUTHOR]

The rise in the productivity of sweet orange in Brazil has been related to the use of superior rootstocks and higher tree density, among other factors. In order to investigate whether the cropping system and the land use efficiency would benefit from more intensive cultivation, the performance of Valencia sweet orange was evaluated over nine years on four rootstocks, which induced contrasting vigor, at 513, 696 and 1000 trees·ha−1. Agronomic Institute of Campinas (IAC) 1697 and IAC 1710 citrandarins, and diploid and allotetraploid (4×) Swingle citrumelos were classified as semi-dwarfing, super-standard, standard, and dwarfing rootstocks, respectively. The fruit yield per tree was decreased at higher tree densities, notably for more vigorous rootstocks. Conversely, the cumulative productivity was increased over the evaluation period by 27% at 1000 trees·ha−1, irrespective of the rootstock, and the most vigorous rootstock resulted in 2.5 times higher production than the dwarfing one on average. Most fruit quality parameters were seldom influenced by the tree density, while the rootstock was a decisive factor in improving the quality and the soluble solids content. Dwarfing rootstocks allowed for harvesting 17% more fruit per minute by manual pickers. Because the tree row volume per area is lower with such rootstocks, even at higher tree density, spray volume can be reduced, although appropriate equipment should be developed for better spray coverage on smaller trees. Nine years after planting under strict vector control, the cumulative incidence of huanglongbing-symptomatic trees on IAC 1710 was double that on Swingle 4×. Taken together, the results suggested that the land use efficiency in the citrus industry can be further improved by planting vigorous rootstocks at moderate to high tree densities. Nevertheless, obtaining highly productive semi-dwarfing and dwarfing rootstocks is the sine qua non for making high-density pedestrian sweet orange orchards more profitable. [ABSTRACT FROM AUTHOR]

The Citrus genus comprises some of the most important and commonly cultivated fruit plants. Within the last decade, citrus greening disease (also known as huanglongbing or HLB) has emerged as the biggest threat for the citrus industry. This disease does not have a cure yet and, thus, many efforts have been made to find a solution to this devastating condition. There are challenges in the generation of high-yield resistant cultivars, in part due to the limited and sparse knowledge about the mechanisms that are used by the Liberibacter bacteria to proliferate the infection in Citrus plants. Here, we present GreeningDB, a database implemented to provide the annotation of Liberibacter proteomes, as well as the host–pathogen comparactomics tool, a novel platform to compare the predicted interactomes of two HLB host–pathogen systems. GreeningDB is built to deliver a user-friendly interface, including network visualization and links to other resources. We hope that by providing these characteristics, GreeningDB can become a central resource to retrieve HLB-related protein annotations, and thus, aid the community that is pursuing the development of molecular-based strategies to mitigate this disease's impact. The database is freely available at http://bioinfo.usu.edu/GreeningDB/ (accessed on 11 August 2021). [ABSTRACT FROM AUTHOR]

Citrus huanglongbing (HLB) disease associated with the 'Candidatus Liberibacter asiaticus' (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays. [ABSTRACT FROM AUTHOR]

The advantages from exogenously applied RNAi biopesticides have yet to be realized in through commercialization due to inconsistent activity of the dsRNA trigger, and the activity level of RNAi suppression. This has prompted research on improving delivery methods for applying exogenous dsRNA into plants and insects for the management of pests and pathogens. Another aspect to improve RNAi activity is the incorporation of modified 2′-F pyrimidine nucleotides into the dsRNA trigger. Modified dsRNA incorporating 32–55% of the 2′-F- nucleotides produced improved RNAi activity that increased insect mortality by 12–35% greater than non-modified dsRNA triggers of the same sequence. These results were repeatable across multiple Hemiptera: the Asian citrus psyllid (Diaphorina citri, Liviidae); whitefly (Bemisia tabaci, Aleyroididae); and the glassy-winged sharpshooter (Homalodisca vitripennis, Cicadellidae). Studies using siRNA with modified 2′-F- pyrimidines in mammalian cells show they improved resistance to degradation from nucleases, plus result in greater RNAi activity, due to increase concentrations and improved binding affinity to the mRNA target. Successful RNAi biopesticides of the future will be able to increase RNAi repeatability in the field, by incorporating modifications of the dsRNA, such as 2′-F- pyrimidines, that will improve delivery after applied to fruit trees or crop plants, with increased activity after ingestion by insects. Costs of RNA modification have decreased significantly over the past few years such that biopesticides can now compete on pricing with commercial chemical products. [ABSTRACT FROM AUTHOR]

Oxytetracycline (OTC) has been used for the control of several plant diseases and was recently approved for the control of Huanglongbing, the citrus greening disease. Huanglongbing is caused by the phloem limited 'Candidatus Liberibacter asiaticus'. Determination of OTC in the xylem and phloem of citrus plants is of great interest as they are the main routes of translocation in citrus. In addition, the determination of the level of OTC in the phloem sap is necessary for the control of the 'Ca. L. asiaticus' pathogen, which resides in the phloem. Herein, we demonstrated that the level of OTC in the citrus phloem and xylem saps obtained using the centrifugation method can be successfully measured using the europium (Eu) method directly or with cleanup by solid-phase extraction (SPE). Recovery of OTC from spiked sap samples purified by solid-phase extraction (SPE) was higher than 90%, while recovery from saps without SPE cleanup were nearly 100%. The 'Ca. L. asiaticus'-infected leaf and phloem sap samples showed higher inhibition of the fluorescence intensity of the OTC standard compared to non-infected control leaf and phloem samples. In agreement with this finding, the levels of phenols and flavonoids in 'Ca. L. asiaticus'-infected leaves were higher than those controls and were shown to interfere with the Eu method. Therefore, the SPE cleanup step only improved OTC recovery from leaf samples containing the interfering compounds. The Eu method was then used to determine OTC levels in the phloem and xylem sap of OTC-treated plants, and the results were similar whether measured directly or after SPE. Visualization under ultraviolet light (400 nm) showed the presence of OTC in citrus xylem and phloem saps with and without the use of SPE. [ABSTRACT FROM AUTHOR]