Citrus Tissues Research Articles

Three-dimensional fluorescence spectral characteristic of flavonoids for citrus Huanglongbing disease early detection

To explore the potential of using flavonoid fluorescence characteristics in citrus leaves and peels for detecting citrus Huanglongbing (HLB), this study focused on Citrus reticulata ‘Shiyue Ju’ and utilized three-dimensional fluorescence spectroscopy for data collection. Excitation-Emission matrices (EEM) of flavonoid standards and citrus leaves and peels were acquired and comparatively analyzed. The concentration of HLB pathogens in citrus tissues was also assessed using qPCR. In order to identify the range of fluorescence bands that are sensitive to HLB, different machine-learning algorithms were used to construct classification models. Support Vector Machine (SVM) was proved most precise, with 93.1 % accuracy for leaves and 86.67 % for peels. The sensitive bands for leaves were EX (Excitation) = 375–455 nm, EM (Emission) = 490–570 nm and for peels were EX = 300–340 nm, EM = 430–470 nm and EX = 330–370 nm, EM = 425–465 nm. These bands correspond closely with the fluorescence signatures of nobiletin, hesperidin, and narirutin. It was further verified that HLB stress affected the changes in the content of flavonoids in the leaves and peels of ‘Shiyue Ju’. This study provides a reference for using flavonoid fluorescence in citrus HLB detection and presents a novel approach for applying spectral imaging techniques in citrus HLB detection.

Transcriptomic Profiling of 'Candidatus Liberibacter asiaticus' in Different Citrus Tissues Reveals Novel Insights into Huanglongbing Pathogenesis.

'Candidatus Liberibacter asiaticus' (Las) is a gram-negative bacterial pathogen associated with citrus huanglongbing (HLB) or greening disease. Las is transmitted by the Asian citrus psyllid (ACP) where it colonizes the phloem tissue, resulting in substantial economic losses to the citrus industry worldwide. Despite extensive efforts, effective management strategies against HLB remain elusive, necessitating a deeper understanding of the pathogen's biology. Las undergoes cell-to-cell movement through phloem flow and colonizes different tissues in which Las may have varying interactions with the host. Here, we investigate the transcriptomic landscape of Las in citrus seed coat vasculatures, enabling a complete gene expression profiling of Las genome and revealing unique transcriptomic patterns compared with previous studies using midrib tissues. Comparative transcriptomics between seed coat, midrib, and ACP identified specific responses and metabolic states of Las in different host tissue. Two Las virulence factors that exhibit higher expression in seed coat can suppress callose deposition. Therefore, they may contribute to unclogging sieve plate pores during Las colonization in seed coat vasculature. Furthermore, analysis of regulatory elements uncovers a potential role of LuxR-type transcription factors in regulating Liberibacter effector gene expression during plant colonization. Together, this work provides novel insights into the pathogenesis of the devastating citrus HLB. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

WRKY75 regulates anthocyanin accumulation in juvenile citrus tissues.

The online version contains supplementary material available at 10.1007/s11032-024-01490-9.

Identification of 'Candidatus Liberibacter asiaticus', the Huanglongbing Bacterium, in Citrus from Colombia.

'Candidatus Liberibacter' spp. are the most prevalent microorganisms in the citrus plant, associated with citrus huanglongbing, which are transmitted by psyllid vectors. In Colombia, the vector Diaphorina citri Kuwayama has been reported in different regions, but 'Ca. Liberibacter asiaticus' (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1/OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a/CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we used droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the β-subunit of the ribonucleotide reductase gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Analysis of sugar components and identification of SPS genes in citrus fruit development.

Sugar is a primary determinant of citrus fruit flavour, but undergoes varied accumulation processes across different citrus varieties owing to high genetic variability. Sucrose phosphate synthase (SPS), a key enzyme in glucose metabolism, plays a crucial role in this context. Despite its significance, there is limited research on sugar component quality and the expression and regulatory prediction of SPS genes during citrus fruit development. Therefore, we analysed the sugar quality formation process in 'Kiyomi' and 'Succosa', two citrus varieties, and performed a comprehensive genome-wide analysis of citrus CsSPSs. We observed that the accumulation of sugar components significantly differs between the two varieties, with the identification of four CsSPSs in citrus. CsSPS sequences were highly conserved, featuring typical SPS protein domains. Expression analysis revealed a positive correlation between CsSPS expression and sugar accumulation in citrus fruits. However, CsSPS expression displays specificity to different citrus tissues and varieties. Transcriptome co-expression network analysis suggests the involvement of multiple transcription factors in shaping citrus fruit sugar quality through the regulation of CsSPSs. Notably, the expression levels of four CsWRKYs (CsWRKY2, CsWRKY20, CsWRKY28, CsWRKY32), were significantly positively correlated with CsSPSs and CsWRKY20 might can activate sugar accumulation in citrus fruit through CsSPS2. Collectively, we further emphasize the potential importance of CsWRKYs in citrus sugar metabolism, our findings serve as a reference for understanding sugar component formation and predicting CsSPS expression and regulation during citrus fruit development.

Direct Organogenesis of Citrus Cultivars from Shoot Tip Nodal Segments.

Citrus cultivar improvement via conventional breeding strategies is impeded by factors related to its reproductive biology. The orange is a hybrid between pomelo (Citrus maxima) and mandarin (Citrus reticulata). Among various orange cultivars, Valencia oranges have a bit of bitter tang mixed in with their sweetness, as Navel oranges are, the most widely cultivated citrus species, quite sweeter, and also don't contain any seeds. Tangelo mandarin orange cultivar is a hybrid of C. reticulata × C. maxima or × C. paradisi. The present study was undertaken to optimize the hormonal composition of the media with regard to plant growth regulators for in vitro propagation of sweet orange cultivars from nodal segment explants. The nodal segment explants were collected from three citrus cultivars, Washington Navel, Valencia and Tangelo. Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of growth regulators was used for shoot proliferation and root induction, and the optimum medium composition was assessed. The patent for Citrus Tissue Culture was obtained from the Office of Research Affairs, Haramaya University. The results indicate that the highest shoot response was recorded for Washington's navel with maximum shoot proliferation rate (99.75%), shoot number per explant (1.76), shoot length (10.70 cm), leaf number per explants (3.54) after three weeks of culture. In all experiments, no growth was observed for the basal MS medium. Phytohormone combinations of IAA (1.2 mg/L) and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Among the cultivars, there were significant differences for the highest rooting rate (81.255), root number (2.22), and root length (2.95 cm) variables for Washington Navel. The lowest rooting rate (48.45%), root number (1.47) and root length (2.26 cm) were observed for Valencia. The highest rooting rate (84.90%), root number per microshoot (2.22) and root length (3.05 cm) was on MS medium supplemented with 1.5 mg/L NAA. A comparison of different concentrations of IAA and NAA on root induction of microshoots from nodal segments of citrus cultivars demonstrated NAA was a more effective hormone than IAA.

Simplified extraction protocol of citrus tissues for rapid detection of ‘Candidatus Liberibacter asiaticus’ using isothermal recombinase polymerase amplification assay and its application for prevalence studies

Huanglongbing disease caused by ‘Candidatus Liberibacter asiaticus’ is a serious threat to citrus production at global level. Its management largely relies on accurate detection and taking preventive measures to check its spread through sensitive and reliable diagnosis. The available standard nucleic acid-based detection assays rely on use of purified DNA as template and are cumbersome. This study reports a simplified extraction protocol of citrus phloem tissues for rapid recombinase polymerase amplification (RPA) assay-based detection of ‘Candidatus Liberibacter asiaticus’ (CLas)-associated with huanglongbing (HLB). Simplified crude plant extract prepared in phosphate buffer and NaOH:EDTA (using mortar pestle or extraction bag without the use of liquid nitrogen) was successfully employed in the RPA assay. Developed assay targeted specific outer membrane protein (OMP) and ribosomal protein (RP) genomic regions of CLas and could detect its infection up to 10−9 to 10−11 dilution of plant extract and up to 1−10 ag µl−1 of DNA of CLas positive citrus tissues and plasmid DNA having respective gene insert, thus exhibiting sufficient sensitivity. Assay was highly specific and showed no cross-reactivity with the common pathogens infecting citrus exhibiting high reliability. Developed RPA assay was validated using 447 citrus samples collected across the Indian citrus belts, where 79.19 % were detected CLas positive and was more sensitive as compared to PCR wherein 0.45 % to 3.80 % samples were detected false negative. The developed RPA assay rapidly detected CLas infection in simply prepared crude plant extract in cost-effective manner and can be performed at low isothermal temperature in resource-poor laboratories for prevalence studies, bud wood certification and production of CLas-free planting materials of citrus.

Complete Genome of Agrobacterium fabrum Strain 1D1416.

This work reports the draft genome of Agrobacterium fabrum strain 1D1416. The assembled genome is composed of a 2,837,379-bp circular chromosome, a 2,043,296-bp linear chromosome, a 519,735-bp AT1 plasmid, a 188,396-bp AT2 plasmid, and a 196,706-bp Ti virulence plasmid. The nondisarmed strain produces gall-like structures in citrus tissue.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Genome-Informed Multiplex Conventional PCR for Identification and Differentiation of Xanthomonas citri pv. citri Subpathotypes, the Causal Agents of Asiatic Citrus Canker

Asiatic citrus canker, one of the most important diseases of citrus, is caused by Xanthomonas citri pv. citri. It has high economic impact and can spread easily, and the disease is difficult to manage; it is a quarantine organism in many citrus-producing countries. X. citri pv. citri has been separated into three subpathotypes (A, A*, Aw) that differ in host range and geographical distribution, thus creating a need to differentiate subpathotypes for surveillance and disease management. Availability of useful diagnostic tools is the cornerstone of successful surveillance, quarantine, and eradication measures. In this study, a multiplex conventional PCR (cPCR) assay was developed for detection and subpathotype determination of X. citri pv. citri. Assay specificity was assessed by four different labs on a total of 146 X. citri pv. citri and 58 other Xanthomonas isolates. The assay demonstrated high analytical sensitivity, specificity, and selectivity. False negatives were observed with A Lineage 2 strains, and potential false positives were observed for X. citri pv. bilvae. Combined with a simple extraction protocol, the assay has been deployed successfully at the Plant Protection and Quarantine Plant Pathogen Confirmatory Diagnostics Laboratory. This assay has proven useful for differentiating Asiatic citrus canker subpathotypes from symptomatic citrus tissue. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Root Absorption and Limited Mobility of Mandipropamid as Compared to Oxathiapiprolin and Mefenoxam After Soil Treatment of Citrus Plants for Managing Phytophthora Root Rot.

Phytophthora root rot can greatly impact citrus production worldwide, especially in newly established orchards by reducing crop yield and increasing the cost of disease management. Mandipropamid is an Oomycota fungicide that is currently registered as a soil treatment for citrus nursery container plants to manage Phytophthora root rot. In this study, we investigated the uptake of mandipropamid into citrus roots and its translocation to stems and leaves after soil application and evaluated its mobility in roots as compared to oxathiapiprolin and mefenoxam using split-root potted plants and trees in the field. A bioassay and liquid chromatography-tandem mass spectrometry were used to detect and quantify fungicides in citrus tissues, and overall, similar results were obtained using the two methods. When applied to the soil of potted, 6- to 7-month-old citrus plants using labeled rates, the majority of mandipropamid was found in root tissues (4.9 to 18.1 μg/g), but small amounts were also present in stems (0.18 to 0.32 μg/g) and leaves (0.03 to 0.22 μg/g). There was no significant increase in concentrations in all three tissues between 1 and 4 weeks after application. Concentrations in all tissues exceeded established EC50 values for mycelial growth inhibition of P. citrophthora and P. nicotianae, the main citrus root rot pathogens in California. In a split-root study where the root systems of single plants were separated, no basipetal phloem-based mobility of mandipropamid or oxathiapiprolin was observed, but relative uptake into roots was higher for mandipropamid. In contrast, low amounts of mefenoxam were also present in roots in the untreated soil. Similar results were obtained in a field study where part of the root system was treated, and fungicides were extracted from nontreated roots. All three fungicides persisted inside roots over the 8-week period of this study. Uptake and persistence inside roots, as well as the previously reported high efficacy against citrus root rot in greenhouse and field studies support the use of mandipropamid in citrus nurseries and potentially in the orchard.

Spatial Distribution and Antioxidant Activity of Extracts from Citrus Fruits.

Citrus fruits are recommended components of the human diet because of their enriched composition in bioactive compounds and health benefits. Among their notable components are phenols, with a special emphasis on flavonoids, limonoids, and carboxylic acids. In this research, we have carried out a spatial metabolomics analysis for the characterization of these bioactive families in three citrus fruits, namely, lemons, limes, and mandarins. Sampling was undertaken, for which the juices and three fruit tissues, namely, albedo, flavedo, and segments, were analyzed. This characterization allowed for the determination of 49 bioactive compounds in all the samples. The composition of the different extracts was correlated with the antioxidant capacity measured by the DPPH radical scavenging activity and β-carotene bleaching assays. Flavonoids, found in the albedo and flavedo at higher concentrations, were the main components responsible for DPPH radical scavenging activity. On the other hand, the combined action of flavonoids and limonoids contributed to explaining the antioxidant activity measured by the β-carotene bleaching assay. Generally, the antioxidant capacity of juices was lower than that estimated for extracts from citrus tissues.

Survey and detection for citrus tristeza virus in Florida groves with an unconventional tool: The Asian citrus psyllid.

The citrus industry of Florida faces insurmountable challenges against the destructive diseases citrus tristeza and Huanglongbing (HLB, or citrus greening). Though the tristeza causal agent, citrus tristeza virus (CTV), has been in Florida decades longer than HLB, growers have concentrated most of their efforts on combating the more detrimental HLB. The Asian citrus psyllid (Diaphorina citri; ACP) is the insect vector of the bacterial pathogen Candidatus Liberibacter asiaticus and transmits the incurable HLB to all commercial citrus. During our searches for biological and viral controls against the ACP, we consistently detected sequences of CTV in Florida field populations of ACP. This unexpected finding led us to investigate whether ACPs collected from young shoots could be used as a tool to survey CTV in Florida citrus groves. We first surveyed for the most common CTV strains in Florida (T30, T36, and VT/T68) in citrus trees on mostly sour orange (Citrus aurantium) rootstock, the rootstock susceptible to CTV decline. Out of 968 trees sampled across five years (2018-2022), approximately 8.2% were positive for CTV, with more than half of the CTV-positive trees infected with strain T30. Simultaneously, we looked at CTV strains in ACPs during this time and found that approximately 88% of pooled adult and nymph ACPs also had CTV, with over half the positive samples having the T36 strain. As a result of the much higher CTV incidences in the ACPs, we conducted a second investigation into whether we could more easily detect the same CTV strains in ACP nymphs as in CTV-infected citrus tissue. After individually sampling 43 trees and pooling the nymphs from each tree, we detected CTV at about the same incidence in the citrus tissue and the nymphs, but with much less ACP tissue, time, and resources required for detection compared to citrus tissue. Results from this study illustrate the sustained threat of CTV to Florida citrus and demonstrate the ACP as a potential bioindicator for CTV.

Phytochemical content and antioxidant activity in eight citrus cultivars grown in Jeju Island according to harvest time

ABSTRACT This study aimed to compare phytochemical content and antioxidant activities of eight diverse citrus cultivars grown in Jeju Island depending on their harvest time owing to their health benefits. Plant peels, flesh, and leaves were extracted, and their flavonoid contents were measured by HPLC. Flavonoid levels were high in peel followed by leaves and juices. It was highest in immature fruits and rapidly declined during ripening. While total phenolic content in all citrus peels and fleshes reduced during ripening, those of leaves did not significantly decrease. Antioxidant activity matched the total phenolic content. This indicates that immature citrus tissues exhibited the highest flavonoid and phenolic content with high antioxidant activity and can be potentially employed as a readily accessible natural antioxidant source.

Bidirectional mRNA transfer between Cuscuta australis and its hosts.

The holoparasitic dodder (Cuscuta spp.) is able to transfer mRNA and certain plant pathogens (e.g., viruses and bacteria) from the host plant. “Candidatus Liberibacter asiaticus,” the phloem-limited causative agent of citrus Huanglongbing, can be transferred from citrus to periwinkle (Catharanthus roseus) mediated by dodder. However, characterization of mRNA transport between dodder and citrus/periwinkle remains unclear. In this study, we sequenced transcriptomes of dodder and its parasitizing host, sweet orange (Citrus sinensis “Newhall”) and periwinkle (Catharanthus roseus), to identify and characterize mRNA transfer between dodder and the host plant during parasitism. The mRNA transfer between dodder and citrus/periwinkle was bidirectional and most of the transfer events occurred in the interface tissue. Compared with the citrus–dodder system, mRNA transfer in the periwinkle–dodder system was more frequent. Function classification revealed that a large number of mRNAs transferred between dodder and citrus/periwinkle were involved in secondary metabolism and stress response. Dodder transcripts encoding proteins associated with microtubule-based processes and cell wall biogenesis were transferred to host tissues. In addition, transcripts involved in translational elongation, plasmodesmata, and the auxin-activated signaling pathway were transmitted between dodder and citrus/periwinkle. In particular, transcripts involved in shoot system development and flower development were transferred between the host and dodder in both directions. The high abundance of dodder-origin transcripts, encoding MIP aquaporin protein, and S-adenosylmethionine synthetase 1 protein, in citrus and periwinkle tissues indicated they could play an important biological role in dodder–host interaction. In addition, the uptake of host mRNAs by dodder, especially those involved in seed germination and flower development, could be beneficial for the reproduction of dodder. The results of this study provide new insights into the RNA-based interaction between dodder and host plants.

A comparative analysis of RNA isolation methods optimized for high-throughput detection of viral pathogens in California’s regulatory and disease management program for citrus propagative materials

Citrus germplasm programs can benefit from high-throughput polymerase chain reaction (PCR)-based methods for the detection of graft-transmissible pathogens in propagative materials. These methods increase diagnostic capacity, and thus contribute to the prevention of disease spread from nurseries to citrus orchards. High quality nucleic acids, as determined by purity, concentration, and integrity, are a prerequisite for reliable PCR detection of citrus pathogens. Citrus tissues contain high levels of polyphenols and polysaccharides, which can affect nucleic acid quality and inhibit PCR reactions. Various commercially available RNA isolation methods are used for citrus and include: phenol-chloroform (TRIzol®, Thermo Fisher Scientific); silica columns (RNeasy® Plant Mini Kit, Qiagen); and magnetic beads-based methods (MagMAX™-96 Viral RNA Isolation Kit, Thermo Fisher Scientific). To determine the quality of RNA and its impact on the detection of graft-transmissible citrus pathogens in reverse transcription (RT) PCR-based assays, we compared these three RNA isolation methods. We assessed RNA purity, concentration, and integrity from citrus inoculated with different viruses and viroids. All three RNA isolation methods produced high quality RNA, and its use in different RT-PCR assays resulted in the detection of all targeted citrus viruses and viroids with no false positive or negative results. TRIzol® yielded RNA with the highest concentration and integrity values but some samples required serial dilutions to remove PCR inhibitors and detect the targeted pathogens. The RNeasy® kit produced the second highest concentration and purity of RNA, and similar integrity to TRIzol®. MagMAX™ isolation also provided high quality RNA but most importantly produced RNA with consistent results clustered around a median value for concentration, purity, and integrity. Subsequently, MagMAX™-96 was combined with the semi-automated MagMAX™ Express-96 Deep Well Magnetic Particle Processor, for high-throughput sample processing. MagMAX™-96 enabled the diagnostic laboratory of the Citrus Clonal Protection Program-National Clean Plant Network at the University of California, Riverside to process over 16,500 samples from citrus budwood source trees between 2010 and 2019. This high-throughput approach dramatically reduced the incidence of viroids in citrus nurseries and was key to the successful implementation of the mandatory Citrus Nursery Stock Pest Cleanliness Program in California.

Spectrum classification of citrus tissues infected by fungi and multispectral image identification of early rotten oranges

Citrus fruit is susceptible to postharvest rot by fungal infection. The detection of early rot is difficult due to similar skin characteristics to sound area, which limits the ability of the grading system to evaluate the comprehensive quality of citrus. In this study, the visible and near infrared hyperspectral imaging system with the wavelength range of 325–1000 nm was used to collect hyperspectral images of oranges. Hyperspectral data of three types of tissues including sound tissue from 80 samples, rotten tissue infected by Penicillium digitatum from 100 samples and rotten tissue infected by Penicillium italicum from 100 samples were extracted. The bootstrapping soft shrinkage (BOSS) and BOSS-SPA (BOSS-Successive Projections Algorithm) combination algorithm were separately used to optimize spectrum variables. The partial least squares discriminant analysis (PLS-DA) model for classifying three types of tissues and PLS-DA model for classifying two types of tissues (sound tissue and rotten tissue) were constructed based on full-spectrum and the selected informative variables. Model comparisonshowed that the BOSS-PLS-DA model can effectively identify three types of tissues with the classification accuracy of 97.1%, while the BOSS-SPA-PLS-DA model was more effective for the binary classification of sound and rotten citrus tissues with the accuracy of 100%. Furthermore, the wavelength images corresponding to the nine informative variables extracted by BOSS-SPA were performed the principal component analysis (PCA), and four feature wavelength images (508, 568, 578 and 614 nm) were obtained by analyzing the weighting coefficients of each single-wavelength images constituting the optimal principal component (PC) image. Finally, a fast multispectral image processing algorithm combined with the global threshold theory was proposed for the rotten orange detection based on the extracted four wavelength images. A total of 280 samples including 80 sound and 200 rotten samples were used to evaluate the classification ability, which showed the proposed multispectral image detection algorithm can successfully differentiate between sound and rotten oranges with an overall classification accuracy of 98.6%.

DISTRIBUTION AND QUALITATIVE AND QUANTITATIVE DETERMINATION OF PESTICIDE RESIDUES FROM A SAMPLE OF RED GRAPEFRUIT, AFTER TREATMENT WITH FUNGICIDE SOLUTIONS

"The Most citrus fruits are compromised each year due to post-harvest fungal infections. To reduce fungal infections, packing centers use fungicide solution mixtures to prevent infections that occur during harvest and then during storage. The most common fungal pathogens of citrus are commonly treated with the fungicides imazalil, Pyrimethanil due to their effectiveness in controlling these pathogens at low cost and ease of handling. However, little is known about how it alters tissues in citrus physiology. In this study we will investigate the behavior and retention of imazalil and pyrimethanil in the tissues of the red grapefruit fruit (peel and core before washing and after washing with hot water and simple dish detergent). This work demonstrates a viable approach for assessing the quality of citrus fruits, and we can even proceed to thoroughly wash each fruit before consumption, as we remove a quantity of residue. This work demonstrates that the treatment affects the citrus tissues and the substances migrate throughout the pulp, although the treatment was applied to the peel. Washing, which is the first step in food processing on a domestic and industrial scale, helps to reduce pesticide residues on the fruit surface. In this study, the effectiveness of washing the peel was also investigated. Although many chemicals are applied to citrus to prevent certain diseases or control pests, there is little literature available on how these treatments alter the physiology of the fruit. Usually, the packaging only analyzes the level of treatments on the products."

Species of Botryosphaeriaceae associated with citrus branch diseases in China.

Citrus is an important and widely cultivated fruit crop in South China. Although the species of fungal diseases of leaves and fruits have been extensively studied, the causal organisms of branch diseases remain poorly known in China. Species of Botryosphaeriaceae are known as important fungal pathogens causing branch diseases on citrus in the USA and Europe. To determine the diversity of Botryosphaeriaceae species associated with citrus branch diseases in China, surveys were conducted in the major citrus-producing areas from 2017 to 2020. Diseased tissues were collected from twigs, branches and trunks with a range of symptoms including cankers, cracking, dieback and gummosis. Based on morphological characteristics and phylogenetic comparison of the DNA sequences of the internal transcribed spacer region (ITS), the translation elongation factor 1-alpha gene (tef1), the β-tubulin gene (tub2) and the DNA-directed RNA polymerase II second largest subunit (rpb2), 111 isolates from nine provinces were identified as 18 species of Botryosphaeriaceae, including Botryosphaeria dothidea, B. fabicerciana, Diplodia seriata, Dothiorella alpina, Do. plurivora, Lasiodiplodia citricola, L. iraniensis, L. microconidia, L. pseudotheobromae, L. theobromae, Neodeightonia subglobosa, Neofusicoccum parvum, and six previously undescribed species, namely Do. citrimurcotticola, L. guilinensis, L. huangyanensis, L. linhaiensis, L. ponkanicola and Sphaeropsis linhaiensis spp. nov. Botryosphaeria dothidea (28.8 %) was the most abundant species, followed by L. pseudotheobromae (23.4 %), which was the most widely distributed species on citrus, occurring in six of the nine provinces sampled. Pathogenicity tests indicated that all 18 species of Botryosphaeriaceae obtained from diseased citrus tissues in this study were pathogenic to the tested Citrus reticulata shoots in vitro, while not all species are pathogenic to the tested Cocktail grapefruit (C. paradisi × C. reticulata) shoots in vivo. In addition, Lasiodiplodia was the most aggressive genus both in vitro and in vivo. This is the first study to identify Botryosphaeriaceae species related to citrus branch diseases in China and the results provide a theoretical basis for the implementation of prevention and control measures. Citation: Xiao XE, Wang W, Crous PW, et al. 2021. Species of Botryosphaeriaceae associated with citrus branch diseases in China. Persoonia 47: 106-135. https://doi.org/10.3767/persoonia.2021.47.03.

An In Silico Detection of a Citrus Viroid from Raw High-Throughput Sequencing Data.

E-probe Diagnostic for Nucleic Acid Analysis (EDNA) is a user-friendly bioinformatic tool that has been adapted for the detection and identification of citrus exocortis viroid (CEVd). Here, we describe the procedures for RNA extraction from citrus tissues, library and sequencing preparation, and the utilization of EDNA Mi-Finder online platform on raw high-throughput sequencing (HTS) data.