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The current investigation assessed the effectiveness of 30 citrus germplasms in combating citrus canker pathogenesis in Assam, North‐East India. Significant differences were observed among the examined citrus germplasms in terms of per cent disease severity and disease reduction against Xanthomonas citri pv. citri. Among the 30 different germplasms, eight germplasms, namely, Jora tenga (14.67%), Bonjora (15.58%), Holong tenga (15.75%), Latipes (17.34%), Patijora (17.75%), Birajora (18.50%), Cleopatra mandarin (20.00%) and Ada jamir (20.18%), exhibited highest disease resistance potential. The study recorded that the germplasm Latipes, which are resistant, had the highest total phenol content (21.26 mg/g dry weight), while the susceptible one, Grapefruit (7.85 mg/g dry weight), had the lowest total phenol content. Jora tenga exhibited the highest flavonoid content, measuring at 6.48 mg/g dry weight, whereas White pummelo had the lowest flavonoid content, measuring at 1.88 mg/g dry weight. Similarly, the highest alkaloid percentage was recorded in resistant germplasm, Adajamir (10.52%), while the lowest was in susceptible germplasm, Volkameriana (2.30%). The levels of total phenol, alkaloid and flavonoid contents exhibited a significant negative correlation with the per cent disease severity of citrus canker disease in both Pearson correlation and Spearmen correlation analysis. The current research on bioprospecting of citrus germplasms in relation to citrus canker pathogenesis has the potential to offer valuable insights into mitigating the transmission of bacterial pathogens and eliminating sources of infection by employing resistant cultivars and implementing approved doses of plant protection formulations. [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]

An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘Candidatus Liberibacter species’ (CLsp), ‘Ca. Liberibacter asiaticus’ (CLas), ‘africanus’ (CLaf), and ‘americanus’ (CLam), associated with the huanglongbing disease of citrus. The multiplex assay was designed based on the qPCR assay published in 2006 by Li et al., considering all available CLsp 16S rRNA gene sequences in GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared with singleplex qPCR, no significant increase in quantitative cycle (Cq) values was detected. The specificity and sensitivity of the updated qPCR assay was optimal, and measuring the intra- and interassay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Huanglongbing (HLB), associated with the proteobacterium 'Candidatus Liberibacter asiaticus'(CLa), is a destructive disease in citrus. Resistance/tolerance has been identified in some citrus species, wild relatives, and transgenic citrus genotypes. The objectives of this study were to: (1) evaluate the response of the citrus germplasm collection from Puerto Rico (namely 'Rico') to HLB, (2) determine the most appropriate rootstock-genotype combination, and (3) quantify the presence of the bacterium in the plant tissue by quantitative polymerase chain reaction (qPCR). Seventeen genotypes were grafted onto 'HRS 812' and 'Swingle' citrumelo rootstocks. The plants were inoculated with HLB infected buds and evaluated at 12 months after inoculation in a greenhouse in Adjuntas, Puerto Rico. The genotypes 'Lares' and 'Padilla' had significantly higher incidence (>65.0%) and severity (3.2–4.3) compared to 'Mexican Lime' and 'Tahiti Lime' that had lower scores (<18.0%, 1.1–1.3) using both rootstocks. In contrast, the genotypes 'Fronton', 'PR-Sel-1', and 'Rico 7–376' grafted on HRS 812; and 'Pietri 334' grafted on Swingle citrumelo had lower HLB incidence (<10%) and severity (<1.5). Significantly higher mean cycle threshold (Ct) values were only observed for the genotypes Fronton (37.0), Tahiti Lime (36.5), and Pietri 334 (36.2) compared to Padilla (34.7 and 32.7 when HRS 812 and Swingle citrumelo was used as rootstock, respectively). Citrus genotypes having lower HLB scores were identified, however, their reaction varied according to the rootstock used. [ABSTRACT FROM AUTHOR]

In the past decade Colletotrichum gloeosporioides, C. karstii, and Alternaria alternata represent emerging fungal pathogens on citrus in the Mediterranean basin. Selection of tolerant Citrus germplasm offers evaluable long-term solution and should be considered as promising alternative to limit synthetic fungicide application to manage Alternaria and Colletotrichum infections in Citrus groves. In this study, the high variability of pre-harvest disease symptoms among 37 Citrus accessions was investigated in Italy in the most representative Sicilian production districts covering 1500 ha and including 20 homogeneous areas over an eleven-year survey period (2010–2020). Early fruit drop, brown spot, and anthracnose on fruit and leaves, pre-harvest fruit drop, and twig blight and defoliation associated to Alternaria and Colletotrichum spp. were identified on oranges, mandarins, and lemons. Comprehensively, first extensive data obtained herein on field susceptibility within “Tarocco” blood orange group to above disease symptoms clearly indicate as “Tarocco Nucellare 57-1E-1,” “Tarocco Tapi,” “Tarocco Sant’Alfio,” and “Tarocco Catania” accessions should be preferred to remaining Tarocco ones. A broad degree of tolerance or susceptibility was also observed within other Citrus group as it happens for tolerant lemon “Femminello Zagara Bianca,” thus demonstrating a putative resource for further studies to employ in a breeding program for genetic improvement of Citrus.

The volatile profiles of fruit peels and juice sacs from 108 citrus accessions representing seven species were analyzed. Using GC–MS 162 and 107 compounds were determined in the peels and juice sacs, respectively. In the peels, monoterpene alcohols were accumulated in loose-skin mandarins; clementine tangerines and papedas were rich in sesquiterpene alcohols, sesquiterpenes, monoterpene alcohols and monoterpene aldehydes. β -pinene and sabinene were specifically accumulated in 4 of 5 lemon germplasms. Furthermore, concentrations of 34 distinctive compounds were selected to best represent the volatile profiles of seven species for HCA analysis, and the clustering results were in agreement with classic citrus taxonomy. Comparison of profiles from different growing seasons and production areas indicated that environmental factors play important roles in volatile metabolism. In addition, a few citrus germplasms that accumulated certain compounds were determined as promising breeding materials. Notably, volatile biosynthesis via MVA pathway in C. ichangensis ‘Huaihua’ was enhanced. [ABSTRACT FROM AUTHOR]

A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory “Section 3701: Citrus Nursery Stock Pest Cleanliness Program”. Adopting a compatible multiplex RT-qPCR testing protocol for these viruses as well as other RNA and DNA regulated pathogens will provide a valuable alternative tool for virus detection and efficient program implementation. [ABSTRACT FROM AUTHOR]

Seventy four percent of the budwood tree sources samples from the Greek national citrus germplasm foundation collection were positive for one or more viroids. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the two potentially damaging viroids for the Greek citriculture, especially after transitioning to Citrus tristeza virus resistant/tolerant rootstocks and scions, were detected along with Citrus bark cracking viroid, Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). All samples tested negative for Citrus viroid V (CVd-V), CVd-VI and CVd-I-LSS (CBLVd variant). An HSVd isolate related to the non-cachexia variant contained two critical cachexia-related nucleotide changes, while two more isolates were unique among the previously reported HSVds. Unusual CDVd isolates with altered RNA secondary structure were identified in trees additionally co-infected with CEVd and HSVd. Budwood sources that had previously undergone therapy tested negative for all targeted viroids, suggesting that budwood sources in Greece can be protected against graft-transmissible pathogens, even under severe inoculum pressure. Therapied and tested citrus propagative material requires a comprehensive program not available currently in Greece, involving regulators, scientists, and the private sector, for the establishment and successful operation of a national citrus germplasm collection. [ABSTRACT FROM AUTHOR]

• Sour limetta and Marrakesh limetta (Citrus x limetta) are different from lemons. • Ancestry ratio Citrus maxima / C. medica lower in limettas than C. x meyerii. • Ancestry ratio Citrus maxima / C. reticulata higher in common lemons than limettas. • Sour limetta and Marrakesh limetta (Citrus x limetta) threatened citrus varieties. • Sour limetta and Marrakesh limetta (Citrus x limetta) a relevant cultural heritage. Citrus (Rutaceae) species have a relevant role in the culture of the Mediterranean countries, not only for their gastronomic or medicinal value, but especially for their ornamental and symbolic importance. The interest on Citrus diversity led to collect and cultivate numerous cultivars around the Mediterranean Sea, at least since the Roman period. Their image is documented in mosaics, paintings, sculptures and coins since the first millennium BC. Among these, sweet and sour limettas Citrus × limon var. limetta (Risso) Ollitrault, Curk & R.Krueger stand for their history and conservation status. Presently sour limetta is cultivated in Morocco, especially in Marrakesh, and in the Valle de Ricote (Murcia, Spain). Our work has three main objectives: to characterize sour limetta on the basis of Spanish and Moroccan populations, as well as its cultural relevance throughout history, especially in painting, gastronomy, agriculture and gardening, to determine their relationships with limes and lemons and to evaluate the availability and state of conservation of that legacy. We analyze and illustrate the morphology of the plant, flowers and fruits of sour limetta and their differences with other related citrus species and cultivars: sweet limetta, lemon, common lime, Persian lime, bergamot, mellarosa, Meyer lemon, rough lemon, Pursha Romana lime, Palestine sweet lime, Pomum Adami lemon and Rangpur lime. The above species and cultivars were selected on the base of their known ancestry and pomological characters. The distinctive characters are useful for the identification of sour limetta in the field but also for the identifying images of this plant in paintings. We analyzed the ancestry of the different taxa involved and compared the classification based on phenotypes with the classification based on the proportion of ancestors' genomes in each individual taxa. The image of sour limetta appears in different paintings, from the van Eyck's Ghent Polyptych (early 15th century), and numerous still life works by Italian and Spanish authors of the 16th to 18th centuries. A peculiar type of evidence is provided by presence of limetta in the pictorial catalogues of the Medici's fruit collections displayed in a series of paintings by Bartolomeo Bimbi. This distinctly sour limetta is sold in the markets of Rabat (and other cities of Morocco), either fresh, candied or brined and canned. It is an important ingredient of traditional Moroccan cuisine. Although sour lime was a widely used ingredient in Italian cuisine during the 17th century, its state of preservation is deplorable outside of Morocco, and even there it is gradually replaced by lemons. We recommend its cultivation as a resource both for obtaining rootstocks as well as for its fruits, both for Moroccan cuisine and that of other parts of the world for the excellent aromas and flavors that they contribute to the dishes. In this way, by promoting its crop, we will prevent it from disappearing. It is also important to preserve sour lime in germplasm banks. [Display omitted] [ABSTRACT FROM AUTHOR]

• Eleven PMFs in 116 citrus accessions were determined by UPLC–ESI-QTOF-MS/MS and HPLC–DAD. • The spatiotemporal and species-specific distribution of PMFs in citrus was revealed. • The clustering of eleven PMFs by HCA agreed with classic citrus taxonomy. • Wild or semi-wild mandarins contained large amounts of total or specific PMFs. • PMF accumulation may be a phenotype inherited from mandarin ancestors. Citrus fruits are the main dietary source of polymethoxylated flavones (PMFs) with significant effects on consumer health. In this study, eleven main PMFs were evaluated in the fruit flavedo or leaves of 116 citrus accessions via UPLC–DAD–ESI-QTOF-MS/MS combined with HPLC–DAD analysis, which revealed significant species-specific and spatiotemporal characteristics. All Citrus reticulata and their natural or artificial hybrids were found to have detectable PMFs, especially in the fruit flavedo of the wild or early-cultivated mandarins at early fruit development stages. However, PMFs were not detected in citrons, pummelos, kumquats, trifoliata oranges, papedas, Chinese box oranges and 'Mangshanyegan'. The results enlightened that PMF accumulation only in mandarins and mandarin hybrids is a phenotype inherited from mandarin ancestors. This study provides a comprehensive PMF profile in various citrus germplasms and will benefit future functional citrus breeding practices aimed at designing plants rich in total or specific PMFs for health benefits. [ABSTRACT FROM AUTHOR]

In the past decade Colletotrichum gloeosporioides, C. karstii, and Alternaria alternata represent emerging fungal pathogens on citrus in the Mediterranean basin. Selection of tolerant Citrus germplasm offers evaluable long-term solution and should be considered as promising alternative to limit synthetic fungicide application to manage Alternaria and Colletotrichum infections in Citrus groves. In this study, the high variability of pre-harvest disease symptoms among 37 Citrus accessions was investigated in Italy in the most representative Sicilian production districts covering 1500 ha and including 20 homogeneous areas over an eleven-year survey period (2010–2020). Early fruit drop, brown spot, and anthracnose on fruit and leaves, pre-harvest fruit drop, and twig blight and defoliation associated to Alternaria and Colletotrichum spp. were identified on oranges, mandarins, and lemons. Comprehensively, first extensive data obtained herein on field susceptibility within "Tarocco" blood orange group to above disease symptoms clearly indicate as "Tarocco Nucellare 57-1E-1," "Tarocco Tapi," "Tarocco Sant'Alfio," and "Tarocco Catania" accessions should be preferred to remaining Tarocco ones. A broad degree of tolerance or susceptibility was also observed within other Citrus group as it happens for tolerant lemon "Femminello Zagara Bianca," thus demonstrating a putative resource for further studies to employ in a breeding program for genetic improvement of Citrus. [ABSTRACT FROM AUTHOR]