The Citrus Exocortis Disease: A Complex of Viroid-RNAs

Citrons inoculated with different field sources, displayed a variety of symptoms ranging from very mild leaf bending and necrosis to the severe reaction normally associated with exocortis disease. Nucleic acid preparations from shoot samples were analyzed by sequential polyacrylamide gel electrophoresis. All source from both California and Spain contained one to four viroids with distinct physical and biological properties. The size range was estimated from 371 nucleotides for the citrus exocortis viroid (CEV) to 275 for the smallest viroid. The recovery of single viroids suggested a relationship between the distinct viroids and the symptom reaction expressed in citron

Shifting from Seedling Mandarin Trees to Grafted Trees and Controlling Huanglongbing and Viroids: a Biotechnological Revolution in Nepal

Poverty in Nepal, largely a rural phenomenon, is widespread, with 30.8% of the population living below the poverty line. Agriculture is the main source of livelihood of the Nepalese people who are living below poverty. Citrus, the first cash crop, is grown in small to very small orchards producing less than 10 tons/ha. More than 90% of the trees are seedlings of a local mandarin and therefore they are essentially free of most graft-transmissible diseases. Huanglongbing (HLB) and the Asian psyllid vector, Diaphorina citri, were reported in Nepal in the mid-1960s and, in the absence of any control measures, have continued to spread ever since. Here we report the information available regarding the presence of HLB in several important citrus-growing areas (Armalakur, Bandipur, Dhankuta/Karmitar, Kathmandu, Lamjung, Paripatle, Pokhara, Sindulimadi and Syangja) and the identification of four citrus viroids (Citrus exocortis viroid, Hop stunt viroid, Citrus viroid-III, and Citrus viroid-V) in the experimental citrus station of Pokhara. Citrus rehabilitation, as part of a program to improve food security for the Nepalese population, was started in 2004, and is based on (I) producing disease-free citrus trees, grafted on adequate rootstocks, in covered, insect-proof nursery facilities, (II) demonstration orchards with grafted trees, (III) control of HLB by trunk applications of systemic insecticides and/or guava interplants, (IV) selection of viroid-free budwood sources, and (V) transfer of technology to the farmers

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

[EN] Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplastreplicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.This work was supported by the European Research Council (erc.europa.eu; ERC-2011-StG-281191-VIRMUT to RS), the Spanish Ministerio de Economia y Competitividad (www.mineco.gob.es; BFU2013-41329 grant to RS, BFU2014-56812-P grant to RF, and a predoctoral fellowship to ALC), and the Spanish Junta de Comunidades de Castilla-La Mancha (www.castillalamancha.es;postdoctoral fellowship to CB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.López-Carrasco, MA.; Ballesteros Martínez, C.; Sentandreu, V.; Delgado Villar, SG.; Gago Zachert, SP.; Flores Pedauye, R.; Sanjuan Verdeguer, R. (2017). Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing. PLoS Pathogens. 13(9):1-17. https://doi.org/10.1371/journal.ppat.1006547S117139Ganai, R. A., & Johansson, E. (2016). DNA Replication—A Matter of Fidelity. Molecular Cell, 62(5), 745-755. doi:10.1016/j.molcel.2016.05.003Lynch, M. (2010). Evolution of the mutation rate. Trends in Genetics, 26(8), 345-352. doi:10.1016/j.tig.2010.05.003Sanjuán, R., & Domingo-Calap, P. (2016). Mechanisms of viral mutation. Cellular and Molecular Life Sciences, 73(23), 4433-4448. doi:10.1007/s00018-016-2299-6Gago, S., Elena, S. F., Flores, R., & Sanjuan, R. (2009). Extremely High Mutation Rate of a Hammerhead Viroid. Science, 323(5919), 1308-1308. doi:10.1126/science.1169202Flores, R., Gago-Zachert, S., Serra, P., Sanjuán, R., & Elena, S. F. (2014). Viroids: Survivors from the RNA World? Annual Review of Microbiology, 68(1), 395-414. doi:10.1146/annurev-micro-091313-103416Flores, R., Minoia, S., Carbonell, A., Gisel, A., Delgado, S., López-Carrasco, A., … Di Serio, F. (2015). Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection. Virus Research, 209, 136-145. doi:10.1016/j.virusres.2015.02.027Steger, G., & Perreault, J.-P. (2016). Structure and Associated Biological Functions of Viroids. Advances in Virus Research, 141-172. doi:10.1016/bs.aivir.2015.11.002Diener, T. O. (1989). Circular RNAs: relics of precellular evolution? Proceedings of the National Academy of Sciences, 86(23), 9370-9374. doi:10.1073/pnas.86.23.9370Ambrós, S., Hernández, C., & Flores, R. 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D., Benenfeld, B. J., & Robertson, H. D. (1988). Evidence for a single rolling circle in the replication of potato spindle tuber viroid. Proceedings of the National Academy of Sciences, 85(23), 9128-9132. doi:10.1073/pnas.85.23.9128Daros, J.-A., & Flores, R. (2004). Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae. Proceedings of the National Academy of Sciences, 101(17), 6792-6797. doi:10.1073/pnas.0401090101Feldstein, P. A., Hu, Y., & Owens, R. A. (1998). Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid. Proceedings of the National Academy of Sciences, 95(11), 6560-6565. doi:10.1073/pnas.95.11.6560Gas, M.-E., Hernández, C., Flores, R., & Daròs, J.-A. (2007). Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations. PLoS Pathogens, 3(11), e182. doi:10.1371/journal.ppat.0030182Nohales, M.-A., Flores, R., & Daros, J.-A. (2012). Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase. Proceedings of the National Academy of Sciences, 109(34), 13805-13810. doi:10.1073/pnas.1206187109Brass, J. R. J., Owens, R. A., Matoušek, J., & Steger, G. (2017). Viroid quasispecies revealed by deep sequencing. RNA Biology, 14(3), 317-325. doi:10.1080/15476286.2016.1272745Bull, J. J., Sanjuán, R., & Wilke, C. O. (2007). Theory of Lethal Mutagenesis for Viruses. Journal of Virology, 81(6), 2930-2939. doi:10.1128/jvi.01624-06Cuevas, J. M., González-Candelas, F., Moya, A., & Sanjuán, R. (2009). Effect of Ribavirin on the Mutation Rate and Spectrum of Hepatitis C Virus In Vivo. Journal of Virology, 83(11), 5760-5764. doi:10.1128/jvi.00201-09Ribeiro, R. M., Li, H., Wang, S., Stoddard, M. B., Learn, G. H., Korber, B. T., … Perelson, A. S. (2012). Quantifying the Diversification of Hepatitis C Virus (HCV) during Primary Infection: Estimates of the In Vivo Mutation Rate. 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Proceedings of the National Academy of Sciences, 55(6), 1504-1511. doi:10.1073/pnas.55.6.1504López-Carrasco, A., Gago-Zachert, S., Mileti, G., Minoia, S., Flores, R., & Delgado, S. (2015). The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in theirin vivoRNA conformations. RNA Biology, 13(1), 83-97. doi:10.1080/15476286.2015.1119365Keese, P., & Symons, R. H. (1985). Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proceedings of the National Academy of Sciences, 82(14), 4582-4586. doi:10.1073/pnas.82.14.4582López-Carrasco, A., & Flores, R. (2016). Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A «naked» rod-like conformation similar but not identical to that observed in vitro. RNA Biology, 14(8), 1046-1054. doi:10.1080/15476286.2016.1223005Flores, R., Hernandez, C., de la Peña, M., Vera, A., & Daros, J.-A. (2001). Hammerhead Ribozyme Structure and Function in Plant RNA Replication. Ribonucleases - Part A, 540-552. doi:10.1016/s0076-6879(01)41175-xMartick, M., & Scott, W. G. (2006). Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis. Cell, 126(2), 309-320. doi:10.1016/j.cell.2006.06.036Ruffner, D. E., Stormo, G. D., & Uhlenbeck, O. C. (1990). Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry, 29(47), 10695-10702. doi:10.1021/bi00499a018Flores, R., Serra, P., Minoia, S., Di Serio, F., & Navarro, B. (2012). Viroids: From Genotype to Phenotype Just Relying on RNA Sequence and Structural Motifs. Frontiers in Microbiology, 3. doi:10.3389/fmicb.2012.00217Owens, R. A., Chen, W., Hu, Y., & Hsu, Y.-H. (1995). Suppression of Potato Spindle Tuber Viroid Replication and Symptom Expression by Mutations Which Stabilize the Pathogenicity Domain. Virology, 208(2), 554-564. doi:10.1006/viro.1995.1186Takeda, R., Petrov, A. I., Leontis, N. B., & Ding, B. (2011). A Three-Dimensional RNA Motif in Potato spindle tuber viroid Mediates Trafficking from Palisade Mesophyll to Spongy Mesophyll in Nicotiana benthamiana. The Plant Cell, 23(1), 258-272. doi:10.1105/tpc.110.081414Zhong, X., Leontis, N., Qian, S., Itaya, A., Qi, Y., Boris-Lawrie, K., & Ding, B. (2006). Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication. Journal of Virology, 80(17), 8566-8581. doi:10.1128/jvi.00837-06Zhong, X., Tao, X., Stombaugh, J., Leontis, N., & Ding, B. (2007). Tertiary structure and function of an RNA motif required for plant vascular entry to initiate systemic trafficking. The EMBO Journal, 26(16), 3836-3846. doi:10.1038/sj.emboj.7601812Zhong, X., Archual, A. J., Amin, A. A., & Ding, B. (2008). A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking. The Plant Cell, 20(1), 35-47. doi:10.1105/tpc.107.056606Thomas, M. J., Platas, A. A., & Hawley, D. K. (1998). Transcriptional Fidelity and Proofreading by RNA Polymerase II. Cell, 93(4), 627-637. doi:10.1016/s0092-8674(00)81191-5Gout, J.-F., Thomas, W. K., Smith, Z., Okamoto, K., & Lynch, M. (2013). Large-scale detection of in vivo transcription errors. Proceedings of the National Academy of Sciences, 110(46), 18584-18589. doi:10.1073/pnas.1309843110Hedtke, B. (1997). Mitochondrial and Chloroplast Phage-Type RNA Polymerases in Arabidopsis. Science, 277(5327), 809-811. doi:10.1126/science.277.5327.809Lerbs-Mache, S. (1993). The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes? Proceedings of the National Academy of Sciences, 90(12), 5509-5513. doi:10.1073/pnas.90.12.5509Oldenkott, B., Yamaguchi, K., Tsuji-Tsukinoki, S., Knie, N., & Knoop, V. (2014). 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Role of age and comorbidities in mortality of patients with infective endocarditis

Purpose: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. Methods: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015. Patients were stratified into three age groups:<65 years, 65 to 80 years, and = 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. Results: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 = 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients =80 years who underwent surgery were significantly lower compared with other age groups (14.3%, 65 years; 20.5%, 65-79 years; 31.3%, =80 years). In-hospital mortality was lower in the <65-year group (20.3%, <65 years;30.1%, 65-79 years;34.7%, =80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%, =80 years; p = 0.003).Independent predictors of mortality were age = 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI = 3 (HR:1.62; 95% CI:1.39–1.88), and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared, the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. Conclusion: There were no differences in the clinical presentation of IE between the groups. Age = 80 years, high comorbidity (measured by CCI), and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Fungal Planet description sheets: 1284–1382

Novel species of fungi described in this study include those from various countries as follows: Antartica, Cladosporium austrolitorale from coastal sea sand. Australia, Austroboletus yourkae on soil, Crepidotus innuopurpureus on dead wood, Curvularia stenotaphri from roots and leaves of Stenotaphrum secundatum and Thecaphora stajsicii from capsules of Oxalis radicosa. Belgium, Paraxerochrysium coryli (incl. Paraxerochrysium gen. nov.) from Corylus avellana. Brazil, Calvatia nordestina on soil, Didymella tabebuiicola from leaf spots on Tabebuia aurea, Fusarium subflagellisporum from hypertrophied floral and vegetative branches of Mangifera indica and Microdochium maculosum from living leaves of Digitaria insularis. Canada, Cuphophyllus bondii fromagrassland. Croatia, Mollisia inferiseptata from a rotten Laurus nobilis trunk. Cyprus, Amanita exilis oncalcareoussoil. Czech Republic, Cytospora hippophaicola from wood of symptomatic Vaccinium corymbosum. Denmark, Lasiosphaeria deviata on pieces of wood and herbaceousdebris. Dominican Republic, Calocybella goethei among grass on a lawn. France (Corsica) , Inocybe corsica onwetground. France (French Guiana) , Trechispora patawaensis on decayed branch of unknown angiosperm tree and Trechispora subregularis on decayed log of unknown angiosperm tree. Germany, Paramicrothecium sambuci (incl. Paramicrothecium gen. nov.)ondeadstemsof Sambucus nigra. India, Aureobasidium microtermitis from the gut of a Microtermes sp. termite, Laccaria diospyricola on soil and Phylloporia tamilnadensis on branches of Catunaregam spinosa. Iran, Pythium serotinoosporum from soil under Prunus dulcis. Italy, Pluteus brunneovenosus on twigs of broad leaved trees on the ground. Japan, Heterophoma rehmanniae on leaves of Rehmannia glutinosa f. hueichingensis. Kazakhstan, Murispora kazachstanica from healthy roots of Triticum aestivum. Namibia, Caespitomonium euphorbiae (incl. Caespitomonium gen. nov.)from stems of an Euphorbia sp. Netherlands, Alfaria junci, Myrmecridium junci, Myrmecridium juncicola, Myrmecridium juncigenum, Ophioceras junci, Paradinemasporium junci (incl. Paradinemasporium gen. nov.), Phialoseptomonium junci, Sporidesmiella juncicola, Xenopyricularia junci and Zaanenomyces quadripartis (incl. Zaanenomyces gen. nov.), fromdeadculmsof Juncus effusus, Cylindromonium everniae and Rhodoveronaea everniae from Evernia prunastri, Cyphellophora sambuci and Myrmecridium sambuci from Sambucus nigra, Kiflimonium junci, Saro cladium junci, Zaanenomyces moderatricis academiae and Zaanenomyces versatilis from dead culms of Juncus inflexus, Microcera physciae from Physcia tenella, Myrmecridium dactylidis from dead culms of Dactylis glomerata, Neochalara spiraeae and Sporidesmium spiraeae from leaves of Spiraea japonica, Neofabraea salicina from Salix sp., Paradissoconium narthecii (incl. Paradissoconium gen. nov.)from dead leaves of Narthecium ossifragum, Polyscytalum vaccinii from Vaccinium myrtillus, Pseudosoloacrosporiella cryptomeriae (incl. Pseudosoloacrosporiella gen. nov.)fromleavesof Cryptomeria japonica, Ramularia pararhabdospora from Plantago lanceolata, Sporidesmiella pini from needles of Pinus sylvestris and Xenoacrodontium juglandis (incl. Xenoacrodontium gen. nov. and Xenoacrodontiaceae fam. nov.)from Juglans regia. New Zealand, Cryptometrion metrosideri from twigs of Metrosideros sp., Coccomyces pycnophyllocladi from dead leaves of Phyllocladus alpinus, Hypoderma aliforme from fallen leaves Fuscopora solandri and Hypoderma subiculatum from dead leaves Phormium tenax. Norway, Neodevriesia kalakoutskii from permafrost and Variabilispora viridis from driftwood of Picea abies. Portugal, Entomortierella hereditatis from abio film covering adeteriorated limestone wall. Russia, Colpoma junipericola from needles of Juniperus sabina, Entoloma cinnamomeum on soil in grasslands, Entoloma verae on soil in grasslands, Hyphodermella pallidostraminea on a dry dead branch of Actinidia sp., Lepiota sayanensis onlitterinamixedforest, Papiliotrema horticola from Malus communis , Paramacroventuria ribis (incl. Paramacroventuria gen. nov.)fromleaves of Ribes aureum and Paramyrothecium lathyri from leaves of Lathyrus tuberosus. South Africa, Harzia combreti from leaf litter of Combretum collinum ssp. sulvense, Penicillium xyleborini from Xyleborinus saxesenii , Phaeoisaria dalbergiae from bark of Dalbergia armata, Protocreopsis euphorbiae from leaf litter of Euphorbia ingens and Roigiella syzygii from twigs of Syzygium chordatum. Spain, Genea zamorana on sandy soil, Gymnopus nigrescens on Scleropodium touretii, Hesperomyces parexochomi on Parexochomus quadriplagiatus, Paraphoma variabilis from dung, Phaeococcomyces kinklidomatophilus from a blackened metal railing of an industrial warehouse and Tuber suaveolens in soil under Quercus faginea. Svalbard and Jan Mayen, Inocybe nivea associated with Salix polaris. Thailand, Biscogniauxia whalleyi oncorticatedwood. UK, Parasitella quercicola from Quercus robur. USA , Aspergillus arizonicus from indoor air in a hospital, Caeliomyces tampanus (incl. Caeliomyces gen. nov.)fromoffice dust, Cippumomyces mortalis (incl. Cippumomyces gen. nov.)fromatombstone, Cylindrium desperesense from air in a store, Tetracoccosporium pseudoaerium from air sample in house, Toxicocladosporium glendoranum from air in a brick room, Toxicocladosporium losalamitosense from air in a classroom, Valsonectria portsmouthensis from airinmen'slockerroomand Varicosporellopsis americana from sludge in a water reservoir. Vietnam, Entoloma kovalenkoi on rotten wood, Fusarium chuoi inside seed of Musa itinerans , Micropsalliota albofelina on soil in tropical evergreen mixed forest sand Phytophthora docyniae from soil and roots of Docynia indica. Morphological and culture characteristics are supported by DNA barcodes

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Fungal Planet description sheets: 1284-1382

Novel species of fungi described in this study include those from various countries as follows: Antartica, Cladosporium austrolitorale from coastal sea sand. Australia, Austroboletus yourkae on soil, Crepidotus innuopurpureus on dead wood, Curvularia stenotaphri from roots and leaves of Stenotaphrum secundatum and Thecaphora stajsicii from capsules of Oxalis radicosa. Belgium, Paraxerochrysium coryli (incl. Paraxerochrysium gen. nov.) from Corylus avellana. Brazil, Calvatia nordestina on soil, Didymella tabebuiicola from leaf spots on Tabebuia aurea, Fusarium subflagellisporum from hypertrophied floral and vegetative branches of Mangifera indica and Microdochium maculosum from living leaves of Digitaria insularis. Canada, Cuphophyllus bondii fromagrassland. Croatia, Mollisia inferiseptata from a rotten Laurus nobilis trunk. Cyprus, Amanita exilis oncalcareoussoil. Czech Republic, Cytospora hippophaicola from wood of symptomatic Vaccinium corymbosum. Denmark, Lasiosphaeria deviata on pieces of wood and herbaceousdebris. Dominican Republic, Calocybella goethei among grass on a lawn. France (Corsica) , Inocybe corsica onwetground. France (French Guiana) , Trechispora patawaensis on decayed branch of unknown angiosperm tree and Trechispora subregularis on decayed log of unknown angiosperm tree. [...]P.R. Johnston thanks J. Sullivan (Lincoln University) for the habitat image of Kowai Bush, Duckchul Park (Manaaki Whenua – Landcare Research) for the DNA sequencing, and the New Zealand Department of Conservation for permission to collect the specimens; this research was supported through the Manaaki Whenua – Landcare Research Biota Portfolio with funding from the Science and Innovation Group of the New Zealand Ministry of Business, Innovation and Employment. V. Hubka was supported by the Czech Ministry of Health (grant number NU21-05-00681), and is grateful for the support from the Japan Society for the Promotion of Science – grant-in-aid for JSPS research fellow (grant no. 20F20772). K. Glässnerová was supported by the Charles University Grant Agency (grant No. GAUK 140520). J. Trovão and colleagues were financed by FEDERFundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operational Programme for Competitiveness and Internationalisation (POCI), and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia in the framework of the project POCI-01-0145-FEDER-PTDC/ EPH-PAT/3345/2014. This work was carried out at the R&D Unit Centre for Functional Ecology – Science for People and the Planet (CFE), with reference UIDB/04004/2020, financed by FCT/MCTES through national funds (PIDDAC). J. Trovão was also supported by POCH – Programa Operacional Capital Humano (co-funding by the European Social Fund and national funding by MCTES), through a ‘FCT – Fundação para a Ciência e Tecnologia’ PhD research grant (SFRH/BD/132523/2017). D. Haelewaters acknowledges support from the Research Foundation – Flanders (Junior Postdoctoral Fellowship 1206620N). M. Loizides and colleagues are grateful to Y. Cherniavsky for contributing collections AB A12-058-1 and AB A12- 058-2, and Á. Kovács and B. Kiss for their help with molecular studies of these specimens. C. Zmuda is thanked for assisting with the collection of ladybird specimens infected with Hesperomyces parexochomi. A.V. Kachalkin and colleagues were supported by the Russian Science Foundation (grant No. 19-74-10002). The study of A.M. Glushakova was carried out as part of the Scientific Project of the State Order of the Government of Russian Federation to Lomonosov Moscow State University No. 121040800174-6. S. Nanu acknowledges the Kerala State Council for Science, Technology and Environment (KSCSTE) for granting a research fellowship and is grateful to the Chief Conservator of Forests and Wildlife for giving permission to collect fungal samples. A. Bañares and colleagues thank L. Monje and A. Pueblas of the Department of Drawing and Scientific Photography at the University of Alcalá for their help in the digital preparation of the photographs, and J. Rejos, curator of the AH herbarium for his assistance with the specimens examined in the present study. The research of V. Antonín received institutional support for long-term conceptual development of research institutions provided by the Ministry of Culture (Moravian Museum, ref. MK000094862). The studies of E.F. Malysheva, V.F. Malysheva, O.V. Morozova, and S.V. Volobuev were carried out within the framework of a research project of the Komarov Botanical Institute RAS, St Petersburg, Russia (АААА-А18-118022090078-2) using equipment of its Core Facility Centre ‘Cell and Molecular Technologies in Plant Science’.The study of A.V. Alexandrova was carried out as part of the Scientific Project of the State Order of the Government of Russian Federation to Lomonosov Moscow State University No. 121032300081-7. The Kits van Waveren Foundation (Rijksherbariumfonds Dr E. Kits van Waveren, Leiden, Netherlands) contributed substantially to the costs of sequencing and travelling expenses for M.E. Noordeloos. The work of B. Dima was partly supported by the ÚNKP- 20-4 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund. The work of L. Nagy was supported by the ‘Momentum’ program of the Hungarian Academy of Sciences (contract No. LP2019- 13/2019 to L.G.N.). G.A. Kochkina and colleagues acknowledge N. Demidov for the background photograph, and N. Suzina for the SEM photomicrograph. The research of C.M. Visagie and W.J. Nel was supported by the National Research Foundation grant no 118924 and SFH170610239162. C. Gil-Durán acknowledges Agencia Nacional de Investigación y Desarrollo, Ministerio de Ciencia, Tecnología, Conocimiento e Innovación, Gobierno de Chile, for grant ANID – Fondecyt de Postdoctorado 2021 – N° 3210135. R. Chávez and G. Levicán thank DICYT-USACH and acknowledges the grants INACH RG_03-14 and INACH RT_31-16 from the Chilean Antarctic Institute, respectively. S. Tiwari and A. Baghela would like to acknowledge R. Avchar and K. Balasubramanian from the Agharkar Research Institute, Pune, Maharashtra for helping with the termite collection. S. Tiwari is also thankful to the University Grants Commission, Delhi (India) for a junior research fellowship (827/(CSIR-UGC NET DEC.2017)). R. Lebeuf and I. Saar thank D. and H. Spencer for collecting and photographing the holotype of C. bondii, and R. Smith for photographing the habitat. A. Voitk is thanked for helping with the colour plate and review of the manuscript, and the Foray Newfoundland and Labrador for providing the paratype material. I. Saar was supported by the Estonian Research Council (grant PRG1170) and the European Regional Development Fund (Centre of Excellence EcolChange). M.P.S. Câmara acknowledges the ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq’ for the research productivity fellowship, and financial support (Universal number 408724/2018-8). W.A.S. Vieira acknowledges the ‘Coordenação de Aperfeiçoamento Pessoal de Ensino Superior – CAPES’ and the ‘Programa Nacional de Pós-Doutorado/CAPES – PNPD/CAPES’ for the postdoctoral fellowship. A.G.G. Amaral acknowledges CNPq, and A.F. Lima and I.G. Duarte acknowledge CAPES for the doctorate fellowships. F. Esteve-Raventós and colleagues were financially supported by FEDER/ Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación (Spain)/ Project CGL2017-86540-P. The authors would like to thank L. Hugot and N. Suberbielle (Conservatoire Botanique National de Corse, Office de l’Environnement de la Corse, Corti) for their help. The research of E. Larsson is supported by The Swedish Taxonomy Initiative, SLU Artdatabanken, Uppsala. Financial support was provided to R.J. Ferreira by the National Council for Scientific and Technological Development (CNPq), and to I.G. Baseia, P.S.M. Lúcio and M.P. Martín by the National Council for Scientific and Technological Development (CNPq) under CNPq-Universal 2016 (409960/2016-0) and CNPq-visiting researcher (407474/2013-7). J. Cabero and colleagues wish to acknowledge A. Rodríguez for his help to describe Genea zamorana, as well as H. Hernández for sharing information about the vegetation of the type locality. S. McMullan-Fisher and colleagues acknowledge K. Syme (assistance with illustrations), J. Kellermann (translations), M. Barrett (collection, images and sequences), T. Lohmeyer (collection and images) and N. Karunajeewa (for prompt accessioning). This research was supported through funding from Australian Biological Resources Study grant (TTC217-06) to the Royal Botanic Gardens Victoria. The research of M. Spetik and co-authors was supported by project No. CZ.02.1.01/0.0/0.0 /16_017/0002334. N. Wangsawat and colleagues were partially supported by NRCT and the Royal Golden Jubilee Ph.D. programme, grant number PHD/0218/2559. They are thankful to M. Kamsook for the photograph of the Phu Khiao Wildlife Sanctuary and P. Thamvithayakorn for phylogenetic illustrations. The study by N.T. Tran and colleagues was funded by Hort Innovation (Grant TU19000). They also thank the turf growers who supported their surveys and specimen collection. N. Matočec, I. Kušan, A. Pošta, Z. Tkalčec and A. Mešić thank the Croatian Science Foundation for their financial support under the project grant HRZZ-IP-2018-01-1736 (ForFungiDNA). A. Pošta thanks the Croatian Science Foundation for their support under the grant HRZZ-2018-09-7081. A. Morte is grateful to Fundación Séneca – Agencia de Ciencia y Tecnología de la Región de Murcia (20866/ PI/18) for financial support. The research of G. Akhmetova, G.M. Kovács, B. Dima and D.G. Knapp was supported by the National Research, Development and Innovation Office, Hungary (NKFIH KH-130401 and K-139026), the ELTE Thematic Excellence Program 2020 supported by the National Research, Development and Innovation Office (TKP2020-IKA-05) and the Stipendium Hungaricum Programme. The support of the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and the Bolyai+ New National Excellence Program of the Ministry for Innovation and Technology to D.G. Knapp is highly appreciated. F.E. Guard and colleagues are grateful to the traditional owners, the Jirrbal and Warungu people, as well as L. and P. Hales, Reserve Managers, of the Yourka Bush Heritage Reserve. Their generosity, guidance, and the opportunity to explore the Bush Heritage Reserve on the Einasleigh Uplands in far north Queensland is greatly appreciated. The National Science Foundation (USA) provided funds (DBI#1828479) to the New York Botanical Garden for a scanning electron microscope used for imaging the spores. V. Papp was supported by the ÚNKP-21-5 New National Excellence Program of the Ministry for Innovation and Technology from the National Research, Development and Innovation Fund of Hungary. A.N. Miller thanks the WM Keck Center at the University of Illinois Urbana – Champaign for sequencing Lasiosphaeria deviata. J. Pawłowska acknowledges support form National Science Centre, Poland (grant Opus 13 no 2017/25/B/NZ8/00473). The research of T.S. Bulgakov was carried out as part of the State Research Task of the Subtropical Scientific Centre of the Russian Academy of Sciences (Theme No. 0492-2021- 0007). K. Bensch (Westerdijk Fungal Biodiversity Institute, Utrecht) is thanked for correcting the spelling of various Latin epithets.Peer reviewe

Morphogenesis and regeneration of whole plants of grapefruit (Citrus paradisi), sour orange (C-aurantium) and alemow (C-macrophylla)

Studies were performed to define tissue culture techniques for morphogenesis and production of whole plants of grapefruit, sour orange and alemow. The NAA and BA concentrations to induce root formation, and bud and shoot regeneration on stem segments were determined. No roots formed in sour orange over the range of 0-162 mu M NAA. All species regenerated buds in response to BA but only those of alemow elongated sufficiently to root them. Of these, 84% rooted but only 16% of these plantlets survived weaning and transplanting to soil. Plants were consistently obtained by grafting apical tips from regenerated shoots and buds of the three species. Plants with successful grafts were transplanted to soil with survival rates of 90-100%

Biological characterization of citrus tristeza virus isolates by in vitro tissue cultures

Stem segments from Mexican lime, sweet orange, grapefruit, Citrus excelsa and alemow, infected with five citrus tristeza virus (CTV) isolates, were cultured in vitro. Regeneration of roots and shoots were modified as a result of infection. The effect of CTV on the morphogenesis of stem segments cultured in vitro depended on the CTV isolate and the plant host, and showed a correlation with the in vivo effects observed in biological indexing. Evaluation of the morphogenic response of stem segments of Mexican lime and grapefruit can be used as an additional tool for the biological characterization of CTV isolates. The symptoms on sweet orange plants obtained from regenerated shoots indicated that CTV is unevenly distributed in the host plant cells and that the regeneration process may be utilized as a tool to separate strains from complex field isolates