Showing papers on "Ribosomal DNA published in 2022"

Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly

Abstract: High content image analysis, single molecule tracking, modeling, and DBA patient analysis revealed that ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly. The nucleolus is the site of ribosome assembly and formed through liquid–liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid–liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond–Blackfan anemia patient harboring a heterozygous, large deletion in RPL5. Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.

Genetic variation at mouse and human ribosomal DNA influences associated epigenetic states

Abstract: Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed.Using a combination of long- and short-read sequencing, we establish that 45S rDNA units in the C57BL/6J mouse strain exist as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. DNA methylation dynamics at these haplotypes are dichotomous and life-stage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during aging only. On the other hand, individual rDNA units in human show limited evidence of genetic haplotypes, and hence little discernible correlation between genetic and epigenetic states. However, in both species, adjacent units show similar epigenetic profiles, and the overall epigenetic state at rDNA is strongly positively correlated with the total rDNA copy number. Analysis of different mouse inbred strains reveals that in some strains, such as 129S1/SvImJ, the rDNA copy number is only approximately 150 copies per diploid genome and DNA methylation levels are < 5%.Our work demonstrates that rDNA-associated genetic variation has a considerable influence on rDNA epigenetic state and consequently rRNA expression outcomes. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.

Genetic variation at mouse and human ribosomal DNA influences associated epigenetic states

Abstract: Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed.Using a combination of long- and short-read sequencing, we establish that 45S rDNA units in the C57BL/6J mouse strain exist as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. DNA methylation dynamics at these haplotypes are dichotomous and life-stage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during aging only. On the other hand, individual rDNA units in human show limited evidence of genetic haplotypes, and hence little discernible correlation between genetic and epigenetic states. However, in both species, adjacent units show similar epigenetic profiles, and the overall epigenetic state at rDNA is strongly positively correlated with the total rDNA copy number. Analysis of different mouse inbred strains reveals that in some strains, such as 129S1/SvImJ, the rDNA copy number is only approximately 150 copies per diploid genome and DNA methylation levels are < 5%.Our work demonstrates that rDNA-associated genetic variation has a considerable influence on rDNA epigenetic state and consequently rRNA expression outcomes. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.

Assessment of ITS1, ITS2, 5′-ETS, and trnL-F DNA Barcodes for Metabarcoding of Poaceae Pollen

Abstract: Grass pollen is one of the major causes of allergy. Aerobiological monitoring is a necessary element of the complex of anti-allergic measures, but the similar pollen morphology of Poaceae species makes it challenging to discriminate species in airborne pollen mixes, which impairs the quality of aerobiological monitoring. One of the solutions to this problem is the metabarcoding approach employing DNA barcodes for taxonomical identification of species in a mix by high-throughput sequencing of the pollen DNA. A diverse set of 14 grass species of different genera were selected to create a local reference database of nuclear ITS1, ITS2, 5′-ETS, and plastome trnL-F DNA barcodes. Sequences for the database were Sanger sequenced from live field and herbarium specimens and collected from GenBank. New Poaceae-specific primers for 5′-ETS were designed and tested to obtain a 5′-ETS region less than 600 bp long, suitable for high-throughput sequencing. The DNA extraction method for single-species pollen samples and mixes was optimized to increase the yield for amplification and sequencing of pollen DNA. Barcode sequences were analyzed and compared by the barcoding gap and intra- and interspecific distances. Their capability to correctly identify grass pollen was tested on artificial pollen mixes of various complexity. Metabarcoding analysis of the artificial pollen mixes showed that nuclear DNA barcodes ITS1, ITS2, and 5′-ETS proved to be more efficient than the plastome barcode in both amplification from pollen DNA and identification of grass species. Although the metabarcoding results were qualitatively congruent with the actual composition of the pollen mixes in most cases, the quantitative results based on read-counts did not match the actual ratio of pollen grains in the mixes.

Multi-Gene Phylogenetic Approach for Identification and Diversity Analysis of Bipolaris maydis and Curvularia lunata Isolates Causing Foliar Blight of Zea mays

Abstract: Bipolaris species are known to be important plant pathogens that commonly cause leaf spot, root rot, and seedling blight in a wide range of hosts worldwide. In 2017, complex symptomatic cases of maydis leaf blight (caused by Bipolaris maydis) and maize leaf spot (caused by Curvularia lunata) have become increasingly significant in the main maize-growing regions of India. A total of 186 samples of maydis leaf blight and 129 maize leaf spot samples were collected, in 2017, from 20 sampling sites in the main maize-growing regions of India to explore the diversity and identity of this pathogenic causal agent. A total of 77 Bipolaris maydis isolates and 74 Curvularia lunata isolates were screened based on morphological and molecular characterization and phylogenetic analysis based on ribosomal markers—nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region, 28S nuclear ribosomal large subunit rRNA gene (LSU), D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA), and protein-coding gene-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Due to a dearth of molecular data from ex-type cultures, the use of few gene regions for species resolution, and overlapping morphological features, species recognition in Bipolaris has proven difficult. The present study used the multi-gene phylogenetic approach for proper identification and diversity of geographically distributed B. maydis and C. lunata isolates in Indian settings and provides useful insight into and explanation of its quantitative findings.

Discrimination of 15 Amazonian Anopheline Mosquito Species by Polymerase Chain Reaction—Restriction Fragment Length Polymorphism

Abstract: Abstract Precise identification of anopheline species is paramount for incrimination of malaria vectors and implementation of a sustainable control program. Anopheline mosquitoes are routinely identified morphologically, a technique that is time-consuming, needs high level of expertise, and prone to misidentifications especially when considering Amazonian species. The aim of this study was therefore to develop a DNA-based identification technique to supplement traditional morphological identification methods for the discrimination of anopheline mosquitoes collected in French Guiana. The internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA) for anopheline species was amplified by polymerase chain reaction (PCR), and digested with AluI/MspI restriction enzymes. PCR-restriction fragments length polymorphism (RFLP) assay was compared to sequencing of the ITS2 region for validation. Fifteen Anopheles species have shown distinct PCR-RFLP profiles. A concordance of 100% was obtained when identification by PCR-RFLP was compared to sequencing of ITS2. A high throughput, fast, and cost-effective PCR-RFLP assay has been developed for unambiguous discrimination of fifteen anopheline mosquito species from French Guiana including primary and suspected secondary malaria vectors.

A New Oomycete Metabarcoding Method Using the <i>rps10</i> Gene

Abstract: Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause plant and animal diseases. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have evaluated the mitochondrial 40S ribosomal protein S10 ( rps10) gene as a locus for oomycete metabarcoding and provide primers predicted to amplify this region from all oomycetes based on a wide range of available reference sequences. We evaluated its utility relative to the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Observed sequences and predicted taxonomy of ASVs and OTUs were compared with the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1-bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest that the rps10 locus results in substantially less amplification of nontarget organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing.

The yeast lichenosphere: high diversity of basidiomycetes from the lichens Tephromela atra and Rhizoplaca melanophthalma.

Abstract: Lichens are well-known examples of complex symbiotic associations between organisms from different Kingdoms. Microfungi in particular, establish diverse associations with the hosting lichen thallus, as species-specific parasites or transient co-inhabitants. The whole community of lichen-associated fungi constitute the 'lichen mycobiome' comprising both ascomycetes and basidiomycetes, including filamentous and yeast taxa. Metabarcoding results and microscopy analyses show that in some thalli, basidiomycetes are frequent lichen-associated fungi but still only a few species could be axenically isolated and morphologically characterized. Within a broad project aiming at characterizing the mycobiome diversity by culture-dependent and independent approaches in two lichen species selected as reference models - Rhizoplaca melanophthalma and Tephromela atra, we succeed in isolating and culturing 76 new strains of basidiomycetous yeasts. The lichen thalli were collected in different mountain regions worldwide and at relatively high elevation. The yeast strains were isolated on different growth media and were studied for their morphological and genetic diversity. Nuclear internal transcribed spacer (ITS) and ribosomal large subunit (LSU) sequence analyses identified them to belong to ten families within the orders Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes. The yeasts here detected showed patterns of host-preference in a few cases and they are potentially related to the ecological conditions.

The Ribosomal DNA Loci of the Ancient Monocot Pistia stratiotes L. (Araceae) Contain Different Variants of the 35S and 5S Ribosomal RNA Gene Units

Abstract: The freshwater plant water lettuce (Pistia stratiotes L.) grows in warm climatic zones and is used for phytoremediation and biomass production. P. stratiotes belongs to the Araceae, an ecologically and structurally diverse early monocot family, but the phylogenetic relationships among Araceae members are poorly understood. Ribosomal DNAs (rDNAs), including the 35S and 5S rDNA, encode the RNA components of ribosomes and are widely used in phylogenetic and evolutionary studies of various plant taxa. Here, we comprehensively characterized the chromosomal locations and molecular organization of 35S and 5S rDNA genes in water lettuce using karyological and molecular methods. Fluorescence in situ hybridization revealed a single location for the 35S and 5S rDNA loci, each on a different pair of the species’ 28 chromosomes. Molecular cloning and nucleotide sequencing of 35S rDNA of P. stratiotes, the first representative Araceae sensu stricto in which such a study was performed, displayed typical structural characteristics. The full-length repeat showed high sequence conservation of the regions producing the 18S, 5.8S, and 25S rRNAs and divergence of the internal transcribed spacers ITS1 and ITS2 as well as the large intergenic spacer (IGS). Alignments of the deduced sequence of 18S rDNA with the sequences available for other Araceae and representatives of other clades were used for phylogenetic analysis. Examination of 11 IGS sequences revealed significant intra-genomic length variability due to variation in subrepeat number, with four types of units detected within the 35S rDNA locus of the P. stratiotes genome (estimated size 407 Mb/1C). Similarly, the 5S rDNA locus harbors gene units comprising a conserved 119-bp sequence encoding 5S rRNA and two types of non-transcribed spacer (NTS) sequences. Type I was classified into four subtypes, which apparently originated via progressive loss of subrepeats within the duplicated NTS region containing the 3’ part of the 5S rRNA gene. The minor Type II NTS is shorter than Type I and differs in nucleotide composition. Some DNA clones containing two or three consecutive 5S rDNA repeats harbored 5S rDNA genes with different types of NTSs, confirming the mosaic composition of the 5S rDNA locus.

Yeast Biodiversity in Vineyard during Grape Ripening: Comparison between Culture Dependent and NGS Analysis

Abstract: In this study, the evolution of the yeast microflora present on the berry surface, during the ripening of Barbera grapes, was monitored. Sampling was performed in three vineyards located in the “Nizza” Barbera d’Asti DOC zone and different methodologies have been employed. A culture-dependent method based on the identification of strains grown on solid media by ARDRA (Amplified Ribosomal DNA Restriction Analysis) and the D1-D2 domain of ribosomal 26S DNA capillary sequencing was coupled to NGS (Next Generation Sequencing) targeting ITS (Internal Transcribed Sequence) amplicons with the Illumina MiSeq platform. By using culture-dependent techniques, the most frequently detected species was the yeast-like fungus Aureobasidium pullulans, which was dominant in the culturable fraction. Among yeasts, the presence of oligotrophic basidiomycetes such as Cryptococcus spp., Rhodotorula graminis and Sporidiobolus pararoseus was observed at the beginning of ripening. Afterward, upon approaching the harvest, a succession of oxidative or weakly fermentative copiotrophic species occurs, such as Saturnispora diversa, Issatchenkia terricola, Hanseniaspora opuntiae, Starmerella bacillaris and Hanseniaspora uvarum. The massive sequencing revealed a larger number of species, respect to the culture-dependent data. Comparing the two different approaches used in this work, it is possible to highlight some similarities since Aureobasidium, Rhodotorula and Sporobolomyces were detected by both methods. On the contrary, genera Hanseniaspora, Issatchenkia and Saturnispora were revealed by culture-dependent methods, but not by NGS, while Saccharomyces spp. were identified, with low frequency, only by NGS. The integrated application of NGS sequencing and culture-dependent techniques provides a comprehensive view of mycodiversity in the wine-growing environment, especially for yeasts with low abundance.

Towards Improved Detection and Identification of Rust Fungal Pathogens in Environmental Samples Using a Metabarcoding Approach

Abstract: The dispersion of fungal inocula such as the airborne spores of rust fungi (Pucciniales) can be monitored through metabarcoding of the internal transcribed spacer 2 (ITS2) of the rRNA gene in environmental DNAs. This method is largely dependent on a high-quality reference database (refDB) and primers with proper taxonomic coverage and specificity. For this study, a curated ITS2 reference database (named CR-ITS2-refDB) comprising representatives of the major cereal rust fungi and phylogenetically related species was compiled. Interspecific and intraspecific variation analyses suggested that the ITS2 region had reasonable discriminating power for the majority of the Puccinia species or species complexes in the database. In silico evaluation of nine forward and seven reverse ITS2 primers, including three newly designed, revealed marked variation in DNA amplification efficiency for the rusts. We validated the theoretical assessment of rust-enhanced (Rust2inv/ITS4var_H) and universal fungal (ITS9F/ITS4) ITS2 primer pairs by profiling the airborne rust fungal communities from environmental samples via a metabarcoding approach. Species- or subspecies-level identification of the rusts was improved by use of CR-ITS2-refDB and the Automated Oligonucleotide Design Pipeline (AODP), which identified all mutations distinguishing highly conserved DNA markers between close relatives. A generic bioinformatics pipeline was developed, including all steps used in this study from in silico evaluation of primers to accurate identification of short metabarcodes at the level of interest for defining phytopathogens. The results highlight the importance of primer selection, refDBs that are resolved to reflect phylogenetic relationships, and the use of AODP for improving the reliability of metabarcoding in phytopathogen biosurveillance.

DNA Methylation Analysis of Ribosomal DNA in Adults With Down Syndrome

Abstract: Control of ribosome biogenesis is a critical aspect of the regulation of cell metabolism. As ribosomal genes (rDNA) are organized in repeated clusters on chromosomes 13, 14, 15, 21, and 22, trisomy of chromosome 21 confers an excess of rDNA copies to persons with Down syndrome (DS). Previous studies showed an alteration of ribosome biogenesis in children with DS, but the epigenetic regulation of rDNA genes has not been investigated in adults with DS so far. In this study, we used a targeted deep-sequencing approach to measure DNA methylation (DNAm) of rDNA units in whole blood from 69 adults with DS and 95 euploid controls. We further evaluated the expression of the precursor of ribosomal RNAs (RNA45S) in peripheral blood mononuclear cells (PBMCs) from the same subjects. We found that the rDNA promoter tends to be hypermethylated in DS concerning the control group. The analysis of epihaplotypes (the combination of methylated and unmethylated CpG sites along the same DNA molecule) showed a significantly lower intra-individual diversity in the DS group, which at the same time was characterized by a higher interindividual variability. Finally, we showed that RNA45S expression is lower in adults with DS. Collectively, our results suggest a rearrangement of the epigenetic profile of rDNA in DS, possibly to compensate for the extranumerary rDNA copies. Future studies should assess whether the regulation of ribosome biogenesis can contribute to the pathogenesis of DS and explain the clinical heterogeneity characteristic of the syndrome.

Varying strength of selection contributes to the intragenomic diversity of rRNA genes

Abstract: Ribosome biogenesis in eukaryotes is supported by hundreds of ribosomal RNA (rRNA) gene copies that are encoded in the ribosomal DNA (rDNA). The multiple copies of rRNA genes are thought to have low sequence diversity within one species. Here, we present species-wide rDNA sequence analysis in Saccharomyces cerevisiae that challenges this view. We show that rDNA copies in this yeast are heterogeneous, both among and within isolates, and that many variants avoided fixation or elimination over evolutionary time. The sequence diversity landscape across the rDNA shows clear functional stratification, suggesting different copy-number thresholds for selection that contribute to rDNA diversity. Notably, nucleotide variants in the most conserved rDNA regions are sufficiently deleterious to exhibit signatures of purifying selection even when present in only a small fraction of rRNA gene copies. Our results portray a complex evolutionary landscape that shapes rDNA sequence diversity within a single species and reveal unexpectedly strong purifying selection of multi-copy genes.

Intact rDNA arrays of Potentilla-origin detected in Erythronium nucleus suggest recent eudicot-to-monocot horizontal transfer.

Abstract: During our initial phylogenetic study of the monocot genus Erythronium (Liliaceae), we observed peculiar eudicot-type internal transcribed spacer (ITS) sequences in a dataset derived from genomic DNA of Erythronium dens-canis. This raised the possibility horizontal transfer of a eudicot alien ribosomal DNA (rDNA) into the Erythronium genome. In this work we aimed to support the hypothesis by carrying out genomic, molecular and cytogenetic analyses. Genome skimming coupled by PacBio HiFi sequencing of a flow-sorted bacterial artificial chromosome (BAC) clone was used to characterise the alien 45S rDNA. Integration of alien rDNA in the recipient genome was further proved by Southern blotting and fluorescence in situ hybridisation (FISH) using specific probes. Alien rDNA, nested among Potentilla species in phylogenetic analysis, likely entered Erythronium lineage in the common ancestor of E. dens-canis and E. caucasicum. Transferred eudicot-type rDNA preserved its tandemly arrayed feature on a single chromosome and was found to be transcribed in the monocot host albeit much less efficiently than the native counterpart. This study adds a new example to the rarely documented nuclear-to-nuclear jumps of DNA between eudicots and monocots while holding the scientific community continually in suspense about the mode of DNA transfer.

Using Culture-Dependent and Molecular Techniques to Identify Endophytic Fungi Associated with Tea Leaves (Camellia spp.) in Yunnan Province, China

Abstract: The association of endophytic fungi with the host plant is called a symbiotic relationship. Studies of the endophytic fungi from tea have been reported in numerous documents, but researchers still largely focus on tea endophytic fungi as they have ability to produce bioactive compounds which have numerous applications. The present work characterizes the fungal endophytic communities associated with healthy tea leaves in Yunnan Province, China. A total of 287 fungal strains were isolated from healthy leaf tissues of tea plants using a culture-dependent approach. Based on nuclear ribosomal DNA internal transcribed spacer (ITS) sequence analyses taken from the fungal cultures, strains were classified into 28 fungal genera with high similarity matches to known sequences in GenBank. The majority of genera (98.25%) belong to the phylum Ascomycota and most of the dominating fungal endophytes are from the genera Colletotrichum and Clonostachys.

Four new species of Diaporthe (Diaporthaceae, Diaporthales) from forest plants in China

Abstract: Abstract Species of Diaporthe inhabit a wide range of plant hosts as plant pathogens, endophytes and saprobes. During trips to collect forest pathogens in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces in China, 16 isolates of Diaporthe were obtained from branch cankers and leaf spots. These isolates were studied by applying a polyphasic approach including morphological, cultural data, and phylogenetic analyses of the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef-1α) and β-tubulin (tub2) loci. Results revealed four new taxa, D.celticola, D.meliae, D.quercicola, D.rhodomyrtispp. nov. and two known species, D.eres and D.multiguttulata.

Molecular Analyses Place the Genus Keraunea Outside Convolvulaceae

Abstract: Abstract. The genus Keraunea was recently described in the Convolvulaceae Juss. family. Two species are currently recognised, both from Brazil. Molecular sequence data using three commonly applied DNA markers (matK, rbcL and the nuclear ribosomal Internal Transcribed Spacer) show that neither species is correctly placed in Convolvulaceae but indicates that the type, K. brasiliensis, should be placed in Malpighiaceae despite several morphological anomalies. The second species, K. capixaba, should be placed in Ehretiaceae. Given the surprising nature of these results, further studies are recommended before formal reclassification of these two taxa is made.

Repeatome Analyses and Satellite DNA Chromosome Patterns in Deschampsia sukatschewii, D. cespitosa, and D. antarctica (Poaceae)

Abstract: Subpolar and polar ecotypes of Deschampsia sukatschewii (Popl.) Roshev, D. cespitosa (L.) P. Beauv, and D. antarctica E. Desv. are well adapted to stressful environmental conditions, which make them useful model plants for genetic research and breeding. For the first time, the comparative repeatome analyses of subpolar and polar D. sukatschewii, D. cespitosa, and D. antarctica was performed using RepeatExplorer/TAREAN pipelines and FISH-based chromosomal mapping of the identified satellite DNA families (satDNAs). In the studied species, mobile genetic elements of class 1 made up the majority of their repetitive DNA; interspecific variations in the total amount of Ty3/Gypsy and Ty1/Copia retroelements, DNA transposons, ribosomal, and satellite DNA were revealed; 12–18 high confident and 7–9 low confident putative satDNAs were identified. According to BLAST, most D. sukatschewii satDNAs demonstrated sequence similarity with satDNAs of D. antarctica and D. cespitosa indicating their common origin. Chromosomal mapping of 45S rDNA, 5S rDNA, and satDNAs of D. sukatschewii allowed us to construct the species karyograms and detect new molecular chromosome markers important for Deschampsia species. Our findings confirmed that genomes of D. sukatschewii and D. cespitosa were more closely related compared to D. antarctica according to repeatome composition and patterns of satDNA chromosomal distribution.

First report of Pestalotiopsis biciliata causing dieback on Quercus coccifera and Pistacia lentiscus in Tunisia

Abstract: Abstract Species of Pestalotiopsis occur commonly as plant pathogens. Accordingly, the aim of this study was to identify the causal agent of a shrub disease in northeastern and northern Tunisian forests. Field surveys showed a progressive dieback of branches, twig blight and trunk cankers on Pistacia lentiscus and Quercus coccifera. Pestalotiopsis spp. were the main fungi consistently isolated from these shrub species. Morphological identification and phylogenetic analysis of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and partial sequence of the translation elongation factor 1-alpha gene (tef1-α) identified the isolated fungi as Pestalotiopsis biciliata. This identification was confirmed by PCR analysis and Sanger sequencing, and demonstrated further using Koch’s postulates. To the best of our knowledge, this is the first report of P. biciliata associated with branch dieback and stem canker on Quercus coccifera and Pistacia lentiscus in Tunisia.

Morphology Characterization, Molecular Identification, and Pathogenicity of Fungal Pathogen Causing Kaffir Lime Leaf Blight in Northern Thailand

Abstract: Thailand is known to be the largest producer of kaffir lime leaf products in the global market. In 2021, leaf blight was found on kaffir lime plants (Citrus hystrix DC.) in Lamphun Province of northern Thailand. This disease has been associated with significant economic losses. However, there have been no prior reports of leaf blight on kaffir lime plants in Thailand or anywhere else in the world. In this study, causal fungi were isolated from lesions of kaffir lime plants and a total of three fungal isolates were obtained. All causal fungi were identified as Lasiodiplodia chinensis based on morphological characteristics and the phylogenetic analysis of combined sequences of the internal transcribed spacer (ITS) of ribosomal DNA, the translation elongation factor 1-alpha (tef-1), β-tubulin (tub), and RNA polymerase II subunit (rbp2) genes. Pathogenicity tests were conducted and the results revealed that all isolated fungi caused symptoms of leaf blight on inoculated leaves. This outcome was similar to symptoms that naturally occur and have been observed in the field. This is the first report on kaffir lime leaf blight caused by L. chinensis. Our study will provide information of high value for the development of effective strategies for the monitoring and prevention of this disease.