Intergenic Spacer Region Research Articles

A real time loop-mediated isothermal amplification (RealAmp) assay for rapid detection of Pleurostoma richardsiae in declining olive plants

Pleurostoma richardsiae is associated with host trunk diseases, known to cause dieback, cankers and wilting of woody trees, and human infections. This fungus was isolated from wood tissues of declining olive trees and grapevines showing esca disease symptoms, in the Apulia region of Italy. Fungus detection has been based on morphological and molecular features, which are time-consuming to identify and require well-trained personnel. Improvement of Pl. richardsiae detection in olive was achieved through development of real time loop-mediated isothermal amplification targeting the intergenic spacer (IGS) region of the fungus. Specificity of the assay was confirmed using ten Pl. richardsiae strains and 36 other fungus strains of species usually isolated from declining olive trees. The achieved limit of detection was 7.5 × 10-2 ng μL-1 of Pl. richardsiae genomic DNA. A preliminary validation of RealAmp was also performed using material from infected olive plants artificially inoculated in a greenhouse.

Fusarium and allied genera from China: species diversity and distribution.

The genus Fusarium includes numerous important plant and human pathogens, as well as many industrially and commercially important species. During our investigation of fungal diversity in China, a total of 356 fusarioid isolates were obtained and identified from diverse diseased and healthy plants, or different environmental habitats, i.e., air, carbonatite, compost, faeces, soil and water, representing hitherto one of the most intensive sampling and identification efforts of fusarioid taxa in China. Combining morphology, multi-locus phylogeny and ecological preference, these isolates were identified as 72 species of Fusarium and allied genera, i.e., Bisifusarium (1), Fusarium (60), and Neocosmospora (11). A seven-locus dataset, comprising the 5.8S nuclear ribosomal RNA gene with the two flanking internal transcribed spacer (ITS) regions, the intergenic spacer region of the rDNA (IGS), partial translation elongation factor 1-alpha (tef1), partial calmodulin (cam), partial RNA polymerase largest subunit (rpb1), partial RNA polymerase second largest subunit (rpb2) gene regions, and partial β-tubulin (tub2), were sequenced and employed in phylogenetic analyses. A genus-level phylogenetic tree was constructed using combined tef1, rpb1, and rpb2 sequences, which confirmed the presence of four fusarioid genera among the isolates studied. Further phylogenetic analyses of two allied genera (Bisifusarium and Neocosmospora) and nine species complexes of Fusarium were separately conducted employing different multi-locus datasets, to determine relationships among closely related species. Twelve novel species were identified and described in this paper. The F. babinda species complex is herein renamed as the F. falsibabinda species complex, including descriptions of new species. Sixteen species were reported as new records from China. Citation: Wang MM, Crous PW, Sandoval-Denis M, et al. 2022. Fusarium and allied genera from China: species diversity and distribution. Persoonia 48: 1-53. https://doi.org/10.3767/persoonia.2022.48.01.

Characterization of the complete mitochondrial genome sequence of Coilia brachygnathus (Clupeiformes, Engraulidae) from Huai River and its phylogenetic position

The complete mitogenome sequence of Coilia brachygnathus (Kreyenberg & Pappenheim, 1908) from Wabu Lake in Huai River Basin was annotated and characterized in this study. This mitochondrial genome is a circular DNA molecule of 16.896 bp in size with 57.52% AT content, including 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an AT-rich region (control region) as other bony fishes. There are a total of 10 overlap locations and 15 intergenic spacer regions throughout the mitogenome of C. brachygnathus. All PCGs employed a standard ATG as a start codon, except cytochrome c oxidase 1 (cox1) with GTG. In addition, TAA or TAG was identified as the typical stop codon. A phylogenetic tree reconstructed with the maximum likelihood method depicted a clone relationship with eight species of genus Coilia and our previous study based on the amino acid sequences of 13 mitochondrial PCGs. The complete mitochondrial genome is a valuable resource in classifying and conserving C. brachygnathus.

Complete Chloroplast Genome of an Endangered Species Quercus litseoides, and Its Comparative, Evolutionary, and Phylogenetic Study with Other Quercus Section Cyclobalanopsis Species.

Quercus litseoides, an endangered montane cloud forest species, is endemic to southern China. To understand the genomic features, phylogenetic relationships, and molecular evolution of Q. litseoides, the complete chloroplast (cp) genome was analyzed and compared in Quercus section Cyclobalanopsis. The cp genome of Q. litseoides was 160,782 bp in length, with an overall guanine and cytosine (GC) content of 36.9%. It contained 131 genes, including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. A total of 165 simple sequence repeats (SSRs) and 48 long sequence repeats with A/T bias were identified in the Q. litseoides cp genome, which were mainly distributed in the large single copy region (LSC) and intergenic spacer regions. The Q. litseoides cp genome was similar in size, gene composition, and linearity of the structural region to those of Quercus species. The non-coding regions were more divergent than the coding regions, and the LSC region and small single copy region (SSC) were more divergent than the inverted repeat regions (IRs). Among the 13 divergent regions, 11 were in the LSC region, and only two were in the SSC region. Moreover, the coding sequence (CDS) of the six protein-coding genes (rps12, matK, atpF, rpoC2, rpoC1, and ndhK) were subjected to positive selection pressure when pairwise comparison of 16 species of Quercus section Cyclobalanopsis. A close relationship between Q. litseoides and Quercus edithiae was found in the phylogenetic analysis of cp genomes. Our study provided highly effective molecular markers for subsequent phylogenetic analysis, species identification, and biogeographic analysis of Quercus.

Plastid Phylogenomics and Plastome Evolution of Nandinoideae (Berberidaceae).

Subfamily Nandinoideae Heintze (Berberidaceae), comprising four genera and ca. 19 species, is disjunctively distributed in eastern North America vs. Eurasia (eastern Asia, Central Asia, Middle East, and southeastern Europe), and represents an ideal taxon to explore plastid phylogenomics and plastome evolution in Berberidaceae. Many species of this subfamily have been listed as national or international rare and endangered plants. In this study, we sequenced and assembled 20 complete plastomes, representing three genera and 13 species of Nandinoideae. Together with six plastomes from GenBank, a total of 26 plastomes, representing all four genera and 16 species of Nandinoideae, were used for comparative genomic and phylogenomic analyses. These plastomes showed significant differences in overall size (156,626–161,406 bp), which is mainly due to the expansion in inverted repeat (IR) regions and/or insertion/deletion (indel) events in intergenic spacer (IGS) regions. A 75-bp deletion in the ndhF gene occurred in Leontice and Gymnospermium when compared with Nandina and Caulophyllum. We found a severe truncation at the 5’ end of ycf1 in three G. altaicum plastomes, and a premature termination of ropC1 in G. microrrhynchum. Our phylogenomic results support the topology of {Nandina, [Caulophyllum, (Leontice, Gymnospermium)]}. Within the core genus Gymnospermium, we identified G. microrrhynchum from northeastern Asia (Clade A) as the earliest diverging species, followed by G. kiangnanense from eastern China (Clade B), while the rest species clustered into the two sister clades (C and D). Clade C included three species from West Tianshan (G. albertii, G. darwasicum, G. vitellinum). Clade D consisted of G. altaicum from northern Central Asia, plus one species from the Caucasus Mountains (G. smirnovii) and three from southeastern Europe (G. odessanum, G. peloponnesiacum, G. scipetarum). Overall, we identified 21 highly variable plastome regions, including two coding genes (rpl22, ycf1) and 19 intergenic spacer (IGS) regions, all with nucleotide diversity (Pi) values > 0.02. These molecular markers should serve as powerful tools (including DNA barcodes) for future phylogenetic, phylogeographic and conservation genetic studies.

Fusarium and allied genera from China: species diversity and distribution

The genus Fusarium includes numerous important plant and human pathogens, as well as many industrially and commercially important species. During our investigation of fungal diversity in China, a total of 356 fusarioid isolates were obtained and identified from diverse diseased and healthy plants, or different environmental habitats, i.e., air, carbonatite, compost, faeces, soil and water, representing hitherto one of the most intensive sampling and identification efforts of fusarioid taxa in China. Combining morphology, multi-locus phylogeny and ecological preference, these isolates were identified as 72 species of Fusarium and allied genera, i.e., Bisifusarium (1), Fusarium (60), and Neocosmospora (11). A seven-locus dataset, comprising the 5.8S nuclear ribosomal RNA gene with the two flanking internal transcribed spacer (ITS) regions, the intergenic spacer region of the rDNA (IGS), partial translation elongation factor 1-alpha (tef1 ), partial calmodulin (cam), partial RNA polymerase largest subunit (rpb1 ), partial RNA polymerase second largest subunit (rpb2 ) gene regions, and partial β-tubulin (tub2 ), were sequenced and employed in phylogenetic analyses. A genus-level phylogenetic tree was constructed using combined tef1 , rpb1 , and rpb2 sequences, which confirmed the presence of four fusarioid genera among the isolates studied. Further phylogenetic analyses of two allied genera (Bisifusarium and Neocosmospora ) and nine species complexes of Fusarium were separately conducted employing different multi-locus datasets, to determine relationships among closely related species. Twelve novel species were identified and described in this paper. The F. babinda species complex is herein renamed as the F. falsibabinda species complex, including descriptions of new species. Sixteen species were reported as new records from China.

First Report of Dickeya fangzhongdai Causing Peduncle Soft Rot of Banana in China.

Banana (Musa spp.) is a popular fruit all over the world, and it's also an important cash crop with a planting area of 358,924 ha in southern China. In July 2020, a peduncle soft rot disease occurred on dwarf banana (Musa sp. cv. Guangfen) in Guigang city (N22°50'29″, E109° 43'34″), Guangxi province, China. More than 20% plants were infected in the banana plantation. The first external sign of the disease appeared on the incisional wound after the flower bud was cut off from the peduncle. The symptom initially appeared as a black lesion on the wound, then extended into the internal tissue of the whole peduncle. In the later stages, the internal tissue became soft and rot, occasionally formed a necrotic cavity, and eventually led to the black rot of the whole peduncle with a foul smell. To isolate the pathogen, the internal lesion tissues of 5 mm × 5 mm were collected between the border of symptomatic and healthy tissue, treated with 75% ethanol for 10 s, and 0.1% HgCl2 for 3 min, then rinsed with sterile water for three times. Sterilized tissue fragments were cut to pieces with sterilized surgical shears and soaked in 5 mL sterile water, then shaken for 10 min in a vortex oscillator. The suspension was diluted 1000 times with sterilized water，then plated on nutrient-agar medium and incubated at 28℃ in darkness for 24 h. Among the 32 isolates, 23 pure bacterial cultures with similar morphology were predominantly obtained from the samples. These bacteria were gram-negative, and their colonies were initially yellowish white with irregular edges and smooth surfaces, then turned to grayish blue after 72 h incubated at 28℃. The representative isolates GZF2-2 and GZF1-8 were selected for further identification. Genomic DNA was isolated from the bacteria and the 16S rDNA was amplified with primers 27F/1492R (Weisburg et al. 1991) and sequenced. The obtained sequences (GenBank Accession No. MZ768922 and OK668082) showed >99% identities to several records of Dickeya fangzhongdai deposited in NCBI GenBank (1400/1404 bps for GZF2-2 to KT992690, 1409/1417 bps for GZF1-8 to MT613398) based on BLAST analysis. In addition, the recA, fusA, gapA, purA, rplB, dnaX genes and the 16S-23S intergenic spacer (IGS) regions of the two isolates were also amplified and sequenced (GenBank Accession Nos. OK634381-OK634382, OK634369- OK634370, OK634373-OK634374, OK634377-OK634378, OK634385-OK634386, OK634365- OK634366 and OK631722-OK631723) as described by Tian et al. (2016). All the DNA sequences matched that of D. fangzhongdai strains JS5T (percent identities>99.06%), PA1 and ECM-1 in GenBank. Neighbor-joining phylogenetic analysis by software MegaX (Kumar et al. 2018) based on the 16S rDNA sequences revealed that the two isolates were in the same clade with reported D. fangzhongdai strains. Multilocus sequence analysis of the other seven regions also showed the two representative isolates were belong to D. fangzhongdai. Therefore, the isolates were identified as D. fangzhongdai. Pathogenicity of isolate GZF2-2 was investigated to demonstrate Koch's postulate. The end of the banana peduncles of 6 healthy plants were cut off, and 10 mL bacterial suspension (108 CFU/mL) was inoculated to the fresh wound on the plants using sterile brushes. Six control plants were inoculated with sterilized water. All the inoculated peduncles were covered with plastic bags to maintain high humidity. After 28 days, all the peduncles inoculated with strain GZF2-2 showed soft rot symptoms similar to those observed in the field, while the controls remained symptomless. The same bacteria were re-isolated from the symptomatic peduncles and confirmed by sequencing the 16S rDNA. D. fangzhongdai has been reported to cause soft rot on onion (Ma et al. 2020) and bleeding cankers on pear trees (Chen et al. 2020). To the best of our knowledge, this is the first report of D. fangzhongdai causing peduncle soft rot on banana in China.

Development of a DNA barcode library of plants in the Thai Herbal Pharmacopoeia and Monographs for authentication of herbal products

Traditional herbal medicine has long been practiced as a method of health care in many countries worldwide. The usage of herbal products has been increasing and is expected to continue to do so in the future. However, admixture and adulteration are concerns regarding the quality of herbal medicine, including its safety and efficacy. We aimed to develop a reference DNA barcode library of plants listed in the Thai Herbal Pharmacopoeia (THP) and Monographs of Selected Thai Materia Medica (TMM) (n = 101 plant species) using four core barcode regions, namely, the ITS2, matK, rbcL and trnH-psbA intergenic spacer regions, for authentication of the plant origin of raw materials and herbal products. Checking sequences from samples obtained from local markets and the Thai Food and Drug Administration (Thai FDA) against our digital reference DNA barcode system revealed the authenticity of eighteen out of twenty tested samples as claimed on their labels. Two samples, no. 3 and 13, were not Cyanthillium cinereum (L.) H.Rob. and Pueraria candollei Wall. ex Benth. as claimed, respectively. They were recognized as Emilia sonchifolia (L.) DC. and Butea superba (Roxb.), respectively. Hence, it is important for the Thai FDA or regulatory agencies to immediately initiate strict enforcement for the development of pharmacopoeial standards as well as revisions or modifications of available regulatory guidelines and to implement close monitoring for the quality control of herbal products in terms of authentication before they enter the herbal market. The centralized digital reference DNA barcode database developed here could play a very important role in monitoring or checking the authenticity of medicinal plants.

Combining DNA and HPTLC profiles to differentiate a pain relief herb, Mallotus repandus, from plants sharing the same common name, "Kho-Khlan".

The pain relief formula "Ya Pa Som Kho-Khlan (YPSKK)" or "ยาผสมโคคลาน" in Thai is officially recorded in the Natural List of Essential Medicines (NLEM) of Thailand. The main component is Mallotus repandus (Willd.) Müll. Arg.; however, Anamirta cocculus (L.) Wight & Arn and Croton caudatus Gleiseler share the same common name: "Kho-Khlan". Confused usage of A. cocculus or C. caudatus can have effects via toxicity or unsuccessful treatment. This study aimed to combine a high-performance thin-layer chromatography (HPTLC) technique and DNA barcoding coupled with high-resolution melting (Bar-HRM) to differentiate M. repandus from the other two species. The M. repandus extract exhibited a distinct HPTLC profile that could be used to differentiate it from the others. DNA barcodes of the rbcL, matK, ITS and psbA-trnH intergenic spacer regions of all the plants were established to assist HPTLC analysis. The rbcL region was selected for Bar-HRM analysis. PCR amplification was performed to obtain 102 bp amplicons encompassing nine polymorphic nucleotides. The amplicons were subjected to HRM analysis to obtain melting curve profiles. The melting temperatures (Tm) of authentic A. cocculus (A), C. caudatus (C) and M. repandus (M) were separated at 82.03±0.09°C, 80.93±0.04°C and 80.05±0.07°C, respectively. The protocol was applied to test crude drugs (CD1-6). The HPTLC profiles of CD2-6 showed distinct bands of M. repandus, while CD1 showed unclear band results. The Bar-HRM method was applied to assist the HPTLC and indicated that CD1 was C. caudatus. While ambiguous melting curves from the laboratory-made formulae were obtained, HPTLC analysis helped reveal distinct patterns for the identification of the plant species. The combination of HPTLC and Bar-HRM analysis could be a tool for confirming the identities of plant species sharing the same name, especially for those whose sources are multiple and difficult to identify by either chemical or DNA techniques.

Comparative analysis of two Korean irises (Iris ruthenica and I. uniflora, Iridaceae) based on plastome sequencing and micromorphology.

Iris ruthenica Ker Gawl. and I. uniflora Pall. ex Link, which are rare and endangered species in Korea, possess considerable horticultural and medicinal value among Korean irises. However, discrimination of the species is hindered by extensive morphological similarity. Thus, the aim of the present study was to identify discriminating features by comparing the species’ complete plastid genome (i.e., plastome) sequences and micromorphological features, including leaf margins, stomatal complex distribution (hypostomatic vs. amphistomatic leaves), anther stomata density, and tepal epidermal cell patterns. Plastome comparison revealed slightly divergent regions within intergenic spacer regions, and the most variable sequences, which were distributed in non-coding regions, could be used as molecular markers for the discrimination of I. ruthenica and I. uniflora. Phylogenetic analysis of the Iris species revealed that I. ruthenica and I. uniflora formed a well-supported clade. The comparison of plastomes and micromorphological features performed in this study provides useful information for elucidating taxonomic, phylogenetic, and evolutionary relationships in Iridaceae. Further studies, including those based on molecular cytogenetic approaches using species specific markers, will offer insights into species delimitation of the two closely related Iris species.

Genetic structure of Sclerotinia sclerotiorum populations from sunflower and cabbage in West Azarbaijan province of Iran

Sclerotinia sclerotiorum is one of the most destructive fungal pathogens infecting a wide array of plant species worldwide. Management of this pathogen relies on the coordinated use of fungicides and resistant host cultivars with other control measures, but the effectiveness of these methods requires knowledge of the genetic variability and structure of the fungal populations. To provide insight into the genetic diversity and structure of this pathogen in West Azarbaijan province of Iran, a total of 136 isolates were collected from symptomatic sunflower and cabbage plants within fields in three regions and analysed using inter-simple sequence repeat (ISSR) markers and intergenic spacer (IGS) region of the rRNA gene sequences. A total of 83 ISSR multilocus genotypes (MLGs) were identified, some of which were shared among at least two regional or host populations but in a low frequency. High genotypic diversity, low levels of clonal fraction, and random association of ISSR loci in a region indicated a low level of clonal reproduction, and possibly a high level of sexually recombining life cycle for the pathogen in the province. Marker analyses revealed that the pathogen was spatially homogeneous among fields, and thus similar control measures, such as the choice of resistant cultivars and fungicides, may effectively manage S. sclerotiorum within the region. Four IGS haplotypes (IGS1–IGS4) were detected within populations with IGS3 being the most prevalent haplotype. The low IGS haplotype diversity, the absence of spatial structure, and shared MLGs among populations may suggest a single introduction and subsequent dispersal of S. sclerotiorum within West Azarbaijan province.

Investigating Influenza Virus Polymerase Activity in Feline Cells Based on the Influenza Virus Minigenome Replication System Driven by the Feline RNA Polymerase I Promoter.

Emerging influenza virus poses a health threat to humans and animals. Domestic cats have recently been identified as a potential source of zoonotic influenza virus. The influenza virus minigenome replication system based on the ribonucleic acid (RNA) polymerase I (PolI) promoter is the most widely used tool for investigating polymerase activity. It could help determine host factors or viral proteins influencing influenza virus polymerase activity in vitro. However, influenza virus polymerase activity has never been studied in feline cells thus far. In the present study, the feline RNA PolI promoter was identified in the intergenic spacer regions between adjacent upstream 28S and downstream 18S rRNA genes in the cat (Felis catus) genome using bioinformatics strategies. The transcription initiation site of the feline RNA PolI promoter was predicted. The feline RNA PolI promoter was cloned from CRFK cells, and a promoter size of 250 bp contained a sequence with sufficient PolI promoter activity by a dual-luciferase reporter assay. The influenza virus minigenome replication system based on the feline RNA PolI promoter was then established. Using this system, the feline RNA PolI promoter was determined to have significantly higher transcriptional activity than the human and chicken RNA PolI promoters in feline cells, and equine (H3N8) influenza virus presented higher polymerase activity than human (H1N1) and canine (H3N2) influenza viruses. In addition, feline myxovirus resistance protein 1 (Mx1) and baloxavir were observed to inhibit influenza virus polymerase activity in vitro in a dose-dependent manner. Our study will help further investigations on the molecular mechanism of host adaptation and cross-species transmission of influenza virus in cats.

Topographic Complexity Facilitates Persistence Compared to Signals of Contraction and Expansion in the Adjacent Subdued Landscape

Topographically heterogeneous areas are likely to act as refugia for species because they facilitate survival during regional climatic stress due to availability of a range of microenvironments. The Stirling Ranges are a topographically complex area in the generally subdued and ancient landscape of south-western Australia. We investigated the influence of these landscape features on the evolutionary history of the rare woody shrub, Banksia brownii through a combined approach using phylogeographic analysis of sequence data from three chloroplast sequences, the trnV–ndhC, trnQ–rps16, and rpl32–ndhF intergenic spacer regions, and species distribution modeling. The Stirling Ranges showed high genetic diversity and differentiation among populations consistent with localized persistence and maintenance of large populations in an area that species distribution modeling identified as providing habitat stability at the Last Glacial Maximum as well as under warmer conditions. In contrast, populations in the adjacent subdued lowlands showed signals of low diversity, suggesting contraction, and subsequent expansion from localized refugia in the west. Cool summers are an important climatic variable for the species and species distribution modeling showed suitable habitat identified at the LGM suggesting expansion at this time following likely contraction during earlier warmer climatic oscillations. The isolated, coastal population at Vancouver Peninsula showed low diversity but no differentiation and it may have been established in more recent historical times, possibly through Aboriginal movement of seed. Our analysis of B. brownii highlights the complex evolutionary history of the species and the influence of topographic complexity and habitat heterogeneity in this global biodiversity hotspot.

Short sequence repeats of the intergenic spacer regions of ribosomal RNA genes in Malassezia globosa and Malassezia restricta colonizing the scalps of male individuals with and without androgenetic alopecia.

We analyzed the short sequence repeats (SSRs) of the intergenic spacer (IGS) region 1 of the ribosomal RNA genes in Malassezia globosa and Malassezia restricta, which predominantly colonize the scalp in androgenetic alopecia (AGA). No AGA-specific SSRs were found in the M. globosa IGS region, whereas a (CT)6 :(AT)8 SSR was predominantly detected in the M. restricta IGS region in the AGA group. Malassezia colonization was higher in the scalps of patients with M. restricta (CT)6 :(AT)8 SSRs than in the scalps of patients without M. restricta (CT)6 :(AT)8 SSRs. These observations suggest that this specific SSR type in M. restricta is involved in the development or exacerbation of AGA.

LABORATORY CONCORDANCE STUDY FOR THE MOLECULAR DETECTION OF MYCOPLASMA OVIPNEUMONIAE.

As part of a respiratory pathogen survey of Alaska wildlife, we conducted a concordance study to assess Mycoplasma ovipneumoniae detection among three different PCR assays using a total of 346 nasal swabs sampled from four species (Dall's sheep, Ovis dalli dalli; mountain goats, Oreamnos americanus; caribou, Rangifer tarandus granti; and moose, Alces alces gigas), and two taxonomic subfamilies (Bovidae subfamily Caprinae and Cervidae subfamily Capreolinae). A federal research laboratory performed two PCR assays (LM40 and intergenic spacer region [IGS]), and a state diagnostic laboratory performed the third (universal Mycoplasma [UM]). Overall concordance was good, ranging from 93% to 99%, which was probably a result of low detection rate of M. ovipneumoniae. Due to differences in positive agreement, the quality of concordance between LM40 and both IGS and UM was considered fair. However, the quality of concordance between IGS and UM was excellent. All three PCR methods detected M. ovipneumoniae in a non-Caprinae species (caribou), and the LM40-PCR assay also detected M. ovipneumoniae in additional Caprinae species. The LM40-PCR assay detected M. ovipneumoniae in a larger number of samples than did the other two assays (IGS, UM). Because of potential differences in detection rates, it is critical to consider test parameters when evaluating a host population for the presence of M. ovipneumoniae.

First Report of Colletotrichum chrysophilum Causing Apple Bitter Rot in Spain.

Bitter rot of apple (Malus × domestica Borkh.) is a cosmopolitan disease affecting fruit and causes considerable losses worldwide. In September 2020, symptoms of bitter rot were observed on 'Pink Lady' apples in two orchards (~2.5 ha each) in Gualta, Catalonia, Spain (42.03803 N, 3.09831 E, and 42.03942 N, 3.10931 E). Early symptoms consisted of light brown and sunken circular lesions (1-4 mm) that enlarged over time, later becoming dark brown and water soaked, and extending cone-shaped toward the core. Sporulation was mostly noticed in larger lesions. Estimated incidence was 2% and 20% of 150 trees surveyed in each orchard, respectively. Twenty-one fungal isolates were obtained from diseased fruit by culturing small pieces of necrotic tissue on potato dextrose agar (PDA) amended with rifampicin at 50 mg/liter. Colonies on PDA looked identical. They were cottony, initially light-gray colored on top and darkening with age; colony reverse initially cream colored and darkening with age. Conidia were produced in orange acervular masses on Spezieller Nährstoffarmer Agar, and were aseptate, hyaline, cylindrical with obtuse ends, and measuring 10.1 to 14.7 × 4.5 to 7.1 μm (average 13.1 ± 1.04 × 5.3 ± 0.67 μm [mean ± SD], n = 50), with a mean length/width ratio 2.6 ± 0.39 (n = 16 isolates). Perithecia were not observed. Based on the conidial morphology, the isolates were tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). Total genomic DNA was extracted from all isolates and six nuclear regions were amplified and partially sequenced: the internal transcribed spacer region of rDNA (ITS), the mating type protein 1-2-1 gene and the Mat1-2-1-Apn2 intergenic spacer region (ApMAT), actin (ACT), calmodulin (CAL), glyceraldehyde 3-P dehydrogenase (GAPDH), and tubulin (TUB2). The sequences for each region were 100% identical across all isolates. BLAST searches in GenBank showed 99-100% identity with sequences of various C. chrysophilum W.A.S. Vieira, W.G. Lima, M.P.S. Câmara & V.P. Doyle strains including the ex-type CMM4268 (Vieira et al. 2017). Sequences of the representative isolate CJL1080 were deposited in GenBank (ACT, MZ488944; ApMAT, MZ442299; CAL, MZ488945; GAPDH, MZ488946; ITS, MZ443972; TUB2, MZ442300). A multilocus phylogenetic analysis through Bayesian inference conducted with the obtained sequences and reference ones (Khodadadi et al. 2020) revealed that our isolates clustered well within C. chrysophilum, as suggested by BLAST results. To confirm Koch's postulates, isolates CJL1080 and CJL1095 were inoculated on 'Pink Lady' apples. Six surface-sterilized fruits per isolate were wound-inoculated four times each with either 20 μl of a conidial suspension (105 conidia/ml) or sterile distilled water (control). After 7 days of incubation in a moist chamber at 22°C, symptoms compatible with Colletotrichum infection were observed around the wounds, whereas control inoculations remained symptomless. The fungus was reisolated from all the lesions and identified through its morphological traits and DNA sequencing (ApMat, CAL, and GAPDH). No fungus was isolated from the controls. Taxa of the C. gloeosporioides species complex causing bitter rot have been recently reported in Europe (Grammen et al. 2019; Nodet et al. 2019). This is the first report of C. chrysophilum causing apple bitter rot in Spain, which expands the knowledge on the geographic distribution of this important pathogen of apple in Europe.

5S Ribosomal DNA of Genus Solanum: Molecular Organization, Evolution, and Taxonomy.

The Solanum genus, being one of the largest among high plants, is distributed worldwide and comprises about 1,200 species. The genus includes numerous agronomically important species such as Solanum tuberosum (potato), Solanum lycopersicum (tomato), and Solanum melongena (eggplant) as well as medical and ornamental plants. The huge Solanum genus is a convenient model for research in the field of molecular evolution and structural and functional genomics. Clear knowledge of evolutionary relationships in the Solanum genus is required to increase the effectiveness of breeding programs, but the phylogeny of the genus is still not fully understood. The rapidly evolving intergenic spacer region (IGS) of 5S rDNA has been successfully used for inferring interspecific relationships in several groups of angiosperms. Here, combining cloning and sequencing with bioinformatic analysis of genomic data available in the SRA database, we evaluate the molecular organization and diversity of IGS for 184 accessions, representing 137 species of the Solanum genus. It was found that the main mechanisms of IGS molecular evolution was step-wise accumulation of single base substitution or short indels, and that long indels and multiple base substitutions, which arose repeatedly during evolution, were mostly not conserved and eliminated. The reason for this negative selection seems to be association between indels/multiple base substitutions and pseudogenization of 5S rDNA. Comparison of IGS sequences allowed us to reconstruct the phylogeny of the Solanum genus. The obtained dendrograms are mainly congruent with published data: same major and minor clades were found. However, relationships between these clades and position of some species (S. cochoae, S. clivorum, S. macrocarpon, and S. spirale) were different from those of previous results and require further clarification. Our results show that 5S IGS represents a convenient molecular marker for phylogenetic studies on the Solanum genus. In particular, the simultaneous presence of several structural variants of rDNA in the genome enables the detection of reticular evolution, especially in the largest and economically most important sect. Petota. The origin of several polyploid species should be reconsidered.

Moleculer study of acenitobacter bumannii associated with corona virus disease

In this current study, 120 samples were taken from Corona virus patients of different ages and of both sexes (males and females), who are in isolation units for Corona virus disease in hospitals in Muthanna Governorate during the period from (September to April) and were cultivated in the Chrom agar Base Acinetobacter. After determine the phenotypic and external characteristics, and conduct chemical tests, Then it was diagnosed by 16S-23S ribosomal DNA intergenic spacer region after conducting chemical and molecular tests , 24 out of 120 isolates were confirmed as Acenitobacter bumannii , Where 10 positive samples were selected for testing 16S-23S ribosomal DNA intergenic spacer region It was diagnosed as Acenitobacter bumannii. The gene was diagnosed PonA gene responsible for the enzyme penicillin-binding protein 1a, The gene responsible for the enzyme Beta-Lactamases PER-1 is the BlaPER1 gene, As the most important virulence factors in Acenitobacter bumannii , Because the two enzymes played an important role in the pathogenesis of these bacteria . And the result was (100% of the PonA gene and 40% of the blaPER1 gene).

Genetic differentiation of two morphotypes of Piper regnellii (Miq.) C. DC. var. regnellii

Piper regnellii (Miq.) C. DC. var. regnellii, popularly known as "pariparoba", is used for therapeutic purposes. However, the morphological similarity among Piper species can lead to misidentifications, so molecular markers have been used to validate the identification of species and their morphotypes. Among the molecular markers used in plants, the intergenic spacer region trnL-trnF has proven effective in identifying plant species. For this reason, this region was used to evaluate two morphotypes of Piper regnellii var. regnellii. Studies with the trnL-trnF region have shown this region as a good marker for establishing phylogenetic relationships, distinguishing species, and identifying new species. We concluded that the trnL-trnF sequenced region show one indel of difference between the two morphotypes. It would be interesting to analyze these two morphotypes with a more variable region than the one used here, aiming to show intraspecific differences.

Comparative analysis of complete Ilex (Aquifoliaceae) chloroplast genomes: insights into evolutionary dynamics and phylogenetic relationships

BackgroundIlex (Aquifoliaceae) are of great horticultural importance throughout the world for their foliage and decorative berries, yet a dearth of genetic information has hampered our understanding of phylogenetic relationships and evolutionary history. Here, we compare chloroplast genomes from across Ilex and estimate phylogenetic relationships.ResultsWe sequenced the chloroplast genomes of seven Ilex species and compared them with 34 previously published Ilex plastomes. The length of the seven newly sequenced Ilex chloroplast genomes ranged from 157,182 bp to 158,009 bp, and contained a total of 118 genes, including 83 protein-coding, 31 rRNA, and four tRNA genes. GC content ranged from 37.6 to 37.69%. Comparative analysis showed shared genomic structures and gene rearrangements. Expansion and contraction of the inverted repeat regions at the LSC/IRa and IRa/SSC junctions were observed in 22 and 26 taxa, respectively; in contrast, the IRb boundary was largely invariant. A total of 2146 simple sequence repeats and 2843 large repeats were detected in the 41 Ilex plastomes. Additionally, six genes (psaC, rbcL, trnQ, trnR, trnT, and ycf1) and two intergenic spacer regions (ndhC-trnV and petN-psbM) were identified as hypervariable, and thus potentially useful for future phylogenetic studies and DNA barcoding. We recovered consistent phylogenetic relationships regardless of inference methodology or choice of loci. We recovered five distinct, major clades, which were inconsistent with traditional taxonomic systems.ConclusionOur findings challenge traditional circumscriptions of the genus Ilex and provide new insights into the evolutionary history of this important clade. Furthermore, we detail hypervariable and repetitive regions that will be useful for future phylogenetic and population genetic studies.