Internal Transcribed Spacer rDNA Research Articles

DNA Barcoding Mushroom Spawn Using EF-1α Barcodes: A Case Study in Oyster Mushrooms (Pleurotus).

Oyster mushrooms (genus Pleurotus) are widespread and comprise the most commonly cultivated edible mushrooms in the world. Species identification of oyster mushroom spawn based on cultural, morphological, and cultivated characteristics is time consuming and can be extraordinarily difficult, which has impeded mushroom breeding and caused economic loss for mushroom growers. To explore a precise and concise approach for species identification, the nuclear ribosomal internal transcribed spacer (ITS), 28S rDNA, and the widely used protein-coding marker translation elongation factor 1α (EF-1α) gene were evaluated as candidate DNA barcode markers to investigate their feasibility in identifying 13 oyster mushroom species. A total of 160 sequences of the candidate loci were analyzed. Intra- and interspecific divergences and the ease of nucleotide sequence acquisition were the criteria used to evaluate the candidate genes. EF-1α showed the best intra- and interspecific variation among the candidate markers and discriminated 84.6% of the species tested, only being unable to distinguish two closely related species Pleurotus citrinopileatus and Pleurotus cornucopiae. Furthermore, EF-1α was more likely to be acquired than ITS or 28S rDNA, with an 84% success rate of PCR amplification and sequencing. For ITS and 28S rDNA, the intraspecific differences of several species were distinctly larger than the interspecific differences, and the species identification efficiency of the two candidate markers was worse (61.5 and 46.2%, respectively). In addition, these markers had some sequencing problems, with 55 and 76% success rates of sequencing, respectively. Hence, we propose EF-1α as a possible DNA barcode marker for oyster mushroom spawn.

Molecular Identification of Isolates of Ustilago Maydis (Dc.) Corda the Causal Common Smut in Maize

Aim of study was to isolate and identify U. maydis races that cause common smut disease using molecular methods. In addition to detect phylogenetic relation among isolates of local races and global races using Polymerase Chain Reaction (PCR) through phylogenetic tree using Internal Transcribed Spacer(ITS). DNA was extracted from 50 isolates of U.maydis and detected 34 bands from the isolates using gel electrophoresis including (8 Baghdad, 5 Babylon, 14 Waset and 7 Erbil ) .Results revealed that detection of isolates was on ITS region and DNA diagnostic was detected at 650 base pair( bp) of all the isolates using ITS1 and ITS4 primers. Also the study included determination of sequencing the nitrogen bases for the isolates. Results indicated that Baghdad isolates of (Um 21,Um 22, Um23, Um24, Um25, Um26, Um27, Um28) have relative relation with global race of FBI.. While Babylon isolates ( Um 5, Um 6, Um7, Um8, Um 10) were had close relation with race 521. And isolates of Waset showed a variation in relationship of a number of global races, Isolate Um41 was closely related to 521, and isolates Um32, Um47, Um48 were closely to the race XA0609, and isolates Um30, Um33, Um36, Um37, Um38, Um43, Um44, Um46, Um49 they are relevant to the races CPO10.020a. However, isolates of Erbil ( Um11, Um 12, Um 14, Um15, Um 16, Um 17, Um19) were in relation with race FB1. Results of genetic origin demonstrated that of Baghdad,Babylon,Waset and Erbil have close relation with global FB1, 521, CPO10.020a, XA0609 which has a1b1 genotype).

Alternaria alternata causing Alternaria Leaf Spot of Cucumis melo (Muskmelon) in Pakistan.

In July 2019, leaf spot symptoms were observed on muskmelon (Cucumis melo L.) cv. Jackball-1 plants in an experimental field of 2.02 ha with a disease incidence of 30% (31°26'05.4"N 73°04'30.3"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms consisted of small, circular, brown, necrotic spots 1 to 2 mm in size covering 10 to 30% of the leaf blade, which gradually enlarged and developed concentric rings. To identify the causal agent of the disease, a total of 20 symptomatic leaves were collected. Small pieces removed from the margin between healthy and diseased tissues were surface disinfected in 70% ethanol for 2 min, rinsed three times with sterile distilled water, plated on Potato dextrose agar and incubated at 25 ± 2°C with a 12-h photoperiod. Morphological observations were made on 7-day-old single-spore cultures. The colonies initially appeared white and then turned olive-green. All 20 fungal isolates were characterized by small, short-beaked, multicellular conidia. The conidia were ellipsoidal or ovoid and measured 11.5 to 30 μm × 7.5 to 15 μm (n = 50) with longitudinal and transverse septa. Conidia were produced on short conidiophores in chains. The beaks were short (often less than one-third the body length) and conical or cylindrical. These morphological features concur with the description of Alternaria alternata (Fr.) Keissler (Woudenberg et al. 2013). For molecular identification, genomic DNA of four representative isolates (HMSMZA 07, 08, 09, 10) were extracted and PCR amplification of the internal transcribed spacer (ITS)-rDNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and translation elongation factor-1 alpha (TEF-1α) gene regions were performed (White et al. 1990, Berbee et al. 1999, Carbone & Kohn, 1999) respectively. The obtained sequences were deposited in GenBank with accession numbers MT253643.1-MT253646.1 (ITS-rDNA), MT318260.1-MT318263.1 (GAPDH), and MT318280.1-MT318283.1 (TEF-1α). BLASTn analysis of HMSMZA 07 sequences showed 100% identity with ITS rDNA (MN615420.1), GAPDH (MK637438.1) and TEF-1α (MN807795.1) sequences of A. alternata. To confirm pathogenicity, 5-6 weeks-old Muskmelon (Cucumis melo L.) cv. Jackball-1 plants (true leaf stage) were sprayed until runoff (1.5 to 2 ml per plant) with A. alternata conidial suspension (106 conidia/ml; obtained from 1 week-old cultures) amended with 0.1% (vol/vol) of Tween 20 using an atomizer in the green house. The experiment included four A. alternata isolates inoculated onto three muskmelon plants per each isolate, whereas control plants (n = 3) were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse and the experiment was conducted twice. After 5 to 7 days post inoculation, necrotic leaf spots were observed on the inoculated plants and A. alternata was reisolated and confirmed by morphological and molecular (ITS) features. No disease was observed on control plants. Previously, A. alternata on muskmelon has been reported in Pakistan (Ahmad et al. 1997), however this study provides a detailed description of disease symptoms, morphological and molecular identity of the causal agent including completion of Koch's postulates. The disease could represent a threat for muskmelon crop in Pakistan due to its increasing cultivation and therefore warrants the need to develop disease management strategies.

Mucor rongii sp. nov., a New Cold-Tolerant Species from China.

Two strains of a new mucoralean fungus (M1 and R1) were harvested from the moist walls of Sufu workshop in Liaocheng city, Shandong province, China. Internal transcribed spacer (ITS) and the large subunit (LSU) rDNA region D1/D2 domain-based phylogenetic analysis, along with phenotypic characteristics, revealed that the strains belonged to a new genus in the Mucor category. This new category belongs to Mucor flavus complex. However, unlike M. flavus CBS 234.25 (the type species of the complex), the novel species could grow well at 25°C and not grow at 28°C. Moreover, the ITS rDNA sequence similarity of the two species was only 96%. Here, we present the new category Mucor rongii sp. nov. Its holotype is HMAS 248,091, the ex-type culture is M1T (= CICC 41725T), and the other culture examined was R1 (= CICC 41,726).

Bipartite molecular approach for species delimitation and resolving cryptic speciation of Exobasidium vexans within the Exobasidium genus

Exobasidium vexans, a basidiomycete pathogen, is the causal organism of blister blight disease in tea. The molecular identification of the pathogen remains a challenge due to the limited availability of genomic data in sequence repositories and cryptic speciation within its genus Exobasidium. In this study, the nuclear internal transcribed spacer rDNA region (ITS) based DNA barcode was developed for E. vexans, to address the problem of molecular identification within the background of cryptic speciation. The isolation of E. vexans strain was confirmed through morphological studies followed by molecular identification utilizing the developed ITS barcode. Phylogenetic analysis based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) confirmed the molecular identification of the pathogen as E. vexans strain. Further, BI analysis using BEAST mediated the estimation of the divergence time and evolutionary relationship of E. vexans within genus Exobasidium. The speciation process followed the Yule diversification model wherein the genus Exobasidium is approximated to have diverged in the Paleozoic era. The study thus sheds light on the molecular barcode-based species delimitation and evolutionary relationship of E. vexans within its genus Exobasidium.

On the biocontrol by Trichoderma afroharzianum against Fusarium culmorum responsible of fusarium head blight and crown rot of wheat in Algeria

BackgroundDurum wheat (Triticum durum Desf.) is one of the most important cereals in the world. Unfortunately, the wheat plant is a target of several species of the genus Fusarium. This genus causes two serious diseases: fusarium crown rot (FCR) and fusarium head blight (FHB). The search for new indigenous strains of Trichoderma with a high potential for biocontrol against these two diseases was the purpose of this study.ResultsBiocontrol potential of 15 isolates of Trichoderma (T1 to T15), isolated from different rhizosphere soils and Algerian ecosystems, was evaluated against 4 strains of Fusarium culmorum (FC11, FC2, FC4, and FC20); the main causative agent of FCR and FHB. The efficacy of biological control by Trichoderma spp., evaluated by in vitro tests (direct and indirect confrontation), was confirmed by in vivo bioassays. The in vitro results showed a significant inhibition of mycelial growth of F. culmorum species than the control. The highest percentages of inhibition were obtained by T9, T12, and T14 isolates causing a maximum inhibition percentage of 81.81, 77.27, and 80.68%, respectively. T14 was selected for biocontrol in in vivo testing. A tube and pot experiments for FCR against F. culmorum showed that T14 decreased the disease severity with 50 and 63.63% reduction, respectively. FHB infection was significantly reduced by T14 in all durum wheat cultivars tested, where %AUDPC (area under the disease progress curve) reduction was 49.77, 43.43, 48.25, and 74.60% for Simeto, Waha, Bousselem, and Setifis genotypes, respectively. Yields also increased significantly for almost all cultivars. The antagonistic T14 was characterized based on molecular tools, using translation elongation factor1-alpha (TEF1-α) and internal transcribed spacers rDNA (ITS1). The results identified T14 as T. afroharzianum with accession numbers attributed by NCBI GenBank as MW171248 and MW159753.ConclusionsTrichoderma afroharzianum, evaluated for the first time in Algeria as biocontrol agent, is a promising biocontrol approach against FCR and FHB.

Phylogeny and taxonomy of a novel species of Pseudocercospora from India

A new species of Pseudocercospora is described causing leaf spot disease on Crotalaria assamica from India. The identity of isolate was based on asexual morphs, cultural characteristics and phylogenetic analyses of partial nuclear ribosomal 28S large subunit and complete internal transcribed spacer rDNA sequence data with high statistical support value. Morphologically this species has characters similar to allied species in genus Pseudocercospora but differs in having catenate and branched conidia. Phylogenetic analysis using LSU and ITS reveals it a new species of >i>Pseudocercospora (Capnodiales, Mycosphaerellaceae).

Mastomys natalensis (Smith, 1834) as a natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 introgressive hybrids

Cercarial emission of schistosomes is a determinant in the transmission to the definitive host and constitutes a good marker to identify which definitive host is responsible for transmission, mainly in introgressive hybridization situations. Our goal was to test the hypothesis that micro-mammals play a role in Schistosoma haematobium, S. bovis, and/or S. haematobium x S. bovis transmission. Small mammal sampling was conducted in seven semi-lacustrine villages of southern Benin. Among the 62 animals trapped, 50 individuals were investigated for Schistosoma adults and eggs: 37 Rattus rattus, 3 Rattus norvegicus, 9 Mastomys natalensis, and 1 Crocidura olivieri. Schistosoma adults were found in four R. rattus and two M. natalensis, with a local prevalence reaching 80% and 50%, respectively. Two cercarial chronotypes were found from Bulinus globosus experimentally infected with miracidia extracted from naturally infected M. natalensis: a late diurnal and nocturnal chronotype, and an early diurnal, late diurnal, and nocturnal chronotype. The cytochrome C oxidase subunit I mtDNA gene of the collected schistosomes (adults, miracidia, and cercariae) belonged to the S. bovis clade. Eleven internal transcribed spacer rDNA profiles were found; four belonged to S. bovis and seven to S. haematobium x S. bovis. These molecular results together with the observed multi-peak chronotypes add M. natalensis as a new host implicated in S. haematobium x S. bovis transmission. We discuss the origin of the new chronotypes which have become more complex with the appearance of several peaks in a 24-h day. We also discuss how the new populations of offspring may optimize intra-host ecological niche, host spectrum, and transmission time period.

The Use of DNA Barcoding and Phylogenetic Analysis to Improve Identification of <i>Usnea</i> spp. Based on ITS rDNA

Lichen of the genus Usnea is quite common being used as a traditional herbal remedy. This genus is characterized by thallus, which is very similar among the species, leads to some difficulties in distinguishing them. In Indonesia, such research report on the availability of this genus based on their morphological characteristic is minimal. This might be due to too high morphological similarities among them. The molecular character, which is based on the DNA Barcode of Internal Transcribed Spacer (ITS) rDNA sequences, with its conserved region (5.8S) and varied region (ITS1 and ITS2), are becoming essential characters on identifying as well as analyzing the phylogenetic. The current study then proposed to identify and draw the species dendrogram of species within the Usnea genus obtained from Mount Lawu Forest of Central Java and Turgo Forest of Yogyakarta based on their phylogenetic and phenetic analysis. The dendrogram was constructed with UPGMA using the simple matching coefficient, whereas the phylogenetic tree was constructed with Maximum Likelihood (ML) using Kimura-2 parameter with 1000 bootstrap. The data were unable to draw phenetic relationships among the subgenus Usnea and Eumitria members. The phylogenetic tree shows the primary two clades, distinguishing the subgenus Usnea and Eumitria. The ITS rDNA sequence was able to identify most of the Usnea species.

Airway bacterial and fungal microbiome in chronic obstructive pulmonary disease

Little is known about airway mycobiome, and its relationship with bacterial microbiome in chronic obstructive pulmonary disease (COPD). Here we report the first simultaneous characterization of sputum bacterial and fungal microbiome in 84 stable COPD and 29 healthy subjects, using 16S ribosomal DNA and fungal internal transcribed spacer DNA sequencing. Ascomycota predominated over Basidiomycota in fungal microbiome both in COPD patients and healthy controls. Meyerozyma, Candida, Aspergillus and Schizophyllum were most abundant at the genus level. There was a significant inverse correlation between bacterial and fungal microbial diversity, both of which altered in opposite directions in COPD patients versus controls, and in frequent versus non-frequent exacerbators. An enhanced bacterial-fungal ecological interaction was observed in COPD patients, which was characterized by higher proportion of co-occurrence intrakingdom interactions and co-exclusive interkingdom interactions. In COPD, four mutually co-occurring fungal operational taxonomic units (OTUs) in Candida palmioleophila, Aspergillus and Sordariomycetes exhibited co-exclusive relationships with other fungal OTUs, which was specifically present in frequent exacerbators but not in non-frequent exacerbators. The perturbed bacterial-fungal interactions in COPD were associated with increased airway inflammatory mediators such as IL-6 and IL-8. The disruption of airway bacterial-fungal community balance, characterized by the loss of commensal bacterial taxa and enriched pathogenic fungal taxa, is implicated in COPD. The emergence of pathogenic fungi such as Candida and Aspergillus could be a marker for the frequent exacerbator phenotype. The airway mycobiome is an important cofactor mediating pathogenic infection and host inflammation in COPD.

Physiological characterization and molecular identification of some rare yeast species causing onychomycosis

IntroductionOnychomycosis are infections with a variety of etiological agents. Although dermatophytes are responsible for most infections, yeasts are gaining importance as agents of these pathologies. The use of antifungals has increased the incidence of what had been considered rare or novel pathogens. We reidentify three rare yeasts from a culture collection of onychomycosis agents by matrix-assisted laser desorption/ionization time of flight/mass spectrometry (MALDI-TOF/MS) and sequencing the internal transcribed spacer (ITS) regions or the intergenic spacer (IGS) 1 region of ribosomal DNA (rDNA), and present their enzymatic and antifungal susceptibility profiles. Material and methodsWe performed a phenotypical characterization and molecular identification of five yeast isolates. We tested the urease, gelatinase, DNase, phospholipase, protease, and esterase activities, as well as the hemolytic activity. We evaluated the antifungal susceptibility to amphotericin B, fluconazole, anidulafungin and caspofungin. ResultsPhenotypic methods could not identify the isolates. MALDI-TOF/MS was able to properly identify Candida duobushameulonii. The five isolates were successfully identified by sequence analysis as Candida duobushaemulonii, Meyerozyma caribbica and Cutaneotrichosporon dermatis. Candida duobushameulonii showed hemolytic, phospholipase, and protease activities. Meyerozyma caribbica was positive for gelatinase and protease activities. All antifungals exhibited minimum inhibitory concentrations (MICs) ≤2μg/mL against both species. The three isolates of Cutaneotrichosporon dermatis showed urease, DNase, and esterase activities, and resistance to echinocandins (MICs ≥8μg/mL), while amphotericin B and fluconazole exhibited low MICs against these isolates (0.50–2μg/mL). DiscussionSequencing of the ITS or IGS1 regions of rDNA remains the best method for identifying cryptic species over other commercially available systems. More reports are needed to define the enzymatic and antifungal profiles for these species. This is the first report of Meyerozyma caribbica and Cutaneotrichosporon dermatis as etiological agents of onychomycosis.

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods

Ethnopharmacological relevanceThe roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. Aim of this studyThis study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. Materials and methodsThe sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. ResultsThe plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. ConclusionThe nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

Isolation, identification and genotyping of Candida albicans from Landes geese

In May 2018, Landes geese raised in Weifang, Shandong Province, China, developed a disease characterized by thickened oesophageal mucosa and white, round ulcers. Based on pathogen isolation and identification, differential culture and morphological observations, Candida albicans (C. albicans) was identified as the causative pathogen from the oesophagus of infected geese, and artificial infection experiments were then performed using the isolated strains. In experimental reproduction, the symptoms of infected geese were consistent with those of natural infection, and gosling morbidity and mortality were 75% and 60%, respectively. Re-isolation of the strain from the dead goslings confirmed C. albicans as the causative pathogen of oesophageal ulcers. We further performed internal transcribed space rDNA sequence analysis, ABC genotyping and multi-locus sequence typing analysis. We observed 100% sequence similarity between the two strains, designated as WFCL and WFLQ, which were isolated from different regions, with 100% homology between the strains isolated in the present study and the human-origin C. albicans strains isolated previously from China. The goose-origin strains isolated in this study and the human-origin C. albicans isolates were included in the same branch in phylogenetic trees analysis, indicating that the strain responsible for oesophageal ulcer in geese is closely related to human-origin C. albicans. In addition, based on eBURST analysis of sequence types, goose-origin C. albicans strains were relatively independent in terms of population evolution. To the best of our knowledge, this is the first detailed report on goose oesophageal ulceration caused by C. albicans infection in geese. Considering that C. albicans is an important zoonotic pathogen, this study provides a reference for further studies on avian C. albicans infections and is important for ensuring public health and safety.

Evolution of Tandem Repeats Is Mirroring Post-polyploid Cladogenesis in Heliophila (Brassicaceae).

The unigeneric tribe Heliophileae encompassing more than 100 Heliophila species is morphologically the most diverse Brassicaceae lineage. The tribe is endemic to southern Africa, confined chiefly to the southwestern South Africa, home of two biodiversity hotspots (Cape Floristic Region and Succulent Karoo). The monospecific Chamira (C. circaeoides), the only crucifer species with persistent cotyledons, is traditionally retrieved as the closest relative of Heliophileae. Our transcriptome analysis revealed a whole-genome duplication (WGD) ∼26.15–29.20 million years ago, presumably preceding the Chamira/Heliophila split. The WGD was then followed by genome-wide diploidization, species radiations, and cladogenesis in Heliophila. The expanded phylogeny based on nuclear ribosomal DNA internal transcribed spacer (ITS) uncovered four major infrageneric clades (A–D) in Heliophila and corroborated the sister relationship between Chamira and Heliophila. Herein, we analyzed how the diploidization process impacted the evolution of repetitive sequences through low-coverage whole-genome sequencing of 15 Heliophila species, representing the four clades, and Chamira. Despite the firmly established infrageneric cladogenesis and different ecological life histories (four perennials vs. 11 annual species), repeatome analysis showed overall comparable evolution of genome sizes (288–484 Mb) and repeat content (25.04–38.90%) across Heliophila species and clades. Among Heliophila species, long terminal repeat (LTR) retrotransposons were the predominant components of the analyzed genomes (11.51–22.42%), whereas tandem repeats had lower abundances (1.03–12.10%). In Chamira, the tandem repeat content (17.92%, 16 diverse tandem repeats) equals the abundance of LTR retrotransposons (16.69%). Among the 108 tandem repeats identified in Heliophila, only 16 repeats were found to be shared among two or more species; no tandem repeats were shared by Chamira and Heliophila genomes. Six “relic” tandem repeats were shared between any two different Heliophila clades by a common descent. Four and six clade-specific repeats shared among clade A and C species, respectively, support the monophyly of these two clades. Three repeats shared by all clade A species corroborate the recent diversification of this clade revealed by plastome-based molecular dating. Phylogenetic analysis based on repeat sequence similarities separated the Heliophila species to three clades [A, C, and (B+D)], mirroring the post-polyploid cladogenesis in Heliophila inferred from rDNA ITS and plastome sequences.

Morphological and PCR-based characterisation of Podosphaera aphanis (Wallr.) U. Braun & S. Takamatsu causing powdery mildew disease in strawberry in Java

The powdery mildew (CHD13) which infected strawberry (Fragaria ×ananassa Duch) in Java was identified using morphological and Internal Transcribed Spacer rDNA region. It revealed that the identity of fungal pathogen was similar to Podosphaera aphanis. The phylogenetic analysis figured that the CHD13 isolate infecting strawberry in Java was clustered with P. aphanis from China, the United Kingdom, and Argentina. The CHD13 isolate was separated from P. macularis and P. xanthii. The pathogenicity test of the CHD13 isolate was done by using ten strawberry cultivars from three different species (F ×ananassa, F. vesca, and F. moschata). Each cultivar showed different disease reactions. This research concluded that the F. moschata cultivar and two cultivars of F. ×ananassa were resistant, while six cultivars of F. ×ananassa were susceptible and F. vesca were highly susceptible to powdery mildew disease.

Species delimitation of Gyrodactylus (Monogenea: Gyrodactylidae) infecting the southernmost cyprinids (Actinopterygii: Cyprinidae) in the New World.

The genus Gyrodactylus von Nordmann, 1832 represents one of the most diverse and widespread taxa within Monogenea, with approximately 500 species described worldwide. Thirty-three species of Gyrodactylus have been recorded in Mexico, and in the last two decades, at least 26 new species have been described mainly from freshwater fish families such as poeciliids, goodeids, profundulids, characids, and cichlids. In this study, we describe two new species of Gyrodactylus infecting freshwater cyprinids based on morphological and molecular characteristics. Gyrodactylus ticuchi n. sp. and Gyrodactylus tobala n. sp. were recovered from Notropis moralesi de Buen and N. imeldae Cortés, respectively, captured in five localities from the State of Oaxaca, Mexico. The new species differ slightly from their congeners in the morphology of the haptoral hard parts and the male copulatory organ. Sequences of the Internal Transcribed Spacers rDNA (ITS1-5.8S-ITS2), cytochrome oxidase subunit I (cox1), and the D2 + D3 domains of the large subunit (28S rDNA) were obtained from multiple specimens and analyzed using Maximum Likelihood (ML) and Bayesian Inference (BI). Phylogenetic hypotheses using ITS rDNA, cox1, and 28S rDNA genes recovered two new species of Gyrodactylus from N. moralesi and N. imeldae; we briefly discuss their phylogenetic relationship with other congeners. These gyrodactylids represent the first species described in species of Notropis from southern Mexico, the cyprinids exhibiting the southernmost distribution in the New World.

A global meta-analysis of ITS rDNA sequences from material belonging to the genus Ganoderma (Basidiomycota, Polyporales) including new data from selected taxa.

Ganoderma P. Karst. is a cosmopolitan genus of white-rot fungi which comprises species with highly-prized pharmaceutical properties, valuable biotechnological applications and of significant phytopathological interest. However, the status of the taxonomy within the genus is still highly controversial and ambiguous despite the progress made through molecular approaches. A metadata analysis of 3908 nuclear ribosomal internal transcribed spacer (ITS) rDNA sequences obtained from GenBank/ENA/DDBJ and UNITE was performed by targeting sequences annotated as Ganoderma, but also sequences from environmental samples and from material examined for the first time. Ganoderma taxa segregated into five main lineages (Clades A to E). Clade A corresponds to the core of laccate species and includes G. shanxiense and three major well-supported clusters: Cluster A.1 (‘G. lucidum sensu lato’) consists of taxa from Eurasia and North America, Cluster A.2 of material with worldwide occurrence including G. resinaceum and Cluster A.3 is composed of species originating from all continents except Europe and comprises G. lingzhi. Clade B includes G. applanatum and allied species with a Holarctic distribution. Clade C comprises taxa from Asia and Africa only. Clade D consists of laccate taxa with tropical/subtropical occurrence, while clade E harbours the highest number of non-laccate species with a cosmopolitan distribution. The 92 Ganoderma-associated names, initially used for sequences labelling, correspond to at least 80 taxa. Amongst them, 21 constitute putatively new phylospecies after our application of criteria relevant to the robustness/support of the terminal clades, intra- and interspecific genetic divergence and available biogeographic data. Moreover, several other groups or individual sequences seem to represent distinct taxonomic entities and merit further investigation. A particularly large number of the public sequences was revealed to be insufficiently and/or incorrectly identified, for example, 87% and 78% of entries labelled as G. australe and G. lucidum, respectively. In general, ITS demonstrated high efficacy in resolving relationships amongst most of the Ganoderma taxa; however, it was not equally useful at elucidating species barriers across the entire genus and such cases are outlined. Furthermore, we draw conclusions on biogeography by evaluating species occurrence on a global scale in conjunction with phylogenetic structure/patterns. The sequence variability assessed in ITS spacers could be further exploited for diagnostic purposes.

Occurrence of Leaf Spot Caused by Alternaria alternata on Eggplant (Solanum melongena) in Pakistan.

Eggplant (Solanum melongena L.) is a popular vegetable that is grown in both tropical and subtropical regions all year long. The crop is cultivated on small family farms and is a good source of income for resource-limited farmers in Pakistan. In early May 2019, leaf spots on eggplant (cv. Bemisaal) were observed in an experimental field (31°26'14.0"N 73°04'23.4"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, circular, brown, necrotic spots uniformly distributed on leaves. The spots gradually enlarged and coalesced into large, nearly circular or irregularly shaped spots that could be up to 3 cm in length. The center of the spots was light tan, surrounded by a dark brown ring, a chlorotic halo, and tended to split in the later developmental stages. Disease incidence was approximately 35% in the infected field. The causal agent of this disease was isolated consistently by plating surface sterilized (1% NaOCl) sections of symptomatic leaf tissue onto potato dextrose agar (PDA). After 6 days incubation at 25°C with a 12-h photoperiod, fungal colonies had round margins and the cottony mycelia were dark olivaceous with a mean diameter of 7.5 cm. For conidial production, the fungus was grown on potato carrot agar (PCA) and V8 agar media under a 16-h/8-h light/ dark photoperiod at 25°C. Conidiophores were septate, light to olive golden brown with a conidial scar, from which conidia were produced. Conidia were borne singly or in short chains and were obpyriform to obclavate, measured 29 ± 4.8 × 13.25 ± 2.78 μm (n=30) with zero to three longitudinal and two to six transversal septa. The morphological characters matched those of Alternaria alternata (Fr.) Keisel (Simmons et al. 2007). DNA was extracted using the DNAzol reagent (Thermo Fisher Scientific MA, USA). For molecular identification, internal transcribed spacer (ITS) region between ITS1 and ITS2, actin gene (β-Actin), translation elongation factor (TEF-1α) gene, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of two representative isolates (JLUAF1 and JLUAF2) were amplified with primers ITS1/ITS4 (White et al. 1990), β-Actin 512 F/783 R, EF1-728F/-986R (Carbone et al. 1999), and gpd1/gpd2 (Berbee et al. 1999), respectively. The sequences were deposited in GenBank (accession nos. MT228734.1 and MT228735.1 for ITS; MT260151.1 and MT260152.1 for β-Actin, MT260163.1 and MT260164.1, for TEF-1a, and MT260157.1 and MT260158.1 for GAPDH). BLASTn analysis of these sequences showed 100% identity with the sequences of A. alternata for ITS rDNA, β-Actin, TEF-1α, and GAPDH, respectively. Based on the morphological characters and DNA sequences, the leaf spot isolates of eggplant were identified as A. alternata. To confirm the pathogenicity on eggplant, six-week old healthy potted eggplants of cv. Bemisaal were sprayed at the true leaf stage with conidial suspensions of A. alternata (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) using an atomizer in the greenhouse. Three plants were inoculated with each of the two isolates (JLUAF1 and JLUAF2), whereas three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots which were similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling Koch's postulates. Previously, A. alternata has been reported to cause leaf spots on eggplant in India (Raina et al. 2018). To our knowledge, this is the first report of A. alternata causing leaf spot on eggplant in Pakistan. The disease could represent a threat for eggplant crops due to its increasing cultivation. It is important to develop disease management strategies for Alternaria alternata causing leaf spot of Eggplant in Pakistan.

Pathological, biochemical and molecular variability of Colletotrichum truncatum incitant of anthracnose disease in chilli (Capsicum annuum L.)

The present study aims to establish pathogenic variability among Colletotrichum truncatum, an incitant of anthracnose disease across different chilli growing regions of Karnataka. Thirty suspected C. truncatum isolates were identified based on their morphological and conidial characteristics and further confirmed by Internal Transcribed Spacer DNA sequence analysis. Pathogenicity test was carried out by in vitro detached leaf and fruit assay, and also under greenhouse conditions using 20 different chilli cultivars grown across Karnataka. Colletotrichum truncatum isolates recorded the varied degree of pathogenicity index (PI) on different chilli cultivars. Isolate UOM-02 was found highly virulent (PI > 80 against 12 tested cultivars) and cultivar cv. 4 was found highly resistant to C. truncatum infection (Average PI, 48.21). Further, the involvement of enzymes such as cellulase, pectin methylesterase and ascorbate peroxidase in determining the virulence of the pathogen was established. The highest activity of catalase (UOM-24; 7.38 units), ascorbate peroxidase (UOM-02; 2.9 units), cellulase (UOM-02; 0.58 units), and pectin methylesterase (UOM-02; 6.7 units), was recorded by different C. truncatum isolates. Cellulase and pectin methylesterase activities were positively correlated with their pathogenicity, while catalase activity was found least correlated. Results of RAPD and ISSR analysis recorded higher polymorphism among the isolates. Interestingly these isolates were not clustered based on their geographical origin, Pathogenicity index and biochemical characters. From this study, the existence of highly virulent C. truncatum isolate (UOM-02), which can cause severe loss under favourable conditions, was revealed. Further, possible use of specific enzymes as an indicator of virulence of the pathogen is discussed.

ANTI-STAPHYLOCOCCAL POTENTIAL OF FUSARIUM SP: FUNGAL ENDOPHYTE ISOLATED FROM RAUWOLFIA SERPENTINA

Fungal endophytes are considered as repository of potent novel bioactive compounds with profuse chemical diversity. These bioactive compounds have been widely utilized in pharmaceutical and agrochemical industries. The present work was undertaken to bio-prospect the fungal endophytes from Rauwolfia serpentina, a medicinal plant used in multiple indigenous preparations, for possible therapeutic interventions. 21 fungal endophytes were isolated from Rauwolfia serpentina with maximum colonization in leaf (42.8 %) followed by bark (33.3%) and stem (23.8%). In the enzymatic activity screening, #15RSBNEY was found to be potent amylase producer and #3RSSTNEY as potential cellulase producer. Further, three isolates viz. #16RSLBRT, #10RSLBRT and #23RSSTNEY exhibited promising antibacterial activity with maximum activity exhibited by #10RSLBRT against all test strains of Staphylococcus aureus. Interestingly, it was observed that antibacterial activity of #10RSLBRT increased by two fold after fractionation with acetone and chloroform. Based on above result, the culture filtrate of #10RSLBRT was extracted with hexane, chloroform and ethyl acetate followed by agar well gel diffusion method for antibacterial activity. The crude ethyl acetate residue of #10RSLBRT hereby exhibited potent antibacterial activity. The potential isolate i.e. #10RSLBRT was identified by molecular taxonomic tools using internal transcribed spacer rDNA analysis and was clustered in the Fusarium incarnatum-equiseti complex. Further studies on the characterization of bioactive compounds of ethyl acetate extract of # 10RSLBRT are warranted for the development of anti-staphylococcal therapy.