Loquat [Eriobotrya japonica (Thunb.) Lindl.] is an important fruit crop in Pakistan; however, a constant decline in its production is noted due biotic and abiotic stresses, particularly disease infestation. Fungal pathogens are the major disease-causing agents; therefore, their identification is necessary for devising management options. This study explored Taxila, Wah-Cantt, Tret, Chatar, Murree, Kalar-Kahar, Choa-Saidan-Shah and Khan-Pur districts in the Punjab and Khyber Paktoon Khawa (KPK) provinces of Pakistan to explore the diversity of fungal pathogens associated with loquat. The samples were collected from these districts and their microscopic characterizations were accomplished for reliable identification. Alternaria alternata, Curvularia lunata, Lasiodiplodia theobromae, Aspergilus flavis, Botrytis cinerea, Chaetomium globosum, Pestalotiopsis mangiferae and Phomopsis sp. were the fungal pathogens infesting loquat in the study area. The isolates of A. alternata and C. lunata were isolated from leaf spots and fruit rot, while the isolates of L. theobromae were associated with twig dieback. The remaining pathogens were allied with fruit rot. The nucleotide evidence of internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) were computed from all the pathogens and submitted in the database of National Center for Biotechnology Information (NCBI). For multigene analysis, beta-tubulin (BT) gene and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions were explored for A. alternata and C. lunata isolates, respectively. The virulence scales of leaf spots, fruit rot, and twig dieback diseases of loquat were developed for the first time through this study. It is the first comprehensive study with morpho-molecular identification, and newly developed virulence scales of the fungal pathogens associated with loquat, which improves the understanding of these destructive diseases.
The worldwide-distributed aquatic fungus Articulospora tetracladia Ingold is a dominant sporulating species in streams of the Northwest Iberian Peninsula. To elucidate the genetic diversity of A. tetracladia, we analyzed isolates collected from various types of plant litter or foam in streams from North and Central Portugal and North Spain, between 2000 and 2010. Genetic diversity of these fungal populations was assessed by denaturing gradient gel electrophoresis (DGGE) fingerprints and by using ITS1-5.8S-ITS2 barcodes. Moreover, ITS1-5.8S-ITS2 barcodes of A. tetracladia reported in other parts of the world (Central Europe, United Kingdom, Canada, Japan and Malaysia) were retrieved from the National Center for Biotechnology (NCBI) and the National Institute of Technology and Evaluation Biological Resource Center (NBRC) to probe into genetic diversity of A. tetracladia. PCR-DGGE of ITS2 region of 50 Iberian fungal isolates distinguished eight operational taxonomic units (OTUs), which were similar to those obtained from neighboring trees based on ITS2 gene sequences. On the other hand, ITS1-5.8S-ITS2 barcodes of 68 fungal isolates yielded nine OTUs, but five fungal isolates were not assigned to any of these OTUs. Molecular diversity was highest for OTU-8, which included only European isolates. Two haplotypes were observed within OTU-8 and OTU-9, while only one haplotype was found within each of the remaining OTUs. Malaysia did not share haplotypes with other countries. Overall results indicate that, apart from the Malaysian genotypes, A. tetracladia genotypes were geographically widespread irrespective of sampling time, sites or substrates. Furthermore, PCR-DGGE appeared to be a rapid tool for assessing intraspecific diversity of aquatic hyphomycetes.
Hortaea werneckii is an environmental dematiaceous fungus found in the halophilic environment. It causes tinea nigra. We report the isolation of H. werneckii from blood and splenic abscess of two patients with acute myelomonocytic leukaemia. H. werneckii grew at room temperature but not at 37 degrees C, it was identified by biochemical tests, growth characteristics and the presence of conspicuous collarette intercalary on dividing yeast cells. The use of specific oligonucleotide primer Hor-F (5'-TGGACACCTTCA TAACTCTTG-3') and Hor-R (5'-TCACAACGCTTAGAGACGG-3') confirmed the two isolates were H. werneckii. The sequence for 281 nucleotide of HW299 and HW403 were 99% identical but differed only in one nucleotide. In vitro anti-fungal susceptibility testing showed that the isolates were resistant to amphotericin B and flucytosine.