Morpho-molecular study of entomopathogenic fungi associated with citrus orchard pests in Northern Iran.

Entomopathogenic fungi play a significant role in regulating insect populations in nature and have potential applications in pest management strategies in different regions. Citrus spp. are among the important horticultural products in northern Iran, and the orchards are affected by different insect pests, especially mealybugs. This study aimed to isolate and identify entomopathogenic fungi associated with citrus orchard pests in northern Iran, focusing on Akanthomyces and Lecanicillium species on mealybugs. Through the samples collected from different regions within Guilan province, 12 fungal isolates were collected and identified based on the combination of morphological characteristics and molecular data. Akanthomyces lecanii, A. muscarius, Engyodontium rectidentatum, Lecanicillium aphanocladii and Lecanicillium rasoulzarei sp. nov. were identified. Of these, A. muscarius on Lepidosaphes sp., E. rectidentatum on Coccidae, and L. aphanocladii on Tetranychus urticae are reported as new fungal-host records from Iran. Moreover, a new species, Lecanicillium rasoulzarei, is illustrated, described, and compared with closely related species.

Utilization of Low Molecular Weight Carbon Sources by Fungi and Saprolegniales: Implications for Their Ecology and Taxonomy.

Contributions of fungal and oomycete communities to freshwater carbon cycling have received increasing attention in the past years. It has been shown that fungi and oomycetes constitute key players in the organic matter cycling of freshwater ecosystems. Therefore, studying their interactions with dissolved organic matter is crucial for understanding the aquatic carbon cycle. Therefore, we studied the consumption rates of various carbon sources using 17 fungal and 8 oomycete strains recovered from various freshwater ecosystems using EcoPlate™ and FF MicroPlate™ approaches. Furthermore, phylogenetic relationships between strains were determined via single and multigene phylogenetic analyses of the internal transcribed spacer regions. Our results indicated that the studied fungal and oomycete strains could be distinguished based on their carbon utilization patterns, as indicated by their phylogenetic distance. Thereby, some carbon sources had a higher discriminative strength to categorize the studied strains and thus were applied in a polyphasic approach. We concluded that studying the catabolic potential enables a better understanding of taxonomic relationships and ecological roles of fungal vs. oomycete strains.

Phylogeny and taxonomy of Erysiphe berberidis (s. lat.) revisited.

Phylogenetic and morphological analyses have been conducted on powdery mildew specimens on different Berberis and Mahonia spp. from Asia, Europe and North America. The present study showed that collections of Erysiphe berberidis exhibit a high degree of morphological plasticity of the sexual morph, in contrast to their morphologically, rather uniform, asexual morph. In phylogenetic tree, all sequences cluster in a large strongly supported clade, without any indication and support for further differentiation into cryptic species. There are three morphological types within E. berberidis s. lat. that contain consistent differences. Until future multi-locus analyses will be available, we prefer to treat these 'morphological types' as varieties. These include Erysiphe berberidis var. berberidis, E. berberidis var. asiatica, and E. berberidis var. dimorpha comb. nov. (≡ Microsphaera berberidis var. dimorpha, M. berberidicola, and M. multappendicis). To fix the application of species name E. berberidis, an appropriate epitype was designated, with an ITS sequences.

Note on the genus Nemania (Xylariaceae) - first records and a new species of the genus from Iran.

In a survey of xylarialean fungi in northern Iran, some specimens attributable to the genus Nemania were collected, cultured and sequenced. Morphological evidence and phylogenetic analyses of a combined ITS, LSU, RPB2 and TUB2 gene dataset confirmed the presence of Nemaniadiffusa and N.serpens in Iran for the first time. Furthermore, the new species N.hyrcana, which shows similarities to N.subaenea and its putative synonym N.plumbea, but significantly differs from the latter in its DNA sequences, was encountered. All species are illustrated, described and discussed. In the phylogenetic analyses, for the first time, the overlooked ex-type ITS sequences of the neotype of the generic type, N.serpens and that of the holotype of N.prava, were added to a multi-gene matrix of Nemania. This revealed that the two accessions of N.serpens (HAST 235 and CBS 679.86), for which multigene data are available in GenBank, are misidentified, while the Iranian accession of N.serpens has an almost identical ITS sequence to the neotype, confirming its morphological species identification. The two previously accepted species of Euepixylon, E.udum and E.sphaeriostomum, are embedded within Nemania and are revealed as close relatives of N.serpens, supporting the inclusion of Euepixylon in Nemania.

Phylogenetic and Functional Diversity of Saprolegniales and Fungi Isolated from Temperate Lakes in Northeast Germany.

The contribution of fungi to the degradation of plant litter and transformation of dissolved organic matter (humic substances, in particular) in freshwater ecosystems has received increasing attention recently. However, the role of Saprolegniales as one of the most common eukaryotic organisms is rarely studied. In this study, we isolated and phylogenetically placed 51 fungal and 62 Saprolegniales strains from 12 German lakes. We studied the cellulo-, lignino-, and chitinolytic activity of the strains using plate assays. Furthermore, we determined the capacity of 10 selected strains to utilize 95 different labile compounds, using Biolog FF MicroPlates™. Finally, the ability of three selected strains to utilize maltose and degrade/produce humic substances was measured. Cladosporium and Penicillium were amongst the most prevalent fungal strains, while Saprolegnia, Achlya, and Leptolegnia were the most frequent Saprolegniales strains. Although the isolated strains assigned to genera were phylogenetically similar, their enzymatic activity and physiological profiling were quite diverse. Our results indicate that Saprolegniales, in contrast to fungi, lack ligninolytic activity and are not involved in the production/transformation of humic substances. We hypothesize that Saprolegniales and fungi might have complementary roles in interacting with dissolved organic matter, which has ecological implications for carbon cycling in freshwater ecosystems.

Resolution of the Hypoxylon fuscum Complex (Hypoxylaceae, Xylariales) and Discovery and Biological Characterization of Two of Its Prominent Secondary Metabolites.

Hypoxylon, a large, cosmopolitan genus of Ascomycota is in the focus of our current poly-thetic taxonomic studies, and served as an excellent source for bioactive secondary metabolites at the same time. The present work concerns a survey of the Hypoxylon fuscum species complex based on specimens from Iran and Europe by morphological studies and high performance liquid chromatography coupled to mass spectrometry and diode array detection (HPLC-MS-DAD). Apart from known chemotaxonomic markers like binaphthalene tetrol (BNT) and daldinin F, two unprece-dented molecules were detected and subsequently isolated to purity by semi preparative HPLC. Their structures were established by nuclear-magnetic resonance (NMR) spectroscopy as 3'-malonyl-daldinin F (6) and pseudofuscochalasin A (4). The new daldinin derivative 6 showed weak cytotoxicity towards mammalian cells but bactericidal activity. The new cytochalasin 4 was compared to cytochalasin C in an actin disruption assay using fluorescence microscopy of human osteo-sarcoma U2OS cells, revealing comparable activity towards F-actin but being irreversible compared to cytochalasin C. Concurrently, a multilocus molecular phylogeny based on ribosomal and proteinogenic nucleotide sequences of Hypoxylon species resulted in a well-supported clade for H. fuscum and its allies. From a comparison of morphological, chemotaxonomic and phylogenetic evidence, we introduce the new species H. eurasiaticum and H. pseudofuscum.

Discovery of a new species of the Hypoxylon rubiginosum complex from Iran and antagonistic activities of Hypoxylon spp. against the Ash Dieback pathogen, Hymenoscyphus fraxineus, in dual culture.

During a survey of xylarialean fungi in Northern Iran, several specimens that showed affinities to the Hypoxylon rubiginosum complex were collected and cultured. A comparison of their morphological characters, combined with a chemotaxonomic study based on high performance liquid chromatography, coupled with diode array detection and mass spectrometry (HPLC-DAD/MS) and a multi-locus phylogeny based on ITS, LSU, rbp2 and tub2 DNA sequences, revealed a new species here described as Hypoxylon guilanense. In addition, Hypoxylon rubiginosum sensu stricto was also encountered. Concurrently, an endophytic isolate of the latter species showed strong antagonistic activities against the Ash Dieback pathogen, Hymenoscyphus fraxineus, in a dual culture assay in our laboratory. Therefore, we decided to test the new Iranian fungi for antagonistic activities against the pathogen, along with several cultures of other Hypoxylon species that are related to H. rubiginosum. Our results suggest that the antagonistic effects of Hypoxylon spp. against Hym. fraxineus are widespread and that they are due to the production of antifungal phomopsidin derivatives in the presence of the pathogen.

Association Analysis of Charcoal Rot Disease Resistance in Soybean.

In this research, the relationships among the 31 microsatellite markers with charcoal rot disease resistance related indices in 130 different soybean cultivars and lines were evaluated using association analysis based on the general linear model (GLM) and the mixed linear model (MLM) by the Structure and Tassel software. The results of microsatellite markers showed that the genetic structure of the studied population has three subpopulations (K=3) which the results of bar plat also confirmed it. In association analysis based on GLM and MLM models, 31 and 35 loci showed significant relationships with the evaluated traits, respectively, and confirmed considerable variation of the studied traits. The identified markers related to some of the studied traits were the same which can probably be due to pleiotropic effects or tight linkage among the genomic regions controlling these traits. Some of these relationships were including, the relationship between Sat_252 marker with amount of charcoal rot disease, Satt359, Satt190 and Sat_169 markers with number of microsclerota in stem, amount of charcoal rot disease and severity of charcoal rot disease, Sat_416 marker with number of microsclerota in stem and amount of charcoal rot disease and the Satt460 marker with number of microsclerota in stem and severity of charcoal rot disease. The results of this research and the linked microsatellite markers with the charcoal rot disease-related characteristics can be used to identify the suitable parents and to improve the soybean population in future breeding programs.

Comprehensive molecular phylogenetic analysis and evolution of the genus Phyllactinia (Ascomycota: Erysiphales) and its allied genera.

Phyllactinia is a unique genus within the Erysiphales (Ascomycota) having a partly endo-parasitic nature of the mycelium within the host plant tissues. We constructed phylogenetic trees for the genus Phyllactinia and its allied genera based on a total of 120 nucleotide sequences of the 28S rDNA and ITS regions to discuss their phylogenetic relationships with special references to host plants, biogeography, evolutionary dating, and taxonomy. The analysis of the Erysiphales confirmed the monophyly of the endo-parasitic genera, i.e. Leveillula, Phyllactinia, and Pleochaeta. Phyllactinia specimens used in this study were divided into six distinctive groups and three subgroups. Interestingly, Leveillula, an obligately endo-parasitic genus of the Erysiphales, grouped together with Phyllactinia, although this was not significantly supported by the Kishino-Hasegawa and Shimodaira-Hasegawa tests. This suggests that the evolution within this group of fungi occurred from partial endo-parasitism to obligate endo-parasitism. The host range of Phyllactinia is mostly confined to woody plants, especially deciduous trees. Betulaceae, Fagaceae, Ulmaceae, Moraceae, and Rosaceae may have close connections to the divergence of the groups and subgroups of Phyllactinia concerned. Most of these plant families are known as major members of the boreotropical flora of the Tertiary, which suggests an early Tertiary origin of this genus. A comparison of the phylogenies of hosts and parasites revealed that host range expansion at higher taxonomic levels (higher than family level) is independent of the phylogeny of plants. Conversely, host range expansions in lower taxonomic levels (infrafamilial or infrageneric) tend to occur within a single family or genus. An estimation of the evolutionary timing using a molecular clock approach suggested that Phyllactinia split from Pleochaeta about 60 M years ago (Ma) in the early Tertiary and divergence of the six major clades of Phyllactinia occurred between 5 and 40 Ma during the Oligocene and Miocene. Divergence within the major clades and within Leveillula occurred maybe from more than 5 Ma onwards during the Pliocene and Quaternary. This is the first comprehensive phylogenetic study of Phyllactinia and other endo-parasitic genera of the Erysiphales.

Molecular and morphological characterization of Leveillula (Ascomycota: Erysiphales) on monocotyledonous plants.

Leveillula on monocotyledonous plants have been recorded as L. taurica by several authors, whereas the fungus on Allium has been described as an independent species, namely L. allii, by some authors. We sequenced ca 600bp of the rDNA ITS region for two Leveillula specimens from Allium and Polianthes (both from monocotyledons) and compared them with several already published sequences from Leveillula isolates from dicotyledons. Pair-wise percentages of sequence divergences were calculated for all Leveillula isolates. The ITS sequence of the Polianthes isolate was identical to L. taurica on Helianthus and Vicia. The sequence of the Allium isolate was 99.5% identical to L. taurica on Euphorbia, Haplophylum, Peganum, etc. These results suggest close relationships between monocot and dicot pathogenic Leveillula species. The identity between two monocot isolates was 98.4%. Phylogenetic analysis revealed that the two monocot isolates do not group into a clade together. This result suggests that Leveillula acquired parasitism to monocots at least twice independently.

Multilocus phylogenetic analyses within Blumeria graminis, a powdery mildew fungus of cereals.

Blumeria graminis, a powdery mildew fungus, is an important plant pathogen that causes serious damage to a variety of cereal crops. In spite of the importance of the pathogen, information on phylogenetic structure within B. graminis is scarce. In this study we conducted phylogenetic analyses of B. graminis based on the DNA sequences of four different DNA regions (ITS, 28S rDNA, chitin synthase 1, and beta-tubulin). The analyses revealed that the protein-coding regions have higher amounts of phylogenetic signals than rDNA regions and are useful for phylogenetic analyses of B. graminis. The present phylogenetic analyses revealed nine distinct groups in the B. graminis isolates used in this study, a result which was commonly supported by all trees constructed from the four DNA regions. Isolates from a single host genus belonged to a single group except for isolates from Lolium and Bromus, in which the isolates were split into two and three groups, respectively. Isolates from Agropyron, Secale and Triticum formed a distinct clade (Triticum clade) with identical or similar DNA sequences. The Hordeum clade was a sister of the Triticum clade, and Poa and Avena clades were distantly related to the Triticum and Hordeum clades. This phylogenetic relationship of B. graminis is well concordant with the level of reproductive isolation between formae speciales and also with phylogeny inferred from a cytological study. Shimodaira-Hasegawa and Templeton tests using sequences of four different DNA regions significantly rejected the tree topology of plants. Therefore, possibility of co-speciation between B. graminis and its host plants was obscure in this study.