Rabenchromenone and Rabenzophenone, Phytotoxic Tetrasubstituted Chromenone and Hexasubstituted Benzophenone Constituents Produced by the Oak-Decline-Associated Fungus Fimetariella rabenhorstii.

A new phytotoxic tetrasubstituted chromen-4-one (1) and a new hexasubstituted benzophenone (2), named rabenchromenone and rabenzophenone, respectively, were isolated from the culture filtrates of Fimetariella rabenhorstii, an oak-decline-associated fungus in Iran. Rabenchromenone and rabenzophenone, isolated together with known moniliphenone (3) and coniochaetone A (4), were characterized as methyl 3-chloro-1,8-dihydroxy-6-methyl-9-oxo-1,9-dihydrocyclopenta[b]chromene-1-carboxylate and methyl 4-chloro-2-(2,6-dihydroxy-4-methylbenzoyl)-3-hydroxybenzoate, respectively, by spectroscopic methods (primarily nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry). The R absolute configuration at C-1 of rabenchromenone was determined by quantum chemical calculations and electronic circular dichroism experiments. All metabolites (1-4) were tested by leaf puncture on tomato and oak plants. All compounds were active in this assay by causing in both plants a necrosis diameter in the range of 0.2-0.7 cm. Specifically, rabenzophenone (2) was found to be the most phytotoxic compound in both plants.

Molecular and morphological characterization of three new species of Pythium from Iran: P. ershadii, P. pyrioosporum, and P. urmianum.

Three new species of Pythium: P. ershadii, P. pyrioosporum, and P. urmianum from soils of various regions in Iran are described and illustrated. These species are morphologically distinct from all other known species. Pythium ershadii is morphologically characterized by pyriform ornamented oogonia and rarely production of pyriform oospores. Pythium pyrioosporum differs from other species of the genus by the production of pyriform oospores and smooth walled oogonia, oospores with a tapering elongation toward a hypogynous antheridium and intercalary hypogynous antheridia. Pythium urmianum is distinguished by the presence of intercalary hypogynous antheridia, smooth walled oogonia formed laterally on hyphae or on short side branches and peanut-shaped oospores. Phylogenetic relationships of these new taxa with other Pythium species were investigated using internal transcribed spacers rDNA and partial coxI sequence data. The three species reside in clade E1 and are separated from closely related species.

Taxonomy and pathogenicity of fungi associated with oak decline in northern and central Zagros forests of Iran with emphasis on coelomycetous species.

Oak decline is a complex disorder that seriously threatens the survival of Zagros forests. In an extensive study on taxonomy and pathology of fungi associated with oak decline in the central and northern part of Zagros forests, 462 fungal isolates were obtained from oak trees showing canker, gummosis, dieback, defoliation, and partial or total death symptoms. Based on inter-simple sequence repeat (ISSR) fingerprinting patterns, morphological characteristics, and sequences of ribosomal DNA (28S rDNA and ITS) and protein coding loci (acl1, act1, caM, tef-1α, rpb1, rpb2, and tub2), 24 fungal species corresponding to 19 genera were characterized. Forty percent of the isolates were placed in eight coelomycetous species from seven genera, namely, Alloeutypa, Botryosphaeria, Cytospora, Didymella, Gnomoniopsis, Kalmusia, and Neoscytalidium. Of these, four species are new to science, which are introduced here as taxonomic novelties: Alloeutypa iranensis sp. nov., Cytospora hedjaroudei sp. nov., Cytospora zagrosensis sp. nov., and Gnomoniopsis quercicola sp. nov. According to pathogenicity trials on leaves and stems of 2-year-old Persian oak (Quercus brantii) seedlings, Alternaria spp. (A. alternata, A. atra, and A. contlous), Chaetomium globosum, and Parachaetomium perlucidum were recognized as nonpathogenic. All coelomycetous species were determined as pathogenic in both pathogenicity trials on leaves and seedling stems, of which Gnomoniopsis quercicola sp. nov., Botryosphaeria dothidea, and Neoscytalidium dimidiatum were recognized as the most virulent species followed by Biscogniauxia rosacearum.

Untargeted metabolomics and molecular docking studies on green silver nanoparticles synthesized by Sarocladium subulatum: Exploring antibacterial and antioxidant properties.

The biological synthesis of silver nanoparticles (Ag-NPs) with fungi has shown promising results in antibacterial and antioxidant properties. Fungi generate metabolites (both primary and secondary) and proteins, which aid in the formation of metal nanoparticles as reducing or capping agents. While several studies have been conducted on the biological production of Ag-NPs, the exact mechanisms still need to be clarified. In this study, Ag-NPs are synthesized greenly using an unstudied fungal strain, Sarocladium subulatum AS4D. Three silver salts were used to synthesize the Ag-NPs for the first time, optimized using a cell-free extract (CFE) strategy. Additionally, these NPs were assessed for their antimicrobial and antioxidant properties. Various spectroscopic and microscopy techniques were utilized to confirm Ag-NP formation and analyze their morphology, crystalline properties, functional groups, size, stability, and concentrations. Untargeted metabolomics and proteome disruption were employed to explore the synthesis mechanism. Computational tools were applied to predict metabolite toxicity and antibacterial activity. The study identified 40 fungal metabolites capable of reducing silver ions, with COOH and OH functional groups playing a pivotal role. The silver salt type impacted the NPs' size and stability, with sizes ranging from 40 to 52 nm and zeta potentials from -0.9 to -30.4 mV. Proteome disruption affected size and stability but not shape. Biosynthesized Ag-NPs using protein-free extracts ranged from 55 to 62 nm, and zeta potentials varied from -18 to -27 mV. Molecular docking studies and PASS results found no role for the metabolome in antibacterial activity. This suggests the antibacterial activity comes from Ag-NPs, not capping or reducing agents. Overall, the research affirmed the vital role of specific reducing metabolites in the biosynthesis of Ag-NPs, while proteins derived from biological extracts were found to solely affect their size and stability.

Phylogeny and taxonomy of Botryosphaeria and Neofusicoccum species in Iran, with description of Botryosphaeria scharifii sp. nov.

Species of Botryosphaeriaceae are important pathogens and endophytes associated with woody plants. Botryosphaeria and Neofusicoccum are two well known genera of the family. In this study 125 isolates morphologically resembling members of this family were collected from about 20 different fruit and forest trees in Iran. Based on morphology, MSP-PCR profile and DNA sequence data (ITS and tef1-a), four species were identified. Of these, Botryosphaeria dothidea, Neofusicoccum mediterraneum and N. parvum are known while Botryosphaeria scharifii is described here as new. N. mediterraneum is a new record for Iran and is reported here for the first time on mango trees. High diversity within Iranian population of N. parvum suggests the need to revise and reassess the morphological species description of N. parvum and closely related species.

Phytotoxins produced by Didymella glomerata and Truncatella angustata, associated with grapevine trunk diseases (GTDs) in Iran.

Didymella glomerata and Truncatella angustata associated with grapevine trunk diseases (GTDs) in Iran, were grown in vitro to evaluate the production of phytotoxic metabolites as potential pathogenicity determinants. 2,5-Dihydroxymethylfuran and (+)-6-hydroxyramulosin were isolated from the culture filtrates of D. glomerata and T. angustata, respectively. They were identified by physical and spectroscopic (essentially 1 D and 2 D 1H and 13C NMR and ESIMS) methods and X ray analysis. Both compounds induced significant necrosis and curling on the leaves of the host plant Vitis vinifera L. and the effects were concentration dependent. No effect was observed on the leaves of the non-host Solanum lycopersicum L.. plant.

Biscogniauxia rosacearum the charcoal canker agent as a pathogen associated with grapevine trunk diseases in Zagros region of Iran.

Grapevine trunk diseases (GTDs) are well-known and significant fungal diseases of Vitis vinifera with a worldwide distribution. During August to November 2016 in a survey to characterize fungi associated with grapevine trunk diseases in Kermanshah Province (west of Iran) vineyards, 286 fungal isolates were obtained. Based on morphology and DNA sequences data eight species were identified, of which Biscogniauxia rosacearum, Neoscytalidium hyalinum and Phaeoacremonium minimum were the most aggressive fungal pathogenic species characterized in this research. N. hyalinum was the most prevalent species. N. hyalinum and Ph. minimum have previously been reported from Vitis vinifera. Thus far, there are two records of Biscogniauxia mediterranea and Biscogniauxia capnodes on grapevine in the world with no data on pathology aspects. To our knowledge, it is the first time B. rosacearum is reported from grapevine across the globe. Pathogenicity test with three strains of B. rosacearum on 2-year-old potted grapevines confirmed the pathogenicity of B. rosacearum on grapevine. The proximity of vineyards to the oak trees in Zagros forests as one of the plant hosts of Biscogniauxia spp. further highlights the need for extensive studies on B. rosacearum as a new fungal pathogen.

Development of an efficient Tef-1α RNA hairpin structure to efficient management of Lasiodiplodia theobromae and Neofusicoccum parvum.

Lasiodiplodia theobromae and Neofusicoccum parvum are serious worldwide-distributed plant pathogenic fungi with a wide host range in tropical and temperate climates. They cause fruit rot, canker, and dieback of twigs in various woody plants. Protection of pruning wounds using fungicides is the prevalent strategy for the management of the diseases caused by these fungi. Chemical control of plant diseases is not environmentally safe and the residues of fungicides are a threat to nature. Furthermore, genetic resources of resistance to plant diseases in woody plants are limited. The aim of this study was to investigate the efficiency of RNA silencing using an efficient hairpin structure based on Tef-1α gene for the management of L. theobromae and N. parvum. Hairpin structure of Tef-1α was cloned in pFGC5941 binary vector and the recombinant construct was named pFGC-TEF-d. Transient expression of pFGC-TEF-d using Agrobacterium LBA4404 in grapevine (Bidaneh Sefid cv.) and strawberry cultivars (Camarosa and Ventana) led to a reduction in disease progress of L. theobromae. The disease reduction in grapevine was estimated by 55% and in strawberries cultivars Camarosa and Ventana by 58% and 93%, respectively. Further analysis of transient expression of pFGC-TEF-d in strawberry (Camarosa) shown disease reduction using Neofusicoccum parvum. Here we introduce RNAi silencing using pFGC-TEF-d construct as an efficient strategy to the management of L. theobromae and N. parvum for the first time.

Fungal canker agents in apple production hubs of Iran.

To identify apple canker casual agents and evaluate their pathogenicity and virulence in apple production hubs including West Azarbaijan, Isfahan and Tehran provinces; samples were collected from symptomatic apple trees. Pathogenic isolates on the detached branches were identified as Cytospora cincta, Diplodia bulgarica, Neoscytalidium dimidiatum and Eutypa cf. lata. E. cf. lata was reported as a potential apple canker causal agent in Iran for the first time based on the pathogenicity test on the detached branches, whereas it caused no canker symptoms in apple trees until 6 months after inoculation. Currently, E. cf. lata seems to be adapted to a single city. C. cincta, D. bulgarica and N. dimidiatum caused canker symptoms in apple trees. "C. cincta" and also "C. cincta and N. dimidiatum" were the most widespread and aggressive apple canker species, respectively, associated with apple canker in Iran. Therefore, they are considered to be the main threat to apple production in Iran and should be carefully monitored. Disease progress curve, area under the disease progress curve and optimum temperatures were determined for mentioned species. It is concluded that the establishment of each species occurs in appropriate areas and times in terms of the optimum temperature for their growth.

Phytotoxins Produced by Two Biscogniauxia rosacearum Strains, Causal Agents of Grapevine Trunk Diseases, and Charcoal Canker of Oak Trees in Iran.

Biscogniauxia rosacearum, recognized for the first time as a pathogen involved in grapevine trunk diseases in Paveh (west of Iran) vineyards, produced meso-2,3-butanediol (1) as the only phytotoxin. Nectriapyrone (2), (3R)-5-methylmellein (3), (3R)-5-methyl-6-methoxymellein (4), and tyrosol (5) were instead produced as phytotoxins from a strain of the same fungus isolated from oak trees in Zagros forests of Gilan-e Gharb, Kermanshah Province. They were identified comparing their 1H and 13C NMR, ESIMS, and specific optical rotation data with those already reported in the literature. The phytotoxicity of metabolites (1-5) was estimated by leaf puncture assay on Quercus ilex L. and Hedera helix L., and by leaf absorption assay on grapevine (Vitis vinifera L.) at a concentration of 5 × 10-3 and 10-3 M. Tested on grapevine, meso-2,3-butanediol (1) and (3R)-5-methyl-6-methoxymellein (4) resulted to be the most phytotoxic compounds. On Q. ilex, nectriapyrone (2) and tyrosol (5) showed severe necrosis at the highest concentration while none of the compounds (1-5) was active on H. helix. Furthermore, the phytotoxicity of compounds 3 and 4 was also compared with that of some related natural melleins to perform a structure-activity relationship (SAR) study. The results of this study were also discussed.

Phytotoxic metabolites from Stilbocrea macrostoma, a fungal pathogen of Quercus brantii in Iran.

Two phytotoxic furan derivatives were isolated, together with the well-known fungal and plant phytotoxin tyrosol, from the culture filtrates of Stilbocrea macrostoma. This fungal pathogen isolated from Quercus brantii trees induced wood necrosis and decline symptoms on the host plant in Iran. The two furan derivatives, isolated for the first time from Stilbocrea macrostoma, were identified by spectroscopic methods (essentially 1 D and 2 D 1H and 13C NMR and ESIMS spectroscopy) as 5-hydroxymethylfuraldehyde and 2,5-dihydroxymethylfuran. The phytotoxic activity of the three metabolites was evaluated by leaf puncture assay on holm oak (Quercus ilex L.) and tomato (Lycopersicon esculentum L.) leaves. All compounds induced necrosis on holm oak leaves while very low toxicity was showed against tomato leaves. The two furan derivatives were more toxic than tyrosol and particularly 5-hydroxymethylfuraldehyde was the most phytotoxic compound.

Phenazine-1-Carboxylic Acid (PCA), Produced for the First Time as an Antifungal Metabolite by Truncatella angustata, a Causal Agent of Grapevine Trunk Diseases (GTDs) in Iran.

The phytopathogenic fungus Truncatella angustata, associated with grapevine trunk diseases (GTDs) in Iran, produces the well-known secondary metabolite isocoumumarin (+)-6-hyroxyramulosin and surprisingly also phenazine-1-carboxylic acid (PCA). PCA, identified by spectroscopic (essentially 1H NMR and ESI MS) spectra, is a bacterial metabolite well known for its antifungal activity and was found for the first time in T. angustata culture filtrates. The antifungal activity of PCA was assayed against four different fungi responsible for GTDs, Phaeoacremonium minimum, Phaeoacremonium italicum, Fomitiporia mediterranea, involved in grapevine esca disease, and Neofusicoccum parvum, responsible for Botryosphaeria dieback. The activity was compared with that of the known commercial fungicide, pentachloronitrobenzene, and the close phenazine. PCA and phenazine exhibited strong antifungal activity against all phytopathogenic fungi, inhibiting the fungal growth by about 90-100% and 80-100%, respectively. These results suggested that T. angustata could use PCA to compete with other phytopathogenic fungi that attack grapevine and thus PCA could be proposed as a biofungicide against the fungi responsible for grapevine esca and Botryosphaeria dieback diseases.

Massarilactones D and H, phytotoxins produced by Kalmusia variispora, associated with grapevine trunk diseases (GTDs) in Iran.

A strain of Kalmusia variispora associated with grapevine trunk diseases (GTDs) was identified in Iran and induced disease symptoms on the host in greenhouse conditions. The grapevine pathogens are able to produce a plethora of toxic metabolites belonging to different classes of naturally occurring compounds. Two homogeneous compounds were isolated from the organic extract of K. variispora culture filtrates. They were identified by physic (specific optical rotation), and spectroscopic (essentially 1D 1H and 13C NMR and HR ESIMS) methods as the fungal polyketides massarilactones D and H (1 and 2). The unassigned absolute configuration of massarilactone D was unambiguously determined by X-ray diffractometric analysis. Massarilactones D and H showed phytotoxic activity on Vitis vinifera L. at two concentrations used and depending from the days of inoculation. Phytotoxicity is also increased when the 3,4,7-O,O',O"-triacetyl derivative of massarilactone D (3) was assayed on the host plant. This is the first report on the investigation of phytotoxic metabolites produced by K. variispora isolated from infected grapevine in Iran and they seem to be involved in the development of disease symptoms.

Families, genera, and species of Botryosphaeriales.

Members of Botryosphaeriales are ecologically diverse, but most commonly associated with leaf spots, fruit and root rots, die-back or cankers of diverse woody hosts. Based on morphology and DNA sequence data, the Botryosphaeriales have to date been shown to contain eight families, with an additional two, Endomelanconiopsisaceae (Endomelanconiopsis) and Pseudofusicoccumaceae (Pseudofusicoccum) being newly described in this study. Furthermore, Oblongocollomyces is introduced as new genus, while Spencermartinsia is reduced to synonymy under Dothiorella. Novel species include Diplodia pyri (Pyrus sp., the Netherlands), Diplodia citricarpa (Citrus sp., Iran), Lasiodiplodia vitis (Vitis vinifera, Italy), L. sterculiae (Sterculia oblonga, Germany), Neofusicoccum pistaciarum (Pistacia vera, USA), N. buxi (Buxus sempervirens, France), N. stellenboschiana (Vitis vinifera, South Africa), and Saccharata hawaiiensis (Protea laurifolia, Hawaii). New combinations are also proposed for Camarosporium pistaciae (associated with fruit rot of Pistacia vera) in Neofusicoccum, and Sphaeria gallae (associated with galls of Quercus) in Diplodia. The combination of large subunit of the nuclear ribosomal RNA gene (LSU)-rpb2 proved effective at delineating taxa at family and generic level. Furthermore, rpb2 also added additional resolution for species delimitation, in combination with ITS, tef1 and tub2. In this study we analysed 499 isolates, and produce an expanded phylogenetic backbone for Botryosphaeriales, which will help to delimit novelties at species, genus and family level in future.

Efficiency of rep-PCR fingerprinting as a useful technique for molecular typing of plant pathogenic fungal species: Botryosphaeriaceae species as a case study.

Progress in molecular biology and the advent of rapid and accurate molecular techniques have contributed to precise and rapid detection and differentiation of microbial pathogens. Identification of the Botryosphaeriaceae species based on morphology has been problematic over time. In this study, we used rep-PCR technique as a molecular tool for typing and differentiation of the Botryosphaeriaceae species, well-known and cosmopolitan fungal pathogens on woody plants. Three primer sets BOX, ERIC and REP were used to differentiate 27 species belong to eight genera. The majority of them were examined in terms of typing and differentiation using molecular methods for the first time. All the primer sets were able to generate species-specific DNA fingerprints from all the tested strains, with two exceptions in the genera Diplodia and Spencermartinsia. Despite the deficiency of each primer sets to separate a few species, cluster analysis of combined data sets indicated the ability of rep-PCR technique to separate 26 out of 27 examined species in highly supported clusters corresponded to the species recognized based on DNA sequence data. Our findings revealed the efficiency of rep-PCR for detection and differentiation of the Botryosphaeriaceae species, especially cryptic species with the same ITS sequences and similar morphology.