A reference transcriptome for walnut anthracnose pathogen, Ophiognomonia leptostyla, guides the discovery of candidate virulence genes.

Despite the economic losses due to the walnut anthracnose, Ophiognomonia leptostyla is an orphan fungus with respect to genomic resources. In the present study, the transcriptome of O. leptostyla was assembled for the first time. RNA sequencing was conducted for the fungal mycelia grown in a liquid media, and the inoculated leaf samples of walnut with the fungal conidia sampled at 48, 96 and 144 h post inoculation (hpi). The completeness, correctness, and contiguity of the de novo transcriptome assemblies generated with Trinity, Oases, SOAPdenovo-Trans and Bridger were compared to identify a single superior reference assembly. In most of the assessment criteria including N50, Transrate score, number of ORFs with known description in gene bank, the percentage of reads mapped back to the transcript (RMBT), BUSCO score, Swiss-Prot coverage bin and RESM-EVAL score, the Bridger assembly was the superior and thus used as a reference for profiling the O. leptostyla transcriptome in liquid media vs. during walnut infection. The k-means clustering of transcripts resulted in four distinct transcription patterns across the three sampling time points. Most of the detected CAZy transcripts had elevated transcription at 96 hpi that is hypothetically concurrent with the start of intracellular growth. The in-silico analysis revealed 103 candidate effectors of which six were members of Necrosis and Ethylene Inducing Like Protein (NLP) gene family belonging to three distinct k-means clusters. This study provided a complex and temporal pattern of the CAZys and candidate effectors transcription during six days post O. leptostyla inoculation on walnut leaves, introducing a list of candidate virulence genes for validation in future studies.

In vitro antifungal susceptibility profile of Iranian Fusarium isolates: Emphasising on the potent inhibitory effect of efinaconazole compared to other drugs.

BACKGROUND: Fusarium species are opportunistic human pathogens that remarkably cause fungal infections ranging from superficial to fatal invasive disseminated infections. Fusarium species are notoriously resistant to the majority of antifungal agents. OBJECTIVES: Therefore, detailed studies regarding in vitro susceptibility are required and may lead to a better prognosis of severe infections. METHODS: We evaluated 25 antifungal drugs in vitro against 282 clinical and environmental Fusarium isolates. RESULTS: Fusarium species demonstrated high MICs/MECs values to the most commonly used antifungal drugs in clinical practice. The geometric mean (GM) MICs for luliconazole (0.004 µg/ml) and lanoconazole (0.012 µg/ml) were the lowest, followed by efinaconazole (0.98 µg/ml) and amphotericin B (1.04 µg/ml). CONCLUSIONS: Efinaconazole, a novel triazole, may be a promising candidate for the treatment of superficial Fusarium infections. Furthermore, the development of systemic formulations of these drugs as well as further in vitro and in vivo investigations could aid in the treatment of systemic fusariosis.

Stb6 mediates stomatal immunity, photosynthetic functionality, and the antioxidant system during the Zymoseptoria tritici-wheat interaction.

This study offers new perspectives on the biochemical and physiological changes that occur in wheat following a gene-for-gene interaction with the fungal pathogen Zymoseptoria tritici. The Z. tritici isolate IPO323, carries AvrStb6, while ΔAvrStb6#33, lacks AvrStb6. The wheat cultivar (cv.) Shafir, bears the corresponding resistance gene Stb6. Inoculation of cv. Shafir with these isolates results in two contrasted phenotypes, offering a unique opportunity to study the immune response caused by the recognition of AvrStb6 by Stb6. We employed a variety of methodologies to dissect the physiological and biochemical events altered in cv. Shafir, as a result of the AvrStb6-Stb6 interaction. Comparative analysis of stomatal conductance demonstrated that AvrStb6-Stb6 mediates transient stomatal closures to restrict the penetration of Zymoseptoria tritici. Tracking photosynthetic functionality through chlorophyll fluorescence imaging analysis demonstrated that AvrStb6-Stb6 retains the functionality of photosynthesis apparatus by promoting Non-Photochemical Quenching (NPQ). Furthermore, the PlantCV image analysis tool was used to compare the H2O2 accumulation and incidence of cell death (2, 4, 8, 12, 16, and 21 dpi), over Z. tritici infection. Finally, our research shows that the AvrStb6-Stb6 interaction coordinates the expression and activity of antioxidant enzymes, both enzymatic and non-enzymatic, to counteract oxidative stress. In conclusion, the Stb6-AvrStb6 interaction in the Z. tritici-wheat pathosystem triggers transient stomatal closure and maintains photosynthesis while regulating oxidative stress.

In Vitro Antifungal Susceptibility Profile of Miltefosine against a Collection of Azole and Echinocandins Resistant Fusarium Strains.

Fusarium species are filamentous fungi that cause a variety of infections in humans. Because they are commonly resistant to many antifungal drugs currently available in clinical settings, research into alternative targets in fungal cells and therapeutic approaches is required. The antifungal activity of miltefosine and four comparators, amphotericin B, voriconazole, itraconazole, and caspofungin, were tested in vitro against a collection of susceptible and resistant clinical (n = 68) and environmental (n = 42) Fusarium isolates. Amphotericin B (0.8 µg/mL) had the lowest geometric mean (GM) MICs/MECs values followed by miltefosine (1.44 µg/mL), voriconazole (2.15 µg/mL), caspofungin (7.23 µg/mL), and itraconazole (14.19 µg/mL). Miltefosine was the most effective agent against Fusarium isolates after amphotericin B indicating that miltefosine has the potential to be studied as a novel treatment for Fusarium infections.

Key criteria for engineering mycotoxin binding aptamers via computational simulations: Aflatoxin B1 as a case study.

Due to the difficulties in monoclonal antibody production specific to mycotoxins, aptameric probes have been considered as suitable alternatives. The low efficiency of the SELEX procedure in screening high affinity aptamers for binding mycotoxins as small molecules can be significantly improved through computational techniques. Previously, we designed five new aptamers to aflatoxin B1 (AFB1) based on a known aptamer sequence (Patent: PCT/CA2010/001 292, Apt1) through a genetic algorithm-based in silico maturation strategy and experimentally measured their affinity to the target toxin. Here, integrated molecular dynamic simulation (MDs) studies with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis to clarify the binding modes, critical interacting nucleic bases and energy component contributions in the six AFB1-binding aptamers. The aptamer F20, which was selected in the first work, showed the best free binding energy and complex stability compared to other aptamers. The trajectory analysis revealed that AFB1 recognized F20 through the groove binding mode along with precise shape complementarity. The MD simulation results revealed that dynamic water intermediate interactions also play a key role in promoting complex stability. According to the MM-PBSA calculations, van der Waals contacts were identified as dominant energy components in all complexes. Interestingly, a high consistency is observed between the experimentally obtained binding affinities of the six aptamers with their free energy solvation. The computational findings, confirmed via previous experiments, highlighted the binding modes, the dynamic hydration of complex components and the total free interacting energy as the crucial criteria in discovering high functional aptameric probes.

Genetic structure of Fusarium verticillioides populations from maize in Iran.

Fusarium verticillioides is one of the most important fungal pathogens of maize since it causes severe yield losses and produces the mycotoxins fumonisins that represent a major concern for human and animal health. Information about genetic diversity and population structure of fungal pathogens is essential for developing disease management strategies. The aim of this research was to investigate the genetic structure of F. verticillioides isolated from different provinces of Iran through determination of mating type idiomorphs, phylogenetic analyses based on translation elongation factor-1 alpha (EF-1α), RNA Polymerase II Subunit (RPB2), beta-tubulin (tub2) and Calmodulin (cmdA) genes and genetic diversity analyses based on 6 simple-sequence repeats (SSRs). Both mating types were detected in Iranian populations of F. verticillioides, particularly in Qazvin and Khuzestan, with equal frequency, which highlighted that sexual reproduction is favorable under field conditions. However, the linkage disequilibrium indices did not support the hypothesis of random mating in Khuzestan and Fars. Although assessment of nucleotide diversity based on housekeeping genes showed low level of variation among strains, genotype diversity based on SSRs revealed a high level of genetic diversity within Iranian populations. AMOVA analysis highlighted that the genetic variation of F. verticillioides in Iran was mainly distributed within population of a single area (97%), while a small proportion of genetic variation (3%) resided among populations. These patterns of variation are likely explained by the continuous gene flow among populations isolated from different areas. On the other hand, principal coordinate analysis indicated that the distribution of genetic variation among populations could be explained by the geographical distances. Consequently, to reduce pathogen gene flow among regions, the quarantine processes in Iran should be intensified.

Tracing the Origin and Evolutionary History of Pyricularia oryzae Infecting Maize and Barnyard Grass.

Blast disease is a notorious fungal disease leading to dramatic yield losses on major food crops such as rice and wheat. The causal agent, Pyricularia oryzae, encompasses different lineages, each having a different host range. Host shifts are suspected to have occurred in this species from Setaria spp. to rice and from Lolium spp. to wheat. The emergence of blast disease on maize in Iran was observed for the first time in the north of the country in 2012. We later identified blast disease in two additional regions of Iran: Gilan in 2013 and Golestan in 2016. Epidemics on the weed barnyard grass (Echinochloa spp.) were also observed in the same maize fields. Here, we showed that P. oryzae is the causal agent of this disease on both hosts. Pathogenicity assays in the greenhouse revealed that strains from maize can infect barnyard grass and conversely. However, genotyping with simple sequence repeat markers and comparative genomics showed that strains causing field epidemics on maize and on barnyard grass are different, although they belong to the same previously undescribed clade of P. oryzae. Phylogenetic analyses including these strains and a maize strain collected in Gabon in 1985 revealed two independent host-range expansion events from barnyard grass to maize. Comparative genomics between maize and barnyard grass strains revealed the presence or absence of five candidate genes associated with host specificity on maize, with the deletion of a small genomic region possibly responsible for adaptation to maize. This recent emergence of P. oryzae on maize provides a case study to understand host range expansion. Epidemics on maize raise concerns about potential yield losses on this crop in Iran and potential geographic expansion of the disease.

Strain improvement of Trichoderma spp. through two-step protoplast fusion for cellulase production enhancement.

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates were screened for Avicel and carboxymethyl cellulose degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of the resulting hybrids with improved cellulase activity, C1-3, was fused with the hyperproducer Trichoderma reesei Rut-C30. A new selected hybrid, F7, was increased in cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of the cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. The amount of mRNA of cbh1 and cbh2 in F7 resembled that of Rut-C30 while the bgl1 mRNA level was similar to that of C1-3. AFLP (amplified fragment length polymorphism) fingerprinting and GC-MS (gas chromatography - mass spectrometry) analysis represented variations in parental strains and fusants. In conclusion, the results demonstrate that a 3-interspecific hybrid strain was isolated, with improved characteristics for cellulase degradation and showing genetic polymorphisms and differences in the volatile profile, suggesting reorganizations at the genetic level.

In silico maturation of affinity and selectivity of DNA aptamers against aflatoxin B1 for biosensor development.

A high affinity and selectivity DNA aptamer for aflatoxin B1 (AFB1) was designed through Genetic Algorithm (GA) based in silico maturation (ISM) strategy. The sequence of a known AFB1 aptamer (Patent: PCT/CA2010/001292, Apt1) applied as a probe in many aptasensors was modified using seven GA rounds to generate an initial library and three different generations of ss DNA oligonucleotides as new candidate aptamers. Molecular docking methodology was used to screen and analyze the best aptamer-AFB1 complexes. Also, a new pipeline was proposed to faithfully predict the tertiary structure of all single stranded DNA sequences. By the second generation, aptamer Apt1 sequence was optimized in the local search space and five aptamers including F20, g12, C52, C32 and H1 were identified as the best aptamers for AFB1. The selected aptamers were applied as probes in an unmodified gold nanoparticles-based aptasensor to evaluate their binding affinity to AFB1 and their selectivity against other mycotoxins (aflatoxins B2, G1, G2, M1, ochratoxin A and zearalenone). In addition, a novel direct fluorescent anisotropy aptamer assay was developed to confirm the binding interaction of the selected aptamers over AFB1. The ISM allowed the identification of an aptamer, F20, with up to 9.4 and 2 fold improvement in affinity and selectivity compared to the parent aptamer, respectively.

Isolation, Molecular Identification and Mycotoxin Profile of Fusarium Species Isolated from Maize Kernels in Iran.

Fusarium species are among the most important fungal pathogens of maize, where they cause severe reduction of yield and accumulation of a wide range of harmful mycotoxins in the kernels. In order to identify the Fusarium species and their mycotoxin profiles associated to maize ear rot and kernel contamination in Iran, a wide sampling was carried out from field in ten major maize-producing provinces in Iran, during 2015 and 2016. From 182 samples of maize kernels, 551 strains were isolated and identified as belonging to Fusarium genus. Among the 234 representative strains identified at species level by translation elongation factor (EF-1α) sequences, the main Fusarium species were F. verticillioides and F. proliferatum, together representing 90% of the Iranian Fusarium population, and, to a lesser extent, F. incarnatum equiseti species complex (FIESC), F. thapsinum and F. redolens. Fumonisin (FBs) production by F. verticillioides and F. proliferatum representative strains was analysed, showing that all strains produced FB1. None of F. verticillioides strains produced FB2 nor FB3, while both FB2 and FB3 were produced only by F. proliferatum. Total mean of FBs production by F. verticillioides was higher than F. proliferatum. The occurrence of different Fusarium species on Iranian maize is reason of great concern because of the toxigenic risk associated to these species. Moreover, the diversity of the species identified increases the toxigenic risk associated to Fusarium contaminated maize kernels, because of the high possibility that a multi-toxin contamination can occur with harmful consequences on human and animal health.

Correction: Population Genetic Structure of Apple Scab (Venturia inaequalis (Cooke) G. Winter) in Iran.

[This corrects the article DOI: 10.1371/journal.pone.0160737.].

Population Genetic Structure of Apple Scab (Venturia inaequalis (Cooke) G. Winter) in Iran.

The population genetic structure of 278 Venturia inaequalis isolates, collected from different apple cultivars of eighteen different provinces in Iran, was investigated using 22 polymorphic microsatellite markers. Analysis of molecular variation, Bayesian clustering and Nei's genetic distance analyses based on 88 microsatellite alleles indicated substantial levels of gene flow among the collection sites. Ninety three percent of the variation was observed among the individuals within the populations and only 7% variation was observed among the populations. Structure analysis grouped the isolates into two populations. Maximum number of pathogen genotypes (44) was observed in the North of Iran that grows various different apple cultivars. Investigation on the variation of the pathogen on different cultivars in the North of Iran suggested a significant differentiation of the pathogen populations between wild apple and commercial cultivars. During sampling, varying ranges of scab infection were observed on various apple cultivars in forests, monoculture and mix orchards. Wild type apple (Malus orientalis) along the Caspian Sea Coast had the most infection in comparison with the Iranian endemic and commercial cultivars. Based on the genetic analysis and host tracking scenario of the pathogen, it was presumed that Iran could potentially be the center of origin of V. inaequalis, which requires further detailed studies with isolates collected from different parts of central Asia and world for confirmation.

Effects of dog rose and watercress extracts on control of green mould decay and postharvest quality of orange fruits.

Minimum inhibitory concentration (MIC) and IC50 values and total phenolics of dog rose fruits were 72.5-80 µL mL(- 1), 130 µg mL(- 1) and 5.7 mg GA g(- 1), respectively. The Fashand watercress population and dog rose extracts exhibited mycelia growth inhibition values of 45.08% and 37.12%, respectively. The results of in vivo study indicated that the treatment of inoculated fruits with both methanol extracts especially the watercress plant extract considerably controls the citrus fruits decay (Penicillium digitatum) up to 73%. In conclusion, methanol extracts of dog rose fruits and especially watercress plant had the potential to be used against citrus green mould and even for the improvement of postharvest quality of orange fruits.

Antifungal activity of some essential oils against toxigenic Aspergillus species.

Increasing attentions have been paid on the application of essential oils and plant extracts for control of postharvest pathogens due to their natural origin and less appearance of resistance in fungi pathogens. Some Aspergillus species are toxigenic and responsible for many cases of food and feed contamination. Some Toxins that produce with some Aspergillus species are known to be potent hepatocarcinogens in animals and humans. The present work evaluated the parameters of antifungal activity of the essential oils of Zataria multiflora, Thymus migricus, Satureja hortensis, Foeniculum vulgare, Carum capticum and thiabendazol fungicide on survival and growth of different species of Aspergillus. Aerial part and seeds of plant species were collected then dried and its essential oils isolated by means of hydrodistillation. Antifungal activity was evaluated in vitro by poisonous medium technique with PDA medium at six concentrations. Results showed that all essential oils could inhibit the growth of Aspergillus species. The essential oil with the best effect and lowest EC50 and MIC (Minimum Inhibitory Concentration) was Z. multiflora (223 microl/l and 650 microl/l, respectively). The chemical composition of the Z. multiflora essential oil was analyzed by GC-MS.

Survey of Thymus migricus essential oil on aflatoxin inhibition in Aspergillus flavus.

Essential oil components as result of non host disease resistance of plants have high capability to introduce as alternative of chemical pesticides. Thymus migricus essential oil was selected to investigation of its antifungal activity on survival and growth of Aspergillus flavus. For obtain essential oil first Leaves and flowers of Th. migricus collected then dried. The Essential oil was extracted by means of hydro-distillation and afterwards GC-MS analysis was performed to identify their components. The main constituents that resulted were Thymol (44.9%), Geraniol (10.8%), gamma-Terpinene (10.3%), Citronellol (8.5%) and p-Cymene (7.2%). EC50 and MIC (Minimum Inhibitory Concentration) of Th. migricus oil against A. flavus was 324.42 microl/l and 451.62 microl/l, respectively. Whereas EC50 and MIC for chemical thiabendazol was 650 microl/l and 1635 microl/l, respectively. The EC50 and MIC concentrations of Th. migricus oil in antifungal activity examination were used in aflatoxin inhibition test. Result of HPTLC measurement showed that both of concentrations inhibit aflatoxin production completely compares to control with 7.63 ppm aflatoxin production. In other word, Th. migricus oil can suppress aflatoxin production in concentrations lower than EC50 for mycelium growth.

Differentiation of Magnaporthe species complex by rep-PCR genomic fingerprinting.

Rice blast disease, caused by the fungus Magnoporthe grisea is responsible for considerable damages on rice and leaf spot on some weeds in Iran and in other parts of the world. Infected samples were collected from rice and weeds including Digitaria sanguinalis (crabgrass), Setaria italica (foxtail millet), Echinochloa crus-galli (barnyard millet), and some unknown weeds during 1997-2005 and were preserved in collection of Mycology at the University of Tehran, Iran. In this study, genetic diversity of Magnaporthe grisea species complex isolates was studied based on DNA fingerprinting by rep-PCR, using of two primers including ERIC and BOX. The total DNA of 75 isolates was extracted and DNA fragments were amplified in a thermal cycler program using mentioned primers. Therefore, DNA fragments from 400 bp to 3000 bp were amplified. Based on cluster analysis for two primers (ERIC and BOX), eight fingerprinting groups (ctonal lineages) and sixty haplotypes were identified. "A" clonal lineage was containing the highest number of isolates and became dominant clonal lineages with 35 isolates from rice and 3 isolates from S. italica, whereas the highest number of isolates obtained from D. sanguinalis belonged to "E" clonal lineage and was the second largest clonal lineage. Approximately all of the M. grisea species complex isolates from crabgrass and some of unknown weeds were separated from other isolates in 42% similarity. As a result, asexual fertility causes low diversity in populations of M. grisea species complex and speciation could be one of the reasons of differentiation between isolates from D. sanguinalis with other isolates. Overall, these data indicated a low level of genetic diversity in the Iranian M. grisea species complex population similar to that reported in other countries.

Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation.

The Fertile Crescent represents the center of origin and earliest known place of domestication for many cereal crops. During the transition from wild grasses to domesticated cereals, many host-specialized pathogen species are thought to have emerged. A sister population of the wheat-adapted pathogen Mycosphaerella graminicola was identified on wild grasses collected in northwest Iran. Isolates of this wild grass pathogen from 5 locations in Iran were compared with 123 M. graminicola isolates from the Middle East, Europe, and North America. DNA sequencing revealed a close phylogenetic relationship between the pathogen populations. To reconstruct the evolutionary history of M. graminicola, we sequenced 6 nuclear loci encompassing 464 polymorphic sites. Coalescence analyses indicated a relatively recent origin of M. graminicola, coinciding with the known domestication of wheat in the Fertile Crescent around 8,000-9,000 BC. The sympatric divergence of populations was accompanied by strong genetic differentiation. At the present time, no genetic exchange occurs between pathogen populations on wheat and wild grasses although we found evidence that gene flow may have occurred since genetic differentiation of the populations.

Detection of phlD gene in some fluorescent pseudomonads isolated from Iran and its relative with antifungal activities.

Fluorescent pseudomonads that produce antibiotic 2,4-diacetylphloroglocinol (2,4-DAPG) are important group of PGRP that inhibit a broad spectrum of plant pathogenic fungi. Studying on genetic diversity of 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads has been shown with special importance. The first step to investigate the genetic diversity of these bacteria is detecting of the genes required for the biosynthesis of this antibiotic. The objectives of the current study were detection of phlD gene within fluorescent pseudomonads by a PCR-based assay, and comparison of phenotypic and genotypic characteristics of fluorescent pseudomonads with proven biocontrol potential against some soil-borne phytopathogenic fungi. We used a collection of 47 fluorescent Pseudomonas spp. some with known biological control activity against Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var. parasitica, Pythium sp. and Fusarium sp. in vitro and the potential to produce known secondary metabolites such as, siderophore, HCN and protease. The results indicated that 66, 40.42, 63.82,48.94 and 27.65% of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively. Rhizoctonia solani recognized as the most vulnerable fungus. Among 47 strains, 76.59, 97.87 and 17% of strains produced protease, siderophore and HCN, respectively. We could detect phlD gene in strains P-5, P-32, P-47. Strain CHA0 was used as positive control for the detection this gene. Overall, there was no obvious link between the existence of phlD gene and inhibition of fungal growth or production of the antifungal metabolites in vitro. But in some strains such as CHA0 and P-5, we saw a link between the existence of phlD and antifungal activities. Studying on detection and diversity of phlD provides a fundamental knowledge for developing a rapid genetic screening system to identify a potential biocontrol strains.