Potential biodegradation of polycyclic aromatic hydrocarbons (PAHs) and petroleum hydrocarbons by indigenous fungi recovered from crude oil-contaminated soil in Iran.

A total of 265 fungal individuals were isolated from soils exposed to heavy oil spills in the Yadavaran oil field in Iran to discover indigenous fungal species with a high potential to biodegrade petroleum hydrocarbon pollutants. Morphological and molecular identification of obtained fungal species led to their assignment into 16 genera and 25 species. Alternaria spp. (78%), Fusarium spp. (5%), and Cladosporium spp. (4%) were the most common genera, along with Penicillium spp., Neocamarosporium spp., Epicoccum sp., Kotlabaea sp., Aspergillus sp., Mortierella sp., and Pleurotus sp. A preliminary screening using the DCPIP indicator revealed that approximately 35% of isolates from Alternaria, Epicoccum, Neocamarosporium, Cladosporium, Fusarium, Stachybotrys, Penicillium, and Stemphylium demonstrated promising tolerance to crude oil. The best-performing isolates (12 fungal individuals) were further investigated for their capacity to mineralize a mixture of four polycyclic aromatic hydrocarbons (PAH) for 47 days, quantified by GC-MS. Eventually, two top-performing isolates, namely 5c-12 (Alternaria tenuissima) and 3b-1 (Epicoccum nigrum), were applied to petroleum-contaminated soil. The GC-MS analysis showed that 60 days after inoculation, these isolates successfully degraded more than 70% of the long-chain hydrocarbons in the soil, including C8-C16 n-alkanes, C36 n-alkane, and Pristane. This study introduces two fungal species (5c-12 and 3b-1) with high potential for biodegrading petroleum compounds and PAHs, offering promising prospects for the decontamination of oil-contaminated soil.

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.

Efficacy of Disinfectants Against Tropical Race 4 Causing Fusarium Wilt in Cavendish Bananas.

Fusarium wilt of banana (FWB), caused by a suite of Fusarium fungi, is among the most devastating plant diseases. The iconic FWB epidemic in the previous century lasted decades and was caused by so-called Race 1 strains that wiped out the dominant 'Gros Michel' banana plantations across Central America. Eventually, it was stopped because the Race 1-resistant 'Cavendish' banana variety replaced 'Gros Michel', which dominates global production (>50%) and trade (>95%). However, presently, the so-called Tropical Race 4 (TR4) threatens plantations of 'Cavendish' and many other banana varieties around the globe. Prevention is the first line of defense against the spread of TR4. Therefore, many disinfection units are installed to prevent the entry of TR4 in banana plantations. These foot and tire baths are filled with disinfectants, but limited knowledge is available on their efficacy. In this project, we evaluated 13 disinfectants commonly used in the Philippines. Our results show that the efficacy of these products depends on the type of fungal spores, the exposure time, and the replenishment frequency of the disinfection units. The resting spores of TR4 were resistant to all but one - unfortunately corrosive - disinfectant. Furthermore, we show that the actual contact time with disinfectants was far below the thresholds determined in laboratory experiments. Finally, muddy disinfection units reduced the efficacy of disinfectants. Taken together, we conclude that practices are inadequate to prevent the dissemination of TR4.

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.

MADS-Box Transcription Factor ZtRlm1 Is Responsible for Virulence and Development of the Fungal Wheat Pathogen Zymoseptoria tritici.

Zymoseptoria tritici is one of the most economically destructive wheat diseases all over the world and is a model fungal plant pathogen within the ascomycetes. In this study, the instrumental role of the ZtRlm1 gene encoding a MADS-box transcription factor (TF) in the infection process of Z. tritici was functionally characterized as these proteins play critical roles in the global gene regulation required for various developmental and physiological processes. Our infection assays showed that ZtRlm1 mutants were attenuated in disease development as a 30 and 90% reduction in chloro-necrotic lesions and pycnidia formation, respectively, were observed in plants inoculated with ZtRlm1 mutant strains demonstrating that ZtRlm1 is a crucial factor playing a significant role in the late stage of infection corresponding with pycnidial formation. Our expression analysis demonstrated that the transcript level of ZtRlm1 is induced at 2 and 20 days post-inoculation, coinciding with pycnidial sporulation. In addition, microscopic analyses showed that branch intensity and biomass production were significantly reduced, indicating that impaired pycnidia formation is a result of impaired differentiation and biomass production in the ZtRlm1 mutants. Furthermore, melanization, a phenomenon required for fruiting body formation, was significantly hampered in ZtRlm1 mutants as they were not melanized under all tested temperature and media conditions. Overall, our data showed that impaired disease development of the ZtRlm1 mutants is mainly due to the significant impact of ZtRlm1 in different cellular processes, including differentiation, branching, fungal biomass production, and melanization, in which identification of downstream genes are of interest to increase our understanding of this pathosystem.

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.

Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance.

Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici-wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.

The ZtVf1 transcription factor regulates development and virulence in the foliar wheat pathogen Zymoseptoria tritici.

The dimorphic fungal pathogen, Zymoseptoria tritici undergoes discrete developmental changes to complete its life cycle on wheat. Molecular mechanisms underlying morphogenesis during infection process of Z. tritici are poorly understood. In this study, we have investigated the role of ZtVf1 gene encoding a transcription factor belonging to C2-H2 subfamily. In planta assays revealed that ZtVf1 is required for virulence. Reduced necrotic lesions and low pycnidia density within the lesions resulted in significantly reduced virulence of ZtVf1 mutants. Cytological analysis showed that the impaired virulence of ZtVf1 mutants attributed to reduced penetration and colonization along with hampered pycnidia differentiation. In vitro phenotyping showed that ZtVf1 deletion affects hyphal branching and biomass production suggesting that the reduced tissue colonization by the ZtVf1 mutant might be due to lower hyphal branching and less fungal biomass production. In addition, the majority of infected substomatal cavities by the ZtVf1 mutant filled with compacted mycelia mat that did not differentiate to mature pycnidia indicating that the impaired melanization negatively affected pycnidia formation and maturation. The ZtVf1 might target multiple genes belonging to different cellular processes whose identification is of eminent interest to increase our understanding of this pathosystem. Overall, the data provided in this study indicates that attenuated pathogenicity of ZtVf1 mutant is due to involvement of this gene in the regulation of both early and late stages of infection.

Karyotype Variability in Plant-Pathogenic Fungi.

Recent advances in genetic and molecular technologies gradually paved the way for the transition from traditional fungal karyotyping to more comprehensive chromosome biology studies. Extensive chromosomal polymorphisms largely resulting from chromosomal rearrangements (CRs) are widely documented in fungal genomes. These extraordinary CRs in fungi generate substantial genome plasticity compared to other eukaryotic organisms. Here, we review the most recent findings on fungal CRs and their underlying mechanisms and discuss the functional consequences of CRs for adaptation, fungal evolution, host range, and pathogenicity of fungal plant pathogens in the context of chromosome biology. In addition to a complement of permanent chromosomes called core chromosomes, the genomes of many fungal pathogens comprise distinct unstable chromosomes called dispensable chromosomes (DCs) that also contribute to chromosome polymorphisms. Compared to the core chromosomes, the structural features of DCs usually differ for gene density, GC content, housekeeping genes, and recombination frequency. Despite their dispensability for normal growth and development, DCs have important biological roles with respect to pathogenicity in some fungi but not in others. Therefore, their evolutionary origin is also reviewed in relation to overall fungal physiology and pathogenicity.

Proteome catalog of Zymoseptoria tritici captured during pathogenesis in wheat.

Zymoseptoria tritici is an economically important pathogen of wheat. However, the molecular basis of pathogenicity on wheat is still poorly understood. Here, we present a global survey of the proteins secreted by this fungus in the apoplast of resistant (cv. Shafir) and susceptible (cv. Obelisk) wheat cultivars after inoculation with reference Z. tritici strain IPO323. The fungal proteins present in apoplastic fluids were analyzed by gel electrophoresis and by data-independent acquisition liquid chromatography/mass spectrometry (LC/MS(E)) combined with data-dependent acquisition LC-MS/MS. Subsequent mapping mass spectrometry-derived peptide sequence data against the genome sequence of strain IPO323 identified 665 peptides in the MS(E) and 93 in the LC-MS/MS mode that matched to 85 proteins. The identified fungal proteins, including cell-wall degrading enzymes and proteases, might function in pathogenicity, but the functions of many remain unknown. Most fungal proteins accumulated in cv. Obelisk at the onset of necrotrophy. This inventory provides an excellent basis for future detailed studies on the role of these genes and their encoded proteins during pathogenesis in wheat.

FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici.

Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars.

Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi.

Genetic manipulation of fungi requires quick, low-cost, efficient, high-throughput and molecular tools. In this paper, we report 22 entry constructs as new molecular tools based on the Gateway technology facilitating rapid construction of binary vectors that can be used for functional analysis of genes in fungi. The entry vectors for single, double or triple gene-deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, entry vectors containing green fluorescent (GFP) or red fluorescent (RFP) in combination with hygromycin, geneticin or nourseothricin selection markers were generated. The latter vectors provide the possibility of gene deletion and simultaneous labelling of the fungal transformants with GFP or RFP reporter genes. The applicability of a number of entry vectors was validated in Zymoseptoria tritici, an important fungal wheat pathogen.

Effector discovery in the fungal wheat pathogen Zymoseptoria tritici.

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem.

Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici.

Zymoseptoria tritici causes the major fungal wheat disease septoria tritici blotch, and is increasingly being used as a model for transmission and population genetics, as well as host-pathogen interactions. Here, we study the biological function of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans, as a representative of a superfamily of regulatory proteins involved in dimorphic switching. In Z. tritici, this gene is pivotal for pathogenesis, as ZtWor1 mutants were nonpathogenic and complementation restored the wild-type phenotypes. In planta expression analyses showed that ZtWor1 is up-regulated during the initiation of colonization and fructification, and regulates candidate effector genes, including one that was discovered after comparative proteome analysis of the Z. tritici wild-type strain and the ZtWor1 mutant, which was particularly expressed in planta. Cell fusion and anastomosis occur frequently in ZtWor1 mutants, reminiscent of mutants of MgGpb1, the ß-subunit of the heterotrimeric G protein. Comparative expression of ZtWor1 in knock-out strains of MgGpb1 and MgTpk2, the catalytic subunit of protein kinase A, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenesis in many fungal plant pathogens.