Recent reactivation of a pathogenicity-associated transposable element is associated with major chromosomal rearrangements in a fungal wheat pathogen.

Transposable elements (TEs) are key drivers of genomic variation contributing to recent adaptation in most species. Yet, the evolutionary origins and insertion dynamics within species remain poorly understood. We recapitulate the spread of the pathogenicity-associated Styx element across five species that last diverged ∼11 000 years ago. We show that the element likely originated in the Zymoseptoria fungal pathogen genus and underwent multiple independent reactivation events. Using a global 900-genome panel of the wheat pathogen Zymoseptoria tritici, we assess Styx copy number variation and identify renewed transposition activity in Oceania and South America. We show that the element can mobilize to create additional Styx copies in a four-generation pedigree. Importantly, we find that new copies of the element are not affected by genomic defenses suggesting minimal control against the element. Styx copies are preferentially located in recombination breakpoints and likely triggered multiple types of large chromosomal rearrangements. Taken together, we establish the origin, diversification and reactivation of a highly active TE with likely major consequences for chromosomal integrity and the expression of disease.

The expression landscape and pangenome of long non-coding RNA in the fungal wheat pathogen Zymoseptoria tritici.

Long non-coding RNAs (lncRNAs) are regulatory molecules interacting in a wide array of biological processes. lncRNAs in fungal pathogens can be responsive to stress and play roles in regulating growth and nutrient acquisition. Recent evidence suggests that lncRNAs may also play roles in virulence, such as regulating pathogenicity-associated enzymes and on-host reproductive cycles. Despite the importance of lncRNAs, only a few model fungi have well-documented inventories of lncRNA. In this study, we apply a recent computational pipeline to predict high-confidence lncRNA candidates in Zymoseptoria tritici, an important global pathogen of wheat impacting global food production. We analyse genomic features of lncRNAs and the most likely associated processes through analyses of expression over a host infection cycle. We find that lncRNAs are frequently expressed during early infection, before the switch to necrotrophic growth. They are mostly located in facultative heterochromatic regions, which are known to contain many genes associated with pathogenicity. Furthermore, we find that lncRNAs are frequently co-expressed with genes that may be involved in responding to host defence signals, such as oxidative stress. Finally, we assess pangenome features of lncRNAs using four additional reference-quality genomes. We find evidence that the repertoire of expressed lncRNAs varies substantially between individuals, even though lncRNA loci tend to be shared at the genomic level. Overall, this study provides a repertoire and putative functions of lncRNAs in Z. tritici enabling future molecular genetics and functional analyses in an important pathogen.

Genome-wide association mapping reveals genes underlying population-level metabolome diversity in a fungal crop pathogen.

BACKGROUND: Fungi produce a wide range of specialized metabolites (SMs) involved in biotic interactions. Pathways for the production of SMs are often encoded in clusters of tightly arranged genes identified as biosynthetic gene clusters. Such gene clusters can undergo horizontal gene transfers between species and rapid evolutionary change within species. The acquisition, rearrangement, and deletion of gene clusters can generate significant metabolome diversity. However, the genetic basis underlying variation in SM production remains poorly understood. RESULTS: Here, we analyzed the metabolite production of a large population of the fungal pathogen of wheat, Zymoseptoria tritici. The pathogen causes major yield losses and shows variation in gene clusters. We performed untargeted ultra-high performance liquid chromatography-high resolution mass spectrometry to profile the metabolite diversity among 102 isolates of the same species. We found substantial variation in the abundance of the detected metabolites among isolates. Integrating whole-genome sequencing data, we performed metabolite genome-wide association mapping to identify loci underlying variation in metabolite production (i.e., metabolite-GWAS). We found that significantly associated SNPs reside mostly in coding and gene regulatory regions. Associated genes encode mainly transport and catalytic activities. The metabolite-GWAS identified also a polymorphism in the 3'UTR region of a virulence gene related to metabolite production and showing expression variation. CONCLUSIONS: Taken together, our study provides a significant resource to unravel polymorphism underlying metabolome diversity within a species. Integrating metabolome screens should be feasible for a range of different plant pathogens and help prioritize molecular studies.

Streptomyces spp. Isolated from Marine and Caatinga Biomes in Brazil for the Biological Control of Duponchelia fovealis.

Actinobacteria have been drawing attention due to their potential for the development of new pest control products. We hereby assess the effects of Streptomyces isolated from marine and caatinga biomes against Duponchelia fovealis Zeller (Lepidoptera: Crambidae), a pest associated with the strawberry culture at a global scale. To this end, eggs deposited by adults were immersed for 5 s in a bacterial suspension, and the larvae were fed on leaflets placed in glass tubes containing bacterial suspensions. In both treatments, the control was a saline solution. The bioassays demonstrated that the Streptomyces strains were able to cause the death of D. fovealis eggs (≈ 40%) and larvae (≈ 65%) compared to untreated eggs (1.4%) and larvae (2.0%). The crude extract of strain T49 and the chitinase extract of strain T26 affected larval growth when applied directly to the thorax of first-instar larvae (larval-adult lifespan of 65.3 ± 0.5 days and 67.5 ± 0.7 days, respectively; survival of 61.2 ± 1.2%) in relation to the control treatment (larval-adult lifespan of 41.75 ± 0.2 days and survival of 83.7 ± 2.6%). The Streptomyces spp. strains T41, T49, and T50 caused antifeeding activity. Apart from larval mortality, the adults that emerged from the larvae exposed to the extracts presented morphological abnormalities, and the moths' chitin spectra showed clear alterations to the pupa and wings. Our studies show, for the very first time, that Streptomyces isolated from the marine environment and the Caatinga biome are effective at provoking the mortality of D. fovealis and are promising agents for developing new products with biological control properties.

Wild rice (O. latifolia) from natural ecosystems in the Pantanal region of Brazil: Host to Fusarium incarnatum-equiseti species complex and highly contaminated by zearalenone.

We assessed the mycobiota diversity and mycotoxin levels present in wild rice (Oryza latifolia) from the Pantanal region of Brazil; fundamental aspects of which are severely understudied as an edible plant from a natural ecosystem. We found multiple fungal species contaminating the rice samples; the most frequent genera being Fusarium, Nigrospora and Cladosporium (35.9%, 26.1% and 15%, respectively). Within the Fusarium genus, the wild rice samples were mostly contaminated by the Fusarium incarnatum-equiseti species complex (FIESC) (80%) along with Fusarium fujikuroi species complex (20%). Phylogenetic analysis supported multiple FIESC species and gave support to the presence of two putative new groups within the complex (LN1 and LN2). Deoxynivalenol (DON) and zearalenone (ZEN) chemical analysis showed that most of the isolates were DON/ZEN producers and some were defined as high ZEN producers, displaying abundant ZEN levels over DON (over 19 times more). Suggesting that ZEN likely has a key adaptive role for FIESC in wild rice (O. latifolia). Mycotoxin determination in the rice samples revealed high frequency of ZEN, and 85% of rice samples had levels >100 µg/kg; the recommended limit set by regulatory agencies. DON was only detected in 5.2% of the samples. Our data shows that FIESC species are the main source of ZEN contamination in wild rice and the excessive levels of ZEN found in the rice samples raises considerable safety concerns regarding wild rice consumption by humans and animals.

Complex Evolutionary Origins of Specialized Metabolite Gene Cluster Diversity among the Plant Pathogenic Fungi of the Fusarium graminearum Species Complex.

Fungal genomes encode highly organized gene clusters that underlie the production of specialized (or secondary) metabolites. Gene clusters encode key functions to exploit plant hosts or environmental niches. Promiscuous exchange among species and frequent reconfigurations make gene clusters some of the most dynamic elements of fungal genomes. Despite evidence for high diversity in gene cluster content among closely related strains, the microevolutionary processes driving gene cluster gain, loss, and neofunctionalization are largely unknown. We analyzed the Fusarium graminearum species complex (FGSC) composed of plant pathogens producing potent mycotoxins and causing Fusarium head blight on cereals. We de novo assembled genomes of previously uncharacterized FGSC members (two strains of F. austroamericanum, F. cortaderiae, and F. meridionale). Our analyses of 8 species of the FGSC in addition to 15 other Fusarium species identified a pangenome of 54 gene clusters within FGSC. We found that multiple independent losses were a key factor generating extant cluster diversity within the FGSC and the Fusarium genus. We identified a modular gene cluster conserved among distantly related fungi, which was likely reconfigured to encode different functions. We also found strong evidence that a rare cluster in FGSC was gained through an ancient horizontal transfer between bacteria and fungi. Chromosomal rearrangements underlying cluster loss were often complex and were likely facilitated by an enrichment in specific transposable elements. Our findings identify important transitory stages in the birth and death process of specialized metabolism gene clusters among very closely related species.

Correction: Multi-Method Approach for Characterizing the Interaction between Fusarium verticillioides and Bacillus thuringiensis Subsp. Kurstaki.

[This corrects the article DOI: 10.1371/journal.pone.0092189.].

Expression of Genes by Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus flavus Isolated from Brazil Nuts.

The aims of the present study were to monitor the production of aflatoxin B1 (AFB1) and mycelial growth, and to evaluate the expression of genes directly and indirectly involved in the biosynthesis of aflatoxins by Aspergillus flavus isolated from Brazil nuts. Six previously identified A. flavus strains were grown on coconut agar at 25°C for up to 10 days. Mycotoxins were separated by high-performance liquid chromatography and fungal growth was measured daily using the diametric mycelial growth rate. Transcriptional analysis was performed by real-time polymerase chain reaction (PCR) after 2 and 7 d of incubation using specific primers (aflR, aflD, aflP, lipase, metalloprotease, and LaeA). Three (50%) of the six A. flavus isolates produced AFB1 (ICB-1, ICB-12, and ICB-54) and three (50%) were not aflatoxigenic (ICB-141, ICB-161, and ICB-198). Aflatoxin production was observed from d 2 of incubation (1.5 ng/g for ICB-54) and increased gradually with time of incubation until d 10 (15,803.6 ng/g for ICB-54). Almost all A. flavus isolates exhibited a similar gene expression pattern after 2 d of incubation (p > 0.10). After 7 d of incubation, the LaeA (p < 0.05) and metalloprotease (p < 0.05) genes were the most expressed by nonaflatoxigenic strains, whereas aflatoxigenic isolates exhibited higher expression of the aflR (p < 0.05) and aflD genes (p < 0.05). Our results suggest that the expression of aflR and aflD is correlated with aflatoxin production in A. flavus and that overexpression of aflR could affect the transcriptional and aflatoxigenic pattern (ICB-54). Elucidation of the molecular mechanisms that regulate the secondary metabolism of toxigenic fungi may permit the rational silencing of the genes involved and consequently the programmed inhibition of aflatoxin production. Knowledge of the conditions, under which aflatoxin genes are expressed, should contribute to the development of innovative and more cost-effective strategies to reduce and prevent aflatoxin contamination in Brazil nuts.

Fungal diversity and natural occurrence of deoxynivalenol and zearalenone in freshly harvested wheat grains from Brazil.

This study investigated the fungal diversity and presence of deoxynivalenol and zearalenone in 150 samples of freshly harvested wheat grains collected in three regions of Brazil (Sao Paulo, Parana, and Rio Grande do Sul). Analysis of the mycobiota showed a predominance of Alternaria sp., Fusarium sp. and Epicoccum sp. Microdochium nivale (23%), a fungus rarely found in Brazilian crops, was detected in Sao Paulo. Four members of the Fusarium graminearum species complex were isolated: F. graminearum s.s. (37%), Fusarium meridionale (46%), Fusarium cortaderiae (13%), and Fusarium austroamericanum (3%). Toxin analysis revealed 99% contamination with deoxynivalenol (mean 706 µg/kg). The frequency of zearalenone varied greatly across regions: wheat grains from Rio Grande do Sul (84%) and Sao Paulo (12%) had median concentrations of 70.9 and 57.9 µg/kg, respectively. ZEA was not detected in the samples from Parana. A total of six samples were above the maximum tolerated level recommended by the European Commission for ZEA in wheat grains. This study provided new insights into the natural mycobiota of Brazilian wheat, demonstrating contamination of most samples with deoxynivalenol and high frequency of zearalenone in samples from Rio Grande do Sul.

Effect of natural compounds on Fusarium graminearum complex.

BACKGROUND: A search is underway for new solutions to counter farm loss caused by fungal contamination of grains, since the active agents of fungicides can remain in the environment and contribute to the development of resistant and toxigenic species. In this study, the antifungal activity of natural compounds (γ-oryzanol, phenolic extract of neem seeds and of rice bran) was assessed on three toxigenic strains of Fusarium graminearum isolated from wheat, rice and barley. Their efficacy was compared to that of synthetic fungicides. The halo diameters were measured and the susceptible pathways were determined by the levels of structural compounds and activities of enzymes involved in the primary metabolism of the microorganisms. Moreover, mycotoxin production and gene expression were examined. RESULTS: Phenolic extracts were more effective at inhibiting F. graminearum than was γ-oryzanol, as evidenced by the minimum inhibitory concentration. This work contributed to the elucidation of the mechanism of action of natural antifungal agents. CONCLUSION: Natural antifungals effectively inhibited fungal growth, especially via the inactivation of the enzymatic systems of F. graminearum. Natural antifungals inhibited mycotoxin production by the fungi. A correlation between the levels of deoxynivalenol and the expression of Tri5 gene was observed, indicating that the natural compounds could be considered alternatives to synthetic antifungals. © 2015 Society of Chemical Industry.

Multi-method approach for characterizing the interaction between Fusarium verticillioides and Bacillus thuringiensis subsp. Kurstaki.

Bacterial antagonists used as biocontrol agents represent part of an integrated management program to reduce pesticides in the environment. Bacillus thuringiensis is considered a good alternative as a biocontrol agent for suppressing plant pathogens such as Fusarium. In this study, we used microscopy, flow cytometry, indirect immunofluorescence, and high performance liquid chromatography to determine the interaction between B. thuringiensis subsp. kurstaki LFB-FIOCRUZ (CCGB) 257 and F. verticillioides MRC 826, an important plant pathogen frequently associated with maize. B. thuringiensis showed a strong in vitro suppressive effect on F. verticillioides growth and inhibited fumonisin production. Flow cytometry analysis was found to be adequate for characterizing the fungal cell oscillations and death during these interactions. Further studies of the antagonistic effect of this isolate against other fungi and in vivo testing are necessary to determine the efficacy of B. thuringiensis subsp. kurstaki in controlling plant pathogens. This is the first report on the use of flow cytometry for quantifying living and apoptotic F. verticillioides cells and the B. thuringiensis Cry 1Ab toxin.

Black yeasts-like fungi isolated from dialysis water in hemodialysis units.

Hemodialysis in patients with chronic renal failure promotes the removal of toxic substances, water, and minerals from the body and often takes place in specialized clinics. Microbial contamination of dialysis fluid is a serious problem in therapy. One of the sources of contamination is the water used to prepare the dialysate. In Brazil, legislation regulating the microbiological quality of water for dialysis does not cover waterborne microbes such as Pseudomonas, mycobacteria, and fungi. The aim of the present study was to quantify, isolate, and identify fungi present in water systems in six hemodialysis units in Curitiba, Paraná state, Brazil. Fungi were analyzed by surface plating and membrane filtration. Isolates were identified by morphology, while the dematiaceous fungi were identified by sequencing the rDNA ITS region. It was found that 66 % of the samples presented fungi, while black fungi were present in 46 % of all samples. Twenty-eight isolates from treated water for dialysis and dialysate were identified by sequencing and were found to be Exophiala pisciphila, E. cancerae, E. equina, and Rhinocladiella similis. The presence of dematiaceous fungi may pose a risk for debilitated hospitalized patients.

Potential of Fourier transform infrared spectroscopy (FT-IR) to differentiate environmental Aspergillus fungi species A. niger, A. ochraceus, and A. westerdijkiae using two different methodologies.

We assessed the ability of Fourier transform infrared spectroscopy (FT-IR) to differentiate three important and morphologically similar Aspergillus species: A. ochraceus and A. westerdijkiae, and A. niger. Fungi were processed by two methods, powdered mycelia and conidiospore-saline solution, and then recorded in a spectrometer. Second derivatives with nine points of smoothing were applied as spectra data pretreatment. Partial least squares regression was used for the species comparison models and a prediction test was used to evaluate the models. The powdered-mycelia methodology correctly identified 100% of the prediction test set to discriminate A. niger from A. ochraceus and A. westerdijkiae; in addition, it had a 86.6% success rate in discriminating A. ochraceus and A. westerdijkiae. This is the first time a study assessed the ability of FT-IR to differentiate A. niger, A. ochraceus, and A. westerdijkiae, and we believe this technique is very promising for classifying and distinguish fungi isolates.