Genetic diversity and pathogenicity of the Fusarium species complex on soybean in Serbia.

Using morphological and cultural characteristics for identification, 36 Fusarium isolates were recovered from diseased roots, stems, and seeds of soybean from several localities throughout Vojvodina Province, Serbia, were identified as Fusarium spp. Based on molecular characterization, 12 Fusarium species were identified: F. acuminatum, F. avenaceum, F. commune, F. equiseti, F. graminearum, F. incarnatum, F. oxysporum, F. proliferatum, F. solani, F. sporotrichioides, F. subglutinans, and F. tricinctum. The EF-1α based-phylogeny grouped the isolates into 12 well-supported clades, but the polymorphisms among sequences in some clades suggested the use of the species complex concept: (1) FIESC - F. incarnatum and F. equiseti; (2) FOSC - F. oxysporum; (3) FSSC - F. solani; and (4) FAATSC - F. acuminatum, F. avenaceum and F. tricinctum. Pathogenicity tests showed that the most aggressive species causing soybean seed rot were F. sporotrichioides, F. graminearum, FIESC, and F. avenaceum. Furthermore, F. subglutinans, FSSC, and F. proliferatum, showed a high percentage of pathogenicity on soybean seeds (80-100%), while variability in pathogenicity occurred within isolates of F. tricinctum species has occurred variability in the virulence of different isolates. FOSC, F. commune and F. acuminatum had the lowest pathogenicity degree. To our knowledge, this is the first study of the characterization of Fusarium complex species on soybean in Serbia. This study provides valuable information about the structure composition of Fusarium complex species and pathogenicity that will be used in further research on soybean resistance to Fusarium-based diseases.

Rapid molecular assay for the evaluation of clove essential oil antifungal activity against wheat common bunt.

Common bunt of durum wheat (DW), Triticum turgidum L. ssp. durum (Desf.) Husn., is caused by the two closely related fungal species belonging to Tilletia genus (Tilletiales, Exobasidiomycetes, Ustilaginomycotina): Tilletia laevis Kühn (syn. T. foetida (Wallr.) Liro.) and T. caries (DC) Tul. (syn. T. tritici (Bjerk.) G. Winter). This is one of the most devastating diseases in wheat growing areas worldwide, causing considerable yield loss and reduction of wheat grains and flour quality. For these reasons, a fast, specific, sensitive, and cost-effective method for an early diagnosis of common bunt in wheat seedlings is urgent. Several molecular and serological methods were developed for diagnosis of common bunt in wheat seedlings but at late phenological stages (inflorescence) or based on conventional PCR amplification, with low sensitivity. In this study, a TaqMan Real Time PCR-based assay was developed for rapid diagnosis and quantification of T. laevis in young wheat seedlings, before tillering stage. This method, along with phenotypic analysis, was used to study conditions favoring pathogen infection and to evaluate the effectiveness of clove oil-based seed dressing in controlling the disease. The overall results showed that: i) the Real Time PCR assay was able to quantify T. laevis in young wheat seedlings after seed dressing by clove oil in different formulations, greatly reducing times of analysis. It showed high sensitivity, detecting up to 10 fg of pathogen DNA, specificity and robustness, allowing to directly analyze crude plant extracts and representing a useful tool to speed up the tests of genetic breeding for disease resistance; ii) temperature was a critical point for disease development when using wheat seeds contaminated by T. laevis spores; iii) at least one of the clove oil-based formulations tested was able to efficiently control wheat common bunt, suggesting that clove oil dressing could represent a promising tool for managing the disease, especially in sustainable farming.

New-Generation Sequencing Technology in Diagnosis of Fungal Plant Pathogens: A Dream Comes True?

The fast and continued progress of high-throughput sequencing (HTS) and the drastic reduction of its costs have boosted new and unpredictable developments in the field of plant pathology. The cost of whole-genome sequencing, which, until few years ago, was prohibitive for many projects, is now so affordable that a new branch, phylogenomics, is being developed. Fungal taxonomy is being deeply influenced by genome comparison, too. It is now easier to discover new genes as potential targets for an accurate diagnosis of new or emerging pathogens, notably those of quarantine concern. Similarly, with the development of metabarcoding and metagenomics techniques, it is now possible to unravel complex diseases or answer crucial questions, such as "What's in my soil?", to a good approximation, including fungi, bacteria, nematodes, etc. The new technologies allow to redraw the approach for disease control strategies considering the pathogens within their environment and deciphering the complex interactions between microorganisms and the cultivated crops. This kind of analysis usually generates big data that need sophisticated bioinformatic tools (machine learning, artificial intelligence) for their management. Herein, examples of the use of new technologies for research in fungal diversity and diagnosis of some fungal pathogens are reported.

Antibacterial Activity of Essential Oils and Trametes versicolor Extract against Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum for Seed Treatment and Development of a Rapid In Vivo Assay.

Clavibacter michiganensis subsp. michiganensis (Smith) Davis et al. (Cmm) and Ralstonia solanacearum Yabuuchi et al. (Smith) (Rs) are important seed-borne bacterial pathogens of tomato (Solanum lycopersicum) listed as A2 pests in the EPPO (European and Mediterranean Plant Protection Organization) region. At present, there are few strategies to control these pathogens, and seed control with eco-compatible approaches is widely encouraged. In this work, the essential oils (EOs) of oregano (Origanum vulgare), garlic (Allium sativum), basil (Ocimum basilicum), cinnamon (Cinnamomum zeylanicum), clove buds (Syzygium aromaticum), thyme (Thymus vulgaris), and Trametes versicolor extract (Tve) were tested in vitro for their antimicrobial activity against Cmm and Rs (broth microdilution method). The tested EOs and the Tve extract caused a significant inhibition of bacterial growth, with very promising MBC (minimum bactericidal concentration) and MIC90 (minimum inhibitory concentration causing a 90% growth inhibition) values. Moreover, an in vivo germination test showed no major reduction in seed germination when the substances were applied as seed treatment. A rapid molecular screening method has been developed, through real-time PCR, for the specific quantification of Cmm in the presence of a vegetable matrix to test in vivo the antimicrobial efficacy of oregano and cinnamon oil on seed treatment without resorting to whole plant essays, which are time- and space-consuming.

Clonality, spatial structure, and pathogenic variation in Fusarium fujikuroi from rain-fed rice in southern Laos.

Bakanae disease, caused by the fungal phytopathogen Fusarium fujikuroi, can be detected in most rice (Oryza sativa L.) growing areas worldwide. In this study, we investigated the population structure of this fungus in southern Lao PDR, a country located near the geographic origin of rice domestication. Microsatellites (SSRs) and mating type (MAT) analyses, pathogenicity and fungicide sensitivity tests were integrated in the study. The first key finding is that the population genetic structure of F. fujikuroi in Lao PDR is consistent with high clonal reproduction. Indeed, (i) "true" clones were identified; (ii) within populations, MAT types were frequently skewed from 1:1 ratio, (iii) linkage disequilibrium (among SSRs as also among SSRs and MAT) was present, and (iv) gene-flow between opposite MAT types within the same population is restricted. The presence of genetic divergence among areas and populations and the occurrence of positive spatial autocorrelation of genetic variation, indicate that migration is restricted, and that genetic drift plays an important role in the evolution of this fungus. Two main well-defined groups of isolates were detected (FST = 0.213) that display a non-random spatial distribution. They differ in the ability to induce seedlings death but not seedlings elongation (the typical Bakanae symptom) suggesting that the pathogen's ability to induce the two symptoms is under different genetic control. Finally, we compared two agroecosystems with contrasting characteristics: low-input and traditional (Lao PDR) vs high-input and modern (Italy). We found differences in the level of population structuring and of spatial autocorrelation. This suggests that the evolutionary potential of the fungus not only depends on its intrinsic characteristics, but is strongly influenced by other external factors, most likely by the dynamics of infested seed exchange. Thus, quarantine and chemical treatments are a way to reduce population connectivity and hence the evolutionary potential of this pathogen.

Acquired Enamel Pellicle Engineered Peptides: Effects on Hydroxyapatite Crystal Growth.

The aim of this study was to test the hypothesis that duplication/hybridization of functional domains of naturally occurring pellicle peptides amplified the inhibitory effect of hydroxyapatite crystal growth, which is related to enamel remineralization and dental calculus formation. Histatin 3, statherin, their functional domains (RR14 and DR9), and engineered peptides (DR9-DR9 and DR9-RR14) were tested at seven different concentrations to evaluate the effect on hydroxyapatite crystal growth inhibition. A microplate colorimetric assay was used to quantify hydroxyapatite crystal growth. The half-maximal inhibitory concentration (IC50) was determined for each group. ANOVA and Student-Newman-Keuls pairwise comparisons were used to compare the groups. DR9-DR9 increased the inhibitory effect of hydroxyapatite crystal growth compared to single DR9 (p < 0.05), indicating that functional domain multiplication represented a strong protein evolution pathway. Interestingly, the hybrid peptide DR9-RR14 had an intermediate inhibitory effect compared to DR9 and DR9-DR9. This study used an engineered peptide approach to investigate a potential evolution protein pathway related to duplication/hybridization of acquired enamel pellicle's natural peptide constituents, contributing to the development of synthetic peptides for therapeutic use against dental caries and periodontal disease.

Identification of bakanae disease resistance loci in japonica rice through genome wide association study.

BACKGROUND: Bakanae disease, caused by seed-borne Fusarium species, mainly F. fujikuroi, is a rice disease whose importance is considerably increasing in several rice growing countries, leading to incremental production losses. RESULTS: A germplasm collection of japonica rice was screened for F. fujikuroi resistance, allowing the identification of accessions with high-to-moderate levels of resistance to bakanae. A GWAS approach uncovered two genomic regions highly associated with the observed phenotypic variation for response to bakanae infection on the short arm of chromosome 1 (named as qBK1_628091) and on the long arm of chromosome 4 (named as qBK4_31750955). High levels of phenotypic resistance to bakanae were associated to the cumulated presence of the resistant alleles at the two resistance loci, suggesting that they can provide useful levels of disease protection in resistance breeding. A fine comparison with the genomic positions of qBK1_628091 and qBK4_31750955 with respect to the QTLs for bakanae resistance reported in the literature suggests that the resistant loci here described represent new genomic regions associated to F. fujikuroi resistance. A search for candidate genes with a putative role in bakanae resistance was conducted considering all the annotated genes and F. fujikuroi-related DEGs included in the two genomic regions highlighting several gene functions that could be involved in resistance, thus paving the way to the functional characterization of the resistance loci. CONCLUSIONS: New effective sources for bakanae resistance were identified on rice chromosomes 1 and 4 and tools for resistance breeding are provided.

Peptídeos peptidomiméticos da película adquirida do esmalte: efeitos no crescimento de cristal de hidroxiapatita / Peptidomimetics of acquired enamel pellicle peptides: effects on hydroxyapatite crystal growth

Os peptídeos da estaterina (DR9) e da histatina 3 (RR14), que ocorrem naturalmente na película in vivo, amplificam o efeito inibitório do crescimento de cristais de hidroxiapatita, função relacionada à remineralizarão do esmalte e formação de cálculos dentários. A hipótese da duplicação/hibridação de domínios funcionais dos peptídeos DR9 da estaterina e RR14 da histatina 3 foi testada. Para isto, os peptídeos peptidomiméticos (DR9-DR9, DR9-RR14), além deles individualmente e suas proteínas intactas (DR9, RR14, estaterina e histatina 3) foram estudados em sete concentrações diferentes para avaliar o efeito da inibição do crescimento de cristais de hidroxiapatita. Foi utilizado um ensaio colorimétrico de microplaca para quantificar o crescimento de cristais de hidroxiapatita. As experiências foram feitas em triplicata e a concentração inibitória (IC50) foi estabelecida para cada grupo. A IC50 foi calculada para todos os peptídeos e proteínas testados. A histatina 3 e o RR14 não atingiram o valor de IC50. O DR9- RR14 atingiu o valor de IC50 a 3,80 M. Como esperado, DR9 e DR9-DR9 demonstraram um efeito inibitório significativo na atividade de crescimento de cristais, atingindo o valor de IC50 a 2,82 M e 1,07 M, respectivamente. A estaterina atingiu o valor de IC50 a 2,50 M. Na análise estatística, foram aplicados os testes ANOVA e Student-Newman-Keuls para comparações por pares, para comparar os valores entre os grupos. O DR9-DR9 amplificou o efeito inibitório do crescimento de cristais de hidroxiapatita quando comparado com DR9 único (p <0,05), demonstrando que a multiplicação do domínio funcional é uma forte tendência evolutiva da proteína. De forma interessante, o peptídeo híbrido DR9-RR14 demonstrou um efeito inibitório intermediário quando comparado com outros dois grupos: DR9 único e DR9-DR9. Este estudo utilizou a abordagem peptidomimética para investigar uma via potencial de evolução da proteína relacionada com a duplicação/hibridação dos constituintes peptídicos naturais da película adquirida de esmalte. O conhecimento obtido por meio dos resultados deste trabalho pode fornecer uma base para o desenvolvimento de peptídeos sintéticos para uso terapêutico, tanto contra cárie dentária, como para a doença periodontal.(AU)

The statherin and histatin 3 peptides (DR9 and RR14 respectively), which occur naturally in the film in vivo, amplify the inhibitory effect for the growth of hydroxyapatite crystals, a function related to remineralization of the enamel and formation of dental calculi. The hypothesis of duplication/hybridization of functional domains of the DR9 peptides of the statherin and RR14 of histatin 3 was tested. For this, the peptidomimetic peptides (DR9-DR9, DR9-RR14), in addition to them individually and their intact proteins (DR9, RR14, statherin and histatin 3) were studied at seven different concentrations to evaluate the effect of growth inhibition of hydroxyapatite crystals. A colorimetric assay of microplate was used to quantify the growth of hydroxyapatite crystals. The experiments were done in triplicate and the inhibitory concentration (IC50) was established for each group. The IC50 was calculated for all peptides and proteins tested. Histatin 3 and RR14 did not reach the IC50 value. DR9-RR14 reached the IC50 value at 3.80 M. As expected, DR9 and DR9-DR9 demonstrated a significant inhibitory effect on crystal growth activity, reaching the IC50 value at 2.82 M and 1.07 M, respectively. Statherin reached the IC50 value at 2.50 M. ANOVA and Student-Newman-Keuls tests for paired comparisons were applied to compare the values between the groups. DR9-DR9 amplified the inhibitory effect of hydroxyapatite crystal growth when compared to single DR9 (p <0.05), demonstrating that the multiplication of the functional domain is a strong protein evolution pathway. Interestingly, the hybrid peptide DR9-RR14 demonstrated an intermediate inhibitory effect when compared to other two groups: single DR9 and DR9-DR9. This study utilized the peptidomimetic approach to investigate a potential pathway of protein evolution related to duplication/hybridization of the natural peptidic constituents of the acquired enamel film. The knowledge obtained through the results of this work can provide a basis for the development of synthetic peptides for therapeutic use, both against dental caries and for periodontal disease.(AU)

Genetic transformation of the tomato pathogen Pyrenochaeta lycopersici allowed gene knockout using a split-marker approach.

Pyrenochaeta lycopersici, as other soil-transmitted fungal pathogens, generally received little attention compared to the pathogens affecting the aerial parts of the plants, although causing stunt and important fruit yield reduction of agronomic relevant crops. The scope of this study was to develop a system allowing to investigate the functional role of P. lycopersici genes putatively involved in the corky root rot of tomato. A genetic transformation system based on a split-marker approach was developed and tested to knock out a P. lycopersici gene encoding for a lytic polysaccharide monooxygenase (Plegl1) induced during the disease development. The regions flanking Plegl1 gene were fused with the overlapping parts of hygromycin marker gene, to favour homologous recombination. We were able to obtain four mutants not expressing the Plegl1 gene though, when tested on a susceptible tomato cultivar, Plegl1 mutants showed unaltered virulence, compared with the wild-type strain. The strategy illustrated in the present work demonstrated for the first time that homologous recombination occurs in P. lycopersici. Moreover, a transformation system mediated by Agrobacterium tumefaciens was established and stable genetic transformants have been obtained. The transformation systems developed represent important tools for investigating both the role of genes putatively involved in P. lycopersici interaction with host plant and the function of other physiological traits which emerged to be genetically expanded from the recent genome sequencing of this fungus.

De novo genome assembly of the soil-borne fungus and tomato pathogen Pyrenochaeta lycopersici.

BACKGROUND: Pyrenochaeta lycopersici is a soil-dwelling ascomycete pathogen that causes corky root rot disease in tomato (Solanum lycopersicum) and other Solanaceous crops, reducing fruit yields by up to 75%. Fungal pathogens that infect roots receive less attention than those infecting the aerial parts of crops despite their significant impact on plant growth and fruit production. RESULTS: We assembled a 54.9Mb P. lycopersici draft genome sequence based on Illumina short reads, and annotated approximately 17,000 genes. The P. lycopersici genome is closely related to hemibiotrophs and necrotrophs, in agreement with the phenotypic characteristics of the fungus and its lifestyle. Several gene families related to host-pathogen interactions are strongly represented, including those responsible for nutrient absorption, the detoxification of fungicides and plant cell wall degradation, the latter confirming that much of the genome is devoted to the pathogenic activity of the fungus. We did not find a MAT gene, which is consistent with the classification of P. lycopersici as an imperfect fungus, but we observed a significant expansion of the gene families associated with heterokaryon incompatibility (HI). CONCLUSIONS: The P. lycopersici draft genome sequence provided insight into the molecular and genetic basis of the fungal lifestyle, characterizing previously unknown pathogenic behaviors and defining strategies that allow this asexual fungus to increase genetic diversity and to acquire new pathogenic traits.

Molecular and functional characterization of an endoglucanase in the phytopathogenic fungus Pyrenochaeta lycopersici.

Many fungal plant pathogens secrete an array of cell wall degrading enzymes mainly involved in the pathogenesis. In this work, a cDNA clone encoding an extracellular endo-1,4-ß-glucanase (named PlEGL1) from the causal agent of the Corky Root Rot of tomato, Pyrenochaeta lycopersici, was isolated and characterized, in order to understand its putative role in the pathogenesis and its mechanism of action. Multiple alignment of the deduced amino acidic sequence shows a high homology with other endoglucanases from different phytopathogenic fungi and detects a well-defined conserved domain of the Glycosyl Hydrolase family 61 (GH61). In vitro, Plegl1 gene transcription is correlated to a cellulolytic activity of the fungus, regulated, in its turn, by the presence of sugar and/or cellulose in the culture medium. In the infected plants, expression level of Plegl1 is positively correlated to the development of the disease. PlEGL1 was heterologously expressed in Escherichia coli and the recombinant protein was purified and tested for its cellulolytic ability, showing a very weak activity, in agreement with all the endoglucanases belonging to GH61 family. The finding in this paper will provide the basis for further determination of biochemical properties of the PlEGL1 protein and its possible involvement in the host-pathogen interaction.

Avaliaçäo nutricional em pacientes portadores de insuficiência renal crônica / Nutritional assessment of chronic uremic patients

No presente trabalho foram avaliados 12 indivíduos (nove homens e três mulheres) que recebiam atendimento na Unidade de Tratamento Dialítico da Clínica Médica do hospital das Clínicas da Faculdade de Medicina de Botucatu. Estes pacientes foram submetidos à avaliaçäo do estado nutricional, mediante história alimentar, métodos antropométricos e bioquímicos, sendo subdivididos em três grupos: pacientes sem tratamento dialítico, pacientes em diálise peritoneal e pacientes em hemodiálise. Desses 12 pacientes, seis näo recebiam tratamento dialítico, sendo que foram avaliados uma única vez, e os que passavam por tratamento dialítico foram avaliados antes e depois mesmo. Através dos resultados, pode-se observar que os indivíduos com insuficiência renal crônica apresentam ingestäo calórica bastante inadequada, além dos dados antropométricos e bioquímicos que também indicaram significativos graus de deficiência do estado nutricional. Através destes dados, este trabalho também pretende demonstrar a importância de maiores cuidados dietéticos, para que esses indivíduos possam ter uma manutençäo substancial de seu estado nutricional. Também foi sugerido um exemplo de dieta para pacientes renais crônicos näo-submetidos a tratamento dialítico