Influence of H2O2-Induced Oxidative Stress on In Vitro Growth and Moniliformin and Fumonisins Accumulation by Fusarium proliferatum and Fusarium subglutinans

Fusarium proliferatum and Fusarium subglutinans are common pathogens of maize which are known to produce mycotoxins, including moniliformin (MON) and fumonisins (FBs). Fungal secondary metabolism and response to oxidative stress are interlaced, where hydrogen peroxide (H2O2) plays a pivotal role in the modulation of mycotoxin production. The objective of this study is to examine the effect of H2O2-induced oxidative stress on fungal growth, as well as MON and FBs production, in different isolates of these fungi. When these isolates were cultured in the presence of 1, 2, 5, and 10 mM H2O2, the fungal biomass of F. subglutinans isolates showed a strong sensitivity to increasing oxidative conditions (27–58% reduction), whereas F. proliferatum isolates were not affected or even slightly improved (45% increase). H2O2 treatment at the lower concentration of 1 mM caused an almost total disappearance of MON and a strong reduction of FBs content in the two fungal species and isolates tested. The catalase activity, surveyed due to its crucial role as an H2O2 scavenger, showed no significant changes at 1 mM H2O2 treatment, thus indicating a lack of correlation with MON and FB changes. H2O2 treatment was also able to reduce MON and FB content in certified maize material, and the same behavior was observed in the presence and absence of these fungi, highlighting a direct effect of H2O2 on the stability of these mycotoxins. Taken together, these data provide insights into the role of H2O2 which, when increased under stress conditions, could affect the vegetative response and mycotoxin production (and degradation) of these fungi.

Divergence and Gene Flow Between Fusarium subglutinans and F. temperatum Isolated from Maize in Argentina

Fusarium subglutinans and F. temperatum are two important fungal pathogens of maize whose distinctness as separate species has been difficult to assess. We isolated strains of these species from commercial and native maize varieties in Argentina and sequenced >28,000 loci to estimate genetic variation in the sample. Our objectives were to measure genetic divergence between the species, infer demographic parameters related to their split, and describe the population structure of the sample. When analyzed together, over 30% of each species’ polymorphic sites (>2,500 sites) segregate as polymorphisms in the other. Demographic modeling confirmed the species split predated maize domestication, but subsequent between-species gene flow has occurred, with gene flow from F. subglutinans into F. temperatum greater than gene flow in the reverse direction. In F. subglutinans, little evidence exists for substructure or recent selective sweeps, but there is evidence for limited sexual reproduction. In F. temperatum, there is clear evidence for population substructure and signals of abundant recent selective sweeps, with sexual reproduction probably less common than in F. subglutinans. Both genetic variation and the relative number of polymorphisms shared between species increase near the telomeres of all 12 chromosomes, where genes related to plant−pathogen interactions often are located. Our results suggest that species boundaries between closely related Fusarium species can be semipermeable and merit further study. Such semipermeability could facilitate unanticipated genetic exchange between species and enable quicker permanent responses to changes in the agro-ecosystem, e.g., pathogen-resistant host varieties, new chemical and biological control agents, and agronomic practices.

Fumonisin and Beauvericin Chemotypes and Genotypes of the Sister Species Fusarium subglutinans and Fusarium temperatum

ABSTRACT Fusarium subglutinans and Fusarium temperatum are common maize pathogens that produce mycotoxins and cause plant disease. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant. Our objective was to clarify this situation by determining both the chemotypes and genotypes for strains from both species. We analyzed 25 strains from Argentina, 13 F. subglutinans and 12 F. temperatum strains, for toxin production by ultraperformance liquid chromatography mass spectrometry (UPLC-MS). We used new genome sequences from two strains of F. subglutinans and one strain of F. temperatum, plus genomes of other Fusarium species, to determine the presence of functional gene clusters for the synthesis of these toxins. None of the strains examined from either species produced fumonisins. These strains also lack Fum biosynthetic genes but retain homologs of some genes that flank the Fum cluster in Fusarium verticillioides. None of the F. subglutinans strains we examined produced beauvericin although 9 of 12 F. temperatum strains did. A complete beauvericin (Bea) gene cluster was present in all three new genome sequences. The Bea1 gene was presumably functional in F. temperatum but was not functional in F. subglutinans due to a large insertion and multiple mutations that resulted in premature stop codons. The accumulation of only a few mutations expected to disrupt Bea1 suggests that the process of its inactivation is relatively recent. Thus, none of the strains of F. subglutinans or F. temperatum we examined produce fumonisins, and the strains of F. subglutinans examined also cannot produce beauvericin. Variation in the ability of strains of F. temperatum to produce beauvericin requires further study and could reflect the recent shared ancestry of these two species. IMPORTANCE Fusarium subglutinans and F. temperatum are sister species and maize pathogens commonly isolated worldwide that can produce several mycotoxins and cause seedling disease, stalk rot, and ear rot. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant at the species level. Our results are consistent with previous reports that strains of F. subglutinans produce neither fumonisins nor beauvericin. The status of toxin production by F. temperatum needs further work. Our strains of F. temperatum did not produce fumonisins, while some strains produced beauvericin and others did not. These results enable more accurate risk assessments of potential mycotoxin contamination if strains of these species are present. The nature of the genetic inactivation of BEA1 is consistent with its relatively recent occurrence and the close phylogenetic relationship of the two sister species.

IDENTIFICACIÓN DE ESPECIES DE Fusarium AISLADAS DE SEMILLAS SINTOMÁTICAS Y ASINTOMÁTICAS DE MAÍZ CON BASE EN EL GEN TEF-1α

Un problema fitosanitario en el cultivo de maíz (Zea mays L.) es la pudrición de mazorca a causa de hongos del complejo Fusarium fujikuroi. El síntoma típico de la enfermedad es la presencia de semillas con pudrición distribuidas aleatoriamente en la mazorca. Con frecuencia, el fitomejorador selecciona plantas con mazorcas sanas, que en ocasiones contienen pocas semillas con síntomas y suponen que las semillas asintomáticas están libres del patógeno. El objetivo del presente estudio fue identificar especies de Fusarium tanto en semillas sintomáticas como asintomáticas procedentes de la misma mazorca con base en la filogenia de una región del factor de elongación de la traducción 1α (TEF-1α). Se utilizaron mazorcas de lotes de producción de semilla ubicados en 14 localidades de cinco entidades federativas de México; 28 mazorcas de Chiapas, 16 de Chihuahua, 10 de Guanajuato, 14 del Estado de México y 16 de Jalisco. Las semillas de cada tipo se desinfestaron y se colocaron en medio de cultivo. Después de 5 d se observó crecimiento del micelio y de conidios, de los cuales se seleccionaron tres por cada aislamiento, se transfirieron a medio PDA y se registró su coloración (blanco, salmón, morado, violeta o rojo). Se obtuvieron 439 cepas. El color predominante de los micelios fue el blanco. El 54 % del total de cepas provino de semillas sintomáticas y el resto de asintomáticas. En cada estado, el número de cepas procedente de cada tipo de semillas fue similar. En ambos casos se identificó a Fusarium subglutinans, F. temperatum y F. verticillioides; además, por primera vez en México se detectó la presencia de los Haplotipos 10 y 11 de esta última especie. El fitomejorador corre el riesgo de seleccionar germoplasma susceptible a la pudrición cuando elige plantas que producen mazorcas en las que coexisten ambos tipos de semilla.

ASPECTOS BIOMÉTRICOS, GRAU DE UMIDADE EM SEMENTES DE SUCUPIRA E DETERMINAÇÃO DO EFEITO FUNGITÓXICO DO SEU ÓLEO ESSENCIAL

A busca por métodos naturais que viabilizam a eficiência da atividade biológica sobre vários microrganismos vem sendo destaque no cenário atual. A exploração da atividade biológica de compostos secundários presentes no extrato bruto ou óleos essenciais de plantas pode constituir, ao lado da indução da resistência, em uma forma efetiva de controle de doenças em plantas cultivadas. Diante disso, o presente estudo teve por objetivo avaliar a eficiência do óleo essencial de sucupira em suas propriedades de rendimento, bem como sua atividade biológica antifúngica sobre determinados patógenos que afetam culturas promissoras no Estado do Tocantins. Instalou-se o experimento no Campus de Ciências Agrárias da Faculdade Católica do Tocantins no laboratório de Bromatologia e Fitopatologia, para extração do óleo essência, avaliação do grau de umidade e biometria de sementes, obtenção dos fitopatógenos e avaliação dos efeitos fungitóxicos do óleo essencial. O delineamento experimental utilizado foi inteiramente casualizado com quatro tratamentos e cinco repetições. Os tratamentos, adicionados ao meio de cultura, após a autoclavagem e o resfriamento, foram as seguintes: sucupira (BDA + 25% de extrato de sucupira + 0,016% de polissorbato), sucupira (BDA + 50% de extrato de sucupira + 0,016% de polissorbato), sucupira (BDA + 75% de extrato de sucupira + 0,016% de polissorbato), sucupira (BDA + 100% de extrato de sucupira + 0,016% de polissorbato) e testemunha (BDA). Os resultados foram submetidos ao teste Tukey (5%) de probabilidade. O óleo essencial de sucupira inibiu o desenvolvimento micelial de Fusarium subglutinans, Sclerotinia sclerotiurum, Didymella bryoniae, Pyricularia oryzae.

Genomic sequencing of the aquatic Fusarium spp. QHM and BWC1 and their potential application in environmental protection

ABSTRACTFusarium species are distributed widely in ecosystems of a wide pH range and play a pivotal role in the aquatic community through the degradation of xenobiotic compounds and secretion of secondary metabolites. The elucidation of their genome would therefore be highly impactful with regard to the control of environmental pollution. Therefore, in this study, two indigenous strains of aquatic Fusarium, QHM and BWC1, were isolated from a coal mine pit and a subterranean river respectively, cultured under acidic conditions, and sequenced. Phylogenetic analysis of these two isolates was conducted based on the sequences of internal transcript (ITS1 and ITS4) and encoding β-microtubulin (TUB2), translation elongation factors (TEFs) and the second large sub-unit of RNA polymerase (RPB2). Fusarium, QHM could potentially represent a new species within the Fusarium fujikuroi species complex. Fusarium BWC1 were found to form a clade with Fusarium subglutinans NRRL 22016, and predicted to be Fusarium subglutinans. Shot-gun sequencing on the Illumina Hiseq×10 Platform was used to elucidate the draft genomes of the two species. Gene annotation and functional analyses revealed that they had bio-degradation pathways for aromatic compounds; further, their main pathogenic mechanism was found to be the efflux pump. To date, the genomes of only a limited number of acidic species from the Fusarium fujikuroi species complex, especially from the aquatic species, have been sequenced. Therefore, the present findings are novel and have important potential for the future in terms of environmental control.IMPORTANCEFusarium genus has over 300 species and were distributed in a variety of ecosystem. Increasing attention has been drawn to Fusarium due to the importance in aquatic community, pathogenicity and environmental protection. The genomes of the strains in this work isolated in acidic condition, were sequenced. The analysis has indicated that the isolates were able to biodegrade xenobiotics, which makes it potentially function as environmental bio-agent for aromatic pollution control and remediation. Meanwhile, the virulence and pathogenicity were also predicted for reference of infection control. The genome information may lay foundation for the fungal identification, disease prevention resulting from these isolates and other “-omics” research. The isolates were phylogenetically classified into Fusarium fujikuroi species complex by means of concatenated gene analysis, serving as new addition to the big complex.