Sebastián Stenglein

ISSR markers detect high genetic variation among Fusarium poae isolates from Argentina and England

Fusarium poae is one of the Fusarium species isolated from cereal grains infected by Fusarium head blight (FHB), and in recent years it has been identified as a major FHB component. In this study, 97 F. poae isolates from Argentina (n = 62) and England (n = 35) were analysed by inter-simple sequence repeats (ISSR) to examine the genetic diversity and to determine whether intraspecific variation could be correlated with geographic and/or host origin. The molecular analysis showed high intraspecific variability within F. poae isolates, but did not reveal a clear relationship between variability and the host/geographic origin. Fusarium poae isolates from the same geographic region or host appeared in different subclusters. Conversely, isolates with the same haplotype were also collected from different geographic regions. However, we did observe subclusters consisting of isolates from Argentina only or from England only. Furthermore, a single seed sample was found to host different haplotypes. Analysis of molecular variance (AMOVA) indicated a high genetic variability in F. poae, with most of the genetic variability explained by differences within, rather than between Argentinean and English populations. This is the first report on genetic diversity of F. poae using ISSR markers. Moreover, ISSR fingerprinting generates highly polymorphic markers for F. poae and proved to be a useful and reliable assay for genetic variability studies.

Trichothecene genotypes and production profiles of Fusarium graminearum isolates obtained from barley cultivated in Argentina

Fusarium graminearum is one of the most important pathogens isolated from small cereal grains with Fusarium Head Blight symptoms. The presence of this fungus is often linked to the occurrence of several mycotoxins in barley and wheat. The aim of our study was to characterize trichothecene genotypes and production profiles of F. graminearum sensu stricto isolates obtained from barley grains in Argentina. A total of 110 F. graminearum s.s. isolates were analyzed by PCR assays to predict deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) production, and all isolates were found to belong to the same molecular 15-ADON genotype. Trichothecene production in autoclaved rice was analyzed by using gas chromatography (GC) and confirmed by GC-MS. Of the 110 isolates, 95% were able to produce DON, 71% produced 15-ADON, 63% 3-ADON and 52% NIV. With the exception of a single isolate, all isolates that produced NIV, also produced DON. However, the NIV production was very low, ranging from 0.13 to 0.30 μg/g. Six different production profiles of DON and its acetyl-derivatives were detected, the predominant being simultaneous production of DON, 3-ADON and 15-ADON, followed by DON production, and DON and 15-ADON co-production. This work is the first attempt to characterize the trichothecene genotypes and production profiles of F. graminearum s.s. isolates from Argentinean barley.

Pathogenic and molecular variability among isolates of Pyrenophora tritici-repentis , causal agent of tan spot of wheat in Argentina

Tan spot, caused by Pyrenophora tritici-repentis, is a common disease of wheat (Triticum aestivum) responsible for economic losses in some wheat growing areas worldwide. In this study the pathogenic and genetic diversity of 51 P. tritici-repentis isolates collected from different ecological regions of Argentina were analyzed. Virulence tests were conducted on 10 selected wheat cultivars: Buck Halcón, Chris, Gabo, Glenlea, Klein Dragón, Klein Sendero, Max, ND 495, ProInta Guazú and ProInta Imperial. Data revealed significant differences between all main factors evaluated and the interactions for 19 of the isolates analyzed. Based on the reaction type of each isolate/cultivar combination, 48 different pathogenic patterns were detected. The molecular analysis using Inter-Simple Sequence Repeats (ISSR) revealed the existence of 36 different haplotypes among 37 isolates of P. tritici-repentis originally selected for this study. These results indicate that P. tritici-repentis on wheat in Argentina is a heterogeneous fungus, implying that screening wheat germoplasm for resistance for tan spot disease requires a wide range of pathogen isolates.

Endophytic colonisation of tobacco, corn, wheat and soybeans by the fungal entomopathogen Beauveria bassiana (Ascomycota, Hypocreales)

We demonstrate the effectiveness of three inoculation methods (foliar spray, seed immersion and root immersion) in establishing fungal the entomopathogen Beauveria bassiana as an endophyte in tobacco, corn, wheat and soybean. Colonisation of leaves by B. bassiana was assessed 7, 14, 21 and 28 days post-inoculation. There were significant differences (p < 0.001) in endophytic colonisation among the different inoculation techniques.

Cytokinin production by Pseudomonas fluorescens G20-18 determines biocontrol activity against Pseudomonas syringae in Arabidopsis

Plant beneficial microbes mediate biocontrol of diseases by interfering with pathogens or via strengthening the host. Although phytohormones, including cytokinins, are known to regulate plant development and physiology as well as plant immunity, their production by microorganisms has not been considered as a biocontrol mechanism. Here we identify the ability of Pseudomonas fluorescens G20-18 to efficiently control P. syringae infection in Arabidopsis, allowing maintenance of tissue integrity and ultimately biomass yield. Microbial cytokinin production was identified as a key determinant for this biocontrol effect on the hemibiotrophic bacterial pathogen. While cytokinin-deficient loss-of-function mutants of G20-18 exhibit impaired biocontrol, functional complementation with cytokinin biosynthetic genes restores cytokinin-mediated biocontrol, which is correlated with differential cytokinin levels in planta. Arabidopsis mutant analyses revealed the necessity of functional plant cytokinin perception and salicylic acid-dependent defence signalling for this biocontrol mechanism. These results demonstrate microbial cytokinin production as a novel microbe-based, hormone-mediated concept of biocontrol. This mechanism provides a basis to potentially develop novel, integrated plant protection strategies combining promotion of growth, a favourable physiological status and activation of fine-tuned direct defence and abiotic stress resilience.

Phylogenetic relationships of Fusarium poae based on EF-1α and mtSSU sequences

A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations.

Indigenous filamentous fungi on the surface of Argentinean dry fermented sausages produced in Colonia Caroya (Córdoba)

Some producers of dry fermented sausages use fungal starter cultures with the aim to achieve a desirable surface appearance and avoid the growth of mycotoxigenic fungi. These commercial cultures are mainly composed of Penicillium nalgiovense biotype 6. In contrast, in the case of producers who do not use starters, sausages are spontaneously colonized by the house mycobiota, which generally consists of heterogeneous molds corresponding to different genera and species. In this work, the surface mycobiota of dry fermented sausages produced in Colonia Caroya (Córdoba, Argentina) was determined in both summer and winter seasons. All the sausages sampled had been made without the use of surface fungal starters. In the 57 sausages analyzed in the two winter seasons studied (2010 and 2012), we found a total of 95 isolates of filamentous fungi belonging to six genera (Penicillium, Aspergillus, Mucor, Cladosporium, Scopulariopsis and Eurotium) and ten fungal species, whereas in the 36 sausages analyzed in the two summer seasons studied (2011 and 2012), we found 89 isolates belonging to five genera (Penicillium, Aspergillus, Mucor, Cladosporium and Geotrichum) and ten fungal species. Although 16 different species were found in both winter and summer seasons, only 2 of them predominated completely. P. nalgiovense was found in almost 100% of the sausages analyzed, where biotype 4 was the most frequent. This species gives a whitish gray coloration to the sausages. Considering that the factories sampled do not use fungal starter cultures, this predominance is very interesting since mycotoxin production by this fungus has not been reported. Aspergillus ochraceus was isolated with a frequency of 80-90% in the summer seasons, but in none of the winter samples. The presence of this fungus in sausages produced in the summer was attributed to the high environmental temperatures and the uncontrolled temperature in the ripening rooms during the night. In all cases, A. ochraceus was responsible for the undesirable yellowish gold color of the casing. This fungus thus causes significant economic losses to the producers of Colonia Caroya during the months of high temperatures.

First Record of Fusarium verticillioides as an Entomopathogenic Fungus of Grasshoppers

Abstract Fusarium verticillioides (Saccardo) Nirenberg (Ascomycota: Hypocreales) is the most common fungus reported on infected corn kernels and vegetative tissues, but has not yet been documented as being entomopathogenic for grasshoppers. Grasshoppers and locusts represent a large group of insects that cause economic damage to forage and crops. Tropidacris collaris (Stoll) (Orthoptera: Acridoidea: Romaleidae) is a large and voracious grasshopper that in recent years has become an increasingly recurrent and widespread pest in progressively more greatly extended areas of some of in Argentinas northern provinces, with chemical insecticides being currently the only means of control. During February and March of 2008–09, nymphs and adults of T. collaris were collected with sweep nets in dense woodland vegetation at a site near Tres Estacas in western Chaco Province, Argentina, and kept in screened cages. F. verticillioides was isolated from insects that died within 10 days and was cultured in PGA medium. Pathogenicity tests were conducted and positive results recorded. Using traditional and molecular-biological methods, an isolate of F. verticillioides was obtained from T. collaris, and its pathogenecity in the laboratory was shown against another harmful grasshopper, Ronderosia bergi (Stål) (Acridoidea: Acrididae: Melanoplinae). The mortality caused by F. verticillioides on R. bergi reached 58 ± 6.53% by 10 days after inoculation. This is the first record of natural infection caused by F. verticillioides in grasshoppers.

Endophytic fungi from selected varieties of soybean (Glycine max L. Merr.) and corn (Zea mays L.) grown in an agricultural area of Argentina.

Endophytic fungi are ubiquitous and live within host plants without causing any noticeable symptoms of disease. Little is known about the diversity and function of fungal endophytes in plants, particularly in economically important species. The aim of this study was to determine the identity and diversity of endophytic fungi in leaves, stems and roots of soybean and corn plants and to determine their infection frequencies. Plants were collected in six areas of the provinces of Buenos Aires and Entre Ríos (Argentina) two areas were selected for sampling corn and four for soybean. Leaf, stem and root samples were surface-sterilized, cut into 1cm(2) pieces using a sterile scalpel and aseptically transferred to plates containing potato dextrose agar plus antibiotics. The species were identified using both morphological and molecular data. Fungal endophyte colonization in soybean plants was influenced by tissue type and varieties whereas in corn plants only by tissue type. A greater number of endophytes were isolated from stem tissues than from leaves and root tissues in both species of plants. The most frequently isolated species in all soybean cultivars was Fusarium graminearum and the least isolated one was Scopulariopsis brevicaulis. Furthermore, the most frequently isolated species in corn plants was Aspergillus terreus whereas the least isolated one was Aspergillus flavus. These results could be relevant in the search for endophytic fungi isolates that could be of interest in the control of agricultural pests.

Characterization of a Fusarium poae world-wide collection by using molecular markers

Fusarium poae has been considered as a minor species among those that cause Fusarium Head Blight (FHB) disease but in recent years several researchers have documented a high frequency of occurrence of this species. In this study, a total of 173 F. poae isolates from Argentina, Belgium, Canada, England, Finland, France, Germany, Hungary, Italy, Luxembourg, Poland, Switzerland and Uruguay were evaluated by using inter simple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP) to evaluate genetic variability within F. poae and to amplify MAT idiomorphs as a possible mechanism that could explain part of the variability found in this species. The molecular analysis obtained from both molecular markers showed a high intraspecific variability. However, a partial clustering between F. poae isolates and their geographic origin was obtained by ISSR markers while AFLP showed isolates from different geographic locations distributed throughout the dendrogram. Moreover, ISSR grouped all the F. poae isolates into a different cluster from the F. langsethiae and F. sporotrichioides isolates used as outgroups compared with the dendrogram obtained using AFLP markers. Analysis of molecular variance (AMOVA) indicated a high genetic variability in the F. poae collection, with most of the genetic variability resulting from differences within, rather than between, American and European populations by using both molecular markers. Regarding MAT idiomorphs, for most F. poae isolates both MAT-1 and MAT-2 were present from each isolate.