Molecular and phenotypic analyses of pathogenicity, aggressiveness, mycotoxin production, and colonization in the wheat-Gibberella zeae pathosystem

Fusarium head blight (FHB), caused by Gibberella zeae (Schwein.) Petch (anamorph: Fusarium graminearum Schwabe), is one of the principal diseases responsible for extensive damage in wheat fields and contamination of grain with the mycotoxins deoxynivalenol (DON) and nivalenol (NIV), rendering the harvest unsafe for human and animal consumption. Control of FHB is difficult because of the complex nature of host-pathogen-environment interaction and the nonavailability of highly effective fungicides. Agronomic practices and resistance breeding, therefore, offer the best strategies for disease management. Mapping by molecular markers provides an accurate approach for genetic analyses of simple and complex traits particularly pathogenicity, aggressiveness, and mycotoxin production. Pathogenicity, as defined here, is the ability to cause disease whereas aggressiveness is the quantity of disease induced by a pathogenic isolate on a susceptible host in which isolates do not interact differentially with host cultivars. The project aims to (1) map pathogenicity and aggressiveness of G. zeae based on a published genetic map (2) estimate genetic diversity of four parent isolates by PCR-based markers (3) examine the inheritance of pathogenicity, aggressiveness, mycotoxin type (DON/NIV), and DON production on a phenotypic basis, (4) analyse genetic covariation among aggressiveness, DON, and fungal colonization, (5) and compare aggressiveness of 42 isolates in greenhouse and field environments. Two crosses of G. zeae using nit (nitrate nonutilizing) marker technique were performed: (1) pathogenic DON-producing Z-3639 (Kansas, USA) x nonpathogenic NIV-producing R-5470 (Japan) belonging to lineage 7 and 6, respectively, and (2) DON-producing FG24 (Hungary) x FG3211 (Germany), both aggressive lineage 7 isolates. For the first cross, 99 progeny segregated in a consistent 61:38 for pathogenicity: nonpathogenicity in a two-year greenhouse experiment. Among the 61 pathogenic progeny, disease severity, measured as percentage infected spikelets, varied significantly (P = 0.01). Heritability for aggressiveness was high. Pathogenicity locus was mapped on linkage group IV near loci PIG1 (red pigment production), TOX1 (trichothecene toxin amount), and PER1 (perithecial production) explaining 60%, 43%, and 51% of the phenotypic variation, respectively. Two large aggressiveness QTLs were mapped on linkage group I linked to the locus TRI5 (trichodiene synthase in the trichothecene gene cluster) and an amplified fragment length polymorphism (AFLP) marker (EAAMTG0655K), explaining 51% and 29% of the observed phenotypic variation, respectively. These unlinked loci suggest that genetic basis between pathogenicity and aggressiveness were different. TRI5 is located in the same gene cluster as a previously identified gene known as TRI13, which determines whether DON or NIV will be produced. DON-producing progeny were, on average, twice as aggressive as were those producing NIV. Loci were only detected in the two linkage groups mentioned from the nine linkage groups present in the map. For the second cross FG24 x FG3211 with 153 progeny, head blight rating and relative plot yield were used as aggressiveness traits. DON production was measured by a commercial kit enzyme immunoassay. These three traits were quantitatively inherited among 153 progeny across three environments. Repeatabilities within each environment were medium to high but heritabilities across environments were medium only due to high progeny-environment interaction. DON was a less environmentally stable trait than aggressiveness. Transgressive segregants were detected frequently. This implies that even a cross within a lineage could lead to an increase in aggressiveness. Mapping of this cross was not initiated because the parents were not polymorphic enough to construct a genetic map. Instead, the parents were analysed for polymorphism in comparison to the parents of the first cross using 31 AFLP primer combinations and 56 random amplified polymorphic DNA (RAPD) primers. Polymorphism between Z-3639 and R-5470 was about three to four times higher than between FG24 and FG3211. Cluster analysis revealed that R-5470 was genetically separated from the other three parents, thus confirming the lineage assignments. Among preselected 50 progeny from the same field experiments that showed normal distribution for aggressiveness - head blight rating, fungal colonization, and DON production were correlated (r = 0.7, P = 0.01). Fungal colonization measured as Fusarium exoantigen (ExAg) content using enzyme-linked immunosorbent assay (ELISA) varied also quantitatively, but heritability was lower due to high progeny-environment interaction and error. Strong correlations among all traits indicate control by similar genes or gene complexes. No significant variation was observed for DON/ExAg ratio. Aggressiveness traits and DON production were more environmentally stable compared to Fusarium ExAg content. Our findings imply that aggressiveness may have other components apart from mycotoxin production. Genotypic variation for aggressiveness among the 42 progeny in one greenhouse and three field environments was significant and their correlation was moderate (r = 0.7, P = 0.01). High heritability in both environments again indicates that aggressiveness was a relatively stable trait, although methods of inoculation differed, i.e., injection for greenhouse and spraying for field experiments. Greenhouse aggressiveness could predict aggressiveness in the field, and thereby should reduce costs for resistance and phytopathological studies. In conclusion, we consider G. zeae as medium-risk pathogen with the potential to evolve to a higher level of aggressiveness due to sexual recombination. Erosion of quantitative resistance in FHB cannot be ignored, especially if host resistances with oligogenic inheritance, e.g. Sumai 3 from China, are used on a large acreage. Consequently, the rather simple inheritance of pathogenicity and aggressiveness in G. zeae could lead to a gradual increase of aggressiveness. These results should enhance efforts of plant breeders to use several, genetic distinct sources of resistance in order to avoid possible FHB outbreaks in the future.Ährenfusariosen, die im Wesentlichen von Gibberella zeae (Schwein.) Petch (anamorph: Fusarium graminearum Schwabe) verursacht werden, können zu hohen Ertragsverlusten und der Kontamination mit Mykotoxinen führen. Hauptsächlich werden die Mykotoxine Deoxynivalenol (DON) und Nivalenol (NIV) produziert, die eine gesundheitsschädigende Wirkung auf Mensch und Tier haben. Wegen der hohen Wirt-Pathogen-Umwelt-Interaktion sowie wenig effizienter Fungizide ist die Bekämpfung von Ährenfusariosen problematisch. Pflanzenbauliche Maßnahmen und Resistenzzüchtung können zu einer Verminderung des Pilzbefalls führen. Die Kartierung mit molekularen Markern bietet die Möglichkeit, sowohl einfach wie auch komplex vererbte Merkmale zu untersuchen. Pathogenität und Aggressivität stellen zwei wichtige Bedingungen der Infektion und der Befallsentwicklung von Ährenfusariosen dar. Pathogenität wird dabei als die Fähigkeit eines Isolats definiert, Krankheitssymptome auf einem anfälligen Wirt hervorzurufen. Aggressivität beschreibt die Stärke des Befalls, wenn die Isolate nicht differentiell mit den Wirtsgenotypen interagieren. Zielsetzung des Projektes war (1) die Kartierung der Pathogenität und der Aggressivität von G. zeae, aufbauend auf einer bereits publizierten genetischen Karte, (2) die Analyse der Diversität aller Eltern-Isolate mit PCR-basierenden Markern, (3) die Untersuchung der Vererbung von Pathogenität, Aggressivität, Mykotoxin-Typ (DON/NIV) und DON-Produktion, (4) die Analyse der genetischen Kovariation zwischen Aggressivität, DON und Myzelwachstum und (5) der Vergleich der Aggressivität von 42 Isolaten im Gewächshaus und Feld. Es wurden zwei Kreuzungen mittels nit (nitrate nonutilizing)-Markern durchgeführt, zum einen wurde das pathogene, DON-produzierende Isolat Z-3639 aus Kansas (USA) mit dem nicht-pathogenen, NIV- produzierenden Isolat R-5470 (Japan) der Abstammungslinien 7 bzw. 6 gekreuzt, zum anderen wurden die beiden DON-produzierenden, aggressiven Isolate FG24 (Ungarn) und FG3211 (Deutschland) der Abstammungslinie 7 gekreuzt. Die 99 Nachkommen der ersten Kreuzung spalteten in einem zweijährigen Gewächshausversuch qualitativ in einem Verhältnis 61:38 für das Merkmal Pathogenität. Die Aggressivität, gemessen als Prozent infizierter Ährchen, variierte signifikant (P=0,01) zwischen den 61 pathogenen Nachkommen. Die Heritabilität für die Aggressivität war in dieser Kreuzung hoch. Die Pathogenität wurde auf der Kopplungsgruppe IV nahe den Loci PIG1 (Pigmentierung), TOX1 (Trichothecen-Gehalt), und PER1 (Perithezienproduktion) lokalisiert. Sie erklärten 60%, 43% bzw. 51% der phänotypischen Varianz. Zwei Loci für die Aggressivität waren auf der Kopplungsgruppe I mit TRI5 (Trichodiensynthase) und einem AFLP- (amplified fragment length polymorphism) Marker (EAAMTG0655K) gekoppelt und erklärten 51% bzw. 29% der phänotypischen Varianz. TRI5 befindet sich im gleichen Gen-Cluster wie TRI13, das über die Bildung von DON oder NIV entscheidet. Die DON-produzierende Teilpopulation war im Durchschnitt zweimal so aggressiv wie die NIV-produzierende Teilpopulation. Es wurden keine weiteren Loci für die Pathogenität oder Aggressivität auf den restlichen sieben Kopplungsgruppen dieser Karte gefunden. Daraus lässt sich schlussfolgern, dass beide Merkmale in dieser Kreuzung auf unterschiedliche Weise vererbt werden. Für die zweite Kreuzung FG24 x FG3211 mit 153 Nachkommen wurden die Ährenbonitur und das relative Ährengewicht als Merkmal für die Aggressivität in einem Feldversuch über drei Umwelten ermittelt. Zusätzlich wurde die DON-Produktion mittels eines handelsüblichen Immunotestes gemessen. Alle drei Merkmale zeigten eine quantitative Verteilung. Trotz einer mäßigen bis hohen Wiederholbarkeit in den Einzelumwelten, war die Heritabilität in der Serienverrechnung wegen der hohen Genotyp-Umwelt-Interaktion nur mäßig. Die DON-Produktion zeigte eine geringere Umweltstabilität als die Aggressivität. Transgressionen wurden häufig beobachtet. Dies zeigt, dass auch eine Kreuzung innerhalb einer Abstammungslinie zu einer höheren Aggressivität führen kann. Die Kartierung der zweiten Kreuzungsnachkommenschaft konnte nicht durchgeführt werden, weil der Polymorphismus zwischen den Eltern zu gering war. Statt dessen wurden die Eltern dieser Kreuzung mittels 31 AFLP-Primerkombinationen und 56 RAPD- (random amplified polymorphic DNA)-Primern mit den beiden Eltern der ersten Kreuzung verglichen. Der Polymorphiegrad zwischen Z-3639 und R-5470 war etwa drei bis viermal höher als zwischen FG24 und FG3211. Die Cluster-Analyse ergab, dass R-5470 von den anderen drei Eltern genetisch stark verschieden war, wodurch die Zugehörigkeit zu einer anderen Abstammungslinie als die der restlichen Isolate gerechtfertigt werden kann. Bei fünfzig selektierten Nachkommen, die im Feldversuch über drei Umwelten eine Normalverteilung für die Aggressivität zeigten, korrelierten die Ährenbonitur, das Myzelwachstum und die DON Produktion relativ gut (r = 0,7; P = 0,01). Auch das Myzelwachstum, gemessen als Fusarium-Exoantigen (ExAg) mittels ELISA (enzyme-linked immunosorbent assay), variierte quantitativ. Die Heritabilität für dieses Merkmal war jedoch aufgrund der hohen Bedeutung von Genotyp-Umwelt-Interaktion und Fehler niedrig. Die Korrelation zwischen allen Merkmalen zeigt, dass sie von ähnlichen Genen oder Genkomplexen kontrolliert werden. Es wurde keine signifikante Variation für das DON/ExAg-Verhältnis nachgewiesen. Die Aggressivitätsmerkmale und die DON-Produktion waren umweltstabiler als der Fusarium-ExAg-Gehalt. Diese Ergebnisse belegen, dass neben der Mykotoxinproduktion noch andere Merkmale zur Aggressivität beitragen. Die genotypische Variation für die Aggressivität bei 42 Nachkommen im Gewächshaus und an drei Feldumwelten war signifikant (P = 0,01), die Korrelation war mäßig hoch (r = 0,7; P = 0,01). Dies zeigt, dass die Aggressivität stabil vererbt wurde, obwohl zwei unterschiedliche Inokulationsmethoden angewandt worden waren. Die Einzelährcheninjektion im Gewächshaus einerseits und die Sprühinfektion im Feld andererseits. Daher können die Ergebnisse aus den Gewächshausversuchen zur Vorhersage der Aggressivität im Feld genutzt und damit die Kosten für solche Untersuchungen gesenkt werden. Zusammenfassend sollte G. zeae als Pathogen mit einem mittleren Risiko zur Überwindung von Resistenzen eingestuft werden. Allerdings kann evolutionär durchaus ein höheres Aggressivitätsniveau durch sexuelle Rekombination erreicht werden. Die Erosion quantitativer Resistenz bei Ährenfusariosen sollte dabei nicht vernachlässigt werden. Dies gilt besonders dann, wenn Wirtsgenotypen mit oligogenischer Vererbung der Resistenz, wie beispielsweise die chinesische Sorte Sumai 3, einen großen Anteil der Anbaufläche haben. Die einfache Vererbung der Pathogenität und Aggressivität bei G. zeae kann dann zu einer graduellen Erhöhung der Aggressivität führen. Daher sollten Pflanzenzüchter mehrere, genetisch unterschiedliche Resistenzquellen für die Sortenentwicklung nutzen, um in Zukunft größer Epidemien durch Ährenfusariosen zu vermeiden

Phylogeny and taxonomy of obscure genera of microfungi

The recently generated molecular phylogeny for the kingdom Fungi, on which a new classification scheme is based, still suffers from an under representation of numerous apparently asexual genera of microfungi. In an attempt to populate the Fungal Tree of Life, fresh samples of 10 obscure genera of hyphomycetes were collected. These fungi were subsequently established in culture, and subjected to DNA sequence analysis of the ITS and LSU nrRNA genes to resolve species and generic questions related to these obscure genera. Brycekendrickomyces (Herpotrichiellaceae) is introduced as a new genus similar to, but distinct from Haplographium and Lauriomyces. Chalastospora is shown to be a genus in the Pleosporales, with two new species, C. ellipsoidea and C. obclavata, to which Alternaria malorum is added as an additional taxon under its oldest epithet, C. gossypii. Cyphellophora eugeniae is newly described in Cyphellophora (Herpotrichiellaceae), and distinguished from other taxa in the genus. Dictyosporium is placed in the Pleosporales, with one new species, D. streliziae. The genus Edenia, which was recently introduced for a sterile endophytic fungus isolated in Mexico, is shown to be a hyphomycete (Pleosporales) forming a pyronellea-like synanamorph in culture. Thedgonia is shown not to represent an anamorph of Mycosphaerella, but to belong to the Helotiales. Trochophora, however, clustered basal to the Pseudocercospora complex in the Mycosphaerellaceae, as did Verrucisporota. Vonarxia, a rather forgotten genus of hyphomycetes, is shown to belong to the Herpotrichiellaceae and Xenostigmina is confirmed as synanamorph of Mycopappus, and is shown to be allied to Seifertia in the Pleosporales. Dichotomous keys are provided for species in the various genera treated. Furthermore, several families are shown to be polyphyletic within some orders, especially in the Capnodiales, Chaetothyriales and Pleosporales

Comparative study between biological and chemical agents for control sheath blight disease of rice

Biological control measures are indispensable to sustain global food security, due to it being economically profitable and environmentally sound. A comparative study was conducted to know the effectiveness of biological control measures compared with contact fungicide. Trichoderma spp. based bio fungicides Bioquick and Biospark were applied as preventive measures and contact fungicide as a curative measure for controlling sheath blight disease in rice varieties BR 71 and IR 24. Biospark and Bioquick were applied before disease development while, contact fungicide was used after the initiation of sheath blight disease. At the early stage of disease development, the effect of Bioquick, Biospark, and fungicide in terms of reducing percent relative lesion height and percent tiller infection are comparable. At 14 DAI and 18 DAI, contact fungicide performed best among the three control measures based on the two parameters. The genotypes of the rice accessions used in the study also appeared to be a factor in disease development, as evidenced by higher horizontal and vertical disease severity in BR71 than in IR24. Between comparison of Bioquick and Biospark in terms of reducing percent relative lesion height, percent tiller infection, and percent disease control, appeared to be higher in Biospark in both varieties. From this study, we can conclude that farmers can use Biospark as a biofungicide to get maximum benefit considering rice yield and ecology. However, its efficacy is slightly lower than chemical fungicides for controlling sheath blight disease of rice

Geographic Distribution of Avirulence Genes of the Rice Blast Fungus Magnaporthe oryzae in the Philippines

A total of 131 contemporary and 33 reference isolates representing a number of multi-locusgenotypes of Magnaporthe oryzae were subjected to a PCR test to detect the presence/absence ofavirulence (Avr) genes. Results revealed that the more frequently occurring genes were Avr-Pik(81.50%), Avr-Pita (64.16%) and Avr-Pii (47.98%), whereas the less frequently occurring genes wereAvr-Pizt (19.08%) and Avr-Pia (5.20%). It was also laid out that the presence of Avr genes in M. oryzaeis strongly associated with agroecosystems where the complementary resistant (R) genes exist.No significant association, however, was noted on the functional Avr genes and the major geographiclocations. Furthermore, it was identified that the upland varieties locally known as “Milagrosa” and“Waray” contained all the R genes complementary to the Avr genes tested.<br /

Identification and quantification of fumonisin-producing Fusarium species in grain and soil samples from Egypt and the Philippines

Fumonisins are considered among the important mycotoxins associated with human esophageal cancer and livestock diseases. These mycotoxins are mainly produced by Fusarium verticillioides in tropical and subtropical regions such as the Philippines and Egypt and humid temperate regions of the world. The classical taxonomy of fumonisin-producing fungi is challenging, and species-specific PCR reactions are commonly used to clearly identify species within these complexes. The aim of this study was to isolate, identify and quantify fumonisin-producing species in maize, wheat and soil samples from Egypt and the Philippines, and to test Eppendorf-Agar as a long term preservation method. We isolated 44 single spore isolates (39 from Egypt and five from the Philippines) from the collected samples (25 isolates from maize, five from wheat and 14 from soil). In addition, we quantified the content of fumonisin-producing fungi DNA from 15 maize samples and six wheat samples from Egypt, and from six maize samples from the Philippines. morphological and microscopic identification indicated that 21 isolates from Egypt and five from the Philippines were F. verticillioides, one isolate was F. proliferatum and two isolates were F. nygamai. Molecular identification indicated that all these isolates belonged to F. verticillioides. Most were from maize, four were from soil and only one was from wheat. Other Fusarium species isolated included F. oxysporum and F. solani. No F. graminearum isolates were found. The quantitative PCR (qPCR) results obtained using the Taqfum-2f, Vpgen-3R primer pair and the FUMp probe for quantification of fumonisin-producing Fusarium species showed that fumonisin-producing Fusarium isolates were present in four maize samples from the Philippines and eight maize samples from Egypt. The Fusarium DNA levels from fumonisin-producing isolates were in the range of 13 × 10-3 to 61 × 10-1 ng ng-1 total DNA in positive samples, except in one maize sample from the Philippines with high concentration of &gt;0.5 ng ng-1 total DNA. This indicates that &gt;50 % of all DNA was Fusarium DNA. No fumonisin-producing Fusarium DNA was detected in the wheat samples and in the remaining maize samples. These results showed that PCR-techniques based on qPCR can be used to identify fumonisin-producing Fusarium species and quantify risks of mycotoxin contaminated grains

Identification and quantification of fumonisin-producing Fusarium species in grain and soil samples from Egypt and the Philippines

Fumonisins are considered among the important mycotoxins associated with human esophageal cancer and livestock diseases. These mycotoxins are mainly produced by Fusarium verticillioides in tropical and subtropical regions such as the Philippines and Egypt and humid temperate regions of the world. The classical taxonomy of fumonisin-producing fungi is challenging, and species-specific PCR reactions are commonly used to clearly identify species within these complexes. The aim of this study was to isolate, identify and quantify fumonisin-producing species in maize, wheat and soil samples from Egypt and the Philippines, and to test Eppendorf-Agar as a long term preservation method. We isolated 44 single spore isolates (39 from Egypt and five from the Philippines) from the collected samples (25 isolates from maize, five from wheat and 14 from soil). In addition, we quantified the content of fumonisin-producing fungi DNA from 15 maize samples and six wheat samples from Egypt, and from six maize samples from the Philippines. morphological and microscopic identification indicated that 21 isolates from Egypt and five from the Philippines were F. verticillioides, one isolate was F. proliferatum and two isolates were F nygamai. Molecular identification indicated that all these isolates belonged to F. verticillioides. Most were from maize, four were from soil and only one was from wheat. Other Fusarium species isolated included F. oxysporum and F. solani. No F. graminearum isolates were found. The quantitative PCR (qPCR) results obtained using the Taqfum-2f, Vpgen-3R primer pair and the FUMp probe for quantification of fumonisin-producing Fusarium species showed that fumonisin-producing Fusarium isolates were present in four maize samples from the Philippines and eight maize samples from Egypt. The Fusarium DNA levels from fumonisin-producing isolates were in the range of 13 x 10(-3) to 61 x 10(-1) ng ng(-1) total DNA in positive samples, except in one maize sample from the Philippines with high concentration of >0.5 ng ng(-1) total DNA. This indicates that >50 % of all DNA was Fusarium DNA. No fumonisin-producing Fusarium DNA was detected in the wheat samples and in the remaining maize samples. These results showed that PCR-techniques based on qPCR can be used to identify fumonisin-producing Fusarium species and quantify risks of mycotoxin contaminated grains

New genotypes of aflatoxigenic fungi from Egypt and the Philippines

Aflatoxins (AFs), mainly produced by Aspergillus section Flavi, are the major natural toxins of crops and commodities in hot climatic geographic regions. These toxins are considered as type A carcinogens. One hundred and sixty single spore isolates of A. section Flavi were isolated from two different geographical places, Egypt and the Philippines. A quarter (26.5%) of the isolates was able to produce AFs. Four chemotypes of aflatoxin-producing fungi were obtained. Surprisingly, all aflatoxin-producing A. nomius isolates produced higher amounts (2400-40400 ng ml-1) of total AFs (AFB1, AFB2, AFG1 and AFG2) than the toxigenic A. flavus isolates (</p

Molecular Quantification and Genetic Diversity of Toxigenic Fusarium Species in Northern Europe as Compared to Those in Southern Europe

Fusarium species produce important mycotoxins, such as deoxynivalenol (DON), nivalenol (NIV) and T-2/HT-2-toxins in cereals. The highest DON and T-2/HT-2 toxin levels in northern Europe have been found in oats. About 12%&ndash;24% of Finnish oat samples in 2012 contained &gt;1.75 mg&middot;kg&minus;1 of DON, which belongs to type B trichothecenes. Fusarium graminearum is the most important DON producer in northern Europe and Asia and it has been displacing the closely related F. culmorum in northern Europe. The 3ADON chemotype of F. graminearum is dominant in most northern areas, while the 15ADON chemotype of F. graminearum is predominating in Central and southern Europe. We suggest that the northern population of F. graminearum may be more specialized to oats than the southern population. Only low levels of F. culmorum DNA were found in a few oat samples and no correlation was found between F. culmorum DNA and DON levels. DNA levels of F. graminearum were in all cases in agreement with DON levels in 2011 and 2012, when DON was measured by gas chromatography-mass spectrometry (GC-MS). When the RIDA&reg; QUICK SCAN kit results (DON) were compared to DNA levels of F. graminearum, the variation was much higher. The homogenization of the oats flour by grinding oats with 1 mm sieve seems to be connected to this variation. There was a significant correlation between the combined T-2 and HT-2 and the combined DNA levels of F. langsethiae and F. sporotrichioides in Finland in 2010&ndash;2012. Keywords: Fusarium; mycotoxins; diversity; Europe; qPCR <! [endif] --