Outstanding : the dispensable chromosomes of Mycosphaerella graminicola

Bijdrage aan de KNPV-voorjaarsvergaderin

DNA-extractie zonder remming

Moleculaire technieken voor de detectie en identificatie van plantenpathogenen maken gebruik van het DNA of RNA van de ziekteverwekker. Voor een aantal substraten, zoals grond, is de extractie van amplificeerbaar nucleïnezuur een probleem. Tijdens de DNA-extractie uit sommige moeilijke substraten worden ook andere stoffen meegezuiverd, die vervolgens de PCR remmen en daarmee vals-negatieve reacties opleveren

CSI ook in de Plantenwereld

In de land- en tuinbouw heeft de ontwikkeling van (moleculaire) detectiemethoden van plantenpathogenen de laatste jaren een hoge vlucht genomen. Inmiddels worden deze methoden al grootschalig toegepast in de praktijk. Werd in het begin alleen conventionele polymerase chain reaction (PCR) ingezet voor moleculaire detectie, momenteel vindt ook real-time PCR meer en meer ingang. Binnen het FES-programma ‘Versterking infrastructuur plantgezondheid’ zijn binnen het werkpakket ‘Identificatie- en Detectiemethoden’ vele projecten uitgevoerd om de ‘daders’ van aantastingen te kunnen identificeren. De focus was hierbij gericht op quarantaineorganismen

Genetische analyse van Phytophthora infestans

Samenvatting van proefschrift: 'Genetic analysis of Phytophthora infestans

Ga and Gß Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen Mycosphaerella graminicola

We identified and functionally characterized genes encoding three G alpha proteins and one G beta protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian G alpha(i) and G alpha(s) families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola

Variation in sequence and location of the fumonisin mycotoxin niosynthetic gene cluster in Fusarium

In Fusarium, the ability to produce fumonisins is governed by a 17-gene fumonisin biosynthetic gene (FUM) cluster. Here, we examined the cluster in F. oxysporum strain O-1890 and nine other species selected to represent a wide range of the genetic diversity within the GFSC

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp)

ABSTRACT. We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence. Key words: Molecular markers; VNTRs; Genetic diversity; Population genetics; Black Sigatok

Levend of dood, dat is de vraag!

In de literatuur zijn voor de detectie van plantenpathogenen diverse methodieken beschreven. De biologische methodieken detecteren alleen levende organismen. Morfologische, serologische en moleculaire technieken maken mmestal geen onderscheid tussen dood en levend of infectieus en niet infectieus. Met name voor quarantaineorganismen is het onderscheid tussen levende en dode pathogenen van essentieel belang. Binnen het FES-programma 'Versterking infrastructuur plantgezondheid' is binnen werkpakket 3 'Ontwikkeling van methoden voor het aantonen van vitaliteit van plantenpathogenen' gewerkt aan de detectie van vitaliteit in nematoden, schimmels en bacteriën