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Plasmodiophora brassicae, the cause of clubroot, is a very serious problem preventing from successful and profitable cultivation of oilseed rape in Poland. The pathogen was found in all main growing areas of oilseed rape; it also causes considerable problems in growing of vegetable brassicas. The aim of this work was to elaborate fast, cheap and reliable screening method to detect P. brassicae. To achieve this aim the Loopmediated isothermal DNA amplification (LAMP) technique has been elaborated. The set of three primer pairs was designed using LAMP software. The detection was performed with the GspSSD polymerase, isolated from bacteria Geobacillus sp., with strand displacement activity. DNA extraction from clubbed roots obtained from farmers’ fields of oilseed rape infected by P. brassicae was done using a modified CTAB method. The reaction was performed for 60 min at 62oC. The visual detection was done using CFX96 Real Time PCR Detection System (BioRad) or Gerie II Amplicatior (Optigen). The detection with LAMP proved its usefulness; it was easy, fast and accurate and independent of plant age. The detection limit was 5 spores per 1 μl of the spore suspension, so LAMP was less sensitive than quantitative PCR tests reported in the literature. However, the method is cheap and simple, so it is a good alternative, when it comes to practical use and the assessment of numerous samples.
Stem canker of brassicas is one of the most damaging diseases of oilseed rape worldwide. The disease is caused by two related Leptosphaeria species, and L. maculans is regarded as the more damaging one. Being an ascomycete, the pathogen is able to quickly create new variants that can overcome new resistance genes introduced by researchers and breeding companies. The aim of this work was to study polymorphism of L. maculans populations using 10 recently developed minisatellite markers. The studied subpopulations differed with metconazole treatment. Seven minisatellite markers showed polymorphisms and formed alleles varying from 2 to 10 different core motifs, with 5 alleles on average. In total 36 alleles were found. The majority of alleles (72%) were found in both studied subpopulations of L. maculans. There were 28 alleles in the group of L. maculans isolates originating from plants not treated with any fungicide and 32 in the subpopulation treated with metconazole. Ten unique alleles and imbalanced ratios between some alleles contributed to differences between L. maculans subpopulations. The minisatellites MinLm555, MinLm935-2, MinLm939, MinLm1139 and MinLm2451 showed 6 new variants as compared to the isolates described so far.
Variability and genetic structure of a novel Turnip mosaic virus (TuMV) population from horseradish (Cochlearia armoracia L.) were examined. Over 60 horseradish plants were tested to identify a total of 28 TuMV isolates, constituting the Cochlearia ARmoracia (CAR) TuMV population. Two subgroups of the CAR TuMV isolates could be distinguished: subgroup N did not infect oilseed rape (Brassica napus var. oleifera) cv. Westar plants, while subgroup A infected these plants systemically. Two types of infection of oilseed rape plants were induced by inoculation with the CAR TuMV isolates: systemic mosaic infection and systemic necrotic lesions. The complete sequences of isolates CAR37 (subgroup N) and CAR37A (subgroup A) were determined and compared. The sequences of HC-Pro and CP genes of CAR37 and CAR37A and other isolates of TuMV from other countries were compared to provide some insight into their relatedness. CAR37A, initially regarded as a variant, proved to be very different from CAR37. Re-sequencing after repeated passages confirmed the genetic stability of both isolates.
Grzyb workowy Leptosphaeria maculans jest jednym z najgroźniejszych patogenów rzepaku na świecie, przyczyniających się do znacznych strat ekonomicznych. Poznanie sekwencji genomu L. maculans umożliwiło wyodrębnienie licznych sekwencji mini- (MinLm) i mikrosatelitarnych (Lema). Ustalenie zakresu zmienności tych sekwencji oraz frekwencji poszczególnych alleli umożliwia charakteryzowanie i porównywanie populacji L. maculans występujących w różnych latach i regionach geograficznych. Badania dotyczyły porównania frekwencji alleli minisatelitarnego markera MinLm1 w trzech kolekcjach izolatów L. maculans zgromadzonych na terenie Polski w latach 2000-2002 (kolekcja KBN, 94 izolaty), 2003-2004 (kolekcja SECURE, 186 izolatów) oraz 2005 (kolekcja WLKP, 24 izolaty). Udział poszczególnych alleli w danej populacji określono metodą PCR i rozdziału produktów w żelu agarozowym w obecności markerów wewnętrznych o znanej wielkości lub poprzez porównanie ze standardami o znanej wielkości i sekwencji DNA. W kolekcjach stwierdzono obecność siedmiu alleli (od 1x do 7x). Wykazano malejący udział allelu 2x, którego frekwencja co roku zmniejszała się średnio o ok. 8,5%, gdy tymczasem udział allelu 5x co roku średnio rósł o ok. 8%. W kolekcji z terenu Wielkopolski zebranej w 2005 roku stwierdzono jedynie 8,3% udział allelu 2x, który często występował w populacjach zgromadzonych w latach 2000-2004 i stanowił średnio 30,4% izolatów. Taki wynik sugeruje znaczne zmiany zachodzące w składzie populacji L. maculans porażającej rzepak w naszym kraju.
The fungi Leptosphaeria maculans and L. biglobosa cause stem canker – one of the most damaging diseases of oilseed rape in Poland and worldwide. The aim of this study was to characterize the composition of avirulence genes and mating types present in current populations of L. maculans in Poland. The study was done in autumn 2010. The isolates of L. maculans (254) were obtained from infected winter rapeseed leaves collected at six locations. DNA (deoxyribonucleic acid) of each isolate was extracted using a CTAB (cetyltrimethyl ammonium bromide) method. The taxonomic identity of isolates was checked by RAPD (random amplification of polymorphic DNA) using OPJ-10 primer and compared with specific banding patterns characteristic for the representatives of L. maculans and L. biglobosa. Isolates of L. maculans were studied to identify a mating type and avirulence alleles AvrLm1 and AvrLm6. Mating types MAT1.1 and MAT1.2 were found in similar frequencies at all sites, what suggests that both types are well adapted to environment. The AvrLm1 avirulence allele was observed only in isolates obtained at one collection site. For the first time in Poland the avrLm6 virulence allele has been found.
Thirty single-spore isolates of a toxigenic fungus, Fusarium oxysporum, were isolated from asparagus spears and identified by species-specific polymerase chain reaction (PCR) and translation elongation factor 1-? (TEF) sequence analysis. In the examined sets of F. oxysporum isolates, the DNA sequences of mating type genes (MAT) were identified. The distribution of MAT idiomorph may suggest that MAT1-2 is a predominant mating type in the F. oxysporum population. F. oxysporum is mainly recognised as a producer of moniliformin—the highly toxic secondary metabolite. Moniliformin content was determined by high-performance liquid chromatography (HPLC) analysis in the range 0.05–1,007.47 µg g-1 (mean 115.93 µg g-1) but, also, fumonisin B1 was detected, in the concentration range 0.01–0.91 µg g-1 (mean 0.19 µg g-1). There was no association between mating types and the mycotoxins biosynthesis level. Additionally, a significant intra-species genetic diversity was revealed and molecular markers associated with toxins biosynthesis were identified.
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