Mursel Catal

Strong genetic differentiation between North American and European populations of Phytophthora alni subsp. uniformis.

Alder decline caused by Phytophthora alni has been one of the most important diseases of natural ecosystems in Europe during the last 20 years. The emergence of P. alni subsp. alni -the pathogen responsible for the epidemic-is linked to an interspecific hybridization event between two parental species: P. alni subsp. multiformis and P. alni subsp. uniformis. One of the parental species, P. alni subsp. uniformis, has been isolated in several European countries and, recently, in North America. The objective of this work was to assess the level of genetic diversity, the population genetic structure, and the putative reproduction mode and mating system of P. alni subsp. uniformis. Five new polymorphic microsatellite markers were used to contrast both geographical populations. The study comprised 71 isolates of P. alni subsp. uniformis collected from eight European countries and 10 locations in North America. Our results revealed strong differences between continental populations (Fst = 0.88; Rst = 0.74), with no evidence for gene flow. European isolates showed extremely low genetic diversity compared with the North American collection. Selfing appears to be the predominant mating system in both continental collections. The results suggest that the European P. alni subsp. uniformis population is most likely alien and derives from the introduction of a few individuals, whereas the North American population probably is an indigenous population.

Detection of Eutypa lata and Eutypella vitis in Grapevine by Nested Multiplex Polymerase Chain Reaction

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.

Heterokaryotic nuclear conditions and a heterogeneous nuclear population are observed by flow cytometry in Phytophthora infestans

A simple and reliable method for preparation of whole nuclei of a common oomycete, Phytophthora infestans, is described for laser flow cytometry. The ease of preparation, the absence of detectable debris and aggregates, and the precision in determinations of DNA content per nucleus improve interpretation and understanding of the genetics of the organism. Phytophthora infestans is the pathogen that causes potato and tomato late blight. The genetic flexibility of P. infestans and other oomycete pathogens has complicated understanding of the mechanisms of variation contributing to shifts in race structure and virulence profiles on important agricultural crops. Significant phenotypic and genotypic changes are being reported in the apparent absence of sexual recombination in the field. Laser flow cytometry with propidium iodide is useful in investigating the nuclear condition of the somatic colony of field strains of P. infestans. The majority of the studied strains contain a single population of nuclei in nonreplicated diplophase. However, mean DNA content per nucleus varies considerably among isolates confirming the heterogeneity of the nuclear population in regard to C‐value, for field isolates. Nuclear DNA content varies from 1.75× to 0.75× that of nuclei in a standard strain from central Mexico. Some strains contain two to three populations of nuclei with differing DNA contents in the mycelium and are heterokaryons. Such a range in DNA content suggests DNA‐aneuploidy, but direct confirmation of aneuploidy will require microscopy of chromosomes. Heterokaryosis and populations of nuclei of differing DNA content necessarily confound standardized assays used worldwide in crop breeding programs for determination of race profiles and virulence phenotypes of this important pathogen.

Evaluation of resistance to Rhabdocline needlecast in Douglas fir variety Shuswap, with quantitative polymerase chain reaction.

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

Real-time quantitative PCR assays for evaluation of soybean varieties for resistance to the stem and root rot pathogen Phytophthora sojae

Phytophthora root and stem rot caused by Phytophthora sojae is one of the most destructive disease of soybeans in the world. Effective management of the disease depends on selection and use of soybean varieties resistant to the disease. Fast and reliable procedures are vital to screen soybean varieties against the pathogen. Novel real-time quantitative (qPCR) assays were developed for both absolute and relative quantification of P. sojae in infected root tissues. QPCR assays were based on the detection of the internal transcribed spacer (ITS) gene of the pathogen and 18S ribosomal gene of the host plant. Absolute qPCR allowed the detection of as low as 10 femtograms (fg) of P. sojae DNA in soybean roots. Relative qPCR, employing the comparative threshold cycle (Ct) method, was effective and reliable for quantification of P. sojae DNA normalized to plant DNA in infected soybean root tissues. P. sojae DNA quantities detected in both qPCR assays had high correlations with disease severity index (DSI) ratings of soybean varieties. QPCR assays developed in this study were useful for determination of the levels of P. sojae DNA in different varieties of soybean and for evaluation of them for relative resistance to the pathogen.

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

İstiridye mantarının (Pleurotus ostreatus) tohumluk misel üretimi üzerine bir ön çalışma

Istiridye mantari ( Pleurotus ostreatus ), Turkiye’de kultur mantarindan ( Agaricus bisporus) sonra en yaygin yetistirilen mantar turudur. Ulkemizde istiridye mantari yetistiriciligi 1980’li yillarda baslamistir. Ancak, Bu yillarda kultur mantari yetistiriciligi de yayginlasmaya baslamis, aslinda yetistiriciligi kultur mantarina gore daha kolay olmasina ragmen Istiridye mantari yetistiriciligi yeteri kadar ilgi gormemis ve yayginlasmamistir . Kompost hazirlama gibi zahmetli bir islemi gerektirmeyen istiridye mantari mese, kayin, gurgen gibi agac kutukleri ile lignin ve selulozca zengin her turlu tarimsal bitki artiklari kullanilarak kolaylikla yetistirilebilmektedir. Istiridye mantari yetistiriciligi sonunda ortaya cikan bitkisel atiklar hayvan beslenmesi ve tarimsal uretimde farkli amaclar icin kullanilabilmektedir. Bu ozelliklerin yaninda, gida olarak tuketilmesi durumunda besleyici ozelliginin fazla olmasi ve tibbi amacla kullanilabilmesinden dolayi, onemli bir gida ve ham madde kaynagi olarak degerlendirmektedir. Ulkemizde son yillarda istiridye mantari yetistiriciligine olan ilgi dikkate deger sekilde artis gostermektedir. Bununla birlikte kultur mantarinda oldugu gibi istiridye mantari uretiminin en onemli girdisi olan tohumluk misel (spawn) yurtdisi kaynaklardan saglanmakta ve her iki mantarin tohumluk misel uretimi icin gunumuze kadar tespit edilmis ve ticarilestirilmis yerli izolat veya kulturleri bilinmemektedir. Yurtdisi kokenli izolat veya kulturlerden elde edilen tohumlarin ithali ulkemiz mantar yetistiricilerine ciddi bir ekonomik yuk getirmektedir. Bu calismada yerli P. ostreatus izolati kullanilarak tohumluk misel uretimi gerceklestirilmis ve uretilen tohumlar saman substratina sardirilarak istiridye mantari uretimi ve verimi degerlendirilmistir. Bu arastirma ile yerli tohumluk uretiminde bir adim atilarak bu konuda disa bagimliligin azaltilmasi yonunde oncu bir calisma yapilmistir.

Screening, selection and real-time qPCR validation for phytoplasma resistance in sesame (Sesamum indicum L.)

Phyllody is one of the most destructive diseases of sesame and causes serious yield losses worldwide. The present research was conducted to identify phyllody resistant genotypes in sesame. A total of 542 sesame genotypes were screened for the disease resistance in the field using a disease incidence scale of 1–5 in the year 2012. Three hundred four genotypes showing high disease intensity were eliminated under artificially infected field conditions. In the year 2013, only 30 out of 238 accessions were determined as potential resistant genotypes based on the disease incidence scale. These selected genotypes were further evaluated for confirmation of the resistance in greenhouse conditions using the phytoplasma-infected vector insects under choice and no-choice conditions. Furthermore, real-time qPCR was employed for detection and quantification of phytoplasmas to select true resistant genotypes. The sesame accessions ACS38 and ACS102 were identified as resistant to the disease after evaluation in field, greenhouse and qPCR assays. This work is one of the most comprehensive studies to select genotypes resistant to the diseases caused by phytoplasmas.