Soil Microorganisms Quantitative Dynamic Characterizing the Overall Biological State in Rhizosphere of Tobacco and Tomato Plants Infected by Broomrape

The quantitative dynamics of soil microorganisms in the rhizosphere of tobacco and tomato plants infected by broomrape %252FPhelipanche ramosa L.%252F were investigated. Parallel pot experiments with the two hosts of the parasite - Oriental type of tobacco and tomato were presented. The variants were%253A by introducing contamination into the soil - seeds by broomrape and control pots - without the seeds by broomrape. Experiments were carried out under controlled conditions. Soil samples for microbiological analyzes were taken from the rhizosphere, in dynamics. The microbiological analyses were executed according to Kohs, in three replications (MPN%252Fg a.d.s), with confidence level 0.05. The surveyed set of soil microorganisms has been comprised two indicators groups characterizing the overall biological state of the microbial communities%253A Autochthonous (on soil extract agar) and Oligotrophic (on diluted soil extract agar). Diversity indexes Shannon (H) and Simpson (D) and the distribution evenness (EH) in the microbial communities were determined. A statistical analysis has been made. The obtained results showed that the quantities of the two groups of microorganisms begun to change visibly around and after 20th day, compared to the amount at the rhizosphere of uninfected by broomrape plants. There was an increase at the population density of autochthonous microorganisms between 20 and 60 days and reduced significantly after this period. The trend was reversed at oligotrophic microorganisms, but the quantities were higher throughout the study period. These dynamics coincide with the phases of broomrape development - germination, formation of a haustorium (about 10-20th day) and attachment to the root of the host (up to about 40-60 days) and the above-ground phases (80-100 days). The analysis showed that microbial communities formed in rhizospheres in the presence of a broomrape had been an oligotrophic character. The relative share of oligotrophic microorganisms in these microbial communities was over 50%25 at both host rhizospheres

EVALUATION OF THREE SPECIES FROM PYROLACEAE FAMILY USING ISSR MARKERS

The family Pyrolaceae is represented in Bulgaria by six species, belonging to three genera. The viewpoints for the taxonomical position of the species in this family are not clearly unilateral. The aim of this study is to find molecular markers, allowing the objectively differentiation of the genera in this family, without use of morphological characters. Three species of the family were examined, representing the tree genera distributed in Bulgaria: Moneses uniflora (L.) A.Gray, Orthilia secunda (L.) House and Pyrola minor L. DNA was isolated from leafs and amplified via PCR reactions using 4 primers. The results were processed using cluster analysis and non-metric multidimensional scalling. The consequent cladogram, obtained by the mean euclidean distances, displayed grouping of the samples by genera, independently of their locality. The independent multitudes confirm the accuracy of the method for determination of the species in Pyrolaceae

Identification of seeds of Phelipanche ramosa, Phelipanche mutelii and Orobanche cumana in the soils from different agricultural regions in Bulgaria by molecular markers

Broomrapes are root holoparasites from Orobanchaceae family parasitizing other flowering plants. Several of the most aggressive broomrape species are widely spread in Bulgaria often causing serious yield losses of important crops. It is important to be able to detect the presence of broomrape seeds in the soil. In the present study, we combined a proven PCR-based assay for detection of broomrape seeds with methods to distinguish species based on nuclear ITS sequences to identify broomrape seeds isolated from soil samples and to study the population structure and the intraspecific variations within the three species. Fifty-six soil samples from 23 different regions in Bulgaria were studied. Based on molecular and bioinformatics analyses we found that 22 samples contained seeds of Orobanche cernua var. cumana and six samples, seeds of Phelipanche ramosa. Phylogenic and bioinformatics analyses surprisingly revealed that the isolated sequences from supposedly P. mutelii seeds diverge from those annotated in NCBI by other authors on 16 different nucleotide positions and formed two distant branches of the phylogenic tree. On the other hand, the isolated sequences were almost identical with P. rosmarina. Tajimas’ D-test revealed that O. cumana populations are currently stable. Regarding the Phelipanche representatives, based on the D-test we can hypothesize that P. mutelii/P. rosmarina populations are in a period of active expansion, while P. ramosa populations are contracting. All isolated sequences were deposited in NCBI Genbank database

Molecular taxonomy study of representatives of the genus Gobius inhabiting coastal waters of Black Sea region

According to their origin the Black Sea gobies can be divided to Ponto-Caspian relicts and Mediterranean immigrants. The increase of species diversity in the Black Sea is a consequence of Mediterranean immigrants. The present study investigated the variability in Cytochrome b sequences isolated from two former Mediterranean immigrant species: Gobius niger and Zosterisessor ophiocephalus. The annotated at NCBI Cyt b sequences of the species were also used to extend the reach of the study. The analyses demonstrated that Black Sea inhabited by four haplotypes of G. niger. Four other haplotypes were found in sequences of Mediterranean black gobies. In the Black Sea haplotype, 6 is predominant and 83.5% of all G. niger representatives belong to it. Three other haplotypes (H_1; H_7 and H_8) were found to form isolated populations. The Tajima D-test indicated that in the Black Sea G. niger in a stage of expansion and significant evolutionary pressure according to data from Maximum Composite Likelihood model of Tamura-Nei, which can explain the accumulation of mutation and appearance of new haplotypes. Unlike G. niger, Z. ophiocephalus populations are shrinking according to Tajima D-test and only one haplotype is still surviving in isolated locations in the Black Sea. These data are in agreement with previous reports of other authors which alarmed that the grass goby is a critically endangered species close to extinction

A Simple and Powerful Approach for Isolation of Arabidopsis Mutants with Increased Tolerance to H2O2-Induced Cell Death

A genetic approach is described to isolate mutants more tolerant to oxidative stress. A collection of T-DNA activation tag Arabidopsis thaliana mutant lines was screened for survivors under conditions that trigger H2O2-induced cell death. Oxidative stress was induced by applying the catalase (CAT) inhibitor aminotriazole (AT) in the growth media, which results in decrease in CAT enzyme activity, H2O2 accumulation, and subsequent plant death. One mutant was recovered from the screening and named oxr1 (oxidative stress resistant 1). The location of the T-DNA insertion was identified by TAIL-PCR. Oxr1 exhibited lack of cell death symptoms and more fresh weight and chlorophyll content compared to wild type. The lack of cell death correlated with more prominent induction of anthocyanins synthesis in oxr1. These results demonstrate the feasibility of AT as a screening agent for the isolation of oxidative stress-tolerant mutants and indicate a possible protective role for anthocyanins against AT-induced cell death. The chapter includes protocols for ethyl methanesulfonate mutagenesis, mutant screening using AT, T-DNA identification by TAIL-PCR, CAT activity measurements, and determination of malondialdehyde, chlorophyll, and anthocyanins.

Genetic diversity of Orobanche cumana and Orobanche cernua populations as revealed by variability of Internal Transcribed Spacers1/2 of ribosomal cistron and ribulose-bisphosphate carboxylase pseugene

Kirilova, Ina et al.-- Trabajo presentado en el Third Internacional Symposium on broomrape (Orobanche spp.) in Sunflower, celebrado en Córdoba (España) del 3 al 6 de junio de 2014.The sunflower broomrape - Orobanche cumana (Wallr) parasitizes on roots of sunflower plants and is a serious constraint on sunflower production, causing yield losses of up to 60%. The variability of Internal Transcribed Spacers1/2 of ribosomal cistron (ITS1/2) and ribulose-bisphosphate carboxylase pseugene (RbcL) in 32 samples of O. cumana and 4 samples O. crenata collected from different European locations were studied. The results showed that O. cumana can be differentiated from O. cernua, by single C/T transition located in ITS2 (rel. position 423). Rubisco large subunit in O cumana differs from O. cernua with two transversion: T/G (rel. position 15) and A/C (rel. position 84). The genetic diversity observed in O. cumana was lower than in O. cernua. When comparing the ITS and rbcL sequences isolated from O. cernua two single SNPs were found that can discriminate different origins. Both ITS1/2 and rbcL sequences isolated from O. cumana however were completely homogeneous, despite the fact that samples were collected form very distant locations: from Volgograd, Russia to the East to Spain to the West. This observation is in favor of hypothesis that the move of O. cumana from wild hosts on sunflower was a single act that occurred once and all invasive races are descendants from ancient Caucasus population. Probably genes related to O. cumana aggressiveness should be identified and used for molecular markers to determine genetic relationships within and among O. cumana populations.N

Reactive oxygen species as signals that modulate plant stress responses and programmed cell death

International audienc

Recent advances in Broomrapes research

Orobanchaceae (broomrapes) is a morphologically diverse family of predominantly herbaceous, parasitic plants. The majority of species are facultative or obligate root parasites that subsist on broad-leaf plants, thereby depleting them of nutrients, minerals and water. The taxonomy status of the family Orobanchaceae among other flowering plants is often subject of debate. They possess only a few morphological features suitable for taxonomy purposes and yet even they are quite changeable. The variability within the species is too high and hampers the attempts to create proper determination keys. During last two decades several molecular markers were used for reevaluate taxonomy, biodiversity and phylogenetic relationships within the family. Recent investigations supported by molecular taxonomy analyses have resulted in re-definition of Orobanchaceae family. According to this classification Orobanchaceae consists of 89 genera, containing 2061 species. On the Balkans the family Orobanchaceae is represented by 3 genera: Orobanche includes 25 species; Phelipanche comprises of 9 species and some putative hybrids; Diphelypaea occurs with single species, Diphelypaea boissieri, in Macedonia and Greece. Only a few recent studies based on modern methods took place during last decade. Their findings confirmed differences between Phelipanche and Orobanche genera, but raised new question about their internal structure. Several broomrape species parasitize important crops. They are widely spread in Bulgaria, Southern Europe, Russia, Middle East and Northern Africa. They cause losses in crop productivity estimated at hundreds of millions of dollars annually than affect the livelihoods of 100 million farmers. A wide variety of approaches have been explored to control broomrapes, but none have been found to be sufficiently effective and affordable. The new findings about their life cycle and the recent genomic project focused on sequences of Ph. aegyptiaca genome open new perspectives for management of the harmful broomrape species and for understanding of their biology and evolution as well