Sasa Mitrev

Assessment of the genotoxicity of heavy metals in Phaseolus vulgaris L. as a model plant system by Random Amplified Polymorphic DNA (RAPD) analysis

Impact assessments of environmental pollutants are important in eco-genotoxicology. A random amplified polymorphic DNA (RAPD) technique was used to detect genotoxicity-induced DNA damage in Phaseolus vulgaris L. from heavy metals at two different concentrations. The results from six 10-base pair (bp) random RAPD primers with 60–70% GC content used, showed a total of 295 RAPD fragments of 700–4000 bp in molecular size in the seedlings of untreated and treated samples, of which only 163 fragments were polymorphic. Polymorphisms became evident as the disappearance and/or appearance of DNA fragments in treated samples compared to the control. A dendrogram constructed using the Numerical Taxonomy and Multivariate Analysis System (NTSYSps), showed that the control group merged with groups treated with CuSO4·5H2O (150 mg L−1) and MnSO4·H2O (150 mg L−1) in a separate cluster. These groups were linked with all of the other samples treated with metals at concentrations of 150 mg L−1 and CuSO4·5H2O and Cd(NO3)2 at concentrations of 350 mg L−1. Finally, the samples treated with metals at concentrations of 350 mg L−1 together with NiSO4 at the concentration of 150 mg L−1, clustered separately. The DNA polymorphism detected by RAPD analysis offered a useful biomarker assay for the detection of toxic chemicals genotoxicity in plant model systems.

Gibberella fujikuroi (Sawada) Wollenweber, the new parasitical fungus on rice in the Republic of Macedonia.

The recent observation of Gibberella fujikuroi (Sawada) Wollenworth (teleomorph) (anamorf: Fusarium moniliforme Sheld.) Fusarium fujikuroi Nirenberg (ana- morph), F. moniliforme J. Sheld. (synonym), the causal agent of Bakanae disease in rice fields, provides an opportunity to observe the characteristics of the pathogen and the possibility of prevention in Kocani area. Plant material with Gibberella fujikuroi symptoms was collected from rice (Oryza sativa) over the period of 3 years (from 2006 to 2008). Within this study, the presence and damage caused by this pathogenic fungus were con- firmed. The objective of this study was a continuous field observation of symptoms in order to establish the percentage of infection and use of laboratory methods for proper protection.

Bipolaris sorokiniana (Teleomorph Cochliobolus sativus) - causer of barley leaf lesions and root rot in Macedonia.

Diseased barley plants (Hordeum vulgare) were noticed in the area of Kumanovo, Bitola, Probistip, Skopje and Kocani, at the beggining of March 2006. The plants were highly diseased, probably in the stage of germination, dwarfed with necrotic leaves and with poorly developed root. A rotten root collar was noticed notice in some plants, which could be easily pulled out from the soil. Plants infected in a later developing stage became yellow from the top of the leaf, and many brown olive, oval shape lession were noticed. Conidia of Bipolaris sorokiniana (Sacc.) Shoen., were isolated from symptomatic lesions. Pseudotecia with asci and ascospores from teleomorph Cochliobolus sativus, were found on the barley straw in the same field as the previous year.

FIRST APPEARANCE OF WHITE MOULD ON SUNFLOWER CAUSED BY Sclerotinia minor IN THE REPUBLIC OF MACEDONIA

SUMMARY Sclerotinia spp. a very destructive fungus causing “white mould” became one of the biggest problems in sunflower breeding in the Republic of Macedonia in 2010. Field monitoring in the region of Bitola show very high infection of around 20-30%. Two types of symptoms where observed during the field monitoring. First symptoms were observed on the leaves of the infected plants in the form of wilting, prior to flowering stage. The most characteristic symptoms were observed, at the lower part of the stem in the form of a stem cancer. Big variable sclerotia in size and shape were observed inside the stem. The appearance of white mycelium on the infected lower parts of the plant was often observed during the wet weather. Other infected plants showed different symptoms. The stem was longer and thinner than in uninfected plants, and the pit was very small, around 9 cm. Sclerotia observed inside the stem were not bigger than 2.5 mm. In vitro investigations confirmed the presence of ascomycetes Sclerotinia sclerotiorum (Lib.) de Bary and Sclerotinia minor Jagger, for the first time in the Republic of Macedonia. In vitro investigation of antifungal ability of Pseudomonas mediterranea Cattara et al., 2002 against S. sclerotiorum and S. minor showed that it can be a possible antifungal agent against these ascomycetes.

Correlation and path analysis in sunflower (Helianthus annuus L.)

Abstract This study was carried out to determine the correlation between agronomic traits and seed yield of sunflower hybrids and also to determine the direct and indirect effects of analyzed traits on seed yield. The primary task in sunflower breeding programs is to increase the seed yield, as well as create new hybrids with high potential for seed and oil yield. The experiment was set up according to randomized block design during two growing seasons (2013 and 2014), on research area of the Faculty of Agriculture, “Goce Delchev” University – Shtip, in Ovche Pole locality, Republic of Macedonia. As an experimental material 20 sunflower hybrids were used. 1,000 seed weight, seed length and width, oil content, oleic acid content and seed yield were analyzed. 1,000 seed weight showed positive and significant correlation with seed width, seed length and seed yield. On the other hand, negative and significant correlation was obtained between 1,000 seed weight and oil content. 1,000 seed weight expressed the highest positive direct effect on seed yield. The results obtained from correlation and path analysis showed that the efficiency in the selection of seed yield in sunflower hybrids should increased through the selection of 1,000 seed weight.

PSEUDOMONAS POPULATION CAUSING TOMATO PITH NECROSIS IN THE REPUBLIC OF MACEDONIA

Pseudomonas species causing pith necrosis on tomato were collected from 2005 to 2012 in different regions of the Republic of Macedonia and identified as Pseudomonas mediterranea and P. viridiflava. Other Pseudomonas spp. were also recovered, but they are still under identification. The obtained bacterial isolates were compared with reference strains on the basis of phenotypic characteristics, pathogenicity, sensitivity to antibiotics, BIOLOG tests, DNA fingerprinting by multiplex PCR using two specific primers [type I (PC5/1 and PC 5/2) and type II (PC1/1 and PC 1/2)], and 16S rRNA-RFLP analysis. Unique DNA bands from 10 representative strains isolated from symptomatic tomato plants disclosed the presence of P. mediterranea by amplification of their genomic DNA. All strains produced one specific band (600 bp) compared with the representative samples IPVCT9.1 (P. mediterranea). Also, we used another representative sample IPVCT10.3 (P. corrugata), to check if we already have P. corrugata or no, in our samples. In order to investigate the bacterial populations causing tomato pith necrosis in Macedonia, a total of 150 isolates were collected and compared with reference strains. This is the first report on Pseudomonas population causing pith necrosis of tomato in the Republic of Macedonia.

Present status of Fire Blight Caused by Erwinia amylovora on Quince Trees in the Republic of Macedonia

Erwinia amylovora, causal agent of fire blight disease, is an important and destructive bacterial pathogen, mainly on Rosaceae family such as pears, apples and quinces. Present status of E. amylovora, isolated from quince trees (Cydonia oblonga) from different localities in the Republic of Macedonia, included the period 2015-2016, are presented in this study. Severe occurrence of symptomatic quince threes, was recorded in almost all observing localities in Macedonia. After blooming period appeared first symptoms, most expressed symptoms were recorded during intensive growth of shoots. Some resulted from isolated strains elicted HR in tobacco, necrosis of pear seedlings and immature pear fruits. Strains have several biochemical characteristics typical and compare with positive control, such as: non-fluorescent, produce levan; oxidase negative, catalase positive; hydrolysis of gelatine positive; esculin and starch tests were negative; strains metabolise sucrose, sucrose, fructose and galactose, etc. Amplification of total DNA with PCR followed by agarose electrophoresis, originating from quince showed indistinguishable PCR patterns for all investigated native strains that were identical with European reference strains (LMG 1883). The aim of this study was to: i) check the status of E. amylovora on quince and ii) laboratory diagnostics and determination of isolated strains. The importance of the results is that disease incidence is sporadic and crop damages are of medium economic significance and it’s important to know that E. amylovora is present on quince only to be careful of serious outbreaks of fire blight on quince in Macedonia..

Groundwater quality in alluvial and prolluvial areas under the influence of irrigated agriculture activities.

ABSTRACT The aim of this study was to investigate the groundwater pollution from alluvial aquifers lying under surface agriculture activities in two geologically different areas: alluvial and prolluvial. The groundwater in investigated areas is neutral to alkaline (pH 7.05–8.45), and the major dissolved ions are bicarbonate and calcium. Groundwater samples from the alluvial area are characterized by nitrate concentration above the national maximum concentration limit (MCL) at 20.5% of samples [mean value (Me) 6.31 mg/L], arsenic concentrations greater than national MCL at 35.6% of investigated samples (Me 12.12 µg/L) and elevated concentrations of iron (Me 202.37 µg/L) and manganese (Me 355.22 µg/L) at 22.7% and 81% of investigated samples, respectively. Groundwater samples from the prolluvial area did not show significantly elevated concentrations of heavy metals, but the concentration of nitrate was considerably higher (Me 65.06 mg/L). Factor analysis positively correlates As with Mn and Fe, suggesting its natural origin. Nitrate was found in positive correlation with SO42− and Ni but in negative with NH4+, suggesting its anthropogenic origin and the relationship of these ions in the process of denitrification. The t-test analysis showed a significant difference between nitrate pollution of groundwater from alluvial and prolluvial areas. According to the chemical composition of groundwater, the process of denitrification is considered to be the main reason for the reduced presence of nitrate in the groundwater lying under alluvial deposits represented by chalk and sandstones. Denitrification in groundwater lying under prolluvial deposits represented by magmatic and metamorphic rock formations was not observed.

Evaluation of Sunflower (Helianthus annuus L.) Hybrids Using Multivariate Statistical Analysis

Abstract Evaluation and characterization of sunflower hybrids using morphological, physiological and biochemical data are necessary and essential in sunflower breeding programs. The aim of this paper was to evaluate the productive possibilities of some sunflower hybrids grown in Macedonian environmental conditions. The experiment was carried out during the period 2013 and 2014 on the research field of the Faculty of Agriculture, “Goce Delchev” University – Shtip, in Ovche Pole locality, Republic of Macedonia. Total 20 sunflower hybrids were used as an experimental material. The trial was arranged as randomized complete block design (RCBD) with three replications. The average content of oil and oleic acid were 45.6 and 65.2%, respectively. The highest seed yield from all sunflower hybrids was obtained for the hybrid NLK12M144 (3,344 kg ha–1) and the lowest for hybrid NLK12S126 (2,244 kg ha–1). Cluster analysis classified the sunflower hybrids into four groups based on agronomic traits and seed yield. Most of the hybrids were included in cluster I and III (7 hybrids) followed by cluster IV. Using principal component analysis two main components have been extracted, with eigenvalue greater than one. They account for 72.99% of the variability in the original data. Only four hybrids had positive values for both main components (NLK12M144, NLK12S070, NLK12S125 and NLN12N011 DMR). The results from this study can be used to identify the best sunflower hybrids grown under Macedonian environmental conditions and to select the superior hybrids for future exploitation.