Main fields of research
- the detection and identification of plant diseases caused by viruses, bacteria or fungi using traditional research methods, immunoelectronmicroscopy and molecular biology techniques;
- the etiology of plant pathogens;
- the pathogenesis, especially the role of mycotoxins and mycotoxin-forming fungi in the disease process;
- the epidemiology of infectious plant diseases;
- the variability of plant pathogens and the response of plant species or varieties to diseases;
- the hyperparasitism of various fungal species.
Today the faculty members of the Department are working on 2 projects which are funded by the Ministry of Science and Higher Education. Five PhD students are working on their own projects which are financed by The Dean of the Faculty of Horticulture and Landscape Architecture. The research results are being published in international journals with IF.
Since 2009 regular meetings with the Mazovia Region high school students have been organised. The meetings take place during WULS Open Days and popularize knowledge on plant disease factors.
The faculty members of Department run classes for both full-time and extramural students of Engineer and Master degree Courses in Biotechnology, Biology, Horticulture and Agriculture, including English classes for the Socrates/Erasmus/Mundus Programme students. They also supervise numerous bachelor, engineer and master degree thesis projects of students.
Abstracts of major research projects
CHODORSKA M., PADUCH-CICHAL E., KALINOWSKA E., GACZKOWSKA O., LIS M., SIERANT B., SZYNDEL M.S.
"Leek yellow stripe virus in Poland".
Leek yellow stripe virus (LYSV), genus Potyvirus, family Potyviridae (King et al., 2011) is the most common and important virus infecting a wide range of Allium worldwide. The aim of this study was to detect and identify LYSV in leek and garlic plants originating from central Poland, and also in materials from Belgium, Egypt, and Spain purchased in Polish markets in April 2014. Randomly collected 178 samples were tested by a double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA), according to the manufacturer's instructions (DSMZ, Braunschweig, Germany). All leek plants tested negative for LYSV, whereas 31 of 120 garlic bulbs tested positive. The presence of LYSV was confirmed by reverse transcription RT-PCR using total RNA extracted with the silica capture method (Boom et al., 1990; Malinowski, 1997) and primers 1-LYSV/2-LYSV (Parrano et al., 2012) designed to amplify a part of the N-terminal domain of the coat protein (CP) gene of the virus (363 bases). A sequence of the partial CP genes of the 12 LYSV isolates was submitted to GenBank (Accession Nos KM032272-KM032283). BLAST analysis of Polish sequences showed 96-99% identity at the nucleotide and amino acid levels. Sequences of Egyptian isolates, first representatives from this locations, showed 92 and 95% nucleotide and amino acid identities, respectively. Spanish isolates revealed 95% and 97% nucleotide and amino acid identities, respectively. To the best of our knowledge, this is the first report of LYSV in foreign and Polish garlic plants available for purchase in central Poland. The accurate identification of viruses present in garlic plants, especially in the imported plant material, will help to use the appropriate strategies to reduce viral incidence in garlic-growing areas.
CHODORSKA M., PADUCH-CICHAL E., KALINOWSKA E., SZYNDEL M.S.
"Allexiviruses infection in garlic plants in Poland".
Garlic plants may be infected in the field by viruses of the genera Potyvirus, Carlavirus and Allexivirus. These viruses are transmitted by vegetative propagation and by vectors. Detection and identification of allexiviruses based on ELISA and RT-PCR assays was carried out in 2011-2012. Samples of plant material were collected from 26 garlic production fields located in different regions of Poland. Garlic virus D, Garlic virus B and Garlic virus X were the most frequent viruses in all examined regions. They were identified in 79, 64 and 59 % of all garlic samples, respectively. Garlic virus A and Garlic virus C were identified in all studied regions with low frequency. Garlic virus E was detected with 100% frequency in east-central Poland. None of the tested garlic samples were infected with Shallot virus X. Allexiviruses were always present in garlic plants in mixed infections.
CHODORSKA M., PADUCH-CICHAL E., KALINOWSKA E., SZYNDEL M.S.
"Garlic virus D, E and X on garlic in Poland".
Several viruses from the genus Allexivirus, family Alphaflexiviridae, infect garlic and other species of Allium. They commonly occur in multiple infections and are thought to be mite-transmitted (Lu et al., 2008). A survey for viruses was conducted in garlic plants showing mosaic, deformation, and yellow stripes in 13 fields located in different regions of Poland during 2011-2012 growing season. Total RNA was extracted from bulb tissue using a Spectrum plant total RNA kit (Sigma-Aldrich, USA) and tested for Garlic virus D (GarV-D), Garlic virus E (GarV-E), and Garlic virus X (GarV-X) by reverse RT-PCR using the transcriptor one-step RT-PCR kit (Roche, USA) and primers: (i) DCPF/DCPR 5‟-AAGGAGCTACACCGAAGGAC-3‟and 5‟-TAAAGTCGTGTGGATGCATCAGA-3‟; (ii) EF2/ER2 5‟-TTGCTAGACCACCTCAGTATTGAGAA-3‟ and 5‟-TATTGGGCG TACATCGGTGACTGT-3‟; (iii) XF/XR 5‟-GCGGTAATATCTGACACGCTCCA-3‟ and 5‟-ACGTTAGCTTCACTGGGGTAGAATAT-3‟, respectively. Products of the expected size (456 bp for GarV-D, 458 bp for GarV-E, and 386 bp for GarV-X) were amplified from 27 (87%), 16 (52%) and 26 (84%) samples, respectively. Twelve samples were infected by the three viruses and 12 by GarV-D and GarV-X. To our knowledge, this is the first report of GarV-D, GarV-E and GarV-X in garlic in Poland.
CHODORSKA M., PADUCH-CICHAL E., KALINOWSKA E., SZYNDEL M.S.
"Onion yellow dwarf virus, Garlic common latent virus and Shallot latent virus on garlic in Poland".
Garlic is vegetatively propagated and can be affected by a virus complex consisting of two potyviruses, Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), and two carlaviruses, Garlic common latent virus (GCLV) and Shallot latent virus (SLV). OYDV, GCLV, and SLV are economically important viral pathogens of bulb garlic crops in many garlic-growing areas of the world. A general mosaic and yellowing of leaves of four garlic cultivars (Blanko, Harnaś, Jarus, and Mega) was observed in 11 garlic-production fields in the Lodz, Mazowieckie, Małopolskie, and Pomorskie regions of Poland. ELISA was carried out with extracts from 29 collected garlic leaf samples to detect OYDV, GCLV, and SLV using commercial antiserum (DSMZ, Braunschweig, Germany). Results indicated that 6 samples (20.7%) were infected with OYDV, 25 samples (86.2%) were infected with GCLV, and 23 samples (79.3%) were infected with SLV. The presence of these viruses in garlic leaf samples was confirmed by reverse transcription RT-PCR using total RNA extracted using the Spectrum Plant Total RNA kit (Sigma-Aldrich, Munich, Germany) and primers, designed in this study, specific to the whole coat protein gene of OYDV (OYDVF 5‟-TAGGGTTGGATTATGATTTCTCGA-3‟ and OYDVR 5‟-TAGTGGTACACCACATTTCGT-3‟), GCLV (GCLVF 5‟-TTATAGGGACGGCAC AAAATCAATCA-3‟ and GCLVR 5‟-AATAGCACTCCTAGAACAACCATT-3‟) and SLV (SLVF 5‟-AATYATTTACAATCGTCCAGCTA-3‟ and SLVR 5‟-ATAATATCA ATCAAATMCACACAATT-3‟). Amplicons of the expected size were obtained for each virus. The amplified products were purified and sequenced in both directions. Sequence information of the CP genes of 9 OYDV, 12 GCLV, and 7 SLV isolates has been submitted to NCBI-GenBank with accession numbers KF862683 to KF862710. Sequence analysis showed that the coat protein gene of OYDV shared 86% identity with the coat protein gene of OYDV isolate MS/SW1 from Australia (GenBank Accession No. HQ258894). Comparison of the coat protein gene sequences of Polish GCLV isolates with those available in GenBank showed 85 to 91% sequence identities. Multiple sequence alignment revealed 84% nucleotide identity between the Polish isolate of SLV and an SLV isolate from Chinese garlic (AF314147) formerly referred to as Garlic latent virus. To the best of our knowledge, this is the first report of OYDV, GCLV, and SLV in garlic plants in Poland. The accurate identification of viruses present in garlic plants will help to use the appropriate strategies to reduce viral incidence in garlic-growing areas.
GRZYB M., SCHOLLENBERGER M.
"The influence of bacterium Bacillus subtilis on yield and health of red pepper and eggplant".
The aim of the study was to evaluate the influence of biopesticide containing bacterium Bacillus subtilis on red pepper and eggplant crop. The biopesticide was applied during watering (fertigation) of plants in the greenhouse. The results showed that after bacterium application plant infection by fungi from genera Verticillium and Fusarium lowered but there were some differences in gray mold occurring. The average yield of red pepper and eggplant increased by about 10% what was connected with low fungi infection and development of root system. The roots of protected plants were longer, with many branches, with no discoloration nor cracks. Root clods of control plants were much smaller than those of protected plants.
KALINOWSKA E., MROCZKOWSKA K., PADUCH-CICHAL E., CHODORSKA M.
"Molecular characterization of Prune dwarf virus isolates".
Prune dwarf virus (PDV) isolates have been investigated for genetic diversity. Full-length nucleotide and amino acid sequences of viral coat protein of 23 isolates collected from different stone fruit trees (sour and sweet cherry trees, wild cherry tree, plum tree, almond tree, peach tree) and different countries (Poland, Italy, Germany, USA, Israel) were analysed and compared to 57 others available in GenBank. Comparison of all sequenced virus isolates revealed diversity of 86-100% at nucleotide level and 79-100% at amino acid level. The ratio of non-synonymous to synonymous polymorphic sites indicated that purifying selection dominated in case of PDV. However, six codons showed to be under strong positive selection, including the codon located inside the structure involved in RNA binding activity.
MIRZWA-MRÓZ E., KUKUŁA W., DZIĘCIOŁ R., WIT M., BĄCZEK K., WĘGLARZ Z., PAWEŁCZAK A.
"First report of Colletotrichum graminicola (Ces.) Wilson on southern sweet-grass leaves in Poland".
Southern sweet-grass (Hierochloë australis) is a perennial tuft-grass the leaves of which are used for aromatization of alcohol and tobacco products. In 2013 in Warsaw-Wilanów (Poland) oblong, irregular lesions with black border and yellow, reddish-brown discoloration, and black acervuli with setae were observed on H. australis leaves. Seven isolates of the fungus were obtained from infected leaves on potato dextrose agar (PDA). Cultures were black-gray with white mycelium. After seven days of incubation on PDA 100 spores of each isolate were measured; conidia hyaline, 1-celled, lunate to falcateshaped, average size 22.4 x 4.4 μm, developed in black acervuli. All isolates produced melanized appressoria. Linear growth on: PDA, Czapek solution agar, CMA (corn meal agar), MEA (malt extract agar) and SNA (salt nutrient agar) media was examined. The best growth of fungus after ten days of incubation was observed on PDA (diameter 7.3 cm) and Czapek solution agar (6.2 cm) media. The slowest one was observed on SNA (3.4 cm). Total DNA was extracted from 10 day-old cultures. Amplification of rDNA fragments (ITS1, 5.8S, ITS2) with primers ITS1 and ITS4 was performed according to Hsiang and Goodwin (2001). Amplicons were sequenced and analysed by ClustalW and compared with sequences in GeneBank using BLAST. Sequences showed 99% homology to Colletotrichum graminicola. Sequences from two isolates were submitted to GeneBank (Accession Nos KM040784, KM040785). To our knowledge, this is the first report of C. graminicola on H. australis in Poland. So far, in Poland this pathogen has been noted on maize (2006) and bentgrass (Agrostis stolonifera).
MIRZWA-MRÓZ E., WIŃSKA-KRYSIAK M., DZIĘCIOŁ R., MIĘKUS A.
"Characteristics of Aureobasidium pullulans (De Bary et Löwenthal) G. Arnaud isolated from apples and pears with symptoms of sooty blotch in Poland".
Sooty blotch is a disease of apple and pear caused by a complex of fungi that blemish the fruit surface. Results of molecular studies indicated approximately 30 different fungi species associated with this disease. Apples and pears with symptoms of sooty blotch were collected in summer and early autumn 2006-2010 from trees grown in orchards and small gardens non-treated with fungicides located in various regions of Poland. Fungi causing sooty blotch were isolated from fruits and the isolates were divided into six groups, according to their morphological characters. Growth of the fungi colonies was measured on different agar media (PDA, CMA, MEA and Czapek‟s). The ITS region of rDNA from 16 isolates from the first group was amplified by PCR technique and one representative sequence of these isolates was used to alignment in Gene Bank. This isolate was identified as Aureobasidium pullulans and isolates from this group were compared with it on the base of morphological features.
PADUCH-CICHAL E., CHODORSKA M., KALINOWSKA E., KOMOROWSKA B.
"Blueberry scorch virus detection in highbush blueberry".
Viral diseases are worldwide problem of blueberry as a major limiting factor for production. A survey for Blueberry scorch virus (BlScV) by DAS-ELISA in various organs of highbush blueberry conducted from May 2010 to April 2011, showed the occurrence of this virus in cvs. Bluecrop and Herbert, which showing virus-like symptoms. Samples of plant material (bud flower, flower, leaf, bark) were collected individually from each highbush blueberry plant of each cultivar. It was established that the detection of virus in individual investigated bushes cvs. Bluecrop and Herbert depended on the tested plant material as well as the period in which the tests were performed. The effectiveness of virus detection varied for the investigated cultivars. The presence of BlScV was confirmed in leaf samples with specific primer pair which amplifies a 430 bp fragment of the 5‟-proximal ORF I RNA-dependent RNA polymerase (RdRp).
SCHOLLENBERGER M., DRYKA S.
"Bacterial diseases of selected ornamental plants".
The objective of the study was the isolation and identification of pathogenic bacteria from the leaves of 16 species of ornamental shrubs. A characteristic symptoms observed on all of the collected leaves were dark brown or black spots seen clearly on the upper as well as on the lower side of the leaf blade. A total of 18 isolates were obtained from the samples collected from 12 different species of shrubs. In LOPAT tests three isolates were identified as Pseudomonas syringae pv. syringae, this patovar was the cause of Clematis Atragene group, Cornus mas and Prunus laurocerasus leaf blight. Thanks to the results of additional physiological and biochemical tests other isolates were classified into four genera: Burkholderia, Flavobacterium, Pantoea and Xanthomonas.
SCHOLLENBERGER M., REDLICKA M.
"Etiology of Tilia x europaea L. leaf spot".
The aim of the study was the identification of the cause of spots, which occurred only on one species - common linden in nursery near Warsaw. Five type of bacterial colony were isolated from leaf spots and buds collected from infected linden and analyzed using standard microbiological tests. Among obtained bacterial isolates dominated type identified as Pseudomonas syringae.
WIT M., WAKULIŃSKI W., JABŁOŃSKA E.
"Fusarium temperatum as a new main factor of ear rot of maize in Poland".
The molecular studies conducted in 2014 were aimed on estimation of the frequency of Fusarium temperatum among selected isolates of Fusarium spp., collected in Głuchów and Radzików. Previous mycological analyses (2005-2012) showed that Fusarium subglutinans was predominant, but recent EF1& gene analyses showed high frequency (55 isolates) of new species - Fusarium temperatum. Among all tested strains just only one was identified as Fusarium subglutinans. The preliminary results of breeding demonstrate new look at etiology of corn cob fusariosis. Both species from Liseola section are morphologically very similar, and polymorphism of EF1& gene allowed us to recognize the difference.
DOLIŃSKA T.M., BARTKOWSKA A., SCHOLLENBERGER M.
"Light and scanning microscope observations of Cladosporium uredinicola growth on rust fungi".
In the years 2009-2010 relationship between the hyperparasite Cladosporium uredinicola and various rust fungi was investigated. Fourteen various rusts were tested under light stereomicroscope and scanning microscope in order to confirm the relations between the fungi. The hyperparasite produced olive green mycelium on 10 species of rust fungi. Gymnosporangium spp. and Tranzschelia spp. were infected most frequently. The type of the sori also proved important - the most intensive infection occurred on spermogonia and aecia. Directly close to the host spore surface, C. uredinicola produced infection cushions as well as structures similar to appressorium. The hyperparasite often caused spore deformation leading to their destruction. In some cases the hyphae of the fungus plunged into the rust spore surface, which may confirm the fact that it produced lytic enzymes.
DOLIŃSKA T.M., SCHOLLENBERGER M.
"Parasiting of Cladosporium species on Puccinia arenariae".
The aim of this study was the assessment of interaction between hyperparasites from Cladosporium species and rust Puccinia arenariae which infected carnation (Dianthus barbatus). The isolates of Cladosporium were collected from rusts and powdery mildews on annual and perennial plants. In laboratory conditions carnation leaves with rust symptoms were inoculated with hyperparasites spores suspension. Leaves were observed by stereomicroscope, as well as by scanning and transmission electron microscope. The relationship between spores of P. arenariae and Cladosporium spp. was also tested. After three days olive-green conidiophores of hyperparasites were noticed on infected telial sori. The observations showed that hyperparasites formed thickening similar to appressorium on the surface of rust spores. Cladosporium spp. led to deformation and degradation of teliospores. The most effective hyperparasites were C. aecidicola, C. herbarum, C. tenuissimum and C. uredinicola.
KALINOWSKA E., CHODORSKA M., PADUCH-CICHAL E., MROCZKOWSKA K.
"An improved method for RNA isolation from plants using commercial extraction kits".
Isolation of RNA from plants rich in secondary metabolites using commercial kits often results in contaminated preparations which are not suitable for downstream applications. Although many specific protocols appropriate for plants with a high content of phenolics, anthocyanins and polysaccharides have been developed, these are often expensive, time consuming and not applicable to different types of tissues. This study presents a simple and efficient modification of RNA extraction from different types of tissues using two commercial reagent kits. By simple improvement, we routinely obtained high-quality RNA of the following plants: the blackcurrant bush, black chokeberry bush, pear tree apricot tree, apple tree, hardy kiwi, tangerine tree, highbush blueberry and cranberry plant.
KALINOWSKA E., PADUCH-CICHAL E., CHODORSKA M.
"Molecular characterization of Polish isolate of Blueberry red ringspot virus".
In this study, we determined the complete sequence of the genomic DNA of a Polish isolate of Blueberry red ringspot virus (BRRSV24) and compared it with Czech (Darrow 5), and US isolates of the virus and those of other Caulimoviridae family. The genomic DNA of BRRSV24 consists of 8,265 nucleotides and encodes eight open reading frames (ORFs). The sequence homologies of the eight ORFs of BRRSV24 were from 95 to 98% in respect of Darrow 5 and from 91 to 98% in respect of the US isolates at the amino acid level. This high level of amino acid sequence identity within the coding regions among the Czech, the US and Polish BRRSV isolates is suggestive for their common origin.
KALINOWSKA E., PADUCH-CICHAL E., CHODORSKA M.
"Low genetic diversity of the coat protein gene among Blueberry red ringspot virus isolates".
Blueberry red ringspot virus (BRRSV) isolates have been investigated for genetic diversity. Nucleotide sequences of the coat protein (CP) gene of 19 isolates from Poland, Czech Republic, Slovenia and the United States were analyzed. The nucleotide and amino acid sequence identity were 92-100% and 89-100%, respectively. Estimations of the distribution of synonymous and non-synonymous changes indicated negative selection within the analyzed CP gene and confirmed the genetic stability of the virus. At a capsid protein level, our results revealed BRRSV to be distinct from other, recombination-prone pararetroviruses.
MIRZWA-MRÓZ E., WIŃSKA-KRYSIAK M., DZIĘCIOŁ R.
"Diversity of sooty blotch fungi in Poland".
Sooty blotch is one of the most common disease of apples in organic orchards in many countries. Results of molecular studies performed in USA indicated approximately 30 different fungi species associated with this disease. Fungus species causing sooty blotch in Northern, Central and Eastern Poland were identified on the basis of morphology and nucleotide sequence of the rDNA internal transcribed spacer region (ITS). A total 245 isolates were collected in spring and early summer in the years 2006-2009 from fruits with visible symptoms of the disease. Isolates were grown on PDA medium and identified on the basis of morphological characters. DNA was extracted from representative isolates and used as matrices for PCR amplification with ITS1F and ITS4 primers. Fragments of amplified rDNA ITS were sequenced. It was found that 66.53% of all isolates causing sooty blotch were species from genera Microcyclosporella, followed by Aureobasidium pullulans - 22.86%, Microcyclospora sp. - 6.12%, Phialophora sessilis - 3.67%, Peltaster sp. and P. fructicola - 0.41%.
SALA-REJCZAK K., PADUCH-CICHAL E., LATOCHA P.
"Cucumber mosaic virus in Actinidia spp. plants".
Actinidia spp. plants (Actinidia arguta, A. chinensis, A. deliciosa, A. kolomikta, A. melanandra, A. purpurea), were collected from different regions of central Poland during 2011-2012. The actinidia leaves were tested by DAS-ELISA (double antibody sandwich - enzyme linked immunosorbent assay) (Clark and Adams, 1977) using Loewe Biochemica GmbH reagents. The investigated plants were infected with Cucumber mosaic virus (15%). In electron microscope preparations (‘sap-dip’ EM technique; Szyndel, 1997) obtained from actinidia leaves isometric Cucumber mosaic virus particles were observed.
SALA-REJCZAK K., SZYNDEL M.S., MROCZKOWSKA K., NOWAK P., CHODORSKA M., PADUCH-CICHAL E.
"Transmission electron microscope in Prune dwarf virus (PDV) and Allexiviruses detection".
The electron microscope techniques were used to identify Prune dwarf virus (PDV) isolates originating from Department of Plant Pathology collection. ‘Sap-dip’ EM, ISEM (immunosorbent electron microscopy) and ‘decoration’ techniques (Szyndel, 1997) were used. Samples were prepared using cotyledons and mature leaves of Cucumis sativus cv. Wisconsin test plants inoculated with different isolates of PDV. In EM preparations characteristic isometric virus particles were observed.
Garlic plants showing viral disease symptoms were randomly collected from garlic production fields located in different regions of Poland during the 2011-2012 growing season. In electron microscope preparations obtained from symptomatic garlic leaves and prepared using ‘sap-dip’ EM technique characteristic viral particles about 600-800 nm in length characteristic for Allexivirus genus were observed.
SCHOLLENBERGER M., FELCZAK K., BOROWSKA K.
"The effect of epiphytic bacteria on selected rust fungi".
The aim of the study was to characterize the relation of epiphytic bacteria, which colonized the phyllosphere of daisy, carnation and rose, and check their antagonism against spores of Puccinia distincta (daisy rust), P. arenariae (carnation rust) and Phragmidium mucronatum (rose rust). Among 107 obtained bacterial isolates 23 showed the effects on rusts spores in laboratory tests. This impact was manifested in deformations of spores, the abnormal growth of fungal hyphae and unusual distribution and concentration of pigment in both: spores and newly emerging hyphae. Based on phenotypic identification isolates were classified into genera: Aureobacterium, Bacillus, Cellulomonas, Micrococcus and Pseudomonas.
WAKULIŃSKI W., WIT M., WARZECHA R., OCHODZKI P., WAŚKIEWICZ A., JABŁOŃSKA E.
"Susceptibility of maize varieties to opposite mating type strains of Fusarium verticillioides".
Fusarium verticillioides the causal agent of maize ear rot is world wide distributed heterothalic species with dimictic mating system of reproduction. Presented paper summarizes results of fourth years (2007-2010) field studies analyzing disease severity of maize plant after inoculation with opposite mating type strains of the fungus. Genotypes of fourth maize varieties (Zea mays var. indentata, Zea mays var. indurata, Zea mays var. everta and Zea mays var. saccharata) were inoculated with two F. verticillioides isolates KFI 2856 and KFI 3011 representing respectively MAT1-1 and MAT1-2 subpopulations of the species. Infection degree was evaluated using six degree (0-5) rating scale at seven days intervals after inoculation. Obtained results revealed that mating type of analyzed species had not significant impact on maize ear infection in any of growing seasons. Reaction of particular maize varieties against ear rot due to F. verticillioides in particular years were stable. The lowest infection degree exhibited plants of Zea mays var. everta while the highest susceptibility Zea mays var. saccharata. Regardless of cropping seasons genotypes of sweet maize (Su1) were significantly less affected than super sweet genotypes (Sh2).
CHODORSKA M., PADUCH-CICHAL E., KALINOWSKA E., SZYNDEL M.S.
"Allexivirus on garlic plants in Poland".
Garlic (Allium sativum L.) can be infected by numerous viruses that are spread by the seed bulbs during the vegetative propagation of the plants. Eight garlic viruses that belong to the Allexivirus genus are transmitted by mites. The aim of this study was to detect and identify allexiviruses infecting garlic in Poland. Identification of Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C) and Shallot virus X (ShVX) was based on ELISA test and confirmed by RT-PCR technique. Garlic virus D (GarV-D), Garlic virus E (GarV-E) and Garlic virus X (GarV-X) were detected using RT-PCR. The samples of garlic plants were collected in September 2011 and July 2012 from garlic production field located in different part of Poland. GarV-D, GarV-X and GarV-B were the most frequent garlic viruses in all examined regions and were identified in 84, 67 and 54% of all samples, respectively. GarV-A and GarV-C were detected in all regions with low frequency. None of the tested garlic samples were infected with ShVX. Allexiviruses that were present in garlic plants occurred always in mixed infections.
DOLIŃSKA T.M., SCHOLLENBERGER M.
"Molecular technique useful for identification of fungal communities colonizing rust and powdery mildew".
Traditional methods are in use for identification of the well-known hyperparasites as Cladosporium spp., Lecanicillium lecanii, Sphaerellopsis filum or Ampelomyces quisqualis. However, the use of conventional, traditional microscope-based research is laborious and time-consuming and often not satisfactory. More precise molecular technique are used nowadays for fungus identification. Molecular technology was applied in this work for detection and identification of Cladosporium spp hyperparasites.
The isolates of fungi colonizing rusts and powdery mildews were collected in Poland. Single spore cultures grew on PDA medium as axenic cultures. In first step mycelium samples from pure cultures were crushed in liquid nitrogen. All fungal DNA was extracted using two techniques - CTAB/chloroform extraction and DNA extraction kit (Wizard Genomic DNA Purification Kit). To establish the concentration of DNA spectrophotometric quantification method was used. PCR analyses were performed according to the method described by Batzer et al. (2005). The PCR product generated using the ITS1 and ITS4 primers were used as a template for DNA sequencing. Sequencing data were determined and compared in Gen Bank.
The tested molecular method was useful to identify hyperparasitic Cladosporium spp. Techniques using for DNA extraction significantly influenced the quantity and quality of nucleic acid. DNA extracted using instant kit was purer than DNA from CTAB/chloroform extraction. In general, with the use of PCR Cladosporium species were identified in more than 90% of specimens. It means that this molecular method allows the reliable detection of hyperparasites.
DZIĘCIOŁ R., MIRZWA-MRÓZ E., ZIELIŃSKA E., WIŃSKA-KRYSIAK M., WAKULIŃSKI W.
"Valdensinia heterodoxa Peyronel - new patogen of blueberry in Poland".
Valdensia leaf blight on blueberry in Poland was noted in one commercial nursery plantation near Prażmów, Mazovia voivodship, where heavy defoliation was observed in ‘Bluecrop’, grown in nursery pots, in August 2011. Older fruiting bushes were only slightly affected by the disease. Initial symptoms of the disease were small, oval to circular, concentrically zonal necroses surrounded with dark-brown borders that enlarged on the leaves throughout the canopy. Multicellular, hyaline or light brown, star-shaped conidiospores were observed on the necrotic areas. The mean length of 50 conidiospores from the end of head to the end of arm apex was 307-348 μm. Eight single spore isolates of the fungus were obtained. Single conidiospores were picked up from necrotic spots on leaves, transferred to potato dextrose agar (PDA) and incubated at 20°C under ambient light. After 10 days of incubation, total DNA was extracted. Amplification of the internal transcribed spacer (ITS) region of rDNA was done using primers ITS1F and ITS4A. Amplicons, which were approximately 520 bp, were sequenced and nucleotide sequences were analyzed by Clustal W2EBI. The sequences of all eight isolates showed 100% similarity to each other and were compared with sequences stored in GenBank using BLAST. Sequences were 525 bp long and showed 100% homology to Valdensinia heterodoxa Peyronel, Sclerotiniaceae (anamorph: Valdensia heterodoxa Peyronel) from Japan and Norway (Accession Nos. respectively AB663682, Z81447). The sequence from one isolate was submitted to GenBank (Accession No. KF212190). To fulfil Koch’s postulates each of the eight isolates was used to inoculate 20 healthy young leaves of Vaccinium corymbosum L. ‘Bluecrop’ and bilberry (V. myrtillus L) (10 leaves per plant). After 5 days small necrotic lesions consistent with initial symptoms of the disease were observed. Isolates obtained from this symptoms were morphologically identical to those used for inoculation. To our knowledge, this is the first report of Valdensia leaf blight on highbush blueberry in Poland.
FELCZAK K., SCHOLLENBERGER M., WAKULIŃSKI W.
"Typical reaction of rye inbred lines to Puccinia recondita f.sp. secalis infection".
The objective of these studies was the evaluation of the response of rye inbred lines to infection by brown rust under field conditions in two localities - province of Wielkopolska (Great Poland) and West Pomerania. The infection was scored based on a scale from 0 (healthy plants) to 5 (severely infected plants). Most of inbred lines were highly susceptible to infection by Puccinie recondita f.sp. secalis, so disease index reached 5. About 15%, from 318, of estimated inbred lines showed atypical response to infection by urediniospores of brown rust. Leaf chlorosis, chlorosis and necrosis, and necrosis in the form of streaks, stripes and spots occurred. Sometimes few, small uredinia were forming on that leaves. Average measurements of those small uredinia resulted 0.43x0.19 mm while size of regular uredinia on heavily infected leaves was 2.32x0.36 mm. The types of immune response observed on rye leaves were similar to those described on wheat infected by brown rust under field condition.
KALINOWSKA E., PADUCH-CICHAL E., CHODORSKA M.
"Blueberry scorch virus in elderberry plants in Poland".
Blueberry scorch virus (BlScV), a member of the genus Carlavirus is one of the most widespread pathogens of highbush blueberry (Vaccinium corymbosum L.). The virus was first reported in the United States and has been reported in several countries in Europe, including Italy, Germany, the Netherlands, and Poland. Cranberry (V. macrocarpon L.) and wild black huckleberry (V. membranaceum L.) are known as natural symptomless hosts of BlScV. In June 2012, during the research concerning the occurrence of BlScV in plants outside Vaccinium sp., 15 leaf samples from five elderberry bushes (Sambucus nigra L., family Adoxaceae) were randomly collected in Łódź region in Central Poland and three of them were positive in double antibody sandwich (DAS)-ELISA using specific antiserum (Agdia Inc., Elkhart, IN). To confirm the presence of the virus, total nucleic acid was extracted from ELISA-positive elderberry samples according to protocol established by dr. T. Malinowski (1996) and used in one step reverse transcription PCR. Primers were developed against the published NJ-2, BC-1, and BC-2 sequences of BlScV (GenBank Accession Nos. NC_003499, AY941198, and AY941199, respectively). The forward primer, RDP_1 (5′-ATGGCACTCACATACAGAAGTCC-3′), and the reverse primer, RDP_2 (5′-TGCCTCTTCAATGCACGATGTTC-3′), were used to amplify a 420-bp fragment of the RNA-dependent RNA polymerase gene of the virus. Amplicons of expected size were obtained from three DAS-ELISA-positive samples, while no products were observed for the negative control (DAS-ELISA-negative elderberry tissues). Sequence of one selected PCR product revealed 100, 88, and 87% nucleotide sequence identity and 100, 96, and 96% amino acid sequence identity with BC-2, NJ-2, and BC-1, respectively. BlScV-infected elderberry bushes were asymptomatic. As BlScV is transmitted by aphids in a non-persistent manner, infected elderberry bushes near highbush blueberry plantings may play some role in virus spread. The potential for BlScV infection of plants outside family Ericaceae should be investigated. To our best knowledge, this is the first report of BlScV infecting elderberry.
"Plant diseases in Poland, history of studying and controlling them".
The author has finished to write the extensive and comprehensive (over 500 pages) book “Choroby roślin w Polsce, dzieje ich poznania i zwalczania” [Plant diseases in Poland, history of studying and controlling them] and passed the manuscript for printing to Institute for History of Science, Polish Academy of Science. The following subjects are treated in the book: (1) the progress of knowledge on plant diseases in the entire world back from the ancient times; (2) first communications on plant diseases in Polish agricultural and historical literature to the end of 18th century; (3) the influence of the progress in medicine, botanic and mycology at the turn of 18th to 19th century on the attempts to clarify the causes of plant diseases observed in the Polish territory; (4) the change of the ideas about plant diseases from 1846 (epidemics of potato late blight) until 1880 (1st Polish handbook of plant pathology by Felix Kudelka); (5) progress in plant pathological research in Poland till the end of 1st World War, in the period between two World Wars, and after the 2nd World War with special emphasis of the most important groups of plant pathogens; (6) the formation and reorganization of various scientific and administrative institutions dealing with research and control of plant diseases; (7) the progress in chemical control of plant diseases and improving the plant protection equipment; (8) the list of most important papers published by Polish scientists including plant pathology handbooks.
MIRZWA-MRÓZ E., BAGŁAJ B.
"Identification and characteristics of Colletotrichum acutatum J.H. Simmonds - causal agent of highbush blueberry anthracnose".
The aim of this study was the identification and characterization of Colletotrichum acutatum causing anthracnose of highbush blueberry in selected plantations in Masovian and Lublin voivodships. Fungus isolates were obtained from infected stems and fruits of highbush blueberry. According to morphological characteristics all isolates were identified as C. acutatum. Culture of this fungus on PDA, MEA, CMA, SNA and Czapek media formed the mycelium of a pink colour. The best growth and sporulation of the examined fungi were observed on PDA and MEA media and the poorest on Czapek. The isolate Earlib1 produced the largest conidia, while Nel1 produced the shortest ones and Duk1 the thinnest. ‘Earliblue’ appeared the most susceptible cultivar to infection caused by C. acutatum and ‘Bluecrop’ was the least susceptible. The largest number of acervuli on the stems and the highest percentage of infested fruits was observed in ‘Earliblue’.
SALA-REJCZAK K., PADUCH-CICHAL E.
"Molecular characterization of the CP gene of Prunus necrotic ringspot virus isolates".
The nucleotide sequences corresponding to ilarvirus Prunus necrotic ringspot virus (PNRSV) coat protein (CP) gene in eleven Polish isolates, from different Prunus and Rosa species and four isolates provided in plant material from Australia, Hungary and Italy were obtained and described. Virus identification was possible using specific primers, which allowed to amplify the 700 bp amplicon of coat protein gene. The product was obtained using IC-RT-PCR. The results indicated no association between the host species or the geographic origin of the virus and CP sequence specificity. The CP gene nucleotide sequence of studied isolates enabled to cluster them into the previously reported PV32-I, PV96-II and PE5-III phylogroups.
SCHOLLENBERGER M., KOTWICA A.
"Evaluation of the effectiveness of Coniothyrium minitans in control of Sclerotinia sclerotiorum, white mould".
Sclerotinia sclerotiorum (Lib.) de Bary, the cause of white mould occurs on field crops and at storage. This fungus contributes to a large decrease of yields because infected plant organs become unsuitable for consumption or processing. The new possibility of protection is biological control based on hyperparasitism among fungi, especially Coniothyrium minitans. This hyperparasite is used in the commercially available biopesticide Contans XX (Betchim Crop Protection). Its effectiveness was determined under the laboratory and field conditions. In the laboratory the hyperpasite from Contans XX colonized S. sclerotiorum sclerotia and during 3 weeks mummified them. It was also found that the biopreparation limited the occurrence of white mould during the five months period of carrots storage.
WAKULIŃSKI W., WIT M.
"Fumonisins in maize cobs infected with F. verticillioides".
Fusarium verticillioides is along with F. subglutinans and F. proliferatum the principal causative factors of pink ear rot. Occurrence of the species is variable and strongly related to high temperature and limited precipitations. F. verticillioides is heterothallic fungus with dimictic mating system governed by two idiomorphs MAT1-1 and MAT1-2. The strains of opposite mating types are widespread in Poland and frequency of their occurrence is statistically equivalent. Diverse phenotypic traits of opposite mating strains of some fungi inclined us to verify fumonisins accumulation in maize cobs inoculated with MAT1-1 and MAT1-2 F. verticillioides strains.
Ten genotypes of four maize varieties (Zea mays var. indentata, Zea mays var. indurata, Zea mays var. saccharata and Zea mays var. everta) were used in these studies. Cobs were inoculated 7 days after silk emergence with 2 ml conidial suspension of opposite mating type of the fungus. Cobs Infection degree was evaluated according to 6 degree scale while ergosterol and fumonisins content were determined by HPLC method. During four years studies, it was found that neither infection degree of particular Zea mays variety determined phenotypically and by ergosterol content nor fumonisins level were not dependent on MAT type. All the factors were significantly influenced by environmental condition of cropping season, maize variety and kernel composition (especially water, amylose, amylopectin content).
WIT M., WAKULIŃSKI W., JABŁOŃSKA E.
"Occurrence of 1 and 2 mating type among population Fusarium verticillioides (Sacc.) Nirenberg".
Fusarium verticillioides is associated with a main factor of corn cob rot. The heterothallic fungus from Liseola section is characterized by dimictic mating type reproduction. The three years analyzes of Polish population of F. verticillioides showed prevalence of two complementary mating types of the pathogen. Frequent occurrence of MAT1-1 and MAT1-2 idiomorphs and high percentage close to 50% of fertile female forms among isolates of F. verticillioides, indicate on possibility of teleomorph occurrence invivo. Intraspecies MAT1-1 and MAT1-2 alleles analysis showed high variability. The analysis of MAT1-1 fragment sequence showed 70 number of segregating sites (s). Nucleotide diversity (π) analysis of two selected sequence among the population was 0.0821. Test of MAT1-2 fragment showed 11 number of segregating sites (s). Nucleotide diversity (π) analysis was 0.0168. The Tajima’s D statistic was under zero and respectively for MAT1-1 and MAT1-2 alleles and showed a probability above 0.05. It means that evolution of all analyzed sequences followed according to the same model of substitution. Although high diversity among tested mating types has been found, after crossing of isolates in-vitro fully developed perithecia typical for teleomorph F. verticillioides were obtained. In conclusion there is no spatial isolation of mating types of associative F. verticillioides. It is possible that sexual process also occurs in nature and is the so-called hidden (cryptic) of the sexual process.