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Study of alternative oxidase as possible molecular marker for phylogenetic analysis of the Botrytis cinerea
Published March 11, 2014

Botrytis cinerea (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is able to attack several economically important plants causing gray rot. Botrytis cinerea species complex includes two cryptic species (B. cinerea and B. pseudocinerea) that tolerate fungicides differently. On the basis of classical taxonomic markers, the two related specie...s are very difficult to be distinguished; therefore, their separation is usually performed using molecular methods based on the time-consuming molecular analysis of several markers. Our goal was to find markers, which are suitable for the differentiation. Testing the nucleotide sequences of the alternative oxidase encoding gene, B. cinerea and B. pseudocinerea strains were clearly differentiated. Moreover, the analysis of the protein sequences of the enzyme with the maximum likelihood method reflected well the taxonomic relationships of the different fungi.

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Isolation and identification of endophytic fungi connected to Grapevine Diseases, from the Tokaj wine region, Hungary
Published March 11, 2014

Grapevine Trunk Diseases (GTD) is one of the most important diseases in vineyards worldwide, which can be found in Hungarian vineyards as well. In Hungarian wine regions there is very little information about the occurrence of pathogens which cause GTD, in case of Tokaj wine region there is no knowledge about that, what kind of pathogens can be... found in the vineyards.

The objective of our research is to assess the situation and occurrence of GTD in Tokaj wine region in cooperation with local specialists, as well as identification of pathogens which were isolated from the diseased trunks by morphological and genetic basis.

We were able to isolate endophytic fungi from all sampled grape trunk. The majority of them were determined as Diplodia seriata not only based on colony morphology, but also determined by rDNA sequences.

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Application of AquaCrop in processing tomato growing and calculation of irrigation water
Published June 30, 2018

The area and volume of processing tomato production is increasing in Hungary. Irrigation is crucial for processing tomato growing. To save water and energy, it is important to know exactly how much water is needed to reach the desirable quality and quantity. AquaCrop is a complex software, developed by FAO, which is able to calculate irrigation... water needs, several stress factors and to predict yields. A field experiment was conducted in Szarvas in processing tomato stands, under different irrigation treatments. These were the following: fully irrigated plot with 100% of evapotranspiration (ET) (calculated by AquaCrop), deficit irrigated plot with 50% of ET (D) and control (K) plot with basic water supply was also examined. Dry yield, crop water stress index and soil moisture were compared to modelled data. The yields in the plots with different access to water were not outstanding in the experiment. The model overestimated the yields in every case, but the actual and modelled yields showed good correlation. AquaCrop detected stomatal closure percentages only in the unirrigated plot. These values were compared to CWSI – computed from leaf surface temperature data, collected by a thermal cam in July – and showed moderately strong correlation. This result suggests that Aquacrop simulates water stress not precisely and it is only applicable in the case of water scarcity. Soil moisture data of the three plots were only compared by means. The measured and modeled data did not differ in the case of K and ET plots, but difference appeared in the D plot. The obtained results suggest that the use of AquaCrop for monitoring soil moisture and water stress has its limits when we apply the examined variables. In the case of dry yield prediction overestimation needs to be considered.

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Determination methods of quality parameters of Prunus cerasus
Published May 16, 2017

Sour cherry (Prunus cerasus) is a non-climacteric fruit. Storage optimization would enable the expansion of fresh consumption. The quality parameters are required for storage optimization. Those parameters are sugar content, acid composition, mineral content and alcohol content,
moreover it is also planned to determine antioxidants, vitamins... and patulin mikotoxin under different conditions. In this paper, we demonstrate the measurement options of these parameters.

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Pathogenicity differences between group I and group II of Botrytis cinerea
Published October 30, 2011

Botrytis cinerea has been reported as a species complex containing two cryptic species, groups I (Botrytis pseudocinerea) and II (B. cinerea sensu stricto). In order to compare the pathogenicity of group I and group II of B. cinerea, we have selected 4 strains of group I and 4 strains of group II. The results demonstrated that competitive infec...tion of group II was more on grape, cucumber and paprika leaves, than group I. However the results on bean leaves did not correlate the applied B. cinerea group.

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Q-PCR analysis of the resistance of Hungarian Botrytis cinerea isolates toward azoxystrobin
Published October 30, 2011

The genes being in the mitochondrial DNA primarily encode the enzymes of cellular respiration. Fungicides belonging to the family of quinol oxidase inhibitors (QoIs) play on important role in the protection against several plant diseases caused by fungi. These fungicides bind to the cytochrome bc1 complex so they block electron transport betwee...n cytochrome b and cytochrome c1. This way these fungicides inhibit the ATP synthesis consequently they inhibit the mitochondrial respiration. The QoI resistance has two mechanisms. One of them is the point mutation of the cytochrome b gene (CYTB), e.g. the substitution of a single glycine by alanine at position 143 results in high-resistance. The other is the cyanide-resistant alternative respiration sustained by the alternative oxidase.
In a cell there are several mitochondria. The phenomenon when the genomes of all mitochondria in the cell are identical is called homoplazmy. If in the cell there is wild and mutant mitochondrial DNA this is called heteroplasmy. Whether the mutation in the mitochondria causes fenotypical diversity or does not depend on the dose, i.e. it depends on the percentage of the changed mitochondrials. During our work we investigated Botrytis cinerea single spore isolates which have been collected in 2008-2009 on different host plants. Our goal was to decide whether heteroplasmy influences the level of resistance. We managed to detect the change of the level of heteroplasmy, so the change the level of the resistance due to the treatment with fungicide.

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Pre- and postharvest technologies to extend the shelf life of Prunus cerasus
Published May 23, 2019

Fresh tart cherry consumption cannot be increased without the development of an appropriate technology for its elongated storage. This requires the development and optimization of the pre- and postharvest treatments. Currently, we have only limited knowledge about tart cherry (Prunus cerasus L.) postharvest technology, however, related studies ...on sweet cherry (Prunus avium L.) may be adopted. In this article, we have collected the most important research results in this topic.

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Cytochrome b diversity of Hungarian Botrytis cinerea strains
Published November 10, 2010

In the mitochondrion of eukaryotes, cytochrome b is a component of respiratory chain complex III. Cytochrome b is encoded by the
cytochrome b (CYTB) gene located in the mitochondrial genome. The fungicidal activity of QoIs relies on their ability to inhibit mitochondrial respiration by binding at the so-called Qo site (the outer quinol-oxida...tion site) of the complex III. Since their introduction, QoIs (like azoxystrobin) have become essential components of plant disease control programs because of their wide-ranging efficacy against many agriculturally important fungal diseases like grey mould on various crops. QoI resistance primarily arises from a target-site-based mechanism involving mutations in the mitochondrial CYTB. As the management of grey mould is often dependent on chemicals, the rational design of control programs requires the information about the diversity of genes connected with resistance in field populations of the pathogen.
Monospore B. cinerea field isolates has been collected during 2008-2009 from different hosts in Hungary. PCR fragment length analysis
indicated the high frequency presence of type large intron in the isolates while in a few strains G143A substitution could also be detected.
These results indicated the heterogeneity of CYTB in the Hungarian B. cinerea populations, which possibly involve the heteroplasmy of this
mitochondrial gene, moreover indicates the existence op azoxystrobin resistant populations in Hungary.
This work was supported by NKFP-A2-2006/0017 grant. Erzsébet Fekete is a grantee of the János Bolyai Scholarship (BO/00519/09/8).

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The role of cultivar susceptibility and vineyard age in GTD: examples from the Carpathian Basin
Published December 1, 2020

Grapevine trunk diseases (GTDs) are among the most severe problems in viticulture worldwide. The exact etiology and the role of endophytic microorganisms is not known yet and there is no adequate protection or curative treatment against the disease. Hungarian wine regions are also affected by the disease, and there is restricted information... about the rate of infection nation-wide and about the susceptibility of the Hungarian cultivars.

The main objectives of our research are to measure the symptom expression and the damage caused by GTDs, to understand the epidemiology and etiology of the disease to establish a foundation of a proper disease management.

Cultivar susceptibility groups were created with the aim to allocate some Hungarian cultivars and the role of vineyard age was also examined in symptom expression.

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Investigation of host-specificity of phytopathogenic fungi isolated from woody plants
Published May 20, 2020

Host-specificity is an important characteristic of fungal pathogens. Changing climate could create more appropriate environmental conditions for phytopathogens, thus formerly host-specify fungi could be able to colonize new hosts. Noxious plant pathogen fungi, which can infect several plant species are well-known worldwide. These genera may... expand their range of hosts because of the appearance in new geographic areas due to climate change. This new exposure can result in serious problems in agriculture because of the lack of immunity. The susceptibility of apple tree was studied through testing pathogenicity in vitro with species isolated from walnut twigs and nuts, and identified by ITS sequences. Three of four tested species, Botryosphaeria dothidea, Diaporthe eres and Diplodia seriata colonized and necrotized the infected apple branches, while Juglanconis juglandina was not able to infect the twigs. Members of Botryosphaeriaceae were the most virulent, causing the largest lesions in the fastest way. This experiment draws attention to the threat of new host-pathogen connections, which can arise because of the favourable weather conditions and can spread between neighbouring cultures. 

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Qualitative detection of genetically modified organisms in plant samples
Published July 16, 2007

We analysed the GMO content of corn samples by polymerase chain reaction following the appropriate optimization of the reaction. The analysis included two main steps: extraction of DNA from the sample, and detection of the GMO content by polymerase chain reaction. The polymerase chain reaction is an in vitro method to multiply chromosomatic or ...cloned DNA (cDNA) sequences through the enzymatic pathway. The reaction is sensitive enough to produce DNA in sufficient amount for the analysis from a single DNA. We identified the PCR products by agarose gel electrophoresis. When optimizing the reaction, the MgCl2 concentration, reaction time and temperature have to be taken into consideration. The temperature of the anellation has to be increased until the highest specificity and yield is reached. If the temperature of the anellation is too high, the primer is linked to non-specific sites as well; in the gel visualization, more lines can be seen at one sample. If the temperature of the anellation is too high, the primer is insufficiently linked or is not linked at all (too few lines in the gel visualization). After optimization, the GMO content in the unknown sample can be determined along with the appropriate positive and negative controls.

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Biomass production estimation of processing tomato using AquaCrop under different irrigation treatments
Published December 15, 2019

The wiser usage of irrigation water is inevitable in the future. Irrigation has very high input cost; therefore, farmers must carry out irrigation with care. Also, the effect of irrigation on crops has a big role in decision making. Modeling provides a possibility to evaluate this effect. AquaCrop, as a crop production simulation model has grea...t potential in this field. The accuracy of tomato biomass yield prediction of the model was tested in this research. For collecting the necessary data, a field experiment was conducted at Szarvas on processing tomato with different water supplies, such as 100% (I100), 75% (I75), 50% (I50) of potential evapotranspiration and a control with basic water supply (C). The relation of the simulation and actual biomass yields was evaluated during the season. Very good correlation was found between the modelled and the actually harvested data. The data for the control and I100 treatments showed higher correlation than the I75 and I50. The relationship for all of the data was moderately strong. Miscalculations occur mostly when the dry biomass yield reaches
7 t ha-1. The accuracy of the model was evaluated with the use of mean absolute error (MAE) and root mean squared error (RMSE) values. The least error was found in the C treatment, which means 0.34 MAE and 0.45 t ha-1 RMSE. The simulation resulted in higher errors in the I75 and I50 treatments.

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Phylogenetic analysis of Phoma species
Published July 16, 2007

The cosmopolitan Phoma genus contains mainly phytopathogenic, opportunistic parasites, and saprophyte fungal species. Up to now, the characterization of Phoma species and other taxa of Phoma has been determined on the basis of morphology on standardized media, and gene sequence analysis was only used as a confirmative or distinctive complement....
In this study, we tried to find molecular markers which can be used as phylogenetics markers in the molecular based classification in the Phoma genus.
We employed a part of the translation elongation factor 1 subunit alpha (EF-1α=tef1) containing both introns and exons and ITS region containing the internal transcribed spacer regions 1 and 2 and the 5.8S rDNA, as potential genetic markers to infer phylogenetic relationships among different Phoma taxa. Twelve different Phoma species sequences were analysed together with the closely related Ascochyta ones. The constructed phylogenetic trees, based on tef1 and ITS sequences, do not support the traditional Phoma sections based on morphological characterization. However, we managed to distinguish between the Phoma strains and Ascochyta species by comparing their tef1 sequences through parsimony analysis. We proved that a tef1 can be a useful phylogenetic marker to resolve phylogenetic relationships at species level in Phoma genus.
Both parsimony sequence analyses confirmed that the Phyllosticta sojicola species is identical to the Phoma exigua var. exigua species as Kövics et al. (1999) claimed. However, the evolutionary distance by ITS sequences within Phoma species is too small to get well based consequences for the phylogenetic relationships of Phoma genus.
Further investigations would be necessary to clarify whether the tef1 and ITS sequences as phylogenetic molecular markers are well suited for the classification of Phoma species.

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Phylogenetic studies of Phoma species by maximum likelihood analysis
Published October 10, 2008

The cosmopolitan Phoma genus contains mainly phytopathogenic, opportunistic parasite, and saprophyte fungal species. Up to now the characterization of Phoma species and other taxa of Phoma has so far been determined on the basis of morphology on standardized media, and gene sequence analysis was only used as a confirmative or distinctive comple...ment.
In this study we have tried to study phylogenetic relationships by maximum likelihood method in the Phoma genus. We employed a part of the gene responsible for the synthesis of translation elongation factor 1 subunit alpha protein (tef1) containing both introns and exons, a part of the gene responsible for synthesis of tubulin protein and ITS region containing the internal transcribed spacer regions 1 and 2 and the 5.8S rDNA as potential genetic markers to infer phylogenetic relationships among different Phoma taxa. Twenty-four isolates of eleven different Phoma species were firstly characterised by morphologically, and then their tef1, tubulin and ITS sequences were sequenced and analysed by maximum likelihood method carried out by PAUP*4.0b program. According to constructed phylogenetic trees, the different Phoma taxons are well separated. However these trees do not support the traditional Phoma sections based on morphological characterization.
The maximum likelihood analyses of all three sequences confirmed that the Phyllosticta sojicola species is clustered with the Phoma exigua var. exigua group and the Phoma sojicola is grouped with Phoma pinodella group. The experienced molecular evidences initiate the demand of reclassification of formerly mentioned soybean pathogens. 

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Studies of Expression of Peptaibol Synthetase of Trichoderma reesei
Published December 6, 2005

Because of the potential importance of peptaibols in the biological control of plant diseases, a transgenic, a T. reesei strain carrying a tex1-promoter: goxA fusion plasmid was constructed for furthur studies. The peptaibol synthetase gene (which is highly similar to T. virens tex1) was identified in the genome sequence of T. reesei. A 900 bp ...5’ upstream noncoding fragment, presumed to include the promoter region of tex1, was cloned into the pSJ3 plasmid (which contains the Aspergillus niger goxA gene encoding glucose oxidase). Finally, we transformed T. reesei with the tex1-promoter: goxA fusion containing pSJ3 plasmid.

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Population genetic results of Hungarian Botrytis cinerea isolates establishing new technologies with decreased chemical usage against grey mould
Published November 13, 2012

Botrytis cinerea causes gray mold on a high number of crop plants. Information about the populations of plant pathogen fungi may help to develop new strategies for the effective and economic crop protection with reduced fungicide usage. Hungarian B. cinerea populations were characterized with using different molecular genetic parameters. B. cin...erea group I strains, characterized with high rate of fenhexamid resistance, could be detected only in restricted number. The Hungarian B. cinerea populations were characterized with high genetic diversity, and the regular occurrence of sexual reproduction. These results highlight the importance of rotating different type of fungicide in the plant protection technology against grey mould.

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Phylogenetic studies of soybean pathogen Phoma species by Bayesian analysis
Published October 20, 2009

We carried out phylogenetic study analyzing sequences of genetic markers in the taxonomy of Phoma and Phoma-like fungi. Different species of Phoma and Phoma-like fungi occurring on soybean (Phoma pinodella, Phoma sojicola, Phyllosticta sojicola, Phoma exigua var. exigua) are difficult to identy because of their high morphological and symptomati...c similarities.
Twenty-two isolates of nine different Phoma species were obtained from reference culture collections. Seven of them were isolated from soybean, the others were collected from different hosts.
The Phoma isolates were firstly characterised by morphologically, and then we employed a part of the gene responsible for the synthesis of translation elongation factor 1 subunit alpha protein (tef1), ITS region, as well as β-tubulin partial sequences as potential genetic markers to infer
phylogenetic relationships among different Phoma species..Finally, their ITS and tef1 sequences were sequenced and analysed by Bayesian approaches.
According to phylogenetic trees inferred by Bayesian analysis of tef1, ITS and β-tubulin sequences, different Phoma species can be separated proving that these phylogenetic markers are well suited for phylogenetic studies of Phoma species. However, the phylogenetic tree does not support the traditional Phoma sections based on morphological characterization.
Bayesian analyses of the three sequences confirmed that the Phyllosticta sojicola species is clustered with the Phoma exigua var. exigua group and the Phoma sojicola is grouped with Phoma pinodella group. The molecular data provide evidence for reclassification of formerly mentioned soybean pathogens.

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