Elisaveta Stoimenova

Metabolic changes in tomato fruits and seeds after viral, bacterial and fungal infections

The metabolic changes in tomato fruits and seeds separately infected with cucumber mosaic virus, Pseudomonas syringae pv. tomato or Botrytis cinerea were investigated cytochemically. The changes of peroxidase (E.C. 1.11.1.7) and β-glucosidase (E.C. 3.2.1.21) were investigated biochemically as well. Tomato fruits were involved in the study because of their high economic value. Tomato seeds were investigated since they have been used most extensively as a model system for studying the physiology and biochemistry of seed development. The diseases caused by the pathogens under study are of special importance for yield reduction in tomato. The three pathogens provoked local changes in the activities of enzymes under study that affect the infected pericarp tissues and neighboring seeds. It was established non-specific and specific responses. The non-specific responses of invaded tissues were expressed as a local enhancement of peroxidase activity in both pericarp tissues and seeds as well as a decrease in activities of: i. enzymes taking part in aerobic and anaerobic respiration, ii. hydrolases esterase and acid phosphatase involved in the basic metabolism as well as an enhancement of their activities in neighboring tissues. Furthermore, it was observed an enhancement of α-galactosidase activity in infected area was observed. The specific responses depending on the type of the pathogen consisted in an enhancement of glucose-6-phosphate dehydrogenase activity by virus infection and an increase of β-glucosidase activity by fungal invasion.

Application of accelerated storage test to lyophilized plant viruses

The preservation of nine plant virus strains of tobamovirus and cucumovirus groups after freeze-drying in different lyophilic forms was examined. Quantitative studies on survival were performed. In tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV), accelerated storage test at 70 – 100°C was applied for screening 20 protecting media. A perspective medium, 5% sorbitol, 3.6% dextran, for plant viruses lyophilization with high cryo- and xeroprotective effects was found.

Determination of parameters for tobacco mosaic virus cryogenic treatment and freeze-drying

Studies on determination of appropriate conditions for cryogenic treatment and lyophilization of tobacco mosaic virus (TMV) in infected leaf tissue, plant sap and purified preparation were carried out. The found parameters were: freezing to minus 22°C (for 1.5 hours); twelve-hours-sublimation at 50 Pa vacuum until 20°C temperature of the sample and secondary drying at 1–2 Pa for 5–6 hours. Fifteen variants of cryoprotection were applied. TMV showed a survival higher than 89% after freeze-drying in glucose, fructose, maltose, lactose, sorbitol and the combinations with dextran. A technological scheme for automatic processing and standard work was developed.

Accelerated leaf senescence takes part in enhanced resistance in cucumber mosaic virus inoculated pepper leaves

Altered photosynthetic reactions in cucumber mosaic virus (CMV) inoculated leaves of virus resistant lines L113 and L57 and susceptible pepper (Capsicum annuum L.) plants cv. Albena grown in controlled environment and in the field were investigated. The CMV inoculated leaves of virus resistant lines developed different symptoms—necrotic local lesions on L113 and chlorotic spots on L57 while the same leaves of susceptible cv. Albena were symptomless. The changes in Photosystem II (PSII) and PSI electron transport were evaluated by chlorophyll fluorescence, and far-red (FR) light induced leaf absorbance A810–860. CMV infection caused a decrease in maximal PSII quantum yield, Fv/Fm, in susceptible leaves. Increased non-photochemical fluorescence quenching in CMV-inoculated leaves of both resistant lines were observed. In CMV-inoculated leaves of all tested plants FR light induced P700 oxidation was decreased. In the present study, the viral-infected pepper plants grown in controlled environment to avoid the effects of abiotic factors were used as model system that allow us to investigate the differences in leaf senescence in CMV-inoculated leaves of susceptible and resistant pepper lines expressing different symptoms. Earlier leaf falls of inoculated leaves as a result of accelerated leaf senescence is important for building successful secondary virus resistance strategy following fast responses such as hypersensitive reaction.

A model for longevity prediction of freeze-dried tobamoviruses

An accelerated storage test was carried out on freeze-dried samples of five variants of tomato mosaic virus – from leaf material, plant sap and purified preparation with and without protectant, at 28, 37 and 45 °C. A model for longevity prediction of lyophilized tobamoviruses was developed. The preservation of tomato mosaic virus decreased to 10% after 0.7–2.8 years in unprotected variants and after 7.5–11 years in protected samples under real storage conditions. Experimental data have confirmed the predicted values.

Differential expression of β-glucosidase in tomato - stress stimuli systems.

In the present work the responses of β-glucosidase in leaves of tomato plants subjected to various stress factors of both pathogenic (fungi, bacteria, viruses) and abiotic origin (heat shock) were studied. Biochemical and cytochemical methods were applied. It was established that an increase of β-glucosidase activity is induced uniquely by fungal pathogens. The cytochemical tests confirm the finding. Hence, the conclusion can be drawn that β-glucosidase response is a specific character of fungal pathogenesis in tomato; probably, the enzyme is involved in plant — fungi recognition. The data are in accordance with our previous results on tobacco and wheat — stress stimuli systems.

PREDICTION OF THE PRESERVATION OF LYOPHILIZED TOBAMOVIRUSES

ABSTRACT Eleven tobamovirus strains were lyophilized in the form of leaves, plant sap with and without protectant, as well as protected purified preparation. Accelerated storage test at 28–81 °C for 30 to 240 days was applied to the freeze-dried samples. The infectivity was tested on a local host. The survival decrease was represented as pseudo-first order reaction and predictions for stability of lyophilizates at real storage conditions (4 °C) were developed by means of Arrhenius equation. The preservation depended on the lyophilic form and was strain specific. Some strains were expected to loose 90% of the survival after 1–3 years while others—after more than 25 years in unprotected variants. The preservation as protected purified preparations will be more than 60–70 years. Among the tested strains of tomato mosaic virus the highest longevity for the strains of group “2” after Pelham was predicted. The accelerated storage test and the prediction facilitate the maintenance of a plant virus collection.

Tomato lines segregation for resistance to cucumber mosaic virus

Abstract15 lines were bred by interspecific hybridisation and I1 to I6 generation of three of them were tested for resistance to CMV. In spite of the selection by CMV resistance in the progenies the number of the resistant plants did not always increase. The progenies having 100 % symptomless plants for two or more consecutive years were not selected in the studied lines. A large spectrum of variations in the percentage of symptomless plants in the progenies per year and the presence of disease and symptomless parts in one plant were established. These results are possibly due to the complex mechanism of inheritance of CMV resistance as well as to the influence of environmental factors on the expression of the resistance observed

Electrophoretic mobility of different in pathogenicity strains of Tobamovirus group

Twenty four Tobamovirus strains divided according to their ability to overcome the resistance genes in tobacco and tomato as well as in their severity of systemic symptoms were tested. Significant differences between means of particle mobility of the virus groups were found especially about yellow mosaic strains which were distinguishable enough by the electrophoretic analysis.