brechignac_f_desmet_g_eds_equidosimetry_ecological_standardi

SYNERGISM OF LOW DOSE CHRONIC RADIATION AND BIOTIC STRESS FOR PLANTS

A. DMITRIEV, N. GUSCHA, M. KRIZANOVSKA

Institute of Cell Biology and Genetic Engineering, 148 Zabolotnogo St., Kiev 03143 UKRAINE

1. Introduction

Changes in plant disease resistance and in virulence of plant pathogenic organisms under low dose chronic radiation may pose a threat to agriculture. Since 1986 we carried out studies on plant-pathogen interactions in 30and 10-km Chernobyl nuclear power plant (ChNPP) zone. Biochemical studies are related mainly to low dose chronic radiation effects on disease resistance of cereals and corn. Test-systems, which could be useful for the estimation of low dose effects, are developed. Our long-term goal is to investigate the population structure of plant pathogenic fungi in the 10-km ChNPP zone. Puccinia graminis, the causal agent of stem rust of wheat, rye and oat, is the most damaging disease of these crops. Rust diseases of small grain crops are difficult to control with resistant cultivars, because there are many different pathogenic varieties of the rust fungi. Genes for resistance to wheat and rye rust diseases may be very effective against some varieties, but fail completely against other varieties.

Plants do not have an immune system of the kind known in animals but possess different inducible defences against microbial pathogens. Among the defence responses there are mechanical strengthening of cell walls, formation of pathogenesis-related (PR) proteins, synthesis of inducible antibiotic substances - phytoalexins, increasing of enzyme activities, for example, proteinase inhibitors [1]. Some of these defences can be used as quantitative parameters of plant resistance to biotic and abiotic stress factors of the environment.

2. Decrease of plant disease resistance under low dose chronic irradiation

The results obtained suggest that low dose chronic radiation decreased the plant immunity potential. Analysis of wheat powdery mildew disease of three cultivars (Mironovska-808, Polesska 70, Kiyanka) has shown that the extent of disease was 2- fold higher in plants grown from the seeds, collected in 10-km ChNPP zone, than that in plants grown from control seeds.

The data were confirmed in a set of experiments with artificial inoculation of wheat plants in greenhouse. Seedlings of wheat Kiyanka grown from the seeds, collected in the ChNPP zone, were infected at a second leaf stage by brown rust spores. It turned out that incidence and disease development in the seedlings were 2.6-fold higher than that in seedlings, grown from control seeds. Similar results were obtained for two other cultivars seedlings grown from radionuclide-contaminated seeds.

To elucidate alterations in cereals disease resistance field trials were carried out

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plots. Leaves were artificially inoculated at the beginning of milk ripeness phase with brown rust spores. Phytopathological analysis carried out 5 and 10 days after inoculation revealed that the incidence and extent of brown rust was more severe on plants grown on heavily contaminated plots. The extent of brown rust disease 5 days after inoculation was 2-fold higher on plot 3 with maximal gamma-background than on plot 1 (Table 1). Ten days after inoculation the extent of the disease increased on all three plots, however it still remained highest (68 %) on plot 3.

Table 1. Incidence and extent of brown rust on wheat plants grown on plots with different level of radionuclide contamination (cv. Kiyanka)

It appeared that the differences in wheat brown rust resistance were a result of differences in absorbed dose of ionizing radiation. External radiation dose for plants on plot 3 was 27-fold higher than that for plants on low contaminated plot 1. However, based on the data about specific radioactivity of plants (data not shown), it was found that internal absorbed dose for plants grown on plot 3 was 100-fold higher than that for plants on plot 1.

3. Biochemical mechanisms of decreasing in plant disease resistance

We have also analysed biochemical mechanisms underlying the decrease in plant disease resistance. Three wheat cultivars, rye cv. Saratov, and two lines of corn were grown on the experimental plots. The lines of corn were original (W64A+/+) and high lysine opaque-mutation (W64A o2/o2), which reveals increased sensitivity to stress factors of the environment [2]. The absorbed dose during vegetation period was about 7-8 cGy for cereals and 3 cGy for corn. The activity of plant proteinase inhibitors was determined in the albumine fraction of corn leaves and seeds, and in wheat and rye grains by inhibition of serine proteinases proteolytic activity, estimated by casein method.

The data show that the activity of proteinase inhibitors (trypsin, chemotrypsin, subtilysine) in plants grown on radionuclide-contaminated plots decreased. In wheat and rye grains the activity was decreased up to 15-60 % as compared to the control. The inhibitors could form stable complexes with proteolytic enzymes of pathogens and thus restrict the disease development [3]. It is not clear whether other plant defence responses (phytoalexin synthesis, accumulation of PR-proteins) could be also affected by low dose chronic radiation. But decreasing of proteinase inhibitors activity appear to diminish plant disease resistance.

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This assumption was confirmed by experiments with high lysine opaquemutation of corn. Activity of proteinase inhibitors in the mutant (W64A o2/o2) leaves was decreased (data not shown). Moreover, in the mutant grains the proteinase inhibitors activity was 3-4-fold less than in unirradiated plants and 2-fold less than in irradiated plants of original corn line (Table 2). Therefore, the corn mutation is highly sensitive to ionizing radiation and as well as the waxy-mutation of barley [4] it could be a useful tool in understanding low dose effects.

Table 2. Specific activity of proteinase inhibitors in corn grains

Thus results obtained both in greenhouse and field trials demonstrate the decrease in plant disease resistance under low dose chronic radiation.

Simultaneously, changes in virulence and in aggressivity of plant pathogenic fungi could take place in the 10-km zone of ChNPP. We analysed such a possibility using Puccinia graminis Pers. (Uredinales, Basidiomycetes), a causal agent of stem rust of wheat and other cereals.

4. Changes in population structure of plant pathogenic fungi

Rust fungi are highly variable and well adapted to rapid spread over long distances. New rust varieties may arise by mutations and spread from the 10-km ChNPP zone into other parts of the country and overcome the resistance of cultivars that was effective against the old varieties. Two approaches can improve the durability of rust resistance in wheat and rye: 1) improve our understanding of how rust varieties compete within rust populations in order that we can anticipate virulence shifts in rust populations or manipulate the populations to delay shifts in virulence, and 2) identify new types of rust resistance that are either not variety specific or that do not select for rapid increases in virulence in rust populations.

Determination of areal and nutritional plant species for Puccinia graminis was performed by analysis of grain crops on experimental plots. In parallel, we made a search for wild cereals in natural biocenosis’ neighbouring to common barberry (Berberis vulgaris), the host for the sexual stages of the stem rust fungus, and grain crops. All stages of fungus development – aeciospores, urediniospores and teliospores were analysed. Identification of physiological varieties of P. graminis tritici populations was performed on classical Stackman varietiesdifferentiators with known resistance genes to stem rust [5]. Three types of response reactions were taken into account: resistant (0-2 points), susceptible (3-4

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points) and heterogeneous (Õ). Virulence was determined on the basis of monogenic line reactions with resistance genes [6].

Puccinia graminis regularly caused severe epidemics until the mid-1950s. Wheat leaf rust and oat crown rust caused countrywide losses of up to 10% in recent years. These epidemics occurred when new virulent varieties of rust suddenly increased to destructive levels before new cultivars could be developed with resistance to the new varieties.

Table 3. Stem rust lesions of cereal species on experimental plots

The advantage of the research was that since 1966 we studied the sexual stage population of P. graminis tritici in “Maneve” region near Kaniv in Ukraine. That allows us to have a distinctive “zero point” to analyze changes in stem rust population structure as a result of ChNPP accident. To characterize the population structure it was necessary to identify varieties with various virulence phenotypes and to determine frequencies of virulent genes. Analysis of wheat, rye, barley, oat, and grasses on experimental plots in 10-km ChNPP zone revealed stem rust development on 12 varieties of cereals. The incidence of disease was about 50-85 % at practically 100 % damaged crop (Table 3).

Three main forms of the fungus were found: 1) wheat (P. graminis tritici), which damages both wheat and barley; 2) rye (P. graminis secale); 3) oat (P. graminis avenae). All the forms were capable to develop on many cereal grass species, which serve as reservoir of infection accumulation duringof the vegetation period. 642 monopustul clones of stem rust were isolated. Among them 8 physiological varieties of the pathogen were revealed: 11, 21, 34, 40, 100, 189, 3ê, but also the variety, whose characteristic was absent in the International register. We named it variety “X”.

It was found in 1992 that varieties 3ê (27 %) and 100 (23 %) were dominant in the “Chernobyl ” population. Three years after, the most isolates belonged to variety 34 (24 %), but also varieties 11 (18 %), 21 (12 %) and 40 (6 %) were present. Thus only widespread varieties 34, 3ê and the rare variety 189 retained during three years. All varieties demonstrate a high virulence based on their reactions on a set of 12 wheat lines with different genes for rust resistance (Table 4). Analysis of three wheat cultivars (Mironovska-808, Polesska 70 and Kiyanka) inoculated with different varieties of P. graminis revealed a high

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susceptibility (often “4” points, rarely “3”). Varieties 11, 21 and 34 are worldwide known. Variety 21 was dominant during a few years in ex-USSR countries. Variety 189 was of special interest. It induced very high sensitivity on all varieties-differentiators with known resistance genes to stem rust.

Table 4. Reactions of physiological varieties of stem rust revealed in 10-km ChNPP zone

Analysis of genotypes of P. graminis on monogenic lines showed that more virulent clones were present with high frequency in the “Chernobyl” population.

5. Conclusions

The results obtained independently both in greenhouse and field trials in 10km zone of ChNPP demonstrate the decrease in plant disease resistance. Analysis of biochemical mechanisms underlying the decrease in wheat and rye disease resistance revealed reduction in activity of proteinase inhibitors in the plants. An opaquemutation of corn could be a useful tool in understanding low dose radiation effects. The population structure of Puccinia graminis, a causal agent of stem rust and one of the most severe pathogens, has been changed in 10-km ChNPP zone by appearance of a «new» population with high frequency of more virulent clones.

6. References

1.A. Dmitriev. Phytoalexins and their role in plant disease resistance. Kiev: Nauk. dumka, 1999. 208 p.

2.F. Vinnitsenko et al. Influence of a gene opaque-2 on activity of proteinase inhibitors in corn grain // Physiol. Biochem Cult. Plants. 1988. -20. N 5. P. 493-497.

3.L. Metlitskiy et al. Biochemistry of plant immunity, rest and aging. Moscow: Nauka, 1984. 264 p.

4.I. Boubryak, E. Vilensky, V. Naumenko et al. Influence of combined alpha, beta and gamma radionuclide contamination on the frequency of waxy-reversions in barley pollen. Sci. Total

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Environ., 1992. 112: 29-36.

5.E. Stackman and J. Harrah. Plant pathology. NY: Academic Press.1969.-514 p.

6.L. Semenova L. The methodical recommendations for studies on race structure of stem rust fungi. Moscow: VASCHNIL, 1977. – 144p.

COMPARATIVE STUDY OF THE EFFECTS OF ENDOCRINE DISRUPTOR AND IONIZING RADIATION WITH PLANT BIOASSAY

Jin Kyu KIM

Korea Atomic Energy Research Institute, 150 Dukjin-dong, Yusong-gu,

Taejon 305-353, KOREA

Hae Shick SHIN and Jin-Hong LEE

Chungnam National University, Gung-dong, Yusong-gu, Taejon, 305-764,

KOREA

Vladislav G. PETIN

Medical Radiological Research Center, 249036 Obninsk, Kaluga Region,

RUSSIA

1. Introduction

The use of synthetic chemicals exerts a great influence on the daily life of human beings. The recent reports that some of those synthetic chemicals can act as endocrine disrupting substances in various organisms have drawn special attention in environmental research. Among others, dioxins, DDT, PCBs and bisphenol A (BPA) are classified into endocrine disruptors and are under a strict control in many developed countries. Since a small amount of them can disturb biological systems, the ecological problem can also arise when they exist in excess in the environment. They can give rise to such adverse effects as morphological anomaly, decreased number of sperms, cancer and change of sex [1]. Since the use of the synthetic chemical will steadily increase in modern life, it is necessary to establish assay techniques for monitoring them, and to assess their biological risks.

The reproductive cells are the most sensitive to toxic environmental materials and ionizing radiations. The pollen mother cells, male reproductive cells of Tradescantia are highly synchronized in their prophase I and tetrad stages. Chromosomes of this stage are sensitive to physical or chemical mutagens [2]. These induced chromosome aberrations become micronuclei in the synchronized tetrads and they can be easily identified and scored. Based upon these features, the Tradescantia micronucleus bioassay was established. The meiotic pollen mother cells have quite long been known to be more sensitive than mitotic chromosomes to radiation [3]. But the fact that the chromosomes of the pollen mother cells were loose in the stage of metaphase I and thus it is very difficult to be scored had prevented the cells to be used for chromosome aberration assay. However, if the treatment is applied to the early prophase I stage and allow the treated meiotic chromosome to go through a proper recovery time [4], the acentric fragments or the sticky chromosome complex would become micronuclei in tetrad stage of meiosis. This was the basis for establishing the Tradescantia micronucleus (Trad-MCN) bioassay. This bioassay was first utilized in the study of mutagenic effects of 1,2-dibromoethane [5] and followed by X-rays and known mutagens [6].

Since the micronucleus production was understood as a useful indicator for chromosome damage, the micronucleus assay has been widely applied to plant and animal cells by many workers [7-9].

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This research was designed to demonstrate the biological effects of BPA, which is one of the well-known endocrine disruptors, by means of the Tradescantia micronucleus (Trad-MCN) assay. The biological effect of gamma rays on the same cell system was also evaluated for comparison with that of BPA to estimate the equidosimetric effectiveness of BPA.

2. Materials and methods

Fresh cuttings of Tradescantia clone 4430 (an interspecific hybrid between T. hirsutiflora and T. subacaulis) were used in this study. Each experimental group consisted of 20 cuttings. The groups for studying radiation effects were irradiated with gamma-rays from 60Co (source strength 150 TBq, Panoramic Irradiator, Atomic Energy Canada Ltd.). During irradiation the stems of the cuttings were immerged in Hoagland’s No. 2 (x1/6) solution to prevent them from wilting. Radiation doses are 0, 10, 20, 30, 40 and 50 cGy.

Bisphenol A (4,4'-isopropylidenediphenol, Sigma Co.) was dissolved in absolute ethanol. The solution was diluted with distilled water to the experimental concentrations, which were expressed in mM. Plant cuttings were treated with BPA solution in a beaker for 6 hours, and during this period, the solution was continuously aerated and the plants were illuminated with artificial lights. The negative control DW, solvent control 1% ethanol and tap water were also tested for comparison in these experiments. Right after the treatment, the chemical solution was removed and the stems of the cuttings were rinsed in tap water to remove the remaining BPA solution and then transferred into aerated fresh Hoagland’s No. 2 solution (x 1/6) during 24 hours for recovery.

The slide preparation and MCN scoring were carried out according to the standard protocol described in an earlier publication by Ma et al. [10].

3. Results

The results of gamma-ray treatment are given in Figure 1. Linear regression analysis of the highest MCN frequencies and radiation doses gave the following equation. Data for the control group will be separately discussed later.

where,

FMCN = the maximum MCN frequencies (MCN/100 tetrads), D = radiation dose in cGy.

The correlation coefficient of the regression was 0.955. No physical damage was observed in the pollen mother cells at all dose levels. According to the above equation, two micronuclei in 100 tetrads can ascribe to 1 cGy of gamma-radiation.

Micronucleus frequencies obtained by scoring slides from the experimental groups are depicted in Figure 2. The MCN frequencies were 8.06±0.70, 12.76±1.06, and 19.67±1.52 in 1, 2, and 4 mM BPA-treated groups, respectively. For the highest MCN frequencies in the BPA-treated groups, linear regression gave the following equation.

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Fig. 1. Radiation dose-response relationship of micronucleus frequencies in pollen mother cells of T. BNL 4430.

Fig. 2. Bisphenol A concentration-response of micronucleus frequencies in pollen mother cells of T. BNL 4430.

FMCN = the maximum MCN frequencies (MCN/100 tetrads), C = concentration of BPA in mM.

According to the above equation (2), roughly four micronuclei in 100 tetrads can be additively induced by one mM increase in BPA concentration. In the preliminary experiment, the authors found that high concentration (>8 mM) of BPA could affect physically the pollen mother cells themselves, and that such physical damage would be followed by apoptosis of the cells (unpublished data).

For the negative controls, distilled water, solvent (1% Et-OH) and tap water were also tested for their micronucleus induction in the pollen mother cells (Figure 3). The spontaneous MCN frequency in the group without any treatment was 2.75±0.55, and that of 1% EtOH treated group was 2.33±0.62, which were in good accordance with the previously reported background value [11]. In contrast tap