Корневые нематоды (Meloidogyne spp.) являются основными представителями класса нематод, паразитирующих на растениях. Нематоды наносят большой экономический ущерб, который выражается в снижении продуктивности и ухудшении качества урожаев сельскохозяйственных культур и дикорастущих растений, произрастающих во всех климатических зонах (тропических, субтропических, а также в регионах с умеренным климатом). Цель настоящего исследования состоит в изучении изменений, происходящих в галловой нематоде Meloidogyne arenaria и ее хозяине – растении томатов Tiny Tim («Крошка Тим») под воздействием радиации. Было изучено влияние различных доз гамма-излучения – 90, 700 и 1800 мГр на коэффициент изменчивости растений томатов Tiny Tim и развитие яиц галловой нематоды M. arenaria. Ионизирующее излучение в малых дозах (90 мГр) стимулирует рост и развитие растений, в то время как высокие дозы гамма-излучения (700 и 1800 мГр) подавляют развитие томатов, замедляя скорость роста побегов и корней. Использование ионизирующей радиации в высоких дозах (700 и 1800 мГр) способствует замедлению роста нематод. По метрическим данным, в основном это сказывается на уменьшении размеров тела самки M. аrenaria. Применение самой высокой экспериментальной дозы излучения (1800 мГр) препятствовало переходу самок M. arenaria (J4) ко взрослой стадии. Наблюдались изменения численного соотношения самцов и самок под влиянием гамма-излучения, приводящие к снижению числа самцов. Данные результаты открывают перспективы для дальнейших исследований воздействия гамма-излучения на развитие корневой нематоды.
гамма-излучение, нематоды, Meloidogyne arenaria, коэффициент изменчивости, Tiny Tim, растения томатов.
Parasitism as an interrelation between two subjects and is distributed among all alive organisms-plants, invertebrate and vertebrate animals. Root-knot nematodes (Meloidogyne spp.) are one of the most important plant parasitic nematodes of great economic importance. As a root-knot nematodes, the obligate endoparasite Meloidogyne arenaria (Neal, 1889) Chitwood, 1949 are economically very important plants parasites which reduce the quantity and the quality of the yields by causing formation of galls on the host plant roots and consequently disruption of their vascular system.They are pests for many cultivated and wild plants everywhere (in tropical, subtropical and temperate regions). Losses due to these parasites are estimated at hundreds of dollars annually and their adaptation to parasitism is so perfect that in many cases control strategies nave been unsuccessful [17].
At present the number of species belonging to Meloidogyne is about 80. They infect more than 2000 plant species [12]. Meloidogyne invasion causes heavy qualitative and quantitative damages of the plant production up to 70-80% in many countries including Bulgaria [16]. The numerous species belonging to Meloidogyne, their ecological and biological abilities to survive under unfavorable conditions make their control very difficult. The contemporary requirements to the quality and quantity of the plant production as well as to the preservation of the environment strongly restrict use of pesticides.Elaboration development and probation of new methods for control (suppression of the nematode development and reproduction) are necessary.
The parasitology could be an area where the ionizing radiation could have an application and could be used for control of the parasites [6]. Nematode species vary in their sensitivity to irradiation [8, 11, 13]. It is proved that the irradiation retarded the growth of nematodes according to the dose used. Irradiation of second stage juveniles (J2) retarded the growth more that the irradiation of later-stage juveniles. In 1986 the Chernobyl accident gave a wide field for studies in the Chernobyl accident zone of the parasites and parasitic systems in radiation biocenosis. Since the accident the regular studies on the parasitic system have been carried out in the Chernobil accident zone [2].
It is known that γ-irradiation in small doses stimulate increases the germination rate and seed vigour, growth and development, the processes of respiration and photosynthesis of plants, and, ultimately, to improve their productivity [7]. This is due to changes in the metabolism of germinating seeds exposed at the earliest stages of development — accelerated mobilization of nutrients, increased oxidative processes, changes to the nucleotide composition of DNA and RNA [5]. Under the influence of high dose γ-oppressive industrial irradiation slowdown comes the appearance of ugly forms and subsequent destruction of plants. This is due to the formation of free radicals, reduce the content of nucleic acid, DNA mutations, oxidative stress is a significant activation of oxidative processes affecting and enzyme systems. Inhibition of growth also was associated with inhibition of the synthesis of auxin-physiological hormones growth, but have not been finally clarified the mechanisms of action of radiation on synthetic auxins IAA, inter alia, promoting cell growth through stretching [1].
Our previous investigations established the differences in using ɑ- and γ- irradiation on some parasitic nematodes [19, 10].
The present work has a purpose to investigate the influence of increasing doses of γ-radiation on the life cycle of root-knot nematode M. arenaria and on its host – tomato plants, cv. Tiny Tim.
Material and methods
The experiments were carried out under laboratory conditions. Population of plant-parasitic nematode M. arenaria was used in the experiment. Egg sacks with eggs containing larvae (J2) were put into small test-tubes. Every test-tube contained 10 egg sacks. The tubes were irradiated.
For study of irradiation effect on Tiny Tim tomato plants, seeds were irradiated with the same doses of gamma rays. The seeds were sown in soil sterilized by heating under laboratory conditions (t = 20° C). The planting of the irradiated seeds was made on the next day after irradiation. The development of the tomato plants was observed during the following 60 days. The experiment was carried out in three replications.
The irradiation was performed by using a source of gamma-rays was 60Co with activity of 281 µCi and dose rate of 0.1mGy/h. The dosage of γ-irradiation used in this experiment included 90, 700 and 1800 mGy. The parasites and seeds were given a single γ-irradiation treatment.
Resultsand discussion
Influence of γ-irradiation on development of the nematodes M. arenaria.
Root-knot nematodes spend most of their active lives within plant roots, feeding on dramatically modified host cells. Their life cycle involves passage through a series of four juvenile stages, separated by molts, during which the cuticle is replaced. The infective stage is the motile, juvenile (J2) that penetrates the root and migrates to a site near the vascular tissue to establish a permanent feeding site. After feeding is initiated, the nematode becomes sedentary and then undergoes three molts during development to the adult stage. Adult females are bulbous and nonmotile. Egg production begins at 3 to 6 weeks after the initial infection, depending on the species and environmental conditions. Gender is determined epigenetically, with males increasing in frequency under conditions of crowding or poor nutrition [18]. Males also pass through a nonmotile developmental stage but regain motility during the third molt before leaving the root. After development of the female eggs are released on the root surface in a protective, gelatinous matrix (egg sack).
In Figure 1 the data on hatching larvae from eggs in the control and experimental variants (the number of investigated eggs for every variant is 400).
Figure 1. Effect of different doses of irradiation on hatching larvae
The data obtained have shown that irradiation effect on of the larvae hatching. The number of the larvae hatching in control variant (nonirradiated) sharply increased the first day, reached maximum on the fifth day and slowly decreased to the seventh day. Hatching of the eggs irradiated with 90 mGy in general follows the trends of the control variant (Fig 1). Irradiation with 700 mGy leaded to considerable breaking of the egg hatching rhythm compared with the control. Irradiation with 1800 mGy strongly decreased egg hatching to the end on the seventh day.
Metric characteristics of males and females of M. arenaria are made after De Man indices. For statistical reliability each variant was performed in three repetitions. Effect of gamma rays on the metric characteristics of nematodes is given in Table 1, 2.
Table 1.
Metric characteristics of M. arenaria (males)
|
|
Measurments |
Experimental variants |
||||||||
|
|
Control (N=20) |
90 mGy (N=20) |
700 mGy (N=15) |
1800 mGy (N=0*) |
|
|||||
|
|
L (body length) |
1,33 (1,20–1,90) |
1,40 (1,25–1,90) |
1,12 (0,95–1,90) |
* |
|||||
|
|
a (body length/ body diameter) |
57 (42–61) |
55 (43–65) |
50 (40–67) |
* |
|||||
|
|
b (body length/ oesophagus) |
12,5 (11,8–15,4) |
13,0 (12,1–16) |
12 (11,0–17) |
* |
|||||
|
|
c (body length/cauda) |
8,5 (7,1–10,3) |
9 (7,5–11,2) |
10 (7,0–13) |
* |
|||||
|
|
st (length of stylet) |
21 (19,5–22,1) |
21 (20–23) |
19,5 (18–22) |
* |
|||||
* males are not found
Treatment with 90 mGy does not affect on metric characteristics of males. Irradiation with 700 mGy evidently reflexes on metric characteristics of males (shorter body length and larger variations of the metric values). Irradiation with 1800 mGy stops the development of male specimens (Tab. 1).
Higher doses of gamma rays (700 and 1800 mGy) influence on metric characterisrics of M. arenaria females, mainly on body size (after irradiation with 700 mGy body diameter occurs lower values and female body is more elongate).
The highest experimental dose (1800 mGy) destroys development of female specimens (J4) to mature forms (Tab. 2).
Table 2
Metric characteristics of Meloidogyne arenaria (females)
|
Measurments
|
Experimental variants |
|||
|
Control N=20 |
90 mGy (N=20) |
700 mGy (N=20) |
1800 mGy (N=20) |
|
|
L (body length) |
0,780 (0,630–0,850) |
0,800 (0,700–0,900) |
0,700 (0,580–0,800) |
* |
|
d (body diameter) |
0,430 (0,400–0,520) |
0,400 (0,350–0,480) |
0,350 (0,300–0,500) |
* |
|
st (length of stylet) |
21 (19,5–22,1) |
21 (20–23) |
19,5 (18–22) |
* |
*Only J4 were found
2. Data for the characterization of the Tiny Tim plants
During the experiment a data were recorded on the following trials on the parameters of plants – height (cm), shoot weight and dry weight of the experimental plants, root and shoot weight and dry weight. The data obtained for the height changes of Tiny Tim control and experimental plants are shown in Table 3.
Table 3
Root and shoot weight and dry weight of the plants
|
Irradiation dose, mGy |
Root weight,g |
Shoot weight, g |
Dry weight, g |
|
|
roots |
shoots |
|||
|
Control plants |
25,3±1,9 |
46,7±3,5 |
17,5±1,4 |
22,3±1,9 |
|
90 mGy |
27±2,5 |
43±3,9 |
18±1,3 |
22±2,1 |
|
700 mGy |
17,5±1,5 |
22,8±2,0 |
9,0±0,7 |
12±1,1 |
|
1800 mGy |
11,5±1,0 |
16,0±1,2 |
7,0±0,5 |
10,5±0,9 |
Dose of 90 mGy does not affect growth of the plants. Considerable differences were found in variant irradiated with dose of 700 mGy and the most negative effect after treating the seeds with gamma rays were observed in variant with dose of 1800 mGy.
The sensitivity of plants to ionizing radiation depends of radiation doses. Very few doses have been shown to stimulate plant growth [14]. High doses of radiation disturb the synthesis of DNA, RNA and protein and also enzyme activity [15]. It is proved that seeds irradiated with lower doses (0,5 kGy) showed 10% germination. Radiosensitivity varies from species to species even among genotypes of the same species [4].
Another parameters which have been measured are root and shoot weight (Tab. 3). Depending on the radiation dose the root and shoot weight has changed. With the increasing of doses the weight of the root and shoot decreases.
Change of correlation between females and males specimens under influence of ionizing radiation to decreasing of males is proved in the experiment. Decreasing of male numbers is strongly depended on irradiation doses [2]. It is also proved that males of helminths are more sensitive to ionizing radiation than females. Increasing of ionizing radiation doses leads to considerable decreasing of males [3]. Irradiation of eggs (Ascarids) with 60–80 kR decreases their embrional development and 200 kR absolutely stops migratory activity of the larvae [2]. Irradiation of the seeds of mung been with gamma rays increments plant height and weight and suppress infection of the roots with plant pathogenic fungi [15].
Conclusion
The great number of root-knot nematodes (Meloidogyne spp.), their worldwide distribution and economic importance require their detail investigation in different aspects.
The irradiation retarded the growth of nematodes in proportion to the doses as higher doses of gamma rays (700 and 1800 mGy) influence on metric characterisrics of M. arenaria females, mainly on body size (after irradiation with 700 mGy body diameter occurs lower values and female body is more elongate. Change of correlation between females and males specimens under influence of γ -ionizing irradiation has been observed and is express in decreasing of males. The highest experimental dose (1800 mGy) destroys development of female specimens of M. arenaria (J4) to mature forms. The number of incubated J2 forms controls and irradiated with 90 mGy sharply increased after the first day after irradiation, reached maximum on the fifth day and slowly decreased to the seventh day. Irradiation with 700 mGy leaded to considerable breaking of the egg incubation rhythm compared with the controls. Irradiation with 1800 mGy strongly decreased incubation to the end on the seventh day.
Experimental investigations of ionizing radiation effect on the host plants show the seeds effect of high doses gamma rays. Ionizing radiation at low doses stimulate the development of plants. The present investigation established that γ-irradiation in doses (700 and 1800 mGy) suppress the development (height, root and shoot weight) of host plants.
Investigations of the effect of ionizing radiation on the host-parasite systems make clear intime interactions between parasite and its host and is of great importance to increase the plant resistance to parasitic attacks. Detail studies of these processes are important to understand number of changes in the nature and on this base to work out programs for prevention of the environment.
Irradiation may be an alternative way to fumigation for desinfectation of plant material. These results show that up-regulation of some physiological characteristics and seedling growth of wheat which follow gamma radiation treatment may be used for abiotic control such as drought and salt stress.
1. Cojocaru A. F., Revin A. F. Study of mechanism of γ-radiation action on the biosynthesis of indol derivativs and their role in energy formation in plant seedling. Sovremennye problemy nauki i obrazovaniya [Modern problems of education and science]. 2010, no. 4, pp. 8-18. (in Russian)
2. Pelgunov A. N. Parazity i parazitarnye sistemy v radiacionnyh biocenozah. Zona avarii Chernobyl´skoj AES [The parasites and parasitic systems in radiation biocenoses. The Chernobyl accident zone]. Moscow, Nauka, 2005. 207 p. (in Russian).
3. Scihobalova N. P. Study effects of ionizing radiation on eggs and larvae of parasitic nematodes. Trudy gel´mintologicheskoj laboratorii Akademii nauk [Proceedings Helminthol. Lab. Acad. Sc. USSR].1976, no. 26, pp. 240-249. (in Russian)
4. Ahmed S., Quireshi, S. Comparative study of two cultivars Zea mays L., after seed irradiation. Sarhad Journal of Agriculture, 1992, vol. 8, pp. 441-447.
5. Borzouei A., Kafi M., Khazaei H., Nazeriyan B. Effect of gamma radiation on germination and physioligical aspects of wheat (Triticum aestivum L.) seedlings. Pakistan Journal of Bot., 2010, vol. 42, no. 4, pp. 2281-2290.
6. Casarosa L. La radiacioni ionnizanti applicate in helminthologia Stab-Grafico, Grafico F. Lli Lega Faenza, 1964. 137p.
7. Chaudhuri K. S. A simple and reliable method to detect gamma irradiated lentil (Lens culinaris Medik.) seeds by germination efficiency and seedling growth test. Rasiat, Phys. Chem., 2002, vol. 64, pp.131-136.
8. Cinnasri R. H., Moy J. H., Sipes B. S., Shmitt D. P. Effect of gamma -irradiation and heat on root-knot nematode, Meloidogyne javanica. J. of Nematology, 1997, vol. 29, no.1, pp. 30-34.
9. Damianova A., Baicheva O., Sivriev I., Salkova D. Investigation of radiation effect on the life cycle of Meloidogyne arenaria (Neil 1899) Chitwood 1949.J. of Experimental Pathology and Parasitology, 2006, vol. 9, no. 3, pp.20-24.
10. Damianova A., Baicheva O., Sivriev I., Ivanova I. Study of enhanced radiation impact on the resistance of plant parasites system, Mat. INSINUME 2004, Albena 27-30 Sept., Bulgaria.
11. Evans K. The effect of gamma radiation on Heterodera rostochiensis // Nematologica, 1970, vol.16, pp. 284-294.
12. Hussey R. S., Janssen G. J.W. Root-knot nematode: Meloidogvne species. In J. L. Starr, R. Cook, and J. Bridge, eds. Plant Resistance to Parasitic Nematodes. Wallingford, UK: CAB International., 2002, pp. 43-70.
13. Myers R. F. The sensitivity of some plant-parasitic and free living nematodes to gamma and x-irradiation. Nematologica, 1960, vol. 5, pp. 56-63.
14. Munjeeb K. A., Greij J. A. Growth stimulation in Phaseolus vulgaris L. induced by gamma irradiation of seeds. Biol. Plant., 1974, vol. 18, pp. 301-303.
15. Naheed I., Dawar S., Abbas Z., Zaki J. Effect of (60Cobalt) gamma rays on growth and root rot diseases in mungbeen (Vigna radiata). Pakistan Journal of Bot., 2010, vol. 42, no. 3, pp. 2165-2170.
16. Stoyanov D. Rastitelni nematodi i borbata s tiah. Sofia, Zemizdat, 1980. (in Bulgarian).
17. Opperman C. H., Bird D. M. The soybean cyst nematode Heterodera glycines. A genetic model system for the study of plant-parasitic nematodes. Curr. Oppinions Plant Biology, 1998, vol.1, pp. 342-346.
18. Triantaphyllou A. C. Environmental sex differentiation of nematodes in relation to pest management // Annual Review of Phytopathology, 1973, V. 11, P.441-462.
