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Saidova Shoira Olimovna, Institute of Zoology, AS RUz, Tashkent E-mail: saidova.shoira@gmail.com Eshova Kholisa Saidovna, National University of Uzbekistan, Tashkent E-mail: eshova.kholisa@gmail.com
STUDY OF THE PATHOGENIC IMPACT OF NEMATODE MELOIDOGYNE ARENARIA CHITWOOD, 1949 ON THE TISSUE SYSTEMS OF THE HOST PLANT
Abstract: When the root system of aubergine is infected experimentally with Meloidogyne arenaria Chitwood, 1949, certain functional and structural changes occur in the plant's cells and tissues. At the first stages after infection the nematode mechanically damages the cells of the host plant, while at the second stage pathologic changes in the cells and tissues of the root system can be observed.
Keywords: Meloidogyne arenaria, meloydoginosis, pathology, plants, host, mechanisms.
1. Introduction
Parasitic nematodes rank among the most dangerous parasites of agricultural plants. The damage caused annually by parasitic nematodes to the global economy is estimated at 77 billion US dollars [5]. Particularly threatening are nematodes from the genus Meloidogyne Goldi, 1887 (Nematoda: Meloidogyninae) parasitising plants on both open and protected ground. This is why to study the morphofunctional mechanisms of interaction between parasitic nematodes, including the genus Meloidogyne, and the host plant is highly important from the scientific and practical aspects, contributing to the development of new methods of treating and preventing helminthiases of plants.
The goal of the research is to study the mechanisms of pathogenic impact of Meloidogyne arenaria Chitwood, 1949 on the tissue systems of the host plant represented by aubergine Solanum melongena L. in our research, and to describe the morphology of the cells and tissues of an infected plant.
2. Material and methods
The object ofthe research was aubergine (Solanum melongena L.) of the "diamond" variety, which was experimentally infected with Meloidogyne arenaria Chitwood, 1949. The plant was infected at the early vegetative stage, on an experiment site in the Botanical Garden of the Faculty of Biology, National University of Uzbekistan. For the experiment we took 20 pots (10 for reference samples and 10 for experimental ones), 0.6 m3 in volume each. We planted 5 seedlings into each pot, and 7 days later we infected the experimental samples with nematodes. Each of the experimental plants was treated with water suspension containing 3.500 ± 200 larvae. The larvae and eggs of the nematode for the experiment were taken from a laboratory, where they had been bred for the purpose in the roots of tomato plants. The nematodes were extracted from the tomato plants using the method developed by O. Z. Metlitsky (1979).
We first cut the infected roots into segments (2-3 cm), put the segments into Petri dishes, dissected them with a sharp scalpel and needle controlling the process with a binocular microscope, and then collected the nematodes (Motic B1-220A-2). We grew all the experimental groups of objects in a hothouse on the same plot in equal conditions. The reference group of plants uninfected with the nematodes was cultivated on the same plot, where they had no contact with the infected plants. For each object of research we took samples from 50 infected plants, which had been selected beforehand by similar external anatomic features. We collected the material 3 months later, when the plants were at the fruiting stage.
The anatomic structure of the plant's roots was studied using material kept in 70% ethanol. The preparations were made up out of the main and lateral roots of the aubergine of the diamond variety cut longitudinally and across. The cuts 5-6 microns thick were made with the use of a microtome (Microm HM 340 E) out of the middle part of the lateral roots of the infected plants, where the galls had formed. A total of 1.600 cuts were prepared and then painted with methyl blue and safranin. Separately, 35 micropreparations were made out of them following the common method [1].
3. Results and discussion
Nematodes from the genus Meloidogyne are obligate parasites of plants, whose life cycle includes four juvenile stages. The first stage (Jx) and the evolution into a larva take place inside the egg. The larva that exits the egg is capable of infecting plant roots. A part of the infective larvae that develop at stage 2 (J2) leave the infected root into the ground, while the other part may stay within the root system. From the ground they actively penetrate into the sprouting root of a plant near the root cap. Then they move head first to the vessels of the vascular bundle, where they feed and grow into the third-stage (J3) and then fourth-stage (J4) larvae. The latter evolve into
females, which are 1 mm long and have the form of a bottle, sphere or pear [3].
The process of the infection of a plant with the nematode consists of two phases. At the first phase the parasite penetrates into the plant's tissues, while the second stage is characterised by the development of certain relations between the parasite and the host. The host plant responds to the infection in different ways: galls formation, tissue maceration or necrosis. The external display of the parasite-host relations depends on the parasite's specific features [4]. In our research the commonest reaction of the aubergine infected with nematode Meloidogyne arenaria was the formation of galls in the main and lateral roots. As we studied the mechanisms of the pathogenic impact of nematodes from the genus Meloidogyne on aubergine's tissue structure, we established that pathologic changes occur in the root tissues of the host plant. When the nematodes penetrate into and migrate within a plant, nonspecific changes take place in the tissues of their roots, such as the damage of the epidermis, loosening of the parenchyma, maceration of the tissues, formation of cavities, hypertrophic growth and subsequent decomposition of the parenchyma. These are basic changes that occur in areas with the strongest concentration of the infection [5; 6]. As it enters the root of a host plant, the second-stage larva initiates the formation of a feeding zone. Most pathologic changes take place in the cells
and tissues of the vascular bundle and parenchyma around it, as well as in the walls of the phloem and xylem. Analysing the results of our study and comparing aubergine roots infected and uninfected with nematodes from the genus Meloidogyne we came to a conclusion that the plant's root system changes greatly. The changes consist in the rupture of cellular walls, hypertrophy of parenchymal cells which includes thickening of their walls and specific destruction, all of which may be observed in various sections of the tissues of plants' roots. As the plant develops, the damaged area in these organs extends, involving intergumentary and strengthening tissues and the conducting system.
As we studied the transverse section of the lateral roots of infected plants we found out that externally it is covered with 3-4 layers of phellogen, under which the cortex parenchyma constitutes 50% of the central cylinder. Cambium functions in the central cylinder and forms secondary xylem, while between the vessels there is thin-walled parenchyma consisting of 5-6 granules. The radial rays consist of 1-3 rows. The phloem constitutes around 10% of the central cylinder. The root conducting vessels are not deformed (fig. 1a). The nematode second-stage larvae (J2) penetrate through the cellular walls into the vascular cylinder and migrate to the root parenchyma, where they create for themselves a growth and feeding zone. This can be easily seen on the picture (fig. 1b).
Figure 1. Transverse cut of aubergine roots uninfected a) and infected with Meloidogyne arenaria; b) CP - cortex parenchyma, PR - periderm, PHG - phellogen, XL - xylem, RR - radial rays, N - nematode, E - eggs
The head of the nematode larva is usually situated in the centre of the gall. Not far from the head gigantic multinucleated cells (rK) can be observed in the xylem. From 4 to 7 gigantic cells can be found near one nematode. Hydrocytes (water cells) can also be observed around the head (fig. 2 a, b). Pathologic changes in tissues and cells in plant roots occur initially in root ducts and the parenchyma. The walls of the
phloem and xylem in infected roots are considerably thicker and deformed.
Transverse cuts in infected roots are highly important for the study of plant tissue and cells, while longitudinal cuts are necessary to visualise areas where the nematode penetrates, settles and acts.
Figure 2. Transverse cut of the infected roots of aubergine: gigantic cells a) and hydrocytes; b) N - nematode, HC - hydrocytes, GC - gigantic cells, E - eggs
Analyzing the longitudinal cuts of a damaged root we found in the root parenchyma several female nematodes and an ootheca with eggs. Each nematode larva forms a special feeding zone, which leads to pathologic changes in the root parenchyma of the vessels around the larvae. Nematode larvae are usually located near parenchymatous cells, close to the central cylindrical area of the root. The larvae's growth and development, when the gigantic cells are forming, leads to the tightening and deformation of root conducting vessels, which results in larger distances between the vessels. The infectious larva of nematode Meloidogyne arenaria migrates within the root, where it evolves into an egg-laying female. This process repeats several times, which results in an increase of the number of females. Mature females are fertilized. After being fertilized the female first discharges some jelly-like sub-
stance, which forms into an ootheca - an egg sack. After that the female lays eggs into the ootheca. Figure 3 clearly shows an ootheca and mature eggs (fig. 3a). When nematode larvae develop and the gigantic cells form on plant roots, it leads to a malfunction of the root's conducting tissues. A large number of nematodes can be found near the vessels of the conducting bundle, whose walls are thickened. Nematodes from the genus Meloidogyne directly impact the plant in the course of the entire period of its growth. A few individuals can always be found in infected organs. This also results in the break of correlations in physiological processes going on in the organs of a plant, and affects the differentiation of cells and tissues. The host plant's response to the penetration of nematodes can be seen in the formation of galls, maceration of tissues and necrosis (fig. 3b).
Figure 3. Longitudinal cut in the infected root of an aubergine of the diamond variety: ootheca: a) and galls, tissue maceration and necrosis; b) PR - periderm, XL - xylem, CP - cortex parenchyma, N - nematode, L - larva, E - eggs
4. Conclusion At the first stage the nematodes mechanically damage the
To summarise, the experimental infection of aubergine cells of the host plant, while at the second stage pathologic
roots with nematode Meloidogyne arenaria results in struc- changes can be observed in the cells and tissues of plant's
tural and functional changes in the plant's cells and tissues. root system.
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