Histopathological Response of Lens culinaris Roots Towards Root-knot Nematode, Meloidogyne incognita
Lens culinaris (lentil) is an important pulse crop. The yield of the crop is reduced if grown in root-knot nematode (Meloidogyne incognita) infested field. Meloidogyne incognita caused infection in primary and the secondary roots leading to the anomalies in the affected part of the root. The study revealed that the second stage juveniles (J2) of Meloidogyne incognita entered the growing roots and their branches inter and intracellularly. The immediate response was hypertrophy and hyperplasia in the root tissue near the nematode head. In response to hypertrophy some cells became very large and contained dense and granular cytoplasm. Adjacent to the giant cells, the vascular tissue was found to be disturbed. Shape, size and orientation of the vascular elements was so much altered that it had become difficult to trace the normal course of vascular strands. In various sections vascular strands were found disrupted. The vessel elements had the shapes resembling the shapes of parenchyma cells. Similarly sieve tube elements of the phloem, near the giant cells were shorter and resembled with nearby parenchyma cells. Abnormalities in xylem and phloem favored transport water, minerals and metabolites towards the giant cells. From this study, it might be inferred that alteration in the cells of galled tissue was essential for the sustenance of giant cells and for the survival of the nematode.
Received: January 04, 2013;
Accepted: February 13, 2013;
Published: March 22, 2013
Parasitism by root-knot nematode is characterized by establishment of permanent
feeding sites comprising giant cells in the cortex, endodermis, pericycle and
vascular tissue of the host roots. The root cells of different plants respond
quickly and characteristically to Meloidogyne incognita juveniles. The
juveniles move intercellularlly towards the region of vascular differentiation
where they induce the formation of giant cells (Niyaz and
Hisamuddin, 2008; Bhat et al., 2009; Niyaz
et al., 2011). The giant cells are generally transformed from undifferentiated
vessel elements or from xylem parenchyma (Christie, 1936)
or from provascular strand (Krusberg and Nielsen, 1958;
Littrell, 1966), or have been reported to arise from
protophloem cells (Byrne et al., 1977; Yasmeen,
In addition to giant cell formation, the root-knot nematode causes hypertrophy
and hyperplasia in the cells adjacent to the giant cells that lead to the formation
of galls. Meloidogyne infections accompany cortical and stelar proliferations
(Davis and Jenkins, 1960), hypertrophy and hyperplasia
in the cortex, pericycle and stele of the roots (Azam and
Hisamuddin, 2008; Azam et al., 2011).
Histological studies appear to be the key to unraveling the association between injuries caused to plants by plant parasitic nematodes. Yet no work so far has been reported on histopathological studies of lentil roots.
Keeping in view, the damage caused by root-knot nematode, M. incognita
to lentil roots, the present study was conducted to ascertain the histological
changes that lead to the formation of galls on the roots of Lens culinaris,
formation of giant cells and also to assess the abnormalities in orientation
of xylem and phloem and to study the relationship between the giant cells and
the vascular elements.
MATERIALS AND METHODS
Maintenance of Meloidogyne incognita Culture: Pure inoculum of
M. incognita was maintained on egg plants by using single egg mass culture.
Heavily infested roots of egg plant, on which pure culture of M. incognita
was maintained, served as the source of nematode inoculum. The egg masses from
these infected plants were handpicked with the help of sterilized forceps. The
egg masses after being washed with distilled water were placed in 10 cm diameter
sieves, lined with two layers of tissue paper, placed in a Petri plate containing
sufficient amount of double distilled water. Freshly hatched second-stage juveniles
were collected regularly after every 24 h and transferred to a beaker, incubated
at room temperature 27+5°C (Den Ouden, 1958).
Water suspension of second-stage juveniles was thoroughly stirred to homogenize
and the total number of nematodes per milliliter was counted with the help of
a counting dish (Doncaster, 1962) under stereoscopic microscope.
Raising the test plant: Lentil seeds (cultivar K-75) were surface sterilized
with 0.1 sodium hypochlorite (NaOCl) (Koenning and Barker,
1985) poured into sterilized beaker filled with 1:1 mixture of 95% ethanol
and 5.25% NaOCl. The mixture was drained off and the seeds were rinsed with
sterilized distilled water. About 3-4 axenized seeds, pre-soaked in water, were
sown in 15 cm clay pots, each filled with one kg steam sterilized soil (7 clay:
3 sand: 1 farmyard manure). One week after germination thinning was done to
maintain single seedling per pot.
Inoculation technique: When the seedlings became one week old, holes of 5-7 cm depth around the plants within the radius of 2 cm from the plant were made. Through these holes the suspension of second-stage juveniles was inoculated at the rate of 1,000 J2 per plant. The holes were plugged with sterilized soil. To maintain the soil moisture, the pots were regularly watered. The non-inoculated plants served as control.
Harvesting: Plants (one from each treated pot) were harvested after 45 days of inoculation. The roots were washed thoroughly and gently to remove soil particles. The galled roots were then cut into one cm long pieces and processed for histopathological studies.
Processing for histopathological studies: From infected roots, few galled
portions were selected from each treatment for performing histopathological
studies. The galled tissues were cut and fixed in Formalin Aceto-Alcohol (F.A.A)
and then dehydrated through Tertiary Butyl Alcohol (TBA) schedule (Johansen,
1940). The galls were infiltrated with paraffin oil and then embedded in
paraffin wax. The wax embedded galls were trimmed to small blocks and then fixed
on wooden blocks. Sections of 10 μm thickness were obtained in the form
of ribbon with the help of rotary microtome. The ribbons were cut and mounted
on the slides, which were kept in an incubator at 40°C for 24 h (Johansen,
1940). The sections were stained with safranin and fast green as described
by Sass (1951) and mounted in Canada balsam. For anatomical
details, the sections were observed under light microscope and necessary photographs
Anatomy of normal root: The normal primary root of Lens culinaris is tetrarch. It consists of epidermis, cortex, endodermis, pericycle, radial vascular bundles and the pith. The secondary growth pattern is typically of dicotyledonous root type; secondary xylem (wood) is produced towards the periphery. Secondary xylem consists of prominent vessel elements and parenchymatous rays (Fig. 1).
Giant cell and gall formation: The second-stage juveniles of Meloidogyne incognita penetrated the young roots of lentil and moved intercellularly and settled in the region of vascular differentiation (Fig. 2). The juveniles while penetrating, pushed the root cap cells apart and made a narrow passage to move through it. The entrance formed by a juvenile was also used by others reaching at the same point (Fig. 3).
Cellular injury at the root cap was not observed but the cells were just pushed
apart and thus, a narrow passage was established.
|| Normal root anatomy
|| (J2) second stage juvenile
||Second stage juvenile moving through the path made by other
Few cells of epidermis and outer cortex were found severely damaged at the
point of entry. The cells along the length of the nematode appeared small and
compactly arranged. In some cells, two hypertrophied nuclei were noticed. In
each hypertrophied two or more hypertrophied nucleoli were found.
Hypertrophy and hyperplasia occurring simultaneously were observed in the provascular region. The cells near the head of the nematode were severely hypertrophied. These incipient giant cells contained a large central vacuole and parietally distributed, granular cytoplasm.
With the development of the root and formation of secondary xylem, infected zone was found pushed towards the periphery. The giant cell complexes and the nematodes occupied ray regions of the root.
Xylem: The second-stage juveniles of Meloidogyne incognita penetrated the roots of Lens culinaris and after moving through the region of vascular differentiation settled in between xylem and phloem (Fig. 4). The vessel elements and xylem parenchyma were found distorted when observed in transverse and longitudinal sections. The vessel elements of secondary xylem, near the giant cells, were enlarged. Their diameter was larger than the normal vessel elements (Fig. 5).
Abnormalities in xylem were more prominent near the giant cell complex (Fig. 6). The giant cells containing dense cytoplasm, hypertrophied nuclei and nucleoli were observed away from the xylem elements (Fig. 7). The vessel elements exhibited abnormalities in shapes, sizes and orientation. In a transverse section longitudinally arranged abnormal vessel elements were more frequent (Fig. 8).
Abnormal xylem was found differentiated towards the cortex, in the infected part (Fig. 9). The Giant Cell Complexes (GCC) in the wood region and cortical region were observed when the same part of the root was attacked by several nematodes (Fig. 10).
In infected root the wood part and in the cortical part parenchyma exhibited abnormalities in shapes and sizes. The affected part of xylem parenchyma, was found changing into vessel like elements. The abnormal vessel elements differentiated from ray parenchyma in the affected portion were frequently seen (Fig. 7-9).
Phloem: Primary phloem is the product of fascicular cambium and secondary
phloem of vascular cambium. The second stage juveniles were found in the zone
of cell division and differentiation. The giant cells were observed in the vascular
region usually at the places where xylem and phloem elements are synthesized
and differentiated into respective elements.
||Giant cell (GC), giant cell complex (GCC), mature nematode
(N) and ray (R)
||Abnormal Phloem (AP), Abnormal Xylem (AX) and Giant Cell Complex
||Normal Xylem (NX), Normal Ray Parenchyma (NRP), distortion
in ray parenchyma (DR), Abnormal Phloem (AP) and Giant Cell Complex (GCC)
||Abnormal Phloem (AP), Giant Cell (GC), Nucleus (N), Nucleolus
(Nu) and Juvenile (J2)
||Giant Cell (GC), Abnormal Phloem (AP) and Abnormal Xylem (AX)
||Normal Xylem (NX), Abnormal Phloem (AP), Abnormal Xylem (AX),
Giant Cell Complex (GCC)
Phloem elements exhibited changes in their morphology and orientation.
||Abnormal Phloem (AP), Giant Cell Complex (GCC) and mature
The sieve tube elements had larger diameter and shorter length and were termed
as Abnormal Phloem (AP) (Fig. 5). Abnormal sieve tube elements
adjacent to the giant cells had no any definite orientation (Fig.
8). Parenchyma cells and abnormal sieve tube elements near the giant cell
complex were indistinguishable (Fig. 6, 7
The second-stage juveniles of Meloidogyne incognita are probably attracted towards the roots of lentil and gain entry into the inner tissue. The anatomical studies revealed that the juveniles entered into the cortex or the stele through the zone of cell division, elongation and differentiation. The mode of entry was axial (through the root cap), radial or tangential (through the cortex). Formation of channels in the inner tissue of the roots confirmed their intercellular migration.
Occurrence of larger cells, near the nematode, or along the passage of nematode
was probably due to hypertrophic response. The hypertrophic response was also
observed in the nuclei of the affected cells. Entry of the juveniles anywhere
from the root cap to zone of cell differentiation has been reported earlier
(Christie, 1936; Krusberg and
Nielsen, 1958; Siddiqui and Taylor, 1970; Ismail
et al., 2004). The juveniles of root-knot nematode, M. incognita,
migrated intercellularly by separating the cell walls along the middle lamella
(Endo and Wergin, 1973; Jones
and Payne, 1978).
Movement of the nematode inside the root without damaging the inner cells and
tissues was favorable for the nematode as it could be harmful for its own survival.
Any injury to the root tissue may lead to death of the plant that would also
be deleterious for the nematode as a consequence the nematode could die. The
immediate response of the inner tissue towards the nematode was enlargement
of certain cells. It is supposed that the secretions of nematode contained some
enzymes and hormones, like pectinases, cellulases and auxins etc. Enlargement
and fragmentation of the nucleoli of the affected cells occurred in various
plants due to root-knot infection (Hisamuddin, 1992;
Yasmeen, 2002; Parveen, 2006;
Niyaz and Hisamuddin, 2009).
In addition to hypertrophy, the affected cells exhibited nuclear divisions
without cell wall formation. Occurrence of 5-6 giant cells, indicated that the
host parasite relationship has been successfully established during this period.
The giant cells possessed upto six nuclei and each nucleus had one or more nucleoli
(Jones and Payne, 1978; Yousif,
1979; Hisamuddin, 1992; Hisamuddin
and Siddiqui, 1992).
In the affected roots of lentil (Lens culinaris), the root-knot nematode
(Meloidogyne incognita) caused various abnormalities in the cortical
and the stelar region (Chandel et al., 2001;
Sayed et al., 2010). During the development,
the nematode gets oriented radially. When the roots are young, then the nematode
head lies in meristematic (cambial) zone and the body in the cortical region.
This orientation favors the pushing out of egg masses out of the root tissues.
The most affected tissues in the roots appeared to be vascular tissues. The
giant cells were found adjacent to the xylem elements which resulted in various
kinds of abnormalities in xylem elements. The normal vessel elements got enlarged
and disoriented. Both of these anomalies resulted in hindrance in transport
of water and minerals towards the aerial part. The other alteration was that
the parenchyma cells adjacent to the giant cells transformed into vessel like
elements. All these changes in the xylem tissue were responsible in transport
of water towards the giant cells (Singh et al.,
2007). The multinucleate giant cells require more amount of water for enhanced
rate of synthesis of cytoplasmic materials which was fulfilled by modification
of the cells of xylem strand in the galled portion.
The ordinary parenchyma cells, since are transformed into abnormal vessel like
elements, therefore, their function of storage of food is taken up by the giant
cells. Thus, the photosynthates that were to be stored in root parenchyma under
normal conditions, are diverted towards the giant cells. The giant cells are
mimicked as storage cells, functioning as sink for the photosynthetic metabolites.
This kind of modification is favorable for the nematode, as it obtains its nutrition
from the giant cells (Hisamuddin, 1992) on Lufa cylindrica;
(Sosa-Moss et al., 1983) on tobacco; (Vovlas
and Ekanayake, 1985) on banana; (Khan et al.,
2007) on jujube.
In case of severe infection a large number of galls on the roots are formed.
In each gall several giant cell complexes become functional towards which mineral
nutrients, water and metabolites are diverted (Senthilkumar
et al., 2007; Azam, 2009; Niyaz
et al., 2011). Transport of metabolites towards the giant cells is
performed by the phloem that always remains connected with the giant cells.
The phloem elements, near the giant cells become deformed and on the basis of
their shapes, these cannot be identified easily. It was found that phloem strand
got diverted towards the giant cells. Completely suppressed phloem was found
by (Swamy and Krishnamurthy, 1971) in Meloidogyne
infected Basella alba roots. Abnormal sieve tube elements with unusual
orientations were formed in Lagenaria roots only after the destruction
of primary phloem as a result of root-knot nematode infection (Siddiqui
and Ghouse, 1975).
From the study it might be concluded that in the roots of lentil plant, the nematodes modified the functions of developing cells. The parenchyma cells were diffentiated into different kinds of vascular elements. The vascular tissue, in the infection court, transported water, minerals and nutrition in greater amount for sustaining the giant cells. The normal mode of translocation of water and minerals was interfered by the elements of abnormal xylem. A large amount of metabolites were tranlocated towards the giant cells, formation of gall and differentiation of abnormal xylem elements contributed in sustaining giant cells on which the survival of nematode is depended.
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