Studies on the Growth and Development of Root-Knot Nematode Meloidogyne incognita of Phaseolus vulgaris in Tissue Culture Media

Introduction

Common bean (Phaseolus vulgaris L.) is an economically important crop and one of the major grain legumes for human consumption in Latin America, Africa and Asia [1, 2]. Despite its importance, production growth rates are limited by viral, nematode, fungal, and bacterial pathogens, insects, lack of drought tolerances and nutritional deficiencies [3]. Therefore, there is considerable interest in the development of new bean cultivars with useful agronomical traits [3]. Plant biotechnology, together with conventional breeding methods, could facilitate common bean improvement since resistance or tolerance to biotic and abiotic stress could be increased and seed quality, plant architecture, and reproduction modes could be altered [4]. Nevertheless, a reliable and efficient in vitro culture Root-knot nematodes invade an array of important crops and have been found more damaging to vegetables, especially in the tropical and subtropical countries of the world [5]. So far, more than 100 species of Meloidogyne have been described throughout the world and, among these, four species viz. Meloidogyne incognita, M. javanica, M. arenaria and M. hapla, which are commonly found. More than 3000 plant species, almost all cultivated plants, have been recorded as the hosts of root- knot nematodes. The infested plants manifest symptoms of chlorosis, stunting, and unthrifty growth [6].

The life cycle of Meloidogyne includes six developmental stages: the egg stage, four juvenile stages (J1–J4), and the adult stage. The life cycle begins when the only infective and mobile stage, J2, penetrates the roots. Then, the nematode migrates through intercellular spaces to establish a permanent feeding site in the vascular cylinder, in which it will induce the differentiation of five to seven cells, named giant cells, that experience morphological, physiological, and molecular modifications caused by the J2 infection and will supply food to the nematode. The root tissue becomes distorted due to the hyperplasia around the cell, forming the root gall. Once the nematode establishes its feeding site, it will become sedentary and will grow in length and width (J3– J4), until reaching the adult stage. In favorable conditions, J2 develops in pear-shaped females. They lay eggs into a gelatinous matrix (egg mass), generally outside the gall. Embryogenesis will lead to a J1 developing inside the egg, which will molt to J2 that will emerge and migrate into the soil. Only males appear under unfavorable conditions, and they will migrate into the soil with no evidence of feeding on plants [7].

Plant tissue cultures have been utilized to propagate some species of tissue invading nematodes. In most studies on Meloidogyne spp., investigators have employed larvae or eggs from roots growing in infested soil. This technique is a simple, inexpensive method for maintaining an abundant supply of M. incognita in the laboratory. By utilizing glass capillary tubes for inoculations, single egg sacs can be isolated to establish and perpetuate pure nematode cultures. Most of the protocols for in vitro regeneration of common bean are based on direct organogenesis or shoot development of different types of exploits, viz. shoot apical meristems, cotyledonary and primary leaf node explants, petiole etc. [8, 9]. However, these procedures yield very low regeneration efficiency [10]. Another approach has been done through indirect in vitro regeneration. To date, there are only four reports on indirect regeneration of P. vulgaris. However, the frequency of shoot regeneration from callus is extremely low [11, 12] or higher depends on cultivars [13, 14]. Therefore, the objective of the present work was to study the growth and development of root-knot nematode Meloidogyne incognita of Phaseolous vuligaris in tissue culture media.

Materials and Methods

In vitro experiments were conducted at the Department of Agricultural Botany, Faculty of Agric., Suez Canal Univ. The cultivar cv. Paulista was obtained from the Vegetable Crop Res. Depar., Horticul. Res. Inst., Agric. Res. Center, Giza. Culturing of Nematode: The pure culture of M. incognita from single egg mass has been maintained on tomato seedling (Lycopersicon esculantum Mill cv. G.S) at 25±2°C in order to obtain sufficient numbers of second stage juveniles.

Results and Discussion

Developmental Stages of in Vitro Propagation of Common Bean Bean Cv. Paulista by Direct Organogenesis from the Embryonic Axis of Mature Seed The developmental common bean cv. Paulista plant stages of M.S media revealed in Figure 1 as the following: the seed stage in Figure 1A, the seed swelling stage after 3 days Figure 1B, the radical appearance stage after 5 days Figure 1C. The cotyledons leaves appear after 6 days Figure 1D, the cotyledon leaves appear after 8 days Figure 1E, the seedling stage after 12 days Figure 1F. The use of tissue culture for plant regeneration and the introduction of foreign genes that confer pathogens resistance or drought tolerance are good options for bean cultivation. Most of the published protocols are based on direct organogenesis or shoot development from meristematic cells [11].

Click to enlarge

Figure 1: The developmental plant stages of common bean cv. Paulista on M.S. med. A: Seeds on M.S media; B: Seed swelling; C: Radical appearance; D: Cotyledon leaf appearance; E: Cotyledon leaf appearance; F: Seedlings; H: Galls on the root Some recent examples of direct organogenesis may be found in the literature [10]. Until now several types of cells, tissues and organs (cotyledonary nodes, embryonic axes, auxiliary shoots, cotyledon with split embryo axis, internodes, hypocotyls, leaves, leaf petioles or intact seedlings) have been used to induce the regeneration pathways [1, 12, 19]. Cotyledons with part of the embryonic axis as explained have been used to regenerate whole plants [20]. These protocols are very efficient.

Conclusion

Root-knot nematode, Meloidogyne incognita completed its life cycle about thirty seven days in root tissues of Paulista cultivar on tissue culture media. Second-stage juveniles of M. incognita penetrated the roots within 24 hrs., and third- stage juveniles were observed in roots (six-eight) days after nematode inoculation. Fourth-stage juveniles were observed in roots 12-14 days, and the pre-adult females were seen inside roots within twenty days after nematode inoculation. The adult females were seen inside roots within twenty-seven days after nematode inoculation. While, egg laying females with deposit egg masses of M. incognita was observed thirty-seven days after nematode inoculation.