Antinemic activity of Plant Growth Promoting Rhizobacteria, Pseudomonas fluorescens against root knot nematode, Meloidogyne incognita infesting tomato, Lycopersicon esculentum Mill.

Tomato

Tomatoes (Lycopersicon esculentum) are origin of South America and were first used as food in Southern Mexico. Since the fruit has seeds and grows from flowering plant, botanically it is classified under fruits. It is the state vegetable of New Jersey and official state fruit of Ohio and its juice is the official beverage of Ohio.

Largest producer of tomato in the world is China which accounts for the production of 5,00,00,000 MT/ha in the year 2012. Second highest producers of tomato is India which contributed 1, 75, 00,000 MT/ha.

There are more than 7,500 tomato varieties grown around the world and most of the varieties exhibit red colour although colours like green, yellow, orange, pink, black, brown, white and purple are available in the world market. Size of the tomato determines its use as slicing (globe) tomatoes are used for processing in food industries, beefsteak tomatoes (large) are used for making sandwiches, plum tomatoes (oblong) are used for the preparation of sauce and paste. Preparation of salads includes the cherry tomatoes which are small round and often sweet.

The fruit is rich in lycopene which is an antioxidant found to be good for the heart and effective against certain cancer diseases. Also the fruits are rich in vitamin A and C, calcium and potassium. The best sources of lycopene are found in processed tomato products. Eighty per cent of the tomatoes harvested are industrially processed. Increase in demand of the processed products of tomato in domestic and international markets make an upward trend in the cultivation of this crop. Now days, tomatoes are important ingredient in pizza and pasta sauces. High acidic content in the fruit makes it a prime candidate in the canning industries.

The factors that are attributed for the reduction of fruit yield of the crop include both biotic and abiotic stresses. Among the biotic stresses, plant parasitic nematodes play a major role in reducing the crop yield as tomatoes are excellent host for the nematodes. In general, Crop loss due to these nematodes worldwide was estimated to be greater than 11 per cent and among vegetables, infestation of root knot nematode contributes major loss for the crops (Ayyar, 1926).

Generally, plant parasitic nematodes are microscopic; colorless which are vermiform, triploblastic, bilaterally symmetrical, unsegmented and pseudocoelomatic organisms. They inhabit marine, fresh water and terrestrial environment as free living and plant parasites.

In agro ecosystem, a hectare of soil contains about billion of plant parasitic and free living form of nematodes. Damage caused by the plant parasitic nematodes are often unnoticed because the symptoms caused due to nematode damage are similar to that of nutrition deficiency disorder which include slow growth of the plant associated with stunting and yellowing.

Annual yield loss of crops due to plant parasitic nematodes have been estimated about dollar 78 billion worldwide and dollar 8 billion for US growers. In Tamil Nadu, the loss incurred was estimated about Rs. 200 crores (Jonathan et al., 2005).

Root knot nematodes are important parasitic nematodes of tomato worldwide and the crop is most seriously affected with root knot nematodes (Meloidogyne spp.) causing problems throughout the world which reduce the crop yield both in quantity and quality (Plate 1). Although the annual loss reported due to nematodes in tomato is 60-75 per cent in India, the estimated loss due to the root knot nematodes in tomato is about 40 per cent (Dasgupta, 1998). In India, this plant is commonly infested with the root knot nematode species viz., Meloidogyne incognita, M. javanica, M. arenaria and M. hapla.

Root knot nematodes can be easily detectable by their characteristic symptom of producing galls in the roots of the plant. Nematode infected plants show the symptoms of stunted growth, chlorosis and wilting. In tropical countries, the loss incurred due to M. incognita on tomato was estimated as 29 per cent.

Root knot nematodes develop feeding cells called as giant cells which are very important for the successful host - parasitic relationship. The plant tissues around the feeding site of nematode female undergo hypertrophy and hyperplacia to form characteristic root galls (Plate 2). The giant cells are formed due to repeated mitosis without subsequent cytokinensis and these giant cells act as transfer cells which pass nutrients to the nematodes (Plate 3).

Plate 1. Root knot nematode females and juveniles

illustration not visible in this excerpt

Plate 2

illustration not visible in this excerpt

Plate 3. Giant cells formed due to root knot nematode infestation in tomato roots-A microtome section view

illustration not visible in this excerpt

1, 2, 3. Giant cells formed in tomato roots 4. Root knot nematode female

Apart from the direct damage, nematodes also predisposes the plants to secondary invasion by the fungal pathogens like Fusarium spp., Rhizoctonia spp. and the bacterial pathogens like Pseudomonas solanacearum, Corynebacterium michiganense pv. michiganense, etc., which aggravate the disease severity to the crop and finally leads to death of the plant.

Nematode management with nematicides in farmer’s field has limitations due to their high cost, adverse effects on environment as well as difficulties in applying them in the fields. Moreover, very little is known about the mode of action of chemical nematicides and the fate of volatile chemicals applied to the soil is not well known.

Generally, nematode management success over a long term requires several alternatives and integration of approaches which should be economically feasible. This is especially true for horticultural crops. Therefore, safe and alternative methods for managing plant parasitic nematodes in horticultural crops are critically needed for the development of sustainable cropping systems.

A promising alternative is the use of microbial antagonists against plant parasitic nematodes which are ecofriendly and economically feasible and it does not allow the nematodes to develop into new races or biotypes. In addition to that, they are easily amenable for mass production, formulation technology and easy to deliver in the field.

In the recent years, plant growth promoting rhizobacteria (PGPR) viz., Pseudomonas fluorescens and Bacillus subtilis (Oostendorp and Sikora, 1989; Sankari Meena et al., 2011; Ramyabharathi and Raguchander, 2014) which live in close proximity of plant roots have been reported to be effective in boosting the plant growth and vigour and are also deleterious to the plant pathogens in the soil including the ubiquitous tiny worms, nematodes (Rodriguez Kabana et al., 1965; Singh et al., 1990; Sankari Meena et al., 2012; ). The bacterium achieves this mainly by competition, antibiosis and induced systemic resistance.

Induced systemic resistance (ISR) of the plants against pathogens is a widespread phenomenon that has been intensively used in plant protection with biocontrol agents

(Wei et al., 1996). Elicited by a local infection, plants respond with a salicylic dependent

illustration not visible in this excerpt

composition, de novo production of pathogenesis related proteins such as chitinases and glucanases, synthesis of phytoalexins are associated with resistance due to the application of

P. fluorescens.

Increased fresh weight of tomato, cucumber, lettuce and potato were resulted due to bacterization of the plants with Pseudomonas strains (Vanpeer and Schippers, 1988). Similarly, Jonathan et al. (2004) reported that P. fluorescens and B. subtilis induced root development in tomato, banana and betelvine and decreased M. incognita population both in soil and roots. The bacterium has been used as soil inoculants and seed dressing materials because of their potential for rapid and aggressive root colonization which will minimize nematode population.

Hence, a study has been proposed to isolate the native strains of P. fluorescens from tomato fields belonging to different districts of Tamil Nadu and to assess the efficacy of bacteria against root knot nematode, M. incognita in vitro, pot culture and under field condition.

Plant Growth Promoting Rhizobacteria

Plant growth promoting rhizobacteria (PGPR) are a group of free living bacteria that colonize the rhizosphere and benefit the root growth. Bacteria of diverse genera were identified as PGPR of which Bacillus and Pseudomonas are predominant. PGPR exert a direct effect on plant growth by the production of phytohormones, solubilization of inorganic phosphates, increased iron nutrition through iron chelating siderophores and production of volatile compounds that affect the plant signaling pathways. Additionally, by antibiosis, competition (for space and nutrients) and induction of systemic resistance in plants against a broad spectrum of root and foliar pathogens, PGPR reduce the populations of root pathogens and other deleterious microorganisms in the rhizosphere, thus benefiting the plant growth.

Pseudomonas fluorescens

Pseudomonas are gram negative, rod shaped bacterium and their cells are single, straight and measures about 0.7-0.8 X 2.3- 2.8 micron in size during exponential growth and are motile with flagellation (Plate 4). Pseudomonas culture produces diffusible fluorescent (yellow-green) pigment on King’s B medium which is one of the characteristic features for identification the bacterium. The bacterium grows with optimum temperature range of 25 - 30oC.

Plate 4. Pseudomonas fluorescens

illustration not visible in this excerpt

1. Antibiotic production
2. Siderophore production
3. Induced Systemic Resistance
4. Competition
5. Hydrogen Cyanide Production
6. Plant Growth Promotion

1. Antibiotic Production

P. fluorescens is very effective antibiotic producer. Secondary metabolites produced by

P. fluorescens acts as antibiotics against plant pathogens and nematodes. Some of the antibiotics produces by P. fluorescens include Phenazine-1-Carboxylic Acid (PCA), 2,4 - Diacetylphloroglucinol (DAPG), Pyocynine, Pyrrolnitrin, Pyoluteorin and Oomycin-A.

2. Siderophore Production

Siderophore are extra cellular, low-molecular weight compounds with very high affinity for ferric iron. Pseudomonas produces siderophore that is capable of sequestering the available soluble iron, which could interfere with plant growth and function. However, plant roots are sometimes capable of taking up ferric complexes of siderophore and using these as sources of iron (Powell et al., 1982). Thus, siderophore may play an important role in the competition between microorganisms and may also act as growth promoters (Omar and Abd-Alla, 1998). Microbial siderophore may stimulate plant growth directly by increasing the availability of iron in the soil surrounding the roots or indirectly by competitively inhibiting the growth of plant pathogens with less efficient iron-uptake systems (Joseph et al., 2007). The types of siderophore produced by P. fluorescens are Ferribactin, Ferrichrome, Ferroxamine B, Pseudobactin, Pyochelin and Pyoverdine.

3. Induced Systemic Resistance:

The phenomenon of systemic acquired resistance or induced systemic acquired resistance has attracted increased interest as a novel approach to the integrated protection of crop plants (Ryals et al., 1996). P. fluorescens induce systemic resistance in plants that is phenotypically similar to systemic acquired resistance (SAR). Induction of resistance by P. fluorescens is mainly through the production of defense related enzymes and phytoalexins.

4. Competition:

P. fluorescens prevent the establishment of other rhizosphere microorganisms through competition for favored sites or nutrition on the roots and in the rhizosphere region.

5. Hydrogen cyanide production:

Hydrogen cyanide (HCN) is representative of class of volatile inhibitors. Production of volatile cyanide is very common among the rhizosphere pseudomonads (Dowling and O’gara, 1994) which involve in the disease resistance.

6. Plant Growth Promotion:

PGPR are a heterogeneous group of bacteria that can be found in the rhizosphere, at root surfaces and in association with roots, which can improve the extent or quality of plant growth directly and/or indirectly. P. fluorescens promotes plant growth by the production of phytohormones such as auxins and gibberellins and also by the phosphate solubilization. It promotes plant growth directly by production of plant growth regulators or indirectly by stimulating nutrients uptake, by producing siderophore or antibiotics to protect plants from soil borne pathogens.

Overview of plant protection mechanisms of P. fluorescens has been described in Figure 1. These pseudomonads may act directly on the plants, noticeably via production of various signals (phytohormones) and triggering induced systemic resistance (ISR) pathways. They may also inhibit the phytopathogens by competition and ⁄ or antagonism mediated by secondary metabolites such as DAPG. In addition to these effects, the action of certain non Pseudomonas members of the microbial community, may also have direct or indirect biocontrol effects and interfere with the functioning of biocontrol agents from P. fluorescens and related species.

illustration not visible in this excerpt

Figure 1. Overview of plant protection mechanisms exhibited by Pseudomonas fluorescens

[...]