Water Soluble Formulation of Nematode Antagonistic Bacterium, Pasteuria penetrans for the Management of Root-knot Nematode, Meloidogyne incognita in Tuberose

Author: M. Janani*, N. Swarnakumari, A. Shanthi and M. Gnanachitra

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Abstract

Root-knot nematode, Meloidogyne incognita is a serious damage causing nematode in both sub-tropical and tropical regions. It causes global crop losses in agricultural and horticultural crops. Recent reports indicate that this nematode become an issue to farmers due to its parasitism. Pasteuria penetrans is a potential hyperparasitic bacterium against M. incognita. Though it is a successful biocontrol agent against nematodes, mass production and commercial formulation of P. penetrans is still unavailable. Based on these information, a water soluble formulation of P. penetrans was prepared using emulsifiers and surfactants. This formulation was tested against M. incognita in tuberose under pot culture condition. Different concentrations of the formulation viz., 0.5ml, 1ml, 2ml and 2.5ml were prepared and tested its efficacy against M. incognita. Results showed that the formulation 2ml per plant recorded with highest reduction in number of gall (5.1/plant) as well as egg masses production (3.1/plant; 82% reduction over control). The same treatment showed highest plant growth parameters in terms of shoot weight as well as number of tubers. The outcome of this study showed that the formulation 2ml/plant possesses highest potential in suppressing nematode infestation in tuberose. Hence, this treatment was further were forwarded for field study.

Keywords

Meloidogyne incognita, Pasteuria penetrans, water soluble formulation; Shelf-life, Parasitization, Tuberose, Pot culture study

Conclusion

P. penetrans is a successful and potential biocontrol agent which is very effective in controlling the most damage causing root-knot nematode M. incognita in tuberose plants. Water soluble formulation of P. penetrans was developed and tested against M. incognita in tuberose. Application of this formulation at the rate of 2ml / plant showed highest reduction in nematode infestation that was below economic thresh hold level. Hence this formulation may be further refined and tested in other crops in future.

References

INTRODUCTION Root-knot nematode, Meloidogyne sp. is one of the most devastating pests of many field and horticultural crops (Khan, 2015). They exist in soil in areas with hot climates or short winters (Khan et al. 2021). About 2000 plants worldwide are susceptible to infection caused by root-knot nematodes and they contribute approximately 5% of global crop loss (Saha et al., 2016). If root-knot nematodes become established in deep-rooted, perennial crops, control is difficult and options will be limited. Bio-control potential of P. penetrans against Meloidogyne incognita was documented (Mukhtar et al., 2013). Tuberose (Polianthes tuberose L) is one of the most important tropical ornamental bulbous flowering plants cultivated for production of long lasting flower spikes. It is popularly known as Rajanigandha or Nishigandha. It belongs to the family Amaryllidaceae and it is native of Mexico. Tuberose can successfully be grown in pots, borders, beds and commercially cultivated for various uses (Johnson 1970). It's spikes produce about 20 fragrant white florets. Nowadays, root-knot nematode infestation that is particularly M. incognita in tuberose is a challenging one for farmers (Gowda and Chawla 2013). It is the most economically damaging genera of plant parasitic nematodes (Grace et al., 2019). Root-knot nematode infection has been reported from all tuberose growing areas in India (Sellaperumal et al., 2015). Most of the commercially grown varieties of tuberose have been reported to be susceptible to M. incognita (Chawla and Singh, 2006). Understanding the interaction between an obligate hyperparasitic bacterium, P. penetrans and its obligate plant‐parasitic nematode host, Meloidogyne spp (Davies, 2009). So in such way, a biocontrol agent can be used to control its infestation and the disease caused by them (Abdullah, 2012; Grace et al., 2019). P. penetrans is a potential biocontrol agent and studied by several authors (Chen and Dickson 1998; Swarnakumari and Sivakumar 2005). It is a good bacterial parasite of nematode. It was first described as P. romosa a parasite of water fleas, Daphina magna (Skerman et al., 1980) followed by many scientists renamed differently, finally it was reported as Pasteuria penetrans (Sayre and Starr 1985). Juvenile of Meloidogyne nematode hatch from eggs and have a short-free living second stage (J2 stage) in soil after which they invade the rhizosphere and attach to host-plants. The form galls around developing juveniles where they complete their life cycle (Danlei et al. 2004). A study by Chen and Dickson (1997) developed binomial sampling plans to estimate P. penetrans endospore attachment to J2 M. arenaria. Ayanaba (1993) invented the slow-release bio-degradable granules containing endospores of P. penetrans in order to suppress nematode reproduction. Swarnakumari (2021) have entrapped P. penetrans endospores in alginate beads and tested its efficacy in cucumber. EC formulation of this bacterium was developed and tested by Srishalini et al. (2021). In the current study a water soluble formulation was developed and tested in tuberose. The methodology adopted and results obtained are described in this paper. MATERIALS AND METHODS Pure culture maintenance of P. penetrans on root-knot nematode Meloidogyne incognita. Pure culture of P. penetrans was maintained in bhendi (variety Co 4) plants. Egg masses of M. incognita were collected from the infected field (Fig. 1) and incubated at room temperature (27±2 ℃) in water for hatching. After egg hatching endospores of P. penetrans were added to the suspension containing J2 of M. incognita and incubated for 3 days for spore attachment. Then the endospore attached J2 were inoculated into the bhendi plants and maintained in the sterile pot mixture (sand 1: redsoil 2: FYM 2). These plants were pulled out 30 days after inoculation to confirm the multiplication of P. penetrans (Fig. 2). These cultures were used for experimental purpose. After that bhendi plants were uprooted and infested gravid female of M. incognita were dissected to record the presence of endospores. Preparation of water soluble formulation of P. penetrans. Endospores of P. penetrans were collected and stored in sterile microfuge tubes containing sterile water (2ml) by crushing infected female nematodes manually. Water soluble formulation was prepared by using lecithin (Van Nieuwenhuyzen, 1976), Triton –X, tween 80, glycerol, tween 20. Lecithin (1ml) was added to the hot water and mixed thoroughly. Tween 80 (3ml), glycerol (1ml) Triton- X (3ml) and tween 20 (3ml) were added to this prepared mixture (Fig.3). Finally 1 ml of endospore suspension (1 × 106/ml) was added to this mixture and stored in a glass vial at room temperature (27±2 ℃). Assessment of shelf-life of the formulation. The formulation was transferred into sterilized microfuge tubes and stored in room temperature (27±2 ℃). Observations on viability of endospores and condition of the formulation were recorded on 30th and 50th day after inoculation (DAI). Testing the efficacy of water soluble formulation of P. penetrans against M. incognita in tuberose. Seed tubers of tuberose plant were planted in pots filled with sterile pot mixture (sand : vermicompost: red soil - 2:1:1). Various concentrations of each formulation viz., 0.5ml, 1ml, 2ml and 2.5ml were inoculated separately to pots. A chemical check was maintained with Nimitz (1.5g/plant) and the fungal bioagent, Pochonia chlamydosporia (1ml/plant) as positive check. One set of pots were maintained without any treatment as untreated (Control) for comparison. Three replications were maintained for each treatment. Healthy J2 of M. incognita were collected from freshly hatched eggs and were inoculated into the pots (100 J2/pot). Observations were taken on nematode infestation in soil and root and plant biometric characters were also recorded. Statistical analysis. The data obtained from various experiments described above were analyzed using ANOVA DMRT (Panse and Sukhatme 1954) and analyzed using AGRES statistical software. RESULTS AND DISCUSSION Properties of the formulation. The physical characters of the formulation showed that the formulation was dull white in colour and was stable upto 2 months. The formulation was also water soluble. It contained an average of 1.5 × 106 spores/1.5ml (Table 1). Endospores retained their shape and were intact in the formulation. Findings of Raut et al. (2012) also revealed that the formulation was more effective and consistent. Zhou et al. (2010) confirmed that the role of lecithin in formulation preparation and they have a synthesized a lecithin-based nanoemulsion and measured particle size, viscosity, stability and skin hydration. Swarnakumari (2021) have entrapped P. penetrans endospores in alginate beads and tested its efficacy in cucumber. EC formulation of this bacterium was developed and tested by Srishalini et al. (2021). These findings were in agreement with the current observations. Results of these experiments confirmed that the water soluble formulation was effective against M. incognita in tuberose. Shelf- life assessment. Viability of endospores in the formulation was observed on 30 and 60 days after storage. There was no change in the physical condition of the formulation for 2 months (60 days). The parasitization potential of P. penetrans on J2 of M. incognita was normal on 30 and 60 days after storage at room temperature. Pot culture. Water soluble formulation of P. penetrans with different concentrations was applied to tuberose plants. A check of an untreated control was also maintained for comparison. The whole plants were uprooted 45 days after planting (Fig. 6). Growth parameters with each treatment were observed and recorded (Table 3, Fig. 5) This was in accordance with Weibelzahl-Fulton et al. (1996) who tested the efficacy of P. penetrans against M. incognita and M. javanica in tobacco plant. Application of water soluble formulation with concentration of 2ml /plant recorded the lowest gall formation that was 66% reduction over control. The same treatment was on par with P. chlamydosporia (49.3% reduction over control) followed by Nimitz (48.3%). Whereas, formulation at 1ml and 2.5ml / plant was on par with each other and reduced the gall formation by 46% when compared to control. Highest reduction of egg masses was recorded in 2.5ml (3 egg masses /plant that is 82.3% reduction over control). Application of P. chlamydosporia recorded 4 egg masses / plant that was 76.4% reduction over control followed by Nimitz (Table 2, Fig. 4). Highest reduction of J2 population in soil was observed in pots received 2ml of the formulation followed by P. chlamydosporia. Number of infested females was higher in plants applied with the formulation 2ml followed by 2.5ml. Tzortzakakis et al. (1997) conducted the interaction study between P. penetrans and M. incognita under pot culture condition. The spore load was sufficient in females and successfully controlled the M. incognita.

How to cite this article

M. Janani, N. Swarnakumari, A. Shanthi and M. Gnanachitra (2022). Water Soluble Formulation of Nematode Antagonistic Bacterium, Pasteuria penetrans for the Management of Root-knot Nematode, Meloidogyne incognita in Tuberose. Biological Forum – An International Journal, 14(2a): 533-538.