THE INVESTIGATION OF INFLUENCE OF STATIC PRESSURE PERIODS IN SPRAYERS ON ENTOMOPATHOGENIC NEMATODES VIABILITY

Jerzy Chojnacki
Department of Agricultural Engineering, Koszalin University of Technology, Poland

ABSTRACT

There were performed an experiments with entomopathogenic nematodes of four species: Heterorhabditis bacteriophora, Heterorhabditis megidis, Steinernema feltiae and Pharmarhabditis hermaphrodita that were subjected to the liquid static pressure of 52.5 MPa. The static pressure was intermitted. The times of interval and compressive periods were equal 1 minute. The duration of each experiment was 30 minutes. There were counted a relative viability of nematodes before and after treatment. It was not observed that the intermitted pressure, caused death of the nematodes.

Key words: Entomopathogenic nematodes, biopesticides, liquid pressure.

INTRODUCTION

Entomopathogenic nematodes (EPN) naturally occur in soils. Some species of EPN are used as a natural plant protection control agents both in organic agriculture as well in conventional agriculture [10,11,14,15,20,27]. The nematodes are applied mainly in horticulture on a fields, in glasshouses [26] and in mushroom production [24]. Biopesticides including entomopathogenic nematodes can be used also to the protection of trees: in wood [28] and in urban parks and gardens [25]. Beneficial nematodes have been used against soil inhabiting insects as soil application and against above-ground insects as foliar application [14,22,23]. Except controling the insect grubs beneficial nematodes, are effective in destroying the slug pest [30].

The entomopathogenic nematodes mixed with water can be applied by means of an irrigation systems [29] but most frequent the nematodes are spraying by the means of sprayer machines. During this application, the nematodes are pumped through a hydraulic installation of the sprayers, where they can be destroyed [3,16,18]. The causes of nematode destruction can be changes of liquid dynamic and static pressures inside the installation [8]. The dynamic pressure is associated with kinetic energy in the flowing liquid. It's value depends on the speed of liquid passing through individual elements of the sprayer. Static liquid pressure pushes on the walls of the sprayer installation and presses on the nematodes. During the spraying value of the operating pressures for some nematode species should be not more upper than 2 MPa [23].

Because efficiency of sprayer pump is much more then liquid flow rate from the sprayer nozzles part of pumped liquid is not sprayed and returnes through pipes, control valve and hydraulic agitator into container and next it is pumped back into the installation. This process can be repeated many times. It causes that the nematodes mixed with water undergo pressure changes [9,13].

Fife at al. [8] studied an effects of static pressure differentials on a survival of entomopathogenic nematodes Heterorhabditis bacteriophora. The nematodes, contained in the water, were compressed by means of a piston in a press chamber. Suspension with nematodes was exposed to a pressure of 3421 kPa for 0, 5, 10, and 30 minutes. After the designated time period, the chamber of press was decompressed back to atmospheric pressure. No significant difference in relative viability was detected. The pressure of 3421 kPa was to little to be able to cause the death of nematodes. But, there was expressed a hypothesis that there exists a critical static pressure above which an organism of nematodes can be destroyed.

The tests of determining of critical static pressure for entomopathogenic nematodes were described in papers [4 and 5]. The EPN species: Heterorhabditis bacteriophora, Heterorhabditis megidis, Pharmarhabditis hermaphrodita were exposed to 50 MPa constant static pressure. Time of testing was 30 minutes. It was not noted that that pressure destroyed nematodes [4]. The influence of high pressure of 55 MPa, with three-hour operating time on the viability of nematodes Steinernema feltiae has been investigated but only in the liquid samples taken after third hour the nematodes viability was decreased [5].

Hight static pressure can be used in food production to the meat sterilisation. The pressure from 100 to 1000 MPa is used for killing the food parasites [12]. These parasites after consumption of infected meat may become dangerous human parasites [2,12]. Nematodes Trichinella spirali included in a pork, were subjected to pressure amounting to 200 MPa. The pressure killed them [17]. Larvae of nematodes Anisakis simple included in mackerel were treated the pressure of 300 MPa for 5 minutes [1]. The pressure annihilated them in 100%. High pressure can be used also to sterylization bacteria and their spores. The effectiveness of the adverse effect on bacteria could be increased by raising the temperature [19]. The destructive effect of high pressure to micro and macro organisms is difficult to be explained. Probably it is connected with morphological changes in organic cells (deformation and changes in plasma lemma structure, changes in nuclei) [6,7]. The destructive effect of high pressure is also result its influence on proteins, enzymes and polysaccharides [2].

A general goal of the carried out experiments, was to determine whether the breakes of static pressure in the installation of the sprayers can cause destruction of entomopathogenic nematodes mixed with water, during the multiple flowing through installation.

MATERIALS AND METHODS

The materials used in the experiments were biological plant protection agents: B-green, Heterorhabditis System, Steinernema system and Phasmarhabditis System, manufactured by Biobest N.V. Biopesticide B-green contains nematodes Heterorhabditis bacteriophora is used in the destruction of white grubs in the soil. Active ingredients of Heterorhabditis System are insect parasitic nematodes Heterorhabditis megidis, which seek out and destroy vine weevil larvae in compost. Steinernema system, containing Steinernema feltiae species of nematodes is used to control Sciarids larvaes. Phasmarhabditis System is based on a selected strain of the mollusc parasitizing nematode Pharmarhabditis hermaphrodita, which searches the soil for slugs in order to subsequently destroy them. Before the commencing of the experiment, each species of nematodes were mixed with water. Concentration of nematodes Heterorhabditis bacteriophora was 5000 elements in 1 ml of suspension, Heterorhabditis megidis 4000, Steinernema feltiae – 10000 and Pharmarhabditis hermaphrodita 2000. Everage lenght of stage Infective Juveniles of Heterorhabditis bacteriophora is 588 µm, width – 23 µm, for Heterorhabditis megidis length – 768 µm, width – 29 µm, Steinernema feltiae length – 849 µm, width – 26 μm [21].

There was assumed in the investigation that the influence of static pressure on the damage of nematodes has a linear correlation, and hence it was decided to multiply the static pressure in the testing equipment, as compared to the pressure in the sprayer. The value of experimental static pressure was set to 52.5 MPa.

There are two methods to obtain the liquid high pressure. As a first is a direct system – by compressing the liquid with a piston and as a second is an indirect system – by pumping the liquid into a pressure chamber filled with sample. The application of the direct method to pressure a suspension, nematodes and water, may additionally contribute to failure of pressed nematodes due to direct, mechanical contact with a piston.

Because in the sprayer installation exists static and dynamic pressures, in order to avoid influence of dynamic pressure on nematodes damage a special testing device had to be constructed. Construction of the testing equipment made possible to perform an experiments with intermitted static pressure – with timed intervals of the pressure treatment. The testing device is shown on Figure 1. It consisted of a pump, which was powered by an electrical motor, and generated high water pressure. The pressure inside of the installation was controlled by a pressure valve.

The values of pressure were displayed both on a manometer and on a computer monitor because an electronic pressure sensor was built in the testing equipment. Analog data from the sensor were collected and digitalised by means of measuring card and computer program LabView – products of National Instruments Corporation. Instantaneous pressure values were presented on the Front Panel of LabView program. The Front Panel purpose-built to the experiments is showed on Figure 2.

The breakes of pressure action were made by means of hand-operated cut-off valve and an upper ball valve. There were no liquid pressure in measuring pipe of testing equipment when the cut-off valve was closed and upper valve was open. The pressure resurged when the cut-valve was opened and the others valves were closed. The interval periods of pressure and the action periods of pressure were equal one minute. Total time of each experiment, with interruptions in pressure, was equal 30 minutes. Temperature of water inside of the installation was 18°C.

The samplers were transparent, plastic pipes with openings from both sides (Fig. 3). To prevent any nematode damages during the filling of the sampler, at the begining of the filling the piston was placed inside of the sampler. Opening of the pipe was inserted into a dish with the nematode suspension. Liquid with nematodes was hoisted into the pipe by means of piston. When the pipe has been filled up then opening the liquid drawn into the pipe was closed by using a removable plug and the piston was taken out. The capacity of suspension placed inside of the samplers was 2.5 ml.

Samplers with nematodes were introduced into the installation through the opening in the upper ball valve, while it stayed open. After the placement of a sample in the installation and filling out free space with water, the upper valve was closed. Then the cut-off valve was opened to allow the pumped water to reach the sample and raise its pressure. The testing device was constructed in such a way not to allow any contact between nematodes and any of its moving elements, which could possibly carry away the nematodes with the stream of moving liquid. A sampler was placed on the bottom of the conduit with valves. Its height was small adequate to be placed below the crossing of the pipes feeding the installation with the liquid. After high pressure treatment the samplers were removed from the testing device through the lower ball valve after opening it.

The resistance of beneficial nematodes to liquid static pressure was assessed through their changes of relative nematode viability. The relative nematode viability was counted through the following formula (1):

(1)

The relative nematode viability was determined from liquid samples with nematodes taken before subjecting them to pressure and afterwards. The samples taken before experiment and after, were left in the thermostatic box (at 18°C for 24 hours). It was assumed that during that time, those nematodes which sustained damage from pressure would die. The experiment was repeated twice for each species of nematodes. In each experiment, six samples of liquid 0.050 ml were taken to asses viability. The assessment was made by observations of samples under a microscope and counting live and dead nematodes in each one. The results were assessed using ANOVA.

RESULTS

The values of relative viability of each nematode species after experiencing pressure treatments is displayed in figure 4. Basing on the results from the experiments a standard deviation and significance level were assessed for each species of nematodes by means of the ANOVA. Values of the standard deviation were marked in a charts.

a)

b)

c)

d)

The ANOVA did not show any significant influence of static pressure on relative viability (significance level > 0.05). For each nematodes species the differences between mean relative viability of control sample and any one pressed sample is within the value of standard deviation.

CONCLUSION

1. The entomopathogenic nematodes of species: Heterorhabditis bacteriophora, Heterorhabditis megidis, Steinernema feltiae and Pharmarhabditis hermaphrodita were resistanced to the static pressure of 52.5 MPa, intermitted with one-minutes time of periods, for 30 minutes.

2. The breakes of static pressure can not cause any damage to the entomopathogenic nematodes mixed with water, during the multiple flowing through installation of agricultural sprayers.

ACKNOWLEDGEMENTS

Scientific works were carried out with funds for science in the years 2007–2010 as a research project No. N 310 049 32/2537.

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Accepted for print: 8.04.2011

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