Distribution of Entomopathogenic Nematodes in a Biopesticide Spray

Transactions of the ASABE

Vol. 54(6): 1981-1989 � 2011 American Society of Agricultural and Biological Engineers ISSN 2151-0032 1981

DISTRIBUTION OF ENTOMOPATHOGENIC

NEMATODES IN A BIOPESTICIDE SPRAY

E. Brusselman, B. Beck, F. Temmerman, S. Pollet, W. Steurbaut, M. Moens, D. Nuyttens

ABSTRACT. Application technology for biological insecticides like entomopathogenic nematodes (EPN) has been relativelyneglected. One of the major considerations related to the selection and use of an application system is the applicationdistribution pattern. A completely uniform distribution of the nematodes in soil applications is not essential because thenematodes can move over short distances; however, a uniform distribution is important in foliar EPN applications. Thepresent study examined the volumetric distribution pattern of Steinernema feltiae beneath single standard flat‐fan,air‐induction, deflector, and TwinJet spray nozzles. A comparison with the distribution of a chemical tracer was made to revealpossible distribution problems. Droplet size spectra of the nozzles were measured and linked with the distribution results. Atheoretical calculation of the coefficient of variation of the nematode distribution beneath a spray boom was performed.Finally, the actual spray pattern of the EPN on a horizontal surface beneath a spray boom was studied. We can conclude thatthe volumetric distribution pattern of EPN is influenced by nozzle type and is different from the volumetric distribution patternof a chemical compound beneath a TT 110 08 deflector nozzle. The spray overlap from a spray boom decreases differencesin nematode distribution to an acceptable level. Nozzle type significantly influences the number of nematodes deposited ona horizontal Petri dish and their distribution within the droplets. Future experiments are needed to reveal if the measureddifferences in deposition and coverage due to nozzle type will result in significant differences in pest control.

Keywords. Droplet characteristics, Entomopathogenic nematodes, Foliar application, Nozzle type, Spray distribution, Spraypattern.

ince the early 1980s, entomopathogenic nematodes(EPN) classified in the families Steinernematidaeand Heterorhabdititae have received considerableattention because they possess many favorable at‐

tributes as biological pest control agents (Kaya and Gaugler,1993). The steinernematid and heterorhabditid nematodeshave similar life cycles. At the end of their free‐living stage,infective juveniles (IJ) of the nematode enter a suitable insecthost either through natural openings of the insect or via open‐ings made by the nematode itself. Once in the insect's bodycavity, the IJ release a mutualistic bacterium on which theywill further develop. The bacteria multiply rapidly, causinghost mortality within 48 h (Kaya and Koppenhöfer, 1999).

Submitted for review in November 2010 as manuscript number PM8935; approved for publication by the Power & Machinery Division ofASABE in November 2011.

The authors are Eva Brusselman, Agricultural Engineer, Technologyand Food Science Unit ‐ Agricultural Engineering, Institute for Agriculturaland Fisheries Research (ILVO), Merelbeke, Belgium; Bert Beck,Agricultural Engineer, Department of Crop Protection, Ghent University,Ghent, Belgium; Femke Temmerman, Agricultural Engineer, and SabienPollet, Agricultural Engineer, Inagro, Rumbeke, Belgium; WalterSteurbaut, Professor, Department of Crop Protection, Ghent University,Ghent, Belgium; Maurice Moens, Professor, and David Nuyttens,ASABE Member, Agricultural Engineer, Technology and Food ScienceUnit—Agricultural Engineering, ILVO, Merelbeke, Belgium. Corres-ponding author: Eva Brusselman, Technology and Food ScienceUnit—Agricultural Engineering, Institute for Agricultural and FisheriesResearch (ILVO), Burg. Van Gansberghelaan 115, bus 1, 9820 Merelbeke,Belgium; phone: +32‐9‐272‐27‐84; fax: +32‐9‐272‐28‐01; e‐mail: [email protected].

Several attempts have been made to use EPN as biocontrolagents against target pests located on crop foliage. Early re‐sults, however, were not encouraging both in glasshouse(Hara et al., 1993) and field conditions (Kaya et al., 1981;Hara et al., 1993; Mason and Wright, 1997; Bélair et al.,1998). Failure of EPN in foliar application is mostly attrib‐uted to the sensitivity of EPN to three major abiotic factors,i.e., desiccation, temperature, and UV radiation (Begley,1990; Gaugler et al., 1992; Nickle, 1992; Grewal, 2002).However, the use of sub‐optimal application methods hasalso contributed significantly to these failing applications(Georgis, 1990; Wright et al., 2005; Shapiro‐Ilan et al.,2006). Together with the addition of adjuvants to increaseleaf coverage and persistence of the IJ on foliage (Mason etal., 1998a; Head et al., 2004; Schroer et al., 2005a, 2005b;Qiu et al., 2008), the development of more effective applica‐tion techniques might improve nematode efficacy (Tomalaket al., 2005).

Only a few researchers have studied the effect of applica‐tion techniques on the efficacy of foliar‐applied EPN. In mosttrials, standard hydraulic equipment has been used to applynematodes on foliage (Wright et al., 2005), while previous re‐search has shown that this equipment does not perform well(Lello et al., 1996; Mason et al., 1998b, 1999). Hydraulic flat‐fan and full‐cone nozzles produce a wide range of dropletsizes, many of which are too small to carry a nematode (Lelloet al., 1996). These nozzles are inefficient for application ofIJ using large quantities of water with a high ratio of water tonematode (Lello et al., 1996). Comparing the efficacy of dif‐ferent spray methods in the laboratory, Lello et al. (1996)found that higher‐output nozzles gave the best coverage ordeposition of nematodes and greater insect control. Lower

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1982 TRANSACTIONS OF THE ASABE

deposition rates and poorer insect control were observed us‐ing an ultra‐low‐volume spinning disc. However, because thespinning disc gave nearly 50% mortality while applying lessthan 9% of the nematodes, the authors advised further inves‐tigation of low‐volume systems. Additional studies showedthat the existing spinning discs failed to produce a dropletspectrum carrying sufficient EPN (Mason et al., 1998b,1999). Moreover, Piggott et al. (2003) reported that EPNsometimes aggregate in the disc grooves and are emitted insemi‐dry clumps. A new prototype spinning disc eliminatedclumping, but EPN were emitted in clusters in larger, moredispersed droplets compared to the original system, whichcould reduce the effectiveness (Piggott et al., 2003). Wrightet al. (2005) stated that, even if design problems can be over‐come, such novel application methods are probably commer‐cially unviable since growers may be unwilling to replacetheir existing systems.

No reports were found in the literature about the uniformi‐ty of nematode distribution beneath a spray boom, despite itspossible influence on nematode efficacy. Spray uniformitydepends on the individual spray pattern profiles (Azimi et al.,1985). Commonly used field spray equipment is developedto apply a uniform deposition of chemicals. A minimumoverlap of the individual spray patterns results in a uniformdeposition if the right boom height is chosen (Matthews,2000). However, Krueger and Reichard (1985) and Spanogheet al. (2007) have shown that the chemical composition of thespray liquid can affect the shape of the pattern. The additionof nematodes in the spray liquid might also affect the spraypattern and hence possibly result in a non‐uniform nematodedistribution beneath the spray boom. The present study,therefore, examined the volumetric distribution pattern of theEPN Steinernema feltiae beneath single standard flat‐fan,air‐induction, deflector, and TwinJet spray nozzles. A com‐parison with the distribution of a chemical tracer was madeto reveal possible distribution problems. The uniformity ofnematode distribution beneath a spray boom was assessed bycalculating the coefficient of variation of the nematode dis‐tribution for the different nozzle types, which was comparedwith the corresponding coefficient of variation of the dis‐tribution of the chemical tracer.

A uniform spray distribution of a chemical beneath thespray boom results in a uniform deposition on the sprayedsurface since chemicals are dissolved in water and hence arepresent in every droplet. Nematodes, however, are suspendedin water and are aggregated in a very small number of drop‐lets. Lello et al. (1996) found that 89% to 97% of the dropletscollected on Perspex grids did not contain nematodes. Forthis reason, the actual deposition and spray pattern of the EPNon a horizontal surface beneath a spray boom were studied.In the study by Lello et al. (1996), low‐output flat‐fan andfull‐cone nozzles with ISO 02, 03, and 04 nozzle sizes wereused. The nozzles with the highest output deposited the great‐est number of IJ onto leaves and yielded the greatest insectmortality. Moreover, the high flow rates (1000 L ha‐1) ad‐vised for EPN application demand high‐output nozzles to en‐able field applications. Spray nozzles with an ISO 08 nozzlesize were, therefore, used in the present study.

Droplet size and velocity characteristics of the nozzleswere measured using either water or the nematode‐chemicalsuspension similar to the one used in the volumetric distribu‐tion pattern experiment. The distribution results were ana‐lyzed using these droplet size spectra.

Table 1. Nozzle types used in the experiments with their BCPCclassification and flow rate at a spray pressure of 4.0 bar.

NozzleBCPC

Classification[a]Flow Rate[b]

(L min‐1)

XR 110 08 M 3.67 ±0.04AI 110 08 VC 3.58 ±0.05TT 110 08 M 3.69 ±0.04

TJ60 110 08 F 3.61 ±0.01[a] F = fine, M = medium, and VC = very coarse.[b] Measured in the BELAC‐accredited Spray Tech Lab using a flow rate

test bench according to ISO 5682‐1 and ISO 17025 (ISO, 1996,2005).

MATERIALS AND METHODSSPRAY APPLICATION EQUIPMENT

The effect of nozzle type was studied in all experiments.Four nozzle types were used, i.e., a standard flat‐fan nozzle(TeeJet XR 110 08), an air‐induction flat‐fan nozzle (TeeJetAI 110 08), a drift‐reducing deflector type nozzle (TeeJet TT110 08), and a TwinJet spray nozzle (TeeJet TJ60 110 08)(TeeJet, Springfield, Ill.). All spray applications were per‐formed at 4.0 bar. The actual flow rates of the nozzles andtheir BCPC classifications are provided in table 1. The BCPCclassification is based on the comparison of the droplet sizespectrum (DV0.1, DV0.5, and DV0.9) produced by the nozzle‐pressure combinations with the BCPC reference nozzle‐pressure combinations as defined by Southcombe et al.(1997) and measured with the PDPA laser‐based measuringsetup and protocols used by Nuyttens et al. (2007).

All spray applications were performed using a greenhousesprayer (Delvano, Harelbeke, Belgium). The sprayer has aspray tank with a maximum volume of 200 L, a motor‐drivendiaphragm pump (AR 813, Annovi Reverberi, Modena, Ita‐ly), and a pressure regulator (VDR50, Annovi Reverberi,Modena, Italy). The filter was removed before spraying.

DROPLET SIZE AND VELOCITY CHARACTERISTICS

Droplet spectra were measured with a PDPA laser (phaseDoppler particle analyzer). The measurement protocol usedwas described by Nuyttens et al. (2007, 2009). The spraycloud was scanned on the horizontal long axis at 0.50 m be‐neath the spray nozzle in 20 cm intervals. The percentage ofliquid by volume produced for each droplet size interval, thevolume median diameter (VMD), and the Vvol50 were deter‐mined. The VMD is commonly used to describe droplet sizeand provides an indication of the size of droplets produced inrelation to the proportion of the total volume (Matthews,2000). The Vvol50 is the vertical droplet velocity below whichslower droplets constitute 50% of the total volume (Nuyttenset al., 2009).

The droplet spectra were measured with water and with asuspension of 2500 S. feltiae mL‐1 and 0.01 g L‐1 BrilliantSulfo Flavine (BSF, Waldeck GmbH, Muenster, Germany) asspray solution. For every nozzle type and spray suspension,three repetitions were performed using three differentnozzles.

VOLUMETRIC DISTRIBUTION PATTERN BENEATH ONENOZZLE

Entomopathogenic NematodesInfective juveniles of S. feltiae (Koppert B.V., Berkel en

Rodenrijs, The Netherlands) with a size of 736 to 950 �m(Grewal et al., 1999) were used in the experiments. Approxi‐

1983Vol. 54(6): 1981-1989

mately 30 min before every test, a package of 250 million IJwas suspended in 10 L of water before it was added to 50 Lof water in the spray tank of the greenhouse sprayer. Eventu‐ally, the tank was filled to a volume of 100 L to obtain a sus‐pension of approximately 2500 IJ mL‐1. Before every test, thespray suspension was hydraulically mixed for 5 min using thesprayer pump to ensure an equal distribution of EPN in thespray tank (Brusselman et al., 2010a), after which a samplewas taken at the top of the tank. For every sample, the nema‐tode concentration was estimated by counting the number ofnematodes in three 125 �L subsamples using a microscope.

Chemical TracerBrilliant Sulfo Flavine was used as a chemical tracer. By

adding 1 g of BSF to the 100 L nematode suspension, a con‐centration of approximately 0.01 g L‐1 was achieved. The ex‐act concentration of BSF in the spray tank was measuredusing a fluorimeter (Fluostar Optima, BMG Labtech GmbH,Offenburg, Germany).

Spray PatternatorThe volumetric distribution pattern of the EPN and BSF

beneath a spray nozzle was measured using a spray patterna‐tor according to ISO 5682‐1 (ISO, 1996). The sprayed liquidwas intercepted in channels with a width of 0.025 m and col‐lected in graduated collecting tubes. In the center of the table,spray liquid was collected by 40 tubes with a maximum vol‐ume of 100 mL. Adjacent on both sides, 40 smaller tubes witha maximum volume of 50 mL collected the spray liquid.

The EPN‐BSF suspension was sprayed at 50 cm above thespray patternator at a pressure of 4.0 bar. When a stable sprayfan was formed, the collecting tubes were put in measuringposition. The measurement was ended when the central tubeswere almost completely full. The duration of each measure‐ment was timed, and the volume of spray liquid collected inevery tube was recorded. The content of each tube was col‐lected in a recipient. To restrict the number of analyses, the100 mL tubes were joined per two, and the 50 mL tubes werejoined per three. To prevent the loss of sedimented nema‐todes, the tubes were rinsed. The rinsing water, i.e. 50 mL forthe 100 mL tubes and 25 mL for the 50 mL tubes, was addedto the respective recipient.

For every recipient, the number of nematodes was countedin three 125 �L subsamples using a microscope and then col‐lected with a micropipette after thoroughly shaking. A mini‐mum number of 200 EPN in every subsample was aimed for.In the samples at the outer regions of the spray fan, the vol‐ume of the subsample had to be increased to achieve this lim‐it. If the deviation of the three counted numbers was largerthan 20%, then an additional counting was performed. TheBSF concentration in the samples was measured using afluorimeter. Based on the spray volume in the tubes and thevolume of rinsing water added, the original nematode andBSF concentration in the tubes could be calculated. Relativeconcentrations of EPN and BSF were calculated using thefollowing equations:

100(%)

,

, ×=

V

NV

N

RCEPN

x

xEPN

xEPN (1)

100(%)

,

, ×=

V

MV

M

RCBSF

x

xBSF

xBSF (2)

where RCEPN, x and RCBSF, x are the relative concentrationsof S. feltiae and BSF, respectively, at distance x from the cen‐ter of the nozzle, NEPN, x is the estimated total number ofnematodes found at x, MBSF, x is the estimated weight of BSFcollected at x, Vx is the volume of spray suspension measuredat x, V is the total volume of spray suspension collected be‐neath the nozzle, NEPN is the total number of nematodesfound beneath the nozzle, and MBSF is the total weight of BSFfound beneath the nozzle. For every nozzle type, three repeti‐tions were performed using the same nozzle.

THEORETICAL NEMATODE DISTRIBUTION BENEATH A SPRAY

BOOMSpray liquid distribution over the width of a spray boom

depends on nozzle spacing, boom height, and the singlenozzle spray distribution. Ideally, the distribution of EPN be‐neath the spray boom should be uniform. According to Euro‐pean Standard EN 12761‐2 (EU, 2001), which specifies therequirements for design and performance of a field cropsprayer, a spray distribution is uniform if the coefficient ofvariation (CV) does not exceed 7% at one boom height andone pressure. CV was calculated on the flow of EPN and BSFfor a width of 1.4 m at 0.50 m beneath a theoretical sprayboom consisting out of five nozzles mounted at a distance of0.50 m, using the measured single‐nozzle distribution pat‐tern.

DEPOSITION AND SPRAY PATTERN BENEATH A SPRAY BOOMA nematode suspension of approximately 2500 IJ mL‐1

was prepared in the spray tank of the greenhouse sprayer ina similar way as in the volumetric distribution experiment.The nematodes were applied using a five‐nozzle spray boommounted on a spray track (Foqué and Nuyttens, 2011a,2011b) and traveling at a speed of 1.1 m s‐1. Spray depositswere collected into five empty Petri dishes with a diameterof 3.4 cm and in five dishes filled with 3 mL of water. Thesedishes were placed in pairs (one empty and one filled withwater) in the shape of a cross, with one axis parallel and theother perpendicular to the spray boom (fig. 1), on top of a hor‐izontal rolling bench. The distance between the two centraldishes and the others was 70 cm. All nozzle types were testedusing the same spray pressure (4.0 bar) and height (0.50 m).The distance between the nozzles on the spray boom was50�cm. For every nozzle type, three repetitions were per‐formed with the same nozzle set.

After spraying, the total number of nematodes wascounted microscopically in the dishes filled with water. Therelative deposition (RD, %) was calculated based on thetheoretical maximum deposition that could be obtained in thePetri dishes using the following equation:

100(%)RD ×=ca

s

n

(3)

where n is the number of nematodes counted in the Petri dish,s is the surface area of the Petri dish (cm2), a is the application


Figure 1. Schematic of the experimental setup to measure the spray pat‐tern of entomopathogenic nematodes on a horizontal surface at 0.50 m be‐neath a horizontal spray boom.

rate (mL cm‐2), and c is the concentration of nematodes mea‐sured in the spray tank (EPN mL‐1).

The initial empty dishes were analyzed using an imageprocessing system (Brusselman et al., 2010b). With the time‐lapse function of the camera, a sequence of five images at 2�sintervals was taken of all droplets in the Petri dish. The imag‐es were analyzed using the freeware program ImageJ (Ras‐band, 2008). They were converted into 8‐bit pictures and puttogether in a stack. (ImageJ can display multiple spatially ortemporally related images in a single window. These imagesets are called stacks.) Displaying the five images in se‐quence allowed for the easy recognition of mobile nema‐todes. A grid was placed with area per point of 74,272 pixels2.A square of 4 × 4 points in the center of the image, corre‐sponding with a surface of 1.96 cm2, was used for the analy‐sis. All droplets containing at least one EPN were cleared(turned white), and then all nematodes without surroundingwater were counted (Z) and then filled (turned black). Drop‐lets without nematodes were also filled. After manually set‐ting a threshold value, a particle analysis was performed,resulting in the number of droplets containing one or morenematodes (N), the mean size of these droplets (S), and thesurface fraction covered with droplets containing one ormore nematodes (O) in an area of 1.96 cm2.

STATISTICSThe experiments were statistically analyzed using Statis‐

tica (ver. 8.0., StatSoft, Tulsa, Okla.). The calculated relativeconcentration data for BSF and S. feltiae were analyzed forevery nozzle separately by a factorial ANOVA with tracer(BSF, EPN) and position as fixed factors with an interactionterm.

The measurement of the droplet size spectra resulted inVMD and Vvol50 results for different positions along the longhorizontal axis of the spray fan. The effects of nozzle type andtype of suspension on these parameters were analyzed usinga factorial ANOVA.

In the spray pattern experiment, relative deposition wasanalyzed using a factorial ANOVA with position and nozzletype as fixed factors with an interaction term. The parametersresulting from the image processing (Z, N, S, and O) wereanalyzed by a factorial ANOVA with position and nozzletype as fixed factors with an interaction term.

Significant differences were assessed by Tukey's post‐hoctest. A p‐value <0.05 was considered statistically significant.

RESULTSDROPLET SIZE AND VELOCITY CHARACTERISTICS

The addition of nematodes and BSF in the spray tank didnot affect the VMD (F = 2.21, df = 1, p = 0.1385) nor theVvol50 (F = 1.75, df = 1, p = 0.1870) of the spray, while nozzletype significantly affected both (VMD: F = 134.61, df = 3, p�<0.000001; Vvol50: F = 47.67, df = 3, p < 0.000001) (table 2).

For the same nozzle size (ISO 08) and pressure (4.0 bar),the AI 110 08 nozzle produced a coarser spray compared withthe other nozzles (table 2, fig. 2). The XR 110 08 and the TT110 08 nozzles produced a very similar spray, while the TJ60110 08 nozzle produced the smallest droplets. The differencein VMD between the AI 110 08 nozzle and the other nozzleswas significant for every position along the fan width(fig.�3a). In contrast, the difference between VMD of the XR110 08, TT 110 08, and TJ60 110 08 nozzles was only signifi‐cant in the close region below the center of the nozzle. Nearthe edges of the fans, i.e., more than 60 cm from the center,the differences in VMD disappeared.

The droplets produced by the TJ60 110 08 nozzle had thelowest speed. The speed of the droplets produced by the XR110 08 nozzle was comparable to that of the AI 110 08 nozzle(table 2). For all nozzles, except for the TJ60 110 08 nozzle,droplets had the highest speed in the region below the centerof the nozzle (fig. 3b). Corresponding with the VMD results,the differences in Vvol50 disappeared more than 60 cm fromthe center of the fan.

VOLUMETRIC DISTRIBUTION PATTERN BENEATH ONE

NOZZLE

All nozzle types showed a similar or a narrower distribu‐tion of EPN compared with the chemical tracer BSF (fig. 4).The differences between the distribution of BSF and EPNwere very small for the XR 110 08 and TJ60 110 08 nozzles.For the XR 110 08 nozzle, only one position of the 32 sam‐pling positions revealed a significant difference in relativeconcentration. No significant differences were found for theTJ60 110 08 nozzle. For the AI 110 08 nozzle, significant dif‐ferences were found in 14.8% of the sampling positions. Thedifference between the deposition of nematodes and BSF was

Table 2. Volume median diameter (VMD) (mean ±SE) and verticaldroplet velocity (Vvol50) below which slower droplets constitute 50%of the total spray volume (mean ± SE) for different TeeJet nozzlesmeasured using water and EPN‐BSF suspension at a pressure of4.0 bar. Means followed by the same letter are not significantly

different (VMD: F = 0.95493, df = 3, p = 0.41448;Vvol50: F = 1.2266, df = 3, p = 0.30031).

NozzleType

VMD (μm) Vvol50 (m s‐1)

Water EPN‐BSF Water EPN‐BSF

XR 110 08 335.78 a±5.22

352.52 a±9.44

8.29 a±0.57

8.33 a±0.81

AI 110 08 513.62 b±5.49

522.18 b±7.06

7.04 a±0.39

6.87 ac±0.57

TT 110 08 314.45 ab±9.46

308.03 ab±15.99

5.24 bc±0.35

3.71 bd±0.29

TJ60 110 08 244.70 c±15.56

279.77 bc±24.40

3.33 d±0.18

3.18 d±0.19

1985Vol. 54(6): 1981-1989

Figure 2. Volumetric droplet size spectrum for different TeeJet nozzlesmeasured with a PDPA laser using EPN‐BSF suspension at a pressure of4.0 bar and by scanning along the horizontal long axis at 50 cm beneaththe spray nozzle in 20 cm intervals.

highest for the TT 110 08 nozzle. Nematode distribution wasclearly more concentrated in the center of the spray cone ofthis nozzle. At 50% of the sampling positions, a significantdifference between relative BSF and EPN concentrations wasmeasured.

THEORETICAL NEMATODE DISTRIBUTION BENEATH A SPRAYBOOM

The coefficient of variation varied between 3.64% for thedistribution of BSF beneath a spray boom with TT 110 08nozzles and 7.07% for the distribution of EPN beneath aspray boom with TJ60 110 08 nozzles (table 3). For the XR110 08 and the AI 110 08 nozzles, a lower coefficient of varia‐tion was calculated for the distribution of EPN comparedwith BSF. The opposite was found for the TT 110 08 and TJ60110 08 nozzles.

(a) (b)

Figure 3. (a) Volume median diameter (VMD, �m) and (b) vertical droplet velocity (Vvol50, m s‐1) below which slower droplets constitute 50% of thetotal spray volume for different TeeJet nozzles at different horizontal positions within the spray fan, measured with a PDPA laser using EPN‐BSF sus‐pension at a pressure of 4.0 bar.

Figure 4. Concentration of BSF and EPN relative to the concentration in the spray tank, measured 0.50 m beneath the nozzle at different horizontalpositions within the spray fan, after spraying at 4.0 bar.


Table 3. Coefficient of variation calculated on the single nozzledistribution of BSF and EPN over a length of 1.4 mbeneath a theoretical spray boom with five nozzles.

Nozzle Type BSF (%) EPN (%)

XR 110 08 5.68 5.40AI 110 08 4.05 3.85TT 110 08 3.64 6.94

TJ60 110 08 5.99 7.07

DEPOSITION AND SPRAY PATTERN BENEATH A SPRAY BOOMRelative Deposition

Relative deposition was significantly influenced by posi‐tion (F = 12.21, df = 4, p = 0.000001) and nozzle type (F =5.73, df = 3, p = 0.0023). At position 3, a significantly higherdeposition (102.66% ±5.16%) was measured compared tothe other positions. Position 3 was situated at the end of thespray track. When the spraying machine was not turned offimmediately after spraying, the collectors at this position re‐ceived more spray liquid than intended. The results for thisposition were therefore considered outliers and removed forthe analysis of the effect of nozzle type on relative deposition(table 4) (F = 3.05, df = 3, p = 0.0428). The highest relativedeposition, i.e., 88.43% ±3.43%, was obtained with the AI110 08 nozzle. This deposition was significantly higher thanthe deposition obtained with the XR 110 08 nozzle (76.88%±3.26%).

Image ProcessingThe position of the dishes did not significantly affect any

parameter of N, S, O, and Z. Nozzle type significantly af‐fected the number of droplets containing a minimum of onenematode (N) (F = 15.49, df = 3, p = 0.000001), the mean sizeof these droplets (S) (F = 17.19, df = 3, p < 0.000001), and thesurface covered with these droplets (O) (F = 19.98, df = 3, p�<0.000001) (table 5). The spray booms equipped with TT 11008 and TJ60 110 08 nozzles delivered significantly moredroplets with a minimum of one nematode than the othernozzles. The TT 110 08 nozzle, however, produced smallerdroplets, resulting in a covered surface fraction that was notdifferent from that of the XR 110 08 and AI 110 08 nozzles.The surface of the dishes covered with these droplets was sig‐nificantly higher with the TJ60 110 08 nozzle. No effect of

Table 4. Relative deposition (RD) (mean ±SE) of S. feltiae on horizontalPetri dishes measured beneath a spray boom equipped with different

TeeJet nozzle types. Means followed by the same letter arenot significantly different (F = 3.05, df = 3, p = 0.0428).

Nozzle Type RD (%)

XR 110 08 76.88 ±3.26 aAI 110 08 88.43 ±3.43 bTT 110 08 79.67 ±2.03 ab

TJ60 110 08 78.38 ±3.44 ab

Table 5. Number of droplets with minimum one nematode (N) in anarea of 1.96 cm2, the mean size of these droplets (S), and the fraction ofthe surface covered with droplets containing minimum one nematode

(O) for different TeeJet nozzle types. Means followedby the same letter are not significantly different.

Nozzle Type N S (pixels2)[a] O (%)

XR 110 08 19.36 ±0.99 a 9075 ±683 a 14.17 ±0.67 aAI 110 08 16.67 ±0.73 a 10830 ±562 a 15.01 ±0.76 aTT 110 08 23.53 ±1.01 b 5600 ±231 b 11.07±0.53 a

TJ60 110 08 26.20 ±1.10 b 10120 ±616 a 22.58 ±1.82 b[a] 10000 pixels2 ~ 0.0165 cm2.

nozzle type on the number of nematodes without surroundingwater (Z) was found. Z varied from 0 to 7 with a mean of2�nematodes.

DISCUSSIONConventional boom sprayers equipped with hydraulic

nozzles are used to apply entomopathogenic nematodes inbroad‐acre crops. According to Chapple (1999), the spraycloud produced by these nozzles has two components of in‐terest to the applicator: droplet size distribution and spraypattern. When applying nematodes, the primary requirementis an even deposition pattern across the spray swath. Al‐though some nematode species can travel substantial dis‐tances through the soil, others do not. In either case, evendistribution across the target surface is important, and ho‐mogenous distribution is even more important in foliar ap‐plications (Chapple, 1999). On a leaf, the nematodes need topenetrate a host and escape from the detrimental conditionson the foliage as quickly as possible (Chapple, 1999).

The application of foliar sprays is a complex process thatoccurs in several steps: after spray atomization, the dropletstravel to the plant surface where they can impact on leavesand be retained (Reichard et al., 1998). This research investi‐gated spray atomization of a nematode suspension and theretention of nematode‐containing droplets.

Measurement of the volumetric distribution pattern re‐vealed a significant effect of nozzle type on the distributionof S. feltiae beneath a spray nozzle. The differences betweenthe nematode distribution and the distribution of the chemi‐cal tracer seem negligible for the standard flat‐fan and theTwinJet nozzle. Small differences were measured for the air‐inclusion nozzle, while a remarkable difference in EPN‐BSFdistribution was found for the deflector nozzle. The nema‐tode concentration showed a sharp peak in the center of thespray cone and declined much faster toward the edges ascompared with the BSF concentration.

No correlation could be found between the droplet size orvelocity characteristics and the high nematode concentrationbeneath the deflector nozzle (TT 110 08). In the center of thespray cone, the deflector nozzle produced droplet sizes com‐parable to those of the standard flat‐fan nozzle. The velocityof these droplets is intermediate between the velocity of thedroplets produced by the air‐inclusion and the TwinJetnozzle. At the edges of the spray fan, the differences in drop‐let size between the standard flat‐fan, deflector, and TwinJetnozzle disappeared. Differences in droplet velocity disap‐peared at the edges for all nozzle types. This means that theeffect of nozzle type on the distribution of nematodes beneatha nozzle cannot be attributed to the different nozzle spectra.We therefore conclude that the reason for this effect shouldbe found prior to the spray formation process.

No reports were found on the distribution of nematodes ina pressure‐driven channel flow; studies on long‐chain flex‐ible polymers in this situation have received much moreattention (Jendrejack et al. 2003, 2004; Chen et al., 2005;Fang et al., 2005; Usta et al., 2005). The consensus of thesestudies is that flexible polymers in a pressure‐driven channelflow migrate away from the walls toward the center line,leading to the formation of a depletion layer (Saintillan et al.,2006). If this is also the case for nematodes, then this couldresult in a higher concentration of nematodes in the center of

1987Vol. 54(6): 1981-1989

the channel connected to the spray nozzle. In a deflectornozzle, a jet of liquid passes through a relatively large orificeand impinges at high velocity on a smooth surface at a highangle of incidence (Matthews, 2000). As no mixing occursduring passage through the nozzle, this could lead to highernematode concentrations in the center of the nozzle's spraycone. The internal shape of a fan nozzle causes liquid froma single direction to curve inwards, so the two streams of liq‐uid meet at the exit orifice (Matthews, 2000), which resultsin the mixing of the spray suspension. This could explain themore uniform distribution of nematodes beneath the othernozzles.

The measurement of volume distribution patterns for indi‐vidual nozzles is important for assessing the variation in liq‐uid volume and consequently in number of nematodesapplied across the boom (Miller and Butler Ellis, 2000).Ideally, the distribution of EPN beneath the spray boomshould be uniform. An acceptable value for the coefficient ofvariation was found for all nozzles, except for the TwinJetnozzle where the CV was slightly above 7%. The comparabledeposition of Petri dishes with nematodes placed at three dif‐ferent positions (2, 4, and 5) beneath the spray boom con‐firmed that the distribution of the nematodes is more or lessuniform for all nozzle types tested.

The addition of BSF and nematodes to the spray suspen‐sion did not result in different droplet spectra. Chapple (1999)stated that droplet size spectra can largely be ignored whenapplying nematodes: “The nematodes do not `queue up' atthe edge of the sheet of liquid produced by the nozzle, butthemselves act as the foci for drop production.” The latterstatement is clearly not applicable for 08 or 04 standard flat‐fan nozzles at a spray pressure of 4.0 bar (e.g., the TwinJet 08nozzle, which is composed of two 04 spray cones). In our ob‐servations, the large nematodes (736 to 950 �m) did not inter‐fere with the spray atomization, not even in the center of theTwinJet spray fans with a mean VMD of 93 �m. This resultis in compliance with the observations of Mason et al.(1998b). In their study, the addition of IJ with sizes varyingfrom 611 to 1066 �m did not alter the droplet spectra pro‐duced by two spinning disc systems (VMD: Herbaflex200��m; Ulva+ 50‐150 �m), irrespective of the flow or theinitial concentration of the IJ. We therefore assume that thenematodes that arrive at the break‐up site are incorporated ina droplet that is produced as a result of the physical propertiesof the spray liquid. This implies that the number of nema‐todes within a deposited droplet will be related to the initialvolume of the droplet at the time of its formation at the break‐up site. If a nematode arrives at the break‐up site at a positionwhere a droplet is formed, but the droplet is too small to con‐tain a nematode in its most curled position, then that nema‐tode will be deposited bulging out of the droplet or evenwithout surrounding water due to evaporation of the smalldroplet in the air. We presume that the number of nematodesemerging from the nozzles corresponds with the number ofnematodes delivered from the spray tank, regardless of thedroplet size. The number of nematodes actually deposited onthe target, in this case the water surface in a Petri dish, de‐pends on the number of nematode‐containing droplets re‐tained on this surface after impact and the number ofnematodes in those droplets.

The highest relative nematode deposition on the horizon‐tal Petri dishes beneath the spray boom was obtained with theair‐induction nozzle and was significantly different from the

lowest deposition, which was obtained with the standard flat‐fan nozzle. This difference could be attributed to an effect ofdroplet reflection on the water in the Petri dish. Whether adroplet is retained or reflected by a plant or an artificial sur‐face is a function of the properties of the spray solution (sur‐face tension and viscosity), spray pattern (droplet size andvelocity), and the surface morphology and chemistry of sur‐face functional groups of the target surface (Reichard et al.,1998). Since the tank solution or collecting surface did notchange during our experiment, the significance of dropletsize and velocity can be retained. It has been suggested thatthe presence of air bubbles in the large droplets, produced bythe air‐induction nozzle, reduces the risk of droplets bounc‐ing off a leaf surface (Matthews, 2000). Cooper and Taylor(1999) found higher deposition on horizontal targets with air‐induction nozzles compared with standard fan nozzles. Thiseffect depends on the surface morphology and the position ofthe target. Therefore, in future research, deposition experi‐ments should be performed with the crop of interest.

No differences in relative nematode deposition could befound between the standard flat‐fan, deflector, and TwinJetspray nozzles. Since the TwinJet nozzle produces the finestspray compared with the other nozzles, droplet size cannot beassigned as the restricting factor for the number of EPN deliv‐ered in this case, i.e., nozzles with a VMD of 245 �m. Dueto differences in nozzle flow rate and hence application dose,it is difficult to compare our results with the results obtainedearlier by Lello et al. (1996), which were expressed in termsof IJ deposited per cm2. Calculating the relative depositionreveals that the higher‐output flat‐fan nozzles with a smallerVMD (04‐F80: 251 �m at 3 bar) delivered a higher relativenematode deposition, i.e., 116%, than the lower‐output flat‐fan nozzle 03‐F80 with a higher VMD (RD: 85%; VMD:315��m at 2 bar) (Lello et al., 1996). In present study, a rela‐tive deposition of 77% was measured after spraying with thestandard flat‐fan XR 110 08 nozzle (VMD: 352 �m at 4 bar).The increasing relative deposition with decreasing VMDcould be related to the lower number of small droplets bounc‐ing from the collector after impact (Smith et al., 2000).

The differences in relative deposition after spraying withdifferent nozzle types delivering the same flow rate, in thestudy by Lello et al. (1996), should be attributed to differ‐ences in the path of the EPN‐containing droplets or to the cap‐ture efficacy of these droplets by the collectors or to both. Therelative deposition was much higher for the flat‐fan nozzlescompared with the full‐cone nozzles, in which the liquid isswirled through the orifice (Matthews, 2000).

Image processing of the spray deposits on horizontal Petridishes revealed that the nematode distribution in the spraydeposits depends on the nozzle type used. The highest frac‐tion of the surface covered with droplets containing a mini‐mum of one nematode was obtained with the TwinJet nozzle.This is a result of the higher number of droplets with a mini‐mum of one nematode produced by this nozzle in combina‐tion with the large size of these droplets. The size of thenematode‐containing droplets cannot be related with theoverall droplet size characteristics. The TwinJet nozzle, witha BCPC classification of “fine,” delivers nematodes in drop‐lets of the same size as those of the standard flat‐fan and air‐inclusion nozzles with “medium” and “very coarse”classifications, respectively. This observation shows the sig‐nificance of the last steps in the foliar spray process, i.e., theretention of the droplets after impact.


Future experiments are needed to reveal if the measureddifferences in deposition due to nozzle type will result in sig‐nificant differences in pest control. This will probably de‐pend on the nematode species selected, pest characteristics,and crop type.

CONCLUSIONWe can conclude that the volumetric distribution pattern

of EPN is influenced by nozzle type and can be different fromthe volumetric distribution pattern of a chemical compound.However, the spray overlap using a spray boom reduces thedifferences in nematode distribution to an acceptable level.Nozzle type significantly influences the number of nema‐todes deposited on a horizontal Petri dish and their distribu‐tion within the droplets. Future experiments are needed toreveal whether or not the measured differences in depositiondue to nozzle type will result in significant differences in pestcontrol.

ACKNOWLEDGEMENTS

This research was funded by the Ministry of the FlemishCommunity IWT‐Vlaanderen. We wish to thank ILVO'stechnical staff and Donald Dekeyser for technical supportand Miriam Levenson for reviewing the manuscript.

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Distribution of Entomopathogenic Nematodes in a Biopesticide Spray