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Screening and selection of adjuvants for the spray application of entomopathogenic nematodes against a foliar pest
PII: SO261-2194(98)00043-X ELSEVIER
Screening and selection of adjuvants for the spray application of entomopathogenic nematodes against a foliar pest Judy M. Mason*, Graham A. Matthews+ and Denis J. Wright** *Department of Biology, Imperial College of Science, Technology and Medicine, Silwood Park, Ascot, Berkshire, SL5 7PY, UK +lnternational Pesticide Application Research Centre, Imperial College of Science, Technology and Medicine, Silwood Park, Ascot, Berkshire, SL5 7PY, UK
The effects of adding a number of adjuvants to sprays containing infective juveniles (IJS) of Heteronsp. and Steinemema sp. (M87:45) was examined. The adjuvants were not directly toxic to IJS of either nematode species or to larvae of the diamondback moth, Plutella xylostella. However, infectivity of the IJS to larvae of the wax-moth, Galleria mellonella, in terms of mortality and intensity of infection, was affected by the addition of the adjuvants, compared with IJS in water alone. Deposition studies showed that the proportion of droplets carrying IJS was not affected by the addition of the adjuvants following application with spinning disc sprayers (Micron Ulva+ and Micron Herbaflex). However, addition of the adjuvants resulted in a significant increase in deposition, in terms of the mean numbers of IJS per cm ‘. This was noted following application with both sprayers and for both nematode species. The spray spectra produced by either sprayer was generally not affected much by the addition of any of the adjuvants to the spray solutions. 0 1998 Elsevier Science Ltd. All rights reserved
rhabditis
Keywords: adjuvants; spray application; Steinernema spp.; Heterorhabditis spp.
Introduction Application of infective juveniles (IJS) of entomopathogenic nematodes (EPNs) belonging to the families Steinernematidae and Heterorhabditidae has generally employed standard spraying equipment used in the application of pesticides (Georgis, 1990). While this is not so important in the application of EPNs against soil-dwelling pests, choice of application method is more critical for their successful application against foliar pests. Such differences between a variety of spraying methods were highlighted by Lello et al. (1996), when they examined a range of hydraulic nozzles (standard fan and full cone) and a spinning disc. Their work suggested that the development of low volume application systems for the application of IJS against targets such as larvae of the diamondback moth (DBM), Plutella xylostella, was warranted. A recent study on the development of two spinning disc sprayers for such applications has confirmed this (Mason et al., 1998a). In the latter work, deposition of IJS was influenced by both flow rate and the initial concentration of IJS in the spray mixture. *Corresponding author. Tel.: +44 (0) 1344 294248; Fax: +44 (0) 1344 294339; E-mail: [email protected]
Adjuvants are commonly added to pesticide formulations to enhance pesticide performance. The types and range of adjuvants available is extensive (e.g., Thomson, 1992). In the application of IJS of EPNs against foliar pests, the major problems are the limited tolerance of the IJS to extremes of temperature, UV radiation and desiccation (Baur et al., 1995; Mason and Wright, 1997). The latter two are the most critical and have received considerable attention, with a wide range of chemicals having been tested in an attempt to reduce the negative impacts of desiccation (Webster and Bronskill, 1968; MacVean et al., 1982; Shapiro et al., 1985; Glazer et al., 1992; Baur et al., 1998) and UV radiation (Gaugler and Boush, 1979; Nickle and Shapiro, 1992; Nickle and Shapiro, 1994). However, no one adjuvant is suitable for all situations and the results from these numerous studies highlight the need for screening of adjuvants for use in a particular situation (Baur et al., 1998). This is the result of a number of interacting factors, including the insect target (e.g., feeding habit, amount of movement on substrate), host plant (e.g., waxy or non-waxy surface) and method of application (e.g., hydraulic nozzles or spinning disc); it is thus essential to screen potential adjuvants with these factors in mind. In the present work, we have examined the toxicity and resultant deposition of IJS
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J.M. Mason et al.
using a range of potential adjuvants for incorporation into sprays with IJS for application against DBM larvae on crucifers. Materials and methods Nematodes Two species of entomopathogenic nematode were used in the present study, Steinemema sp. (M87: 45) and a new Heterorhabditis sp. (Mason et al., 1996). Results from previous studies had suggested that these two species had greatest potential for application against DBM larvae on crucifers in Malaysia (Mason and Wright, 1997). The nematodes were maintained at 20°C in late instar larvae of Galleria mellonella (The Mealworm Co., Sheffield, UK), with IJS being used within 6 days of emergence. Cabbage plants Chinese cabbage, Brussicu pekinensis (cv. ‘Tip Top’), was grown in individual pots of Levingtons MultiPurpose compost in a greenhouse maintained at 15-20°C with a light regime of approximately 16:8 (L:D). Plants were 4 to 6 weeks old at the time of use. DBM culture A UK isolate of DBM (Rothamsted insecticide susceptible strain) was used (Mason and Wright, 1997). Cultures were maintained at 20°C on 4- to 6-week-old Chinese cabbage. Adjuvants Five adjuvants were used in the present study: Triton X-100 (Merck Ltd, Poole, Dorset, UK), Glycerol, Croduvant (Glycerol-based), Crovol L27 and Crovol L40 (both Linseed oil-based) (Croda Chemicals Ltd, Cowick Hall, Snaith, Goole, North Humberside, UK). Glycerol and Croduvant have anti-evaporant properties, whereas both Crovols and Triton X-100 are non-ionic surfactants. Solutions of the adjuvants were tested at concentrations of 2% and 4% (v/v). Sprayers Two spinning disc sprayers were used: the Micron Herbaflex and the Micron Ulva+ (Micron Sprayers Ltd, Bromyard, Hereford, UK). Unless otherwise stated, the Ulva+ was operated at 6 V with a flow rate of 200 ml min -l and the Herbaflex was operated at 6 V with a flow rate of 30 ml min -’ (Mason et al., 1998a). All spraying was conducted using the sprayers inside a Mardrive linear track chamber (Mason et al., 1998a). Assessment of adjuvants Effect of adjuvants on infectivity of IJS The effect of the five adjuvants on the infectivity of IJS of Heterorhabditis nsp. and Steinemema sp. (M87: 45) was assessed using a sand tube assay with late
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instar larvae of G. mellonella (Fan and Hominick, 1991). Nematode/adjuvant suspensions were prepared using distilled water, to give a final nematode concentration of 200 IJS ml -l with adjuvant concentrations of 2% and 4%. To 30 ml Universal tubes, 12 ml of dry, sterile sand (all particles 5 1 mm diameter) was added. One millilitre of the nematode/adjuvant suspension was then added to each sand tube and the tubes were shaken to ensure even mixing. The final moisture concentration in each sand tube was approximately 8% (v/v). Controls consisted of sand tubes with nematode suspension with no adjuvants or distilled water only. One G. mellonella larvae was added to each tube and the sand tubes inverted following fitting with a screw top lid. There were 20 replicates per adjuvant concentration. Following 48 h incubation at 25°C the G. mellonella larvae were removed, rinsed with water and further incubated at 25°C for 24 h to allow adult nematodes to develop. Both mortality and intensity of infection were assessed. EfSect of adjuvunts on survival of IJS
The toxicity of the five adjuvants to IJS of Heteroand Steinemema sp. (M87: 45) was assessed by incubating IJS in solutions (2% and 4%) of the adjuvants. Solutions of the IJS (200 IJS ml -‘) and adjuvants were prepared with distilled water. Five millilitres of each solution were placed in 25 ml glass vials. Following incubation at 25°C for 0, 6, 12 or 24 h, 0.5 ml of test solution was removed and the numbers of dead and alive IJS assessed. All IJS that did not respond to mechanical stimulation were considered dead. There were five replicates per adjuvant concentration. Controls consisted of adding IJS to distilled water and incubation as described above.
rhubditis n.sp.
Adjuvunt toxicity to DBM larvae
The toxicity of the five adjuvants at concentrations of 2% and 4% to third instar DBM larvae was assessed. Leaf discs (5 cm in diameter) of Chinese cabbage were dipped in solutions of the adjuvants for 10 s and then allowed to dry. The dry leaf discs were placed in 5 cm diameter Petri dishes which had been lined with a piece of moist filter paper. Two third instar DBM larvae were placed on each leaf disc, and the Petri dishes sealed with Parafilm ‘M’@ prior to incubation at 25°C (16:8 L:D cycle). There were ten replicates per treatment. Mortality of the DBM larvae was assessed at 24, 48 and 72 h. Controls consisted of Chinese cabbage leaf discs which had not been dipped in adjuvant. Deposition of IJS in udjuvunt solutions
Deposition of IJS in solutions of the adjuvants was assessed using the Micron Herbaflex and Ulva+ in the Mardrive linear spray track. Both sprayers were set up in the Mardrive chamber, as described previously (Mason et al., 1998a). Deposition was assessed for IJS of Steinememu sp. (M87: 45) and Heterorhabditis n.sp. at concentrations of 3000 and 6000 IJS ml -I, respectively. The Herbaflex was used at a flow rate of 30 ml min-‘, while the Ulva+ was
Screening and selection of adjuvants: J.M. Mason et al.
run at both 3 and 6 V, with a flow rate of 200 ml min -. ’ used at each voltage. The five adjuvants were tested at both 2% and 4% (v/v). Additionally, solutions of IJS in distilled water only were tested. Spray spectra analyses of adjuvant solutions
Spray droplet spectra for solutions of the adjuvants at two concentrations (2% and 4%) were assessed using a Malvern@ Particle Size Analyser 2600 (Malvern, Worcester, UK) using both sprayers. Details of the set-up of the sprayers in relation to the Malvern Particle Size Analyser are given elsewhere (Mason et al., 1998a). Both nematode species were assessed with each adjuvant.
Table 2. Effect of adjuvants on infectivity of IJS of Steinernema in G. me//one//alarvae
68.35 k5.74Aa 40.22 i 6.2OAb 36.23 i 7.49Ac 66.3 I7.73Aa 56.68 F 4.88Ad 98.45 * 1I .OOe
XSAabc YOAac 6SAb 1OOAc 9SAc 1ooc
Crovol L27 4%’ Crovol L40 4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
65.32 & 6.52Aa 53.18 + 7.3SBh 19.11 f4.02Bc 5 1.50 +6.09Bb 32.53 + 2.9SBd 98.45 f I I .ooe
YSAa 85Aa 9UBa YOAa YSAa 1OOa
‘Intensity
of infection
adjuvant.
cantly
different
the samr
Generalised linear modelling was used for all statistical analyses using the statistical package GLIM (Numerical Algorithms Group, 1985). Mortality data were analysed using a binomial error structure, and intensity of infection data with a Poisson error structure. Overdispersion was corrected for accordingly (Crawley, 1993). Survival data were analysed by ANCOVA using a binomial error structure. The negative binomial distribution as a model for the distribution of IJS within droplets was assessed using a goodness of fit statistic (G-test). ANOVA, with contrasts, was used with all remaining data analyses. Significance is reported at the 5% level.
Mortality ’ (S)
Crovol L27 2% Crovol IA0 2% Triton X-100 2% Croduvant 2% Glycerol 2% Water
each
Statistical analyses
Intensity ’ (mean?SE)
Treatment
sp.
means
is defined
as mean
followed
by the same
(P > 0.05).
lowercase
letter
numhcr
For each adjuvant arc not signifcantly
of IJS per infected
uppcrcasc concentration.
letter
are
larva. not
For
signifi-
means followed
hy
diftcrcnt.
(PcO.05) following the addition of Crovol L27. Crovol L40 or Triton X-100. Additionally, mean intensity of infection was significantly lower (PcO.05) compared with that obtained with IJS in water only (Table I). For each adjuvant, increasing the concentration from 2 to 4% resulted in lower mortality of G. mellonella larvae and lower mean intensity of infection. Indeed, only the addition of Croduvant resulted in increased mortality and mean intensity of infection (Table I).
for IJS of Steinerof the generally resulting in a reduction in mean of infection in G. mellonella larvae. addition of the adjuvants to the solutions of not generally have a significant effect on of G. mellonella larvae (Table 2).
Similar results were obtained
nema sp. (M45) (Table 2), with the addition
adjuvants intensity However, IJS did mortality
Results Effect of adjuvants
on infectivity
of IJS
The addition
of adjuvants to the solution of IJS of n.sp. generally resulted in a subsequent reduction in mortality of G. mellonella larvae compared with IJS in water only (Table I). This was more marked at the higher concentration of each adjuvant used, where mortality was significantly lower
Heterorhabditis
Table 1. Effect of adjuvants on infectivity of IJS of Heterorhabdifis nsp. in G. me//one//a larvae Intensity ’ (mean k SE)
Treatment
Mortality ’ (S)
Crovol L27 2% Crovol L40 2% Triton X-100 2% Croduvant 2% Glycerol 2% Water
52.88 + 5.91Aa 43.73 + 6.63Ab 48.00 + 12.60Aab 23.71 f4.14Ac 36.53 +3.19Ad 80.65 +8.07e
8OAa 7SAa 30Ab 8SAa 95Aa 1OOa
Crovol L27 4% Crovol L40 4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
33.44 + 11.3SBa 43.38 + 6.68Ab 57.00 + O.OOAc 39.30 + 6.22Bd 29.72+5.01Be 80.65 + 8.07f
4SBa 65Aa 5Ab 1OOAc 90Ac 1ooc
‘Intensity
of infection
each
adjuvant,
cantly
different
means
is delined followed
(P > 0.05).
the stame lowercase
letter
as mean by the
number same
For each adjuvant are not significantly
of IJS per infected
uppercase concentration, different.
letter
are
larva. not
For
signifi-
means followed
hy
Effect of adjuvants
on survival of IJS
The adjuvants had no significant effect (P > 0.05) on the survival of IJS of either Heterorhabditis n.sp. or Steinemema sp. at either concentration of adjuvant tested. Increasing incubation time of the IJS in the adjuvant solution generally resulted in a slight, although not significant (P > O.OS), reduction in survival. This was again noted for each of the adjuvants and at both concentrations tested with both nematode species. The mean survival of IJS of both species following 24 h incubation is shown in Table 3. However, it was noted that IJS incubated in Crovol L27 and, especially, Crovol LAO, became increasingly inactive and assumed a straight posture with increased incubation time. This was noted for both nematode species, with approximately 94-100% of the IJS requiring mechanical stimulation to establish if they were dead or alive. Incubation of IJS of both species in the remaining adjuvants generally resulted in the IJS retaining their activity. If they became inactive, they assumed their characteristic curved or kinked posture rather than a straight posture which is characteristic when IJS of both of these species are dead.
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J.M. Mason et al.
Table 3. Mean (-&SE) survival at 24 h following
incubation
of IJS of Heterorhabditis
n.sp. and Steinernema
Nematode
2%
Glycerol Croduvant Crovol L27 Crovol IA0 Triton X-100 Control
97.1 96.7 93.9 96.5 95.8 97.1
kO.3 k 1.0 + 1.9 k 0.5 kO.9 k 0.5
Adjuvant toxicity to DBM larvae The adjuvants had no significant effect on the survival of DBM larvae (P > 0.05) with survival generally being 100% after 72 h. A slight reduction in survival was noted at the 24 h assessment for 4% Crovol L27 (85% survival), 4% Crovol L40 and Triton X-100 (90% and 85% survival at 2% and 4% concentration, respectively). No further mortality was recorded at 48 or 72 h.
Deposition of IJS in adjuvant solutions: Micron Herbaflex The negative binomial distribution was generally a good model for the deposition of IJS in the droplets (Table 4). The addition of adjuvants to the spray solution caused a significant increase (PcO.05) in the mean number of droplets deposited per cm* following application of IJS of Heterorhabditis n.sp. (Table 4). This increase in numbers of droplets was more marked at the higher adjuvant concentration, for each adjuvant, where the numbers deposited generally doubled. However, the percentage of droplets carrying IJS was not significantly affected by the addition of the adjuvants at either 2% or 4%. In contrast, addition of adjuvants had a marked effect on the mean number of IJS deposited per cm*
at 25°C
species Steinernema sp. (M87: 45)
Heterorhabditis nsp. Treatment
sp. in adjuvants
4%
2%
4%
96.8 +0.5 97.lkO.6 92.5 k 0.9 90.4k2.4 97.2kO.6 97.lkO.5
99.3 kO.6 99.9kO.2 99.3 kO.6 98.8 k 0.6 98.8 kO.5 100 5 0.0
99.1 kO.6 99.8kO.2 99.lkO.6 95.3 + 1.1 99.6 +0.2 100 + 0.0
(Table 4), with, generally, deposition being significantly higher. Increasing adjuvant concentration generally resulted in a slight increase in the numbers of IJS deposited, although no significant changes were noted. Addition of the adjuvants at either concentration also generally resulted in an increase in the numbers of droplets deposited following application of IJS of Steinemema sp. (Table 5). However, the proportion of droplets carrying IJS was generally significantly lower (PcO.05) following addition of adjuvants to the spray solution. The effect of adding the adjuvants to the spray solution on subsequent deposition of IJS of Steinemema sp., in terms of IJS per cm*, was less clear than that for Heterorhabditis n.sp. Adjuvant concentrations of 2% generally resulted in a reduction in the mean numbers of IJS per cm*, whereas, at 4%, deposition was generally higher than spraying with no adjuvant (Table 5). Deposition of IJS in adjuvant solutions: Micron Ulva+ The distribution of IJS within droplets remained highly aggregated following the addition of adjuvants to the spray solution, with the negative binomial distribution generally a good model for this aggregation. This was noted for IJS of both Heterorhabditis n.sp. and Steinemema sp. (Tables 6 and 7, respec-
Table 4. Effects of addition of adjuvants on the distribution of IJS of Heterorhabditis droplets generated with a Micron Herbaflex (flow rate 30 ml min -I) % of droplets % of droplets with IJS
n.sp. (initial concentration
6000 IJS ml-l)
Mean ’ (+ SE) no. of droplets cm --2
G-test value 3
in spray
Mean4 ( f SE) no. of IJS cm --2
0’
1
2
3
Crovol L27 2% Crovol LAO 2% Triton X-100 2% Croduvant 2% Glycerol 2% Water
95.73 95.78 94.05 95.87 96.56 94.73
3.76 3.49 5.00 3.70 2.81 4.61
0.40 0.51 0.68 0.38 0.63 0.49
0.11 0.22 0.27 0.05 _
4.27 4.22 5.95 4.13 3.44 5.27
58.53 k 2.96 45.87f2.11 49.33 * 1.73 61.33+ 1.95 58.13 + 1.53 30.38 + 1.49
1.05* 2.02* 2.58* 0.29* 5.07 0.88*
2.87 k 0.67Aab 2.37 k 0.45Aad 3.53 +0.82Abc 2.83 + 0.67Aac 2.37 k 0.43Aad 1.9k0.38d
Crovol L27 4% Crovol I_40 4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
96.26 95.36 95.14 95.63 94.77 94.73
3.44 4.15 4.28 3.85 4.53 4.61
0.25 0.31 0.53 0.34 0.57 0.49
0.05 0.18 0.05 0.17 0.13 _
3.74 4.64 4.86 4.37 5.23 5.27
66.80 + 1.72 54.57* 1.41 63.13 + 1.80 58.00 k2.09 52.30 kO.80 30.38 + 1.49
oso* 3.78* 0.55* 2.92* 0.80* 0.88*
2.73 k 0.43Aa 2.90 + 0.5 1Aa 3.47 i 0.55Aa 2.93 k 0.65Aa 3.17 k0.43Aa 1.9 k0.38b
Treatment
‘Number of IJS per droplet. ZMeans of the 40 x 1 cm2 grids originally counted, as the frequency distribution is based on this total. ?The G-test was used to test for goodness-of-fit of the data to the negative binomial distribution. G-test values with an asterisk indicate a significant fit. “For each adjuvant, means followed by the same uppercase letter are not significantly different (P z 0.05). For each adjuvant concentration, means followed same lowercase letter are not significantly different.
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by the
Screening Table 5. Effects of addition of adjuvants on the distribution of IJS of generated with a Micron Herbaflex (flow rate 30 ml min -I)
Sfeinernemasp.
and selection
of adjuvants:
(initial concentration
% of droplets
J.M. Mason et al.
3000 IJS ml ml) in spray
Mean 2 ( i SE) no. of droplets cm -z
droplets
0’
1
2
3
4
% of droplets with IJS
Crovol L27 2% Crovol L40 2% Triton X-100 2% Croduvant 2% Glycerol 2% Water
98.78 98.44 97.43 96.13 97.23 94.73
1.17 1.48 2.34 3.26 2.43 4.61
0.05 0.00 0.23 0.53 0.34 0.49
_ 0.00 _ 0.09 _ -
0.07 _ _ _ -
1.56 2.57 3.87 2.77 5.27
70.93 + 3.57 47.20 f 2.78 42.8 +_1.07 31.87& 1.49 49.33 i 1.72 30.38 f 1.49
0.10* 1.70’O.h7* 0.56* 1.51* 0.88h
0.90 & 0.24Aa 0.83 _+0.2SAa 1.2 kO.38Aac 1.73+0.41Ab 1.53 i 0.30Abc 1.45 f 0.35bc
Crovol L27 4% Crovol L40 4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
97.90 96.15 95.50 96.02 96.59 94.73
1.95 3.70 4.02 3.21 3.00 4.61
0.15 0.15 0.29 0.69 0.41 0.49
_
_ _ 0.10 _ _ _
2.10 2.57 4.50 3.98 3.41 5.27
44.53k1.11 45.07 k 1.24 34.80 * 0.94 43.6Ok 1.56 41.07& 1.21 30.38 * 1.49
0.35 O.lSf 2.15* 1.55* 1.45* 0.8x*
1.00 +0.24Aa 1.80 k 0.39Bbc 1.83 f 0.48Bbc 2.10+0.38Ab 1.57 k 0.33Abc 1.J5 * 0.35c
Treatment
o.lo 0.08 _ _
1.22
G-test value ’
Mean’ (*SE) no. of IJS cm mz
‘Number of IJS per droplet. ZMean number of the 40 x 1cm*grids originally counted, as the frequency distribution of based on this total. ‘The G-test was used to test for goodness-of-fit of the data to the negative binomial distribution. G-test values with an asterisk indicate a signiticant fit
tively). For both nematode species, the addition of adjuvants resulted in an increase in the numbers of droplets deposited at 3 V. In contrast, at 6 V, the numbers of droplets were lower than those after spraying the IJS in water alone. This partially accounts for the observation that the proportion of droplets carrying IJS following the addition of adjuvants is lower than for water alone at 3 V, whereas at 6 V, the proportion of droplets carrying IJS is higher than water alone. This was observed for
both Heterorhabditis n.sp. and Steinemema sp. (Tables 6 and 7). Following addition of the adjuvants to the spray solution, deposition of IJS, in terms of the mean numbers of IJS per cm2, was generally significantly higher than that in water alone. This was noted for both Heterorhabditis n.sp. and Steinemema sp. (Tables 6 and 7), and was especially marked at the lower adjuvant concentration. Deposition of IJS of Heterorhabditis n.sp. generally increased with
Heferorhabditisn.sp.
Table 6. Effects of addition of adjuvants on the distribution of IJS of droplets generated
with a Micron Ulva+
% of droplets 0’
1
2
3
4
3v 6V 3v 6V 3v hV 3v 6V 3v 6V 3V 6V
92.04 98.43 92.25 98.75 91.98 98.56 94.20 98.98 91.33 99.37 91.05 99.60
6.92 1.44 6.27 1.19 7.20 1.37 4.81 0.97 7.23 0.62 7.33 0.37
0.93 0.12 1.41 0.05 0.67 0.06 0.81 0.04 1.36 0.01 1.51 0.01
0.11 0.01 0.06 0.10 0.17 0.07 0.12 0.01
0.05 -
3v 6V 3v 6V 3v 6V 3v 6V 3v 6V 3V 6V
94.34 98.27 93.27 97.72 91.58 97.62 95.57 98.95 92.82 99.17 91.05 99.60
4.68 1.70 6.04 2.23 7.47 2.25 3.73 0.98 6.29 0.79 7.33 0.37
0.68 0.03 0.69 0.05 0.87 0.10 0.71 0.06 0.81 0.04 1.51 0.01
0.23 0.09 0.03 0.02 0.08 0.12 0.01
0.00 -
Treatment Crovo! L27 2% Crovol L40 2% Triton
X-100 2%
Croduvant Glycerol
2% 2%
Water Crovol L27 4% Crovol L40 4% Triton
X-100 4%
Croduvant Glycerol Water
4% 4%
(initial concentration
6000 IJS ml --I) in spray
(flow rate 200 ml min -I)
5
-
0.08
-
Mean? ( f. SE) no. of droplets cm ~’
G-test value’
7.96 1.57 7.75 1.25 8.02 1.44 5.80 1.02 8.67 0.63 8.95 0.40
61.17 t2.79 231.87 + 10.54 52.07 + 2.32 248.53 + 8.02 62.67 i_ 3.99 238.13 k9.22 57.47 k 2.43 223.07k5.60 46.53i 1.19 265.47 i 4.58 21 so * 1.40 353.70* 19.27
0.87* 0.25* 5.41* 0.43* 0.70* 0.38* 1.51* 0.34* 3.23 0.05* 2.12* 0.34*
5.57 k 0.79Aab 3.97) 0.57Ba 4.83 + 0.70Aac 3.23 &-0.43Ba 6.27 f 0.99Ab 3.53i0.31Ba 4.00 + OS8Acd 2.37 & 0.43Bb 4.73 f0.79Aad 1.70i_0.34Bc 2.30 + 0.52Ac 1.50+ 0.48Bbc
5.66 1.73 6.13 2.28 2.38 2.38 4.43 1.05 7.18 0.83 8.95 0.40
44.13k2.14 133.20 k 7.33 43.60* 1.63 139.07+4.80 38.40 + 1.01 129.07 + 2.99 51.87k2.10 218.27 +4.20 41.33 f 1.37 218.27k4.20 21.50+1.40 353.70 * 19.27
0.77% 0.03’ 2.00% O.OP 0.36* 1.08* 4.25’ 0.1s+ 0.5 1* 0.37” 2.12* 0.34”
3.13 &0.66Aab 2.33 + 0.47Ba 3.23 + 0.67Aab 3.23i0.51Ab 3.63 F 0.48Aa 3.27 i 0.60Ab 2.67 i 0.47Abc 2.50 i 0.5 1Aa 3.03 + 0.49Aab 2.17fO.38Ba 2.30 i0.52Ac 1SO + 0.48Bc
% of droplets with IJS
Mean’ (*SE) no. of IJS cm -?
‘Number of IJS per droplet. ‘Means of the 40 x 1 cm* grids originally counted, as the frequency distribution of based on this total. 3The G-test was used to test for goodness-of-fit of the data to the negative binomial distribution. G-test values with an asterisk indicate a significant fit. 4Within each adjuvant concentration, for voltage, means followed by the same uppercase letter are not significantly different. Within each voltage, for adjuvant concentration, means followed by the same lowercase letter are not significantly different.
Crop Protection
1998 Volume 17 Number 5
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Screening
and selection
of adjuvants:
J.M.
Mason et al. of IJS of Steinemem
Table 7. Effects of addition of adjuvants on the distribution generated with a Micron Ulva+ (flow rate 200 ml min -‘)
sp. (initial concentration
0’
1
2
3
Crovol L27 2%
3v 6V 3v 6V 3v 6V 3v 6V 3v 6V 3v 6V
96.58 99.12 95.60 99.36 97.05 99.48 96.62 99.30 95.09 99.22 90.16 99.71
3.04 0.79 3.87 0.64 2.79 0.52 3.00 0.64 4.70 0.73 8.52 0.28
0.25 0.09 0.44
0.13 0.09
Crovol LAO2% Triton X-100 2% Croduvant 2% Glycerol 2% Water Crovol L27 4%
3v 6V 3v 6V 3v 6V 3v 6V 3v 6V 3v 6V
96.26 99.22 95.13 98.76 94.41 97.96 97.22 99.31 97.31 99.15 90.16 99.71
3.28 0.73 4.39 1.22 5.23 1.88 2.43 0.63 2.33 0.82 8.52 0.28
0.46 0.04 0.40 0.02 0.35 0.13 0.35 0.04 0.36 0.02 0.98 0.01
Crovol LAO4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
016 0.32 0.05 0.07 0.04 0.98 0.01
4 -
-
0.00
0.14
0.06 -
0.08
0.03 0.01 0.02 0.33 -
-
-
in spray droplets
% of droplets with IJS
Mean ’ ( k SE) no. of droplets cm e-2
G-test value 3
Mean4 (*SE) no. of IJS cm -’
3.42 0.88 4.40 0.64 2.95 0.52 3.38 0.70 4.91 0.78 8.84 0.29
52.60+ 1.92 182.27f7.98 37.87 k2.05 196.80 + 8.80 62.20 & 2.14 257.60 k 6.89 52.27 i. 1.59 269.87 k2.66 46.13k1.36 231.60+7.84 30.5 + 2.92 245.80 + 12.86
2.03* 1.34* 0.36* 0.26* 0.37* 0.22* 0.28* 0.76* 0.97 0.43* 0.22* 0.09*
2.07* 0.31Aab 1.77_f0.30Aab 1.90 + 0.46Aab 1.27 f0.33Ba 1.90 f 0.40Aa 1.33 + 0.34Ba 2.03 kO.37Aab 2.00 &0.29Ab 2.43 + 0.43Ab 1.9070.3OAb 0.93 k0.31Ac 0.75 k 0.21Ac
3.74 0.78 4.87 1.24 5.59 2.04 2.78 0.69 2.69 0.85 9.84 0.29
50.80f2.11 231.6Ok7.84 41.73 + 1.55 169.47 I5.31 37.60k1.41 104.53 53.17 48.00 & 1.45 247.20 k3.500 45.87 f 1.52 211.07f3.15 30.5 +2.92 245.80 k 12.86
2.08* 0.43* 0.40* 0.03* 0.52* 0.19* 1.55* 2.20* 1.68* 0.43* 0.22* 0.09*
2.13 +0.41Aac 1.90f0.30Aa 2.27 k 0.35Aa 2.13 i 0.36Aa 2.23 +0.42Aa 2.33 f.0.41Aa 1.50 +0.35Ab 1.87z0.40Aa 1.40 k 0.33Ab 1.90+0.36Aa 0.93 & 0.31Ac 0.75 i0.21Ac
% of droplets
Treatment
6000 IJS ml -I)
‘Number of IJS per droplet. 2Means of the 40 x 1 cm2 grids originally counted, as the frequency distribution of based on this total. 3The G-test was used to test for goodness-of-fit of the data to the negative binomial distribution. G-test values with an asterisk indicate a significant fit. 4Within each adjuvant concentration, for voltage, means followed by the same uppercase letter are not significantly different. Within each voltage, for adjuvant concentration, means followed by the same lowercase letter are not significantly different.
increased adjuvant concentration (Table 6). This was noted at 3 and 6 V. In contrast, an increase in adjuvant concentration generally resulted in a decrease in deposition of IJS of Steinemema sp. at both voltages used (Table 7).
solutions of the IJS of both species resulted in a reduction in volume median diameter (vmd) and a slight narrowing of the width of the main droplet band. However, increased production of larger droplets accounted for an increased proportion of the volume. Production of foam was not noted with this spraying system.
Spray spectra analyses of adjuvant solutions: Micron Herbaflex The addition of Croduvant and Glycerol (2% and 4% concentration) to the solution of IJS of both nematode species had no effect on the spray spectra produced compared with spraying in water only. In contrast, the addition of Crovol L27 or Crovol LAO, at 2% or 4%, to solutions of IJS of both nematode species resulted in a narrower band width of droplets and a reduction in the vmd values compared with IJS in water. Addition of Triton X-100 resulted in the production of a large volume of foam, with subsequent inefficient droplet production and many large droplets formed. Representative results for the spray spectra are shown in F@re I for Heterorhabditisn.sp. Spray spectra analyses of adjuvant solutions: Micron Ulva+ The addition of Crovol L27, Crovol LAO, Croduvant or Glycerol at 2% or 4% to solutions of IJS of both nematode species had little effect on the resultant spray spectra or vmd values compared with the spectra produced for IJS in water (Mason et al., 1998a). Addition of Triton X-100 at 2% and 4% to
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Discussion None of the adjuvants tested were toxic to DBM larvae, with survival generally being 100% after 72 h. However, reduced feeding levels of the DBM larvae were noted, especially on leaf discs dipped in Triton X-100 and Crovol L27 (J. M. Mason, unpublished results). This result for Triton X-100 was not entirely unexpected as it is normally used as an adjuvant at concentrations ~1%. The adjuvants tested in the present study were shown to have minimal direct toxicity to IJS of both Heterorhabditis n.sp. and Steinemema sp., although it was noted that increasing incubation time in both Crovol L27 and Crovol IA0 resulted in reduced nematode motility. The reasons for this are not clear. However, in terms of field applications, this is of minimal significance as IJS in droplets will not survive for this length of time, irrespective of timings of application. Although direct toxicity was minimal, infectivity of IJS was affected. Both mortality and intensity of infection in larvae of G. melionetta was significantly reduced compared with infectivity of IJS, of either nematode species, in water. There are a number of possible mechanisms
Screening
and selection
of adjuvants:
J.M.
Mason
et al.
water; vmd = 218 pm CrovolL27;
vmd = 182 pm
Crovol L40; vmd = 191 pm Croduvant; Glycerol;
200
300
vmd = 221 pm vmd = 217 pm
400
500
Band size (pm) Figure 1. Effects of adjuvants (2%) on spray droplet Heferorhabditis n.sp. (6000 IJS ml -I).
spectra
produced
for this, including direct toxic effect on the IJS, toxicity to G. mellunellu larvae or change in the surface tension of the fluid surrounding the sand particles. The latter two mechanisms are the more plausible, as our results suggest minimal direct toxic effect on the IJS. In a similar bioassay, assessing the toxicity of a number of potential adjuvants on IJS of S. catpocupsae, Lello et al. (1996) noted no significant difference in either mortality or intensity of infection of G. mellonella larvae compared with IJS in water. One of the adjuvants used in the present study, Triton X-100, is the same as that used by Lello et al. (1996). Differences noted between the two studies may be a result of differences in adjuvant concentration, nematode species or incubation time. However, efficacy against larvae of l? xylostella is more important (see Mason et al., 1998b). Deposition of IJS was significantly affected by the addition of the adjuvants to the spray solution. Generally, addition of the adjuvants resulted in an increase in the mean numbers of IJS deposited per cm2. This increase in deposition of IJS has also been noted following application on leaf discs of Chinese cabbage (J. M. Mason, unpublished results). That addition of the adjuvants results in higher numbers of IJS being deposited within the defined catchment area suggests that there is a change in the swath pattern as the total output of IJS from either sprayer is not significantly affected by the addition of any of the adjuvants compared with spraying the IJS in water (Mason et al., 1998a), although swath pattern was not measured in the present study. Furthermore, this change in swath pattern may be a result of a reduction in dynamic surface tension at the time of atomisation, although changes in fluid viscosity and other undefined variables (Hall et al., 1993) may also be involved. That the addition of adjuvants does affect swath pattern has been shown, for example, for
by a Micron
Herbaflex
(flow rate 30 ml min -‘)
spraying
IJS of
a flat-fan hydraulic nozzle (Chapple et al., 1993). While it is appreciated that the study by Chapple et al. (1993) was carried out while the nozzles were stationary and also that the total output volume from a centrifugal energy nozzle (in this case, spinning discs) and a hydraulic nozzle are on completely different scales, it is conceivable that the addition of IJS and/or adjuvants could significantly affect the swath pattern produced by spinning discs. This is worthy of further investigation as it may have significant bearing on subsequent deposition of the IJS, dependent on the plant substrate/architecture, and be open for manipulation. The major reason for considering incorporation of adjuvants in sprays of IJS was primarily to limit the damaging effects of desiccation. Although UV radiation is also known to be lethal to IJS of a number of species of EPNs (Gaugler and Boush, 1978) judicious timings of field applications can minimise such effects (e.g., MacVean et al., 1982). Incorporation of the adjuvants was not intended to directly enhance desiccation survival of the IJS, but rather to reduce the evaporative rate of the droplets. To this effect, the adjuvants we tested, with the exception of Triton X-100, promote the maintenance of the integrity of the droplet. The decision not to test spreaders, such as organosilicone compounds, was based on the fact that although surface tension is reduced, which we considered a requirement for potential enhancement of infection by allowing IJS to ‘break’ the surface tension, and adhesion is greater, the sprayed liquid spreads which can lead to reduced retention due to coalescence of the droplets and, hence, increased run-off (Stevens and Kimberley, 1993). Overall, the results obtained with these adjuvants are very encouraging. Although infection of G. mellonella larvae was significantly reduced, this standard
Crop Protection
1998 Volume 17 Number
5
469
Screening
and selection
of adjuvants:
J.M. Mason et
al.
infectivity assay for EPNs is not representative of the final intended host insect and plant. However, it enabled an initial screening without the use of spraying equipment, as at that time the effect of the adjuvants on spray quality was not known. Obviously, a more realistic assay will be against DBM larvae on crucifers (Mason et al., 1998b). The adjuvants were ranked based on the results from each of the studies presented here. On the basis of these combined rankings, the adjuvants scored as follows. For IJS of Heterorhabditis n.sp., the ‘best’ adjuvant for use with the Ulva+ and Herbaflex is Triton X-100, followed by Crovol L27, Crovol L40, Croduvant and Glycerol, with IJS in water alone being the least efficacious. However, for Steinemema sp., the best adjuvant for use with either sprayer is Croduvant, followed by Triton X-100 and Glycerol, Crovol LAO and Crovol L27, with IJS in water again being the worst option. Results from further studies examining infectivity of IJS against DBM larvae are presented elsewhere (Mason et al., 1998b).
Gaugler, R. and Boush, G. M. (1979) Laboratory tests on ultraviolet protectants of an entomogenous nematode. Environ. Entomol. 8, MO-813
Georgis,
R. (1990) Formulation
Entomopathogenic
and application
technology.
In
Nematodes in Biological Control, ed. R. Gaugler
and H. K. Kaya. CRC Press, Boca Raton, FL, pp, 173-191 Glazer, I., Klein, M., Navon, A. and Nakache, Y. (1992) Comparison of efficacy of entomopathogenic nematodes combined with antidesiccants applied by canopy sprays against three cotton pests (Lepidoptera: Noctuidae). J. Econ. Entomol. 85, 1636-1641 Hall, F. R., Chapple, A. C., Downer, R. A., Kirchener, L. M. and Thacker, J. R. M. (1993) Pesticide application as affected by spray modifiers. Pestic. Sci. 38, 123-133 Lello, E. R., Patel, M. N., Matthews, G. A. and Wright, D. J. (1996) Application technology for entomopathogenic nematodes against foliar pests. Crop Prot. 15,567-574 MacVean, C. M., Brewer, J. W. and Capinera, J. L. (1982) Field tests of antidesiccants to extend the infection period of an entomogenous nematode, Neoaplectana carpocapsae, against the Colorado potato beetle. J. Econ. Entomol. 75, 97-101 Mason, J. M. and Wright, D. J. (1997) Potential for the control of with entomopathogenic nematodes. .I.
Plutella xylostella larvae Invert. Pathol. 70, 234-242
Mason, J. M., Matthews, G. A. and Wright, D. J. (1998a) Appraisal of spinning disc technology for the application of entomopathogenic nematodes. Crop Protection 17, 453-461
Acknowledgements This research is funded by The Leverhulme Trust. We wish to thank Micron Sprayers Ltd for generously supplying the Micron Ulva+ and Micron Herbaflex sprayers, and Croda Chemicals Ltd for generously supplying adjuvants. References
Mason, J. M., Matthews, G. A. and Wright, D. J. (1998b) Evaluation of spinning disc technology for the application of entomopathogenic nematodes against a foliar pest. J. Invert. Pathol. (in press) Mason, J. M., Razak, A. R. and Wright, D. J. (1996) The recovery of entomopathogenic nematodes from selected areas within Peninsular Malaysia. J. Helminthol. 70, 303-307 Nickle, W. R. and Shapiro, M. (1992) Use of a stilbene brightener, Tinopal LPW, as a radiation protectant for Steinemema carpo-
Baur, M. E., Kaya, H. K. and Thurston, G. S. (1995) Factors affecting entomopathogenic nematode infection of Plutella xylostella on a leaf surface. Entomol. Exp. Applic. 77, 239-250 Baur, M. E., Kaya, H. K., Gaugler, R. and Tabashnik, B. (1998) Effects of adjuvants on entomopathogenic nematodes and efficacy against Plutella xylostella. Bioc. Sci. Technol., in press Chapple, A. C., Downer, R. A. and Hall, F. R. (1993) Effects of spray adjuvants on swath patterns and droplet spectra for a flatfan hydraulic nozzle. Crop Prot. l&579-590 Crawley, M. J. (1993) GLIM for Ecologists. Blackwell Scientific Publications, Oxford, 379 pp Fan, X. and Hominick, W. M. (1991) Efficiency of the Galleria (wax moth) baiting technique for recovering infective stages of entomopathogenic rhabditids (Steinernematidae and Heterorhabditidae) from sand and soil. Rev. Nematol. 14, 381-387 Gaugler, R. and Boush, G. M. (1978) Effects of ultraviolet radiation and sunlight on the entomogenous nematode Neoaplectana carpocapsae. J. Invert. Pathol. 32, 291-296
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1998 Volume 17 Number 5
capsae. .I. Nematol. 24,371-373
Nickle, W. R. and Shapiro, M. (1994) Effects of eight brighteners as solar radiation protectants for Steinemema carpocapsae, All Strain. _I.Nematol. (Suppl.) 26, 782-784 Shapiro, M., McLane, W. and Bell, R. (1985) Laboratory evaluation of selected chemicals as antidesiccants for the protection of the entomogenous nematode Steinemema feltiae (Rhabditidae: Steinernematidae), against Lymanttia dispar (Lepidoptera: Lymantriidae). J. Econ. Entomol. 78, 143771441 Stevens, P. J. G. and Kimberley, M. 0. (1993) Adhesion of spray droplets to foliage: the role of dynamic surface tension and advantages of organosilicone surfactants. Pestic. Sci. 38,237-245 Thomson, L. A. (1992) A Guide to Agricultural Spray Adjuvants used in the United States. Thomson Publications, Fresno, CA Webster, J. M. and Bronskill, J. F. (1968) Use of Gelard M and an evaporation retardant to facilitate control of larch sawfly by a nematode-bacterium complex. J. Econ. Entomol. 61, 1370-1373
Screening and selection of adjuvants for the spray application of entomopathogenic nematodes against a foliar pest Judy M. Mason*, Graham A. Matthews+ and Denis J. Wright** *Department of Biology, Imperial College of Science, Technology and Medicine, Silwood Park, Ascot, Berkshire, SL5 7PY, UK +lnternational Pesticide Application Research Centre, Imperial College of Science, Technology and Medicine, Silwood Park, Ascot, Berkshire, SL5 7PY, UK
The effects of adding a number of adjuvants to sprays containing infective juveniles (IJS) of Heteronsp. and Steinemema sp. (M87:45) was examined. The adjuvants were not directly toxic to IJS of either nematode species or to larvae of the diamondback moth, Plutella xylostella. However, infectivity of the IJS to larvae of the wax-moth, Galleria mellonella, in terms of mortality and intensity of infection, was affected by the addition of the adjuvants, compared with IJS in water alone. Deposition studies showed that the proportion of droplets carrying IJS was not affected by the addition of the adjuvants following application with spinning disc sprayers (Micron Ulva+ and Micron Herbaflex). However, addition of the adjuvants resulted in a significant increase in deposition, in terms of the mean numbers of IJS per cm ‘. This was noted following application with both sprayers and for both nematode species. The spray spectra produced by either sprayer was generally not affected much by the addition of any of the adjuvants to the spray solutions. 0 1998 Elsevier Science Ltd. All rights reserved
rhabditis
Keywords: adjuvants; spray application; Steinernema spp.; Heterorhabditis spp.
Introduction Application of infective juveniles (IJS) of entomopathogenic nematodes (EPNs) belonging to the families Steinernematidae and Heterorhabditidae has generally employed standard spraying equipment used in the application of pesticides (Georgis, 1990). While this is not so important in the application of EPNs against soil-dwelling pests, choice of application method is more critical for their successful application against foliar pests. Such differences between a variety of spraying methods were highlighted by Lello et al. (1996), when they examined a range of hydraulic nozzles (standard fan and full cone) and a spinning disc. Their work suggested that the development of low volume application systems for the application of IJS against targets such as larvae of the diamondback moth (DBM), Plutella xylostella, was warranted. A recent study on the development of two spinning disc sprayers for such applications has confirmed this (Mason et al., 1998a). In the latter work, deposition of IJS was influenced by both flow rate and the initial concentration of IJS in the spray mixture. *Corresponding author. Tel.: +44 (0) 1344 294248; Fax: +44 (0) 1344 294339; E-mail: [email protected]
Adjuvants are commonly added to pesticide formulations to enhance pesticide performance. The types and range of adjuvants available is extensive (e.g., Thomson, 1992). In the application of IJS of EPNs against foliar pests, the major problems are the limited tolerance of the IJS to extremes of temperature, UV radiation and desiccation (Baur et al., 1995; Mason and Wright, 1997). The latter two are the most critical and have received considerable attention, with a wide range of chemicals having been tested in an attempt to reduce the negative impacts of desiccation (Webster and Bronskill, 1968; MacVean et al., 1982; Shapiro et al., 1985; Glazer et al., 1992; Baur et al., 1998) and UV radiation (Gaugler and Boush, 1979; Nickle and Shapiro, 1992; Nickle and Shapiro, 1994). However, no one adjuvant is suitable for all situations and the results from these numerous studies highlight the need for screening of adjuvants for use in a particular situation (Baur et al., 1998). This is the result of a number of interacting factors, including the insect target (e.g., feeding habit, amount of movement on substrate), host plant (e.g., waxy or non-waxy surface) and method of application (e.g., hydraulic nozzles or spinning disc); it is thus essential to screen potential adjuvants with these factors in mind. In the present work, we have examined the toxicity and resultant deposition of IJS
Crop Protection
1998 Volume 17 Number 5
463
Screening
and selection
of adjuvants:
J.M. Mason et al.
using a range of potential adjuvants for incorporation into sprays with IJS for application against DBM larvae on crucifers. Materials and methods Nematodes Two species of entomopathogenic nematode were used in the present study, Steinemema sp. (M87: 45) and a new Heterorhabditis sp. (Mason et al., 1996). Results from previous studies had suggested that these two species had greatest potential for application against DBM larvae on crucifers in Malaysia (Mason and Wright, 1997). The nematodes were maintained at 20°C in late instar larvae of Galleria mellonella (The Mealworm Co., Sheffield, UK), with IJS being used within 6 days of emergence. Cabbage plants Chinese cabbage, Brussicu pekinensis (cv. ‘Tip Top’), was grown in individual pots of Levingtons MultiPurpose compost in a greenhouse maintained at 15-20°C with a light regime of approximately 16:8 (L:D). Plants were 4 to 6 weeks old at the time of use. DBM culture A UK isolate of DBM (Rothamsted insecticide susceptible strain) was used (Mason and Wright, 1997). Cultures were maintained at 20°C on 4- to 6-week-old Chinese cabbage. Adjuvants Five adjuvants were used in the present study: Triton X-100 (Merck Ltd, Poole, Dorset, UK), Glycerol, Croduvant (Glycerol-based), Crovol L27 and Crovol L40 (both Linseed oil-based) (Croda Chemicals Ltd, Cowick Hall, Snaith, Goole, North Humberside, UK). Glycerol and Croduvant have anti-evaporant properties, whereas both Crovols and Triton X-100 are non-ionic surfactants. Solutions of the adjuvants were tested at concentrations of 2% and 4% (v/v). Sprayers Two spinning disc sprayers were used: the Micron Herbaflex and the Micron Ulva+ (Micron Sprayers Ltd, Bromyard, Hereford, UK). Unless otherwise stated, the Ulva+ was operated at 6 V with a flow rate of 200 ml min -l and the Herbaflex was operated at 6 V with a flow rate of 30 ml min -’ (Mason et al., 1998a). All spraying was conducted using the sprayers inside a Mardrive linear track chamber (Mason et al., 1998a). Assessment of adjuvants Effect of adjuvants on infectivity of IJS The effect of the five adjuvants on the infectivity of IJS of Heterorhabditis nsp. and Steinemema sp. (M87: 45) was assessed using a sand tube assay with late
464
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instar larvae of G. mellonella (Fan and Hominick, 1991). Nematode/adjuvant suspensions were prepared using distilled water, to give a final nematode concentration of 200 IJS ml -l with adjuvant concentrations of 2% and 4%. To 30 ml Universal tubes, 12 ml of dry, sterile sand (all particles 5 1 mm diameter) was added. One millilitre of the nematode/adjuvant suspension was then added to each sand tube and the tubes were shaken to ensure even mixing. The final moisture concentration in each sand tube was approximately 8% (v/v). Controls consisted of sand tubes with nematode suspension with no adjuvants or distilled water only. One G. mellonella larvae was added to each tube and the sand tubes inverted following fitting with a screw top lid. There were 20 replicates per adjuvant concentration. Following 48 h incubation at 25°C the G. mellonella larvae were removed, rinsed with water and further incubated at 25°C for 24 h to allow adult nematodes to develop. Both mortality and intensity of infection were assessed. EfSect of adjuvunts on survival of IJS
The toxicity of the five adjuvants to IJS of Heteroand Steinemema sp. (M87: 45) was assessed by incubating IJS in solutions (2% and 4%) of the adjuvants. Solutions of the IJS (200 IJS ml -‘) and adjuvants were prepared with distilled water. Five millilitres of each solution were placed in 25 ml glass vials. Following incubation at 25°C for 0, 6, 12 or 24 h, 0.5 ml of test solution was removed and the numbers of dead and alive IJS assessed. All IJS that did not respond to mechanical stimulation were considered dead. There were five replicates per adjuvant concentration. Controls consisted of adding IJS to distilled water and incubation as described above.
rhubditis n.sp.
Adjuvunt toxicity to DBM larvae
The toxicity of the five adjuvants at concentrations of 2% and 4% to third instar DBM larvae was assessed. Leaf discs (5 cm in diameter) of Chinese cabbage were dipped in solutions of the adjuvants for 10 s and then allowed to dry. The dry leaf discs were placed in 5 cm diameter Petri dishes which had been lined with a piece of moist filter paper. Two third instar DBM larvae were placed on each leaf disc, and the Petri dishes sealed with Parafilm ‘M’@ prior to incubation at 25°C (16:8 L:D cycle). There were ten replicates per treatment. Mortality of the DBM larvae was assessed at 24, 48 and 72 h. Controls consisted of Chinese cabbage leaf discs which had not been dipped in adjuvant. Deposition of IJS in udjuvunt solutions
Deposition of IJS in solutions of the adjuvants was assessed using the Micron Herbaflex and Ulva+ in the Mardrive linear spray track. Both sprayers were set up in the Mardrive chamber, as described previously (Mason et al., 1998a). Deposition was assessed for IJS of Steinememu sp. (M87: 45) and Heterorhabditis n.sp. at concentrations of 3000 and 6000 IJS ml -I, respectively. The Herbaflex was used at a flow rate of 30 ml min-‘, while the Ulva+ was
Screening and selection of adjuvants: J.M. Mason et al.
run at both 3 and 6 V, with a flow rate of 200 ml min -. ’ used at each voltage. The five adjuvants were tested at both 2% and 4% (v/v). Additionally, solutions of IJS in distilled water only were tested. Spray spectra analyses of adjuvant solutions
Spray droplet spectra for solutions of the adjuvants at two concentrations (2% and 4%) were assessed using a Malvern@ Particle Size Analyser 2600 (Malvern, Worcester, UK) using both sprayers. Details of the set-up of the sprayers in relation to the Malvern Particle Size Analyser are given elsewhere (Mason et al., 1998a). Both nematode species were assessed with each adjuvant.
Table 2. Effect of adjuvants on infectivity of IJS of Steinernema in G. me//one//alarvae
68.35 k5.74Aa 40.22 i 6.2OAb 36.23 i 7.49Ac 66.3 I7.73Aa 56.68 F 4.88Ad 98.45 * 1I .OOe
XSAabc YOAac 6SAb 1OOAc 9SAc 1ooc
Crovol L27 4%’ Crovol L40 4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
65.32 & 6.52Aa 53.18 + 7.3SBh 19.11 f4.02Bc 5 1.50 +6.09Bb 32.53 + 2.9SBd 98.45 f I I .ooe
YSAa 85Aa 9UBa YOAa YSAa 1OOa
‘Intensity
of infection
adjuvant.
cantly
different
the samr
Generalised linear modelling was used for all statistical analyses using the statistical package GLIM (Numerical Algorithms Group, 1985). Mortality data were analysed using a binomial error structure, and intensity of infection data with a Poisson error structure. Overdispersion was corrected for accordingly (Crawley, 1993). Survival data were analysed by ANCOVA using a binomial error structure. The negative binomial distribution as a model for the distribution of IJS within droplets was assessed using a goodness of fit statistic (G-test). ANOVA, with contrasts, was used with all remaining data analyses. Significance is reported at the 5% level.
Mortality ’ (S)
Crovol L27 2% Crovol IA0 2% Triton X-100 2% Croduvant 2% Glycerol 2% Water
each
Statistical analyses
Intensity ’ (mean?SE)
Treatment
sp.
means
is defined
as mean
followed
by the same
(P > 0.05).
lowercase
letter
numhcr
For each adjuvant arc not signifcantly
of IJS per infected
uppcrcasc concentration.
letter
are
larva. not
For
signifi-
means followed
hy
diftcrcnt.
(PcO.05) following the addition of Crovol L27. Crovol L40 or Triton X-100. Additionally, mean intensity of infection was significantly lower (PcO.05) compared with that obtained with IJS in water only (Table I). For each adjuvant, increasing the concentration from 2 to 4% resulted in lower mortality of G. mellonella larvae and lower mean intensity of infection. Indeed, only the addition of Croduvant resulted in increased mortality and mean intensity of infection (Table I).
for IJS of Steinerof the generally resulting in a reduction in mean of infection in G. mellonella larvae. addition of the adjuvants to the solutions of not generally have a significant effect on of G. mellonella larvae (Table 2).
Similar results were obtained
nema sp. (M45) (Table 2), with the addition
adjuvants intensity However, IJS did mortality
Results Effect of adjuvants
on infectivity
of IJS
The addition
of adjuvants to the solution of IJS of n.sp. generally resulted in a subsequent reduction in mortality of G. mellonella larvae compared with IJS in water only (Table I). This was more marked at the higher concentration of each adjuvant used, where mortality was significantly lower
Heterorhabditis
Table 1. Effect of adjuvants on infectivity of IJS of Heterorhabdifis nsp. in G. me//one//a larvae Intensity ’ (mean k SE)
Treatment
Mortality ’ (S)
Crovol L27 2% Crovol L40 2% Triton X-100 2% Croduvant 2% Glycerol 2% Water
52.88 + 5.91Aa 43.73 + 6.63Ab 48.00 + 12.60Aab 23.71 f4.14Ac 36.53 +3.19Ad 80.65 +8.07e
8OAa 7SAa 30Ab 8SAa 95Aa 1OOa
Crovol L27 4% Crovol L40 4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
33.44 + 11.3SBa 43.38 + 6.68Ab 57.00 + O.OOAc 39.30 + 6.22Bd 29.72+5.01Be 80.65 + 8.07f
4SBa 65Aa 5Ab 1OOAc 90Ac 1ooc
‘Intensity
of infection
each
adjuvant,
cantly
different
means
is delined followed
(P > 0.05).
the stame lowercase
letter
as mean by the
number same
For each adjuvant are not significantly
of IJS per infected
uppercase concentration, different.
letter
are
larva. not
For
signifi-
means followed
hy
Effect of adjuvants
on survival of IJS
The adjuvants had no significant effect (P > 0.05) on the survival of IJS of either Heterorhabditis n.sp. or Steinemema sp. at either concentration of adjuvant tested. Increasing incubation time of the IJS in the adjuvant solution generally resulted in a slight, although not significant (P > O.OS), reduction in survival. This was again noted for each of the adjuvants and at both concentrations tested with both nematode species. The mean survival of IJS of both species following 24 h incubation is shown in Table 3. However, it was noted that IJS incubated in Crovol L27 and, especially, Crovol LAO, became increasingly inactive and assumed a straight posture with increased incubation time. This was noted for both nematode species, with approximately 94-100% of the IJS requiring mechanical stimulation to establish if they were dead or alive. Incubation of IJS of both species in the remaining adjuvants generally resulted in the IJS retaining their activity. If they became inactive, they assumed their characteristic curved or kinked posture rather than a straight posture which is characteristic when IJS of both of these species are dead.
Crop Protection
1998 Volume 17 Number 5
465
Screening
and selection
of adjuvants:
J.M. Mason et al.
Table 3. Mean (-&SE) survival at 24 h following
incubation
of IJS of Heterorhabditis
n.sp. and Steinernema
Nematode
2%
Glycerol Croduvant Crovol L27 Crovol IA0 Triton X-100 Control
97.1 96.7 93.9 96.5 95.8 97.1
kO.3 k 1.0 + 1.9 k 0.5 kO.9 k 0.5
Adjuvant toxicity to DBM larvae The adjuvants had no significant effect on the survival of DBM larvae (P > 0.05) with survival generally being 100% after 72 h. A slight reduction in survival was noted at the 24 h assessment for 4% Crovol L27 (85% survival), 4% Crovol L40 and Triton X-100 (90% and 85% survival at 2% and 4% concentration, respectively). No further mortality was recorded at 48 or 72 h.
Deposition of IJS in adjuvant solutions: Micron Herbaflex The negative binomial distribution was generally a good model for the deposition of IJS in the droplets (Table 4). The addition of adjuvants to the spray solution caused a significant increase (PcO.05) in the mean number of droplets deposited per cm* following application of IJS of Heterorhabditis n.sp. (Table 4). This increase in numbers of droplets was more marked at the higher adjuvant concentration, for each adjuvant, where the numbers deposited generally doubled. However, the percentage of droplets carrying IJS was not significantly affected by the addition of the adjuvants at either 2% or 4%. In contrast, addition of adjuvants had a marked effect on the mean number of IJS deposited per cm*
at 25°C
species Steinernema sp. (M87: 45)
Heterorhabditis nsp. Treatment
sp. in adjuvants
4%
2%
4%
96.8 +0.5 97.lkO.6 92.5 k 0.9 90.4k2.4 97.2kO.6 97.lkO.5
99.3 kO.6 99.9kO.2 99.3 kO.6 98.8 k 0.6 98.8 kO.5 100 5 0.0
99.1 kO.6 99.8kO.2 99.lkO.6 95.3 + 1.1 99.6 +0.2 100 + 0.0
(Table 4), with, generally, deposition being significantly higher. Increasing adjuvant concentration generally resulted in a slight increase in the numbers of IJS deposited, although no significant changes were noted. Addition of the adjuvants at either concentration also generally resulted in an increase in the numbers of droplets deposited following application of IJS of Steinemema sp. (Table 5). However, the proportion of droplets carrying IJS was generally significantly lower (PcO.05) following addition of adjuvants to the spray solution. The effect of adding the adjuvants to the spray solution on subsequent deposition of IJS of Steinemema sp., in terms of IJS per cm*, was less clear than that for Heterorhabditis n.sp. Adjuvant concentrations of 2% generally resulted in a reduction in the mean numbers of IJS per cm*, whereas, at 4%, deposition was generally higher than spraying with no adjuvant (Table 5). Deposition of IJS in adjuvant solutions: Micron Ulva+ The distribution of IJS within droplets remained highly aggregated following the addition of adjuvants to the spray solution, with the negative binomial distribution generally a good model for this aggregation. This was noted for IJS of both Heterorhabditis n.sp. and Steinemema sp. (Tables 6 and 7, respec-
Table 4. Effects of addition of adjuvants on the distribution of IJS of Heterorhabditis droplets generated with a Micron Herbaflex (flow rate 30 ml min -I) % of droplets % of droplets with IJS
n.sp. (initial concentration
6000 IJS ml-l)
Mean ’ (+ SE) no. of droplets cm --2
G-test value 3
in spray
Mean4 ( f SE) no. of IJS cm --2
0’
1
2
3
Crovol L27 2% Crovol LAO 2% Triton X-100 2% Croduvant 2% Glycerol 2% Water
95.73 95.78 94.05 95.87 96.56 94.73
3.76 3.49 5.00 3.70 2.81 4.61
0.40 0.51 0.68 0.38 0.63 0.49
0.11 0.22 0.27 0.05 _
4.27 4.22 5.95 4.13 3.44 5.27
58.53 k 2.96 45.87f2.11 49.33 * 1.73 61.33+ 1.95 58.13 + 1.53 30.38 + 1.49
1.05* 2.02* 2.58* 0.29* 5.07 0.88*
2.87 k 0.67Aab 2.37 k 0.45Aad 3.53 +0.82Abc 2.83 + 0.67Aac 2.37 k 0.43Aad 1.9k0.38d
Crovol L27 4% Crovol I_40 4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
96.26 95.36 95.14 95.63 94.77 94.73
3.44 4.15 4.28 3.85 4.53 4.61
0.25 0.31 0.53 0.34 0.57 0.49
0.05 0.18 0.05 0.17 0.13 _
3.74 4.64 4.86 4.37 5.23 5.27
66.80 + 1.72 54.57* 1.41 63.13 + 1.80 58.00 k2.09 52.30 kO.80 30.38 + 1.49
oso* 3.78* 0.55* 2.92* 0.80* 0.88*
2.73 k 0.43Aa 2.90 + 0.5 1Aa 3.47 i 0.55Aa 2.93 k 0.65Aa 3.17 k0.43Aa 1.9 k0.38b
Treatment
‘Number of IJS per droplet. ZMeans of the 40 x 1 cm2 grids originally counted, as the frequency distribution is based on this total. ?The G-test was used to test for goodness-of-fit of the data to the negative binomial distribution. G-test values with an asterisk indicate a significant fit. “For each adjuvant, means followed by the same uppercase letter are not significantly different (P z 0.05). For each adjuvant concentration, means followed same lowercase letter are not significantly different.
466
Crop Protection
1998 Volume 17 Number 5
by the
Screening Table 5. Effects of addition of adjuvants on the distribution of IJS of generated with a Micron Herbaflex (flow rate 30 ml min -I)
Sfeinernemasp.
and selection
of adjuvants:
(initial concentration
% of droplets
J.M. Mason et al.
3000 IJS ml ml) in spray
Mean 2 ( i SE) no. of droplets cm -z
droplets
0’
1
2
3
4
% of droplets with IJS
Crovol L27 2% Crovol L40 2% Triton X-100 2% Croduvant 2% Glycerol 2% Water
98.78 98.44 97.43 96.13 97.23 94.73
1.17 1.48 2.34 3.26 2.43 4.61
0.05 0.00 0.23 0.53 0.34 0.49
_ 0.00 _ 0.09 _ -
0.07 _ _ _ -
1.56 2.57 3.87 2.77 5.27
70.93 + 3.57 47.20 f 2.78 42.8 +_1.07 31.87& 1.49 49.33 i 1.72 30.38 f 1.49
0.10* 1.70’O.h7* 0.56* 1.51* 0.88h
0.90 & 0.24Aa 0.83 _+0.2SAa 1.2 kO.38Aac 1.73+0.41Ab 1.53 i 0.30Abc 1.45 f 0.35bc
Crovol L27 4% Crovol L40 4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
97.90 96.15 95.50 96.02 96.59 94.73
1.95 3.70 4.02 3.21 3.00 4.61
0.15 0.15 0.29 0.69 0.41 0.49
_
_ _ 0.10 _ _ _
2.10 2.57 4.50 3.98 3.41 5.27
44.53k1.11 45.07 k 1.24 34.80 * 0.94 43.6Ok 1.56 41.07& 1.21 30.38 * 1.49
0.35 O.lSf 2.15* 1.55* 1.45* 0.8x*
1.00 +0.24Aa 1.80 k 0.39Bbc 1.83 f 0.48Bbc 2.10+0.38Ab 1.57 k 0.33Abc 1.J5 * 0.35c
Treatment
o.lo 0.08 _ _
1.22
G-test value ’
Mean’ (*SE) no. of IJS cm mz
‘Number of IJS per droplet. ZMean number of the 40 x 1cm*grids originally counted, as the frequency distribution of based on this total. ‘The G-test was used to test for goodness-of-fit of the data to the negative binomial distribution. G-test values with an asterisk indicate a signiticant fit
tively). For both nematode species, the addition of adjuvants resulted in an increase in the numbers of droplets deposited at 3 V. In contrast, at 6 V, the numbers of droplets were lower than those after spraying the IJS in water alone. This partially accounts for the observation that the proportion of droplets carrying IJS following the addition of adjuvants is lower than for water alone at 3 V, whereas at 6 V, the proportion of droplets carrying IJS is higher than water alone. This was observed for
both Heterorhabditis n.sp. and Steinemema sp. (Tables 6 and 7). Following addition of the adjuvants to the spray solution, deposition of IJS, in terms of the mean numbers of IJS per cm2, was generally significantly higher than that in water alone. This was noted for both Heterorhabditis n.sp. and Steinemema sp. (Tables 6 and 7), and was especially marked at the lower adjuvant concentration. Deposition of IJS of Heterorhabditis n.sp. generally increased with
Heferorhabditisn.sp.
Table 6. Effects of addition of adjuvants on the distribution of IJS of droplets generated
with a Micron Ulva+
% of droplets 0’
1
2
3
4
3v 6V 3v 6V 3v hV 3v 6V 3v 6V 3V 6V
92.04 98.43 92.25 98.75 91.98 98.56 94.20 98.98 91.33 99.37 91.05 99.60
6.92 1.44 6.27 1.19 7.20 1.37 4.81 0.97 7.23 0.62 7.33 0.37
0.93 0.12 1.41 0.05 0.67 0.06 0.81 0.04 1.36 0.01 1.51 0.01
0.11 0.01 0.06 0.10 0.17 0.07 0.12 0.01
0.05 -
3v 6V 3v 6V 3v 6V 3v 6V 3v 6V 3V 6V
94.34 98.27 93.27 97.72 91.58 97.62 95.57 98.95 92.82 99.17 91.05 99.60
4.68 1.70 6.04 2.23 7.47 2.25 3.73 0.98 6.29 0.79 7.33 0.37
0.68 0.03 0.69 0.05 0.87 0.10 0.71 0.06 0.81 0.04 1.51 0.01
0.23 0.09 0.03 0.02 0.08 0.12 0.01
0.00 -
Treatment Crovo! L27 2% Crovol L40 2% Triton
X-100 2%
Croduvant Glycerol
2% 2%
Water Crovol L27 4% Crovol L40 4% Triton
X-100 4%
Croduvant Glycerol Water
4% 4%
(initial concentration
6000 IJS ml --I) in spray
(flow rate 200 ml min -I)
5
-
0.08
-
Mean? ( f. SE) no. of droplets cm ~’
G-test value’
7.96 1.57 7.75 1.25 8.02 1.44 5.80 1.02 8.67 0.63 8.95 0.40
61.17 t2.79 231.87 + 10.54 52.07 + 2.32 248.53 + 8.02 62.67 i_ 3.99 238.13 k9.22 57.47 k 2.43 223.07k5.60 46.53i 1.19 265.47 i 4.58 21 so * 1.40 353.70* 19.27
0.87* 0.25* 5.41* 0.43* 0.70* 0.38* 1.51* 0.34* 3.23 0.05* 2.12* 0.34*
5.57 k 0.79Aab 3.97) 0.57Ba 4.83 + 0.70Aac 3.23 &-0.43Ba 6.27 f 0.99Ab 3.53i0.31Ba 4.00 + OS8Acd 2.37 & 0.43Bb 4.73 f0.79Aad 1.70i_0.34Bc 2.30 + 0.52Ac 1.50+ 0.48Bbc
5.66 1.73 6.13 2.28 2.38 2.38 4.43 1.05 7.18 0.83 8.95 0.40
44.13k2.14 133.20 k 7.33 43.60* 1.63 139.07+4.80 38.40 + 1.01 129.07 + 2.99 51.87k2.10 218.27 +4.20 41.33 f 1.37 218.27k4.20 21.50+1.40 353.70 * 19.27
0.77% 0.03’ 2.00% O.OP 0.36* 1.08* 4.25’ 0.1s+ 0.5 1* 0.37” 2.12* 0.34”
3.13 &0.66Aab 2.33 + 0.47Ba 3.23 + 0.67Aab 3.23i0.51Ab 3.63 F 0.48Aa 3.27 i 0.60Ab 2.67 i 0.47Abc 2.50 i 0.5 1Aa 3.03 + 0.49Aab 2.17fO.38Ba 2.30 i0.52Ac 1SO + 0.48Bc
% of droplets with IJS
Mean’ (*SE) no. of IJS cm -?
‘Number of IJS per droplet. ‘Means of the 40 x 1 cm* grids originally counted, as the frequency distribution of based on this total. 3The G-test was used to test for goodness-of-fit of the data to the negative binomial distribution. G-test values with an asterisk indicate a significant fit. 4Within each adjuvant concentration, for voltage, means followed by the same uppercase letter are not significantly different. Within each voltage, for adjuvant concentration, means followed by the same lowercase letter are not significantly different.
Crop Protection
1998 Volume 17 Number 5
467
Screening
and selection
of adjuvants:
J.M.
Mason et al. of IJS of Steinemem
Table 7. Effects of addition of adjuvants on the distribution generated with a Micron Ulva+ (flow rate 200 ml min -‘)
sp. (initial concentration
0’
1
2
3
Crovol L27 2%
3v 6V 3v 6V 3v 6V 3v 6V 3v 6V 3v 6V
96.58 99.12 95.60 99.36 97.05 99.48 96.62 99.30 95.09 99.22 90.16 99.71
3.04 0.79 3.87 0.64 2.79 0.52 3.00 0.64 4.70 0.73 8.52 0.28
0.25 0.09 0.44
0.13 0.09
Crovol LAO2% Triton X-100 2% Croduvant 2% Glycerol 2% Water Crovol L27 4%
3v 6V 3v 6V 3v 6V 3v 6V 3v 6V 3v 6V
96.26 99.22 95.13 98.76 94.41 97.96 97.22 99.31 97.31 99.15 90.16 99.71
3.28 0.73 4.39 1.22 5.23 1.88 2.43 0.63 2.33 0.82 8.52 0.28
0.46 0.04 0.40 0.02 0.35 0.13 0.35 0.04 0.36 0.02 0.98 0.01
Crovol LAO4% Triton X-100 4% Croduvant 4% Glycerol 4% Water
016 0.32 0.05 0.07 0.04 0.98 0.01
4 -
-
0.00
0.14
0.06 -
0.08
0.03 0.01 0.02 0.33 -
-
-
in spray droplets
% of droplets with IJS
Mean ’ ( k SE) no. of droplets cm e-2
G-test value 3
Mean4 (*SE) no. of IJS cm -’
3.42 0.88 4.40 0.64 2.95 0.52 3.38 0.70 4.91 0.78 8.84 0.29
52.60+ 1.92 182.27f7.98 37.87 k2.05 196.80 + 8.80 62.20 & 2.14 257.60 k 6.89 52.27 i. 1.59 269.87 k2.66 46.13k1.36 231.60+7.84 30.5 + 2.92 245.80 + 12.86
2.03* 1.34* 0.36* 0.26* 0.37* 0.22* 0.28* 0.76* 0.97 0.43* 0.22* 0.09*
2.07* 0.31Aab 1.77_f0.30Aab 1.90 + 0.46Aab 1.27 f0.33Ba 1.90 f 0.40Aa 1.33 + 0.34Ba 2.03 kO.37Aab 2.00 &0.29Ab 2.43 + 0.43Ab 1.9070.3OAb 0.93 k0.31Ac 0.75 k 0.21Ac
3.74 0.78 4.87 1.24 5.59 2.04 2.78 0.69 2.69 0.85 9.84 0.29
50.80f2.11 231.6Ok7.84 41.73 + 1.55 169.47 I5.31 37.60k1.41 104.53 53.17 48.00 & 1.45 247.20 k3.500 45.87 f 1.52 211.07f3.15 30.5 +2.92 245.80 k 12.86
2.08* 0.43* 0.40* 0.03* 0.52* 0.19* 1.55* 2.20* 1.68* 0.43* 0.22* 0.09*
2.13 +0.41Aac 1.90f0.30Aa 2.27 k 0.35Aa 2.13 i 0.36Aa 2.23 +0.42Aa 2.33 f.0.41Aa 1.50 +0.35Ab 1.87z0.40Aa 1.40 k 0.33Ab 1.90+0.36Aa 0.93 & 0.31Ac 0.75 i0.21Ac
% of droplets
Treatment
6000 IJS ml -I)
‘Number of IJS per droplet. 2Means of the 40 x 1 cm2 grids originally counted, as the frequency distribution of based on this total. 3The G-test was used to test for goodness-of-fit of the data to the negative binomial distribution. G-test values with an asterisk indicate a significant fit. 4Within each adjuvant concentration, for voltage, means followed by the same uppercase letter are not significantly different. Within each voltage, for adjuvant concentration, means followed by the same lowercase letter are not significantly different.
increased adjuvant concentration (Table 6). This was noted at 3 and 6 V. In contrast, an increase in adjuvant concentration generally resulted in a decrease in deposition of IJS of Steinemema sp. at both voltages used (Table 7).
solutions of the IJS of both species resulted in a reduction in volume median diameter (vmd) and a slight narrowing of the width of the main droplet band. However, increased production of larger droplets accounted for an increased proportion of the volume. Production of foam was not noted with this spraying system.
Spray spectra analyses of adjuvant solutions: Micron Herbaflex The addition of Croduvant and Glycerol (2% and 4% concentration) to the solution of IJS of both nematode species had no effect on the spray spectra produced compared with spraying in water only. In contrast, the addition of Crovol L27 or Crovol LAO, at 2% or 4%, to solutions of IJS of both nematode species resulted in a narrower band width of droplets and a reduction in the vmd values compared with IJS in water. Addition of Triton X-100 resulted in the production of a large volume of foam, with subsequent inefficient droplet production and many large droplets formed. Representative results for the spray spectra are shown in F@re I for Heterorhabditisn.sp. Spray spectra analyses of adjuvant solutions: Micron Ulva+ The addition of Crovol L27, Crovol LAO, Croduvant or Glycerol at 2% or 4% to solutions of IJS of both nematode species had little effect on the resultant spray spectra or vmd values compared with the spectra produced for IJS in water (Mason et al., 1998a). Addition of Triton X-100 at 2% and 4% to
468
Crop Protection
1998 Volume 17 Number 5
Discussion None of the adjuvants tested were toxic to DBM larvae, with survival generally being 100% after 72 h. However, reduced feeding levels of the DBM larvae were noted, especially on leaf discs dipped in Triton X-100 and Crovol L27 (J. M. Mason, unpublished results). This result for Triton X-100 was not entirely unexpected as it is normally used as an adjuvant at concentrations ~1%. The adjuvants tested in the present study were shown to have minimal direct toxicity to IJS of both Heterorhabditis n.sp. and Steinemema sp., although it was noted that increasing incubation time in both Crovol L27 and Crovol IA0 resulted in reduced nematode motility. The reasons for this are not clear. However, in terms of field applications, this is of minimal significance as IJS in droplets will not survive for this length of time, irrespective of timings of application. Although direct toxicity was minimal, infectivity of IJS was affected. Both mortality and intensity of infection in larvae of G. melionetta was significantly reduced compared with infectivity of IJS, of either nematode species, in water. There are a number of possible mechanisms
Screening
and selection
of adjuvants:
J.M.
Mason
et al.
water; vmd = 218 pm CrovolL27;
vmd = 182 pm
Crovol L40; vmd = 191 pm Croduvant; Glycerol;
200
300
vmd = 221 pm vmd = 217 pm
400
500
Band size (pm) Figure 1. Effects of adjuvants (2%) on spray droplet Heferorhabditis n.sp. (6000 IJS ml -I).
spectra
produced
for this, including direct toxic effect on the IJS, toxicity to G. mellunellu larvae or change in the surface tension of the fluid surrounding the sand particles. The latter two mechanisms are the more plausible, as our results suggest minimal direct toxic effect on the IJS. In a similar bioassay, assessing the toxicity of a number of potential adjuvants on IJS of S. catpocupsae, Lello et al. (1996) noted no significant difference in either mortality or intensity of infection of G. mellonella larvae compared with IJS in water. One of the adjuvants used in the present study, Triton X-100, is the same as that used by Lello et al. (1996). Differences noted between the two studies may be a result of differences in adjuvant concentration, nematode species or incubation time. However, efficacy against larvae of l? xylostella is more important (see Mason et al., 1998b). Deposition of IJS was significantly affected by the addition of the adjuvants to the spray solution. Generally, addition of the adjuvants resulted in an increase in the mean numbers of IJS deposited per cm2. This increase in deposition of IJS has also been noted following application on leaf discs of Chinese cabbage (J. M. Mason, unpublished results). That addition of the adjuvants results in higher numbers of IJS being deposited within the defined catchment area suggests that there is a change in the swath pattern as the total output of IJS from either sprayer is not significantly affected by the addition of any of the adjuvants compared with spraying the IJS in water (Mason et al., 1998a), although swath pattern was not measured in the present study. Furthermore, this change in swath pattern may be a result of a reduction in dynamic surface tension at the time of atomisation, although changes in fluid viscosity and other undefined variables (Hall et al., 1993) may also be involved. That the addition of adjuvants does affect swath pattern has been shown, for example, for
by a Micron
Herbaflex
(flow rate 30 ml min -‘)
spraying
IJS of
a flat-fan hydraulic nozzle (Chapple et al., 1993). While it is appreciated that the study by Chapple et al. (1993) was carried out while the nozzles were stationary and also that the total output volume from a centrifugal energy nozzle (in this case, spinning discs) and a hydraulic nozzle are on completely different scales, it is conceivable that the addition of IJS and/or adjuvants could significantly affect the swath pattern produced by spinning discs. This is worthy of further investigation as it may have significant bearing on subsequent deposition of the IJS, dependent on the plant substrate/architecture, and be open for manipulation. The major reason for considering incorporation of adjuvants in sprays of IJS was primarily to limit the damaging effects of desiccation. Although UV radiation is also known to be lethal to IJS of a number of species of EPNs (Gaugler and Boush, 1978) judicious timings of field applications can minimise such effects (e.g., MacVean et al., 1982). Incorporation of the adjuvants was not intended to directly enhance desiccation survival of the IJS, but rather to reduce the evaporative rate of the droplets. To this effect, the adjuvants we tested, with the exception of Triton X-100, promote the maintenance of the integrity of the droplet. The decision not to test spreaders, such as organosilicone compounds, was based on the fact that although surface tension is reduced, which we considered a requirement for potential enhancement of infection by allowing IJS to ‘break’ the surface tension, and adhesion is greater, the sprayed liquid spreads which can lead to reduced retention due to coalescence of the droplets and, hence, increased run-off (Stevens and Kimberley, 1993). Overall, the results obtained with these adjuvants are very encouraging. Although infection of G. mellonella larvae was significantly reduced, this standard
Crop Protection
1998 Volume 17 Number
5
469
Screening
and selection
of adjuvants:
J.M. Mason et
al.
infectivity assay for EPNs is not representative of the final intended host insect and plant. However, it enabled an initial screening without the use of spraying equipment, as at that time the effect of the adjuvants on spray quality was not known. Obviously, a more realistic assay will be against DBM larvae on crucifers (Mason et al., 1998b). The adjuvants were ranked based on the results from each of the studies presented here. On the basis of these combined rankings, the adjuvants scored as follows. For IJS of Heterorhabditis n.sp., the ‘best’ adjuvant for use with the Ulva+ and Herbaflex is Triton X-100, followed by Crovol L27, Crovol L40, Croduvant and Glycerol, with IJS in water alone being the least efficacious. However, for Steinemema sp., the best adjuvant for use with either sprayer is Croduvant, followed by Triton X-100 and Glycerol, Crovol LAO and Crovol L27, with IJS in water again being the worst option. Results from further studies examining infectivity of IJS against DBM larvae are presented elsewhere (Mason et al., 1998b).
Gaugler, R. and Boush, G. M. (1979) Laboratory tests on ultraviolet protectants of an entomogenous nematode. Environ. Entomol. 8, MO-813
Georgis,
R. (1990) Formulation
Entomopathogenic
and application
technology.
In
Nematodes in Biological Control, ed. R. Gaugler
and H. K. Kaya. CRC Press, Boca Raton, FL, pp, 173-191 Glazer, I., Klein, M., Navon, A. and Nakache, Y. (1992) Comparison of efficacy of entomopathogenic nematodes combined with antidesiccants applied by canopy sprays against three cotton pests (Lepidoptera: Noctuidae). J. Econ. Entomol. 85, 1636-1641 Hall, F. R., Chapple, A. C., Downer, R. A., Kirchener, L. M. and Thacker, J. R. M. (1993) Pesticide application as affected by spray modifiers. Pestic. Sci. 38, 123-133 Lello, E. R., Patel, M. N., Matthews, G. A. and Wright, D. J. (1996) Application technology for entomopathogenic nematodes against foliar pests. Crop Prot. 15,567-574 MacVean, C. M., Brewer, J. W. and Capinera, J. L. (1982) Field tests of antidesiccants to extend the infection period of an entomogenous nematode, Neoaplectana carpocapsae, against the Colorado potato beetle. J. Econ. Entomol. 75, 97-101 Mason, J. M. and Wright, D. J. (1997) Potential for the control of with entomopathogenic nematodes. .I.
Plutella xylostella larvae Invert. Pathol. 70, 234-242
Mason, J. M., Matthews, G. A. and Wright, D. J. (1998a) Appraisal of spinning disc technology for the application of entomopathogenic nematodes. Crop Protection 17, 453-461
Acknowledgements This research is funded by The Leverhulme Trust. We wish to thank Micron Sprayers Ltd for generously supplying the Micron Ulva+ and Micron Herbaflex sprayers, and Croda Chemicals Ltd for generously supplying adjuvants. References
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