3 Red River Research Station, Louisiana Agricultural Experiment Station, Louisiana State University. Agricultural Center, Bossier City, LA 71113, USA.
MICROBIAL ECOLOGY Microb Ecol (2001) 41:222–232 DOI: 10.1007/s002480000088 © 2001 Springer-Verlag New York Inc.
Persistence and Distribution of Wild-Type and Recombinant Nucleopolyhedroviruses in Soil J.R. Fuxa,1 M.M. Matter,2 A. Abdel-Rahman,1,2 S. Micinski,3 A.R. Richter,1 J.L. Flexner4 1
Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA 2 Pests and Plant Protection Department, National Research Centre, Dokki–Cairo, Egypt 3 Red River Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Bossier City, LA 71113, USA 4 Stine-Haskell Research Center, E. I. DuPont de Nemours & Co., Newark, DE 19714, USA Received: 24 July 2000; Accepted: 3 October 2000; Online Publication: 23 February 2001
A
B S T R A C T
Persistence of recombinant and wild-type nucleopolyhedroviruses (NPV) was compared in field and laboratory microcosm experiments. Horizontal and vertical distribution of the viruses also was monitored in the field agricultural soil. Mixed populations of the bollworm, Helicoverpa zea, and tobacco budworm, Heliothis virescens, in cotton were sprayed five times during a growing season with wild-type H. zea NPV (HzSNPV.WT) or with a genetically modified H. zea NPV expressing an insect-specific neurotoxin (HzSNPV.LqhIT2). HzNPV.WT accumulated 2.3 times as many occlusion bodies (OB) as HzSNPV.LqhIT2 in soil by the end of the growing season in October 1997. Both NPVs were detected at all soil depths down to 26–35 cm. Both NPVs were randomly distributed among 0–2 cm soil samples throughout the plots according to analysis with Taylor’s power law. By 4 August 1998, soil concentration of HzSNPV.WT was only 11–13 OB/g at depths from 0 to 14 cm, and the wild-type virus was not detected below 14 cm. HzSNPV.LqhIT2 was detected only in trace amounts at 0–2 cm at this time. Neither NPV was detected in bioassays of cotton leaves nor in insects sampled from the plots in 1998. Viral persistence also was monitored in laboratory soil microcosms. Three viruses—wild-type Autographa californica NPV (AcNPV.WT), A. californica NPV expressing a scorpion toxin (AcNPV.AaIT), and A. californica NPV expressing juvenile hormone esterase (AcNPV.JHE-S201G)—were introduced into soil microcosms by each of two methods, in water suspension or in host cadavers, for a total of six treatments plus controls. After 17 months, the number of viable OB remaining did not differ among the treatments. The results indicate that the only differences in soil populations of wild-type versus recombinant NPVs are due to the greater amounts of the wild-type viruses that accumulate, probably because they have a greater capacity to replicate in the host insect population.
Correspondence to: J.R. Fuxa; Fax: (225) 388-1643; E-mail: jfuxa@unix1. sncc.lsu.edu
Wild-Type and Recombinant Viruses in Soil
Nucleopolyhedroviruses (NPVs) have been used successfully for insect pest management, but certain weaknesses such as slow death of the target host have hindered their development as microbial insecticides [12, 27]. Genetic modification of these viruses has been pursued as a means to correct certain of these weaknesses [3]. For example, insect survival time has been reduced [10, 21] when NPVs express modified juvenile hormone esterase (JHE) [4, 5] or highly specific insect toxins from the scorpions Androctonus australis Hector [26, 32] or Leiurus quinquestriatus hebraeus (Birula) [7, 35, 36]. Ecology of DNA-recombinant NPVs is of practical as well as theoretical interest. The primary rationale for ecology of these viruses has been as a contribution to environmental risk assessment [11]. Knowledge of the capability of these viruses for persistence, population growth, and spread can reduce their risk of environmental release even when the possibility of harmfulness to nontarget organisms is difficult or imposssible to ascertain. Ecology is only beginning to be studied from the standpoint of improving efficacy, as it has been for the wild-type NPVs [25]. Of more theoretical interest, recombinant NPVs are providing an opportunity to test hypotheses about the effects of the addition of a single gene to increase virulence on basic ecological characteristics of viruses, such as population growth, density, distribution, and dispersal [10, 23, 24]. One of the more important gaps in our knowledge of the ecology of recombinant NPVs is their population dynamics in soil. NPVs expressing scorpion toxins and, to a lesser degree, those expressing modified JHE produce fewer viruscontaining occlusion bodies (OB) than the parental, wildtype NPVs [10, 19, 20, 22]. Furthermore, these recombinant NPVs, particularly those expressing scorpion toxins, do not liquefy or disintegrate the dead host insect, which is a characteristic sign of infection by wild-type NPV [8, 10, 16]. Thus, lepidopterous insects killed by the recombinant NPVs tend to fall off their host plant to the ground without disintegrating and releasing OB, whereas those killed by wildtype NPVs tend to disintegrate rapidly, releasing OB onto host plant surfaces. Not unexpectedly, this results in a lower population density of recombinant NPV than wild-type NPV on the insect’s host plant [10]. Prediction of which type of NPV will increase to greater numbers in soil, their natural reservoir where they can persist for years, is not as simple. More wild-type than recombinant NPV could accumulate in soil, because the greater numbers of OB of wild-type virus than of recombinant NPV on leaves infect more hosts,
223
thereby increasing replication in subsequent host generations. Alternatively, more recombinant than wild-type NPV could accumulate in soil, because wild-type virus released from cadavers disintegrating on leaves is more exposed to ultraviolet (UV) radiation from sunlight, which inactivates NPVs within hours [1], than recombinant NPV in intact cadavers that have fallen to the soil surface beneath plants. If population densities of the two types of NPV differ in soil, this could affect subsequent contamination of soil at different depths as well as infection of new generations of insects in subsequent growing seasons. In the current research, we treated mixed populations of bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (Fabricius), larvae over a cotton-growing season with recombinant or wild-type NPV. The objectives of this experiment were (1) to periodically compare population densities of the two types of virus in soil from the conclusion of a growing season until the next season was underway; and (2) to compare their horizontal and vertical distribution in soil. A second experiment, in laboratory soil microcosms, tested whether viral inoculation into soil in water suspension or host cadavers affected persistence of recombinant and wild-type NPVs. If not, then any difference in field persistence between the two types of virus probably would be due to quantity of NPV reaching the soil rather than to retention of virus in host cadavers falling to the ground.
Methods Viruses The two viruses in the field experiment were variants of the Heliothis/Helicoverpa NPV (HzSNPV) provided by DuPont Agricultural Products. These included wild-type HzSNPV (HzSNPV.WT) and a genetically modified HzSNPV expressing an insect-specific neurotoxin from the scorpion L. quinquestriatus hebraeus (HzSNPV. LqhIT2). The three viruses in the laboratory microcosm experiment were variants of Autographa californica (Speyer) NPV (AcNPV), all of which were grown in laboratory-reared cabbage loopers, Trichoplusia ni (Hu¨bner). Wild-type AcNPV (AcNPV.WT) was a plaque-purified isolate, AcNPV-C6, provided by BioSys Inc. (now Thermo Trilogy Corporation). B. D. Hammock (Dept. of Entomology and Environmental Toxicology, University of California, Davis) provided variants of AcNPV expressing insect-specific neurotoxin from the scorpion A. australis (AcNPV.AaIT) or modified juvenile hormone esterase (AcNPV.JHE-S201G). Field Persistence Experiment. Cotton (DPL 5409) was planted on 19 June 1997 on the Louisiana State University Agricultural Center Red River Research Station near Bossier City. The soil was 61.0%
224 sand, 15.9% silt, and 23.1% clay, with 0.22% organic matter and a pH of 7.1. The plots, each of which measured 16 rows (total 16.3 m) × 12.2 m, were arranged in a randomized block design with four replications. The three treatments were an untreated control, HzSNPV.WT, and HzSNPV.LqhIT2. Both viruses were sprayed on five dates (August 12, 16, 21, 27, and September 3) at 4.94 × 1012 OB/ha in 55.2 liter water/ha. Viral treatments were sprayed with a high-clearance CO2 sprayer at 4.22 kg/cm2 pressure through TX-3 hollow-cone nozzles spaced 51 cm apart (two nozzles/row). Soil was randomly sampled from one site within each plot on each of eight sampling dates. At each site, the top 2 cm soil was collected from a 10-cm diameter sampler, then a 1.7-cm diameter core sampler took soil down to 38 cm. The core sample was separated into three subsamples (2–14, 14–26, and 26–38 cm). Additionally, on 10 September 1997, samples were collected to ascertain horizontal distribution of each NPV in soil. Two 1-m2 sampling frames were placed randomly in each virus-treated plot, and four random 0–2 cm soil samples were collected within each frame with the 10-cm diameter sampler. Insects and cotton leaves were sampled at various times from the field plots. Bollworm (H. zea) and tobacco budworm (H. virescens) numbers were determined by examination of 10 cotton terminals and 25 squares randomly selected in each plot on 21 and 27 August and 2 September 1997. Live bollworms/tobacco budworms (mean of 21 per plot per date) were randomly sampled on 12, 20, and 26 August and 5 September 1997, placed individually in 30-ml cups with artificial diet, and returned to the laboratory to be observed for signs and symptoms of nuclear polyhedrosis, which was confirmed by detection of OB by phase microscopy of smears of the dead insects. The larvae that survived to the adult stage were identified to species. The plots were planted again with cotton in 1998 but were not treated with virus. Ten live bollworms/tobacco budworms per plot were randomly sampled on 11 September 1998 and returned to the lab for observation, as in 1997. Additionally, two cotton leaves from the bottom and two from the top of each of three randomly selected plants per plot (12 leaves per plot) were sampled on 28 July 1998, when the plants were approximately 25 cm high, and returned to the laboratory in petri dishes (one leaf per dish) to be bioassayed for NPV. The soil and leaf samples were bioassayed with H. zea larvae. Soil samples were air-dried and ground with a mortar and pestle. A 30-g random subsample from each sample was then mixed into 270 ml artificial diet when it was still liquefied but had cooled to 1.0 indicates a clumped distribution [9] of virus among the soil samples. A mean and variance were calculated for the four random soil samples within each sampling flame (n = 8 frames per virus), and the log-transformed parameters were analyzed by the SAS regression and GLM procedures [30].
Results Field Persistence Experiment Sprays of HzSNPV.WT and HzSNPV.LqhIT2 caused seasonlong epizootics in mixed H. zea and H. virescens populations in cotton in 1997, but neither virus reduced (P > 0.05) the numbers of insects (Tables 1, 2). These epizootics, or unusually large numbers of cases of the disease, established populations of the two viruses in soil of the treated plots, and these viral populations reached their peak densities on 7 October 1997, at the end of the cotton growing season (Tables 3, 4). The concentration of HzSNPV.WT was greater (P < 0.05) than that of HzSNPV.LqhIT2 at 0–2 and 2–14 cm on every post-treatment sampling date in 1997 and 1998. The concentration of both NPVs decreased as soil depth increased, although differences among depths were signifi-
225 Table 1. Effects of recombinant and wild-type nucleopolyhedroviruses on a mixed population of Helicoverpa zea and Heliothis virescens larvae in cotton Virus treatment Data set
Control
HzSNPV.WT
HzSNPV.LqhIT2
1997 seasona Mean no. live larvae per 10 terminals on: Aug. 21 2.0 0.8 Aug. 27 2.0 1.5 Sep. 2 5.8 3.8 Overall mean 3.3 a 2.0 a Mean no. live larvae per 25 squares on: Aug. 21 1.5 0.8 Aug. 27 1.3 1.5 Sep. 2 3.8 4.3 Overall mean 2.2 a 2.2 a Mean % infection (n), live larvaeb sampled on: Aug. 20 6.0 (82) 48.5 (75) Aug. 26 7.5 (80) 35.0 (77) Sep. 5 3.3 (88) 29.0 (104) Overall mean 5.5 c 37.5 a 1998 season Mean % infection (n), bioassay of cotton leaves July 28 0 (877) 0 (895) Mean % infection (n), live larvae sampled on: Sep. 11c 0 (30) 0 (30)
1.8 0.5 3.8 2.0 a 1.3 0.5 3.0 1.6 a 16.8 (89) 15.8 (77) 26.3 (103) 19.5 b sampled on: 0 (882) 0 (39)
a Plots were treated with their respective viruses on 8/12, 8/16, 8/21, 8/27, and 9/3. b Proportion of surviving adults, H. zea: H. virescens (n), were 49:51 (49) on 8/20, 27:73 (56) on 8/26, and 8:92 (36) on 9/5. n is the total number of insects in four replications (plots). c One plot each in the control and HzSNPV.WT treatments had been destroyed by drought by this date. Means in each row followed by the same letter are not significantly different (P < 0.05, Tukey HSD).
cant (P < 0.05) on more sampling dates for HzSNPV.WT than for HzSNPV.LqhIT2. Both viruses were detected at the greatest depth sampled, 26–38 cm, on at least three sampling dates but not after 6 April 1998. Overall amounts of both NPVs in soil began to decrease after 7 October 1997, although OB concentration at depths greater than 14 cm increased for both NPVs in sampling of early 1998, probably because of percolation from upper soil layers. NPV increased in the soil of control plots as the experiment progressed until the 17 February 1998 sample, probably as a result of movement of agricultural machinery. By 4 August 1998, 11–13 ob/g of HzSNPV.WT and trace amounts of HzSNPV.LqhIT2 remained in the uppers layers of the soil, but these population densities apparently were insufficient to be transported to cotton leaves or to infect 1998 populations of H. zea and H. virescens (Table 1). On the last soil sampling date, HzSNPV.WT and HzSNPV.LqhIT2 were not detected below
226
J.R. Fuxa et al.
Table 2. General linear models procedure, repeated measures ANOVA [30], indicating sources of variation in 1997 insect sampling for the field soil persistence experiment Source
df
SS
F
Sampling of larvae per 10 terminals Among-treatment effects Plot treatment 2 12.5 1.7 Error 9 32.3 Within-treatment effects Sampling date 2 72.2 30.2 Treatment*date 4 6.3 1.3 Error (time) 18 21.5 Sampling of larvae per 25 squares Among-treatment effects Plot treatment 2 2.7 1.5 Error 9 8.3 Within-treatment effects Sampling date 2 51.7 22.7 Treatment*date 4 3.8 0.8 Error (time) 18 20.5 Percentage infection of larvae Among-treatment effects Plot treatment 2 6,153.8 85.9 Error 9 276.1 Within-treatment effects Sampling date 2 141.0 2.2 Treatment*date 4 947.3 7.4 Error (time) 18 575.5
P>F
0.2290
0.0001 0.2984
0.2772
0.0001 0.5238
0.0001
0.1392 0.0010
14 cm and 2 cm, respectively (Tables 3, 4). None of the no-soil bioassay control insects died because of NPV during the experiment. The interactions of treatment × soil depth, treatment × sampling date, and depth × date significantly (P = 0.0001) affected concentrations of OB in soil (Table 5). The slopes of Taylor’s power law regressions (Table 6) for the two NPVs were not greater than 1.0 (P > 0.05). Analysis of covariance indicated that the intercepts of these two regression lines were different (F = 9.16, df = 1, P = 0.0105) but the slopes were the same (F = 0.02, df = 1, P = 0.8854).
Laboratory Microcosm Persistence Experiment Neither the type of NPV nor method of viral inoculation into soil affected overall viral persistence (P > 0.05), although persistence among viral and inoculation treatments differed (P < 0.05) occasionally on certain dates (Tables 5, 7). More than 20,000 OB per gram soil remained in all virus/inoculation treatments after 17 months, but this was less than 1% of the original active virus in each treatment (Table 8). None of the no-virus or no-soil control insects died because of NPV during the experiment.
Discussion HzSNPV.WT accumulated greater numbers of OB in soil than HzSNPV.LqhIT2 during and immediately after the cotton growing season, an effect that persisted until the termination of soil sampling 10 months later (Tables 3–5). This difference probably can be attributed to the greater replication of the wild-type virus in host insects. Wildtype NPVs produce up to 3–5 times more OB per insect than their corresponding scorpion-toxin recombinant NPVs [10, 22]. Prevalence of HzSNPV.WT also was greater than that of HzSNPV.LqhIT2 in host insects sampled from cotton plants (Tables 1, 2), though this difference probably was due to the more rapid mortality and dropping of intact host cadavers to the ground associated with the scorpion-toxin NPVs [16]. The only way, beside viral replication, in which insertion of a gene expressing scorpion toxin or a modified JHE might affect NPV persistence is through characteristics of the host cadavers. There should be no inherent difference in persistence of wild-type versus recombinant NPV OB in themselves, because insertion of a scorpion-toxin or JHE gene should not alter the polyhedrin protein comprising the OB [3], which in turn allows NPVs to persist for long periods in soil. However, insects killed by scorpion-toxin NPVs and, to a lesser degree, those killed by JHE recombinant NPVs tend to remain intact [10] and fall to the ground [16], whereas those killed by wild-type NPVs characteristically disintegrate, liberating OB onto foliage and soil [17]. Thus, OB reaching the soil in intact host cadavers might be more protected from environmental conditions than those falling naked to the soil surface. The soil in our microcosms, which was not autoclaved, gave indications in previous field and microcosm studies [29] of containing one or more agents, probably biotic, that contributed to degradation of a wildtype NPV. The recombinant and wild-type AcNPVs were equally persistent in this agricultural soil harmful to NPV, regardless of whether their OB were inoculated in water suspensions or in host cadavers (Tables 7, 8). Previous research of soil persistence of genetically modified NPVs has dealt only with genetically marked virus or with virus engineered to produce weakened OB or no OB at all [2, 28], as opposed to our study of NPV modified for improved insect control with normal OB. Further research is necessary to determine whether the type of inoculation would affect survival of scorpion-toxin and JHE recombinant NPVs in sunlight on the soil surface. An important result of the current field-persistence study
0c
2.5 d
1.9 b
Sep. 10
Oct. 7
Dec. 7
0.3 b
£ ±0.6 0b ±0.3
4.2 c ±1.7 0c
1.3 bc ±0.7 0d ±1.0 0b ±0.8
0a
2–14 cm
0b
£
0.7 c ±0.7 0c
0b
0d
0c
0a
14–26 cm
0b
£
0.6 c ±0.6 0c
†
0d
0c
§
26–38 cm
3.2 a
21.2 a ±2.7 10.0 a ±3.5 9.4 a
26.0 a
22.8 a ±7.5 42.2 a
0a
0–2 cm
11.8 b ±3.3 6.5 ab ±3.1 £ ±1.5 2.6 a ±1.1
16.4 a ±4.6 27.5 b ±5.9 23.8 a ±2.7
0a
0b ±1.1
6.9 bc ±1.9 0.6 c ±0.6 £
1.3 d ±9.8 1.3 b ±8.3
0c
0.6 a
14–26 cm
HzSNPV.WT 2–14 cm
* Cotton plots (4 replications) were treated on August 12, 16, 21, 27, and September 3, 1997. § Not sampled † Soil too wet to sample lowest depth £ Soil too dry to sample below 2 cm Means in each row followed by the same letter are not significantly different (P < 0.05, Tukey HSD).
Aug. 4
June 6
Apr. 6
3.4 c ±2.1 3.2 bc ±1.4 0.9 b
0a
1997 July 29
1998 Feb. 17
0–2 cm
Sample date
Control
0b
5.5 c ±2.5 0.6 c ±0.6 £
§ ±0.6 1.9 bc ±1.9 10.0 cd ±1.3 † ±0.8
26–38 cm
0.3 b
11.4 b ±2.0 4.1 bc ±2.1 1.0 b
8.1 b ±1.9 13.4 c ±4.1 7.6 b
0a
0–2 cm
£ ±0.6 0b ±0.3
4.7 c ±2.1 0c
5.0 bc ±1.0 3.1 d ±6.4 2.0 b ±2.4
0.7 a
2–14 cm
0b
£
0.7 c ±0.7 0c
0a ±0.7 0.6 c ±0.6 1.9 d ±1.6 0.7 b ±1.3
14–26 cm
HzSNPV.LqhIT2
0b
1.4 c ±0.8 1.9 bc ±1.2 £
1.9 bc ±1.9 0d ±1.2 † ±0.7
§
26–38 cm
Table 3. Mean percentage mortality (±SE) by nucleoplyhedrovirus in bioassay of soil at different depths in untreated (control) plots and in plots treated with HzSNPV.WT or with HzSNPV.LqhIT2*
Wild-Type and Recombinant Viruses in Soil 227
0 5.9 0 0 16.4 0 @ 0
13.5 13.0
