Persistence and Distribution of Wild-Type and Recombinant Nucleopolyhedroviruses in Soil.

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 MICROBIAL ECOLOGY Microb Ecol (2001) 41:222–232 DOI: 10.1007/s002480000088 © 2001 Springer-Verlag New York Inc. 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, 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.