Bioresponse of Nontarget Organisms Resulting from the Use of Chloropicrin To Control Laminated Root Rot in a Northwest Conifer Forest:

Part 2. Evaluation of Bioresponses

by

Elaine R. Ingham, Walter G. Thies, Daniel L. Luoma 2 , Andrew R. Moldenke3 and Michael A. CasteIland

Author affiliation: Department of Botany and , Oregon State University, Pacific Northwest Research Station, USDA Forest Service; Department of Entomology, Oregon State University, OR.

Known impacts of chloropicrin choices, and position in the food populations are web will expose the diversity of reduced by chloropicrin alone and species to different concentrations Most research has in combination with methyl of fumigant. concentrated on the effects of bromide or dazomet. Research combinations of methyl bromide has concentrated on the and chloropicrin fumigants on commercially important parasites Objectives disease-causing organisms. Little of plant roots and no information research has been reported on the was found on free-living fungal or One objective of this study was response of saprophytic fungi to bacterial feeding . to determine changes in diversity chloropicrin. Some evidence exists Fumigation reduced the nematode of specific nontarget organism for the escape of some species of root pathogens Meloidogyne spp. components of a coastal ecosystem fungi during fumigation efforts and minor which occur as a result of thus positioning the survivors for (Sumner et al. 1985) and application of chloropicrin to rapid recolonization of the chloropicrin in combination with stumps to control laminated root available substrate. The ethylene dibromide reduced rot. A major challenge was to occurrence of Trichoderma spp. in Belonolimus longicaudatus, evaluate the impact of chloropicrin roots of fumigated Douglas-fir Meloidogyne incognita, and on a range of organism groups. A stumps was noted during the Hoplolaimus galeatus (Rhoades sample scheme capable of evaluation of several fumigants for 1983). distinguishing at least two levels of the control of laminated root rot spatial variability was designed. (Thies and Nelson 1982). As a measure of species Background variability of the Following fumigation, increased richness, soil arthropods in conifer organisms resulting from numbers of Trichoderma spp. were forests of the Pacific Northwest heterogeneity in soil microsites found in some soils (Mughogho average nearly 200 species/m2 and seasonal shifts is a common 1968). Combinations of methyl (personal communications Andrew difficulty in studies of soil bromide and chloropicrin control a R. Moldenke, Oregon State organisms. Sampling intensity had variety of soil-borne diseases in University, Corvallis, OR). Thus to be great enough that treatment forest tree nurseries (Peterson and soil arthropods may be an effects could be assessed, without Smith 1975) and can reduce important and sensitive indicator increasing the work load beyond populations of various other fungi. to evaluate potential ecosystem that of a limited budget. These include VA mycorrhizal effects of chloropicrin on fungi (Jones and Hendrix 1987, nontarget soil organisms. Soil The approach described here McGraw and Hendrix 1986), arthropods are sensitive indicators can be applied to any ecosystem, ectomycorrhizal fungi (Trappe et for soil moisture, successional and to a number of situations al. 1984), as well as root stages, plant communities, and where effects of pesticide disease-causing species of Pythium, mycorrhizal biomass (Cromack et application need to be assessed. Rhizoctonia, Phoma (Sumner et. al. 1988, Moldenke and Fichter With this approach, the effect of al. 1985), Helicobasidum momta, 1988). All arthropod species are pesticide on plants, soil detrital (Sakuwa et al. 1984), Verticillium presumed to be sensitive to foodweb organisms, mycorrhizal albo-atrum, and Sclerotium chloropicrin, although differing colonization of dominant plants, (Himelrick 1986). feeding preference, microhabitat and survival of sensitive bioassay

HOW CAN THEIR EFFECTS BE MONITORED? 85 NONTARGET BIORESPONSE TO CHLOROPICRIN: 2. EVALUATION plants can be interpreted in a three mature trees in adjacent un- (Ingham et al. 1985; Ingham and biologically meaningful manner at cut stands were mapped. Circular Horton, 1987). Mycorrhizal fungi a number of spatial scales. For treatment plots 0.4 ha in size were are extremely important in survival example, the pesticide may reduce established in the clearcut area of Douglas-fir; in field sites, root rot, but destroy those and blocked into groups of four Douglas-fir is not found without organisms responsible for nutrient based on similar inoculum rating the symbiotic fungus (Trappe et al. cycling, or could allow another (see part 1 for explanation). Each 1984). Thus, monitoring pathogen to become a problem, of the four plots in each block mycorrhizal fungi can be extremely exchanging one pathogen problem received one of the following important in determining if for another. Root-feeding treatments: all stumps treated with pesticides have an effect. nematodes might be advantaged, 100% chloropicrin, only infected resulting in seedling death. stumps treated with 100% Tomatoes and alfalfa were Alternatively, greater mycorrhizal chloropicrin, all stumps treated planted in the field sites. colonization of roots might occur, with 20% chloropicrin, and a is extremely sensitive to and benefit the survival of desired control plot with no application chloropicrin (Rhoades 1983), and plant species. Before we continue chloropicrin. The 100% label alfalfa is symbiotic with N-fixing with the use of pesticides, we dosage was 3.3 ml of chloropicrin rhizobium. Release of should attempt to understand per kilogram of stump and root chloropicrin in field soils could be beneficial or detrimental biomass. monitored with tomato, and interactions which may be natural populations of rhizobia produced. Our approach allows us could be assessed by examining the to assess these possible beneficial Choice of bio-response parameters roots of the alfalfa. or detrimental effects. Bio-response assessment was initiated in the spring of 1989. Spatial scales Experimental site Five major component groups of organisms were assessed; the Within the 0.4 ha circular plots, When the experimental site above ground plant community, responses were monitored on was cut early in the fall of 1988, detrital foodweb organisms, several spatial scales, as well as see Part 1. of this study, p. 81) soil mycorrhizal colonization of over time. consisted of a moss (bryophyte) Douglas-fir roots, and the response Aboveground higher plant layer overlying poorly developed of chloropicrin-sensitive plants. responses were assessed each litter, and fermentation layers, with These assessments will continue spring and fall by monitoring a 1-5 cm depth humus soil horizon until the fall of 1991. percent cover of each species resulting from the rapid present, decomposition rates in these Soil foodweb organisms systems. When soil development respond more rapidly than plants in the entire plot, is limited, nutrient cycling is to environmental change and usually tightly coupled to organism disturbance. Responses of in six 2 m X 3 m plots dynamics (Read and Birch, 1988; bacteria and fungi often reflect arranged sequentially at 1, 2 and 3 Perry et al. 1989). When litter day-by-day fluctuations in meters from four individual stumps falls to the forest floor, it is rapidly temperature, moisture, grazing, at the edge of the plot, and decomposed, and the nutrients and nutrient availability, and thus converted to microbial biomass. are not useful as measures of (3) in six 1 meter square plots These nutrients are then released ecosystem response to disturbance. located between two stumps within by arthropod, nematode and However, by examining changes in 2 meters of each other. protozoan grazing of decomposers, activity, in ratios of fungal to resulting in high soil fertility and bacterial biomass, or important maximum nutrient availability to populations of microorganisms, Detrital foodweb responses plants during times of rapid plant longer term impacts on the system (numbers and activity of bacteria, utilization (Coleman 1985). can be assessed. active and total fungal biomass, However, with clearcutting, much and numbers and community of the thin soil layer was destroyed Protozoa, nematodes and structure of protozoa, nematodes, and mineral soil revealed. microarthropods are intimately and microarthropods) were involved in nutrient cycling monitored over time. Samples The location of each infected (Coleman 1985) and thus are good were taken mid-spring, early stump in the clearcut stand and indicators of ecosystem health 86 PESTICIDES IN NATURAL SYSTEMS: Ingham, et. al. summer, late summer, and mid- microscopic viewing (Darbyshire, stand. The blocking initiated at fall each year. et al. 1974). Nematodes were the beginning of the study based assessed by Baerman extraction on Phellinus inoculum density in To determine if soil organism and microscopic observation stumps reflected changes in soil numbers, activity or community (Anderson and Coleman, 1977). organism community structure, structure changed on a whole plot Microarthropod numbers were and was related to surface soil spatial scale, twelve random points assessed by high efficiency characteristics. We use these were sampled in each of five plots Tullgren extraction and block effects as covariates in per treatment on the four sample observation (Merchant and statistical analyses. dates each year. The same four Crossley 1970). random sets of coordinates were Pesticide application did not designated in each third of the plot Chloropicrin-sensitive bioassay impact establishment, growth or to maximize coverage of the area. plants (tomatoes and alfalfa) were survival of any plant species in the planted each year in the spring at first year after application of 1 and 2 meters distance from chloropicrin. To determine if a localized stumps of five trees of each effect of chloropicrin treatment treatment type and were randomly In the first year, chloropicrin occurred, soil samples were taken placed in another five plots of each impacted soil bacteria, fungi, at 4 distances (0.5, 1.0, 1.5, and 2.0 treatment. Survival was assessed protozoa, and nematodes only in a m) along three equally spaced on each sample date. In the few isolated points near stumps: radii extending from tree stumps second year, a ring of alfalfa was area 8, point 2, 20% (five separate tree stumps per planted at 1 meter distance from chloropicrin treatment, treatment). While soil samples for each stump. All alfalfa plants microarthropods were taken from were examined for N-fixing plot 2, 20% treatment, 2.0 each point, samples for the other bacteria nodules on their roots at distance, organisms were bulked by the end of the second year. plot 602, 100% treatment, 2.0 distance. Since it was chloropicrin distance, and treatment, not soil heterogeneity, that was being assessed, averaging RESULTS plot 736, 100% treatment, 2.0 the differences resulting from soil distance. microsite heterogeneity was Chloropicrin application was acceptable. not the only environmental When these single points were variable to which these sites were averaged with all five replicates On each sample day, the actual responding. Two extremely from a treatment, variance was point from which soil was removed important correlated variables significantly increased, but no was repositioned from the original were (1) removal of canopy cover significant treatment effect was marker by pre-determined and (2) compaction of the soil by observed. distances. A 7.5 cm diameter, 7.5 heavy machinery. All the cm deep sample of soil was experimental sites were exposed to In these isolated cases, removed from each point for both, either of which may have reductions in numbers of microarthropod estimates. An ecosystem effects equal to or organisms were considerable, from approximately 2 cm diameter, 5 greater than the chloropicrin around 101 , or 10 million total cm deep soil sample was taken treatments. The three types of number of bacteria per gram soil from each point, but soils from plant plots, the two types of to less than 100,000 per gram soil each of the three quadrants (i.e., 4 detrital foodweb organism plots, in impacted points. Fungi soil samples) were bulked for placement of chloropicrin- normally measured about 600 m of bacteria, fungi, nematodes, and senstitive plants, and placement of hyphae per g with between 10 and protozoa assessments. Douglas-fir seedlings to assess 50% of those hyphae active, mycorrhizal colonization allowed dependent on season. In impacted Active bacteria, active fungi, assessment of bio-responses to areas, less than 5 m of hyphae per and total fungal biomass were these environmental variables. g were found, with no active assessed by the FDA method of hyphae present. Protozoa tended Ingham and Klein (1984). Total In addition to compaction and to number around 10,000 per g bacterial numbers were assessed canopy removal, we observed a soil, but in impacted soils, were by FITC staining (Babiuk and gradient of organism numbers and less than 10 per g. We have not Paul, 1970). Protozoa were community structure from north to finished assessing protozoan determined by MPN and direct south and east to west in this community structure, but no

HOW CAN THEIR EFFECTS BE MONITORED? 87 NONTARGET BIORESPONSE TO CHLOROPICRIN: 2. EVALUATION immediately obvious changes were There was no difference The increase in plant-feeding detected in the first year. In between survival of tomato plants nematodes has coincided with control soils, nematodes tended to in year one in any treatment, but increase in exotic weed species on number around 100 per 5 g soil, in year two, more tomato plants these sites. Plant-feeding but in impacted soil, less than 10 died in the 100% treatments than nematodes may be attacking the per 5 g soil were found. However, in other treatments. There was no roots of these weedy species and no significant changes in nematode significant difference in nodulation thus be reducing competition community structure were of alfalfa roots in any plot in any between the weed species and observed in the first year. year. Douglas-fir seedlings. In areas impacted by chloropicrin, it may Microarthropod community be that the weeds dont suffer structure has been assessed for the CONCLUSIONS limitation by root-destroying first year, and it is clear that nematodes and the Douglas-fir though individual species respond In the first year after seedlings will experience increased both positively and negatively to a chloropicrin application, reductions competition. We will be able to chloropicrin-fumigated in organism numbers in a few assess this interaction by continued environment, these responses were isolated points and changes in examination of the soil and the not of great enough magnitude to individual species of plant communities over time. radically alter total densities or microarthropods were significant biomass either around individual on the spatial scale of a single All of our information supports trees, or in whole plots. However, stump. These impacts could be the conclusion that very little in tree-centered samples, guild important to a new seedling trying chloropicrin escaped from roots in diversity was decreased in the 20% to obtain nutrients from the soil in the first year. Only at a few treatments, and comparisons of an impacted area. On an larger points, not significant on an individual species showed that 15 spatial scale, such as the 0.4 ha ecosystem scale, were effects of the commonest species plots, and certainly on an detected. In the second year, decreased with increasing dosage, ecosystem-level, the impact was responses of soil organisms and 8 increased with dosage, 2 were not significant, based on plant chloropicrin-sensitive plants highest in 20%, and 3 were lowest response (no impact on plant provided information that more in 20% chloropicrin treatments. community structure or on chloropicrin escaped from roots, The commonest species of chloropicrin-sensitive bioassay but still not enough to result in an springtail was unaffected by plants) or on numbers of bacteria, effect on the plant community. chloropicrin, whereas most fungi, protozoa, nematodes or Early warning that potential effects oribatid species were affected, microarthropods. may occur is being provided by the although increases and decreases soil organisms, but we dont have tended to cancel out overall Will the impact be detrimental the database to tell us if this effects. Coupling these analyses or beneficial, in the long term? In means an overall detrimental with covariate information the second year, numbers of plant- effect on the ecosystem, or an (removal of canopy cover, feeding nematodes have increased overall beneficial effect on the compaction, plant diversity, etc.), from barely detectable numbers ecosystem. should lead to identification of a present in soil to comprising up to small set of indicator taxa that can 50% of the nematode population We know that the fungus that be used for more efficient in some samples in the second causes laminated root rot is being monitoring of similar situations. year. This could be detrimental to killed in these stumps (Thies and Douglas-fir seedlings, but this is a Nelson, 1985). Within the stump, In the second year, the areas stand-wide effect, not an effect of other organisms are being killed, impacted in the first year chloropicrin application. In fact, and it is likely that wood expanded, and four to five new those points impacted by the decomposition is being slowed. Is points of impact were observed. chloropicrin have below-detection that positive or negative? We The position of newly impacted level numbers of nematodes, and dont know. What is the balance points appear random. Complete so, chloropicrin application could sheet going to indicate in ten analysis of variance has not been be beneficial for Douglas-fir years? Will the detrimental effects performed at this time, because seedlings growing in those areas, outweigh the positive? not all samples have been because the plant-feeding completely analysed. nematodes have been negatively Whatever happens in this impacted in those places. particular ecosystem, however, the type of sampling being done, 88 PESTICIDES IN NATURAL SYSTEMS: Ingham, et. al.

.limited as it is, allows biologically between another system and this Fitter, A.H. (ed.) Ecological meaningful conclusions to be made one must be understood in Interactions in Soil: Plants, about the impact of this pesticide predicting possible impacts. Microbes and . Brit. Ecol. in this ecosystem. This approach Soc. Special Publication #4, of assessing bio-responses gives us Blackwell, Oxford. the ability to make predictions COOPERATION about the possible trajectories this Perry, DA., M.P. Amaranthus, J. ecosystem may take. We have the The following organizations are G. Borchers, S.L. Borchers and R. means to make useful predictions, cooperating in support of this E. Brainerd. 1989. Bootstrapping and by looking at this suite of study. Simpson Timber Co.; Great in ecosystems: Internal interactions organism responses, we can Lakes Chemical Co.; National largely determine productivity and indicate problem areas before a Agricultural Pesticide Impact stability in biological systems with situation may result in irreversible Assessment Program (NAPIAP), strong positive feedback. loss of a particular habitat or US Department of Agriculture; Bioscience 39:230-237 species. Pacific Northwest Research Station, US Department of Read, Di. and C.P.D. Birch. So, is chloropicrin use Agriculture, Forest Service; and 1988. The effects and implications detrimental? If the points where the departments of Forest Science, of disturbance of mycorrhizal organism numbers have been Botany and Plant Pathology, and mycelia systems. Proc. Royal Soc. negatively impacted dont spread Entomology, Oregon State (Edinburgh) 94B:13-24. farther than they have in the University. second year, and if the pathogens Coleman D.C., Oades J.M. and dont cause a problem as impacted Uchara G. 1989. Dynamics of Soil areas are re-colonized, the SELECTED LITERATURE Organic Matter in Tropical liklihood is that the pesticide Ecosystems. NifTAL, University should continue to be registered Anderson, R.V. and D.C. of Hawaii Press, Honolulu. for this use, with the clear Coleman. 1977. The use of glass explanation that higher doses, microbeads in ecological Cromack, K., B. L. Fichtcr, A. R. applied in a different manner, experiments with bacteriophagic Moldenkc, J. A. Entry, and E. R. might be detrimental. However, nematodes. J. Nematol. 9:319-322. Ingham. 1988. Interactions impacted areas are likely to between soil animals and continue to expand, because not all Babiuk L.A. and Paul EA. 1970. ectomycorrhizal fungal mats. In: the pesticide has volatilized from The use of fluorescein isothiocya- C. A. Edwards (ed), Proceedings the stumps. How far will the nate in the determination of the of the workshop on interactions affected areas expand? Will bacterial biomass of a grassland between soil-inhabiting pathogens colonize the center of soil. Can. J. Microbiol. 16:57-62. invertebrates and microorganisms these impacted areas and cause in relation to plant growth. Ohio problems? Will we select for Borchers, J.G. and D.A. Perry. State University, Columbus. worse disease problems by using 1989. Organic matter content and this pesticide? Once all the aggregation of forest soils with Darbyshire, J.F., R.E. Wheatley, chloropicrin is out of the stumps, different textures in southwest M.P. Grcaves, and R.H.E. Inkson. how long before affected areas will Oregon clearcuts. pp. 245-250. In: 1974. A rapid micromethod for be re-colonized by the normal DA. Perry (ed.). Maintaining the estimating bacterial and protozoan organisms? Or will these areas be Longterm Productivity of Pacific populations in soil. Ecology pushed into completely different Northwest Forest Ecosystems. 61:764-771. ecosystem trajectories, similar to Timber Press, Portland, Oregon. what has occurred in some plots in Himelrick, D. G. 1986. The effect southern Oregon with completely Castro, C. E., R. S. Wade, and N. of methyl bromide and different disturbances (Borchers 0. Belser. 1983. Biohalogenation: chloropicrin soil fumigation on 1 and Perry, 1989)? Answers to The metabolism of chloropicrin by strawberry (Fragaria ananassa) these questions are not available. Pseudomonas sp. J. Agric. Food yields. VA. J. Science 37:23-27. 1 But continued monitoring in this Chem. 31:1184-1187. system will allow these questions Ingham E.R. and Horton KA. to be answered for this system. Coleman, D.C. 1985. Through a 1987. Bacterial, fungal and proto- Extrapolations can be made to ped darkly an ecological zoan responses to chloroform other systems with the assessment of root-soil-microbial- fumigation in stored prairie soil. understanding that differences faunal interactions. pp. 1-21. In:

HOW CAN THEIR EFFECTS BE MONITORED? 89

,,,, t `, NONTARGET BIORESPONSE TO CHLOROPICRIN: 2. EVALUATION

Soil Biology Biochemistry alcohol, Vapam, or Vorlex. Can. J. 19:545-550. Mughogho, L. K. 1968. The For. Res. 12:528-532. fungus flora of fumigated soils. Ingham E.R. and Klein DA. Trans. Brit. Myco. Soc. 51:441-459. Thies, W. G. and E. E. Nelson. 1984. Soil fungi: Relationships 1987. Reduction of Phellinus weirii between hyphal activity and Ono, K. 1985. The application of inoculum in Douglas-fir stumps by staining with fluorescein diacetate. soil sterilants for controlling the fumigants chloropicrin, Vorlex, Soil Biology Biochemistry tobacco wildfire and angular leaf or methylisothiocyanate. Forest 16:273-278. spot caused by Pseudomonas Science 33:316-329. syringae pathovar tabaci. Bull. Ingham E.R., Trofymow JA, Okayama Tob. Exp. Stn. pp. Trappe, J. M., R. Molina, and M. Ames R.N., Hunt H.W., Morley 93-100. A. Castellano. 1984. Reactions of C.R., Moore J.C., and Coleman mycorrhizal fungi and mycorrhiza D.C. 1985. Tophic interactions Peterson, G. W. and R. S. Smith, formation to pesticides. Ann. Rev. and nitrogen cycling in a semi-arid Jr. 1975. Forest nursery diseases Phytopatho. 22:331-59. grassland soil. II. System responses in the United States. USDA Ag. to removal of different groups of Handbook No. 470, 125 p. soil microbes or fauna. Journal of Applied Ecology 23:615-630. Rhoades, H. L. 1983. Efficacy of soil fumigants and nonfumigants Jones, K. and J. W. Hendrix. for controlling plant nematodes 1987. Inhibition of root extension and increasing yield of snap beans in tobacco by the mycorrhizal (Phaseolus vulgaris). Nematropica fungus Glomus macrocarpun: and 13:239-244. its prevention by benomyl. Soil Biol. Biochem. 19:297-300. Sakuwa, T., H. Miyagawa, and H. Koganezawa. 1984. Effect of Martin, J. K. and J. R. Kemp. chloropicrin applied mechanically 1986. The measurement of carbon for control of Helicobasidium transfer within the rhizosphere of monua, causal fungus of apple wheat grown in field plots. Soil violet root rot and its evaluation by Biol. Biochem. 18:103-108. using a susceptable plant, Medicago saliva. Bull. Fruit Tree McGraw, A. C. and J. W. Hendrix. Res. Stn. Ser. C.(MORIOKA) vol 1986. Influence of soil fumigation 0(11):39-48. and source of strawberry (Fragaria ananassa) plants on population Sumner, D. R., Dowler, C. C., densities of spores and active Johnson, A. W., Chalfant, R. B., propagules of endogonaceous Glaze, N. C., Phatak, S. C. and mycorrhizal fungi. Plant Soil Epperson, J. E. 1985. Effect of 94:425-434. root diseases and nematodes on yield of corn (Zea mays) in an Merchant, V.A. and DA. Crossley, irrigated multiple-cropping system Jr. 1970. An inexpensive high- with management. Plant Dis. efficiency Tulgren extractor for soil 69:382-387. microarthropods. J. Georgia Entomol. Soc. 5:83-87. Tanagawa, S., T. Irimajiri, and M. Oyamada. 1985. Persistence of Moldenke, A. R. and B. L. Fichter. chloropicrin in soil and 1988. Invertebrates of the H. J. environmental effect on it. J. Andrews Experimental Forest, Pestic. Science 10:205-210. western Cascade Mountains, Oregon: IV The orbatid mites Thies, W. G., and E. E. Nelson. (Acari: Cryptostigmata). USDA 1982. Control of Phellinus weirii in Forest Service, Pacific Northwest Douglas-fir stumps by the Research Station, General fumigants chloropicrin, ally Technical Report PNW-217, 112 p.

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