BIOLOGICAL CONTROL Calibration of a Sunlight Simulator for Determining Solar Stability of Bacillus thuringiensis and Nuclear Polyhedrovirus

2 3 4 MICHAEL R. McGUIRE!, ROBERT \\T. BEHLE , HOLLY N. GOEBEL , A,,,n TED C. FRy

Bioactive Agents Research Unit, USDA-ARS, 1815 N. University, Peoria, II.. 61604

Environ. EntomoI. 29(5): 1070-1074 (2000) ABSTRACT The effect of light on survival of entomopathogens is well described and efforts are underway to develop formulations that may protect an entomopathogen from damage by sunlight. The availability of solar simulators allows for year-round testing ofsolar protectants. A commercial formulation of Bacillus thuringiensis Berliner and an unformulated baculovirus isolated from Ana­ grapha fakifera (Kirby) were exposed to various amounts oflight from a solar simulator or the sun to determine the relative effect of each source on loss of insecticidal activity. Rate of pathogen degradation was essentially the same for both light sources when original activity remaining was 2 regressed against total energy (as measured by joules/m ). The amount of time required to reduce activity was different, however, because of a difference in total energies produced by the solar simulator and natural sunlight. Virus was approximately two times more sensitive to light than bacteria. To obtain 50% reduction ofvirus activity, exposure to 1.8 X 107jouleswas required, whereas 3.2 X 107 joules was necessary to achieve a similar loss ofactivity for B. thuringiensis. The importance of reporting energy levels from various solar simulators is discussed.

KEY WORDS Light, Solar Simulator, Entomopathogen, Residual Activity

IT IS WELL known that entomopathogens lose activity used by Pozsgay et al. 1987) and the Atlas CPS Suntest when exposed to sunlight. Numerous studies and re­ that we currently use. These machines use a xenon views (e.g., Ignoffo 1992) have examined the effect of light source and glass filters are inserted between simulated sunlight, UV light, and natural sunlight on the light bulb and exposure surface to provide an the activity ofbacteria, viruses, and fungi (e.g., Pozs­ energy spectrum that is a reasonable approximation of gay et al. 1987, Shapiro and Robertson 1990, McGuire sunlight. Light sources that emit a narrower spectrum et al. 1996, Fargues et al. 1997, Ignoffo et al. 1997). of light may not necessarily prOvide as complete a Currently, efforts are underwayin severallaboratories response as full spectrum simulators. However, even and companies to develop formulations for ento­ full spectrum simulators may give misleading results mopathogens that can protect the microorganisms unless itis known how accurately sunlightis mimicked from the detrimental effects of sunlight in the field. and whetherthe differences in the intensity ofvarious The use of artificial light generated from a variety of wavelengths affect the entomopathogen dispropor­ sources allows for year-round testing ofmaterials that tionately. may protectentomopathogensfrom solar degradation. The purpose ofthe research reported herein was to Because light sources vary from expensive manufac­ compare the effect of the Atlas Suntest CPS solar tured products to laboratory-built devices, it is essen­ simulatorwith the effect ofnatural sunlight on survival tial to know the relationship between natural sunlight of a formulation of Bacillus thuringiensis Berliner and intensity and the energy generated by other light an unformulated nuclearpolyhedrovirus isolated from sources. Anagrapha falcifera (Kirby) (AfMNPV). The recent availability ofaffordable light generators that mimic sunlight in intensity and spectrum has Materials and Methods made itpossible to conduct indoor solar stability stud­ ies. Examples include the Oriel solar simulator (as Entomopathogens. B. thuringiensis was supplied by Abbott (Chicago, IL) as formulated Dipel 2X. For­ This article reports the results of research only. Mention of a mulated material was chosen because it spreads more proprietary product does not constitute an endorsement or recom­ evenly on a leafsurface than technical powder. It is a mendation by the USDA for its use. I Current address: Western Integrated Cropping Systems Research spore crystalpreparation with32,000 IUt mg. Although Unit, 17053 Shafter Avenue, Shafter, CA 93263. formulation ingredients may help protect insecticidal 2 To whom reprint requests should be addressed. activity from sunlight, the purpose of these experi­ 3 Current address: Department of Cell and Molecular Physiology, ments was to compare light sources, not formulations. University of North Carolina, Chapel Hill, NC 27514. 4 Current address: University of Missouri Extension of Miller A suspension of 0.5 mgtml water that provides =85% County, P.O. Box 20, Tuscumbia, MO 65082. mortality in neonate Ostrinia nubilalis (Hubner) October 2000 McGUIRE ET AL.: CALIBRATION OF A SUNlJGHT SIMULATOR 1071 when spread onto a leafsurface (see below) was used for testing. Virus originally isolated from A. fakifera was obtained from biosys (now Thermo Trilogy, Co­ lumbia, MD) and propagated in our laboratory by feeding third-instar Trichoplusia ni (Hubner). After 1 wk, virus was harvested by blending larvae, illtering through two layers ofcheesecloth, rinsing the cheese­ cloth and filtering again. A virus pellet was obtained after centrifugation and the number of polyhedral inclusion bodies (PIB) was determined using a Neubauer hemacytometer (Hausser Scientific Com­ pany, Horsham, PA). Although cleaned, the virus preparations still retained remnants of the host that 4DG 5()() soo iCO c:l-J 900 100Q 11O'J may affect solar stability. Virus was stored frozen until Wavelength (ran) 6 needed. A suspension of3 X 10 PIB/ml water, which Fig. 1. Comparison of irradiance from sunlight and two causes =85% mortality to neonate T. ni larvae, was settings on an Atlas CPS Suntest solar simulator. (a) Irradi­ used for testing. ance from sunlight. (b) Irradiance recorded at setting5 (used Test Plants. Cotton (cultivar'DES 119') was used for exposure of virus) (c) Irradiance at setting 7.5 (used to =30 d after planting. Two plants were grown in each expose B. thuringiensis). 15-cm-diameter pot to =40 em tall and all but the uppermost two fully expanded leaves were trimmed to light, they were kept in the laboratory under am­ from each plant. These two leaves were flat and ori­ bient fluorescent light. To correct for loss of activity ented parallel to the ground. Plants were rinsed with causedby ambient light on leafsurfaces, treated plants tap water to remove debris and plant exudates that not exposed to either light source were kept under 2 may interfere with larval survival. Circles (32 cm ) similar conditions. Leaves from these plants were used were marked on the leaves and 100 ILl of pathogen in bioassays to determine original activity for each suspension was applied to the circles and spread assay. For sunlight exposures, plants were moved out­ evenly with a glass rod. For B. thuringiensis, this cor­ side at =0700 hours and brought back into the labo­ responds to 1,600 ill/ cm2 and for virus, 1.1 X 104 ratory no later that 1600 hours. For each replication, 2 PIB/ cm . Preparations were allowed to dry before the bioassay with all leafdisks was initiated on the day exposure to natural or simulated light. after the last exposure. Light Sources and Detectors. A Suntest CPS (Atlas, To quantify the amount of energy received by the Gainesville, FL) was used for solar simulation. An lJ leaf surfaces, a U-1800 scanning spectroradiometer 1400 pyranometer (LiCor, Lincoln, NE) that mea­ (LiCor) was used to measure theirradiance from each sured energy from 250-310 nm was used to determine light source. Irradiance is a measure ofthe radiometric 2 that no light below 300 nm was emitted by the simu­ flux perunit areaexpressed in W / m / nm (Ryer1997). lator. To avoid leaf burning, the floor of the solar The spectroradiometer was programmed to measure simulator was removed and the machine was raised irradiance every 2 nm between 300 and 1,100 nm. above the laboratory bench and supported by a spe­ Readings were taken twice and the average was used cially prepared stand. Temperature under the light for data analyses. To measure irradiance from the solar was =35"C. Potted plants were then placed under­ simulator, the spectroradiometer was placed below neath the machine for exposure (see McGuire et al. the plastic at the same distance from the light where 1997 for a picture ofthe apparatus). A piece ofTefcel the leaves would normally be. Because the solar sim­ (American Duraillm, Holliston, MA) plastic was ulatorproduces a constant light, only one average was placed between the light source and plant leaves. The acquired (Fig. 1). When plants were exposed outside, plastic acted to reduce the heat effects and to reduce the spectroradiometer was placed beside the plants movement of the leaves caused by the cooling fan in and programmed to take irradiance measurements the machine during the exposure period but did not each hour. Each measurement took =1 min to obtain. 2 reduce energy in the UV wavelengths (McGuire et al. To obtain total irradiance (in W/ m ) across a range of 1996). Treatedplants were exposed to natural sunlight wavelengths, a program internal to the spectroradi­ by placing plants outside on mostly cloudless days and ometer was used to integrate readings. Relative exposing them to full sunlight. Temperatures during amounts of irradiance in bands of interest (i.e., UV, exposure did not exceed 33°C. Plants were exposed to visible, infrared) can then be compared among light eitherlight source for varying lengths oftime based on sources (Table 1). In addition, a pyranometer (model the pathogen. In general, plants treated with B. thu­ 200SA, Licor) that quantifies energybetween 400 and ringiensis were exposed for 4,8, or16 h in the simulator 1,100 nm was used to measure sunlight during one of (three replicates) or4, 8, 16, or32h (overa3-dperiod) the days of virus exposure. A data logger (model U­ (four replicates) to direct sunlight. Plants treated with 1000, Licor) was used to record the readings (Table 1). virus were exposed for 1, 2, 3, 4, or 5 h (three repli­ Bioassay. Afterlight exposure, circles wereremoved cates) under the simulator or 2,4,6,8, or 10 h (three from plants and placed individually into a sealing plas­ replicates) or 1, 2, 3, 4, or 5 h (three replicates) to tic petri dish. For plants treated with B. thuringiensis, direct sunlight. When plants were not being exposed 10 neonate European corn borer, O. nubilalis, were 1072 R'NIRONMENTAL R1IlTOMOLOGY Vol. 29, no. 5

2 Table 1. Comparison ofirradiance (W/m ) in different categories of natnralsunlight aod light from ao Atlas CPS Snntest measured with a scanning spectroradiometer aod a pyraoometer, 28 May 1999, Peoria, IL

Spectroradiometer Time of Pyranometer day, hours UV % of Visible %of IR % of Total (400-1,100 nm) 2 (Sunlight) (300-400 nm) total (400-800 nm) total (800-1,100 nm) total (300-1,100 nm) (w/m ) 2 2 2 2 (w/m ) energy (w/m ) energy (w/m ) energy (w/m ) 0700 4.0 3.5 75.5 66.7 33.7 29.8 113.2 21.53 0800 8.8 3.3 180.7 66.9 80.7 29.9 270.2 116.2 0900 14.8 3.6 279.8 67.5 119.7 28.9 414.3 228.9 1000 20.7 3.8 369.6 67.7 155.2 28.5 545.5 323.8 1100 25.6 3.9 438.1 67.6 184.7 28.5 648.4 417.4 1200 28.3 4.0 476.9 67.5 201.1 28.5 706.4 498.6 1300 29.3 4.0 487.8 67.4 206.8 28.6 723.9 466.3 1400 27.9 4.0 468.8 67.4 198.8 28.6 695.6 448.2 1500 24.5 3.9 419.8 67.4 178.7 28.7 623.0 525.6 1600 19.3 3.8 34..2.0 67.5 145.6 28.7 506.9 470.1 1700 13.2 3.5 254.2 67.2 111.1 29.3 378.6 380.3 Simulator Setting 7.5a 26.5 3.0 384.6 43.1 480,4 53.9 891.5 Setting 5.0b 24.3 3.1 321.2 40.7 444.2 56.3 789.6

a Atlas Suntest CPS setting used for exposure of leaves treated with B. thuringiensis. b Atlas Suntest CPS setting used for exposure of leaves treated with baculovirus.

placed in each dish. A total of 10 dishes per exposure from treated leaves that were not exposed to light. The time per replication were set up. Mortality was re­ transformation to original activity remaining was nec­ corded after 3 d incubation at 28°C. The overall per­ essary because ofthe day to day differences in larvae centage ofmortality from the 10 dishes was averaged used for bioassay that resulted in different percentage and then transformed to original activity remaining for mortality from unexposed treatments. Linear regres­ use in regression analysis (see below). For plants sion analysis (Statistix 1997) was done with original treated with virus, 10 neonate T. ni were placedin each activity remaining as the dependent variable and total offive dishes per exposure time per replication. After joules/m2 of exposure as the independent variable. 24 h, six larvae from each dish (30 larvae pertreatment Slopes and intercepts were calculated for each line per replication) were transferred to individual cups (pathogen X light source) individually. Then, for each containing artificial diet and mortality was recorded pathogen, tests were made to determine if slopes or 7 d after initial exposure to leaves. To determine the intercepts were different between the two light effect ofassay conditions on survival, 10 dishes, sources. Forthis test the data were combinedinto one each containing an untreated leaf disk and 10 larvae, model, which allowed for two slopes and two inter­ were set up for each assay employing B. thuringiensis, cepts. Then the fitted slope or intercept parameters and 30 larvae were collected from untreatedleaves for were reduced from two to one and F tests were con­ each virus assay. Ifcontrolmortality exceeded 15% the structed according to the "extra sum ofsquares" prin­ assay was discarded and omitted from analysis. Treat­ ciple (Draper and Smith 1966) to determine ifa single ment means were not corrected for untreated control parameter could describe the data as effectively as mortality. two. A small Fvalue would indicate that the regression DataAnalysis. Because the solar simulator provided parameters for the two light sources were effectively constant light at a flux level higher than sunlight and "the same" and that a single parameter was sufficient. because sunlight intensity varied through the day, the appropriate measure for effect oflight is accumulated joules/m2 rather than time of exposure. The funda­ mentalunit oflight (watt) is defined as arate ofenergy of 1 joule per second (Ryer 1997). For the solar sim­ ulator, which provided a constant light energy, total joules/m2 was calculated simply by multiplying the W/m2 (300-1,100 nm) by the number of seconds of exposure. However, because intensity ofsunlight var­ ies throughout the day (Fig. 2), a quadratic regression model was fitted to approximate energy throughout the day. Total joules were obtained by integrating under the curve for each exposure period. Percentage ofmortality for each treatment within a Fig. 2. Irradiance (300-1,100 nm) recorded on a cloud­ bioassay day was transformed to original activity re­ less day in Peoria, II. (28 May 1999), (a) and resulting ac­ maining by dividing each treatment mean of samples cumulation of joules throughout the day (b). Time at first exposed to light by the percentage mortality obtained recording (time 0) is 0700 hours. October 2000 MCGUIRE IT AL.: CALIBRATION OF A SUNLIGHT SIMULATOR 1073

100 110 90 100 go g> 80 c 90 ·S ·S." 'I.'. E 80 E 70 a 0 c 0: 70 0: ...... / .?> .?> 60 ·5 ·S 13 60 =cr 50 ./ < < / 50 iIi 40 ~ ~ Il '> 40 30 g 0> .6> 30 a 20 .. oft a 20 '#. 10 10 0 0 0 3 0.0 0.5 1.0 1.5 2.0 2.5 Joules (><10'ym' Joules (x10'Vm2

Fig. 3. Loss of activity of B. thuringiensis in response to Fig. 4. Loss ofactivity ofvirus in response to sunlight (a, sunlight (a, dotted line and circles) and artificial light (b, dotted line and circles) and artiliciallight (b, solid line and solid line and squares). Joules were calculated between 300 squares). A third regression line (c) was calculated for sun­ and 1,100 nm. See text for details. light energy <1.5 X 107 joules. Joules were calculated be­ tween 300 and 1,100 nm. See text for details. Results Bacteria. Insecticidal activity ofB. thuringiensis sig­ (111.85) were essentially the same (F = 3.08, df == 2, nificantly declined with increasing exposure to light 35; P = 0.06). (Fig. 3). Significant linear regressions were observed In practicality, to get 50% loss ofinsecticidal activity for each light source and accounted for 64% of the of a B. thuringiensis spray deposit, leaves need to be variability in insect mortality from samples exposed to exposed to an accumulation of =3.2 X 107 joules of sunlight and 63% from samples exposed to the solar light (or =9 h at setting 7.5 from the Suntest CPS). simulator (Table 2). When the data were combined Similarly, to get 50% loss of activity, virus deposits into a single model, thus allowing comparisons of would need to be exposed to only 1.8 X 107 joules of slopes and intercepts, the two lines were not signifi­ light (or =6.3 h at setting five from the Suntest CPS). cantly different (overall test for differences in slope and intercept: F = 0.71; df = 2,17; P = 0.50). Thus, Discussion natural sunlight and simulated sunlight exhibited es­ sentially the same linear relationship between loss of The ability to determine the influence of light on activity and total joules of exposure. preparations of microbial pesticides is critical to the Virus. Insecticidal activity of AfMNPV also de· development offormulations designed to provide so­ creased in response to light (Fig. 4), although at a rate lar stability. In addition, knowledge of the half-life of approximately two times faster than B. thuringiensis. microbial pesticide formulations is essential to their Each regression accounted for 73% ofthe variabilityin proper timing of application and use in the field. The insect mortality in response to virus exposed to either emergence of affordable laboratory solar simulators light source. However, the combined regression enables the determination of the effect of light on model indicated that although slopes were not signif­ biopesticide activity on a year-round basis. However, icantly different (F = 0.74; df = 1, 41; P = 0.4), the relationship between loss of insecticidal activity intercepts were significantly different (F= 14.42; df= from sunlight and simulated light must be determined 1,42; P< 0.001). A noticeable change in loss ofactivity to accurately use the simulator as a predictive device. occurred at exposures>1.5 X 107 joules in samples Extensive literature exists detailing very careful exposed to sunlight. This is essentially the same "bi­ studies of the effect of artificial light sources on via­ segmented" survival curve described by Huber and bility ofinsectpathogens. Ignoffo and colleagues have Ludcke (1996) and Jones et al. (1993). Therefore,data done an extensive amount ofwork on the effect of a from exposures>1.5 X 107 joules were omitted (Fig solar simulator on viruses. Although they report that 4, line c) and the combined analysis was repeated. This 1 h of simulated light is equivalent to 4 h natural 6 time, both slope (-6.03 X 10- ) and intercept sunlight (Ignoffo et al. 1997), no mention is made of

Table 2. Linear regre8sion analysi8 of energy of expo8ure and original inHectieidal acti.it}· remaining of B. lhuringknois (Bt) or A. falcifera nuclear polyhedronrn.

Pathogen Light Source R2 Slope X 10 6 (t) Intercept (t) F(df) Bt Sunlight 0.64 - 1.3 (-4.23) 90.7 (10.2) 17.92 (1, 10) Bt Simulator 0.63 -0.8 (-3.42) 76.6 (9.4) 11.72 (1,7) Virus Sunlight 0.73 -4.0 (-8.77) 106.4 (24.7) 76.87 (1,28) Virus Sintulator 0.73 -4.9 (-5.95) 96.7 (12.5) 35.35 (1,13)

All Student t and F tests are significant at P < 0.01. 1074 ENVIRONMENTAL RlIITOMOWGY VoL 29, no. 5 the spectrum ofartificial light used to expose the virus eter when the model was forced through the origin preparations. They do report that UV in the simulator (correlation coefficient = 0.98; P < 0.0001). Although is very similar to UV in natural sunlight (Ignoffo and not a perfect representation, these instruments can Batzer 1971). Similarly, Fargues et al. (1997) report provide a reasonable approximation ofsunlight energy degradation of Paecilomyces jUmosoroseus (Wize) for field studies. Reporting light exposure in terms of A.H.S. Br. & G. Sm. conidia by high pressure metallic total energy and spectra in the future should enable halogen lamps. Although they reported spectral irra­ scientists to more accurately compare, among labora­ diance in the 300-400 nm wavelengths, higher wave­ tories, results of solar stability assays. lengths were omitted. These groups and others (e.g., Shapiro 1985) concentrated almost exclusively on the UV spectra produced by simulators. This begs the Acknowledgments question ofDNA repair mechanisms that may occur at We thank R. Bartelt for assistance with statistics and re­ higher wavelengths thus ameliorating inactivation view of the manuscript and P. Hughes (Boyce Thompson mechanisms as suggested by Huber and Liidcke Institute) for review of the manuscript. M. Trepanier, E. (1996). It may be important to consider these higher Bailey, andK. Girsch provided excellent technical assistance. wavelengths in future research. The Atlas Suntest CPS produces a spectrum oflight References Cited that accurately represents the spectrum observed in natural sunlight in the wavelengths of 300-800 nm Draper, N. Rand H. Smith. 1966. Applied regression anal­ (Fig. 1; Table 1). However, this simulator produces ysis. Wiley, New York. Fargues,J., M. Rougier, R Goujet, N. Smits, C. Coustere, and high spectral irradiance above 800 nm. In fact, the B. Itier. 1997. Inactivation of conidia of Paecilomyces percentage energy represented from 300 to 400 nm fumosoroseus by near-ultraviolet (UVB and UVA) and (UV B and A) is =3% ofthe total energy produced by visible radiation. J. Invertebr. Pathol. 69: 70-78. the simulator; whereas, in natural sunlight, =3.5-4% Huber, J., and C. Ludeke. 1996. UV-inactivation ofbaculo­ (depending on the time ofday) ofthe energy is in the virus: the bisegmented survival curve. Bull. IOBC/WPRS UV spectrum. In the morning and evening, relatively 19: 253-256. less UV occurs than in the middle parts of the day. Ignoffo, C. M. 1992. Environmental factors affecting persis­ Presumably, more UV is reflected or absorbed by the tence of entomopathogens. Fla. Entomol. 75: 516-525. atmosphere when the sun is closer to either horizon. Ignoffo, C. M., and O. F. Batzer. 1971. Microencapsulation and ultraviolet protectants to increase sunlight stability of This could, in part be responsible for very little loss of an insect virus. J. Econ. Entomo!. 64: 850-853. activity of our virus preparations exposed to sunlight Ignoffo, C. M., C. Garcia, and S. G. Saathoff. 1997. Sunlight for the shorter periods. Plants were moved outside stability and rain-fastness of formulations of Baculovirus between 0700 and 0800 hours and the first samples heliothis. Environ. Entomol. 26: 1470-1474. were taken 1 or 2 h later. Although the UV wave­ Jones, K A., G. Moawad, D. J. McKinley, and G. Grzywocz. lengths make up a relatively small fraction ofthe total 1993. The effects of natural sunlight on Spodoptera lit­ energy in sunlight, the photons have much more en­ taralis nuclear polyhedrosis virus. Biocontrol Sci. Tech­ ergy than those in higher wavelength bands (Ryer no!. 3: 189-197. 1997). Therefore, even a small shift in percentage of McGuire,M. R., B. S. Shasha, C. E. Eastman, andH. Oloumi­ Sadeghi. 1996. Starch- and flour-based sprayable formu­ energy could make a significantimpact ondegradation lations: effect on rainfastness and solarstability ofBacillus of virus or bacteria and could lead to the observed thuringiensis. J. Econ. Entomol. 89: 863-869. curvature and different intercepts in the virus regres­ McGuire, M. R, L. J. Galan-Wong, and P. Tamez-Guerra sion calculations. 1997. Bacteria: bioassay of Bacillus thuringiensis against It is also important to differentiate between time of lepidopteran larvae, pp. 91-99. In L. Lacey [ed.], Manual exposure and total energy of exposure. Most studies, oftechniques in insect pathology. Academic, San Diego, conducted outside, use days or hours of exposure CA. when reporting loss ofactivity causedby sunlight. This Pozsgay, M., P. Fast, H. Kaplan, and P. R Carey. 1987. The fulfills most practical expectations for observing the effect of sunlight on the protein crystals from Bacillus thuringiensis var. kurstaki HD1 and HD12: a raman spec­ effects oflight exposure on loss ofactivity. However, troscopic study. J. Invertebr. Pathol. 50: 246-253. when comparing different sites, times ofyear, or solar Ryer, A. 1997. Light measurement handbook. International simulators, the use of total energy is more helpful in Light, Newbury Port, MA making meaningful comparisons. A variety of equip­ Shapiro, M. 1985. Effectiveness ofB vitamins as UV screens ment is available for measuring light energy. Because for the gypsy (: Lymantriidae) nucleo­ scanning spectroradiometers are expensive (>10,000 polyhedrosis virus. Environ. Entomol. 14: 705-708. USD) , pyranometers, which measure global solar ir­ Shapiro, M., and J. L. Robertson. 1990. Laboratory evalua­ radiation, are commonly available, inexpensive (less tion of dyes as ultraviolet screens for the gypsy moth than $1,000 U.S.), and can be attached to data loggers (Lepidoptera: Lymantriidae) nuclear polyhedrosis virus. J. Econ. Entomol. 83: 168-172. to provide useful information over the course of an Statistix for Windows. 1997. User's manual. Analytical Soft­ experiment. A LiCor 200SA pyranometer recording ware, Tallahassee, FL. light in the range of 400-1,100 nm during one of the days of exposure (Table 1) provided readings that Receivedfor publictition 2 ~(n:'e1nber 1999; accepted 15 June were Significantly correlated with the spectroradiom- 2000. Supplied by Ul.Sa Dept IIJf Agriculture National Cemer for Agricultural

Utilization Research? Peoria F Illinois