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Crop Science 42:859-868 (2002)
© 2002 Crop Science Society of America

TURFGRASS SCIENCE

UV-B Biodosimetry in Turfgrass Canopies

G. Y. Yuen*,a, C. C. Jochumb, L. J. Gieslerc, M. D. Shulskid, E. A. Walter-Sheae, K. G. Hubbardf and G. L. Horstg

a Dep. of Plant Pathology, Univ. of Nebraska, Lincoln, NE 68583-0722
b Dep. of Plant Pathology, Univ. of Nebraska, Lincoln, NE 68583-0722
c Dep. of Plant Pathology, Univ. of Nebraska, Lincoln, NE 68583-0722
d Dep. of Soil, Water and Climate, Univ. of Minnesota, St. Paul, MN 55108
e School of Natural Resource Sciences, Univ. of Nebraska, Lincoln, NE 68583-0728
f School of Natural Resource Sciences, Univ. of Nebraska, Lincoln, NE 68583-0728
g Dept of Agronomy and Horticulture, Univ. of Nebraska, Lincoln, NE 68583-0724

* Corresponding author (gyuen1{at}unl.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phylloplane microorganisms are affected by ultraviolet (UV) radiation penetrating into plant canopies, but data as to the relationships between microorganism activity and canopy UV levels is lacking. Current instrumentation and modeling systems are inadequate to analyze canopy radiation environments at scales relevant to microorganisms. A biological dosimeter system was developed for measuring UV-B in turfgrass and other compact canopies. Cell suspensions of a DNA repair-deficient strain of Escherichia coli (CSRO6) were enclosed in packets (0.2-mL volume) of UV-transmissible polyethylene. After the packets were exposed to sunlight, numbers of surviving bacteria were determined. The log percent survival was found to be linearly related to accumulative UV-B dosage, as measured with a broad-band UV-B radiometer, but was not related to UV-A dosage. In one experiment, the performance of the biodosimeter system was compared with that of a miniature UV-B radiometer mounted in soil-level tracks in eight plots of tall fescue (Festuca arundinacea Schreb.) that varied in leaf area index (LAI). The two methods yielded similar mean transmittance values that decreased with increasing LAI, closely fitting Beer's law. A similar relationship was found in a second experiment, in which biodosimeter packets were placed at the base of undisturbed tall fescue canopies. The packets also revealed considerable variation in transmittance possibly because of localized shading and sun flecks in the natural canopies. In a third experiment, direct and diffuse UV-B at different heights within a tall fescue canopy was measured by packets attached to narrow, flat wooden sticks simulating grass leaves. This method has potential as a tool to capture the variability in UV levels related to nonuniformity in canopy structure, depth in a canopy, and leaf orientation.

Abbreviations: CFU, colony-forming units • DOY, day of year • LAI, leaf area index • UV, ultraviolet


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MICROORGANISMS (bacteria, fungi, and nematodes) are being developed and used for the control of insect pests, plant pathogens, and weeds. Although most of the effective biological control agents were selected in part for their ability to colonize leaf surfaces, they are often introduced to foreign plant environments and subjected to unfavorable microenvironmental conditions to which they are not adapted. This reduces the applied agent's population size, restricts its distribution on leaf surfaces, and alters its metabolic activity, ultimately causing reduced biocontrol efficacy (Andrews, 1992; Weller, 1988). Ultraviolet radiation is thought to be an environmental factor that limits the survival and growth of microorganisms on the phylloplane (Beattie and Lindow, 1995). Radiation in the UV spectrum accounts for less than 9% of the total incoming solar energy, but it has deleterious effects on all biological systems. Wavelengths shorter than 280 nm (UV-C) are effectively absorbed by the earth's atmosphere, and therefore, only UV-A (320-400 nm) and UV-B (280-320 nm) are considered to be biologically important at the earth's surface (Young et al., 1993). Although there is a greater amount of UV-A than UV-B (roughly 10:1) and both can cause cell death and mutation, UV-B causes more direct DNA damage and thus is the more harmful of the two.

The response of microorganisms to UV varies among taxa. Most bacteria and fungi isolated from leaves are chromogenic, suggesting that pigmentation may be a protective adaptation to UV (Ayers et al., 1996; Sundin and Jacobs, 1999). Genes that confer UV tolerance in some phylloplane bacteria have been identified (Sundin et al., 1996; Willis et al., 1988). Foliar pathogens and phylloplane saprophytes that also have an endophytic existence can survive unfavorable phylloplane conditions such as UV through escape, i.e., gain entry into the plant tissues (Beattie and Lindow, 1995; Wilson et al., 1999). Our understanding of the role of UV in regulating microorganism numbers and distributions on the phylloplane, however, is based primarily on laboratory research. The response to UV by phylloplane microbes, including those used for biological control (Gaugler et al., 1992; Inglis et al., 1995; Ignoffo and Garcia, 1978), was described in numerous reports, but few examined microorganism populations on leaves in relation to natural UV irradiation. In a study by Newsham et al. (1997), numbers of phylloplane fungi on adaxial and abaxial leaf surfaces varied with changes in levels of natural UV radiation and natural radiation supplemented by lamps. In another study, Sundin and Jacobs (1999) found high numbers of pigmented, and thus UV tolerant, bacteria on leaves in the field and higher bacterial populations on abaxial surfaces than on adaxial surfaces. They suggested that UV tolerance and avoidance strategies were necessary for surviving solar radiation. Although UV fluxes above canopies were measured in both studies, UV irradiance on plant surfaces was not assessed. In our own studies on bacterial biocontrol agents of turfgrass pathogens, the relative ability of bacterial strains to colonize field turfgrass was related to their tolerance of UV-A and UV-B irradiation demonstrated in the laboratory (Giesler, 1998). The bacteria, when applied to field turf, typically established higher numbers in the lower canopy region as opposed to the upper canopy, and this differential was negated when the turf was shaded (Giesler et al., 2000). These colonization patterns could not be explained solely by measured leaf wetness conditions, thus suggesting the involvement of a radiation mechanism. However, there were no measurements of the light environment within the turf canopies to support this hypothesis.

The lack of methods to measure and analyze the UV environment within plant canopies at a scale relevant to microorganisms is the primary reason why research on the influence of radiation on phylloplane microorganisms has not been supported by in-canopy measurements of UV. Nonuniformity in canopy structure, differences in depth within a canopy, and the orientation of leaf surfaces to the sun are all factors that affect the light environment at a particular leaf surface, and thus, are important to the establishment of microbes. There is some limited research regarding UV radiation regimes within agronomic crop canopies such as in corn, Zea mays L., (Grant, 1991, 1993), winter wheat, Triticum aestivum L., and soybean, Glycine max (L.) Merr., (Grant, 1993), and alfalfa, Medicago sativa L., (Durr, 1998). The decline of incident radiation with increased depth in canopies has been shown, and models have been developed to estimate irradiance within the canopy given other information. A general form of Beer's law predicts light transmittance in canopies as a function of LAI, which is defined as half the total leaf area per unit ground surface area (Campbell and Norman, 1998). The scale of these predictions, however, are dependent upon the precision of the methods used for assessing LAI. The light environment in grasses was analyzed directly with sensors placed in artificial canopies (Dekmyn and Impens, 1998) and with sensors mounted in subsurface tracks (Durr, 1998). Sensors used for direct measurement of UV levels are either too large for placement in small canopies, such as turfgrass, without disruption of the canopy structure, or too costly for simultaneous, replicated measurements within and across canopies.

Opportunities for direct assessment of UV radiation on leaf surfaces came from the development of biological dosimeters. Comprised of DNA, bacteriophage, or bacteria, biological dosimeters have been employed to measure UV radiation in aquatic (Karentz and Lutze, 1990; Regan et al., 1992; Ronto et al., 1994) and terrestrial environments (Puskeppeleit et al., 1992). In addition to low cost and feasibility of use in most types of field environments, another advantage of biological dosimeters is that they provide measurements that integrate biologically harmful energy across all UV wavelengths. Karentz and Lutze (1990) described the use of Escherichia coli strain CSRO6, a DNA repair-deficient derivative of strain K12, as a UV-B dosimeter in the ocean. We embarked on modifying this bacterial dosimeter system for use in small plant canopies. Our first objective was to design a packaging protocol for placement of strain CSRO6 as a biodosimeter in turfgrass. The primary criteria for evaluating the design were cost, reproducibility, and nondisruption of the plant canopy. Our second objective was to compare the biodosimeter system with an electronic sensor for measuring UV-B. A third objective was to validate the precision of the biodosimeter system in discerning differences in canopy irradiance because of density of leaf cover, leaf orientation, and depth within turfgrass canopies.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culturing and Packaging of Dosimeter Strain
Escherichia coli strain CSRO6 was provided courtesy of D. Karentz, University of San Francisco. The strain contains mutations that render it deficient in photoreactivation (phr-1) and excision repair (uvrA6) (Karentz and Lutze, 1990), and thus, highly sensitive to UV. It was cultured in Luria-Bertani broth (Difco Laboratories, Detroit, MI) in a side-arm flask with shaking at 37°C until the cell population reached mid-log phase. Cells were harvested by centrifugation at 3000 g for 10 min and resuspended in normal saline [0.85% (w/v) NaCl] to 5 x 107 CFU mL-1, as measured by turbidity on a spectrophotometer (Spectronic 20, Spectronic Instruments, Rochester, NY) at 590 nm.

To construct biodosimeter packets, cell suspensions of strain CSRO6 were dispensed into sterile sample bags (Whirl-Pak, Weatherby/Nasco. Inc., Fort Atkinson, WI). The polyethylene used in manufacturing the bags was reported to transmit UV-A and UV-B (Karentz and Lutz, 1990). Transmittance of the batch of sample bags used in our study was confirmed with a spectrometer (Ocean Optics, Inc., Dunedin, FL); the material transmitted approximately 90% of the solar radiation incident on the material, including UV-A and UV-B wavelengths. Double seams were formed down the length and across the width of cell suspension-filled bags with a heat sealer. Care was taken to exclude air bubbles during the process. The procedure produced compartments, each with 150 to 200 mL of cell suspension, which were cut apart into individual biodosimeter packets. Each packet was 2 cm long by 1 cm wide (to approximate the width of grass leaf blades) and 0.2 cm thick (to reduce the potential for shadowing of bacterial cells within the suspension). The packets were stored in the dark at 4°C to reduce cell division and were used within 2 d of assembly. Technicians wore gloves during packet assembly to prevent marring of packet surfaces.

Calibrating CSRO6 Survival to UV-B Dose
Initial experiments tested the response of strain CSRO6 in biodosimeter packets to increasing UV-B dose and evaluated the effects of packet age, packet temperature, and support material. The experiments were conducted from mid-June through mid-September of 1997 at the University of Nebraska-Lincoln East Campus. All exposures occurred within 2 h of solar noon under cloud-free conditions. Before and after exposure, packets were wrapped in aluminum foil to exclude light and kept cool in an ice chest to minimize changes in CSRO6 cell numbers. Packets were placed on the surface of chilled agar in 9-cm Petri dishes to keep them cool during exposure. Alternatively, they were attached by rubber cement to flat, wooden craft sticks (1 cm wide, 0.2 cm thick, and 11.5 cm long; Penley Corp., West Paris, ME), which served as simulated grass blades. The plates and sticks were placed horizontally on level, low-reflective surfaces (wood or bare soil) and exposed to full sunlight. Exposure periods increased in 5-min increments up to 40 min. Control packets were covered with foil during the entire 40-min period. For each duration of exposure, there were three replicate packets. Miniature chromel/constantan thermocouples (Type E, Omega Scientific, Stamford, CT), with 0.08-mm wire diameter, were implanted into several packets filled with distilled water to monitor temperatures during exposure. Temperatures were measured every 10 s and 1-min averages were recorded in a datalogger (CR-10, Campbell Scientific, Logan, UT).

During packet exposure, UV-B irradiance (J s-1 m-2) was measured with a broad band UV-B radiometer (UVB-1, Yankee Environmental Systems, Inc., Turners Falls, MA), hereafter referred to as the YES radiometer, having a spectral response primarily in the 280- to 330-nm range. The horizontal sensor face was positioned 15 cm above the soil surface. Measurements were made every 10 s, and 1-min averages were recorded with a CR-10 datalogger. Irradiance measurements were integrated over time to give a dose value (J m-2) for a particular exposure period.

Cell concentration in biodosimeter packets was determined by dilution plating on tryptic soy agar (Difco) amended with streptomycin at 100 mg L-1. An automated spiral plater (Autoplate 4000, Spiral Biotech, Bethesda, MD) and laser plate reader (Model 500A, Spiral Biotech) were used for plating and colony enumeration. The survival level (S) of CSRO6 within an exposed packet was determined by:

[1]
where Ns and No were the concentrations of viable bacteria in the exposed packet and in control packets protected from sunlight, respectively. The relationship between S and accumulative UV-B dose (J m-2) measured by the YES radiometer was determined by linear regression analysis.

A separate experiment was conducted to determine the relative effects of UV-A, UV-B, and visible light wavebands on viability of strain CSRO6 in biodosimeter packets. Packets placed on chilled agar in Petri dishes were exposed to sunlight for up to 40 min, during which the packets were (i) covered with a filter that blocked UV-B and UV-C (<330 nm; filter 51258; Oriel Corp., Stratford, CT), (ii) covered with a bandpass filter that transmitted UV-B and UV-A (approximately 250 to 390 nm) but blocked longer wavelengths (filter 51650; Oriel Corp., Stratford, CT), (iii) covered with foil to exclude all light, or (iv) left uncovered. The experiment was conducted on DOY (day of year) 163 and repeated on DOY 164, 1997.

UV-B Measurements in Turfgrass Canopies—General Procedures
Experiments in turfgrass canopies were conducted in 1998 at the University of Nebraska-Lincoln Agricultural Research and Development Center, near Mead, NE, in a 5-yr-old sward of tall fescue ‘Kentucky-31’. Plantings were irrigated and fertilized following standard cultural regimes to maintain vigorous growth. Because canopies varying over a wide range of LAI were desired, plots (minimum 2 by 3 m) were maintained at different heights, from 3 to approximately 20 cm. Plots were mowed on different schedules and biodosimeter measurements were also taken at various times after mowing.

Biodosimeter packets, attached to simulated leaf blades (wooden sticks), were inserted into the canopies and exposed for 30 min. Above-canopy UV-B was monitored concurrently with the YES radiometer placed over the same, or adjacent, canopy with the sensor face at 15 cm above the soil surface. The linear relation of S (Eq. [1]) in packets placed above the canopy to accumulative above-canopy UV-B dose (UVBA), generated as described above, was used to estimate in-canopy UV-B dose (UVBC), from in-canopy S data. Estimated UVBC was expressed as transmittance (T), calculated as

[2]

Comparison of Biodosimeter and In-Canopy Radiometer
In one experiment, UV-B transmittance determined by biodosimeter packets were compared with transmittance measured with a miniature (3-cm-diam) UV-B photodiode-based sensor (BW-20-UVB, Vital Technologies, Bolton, ON, Canada), referred to hereafter as the Vital radiometer. The spectral response of this radiometer is primarily in the 275- to 320-nm range. Measurements were taken between 1000 and 1300 h CST under clear sky conditions on 3 d (DOY 230, 237, and 288, 1998) in eight plots varying in LAI from 0.4 to 5.2. The procedure for using the Vital radiometer to measure UV-B irradiance in turfgrass canopies was described by Durr (1998). Briefly, narrow trenches (1.6 m long, 12 cm deep, 7 cm wide), lined with aluminum sheeting, were installed in turf plots at least 2 mo prior to the experiment. A 1-m-long track was placed in a trench, and a sliding carriage, on which the radiometer was mounted, was pulled down the length of the track with a cord. The depth of the track was adjusted so that the radiometer sensor face was level with the soil surface. The carriage was stopped at 5-cm intervals, and a UV-B irradiance measurement (Wm-2) was made at each stop, yielding 20 measurements along the track. Measurements were recorded on a data logger. A track also was installed above the turf in an area away from the in-canopy track for measurement of ambient above-canopy UV-B irradiance. Ambient conditions were measured immediately before and after in-canopy measurements. The transmittance value for the plot was calculated as the ratio of the mean irradiance of the 20 measurements taken along the in-canopy track to the mean of the above-canopy irradiance.

Measurements were taken with biodosimeter packets in the same trench within 24 h of the Vital radiometer measurements, both sets of measurements being collected within 2 h of solar noon. Twenty packets were attached to one face of a wooden meter stick at 5-cm intervals. The assembly was housed in a length of PVC pipe capped at both ends to exclude light and then transported into the field in an ice chest. The stick assembly was removed from the PVC housing in the field and was inserted into the trench with the packets facing upwards and level with the soil surface. The packets were exposed for 30 min and then returned to the polyvinylchloride housing for transport to the laboratory. Transmittance (Tbio) detected by each packet was calculated from S (Eq. [1]) and concurrent above-canopy measurements of UV-B irradiance taken with the YES radiometer as described in Eq. [2]. The transmittance value for the plot was the mean of the 20 measurements taken along the track.

To evaluate agreement between biodosimeter and Vital radiometer measurements, bio and rad values determined within each canopy were plotted against each other, and linear correlation analysis was performed. The methods also were compared by applying analysis of variance of a randomized complete block design to transmittance averaged across the eight canopies, with each set of measurements within a canopy being treated as a block. A difference at the 95% probability level was considered significant.

The LAI of each plot was measured with a leaf area meter (LAI 2000 Plant Canopy Analyzer, LI-COR, Inc., Lincoln, NE) placed in the trench at soil level on the same day as the Vital radiometer measurements. For each of the two methods of UV-B measurement, the relationship between mean transmittance and LAI was derived according to a form of Beer's law:

[3]
in which 100 represents 100% incident UV-B irradiance or dose above the canopy, k is the canopy extinction coefficient, and i represents the method. k, in this case, was the unknown; it was determined by:

[4]

Predicted i values were compared with observed i by regression analysis for evaluation of the fit of the equation to observed data.

Relationship of Transmittance to LAI in Undisturbed Canopies
UV-B transmittance at the soil surface within natural tall fescue canopies as a function of LAI was measured with biodosimeter packets. On DOY 194, 244, and 260 in 1998, at least 20 canopy sampling locations were chosen at random each day. Pairs of packets were placed next to each other on the soil or thatch surface in each location and exposed for 30 min facing upward. S values were averaged for each pair of packets to determine T. The LAI at each sampling location was determined by extracting an 11-cm-diam. sod plug, centered on the sampling location, with a standard cup cutter. All of the aboveground growth on the plug was removed, dried in a 60°C oven and weighed. Dry weight of the foliage was converted to leaf area by means of an equation determined with samples of foliage from the same plot measured with a leaf area meter (LI-3000, LI-COR, Inc.). The Beer's law relationship between T and LAI, and the extinction coefficient k, were determined (Eq. [3] and [4]). In the calculation of k, T values of 0% were assigned the value of 1% to allow for natural log transformation.

Influence of Height and Orientation
Biodosimeter packets were used to measure UV-B dose received by vertically oriented, simulated leaf blade surfaces at different heights within a canopy. The experiment was conducted on DOY 286 and 288, 1998. The canopy was approximately 18 cm tall, and LAI was 2.9, as determined at the base of the canopy with a LAI 2000 Plant Canopy Analyzer. Biodosimeter packets were attached at 5-cm intervals to both faces of 18-cm-long simulated leaves, made by gluing two craft sticks end to end. The simulated leaves were inserted vertically into the canopy such that packets were positioned at 0, 5, 10, and 15 cm from the soil surface and normal to the horizon. One surface of each leaf was oriented toward the sun, and thus, was exposed to direct and diffuse radiation. The opposite leaf surface was exposed only to diffuse radiation. Six replicate measurements were made each day. Horizontally oriented packets placed above the canopy were used to generate the calibration line for calculating UV-B dose from S in the in-canopy packets. UV-B dose measured by an in-canopy (vertical) packet was expressed as a percentage of the above-canopy UV-B dose measured with the (horizontal) YES UV-B radiometer, and thus, was termed a relative transmittance. Relative transmittance data were analyzed by analysis of variance of a split-plot design, with leaf surface and height within the canopy being the main and subplot factors, respectively. Polynomial regression analysis, done by SigmaPlot (Version 3.02, Jandel Corp., Aurora, CO), was used to determine equations that best described the relationship between relative UV-B transmittance and height within the canopy.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Relationship of CSRO6 Survival to UV-B
The survival of strain CSRO6 in biodosimeter packets exposed to full sunlight was inversely related to UV-B dosages measured above the canopy with the broadband YES radiometer (Fig. 1, 2B, and 3) . In 23 experiments, survival levels (S) after 0 to 30 min exposures (corresponding to doses of less than 6000 J m-2) plotted against radiometric dosages yielded linear relationships with coefficient of determination (r2) generally in the range of 0.7 to 0.9 (data not shown). Y-intercepts and regression line slopes varied among days of exposure. Nevertheless, survival levels determined on different days tended to follow a similar relationship with UV-B dose. Pooled data from eight days in 1997, in which dosimeter packets varied in age, revealed a linear relationship between survival and UV-B dose, with r2 = 0.66, P < 0.001 (Fig. 1). When data from packets up to 3 d old and those from older packets were partitioned, the relationship of UV-B dose to the former group (r2 = 0.81, P < 0.001) was closer than that of the older (r2 = 0.41, P < 0.001). Survival values following exposures exceeding 30 min were highly variable and deviated from the linear relationship (data not shown).



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  		Fig. 1. Relationship between survival (S) of E. coli strain CSRO6 after sunlight exposure in dosimeter packets differing in age and solar UV-B dose (D) measured with a YES broadband radiometer. For packets 3 d old or less, n = 103; for packets over 3 d old, n = 128. Data were collected over 8 d in 1997.

 


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  		Fig. 2. (A) Temperatures measured with thermocouples in three dosimeter packets placed above or in a tall fescue canopy; and (B) relationship of survival (S) of E. coli strain CSRO6 in dosimeter packets on chilled agar or on wooden sticks to UV-B dose (D) measured with a YES broad-band radiometer during full sunlight exposure. All measurements were made on DOY 183, 1997. In (A), packets were mounted on wooden sticks. At ‘0’ time (1107 h CST), packet 1 was placed above the canopy while Packets 2 and 3 were placed on the soil surface. At time ‘a’, the positions were interchanged. At time ‘b’ all of the packets were transferred to an ice chest. In (B), exposure began at 1200 h CST and sets of packets were removed from sunlight at 5-min intervals.

 


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  		Fig. 3. Survival (S) of E. coli strain CSRO6 in dosimeter packets following exposure to various wavebands in relation to UV-B dose (D) measured with a YES broad-band radiometer. Packets were exposed to sunlight for up to 40 min while covered with foil (No light), a filter that blocked UV-B and UV-C (+UV-A/no UV-B), a band-pass filter that transmitted UV-B and UV-A but blocked visible wavelengths (+UV-A/+UV-B), or no covering (Full sunlight). The experiment was conducted on DOY 163, 1997. n = 3 at each dose.

 
The survival of CSRO6 in the biodosimeter packets was not affected by field ambient temperatures or by the material used to support dosimeter packets. At solar noon on hot days (air temperature >33°C), temperatures in packets exposed to full sun above plant canopies for 30 min did not exceed 40°C, as illustrated by results from DOY 183, 1997 (Fig. 2A). There were no significant differences in survival levels between packets placed on chilled agar and packets attached to wooden craft sticks used to simulate grass blades, when the units were exposed to full sun at noon that day (Fig. 2B).

Inactivation of strain CSRO6 in biodosimeter packets during sunlight exposure was due exclusively to UV-B. No loss of cell viability was found in packets exposed for up to 40 min (maximum UV-B dose of 5230 J m-2) when they were covered with a filter that blocked UV-C and UV-B wavelengths (Fig. 3). There was no difference in the survival of strain CSRO6 in packets fully-exposed to sunlight as compared with packets covered with a bandpass filter that transmitted UV-B and UV-A only. The same results were obtained when the experiment was repeated (data not shown).

Comparison of Biodosimeter and Radiometer Measurements in Turfgrass Canopies
There was a high positive correlation [correlation coefficient (r) was 0.951, P < 0.001] between transmittance calculated from doses estimated by biodosimeter packets and transmittance calculated from in-canopy irradiance values measured with the Vital radiometer (Fig. 4A) . Variability among the 20 measurements along a track was very high regardless of the method, but generally was consistent across the eight canopies. When transmittance averaged across the eight canopies were compared by analysis of variance, the radiometer yielded a higher mean value (35%) than the biodosimeter (28%), but the difference was not significant (P = 0.07; data not shown).



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  		Fig. 4. UV-B transmittance measured by biodosimeter packets (bio) and a Vital radiometer (rad) at soil level in eight tall fescue canopies: (A) correlation between the methods; (B) relationship of transmittance values to leaf area index (LAI) in each canopy. In A and B, each value is a mean of 20 measurements taken along a 1-m-long track at 5-cm intervals. In A, error bars denote standard deviation. In B, vertical bars indicate the range of measurements, and data are offset on the X scale for clarity.

 
Transmittance values determined by both measurement methods plotted against eight LAI levels followed the Beer's law relationship, with k values determined by the biodosimeter and the Vital radiometer being 0.51 and 0.44, respectively (Fig. 4B). The coefficient of determination (r2) exceeded 0.91 (P < 0.001) for both methods.

UV-B Transmittance in Relationship to LAI in Undisturbed Canopies
Transmittance calculated from biodosimeter estimates of UV-B doses at the soil surface in 68 sites in natural turfgrass canopies decreased with increasing canopy LAI following the Beer's law relationship (r2 = 0.76, P < 0.001), with a k value of 1.19 (Fig. 5) . Nearly all of the deviation of estimated T values from the Beer's law curve was found at LAI <3. Even at low LAI (<1), some assay points fell in shaded areas of the canopy, and thus, the associated transmittance values were very low.



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  		Fig. 5. UV-B transmittance (T) measured with biodosimeter packets at the base of tall fescue canopies as a function of leaf area index (LAI). Each value (n = 68) is a mean of two measurements.

 
Influence of Height and Leaf Orientation
Using dosimeter packets attached to surfaces of simulated grass leaves directed toward and away from the sun, we found a significant (P = 0.03) leaf surface x height interaction in the relative UV-B transmittance. Relative transmittance determined on vertical surfaces facing the sun was higher than on corresponding surfaces facing away from the sun at 10 and 15 cm above the soil in the 18-cm-tall canopy (Fig. 6) . Similar values were found between the two surfaces at the lower heights, with doses received at the soil surface being approximately 5% of that detected horizontally above the canopy. Relative transmittance of direct and diffuse UV-B, detected by packets facing toward the sun, was related to height above the soil surface following a linear relationship, whereas relative transmittance of diffuse UV-B, measured by packets facing away from the sun, was best described by a quadratic relationship.



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  		Fig. 6. Relative UV-B transmittance (T) detected by biodosimeter packets on surfaces of vertical simulated grass blades placed at various heights (H) in a tall fescue canopy of 18 cm height. T was determined relative to UV-B irradiance measured horizontally above the canopy by a YES broad-band radiometer. Each value is a mean of six measurements made in each of 2 d. Error bars denote standard deviation.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrated in this study that it is feasible to use CSRO6 as a biological dosimeter for measuring UV-B transmittance in turfgrass canopies. Karentz and Lutze (1990) showed that UV-A could contribute a small portion to the mortality of CSRO6 upon sunlight exposure. However, we found that UV-A did not affect the survival of CSRO6 in the packets during 40-min exposures. Although we cannot discount a possible influence from UV-A upon longer exposures, it appears that the biodosimeter system was responding to UV-B in this study. We identified several factors that could affect the dose-response relationship, and thus, the accuracy of the biodosimeter system. A separate dose-response calibration line needs to be generated for each 30-min exposure period to have the greatest accuracy. Storage can affect variability; cells within packets clumped and settled and upon extended storage, and thus, best results were obtained when packets were used within 3 d after assembly. Gravitational settling of cells, particularly in vertically oriented packets, might cause errors when packets are exposed for long periods, but is likely a minor factor over short durations. Temperature does not appear to be an important factor when exposures were limited to 30 min, as temperatures during full sun exposure did not exceed 40°C, a level that had no effect on viability of CSRO6 in the laboratory (unpublished data). Under higher temperature conditions or for longer exposures, spores of Bacillus lacking in UV repair (Puskeppeleit et al., 1992) might serve as an alternative to CSRO6. The protocol for assembling the bacterial packets requires considerable hand labor. Methods to mechanize the process are needed to allow rapid mass production of packets and provide greater consistency in packet size and suspension volume.

The biodosimeter system yielded transmittance data comparable to that calculated from UV-B irradiance measured by the Vital radiometer that is among the smallest currently available. Readings with the two methods along any given track in tall fescue varied to a similar extent, the variation being created by localized areas with high sunlight penetration (sun flecks) or more intense shading. Mean transmittance values determined by the Vital radiometer tended to be higher than those of the biodosimeter, perhaps because of spectral range differences between the in-canopy Vital radiometer and the YES radiometer used to calibrate the biodosimeter. In addition, the differences could be related to the Vital sensor being used in canopies when, in fact, it was calibrated in full sunlight. Finally, the lower transmittance determined with the biodosimeter could be due to the spectral response of CSRO6, but this characteristic has not yet been examined.

The biodosimeter system clearly has some advantages over current instrumentation. The low relative cost of the packets allows replicate sampling of many areas within a plant canopy and simultaneous assay of multiple plant canopies. The packet material is more cost effective than other small containment devices, e.g., quartz tubes (Regan et al., 1992), reported in other UV dosimeter systems. The small size of the dosimeter packets allows site-specific measurements with minimal disruption of the canopy structure. Measurements with instruments in turfgrass are difficult without creating artificial canopies, as in Dekmyn and Impens (1998), or installing semipermanent equipment, such as the subterranean tracks in this study and that of Durr (1998), to accommodate the devices. We found using the biodosimeter system that transmittance in natural canopies had a higher extinction coefficient, k, than in canopies with the tracks. In addition, variation in transmittance in natural canopies was greatest at low LAI and was extremely low at LAI levels above 3, whereas variation in canopies with the tracks was consistent across LAI levels. The differences could be attributed in part to the use of different LAI measurement techniques, with destructive sampling used in the former case and an electronic sensor in the latter. We suggest, however, that a more important factor could be the nature of the two types of canopies. In the natural canopies, biodosimeter packets were often placed by chance immediately next to grass stems or under lower, horizontal grass blades, accounting for low transmittance measurements at high LAI. In the tracks, UV-B attenuation was due to over-arching leaves that were at least 3 cm away from any given biodosimeter packet.

The small size of the dosimeter packets also makes it possible to detect variation in transmittance related to canopy height and to leaf orientation. The data we presented in this regard represent snap-shots of conditions in tall fescue canopies. Much more data of this type need to be collected over time and locations before a true picture of the temporal and spatial heterogeneity of UV-B irradiance in turfgrass canopies can be drawn. Our data nevertheless support findings from other studies that minimum and maximum irradiance levels may be as important as the mean for describing the radiation environment in a plant canopy and that diffuse UV-B is an important component in grass canopies (Dekmyn and Impens, 1998; Durr, 1998).

We echo Jagger's (1985) caveat that biological dosimeters can be useful to measure relative amounts of lethal sunlight, but cannot be used to predict lethality to microorganisms in general because microorganisms vary in their sensitivity to different UV wave bands. The development of this biodosimeter system represents a first step in understanding the UV-B environment at the single leaf level; along with data on the sensitivity of microbial strains to solar UV, the impact of solar UV on microbe populations within a particular canopy site eventually can be predicted. In our previous studies in tall fescue, we found that applied bacteria colonize turf canopies in spatial patterns that suggest a UV influence (Giesler et al., 2000). The biodosimeter system will be an important tool in determining the relationships between UV irradiance and colonization by applied microorganisms.


   ACKNOWLEDGMENTS
 
We thank Mischell Craig, Mark Mesarch, Heather Root, Frank Soto, and Lanny Wit for technical assistance. We also thank Dr. Tyler Kokjohn, Midwestern University, for advice and discussions on UV effects on microbes. This research was supported in part by grants from the University of Nebraska Agricultural Research Division Interdisciplinary Research Program and the Nebraska Turfgrass Foundation.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Journal number 13386 in the Univ. of Nebraska Agricultural Research Division journal series.

Received for publication May 16, 2001.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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M. D. Shulski, E. A. Walter-Shea, K. G. Hubbard, G. Y. Yuen, and G. Horst
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