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a University of Nebraska-Lincoln, 448 Plant Sciences Hall, Lincoln, NE 68583-0722 USA
b Univ. of Nebraska-Lincoln, 406 Plant Sciences Hall, Lincoln, NE 68583-0722 USA
c Univ. of Nebraska-Lincoln, 377 Plant Sciences Hall, Lincoln, NE 68583-0724 USA
lgiesler1{at}unl.edu
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Abbreviations: CFU, colony-forming units • LAI, leaf area index • PAR, photosynthetically active radiation • PB, phosphate buffer • RH, relative humidity • UV, ultraviolet
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Within all turf canopies, there also is an internal shading element imposed by the foliage in the upper canopy. The level of internal shading is determined by the leaf area index (LAI; i.e. total leaf surface over a given measurement area) with greater light interception and evaporative barrier occurring under higher LAI. Attenuation of PAR and UV wavelengths in grass canopies with different LAI has been demonstrated (Deckmyn and Impens, 1998). Leaf area index as determined by shoot density was found to affect moisture conditions in tall fescue canopies, and in turn, the severity of brown patch disease caused by Rhizoctonia solani Kühn (Giesler et al., 1996c). Internal shading increases as a function of depth within the canopy, with greater LAI occurring over the soil surface than in the upper canopy region (Rosenberg et al., 1983). Higher humidity levels, longer leaf wetness durations, and more moderate temperatures are predicted in the lower canopy regions compared with the top portions. This may result in foliar pathogens being more active in the lower canopy regions, but the issue of microclimate conditions and microbial populations along the vertical axis has not been investigated in turfgrass canopies.
Given that turfgrass diseases are particularly important in shaded environments, control measures must be effective under those conditions. Biological control agents are being investigated as an alternative to fungicides for the management of foliar turfgrass pathogens (Giesler and Yuen, 1998; Goodman and Burpee, 1991; Hodges et al., 1993, 1994; Kobayashi et al., 1995; Lo et al., 1996; Nelson and Craft, 1991). Bacteria are particularly attractive as potential biocontrol agents for turfgrass pests because of their ease of propagation, compatibility with fungicides, and delivery feasibility through conventional liquid systems (Weller, 1988). Poor survival or colonization by applied biocontrol agents has been reported to be the primary barrier to field efficacy (Spurr and Knudsen, 1985). Repeated applications of biological control agents to maintain effective population levels is compatible with current turfgrass management methods; therefore, long-term colonization by applied strains is not as critical as in other crop systems. Nevertheless, environmental conditions occurring in the turf canopy may affect short-term colonization, and thus it is important to understand the influence of shading on biological control agents. The scarce amount of data pertaining to moisture and UV effects on phylloplane bacteria suggests that bacterial biocontrol agents would also thrive in shaded (and thus, more disease-prone) conditions as opposed to environments with exposure to full sun. However, the effects of shading on applied bacterial population levels and distributions in turfgrass have not been investigated. The objectives of this investigation were (i) to determine the effects of externally imposed shade on turfgrass colonization by applied bacterial biocontrol agents, (ii) to record microenvironmental changes in tall fescue canopies caused by external shading and to identify conditions associated with changes in applied bacterial populations, and (iii) to ascertain the extent to which applied bacterial agents respond to internal shading. Preliminary results have been reported (Giesler and Yuen, 1995; Giesler et al., 1996a).
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Materials and methods |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Shading was imposed on the plots for the duration of each experiment by suspending two layers of black polypropylene shade fabric rated at 50% shade (Hummert Int., Earth City, MO) 1 m above the plot with aluminum tent rods and polyvinyl chloride pipe. The suspended shade cloth resulted in a 2 m wide by 5 m long shadow band. The length of the shade cloth was oriented east to west so that a 1.5 by 3 m strip was permanently shaded inside the plot areas as described above. In 1994 and 1995, there were two and three replicated blocks arranged in a randomized complete block design. Shade cloth was removed only during mowing, which occurred between 1000 and 1800 h.
Plot Maintenance
All experiments were conducted at the John Seaton Anderson Turfgrass Research Facility near Mead, NE. All plots were maintained at an 8-cm height by weekly mowing, and clippings were removed. High soil moisture was provided by daily sprinkler irrigation while the shade cloth was in place, as shade cloth structures were 2 m or more away from the irrigation system heads. In 1994, irrigation was applied for two 10-min intervals between 1900 h and midnight to supply 3.8 cm water wk-1. In 1995, irrigation was applied between 0500 and 0700 h to replace evapotranspiration estimated from weather station data. All plot areas were fertilized monthly from May through September with 50 kg of urea N ha-1 mo-1.
Environmental Measurements
A previously described microenvironmental monitoring system was used to measure canopy RH and canopy air and foliage temperatures (Giesler et al., 1996c). Leaf wetness duration was detected with an impedance sensor similar to one described by Weiss et al. (1989). In addition, incoming solar radiation in both shaded and full sun plots was monitored with a pyranometer with 400- to 1100-nm sensitivity (Model LI-200SZ, LI-COR, Lincoln, NE) set at 0.5 m above the soil surface. Data loggers scanned every minute and averaged values every 10 min.
Instrumentation was available to monitor two plot areas (shaded and full sun) at one time. Therefore, instrumentation was moved from one block to another at 4-d intervals immediately after mowing. The data were analyzed by first calculating summary statistics—maximum, minimum, average, and range (maximum minus minimum)—for each 24-h period. Relative humidity also was analyzed by calculating the duration in which RH exceeded 90% within each 24-h period. Leaf wetness was summarized by calculating leaf wetness duration within each 24-h period. Each 24-h period began at 1700 h, this time being chosen because leaf wetness typically was not present at this time, and therefore, full leaf wetness to drying cycles could be evaluated. Summary data were then analyzed using analysis of variance of a randomized complete block design, with dates considered to be subsamples. All statistical analyses were performed with Statistical Analysis Software (SAS Institute, 1985) using the Proc GLM option.
Data for ambient conditions during the experiments were obtained from a recording weather station located within 100 m of the field plots (High Plains Climate Center, 1994-1995). The data included hourly measurements of RH, air temperature, wind speed, and solar radiation. Summary statistics for ambient conditions were calculated for canopy microclimate conditions.
Bacterial Application and Colonization Assessments
Three bacterial biological control agents, Bacillus megaterium strain B153-2-2 (Liu and Sinclair, 1991), Stenotrophomonas maltophilia strain C3 (Giesler and Yuen, 1998; Zhang and Yuen, 1999), and Enterobacter cloacae strain EcCT-501 (Nelson and Craft, 1991) were applied. They were selected because they inhibited disease caused by fungal pathogens in previous studies and are representative of the diversity of bacterial species being developed for biological control. The strains were cultured on KG medium (Yuen et al., 1991) in 9-cm petri dishes for 48 h at 25°C. Cells were suspended in 0.01 M phosphate buffer, pH 7 (PB) to 
7.5 log colony-forming units (CFU) mL-1. Suspensions of the three strains were combined in equal volumes and then applied to all plot areas using a Solo backpack sprayer (Solo, Newport News, VA). Applications were made on 27 July 1994 and 26 Aug. 1995 at 1500 and 1130 h, respectively.
Population levels of the applied bacteria were monitored during the course of each experiment. In 1994, canopies were sampled by clipping shoots near the soil surface with scissors. Two clippings (1 g dry tissue) were collected at random within a plot and pooled into a subsample; two subsamples were collected per plot and processed separately. In the 1995 experiment, subsamples were collected by removing 11-cm-diam. cores with a cup cutter. All samples were collected between 1100 and 1900 h. The foliage was divided into top and bottom 4-cm fractions, and each fraction was processed separately to assess bacterial populations within the two strata. Three subsamples per plot were collected. Cores were immediately replaced into their original locations, and regrowth from crowns occurred within 1 wk. Day 0 samples were collected after the foliage had dried for 2 and 4 h after application in 1994 and 1995, respectively. Subsequent samples were collected on Day 2 and 8 in 1994, and every 4 d for 16 d in 1995.
Clippings were assayed for numbers of applied bacteria using the plate-dilution frequency technique described by Harris and Sommers (1968) and modified by Yuen et al. (1991). Foliage samples were washed in PB, and then dilutions of the wash were applied to plates of selective agar media. To detect B153-2-2, one-tenth tryptic soy agar (Sigma Chemical, St. Louis, MO) amended with 100 µg mL-1 each of cycloheximide and rifampicin and 25 µg mL-1 polymyxin sulfate was used. Strain C3 was detected on one-tenth tryptic soy agar amended with 100 µg mL-1 each of cycloheximide and rifampicin, 20 µg mL-1 basic fuchsin, and 10 µg mL-1 naladixic acid. The strain EcCT-501 was detected on Ayers, Rupp, and Johnson minimal salts medium (Ayers et al., 1919) amended with 10 mg mL-1 maltose and 100 µg mL-1 each of cycloheximide and rifampicin. All cultures were incubated for 4 to 6 d at 25°C before colony counts were taken. Foliage samples were dried in a convection oven for 48 h at 60°C after bacterial assays were completed. The samples were then weighed so that bacterial population numbers could be expressed on the basis of dry weight of the sample. Data were log10 transformed prior to statistical analysis (Hirano et al., 1982), and all samples with no bacteria detected were defined as having 1 CFU g-1 to permit log transformation. Data from 1994 were analyzed using a split plot design with shade being the main treatment and sampling date being the subplot treatment. In 1995, data were analyzed with a split-split plot design with shade being the main treatment, foliage part being the first split-plot, and time being the second split-plot.
In addition to measuring bacterial population levels, numbers of phylloplane sites colonized by the applied bacteria also were determined. The shoot imprinting method used for this purpose was similar to procedures described for studies on bacterial colonization of leaf surfaces (Hirano and Upper, 1993; Leben, 1965). Ten shoots were collected randomly from each plot and one side of the shoot was gently pressed onto each of the selective agar media to leave an impression. The same media described above were used except that each also was amended with 10 µg mL-1 amphotericin to suppress fungal growth. Plates were incubated at 25°C for 4 to 6 d. Numbers of colonies growing from impressions of plant parts above and below the lowest leaf collar (Fig. 1) , representing upper and lower foliage parts, respectively, were counted. Each colony was assumed to represent a site of colonization. Numbers of colonization sites were determined on Day 0, 2, and 8 in 1994, and only on Day 13 in 1995. Because the colonization site data did not fit a normal distribution according to the Shapiro-Wilk statistic (Shapiro and Wilk, 1965), the data were transformed using a rank transformation (Conover and Ronald, 1981) prior to analysis. This allowed use of parametric methods by replacing the raw numeric data with their ranks. The selection of this transformation was based on comparisons of several methods for analyzing leaf imprint data (Giesler et al., 1996b). Following transformation, colonization site data were subjected to the same statistical analysis as the population size data.
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Results |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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70% and caused changes in other microenvironmental conditions (Table 1)
. In comparisons of shaded and full sun canopies in 1994 and 1995, there were significant differences (P 
0.05) for all temperature parameters (maximum, minimum, average, and range), with the exception of maximum canopy air temperature in 1994. Shaded canopies were cooler during the day and warmer at night (data not shown). Maximum foliage temperatures in shaded canopies were 5°C lower than in full-sun canopies in 1994 and more than 3°C lower in 1995. Minimum foliage temperature in shaded canopies was 1°C higher in 1994 and 1.5°C higher in 1995 than in full-sun canopies. Similar trends were found for canopy air temperature parameters, but the differences among the treatments were not as great in magnitude as those observed for foliage temperatures.
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Influence of Shading on Bacterial Population Levels
Each of the applied bacterial strains was detected in higher population levels in shaded turf than in turf exposed to full sun in one or both experiments (Fig. 2)
. In 1994, only strain C3 was significantly influenced by shading
, with shaded canopies having up to 0.7 log units higher C3 population levels than full sun canopies on all sampling dates. All strains were significantly (P 0.10) affected by shading in 1995. Population levels of B153-2-2, C3, and EcCT-501 in shaded canopies averaged across the experiment were 0.4, 1.0, and 0.3 log units higher, respectively, than in full sun. In 1995, there also was a significant shade x time interaction for all three strains. For strain B153-2-2, differences between shaded and full sun canopies were not apparent until 8 d after application. For strain C3, population levels in shaded canopies were consistently more than 0.5 log units higher than in full sun canopies. Population levels of strain EcCT-501 were higher in shaded canopies on all sampling dates except Day 16, when population levels were equal.
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. Numbers of C3 colonization sites in shaded and full-sun canopies were statistically similar in 1994. In 1995, there were four times as many C3 colonization sites in the shaded canopies as in full-sun canopies
, with an average of 19 colonization sites being detected on shoots in the shade, compared with five sites per shoot in full sun. There were more EcCT-501 colonization sites
in shaded canopies than in full-sun canopies in 1994. The greatest difference was detected on the second day of the experiment, when there were twice as many EcCT-501 colonization sites in shaded canopies as in full sun canopies. Similar results were observed in 1995, when seven more
EcCT-501 colonization sites per shoot were detected in shaded canopies than in full sun canopies.
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in the bottom than the top foliage part of the canopy. However, average C3 population levels across the entire experiment were 0.5 log units higher
in the top foliage part of the canopy. Average population levels of EcCT-501 in the top and bottom canopy regions were similar across the entire experiment, but there was a foliage part x time interaction
. This was because populations decreased more rapidly in the first 8 d in the upper foliage part than the lower foliage part of the canopy.
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for population levels of strain B153-2-2. The greatest difference in population sizes between shaded and full sun canopies was found in the top foliage part, with population levels in the shaded canopies being more than 1 log unit higher than in the full-sun canopies.
Stratification of bacterial colonization also was observed with colonization sites detected by shoot imprinting (Fig. 5)
; however, the results from shoot imprinting were not consistent with the population numbers measured by dilution plating. Numbers of B153-2-2 colonization sites did not vary between the top and bottom canopy regions. There was a significant foliage part x sampling time interaction for strain C3
in 1994. In 1994, colonization sites initially were greater at the upper canopy region, but were greater in the bottom canopy region by Day 8. In 1995, there were more C3 colonization sites
in the top than in the bottom foliage part of the canopy. In 1994, there were higher
numbers of EcCT-501 colonization sites in the upper foliage part initially, but numbers of colonization sites were similar by Day 8 (
for foliage part x time interaction).
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Discussion |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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The 70% reduction in solar radiation caused by the shade cloth in this study is similar to that found under a typical shade tree (Salisbury and Ross, 1992). Although UV was not measured in this study, we would expect UV levels arriving at the shaded turf to be reduced by roughly the same proportion. The involvement of UV radiation also could explain in part why bacterial population levels in shaded canopies were generally higher than in nonshaded canopies. Starting population levels were determined several hours after application. In the interim, solar radiation was high and the UV component may have been deleterious. We did detect changes in canopy temperature and moisture conditions associated with shading. Temperature differentials between shaded and nonshaded turf were comparable with those reported in other types of plant canopies (Blad and Lemeur, 1979). The differences in moisture parameters (i.e., RH and leaf wetness duration) were of relatively low magnitude, which was not what we had expected based on observations in other crops. It is possible that local advection minimized the influence of shading on moisture conditions, as the shaded areas were isolated within large expanses of nonshaded turf. The shade cast was typical of isolated trees or bushes. In landscapes in which shade is associated with topography or tree groups, air mixing would be important primarily at the shade margins. Thus, conditions in the center of the shaded areas could be considerably different than in full sun areas.
It is uncertain whether any one of the measured changes in microclimatic conditions alone could have accounted for the elevated bacterial populations found in the shaded turf. Daytime temperatures in both shaded and nonshaded turf tended to be near the optimum for growth of the applied strains, and nighttime low temperatures did not differ between shading treatments by more than two degrees. Leaf wetness persisted in shaded canopies for 1 h longer than in nonshaded areas; however, the additional period of wetness may not have had a significant effect on bacterial multiplication considering that leaf wetness in both shaded and nonshaded canopies persisted for >10 h. Under growth chamber conditions, multiplication of the same three bacterial strains on tall fescue was not appreciably influenced by leaf wetness durations when 5 to 18 h of wetness were provided (Giesler, 1998). The additional wetness could have aided bacterial dispersal, and thus, could have contributed to the higher numbers of colonization sites being detected in shaded canopies by the shoot imprinting method. This effect would have been particularly important to strains EcCT-501 and B153-2-2, which are motile. Strain C3 is non-motile. Minimum RH levels were the same for shaded and full sun canopies in both years, and thus, desiccation did not appear to be a factor affecting survival. Minimum RH levels were lower during the 1995 experiment, which was performed later in the year than the 1994 experiment. This could have affected population levels and thus account for increased effects of shading in the 1995 experiment. It is unknown, however, how RH could interact with temperatures in relation to desiccation tolerance for these organisms.
Although environmental conditions in the upper and lower canopy regions were not directly measured in this study, results from some of our other studies would suggest that considerable differences exist between the two levels. We found leaf wetness at the 2-cm level to last as much as 3 h longer than at the 5-cm level in perennial ryegrass (Lolium perenne L.) mowed to a height of 8 cm (Giesler, 1998). Using a biological dosimeter to measure UV-B, we found that UV-B transmission into tall fescue canopies decreased with depth along steep gradients (Yuen and Giesler, 1998).
There is insufficient information to determine whether the differential response among the bacterial strains to shading observed in this study was related to the bacterial species or was strain specific. Strain B153-2-2 has not been tested in foliage until this study, as it is a rhizosphere colonizer isolated originally from a soybean (Glycine max L.) root (Liu and Sinclair, 1991). Ultraviolet LD50 values, determined with a solar simulator, were two and four times higher for strains C3 and EcCT-501 than for B153-2-2, respectively (Giesler, 1998). There may be similarities in environmental conditions between the lower turf canopy region and the upper soil layer, as population levels of B153-2-2 found in the lower turf canopy region were similar to those recovered from soybean hypocotyls (5.0–4.5 log CFU g-1 hypocotyl) (Liu and Sinclair, 1993). These population levels also may reflect the ecological competitiveness of B153-2-2, as similar maximum population levels were observed in both environments.
Population trends for strains C3 and EcCT-501 suggest that they are more adapted to phyllosphere existence. The detection of strains C3 and EcCT-501 in the upper canopy regions in numbers equal to or greater than the lower canopy regions was very surprising. This would suggest that the two strains are highly effective colonizers of grass foliage and might prefer young, green foliage as a niche, as opposed to older, senescing foliage. Population levels of the two strains detected in this study are comparable to those previously reported. In previous studies with strain EcCT-501, which was originally isolated from cotton hypocotyls (Nelson, 1988), populations in bentgrass (Agrostis palustris Huds.)–annual bluegrass (Poa annua L.) putting greens were 6 to 7 log CFU g-1 dry weight in 3-cm-deep cores containing leaves, roots, rhizomes, thatch, and soil up to 12 wk after application (Nelson and Craft, 1991). Strain C3 maintained stable populations of 6 log CFU g-1 foliage on four tall fescue cultivars for a 16-d period (Giesler and Yuen, 1998). These population levels are similar to populations detected in this study in 1995, with populations in 1994 being 1 log unit lower. The difference between years could be due to differences in ambient conditions. As noted above, the 1995 experiment was conducted later in the year when conditions were dryer and slightly cooler.
Relating these results to disease biological control, the increase in bacterial population levels and colonization sites associated with shading would suggest that applied biocontrol strains of bacteria may be more efficacious in shaded areas. The effectiveness of strain C3 in suppressing Bipolaris leaf spot [Bipolaris sorokiniana (Sacc.) Shoemaker], is directly related to the population density of the bacterium on leaf surfaces (Zhang and Yuen, 1999). An increase in population numbers of strain C3 by 1 log unit, as found resulting from shading, could provide an incremental improvement in efficacy given that all other conditions are kept constant. In order to predict the effects of shading on biological control efficacy, pathogen x biocontrol interactions need to be studied directly under shaded conditions in the field. Results from this study suggest that laboratory and greenhouse procedures that evaluate ecological compatibility may be useful in predicting the effectiveness of potential biocontrol agents for turfgrass, and thus, facilitate the search for efficacious strains.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Received for publication October 11, 1999.
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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