Potential for Use of Stenotrophomonas maltophilia and a Related

Chapter 22 Potential for Use of Stenotrophomonas maltophilia and a Related Bacterial Species for the Control of Soilborne Turfgrass Diseases

Downloaded by SUFFOLK UNIV on January 19, 2018 | http://pubs.acs.org Publication Date: December 29, 1999 | doi: 10.1021/bk-2000-0743.ch022

D. Y . Kobayashi, J . D. Palumbo, and M . A . Holtman Department of Plant Pathology, Cook College, Rutgers University, New Brunswick, N J 08901

Strains of Stenotrophomonas maltophilia and a related bacterium, proposed as Lytobacter mycophilus gen. nov., sp. nov., are capable of controlling summer patch, a turfgrass disease caused by Magnaporthe poae. Strains of these bacteria produce an abundance of extracellular enzymes that have the potential to degrade fungal cell walls, and are capable of colonizing the turfgrass rhizosphere. Growth chamber studies indicate repeated application of S. maltophilia improves disease suppression compared to standard applications. S. maltophilia populations are reestablished above 10 colony forming units/g rhizosphere sample following each repeated application, suggesting these higher populations are critical for disease suppression. Field studies indicate that S. maltophilia populations can be established in turfgrass at these levels, providing support that disease control in the field can be achieved. 7

Growing concerns for environmental and health safety issues have led to the anticipation of stricter regulations for the use of chemical pesticides to control diseases. As a result, investigations into the development of alternatives to fungicides for disease control have increased. Biological control provides one potential alternative; however, its development has been faced with several challenges. One of the most significant challenges is overcoming inconsistencies in disease control performance (/), a problem that is accentuated for turfgrass diseases due to the lack of tolerance for disease by the industry. Maintaining high aesthetic quality of turfgrass is often further complicated by intense management practices that cause severe environmental and physical stress to the plant.

© 2000 American Chemical Society

Clark and Kenna; Fate and Management of Turfgrass Chemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

353

354


The rigorous requirements for control of turfgrass diseases can contribute to frequently reported failures of biocontrols. Furthermore, the nature of biocontrol hinders its effective use within traditional paradigms for pesticide usage, where methods for application and evaluation of performance are often similar to those used for evaluating chemical products. However, the expectation of biocontrols to perform in the same manner and at levels similar to chemicals is unlikely. Therefore, it is necessary to develop alternative models to those used for chemicals for the use of biocontrol of turfgrass diseases. Alternative approaches can be envisioned to incorporate all levels of the development of biocontrol, including developing new methods for their application, to incorporating its use with other management practices to reduce disease. Diseases of Turfgrass Caused by Root-Infecting Fungi Summer patch, caused by Magnaporthe poae, is an important disease that affects cool season turfgrass species such as Kentucky bluegrass (Poa pratensis) and fine fescuses (Festuca spp.) (2). The disease occurs during periods of high soil temperatures and sustained high water potential in the soil, that are combined with other environmental conditions leading to root stress, such as soil compaction and low mowing heights (3). Summer patch is similar to other patch diseases caused by root-infecting pathogens, including take-all patch, necrotic ring spot and spring dead spot (2). The life cycles of the pathogens causing these diseases are presumed to be similar. Ectotrophic colonization of the host roots by the pathogen is followed by infection of roots and subsequent colonization of the vascular tissue. Under the appropriate conditions, infection leads to foliar symptom development, and eventual death of the plant. Pathogen spread between plants likely occurs through ectotrophic growth on roots. For summer patch, ectotrophic growth can begin during the early spring, well before disease symptoms appear during summer months. Ectotrophic growth, as well as infection, can continue throughout the disease season into late fall. The pathogen is presumed to overwinter either ectotrophically on roots, or within the roots of infected plants. The endophytic and ectotrophic stages of the pathogen represent two different stages of the M poae life cycle, and thus represent two different stages targeted for disease control. Current methods for control of summer patch and related diseases rely heavily on the use of systemic fungicides. Contact fungicides have proven less effective (3), since they do not function as curative controls, presumably due to the inability to affect the pathogen after infection has occurred. In addition to chemical controls, some cultural and management practices have been demonstrated to alleviate symptoms (3,4), which indicate promise for the development of integrated disease management strategies in the future.


355


Patch Disease Severity Evaluations in Relation to Biocontrol Efficacy Some success has been reported for biocontrol efficacy of patch diseases (e.g. 5,6), although control levels are not comparable to the most effective fungicides. It is likely that many biocontrols function similarly to contact fungicides, in which control results from antagonism occurring during the ectotrophic growth phase of the pathogen life cycle. Disease control at this level may not be as easily detected using standard rating methods used for evaluating chemical products. A variety of methods are used to measure the severity of summer patch and other turfgrass patch diseases. Two methods involve visual ratings that either estimate the percentage of foliar necrosis within the affected area or determine the size of the area affected by disease. Disease severity, however, is more accurately measured as the product of both measurements, i.e., the percent necrosis within the affected patch area (% necrosis/measurement ). Significant differences in disease severity among treatments that are not detectable on a given observation date can sometimes be detected by comparing disease severity over time, as measured by calculating the area under the disease progress curve (AUDPC). For example, in field studies evaluating summer patch disease control using treatments of bacterial biocontrol agents, significant differences in patch severity were either not detected on any observation date, or detected on only one of three observation dates. However, significant differences were detected for these same treatments when total disease as measured by A U D P C was compared (5,6). The observed differences in total disease using more sensitive methods of disease evaluation such as AUDPC may indeed reflect effects of treatments that represent significant reduction in pathogen populations and/or infections that are not as easily detected on single observation dates. Observations such as these may prove significantly useful in devising effective disease management strategies that integrate the use of biocontrols with other practices, while at the same time reducing chemical input. 2

Isolation and Taxonomic Characterization of Bacterial Strains for Biocontrol of Summer Patch Disease A modified method that used mycelia of the pathogen as bait, originally designed by Scher and Baker (7), was combined with an enrichment culture procedure to isolate bacteria with parasitic traits to the summer patch pathogen, M poae (8,9). The objective of this strategy was to isolate potential biocontrol bacteria which function by directly attacking the fiingal pathogen. Using this approach, several bacterial strains were isolated from sources originating from golf course turf that demonstrated good biocontrol activity against summer patch under controlled environmental conditions (8,9, Kobayashi, D. Y. and El-Barrad, N . , Rutgers University, unpublished data). Stenotrophomonas (formerly Xanthomonas) maltophilia has been identified as a potential biocontrol agent for several agronomic crops. This bacterium is also known



356 to possess several traits important in biocontrol. For example, S. maltophilia is competent within the rhizosphere of a variety of plant species (10,11 J2), and is known to produce several extracellular enzymes, including chitinase, protease and lipase, that have the potential to degrade fungal cell wall components (8,13,14). Furthermore, this bacterium has been previously shown to form parasitic relationships with fungi (15). One bacterial strain, 34S1, demonstrated to have good biocontrol activity for summer patch disease, was identified as Stenotrophomonas maltophilia based on a variety of tests, including comparisons to the Biolog nutritional utilization database (Microlog, Hayward, CA), and the fatty acid methyl ester (FAME) profile database (Microbial ID, Newark, DE) (8). Taxonomic characterization of a second strain, N4-7, revealed species similarity matches to strain 34S1 based on the results of Biolog. The near identical nutritional utilization profile suggests that the two strains occupy the same nutritional niches. However, distinct differences were determined between the two strains based on F A M E profiles, 16S rDNA sequence, and serological comparisons. Physiological and morphological differences are also detected between the two strains. Strain N4-7 is nonmotile and lacks flagella, whereas strain 34SI is motile and possesses polar flagella. Furthermore, strain N4-7 grows optimally between 25-30 C, with essentially no detectable growth at 37 C, while strain 34S1 grows well at 37 C. Based on these significant differences, strains N4-7 and 34S1 warrant taxonomic separation at the genus level (16, Holtman, Μ. Α., Goyal, Α., Zylstra, G. and Kobayashi, D. Y . , Rutgers University, manuscript submitted), for which we propose the name Lytobacter mycophilus gen. nov., sp. nov., for strain N4-7. Strain N4-7 shares a variety of traits with strain 34S1 that are thought to contribute to biocontrol activity. Both strains are capable of colonizing the turfgrass rhizosphere at populations greater than 10 colony forming units (cfu)/g (fresh weight) rhizosphere sample (8,9). In addition, both strains produce a variety of extracellular enzyme activities, including chitinase, protease and lipase. Strain N4-7 also produces significant p-l,3-glucanase activity, as detected by hydrolysis of the P-l,3-glucan substrates laminarin and zymosan. The substrates that are degraded by these enzymes constitute major components of the cell wall of M poae as well as other fungal pathogens. The extensive work on biocontrol activity of S. maltophilia 34S1, along with other isolates of the same species, suggests that environmental isolates of this species offer potential as biocontrol agents. However, their use beyond research purposes as biocontrol strains is subject to regulatory scrutiny due to their association with clinical strains that bear the same species name. Since significant similarities are observed between S. maltophilia 34S1 and strain N4-7, including traits involved in biocontrol activity as well as field efficacy data (Kobayashi, D. Y., El-Barrad, N . and MacDonald, G., Rutgers University, unpublished data), the taxonomic distinction of strain N4-7 reflects the importance and necessity to more clearly define potentially useful environmental isolates at taxonomic levels for use as bacterial biocontrol agents, 6


357 thereby separating them from bacterial species classified as potentially hazardous clinical isolates.


Repeated Applications of Biocontrol Strains Improve Summer Patch Disease Control Efficacy in Controlled Environment Studies Disease control efficacy studies in growth chambers indicate that strains N4-7 and 34S1 are both capable of significantly reducing summer patch disease by greater that 70% compared to disease in untreated control plants on single observation dates (8,9). Examination of disease progress curves indicates that treatment of pathogen-inoculated Kentucky bluegrass plants with strain 34S1 significantly delays the onset of summer patch, but does not change the rate of disease progression (8), In these studies, increasing bacterial concentrations further delays the onset of disease. Therefore, disease suppression results from a delay in disease onset, or a shift in time of disease progress curves. Similar observations are observed with strain N4-7 (Kobayashi, D.Y., El-Barrad, N . and MacDonald, G., Rutgers University, unpublished data), indicating that strains N4-7 and 34S1 both have similar effects on the suppression of summer patch. The observations that onset of summer patch disease in pathogen-inoculated plants is delayed by treatment with biocontrol bacteria, and that increased concentrations of bacteria further delay disease onset, suggest that repeated application of biocontrol bacteria may continuously delay onset of disease. To test this hypothesis, strain 34S1 was repeatedly applied to Magnaporthe /wxre-inoculated Kentucky bluegrass var. Baron grown in 9" containers in controlled environmental conditions similar to the method described (8,9). Briefly, 25 ml of strain 34S1, at a concentration of 10 cfii/ml, was applied to each container on a bimonthly schedule beginning two weeks after seeding and continuing to the end of the experiment. This treatment was compared to a standard treatment of strain 34S1 applied two and three weeks after seeding. Plants were moved to the growth chamber, set at disease conducive conditions of 28 C and 70% humidity, four weeks after seeding and were scored regularly for disease symptom development by rating the percent foliar necrosis in each container as previously described (8,9). Figure l a shows the results of mean rating values of 10 replicates for each treatment four weeks after plants were moved to disease conducive conditions. Summer patch disease suppression was significantly improved by repeated applications of strain 34S1 when applied on a bimonthly schedule over the eight week experimental period, compared to a standard experimental treatment of two single applications at weeks 2 and 3 of the experiment. The experiment was repeated a second time with similar results (data not shown). Examination of bacterial populations in the rhizosphere of turfgrass, according the method described previously (8,9) indicated that repeated applications boosted strain 34S1 populations to above 10 cfu/g rhizosphere sample upon each application. In contrast, populations of strain 34S1 on plants treated by standard applications 8

7



358

Figure 1. Effect of repeated and standard applications of Stenotrophomonas maltophilia 34S1 to Magnaporthe /?oae-inoculated Kentucky bluegrass var. Baron. A) Foliar disease symptom severity. Con = pathogen-inoculated, untreated disease control plants; std = standard treatment application of strain 34S1; rep = repeated application of strain 34S1. Different letters above bars indicate significant differences according to Duncan's multiple range test (P=0.05). B) Rhizosphere populations of strain 34S1 sampled on a weekly basis for standard application (solid line with close circles) compared to bimonthly (repeated) applications (dotted line with open squares). Error bars represent one standard deviation from the mean.


359 5

decreased to 10 cfu/g rhizosphere sample after initial applications. These observations suggest that establishing the bacterium at populations of 10 cfu/g rhizosphere sample is necessary and critical to achieve significant disease control. 7

Field Trials for Summer Patch Disease Control 8

During the summer of 1995, strain 34S1 was applied at a concentration of 10 cfu/ml at a rate of 1 L/m on a weekly and bimonthly basis in field trials of 3-year-old stand of Kentucky bluegrass var. Baron inoculated with M poae in New Brunswick, NJ, similar to the procedure described (5,6). The plots were maintained at conditions similar to those for landscape turf, with a mowing height of 1.5 inches. Summer patch disease was not significantly reduced by any treatment within this field trial. However, enumeration of strain 34S1 populations in the rhizosphere indicated that the bacterium did not reach critical levels at any point during the season, even 24 h after application (Figure 2a). During the summer of 1996, field application of strain 34S1 was repeated at a separate site from 1995 on turfgrass more intensely managed under golf course greens conditions (mowing height of 1/16") in New Brunswick, NJ. In this study, treatments of weekly and bimonthly bacterial applications were conducted on a mixed stand of pathogen-inoculated bentgrass and annual bluegrass. No summer patch disease was observed on any pathogen-inoculated plots used for the experiment. However, mean population levels of strain 34S1 in the turfgrass rhizosphere generally reached higher levels in 1996 than those observed in 1995 (Figure 2b). On a few occasions, populations reached above 10 cfu/g rhizosphere sample. However, values still decreased to below 10 cfu/g sample on a regular basis. Nonetheless, overall population sizes from weekly applications appeared to be greater than those from bimonthly applications. In an effort to increase rhizosphere populations, field studies were repeated in 1997, in which a single treatment of strain 34S1 was applied on a weekly basis at a higher concentration of 5* 10 cfii/ml. The experiment was conducted on pathogeninoculated plots of annual bluegrass maintained at identical conditions to plots used in 1996. Under these conditions, populations repeatedly achieved levels above 10 cfii/g rhizosphere sample, and on only a few occasions dropped below 10 cfu/g sample (Figure 2c). These observations indicate that under the appropriate conditions, rhizosphere population levels deemed critical for biocontrol in growth chamber studies can be established in the field.


2

7

5

8

7

s

Future Directions for Biocontrol on Turfgrass The expectation of biocontrols to perform at consistent levels equivalent to current chemical disease control methods, using similar application methods, are major impediments for the development of biological controls. As a consequence, few


360


10" ίο

7

ίο

6

ίο

5

i ίο

4

8

8

y

,

,

1

H

1

f-

,

,

1

H

H

1

1

(.

& ίο w eu

7

•2 10 iio

e

CO

S

O

g io

g

ίο

N

•S:

7

10* io

5

10 10 0

10 20 30 40 50 60 70 80 90 100

days after first treatment application Figure 2. Turfgrass rhizosphere populations of Stenotrophomonas maltophilia 34S1 applied to field plots. A) 1995 field study comparing weekly applications (solid line with closed circles) and bimonthly applications (dotted line with open squares). B) 1996 field study comparing weekly applications (solid line with closed circles) and bimonthly applications (dotted line with open squares). C) 1997 field study of weekly applications. Error bars represent one standard deviation from the mean.



361 commercial biocontrol products are currently available for use on turfgrass. Infrequent application rates and poor establishment of biocontrol agent populations most likely contribute to failures that are often observed in the field. It is likely that the successful contribution of biocontrol for control of turfgrass diseases will require management practices that are adapted to its use and maximize its potential. Methods to apply biocontrols are already being developed and focus on integration with current management practices, such as delivering the agent through methods similar to those developed for fertigation and chemigation. There is ample evidence that management practices, such as aerifying soilcompacted areas, raising mowing heights and using different nitrogen fertilization sources, can reduce diseases such as summer patch. The development of disease management programs that incorporate these practices, along with the use of biologicals, can very likely lead to disease control at levels acceptable to the industry. In efforts to develop disease management programs that successfully incorporate biocontrol, strong efforts will be needed to educate both users and the public concerning its benefits and safety. This includes clear taxonomic distinction of new beneficial biocontrol strains, as well as acceptance for the use of biocontrols under specific conditions. A clear understanding of how biocontrols can function as components of integrated disease management approaches is important to their success. Under these conditions, it is likely that biologicals can be utilized for turfgrass diseases, not necessarily to replace chemical pesticides, but to reduce their input, while still maintaining high efficacies for disease control expected by the industry. Acknowledgments Portions of this work was funded in part by United States Golf Association, Rutgers University Center for Turfgrass Sciences, The New Jersey Turfgrass Association, and The New Jersey Agricultural Experiment Station. We thank N. El-Barrad and G. MacDonald for excellent technical assistance with disease assays, and B. Dickson and J. Clarke for excellence assistance with maintenance of field plots. We also are grateful to B. Clarke for helpful suggestions throughout these studies.

Literature Cited 1. 2. 3. 4. 5. 6.

Weller, D. Ann. Rev. Phytopathol. 1988, 26, 379-407. Turfgrass Patch Diseases caused by ectotrophic root-infectingfungi; Clarke, B.B.; Gould, A.B., Eds.; APS Press. St. Paul, M N , 1993. Landschoot, P.J.; Clarke, B.B.; Jackson, N . ; Golf Course Manag. 1989, 57, 38-41. Thompson, D.C.; Clarke, B.B.; Heckman, J.R.; Plant Dis. 1995, 79, 5156. Thompson, D.C.; Clarke, B.; Kobayashi, D. Y.; Plant Dis. 1996, 80, 856862. Thompson, DC.; Kobayashi, D.Y.; Clarke, B.B.; SoilBiol.Biochem. 1997, 30, 257-263.


362 7. 8. 9. 10. 11. 12.


13. 14. 15.

Scher, F.; Baker, R.; Phytopathology 1980, 70, 412-417. Kobayashi, D.Y.; Guglieimoni, M.; Clarke, B.; Soil Biol. Biochem. 1995, 27, 1479-1487. Kobayashi, D.Y.; El-Barrad, N.; Cur. Microbiol. 1996, 32, 106-110. Debette, J.; Blondeau, R.; Can. J. Microbiol. 1980, 26, 460-463. Lambert, B.; Leyns, F.; Van Rooyen, L.; Gosselé, F.; Papon, Y . ; Swings, J.; Appl. Environ. Microbiol. 1987, 53, 1866-1871. Lambert, B.; Meire, P.; Joos, H . ; Lens, P.; Swings, J.; Appl. Environ. Microbiol. 1990, 56, 3375-3381. Nord, C.-E.; Sjöberg, L.; Wadström, T.; Wretlind, B.; Med. Microbiol. Immunol. 1975, 161, 79-87. O'Brien, M.; Davis, G.H.G.; J. Clin.Microbiol.1982, 16, 417-421. Willoughby, L . G . ; Trans. Br. Mycol. Soc. 1983, 80, 91-97.15.12.


Potential for Use of Stenotrophomonas maltophilia and a Related

Recommend Documents