Synergizing Weed Biocontrol Agents with Chemical Herbicides

Chapter 7

Synergizing Weed Biocontrol Agents with Chemical Herbicides A. L. Christy, K. A. Herbst, S. J. Kostka, J. P. Mullen, and P. S. Carlson

Downloaded by FUDAN UNIV on March 2, 2017 | http://pubs.acs.org Publication Date: March 12, 1993 | doi: 10.1021/bk-1993-0524.ch007

Crop Genetics International, 7170 Standard Drive, Hanover, MD 21076

Enhanced weed control can occur when chemical herbicides are applied in combination with microbial agents. Synergy between chemicals and biologicals was first observed with fungal bioherbicides. Use of a herbicide with a fungal-bioherbicide does not appear to widen the weed spectrum of the bioherbicide but does increase the efficacy. In some cases the synergy may result from a suppression of phytoalexin synthesis by the chemical herbicide. Use of synthetic herbicides to synergize mycoherbicides will in some cases reduce herbicide use rate and improve mycoherbicide efficacy. We report here that synergy occurs between chemical herbicides and bacterial agents. We have discovered that bacteria which cause little or no injury to weeds when applied alone can enhance the stability of low levels of chemical herbicides to control a broad spectrum of weeds. We term this synergistic combination the X-tend system. In the first year of X-tend field trials, the level of weed control required for commercialization was not obtained consistently across geographic locations. Consistency will need to be improved through selection of more robust microbial strains and/or improvements in formulations. The use of biocontrol agents to control pests in the United States celebrated a 100 year anniversary in 1989. While there are a number of examples of very successful biocontrol projects, the widespread use of biological-based pest control technology has not been adapted to major agricultural production systems. The growing concern over the continued use of synthetic chemical pesticides has promoted efforts to expand biocontrol technologies for major pest problems. To date, however, biological pest control technologies have not proven as effective or economical as chemical control. Many biocontrol

0097-6156y93/0524-O087$06.00/0 © 1993 American Chemical Society Duke et al.; Pest Control with Enhanced Environmental Safety ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


PEST CONTROL WITH ENHANCED ENVIRONMENTAL SAFETY

products targeted for control of major pests are still in the research and development stage. Some of the problems with using biocontrol to manage pest problems include: 1) slow and/or insufficient activity, 2) limited pest control spectrum, 3) inconsistent activity across different environments, and 4) high cost of production. One approach to overcome some of these problems may be to combine chemical and biological control agents in an integrated pest management system. Synergy between synthetic chemical and biological agents may result in both a reduction in chemical application rate and an increase in efficacy of the biological agent. Such a system may both reduce the chemical load on the environment and reduce the cost of pest control to the grower. Several recent studies have reported possible synergy between synthetic chemical herbicides and fungal plant pathogens for use in weed control (1, 2). In addition, we have been developing a weed control system known as X-tend that combines bacterial agents and reduced rates of herbicides to control major agriculturally important weeds. Fungal-Based Bioherbicides The majority of the bioherbicides studied to date have involved the use of fungal pathogens to control weeds. Two commercially available bioherbicides, Collego (Colletotrichum gloeosporioides (Penz). Sacc. f. sp. aeschvnomene) and DeVine (Phytophthora palmivora) are each specific for only one weed; northern jointvetch, Aeschynomere virginica, and milkweed vine, Morrenia odorata, respectively. In addition, fungal bioherbicides require specific conditions for infection and disease development. For example, a dew period in excess of 24 h is often required for spore gerrnination. Variable environmental conditions may severely hamper effective control. Some examples of synergy observed between fungal pathogens and herbicides are summarized in Table I. Holliday and Keen (f) were perhaps the first to demonstrate that a herbicide could block the expression of resistance to a fungal pathogen. Treating soybean plants with glyphosate appeared to promote infection by an incompatible race of the fungus Phytophthora megasperma f. sp. glvcinea (1, 3). These data suggest that compromising the plant in some way with a chemical agent reduces the plant's ability to mount a defense to pathogen attack and improves the efficacy of the bioherbicide. Keen et al. (3) found that low levels of glyphosate blocked production of glyceollin, the phytoalexin in soybean thought to provide some resistance to Phytophthora megasperma infection. While an undesirable effect in a crop, it is a desirable synergy in weed control. For example, Sharon et al. (4) demonstrated that glyphosate inhibited phytoalexin synthesis in treated sicklepod, possibly reducing plant resistance to fungal attack and enhancing the activity of the bioherbicide Alternaria cassiae.

Duke et al.; Pest Control with Enhanced Environmental Safety ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

7. CHRISTY ETAL.

Synergizing Biocontrol Agents with Chemical Herbicides


Table I. Some Examples of Synergistic Fungal Pathogen and Chemical Combinations Pathogen

Herbicide/PGR

Plant

Reference

Phytophthora megasperma

glyphosate

soybean

Keen et al., 1982 (3)

Colletotrichum coccodes

thidiazuron

velvetleaf

Wymore et al., 1987 (3) Hodgson et al., 1988 (8)

Alternaria cassiae

numerous herbicides

several

Caulder and Stowell, 1988 (9)

Fusarium lateritiun

bentazon and acifluorfen

Florida beggarweed

Caulder and Stowell, 1988 (9)

Pvthium and Fusarium ssp.

glyphosate

black bean

Rahe et al., 1990 (10)

Alternaria cassiae

glyphosate

sicklepod

Sharon et al., 1991 (4)

Alternaria cassiae

invert emulsion

sicklepod

Amsellem et al., 1990(11)

Two research groups have reported synergy between the fungus Colletotrichum coccodes and the plant growth regulator (PGR) thidiazuron for the control of velvetleaf (Abutilon theophrasti) (5, 6). Wymore et al. (5) showed that separate applications of C coccodes and thidiazuron reduced velvetleaf growth and provided some level of weed control. However, application of tank mixes of thidiazuron and C. coccodes spores resulted in as much as 100% mortality of velvetleaf in both greenhouse (5) and field studies (7). Hodgson et al. (8) confirmed these results in field studies in both Canada and Maryland (Figure 1). The mode of action of the synergy between thidiazuron and C. coccodes was investigated by Hodgson and Synder (6). Application of thidiazuron or an extract of C. coccodes mycelium to velvetleaf petioles enhanced ethylene production. However, it is not clear from this research whether the ethylene was produced as a result of thidiazuron application. The increased ethylene level may contribute to the observed synergy or may be a response of the plant tissue to the application of the PGR (6). Caulder and Stowell (9) received a US patent in 1988 for the use of four fungal plant pathogens in combination with a number of herbicides and PGR's. Table II summarizes the synergy they observed with various fungal and chemical combinations. It is interesting to note that the chemical agents represent a variety of chemical classes with different modes of action. Synergy was observed with the fungal pathogen on the target (susceptible)


89


90

| TDZ a l o n e

TRIAL 1

MARYLAND

QUEBEC


TDZ + cc

I

I

TRIAL 2 MARYLAND

QUEBEC

I

0 1 0.0 0.1 0.2 0.3 0.4

i

o.O 0.1 0.2 0.3 0.4

T H I D I A Z U R O N [TDZ] ( k g / h a ) Figure 1. Control ratings for velvetleaf tested with thidiazuron (TDZ) and Colletotrichum coccodes (Cc), Trial 1 treated at 1 to 2 leaf stage, Trial 2 treated at 4 to 6 leaf stage. (Reproduced with permission from reference 6. Copyright 1989, Weed Science Society of America.)


CHRISTY ET AL.


Table II. Summary of Synergy Between Four Fungal Pathogens and a Series of Chemical Agents Synergy with a

b


Herbicide/PGR

AC

0

CC

CT

FL!

bentazon

+

+

+

+

acifluorfen

+

+

+

+

chlorimuron

-

+

-

fluazifop

+

+

diolofop

+

-

sethoxydim

+

-

imazaquin

+

+

metribuzin

+

-

oryzalin

+

+

thidiazuron

-

diaminozide

-

+

mefluidide

+

+

+

+

a

From US Patent 4,775,405, to J.D. Caulder and L . Stowell (Mycogen Corp.), 1988. b

M

"+ indicates synergy, "-" indicates a lack of synergy, and a blank indicates that the combination as not tested. C

AC CC CT FL

= Alternaria cassiae = Colletotrichum coccodes = Colletotrichum truncatum = Fusarium lateritium



92


host, however, the herbicide did not expand the weed spectrum of the biocontrol fungus (9). Sharon et al. (4) showed that glyphosate suppressed the accumulation of total phenolic compounds, flavonoids, and the resulting phytoalexins in sicklepod (Cassia obtusifolia L.) (Figure 2). This resulted in glyphosatetreated sicklepod seedlings being highly susceptible to the fungal pathogen Alternaria cassiae (4). However, when soybean plants were treated with glyphosate, no increase in susceptibility to A . cassiae was observed. These results are consistent with earlier reports in which herbicide-mycoherbicide combinations did not expand the host range of the mycoherbicide. Other herbicides and PGR's have been reported to synergize fungal pathogens. The role of these herbicides and PGR's on the inhibition of phenolics, flavonoids, and phytoalexins has not been investigated at this time. Perhaps chemicals that stress the plant and reduce growth and metabolism may also reduce phytoalexin synthesis either directly or indirectly and synergize the bioherbicide. It is equally plausible that other physiological processes may be influenced and yield similar phenomenology. Bacteria-Based Bioherbicides Research on bacteria as bioherbicides has focused primarily on the use of rhizobacteria (12, 13, 14). Kennedy et al. (12) characterized rhizosphere strains of fluorescent pseudomonads that, when applied to soils, inhibited the germination and growth of downy brome (Bromus tectorum) without affecting the growth of wheat. Similarly, Harris and Stahlman (15) identified rhizosphere strains that inhibited root elongation in one or more grasses without significantly affecting winter wheat root growth. Rhizobacteria representing diverse gram-negative bacterial genera have been demonstrated by Kramer et al. (13, 14) as potential biocontrol agents for broadleaf weeds. Although examining expanded host ranges within either monocots or dicots, none of the researchers screened candidate strains against both groups of plants. A novel approach to control of annual bluegrass through the exploitation of a vascular, phytopathogenic bacterium is under development. (16). Xanthomonas campestris, the causal agent of bacterial wilt of annual bluegrass (Poa annua L.) is applied in the early spring to newly mown grass. The bacterium rapidly colonizes the xylem; reaching populations of up to l x l O colony forming units(cfu)/ml of tissue, the foliage wilts and the plants die within six weeks. This strain is being developed for use in controlling bluegrass in turf and does not harm desirable turf species. Another novel approach, combinations of bacterial and chemical agents for enhanced weed control, the X-tend bioherbicide system, is under development by our Company. Research has involved both greenhouse evaluations and field trials with a range of bacteria and herbicide combinations. Bacteria with X-tend activity are representative of a range of well characterized genera and species, as well as, several unidentified plantassociated strains. A summary of this research is included here as an 10



7. CHRISTY ET AL.

o l 0


1

1

1

1

20

40

60

80

GLYPHOSATE

93

1

100

C O N C E N T R A T I O N (LXM)

Figure 2. Glyphosate suppression of phytoalexin accumulation in detached leaves of sicklepod (Cassia tectorum). (Reproduced with permission from reference 4. Copyright 1992, American Society of Plant Physiologists.)




example of synergy between bacterial agents and synthetic chemical herbicides. A l l bacteria are referred to by either a strain number or preparation number. Preparation of Bacterial/Herbicide Tank Mixes. Strains of bacteria were obtained from commercial culture collections, academic and governmental research laboratories, or from isolations made from plant and environmental samples collected throughout the continental United States. Strains were archivally stored by lyophilization and in glycerol (-80°C). Working stocks of specific strains were stored in glycerol at -20°C. Bacteria for plant treatments were produced by growing strains of interest in solid phase or liquid fermentations using one or more culture media incubated at 28°C for 24 to 26 h. Solid phase fermentations were prepared in Petri dishes by streaking the surface of a semi-solid culture medium with a sterile swab soaked with cells from a log phase broth culture. Liquid fermentations were normally conducted in 2 liter, triple baffled, culture flasks containing 1 liter of broth medium and seeded with bacteria from log phase broth cultures. Depending on the strain, cultures were incubated for 48 to 64 h at 20°C, 25°C, or 28°C on a rotary shaker (180 to 190 rpm). Cells were harvested from Petri dishes with a rubber policeman or from liquid culture by centrifugation using a Sorval RC5C centrifuge at 8000 rpm (10°C) for 8 to 10 min. Spray solutions were prepared by resuspending cell pellets in either decanted, spent fermentation medium, or sterile distilled water. Cell concentrations were standardized spectrophotometrically (600 nm) to approximately 1 x 10 cfu/ml. Spray solutions were prepared by adding the chemical herbicides and appropriate surfactant directly into the bacterial preparations. Treatments were applied using a Devries SB-8 spray booth equipped with a 8004E flat fan spray nozzle traveling at 80.5 m/min delivering 233.7 1/ha. One replication consisted of four broadleaf species at the 2 to 3 true leaf stage and four narrowleaf species 8 to 10 cm in height grown in a spaghnum peat: vermiculite: bark ash mix. A randomized complete block design was used with four replications. Field treatments were applied using a C 0 pressurized backpack sprayer equipped with 8004 flat fan nozzles delivering 233.7 1/ha at 93.9 m/min. Field studies typically included three to four broadleaf weed species having 4 to 6 true leaves and three to four narrowleaf species 5 to 10 cms in height. Plots were 1.5 m x 3 m with 25 cm row spacing. 9

2

Greenhouse Screens. Numerous pathogenic and nonpathogenic bacteria have been evaluated with several herbicides for weed control in the greenhouse. Figure 3 shows the response typically observed with sulfosate (Trimethylsulfonium-carboxymethylamino-methyl phosphonate) applied alone and in combination with a bacterial preparation. Unlike mycoherbicidal fungi, the bacterial preparation applied alone caused no injury to the test weeds. However, the bacterial preparation plus 0.067 kg




CHRISTY ET AL.

Figure 3. Results of a typical greenhouse screen showing the synergy between sulfosate 0.067 kg ai/ha and a bacterial preparation.




ai/ha sulfosate demonstrated greater injury than the herbicide alone. Moderate to good control of morningglory (Ipomoea hederacea L.) and pigweed (Amaranthus retroflexus L.), two difficult to control weeds, was observed. The level of injury on narrowleaf weeds was more than twice that of the herbicide alone. Since no injury was observed in treatments where the bacteria was applied alone, the dramatic increase in activity observed with the bacteria-herbicide combination demonstrates synergy. Field Trials. Maryland field trials of the X-tend bioherbicide preparation 400S plus 0.56 kg ai/ha sulfosate demonstrated a 40 to 50 percentage point increase in control of pigweed, morningglory, and velvetleaf (Abutilon theophrasti) over treatments that received only sulfosate (Figure 4). Preparations of 1518S and 36IS in combination with sulfosate improved broadleaf weed control but were less effective than 400S. No synergistic effect on grasses was observed in this study due to complete control by the 0.56 kg ai/ha rate of sulfosate alone. Similar field results were obtained with glufosinate and two bacterial preparations (Figure 5). Most of the synergy was observed at a rate of 0.224 kg ai/ha glufosinate. This is approximately 1/4 to 1/5 the recommended field rate on the proposed glufosinate label. Other field trials at our Maryland research farm provided similar results with the herbicides fluazifop, sethoxydim, and nicosulfuron. In most cases as the herbicide rate approached the recommended field rate less synergy with the bioherbicide was observed. Field trials at other locations revealed a similar pattern (Figure 6). Application of sulfosate at 0.28 kg ai/ha in combination with preparation 400S was synergistic on barnyardgrass at three locations. The herbicide rate was one third the recommended rate and obviously below the rate needed to provide consistent control of barnyardgrass (Echinochloa crus-galli L.). This was particularly evident in tests in Indiana (IN) and Kansas (KS), where the weeds were larger or were grown under drought conditions (IN). It appears that the bioherbicide was strongly dependent on the level of control or injury provided by the chemical herbicide. The results indicate that a more robust strain of the bacteria will be required to obtain commercial levels of control. Development of surfactant and formulation technology may also aid in improving control. Summary. Synergy has been observed between chemical herbicides and bacterial agents in both greenhouse and field trials. Combinations of herbicides and bacteria can significantly reduce herbicide use rate to control a broad spectrum of weed species. However, the present level of control achieved with these tank mixes in the field indicates that either more robust strains or improved formulations will be needed before commercialization is realized.




CHRISTY ET AL.

Figure 4. Field study of synergy between sulfosate (0.56 kg ai/ha) and three bacterial preparations.



98


100

Johnsongrass Yellow Foxtail Pigweed Morningglory Barnyardgrass Fall Panicum Jimsonweed

Glufosinate ^

Glufosinate + 400S J

Glufosinate + 300C

Figure 5. Synergy between glufosinate (0.28 kg ai/ha) and two bacterial preparations in field trials.




CHRISTY ET AL.

MD-4

MD-9

KS

Location Sulfosate

Sulfosate + 400S

Figure 6. Synergy between sulfosate (0.28 kg ai/ha) and a bacterial preparation (400S) on barnyardgrass at three field locations.



100


Literature Cited 1. Holliday, M.J.; Keen, N.T. Phytopathology 1981, 71, 881. 2. Quimby, P.C. Proc. South Weed Sci. Soc. 1985, 38, 365-371. 3. Keen, N.T.; Holliday, M.J.; Yoshikawa, M . Phytopathology 1982, 72, 1467-1470. 4. Sharon, A.; Amsellem, Z; Gressel, J. PlantPhysiol.1992, 98, 654659. 5. Wymore, L.A.; Watson, A.K.; Gotlieb, A.R. WeedSci.1987, 35, 377-383. 6. Hodgson, R.H.; Snyder, R.H. WeedSci.1989, 37, 484-489. 7. Wymore, L.A.; Watson, A.K.; WeedSci.1989, 37, 498-483. 8. Hodgson, R.H.; Wymore, L.A.; Watson, A.K.; Snyder, R.H.; Collette, A. Weed Technol. 1988, 2: 473-480. 9. Caulder, J.D.; Stowell, L. U.S. Patent #4,766,873, 1988, 18PP. 10. Rahe, J.E.; Levesque, C.A.; Johal, G.S. In Microbes and Microbial Products as Herbicides, edited by R.E. Hoagland. ACS Symposium Series No. 439; American Chemical Society: Washington, DC 1990, PP. 260-275. 11. Amsellem, Z.; Sharon, A.; Gressel, J.; Quimby, P.C., Jr. Phytopathology 1990, 80, 925-929. 12. Kennedy, A.C.; Elliott, L.F.; Young, F.L.; Douglas, C.L. SoilSci.Soc. Am. J. 55, 722-727. 13. Kremer, R.J. Microbial Ecology 1987, 14, 29-37. 14. Kremer, R.J.; Begonia, M-F.T; Stanley, L; Lanham, E.T. 1990. Appl. Eviron. Microbiol. 56, 1649-1655. 15. Harris, P.A.; Stahlman, P.W. Weed Sci. Soc. Amer. Abstracts 1992, 32, 50. 16. Baragona, J.S.; White, J.C. Weed Sci. Soc. Amer. Abstracts 1992, 32, 50. RECEIVED September 14, 1992


Synergizing Weed Biocontrol Agents with Chemical Herbicides

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