Allelopathic Influences on No Tillage Versus Conventional Tillage in

C h a p t e r 34

Allelopathic Influences on No Tillage Versus Conventional Tillage in Wheat Production George R. Waller , E. G. Krenzer, Jr. , James K. McPherson , and Steven R. McGown 1

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Departments of Biochemistry , Agronomy , Botany , and Microbiology , Oklahoma State University, Stillwater, OK 74078 Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 1, 2016 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch034

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Incorporating allelopathy into agricultural management may reduce the use of herbicides, cause less pollution, and diminish autotoxic hazards. Authentic inhibitors isolated from plant material have been subjects for examination in vitro, but attempts to compare their effects in soils are limited. Soils contain a heterogeneous collection of organic matter of various origins. Organic solvents and water extracts prepared from monoculture wheat soils under conventional tillage (CT) and no tillage (NT) indicated that both soils contain some inhibitory compounds. The CGC/MS/DA of some of the organics is presented. Selected organics from CT and NT as well as allelopathic and autotoxic effects are described and discussed. The relationship between the wheat yields in CT and NT and the possible biological stress is indicated. Conservation tillage practices in wheat production have increased steadily from about 15% in 1971 to 30% in 1985. These practices are attractive to growers for soil and moisture conservation and fuel savings, but there may be drawbacks. Variations in yields of forage and grain compared with those of conventional tillage differ according to rainfall conditions and geographic location. These erratic results are probably also partly attributable to biological factors such as diseases, insect damage, and allelopathy, which can vary substantially from season to season. Researchers elsewhere have generally shown that allelopathy from wheat residue reduces the subsequent wheat yield (1-3). There is reason to believe that the same phenomenon is occurring in the Great Plains Area as reported here, though under some circumstances it may be masked by the favorable effects of no-till growing such as greater retention of soil moisture. Allelopathic chemicals from soils, crop residues, and weeds are known to reduce the growth of several crops, and there are numerous examples of allelopathy among wild plants. We are studying the allelopathic effects of wheat residue and soil on the germination and growth of wheat under Oklahoma conventional-tillage and no-tillage conditions. We see this as basic research aimed at determining the presence and magnitude of any allelopathic effects of wheat on itself, as well as the identity of the chemicals causing them. This would, of course, be a necessary first step in remedying allelopathic effects and increasing wheat yield. Whittaker (4), Waller and Nowacki (5.), and Rabotnov (£) discussed the evolution of stable plant communities and species susceptible to allelopathic chemicals that were released by other plants. Such plants would have been eliminated by natural selection, and allelopathically neutral or allelopathically 0097-6156/87/0330-0371$06.00/0 © 1987 American Chemical Society

Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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372

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

tolerant plant communities would result. Allelopathy is often more evident in disturbed plant communities, such as agricultural ones. M c C a l l a and coworkers (1-3. 7) did pioneer research on the wheat crop i n the eastern Nebraska area. They showed that water-soluble substances i n crop residues reduced the germination and growth o f seedlings o f wheat, c o r n , sorghum, and other crops. The water extracts o f the seeds had the least effect and the stem extracts had the greatest inhibitory effect on wheat seedlings. Wheat as well as other crops were shown to contain a number of phenolic acids and the five most dominant ones were: ferulic, p_-coumaric, syringic, v a n i l l i c , and p_hydroxybenzoic acids. These were quantitatively estimated i n the crop residues; e.g., the total amount o f phenolic acids from wheat left on the field was 1.5 tons/acre under no-tillage conditions. M c C a l l a and Norstadt (3) worked extensively o n the antibiotic patulin (CjHgO^; M . W . 156) produced by Pénicillium urticae Bainer, which was found i n wheat soil, and found that the severity o f visible symptoms o f phytotoxicity to winter wheat (no-tillage) corresponded to the concentration of patulin. Elliott et al. provided a review of phytotoxicity in 1978 (£) and Elliott et al. (9) showed that bacterial colonization of plant roots can cause a 2 5 % increase i n the release of allelochemical compounds produced. Putman and DeFrank (10) made use o f phytotoxic plant residues for selective weed control. Lehle and Putman (11) used sorghum plants to show that the self-inhibitory activity (autotoxicity) varied widely depending upon the stage of development. Schilling et al. (12) snowed that compounds, some identified (βphenyllactic acid, β-hydroxybutyric acid) and some not identified, were effective in the suppression o f certain weeds by rye and wheat mulches in no-till crops. The data otherwise accumulated by the Oklahoma Agricultural Experiment Station so far indicate that for 6 years the wheat y i e l d obtained i n studies comparing conventional-tillage and no-tillage averages is 42 bu/acre for each. This is independent o f location. However, there is an indication that the soil systems have not stabilized with the change i n tillage practices; thus, one would not expect to see any real differences i n the soil as it relates to the wheat crop yields between the tillage treatments. Factors essential to maintain wheat crop yields are the availability o f water and nutrients, control o f insects, and disease. W e have found that the forage yield (hay) from the wheat crop taken at the early jointing stage for the conventional-tillage system is twice that for no-tillage. If this difference continues to be found i n future years then it provides some evidence for an allelopathic effect occurring early i n the growing season. O u r objective was to study i n detail these systems by identifying the allelochemicals involved, their primary modes o f action as mediated by soils, and as appropriate the microflora, microclimate, soil moisture and other factors that may influence allelopathy. Experimental Sampling o f the Soil. Representative soil samples were taken from conventional and no-tillage plots before planting and afterward at intervals o f 1 month since A p r i l , 1985. The soil samples were placed i n quart jars, frozen immediately by using dry ice, and stored at -18 °C. Extraction o f Organic Compounds from the S o i l . S o i l biochemicals that are free or absorbed loosely, but not bound to the humus, were extracted by the following procedures: A)

A 100-200-g sample o f soil was thawed, placed i n an extraction thimble, and extracted with redistilled isopropyl alcohol in a Soxhlet extractor for 48 h. Some of these alcohol extracts were analyzed for


34.

WALLER ET AL.


B)

C)

Tillage in Wheat

Production

373

their biological activity and others were evaporated and the residue was extracted with water at 60 °C, 3 times, and the mixture filtered through 7-mm Whatman 41 filter paper; the insoluble part was extracted with methyl alcohol at r o o m temperature, and those compounds that remained were redissolved i n isopropyl alcohol. Each extract was weighed and bioassayed. A 200-g sample of soil was loosely packed i n a 24 χ 40 c m chromatography column and sequentially extracted with redistilled organic solvents at room temperature and a flow rate of 0.5 m L per minute i n the following order; 200 m L hexane; 100 m L hexane + 100 m L methylene chloride; 200 m L methylene chloride; 100 m L methylene chloride + 100 m L ethyl acetate; 200 m L ethyl acetate; 100 m L ethyl acetate + 100 m L methyl alcohol; 200 m L methyl alcohol; 100 m L methyl alcohol + 1 0 0 m L triply distilled water; 800 m L triply distilled water. E a c h extract was taken to dryness over nitrogen gas except the aqueous one, which was evaporated with a rotary evaporator at 45 °C. E a c h residue was weighed and bioassayed. Steam distillation, extraction, and evaporation (Waller et al. [13. 14]) were completed and the resulting mixtures bioassayed.

Analysis of the M i x t u r e of Organic Compounds from the S o i l . The crude fractions were analyzed using a L K B - 2 0 9 1 capillary gas chromatograph/mass spectrometer/data analysis system ( C G C / M S / D A ) . The capillary column used was a J & W D B - 1 , 60 m χ 0.32 m m , connected directly to the ion source of the mass spectrometer. U p to 1.0 u L of a solution of the sample i n an appropriate solvent was injected directly onto the column at 40 °C, whereupon the column temperature was immediately raised to 100 °C for 4 min, and programmed to 310 °C at a rate of 10°/min. and held there for 30 min. Bioassay of Organic Compounds from the S o i l . Bioassay experiments measured the germination and early growth (generally the most sensitive time of any plant's life) of wheat. The methods are similar to those of McPherson and M u l l e r (15) and others i n the field, and are summarized below. Containers, media and seeds. Glass Petri dishes, 100 χ 15 cm, were used with two sheets of 75-mm Whatman 41 filter paper as the absorptive medium. Ten seeds of Τ Α Μ 105 wheat were placed i n a radial pattern with the micropyle end toward the center between the two sheets of filter paper. Seeds were hand-selected for normal size and absence of damage. TÀM105 was selected because it is the variety used i n the ongoing field research on conservation tillage practices. The bottom section of each Petri dish cover was covered with a square of kitchen-type plastic wrap to retard moisture loss before the l i d was pressed on. Allelopathic test materials and controls. Some 2.5 m L of aqueous or organic extracts were required for thorough saturation. Water-soluble or partially water-soluble extracts were applied directly to the filter paper. Distilled water controls were used. W i t h organic solvent-soluble extracts, the solution was applied to the filter paper and allowed to dry, then distilled water was added to support germination. Controls having pure solvent applied were similarly allowed to dry before the distilled water was added. Quantification of the amount of allelopathic material applied to each sample



374


was made by weighing the amount o f extract so that a consistent ratio could be maintained. Records o f the amounts were kept so that a consistent calculation o f concentrations could be made. Incubation conditions. Preliminary trials indicated that incubation at 20 °C for 72 h i n darkness is optimal. This relatively l o w temperature allows adequate wheat growth while retarding mold development. Replication. Six Petri dishes each containing ten seeds were used for each control and for each treatment Controls accompanied all experiments. Results, parameters, and measurements. Counts o f germinated vs. ungerminated seed, length and width of coleoptile, and length of central root and stem were recorded. Means per dish and per treatment (four to six dishes) were calculated and standard statistical tests were used i n the analysis. Results This paper reports the initial results of a new study. W e collected soil samples from the Agronomy Farm at Stillwater at monthly intervals (April-July, 1985) and extracted them with isopropyl alcohol i n a Soxhlet apparatus. The bioassay o f the dried extracts o f soil from no-tillage vs conventional-tillage plots is shown i n Tables I, l i a and III. Suprisingly both types o f soils were inhibitory to the growth o f wheat. The Soxhlet extract o f June soil was dried i n a rotary evaporator and then subjected to successive methyl alcohol and water extractions. The bioassay results are shown in Table l i b and show that the conventional-tillage soil contained no more allelopathic material than did the no-tillage soil. In view o f a laboratory error that influenced these results, we believe that the extract o f notillage soil may have equalled or exceeded the conventional-tillage s o i l i n allelopathic potency. S o i l collected at other locations ( E l Reno and Altus) were extracted and steam distilled as described i n Experimental. No-tillage samples o f soil were used; however, they varied from Altus plots devoted to monoculture for 10 years previously, to soil that had been used to grow wheat only one previous year ( E l Reno) which had been part o f a virgin prairie until cultivation. The results, shown i n Table I V , again indicate that both these no-tillage soils are allelopathic toward wheat. Products o f steam distillation, although this is a severe treatment, showed slight growth inhibition by the initial (pH 6.0) fraction, whereas those form the highly basic soil suspensions were markedly inhibitory. Table V shows the number o f compounds obtained from T i l l m a n s o i l obtained by steam distillation as identified by the C G C / M S / D A system; the soil has quite an array of organic compounds ~ some quite complex (12,14). In the milder treatment by solvent extraction for E l Reno soil, shown in Table IVb, the aqueous fraction was more allelopathic than the ethyl acetate/methylene chloride fraction. The duplicate plots (Table IVb, no-till I & II) showed some difference in bioassay results which cannot be explained just now. A l s o shown i n Tables I-IV are the amounts o f crude organic extract and the amount o f soil extracted; each represents the quantity that was bioassayed per wheat seed. These amounts represent less than the soil mass i n the normal seedling environment. The quantity o f organic matter that is present i n the soil around the germinating seed and seedling is striking. It strongly suggests that this soil organic matter is a subject about which scientists should be concerned. In fact, solvent extraction (Table IVb) shows the quantity of organics in the soil and is representative o f what would be found i n nature. Presented i n Figures l a and 2a are reconstructed partial total ion current chromatograms obtained by the C G C / M S / D A run on the M a y , 1985 samples. Figures l b and 2b show mass spectra taken at a certain specified time and peak number. Figure l b shows the mass spectrum o f phthalate plasticizer i n the soil


Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987. C

10

32 2

19

Significantly different from control at 9 5 % level of confidence or better (i-test).

6.5 ± 0.8

b

Not significantly different from control (t-test).

C

5.3 ± 0 . 7

Inhibition % Root Shoot

b

20.7 ± 4.2

C o n v - T i l l , A q . Ext.

b

6.6 ± 0.2

Shoot Length

c

15.5 ± 2.9

N o - T i l l , A q . Ext.

(mm)

23.1± 0.9

2

Root Length

Control, Dist. H 0

Experimental Soil

Collected A p r i l 9,1985

0.75

0.76

(mg/seed)

Amount of Crude Organic Extract

Table L Wheat Bioassay O f Soxhlet Extracts of Wheat Soil


3.6

3.5

(g/seed)

Amount of Soil Extracted


A

3.3 ± 0 . 5

C o n v - T i l l , A q . Ext. a

a

4.9 ± 1.3

N o - T i l l , A q . Ext.

{mm)

18.0 ± 1.7

2

Root Length

Control, Dist. H 0

Experimental Soil

a

2.9 ± 0 . 3

3.5 ± 0 . 5

5.7 ± 0 . 1

Shoot Length

a

92

83 49

39

Inhibition % Shoot Root

Collected June 10,1985

1.4

0.7*

(mg/seed)


Table Π. Wheat Bioassay O f Soxhlet Extracts of Wheat Soil


2.5

2.8

(g/seed)

Amount of S o i l Extracted


Β

22.8 ± 1.7

18.2 ± 3 . 3

N o - T i l l , M e O H Ext.

Conv-Till,MeOHExt.

5.0 ± 0.9

6.2 ± 0.5 b

C

6.5 ± 0.7

24

4 22

5


c

b

a

*Lost about one-half of extract by accident. Significantly different from control at 99.9% level of confidence or better ft-test). Significantly different from control at 9 5 % level of confidence or better (i-test). Not significantly different from the control (t-test).

b

C

23.8 ± 4.6

2

Control, Dist. H 0 andMeOHExt.

(mm)

Root Length

Experimental Soil

Shoot Length

1.4

0.7*

(mg/seed)



2.5

2.8

(g/seed)



18.7 ± 2 . 5

Conv-Till, A q . Ext.

b

16.6 ±1.9

N o - T i l l , A q . Ext. b

b

W

5.2 ± 0 . 6

5.2 ± 0 . 3

6.5 ± 0.7

Shoot Length

b

b

22

30 20

20


Significantly different from control at 9 5 % level of confidence or better (t-test).

b

23.8 ± 4.6

2

Root Length

Control, Dist. H 0

Experimental Soil

Collected July 9,1985

0.58

0.45

(mg/seed)


Table ΠΙ. Wheat Bioassay O f Soxhlet Extract of Wheat Soil


2.0

1.8

(g/seed)


Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987. Root Length (mm)

23.7 ± 1.7

3.9 ± 1.4a

No-Till Made at p H 5.9 (natural)

No-Till Made at p H 11

C

24.4± 0.7

2

Control, D i s t . H 0

Α-Steam Distillation, Extraction, Evaporation (2-kg sample)

Method of Obtaining Organics from Experimental Soil

2.7 ± 0 . 6

6.5 ± 0.3

7.2 ± 0.6

Shoot Length

a

C

85

10

10

10


67 3.5

Continued on next page

67

Amount of Soil Extracted (g/seed)

2.9

Amount of Crude Organic Extract (mg/seed)

Table IV. Wheat Bioassay O f (A) Fractions O f Steam Distillates of Altus, O K and (B) Solvent Extracts of E l Reno, O K Wheat Soil



Continued

Root Length

Significantly different from control at 9 5 % level of confidence or better (i-test).

0

Not significantly different from control ft-test).

11

0

13

e

e

17

39

Significantly different from control at 99.9% level of confidence or better (t-test).

7.2 ± 0.6

e

b


b

e

7.3 ± 0.7

6.3 ± 0.7

7.2 ± 0.6

Shoot Length

a

2

23.4 ± 0.2

No-Till-Plot I EtOAc + CH C1

2

21.8 ± 5.2

No-Till-Plot Π A q . Ext.

b

e

16.2±2.5

No-Till-Plot I A q . Ext.

2

24.4 ± 0.7

(mm)

Control, Dist. H 0

B-Solvent E x t r a c t i o n and Evaporation (200-g s a m p l e )

Method of Obtaining Organics from Experimental Soil

Table IV.

2.5

2.5

Amount of Crude Organic Extract (mg/seed)


6.7

6.7

6.7

Amount of Soil Extracted (g/seed)


*A11 ethyl esters

Totals

Fatty acids Fatty acid esters Alcohols Aldehydes Ketones C - N and other N-contg, compounds S-contg. compounds Cl-contg. compounds Aromatics not otherwise included Aliphatics not otherwise included

39

8 0 1 3 1 4 1 2 7 11

Initial

75

0 0 3 8 2 17 2 0 23 16

20 3* 2 6 4 5 1 0 1 33

75

Basic

Acidic

Table V . Compound Groups Obtained From Soil B y Steam Distillation A s Identified B y C G C / M S / D A System


382


400 ' 500 ' 600 ' 760 860960' 100θ' ϊίββ' 1200" 1300' ΐ4θβ' 1*00 1600 Time,

Seconds

!


b

' 2 5 ' W ' ' ''' 75^ i00 J

J,,

,,,ml

US • ' lié'" i?S " 260 " ÏH ' 256 " 1275 " 360 m/z

Figure 1. a) Reconstructed Part of the Total Ion Current Chromatogram of a No-Tillage Soil Extract (LKB-2091 C G C / M S / D A ) : Peaks Represent Compounds. Soil Sample: M a y 9,1985 b) Mass Spectrum o f a Phthalate Plasticizer Present i n the Soxhlet Soil Extract That Corresponds to Peak (750 s) Marked with the Cursor.

m/z

Figure 2. a) Reconstructed Part of the Total Ion Current Chromatogram of a Conventional-Tillage Soil Extract (LKB-2091 CGC/MS/DA): Peaks Represent Compounds. S o i l Sample: M a y 9,1985 b) Mass Spectrum of a Hydrocarbon Present i n the Soxhlet Soil Extract That Corresponds to Peak (1338 s) Marked with the Cursor.


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WALLER ETAL.

Tillage in Wheat

Production

383


extract giving the peak at 749 s. We believe it to be an actual soil component and not an artifact of our work since our handling of the sample used all-glass equipment with Teflon stopcocks and closures without stopcock grease. We have not determined whether or not it is phytotoxic. Figures 2b is a mass spectrum of a hydrocarbon from the soil extract It is probably not an allelopathic compound. Allelopathic activity toward germinating wheat Was clearly demonstrated with extracts of Oklahoma soils. However, a convincing difference in activities in notill and conventional-till soil has not yet appeared. The total ion current chromatograms (Figures la and 2a) of the CGC/MS/DA illustrate the complexity of the soil extracts. Some of these compounds, if isolated, may serve as new effective biodegradable insecticides, herbicides, or fungicides. Acknowledgment The critical review of this manuscript by Otis C. Dermer is appreciated. This is Journal Article No. 4933 of the Oklahoma Agricultural Experiment Station, Oklahoma State University Stillwater, Oklahoma, 74078. We acknowledge with appreciation the asistance of Thanh Dao of the USDA, Livestock and Forage Research Laboratory, El Reno, Oklahoma, in collecting the soil samples. Literature Cited 1. McCalla, T. M.; Haskins, F. A. Bacteriol. Rev. 1964, 28, 181-207. 2. McCalla, T. M. In Biochemical Interactions Among Plants: Natl. Acad. Sci. U.S.A.: Washington, DC, 1971; pp. 39-43. 3. McCalla, T. M.; Norstadt, F. A. Agric. Environ. 1974, 1, 153-174. 4. Whittaker, R. N. "The Biochemical Ecology of Higher Plants. Chemical Ecology" Sondheimer; E.; Simeone, J. B., Eds.; Academic Press: New York, 1970; pp. 43-70. 5. Waller, G. R.; Nowacki, Ε. K. "Alkaloid Biology and Metabolism in Plants" Plenum Press: New York, 1978; 294 pp. 6. Rabotnov, Τ. Α., Soviet J. Ecol. 1981, 12, 127-131. 7. McCalla, T. M., Studies on Phytotoxic Residues from Soil, Microorganisms and Crop Residues, Lincoln, Nebraska, Nebr. Agric. Exp. Stn. Bull No. 2257, 1-8. 8. Elliott, L. F.; McCalla, T. M.; Waiss, Α., Jr. Crop Residue Management Systems: Am. Soc. Agron., Spec. Pub. No. 31, 1978; Chap. 7, pp. 131146. 9. Elliott, L. F.; Gilmour, C. M.; Lynch, J. M.; Titlemorre, D. Microbial-Plant Interactions: Am. Soc. Agron., Spec. Pub. No. 47, 1984; Chap. 1, pp. 124. 10. Putnam, A. R.; DeFrank, J. Crop Protect. 1983, 2, 173-181. 11. Lehle, F. R.; Putnam, A. R. Plant Physiol., 1982, 69, 1212-1216. 12. Schilling, D. G.; Liebl, R. Α.; Worsham, D. A. "The Chemistry of Allelopathy: Interaction Between Plants" Thompson, A. C., Ed.; ACS SYMPOSIUM SERIES No. 268, American Chemical Society: Washington, DC, 1985; pp. 243-71. 13. Waller, G. R.; Ritchey, C. R.; Krenzer, E. G., Jr.; Smith, G.; Hamming, M. 30th Ann. Conf. Am. Soc. Mass Spectrom. Allied Topics, San Antonio, TX, 1984; Abstract MPB14, pp. 144-145. 14. Waller, G. R.; McPherson, J. K.; Ritchey, C. R.; Krenzer, E. G., Jr. Smith, G.; Hamming, M. Abstracts of Papers, 190th American Chemical Society Meeting, Chicago, IL, September 9-14, 1985; AGFD 110. 15. McPherson, J. K.; Muller, C. H. Ecol. Monog. 1969, 39, 198 pp. RECEIVED June 16, 1986


Allelopathic Influences on No Tillage Versus Conventional Tillage in

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