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RELATIVE CONCENTRATION OF ISOTHIOCYANATES IN WATER AND IN SOIL AND THE IMPLICATIONS FOR SOILBORNE PATHOGEN CONTROL

J.W. NichollsA, V. BiancoA, D. AllenB and I.J. PorterA

A Agriculture Victoria, Institute for Horticultural Development Knoxfield, Private Bag 15, South Eastern Mail Centre, 3176, VIC
B
Agriculture Victoria State Chemistry Laboratory, Cnr. Sneydes and South Roads, Werribee, 3030, VIC

ABSTRACT

Allelochemicals, such as those released by plants of the Brassicaceae family (biofumigation), have been proposed as an alternative to synthetic chemical fumigants for the control of soilborne pathogens. Five isothiocyanates (ITCs) were identified and quantified from freeze-dried root tissues of a composite fodder mix of Brassica napus + B. campestris and B. juncea. It was shown in the laboratory, that ITC concentrations varied eightfold (0.8 to 7.5 Ámol ITC/g root tissue). However, between 4 and 66% of the ITC concentrations were present within 24 hours of incorporation. This dramatic reduction in ITC concentration may affect the potential soilborne pathogen control. In-situ efficacy studies are required to confirm these observations.

KEYWORDS Allelochemicals, biofumigation, glucosinolate hydrolysis products, metham sodium, Brassica spp.

INTRODUCTION

Biofumigation is the practice of using plants of the Brassicaceae family (eg. Brassica and Raphanus spp.) that produce toxic chemicals (allelochemicals) to suppress soilborne pathogens such as Aphanomyces euteiches (Smolinska et al. 1997), Pythium ultimum and Sclerotium rolfsii (Gamliel and Stapleton 1993). The allelochemicals are the hydrolysis products of a group of chemicals known as glucosinolates (GLCs). When these plants are incorporated into soil, myrosinase enzymes hydrolyse the GLCs to form isothiocyanates (ITCs), nitriles and oxazolidinethiones. One of the volatile allelochemicals produced by some biofumigant species (methyl ITC) is the same as the hydrolysis product of the synthetic chemical fumigant, metham sodium, used in intensive horticulture for the control of soilborne pathogens.

Biofumigation offers a potential biological alternative to synthetic chemical fumigants. It may have the potential to provide a sustainable pest and disease control option in integrated pest management systems and improve soil health. The incorporation of biofumigation crops into soil provides valuable organic matter for the following crop, possibly reducing the dependence on artificial fertilisers. Part of the process of determining the efficacy of biofumigation on soilborne pathogens, requires in-situ work to determine both the identity and quantity of GLC hydrolysis products. Much of the work to date has determined empirical levels of volatile or water soluble hydrolysis products in petri dishes (Ámol/mL agar, Sexton et al. 1997) or in the headspace of flasks (Ámol/cm3, Vaughn and Boydston 1997). Such situations bear little resemblance to that experienced in the field.

The possible changes in identity and quantity of GLC hydrolysis products following the incorporation of biofumigant crops into a sandy soil, compared to that potentially available in crop tissues, has been suggested by Brown et al. (1991). Such studies however, have not been investigated in light clay loams, a soil type commonly used to grow Australian horticultural crops in. This paper reports on a laboratory evaluation that identifies and quantifies the ITCs present prior to (potential) and following incorporation of freeze-dried Brassica tissues with a light clay loam. Investigations using Brassica root tissues will be reported in these studies. These results are discussed in terms of their relative success in controlling soilborne pathogens using literature values.

MATERIALS AND METHOD

Potential level of ITCs present in Brassica root tissues

Freeze-dried root tissue of Brassica juncea and a composite fodder mix of B. napus + B. campestris (Bn/Bc) (0.5 g) was added to separate extraction solutions (15 mL ethyl acetate and 15 mL distilled and deionised water) in duplicate 250 mL centrifuge tubes. Samples were shaken for 1 hour, then centrifuged (Leistra et al. 1974) and the supernatant retained in a sealed gas chromatograph vial.

ITCs present after incorporation of Brassica root tissues into soil

Freeze-dried root tissue of B. juncea and the Bn/Bc mix. (2.5 g) was incorporated with 132 g light clay loam (14.6% (w/w) moisture) in duplicate 500 mL Schott bottles. This is equivalent to a 2.0% (w/w) mixture of Brassica tissue in soil. Bottles were rolled at regular intervals to mix the contents. At 1, 4 and 24 hours after incorporation, 22.0 g of soil + root tissue was removed and placed in extraction solutions and treated as described above.

Gas chromatography

Isothiocyanates were identified from the extraction solution by gas chromatography mass spectrometry. Electron impact mass spectra in the range 35-250 amu were recorded on a Shimadzu QP5000 instrument using a 1ÁL splitless injection (at 220░C) of concentrated freeze-dried Brassica tissue extract onto a 30m x 0.25mm ID DB-5MS 0.25Ám column, being temperature programmed from 40░C to 250░C. Benzyl, 3-butenyl, 2-phenylethyl, 4-methylthiobutyl, and 5-methylthiopentyl ITC were identified by mass spectral library search and 4-pentenyl ITC was tentatively identified by mass spectral interpretation with reference to the occurrence and the characteristic ions reported by Gardiner et al. (1999).

Isothiocyanates were quantified by gas chromatography with flame photometric detection in sulphur mode, after separation on a 15m x 0.32mm ID Rtx-50 0.25Ám column using a 2ÁL split injection at 220░C. The oven of the Perkin Elmer 8500 GC was temperature programmed after 2 minutes from 45░C to 250░C at 10░C/minute. Isothiocyanate concentrations were estimated from a non-linear calibration curve constructed for methyl ITC standards at concentrations of 0.4, 1, 4 and 10 Ág/mL.

RESULTS

Potential level of ITCs present in Brassica root tissues

Overall, the Bn/Bc mix contained more than eight times the quantity of ITC (7.5 Ámol ITC/g root tissue) than B. juncea (0.9 Ámol ITC/g root tissue) (Figure 1). The concentrations of individual ITCs varied from 1.9 Ámol ITC/g root tissue in the Bn/Bc mix to less than 0.1 Ámol ITC/g root tissue in B. juncea. The relative concentrations of 3-butenyl, 4-pentenyl and 2-phenylethyl ITC in the Bn/Bc mix were respectively 8.4, 22 and 3.4 times that in B. juncea. Benzyl, 4-methylthiobutyl and 5-methylthiopentyl ITC were present only in the Bn/Bc mix.

ITCs present after incorporation of Brassica root tissues with soil

On adding to soil, between 4 and 66% of ITCs potentially available, were present during the 24 hour period (Figure 2). The total concentration of ITCs present in the Bn/Bc mix was 2.98 Ámol ITC/g root tissue and for B. juncea 0.60 Ámol ITC/g root tissue. The proportion of ITC present in soil (as a percentage of that present without soil) was twice as high for B. juncea than the Bn/Bc mix (Table 1). In B. juncea, the highest proportion of ITC present was from 1 to 4 hours after incorporation, with 86% of 2-pentenyl and 65% of 3-butenyl and 2-phenylethyl present. In the same time period, the Bn/Bc mix had 28% of 2-pentenyl and between 35 and 53% of 3-butenyl and 2-phenylethyl present compared to that without soil. After 24 hours, the ITCs present in soil were less than 10% that without soil, except that of 4-pentenyl ITC, which remained above 70%. The ITCs identified from Brassica root tissue was similar to that without soil, however benzyl ITC was not identified in either the Bc/Bn mix or B. juncea.

Figure 1. Level of ITCs present in Brassica root tissues

Figure 2. Level of ITCs present following incorporation of Brassica root tissues into soil

Table 1. Concentration and percentage ITC present in Brassica tissues combined with soil

   

μmol ITC/g root tissue combined with soil

% ITC (of potential) that remains in soil

Species

Hrs

3-b

4-p

2-p

5-m

4-m

Tot

3-b

4-p

2-p

5-m

4-m

Av

B. juncea

1

0.17

0.06

0.31

-

-

0.54

66

80

52

-

-

58

B. juncea

4

0.17

0.06

0.37

-

-

0.60

65

86

65

-

-

66

B. juncea

24

-

0.05

0.05

-

-

0.10

-

72

9

-

-

11

B.n + B.c

1

0.51

0.40

0.69

0.32

0.09

2.02

25

22

35

24

34

27

B.n + B.c

4

0.72

0.51

1.03

0.57

0.14

2.98

35

28

53

43

53

40

B.n + B.c

24

0.07

-

0.15

0.10

-

0.32

3

-

7

-

-

4

B.n + B.c = B. napus + B. campestris, Hrs = hours after incorporation of Brassica tissue with soil, Tot = Total ITC concentration, Av = Average ITC concentration, 3-b = 3-butenyl, 4-p = 4-pentenyl, 2-p = 2-phenylethyl, 5-m = 5-methylthiopentenyl, 4-m = 4-methylthiobutyl

DISCUSSION

Potentially, more than eight times the quantity of ITCs were available in the Bn/Bc mix than in B. juncea. After incorporation into soil, between 4 and 66% of these ITCs were identified in the Brassica tissues. The highest ITC concentrations were present within the first 4 hours of incorporation, with dramatic reductions between 4 and 24 hours. This suggests that ITC concentrations in isolated Brassica root tissues are not reliable indicators of the ITC concentration after these tissues have been incorporated into soil.

The variation in the identity and quantity of ITCs present in Brassica tissues, indicates the GLC degradation products are species dependent and are influenced by the edaphic environment in which the tissues are incorporated. This is consistent with other studies (Brown and Morra 1997). In addition to the GLC hydrolysis products identified in this work, a number of nitriles and oxazolidines have been identified in B. napus tissues (Gardiner et al. 1999, Smolinska et al. 1997), adding to the potential efficacy on soilborne pathogens.

The identity and quantity of the individual ITCs present in plant tissues will determine efficacy on soilborne pathogens. For example, methyl, propenyl, 3-butenyl and 4-pentenyl ITC are between 13-20, 9-20, 3-10 and 1-5 times more effective against the soilborne pathogens Pythium graminearum, Fusarium oxysporum, Rhizoctonia solani and Biopolaris sorokiniana compared to methyl bromide (MB) (Desmarchelier and Vu, 1998). This indicates that of the biofumigant species available, not all ITCs present will be equal in their effect on soilborne pathogens, therefore influencing the selection of species as a biofumigant. The relatively high efficacy of methyl ITC compared to MB suggests that application technologies and timing of fumigation could increase the situations where metham sodium replaces MB.

The total ITC concentration reported in this current work, 3.0 μmol ITC/g root tissue (156 nmol/g soil), and that reported by Gardiner et al. (1999) in a field study with B. napus (30 nmol ITC/g soil), is much less than the estimated ITC concentration released by the commercial fumigant, metham sodium (1241 nmol methyl ITC/g soil) applied at the label rate (450 kg product/ha equivalent to 218 kg methyl ITC/ha) and incorporated to a depth of 20 cm (soil bulk density 1.2 g/cm3). This suggests that for the quantities of Brassica tissue used, biofumigation will probably not produce the required quantity of allelochemicals required to control soilborne pathogens, compared to that from a short-term exposure associated with a chemical fumigation. However Gardiner et al. (1999) were careful to point out that the concentrations quoted are only moments in time and the effect continuous exposure to sub-lethal levels of ITCs has on soilborne pathogens is not fully understood. In reality, biofumigation crops are grown for extended periods of time, and the concentration at a particular instant may be less important that the total amount of GLC hydrolysis products present.

CONCLUSION

In this study, the amount of ITCs present in soil after incorporation of Brassica root tissues was between 4% to 66% of that present prior to incorporation and was thought to be below the concentration required for control of soilborne pathogens. This reduction in ITC concentration must be taken into account when evaluating the efficacy of biofumigation against soil borne pathogens in the field. In-situ measurements of GLC hydrolysis products from Brassica spp. incorporated into soil need to establish the relationship between those hydrolysis products available prior to and after incorporation and the potential efficacy on soilborne organisms. Care must be taken to interpret these data, as climatic and edaphic conditions will influence the result enormously.

ACKNOWLEDGMENTS

The authors would like to thank Environment Australia for their funding of this project and Wrightsons Research for supplying the freeze-dried Brassica tissues.

REFERENCES

1. Brown, PD. and Morra, J.M. (1997). Control of soil borne plant pests using glucosinolate-containing plants. In ‘Advances in Agronomy,’ Vol. 61, ed. D.L. Sparks, pp 167-231. (Academic Press, New York, USA).

2. Desmarchelier, J.M. and Vu, L.T. (1998). Determination of effective fumigant concentrations in different soil types for methyl bromide and other fumigants. Final RIRDC Report. Project PWD 38. CSIRO Canberra.

3. Gamliel, A. and Stapleton, J.J. (1993). Characterisation of antifungal volatile compounds evolved from solarised soil amended with cabbage residues. Phytopathology. 83. 899-905.

4. Gardiner, J.B., Morra, M.J., Eberlein, C.V., Brown, P.D. and Borek, V. (1999). Allelochemicals released in soil following incorporation of rapeseed (Brassica napus) green manures. Journal of Agriculture and Food Chemistry. 47. (in press).

5. Leistra, M., Smelt, J.H. and Nollen, H.M. (1974). Concentration-time relationships for methyl isothiocyanate in soil after injection of metham sodium. Pesticide Science. 5. 409-417.

6. Sexton, A.C., Kirkegaard, J.A. and Howlett, B.J. (1999). Glucosinolates in Brassica juncea and resistance to Australian isolates of Leptosphaeria maculans, the blackleg fungus. Australasian Plant Pathology. 28. 95-102.

7. Smolinska, U., Morra, M.J., Knudsen, G.R. and Brown, P.D. (1997). Toxicity of glucosinolate degradation products from Brassica napus seed tissue toward Aphanomyces euteiches f.sp. pisi. Biological Control. 87(1). 77-82.

8. Vaughn, S.F. and Boydston, R.A. (1995). Phytotoxicity of Brassica species. volatiles to weed and crop seed germination and growth. In. ‘WSSA Abstracts’ (C.C. Kupatt. Ed.). p. 55. Weed Science Society of America. Champaign, IL.

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