Biocidal plant dried pellets for biofumigation

Industrial Crops and Products 20 (2004) 59–65

Biocidal plant dried pellets for biofumigation L. Lazzeri∗ , O. Leoni, L.M. Manici Research Institute for Industrial Crops (ISCI—MIPAF), Via di Corticella 133, Bologna 40128, Italy Received 1 April 2002; accepted 22 December 2003

Abstract The presence in all Brassicaceae plant organs of high amounts of glucosinolates, and of the enzyme myrosinase that catalyses their hydrolysis, linked to the high biocidal activity of some glucosinolate enzymatic hydrolysis derivative products (mainly isothiocyanates and nitriles) have suggested the practical possibility of amending soil with these natural biocidal compounds by the cultivation and green manure of selected species of this family. The application of this technique even at full field level has given, in recent years, interesting applicative results, with clear advantages for the following crop yield (e.g. strawberry), if compared with an untreated soil or a conventional green manure, evidencing that the glucosinolate–myrosinase system may provide a natural alternative for methyl bromide soil fumigation. In this paper, some preliminary results of a study on the possibility of drying some of these selections to produce biocidal pellets to be used as organic treatments in addition or in alternative to biocidal green manure are reported and discussed. The first target was to limit, during drying, glucosinolate leakage and myrosinase activity loss; in this way, in fact, dry plants should be able to produce in soil the biocidal compounds when watered by irrigation. Using a simple drying up lab technique, it was possible, for some selections, to lose <40% of starting glucosinolate and to preserve myrosinase activity at a sufficient level. These results could be further improved using industrial dehydration plant. The dried plants, after water addition, showed, in vitro, a good fungitoxic activity on Pythium ssp. and Rhizoctonia solani, confirming the results obtained in similar tests with pure glucosinolate and pure myrosinase. These results open interesting applicative perspectives for this new raw material probably even as a natural alternative to methyl bromide. © 2004 Elsevier B.V. All rights reserved. Keywords: Glucosinolate; Myrosinase; Cleome hassleriana; Pythium ssp.; Rhizoctonia solani

1. Introduction The production of several vegetable crops depends on the use of methyl bromide (MB) soil fumigation to control a wide array of soil-borne fungi, nematodes, ∗ Corresponding author. Tel.: +39-051-6316835; fax: +39-051-374857. E-mail address: [email protected] (L. Lazzeri).

insects and weeds, but, in accordance with the US Clean Air Act, the use of MB as a fumigant will be banned in developed countries by 2005 (Anonymous, 1992). This decision has prompted increased interest in the development of alternative strategies to control soil-borne pathogens with a low environmental impact. One alternative approach might be provided by the well-known biocidal activity of enzymatic (via myrosinase) hydrolysis derived products (DPs) of glu-

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cosinolates (GLs) (Walker et al., 1937), typical natural compounds of the Brassicaceae and other minor families (Rosa et al., 1997). The high biological activity of these compounds (mainly isothiocyanates and nitriles) on various fungal agents and nematodes was clearly observed in vitro using both pure compounds (Manici et al., 1997; Lazzeri et al., 1993) and different plant materials (Lazzeri and Manici, 2001). These results suggested the possibility of cultivating selected Brassicaceae plants as biocidal green manure crops to amend soil with high levels of organic matter and natural bioactive compounds. The application of this technique even at full field level in recent years evidenced some advantages for the following crop yield (Kirkegaard et al., 1993) even if compared with a not treated soil and a conventional green manure (Lazzeri et al., 2003). The full field application of this technique is easy and practical, but it presents the typical green manure disadvantages, that could be overcome by the production of organic dried plants containing high levels of glucosinolate–myrosinase system. In fact, the dried materials could be used as organic treatments for biofumigation, in addition or even in alternative to biocidal green manure, opening interesting applicative perspectives in several horticultural fields. The aim of this study was to explore the possibility of dehydrating biocidal plants. These dried plants should contain a high level of GLs and a quantity of myrosinase (MYR) sufficient to hydrolyze them; in this way, in fact, dehydrated plants, only after water addition, should be able to produce the biocidal compounds. The potential suppressive activity of these dried materials against Pythium spp. and Rhizoctonia solani, two widespread soil-borne pathogens that cause significant losses in agricultural and horticultural crops world-wide, was also, in vitro, evaluated and discussed.

2. Materials and methods 2.1. Biocidal plants cultivation In 2000, several plant selections characterized by a high content of GLs able to produce highly fungitoxic DPs (Rapistrum rugosum sel. ISCI4, Cleome hassleriana sel. ISCI2, Brassica juncea sel. ISCI20, B. juncea sel. ISCI61, Iberis amara sel. ISCI31, Le-

pidium sativum sel. ISCI101) were cultivated in the Po Valley (Budrio, Bologna) (continental climate), in 15 m2 plots. The soil was medium clayey with a good phosphorous and potassium content, and was amended with 80 U ha−1 of N and 100 U ha−1 of sulfur to stimulate GLs synthesis. Sowing was performed in the first week of March, with a plant density of approximately 200 seeds m−2 , a distribution in continuous rows and an inter-row spacing of 18 cm. During cultivation, irrigation and defense treatments were, as expected, unnecessary. 2.2. Dehydration process At full flowering time, plants were manually cut and dehydrated in a ventilated oven at a temperature of 40 ◦ C for 3 days. Some preliminary trials of dehydration process in full field conditions and in an oven by an industrial plant at high temperatures (from 100 to 250 ◦ C) for 10–20 min were also performed. After dehydration, plants were grinded and passed through a press for pellets Mod. VM. 535/2 of Tekno (S. Vito di Bussolengo, VR). 2.3. Glucosinolate and myrosinase determination At full flowering time, a sample of each GLs containing plant was collected and immediately frozen in liquid nitrogen, stored at −20 ◦ C and subsequently freeze-dried using an Edwards Minifast Do. 1 freeze-drier (from −40 to 18 ◦ C in 8 h with a vacuum of 10−1 mbar). The same sampling procedure was used also to follow the GLs level in cut plants during dehydration process. Dehydration process was considered to be finished when the residual humidity became lower than 10%. To determine the GLs content, the freeze-dried materials were homogenized in a mortar and analyzed essentially following the procedure proposed for seed analysis in the Official Journal of European Community Regulation (1990), with some minor modifications. GLs were determined as desulfo-GLs by HPLC analysis, using a Hewlett Packard chromatograph Model 1090L equipped with a diode array detector and a 200 mm ×4.6 mm column HP ODS Hypersil C18, 5 ␮m. Elution was performed with a CH3 CN gradient in water 1–22% in 22 min at a flow of 2 ml min−1 , with a column temperature of 35 ◦ C.

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To confirm the presence of myrosinase after dehydration process, dried plants were homogenized in a vial with Phosphate buffer 50 mM pH 6.5 (1:8 (w/v)) by a homogenizer Ultra-Turrax Ika Werk Staufen TP18/2K, Germany. The screw-capped vials were kept in a water bath at 37 ◦ C for 8 h. The homogenates were shaken with CH2 Cl2 to extract DPs (four extractions), and the extracts were analyzed by GC–MS with a GCD Hewlett Packard, mod. G1800A, using a 30 m × 0.25 mm capillary column HP-5. The flow rate of the carrier gas (He) was 1 ml min−1 and the sample (1 ␮l) was injected in the splitless mode. Column temperature was 40 ◦ C at the start and 220 ◦ C at the end of the analysis, with an increase rate of 10 ◦ C min−1 . The temperature of injector and detector was 220 and 260 ◦ C, respectively. Mass spectra were scanned in the 10–425 m/z range. The same procedure was repeated after addition to the dried plants of exogenous epi-progoitrin (2-hydroxy-but-3-enyl glucosinolate), isolated from Crambe abyssinica seeds at a purity level higher than 85%, to verify the production of the corresponding degradation products (nitrile and vinyl-oxadolidine-thione (VOT)). 2.4. Dried plant fungitoxicity The in vitro fungitoxic activity of B. juncea ISCI61, I. amara ISCI4 and C. hassleriana ISCI2 dried plant tissues were determined. B. juncea ISCI20 dried tissues, characterized by a low GL content, and sterile water were also included in the trials as controls. The amount of GLs was fixed at 30 ␮mol per plate on the basis of previous experiences carried out with pure GLs (Manici et al., 2000). Consequently, the amount of dry tissues used for tests was calculated on the basis of their GLs content (Table 1).

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Two soil borne pathogens, well-known as two of the main agents of “root rot”, Pythium irregulare and R. solani AG-5, were chosen as test fungi. Fungitoxic activity was assayed both on poisoned media (using water extract of dry tissues) and in vapor phase. Each experiment was organized in a two factor analysis: GLs activity of chopped dry tissues of four Brassicaceae selection, after water addition, with and without MYR addition. 2.5. Fungitoxicity of soluble compounds The GLs degradation product fungitoxicity of selected biocidal dried plants was tested by poisoned media method (Manici et al., 1997). The experiments were organized in a randomized design with three replicates. Dried plant tissues, calculated to obtain 3 ml of water extract containing the fixed GLs amount per each plate (Table 1) were maintained under shaking in water at 37 ◦ C overnight in a sealed 40 ml cap tube. Water extracts were centrifuged at 17,000 rpm for 30 min, filtered on Whatman 25 mm GD/X sterile syringe filters (0.2 ␮m pore size). Two hours before test starting, the procedure with MYR addition were enriched with exogenous MYR (25 U ml−1 ) previously purified starting from Sinapis alba seed (Palmieri et al., 1986). Water extract (3 ml per plate, diameter: 9 cm) was added with 7 ml of melted potato sucrose agar (PSA) + 100 mg l−1 of streptomycin sulfate cooled at 40 ◦ C. Four millimeter disks from 4-day-old colonies of P. irregulare and R. solani were inoculated at the center of each plate with poisoned media. 2.6. Fungitoxicity of volatile compounds The chopped dried tissues amount per plate, arranged on the plate base, were added with a water

Table 1 GLs content in dehydrated tissues and their amount in each plate for fungitoxicity trials

B. juncea ISCI61 I. amara ISCI4 C. hassleriana ISCI2 B. juncea ISCI20 (control) a

GLs content in dried tissues (␮mol g−1 )

Dried tissues per plate (g)

GLs per plate (␮mol)

6.5 11.1 16.6 1.0

4.6 2.7 1.8 4.6a

30 30 30 4.6

The same amount of B. juncea ISCI61 dried tissues.

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amount sufficient to obtain tissues wetting. The procedures with exogenous MYR addition were enriched with 20 ␮l of the above reported enzyme solution. Plates with 10 ml of PSA were inoculated with Pythium and Rhizoctonia, reversed on plate base containing dried tissues and sealed with parafilm “M” (American National Can, Chicago). In each trial (soluble and volatile released compounds), plates were incubated at 24 ◦ C for 3 days (Pythium) and 4–5 days (Rhizoctonia); colony diameters were measured when the control colony (water) had reached the edge of the Petri plate. Data were expressed as fungal growth inhibition: effectiveness index (EI, %) = 100(diameter control − diameter treatment)/diameter control. In each experiment, arcsin transformed data were subjected to two way analysis of variance by using Statgraphic Plus Program, version 2 (Manugistatic Inc. Rockville, Maryland, USA).

3. Results and discussion During cultivation, the selected species confirmed their good adaptation to spring sowing in the Po Valley pedoclimatic conditions, with sufficient vegetative growth and abundant flowering. The cycle length from sowing to full flowering varied from 55 to 106 days, depending on the sowing time and the species. Laboratory dehydration process maintained a residual GLs content (percentage of initial GLs content in intact fresh plants) that significantly varied with the species (Table 2). Dried Cleome (16.6 ␮mol g−1 on dry matter (DM)) and Iberis (11.1 ␮mol g−1 on DM) plants,

in fact, showed the highest GLs residual content (respectively, 62.9 and 60.9% of initial GLs content in fresh plants). B. juncea ISCI20(6.5 ␮mol g−1 on DM) gave the lowest residual GLs content (16.1%), probably due to the high biomass content of these plants that made their dehydration longer and difficult. Cleome and Iberis applicative perspectives seem interesting even from an agro-technological point of view. These plants, in fact, can be cultivated in spring or in summer cultivation cycle, with a biomass yield that, in same trials carried out in the Po Valley, ranged from 35 to 45 t ha−1 on fresh matter, following the different sowing times. Preliminary trials on full field dehydration did not show interesting applicative perspectives, particularly in the presence of rains during the process (data not published). The plants dehydrated in full field, in fact, had a very low GLs content (1 ␮mol g−1 on DM), and for this reason they were used as a control in fungitoxicity tests of these materials. On the contrary, similar trials carried out by an industrial dehydration plant at temperatures ranging from 100 to 250 ◦ C for 10–20 min gave dehydrated plants with a residual GLs content as high as 80% if compared to fresh plant content (data not shown). The GC–MS analysis clearly evidenced the production of GLs degradation products after dehydrated plants wetting, confirming that dried plants contain a sufficient residual MYR amount to catalyze GLs hydrolysis. In particular, after the addition of 50 mM phosphate buffer pH 6.5 to Cleome dried plants methyl isothiocyanate (MIT) (from glucocapparin) and methyl-ethyl-oxazolidine-thione (MEOT) (from glucocleomin) (Fig. 1) were formed. It is interesting

Table 2 Glucosinolate residual content after dehydration of some biocidal selections Species

Gls content of dehydrated plants (␮mol g−1 )

Residual GLs content (%)

Main glucosinolate and purity level (%)

B. juncea sel. ISCI20 C. hassleriana sel. ISCI2 I. amara sel. ISCI14 R. rugosum sel. ISCI4 L. sativum sel. ISCI120 Treatments

6.5 16.6 11.1 12.1 12.7

16.1 62.9 60.9 41.3 46.2

SIN (96) GCA (90) GIB (98) GCH (95) GTL (99) –

∗∗

c a b b b

∗∗

c a a b b

Mean values in the same column with the same letter do not differ significantly for P ≤ 0.05 in the LSD Test. SIN: sinigrin; GCA: glucocapparin; GIB: glucoiberin; GCH: glucocheirolin; GTL: glucotropaeolin. ∗∗ P ≤ 0.01.

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Fig. 1. Glucosinolate degradation products released from Cleome pellets after 50 mM phosphate buffer pH 6.5 addition. MIT: methyl-isothiocyanate; MEOT: methyl-ethyl-oxazolidine-thione.

to emphasize that MIT serves as active compound of several synthetic fumigants (Lazzeri et al., 2000). Even the addition of exogenous GL (epi-progoitrin from C. abyssinica) to the dried plants confirmed the presence of a sufficient residual MYR amount. In the reaction mixture, in fact, together with Cleome GLs degradation products it was possible to detect the typical degradation products of epi-progoitrin (vinyl-oxazolidine thione (VOT) and 2-hydroxybut-3-enyl cyanide) (Fig. 2).

GLs degradation products were present only in traces in dried plants (data not shown), confirming that their production occurs only after water or buffer addition. To make the dried plants management and field distribution easier, they were passed through a press for producing pellets (Fig. 3). The procedure was easy and practical, did not request any additives and no significant variation in dried plant composition occurred during its application.

Fig. 2. Glucosinolate degradation products released from Cleome pellets after 50 mM phosphate buffer pH 6.5 and pure epi-progoitrin addition. MIT: methyl-isothiocyanate; MEOT: methyl-ethyl-oxazolidine-thione; VOT: vinyl-oxazolidine-thione epi-NIT 2-hydroxybut-3-enyl-cyanide.

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L. Lazzeri et al. / Industrial Crops and Products 20 (2004) 59–65 Table 4 Mean separation by LSD test (P = 0.05) of Effectiveness Index of water soluble compounds in fungitoxicity tests Contact treatments Dry tissues of I. amara ISCI4 B. juncea low GLs B. juncea ISCI61 C. hassleriana ISCI2

P. irregulare (EI (%))

R. solani (EI (%))

89 12 87 100

73 22 81 100

b c b a

b c ab a

Means followed by a common letter are not significantly different. Table 5 Mean separation by LSD test (P = 0.05) of Effectiveness Index of volatile compounds in fungitoxicity tests Volatile treatments

Fig. 3. Photograph of pellets of C. hassleriana ISC12 dehydrated plants.

The fungitoxic activity toward Pythium and Rhizoctonia of four dehydrated plants differed in a highly significant way (P ≤ 0.01) both in trials with soluble and volatile compounds (Table 3). In both trials, MYR enrichment did not cause any significant increase of fungitoxicity toward Pythium and Rhizoctonia (not significant interaction treatment × MYR). This result confirmed again that dried plants contain a MYR amount, that is sufficient, after water addition, to catalyze the hydrolysis of GLs present in their tissues. Pythium and Rhizoctonia response to volatile and to contact treatments varied with DPs chemical and physTable 3 Factorial analysis of variance of Effectiveness Index obtained in two fungitoxicity tests (vapour phase and contact in water extract) with four tested dried plant tissues, with or without myrosinase addition P. irregulare

R. solani

∗∗

∗∗

ns ns

Treatments MYR addition Treatment × MYR

Volatile Volatile Volatile

ns ns

Treatments MYR addition Treatment × MYR

Contact Contact Contact

∗∗

∗∗

ns ns

ns ns

ns: not significant. ∗∗ P < 0.01.

Dehydrated tissues I. amara ISCI4 B. juncea low GLs B. juncea ISCI61 C. hassleriana ISCI2

P. irregulare (EI (%))

R. solani (EI (%))

22 45 100 100

50 60 100 100

c b a a

c b a a

Means followed by a common letter are not significantly different.

ical characteristics that play a well-known role on DPs biological activity (Nastruzzi et al., 1996; Manici et al., 1997). Mean separation of results on poisoned media gave C. hassleriana ISC12 dried tissues as the most active, followed by B. juncea ISCI61 and I. amara (not significantly different), while B. juncea ISCI20, with a low GLs content, showed the lowest activity (Table 4). In the test on volatile compounds released by dried tissues, the lowest activity was that of I. amara, containing glucoiberin, and the higher was that of C. hassleriana e B. juncea ISCI61 containing, respectively, glucocapparin and sinigrin (Table 5). These differences are in accordance with the different volatility of their corresponding DPs (Leoni et al., 2002).

4. Conclusions The preliminary results presented in this paper confirm that it is possible and practical to perform the dehydration of several Brassicaceae selected for high GLs content, maintaining their main biological properties. In fact, the dried plants had a sufficient residual content of GLs and of MYR activity to produce, after

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water addition, the GL DPs. The dried plants, showed in vitro a fungitoxic activity on two of the main agents of “root rot”, P. irregulare and R. solani AG-5. These results, moreover if they will confirm even at an industrial scale, open interesting applicative perspectives for these new raw materials as an ecological alternative to MB for the control of soil-borne fungi, both in organic as well as in conventional agriculture. Some other trials with a dehydration industrial plant have been planned for next years in cooperation with an Italian private company to enlarge the application of this method.

Acknowledgements This research was supported by the Regione Emilia Romagna in the ambit of the Project “Utilizzazione di piante Crucifere e di alcuni composti secondari ad attività biocida per la lotta agronomica a funghi patogeni in piante orticole”. The authors are grateful for the technicians L. Malaguti and S. Cinti for their fundamental collaboration in analysis. This paper won the Industrial Crops and Products Award at the 5th European Symposium “Industrial Crops and Products”, Floriade, The Netherlands, 24–26 April 2002.

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