HORTSCIENCE 36(7):1283–1289. 2001. varies according to the characteristics of the GL side chain. The DPs from glucoiberin, glucocheirolin, and glucoerucin {GLs with a Allelopathic Effect of Glucosinolate- extra S atom in their side chain [thiofunction- alized glucosinolates (GLThio)]} and from containing Plant Green Manure on glucocapparin (methyl-glucosinolate) have shown significantly higher fungicidal activity Pythium sp. and Total Fungal than DPs obtained from aliphatic or hydroxy GLs (Manici et al., 1997). Again in vitro, these GL-DPs showed suppressive activity against Population in Soil some widespread soilborne fungi (Rhizocto- 1 2 nia solani, Pythium sp., and others) (Manici et Luca Lazzeri and Luisa M. Manici al., 1999), supporting the hypothesis that these Istituto Sperimentale per le Colture Industriali, Via di Corticella 133, 40129 molecules can be used as natural biofumigants. Bologna, Italy The quality and quantity of GLs in cruciferous plant organs vary according to the genera, Additional index words. biofumigation, Cleome hassleriana, Iberis amara, Rapistrum species, and, in many cases, the variety (Rosa rugosum, soilborne pathogens et al., 1997). Therefore, the biocidal activity of a green manure may vary with the quality and Abstract. Two Brassicaceae (Iberis amara L. selection ISCI14 and Rapistrum rugosum All. quantity of the GL content of the green manure selection ISCI4) and a Capparidacea (Cleome hassleriana L. selection ISCI2) possessing plant species (Smolinska and Horbowicz, glucosinolates whose degradation products exhibit high fungitoxic activity in vitro were 1999). In recent years, at the Research Insti- assayed as biocidal plants in a green manure simulation. The trials were carried out in pots, tute for Industrial Crops of Bologna, some using aboveground fresh plant tissues incorporated at a realistic field rate into soil ecotypes of the Brassicaceae family (Iberis naturally infected by Pythium sp. The effect of these plant tissues on total fungal amara L., Rapistrum rugosum All.) and one of populations and Pythium sp. were compared with Crambe abyssinica H. cv. Mario, a the Capparidaceae (Cleome hassleriana L.) Brassicacea containing glucosinolates whose degradation products exhibit low fungitoxic were selected for their high content of GL activity in vitro, and a plant (Helianthus annuus L.) not containing glucosinolates. All whose DPs have exhibited strong activity in green manure treatments induced increases in total fungi over a 10-week period, showing vitro. an enhanced microflora level compared with untreated soil. Pythium sp. was strongly The aim of this study was to investigate the suppressed by the C. hassleriana, I. amara, and R. rugosum selections, while sunflower and allelopathic effect in soil of GL-containing crambe treatments increased Pythium sp. in a manner similar to that observed for total plant green manure. It remains to be clarified fungal population. These findings indicate that the green manures assayed suppress as to how much the suppressive effect of Pythium sp. and also induced an increase in total soil microbial activity. cruciferous amendments on soilborne patho- gens is due to the fungitoxic effect of GL-DPs The production of several vegetable crops ous meal (Smolinska et al., 1997), and also (Manici et al., 2000) or the stimulation of depends on the use of methyl bromide (MB) purified GLs hydrolyzed by pure myrosinase microbial activity (Gamliel and Stapleton, soil fumigation to control a wide array of (MYR) (Mari et al., 1993). The presence of 1993; Mazzola, 2000). Therefore, we evalu- soilborne fungi, nematodes, insects, and weeds. GLs (Sang et al., 1984) and MYR in ated variations in Pythium sp. population, a In accordance with the U.S. Clean Air Act, the Brassicaceae plant organs suggests the possi- pathogen very sensitive to GL-DPs (Manici et use of MB as a fumigant will be banned in bility of amending soil with fungicidal com- al., 1999), and those of the total fungal popu- developed nations by 2005 (United Nations pounds by using these plants as green manure. lation, one of the several components of the Environment Programme, 1992). This deci- In healthy plants, GLs and MYR are located soil microbial activity. sion has prompted increased interest in the in different cell compartments and come into development of alternative strategies with a contact only after cell collapse, such as occurs Materials and Methods lower environmental impact to control soil- following a pathogen attack or when plants are borne pathogens. chopped up, with the consequent production In 1997 and 1998, fresh plant tissues of One alternative approach might be pro- of fungicidal GL-DPs (isothiocyanates, thio- Iberis amara selection sel. ISCI14 (iberis), vided by the well-known fungitoxic activity of cyanates, and/or nitriles) in soil. Brassicaceae Rapistrum rugosum sel. ISCI4 (rapistrum), enzymatic hydrolysis-derived products (DPs) green manures have been shown to be active and Cleome hassleriana sel. ISCI2 (cleome), of glucosinolates (GLs), natural compounds against Fusarium oxysporum f.sp. conglutinans with a high content of strongly active GL-DPs, of the Brassicaceae (Walker et al., 1937). (Ramirez-Villapudua and Munnecke, 1988), were incorporated into a naturally infested soil Their high biological activity has been ob- Verticillium dahliae (Subbarao and Hubbard, in pots. Crambe abyssinica H. cv. Mario served against various fungi using parts of 1996), Aphanomyces euteiches (Chan and (crambe), a species containing GLs producing cruciferous plants (Charron and Sams, 1999), Close, 1987; Muehlchen et al., 1990), low fungitoxic DPs, and Helianthus annuus L. crude extracts (Mayton et al., 1996), crucifer- Thielaviopsis basicola (Adams, 1971), cv. Gloriasol (sunflower), a plant that does not Pythium ultimum and Sclerotium rolfsii contain GLs, were inserted in the experiments (Gamliel and Stapleton, 1993), and to straw- as nonbiocidal green manure controls. We gratefully acknowledge S. Palmieri and C. Cerato berry rot root (Lazzeri et al., 1999). Many Green manure crop production. In both for their helpful discussion. In addition, we thank G. Brassicaceae used in these studies are com- years, the green manure crops were cultivated Baruzzi and L. Malaguti very much for their skillful monly available Brassica species, such as in the Po Valley (Budrio, Bologna, Italy; con- technical assistance. This research was supported in cabbage (Brassica oleracea L. var. capitata tinental climate), in plots (15 m2) arranged in part by the project “Utilizzazione di piante crucifere L.), Indian mustard (B. juncea L.), yellow seed a randomized block design, with three repli- e di alcuni loro composti secondari ad attività biocida mustard (B. juncea L. ssp. trilocularis Olsson), cates. The trials were performed on a silt loam per la lotta a funghi patogeni in piante orticole” and rapeseed (B. campestris L.), as well as soil (clay 26%, silt 54%, sand 20%) with a high financed by the Emilia-Romagna region and in part other common Brassicaceae, like white mus- content in available potassium (240 ppm) and by the Project POM B30 “Linee innovative di tecnica colturale e difesa a basso impatto ambientale dei tard (Sinapis alba L.) and oil radish (Raphanus average content in phosphate (35 ppm) and fragoleti meridionali.” sativus L.). organic matter (1.8%). At sowing time, 80 1Technologist; to whom reprint requests should be Recent studies carried out in vitro with kg·ha–1 of N (ammonium nitrate) and 100 addressed. E-mail address: [email protected] purified GLs and MYR on Fusarium culmorum kg·ha–1 of sulfur were applied. Biocidal plants 2Plant Pathologist. have shown that the fungicidal activity of DPs were sown in the first week of March, with a HORTSCIENCE, VOL. 36(7), DECEMBER 2001 1283 6547, p. 1283-1289 1283 12/19/01, 1:10 PM PLANT PATHOLOGY density of ≈250 seeds/m2, in rows 28 cm wide was 70 W and mass spectra were scanned in lations were recorded 3, 6, and 10 weeks after for rapistrum and crambe and 14 cm wide for the 10–425 m/z range. Peaks of GL-DPs were plant incorporation. Twenty-five-gram soil iberis and cleome. During cultivation, no irri- identified using the NBS75K library. samples were collected from each pot and gation water and pesticide treatments were Green manure simulation. Trials were car- mixed with 225 mL of sterile water with a applied. At full flowering, the fresh biomass ried out using a silty clay loam soil (39% clay, magnetic stirrer for 10 min to obtain a 10–1 yield (t·ha–1) of aboveground parts was evalu- 49% silt, 12% sand), series Medicina, fine, diluted soil solution, and analyzed by the soil ated and the dry matter (DM) content (%) was mixed, mesic Vertic Ustochrepts (Soil Tax- dilution plate method on selective media measured by oven-drying a plant sample at onomy, 1994), naturally infested with Pythium (Dhiangra and Sinclair, 1986). 105 °C overnight. sp., collected in May 1997 and 1998 in Cesena Pythium sp. Three plates of selective me- Glucosinolate and myrosinase determina- (eastern Po Valley) from nonfumigated fields, dia were spread with 0.5 mL of 10–1 and 10–2 tion. At full flowering, a sample of each GL- which had been cultivated with strawberry for soil dilutions according to the modified Jeffers containing plant was weighed, immediately several years. The field was fallow in 1997. In and Martin (1986) method (cornmeal amended frozen at –20 °C, and subsequently freeze- autumn 1997, burley green manure was planted, with 5 mg·L–1 Pimaricin, 250 mg·L–1 Ampicil- dried using an Edwards Minifast Do.1 freeze- followed by strawberry in summer 1998. Soil lin, 10 mg Rifampicin, 100 mg of PCNB, and 1 drier (from –40 to +18 °C in 8 h with a vacuum (pH 8.2) was collected at random from the top g·L–1 Ox-gall). After an incubation time of 24 of 10–1 mbar). The freeze-dried materials were 20 cm, partially dried at room temperature for h at 18 °C in the dark, the soil dilution was then homogenized with a mortar and their GL 3 d, and then stored at 8 to 10 °C for 4 d.