Effect of some cover crops and their secondary metabolites on nitrous oxide (N2O) emission by Pseudomonas denitrifiers isolated from chemically fertilized corn farm soil
Abstract
Using a Pseudomonas denitrifying bacterium, which had been isolated from Japanese Andisol corn farm as an active nitrous oxide (N2O) emitter and likely to be missing nosZ gene, we investigated denitrification-regulating activity of some cover crops or green manure plants against this N2O emitter. In the preliminary screening, root exudates from the 10 seedlings of yellow flowering leaf mustard (Brassica juncea) and crimson clover (Trifolium incarnatum) showed 50% repression of the N2O emission by an incomplete denitrifier Pseudomonas sp. 05CFM15-6D. When direct extracts of the seedling roots with MeOH were assayed, however, only the seedlings of B. juncea showed a remarkable inhibition of bacterial cell growth and N2O emission at concentration equivalent to 10 seedlings. The root extract from B. juncea equivalent to 2 to 4 seedlings maintained inhibiting activity toward N2O emission, while it did not affect bacterial cell growth. Conversely, water-soluble fraction from aboveground of European small radish (Raphanus sativus var. sativus) sprouts showed statistically significant acceleration of N2O emission (P<0.01) with slight but insignificant cell growth activation. As some secondary metabolites are uniquely contained in these cover crops, suppressing or accelerating activity of such phytochemicals in N2O emission was also investigated. Allyl isothiocyanate at 30 µM markedly inhibited N2O emission of the Pseudomonas denitrifier but not suppressed its cell growth. In contrast, methyl isothiocyanate sinigrin, cyanamide, and betanin did not affect on N2O emission of the denitrifier at 150-300 µM.
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Introduction
Nitrous oxide (N2O) is an active greenhouse gas contributing more than 7% of global warming [1]. N2O, one of the most active greenhouse gases, has a strong global warming potential and contributes to depleting ozone layer in atmosphere [2]. In global scale, agricultural soils are recognized as major anthropogenic N2O emission sources. In particular, agriculture soil plays dominant roles in N2O emission with widespread use of nitrogenous fertilizers and manure to drive emission increment [3,4], in which N cycle, biological denitrification is the most important process [5]. Denitrification is a microbial nitrate respiration process, in which the oxidized nitrogen compounds served as electron acceptors for energy production in anaerobic conditions [6,7].
Cover crops, including some Brassicaceae and Fabaceae plants are often introduced into farmland to be subjected to crop rotation or maintain soils before leaving idle. The cover crops often play a role of green manure to reserve mineral nutrition in the soil, while they are often cultivated for the purpose of repression and regulation of weeds [8,9] or soil-borne disease causative soil microorganisms [10,11]. In particular, yellow flowering leaf mustard (Brassica juncea), white mustard (Sinapis alba) [12], and hairry vetch (Vicia villosa) [13] are cover crops used worldwide for biofumigation and weed control. If any annual plants can suppress N2O emission from the farmland soil, such cover crops are useful for cleaning of denitrifiers from the cropland soils. As cover crops are generally plowed into soil for fertilization, it may affect on denitrification processes in soil if plant tissues contained secondary metabolites active against denitrification-associated enzymes. In this paper, we wish to report unique responses of methanolic extracts from seedlings of some cover crops on an active N2O emitter isolated from Andisol of corn farmland in Shicizunai, Japan. Also, we further discuss on soil management strategy to minimize N2O emission from fertilized soils in croplands.
Conclusion
The N2O emitter Pseudomonas sp. 05CFM15-6D is a facultative anaerobe, and its N2O emission progresses under anaerobic conditions with an excessive organic matter utilizable for the copiotrophic and saprophytic denitrifier [19]. Therefore, green manure as a possible accelerator for N2O emission from the tilled soil is great concern. Indeed, we found that MeOH extract from the aboveground of R. sativum var. sativum seedlings accelerated N2O emission by the incomplete denitrifiers in this study (Fig. 3), while allyl isothiocyanate from B. juncea showed marked suppression of N2O emission by an incomplete denitrifier in the culture-based bioassay (Fig. 4). We therefore imply necessity of risk management for such tilling the green manure and pre-harvest vegetable crops.