Biological Activities of Rice Allelochemicals Momilactone A and B

Many plants were found to secrete a wide range of compounds into the rhizosphere and to change the chemical and physical properties of the rhizosphere soil, which affect the community of microbial, fungi and plants [1-4]. Through the secretion of compounds such as allelochemicals, plants inhibit the germination and growth of neighboring plants to compete more effectively for the resources [3,5,6].


Introduction
Many plants were found to secrete a wide range of compounds into the rhizosphere and to change the chemical and physical properties of the rhizosphere soil, which affect the community of microbial, fungi and plants [1][2][3][4]. Through the secretion of compounds such as allelochemicals, plants inhibit the germination and growth of neighboring plants to compete more effectively for the resources [3,5,6].
The negative impacts of commercial herbicide use on the environment make it desirable to diversify weed management options [7][8][9]. Many investigations have been attempted to exploit allelopathy of plants for weed control purposes in a variety of agricultural settings [10][11][12]. Rice has also been extensively studied with respect to its allelopathy as part of a strategy for sustainable weed management, such as breeding allelopathic rice strains [13][14][15].
Rice plants secrete momilactones from their roots into the rhizosphere over their entire life cycle at phytotoxic levels, and momilactones are able to account for the majority of the observed rice allelopathy [30][31][32]. In addition, genetic studies have shown that selective removal of only the momilactones from the complex mixture found in rice root exudates significantly reduces allelopathy, demonstrating that these serve as allelochemicals, the importance of which is reflected in the presence of a dedicated momilactone biosynthetic gene cluster in the rice genome [33,34]. However, allelopathic activities of momilactones were determined on only a few test plant species such as lettuce and barnyard grass [25,32]. Therefore, in the present study, the allelopathic activities of momilactone A and B were determined nine test plant species including weed plants, and toxicities of momilactone A and B on four rice cultivars were also determined.

Momilactone A and B
Momilactone A and B were isolated from husks of rice (cv. Koshihikari) as described by Kato-Noguchi et al. [35,36]. Husks (1 kg) of rice were extracted with 4 L methanol for three days. After filtration using filter paper (No. 2; Toyo ltd, Tokyo) filtrate was concentrated at 40°C in vacuo to produce an aqueous extract. The aqueous extract was Volume 1 • Issue 2 • 1000108 J Rice Res ISSN: JRR, an open access journal adjusted to pH 7.0 with 1 M phosphate buffer and the extract was then partitioned three times against an equal volume of ethyl acetate. The ethyl acetate phase was evaporated and separated with columns of silica gel and Sephadex LH-20. Momilactone A and B were finally purified by HPLC and identified by 1 H-NMR spectra.

Bioassay of momilactone A and B
Momilatone A and B were dissolved in 0.2 mL methanol, added to two sheets of filter paper (No. 2) in a 5.5-cm Petri dish. Methanol was subsequently evaporated and the filter paper in the Petri dishes was moistened with 3 mL of 1 mM MES buffer. The final concentrations of momilactone A and B were 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000 and 10000 μM. Seeds of cress, lettuce, alfalfa, ryegrass, timothy, barnyard grass, E. colonum, crabgrass, Arabidopsis and rice cultivars (Koshihikari, Nipponbare, Norin 8 and Sasanishiki) were surface sterilized in a 2% (w/v) solution of sodium hypochlorite for 15 min, rinsed four times in distilled water and germinated in the darkness at 25°C for 16-72 h.
Then, 10 germinated seeds of each test plant were individually placed on the filter paper in Petri dishes. The length of roots and shoots of these seedlings was measured after 48 h of incubation in the darkness at 25°C. For control treatments, methanol was added to the filter paper in the Petri dish and evaporated. Control germinated seeds of each test plants were then placed on the filter paper moistened with 3 mL of 1 mM MES buffer as described above. Percentage inhibition was determined by the formula: [(control plant length-plant length incubated with momilactone A or B)/control plant length]×100. These were three replicates per treatment and the experiment was repeated six times.

Inhibitory activities of momilactone A and B on nine plant species
Momilactone A inhibited the growth of roots and shoots of ryegrass at concentrations greater than 10 μM (Figure 1). The inhibition was increased with increased concentration of momilactone A. When inhibition of ryegrass roots and shoots were plotted against the logarithm of momilactone A concentrations as described by Streibig [37]  In addition, it was reported that momilactone A and B inhibited the growth of Amaranthus lividus and Poa annua at concentrations greater than 20 ppm (ca. 60 µM) and 4 ppm (ca. 12 µM), respectively [38]. The growth inhibitory activities of momilactome B are also greater than those of momilactone A under other bioassay systems [25,32,[39][40][41][42].

Inhibitory activities of momilactone A and B on rice
Momilactone A and B inhibited the growth of all plant species including the weed plants at μM level (Figures 1 and 2 and Table 1). Rice plants produce momilactone A and B and secret momilactone A and B into the rhizosphere [31,32,34]. Thus, the growth inhibitory activities of momilactone A and B against rice plants themselves were determined at concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000 and 10000 μM. Effectiveness of momilactone A and B on rice cultivars, Koshihikari, Nipponbare, Norin 8 and Sasanishiki was weak. Momilactone A and B only inhibited root and shoot growth of all rice cultivars at concentrations greater than 100 and 300 μM, respectively.
I 50 values of momilactone A and B on rice root and shoot were not obtained because of their weak inhibitory activities. Thus, the concentrations required for 25% growth inhibition (defined as I 25 ) for roots and shoots of rice were calculated from the equations of the logistic functions (Table 2). Comparing I 25 values, the inhibitory activities of momilactone B on the rice root and shoot growth, respectively, were 3.1-to 4.1-fold and 2.4-to 3.7-fold greater than those of momilactone A, which was consistent with results obtained with other plant species (Table 1).   (Table 3). Comparing I 25 values, the effectiveness of momilactone A and B on the root and shoot growth of rice cultivars was much less than that of weed plant species, barnyard grass, E. colonum, crabgrass and ryegrass. Barnyard grass is the most significant biological constrain to rice production [43].
The effectiveness of momilactone A and B on the growth of those rice cultivars was much less than that on the growth of barnyard grass. In addition, no visible damage to rice cultivars by momilactone A and B was observed at levels that are cytotoxic to these other plant species. These results suggest that the toxicities of momilactone A and B to rice cultivars may be much less than those to other plant species. The basis for rice resistance is currently unknown, but presumably involves either efflux (e.g. via the same transport mechanism responsible for momilactone secretion), insensitivity of the molecular target, and/or degradation.

Conclusion
Rice allelochemicals momilactone A and B inhibited Arabidopsis, alfalfa, lettuce, cress, timothy, barnyard grass, E. colonum, crabgrass and ryegrass by concentration dependently (Figures 1 and 2 and Table 1). However, the ability of momilactones A and B to suppress the growth of rice was by far less than their effects on other plant species (Table 2). Allelopathic substances have potential as either herbicides or templates for new synthetic herbicide classes [6,7,10,12,44,45].
Natural compounds are considered to be more environmentally benign than most synthetic herbicides [12]. In many cases, the natural compounds are also highly active at a molecular target site [45]. Momilactone A and B may have potential as a template for the development of new plant control substances because their selective inhibitory activities for weed plant species. More importantly, identification of momilactone A and B as allelochemicals in rice provides a molecular marker for breeding and/or engineering efforts directed at increasing allelopathic activity of this critical staple food crop.