Rice Allelopathy and Allelochemicals in Japan and Bangladesh

Transcription

1 W3-06 Rice Allelopathy and Allelochemicals in Japan and Bangladesh Hisashi Kato-Noguchi, Md. A. Salam, Takeshi Ino Department of Applied Biological Science, Faculty of Agriculture Kagawa University, Miki, Kagawa , Japan. Abstract: Allelopathic activities of 8 Japanese rice and 102 Bangladeshi rice (42 high yielding and 60 traditional cultivars) were determined by donor-receiver bioassay. cv. Koshihikari marked the greatest inhibitory activity in Japanese rice with an average of 67% of the growth inhibition on roots, shoots and fresh weight of alfalfa, cress and lettuce. High yielding rice cultivar, BR17 marked the greatest inhibitory activity in Bangladeshi rice with an average of 59% of the growth inhibition on roots and shoots of cress, lettuce, E. crus-galli and E. colonum. Allelochemicals of cv. Koshihikari were isolated from the root exudates and determined by spectral data as momilactone A and B. Momilactone A and B, respectively, inhibited the root and shoot growth of E. crus-galli and E. colonum at concentrations greater than 10 and 1 μm. Allelochemical of cv. BR17 was also isolated and identified as 9-hydroxy-4-megastigmen-3-one. This substance inhibited root and shoot growth of cress and E. crus-galli seedlings at concentrations greater than 0.03 and 3 μm, respectively. These allelochemicals may play important roles in rice allelopathy. Keywords: Allelopathy, Bangladesh, Donor-receiver bioassay, Momilactone, Echinochloa crus-galli. Echinochloa colonum, Oryza sativa. 1. Introduction Rice is one of the most important crops in the world and also the staple food for peoples in Bangladesh. Crop productivity of Bangladesh is low in comparison to other rice producing countries. Severe weed infestation is one of the major reasons for such low yield of rice in Bangladesh [16]. Weeds reduced the grain yield of direct seeded summer (aus) rice by %, autumn (aman) rice by 16-48% and modern winter (boro) rice by 22-36% [17]. Hand weeding is the most commonly used weed control method in this country, but it is often imperfect and/or delayed because of limited budgets for hiring labor and availability of labor during peak periods. Since Dilday et al. [4] reported that some rice accessions possess allelopathic activity in weed suppression, rice allelopathy has been received a great deal of attention [1, 5, 14, 19]. The allelopathic effect of rice on weeds could be applied to reduce use of chemical herbicides, which might results in improved water quality and less environmental contamination and might also reduce labor for hand weeding. It was therefore of interest to assess the allelopathic potential of Bangladeshi rice cultivars for weed control purpose. The objective of this research was to identify allelochemicals in Japanese rice and Bangladeshi rice cultivars. Therefore, allelopathic activities of Japanese rice and Bangladeshi rice cultivars were determined and allelochemicals in Japanese rice and Bangladeshi rice cultivars were isolated and identified. Biological activities of these allelochemicals were also determined. 2. Materials and Methods 2.1. Rice seeds Seeds of Bangladeshi rice were collected by permission of the authorities in Genetics Resource Division, Bangladesh Rice Research Institute (Joydebpur, Gazipur), Bangladesh Institute of Nuclear Agriculture (BAU campus, Mymenthingh), and farmers of Patuakhali and Dinajpur districts. Seeds of Japanese rice were obtained from research farm of Kagawa University Donor-Receiver Bioassay Allelopathic activities of 8 Japanese rice cultivars against the growth of roots, shoots and fresh weight of alfalfa (Medicago sativa L.), cress (Lepidium sativum L.) and lettuce (Lactuca sativa L.) were determined by donor-receiver bioassay as described by Kato-Noguchi et al. [9]. Allelopathic activities of 102 (42 high yielding and 60 traditional cultivars) Bangladeshi rice against cress, lettuce, Echinochloa crus-galli (L.) Beauv. and E. colonum (L.) Link were also determined by donor-receiver bioassay as described by Kato-Noguchi et al. [13]. Percentage inhibition was then calculated by the formula: [(control plant length - plant length incubated with rice) / control plant length] x 100.

2 2.3. Isolation and Determination of Allelochemicals in cv. Koshihikari Seeds of rice (Oryza sativa cv. Koshihikari) were germinated and hydroponically grown for 14 days as described by Kato-Noguchi et al. [9]. Then, the culture solution was filtrated and separated by several chromatographic fractionations keeping track of the biological activity and two putative compounds, 1 and 2, causing the inhibitory effect of the rice seedlings were isolated. The active compounds were characterized by MS, 1 H NMR and 13 C NMR spectra Isolation and Determination of Allelochemical in cv. BR17 Rice plants (cv. BR 17) were extracted with 80% (v/v) aqueous methanol and the extract was concentrated in vacuo to produce an aqueous residue. The aqueous residue was adjusted to ph 7.0 with 1 M phosphate buffer and partitioned against ethyl acetate. The ethyl acetate fraction was separated by several chromatographic fractionations keeping track of the biological activity and one putative compound causing the inhibitory effect of the rice seedlings was isolated. The active compound was characterized by 1 H-NMR spectra Bioassay Allelochemicals were dissolved in a 0.2 ml of methanol and added to a sheet of filter paper (No. 2) in a 5-cm Petri dish. Methanol was evaporated in a draft chamber. Then, the filter paper in the Petri dishes was moistened with 3 ml of a 0.05% (v/v) aqueous solution of Tween 20, and 10 seeds of cress, rice, E. crus-galli and E. colonum were sown on the Petri dishes. The length of their roots and shoots was measured after 48 h of incubation in the darkness at 25 C. 3. Results and Discussion 3.1. Allelopathic Activity of Japanese Rice The effectiveness of cv. Koshihikari was the greatest with an average of 67% growth inhibition on shoots, roots and fresh weight of alfalfa, cress and lettuce (Table 1). More than 40% inhibition was also recorded for cv. Norin Allelopathic Activity of Bangladeshi Rice One hundred and two Bangladeshi rice cultivars were divided into two groups such as high yielding rice (42 cultivars) and traditional rice (60 cultivars, Table 2), and their allelopathic effects have been determined and summarized in Table 3. Traditional rice cultivars are long stature and lodge easily. Grain yield is half of straw weight. High yielding rice cultivars are short stature and leaves are dark-green and vertical. Grain yield is almost equal with straw weight. Effects of 102 Bangladeshi rice cultivar, four test plant species and their interactions were significant both for shoot and root growth (P < ). Significant effects of cultivar and the interactions indicated that there was variation in allelopathic activity among 102 rice cultivars (Table 4). This result suggests that rice cultivars which were allelopathic against one plant species were not always allelopathic towards other plant species. However, high yielding rice cultivar, BR17 marked the greatest inhibitory activity with an average of 59% growth inhibition on shoots and roots of cress, lettuce, barnyardgrass and E. colonum (Table 3) Identification of Allelochemicals in cv. Koshihikari ESI-MS of inhibitor 1 gave positive-mode molecular ion peak [M+H] + and [M+Na] + at m/z 315 and 337, respectively, suggesting a molecular weight of 314. The 1 H NMR spectrum of inhibitor 1 (600 MHz, CD 3 OD, TMS as internal standard) showed δ 5.89 (1H, dd, J = 18.0 and 10.8 Hz, H-15), 5.75 (1H, d, J = 5.4 Hz, H-7), 5.00 (1H, dd, J = 18.0 and 1.2 Hz, H-16a), 4.98 (1H, dd, J = 5.4 and 4.8 Hz, H-6), 4.92 (1H, dd, J = 10.8 and 1.2 Hz, H-16b), 2.65 (1H, ddd, J = 19.8, 7.2, and 1.8 Hz, H-2a), 2.55 (1H, d, J = 4.8 Hz, H-5), 2.54 (1H, m, H-2b), 2.23 (1H, d, J = 12.0 Hz, H- 14a), 2.07 (1H, d, J = 12.0 Hz, H-14b), 2.03 (1H, m, H-1a), 1.86 (1H, dd, J = 12.6 and 3.6 Hz, H-9), 1.77 (1H, ddd, J = 12.6, 7.2, and 3.6 Hz, H-11a), 1.61 (1H, m, H-1b), 1.60 (2H, m, H-12), 1.50 (3H, s, H-18), 1.43 (1H, ddd, J = 12.6, 12.6, and 5.4 Hz, H-11b), 0.93 (3H, s, H-17 or H-20), and 0.92 (3H, s, H-17 or H-20). The 13 C NMR spectrum of inhibitor 1 (150 MHz, CD 3 OD, TMS as internal standard) showed δ (C-3), (C-19), (C-15), (C-8), (C-7), (C-16), 78.3 (C-6), 55.2 (C-4), 51.5 (C-9), 48.7 (C-14), 47.1 (C-5), 41.3 (C-13), 38.5 (C- 12), 35.5 (C-2), 33.6 (C-10), 31.7 (C-1), 25.1 (C-11), 22.4 (C-17), 22.2 (C-20), and 21.6 (C-18). 2

3 Table 1. Effects of Japanese rice cultivars on the growth of shoots, roots and fresh weight of alfalfa, cress and lettuce. *; P < 0.05, **; P < 0.01, ***; P < Shoot Root Fresh weight Cultivar Alfalfa Cress Lettuce Alfalfa Cress Lettuce Alfalfa Cress Lettuce Yukihikari 31.2** 24.6** 41.8*** 25.4** 35.6** 41.3*** 26.4** 31.8** 38.2** Hinohikari 10.9* 21.6** 58.7*** ** 53.2*** ** 44.8** Koshihikari 65.2*** 68.6*** 84.6*** 59.3*** 65.8*** 76.3*** 58.7*** 59.6*** 65.8*** Kinuhikari 33.7** 48.3*** 54.3*** 15.5* 23.4** 57.4*** 21.3* 47.3** 41.2*** Sasanishiki 34.1*** 59.1*** 61.3*** 32.0** 38.8** 61.8*** 23.4** 29.8** 35.8** Nipponbare 21.2** 42.7*** 34.8** 28.8* 34.6** 31.2** 11.3* 18.7* 24.8** Kamenoo 27.7** 53.7*** 63.5*** 23.7** 51.2*** 59.1*** 18.7* 36.4** 39.4** Norin *** 56.7*** 51.2*** 48.2*** 65.2*** 57.6*** 42.8*** 41.2*** 54.3*** Table 2. Bangladeshi rice cultivars used in the present research. Traditional cultivar High yielding cultivar Badshabhog Deshibalam Jhingashail Manikjour BR1 BR17 BRRI dhan35 Baron Dhepa Jogly Marichbati BR2 BR19 BRRI dhan36 Bashful Dhepa2 Jotabalam Matiagorol BR3 BR20 BRRI dhan37 Bashiraj Dudhkalam Kachamota Mohanbhog BR4 BR21 BRRI dhan38 Bashmoti Dudhlaki Kalamanik Motaman BR5 BR22 BRRI dhan39 Binnaful Dudshor Kalizira Nizershial BR6 BR23 BRRI dhan40 Biroi Dular Kartikshail Pajam BR7 BR26 BRRI dhan42 Buta Gabura Kataribhog Pashushail BR8 BRRI dhan27 BRRI dhan43 Chandon Gangasagor Kazliboro Patnai BR9 BRRI dhan28 BRRI dhan45 Chikon aman Ganjia-3 Khoiyaboro Pusur BR10 BRRI dhan29 Binadhan-4 Chiniatop397 Goai Kumari Rajashail BR11 BRRI dhan30 Binadhan-5 Chiniatop398 Gobolshail Lakkhidigha Sadajira BR12 BRRI dhan31 Iratom-24 Chinigura Gobrijoshua Lalaman Shakkhorkona BR14 BRRI dhan32 Chinisagor Hashikalmi Madhabjota Shorna BR15 BRRI dhan33 Choiyamura Jamainaru Maliabhangor Tepiboro BR16 BRRI dhan34 Table 3. Effects of Bangladeshi rice cultivars on the growth of shoots and roots of cress, lettuce, E. crus-galli and E. colonum. NS; not significant, *; P < 0.05, **; P < 0.01, ***; P < Cress Lettuce E. crus-galli E. colonum Total Cultivar Shoot Root Shoot Root Shoot Root Shoot Root average BR1 5.75NS 23.5NS -10.5NS -81.4*** -10.3NS 10.5NS 14.3* 0.70NS BR2 20.6** 26.5NS 44.4NS 64.8*** 8.27NS 35.6** 4.03NS 39.9*** BR3 13.4NS 24.2NS 46.7** 41.9*** 19.4*** 32.4*** 8.79NS 13.6NS BR4-5.49NS -23.7NS 6.67NS -83.3** 9.86NS 30.6* 19.1* -9.38NS BR5 29.8** 53.2*** 16.7NS 5.71NS 10.7NS 18.4* 4.84NS 14.7NS BR6 8.42NS 4.86NS 25.0* 3.33NS 6.99NS 26.1* 1.77NS -17.7NS 7.34 BR7 9.89NS -1.68NS 33.3* 28.3* -9.85NS 21.6NS 14.4** 34.8NS BR8 21.6* 29.8NS 18.2NS -4.88NS 11.8NS 23.4NS 12.1NS 23.1NS BR9 19.0** 35.2** 21.4* 48.7*** 15.9** 43.1*** 16.8** 30.9** BR NS -23.8NS 30.8* 61.9*** 1.59NS 18.3NS 6.73NS -36.0NS 7.80 BR NS 39.4** -6.67NS 20.5* 7.25NS 15.0NS 8.43NS 28.6**

4 BR NS 49.2** -6.25NS -9.86NS 5.29NS 19.7** 6.58NS 4.73NS BR ** -7.02NS 25.0* NS 21.4** 40.7*** 15.2* 0.00NS 9.00 BR NS 33.1** 15.8* 22.3NS 12.7NS 33.2** 15.4NS 51.3** BR ** 39.9** 44.8*** 22.5NS 5.33NS 19.1NS 20.1* 39.8*** BR ** 47.4*** 56.5*** 58.4*** 14.4NS 46.6*** 26.5*** 43.2*** BR NS 15.7NS 37.5** 28.1NS 7.63NS 25.8* 10.1* 29.1* BR * 53.2*** 23.1NS 30.8NS 4.93NS 8.91NS 25.6*** 34.8NS BR NS 33.7* 28.6NS 18.6NS 4.35NS 11.9NS 8.74NS 45.0*** BR NS 38.9** 83.3* 90.3* 16.2NS 22.0NS 5.16NS -2.6NS BR ** 49.8** 39.1*** 62.0*** 19.5* 26.9*** 1.92NS 19.1NS BR * 35.9* 37.5** 40.4** 20.0** 45.6*** 11.0NS 43.9*** BRRI dhan NS 14.4NS 5.88NS -59.3** 15.0NS 13.3NS 14.2* 4.64NS 2.94 BRRI dhan ** 48.6*** 41.2*** 56.73*** 19.2* 26.2* 18.6* 32.7* BRRI dhan * 46.2** 18.2NS -38.3* 19.9** 39.7*** 25.6*** 40.9*** BRRI dhan ns -2.92NS 90.0*** 94.0*** 14.1NS 19.4NS 22.8* 15.3NS BRRI dhan NS 40.2* 5.00NS 16.0NS 15.7NS 38.2*** 2.82NS 7.78NS BRRI dhan NS 21.6NS 50.0* 47.7* -3.38NS -8.86NS 2.27NS 4.68NS BRRI dhan * 22.8NS 25.0* 17.8NS 17.7* 25.3* 13.1NS -13.6NS BRRI dhan ** 33.2*** 0.00NS 9.80NS 18.8** 26.1*** 22.1** 2.88NS BRRI dhan NS 20.0NS 6.25NS -52.2** 17.7NS 37.4*** 5.56NS 9.20NS 7.26 BRRI dhan ns 37.9** 14.3NS -16.0NS 1.91NS 24.0* 9.49NS 9.49NS 9.56 BRRI dhan *** 53.3*** -7.14NS -26.9NS -2.9NS 1.22NS 7.87NS 12.0NS 8.98 BRRI dhan NS 38.2* -40.0NS 12.7NS -0.82NS 38.3** 10.6NS 75.3*** BRRI dhan NS 44.4*** -33.3* -142*** -15.8NS 20.3NS -7.63NS 19.7NS BRRI dhan ** 36.6** -11.8NS -65.4*** 8.28NS 10.7NS 15.8** 20.9NS 5.15 BRRI dhan * 43.3*** -25.0NS -50.8* 2.88NS 6.0NS 13.6* 3.55NS 1.26 BRRI dhan * 37.4* 7.14NS 41.8*** 18.4** 37.7** 21.7* 37.2** BRRI dhan * 33.2** 11.8NS -29.1* 13.4NS 1.39NS 5.97NS 2.21NS 6.53 Binadhan NS 17.7NS 77.8*** 58.3* 5.41NS 0.00NS 10.8NS -18.6NS Binadhan NS 36.0** 25.0NS 34.0NS 18.0* 26.4* 27.5NS 16.7NS Iratom ** 49.8*** 5.6NS 9.09NS 1.37NS 1.83NS 2.94NS 13.0NS Badshabhog 15.9NS 36.5** 35.3*** 51.1*** 7.34NS 6.55NS 33.0*** 41.0** Baron 10.4NS -24.0NS 63.6** 65.4NS -9.24NS -3.03NS 2.86NS 24.0NS Bashful 6.67NS 45.7*** 15.4NS 51.1** -3.85NS 24.4** 24.4NS 12.3NS Bashiraj -8.64NS 28.1* 29.4** 40.5** -4.76NS 10.6NS 14.6NS 7.94NS Bashmoti 3.41NS 31.8NS 0.00NS ** 10.3NS 13.3NS 7.14NS 4.31NS Binnaful -6.98NS -19.7NS 16.7NS 31.0* -6.47NS 14.5NS 6.87NS 6.41NS 5.28 Biroi -2.13NS 39.7** 21.1* 62.0*** -2.04NS 13.5NS -4.96NS 15.5NS Buta 10.7NS 27.1NS 50.0** 60.1*** 15.5* 15.8* 13.0NS 33.5** Chandon 9.78NS 34.1*** 27.9NS 48.9NS 4.76NS 33.9** -11.4NS 32.8NS Chikon aman -24.0NS -13.6NS 57.1** 57.1*** 8.96NS 25.5* 8.13NS 13.2NS Chiniatop NS 32.9** -57.9*** -20.0NS 11.7NS 27.2** 9.68NS 53.3NS 7.62 Chiniatop NS 32.1** -13.3NS -38.1** 2.68NS 23.0** -9.30NS 19.0* 3.06 Chinigura 15.8* 42.3** 5.56NS 9.73NS 8.63NS 21.9** 18.6** -7.07NS Chinisagor -2.22NS 14.4NS 0.00NS -18.4NS 9.58NS 24.2** 9.52NS 30.3* 8.42 Choiyamura 9.28NS 34.1* 11.8NS -12.8NS 10.9NS 30.4** 12.6NS 20.8NS Deshibalam 6.90NS 16.6NS 5.88NS -16.7NS 4.73NS 12.4NS 16.4NS 24.2NS 8.80 Dhepa -2.33NS 16.2NS 88.9*** 47.6*** 7.63NS 20.2** 3.06NS -19.0NS Dhepa2 5.88NS 41.5* 0.00NS -1.88NS -6.25NS 15.4NS 10.0NS 42.9NS Dudhkalam 12.8NS 29.2* 11.1NS 14.1NS 4.14NS 25.5* 4.88NS 12.9NS Dudhlaki 19.3* 51.2*** 5.56NS 3.85NS 9.22NS 18.3NS 4.46NS 29.1* Dudshor -4.05NS -19.3NS 0.00NS -21.4NS 6.33NS 13.7NS 7.56NS 16.1NS Dular 19.2* 45.5*** 6.67NS 27.9NS 25.2** 44.0*** 9.23NS 29.9NS Gabura 7.07NS 40.6*** 0.00NS 11.0NS 6.67NS 39.3*** 0.00NS 12.7NS

5 Gangasagor -1.35NS -20.2NS -6.25NS -8.77NS 5.8NS 8.21NS -6.9NS -17.1NS Ganjia NS -34.9NS 0.00NS -85.1*** 0.97NS -13.7NS 6.06NS 15.0NS Goai 22.5* 55.8*** 9.09NS -17.7NS 9.02NS 27.8** -17.1NS 0.00NS Gobolshail 10.9NS 40.0** 11.1NS -7.34NS 6.76NS 23.6NS 12.5NS 2.38NS Gobrijoshua 6.82NS 42.7*** 0.00NS 36.9** 7.69NS 26.7* 8.51NS 2.90NS Hashikalmi 9.8NS 39.8** 26.3* 31.1* 7.75NS 30.6** -2.56NS -11.5NS Jamainaru 15.7NS 31.5* -43.8*** -11.7NS 0.00NS 25.0** -22.5NS 38.4* 4.08 Jhingashail 1.20NS 26.9* -9.09NS 16.7NS -1.59NS 24.8* -23.3NS 4.35NS 5.00 Jogly 16.7* 39.0** 69.2*** 71.4*** 6.09NS 27.9** -2.00NS -13.5NS Jotabalam 1.03NS 22.5NS -13.3NS -71.2*** 10.1NS 22.3NS -22.5NS -27.2NS Kachamota 21.5* 53.4*** 15.8NS 7.89NS 0.67NS 9.57NS -8.89NS -8.06NS Kalamanik 16.00NS 14.0NS 50.0* 45.0** -1.61NS 6.35NS 7.34NS 2.04NS Kalizira 3.64NS -15.9NS 23.1* 5.69NS 6.18NS 19.0* 11.9NS 30.4* Kartikshail 10.5NS 14.8NS 13.3NS 42.1* 13.6NS 70.5*** 16.9NS 66.1*** Kataribhog 21.8** 40.3*** -10.5NS -30.9* 1.91NS 24.8* 13.3NS 25.0NS Kazliboro 7.60NS 51.5*** 7.69NS 22.9NS 23.6*** 46.8* 10.8NS 18.5NS Khoiyaboro 13.3NS 40.1** 14.3NS -33.3NS -1.38NS 30.7*** 20.0NS 24.1NS Kumari 22.0** 40.6** 0.00NS -29.6NS -4.79NS 17.0NS 9.09NS 18.6NS 9.10 Lakkhidigha -10.8NS -23.1NS 0.00NS 23.2* 9.09NS 27.8NS 12.1NS 40.0*** Lalaman 12.1NS 50.0*** 0.00NS -3.27NS 14.8* 40.8*** 15.4NS 47.1NS Madhabjota 5.21NS 37.9** 18.5NS 36.6*** 10.1NS 19.4NS -2.63NS 10.0NS Maliabhangor 8.73NS 35.4*** 4.76NS -17.5NS 11.1NS 20.9* 15.2NS -12.9NS 8.20 Manikjour 11.0NS 50.6*** 22.2* 30.5* 11.6NS 30.8** 3.29NS 12.7NS Marichbati 13.5* 37.7** 0.00NS 4.92NS 7.14NS 39.7*** 21.1NS 27.3NS Matiagorol -2.06NS 22.1* 0.00NS -29.6* 7.91NS 28.6** 8.62NS 11.5NS 5.88 Mohanbhog 12.1NS 5.83NS 9.09NS 0.00NS 4.48NS 4.80NS 7.32NS -8.24NS 4.43 Motaman -5.68NS -16.9NS 13.3NS -2.38NS 7.83NS 17.4NS 0.00NS -15.1NS Nizershial 17.1NS 32.4NS 7.69NS 16.8NS 16.2** 4.96NS -3.88NS 12.9NS Pajam 19.4* 39.5** 28.6NS -9.02NS -1.98NS 18.6NS 11.7* 17.3NS Pashushail 18.3* 31.2** -12.5NS -15.8NS 12.2* 25.4** 1.82NS -13.9NS 5.84 Patnai NS 7.91NS 13.0* -14.4NS 20.7** 11.0NS 23.3* -26.1NS 4.88 Pusur 17.0NS 41.7* 0.00NS 38.9*** 4.07NS 26.8** 11.1NS 11.0NS Rajashail 2.46NS -7.17NS 27.8* 20.0NS 4.30-NS 8.98NS -1.12NS -6.36NS 6.11 Sadajira NS -52.4** 21.4NS 4.86NS 16.2NS 5.08NS 11.8* -26.2NS Shakkhorkona -7.92NS -10.1NS 30.0NS 52.9* 14.8NS 38.6*** 3.70NS 6.94NS Shorna 12.8* 39.8** -7.14NS 3.81NS 4.51NS 32.8*** 7.41NS 27.4** Tepiboro 17.1* 42.3** 0.00NS 29.9* 7.91NS 20.9NS 2.70NS 20.8NS Table 4. Two-way analysis of variance (ANOVA) for relative shoot or root length of treated plants (%) to control plants of cress, lettuce, E. crus-galli and E. colonum. d.f., degrees of freedom; SS, sum of squares; MS, mean square; P, probability. Factor d.f. Shoot Root SS MS F P SS MS F P Rice cultivar (C) < < Test plant species (T) < < C x T < < Error

6 ESI-MS of inhibitor 2 gave positive-mode molecular ion peak [M+H] + and [M+Na] + at m/z 331 and 353, respectively suggesting a molecular weight of 330. The 1 H NMR spectrum of inhibitor 2 (600 MHz, CD 3 OD, TMS as internal standard) showed δ 5.86 (1H, dd, J = 18.0 and 10.8 Hz, H-15), 5.67 (1H, d, J = 4.8 Hz, H-7), 5.01 (1H, dd, J = 6.0 and 4.8 Hz, H-6), 4.98 (1H, dd, J = 18.0 and 1.2 Hz, H-16a), 4.90 (1H, dd, J = 10.8 and 1.2 Hz, H-16b), 4.00 (1H, d, J = 9.0 Hz, H-20a), 3.57 (1H, d, J = 9.0 Hz, H-20b), 2.38 (1H, d, J = 6.0 Hz, H-5), 2.15 (1H, d, J = 12.6 Hz, H- 14a), 2.10 (1H, ddd, J = 13.8, 8.4, and 5.4 Hz, H-2a), 2.01 (1H, d, J = 12.6 Hz, H-14b), 1.87 (1H, ddd, J = 13.8, 7.8, and 4.3 Hz, H-2b), 1.77 (1H, m, H-1a), 1.74 (1H, m, H-11a), 1.71 (1H, m, H-1b), 1.56 (2H, m, H-12), 1.53 (1H, dd, J = 13.2 and 3.6 Hz, H-9), 1.39 (3H, s, H-18), 1.36 (1H, m, H-11b), and 0.90 (3H, s, H-17). The 13 C NMR spectrum of inhibitor 2 (150 MHz, CD 3 OD, TMS as internal standard) showed δ (C-19), (C-15), (C-8), (C-7), (C-16), 96.7 (C-3), 73.8 (C-6), 72.6 (C-20), 50.9 (C-4), 48.6 (C-14), 44.4 (C-5), 42.9 (C-9), 39.9 (C-13), 37.2 (C-12), 30.6 (C-10), 28.6 (C-1), 27.4 (C-2), 24.6 (C-11), 21.1 (C-17), and 17.7 (C-18). From the comparison of these data with those reported in the literature [2, 8, 9], these inhibitors were identified as momilactone A (1) and momilactone B (2), respectively Biological Activity of Momilactone A and B The biological activities of momilactone A and B were determined with two weed species, E. crus-galli and E. colonum. Momilactone A and B, respectively, inhibited the growth of roots and shoots of E. crus-galli at concentrations greater than 10 and 1 μm (Fig. 1). Momilactone A and B, respectively, also inhibited the growth of roots and shoots of E. colonum at concentrations greater than 10 and 1 μm (Fig. 2). Increasing the concentrations increased the inhibition of both root and shoot growth of the two weed species. The concentrations required for 25 and 50 % inhibition of the growth of E. crus-galli and E. colonum in the assay (defined as I 25 and I 50, respectively), as calculated from the regression equation of the concentration-response curves, were summarized in Table 5. Comparing I 25 and I 50 values, the inhibitory activities of momilactone B on the root and shoot growth of E. crus-galli were 4.7- to 7.4-fold greater than those of momilactone A, and the inhibitory activities of momilactone B on the root and shoot growth of E. colonum were 6.0- to 19.2-fold greater than those of momilactone A. Thus, effectiveness of momilatone B on the growth inhibition is much greater than that of momilactone A. Fig. 1. Effects of momilactone A and B on root and shoot growth of E. crus-galli seedlings. 6

7 Fig. 2. Effects of momilactone A and B on root and shoot growth of E. colonum seedlings. Table 5. I 25 and I 50 of momilactone A and B for root and shoot growth of E. crus-galli, E. colonum and rice seedlings. I 25 and I 50, the concentration required for 25 and 50% growth inhibition, respectively. E. crus-galli E. colonum Rice Root Shoot Root Shoot Root Shoot Momilactone A I I Momilactone B I I Fig. 3. Effects of momilactone A and B on root and shoot growth of rice, cv. Koshihikari seedlings Toxicities of Momilactone A and B on Rice Seedlings Momilactone A and B were synthesized in rice plants by biotic and abiotic stress conditions [15, 11, 20] and possess growth inhibitory activities (Fig. 1 and 2), thus growth inhibitory activities of momilactone A and B to rice seedlings themselves were determined. Momilactone A and B, respectively, inhibited root and shoot growth of rice seedlings at concentrations greater than 100 and 300 μm (Fig. 3). I 50 values of momilactone A and B on rice root and shoot were not obtained because of their weak inhibitory activities. Comparing I 25 values, the inhibitory activi- 7

8 ties of momilactone B on the rice root and shoot growth, respectively, were 4.6- and 3.7-fold greater than those of momilactone A, which is consistent with results obtained with E. crus-galli and E. colonum. However, the inhibitory activities of momilactone A and B, respectively, on the root and shoot growth of rice seedlings were 1-2 % and % of those on the root and shoot growth of E. crus-galli and E. colonum. Thus, the effectiveness of momilactone A and B on the growth of rice seedlings was much less than that on the growth of E. crus-galli and E. colonum. In addition, no visible damage to rice seedlings by momilactone A and B was observed. Therefore, these results suggest that the toxicities of momilactone A and B to rice seedlings may be much less than those to the two weed species Identification of Allelochemical in cv. BR17 The 1 H NMR (CD 3 OD, 400 MHz) spectrum of the substance obtained from cv. BR 17 showed; δ: 1.00 (3H, s), 1.05 (3H, s), 1.20 (3H, d, J = 5.9 Hz), 1.86 (1H, t, J = 4.4 Hz), 1.99 (1H, d, J = 1.2 Hz), 2.03 (1H, d, J = 17.1 Hz), 2.38 (1H, d, J = 17.1 Hz), 3.75 (1H, m), 5.81 (1H, s). From the comparison of these data with those reported in the literature [3], the substance was identified as 9-hydroxy-4-megastigmen-3-one. This substance was first isolated from Cestrum parqui as a new C 13 nor-isoprenoid by D Abrosca et al. [3] Biological Activity of Allelochemicals 9-Hydroxy-4-megastigmen-3-one inhibited the root and shoot growth of cress at concentrations greater than 0.03 and 0.1 μm, respectively, and the root and shoot growth of E. crus-galli at concentrations greater than 3 and 10 μm, respectively. I 50 for cress roots and shoots, respectively, was 0.22 and 0.47 μm, and that for E. crus-galli roots and shoots, respectively, was 36 and 133 μm. Comparing I 50 values, effectiveness of 9-hydroxy-4-megastigmen-3-one on the root and shoot growth of cress, respectively, was 160- and 280-fold greater than on the root and shoot growth of E. crus-galli [23]. 4. Conclusions cv. Koshihikari marked the greatest inhibitory activity in Japanese rice with average of 67% of the growth inhibition and allelochemicals were isolated from root exudates of cv. Koshihikari and determined by spectral data as momilactone A and B. Momilactone A and B, respectively, inhibited the growth of E. crus-galli and E. colonum, at concentrations greater than 10 and 1 μm (Fig. 1 and 2). The toxicities of momilactone A and B to rice are much less than those to the weed species (Fig. 3). Rice plants secreted momilactone A and B from their roots into rice rhizosphere over the life cycle [10, 12]. These results suggest that rice plants may be able to inhibit the growth of their neighboring plants through the secretion of momolactone A and B into their rhizosphere without serious toxicity of momilactone A and B to rice plants themselves. Momilactone A and B also exhibit antimicrobial properties and are synthesized as a part of defensive response to the bacterial and antifungal activities [15, 20]. Therefore, secretion of momilactone A and B into rice rhizosphere may provide a competitive advantage for root establishment through local suppression of soil microorganism and inhibition of the growth of competing plant species. However, the involvement of momilactone B for the defense mechanism may be greater than momilactone A because growth inhibitory activity and secretion rate of momilactone B were much grater than those of momilactone A. A growth inhibitory substance was isolated from the aqueous methanol extract and its chemical structure was determined as 9-hydroxy-4-megastigmen-3-one. This substance was active at concentrations greater than 0.03 μm. Under certain conditions allelopathic compounds are released into the plant rhizosphere, either as exudates from living tissues or by decomposition of plant residues in sufficient quantities to inhibit the growth of neighboring plants [7, 21, 22, 24]. The endogenous concentration of 9-hydroxy-4-megastigmen-3-one was at least 3.7 μm/kg because of 2.5 mg of the substance (MW 223) was isolated from 3 kg rice plants. Considering the endogenous level and the inhibitory activity, 9-hydroxy-4-megastigmen-3-one may provide the competitive advantage to rice plants in the rhizosphere as an allelopathic substance through the growth inhibition of neighboring plant species. Many attempts have been made to exploit allelopathy of plants for weed control in a variety of agricultural settings [6, 24]. Synthetic chemical herbicides may continue to be a key component in many integrated weed management systems, but controlling weeds through allelopathy is one strategy to reduce herbicide dependency [6, 7, 18, 25]. Thus, Bangladeshi rice BR 17 may be potentially useful for weed management in a field setting. References [1] Azmi, M., Abdullah, M.Z., Fujii, Y., Exploratory study on allelopathic effect of selected Malaysian rice varieties and rice field weed species. J. Trop. Agric. Food. Sci. 28: [2] Cartwright, D.W., Langcake, P., Pryce, R.J., Leworthy, D.P., Ride, J.P., Isolation and characterization of two phytoalexins from rice as momilactones A and B. Phytochemistry 20:

9 [3] D'Abrosca, B., DellaGreca, M., Fiorentino, A., Monaco, P., Oriano, P., Temussi, F., Structure elucidation and phytotoxicity of C 13 nor-isoprenoids from Cestrum parqui. Phytochemistry 65: [4] Dilday, R.H., Lin, J., Yan, W., Identification of allelopathy in the USDA-ARS rice germplasm collection. Aust. J. Exp. Agric. 34: [5] Dilday, R.H., Yan, W.G., Moldenhauer, K.A.K., Gravois, K.A., Allelopathic activity in rice for controlling major aquatic weeds. In: Olofsdotter, M. (ed.) Allelopathy in Rice, International Rice Research Institute; Manila, pp [6] Duke, S.O., Dayan, F.E., Romagni, J.G., Rimando, A.M., Natural products as sources of herbicides, current status and future trends. Weed Res. 40: [7] Einhellig, F.A., An integrated view of allelochemicals amid multiple stresses. In: Inderjit, Dakshini, K.M.M., Foy, C.L. (eds). Principals and Practices in Plant Ecology: Allelochemical Interactions, CRC Press; Boca Raton, Florida, pp [8] Kato, T., Kabuto, C., Sasaki, N., Tsunagawa, M., Aizawa, H., Fujita, K., Kato, Y., Kitahara, Y., Momilactones, growth inhibitors from rice, Oryza sativa L. Tetrahedron Lett. 39: [9] Kato-Noguchi, H., Ino, T., Sata, N., Yamamura, S., Isolation and identification of a potent allelopathic substance in rice root exudates. Physiol. Plant. 115: [10] Kato-Noguchi, H., Ino, T., Rice seedlings release momilactone B into the environment. Phytochemistry 63: [11] Kato-Noguchi, H., Allelopathic substance in rice root exudates: Rediscovery of momilactone B as an allelochemical. J. Plant Physiol. 161: [12] Kato-Noguchi, H., Ota, K., Ino, T., Release of momilactone A and B from rice plants into the rhizosphere and its bioactivities. Allelopathy J. 22: [13] Kato-Noguchi, H., Salam, M.A., Kobayashi, T., A quick seeding test for allelopathic potential of Bangladesh rice cultivars. Plant Prod. Sci. 12: [14] Kim, K.U., Shin, D.H., Kim, H.Y., Lee, Z.L., Olofsdotter, M., Evaluation of allelopathic potential in rice germplasm. Korean J. Weed Sci. 19: 1-9. [15] Kodama, O., Suzuki, T., Miyakawa, J., Akatsuka, T., Ultraviolet-induced accumulation of phytoalexins in rice leaves. Agric. Biol. Chem. 52: [16] Mamun, A.A., Crop-ecosystem: Weed Vegetation and Weed Management in Dakshin Chamuria and Jawar. Agricultural and Rural Development in Bangladesh. JICA Dhaka, Bangladesh. JSARD, Pub. No. 6: 334. [17] Mamun, A.A., Weed and their control: A review of weed research in Bangladesh. Agricultural and Rural Development in Bangladesh. JSARD Japan International Cooperation Agency, Dhaka, Bangladesh. Pub. No. 19: [18] Narwal, S.S., Allelopathy in weed management. In: Narwal, S.S. (ed) Allelopathy Update. Vol. 2. Basic and Applied Aspects, Science Publishers Inc.; Enfield, New Hampshire, pp [19] Olofsdotter, M., Navarez, D., Rebulalan, M. and Streibig, J.C., Weed-suppressing rice cultivars: Does allelopathy play a role? Weed Res. 39: [20] Otomo, K., Kenmoku, H., Oikawa, H., König, W.A., Toshima, H., Mitsuhashi, W., Yamane, H., Sassa, T., Toyomasu, T., Biological functions of ent- and syn-copalyl diphosphate synthases in rice: key enzymes for the branch point of gibberellin and phytoalexin biosynthesis. Plant J. 39: [21] Putnam, A.R., Tang, C.-S., Allelopathy: State of the science. In: Putnam AR, Tang C-S (eds) The Science of Allelopathy. John Wiley & Sons; New York, pp [22] Rice, E.L., Allelopathy, 2nd ed. Academic Press; Orlando, Florida. [23] Salam, M.A., Morokuma, M., Teruya, T., Suenaga, K., Kato-Noguchi, H., Isolation and identification of a potent allelopathic substance in Bangladesh rice. Plant Grow. Regul. 58: [24] Seigler, D.S., Chemistry and mechanisms of allelopathic interactions. Agron J. 88: [25] Weston, L.A., Utilization of allelopathy for weed management in agroecosystems. Agron J. 88: