THE 4th TROPICAL WEED SCIENCE CONFERENCE 2013

THE 4th TROPICAL WEED SCIENCE
CONFERENCE 2013

Weed Management and Utilization in the Tropics

January 23-25, 2013
The Empress Hotel, Chiang Mai, Thailand

PROCEEDING

THE 4th TROPICAL WEED SCIENCE CONFERENCE

(TWSC 2013)

ORGANIZED BY
WEED SCIENCE SOCIETY OF THAILAND

AND
DEPARTMENT OF AGRICULTURE

Address : Weed Science Society of Thailand
2nd Floor Weed Science Building
Plant Protection Research and Development Office
Department of Agriculture
Chatuchak , Bangkok 10900
Thailand
+66-02-561-1785
Tel/ Fax:

CONFERENCE INFORMATION

REGISTRATION AND HOSPITALITY DESK

The registration and hospitality desks will be situated at “1st FLOOR OF CONVENTION
Building ” The Empress Hotel, Chiang Mai. The registration will be operated from 14:00 -19:00
p.m. on January 22, 2013 and from 8:00-10:00 a.m. on January 23, 2013. The 4th Tropical Weed
Science Conference (TWSC 2013) bag in which the full registered participants will receive
upon registration comprising of a copy of program & abstracts, pen, handy drive and name
badge.

THE CONFERENCE VENUE

The main Conference activities will take place at the CONVENTION Building, The
Empress Hotel, Chiang Mai, THAILAND. This hotel is near Night Bazaar at 10 minute distance
walk, 30-40 minutes from the Chiang Mai International Airport.

THE SECRETARIAT OFFICE

The Secretariat office will be at “1st FLOOR OF CONVENTION Building”, set up
near the main conference room to facilitate and assist delegates and participants during the
Conference. Services will be available from 13.00-18.00 on January 22 and 08:00-16:00 on
January 23, 2013. Arrangements for checking PowerPoint presentation by the speakers will
also be available at the secretariat office.

POSTERS AND TRADE EXHIBITION

20 Posters, 14 Trade and Professional Exhibition will run concurrently with the
Conference display adjacent to the conference room for the duration of the Conference.
Presenters are requested to check the program for the board number assigned to them and use
the board with the same number. One mounting board measuring 1.8 m high and 1.2 m wide
will be available for each poster exhibit. Poster and Trade Exhibition should be mounted from
13:00-19:00 on January 22 and from 08:00-10:00 on January 23, 2013. Presenters are requested
to be present at their posters from 08:00-10:00, 16:30-17:30 on January 23-24, 2013.

LANGUAGE

English will be used in all scientific sessions and all Conference communications and
publications.

VISUAL AIDS

The following facilities will be available to assist the speakers in their presentation of
oral papers.

- LCD projectors
- Computers to read CDs

THE HOTEL

The Empress Hotel, the venue of the Conference is situated at The Empress Hotel &
Convention Centre 199/42 Chang Klan Road, Chiang Mai 50100 Thailand Tel. (053) 253 199,
Fax: (053) 272 467.

Le Méridien Chiang Mai 108 Chang Klan Road, Tambol Chang Klan, Amphur Muang,
Chiang Mai, 50100 Thailand Tel. : (66)(53) 253 666

All participants are requested to stay both hotels for convenience in attending the
Conference.

TRANSPORTATION

All foreign participants may enter Thailand through either Bangkok or Chiang Mai
International Airports. Transfers to domestic flights are available at Bangkok with Chiang Mai.
Transportation service ic, airport taxi is available all the time for serving at the taxi counter and
hotel limousine need booking prior to the National Organizing Committee. Train travel and
air-conditioned first-class sleepers are also convenient for participants. It can also be reached
comfortably both day and night by deluxe air-conditioned coaches.

SOCIAL EVENTS

Welcome reception dinner will be organized on Wednesday, January 23, 2013 at 18.30-
21.00 p.m. at The Imperial Ballroom, The Empress Hotel.

Farewell dinner will be organized on Friday, January 25, 2013 at 18.30-21.00 p.m. at
Imperial Ballroom, The Empress Hotel.

PROFESSIONAL TOUR TO GROWING SITES

A one-day professional excursion is being organized on January 25, 2013 to enable
participants to visit Rice Research Center or Mae Kuang Udomthara Dam, Royal Project or
Chiang Mai’s Tiger Kingdom. Refreshments and lunch box will be provided. Admission fee of
this excursion for an accompanying person is free of charge. Those who wish to join this field
trip please sign your name at the registration desk to secure a seat in the bus or van.

MEALS

For vegetarians, please inform the National Organizing Committee for preparing your
meals earlier.

ACKNOWLEDGEMENT

The National Organizing Committee wishes to acknowledge the following organizing
companies and persons whose generous support has made this conference and the professional
tour possible.

- Royal Project Foundation
- Chiang Mai Rice Research Center
- Mae Kuang Udomthara Dam
- Office of Agricultural Research and Development Region 1
- Chiang Mai Royal Agricultural Research center

Contents Page

Organizing Committee............................................................................................................ 7

Allelopathy in Sustainable Agriculture: Rice Allelopathy and Momilactone..................... 8

Hisashi Kato-Noguchi

Allelochemicals in Cuscuta campestris Yuncker......................................................... 23

BakiHj Bakar, Sow Tein Leong, Muhammad Remy Othman, MohamadSuffianMohamad Annuar and
Khalijah Awang

Allelopathic Potential of Jasminum officinale f. var. grandiflorum (Linn.) Kob. and Its

Physiological Mechanisms on Bioassay Plants.......................................................... 29

Montinee Teerarak, Patchanee Charoenying and Chamroon Laosinwattana

Studies on Natural Herbicide Resistance (HR) among traditional and developed rice
(Oryza sativa L.) varieties cultivated in Sri Lanka and inducing HR with Chemical

mutagens, NaN3 and EMS...................................................................................... 26

Shyama Ranjani Weerakoon, R. G. Danushka Wijeratne and Seneviratne Somaratne

Rapid bioassay method for herbicide dose-response study and herbicide resistance

diagnosis................... ......................................................................................... 37

Chuan-Jie Zhang, Soo-Hyun Lim and Do-Soon Kim

Efficacy and Rice Crop Tolerance to Mixtures of Penoxsulam+Cyhalofop as

One-Shot Rice Herbicide in ASEAN Countries.......................................................... 41

N. Lap, S. Somsak, I.M. Yuli, Le Duy, Lee Leng Choy, Ermita, Bella Victoria, B.V. Niranjan, R.K.Mann

Potential of Organic Herbicide from Aglaia odorata Lour........................................... 48

Chamroon Laosinwattana Montinee Teerarak and Patchanee Charoenying

Diversity of Hyphomycetes Fungi from Diseased Weeds............................................. 55

Duangporn Suwanagul, Jitra Kokae and Anawat Suwanagul

Impacts of meadowfoam seed meal amendment on weeds and soil microbial activity....... 61

Suphannika Intanon, Andrew Hulting, David Myrold, and Carol Mallory-Smith

Imidazolinone tolerance variety for weedy rice control in direct-seeded rice:

The Malaysian Experience.......................................................................................69

Azmi, M, Yim, K.M. and George, T.V.

Phylogenetic relationships of Echinochloa species based on phenotypic and

SSRs markers....................................................................................................... 74

Eun-Jeong Lee, Min-Jung Yook, Do-Soon Kim

Eco-efficient weed management approaches for rice in tropical Asia............................. 78

A.N. Rao and A. Nagamani

Morphological and physiological responses of Miscanthus spp. to varying temperature

and light intensity....................................................... ......................................... 88

Jastin Edrian Revilleza, Soo-Hyun Lim, Ji-Hoon Chung, Do-Soon Kim

Flucetosulfuron performance improved by adjuvant................................................... 91

Jin Won Kim, Seong-Hyu Shin, Jong-Nam Lee, Se-Eun Lim, Soo-Hyun Lim, Do-Soon Kim

Baseline sensitivity of Echinochloa crus-galli to alternative herbicides selected for

managing herbicide resistant Echinochloa species..................................................... 95

Ji-Soo Lim, Soo-Hyun Lim, Do-Soon Kim

Weed and Weedy Rice Control by Imidazolinone Herbicides in ClearfieldTM Paddy in

Vietnam............................................................................................................. 98

Duong Van Chin, Tran Cong Thien, Huynh Hong Bi, Nguyen Thi Nhiem and Tran Thi Ngoc Son

Utilization of weeds in Thailand............................................................................ 103

Pensee Nantasomsaran, Komson Nakornsri and Patpitcha Rujirapongchai

A General View of Weeds in Lowland Rice and Up-Land Crops in The South of

Vietnam............................................................................................................. 113

Ho Van Chien

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 7

Organizing Committee

Organizer: Weed Science Society of Thailand

Department of Agriculture, Ministry of Agriculture and Cooperatives

Advisory Board:

Mr. Dumrong Jirasuthas
Dr. Manthana Milne
Prof. Umporn Suwannamek
Prof. Rungsit Suwanmankha

International Organizing Committee

Prof. Stephen B. Powles (Australia)
Prof. Carol Mallory-Smith (USA)
Prof. Koichi Yoneyama (Japan)
Prof. Nilda R. Burgos (USA)
Prof. Do Soon Kim (Korea)
Prof. Hisashi Kato-Noguchi (Japan)
Prof. Steve Adkins (Australia)
Prof. Michael Braverman (USA)
Assoc. Prof. Sansanee Jamjod (Thailand)
Dr. David Johnson (IRRI)

National Organizing Committee

Chairman: Dr. Chanya Maneechote

Vice-Chairman: Dr. Sarawut Rungmekarat

Secretary: Ms. Nongnuch Yokyongsakul

Proceedings: Dr. Chamroon Laosinwattana

Registration: Ms. Wanida Thantawin

Ceremony: Dr. Acharaporn Na Lampamg Noenplub

Reception: Mr. Virach Chantarasmee

Public Relations: Ms. Punnee Witchachoo

Treasurer: Dr. Sujin Jenweerawat

Venue: Mr. Sirichai Sathuwijarn

Excursion: Ms. Jeerawan Petpaisit

Sponsor: Mr. Komsan Nakornsri

Field trials: Mr. Jeerawat Jitprom

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 8

Allelopathy in Sustainable Agriculture: Rice Allelopathy and Momilactone

Hisashi Kato-Noguchi
Department of Applied Bioresource Science, Faculty of Agriculture, Kagawa University, Miki,

Kagawa 761-0795, Japan,
E-mail address: [email protected]

Abstract

Given the agricultural importance of rice, it has been extensively studied with respect to its
allelopathy as part of a strategy for sustainable weed management options. All available information
indicates that rice plants possibly release allelochemicals into the neighbouring environments, and a
number of compounds, such as phenolic acids, fatty acids, phenylalkanoic acids, hydroxamic acids,
terpenes and indoles, have been identified in rice plant extracts, root exudates, and decomposing residues
as potential rice allelochemicals. However, the studies demonstrate that the diterpenoid momilactones
are the most important rice allelochemicals, with momilactone B playing a particularly critical role.
Rice plants secrete momilactone B into the neighboring environments over their entire life cycle at
phytotoxic levels, and momilactone B seems to account for the majority of the observed rice allelopathy.
Allelochemicals can provide a competitive advantage for host-plants through suppression of soil
microorganism and inhibition of the growth of competing plant species because of their antibacterial,
antifungal, and growth inhibitory activities. The use of allelopathic rice can definitely reduce the
ecological impact in rice cultivation, particularly by reducing the amount of herbicide used. The rice
allelopathy may be one of the options in the sustainable weed management strategies.

Keywords: Allelopathy, Allelochemical, Momilactone, Root exudates, Sustainable weed management.

Introduction

Weeds cause reductions in rice yield and quality and remain one of the biggest problems in rice
production. The negative impacts of commercial herbicide use on the environment make it desirable
to diversity weed management options. Allelopathy is one of the options (Rimando and Duke, 2003;
Macías et al., 2007; Kong, 2008; Tesio and Ferrero, 2010). Allelopathy is the direct influence of an
organic chemical released from one living plant on the growth and development of other plants (Inderjit
and Duke, 2003; Belz, 2007; Macías et al., 2007). Allelochemicals are such organic chemicals involved
in the allelopathy (Rice 1984; Putnam and Tang 1986;Inderjit 1996). Allelochemicals can provide a
competitive advantage for host-plants through suppression of soil microorganism and inhibition of
the growth of competing plant species because of their antibacterial, antifungal, and growth inhibitory
activities (McCully, 1999; Hawes et al., 2000; Bais et al., 2004). 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. A large number of rice varieties were found to inhibit the growth of several
plant species when these rice varieties were grown together with these plants under the field or/and
laboratory conditions (Dilday et al., 1994; 1998; Kim et al., 1999; Olofsdotter et al., 1999; Azmi et
al., 2000; Gealy et al., 2003; Seal et al., 2004a; Kim et al., 2005). These findings suggest that rice may
produce and release allelochemicals into neighboring environment, thus encouraging the exploration
of allelochemicals in rice. Many secondary compounds, such as phenolic acids, fatty acids, indoles and
terpenes were identified in rice root exudates and decomposing rice residues as putative allelochemicals
(Takeuchi et al., 2001; Rimando and Duke, 2003; Khanh et al., 2007). However, these compounds are
almost ubiquitous in plants and rice allelopathy can not be explained by these compounds (Olofsdotter
et al., 2002b; Seal et al., 2004b). Tricyclic diterpen, known as momilactone B, which are unique to
rice, have been isolated (Kato-Noguchi et al., 2002). Momilactone B inhibits the growth of typical

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 9

rice weeds like Echinochloa crus-galli and E. colonum at concentrations greater than 1 μM (Kato-
Noguchi et al., 2008). Rice plants secrete momilactone B from the roots into the rhizosphere over their
entire life cycle (Kato-Noguchi et al., 2003). These observations suggest that rice plants may inhibit
the growth of the neighboring plants through the secretion of momilactone B into their rhizosphere.
Although considerable progress in our understandings has been made, much work remains to be done
before we fully understand this process in rice and can utilize it for more environmentally benign
weed management. This chapter summarized the research history of rice allelopathy and putative
allelochemicals found and discussed possible involvement of these compounds in rice allelopathy.

The first observation of rice allelopathy
The first observation of allelopathy in rice was made in field examinations in Arkansas, U.S.A.

in which about 191 of 5,000 rice accessions inhibited the growth of Heteranthera limosa (Dilday et
al., 1989). This finding led to a large field screening program. More than 16,000 rice accessions from
99 countries in the USDA-ARS germplasm collection have been screened. Of these, 412 accessions
inhibited the growth of H. limosa and 145 accessions inhibited the growth of Ammannia coccinea
(Dilday et al., 1994, 1998). In Egypt, 1,000 rice varieties were screened for suppressive ability against
Echinochloa crus-galli and Cyperus difformis under field conditions, and inhibitory activity was found
in more than 40 of them (Hassan et al., 1998). Similar attempts have been conducted in some other
countries, and many rice varieties were found to inhibit the growth of several plant species (Kim and
Shin, 1998; Olofsdotter et al., 1999; Pheng et al., 1999). It is obscure, however, whether the inhibition was
caused by only allelopathic effects. Plant-to-plant interference is a complex combination of competitive
interference for resources such as light, nutrients and water, and allelopathic interactions (Qasem and
Hill, 1989; Einhellig, 1996; Belz, 2007). Competitive interference and allelopathy cannot be separated
under field conditions (Fuerst and Putnam, 1983; Leather and Einhellig, 1998). Considering the
allelopathic potential of plants, however, it is essential to distinguish between the effects of competitive
interference and allelopathy (Fuerst and Putnam, 1983; Leather and Einhellig, 1986; Inderjit and
Olofsdotter, 1998). Thus, bioassays in allelopathy research should be designed to eliminate the effects
of competitive interference from their experimental systems. Many scientists have also paid attention
to test solution characteristics for bioassays in allelopathy research because the growth of roots and
shoots of several plants as well as germination are inhibited by extreme pH and osmotic potential in test
solutions (Wardle et al., 1992; Haugland and Brandsaeter, 1996; Hu and Jones, 1997).

Rice allelopathy in controlled environments
Well-designed bioassays under controlled environments can only evaluate the allelopathic

potential of plants (Leather and Einhellig, 1986; Inderjit and Olofsdotter, 1998). A laboratory whole-
plant bioassay for allelopathic rice screening, called “relay-seedling assay”, was developed at the
International Rice Research Institute in the Philippines (Navarez and Olofsdotter et al., 1996). This
bioassay may eliminate the effects of competitive interference for resources between rice and test
plants from the experimental system, and may evaluate the allelopathic potential of rice. By using this
bioassay, several rice varieties were found to possess strong growth inhibitory activity. In addition, the
111 rice varieties were tested for their growth inhibitory activity under laboratory and field conditions,
but the results were inconsistent (Olofsdotter et al., 1999). Screenings for allelopathic rice have also
been undertaken in several other laboratories. These studies shown that there was a marked difference
among rice varieties in growth inhibitory activity and that about 3-4% of tested rice varieties had strong
allelopathic potential (Fujii, 1992; Hassan et al., 1998; Kim et al., 1999; Olofsdotter et al., 1999; Azmi
et al., 2000). These results suggest that some rice varieties may possess allelopathic potential.

The allelopathic potential of rice seedlings of eight cultivars was determined at an early
developmental stage in Petri dishes under controlled laboratory conditions (Kato-Noguchi and Ino,
2001). Three plants, alfalfa (Medicago sativa), cress (Lepid­ ium sativum) and lettu­ ce (Lactuca sativa)
were chosen for the bioassay as test plants because of their known germination behaviors. According
to the test solution of Weidenhamer et al. (1987), phosphate buffer (pH 6.0) was chosen as the test
solution, which did not affect the germination and growth of cress, lettuce, alfalfa or rice, and did not

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 10

cause any significant pH changes during the bioassay. In addition, no effect of osmotic potential of the
test solutions in all dishes was detected on the germination and growth of these plant species. The trial
indicated that all rice cultivars tested inhibited the growth of roots, shoot and fresh weight of these test
plants. However, the effectiveness of cv. Koshihikari on growth inhibition was greatest among these
rice cultivars and more than 60% inhibition was recorded by cv. Koshihikari in all bioassays. Test
plants could germinate and grow with the rice seedlings without competition for nutrients and water
because no nutrients were added in the bioassay and water was supplied regularly (Kato-Noguchi and
Ino, 2001). Light is also unnecessary in the developmental stages of these seedlings, since seedlings
mostly withdraw nutrients from the reserve of their seeds during early developmental stages (Fuerst
and Putnam, 1983). Thus, the inhibitory effects of these rice seedlings may not be due to competitive
interference, suggesting that rice seedlings may have allelopathic potential. Allelopathic potential of
these rice seedlings against typical rice weeds, Echinochloa crus-galli, were also determined by a
“donor-receiver bioassay” (Kato-Noguchi et al., 2010). All rice cultivars inhibited the growth of shoots
and roots of E. crus-galli seedlings, but with a different level of inhibitory activity. Koshihikari showed
the greatest inhibitory activity on both shoot and root growth of E. crus-galli.

Phenolic acids
Phenolic acids are often mentioned as putative allelochemicals and the most commonly

investigated compounds among potential allelochemicals since they have been found in a wide range
of soils (Hartley and Whitehead, 1985; Inderjit, 1996; Dalton, 1999). Hsu et al. (1989) evaluated the
inhibitory activities of phenolic acids against germination of lettuce and alfalfa. 4-Hydroxybenzoic
acid and salicylic acid were the most active and inhibited the germination at a concentration greater
than 0.5-1.5 mM. Olofsdotter et al. (2002b) evaluated whether phenolic acids are responsible for rice
allelopathy. They found that allelopathic rice cultivars did not release a significantly greater amount
of phenolic acids than non-allelopathic cultivars. The maximum release rate of phenolic acid from
rice plants was approximately 10 μg/plant /day. Therefore, at a conventional plant density (100 rice
plants/m2), the release rate of phenolic acids would be approximately 1 mg/m2 day. Considering the
inhibitory activity of phenolic acids, it was concluded that, even if all phenolic acids were as phytotoxic
as 4-hydroxybenzoic acid, the release level of phenolic acids from rice is not sufficient to cause growth
inhibition of neighboring plants (Olofsdotter et al., 2002b). Five major phenolic acids in rice root exudates,
4-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid and caffeic acid, were mixed and
their biological activities were determined against Sagittaria monotevidensis (Seal et al., 2004b). The
concentration required for 50% growth inhibition (IC50) of the mixture of these five phenolic acids was
502 μM. The concentrations of these phenolic acids detected in rice roots exudates were by far less than
500 μM (Seal et al., 2004b). The inhibitory activity of a mixture of all 15 compounds identified in rice
roots exudates, resorcinol, 2-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 4-phenylbutyric
acid, 4-hydroxybenzoic acid, vanillic acid, syringic acid, salicylic acid, cinnamic acid, p-coumaric acid,
caffeic acid, ferulic acid, 5-hydroxyindole-3-acetic acid and indole-5-carboxylic acid and abietic acid,
was also determined and IC50 of the mixture was found to be 569 μM (Seal et al., 2004a, 2004b). In
addition, it was clarified that synergistic action of phenolic acids on growth inhibition did not work well
(Seal et al., 2004b). These studies indicate that any compounds found in rice root exudates including
phenolic acids are not responsible for the allelopathy of rice. All information available suggests that
phenolic acid concentrations in rice root exudates were much lower than the required threshold of these
phytotoxic levels, and phenolic acids seem not to act as rice allelochemicals.

Momilactones
Momilactone A and B were first isolated from rice husks as growth inhibitors (Kato et al., 1973;

Takahashi et al., 1976). Momilactone A and B were later found in rice leaves and straw as phytoalexins
(Cartwright et al., 1977; 1981; Kodama et al., 1988; Lee et al., 1999). Thereafter, the function of
momilactone A as a phytoalexin has been extensively studied and several lines of evidence indicate that
momilactone A has an important role in rice defense system against pathogen attacks (Nojiri et al., 1996;

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 11

Araki and Kurahashi, 1999; Takahashi et al., 1999; Tamogami and Kodama, 2000; Agrawal et al., 2002).
Although the growth inhibitory activity of momilactone B was much greater than that of momilactone
A (Takahashi et al., 1976; Kato et al., 1977), the efforts to find the function of momilactone B were
limited. Momilactone A and B, respectively, inhib­ite­ d the growth of Amaranthus lividus, Digitaria
sanginalis and Poa annua at concentrations greater than 20 ppm (ca. 60 µM) and 4 ppm (ca. 12 µM)
(Chung et al., 2005). Momilactone A and B were also reported to inhibit the growth of Echinochloa
crus-galli and E. colonum, which are the most noxious weeds in rice fields, at concentrations greater
than 10 and 1 µM, respectively. Thus, effectiveness of momilatone B on growth inhibition is much
greater than that of momilactone A. The growth inhibitory activities of momilactome B are also greater
than those of momilactone A under other bioassay systems, (Takahashi et al., 1976; Kato et al., 1977;
Fukuta et al., 2007; Toyomasu et al., 2008). Momilactone A and B, respectively, inhibited root and shoot
growth of rice seedlings at concentrations greater than 100 and 300 µM. IC50 values of momilactone
A and B on rice root and shoot were not obtained because of their weak inhibitory activities against
rice. The inhibitory activities of momilactone A and B, respectively, on the root and shoot growth of
rice seedlings were 1 - 2 % and 0.6 - 2 % 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. These results suggest that the toxicities of
momilactone A and B to rice seedlings are much less than those to the two weed species (Kato-Noguchi
et al., 2008). Momilactone A and B were secreted from rice plants into the rhizopsphere throughout all
life cycle stage of rice (Kato-Noguchi et al., 2003; 2008). The secretion level of momilactone A and B
increased until flowering initiation, and decreased thereafter. The level of momilactone A and B at day
80 (around flowering) was 1.1 and 2.3 μg/plant/day, which was 55- and 58-fold greater than that at day
30. Although concentration of momilactone A in rice was greater than that of momilactone B, secretion
level of momilactone B was greater than that of momilactone A, which suggests that momilactone B
may be selectively secreted into the rhizophere than momilactone A. Considering the growth inhibitory
activity and concentrations found in the bioassay medium, momilactone A may cause only 0.8 - 2.2%
of the observed growth inhibition of E. crus-galli roots and shoots by rice. However, momilactone
B in the medium was estimated to cause 59 - 82% of the observed growth inhibition of E. crus-galli
roots and shoots by the rice seedlings. In addition, the concentrations of momilactone B in the medium
reflected the observed differences in the growth inhibition of E. crus-galli by the eight rice cultivars
investigated (Kato-Noguchi et al., 2010). This suggests that the allelopathic activity of rice may be
primarily depend on the secretion level of momilactone B. Therefore, momilactone B may play a very
important role in rice allelopathy.

Rice allelopathy and allelochemicals
Since the first observation of allelopathy in rice by Dilday et al. (1989), more than 16,000 rice

accessions from 99 countries in the USDA-ARS germplasm collection have been screened. Of these,
412 accessions inhibited the growth of Heteranthera limosa and 145 accessions inhibited the growth
of Ammannia coccinea (Dilday et al., 1994; 1998). Similar attempts have been conducted in some
other countries, and a large number of rice varieties were found to inhibit the growth of several plant
species when these rice varieties were grown together with these plants under field and/or laboratory
conditions (Kim et al., 1999; Olofsdotter et al., 1999; Azmi et al., 2000; Gealy et al., 2003; Seal et al.,
2004a; Kim et al., 2005). These findings suggest that rice may produce and secrete allelochemicals into
its neighboring environments. Although mechanisms of the exudation are not well understood, it is
suggested that plants are able to secrete a wide variety of compounds from root cells by plasmalemma-
derived exudation, endoplasmic-derived exudation, and proton-pumping mechanisms (Hawes et al.,
2000; Bais et al., 2004). Through the root exudation of compounds, plants are able to regulate the soil
microbial community in their immediate vicinity, change the chemical and physical properties of the
soil, and inhibit the growth of competing plant species (MuCully, 1999; Hawes et al., 2000; Bais et
al., 2004). Momilactone B was secreted from rice plants into the rhizopsphere throughout all life cycle
stage of rice (Kato-Noguchi et al., 2003; 2008). Considering the inhibitory activity and the secretion
level, momilactone B may play a very important role in rice defense mechanism in the rhizosphere as
an allelochemical.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 12

Rice allelopathy for sustainable agriculture
Sustainable agriculture has now received more attention from agricultural scientists, ecologist

and social economists. Sustainable agriculture requires making efficient use of resources internal to the
farm, and relying on a minimum of essential external inputs (Tesio and Ferrero, 2010). Putnam and Duke
(1974) have first evaluated the possibility of using allelopathic crops to manage weeds in agricultural sites
to minimize serious problems in the present agricultural production, such as environmental pollution,
human health concern and depletion of crop diversity. Allelopathy may represent a new frontier for
the implementation of the practices applicable in the integrated weed management strategies by using
suppressive cover crops, crop rotation and selection of varieties with strong allelopathic potential to
biologically reduce the intensity of weeds. Another important approach is the identification of genes with
allelopathic activities, and the application of breeding and transgenic techniques to place allelopathic
traits into useful crops. Progress made in understanding mechanisms of allelochemicals selectively,
physiological modes of action, and genetic regulation of the biosynthesis should represent the basis for
manipulation of germplasm resources. In addition, allelochemicals have potential as either herbicides
or templates for new herbicide classes because of strong herbicidal potential (Duke, 1986; Dodge, 1987;
Putnam, 1988; Gross and Parthier, 1994; Seigler, 1996; Duke et al., 2000; Macías et al., 2007).

The use of allelopathic rice cultivars and allelochemicals can definitely reduce the ecological
impact, particularly by reducing the amount of herbicide used. Allelopathic rice cultivars combined
with cultural management options is, therefore, an interesting and potential technique, contributing
to alternative chemical control of weeds in paddy ecosystems (Weston, 1996; Olofsdotter, 2001;
Olofsdotter et al., 2002a). Such an allelopathy-based technique for paddy weed control is the most
easily transferable to the low-input management systems prevailing in most Asian rice farming systems
(Kong, 2008). Therefore, the rice allelopathy may be one of the options in the sustainable weed
management strategies.

References

Agrawal, G.K., Rakwal, R., Tamogami, S., Yonekura, M., Kubo, A. and Saji, H. (2002) Chitosan
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Allelochemicals in Cuscuta campestris Yuncker

BakiHj Bakar1, Sow Tein Leong2, Muhammad Remy Othman1, MohamadSuffianMohamad Annuar1 and
Khalijah Awang2

1Institute of Biological Sciences, University of Malaya, 50603Kuala Lumpur, Malaysia
2Department of Chemistry, Faculty of Science, University of Malaya, 50603Kuala Lumpur, Malaysia

Email: [email protected]

Abstract

Golden dodder (Cuscuta campestris Yuncker), commonly known as Rumput Emas in Malaysia
is a parasitic weed infesting many crops and weed species alike. A phytochemical study on the chemical
constituents of C. campestris was carried out. Six compounds were isolated through chromatographic
method and identified as sitosterol1, pinoresinol2, arbutin3, kaempferol4, quercetin5, and astragalin6.
The ethanolic extract of C. camprestris displayed inhibitory allelopathic effects at doses 500 ppm and
above by inhibiting seeds germination and seedling growth of lettuce, radish and weedy rice as the
test plants. The allelopathic potential of three compounds isolated, viz. kaempferol4, sitosterol1 and
pinoresinol2 were investigated. All the three compoundsat doses of 1-100μM showed stimulatory
effects on plant growth of radish, lettuce and weedy rice seedlings. The response of all assayed species
was dose-dependent.

Keywords: Cuscuta campestris, seed germination, seedling growth, flavonoids, sitosterol,
pinoresinol, arbutin

Introduction

Synthetic herbicides when used repetitively not only are likely to persist in the environment
as bound residues and damage other organisms, but also may lead to the emergence of resistantweed
species (Adam et al. 2010).The release of bioactive compounds or allelochemicals from natural sources
has the ability to suppress the growth of weeds (Baki et al. 2009). Natural products are a source of
compounds that might be used as herbicide directly or as lead structure for herbicide discovery(Duke et
al., 2000). Our preliminary screening study showed that the crude aquous extracts of C. campestris as
a parasitic weed with special features – the haustoria infesting and growing on different types of weeds
and plant crops alike, possess some potent bioactive constituents that are herbicidal in nature, and
strongly inhibited the growth of lettuce and radish seedlings (Othman et al. 2012). Cuscuta campestris
Yuncker from the family Cuscutaceae (Convolvulaceae) is an annual obligate angiosperm parasite with
golden yellow colour (Shen et al. 2005). This parasite twines on other plants and attaches to the above-
ground parts of a wide range of host plants. A single plant of C. campestris may attack varieties of host
plants at a time and the host can be weeds or crops (Bungard et al., 1999; Baki et al. 2009, Othman et
al. 2012). It grows in abandoned area like shrubs and bushes on roadsides and open spaces in Malaysia
(Baki et al. 2009).

In this communication we investigated the allelopathic activities of C. campestris and assess its
potential as a natural herbicide, primarily focusing on its bioactive chemical constituents and chemical
bioassay.

Materials and Methods

Extraction, Isolation and Separation
The Cuscuta samples were separated from the hosts after collection from Johore, Malaysia in

October–December 2010. All the samples were dried in the oven for 24 - 48 hours at 60°C.About 200g
of the dried samples of Cuscuta was dismembered and extracted with ethanol at room temperature

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 17

for three times. The ethanol extract was freed from the solvent by rotary evaporator and freeze dried.
About 5g of the crude was subjected to column chromatography (CC) with silica gel as the adsorbent.
Gradient elutions with three solvents were employed as the mobile phase; hexane, ethyl acetate (EA),
and methanol. Every fraction was collected in 200 mL of eluents. A total of 28 fractions were collected
from the first column. Each fraction was tested on TLC to check for purity. Fraction II (Hexane: EA;
100: 0), VI (Hexane: EA; 80: 20), and X (Hexane: EA; 20: 80) were further subjected into CC in order
to obtain single spot on TLC. Fraction II (245.7mg) was fractionated in hexane-ethyl acetate. Of 38
fractions that were tested with TLC and NMR, fractions 19-23 gavesitosterol1 (1.3mg).Fraction VI
(71.4mg) was subjected to CC using hexane: ethyl acetate solvent system to give two compounds;
pinoresinol2(3.5mg, 50:50) and kaempferol4(5.6mg, 0:100). From fraction X, 27 sub fractions were
obtained and sub fraction 10 was subjected again to CC with dichloromethane and ethyl acetate as
solvent systems. From this fraction, eluted by a mixture of dichloromethane-ethyl acetate (50: 50), was
purified quercetin5(0.5mg), while Fraction XII was further purified using HPLC to give arbutin3(2.1mg)
and kaempferol-3-O-glucoside 6(1.3mg). All the structures were identified by comparing their NMR
and UV spectra with those reported in the literature.

Bioassays
Bioassays based on seed germination, shoot and root growths of radish (Raphanus sativus),
lettuce (Lactuca sativa) and weedy rice (Oryza sativa) were used to study the allelopathic potential of
C. campestris. About 1 ml of ethanol extract of dried C. campestris was dissolved in 200 ml deionized
water with 1 % of ethanol. The extract was set as original concentration (5000 ppm) and diluted into
100 ppm, 200 ppm, 500 ppm, and 1,000 ppm. An 8 ml aliquot of the extract was pipette in to petri dish
that was lined with filter paper and previously sown with 20 seeds of radish (R. sativus). The control
used was deionized water with 1% of ethanol. These petri-dishes were placed in Precision Plant Growth
Chamber Model 818 (230 V, 860 watts) for 7 days. Three replicates were prepared for each treatment. The
plants were frozen seven days after treatment in order to avoid subsequent growth until measurements
were recorded. The seed germination, shoot and root lengths, and dry mass were recorded. Selected
constituents, namely, kaempferol, sitosterol and pinoresinol from C. campestris were prepared with
1% of ethanol in deionized water (100μM) and the rest (1 and 10μM) were obtained by dilution. The
same bioassay method was used to determine the allelopathic potential of these chemical constituents
on lettuce seed germination and root and shoot lengths following exposures as such.

Statistical Analysis
The statistical analyses were performed on the data using analysis of variance (ANOVA) using

the SPSS Program version 15.0. Any significant difference between treatment means was tested with
Tukeys’ test at p<0.05. The percentages of shoot and root growths were calculated according to the
following formula:

where Pc and Pt are the shoot or root lengths of the control and the treated samples, respectively.

Results and Discussion

Identification of Chemical Constituents from Cuscuta campestris
Six different chemical constituents were isolated from C. campestris: three were flavonoids,

one sterol, one lignan and one arbutin. The three flavonoids were kaempferol4, kaempferol-3-O-
glycoside6 and quercetin5. The concentration of kaempferol4 was relatively high vis-a-vis the six
chemical constituents in the parasitic weed. All these constituents are putative allelochemicals which
show allelopathic effects on the test plants (see section 2.2). The identifications of these constituents are
based on the comparison of the spectral data with those reported. The chemical structures of sitosterol1,
pinoresinol2, arbutin3, kaempferol4, quercetin5, and astragalin6 are shown below.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 18

29 HO 5
28 4

6

21 22 24 27
25
18 20 23 H3CO 3 2 1 O
26 7
12 17 H
9
11 13 8
19 H 14
19 16 8'

9' 2'

2 10 8 15 O 7' 1' 3' OCH3
H H
3
HO 5 7
46
6' 4' OH
5'

1 2

OH 3' OH
2' 4'
4
3 5 HO 8
7
9 O 1' 5'
6 2
5
OH 6 6'

2

6' 1 3

4' 5' O O 10 OH
4
HO HO 1'

3' 2' OH O

OH

3 4

OH

3' OH 3' OH

2' 4' 4'
2'

HO 8 O 1' 5' HO 8 O 1' 5'
9 9 6'
7 2 7 2
10 10
6 5 6' 6 5

3 OH 3 2'' OH 4''

4 OH 4 O HO O 3'' OH
6'' OH
1'' 5''

OH O O

5 6

Sitosterol (1) (De-Eknamkul and Potduang, 2003; Rawat et al., 1998), C29H50O : 1H NMR (CDCl3)
δ3.49 (1H, m, H-3), 5.32 (1H, m, H-6), 0.65 (3H, s, H-18), 0.98 (3H, s, H-19), 0.89 (3H,d, J=6.4Hz,

H-21), 0.80 (3H, d, J=1.8Hz, H-26), 0.78 (3H, d, J=, H-27), 0.84 (3H, s, H-29). 13C NMR (CDCl3) δ37.3
(C-1), 31.7 (C-2), 71.9 (C-3), 42.3 (C-4), 140.8 (C-5), 121.8 (C-6), 32.0 (C-7), 32.0 (C-8), 50.2 (C-9),

36.6 (C-10), 21.2 (C-11), 39.8 (C-12), 42.4 (C-13), 56.9 (C-14), 24.4 (C-15), 28.3 (C-16), 56.1 (C-17),

11.9 (C-18), 19.5 (C-19), 36.2 (C-20), 18.9 (C-21), 34.0 (C-22), 26.1 (C-23), 45.9 (C-24), 29.2

(C-25), 19.9 (C-26), 19.1 (C-27), 23.1 (C-28), 12.1 (C-29),.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 19

Pinoresinol (2) (Do et al., 2009; Li Hui et al., 2004), C20H22O6 : 1H NMR (CDCl3) δ6.86 (1H, s, H-2 and
H-2’), 6.87 (1H, d, J=2.28Hz, H-5 and H-5’), 6.81 (1H, dd, J=1.84, 6.64Hz, H-6 and H-6’), 4.71 (1H,
d, J=4.12Hz, H-7 and H-7’), 3.07 (1H, m, H-8 and H-8’), 3.84 (2H, dd, J=4.12, 5.52Hz, H-9 and H-9’),
4.21 (2H, dd, J=6.88, 2.28Hz, H-9 and H-9’), 3.89 (3H, s, 3-OCH3 and 3’-OCH3). 13C NMR (CDCl3)
δ132.95 (C-1 and C-1’), 108.67 (C-2 and C-2’), 146.78 (C-3 and C-3’), 145.31 (C-4 and C-4’), 114.35
(C-5 and C-5’), 119.06 (C-6 and C-6’), 85.97 (C-7 and C-7’), 54.23 (C-8 and C-8’), 71.75 (C-9 and
C-9’), 56.05 (3-OCH3 and 3’-OCH3).

Kaempferol (4) (Hadizadeh et al., 2003; Rawat et al., 1998), C15H10O6 : 1H NMR (CD3OD) δ6.15 (1H,
d, J=1.84Hz, H-6), 6.37 (1H, d, J=2.28Hz, H-8), 8.05 (1H, d, J=9.16Hz, H-2’ and H-6’), 6.87 (1H, d,
J=9.16Hz, H-3’ and H-5’). 13C NMR (CD3OD) δ146.7 (C-2), 135.8 (C-3), 176.0 (C-4), 161.2 (C-5),
97.9 (C-6), 164.2 (C-7), 93.1 (C-8), 156.9 (C-9), 103.2 (C-10), 122.4 (C-1’), 129.3 (C-2’ and C-6’),
115.0 (C-3’ and C-5’), 159.2 (C-4’).

Quercetin (5) (Guvenalp and Demirezer, 2005; Rawat et al., 1998), C15H10O7 : 1H NMR (CD3OD)
δ6.17 (1H, d, J=2.0Hz, H-6), 6.38 (1H, d, J=2.0Hz, H-8), 7.72 (1H, d, J=2.2Hz, H-2’), 6.87 (1H, d,
J=8.52Hz, H-5’), 7.61 (1H, dd, J=2.0, 6.6Hz, H-6’). 13C NMR (CD3OD) : δ146.4 (C-2), 136.0 (C-3),
176.1 (C-4), 161.2 (C-5), 97.9 (C-6), 164.2 (C-7), 93.1 (C-8), 156.9 (C-9), 103.0 (C-10), 120.3 (C-1’),
114.6 (C-2’), 144.9 (C-3’), 147.3 (C-4’), 114.9 (C-5’), 122.7 (C-6’).

Kaempferol-3-O-glucoside (6)(Lee et al., 2004), C21H20O11 : 1H NMR (CD3OD) δ6.20 (1H, d, J=1.96Hz,
H-6), 6.39 (1H, d, J=1.96Hz, H-8), 8.05 (1H, d, J=8.8Hz, H-2’ and H-6’), 6.88 (1H, d, J=8.8Hz, H-3’
and H-5’), 5.25 (1H, d, J=7.1Hz, H-1’’), 3.33-3.96 (5H, m, H-2’’, H-3’’, H-4’’, H-5’’, H-6’’). 13C NMR
(CD3OD) δ159.2 (C-2), 135.5 (C-3), 179.6 (C-4), 161.7 (C-5), 100.2 (C-6), 166.6 (C-7), 95.0 (C-8),
158.7 (C-9), 105.7 (C-10), 122.9 (C-1’), 132.4 (C-2’ and C-6’), 116.2 (C-3’ and C-5’), 161.7 (C-4’),
104.2 (C-1’’), 75.8 (C-2’’), 78.1 (C-3’’), 71.5 (C-4’’), 78.5 (C-5’’), 72.7 (C-6’’).

Arbutin (3)(Cepanec and Litvi, 2008), C12H16O7 : 1H NMR (CD3OD) δ6.93 (1H, d, J=8.8Hz, H-2 and
H-6), 6.66 (1H, d, J=8.8Hz, H-3 and H-5), 4.70 (1H, d, J=7.2Hz, H-1’), 3.25-3.88 (5H, m, H-2’, H-3’,
H-4’, H-5’, H-6’). 13C NMR (CD3OD) : δ152.4 (C-1), 116.6 (C-2 and C-6), 119.4 (C-3 and C-5), 152.4
(C-4), 103.7 (C-1’), 75.0 (C-2’), 78.0 (C-3’), 71.4 (C-4’), 78.0 (C-5’), 62.6 (C-6’).

Bioassay of Ethanol Extract of Cuscuta campestris
Table 1 shows the results on the effect of different concentration of ethanol extract of C.

campestris on the seeds of lettuce, radish and weedy rice. The effect of the extract of C. campestris on
seed germination was not significant except lettuce. Interestingly, the growth of shoot and root of all
three assayed species were severely affected. The efficacy of the extract was measurably higher on the
roots than shoots of the lettuce and weedy rice seedlings. The responses of all assayed species were
dose-dependent (Fig. 1 and Fig. 2). At low concentration (100-1000 ppm), there was negligible and
non-significant reduction in germination of lettuce. At higher dose (5000 ppm), germination of lettuce
seeds was markedly decreasing (45% inhibition). The inhibitory effectsof the extract at 5000 ppm on
the growth of lettuce shoots were highly significant at 89% compared with the control. Synergistic
effects on shoot growth of lettuce seedlings were observed at other applied doses, explaining perhaps
the hormonal role of the extracts at low concentrations. The roots of lettuce displayed higher sensitivity
than the shoots to the ethanol extracts of C. campestris. A significant synergistic effect with 14-47%
stimulation on root growth obtained when low concentrations of 100 -200 ppm. However, when treated
with higher dosage, root growth decreased considerably at 43-95% (Table 1). The ethanol extract of C.
campestris did not show any significant effect on radish seed germination. However, the root growth
appeared greatly affected. With parallel increase in concentration, root growth was inhibited with values
ranging from 27% to 84%, although there was no obvious inhibition at low concentration (100 -200
ppm) (Fig. 2). The effect of C. campestris extract on radish shoot growth was not as great as the effect
on roots. In fact, the growth of radish shoot was inhibited only when higher dosage (1000 -5000 ppm)
applied (Table 1). There were negligible effects of C. campestris extract on weedy rice germination.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 20

Yet, the growth of weedy rice seedlings is sensitive to lower concentration of C. campestris extract.
The shoot and root growthsof weedy rice seedlings were inhibited by C. campestris ethanol extract at
200ppm, with differential sensitivity of 5-63% reductions of shoot growth and 15-93% reductions of
growth compared with the respective controls (Table 1).

Allelopathic Potential of Chemical Constituents of Cuscuta campestris on Lettuce
Three bioactive compounds, kaempferol, pinoresinol and sitosterol were selected to determine

their allelopathic potentials against the test plants. The results were reported as percentage differences
in germination (Table 2), root growth and shoot growths and dry weight (Table 2) compared with
the respective controls. Overall, exposures to each of the three chemical constituents failed to have
any significant effect on the germination of lettuce seeds. Similar exposures to 1 – 100 μM showed
synergistic effects on the growth of shoots and roots of lettuce seedlings vis-à-vis the control. The dose-
mediated synergistic effects on the growth of lettuce seedlings were observed when the concentrations
were increased. This shows that the responses of lettuce to kaempferol, pinoresinol and sitosterol were
dose-dependent with the roots displaying greater sensitivity than the shoots.

Pinoresinol2showedthe greatest stimulatory effect on the growth of rootsand shoots of lettuce.
Exposures to 1 – 100 μM of pinoresinol resulted in 60-76% increase in root growth of lettuce, and 21-
64% in the shoot growth (Table 2). The parallel figures for exposures to kaempferol4 were 20-60%
increase in shoot growth and for the root growth these were 58-67% (Table 2). Sitosterol1 showed the
least growth synergistic effects on lettuce growth with only 13-49% in shoot growth the root growth
increased up to 46-63% following exposures to 1 – 100 μM (Table 2). However, the beneficial effects
of these three constituents on seedling growth of lettuce were reduced with parallel increase in doses in
excess of 100 μM suggesting inhibitory effects at higher concentration.

Conclusions

The biological and pharmacological activities of C. campestris are remarkable (Agha et al.,
1996; Istudor et al., 1984). Nevertheless, not much research was done on the allelopathic potentials of
the weed against other weed species and crops. The preliminary results in this research suggested that
application of C. campestris extract at 500 ppm and above has allelopathic potential on other weeds (e.g.
weedy rice) and crops (lettuce and radish). However, the pure compounds did not exhibit measurable
allelopathic activity against the test plants at concentration of 100 μM and below. This observation
on shoot and root growths of lettuce suggest that the allelopathic potential of these constituents may
be synergistic at low doses, but inhibitory effects on growth may prevail at higher doses. Further
investigation on a broader range of doses and application times of C. campestris in petri-dishes and
soils, on different plants is commendable to be carried out to improve their efficacies for weed control.

Table 1. Effects of ethanol extract of Cuscuta campestris on the germination and growth of lettuce,
radish, and weedy rice seedlings.

Concentration Germination Shoot Length Root Length (mm) Dry Weight (g)
(ppm) (%) (mm)
39.3d (0.0) 0.010a (0.0)
0 100.0b (0.0) Lettuce 57.8f (-47.1) 0.011a (-10.0)
100 100.0b (0.0) 10.0b (0.0) 44.7e (-13.8) 0.012a (-20.0)
200 100.0b (0.0) 14.0c (-39.0) 22.4c (43.0) 0.013 a (-30.0)
500 98.3b (1.7) 13.0c (-30.0) 11.2b (71.6) 0.011a (-10.0)
1000 100.0 b (0.0) 12.4bc (-23.1) 1.9a (95.3) 0.017b (-70.0)
5000 55.0a (45.0) 12.5bc (-24.1)
1.1a (88.9)

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 21

Concentration Germination Shoot Length Root Length (mm) Dry Weight (g)
(ppm) (%) (mm)
71.9c (0.0) 0.15a (0.0)
0 100.0a (0.0) Radish 71.0c (1.2) 0.17a (-13.3)
100 98.3a (1.7) 62.2bc (13.5) 0.15a (0.0)
200 98.3a (1.7) 32.6b (0.0) 52.2b (27.4) 0.16a(-6.7)
500 100.0a (0.0) 41.2c (-26.5) 16.1a (77.6) 0.16a (-6.7)
1000 100.0a (0.0) 40.4c (-23.9) 11.6a (83.8) 0.18a (-20.0)
5000 95.0a (5.0) 29.6b (9.2)
15.9a (51.1)
11.4a (65.1)

0 100.0a (0.0) Weedy rice 51.2c (0.0) 0.34a (0.0)
100 100.0a (0.0) 52.4d (0.0) 50.3c (1.8) 0.33a (2.9)
200 100.0a (0.0) 59.3e (-13.1) 43.2b (15.6) 0.35a (-2.9)
500 98.3a (1.7) 49.8cd (5.0) 43.6b (14.8) 0.36ab (-5.9)
1000 98.3a (1.7) 45.6bc (12.9) 40.6b (20.8) 0.36ab (-5.9)
5000 100.0a (0.0) 41.7b (20.4) 3.5a (93.1) 0.39b (-14.7)
19.4a (63.0)

Values in the column with the same letter are not significantly different at p<0.05.
Values in the parentheses are inhibition percentages over control.
Values in the parentheses with (-) are promotion percentages over control.

Table 2. Effect of three constituents from Cuscuta campestris on the germination and growth of
lettuce (Lactuca sativa) seedlings

Concentration Germination (%) Shoot Length Root Length (mm) Dry Weight (g)
(μM) (mm)
11.85a (0.0) 0.032a (0.0)
0 96.67a (0.0) Kaempferol 19.76b (-66.70) 0.010a (69.29)
1 100.00a(-3.44) 8.52a (0.0) 18.94b (-59.83) 0.010a (68.97)
10 98.33a(-1.72) 13.66b (-60.27) 18.71b (-57.89) 0.009a (73.04)
100 96.67a (0.0) 12.56b (-47.43)
10.25a (-20.31)

Pinoresinol

0 96.67a (0.0) 8.52a (0.0) 11.85a (0.0) 0.032a (0.0)

1 100.00a (-3.44) 13.99c (-64.18) 20.91b (-76.48) 0.011a (64.58)

10 98.33a(-1.72) 12.78c (-49.93) 22.48b (-89.69) 0.010a (67.40)

100 100.00a (-3.44) 10.33b (-21.18) 18.97b (-60.06) 0.010a (69.59)

Sitosterol

0 96.67a (0.0) 8.52a (0.0) 11.85a (0.0) 0.032a (0.0)
0.010a (68.97)
1 98.33a (-1.72) 12.74b (-49.48) 19.36b (-63.36) 0.010a (70.22)
0.009a (70.85)
10 100.00a (-3.44) 12.34b (-44.85) 20.67b (-74.40)

100 96.67a (0.0) 9.62a (-12.83) 17.30b (-45.98)

Values in the column with the same letter are not significantly different at p<0.05.
Values in the parentheses are inhibition percentages over control.
Values in the parentheses with (-) are promotion percentages over control

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 22

Acknowledgements

The authors acknowledge the financial support from University of Malaya to the senior author
through the UMRG Research Grant No. RG007-09SUS.

References

Agha, A. M., Sattar, E. A., Galal, A., 1996.Pharmacological Study of Cuscuta campestris Yuncker.
Phytotherapy Research 10:117-120.

Adam, J., Jeremy, N., Baki, B.B. & Alias, Z. 2010. Resistant Goosegrass (Eleusineindica
[L.]Gaertn.)Biotypes to Glufosinate and Glyphosate in Malaysia.Weed Biology and
Management 10(4):256-260.

Baki, B. B., Remy, M. O., Aini, H., Khalijah, A., Fujii, Y., Annuar, M. S. M., Zazali, A., 2009. Distribution
Patterns, Host Status and Damage Susceptibility of Crop Plants and Weed Species to Cuscuta
campestris Yuncker in Malaysia. Korean Journal of Weed Science 29(3):185-193

Bungard, R. A., Ruban, A. V., Hibberd, J. M., Press, M. C., Horton, P., Scholes, J. D., 1999. Unusual
carotenoid composition and a new type of xanthophyll cycle in plants. Proc. National Academy
of Sciences 96:1135-1139.

Cepanec, I., Litvi, M., 2008.Simple and efficient synthesis of arbutin. Arkivoc 2:19-24.
De-Eknamkul, W., Potduang, B., 2003. Biosynthesis of β-sitosterol and stigmasterol in Croton sublyratus

proceeds via a mixed origin of isoprene units. Phytochemistry 62:389-398.
Do, K. H., Choi, Y. W., Kim, E. K., Yun, S. J., Kim, M. S., Lee, S. Y., Ha, J. M., Kim, J. H., Kim, C. D.,

Son, B. G., Kang, J. S., Khan, I. A., Bae, S. S., 2009. Pinoresinol-4,4’-di-O-β-D-glucoside from
Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse
embryo fibroblasts. Phytomedicine 16: 530-537.
Duke, S. O., Romagni, J. G., Dayan, F. E., 2000. Natural products as sources for new mechanisms of
herbicidal action. Crop Protection 19:583-589.
Guvenalp, Z., Demirezer, L. O., 2005. Flavonol Glycosides from Asperulaarvensis L. Turkish Journal
of Chemistry 29:163-169.
Hadizadeh, F., Khalili, N., Hosseinzadeh, H., Khair-Aldine, R., 2003.Kaempferol from saffron petals.
Iranian Journal of Pharmaceutical Research 2:251-252.
Istudor, V., Predescu, I., Popa, E., Badoi, F., Sialvara, S., 1984. Study of polyphenol and saponoside
derivatives in Cuscuta campestris var. typica F. orsoviana Buia. Farmacia (Bucharest) 32:173-182.
Lee, J., Ku, C., Baek, N.-l., Kim, S.-H., Park, H., Kim, D., 2004. Phytochemical constituents from
Diodiateres. Archives of Pharmacal Research 27:40-43.
Rawat, M. S. M., Pant, G., Prasad, D., Joshi, R. K., Pande, C. B., 1998. Plant growth inhibitors
(Proanthocyanidins) from Prunus armeniaca. Biochemical Systematics and Ecology 26:13-23.
Rice, E. L., 1984. Allelopathy.Academic Press, London.
Rizvi, S. J. H., Rizvi, V., 1992. Allelopathy: Basic and Applied Aspects. Chapman & Hall, London.
Shen, H., Ye, W., Hong, L., Cao, H., Wang, Z., 2005. Influence of the obligate parasite Cuscuta campestris
on growth and biomass allocation of its host Mikania micrantha. Journal of Experimental Botany
56:1277-1284.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 23

Allelopathic Potential of Jasminum officinale f. var. grandiflorum (Linn.)
Kob. and Its Physiological Mechanisms on Bioassay Plants

Montinee Teerarak1, Patchanee Charoenying1 and Chamroon Laosinwattana1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of

Technology Ladkrabang, Bangkok 10520, Thailand
2 Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok

10520, Thailand

Abstract

Higher plants are a rich source of valuable allelochemicals used for weed control technologies
based on natural products. Jasminum officinale f. var. grandiflorum (Linn.) Kob. belongs to the plant
family Oleaceae. The main active compound of oleuropein was isolated from methanolic crude extracts
of J. officinale dried leaves. The pure active compound of oeuropein exhibited lower growth inhibition
compared to methanolic extracts from J. officinale leaves (mixture of allelochemical compound). In
cytogenetic bioassay, methanolic crude extract produced lower mitotic index, alternation of phase index
and induction of mitotic abnormalities. Allelochemicals found in J. officinale adversely affected seed
germination and seedling growth of Echinochloa crus-galli (L.) seeds treated with wettable powder
formulation of J. officinale crude extract inhibited seed germination by impeding seed imbibition and
induction of α-amylase activity. Taken together, the findings presented in this study strongly suggest that
J. officinale may harbor biologically active products the natural properties of which may be exploited
to create a successful biorational herbicide.

Keywords: Allelopathy, α-amylase, biorational herbicide, cytogenetics, imbibition, Jasminum
officinale,

Introduction

The indiscriminate use of new technology of agrochemicals for success for modern agriculture
has made soil sick, caused environmental pollution, development of resistance in pest, toxic residues in
our food and quality of life. This indicates that the new technology is not sustainable over long periods.
Therefore, the recent emphasis in agriculture has shifted from a primary goal of maximizing yields over
the short term, to a sustainable productivity over long periods of time. Sustainability can be achieved
in an agriculture that is ecologically sound, resource conserving and not environmentally degrading.
Natural products have historically been a valuable source of many pesticides, used either directly as
crude preparations, as pure compounds, or as structural leads for the discovery and development of
natural product based pesticides. The impact of natural products have historically been greater on
the development of fungicides and insecticides than on herbicide (Vyvyan, 2002), but the potential
benefits of natural product based herbicide remain underestimated. Higher plants are rich source of
valuable allelochemicals used for weed control technologies based on natural products. The initiation
of laboratory bioassays of allelochemicals on seed germination efficiency and seedling growth is an
important component of modes of action and predict the ability of allelochemicals to field performance
potential for improving weed management. The aims of this study were focused on (i) isolation and
identification of the main active compound from J. officinale leaves; (ii) examination of the growth
inhibitory effect of the main active compound compared with its compound in mixture; and, (iii)
determination of modes of action of the compound in mixture.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 24

Material and methods

Plant materials and methanol extract
J. officinale leaves were harvested, chopped into 1-cm-long pieces and oven-dried at 45°C for

5 days. A hundred grams of dried leaves were used to make methanol extraction by soaking in 1 liter of
methanol at 12°C for 48 h to yield a final concentration of 100 g dry leaf per liter (g/L). The resultant
extract was filtered through four layers of cheesecloth to remove any fiber debris, followed by a second
filtration through Whatman No. 1 filter paper, and set as original concentration (100 g/L). This original
stock extract was kept at 5°C until used.

Methanol extract of J. officinale leaves on seed bioassay
Echinochloa crus-galli L. Beauv. and Phaseolus lathyroides L. seeds were transferred to Petri-

dishes containing filter paper moistened with 5 mLof distilled water and 25-100 g/L of J. officinale
methanol extract. Petri dishes were kept in a growth chamber. The number of germinating seeds was
counted and seedling growth was measured as the root and shoot lengths at seven days after treatment.

Prelimary isolation of active compound
Crude extracts were prepared from J. officinale dried leaves by extract with 90% methanol in

water. A 90% methanol crude extract (OR) from J. officinale leaves was partitioned into aqueous (AQ)
fraction, acidic (AE) and neutral (NE) fractions. Each fraction of OR, AQ, NE and AE fractions in
wettable powder form was prepared to contain four concentrations of each fraction from 1000 to 8000
ppm. Bioassays on seed germination and seedling growth were tested as previously described. The
fraction showing the highest inhibition activity was isolated active compound by column chromatography
and the active substance was characterized by detailed spectroscopic analysis.

Cytogenetic bioassay
Bulbs of Allium cepa L. were used for cytogenetic experiment. After outer scales were removed

and basal ends were cut, the bulbs of A. cepa L. were placed in containers, with their basal ends dipped
in distilled water, and germinated under standard laboratory conditions. When the newly emerged roots
reached 1.50–2.00 cm in length, they were used in the test. The newly emerged roots were treated with a
series of concentrations of the J. officinale extracts (12.5, 25, 50, 100, 200 and 400 ppm) for 18 h. Root
tips of A. cepa were cut off and fixed in ethanol:acetic acid (3:1). The fixed root tips were macerated
in a mixture of hydrolytic enzymes, squashed stained with Giemsa solution for 10 min. Mitotic index,
phase index and chromosomal abnormalities were recorded.

Preparation of methanol extract of J. officinale leaves in wettable form (WP)
One kilogram of 100 mesh J. officinale leaf powder was extracted (ratio 1 kg: 10 L), with

methanol at 25°C constant temperature. After 24 hours of extraction, the brown supernatants were
filtered through four layers of cheesecloth and re-filtered through Whatman no. 1 filter paper (Whatman
Inc. Clifton, NI, USA.). Following filtration, the brown supernatants were dried by evaporation of the
solvent using a rotary evaporator (BUCHI Rotavapor R255), BUCHI, Lausanne, Switzerland) under a
partial vacuum at 45°C until a constant crude extract weight was reached. Wettable powder formulation
of crude extract (JWP) was prepared by dissolving sticky crude extract with acetone in a mortar jar and
then wettable powder (kaolinite:anionic surfactant; 97:3 (w/w)) was added into the mortar jar in a 3:7
ratio (crude extract:wettable powder). The mixture was slowly pulverized until completely dry. Acetone
was added three times and kept in the dark at a low temperature until used.

JWP on seed germination, seed imbibition and α-amylase activity
The JWP was dissolved in distilled water to contain five concentrations of 500, 1000, 2000, 4000

and 8000 ppm. Bioassays on seed germination and seedling growth were tested as previously described.
Measurement of seed imbibition was done by following the method of Turk and Tawaha (2002) whereas
extraction and measurement of activity of α-amylase was done by following the methods of Bernfield
(1955) and Sadasivam and Manickam (1996).

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 25

Statistic analysis
Each treatment consisted of four replications in completely randomized design. Analysis of

variance was calculated for all data and comparisons between treatments were made at probability level
p ≤ 0.05 using Tukey’s Studentized Range Test.

Results

The results indicated that methanolic extracts of J. officinale at all concentrations markedly
reduced the percentage of seed germination of wild pea weed compared with that of the distilled water
control, while barnyardgrass had no significant effect at the 25 and 50 g/L concentrations. For initial
seedling growth, root length of both weed seedlings was inhibited by a magnitude greater than that of
the shoot length (Table 1). The main active compound from J. officinale was isolated and determined
by spectral data as a secoiridoid glucoside named oleuropein. The pure active compound of oeuropein
exhibited lower growth inhibition compared to methanolic extracts from J. officinale leaves (mixture
of allelochemical compound) which may well act synergistically. In cytogenetic bioassay, Allium root
treated with J. officinale extract induced a decrease in mitotic index and alternation of phase index (Fig.
1). In addition, the toxic effect of J. officinale extract induced cell division aberrations.

Table 1 Allelopathic effect of the methanolic extract of Jasminum officinale f. var. grandiflorum (Linn.
Kob.) on the germination, shoot length and root length of Echinochloa cruss-galli (L.) Beauv. and
Phaseolus lathyroides (L.).

Echinochloa cruss-galli (L.) Beauv Phaseolus lathyroides (L.).

Conc. -----------------------------% of control-----------------------------
(g/L)
Root Shoot
germination Root length shoot length germination length length

0 100a 100a 100a 100a 100a 100a
25 97a 87b 98a 85a 50b 75ab
50 75a 28c 95a 62b 15c 52b
75 33b 5c 63b 10c 2d 20c
100 0c 0d 0c 0d 0d 0d

Different letters within the same column indicate significance differences (p<0.05) between
treatments.

Allelochemicals found in J. officinale adversely affected seed germination and seedling growth of E.
crus-galli. seeds treated with JWP. The inhibition percentages on E. crus-galli seed germination treated
with 500 to 8,000 ppm for 7 days was about 0 to 70%, respectively, whereas shoot length was inhibited
19.04 to 71.82% and root length was 76.31 to 100% inhibition, respectively (Table 2). Further studies
were extended to explore the impact of JWP on imbibition and α-amylase activities of E. crus-galli seeds
(Table 3). For all treatment concentrations, no significant differences in imbibition after the 12 and 24
h imbibition time were observed. After the 48 h imbibition period, the percentage of imbibition caused
marked changes for all concentrations used. Application of 500 ppm JWP had a stimulatory activity of
α-amylase on E. crus-galli. An increased concentration of JWP inhibited α-amylase activity. However,
the activity of α-amylase was not significantly inhibited at concentrations of 1000 and 2000 ppm crude
methanolic extract in wettable powder during whole experiment. It was significantly inhibited when
imbibing the seeds in JWP at concentrations of 4000 and 8000 ppm for a period of 12 h, 24 h and 48 h.
Generally, the imbibition and α-amylase activities in the treated E. crus-galli seeds were progressively
depressed with increasing JWP concentrations (Table 3).

percent 26

TWSC 2013
The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand

100 12.5 25 50 100 200 400
90
80
70
60
50
40
30
20
10
0

0

concentration (ppm)

% Prophase % Metaphase
% Anaphase-Telophase Mitotic index

Fig. 1 Mitotic index and mitotic phases of onion root-meristem cells exposed to different concentrations
of Jasminum officinale f. var. grandiflorum (Linn.) Kob. crude methanolic extracts for 18 h.

Table 2 Effects of crude metanolic extract from J. officinale in wettable powder form (JWP) on seed
germination and seedling growth of E. crus-galli seeds.

Concentration Germination Inhibition (% of control) Root length
(ppm) 0.00c 0.00c
0.00c Shoot length
Control 7.50c 0.00cd -15.29d
500 5.00c -8.00d 76.31b
1000 40.00b 97.12a
2000 70.00a 19.04bc
4000 20.81bc 100.00a
8000 30.20b 100.00a
60.18a

The values represent the means. Different letters within the same column indicate significance
differences (p<0.05) between treatments.

Table 3 Effects of crude methanolic extract from J. officinale in wettable powder form (JWP) on seed
imbibition and α-amylase activities of E. crus-galli seeds at different imbibition periods.

Concentration Seed imbibition α-amylase activities
(ppm) (%) (µmol maltose min-1 g-1(FW))

control 12h 24h 48h 12h 24h 48h
500
1000 32.84a 45.11a 79.54a 3.15a 4.37a 9.49a
2000 32.99a 45.83a 66.37b 3.15a 4.82a 9.52a
4000 34.78a 46.85a 50.62c 2.63ab 4.17ab 8.08ab
8000 34.00a 46.61a 49.18c
31.97a 40.40a 47.44c 2.12ab 4.09ab 6.81abc
29.50a 35.31a 41.45c
1.69bc 3.48b 5.38bcd

1.34c 2.89b 4.39cd

The values represent the means. Different letters within the same column indicate significance
differences (p<0.05) between treatments.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 27

Discussion

Allelochemicals from J. officinale suppressed growth plant bioassays. The main active
compound of oleuropein was isolated from methanolic crude extracts of J. officinale dried leaves.
Oleuropein was less inhibition effect on germination and seedling growth of bioassay plants than
crude extract which may well act synergistically. This finding is congruent with the observation of
synergistic of biological compounds reported by Lydon et al., (1997), Inderjit et al., (2002) and Kilani
et al., (2008). In cytogenetic bioassay, methanolic extract produced lower mitoitc index, alternation of
phase index and induction of mitotic abnormalities. A similar observation of commercial herbicides
like pentachlorophenol, 2,4-D and butachlor was reported by Ateeq et al. (2002). In addition, the toxic
effect of J. officinale extract induced cell division aberrations. These findings correspond to others in the
published literature which have examined the effects of several herbicides, including dimethyl 2,3,5,6-
tetrachloro-1,4-benzenedicarboxylate (DCPA), propham, chloropropham (carbamates), dithiopyr and
thiazopyr, all of which have shown interference with mitosis (Dyer and Weller, 2005).

Allelochemicals found in J. officinale adversely affected seed germination and seedling growth
of E. crus-galli seeds treated with JWP inhibited seed germination by impeding seed imbibition and
induction of α-amylase activity. Seed which inhibited imbibition may be limited in specific enzymes
required for metabolism of reserved food and hence have poor seed germination. The decrease in α-
amylase activity as a result of exposure to JWP could suggest the retardation of substrate production for
respiration and consequently limited energy production (Taiz and Zeiger, 2006). For this reason, JWP
may adversely affect seed germination. It was shown that the activity of α-amylase was inhibited by the
presence of allelochemicals. Kato-Noguchi and Macías (2005) previously reported that lettuce (Lactuca
sativa L. cv. Grand Rapids) seeds treated with 6-methoxy-2-benzoxazolinone (MBOA) inhibited seed
germination by impeding induction of α-amylase activity.

Conclusion

Allelochemical, oeuropein was isolated from J. officinale.The pure active compound of oeuropein
exhibited lower growth inhibition compared to crude form (mixture of allelochemical compound) which
may well act synergistically. Crude forms of J. officinale adversely affected germination and growth
tested weeds and showed the modes of negative action on physiological and cellular processes.

Acknowledgements

The authors acknowledge The Thailand Research Fund (TRF, Grant number DBG-5080019)
for financial support.

References

Ateeq, B., Farah, M.A., Ali, M.N. and Ahmad, W. (2002). Clastogenicity of Pentachlorophenol, 2,4-D
and Butachlor Evalualted by Allium Root Tip Test. Mutat. Res. 514: 105-113.

Bernfeld, P. (1955). Amylases α and β. in: Colowick, S.P., Kaplan, N.O. (Eds.), Method in
Enzymology. Academic Press, New York, 149-158.

Dyer, W.E. and Weller, S.C. (2005). Plant Response to Herbicides, in: Jenks, M.A., Hasegawa, P.M.
(Eds.), Plant Abiotic Stress. Blackwell Publishing, Oxford, pp. 171-214.

Inderjit, Streibig, J.C. and Olofsdotter, M. (2002). Joint of Action of Phenolic Acid Mixtures and Its
Significance in Allelopathy Research. Physiol. Plant. 144: 422-428.

Kato-Noguchi, H. and Macías, F.A. (2005). Effects of 6-Methoxy-2-Benzoxazolinone on the
Germination and α-Amylase Activity in Lettuce Seeds. J. Plant Physiol. 162: 1304–1307.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 28

Kilani, S., Sghaier, M.B., Limem, I., Bouhlel, I., Boubaker, J., Bhouri, W., Skandrani, I., Neffatti,
A., Ammar, R.B., Dijoux-Franca, M.G., Ghedtra, K. and Chekir-Ghedira, L. (2008). In vitro
Evaluation of Antibacterial, Antioxidant, Cytotoxic and Apoptotic Activities of The Tubers
Infusion and Extracts of Cyperus rotundus. Bioresour. Technol. 99: 9004-9008.

Lydon, J., Teasdale, J.R. and Chen, P.K. (1997). Allelopathic Activity of Annual Wormwood
(Artemisia annua) and The Role of Artemisinin. Weed Sci. 45: 807-811.

Sadasivam, S. and Manickam, A. (1996). Biochemical Methods. New Age International (P) Ltd., New
Delhi.

Taiz, L. and Zeiger, E. (2006). Plant Physiology, 4th edn. Sinauer Associates, Massachusetts.
Turk, M.A. and Tawaha, A.M. (2002). Inhibitory Effects of Aqueous Extracts of Black Mustard on

Germination and Growth of Lentil. Agron. J. 1: 28-30.
Vyvyan, J.R. 2002. Allelochemicals as Leads for New Herbicides and Agrochemicals. Tetrahedron 58:

1631-1646.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 29

Studies on Natural Herbicide Resistance (HR) among traditional and
developed rice (Oryza sativa L.) varieties cultivated in Sri Lanka and
inducing HR with Chemical mutagens, NaN3 and EMS

Shyama Ranjani Weerakoon1*, R. G. Danushka Wijeratne1 and Seneviratne Somaratne1
1Department of Botany, Faculty of Natural Sciences, The Open University of Sri Lanka, P. O. Box 21, Nawala,

SRI LANKA.

Abstract

At present, weeds are the major biotic constraint to increased rice production worldwide
affecting growth and causing a considerable reduction in yield. Application of high concentrations
of pre-emergent-broad-spectrum systemic herbicide, Glyphosate is prevalently used to control rice
weeds in South East Asian countries including Sri Lanka and which intern cause severe damages to
cultivated rice too. Therefore, inducing herbicide resistance (HR) in cultivated rice is a novel measure
used to increase selectivity and enhance crop safety and production. Hitherto, HR in Sri Lankan rice
varieties has neither been evaluated nor been attempted to induce. Therefore, varying concentrations
of chemical mutagens, Sodium Azide (NaN3) 1.5, 3.0 and 6.0 mmol l-1 and Ethyl Methyl Sulphonate
(EMS) 4.5 mmol l-1 concentrations were used to induce HR against Glyphosate at 0.25 and 0.5 g l-1
concentrations. Six traditional and eighteen developed-cultivated rice varieties (Bg, Bw, At and Ld series
developed by Rice Research Development Institute, Sri Lanka) were used in the study. Experimental
design used was RCBD with five replicates and three blocks in each treatment-combination. Plants
with ≥ 40% resistance were considered as resistant to Glyphosate. Observations on time taken-to seed
germination, -to flowering; measurements of plant height and number of leaves at 12-weeks after
sawing, leaf-length, breadth, panicle-length, number of seeds/panicle of resistant plants and controls
were recorded.Ten developed-cultivated varieties (Bg250, Bg94-1, Bg304, Bg359, Bg406, Bg379-2,
Bg366, Bg300, Bw364, At362) and three traditional rice varieties (“Kalu Heenati”, “Sudu Heenati”,
“Pachchaperumal’) were found to be naturally resistant to 0.25 g l-1 Glyphosate concentration. NaN3-
treatment enhanced HR in four varieties (Bg406, Bg379-2, Bg300, Bw364) from 40% to 56% and a
susceptible variety (Bg352) developed HR up to 56%. In EMS-treatment, five varieties of developed-
cultivated rice (Bg300, Bg359, Bg304, Bg403, Bw364) and two traditional varieties (“Suduru Samba”,
“Murungakayan”) were induced HR and four varieties (Bg300, Bg359, Bg304 and “Suduru Samba”)
enhanced their HR against Glyphosate at 0.25 g l-1 concentration. Mutagenic chemicals NaN3 or EMS
could successfully induce and enhance Glyphosate-resistance in traditional and developed-cultivated rice
varieties. Mutant varieties exhibited a considerably higher resistance to Glyphosate. NaN3-mutant and
EMS-mutant rice were morphologically different from corresponding parental varieties. EMS showed
more effective mutagenic action than NaN3 by showing a decreasing trend and significant difference
in agro-morphological characters compared to their respective parental varieties. EMS induced HR
in most rice varieties showed a considerable yield penalty associated with growth stunting trends in
agro-morphological characters. However, there was no indication of yield penalty in rice varieties with
NaN3 induced HR via affecting agro-morphological characters. Increasing concentrations of NaN3 and
Glyphosate have shown a negative effect on agro-morphological characters of rice varieties. Mutated
rice varieties yielded the higher Glyphosate resistance and they possess higher candidacy to incorporate
them in rice breeding programs and to develop HR rice varieties in near future. However, further
studies using varying concentrations of NaN3 and EMS with/without gamma irradiation with several
traditionally-cultivated and developed-cultivated rice varieties are recommended for better results.

Keywords: Glyphosate, Herbicide Resistance, Oryza sativa, Sodium Azide, Ethyl Methyl Sulphonate,
Sri Lanka

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 30

Introduction

Weeds are the major biotic constraint to increased rice production worldwide. Weeds can
cause severe yield losses to cultivated rice in relation to the density, type of weeds and cultivated
rice varieties (Diarra et al. 1985a; Diarra et al. 1985b; Fisher and Ramirez, 1993; Eleftherohorinos et
al. 2002). Therefore, weed control is an essential component of profitable crop production and weeds
can be controlled mechanically; chemically or by crop rotation and most farmers rely on a blend of
these methods. Application of high concentrations of pre-emergent-broad-spectrum systemic herbicide,
Glyphosate is required to control rice weeds in the rice fields of South East Asian countries including
Sri Lanka. Glyphosate causes damages to the cultivated rice as well (Labrada, 2007; Davis et al, 2009).
It inhibits 5-enolpyruvylshikimate-3-phosphate synthase, an enzyme involved in the shikimic acid
pathway of plants (Cioppa et al., 1986). Glyphosate can cause a significant damage to rice yield with
a reduction of yield up to 80% (Davis et al., 2009). Inducing herbicide resistance (HR) into rice is a
new means to confer selectivity and enhance crop safety and production (Guttieri et al., 1996). Over
the last two decades, mutational techniques have become one of the most important tools available to
progressive rice-breeding programs. Imidazolinone-resistant rice was developed through chemically
induced seed mutagenesis with Ethyl Methyl Sulfonate (EMS) (Gealy et al., 2003) and “Clear field”
rice variety was also developed through EMS mutagen against herbicides Imidazolinone (Lang and
Buu, 2007). Similarly, Sodium Azide (NaN3) has been used to produce rice mutant for the enhancement
of agronomic traits (Nakata et al., 2008; Young Seop et al., 2009). Screening for herbicide resistance
is often based on visual evaluation, mortality or growth inhibition compared to untreated plants. There
are evidences that HR crops can bring significant benefit to farmers, consumers and the environment.
Farmers are benefited from the excellent broad-spectrum weed control provided by such herbicides
and from substantially lower costs of growing some HR crops. HR crops provide additional crop
choice, enabling implementation of alternate weed management tactics to target specific weeds while
maintaining crop sequences. Therefore, inclusion of an HR crop in a cropping program along with
a range of weed management tactics can ensure to control hard-to-control weeds (Devine and Buth,
2001).

There is an inadequacy of research efforts on screening of the herbicide resistance among
cultivated rice varieties and development of HR rice varieties up to date in Sri Lanka. Therefore, in the
present study six traditionally cultivated and eighteen developed-cultivated (Bg, At, Bw, Ld series) rice
(Oryza sativa L.) varieties were chosen to screen the herbicide resistance (HR) against pre-emergent-
broad-spectrum herbicide, Glyphosate and to induce HR against Glyphosate via mutagenesis using the
chemical mutagens, Sodium Azide (NaN3) and Ethyl Methyl Sulphonate (EMS).

Materials and Methods

Materials - Rice varieties
Twenty four rice varieties with germination percentage of > 85% were selected for the study.

Six traditionally cultivated varieties (“Kalu Heenati”, “Sudu Heenati”, “Suwadal”, “Suduru Samba”,
“Pachchaperumal” and “Murungakayan”) and eighteen inbread-developed(cultivated) rice varieties
(Bg94-1, Bg250, Bg300, Bg304, Bg305, Bg352, Bg357, Bg358, Bg359, Bg360, Bg366, Bg379-2,
Bg403, Bg406, Ld365, At362, At308, Bw364) were collected from Research Centers of Rice Research
Development Institute at Batalagoda, Ambalanthota, Bombuwela and Labuduwa, Sri Lanka for the
study. These lines were maintained in a greenhouse in the premises of the Open University of Sri Lanka,
Nawala, Sri Lanka.

Method 1 - Natural Glyphosate resistance among traditional and developed(cultivated) rice
varieties

Seeds of the twenty four rice varieties mentioned above were surface sterilized and placed in
moist chambers for germination. The germinated rice seedlings (height about 4 cm) were immersed in

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 31

Glyphosate solutions with two different concentrations, 0.25 g l-1 and 0.5 g l-1 (360 g l-1 Glyphosate) for
4 days. Randomized Complete Block Design (RCBD) was used in the experiment and there were five
replicates used for each treatment and three blocks in each treatment combination. Control treatment was
carried out without Glyphosate. All seedlings were subsequently transferred to soil medium (sterilized
mud) and allowed to grow and observations were taken for ten weeks. Dead plants were considered
as susceptible to the herbicide and surviving plants with a substantial growth were considered as
resistant to the herbicide. For each rice variety, number of resistant plants and percentage resistance was
calculated. The plants with percentage resistance of ≥ 40% were considered as resistant to Glyphosate.
The percentage (%) of resistance was calculated using the following equation.

Method 2 - Mutation Studies using NaN3
The seeds of inbred-developed (cultivated) varieties were exposed to the chemical mutagen

Sodium Azide (NaN3) at 1.5 mmol l-1, 3.0 mmol l-1 and 6.0 mmol l-1 concentrations for a day and
followed the same steps of the Method 1. Agro-morphological characters were measured or counted
at 12 weeks after sawing (WAS). These characters were time-to seed germination, time-to flowering,
plant height at, number of leaves, leaf length and width, length of panicle and number of seeds/panicle
of the plants with herbicide treatment and the controls without herbicide treatment were also recorded.

Method 3 - Mutation Studies using EMS
In the mutational studies with EMS, seeds of each rice variety were exposed to the chemical

mutagen Ethyl Methyl Sulfonate (EMS) at 4.5 mmol l-1 concentration for a day and followed same steps
described in Method 1 and Method 2. Agro-morphological characters of the rice plants at 12 WAS were
recorded.

Statistical analyses
A descriptive statistics were performed on the data set (mean, standard deviation). The GLM

(General Linear Models) was used to test the effects of factors (rice variety, NaN3 and Glyphosate
concentrations) on agro-morphological characters. Since there were no significant interactions among
the factors, One-way-analysis of variance (ANOVA) was performed on agro-morphological characters.
All statistical analyses were carried out using SAS Version 9.2 (SAS, 2008).

Results

The comparison of natural Glyphosate resistance among traditional and developed (cultivated)
rice varieties revealed that thirteen varieties (Bg250, Bg94-1, Bg304, Bg359, Bg406, Bg379-2, Bg 366,
Bg300, Bw364, At362, “Kalu Heenati”, “Sudu Heenati”, “Pachchaperumal’) were naturally resistant
to 0.25 g l-1 Glyphosate. The higher percentage of resistance was resulted in 0.25 g l-1 Glyphosate
concentration, and 0.5 g l-1 concentration caused reduction in the percentages of resistance due to
inhibited seed germination. Certain herbicide resistant rice varieties which were mutated with NaN3
(Bg406, Bg379-2, Bg300, Bw364) have enhanced their percentages of resistance from 40% to 56%
while a susceptible variety (Bg352) developed HR up to 56% under 0.25 g l-1 Glyphosate concentration.
Seven varieties after mutation with EMS (Bg300, Bg359, Bg304, Bg403, Bw364, “Suduru Samba”,
“Murungakayan”) were resistant to 0.25 g/l Glyphosate. EMS treatment has enhanced the percentages
of resistance in Bg300, Bg359, Bg304 and “Suduru Samba”.

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A combined ANOVA (results not given in the manuscript) revealed that all main effects (rice
variety, NaN3 and Glyphosate concentration) were not statistically significant (p ≥ 0.05). Further, in
relation to all agro-morphological characters observed, no statistically significant differences between
NaN3 mediated-mutated rice plants were observed compared to non-mutated rice plants under treatment
of different Glyphosate concentrations (Table 1). However, an increase in number of days taken to
seed germination was observed for rice plants mutated with higher concentration of NaN3 (Table 2).
Decreasing trends were observed in time-to flowering, number of leaves/plant, leaf length, leaf width
and panicle length in the mutated rice plant along the increasing Glyphosate concentrations. Apparently,
number of seed/panicle indicated a considerable reduction across increasing Glyphosate concentrations
(Table 2). In contrary, there were statistically significant differences (p ≤ 0.05) between the EMS-
mediated-mutated rice plants with non-mutated rice plants related to agro-morphological characters
such as plant height and number of leaves/ plant at 12 WAS, leaf length and leaf width, under treatment
of different Glyphosate concentrations (Table 1). However, time-to flowering, time-to seed germination,
panicle length and number of seed/panicle indicated no statistically insignificance when compared
mutated rice plants with non-mutated rice plants (Table 1). Mutational study with EMS revealed that
mutated rice plants showed a considerable reduction in the agro-morphological characters, plant height
and number of leaves/ plant leaf length and leaf width, panicle length and number of seed/panicle across
the increasing Glyphosate concentrations (Table 3).

Discussion

There were no previous records on the existence of natural herbicide resistance in traditional
and inbred-developed rice varieties in Sri Lanka. The present study revealed that several traditional and
developed rice varieties were possessing considerably high HR against Glyphosate. Further, the study
showed that NaN3 and EMS could successfully be used to induce HR in Glyphosate susceptible rice
varieties as well as to enhance the existing HR. The chemical mutagen EMS has been used to develop
HR varieties against the herbicide Imidazolinone (Gealy et al., 2003; Lang and Buu, 2007).

The NaN3-mutant rice varieties were not morphologically different from their parent varieties.
Meanwhile, increasing NaN3 concentrations indicated a negative effect on agro-morphological
characters of rice varieties. A similar observation has made by Awan et al.,1980 and YoungSeop et al.
2009 with NaN3, reported that there was a decreasing trend in germination rate and seedling height with
the increasing NaN3 concentration in Oryza sativa L. ssp. Japonica and few other varieties. However, in
the present study no yield penalty was observed in NaN3-mutant rice varieties because yield parameters
such as time-to flowering and number of seeds/panicle was not significantly differ compared to parental
varieties. EMS seems to be having more effective mutagenic action than NaN3 by showing a decreasing
trend and significantly difference in agro-morphological characters such as plant height, number of
leaves/plant, leaf length and leaf width compared to their respective non-mutated varieties. Though
EMS induced HR in most of the rice varieties studied, there was a considerable yield penalty associated
with growth stunting trends in agro-morphological characters. However, more studies with different
concentrations of EMS and NaN3 are necessary to make a solid conclusion.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 33

Table 1. Summary of ANOVA performed on the agro-morphological characters along the different
concentrations of EMS, NaN3. S = Significant at p ≤ 0.05; NS = Not significant, p ≥ 0.05

Source Sum of Squares df Mean F Significance
Square
EMS treatments
Germinating time 0.028 2 0.014 0.301 NS
Height 9185.002
Number of leaves/plant 149.433 2 4592.501 16.673 S
Leaf width 267.159
Leaf length 2220.196 2 74.717 15.52 S
Flowering time 1041.436
Panicle length 2580.56 2 133.579 8.181 S
Number of seeds/plant 11934.44
2 1110.098 7.804 S

2 520.718 1.448 NS

2 1290.280 39.480 S

2 5967.220 36.887 S

NaN3 treatments 6.786 2 3.393 3.791 NS
Germinating time 952.84
Height 57.371 2 476.42 1.612 NS
Number of leaves/plant 0.123
Leaf width 701.198 2 28.686 3.326 NS
Leaf length 1999
Flowering time 169.633 2 0.061 1.8 NS
Panicle length 249.018
Number of seeds/plant 2 350.599 2.697 NS

2 999.5 0.571 NS

2 84.817 1.948 NS

2 124.509 1.518 NS

Table 2.Summary of the morphological and yield characters of parental var
Mean values and standard error of mean is given within parenthesi

Glyphosate Sodium aside Variety No. of days No. of days
concentration Concentration taken to taken to
flowering germinate
(g/l) (mmol/l)
NA M
0.25 6 Bg 304 38.0 (53.7)
0.25 3 Bg 352 5 (0)
0.5 3 Bg 366 NA 3 (0)
0.25 1.5 Bg 406 NA 4 (0)
0.25 3 Bg 406 NA) 3 (0)
0.25 1.5 Bw 364 74.5 (2.1) 5 (0)
3 (0)
control control Bg 304 47.5 (0.7)
control control Bg 352 55.5 (2.1) Pa
control control Bg 366 60.5 (2.1) 2 (0)
control control Bg 406 69.5 (0.7) 2 (0)
control control Bw 364 57.5 (2.1) 3 (0)
3 (0)
3 (0)

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 34

rieties and Glyphosate resistant rice produced by different concentrations of NaN3.
is. NA = Not available

Height (cm) No. of Leaf length Leaf width Panicle No. of seeds/
leaves (cm) (cm) length (cm) panicle

Mutated line 6.0 (1.4) 44.0 (2.8) 1.0 (0.1) NA NA
56.5 (5.0) 11.0 (1.4) 19.5 (13.4) 0.6 (0.3) 1.3 (1.8) 2.0 (2.8)
32.3 (18.0) 13.0 (6.0) 0.6 (0)
19.0 (2.3) 4.0 (0) 0.6 (0) NA NA
19.0 (2.3) 6.0 (1.4) 12.0 (0) 0.7 (0.1) NA NA
33.5 (6.4) 13.0 (2.8) 16.0 (1.4) 0.8 (0.1) NA NA
57.0 (12.7) 12.5 (0.7) 19.3 (6.0) 12.0 (5.7) 14.5 (10.7)
1.1 (0.1)
arental line 11.0 (1.4) 47.5 (2.1) 1.4 (0.1) 29.5 (3.6) 50.0 (1.4)
59.0 (1.4) 14.0 (1.4) 51.5 (2.1) 1.2 (0.2) 29.5 (3.6) 57.5 (2.1)
67.5 (3.5) 13.0 (1.4) 47.5 (6.4) 1.2 (0.3) 35.0 (1.4) 65.0 (0)
66.5 (0.7) 14.5 (0.7) 52.0 (5.7) 1.4 (0.2) 33.5 (6.4) 64.0 (4.2)
73.0 (5.7) 11.5 (0.7) 55.0 (2.8) 34.0 (1.4) 58.5 (6.4)
64.5 (5.0)

Table 3. Summary of the morphological and yield characters of parental va
Mean values and standard error of mean is given within parenthes

Glyphosate Ethyl Methyl Variety No. of days taken No. of days taken
concentration (g/l) Sulphonate
Concentration
(mmol/l) to flowering to germinate

M

Control 4.5 Bg 300 59.0(1.4) 3.0(0)

Control 4.5 Bg 304 54.0(1.4) 3.0(0)

Control 4.5 Bg 359 60.0(1.4) 3.0(0)

Control 4.5 Bg 403 66.5(0.7) 3.0(0)

Control 4.5 Bw 364 57.5(0.7) 3.0(0)

Control 4.5 Murungakayan 63.5(0.7) 3.0(0)

Control 4.5 Suduru Samba 70.5(2.1) 3.0(0)

0.25 4.5 Bg 300 62.5(0.7) 3.0(0)

0.25 4.5 Bg 304 61.5(0.7) 3.0(0)

0.25 4.5 Bg 359 62.0(1.4) 3.0(0)

0.25 4.5 Bg 403 74.0(1.4) 3.0(0)

0.25 4.5 Bw 364 62.0(2.8) 3.0(0)

0.25 4.5 Murungakayan 67.5(2.1) 3.0(0)

0.25 4.5 Suduru Samba 75.5(2.1) 3.0(0)

0.5 4.5 Bg 300 61.5(3.5) 3.0(0)

0.5 4.5 Bg 359 64.5(0.7) 3.0(0)

0.5 4.5 Bg 403 NA 3.0(0)

0.5 4.5 Bw 364 NA 3.0(0)

0.5 4.5 Murungakayan NA 3.0(0)

0.5

4.5 Suduru Samba 77.5(5.0) 3.0(0)

P

Bg 300 52.0(1.4) 2.0(0)

Bg 304 50.0(2.8) 2.0(0)

Bg 359 57.0(2.8) 3.0(0)

Bg 403 69.0(2.8) 3.0(0)

Bw 364 57.5(2.1) 3.0(0)

Murungakayan 56.5(3.5) 2.0(0)

Suduru Samba 68.0(1.4) 2.0(0)

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 35

arieties and Glyphosate resistant rice produced by different concentrations of EMS.
sis. NA = Not available

n Height (cm) No. of leaves Leaf length Leaf breadth Panicle length No. of seeds/
(cm) (cm) (cm) panicle

Mutated line 11. 5(0.7) 1.2(0.1) 43.5(3.5) 22.5(2.1) 35.5(5.0)
66.5(2.1) 11. 5(0.7) 1.8(0) 48.5(3.5) 32.5(5.0) 81.0(22.6)
94.0(2.9) 10.5(0.7) 1.4(0.1) 39.5(2.1) 26.0(1.4) 33.0(1.4)
64.5(3.5) 12.5(0.7) 1.4(0) 42.5(2.1) 26.5(3.5) 52.0(5.7)
67.0(2.8) 12.5(0.7) 1.3(0.1) 45.0(4.2) 25.5(0.7) 32.0(2.8)
79.0(2.8) 10.5(0.7) 1.4(0) 58.5(5.0) 23.0(1.4) 33.0(1.4)
91.7(2.4) 9.5(0.7) 1.7(0.1) 71.1(7.1) 31.5(3.5) 58.5(3.5)
129.0(4.2) 0.8(0) 20.0(1.4) 11.5(0.7) 12.5(3.5)
38.0(1.4) 9.0(0) 0.9(0) 23.5(3.5) 14.5(2.1) 13.5(5.0)
8.0(0) 0.8(0.1) 21.0(2.8) 13.0(2.8) 10.5(7.8)
54.5(0.7) 8.0(0) 1.0(0) 27.0(2.8) 14.5(0.7) 16.5(0.7)
46.5(0.7) 10.5(0.7) 0.9(0) 28.5(3.5) 14.5(2.1) 12.5(3.5)
55.5(2.1) 7.5(0.7) 1.1(0.1) 43.0(4.2) 13.5(2.1) 12.5(3.5)
57.5(0.7) 8.0(0) 1.4(0) 53.0(8.5) 25.5(0.7) 31.5(3.5)
74.0(1.4) 7.5(0.7) 0.9(0) 20.0(2.8) 10.0(1.4) 8.5(0.7)
96.5(3.5) 7.0(0) 0.7(0) 23.0(2.8) 13.0(2.8)
37.5(2.1) 7.5(0.7) 0.7(0) 27.3(4.6) 10.0(0)
44.5(2.1) 7.5(0.7) 0.8(0.1) 21.5(5.0) NA NA
45.3(1.8) 6.5(0.7) 0.7(0) 34.0(7.1) NA NA
42.5(2.1) 7.5(0.7) NA NA
62.5(2.1) 0.8(0) 29.0(2.8)
6.5(0.7) 19.0(2.8) 17.0(2.8)
52.0(1.4) 1.2(0.1) 52.0(4.2)
Parental line 12.5(0.7) 1.1(1.1) 49.5(5.0) 28.5(3.5) 86.5(6.4)
11.0(1.4) 1.3(0.1) 53.5(3.5) 30.5(2.1) 59.0(11.3
64.0(1.4) 14.5(2.1) 1.4(0.1) 60.0(5.7) 30.5(2.1) 41.0(2.8)
63.0(4.2) 12.0(0) 1.5(0.1) 57.0(5.7) 32.0(0) 49.5(9.2)
66.0(2.8) 12.5(0.7) 1.5(0.1) 70.5(6.4) 36.0(1.4) 72.0(12.7)
70.5(2.1) 14.0(0) 1.5(0.1) 94.0(4.2) 34.0(4.2) 63.5(10.6)
64.5(5.0) 10.0(0) 62.5(6.4) 108.5(16.3)
98.0(8.5)
149.5(3.5)

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 36

Conclusion

The results of the present study led to reach the following conclusions: (a) Several traditional
and inbred-developed rice varieties possesses HR against Glyphosate (b) The mutant varieties showed
considerably higher resistance to the Glyphosate, (c) Chemically mutated varieties using NaN3 was
morphologically different from respective parental varieties (d) Increasing NaN3 and Glyphosate
concentrations have a negative impact on agro-morphological characters of rice varieties (e) EMS
seems to be having more effective mutagenic action than NaN3 (f) Mutated rice varieties with high
Glyphosate resistance have higher potential to incorporate in rice breeding programs as well as could
lead to develop herbicide resistant rice varieties in future.

Acknowledgements

The research grant provided by the Faculty of Natural Sciences, The Open University of Sri
Lanka is greatly appreciated.

References

Awan, M.A., Konzak,C.F.,  Rutger, J. N.  and Nilan, R.A. (1980). Mutagenic Effects of Sodium Azide
in Rice. Crop Science  20(5): 663-668.

Cioppa, G. D., Bauer, B.K., Klein, B.K., Shah, R.T., Fraley, S.C. and Kishore, G. (1986).
Translocation of the Precursor of 5-Enolpyruvylshikimate-3-Phosphate Synthase into
Chloroplasts of Higher Plants in vitro, Proceedings of National Academy of Science, USA,
83: 6873-6877.

Davis, B.M., Scott, R.C. Norsworthy, J.K. and Smith, K.L. (2009). Effects of Low Rates of Glyphosate
and Glufosinate on Rice, AAES. Research Series 581p.

Devine, M.D. and Buth, J.L. (2001). Advantages of Genetically Modified Canola: a Canadian
Perspective. Proceedings of Brighton Crop Prot Conf-Weeds, BCPC, Farnham, Surrey, UK.
Pp. 367-372.

Diarra, A.R.J., Smith, R.J. and Talbert, R.E. (1985a). Growth and Morphological Characteristics of Red
Rice (Oryza sativa) Biotypes. Weed Science. 33: 310-314.

Diarra, A.R.J., Smith, R.J. and Talbert, R.E. (1985b). Interference of Red Rice (Oryza sativa) with Rice
(O. sativa). Weed Science. 33: 644-649.

Elefhterohorinos, I.G., Dhima, K.V. and Vasilakoglou, I.B. (2002). Interference of Red Rice in Rice
Grown in Greece. Weed Science. 50: 167-172.

Fischer, A.J. and Ramirez, A.  (1993). Red Rice (Oryza sativa): Competition Studies for
Management Decisions. International Journal of Pest Management. 39: 133-138.

Gealy, D.R., Mitten, D.I.L. and Putger, J.N. (2003). Gene Flow Between Red Rice (Oryza sativa) and
Herbicide-Resistant Rice (O. sativa). Weed Technology. 17: 627-645.

Guttieri, M.J., Eberlein, C.A. and Smith, D.C.M. (1996). Molecular Genetics of Target-site Resistance
to Acetolactate Synthase Inhibiting Herbicides, pp. 645.

In Brown, T.M. and Washington, D.C. (eds.). Molecular Genetics and Evolution of Pesticides Resistance.
ACS Symposium Series. ACS.

Labrada, R. (2007). Weedy and Wild Rice. Their Impact and Management. 21st Asian Pacific Weed
Science Society (APWSS) Conference, Colombo, Sri Lanka. Pp. 8-15.

Lang, N. T. and Buu, B.C. (2007). Rice Breeding and Inheritance of Herbicide Resistance in
Clearfield Rice (Oryza sativa L.).Omonrice. 15: 36-45.

Nakata Y., Ueno, M., Kihara, J., Ichii, M., Taketa, S. and Arase, S. (2008). Rice Blast Disease and
Susceptibility to Pests in a Silicon Uptake-Deficient Mutant Isil of Rice. Crop protection, 27:
865-868.

YoungSeop, S., YongHee, J., KyungHo, K., YongWeon, S. and JiUng, J. (2009). Variation of
Agronomic Traits of Rice Mutant lines Induced by Sodium Azide.Korean Journal of Breeding
Science, 41 (2): 92-100.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 37

Rapid bioassay method for herbicide dose-response study and herbicide
resistance diagnosis

Chuan-Jie Zhang, Soo-Hyun Lim and Do-Soon Kim
Department of Plant Science, Seoul National University, Seoul 151-742, Korea

Corresponding author: E-mail: [email protected]

Abstract

This study was conducted to develop a rapid bioassay method for herbicide dose-response
of Echinochloa species and herbicide resistance diagnosis in Echinochloa spp. Germinated seeds of
Echinochloa spp. were placed on the paper wick of 18 cm x 16.5 cm growth pouch containing herbicide
solution at a range of concentrations. Shoot and root lengths of Echinochloa spp. were then measured
after incubation for 6 days. Dose-responses in root length by the growth pouch method were well
described by logistic function and confirmed to be similar to those of whole plant assay regardless of
herbicide modes of action, suggesting that the growth pouch method can be used for herbicide bioassay.
The growth pouch method was then applied to rapid diagnosis of ACCase or ALS inhibitor resistance
in Echinochloa spp. Resistant and susceptible biotypes were discriminated at 180 to 300 mg and 80 to
120 mg ai L-1 of cyhalofop-butyl for E. crus-galli and E. oryzicola, respectively, and at 350 to 500 mg
and 650 to 1000 mg ai L-1 of penoxsulam for E. crus-galli and E. oryzicola, respectively. Therefore, the
growth pouch method can be used for herbicide resistance in Echinochloa spp. with significant time and
cost-savings as compared with the conventional whole-plant assay.

Keywords: Diagnosis, Echinochloa species, growth pouch, herbicide resistance, rapid bioassay

Introduction

Echinochloa species is one of the most troublesome weeds in rice cultivations both transplanted
and direct-seeded. Reliance upon herbicides and continuous use of herbicides with the same mode
of action has led to the development of herbicide resistance in weed populations (Holt et al., 1993).
Studies have been confirmed to be resistant to several herbicides in Echinochloa species. Several dose-
response methods of weed species have been developed, including whole-plant assay which has been
the most widely used for herbicide dose-response and resistance diagnosis. Other methods, such as Petri
dish assay (Moss, 1990), trimmed seedling, tiller and stem node tests (Kim et al., 2000), chlorophyll
fluorescence measurement (Norsworthy et al., 1998), leaf disc flotation test (Kemp et al., 1990), and
pollen germination test (Richter and Powles, 1993) were also developed. However, many of these
methods are expensive, limited, difficult to assess, time-consuming, or limited for a certain growth
stage. The methods for herbicide dose-response test and resistance diagnosis should be rapid, accurate,
cheap, reproducible and readily available and should provide a steady result on herbicide performance.
Therefore, this study was conducted to develop a new method by using growth pouch for dose-response
test of herbicides with different modes of action. The method was then further applied to herbicide
resistance diagnosis in Echinochloa specie.

Materials and Methods

Whole-plant assay of herbicides with different modes of action
Pot experiments were conducted to evaluate dose-responses of whole plants of E. crus-galli

to herbicides with different modes of action in the glasshouse at Experimental Farm Station of Seoul
National University, Suwon, Korea. Herbicides, bentazone, cyhalofop-butyl, bispyribac-sodium,
penoxulam, glufosinate and glyphosate were applied to E. crus-galli (Suwon biotype) at 5th to 6th leaf

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 38

stage at different dosage ranges. All the treatments were replicated three times and were arranged in a
completely randomized design. Above-ground fresh weight was assessed at 30 days after application
(DAA), and data were expressed as percentage of the untreated control. GR50 values, dose required to
inhibit plant growth by 50% of the untreated control were determined by fitting the data to the standard
dose-response model (Streibig, 1980).

Growth pouch test of herbicides with different modes of action
Germinated seeds of E. crus-galli (Suwon biotype) were placed in the growth pouch containing

a range of concentrations of bentazone, cyhalofop-butyl, bispyribac-sodium, penoxsulam, glufosinate,
glyphosate and then kept in an incubation room maintained at 35/25oC (day/night) with supplementary
light where the roots were being kept in the dark. De-ionized water was added to the growth pouch
every day to replace water loss due to evaporation. The root and shoot length were measured at 6 days
after treatment (DAT). The test consisted of 3 replications of a completely randomized block design.

Diagnosis of ACCase and ALS inhibitor resistant Echinochloa spp.
Origin of Echinochloa species. E. crus-galli, Seosan-5, Seosan-152, and Suwon (reference
susceptible) biotypes, previously determined as resistant or susceptible to cyhalofop-butyl (Im et al.,
2009) were used for whole plant and growth pouch tests with cyhalofop-butyl. E. oryzicola, Gimje,
Iksan, and Suwon (reference susceptible) biotypes, previously determined as resistant or susceptible to
penoxulam (Kim, 2010) were also used for whole plant and growth pouch tests with penoxulam.

Whole-plant assay. Pot experiments were conducted to evaluate dose-responses of E. crus-galli
and E. oryzicola biotypes to cyhalofop-butyl and penoxsulam at a range of their doses. Fresh weight
was assessed at 30 DAA and the data were fitted to the standard dose-response model (Streibig, 1980) to
estimate GR50 value of each biotype. The R/S ratio was calculated by dividing GR50 values by the GR50
of the reference susceptible Suwon biotype.

Growth pouch test. Germinated Echinochloa seeds were placed in the growth pouch containing
a range of concentrations of cyhalofop-butyl and penoxsulam and then kept in the incubation room
maintained at 35/25oC with supplementary light with root being kept in the dark. Germination pouches
were topped up with de-ionized water every day to replace water loss due to evaporation. The root
length was measured at 6 DAT. The test consisted of 3 replications of a completely randomized block
design.

Results and Discussion

Growth pouch method for herbicide dose-response study
Shoot and root growths of E. crus-galli were significantly affected by herbicides in both whole-

plant assay and growth pouch tests. GR50 values were estimated using shoot fresh weights in the whole-
plant assay and root length in the growth pouch test.

Based on the GR50 values we can concluded that the values for shoot length was much bigger
than those of root length except glufosinate and were not calculable for cyhalofop-butyl and penoxulam,
indicating that roots of Echinochloa species are more sensitive to the herbicides tested in this study than
shoot except glufosinate. Comparison between the two testing methods thus suggests that the growth
pouch test can be used to test herbicide with different modes of action by monitoring root length within
a week.

Diagnosis of ACCase inhibitor resistant Echinochloa spp.
In E. crus-galli biotypes, the GR50 values by the whole-plant assay of Seosan-5 and Seosan-

152 biotypes were 4.3 and 3.6 times as high as that of Suwon biotype, respectively (Table 2). The
GR50 values by the growth pouch test of Seosan-5 and Seosan-152 were 5.4 and 3.6 times greater
than that of Suwon biotype, respectively (Table 2). In E. oryzicola biotypes, the GR50 values by the
whole-plant assay of Gimje and Iksan were 4.2 and 5.9 times as high as that of Suwon (Table 2). The

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 39

GR50 values by the growth pouch test of Gimje and Iksan were 3.4 and 4.9 times greater than that of
Suwon biotype, respectively (Table 2). The optimum concentrations of cyhalofop-butyl to discriminate
between resistant and susceptible biotypes are 180-300 mg and 80-120 mg a.i. L-1 for E. crus-galli and
E. oryzicola, respectively.

Diagnosis of ALS inhibitor resistant Echinochloa spp.
In E. crus-galli biotypes, the GR50 values by the whole-plant assay of Seosan-5 and Seosan-152

biotypes were 7.8 and 4.1 times greater than that of Suwon biotype, respectively (Table 2). The GR50
values by the growth pouch test of Seosan-5 and Seosan-152 were 7.4 and 5.3 times greater than that
of Suwon biotype, respectively (Table 2). In E. oryzicola biotypes, the GR50 values by the whole-plant
assay of Gimje and Iksan were 7.4 and 11.6 times greater than that of Suwon, respectively (Table 2).
The GR50 values by the growth pouch test of Gimje and Iksan were 6.1 and 9.8 times greater than that
of Suwon biotype, respectively (Table 2). The optimum concentrations of penoxsulam to discriminate
between resistant and susceptible biotypes are 350-500 mg and 650-1000 mg a.i. L-1 for E. crus-galli
and E. oryzicola, respectively.

Table 1. GR50 values of herbicides tested by the whole-plant and the growth pouch methods.

Mode of action Herbicide Whole-plant assay GR50 values
(g a.i. ha-1)
Growth pouch test
Shoot weight (mg a.i. L-1)

13095.2 Root length Shoot length
15.7
PS II Bentazone 13.6 2479.0 6066.3
ACCase Cyhalofop-butyl 5.5 21.4 NA
Bispyribac-sodium 113.1 193.3
ALS 303.4 6148.7 1278.0
Penoxsulam 187.2 NA
GS Glufosinate 71.7 108.0
EPSPS Glyphosate 345.9

Table 2. R/S ratios in the whole-plant assay and the growth pouch tests of resistant (Seosan-5, Seosan-
152, Gimje, Iksan) biotypes of Echinochloa species to cyhalofop-butyl and penoxsulam.

Test method Herbicide E. crus-galli E. oryzicola

Whole plant Cyhalofop-butyl Seosan-5 Seosan-152 Gimje Iksan
Growth Penoxsumal
pouch 4.3 3.6 4.2 5.9
Cyhalofop-butyl 7.8 4.1 7.4 11.6
Penoxsumal 5.4 3.6 3.4 4.9
7.4 5.3 6.1 9.8

In summary, the growth pouch method produced accurate and consistent results with significant
time and cost-savings as compared with the whole-plant assay. It is cheap and can be conducted with a
minimum facility without spray application equipment. Moreover, the method using germinated seeds
requires very little amount of herbicide molecule. Therefore, this method can be used not only for
herbicide resistance diagnosis but also for early screening of a new molecule with herbicidal activity in
herbicide discovery.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 40

References

Holt, J.S., Holtum, J. A.M. and Powles, S.B. (1993). Mechanisms and Agronomic Aspects of Herbicide
Resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 203-229.

Kim, D.S., Caseley, J.C., Brain, P., Riches C.R. and Valverde, B.E. (2000). Rapid detection of propanil
and fenoxaprop resistance in junglerice (Echinochloa colona). Weed Science 48: 695-700.

Kim, D.S. (2010). Evolution and Impact of Herbicide Resistant Echinochloa Species. Proceeding of
Workshop on Effective Management of Herbicide Resistant Weeds, RDA, Suwon, Korea (in
Korean).

Im, S.H., Park, M.W., Yook, M.J. and Kim, D.S. (2009). Resistance to ACCase Inhibitor Cyhalofop-
Butyl in Echinochloa crus-galli var. crus-galli Collected in Seosan, Korea. Kor. J. Weed Sci. 29:
178-184.

Moss, S.R. (1990). Herbicide Cross-Resistance in Slender Foxtail (Alopecurus myosuroides). Weed Sci.
38: 492-496.

Norsworthy, J.K., Talbert, R.E. and Hoagland R.E. (1998). Chlorophyll Fluorescence for Rapid Detection
of Propanil-Resistant Barnyardgrass (Echinochloa crus-galli). Weed Sci. 46: 163-169.

Richter, J. and Powles, S.B. (1993). Pollen Expression of Herbicide Target Site Resistance Genes in
Annual Ryegrass (Lolium rigidum). Plant Physiol. 102: 1037-1041.

Streibig, J.C. (1980). Models for Curve-Fitting Herbicide Dose Response Data. Acta Agric. Scand. 30:

59-63.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 41

Efficacy and Rice Crop Tolerance to Mixtures of Penoxsulam+Cyhalofop as
One-Shot Rice Herbicide in ASEAN Countries.

N. Lap1, S. Somsak2, I.M. Yuli 3, Le Duy 4, Lee Leng Choy 5, Ermita, Bella Victoria 6 ,
B.V. Niranjan 7 , R.K.Mann 8

1&4DowAgroSciences Vietnam, 2DowAgroSciences Thailand, 3DowAgroSciences Indonesia 5 Dow AgroSciences
Malaysia, 6Dow AgroSciences Philippines, 7Dow AgroSciences ASEAN & 8Dow AgroSciences LLC USA.

Abtract

Penoxsulam, a triazolopyrimidine sulfonamide rice herbicide, provides good control
of Echinochloa spp., annual sedges and many broadleaf weeds. Cyhalofop-butyl, an aryloxy
phenoxypropionate rice herbicide, provides good control of many grass weeds such as Echinochloa spp.
and Leptochloa chinensis. The pre-mix formulation of 10g ai Penoxsulam + 50g ai Cyhalofop-butyl/liter
(Accept60OD/TopShot60OD) and the tank-mix of penoxsulam (Clipper25OD/Rainbow25OD)
+ Cyhalofop-butyl(Clincher100EC/Cranstan100EC) are broad-spectrum herbicide products that
are applied post-emergence and have residual weed control activity to control many grass, broadleaf
and sedge weeds with excellent rice tolerance in ASEAN countries. Combination products containing
penoxsulam + cyhalofop-butyl can increase rice productivity in a wide diversity of rice production
systems in direct-seeded, water-seeded and transplanted rice. Small plot field research trials and on-
farm demonstration trials were completed from 1998 to 2011. Trials were conducted in many locations
across ASEAN countries over a 13 year period. In large plot on-farm demonstrations from 2003 to 2011,
premixes or tank mixes of penoxsulam + cyhalofop-butyl at 10 g ai+50 g ai/ha to12.5 g ai+62.5 g ai/ha,
respectively, applied as a foliar post-emergence treatment at 7 to18 days after sowing or transplanting
provided >90% control of common weeds in rice. This high level of weed control resulted in a 20 to 50
% yield increase when compared to rice production in untreated areas. Both active ingredients in the
mixtures are highly efficacious on Echinochloa spp. Each herbicide has a different mode of action, so
these pre-mix products are a good tool for Echinochloa spp. resistance management in rice. Pre-mixes
and tank mixes of penoxsulam + cyhalofop-butyl demonstrated excellent rice crop safety. The herbicide
mixtures were applied post-emergence at rates up to 5 times the labeled use rate ( 300 g/ha) at 7 to 18
days after sowing or transplanting and did not injure the rice crop or reduce yields.

Keywords: penoxsulam, cyhalofop–butyl, efficacy, yield, direct-seed rice, transplanted rice, grasses,
sedges, broadleaf weeds.

Introduction

Rice is a major staple food with a planted area of 29 MM ha in ASEAN countries in 2009, which
provides for 600 million people in the area. Rice production should be increased to meet the demand of
the increasing population. One of the most important methods to increase rice production is to minimize
the loss caused by weed competition in rice fields. These weeds are not only reducing rice production
but also affecting rice seed quality. Since the beginning of agriculture, growers have tried to control
rice weeds by any means. One of the means is using synthetic herbicides. Many different herbicides
have been commercialized; however, farmers still prefer a one shot treatment that will provide broad-
spectrum weed control.

The premix of Penoxsulam + Cyhalofop-butyl has been developed by Dow AgroSciences
since 2005 and commercialized under the trade name of TopShot60OD in Indonesia, Philippine,
Vietnam and Accept60OD in Thailand. Penoxsulam is an ALS inhibitor that belongs to group

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 42

B1 (triazolopyrimidine sulfonamides), and penoxsulam is broad spectrum herbicide that controls
Echinochloa spp, broadleaf and annual sedge weeds. Cyhalofop-butyl is an ACCase inhibitor in Group
A (Aryloxyphenoxypropionates) which provides very high control of grassy weeds like Echinochloa
spp and Leptochloa spp. Both active ingredients are very safe to rice and have broad spectrum control
efficacy. This report is a summary of 106 field trials across Vietnam, Philippine, and Thailand since
1998-2011.

Materials and Methods

Field studies were conducted on field stations of Rice Research Institutes in Malaysia, Philippine,
Indonesia and Vietnam and on farmer fields in Thailand. Trials were randomized complete block design
(RCBD) with 3 or 4 replications with plot area of 16-25 m2. Target crop was Oryza sativa (Indica)
cultivated by direct-seeding or transplanting. In wet-seeded rice, water was partially drained from the
field, with post emergence foliar application made to exposed weeds, and the paddy reflooded within
48 hours after application. Other rice cultivation followed local farming practice.

For the tank mixture trials, Penoxsulam 25 OD (25 g/l) and Cyhalofop-butyl 100 EC (100 g/l)
were used at early stage of development. The herbicide premix was Topshot 60 OD (10 g/l Penoxsulam
+ 50 g/l Cyhalofop-butyl); tested rates were 1, 1.25 and 1.5 liters/hectare. Each treatment was diluted in
a spray volume of 320-400 liter water per hectare, and applied by knapsack sprayer with fan nozzle.

Individual weed control evaluation was made at 14 DAA (Days After Application), 28 DAA
and 42 DAA by visual observation on biomass reduction of weed compared with untreated plot as
percentage of control. Phytotoxicity was recorded at 1, 3, 5,7,14 and 28 DAA by visual assessment
based on 1-9 injury scale level. Rice yields were harvested in 5m2 frame in each plot then calculated
into theoretical rice yield per hectare. Collected data were statistical analyzed by ARM7 (owned by
Dow AgroSciences).

Results and Discussions

Rice Crop Phytotoxicity
The tested rates from 5-50 g ai + 25-250 g ai/ha of the tankmix or premix of penoxsulam +

cyhalofop, respectively, did not cause any injury symptom to indica rice when applied at 3-18 days
after sowing or transplanting. Table 1 shows that up to 5 times label rate, e.g. 300 g ai/ha applied at 7-18
days after sowing/transplanting did not injure the rice crop.

Efficacy of tank-mixture Penoxsulam + Cyhalofop on Leptochloa chinenses (LEFCH)
Penoxsulam at 10-30 g ai/ha does not provide commercial control of LEFCH. However,

Cyhalofop-butyl makes an excellent tank mix partner for control of LEFCH without antagonizing the
control of ECHCG and other weeds listed. The result of this mixture as shown in Table 2 demonstrates
that 10+50 to 12.5+62.5 g ai/ha of Penoxsulam + Cyhalofop-butyl, respectively, applied at 3-16 DAS
provided more than 85% control of LEFCH without antagonizing ECHCG control.

Efficacy of premix Penoxsulam+Cyhalofop_butyl on Leptochloa chinensis (LEFCH)
Table 3 shows that at 4-18 DAS (Day After Seeding) application timing, the premix at rates of

0.75-1.5 l/ha provided very high herbicidal activity on LEFCH. In general, the 1 l/ha rate provided equal
efficacy on LEFCH compared with rates of 1.25 and 1.5 l/ha, at same application timing, with results
similar among ASEAN countries.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 43

Table 1 Crop Injury of Penoxsulam + Cyhalofop-butyl to rice at 1-28 DAA when applied 3-18 days
after sowing across ASEAN countries

Days Rates No. of 1DAA Crop injury level*
after (g ai/ha) observation 3DAA 5DAA 7DAA 14DAA 28DAA
seeding

60 g mixtures N=60 111 1 1 1
1 1 1
120 g mixtures N=60 1 1 1 1 1 1
4-9 300 g mixtures N=60 1 1 1
1 1 1
225 g Butachlor + N=52 1 3 2
225 g Propanil 1 1 1
1 1 1
60 g mixtures N=54 111 1 1 1

10-14 120 g mixtures N=54 1 1 1 3 1 1

300 g mixtures N=54 1 1 1

36 g Fenoxaprop _ N=48 3 4 5
ethyl

* crop injury based on 1-9 scale with 1 = totally safe and 9 = complete kill.

Table 2. Efficacy of Penoxsulam + Cyhalofop on LEFCH at 28 DAA (% Biomass Reduction)

Days after Country No. of Penoxsulam + Cyhalofop (g ai/ha)
seeding observation
3 -5 Vietnam 10 + 50 12.5 + 62.5 15 + 75
6 -9 Thailand n = 12
Vietnam n = 12 90.9 94.2 99.6
10 –12 Philippines n = 12
Thailand n = 12 94.4 100.0 100.0
13-16 Vietnam n=8
Philippines 89.6 94.2 98.0
Thailand n = 12
Vietnam n = 20 96.4 99.7 95.0
n=8 89.2 91.7 95.0
n = 16
90.7 94.2 97.5

96.2 100.0 98.4
70.0 85.0 91.7
95.3 95.0 99.1

From the data of these trials, the premix of Penoxsulam + Cyhalofop-butyl was developed, results of
the premix are presented in the next section.

Efficacy of premix Penoxsulam+ Cyhalofop-butyl on Echinochloa crus- galli (ECHCG)
Table 4 demonstrates that 1 l/ha of Penoxsulam + Cyhalofop-butyl premix provided >90%

control of ECHCG with an application window of 0-18 DAS across ASEAN countries. Very high
results were seen on ECHCH, because both active ingredients can provide effective control of ECHCG.
Thus, the premix of Penoxsulam + Cyhalofop-butyl at 1 l/ha could be a good choice for ECHCG
resistance management across ASEAN. It not only provides very high control but has a wide application
window.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 44

Table 3. Efficacy of premix Penoxsulam + Cyhalofop-butyl on LEFCH at 28 DAA (Averaged %
Biomass Reduction)

Days after Country No. of Rate of product (liter/ha)
seeding observation 1 1.25 1.5
4-9 Thailand 93 95 96
Vietnam N=52 92 99 96
10-14 Philippines N=24 94 96 97
Thailand N=40 96 95 97
15-18 Vietnam N=60 95 99 98
Philippines N=64 95 95 98
Vietnam N=32 - 88 100
N=12

Table 4. Efficacy of premix Penoxsulam + Cyhalofop-butyl on ECHCG at 28 DAA (% Biomass
Reduction)

Days after Country No. of Rate of product (liter/ha)
seeding observation
0-3 Vietnam 1 1.25 1.5
4-9 Thailand N=48 98 97 96
Vietnam N=52
10-14 Philippines N=32 91 94 97
Thailand N=40
15-18 Vietnam N=40 95 97 99
Philippines N=64
Vietnam N=24 94 93 96
N=8
98 97 99

97 99 99

93 98 91

- 90 100

Efficacy of premix Penoxsulam + Cyhalofop-butyl on Echinochloa colona (ECHCO)
As shown in Table 5, control efficacy of the premix on ECHCO is similar to ECHCG. At 10-14

DAS, 1 l/ha provided >92% control of ECHCO. In Philippines, 1-1.25 l/ha at 15-18 DAS application
timing provided lower control than at 10-14 DAS, possibly due to weeds being larger and having more
tolerance at later timing. However, 1.5 l/ha was similar between 2 application timings.

Efficacy of premix Penoxsulam + Cyhalofop-butyl on Cyperus difformis (CYPDI)
As presented in Table 6, 1 l/ha of premix penoxsulam + cyhalofop-butyl at 0-14 DAS provide

very good control of CYPDI, higher rate at 1.25-1.5 l/ha did not show significantly better efficacy.

Table 5. Efficacy of premix Penoxsulam + Cyhalofop-butyl on ECHCG at 28 DAA (% Biomass
Reduction)

Days after Country No. of Rate of product (liter/ha)
seeding observation 1 1.25 1.5

10-14 Philippines N=16 96 99 96
Thailand N=8 93 90 97

15-18 Philippines N=8 91 95 97

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 45

Table 6. Efficacy of premix Penoxsulam + Cyhalofop_butyl on CYPDI at 28 DAA (% Biomass
Reduction

Days after seeding Country No. of Rate of product (liter/ha)
observation 1 1.25 1.5

0-3 Vietnam N=40 98 96 100

4-9 Thailand N=30 91 92 98

Vietnam N=8 100 99 99

10-14 Thailand N=70 94 95 95
Vietnam N=32 97 98 99

Efficacy of premix Penoxsulam + Cyhalofop-butyl on Cyperus iria (CYPIR)
Similar to CYPDI, result in Table 7 demonstrates that at 4-18 DAS application timing, 1 l/

ha provided >90% control of CYPIR in Thailand and Philippines. In Thailand, control efficacy of the
premix on CYPIR was really high across the application window of 4-14 DAS, it implied that the
premix of Penoxsulam + Cyhalofop-butyl can provide very good control of CYPIR in Thailand.

Efficacy of premix Penoxsulam + Cyhalofop-butyl on Fimbristylis miliacea (FIMMI)
From the data in Table 8, it is clear that 1 l/ha of premix Penoxsulam + Cyhalofop-butyl can

provide effective control of FIMMI at 0-18 DAS in Thailand, Philippines and Vietnam. The differences
between 1, 1.25 and 1.5 l/ha is minor, demonstrating that the FIMMI was very susceptible to the premix
in those countries. Moreover, according to N.Lap et al (2003) 10 g a.i/ha Penoxsulam at 3-18 DAS
provides 87-90 % control of FIMMI in Vietnam, Thailand and Philippine, it showed that there is no
antagonism between penoxsulam and cyhalofop-butyl on FIMMI in this area.

Table 7 Efficacy of premix Penoxsulam + Cyhalofop-butyl on CYPIR at 28 DAA (% Biomass
Reduction)

Days after Country No. of Rate of product (liter/ha)
seeding Thailand observation
1 1.25 1.5
4-9 N=9
97 98 98

10-14 Philippines N=12 85 - 90
Thailand N=24 96 97 97

15-18 Philippines N=48 93 95 -

Table 8 Efficacy of premix Penoxsulam + Cyhalofop-butyl on FIMMI at 28 DAA (% Biomass
Reduction)

Days after Country No. of Rate of product (liter/ha)
seeding observation 1 1.25 1.5

0-3 Vietnam N=48 99 97 97

4-9 Thailand N=20 96 99 99

Vietnam N=40 97 99 98

Philippines N=24 97 98 95

10-14 Thailand N=90 98 98 99

Vietnam N=72 95 97 98

15-18 Philippines N=48 94 96 97

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 46

Efficacy of premix Penoxsulam + Cyhalofop-butyl on Sphenoclea zeylanica (SPDZE)
Table 9 shows that the premix at 1 l/ha provided very high control of SPDZE with application

window at 0-14 DAS.

Efficacy of premix Penoxsulam+ Cyhalofop-butyl on Monochoria vaginalis (MOOVA)
Table 10 showed that premix penoxsulam + cyhalofop at 1 l/ha provided very high control

of MOOVA at 4-18 DAS in Philippine and Vietnam, higher rate at 1.25 and 1.5 l/ha did not increase
control efficacy on this weed.

Effect of premix Penoxsulam + Cyhalofop-butyl on rice yield
The data in Table 11 shows that the premix at a rate of 1 l/ha helps to increase yield dramatically

up to 106 to 121% compared to the untreated plot. Rate of 1.25-1.5 l/ha did not show any difference to
the response from the 1 l/ha rate.

Table 9. Efficacy of premix Penoxsulam + Cyhalofop-butyl on SPDZE at 28 DAA (% Biomass
Reduction)

Days after Country No. of Rate of product (liter/ha)
seeding observation
1 1.25 1.5

0-3 Vietnam N=48 97 99 98
4-9 Thailand N=52 96 91 96
10-14 Vietnam N=24 95 98 96
Philippines N=24 100 100 100
Vietnam N=40 97 100 99

15-18 Philippines N=40 100 100 100

Table 10 Efficacy of premix Penoxsulam + Cyhalofop-butyl on SPDZE at 28 DAA (% Biomass

Reduction)

Days after seeding Country No. of Rate of product (liter/ha)
observation 1 1.25 1.5

4-9 Vietnam N=8 100 98 99

10-14 Philippines N=24 100 100 100
Vietnam N=32 95 98 100

15-18 Philippines N=24 100 100 100

Table 11 Average rice yield of Premix Penoxsulam + Cyhalolop-butyl compared with commercial
standards and untreated plots across ASEAN countries

Days after Rates No. of Average yield* Yield increased
seeding (g ai/ha) observation (ton/ha) compared to
untreated
4-9 60 g Premix N=60 7.59 121 %
75 g Premix N=60 7.61 122 %
90 g Premix N=60 7.68 124 %
225 g Butachlor + 225 g
N=52 6.73 96 %
Propanil
Untreated N=60 3.43 0%

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 47

Days after Rates No. of Average yield* Yield increased
seeding (g ai/ha) compared to
observation (ton/ha) untreated
106 %
60 g Premix N=54 7.28 113 %
7.54 114 %
10-14 75 g Premix N=54 7.58
90 g Premix N=54 85 %
6.52
34.5 g Fenoxaprop_ethyl + N=48 0%
10 g Ethoxysulfuron N=54 3.64

Untreated

* Theoretical yield, calculated from harvested yield in 5m2/ plot.

Acknowledgements

We thank Dr.DuongVan Chin of the Cuu Long Rice Research Institute (Vietnam); Mr. Henry
Dupo, Sr. (Philippines); Hj Muhammad Harun and En Hassan Ahmad (Malaysia Agriculture Research
and Development institute) and Dr. Hamdan Pane and Dr.Zainal Lamid (Indonesia) for cooperation in
carrying out these trials in respective country for this study.

References

Mann, R.K. et al. Penoxsulam and cyhalofop butyl_Technical Bulletin. Dow AgroSciences internal
document

N. Lap, S.Pornkulwat, S.N.A Sayomchai, C. Antipas, M.A. Jaafar, S. Djoko,Vasant L.Patil, S. Sonoredjo
and R.K. Mann. 2003. Manila weed conference.

Sujitno, S et al. premix Penoxsulam + Cyhalofop_butyl Postemergence FoliarApplications at 3 to 27 Days
After Seeding in ASEAN Direct-Seeded Indica Rice in 2000 – 2001. DERBI DowAgroSciences
internal report.

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand TWSC 2013 48

Potential of Organic Herbicide from Aglaia odorata Lour

Chamroon Laosinwattana1 Montinee Teerarak1 and Patchanee Charoenying 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of

Technology Ladkrabang, Bangkok 10520, Thailand
2 Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok

10520, Thailand
[email protected]

Abstract

This study was undertaken to explore the potential of Aglaia odorata Lour.) organic herbicide
under laboratory, pot culture and natural field conditions. The aqueous extracts of leaf and branches
of Aglaia odorata inhibited the germination and seedling growth of Echinochloa crus-galli and
Phaseolus lathyroides and the leaf extract was slightly more inhibitory than the branch extract at the
same concentration. These results indicated that leaf and branch from A. odorata contained certain
allelochemicals inhibitory to E. crus-galli and P. lathyroides germination and growth. Subsequent
research had examination of the different A. odorata forms on the growth of E. crus-galli and P.
lathyroides, under laboratory and pot culture conditions. The results showed that the degree of toxicity
of different A. odorata forms can be classified in order of decreasing inhibition as pellet > dried leaf
powder > aqueous extract. Addition, effects of A. odorata granules was study on seedling growth of major
maize weeds and the influence of soil type on its residue’s efficacy. Under experimental greenhouse,
emergence and seedling growth of two major maize weeds (large crabgrass (Digitaria adscendens) and
horse purslane (Trianthema portulacastrum L.)) was inhibited but varied with soil type. The degree of
toxicity of different soil types can be classified in order of decreasing inhibition as sand > sandy loam
>clay. Under natural field conditions, an organic herbicide produced from A. odorata in the granule
form could be used to suppress D. adscendens weed emergence and growth in maize field and had no
adverse effect on maize growth and silage yield.

Keywords: Allelopathy, herbicide formulation, inhibition, organic herbicide, weed control

Introduction

The use of allelopathic plants as mulch or soil incorporated has been suggested for alternative
weed management in sustainable agriculture (Fujii, 2001; Singh et al., 2003a; Xuan et al., 2005; Batish
et al., 2006a; Khanh et al., 2006). For example, hairy vetch (Vicia vilosa L.) is promising cover crop for
weed control in fields, grasslands and orchards in Japan (Fujii, 2001). Dried Saururaceae (Houttuynia
cordata Thunb.) powder significantly reduces the Echinochloa and Monochoria paddy weeds at 150
g m–2 and increases the grain yield of rice than control (Lin et al., 2006). However, there are many
limitations for using plant residues such as mulch or incorporating them due to heavy fieldwork for
applying large amount of plant residues, which is often cost prohibitive. Over many years, various
types of allelochemicals have been isolated and characterized from hundreds of plants. Some of the
allelochemicals products exploited as commercial herbicides are cineole (shell), benzoxazinones
(BASF), quinolic acid (BASF) and leptpspermones (Zeneca) (Kohli et al., 1998). Natural plant products
may provide clues to new and safe herbicide chemistry (Duke, 1986). However, most of allelochemicals
having potential herbicidal activity but not commercially used because of there are extremely expensive
to manufacture. Einhellig (1995) suggested that nearly all allelopathic activities are due to the presence of
several compounds in a mixture. The concentration of each compound in a mixture might be significantly
less than the concentration of individual compounds needed to cause growth inhibition.This illustrates
the significance of joint action of allelochemicals in mixtures (Inderjit et al., 2002). Another challenge
is that the use of products formulated from crude extract of joint action of allelochemicals in mixtures