25.

2/2000

August 2000

International Rice Research Institute IRRI home page: http://www.cgiar.org/irri Riceweb: http://www.riceweb.org Riceworld: http://www.riceworld.org IRRI Library: http://ricelib.irri.cgiar.org IRRN: http://www.cgiar.org/irri/irrn.htm

International Rice Research Notes

Copyright International Rice Research Institute 2000

The International Rice Research Notes (IRRN) expedites communication among scientists concerned with the development of improved technology for rice and rice-based systems. The IRRN is a mechanism to help scientists keep each other informed of current rice research findings. The concise scientific notes are meant to encourage rice scientists to communicate with one another to obtain details on the research reported. The IRRN is published three times a year in April, August, and December by the International Rice Research Institute.

Contents
4
MINI REVIEW

Bt rice: practical steps to sustainable use

M.B. Cohen, F. Gould, and J.S. Bentur

Plant breeding

11 Differentiation of rice varieties cultivated in

Yunnan, China, using PCR markers corresponding to conserved motifs of disease resistance genes
Z.H. Wang, E.S. Borromeo, P. Teng, H. Leung, and Y.Y. Zhu

13 Genetic variability in photosynthesis and chlorophyll content of various landraces of upland rice
R.K. Agnihotri, S. Chandra, S. Sharma, and L.M.S. Palni

15 Inheritance of aroma in four rice cultivars

(Oryza sativa L.)
Y.J. Dong, E. Tsuzuki, and H. Terao

Genetic resources

16 Variation in RFLP markers associated with rootgrowth QTLs in Indian upland rice
K.A. Steele, B.J. Moore, J.R. Witcombe, D.S. Virk, and A.H. Price

19 Identification of genomic constitution of three

tetraploid Oryza species through two-probe genomic in situ hybridization
C.B. Li, D.M. Zhang, S. Ge, D.Y. Hong, and B.R. Lu

17 Evaluation of in situ conservation of Oryza

rufipogon populations using RAPD markers
Z.-W. Xie, S. Ge, K.-Q. Wang, D.-Y. Hong, and B.R. Lu

22 Collection and evaluation of hill germplasm

from Karbi Anglong and North Cachar Hill districts of Assam, India
A. Roy and K. Das

23 WEB NOTES

August 2000

Pest science & management

24 Improvement of conjugation methods for

Xanthomonas oryzae pv. oryzae strain DY89031 to identify avrXa21 clones
P.K. Sharma, F.G. da Silva, Y. Shen, and P.C. Ronald

Soil, nutrient, & water management

25 Comparing management techniques to optimize

fertilizer N application in rice in the Cauvery Delta of Tamil Nadu, India
P. Stalin, T.M. Thiyagarajan, S. Ramanathan, and M. Subramanian

30 Efficiency of controlled-release urea and coated

urea fertilizers in irrigated transplanted rice in Andhra Pradesh, India
K. Padmaja, R.M. Kumar, S.P. Singh, A.G.K. Murthy, and S.V. Subbaiah

27 Optimizing chlorophyll meter threshold values for

different seasons and varieties in irrigated lowland rice systems of the Cauvery Delta zone, Tamil Nadu, India
M. Babu, R. Nagarajan, S.P. Ramanathan, and V. Balasubramanian

32 Varietal response to different nitrogen

management methods in irrigated transplanted rice ecosystem in a Vertisol, Andhra Pradesh, India
R.M. Kumar, K. Padmaja, S.V. Subbaiah, and V. Balasubramanian

34 Assessment of chlorophyll meter-based N 28 On-farm evaluation of chlorophyll meter-based

N management in irrigated transplanted rice in the Cauvery Delta, Tamil Nadu, India
M. Babu and R. Nagarajan, S. Mohandass, C. Susheela, P. Muthukrishnan, M. Subramanian, and V. Balasubramanian

application at critical growth stages of irrigated transplanted rice

S.P. Ramanathan, R. Nagarajan, and V. Balasubramanian

35 Chlorophyll meter threshold values for N management in wet direct-seeded irrigated rice
V. Balasubramanian, A.C. Morales, and R.T. Cruz

Crop management & physiology

38 Chlorophyll stability index (CSI): its impact on

salt tolerance in rice
M. Madhan Mohan, S. Lakshmi Narayanan, and S.M. Ibrahim

39 Exogenous glycinebetaine reduces sodium

accumulation in salt-stressed rice plants
S. Lutts

Socioeconomics

41 Partnership in agricultural extension

G.P. Ojha

42 NEWS 43 NOTES FROM THE FIELD

44 RESEARCH HIGHLIGHTS 47 INSTRUCTIONS TO CONTRIBUTORS

About the cover Background: healthy panicle (right) and whitehead (left). Foreground: live yellow stem borer larva in nontransgenic rice (left) and dead larva in Bt rice (right).

Cover photos: Joachim Wnn (panicles), Behzad Ghareyazie (larvae).

Editorial Board Michael Cohen (pest science and management), Editor-in-Chief Zhikang Li (plant breeding; molecular and cell biology) David Dawe (socioeconomics; agricultural engineering) Bas Bouman (soil, nutrient, and water management; environment) Bao-Rong Lu (genetic resources) Shaobing Peng (crop management and physiology)

Production Team Katherine Lopez, Managing Editor Editorial Bill Hardy and Tess Rola Design and layout CPS design team, Grant Leceta, and Arleen Rivera Artwork Grant Leceta, Juan Lazaro Word processing Arleen Rivera

IRRN 25.2

MINI REVIEW

Bt rice: practical steps to sustainable use

M.B. Cohen, Entomology and Plant Pathology Division, IRRI; F. Gould, Department of Entomology, North Carolina State University, Raleigh, NC 27695-7634, USA; and J.S. Bentur, Department of Entomology, Directorate of Rice Research, Rajendranagar, Hyderabad 500030, India E-mail: m.cohen@cgiar.org

Introduction n 1996, maize, cotton, and potato farmers in the USA became the first farmers in the world to grow transgenic insect-resistant cultivars. These cultivars contain genes from the bacterium Bacillus thuringiensis (Bt) that encode insecticidal proteins known as delta-endotoxins. Delta-endotoxins have two properties that are essential for toxins used in transgenic crops: they are highly toxic to certain insect pests, and they are generally safe for human consumption (NRC 2000). Bt crops have been a large commercial success. In 1999, 11.7 million ha of Bt crops were grown by farmers in 10 countries, including Australia, Canada, China, South Africa, Spain, and the USA (James 1999). Many laboratories around the world have transformed rice with Bt genes and have evaluated their effectiveness under greenhouse conditions (e.g., Cheng et al 1998, Datta et al 1998, Maqbool et al 1998). No Bt rice varieties have yet been released to farmers, but field testing began at two sites in China in 1998 (Datta 1999, Ye et al 2000). The target pests for control by Bt rice are caterpillars, most importantly the yellow stem borer (YSB, Scirpophaga incertulas), the striped stem borer (SSB, Chilo suppressalis), and leaffolders such as Cnaphalocrocis medinalis. It has not been possible to produce rice with high resistance to these pests through conventional breeding, although short-duration semidwarf varieties are generally less damaged by stem borers than are traditional varieties (Khan et al 1991).

August 2000

Genetic engineering with Bt genes is a powerful technology for protecting crops against some kinds of insects. Bt cultivars, however, have the same weakness as many other insect control technologies: insects can evolve resistance to them, thereby eliminating their effectiveness.1 Insects have evolved resistance to all classes of widely used insecticides, including Bt products that are applied as sprays (Frutos et al 1999). Insects have also adapted to numerous resistant crop varieties produced through conventional breeding. Notable examples in rice include the brown planthopper, Nilaparvata lugens (Heinrichs 1986), and the Asian rice gall midge, Orseolia oryzae (Bennett et al 2000). The problem of insect adaptation to insecticides and resistant cultivars results in substantial costs to society, as crop failures, environmental damage, and loss of useful products. This has stimulated extensive research on the genetic and biochemical basis of resistance, and the development of resistance management strategies that can extend the effectiveness of insecticides and cultivars. In this review, we describe some practical steps that can be taken to delay the evolution of pest resistance to Bt rice. The high-dose/refuge resistance management strategy Over the past 10 years, scientists, government regulators, environmentalists, and the private sector have vigorously debated if and how the evolution of pest resistance to Bt crops can be delayed (e.g., Mellon and Rissler 1998, Gould 1998). There is widespread agreement that one strategythe high-dose/refuge strategyis the most promising and practical approach to prolonging the effectiveness of Bt crops. The high-dose/refuge strategy is being enforced for Bt cotton, maize, and potato in the USA (EPA-USDA 1999), Bt maize in Canada (CFIA 1999), and Bt cotton in Australia (ACGRA-TIMS 1997). To understand how the high-dose/refuge strategy works, it is necessary to examine the genetic basis of resistance. In many cases, resistance to an insecticide is caused by a mutation in one gene of an insect. If there are two possible forms of the gene (alleles), i.e., R (the mutant allele, conferring resistance) and S (the normal allele, conferring susceptibility), and each insect has two copies of the gene, then there are three possible genetic types (genotypes) of insects: SS, RS, and RR. Figure 1 illustrates the response of each genotype to increasing concentrations of an insecticide. In this example, the response of RS insects to the insecticide is intermediate between that of the SS and RR insects, but is more similar to that of the SS insects, indicating that the R allele is partially recessive [as it is in many cases of resistance to Bt toxins (Frutos et al 1999)]. To implement the high-dose/refuge strategy, it is necessary to have a titer of toxin in the Bt cultivar

that is high enough to kill almost all of the RS insects (indicated by the dotted line in Figure 1). Refuges are non-Bt crop plants that serve to maintain Btsusceptible insects in the population. Refuges can consist of fields planted with non-Bt plants or of non-Bt plants within fields of Bt plants. The large number of insects with the SS genotype that survive on the refuge plants are then available to mate with the small number of RR insects that survive on the Bt plants (Fig. 2). The offspring of SS RR matings will be RS, and therefore will not survive when they feed on high-dose Bt plants. It might seem that a high dose of toxin in Bt cultivars would accelerate the evolution of pest resistance rather than delay it. The output of a simple population genetics model, however, shows that if refuges are maintained, high-dose plants are more durable than low-dose plants (Fig. 3). Which spatial arrangement of refuge plants is best depends on the biology of the pest. Mixtures of Bt and non-Bt plants within fields can be established by sowing seed mixtures or by planting rows of refuge plants within fields of Bt plants. Within-field mixtures are not the best type of refuge for insects that move between plants during development. This is because some of the insects will feed on Bt and non-Bt plants, thereby diluting the high-dose titer in Bt plants (Gould 1998). Most larvae of YSB and SSB move between plants during development (Cohen et al 2000). If separate refuge fields are established, then Bt fields must not be too far from a refuge field, or else there might not be random mating between the RR and SS insect genotypes. Therefore, it is important to know approximately how far the adult pest insects will move before mating. This question is being studied at IRRI for YSB and SSB (A.M. Dirie, N.L. Cuong, F. Gould, and M.B. Cohen, unpubl.). Based on our current knowledge of YSB and SSB biology, it appears that maintaining separate refuge fields within 1 km of Bt rice fields would be a suitable form of refuge.

SS
% mortality

RR

RS

Log toxin concentration

Fig. 1. Dose-response lines indicating the mortality of three insect genotypes at increasing concentrations of an insecticidal toxin. The dotted line indicates the concentration required for a high dose. S, allele conferring susceptibility; R, allele conferring resistance.

1 Note that the word resistance is used in two ways, which can sometimes be confusing. Cultivars can be produced that have resistance to insects; and insects can evolve resistance to insecticides, such as Bt toxins in transgenic plants.

IRRN 25.2

Almost all RR moths mate with SS moths.

RR Very few moths emerge. Almost all are RR.

SS Many moths emerge. Almost all are SS (a few are RS).

All offspring are RS, which will be killed by high-dose plants.

Bt rice field

Non-Bt rice field

Fig. 2. How the high-dose/refuge strategy works to delay the increase in highly resistant (RR) insects in a pest population.

A. Low-dose plants

B. High-dose plants 1.0 Without refuge With 10% refuge 0.8 0.6 0.4 0.2 0

Frequency of R allele

1.0 0.8 0.6 0.4 0.2 0 0 10 20 30 40 50

10

20

30

40

50

Generations after release of Bt cultivar Fig. 3. Rate of evolution of pest resistance to Bt plants under various conditions, as simulated by a population genetics model. Settings of model parameters are as follows: Both low-dose (panel A) and high-dose (panel B) plants kill 100% of the SS insects and 0% of the RR insects. On lowdose plants, 5% of the RS insects survive to maturity; while on high-dose plants, only 0.5% of the RS insects survive. The initial frequency of the R allele is 103. The model output indicates that if the low-dose cultivar is grown in 90% of the crop fields and the remaining fields serve as a non-Bt refuge, resistance to the Bt toxin evolves in about 15 pest generations. Under the same conditions, the high-dose cultivar remains effective for about 45 pest generations. If no refuge is maintained, resistance evolves in less than five generations on both cultivars. In tropical irrigated areas, there are approximately six generations of rice stem borers per year.

How can high-dose plants be identified? When plants are transformed, transgenes become incorporated into plant chromosomes at random locations. For this and other reasons, there is great variability among independent transgenic lines in the level of transgene expression. Because toxin titers
6

can differ substantially among plant lines that are transformed with the same Bt gene construct (e.g., Cheng et al 1998, Datta et al 1998, Maqbool et al 1998), many transformed lines must be screened to identify a few that produce adequate quantities of Bt toxin and do not have agronomic problems such as stunted growth.
August 2000

Dose-response data. Plants have a high dose of toxin when they are able to kill almost all insects of the RS genotype (Fig. 1). To make practical use of this definition, a resistant colony of the pest must be available so that RS insects can be tested. Unfortunately, Bt-resistant colonies of YSB or SSB have not been developed. A high dose has also been defined as one that is 25X higher than that required to kill 99% of homozygous susceptible insects (EPA 1998). To make practical use of this definition, it is necessary to have an accurate estimate of the LD99, determined from dose-response experiments using purified Bt toxin. No rigorous estimates of the LD99 for Bt toxins against YSB or SSB have been published. We have not been able to obtain reliable estimates of LD50 or LD99 values at IRRI due to very high variability between experiments (R. Aguda, F. Gould, and M.B. Cohen, unpubl.). Plant bioassays. Another way to determine if a Bt rice line has a high dose of toxin is to test it against a large number of stem borer larvae and verify that very few insects survive to maturity. A large number of test larvae is necessary because RR insects are likely to be rare. A study of the tobacco budworm in the USA found that the frequency of an allele conferring high resistance to Cry1Ab and other Bt toxins was 1.5 103 (Gould et al 1997). A population of YSB in the Philippines was found to have a frequency of alleles conferring high resistance to Cry1Ab of less than 3.6 103, with 95% confidence (Bentur et al 2000). If the frequency of an allele for Bt resistance in a pest population is 103 (meaning that 1 out of 1,000 copies of the resistance gene has the R allele), then the frequency of RR insects of the population would be 106. If such a population were in Hardy-Weinberg equilibrium, then the frequency of RS insects would be approximately 2 103 and that of SS insects would be about 0.998. On a high-dose Bt cultivar that killed all of the SS insects, 99.5% of the RS insects, and none of the RR insects, only about 10 out of 1 million insects would survive to maturity!

Typically, greenhouse evaluations of Bt rice lines use only a few dozen or perhaps a few hundred larvae. With these small numbers of test insects, even plants with a very low dose of toxin will probably kill 100% of test insects, almost all of which will be of the SS genotype. Thus, 100% larval mortality in a typical greenhouse bioassay does not indicate that the Bt line has a high dose of toxin, as defined in terms of the high-dose/refuge strategy. What about field evaluations of Bt rice plants? Imagine a 100-m2 test plot of Bt rice, transplanted at 20 20 cm spacing, and one seedling per hill. Such a field would contain 2,500 plants. If every plant were artificially infested with one stem borer egg mass at vegetative stage, and a second egg mass at booting stage, and each egg mass contained 100 eggs, and there were no natural infestation, then there would be 500,000 larvae in the plot during the growing season. If the plant killed all of the SS insects, 99.5% of the RS insects, and none of the RR insects, and there were no mortality caused by natural enemies or other non-Bt factors, then about 5 insects would survive to maturity. This would result in about 10 deadhearts or whiteheads in the entire plot. If there were 10 panicles per plant, this would be equivalent to a damage level of about 0.04% whiteheads. Calculations such as these should be made when researchers evaluate the performance of Bt rice in field tests. Quantification of toxin titer. Comparisons with commercially released Bt cotton, maize, and potato cultivars can provide guidance on what is a high-toxin titer dose in Bt rice. Three released cultivars in the USA are considered to have a high dose of toxin against all target pests, based on extensive experimental evaluation and experience in farmers fields (see table). The toxin titer in these cultivars ranges from 1 to 11 g g-1 of leaf fresh weight or 0.1% to 0.2% of soluble leaf protein. A Bt toxin titer of 0.2% of soluble leaf protein is well within reach of what can be achieved with rice (e.g., Cheng et al 1998,

Characteristics of commercially available Bt cultivars. Approximate maximum toxin titer Crop Transformation event (company) nac (Monsanto) Events 531 and 931 (Monsanto) Event Bt11 (Novartis) Event MON810 (Monsanto) Event 176 (Novartis, Mycogen) Bt toxin g g1 leaf fresh weightb na 12 Principal target pests % soluble leaf protein 0.10.2d na Colorado potato beetle Cotton bollworm Tobacco budworm Pink bollworm European corn borer European corn borer European corn borer Does the event have a high dose?a Yes No Yes Yes Yes Yes Vegetative stage: yes Reproductive stage: no

Potato Cotton

Cry3A Cry1Ac

Maize Maize Maize

a e

Cry1Ab Cry1Ab Cry1Ab

3 511 4

na na 0.2e

Gould 1998, Mellon and Rissler 1998. bUS Environmental Protection Agency fact sheets (http://www.epa.gov/pesticides/biopesticides/). cna = not available. dFeldman and Stone 1997. Koziel et al 1993.

IRRN 25.2

Datta et al 1998, Maqbool et al 1998). This toxin titer could be considered as a guideline for the minimum titer for high-dose Bt rice plants. For successful resistance management, it must also be demonstrated that the high-toxin titer is maintained over the entire period of pest attack. Toxin titer declines substantially at reproductive stage in a maize line (Mellon and Rissler 1998) and a rice line (Alinia et al 2000) with a cry1Ab gene under control of the PEP C promoter. Toxin decline at later stages of plant growth has also been observed under some environmental conditions for Bt cotton with the CaMV 35S promoter (Fitt et al 1998). How can refuges be maintained? Farmers growing Bt crops in the USA must plant 420% of their land to non-Bt cultivars, and these refuge fields must be within approximately 0.8 km of their Bt fields (EPA-USDA 1999). Obviously, enforcing a similar system for small rice farmers will not be possible in most parts of Asia. This does not mean, however, that a functional high-dose/refuge strategy cannot be achieved for Bt rice. In a typical village, it is unlikely that all farmers will plant Bt rice on all their land. Bt genes will be one of many factors that farmers will consider when choosing which rice varieties to grow. Governments can promote the maintenance of refuges by restricting the number and diversity of Bt cultivars that can be released. For example, in the Indian state of Punjab, farmers grow traditional Basmati varieties and modern semidwarf varieties. Stem borer damage is higher in Basmati varieties, and thus the government could authorize the release of Bt-transformed Basmati varieties but not Bt-transformed semidwarfs. Because of the great importance of maintaining refuges, governments should also consider implementing a refuge system where this would be practical. Examples include large ricegrowing estates or collectives, villages with well-organized farmers organizations, and areas with strong extension services. The concern is often raised that stem borer damage in nonBt fields will increase after introducing Bt rice. The implication is that farmers will be even less likely to grow non-Bt rice because of the increased damage, and therefore there will be even fewer refuge fields. In fact, after the introduction of Bt rice, the level of stem borer damage in non-Bt rice fields will probably be unchanged or will decline. Moths do not prefer to lay their eggs on non-Bt plants (Riggin-Bucci and Gould 1997, Hellmich et al 1999), and there is no evidence that moths can detect whether or not a plant contains Bt toxin. In some studies, it has been found that, after feeding begins, caterpillars move away from Bt plants faster than from non-Bt plants (e.g., Dirie et al 2000), but very few rice stem borer larvae crawl far enough to move from one field to another (Cohen et al 2000). Stem borer moths are good fliers and are well capable of moving from one field to another (Khan et al 1991; A. Dirie, F. Gould, and M.B. Cohen, unpubl.). Many of the moths that emerge from fields of non-Bt rice will disperse and lay their eggs in fields of Bt rice. In contrast, because very few moths will emerge from
8

Bt fields, very few moths will move from Bt fields to non-Bt fields. As a consequence, the amount of stem borer damage in non-Bt fields may decline if most fields are planted to Bt rice. This decline in damage in refuge fields, called the halo effect, has been observed in experiments with the diamondback moth on Bt collards (Riggin-Bucci and Gould 1997) and the European corn borer on Bt maize (Andow and Hutchinson 1998). The importance of two-toxin Bt rice Another practical step that governments can take to promote the sustainable use of Bt rice is to release Bt cultivars only if they contain two Bt toxins, both at a high dose. If insects that are able to survive on a plant with one high-dose toxin are rare, then insects that are able to survive on plants with two high-dose toxins will be very rare indeed. If such insects must be homozygous for resistance alleles for two different genes, and if the frequency of the allele for resistance of each gene is 103, then insects of the genotype R1R1R2R2 will occur at a frequency of only 1012, i.e., 1 out of 1 trillion. Because such insects will be very rare, fewer susceptible insects will be needed to ensure that resistant insects do not mate with each other. Therefore, fewer refuge fields will be necessary (although it is still very important to have some refuge fields). More than 100 Bt toxin genes have been cloned and sequenced; the toxins are highly divergent in amino acid sequence and some biochemical properties (Frutos et al 1999). Any two Bt toxins that are used in combination must not be too similar to each other, otherwise, a single mutation could confer cross-resistance to both toxins. In most cases that have been studied, insect resistance to Bt toxins is caused by mutations in receptor proteins in the insect gut (Frutos et al 1999). Toxins must bind to receptors to initiate the process that will ultimately kill the insect, and mutations in receptors can eliminate toxin binding. Biochemical tests can determine whether two Bt toxins bind to the same receptor, and thus whether one mutation might disrupt the effectiveness of both toxins. Based on studies done by Fiuza et al (1996) and Lee et al (1997), it is known that the following are good toxin combinations for both YSB and SSB: Cry1Aa or Cry1Ac with Cry1C or Cry2A. Both studies also found that Cry1Aa and Cry1Ac should not be used in combination. Recently, it has been found that Cry1Ab with Cry1Ac would not be a good combination for either YSB or SSB, but that Cry1Ab with Cry1C or Cry2A would be effective (E. Alcantara, R. Aguda, D. Dean, and M.B. Cohen, unpubl.). Cotton producing the Cry1Ac and Cry2A toxins has been produced by Monsanto and approved for field testing by the US Environmental Protection Agency (http://www.epa.gov/ oppbppd1/biopesticides/reg_activ/reg_act_all_byAI.htm). Maqbool and Christou (1999) transformed rice with genes for the Cry1Ac and Cry2A toxins and found several lines in which the levels of both toxins were >0.5% of soluble leaf protein. Additional laboratories are expected to produce two-toxin Bt rice soon.
August 2000

A computer model of the evolution of Bt resistance shows that much of the long-term utility of cultivars with two toxins can be lost if both one- and two-toxin cultivars are grown in the same region, or if one-toxin cultivars are widely grown prior to the release of two-toxin cultivars (F. Gould, J. Van Duyn, and G. Kennedy, unpubl.). Therefore, it is critical to develop appropriate two-toxin cultivars and not to first deploy one-toxin cultivars. An important long-term goal is transformation of plants with combinations of a Bt toxin gene and a gene encoding an unrelated toxin because some insect mutations can confer cross-resistance to multiple, distantly related Bt toxins (Frutos et al 1999). Summary and recommendations Four practical recommendations for promoting the sustainable use of Bt rice can be made, based on existing knowledge of the biology of YSB and SSB and the principles of resistance management: 1. Do not release Bt varieties that do not have a high dose of toxin. Toxin titers of 2 g g1 of leaf fresh weight or 0.2% of soluble leaf protein are attainable in rice, and have been shown to act as high doses against most pests in other crops. 2. Release only Bt cultivars that have two Bt toxin genes. The genes should not be closely related to each other, and both should be expressed at a high dose. Two-toxin cultivars require smaller refuges to achieve successful resistance management. 3. Do not release Bt-transformed versions of all popular rice varieties. Some popular non-Bt varieties should remain available to improve chances that some non-Bt rice fields (refuges) will exist. Sufficient seed supplies of non-Bt varieties should be maintained. 4. Implement a resistance monitoring program. Several methodologies can be used to monitor pest populations for the evolution of resistance to Bt cultivars (Andow and Hutchinson 1998). The use of sentinel plots, in which insect damage is monitored in unsprayed fields of Bt cultivars, is perhaps the most practical for rice-growing areas. Resistance monitoring programs can serve as an early warning system for governments and farmers and provide valuable information for improved deployment of future pest-resistant cultivars. More than 20 research groups in many countries have produced Bt rice. Most of the lines produced do not meet recommendation 1, and very few lines meet recommendation 2. Scientists and governments will soon be faced with decisions of whether to release single-toxin and/or low-dose Bt rice cultivars to farmers. Their decisions should be based on long-term as well as shortterm benefits. Many farmers would benefit from growing Bt rice, but in most areas yield increases would be modest. Stem borers are usually low-level, chronic pests. Based on an extensive survey
IRRN 25.2

of farmers fields in Asia, stem borers were estimated to cause an overall mean yield loss of 2.4% (Savary et al 2000). In areas where stem borers are not an urgent problem, it would seem sensible to delay the release of Bt rice until a two-toxin, high-dose cultivar is available. In areas where stem borer damage is high, it must be realized that low-dose and/or single-toxin cultivars might not remain effective for very long. There are currently no proven alternatives to Bt toxins for use in transgenic plants to control caterpillar pests. Clearly, Bt toxins are a valuable natural resource that must be used with great care. References
ACGRA-TIMS (Australian Cotton Growers Research AssociationTransgenic and Insect Management Strategy Committee). 1997. Insect management plan for InGard Cotton, 1997-98. Wee Waa, Australia: ACGRA (Online: http://www.mv.pi.csiro.au/publicat/pest/ ingres97.htm) Alinia F, Ghareyazie B, Rubia LG, Bennett J, Cohen MB. 2000. Effect of plant age, larval age, and fertilizer treatment on resistance of a cry1Abtransformed aromatic rice to lepidopterous stem borers and foliage feeders. J. Econ. Entomol. 93:484493. Andow DA, Hutchinson WD. 1998. Bt-corn resistance management. In: Mellon M, Rissler J, editors. Now or never: serious new plans to save a natural pest control. Cambridge, MA.: Union of Concerned Scientists. p 1966. Bennett J, Bentur JS, Pasalu IC, Krishnaiah K, editors. 2000. New approaches to gall midge resistance in rice. Makati City (Philippines): International Rice Research Institute. Bentur JS, Andow DA, Cohen MB, Romena AM, Gould F. 2000. Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of a rice stem borer, Scirpophaga incertulas (Lepidoptera: Pyralidae). J. Econ. Entomol. (in press) CFIA (Canadian Food Inspection Agency). 1999. Responsible deployment of Bt corn technology in Ontario. Nepean, Ontario, Canada: CFIA. (Online: http://www/cfia-acia.agr.ca/english/plaveg/pbo/old/ btweb2_e.shtml) Cheng X, Sardana R, Kaplan H, Altosaar I. 1998. Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer. Proc. Natl. Acad. Sci. USA 95:27672772. Cohen MB, Romena AM, Gould F. 2000. Dispersal by larvae of the stem borers Scirpophaga incertulas (Lepidoptera: Pyralidae) and Chilo suppressalis (Lepidoptera: Crambidae) in plots of transplanted rice. Environ. Entomol. (in press) Datta K, Vasquez A, Tu J, Torrizo L, Alam MF, Oliva N, Abrigo E, Khush GS, Datta SK. 1998. Constitutive and tissue-specific differential expression of the cry1Ab gene in transgenic rice plants conferring resistance to rice insect pest. Theor. Appl. Genet. 97:2030. Datta SK. 1999. Transgenic rice products: roadmap for future impact. Abstracts, General Meeting of the International Program on Rice Biotechnology, 20-24 Sep 1999, Phuket, Thailand. NY: Rockefeller Foundation. p 125. Dirie AM, Cohen MB, Gould F. 2000. Larval dispersal and survival of two stem borer (Lepidoptera: Crambidae) species on cry1Ab-transformed and non-transgenic rice. Environ. Entomol. (in press) EPA (US Environmental Protection Agency). 1998. FIFRA Scientific Advisory Panel. Subpanel on Bacillus thuringiensis (Bt) plant-pesticides and resistance management. Washington, D.C.: EPA. (Online: http:// www.epa.gov/scipoly/sap/1998/february/finalfeb.pdf)

EPA-USDA (US Environmental Protection Agency, US Department of Agriculture). 1999. EPA and USDA position paper on insect resistance management in Bt crops. Washington, D.C.: EPA. (Online: http:// www.epa.gov/oppbppd1/biopesticides/otherdocs/ bt_position_paper_618.htm) Feldman J, Stone T. 1997. The development of a comprehensive resistance management plan for potatoes expressing the Cry3A endotoxin. In: Carozzi N, Koziel M, editors. Advances in insect control: the role of transgenic plants. London: Taylor & Francis. p 4961. Fitt GP, Daly JC, Mares CL, Olsen K. 1998. Changing efficacy of transgenic Bt cottonpatterns and consequences. In: Zalucki MP, Drew RAI, White GG, editors. Proceedings of the 6th Australian Applied Entomology Conference, Brisbane, 29 Sep2 Oct. Vol. 1. Brisbane: University of Queensland. p 189196. Fiuza L-M, Nielsen-Leroux C, Goz E, Frutos R, Charles J-F. 1996. Binding of Bacillus thuringiensis Cry1 toxins to the midgut brush border membrane vesicles of Chilo suppressalis (Lepidoptera: Pyralidae): evidence of shared binding sites. Appl. Environ. Microbiol. 62:1544 1549. Frutos R, Rang C, Royer M. 1999. Managing insect resistance to plants producing Bacillus thuringiensis toxins. Crit. Rev. Biotechnol. 19:227276. Gould FA, Anderson A, Jones D, Sumerford D, Heckel G, Lopez J, Micinski S, Leanard R, Laster M. 1997. Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens. Proc. Natl. Acad. Sci. USA 94:35193523. Gould F. 1998. Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annu. Rev. Entomol. 43:701726. Heinrichs EA. 1986. Perspectives and directions for the continued development of insect-resistant rice varieties. Agric. Ecosyst. Environ. 18:936. Hellmich RL, Higgins LS, Witkowski JF, Campbell FE, Lewis LC. 1999. Oviposition by European corn borer (Lepidoptera: Crambidae) in response to various transgenic corn events. J. Econ. Entomol. 92:1014 1020. James C. 1999. Global review of commercialized crops: 1999 (Preview). ISAAA Briefs No. 12.

Khan ZR, Litsinger JA, Barrion AT, Villanueva FFD, Fernandez NJ, Taylor LD. 1991. World bibliography of rice stem borers, 1794-1990. Manila (Philippines): International Rice Research Institute. Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E, Rhodes R, Warren GW, Wright M, Evola SV. 1993. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11:194200. Lee MK, Aguda R, Cohen MB, Gould FL, Dean DH. 1997. Determination of receptor binding properties of Bacillus thuringiensis -endotoxins to rice stem borer midguts. Appl. Environ. Microbiol. 63:14531459. Maqbool SB, Husnain T, Riazuddin S, Masson L, Christou P. 1998. Effective control of yellow stem borer and rice leaffolder in transgenic rice indica varieties Basmati 370 and M7 using the novel -endotoxin cry2A Bacillus thuringiensis gene. Mol. Breed. 4:501507. Maqbool SB, Christou P. 1999. Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns, expression levels and stability. Mol. Breed. 5:471480. Mellon M, Rissler J, editors. 1998. Now or never: serious new plans to save a natural pest control. Cambridge, MA.: Union of Concerned Scientists. NRC (National Research Council, USA). 2000. Genetically modified pestprotected plants: science and regulation. Washington, D.C.: National Academy Press. Riggin-Bucci TM, Gould F. 1997. Impact of intraplot mixtures of toxic and non-toxic plants on population dynamics of diamondback moth (Lepidoptera: Plutellidae) and its natural enemies. J. Econ. Entomol. 90:241251. Savary S, Willocquet L, Elazegui FS, Castilla NP, Teng PS. 2000. Rice pest constraints in tropical Asia: quantification of yield losses due to rice pests in a range of production situations. Plant Dis. 84:357369. Ye G-Y, Shu Q-Y, Yao H-W, Cui H-R, Cheng X-Y, Hu C, Xia Y-W, Gao M-W, Altosaar I. 2000. Field evaluation of resistance of transgenic rice containing a synthetic cryIAb gene from Bacillus thuringiensis Berliner to two stem borers. J. Econ. Entomol. (in press)

About IRRIs Library and Documentation Service

Did you know... ...that IRRI has the worlds biggest rice library? ...that it provides a free photocopy service1 to rice scientists everywhere? ...that its catalogue and rice bibliography are available on the World Wide Web for searching, 24 hours a day2? ...that IRRI Library staff are waiting right now to receive your requests3? We hope to be of service to you soon!

1 2 3

Maximum of 50 pages per request Web address: http://ricelib.irri.cgiar.org Write to Carmelita Austria at this address: Library and Documentation Service, International Rice Research Institute, MCPO Box 3127, Makati City 1271, Philippines. E-mail address: c.austria@cgiar.org

10

August 2000

Plant breeding

Differentiation of rice varieties cultivated in Yunnan, China, using PCR markers corresponding to conserved motifs of disease resistance genes
Z.H. Wang, E.S. Borromeo, P. Teng, H. Leung, Entomology and Plant Pathology Division, IRRI; and Y.Y. Zhu, Yunnan Agricultural University, Kunming, China E-mail: e.s.borromeo@cgiar.org

Many disease resistance genes in plants are found to have similar sequences that are presumably involved in pathogen recognition and signal transduction. This sequence conservation offers opportunities to develop markers that are indicative of function. Using polymerase chain reaction (PCR), primers can be designed to amplify sequences that correspond to conserved motifs of resistance genes. These PCR markers, called resistance gene analog (RGA) markers, have been used to characterize germplasm and breeding lines in barley, wheat, and rice (Chen et al 1998). This study tested this approach to evaluate the diversity of a set of varieties from Yunnan Province, China. RGA markers were applied to analyze 41 modern rice varieties and landraces cultivated in Luxi county in Yunnan and to test the minimal number of primer combinations needed to reveal the informative relationship among varieties. Rice genome DNA was extracted at Yunnan Agricultural University using miniprep protocol (Zheng et al 1995). Molecular characterization was done at IRRI. The PCR primers were designed based on conserved motifs of disease resistance genes: leucine-rich repeats, nucleotide-binding site, and kinase of a broad collection of disease resistance genes (see table). Genomic DNA (20 ng) from each variety was PCR-amplified using a primer pair as described by Chen et al (1998). Each 25 mL reaction contains 50 mM KCl, 20 mM TrisHCl, 5 mM MgCl2, 0.2 mM of each dNTP, 60 ng of each primer, 20 ng template DNA, and 1 unit of Taq. The reaction was conducted using the following conditions: denature 5 min at 95 C; 40 cycles of 1 min at 94 C, 1 min at 45 C, 2 min at 72 C;
IRRN 25.2

and a final extension of 7 min at 72 C. Amplified fragments were separated by 4% polyacrylamide gel electrophoresis and silver-stained. RGA banding patterns produced by each of the five primers revealed a high degree of intervarietal polymorphism. The composite banding patterns generated from all PCR primers were scored as binary data and analyzed using NTSYS version 1.8 (Exeter Software, Setauket, New York). Dice similarity coefficients were calculated to generate a dendrogram. Statistical stability of the branches in the cluster was estimated by bootstrap analysis with 2,000 replicates using the Winboot computer program (Yap and Nelson 1996). A consensus dendrogram based on markers generated from five primer pairs is shown in the figure.

Most of the branches had high bootstrap values, indicating the robustness of the branching pattern. The consensus dendrogram divided the Yunnan varieties into two groups corresponding to indicajaponica differentiation. With a few exceptions, modern and landrace varieties tended to form separate groupings. Lower bootstrap values were noted within the landrace group, suggesting that diversity within groups was higher among landraces. The degree of relatedness was higher among newly bred and released varieties. This may reflect the common pedigrees shared by improved varieties, e.g., Chujing and Hexi series of varieties. Three improved varietiesChujing 14, Gangyou 12, and Xianyou 22appear to be distinct from the others.

Primers used and the corresponding number of amplified and polymorphic bands generated. Primera XLRR for XLRR rev NLRR inv1 NLRR inv2 LM637 LM638 S1 AS3 Pto kin1 Pto kin2
a

Sequence (53)b CCG TTG GAC AGG AAG GAG CCC ATA GAC CGG ACT GTT TGC TAC GTT CTC CGG G TGA GGC CGT GAA AAA TAT ARI GCT ARI GGI ARI CC GGI GGI GTI GGI AAI ACI AC GGT GGG GTT GGG AAG ACA ACG IAG IGC IAG IGG IAG ICC GCA TTG GAA CAA GGT GAA AGG GGG ACC ACC ACG TAG

Amplified bands 60

Polymorphic bands 40

74

59

55

43

51

36

51

38

The primer pairs, XLRR for and XLRR rev and NLRR inv1 and NLRR inv2 were designed based on leucine-rich repeat regions of genes Xa21 and N (N. Naqvi, IRRI). The primer pairs LM637 and LM638 and S1 and AS3 were designed based on the P-loop nucleotide-binding sites and LRR regions of genes RPS2, N, and L6. The primer pair Pto kin1 and Pto kin2 were designed based on the DNA sequence encoding for protein kinase in the tomato Pto gene conferring resistance to Pseudomonas syringae pv. tomato.All primers were made by GIBCO. bCodes for mixed bases: I = isonine, R = A/G. Degenerate primers were designed with isonine or more than one residue at the third codon position to fit the consensus amino acid sequence.

11

69.5 37.2 38.5 25.0 20.0 29.3 99.9 44.4 48.4 60.0 39.0 26.2 13.4 38.0 88.1 63.2 90.3

92.2 91.3 62.0 16.4 22.5 34.5 43.0 88.3 60.5 22.0 73.1 94.8 65.0 85.5 81.9 83.4 56.5 61.0 90.6 73.1

99.8 18.5

Ai zhegu (Landrace) Duodiangu (Landrace) Laojingnuo (Landrace) Baijiaolaojing (Landrace) Yinggu (Landrace) Huagu (Landrace) Zhongyanggu (Landrace) Yuelianggu (Landrace) Chujing No. 14 Zhaogu (Landrace) Hongzhaogu (Landrace) Longshalingu (Landrace) Yuenangu (Landrace) Duodiangu (Landrace) Niangduogu (Landrace) Erlaojing (Landrace) Mazhagu (Landrace) Gangyou 12 Shanyou 22 260-1 260 Xianhuanuo (Landrace) Gaoshanrenshuigu (Landrace) Liming 251 Hexi 42 Zhangjing No. 8 Hexi 32 Hexi 39 Xinan 175 Dianyu No. 1 Chujing No. 9 Chujing No. 11 Chujing No. 3 88010-1 251 Xuan Luxuan No. 1 1138 Hexi 24 Hexi 30 Hexi 22-2 Chujing No. 16

0.51

0.61

0.71

0.81

0.91

Coefficient
Dendrogram of 41 rice varieties based on polymorphic bands generated by five primer pairs. Numbers in branches indicate the percentage of times the group of lines in a branch occurred, based on 2,000 cycles in bootstrap analysis using the Winboot program.

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August 2000

Similarity matrices generated by different combinations of primers using MXCOMP program in NTSYS-pc were used to determine the minimal primer combinations needed to obtain a good assessment of the genetic relationship among rice varieties. Matrix comparison was conducted by pooling data in a stepwise manner (e.g., comparing the matrix produced by five primer pairs with matrices from combinations of two, three, and four primer pairs). Matrix comparison showed that the similarity matrix produced by three primer pairs was correlated (0.87 0.97) with that generated by five primer pairs (data not shown). The result suggests that using three primer pairs can provide

a quick assessment of genetic diversity. Since RGA polymorphic markers may reflect diversity in domains of resistance genes, observed varietal groups may have different spectra of disease resistance. There has been a growing interest in increasing the level of genetic diversity in modern agricultural systems to sustain productivity. This approach is based on the premise that greater diversity in an agroecosystem would provide greater buffering capacity against biotic and abiotic stresses. The diversity information will be used to test whether mixing varieties that are agronomically similar but functionally diverse in disease resistance would provide enhanced protection against blast in the field.

References
Chen XM, Line RF, Leung H. 1998. Genome scanning for conserved motifs of disease resistance gene in rice, barley, and wheat by high resolution electrophoresis. Theor. Appl. Genet. 97:345355. Yap IV, Nelson RJ. 1996. Winboot: a program for performing bootstrap analysis of binary data to determine the confidence limits of UPGMA-based dendrograms. IRRI Discuss. Pap. Ser. 14. Zheng KL, Subudi PK, Domingo J, Magpantay G, Huang N. 1995. Rapid DNA isolation for marker-assisted selection in rice breeding. Rice Genet. Newsl. 12-255-258.

Genetic variability in photosynthesis and chlorophyll content of various landraces of upland rice
R.K. Agnihotri, S. Chandra, S. Sharma, and L.M.S. Palni, G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora 263643, India

After an extensive survey of the Kumaun region in the Central Himalaya, 28 different landraces of upland rice were collected. These landraces were studied for variation in chlorophyll content and photosynthetic rate and were compared with an introduced high-yielding variety (VL 206) released by the Vivekananda Hill Agriculture Research Laboratory, Almora (Indian Council of Agricultural Research), for rainfed areas. Landraces were grown in earthen pots (30 cm diameter and 30 cm high) containing farmyard manure, sand, and soil

(1:1:1, v/v) during kharif (wet season) in 1998. Three sets of plants (15 plants per set) per landrace were kept under similar environmental conditions in the Institute nursery at Kosi (1,150 m asl; geographical coordinates 79 38 10 E and 29 38 15 N). Pots were irrigated to maintain soil moisture (around 2325%) for seed germination. No watering was done after germination and plants were kept under rainfed conditions. Observations were made before the flowering stage (90 5 d after seed sowing). Optimum photosynthetic activities

during this stage have been noted (Chauhan et al 1996). The photosynthesis rate was measured in flag leaves of three uniform plants from each set, using a closed portable photosynthetic system model LI-6400 (LI-COR, USA). Measurements were carried out at 25 C and at 800 mmol m2 s1 to avoid possible fluctuations due to temperature and light variations. The chlorophyll contents (chlorophyll a & b and total) of the flag leaves of various landraces were estimated following the method of Holm (1954).

Photosynthesis, stomatal conductance, and chlorophyll content of various landraces of rice (SD). Landrace/ introduced variety Landraces Anjani Bauran Bauriya Bindudhan Chhatuli Chhotiya Dalbadal Danbasmati Dehradoonibasmati Dudhikapkoti Dutiau Jhungia Kaladur Kaunkaun Khudinandhani Kururhidhan Laldhan Nandhani Nauli Patoli Sabhawati Sailani Santoli Saunji Saurajubawan Syaudhan Taichin Tilansi Introduced variety VL 206 Net photosynthesis (mmol m2 s1) Stomatal conductance (mmol m2 s1) Chlorophyll (mg g1) a b Total

2.21 0.12 4.70 0.50 4.80 0.03 2.53 0.57 2.61 0.47 2.27 0.15 3.12 0.50 3.35 0.46 2.60 0.41 3.76 0.44 2.44 0.38 2.46 0.36 2.60 0.55 2.30 0.24 2.26 0.08 2.73 0.70 4.58 0.39 2.42 0.47 5.37 0.45 4.15 0.41 3.18 0.41 5.27 0.28 2.36 0.12 3.64 0.44 8.10 0.58 6.30 0.40 9.45 0.44 2.29 0.45 7.22 0.90

34.80 3.91 127.20 12.60 73.50 1.26 5.30 5.80 53.91 2.76 32.60 2.30 67.70 4.00 66.70 1.30 56.71 3.50 72.70 5.48 50.20 2.81 63.11 2.82 88.81 4.94 45.20 2.87 43.40 2.00 65.60 2.98 116.00 2.34 53.00 2.60 122.80 4.10 78.00 1.80 59.81 2.61 136.80 10.70 68.51 5.01 116.50 8.03 122.00 6.07 149.00 10.46 230.50 13.90 71.10 4.90 161.10 2.34

0.591 0.062 0.662 0.194 0.707 0.174 0.338 0.046 0.614 0.151 0.504 0.143 0.826 0.202 0.879 0.153 0.583 0.061 0.702 0.030 0.336 0.057 0.913 0.145 0.850 0.024 1.060 0.279 0.683 0.050 0.500 0.120 0.549 0.107 0.638 0.258 1.264 0.040 0.878 0.068 0.640 0.078 0.896 0.115 0.840 0.103 0.502 0.178 1.133 0.110 0.678 0.091 1.325 0.123 0.731 0.108 0.836 0.154

0.253 0.030 0.259 0.061 0.324 0.067 0.146 0.146 0.234 0.051 0.217 0.057 0.313 0.075 0.339 0.054 0.256 0.015 0.136 0.018 0.140 0.012 0.452 0.063 0.411 0.031 0.400 0.095 0.282 0.007 0.208 0.045 0.229 0.044 0.173 0.200 0.281 0.031 0.189 0.025 0.352 0.065 0.242 0.045 0.254 0.086 0.214 0.060 0.212 0.031 0.335 0.032 0.231 0.056 0.314 0.056 0.384 0.137

0.843 0.090 0.922 0.255 1.031 0.240 0.484 0.066 0.848 0.202 0.723 0.199 1.139 0.277 1.218 0.207 0.839 0.062 0.838 0.049 0.476 0.070 1.365 0.202 1.261 0.044 1.460 0.374 0.965 0.058 0.708 0.165 0.778 0.152 0.811 0.112 1.545 0.070 1.061 0.138 1.082 0.064 1.138 0.161 1.095 0.173 0.716 0.238 1.320 0.142 1.014 0.119 1.556 0.179 1.046 0.165 1.220 0.275

IRRN 25.2

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Digital Literacy for Rice Scientists

To help rice scientists take advantage of new information and communication technologies, the IRRI Training Center has developed the Digital Literacy Course for Rice Scientists. The course aims to provide scientists with information about what resources are available on the Internet and how they can go about accessing these resources. The course is unique in that it focuses on the needs of rice scientists, it provides a forum for rice scientists to share their experiences and Internet resources with other rice scientists online, and it establishes a learner-centered knowledge network in the form of an online community centered on rice research. The topics covered by the course include What is the Internet What is the World Wide Web and what makes it work Key Internet terminology How to use the Internet for communication with other scientists How to use Web browsers How to search for information efficiently and effectively What are some of the good sources of information for rice scientists available on the Internet How to cite Internet documents What training opportunities are available online Connection to the Internet offers national scientists a low-cost communication medium with other scientists linked to the Internet, gives them access to the ever-growing body of information available on and through interlinked computers throughout the world, and provides access to formal and informal training offered online from virtually anywhere.

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August 2000

Data on photosynthesis (see table) reveal that various landraces differed significantly in photosynthetic rates (CO2 uptake ranged from 2.21 to 9.45 mmol m2 s1) and stomatal conductance (between 34.8 and 230.5 mmol m2 s1). The leaf chlorophyll content also showed significant variation among different landraces: chlorophyll a = 0.3361.325, chlorophyll b = 0.1360.452, and total chlorophyll = 0.4761.556 mg g1 of fresh weight. Several landraces (18% of total) exhibited high photosynthetic rate (CO2 uptake >5 mmol m2 s1). Total chlorophyll content was also greater (1.3381.556 mg g 1 ) in these landraces; 32% of the landraces could be placed in an intermediate group for which CO2 uptake ranged between 3 and 5 mmol m2 s1. For this intermediate group, total chlorophyll

content ranged from 0.716 to 1.218 mg g 1. Half of the landraces showed low photosynthesis (CO2 uptake <3 mmol m 2 s 1). A similar trend was seen for stomatal conductance in the landraces. Introduced variety VL 206 exhibited physiological characteristics (photosynthesis, stomatal conductance, and chlorophyll content) similar to those of landraces with high photosynthesis. The rate of photosynthetic CO2 uptake is an important component of plant growth (Leding and Botkin 1974). Our results indicate a positive correlation of net photosynthesis with stomatal conductance (P<0.01) and chlorophyll content (P<0.02). These characteristics can thus be used for the primary selection of appropriate landraces for a particular environment. Further, these landraces can serve as donors of genes for introgressing

from landraces with high photosynthesis to those with low photosynthesis through suitable genetic approaches. Acknowledgments We acknowledge the financial assistance from the International Development Research Centre (IDRC) for the Agricultural Diversity Project. References
Chauhan JS, Singh CV, Singh RK, Chauhan VS, Tomar RK. 1996. Physiological characteristics of some rainfed upland rice cultivar. Oryza 33:103106. Holm G. 1954. Chlorophyll mutation in barley. Acta Agric. 3:457. Leding FT, Botkin DB. 1974. Photosynthetic CO2 uptake and distribution of photosynthate as related to growth of large hemlock seedling. Can. J. Bot. 47:519527.

Inheritance of aroma in four rice cultivars (Oryza sativa L.)

Y.J. Dong, E. Tsuzuki, and H. Terao, Crop Sciences Laboratory Agricultural Faculty, Miyazaki University, Miyazaki City 8892155, Japan

Behavior of parent, F1, and F2 populations with regard to aroma. Cross Parental leaf aroma ratinga +/ /+ +/ /+ +/+ +/+ +/+ +/+ Aromatic nature of F1 plants + + + + Observed F2 plants Total 120 109 144 182 81 90 24 76 Nonaromatic 90 81 109 100 0 0 0 0 Aromatic 30 28 35 82 81 90 24 76 0.011 (3:1) 0.031 (3:1) 0.009 (3:1) 0.079 (9:7) nab na na na >0.90 0.75~0.90 >0.90 0.75~0.90 na na na na 2 P

Della/Nipponbare Koshihikari/Shiroikichi Hokkaido 270/Koshihikari Koshihikari/HB1 Della/Shiroikichi Della/HB1 Della/Hokkaido 270 Shiroikichi/HB1
a

+ = aromatic, = nonaromatic. b na = chi-square test does not apply.

IRRISTAT Windows 4.0

Now available online and on CD!

IRRISTAT is a computer program for data management and basic statistical analysis of experimental data. It can be run in any 32-bit Windows operating system. IRRISTAT has been developed primarily for analyzing data from agricultural field trials, but
IRRN 25.2

many of the features can be used for analysis of data from other sources. The main modules and facilities are: Data management with a spreadsheet, Text editor, Analysis of variance, Regression, Genotype environment interaction analysis, Quantitative trait analysis, Single site analysis, Pattern analysis, Graphics, and Utilities for randomization and layout, general factorial EMS, and orthogonal polynomial. The software (including tutorial in zip and pdf files) can be downloaded from the IRRI site at http://www.cgiar.org/irri/irristat.htm

The software is also available on CD for US$19 for highly developed countries and $5 for developing countries, with $7 for handling costs. Send suggestions, comments, or problems in using the software to: Biometrics International Rice Research Institute MCPO Box 3127 Makati City 1271, Philippines or e-mail: biometrics@irri.cgiar.org To order a CD, e-mail: e.ramin@cgiar.org

15

We studied aroma inheritance in four rice cultivarsDella (USA), Shiroikichi (Japan), Hokkaido 270 (Japan), and HB1 (China). These were crossed with nonaromatic cultivars (Koshihikari, Nipponbare) and with each other. Four crosses each for aromatic/ nonaromatic and aromatic/aromatic were obtained in 1998. P1, P2, and F1 plants from all crosses were grown in pots in the greenhouse during the 1998-99 winter season; P1, P2, and F2 plants were cultivated in the field in 1999. Ten leaves were sampled from individual plants at tillering and cut into 5-mmlong pieces, put into a capped glassware, and stored at 20 C before aroma evaluation. No more than 20 samples were evaluated in a single day. In this process, 1 g

was measured from each frozen leaf sample, put into a capped test tube, and mixed with 5 mL of 1.7% KOH solution for 10 min at 50 C. Four to five panelists were asked to classify the samples as either aromatic or nonaromatic by their smell. The F 1 plants from crosses of nonaromatic/aromatic cultivars were nonaromatic, indicating that aroma in the four aromatic cultivars was recessive (see table). Segregating ratios of nonaromatic to aromatic plants in the F2 populations from the crosses of Della/Nipponbare (2 = 0.011, P>0.90), Koshihikari/Shiroikichi (2 = 0.031, P = 0.750.90), and Hokkaido 270/Koshihikari (2 = 0.009, P>0.90) were all 3:1, which indicated the inheritance of a single recessive gene for aroma in Della, Shiroikichi, and Hokkaido 270. In the F2

population from the cross Koshihikari/ HB1 (2 = 0.079, P = 0.750.90), the segregating ratio was 9:7, indicating that two recessive genes control aroma in HB1. The F1 plants from crosses between any two aromatic cultivars (Table 1) were aromatic; the F2 population did not segregate for aroma, which indicated that the parents contained at least one common aroma gene. The F2 data show that Della, Shiroikichi, and Hokkaido 270 each contains a single recessive gene and HB1 has two recessive genes for aroma. Accordingly, the aroma genes in Della, Shiroikichi, and Hokkaido 270 were allelic to each other and allelic to one of two aroma genes in HB1.

Genetic resources

Variation in RFLP markers associated with rootgrowth QTLs in Indian upland rice
K.A. Steele, B.J. Moore, J.R. Witcombe, and D.S.Virk, Centre of Arid Zone Studies, University of Wales, Bangor, LL57 2UW; and A.H. Price, Department of Plant and Soil Science, University of Aberdeen, Cruickshank Building, Aberdeen, AB24 3UU, UK E-mail: k.a.steele@bangor.ac.uk

Kalinga III, bred at the Central Rice Research Institute in Cuttack, is an improved variety popular in drought-prone areas of India. It normally escapes the end-of-season drought because of its extreme earliness, but it is drought-susceptible. Sathi 3436, an improved variety from Gujarat, India, derived from landraces, has longer and thicker roots than Kalinga III. Quantitative trait loci (QTLs) influencing root length and thickness in rice have been identified in previous studies (Price and Tomos 1996, Price et al 2000, Yadav et al 1997). Molecular markers located near QTLs may correlate with the same trait in different varieties, and these markers could be used in marker-assisted selection (MAS) to improve drought-sensitive varieties. Restriction fragment length polymorphism (RFLP) markers on chromosomes with root QTLs were used to compare genetic variation within and between Indian landraces and improved varieties. Sixteen rice varieties were screened with 55 RFLP probes (from chromosomes 2, 5, 7, 8, 9, and 11), 14 of which were linked to QTLs for root growth. Four varieties were landraces and six were released varieties derived from landraces, all widely grown in India (see table). Other varieties which have been used for mapping were included: Azucena (japonica), Bala (indica, an Aus variety), CO 39 (indica), and Moroberekan (japonica). DNA extraction and RFLP analysis were carried out as described in Price et al (2000). Five restriction enzymes were tested and polymorphic bands were scored for presence or absence in all 16 varieties. Data were analyzed by cluster analysis using SPSS (version 8). Similarity was estimated using Jaccards coefficient. Hierarchical clustering was performed on
16

the squared Euclidean distance matrix to produce a dendrogram. Twelve probes gave no polymorphisms while 43 probes gave 110 scorable polymorphic bands (30 of which have been mapped in the Bala/Azucena F6 population). Cluster analysis using all 110 polymorphic markers separated indicas from japonicas (see figure). Average linkage and single linkage gave similar results. Simi-

larity ranged from 82 4% between Azucena and Moroberekan to 194% between Azucena and Bala. The four Indian landraces and Sathi 34-36 grouped together and were separated from the improved varieties (Sathi 34-36 and Kalinga III had only 37 5% similarity). Despite being geographically diverse, the landraces were similar at many marker loci and were less diverse than improved varieties (e.g.,

Indian varieties used in molecular marker studies. Germplasm Kalinga III Desirable trait Earliness, medium-tall plants, fine grains, good cooking quality Type Released for uplands in Orissa Basis of selection Most farmer-preferred variety in participatory varietal selection in India.a,b Most widely adopted in droughtprone uplands High yield, multiple resistances High yield, multiple resistances Performance in westerna India Performance in westerna India Performance in onstation trials Performance in farmers field trials in westerna and easternb India Performance in farmers field trials in easternb India Performance in farmers field trials in easternb India Observations in farmers fields in westerna India Observations in farmers fields in westerna India Observations in farmers fields in westerna India

IR36 IR64 Sathi 34-36

High yield, stiff straw High yield, fine grain High yield, stiff straw, long roots, drought tolerance Drought resistance, delayed senescence Grain quality

Nanisal Vaghardha

Released variety for irrigated environments Released variety for irrigated environments Released variety in Gujarat (derived from a landrace) Landrace Released variety for Banswara area of Rajasthan Released for uplands in India Released for uplands in India Released in Bihar, developed by selection from a local landrace of upland rice, Brown Gora Landrace in western India Landrace in western India Landrace in western India

Vandana

Vanprabha

Birsa Dhan 102

High yield, earliness, long roots, drought resistance High yield, earliness, long roots, drought resistance Earliness, drought resistance, long roots Earliness, drought resistance Drought resistance, stiff straw, tall plants Drought resistance, earliness

Dabra Ratta Chawal Pathara

a Data from KRIBP (W): Krishak Bharati Co-operative Indo-British Rainfed Farming Project, West. bData from KRIBP(E): Krishak Bharati Co-operative Indo-British Rainfed Farming Project, East.

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0 Azucena Moroberekan Pathara Sathi 34-36 Ratta Chawal Dabra Nanisal IR36 Vanprabha Birsa Dhan 102 Vaghardha Kalinga III Vandana Bala IR64 CO 39

Rescaled combined cluster distance 10 15

20

25

Acknowledgment This document is an output from a project funded by the UK Department for Inter-

Dendrogram showing hierarchical (average linkage, between-group) clustering of varieties based on 110 RFLP bands.

Evaluation of in situ conservation of Oryza rufipogon populations using RAPD markers

Z.-W. Xie, S. Ge, K.-Q. Wang, D.-Y. Hong, Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences (CAS), Beijing 100093; and B.R. Lu, GRC, IRRI E-mail: xiezw@public.east.cn.net

Oryza rufipogon Griff. is a perennial species widely distributed in the tropics and subtropics of monsoon Asia. It constitutes a valuable genetic resource for rice improvement (Chang 1984). This species once occurred in eight provinces and autonomous regions of China but it is now extinct in Fujian and is nearly extinct in Hunan and Jiangxi. An intensive biodiversity conservation program for O. rufipogon has recently been initiated in China, using the in situ approach, which has been regarded as an effective method
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to maintain diversity of wild rice species in their original habitats (Vaughan and Chang 1992). Knowledge on genetic diversity, population structure, and dynamics of the target O. rufipogon populations is important for strategic design and effective management and conservation. Such information, however, is still very limited. This study aimed to estimate the genetic diversity of O. rufipogon populations and to confirm the in situ conservation sites for this species using random amplified polymorphic DNA (RAPD) markers.

Six O. rufipogon populations from in situ conservation sites in China were investigated through field surveys during 1997-98. These populations were classified into four groups according to their origins (Table 1). Young and clean leaves of 15 18 individuals were sampled from different populations (Table 1). The method for sample collection, leaf preservation, and DNA extraction followed those described by Xie et al (1999). To prevent overestimating the genetic diversity by polymorphic loci variation, 20 primers (OPB 120
17

Dabra and Pathara shared 73 4% similarity). Cluster analysis using only the 14 mapped markers linked to QTLs for root traits gave identical first- and second-level groups, as did analysis using only 16 mapped markers not linked to root QTLs. A further 34 markers, covering all 12 chromosomes, were screened on the two released varieties, Kalinga III and Sathi 3436; 66% of them were polymorphic. In the mapping population derived from Bala/Azucena, Azucena was the donor of positive alleles for four QTLs, while Bala was the donor for one QTL on chromosome 5. Kalinga III had the Bala-type locus for nine markers at QTLs on chromosomes 2, 7, 9, and 11. Because most Indian landraces had the Azucena locus in up to five of these markers, they could be used as donor parents for MAS to improve Kalinga III for root growth. Dabra, Ratta Chawal, and Sathi 34-36 had the Azucena allele at markers C601 (chromosome 2), RG650 (chromosome 7), and RG2 (chromosome 11).

national Development (DfID) (project No. R7080, Plant Sciences Programme) for the benefit of developing countries. The views expressed are not necessarily those of DfID. References
Price AH, Steele KA, Moore BJ, Barraclough PB, Clarke LJ. 2000. A combined RFLP and AFLP linkage map of upland rice (Oryza sativa L.)

used to identify QTLs for root penetration ability. Theor. Appl. Genet. (in press) Price AH, Tomos AD. 1996. Genetic dissection of root growth in rice (Oryza sativa L.). II: Mapping quantitative trait loci using molecular markers. Theor. Appl. Genet. 95:143152. Yadav R, Courtois B, Huang N, McLaren G. 1997. Mapping genes controlling root morphology and root distribution on a double-haploid population of rice. Theor. Appl. Genet. 96:619632.

of kit B of Operon Technologies Inc.) were randomly chosen to analyze leaf samples, regardless of the polymorphism of amplified fragments. Only one primer (OPB-06) amplified unclear bands and was excluded from the experiment. The other 19 primers produced strong and reproducible fragments and thus were used for further analysis. RAPD fragments were scored as present (1) or absent (0) for each DNA sample to estimate genetic distance between individuals. Genetic diversity was measured by the percent polymorphic bands (PPB) at population and region levels. An analysis of molecular variation (AMOVA) was used to partition variance components among individuals of a population, among populations within a region, and among regions. All the analyses were

performed with the RAPDistance program (version 1.04) and WINAMOVA program (version 1.5). An unweighted paired group method for cluster analysis (UPGMA) using the genetic distance estimates was also used. A total of 241 fragments ranging from 190 to 2,800 bp in size were scored in 100 individuals of the six populations studied. Each individual had a unique haplotype. PPBs for each population and region are shown in Table 2. AMOVA results showed highly significant genetic differentiation among regions and among populations of the same regions (Table 3). Considerable variation was detected within each of the six populations. The 100 individuals were clustered into six groups that corresponded to the six populations in-

Table 1. Populations of Oryza rufipogon used in this study. Group code GP1 GP2 GP3 GP4 Population code PO2 PO3 PO5 P19 P59 P47 Locality Dongxiang, Jiangxi Province Dongxiang, Jiangxi Province Yangtianqiao, Guangdong Province Hanguang, Guangdong Province Heping, Guangxi Province Sanya, Hainan Province Individuals sampled (no.) 15 15 18 18 16 18

cluded in this study. The major portion of the total genetic variation was identified within rather than between populations. These data can help in designing more strategic in situ conservation programs for O. rufipogon in China. China established in situ conservation sites of O. rifupogon in Dongxiang county (populations PO2 and PO3) in Jiangxi Province in 1992. The Dongxiang populations are the northernmost populations in the entire distribution of this species. The two populations are located about 5 km from each other and have special characteristics such as cold tolerance. Five population-specific RAPD bands were identified in each population. The two populations also showed some differentiation based on cluster analysis of RAPD bands. The levels of genetic diversity of the two populations were moderately high among 29 Chinese O. rufipogon populations analyzed by Xie (1999). Therefore, it is necessary to independently conserve the two populations in situ. Population PO5, located in Yangtianqiao of Guangdong Province, is particularly valuable. Individuals from this population have been extensively used for

Table 2. Number and percentage of polymorphic bands detected in each population and region.a Primer OPB-01 OPB-02 OPB-03 OPB-04 OPB-05 OPB-07 OPB-08 OPB-09 OPB-10 OPB-11 OPB-12 OPB-13 OPB-14 OPB-15 OPB-16 OPB-17 OPB-18 OPB-19 OPB-20 Total PPB
a

Bands (no.) PO2 14 12 15 15 15 12 14 10 10 13 12 13 8 12 6 18 12 14 16 241 3 3 6 4 2 4 4 2 5 4 5 4 0 4 1 3 5 5 4 72 29.88 PO3 6 3 5 5 3 5 4 2 4 7 3 3 2 5 1 6 5 4 5 78 32.37 GP1 6 3 7 6 3 5 6 3 6 8 6 5 2 6 1 7 5 7 6 98 41.08 PO5 5 6 9 3 1 3 5 3 5 7 6 5 2 5 2 9 4 6 5 91 37.76

Polymorphic bands (no.) P19 8 7 7 4 3 4 7 3 4 6 6 6 3 7 2 9 4 4 6 100 41.49 GP2 8 8 11 5 4 6 9 4 5 7 8 9 4 9 3 12 5 8 7 132 54.77 P59 2 6 3 5 3 5 4 4 4 5 2 3 1 3 3 6 5 2 2 68 28.22 GP3 2 6 3 5 3 5 4 4 4 5 2 3 1 3 3 6 5 2 2 68 28.22 P47 5 9 5 4 8 6 7 4 5 5 4 5 3 5 3 6 4 5 9 102 42.32 GP4 5 9 5 4 8 6 7 4 5 5 4 5 3 5 3 6 4 5 9 102 42.32 Total 9 12 12 13 12 10 13 9 9 12 8 10 7 10 6 15 10 11 11 200 82.99

Population codes = PO2, PO3, PO5, P19, P59, and P47; group codes = GP1, GP2, GP3, and GP4. PPB = percent polymorphic bands.

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Table 3. Analysis of molecular variance (AMOVA) for 100 individuals of Oryza rufipogon from four different regions. Source of variation Global Between regions Populations/region Individuals/population Within regions Between populations Within populations Among regions Among regions Within regions df SSD MSD Variance component 0.024 0.063 0.094 0.084 0.094 0.067 0.115 % total variance 13.40 34.73 51.87 47.20 52.80 36.73 63.27 P value Bartletts statistic 2.638 2.608

3 2 94 5 94 3 96

5.175 2.256 8.799 7.431 8.799 5.175 11.054

1.725 1.128 0.094 1.486 0.094 1.725 0.115

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

SSD = sum of squared deviation, MSD = mean square deviation.

Identification of genomic constitution of three tetraploid Oryza species through two-probe genomic in situ hybridization
C.B. Li, D.M. Zhang, S. Ge, and D.Y. Hong, Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China; and B.R. Lu, GRC, IRRI E-mail: licb@public2.east.net.cn

Two tetraploid Oryza species, O. minuta and O. punctata (with diploid and tetraploid types), are reported to contain the BBCC genomes based on the analysis of meiotic chromosome pairing at metaphase I of interspecific F1 hybrids. O. officinalis and O. eichingeri have diploid and tetraploid forms. The genomic constitution of
IRRN 25.2

tetraploid forms is still unclear. The genomic constitution of O. malampuzhaensis (treated as a synonym of O. officinalis by Vaughan 1989) was tentatively designated as BBCC, but no sound cytological data have been provided to support this designation.

Genome analysis based on observation of chromosome pairing has been largely challenged because chromosome pairing is known to be influenced by several factors including environmental and genetic ones, which have led to inaccurate measures of genomic affinities in some
19

rice breeding since 1980. Some high-quality and high-yielding new rice varieties have been produced using O. rufipogon from this population as a parent in crossing programs. Considering its high genetic diversity as revealed by RAPD analysis (PPB = 38.15), this population needs to be conserved in situ. Population P19, found in Hanguang, Guangdong Province, is one of the northern marginal populations in China. It has a much higher genetic diversity (PPB = 40.16%) than many of the 29 other populations studied by Xie (1999) and therefore is also a key population that needs to be conserved in situ. One of the southernmost populations of O. rufipogon in China is population P47, located in Sanya of Hainan Prov-

ince. It occurs in a large pond near the sea and grows in brackish water. Because of the importance of maintaining some populations with special characteristics such as salinity tolerance, this population should be conserved in situ. In addition, the genetic diversity (PPB = 46.18%) of this population is the highest among the 29 populations studied. Population P59 occurs on the upper reaches of a river in Heping, Guangxi Province, and is isolated from other O. rufipogon populations. RAPD analysis and previous analysis of data from 29 other populations showed that this population has a relatively low genetic diversity (PPB = 25.70) and no special characters. Therefore, this population could be maintained under ex situ conservation.

References
Chang TT. 1984. Conservation of rice genetic resources: luxury or necessity? Science 224:251256. Vaughan DA, Chang TT. 1992. In situ conservation of rice genetic resources. Econ. Bot. 46:368383. Xie ZW. 1999. Population genetics and conservation strategy of natural populations of Oryza rufipogon Griff. in China. PhD dissertation, Institute of Botany, Chinese Academy of Sciences, Beijing. p 3657. Xie ZW, Ge S, Hong DY. 1999. Preparation of DNA from silica gel dried mini-amount of leaves of Oryza rufipogon Griff. for RAPD study and total DNA bank construction. Acta Bot. Sin. 41:884890.

cases. It is therefore necessary to adopt more reliable and powerful methods to detect and confirm the genomic constitution of these tetraploid wild rice species. Total genomic in situ hybridization (GISH) uses total genomic DNA of an analyzer (genomically known species) as a probe to detect chromosomal homology of the analyzer with a genome of an unknown target species. It provides a more direct approach for genomic studies at the chromosome and DNA levels. This study aimed to determine the genomic constitution of the tetraploid O. malampuzhaensis (IRGC 80764), O. minuta, and O. punctata by two-probe GISH technology using total genomic DNA of three diploid wild rice speciesO. eichingeri, O. officinalis, and O. punctataas probes. Six wild rice species with different ploidy levels and origins were used in this study (Table 1). Diploid species were used as analyzers, from which genomic probes were prepared, with the tetraploid species as target species. Somatic chromosomes of target species were prepared by enzymatic maceration and an air-dry method as described by Fukui and Iijima (1991) and Fukui et al (1994) with modifications. A precooled wet slide covered with polylysine was placed on ice; then a macerated root tip was placed on it with a drop of cold fixative (acetic acid: absolute ethanol = 1:3) at 0 C and smeared with sharp tweezers. The in situ hybridization and probe detection methods followed those of Leitch et al (1994). Chromosome samples were treated in turn with 100 mg mL1 Dnase-free Rnase, 1 mg mL1 proteinase K, 20% acetamide, and 4% (w/v) paraformaldehyde; then dehydrated through a graded ethanol series (70%, 90%, and 100%); and air-dried. Total DNA from the C- and B-genome species was labeled by nick translation with bio-14-dATP (GIBCO BRL Cat. No. 19524-016) and DIG11-dUTP (Boehringer Mannheim, 1093088), respectively. The hybridization mixture consisted of 50% deionized formamide, 2 SSC, 10% (w/v) dextran sulfate, 0.1% (w/v) SDS, 0.25 mg mL1 sheared salmon sperm DNA (about 100
20

bp), and the probes (35 ng mL1 for each probe). Table 2 shows the probes in the hybridization mixture and the stringency at which posthybridization washes were carried out. Detection of the biotin-labeled probe was achieved by using avidin-FITC and of the digoxigenin-labeled probe by using anti-digoxigenin rhodamine conjugate. Results of the two-probe GISH were observed with a fluorescence microscope and photographs were taken with Kodak Ektachrome 400 film. Results clearly showed that the tetraploid O. malampuzhaensis (IRGC 80764) was an allotetraploid and its genomic constitution was BBCC (Figs. 1a & 1b). This taxon was collected from India and provided by the International Rice Genebank (IRG). We carefully examined the specimen of IRGC 80764 deposited at IRRI and found that O. malampuzhaensis was different from O. officinalis in many characters, particularly panicle characters. Results also confirmed that the genomic constitutions of O. punctata and O. minuta were BBCC (Figs. 2a,b & 3a,b). The

identification of the BBCC genomes in the latter two species further confirmed the reliability of the genomic determination of O. malampuzhaensis as BBCC. Although containing the same BBCC genomes, these species showed considerable differences in their karyotypes. One pair of satellite chromosomes belonging to the BB genome was clearly identified in the tetraploid O. malampuzhaensis (Figs. 1a & 1b), but no such satellite chromosomes were found in either O. minuta or O. punctata. Eight visible hybridization sites, which showed co-hybridization signals on both BB and CC genomes, were located on the chromosomes of the BB genome of the tetraploid O. punctata (Fig. 3a). The average chromosome length of the CC genome is larger than that of the BB genome in O. minuta (Figs. 2a & 2b) but not in O. punctata (Figs. 3a & 3b) and the tetraploid O. malampuzhaensis (Figs. 1a & 1b). The ratio of the total chromosome length between the BB and CC genomes was determined as 1:1.5 in O. minuta, 1:0.9 in O.

Table 1.Wild rice species and accessions used in the study with their ploidy level and origin.a Species O. officinalis O. eichingeri O. punctata O. punctata O. minuta O. malampuzhaensis
a b

Accession number Zhou-198 IRGC 101422b IRGC 103896 IRGC 105137 IRGC 101082 IRGC 80764

2n 24 24 24 48 48 48

Genomic constitution CC CC BB BBCC BBCC Unknown

Country China Uganda Tanzania Zaire Philippines India

All living materials are maintained in the greenhouse of the Beijing Institute of Botany, Chinese Academy of Sciences. IRGC = International Rice Genebank Collection stored at IRRI.

Table 2. Probes in the hybridization mixture and the posthybridization wash stringency. Species O. minuta (BBCC) Probe DIG-CCb (O. eichingeri) BIO-BBc (O. punctata) DIG-CC (O. eichingeri) BIO-BB (O. punctata) DIG-BB (O. punctata) BIO-CC (O. officinalis) Stringent washes 50% formamide in 0.1 SSC at 42 C for 10 min 20% formamide in 0.1 SSC at 42 C for 10 min 20% formamide in 2 SSC at 42 C for 10 min Stringency (%)a 99

O. punctata (BBCC)

8287

O. malampuzhaensis (4x)

6063

Calculated by using the equation described by Meinkoth and Wahl (1984). bDIG-CC = total DNA of CC genome labeled with digoxigenin. cBIO-BB = total DNA of BB genome labeled with biotin.

August 2000

Figs. 1-3. Metaphase of Oryza malampuzhaensis, O. minuta, and O. punctata after in situ hybridization with the B- and C-genome species and counterstained with DAPI (blue). Figures 1a and 1b show results of O. malampuzhaensis probed with labeled B genome (diploid O. punctata) and C genome (O. officinalis) DNA. The 24 chromosomes with strong violet signals belong to the BB genome (Fig. 1a) and the other 24 with strong green-blue signals are the C-genome chromosomes (Fig. 1b). Arrows indicate a pair of satellite chromosomes. Figures 2a and 2b show a metaphase of O. minuta hybridized by the B- and C-genome DNA. The chromosomes with green-blue signal belong to the BB genome (Fig. 2a) and the other chromosomes showing violet color belong to the CC genome (Fig. 2b). Figures 3a and 3b show results of tetraploid O. punctata probed with B- and C-genome (O. eichingeri) DNA. The 24 chromosomes with violet color belong to the CC genome (Fig. 3a) and the other 24 chromosomes with green-blue color belong to the BB genome (Fig. 3b). The arrows indicate hybridization signals of the C-genome probe on the B-genome chromosomes (Fig. 3a). Scale bar = 5 mm.

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Collection and evaluation of hill germplasm from Karbi Anglong and North Cachar Hill districts of Assam, India
A. Roy and K. Das, Regional Agricultural Research Station (RARS), Assam Agricultural University, Diphu 782460, Assam, India

In Karbi Anglong and North Cachar Hill districts of Assam, India, local tribes grow several hill rice cultivars on the hilly slopes as jhum (shifting cultivation practiced through slash-and-burn method) and on terraces. These cultivars are highly adaptable to local agroecological conditions and are grown at the onset of monsoon (AprilMay) with minimum or even zero-tillage operation as a direct-seeded rainfed crop. They require less inputs and management practices and possess enormous variability for various quantitative and qualitative traits. Rice is mostly grown as a mixed crop with various crops such as sesame, maize,

cotton, colocasia, chili, ginger, turmeric, and others in this region. These local cultivars, however, face extinction due to recent trends in agricultural development. Hence, the collection and evaluation of these varieties for various quantitative and qualitative traits are important for breeding and conservation programs. This study reports on the collection and evaluation of hill rice germplasm from Assam. Seventy-six rice hill cultivars were collected from 36 sampling sites in the two districts. The farmers field was considered as a unit area. Random samples from populations and biased samples of elite materi-

als were collected. Germplasm samples were also collected from threshing yards and farm stores (Pandravada et al 1996). Each sample was given an accession number; source and date of collection were also recorded. The collected germplasm was grown in a randomized block design with three replications during summer 1998 at RARS in Assam (25 50 N latitude, 90 30 E longitude, and an altitude of 180 m asl). Cultivars were evaluated for 13 yield and yield-attributing traits (see table). A wide range of mean values depicted sufficient variability among cultivars for all the traits.

Variability parameters of hill rice cultivars from Karbi Anglong and North Cachar Hill districts, Assam, India.a Character Days to 50% flowering Days to maturity Plant height (cm) Tillers plant1 (no.) Leaf length (cm) Flag leaf length (cm) Leaf breadth (cm) Flag leaf breadth (cm) Panicle length (cm) Spikelets panicle1 (no.) Grains panicle1 (no.) 100-grain weight (g) Yield plant1 (g)
a

Range 81.0 145.0 113.5 162.5 88.8 161.8 3.4 12.5 40.6 73.7 21.8 49.2 1.2 2.2 1.0 1.9 19.7 31.8 81.8 293.0 51.5 243.2 1.2 3.0 5.0 35.4

Mean SE 101.36 1.46 126.86 1.35 127.62 1.69 8.45 0.26 57.54 0.96 34.30 0.64 1.66 0.03 1.42 0.03 25.60 0.30 181.77 5.24 131.47 4.96 1.84 0.04 15.26 0.92

GCV (%) 12.5 9.2 11.3 26.6 13.9 15.9 13.5 14.5 9.7 24.3 32.1 20.6 52.5

PCV (%) 12.7 9.3 12.0 28.5 14.8 17.0 15.9 16.4 10.6 26.7 34.5 20.8 53.2

Heritability (%) 95.8 98.3 89.2 87.2 87.2 87.1 71.4 77.8 84.1 83.0 86.5 98.4 97.4

GA (%) of mean 25.1 18.9 22.0 51.2 26.7 30.5 23.4 26.3 18.3 45.6 61.5 42.0 106.8

SE = standard error, GCV = genotypic coefficient of variation, PCV = phenotypic coefficient of variation, and GA = genetic advance.

22

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punctata, and 1:1 in O. malampuzhaensis. These ratios strongly suggest differences in BBCC genomes in the three rice species. Possible causes for differences in genomes might be the different sources/origins of the BB and CC genomes for the different species. It is also possible that chromosome differentiation within species occurred after the formation of these species. The former is more likely the mechanism that causes differences in BBCC genomes in the three tetraploid wild rice species.

This study also shows that the twoprobe GISH can give a high resolution in identifying chromosomes of different genomes of an allopolyploid species on the same chromosome spread. The method has a great potential to be used for further genomic studies in the genus Oryza. References
Fukui K, Iijima K. 1991. Somatic chromosome map of rice by imaging methods. Theor. Appl. Genet. 81:589596.

Fukui K, Ohmido N, Khush GS. 1994. Variability in rDNA loci in genus Oryza detected through fluorescence in situ hybridization. Theor. Appl. Genet. 87:893899. Leitch AR, Schwarzacher T, Jackson D, Leitch IJ. 1994. Hybridization, a practical guide. UK: BIOs Scientific Publishers Limited. Meinkoth J, Wahl G. 1984. Hybridization of nucleic acids immobilized on solid support. Anal. Biochem. 138:267284. Vaughan DA. 1989. The genus Oryza L. Current status of taxonomy. IRRI Res. Pap. Ser. 138.

Badan Pusat Statistik (Indonesian Central Bureau of Statistics) http://www.bps.go.id

This is the Web site for the Indonesian Central Bureau of Statistics (Badan Pusat Statistik, BPS, in Indonesian). It presents statistical information at both national and provincial levels organized into different subjects, including population, employment, wages, agriculture, foreign trade, consumer and wholesale price indices, consumption expenditures, and many other topics. For example, one can find data on rice production for the past several years. The statistics are accompanied by explanations of concepts, definitions, methodologies, publications, and tables. More detailed information at the provincial level can be obtained through links to the homepages of individual provinces. Other interesting sites to explore that are linked to the BPS Web page include those of Indonesian government agencies, international organizations, statistical agencies in other countries, and other members of the statistics community within Indonesia. A search engine is provided to make browsing the BPS site easier. There is also a page devoted to explaining how BPS was established, the functions of the institution, and how it undertakes data collection, storage, and management.

High Wire Press http://highwire.stanford.edu

High Wire Press, the Internet imprint of the Stanford University libraries, offers free access to full text articles from dozens of scientific journals. Journals with free access that are of interest to rice scientists include Proceedings of the National Academy of Sciences of the USA, Plant Cell, Plant Physiology, and Microbiology and Molecular Biology Reviews. Although current issues of most journals are not available for free, many journals offer free access to back issues 1 month or 1 year after publication. The site also provides information on obtaining faster Web access for users outside of the USA and Canada.

IRRN 25.2

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A high genotypic coefficient of variation (GCV) and phenotypic coefficient of variation (PCV) were recorded for grain yield per plant (52.2% and 53.2%, respectively), indicating maximum variability among genotypes for this trait. Moderate GCV and PCV values (20% and 50%) were recorded for grains per panicle, tillers per plant, and 100-grain weight. The remaining traits showed relatively low GCV and PCV estimates. Heritability estimates were high for all traits studied, ranging from 71.4% in leaf breadth to 98.4% in 100-grain weight. High estimates of heritability revealed reliability for phenotypic selection for all traits. Genetic advance (GA) as a percentage of mean was high for yield per plant

(106.8%), grains per panicle (61.5%), tillers per plant (51.2%), and spikelets per panicle (45.6%). The remaining traits exhibited relatively low GA. High heritability coupled with high GA was observed in yield per plant, grains per panicle, tillers per plant, and spikelets per panicle. This indicated the predominance of additive gene effects in the inheritance of these traits and revealed relatively high feasibility for phenotypic selection. Similar observations were also reported (Kihupi and Dote 1989, Deb Choudhury and Das 1998). In this work, sufficient variability was observed among cultivars for all traits studied. Hence, these cultivars may serve as an important source of genetic variability for

developing suitable rice varieties for the region. Moreover, these cultivars are likely to possess drought resistance/tolerance and can be used for future breeding programs. References
Deb Choudhary PK, Das PK. 1998.Genetic variability, correlation and path coefficient analysis in deepwater rice. Ann. Agric. Res. 19(2):120124. Kihupi AN, Dote AL. 1989. Genotypic and environmental variability in selected rice characters. Oryza 26(2):129134. Pandravada SR, Sivraj N, Murthy KRK. 1996. Collection of rice germplasm from Adilabad District, Andhra Pradesh, India. Indian J. Plant Genet. Resour. 9(2):255259.

Pest science & management

Improvement of conjugation methods for Xanthomonas oryzae pv. oryzae strain DY89031 to identify avrXa21 clones
P.K. Sharma, Department of Microbiology, CCS Haryana Agricultural University, Hisar, India 125004; F.G. da Silva, Y. Shen, and P.C. Ronald, Department of Plant Pathology, University of CaliforniaDavis, CA 95616, USA E-mail: faculty@hau.hry.nic.in

Conjugation frequency of Xanthomonas oryzae pv. oryzae strain DY89031 with pUFRO27 library clones. Conjugation frequency Insert size (kb) Mating typea Modified method Previous methodb Vector pUFRO27 (No insert) 0.4 2.6 5.6 7.2 10.6 12.0 Vector pUFRO27 (No insert) 2.7 5.9 12.0
a

BP BP BP BP BP BP BP TP TP TP TP

1.80 106 1.65 106 1.08 106 1.02 106 1.05 106 1.06 106 0.96 106 1.04 106 1.50 107 1.20 107 1.10 107

2.5 108 1.3 108 1.9 108 1.0 1010 c 1.2 108 >1010

BP = biparental mating, TP = triparental mating. bChoi and Leach (1994). c = no conjugation.

24

August 2000

Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, causes bacterial blight, a destructive disease of rice. Xoo strain PXO99A (race 6), an avirulent strain on plants carrying the Xa21 gene, is hypothesized to carry the avrXa21 gene. Attempts to identify avrXa21 by transferring genomic library clones (plasmid or cosmid) of Xoo strain PXO99A to Xoo strain DY89031 (virulent on plants carrying the Xa21 gene) were not successful because strain DY89031 was not a good recipient strain. The conjugation methods developed for Xoo strain PXO99A failed to transfer cosmid pHM1 and plasmid pUFRO27 clones to Xoo strain DY89031, thus delaying identification of avrXa21. In this report, conjugation methods described by Choi and Leach (1994) and Simon et al (1989) were modified to give a high frequency of conjugation in Xoo strain DY89031. Xoo strain DY89031 was streaked on peptone sucrose agar (PSA) plates that were incubated at 30 C for 34 d. Cells were scraped from plates and resuspended in 100 mL of nutrient broth (NB) to give O.D.600 = 0.250.3. Cells were incubated for 56 h on a rotary shaker (150 rpm) at 30 C to O.D.600 = 0.50.6. Donors Escherichia coli S17-1 (pUFRO27) for biparental mating and E. coli DH10B (pUFRO27) for triparental mating containing different-size inserts were grown in NB at 37 C for 4 h to give O.D.600 = 0.5. Helper E. coli DH10B (pRK2013) was grown in NB at 37 C for 4 h to O.D.600 = 0.5. For biparental matings, 1.5 mL of Xoo strain DY89031 was mixed with 0.1 mL of donor, while for triparental matings, 1.35 mL of Xoo strain DY89031 was mixed with 0.15 mL of helper and 0.1 mL of donor in an eppendorf tube. The mating mixture was centrifuged for 2 min at 8,000 rpm. The pellet was resuspended in 40 mL of sterilized distilled water, spot-

ted on well-dried PSA plates, and allowed to dry in the laminar flow chamber. After incubation at 30 C for 48 h, the mating mixture spots were resuspended in 0.2 mL sterilized distilled water and were spread on PSA plates containing kanamycin (25 mg mL1) and cephalexin (20 mg mL1). Transconjugants appearing after 72 h were purified on the same medium and stored in 10% glycerol in microtiter plates for inoculation experiments. Conjugation frequency was improved 1,000-fold by manipulating growth conditions, growth stage of the recipient, and proportion of donor, recipient, and helper in the mating mixture (see table). Using this modified protocol, 8095% of the clones of the pUFRO27 library were able to undergo conjugation in contrast to published protocols with a 15% success rate. Thus, using the modified protocol, 400500 matings can be performed in 1 wk and 6,000 clones (with average insert size between 6 and 8 kb) can be transferred in 1215 wk. Recipient cells in the early exponential phase were a better candidate for conjugation compared with cells in the late exponential phase. Dominance of Xoo

(O.D.600 > 0.81.0) in the mating mixture inhibited the conjugation. A minimum threshold number of E. coli (O.D.600 = 0.3 0.4) was required for efficient conjugation. A regulatory mechanism such as quorum sensing appears to be involved in XooE.coli conjugations. Such a phenomenon has been described for conjugation in Agrobacterium tumefaciens (Piper et al 1993). Finally, the modification of conjugation protocols for Xoo strain DY89031 and the use of a plasmid library with small inserts (912 kb) have facilitated the identification of clones carrying the avrXa21 gene. References
Choi SH, Leach JE. 1994. Genetic manipulations of Xanthomonas oryzae pv. oryzae. Int. Rice Res. Newsl. 19:131132. Piper KR, Beck von Bodman S, Farrand SK. 1993. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362:448450. Simon R, Quandt J, Klipp W. 1989. New derivatives for transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram negative bacteria. Gene 80:161169.

Soil, nutrient, & water management

Part 2. All notes published under this section constitute part of the outputs of the second CREMNET (Crop and Resource Management Network) India Workshop-cum-Group Meeting held at the Soil and Water Management Research Institute in Thanjavur, India, on 2427 August 1999. The workshop was organized by the Directorate of Rice Research (DRR), India, and CREMNET-IRRI with the theme Innovative nitrogen and other crop management techniques for intensive rice systems of South Asia.

Comparing management techniques to optimize fertilizer N application in rice in the Cauvery Delta of Tamil Nadu, India
P. Stalin, T.M. Thiyagarajan, Department of Soil Science and Agricultural Chemistry, Tamil Nadu Agricultural University, Coimbatore; S. Ramanathan and M. Subramanian, Tamil Nadu Rice Research Institute (TNRRI), Aduthurai, India

With the present levels of fertilizer N use efficiency (NUE) (<40%) in rice, it is estimated that an increase of about 300% in N use will be required to achieve an average yield of 8 t ha1 by 2025 (Cassman and Pingali 1995). Many fertilizer application methods and fertilizer types can help optimize N use in rice. In this paper, the following techniques and fertilizer types were evaluated on an Udorthentic Chromustert soil at TNRRI in the Cauvery Delta, during the 1997 and 1998 dry seasons (DS): the model Manage-N to optimize dose and timing of N application (Thiyagarajan et al 1994), the chlorophyll or SPAD meter to dose topdressing of N (Peng et al 1996, Balasubramanian et al 1999), controlledrelease fertilizers (Shoji and Gandeza 1992), and a soil test crop response-based N application (STCR-N) developed in India. In 1997, an on-station trial was conducted to compare seven N treatments: (1) zero-N control; (2) STCR-N for a yield target of 8 t ha1 (167 kg N ha1 applied in four equal splits: basal, 15, 30, and 45 d after transplanting, DAT); (3) Manage-N (168 kg N ha1 applied in four equal splits at 13 DAT, 23 DAT, panicle initiation, and flowering); (4) SPAD-guided N application without basal application; (5) same as 4 but with 20 kg N ha1 basal; (6) resin-coated controlled-release urea (CRU) with 4.5% coating at 60% of STCR-N recommendation applied as a single dose at the last puddling; and (7) same as 6 but with CRU with 6% coating. In all but the CRU treatments, the fertilizer N used was prilled urea. The
IRRN 25.2

model Manage-N was calibrated on data from 1996 DS experiments to generate fertilizer N recommendations. In the SPADN treatment, SPAD readings were taken weekly from 14 DAT until flowering. Nitrogen was applied when SPAD readings dropped below 35 (Peng et al 1996, Balasubramanian et al 1999). In 1998, both on-station and on-farm verification trials using the same N treatments as in 1997 except for treatments 1 (zero N) and 5 (SPAD with basal N application) were conducted (four locations). Instead of the control zero-N treatment, a treatment using the current farmers practice was used. On the research farm, all treatments were replicated three times in a randomized complete block design. Each farm formed a replication in on-farm trials. In both 1997 and 1998, irrigated transplanted rice cv

ADT36 was used. Nitrogen use efficiency was expressed as agronomic N efficiency (AEN) and as partial factor productivity for applied N (PFP-N). Results in 1997 showed that the SPAD-guided treatment had a significantly lower grain yield than the Manage-N and STCR-N methods (Table 1). This may be due to the relatively low amount of N applied (75 kg N ha1), suggesting that the SPAD threshold value for ADT36 needs to be increased to maximize grain yield and NUE. There was no substantial increase in yield with an additional basal application of 20 kg N ha1. With CRU application, grain yields obtained were on a par with the SPAD treatment but were significantly lower than yields under the Manage-N and STCR-N methods (Table 1). The high single dose of CRU-N (100 kg N ha1) as basal ap-

Table 1.Total N applied, N uptake, grain yield, and N use efficiency of irrigated transplanted rice (cv ADT36) under various N sources and N management practices in an on-station trial at TNRRI, Aduthurai, Tamil Nadu, India, 1997 dry season.a N treatment Control STCR-N for 8 t ha1 yield targetd Manage-Ne SPAD-35 N SPAD-35 N + 20 kg N ha1 basal CRU 4.5%f CRU 6.0%f CDg (.05)
a

Total N applied (kg ha1) 0.0 167.2 168.0 75.0 95.0 100.3 100.3

Total N uptake (kg ha1) 60.3 d 124.2 b 144.7 a 114.3 c 109.1 c 106.0 c 109.9 c

Grain yield (t ha1) 3.5 c 5.7 a 5.9 a 5.0 b 5.1 b 4.9 b 5.0 b 9.6

AENb 13.1 14.3 20.3 16.5 14.2 15.6 0.34

PFP-Nc 34.0 35.1 66.8 53.3 49.0 50.3

Means followed by the same letter are not significantly different at LSD (5% level). bAEN = additional grain yield over control per kg N applied. cPFP-N = total grain yield divided by total N applied. dApplied in four equal splits: basal, 15, 30, and 45 d after transplanting (DAT). eApplied in four equal splits at 13 DAT, 23 DAT, panicle initiation, and flowering. fApplied as a single dose at last puddling. gCD = critical difference, STCR = soil test crop response, CRU = controlled-release urea.

25

plication resulted in luxurious vegetative growth, which led to higher pest incidence and lower yields. This suggested that the N rate for STCR is high and that using 60% of it as a single basal application of CRU may be too high for the soil of our experiment. In the STCR-N and Manage-N treatments, the target yield of 8 t ha1 was not reached, which indicates the need to refine these techniques further to achieve target yields with high NUE. The NUE values (AEN and PFP-N) were highest for the SPAD method, which could be attributed to improved timing of N application. Although the SPAD method did not have the highest yield, it had the highest NUE because of less N application (75 kg ha1). SPAD-guided N management may be an attractive option for resourcepoor farmers. NUE values were low in the STCR-N and Manage-N methods in spite of high yields. It is therefore important to further improve the STCR-N and ManageN methods to achieve higher NUE values consistent with high yields. N uptake was highest in the Manage-N method, probably due to better timing of split N applications based on the model. The STCR-N method gave the second highest N uptake. All other N treatments gave similar N uptake values (106114 kg N ha1), which were significantly superior to that of the control. Results of the 1998 on-station trial showed that the Manage-N method produced the highest yield (Table 2). All other treatments produced the same yields, which were significantly higher than the yield of the local recommendation (125 kg N ha1). As in 1997, the target yield of 8 t ha1 was not reached in the STCR-N and Manage-N treatments. In on-farm trials,

Table 2. Total N, applied N, and grain yield of irrigated transplanted rice (cv ADT36) under various N sources and N management practices in on-station and on-farm trials at TNRRI, Aduthurai,Tamil Nadu, India, 1998 dry season.a On-station trial N treatment Total N applied (kg ha1) Local N recommendation (farmers practice) STCR-N for 8 t ha1 yield targetb Manage-Nc SPAD-35 N CRU 4.5%d CRU 6.0%d CDe (.05) 125.0 163.1 168.0 105.0 97.9 97.9 Grain yield (t ha1) 4.3 c 4.6 b 5.2 a 4.7 b 4.6 b 4.7 b 0.22 Total N applied (kg ha1) 121.4 124.4 125.0 101.3 74.6 74.6 Grain yield (t ha1) 5.5 5.7 6.3 5.4 6.1 6.4 On-farm trial (mean of 4 farms)

a Means followed by the same letter are not significantly different at LSD (5% level). bApplied in four equal splits: basal, 15, 30, and 45 d after transplanting (DAT). cApplied in four equal splits at 13 DAT, 23 DAT, panicle initiation, and flowering. d Applied as a single dose at last puddling. eCD = critical difference.

mean grain yield with the Manage-N treatment was similar to that with 75 kg N ha1 as CRU 6%. The CRU 6% treatment provided the N needs of the crop throughout its growth period, probably similar to that provided by four equal split applications of N in the Manage-N treatment. On the other hand, the SPAD treatment produced lower yields than the other treatments, suggesting a need to adjust the SPAD threshold to a higher value to optimize rice yields. Our results showed that the Manage-N and STCR-N methods produced high grain yields with low NUE values; further refinement of these methods is needed to improve yields and NUE. The SPAD method has to be evaluated with a higher SPAD threshold value (e.g., 37) to see whether high grain yields can be obtained in the Cauvery Delta. The N rate that will be applied as a single basal dose of CRU before planting requires further study.

References
Balasubramanian V, Morales AC, Cruz RT, Abdulrachman S. 1999. On-farm adaptation of knowledge-intensive nitrogen management technologies for rice systems. Nutr. Cycl. Agroecosyst. 53:59-69. Cassman KG, Pingali PL. 1995. Extrapolating trends from long-term experiments to farmers fields: the case of irrigated rice systems in Asia. In: Barnett V, Payne R, Steiner R, editors. Agricultural sustainability: economic, environmental and statistical considerations. New York: Wiley. p 64-84. Peng S, Garcia FV, Laza RC, Sanico AL, Visperas RM, Cassman KG. 1996. Increased N-use efficiency using a chlorophyll meter on high-yielding irrigated rice. Field Crops Res. 47:243-252. Shoji S, Gandeza AT, editors. 1992. Controlledrelease fertilizers with polyolefin resin coating. Sendai (Japan): Tohoku University and Konno Printing Co. Ltd. Thiyagarajan TM, Sivasamy R, ten Berge HFM. 1994. Time course of leaf nitrogen concentration required to attain target yields in transplanted rice. In: ten Berge HFM, Wopereis MCS, Shin JC, editors. Nitrogen economy of irrigated rice: field and simulation studies SARP Research Proceedings, April 1994. Wageningen (The Netherlands): DLO Research Institute for Agrobiology and Soil Fertility, and Manila (Philippines): IRRI. p 267-282.

Information support system for rice crop management

TropRice is an information support system of best-bet practices designed to provide practical field-level guides for rice crop management in the tropics. It aims to help users make informed decisions related to rice production. It is not a single system for the world. It contains some generic information, but some is site- or region-specific. TropRice is intended to be a template that could be modified for different environments. As improved systems on component technologies become available, they will replace or be linked to the system. TropRice is an ongoing project intended for intermediary technology transfer agents and farmers who have no access to information on how to grow rice.
Web site: http://www.cgiar.org/irri/TropRice

26

August 2000

Optimizing chlorophyll meter threshold values for different seasons and varieties in irrigated lowland rice systems of the Cauvery Delta zone, Tamil Nadu, India
M. Babu, R. Nagarajan, and S.P. Ramanathan, Soil and Water Management Research Institute (SWMRI), Thanjavur 613501, Tamil Nadu, India; and V. Balasubramanian, IRRI

IRRN 25.2

27

Innovative tools such as the chlorophyll or SPAD meter offer a new strategy for optimizing fertilizer nitrogen (N) application in rice (Peng et al 1996, Balasubramanian et al 1999). A simple and portable device, the SPAD meter provides a nondestructive and accurate measurement of rice leaf N status in situ in the field. It thus helps assess the N topdressing requirements of rice crops. Earlier studies indicated that critical SPAD values could be established for different varieties and growing seasons, depending on local growing conditions (Balasubramanian et al 1999). This study was conducted to determine the critical values for optimizing grain yield and N use efficiency for different rice varieties and growing seasons in the Cauvery Delta of Tamil Nadu. On-station trials were conducted at the experimental farms of SWMRI in Thanjavur (representing the New Cauvery Delta area) during the 1996, 1997, and 1998 dry seasons [DS (kuruvai): June to September] and 1997-98 first wet season [WS-I (samba): August to January] and second wet season [WS-II (thaladi): October to February]. Different SPAD threshold levels (29, 31, 33, 35, 37, and 39) were evaluated along with the local N recommendation of 125 (dry season) or 150 (wet season) kg N ha1 and a zero-N control. Treatments were arranged in a randomized complete block design with three replications. Rice varieties used for different seasons and locations are given in the table. SPAD readings were taken weekly in all treatments starting from 14 d after transplanting (DAT) until first flowering, using the youngest fully expanded leaf of 10 randomly selected plants from each plot. For the SPAD-N treatments, N was applied whenever the SPAD reading fell below the set critical threshold value. Dur-

ing the DS, N was applied each time at the rate of 30 kg N ha1 at early to maximum tillering stage, 45 kg N ha1 at maximum tillering to panicle initiation (PI) stage, and 30 kg N ha1 at PI to flowering stage; for the wet season, it was reduced to 20, 30, and 20 kg N ha1, respectively, for the three growth stages. Phosphorus (P) and potassium (K) were applied at 23 kg P and 40 kg K ha1 during the dry season, and at 27 kg P and 48 kg K ha1 during the wet season. A full dose of P and 50% of K were incorporated into the soil at last puddling and the remaining K was topdressed at PI. Zinc at 10 kg ha1 was also applied along with P and K at last puddling in Zn-deficient soils. Two indicators of N use efficiency were used in this study: agronomic efficiency of applied N (AEN) and partial factor productivity of N (PFP-N). The 1996 DS trials showed that cv ADT36 and IR50 produced significantly higher grain yields at a SPAD threshold value of 35 compared with 29, 31, and 33 (see table). No significant difference in grain yields, however, was observed between SPAD 35 and 39 in ADT36, and among SPAD 33, 35, and 37 in IR50 during the 1997 DS. In both varieties, grain yield was highest at SPAD 39, but it was not significantly different from that of SPAD 35 in ADT36 and SPAD 35 and 37 in IR50. In the 1998 DS, grain yields were significantly lower at SPAD 35 than those at SPAD 37, which were on a par with SPAD 39 in varieties ADT36 and ADT42. These results indicate that a SPAD threshold of 35 to 37 will optimize grain yield and N use efficiency for the three test varieties. The respective AEN values for SPAD 35 and 37 were 49 and 31 in 1997 and 80 and 51 in 1998. During the 1996 WS-I and WS-II, yields were relatively low and crop re-

sponse to N was poor (see table). In WS-I, White Ponni had the highest yield with 3.9 t ha1 at SPAD 35, but it was not significantly different from that of SPAD 31 and 33. Similarly, in WS-II, grain yields of cv TRY1 were not significantly different at SPAD 29, 31, 33, and 35. The AEN values were 14 for White Ponni and 1011 for TRY1 at SPAD thresholds of 3135. In the 1997 WS-I and WS-II, when higher SPAD values were used, grain yields of both varieties did not vary significantly at SPAD 35, 37, and 39. The AEN values were 1314 for White Ponni and 1522 for TRY1 at yields of 3.64.2 t ha1. In 1998, rice variety ASD19 produced grain yields of 4.1 t ha1 at SPAD 37 and 39; in WS-I, corresponding AEN values were 26 and 21. The yield of cv ADT38 in WS-II was significantly higher at SPAD 39 than at 37 or 35; corresponding AEN values were 20, 23, and 27. These results indicated that a SPAD threshold value of 37 will optimize grain yield and fertilizer use efficiency of modern varieties grown during the wet season. In conclusion, critical SPAD values vary between 35 and 37 depending on season and rice variety. For modern varieties, a SPAD threshold value of 3537 is optimum during the DS (kuruvai), while SPAD 37 is suitable for the WS (samba and thaladi). References
Balasubramanian V, Morales AC, Cruz RT, Abdulrachman S. 1999. On-farm adaptation of knowledge-intensive nitrogen management technologies for rice systems. Nutr. Cycl. Agroecosyst. 53:5969. Peng S, Garcia FV, Laza RC, Sanico AL, Visperas RM, Cassman KG. 1996. Increased N-use efficiency using a chlorophyll meter on high-yielding irrigated rice. Field Crops Res. 47:243252.

Mean grain yield and N use efficiency of irrigated transplanted rice varieties under different SPAD threshold levels, SWMRI farm,Thanjavur (New Cauvery Delta area),Tamil Nadu, India, 1996-98 dry and wet seasons.a SPAD threshold level/season/variety 1996 1997 1998 DS (kuruvai) Rice variety: ADT36 Control Control Control SPAD 29 SPAD 33 SPAD 33 SPAD 31 SPAD 35 SPAD 35 SPAD 33 SPAD 37 SPAD 37 SPAD 35 SPAD 39 SPAD 39 IR50 IR50 ADT42 Control Control Control SPAD 29 SPAD 33 SPAD 33 SPAD 31 SPAD 35 SPAD 35 SPAD 33 SPAD 37 SPAD 37 SPAD 35 SPAD 39 SPAD 39 LSD 0.05 WS-I (samba) W. Ponni W. Ponni ASD19 Control Control Control SPAD 29 SPAD 33 SPAD 33 SPAD 31 SPAD 35 SPAD 35 SPAD 33 SPAD 37 SPAD 37 SPAD 35 SPAD 39 SPAD 39 WS-II (thaladi) TRY1 TRY1 ADT38 Control Control Control SPAD 29 SPAD 33 SPAD 33 SPAD 31 SPAD 35 SPAD 35 SPAD 33 SPAD 37 SPAD 37 SPAD 35 SPAD 39 SPAD 39 LSD 0.05
a

Grain yield (t ha1) 1996 1997 1998

N applied (kg ha1) 1996 1997 1998

1996

AEN (kg grain kg1 N) 1997 1998

4.1 b 3.7 b 3.8 b 4.0 b 5.7 a 3.3 b 3.6 b 3.4 b 3.5 b 5.1 a 480

2.8 d 4.2 c 5.3 ab 5.2 b 6.1 a 3.2 c 5.3 b 6.2 ab 6.0 ab 6.6 a 467

2.7 d 4.0 c 6.2 b 7.0 a 7.1 a 2.8 d 4.1 c 6.6 b 7.3 a 7.3 a 501

0 0 0 0 45 0 0 0 30 75

0 30 60 75 120 0 75 75 75 120

0 25 45 85 110 0 35 55 75 125

36 6 24

45 49 31 27 15 26 24 20

53 80 51 41 35 68 60 36

2.2 d 3.0 c 3.5 ab 3.6 ab 3.9 a

2.1 d 3.5 bc 3.8 ab 3.8 ab 4.3 a

2.0 d 3.2 c 3.5 c 4.1 b 4.1 b

0 50 100 100 120

0 90 130 140 160

0 50 70 80 100

17 14 14 14

16 14 13 14

23 21 26 21

2.1 d 2.9 c 2.9 c 3.2 bc 3.0 bc

2.0 d 3.1 c 3.6 b 4.0 ab 4.0 ab 558

2.0 d 3.1 c 3.9 bc 4.3 b 4.8 a 923

0 30 80 100 100 400

0 50 70 120 130

0 50 70 100 140

25 10 11 10

21 22 16 15

22 27 23 20

In a column, means followed by the same letter are not significantly different at the 5% level by DMRT. AEN = additional grain yield over control per kilogram N applied.

On-farm evaluation of chlorophyll meter-based N management in irrigated transplanted rice in the Cauvery Delta, Tamil Nadu, India
M. Babu and R. Nagarajan, TNRRI, Aduthurai 612101, Tamil Nadu; S. Mohandass, C. Susheela, P. Muthukrishnan, and M. Subramanian, SWMRI, Thanjavur 613501, Tamil Nadu, India; and V. Balasubramanian, IRRI

Judicious use of nitrogen (N) fertilizer in rice requires synchronizing N fertilizer applications with plant needs. Predicting plant N requirement through soil testing has not proven valuable for rice because of dynamic changes in native N supply and difficulty in predicting climatic variables that control soil N mineralization and crop growth. The chlorophyll meter is a simple, portable device that accurately measures leaf N status of rice plants in situ. It could help improve grain yield and N use efficiency (Peng et al 1996, Balasubramanian et al 1999).
28

The SPAD-guided N technique was evaluated in farmers fields in the new and old Cauvery Delta areas of Tamil Nadu, India, during the 1996-98 wet and dry seasons (DS, kuruvai: June to September; WSI, samba: August to December/January; WS-II, thaladi: October to February). Trials were conducted by TNRRI and SWMRI to compare grain yield and N use efficiency of irrigated transplanted rice under different N management practices. Three N treatmentszero-N control, local N recommendation, and SPAD-guided Nwere

evaluated in the 1996-97 trials. A fourth treatment of SPAD-guided N with a basal application of 20 kg N ha1 was included in the 1997-98 trials. Each farm had one replication in the old area and two in the new area. Soils of the experimental fields in the new Cauvery Delta belong to two seriesMadukkhur (Alfisol) and Pattukkottai (Alfisol)while those of the old Cauvery Delta constitute three major seriesKalathur (Udorthentic Chromustert), Adanur (Entic Chromustert), and Padugai (Typic
August 2000

Table 1. Total N applied, grain yield, and N use efficiency of irrigated transplanted rice under different N management practices in the new Cauvery Delta area of Tamil Nadu, India, 1996-98 cropping seasons.a DS (kuruvai) Treatment N applied (kg ha1) Grain yield (t ha1) AENc N applied (kg ha1) WS-I (samba)b Grain yield (t ha1) AEN N applied (kg ha1) WS-II (thaladi) Grain yield (t ha1) AEN

I996-97 Control Local N recommendation SPAD-based N 1997-98 Control Local N recommendation SPAD-based N Basal + SPAD-N
a

2 farms (2 reps each) 5.3 b 6.4 a 8.8 7.1 a 29.0 6 farms (2 reps each) 0 2.9 b 125 (3) 3.9 a 7.6 75 (3) 4.1 a 13.5 107 (4) 4.4 a 15.9 0 125 (3) 60 (2)

7 farms (2 reps each) 0 3.4 b 150 (4) 4.8 a 56 (2) 4.6 a 3 farms (2 reps each) 0 2.7 b 150 (4) 3.8 a 72 (3) 3.5 a 92 (4) 3.4 a

9.7 20.7 7.3 11.3 8.0

3 farms (2 reps each) 0 3.4 b 150 (4) 5.1 a 11.7 60 (2) 5.1 a 32.0 3 farms (2 reps each) 0 2.5 b 150 (4) 3.4 a 5.6 55 (2) 3.1 a 10.6 65 (3) 3.0 a 6.4

All treatments received 22-42-10 kg ha1 of P, K, and Zn in the dry season and 26-50-10 kg ha1 of P, K, and Zn in the wet season. Means followed by a common letter are not significantly different at the 5% level by DMRT. Number in parentheses refers to number of split N applications. bFlood affected yields during 1997-98. cAEN = additional grain yield over control per kilogram N applied.

Ustifluent). The number of on-farm trials conducted during each season is given in Tables 1 and 2. In SPAD-guided N treatments, leaf N status was monitored weekly starting from 14 d after transplanting (DAT) until first flowering. Measurements were obtained from the youngest fully expanded leaf of 10 randomly selected plants from each plot. Whenever the average SPAD reading fell below the critical value of 35, N fertilizer was applied immediately. During the DS, the amount of N applied each time was 30 kg N ha1 from early to maximum tillering stage (1428 DAT), 45 kg N ha1 from maximum tillering to panicle initiation (PI) stage (2948 DAT), and 30 kg N ha1 from PI to flowering (49 DAT to flowering); it was reduced to 2030-20 kg N ha1 for corresponding stages in the WS. All treatments received a basal application of 22-40-10 kg ha1 of phosphorus (P), potassium (K), and zinc (Zn) in the DS and 26-50-10 kg ha1 of P, K, and Zn in the WS. Zinc was applied only to Zn-deficient soils. Grain yield was determined from three 5-m2 sample areas from each plot at 14% moisture content. The agronomic efficiency of applied N (AEN) was calculated as additional grain yield over the control per kilogram N applied. Trial results indicated no significant differences in crop response to N treatIRRN 25.2

Table 2.Total N applied, grain yield, and N use efficiency of irrigated transplanted rice under different N management practices in the old Cauvery Delta area of Tamil Nadu, India, 199698.a Treatment 1996-98 dry seasonc 1996 DS (mean of 5 farms) Control Local N recommendation SPAD-35 N 1997 DS (mean of 12 farms) Control Local N recommendation SPAD-35 N Basal + SPAD-35 N 1998 DS (mean of 7 farms) Control Local N recommendation SPAD-35 N Basal + SPAD-35 N 1997-98 wet seasond WS-1: samba (mean of 9 farms) Control Local N recommendation SPAD-N Basal + SPAD-N WS-II: thaladi (mean of 6 farms) Control Local N recommendation SPAD-N Basal + SPAD-N
a

N applied (kg ha1)

Grain yield (t ha1)

AENb

0 125 (3) 105 (3) 0 125 (3) 67 (2) 88 (3) 0 125 (3) 61 (2) 67 (3)

5.1 c 5.8 b 6.7 a 4.9 c 7.1 b 8.3 a 7.7 a 4.0 b 6.1 a 6.1 a 5.8 a

5.3 14.6 21.5 50.5 31.9 16.5 36.4 27.3

0 150 (4) 69 (3) 89 (4) 0 150 (4) 70 (3) 90 (4)

4.3 b 7.4 b 7.4 a 7.3 a 3.4 c 5.5 ab 5.7 a 5.4 ab

20.9 45.2 33.7 13.9 32.1 21.7

Means followed by a common letter are not significantly different at the 5% level by DMRT. Number in parentheses refers to number of split N applications. bAEN = additional grain yield over control per kilogram N applied. cAll treatments received 22-42-10 kg ha1 of P, K, and Zn. dAll treatments received 26-50-10 kg ha1 of P, K, and Zn.

ments in various soil series in both zones; hence, data across soil series were pooled for analysis and interpretation. In farms of

the new Cauvery Delta, grain yields were generally higher for all seasons in 1996-97 than in 1997-98 (Table 1) due to heavy rain29

Efficiency of controlled-release urea and coated urea fertilizers in irrigated transplanted rice in Andhra Pradesh, India
K. Padmaja, R.M. Kumar, S.P. Singh, A.G.K. Murthy, and S.V. Subbaiah, Directorate of Rice Research (DRR), Hyderabad 500030, Andhra Pradesh, India

Nitrogen (N) is one of the major nutrients applied to rice. The recovery of N from N applied to wetland rice, however, seldom exceeds 40% (De Datta and Buresh 1989). Thus, new methods or strategies are needed to improve the agronomic and economic efficiency of N fertilizers in rice that would also minimize fertilizer-related environmental pollution. Some of the promising N management techniques include split application, subsurface placement, and the use of slow-release and coated urea fertilizers (Mohanty et al 1999). We evaluated the relative efficiency of selected controlled-release urea (CRU) and coated urea fertilizers compared with a single basal application of prilled urea on irrigated transplanted rice (cv Ajaya) at the DRR farm, Andhra Pradesh, India. Field trials were conducted during the monsoon (kharif: July to October 1997) and winter
30

(rabi: November 1997 to March 1998) seasons on a typic Vertisol soil (pH in water 8.3, cation exchange capacity (CEC) 45 cmol P+ kg1, organic carbon 5.5 g kg1, available KMnO4-extractable N 150 kg ha1, Olsen P 30.4 kg ha1, and NH4Ac-extractable K 244.5 kg ha1). Treatments were four types of fertilizer N and a no-N control (Table 1) arranged in a randomized complete block design with three replications. Types of fertilizer N used were CRU with 4.5% and 6% coating, gypsum-coated urea (GCU), neem-coated urea (NCU), and commonly available prilled urea (PU). CRU, a free-flowing granular fertilizer coated with polyurethane and wax, had 43% N, 1.3% biuret, 120 ppm free ammonia, 0.08% moisture, and 1.8% conditioner. All fertilizers were broadcast in a single dose of 86 kg N ha1 just before

planting. Phosphorus (P) as single superphosphate and potassium (K) as muriate of potash were applied to all plots at locally recommended rates of 27 kg P and 32 kg K ha1. Among different fertilizers, CRU 6% produced the highest grain yields, whereas a single basal application of PU had the lowest yields in both seasons (Table 1). Grain yields obtained with CRU 6% were 38% and 27% higher than those obtained with PU in the monsoon and winter seasons, respectively. In the monsoon season, yield of CRU 6% was significantly higher than that of CRU 4.5%, but, during winter, there was no significant difference in grain yield of the two CRU fertilizers. Grain yields produced by GCU and NCU were similar but significantly lower than those of the two CRUs in the monsoon season. During the winter season, howAugust 2000

fall and flooding at critical crop growth stages in 1997-98. In both good and bad years, the SPAD-guided N treatment recorded a significantly higher grain yield than the control, but it was on a par with the local N recommendation in all three cropping seasons. The AEN values of the SPAD treatment were 1.52.5 times that of the local N recommendation (Table 1). Savings in fertilizer rates of 5065 kg N ha1 were recorded during the DS; 7895 kg N ha1 were saved during the WS. Data also indicated that no basal N application is required for the SPAD method in soils of the new Cauvery Delta because there was no substantial increase in grain yield with a basal application of 20 kg N ha1. In farms of the old Cauvery Delta, the crop response to different N

treatments was similar to that observed in the new Cauvery Delta (Table 2). Likewise, AEN values followed the same trend. Grain yield was highest for the SPAD method, but it was on a par with the local N recommendation and SPAD method with a basal application of 20 kg N ha1. The data again indicated that basal N application is not advantageous for various soil types in the old Cauvery Delta. An average savings of 80 kg ha1 for N fertilizer was obtained in both the DS and WS. These results clearly demonstrated the advantage of using the chlorophyll meter technique to fine-tune N recommendations for various seasons in the Cauvery Delta. There is a potential to save 5095 kg N ha1 if refined N recommendations are used. This study focused on using a

single SPAD threshold value to assess N needs of rice crops. Further research is ongoing to determine the effect of using a range of SPAD values (e.g., 3234, 3537, 3840, etc.) for synchronizing N application with crop demand in rice. References
Balasubramanian V, Morales AC, Cruz RT, Abdulrachman S. 1999. On-farm adaptation of knowledge-intensive nitrogen management technologies for rice systems. Nutr. Cycl. Agroecosyst. 53:59-69. Peng S, Garcia FV, Laza RC, Sanico AL, Visperas RM, Cassman KG. 1996. Increased N-use efficiency using a chlorophyll meter on high-yielding irrigated rice. Field Crops Res. 47:243-252.

Table 1. Effect of controlled-release and coated urea fertilizers on grain yield and N use efficiency of rice (cv Ajaya) in two seasons, 1997-98, Andhra Pradesh, India.a Grain yield (t ha1) Treatment
b

N uptake (kg ha1) Monsoon Winter crop crop 51.5 e 101.5 c 142.1 a 115.9 b 105.2 c 77.9 d 8.34

N recovery efficiency (%)

AEN

PFP-N Mean 59.1 68.2 61.6 60.9 45.8

Monsoon Winter Mean crop crop Control CRU 4.5% CRU 6.0% GCU NCU Prilled urea CD (5%) 2.3 e 4.7 c 6.0 a 5.4 b 5.2 b 3.7 d 0.35 2.1 d 5.5 a 5.7 a 5.2 b 5.1 b 4.1 c 0.48 2.2 5.1 5.9 5.3 5.1 3.9

Mean Monsoon Winter Mean Monsoon Winter Mean Monsoon Winter crop crop crop crop crop crop 58.1 105.3 74.9 58.7 30.7 45.0 53.8 55.2 56.0 8.2 51.6 79.6 65.1 57.4 19.5 39.6 42.0 35.8 34.7 23.9 29.3 45.2 38.2 35.4 18.3 34.5 43.6 37.0 35.1 21.1 54.2 70.4 63.4 60.6 43.5 63.7 66.2 59.9 61.2 48.0

45.9 d 48.7 84.6 b 93.0 92.2 a 117.1 93.4 a 104.6 94.1 a 99.6 53.0 c 65.5 5.78

a In a column, means followed by the same letter are not significantly different at CD 5% probability level. bN rate for all sources was 86 kg ha1. GCU = gypsum-coated urea, NCU = neem oil-coated urea, CRU = controlled-release urea, AEN = additional grain yield over control per kilogram N applied, PFP-N = total grain yield per kilogram N applied, CD = critical difference.

Table 2. Economic analysis of using coated fertilizers. Mean yield (t ha1) 5.1 5.9 5.3 5.1 3.9 Gross value (Rs ha1)a 25,400 29,350 26,500 25,650 19,500 Additional labor costb (Rs ha1) 1,120 1,900 1,340 1,170 Net value (Rs ha1) 24,280 27,450 25,160 24,480 19,500 Net value to investment (Rs Rs1) 33.1 37.1 34.0 33.1 26.6 Additional value to investment (Rs Rs1) 6.5 10.5 7.4 6.5 Additional value (%) 24.4 39.5 27.8 24.4

Treatment CRU 4.5% CRU 6.0% GCU NCU PU

a

US$1 = Rs 42.00. Cost of PU = Rs 8.60 kg1, cost of rough rice = Rs 5.00 kg1. bAdditional labor cost for harvest and postharvest processing of additional produce.

New handbook and CD on nutrient deficiencies

The Handbook and CD titled Rice: nutrient deficiencies and nutrient management will soon be available. For more information about this publication, e-mail: e.ramin@cgiar.org or t.fairhurst@ppi-ppc.org

References
De Datta SK, Buresh RJ. 1989. Integrated nitrogen management in irrigated rice. Adv. Soil Sci. 10:143-169. Mohanty SK, Singh U, Balasubramanian V, Jha KP. 1999. Nitrogen deep-placement technologies for productivity, profitability, and

IRRN 25.2

31

ever, grain yields of CRU 4.5%, GCU, and NCU were on a par and the performance of CRU 6% was significantly superior to that of GCU and NCU. Agronomic efficiency of applied N (AEN) and partial factor productivity for applied N (PFP-N) (Peng et al 1996) were 43.6 and 68.2 for CRU 6%, respectively. Mean AEN values ranged from 34.5 to 37.0 and the PFP-N ranged from 59.1 to 68.2 for other coated fertilizers. The single basal application of PU was the least efficient (AEN = 21.1, PFP-N = 45.8). Total N uptake was highest in CRU 6% with 117 kg N ha1 and lowest in the single basal application of PU (55 kg N ha1). Financial returns to investment in fertilizers were computed for different fertilizer types. When prices of CRUs and modified urea were assumed to be equal to that of commonly available PU (Rs 8.6 per kg N as PU), the analysis indicated a 2440% additional net value for money invested in CRUs and modified urea

(Table 2). This means that, if these fertilizers are priced at less than 124140% of the price of PU, they will be competitive in the market. Mean values over several seasons and sites must be considered to achieve the economic advantage and comparative pricing of CRU fertilizers. These results revealed that CRU 6% was better than all other coated fertilizers in rice yield, N use efficiency, and plant N uptake. CRU 4.5% was comparable with other coated fertilizers (GCU and NCU). If appropriately priced, CRU fertilizers will be economically competitive with the commonly available PU, and may become an important source of N fertilizer for rice with a high N use efficiency.

environmental quality of rainfed lowland rice systems. Nutr. Cycl. Agroecosyst. 53:43-57. Peng S, Garcia FV, Gines HC, Laza RC, Samson MI, Sanico AL, Visperas RM, Cassman KG. 1996. Nitrogen use efficiency of irrigated tropical rice established by broadcast wet-seeding and transplanting. Fert. Res. 45:123-134.

Varietal response to different nitrogen management methods in an irrigated transplanted rice ecosystem in a Vertisol, Andhra Pradesh, India
R.M. Kumar, K. Padmaja, S.V. Subbaiah, Directorate of Rice Research (DRR), Hyderabad 500030, Andhra Pradesh, India; and V. Balasubramanian, IRRI

Nitrogen (N) fertilizer is a major input for rice. Only 3040% of applied N is used by the crop, however, due to losses through volatilization, denitrification, leaching, and runoff (De Datta and Buresh 1989). Deep placement of urea, split N application, preplanting incorporation of coated and controlled-release urea (CRU), and the chlorophyll meter and leaf color chart techniques are some N management strategies that could reduce N losses and improve fertilizer use efficiency in rice (Katyal et al 1985, Kumar et al 1989, Peng et al 1996, Balasubramanian et al 1999). Since rice varieties differ in N use efficiency (NUE), this study was conducted to determine their response to selected N management techniques. Field trials were conducted at the DRR farm, Andhra Pradesh, India, during the 1997 monsoon (kharif, July to October) and 1997-98 winter (rabi, November to February/March) seasons. The soil at the experimental site is a typic Vertisol, with a pH of 8.3, cation exchange capacity (CEC) 45 cmol P+ kg1, organic carbon 5.5 g kg1, available (KMnO4-extractable) N 150 kg ha1, Olsen P 30.4 kg ha1, and NH4Ac-extractable K 244.5 kg ha1. Five N treatments were evaluated: zero-N control, controlled-release urea with 4.5% and 6% coating, chlorophyll meter-guided N, and prilled urea (PU) applied in two equal splits. The CRU fertilizers had 43% N, 1.3% biuret, 120 ppm free ammonia, 0.08% moisture, and 1.8% conditioner. The two CRUs were broadcast at the rate of 86 kg N ha1 and incorporated into the soil just before planting. PU (100 kg N ha1) was applied in two equal splits: 50% basal and 50% topdressing at the maximum tillering stage.

Three rice varieties were used: Rasi (short duration, 105 d, and yield potential of 6.5 t ha1), Ajaya (medium duration, 135 d, and yield potential of 7.5 t ha1), and Pusa Basmati (improved aromatic variety, 145 d, and yield potential of 4.5 t ha1). All varieties were transplanted at 20 10-cm spacing. The two-factor factorial was organized in a randomized block design with three replications. In all treatments, chlorophyll meter readings were taken weekly starting from 14 d after transplanting (DAT) until first flowering using the youngest fully expanded leaf of 10 randomly selected plants in each plot. For the SPAD method, N was applied when measured mean SPAD values fell below the threshold of 35 for each of the three varieties. The amount of N applied in the SPAD treatment varied with season and crop growth stages. The rate per application was 20, 30, and 20 kg N ha1 in the monsoon season and 30, 35, and 20 kg N ha1 in the winter season at the early (1428 DAT), rapid (2948 DAT), and late growth stages (49 DAT-first flowering), respectively. Phosphorus (P) and potassium (K) were applied in all plots at locally recommended rates of 27 kg P and 32 kg K ha1. Locally recommended practices were used for all other field operations and crop care. In the SPAD treatments, the total amount of N applied to varieties Ajaya and Rasi was 70 kg ha1 in the monsoon and 75 kg ha1 in winter. A lower amount was applied to Pusa Basmati50 kg ha1 in the monsoon and 65 kg ha1 in winter. For short-duration Rasi, CRU 6% and SPAD-35 N gave higher grain yields than other treatments in both seasons (Table 1). Yield performances of CRU 4.5% and CRU 6%

were comparable in the winter season. The mean increase in grain yield over PU-2 splits was 26% for SPAD-35 N, 24% for CRU 6%, and 7% for CRU 4.5%. The mean agronomic efficiency of applied N (AEN) and partial factor productivity for N (PFPN) were highest in the SPAD-35 N treatment, followed closely by CRU 6% (Table 1). Plant N uptake was highest (111.8 kg N ha1) in Rasi fertilized with CRU 6%; it had an N recovery of 87.4%. The response of medium-duration Ajaya to N application was better than that of the other two varieties in both seasons (Table 1). Application of CRU 6% produced a mean grain yield over two seasons of 6.3 t ha1, compared with 5.9 t ha1 for SPAD35 N, 5.7 t ha1 for CRU 4.5%, and 5.6 t ha1 for PU-2 splits. In the monsoon season, CRU 6% gave the highest grain yield, which was on a par with CRU 4.5% but significantly superior to the other treatments. In the winter season, grain yields of CRU 6% and SPAD-35 N (6.8 t ha1 each) were significantly higher than those of PU-2 splits and CRU 4.5% (5.9 t ha1 each). The AEN and PFP-N values of these treatments showed a similar trend (Table 1). Mean AEN and PFP-N values over two seasons were highest in the SPAD-35 N treatment, followed closely by CRU 6%. Plant N uptake, however, was highest in CRU 6% (110.0 kg N ha1), followed by the SPAD35 N treatment (93.5 kg N ha1). The aromatic rice variety Pusa Basmati was the least responsive to various N treatments. The CRU 6% and SPAD-35 N treatments produced the highest grain yield in the monsoon and winter, respectively, followed by PU-2 splits. The single application of CRU 4.5% was less efficient than the other methods.

32

August 2000

Table 1. Response of three rice varieties to different N management methods in irrigated transplanted rice at the DRR farm, Andhra Pradesh, India, 1997-98.a Grain yield (t ha1) Treatment 1997 kharif Rasi Control 1.9 c CRU 4.5% 3.5 b CRU 6% 4.3 a SPAD-35 N 4.8 a PU-2 splits 3.3 b Mean 3.6 Ajaya Control 2.4 c CRU 4.5% 5.5 ab CRU 6% 5.7 a SPAD-35 N 5.0 b PU-2 splits 5.4 ab Mean 4.8 Pusa Basmati Control 1.4 c CRU 4.5% 2.6 b CRU 6% 3.3 a SPAD-35 N 2.6 b PU-2 splits 2.6 b Mean 2.5 CDe (5%) Varieties 0.2 Treatments 0.3 Varieties treatments
a

Total N uptake (kg ha1) 1997 kharif 37.1 e 79.3 c 91.4 a 83.7 b 61.6 d 70.6 37.6 d 93.5 b 103.5 a 87.0 c 88.1 c 81.9 25.7 d 64.5 c 75.0 a 70.0 b 60.7 c 59.2 1.8 2.3 1997-98 rabi 36.2e 97.8 d 132.3 a 125.5 b 114.3 c 101.2 28.2 d 80.1 c 116.5 a 100.0 b 79.9 c 80.9 32.4 d 95.2 a 88.4 b 88.8 b 74.0 c 75.7 2.4 3.1 4.0 Mean

Recovery (%)b

AENc

PFP-Nd 1997 1997-98 kharif rabi 40.8 50.0 68.1 32.8 47.9 63.7 66.7 71.4 53.5 63.8 30.5 38.1 52.2 26.2 36.8 64.2 68.4 77.5 50.9 65.2 68.4 78.6 90.0 59.3 74.1 41.7 46.6 69.8 38.3 49.1 Mean

1997-98 rabi 2.0 c 5.5 ab 5.9 a 5.8 a 5.1 b 4.9 2.2 c 5.9 b 6.8 a 6.8 a 5.9 b 5.5 2.6 c 3.6 b 4.0 b 4.5 a 3.8 b 3.7 0.2 0.3 0.6

Mean

1997 1997-98 Mean 1997 1997-98 Mean kharif rabi kharif rabi 49.1 63.1 66.5 24.5 50.8 65.0 76.6 70.6 50.5 65.7 45.1 57.3 88.6 35.0 56.5 71.6 111.7 119.0 78.1 95.1 60.3 102.7 95.7 51.7 77.6 73.0 65.1 86.8 41.6 66.6 60.3 87.4 92.8 51.3 73.0 62.7 89.6 83.2 51.1 71.6 59.1 61.2 87.7 38.3 61.6 18.3 27.4 40.4 13.5 24.9 36.3 39.3 37.7 29.0 35.6 14.7 22.3 25.0 12.6 18.6 40.5 44.7 50.3 30.5 41.5 43.0 53.3 60.9 37.5 48.7 11.7 16.6 30.2 12.5 17.8 29.4 36.1 45.3 22.0 33.2 39.7 46.3 49.3 33.3 42.1 13.2 19.5 27.6 12.6 18.2

2.0 4.5 5.1 5.3 4.2 4.2 2.3 5.7 6.3 5.9 5.6 5.1 2.0 3.1 3.7 3.6 3.2 3.1

36.6 88.6 111.8 104.6 87.9 85.9 32.9 86.8 110.0 93.5 84.0 81.4 29.0 79.8 81.7 79.4 67.4 67.5

52.5 59.2 72.8 41.8 56.6 66.1 72.7 80.7 56.4 69.0 36.1 42.4 61.0 32.3 43.0

0.5

5.4

Treatment means within each variety followed by the same letter are not significantly different at CD 5% probability level. bRecovery efficiency: ( r = plant N uptake/N applied). cAEN = additional grain yield over control per kilogram N applied. dPFP-N = total grain yield per kilogram N applied. eCD = critical difference.

The interaction between rice cultivars and N treatments was significant for grain yield in both seasons. The highest grain yield of 6.8 t ha1 was recorded in Ajaya given CRU 6% and SPAD-35 N treatments during the winter season. The N recovery (mean of two seasons) was highest in SPAD-35 N, followed by CRU 6% in all three cultivars; this was probably due to the congruence of N supply with crop demand. The highest SPAD values were observed at the maximum tillering stage for all varieties (Table 2). SPAD values recorded at critical crop growth stages were higher for CRU 6% and SPAD-35 N treatments than for PU-2 splits. The higher SPAD values indicate a higher foliar N concentration that led to higher grain yield, plant N uptake, fertilizer N recovery, and efficiency with CRU 6% and SPAD-35 N treatments. The highest total N uptake by all varieties recorded in the CRU 6% treatment indicated that this controlledrelease fertilizer maintained a steady N
IRRN 25.2

Table 2. SPAD values at critical growth stages of three rice varieties as influenced by various N treatments, DRR farm, Andhra Pradesh, India, 1997-98 cropping seasons. Maximum tillering Treatment Rasi Control CRU 4.5% CRU 6% SPAD-35 N PU-2 splits PU-basal Ajaya Control CRU 4.5% CRU 6% SPAD-35 N PU-2 splits PU-basal Pusa Basmati Control CRU 4.5% CRU 6% SPAD-35 N PU-2 splits PU-basal 1997 kharif 30.6 44.6 39.7 38.7 39.3 39.5 31.0 40.3 41.1 38.0 38.8 39.8 33.0 42.8 42.8 43.1 40.4 41.1 1997-98 rabi 32.1 38.4 38.2 40.2 38.9 35.9 32.3 38.2 41.1 38.7 39.8 38.6 32.0 39.5 42.1 37.4 34.9 41.1 Mean Panicle initiation 1997 kharif 31.6 36.8 39.9 38.1 37.4 35.6 30.5 32.2 35.5 35.5 33.6 32.9 33.0 37.4 38.8 36.0 36.8 35.7 1997-98 rabi 32.0 37.5 40.3 39.0 37.0 33.1 29.0 31.8 35.0 40.6 32.3 33.5 31.3 36.2 38.0 41.3 35.9 33.8 Mean 1997 kharif 29.7 34.1 36.2 34.7 29.7 29.7 30.8 32.4 35.6 34.0 32.7 30.1 34.0 37.9 36.1 37.1 38.2 36.0 Heading 1997-98 rabi 28.3 33.2 35.7 34.0 28.8 28.8 30.1 32.5 33.2 34.7 30.9 30.0 33.2 35.3 37.4 38.7 33.2 30.4 Mean

31.3 41.5 39.0 39.5 39.1 37.7 31.6 39.3 41.1 38.4 39.3 39.2 32.5 41.2 42.4 40.3 37.7 41.1

31.8 37.2 40.1 38.6 37.2 34.4 29.8 32.0 35.3 38.1 33.0 33.2 32.2 36.8 38.4 38.7 36.3 34.8

29.0 33.7 36.0 34.4 29.3 29.3 30.5 32.5 34.4 34.4 31.8 30.5 33.6 36.6 36.8 37.9 35.7 33.2

33

supply to the crop throughout the growth period. These observations confirm that the use of CRU 6% fertilizer and the chlorophyll meter technique (SPAD-N) produces high grain yields with high N uptake and efficiency. CRU 6% also appeared to be better than CRU 4.5%, probably due to its slower N release profile. If CRU could be priced competitively with the commonly available PU, it could become the preferred N fertilizer for rice.

Real-time N management using the chlorophyll meter and PU represents an attractive alternative for improving grain yields and N use efficiency in rice. References
Balasubramanian V, Morales AC, Cruz RT, Abdulrachman S. 1999. On-farm adaptation of knowledge-intensive nitrogen management technologies for rice systems. Nutr. Cycl. Agroecosyst. 53:59-69. De Datta SK, Buresh RJ. 1989. Integrated nitrogen management in irrigated rice. Adv. Soil Sci. 10:143-169.

Katyal JC, Singh B, Sharma VK, Grasswell ET. 1985. Efficiency of some modified urea fertilizer for lowland rice grown on a permeable soil. Fert. Res. 6:279-290. Kumar V, Shrotriya GC, Kaore SV, editors. 1989. Soil fertility and fertilizer use. Vol. III. Urea supergranules for increasing nitrogen use efficiency. New Delhi (India): Indian Farmers Fertilizer Cooperative. 143 p. Peng S, Garcia FV, Laza RC, Sanico AL, Visperas RM, Cassman KG. 1996. Increased N-use efficiency using a chlorophyll meter on high-yielding irrigated rice. Field Crops Res. 47:243-252.

Assessment of chlorophyll meter-based N application at critical growth stages of irrigated transplanted rice
S.P. Ramanathan and R. Nagarajan, SWMRI, Thanjavur, India; and V. Balasubramanian, IRRI

Synchronizing nitrogen (N) application with crop demand and soil N supply is one strategy for improving N use efficiency (NUE) in rice. Using a variable N rate application based on observed chlorophyll meter readings at critical growth stages may help optimize grain yield and NUE at the same time. The chlorophyll or SPAD meter can accurately measure leaf chlorophyll content that is related to leaf N status. It could help develop need-based N fertilization in rice (Peng et al 1996). One disadvantage of the SPAD technique, however, is the need for weekly observations to monitor crop N status, which could sometimes lead to many (36) split N applications (Balasubramanian et al 1999). Field experiments were conducted during the 1997-98 dry (DS, kuruvai: June to September) and wet (WS-II, thaladi: October to February) seasons at the SWMRI farm in Thanjavur, India. The objective was to determine whether chlorophyll meter readings taken only at critical crop growth stagesearly tillering, active tillering, panicle initiation, and 10% floweringwere sufficient, and to apply variable rates of N based on a range of SPAD threshold values (Table 1) rather than on a single critical SPAD value. The effect of variable rate N application, based on a range of chlorophyll meter readings, on
34

grain yield and NUE was measured in these trials. Treatments included different ranges of SPAD threshold values: <32, 3335, and 3638. Rice varieties ADT42 (115 d) and ADT38 (135 d) were used during the DS and WS-II, respectively. Treatments were arranged in a completely randomized block design with seven replications. SPAD readings were taken in all treatments at critical crop growth stages using the youngest fully expanded leaf of 10 randomly selected plants from each plot. Whenever average SPAD readings fell within a set range of threshold values, N was applied immediately based on the variable rates specified in Table 1. Plant N status was allowed to fall to the specific range of SPAD values for each treatment before N was applied. Grain yield (t ha1) was determined at 14% moisture content from three 5-m2 sample areas taken from each plot. The NUE

is expressed as the partial factor productivity of applied N (PFP-N). Grain yields were low in both the 1997-98 DS and WS due to heavy flooding at critical crop growth stages. Among the SPAD threshold ranges used in the DS, the 3638 range gave the highest grain yield (4.9 t ha1), but it was not significantly different from that of SPAD <32 (4.7 t ha1) or 3335 (4.6 ha1) (Table 2). The NUE as expressed by the PFP-N value was 125 for SPAD <32, 92 for SPAD 3336, and 54 for SPAD 3638. The data seem to indicate that a SPAD threshold range of 3638 could be optimum for DS rice crops. Further research is ongoing to confirm these results. In the WS, the highest grain yield was obtained from SPAD 3638; this was not significantly different from that of SPAD 3335, but significantly different from that of SPAD <32 (Table 2). More N

Table 1. Amount of N applied (kg ha1) based on range of SPAD threshold values observed at critical crop growth stages. Range of SPAD threshold values <32 3335 3638
a

Early tillering (14 DAT)a 30 30 20

Active tillering (28 DAT) 50 40 30

Panicle initiation (42 DAT) 50 40 30

Flowering (56 DAT) 30 30 20

DAT = d after transplanting.

August 2000

Table 2. Effect of variable rate N application based on range of SPAD values observed at critical crop growth stages on yield and N use efficiency of irrigated transplanted rice at the SWMRI farm,Thanjavur, India, 1997-98 dry (DS) and wet seasons (WS).a Range of SPAD threshold values <32 3335 3638 SEc LSDc (5%) Total N applied (kg ha1) DS 38 50 92 WS 58 70 104 DS 4.7 a 4.6 a 4.9 a 347 nsc Grain yield (t ha1) WS 3.3 b 3.7 ab 4.0 a DS 125 92 54 202 530 PFP-Nb WS 57 53 38

tween N topdressing in this treatment. These observations suggested that, for grain yield and NUE, a SPAD threshold range of 3335 could be optimum for the WS rice in the new Cauvery Delta zone. References
Balasubramanian V, Morales AC, Cruz RT, Abdulrachman S. 1999. On-farm adaptation of knowledge-intensive nitrogen management technologies for rice systems. Nutr. Cycl. Agroecosyst. 53:59-69. Peng S, Garcia FV, Laza RC, Sanico AL, Visperas RM, Cassman KG. 1996. Increased N-use efficiency using a chlorophyll meter on high-yielding irrigated rice. Field Crops Res. 47:243-252.

a P, K, and Zn were applied to all treatments at the rate of 22-42-10 kg ha1 in the DS and 26-50-10 kg ha1 in the WS. bPFPN = total grain yield per kilogram N applied. cns = not significant. cSE = standard error, LSD = least significant difference.

(104 kg ha-1), however, was required at this range to produce a yield of 4.0 t ha-1, which resulted in a low PFP-N value of 38. SPAD <32 produced the lowest yield with a to-

tal N use of 58 kg ha-1 (PFP-N = 57). Waiting for SPAD values to fall below 32 before applying N fertilizer may have starved plants for N during the long intervals be-

Chlorophyll meter threshold values for N management in wet direct-seeded irrigated rice
V. Balasubramanian and A.C. Morales, IRRI; and R.T. Cruz, Philippine Rice Research Institute (PhilRice), Maligaya, Nueva Ecija, Philippines

Asian rice farmers are increasingly adopting wet seeding as an alternative method to transplanting for economic and technical reasons. High-density, excessive early vegetative growth and low foliar N concentration (Schnier et al 1990, Peng et al 1996b) characterize wet direct-seeded rice (W-DSR). These characteristics will have an impact on the critical leaf N concentration required for high grain yields in W-DSR. The chlorophyll or SPAD meter offers a new strategy for synchronizing N application with actual crop demand in rice (Peng et al 1996a, Balasubramanian et al 1999). Earlier studies indicated that the SPAD threshold or critical value of 35 established for transplanted rice invariably overestimated the N requirement of and reduced the N use efficiency (NUE) in WDSR (IRRI-CREMNET 1996). This study was undertaken to establish specific SPAD threshold values for maximizing both grain yield and NUE in W-DSR in the Philippines. In collaboration with PhilRice, we conducted on-farm trials at Maligaya in the
IRRN 25.2

1996 dry (DS) and wet seasons (WS) and in the 1997 DS (DS: December/JanuaryApril; WS: July-October). The objective was to compare the grain yield and NUE of irrigated W-DSR under different N management practices. In the 1996 trials, the SPAD method with a single SPAD threshold of 35 was compared with the farmers practice and a zero-N control. The farmers practice consisted of 150 kg ha1 N application in two or three splits in the DS and 90 kg N ha1 in the WS. In the 1997 DS, different SPAD threshold levels (29, 32, and 35) were evaluated along with a fixed N application rate of 120 kg ha1 in two or three splits and a zero-N control. Treatments were arranged in a randomized complete block design with each farm as a replication. The mean amount of P and K applied was 13 kg P ha1 and 25 kg K ha1 in the 1996 DS, 17 kg P ha1 and 28 kg K ha1 in the 1996 WS, and 26 kg P ha1 and 50 kg K ha1 in the 1997 DS. SPAD readings were taken in all treatments weekly starting from 21 d after

seeding (DAS) until first flowering, using the youngest fully expanded leaf of 10 randomly selected plants from each plot. For the SPAD-N treatments, N was applied whenever the mean SPAD reading fell below the chosen threshold values. The N rates applied were 30, 45, and 30 kg ha1 in the DS and 20, 30, and 20 kg ha1 in the WS during the early (2135 DAS), rapid (3656 DAS), and late (57 DAS-flowering) growth stages, respectively. The P and K rates applied followed the local farmers practice. Grain yield was expressed in kg ha1 at 14% moisture content. Two indicators of N use efficiency were used in this study: agronomic efficiency of applied N (AEN) and partial factor productivity of N (PFP-N). In the 1996 DS, grain yields and efficiency values (AEN and PFP-N) were high for 3-N splits in broadcast and 2-N splits in row-seeded rice (Table 1). In spite of the high yields, SPAD-35 N was the least efficient due to a high N rate of 213 kg
35

Table 1. Comparison of the chlorophyll meter method and fixed-schedule N application in irrigated direct-seeded rice, Maligaya, Nueva Ecija, Philippines, 1996 dry (DS) and wet seasons (WS).a Treatmentb N used (kg ha1) Splits (no.) Yield (t ha1) 2.9 c 6.0 a 5.2 b 5.8 a 6.0 a 2.5 c 3.9 a 3.4 b 3.7 ab 3.8 ab AENc 15 b 15 b 19 a 19 a 14 ab 11 b 14 ab 16 a PFP-Nd 28 34 38 40 36 38 42 42

1996 DS: 13 kg P ha1 & 25 kg K ha1 (10 farms) Control, B SPAD-35 N, B 213 7 2-N splits, B 150 2 3-N splits, B 150 3 2-N splits, R 150 2 1996 WS: 17 kg P ha1 & 28 kg K ha1 (9 farms) Control, B SPAD-32 N, B 57 4 2-N splits, B 90 2 3-N splits, B 90 3 2-N splits, R 90 2

a In a column, means followed by the same letter are not different at 5% probability by DMRT. bB = broadcast-sown rice, R = row-seeded rice. cAEN = agronomic efficiency of applied N (grain yield over control per kilogram N applied). dPFP-N = partial factor productivity for N (total grain yield per kilogram N applied).

SPAD value 40 T1 35 T2 T3 T4 T5

30

25

20

15

MT (35 DAS)

PI (56 DAS) Plant growth stage

FL (77 DAS)

Mean SPAD values at critical crop growth stages (MT, PI, FL) of B-WSR and R-WSR (cv IR64), Maligaya, Nueva Ecija, 1996 dry season (mean of 10 farms). Table 2. Comparison between chlorophyll meter method with three thresholds and fixedschedule N application in irrigated direct-seeded rice, Maligaya, Nueva Ecija, Philippines, 1997 dry season.a Treatment N used (kg ha1) Splits (no.) Yield (t ha1) 4.4 c 6.8 ab 6.8 ab 6.5 b 6.5 b 6.9 a 4.7 c 6.1 b 6.8 a 6.6 ab 6.7 a 6.7 ab AENb 25 a 18 b 12 c 18 b 21 b 25 a 19 ab 11 b 16 b 16 b PFP-Nc 68 50 36 54 58 107 60 40 56 55

Broadcast-sown rice: 26 kg P ha1 & 50 kg K ha1 (10 farms) Control SPAD-29 N 100 4 SPAD-32 N 137 45 SPAD-35 N 180 6 2-N splits 120 2 3-N splits 120 3 Row-seeded rice: 26 kg P ha1 & 50 kg K ha1 (10 farms) Control SPAD-29 N 57 4 SPAD-32 N 114 45 SPAD-35 N 165 6 2-N splits 120 2 3-N splits 120 3

a In a column, means followed by the same letter are not different at 5% probability by DMRT. bAEN = agronomic efficiency of applied N (grain yield over control per kg N applied). cPFP-N = partial factor productivity for N (total grain yield per kg N applied).

ha1 applied in seven splits. In the 1996 WS, SPAD-32 N gave the highest grain yield, which was on a par with 3-N splits for broadcast and 2-N splits for row-seeded rice. All three methods were equally efficient in N use. In the DS, SPAD readings at maximum tillering, panicle initiation, and first flowering varied between 3132 for 3-N splits in broadcast and 2-N splits in rowseeded rice and 3233 for the SPAD method (see figure). The trend was similar in the WS (data not shown). This indicated that the SPAD threshold value for W-DSR appears to be around 32 for both DS and WS. In broadcast rice, SPAD-29 N, SPAD32 N, and the 3-N splits gave significantly higher grain yields than the other treatments (Table 2). The AEN was highest in SPAD-29 N. Again, SPAD-35 N was the least efficient. This suggested that, for highdensity (~800 productive tillers m2) broadcast-sown rice, the SPAD critical value is around 29. In row-seeded rice, SPAD-32 N produced the highest grain yield with an AEN of 19. Despite having the highest AEN, SPAD-29 N had the lowest grain yield among fertilized plots. The 2-N or 3-N splits gave similar grain yields and AEN values. The SPAD-35 N was the least efficient due to the high N requirement (165 kg N ha1) for moderate yields. For the high-density (662 panicles m2) row-seeded rice, a SPAD threshold of 32 maximized both grain yield and NUE, which were similar to those of 2-N splits. In conclusion, the SPAD threshold of 35, considered optimum for transplanted rice, overestimated the N requirement and drastically reduced the NUE of broadcast and row-seeded rice. A SPAD threshold of 29 for highdensity broadcast rice (800 panicles m2) and 32 for high-density row-seeded rice (650 panicles m2) appears to maximize both grain yield and NUE. The 3-N splits (at 21, 35, and 49 DAS) for broadcast rice and 2-N splits (at 21 and 42 DAS) for row-seeded rice produced high grain yields with high NUE that
August 2000

36

were similar to those of the SPAD technique with optimum threshold values for the respective sowing methods. Thus, the adequacy of farmers current N fertilization practices or local recommendations can be verified by the SPAD technique and adjusted, if necessary, to optimize rice productivity.

References
Balasubramanian V, Morales AC, Cruz RT, Abdulrachman S. 1999. On-farm adaptation of knowledge-intensive nitrogen management technologies for rice systems. Nutr. Cycl. Agroecosyst. 53:59-69. IRRI-CREMNET (International Rice Research Institute Crop and Resource Management Network). 1996. CREMNET annual report for 1994-95. Manila (Philippines): IRRI. Peng S, Garcia FV, Laza RC, Sanico AL, Visperas RM, Cassman KG. 1996a. Increased N-use

efficiency using a chlorophyll meter on high-yielding irrigated rice. Field Crops Res. 47:243-252. Peng S, Garcia FV, Gines HC, Laza RC, Samson MI, Sanico Al, Visperas RM, Cassman KG. 1996b. Nitrogen use efficiency of irrigated tropical rice established by broadcast wet-seeding and transplanting. Fert. Res. 45:123-134. Schnier HF, Dingkuhn M, De Datta SK, Marqueses EP, Farolina JE. 1990. Nitrogen-15 balance in transplanted and direct-seeded flooded rice as affected by different methods of urea application. Biol. Fertil. Soils 10:89-96.

The leaf color chart (LCC)

Leaf color is generally used as a visual and subjective indicator of the rice crops need for nitrogen (N) fertilizer. Leaf color intensity is directly related to leaf chlorophyll content and leaf N status. Here is a tool that can help farmers improve their decision-making process in N management. The leaf color chart is a simple, easy-to-use, and inexpensive guide that can help farmers determine the right time of N application to the rice crop by measuring leaf color intensity. It is an ideal tool to optimize N use at reasonably high yield levels, irrespective of N source appliedorganic, biological, or chemical fertilizers. The tool was developed from a Japanese prototype by the Crop and Resource Management Network (CREMNET) at IRRI and the Philippine Rice Research Institute (PhilRice). Features Made of high-quality plastic material Consists of six color shades ranging from light yellowish green (no.1) to dark green (no.6). Color strips fabricated with veins resembling those of rice leaves Advantages Inexpensive, simple, easy to use Helps avoid too much N application Reduces lodging problem and pest and disease incidence Environment-friendly Savings in fertilizer use range from 0 to 53 kg N ha1 in flooded rice Limitations Several factors influence LCC readings: varietal group, plant or tiller density, solar radiation differences between seasons, status of nutrients other than N in soil and plant, and biotic and abiotic stresses that induce discoloration of leaves. Using the LCC Start taking LCC readings from 14 days after transplanting or 21 days after seeding. Take the last reading when the crop starts to flower. Select at least 10 disease-free rice plants or hills in a field with uniform population. Under the shade, compare the color of the youngest fully expanded leaf of the selected plant or hill with the color strips of the chart. If more than five leaves read below a set critical value, apply 23 kg N ha1 (if wet season) or 35 kg ha1 (if dry season). The suggested critical values are 4 for transplanted rice (TPR) and 3 for high-density wet-seeded rice (WSR). Repeat the process every 710 days or at critical growth stages (early tillering, active tillering, panicle initiation, and first flowering) and apply N as needed. The LCC is being introduced and distributed to farmers by field researchers and extension staff of government and nongovernment organizations and privatesector agencies in different countries.
For comments and inquiries, contact The CREMNET Coordinator International Rice Research Institute MCPO Box 3127, Makati City 1271 Philippines E-mail: v.balasubramanian@cgiar.org Fax: (63-2) 891-1292/845-0606

IRRN 25.2

37

Crop management & physiology

Chlorophyll stability index (CSI): its impact on salt tolerance in rice

M. Madhan Mohan, S. Lakshmi Narayanan, and S.M. Ibrahim, Department of Agricultural Botany, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625104, India

Salinity is a major constraint to successful agriculture in the arid and semiarid tropics. High-yielding varieties do not perform well under salt stress conditions. Thus, there is a need to develop special varieties that can perform well under saline conditions. The area with potential saline soil is about 20 million ha, and about 7 million ha are severely affected by salinity. This constitutes a major part of problem soils in India. Irregular monsoon and irrigation in red soil cause salinity problems in large areas. The chlorophyll stability index (CSI) is an indication of the stress tolerance capacity of plants. A high CSI value means that the stress did not have much effect on chlorophyll content of plants. A higher CSI helps plants to withstand stress through better availability of chlorophyll. This leads to increased photosynthetic rate, more dry matter production, and higher productivity. This indicates how well chlorophyll can perform under stress. This study was carried out to estimate the CSI of salt tolerance in rice using three salt-tolerant varieties (CSR10, TRY1, and CO 43) and four salt-susceptible varieties (IR20, White Ponni [WP], ADT36, and Jaya) along with their hybrids in two soil types (normal and salt-affected soil). Each treatment was replicated five times in pot cultures using a completely randomized design. The field experiment was conducted in normal soil on the college farm. Salt-affected soil (sodic) was taken from a farmers field at Elanthakulam (25 km away) and transferred to pots for further study. Soil samples were randomly collected from a depth of 30 cm from both the college farm and Elanthakulam and their nutrient status analyzed (Table 1).

CSI in the leaf was estimated using a spectrometer following the method of Koleyoreas (1958). It was calculated as the difference in light transmission percentage between treated and untreated leaf samples (see formula).

Experimental results are presented in Table 2. Under normal soil conditions, all varieties and their hybrids showed good levels of CSI. Salt-tolerant cultivars CO 43, TRY1, and CSR10 registered higher CSI values, whereas susceptible varieties

Treated sample = 1,000 mg of leaf sample kept in a test tube containing water at 55 C for 1 h Untreated/ = 1,000 mg of leaf sample kept in a test tube (control) containing water at room temperature CSI = OD at 652 nm of treated sample 100 OD at 652 nm of control
Table 1. Soil properties of the experimental field. Character Texture pH (1:2.5 soil:water suspension) Electrical conductivity (dS m1) (1:2.5 soil:water suspension) Normal soil Sandy clay loam 7.2 0.4 Saline soil Sandy clay loam 8.9 1.5 (soil) 2.5 (irrigation water)

Table 2. Effect of salt stress on CSI (%). Variety/hybrid CSR10 TRY1 CO 43 IR20 WP ADT36 Jaya IR20/CSR10 IR20/TRY1 IR20/CO 43 WP/CSR10 WP/TRY1 WP/CO 43 ADT36/CSR10 ADT36/TRY1 ADT36/CO 43 Jaya/CSR10 Jaya/TRY1 Jaya/CO 43 SEa CDb
a

Normal soil 74.4 82.8 83.3 72.9 74.5 73.5 71.2 73.3 75.6 75.2 74.2 76.6 78.5 73.3 77.7 78.1 72.5 75.7 77.8 0.156 0.311

Saline soil 71.9 80.3 82.0 68.9 69.5 68.2 67.8 74.7 74.3 79.8 75.8 75.4 69.8 70.9 70.7 81.9 70.6 71.3 73.7 0.3 0.598

% decrease ()/ increase (+) 3.36 3.02 1.56 5.49 6.71 7.21 4.78 +1.91 1.72 +6.12 +2.16 1.57 11.08 3.27 9.01 +4.87 2.62 5.81 5.27

SE = standard error, bCD = critical difference.

38

August 2000

ADT36, WP, IR20, and Jaya showed lower values under saline conditions. This means that under stress conditions, chlorophyll content was affected to a greater extent in susceptible varieties than in tolerant varieties. Hybrids IR20/CO 43, ADT 36/CO 43, WP/CSR10, and IR20/CSR10, however, per-

formed better under saline conditions than under normal conditions. All hybrids were generally found to be more tolerant compared with their more susceptible parents (Table 2). This suggests that salt tolerance is a dominant trait. It is possible to incorporate salt tolerance through hybridization between tol-

erant and susceptible parents. Increased salt tolerance in hybrids is a consequence of increased seedling vigor. Reference
Koleyoreas SA. 1958. A new method for determining drought resistance. Plant Physiol. 33:22.

Exogenous glycinebetaine reduces sodium accumulation in salt-stressed rice plants

S. Lutts, Laboratoire de Cytogntique, Universit Catholique de Louvain, 5 (Bte 13), Place Croix du Sud, 1348 Louvainla-Neuve, Belgium

Glycinebetaine (GB) is a compatible organic solute produced by several plant species to cope with salt or water stress. This compound is involved in osmotic adjustment as well as in protecting several cellular structures. It is well established that rice is unable to synthesize this compound because it lacks choline monooxygenase (CMO), which is involved in the synthesis of betaine aldehyde, the immediate precursor of GB. It has also been reported that exogenous GB protects rice from salt stress by maintaining photosystem II integrity and relative water content (Harinasut et al 1996). Its effect on sodium nutrition in saltstressed rice, however, was never reported.

In a phytotron experiment, a saltsensitive rice cultivarI Kong Paomaintained on nutrient solution (Yoshida et al 1976; renewed each week) was subjected to various NaCl doses (0, 30, 50, and 100 mM), with or without simultaneous exposure to 1 mM GB (Sigma Chemical). Plants were exposed to salt stress at the seedling stage (20 d old; 100 plants per treatment) on 25-L tanks in a complete randomized block. Ten surviving plants were collected each week during a subsequent 1-mo experiment. Relative growth rate was quantified on a dry-weight basis and ion concentration was determined after digestion of organic matter with nitric acid by an in-

ductively coupled argon plasma emission spectrophotometer. The presence of GB in nutrient solution had no deleterious effect on unstressed plants, but it clearly improved surviving plant percentages and growing abilities of salt-treated ones (see table). At the highest NaCl dose (100 mM), exogenous GB affords protection only during the 2 wk of exposure to salinity. The positive effect of exogenous GB was associated with reduced sodium accumulation and with the maintenance of potassium concentration in all parts of salinized plants as shown in the figure for plants exposed to various NaCl doses during 2 wk. Exog-

Surviving percentagesa (S%) and relative growth rates (RGR, expressed on a dry-weight basis in g g1 d1 103) of plants of salt-sensitive cultivar I Kong Pao exposed during 4 wk to various NaCl doses (0, 30, 50, and 100 mM) on nutrient solution in the presence (+) or absence () of 1 mM GB. Parameter Duration of exposure (wk) GB S% 1 2 3 4 1 2 3 4 1 2 3 4 100 a 100 a 98 a 100 a 113.4 a 98.8 a 95.1 a 77.5 b 86.0 a 77.9 a 68.9 b 72.3 b 0 mM +GB 100 a 99 a 100 a 100 a 108.9 a 102.4 a 86.4 b 80.3 b 83.7 a 80.8 a 71.2 b 68.6 b 30 mM GB 100 a 83 b 71 c 65 c 85.2 b 77.7 b 50.2 c 38.2 c 80.1 a 63.9 b 65.5 b 49.7 c +GB 100 a 86 b 86 b 75 c 100.7 a 97.1 a 81.9 b 69.3 c 75.4 a 71.3 b 66.2 b 68.5 b GB 71 c 70 c 54 d 38 e 55.5 c 17.5 e * * 44.4 d 39.7 d 10.4 f * 50 mM +GB 85 b 87 b 73 c 62 d 71.6 b 35.2 c 22.3 d 28.9 d 58.6 c 55.7 c 21.6 e 12.6 f GB 72 c 51 d 32 e 7f 49.0 c 15.6 e * * 40.3 d 11.6 f * * 100 mM +GB 83 b 78 b 45 e 12 f 58.3 c 29.6 d * * 53.8 c 38.4 d 22.8 e *

Shoot RGR (in g g1 d1 103)

Root RGR (in g g1 d1 103)

Values followed by the same letter are not significantly different at P = 0.05 (2 test); * = not significantly different from 0.

IRRN 25.2

39

enous GB had no effect on calcium and magnesium nutrition of stressed plants, whatever the NaCl dose (detailed data not shown). These results suggest that GB may have a positive impact on both absorption and translocation of monovalent cations in salt-stressed rice and that its synthesis by transferring gene coding for CMO may constitute an interesting goal for genetic engineering in this species. References
Harinasut P, Tsutsui K, Takebe T, Nomura M, Takebe T, Kishitani S. 1996. Exogenous glycinebetaine accumulation and increased salt-tolerance in rice seedlings. Biosci. Biotechnol. Biochem. 60:366368. Yoshida S, Forno OA, Cock JH, Gomez KA. 1976. Laboratory manual for physiological studies of rice. Manila (Philippines): International Rice Research Institute.

Na concentration (mmol g1 DW) 1.5 Sodium

Na concentration (mmol g1 DW) 1.4 Potassium 1.2 1.0 0.8 0.6

1.0

0.5

root - GB root + GB shoot - GB shoot + GB

0.4 0.2 0.0 100 0 25 50 75 100

0.0 0 25 50 75

NaCl concentration (mM)

Sodium and potassium concentrations in roots and shoots of rice plants (cv I Kong Pao) exposed during 2 wk to a nutrient solution containing various NaCl doses (0, 30, 50, and 100 mM) in the presence (+) or absence () of 1 mM GB. Each value is the mean of at least four replicates and vertical bars are S.E.

Instructional videos available

The Leaf Color Chart (LCC) (8:20 min)
Farmers generally observe the color of rice leaves to determine a rice crops need for nitrogen fertilizer. Dark green rice leaves mean a high nitrogen content, while pale green rice leaves necessitate the application of more nitrogen fertilizer. Mere observation, however, holds no absolute guarantee of measurement accuracy. Thus, to better help farmers determine their rice crops need for nitrogen, the leaf color chart (LCC) was developed. The Leaf Color Chart (LCC) instructional video was produced to familiarize farmers and extension workers with the proper use of this new and affordable farming implement.

Portable Chlorophyll Meter for Nitrogen Management in Rice (13:30 min)

In agriculture, excess nitrates can actually be highly damaging to crops and the environment. There is thus a need to efficiently manage the application of nitrogen fertilizers on rice crops. The Portable Chlorophyll Meter for Nitrogen Management in Rice introduces the features and use of the chlorophyll or SPAD meter, which is capable of measuring the relative nitrogen content in plant leaves through a simple, quick, and nondestructive procedure.

Go breAk inTo the Code (13:30 min)

Genetic engineering need not be a property of the scientific few. This is what IRRI had in mind when it produced Go breAk inTo the Code: to make the general public grasp and understand the seemingly complicated science through a visually stunning, fast-paced, and entertaining presentation of the genetic code, DNA sequencing, and plant biotechnology. The video also gives the public a glimpse as to how IRRI scientists are redesigning the rice plantthat most important food stapleusing biotechnology tools. These instructional videos are available in English, in the 3/4-inch u-matic and VHS formats, and in the NTSC, SECAM, or PAL systems. For more information about the videos, contact Marketing and Distribution Communication and Publications Services IRRI, MCPO Box 3127, Makati City 1271 Philippines E-mail: e.ramin@cgiar.org 40
August 2000

Socioeconomics

Partnership in agricultural extension

S i i

G.P. Ojha, Ministry of Agriculture, Kathmandu, Nepal

E-mail: ojhasirkutane@hotmail.com

Partnership has been recognized as a strategy for attaining effectiveness and efficiency in agricultural research and development. Research and evaluation studies on this theme, however, are not adequate (Carney 1998). A study was conducted in Chitwan, Nepal, to assess the effectiveness of partnership between and among government (GO), nongovernment (NGO), and private (PO) organizations. Through informal discussion and peer ratings, the District Agriculture Development Office (DADO) was selected as the GO, the Rural Reconstruction Nepal (RRN) as the NGO, and four private entrepreneurs (Inter Nepal Agrovet, Beera Agrovet, Narayani Agrovet, and Kisan Seva Agrovet) dealing with agricultural inputs as POs. Identified partners, together with farmers, selected the research problem and technologies related to the crop variety that farmers preferredrice, hybrid maize, and sunflower. The partners divided responsibilities and agreed to work together through a memorandum of understanding. During the implementation phase, partners reviewed the performance of the past month and developed programs for the ensuing months. After the implementation of the project in seven villages for 21 mo, data obtained from a complete enumeration of 123 adopters and 17 extension workers were used to assess the effectiveness of three individual organizations (GO, NGO, and PO) and four partnership (GO+NGO, GO+PO, NGO+PO, and GO+NGO+PO) patterns. The study showed that partnership was more effective than nonpartnership in terms of crop variety use by farmers. Mean adoption was 8.4% for partnership and 4.3% for individual organizations. Five reasons explained the differences in effectiveness of institutions. Institutions under partnership performed their given responsibilities at a higher rate through compleIRRN 25.2

mentation, provision of more resources (manpower and materials), and more professional use of agents time. Other factors that contributed to effectiveness were peer pressure and mutual benefits. The study also showed additional differences in collaborative efforts. Among partnerships, GO+PO and GO+NGO were more effective than other forms of partnerships (Table 1). The reasons for this include mutual understanding of each others responsibilities, closely located offices at research sites, more frequent participation in monthly joint meetings, and a positive attitude toward each other. Among these patterns, GO+NGO+PO had the lowest adoption rate. The reasons were higher transaction costs and lower participation level in activities by the NGO due to project phaseout and staff reduction. This resulted in fewer farmers being motivated. Consequently, demand for inputs is lower. The POs participation was low because of low demand for inputs.

Partnership was found to be technology-specific. Technology was classified as either high-cost or low-cost. Hybrid seed and associated technology were considered as a high-cost technology, whereas use of open-pollinated variety and associated technology was a low-cost technology. Among the effective patterns, adoption of high-cost technologies took place under GO+PO partnership. With GO+NGO partnership, low-cost and locally available agricultural technologies were adopted (Table 2). Similarly, partnerships were specific to farmer circumstances. Except for the GO+NGO pattern, mean farm size of adopters was larger than the mean farm size of nonadopters. Significant differences, however, were observed only with PO, GO+PO, and GO+NGO+PO patterns. Among the effective partnerships, GO+PO mostly reached large landholders, while the GO+NGO partnership preferred small farmers (Table 3).

Table 1. Effectiveness of seven institutional patterns. Pattern GO NGO PO GO+NGO GO+PO NGO+PO GO+NGO+PO Adopter farmers (no.) 7 17 5 22 52 14 6 Households (no.) 154 287 209 271 311 198 326 Adoption (%) 4.6 5.9 2.4 8.1 16.7 7.1 1.8

Table 2. Adopter farmers and technology types, by pattern. Pattern GO NGO PO GO+NGO GO+PO NGO+PO GO+NGO+PO Adopter farmers (no.) 7 17 5 22 52 14 6 Low-cost technology (%) 100.0 100.0 40.0 90.9 11.5 85.7 83.3 High-cost technology (%) 0.0 0.0 60.0 9.1 88.5 14.3 16.7

41

Table 3. Differences in farm size between adopter and nonadopter farmers, by pattern. Pattern Adopter GO NGO PO GO+NGO GO+PO NGO+PO GO+NGO+PO
a

Mean farm size (ha) Nonadopter 0.69 0.37 0.77 1.04 0.76 0.58 0.62 Differences 0.48 0.14 1.63 0.15 0.47 0.50 1.40 t valuea 0.1250 0.1852 0.0087** 0.6110 0.0470* 0.0856 0.0497*

1.17 0.51 2.40 0.89 1.23 1.08 2.02

This study showed that partnership is an effective strategy but is specific to circumstances. Results suggest that partnership should not be generalized to be equally effective in all situations. Therefore, efforts should be made to develop partnership according to the local context. Reference
Carney D. 1998. Changing public and private roles in agricultural services. London: Overseas Development Institute.

** = significant at 0.01 level, * = significant at 0.05 level.

NEWS

International Rice Symposium Honors Chandler at Cornell

Dr. Robert F. Chandler Jr., IRRIs founding director, who died in March 1999 at the age of 91, was honored in a major scientific gathering held at Cornell University in Ithaca, New York, on 15-17 June 2000. The Symposium, Rice Research and Production in the 21st Century, recognized his lifetime contributions to agricultural science. More than 150 scientists involved in rice research attended the international symposium. Dr. Chandler, considered by many as a visionary, was a scientist of conviction and wisdom. His impact on international agricultural research is still widely felt today, particularly in Asia and in rice research. According to Dr. Ronald Cantrell, current IRRI director general, If it were not for visionaries such as Bob Chandler, there may have never been a CGIAR and the makeup and strength of the national agricultural research systemsor NARSas we see today in Asia and other regions of the world would be quite different. Dr. Chandler was at IRRIs helm until 1972. Dr. Chandler wrote An Adventure in Applied Science: A History of the International Rice Research Institute. Before the Rockefeller Foundation hired him to establish IRRI, he was a professor at Cornell University. He later served as Dean of Agriculture, then President, of the University of New Hampshire. In 1972, Dr. Chandler became the founding director general of the Asian Vegetable Research and Development Center (AVRDC) based in Taiwan, China. He remained active in international agricultural research even after his retirement in 1975. In addition to honoring Dr. Chandler, the symposium focused on rice genetics and production in the context of international agricultural research management. Among the speakers was Dr. Norman Borlaug, who received the 1970 Nobel Peace Prize for developing high-yielding wheat varieties that, along with the IRRI rice semidwarfs, triggered the Green Revolution. Sessions focused on Intensive Rice Production Systems: Implications and Opportunities and Developments in Genetics: Future Opportunities in Rice. The symposium was sponsored by the Office for Research, the International Agriculture Program of the College of Agriculture and Life Sciences of Cornell University; and the Cornell International Institute for Food, Agriculture and Development. Financial assistance was provided by a grant from the Rockefeller Foundation. A 2-day reunion of former and current IRRI scientists, scholars, and dependents followed the symposium.
Source: IRRI hotline and Rice News of PlanetRice.net (http://www.planetrice.net) 42
August 2000

NOTES FROM THE FIELD

Does wild rice Oryza minuta exist in Thailand?

Dayun Tao, Food Crops Research Institute,Yunnan Academy of Agricultural Sciences, Kunming 650205, Peoples Republic of China; and Prapa Scripichitt, Agronomy Department, Kasetsart University, P.O. Box 1097, Bangkok 10903,Thailand

It is generally believed that Oryza minuta is distributed only in the Philippines, Papua New Guinea, and Indonesia (Irian Jaya). Recently, Japanese scientists reported collecting this species from northern Thailand (Sato et al 1996, see map in Fig. 1), and a Thai scientist, Dr. Songkran Chitrakon, claims that the species belongs to O. officinalis because of its tall stature. To confirm the existence of O. minuta in Thailand, one accession of O. minuta (GS No. 8173), supplied by Dr. Songkran of the Pathumthani Rice Research Center, Thailand, was planted in a greenhouse at Kasetsart University. Three plants reached flowering stage. Among these, one was a typical O. officinalis with tall stature (152 cm), rhizomes, and equally long branches at the panicle base. The

lower half of these branches had no spikelets, which is a character of O. officinalis (Fig. 2). The other two plants were typical O. minuta, having short stature (77.5 cm), a panicle base lacking a whorl of branches, spikelets inserted in the entire length of panicle branches, and less spikelets (Fig. 2). Plants were nonrhizomatous, creeping, and stoloniferous. Results suggest that O. minuta exists in Thailand and O. officinalis and O. minuta grow sympatrically in Sukhothai because O. officinalis was collected at the same site. If this is true, Sukhothai is another sympatric site for O. officinalis and O. minuta. To confirm this conclusion, the site should be investigated and the chromosome number of plants should be checked. Reference
Sato YI, Morishima H, Shimamoto Y. 1996. Roles of in situ conservation of wild rice for ecosystem sustainability. In: Seminar on the 80th Anniversary of Pathumthani Rice Research Center; 13-14 Nov 1996, Pathumthani Rice Research Center; Pathumthani, Thailand. p 155175.

Fig. 2. Panicles of (a) Oryza minuta and (b) O. officinalis (GS No. 8173).

Job openings at IRRI

IRRI has openings for the following internationally recruited staff: 1. Public Awareness Editor/Writer 2. Molecular Geneticist/Molecular Taxonomist

China Vietnam

For more information and full position descriptions, visit http://irriwww.irri.cgiar.org/ IRRIHome/irsemp.htm or contact Gene Hettel Chair, Selection Committee IRRI e.hettel@cgiar.org For Public Awareness Editor/Writer (application deadline: 15 October 2000) Michael Cohen Chair, Selection Committee IRRI m.cohen@cgiar.org For Molecular Geneticist/Molecular Taxonomist (application deadline: 31 October 2000)

Myanmar

Chiang Mai

Laos

20 N

Uttaradit Sukhothai

Thailand
COLLECTION SITE

Ayutthaya Bangkok

15 N

Cambodia
100 E 105 E

Fig. 1. Collection site of Oryza minuta population (after Sato et al 1996).

IRRN 25.2

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