RIRDC Completed Projects in 1999-2000 & Research in Progress as at June 2000

3.4 Rice - Completed Projects
Project No.	Project Title	Researcher	Phone	Organisation

Varietal Improvement and Pure Seeds Maintenance

CMB-1A	Molecular methods for hybrid rice production	Dr Richard Jefferson	(02) 6246 4502	Center for the Application of Molecular Biology to International Agriculture (CAMBIA)
DAN-147A	Evaluation of rice grain quality – II	Dr Melissa Fitzgerald	(02) 6951 2656	NSW Agriculture
USC-1A	Genetic engineering of Australian rice varieties for improved cold tolerance and grain quality	Prof Robert Henry	(02) 6620 3010	Southern Cross University
USC-2A	Application of molecular markers to rice breeding in Australia	Prof Robert Henry	(02) 6620 3010	Southern Cross University

Crop Establishment

CST-2A	Seed treatment and foliar application of glycinebetaine to increase crop establishment and cold tolerance in rice	Dr Naidu Bodapati	(07) 3214 2285	CSIRO Tropical Agriculture
DAN-146A	Sustainable control of aquatic earthworms and bloodworms in rice	Dr Mark Stevens	(02) 6951 2611	NSW Agriculture

Agronomy/Crop Physiology

ANU-29A	Investigation of the molecular mechanisms causing cold induced sterility in rice	Prof Barry Rolfe	(02) 6249 4054	Australian National University
ANU-34A	Towards the development of more effective nitrogen-fixing associations between rice cultivars and endophytic bacteria	Prof Barry Rolfe	(02) 6249 4054	Australian National University
DAN-119A	The yield stability of short season rices	Mr Russel Reinke	(02) 6951 2516	NSW Agriculture

Crop Protection

DAN-121A	Further development of a mycoherbicide for control of Alismataceae weeds in rice	Dr Eric Cother	(02) 6391 3886	NSW Agriculture
UCS-23A	Herbicide resistance development in dirty dora (Cyperus difformis) and starfruit (Damasonium minus)	Mr Peter Baines	(02) 6933 2177	Charles Sturt University
UCS-7A	Biological control of starfruit in rice fields using a mycoherbicide	Dr Eric Cother	(02) 6391 3886	NSW Agriculture

Technology Transfer and Communications

RGA-1A

2nd International Temperate Rice Conference and US study tour

Mr Mike Hedditch

(02) 6953 0619

Ricegrowers' Co-operative Ltd

Harvesting, Storage, Handling and Transport

KDI-18A

An evaluation of rice harvesting machinery and systems

Mr Scott Boyle

(02) 6921 5825

Kondinin Group

Varietal Improvement and Pure Seeds Maintenance
 

Project Title	Molecular methods for hybrid rice production
RIRDC Project No:	CMB-1A
Researcher:	Dr Richard Jefferson
Organisation:	CAMBIA GPO Box 3200 CANBERRA ACT 2601
Phone:	(02) 6246 4502
Fax:	(02) 6246 4501
Objectives	· To develop a viable two-line hybrid rice system by provision of a method for facultative, environment-independent nuclear male sterility in transgenic rice. · To provide a molecular toolkit of inducible rice genes specific to female heterotic effects and achieve new yield ceilings.
Background	Self-pollination in rice, posses a barrier to yield ceiling improvements using hybrid seed technology. While manual methods for hybrid rice production are economically impractical in Australian and also result in non-homogenous progenies, available transgenic technologies are laborious in that a three-line system is required to maintain the male sterile (MS) parents. The rationale of the project is to create facultative MS lines based on transgene activation of pro-gametocides. A long-term goal of this project is to fix the heterotic effects obtained from hybridisation by introducing apomixis into rice. Here we provide some of the tools required to achieve this goal.
Research	Most milestones were met, while some activities are still ongoing and will be carried on to fruition. Within the frame of this project, highly reproducible and efficient rice genetic transformation techniques were adopted and further developed for local rice varieties. Versatile vectors for rice transformation, now widely used by the research community, were developed and tested extensively. Hundreds of transgenic rice plants were and are routinely produced to test genetic constructs belonging to this and to other, related projects at CAMBIA. Stable inheritance traits was successfully tested over generations together with phenotypic assessment. Two rice genes with sequence homology to yeast early meiosis regulatory genes were isolated and partially characterised. The promoter regions of one of the genes were used to drive the expression of a marker gene, showing that gene expression was specific to nodal meristems and pollen cells. This and other pollen-specific promoters are now being used to direct the expression of a hydrolase capable of cleaving pro-gametocides in pollen cells. An embryo sac-specific gene regulatory region was isolated from Arabidopsis thaliana. The corresponding enhancer region was narrowed down and reintroduced into the original species in both orientation, one of them giving a stronger expression level of the marker gene than the other. Transgenic rice plants containing these constructs have been produced and are being evaluated. In a different approach, rice lines containing enhancer traps that display ovule-specific expression and other interesting expression patterns have been produced. Some of these regulatory regions will provide useful tools to the project.

	A secretable hydrolase (beta-glucuronidase) – a critical tool required to make the two-line hybrid system work – is now in place and working in rice. Gene switches to control the activity of the hydrolase at will need some refinement. Some preliminary experiments were done to test the suitability of toxic compounds (eg, asulam) as candidates to be converted into pro-toxins (pro-gametocides); these were complemented with experiments on uptake and transport of glucuronides in plants. Two avenues of synthesis of pro-gametocides are being followed through collaborations with a partner in India as well as with a reputed organic chemist in Poland.
Outcomes	Most of the required tools that will allow us to generate a two-line hybrid system for rice are now in place: routine rice transformation; pollen, anther and tapetum-specific promoters; proof of principle for the use of a beta-glucuronidase expressed in a tissue-specific manner and the concomitant activation of a pro-compound have been demonstrated elsewhere; a secretable beta- glucuronidase that works in rice. Transgenic tools for expression in the ovule and in the megagametophyte are well advanced.
Implications	Although RIRDC funding of this project is completed, work is ongoing and well underway. We expect to be able to produce facultative MS plants using the outlined approach in the course of the next year. Experiments should go as far as greenhouse studies. Field trials should be conducted with the assistance of and in conjunction with the rice industry.
Publications	Jefferson RA, Nugroho S (1997) Molecular strategies for hybrid rice: male sterility and apomixis. Proceedings of the 3rd Hybrid Rice Symposium. India. Nguyen TA, Rajagopal S, Badger M, Kilian A, Keese PK, Jefferson RA (1999) A synthetic reporter gene encoding a novel Bacillus ß-glucuronidase: design, construction and expression. Proceedings of the Rockefeller Foundation Meeting in Phuket, Thailand. Nguyen TA, Wenzl P, Rajagopal S, Badger M, Kilian A, Mayer JE, Jefferson RA (1999) Biochemical characterisation and preliminary secretion analysis of a novel Bacillus ß-glucuronidase. Proceedings of the Rockefeller Foundation Meeting in Phuket, Thailand. Nugroho S, Keese PK, Desamero NV, Kilian A, Jefferson RA (1997) Molecular strategies for apomictic rice I: Isolation and analysis of genes controlling the meiotic process. Proceedings of the Rockefeller Foundation Meeting in Malacca, Malaysia. Nugroho S, Keese P, Kilian A, Desamero NV, Cohn PCL, Jefferson RA (1999) Molecular and cellular analysis of a rice gene related to a yeast meiotic regulatory gene; RIM11. Proceedings of the Rockefeller Foundation Meeting in Phuket, Thailand. Ravi KS, Jefferson RA (1997) Development of a glucuronide responsive promoter for field level control of transgene expression. Proceedings of the Rockefeller Foundation Meeting in Malacca, Malaysia. Roberts C, Rajagopal S, Smith LM, Nguyen TA, Yang W, Nugrohu S, Ravi KS, Vijayachandra K, Harcourt RL, Dransfield L, Desamero N, Slamet I, Hadjukiewicz P, Svab Z, Maliga P, Mayer JE, Keese PK, Kilian A and Jefferson RA (2000). pCAMBIA binary vectors: A comprehensive set of modular vectors for advanced manipulations and efficient transformation of plants. Genbank acc nrs AF234290-AF234316 Yang W, Kilian A, Keese PK, Grossniklaus U, Jefferson RA (1997) Molecular strategies for apomictic rice II: Isolation and analysis of megagametophyte-specific regulatory elements and genes. Proceedings of the Rockefeller Foundation Meeting in Malacca, Malaysia. Yang W, Rajagopal S, Kilian A, Badger M, Grossniklaus U, Jefferson RA (1999) Characterisation of a megagametophyte-specific enhancer from Arabidopsis thaliana and its possible applications in rice. Proceedings of the Rockefeller Foundation Meeting in Phuket, Thailand.

Project Title	Evaluation of Rice Grain Quality 2
RIRDC Project No:	DAN-147A
Researcher:	Melissa Fitzgerald, Laurie Lewin, Russell Reinke
Organisation:	Yanco Agricultural Institute NSW Agriculture PMB Yanco NSW 2703
Phone:	02 69 512 656
Fax:	02 69 512 719
Email:	melissa.fitzgerald@agric.nsw.gov.au
Internet:	www.agric.nsw.gov.au
Objectives	· Continued evaluation of crossbreds in the rice-breeding program; · Describing and measuring the factors that influence rice grain quality for specific needs; and  · Development of quality criteria and assessment technology such as NIR and NIT, for incorporation into routine quality evaluation programs.
Background	Australia has an international reputation as a supplier of high-quality, uniform rice. Maintenance of quality within specification continues to be a major aim of the breeding program here at Yanco. Ricegrowers Cooperative LTD, who market the rice grown in NSW, actively pursue niche markets as an export strategy. This marketing strategy increases the number of markets we aim to supply, and therefore broadens the range of quality-attributes the breeding and quality evaluation programs must appraise. Methods to evaluate quality require continual examination and redevelopment as the level of discrimination of quality changes within the market. Evaluation of crossbred lines must now incorporate methods for selecting varieties suitable for the Japanese market, Arborio type rices, Thai and Basmati fragrant lines. The search for new varietal types has led us to seek more efficient methods of analysis that are rapid, non-destructive, objective, and require smaller samples so analysis of characteristics can occur in earlier generations. The use of NIR and NIT technology for assessment of amylose and milling quality is under evaluation.
Research	· Identifying quality parameters required by different markets and developing method to measure those, · NIR and NIT for measuring amylose, · SDS PAG Electrophoresis for determining proteins, · Analysis of enzyme activity for starch synthesising enzymes, · Radiation techniques for measuring deposition of starch in chalky and translucent rice, · HPLC for starch structure · Rapid Visco Analysis for measuring cooking quality.
Outcomes	· A different way to measure the texture of basmati rices was identified, · On-Line Milling, a computer program to collect all data from the Quality Evaluation Program was developed and is now in place; it removes the need for recording data manually, and substantially increases the speed and accuracy of the Program, · NIR and NIT are not possible methods for measuring amylose, · Different varieties of rice contain slightly different protein contents, · Proteins in rice have a large influence on rice cooking quality, · In different varieties, proteins influence cooking quality differently, · Starch structure differs between chalky and translucent grains, · Starch synthesis and deposition is less in chalky than translucent grains · Chalky areas are characterised by deformed starch granules and incomplete packing of single granules into compound structures.  · The RVA is our most valuable method for evaluating cooking quality, and progress has been made to understand the results it generates.

Implications

The Quality Evaluation Program is faster, and new procedures are being incorporated to evaluate quality of rices for different markets. Furthermore, we are making progress in understanding rice quality from the perspective of starch and proteins, and we are making progress in understanding chalk. Understanding chalk and identifying the basis of tolerance will allow us to select actively and more accurately for varieties tolerant to forming chalk.

Publications

Lisle, A.J., Martin, M., and Fitzgerald M. A. (in press Cereal Chemistry) Chalky rice grains differ from translucent rice grains in starch content and structure and cooking properties.  Fitzgerald, M.A. (1999). How does starch structure affect quality of rice? Invited seminar presented to the Southern Regional Research Centre of USDA, Louisiana, USA.  Fitzgerald, M.A. (1999). Varietal/environmental influence on the starch structure of rice. Invited paper presented at Rice Utilisation Workshop, Arkansas. USA.  Fitzgerald M. A., (1998). Rice Cereal Chemistry, Unlocking the keys to rice quality. Chemistry in Australia 65: 32-34. Fitzgerald, MA, Martin M, Lisle, AJ. 1999. G, E, and GxE change the cooking properties of rice, HOW? Paper presented at 2nd Temperate Rice Conference, California, June 1999. Lisle, AJ, Martin, M, Fitzgerald, MA. 1999. Is chalkiness linked to starch synthesis? Paper presented at 2nd Temperate Rice Conference, California, June 1999.  Fitzgerald, MA, Martin, M. 1999. The chemistry of rice, it flows when stirred. Paper presented at 49th Cereal Chemistry Conference, Melbourne September 1999. Lisle, AJ, Martin M, Fitzgerald MA. 1999. Linking chalkiness to starch synthesis in rice. Paper presented at 49th Cereal Chemistry Conference, Melbourne September 1999. Fitzgerald, M.A. (1999). Varietal/environmental influence on the starch structure of rice. Invited paper presented at Rice Utilisation Workshop, Arkansas. USA. Fitzgerald MA, Martin M, White JM, 1998. High temperatures during grain-filling change the chemical attributes of Australian rice. Presented at 48th Australian Cereal Chemistry Conference. Queensland, AUSTRALIA.  Martin M, Fitzgerald MA, 1998. Growing rice hydroponically to manipulate the quality attributes of grains. Presented at 48th Australian Cereal Chemistry Conference. Queensland, AUSTRALIA.  Lisle AJ, White JM, Fitzgerald MA, 1998. The effect of temperature on starch synthesis in Australian rice grains. Presented at 48th Australian Cereal Chemistry Conference. Queensland, AUSTRALIA.  Fitzgerald MA, Martin M, White JW, Lewin LG. 1998. Effect of high temperatures during grain development on the quality of Australian rice. Presented at 27th Rice Technical Working Group, Nevada USA, and at 6 Institutes in the USA.  Lewin, LG, Batten GD, Blakeney AB, Reinke RF, Williams RL, Fitzgerald MA. 1998. Genetic improvement of rice in Australia – aa key factor in sustainable rice production. International Symposium on Rice Germplasm Evaluation and Enhancement, Stuttgart, AR .

Project Title	Genetic engineering of Australian rice varieties for improved cold tolerance and grain quality
RIRDC Project No:	USC 1A
Researcher:	Professor Robert Henry
Organisation:	Centre for Plant Conservation Genetics Southern Cross University PO Box 157 LISMORE NSW 2480
Phone:	02 66203010
Fax:	02 66222080
Email:	rhenry@scu.edu.au
Internet:	www.scu.edu.au/research/cpcg/index.htm
Objective	To identify plants with improved cold tolerance and novel grain qualities by generating and screening large populations of rice plants containing genes from wild rice (Zizania)
Background	The forerunner to this project (DAQ 157A) developed a range of genetic engineering techniques for use in the NSW cultivar Jarrah. These were further developed in this project both for generating large numbers of lines and for extending the techniques for use with other NSW cultivars. These techniques are now available for other characters. Contact between researchers in this project and those involved in the NSW rice improvement program enabled them to remain current with new technology in this emerging field.
Research	The initial focus of the project was to develop an efficient transformation system using the Australian rice variety Jarrah. This was achieved by using selectable marker genes. The result was development of protocols for very efficient transformation of rice based on particle bombardment of mature embryo derived callus and a highly efficient regeneration system, which is currently being used in Australia and overseas. The technique was extended to other Australian varieties in this project.
Outcomes	A valuable patent position is being established in relation to the key enabling technology of rice transformation. This position should be maintained.
Implications	Significant rice germplasm has been developed and should be further evaluated. The rice industry should attempt to negotiate freedom to operate in the production of commercial transgene rices using the patent position now held as a bargaining chip. Analysis of opportunities to exploit transgene rice in the Australian rice industry should be undertaken.
Publications	M Abedinia, RJ Henry, AB Blakeney and L Lewin 1997 An Efficient Transformation System for the Australian Rice cultivar, Jarrah. Aust. J. Plant Physiol, 24, 133-141 M Abedinia, RJ Henry and S Cripps Clarke 1998 Distribution and phylogeny of Potamophila parviflora R.Br. a wild relative of rice from eastern Australia. Genetic resources and crop Evolution 45: 399-406 M Abedinia, R Henry, A Blakeney and L Lewin 2000 Accessing Genes in the Tertiary Gene Pool of Rice by Direct Introduction of Total DNA from Zizania palustris (Wild Rice) Plant Molecular Biology Reporter 18: (in press) Provision Patent application Plant Genome Transfer

Project Title	Application of molecular markers to rice breeding in Australia
RIRDC Project No:	USC-2A
Researcher:	Prof Robert Henry
Organisation:	Southern Cross University Centre for Plant Conservation Genetics PO Box 157 LISMORE NSW 2480
Phone:	(02) 6620 3010
Fax:	(02) 6622 2080
Email:	rhenry@scu.edu.au
Objective	The major objectives of the project were to identify, adapt and evaluate molecular markers for routine use in the Australian rice-breeding program. The major component of the work involved the development of markers for the semi-dwarfing gene sd-1 and the major fragrance gene fgr.
Background	The semi-dwarfing gene (sd-1) is responsible for producing sturdy and moderately short plants with higher harvest index compared to similar tall plants. sd-1 is the source of semi-dwarfism in the Australian rice varieties. The genetics of fragrance in rice is more complicated and involves the production of basmati or jasmine style aroma. Several chemicals are involved with aroma, however a single constituent (2-acetyl-1-pyrroline) is believed to be a major component and is controlled by a single gene. The application of marker assisted selection for these genes, has potential to greatly improve the efficiency of the Australian rice-breeding program. Markers have the capacity to assist in the selection of traits that are expensive or laborious to assess. Molecular markers can be evaluated from a single seedling leaf or seed samples allowing selection to occur before the trait is expressed. The semi-dwarf character, for example, could be detected before maturity or even if only one copy of the gene is present, thus eliminating the need for progeny testing in a backcrossing program. Grain quality characters such as aroma are very difficult to assess accurately. Markers used in preliminary screening would reduce labour costs and assist in selection.
Research	We screened for differences in portions of DNA linked to sd-1 and fgr to develop markers that can be easily produced and identified. In addition, markers already published and near fgr were assessed to provide alternative markers for fragrance.
Outcomes	Four useful markers have been produced for sd-1. Three useful markers have also been produced or identified for fgr. The markers for fgr and sd-1 will be useful in the Australian rice-breeding program and in rice breeding programs in general.
Implications	This project has demonstrated that molecular markers are suitable for variety identification and marker assisted selection within the Australian Industry. The challenge is to maximise the potential benefits of markers within the breeding program. It may be necessary to restructure the breeding program to obtain maximum benefits.
Publications	Garland SH, Lewin L, Abedinia M, Henry R, Blakeney A (1999) The use of microsatellite polymorphisms for the identification of Australian breeding lines of rice (Oryza sativa L.). Euphytica 108: 53-63. Garland S, Lewin L, Blakeney A, Reinke R, Henry R, (in press) PCR based molecular markers for the fragrance gene in rice (Oryza sativa L.). Theor Appl Genet.

Crop Establishment
 

Project Title	Seed treatment and foliar application of glycinebetaine to increase crop establishment and cold tolerance in rice
RIRDC Project No:	CST-2A
Researcher:	Dr Bodapati P. Naidu
Organisation:	CSIRO Tropical Agriculture Cunningham Laboratory 306 Carmody Road ST LUCIA QLD 4067
Phone:	(07) 3214 2285
Fax:	(07) 3214 2288
Email:	naidu.bodapati@tag.csiro.au
Objectives	· To test if seed treatment with glycinebetaine or other osmoprotectants would increase rice seed germination and vigour at low temperature to enhance seedling establishment under controlled conditions.  · To examine if foliar application of these solutes would increase yield of cold stress affected rice plants under laboratory conditions.
Background	Low temperature at rice sowing time contributes to poor crop establishment and this accounts to about $30 million loss annually. Once in 3-4 years rice crop suffers from cold damage at flowering or panicle initiation stage leading to panicle sterility and this accounts for up to $120 million loss. Breeding to develop cold tolerant rice cultivars is a long-term strategy. Recent research shows that other plant species have a greater capacity to tolerate cold stress and this capacity is due mainly due to the accumulation of organic compounds called Osmoprotectants.
Research & Outcomes	Based on the patented previous research of CSIRO Tropical Agriculture, we have tested 5 osmoprotectants. Seed treatment with spermine increased seedling cold stress tolerance by increasing germination % from 80 to 100% and by increasing shoot weight by up to 62% and by almost doubling root growth.  Foliar application of glycinebetaine increased seed set by 94 and 39% in Amaroo and Doongara, respectively. Spermine foliar application increased grain set by 68 and 82 % in Amaroo and Doongara, respectively. The requirement of glycinebetaine or spermine is only about 30g/ha. Glycinebetaine is commercially available and would cost only less than $1/ha.
Implications	We recommend field trials be conducted prior to recommending the agronomic use of osmoprotectants. If proven successful, genetic engineering techniques should be used to introduce the biosynthetic pathways of these osmoprotectants.

Project Title	Sustainable control of aquatic earthworms and bloodworms in rice
RIRDC Project No:	DAN-146A
Researcher:	Dr Mark Stevens
Organisation:	NSW Agriculture Yanco Agricultural Institute Private Mail Bag YANCO NSW 2703
Phone:	(02) 6951 2611
Fax:	(02) 6951 2719
Email:	mark.stevens@agric.nsw.gov.au
Objectives	· Identify chemical treatments that will control the aquatic worm Eukerria saltensis, and quantify the relationship between worm populations and factors associated with crop establishment failure. · Evaluate low drainage residue alternatives to chlorpyrifos for bloodworm control in aerially sown rice crops, and investigate the colonisation of rice fields by species other than Chironomus tepperi.
Background	Aquatic earthworms and bloodworms are two of the most serious pests affecting rice crop establishment in NSW. The aquatic earthworm Eukerria saltensis has been known from Australia for almost 100 years, and has colonised the NSW rice area during the last 2 decades. Bloodworms are the larvae of chironomid midges, and attack the newly sown plants, feeding on both the developing roots and the rice seed itself. Whilst Chironomus tepperi is the most important species affecting crop establishment, other species are also implicated in causing crop damage.
Research	· Commercial-scale trails were conducted confirming the efficacy of fipronil seed treatments for bloodworm control. · Small-scale trials of alphacypermethrin for bloodworm control demonstrated good efficacy at rates below 10 grams active/ha, short-term water column persistence and no detectable crop residues. · Studies on bloodworm colonisation patterns in untreated fields across 2 seasons led to the identification of 18 species in the first 40 days after flooding. · Laboratory studies were used to determine how Eukerria infestations affect plant growth. · Field trials of potential Eukerria control compounds were conducted. Carbofuran applied to the soil before flooding provided only partial control at rates of 1 to 2 kilograms active/hectare. No effective treatments were located for use in flooded fields. · Monitoring of Eukerria populations has shown dryland cropping between rice crops reduces earthworm densities.
Outcomes	Fipronil seed treatments are now registered for commercial use against bloodworms in rice. Commercial scale trials of alphacypermethrin are currently being planned. Recommendations on how to minimise earthworm damage by changing crop management practices have been developed and are being widely promoted. Further work is required on bloodworm colonisation patterns and chemical control of Eukerria, and this is currently being conducted as part of Project DAN184A.

Implications

Growers now have a registered seed treatment for bloodworm control for the first time since 1994, allowing a substantial reduction in aerial spraying during rice crop establishment. Recommendations are now available on how best to minimise Eukerria damage. In the future, commercial development of alphacypermethrin for bloodworm control will provide an alternative to chlorpyrifos for direct spray treatments, whilst further studies on bloodworm colonisation will help to determine which species other than C.tepperi may be contributing to crop damage.

Agronomy/Crop Physiology
 

Project Title:	Investigation of the molecular mechanisms causing cold induced sterility in rice
RIRDC Project No:	ANU-29A
Researcher:	Professor BG Rolfe, Dr JJ Weinman and RL Williams
Organisation:	Genomic Interactions group, Research School of Biological Sciences,  Australian National University, PO Box 475 Canberra ACT 2601 &  NSW Agriculture, Yanco Agricultural Institute, PMB Yanco, NSW 2705
Phone:	02 6249 4054
Fax:	02 6249 0754
Email:	weinman@rsbs.anu.edu.au; rolfe@rsbs.anu.edu.au
Objective	Developing a clearer understanding of the mechanisms that cause cold-induced sterility will enable rice breeders to target specific physiological traits for selection in their ongoing program to develop varieties with lower yield losses due to cold-induced sterility.
Background	Reproductive cold damage is the main environmental limitation to yields in the NSW rice industry, causing an average loss of approximately 2 t/ha over the last 10 years. In extreme years this can cost the rice industry over $60M. The mechanisms by which cold temperatures cause pollen sterility are poorly understood and this lack of knowledge needed to be addressed to accelerate the rate at which resistance to cold-temperature sterility can be introduced into current commercial cultivars. Past research has been unable to identify the causes of the sterility. Recent technological advances offer the ability to discover what causes sterility and provide the breeding program with an understanding of what traits to incorporate for resistance to cold-temperature sterility.
Research	Experimental procedure was growth and 1, 2 and 4 day cold-temperature treatment of rice at the cold-sensitive pollen stage, micro-dissecting the anthers, analysing them for their total protein complement, and characterising proteins altered in amount by the cold-treatment. This was done for both the cold-sensitive Australian cultivar Doongara and the relatively cold-tolerant Hungarian cultivar HSC-55. Proteome analysis established the first two-dimensional anther protein maps of rice cultivars Doongara and HSC-55. Over 4,000 anther proteins at the young microspore stage were reproducibly resolved, representing up to 20% of the estimated total genomic output. The most cold-responsive proteins observed after plants were treated with a 12oC cold treatment were selected and analysed to predict their function.
Outcomes	At least 50 Doongara anther proteins were responsive to cold stress. Among these, 7 anther proteins have been identified with confidence. They include a lipid transfer protein down regulated after cold treatment and a translation initiation factor eIF-5a which is newly induced after cold treatment. The levels of these proteins did not vary in panicle samples or in the anthers of the relatively cold-tolerant cultivar HSC-55 in response to cold treatment. This implicates these proteins in playing a role in the process of cold induced sterility and suggests mechanisms involved in cold damage.
Implications	The cold-responsive Doongara proteins that were identified in this project can be used as molecular markers to identify more cold-tolerant cellular metabolism, and be used to help breed increased cold-tolerance into existing high yield cold-susceptible cultivars.

Publications

Weinman J.J. et al. (1998).2-Dimensional electrophoresis as a tool for protein differential display in biological research. 3rd International 2D Electrophoresis Meeting. September 1998 (Poster and Abstract). Siena, Italy. Imin N. et al. (1999). Differential protein display as a tool to identify condition-dependant or developmentally regulated proteins in rice anthers, mite infested clover, Melilotus nodules and wallaby brain. 3rd International conference on Functional Proteomics. April 1999 (Poster and Abstract). Boston, USA. Imin N., Weinman J.J., Rolfe B.G., and Williams R.L. (2000). Proteomic approaches to understanding the molecular mechanisms causing cold induced sterility in rice. (Manuscript prepared for publication)

`Project Title:	Towards the development of more effective nitrogen-fixing associations between rice cultures and endophytic bacteria
RIRDC Project No:	ANU-34A
Researcher:	Professor BG Rolfe, Dr JJ Weinman
Organisation:	Genomic Interactions Group, Research School of Biological Sciences Australian National University PO Box 475  CANBERRA ACT 2601
Phone:	02 6249 4054
Fax:	02 6249 0754
Email:	rolfe@rsbs.anu.edu.au; weinman@rsbs.anu.edu.au
Objective	Evaluation of endophytic nitrogen-fixing strains for their capacity to colonise and associate with Australian and IRRI rice cultivars and promote growth in these cultivars.
Background	Rice is a major food source, and the financial and environmental costs of nitrogen fertilisers mean that alternative ways of increasing rice yield would be a major benefit. In the Philippines research showed a diverse group of nitrogen-fixing bacteria are capable of colonising the roots to enhance yield. In Egypt, Rhizobium bacteria which nodulated berseem clover (Trifolium alexandrium) and colonised the roots of the non-legume rice were found. Rice inoculated with some of these strains stimulated plant growth and contributed to higher grain yields in three separate field trials.
Research	We have studied in detail the effects of several chosen bacterial strains on the early growth and development of rice. Rice seedlings inoculated with bacteria showed three sources of variability in growth outcome: these were due to the rice cultivar tested, the performance of different bacterial strains, and the growth medium used. Some bacterial strains stimulated rice growth, others had little effect, while others inhibited rice growth and development. Varying micronutrients could abolish the inhibition. Detailed studies of this inhibition phenomenon and bacterial colonisation were done with rice seedlings of cultivar Pelde inoculated with rhizobia labelled with the green fluorescent protein. The most rice-stimulatory strain associated only with the first anchor root of rice seedlings and exhibited a particular colonisation pattern by forming intercellular long lines inside the lateral roots between the plant cells. In contrast, other strains associated with the lateral root junctions. The addition of the phytohormone indole acetic acid (IAA) at various concentrations mimicked the Rhizobium-induced inhibition of rice growth, suggesting a role for phytohormone modulation in the rice growth effects. The genetic basis of growth inhibition was shown to be associated with the plasmids carried by the Rhizobiumtrifolii strains. Other studies indicate that these rice-associating bacteria posses genes that enhance their ability to intimately colonise niches on and within rice tissues and thereby affect rice seedling growth.
Outcomes	The inoculant Company Bio-Care Technology Pty Ltd has formulated a Rhizobium inoculant for yield trails with rice at the Agriculture NSW station at Yanco. Subsequent discussions with Bio-Care have led to an in-principal agreement to evaluate additional Rhizobium strains that may be generated. Genetic regions that contribute, positively or negatively, to rice seedling growth and micronutrients that modulate rice growth, have now been identified.
Implications	Some bacterial strains can significantly alter the growth of rice seedlings. Many of these strains colonise, survive and multiply inside the rice seedling tissues without producing any gross disease effects of plant growth. It is likely that certain soil conditions will enhance the rice growth inhibition or stimulation seen. We are now identifying the genes in these bacteria that are responsible for these characteristics. This should lead to the development and eventual use of specific rice inoculant strains.
Publications	Prayitno et al. 1999, Aust. J. Plant Physiol. 26: 521-535. Rolfe et al. 2000, in "The quest for nitrogen fixation in rice", Eds. Ladha, Reddy, IRRI Publication, pp 291-309, Los Banos, Philippines.

Project Title	Increasing the yield stability of short-season rices
RIRDC Project No:	DAN-119A
Researcher:	Russell Reinke
Organisation:	NSW Agriculture Yanco Agricultural Institute Private Mail Bag YANCO NSW 2703
Phone:	(02) 6951 2516
Fax:	(02) 6955 7580
Email:	Russell.Reinke@agric.nsw.gov.au
Objective	To investigate ways of achieving increased vegetative growth for short-season rice cultivars, thereby providing greater carbohydrate reserves for grain filling and higher, more stable yields across growing seasons.
Background	There is a need for rice varieties that take less time from sowing to harvest under NSW conditions, however current short-season cultivars generally have lower and less stable yields. Reductions in duration are generally achieved through shortening the vegetative phase, resulting in smaller biomass accumulation at the beginning of the reproductive stage, hence fewer carbohydrate reserves. High yields are still possible provided that optimal growth conditions are maintained throughout the latter stages of growth, but during sub-optimal conditions reserves are necessary to maintain carbohydrate supply to the developing grain. Faster vegetative growth, or sustained growth under low temperatures, should increase biomass and carbohydrate reserves available for grain filling, resulting in greater yield potential and less year-to-year variability in yields.
Research	Variation in biomass accumulation during the vegetative stage was noted among 37 rice cultivars from the International Rice Cold Tolerance Nursery, and increased seedling vigour at the three-leaf stage was clearly associated with improved mid-season biomass production. The principal factors affecting seedling vigour were investigated under varying temperature regimes in a smaller sub-set of cultivars in the Canberra phytotron. Of the three factors investigated viz. seed size, speed of emergence and the rate of leaf elongation immediately after emergence, leaf elongation rate (LER) showed most promise as a selection criteria to improve seedling vigour. The inheritance of LER was investigated in three populations varying in LER and seed size. Significant broad-sense heritability for the traits were noted in each population, however narrow-sense heritability was significant in only one population.
Outcomes	Seedling vigour has been identified as pivotal in obtaining rapid vegetative growth. Of the factors affecting seedling vigour, LER was identified as a useful trait to improve vigour without changing seed size, an important quality attribute.
Implications	This research has established the importance of seedling vigour as a necessary pre-cursor to rapid vegetative growth. Additional germplasm will be introduced and the screening protocols developed in this project used to introgress this trait into future short-season varieties.
Publications	Reinke, R.F., Richards, R.A., Angus, J.F., & Lewin L.G. (1999) Early vigour - an important foundation for rapid biomass accumulation. In proc. 2nd Temperate Rice Conference Sacramento Ca. June 13-17, 1999. Reinke, R.F. (2000) Factors influencing rice seedling vigour under temperate conditions In proc. International Rice Research Conference, IRRI, Los Banos, Philippines March 31-April 3, 2000

Crop Protection
 

Project Title	Further development of a mycoherbicide for control of Alismataceae weeds in rice
RIRDC Project No:	DAN-121A
Researcher:	Dr Eric Cother
Organisation:	NSW Agriculture Forest Road ORANGE NSW 2800
Phone:	(02) 6391 3886
Fax:	(02) 6391 3899
Email:	ric.cother@agric.nsw.gov.au
Objective	To further assess the biology of Rhynchosporium alismatis as a biological control agent.
Background	Previous research demonstrated the potential of this fungal pathogen for biocontrol of Alisma lanceolatum and Damasonium minus. For further development, it was necessary to know the host range of the fungus and its nutritional requirements in culture.
Research	Twenty eight species of aquatic plants in the Alismataceae and related families and 39 cultivars of 25 species of agriculturally important plants were tested for their reaction to inoculation with conidial suspensions of R. alismatis under glasshouse conditions. Lesions developed on species of Vallisneria, Triglochin and Marsilea but the fungus was re-isolated only from Vallisneria. Scattered infrequent lesions developed on leaves of barley, oats, triticale, lupin, soybean, lettuce and tomato but the pathogen was only re-isolated from lesions on soybean cv Bowyer. Cucurbits and tomato were the most susceptible plants, based on the frequency of re-isolation of the pathogen. There was no progression of disease in any of the infected plants and infection did not appear to influence plant growth and development. The use of this pathogen as a mycoherbistat for Alismataceae weeds is considered to pose a negligible risk to crops grown adjacent to, or in rotation with, rice crops in southern Australia. Nutrient composition of liquid-shake cultures in which an isolate was grown had a significant effect on the virulence of the resulting conidia There was a significant difference between isolates, produced in the same medium, in the subsequent rate of disease development. Isolate DAR 73158 and lima bean broth are considered to be the combination of choice for further studies to explore the fitness of conidia produced in small-scale biofermentors. Rhynchosporium alismatis produces thick-walled resting structures known as chlamydospores. The relative robustness of chlamydospores is an advantage over conidia for the future production of a mycoherbicide.
Outcomes	The safety of this fungus for use in rice fields has been established and small-scale inoculum production requirements have been defined.
Implications	Two potential development constraints, host range and nutritional requirements, have been removed. Ease of conidial production is assured but a reliable method to produce large quantities of easily harvested chlamydospores in a short period of time must be found. In addition, formulation of the inoculum in a matrix that maximises conidial survival, germination and infection should now be examined.

Project Title	Herbicide resistance development in dirty dora (Cyperus difformis) and starfruit (Damasonium minus)
RIRDC Project No:	UCS-23A
Researcher:	Mr Peter Baines
Organisation:	Charles Sturt University PO Box 588 WAGGA WAGGA NSW 2650
Phone:	(02) 6933 2177
Fax:	(02) 6933 2924
Objectives	The objectives of this preliminary research project were to evaluate specimens of aquatic weeds of rice, namely dirty dora and starfruit, to clarify: · whether resistance to herbicide demonstrated in one generation would be transferred to the subsequent generation; and · where samples have demonstrated herbicide resistance to be developing, whether higher rates of the herbicide would provide an effective control of the weed.
Background	Herbicide resistance represents a threat to Australian rice crops. Resistance to bensulfuron has been found in the aquatic weeds dirty dora, arrowhead and starfruit and as such has reduced the flexibility of weed control. Improved understanding of the transfer of resistance between generations and the value, if any, of higher rates of chemical are needed in order to generate advice to farmers on the management of herbicide resistance.
Research	Three biotypes of each of dirty dora and starfruit with the "developing resistance" status were further screened to the herbicide bensulfuron under glasshouse conditions across a range of rates including some well in excess of those used commercially. In respect of dirty dora, parent lines exhibited similar levels of resistance with F1 populations showing a minor decline in resistance relative to the parents. The difference, however, was not commercially significant. For starfruit, there were large differences in resistance level between biotypes. Again, slightly lower levels of resistance were found in the F1 generation suggesting incomplete dominance or a lower level of fitness among resistants.
Outcomes	The general conclusions from this preliminary study are: · that no conclusions can be drawn from these data about the number of genes involved in the resistance; · that high rates of Londax are not the solution for more effective control of resistant populations of dirty dora; · that the starfruit lines used exhibited lower levels of resistance which may indicate incomplete dominance;  · that low rates of Londax would not appear to be a useful strategy, particularly since most paddocks under rice would have received Londax treatment on more than one occasion. This exercise has probably raised more questions than were answered. For complete resolution of these questions the procedures would need to be applied specifically to an individual population and followed through at least two generations.
Implications	Farmers should have weeds tested for herbicide resistance status. Where resistance is present alternative weed control strategies should be employed rather than increasing the rates of bensulfuron.

Project Title	Biological control of starfruit in rice fields using a mycoherbicide
RIRDC Project No:	UCS-7A
Researcher:	Dr Eric Cother
Organisation:	Charles Sturt University PO Box 588 WAGGA WAGGA NSW 2678
Phone:	(02) 6391 3886
Fax:	(02) 6391 3899
Email:	ric.cother@agric.nsw.gov.au
Objective	To assess the potential of Rhynchosporium alismatis as a biological control agent for Damasonium minus (starfruit) in rice fields.
Background	The pathogen was assessed as a mycoherbicide for integrated management of starfruit by studying growth and sporulation in artificial culture, optimisation of disease development in the plant, microscopy of the infection process, fungal interaction with chemical herbicides, variability of host (starfruit) populations and effect of the disease on starfruit-rice competition.
Research	Rhynchosporium alismatis sporulated on a range of media but those based on lima bean were the most successful in producing large numbers of viable and infective conidia. Sporulation, germination and germ-tube elongation were greatest at 25° C and 30° C. Germ-tube growth was inhibited if the conidia were submerged in water. Infection by R. alismatis affected juvenile and adult starfruit plants differently: necrosis and chlorosis developed on aerial parts, mostly leaves, of adult plants, while juvenile plants were stunted.  Twelve fungal isolates caused different levels of disease that was apparent only on juvenile plants where the minimum inoculum density necessary to cause significant level of disease was 105 conidia mL-1. Water level during inoculation had no effect on disease development of adult plants. Growth suppression did not occur if the plants were submerged during inoculation. A competition experiment showed that when inoculated, the aboveground biomass of rice did not vary as the weed density increased. However, rice biomass declined with increasing weed density in the absence of the fungus. A dew period was not critical for disease development. Lesion development was enhanced at 25° C and reduced at temperatures equal to, or higher than, 30° C. Appressorium formation was the principal means of infection, starting to form after 4 h and infection hyphae were clearly visible within 24 h of inoculation. The rate of appressorium formation was greater at 25° C than at 30° C.  Bensulfuron-methyl (Londaxâ ) and MCPA (MCPA250â ) had the least effect on conidial germination whereas propanil (Ronacilâ ) had the greatest effect. There was a synergistic interaction between the fungus and sublethal doses of Londaxâ . The use of simple sequence repeat primers (SSRs) for PCR analyses to study genetic variability of different populations of the host revealed little variation between starfruit plants from different regions.
Outcomes	R. alismatis has characteristics to be a successful mycoherbicide. It can be produced abundantly in artificial culture, a dew period is not essential for disease development, it reduces the competition with rice by suppressing the weed and it has synergistic effects with at least one chemical herbicide (Londaxâ )
Implications	The fungus has excellent prospects for further development and should be evaluated in field experiments.

Publications

F.G. Jahromi, G.J. Ash and E.J. Cother (1998). Influence of cultural and environmental conditions on conidial production, germination and infectivity of Rhynchosporium alismatis, a candidate mycoherbicide. Australasian Plant Pathology 27: 180-185.

Technology Transfer and Communications
 

Project Title	2nd International Temperate Rice Conference and US Study Tour
RIRDC Project No:	RGA – 1A
Researcher:	Mr Mike Hedditch
Organisation:	Ricegrowers’ Association of Australia PO Box 706 LEETON NSW 2705
Phone:	(02) 6953 0433
Fax:	(02) 6953 4733
Email:	mhedditch@ricegrowers.com.au
Internet:	www.rga.org.au
Objective	To participate in the 2nd International Temperate Rice Conference; to network with R&D personnel from Temperate rice producing countries; to investigate US rice production technology and policy issues
Background	The Australian rice industry hosted the inaugural International Temperate Rice conference in 1994 at Yanco. The conference was a very successful forum for exchange of R&D information and networking. The industry saw merit in participating at the second Conference for the same reasons and in particular to develop young Australian rice R&D personnel and members of the RIRDC Rice R&D Committee
Research	The Conference was attended by 250 delegates from 20 overseas rice producing countries. The Australian delegation presented papers at the two major symposia (Biotechnology and the Environment) and at the six concurrent sessions (Genetics, Agronomy, Entomology, Grain Quality, Weeds, Diseases). Following the Conference, the Australian delegation of 22 persons participated in a US rice industry study tour, examining rice production technology and policy issues in California, Mississippi, Arkansas and Louisiana.
Outcomes	The following conclusions/recommendations were reached by the participating group · The inclusive nature of the Australian Rice R&D Program and its scientific excellence lead the Temperate rice world. · International germplasm exchange will become restricted due to plant variety protection laws/biotechnology. · Australia’s plant variety protection laws are inadequate to properly protect public plant breeding programs. · Environmental guidelines governing pesticide levels in drainage water and rice straw burning are tightening and will probably flow on to Australia. · Serious rice herbicide resistance in the US requires improved adoption of resistance management in Australia.
Implications	The RIRDC Rice R&D Committee will need to focus on appropriate ways to overcome the challenges facing the US industry, which will flow on to Australia. The committee should also consider supporting an Australian delegation to participate in the 3rd International Temperate Rice Conference to be held in Punte del Este, Uruguay, March 2003.
Publications	Reinke R "Conference opens up possibilities for new research techniques" Farmers Newsletter No 154, March 2000 Woodside D "2nd International Temperate Rice Conference – a growers perspective" - Farmers Newsletter No 154, March 2000 Williams R "Ricegrowing US Style" - Farmers Newsletter No 154, March 2000 Fitzgerald M "Research in Rice Quality – What’s happening is America" Farmers Newsletter No 154, March 2000

Harvesting, Storage, Handling and Transport
 

Project Title	An evaluation of rice harvesting machinery and systems
RIRDC Project No:	KDI-18A
Researcher:	Mr Scott Boyle
Organisation:	Kondinin Group 26 The Esplanade WAGGA WAGGA NSW 2650
Phone:	(02) 6921 5825
Fax:	(02) 6921 4182
Email:	scott@kondinin.com.au
Objectives	The objective of this project is to equip rice growers with information that will allow them to reduce harvest costs, improve grain quality and so increase overall profitability. The outcome of this project is a comprehensive assessment of the factors contributing to harvest system efficiency, including machinery and labour management. It has brought together all of the work conducted by the Kondinin Group since the first evaluation in 1996. This work has been disseminated to all growers through the release of the Rice Harvesters Reference.
Background	Correct timing, duration and operation at harvest is critical to rice quality and farmer returns. Rice, unlike other cereals, is a crop that has its greatest value as a whole grain, and is largely governed by harvest management. Delayed or badly managed harvest and incorrect harvester set-up can lower whole grain millout to a fraction of potential yield. The first stage of this project, conducted in 1996, showed that the harvest can be the most expensive field operation in rice production, with a range in costs from $7 to $70 per tonne depending on field conditions. In 1997 the second stage of this project investigated the conduct of the harvest on rice quality and on growers’ incomes.
Research	Harvesting equipment performance was assessed as material-processing performance, derived from field tests. It was also assessed as economic performance, computer as on costs provided by farmers, company and dealer personnel. Comparative harvest costs were then calculated in $/tonne, based on the criterion of throughput at 3% machine loss. Rice sample quality was assessed by the RCL appraisals laboratory. Throughout the three years of projects, more than 30 different combinations of harvester aid fronts were tested on a range of sites over three seasons. Included were John Deere CTS; Case-IH 2166, 2188 and 2388; John Deere 9500 and 9600; Gleaner R62; FNH TX66; Shelboume Reynolds and Harvest Hustler strippers tested against standard fronts; plus the draper front and Vibramat attachments for cutterbars.
Outcomes	The major outcome from this project has been the publishing of the "Rice Harvesters Reference" bringing together three years of rice harvesting research by the Kondinin Group. This is the compilation of all current rice harvesting research by the Kondinin Group. This is the compilation of all current rice harvesting machinery information relevant to the Australian rice industry and results of three years of Kondinin Group research into harvester performance. The 1998 harvesting trial results showed total losses for stripper equipped machines were higher than cutterbar machines and that front losses for strippers were also higher than cutterbar machines. This is consistent with the two previous years of research.

Other findings included that trash levels did not vary with machine type or speed, but did with bays or plots. The average trash level was 0.9%, which compares with 4% for the 1996 harvest and 2.23% for the 1997 harvest. Overall the results show general trends and reinforce the fact that for best performance, regardless of machine type, the harvester should be set up for the crop and weather conditions at that time. Another major component of this project was a study of field efficiency, which was found to have a big impact on the overall profitability of machinery operation. This study showed that a chaser bin (or bins) that can keep a harvester from stopping to unload or wait to unload would increase field efficiency from around 55% to about 85%. Bay shape, corners, machine overlap keeping the front full and breakdowns or stoppages account for as much as 25% of field efficiency. The study also reinforces the importance of keeping the harvester working by keeping the grain away from the machine. The largest harvester is only as good as the worst link in the chaser bin, storage and transport system.

Implications

With every rice grower receiving a copy of this manual, they will be armed with the latest harvesting information to improve the quality of the grain delivered. Harvesting is a critical final stage in a successful rice crop. It has a significant effect on grain quality. The results of the project and farmers’ input has suggested that much more research and extension of harvesting machinery information is needed. Some fresh challenges to the industry are presented with the results of this project, along with proposed methods to meet the challenges.

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