Dehydration responsive element binding protein (DREB)1-type transcription factors in transgenic rice improve tolerance to drought, salt, and freezing

Drought, salt and freezing are environmental conditions that dramatically affect the growth of plants and crop yields. Genetic engineering has high potential for improving the stress tolerance of crops using gene transfer technology. A cis-acting promoter element, DRE/C-repeat (CRT), plays an important role in regulating gene expression in response to these environmental stresses. The Arabidopsis transcription factors DREB1s/C-repeat binding factor (CBF)s bind to DRE and control the expression of many stress-responsive genes. In rice, we have isolated cDNAs for DREB1 homologs and named them OsDREB1s. We have reported previously that overexpression of the DREB1 or OsDREB1 genes induces strong expression of the many stress-responsive genes in transgenic Arabidopsis plants, which in turn boost stress tolerance to high salt and freezing. In this study, we analyzed the functions of the OsDREB1A and OsDREB1B genes in stress tolerance using transgenic rice and compared it with that of DREB1A. We observed improved tolerance to drought, high-salt and cold stresses in the transgenic rice plants. 　　To analyze the function of rice OsDREB1A and OsDREB1B proteins, we generated transgenic rice (Oryza sativa cv. Kita-ake) overexpressing OsDREB1A or OsDREB1B under the control of the constitutive maize ubiquitine promoter. To compare the function of the rice OsDREB1 family with that of Arabidopsis DREB1/CBF family in rice, we also generated transgenic rice overexpressing Arabidopsis DREB1A, DREB1B or DREB1C. These transgenic rice plants showed growth retardation under normal growth conditions in the same way as did the transgenic Arabidopsisplants overexpressing OsDREB1 or DREB1. Both OsDREB1 and DREB1 showed a similar effect of growth retardation under normal growth conditions. The tolerance of the transgenic rice overexpressing OsDREB1 or DREB1 to drought, high-salt and cold stresses was compared with that of the control plants (Fig. 1). Although 0%, 5% and 5% of the wild-type plants survived under drought, high-salinity and cold conditions, respectively, 17 to 80%, 13 to 83% and 25 to 60% of the plants overexpressing OsDREB1 or DREB1 respectively survived under the same conditions. 　　Many plants accumulate a number of compatible osmolytes such as proline and various sugars under drought, high-salt and cold stress conditions, and these osmolytes function as osmoprotectants that contribute to tolerance to stress. Transgenic Arabidopsis plants overexpressing DREB1A have been reported to accumulate proline and various sugars even under unstressed control conditions. We examined whether transgenic rice overexpressing OsDREB1A or DREB1Aalso accumulated proline and various sugars under control conditions. The free proline levels in the wild-type rice under high-salt and cold stress conditions were approximately 5- and 7-fold higher than those under the control conditions, respectively. Even under control conditions, the transgenic plants overexpressing OsDREB1A and DREB1A accumulated 4- to 5-fold and 6- to 15-fold higher levels of proline, respectively, as compared with the wild-type rice. Similarly, wild-type rice plants accumulated high levels of sugars such as raffinose, sucrose, glucose and fructose in response to high salt or cold stress. Even under unstressed conditions, the transgenic rice plants overexpressing DREB1A accumulated high levels of these sugars. 　　The whole genome sequence of rice (cv. Nipponbare) has recently been determined. In addition, approximately 22,000 sequences of independent full-length cDNAs of rice (cv. Nipponbare) were registered in a public database, and an oligoarray containing approximately 21,500 rice probes was produced based on these sequences. To analyze the upregulated genes in the transgenic rice overexpressing OsDREB1A or DREB1A, we generated transgenic rice plants overexpressing OsDREB1A or DREB1A using cv. Nipponbare. We identified 12 target genes of OsDREB1A in the transgenic rice using microarray and RNA gel blot analyses (Fig. 2). These genes encode proteins that are thought to function in stress tolerance in plants and most of these genes were induced by at least one of drought, high-salt and cold stresses; many promoters of the upregulated genes contain DRE sequences. These results indicate that the DREB1/CBF cold-responsive pathway is conserved in rice and the DREB1-type genes are quite useful for improvement of tolerance to environmental stresses in various kinds of transgenic plants, including rice. (Y. Ito, K. Maruyama and K. Yamaguchi-Shinozaki)

Fig. 1. Drought, high-salt and cold stress tolerance of the transgenic rice plants (cv. Kita-ake) overexpressing OsDREB1 or DREB1 genes, and wild-type plants. The stress treatments were conducted as described below. Drought = 17-day-old plants with water withheld for 9 days and then supplied with water again for 13 days. High-salt = 17-day-old plants soaked in 250 mM NaCl solution for 3 days and transferred to pots under normal growth conditions for 19 days. Cold = 17-day-old plants were exposed to a temperature of 2 ˚C for 93 h and then to 28 ˚C for 18 days. ‘Wild’, ‘Os1A’, ‘Os1B’, ‘At1A’, ‘At1B’ and ‘At1C’ mean wild-type plants and transgenic rice plants overexpressing OsDREB1A, OsDREB1B, DREB1A, DREB1B and DREB1C, respectively. Fig. 2. Expression of the OsDREB1Aupregulated genes in the transgenic rice plants (cv. Nipponbare) overexpressing the OsDREB1A or DREB1Agenes and in the wild-type plants. Sixteen-day-old transgenic rice (cv. Nipponbare) seedlings grown hydroponically were sampled under unstressed conditions (control) to prepare total RNAs. The wild-type plants were kept in 250 mM NaCl for 5 h (NaCl), water withheld for 5 h (dry) or kept at 4 ˚C for 24 h (cold). Each lane was loaded with 10 mg of total RNA.

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Spatial identification of the crop-fallow rotation cycle and potential capacity for regeneration of fallow in northern Laos by satellite imagery

　　In northern Laos, shifting cultivation, namely slash-and-burn agriculture, has been conventionally practiced for upland rice (Fig. 1). However, the crop-fallow rotation cycle is tending to shorten due to forest conservation policies and population pressure, causing deterioration of productivity and in turn affecting farmers’ livelihoods in the region. To investigate the land condition in these areas, we developed a robust method of identifying the crop-fallow rotation cycle spatially using periodical observed satellite imagery, i.e., Landsat/Thematic Mapper (TM) and Enhanced Thematic Mapper + (ETM+). The study site was an area of 442,000 ha within N19˚30’; E101˚45’; – N20˚00’; E102˚30’ in Luang Prabang Province. 

　　According to the typical calendar for slash-and-burn agriculture in northern Laos, land is cleared from February to early April, and burned in March and April. Sowing is practiced from late April to June, and harvesting is from September to November. The most drastic change in the land surface is slashing to clear the fallow plants in the late dry season: the area used for cropping in the imminent rainy season changes to non-vegetation as a result of slashing for land preparation, but fallow areas show uninterrupted vegetative growth. The classification of vegetation/non-vegetation is one of the most reliable applications using remote sensing; therefore, land use in each year was identified by the presence of vegetation in the late dry season. In this study, 8-scene imagery of TM and ETM+ acquired annually from 1995 to 2003 were applied.

　　The results of classification of 8 scenes were represented by an 8-digit code to track the land use change year by year. For 1995-2003, approximately 77,000 ha (17.3%) had never been cropped but 41,000 ha (9.2%) had been used for cropping every year. The former was regarded as forest and the later as sedentary farms growing rice. The rotated area between cropping and fallow was classified by crop intensity. The areas cropped 1-2 times, 3-4 times, and 5-6 times occupied 129,000 ha (29.1%), 83,000 ha (18.7%), and 54,000 ha (12.2%), respectively (Fig. 2).

　　Vegetation in fallow shows a succession from shrub to bush, and ultimately to forest. Normalized Difference Vegetation Index (NDVI), derived from TM and ETM+, is known to be related to biomass. Hence, NDVI was applied to assess the process of plant regeneration in fallow. Fallow was classified by fallow length (age) from the 1st to 7th years and the mean NDVI was calculated for each fallow age. The mean NDVI increased constantly with fallow age, and it was estimated that 11 years was needed to reach to the same NDVI as that of forest. However, the cropping intensity mentioned above showed that long-term fallow exceeding 7 years accounted for only 17.3% of the area, implying that a large area might be re-used before vegetation is sufficiently regenerated. In addition, the potentiality for vegetation regeneration in fallow was assessed by comparison to the mean NDVI corresponding to the same fallow age. The results indicated that low-potential areas showed a lower NDVI than the mean for 66,000 ha, confirming that low-potential area increased as cropping intensity increased.

　　Due to restrictions on slash-and-burn agriculture, farmers in northern Laos have been urged to switch to more productive and conservational farming systems. The method and spatial information provided in this study will be useful for regional scaled land resource management.

(Y. Yamamoto)

Fig. 1. View of hills in northern Laos where slash-and-burn agriculture is implemented. Fig. 2. Cropping intensity for 1995-2002.

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Improvement of drought stress tolerance by gene transfer of a transcription factor, AREB1, involved in ABA-responsive gene expression

　　Due to the food crisis and environmental pollution, it is becoming ever more important to breed environmental stress-tolerant crops. Plant productivity is greatly affected by environmental stresses, such as drought and high salinity. Genetic engineering has high potential to improve the stress tolerance of crops using gene transfer technology. Several different approaches to improving the stress tolerance of plants by gene transfer have been attempted. In our strategy, a gene encoding a transcription factor involved in abscisic acid (ABA)-responsible gene expression was used.

　　The plant hormone ABA is produced under drought and high-salinity stress conditions and plays a pivotal role in tolerance to these stresses. Numerous drought- and high-salinity-stress-inducible genes have been reported in plants, and many of them are also activated by ABA. In analyses of the promoters of such ABA-regulated genes, a conserved cis-element designated ABA-responsive element (ABRE; PyACGTGGC), which controls ABA-regulated gene expression, has been identified. The drought-responsive expression of an Arabidopsis gene, rd29B, is mainly mediated by ABA. Two ABREs play a crucial role in the expression of rd29B as cis-elements. Using yeast one-hybrid screening, we cloned three different cDNAs encoding ABRE-binding proteins (AREB1, AREB2, and AREB3) of Arabidopsis. Expression of AREB1 and AREB2 is upregulated by ABA and drought and high-salinity stresses. Both AREB1 and AREB2 function as trans-acting activators, as identified by transient expression analysis in protoplasts. 

　　To determine the temporal and spatial expression patterns of AREB1, we analyzed transgenic Arabidopsis plants expressing an AREB1 promoter-GUS reporter gene. In unstressed plants, weak GUS expression was observed in the roots, leaf vascular tissues and hydathodes. By contrast, drought or ABA treatment of plants enhanced the AREB1 promoter activity in all tissues (Fig. 1). The subcellular localization of the AREB1 protein in plant cells was further analyzed using a Green Fluorescent Protein (GFP):AREB1 fusion protein. GFP fluorescence was detected in the nucleus, indicating that AREB1 is localized in the nucleus of the plant cells.

　　We generated transgenic plants overexpressing the AREB1 cDNA under the control of the CaMV 35S promoter (35S-AREB1). However, constitutive overexpression of intact AREB1 alone is not sufficient to activate its downstream genes such as RD29B under normal growth conditions. The ABA-induced modification of the AREB1 protein seems to be also required for the expression of its downstream genes. To overcome the masked transactivation activity of AREB1, we created an activated form of AREB1 (AREB1∆QT) carrying the AREB1 internal deletion mutants containing the bZIP DNA binding domain and transcriptional active domain of AREB1. We generated transgenic plants overexpressing AREB1∆QT and examined their stress tolerance. When plants grown in pots were not watered, almost all the wild-type plants died within 12 days. In contrast, nearly all the transgenic plants of two independent lines survived this level of drought stress and continued to grow when watering resumed, demonstrating their enhanced drought tolerance (Fig. 2). We analyzed upregulated genes in the transgenic plants using a microarray and found that eight genes in two groups were upregulated: LEA-class genes including RD29B and ABA- and drought-stress-inducible regulatory genes such as HIS1-3 (encoding a linker histone H1), GBF3 and RD20. All eight upregulated genes have at least two ABRE sequences in their promoter regions, and are known to be ABA- and stress-inducible. By contrast, an areb1 null mutant and a dominant loss-of-function mutant of AREB1 (AREB1:RD) with a repression domain exhibited ABA insensitivity. Further, AREB1:RD plants displayed reduced survival under dehydration. These data thus suggest that AREB1 regulates ABRE-dependent ABA-signaling that enhances drought tolerance in vegetative tissues. We believe that the activated form of AREB1 may be useful for improving the stress tolerance of agriculturally important crops by gene transfer. 

(K. Yamaguchi-Shinozaki and Y. Fujita)

Fig. 1. Histochemical localization of AREB1 promoter-driven GUS expression in Arabidopsis: (a) 2-week old plant, (b) 2-week-old plant treated with 50 µM ABA. Bars = 5.0 mm

Fig. 2. Enhanced tolerance to drought in the 35S-AREB1∆QT plants (2 independent transgenic plant lines). Watering was withheld from 3-week-old plants for 12 days before the photograph was taken. Number codes = number of surviving plants out of the total.

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First report on the growth reduction of soybean caused by Heterodera glycines and its distribution in Paraguay

The soybean cyst nematode, Heterodera glycines, one of the most widespread and destructive soybean pests, was identified in Paraguay in 2002. Since soybean is the most important crop in the country, the distribution of this nematode was surveyed. Soil samples were collected from 64 fields in the main soybean producing districts of Alto Parana, Itapua, Canindeyu and Caaguazu, from 2004 to 2006 in Paraguay. A soil sample of approximately 5 kg was collected from the top 0-15 cm soil from 10 points in each field. GPS coordinates of the sampling points were recorded. Nematode cysts were extracted using a flotation sieving technique. The nematode was identified on the basis of the shape and vulval cone structure of the cysts. Heterodera glycines was recovered from a total of 16 fields (Fig. 1), of which eight are new records of this nematode in Paraguay. In Canindeyu, H. glycines was detected for the first time in seven out of the 28 fields surveyed, i.e., a frequency of 25%. The nematode was also detected in two fields in which it had been previously reported. More than 10 cysts/50 g soil were extracted from some of these fields. The results suggest that H. glycines may be distributed over large areas in this district. The nematode was also detected in four fields in Alto Parana and three fields in Caaguazu, respectively, but not in the Itapua district. 　　During the surveying process, an area of stunted plants was observed in late January 2005 in a soybean field at San Alberto, Alto Parana, that had been cropped with soybeans for many years. In this field, the soybean cv. Monsoy 7204 had been sown without tillage on October 22, 2004 and was at the seed-producing stage when sampled. To asses the effects of the nematode on the growth of the plants, a total of 32 and 36 soybean plants were randomly dug up from 10 points in the stunted area and in an area with no apparent stunting. The heights of the plants were measured and degrees of female infection were rated. Soil samples were collected at each of the 10 points where plants had been removed in the stunted and non-stunted areas. Soil attached to the root systems was also collected to examine the effect of the nematode on the growth of soybean. Nematode cysts were extracted using a sugar solution flotation technique.In the stunted area, the average height of soybean plants was 48 cm, less than 60% of that of plants (85 cm tall) from the non-stunted area. Chlorosis was not obvious at the time of survey, but browning of leaf margins was observed on the soybean plants in the stunted area. The female infection index was high (60.1) on the root-systems of stunted plants and fairly low (19) on the roots of the apparently non-stunted plants (Fig. 2). In the soil, the nematode population density was also extremely high in the stunted area (Fig. 3), where more than 12,800 eggs were extracted from 10 g of soil collected from the surface of roots, more than 20 times the density found in the non-stunted area. A similar trend was observed in the rhizosphere soil. Stunting of soybean plants caused by this nematode was detected in four other fields in surveys of 2005/2006 year soybeans. 　　These results indicate that H. glycines is potentially a serious threat to soybean production even in the “Terra rossa” soil type and subtropical climate conditions in Paraguay. More insights are necessary, therefore, on the ecology and pathogenicity of the nematode to allow the implementation of effective management measures. (Z. Sano)

Fig. 1. Distribution of Heterodera glycines in Paraguay surveyed in 2004-2006.
The key to each point indicates place and field code.

Fig. 2. Comparison of plant growth and degrees of infection of Heterodera glycines females between a stunted area and that of a non-stunted in a soybean field in San Alberto. Female infection index: none—0, light—25, moderate—50, considerable—75, and heavy—100. 

Fig. 3. Comparison of Heterodera glycines populations in stunted and non-stunted areaa in a soybean field in San Alberto, Alto Parana.

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Effects of rice straw compost on rice cultivation on alluvial soil area in Mekong Delta, Vietnam

　　Few farmers in the Mekong Delta, Vietnam use organic matter for their rice cultivation. This is quite different from farmers in Vietnam’s Red River Delta. Soil in the alluvial soil area in the Mekong Delta is fertile because nutrients have been supplied by floodwaters that arrive annually. However, it appears that exhaustion of soil fertility has been accelerated by intensive agriculture and the supply of nutrients by floodwaters has decreased due to dyke construction.

　　We conducted field experiments in an alluvial soil area in the Mekong Delta to elucidate the effects of rice straw compost. Rice was cultivated over 10 seasons with replicated management. Rice straw compost (6 Mg ha^–1) and different doses of chemical fertilizer were applied to compare with general fertilizer application without application of organic matter.

　　The results showed that it is possible to decrease chemical fertilizer (N, P and K) input by 60% without decreasing rice yield by applying rice straw compost (Fig. 1). Although the difference was not significantly different except for the 6^th season, rice yields cultivated using chemical fertilizer alone were constantly lower than rice yields where rice straw compost and decreased amounts (up to 60%) of chemical fertilizer were applied.

　　Rice yields with general fertilizer application were significantly lower after outbreak of disease (in the 6^th season). Rice cultivated using general fertilizer application was more severely damaged by rice blast than rice cultivated with rice straw compost and smaller amounts of chemical fertilizer (Fig. 2).
　　Soil organic matter, total-N and available-N did not show a clear difference among treatments after the 10th season. Originally, these concentrations were high in the field (Total-C: 31.7 gC kg^–1, Total-N 3.0 gN kg^–1, Available-N: 145 kgN ha^–1 in the 0-10 cm soil layer). Ten seasons were not enough to cause significant differences in these parameters.

　　The results showed that general fertilizer application should be improved to increase fertilizer efficiency and mitigate pest damage. 

(T. Watanabe)

Fig. 1. Grain yield over 10 seasons. WS: Wet Season, DS: Dry Season Fig. 2. Rice heavily damaged by blast disease after general fertilizer application. Right: General fertilizer application (100-30-30 N-P2O5-K2O kg ha-1). Left: Rice straw compost (6 Mg ha-1) and (80-24-24 N-P2O5-K2O kg ha-1).

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Mitigation of nitrous oxide emissions from agricultural soil by 2-amino-4-chloro-6-methylpyrimidine

　　Agricultural soil is the largest anthropogenic source of nitrous oxide (N₂O), a greenhouse gas. Nitrification inhibitors have been used to increase fertilizer efficiency and to mitigate nitrogen leaching. They also decrease N₂O emissions from agricultural land. 2-chloro-6 (trichloromethyl) pyridine (nitrapyrin) is a strong nitrification inhibitor that has been used with liquid ammonia in the USA. However, it cannot be amended to solid fertilizer because of its low solubility and high volatility. To select an alternative nitrification inhibitor that is easy to handle and which will effectively mitigate N₂O emissions from the soil, 2-amino-4-chloro-6-methylpyrimidine (AM), sulfathiazole and 1-aryl-2-thiourea were compared. These chemicals can be amended into solid fertilizer.

　　The results of soil incubation experiments showed that AM mitigated N₂O emission from soil where nitrogen fertilizer had been applied. The effect of AM on reducing N₂O emissions was weaker than nitrapyrin but stronger than sulfathiazole and 1-aryl-2-thiourea (Table 1). Application of AM did not inhibit soil respiration. The side effects of AM on 7 bacterial species that contribute to agricultural production in the field were tested. AM proved to inhibit the growth of these bacteria to the same degree as other inhibitors (Table 2). These results show that AM selectively inhibits nitrification bacteria and that the side effects on other bacteria are minor. It was concluded that AM can be used to mitigate N₂O emissions from agricultural soil as an alternative to nitrapyrin. 

(T. Watanabe)

Table 1. Effect of nitrification inhibitors on mitigating N₂O emissions (Soil incubation experiment).

Table 2. Inhibitory effects of nitrification inhibitors on 7 bacteria useful in agriculture.  

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Change in nitrogen cycle related to agricultural activity from 1990 to 2000 in Khon Kaen Province, Thailand

　　With the development of the Thai economy from 1990 to 2000, the agricultural situation changed, showing increased chemical fertilizer application rates and lower numbers of buffalo being raised, resulting in a shortage of animal manure for farmers. To clarify the subsequent changes in the nitrogen cycle, we estimated the nitrogen cycle in Khon Kaen province in Northeast Thailand in 1990-92 and 2000-02.

　　The nitrogen cycle in Khon Kaen province was estimated using Thai agricultural statistics, study reports of food consumption and human excreta discharge, animal feed consumption and animal excrement production, survey data on the utilization of crop residues, human excreta, garbage, animal excrement and food factory (rice mill, sugar mill, cassava chips and starch) waste, measurement data of crop residue production and nitrogen content of crop residues, animal excrement and food factory waste, and journal articles on N2 fixation and nitrogen losses from farmland.
　　

Nitrogen cycle related to agriculture activity in 1990-92 and 2000-02 in Khon Kaen province is shown in Fig. 1. Chemical fertilizer application doubled from 16 kg N ha^–1 year^–1 in 1990-92 to 34 kg N ha^–1 year^–1 in 2000-02, caused by an increase in the chemical fertilizer application rate to rice and cassava cultivation by farmers to the recommended rate. Sugarcane was already having chemical fertilizer applied at the recommend rate in 1990. Animal manure input to farmland decreased 40%, from 28 kg N ha^–1 year^–1 in 1990-92 to 16 kg N ha^–1 year^–1 in 2000-02, because animal excrement production fell due to the sharp decrease in numbers of buffalo being raised, although the number of cattle, pigs and poultry increased. The amount of crop residues returned to farmland more than doubled, from 4 kg N ha^–1 year^–1 in 1990-92 to 14 kg N ha^–1 year^–1 in 2000-02, due to sugarcane residue (leaves and tops) production increasing as a result of a more than doubling of sugarcane area cultivated, and because the rate of return of sugarcane residue to farmland increased from 40% to 54%. The nitrogen balance in farmland changed from a negative –23 in 1990-92 to a positive value of +10 kg N ha^–1 year^–1 in 2000-02, caused by an increase in the chemical fertilizer application rate to +18 kg N ha^–1 year^–1 and crop residue input of +10 kg N ha^–1year^–1 in spite of a –12 kg N ha^–1 year^–1 decrease in animal manure input. Nitrogen supply to farmland thus changed mainly to chemical fertilizer, which threatens the sustainability of crop production due to degradation of soil organic matter. Nitrogen loaded from humans to the hydrosphere risks polluting underground water in populated areas. For sustaining crop production and environmental conservation in Khon Kaen province, effective utilization of crop residues is needed, such as the use of sugarcane residue as mulch and rice straw as compost, improvements in animal raising systems to increase manure production, and the development of treatment and effective use of human excreta. 

(N. Matsumoto and K. Paisancharoen)

Fig. 1 Nitrogen cycle-related to agriculture activity in 1990-92 and 2000-02 in Khon Kaen Province, Thailand (kg N ha^-1 year^-1). Values outside parentheses indicate nitrogen flow in 2000-02, and values inside parentheses indicate nitrogen flow in 1990-92. Values in italics are calculated by subtracting output from input.

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Actual situation of local soil fertility management on agricultural land in the Sahel, West Africa

　　The consistently low level of agricultural production in the Sahel zone of West Africa, especially in Niger, due to poor soil characteristics has hampered sustainable development in this region. It is important to make use of local farmers’ knowledge through a better understanding of its context in order to mitigate existing problems that are impeding sustainable technology development. This is the basic and essential approach for a new technology to be more acceptable and sustainable at local farmers’ level.

　　The agricultural lands in the study site are managed in two ways: intensively and extensively. Recycling (use of household waste and livestock excrement) and corralling (application of livestock excrement through the tethering of sheep, goats or cows in the fields in the evening) are systems categorized as intensive management, while fallow is a system practiced under extensive management. Based on a field survey of 2,430 ha, fallow is clearly the major system at the study site, since it accounted for 66% of surveyed fields. The corralling and recycling system account for the remaining 18% and 16%, respectively. Despite their large area, fields under the fallow system have the poorest soil fertility level of all due to the combination of longer cultivation periods (6 years on average) and shorter period of restoration (3 years). This impedes the production of millet, as shown in the results (Table 1). Local farmers adopt a recycling system (transporting household waste and/or animal dung) during the dry season, which lasts from late October to May. Since transport of waste using either animal carts or buckets carried on the head is time-consuming, this system is usually limited to fields adjacent to the village. On the other hand, corralling is done some distance from the village, since the pastoralists who use this system move to where they have better access to water for their livestock: areas which tend to be distant from the village. Consequently, fields located far from the village are managed using the fallow system. This phenomenon was confirmed quantitatively by a study of the distribution pattern of a field under different management systems (Fig. 1). Through factor analysis, the distance of a field from the village proved to be a more important factor than soil type for local farmers when managing soil fertility on their land (Fig. 2).

(K. Hayashi)

Table 1. Productivity of millet and soil fertility characteristics of local fertility management systems.

Fig. 1. Traits of local systems on soil fertility management with distance.

Fig. 2. Factors in the decision-making of local farmers on soil fertility management in the study area

Photo 1. A compost heap in a compound of a village. This is the source for the recycling system and local farmers transport it once every few days.

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Attempt to practically use a lactic acid bacterial strain, SP 1-3, for making good quality silage in Thailand

Feeding of good quality silage (GQS) throughout the year is a suitable way to stably increase raw milk production in spite of its not being in wide use in Thailand. However, making GQS in Thailand is not always ensured if ensilage depends on natural fermentation. Therefore, lactic acid bacteria (LAB) strains to make GQS were screened, and strain SP 1-3 was selected. 　　For the practical use of this strain, a culture method for harvesting large numbers of LAB cells in a low-cost and convenient way was examined, and a culture medium composed of materials that are readily available in Thailand was designed. The medium designed here consisted of molasses 2.0%, rice bran 0.5%, yeast extract 0.2%, and mineral mixture (pH 6.5), and gave a cell mass of 109 cfu/ml after 24 h culture at 35˚C. The cost of this medium was calculated at about 23 yen/L, about 1/60 of that of MRS broth. A silage inoculant (spray-dried granule) from strain SP 1-3 cultured using this medium was prepared for trial. This granule was stable and kept a living cell count of 108~9 cfu/g under storage at 4˚C for 28 days, as shown in Table 1, and reliably improved the fermentation quality of Napier grass silage prepared beforehand in the laboratory. To use these granules for the preparation of practical-scale silage, a low-cost and convenient method of increasing the cell mass of the LAB strain at farm level was developed using a 5-liter plastic drinking water bottle. LAB cell counts in the water (4.5 L), in which had been suspended 45 mg of granules together with the abovementioned medium, rose to 3.85 x 109 cfu/ml after culturing for 24 h. This LAB liquid (4.5 L) is ideal for preparing 4.5 ton of GQS. The additional cost using this system was calculated to be about 50 yen/ton. 　　Based on these results, Pangola grass and Erianthus silages inoculated with the LAB liquid were prepared on a practical scale. After being kept at 30~35˚C for 90 days, the silage pH, the organic acids and volatile basic nitrogen (VBN) content and microbial cell counts were examined. The results are shown in Table 2. Compared with silage without inoculation of LAB, the fermentation quality of silage inoculated with LAB liquid was improved as shown as pH reduced to below 4, increased lactic acid content, and reduced VBN content. These improvements were particularly notable in Erianthus silage. However, the yeast cell count was not reduced by inoculation with LAB. Abundant yeast cells in silage prepared in Thailand were hitherto known, and this trend was marked in the case of Pangola grass silage in this experiment. Abundant yeast cells in silage leads to the outbreak of aerobic deterioration after opening the silo. However, silage pH 48 h after opening the silo rose to almost neutral while the pH was about 3.5 on initial opening. 　　In addition, the palatability of silage inoculated with LAB was compared with non-inoculated silage by the cafeteria method using Brahman steers and native Thai steers. However, no significant difference in intake amount was seen between both types of silage (data not shown). (S. Ohmomo)

Table 1.  Variation in the survival of the cells in granules prepared from strain SP 1-3. Table 2.  Fermentation quality of practical-use silage inoculated with LAB strain SP 1-3.

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The impacts of climate change on the world food market: Mid-term analysis using International Food and Agricultural Policy Simulation Model (IFPSIM)

Global warming caused by concentration of carbon dioxide in the atmosphere will be a major issue in world food markets over the next century. Agricultural production will be affected by climate change, such as rising temperatures or droughts, mainly through changes in crop yields. Environmental changes in a particular country or region will affect agricultural production in other countries or regions indirectly through the trade of food products. Thus, it is likely that climate changes, such as global warming, will cause drastic changes in agricultural markets. This research examines the possible impacts of global warming on world agricultural product markets using IFPSIM, the world food model of JIRCAS. 　　Rainfall and temperature affect crop production. It is assumed that only yield is affected by these climatic variables. The following double-log form yield function is estimated as lnYH t = a + b 1T + b 2 ln PRC t + b3lnTMPt , where YH is yield, T is the time trend, PRC is rainfall measured in millimeters, and TMP is temperature measured in degrees Celsius. The difference function is estimated if the probability that the yield data is non-stationary is higher than ten percent. 　　The rainfall and temperature data are the average values from the Global Historical Climatology Network (GHCN). The climate variables are based on monthly data on the flowering or silking season of each crop. The year term of the estimation period is from 1961 to 2000. Large countries are divided into regions based on the cropping map. The crops in the model are wheat, maize, other coarse grains, rice, and soybeans. The other coarse grains include barley, rye, oats, millet, and sorghum. The yield and production data for each crop is that of Food and Agriculture Organization of the United Nations (FAO)-STAT. The base year of the simulation is 1998 and the term of the projection is from the base year to 2025. 　　The assumptions of the simulation are as follows: (1) the cropping calendar is fixed, (2) the area available for cropping is fixed, (3) the climatic variables directly affect only yields, (4) the temperature measured in degrees Celsius of all countries and regions increases 0.05% per year, and (5) all other parameters are fixed. 　　Table 1 shows the results of the estimation. The parameters are the elasticities of yield for rainfall and temperature. The results show that rainfall has a significantly positive effect on the production of maize and other coarse grains. On the other hand, higher temperatures have a negative effect on the production of most crops except for rice. Figs. 1 and 2 show that the growth rates of production in different countries between 2005 and 2025. Wheat production in the USA will increase due to falling production in other countries. The production of rice in the USA will sharply decrease while that in Japan, South Korea, and many developing countries will increase. Fig. 2 shows that the world production of most farm commodities will not be severely affected by high temperatures, even though each country will be affected by climate change. This is because the sign of elasticity is different for each country, as seen in Table 1. Rice production, however, will be significantly affected by climate change, largely because of the sharp drop in rice yields and production in the USA and South Asian countries. 　　While the scenario is fairly simple (temperature increases 0.05% per year for all countries, or about 0.2 ˚C over the next twenty years), drastic changes in crop production are seen in some countries. Countries that suffer severe damage due to higher temperatures may need to consider changes in cropping patterns and practices.          (J. Furuya)

Table 1. Elasticity of yields for climatic variables. Fig. 1. Growth rate of production in the USA. Fig. 2. World growth rate of production.

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Utilization of sugarcane silage as feed for beef cattle in Northeast Thailand

　　Northeastern Thailand is the biggest region of beef cattle production in Thailand. Most beef cattle are raised on small mixed farms. It is generally recognized that the shortage of forage in the dry season is the chief factor limiting beef cattle production in the region. Additionally, hay and silage preservation is not easy, because of frequently unsuitable weather for forage drying during the rainy season and low fermentable substrate content in tropical grasses for silage fermentation. Sugarcane, which is planted widely in this region, has high amounts of fermentable substrates, i.e., sugars, and gives high yields. Therefore, there is the potential for preparing reliable and high-quality silage that will provide a steady supply of food throughout the year. This study aimed to make and evaluate sugarcane silage, and to investigate the effect of sugarcane feeding on growth performance in beef cattle.

　　 Pangola grass (Digitaria eriantha), which was cut at early bloom, and sugarcane, which was harvested at around six months after regrowth, were chopped and ensiled in plastic bags, then evaluated for their chemical components and quality six months later. Crude protein content in sugarcane silage was lower than that in pangola grass silage (Table 1). On the other hand, the lactic acid content was higher for sugarcane silage than pangola grass silage, and pH and spoilage ratios due to molds were lower for sugarcane silage than pangola grass silage. These results show that the fermentation quality of sugarcane silage was higher than that of pangola grass. Total digestible nutrient content, digestible energy content and metabolizable energy content of the sugarcane silage were respectively 49.6% DM, 9.7 MJ/kgDM and 7.8 MJ/kgDM. A feeding experiment with the beef cattle diet was conducted at a beef cattle raising farm in Khonkaen District. Six American Brahman crossbred bulls (around 18 months old) were separated into two groups, one of which was fed Ruzi grass (Brachiaria ruziziensis) hay with concentrate and the other sugarcane silage with concentrate. Both groups were fed the same roughage-to-concentrate ratio (38:62) for three months. The results showed that an average daily gain of 0.9 kg/day could be achieved with sugarcane silage during the feeding period (Table 2). 

　　It is possible to supply silage which of good and reliable quality using sugarcane, and to raise beef cattle using sugarcane silage as the basic diet. However, sugarcane silage needs to be consumed as soon as possible after opening due to its low aerobic stability.

(T. Suzuki)

Table 1.  Chemical components and fermentation quality of pangola grass silage and sugarcane silage.

Table 2. Feed chemical components and performance of beef cattle fed Ruzi grass hay or sugarcane silage with concentrate†

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Piperine, a component of long pepper, suppresses insect pests of rice storag

　　Under the JIRCAS international research project entitled "Development of low-input technology for reducing postharvest losses of staples in Southeast Asia," the research team studied the growth-inhibitory effect against stored-product insect pests of various botanicals. The target insect pests were the rice weevil (Sitophilus oryzae), maize weevil (S. zeamais), red flour beetles (Tribolium castameum) and rice moth (Corcyra cephalonica), which are commonly found in stored rice worldwide. The project team found a strong growth-inhibitory effect of a spice plant, long pepper (Fig. 1, Piper retrofractum) against weevils and the beetle. Long pepper is a vine in the family Piperaceae; the fruit has a hot taste similar to black pepper. It is widely used in many types of food and medicines in tropical and subtropical regions. In the experiment, dried and powdered long pepper fruits were mixed into feed and its effect against insect pests was examined. Long pepper completely suppressed adult emergence from eggs of weevils at a dose of 0.5% (w/w), and the growth of 1–7-day-old red flour beetle larvae by approximately 90% at a dose of 5% (w/w). 

In addition, long pepper showed a definite suppressive effect against red flour beetles in the model experiment using imitation warehouse-like boxes in which brown rice and starch powder mixed with long pepper powder were separately placed. Long pepper also inhibited the development of another important insect pest, the rice moth (Corcyra cephalonica), but the effect was not as strong as against Coleoptera. The growth-inhibitory component of long pepper was then isolated by activity-guided fractionation using weevils. The obtained pale yellow crystals of the active principle were identified as piperine by mass and nuclear magnetic resonance (NMR) spectrometry. Piperine is an alkaloid found in the black pepper seed that has a hot flavor. Purified piperine substantially suppressed the adult emergence of weevils by 50% (ED50) at a dose of approximately 50 ppm (Fig. 2). The risk of long pepper extract or piperine to human health is likely to be much lower than most other synthetic insecticides, since it has a long history of use as a spice. We therefore believe that long pepper has the potential to provide a safe alternative to synthetic insecticides. The project team is currently studying how to apply long pepper or piperine extracts on an individual scale in rice storage. 

(G. Trakoontivakorn, R. Juntarawimoon, Y. Hanboonsong and K. Nakahara)

Fig. 1. Long pepper (Piper retrofractum), fresh fruit and leaves (left bottom); dried fruits (top right).

Fig. 2. Growth- inhibitory effect of piperine against weevils.

Eggs of weevils were reared in the feed pellet containing purified piperine at the indicated doses. Emerged adult weevils during 8 weeks were counted. The chemical structure of piperine is shown in the panel.

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Arachidonic Acid can Help the Aquaculture Industry to Take off in Developing Countries “Hatcheries in the Tropics require arachidonic acid for stable fry production”

　　Aquaculture contributes significantly to food production, incomes and jobs in developing regions. However, the persistent constraint in aquaculture development is presently the supply of good quality seeds (fry) in quantity. Hatcheries are expected to provide stable fry supply for farmers, but fry production is often highly variable due to low survival rates. During the period 2002–2005, the Southeast Asian Fisheries Development Center - Aquaculture Department (SEAFDEC/AQD, Iloilo, Philippines) and JIRCAS have collaborated on studies aimed at developing advanced diets to improve the quality and production of eggs, larvae and seedstock for tropical marine fish species.
　　

The most striking difference between cold water fish species and tropical fish species is that arachidonic acid, which is found in only minor quantities in cold water fish species with their high EPA levels, is one of the major essential fatty acids in the ovaries, testes, eggs and muscles of tropical fish species from mangrove areas or coral reefs in the Philippines, Malaysia and Ishigaki, Japan. It is notable that the major essential fatty acids in tropical fish species are DHA and arachidonic acid (not EPA) at a ratio of about 2:1. This result suggests that a dietary ratio of DHA/arachidonic acid (not EPA) of about 2 or greater can be recommended as an ideal regimen for broodstock of tropical fish species.

　　Thus, arachidonic acid is a major fatty acid widely distributed in tropical marine fish species and is likely to be important to reproduction and larval/fry performance. However, most studies on essential fatty acids in relation to fry production have been focused on EPA and DHA, and the potential value of arachidonic acid has not been applied to fry production technologies for tropical and subtropical fish species.

　　In the mangrove red snapper, total egg production and total number of spawn can be clearly improved providing a formulated diet containing arachidonic acid at 5 g/kg. Moreover, this diet can increase the percentage of normal larvae and boosts the cumulative survival rate to more than twice the level of those on diets without arachidonic acid. Rabbitfish fry, when fed rotifers or brine shrimps enriched with a combination of DHA and arachidonic acid, always showed the best survival rate. We conclude that adding arachidonic acid to broodstock diets or larval feeds dramatically improves seed production. Improved hatchery production of seedstocks can thereby help aquaculture take off in the developing tropical countries.

(H. Ogata)

Fig.1 Arachidonic acid levels are higher in tropical fishes than in cold water fishes. Fig.2 Dietary arachidonicacid improves egg production. Total egg production (x106) Fig.3 Arachidonic acid improves survival of rabbitfish fry. Survival (%)

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Recycling-oriented aquaculture system utilizing natural ecological functions

　　Coastal aquaculture in mangrove swamp areas has rapidly developed and expanded since the middle of the 1980s, and the production of cultured prawns has greatly increased. During the 25 years from 1975 to 2000, shrimp farming areas increased more than sixfold, from 129 km² to 811 km². However, excessively intensive utilization of brackish water areas has resulted in the destruction of many mangrove ecosystems and water pollution affecting the culture ponds and adjacent coastal areas. Recovery of mangrove ecosystems is of utmost importance for restoring the productivity of coastal aquaculture and fisheries. To develop a rational and sustainable utilization strategy for mangrove ecosystems, it is necessary to carry out studies relating to the practical use of coastal aquaculture systems that are compatible with the preservation of the environment.

　　To investigate rational and sustainable utilization of benthic organisms for prawn aquaculture, the community structure of macrobenthic organisms was examined in Samut Songkhram aquaculture ponds in the innermost part of the Gulf of Thailand. A total of 84 species/taxa of macrobenthos were identified, in which 42 species/taxa were found in the six experimental ponds: four ponds used for prawn culture and two ponds planted with mangrove stands. The dominant species belong to three taxonomic categories; that is, (1) sedentary, tube-dwelling spionids, Polydora sp., free-living nereids, Perinereis sp. and some other polychaetes (Annelida); (2) gastropods such as Cerithidea cingulata, Cerithium coralium, Thiara riqueti, and Stenothyra ovalis, which live on the surface or in the shallow layers of the substratum (Mollusca); and (3) barnacles, Balanus sp., and some small crustaceans such as ostracods, copepods, harpacticoids, amphipods and dipterans (Arthropoda). Population density, biomass and species diversity in the mangrove planted ponds were higher than those in the prawn culture ponds, and depending on their behavioral traits, the depth of sediment utilized.

　　Model experiments of a recycling-oriented prawn aquaculture that exploited the natural purification capacity of the mangrove ecosystem and the productivity of the macrobenthic community were carried out in the experimental ponds in Samut Songkhram. The survival rate and average weight of prawns in the water-circulated culture systems were higher than those in the closed culture system. When prawns were cultured under semi-intensive conditions in mangrove planted ponds, their average size increased about by 25%, and artificial feed could be reduced about by 19% compared to the intensive culture system. Diversity and biomass of the macrobenthos decreased during the culture experiments in the prawn culture ponds, while they increased in the mangrove ponds, indicating the possibility that the prawns fed on the small macrobenthos such as polychaetes and juvenile gastropods as a natural food source under aquaculture conditions. The results of feed preference experiments in the laboratory demonstrated that prawns significantly selected polychaetes and crustaceans over commercially sold pellet feed. It was concluded that macrobenthic invertebrates are an important natural feed for prawn aquaculture and may participate closely in the transfer of energy from the producer organisms to the cultured prawns.

　　On the basis of the results of the present study, we constructed a model of a recycling-oriented aquaculture system by utilizing the natural ecological functions of benthic and planktonic organisms, mangrove stands and seaweeds. 

(Y. Fujioka)

A model of recycling-oriented aquaculture system utilizing natural ecological functions of benthic and planktonic organisms, mangrove stands and seaweeds. 

Overview of recycling-oriented aquaculture system of Samut Songkhram.

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Co-culture of Black tiger shrimp (Penaeus monodon) and Sea grapes (Caulerpa lentillifera)

　　We are attempting to develop co-culture in shrimp farming intended to improve the environment within shrimp ponds by using biofilters. Caulerpa lentillifera, a species of edible seaweed, was chosen for co-culture with Penaeus monodon.
　　It became obvious that C. lentillifera has high water purification abilities (dissolved nutrient uptake and physical filtration) and it also offers a hiding place for the shrimp.
On the other hand, C. lentillifera is expected to guarantee an alternative income in the event that the P. monodon perish in large numbers, making it possible to prevent the abandonment or neglect of shrimp culture ponds.

　　The co-culture of P. monodon and C. lentillifera markedly decreased the ammonia nitrogen (by up to approximately 50%), which exerts the most adverse effect on the cultivation of underwater shrimp. We discovered that favorable water quality could be easily maintained over the long term by co-culture with seaweed, since seaweed grows more steadily than phytoplankton. As a result of C. lentillifera utilizing the nutrients dissolving from leftover shrimp feed, chemical fertilizers are not needed for growth of C. lentillifera. The growth of seaweed by this system showed an almost identical value to that grown independently using chemical fertilizers.

　　Caulerpa lentillifera has the ability to adapt to environments with a salinity of approximately 25~35% and water temperatures between approximately 23~33 ˚C. It was thus thought that C. lentillifera would be a suitable species for shrimp culture ponds where temperature and salinity can readily change. The growth of shrimp is not obstructed by co-cultivation with the C. lentillifera.
　　

Using this co-culture system, the number of bacteria adhering to the gills of the shrimp decreased, and the biomass of bacteria in the water stabilized. It is possible to prevent total annihilation of the shrimp from the normally inescapable yellow head virus (YHV), since 1% of shrimps infected with YHV in several earthen shrimp ponds were observed to survive due to co-culture with C. 

　　When a taste test targeting Thai people was conducted, the unexpected flavor and fragrance of this species of C. lentillifera presented no problems, and the texture was particularly popular. It has the potential to become a popular food throughout Bangkok and subsequently Thailand, and may have a bright future as a secondary means generating income for small-scale shrimp culture farmers.
　　We believe that seaweed has a potentially large market in Thailand, since it can be utilized as human food, in cosmetic and pharmaceutical products, and for animal feed. Our experiments indicate that some seaweeds are effective in improving the aquatic environment. A further series of experiments will be necessary to choose other organisms, like shellfish, to act as effective biofilters in an integrated system.

(K. Hamano)

Changes in ammonia (NH3), nitrite (NO2) and nitrate (NO3) concentration in the water of monoculture and co-culture tanks. Error bars indicate standard deviation with n = 4.

A Black Tiger Shrimp that survived mass death by YHV disease in a culture pond where Caulerpa lentillifera grows abundantly.

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Effect of shuttle breeding by taking advantage of the wide latitude in Japan for heading traits of wheat

　　Usually it takes many years to breed wheat cultivars. Rapid generation advancement is, therefore, one of the more important techniques for shortening the process. On the other hand, photoperiod response (photoperiod-sensitive or insensitive) and vernalization response (winter or spring growth habit), which are controlled by Ppd and Vrn genes, respectively, are particularly important physiological characters, since they are responsible for earliness of heading and are thus closely related to regional adaptability. If the desirable Ppd and Vrn genotypes can be selected by natural and artificial selection during rapid generation advancement, its system should be very efficient. In this study, to establish an efficient method of rapid generation advancement applicable to spring wheat breeding programmes, the impact of shuttle breeding through fall-sown cultivation on Ishigaki (24˚N), Okinawa, in the southernmost part of Japan, followed by the spring-sown cultivation at Memuro (42˚N), Hokkaido in the northernmost part of Japan on the fluctuation in the frequency of the Ppd and Vrn genotypes was investigated.

　　Analysis of heading date in characteristic wheat cultivars and the F2 populations indicated that the earliness of heading in Okinawa and Hokkaido was closely related to both Ppd and Vrngenotypes and the Vrn genotype, respectively. The two hybrid populations segregating for Ppd or Vrn genes, ‘Saitama 27’ (Ppd-S, Vrn-A1) × ‘Haruhikari’ (no Ppd genes, Vrn-A1 Vrn-B1) and ‘Saitama 27’ × ‘Norin 59’ (Ppd-S, no Vrn genes), were treated using rapid generation advancement (F2: Okinawa, F3: Hokkaido). As a control, the same cross combinations were also treated using the single seed descent method in F2 - F3 generations. The analysis of their photoperiod and vernalization responses showed that plants with photoperiod-sensitivity and winter growth habit were eliminated in Okinawa and those with winter growth habit were eliminated in Hokkaido (Table).

　　The present study proved this system of rapid generation advancement to be effective in eliminating the photoperiod-sensitive and winter types, therefore making it useful for photoperiod-insensitive spring wheat breeding. In addition, it was considered that, in a cross combination between distantly related parents, the following shuttle breeding system scheme would be appropriate, taking the degree of fixation into account: 1st year (F2: Okinawa, F3: Hokkaido) and 2nd year (F4: Okinawa, F5: Hokkaido). On the other hand, for photoperiod-sensitive spring wheat breeding, a long-day treatment is necessary to avoid eliminating photoperiod-sensitive types in fall-sown cultivation in Okinawa.

(M. Tanio, K. Kato, N. Ishikawa, T. Tabiki, Z. Nishio, K. Nakamichi,
Y. Tamura, M. Sato, H. Takagi and M. Matsuoka)

Table1. Fluctuation in photoperiod response (A) and vernalization response (B) with rapid generation advancement (RGA) or single seed descent (SSD) in two wheat hybrid populations

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Evaluation of functional components in passionfruit cultivated under drip-fertigation and light culture

　　Passionfruit is produced from South Kyushu to Okinawa in Japan and is consumed as a table fruit or as juice. The flavor is appealing and the taste is subacid to acid. Passionfruit contains many functional components such as carotenoids and ascorbic acid. In open field culture, passionfruit is harvested only in summer and winter. However, it is possible to harvest it in autumn as well by using drip-fertigation and light culture, which comprises drip-fertigation culture that allows optimum control of fertilizer application and ground temperature, and light culture using energy-efficient red LEDs that induce flower bud formation (Fig. 1). We attempt to identify contained in passionfruit and examine the influence of harvesting season on the quantity of the functional components under drip-fertigation and light culture.

　　‘Summer Queen’ passionfruit used in this study was collected from the Kagoshima Fruit Tree Experimental Station. As a result of analyzing the juice of the passionfruit using HPLC, it was found from the absorption spectrum that the main carotenoid in the passionfruit was z- carotene (Fig. 2). The UV-visible spectrum of z-carotene showed lmax at 379, 400 and 425, with high spectral fine structure (%III/II 93). After filtering the passionfruit juice on a Sep-Pak C18 cartridge, the concentration of ascorbic acid was rapidly and simply determined using the Reflectoquant Ascorbic Acid Test (Merck Ltd., Japan) without the need to use HPLC. The quantities of z-carotene and ascorbic acid in the passionfruit juice varied significantly according to harvesting season (Fig. 3). The concentrations of z-carotene and ascorbic acid were significantly higher in fruits harvested in autumn than in summer and winter. There was a direct correlation between z-carotene and ascorbic acid content (Fig. 4) that was not influenced by harvesting season, making it possible to estimate the concentration of z-carotene by measuring the concentration of ascorbic acid using the Reflectoquant Ascorbic Acid Test.

　　These results indicate that drip-fertigation and light culture is useful for harvesting autumn fruit that contains numerous functional contents.

(H. Kato)

Fig. 1. Light culture by red LEDs (A) and drip-fertigation (B) of passionfruit. Fig. 2. UV-visible spectrum of major carotenoid fraction (retention time = 59.29 min). The conditions were as follows: column, YMC carotenoid S5 µm (4.6 x 250 mm); temperature, 40 ˚C; flow rate, 1.0 mL/min; mobile phase, A: 20% (v/v) MeOH in H2O, B: MTBE: MeOH (9:1); gradient, 26% B hold for 15 min, to 34% B in 25 min, to 44% B in 10 min, to 56% B in 15 min, hold for 10 min, then back to 26% in 5 min. The structural formula of z-carotene is illustrated. Fig. 3. Concentration of z-carotene (A) and ascorbic acid (B) in the juice of ‘Summer Queen’ passionfrui The concentration of z-carotene is *measured as b-carotene corresponding. Vertical bars indicate SE (n = 4-15). Different letters are significant at P < 0.05 according to the Tukey-Kramer HSD test. Fig. 4. Relationship between contents of ζ-carotene and ascorbic acid in juice of ‘Summer Queen’ harvested in summer (●), autumn (▲) and winter (□). *** Significant at the 0.1% level.

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Moving distance a day of the released citrus psyllid, Diaphorina citri using a newly developed marking method

　　The Asian citrus psyllid, Diaphorina citri Kuwayama, is a vector of Asian Citrus Greening Disease which is spreading in Asia, including Japan, North and South America. Citrus greening disease or Huanglongbing (HLB) is the most destructive disease affecting citrus production. However, although knowledge of its vector ecology, especially dispersal studies, is needed for its control, data regarding the movement of this psyllid is minimal or entirely lacking. We have recently developed an effective marking method for this psyllid using fluorescent powder. We investigated the moving ability of the psyllids using this newly developed marking method and took into account moving characteristics as related to weather factors in the field.

　　First, we prepared 20,000 marked psyllids (Fig. 1) in a release experiment, reared for mass propagation in a glasshouse on orange jasmine. The reared psyllids used in this experiment were CG-free, since the CG bacterium does not propagate in orange jasmine. These marked psyllids were monitored at 65 points which comprised four orange jasmine trees per point, located concentrically at 50-m intervals up to 350 m from the release point (the center of our station) after one day (Fig. 2). Release experiments were conducted twice, in May and October 2005, at our station on Ishigaki Island, southwest of Japan.

　　The first experiment was conducted on March 21, 2005. The daily mean wind speed and daily mean wind direction at our station were 2.2 m/s and SSW, respectively, on the day of release. One day later, 516 psyllids had taken up residence on the orange jasmine plants at the monitoring points. Of those, 79.5% were observed on the north side and on the leeward of the release point. (Fig. 2).
　　The second experiment was conducted on October 15, 2005. The daily mean wind speed and daily mean wind direction at our station were 5.8 m/s and NNE, respectively, on the day of release. One day later, 118 psyllids were observed on the orange jasmine plants at the monitoring points. Of these, 96.6% were observed on the south side, on the leeward of the release point. The maximal distance traveled after a day of these marked psyllids was measured as 200 m and the mean moving distance was estimated at 66.9 m.

　　These results indicate that the marked vector psyllids moved to the leeward of the release point, and the maximal distance traveled after a day of these marked psyllids was measured as 350 m. The mean distance traveled in the two field experiments was estimated at 73.4 m. Since the psyllids moved 350 m in the first release experiment, some may have father migrated to the leeward side, exceeding the monitoring limit. These results will contribute to basic knowledge of the control strategy of this insect. 

(T. Nakata)

Fig. 1. Large amount of marked psyllids on orange jasmine for release experiments.

Fig. 2. The movement of marked psyllids in a day from a release point.

20,000 marked psyllids were released in the field on March 21, 2005, and a day later the number of psyllids was monitored visually on the orange jasmines. The intervals of the concentric circle were 50 m and the maximum range was up to 350 m.

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