Bait method to detect Pythium species that grow at high temperatures in hydroponic solutions

Pythium helicoides, P. aphanidermatum and P. myriotylum are important pathogens that cause root rot of several crops in hydroponic culture and in ebb-and-flow irrigation systems. These species belong to a group of Pythium species that can grow at temperatures higher than 40°C. We developed a method for baiting these high-temperature Pythium species and evaluated its practicality to monitor their presence in nutrient solutions. Seeds of cucumber, tomato, radish, hemp, perilla and millet and leaves of bent grass and rose were tested as baits in hydroponic systems. Hemp, perilla and radish seeds and bent grass and rose leaves were more effective than the other baits for Pythium zoospores, and bent grass leaves were the most effective. In a sensitivity test, bent grass leaf traps (BLTs) detected three Pythium species after only a 1 day exposure to suspensions of 40 zoospores per liter of water, and the frequency of detection increased with zoospore density and with baiting period. A temperature of 38°C was optimum for the selective reisolation of the high-temperature Pythium species from the BLTs. The BLT was also tested with inoculated and noninoculated miniature roses that shared a recirculating nutrient solution. The pathogen was detected in the nutrient solution 23 days before the disease spread to the noninoculated roses. In addition, P. helicoides was detected 30 days before the disease was evident in a commercial greenhouse. The baiting method described here will be useful for monitoring high-temperature Pythium species in recirculating hydroponic culture systems.     

Quantitative loop-mediated isothermal amplification method for the detection of <Emphasis Type="Italic">Vibrio nigripulchritudo</Emphasis> in shrimp

Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60 min at 63°C. Quantitative analysis was then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification of the initial DNA concentration with a sensitivity of 10² copy numbers equivalent to 2.3 colony forming units/ml or 0.3 fg/μl. The LAMP assay was able to specifically detect two representative strains of V. nigripulchritudo, whereas other Vibrio and non-Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time (T _t) versus the log of bacterial number. A high correlation coefficient (R ² = 0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.     

Identification of Petunia hybrida cultivars that diurnally emit floral fragrances

In order to identify genetic resources for breeding fragrant petunias for use as bedding plants, volatile compounds released by day from the flowers of 40 commercial Petunia hybrida cultivars were analyzed using a solid-phase micro-extraction technique coupled with GC–MS. The three cultivars with solid deep-blue flowers that accumulate malvidin in corollas with high tissue pH were found to emit abundant iso-eugenol as the principal floral fragrance. Several other cultivars that emitted considerable amounts of methylbenzoate and/or benzylbenzoate from the flower were also identified. Association between the floral fragrance and the other floral traits such as floral anthocyanin composition and corolla-tissue pH was discussed.     

Performance of a closed recirculating system with foam separation, nitrification and denitrification units for intensive culture of eel: towards zero emission

The development of a closed recirculating aquaculture system that does not discharge effluents would reduce a large amount of pollutant load on aquatic bodies. In this study, eel were reared in a closed recirculating system, which consisted of a rearing tank, a foam separation unit, a nitrification unit and a denitrification unit. The foam separation unit has an inhalation-type aerator and supplies air bubbles to the rearing water. The growth of eel, which were fed a commercial diet, was satisfactory, with gross weight increases of up three times in 3 months. The survival rate under the congested experimental conditions was 91%. The foam separation unit maintained oxygen saturation in the rearing water at about 80%. Furthermore, fine colloidal substances were absorbed on the stable foam formed from eel mucus and were removed from the rearing water by foam separation. Ammonia oxidation and the removal of suspended solids were accomplished rapidly and simultaneously in the nitrification unit. The ammonia concentration and turbidity were kept at less than 1.2 mg of N per litre and 2.5 units, respectively. When the denitrification process was operated, nitrate that accumulated in the rearing water (151 mg of N per litre) was reduced to 40 mg of N per litre. The sludge was easily recovered from the nitrification and denitrification tanks, and the components were found suitable as compost. Based on these results, the intensive aquaculture of freshwater fish such as eel can be achieved using a closed recirculating system without emission.     

大白菜子叶离体培养再生植株

 探讨了植物激素、AgNO₃ 和琼脂浓度对大白菜子叶培养芽再生和植株再生的影响。结果表明:优化培养基条件为MS 培养基中加入5 mg / L BA、0. 5 mg/ L NAA、2 mg/ L AgNO₃ 和1. 2%~ 1. 6 %琼脂。培养过程中, 子叶的乙烯释放量对芽再生起很重要的作用, 具有高芽再生率的基因型或高浓度琼脂的培养条件下, 子叶产生的乙烯量较少。培养基中有AgNO₃ 存在时, 尽管子叶的乙烯释放量增加, 芽再生率也有提高。对子叶的乙烯释放量和不定芽再生的关系进行了讨论。     

Anther culture in rice proportionally rescues microspores according to gametophytic gene effect and enhances genetic study of hybrid sterility

Background

To investigate plant hybrid sterility, we studied interspecific hybrids of two cultivated rice species, Asian rice (Oryza sativa) and African rice (O. glaberrima). Male gametes of these hybrids display complete sterility owing to a dozen of hybrid sterility loci, termed HS loci, but this complicated genetic system remains poorly understood.

Results

Microspores from these interspecific hybrids form sterile pollen but are viable at the immature stage. Application of the anther culture (AC) method caused these immature microspores to induce callus. The segregation distortion of 11 among 13 known HS loci was assessed in the callus population. Using many individual calli, fine mapping of the HS loci was attempted based on heterozygotes produced from chromosome segment substitution lines (CSSLs). Transmission ratio distortion (TRD) from microspores was detected at 6 of 11 HS loci in the callus population. The fine mapping of S₁ and S₁₉ loci using CSSLs revealed precise distances of markers from the positions of HS loci exhibiting excessive TRD.

Conclusions

We demonstrated that AC to generate callus populations derived from immature microspores is a useful methodology for genetic study. The callus population facilitated detection of TRD at multiple HS loci and dramatically shortened the process for mapping hybrid sterility genes.

     

The transition time from the juvenile phase to the adult phase differs in soybean cultivars ‘Enrei’ and ‘Peking’

Plants develop juvenile phase to adult phase in vegetative stage. Although soybean is a very important crop worldwide, there has been only one study of the juvenile–adult phase change. In this study, we determined that the juvenile–adult phase change occurred at different stages in two soybean cultivars that differ in their photosensitivity. Cultivar ‘Enrei’ (E1e2e3E4) is weakly photosensitive and cultivar ‘Peking’ (E1E2E3E4) is strongly photosensitive. In ‘Enrei’, the leaf size gradually increased at a constant leaf position regardless of the difference in day length. In ‘Peking’ plants transferred to short‐day conditions at several leaf development stages, leaf size gradually increased at different leaf positions. Expression of miR156 by ‘Enrei’ transferred to short‐day conditions had nearly the same pattern as that of ‘Enrei’ grown under long‐day conditions. In ‘Peking’, the expression of miR156 had different patterns in younger leaves of plants subjected to either a short‐day treatment or long‐day conditions. These results indicate that the E2 and E3 loci that regulate photosensitivity also regulate the expression of miR156 and the juvenile–adult phase change in soybean.     

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002