A Microfluidic Diagnostic Device Capable of Autonomous Sample Mixing and Dispensing for the Simultaneous Genetic Detection of Multiple Plant Viruses.

As an efficient approach to risk management in agriculture, the elimination of losses due to plant diseases and insect pests is one of the most important and urgent technological challenges for improving the crop yield. Therefore, we have developed a polydimethylsiloxane (PDMS)-based microfluidic device for the multiplex genetic diagnosis of plant diseases and pests. It offers unique features, such as rapid detection, portability, simplicity, and the low-cost genetic diagnosis of a wide variety of plant viruses. In this study, to realize such a diagnostic device, we developed a method for the autonomous dispensing of fluid into a microchamber array, which was integrated with a set of three passive stop valves with different burst pressures (referred to as phaseguides) to facilitate precise fluid handling. Additionally, we estimated the mixing efficiencies of several types of passive mixers (referred to as chaotic mixers), which were integrated into a microchannel, through experimental and computational analyses. We first demonstrated the ability of the fabricated diagnostic devices to detect DNA-based plant viruses from an infected tomato crop based on the loop-mediated isothermal amplification (LAMP) method. Moreover, we demonstrated the simultaneous detection of RNA-based plant viruses, which can infect cucurbits, by using the reverse transcription LAMP (RT-LAMP) method. The multiplex RT-LAMP assays revealed that multiple RNA viruses extracted from diseased cucumber leaves were successfully detected within 60 min, without any cross-contamination between reaction microchambers, on our diagnostic device.

Ferrigenium kumadai gen. nov., sp. nov., a microaerophilic iron-oxidizing bacterium isolated from a paddy field soil.

An iron-oxidizing bacterium, designated strain An22T, which was isolated from a paddy field soil in Anjo, Japan, was described taxonomically. Strain An22T was motile by a single polar flagellum, curved-rod, Gram-negative bacterium that was able to grow at 12-37 °C (optimally at 25-30 °C) and at pH 5.2-6.8 (pH 5.9-6.1). The strain grew microaerobically and autotrophically by oxidizing ferrous iron, but did not form stalks, a unique structure of iron oxides. The major cellular fatty acids were C16 : 0 and C16 : 1ω7c/C16 : 1ω6c. The major respiratory quinones were UQ-10 and UQ-8. The strain possessed ribulose-1,5-bisphosphate carboxylase/oxygenase indicating an autotrophic nature via the Calvin-Benson-Bassham cycle. The total DNA G+C content was 61.4 mol%. 16S rRNA gene sequence analysis revealed that strain An22T was affiliated with the class Betaproteobacteria and clustered with iron-oxidizing bacteria, Gallionella ferrugineaJohan (94.8 % similarity) and Ferriphaselus amnicola OYT1T (94.4 %) in the family Gallionellaceae. Based on the low 16S rRNA gene sequence similarity to the phylogenetically closest genera and the combination of unique morphological, physiological and biochemical characteristics, strain An22T represents a novel genus and species within the family Gallionellaceae, for which the name Ferrigenium kumadai gen. nov., sp. nov. is proposed. The type strain is An22T (=JCM 30584T=NBRC 112974T=ATCC TSD-51T).

Gene pyramiding enhances durable blast disease resistance in rice.

Effective control of blast, a devastating fungal disease of rice, would increase and stabilize worldwide food production. Resistance mediated by quantitative trait loci (QTLs), which usually have smaller individual effects than R-genes but confer broad-spectrum or non-race-specific resistance, is a promising alternative to less durable race-specific resistance for crop improvement, yet evidence that validates the impact of QTL combinations (pyramids) on the durability of plant disease resistance has been lacking. Here, we developed near-isogenic experimental lines representing all possible combinations of four QTL alleles from a durably resistant cultivar. These lines enabled us to evaluate the QTLs singly and in combination in a homogeneous genetic background. We present evidence that pyramiding QTL alleles, each controlling a different response to M. oryzae, confers strong, non-race-specific, environmentally stable resistance to blast disease. Our results suggest that this robust defence system provides durable resistance, thus avoiding an evolutionary "arms race" between a crop and its pathogen.

Variation in heading date conceals quantitative trait loci for other traits of importance in breeding selection of rice.

To identify quantitative trait loci (QTLs) associated with the primary target traits for selection in practical rice breeding programs, backcross inbred lines (BILs) derived from crosses between temperate japonica rice cultivars Nipponbare and Koshihikari were evaluated for 50 agronomic traits at six experimental fields located throughout Japan. Thirty-three of the 50 traits were significantly correlated with heading date. Using a linkage map including 647 single-nucleotide polymorphisms (SNPs), a total of 122 QTLs for 38 traits were mapped on all rice chromosomes except chromosomes 5 and 9. Fifty-eight of the 122 QTLs were detected near the heading date QTLs Hd16 and Hd17 and the remaining 64 QTLs were found in other chromosome regions. QTL analysis of 51 BILs having homozygous for the Koshihikari chromosome segments around Hd16 and Hd17 allowed us to detect 40 QTLs associated with 27 traits; 23 of these QTLs had not been detected in the original analysis. Among the 97 QTLs for the 30 traits measured in multiple environments, the genotype-by-environment interaction was significant for 44 QTLs and not significant for 53 QTLs. These results led us to propose a new selection strategy to improve agronomic performance in temperate japonica rice cultivars.

Loss of function of a proline-containing protein confers durable disease resistance in rice.

Blast disease is a devastating fungal disease of rice, one of the world's staple foods. Race-specific resistance to blast disease has usually not been durable. Here, we report the cloning of a previously unknown type of gene that confers non-race-specific resistance and its successful use in breeding. Pi21 encodes a proline-rich protein that includes a putative heavy metal-binding domain and putative protein-protein interaction motifs. Wild-type Pi21 appears to slow the plant's defense responses, which may support optimization of defense mechanisms. Deletions in its proline-rich motif inhibit this slowing. Pi21 is separable from a closely linked gene conferring poor flavor. The resistant pi21 allele, which is found in some strains of japonica rice, could improve blast resistance of rice worldwide.