Self-EcoTILLING to identify single-nucleotide mutations in multigene family

TILLING (Targeting Induced Local Lesions IN Genomes) is a low-cost, high-throughput reverse genetic technique that employs a mismatch-specific endonuclease CEL-1 to discover induced point mutations in the genes of interest. The use of the TILLING technique to survey natural variation in genes is called EcoTILLING. Here, we report a modified EcoTILLING method for the discovery of mutations in multigene family, which we coin “Self-EcoTILLING” by using an allotetraploid Monochoria vaginalis ALS multigene family as an example. The mutations could be detected by TILLING of PCR products resulting from the primers specific to both Als1 and Als3 without involving the experimental step of mixture of reference and query DNA. Either of the two co-amplified loci could serve as reference DNA to the other. We demonstrate with this example that Self-EcoTILLING is a fast, reliable and economical technique of detecting single-nucleotide mutations in polyploid plants containing multigene family.     

Electrophoretic karyotyping and mapping of pathotype-specific DNA sequences in Japanese isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

The chromosome number and electrophoretic karyotype of Japanese isolates of Verticillium dahliae were investigated. In a genomic Southern blot analysis of seven isolates probed with a telomere consensus sequence (TTAGGG)₅, 12 or 14 bands were observed. Furthermore, pulsed-field gel electrophoresis (PFGE) of these isolates revealed five or six chromosomal bands. A band (approx. 3.5 Mbp) common to all isolates apparently contained more than two chromosomes. From these results, we concluded that each isolate’s chromosome number is six (an eggplant pathotype isolate) or seven (all isolates of tomato and sweet pepper pathotypes). Although the chromosome sizes differed among isolates, karyotypes were similar within tomato and sweet pepper pathotypes. A small chromosome (approx. 1.8 Mbp) was observed only in the sweet pepper pathotype. Subsequent PFGE-Southern hybridization analyses revealed that the three DNA fragments specific to tomato pathotype are located on the same chromosome. These results suggest that the tomato-pathotype-specific DNA sequences might coexist on one chromosome.     

Practical phytoextraction in cadmium-polluted paddy fields using a high cadmium accumulating rice plant cultured by early drainage of irrigation water

Some indica rice varieties are potential phytoextractors for paddy fields polluted with Cd because of their high biomass and because they can accumulate Cd to moderate levels in their shoots. To establish a practical phytoextraction system, phytoextraction using two indica rice cultivars (MORETSU and IR-8) was carried out in a paddy field polluted with moderate Cd levels (2.91 and 2.52 mg kg⁻¹, respectively). The Cd concentration and Cd uptake of MORETSU increased when irrigation water was drained at the maximum tillering stage, and the paddy soil was under oxidative conditions until harvesting. The Cd uptake of MORETSU and IR-8 increased and reached 516 and 657 g ha⁻¹, respectively, at the beginning of October. After phytoextraction using these high Cd accumulating rice varieties for 2 years, the Cd concentration in the paddy field decreased by 18% compared with the initial Cd concentration. The Cd concentration in the rice grains of a japonica ordinary rice variety (HINOHIKARI) subsequently grown on the field after the phytoextraction was lower than the concentration in rice grown on a non-phytoextracted field. These results suggest that phytoextraction using high Cd accumulating rice varieties with early drainage of irrigation water is a practical remediation system for moderate Cd polluted paddy fields in southwest Japan.     

Physicochemical and morphological properties of termite (Macrotermes bellicosus) mounds and surrounding pedons on a toposequence of an inland valley in the southern Guinea savanna zone of Nigeria

Termites play a significant role in soil-forming processes of the tropics. The influence of termites on pedogenesis as affected by the toposequence, however, has rarely been explored. We investigated the soil physicochemical and morphological characteristics of epigeal mounds constructed by Macrotermes bellicosus (Smethman) compared with those of surrounding pedons along a toposequence (bottom, fringe and upland sites) of an inland valley in central Nigeria. The physicochemical and morphological properties of the mound soils varied according to structural units but were generally different from those of the adjacent pedons. The differences included finer texture, higher electrical conductivity, total N, exchangeable bases (Ca, Mg and K) and effective cation exchange capacity and lower C/N ratio and exchange acidity in the mound than the pedon at each toposequence position. This tendency to modify the soil properties was more prominent in the nest body where the termites actually live, that is, in the hives, royal cell and base-plate, than in the soils below the nest and the other mound parts, that is, the external wall, internal wall and pillars. We found this trend to a greater or lesser degree at all toposequence positions. Our findings suggest that: (1) M. bellicosus can manipulate the mound soils according to functional applications of structure units or environmental requirements for its livelihood, regardless of local soils; (2) M. bellicosus makes ecological patches (hot spots) at all toposequence positions in the same measure; (3) the influence of M. bellicosus on the pedogenesis is reduced in the lowlands compared with the uplands because the number and volume of the mounds were substantially lower in the bottom and fringe sites compared with the upland site.     

Brown root rot of Russell prairie gentian caused by Subplenodomus drobnjacensis

Brown root rot of Russell prairie gentian was observed in the Aomori Prefecture, Japan in April 2011. The fungal isolate from the diseased root was identified as Subplenodomus drobnjacensis on the basis of its morphological characteristics and nucleotide sequences. The isolate induced similar root rot symptoms when inoculated in healthy Russell prairie gentian plants. We proposed the name “brown root rot” for this disease.     

Infection by Magnaporthe oryzae chrysovirus 1 strain A triggers reduced virulence and pathogenic race conversion of its host fungus, <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is associated with an impaired growth phenotype of its host fungus, Magnaporthe oryzae. In this report, we assayed the virulence and pathogenicity of MoCV1-A-infected and MoCV1-A-free M. oryzae on rice plants. MoCV1-A infection did not affect virulence-associated fungal traits, such as conidial germination and appressorium formation. However, after punch inoculation of leaves on rice plants, MoCV1-A-infected strain formed smaller lesions than the MoCV1-A-free strain did on all rice varieties tested, showing that MoCV1-A infection resulted in reduced virulence of host fungi in rice plants. In contrast, after spray inoculation of rice seedlings, in some cases, MoCV1-A-infected and MoCV1-A-free strains caused different lesion types (resistance to susceptible, or vice versa) on individual international differential rice varieties. However, we did not find any gain/loss of the fungal avirulence genes by PCR, suggesting that MoCV1-A infection can convert the pathogenicity of the host M. oryzae from avirulence to virulence, or from virulence to avirulence, depending on the rice variety. We also confirmed the correlation of these race conversion events and invasive hyphae growth of the fungi in a leaf sheath inoculation assay. These data suggested that MoCV1-A infection generally confers hypovirulence to the fungal host and could be a driving force to generate physiological diversity, including pathogenic races.     

Weedy rice represents an emerging threat to transplanted rice production systems in Japan

Weedy rice is a conspecific weed of cultivated rice (Oryza sativa L.) and is a primary weed of direct‐seeded rice production systems worldwide. The shift in the method of establishing rice, that is, from transplanting to direct seeding, is considered the main reason for the emergence of weedy rice in Asia and, more recently, Japan. Japan has increasingly adopted directly seeded rice, even though this practice remains limited. In this study, I confirmed the relationship between weedy red rice emergence and the adoption of direct‐seeded rice in Japan. Twenty‐seven areas from eight prefectures infested with weedy rice were selected to confirm the relationship between the rice planting method and the emergence of weedy rice. Fields that were severely infested with weedy rice were selected as survey fields for this study. I found no relationship between the method of planting rice and weedy rice emergence. Changes to herbicide application, duration of rice production and hand weeding also contribute to the emergence of weedy rice in transplanted rice fields in Japan. The results of this study indicate that weedy rice could become a serious problem in Japan, even in transplanted rice fields.     

Pathogenicity of <Emphasis Type="Italic">Plectosphaerella</Emphasis> species on lettuce and susceptibility of lettuce cultivars

Plectosphaerella rot affects hydroponically grown lettuce, especially those grown using deep flow technique. Plectosphaerella species such as P. pauciseptata and P. cucumerina are reported as causal agents of this disease. However, the relation between fungal lineage and pathogenicity on lettuce has been unclear. From inoculation tests, we discovered that various lineages of Plectosphaerella can infect lettuce tissues. Even strains isolated from non-lettuce plants were pathogenic on lettuce. Furthermore, various lettuce cultivars were equally susceptible to a particular strain. These results indicate that strains from a wide lineage of Plectosphaerella can be pathogenic on various lettuce cultivars.     

Reduction of formaldehyde emission from plywood using composite resin composed of resorcinol–formaldehyde and urea-modified scallop shell nanoparticles

More than 200,000 tons of scallop shells are disposed annually alone in Japan. Nanoparticles derived from scallop shells have the potential to adsorb gaseous formaldehyde; therefore, such discarded shells have now been tested as additive filler in plywood adhesive by mixing high specific surface area, urea-modified shell nanoparticles with a resorcinol–formaldehyde resin. With this procedure, it was found that the emission of formaldehyde from the resulting plywood could be substantially reduced. The urea-modified scallop shell nanoparticles were prepared by two different methods: (1) a dry method where the shells were treated by planetary ball-grinding under ambient conditions—a completely dried powder was obtained after addition of the surface-modifying urea solution; (2) a moist method by treating dry ground shell particles in a wet grinding process with the urea solution, followed by centrifugation to obtain a paste. The specific surface area of the nanoparticles obtained by both treatments was 42 ± 3 m²/g. Measurement of the subsequent formaldehyde emission showed that the addition of the modified scallop shell nanoparticles substantially reduced the formaldehyde emission from plywood. The reduction of the specific mass uptake of urea depends on the nanoparticles which especially was the case when resins containing nanoparticles processed by the moist method were used.