Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil.

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10-2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10-1 J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

Mechanisms of invasive population establishment and spread of pinewood nematodes in China.

This paper summarizes the results of our study of the pinewood nematode (Bursaphelenchus xylophilus). By population genetic analysis, it was determined that there was no genetic bottle caused by the founder effect and genetic drift in the Chinese invasive population. Multiple invasions with large amounts of nematodes from different sources led to rich genetic diversity in the invasive population. Keeping high genetic diversity in the invasive process may be one of the genetic mechanisms in its successful invasion. By testing interspecies competition, it was shown that, with high fecundity and a strong competitive ability, B. xylophilus outcompeted the native species B. mucronatus in the natural ecosystem during the invasion process. Competitive displacement may be one of the ecological mechanisms of B. xylophilus's invasion. In addition, an unequal interspecific hybridization with introgress was in favor of the invasive species which also accelerated the replacement of B. mucronatus by B. xylophilus. The structures, functions and evolutions of a few important genes that are closely related to the ecological adaptation of pinewood nematodes were studied to explore the molecular mechanism of its ecological adaptations. Further, the resistance and resilience mechanism of the pine ecosystem invaded by pinewood nematodes was also investigated. The results of these studies uncovered a portion of the genetic and ecological mechanisms of PWN's successful invasion and laid a foundation for further study to obtain a comprehensive interpretation of the mechanisms of the nematode invasion. The results also provided a scientific basis for effectively controlling the occurrence and spread of pine wilt disease which is caused by nematodes. Various aspects requiring further investigation are considered.

[Cloning and prokaryotic expression of Rhodoblastus acidophilus 5-aminolevlinate synthase gene].

5-aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (ALAS). However, the fidelity of ALAS gene among species is low. The ALAS gene of photosynthetic bacteria Rhodoblastus acidophilus was cloned from its genomic DNA by conventional PCR and Veterette PCR and further sequenced. The identity of ALAS gene among photosynthetic bacteria species is from 64.0% to 95.1% according to phylogenic analysis. Furthermore, the ALAS gene was subcloned into an expression vector pQE30. For the overproduction of ALA, the recombinant ALAS was overexpressed in Escherichia coli strains JM109, M15 and BL21 (DE3), respectively. The expected 44kD protein was detected by SDS-PAGE in three E. coli strains after IPTG induction and further purified by affinity purification on Ni-NTA. The conditions including strain, medium, substrate of ALA synthesize (glycine and succinic acid), and ALA dehydratase inhibitor (levulinic acid) were optimized for attainning the maximum yield of ALA in E. coli. The ALA production was established on E. coli M15, medium 1 supplied with 100mmol/L glycine and 50mmol/L succinic acid, and 40mmol/L levulinic acid. The activity of ALAS was up to 333U/min x mg of protein. Meanwhile, the output of ALA was reached to 5.379g/L, which is the highest yield of ALA up to date by biofermentation. ALA has a variety of agricultural applications not only as an herbicide, insecticide, and growth promoting factor, but also based on its ability to confer salt and cold temperature tolerance in plants. Our recombinant bacteria are of great potential in the production of ALA. Our results offer an easy and simple ALA mass production method and may stimulate the application of ALA in agriculture.