Effects of dazomet combined with Rhodopsesudomonas palustris PSB-06 on root-knot nematode, Meloidogyne incognita infecting ginger and soil microorganisms diversity.

Root-knot nematode, Meloidogyne incognita is one of the most important nematodes affecting ginger crop. Rhodopseudomonas palustris PSB-06, as effective microbial fertilizer in increasing plant growth and suppressing soil-borne disease of many crops has been reported. The combination of R. palustris PSB-06 and dazomet treatments had been proved to inhibit root-knot nematode on ginger and increase ginger yield in our preliminary study. The field experiments were conducted to elucidate the reasons behind this finding, and followed by next-generation sequencing to determine the microbial population structures in ginger root rhizosphere. The results showed that combination of R. palustris PSB-06 and dazomet treatment had a synergetic effect by achieving of 80.00% reduction in root-knot nematode numbers less than soil without treatment, and also could increase 37.37% of ginger yield through increasing the contents of chlorophyll and total protein in ginger leaves. Microbiota composition and alpha diversity varied with treatments and growth stages, soil bacterial diversity rapidly increased after planting ginger. In addition, the combined treatment could increase diversity and community composition of probiotic bacteria, and decrease those of soil-borne pathogenic fungi comparing to the soil treated with dazomet alone. Meanwhile, it could also effectively increase soil organic matter, available phosphorus and available potassium. Analysis of correlation between soil microorganisms and physicochemical properties indicated that the soil pH value and available phosphorus content were important factors that could affect soil microorganisms structure at the harvest stage. The bacterial family was more closely correlated with the soil physicochemical properties than the fungal family. Therefore, the combination of R. palustris PSB-06 and dazomet was considered as an effective method to control root-knot nematode disease and improve ginger soil conditions.

Rapid and Sensitive Detection of Meloidogyne graminicola in Soil Using Conventional PCR, Loop-Mediated Isothermal Amplification, and Real-Time PCR Methods.

Meloidogyne graminicola is one of the major plant-parasitic nematodes (PPNs) that affect rice agriculture. Rapid identification and quantification of M. graminicola in soil is crucial for early diagnosis so that measures can be taken to reduce the impact of PPN diseases and ensure food security. In this study, M. graminicola species-specific primers for conventional PCR, loop-mediated isothermal amplification (LAMP), and real-time PCR were designed based on the sequence-characterized amplified region. The primers were highly specific and sensitive, and only samples containing M. graminicola DNA showed positive results. The sensitivity of LAMP and real-time PCR (two second-stage juvenile [J2] M. graminicola in 100 g of soil) was higher than that of conventional PCR (200 J2s in 100 g of soil). A standard curve (correlation coefficient R2 = 0.970, P < 0.001) was generated by amplifying DNA extracted from 0.5 g of soil, and a significant correlation was observed between the number of M. graminicola determined by microscopic examination and that predicted from the standard curve (R2 = 0.477, P = 0.0160). In quantification analyses of M. graminicola isolated from 31 naturally infested soils, the sensitivity of LAMP and real-time PCR (22 M. graminicola in 100 g of soil) was higher than that of conventional PCR (211 M. graminicola in 100 g of soil). The conventional PCR, LAMP, and real-time PCR methods have the potential to provide a useful platform for rapid species identification according to the experimental conditions. The real-time PCR assay and standard curve can be used for quantification of M. graminicola. These newly developed assays will help to facilitate the control of these economically important PPNs.

Selection of reliable reference genes for gene expression studies in Caenorhabditis elegans exposed to crystals (Cry1Ia36) protein of Bacillus thuringiensis.

Quantitative real time PCR (qRT-PCR) is a nucleic acid quantitative technique and is also considered as a validation tool. The Cry1Ia36 protein isolated from Bacillus thuringiensis (Bt) strain YC-10 has high nematicidal activity against nematodes. Caenorhabditis elegans is one of the major model organisms and a readily accessible source of biological material for gene expression studies. To evaluate the expression stability of 12 candidate reference genes of C. elegans for exposing to different concentrations of Cry1Ia36 protein and different treat time, five statistical approaches (the comparative delta-Ct method, BestKeeper, NormFinder, Genorm and RefFinder) were used to evaluate each individual candidate reference gene. The results indicated that cdc-42 and F35G12.2 were the best reference genes for performing reliable gene expression normalization in the impact of Cry1Ia36 protein. In addition, when C. elegans was exposed to Cry1Ia36 protein and other nematicides, avermectin and 5-aminolevulinic acid, cdc-42 was recommended as the most reliable reference genes. Y45F10D.4 was the least stable reference genes in our experimental settings. Therefore, cdc-42 was reliable reference gene for gene expression studies in C. elegans exposed to Cry1Ia36 protein and other nematicides.

Nematicidal Effects of 5-Aminolevulinic Acid on Plant-Parasitic Nematodes.

Plant-parasitic nematodes are important agricultural pests and often cause serious crop losses. Novel, environmental friendly nematicides are urgently needed because of the harmful effects of some existing nematicides on human health. 5-Aminolevulinic acid (ALA) was reported as a potential biodegradable herbicide, insecticide, or plant-growth promoting agent. Lack of information on ALA against plant-parasitic nematodes prompted this investigation to determine the effects of ALA on Meloidogyne incognita, Heterodera glycines, Pratylenchus coffeae, and Bursaphelenchus xylophilus. A series of in vitro assays and one greenhouse trial were conducted to examine the nematicidal effects of ALA. The results demonstrated that ALA exhibited a strong effect of suppression against the four nematodes tested. ALA also inhibited hatching of M. incognita and H. glycines. Results from the greenhouse experiment indicated that treatment of soil with 6.0 mM ALA significantly reduced the root-gall index (RGI) and egg mass number per root system compared with the uninoculated control (P ≤ 0.05). The metabolism assays indicated that ALA treatment significantly altered the nematode metabolism including the total protein production, malondialdehyde (MDA) content, and oxidase activities. This study suggested that ALA is a promising nematicide against plant-parasitic nematodes.

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.

Complete genome sequence of Bacillus thuringiensis YC-10, a novel active strain against plant-parasitic nematodes.

Bacillus thuringiensis is an important microbial biopesticide for controlling agricultural pests by the production of toxic parasporal crystals proteins.Here,we report the finished annotated genome sequence of B. thuringiensis YC-10,which is highly toxic to nematodes.The complete genome sequence consists of a circular chromosome and nine circular plasmids,which the biggest plasmid harbors six parasporal crystals proteins genes consisting of cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and cryB1. The crystals proteins of Cry1Ia and Cry1Aa have high nematicidal activity against Meloidogyne incognita.

Cometabolic biotransformation of fenpropathrin by Clostridium species strain ZP3.

A novel bacterial strain capable of degrading the pyrethroid pesticide fenpropathrin was isolated from mixed wastewater and sludge samples. Phylogenetic analysis of the 16S rDNA sequence revealed that the organism belongs to the genus Clostridium. The organism can co-metabolically transform fenpropathrin at 100 mg l(-1) at 35°C and pH 7.5 in 12 days. Metabolic products of fenpropathrin from strain ZP3 were examined by gas chromatography/mass spectrometry, and the results showed that the organism degraded fenpropathrin with an oxidization process to yield benzyl alcohol, benzenemethanol, 3,5-dimethylamphetamine. Analyses of cell-free extracts from this strain showed that the optimal degrading conditions for degrading fenpropathrin were 35°C and pH 7.5, and degradation efficiency was 20.0 mg l(-1) day(-1), and it might be potential using for rapid treating fenpropathrin, for example, on the surface of fruits and vegetables.

[Isolation, identification and degrading gene cloning of a pyrethroids-degrading bacterium].

OBJECTIVE: This study aimed to isolate, identify and clone degrading gene of a synthetic pyrethroids degrading bacterium. METHODS: A photosynthetic bacterial strain PSB07-21 capable of degrading several synthetic pyrethroids efficiently was isolated by an enrichment culture. PSB07-21 was identified based on its morphology, physiology and phylogenetic analysis of 16S rDNA sequence. The degradation ability of this strain was evaluated with gas chromatography.The degrading gene was cloned with PCR. RESULTS: PSB07-21 was closely related to Rhodopseudomonas sp. The optimum condition of degrading synthetic pyrethroidss was at 35 degrees C, pH 7 and 3000 lx. PSB07-21 could degrade fenpropathrin, cypermethrin and bipthenthrin by 66.63%, 43.25% and 50.18% in a concentration of 600 mg/L at day 15, respectively. We cloned a putative gene which was 326bp long with 37.0% identical to 20G-Fe (II) oxygenase gene. When compensating low concentration Fe (II) in PSB medium with synthetic pyrethroids, the degradation efficiency of PSB07-21 was enhanced. CONCLUSION: The strain has the potential application to synthetic pyrethroids bioremediation.

[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.