Effective Management of Citrus Melanose Based on Combination of Ecofriendly Chemicals.

Citrus melanose, caused by the ascomycete fungus Diaporthe citri, is one of the most important diseases in China that affects not only the production but also the quality of citrus. In China, mancozeb is recommended to control melanose disease at the dose of 1.34 g/liter. However, it is widely applied in practice at the dose of 2.66 g/liter or even 4 g/liter, because reduced efficacy of the recommended dose was observed in regions severely damaged by melanose. In this study, some ecofriendly chemicals for melanose management were evaluated. First, the sensitivity to fungicides was screened in the laboratory based on the inhibition of mycelial growth and conidial germination of D. citri. Results showed that both quinone outside inhibitor (QoI) fungicides kresoxim-methyl and trifloxystrobin inhibited conidial germination of D. citri up to 100% at 0.1 µg/ml. The in vivo control efficacy on detached fruit indicated that treatments with elastic nanocopolymer film at 2 g/liter, mancozeb at 1 g/liter, and kresoxim-methyl at 0.1 g/liter significantly inhibited the infection process compared with the control treatment of mineral oil alone. In field trials, the efficacy of kresoxim-methyl at 0.1 g/liter and elastic nanocopolymer film at 2 g/liter mixed with mancozeb at 1 g/liter was equal to that of mancozeb at 2.66 g/liter. The use of mancozeb could be reduced greatly, and the newly developed fungicide combinations are more environmentally friendly due to the low toxicity of both QoI fungicides and elastic nanocopolymer film. The newly developed method with ecofriendly chemicals should play an important role in the management of citrus melanose in the future.

Development of a LAMP Method for Detecting SDHI Fungicide Resistance in Botrytis cinerea.

Resistance to succinate dehydrogenase inhibitors (SDHI) in Botrytis cinerea is associated with point mutations in the target gene succinate dehydrogenase subunit B (SdhB). The substitution from histidine to arginine at codon 272 (H272R) is currently the predominant mutation in SDHI-resistant populations in B. cinerea worldwide. In order to monitor the development of resistance to SDHI, a rapid, simple, and efficient method with high specificity to the H272R point mutation was developed based on loop-mediated isothermal amplification (LAMP). To specifically detect the H272R mutation, a set of four primers was designed based on the sequence of SdhB, and the LAMP reaction was optimized. When SYBR Green I was added after reaction, only samples with the H272R mutation showed the color change (from brown to fluorescent yellow), indicating that this set of primers could successfully discriminate the H272R genotype from other genotypes. Specificity and accuracy tests showed that this LAMP assay had high specificity and accuracy. Moreover, the LAMP method was further simplified with fungal mycelia and conidia as the amplification template which could be prepared within 5 min. Due to the low cost, simplicity, high efficiency, and specificity, the developed LAMP assay may contribute to the monitoring of resistance development to SDHI in B. cinerea, especially in field and high-throughput experiments.

Sensitivity of Botrytis cinerea From Nectarine/Cherry in China to Six Fungicides and Characterization of Resistant Isolates.

Botrytis cinerea, the causal agent of gray mold, can result in considerable preharvest and postharvest losses in many economically valuable plant species. Fungicides were widely used to minimize such losses, but fungicide resistances were detected frequently. In the present study, we collected 164 isolates from nectarine and cherry in China and tested the sensitivity to six fungicides. Among the tested isolates, 71 (43.3%) were resistant to azoxystrobin, 14 (8.5%) to cyprodinil, 7 (4.3%) to boscalid, 4 (2.4%) to carbendazim, 1 (0.6%) to iprodione, and no isolates were found to be resistant to fludioxonil. The EC50 value and resistance factor (RF) of resistant isolates were determined. Fitness analysis showed that there were no significant differences between sensitive and resistant isolates for osmotic stress and pathogenicity, while more conidia production was observed for some resistant isolates. Control efficacy of fungicides showed that the resistant isolates could not be controlled efficiently by using corresponding fungicides. The point mutation G143A was detected in the Cyt b gene of the isolates resistant to azoxystrobin, while the point mutation H272R of SdhB gene was confirmed in boscalid-resistant isolates, and mutations E198V/A of TUB2 gene and mutation I365S of BcOs1 occurred in carbendazim-resistant and iprodione-resistant isolates, respectively. These results indicate that the occurrence of fungicide resistance greatly threatens the management of gray mold on stone fruits nectarine and cherry.

[The production and scavenging of reactive oxygen species in plants].

The imposition of environmental stress leads to increased production of reactive oxygen species (ROS) in plant cells, Which can damage proteins, membrane lipids, DNA and other cellular components. Plants have evolved enzymatic and no-enzymatic protection mechanisms that efficiently scavenge ROS. Enzymatic detoxication system includes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione peroxidase (GAX); no-enzymatic antioxidants include ascorbic acid, glutathione, mannitol and carotenoids, which occur in high concentrations in plants. The over-expression and accumulation strategies of these antioxidants in plants have been followed up to now, and have gained many transgenic plants showing increased stress tolerance.

Inversion and transposition of Tc1 transposon of C. elegans in mammalian cells.

Tc1/mariner transposons are widespread in the eukaryotes. In vitro transposition test indicated that the transposase is the only protein that is needed in transpositions. It was shown later that the reconstructed Tc1-like transposon, "sleeping beauty" in fish, and the Tc1 transposon in C. elegans jumps in human cells. This discovery indicates that the Tc1/mariner transposon may be engineered as a somatic gene therapy vector if coupled with an efficient gene delivery system. We introduced the Tc1 transposon from C. elegans into different mammalian cell lines and detected the transposition events, indicating that Tc1 transposon functions in different mammalian cells. Interestingly, a high inversion frequency of the transposon was also detected, suggesting that this type of transposon may add variations to host genome when it is horizontally transferred into a new species.

Targeted correction of the point mutations of beta-thalassemia and targeted mutagenesis of the nucleotide associated with HPFH by RNA/DNA oligonucleotides: potential for beta-thalassemia gene therapy.

An RNA/DNA chimeric oligonucleotide was found to be effective in the targeted correction of point mutations in Escherichia coli, plant, and mammalian genomes. This strategy, named chimeraplasty, has the potential for gene therapy of many genetic diseases caused by point mutations. beta-Thalassemia is a very common human genetic disease and in most cases it is caused by point mutations. To test whether the chimeraplasty can be used to correct the point mutations responsible for beta-thalassemia, we introduced one mutated beta-globin gene, betaE, into MEL cells and successfully corrected the point mutation of the betaE gene with the highest correction efficiency of 1.9%. Furthermore, a targeted -202 C-->G mutation of the Ggamma-globin gene, which is associated with the elevated Ggamma-globin gene expression in the adult stage, was introduced into HeLa and CMK cells by an RNA/DNA oligonucleotide. These results indicated that the chimeraplasty has potential for human beta-thalassemia gene therapy.