Design, Synthesis, and Bioactivity of Novel Quinazolinone Scaffolds Containing Pyrazole Carbamide Derivatives as Antifungal Agents.

In this study, 32 novel quinazolinone-scaffold-containing pyrazole carbamide derivatives were designed and synthesized in a search for a novel fungicide against Rhizoctonia solani. Single-crystal X-ray diffraction of 3-(difluoromethyl)-N-(2-((6,7-difluoro-4-oxoquinazolin-3(4H)-yl)methyl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide (6a11) confirmed the structure of the target compounds. The in vitro antifungal activity of the target compounds against R. solani was evaluated at 100 µg/mL. The structure-activity relationship analysis results revealed that antifungal activity was highest when the substitution activity was at position 6. Moreover, the position and number of chlorine atoms directly affected the antifungal activity. Further in vitro bioassays revealed that 6a16 (EC50 = 9.06 mg/L) had excellent antifungal activity against R. solani that was higher than that of the commercial fungicide fluconazole (EC50 = 12.29 mg/L) but lower than that of bixafen (EC50 = 0.34 mg/L). Scanning electron microscopy), 7.33 (SEM) revealed that N-(2-((6,8-dichloro-4-oxoquinazolin-3(4H)-yl)methyl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (6a16) also affected the mycelial morphology. The findings revealed that molecular hybridization was an effective tool for designing antifungal candidates. Meanwhile, pyrazolecarbamide derivatives bearing a quinazolinone fragment exhibited potential antifungal activity against R. solani.

Effect of the Seasonal Climatic Variations on the Accumulation of Fruit Volatiles in Four Grape Varieties Under the Double Cropping System.

The double cropping system has been widely applied in many subtropical viticultural regions. In the 2-year study of 2014-2015, four grape varieties were selected to analyze their fruit volatile compounds in four consecutive seasons in the Guangxi region of South China, which had a typical subtropical humid monsoon climate. Results showed that berries of winter seasons had higher concentrations of terpenes, norisoprenoids, and C6/C9 compounds in "Riesling," "Victoria," and "Muscat Hamburg" grapes in both of the two vintages. However, in the "Cabernet Sauvignon" grapes, only the berries of the 2014 winter season had higher terpene concentrations, but lower norisoprenoid concentrations than those of the corresponding summer season. The Pearson correlation analysis showed the high temperature was the main climate factor that affected volatile compounds between the summer and winter seasons. Hexanal, γ-terpinene, terpinen-4-ol, cis-furan linalool oxide, and trans-pyran linalool oxide were all negatively correlated with the high-temperature hours in all of the four varieties. Transcriptome analysis showed that the upregulated VviDXSs, VviPSYs, and VviCCDs expressions might contribute to the accumulations of terpenes or norisoprenoids in the winter berries of these varieties. Our results provided insights into how climate parameters affected grape volatiles under the double cropping system, which might improve the understanding of the grape berries in response to the climate changes accompanied by extreme weather conditions in the future.

Effect of the Seasonal Climatic Variations on the Flavonoid Accumulation in Vitis vinifera cvs. 'Muscat Hamburg' and 'Victoria' Grapes under the Double Cropping System.

Under the double cropping system, berries usually showed significant quality variations in the summer and winter seasons. In the two-year/four-consecutive-season study, two table grapes of 'Muscat Hamburg' and 'Victoria' were investigated to determine the phenolic compounds in their berries. Different from those of the summer season, the berries in the winter season suffered no high-temperature stress since veraison to harvest in 2014 and 2015. The variations in the season temperatures led to a higher anthocyanin concentration in the winter season berries of 'Muscat Hamburg' grapes than that in the summer berries, while the summer season berries had higher proportions of acylated and methylated anthocyanins than those in the winter season berries. Similar to the anthocyanins, the winter season berries also had a higher flavonol concentration in both varieties. Transcriptome analysis showed that the upregulated genes involved in the flavonoid pathway in the winter season berries were agreed with the changes found in the metabolites. However, the influence of the growing seasons on the flavanols was not consistent in the two varieties, and the variations in VviLARs between the grapes of 'Muscat Hamburg' and 'Victoria' might be the cause. This research helped us better understand the double cropping system and how the climate factors affected the phenolic compounds in the double cropping system.

Comparison of transcriptional expression patterns of phenols and carotenoids in 'Kyoho' grapes under a two-crop-a-year cultivation system.

To fully utilize the characteristic climatic conditions in the southern region of China, a two-crop-a-year cultivation system technique for 'Kyoho' grape was developed during the past decade. After summer harvest in June, appropriate pruning and chemical treatments promote flowering and fruiting, which enables a second harvest in late December. Due to climatic differences between the two crop growing seasons, grape phenol and carotenoid metabolism differ greatly. The reported study analyzed the transcriptome of the carotenoid and phenylpropanoid/flavonoid pathways in grapes at four different stages during the two growing seasons. Compared with those in summer grapes, expression levels of the majority of genes involved in the carotenoid metabolic pathway in winter grapes were generally upregulated. This result was associated with lower rainfall and much more abundant sunlight during the second growing season. On the other hand, summer cropping strongly triggered the expression of upstream genes in the phenylpropanoid/flavonoid pathway at E-L 33 and E-L 35. Transcript levels of flavonoid 3',5'-hydroxylase (F3'5'H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3-hydroxylase (F3H) and glutathione S-transferase (GST) were upregulated in winter grapes at the mature stage. Together, these results might indicate that more flavonoids would be synthesized in ripe winter grapes during the mature stage of the second crop under much drier conditions, longer sunlight hours and lower temperature. These data provide a theoretical foundation for the secondary metabolism of berries grown under two-crop-a-year cultivation systems.

Evolutionary, interaction and expression analysis of floral meristem identity genes in inflorescence induction of the second crop in two-crop-a-year grape culture system.

The fruitfulness of grapevines (Vitis viniferaL.) is determined to a large extent by the differentiation of uncommitted meristems, especially in the second-crop production of some varieties, where the intermediate of inflorescence and tendril accounts for a significant proportion in two-crop-a-year grape culture system. The differentiation of uncommitted lateral meristem was reported to be regulated by a network, whose backbone was composed of several floral meristem identity genes. In the present study, the phylogenetics of grape floral meristem identity genes with their orthologues in other species, and their conserved domain and interaction networks were analysed. In addition, the effects of chlormequat chloride and pinching treatments on the expression profiles of floral meristem identity genes and content of gibberellic acid (GAs) and zeatin riboside (ZR), as well as the ratio of ZR/GAs in buds that were used to produce the second crop, and the ratio of inflorescence induction of the second crop were studied in 'Summer Black'. The present results showed that floral meristem identity genes of grape and their orthologues in one or more among Malus domastic, Citrus sinensis, heobroma cacaoT, Nicotiana tabacum, Solanum lycopersicum and Glycine hirsutum, probably originated from a common ancestor. Interaction networks of six grape-floral meristem identity genes indicated that the inflorescence induction and floral development were regulated by one more complex network, and expression profiles of genes that involved in this network could be affected by each other. Expression profiles of eight floral meristem identity genes were affected by chlormequat chloride and pinching treatments, and higher expression levels of FT, TFL1A and TFL1B, as well as lower expression levels of LFY from 3 days before full bloom to 11 days after full bloom were thought to play important roles in promoting the formation of inflorescence primordial of the second crop, and higher expression levels of CAL A, SOC1 and TFL1A at 18 days after full bloom (DAF) could promote the development of inflorescence primordial. In addition, lower ratio of ZR/GAs at 3 days before full bloom and 4 days after full bloom could promote the formation of uncommitted lateral meristems in chlormequat chloride and pinching-treated plants, and higher ratio at 11 days after full bloom was the main reason for the formation of more inflorescences after chlormequat chloride treatment.

Dissecting the Variations of Ripening Progression and Flavonoid Metabolism in Grape Berries Grown under Double Cropping System.

A double cropping system has been commercially adopted in southern China, where there is abundant sunshine and heat resources. In this viticulture system, the first growing season normally starts as a summer cropping cycle; then, the vine is pruned and forced, resulting in a second crop in winter. Due to climate differences between the summer and winter growing seasons, grape ripening progression and flavonoid metabolism vary greatly. Here, the metabolites and transcriptome of flavonoid pathways were analyzed in grapes grown under two growing seasons at different stages. Notably, the winter cropping cycle strongly increased flavonoid levels by several times in comparison to summer grapes, while the summer season took a major toll on anthocyanin and flavonol accumulation, since the winter cropping greatly triggered the expression of upstream genes in the flavonoid pathway in a coordinated expression pattern. Moreover, the ratio of VviF3'5'Hs (flavonoid 3'5'-hydroxylase) to VviF3'Hs (flavonoid 3'-hydroxylase) transcript levels correlated remarkably well with the ratio of 3'5'-substituted to 3'-substituted flavonoids, which was presumed to control the flux of intermediates into different flavonoid branches. On the other hand, the phenological phase also varied greatly in the two crops. Compared to summer cropping, winter growing season accelerated the duration from budburst to veraison, therefore advancing the onset of ripening, but also prolonging the duration of ripening progression due to the purposes to harvest high-quality grapes. The differential expression pattern of hormone-related genes between the two cropping cycles might explain this phenomenon.

Complete Genome Sequence of a Chinese Strain of "Candidatus Liberibacter asiaticus".

We report here the complete genome sequence of "Candidatus Liberibacter asiaticus" (strain Guangxi-1). The 1,268,237-bp genome with a 36.5% G+C content comprises 1,141 open reading frames, 44 tRNAs, and 3 complete rRNAs in a circular chromosome.

A new diagnostic system for ultra-sensitive and specific detection and quantification of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing.

An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan PCR in a single tube was developed for detection of "Candidatus Liberibacter asiaticus". The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan is high because the fluorescent signal can only be generated from the TaqMan probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.

Comparative phylogenomics and multi-gene cluster analyses of the Citrus Huanglongbing (HLB)-associated bacterium Candidatus Liberibacter.

BACKGROUND: Huanglongbing (HLB, previously known as citrus greening), is associated with Candidatus Liberibacter species and is a serious threat to citrus production world-wide. The pathogen is a Gram negative, unculturable, phloem-limited bacterium with limited known genomic information. Expanding the genetic knowledge of this organism may provide better understanding of the pathogen and possibly develop effective strategies for control and management of HLB. RESULTS: Here, we report cloning and characterization of an additional 14.7 Kb of new genomic sequences from three different genomic regions of the Candidatus Liberibacter asiaticus (Las). Sequence variation analyses among the available Ca. Liberibacter species sequences as well as the newly cloned 1.5 Kb of rpoB gene from different Ca. Liberibacter strains have identified INDELs and SNPs. Phylogenetic analysis of the deduced protein sequences from the cloned regions characterizes the HLB-associated Candidatus Liberibacter as a new clade in the sub-division of the alpha-proteobacteria. CONCLUSION: Comparative analyses of the cloned gene regions of Candidatus Liberibacter with members of the order Rhizobiales suggest overall gene structure and order conservation, albeit with minor variations including gene decay due to the identified pseudogenes. The newly cloned gene regions contribute to our understanding of the molecular aspects of genomic evolution of Ca. Liberibacter.