RESUMEN
The availability of efficient diagnostic methods is crucial to monitor the incidence of crop diseases and implement effective management strategies. One of the most important elements in diagnostics, especially in large acreage crops, is the sampling strategy as hundreds of thousands of individual plants can grow in a single farm, making it difficult to assess disease incidence in field surveys. This problem is compounded when there are no external disease symptoms, as in the case for the ratoon stunting disease (RSD) in sugarcane. We have developed an alternative approach of disease surveillance by using the crude cane juice expressed at the sugar factory (mill). For this purpose, we optimized DNA extraction and amplification conditions for the bacterium Leifsonia xyli subsp xyli, the causal agent of RSD. The use of nucleic acid dipsticks and LAMP isothermal amplification allows to perform the assays at the mills, even in the absence of molecular biology laboratories. Our method has been validated using the qPCR industry standard and shows higher sensitivity. This approach circumvents sampling limitations, providing RSD incidence evaluation on commercial crops and facilitating disease mapping across growing regions. There is also potential is to extend the technology to other sugarcane diseases as well as other processed crops.
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MAIN CONCLUSION: When gene editing was applied to knockout beta-kafirin, there was a compensatory increase of gamma-kafirin which does not occur in domesticated null varieties, so enhanced grain quality was not achieved. Sorghum bicolor is an important animal feedstock cereal crop throughout Australia and the southern United States, where its use as a food product is limited by issues with low calorific and nutritive value. Qualities such as reduced digestibility and low essential amino acid content are directly attributed to the kafirin grain storage proteins, the major components of protein bodies within the endosperm. Specifically, the ß- and γ-kafirins have few protease cleavage sites and high levels of cysteine residues which lead to a highly cross-linked shell of intra- and inter-molecular disulphide linkages that encapsulate the more digestible α- and δ-kafirins in the core of the protein bodies. Naturally occurring ß-kafirin mutants exist and are known to have improved grain quality, with enhanced protein contents and digestibility, traits which are often attributed to the lack of this cysteine-rich kafirin in the mature grain. However, when CRISPR/Cas9 editing was used to create ß-kafirin knockout lines, there was no improvement to grain quality in the Tx430 background, although they did have unique protein composition and changes to protein body morphology in the vitreous endosperm. One explanation of the divergence in quality traits found the lines lacking ß-kafirin are due to a drastic increase of γ-kafirin which was only found in the gene edited lines. This study highlights that in some germplasm, there is a level of redundancy between the peripheral kafirins, and that improvement of grain protein digestibility cannot be achieved by simply removing the ß-kafirin protein in all genetic backgrounds.
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Sorghum , Sorghum/genética , Cisteína , AustraliaRESUMEN
In most agriculture farmlands, weed management is predominantly reliant on integrated weed management (IWM) strategies, such as herbicide application. However, the overuse and misuse of herbicides, coupled with the lack of novel active ingredients, has resulted in the uptrend of herbicide-resistant weeds globally. Moreover, weedy traits that contribute to weed seed bank persistence further exacerbate the challenges in weed management. Despite ongoing efforts in identifying and improving current weed management processes, the pressing need for novel control techniques in agricultural weed management should not be overlooked. The advent of CRISPR/Cas9 gene-editing systems, coupled with the recent advances in "omics" and cheaper sequencing technologies, has brought into focus the potential of managing weeds in farmlands through direct genetic control approaches, but could be achieved stably or transiently. These approaches encompass a range of technologies that could potentially manipulate expression of key genes in weeds to reduce its fitness and competitiveness, or, by altering the crop to improve its competitiveness or herbicide tolerance. The push for reducing or circumventing the use of chemicals in farmlands has provided an added incentive to develop practical and feasible molecular approaches for weed management, although there are significant technical, practical, and regulatory challenges for utilizing these prospective molecular technologies in weed management.
RESUMEN
KEY MESSAGE: Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals.