Rhizosphere microbial communities are known to be related to plant health; using such an association for crop management requires a better understanding of this relationship. We investigated rhizosphere microbiomes associated with Verticillium wilt symptoms in two cotton cultivars. Microbial communities were profiled by amplicon sequencing, with the total bacterial and fungal DNA quantified by quantitative polymerase chain reaction based on the respective 16S and internal transcribed spacer primers. Although the level of V. dahliae inoculum was higher in the rhizosphere of diseased plants than in the healthy plants, such a difference explained only a small proportion of variation in wilt severities. Compared to healthy plants, the diseased plants had much higher total fungal/bacterial biomass ratio, as represented by quantified total fungal or bacterial DNA. The variability in the fungal/bacterial biomass ratio was much smaller than variability in either fungal or bacterial total biomass among samples within diseased or healthy plants. Diseased plants generally had lower bacterial alpha diversity in their rhizosphere, but such differences in the fungal alpha diversity depended on cultivars. There were large differences in both fungal and bacterial communities between diseased and healthy plants. Many rhizosphere microbial groups differed in their abundance between healthy and diseased plants. There was a decrease in arbuscular mycorrhizal fungi and an increase in several plant pathogen and saprophyte guilds in diseased plants. These findings suggested that V. dahliae infection of roots led to considerable changes in rhizosphere microbial communities, with large increases in saprophytic fungi and reduction in bacterial community.

Crop breeding is an important global strategy to meet sustainable food demand. CRISPR/Cas is a most promising gene-editing technology for rapid and precise generation of novel germplasm and promoting the development of a series of new breeding techniques, which will certainly lead to the transformation of agricultural innovation. In this review, we summarize recent advances of CRISPR/Cas technology in gene function analyses and the generation of new germplasms with increased yield, improved product quality, and enhanced resistance to biotic and abiotic stress. We highlight their applications and breakthroughs in agriculture, including crop de novo domestication, decoupling the gene pleiotropy tradeoff, crop hybrid seed conventional production, hybrid rice asexual reproduction, and double haploid breeding; the continuous development and application of these technologies will undoubtedly usher in a new era for crop breeding. Moreover, the challenges and development of CRISPR/Cas technology in crops are also discussed.

Background: In our previous study, a strain EBS03 with good biocontrol potential was screened out of 48 strains of cotton endophyte Bacillus subtilis by evaluating the controlling effect against cotton Verticillium wilt. However, its mechanism for controlling Verticillium wilt remains unclear. The objective of this study was to further clarify its controlling effect and mechanism against cotton Verticillium wilt. Results: The results of confrontation culture test and double buckle culture test showed that the inhibitory effects of EBS03 volatile and nonvolatile metabolite on mycelium growth of Verticillium dahliae were 70.03% and 59.00%, respectively; the inhibitory effects of sporulation and microsclerotia germination were 47.16% and 70.06%, respectively. In the greenhouse test, the EBS03 fermentation broth root irrigation had the highest controlling effect at 87.11% on cotton Verticillium wilt, and significantly promoted the growth of cotton seedlings. In the field experiment, the controlling effect of EBS03 fermentation broth to cotton Verticillium wilt was 42.54% at 60 days after cotton sowing, and the boll number per plant and boll weight in EBS03 fermentation broth seed soaking, root irrigation, and spraying treatments significantly increased by 19.48% and 7.42%, 30.90% and 2.62%, 15.99% and 9.20%, respectively. Furthermore, EBS03 improved the resistance of cotton leaves against the infection of V. dahliae, and induced the outbreak of reactive oxygen species and accumulation of callose. In addition, the results of real time fluorescent quantitative polymerase chain reaction (RT-qPCR) detection showed that EBS03 significantly induced upregulation expression level of defense-related genes PAL, POD, PPO, and PR10 in cotton leaves, enhanced cotton plant resistance to V. dahliae, and inhibited colonization level of this fungal pathogen in cotton. Conclusion: Bacillus subtilis EBS03 has a good biological defense capability, which can inhibit the growth and colonization level of V. dahliae, and activate the resistance of cotton to Verticillium wilt, thus increase cotton yield.

Cotton is a vital industrial crop that is gradually shifting to planting in arid areas. However, tubby-like proteins (TULPs) involved in plant response to various stresses are rarely reported in cotton. The present study exhibited that GhTULP30 transcription in cotton was induced by drought stress. The present study demonstrated the improvement of plant tolerance to drought stress by GhTULP30 through regulation of stomatal movement. GhTULP30 response to drought and salt stress was preliminarily confirmed by qRT-PCR and yeast stress experiments. Ectopic expression in Arabidopsis and endogenous gene silencing in cotton were used to determine stomatal movement. Yeast two-hybrid and spilt-luciferase were used to screen the interacting proteins. Ectopic expression of GhTULP30 in yeast markedly improved yeast cell tolerance to salt and drought. Overexpression of GhTULP30 made Arabidopsis seeds more resistant to drought and salt stress during seed germination and increased the stomata closing speed of the plant under drought stress conditions. Silencing of GhTULP30 in cotton by virus-induced gene silencing (VIGS) technology slowed down the closure speed of stomata under drought stress and decreased the length and width of the stomata. The trypan blue and diaminobenzidine staining exhibited the severity of leaf cell necrosis of GhTULP30-silenced plants. Additionally, the contents of proline, malondialdehyde, and catalase of GhTULP30-silenced plants exhibited significant variations, with obvious leaf wilting. Protein interaction experiments exhibited the interaction of GhTULP30 with GhSKP1B and GhXERICO. GhTULP30 participates in plant response to drought stress. The present study provides a reference and direction for further exploration of TULP functions in cotton plants.

This study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars. We optimized the polymerase chain reaction (PCR) system for multi-platform compatibility and improving detection efficiency. Based on the reference genome of upland cotton and 10× resequencing data of 48 basic cotton germplasm lines, single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers. The SSR markers were detected by denaturing polyacrylamide gel electrophoresis (PAGE) for initial screening, then fluorescence capillary electrophoresis for secondary screening. The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials. Using bioinformatics techniques, 1 246 SSR markers were designed from 26 626 single-copy SSR loci. Adopting a stepwise (primary and secondary) screening strategy, a set of 60 perfect SSR markers was selected with high amplification efficiency and stability, easy interpretation of peak type, multiple allelic variations, high polymorphism information content (PIC) value, uniform chromosome distribution, and single-copy characteristics. A multiplex PCR system was established with ten SSR markers using capillary electrophoresis detection. A set of perfect SSR markers of cotton was developed and a high-throughput SSR marker detection system was established. This study lays a foundation for large-scale and standardized construction of a cotton DNA fingerprint database for authentication of cotton varieties.