Members of the chalcone synthase (CHS) family participate in the synthesis of a series of secondary metabolites in plants, fungi and bacteria. The metabolites play important roles in protecting land plants against various environmental stresses during the evolutionary process. Our research was conducted on comprehensive investigation of CHS genes in maize (Zea mays L.), including their phylogenetic relationships, gene structures, chromosomal locations and expression analysis. Fourteen CHS genes (ZmCHS01–14) were identified in the genome of maize, representing one of the largest numbers of CHS family members identified in one organism to date. The gene family was classified into four major classes (classes I–IV) based on their phylogenetic relationships. Most of them contained two exons and one intron. The 14 genes were unevenly located on six chromosomes. Two segmental duplication events were identified, which might contribute to the expansion of the maize CHS gene family to some extent. In addition, quantitative real-time PCR and microarray data analyses suggested that ZmCHS genes exhibited various expression patterns, indicating functional diversification of the ZmCHS genes. Our results will contribute to future studies of the complexity of the CHS gene family in maize and provide valuable information for the systematic analysis of the functions of the CHS gene family.

Purpose: To screen endophyticBacillusproducing volatile organic compounds (VOCs) with antifungal activity, and to explore their biocontrol properties toward the growth and pathogenicity ofCurvularia lunata.Methods: Two-sealed-base-plate assays were used to estimate the antifungal activities ofBacillusstrains againstC. lunata. Conjoint analysis of solid-phase microextraction gas chromatography-mass spectrometry and antagonistic experiments were used to identify the VOCs responsible for the antifungal activity. Effects of individual synthetic VOCs were analyzed along with reactive oxygen species (ROS) accumulation inC. lunataconidia. After exposure to individual VOCs, conidia were also sprayed onto maize leaves to evaluate their pathogenicity. Expression levels of virulence-related genes inC. lunatamycelium following exposure to VOCs were analyzed using quantitative real-time PCR.Results: Among the ten endophyticBacillusstrains and two plant growth-promoting rhizobacterial (PGPR) strains, onlyB. subtilisstrain DZSY21 strongly inhibited the growth ofC. lunataby producing VOCs. 2-Methylbutyric acid, 2-heptanone, and isopentyl acetate produced by strain DZSY21 showed inhibitory effects on the mycelia growth and conidial sporulation ofC. lunata. 2-Heptanone and isopentyl acetate also repressed the germination of conidia and the expression levels of virulence-related genes inC. lunatamycelium. Moreover, isopentyl acetate strongly enhanced the accumulation of intracellular ROS in conidia. The disease indexes of maize leaves sprayed with VOC-treatedC. lunataconidia were reduced from 60.52 to 26.64%.Conclusion: EndophyticB. subtilisstrain DZSY21 displayed the potential to controlC. lunataby producing VOCs, especially 2-heptanone and isopentyl acetate.

Bacillus subtilis DZSY21 isolated from the leaves of Eucommia ulmoides oliv. was labelled by antibiotic marker and found to effectively colonize the leaves of maize plant. Agar diffusion assays and biocontrol effect experiments showed that strain DZSY21 and its lipopeptides had antagonistic activity against Bipolaris maydis, as well as high biocontrol effects on southern corn leaf blight caused by B. maydis. Using MALDI-TOF-MS analysis, we detected the presence of antimicrobial surfactin A, surfactin B and fengycin in the strain DZSY21. Signaling pathways mediated by DZSY21 were analyzed by testing the expression of key plant genes involved in regulation of SA or JA/ET pathways, the defense-related genes PR1 and LOX were concurrently expressed in the leaves of DZSY21-treated plants; this corresponded to slight increase in the expression level of PDF1.2 and decreases in ERF gene transcription levels. The results indicated an induced systemic response that is dependent on the SA and JA pathways. Thus, we hypothesized that the strain DZSY21 inhibits Bipolaris maydis by producing antifungal lipopeptides and activating an induced systemic response through SA- and JA-dependent signaling pathways. This work describes a mechanism behind reduced disease severity in plants inoculated with the endophytic bacteria DZSY21.

Natural rhizosphere bacteria has the potential to act as an alternative of chemical pesticides for sustainable agriculture. In the current study, tobacco rhizosphere Bacillus velezensis D exhibited great antibacterial effect against Ralstonia solanacearum, and significantly enhanced the tobacco resistance against bacterial wilt in pot experiments. Then Bacillus velezensis D was labeled with gfp marker and found to stably colonize in tobacco root, the colonization density of strain D in root still remained 5.33 × 10⁴ CFU/gat 30 days post-inoculation. Subsequently, field trials for two years (2021–2022) showed the control effects of the strain D on the tobacco bacterial wilt were 12.26% and 36.37%, respectively, indicating the application of B. velezensis D could improve plant resistance to R.solanacearum. In order to further study the antibacterial activities of strain D, effects of the crude extracts on the swimming ability, cell viability and the morphology of R. solanacearum were analyzed. The results showed that the crude extracts reduced the motility of R. solanacearum, and caused cell wall rupture and cell death. Furthermore, MALDI-TOF-MS and HPLC-QTOF-MS analysis indicated that lipopeptides (fengycin and iturin) and polyketides (bacillaene) were detected in the crude extracts of strain D. Based on these findings, we speculated that Bacillus velezensis D firstly colonized in tobacco root, then produced antibacterial substances at ecological sites to exert antagonistic effects, inhibiting motility traits of R. solanacearum and damaging the cell well. Hence, Bacillus velezensis D could be used as a potential biological control agents against tobacco bacterial wilt.

TLC (TRAM/LAG/CRN8) proteins play important roles in ceramide metabolism and mycotoxin resistance. Herein a comparative genomics analysis of TLCs was performed in 31 plant and 3 species from other kingdoms, with an emphasis mainly on maize. TLCs were conserved across kingdoms and expanded in angiosperms, largely due to whole-genome/segmental duplication (WGD/SD) under purifying selection. Phylogeny reconstruction by maximum-likelihood method uncovered five TLC clades, subsequently named as TRAM/LAG, CLN8, PS-TLC, TM136 and TLCD clades. Each clade of TLCs shared specific transmembrane regions and motif composition. Divisions of conserved motifs to subunits may have occurred in TM136-type TLCs. Focusing on maize, five WGD and two DNA-mediated transposed duplication (TD) pairs were discovered, accounting for 61.11% ZmTLCs. Combined with further expression analysis, significant divergence was found in expression patterns between most maize WGD pairs, indicating subfunctionalization or/and neofunctionalization. Moreover, ZmTLC5, a deduced parental copy in a TD pair, was highly induced under FB₁ and fungus pathogen injection and exhibited potential capacity to respond to environmental stimuli. Additionally, population genetics analysis showed that ZmTLC10 in the CLN8-clade may have experienced significant positive selection and differentiated between wild and inbred maize populations. Overall, our results help to decipher the evolutionary history of TLCs in maize and plants, facilitating further functional analysis of them.