First Report of Bacterial Leaf Spot Disease on Ginger Caused by Enterobacter quasiroggenkampii in China.

In autumn 2023, an unknown leaf spot disease has occurred on ginger (Zingiber officinale Roscoe) in two fields of approximately 1800 m2 in Yongning District (22°49'N; 108°48'E), Nanning, China, with a incidence of 20-30%. The symptoms began as yellow spots on the leaves, expanding into elliptical to irregular lesions with yellow edges, the middle of the lesion turning grey-white in dry weather. Finally, multiple spots caused necrosis of the whole leaf. Twelve diseased leaves from six plants of two fields were collected, surface disinfected and ground. The ground samples were diluted and plated on nutrient agar (NA) medium at 28 °C for 48-72 h. The purified colonies appeared milky white and round, with smooth edges. Three isolates (GL1, GL2 and GL3) were selected for identification and pathogenic determination. They were gram negative, could utilize sorbitol, mannitol, inositol, raffinose, melibiose, disaccharides, and citrate; negative for methyl red, phenylalanine decarboxylase, hydrogen sulfide, urease; positive for voges-proskauer test and ornithine decarboxylase. These characteristics were consistent with Enterobacter genus (Wu et al., 2020). Genomic DNA was extracted from three isolates. The 16S rDNA region was amplified using 27F/1492R primers (Weisburg et al. 1991) and sequenced (accession no. PP837703-PP837705). Blastn analysis revealed that 16S rDNA sequences for GL1 was 99% identical (1373/1387 nt), GL2 96% (1364/1422 nt) and GL3 95% (1365/1435 nt) to Enterobacter quasiroggenkampii WCHECL1060 (NR_179166). To determine the species, the sequences of gyrB, rpoB and atpD genes were amplified using primers gyrB 01-F/gyrB 02-R, rpoB CM7/rpoB CM31b, and atpD 01-F/atpD 02-R, respectively (Lin et al. 2015; Zhu at al. 2010; Zhang et al. 2013). The GenBank accession numbers for the sequences were PP857680-PP857688. A multilocus phylogenetic tree was constructed with the concatenated sequence of 16S rDNA-gyrB-rpoB-atpD by using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA6 software. The three isolates clustered with E. quasiroggenkampii. Fifteen Darou ginger variety plants at the 4-5 leaf stage were tested for pathogenicity. Two to three leaves of each ginger plant were pricked with a syringe needle of 0.36mm in diameter or not and inoculated by spraying the bacterial suspension (108 CFU/mL), sterile water was used as a control. Five plants were inoculated with each isolate and the test was repeated three times. After 3-4 days of inoculation, all wounded leaves and about 10% of the unwounded leaves showed symptoms similar to those observed in the field. Control plants did not develop symptoms. Enterobacter quasiroggenkampii isolates were re-isolated from the inoculated leaves with symptoms, and their identity was confirmed by gyrB sequencing and colony morphology, completing Koch's postulates. Enterobacter quasiroggenkampii is a pathogen of humans that can cause nosocomial infections (Wu et al., 2020). In Guangxi, E. quasiroggenkampii was identified as one of the pathogens causing mulberry wilt (Jiao, 2022). To our knowledge, this is the first report of E. quasiroggenkampii causing bacterial leaf spot disease of ginger. The results of this study not only have practical significance for the control of ginger leaf spot, but also can provide excellent materials for the study of the differentiation and pathogenic mechanism of the genus Enterobacter, which has important academic value.

CLAVATA1-type receptor-like kinase CsCLAVATA1 is a putative candidate gene for dwarf mutation in cucumber.

Dwarf mutations have played vital roles in elucidating the regulatory molecular mechanisms of plant height. In this study, we identified a mutant named Csdw, whose mutagenesis was induced by ethyl methyl sulfonate in cucumber, and this mutant exhibited a dwarf phenotype with a reduced internode length because of the reduction of cell division in the main stem. The dwarf phenotype of Csdw could be partially rescued through GA3 application, and endogenous GA3 levels from the stem of Csdw decreased distinctly. Genetic analysis showed that Csdw was attributed to a recessive gene. The MutMap and Kompetitive Allele Specific PCR genotyping results revealed that Csa3G872760 (CsCLAVATA1), encoding a CLAVATA1-type receptor-like kinase, was a putative candidate gene for dwarf mutation in cucumber. The expression of CsCLAVATA1 in the stem of Csdw was lower than that of wild-type plants. Therefore, CsCLAVATA1 could regulate the dwarf phenotype in cucumber.

CsMYB36 is involved in the formation of yellow green peel in cucumber (Cucumis sativus L.).

KEY MESSAGE: A yellow green peel mutant (ygp) in cucumber was caused by a mutation in Csa2G352940 encoding MYB36 transcription factor. Peel color is one of the important agronomic traits of cucumber (Cucumis sativus L.). However, studies on the molecular regulation mechanism of peel color in cucumber are few. In this study, a cucumber yellow green peel mutant (ygp) of cucumber mutagenized with ethylmethylsulfone by using a wild type cucumber with dark green peel was identified. Pigment measurements indicated that the chlorophyll content of the ygp mutant was less than that of the wild type. Genetic analysis revealed that the phenotype of the ygp mutant was monogenic recessive inheritance. MutMap and genotyping results demonstrated that Csa2G352940 (CsMYB36), encoding the transcription factor MYB36, was the causal gene of the ygp mutant in cucumber. CsMYB36 was downregulated in the fruit of the ygp mutant. Transcriptome profile analysis of the fruit peel of the ygp mutant identified 92 candidate genes including genes that encode Casparian strip (CsCASP1) and pigment synthesis protein (CsMYC2) involved in peel color development in cucumber. CsMYB36 may regulate yellow green coloration in cucumber by interacting with these genes. Overall, these results showed that CsMYB36 can regulate the yellow green peel coloration in cucumber.

Effects of microbial agent application on the bacterial community in ginger rhizosphere soil under different planting years.

Ginger is one of the important spice crops in the world. Due to the prevalence of ginger wilt disease and the lack of effective prevention and control methods, the planting area, total production and value have declined sharply, which have become a key factor restricting ginger industry development in China. Understanding the influence of microbial agents on the rhizosphere microbiota of ginger will facilitate developing novel technologies for the prevention and control of ginger wilt disease. In the new planting and continuous cropping ginger fields, using large-root ginger and microbial agents, two inoculation levels (inoculation and no inoculation) were designed, and high-throughput sequencing technology was used to study the bacterial community structure in the rhizosphere soil at mature stage of ginger. The results showed that newly planted ginger showed a significant yield advantage over continuous cropping ginger, with a yield increase of 39% to 56%, and the lowest ginger wilt disease index. The community structure at the phylum level of soil bacteria in each treatment was very similar to that in the control, but the abundance of some taxonomic units changed significantly. The four dominant phyla of bacteria in mature ginger rhizosphere soil were Proteobacteria, Actinobacteria, Chloroflexi, and Acidobacteria, accounting for 72.91% to 89.09% of the total. The microbial agent treatment of continuous cropping had beneficial microorganisms such as Acidobacteria and Gemmatimonadetes with abundances increased by 12.2% and 17.1%, respectively, compared to the control. The microbial inoculant treatment of newly planted ginger increased the abundance of Acidobacteria and Gemmatimonadetes by 34.4% and 10.7%, respectively, compared to the control. The composition of bacterial communities were affected by changes in soil properties. Redundancy analysis showed that the hydrolysable nitrogen, available phosphorus, available potassium, and organic matter were significantly related to the composition of soil bacterial communities. Therefore, the microbial agents can not only promote the proliferation of beneficial microorganisms in the continuous cropping soil but also further reshape the soil bacterial community structure by changing the soil physicochemical properties such as effective phosphorus. These results provided a reference for related research on the impact of ginger continuous cropping on soil environment and soil management improvement in ginger fields.

Phylogenomics and plastome evolution of Indigofera (Fabaceae).

Introduction: Indigofera L. is the third largest genus in Fabaceae and includes economically important species that are used for indigo dye-producing, medicinal, ornamental, and soil and water conservation. The genus is taxonomically difficult due to the high level of overlap in morphological characters of interspecies, fewer reliability states for classification, and extensive adaptive evolution. Previous characteristic-based taxonomy and nuclear ITS-based phylogenies have contributed to our understanding of Indigofera taxonomy and evolution. However, the lack of chloroplast genomic resources limits our comprehensive understanding of the phylogenetic relationships and evolutionary processes of Indigofera. Methods: Here, we newly assembled 18 chloroplast genomes of Indigofera. We performed a series of analyses of genome structure, nucleotide diversity, phylogenetic analysis, species pairwise Ka/Ks ratios, and positive selection analysis by combining with allied species in Papilionoideae. Results and discussion: The chloroplast genomes of Indigofera exhibited highly conserved structures and ranged in size from 157,918 to 160,040 bp, containing 83 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Thirteen highly variable regions were identified, of which trnK-rbcL, ndhF-trnL, and ycf1 were considered as candidate DNA barcodes for species identification of Indigofera. Phylogenetic analysis using maximum likelihood (ML) and Bayesian inference (BI) methods based on complete chloroplast genome and protein-coding genes (PCGs) generated a well-resolved phylogeny of Indigofera and allied species. Indigofera monophyly was strongly supported, and four monophyletic lineages (i.e., the Pantropical, East Asian, Tethyan, and Palaeotropical clades) were resolved within the genus. The species pairwise Ka/Ks ratios showed values lower than 1, and 13 genes with significant posterior probabilities for codon sites were identified in the positive selection analysis using the branch-site model, eight of which were associated with photosynthesis. Positive selection of accD suggested that Indigofera species have experienced adaptive evolution to selection pressures imposed by their herbivores and pathogens. Our study provided insight into the structural variation of chloroplast genomes, phylogenetic relationships, and adaptive evolution in Indigofera. These results will facilitate future studies on species identification, interspecific and intraspecific delimitation, adaptive evolution, and the phylogenetic relationships of the genus Indigofera.

[Cloning and Evolutionary Analysis of Homologous Sequences of CYP86MF Gene in Cruciferae.].

In order to direct the construction of plant germplasms by elucidating the relatives among plants at the level of gene, CYP86MF gene analogues from 11 species of 6 genera in Cuciferae were respectively obtained by PCR strategy using gene specific primers designed from conserved regions of CYP86MF gene reported. Sequence comparisonindicated that the similarities among the genes at nucleotide level were over 80%, and the similarities at amino acid level remained above 70%. The differences between the genes at nucleotide and amino acid level between species were 1.0% ~ 5.7% and 2.6% ~ 7.3% respectively, while those between genera 5.6% ~ 22.5% and 7.3% ~ 31.2%, respectively. Phylogenetic analysis showed that Brassica was closely related to Raphanus, followed by Rorippa Scop, Arabidopsis Heynh, Capsella Medic orderly, most distantly related to Orychophrogmus. It was concluded that CYP86MF gene was not applicable to specie and subspecie taxon but genus taxon because the differences of sequences in nucleotides and amino acids were lower between species than genera.