First report of Pectobacterium brasiliense causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) belongs to Brassicaceae and is an important leaf vegetable widely cultivated in the Yangtze River basin and various southern provinces in China. In August 2023, the rhizome decay symptoms were observed at the stem base of leaf mustard plants (cv. Huarong) in the field of Changde City (29.05 °N; 111.59 °E), Hunan Province, China. The incidence of symptomatic leaf mustard was approximately 30% in several fields (2 ha in total). Brown and water-soaked symptoms appeared at the base of the outer leaves, and hollow rot at the base of the stem, accompanied by a fishy odor. To identify the causal agent, six infected stem samples were collected and surface sterilized by soaking in 75% ethanol for 60 seconds, rinsed three times with sterile distilled water, and finally cut into pieces (5 × 5 mm) in the sterile water. The extract was streaked on nutrient agar medium. After incubation at 28°C for 24 h, 17 strains were obtained and the colonies of all strains were creamy white, roughly circular, and convex elevation. Six single bacterial strains JC23121001-JC23121006, individually isolated from six different diseased stem samples, were selected as representative strains for further study. For preliminary identification, DNA from the six strains was extracted and identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991), and the sequences (accession nos. PP784484 to PP784489) showed 99% query coverage and 99.65% identity to Pectobacterium brasiliense type strain IBSBF1692T (Nabhan et al. 2012). In addition, five housekeeping genes acnA, mdh, mltD, pgi, and proA of the six strains were amplified with specially designed primers (Ma et al. 2007), and the resulting sequences from all six strains were 100% identical. The sequences of the representative strain JC23121001 were deposited into GenBank with accession numbers PP108247, PP066857, PP108248, PP066858, and PP066860, respectively. The maximum-likelihood phylogenetic tree clustered JC23121001 with P. brasiliense type strain IBSBF1692T (Nabhan et al. 2012). The pathogenicity test of six strains was carried out on the six-week-old leaf mustard (cv. Huarong) plants grown in the greenhouse by inoculating 10 µl of each bacterial suspension (108 CFU/ml) on needle-like wounds on the stem base of three healthy leaf mustard plants (Singh et al. 2013). Control plants were treated with sterile distilled water. After inoculation, the plants were incubated at 28°C and 90% relative humidity in a growth chamber. This trial was repeated three times. All inoculated mustard stems were slightly water-soaked after 24 hours and eventually developed into soft rot symptoms, consistent with the original symptoms observed. The control plants remained symptom-free. The strains were re-isolated from inoculated plants and re-identified as P. brasiliense by sequencing five housekeeping genes, thus fulfilling Koch's postulates. P. brasiliense has a broad host range and has been reported on other Brassica species, such as Bok choy (Brassica rapa var. chinensis) in China (Li et al. 2023). Soft rot of leaf mustard caused by Pectobacterium aroidearum has also been reported previously (Chu et al. 2023). To our knowledge, this is the first report of P. brasiliense causing soft rot on leaf mustard in China. The soft rot poses a significant threat to the local leaf mustard industry and requires further research into epidemiology and disease management options.

First report of Pectobacterium aroidearum causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) is widely planted in China as an important leaf vegetable. In March 2022, water-soaked and mushy rot symptoms were observed on leaf mustard plants in the field of Zhaotong (27.85°N; 105.05°E), Yunnan, China. The incidence of symptomatic leaf mustard was approximately 10%. The isolation of the causal agent followed the method of Peng et al. (2023). Briefly, infected tissues from four diseased plants were mixed and teased apart, and homogenized by vortex shaking. The bacterial suspension was diluted and spread on nutrient agar (NA). About 10 single colonies exhibiting different colony morphologies were picked and purified separately by successive streaking. A pinprick method was used for pathogenicity tests with an inoculum concentration of 108 CFU/ml (Singh et al. 2013). Among 10 isolates, only strain YKX exhibited soft rot symptoms on detached mustard leaves. In addition, ten two-month-old leaf mustard plants grown in the greenhouse were used for in vivo pathogenicity tests. Briefly, sterilized pins were dipped in the bacterial suspension, and then leaf mustard petioles were pricked with these pins. After inoculation, each plant was kept in a plastic bag for 12 hours to maintain high humidity. As expected, strain YKX caused obvious rot symptoms on eight plants at 1-2 days post-inoculation while the control group including two plants treated with sterile water showed no symptoms. The colonies of strain YKX on NA were white, roughly circular, and convex. For a preliminary identification, total DNA was extracted and used as the template in PCR amplification of 16S rDNA with the universal PCR primer pair 27F/1492R (Weisburg et al. 1991). The quality-filtered DNA sequence (871 bp) showed 100% query coverage and 99.47% identity to the 16S rDNA sequences of type strain Pectobacterium aroidearum SCRI 109T (GenBank: NR_159926) found in the NCBI rRNA/ITS database. Whole-genome sequencing of strain YKX was then performed using the Illumina and Nanopore sequencing platforms by Tsingke Biotechnology Co., Ltd. (Beijing, China). A single contig (GenBank: CP129239) with a length of approximately 4.9 Mb was obtained by de novo hybrid assembly using Unicycler v0.5.0 (Wick et al. 2017). The quality of the genomic data was evaluated by BUSCO v5.4.7 (Manni et al. 2021) against the gammaproteobacteria_odb10 dataset. A BUSCO complete score of 99.5% indicated high assembly quality. The genome sequence of strain YKX was uploaded to the Type Strain Genome Server for a genome-based taxonomic analysis (Meier-Kolthoff et al. 2022). The distance-based phylogeny showed that strain YKX and P. aroidearum L6 (GenBank: CP065044) and P. aroidearum PC1 (GenBank: NC_012917) form a clade. When comparing strain YKX with L6 or PC1, the digital DNA-DNA hybridization value (83.5-83.8%) was above the species delineation threshold (70% for DDH), clearly indicating that strain YKX should be classified as P. aroidearum. Additionally, P. aroidearum was reisolated from inoculated leaves and identified based on morphological similarities and 16S rDNA sequencing, thus fulfilling Koch's postulates. It is worth noting that a previous study reported occurrences of soft rot disease on leaf mustard attributed to Rhizopus microsporus var. chinensis (Wang et al. 2020). To our knowledge, this is the first report of P. aroidearum causing soft rot on leaf mustard in China, which expands the known host range of this pathogen and benefits the control of this disease.

Physicochemical properties and fractal characterizations of the functionalized porous clinoptilolites for controlling alginate delivery in growing cauliflower and leaf mustard.

Using natural clinoptilolite (NCP) as a carrier and alginate (Alg)-calcium as an active species, the porous silicon calcium alginate nanocomposite (Alg-Ca-NCP) was successfully fabricated via adsorption-covalence-hydrogen bond. Its structural features and physicochemical properties were detailed investigated by various characterizations. The results indicated that Alg-Ca-NCP presented the disordered lamellar structures with approximately uniform particles in size of 300-500 nm. Specially, their surface fractal evolutions between the irregular roughness and dense structures were demonstrated via the SAXS patterns. The results elucidated that the abundant micropores of NCP were beneficial for unrestricted diffusing of Alg-Ca, which was conducive to facilitate a higher loading and sustainable releasing. The Ca content of leaf mustard treated with Alg-Ca-NCP-0.5 was 484.5 mg/100g on the 21st day, higher than that by water (CK) and CaCl2 solution treatments, respectively. Meanwhile, the prepared Alg-Ca-NCPs presented the obvious anti-aging effects on peroxidase drought stress of mustard leaves. These demonstrations provided a simple and effective method to synthesize Alg-Ca-NCPs as delivery nanocomposites, which is useful to improve the weak absorption and low utilization of calcium alginate by plants.

Correlations between flavor and glucosinolates and changes in quality-related physiochemical characteristics of Guizhou suancai during the fermentation process.

The flavor of Guizhou suancai fermented by the emerging varieties Zhuchang-2 (ZC-2) and Zhuchang Red (ZC Red) is superior to that of ZC-1, a typical local variety of leaf mustard in Guizhou Province, China. Here, changes in phytochemical qualities during the fermentation of three Guizhou suancai were characterized to identify the causes underlying differences in flavor. The content of pigments, antioxidants, gluconapin, gluconasturtiin, and total isothiocyanates, and antioxidant capacity were highest in fresh ZC Red. The content of sinigrin, indolic glucosinolates, and total glucosinolate breakdown products was highest in fresh ZC-2. The content of phytochemicals, with the exception of carotenoids, was significantly decreased after fermentation. Odor was correlated with gluconapin, while taste was correlated with sinigrin. These findings provide detailed insights into the sensory and phytochemical properties of three Guizhou suancai that could facilitate the selection of raw material varieties.

Evaluation and comparison of physicochemical properties, volatile substances, and microbial communities of leaf mustard (Brassica juncea var. multiceps) under natural and inoculated fermentation.

Due to the uncontrolled fermentation process and unstable quality of naturally fermented leaf mustard, inoculated fermentation is receiving more attention. Here, the physicochemical properties, volatile compounds, and microbial community in leaf mustard under natural fermentation (NF) and inoculated fermentation (IF) were analyzed and compared. The contents of total acid, crude fiber, and nitrite of leaf mustard were measured. Headspace-solid phase microextraction-gas chromatography-mass spectrometry and orthogonal projection on latent structure-discriminant analysis were used to analyze the differences of volatile compounds in NF and IF leaf mustard. Moreover, Illumina MiSeq high-throughput sequencing technology was employed to reveal the composition of microbiota. The results showed that the nitrite content in leaf mustard after IF (3.69 mg/kg) was significantly lower than that after NF (4.43 mg/kg). A total of 31 and 25 kinds of volatile components were identified in IF and NF, respectively. Among the detected compounds, 11 compounds caused the differences between IF and NF leaf mustard. The results of inter-group difference analysis showed that there were significant differences in fungal flora between IF and NF samples. Saccharomycetes, Kazachstania, and Ascomycota were the landmark microorganisms in IF leaf mustard and the landmark microorganisms in NF were Mortierellomycota, Sordariomycetes, and Eurotiomycetes. The abundance of probiotics (such as Lactobacillus) in IF leaf mustard (51.22%) was higher than that in NF (35.20%) and the abundance of harmful molds (such as Mortierella and Aspergillus) was opposite. Therefore, IF leaf mustard showed the potential to reduce the content of nitrite and harmful molds and increase the beneficial volatile compounds and probiotics. PRACTICAL APPLICATION: Leaf mustard of inoculated fermentation (IF) showed better fermented characteristics than natural fermentation in terms of lower nitrite content, greater beneficial volatile substances, and better potential for increasing probiotics and reducing harmful molds. These results provided a theoretical basis for IF leaf mustard and contributed to the industrial production of fermented leaf mustard.

Leaf Mustard (Brassica juncea) Germplasm Resources Showed Diverse Characteristics in Agro-Morphological Traits and Glucosinolate Levels.

Leaf mustard, characterized by its purple/red/green leaves with a green/white midrib, is known for its thick, tender, and spicy leaves with a unique taste and flavor. There were only a few studies reported on leaf mustard for its morphological and biochemical traits from Korea. A total of 355 leaf mustard accessions stored at the GenBank of the National Agrobiodiversity Center were evaluated for 25 agro-morphological traits and seven intact glucosinolates (GSLs). The accessions showed a wide variation in terms of most of the traits. The quantitative agro-morphological traits varied from 16.0 (leaf length) to 48.7% (petiole width) of the coefficient of variation (CV). The highest variation was observed in glucoiberin (299.5%, CV), while the total GSL showed a CV of 66.1%. Sinigrin, followed by gluconapin and gluconasturtiin, was the most abundant GSL, accounting for as high as 75% of the total GSLs, while glucobrassicanapin and glucoiberin were the least abundant, contributing 0.7% and 0.1% on average, respectively. Sinigrin had a positive significant correlation with all GSLs but gluconasturtiin, while glucobarbarin and gluconasturtiin were highly positively correlated to each other, but least correlated with other GSLs. The leaf length was negatively correlated with sinigrin and glucoiberin. The width of the petiole showed a positive correlation with gluconapin, glucobrassicanapin, and glucobrassicin, while the length of the petiole had a negative correlation with sinigrin, glucobrassicanapin, glucoiberin, glucobrassicin, and the total GSLs. A higher width of the midrib was associated with higher contents of gluconapin, glucobrassicanapin, and glucobrassicin. A PCA analysis based on the agro-morphological traits showed that the first and second principal components accounted for 65.2% of the overall variability. Accessions that form a head tend to exhibit a longer leaf length, a larger plant weight, a thicker midrib, and higher widths of the midrib, petiole, and leaf. The GSLs showed inconsistent inter-and intra-leaf variation. Accessions that identified for various traits in their performance, such as, for example, Yeosu66 and IT259487 (highest total glucosinolates) and IT228984 (highest plant weight), would be promising lines for developing new varieties.

The optimal mixing ratio of Brassica napus and Brassica juncea meal improve nematode Meloidogyne hapla effects.

The use of rapeseed (Brassica napus L.) or leaf mustard (Brassica juncea L. Czern) meal or both as organic fertilizer not only improves the soil environment and crop productivity by supplying nutrients but also has nematicidal effects. This study aimed to establish the optimal application levels of rapeseed and leaf mustard meal for stronger nematode control in tomato. Tomato is one of the most important solanaceous crops which is severely damaged by nematodes. At first, meal (120 g of varying mixing ratios of rapeseed and leaf mustard meal) was mixed with sterilized soil (1 kg). The optimal ratio of rapeseed:leaf mustard meal for effective nematode control was 20:100 g/kg of soil. Progoitrin and gluconapin were the most abundant glucosinolates found in rapeseed meal, while sinigrin was the most abundant in leaf mustard meal. The amount of sinigrin increased if the leaf mustard meal proportion increased in the meal mixture. Although the content of sinigrin in optimal ratio mixture of rapeseed and leaf mustard meal is lower than only leaf mustard meal, it is presumed that nematocidal effects of the mixture are better than that of the single component due to the high contents of progoitrin and gluconapin. So, we propose that rapeseed and leaf mustard meal mixture at an appropriate ratio can be used as an environmentally friendly nematocide.

Free and bound phenolic compounds in leaves of pak choi (Brassica campestris L. ssp. chinensis var. communis) and Chinese leaf mustard (Brassica juncea Coss).

Eleven pak choi cultivars and two leaf mustard cultivars grown under field conditions in China were investigated for the free polyphenol content in their outer and inner leaves, as well as in their leaf blades and leaf stalks. In most cases, there were no significant differences between the hydroxycinnamic acid derivative and flavonoid derivative contents in the outer and inner leaves for the 13 cultivars. However, the contents of blades and stalks differed: hydroxycinnamic acids and flavonoids were present in greater amounts in the leaf blade than in the leaf stalk. Trace or small amounts of flavonoids were detected in the pak choi and leaf mustard stalks. Additionally, the bound phenolic contents of two pak choi cultivars and two leaf mustard cultivars were investigated. The concentrations of cell wall-bound phenolic compounds were higher in the leaf blade than in the leaf stalk under field conditions in China. These compounds represent only a minor portion of the total phenolic contents (flavonoids and hydroxycinnamic acids) in leaf stalks (0.81-1.18%) and leaf blades (0.05-0.08%) from fresh plant material. The storage of plant samples from four Chinese cabbage cultivars resulted, in most cases, in an increase of phenolic content, within six days, at 4°C and 20°C. The increase might have been triggered by post-harvest plant stresses, which stimulate the biosynthesis of polyphenols.

Antioxidant activity of different parts of Dolsan Leaf Mustard.

This study aimed to investigate the sinigrin content and antioxidant properties of Dolsan mustard seeds (DMS) and Dolsan leaf mustard (DLM) extracted with 50% acetonitrile. The extracted substances were fractionated by HPLC using a preparative ODS column and then applied to measure sinigirn, total polyphenol (TP), and total flavonoid (TF) contents and antioxidant activities using 2,2-azino-bis(3-ethyl-benzothizoline-6-sulfonic acid) (ABTS), electron donating ability (EDA), and ferric reducing antioxidant power (FRAP) assays. The contents of sinigrin (53.77 mg/g), TP (404.33 mg gallic acid equivalents/g extract), TF (395.33 mg quercetin equivalents/g extract) were higher content in DMS than in DLM. Antioxidant activities of DMS were a slightly higher than DLM. ABTS, EDA, and FRAP assays showed that there was a linear correlation between TP and TF contents and antioxidant capacity. These results suggested that the antioxidant effects of DMS and DLM might be mediated through the TP and TF contents, providing potential source of natural antioxidants.