Enhancement of the germination and growth of Panicum miliaceum and Brassica juncea in Cd- and Zn-contaminated soil inoculated with heavy-metal-tolerant Leifsonia sp. ZP3.

The addition of plant-growth-promoting bacteria (PGPB) to heavy-metal-contaminated soils can significantly improve plant growth and productivity. This study isolated heavy-metal-tolerant bacteria with growth-promoting traits and investigated their inoculation effects on the germination rates and growth of millet (Panicum miliaceum) and mustard (Brassica juncea) in Cd- and Zn-contaminated soil. Leifsonia sp. ZP3, which is resistant to Cd (0.5 mM) and Zn (1 mM), was isolated from forest soil. The ZP3 strain exhibited plant-growth-promoting activity, including indole-3-acetic acid production, phosphate solubilization, catalase activity, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging. In soil contaminated with low concentrations of Cd (0.232 ± 0.006 mM) and Zn (6.376 ± 0.256 mM), ZP3 inoculation significantly increased the germination rates of millet and mustard 8.35- and 31.60-fold, respectively, compared to the non-inoculated control group, while the shoot and root lengths of millet increased 1.77- and 4.44-fold (p < 0.05). The chlorophyll content and seedling vigor index were also 4.40 and 18.78 times higher in the ZP3-treated group than in the control group (p < 0.05). The shoot length of mustard increased 1.89-fold, and the seedling vigor index improved 53.11-fold with the addition of ZP3 to the contaminated soil (p < 0.05). In soil contaminated with high concentrations of Cd and Zn (0.327 ± 0.016 and 8.448 ± 0.250 mM, respectively), ZP3 inoculation led to a 1.98-fold increase in the shoot length and a 2.07-fold improvement in the seedling vigor index compared to the control (p < 0.05). The heavy-metal-tolerant bacterium ZP3 isolated in this study thus represents a promising microbial resource for improving the efficiency of phytoremediation in Cd- and Zn-contaminated soil.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

MAIN CONCLUSION: The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against the broad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

Zinc oxide nanoparticles and Klebsiella sp. SBP-8 alleviates chromium toxicity in Brassica juncea by regulation of antioxidant capacity, osmolyte production, nutritional content and reduction in chromium adsorption.

Heavy metals are one of the most damaging environmental toxins that hamper growth of plants. These noxious chemicals include lead (Pb), arsenic (As), nickel (Ni), cadmium (Cd) and chromium (Cr). Chromium is one of the toxic metal which induces various oxidative processes in plants. The emerging role of nanoparticles as pesticides, fertilizers and growth regulators have attracted the attention of various scientists. Current study was conducted to explore the potential of zinc oxide nanoparticles (ZnONPs) alone and in combination with plant growth promoting rhizobacteria (PGPR) Klebsiella sp. SBP-8 in Cr stress alleviation in Brassica juncea (L.). Chromium stress reduced shoot fresh weight (40%), root fresh weight (28%), shoot dry weight (28%) and root dry weight (34%) in B. juncea seedlings. Chromium stressed B. juncea plants showed enhanced levels of malondialdehyde (MDA), electrolyte leakage (EL), hydrogen peroxide (H2O2) and superoxide ion (O2• -). However, co-supplementation of ZnONPs and Klebsiella sp. SBP-8 escalated the activity of antioxidant enzymes i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) in B. juncea grown in normal and Cr-toxic soil. It is further proposed that combined treatment of ZnONPs and Klebsiella sp. SBP-8 may be useful for alleviation of other abiotic stresses in plants.

The correlation between flavor formation and microbial community dynamics during the fermentation of zha cai.

BACKGROUND: Zha cai, a pickled vegetable with unique flavors, is produced by fermenting fresh mustard tubers. In this study, the main physicochemical indices and volatile flavor compounds were determined in three fermentation periods. The bacterial and fungal communities in the three fermentation periods of zha cai were also monitored using high-throughput sequencing. Key microbial communities were identified based on significant correlations with flavor substances. RESULTS: Firmicutes and Proteobacteria were the main bacterial phyla found within the three fermentation periods. Lactic acid bacteria, namely Lactobacillus, was the predominant bacteria found at the genus level. Ascomycetes and Stenotrophomonas were the major fungal phyla found in the three fermentation periods. Yeast, namely Debaryomyces, was the predominant fungus found at the genus level. A total of 42 bacterial genera were negatively correlated with volatile flavor substances of zha cai, and 37 bacterial genera were positively correlated. Meanwhile, a total of 47 genera of fungi were negatively correlated with the volatile flavor substances of zha cai, while 50 genera were positively correlated. Several microbial genera were significantly correlated with volatile flavor compounds, including Lactobacillus, Halomonas, Rhodococcus, and Debaryomyces. CONCLUSION: This study identified the microbial classes that positively regulate the flavor of zha cai which could provide valuable help for flavor modulation in zha cai production. © 2024 Society of Chemical Industry.

Overexpression of flowering locus D (FLD) in Indian mustard (Brassica juncea) enhances tolerance to Alternaria brassicae and Sclerotinia sclerotiorum.

KEY MESSAGE: Overexpression of BjFLD in Brassica juncea imparts resistance against fungal pathogens and increases the yield. These transgenics could lower the use of fungicides, which have detrimental effects on the environment. Productivity of Indian mustard (Brassica juncea) is adversely affected by fungal phytopathogens, Alternaria brassicae and Sclerotinia sclerotiorum. Arabidopsis flowering locus D (FLD) positively regulates jasmonic acid signaling and defense against necrotrophic pathogens. In this study, the endogenous FLD (B. juncea FLD; BjFLD) in Indian mustard was overexpressed in B. juncea to determine its role in biotic stress tolerance. We report the isolation, characterization, and functional validation of BjFLD. The transgene expression was confirmed by qRT-PCR. The constitutive overexpression of BjFLD enhanced the tolerance of B. juncea to A. brassicae and S. sclerotiorum, which was manifested as delayed appearance of symptom, impeded disease progression, and enhanced percentage of disease protection. The transgenic lines maintained a higher photosynthetic capacity and redox potential under biotic stress and could detoxify reactive oxygen species (ROS) by modulating the antioxidant machinery and physiochemical attributes. The BjFLD-overexpressing lines showed enhanced SA level as well higher NPR1 expression. The overexpression of BjFLD induced early flowering and higher seed yield in the transgenic lines. These findings indicate that overexpression of BjFLD enhances the tolerance of B. juncea to A. brassicae and S. sclerotiorum by induction of systemic acquired resistance and mitigating the damage caused by stress-induced ROS.

The elemental defense effect of cadmium on Alternaria brassicicola in Brassica juncea.

BACKGROUND: The elemental defense hypothesis states a new defensive strategy that hyperaccumulators defense against herbivores or pathogens attacks by accumulating heavy metals. Brassica juncea has an excellent ability of cadmium (Cd) accumulation. However, the elemental defense effect and its regulation mechanism in B. juncea remain unclear. RESULTS: In this study, we profiled the elemental defense effect and the molecular regulatory mechanism in Cd-accumulated B. juncea after Alternaria brassicicola infection. B. juncea treated with 180 mg Kg- 1 DW CdCl2 2.5H2O exhibited obvious elemental defense effect after 72 h of infection with A. brassicicola. The expression of some defense-related genes including BjNPR1, BjPR12, BjPR2, and stress-related miRNAs (miR156, miR397, miR398a, miR398b/c, miR408, miR395a, miR395b, miR396a, and miR396b) were remarkably elevated during elemental defense in B. juncea. CONCLUSIONS: The results indicate that Cd-accumulated B. juncea may defend against pathogens by coordinating salicylic acid (SA) and jasmonic acid (JA) mediated systemic acquired resistance (SAR) and elemental defense in a synergistic joint effect. Furthermore, the expression of miRNAs related to heavy metal stress response and disease resistance may regulate the balance between pathogen defense and heavy metal stress-responsive in B. juncea. The findings provide experimental evidence for the elemental defense hypothesis in plants from the perspectives of phytohormones, defense-related genes, and miRNAs.

Genetic and Proteomic Basis of Sclerotinia Stem Rot Resistance in Indian Mustard [Brassica juncea (L.) Czern & Coss.].

Sclerotinia stem rot is one of the utmost important disease of mustard, causing considerable losses in seed yield and oil quality. The study of the genetic and proteomic basis of resistance to this disease is imperative for its effective utilization in developing resistant cultivars. Therefore, the genetic pattern of Sclerotinia stem rot resistance in Indian mustard was studied using six generations (P1, P2, F1, F2, BC1P1, and BC1P2) developed from the crossing of one resistant (RH 1222-28) and two susceptible (EC 766300 and EC 766123) genotypes. Genetic analysis revealed that resistance was governed by duplicate epistasis. Comparative proteome analysis of resistant and susceptible genotypes indicated that peptidyl-prolyl cis-trans isomerase (A0A078IDN6 PPIase) showed high expression in resistant genotype at the early infection stage while its expression was delayed in susceptible genotypes. This study provides important insight to mustard breeders for designing effective breeding programs to develop resistant cultivars against this devastating disease.

Evaluation of the Relationship among Biogenic Amines, Nitrite and Microbial Diversity in Fermented Mustard.

Biogenic amines (BAs) and nitrites are both considered harmful compounds for customer health, and are closely correlated with the microorganisms in fermented mustard (FM). In this study, BAs and nitrite contents in fifteen FM samples from different brands were analyzed. The concentrations of cadaverine in one sample and of histamine in one sample were above the toxic level. Moreover, five FM samples contained a high level of nitrite, exceeding the maximum residue limit (20 mg/kg) suggested by the National Food Safety Standard. Then, this study investigated bacterial and fungal communities by high-throughput sequencing analysis. Firmicutes and Basidiomycota were identified as the major bacteria and fungi phylum, respectively. The correlations among microorganisms, BAs and nitrite were analyzed. Typtamine showed a positive correlation with Lactobacillus and Pseudomonas. Cadaverine and nitrite is positively correlated with Leuconostoc. Furthermore, thirteen strains were selected from the samples to evaluate the accumulation and degradation properties of their BAs and nitrite. The results indicated that the Lactobacillus isolates, including L. plantarum GZ-2 and L. brevis SC-2, can significantly reduce BAs and nitrite in FM model experiments. This study not only assessed the contents of BAs and nitrite in FM samples, but also provided potential starter cultures for BAs and nitrite control in the FM products industry.

Introgression and QTL mapping conferring resistance for Alternaria brassicae in the backcross progeny of Sinapis alba + Brassica juncea somatic hybrids.

KEY MESSAGE: A total of three QTLs, responsible for A. brassicae resistance were introgressed into S. alba - B. juncea introgression lines from S. alba and mapped through donor genome-specific SSR markers. Alternaria brassicae is a key pathogen of the Brassicaceae family causing severe blight disease to oilseed crops that leads to heavy yield losses due to lack of resistance source within cultivated Brassicas. However, the host resistance present in the Sinapis alba, an allied member of the Brassicaceae family is still unexplored precisely due to the unavailability of segregating population for Alternaria blight resistance and scarcity of donor genome-specific genetic markers. The present study was undertaken to identify quantitative trait loci governing resistance to Alternaria blight which was introgressed from S. alba to the backcross population of stable S. alba + B. juncea somatic hybrids (2n = 60; AABBSS). The second backcross population showed significant phenotypic variations for Alternaria blight ranging from immune to highly susceptible phenotype, thus suggesting quantitative nature of resistance for the disease. A subset of 154 BC2F3-4 lines was used for disease screening and genotyping with 234 S. alba genome-specific microsatellite markers. As a result of the study, twelve linkage groups were developed corresponding to 12 chromosomes of S. alba (n = 12) covering a length of 1694.02 cM. The chromosomes 5 and 11 harbored a total of 1 (Abr-01), and 2 (Abr-02, and Abr-03) QTLs detected by ICIM-ADD mapping method at LOD score values 3.7, 5.12, and 6.74, respectively. The QTLs identified during the study have a range of 5.51-10.87 percent phenotypic variations for disease resistance. To the best of our knowledge, this is the first report of QTLs introgression for A. brassicae resistance in cultivated Brassica from an allied member of Brassicaceae.

Extra-large G-proteins influence plant response to Sclerotinia sclerotiorum by regulating glucosinolate metabolism in Brassica juncea.

Heterotrimeric G-proteins are one of the highly conserved signal transducers across phyla. Despite the obvious importance of G-proteins in controlling various plant growth and environmental responses, there is no information describing the regulatory complexity of G-protein networks during pathogen response in a polyploid crop. Here, we investigated the role of extra-large G-proteins (XLGs) in the oilseed crop Brassica juncea, which has inherent susceptibility to the necrotrophic fungal pathogen Sclerotinia sclerotiorum. The allotetraploid B. juncea genome contains multiple homologs of three XLG genes (two BjuXLG1, five BjuXLG2, and three BjuXLG3), sharing a high level of sequence identity, gene structure organization, and phylogenetic relationship with the progenitors' orthologs. Quantitative reverse transcription PCR analysis revealed that BjuXLGs have retained distinct expression patterns across plant developmental stages and on S. sclerotiorum infection. To determine the role of BjuXLG genes in the B. juncea defence response against S. sclerotiorum, RNAi-based suppression was performed. Disease progression analysis showed more rapid lesion expansion and fungal accumulation in BjuXLG-RNAi lines compared to the vector control plants, wherein suppression of BjuXLG3 homologs displayed more compromised defence response at the later time point. Knocking down BjuXLGs caused impairment of the host resistance mechanism to S. sclerotiorum, as indicated by reduced expression of defence marker genes PDF1.2 and WRKY33 on pathogen infection. Furthermore, BjuXLG-RNAi lines showed reduced accumulation of leaf glucosinolates on S. sclerotiorum infection, wherein aliphatic glucosinolates were significantly compromised. Overall, our data suggest that B. juncea XLG genes are important signalling nodes modulating the host defence pathways in response to this necrotrophic pathogen.

The Effect of Photoperiod on Necrosis Development, Photosynthetic Efficiency and 'Green Islands' Formation in Brassica juncea Infected with Alternaria brassicicola.

The main goal of growing plants under various photoperiods is to optimize photosynthesis for using the effect of day length that often acts on plants in combination with biotic and/or abiotic stresses. In this study, Brassica juncea plants were grown under four different day-length regimes, namely., 8 h day/16 h night, 12 h day/12 h night, 16 h day/8 h night, and continuous light, and were infected with a necrotrophic fungus Alternaria brassicicola. The development of necroses on B. juncea leaves was strongly influenced by leaf position and day length. The largest necroses were formed on plants grown under a 16 h day/8 h night photoperiod at 72 h post-inoculation (hpi). The implemented day-length regimes had a great impact on leaf morphology in response to A. brassicicola infection. They also influenced the chlorophyll and carotenoid contents and photosynthesis efficiency. Both the 1st (the oldest) and 3rd infected leaves showed significantly higher minimal fluorescence (F0) compared to the control leaves. Significantly lower values of other investigated chlorophyll a fluorescence parameters, e.g., maximum quantum yield of photosystem II (Fv/Fm) and non-photochemical quenching (NPQ), were observed in both infected leaves compared to the control, especially at 72 hpi. The oldest infected leaf, of approximately 30% of the B. juncea plants, grown under long-day and continuous light conditions showed a 'green island' phenotype in the form of a green ring surrounding an area of necrosis at 48 hpi. This phenomenon was also reflected in changes in the chloroplast's ultrastructure and accelerated senescence (yellowing) in the form of expanding chlorosis. Further research should investigate the mechanism and physiological aspects of 'green islands' formation in this pathosystem.

Amelioration of Chlorpyrifos-Induced Toxicity in Brassica juncea L. by Combination of 24-Epibrassinolide and Plant-Growth-Promoting Rhizobacteria.

Pervasive use of chlorpyrifos (CP), an organophosphorus pesticide, has been proven to be fatal for plant growth, especially at higher concentrations. CP poisoning leads to growth inhibition, chlorosis, browning of roots and lipid and protein degradation, along with membrane dysfunction and nuclear damage. Plants form a linking bridge between the underground and above-ground communities to escape from the unfavourable conditions. Association with beneficial rhizobacteria promotes the growth and development of the plants. Plant hormones are crucial regulators of basically every aspect of plant development. The growing significance of plant hormones in mediating plant-microbe interactions in stress recovery in plants has been extensively highlighted. Hence, the goal of the current study was to investigate the effect of 24-epibrassinolide (EBL) and PGPRs (Pseudomonas aeruginosa (Ma), Burkholderia gladioli (Mb)) on growth and the antioxidative defence system of CP-stressed Brassica juncea L. seedlings. CP toxicity reduced the germination potential, hypocotyl and radicle development and vigour index, which was maximally recuperated after priming with EBL and Mb. CP-exposed seedlings showed higher levels of superoxide anion (O2-), hydrogen peroxide (H2O2), lipid peroxidation and electrolyte leakage (EL) and a lower level of nitric oxide (NO). In-vivo visualisation of CP-stressed seedlings using a light and fluorescent microscope also revealed the increase in O2-, H2O2 and lipid peroxidation, and decreased NO levels. The combination of EBL and PGPRs reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) contents and improved the NO level. In CP-stressed seedlings, increased gene expression of defence enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APOX), glutathione peroxidase (GPOX), dehydroascorbate reductase (DHAR) and glutathione reductase (GPOX) was seen, with the exception of catalase (CAT) on supplementation with EBL and PGPRs. The activity of nitrate reductase (NR) was likewise shown to increase after treatment with EBL and PGPRs. The results obtained from the present study substantiate sufficient evidence regarding the positive association of EBL and PGPRs in amelioration of CP-induced oxidative stress in Brassica juncea seedlings by strengthening the antioxidative defence machinery.

A complex interplay of Gß and Gγ proteins regulates plant growth and defence traits in the allotetraploid Brassica juncea.

KEY MESSAGE: Gene expression analysis coupled with in-planta studies showed that specific Gßγ combination regulates plant growth and defence traits in the allotetraploid Brassica juncea. Plant heterotrimeric G-proteins regulate a wide range of responses despite their limited repertoire of core components. The roles and functional interactions between different G-protein subunits are quite perplexing, which get further complicated with polyploidy. Here, we show that the allotetraploid Brassica juncea comprises multiple homologs of G-protein genes, encoding six BjuGß and ten highly divergent BjuGγ subunit proteins, later being classified into type-A1, type-A2 and type-C Gγ proteins. The encoded BjuGß and BjuGγ proteins shared close evolutionary relationship and have retained distinct spatio-temporal expression patterns during plant developmental stages and in response to the necrotrophic pathogen, Sclerotinia sclerotiorum. RNAi based suppression of BjuGß and BjuGγ genes suggested functional overlap and selectivity of BjuGßs with three distinct BjuGγ type subunits, to regulate plant height (BjuGßγA2 and BjuGßγC), seed weight (BjuGßGγA1 and BjuGßGγC), silique size (BjuGßGγC) and pathogen response (BjuGßGγA1 and BjuGßGγC). Further, the triplicated BjuGß genes, formed due to Brassica specific whole-genome-triplication event, showed differential involvement during pathogen response, wherein overexpression of BjuGß2 displayed higher resistance to Sclerotinia infection. Taken together, our study demonstrates that multiple BjuGß and BjuGγ proteins have retained distinct spatio-temporal expression and functional selectivity to regulate specific plant growth and defence traits in the oilseed B. juncea.

Role of Bacillus cereus in Improving the Growth and Phytoextractability of Brassica nigra (L.) K. Koch in Chromium Contaminated Soil.

Plant growth-promoting rhizobacteria (PGPR) mediate heavy metal tolerance and improve phytoextraction potential in plants. The present research was conducted to find the potential of bacterial strains in improving the growth and phytoextraction abilities of Brassica nigra (L.) K. Koch. in chromium contaminated soil. In this study, a total of 15 bacterial strains were isolated from heavy metal polluted soil and were screened for their heavy metal tolerance and plant growth promotion potential. The most efficient strain was identified by 16S rRNA gene sequencing and was identified as Bacillus cereus. The isolate also showed the potential to solubilize phosphate and synthesize siderophore, phytohormones (indole acetic acid, cytokinin, and abscisic acid), and osmolyte (proline and sugar) in chromium (Cr+3) supplemented medium. The results of the present study showed that chromium stress has negative effects on seed germination and plant growth in B. nigra while inoculation of B. cereus improved plant growth and reduced chromium toxicity. The increase in seed germination percentage, shoot length, and root length was 28.07%, 35.86%, 19.11% while the fresh and dry biomass of the plant increased by 48.00% and 62.16%, respectively, as compared to the uninoculated/control plants. The photosynthetic pigments were also improved by bacterial inoculation as compared to untreated stress-exposed plants, i.e., increase in chlorophyll a, chlorophyll b, chlorophyll a + b, and carotenoid was d 25.94%, 10.65%, 20.35%, and 44.30%, respectively. Bacterial inoculation also resulted in osmotic adjustment (proline 8.76% and sugar 28.71%) and maintained the membrane stability (51.39%) which was also indicated by reduced malondialdehyde content (59.53% decrease). The antioxidant enzyme activities were also improved to 35.90% (superoxide dismutase), 59.61% (peroxide), and 33.33% (catalase) in inoculated stress-exposed plants as compared to the control plants. B. cereus inoculation also improved the uptake, bioaccumulation, and translocation of Cr in the plant. Data showed that B. cereus also increased Cr content in the root (2.71-fold) and shoot (4.01-fold), its bioaccumulation (2.71-fold in root and 4.03-fold in the shoot) and translocation (40%) was also high in B. nigra. The data revealed that B. cereus is a multifarious PGPR that efficiently tolerates heavy metal ions (Cr+3) and it can be used to enhance the growth and phytoextraction potential of B. nigra in heavy metal contaminated soil.

Biochemical changes and microbial community dynamics during spontaneous fermentation of Zhacai, a traditional pickled mustard tuber from China.

Zhacai is a traditional fermented vegetable that has been consumed in China for centuries. It is currently manufactured by spontaneous fermentation and therefore mostly relies on the activities of autochthonous microorganisms. Here, we characterized microbial community dynamics and associated biochemical changes in 12% salted Zhacai during a 90-day spontaneous fermentation process using high-throughput sequencing and chromatography-based approaches to identify associations between microorganisms and fermentation characteristics. Amplicon sequencing targeting bacterial 16S rRNA genes revealed that bacterial communities were dominated by halophilic bacteria (HAB, i.e., Halomonas and Idiomarina) and lactic acid bacteria (LAB, i.e., Lactobacillus-related genera and Weissella) after 30 days of fermentation. In addition, the relative abundances of the fungal genera Debaryomyces, Sterigmatomyces, and Sporidiobolus increased as fermentation progressed. Concomitantly, pH decreased while titratable acidity increased during fermentation, along with associated variation in biochemical profiles. Overall, the levels of organic acids (i.e., lactic and acetic acid), free amino acids (i.e., alanine, lysine, and glutamic acid), and volatiles (i.e., alcohols, esters, aldehydes, and ketones) increased in mature Zhacai. In addition, the abundances of Lactobacillus-related species, Halomonas spp., Idiomarina loihiensis, as well as that of the yeast Debaryomyces hansenii, were strongly correlated with increased concentrations of organic acids, amino acids, biogenic amines, and volatiles. This study provides new detailed insights into the succession of microbial communities and their potential roles in Zhacai fermentation.

Candidate Rlm6 resistance genes against Leptosphaeria. maculans identified through a genome-wide association study in Brassica juncea (L.) Czern.

KEY MESSAGE: One hundred and sixty-seven B. juncea varieties were genotyped on the 90K Brassica assay (42,914 SNPs), which led to the identification of sixteen candidate genes for Rlm6. Brassica species are at high risk of severe crop loss due to pathogens, especially Leptosphaeria maculans (the causal agent of blackleg). Brassica juncea (L.) Czern is an important germplasm resource for canola improvement, due to its good agronomic traits, such as heat and drought tolerance and high blackleg resistance. The present study is the first using genome-wide association studies to identify candidate genes for blackleg resistance in B. juncea based on genome-wide SNPs obtained from the Illumina Infinium 90 K Brassica SNP array. The verification of Rlm6 in B. juncea was performed through a cotyledon infection test. Genotyping 42,914 single nucleotide polymorphisms (SNPs) in a panel of 167 B. juncea lines revealed a total of seven SNPs significantly associated with Rlm6 on chromosomes A07 and B04 in B. juncea. Furthermore, 16 candidate Rlm6 genes were found in these regions, defined as nucleotide binding site leucine-rich-repeat (NLR), leucine-rich repeat RLK (LRR-RLK) and LRR-RLP genes. This study will give insights into the blackleg resistance in B. juncea and facilitate identification of functional blackleg resistance genes which can be used in Brassica breeding.

Metagenomic analysis of microbial community succession during the pickling process of Zhacai (preserved mustard tuber) and its correlation with Zhacai biochemical indices.

BACKGROUND: Industrial Fuling Zhacai is pickled by a method summarized as 'three times pickled and pressed', in which raw mustard tubers are subjected to three stages of pickling in different salt concentrations, with a pressing operation at the end of each stage to remove brine. This study used Illumina MiSeq technology and multivariate statistical analyses to investigate microbial community succession during the pickling process and its correlation with Zhacai biochemical indices. RESULTS: A total of 19 phyla, 208 genera, and 295 species of bacteria were identified. Lactobacillus was the dominant genus of bacteria in all three stages and Lactobacillus sakei was the dominant species in the first and second stages. A total of six phyla, 200 genera and 301 species of fungi were also identified. According to a PICRUSt2 prediction, the main functions of the bacterial and fungal communities were carbohydrate and protein metabolism, while alcohol metabolism was also a function of fungi. Nine bacterial genera closely correlated with Zhacai biochemical indices: Acinetobacter, Pseudomonas, Pedobacter, Erwinia, Lactobacillus, Chryseobacterium, Flavobacterium, Duganella, and Paenarthrobacter. Six genera of fungi correlated closely: Penicillium, Cystobasidium, Cladosporium, Plenodomus, Aspergillus, and Simplicillium. All these genera probably originated from the surface microorganisms of raw mustard tuber. CONCLUSION: This study reveals the succession patterns of microbial community structures during the pickling process of industrial Zhacai and infers the core functional flora, providing reference data for Zhacai pickling process control. © 2020 Society of Chemical Industry.

Evaluation of physiological and molecular effect of variable virulence of Alternaria brassicae isolates in Brassica juncea, Sinapis alba and Camelina sativa.

Brassica genus comprises many prominent species valuable for human nutrition including vegetable crops and oilseed. Production of B. juncea is challenged by many abiotic and biotic stresses, Alternaria blight caused by a necrotrophic fungal pathogen Alternaria brassicae is one of the most prominent diseases of cruciferous crops including B. juncea. However, some closely related wild species like Sinapis alba and Camelina sativa exhibit a variable level of resistance towards the pathogen. Apart from the host resistance, intra-specific pathogen variability also influences disease severity to a larger extent. In this study, we identified and isolated two strains of A. brassicae viz ABS1 and ABS2 exhibiting morphological and pathological variability. These isolates were further used to artificially inoculate B. juncea and two of its wild relatives under in-vitro as well as in-vivo conditions to inspect their pathogenicity in a susceptible, a moderately resistant and a highly resistant host. virulent isolate (ABS2) was able to readily establish infection in all the three species whereas the less virulent isolate (ABS1) readily infected susceptible species B. juncea but delayed and mild infection was noticed in tolerant hosts. Variable physiological and molecular host response towards the differential level of virulence of pathogen were established with many confirmatory experiments like DAB staining study, Disease severity index and microscopic analysis. Real-time PCR results confirm that these two isolates induce a variable level of induction in genes PR1 and PDF1.2 within 48 h of the artificial inoculation in B. juncea and its wild relatives.

Physiological change alters endophytic bacterial community in clubroot of tumorous stem mustard infected by Plasmodiophora brassicae.

BACKGROUND: Endophytic bacteria are considered as symbionts living within plants and are influenced by abiotic and biotic environments. Pathogen cause biotic stress, which may change physiology of plants and may affect the endophytic bacterial communiy. Here, we reveal how endophytic bacteria in tumorous stem mustard (Brassica juncea var. tumida) are affected by plant physiological changes caused by Plasmodiophora brassicae using 16S rRNA high-throughput sequencing. RESULTS: The results showed that Proteobacteria was the dominant group in both healthy roots and clubroots, but their abundance differed. At the genus level, Pseudomonas was dominant in clubroots, whereas Rhodanobacter was the dominant in healthy roots. Hierarchical clustering, UniFrac-weighted principal component analysis (PCA), non-metric multidimensional scaling (NMDS) and analysis of similarities (ANOSIM) indicated significant differences between the endophytic bacterial communities in healthy roots and clubroots. The physiological properties including soluble sugar, soluble protein, methanol, peroxidase (POD) and superoxide dismutase (SOD) significantly differed between healthy roots and clubroots. The distance-based redundancy analysis (db-RDA) and two-factor correlation network showed that soluble sugar, soluble protein and methanol were strongly related to the endophytic bacterial community in clubroots, whereas POD and SOD correlated with the endophytic bacterial community in healthy roots. CONCLUSIONS: Our results illustrate that physiologcial changes caused by P. brassicae infection may alter the endophytic bacterial community in clubroots of tumorous stem mustard.

Expression of N-cycling genes of root microbiomes provides insights for sustaining oilseed crop production.

Agricultural production is dependent on inputs of nitrogen (N) whose cycle relies on soil and crop microbiomes. Crop diversification has increased productivity; however, its impact on the expression of microbial genes involved in N-cycling pathways remains unknown. Here, we assessed N-cycling gene expression patterns in the root and rhizosphere microbiomes of five oilseed crops as influenced by three 2-year crop rotations. The first phase consisted of fallow, lentil or wheat, and the second phase consisted of one of five oilseed crops. Expression of bacterial amoA, nirK and nirS genes showed that the microbiome of Ethiopian mustard had the lowest and that of camelina the highest potential for N loss. A preceding rotation phase of lentil significantly increased the expression of nifH gene by 23% compared with wheat and improved nxrA gene expression by 51% with chemical fallow in the following oilseed crops respectively. Lentil substantially increased biological N2 fixation and reduced denitrification in the following oilseed crops. Our results also revealed that most N-cycling gene transcripts are more abundant in the microbiomes associated with roots than with the rhizosphere. The outcome of our investigation brings a new level of understanding on how crop diversification and rotation sequences are related to N-cycling in annual cropping systems.