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Wild relatives of Brassica are a rich reservoir of genes that are invaluable for the improvement of cultivated species. Sinapis alba is a close relative of crop Brassicas that possesses several desirable traits such as tolerance to Alternaria black spot disease, heat stress, insect pests and nematodes. This study is aimed at developing and characterizing hybrids between Brassica juncea and S. alba with the ultimate goal of transferring genes for tolerance to Alternaria brassicae and heat stress, the traits that are lacking in cultivated Brassica. We generated three hybrids between B. juncea and S. alba through protoplast fusion. The hybridity was confirmed through cytology and molecular markers. While two of the hybrids were symmetric, the third one was asymmetric and had greater resemblance to B. juncea. Hybrids showed some characteristic features of the parents and were fully male and female fertile and also set seeds upon back crossing with the parent species. In vitro leaf assay and field inoculation studies revealed that the hybrids are highly resistant to A. brassicae. Besides, hybrids set seeds at temperature of > 38 °C when parents failed to produce seeds indicating that hybrids possess heat tolerance. These stable hybrids provide a reliable genetic resource for transfer of genes from S. alba into cultivated Brassica species.

In this paper, thermal (8-13 µm) and hyperspectral imaging in visible and near infrared (VNIR) and short wavelength infrared (SWIR) ranges were used to elaborate a method of early detection of biotic stresses caused by fungal species belonging to the genus Alternaria that were host (Alternaria alternata, Alternaria brassicae, and Alternaria brassicicola) and non-host (Alternaria dauci) pathogens to oilseed rape (Brassica napus L.). The measurements of disease severity for chosen dates after inoculation were compared to temperature distributions on infected leaves and to averaged reflectance characteristics. Statistical analysis revealed that leaf temperature distributions on particular days after inoculation and respective spectral characteristics, especially in the SWIR range (1000-2500 nm), significantly differed for the leaves inoculated with A. dauci from the other species of Alternaria as well as from leaves of non-treated plants. The significant differences in leaf temperature of the studied Alternaria species were observed in various stages of infection development. The classification experiments were performed on the hyperspectral data of the leaf surfaces to distinguish days after inoculation and Alternaria species. The second-derivative transformation of the spectral data together with back-propagation neural networks (BNNs) appeared to be the best combination for classification of days after inoculation (prediction accuracy 90.5%) and Alternaria species (prediction accuracy 80.5%).

Cauliflower ( Brassica oleracea L. var. botrytis ) and mustard [ Brassica juncea (L.) Czern] are important cruciferous crops facing serious yield and quality loss in India from dark leaf spot disease caused by Alternaria brassicae (Berk.) Sacc. Genetic variation among 32 pathogenic A. brassicae isolates from both crops was analyzed with random amplified polymorphic DNA and inter-simple sequence repeat primers in which the mean similarity coefficient was found to be 0.73 and 0.84, respectively. Further internal transcribed spacer analysis showed all isolates are 90–100 % similar to each other, indicating genetic similarity among different A. brassicae isolates that vary pathogenically.

Background Alternaria brassicae , a necrotrophic pathogen, causes Alternaria Leaf Spot, one of the economically important diseases of Brassica crops. Many other Alternaria spp. such as A. brassicicola and A. alternata are known to cause secondary infections in the A. brassicae -infected Brassicas. The genome architecture, pathogenicity factors, and determinants of host-specificity of A. brassicae are unknown. In this study, we annotated and characterised the recently announced genome assembly of A. brassicae and compared it with other Alternaria spp. to gain insights into its pathogenic lifestyle. Results We also sequenced the genomes of two A. alternata isolates that were co-infecting B. juncea using Nanopore MinION sequencing for additional comparative analyses within the Alternaria genus. Genome alignments within the Alternaria spp. revealed high levels of synteny between most chromosomes with some intrachromosomal rearrangements. We show for the first time that the genome of A. brassicae , a large-spored Alternaria species, contains a dispensable chromosome. We identified 460 A. brassicae -specific genes, which included many secreted proteins and effectors. Furthermore, we have identified the gene clusters responsible for the production of Destruxin-B, a known pathogenicity factor of A. brassicae . Conclusion The study provides a perspective into the unique and shared repertoire of genes within the Alternaria genus and identifies genes that could be contributing to the pathogenic lifestyle of A. brassicae .

Huperzine A (HupA) is a potent acetylcholinesterase (AChE) inhibitor of a great consideration as a prospective drug candidate for Alzheimer’s disease treatment. Production of HupA by endophytes offers an alternative challenge to reduce the massive plant harvest needed to meet the increasing demand of HupA. In the current study, some endophytic fungal and actinobacterial isolates from the Chinese herb, Huperzia serrata , underwent liquid fermentation, alkaloid extraction, and screening for AChE inhibition and HupA production. Among these isolates, Alternaria brassicae AGF041 strain was the only positive strain for HupA production with the maximum AChE inhibition of 75.5%. Chromatographic analyses verified the identity of the produced HupA. The HupA production was efficiently maximized up to 42.89 μg/g of dry mycelia, after optimization of thirteen process parameters using multifactorial statistical approaches, Plackett–Burman and central composite designs. The statistical optimization resulted in a 40.8% increase in HupA production. This is the first report to isolate endophytic actinobacteria with anti-AChE activity from H. serrata , and to identify an endophytic fungus A. brassicae as a new promising start strain for a higher HupA yield.

Among biotic stresses, Alternaria leaf spots caused by Alternaria brassicae and A. brassicicola and black rot caused by Xanthomonas campestris pv. campestris are major limiting factors in brassica cultivation across the world. Because of seed-borne nature of these pathogens primarily, disease-free conservation as well as exchange of brassica seeds at domestic as well as international level are major challenges. To facilitate disease-free conservation and transboundary movement of brassica germplasm, a highly specific and sensitive method was developed for simultaneous detection of these pathogens. A set of primers namely, AbeABC1F and AbeABC1R based on ABC transporter (Atr1) gene for A. brassicae, Aba28sF and Aba28sR based on SSR marker was developed for A. brassicicola as well as rpf gene-based primers namely, rpfH_F and rpfH_R for X. campestris pv. campestris were used for multiplex PCR. The specific bands of 586, 201 and 304 bp were obtained in multiplex PCR assay for A. brassicae, A. brassicicola and X. campestris pv. campestris, respectively. Therefore, the developed multiplex PCR protocol could be utilized for a reliable diagnosis of these pathogens to facilitate safe conservation, exchange of seeds to the researchers and also by seed certification agencies for ensuring quality seed availability to farmers.

Alternaria leaf blight (ALB), caused by a necrotrophic fungus Alternaria brassicae is a serious disease of oleiferous Brassicas resulting in significant yield losses worldwide. No robust resistance against A. brassicae has been identified in the Brassicas. Natural accessions of Arabidopsis show a spectrum of responses to A. brassicae ranging from high susceptibility to complete resistance. To understand the molecular mechanisms of resistance/ susceptibility, we analysed the comparative changes in the transcriptome profile of Arabidopsis accessions with contrasting responses- at different time points post-infection. Differential gene expression, GO enrichment, pathway enrichment, and weighted gene co-expression network analysis (WGCNA) revealed reprogramming of phenylpropanoid biosynthetic pathway involving lignin, hydroxycinnamic acids, scopoletin, anthocyanin genes to be highly associated with resistance against A. brassicae. T-DNA insertion mutants deficient in the biosynthesis of coumarin scopoletin exhibited enhanced susceptibility to A. brassicae. The supplementation of scopoletin to medium or exogenous application resulted in a significant reduction in the A. brassicae growth. Our study provides new insights into the transcriptome dynamics in A. brassicae-challenged Arabidopsis and demonstrates the involvement of coumarins in plant immunity against the Brassica pathogen A. brassicae.Key messageAlternaria brassicae responsive comparative transcriptomics and analysis of mutants deficient in phenylpropanoid pathway provide evidence for its involvement in resistance against the necrotrophic fungus A. brassicae.

Huperzine-A (HupA) is an emerging, powerful, and promising natural acetylcholinesterase inhibitor. Despite that, the achieved yields of HupA from microbial sources are still far from the industrial applications. Accordingly, this paper was conducted to valorize solid-state fermentation (SSF) as an efficient production platform of HupA. Four agro-industrial wastes, namely rice bran, potato peel, sugarcane bagasse, and wheat bran, were tested and screened as cultural substrates for the production of HupA by the endophytic Alternaria brassica under SSF. Maximum HupA production was attained on using rice bran moistened by Czapex’s dox mineral broth. In the effort to increase the HupA titer, supplementation of the best moistening agent by different carbon and nitrogen sources was successfully investigated. Additionally, factors affecting HupA production under SSF including substrate concentration, moistening level, and inoculum concentration were optimized using response surface methodology. A Box-Behnken design was applied for generating a predictive model of the interactions between these factors. Under the optimum conditions of 15 g rice bran, inoculum concentration of 5 × 106 spores mL−1, and 60% moisture level, HupA concentration was intensified to 518.93 μg g−1. Besides, HupA production by the fungal strain was further enhanced using gamma-irradiation mutagenesis. The final HupA production was significantly intensified following exposure to 0.5 KGy gamma radiation to 1327 μg g−1, which represents a 12.85-fold increase. This is the first report on the successful production of the natural fungal metabolite HupA under SSF. Moreover, the achieved yield in this study using agro-industrial wastes may contribute to reducing the cost of HupA manufacture.Key points• Different agro-industrial by-products were tried as cultural substrates for the production of the acetylcholinesterase inhibitor HupA under SSF for the first time.• Factors affecting HupA production under SSF were optimized using response surface methodology.• The final HupA production was intensified following exposure to gamma radiation recording 1327 μg g−1, which represents a 12.85-fold increase.

Plant pathogens resistant to the commercially available fungicides and bactericides even at higher concentrations are the biggest challenge for the farmers to control the losses due to plant diseases. The antibacterial and antifungal potential of nanomaterials makes them a suitable candidate for the control of plant diseases. Thus, the present study reports the phytofabricated zinc oxide nanoparticles (ZnO Np’s) using aqueous plant leaf extract of Terminalia bellerica (Baheda). Characterization of ZnO nanoparticles was done by ultraviolet–visible (UV–Vis) studies, X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infra-red (FT–IR) analysis, and transmission electron microscopy (TEM). The presence of pure hexagonal wurtzite crystalline structure of ZnO nanoparticles was confirmed by XRD analysis. The TEM images revealed the spherical to hexagonal shaped ZnO nanoparticles with sizes ranging from 20 to 30 nm. The stabilization of synthesized ZnO nanoparticles through the interactions of terpenoids, steroids, phenylpropanoids, flavonoids, phenolic acids, and enzymes present in the leaf extract was suggested by FTIR analysis. The mechanism of the formation of ZnO nanoparticles using Terminalia bellerica (Baheda) (Tb-ZnO Np’s) as a bioactive compound is proposed. These phytofabricated ZnO nanoparticles (Tb-ZnO Np’s) have shown significant antifungal potential against Alternaria brassicae the causal agent of Alternaria blight disease/leaf spot disease in Brassica species. The microscopic results confirm the changes in mycelium morphology and reduction in the number of spore germination at 0.2 mg/mL concentration Tb-ZnO Np’s.

and are two major phytopathogenic fungi which cause blight, a recalcitrant disease on crops throughout the world, which is highly destructive and responsible for significant yield losses. Since no resistant source is available against blight, therefore, efforts have been made in the present study to identify defense inducer molecules which can induce jasmonic acid (JA) mediated defense against the disease. It is believed that JA triggered defense response will prevent necrotrophic mode of colonization of fungus. The JA receptor, COI1 is one of the potential targets for triggering JA mediated immunity through interaction with JA signal. In the present study, few mimicking compounds more efficient than naturally occurring JA in terms of interaction with COI1 were identified through virtual screening and molecular dynamics simulation studies. A high quality structural model of COI1 was developed using the protein sequence of . This was followed by virtual screening of 767 analogs of JA from ZINC database for interaction with COI1. Two analogs viz. ZINC27640214 and ZINC43772052 showed more binding affinity with COI1 as compared to naturally occurring JA. Molecular dynamics simulation of COI1 and COI1-JA complex, as well as best screened interacting structural analogs of JA with COI1 was done for 50 ns to validate the stability of system. It was found that ZINC27640214 possesses efficient, stable, and good cell permeability properties. Based on the obtained results and its physicochemical properties, it is capable of mimicking JA signaling and may be used as defense inducers for triggering JA mediated resistance against blight, only after further validation through field trials.