Botanical herbicide has been a hot topic in the research and development of novel pesticides.  The herbicidal activity and biochemical characteristics of the botanical compound drupacine were studied by evaluating its effects on seed germination, seedling growth, morphological and physiological characteristics of Amaranthus retroflexus.  Drupacine inhibited seed germination and seedling growth, and had a median inhibition concentration (IC₅₀) value of 38.99 mg L⁻¹ against A. retroflexus root.  The α-amylase activity and soluble sugar content in treated plants were significantly lower than that of the control.  The expression of α-amylase gene was dosage-dependently inhibited compared to the untreated control.  This suggested that inhibition of α-amylase activity was a mode of action on seed germination.  The root hairs were significantly decreased and part of the root cap fell off after treatment with drupacine.  The ultrastructure observation showed that cell damage of root tips increased with the treatment time.  Drupacine also increased the relative conductivity and malondialdehyde (MDA) content.  Peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities were significantly enhanced in the treatment compared to the control.  These findings indicated that the physiological and biochemical reaction changes leading to morphological and membrane injuries were the main effects of drupacine on the inhibition of seedling growth.  Drupacine can be developed as a botanical herbicide. 

The fungal pathogen Setosphaeria turcica causes northern corn leaf blight (NCLB), which leads to considerable crop losses.  Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection.  Previously, we demonstrated that the S. turcica triggers an S-phase checkpoint and ATR (Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection.  However, how the regulatory mechanism works was still largely unknown.  Here, we report a genome wide transcriptional profile analysis during appressorium formation in the present of DNA replication stress.  We performed RNA-Seq analysis to identify S. tuicica genes responsive to DNA replication stress.  In the current work, we found that appressorium-mediated maize infection by S. turcica is significantly blocked by S-phase checkpoint.  A large serial of secondary metabolite and melanin biosynthesis genes were blocked in appressorium formation of S. turcica during the replication stress.  The secondary metabolite biosynthesis genes including alcohol dehydrogenase GroES-like domain, multicopper oxidase, ABC-transporter families, cytochrome P450 and FAD-containing monooxygenase were related to plant pathogen infection.  In addition, we demonstrated that autophagy in S. turcica is up-regulated by ATR as a defense response to stress.  We identified StATG3, StATG4, StATG5, StATG7 and StATG16 genes for autophagy were induced by ATR-mediated S-phase checkpoint.  We therefore propose that in response to genotoxic stress, S. turcica utilizes ATR-dependent pathway to turn off transcription of genes governing appressorium-mediated infection, and meanwhile inducing transcription of autophagy genes likely as a mechanism of self-protection, aside from the more conservative responses in eukaryotes.

Sugar is an indispensable source of energy for plant growth and development, and it requires the participation of sugar transporter proteins (STPs) for crossing the hydrophobic barrier in plants.  Here, we systematically identified the genes encoding sugar transporters in the genome of maize (Zea mays L.), analyzed their expression patterns under different conditions, and determined their functions in disease resistance.  The results showed that the mazie sugar transporter family contained 24 members, all of which were predicted to be distributed on the cell membrane and had a highly conserved transmembrane transport domain.  The tissue-specific expression of the maize sugar transporter genes was analyzed, and the expression level of these genes was found to be significantly different in different tissues.  The analysis of biotic and abiotic stress data showed that the expression levels of the sugar transporter genes changed significantly under different stress factors.  The expression levels of ZmSTP2 and ZmSTP20 continued to increase following Fusarium graminearum infection.  By performing disease resistance analysis of zmstp2 and zmstp20 mutants, we found that after inoculation with Cochliobolus carbonum, Setosphaeria turcica, Cochliobolus heterostrophus, and F. graminearum, the lesion area of the mutants was significantly higher than that of the wild-type B73 plant.  In this study, the genes encoding sugar transporters in maize were systematically identified and analyzed at the whole genome level.  The expression patterns of the sugar transporter-encoding genes in different tissues of maize and under biotic and abiotic stresses were revealed, which laid an important theoretical foundation for further elucidation of their functions.

Botrytis cinerea is a typical necrotrophic pathogenic fungus that causes severe diseases in a wide range of plant species, leading to significant economic losses.  Our previous study showed that BcSDR1 positively regulates growth, development, and pathogenicity of B. cinerea.  However, the regulation mechanism of BcSDR1 and the relationship between BcSDR1 and cAMP and MAPK signaling pathways are not well understood.  In this study, transcriptome data showed that BcSDR1 is involved in glucose transmembrane transport, signal transduction, secondary metabolism, and other biological processes.  BcSDR1 mutant (BCt41) showed remarkably weak sensitivity to cAMP and MAPK signaling pathways specific inhibitors, SQ22536 and U0126, and significantly decreased cAMP content.  The key genes of cAMP and MAPK signaling pathways, BcGB1, BcBTP1, BcBOS1, BcRAS1, and BcBMP3 were significantly upregulated, whereas BcPLC1, BcBCG1, BcCDC4, BcSAK1, BcATF1, and BcBAP1 were significantly downregulated (P<0.05).   BcSDR1 was obviously upregulated in BcBCG2, BcBCG3, BcPKA1, and BcPKAR RNA interference (RNAi)  mutants, but significantly downregulated in BcPKA2, BcBMP1, and BcBMP3 RNAi mutants.  Thus, BcBCG2, BcBCG3, BcPKA1, and BcPKAR negatively regulate BcSDR1 expression, whereas BcPKA2, BcBMP1, and BcBMP3 positively regulate BcSDR1 expression.

Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants.  In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall.  The function of another major laccase gene, StLAC6, was studied here.  The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal.  The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains.  In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes.  This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi.   

Setosphaeria turcica (syn. Exserohilum turcicum) is the pathogenic fungus of maize (Zea mays) that causes northern leaf blight, which is a major maize disease worldwide.  Melanized appressoria are highly specialized infection structures formed by germinated conidia of S. turcica that infect maize leaves.  The appressorium penetrates the plant cuticle by generating turgor, and glycerol is known to be the main source of the turgor.  Here, the infection position penetrated by the appressorium on maize leaves was investigated, most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells, and the appressorium structure was necessary for the infection, whether it occurred through epidermal cells or stomata.  Then, to investigate the effects of key factors in the development of the appressorium, we studied the effects of three inhibitors, including a melanin inhibitor (tricyclazole, TCZ), a DNA replication inhibitor (hydroxyurea, HU), and an autophagy inhibitor (3-methyladenine, 3-MA), on appressorium turgor and glycerol content.  As results, appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments.  The three inhibitors had the greatest effects on appressorium turgor pressure at this stage.  Glycogen and liposomes are the main substances producing glycerol.  It was also found inhibitors affected the distribution of glycogen and liposomes, which were detected in the conidia, the germ tube, and the appressorium during appressorium development.  This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content, which was affected by the synthesis of melanin, DNA replication, and autophagy in the developing appressorium during a S. turcica infection.  

The mitogen-activated protein kinase (MAPK), a key signal transduction component in the MAPK cascade pathway, regulates a variety of physiological activities in eukaryotes.  However, little is known of the role MAPK plays in phytopathogenic fungi.  In this research, we cloned the MAPK gene STK1 from the northern corn leaf blight pathogen Setosphaeria turcica and found that the gene shared high homology with the high osmolality glycerol (HOG) MAPK gene HOG1 of Saccharomyces cerevisiae.  In addition, gene knockout technology was employed to investigate the function of STK1.  Gene knockout mutants (KOs) were found to have altered hyphae morphology and no conidiogenesis, though they did show similar radial growth rate compared to the wild-type strain (WT).  Furthermore, microscope observations indicated that STK1 KOs did not form normal appressoria at 48 h post-inoculation on a hydrophobic surface.  STK1 KOs had reduced virulence, a significantly altered Helminthosporium turcicum (HT)-toxin composition, and diminished pathogenicity on the leaves of susceptible inbred corn OH43.  Mycelium morphology appeared to be significantly swollen and the radial growth rates of STK1 KOs declined in comparison with WT under high osmotic stress.  These results suggested that STK1 affects the hyphae development, conidiogenesis, and pathogenicity of S. turcica by regulating appressorium development and HT-toxin biosynthesis.  Moreover, the gene appears to be involved in the hypertonic stress response in S. turcica.

A total of 479 bacterial strains were isolated from brine (Bohai, Qinhuangdao City, Hebei Province, China). Bioassay results indicated that 4 strains named Ha1, Ha17, Ha38, and Ha384 had herbicidal activity. And strain Ha1 had the highest effective herbicidal activity. As a result, this study aims to identify strain Ha1, characterize its physiological and biological activities, evaluate the herbicidal activity of its metabolites, and develop a ‘pesta’ formulation and assess its effectiveness on Digitaria sanguinalis. Ha1 was identified as Serratia marcescens based on 16S rDNA sequencing. This strain has a flagellum, a diameter of 0.5 to 0.8 μm, and a length of 0.9 to 2.0 μm. The indole test shows positive results, and the catalase enzyme exhibits strong positive reactions. Results further showed that the inhibitory concentration (IC50) of the crude extracts to D. sanguinalis radicula and coleoptile were 3.332 and 2.828 mg mL–1, respectively. Both the suppression of D. sanguinalis and the cell viability of the Ha1 formulation in ‘pesta’ were higher when stored at 4°C than at (25±2)°C. These results indicated that S. marcescens Ha1 can potentially be used as a biocontrol agent against D. sanguinalis.

CB-4, a bacterial strain with highly effective herbicidal activity, was isolated from infected corn leaves. Through morphology, physiological and biochemical tests, and 16S ribosomal DNA gene sequencing methods, CB-4 was identified as Pseudomonas aeruginosa. We conducted activity-evaluation experiments in the laboratory to assess the herbicidal potential of metabolites produced by strain CB-4. Crude extracts of strain CB-4 have high inhibition activity on Digitaria sanguinalis. In general, the root and shoot growth parameters of D. sanguinalis were significantly reduced by metabolites of strain CB-4. The IC50 of the culture filtrate extracts for the radicula and coleoptile of D. sanguinalis were 0.299 and 0.210 mg mL-1, respectively. Component 2 of the herbicidal activity of the crude toxin from strain CB-4 was successfully purified for the first time by using high-speed counter current chromatography with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (4:5:4:5, v/v) and high-performance liquid chromatography. We concluded that the metabolites of strain CB-4 have the potential to be developed as a microbe-based herbicide.

A strain of Bacillus subtilis strain YB1, isolated and preserved in our lab., showed a high nicosulfuron-degrading activity. Optimization of culture conditions on production of nicosulfuron-degrading enzyme from Bacillus subtilis strain YB1 was carried out through mono-factor experiments. The characterization of degrading enzyme(s) was studied in this paper. The results showed that B. subtilis YB1 can use nicosulfuron as sole carbon source under aerobic condition. The key enzyme(s) involved in the initial biodegradation of nicosulfuron was localized to extracellular proteins and showed to be induced expressed. Enzyme-specific activity was up to 89.34 U mg-1 at pH 8.0 and 30°C, incubation for 96 h, inoculum 4.5×108 CFU mL-1 in Luria-Bertani liquid medium with nicosulfuron of 40 mg L-1. The maximum degradation rate of extracellular crude enzymes on nicosulfuron was 66% at pH 9.0, 35°C in the enzymatic reaction system with nicosulfuron of 5 mg L-1. This degrading enzyme(s) was sensitive to high temperature, but kept high activity under alkaline conditions.