Distribution and Pathogenicity of Fusarium Species Associated with Soybean Root Rot in Northeast China.

Fusarium root rot is an increasingly severe problem in soybean cultivation. Although several Fusarium species have been reported to infect soybean roots in Heilongjiang province, their frequency and aggressiveness have not been systematically quantified in the region. This study aimed to investigate the diversity and distribution of Fusarium species that cause soybean root rot in Heilongjiang province over two years. A total of 485 isolates belonging to nine Fusarium species were identified, with F. oxysporum and F. solani being the most prevalent. Pot experiments were conducted to examine the relative aggressiveness of different Fusarium species on soybean roots, revealing that F. oxysporum and F. solani were the most aggressive pathogens, causing the most severe root rot symptoms. The study also assessed the susceptibility of different soybean cultivars to Fusarium root rot caused by F. oxysporum and F. solani. The results indicated that the soybean cultivar DN51 exhibited the most resistance to both pathogens, indicating that it may possess genetic traits that make it less susceptible to Fusarium root rot. These findings provide valuable insights into the diversity and distribution of Fusarium species that cause soybean root rot and could facilitate the development of effective management strategies for this disease.

Overexpression of CcFALDH from spider plant (Chlorophytum comosum) enhances the formaldehyde removing capacity of transgenic gloxinia (Sinningia speciosa)1.

Formaldehyde can cause leukemia and nasopharyngeal cancer in humans, and is a major indoor air pollutant. In this study, to improve the ability of flowering plants to purify formaldehyde, we cloned the CcFALDH gene encoding formaldehyde dehydrogenase (FALDH) from the spider plant (Chlorophytum comosum), which encodes 379 amino acids with the alcohol dehydrogenase (ADH) structural domain, and used it to transform the flowering plant gloxinia (Sinningia speciosa). The FALDH activity of transgenic gloxinia was 1.8-2.7 times that of wild-type (WT) with a considerable increase in formaldehyde stress tolerance. The activities of the antioxidant enzymes SOD, POD, and CAT of transgenic gloxinia were 1.5-2.0 times those of the WT under formaldehyde stress; H2O2, O2-, and MDA contents were markedly lower than those in WT. Liquid formaldehyde and gaseous formaldehyde were metabolized at 2.1-2.8 and 2.1-2.7 times higher rates in transgenic gloxinia than in WT. Our findings indicate that overexpression of CcFALDH can enhance the capacity of flowering plants to metabolize formaldehyde, which provides a new strategy to tackle the indoor formaldehyde pollution problem.

Structural feature in dynamical processes accelerated transition state calculations.

Minimum energy path (MEP) search is a vital but often very time-consuming method to predict the transition states of versatile dynamic processes in chemistry, physics, and materials science. In this study, we reveal that the largely displaced atoms in the MEP structures maintain transient chemical bond lengths resembling those of the same type in the stable initial and final states. Based on this discovery, we propose an adaptive semirigid body approximation (ASBA) to construct a physically reasonable initial guess for the MEP structures, which can be further optimized by the nudged elastic band method. Examination of several distinct dynamical processes in bulk, on crystal surface, and through two-dimensional system shows that our transition state calculations based on the ASBA results are robust and significantly faster than those based on the popular linear interpolation and image-dependent pair potential methods.

Role of FoERG3 in Ergosterol Biosynthesis by Fusarium oxysporum and the Associated Regulation by Bacillus subtilis HSY21.

Ergosterol is an important component of the fungal cell membrane and represents an effective target of chemical pesticides. However, the current understanding of ergosterol biosynthesis in the soybean root rot pathogen Fusarium oxysporum remains limited. In addition, the regular use of fungicides that inhibit ergosterol synthesis will seriously harm the ecological environment and human health. Bacillus subtilis is gradually replacing chemical control as a safe and effective biological agent; to investigate its effect on ergosterol synthesis of F. oxysporum, we verified the biological function of the FoERG3 gene of F. oxysporum by constructing knockout mutants. The results showed that knocking out FoERG3 blocked ergosterol biosynthesis, restricted mycelial growth, and increased the sensitivity to external stressors (NaCl, D-sorbitol, Congo Red, and H2O2). The increased permeability of the cell membrane promoted increased extracellular K+ levels and decreased mitochondrial cytochrome C contents. Treatment with suspension of B. subtilis HSY21 cells resulted in similar damage as observed when treating FoERG3-knockout F. oxysporum cells with ergosterol, which was characterised by deformity and swelling of the mycelium surface; increased membrane permeability; decreased pathogenicity to soybeans; and significantly decreased activities of cellulase, ß-glucosidase, amylase, and pectin-methyl galactosylase. Notably, deleting FoERG3 resulted in a significant lag in the defense-response time of soybeans. Our results suggest that FoERG3 strongly influences the virulence of F. oxysporum and may be used as a potential antimicrobial target by B. subtilis HSY21 to inhibit ergosterol synthesis, which supports the use of B. subtilis as a biological control agent for protecting against F. oxysporum infection.

Meta-analysis of fungal plant pathogen Fusarium oxysporum infection-related gene profiles using transcriptome datasets.

Fusarium oxysporum is a serious soil-borne fungal pathogen that affects the production of many economically important crops worldwide. Recent reports suggest that this fungus is becoming the dominant species in soil and could become the main infectious fungus in the future. However, the infection mechanisms employed by F. oxysporum are poorly understood. In the present study, using a network meta-analysis technique and public transcriptome datasets for different F. oxysporum and plant interactions, we aimed to explore the common molecular infection strategy used by this fungus and to identify vital genes involved in this process. Principle component analysis showed that all the fungal culture samples from different datasets were clustered together, and were clearly separated from the infection samples, suggesting the feasibility of an integrated analysis of heterogeneous datasets. A total of 335 common differentially expressed genes (DEGs) were identified among these samples, of which 262 were upregulated and 73 were downregulated significantly across the datasets. The most enriched functional categories of the common DEGs were carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Nine co-expression modules were identified, and two modules, the turquoise module and the blue module, correlated positively and negatively with all the infection processes, respectively. Co-expression networks were constructed for these two modules and hub genes were identified and validated. Our results comprise a cross fungal-host interaction resource, highlighting the use of a network biology approach to gain molecular insights.

Bacillus subtilis HSY21 can reduce soybean root rot and inhibit the expression of genes related to the pathogenicity of Fusarium oxysporum.

Soybean root rot occurs globally and seriously affects soybean production. To avoid the many disadvantages of chemical fungicides, the addition of Bacillus is gradually becoming an alternative strategy to tackle soybean root rot. However, the molecular mechanism of phytopathogenic fungi in this process by Bacillus inhibition is rarely reported. In this study, we isolated a strain of B. subtilis HSY21 from soybean rhizosphere soil, which had an inhibition rate of 81.30 ± 0.15% (P < 0.05) against Fusarium oxysporum. The control effects of this strain against soybean root rot under greenhouse and field conditions were 63.83% and 57.07% (P < 0.05), respectively. RNA-seq analysis of F. oxysporum after treatment with strain HSY21 revealed 1445 downregulated genes and 1561 upregulated genes. Among them, genes involved in mycelial growth, metabolism regulation, and disease-related enzymes were mostly downregulated. The activities of cellulase, ß-glucosidase, α-amylase, and pectin-methyl- galacturonase as well as levels of oxalic acid and ergosterol in F. oxysporum were significantly decreased after HSY21 treatment. These results demonstrated that B. subtilis HSY21 could effectively control F. oxysporum by inhibiting its growth and the expression of pathogenic genes, thus indicating that this strain may be an ideal candidate for the prevention and control of soybean root rot.

Alfalfa (Medicago sativa L.) MsCML46 gene encoding calmodulin-like protein confers tolerance to abiotic stress in tobacco.

KEY MESSAGE: MsCML46 enhances tolerance to abiotic stresses through alleviating osmotic stress and oxidative damage by regulating the expression of stress-related genes to optimize osmolytes levels and antioxidant enzyme activity in transgenic tobacco. Abiotic stresses are major environmental factors that constraint crop productivity worldwide. Various stimuli regulate intracellular calcium levels and calcium-mediated signal transduction, and cellular responses. Ca2+ signals are perceived by different Ca2+ receptors. Calmodulin-like protein (CML) is one of the best-characterized Ca2+ sensors which shares sequence similarity with highly conserved calmodulin (CaM) ubiquitously expressed in plants. Currently, the molecular and physiological functions of CMLs are largely unknown. In this study, the MsCML46 was characterized in alfalfa (Medicago sativa cv. Zhaodong) under freezing stress. Results showed that MsCML46 was localized to the cytoplasm of Arabidopsis, and its expression was strongly elevated by cold, drought, salt, saline-alkali, and ABA treatments. Overexpressing MsCML46 in tobacco enhanced tolerance to freezing, drought, and salt stresses as evidenced by improved contents of osmotic regulatory solutes and antioxidant enzyme activity but decreased reactive oxygen species (ROS) accumulation. Furthermore, cold, drought, and salt stresses increased the expression of stress-related genes in transgenic tobacco. MsCML46 binds free Ca2+ to promote signal transduction and maintain higher K+/Na+ ratio. In this way, it protects intracellular homeostasis under sodium ion toxicity. These results suggest that MsCML46 plays a crucial role in resisting abiotic stresses and can be exploited in genetic engineering for crops.

Systematic Investigation of the Effects of Macro-elements and Iron on Soybean Plant Response to Fusarium oxysporum Infection.

Nutrient manipulation is a promising strategy for controlling plant diseases in sustainable agriculture. Although many studies have investigated the relationships between certain elements and plant diseases, few have comprehensively explored how differing mineral nutrition levels might affect plant-fungal pathogen interactions, namely plant susceptibility and resistance. Here, we systematically explored the effects of the seven mineral elements that plants require in the greatest amounts for normal development on the susceptibility of soybean plants (Glycine max) to Fusarium oxysporum infection in controlled greenhouse conditions. Nitrogen (N) negligibly affected plant susceptibility to infection in the range 4 to 24 mM for both tested soybean cultivars. At relatively high concentrations, phosphorus (P) increased plant susceptibility to infection, which led to severely reduced shoot and root dry weights. Potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), and iron (Fe) induced plant resistance to infection as their concentrations were increased. For K and Ca, moderate concentrations had a positive effect on plant resistance to the pathogen, whereas relatively high doses of either element adversely affected plant growth and promoted disease symptoms. Further experiments were conducted, assessing disease suppression by selected combinations of macro-elements and Fe at screened concentrations, i.e., K (9 mM) plus Fe (0.2 mM), and S (4 mM) plus Fe (0.2 mM). The disease index was significantly reduced by the combination of K plus Fe. In conclusion, this systematic investigation of soybean plant responses to F. oxysporum infection provides a solid basis for future environmentally-friendly choices for application in soybean disease control programs.

Comparative genomics of 151 plant-associated bacteria reveal putative mechanisms underlying specific interactions between bacteria and plant hosts.

Although much work has explored how microbes can benefit plant growth, the mechanisms underlying this intriguing process remain largely unknown, especially considering the diversity of bacteria that surrounds plants. The objective of the present study was to identify bacterial genes contributing to plant-microbe associations, beneficial effects, and host specificities. For this purpose, comparative genomics investigation of 151 plant-associated bacteria was performed. A principal component analysis of seven key genomic features revealed patterns in the specific properties of these bacteria: N2-fixing bacteria were more closely related to pathogenic ones than to beneficial bacteria. A common set of genes over-represented in these plant-associated bacteria were found to be remarkably similar in terms of (1) genetic information processing, (2) amino acid metabolism, (3) metabolism of cofactors and vitamins, (4) nucleotide metabolism, (5) human diseases, and (6) metabolism of terpenoids and polyketides. Although we did not detect a common genetic basis for these beneficial effects, further in-depth analysis revealed that each of five beneficial bacterial groups shared specific gene sets. Functional annotation showed that environmental information processing, genetic information processing and cellular processes predominated in these beneficial groups. Hypothesizing that plant-associated bacteria may have overlapping strategies to colonize their plant hosts, we successfully identified many putative genes that determine host specificities. Most of these genes were classified as transcription factors, enzymes, transporters, and chemotaxis regulators. Comparative genomics provides a powerful tool for helping to identify genes that are common among species. Genome-based views of plant-associated bacteria reveal specific interactions between bacteria and plant hosts.

Overexpression of the alfalfa WRKY11 gene enhances salt tolerance in soybean.

The WRKY transcription factors play an important role in the regulation of transcriptional reprogramming associated with plant abiotic stress responses. In this study, the WRKY transcription factor MsWRKY11, containing the plant-specific WRKY zinc finger DNA-binding motif, was isolated from alfalfa. The MsWRKY11 gene was detected in all plant tissues (root, stem, leaf, flower, and fruit), with high expression in root and leaf tissues. MsWRKY11 was upregulated in response to a variety of abiotic stresses, including salinity, alkalinity, cold, abscisic acid, and drought. Overexpression of MsWRKY11 in soybean enhanced the salt tolerance at the seedling stage. Transgenic soybean had a better salt-tolerant phenotype, and the hypocotyls were significantly longer than those of wild-type seeds after salt treatment. Furthermore, MsWRKY11 overexpression increased the contents of chlorophyll, proline, soluble sugar, superoxide dismutase, and catalase, but reduced the relative electrical conductivity and the contents of malonaldehyde, H2O2, and O2-. Plant height, pods per plant, seeds per plant, and 100-seed weight of transgenic MsWRKY11 soybean were higher than those of wild-type soybean, especially OX2. Results of the salt experiment showed that MsWRKY11 is involved in salt stress responses, and its overexpression improves salt tolerance in soybean.

Pleiotropic effects of TaMYB3R1 on plant development and response to osmotic stress in transgenic Arabidopsis.

In a previous study, we isolated and characterized TaMYB3R1, a MYB3R gene, from wheat (Triticum aestivum L.). In vitro assays showed that the TaMYB3R1 protein is localized to the nucleus, and functions as an MSA-binding transcriptional activator. Expression of TaMYB3R1 is induced by exogenous abscisic acid (ABA) and abiotic stress, which encouraged us to further investigate its function in planta. In the present study, we generated transgenic Arabidopsis plants overexpressing TaMYB3R1. Compared with wild-type plants, the transgenic lines produced more rosette leaves, and thus more inflorescences, but the plants showed delayed development at the reproductive stage. The TaMYB3R1 protein also functions in the osmotic stress response. Transgenic Arabidopsis plants showed enhanced tolerance to drought and salt stresses, and the tolerance phenotype was conveyed by limiting transpiration through increasing stomatal closure as well as reducing water loss. In addition, TaMYB3R1 influenced the expression of both ABA-dependent and ABA-independent responsive genes, implicating TaMYB3R1 in diverse osmotic stress-response mechanisms in Arabidopsis. Our study sheds light on novel functions of a plant MYB3R protein.

Risk assessment of petroleum-contaminated soil using soil enzyme activities and genotoxicity to Vicia faba.

Pollution caused by petroleum is one of the most serious problems worldwide. To better understand the toxic effects of petroleum-contaminated soil on the microflora and phytocommunity, we conducted a comprehensive field study on toxic effects of petroleum contaminated soil collected from the city of Daqing, an oil producing region of China. Urease, protease, invertase, and dehydrogenase activity were significantly reduced in microflora exposed to contaminated soils compared to the controls, whereas polyphenol oxidase activity was significantly increased (P < 0.05). Soil pH, electrical conductivity, and organic matter content were correlated with total petroleum hydrocarbons (TPHs) and a correlation (P < 0.01) existed between the C/N ratio and TPHs. Protease, invertase and catalase were correlated with TPHs. The Vicia faba micronucleus (MN) test, chromosome aberrant (CA) analyses, and the mitotic index (MI) were used to detect genotoxicity of water extracts of the soil. Petroleum-contaminated samples indicated serious genotoxicity to plants, including decreased index level of MI, increased frequency of MN and CA. The combination of enzyme activities and genotoxicity test via Vicia faba can be used as an important indicator for assessing the impact of TPH on soil ecosystem.

Biosafety assessment of GFP transplastomic tobacco to rhizosphere microbial community.

Green fluorescent protein (GFP) is one of the most widely studied and exploited proteins in biochemistry, and has many applications as a marker, especially in plant transformation system. Although a number of studies have been conducted to assess the toxify of this protein to specific organisms, little is known about GFP on rhizosphere microbial community, which is regarded as good indicator for environmental risk assessment. Chloroplast genetic engineering has shown superiority over traditional nuclear genetic engineering, and has been used in many aspects of plant genetic engineering. High levels of chloroplast-based protein accumulation make this technology as an ideal strategy to evaluate biosafety of transgenes. In the present study, the effects of field-released GFP transplastomic tobacco (Nicotiana tabacum) on rhizosphere microbes over a whole growth cycle were investigated by using both culture-dependent and culture-independent methods. Compared to wild-type control, transplastomic tobacco had no significant influence on the microbial population at the seedling, vegetative, flowering and senescing stages. However, developmental stages had more influence than ecotypes (GFP-transformed and wild-type). This was confirmed by colony forming unit, Biolog Eco(TM) and PCR-DGGE analysis. Thus, these results suggest chloroplast transformation with a GFP reporter gene has no significant influence on rhizosphere microbial community, and will be potential platform for plant biotechnology in future.

Transgenic Expression of the Functional Fragment Hpa110-42 of the Harpin Protein Hpa1 Imparts Enhanced Resistance to Powdery Mildew in Wheat.

Powdery mildew, one of devastating diseases of wheat worldwide, is caused by Erysiphe graminis f. sp. tritici, a fungal species with constant population changes, which often poses challenges in disease management with host resistance. Transgenic approaches that utilize broad-spectrum resistance may limit changes of pathogen populations and contribute to effective control of the disease. The harpin protein Hpa1, produced by the rice bacterial blight pathogen, can induce resistance to bacterial blight and blast in rice. The fragment comprising residues 10 through 42 of Hpa1, Hpa110-42, is reportedly three- to eightfold more effective than the full-length protein. This study evaluated the transgenic expression of the Hpa110-42 gene for resistance to powdery mildew in wheat caused by E. graminis f. sp. tritici. Nine Hpa110-42 transgenic wheat lines were generated. The genomic integration of Hpa110-42 was confirmed, and expression of the transgene was detected at different levels in the individual transgenic lines. Following inoculation with the E. graminis f. sp. tritici isolate Egt15 in the greenhouse, five transgenic lines had significantly higher levels of resistance to powdery mildew compared with nontransformed plants. Thus, transgenic expression of Hpa110-42 conferred resistance to one isolate of E. graminis f. sp. tritici in wheat in the greenhouse.

Large scale in silico identification of MYB family genes from wheat expressed sequence tags.

The MYB proteins constitute one of the largest transcription factor families in plants. Much research has been performed to determine their structures, functions, and evolution, especially in the model plants, Arabidopsis, and rice. However, this transcription factor family has been much less studied in wheat (Triticum aestivum), for which no genome sequence is yet available. Despite this, expressed sequence tags are an important resource that permits opportunities for large scale gene identification. In this study, a total of 218 sequences from wheat were identified and confirmed to be putative MYB proteins, including 1RMYB, R2R3-type MYB, 3RMYB, and 4RMYB types. A total of 36 R2R3-type MYB genes with complete open reading frames were obtained. The putative orthologs were assigned in rice and Arabidopsis based on the phylogenetic tree. Tissue-specific expression pattern analyses confirmed the predicted orthologs, and this meant that gene information could be inferred from the Arabidopsis genes. Moreover, the motifs flanking the MYB domain were analyzed using the MEME web server. The distribution of motifs among wheat MYB proteins was investigated and this facilitated subfamily classification.

Identification of a MYB3R gene involved in drought, salt and cold stress in wheat (Triticum aestivum L.).

Abiotic stress seriously affects crop growth and productivity. To better understand the mechanisms plant uses to cope with drought, cold and salt stress, it is necessary to isolate and characterize important regulators response to these stresses. In this study, we cloned a MYB gene from wheat (Triticum aestivum L.) and designated it as TaMYB3R1 based on its conserved three repeats in MYB domain. The sequence of TaMYB3R1 protein shares high identity to other plant MYB3R proteins. Subcellular localization experiment in onion epidermal cells proved that TaMYB3R1 localized in the nucleus. Trans-activation essays in yeast cells confirmed that TaMYB3R1 was a transcriptional activator, and only C-terminal region was able to activate the expression of ß-galactosidase. DNA-binding test showed the MSA cis element-binding activity of TaMYB3R1. After exogenous application of phytohormone ABA, the expression of TaMYB3R1 was induced, and its transcripts accumulated up to 24h; this is also the case for MeJA treatment, but after it peaked at 4h, it decreased to low levels. However, either SA or ET had no obvious effect on the expression of TaMYB3R1. Furthermore, the TaMYB3R1 was initially expressed at low levels and was gradually induced following treatment with salt, and continued to increase up to 72 h. This was similar for the cold treatment. In contrast, the peak appeared at 6h of the PEG treatment, and then gradually decreased to low levels. Our results suggest that TaMYB3R1 is potentially involved in wheat response to drought, salt and cold stress.