•Soil microbial source fungicide-lipopeptides can impact soil fungal community.•Non-ribosomal peptide synthetase (NRPS) gene has a feedback regulation mechanism like the nifH gene.•Basidiomycota was increased and Fusarium oxysporum was decreased after application of lipopeptides.Lipopeptides have extensively been shown to be an efficient antifungal agent against many fungal pathogens. However, so far no studies have been reported focusing on their effect on both the soil fungal community and the abundance of genes encoding functions linked to lipopeptide. In this study, a lipopeptide mixture from Bacillus amyloliquefaciens NJN-6 was applied to soil to investigate these effects. The results showed lipopeptides could reduce the copy number of 18S rRNA gene and the α-diversity of the fungal community in soil using qPCR and ITS rRNA Illumina MiSeq sequencing platform technology. Further evaluation found that lipopeptides significantly increased the relative abundance of Basidiomycota and specifically sub-groups Cryptococcus and Trichosporon. At the same time, Fusarium spp., especially the relative abundance of the plant pathogen Fusarium oxysporum was significantly inhibited. In addition, the native genes encoding functions related to lipopeptide production, specifically genes encoding the non-ribosomal peptides synthase (NRPS), were also reduced in diversity and in abundance after evaluation by sequencing using a 454 GS-FLX Titanium platform. Several genes encoding NRPS annotated to the phyla Proteobacteria were increased in proportion, while some genes encoding NRPS annotated to the phylum Actinobacteria and Cyanobacteria were decreased in proportion at the same time. The results obtained in this study suggest that bacterial-source antifungal agent can change the soil fungal community. Moreover, the application of these antifungal agents may also disturb the balance of the native microbial community responsible for lipopeptide production.Download high-res image (101KB)Download full-size image

•Naturally suppressive soil related to vanilla Fusarium wilt disease was identified.•Suppressive soil was associated with higher fungal diversity.•Suppressive soil was dominated by the fungal genus Mortierella.•Fungal communities showed more co-occurrence relationships in suppressive soil.Characterizing microbial communities associated with disease-suppressive soil is an important first step toward understanding the potential of microbiota to protect crops against plant pathogens. In the present study, we compared microbial communities in suppressive- and conducive-soils associated with Fusarium wilt disease in a vanilla long-term continuous cropping system. Suppressive soil was associated with higher fungal diversity and lower bacterial diversity. The fungal phyla Zygomycota and Basidiomycota, and the bacterial phyla Acidobacteria, Verrucomicrobia, Actinobacteria and Firmicutes were strongly enriched in the suppressive soil. Notably, suppressive soil was dominated by the fungal genus Mortierella, accounting for 37% of the total fungal sequences. The hyper-dominance of Mortierella spp. in suppressive soil suggests that this taxon may serve as an indicator and enhancer of Fusarium wilt disease suppression in vanilla. In addition, Molecular Ecological Network analysis revealed that fungal communities were more connected and showed more co-occurrence relationships in the suppressive versus conducive soils. Our results indicate that fungal communities may be important in the development of soil suppressiveness against vanilla Fusarium wilt disease.

Volatile organic compounds (VOCs) from soil bacteria are likely to have an important role in the interactions among soil microorganisms. However, their effects on the soil microbial community have not been extensively studied. In this study, the effect of bacterial VOCs generated by growing Bacillus amyloliquefaciens NJN-6 on modified MS medium on soil microbial community was evaluated. B. amyloliquefaciens NJN-6 was able to produce 48 volatile compounds as determined by solid-phase microextraction-GC/MS. MiSeq sequencing data showed that bacterial VOCs could alter the composition of both soil bacterial and soil fungal communities and could decrease the alpha-diversity of the soil microbial community. Taxonomic analysis revealed that bacterial VOCs significantly increased the relative abundance of Proteobacteria, Bacteroidetes, and Firmicutes. Moreover, bacterial VOCs significantly increased the relative abundance of Ascomycota. The qPCR data showed that bacterial VOCs of strain NJN-6 decreased the soil fungal biomass and increased the soil bacterial biomass. Further evaluation of the effect of bacterial VOCs on functional genes revealed that VOCs could reduce the copies of nifH, nirS, and a gene encoding nonribosomal peptide synthase, while increasing the copy number of the ammonium-oxidizing bacteria gene. The effect on gene encoding polyketide synthase was insignificant. Results from this study indicated that bacterial VOCs could influence the soil microbial community as well as functional gene abundance.

In the present study, soil bacterial and fungal communities across vanilla continuous cropping time-series fields were assessed through deep pyrosequencing of 16S ribosomal RNA (rRNA) genes and internal transcribed spacer (ITS) regions. The results demonstrated that the long-term monoculture of vanilla significantly altered soil microbial communities. Soil fungal diversity index increased with consecutive cropping years, whereas soil bacterial diversity was relatively stable. Bray-Curtis dissimilarity cluster and UniFrac-weighted principal coordinate analysis (PCoA) revealed that monoculture time was the major determinant for fungal community structure, but not for bacterial community structure. The relative abundances (RAs) of the Firmicutes, Actinobacteria, Bacteroidetes, and Basidiomycota phyla were depleted along the years of vanilla monoculture. Pearson correlations at the phyla level demonstrated that Actinobacteria, Armatimonadetes, Bacteroidetes, Verrucomicrobia, and Firmicutes had significant negative correlations with vanilla disease index (DI), while no significant correlation for fungal phyla was observed. In addition, the amount of the pathogen Fusarium oxysporum accumulated with increasing years and was significantly positively correlated with vanilla DI. By contrast, the abundance of beneficial bacteria, including Bradyrhizobium and Bacillus, significantly decreased over time. In sum, soil weakness and vanilla stem wilt disease after long-term continuous cropping can be attributed to the alteration of the soil microbial community membership and structure, i.e., the reduction of the beneficial microbes and the accumulation of the fungal pathogen.

Particle-size soils were fractionated for evaluating changes in the composition of bacterial community and enzyme activity in response to 13 years of fertilization. This study focused on Mollisol and its particle-size fractions of 200–2,000 μm (coarse sand sized), 63 to 200 μm (fine sand sized), 2 to 63 μm (silt sized), and 0.1 to 2 to μm (clay-sized). Long-term chemical fertilization lowered the pH of all particle fractions, whereas organic fertilizer application mitigated soil acidification. Nutrient concentrations depended on both fertilizer treatment and particle fractions and enzymes were unevenly active throughout the soil. Generally, the highest enzyme activities were observed in the silt and clay fractions of control soil and the soil treated with chemical fertilizer (N, P, and K (NPK)) and in the sand-sized fraction of soil treated with manure and chemical fertilizer (MNPK). Except for acid phosphomonoesterase, the other tested enzyme activities in coarse-sized fractions of MNPK soil were significantly higher than those of the control and NPK soils. Fertilization and soil fraction interactively (p < 0.05) affected the enzyme activity. Denaturing gradient gel electrophoresis analysis showed that the bacterial community structure significantly differed in different particle sizes with a higher bacterial diversity in small-sized than in coarse-sized fractions. Dominant bands were excised and sequenced. We have found the following bacterial groups: Actinobacteria, γ-proteobacteria, and Acidobacteria. In addition, enrichment of organic matter in coarser fractions was related to greater bacterial diversity than any other treatment. Principal component analysis showed a smaller variability among fractions of the organic amended treatment. Redundancy analysis showed that the tested properties significantly affected the composition of bacterial community with the exception of C/N and available P. No significant correlation between enzyme activity and bacterial community composition was detected, whereas positive correlations between other soil properties and enzyme activities were observed to various extents. Probably, enzyme activities might be affected by specific functional bacterial communities rather than by the overall bacterial community. We concluded that the long-term application of organic manures contributed to the increase of soil organic matter content of particles higher than 200 mm, with higher bacterial diversity and increases in most of the enzyme activities.

Watermelon Fusarium wilt is one of the most severe soil-borne diseases caused by Fusarium oxysporum f. sp. niveum. In this study, the population of F. oxysporum was quickly monitored by real-time PCR and DNA array in watermelon Fusarium wilt infected soils treated with Paenibacillus polymyxa SQR21 enhanced bio-organic fertilizer (BIO) at the beginning of nursery growth and/or at the beginning of transplanting. The fungal community composition was investigated by molecular cloning and DGGE techniques. The real-time PCR results showed the F. oxysporum population in the rhizosphere soil decreased from 8.56 × 104 colony-forming units (cfu) g−1 rhizosphere soil to 9.41 × 103 cfu g−1 rhizosphere soil after BIO application and the DNA array detection signals of F. oxysporum population weakened. The difference between F. oxysporum abundance of BIO amended and not amended bulk soils was lower than 104 cfu g−1 soil. DGGE profile indicated that BIO application changed the fungal community structure in the rhizosphere soils; the molecular cloning data revealed that consecutive applications of BIO at nursery and transplanting stages not only decreased Ascomycota and increased Basidiomycota abundance in the rhizosphere soil but also caused the apperance of unique fungal group which were not found in the control. The beneficial fungi Chaetomium sp. Aspergillus penicillioides were found in the BIO amended treatment, while some harmful fungi such as F. oxysporum, Rhizoctonia solani, and Fusarium solani were only detected in the control. Data from this study indicated that BIO application can control watermelon Fusarium wilt by suppressing the population of F. oxysporum and changing the fungal community structure in the rhizosphere soils.

Cotton plants that are continuously subjected to monoculture suffer greatly from Verticillium wilt disease. The application of a novel bioorganic fertilizer (BOF) consisting of organic fertilizer combined with the antagonistic Bacillus subtilis strain HJ5 significantly suppressed Verticillium wilt of cotton. The disease incidence rates in soils that were treated with BOF (1 %, w/w) in the nursery stage, in the transplanted soil stage or in both stages, decreased by 42.9 %, 57.1 %, and 88.0 %, respectively, compared with controls. B. subtilis HJ5 was tagged with a plasmid-borne gfp gene encoding the green fluorescent protein to investigate its colonization behavior on cotton root surfaces. The results indicated that B. subtilis HJ5 predominantly colonizes the elongation and differentiation zones of the roots and forms micro-colonies in hydroponic and soil systems. The population of B. subtilis HJ5 in the rhizosphere and on cotton roots was also monitored. The number of B. subtilis HJ5 cells on the root surface reached a peak value of approximately 107 cfu per gram of roots 3 days after exposure of the cotton seedlings to the bacteria. Probably the colonization of B. subtilis HJ5 on cotton roots is one of the mechanisms involved in protecting cotton plants from fungal infection.

Bacterial wilt caused by Ralstonia solanacearum is one of the most serious tobacco diseases worldwide. Brevibacillus brevis (L-25) and Streptomyces rochei (L-9) with strong inhibitory effects on R. solanacearum in vitro were isolated from the rhizosphere of a healthy tobacco plant in a severely wilt-diseased field. Pot and field experiments were conducted to evaluate the biocontrol effect of the isolated antagonists alone and in combination with organic fertilizer. In pot experiment, the control efficacy was 92.3–100 % in the treatments applied with L-25 and L-9 alone or together with organic fertilizers. When bioorganic fertilizer containing L-9 and L-25 was applied to the soil in field condition, the control efficacies were 95.4 and 30.0 in the Anhui and Guizhou field plots, respectively. The counts of bacteria and actinomycetes in rhizosphere soil were significantly increased (p ≤ 0.05) under all antagonist applications compared with CK (PR). In contrast, fungal and R. solanacearum densities in the rhizosphere soil applied with antagonists were much lower than the CK (PR) rhizosphere. Combined application of the two antagonists had better effect than single antagonist treatments. The antagonists were more effective when they were combined with organic fertilizer as compared with the antagonistic strains only. These results allow us to conclude that a combination of the biocontrol agents, L-25 and L-9, together with organic fertilizers can effectively control bacterial wilt by affecting soil microbial structure.

Fusaricidins produced by Paenibacillus polymyxa are lipopeptide antibiotics with outstanding antifungal activity. In this study, the whole gene cluster responsible for fusaricidin biosynthesis (fusA) was isolated and identified from the cDNA library of one biocontrol agent P. polymyxa SQR-21 (SQR-21). MALDI-TOF MS analysis confirmed that SQR-21 could produce four kinds of fusaricidins: A, B, C, and D. A central promoter that drove the transcription of fusGFEDCBA was revealed by mapping of the fus promoter region by 5′ deletions. The disruption of fusA in SQR-21 led to the abolishment of fusaricidin production and antifungal activity. The direct interaction between a potential regulator, AbrB, and the promoter region of fus gene cluster was confirmed by electrophoretic mobility shift assays. One abrB disruption mutant showed significantly higher antifungal activity compared with the wild type. These results revealed a pathway for the transcriptional regulation of the fus gene cluster in P. polymyxa.

The binding characteristics of organic ligands with Al(III) in soil dissolved organic matter (DOM) is essential to understand soil organic carbon (SOC) storage. In this study, two-dimensional (2D) FTIR correlation spectroscopy was developed as a novel tool to explore the binding of organic ligands with Al(III) in DOM present in soils as part of a long-term (21-year) fertilization experiment. The results showed that while it is a popular method for characterizing the binding of organic ligands and metals, fluorescence excitation–emission matrix–parallel factor analysis can only characterize the binding characteristics of fluorescent substances (i.e., protein-, humic-, and fulvic-like substances) with Al(III). However, 2D FTIR correlation spectroscopy can characterize the binding characteristics of both fluorescent and nonfluorescent (i.e., polysaccharides, lipids, and lignin) substances with Al(III). Meanwhile, 2D FTIR correlation spectroscopy demonstrated that the sequencing/ordering of organics binding with Al(III) could be modified by the use of long-term fertilization strategies. Furthermore, 2D FTIR correlation spectroscopy revealed that the high SOC content in the chemical plus manure (NPKM) treatment in the long term fertilization experiment can be attributed to the formation of noncrystalline microparticles (i.e., allophane and imogolite). In summary, 2D FTIR correlation spectroscopy is a promising approach for the characterization of metal–organic complexes.

The extracellular polysaccharide produced by a newly isolated strain Pseudomonas fluorescens WR-1 was purified and characterized and its production was optimized using response surface methodology. The results showed that the strain WR-1 produced one kind of EPS that was composed of arabinose, glucose and uronic acid. The molecular weight of the EPS was determined to be 6.78 × 106 Da. The preferable culture conditions for EPS production were pH 7.0, temperature 28 °C for 72 h with peptone and maltose as best N and C sources, respectively. The model predicted that the maximum EPS production (39.6 g L−1) was appeared with maltose 48.65 g L−1, Mn2+ 1118 μM and Zn2+ 901 μM. The EPS also showed good H2O2 scavenging activity while moderate free radical scavenging activity and reductive ability were determined. The EPS from WR-1 may be a new source of natural antioxidants with potential value for health, food and industry.Highlights► First report of optimization and antioxidant activity of an EPS by P. fluorescens. ► The EPS was composed of arabinose, glucose and uronic acid. ► Maltose, Zn2+ and Mn2+ were found important factors for its optimum production. ► Good H2O2 scavenging while moderate free radical scavenging and reductive ability. ► The EPS can be a new source of natural antioxidants.

Cotton Verticillium wilt is a destructive soil-borne disease affecting cotton production. In this study, application of bio-organic fertilizer (BIO) at the beginning of nursery growth and/or at the beginning of transplanting was evaluated for its ability to control Verticillium dahliae Kleb. The most efficient control of cotton Verticillium wilt was achieved when the nursery application of BIO was combined with a second application in transplanted soil, resulting in a wilt disease incidence of only 4.4%, compared with 90.0% in the control. Denaturing gradient gel electrophoresis patterns showed that the consecutive applications of BIO at nursery and transplanting stage resulted in the presence of a unique group of fungi not found in any other treatments. Humicola sp., Metarhizium anisopliae, and Chaetomium sp., which were considered to be beneficial fungi, were found in the BIO treatment, whereas some harmful fungi, such as Alternaria alternate, Coniochaeta velutina, and Chaetothyriales sp. were detected in the control. After the consecutive applications of BIO at nursery and transplanting stage, the V. dahliae population in the rhizosphere soil in the budding period, flowering and boll-forming stage, boll-opening stage, and at harvest time were 8.5 × 102, 3.1 × 102, 4.6 × 102, and 1.7 × 102 colony-forming units per gram of soil (cfu g−1), respectively, which were significantly lower than in the control (6.1 × 103, 3.4 × 103, 5.2 × 103, and 7.0 × 103 cfu g−1, respectively). These results indicate that the suggested application mode of BIO could effectively control cotton Verticillium wilt by significantly changing the fungal community structure and reducing the V. dahliae population in the rhizosphere soil.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum J. H. Owen, results in considerable yield losses for cucumber plants. A bio-organic fertilizer (BIO), which was a combination of manure composts with antagonistic microorganisms, and an organic fertilizer (OF) were evaluated for their efficiencies in controlling Fusarium wilt. Application of the BIO suppressed the disease incidence by 83% and reduced yield losses threefold compared with the application of OF. Analysis of microbial communities in rhizosphere soils by high-throughput pyrosequencing showed that more complex community structures were present in BIO than in OF treated soils. The dominant taxonomic phyla found in both samples were Proteobacteria, Firmicutes, Actinobacteria and Acidobacteria among bacteria and Ascomycota among fungi. Abundance of beneficial bacteria or fungi, such as Trichoderma, Hypoxylon, Tritirachium, Paenibacillus, Bacillus, Haliangium and Streptomyces, increased compared to the OF treatment, whereas the soil-borne pathogen, Fusarium, was markedly decreased. Overall, the results of this study demonstrate that the application of the BIO was a useful and effective approach to suppress Fusarium wilt and that the high-throughput 454 pyrosequencing was a suitable method for the characterization of microbial communities of rhizosphere soil of cucumber.

Knowledge on the structure and function of extracellular polymeric substances (EPS) in biofilms is essential for understanding biodegradation processes. Herein, a novel method based on multiple fluorescence labeling and two-dimensional (2D) FTIR–13C NMR heterospectral correlation spectroscopy was developed to gain insight on the composition, architecture, and function of EPS in biofilms during composting. Compared to other environmental biofilms, biofilms in the thermophilic (>55 °C) and cooling (mature) stage of composting have distinct characteristics. The results of multiple fluorescence labeling demonstrated that biofilms were distributed in clusters during the thermophilic stage (day 14), and dead cells were detected. In the mature stage (day 26), the biofilm formed a continuous layer with a thickness of approximately 20–100 μm around the compost, and recolonization of cells at the surface of the compost was easily observed. Through 2D FTIR–13C NMR correlation heterospectral spectroscopy, the following trend in the ease of the degradation of organic compounds was observed: heteropolysaccharides > cellulose > amide I in proteins. And proteins and cellulose showed significantly more degradation than heteropolysaccharides. In summary, the combination of multiple fluorescence labeling and 2D correlation spectroscopy is a promising approach for the characterization of EPS in biofilms.

Rice seedling wilt frequently occurs in upland nurseries under well-aerated conditions and causes considerable economic loss. Whether the wilt is pathogenic or edaphic is not known. We hypothesize the use of composts to alleviate seedling wilt. The severity level of upland rice seedling wilt was significantly (p < 0.05) positively correlated with soil pH (r = 0.499; n = 19), but negatively correlated with soil organic matter (r = −0.745), microbial biomass C (r = −0.669), activities of dehydrogenase (r = −0.589), arylsulfatase (r = −0.272), fluorescein diacetate hydrolysis (r = −0.466), and β-glucosidase (r = −0.280). Correlations between severity level and soil inorganic N and exchangeable potassium K were not significant. Contents of Fe, Zn, Cu, and Mn in healthy seedlings were not significantly (p < 0.05) different from those in infected seedlings. These data suggest that seedling wilts are not associated with nutrient constraints. Compost amendment at the rate of 3% or above in pot experiments significantly improved seedling growth and reduced the wilt symptoms. Field trials further showed that aboveground weight of seedlings in compost-amended treatment ranged from 11.5 to 14.9 mg per plant, significantly higher than the range from 6.38 to 12.1 mg per plant in the control treatment; in addition to rice growth compost significantly increased microbial biomass and enzyme activities of soils. Soil fumigation significantly increased rice growth and alleviation symptoms in 11 out of 19 soils, suggesting the involvement of pathogens. It is concluded that upland seedling wilt is a pathogen-associated disease. Probably high soil pH and low soil biochemical activities may favor pathogen activities.

Fusarium wilt is one of the major constraints on cucumber production worldwide. Several strategies have been used to control the causative pathogen, Fusarium oxysporum f. sp. cucumerinum J. H. Owen, including soil solarization, fungicide seed treatment and biological control. In this study, F. oxysporum f. sp. cucumerinum was successfully controlled by a newly isolated strain, Bacillus subtilis SQR 9, in vitro and in vivo. Greenhouse experiments were carried out to evaluate the effect of inoculation and solid fermentation of organic fertilizer with B. subtilis SQR 9, hereby defined as bio-organic fertilizer (BIO), on the control of Fusarium wilt. In comparison with the control, the wilt incidence was significantly reduced (49–61% reduction) by application of BIO. The rhizosphere population of F. oxysporum f. sp. cucumerinum, as detected both by selective plating and realtime PCR, was significantly lower in BIO-treated plants than the control. The localization of bacterial cells, pattern of colonization and survival of B. subtilis SQR 9 in the rhizsosphere of cucumber, was examined by fluorescent microscopy and explored following recovery of the green fluorescent protein (gfp)-labeled SQR 9 with the new gfp-marked shuttle vector pHAPII through selective plating. The preferential sites of the labeled strain were the differentiation and elongation zone, root hair and the lateral root junctions. The population of the strain was 106 cfu/g root in rhizoplane. These results indicate that the strain was able to survive well in the rhizosphere of cucumber, suppressed growth of F. oxysporum in the rhizosphere of cucumber and protected the host from the pathogen.

Pot experiments were carried out over two growing periods to assay the biocontrol efficacy and rhizosphere colonization of Trichoderma harzianum SQR-T037 (SQR-T037) applied as SQR-T037 conidia suspension (TCS), SQR-T037 conidia suspension blended with organic fertilizer (TBF), or SQR-T037 fermented organic fertilizer (TFF). Each formulation had three T. harzianum numbers. In two experiments, Percent Disease Indexes (PDIs) decreased with the increase of SQR-T037 number added to soils. The TFF treatment consistently exhibited the lowest PDIs at same amendment rate of SQR-T037 and 0–8.9%, 25.6–78.9%, and 4.4–50.0% of PDIs were found in TFF, TCS, and TBF treatment, respectively. Soils treated with TFF showed the highest SQR-T037 population in rhizosphere and bulk soil. Decrease of Fusarium oxysporum population in both bulk and rhizosphere soils occurred in the treatment SQR-T037 at 105 and 106 cfug−1 soil rate. The TFF treatment at the SQR-T037 rate of 103 cfug−1 soil significantly (p < 0.05) increased SQR-T037 population within the rhizoplane but had no effect on F. oxysporum population when compared to TCS and TBF. Generally, TFF treatments were superior to TCS and TBF treatments on disease control by sustaining colonization of SQR-T037 and decreasing F. oxysporum abundance in the rhizosphere soil. We propose that TFF treatment at SQR-T037 rate of 107 cfug−1 (i.e., 105 cfug−1 soil after applied to soil) was the best formulation for controlling Fusarium wilt of cucumber.

An experiment was planned to evaluate the behavior of Paenibacillus polymyxa SQR-21 under differential iron availability. P. polymyxa was grown under three differential iron conditions (0, 2, 20 μM). Iron starvation slowed bacterial growth and at all iron levels, pH of liquid culture was decreased, but maximum decrease was observed at highest iron level. Cell surface ferrireductase activity decreased as culture aged, while extracellular Fe3+-reducing activity constantly increased. Hydroxamates type siderophores production was increased with the decrease in iron levels. Numerous cellular proteins were expressed by P. polymyxa in the range of 5–140 kDa and several of them showed conspicuous differential iron regulation. P. polymyxa seems to have more than one type of iron acquisition mechanism including gradual release of organic acids, cell surface ferrireductases, extracellular reductants, and secretion of low molecular weight hydroxamates chelators. This article is the first to report the kinetic study of P. polymyxa under differential iron availability. The information provided here gives initial information about the iron uptake mechanism of P. polymyxa.

Bioorganic fertilizer containing Paenibacillus polymyxa SQR-21 showed very good antagonistic activity against Fusarium oxysporum. To optimize the role of P. polymyxa SQR-21 in bioorganic fertilizer, we conducted a study of spore germination under various conditions. In this study, l-asparagine, glucose, fructose and K+ (AGFK), and sugars (glucose, fructose, sucrose, and lactose) plus l-alanine were evaluated to determine their ability to induce spore germination of two strains; P. polymyxa ACCC10252 and SQR-21. Spore germination was measured as a decrease in optical density at 600 nm. The effect of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris–HCl. l-Alanine alone showed a slight increase in spore germination; however, fructose plus l-alanine significantly induced spore germination, and the maximum spore germination rate was observed with 10 mmol l−1l-alanine in the presence of 1 mmol l−1 fructose in phosphate-buffered saline (PBS). In contrast, fructose plus l-alanine hardly induced spore germination in Tris–HCl; however, in addition of 10 mmol l−1 NaCl into Tris–HCl, the percentages of OD600 fall were increased by 19.6% and 24.3% for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination was much more strict to germination temperature than that induced by fructose plus l-alanine. For both strains, fructose plus l-alanine-induced spore germination was not sensitive to pH. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination diversity, which will be beneficial for bioorganic fertilizer storage and transportation to improve the P. polymyxa efficacy as biological control agent.

Fusarium wilt of watermelon commonly occurs in locations where the crop has been grown for many seasons. Its occurrence results in a severely decreased watermelon crop. The goal of this study was to assess the capability of a new product (bio-organic fertilizer) to control the wilt in Fusarium-infested soil. Pot experiments were conducted under growth chamber and greenhouse conditions. The results showed that the fertilizer controlled the wilt disease. Compared with control pots, the incidence rates of Fusarium wilt at 27 and 63 days following treatment of the plants with the bio-organic fertilizer at a rate of 0.5% (organic fertilizer + antagonistic microorganisms, including 3 × 109 CFU g−1Paenibacillus polymyxa and 5 × 107 CFU g−1Trichoderma harzianum) were reduced by 84.9 and 75.0%, respectively, in both the growth chamber and greenhouse settings. The activities of antioxidases (catalase, superoxide dismutase and peroxidase) in watermelon leaves increased by 38.9, 150 and 250%, respectively. In the roots, stems and leaves, the activity of β-1,3-glucanase (pathogenesis-related proteins) increased by 80, 1140 and 100% and that of chitinase increased by 240, 80, and 20%, respectively, while the contents of malondialdehyde fell by 56.8, 42.1 and 45.9%, respectively. These results indicate that this new fertilizer formula is capable of protecting watermelon from Fusarium oxysporum f.sp. niveum. The elevated levels of defense-related enzymes are consistent with the induction and enhancement of systemic acquired resistance of plant.

To assess the influence of phenolic acids from plant root exudates on soil pathogens, we studied the effect of sinapic acid added to chemically defined media on the growth and virulence factors of Fusarium oxysporum f. sp. niveum. Sinapic acid inhibited the growth and conidial formation and germination of F. oxysporum f. sp. niveum by 6.7–8.8% and 11.2–37.3%, respectively. Mycotoxin production by F. oxysporum f. sp. niveum was stimulated by 81.6–230.7%. Pectinase, proteinase, cellulase, and amylase activities were stimulated at a lower concentration of sinapic acid, while they were inhibited at a higher concentration. It is concluded that sinapic acid inhibited the growth and conidial germination of F. oxysporum f. sp. niveum and decreased the pathogenic enzymes’ activity at higher doses.

The allelopathic potential of an artificially applied allelochemical, benzoic acid, on in vitro Fusarium oxysporum f.sp. niveum (a soil-borne pathogen causing watermelon wilt) was evaluated. Benzoic acid strongly inhibited its growth, sporulation and conidia germination, whereas it stimulated virulence factors of this pathogen. The biomass was reduced by 83–96 % and the conidia germinating rate and conidia production rate were decreased by 100 % at a concentration of >200 mg/L. However, phytopathogenic enzyme activities and mycotoxin production were stimulated with an increase of 10.2–1250 % for enzyme activities and 610–2630 % for mycotoxin yield.

A bacterial strain was isolated from the rhizosphere of healthy watermelon plants in a heavily wilt-diseased field. This isolate was tentatively identified as Paenibacillus polymyxa (SQR-21) based on biochemical tests and partial 16S rRNA sequence similarity. The purified antifungal compounds were members of the fusaricidin group of cyclic depsipeptides having molecular masses of 883, 897, 947, and 961 Da with an unusual 15-guanidino-3-hydroxypentadecanoic acid moiety, bound to a free amino group. The strain SQR-21 was not able to produce antifungal volatile compounds but was able to produce cellulase, mannase, pectinase, protease, β-1,3-glucanase and lipase enzymes. However, the strain did not show any chitinase activity. Biocontrol potential of this strain was evaluated against Fusarium oxysporum cause of Fusarium wilt disease of watermelon in a greenhouse experiment. This strain combined with organic fertiliser decreased the disease incidence by 70% and increased the dry plant weight by 113% over the control.

Long-term monoculture of watermelon leads to frequent occurrence of watermelon fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON). Some allelochemicals contained in watermelon root exudates and decaying residues are possibly responsible for promoting the wilt disease. The purpose of this study was to evaluate the allelopathic effect of artificially applied cinnamic acid on FON. Results demonstrated that hyphal growth of FON was strongly inhibited by cinnamic acid. At the highest concentration of cinnamic acid, the biomass in liquid culture was decreased by 63.3%, while colony diameter, conidial germination on plates, and conidial production in liquid culture were completely inhibited. However, mycotoxin production and activity of phytopathogenic enzymes were greatly stimulated. Mycotoxin yield, pectinase activity, proteinase activity, cellulase activity, and amylase activity were increased by 490, 590, 760, 2006, and 27.0%, respectively. It was concluded that cinnamic acid dramatically stimulated mycotoxin production and activities of hydrolytic enzymes by FON but inhibited growth and germination of FON. The findings presented here indicate that cinnamic acid is involved in promoting watermelon fusarium wilt.

Two field experiments were conducted to evaluate the effect of organic fertilizer application either with or without antagonistic bacteria (Bacillus subtilis SQR-5 and Paenibacillus polymyxa SQR-21) on the control of Fusarium oxysporum f. sp. Cucumerinum J. H. Owen wilt disease in cucumber. The incidence of Fusarium wilt disease was 5.3–13.5% for cucumber plants treated with bioorganic fertilizer, while it was 30.3–51% in controls (only with organic fertilizer). Higher yields and lower disease incidences were observed in the dry season when compared with the wet season for both types of organic fertilizer treatments. Biolog analysis showed a significant change in soil bacterial composition and activity after bioorganic fertilizer application. The numbers of colony-forming units of F. oxysporum f. sp. Cucumerinum J. H. Owen for bioorganic-fertilizer-treated soils were significantly decreased compared with control. Scanning electron micrographs of cucumber basal stems showed a presence of mycelia-like mini strands accompanied by an amorphous substance within the xylem vessels. This amorphous substance and mini strands were richer in calcium and phosphorus but had low carbon and oxygen than the living mycelia. Reverse-phase high-pressure liquid chromatography and mass spectroscopic analysis showed that the antagonistic bacteria produced the antifungal compounds fusaricidin A, B, C, and D with molecular weights of 883.5, 897.5, 947.5, and 961.5 Da, respectively. The application of bioorganic fertilizer has a great potential for the control of F. oxysporum wilt disease in cucumber plants.

The impact of fusaric acid (FA) phytotoxin on the physiology of root leaf cells in watermelon seedlings was evaluated. Results revealed that the cell membrane potential treated with FA in 12 h was decreased by 61.9–81.8% compared with untreated controls. FA markedly accelerated the lipid peroxidase activity of watermelon leaves. Malondiadehyde in leaves treated with different concentrations of FA in 24 h was 5.2–11.0 fold as much as control. Phenylalanine ammonia-lyase activity (PAL) in leaves treated with FA was first increased and then decreased. The highest PAL activity was obtained after 6–12 h, which was 7.2–10.5 fold as much as control. Activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) were first increased and then decreased when the seedlings were treated with FA. The highest SOD and POD activities were observed after 12 h and then both declined. The β-1,3-glucanase and chitinase activities in leaves treated with FA were first quickly increased and then declined, the highest activities were found after 12 and 3 h. We concluded that FA strongly inhibited root and leaf cells functions physiologically responsible for fusarium wilt of watermelon.

Watermelon production is threatened by fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON) in continuous cultivation system. Some elements, mainly allelochemicals, released from living roots or decayed plants might be associated with the disease. The purpose of this work was to evaluate the possible impact of coumarin, one kind of watermelon allelochemical, on FON. Furthermore, possible new mechanisms might be investigated during the ecological interactions of plant-microbe. Results showed that coumarin strongly inhibited growth of FON leading to a decrease in its biomass, dry weight of mycelia of FON in a liquid culture. The dry weight was decreased by 62.9% compared with control. The hyphal growth of FON on plates was stopped at high (>400 mg l−1) concentrations of coumarin. At 320 mg l−1, sporulation and enzyme activities of FON were also severely suppressed by coumarin. The yield of conidia, and the activities of proteinase, cellulase, and amylase were reduced by 98.9%, 79.7%, 29.8% and 15.9% respectively. However, conidial germination and mycotoxin (MT) production of FON were greatly stimulated, being increased by 55.7% and 14.9 fold at 320 mg l−1 respectively. We conclude that coumarin acted as an allelochemical substance to inhibit growth and pathogenic enzyme activities of FON but to stimulate mycotoxin production and conidial germination. It was suggested that coumarin acted as a signal transduction element bridging plant and pathogen in the process of plant-microbe interactions.

A rhizobox with three compartments and soil slicing followed by quick freezing were used to study the spatiotemporal variations of nitrification of rhizospheric soil of Yangdao 6 (Indica) and Nongken 57 (Japonica). The results obtained revealed that ammonium (\({{NH}}^{ + }_{4}\)) was the main N form in flooded paddy soil. A concentration gradient for \({{NH}}^{ + }_{4}\) was observed with the lowest concentration nearer to the root zone and the concentrations increased with increasing distance from the root zone. No concentration gradient was observed for nitrate (\({{NO}}^{ - }_{3}\)). The nitrification activities of both rice cultivars increased with the development of the incubation time. The nitrification activities were maximal in rhizospheric soil, followed by those in bulk soil and in the root zone. In the rhizosphere, nitrification activities decreased with increasing distance from the root zone. The maximal nitrification activity measured at 44, 51, and 58 days after sowing of Yangdao 6 and Nongken 57 rice cultivars was at a distance of 6 and 2 mm away from the root zone, respectively, and they were 0.88 and 0.73 mg kg−1 h−1, respectively. In this experiment, the nitrification activities were significantly and positively correlated with the ammonia-oxidizing bacteria (AOB) abundance (r=0.86, p<0.01). The nitrification activity, \({{NO}}^{ - }_{3}\) concentration, AOB abundance, dry matter and N accumulation and leaf \({{NO}}^{ - }_{3}\) reductase activity associated with Indica were always higher than those with Japonica. Therefore, nitrification in rhizosphere had more important significance for N nutrition, especially for the Indica rice cultivars.

An hydroponic experiment with a simulated water stress induced by PEG (6000) was conducted in a greenhouse to study the effects of nitrate (NO3−), ammonium (NH4+) and the mixture of NO3− and NH4+, on water stress tolerance of rice seedlings. Rice (Shanyou 63) was grown under non- or simulated water stress condition (10% (w/v) PEG, MW6000) with the 3 different N forms during 4 weeks. Under non-stressed condition no difference was observed among the N treatments. Under simulated water stress, seedlings grown on N-NO3− were stunted. Addition of PEG did not affect rice seedling growth in the treatment of only NH4+ supply but slightly inhibited the rice seedling growth in the treatment of mixed supply of NO3− and NH4+. Simulated water stress, when only N-NH4+ was present, did not affect leaf area and photosynthesis rate, however, both parameters decreased significantly in the NO3− containing solutions. Under water stress, Rubisco content in newly expanded leaves significantly increased in the sole NH4+ supplied plants as compared to that in plants of the other two N treatments. Under water stress, the ratio of carboxylation efficiency to Rubisco content was, respectively, decreased by 13 and 23% in NH4+ and NO3− treatments, respectively. It is concluded that, water stress influenced the Rubisco activity than stomatal limitation, and this effects could be regulated by N forms.

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS–PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60 °C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4–7, and both enzymes demonstrated good thermostability ranging from 30 to 60 °C. The Km and Vmax values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by β-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.Highlights► Two endoglucanase genes were cloned from a lignocellulosic decomposing fungus. ► Both of two genes were successfully expressed in Pichia pastoris X33. ► Several biochemical properties of the two recombinant enzymes were studied. ► These enzymes can attract a great deal of interest for industrial applications.

Real-time polymerase chain reaction analysis was used to compare the effect of NO−3 on the activities of nitrate reductase (NR) and glutamine synthetase (GS), and the transcript levels of two NR genes, OsNia1 and OsNia2, two cytosolic GS1 genes, OsGln1;1 and OsGln1;2, and one plastid GS2 gene OsGln2, in two rice (Oryza sativa L.) cultivars Nanguang (NG) and Yunjing (YJ). Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH+4 nutrition. Supply of NO−3 increased NR activity in both leaves and roots. Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNia1. NO−3 also caused an increase in GS activity, but had a complex effect on the expression of the three GS genes. In roots, the OsGln1;1 transcript increased, but OsGln1;2 decreased. In leaves, NO−3 had no effect on the GS1 expression, but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N. These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes. In addition, the NO−3-induced increase of biomass, NR activity, GS activity, and the transcript levels of NR and GS genes were proportionally higher in NG than in YJ, indicating a stronger response of NG to NO−3 nutrition than YJ.

Real-time polymerase chain reaction analysis was used to compare the effect of NO−3 on the activities of nitrate reductase (NR) and glutamine synthetase (GS), and the transcript levels of two NR genes, OsNia1 and OsNia2, two cytosolic GS1 genes, OsGln1;1 and OsGln1;2, and one plastid GS2 gene OsGln2, in two rice (Oryza sativa L.) cultivars Nanguang (NG) and Yunjing (YJ). Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH+4 nutrition. Supply of NO−3 increased NR activity in both leaves and roots. Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNia1. NO−3 also caused an increase in GS activity, but had a complex effect on the expression of the three GS genes. In roots, the OsGln1;1 transcript increased, but OsGln1;2 decreased. In leaves, NO−3 had no effect on the GS1 expression, but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N. These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes. In addition, the NO−3-induced increase of biomass, NR activity, GS activity, and the transcript levels of NR and GS genes were proportionally higher in NG than in YJ, indicating a stronger response of NG to NO−3 nutrition than YJ.

Under high light conditions, ammonium nutrition has a negative effect on plant growth. This suggests that the adverse effects of ammonium nutrition on plant growth may be related to carbon gain, photosynthesis, and photorespiration. However, there is no consistent evidence of a specific mechanism that could explain the plant growth reduction under ammonium supply. It is generally accepted that during the light reaction, a surplus of nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) is produced, which is not completely used during the assimilation of CO2. Nitrate reduction in the leaf represents an additional sink for NADPH that is not available to ammonium-grown plants. Nitrate and ammonium nutrition may use different pathways for NADPH consumption, which leads to differences in photosynthesis and photorespiration. The morphological (i.e., cell size, mesophyll thickness, and chloroplast volume) and enzymic (i.e., ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPCase), and glutamine synthetase/glutamate synthetase (GS/GOGAT)) differences that develop when plants are treated with either nitrate or ammonium nitrogen forms are related to photosynthesis and photorespiration. The differences in photorespiration rate for plants treated with nitrate or ammonium are related to the conversion of citrate to 2-oxoglutarate (2-OG) and photorespiratory CO2 refixation.

The volatile organic compounds (VOCs) produced by soil microbes have a significant role in the control of plant diseases and plant growth promotion. In this study, we examined the effect of VOCs produced by Pseudomonas fluorescens strain WR-1 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum. The VOCs produced by P. fluorescens WR-1 exhibited concentration dependent bacteriostatic effect on the growth of R. solanacearum on agar medium and in infested soil. The VOCs of P. fluorescens WR-1 also significantly inhibited the virulence traits of R. solanacearum. The proteomics analysis showed that the VOCs of P. fluorescens WR-1 downregulated cellular proteins of R. solanacearum related to the antioxidant activity, virulence, inclusion body proteins, carbohydrate and amino acid synthesis and metabolism, protein folding and translation, methylation and energy transfer, while the proteins involved in the ABC transporter system, detoxification of aldehydes and ketones, protein folding and translation were upregulated. This study revealed the significance of VOCs of P. fluorescens WR-1 to control the tomato wilt pathogen R. solanacearum. Investigation of the modes of action of biocontrol agents is important to better comprehend the interactions mediated by VOCs in nature to design better control strategies for plant pathogens.Download high-res image (174KB)Download full-size image

•An overview of the recent progresses in rhizosphere interaction networks, especially of the crops was provided.•The unprecedented holistic view of the root-soil-rhizomicrobiome interactions was discussed.•The potential strategies to manage the complex rhizosphere interactions for enhancing crop production were proposed.The underground root–soil–microbe interactions are extremely complex, but vitally important for aboveground plant growth, health and fitness. The pressure to reduce our reliance on agrochemicals, and sustainable efforts to develop agriculture makes rhizosphere interactions’ research a hotspot. Recent advances provide new insights about the signals, pathways, functions and mechanisms of these interactions. In this review, we provide an overview about recent progress in rhizosphere interaction networks in crops. We also discuss a holistic view of the root–soil–rhizomicrobiome interactions achieved through the advances of omics and bioinformatics technologies, and the potential strategies to manage the complex rhizosphere interactions for enhancing crop production.

The effect of fusaric acid (FA) on the activity of leaf nitrogen (N) metabolism enzymes in watermelon seedlings supplied with different N forms was studied. The results showed that FA inhibited nitrogen uptake and caused decreased leaf amide and protein but increased the content of ammonium and amino acids. When treated with FA the activities of enzymes in the pathway for the synthesis of amino acid in leaves (GS, GOGAT, and GDH) were decreased by 15–23%, 13–40%, and 71–86%, respectively. The activity of asparagine synthetase was decreased by 34–57%. The proteinase activity was initially increased by 37–125% at 12 h after treatment of FA but then subsequently decreased. The activity of glutamate-pyruvate aminotransferase was increased by 280–400%, though the activity of glutamate-oxaloacetate aminotransferase was decreased by 30–63%. It was suggested that FA inhibited the uptake of ammonium in seedlings and suppressed the activities of amino acid and amide synthases, while stimulating proteinase activity.A new pathogenic mechanism of watermelon infection by Fusarium spp. was found as FA caused the complete disorder and collapse of the host plant's nitrogen metabolism. This work provides a new insight into the progression of watermelon wilting caused by Fusarium oxysporum f.sp. niveum.

Endogenous hormones play an important role in the growth and development of roots. The objective of this research was to study the effect of four types of N fertilizers on the root growth of strawberry (Fragaria ananassa Duchesne) and the endogenous enzymes of indole-3-acetic acid (IAA), abscisic acid (ABA), and isopentenyl adenosine (iPA) in its roots and leaves using enzyme-linked immunosorbent assay. Application of all types of N fertilizers significantly depressed (P ≤ 0.05) root growth at 20 d after transplanting. Application of organic-inorganic fertilizer (OIF) as basal fertilizer had a significant negative effect (P ≤0.05) on root growth. The application of OIF and urea lowered the lateral root frequency in strawberry plants at 60 d (P ≤0.05) compared with the application of two organic fertilizers (OFA and OFB) and the control (CK). With the fertilizer treatments, there were the same concentrations of IAA and ABA in both roots and leaves at the initial growth stage (20 d), lower levels of IAA and ABA at the later stage (60 d), and higher iPA levels at all seedling stages as compared to those of CK. Thus, changes in the concentrations of endogenous phytohormones in strawberry plants could be responsible for the morphological changes of roots due to fertilization.

Efficient use of N in agricultural practice can increase yield, decrease production costs and reduce the risk of environmental pollution. Effects of N fertilizer application rates on grain yield and physiological N use efficiency (PE) in relation to the accumulation and redistribution of biomass and N in rice (Oryza sativa L.) cultivars were studied at two experimental farms of Nanjing Agricultural University, Nanjing, China in 2004. Three high N use efficiency (NUE) rice cultivars (Wuyunjing 7, Nanguang and 4007) and one low NUE rice cultivar (Elio) with similar growth patterns were studied under seven N rates (0, 60, 120, 180, 240, 300 and 360 kg ha−1). Grain yield increased with the N application rate and attained plateau at 180 kg N ha−1 for rice cultivars at each site. Increasing N rate decreased PE for biomass and grain yield. Grain yield and PE of Elio were about 20% and 18% lower than those of high NUE cultivars. Differences in biomass, N accumulation and N redistribution were observed at the post-heading stage among rice cultivars with differing NUEs. The less reproductive tillers of Elio resulted in less demand for C and N during grain filling, thus leading to lower PE of Elio compared with the high NUE rice cultivars.

Developing high-yielding rice (Oryza sativa L.) cultivars depends on having a better understanding of nitrogen (N) accumulation and translocation to the ear during the reproductive stage. Field experiments were carried out to evaluate the genetic variation for N accumulation and translocation in different Japonica rice cultivars at different N rates and to identify any relationship to grain yield in southeast China. Four Japonica cultivars with similar agronomic characteristics were grown at two experimental sites in 2004 with three N rates of 0, 60, and 180 kg N ha−1. Dry weights and N contents of rice plants were measured at tillering, initiation, anthesis, and maturity. Grain yields exhibited significant differences (P < 0.05) among the cultivars and N application rates. Increasing N rates improved N uptake at anthesis and maturity in all four cultivars (P < 0.05). N translocation from vegetative organs to the grains increased with enhanced N rates (P < 0.05). N translocation to the grains ranged from 9 to 64 kg N ha−1 and N-translocation effciency from 33% to 68%. Grain yield was linear to N uptake at anthesis (r2 = 0.78**) and N translocation (r2 = 0.67**). Thus, cultivars with a high N uptake at anthesis, low residual N in the straw at maturity, and appropriate low N fertilizer supply in southeast China should effciently increase N-recovery rate while maintaining grain yield and soil fertility.

Rice is being increasingly cultivated in intermittently irrigated regions and also in aerobic soil in which Nitrate (NO−3) plays important role in nutrition of plant. However, there is no information regarding the influence of nitrate on the overall growth and uptake of nitrogen (N) in rice plant. Solution culture experiments were carried out to study the effects of NO−3 on the plant growth, uptake of N, and uptake kinetics of NH+4 in four typical rice (Oryza sativa L.) cultivars (conventional indica, conventional japonica, hybrid indica, and hybrid japonica), and on plasma membrane potential in roots of two conventional rice cultivars (indica and japonica) at the seedling stage. The results obtained indicated that a ratio of 50/50 NH+4 -N/NO−3-N increased the average biomass of rice shoots and roots by 20% when compared with that of 100/0 NH+4 -N/NO−3-N. In case of the 50/50 ratio, as compared with the 100/0 ratio, total N accumulated in shoots and roots of rice increased on an average by 42% and 57%, respectively. Conventional indica responds to NO−3more than any other cultivars that were tested. The NO−3supply increased the maximum uptake rate (Vmax) of NH+4 by rice but did not show any effect on the apparent Michaelis-Menten constant (Km) value, with the average value of Vmax for NH+4 among the four cultivars being increased by 31.5% in comparison with those in the absence of NO−3. This suggested that NO−3 significantly increased the numbers of the ammonium transporters. However, the lack of effect on the Km value also suggested that the presence of NO−3had no effect on the affnity of the transporters for NH+4. The plasma membrane potential in the roots of conventional indica and japonica were greatly increased by the addition of NO−3, suggesting that NO−3could improve the uptake of N by roots of the rice plant. In conclusion, the mechanisms by which NO−3enhances the growth and N uptake of rice plant was found by the increased value of Vmax of NH+4 and increased plasma membrane potential. Thus promotion of nitrification in paddy soil is of great significance for improving the production of rice.

Composting of animal manures is believed as an alternative way for directly recycling them in farms, and therefore assessment of compost maturity is crucial for achieving high quality compost. Fluorescence excitation–emission matrices (EEMs) combined with regional integration analysis is presented to assess compost maturity. The results showed that the EEM contours of water-extract organic matter (WEOM) from immature composts exhibited four peaks at excitation/emission (Ex/Em) of 220/340 nm, 280/340 nm, 220/410 nm, and 330/410 nm, whereas EEM contour of WEOM from mature composts had only two peaks at Ex/Em of 230/420 nm and 330/420 nm. Pearson correlation demonstrated that peaks intensity rather than their ratios had a significantly correlation with the common indices assessing compost maturity, whereas the normalized excitation–emission area volumes (Φi,ns) from regional integration analysis had a stronger correlation with the common indices assessing compost maturity than peaks intensity. It is concluded that the Φi,ns from regional integration analysis are more suitable to assess the maturity of compost than the intensities of peaks. Therefore, the fluorescence spectroscopy combined with regional integration analysis can be used as a valuable industrial and research tool for assessing compost maturity, given its high sensitivity and selectivity.

A lignocellulosic decomposing fungus Z5 was isolated and identified as Aspergillus fumigatus, its capacity to produce cellulase was assessed under solid-state fermentation (SSF) using lignocellulosic materials as substrates. Cultivation conditions of A. fumigatus Z5 for cellulase production were optimized, results showed that for carboxymethyl cellulase (CMCase) and filter paper enzyme (FPase), the best condition was 50 °C, 80% initial moisture, initial pH 4.0 and 7% initial inoculum, the average activity of CMCase activity, FPase activity reached 526.3 and 144.6 U g−1 dry weight (dw) respectively, much higher than most of previous reports of this genus. Optimal temperature and pH for the CMCase activity of the crude enzyme were found to be 50 °C and 5.0, respectively. Zymogram analysis showed that eight kinds of CMCase were secreted by A. fumigatus Z5 when cellulose-containing materials were supplied in the culture. The crude enzyme secreted by the strain was further applied to hydrolyze pretreated corn stover and the enzymatic hydrolysate was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of bio-ethanol was 0.112 g g−1 dry substrate (gDS), suggesting that it is a promising fungus in the bio-ethanol production process.Highlights► A thermostable lignocellulosic decomposing fungus Z5 was isolated and identified. ► Z5 showed high cellulase activity which ranks top of the same genus. ► Zymogram result showed that eight kinds of CMCase were secreted by A. fumigatus Z5. ► The crude enzyme secreted by Z5 was applied to hydrolyze pretreated corn stover. ► The yield of bio-ethanol was 0.112 g g−1 dry substrate (gDS).

The variation in nitrogen (N) uptake by rice has been widely studied but differences in rice root morphology that may contribute to this variation are not completely understood. Field and greenhouse experiments were carried out to study N accumulation, root dry weights, total root lengths, root surface areas, and root bleeding rates of two rice cultivars, Elio with low N-use efficiency and Nanguang with high N-use efficiency. Low (1 mmol N L−1) and high (5 mmol N L−1)N applications were established in the greenhouse experiment, and the N rates were 0, 120, and 240 kg ha−1 in the field experiments at Jiangning and Jiangpu farms, Nanjing, China. The results showed that the N accumulation, root dry weight, total root length, and root surface area increased with an increase in N application. At the heading stage, N accumulation in the shoots and roots of Nanguang was greater than that of Elio in the field experiments and that of Elio at 5 mmol N L−1 in the greenhouse experiment. After the heading stage, N accumulation was higher for Nanguang at both 1 and 5 mmol N L−1 in the greenhouse experiment. The total root length and root surface area were significantly different between the two cultivars. Over the range of the fertilizer application rates, the root lengths of Nanguang at Jiangning Farm were 49%–61% greater at booting and 26%–39% greater at heading than those of Elio, and at Jiangpu Farm they were 22%–42% and 26%–38% greater, respectively. Nanguang had a greater root bleeding rate than Elio. It was concluded that the N-use efficiency of the two rice cultivars studied depended to a great extent on the root morphological parameters and root physiological characteristics at different growth stages.

The variation in nitrogen (N) uptake by rice has been widely studied but differences in rice root morphology that may contribute to this variation are not completely understood. Field and greenhouse experiments were carried out to study N accumulation, root dry weights, total root lengths, root surface areas, and root bleeding rates of two rice cultivars, Elio with low N-use efficiency and Nanguang with high N-use efficiency. Low (1 mmol N L−1) and high (5 mmol N L−1)N applications were established in the greenhouse experiment, and the N rates were 0, 120, and 240 kg ha−1 in the field experiments at Jiangning and Jiangpu farms, Nanjing, China. The results showed that the N accumulation, root dry weight, total root length, and root surface area increased with an increase in N application. At the heading stage, N accumulation in the shoots and roots of Nanguang was greater than that of Elio in the field experiments and that of Elio at 5 mmol N L−1 in the greenhouse experiment. After the heading stage, N accumulation was higher for Nanguang at both 1 and 5 mmol N L−1 in the greenhouse experiment. The total root length and root surface area were significantly different between the two cultivars. Over the range of the fertilizer application rates, the root lengths of Nanguang at Jiangning Farm were 49%–61% greater at booting and 26%–39% greater at heading than those of Elio, and at Jiangpu Farm they were 22%–42% and 26%–38% greater, respectively. Nanguang had a greater root bleeding rate than Elio. It was concluded that the N-use efficiency of the two rice cultivars studied depended to a great extent on the root morphological parameters and root physiological characteristics at different growth stages.

Fusarium wilt is one of the major constraints on cucumber production worldwide. The introduction of beneficial microorganisms into soil has been widely adopted for suppression of the causative soilborne pathogen Fusarium oxysporum f. sp. cucumerinum J. H. Owen. The goal of this study was to investigate the effects of the new bio-organic fertilizer (BIO A) made from organic fertilizer and Bacillus subtilis SQR 9 on Fusarium wilt control in pot experiments. The results showed that application of BIOs significantly decreased the Fusarium wilt disease incidence and promoted plant biomass. The average disease incidence in BIO-treated plants was reduced by 68%, compared with the control treatment. Application of BIO A strongly reduced the number of the pathogen. The copy number of F. oxysporum DNA in BIO-treated soil declined to 105/g fresh root 60 days after pathogen attack, while those in the control treatment remained high (107/g fresh root). To determine the mechanisms of the antagonistic strain, polymerase chain reaction was used to screen SQR 9 for genes involved in biosynthesis of antibiotics. Amplicons of the expected sizes were detected as yndJ involved in the biosynthesis of Yndj protein, qk involved in subtilisin, sboA involved in subtilosin, bamC involved in bacillomycin, ituA, ituB, ituC and ituD involved in iturin, fenB and fenD involved in fengycin, and srfAB involved in surfactin synthesis. Fengycin and bacillomycin production in its culture filtrate were detected by liquid chromatography coupled with mass spectroscopy. The antifungal compounds significantly inhibited mycelial growth of F. oxysporum and caused a 13.4–83.6% reduction in the number of germinated spores compared with the control treatment. We speculate that the antibiotic production can be linked to the mechanism of protection of plants from pathogen attack by SQR 9.Highlights► A new bio-organic fertilizer was made by combining organic fertilizers with the antagonist B. subtilis SQR 9. ► The bio-organic fertilizer could significantly reduced Fusarium wilt disease incidence and promote plant growth of cucumber. ► Lipopeptide (fengycin and bacillomycin) production could be another mode of action of the strain against Fusarium oxysporum. ► It is the first time to investigate the antibiotics produced by the new antagonist SQR 9.

•We selected seven experiment sites which have different soil fertility in Jiangsu province.•There was a negative relationship between panicles per unit area and spikelets per panicle.•Optimize fertilization could mitigate the contradictions between yield formation factors.•Optimize fertilization improved yield mainly by increasing the spikelets per unit area.•Yield-promotion could be divided into primary and advanced nutrient contribution stages.Recently, many optimized nutrient management strategies have been applied to improve rice yield and nitrogen use efficiency (NUE) in China, most of which achieved higher yield and NUE than farmers’ usual fertilizer practices. The objective of this study was to investigate the aboveground biomass, nitrogen (N) accumulation and contribution of yield factors (i.e., number of effective panicles, number of spikelets per panicle, and grain weight) on rice yield regulated by optimized nitrogen application at different growth stages. Field experiments were conducted in 2009 and 2010 at 7 different sites, and three optimized N treatments (OPT) were compared with local farmers’ fertilizer practices (FFP). Rice yields of the optimized treatments increased 8.2–12.6% over the farmers’ fertilizer practices. Recovery efficiency of N (REN), agronomic N use efficiency (AEN) and partial factor productivity of applied N (PFPN) for OPT1 and OPT2 were all significantly higher than those obtained using the farmers’ fertilizer practices. The aboveground biomass and nitrogen accumulation of OPTs were lower than FFP at the vegetative and earlier reproductive growth stages, but the accumulation rate became faster than FFP at the later reproductive stage. In our result, grain yield was positively correlated with the panicles per unit area for OPTs, but not for FFP. Spikelets per panicles had negative correlation with panicles per unit area but the slop of OPTs was slower than the slope of FFP. We found that the OPTs treatments could mitigate the contradiction between yield formation factors. According to yield responses to the treatments, we defined two dominant yield-promotion stages: the primary nutrient contribution stage and the advanced nutrient contribution stage. Our results illustrated that at the primary nutrient contribution stage, the main promotion factor for yield was panicle number, which was mainly promoted by high N application at the early vegetative stage, and at the advanced nutrient contribution stage, the yield increase depended upon resolving the contradiction between number of effective panicles and spikelet number per panicle, which resulted from appropriate adjustments to the proportions of added N applied at different growth stages. In conclusion, our results indicated that adjusting the proportion of N application at different growth stages may reform the source–sink contradiction of yield, thus further increasing rice yield.

•Organic fertilizers improved the growth of Bacillus amyloliquefaciens strains T-5 and SQR-9.•Organic fertilizers improved the production of volatile compounds by strains T-5 and SQR-9.•Optimum production of volatile compounds was found at 1.5% BOF2 by strain T-5.•Optimum production of volatile compounds was found at 2% BOF3 by strain SQR-9.•Volatile compounds significantly inhibited the growth and virulence traits of Ralstonia solanacearum.Three organic fertilizers made of different animal and plant waste materials (BOFs) were evaluated for their effects on the production of antibacterial volatile organic compounds (VOCs) by two Bacillus amyloliquefaciens strains SQR-9 and T-5 against the tomato wilt pathogen Ralstonia solanacearum (RS). Both strains could produce VOCs that inhibited the growth and virulence traits of RS; however, in the presence of BOFs, the production of antibacterial VOCs was significantly increased. The maximum inhibition of growth and virulence traits of RS by VOCs of T-5 and SQR-9 was determined at 1.5% BOF2 and 2% BOF3, respectively. In case of strain T-5, 2-nonanone, nonanal, xylene, benzothiazole, and butylated hydroxy toluene and in case of strain SQR-9, 2-nonanone, nonanal, xylene and 2-undecanone were the main antibacterial VOCs whose production was increased in the presence of BOFs. The results of this study reveal another significance of using organic fertilizers to improve the antagonistic activity of biocontrol agents against phytopathogens.

Allelochemicals from root exudates or decaying residues of watermelon plant may be related to watermelon fusarium wilt. The aim of this work was to study the effect of an artificially applied allelochemical, ferulic acid, on in vitro Fusarium oxysporum f. sp. niveum (FON), a causal pathogen of plant wilting in the laboratory setting.The results showed that ferulic acid inhibited growth at high concentrations. The biomass was reduced by 71.6% and the conidial germinate rate was decreased by 100%, while mycotoxin production by FON was increased by 227.7% at the highest concentration (1600 mg L−1). Activities of hydrolytic enzymes related to pathogenicity were also affected.It is concluded that ferulic acid at commonly found concentrations inside plants suppressed the growth of pathogen F. oxysporum f. sp. niveum.