Previous studies demonstrated that the Chryseobacterium sp. WR21 could effectively control the bacterial wilt disease caused by Ralstonia solanacearum through effective root colonization. The strain WR21 exhibited a low level of DNA homology with Chryseobacterium strains DSM 15235T (24.1%), DSM 17724T (24.8%), and DSM 18014T (10.4%), suggesting that WR21 may represent a novel species, for which the name Chryseobacterium nankingense sp. nov. is proposed. The in vitro competition experiments with strain WR21 indicated it significantly inhibited growth of the pathogen in co-culture with six of nine tested nutrients (e.g. root exudates) that could be utilized by strain WR21 and R. solanacearum. Similar trends were observed in co-culturing experiments using tissue exudates of tomato. A positive relationship (r = 0.785) was noticed between the differences in the average growth rate of both strains and the disease suppression effects. In conclusion, Chryseobacterium nankingense sp. nov. WR21 exhibits antagonism through nutrient competition that might be used for achieving biocontrol of Ralstonia solanacearum induced wilts.

Plant-derived root exudates modulate plant-microbe interactions and may play an important role in pathogen suppression. Root exudates may, for instance, directly inhibit pathogens or alter microbiome composition. Here, we tested if plants modulate their root exudation in the presence of a pathogen and if these shifts alter the rhizosphere microbiome composition. We added exudates from healthy and Ralstonia solanacearum-infected tomato plants to an unplanted soil and followed changes in bacterial community composition. The presence of pathogen changed the exudation of phenolic compounds and increased the release of caffeic acid. The amendment of soils with exudates from the infected plants led to a development of distinct and less diverse soil microbiome communities. Crucially, we could reproduce similar shift in microbiome composition by adding pure caffeic acid into the soil. Caffeic acid further suppressed R. solanacearum growth in vitro. We conclude that pathogen-induced changes in root exudation profile may serve to control pathogen both by direct inhibition and by indirectly shifting the composition of rhizosphere microbiome.

Investigation of the properties and mechanisms of the interactions of root-colonizing biocontrol bacteria and plant pathogens is necessary to optimize the biocontrol strategies. In the present study, the interaction of a biocontrol strain Bacillus amyloliquefaciens T-5 tagged with a green fluorescent protein marker and a bacterial wilt pathogen Ralstonia solanacearum QL-Rs1115 tagged with red fluorescent protein marker was studied on tomato roots using different inoculation methods. The results showed that in the co-culture experiment, the population of pathogen QL-RFP was decreased by increasing the initial inoculum concentration of biocontrol strain. In the greenhouse experiment, both strains T-5-GFP and QL-RFP colonized tomato roots (root tips, root hairs, primary roots, and root junctions) and formed a biofilm on the root surfaces as determined by dilution plating and confocal laser scanning microscopy (CLSM) techniques. However, the root colonization of pathogen strain QL-RFP was almost completely suppressed in the presence of biocontrol strain T-5-GFP when both soil and plant seedlings were treated with T-5-GFP. The results of this study revealed the effectiveness of strain B. amyloliquefaciens T-5 as a biocontrol agent against tomato wilt pathogen and the significance of inoculation method used to inoculate biocontrol strain.

Ralstonia solanacearum (Smith) is an important soil-borne pathogen worldwide. We investigated the effects of a new bioorganic fertilizer, BIO62, which was made from organic fertilizer and antagonist Bacillus amyloliquefaciens HR62, on the control of bacterial wilt of tomato in greenhouse condition. The results showed that the application of BIO62 significantly decreased disease incidence by 65% and strongly reduced R. solanacearum populations both in the rhizosphere soil (8.04 log cfu g–1 dry soil) and crown sections (5.63 log cfu g–1 fresh plant section) at 28 days after pathogen challenge. Antibacterial compounds produced by HR62 were purified by silica gel, Sephadex LH-20, and HPLC and then identified using HPLC/electrospray ionization mass spectrometry analysis. Macrolactin A and 7-O-malonyl macrolactin A (molecular weights of 402 and 488 Da, respectively), along with surfactin B (molecular weights of 994, 1008, 1022, and 1036 Da), were observed to inhibit the growth of R. solanacearum.

Agricultural residue is more efficient than purified cellulose at inducing lignocellulolytic enzyme production in Penicillium oxalicum GZ-2, but in Trichoderma reesei RUT-C30, cellulose induces a more efficient response. To understand the reasons, we designed an artificially simulated plant biomass (cellulose plus xylan) to study the roles and relationships of each component in the production of lignocellulolytic enzymes by P. oxalicum GZ-2.The changes in lignocellulolytic enzyme activity, gene expression involving (hemi)cellulolytic enzymes, and the secretome of cultures grown on Avicel (A), xylan (X), or a mixture of both (AX) were studied. The addition of xylan to the cellulose culture did not affect fungal growth but significantly increased the activity of cellulase and hemicellulase. In the AX treatment, the transcripts of cellulase genes (egl1, egl2, egl3, sow, and cbh2) and hemicellulase genes (xyl3 and xyl4) were significantly upregulated (P <0.05). The proportion of biomass-degrading proteins in the secretome was altered; in particular, the percentage of cellulases and hemicellulases was increased. The percentage of cellulases and hemicellulases in the AX secretome increased from 4.5% and 7.6% to 10.3% and 21.8%, respectively, compared to the secretome of the A treatment. Cellobiohydrolase II (encoded by cbh2) and xylanase II (encoded by xyl2) were the main proteins in the secretome, and their corresponding genes (cbh2 and xyl2) were transcripted at the highest levels among the cellulolytic and xylanolytic genes. Several important proteins such as swollenin, cellobiohydrolase, and endo-beta-1,4-xylanase were only induced by AX. Bray-Curtis similarity indices, a dendrogram analysis, and a diversity index all demonstrated that the secretome produced by P. oxalicum GZ-2 depended on the substrate and that strain GZ-2 directionally adjusted the compositions of lignocellulolytic enzymes in its secretome to preferably degrade a complex substrate.The addition of xylan to the cellulose medium not only induces more hemicellulases but also strongly activates cellulase production. The proportion of the biomass-degrading proteins in the secretome was altered significantly, with the proportion of cellulases and hemicellulases especially increased. Xylan and cellulose have positively synergistic effects, and they play a key role in the induction of highly efficient lignocellulolytic enzymes.

A new acidophilic xylanase (XYN11A) from Penicillium oxalicum GZ-2 has been purified, identified and characterized. Synchronized fluorescence spectroscopy was used for the first time to evaluate the influence of metal ions on xylanase activity. The purified enzyme was identified by MALDI TOF/TOF mass spectrometry, and its gene (xyn11A) was identified as an open reading frame of 706 bp with a 68 bp intron. This gene encodes a mature protein of 196 residues with a predicted molecular weight of 21.3 kDa that has the 100 % identity with the putative xylanase from the P. oxalicum 114-2. The enzyme shows a structure comprising a catalytic module family 10 (GH10) and no carbohydrate-binding module family. The specific activities were 150.2, 60.2, and 72.6 U/mg for beechwood xylan, birchwood xylan, and oat spelt xylan, respectively. XYN11A exhibited optimal activity at pH 4.0 and remarkable pH stability under extremely acidic condition (pH 3). The specific activity, Km and Vmax values were 150.2 U/mg, 30.7 mg/mL, and 403.9 μmol/min/mg for beechwood xylan, respectively. XYN11A is a endo-β-1,4-xylanase since it release xylobiose and xylotriose as the main products by hydrolyzing xylans. The activity of XYN11A was enhanced 155 % by 1 mM Fe2+ ions, but was inhibited strongly by Fe3+. The reason of enhancing the xylanase activity of XYN11A with 1 mM Fe2+ treatment may be responsible for the change of microenvironment of tryptophan residues studied by synchronous fluorescence spectrophotometry. Inhibition of the xylanase activity by Fe3+ was first time demonstrated to associate tryptophan fluorescence quenching.

Muskmelon (Cucumis melo L.) wilt caused by Fusarium oxysporum f. sp. melonis leads to severe economic losses. A bio-organic fertilizer (BIO) fortified with an antagonistic strain of Bacillus subtilis Y-IVI was used to control this disease. Pot experiments were carried out to investigate the efficacy and to elucidate biocontrol mechanisms for the disease. BIO significantly reduced the disease incidence. Population of F. oxysporum in plant shoots of the BIO treatment were about 1000-fold lower than the control. Population of Y-IVI remained high in muskmelon rhizosphere of the BIO treatment during the experiment. Concentration of antifungal lipopeptides, iturin A, in the BIO treatment was significantly higher than other treatments. Ten days after transplantation, the salicylic acid content in BIO-treated plant leaves was significantly higher than control. In conclusion, BIO effectively controlled muskmelon wilt, possibly because the antagonistic microbes effectively colonize the plant rhizosphere and shoots to preclude pathogen invasion. Furthermore, Y-IVI produces antifungal lipopeptides in the rhizosphere.

Laboratory tests and greenhouse experiments were carried out to investigate the abilities of Bacillus subtilis Y-IVI to promote plant growth and to colonize the rhizosphere and interior tissues of muskmelon. Laboratory tests showed that B. subtilis Y-IVI can produce indole acetic acid, siderophores, and ammonia. The inoculation of soil with green fluorescent protein-tagged Y-IVI (GY-IVI) significantly increased plant shoot and root dry weights as compared with the non-inoculated soils. The inoculation of soil with B. subtilis GY-IVI maintained approximately 108 colony-forming units (cfu) of GY-IVI per gram of dry rhizosphere soil for 1 month. The GY-IVI recovered from the interior of crowns and roots in the inoculated soil were 106 and 107 cfu g−1 dry weight, respectively, suggesting that GY-IVI acted as an endophyte. In the present study, we combined the two important growth promotion ingredients, colonization ability and growth promotion metabolites produced by biological agents, to investigate B. subtilis Y-IVI’s promotion effects on muskmelon growth.

The present study was carried out on pot experiments with rice (Oryza sativa L. cv. Wuyujing 7) and winter wheat (Triticum aestivum L. cv. Yangmai 6) rotation in a sandy and a clayey soil fertilized with 15N-labeled ammonium sulfate (AS) and 15N-labeled rabbit feces so as to study the mechanisms of reduction of fertilizer N loss by organic fertilizers. The treatments included: (1) control without any N fertilizer application; (2) fertilization with 15N-labeled AS (IF); (3) fertilization with labeled rabbit feces (OF); (4) fertilization with either 40% 15N-labeled rabbit feces and 60% unlabeled AS (IOF1) or (5) 40% unlabeled rabbit feces and 60% 15N-labeled AS (IOF2). In the rice season, the IOF treatments compared to the IF treatment decreased the percentage of lost fertilizer N from the sandy and clayey soils, whereas it increased the percentage of fertilizer N, present as mineral N and microbial biomass N (MBN). During the second season, when soils were cropped to winter wheat, the IOF treatments in comparison with the IF or OF treatment increased mineral N and MBN contents of soils sampled at tillering, jointing, and heading stages, and such increases were derived from the organic N fertilizer in the sandy soil and from the inorganic N fertilizer in the clayey soil. The increased MBN in the IOF treatments was derived from inorganic fertilizers applied both soils. Therefore, in the IOF treatment, during the rice season, the organic N increased the immobilization of inorganic N in MBN, while the inorganic N fertilizer applied to both soils stimulated the uptake of organic N and the organic N fertilizer increased the uptake of inorganic N by winter wheat; the inorganic N increased the recovery of organic N in the plant-soil system after harvesting the winter wheat.

The activities of invertase, protease, urease, acid phosphomonoesterase, dehydrogenase, and catalase in different fractions of water-stable aggregates (WSA) were examined in long-term (26 years) fertilised soils. The long-term application of organic manure (OM) with chemical fertiliser (CF) significantly increased macroaggregate and decreased microaggregate percentages, enhanced the mean weight diameter, and significantly increased soil total carbon (TC) and total nitrogen (TN) contents of WSA in different size fractions. Combined fertilisation with OM and CF also increased invertase, protease, urease, acid phosphomonoesterase, dehydrogenase, and catalase activities of WSA in different size fractions. Enzyme activities were higher in macroaggregates than in microaggregates. The distribution of enzyme activities generally followed the distribution of TC and TN in WSA. The geometric mean of the enzyme activities in different WSA of OM-treated soils was significantly higher than that in soils treated with 100% CF or no fertiliser. The results indicated that the long-term combined application of OM with CF increased the aggregate stability and enzyme activity of different WSA sizes, and consequently, improved soil physical structure and increased soil microbial activity.