Strawberry Fusarium wilt, caused by the virulent fungus Fusarium oxysporum formae speciales fragariae (Fof) is a devastating soil-borne disease that causes severe production losses worldwide, including Japan. Fof is one of the top 10 fungal pathogens that threaten global crop security, and a method to effectively control this pathogen has yet to be found. This study aimed to investigate the effectiveness of dark septate endophytic (DSE) fungi against Fof to develop an efficient, effective, and environmentally friendly approach to improve plant health and fitness. A total of 19 fungal isolates were assessed, out of which three (SK47, SK48, and SK51) were selected based on their effectiveness in disease suppression in controlled growth chamber conditions using a soil system. Isolates SK47, SK48, and SK51 suppressed disease severity by 85.71, 61.90, and 90.48%, respectively. Molecular identification based on highly conserved small subunit (SSU), internal transcribed spacer (ITS), and large subunit (LSU) nrRNA regions identified these isolates as DSE Exophiala sp., Exophiala pisciphila, and Cladophialophora chaetospira, respectively. The sequences were deposited under accession numbers MN811693-MN811695 in the GenBank database. Notably, our results revealed that isolate C. chaetospira SK51 possessed superior growth promotion activities as well as disease suppression by significantly increased plant growth parameters (shoot and root dry mass, chlorophyll content, flower bud initiation, and number of fruit) in comparison to control plants and other two fungal candidates. Root colonization by C. chaetospira SK51 was visualized, and it was confirmed that the symbiosis with strawberry plants occurred successfully. Our results provide new insights in the application of DSE fungus C. chaetospira SK51 as a biocontrol agent on

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Crop rotation and intercropping with Allium plants suppresses Fusarium wilt in various crops. However, the mechanisms underlying this phenomenon have not been fully elucidated. This study was designed to assess the role of microorganisms inhabiting Allium rhizospheres and antifungal compounds produced by Allium roots in Fusarium wilt suppression by Allium cultivation. Suppression of cucumber Fusarium wilt and the pathogen multiplication by Allium (Welsh onion and/or onion)-cultivated soils were eliminated by heat treatment at 60 °C, whereas those by Welsh onion-root extract were lost at 40 °C. The addition of antibacterial antibiotics eliminated the suppressive effect of Welsh onion-cultivated soil on pathogen multiplication, suggesting the contribution of antagonistic gram-negative bacteria to the soil suppressiveness. The Illumina MiSeq sequencing of 16S rRNA gene amplicons revealed that genus Flavobacterium was the predominant group that preferentially accumulated in Allium rhizospheres. Flavobacterium species recovered from the rhizosphere soils of these Allium plants suppressed Fusarium wilt on cucumber seedlings. Furthermore, confocal laser scanning microscopy revealed that Flavobacterium isolates inhibited the multiplication of the pathogen in soil. Taken together, we infer that the accumulation of antagonistic Flavobacterium species plays a key role in Fusarium wilt suppression by Allium cultivation.

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Reducing dependence on chemical pesticides is considered as an essential challenge for sustainable crop production. The use of microbial biocontrol agents (MBCAs) is a key component of sustainable pest management. Numerous antagonistic microorganisms are known to suppress plant diseases, but their practical application and commercialization are still limited in part due to poor reliability of their efficacy in the field. Although promising MBCAs achieve remarkable disease control in the laboratory or greenhouse, field control is often unsatisfactory. Thus, for MBCAs to be integrated into crop production, their field performance must be improved to provide the cost-effectiveness and efficacy required by growers. In this review, we highlight recent approaches to enhance the field performance of MBCAs.

The aim of the present study was to identify a strain of endophytic Bacillus species that control tomato bacterial wilt by foliar spray application. Fifty heat-tolerant endophytic bacteria were isolated from the surface-sterilized foliar tissues of symptomless tomato plants that had been pre-inoculated with the pathogen Ralstonia pseudosolanacearum. In the primary screening, we assessed the suppressive effects of a shoot-dipping treatment with bacterial strains against bacterial wilt on tomato seedlings grown on peat pellets. Bacillus sp. strains G1S3 and G4L1 significantly suppressed the incidence of tomato bacterial wilt. In subsequent pot experiments, the biocontrol efficacy of foliar spray application was examined under glasshouse conditions. G4L1 displayed consistent and significant disease suppression, and, thus, was selected as a biocontrol candidate. Moreover, the pathogen population in the stem of G4L1-treated plants was markedly smaller than that in control plants. A quantitative real-time PCR analysis revealed that the foliar spraying of tomato plants with G4L1 up-regulated the expression of PR-1a and LoxD in stem and GluB in roots upon the pathogen inoculation, implying that the induction of salicylic acid-, jasmonic acid-, and ethylene-dependent defenses was involved in the protective effects of this strain. In the re-isolation experiment, G4L1 efficiently colonized foliar tissues for at least 4 weeks after spray application. Collectively, the present results indicate that G4L1 is a promising biocontrol agent for tomato bacterial wilt. Furthermore, to the best of our knowledge, this is the first study to report the biocontrol of bacterial wilt by the foliar spraying with an endophytic Bacillus species.

We previously identified Mitsuaria sp. TWR114 and nonpathogenic Ralstonia sp.TCR112 as potential biocontrol agents to suppress tomato bacterial wilt caused by Ralstonia pseudosolanacearum. Because commercial biocontrol products require a practical cost-effective application method that maximizes their performance, we investigated whether the combined application of TWR114 and TCR112 enhances the biocontrol of bacterial wilt. In pot experiments, all the tested inoculum ratios (i.e., 1:1, 1:2, and 2:1) of the TWR114+TCR112 treatment significantly suppressed the incidence of bacterial wilt, even at 28 days post-challenge inoculation (dpi) (13-47% wilt incidence), while 60% of plants treated with the individual isolates developed bacterial wilt within 10-12 dpi. The pathogen population in the rhizosphere and aboveground regions decreased considerably after the TWR114+TCR112 treatment compared with that in the individual treatments. Moreover, the pathogen population in the aboveground parts of TWR114+TCR112-treated plants had decreased to an undetectable level by 28 dpi. After inoculation with the pathogen, the expression of several tomato defenserelated genes was higher in the TWR114+TCR112-treated plants than in those treated with the individual isolates. Altogether, the results indicate that TWR114 and TCR112 applied together have a synergistic suppressive effect and that stronger defense priming might contribute to the improved biocontrol. The combination of both isolates may be a very promising approach for controlling tomato bacterial wilt in the future.

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