We previously reported that the rhizobacterium Enterobacter pseudoroggenkampii strain GVv1 protects soybeans against Phytophthora root and stem rot (PRSR), caused by Phytophthora sojae. In this study, we explored GVv1's mode of action by comparing its effects on P. sojae with three bacterial strains showing low or no protective activity against PRSR. All strains inhibited hyphal growth, but only GVv1 disrupted multiple infection stages. Exposure to GVv1 cell suspensions or volatiles reduced zoosporangium formation by about 97 % and 92 %, respectively. GVv1 also decreased zoospore migration toward soybean roots by 66 %. Capillary chemotaxis assays revealed that GVv1 significantly reduced zoospore migration toward root exudates, 10 mu M daidzein, and 10 mu M genistein by 88 %, 49 %, and 58 %, respectively. GVv1 culture filtrates also suppressed zoospore migration toward daidzein and inhibited zoospore germination. In contrast, split-root assays showed that GVv1 did not induce systemic resistance in soybean against PRSR. Genome analysis identified several biocontrol-related genes such as hydrolytic enzymes, lipopeptides, volatiles, phenazine derivatives, toxoflavin, bacilysin, as well as six secondary metabolite biosynthetic gene clusters, including a hybrid NRPS-T1PKS, terpene precursor, azolecontaining RiPP, and siderophore biosynthetic genes. Overall, experimental results along with genomic evidence indicate that GVv1 exerts biocontrol primarily through direct inhibition of various P. sojae infection behaviors, likely mediated by diffusible and volatile inhibitory compounds.

Pseudomonas protegens, a member of the P. fluorescens complex, is a key biocontrol bacterium with well-documented potential to protect plants against diverse pathogens. Although P. protegens strains have been widely examined globally, those originating from Japan have not been well described. In this study, we isolated and characterized a new P. protegens strain, GSF-73, from the rhizosphere of Allium fistulosum in Gifu, Japan, and assessed its performance against several major plant diseases. Draft genome sequencing produced a 7.13-Mbp assembly, and average nucleotide identity values of 97.81-98.26% with reference strains (Cab75, CHA0, and Pf-5) confirmed its species identity. A comparative genomic analysis showed that GSF-73 possessed a larger genome than the reference strains, containing eight conserved biosynthetic gene clusters for antimicrobial compounds and an expanded set of strain-specific genes related to metabolism, regulation, mobilome functions, and secretion. GSF-73 exhibited broadspectrum antagonistic activity in vitro against fungal, oomycete, and bacterial pathogens. In biocontrol assays, GSF-73 significantly suppressed spinach Fusarium wilt, cucumber anthracnose on detached cotyledons, tomato bacterial wilt, and cucumber downy mildew. In contrast, root and/or seed treatments enhanced Pythium root rot in spinach and anthracnose in pot-grown cucumbers, indicating pathosystem-dependent efficacy. Despite these contrasting outcomes, GSF-73 shows strong biocontrol potential and merits further study to elucidate the mechanisms underlying both beneficial and adverse effects for its optimized use as a locally adapted biocontrol agent for Japanese agriculture.

Leaf fall disease (LFD) has recently emerged as a serious threat to natural rubber production in Southeast Asia, driven by increasingly frequent and severe outbreaks caused by fungal pathogens belonging to the Pestalotiopsis group and related genera such as Neopestalotiopsis, Pseudopestalotiopsis, and Colletotrichum. To identify effective biocontrol agents (BCAs) against LFD, we isolated more than 200 heat-tolerant, Gram-positive endophytic bacteria from rubber leaves and screened 170 isolates for their ability to suppress disease symptoms caused by Japanese Neopestalotiopsis sp. and Colletotrichum siamense using a detached-leaf assay in Japan. Two strains, GURL-96 and GURL-107, consistently inhibited necrotic lesion development induced by both fungal pathogens. Whole-genome sequencing identified GURL-96 as Siminovitchia fortis and GURL-107 as Priestia megaterium. Under greenhouse conditions in Indonesia, both strains showed significant biocontrol efficacy against Indonesian Neopestalotiopsis sp. on seedlings of three of the four tested rubber clones. Moreover, field trials conducted in Palembang, Indonesia, revealed that GURL-96 and GURL-107 reduced LFD severity by an average of 15% and 11%, respectively, on young trees of the susceptible GT-1 clone. These findings highlight the promise of GURL-96 and GURL-107 as BCAs for managing rubber LFD. Both strains suppressed pathogen spore germination on rubber leaves and restricted mycelial growth through antibiosis mediated by diffusible metabolites and volatile compounds, indicating that direct inhibition contributes to their biocontrol performance. To our knowledge, this is the first report demonstrating the biocontrol potential of S. fortis and P. megaterium against rubber LFD.

Rice bakanae disease, caused by the fungal pathogen Fusarium fujikuroi, results in significant yield reductions in global rice production. Pefurazoate, one of the sterol 14α-demethylase inhibitors (DMIs), has been used to control rice bakanae disease. However, pefurazoate-resistant isolates have also been reported in Japan. The F490V substitution in FfCYP51A or S312T/F511S substitutions in FfCYP51B were found in field isolates with pefurazoate resistance. In the present study, we aimed to confirm the contribution of these amino acid substitutions to pefurazoate resistance; toward this, we created a V-type mutation at the 490th codon in FfCYP51A from an F-type isolate using a gene-editing technique. S/F- and T/S-types at the 312/511th codons in FfCYP51B, created from an S/S-type isolate in a previous study, were also used. A sensitivity test for pefurazoate indicated that F490V in FfCYP51A or S312T/F511S in FfCYP51B independently conferred pefurazoate resistance to F. fujikuroi. Notably, S312T/F511S in FfCYP51B was revealed to confer positive cross-resistance between prochloraz and pefurazoate. The increased expression of FfCYP51A and FfCYP51B in the pefurazoate-treated amino acid substitution mutants suggests the presence of a system regulating FfCYP51A and FfCYP51B expression in response to FfCYP51B activity in F. fujikuroi. These results demonstrate that F490V in FfCYP51A and S312T/F511S in FfCYP51B contribute to pefurazote resistance in F. fujikuroi, although additional mechanisms may also modulate fungicide sensitivity. We also developed the polymerase chain reaction-restriction fragment length polymorphism assay for F490V in FfCYP51A and S312T/F511S in FfCYP51B to manage DMI resistant isolates of F. fujikuroi.

The circular leaf disease, caused by Pestalotiopsis spp., is a new primary foliar disease characterized by yellow-brown lesions that develop into dark-brown lesions on rubber leaves. This disease, named leaf fall disease, has recently led to significant economic losses in countries that produce natural rubber. However, molecular mechanisms that are involved in the interaction between P. microspora and rubber tree (Hevea brasiliensis) remain unclear. In this study, we conducted transcriptome analysis using RNA-seq of a susceptible rubber clone (GT1) after an inoculation with the mycelial plug of P. microspora. Among 84,443 rubber genes, the analysis revealed 3,799, 7,274, and 4,678 differentially expressed genes (DEGs) at 2, 4, and 6 days post-infection (dpi), respectively. Our Gene Ontology analysis of the DEGs detected the GO terms of the general hypersensitive response and systemic acquired resistance, which is known to be mediated by salicylic acid and hydrogen peroxide. We also detected the GO terms related to abscisic acid and ethylene responses. In addition, the detection of these two phytohormone transcriptional responses suggests the possible involvement of abscisic acid and/or ethylene in the process that led to the leaf fall symptom in susceptible rubber. Further, our promoter analysis to extract putative promoter elements responsive to the pathogen supported ethylene signaling activation by the pathogen infection. In addition, we extracted general Regulatory Element Groups that are position-dependent cis-regulatory elements, and established molecular markers for quantitative reverse transcription polymerase chain reaction. Our study provides genomic information and convenient tools for facilitating further exploration of Hevea-Pestalotiopsis interaction.

The Lysinibacillus xylanilyticus strain GIC41 has been previously reported to promote spinach growth. This study evaluated GIC41's potential as a biostimulant by assessing its ability to mitigate Pythium root rot and enhance plant growth across various cultivation systems. In a pot experiment, GIC41 application to potting soil reduced the disease severity index (DSI) by approximately 52% in spinach seedlings 15 days post-pathogen inoculation (dpi). Similarly, introducing GIC41 into hydroponic nutrient solutions decreased the DSI in tomato seedlings from 61% to 15% at 14 dpi. Reisolation experiments and quantitative real-time PCR analysis confirmed that GIC41 significantly suppressed root colonization by Pythium aphanidermatum in both spinach and tomato seedlings. Microscopic analysis showed that GIC41 treatment inhibited pathogen mycelial colonization and caused morphological abnormalities in about 93% of encysted zoospores on the tomato rhizoplane. Although GIC41 exhibited no direct anti-oomycete activity in dual culture, it produced protease. Notably, GIC41 treatment significantly improved plant growth, increasing tomato shoot dry weight and stem diameter by 47% and 43%, respectively. These findings suggest that GIC41 is a promising biostimulant, offering dual benefits of disease mitigation and growth promotion across different crops and cultivation systems.

Phytophthora root and stem rot (PRSR) caused by Phytophthora sojae is a major concern for global soybean production. To identify a bacterial biocontrol agent against PRSR, 73 rhizobacterial strains were isolated from wild and cultivated legumes and screened for their protective activities against PRSR in pot experiments. Strain GVv1 was selected for its consistent protective effect through repeated pot experiments. The protective effect of this strain was similar to that of the fungicide mancozeb-metalaxyl. A dual-culture assay showed that GVv1 produced antifungal metabolites effective against P. sojae. To evaluate the potential adaptability of GVv1 to the soybean rhizosphere environment, its growth was exami-ned in soybean root exudates and nutrient medium, both supplemented with daidzein, an antimicrobial isoflavone secreted by soybean roots. GVv1 proliferated using soybean root exudates and had sufficient tolerance to daidzein to colonize the soybean rhizosphere. The plant growth-promoting effect of GVv1 on soybean plants was also investigated. GVv1 significantly increased shoot and root dry weights, indicating its plant growth-promoting activity. In vitro assays showed that GVv1 produced indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylate deaminase and solubilized insoluble phosphates. A taxonogenomic ana-lysis of the draft genome identified GVv1 as Enterobacter pseudoroggenkampii with high similarity (98.32% average nucleotide identity) to E. pseudoroggenkampii strain 155092T. To the best of our knowledge, this is the first study to report the biocontrol and plant growth-promoting activities of E. pseudoroggenkampii.

Gypsum (calcium sulfate) are commonly used to improve subsoil acidity. Aluminum (Al) toxicity in acid soil reduces crop yields worldwide, especially in the tropical regions. In soil, sulfate decrease Al toxicity and improves plant growth and yield. Here, we aimed to investigate the effects of CaSO4 on Al stress of Al-tolerant accession Col-0 as well as the Al-sensitive accessions, Wei-0 and Ts-5, of in Arabidopsis thaliana using hydroponics. Our research indicates that CaSO4 supply has a significant effect on root growth and malate release compared to CaCl2 under Al stress, especially in Al-sensitive accessions. In response to Al stress, CaSO4 supply enhanced the expression of malate transporter gene AtALMT1 and sulfate transporter gene SULTR3;5, which were regulated by the Al-resistant transcription factor STOP1. Furthermore, knockout lines of SULTR3;5 and SULTR2;1, which are involved in sulfate uptake and translocation, were more Al-sensitive than the wild type under Al stress without CaSO4, and malate release was reduced with decreased AtALMT1 expression. In conclusion, one of the alleviative effects of gypsum application on Al rhizotoxicity is promoted by sulfate through organic acid release mechanism enhancing AtALMT1 expression, alongside the Ca2+ reduction Al3+ activity on the plasma membrane.

BACKGROUNDCucumber Fusarium wilt (CFW), triggered by Fusarium oxysporum f. sp. cucumerinum, leads to substantial yield reductions in global cucumber (Cucumis sativus L.) production. Common management strategies for CFW include soil fumigation, grafting, and crop rotation. However, these methods have limitations regarding safety and efficacy stability, necessitating the development of new, cost-effective, and eco-friendly control strategies. Our prior research demonstrated that L-arabinose, an inexpensive and safe sugar commonly used in food and beverages, effectively suppressed bacterial wilt in tomatoes. This study explores the potential of L-arabinose in managing CFW and investigates its mechanism of action.RESULTSSoil applications of L-arabinose, ranging from 0.00001 to 0.01%, effectively suppressed CFW. The most significant suppressive effect was observed at 0.01%, reducing the disease severity index by 67.5% compared to the control treatment. Microscopic examination of transverse root sections showed that pathogen hyphae colonized the epidermis but seldom penetrated the cortical layer of roots in L-arabinose-treated seedlings. In contrast, the entire root tissue of control seedlings was colonized by the pathogen. Quantitative real-time PCR revealed a significant increase in the expression of defense-related genes dependent on salicylic acid, jasmonic acid, and ethylene in L-arabinose-treated plants compared to control plants, 6 and 10 days post pathogen inoculation.CONCLUSIONThis study demonstrated that soil application of L-arabinose can effectively suppress CFW by priming root tissues for multiple defense signaling pathways. Therefore, L-arabinose holds potential as a new fungicide for managing CFW. (c) 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

In recent years, the stable supply of natural rubber has been threatened by a new leaf fall disease (LFD) caused by filamentous fungi. We screened pesticides to control the growth of Neopestalotiopsis sp. and Colletotrichum sp., which are considered to be the causal agents of LFD in rubber trees. We identified two effective pesticides, Quinondo 80% WP and Topsin M WP. When these two candidate pesticides were used in combination at 10ppm each, there was enhanced inhibition of growth of both fungal species. Furthermore, the use of Quinondo 80% WP was shown to suppress the development of necrotic lesions caused by Neopestalotiopsis in rubber seedlings. These results suggest that Quinondo 80% WP is effective in controlling the spread of damage caused by LFD infection in rubber trees, and further verification of the concentration and method of application is needed to further demonstrate its effectiveness.

Prochloraz has been used to control Fusarium fujikuroi, the causative pathogen of rice bakanae disease. Linkage analysis of FfCYP51 genes in the progenies obtained from crossing prochloraz moderately resistant and sensitive strains suggested that the FfCYP51B gene is involved in prochloraz resistance. Sequence comparison revealed that the prochloraz-resistant strain had an F511S or S312T/F511S substitution in FfCYP51B compared with the sensitive strains. The contribution of the S312T and F511S substitutions in FfCYP51B to prochloraz resistance was investigated by creating S/F-, T/F-, or T/S- types at 312/511 codons from the S/S-type, which is a natural moderately resistant strain, using a gene-editing technique. T/S exhibited the highest prochloraz resistance, followed by S/S-, T/F-, and S/F-types. These results indicated that the S312T and F511S substitutions in FfCYP51B had a synergistic effect on prochloraz resistance in F. fujikuroi.

Streptomyces sp. strain MBCN152-1, isolated from cabbage, has potential as a biocontrol agent for Alternaria brassicicola on cabbage seedlings. The present study examined its mode of action. Light microscopy showed that appressorium formation by A. brassicicola was significantly suppressed on cabbage seedlings bacterized with MBCN152-1. Furthermore, scanning electron microscopy revealed that the mycelia of MBCN152-1, which were epiphytically growing on the cotyledon leaves of cabbage seedlings, intensively coiled around the germinating conidia of A. brassicicola. In vitro co-culture experiments demonstrated that MBCN152-1 is an aggressive mycoparasite of A. brassicicola, but not of A. brassicae or Colletotrichum higginsianum. Biocontrol experiments indicated that MBCN152-1 did not control diseases caused by A. brassicae or C. higginsianum. These results suggest that mycoparasitism is the primary mode of action for MBCN152-1. This is the first study to clearly demonstrate the significance of mycoparasitism in the biocontrol efficacy of endophytic Streptomyces.

Fusarium fujikuroi is the pathogen of rice bakanae disease and is subclassified into gibberellin and fumonisin groups (G and F groups). Thiophanate-methyl (TM), a benzimidazole fungicide, has been used extensively to control F. fujikuroi. Previous investigation showed that F-group strains are TM sensitive (TMS), whereas most G-group strains are TM resistant (TMR) in Japan. The minimum inhibitory concentration in TMS strains was 1 to 10 μg ml-1, whereas that in TMR strains was >100 μg ml-1. E198K and F200Y mutations in β2-tubulin were detected in TMR strains. A loop-mediated isothermal amplification-fluorescent loop primer method was developed for diagnosis of these mutations and applied to 37 TMR strains and 56 TMS strains. The results indicated that 100% of TMR strains were identified as having either the E198K mutation (41%) or the F200Y mutation (59%), whereas none of the TMS strains tested showed either mutation. We found one remarkable TMR strain in the F group that had an F200Y mutation. These results suggest that E198K and F200Y mutations in β2-tubulin contribute to TM resistance in F. fujikuroi.

Methods for increasing soil suppressiveness via soil microflora manipulation have long been explored as an ideal strategy to protect plants from soilborne pathogens. However, viable methods offering consistent disease control effects have not yet been developed.

Members of the Fusarium graminearum species complex are known to cause head blight of wheat. A natural nonpathogenic mutant, strain Fa0233007, was found in the strain collection of the Japanese F. graminearum species complex. In addition, this strain cannot form perithecia. Complete linkage of these deficiencies in the progenies obtained from crossing a wild-type strain and the mutant strain suggested that a single genomic locus was responsible for deficiencies in both traits. The locus was mapped to chromosome 1 using sequence-tagged markers. An NADPH oxidase gene (NoxA, FGSG00739) was identified in the mapped region, and a 2-bp deletion that results in premature termination of the open reading frame was detected in the gene. Both pathogenicity and perithecium formation were recovered by FGSG00739 complementation in the mutant strain.

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay.

BACKGROUND Induced resistance against several plant pathogens was reported using different beneficial plant growth-promoting microorganisms. The potential of five fungal isolates, Trichoderma harzianum GT 3-2, Fusarium equiseti GF 18-3, F. equiseti GF 19-1, Phoma sp. GS 10-1 and Phoma sp. GS 14-1, to stimulate tomato growth and resistance against bacterial speck disease caused by Pseudomonas syringae pathovar (pv.) tomato DC3000 was evaluated. RESULTS Based on the results of disease severity and growth promotion experiments, GF 18-3 exhibited the best results among all fungal isolates. Treatment with barley grain inocula (BGI) and culture filtrate (CF) of the isolates promoted tomato growth and suppressed the pathogen in pot trials. Furthermore, expressions of the pathogenesis-related genes (PR-1, beta-1,3-glucanase A, beta-1,3-glucanase B and LOX) were relatively higher than the control in the leaves of tomato plants treated with both BGI and CF. The transcription levels remained consistently higher than the control plants for 6 days post-inoculation with pathogen. CONCLUSION Taken together, the results indicate that the tested fungal isolates have the potential to promote tomato growth and induce systemic resistance against the bacterial speck disease. Analysis of certain PR gene expression revealed significant activation in both BGI and CF treatments, leading to stimulated resistance against the pathogen.

The use of synthetic chemicals has increased drastically due to industrialization and urbanization. However, the long-term and indiscriminate use of these chemicals has a negative impact on environment and human health; thus, public concerns about the hazardous effects of such synthetic chemicals are increasing day by day. To solve these problems, the exploitation of potential alternatives has become a major challenge, and the admiration of beneficial microbes is increasing due to their safe and environment-friendly nature. Microbes can mitigate the hazardous effects of synthetic chemicals by reducing their use and toxicity. Lysinibacillus species are gram-positive, spore-forming, motile bacteria. This genus was previously designated as Bacillus spp. under the family Bacillaceae of the phylum Firmicutes. For a long period of time, Lysinibacillus is well-known for its insecticidal activity against various insects, including mosquitoes, which are the vector of several human diseases. In addition, some Lysinibacillus species have a potential for heavy metal remediation. In recent years, Lysinibacillus spp. are attracting the researchers’ attention as plant growth-promoting and disease control agents, which would be used as alternatives to agrochemicals. This study gives an overview of the entomopathogenic, bioremediation, plant growth-promoting, and biological disease control abilities of the genus Lysinibacillus.

Mitsuaria sp. TWR114 is a biocontrol agent against tomato bacterial wilt (TBW). We aimed to gain genomic insights relevant to the biocontrol mechanisms and colonization ability of this strain. The draft genome size was found to be 5,632,523 bp, with a GC content of 69.5%, assembled into 1144 scaffolds. Genome annotation predicted a total of 4675 protein coding sequences (CDSs), 914 pseudogenes, 49 transfer RNAs, 3 noncoding RNAs, and 2 ribosomal RNAs. Genome analysis identified multiple CDSs associated with various pathways for the metabolism and transport of amino acids and carbohydrates, motility and chemotactic capacities, protection against stresses (oxidative, antibiotic, and phage), production of secondary metabolites, peptidases, quorum-quenching enzymes, and indole-3-acetic acid, as well as protein secretion systems and their related appendages. The genome resource will extend our understanding of the genomic features related to TWR114's biocontrol and colonization abilities and facilitate its development as a new biopesticide against TBW.