Characterization of Fusarium species causing soybean root rot in Heilongjiang, China, and mechanism underlying the differences in sensitivity to DMI fungicides.

Soybean root rot is a worldwide soil-borne disease threatening soybean production, causing large losses in soybean yield and quality. Fusarium species are the most detrimental pathogens of soybean root rot worldwide, causing large production losses. Fusarium root rot has been frequently reported in Heilongjiang Province of China, but the predominant Fusarium species and the sensitivity of these pathogens to different fungicides remain unclear. In this study, diseased soybean roots were collected from 14 regions of Heilongjiang province in 2021 and 2022. A total of 144 isolates of Fusarium spp. were isolated and identified as seven distinct species: F. scirpi, F. oxysporum, F. graminearum, F. clavum, F. acuminatum, F. avenaceum, and F. sporotrichioide. F. scirpi and F. oxysporum had high separation frequency and strong pathogenicity. The sensitivity of Fusarium spp. to five different fungicides was determined. Mefentrifluconazole and fludioxonil showed good inhibitory effects, and the sensitivity to pydiflumetofen and phenamacril varied between Fusarium species. In particular, the activity of DMI fungicide prothioconazole was lower than that of mefentrifluconazole. Molecular docking showed that mefentrifluconazole mainly bound to CYP51C, but prothioconazole mainly bound to CYP51B. Furthermore, the sensitivity to prothioconazole only significantly decreased in ΔFgCYP51B mutant, and the sensitivity to mefentrifluconazole changed in ΔFgCYP51C and ΔFgCYP51A mutants. The results demonstrated that the predominant Fusarium species causing soybean root rot in Heilongjiang province were F. scirpi and F. oxysporum and DMI fungicides had differences in binding cavity due to the diversity of CYP51 proteins in Fusarium.

First report of soybean root rot caused by Fusarium proliferatum in the Republic of Korea.

Fusarium species are widespread soilborne pathogens that can cause damping-off, root rot, and wilting in soybean [Glycine max (L.) Merrill], subsequently leading to significant yield suppression. Several Fusarium spp. have already been documented for their pathogenicity on soybean plants in the Republic of Korea. The nationwide monitoring of soybean diseases continues to identify new pathogenic Fusarium spp. In 2016, five plant samples at R3-R4 growth stages, showing symptoms of wilting in the upper parts and root rot, were collected in Suwon, Gyeonggi, Republic of Korea. Fungal colonies were obtained from the diseased root samples, with the surface sterilized in 1% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, and placed on water agar at 25°C. Five isolates were collected and purified by single-spore isolation. The fungal mycelium was subsequently cultivated on potato dextrose agar for ten days. The isolates produced abundant, aerial, and white mycelium and became purple in old cultures. Macroconidia were slender, falcate to almost straight, usually 3 to 5 septated, and thin-walled. Microconidia were formed in chains from polyphalides, clavate or oval, usually single-celled with a flattened base. These characteristics of isolates were consistent with the description of F. proliferatum (Leslie and Summerrell 2006), and the representative isolate 16-19 was selected for molecular identification to confirm its identity as F. proliferatum. Two evolutionarily conserved genes, the translation elongation factor 1-alpha (EF-1α) and the second-largest subunit of RNA polymerase II (RPB2) genes, were partially amplified using the primers described by O'Donnell et al. (2008), resulting in nucleotide sequences of 680 and 382 base pairs, respectively. These two sequences (GenBank accession numbers: OQ992720 and OR060666) showed 100 and 99.5% identity to the EF-1α and RPB2 of F. proliferatum A40 (GenBank accession numbers: KP964907 and KP964842). For the Petri-dish pathogenicity assay (Broders et al. 2007), five surface-sterilized seeds were placed on water agar media with either sterile water or actively growing '16-19' culture. After 7 days of incubation in a growth chamber (25°C; 12-hour photoperiod), brown lesions were observed on the roots of the inoculated plants, while no symptoms were observed in the sterile water-treated controls. The experiment was conducted three times. For root-cut pathogenicity assay, conidial suspension (1×106 conidia/ml) of the isolate '16-19' was prepared with harvested mycelia cultured on PDA for 10 days with sterile water. The roots of 10-day-old soybean seedlings were partially cut and soaked in either the suspension or sterile water for 2 hours. The seedlings were transplanted into 12 cm plastic pots (11 cm in height) and grew in a greenhouse (26 ± 3°C, 13-h photoperiod). The experiment followed a completely randomized design with three replicates (i.e. three plants in a pot), and it was repeated twice. The inoculated plants began to wilt 7 days after inoculation, while the sterile water-treated controls remained healthy. Ten days after inoculation, all plants were collected, washed under running tap water, and evaluated for the presence and severity of root rot using a 0-4 scale (Chang et al. 2015). The inoculated plants exhibited reduced vigor and developed dark brown lesions on their roots. F. proliferatum was reisolated from symptomatic root tissues of the infected plants, while not from those of the controls. Its colony and spores were morphologically identical to those of the original isolate. F. proliferatum was previously reported as a causative agent of soybean root rot in the United States (Díaz Arias et al. 2011) and Canada (Chang et al. 2015). This is the first report of soybean root rot caused by F. proliferatum in the Republic of Korea. This finding implies that F. proliferatum may potentially threaten soybean production in the Republic of Korea and suggests that effective disease management strategies should be established for soybean protection against the disease, along with continuous surveillance.

First report of Tobacco Fusarium Root Rot Caused by Fusarium meridionale in China.

Tobacco (Nicotiana tabacum L.) was an important economic crop in China. A survey in Yunnan Province in the last several years showed that the incidence of tobacco root rot was 3 to 30%. In July 2021, root rot symptoms were observed with an average incidence of 5% on tobacco (cultivar Yunyan 87) in Dali (25.61° N, 100.27° E). Typical disease symptoms included plants stunted at early stages, brown-colored withering lower leaves and roots that became brown. Under high humidity conditions, symptoms of rot expanded in the roots, also the whole plant became wilted and stunted, and some plants ultimately died. Infected pieces of stem tissues and root were dissected and then sterilized with 2% NaOCl for 30 s, rinsed three times with sterile distilled water, and dried with sterilized filter paper. Three pieces were plated onto potato dextrose agar (PDA) for 3 days at 25°C with a 12-h light period. Colonies on PDA were characterized by white to pale yellow flocculent aerial mycelium, and a pink to red pigment in the agar. To induce sporulation, mycelium on PDA was transferred to carnation leaf agar (CLA) medium. After incubation for 7 days, a single spore was isolated from representative isolate 21DL16 for morphological and molecular analyses. Macroconidia observed on CLA were falcate, slightly curved, three to five septate, measured 33.1 to 53.7 × 3.2 to 4.6 µm (n=50), with a typical foot shaped basal cell. Morphological characteristics of the fungus were in agreement with the description of Fusarium graminearum (Leslie and Summerell 2006). For further identification, the internal transcribed spacer (ITS) region rDNA, translation elongation factor 1ɑ (EF-1α) and RNA polymerase II second largest subunit (RPB2) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell et al. 2015) and RPB2-5F/RPB2-7cR (Reeb et al. 2004), respectively. Although the ITS sequence (GenBank accession no. OM392025) cannot distinguish F. meridionale from F. graminearum, combined phylogenetic analysis of the sequence of TEF1 (ON062055) and RPB2 (ON211932) clearly showed that the pathogen is F. meridionale that the sequences were 100% similarity, 0.0e-value and 100% query coverage to F. meridionale. Pathogenicity studies were conducted on six-leaf-stage tobacco seedlings cultivar Yunyan 87. A conidial suspension (1×105 spores/mL) was poured over the roots of tobacco seedlings. Three seedlings were treated with sterile water that served as controls. All 10 seedlings were maintained at 25°C at 70% relative humidity. After 5 days, the lower leaves showed symptoms of wilting and the roots of all inoculated seedlings become discolored, that were similar with the original symptoms, whereas the control seedlings did not develop symptoms. The fungus reisolated from the inoculated seedlings was identical to F. meridionale using the EF-1α gene sequence. To date, Fusarium root rot on tobacco in China was caused by F. oxysporium (Chen 2013). However, to the best of our knowledge, this is the first report of F. meridionale causing root rot on tobacco in China. Identification of F. meridionale as a root rot agent might provide important insight for disease management practices on tobacco caused by Fusarium species.

First Report of Fusarium Root Rot of Tobacco Caused by Fusarium fujikuroi in Guangdong Province, China.

From March to June 2022, Fusarium tobacco root rot broke out in Shaoguan Guangdong Province, China, affecting approximately 15% of tobacco production fields, with an incidence of 24% to 66%. In the early stage, the lower leaves showed chlorosis, and the roots became black. In the later stage, the leaves became browned and withered, the root cortices were broken and shed, and only a small number of roots were left. Eventually, the entire plant died. Six diseased plant samples (cv. yueyan 97) from Shaoguan (113.8°E, 24.8°N) were collected as test materials. The diseased root tissues (4×4 mm) were surface-sterilized using 75% ethanol for 30 s and 2% NaOCl for 10 min, rinsed 3 times with sterile water and incubated for 4 days on potato dextrose agar (PDA) medium at 25 °C. Fungal colonies were subcultured on fresh PDA, grown for the next 5 d and purified by single-spore separation. Eleven isolates with similar morphological characteristics were obtained. Their colonies were white and fluffy, and the bottoms of the culture plates were pale pink after 5 days of incubation. The macroconidia were slender, slightly curved and measured 18.54~45.85 µm×2.35~3.84 µm (n=50), with 3 to 5 septa. The microconidia were oval or spindle shaped, with one to two cells, and measured 5.56~16.76 µm×2.32~3.86 µm (n=50). Chlamydospores were absent. Such characteristics are typical of the genus Fusarium (Booth C, 1971). The SGF36 isolate was chosen for further molecular analysis. The TEF-1α and ß-tubulin genes (Pedrozo et al.2015) were amplified. Based on a phylogenetic tree (neighbor-joining method and 1,000 bootstrap values) obtained using multiplex alignments of concatenations of these two genes from 18 Fusarium species, SGF36 was grouped into a clade with Fusarium fujikuroi strain 12-1 (MK443268.1/MK443267.1) and F. fujikuroi isolate BJ-1 (MH263736.1/MH263737.1). To further identity the isolate, five additional gene sequences (rDNA-ITS (OP862807.1), RPB2, histone 3, calmodulin, and mitochondrial small subunit) (Pedrozo et al.2015), were subjected to BLAST searches in GenBank, and the results indicated that they were most similar to F. fujikuroi sequences, with sequence identities greater than 99%. The phylogenetic tree obtained using six genes except mitochondrial small subunit gene showed that SGF36 was grouped together with four F. fujikuroi strains to form a single clade. Pathogenicity was determined by the inoculation of wheat grains with fungi in potted tobacco plants. The SGF36 isolate was inoculated onto sterilized wheat grains, which were then incubated at 25 °C for 7 d. Thirty wheat grains with fungi were added to 200 g of sterilized soil, which was then mixed well and placed into pots. One six-leaf-stage tobacco seedling (cv. yueyan 97) was planted in each pot. A total of 20 tobacco seedlings were treated. Another 20 control seedlings were treated with wheat grains without fungi. All seedlings were placed in a greenhouse at 25 °C with 90% relative humidity. After 5 d, the leaves of all inoculated seedlings showed chlorosis, and the roots became discolored. No symptoms were observed in the controls. The fungus was reisolated from symptomatic roots and confirmed to be F. fujikuroi based on the TEF-1α gene sequence. No F. fujikuroi isolates were recovered from control plants. F. fujikuroi was previously reported to be associated with rice bakanae disease (Ram et al., 2018), soybean root rot (Zhao et al., 2020) and cotton seedling wilt (Zhu et al., 2020). To our knowledge, this is the first report of F. fujikuroi causing root wilt on tobacco in China. The identification of the pathogen may help to establish appropriate measures for controlling this disease.

Effects of Soil Type, Temperature, and Moisture on Development of Fusarium Root Rot of Soybean by Fusarium solani (FSSC 11) and Fusarium tricinctum.

Fusarium solani FSSC 11 and F. tricinctum are important root rot pathogens of soybean in North Dakota. The roles of soil type, temperature, and moisture in disease development by both species are poorly documented. To assess the effect of soil type on disease, three types of soil (Glyndon sandy loam, La Prairie silt loam, and Fargo clay) that represent soils of the soybean production region in the Red River Valley were examined in greenhouse, microplot, and growth chamber studies. Disease incidence and lesion length on roots were evaluated at growth stages V3 and R6. Soil type significantly affected disease development, with higher severity in the lighter soils of Glyndon sandy loam and La Prairie silt loam compared with Fargo clay. Soil type also interacted with Fusarium species, in which the maximum severity was observed in Glyndon sandy loam for F. solani, and in La Prairie silt loam for F. tricinctum. In addition, the cumulative effects of soil type, temperature, and soil moisture were tested in a growth chamber. Emergence and disease on seedlings were evaluated at growth stage V3. Significant reductions in emergence occurred at 10°C in treatments with F. solani and F. tricinctum, but there was no significant difference among the three soils. Infection was visible at temperatures of 10 to 20°C for F. solani and 15 to 20°C for F. tricinctum. F. solani caused the greatest infection at 20°C in Glyndon sandy loam, while it was at 15°C in La Prairie silt loam for F. tricinctum. The isolates of the two Fusarium species caused root rot in soil moisture ranging from 20 to 100% water holding capacity (WHC). The greatest reduction in emergence caused by the Fusarium spp. was observed at 80% WHC in silt loam and clay soils and 40% WHC in sandy loam soil, when compared with the same WHC in noninfested soils. Ranges of soil moisture causing infection were negatively correlated with temperature. At the lower temperature there was a broader range of soil moistures resulting in infection compared with higher temperatures. At 18°C, most infection occurred at soil moistures of 20 to 80% WHC, while it was 40 to 80% WHC at 28°C. Disease caused by F. solani was favored by a temperature of 18°C with high soil moisture (60 to 80% WHC) or 28°C with low soil moisture (20 to 40% WHC), while F. tricinctum was favored by cooler temperature and lower soil moisture.

Application of vermicompost and biochar suppresses Fusarium root rot of replanted American ginseng.

Soil sterilization integrated with agronomic measures is an effective method to reduce soilborne replant diseases. However, the effect of vermicompost or biochar application after soil sterilization on soilborne diseases is poorly understood. A pot experiment was conducted in American ginseng to investigate the effects of vermicompost (VF), biochar (BF), and a combination of vermicompost and biochar (VBF) applied after soil sterilization on the incidence of Fusarium root rot using natural recovery (F) as control. After one growing season, the disease index of root rot, the phenolic acids, and the microbial communities of American ginseng rhizosphere soil were analyzed. The disease index of VF, BF, and VBF decreased by 33.32%, 19.03%, and 80.96%, respectively, compared with F. The highest bacterial richness and diversity were observed in the rhizosphere soil of VBF. Besides, VF and VBF significantly increased the relative abundance of beneficial bacteria (Pseudomonas, Lysobacter, and Chryseolinea) in the rhizosphere soil. Higher concentrations of vanillin, one of the phenolic acids in the roots exudates, were recorded in the rhizosphere soils of BF and VBF. The vanillin concentration showed a significant negative correlation with the disease index. To conclude, vermicompost improved the beneficial bacteria of the rhizosphere soil, while biochar regulated the allelopathic effect of the phenolic acids. The study proposes a combined application of biochar and vermicompost to the rhizosphere soil to control Fusarium root rot of replanted American ginseng effectively. KEY POINTS: Vermicompost improves the relative abundance of rhizosphere beneficial bacteria. Biochar inhibits the degradation of phenolic acids by adsorption. The combination of vermicompost and biochar enhances the disease control effect.

Fusarium spp. Associated With Root Rot of Pulse Crops and Their Cross-Pathogenicity to Cereal Crops in Montana.

Root rot caused by Fusarium species is a major problem in the pulse growing regions of Montana. Fusarium isolates (n = 112) were obtained from seeds and roots of chickpea, dry pea, and lentil. Isolates were identified by comparing the sequences of the internal transcribed spacer region and the translation elongation factor 1-α in Fusarium-ID database. Fusarium avenaceum was the most abundant species (28%), followed by F. acuminatum (21%), F. poae (13%), F. oxysporum (8%), F. culmorum (6%), F. redolens (6%), F. sporotrichioides (6%), F. solani (4%), F. graminearum (2%), F. torulosum (2%), and F. tricinctum (0.9%). The aggressiveness of a subset of 50 isolates that represent various sources of isolation was tested on three pulse crops and two cereal crops. Nonparametric analysis of variance conducted on ranks of disease severity indicated that F. avenaceum and F. solani isolates were highly aggressive on pea and chickpea. In lentil, F. avenaceum and F. culmorum were highly aggressive. In barley, F. avenaceum, F. solani, F. culmorum, and F. graminearum were highly aggressive. In wheat, F. avenaceum, F. graminearum, and F. culmorum were highly aggressive. Two F. avenaceum isolates were highly aggressive across all the crops tested and found to be cross-pathogenic. One isolate of F. culmorum and an isolate of F. graminearum obtained from chickpea and lentil seed were highly aggressive on barley and wheat. The results indicate that multiple Fusarium spp. from seeds and roots can cause root rot on both pulse and cereal crops. Rotating these crops may still lead to an increase in inoculum levels, making crop rotation limited in efficacy as a disease management strategy.

Fusarium graminearum KP4-like proteins possess root growth-inhibiting activity against wheat and potentially contribute to fungal virulence in seedling rot.

The virally encoded KP4 killer toxin protein was first identified from Ustilago maydis (Um), and its homologues are present in diverse fungi and in one species of moss. No KP4-like (KP4L) proteins have been functionally characterized. Here, we report the identification and functional analysis of four KP4L proteins from Fusarium graminearum (Fg), the primary causal pathogen of Fusarium head blight (FHB), which is also known to associate with seedling rot of wheat. The four FgKP4L proteins (FgKP4L-1, -2, -3 and -4) are encoded by small open reading frames (378-825 bp) located on chromosome 1 with the FgKP4L-1, -2 and -3 genes clustering together. Sequence analysis indicated that FgKP4L proteins have conserved domains predicted to form a three-dimensional alpha/beta-sandwich structure as first reported for UmKP4, with FgKP4L-4 featuring double Kp4 domains. Further analyses revealed that the FgKP4L genes are expressed in vitro under certain stress conditions, and all up-regulated during FHB and/or seedling rot development, the recombinant FgKP4L-2 protein does not induce cell death in wheat leaves or spikelets, but inhibits root growth of young seedlings, and the elimination of the FgKP4L-1/-2/-3 gene cluster from the fungal genome results in reduced virulence in seedling rot but not in FHB. Database searches revealed KP4L proteins from ∼80 fungal species with more than half from human/animal pathogens. Phylogenetic analysis suggested that UmKP4 and the moss KP4L proteins are closely related to those from a zygromycete and Aspergillus, respectively, implying cross-kingdom horizontal gene transfer.

Pseudomonas chlororaphis IRHB3 assemblies beneficial microbes and activates JA-mediated resistance to promote nutrient utilization and inhibit pathogen attack.

Introduction: The rhizosphere microbiome is critical to plant health and resistance. PGPR are well known as plant-beneficial bacteria and generally regulate nutrient utilization as well as plant responses to environmental stimuli. In our previous work, one typical PGPR strain, Pseudomonas chlororaphis IRHB3, isolated from the soybean rhizosphere, had positive impacts on soil-borne disease suppression and growth promotion in the greenhouse, but its biocontrol mechanism and application in the field are not unclear. Methods: In the current study, IRHB3 was introduced into field soil, and its effects on the local rhizosphere microbiome, disease resistance, and soybean growth were comprehensively analyzed through high-throughput sequencing and physiological and molecular methods. Results and discussion: We found that IRHB3 significantly increased the richness of the bacterial community but not the structure of the soybean rhizosphere. Functional bacteria related to phosphorus solubilization and nitrogen fixation, such as Geobacter, Geomonas, Candidatus Solibacter, Occallatibacter, and Candidatus Koribacter, were recruited in rich abundance by IRHB3 to the soybean rhizosphere as compared to those without IRHB3. In addition, the IRHB3 supplement obviously maintained the homeostasis of the rhizosphere microbiome that was disturbed by F. oxysporum, resulting in a lower disease index of root rot when compared with F. oxysporum. Furthermore, JA-mediated induced resistance was rapidly activated by IRHB3 following PDF1.2 and LOX2 expression, and meanwhile, a set of nodulation genes, GmENOD40b, GmNIN-2b, and GmRIC1, were also considerably induced by IRHB3 to improve nitrogen fixation ability and promote soybean yield, even when plants were infected by F. oxysporum. Thus, IRHB3 tends to synergistically interact with local rhizosphere microbes to promote host growth and induce host resistance in the field.

Biological potential of Bacillus subtilis BS45 to inhibit the growth of Fusarium graminearum through oxidative damage and perturbing related protein synthesis.

Fusarium root rot (FRR) caused by Fusarium graminearum poses a threat to global food security. Biological control is a promising control strategy for FRR. In this study, antagonistic bacteria were obtained using an in-vitro dual culture bioassay with F. graminearum. Molecular identification of the bacteria based on the 16S rDNA gene and whole genome revealed that the species belonged to the genus Bacillus. We evaluated the strain BS45 for its mechanism against phytopathogenic fungi and its biocontrol potential against FRR caused by F. graminearum. A methanol extract of BS45 caused swelling of the hyphal cells and the inhibition of conidial germination. The cell membrane was damaged and the macromolecular material leaked out of cells. In addition, the mycelial reactive oxygen species level increased, mitochondrial membrane potential decreased, oxidative stress-related gene expression level increased and oxygen-scavenging enzyme activity changed. In conclusion, the methanol extract of BS45 induced hyphal cell death through oxidative damage. A transcriptome analysis showed that differentially expressed genes were significantly enriched in ribosome function and various amino acid transport pathways, and the protein contents in cells were affected by the methanol extract of BS45, indicating that it interfered with mycelial protein synthesis. In terms of biocontrol capacity, the biomass of wheat seedlings treated with the bacteria increased, and the BS45 strain significantly inhibited the incidence of FRR disease in greenhouse tests. Therefore, strain BS45 and its metabolites are promising candidates for the biological control of F. graminearum and its related root rot diseases.

Genome-wide association study to identify novel loci and genes for Fusarium root rot resistance in sweet potato using genotyping-by-sequencing.

Fusarium root rot, caused by Fusarium solani, is a major post-harvest disease in sweet potatoes (Ipomoea batatas (L.) Lam.). An effective strategy for controlling this disease is the development of resistant varieties. In this study, a genome-wide association study (GWAS) was conducted on 96 sweet potato genotypes to identify novel candidate loci and dissect the genetic basis of Fusarium root rot resistance. Genotyping was performed using genotyping-by-sequencing (GBS), and 44,255 SNPs were identified after filtering. The genotypes (n = 96) were evaluated through resistance tests in 2021 and 2022, separately and combined. The GWAS identified two significant SNP markers (LG3_22903756 and LG4_2449919) on chromosomes 3 and 4 associated with Fusarium root rot resistance, respectively. Lesion length showed significant differences between homozygous A and G alleles of LG3_22903756, which can potentially be used to develop molecular markers for selecting accessions resistant to Fusarium root rot. Expression analysis of 11 putative genes flanking the significant SNPs revealed the alteration in the expression of nine genes, indicating their possible involvement in Fusarium root rot resistance. The results of this study will aid in the marker-assisted selection and functional analysis of candidate genes for Fusarium root rot resistance in sweet potatoes.

Agricultural Jiaosu: An Eco-Friendly and Cost-Effective Control Strategy for Suppressing Fusarium Root Rot Disease in Astragalus membranaceus.

Root rot caused by the pathogenic fungi of the Fusarium genus poses a great threat to the yield and quality of medicinal plants. The application of Agricultural Jiaosu (AJ), which contains beneficial microbes and metabolites, represents a promising disease control strategy. However, the action-effect of AJ on Fusarium root rot disease remains unclear. In the present study, we evaluated the characteristics and antifungal activity of AJ fermented using waste leaves and stems of medicinal plants, and elucidated the mechanisms of AJ action by quantitative real-time PCR and redundancy analysis. The effects of AJ and antagonistic microbes isolated from it on disease suppression were further validated through a pot experiment. Our results indicate that the AJ was rich in beneficial microorganisms (Bacillus, Pseudomonas, and Lactobacillus), organic acids (acetic, formic, and butyric acids) and volatile organic compounds (alcohols and esters). It could effectively inhibit Fusarium oxysporum and the half-maximal inhibitory concentration (IC50) was 13.64%. The antifungal contribution rate of the microbial components of AJ reached 46.48%. Notably, the redundancy analysis revealed that the Bacillus and Pseudomonas genera occupied the main niche during the whole inhibition process. Moreover, the abundance of the Bacillus, Pseudomonas, and Lactobacillus genera were positively correlated with the pH-value, lactic, formic and butyric acids. The results showed that the combined effects of beneficial microbes and organic acid metabolites increased the efficacy of the AJ antifungal activity. The isolation and identification of AJ's antagonistic microbes detected 47 isolates that exhibited antagonistic activities against F. oxysporum in vitro. In particular, Bacillus subtilis and Bacillus velezensis presented the strongest antifungal activity. In the pot experiment, the application of AJ and these two Bacillus species significantly reduced the disease incidence of Fusarium root rot and promoted the growth of Astragalus. The present study provides a cost-effective method to control of Fusarium root rot disease, and establishes a whole-plant recycling pattern to promote the sustainable development of medicinal plant cultivation.

Resistance Induction by Salicylic Acid Formulation in Cassava Plant against Fusarium solani.

Fusarium root rot caused by the soil-borne fungus Fusarium solani is one of the most important fungal diseases of cassava in Thailand, resulting in high yield losses of more than 80%. This study aimed to investigate if the exogenous application of salicylic acid formulations (Zacha) can induce resistance in cassava against Fusarium root rot and observe the biochemical changes in induced cassava leaf tissues through synchrotron radiation based on Fourier-transform infrared (SR-FTIR) microspectroscopy. We demonstrated that the application of Zacha11 prototype formulations could induce resistance against Fusarium root rot in cassava. The in vitro experimental results showed that Zacha11 prototype formulations inhibited the growth of F. solani at approximately 34.83%. Furthermore, a significant reduction in the disease severity of Fusarium root rot disease at 60 days after challenge inoculation was observed in cassava plants treated with Zacha11 at a concentration of 500 ppm (9.0%). Population densities of F. solani were determined at 7 days after inoculation. Treatment of the Zacha11 at a concentration of 500 ppm resulted in reduced populations compared with the distilled water control and differences among treatment means at each assay date. Moreover, the SR-FTIR spectral changes of Zacha11-treated epidermal tissues of leaves had higher integral areas of lipids, lignins, and pectins (1,770-1,700/cm), amide I (1,700-1,600/cm), amide II (1,600-1,500/cm), hemicellulose, lignin (1,300-1,200/cm), and cellulose (1,155/cm). Therefore, alteration in defensive carbohydrates, lipids, and proteins contributed to generate barriers against Fusarium invasion in cassava roots, leading to lower the root rot disease severity.

Transcriptome Analysis of Fusarium Root-Rot-Resistant and -Susceptible Alfalfa (Medicago sativa L.) Plants during Plant-Pathogen Interactions.

Alfalfa (Medicago sativa L.) is a perennial leguminous forage cultivated globally. Fusarium spp.-induced root rot is a chronic and devastating disease affecting alfalfa that occurs in most production fields. Studying the disease resistance regulatory network and investigating the key genes involved in plant-pathogen resistance can provide vital information for breeding alfalfa that are resistant to Fusarium spp. In this study, a resistant and susceptible clonal line of alfalfa was inoculated with Fusarium proliferatum L1 and sampled at 24 h, 48 h, 72 h, and 7 d post-inoculation for RNA-seq analysis. Among the differentially expressed genes (DEGs) detected between the two clonal lines at the four time points after inoculation, approximately 81.8% were detected at 24 h and 7 d after inoculation. Many DEGs in the two inoculated clonal lines participated in PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) mechanisms. In addition, transcription factor families such as bHLH, SBP, AP2, WRKY, and MYB were detected in response to infection. These results are an important supplement to the few existing studies on the resistance regulatory network of alfalfa against Fusarium root rot and will help to understand the evolution of host-pathogen interactions.

Changes in the Density and Composition of Rhizosphere Pathogenic Fusarium and Beneficial Trichoderma Contributing to Reduced Root Rot of Intercropped Soybean.

The dynamic of soil-borne disease is closely related to the rhizosphere microbial communities. Maize-soybean relay strip intercropping has been shown to significantly control the type of soybean root rot that tends to occur in monoculture. However, it is still unknown whether the rhizosphere microbial community participates in the regulation of intercropped soybean root rot. In this study, rhizosphere Fusarium and Trichoderma communities were compared in either healthy or root-rotted rhizosphere soil from monocultured and intercropped soybean, and our results showed the abundance of rhizosphere Fusarium in intercropping was remarkably different from monoculture. Of four species identified, F. oxysporum was the most aggressive and more frequently isolated in diseased soil of monoculture. In contrast, Trichoderma was largely accumulated in healthy rhizosphere soil of intercropping rather than monoculture. T. harzianum dramatically increased in the rhizosphere of intercropping, while T. virens and T. afroharzianum also exhibited distinct isolation frequency. For the antagonism test in vitro, Trichoderma strains had antagonistic effects on F. oxysporum with the percentage of mycelial inhibition ranging from 50.59-92.94%, and they displayed good mycoparasitic abilities against F. oxysporum through coiling around and entering into the hyphae, expanding along the cell-cell lumen and even dissolving cell walls of the target fungus. These results indicate maize-soybean relay strip intercropping significantly increases the density and composition proportion of beneficial Trichoderma to antagonize the pathogenic Fusarium species in rhizosphere, thus potentially contributing to the suppression of soybean root rot under the intercropping.

Decoding the Plant Growth Promotion and Antagonistic Potential of Bacterial Endophytes From Ocimum sanctum Linn. Against Root Rot Pathogen Fusarium oxysporum in Pisum sativum.

The present study demonstrates plant growth promotion and induction of systemic resistance in pea (Pisum sativum) plant against Fusarium oxysporum f.sp. pisi by two bacterial endophytes, Pseudomonas aeruginosa OS_12 and Aneurinibacillus aneurinilyticus OS_25 isolated from leaves of Ocimum sanctum Linn. The endophytes were evaluated for their antagonistic potential against three phytopathogens Rhizoctonia solani, F. oxysporum f. sp. pisi, and Pythium aphanidermatum by dual culture assay. Maximum inhibition of F. oxysporum f. sp. pisi was observed by strains OS_12 and OS_25 among all root rot pathogens. Scanning electron microscopy of dual culture indicated hyphal distortion and destruction in the case of F. oxysporum f. sp. pisi. Further, volatile organic compounds (VOCs) were identified by gas chromatography-mass spectrometry (GC-MS). The GC-MS detected eight bioactive compounds from hexane extracts for instance, Dodecanoic acid, Tetra decanoic acid, L-ascorbic acid, Trans-13-Octadecanoic acid, Octadecanoic acid. Both the endophytes exhibited multifarious plant growth promoting traits such as indole acetic production (30-33 µg IAA ml-1), phosphate solubilization, and siderophore and ammonia production. Pot trials were conducted to assess the efficacy of endophytes in field conditions. A significant reduction in disease mortality rate and enhancement of growth parameters was observed in pea plants treated with consortium of endophytes OS_12 and OS_25 challenged with F. oxysporum f.sp. pisi infection. The endophytic strains elicited induced systemic resistance (ISR) in pathogen challenged pea plants by enhancing activities of Phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), ascorbate oxidase (AO), catalase (CAT) and total phenolic content. The endophytes reduced the oxidative stress as revealed by decrease in malondialdehyde (MDA) content and subsequently, lipid peroxidation in host plant leaves. Robust root colonization of pea seedlings by endophytes was observed by scanning electron microscopy (SEM) and fluorescence microscopy. Thus, plant growth promoting endophytic P. aeruginosa and A. aneurinilyticus can be further exploited through bio-formulations for sustainable protection of crops against root rot diseases as bio-control agents.

Tissue-specific transcriptome responses to Fusarium head blight and Fusarium root rot.

Fusarium head blight (FHB) and Fusarium root rot (FRR) are important diseases of small-grain cereals caused by Fusarium species. While host response to FHB has been subject to extensive study, very little is known about response to FRR and the transcriptome responses of FHB and FRR have not been thoroughly compared. Brachypodium distachyon (Bd) is an effective model for investigating host responses to both FHB and FRR. In this study the transcriptome response of Bd to F. graminearum (Fg) infection of heads and roots was investigated. An RNA-seq analysis was performed on both Bd FHB and FRR during the early infection. Additionally, an RNA-seq analysis was performed on in vitro samples of Fg for comparison with Fg gene expression in planta. Differential gene expression and gene-list enrichment analyses were used to compare FHB and FRR transcriptome responses in both Bd and Fg. Differential expression of selected genes was confirmed using RT-qPCR. Most genes associated with receptor signalling, cell-wall modification, oxidative stress metabolism, and cytokinin and auxin biosynthesis and signalling genes were generally upregulated in FHB or were downregulated in FRR. In contrast, Bd genes involved in jasmonic acid and ethylene biosynthesis and signalling, and antimicrobial production were similarly differentially expressed in both tissues in response to infection. A transcriptome analysis of predicted Fg effectors with the same infected material revealed elevated expression of core tissue-independent genes including cell-wall degradation enzymes and the gene cluster for DON production but also several tissue-dependent genes including those for aurofusarin production and cutin degradation. This evidence suggests that Fg modulates its transcriptome to different tissues of the same host.

Green Synthesized of Thymus vulgaris Chitosan Nanoparticles Induce Relative WRKY-Genes Expression in Solanum lycopersicum against Fusarium solani, the Causal Agent of Root Rot Disease.

Fusarium solani is a plant pathogenic fungus that causes tomato root rot disease and yield losses in tomato production. The current study's main goal is testing the antibacterial efficacy of chitosan nanoparticles loaded with Thyme vulgaris essential oil (ThE-CsNPs) against F. solani in vitro and in vivo. GC-MS analysis was used to determine the chemical constituents of thyme EO. ThE-CsNPs were investigated using transmission electron microscopy before being physicochemically characterized using FT-IR. ThE-CsNPs were tested for antifungal activity against F. solani mycelial growth in vitro. A pot trial was conducted to determine the most effective dose of ThE-CsNPs on the morph/physiological characteristics of Solanum lycopersicum, as well as the severity of fusarium root rot. The relative gene expression of WRKY transcript factors and defense-associated genes were quantified in root tissues under all treatment conditions. In vitro results revealed that ThE-CsNPs (1%) had potent antifungal efficacy against F. solani radial mycelium growth. The expression of three WRKY transcription factors and three tomato defense-related genes was upregulated. Total phenolic, flavonoid content, and antioxidant enzyme activity were all increased. The outfindings of this study strongly suggested the use of ThE-CsNPs in controlling fusarium root rot on tomatoes; however, other experiments remain necessary before they are recommended.

Biochemical Characterization, Antifungal Activity, and Relative Gene Expression of Two Mentha Essential Oils Controlling Fusarium oxysporum, the Causal Agent of Lycopersicon esculentum Root Rot.

Tomato (Lycopersicon esculentum Mill.) is important food in daily human diets. Root rot disease by Fusarium oxysporum caused huge losses in tomato quality and yield annually. The extensive use of synthetic and chemical fungicides has environmental risks and health problems. Recent studies have pointed out the use of medicinal plant essential oils (EOs) and extracts for controlling fungal diseases. In the current research, Mentha spicata and Mentha longifolia EOs were used in different concentrations to control F. oxysporum. Many active compounds are present in these two EOs such as: thymol, adapic acid, menthol and menthyl acetate. These compounds possess antifungal effect through malformation and degradation of the fungal cell wall. The relative expression levels of distinctly upregulated defense-related WRKY genes (WRKY1, WRKY4, WRKY33 and WRKY53) in seedling root were evaluated as a plant-specific transcription factor (TF) group in different response pathways of abiotic stress. Results showed significant expression levels of WRKY, WRKY53, WRKY33, WRKY1 and WRKY4 genes. An upregulation was observed in defense-related genes such as chitinase and defensin in roots by application EOs under pathogen condition. In conclusion, M. spicata and M. longifolia EOs can be used effectively to control this plant pathogen as sustainable and eco-friendly botanical fungicides.

Enniatin Production Influences Fusarium avenaceum Virulence on Potato Tubers, but not on Durum Wheat or Peas.

Fusarium avenaceum is a generalist pathogen responsible for diseases in numerous crop species. The fungus produces a series of mycotoxins including the cyclohexadepsipeptide enniatins. Mycotoxins can be pathogenicity and virulence factors in various plant-pathogen interactions, and enniatins have been shown to influence aggressiveness on potato tubers. To determine the role of these mycotoxins in other F. avenaceum-host interactions, enniatin synthase 1 (ESYN1) disruption and overexpression mutants were generated and their ability to infect wheat and peas investigated. As a preliminary study, the transformants were screened for their ability to cause potato tuber necrosis and, consistent with a previous report, enniatin production increased necrotic lesion size on the tubers. By contrast, when the same mutants were assessed in their ability to cause disease in pea roots or durum wheat spikes, no changes in disease symptoms or virulence were observed. While it is known that, at least in the case of wheat, exogenously applied enniatins can cause tissue necrosis, this group of mycotoxins does not appear to be a key factor on its own in disease development on peas or durum wheat.