Intercropping improves faba bean photosynthesis and reduces disease caused by Fusarium commune and cinnamic acid-induced stress.

Modern intensive cropping systems often contribute to the accumulation of phenolic acids in the soil, which promotes the development of soilborne diseases. This can be suppressed by intercropping. This study analyzed the effects of intercropping on Fusarium wilt based on its effect on photosynthesis under stress by the combination of Fusarium commune and cinnamic acid. The control was not inoculated with F. commune, while the faba bean plants (Vicia faba L.) were inoculated with this pathogen in the other treatments. The infected plants were also treated with cinnamic acid. This study examined the development of Fusarium wilt together with its effects on the leaves, absorption of nutrients, chlorophyll fluorescence parameters, contents of photosynthetic pigments, activities of photosynthetic enzymes, gas exchange parameters, and the photosynthetic assimilates of faba bean from monocropping and intercropping systems. Under monocropping conditions, the leaves of the plants inoculated with F. commune grew significantly less, and there was enhanced occurrence of the Fusarium wilt compared with the control. Compared with the plants solely inoculated with F. commune, the exogenous addition of cinnamic acid to the infected plants significantly further reduced the growth of faba bean leaves and increased the occurrence of Fusarium wilt. A comparison of the combination of F. commune and cinnamic acid in intercropped wheat and faba bean compared with monocropping showed that intercropping improved the absorption of nutrients, increased photosynthetic pigments and its contents, electron transport, photosynthetic enzymes, and photosynthetic assimilates. The combination of these factors reduced the occurrence of Fusarium wilt in faba bean and increased the growth of its leaves. These results showed that intercropping improved the photosynthesis, which promoted the growth of faba bean, thus, reducing the development of Fusarium wilt following the stress of infection by F. commune and cinnamic acid. This research should provide more information to enhance sustainable agriculture.

The interplay of suppressive soil bacteria and plant root exudates determines germination of microsclerotia of Verticillium longisporum.

Dormant microsclerotia play a vital role in the survival and spread of Verticillium longisporum, as they can stay viable in the soil and maintain their infectivity for many years. In our previous work, we revealed that soil bacterial volatiles are a key inhibitory factor causing microsclerotia dormancy in the soil. In this study, we further demonstrate that root exudates collected from both host and non-host plants can effectively rescue microsclerotia from bacterial suppression and initiate germination. To identify the specific compounds in root exudates responsible for microsclerotia germination, we fractionated the collected root exudates into polar and non-polar compounds. Subsequently, we conducted comprehensive bioassays with each fraction on germination-suppressed microsclerotia. The result revealed a pivotal role of primary metabolites in root exudates, particularly glutamic acid, in triggering microsclerotia germination and overcoming bacterial inhibition. Moreover, our studies revealed a decrease in inhibitory bacterial volatile fatty acids when bacteria were cultured in the presence of root exudates or glutamic acid. This suggests a potential mechanism, by which root exudates set-off bacterial suppression on microsclerotia. Here, we reveal for the first time that plant root exudates, instead of directly inducing the germination of microsclerotia, enact a set-off effect by counteracting the suppressive impact of soil bacteria on the microsclerotia germination process. This nuanced interaction advances our understanding of the multifaceted dynamics governing microsclerotia dormancy and germination in the soil environment. IMPORTANCE: Our research provides first-time insights into the crucial interaction between plant root exudates and soil bacteria in regulating the germination of Verticillium longisporum microsclerotia, a significant structure in the survival and proliferation of this soil-borne pathogen. We describe so far unknown mechanisms, which are key to understand how root infections on oilseed rape can occur. By pinpointing primary metabolites in root exudates as key factors in overcoming bacteria-induced dormancy and promote microsclerotia germination, our study highlights the potential for exploiting plant - as well as soil microbe-derived - compounds to control V. longisporum. This work underscores the importance of elucidating the nuanced interactions within the soil ecosystem to devise innovative strategies for managing root infective plant diseases, thereby contributing to the resilience and health of cropping systems.

Facilitative and competitive interactions between mycorrhizal and nonmycorrhizal plants in an extremely phosphorus-impoverished environment: role of ectomycorrhizal fungi and native oomycete pathogens in shaping species coexistence.

Nonmycorrhizal cluster root-forming species enhance the phosphorus (P) acquisition of mycorrhizal neighbours in P-impoverished megadiverse systems. However, whether mycorrhizal plants facilitate the defence of nonmycorrhizal plants against soil-borne pathogens, in return and via their symbiosis, remains unknown. We characterised growth and defence-related compounds in Banksia menziesii (nonmycorrhizal) and Eucalyptus todtiana (ectomycorrhizal, ECM) seedlings grown either in monoculture or mixture in a multifactorial glasshouse experiment involving ECM fungi and native oomycete pathogens. Roots of B. menziesii had higher levels of phytohormones (salicylic and jasmonic acids, jasmonoyl-isoleucine and 12-oxo-phytodienoic acid) than E. todtiana which further activated a salicylic acid-mediated defence response in roots of B. menziesii, but only in the presence of ECM fungi. We also found that B. menziesii induced a shift in the defence strategy of E. todtiana, from defence-related secondary metabolites (phenolic and flavonoid) towards induced phytohormone response pathways. We conclude that ECM fungi play a vital role in the interactions between mycorrhizal and nonmycorrhizal plants in a severely P-impoverished environment, by introducing a competitive component within the facilitation interaction between the two plant species with contrasting nutrient-acquisition strategies. This study sheds light on the interplay between beneficial and detrimental soil microbes that shape plant-plant interaction in severely nutrient-impoverished ecosystems.

The biocontrol potentials of rhizospheric bacterium Bacillus velezensis K0T24 against mulberry bacterial wilt disease.

Mulberry bacterial wilt disease, caused by Ralstonia pseudosolanacearum, is a devastating soil-borne disease in the silk-mulberry-related industry. In this study, through high-throughput sequencing, we compared the rhizosphere bacterial composition of the mulberry-resistant cultivar (K10) and susceptible cultivar (G12), confirming Bacillus as a genus-level biomarker for K10. Next, twelve Bacillus spp. isolates, derived from the rhizosphere of K10, were screened for their antagonistic activity against R. pseudosolanacearum. The isolate showing strong antagonism was identified as B. velezensis K0T24 and selected for further analysis. The fermentation supernatant of B. velezensis K0T24 significantly inhibited the growth of R. pseudosolanacearum (82.47%) and the expression of its pathogenic genes. Using B. velezensis K0T24 in mulberry seedlings also increased defense enzyme activities and achieved a control efficacy of up to 55.17% against mulberry bacterial wilt disease. Collectively, our findings demonstrate the potential of B. velezensis K0T24 in suppressing mulberry bacterial wilt disease.

Transcriptome analysis reveals the humic acids and chitosan suppressing Alternaria solani growth.

AIMS: Understanding the inhibitory effects of natural organic substances on soil-borne pathogenic fungi and the relevant molecular mechanisms are highly important for future development of green prevention and control technology against soil-borne diseases. Our study elucidates the inhibitory effect of the combined application of humic acids (HAs) and chitosan on Alternariasolani and the light on the corresponding mechanism. METHODS AND RESULTS: The effect on A. solani growth by HAs incorporated with chitosan was investigated by plate culture and the corresponding mechanism was revealed using transcriptomics. The colony growth of A. solani was suppressed with the highest inhibition rate 33.33% when swine manure HAs was compounded with chitosan at a ratio of 1:4. Chitosan changed the colony morphology from round to irregularly. RNA-seq in the HAs and chitosan (HC) treatment revealed 239 differentially expressed genes compared with the control. The unigenes associated with enzymes activities related to growth and biological processes closely related to mycelial growth and metabolism were downregulated. RNA-seq also revealed that chitosan altered the expression of genes related to secondary metabolism, fungal cell wall formation and polysaccharide synthesis, and metabolism. Meanwhile, weighted gene co-expression network analysis showed that, genes expression in the module positively correlated with mycelial growth was significantly reduced in the HC treatment; and the results were verified by real-time quantitative polymerase chain reaction. CONCLUSIONS: The co-inhibition effect of HAs and chitosan on A. solani is associated with downregulated genes expression correlated with mycelial growth.

First Report of Rhizoctonia solani AG 4 HG-III Causing Stem Lesion of Watermelon in Idaho.

In July 2022, stem lesions, approximately 4 to 5 cm in length as well as leaf wilt and dark brown necrosis on stems and roots were observed in two fields in Southwest Idaho on 20 to 30% of watermelons (Citrullus lanatus). To determine the causal agent, isolations were attempted from symptomatic tissue. The surface of the affected material was disinfected with 0.6% sodium hypochlorite for 1 min and rinsed three times with sterile water. Approximately 2 mm3 sections of tissue were plated on water agar amended with 0.02% penicillin and 0.08% streptomycin and incubated at room temperature for 7 days. Fungal colonies were tentatively identified as Rhizoctonia from right-angle branching and septate hyphal structures, slight constriction and septum near the branch base, and the production of 1 to 2 mm white to light brown irregularly shaped sclerotia. Single hyphal tips were transferred to potato dextrose agar (PDA) and grown at room temperature. Approximately ten isolates from each field with a consistent macromorphology were observed. These isolates had light brown mycelia, produced sclerotia at ambient temperature with no exposure to continuous light, and a representative isolate, designated D22-110 was selected for sequencing and pathogenicity testing. For isolate D22-110, mycelia were removed with a scalpel after 7 days of growth, for DNA extraction and sequencing of the rDNA internal transcribed spacer (ITS) region as previously described (White et al., 1990). A 726 bp product was generated and the sequence was submitted to GenBank (Accession No. OQ794049). NCBI-BLAST indicated this sequence was 99% identical (631 of 634 bp and 632 of 634 bp identical) with known reference isolates previously identified as R. solani AG 4 HG-III (Accession No. AF354075 and AF354076, respectively) from a phylogenetic study (Gonzalez et al., 2001). Pathogenicity testing was performed twice on two-week-old seedlings of watermelon cultivars Endless Summer and Wingman in greenhouse conditions (29oC, 12 h daylight). Two disks (3 mm diam) from 7-day-old plates of PDA were placed around each seedling at the root and stem convergence point. Ten seedlings were mock-inoculated with sterile PDA plugs as a control. Approximately 35% damping-off incidence was observed on inoculated seedlings six days post-inoculation, while control seedlings remained healthy. At 20 days post-inoculation, 20 (first trial) and 34 seedlings (second trial) were assessed for visible stem and root lesions. Incidence of stem lesions occurred on 90% of seedlings, with 80% of seedlings possessing lesions greater than 10 mm in diameter. Seedlings without R. solani inoculation were free of stem and root lesions. R. solani was re-isolated from symptomatic tissue, with 40% frequency of isolation, identified by right-angle branching of the hyphae thus confirming Koch's postulates. R. solani AG 4 has been reported in watermelon in the US since 1994 (Hall and Summer, 1994) but the AG 4 subgroup was not reported. AG 4 HG-III was reported in melon seedlings causing damping-off in Kyrgyzstan (Erper et al. 2016). In other hosts, AG 4 HG-III was found in potatoes in South Africa (Muzhinji et al., 2014), buckwheat and foxtail millet in China (Zhou et al., 2015; Hao et al., 2023), broccoli and spinach (Kuramae et al., 2003) and turnip green (Sekiguchi et al., 2015). To the best of our knowledge, this is the first report of R. solani AG 4 HG-III causing disease in watermelon in Idaho. Given the the rate of disease incidence observed in the field, growers should consider avoiding planting alternative host crops to minimize inoculum buildup.

First report of Macrophomina phaseolina causing сharcoal rot in soybean (Glycine max (L.) Merr.) plants in Uzbekistan.

The soybean production area is expanding in Uzbekistan. Soybeans were planted on an area of 10 thsd ha and the harvest amounted to 30 thsd metric tons in 2023 (IPAD, https://ipad.fas.usda.gov/countrysummary). Macrophomina phaseolina (Mp) is a soil- and seed-borne fungal pathogen causing economically important diseases of legume crops (Pennerman et al. 2024). Drought stress and a warm climate are favorable to this pathogen (Irulappan et al. 2022). Under these conditions, its microsclerotia survive for a longer period and become more virulent (Chamorro et al. 2015). In August 2022, typical symptoms of charcoal rot were observed in about 25% of "Orzu" soybean cultivar affecting 6 ha located on the experimental base "Durmon" of our institute. Diseased plants displayed the following charcoal rot symptoms: leaves turn yellow, then wilt, die, and remain attached to the plant; the lower portion of the stem and tap root have a light gray or ashy black discoloration; tiny black specks on the lower stem and root; after splitting the stem, it has the appearance of fine charcoal powder. In order to determine the causal agent of these symptoms, a total of 17 diseased plants were collected from focal lesions in soybean plantings. From each plant, twelve sections of stem and root tissue were selected, cut into small 5-mm pieces, and surface sterilized with 1% sodium hypochlorite for four minutes, then rinsed three times with sterile distilled water. The disinfected tissues were dried on sterile filter paper for 5 min and placed on PDA Petri plates, which were incubated in an incubation chamber for 3 days (16 h light (26oC) and 8 h dark (18oC)). Fungi were subsequently subcultured on PDA and incubated for 7 days to obtain pure cultures. Six monohyphal colonies were purified. The colonies showed dense growth, with a gray initial mycelium becoming darker with aging. After 8 days on PDA, black-colored microsclerotia with spherical to oblong shapes were observed. On average, they measured 60 µm in width and 130 µm in length (n = 30). From six isolated monohyphal colonies, one has been chosen for molecular-genetic identification. Molecular-genetic analysis was conducted by amplification and sequencing of the ITS region with the ITS1 and ITS4 primers (White et al. 1990). The resulting sequence was deposited in the NCBI database under accession number OQ073450. After BLAST analysis (Altschul et al. 1990) it was 100% identical with the reference sequences of Mp (accession MT039671, MT039663 and MH496040) isolated in sugar beet, maize and sunflower, respectively, from Serbia. In order to verify the pathogenicity, soybean seedlings (cv. Orzu) were dipped into spore suspension (1 × 107 spores/ml) of sequenced strain R-17 for 1 minute and transferred to a 15 cm diameter plastic pot with 350 g of sterilized soil mix. After 25 days, the inoculated plants showed classic charcoal rot symptoms, while the control plants remained healthy. The pathogen was successfully reisolated from the infected seedlings onto PDA, fulfilling Koch's postulate. The identity of the re-isolated strain was confirmed by morphological features and sequencing of the ITS region. It should be noted that in Uzbekistan, Mp has not been documented in any plants. Therefore, according to our knowledge, this is the first report of this fungus affecting soybean plants in Uzbekistan. Since molecular-genetic analysis of the R-17 strain showed clustering with strains from Serbia, we speculate that there may have been a recent introduction of Mp from Serbia into Uzbekistan. This assumption is additionally confirmed by the fact that Serbia is the largest seed exporter in Uzbekistan. The increase in charcoal rot disease poses a major challenge to soybean production in Uzbekistan. Understanding the genetic diversity of Mp can be utilized to manage this disease, improve soybean yield, and help soybean breeding programs in Uzbekistan.

Rapid and Sensitive Detection of Verticillium dahliae from Soil Using LAMP-CRISPR/Cas12a Technology.

Cotton Verticillium wilt is mainly caused by the fungus Verticillium dahliae, which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of microsclerotia, making it a destructive soil-borne disease. The accurate, sensitive, and rapid detection of V. dahliae from complex soil samples is of great significance for the early warning and management of cotton Verticillium wilt. In this study, we combined the loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology to develop an accurate, sensitive, and rapid detection method for V. dahliae. Initially, LAMP primers and CRISPR RNA (crRNA) were designed based on a specific DNA sequence of V. dahliae, which was validated using several closely related Verticillium spp. The lower detection limit of the LAMP-CRISPR/Cas12a combined with the fluorescent visualization detection system is approximately ~10 fg/µL genomic DNA per reaction. When combined with crude DNA-extraction methods, it is possible to detect as few as two microsclerotia per gram of soil, with the total detection process taking less than 90 min. Furthermore, to improve the method's user and field friendliness, the field detection results were visualized using lateral flow strips (LFS). The LAMP-CRISPR/Cas12a-LFS system has a lower detection limit of ~1 fg/µL genomic DNA of the V. dahliae, and when combined with the field crude DNA-extraction method, it can detect as few as six microsclerotia per gram of soil, with the total detection process taking less than 2 h. In summary, this study expands the application of LAMP-CRISPR/Cas12a nucleic acid detection in V. dahliae and will contribute to the development of field-deployable diagnostic productions.

Fengycin produced by Bacillus subtilis XF-1 plays a major role in the biocontrol of Chinese cabbage clubroot via direct effect and defense stimulation.

Bacillus subtilis XF-1 is a well-investigated biocontrol agent against the biotrophic Plasmodiophora brassicae Woron., the causal agent of clubroot disease of cruciferous crops. The present study demonstrates that XF-1 could efficiently control clubroot disease via leaf spraying and provides an understanding of the biocontrol mechanisms. High-performance thin-layer chromatography (HTPLC) analysis indicated the presence of fengycin-type cyclopeptides in the supernatant. A ppsB deletion mutant of XF-1 resulted in no fengycin production, significantly reduced the lysis rate of testing spores in vitro and the primary infection rate of root hair in vivo, and decreased the protection value against clubroot disease under the greenhouse conditions. Confocal laser scanning microscopy proved that fengycin was not required for leaf internalization and root colonization. Moreover, the expression level of the ppsB gene in XF-1 was regulated by its cell density in root during interaction with P. brassicae. In addition, the ΔppsB mutant of XF-1 could not efficiently control disease because it led to a lower activation level of the jasmonic acid and salicylic acid signaling pathways in roots, which are necessary for the plant defense reaction upon pathogen invasion. Altogether, the present study provides a new understanding of specific cues in the interaction between B. subtilis and P. brassicae as well as insights into the application of B. subtilis in agriculture.

A screening of inhibitors targeting the receptor kinase FERONIA reveals small molecules that enhance plant root immunity.

Receptor-like kinases (RLKs) constitute the largest receptor family involved in the regulation of plant immunity and growth, but small-molecule inhibitors that target RLKs to improve agronomic traits remain unexplored. The RLK member FERONIA (FER) negatively regulates plant resistance to certain soil-borne diseases that are difficult to control and cause huge losses in crop yields and economy. Here, we identified 33 highly effective FER kinase inhibitors from 1494 small molecules by monitoring FER autophosphorylation in vitro. Four representative inhibitors (reversine, cenisertib, staurosporine and lavendustin A) inhibited the kinase activity of FER and its homologues in several crops by targeting the conserved ATP pocket in the kinase structure. FER contributes to the physiological impact of representative inhibitors in plants. The treatment of roots with reversine, staurosporine and lavendustin A enhanced innate immunity in plant roots and thus alleviated soil-borne diseases in tobacco, tomato and rice without growth penalties. Consistently, RNA sequencing assays showed that lavendustin A and reversine exert profound impacts on immunity-related gene expression. Our results will set a new milestone in the development of the plant RLK kinase regulation theory and provide a novel strategy for the prevention and control of plant soil-borne diseases without growth penalties.

Cell Density-Regulated Adhesins Contribute to Early Disease Development and Adhesion in Ralstonia solanacearum.

Adhesins (adhesive proteins) help bacteria stick to and colonize diverse surfaces and often contribute to virulence. The genome of the bacterial wilt pathogen Ralstonia solanacearum (Rs) encodes dozens of putative adhesins, some of which are upregulated during plant pathogenesis. Little is known about the role of these proteins in bacterial wilt disease. During tomato colonization, three putative Rs adhesin genes were upregulated in a ΔphcA quorum-sensing mutant that cannot respond to high cell densities: radA (Ralstonia adhesin A), rcpA (Ralstonia collagen-like protein A), and rcpB. Based on this differential gene expression, we hypothesized that adhesins repressed by PhcA contribute to early disease stages when Rs experiences a low cell density. During root colonization, Rs upregulated rcpA and rcpB, but not radA, relative to bacteria in the stem at mid-disease. Root attachment assays and confocal microscopy with ΔrcpA/B and ΔradA revealed that all three adhesins help Rs attach to tomato seedling roots. Biofilm assays on abiotic surfaces found that Rs does not require RadA, RcpA, or RcpB for interbacterial attachment (cohesion), but these proteins are essential for anchoring aggregates to a surface (adhesion). However, Rs did not require the adhesins for later disease stages in planta, including colonization of the root endosphere and stems. Interestingly, all three adhesins were essential for full competitive fitness in planta. Together, these infection stage-specific assays identified three proteins that contribute to adhesion and the critical first host-pathogen interaction in bacterial wilt disease. IMPORTANCE Every microbe must balance its need to attach to surfaces with the biological imperative to move and spread. The high-impact plant-pathogenic bacterium Ralstonia solanacearum can stick to biotic and abiotic substrates, presumably using some of the dozens of putative adhesins encoded in its genome. We confirmed the functions and identified the biological roles of multiple afimbrial adhesins. By assaying the competitive fitness and the success of adhesin mutants in three different plant compartments, we identified the specific disease stages and host tissues where three previously cryptic adhesins contribute to success in plants. Combined with tissue-specific regulatory data, this work indicates that R. solanacearum deploys distinct adhesins that help it succeed at different stages of plant pathogenesis.

Modeling impacts of saltwater intrusion on methane and nitrous oxide emissions in tidal forested wetlands.

Emissions of methane (CH4 ) and nitrous oxide (N2 O) from soils to the atmosphere can offset the benefits of carbon sequestration for climate change mitigation. While past study has suggested that both CH4 and N2 O emissions from tidal freshwater forested wetlands (TFFW) are generally low, the impacts of coastal droughts and drought-induced saltwater intrusion on CH4 and N2 O emissions remain unclear. In this study, a process-driven biogeochemistry model, Tidal Freshwater Wetland DeNitrification-DeComposition (TFW-DNDC), was applied to examine the responses of CH4 and N2 O emissions to episodic drought-induced saltwater intrusion in TFFW along the Waccamaw River and Savannah River, USA. These sites encompass landscape gradients of both surface and porewater salinity as influenced by Atlantic Ocean tides superimposed on periodic droughts. Surprisingly, CH4 and N2 O emission responsiveness to coastal droughts and drought-induced saltwater intrusion varied greatly between river systems and among local geomorphologic settings. This reflected the complexity of wetland CH4 and N2 O emissions and suggests that simple linkages to salinity may not always be relevant, as non-linear relationships dominated our simulations. Along the Savannah River, N2 O emissions in the moderate-oligohaline tidal forest site tended to increase dramatically under the drought condition, while CH4 emission decreased. For the Waccamaw River, emissions of both CH4 and N2 O in the moderate-oligohaline tidal forest site tended to decrease under the drought condition, but the capacity of the moderate-oligohaline tidal forest to serve as a carbon sink was substantially reduced due to significant declines in net primary productivity and soil organic carbon sequestration rates as salinity killed the dominant freshwater vegetation. These changes in fluxes of CH4 and N2 O reflect crucial synergistic effects of soil salinity and water level on C and N dynamics in TFFW due to drought-induced seawater intrusion.

Revising Clonostachys and allied genera in Bionectriaceae.

Clonostachys (Bionectriaceae, Hypocreales) species are common soil-borne fungi, endophytes, epiphytes, and saprotrophs. Sexual morphs of Clonostachys spp. were placed in the genus Bionectria, which was further segregated into the six subgenera Astromata, Bionectria, Epiphloea, Myronectria, Uniparietina, and Zebrinella. However, with the end of dual nomenclature, Clonostachys became the single depository for sexual and asexual morph-typified species. Species of Clonostachys are typically characterised by penicillate, sporodochial, and, in many cases, dimorphic conidiophores (primary and secondary conidiophores). Primary conidiophores are mononematous, either verticillium-like or narrowly penicillate. The secondary conidiophores generally form imbricate conidial chains that can collapse to slimy masses, particularly on sporodochia. In the present study, we investigated the species diversity within a collection of 420 strains of Clonostachys from the culture collection of, and personal collections at, the Westerdijk Fungal Biodiversity Institute in Utrecht, the Netherlands. Strains were analysed based on their morphological characters and molecular phylogeny. The latter used DNA sequence data of the nuclear ribosomal internal transcribed spacer regions and intervening 5.8S nrDNA (ITS) and partial 28S large subunit (LSU) nrDNA and partial protein encoding genes including the RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1) and ß-tubulin (TUB2). Based on these results, the subgenera Astromata, Bionectria, Myronectria and Zebrinella are supported within Clonostachys. Furthermore, the genus Sesquicillium is resurrected to accommodate the former subgenera Epiphloea and Uniparietina. The close relationship of Clonostachys and Sesquicillium is strongly supported as both are inferred phylogenetically as sister-genera. New taxa include 24 new species and 10 new combinations. Recognition of Sesquicillium distinguishes species typically forming a reduced perithecial stroma superficially on plant tissue from species in Clonostachys often forming well-developed, through bark erumpent stromata. The patterns of observed perithecial wall anatomies, perithecial wall and stroma interfaces, and asexual morph diversifications described in a previously compiled monograph are used for interpreting ancestral state reconstructions. It is inferred that the common ancestor of Clonostachys and Sesquicillium may have formed perithecia superficially on leaves, possessed a perithecial wall consisting of a single region, and formed intercalary phialides in penicilli of conidiophores. Character interpretation may also allow hypothesising that diversification of morphs occurred then in the two genera independently and that the frequently stroma-linked Clonostachys morphs evolved together with the occupation of woody host niches and mycoparasitism. Taxonomic novelties: New species: Clonostachys aurantiaca L. Zhao & Crous, Clonostachys australiana L. Zhao & Crous, Clonostachys bambusae L. Zhao & Crous, Clonostachys buxicola L. Zhao & Crous, Clonostachys cylindrica L. Zhao & Crous, Clonostachys ellipsoidea L. Zhao & Crous, Clonostachys flava L. Zhao, Crous & Schroers, Clonostachys fujianensis L. Zhao & Crous, Clonostachys fusca L. Zhao, Crous & Schroers, Clonostachys garysamuelsii L. Zhao & Crous, Clonostachys hongkongensis L. Zhao & Crous, Clonostachys longiphialidica L. Zhao, Crous & Schroers, Clonostachys obovatispora, L. Zhao & Crous, Clonostachys palmae L. Zhao, Crous & Schroers, Clonostachys parasporodochialis L. Zhao & Crous, Clonostachys penicillata L. Zhao, Crous & Schroers, Clonostachys reniformis L. Zhao & Crous, Clonostachys vacuolata L. Zhao, Crous & Schroers, Clonostachys venezuelae L. Zhao, Crous & Schroers, Mycocitrus synnematus L. Zhao & Crous, Nectriopsis didymii L. Zhao & Crous, Sesquicillium intermediophialidicum L. Zhao & Crous, Sesquicillium neerlandicum L. Zhao & Crous, Sesquicillium symmetricum L. Zhao & Crous. New combinations: Mycocitrus coccicola (J.A. Stev.) L. Zhao & Crous, Mycocitrus coxeniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium essexcoheniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium lasiacidis (Samuels) L. Zhao, Crous & Schroers, Sesquicillium phyllophilum (Schroers) L. Zhao, Crous & Schroers, Sesquicillium rossmaniae (Schroers) L. Zhao, Crous & Schroers, Sesquicillium saulense (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium sesquicillii (Samuels) L. Zhao, Crous & Schroers, Sesquicillium spinulosisporum (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium tornatum (Höhn.) Schroers. New synonyms: Clonostachys aranearum W.H. Chen et al., Clonostachys chuyangsinensis H. Yu & Y. Wang, Clonostachys eriocamporesiana R.H. Perera & K.D. Hyde, Clonostachys granuligera (Starbäck) Forin & Vizzini, Clonostachys indica Prasher & R. Chauhan, Clonostachys spinulosa R.H. Perera et al., Clonostachys squamuligera (Sacc.) Forin & Vizzini, Clonostachys wenpingii (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y. Zhuang. Epitypes (basionyms): Fusidium buxi J.C. Schmidt ex Link, Verticillium candelabrum Bonord. Citation: Zhao L, Groenewald JZ, Hernández-Restrepo M, Schroers H-J, Crous PW (2023). Revising Clonostachys and allied genera in Bionectriaceae. Studies in Mycology 105: 205-266. doi: 10.3114/sim.2023.105.03.

Bacillus velezensis RC116 Inhibits the Pathogens of Bacterial Wilt and Fusarium Wilt in Tomato with Multiple Biocontrol Traits.

Soil-borne plant diseases seriously threaten the tomato industry worldwide. Currently, eco-friendly biocontrol strategies have been increasingly considered as effective approaches to control the incidence of disease. In this study, we identified bacteria that could be used as biocontrol agents to mitigate the growth and spread of the pathogens causing economically significant diseases of tomato plants, such as tomato bacterial wilt and tomato Fusarium wilt. Specifically, we isolated a strain of Bacillus velezensis (RC116) from tomato rhizosphere soil in Guangdong Province, China, with high biocontrol potential and confirmed its identity using both morphological and molecular approaches. RC116 not only produced protease, amylase, lipase, and siderophores but also secreted indoleacetic acid, and dissolved organophosphorus in vivo. Moreover, 12 Bacillus biocontrol maker genes associated with antibiotics biosynthesis could be amplified in the RC116 genome. Extracellular secreted proteins of RC116 also exhibited strong lytic activity against Ralstonia solanacearum and Fusarium oxysporum f. sp. Lycopersici. Pot experiments showed that the biocontrol efficacy of RC116 against tomato bacteria wilt was 81%, and consequently, RC116 significantly promoted the growth of tomato plantlets. Based on these multiple biocontrol traits, RC116 is expected to be developed into a broad-spectrum biocontrol agent. Although several previous studies have examined the utility of B. velezensis for the control of fungal diseases, few studies to date have evaluated the utility of B. velezensis for the control of bacterial diseases. Our study fills this research gap. Collectively, our findings provide new insights that will aid the control of soil-borne diseases, as well as future studies of B. velezensis strains.

Deciphering the Mechanism of Tolerance to Apple Replant Disease Using a Genetic Mapping Approach in a Malling 9 × M. × robusta 5 Population Identifies SNP Markers Linked to Candidate Genes.

Apple replant disease (ARD) is a worldwide economic risk in apple production. Although several studies have shown that the wild apple accession Malus × robusta 5 (Mr5) is ARD-tolerant, the genetics of this tolerance have not yet been elucidated. A genetic mapping approach with a biparental population derived from contrasting parents involving molecular markers provides a means for marker-assisted selection of genetically complex traits and for determining candidate genes. In this study, we crossed the ARD-tolerant wild apple accession Mr5 and the ARD-susceptible rootstock 'M9' and analyzed the resultant progeny for ARD tolerance. Hence, a high-density genetic map using a tunable genotyping-by-sequencing (tGBS) approach was established. A total of 4804 SNPs together with 77 SSR markers were included in the parental maps comprising 17 linkage groups. The phenotypic responses to ARD were evaluated for 106 offspring and classified by an ARD-susceptibility index (ASI). A Kruskal-Wallis test identified SNP markers and one SSR marker on linkage groups (LG) 6 and 2 that correlated with ARD tolerance. We found nine candidate genes linked with these markers, which may be associated with plant response to ARD. These candidate genes provide some insight into the defense mechanisms against ARD and should be studied in more detail.

Plant neighbours can make or break the disease transmission chain of a fungal root pathogen.

Biodiversity can reduce or increase disease transmission. These divergent effects suggest that community composition rather than diversity per se determines disease transmission. In natural plant communities, little is known about the functional roles of neighbouring plant species in belowground disease transmission. Here, we experimentally investigated disease transmission of a fungal root pathogen (Rhizoctonia solani) in two focal plant species in combinations with four neighbour species of two ages. We developed stochastic models to test the relative importance of two transmission-modifying mechanisms: (1) infected hosts serve as nutrient supply to increase hyphal growth, so that successful disease transmission is self-reinforcing; and (2) plant resistance increases during plant development. Neighbouring plants either reduced or increased disease transmission in the focal plants. These effects depended on neighbour age, but could not be explained by a simple dichotomy between hosts and nonhost neighbours. Model selection revealed that both transmission-modifying mechanisms are relevant and that focal host-neighbour interactions changed which mechanisms steered disease transmission rate. Our work shows that neighbour-induced shifts in the importance of these mechanisms across root networks either make or break disease transmission chains. Understanding how diversity affects disease transmission thus requires integrating interactions between focal and neighbour species and their pathogens.

Two dominant genes in barley (Hordeum vulgare L.) complementarily encode perfect resistance to Japanese soil-borne wheat mosaic virus.

Japanese soil-borne wheat mosaic virus (Furovirus) is a damaging pathogen of wheat and barley. This virus can survive in the soil for several decades, so the deployment of resistant cultivars represents the only practical control measure. Here, a genetic analysis has identified two regions of the barley genome-one on chromosome 2H and the other on chromosome 3H-as harboring gene(s) encoding resistance to this virus. The joint presence of both loci, termed Jmv1 and Jmv2, made the plants essentially immune, with resistance being dominant over susceptibility at each locus. Phylogenetic analysis showed that the virus is not closely related to the type Furovirus species Soil-borne wheat mosaic virus. There was a difference between the RNA1- and RNA2-based phylogenies of the virus species in Furovirus implying the independent segregation of the virus subgenomes.

First Report of Crown Rot Caused by Fusarium algeriense on Wheat in Kyrgyzstan.

Fusarium crown rot of wheat is an economically important disease that leads to significant yield and quality losses, especially in many arid and semi-arid wheat-growing areas worldwide. In June 2020, winter wheat (Triticum aestivum L.) plants exhibiting crown rot symptoms were identified in a commercial field located in the Tokbay location (43.033719°N, 74.325623°E), Chuy Province, Kyrgyzstan. The diseased plants were stunted and had brown discoloration on internodes of the stem bases and roots. Disease incidence was about 3%. A total of 10 plants were sampled at the ripening stage from the field to identify the causal agent. Symptomatic tissues were excised, surface disinfected with 1% NaOCl, rinsed three times with distilled water, and placed on one-fifth strength potato dextrose agar (PDA) followed by incubation at 23°C in the dark for 5 days. A total of 8 Fusarium isolates were recovered from tissues and purified by the hyphal tips method onto fresh PDA and Spezieller-Nährstoffarmer agar (SNA) plates (Leslie and Summerell 2006). Sequence analysis of the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were performed with primers EF1 and EF2 (O'Donnell et al. 1998), and 5f2 (Reeb et al. 2004) and 7cr (Liu et al. 1999), respectively. The sequences of three isolates showed 100% identities with the corresponding sequences of the strain NRRL 66652 of Fusarium algeriense Laraba & O'Donnell (TEF1: MF120515 and RPB2: MF120504), and the sequences of a representative isolate (KyrFa01) were deposited in GenBank (TEF1: OM135603 and RPB2: OM135604). On PDA, fungal colonies were initially yellowish-white but gradually turned yellowish-brown. Ellipsoidal microconidia produced in false heads on monophialides were usually aseptate (8.30 ± 1.17 µm, n = 50) and occasionally one-septate (21.89 ± 2.01 µm, n = 50). Sporodochial macroconidia were mostly 3-4 septate measuring 43.41 ± 2.83 µm (n = 50), slightly curved and formed generally on monophialides on SNA. No chlamydospores formation was detected after 15 days on SNA or PDA. Morphological characteristics described above were consistent with the morphology of F. algeriense, as reported by Laraba et al. (2017). To confirm pathogenicity, seeds of wheat cultivar Seri 82, Fusarium crown rot susceptible, were treated in 1% NaOCl for 2 min, rinsed twice, and placed in plates containing a piece of sterile filter paper saturated with water to induce germination for 3 days. Five pregerminated seeds were placed on the soil surface for each 9-cm-diameter pot, which was filled with a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v). A 1-cm-diameter mycelial plug taken from the margin of actively growing colonies (PDA) of the representative isolate KyrFa01 was contacted with each seed, and then seeds were covered with the same potting mix. The seeds in control pots were treated with sterile PDA plugs. The experiment was conducted in a growth chamber in a completely randomized design with five replicated pots at 23°C with a 12-h photoperiod. Disease assessment was made after 4 weeks of fungal inoculation. The isolate KyrFa01 induced discoloration on the crown and root tissues of inoculated plants similar to those observed in the field-grown plants, whereas no symptoms were observed on plants grown in the control pots. The pathogen was successfully reisolated from the symptomatic tissues, confirming Koch's postulates. To the best of our knowledge, this is the first report of crown rot caused by F. algeriense on wheat in Kyrgyzstan. Fusarium algeriense was firstly described within the Fusarium burgessii species complex by Laraba et al. (2017) as a crown rot pathogen of wheat in Algeria. The pathogen was secondly reported from wheat-growing areas in Azerbaijan (Özer et al. 2020a) and thirdly from Kyrgyzstan in this report. Özer et al. (2020b) confirmed the coexistence of this pathogen with other Fusarium species. The result warrants the need to further investigate the potential of this species in the Fusarium crown rot complex of wheat.

First Report of Fusarium incarnatum-equiseti Species Complex Causing Fruit Rot on Muskmelon in Taiwan.

Muskmelon (Cucumis melo L.) is an economically important fruit crop in Taiwan. In March 2020, the symptoms of fruit rot were observed in approximately 10% of mature muskmelon fruits in a field located in Wuri (24.043585, 120.657588), Taichung City, Taiwan. Symptoms including water-soaked lesions were initially observed on the lwer side of fruit, extending with time to cover most of the fruit area, and internal dissolution with white to brown mycelia on the surface was also observed. Ten rotted fruits were disinfested with 70% ethanol for 1 min followed by 1% NaOCl for 5 min, then rinsed three times with sterile distilled water (SDW). Fifteen sterilized symptomatic fruit fragments were cut into 1-cm3 pieces, placed on potato dextrose agar (PDA) amended with 35 mg/liter of streptomycin sulfate and incubated at 28°C in the dark for 1 week. Ten isolates with similar morphology were obtained and the representative isolate FOS-1 was characterized further. Single-spore isolates were used for morphological and molecular analyses. Isolates grown on PDA had dense, cottony white aerial mycelium, changed to light brown, and with the time yellowish-brown pigmentation appeared. Microconidia were ovoid, fusiform, or slightly curved, 0 to1 septate, and ranged between 7.9 to 16.5 × 2.8 to 3.5 µm. Macroconidia were 3 to 5 septate, with a slightly curved and tapering apical cell, and ranged between 18.7 to 35.1 × 3.3 to 4.1 µm. Spherical chlamydospores with thick walls were abundant and single, being produced in terminal or intercalary position. Based on morphological characteristics, the fungus was identified as Fusarium sp. (Leslie and Summerell 2006). PCR amplification and DNA sequencing were performed using primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) to amplify the complete internal transcribed spacer (ITS) region and the partial translation elongation factor 1-alpha (TEF1-α) gene, respectively. The ITS and TEF1-α gene sequences of Isolate FOS-1 were deposited in GenBank database with acc. nos. MZ749694.1 and MZ782277.1, respectively. BLAST analysis showed 99.64% and 100% sequence identity with F. incanatum-equiseti species complex (FIESC) with MT563419.1 for ITS and MW034437.1 for EF-1α, respectively. BLAST analysis of TEF1-α gene sequence in FUSARIUM-ID database (Geiser et al. 2004), showed 99.31% sequence identity with FIESC (NRRL34070). Pathogenicity was confirmed by fulfilling Koch's postulates. Three healthy muskmelon fruit were disinfested using 70% ethanol for 30 s and 1% NaOCl for 5 min, and followed by three rinses with SDW. Then, the fruit were wounded using a sterile needle and inoculated with an 8 mm-mycelium agar plug. Three sites per fruit were inoculated, and three other fruits treated with mycelium-free PDA plugs served as the controls. The inoculated and control fruit were placed in a plastic box and incubated at 25°C under a 12 h photoperiod for 1 week. All inoculated fruit showed symptoms similar to those observed in the field, whereas no symptoms occurred on the controls. The fungus was re-isolated from the infected fruit, and identified as FIESC by the morphological and molecular methods described above. This pathogen could cause great losses in muskmelon. Members of the FIESC have been reported to cause leaf spot and fruit rot in muskmelon (Cao et al. 2019; Ismail et al. 2021). To our knowledge, this is the first report of the FIESC causing fruit rot of muskmelon in Taiwan. References: Cao, P., et al. 2019. Plant Dis.103:1768. Carbone, I., and Kohn, L. M. 1999. Mycologia. 91:553. Geiser, D.M., et al. 2004. Eur. J. Plant Pathol. 110:473. Ismail, S. I., et al. 2021. Plant Dis. 105:1197. Leslie, J. F., and Summerell, B. A. 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, U.K. White, T. J., et al. 1990. PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA. 315.

Biological control of fungal pathogens of tomato (Lycopersicon esculentum) by chitinolytic bacterial strains.

The need to increase food production and to reduce the pollution caused by synthetic chemicals has led to a search for biocontrol agents against plant pathogens. In the present study, a total of 37 chitinolytic bacteria were isolated from the rhizospheric soil of tomatoes using a chitin agar medium. In vitro bacterial isolates, that is, TD9, TD11, TD15, and TD24 showed strong antagonistic and enzymatic activities against Rhizoctonia (8%-55%), Fusarium (31%-48%), Colletotrichum (24%-49%), and Aspergillus on a dual culture plate and enzyme assay. Furthermore, these putative antagonistic bacterial isolates were identified as Pantoea agglomerans (TD9), Bacillus subtilis (TD11), Bacillus cereus (TD15 and TD24) using 16S rRNA sequence analysis. Additionally, in culture filtrate in vivo assay, the isolates TD11 and TD15 inhibited the growth of Rhizoctonia solani about 40% and Fusarium oxysporum about 80%. However, in the pot trials, these two bacterial isolates (TD11 and TD15) considerably suppressed the disease rate in tomatoes caused by Fusarium and Rhizoctonia fungal species. Moreover, it was concluded that B. subtilis (TD11) was found to be the most promising putative biocontrol agent, inhibiting the fungal diseases of tomatoes by 50% and showing versatile antagonistic potential.