Planta Growth Research Articles

Effective Control of Neofusicoccum parvum in Grapevines: Combining Trichoderma spp. with Chemical Fungicides

Vitis vinifera is highly susceptible to grapevine trunk diseases, with Neofusicoccum parvum recognized as a highly destructive pathogen. This study investigates the biocontrol potential of five Trichoderma species (T. harzianum, T. viride, T. asperellum, and T. virens) against N. parvum, evaluating multiple biocontrol mechanisms (mycoparasitism, competition for nutrients, production of volatile organic compounds (VOCs), and antibiosis) as well as their compatibility with the fungicides copper oxychloride and sulfur. Results demonstrated that (1) Trichoderma harzianum effectively suppressed N. parvum through VOC production, mycoparasitism, and nutrient competition, significantly reducing pathogen growth in planta while showing compatibility with both fungicides, highlighting its suitability for integrated disease management; (2) Trichoderma viride showed high inhibition of N. parvum in vitro, but its phytotoxicity in planta limits its field application. These findings support T. harzianum as a promising agent within integrated disease management strategies, offering a sustainable alternative to reduce chemical fungicide reliance in controlling grapevine trunk pathogens.

1H NMR spectroscopic analysis of metabolite changes in kauri leaf apoplastic washing fluid following inoculation with Phytophthora agathidicida

Phytophthora agathidicida is responsible for a devastating dieback disease that threatens the survival of Agathis australis (kauri), an ancient conifer species endemic to New Zealand. To develop durable control strategies against kauri dieback disease, a better understanding of the host metabolites necessary for the growth and survival of P. agathidicida during in planta growth, particularly during colonisation of the apoplastic environment, where early contact between host and pathogen cells is made, is required. As a starting point to address this knowledge gap, we investigated changes in the metabolite profile of apoplastic washing fluid (AWF) samples harvested from kauri leaves following either mock inoculation or inoculation with P. agathidicida. AWF was extracted from leaves of kauri saplings and inoculated with P. agathidicida on cellophane membranes or cellophane membranes without the pathogen as a control. The metabolite profile of the AWF samples was then analysed via proton nuclear magnetic resonance (1H NMR) spectroscopy at 24 hours and 10 days post-inoculation, and changes investigated relative to the control. Some changes in the metabolite profile of kauri AWF samples following P. agathidicida inoculation were observed using 1H NMR spectroscopy, including a decrease in sucrose and an increase in glucose resulting from the breakdown of more complex carbohydrates. Our results suggest that P. agathidicida modifies or utilises metabolites present in the leaf apoplast of kauri, including carbohydrates that serve as a source of nutrition. These results provide possible new insights into the nutritional requirements of P. agathidicida during apoplastic colonisation of kauri.

MoNOT3 Subunit Has Important Roles in Infection-Related Development and Stress Responses in Magnaporthe oryzae.

The multifunctional carbon catabolite repression negative on TATA-box-less complex (CCR4-NOT) is a multi-subunit complex present in all eukaryotes, including fungi. This complex plays an essential role in gene expression; however, a functional study of the CCR4-NOT complex in the rice blast fungus Magnaporthe oryzae has not been conducted. Seven genes encoding the putative CCR4-NOT complex were identified in the M. oryzae genome. Among these, a homologous gene, MoNOT3, was overexpressed during appressorium development in a previous study. Deletion of MoNOT3 in M. oryzae resulted in a significant reduction in hyphal growth, conidiation, abnormal septation in conidia, conidial germination, and appressorium formation compared to the wild-type. Transcriptional analyses suggest that the MoNOT3 gene affects conidiation and conidial morphology by regulating COS1 and COM1 in M. oryzae. Furthermore, Δmonot3 exhibited a lack of pathogenicity, both with and without wounding, which is attributable to deficiencies in the development of invasive growth in planta. This result was also observed in onion epidermal cells, which are non-host plants. In addition, the MoNOT3 gene was involved in cell wall stress responses and heat shock. Taken together, these observations suggest that the MoNOT3 gene is required for fungal infection-related cell development and stress responses in M. oryzae.

Rapid dissemination of host metabolism–manipulating genes via integrative and conjugative elements

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.

The effect of LED light quality on the carotenoid metabolism and related gene expression in the genus Brassica

BackgroundNew vegetable production systems, such as vertical farming, but also well-established in-door production methods led to the implementation of light emitting diodes (LEDs). LEDs are the most important light sources in modern indoor-production systems and offer the possibility for enhancing growth and specific metabolites in planta. Even though the number of studies investigating the effects of LED lighting on vegetable quality has increased, the knowledge about genus variability is limited. In the present study, the effect of different LED spectra on the metabolic and transcriptional level of the carotenoid metabolism in five different Brassica sprouts was investigated. Cruciferous vegetables are one of the main food crops worldwide. Pak choi (Brassica rapa ssp. chinensis), cauliflower (Brassica oleracea var. botrytis), Chinese cabbage (Brassica rapa ssp. pekinensis), green kale (Brassica oleracea ssp. sabellica) and turnip cabbage (Brassica oleracea spp. gongylodes) sprouts were grown under a combination of blue & white LEDs, red & white LEDs or only white LEDs to elucidate the genus-specific carotenoid metabolism.ResultsGenus-specific changes in plant weight and on the photosynthetic pigment levels as well as transcript levels have been detected. Interestingly, the transcript levels of the three investigated carotenoid biosynthesis genes phytoene synthase (PSY), β-cyclase (βLCY) and β-carotene hydroxylase (βOHASE1) were increased under the combination of blue & white LEDs in the majority of the Brassica sprouts. However, only in pak choi, the combination of blue & white LEDs enhanced the carotenoid levels by 14% in comparison to only white LEDs and by ~ 19% in comparison to red & white LEDs.ConclusionsThe effects of light quality differ within a genus which leads to the conclusion that production strategies have to be developed for individual species and cultivars to fully benefit from LED technology.

Optimization of culture medium and bioformulation of rhizobial actinomycetes to enhance soybean plant growth

Abstract. Fatmawati U, Qurrata’aini MR, Rosyidi NW, Sari DP, Santosa S. 2023. Optimization of culture medium and bioformulation of rhizobial actinomycetes to enhance soybean plant growth. Biodiversitas 24: 2912-2918. Actinomycetes are a group of Gram-positive bacteria that are well-known for having plant growth-promoting activity. In an earlier study, Streptomyces sp. strain ASR 58 and Streptomyces sp. strain ASR 67, isolated from soybean rhizosphere, were found to be capable of producing Indole-Acetic Acid (IAA), dissolving phosphate, and stimulating soybean growth. Optimization of culture media and selection of carrier materials for formulation are necessary to maintain actinomycetes' cell viability and plant growth property stability. This study aimed to evaluate several culture media, determine the optimal carrier materials in actinomycetes formulation, and evaluate the effect of actinomycete bioformulation on the soybean plant growth in planta. The sugar potato broth medium was the best in increasing the cell biomass of ASR 67 and co-culture of ASR 58 and ASR 67 compared to other mediums (skim molasses and rice bran broth). The viability of ASR 58 cell (5.3 x 107 CFU/mL), ASR 67 (3.8 x 107 CFU/mL), and its combination of both (4.3 x 107 CFU/mL) was best maintained in talc powder carrier during 10 weeks of storage as revealed by the number of viable cells decreases. Applying ASR 58 and ASR 67 co-culture on talc powder formula enhanced soybean plant biomass four weeks after planting, including plant height, the number of leaves, and root dry weight.

Nitric Oxide Regulates the Ralstonia solanacearum Type III Secretion System.

Ralstonia solancearum causes bacterial wilt disease on diverse plant hosts. R. solanacearum cells enter a host from soil or infested water through the roots, then multiply and spread in the water-transporting xylem vessels. Despite the low nutrient content of xylem sap, R. solanacearum grows very well inside the host, using denitrification to respire in this hypoxic environment. R. solanacearum growth in planta also depends on the successful deployment of protein effectors into host cells via a type III secretion system (T3SS). The T3SS is absolutely required for R. solanacearum virulence, but it is metabolically costly and can trigger host defenses. Thus, the pathogen's success depends on optimized regulation of the T3SS. We found that a byproduct of denitrification, the toxic free-radical nitric oxide (NO), positively regulates the R. solanacearum T3SS both in vitro and in planta. Using chemical treatments and R. solanacearum mutants with altered NO levels, we show that the expression of a key T3SS regulator, hrpB, is induced by NO in culture. Analyzing the transcriptome of R. solanacearum responding to varying levels of NO both in culture and in planta revealed that the T3SS and effectors were broadly upregulated with increasing levels of NO. This regulation was specific to the T3SS and was not shared by other stressors. Our results suggest that R. solanacearum may experience an NO-rich environment in the plant host and that this NO contributes to the activation of the T3SS during infection. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Endophytic fungi related to the ash dieback causal agent encode signatures of pathogenicity on European ash

Tree diseases constitute a significant threat to biodiversity worldwide. Pathogen discovery in natural habitats is of vital importance to understanding current and future threats and prioritising efforts towards developing disease management strategies. Ash dieback is a fungal disease of major conservational concern that is infecting common ash trees, Fraxinus excelsior, in Europe. The disease is caused by a non-native fungal pathogen, Hymenoscyphus fraxineus. Other dieback causing-species have not previously been identified in the genus Hymenoscyphus. Here, we discover the pathogenicity potential of two newly identified related species of Asian origin, H. koreanus and H. occultus, and one Europe-native related species, H. albidus. We sequence the genomes of all three Hymenoscyphus species and compare them to that of H. fraxineus. Phylogenetic analysis of core eukaryotic genes identified H. albidus and H. koreanus as sister species, whilst H. occultus diverged prior to these and H. fraxineus. All four Hymenoscyphus genomes are of comparable size (55–62 Mbp) and GC contents (42–44%) and encode for polymorphic secretomes. Surprisingly, 1133 predicted secreted proteins are shared between the ash dieback pathogen H. fraxineus and the three related Hymenoscyphus endophytes. Amongst shared secreted proteins are cell death-inducing effector candidates, such as necrosis, and ethylene-inducing peptide 1-like proteins, Nep1-like proteins, that are upregulated during in planta growth of all Hymenoscyphus species. Indeed, pathogenicity tests showed that all four related Hymenoscyphus species develop pathogenic growth on European ash stems, with native H. albidus being the least virulent. Our results identify the threat Hymenoscypohus species pose to the survival of European ash trees, and highlight the importance of promoting pathogen surveillance in environmental landscapes. Identifying new pathogens and including them in the screening for durable immunity of common ash trees is key to the long-term survival of ash in Europe.

The endophytobiome of wild Rubiaceae as a source of antagonistic fungi against the American Leaf Spot of coffee (Mycena citricolor).

The American leaf spot, caused by Mycena citricolor, is an important disease of coffee (Coffea arabica), mostly in Central America. Currently, there are limited pathogen control alternatives that are environment friendly and economically accessible. The use of fungi isolated from the plant endomycobiota in their native habitats is on the rise because studies show their great potential for biological control. To begin to generate a green alternative to control M. citricolor, the objectives of the present study were to (i) collect, identify, screen (in vitro and in planta), and select endophytic fungi from wild Rubiaceae collected in old-growth forests of Costa Rica; (ii) confirm endophytic colonization in coffee plantlets; (iii) evaluate the effects of the endophytes on plantlet development; and (iv) corroborate the antagonistic ability in planta. Through in vitro and in planta antagonism assays, we found that out of the selected isolates (i.e. Daldinia eschscholzii GU11N, Nectria pseudotrichia GUHN1, Purpureocillium aff. lilacinum CT24, Sarocladium aff. kiliense CT25, Trichoderma rifaii CT5, T. aff. crassum G1C, T. aff. atroviride G7T, T. aff. strigosellum GU12, and Xylaria multiplex GU14T), Trichoderma spp. produced the highest growth inhibition percentages in vitro. Trichoderma isolates CT5 and G1C were then tested in planta using Coffea arabica cv. caturra plantlets. Endophytic colonization was verified, followed by in planta growth promotion and antagonism assays. Results show that Trichoderma isolates CT5 and G1C have potential for plant growth promotion and antagonism against Mycena citricolor, reducing incidence and severity, and preventing plant mortality.

Contrasting effector profiles between bacterial colonisers of kiwifruit reveal redundant roles converging on PTI-suppression and RIN4.

Testing effector knockout strains of the Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) for reduced in planta growth in their native kiwifruit host revealed a number of nonredundant effectors that contribute to Psa3 virulence. Conversely, complementation in the weak kiwifruit pathogen P.syringae pv. actinidifoliorum (Pfm) for increased growth identified redundant Psa3 effectors. Psa3 effectors hopAZ1a and HopS2b and the entire exchangeable effector locus (ΔEEL; 10 effectors) were significant contributors to bacterial colonisation of the host and were additive in their effects on virulence. Four of the EEL effectors (HopD1a, AvrB2b, HopAW1a and HopD2a) redundantly contribute to virulence through suppression of pattern-triggered immunity (PTI). Important Psa3 effectors include several redundantly required effectors early in the infection process (HopZ5a, HopH1a, AvrPto1b, AvrRpm1a and HopF1e). These largely target the plant immunity hub, RIN4. This comprehensive effector profiling revealed that Psa3 carries robust effector redundancy for a large portion of its effectors, covering a few functions critical to disease.

Bioassessment of Phylogenetic Relatedness and Plant Growth Enhancement of Endophytic Bacterial Isolates from Cowpea (Vigna unguiculata) Plant Tissues

Cowpea is of great importance to people in most tropical countries of the world. It is the preeminent indigenous African legume and a frontline option for meeting the nutritional protein demands of people and livestock. The use of an eco-friendly alternative to synthetic fertilizers and agro-pesticides has, in recent times, become an attractive research theme. Therefore, bioprospecting for effective endophytic bacteria isolates as potential bioinoculants for enhancing cowpea productivity makes this research a priority. In this study, cowpea tissues were used to isolate and characterize endophytic bacterial strains through morpho-genotypic techniques and then assessed for their in vitro growth promotion, as well as their in planta growth potential in chamber experiments. In all, 33 endophytic bacterial strains were authenticated by sequencing the 16S rRNA and through further bioinformatics analysis. Also, plant-growth promoting (PGP) genes and seed germination percentage improvements were confirmed in the endophytic bacteria isolates. The research findings highlight that the bacterial strains are molecularly diverse and some of the authenticated endophytic bacteria isolates are potential bioinoculants that can be applied in further studies to improve the agronomic productivity of cowpea plant.

Bacterial outer membrane vesicles induce a transcriptional shift in arabidopsis towards immune system activation leading to suppression of pathogen growth in planta.

Gram-negative bacteria form spherical blebs on their cell periphery, which later dissociate from the bacterial cell wall to form extracellular vesicles. These nano scale structures, known as outer membrane vesicles (OMVs), have been shown to promote infection and disease and can induce typical immune outputs in both mammal and plant hosts. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated plants at 2, 6 and 24h post challenge. The most pronounced transcriptional shift occurred at the first two time points tested, as reflected by the number of differentially expressed genes and the average fold change. OMVs induce a major transcriptional shift towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors. Comparing the response of Arabidopsis to OMVs and to purified elicitors, revealed that OMVs induce a similar suite of genes and pathways as single elicitors, however, pathways activated by OMVs and not by other elicitors were detected. Pretreating Arabidopsis plants with OMVs and subsequently infecting with a bacterial pathogen led to a significant reduction in pathogen growth. Mutations in the plant elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) co-receptor, did not significantly affect the immune priming effect of OMVs. All together these results show that OMVs induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change may facilitate resistance to bacterial infection.

Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

Development of siderophore-based rhizobacterial consortium for the mitigation of biotic and abiotic environmental stresses in tomatoes: An in vitro and in planta approach.

Tomato-associated plant-growth-promoting rhizosphere bacteria were screened for effective antagonistic activity against the fungal vascular wilt pathogens; tolerance to heavy metals; and enhancing the bioavailability of iron for tomato plants through in vitro and in vivo approaches. Among the 121 rhizobacteria screened for siderophores, 25 isolates were observed to be siderophore producers and out of these, seven isolates chelate copper and iron thus exhibiting in vitro antagonism against the virulent strains of Fusarium oxysporum f. sp. lycopersici MTCC10270 (Fol), Fusarium equiseti MFol and Sarocladium sp. SWL isolated from infected tomatoes. Pseudomonas stutzeri KRP8 was identified to be the most potent strain among the siderophore producers and its siderophores were chemically characterized by mass spectra as metal bound and metal-free forms. Upon bio-inoculation of fortified bacterial consortium (siderozote) into the rhizosphere of vermiculite pot cultured tomatoes supplied with varying concentrations of iron and copper ions, we observed in planta growth improvements, antagonism, enhancement of bioavailability of iron and heavy metal tolerance using Inductively Coupled Plasma-Optical Emission Spectrometry. Our rhizobacterial consortium provides an opportunity for soil reclamation through an ecofriendly method for a heavy metal-free agricultural landscape.

OxyR contributes to virulence of Acidovorax citrulli by regulating anti-oxidative stress and expression of flagellin FliC and type IV pili PilA.

In many bacteria, OxyR acts as a transcriptional regulator that facilitates infection via degrading hydrogen peroxide (H2O2) generated by the host defense response. Previous studies showed that OxyR also plays an important role in regulating biofilm formation, cell motility, pili relate-genes expression, and surface polysaccharide production. However, the role of OxyR has not been determined in Acidovorax citrulli strain xjl12. In the current study, the qRT-PCR and western blot assays revealed that the expression level of oxyR was significantly induced by H2O2. The oxyR deletion mutant of A. citrulli was significantly impaired bacterial tolerance to oxidative stress and reduced catalase (CAT) activity. In addition, oxyR mutant resulted in reduced swimming motility, twitching motility, biofilm formation, virulence, and bacterial growth in planta by significantly affecting flagellin and type IV pili-related gene (fliC and pilA) expression. The qRT-PCR assays and western blot revealed that OxyR positively regulated the expression of fliC and pilA. Furthermore, bacterial one-hybrid assay demonstrated that OxyR directly affected pilA and fliC promoter. Through bacterial two-hybrid assay, it was found that OxyR can directly interact with PilA and FliC. These results suggest that OxyR plays a major role in the regulating of a variety of virulence traits, and provide a foundation for future research on the global effects of OxyR in A. citrulli.

Pseudomonas fluorescensNK4 siderophore promotes plant growth and biocontrol in cucumber.

To test the effect of zinc oxide nanoparticle (ZnO-NP) supplementation for enhancing the efficacy of Pseudomonas fluorescens NK4 siderophore as a biocontrol agent against P. viridiflava NK2 and a plant growth promoter. Cucumber seedlings were treated with a suspension of P. fluorescens NK4 and its siderophore generated in siderophore-inducing medium (SIM), SIM supplemented with ZnO-NP (<100 nm) and SIM supplemented with Zn2+ ions from Zn(NO3 )2 . Supplementing SIM with ZnO-NP increased siderophore secretion in P. fluorescens NK4, and irrigation of cucumber seedlings with a filtrate containing the ZnO-NP-supplemented siderophore increased survival, improved vegetative and root growth, and thus increased yield similar to the effects of dipping seedlings in a P. fluorescens NK4 suspension. Both P. fluorescens NK4 and its ZnO-NP-supplemented siderophore inhibited P. viridiflava NK2 population growth in planta. The siderophore of P. fluorescens NK4 produced by ZnO-NP supplementation can be employed as a biocontrol agent and biofertilizer. ZnO-NPs can boost the synthesis of siderophores, which can then be employed as biofertilizers to boost iron bioavailability in iron-deficient soils.

Sulforaphane, a secondary metabolite in crucifers, inhibits the oxidative stress adaptation and virulence of Xanthomonas by directly targeting OxyR.

Plant secondary metabolites perform numerous functions in the interactions between plants and pathogens. However, little is known about the precise mechanisms underlying their contribution to the direct inhibition of pathogen growth and virulence in planta. Here, we show that the secondary metabolite sulforaphane (SFN) in crucifers inhibits the growth, virulence, and ability of Xanthomonas species to adapt to oxidative stress, which is essential for the successful infection of host plants by phytopathogens. The transcription of oxidative stress detoxification‐related genes (catalase [katA and katG] and alkylhydroperoxide‐NADPH oxidoreductase subunit C [ahpC]) was substantially inhibited by SFN in Xanthomonas campestris pv. campestris (Xcc), and this phenomenon was most obvious in sax gene mutants sensitive to SFN. By performing microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA), we observed that SFN directly bound to the virulence‐related redox‐sensing transcription factor OxyR and weakened the ability of OxyR to bind to the promoters of oxidative stress detoxification‐related genes. Collectively, these results illustrate that SFN directly targets OxyR to inhibit the bacterial adaptation to oxidative stress, thereby decreasing bacterial virulence. Interestingly, this phenomenon occurs in multiple Xanthomonas species. This study provides novel insights into the molecular mechanisms by which SFN limits Xanthomonas adaptation to oxidative stress and virulence, and the findings will facilitate future studies on the use of SFN as a biopesticide to control Xanthomonas.

A Short Review of Anti-Rust Fungi Peptides: Diversity and Bioassays

Pucciniales are fungal pathogens of plants that cause devastating rust diseases in agriculture. Chemically-synthesized pesticides help farmers to control rust epidemics, but governing bodies aim at limiting their use over the next decade. Defense peptides with antimicrobial activities may help to innovate a next generation of phytosanitary products for sustainable crop protection. This review comprehensively inventories the proteins or peptides exhibiting a biochemically-demonstrated antifungal activity toward Pucciniales (i.e., anti-rust proteins or peptides; hereafter ‘ARPs’), and also analyses the bioassays used to characterize them. In total, the review scrutinizes sixteen publications, which collectively report 35 ARPs. These studies used either in vitro or in planta bioassays, or a combination of both, to characterize ARPs; mostly by evaluating their ability to inhibit the spore germination process in vitro or to inhibit fungal growth and rust disease development in planta. Also, the manuscript shows that almost no mode of action against rust fungi was elucidated, although some might be inferred from studies performed on other fungi. This short review may serve as a knowledge and methodological basis to inform future studies addressing ARPs.

Evaluating the Antagonistic Potential of Actinomycete Strains Isolated From Sudan's Soils Against Phytophthora infestans.

Soil microorganisms play crucial roles in soil fertility, e.g., through decomposing organic matter, cycling nutrients or through beneficial interactions with plants. Actinomycetes are a major component of soil inhabitants; they are prolific producers of specialized metabolites, among which many antibiotics. Here we report the isolation and characterization of 175 Actinomycetes from rhizosphere and bulk soil samples collected in 18 locations in Sudan. We evaluated the strains’ metabolic potential for plant protection by testing their ability to inhibit the mycelial growth of the oomycete Phytophthora infestans, which is one of the most devastating plant pathogens worldwide. Most strains significantly reduced the oomycete’s growth in direct confrontational in vitro assays. A significant proportion of the tested strains (15%) were able to inhibit P. infestans to more than 80%, 23% to 50%–80%, while the remaining 62% had inhibition percentages lesser than 50%. Different morphologies of P. infestans mycelial growth and sporangia formation were observed upon co-inoculation with some of the Actinomycetes isolates, such as the production of fewer, thinner hyphae without sporangia leading to a faint growth morphology, or on the contrary, of clusters of thick-walled hyphae leading to a bushy, or “frozen” morphology. These morphologies were caused by strains differing in activity levels but phylogenetically closely related with each other. To evaluate whether the isolated Actinomycetes could also inhibit the pathogen’s growth in planta, the most active strains were tested for their ability to restrict disease progress in leaf disc and full plant assays. Five of the active strains showed highly significant protection of potato leaves against the pathogen in leaf disc assays, as well as substantial reduction of disease progress in full plants assays. Using cell-free filtrates instead of the bacterial spores also led to full protection against disease on leaf discs, which highlights the strong crop protective potential of the secreted metabolites that could be applied as leaf spray. This study demonstrates the strong anti-oomycete activity of soil- and rhizosphere-borne Actinomycetes and highlights their significant potential for the development of sustainable solutions based on either cell suspensions or cell-free filtrates to safeguard potatoes from their most damaging pathogen.

A comparison of ImageJ and machine learning based image analysis methods to measure cassava bacterial blight disease severity

BackgroundMethods to accurately quantify disease severity are fundamental to plant pathogen interaction studies. Commonly used methods include visual scoring of disease symptoms, tracking pathogen growth in planta over time, and various assays that detect plant defense responses. Several image-based methods for phenotyping of plant disease symptoms have also been developed. Each of these methods has different advantages and limitations which should be carefully considered when choosing an approach and interpreting the results.ResultsIn this paper, we developed two image analysis methods and tested their ability to quantify different aspects of disease lesions in the cassava-Xanthomonas pathosystem. The first method uses ImageJ, an open-source platform widely used in the biological sciences. The second method is a few-shot support vector machine learning tool that uses a classifier file trained with five representative infected leaf images for lesion recognition. Cassava leaves were syringe infiltrated with wildtype Xanthomonas, a Xanthomonas mutant with decreased virulence, and mock treatments. Digital images of infected leaves were captured overtime using a Raspberry Pi camera. The image analysis methods were analyzed and compared for the ability to segment the lesion from the background and accurately capture and measure differences between the treatment types.ConclusionsBoth image analysis methods presented in this paper allow for accurate segmentation of disease lesions from the non-infected plant. Specifically, at 4-, 6-, and 9-days post inoculation (DPI), both methods provided quantitative differences in disease symptoms between different treatment types. Thus, either method could be applied to extract information about disease severity. Strengths and weaknesses of each approach are discussed.