Screening of the High-Rhizosphere Competent Limoniastrum monopetalum' Culturable Endophyte Microbiota Allows the Recovery of Multifaceted and Versatile Biocontrol Agents.
ABSTRACT: Halophyte Limoniastrum monopetalum, an evergreen shrub inhabiting the Mediterranean region, has well-documented phytoremediation potential for metal removal from polluted sites. It is also considered to be a medicinal halophyte with potent activity against plant pathogens. Therefore, L. monopetalum may be a suitable candidate for isolating endophytic microbiota members that provide plant growth promotion (PGP) and resistance to abiotic stresses. Selected for biocontrol abilities, these endophytes may represent multifaceted and versatile biocontrol agents, combining pathogen biocontrol in addition to PGP and plant protection against abiotic stresses. In this study 117 root culturable bacterial endophytes, including Gram-positive (Bacillus and Brevibacillus), Gram-negative (Proteus, Providencia, Serratia, Pantoea, Klebsiella, Enterobacter and Pectobacterium) and actinomycete Nocardiopsis genera have been recovered from L. monopetalum. The collection exhibited high levels of biocontrol abilities against bacterial (Agrobacterium tumefaciens MAT2 and Pectobacterium carotovorum MAT3) and fungal (Alternaria alternata XSZJY-1, Rhizoctonia bataticola MAT1 and Fusarium oxysporum f. sp. radicis lycopersici FORL) pathogens. Several bacteria also showed PGP capacity and resistance to antibiotics and metals. A highly promising candidate Bacillus licheniformis LMRE 36 with high PGP, biocontrol, metal and antibiotic, resistance was subsequently tested in planta (potato and olive trees) for biocontrol of a collection of 14 highly damaging Fusarium species. LMRE 36 proved very effective against the collection in both species and against an emerging Fusarium sp. threatening olive trees culture in nurseries. These findings provide a demonstration of our pyramiding strategy. Our strategy was effective in combining desirable traits in biocontrol agents towards broad-spectrum resistance against pathogens and protection of crops from abiotic stresses. Stacking multiple desirable traits into a single biocontrol agent is achieved by first, careful selection of a host for endophytic microbiota recovery; second, stringent in vitro selection of candidates from the collection; and third, application of the selected biocontrol agents in planta experiments. That pyramiding strategy could be successfully used to mitigate effects of diverse biotic and abiotic stresses on plant growth and productivity. It is anticipated that the strategy will provide a new generation of biocontrol agents by targeting the microbiota of plants in hostile environments.
Project description:Fusarium is a complex genus of ascomycete fungi that consists of plant pathogens of agricultural relevance. Controlling Fusarium infection in crops that leads to substantial yield losses is challenging. These economic losses along with environmental and human health concerns over the usage of chemicals in attaining disease control are shifting focus toward the use of biocontrol agents for effective control of phytopathogenic Fusarium spp. In the present study, an analysis of the plant-growth promoting (PGP) and biocontrol attributes of four bacilli (Bacillus simplex 30N-5, B. simplex 11, B. simplex 237, and B. subtilis 30VD-1) has been conducted. The production of cellulase, xylanase, pectinase, and chitinase in functional assays was studied, followed by in silico gene analysis of the PGP-related and biocontrol-associated genes. Of all the bacilli included in this study, B. subtilis 30VD-1 (30VD-1) demonstrated the most effective antagonism against Fusarium spp. under in vitro conditions. Additionally, 100 ?g/ml of the crude 1-butanol extract of 30VD-1's cell-free culture filtrate caused about 40% inhibition in radial growth of Fusarium spp. Pea seed bacterization with 30VD-1 led to considerable reduction in wilt severity in plants with about 35% increase in dry plant biomass over uninoculated plants growing in Fusarium-infested soil. Phase contrast microscopy demonstrated distortions and abnormal swellings in F. oxysporum hyphae on co-culturing with 30VD-1. The results suggest a multivariate mode of antagonism of 30VD-1 against phytopathogenic Fusarium spp., by producing chitinase, volatiles, and other antifungal molecules, the characterization of which is underway.
Project description:Oomycete and fungal pathogens, mainly Phytophthora and Fusarium species, are notorious causal agents of huge economic losses and environmental damages. For instance, Phytophthora ramorum, Phytophthora cryptogea, Phytophthora plurivora and Fusarium solani cause significant losses in nurseries and in forest ecosystems. Chemical treatments, while harmful to the environment and human health, have been proved to have little or no impact on these species. Recently, biocontrol bacterial species were used to cope with these pathogens and have shown promising prospects towards sustainable and eco-friendly agricultural practices. Olive trees prone to Phytophthora and Fusarium disease outbreaks are suitable for habitat-adapted symbiotic strategies, to recover oomycetes and fungal pathogen biocontrol agents. Using this strategy, we showed that olive trees-associated microbiome represents a valuable source for microorganisms, promoting plant growth and healthy benefits in addition to being biocontrol agents against oomycete and fungal diseases. Isolation, characterization and screening of root microbiome of olive trees against numerous Phytophthora and other fungal pathogens have led to the identification of the Bacillus velezensis OEE1, with plant growth promotion (PGP) abilities and strong activity against major oomycete and fungal pathogens. Phylogenomic analysis of the strain OEE1 showed that B. velezensis suffers taxonomic imprecision that blurs species delimitation, impacting their biofertilizers' practical use. Genome mining of several B. velezensis strains available in the GenBank have highlighted a wide array of plant growth promoting rhizobacteria (PGPR) features, metals and antibiotics resistance and the degradation ability of phytotoxic aromatic compounds. Strain OEE1 harbours a large repertoire of secreted and volatile secondary metabolites. Rarefaction analysis of secondary metabolites richness in the B. velezenis genomes, unambiguously documented new secondary metabolites from ongoing genome sequencing efforts that warrants more efforts in order to assess the huge diversity in the species. Comparative genomics indicated that B. velezensis harbours a core genome endowed with PGP features and accessory genome encoding diverse secondary metabolites. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of OEE1 Volatile Organic Compounds (VOCs) and Liquid Chromatography High Resolution Mas Spectrometry (LC-HRMS) analysis of secondary metabolites identified numerous molecules with PGP abilities that are known to interfere with pathogen development. Moreover, B. velezensis OEE1 proved effective in protecting olive trees against F. solani in greenhouse experiments and are able to inhabit olive tree roots. Our strategy provides an effective means for isolation of biocontrol agents against recalcitrant pathogens. Their genomic analysis provides necessary clues towards their efficient implementation as biofertilizers.
Project description:Plant growth promoting rhizobacteria (PGPR) are potential natural alternatives to chemical fungicides in greenhouse production via inducing plant immune system against biotic stresses. In this research, 126 Streptomyces isolates were recovered from rhizosphere soils of 13 different commercial vegetable greenhouses in Iran. Streptomyces isolates were screened for in vitro Plant growth promoting (PGP) traits and ability to antagonize Fusarium oxysporum f. sp. lycopersici race 3 (FOL), the causal agent of Fusarium wilt of tomato (FWT). Six isolates with the highest antagonistic activity and at least three PGP traits were selected and compared with chemical fungicide Carbendazim® in a greenhouse experiment. All bacterial treatments mitigated FWT disease symptoms like chlorosis, stunting and wilting at the same level or better than Carbendazim®. Strains IC10 and Y28 increased shoot length and shoot fresh and dry weight compared to not inoculated control plants. Phenotypic characterization and 16S rRNA gene sequencing showed, strains IC10 and Y28 were closely related to S. enissocaesilis and S. rochei, respectively. The ability of the superior biocontrol strains to induce antioxidant enzymes activity and systemic resistance (ISR) was investigated. Increased activity of catalase (CAT) in plant treated with both strains as well as an increase in peroxidase (POX) activity in plants treated with Y28 pointed to a strain specific-induced systemic resistance (ss-ISR) in tomato against FOL. The differential induced expression of WRKY70 and ERF1 (two transcription factors involved in plant defense) and LOX and TPX by the analyzed Streptomyces strains, especially after inoculation with FOL, suggests that ss-ISR is triggered at the molecular level.
Project description:Plants are exposed to biotic stresses caused by pathogen attack and complex abiotic stresses including heat and drought by dynamic climate changes. To alleviate these stresses, we investigated two bacterial stains, H26-2 and H30-3 in two cultivars ('Ryeokkwang' and 'Buram-3-ho') of Chinese cabbage in plastic pots in a greenhouse. We evaluated effects of bacterial strains on plant growth-promotion and mitigation of heat and drought stresses; the role of exopolysaccharides as one of bacterial determinants on alleviating stresses; biocontrol activity against soft rot caused by <i>Pectobacterium carotovorum</i> subsp. <i>carotovorum</i> PCC21. Strains H26-2 and H30-3 significantly increased fresh weights compared to a MgSO<sub>4</sub> solution; reduced leaf wilting and promoted recovery after re-watering under heat and drought stresses. Chinese cabbages treated with H26-2 and H30-3 increased leaf abscisic acid (ABA) content and reduced stomatal opening after stresses treatments, in addition, these strains stably colonized and maintained their populations in rhizosphere during heat and drought stresses. As well as tested bacterial cells, exopolysaccharides (EPS) of H30-3 could be one of bacterial determinants for alleviation of tested stresses in Chinese cabbages, however, the effects were different to cultivars of Chinese cabbages. In addition to bacterial activity to abiotic stresses, H30-3 could suppress incidence (%) of soft rot in 'Buram-3-ho'. The tested strains were identified as <i>Bacillus aryabhattai</i> H26-2 and <i>B. siamensis</i> H30-3 based on 16S rRNA gene sequence analysis. Taken together, H26-2 and H30-3 could be candidates for both plant growth promotion and mitigation of heat and drought stresses in Chinese cabbage.
Project description:Given the major threat of phytopathogenic bacteria to food production and ecosystem stability worldwide, novel alternatives to conventional chemicals-based agricultural practices are needed to combat these bacteria. The objective of this study is to evaluate the ability of Pseudomonas segetis strain P6, which was isolated from the Salicornia europaea rhizosphere, to act as a potential biocontrol agent given its plant growth-promoting (PGP) and quorum quenching (QQ) activities. Seed biopriming and in vivo assays of tomato plants inoculated with strain P6 resulted in an increase in seedling height and weight. We detected QQ activity, involving enzymatic degradation of signal molecules in quorum sensing communication systems, against a broad range of N-acylhomoserine lactones (AHLs). HPLC-MRM data and phylogenetic analysis indicated that the QQ enzyme was an acylase. The QQ activity of strain P6 reduced soft rot symptoms caused by Dickeya solani, Pectobacterium atrosepticum and P. carotovorum on potato and carrot. In vivo assays showed that the PGP and QQ activities of strain P6 protect tomato plants against Pseudomonas syringae pv. tomato, indicating that strain P6 could have biotechnological applications. To our knowledge, this is the first report to show PGP and QQ activities in an indigenous Pseudomonas strain from Salicornia plants.
Project description:Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1) homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea (BC) in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC) with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1). Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the necrotic leaf lesions caused by BC while plants treated with the Thmbf1-overexpressing transformants exhibited an increased susceptibility to this pathogen. The percentages of Fusarium wilt disease incidence and values of aboveground dry weight showed that T34 did not have biocontrol activity against FO, at least in the 'Moneymaker' tomato variety, and that Thmbf1 overexpression increased the incidence of this disease. Our results show that the Thmbf1 overexpression in T34 negatively affects its biocontrol mechanisms.
Project description:Animals and plants respond to bacterial infections and environmental stresses by inducing overlapping repertoires of defense genes. How the signals associated with infection and abiotic stresses are differentially integrated within a whole organism remains to be fully addressed. We show that the transcription of a Caenorhabditis elegans ABC transporter, pgp-5 is induced by both bacterial infection and heavy metal stress, but the magnitude and tissue distribution of its expression differs, depending on the type of stressor. PGP-5 contributes to resistance to bacterial infection and heavy metals. Using pgp-5 transcription as a read-out, we show that signals from both biotic and abiotic stresses are integrated by TIR-1, a TIR domain adaptor protein orthologous to human SARM, and a p38 MAP kinase signaling cassette. We further demonstrate that not all the TIR-1 isoforms are necessary for nematode resistance to infection, suggesting a molecular basis for the differential response to abiotic and biotic stress.
Project description:Selection of biological control agents (BCA) profits from an integrated study of the tripartite interactions occurring among the BCA, the plant and the pathogen. The environment plays a crucial role in the efficacy of BCA, therefore, the selection process shall utmost mimic naturally occurring conditions. To identify effective biocontrol strains against Fusarium graminearum, the major cause of Fusarium head blight (FHB) in wheat and deoxynivalenol (DON) accumulation in grains, a workflow consisting of in vitro and in vivo assays was set up. Twenty-one Streptomyces strains, 16 of which were endophytes of different plants, were analyzed. In vitro and in vivo tests characterized their plant growth promoting (PGP) traits. Biocontrol activity against F. graminearum was firstly assessed with a dual culture assay. An in vivo germination blotter assay measured Fusarium foot rot and root rot symptoms (FFR-FRR) reduction as well as growth parameters of the plant treated with the Streptomyces strains. A selected subset of Streptomyces spp. strains was then assessed in a growth chamber measuring FFR symptoms and growth parameters of the wheat plant. The approach led to the identification of an effective Streptomyces sp. strain, DEF09, able to inhibit FHB on wheat in controlled conditions by blocking the spread of the pathogen at the infection site. The results were further confirmed in field conditions on both bread and durum wheat, where DEF09 decreased disease severity up to 60%. This work confirms that FRR and FFR pathosystems can be used to identify BCA effective against FHB.
Project description:Native Bacillus sp. strain D5 coded as (Bar D5) has been isolated from the saffron corm that showed plant growth promotion (PGP) properties and also inhibits the growth of corm rot causing Fusarium oxysporum R1 (Fox R1) in-vitro. Bar D5 was more efficient PGP bacterium in comparison to earlier reported native bio-formulations by our group. Pot assays and field evaluation of Bar D5 confirmed its in-vivo efficacy for PGP traits and biocontrol activity as well. Pot trials were followed by field trials at traditional (Kishtwar) and non-traditional (R.S Pura) saffron cultivation areas in Jammu and Kashmir. At both places, Bar D5 bio-formulation treatment led to the increase in root number & length, shoot number & length, flower number and number & weight of daughter corms. Additionally, it also decreased the corm rot disease incidence significantly. Priming of corms with bio-formulation resulted in the reduction of pathogenic fungal load by three fold at the depth of corm sowing from ground level. The shelf life/viability of Bar D5 based bio-formulation was found to be 52% (viable spores) for one year at room temperature. Draft genome sequence of Bar D5 revealed the presence of genes necessary for PGP and biocontrol activity. Further, confirmation of gene sequences and annotation was done by amplification, re-sequencing and mapping of PGP and biocontrol genes on draft genome. Bar D5 based bio-formulation can be provided to companies/researchers interested in saffron cultivation or bio-formulation production for commercial exploitation, since saffron is grown as revenue crop across continents. The present study bridges the gap between genomics and its field application.
Project description:A bacterium, isolated from nodules of chickpea grown in alluvial soils of Haryana state of India, designated as IC-76 was characterized for in vitro plant growth-promoting (PGP) properties and further evaluated under greenhouse, on-station and on-farm field conditions for PGP activity in chickpea. The isolate IC-76 produced indole acetic acid, siderophore, hydrocyanic acid, cellulase, protease, and β-1,3-glucanase. When the bacterium was evaluated individually for their PGP potential in the greenhouse on chickpea and in combination with five Streptomyces sp. (strains CAI-24, CAI-121, CAI-127, KAI-32, and KAI-90; demonstrated earlier as biocontrol potential against Fusarium wilt disease in chickpea), the traits, including nodule number and weight, shoot, and root weight, pod number and weight, seed number and weight, available phosphorus and % organic carbon were found significantly, enhanced over un-inoculated control. In the on-station and on-farm field conditions, IC-76 significantly enhanced nodule number and weight, shoot, and root weight, stover and grain yield and total dry matter. In the rhizosphere (0-15 cm soil), the bacterium also significantly enhanced the total nitrogen, available phosphorus and % organic carbon. The sequence of 16S rDNA gene of the IC-76 was matched with Pseudomonas geniculata in BLAST analysis. This study demonstrates that IC-76 has the potential for PGP in chickpea.