Antagonism Assays Research Articles

Isolation and screening of rhizobacteria as biocontrol agents against Fusarium oxysporum f.sp vanillae

One of the major diseases that can reduce vanilla growth and production is stem rot caused by Fusarium oxysporum f.sp. vanillae. The objective of this study is to evaluate the rhizobacteria as biocontrol agents against Fusarium oxysporum f.sp vanillae. This research consists of isolation of rhizobacteria, antagonism assays, and identification of the rhizobacteria. A total of 263 isolates were evaluated in antagonism assays. There were 250 isolates isolated from vanilla, and 13 isolates were the collection of Plant Protection Laboratory, Indonesian Spice and Medicinal Crops Research Institute. The results showed that seven rhizobacteria isolates, i.e., L34, PS4, V116, KB7, KB10, M117a, and V112, could inhibit the mycelium growth by more than 60%. Rhizobacteria isolate L35, PS4, and V112 showed the highest inhibition, i.e., 64.92%, 72.72%, 68.72%, respectively. Based on the 16S rRNA sequence, isolate L35, PS4, and V112 were identified as Burkholderia vietnamiensis, B. ambifaria, and B lata, respectively. Further studies are needed to investigate the interaction between rhizobacteria and the host plants and formulate products to evaluate their effectiveness in the fields.

Isolation and Identification of Lipopeptide-Producing Bacillus velezensis Strains from Wheat Phyllosphere with Antifungal Activity against the Wheat Pathogen Zymoseptoria tritici

Septoria tritici blotch, caused by the fungal pathogen Zymoseptoria tritici, is a highly significant disease on wheat crops worldwide. The objective of the present study was to find out new bacterial strains with bio-antimicrobial activity against Z. tritici. Two phyllospheric bacteria (S1 and S6) were isolated from wheat ears and identified as Bacillus velezensis strains according to 16S rRNA Sanger sequencing. Antagonistic assays performed with either living strains or cell-free culture filtrates showed significant in vitro antifungal activities against Z. tritici. For the culture filtrates, the half-maximal inhibitory dilution and the minimal inhibitory dilution were 1.4% and 3.7% for the strain S1, and 7.4% and 15% for the strain S6, respectively. MALDI—ToF analysis revealed that both strains synthesize cyclic lipopeptides but from different families. Interestingly, only strain S1 produces putative bacillomycin D. Such differential lipopeptide production patterns might explain the difference observed between the antifungal activity of the culture filtrates of the two strains. This study allows the identification of new lipopeptide-producing strains of B. velezensis with a high potential of application for the biocontrol of Z. tritici.

Evaluation of Streptomyces saraciticas as Soil Amendments for Controlling Soil-Borne Plant Pathogens.

Soil-borne diseases are the major problems in mono cropping. A mixture (designated LTM-m) composed of agricultural wastes and a beneficial microorganism Streptomyces saraceticus SS31 was used as soil amendments to evaluate its efficacy for managing Rhizoctonia solani and root knot nematode (Meloidogyne incognita). In vitro antagonistic assays revealed that SS31 spore suspensions and culture broths effectively suppressed the growth of R. solani, reduced nematode egg hatching, and increased juvenile mortality. Assays using two Petri dishes revealed that LTM-m produced volatile compounds to inhibit the growth of R. solani and cause mortality to the root knot nematode eggs and juveniles. Pot and greenhouse tests showed that application of 0.08% LTM-m could achieve a great reduction of both diseases and significantly increase plant fresh weight. Greenhouse trials revealed that application of LTM-m could change soil properties, including soil pH value, electric conductivity, and soil organic matter. Our results indicate that application of LTM-m bio-organic amendments could effectively manage soil-borne pathogens.

First report of Serratia marcescens associated with black rot of Citrus sinensis fruit , and evaluation of its biological control measures in Bangladesh

Background: The present study was designed to isolate and identify the phyto-pathogen responsible for black rot of Citrus sinensis, and to determine its biological control measures. Methods: The pathogen was isolated from infected oranges and cultured on Luria-Bertani medium. Gram staining method was used to identify the morphological characteristics of the causal agents of the black rot. Advanced molecular technique was applied to facilitate proper detection of the isolated bacteria. Phylogenetic trees were analyzed using the Neighbor-Joining method. Antimicrobial screening was conducted by disc diffusion method. Antagonistic activity was evaluated by well diffusion method. Results: Gram staining of the causal agent showed rod shaped, small and pink bacteria. Polymerase chain reaction of the 16S ribosomal RNA gene amplified an approximately 1465 bp product. The nucleotide sequences of the isolated bacterial sample 1 (BS1) and bacterial sample 2 (BS2) had 99.34% and 99.45% similarities with the reference of Serratia marcescens sequence in NCBI GenBank. The obtained sequences were deposited in GenBank. Two isolates showed virulence capability on some fresh fruits, which confirmed the stain detection and Koch’s postulates. Allium sativum extract showed the largest (27.33±1.5 mm) diameter of zone of inhibition against BS1, at 30µg/disc concentration. In the antagonistic assay, Rhizobium leguminosarum showed largest (19±1 mm) zone of inhibition against BS1. Conclusions: Findings of the current investigations are constructive for identification of causative pathogens in Citrus sinensis black rot disease and their biological control measures.

Fungi Associated with Postharvest Diseases of Sweet Potato Storage Roots and In Vitro Antagonistic Assay of Trichodermaharzianum against the Diseases.

Sweet potato is the 11th most important food crop in the world and an excellent source of nutrition. Postharvest diseases were monitored in sweet potato storage roots collected from the local markets in Korea during 2021. Several diseases including Fusarium surface and root rot, charcoal rot, dry rot, and soft rot were observed in the postharvest sweet potatoes. A total of 68 fungal isolates were obtained from the diseased samples, and the isolates were grouped into 8 different fungal colony types. Based on multilocus phylogeny and morphological analysis of 17 representative isolates, the isolates were identified as Fusarium oxysporum, F. ipomoeae, F. solani, Penicillium citrinum, P. rotoruae, Aspergillus wentii, Mucor variicolumellatus (Mu. circinelloides species complex), and Macrophomina phaseolina. F. oxysporum was the predominant pathogen as this is the most common pathogen of sweet potato storage roots causing the surface rot disease, and M. phaseolina caused the most severe disease among the pathogens. Dual culture antagonistic assays were evaluated using Trichoderma harzianum strains CMML20–26 and CMML20–27. The results revealed that the two strains showed strong antifungal activity in different ranges against all tested pathogens. This study provides an understanding of diverse postharvest diseases in sweet potatoes and suggests potential biocontrol agents to manage the diseases. In addition, this is the first report of sweet potato storage root rot diseases caused by A. wentii, and P. rotoruae worldwide.

Draft genome sequence of Staphylococcus agnetis 4244, a strain with gene clusters encoding distinct post-translationally modified antimicrobial peptides

Here we report the draft genome sequence of Staphylococcus agnetis 4244, a strain involved in bovine mastitis, and its ability to inhibit different species of antibiotic-resistant Gram-positive bacteria owing to bacteriocin production. An Illumina MiSeq platform was used for genome sequencing. De novo genome assembly was done using the A5-miseq pipeline. Genome annotation was performed by the RAST server, and mining of bacteriocinogenic gene clusters was done using the BAGEL4 and antiSMASH v.5.0 platforms. Investigation of the spectrum of activity of S. agnetis 4244 was performed on BHI agar by deferred antagonism assay. The total scaffold size was determined to be 2 511 708 bp featuring a G+C content of 35.6%. The genome contains 2431 protein-coding sequences and 80 RNA sequences. Genome analyses revealed three prophage sequences inserted in the genome as well as several genes involved in drug resistance and two bacteriocin gene clusters (encoding a thiopeptide and a sactipeptide) encoded on the bacterial chromosome. Staphylococcus agnetis 4244 was able to inhibit all 44 strains of antibiotic-resistant Gram-positive bacteria tested in this study, including vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and other antibiotic-resistant staphylococcal strains. This study emphasises the potential biotechnological application of this strain for production of bacteriocins that could be used in the food industry as biopreservatives and/or in medicine as alternative therapeutic options against VRE, MRSA, vancomycin-intermediate S. aureus and other antibiotic-resistant Gram-positive bacteria, including biofilm-forming isolates. It also provides some genetic features of the draft genome of S. agnetis 4244.

Antifungal activity, identification and biosynthetic potential analysis of fungi against Rhizoctonia cerealis

PurposeWheat sheath blight mainly infected by Rhizoctonia cerealis is one of the soil-borne fungal diseases of wheat worldwide and prevalent in major wheat growing areas in China at present. This study aimed to evaluate the antifungal activity of 163 endophytic fungi on R. cerealis. Antifungal strains were identified and their biosynthetic potential was analysed.MethodsThe antifungal activity of the strains was evaluated via dual-culture antagonism assay. The antifungal strains were identified on the basis of morphological characteristics and internal transcribed spacer gene sequencing. The polyketide synthases (PKSs) and nonribosomal peptide synthetase (NRPS) genes in antifungal strains were detected via specific amplification of chromosomal DNA.ResultTwelve out of 163 fungal strains, including seven strains with matrix competition and five strains with antibiosis, were obtained. The twelve antifungal strains belonged to four genera: Alternaria, Ascochyta, Botryosphaeria, and Talaromyces. The inhibition rate of the seven strains with matrix competition was greater than 50%, with that of Botryosphaeria dothidea S2-33 being the highest at 84.6%. The inhibition zone of Talaromyces assiutensis R-03 amongst the five strains with antibiosis was the widest at up to 7 mm. Among the twelve antifungal strains, the strain S2-16 contained all the genes tested, five B. dothidea strains contained PKS-II and NRPS genes, two Alternaria alternata strains only contained PKS-II gene and the remaining four strains did not contain any.ConclusionResults demonstrated twelve potential strains for the biocontrol of wheat sheath blight. In particular, T. assiutensis R-03 was determined as a promising agent. The active substances secreted by antifungal strains may be produced by other biosynthetic pathways.

Bacterias con actividad antifúngica asociadas a Ocimum basilicum L.

The presence of pathogens, like fungi, is one of the most important causes of basil crop loss around the world; however, many microorganisms have a crucial role on plant development including protection against pathogenic ones. In the present study, basil associated bacteria were isolated, quantified and preserved. Characterization of isolated bacteria showed 165 Gram positive strains, 152 with bacillary and 13 with coccoid morphology. Later, in vitro antagonism assays were performed, first against Aspergillus spp. and then against Neoscytalidium dimidiatum, Alternaria spp. and Aspergillus spp. Finally, the effect of the isolated bacteria on basil seed germination and first stages of development were carried out. Sampled basil plants, produced around La Paz, Baja California Sur, Mexico, were colonized by known antagonistic bacteria of the Bacillus genus. Bacillus amyloliquefaciens strains were the prevailing species with antifungal activity. Moreover, strains ALMH42, ALMR73 and ALAH91 did not show any deleterious effect on basil seedling development. Biotechnological potential exploration of these isolated strains from healthy basil plants is of great interest for future applications on this and other crops.

Soil Bacteria as Potential Biological Control Agents of Fusarium Species Associated with Asparagus Decline Syndrome

To avoid the use of agrochemicals in agriculture, alternative methods are emerging to control plant pathogens. Some plant growth-promoting rhizobacteria (PGPR) can be used as biocontrol agents since they can induce protection against pathogens. The aim of this study was to evaluate the protective effect of several PGPR strains against the main Fusarium species involved in asparagus decline syndrome (ADS): F. proliferatum, F. oxysporum f. sp. asparagi and F. redolens. In vitro antagonism assays showed that all the bacteria inhibited the mycelium growth of the three Fusarium species. The most effective strains (Streptomyces fradiae Hvs6, Bacillus paralicheniformis Hvs2 and Bacillus velezensis FC37) were tested to evaluate their protective effect on asparagus plants inoculated with pathogenic Fusarium isolates. Strains FC37 and Hvs2 were the most effective in controlling pathogenic F. proliferatum and F. oxysporum f. sp. asparagi, but neither could protect against F. redolens isolates. The production of hydrolytic enzymes such as β-glucosidase, amylase and protease by these bacterial strains could be involved in the structural degradation of the fungal cell wall. In addition, the production of toxic volatile compounds, such as hydrogen cyanide, may inhibit the fungal growth, and the production of phosphate solubilizers could be related to the plant growth promotion. These results suggest that strains FC37 and Hvs2 could be used as potential biocontrol agents as a sustainable and environmentally friendly control strategy for ADS-affected fields.

Rhizospheric bacteria as potential biocontrol agents against Fusarium wilt and crown and root rot diseases in tomato

The discovery of novel biocontrol agents requires the continuous scrutiny of native microorganisms to ensure that they will be useful on a regional scale. The goal of the present work was to discover novel antagonistic bacteria against Fusarium oxysporum ff. spp. lycopersici race 3 (Fol R3) and radicis-lycopersici (Forl) causing Fusarium wilt disease and Fusarium crown and root rot of tomatoes, respectively. High-throughput liquid antagonism screening of 1,875 rhizospheric bacterial strains followed by dual confrontation assays in 96-well plates was used to select bacteria exhibiting > 50% fungal growth inhibition. In a second dual confrontation assay in 10-cm Petri dishes, bacteria showing > 20% Fol R3 or Forl growth inhibition were further screened using a blood hemolysis test. After discarding β-hemolytic bacteria, a seedling antagonistic assay was performed to select five potential antagonists. A phylogenetic analysis of 16S rRNA identified one strain as Acinetobacter calcoaceticus (AcDB3) and four strains as members of the genus Bacillus (B. amyloliquefaciens BaMA26, Bacillus siamensis BsiDA2, B. subtilis BsTA16 and B. thuringiensis BtMB9). Greenhouse assays demonstrated that BsTA16 and AcDB3 were the most promising antagonists against Fol R3 and Forl, respectively. Pathogen biocontrol and growth promotion mechanisms used by these bacteria include the production of siderophores, biofilm, proteases, endoglucanases and indole acetic acid, and phosphate solubilization. These five bacteria exerted differential responses on pathogen control depending on the tomato hybrid, and on the growth stage of tomatoes. We report for the first time the use of an Acinetobacter calcoaceticus isolate (AcDB3) to control Forl in tomato under greenhouse conditions.

Endophytic Fungi from Alstonia boonei De Wild and Greenwayodendron suaveolens (Engl. and Diels) Verdc. subsp. Suaveolens Possess Inhibitory Activity against Pneumonia Causing Bacteria.

This study evaluated the antibacterial efficacy of methanolic extracts of isolated endophytic fungi from stem barks and leaves of Alstonia boonei De Wild and Greenwayodendron suaveolens (Engl. and Diels) Verdc. subsp. Suaveolens against Klebsiella pneumoniae ATCC 43816, Haemophilus influenzae ATCC 49247, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 35218, responsible for causing pneumonia. The endophytic fungi were isolated and characterized in the Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), and Czapek Dox Agar (CDA) media. The fungi and their methanolic extracts were tested for in vitro antibacterial potential by antagonistic assay for endophytic fungi against bacterial pathogens and microdilution method. The phytochemical screening of extracts was carried out according to the colorimetric and precipitation methods to reveal the presence of secondary metabolites. The results showed that 24 macroscopically and microscopically distinct endophytic fungi were isolated, identified, and stored. These endophytic fungi possessed antibacterial activity against the selected bacterial strains with inhibition zones ranging from 7.00 to 25.00 mm. The endophytic fungi GS15 and AB24 have presented the inhibitions zones of 20.33 mm and 25.00 mm, respectively, and these were better than the ones obtained for Levofloxacin®. The endophytes with inhibition zones greater than 10 mm were used for extraction of their secondary metabolites. The endophytic fungi extracts showed antibacterial activity with the minimum inhibitory concentrations (MICs) ranging from 6.25 × 10−4 to 2 × 10−2 g/L and the minimum bactericidal concentrations (MBCs) ranging from 2.5 × 10−3 to 2 × 10−2 g/L. The endophytic fungi GS15 extract was the most effective extract; it showed bactericidal effects on the tested bacterial strains. The phytochemical screening of the extracts revealed the presence of secondary metabolites classes, responsible for causing the obtained antibacterial activity. Thus, the endophytic fungi methanolic extracts from A. boonei and G. suaveolens have the potential to inhibit the growth of bacteria responsible for nosocomial pneumonia.

English

The aim of this study was to screen the potential of locally grown sugarcane genotypes for red rot resistance and activity of native microbial strains against the pathogen. Field trials were conducted with 70 genotypes for consecutive years and results showed that only two genotypes viz., SSRI-1 and CO-0238 showed resistant behavior towards red rot of sugarcane. The red rot pathogen Colletotrichum falcatum Went was characterized and pathogenicity tests on two susceptible genotypes (NSG-59 and CPSG-2923) showed high virulence of SUCF04 isolate to develop severe disease lesions. The native rhizospheric microbiota was screened for microbial consortia exhibiting fine antifungal activity against the highly virulent pathogenic strain. The antagonism assay exposed that 10 bacterial isolates out of 46 showed great potential for antifungal activity. The selected bacterial isolates revealed 68–99% pathogen inhibition during the assay. The fungal strains with biological control potential inhibited the pathogen growth by 20–80% and a group of three strains with more than 50% antifungal activity were characterized. The molecular characterization of these microbes revealed that the isolates were belonging to Bacillus subtilus, Pseudomonas putida, Pseudomonas fluorescence, Trichoderma harzianum and several other important taxa. This study revealed that only two sugarcane genotypes were found as resistant against red rot pathogen, while most of the genotypes showed susceptible to moderately susceptible response. Moreover, the native residential microbiota associated with sugarcane exhibited great antifungal potential and can be utilized for disease protection and improved crop productivity. © 2021 Friends Science Publishers

Endophytic bacteria isolated from a weed plant as a potential biocontrol agent against stem end rot pathogen of pitaya in Vietnam

BackgroundStem end rot (SER) disease caused by Alternaria alternata is one of the main fungal diseases in pitaya and other crops in Nam Dinh, Vietnam, that leads to extensive yield and economic losses. Biocontrol of SER, using endophytic bacteria, is environmentally friendly and compatible with other control measures. Hence, it is emerging as an alternative disease management strategy in sustainable agriculture. This study aimed to screen antagonistic bacteria isolated from the weed, Eleusine indica, with the potential to manage SER.ResultsA total of 16 endophytes were isolated from the stems, leaves, and roots of the weed, E. indica. Of those, 6 strains presented antagonistic effects against A. alternata growth, and one isolate, EI-15, showed a significant inhibitory effect on SER. In addition, analyzing the 16S rDNA sequence indicated that EI-15 was a strain of Bacillus amyloliquefaciens. Moreover, the results of the antagonistic spectrum assay showed that EI-15 significantly inhibited some plant and fruit tree pathogens, especially the suppression of A. alternata. Notably, the culture filtrate of strain EI-15 exhibited in vitro apparent activity against A. alternata. Furthermore, an in vivo antagonistic experiment of EI-15 on pitaya twig showed a significant reduction of lesion on twigs than the control.ConclusionsOverall, this study suggested the potential application of the EI-15 strain as a biological agent and needs to be further studied in the field to control SER.

Biocontrol Activity of Aureubasidium pullulans and Candida orthopsilosis Isolated from Tectona grandis L. Phylloplane against Aspergillus sp. in Post-Harvested Citrus Fruit

This study aimed to isolate and identify moulds from rotten Citrus sinensis post-harvests and to investigate the activity of antagonist and biocontrol activity moulds that cause citrus fruit rotting. A total of 12 mould isolates were obtained. Following the pathogenicity test, two representative mould isolates were selected and identified based on the sequence analyses of internal transcribed spacer (ITS) regions of the rDNA. Methods used in this study include isolation of fungal postharvest diseases, pathogenicity assay, antagonism assay, growth curve analysis, in vitro biocontrol assay, and molecular phylogenetic analysis. Two isolates of fungal postharvest diseases were determined as the most destructive pathogens. The biocontrol assay showed that isolates of Y1 and Y10 were capable to reduce the growth of fungal isolates K6 and K9 and mitigate up to 100% of the damage of sweet citrus fruits after 7 days of incubation. The moulds were identified as K6 (Aspergillus flavus sensu lato) and K9 (Aspergillus niger sensu lato). Phylogenetic analysis showed that the Y10 yeast isolate was identified as Candida orthopsilosis, whereas the Y1 isolate had a close genetic relationship with Aureobasidium pullulans and possibly belongs to a new species. Further analysis is necessary to confirm this finding.

Abstract 1370: ADME and toxicology profiles of first-in-class DRD2/ClpP-targeted imipridone ONC201

Abstract Imipridone ONC201 is a first-in-class DRD2 antagonist and ClpP agonist that is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients with once weekly dosing. We evaluated the toxicology, absorption, distribution, metabolism and excretion of ONC201 in animals. Repeat-dose 28-day studies with weekly oral ONC201 in rats and dogs revealed NOAELs (75 and 60 mg/kg, respectively) that represent higher dose levels compared to the 625 mg clinical dose. Genotoxicity (AMES, in vitro and in vivo micronucleus assays) assays demonstrated that ONC201 is not mutagenic at exposures above the RP2D and 2µM average Cmax in patients. The 3T3 neural red uptake assay showed ONC201 is not a phototoxin (photo-irritant factor 1.1). In embryofetal development studies, ONC201 did not lead to maternal effects in rats or rabbits and caused fetal malformations in 3/61 rabbit fetuses with daily dosing (from gestation day 7-17 or 19) at 62.5 and 25 mg/kg, respectively. A quantitative whole-body autoradiography study conducted in rats with one dose of [14C]-ONC201 showed rapidly tissue distribution peaking at 1h in most tissues and exhibiting a plasma terminal half-life of 5.6h. The endocrine, metabolic/excretory, ocular and GI tract tissues contained the highest distribution of [14C]ONC201. [14C]-ONC201 was distributed evenly across CNS substructures, including the midline region. Consistent with the uniform distribution, ONC201 displayed high permeability (23-31×10-6 cm/s, 7-700 μM) and was not a substrate of efflux transporters in Caco-2 cells (efflux ratio 0.46-0.79). ONC201 exerted inhibitory potential on MDR1- and BCRP-mediated transport at 200μM. Clinical trials with ONC201 restrict use of concomitant medications that induce or inhibit CYP enzymes, but several supportive medications can affect these enzymes. In vitro studies revealed that ONC201 is not an inducer of 7 major human CYP enzymes but does inhibit and is a substrate of CYP 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 (IC50 20-90µM). Analysis of dexamethasone, a known inducer of CYP3A4, did not show a correlation between baseline dose level and ONC201 plasma concentrations in H3 K27M-mutant glioma patients (n=22). Metabolic profiling in human hepatocytes revealed only one metabolite, ONC207, that was >10% in abundance. ONC207 did not achieve an IC50 in cancer cell viability and DRD2 antagonism assays. Assaying by LC-MS/MS of plasma samples obtained from ONC201-treated H3 K27M-mutant glioma patients (n=20) confirmed the presence of ONC207 as the major metabolite. A mass balance study conducted in rats with one dose of [14C]-ONC201 showed that the main route of excretion was biliary (61.18%) via the feces (69.78%), with urine (24.99%) also contributing to overall recovery. Together, these data inform the administration of ONC201 to advanced cancer patients who often require a range of comedications and comorbidities. Citation Format: Sara Morrow, Rohinton Tarapore, Ailan Guo, Magdalena Nej, Yunlan Fang, Gina Theerman, Leah Lake, Martin Stogniew, Varun V. Prabhu, Joshua E. Allen. ADME and toxicology profiles of first-in-class DRD2/ClpP-targeted imipridone ONC201 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1370.

Potency of Endophytic and Rhizospheric Bacteria of Akar Kucing (Acalypha indica Linn.) as Antibacteria against Klebsiella pneumoniae

The prevalence of pneumonia in Indonesia was 2% in 2018. Treatment of pneumonia using antibiotics caused resistance to pathogenic bacteria. Endophytic and rhizospheric bacteria from the medicinal plant Acalypha indica Linn., is a new type of bacteria that produces antibacterial compounds against Klebsiella pneumoniae. This study aims to analyze the potency and identify endophytic and rhizospheric bacteria of the A. indica Linn as an antibacterial of K. pneumoniae. The research includes isolation and purification of endophytic and rhizospheric bacteria of the A. indica Linn, antagonistic assay of bacteria cell culture, antibacterial assay of bacteria metabolites, and identification of potential isolates based on 16S rDNA sequence similarity. The results showed that number of rhizosphere bacteria 6.83 × 105 CFU/g was more than endophytic bacteria 1.78 × 104 CFU/g. Diversity of rhizosphere bacterial 0.72 was higher than endophytic bacteria 0.62. The rhizospheric bacteria RU112B and RU315B had the highest activity to inhibit the growth of K. pneumoniae. Both isolates RU112B and RU315B were identified as Staphylococcus saprophyticus with a similarity 99.83% and Luteimonas terrae with a similarity 99.67% respectively

Novel and rapid agar plate methods for in vitro assessment of bacterial biocontrol isolates' antagonism against multiple fungal phytopathogens.

Biological control of plant diseases with antagonistic bacteria is a promising alternative to conventional chemical control strategies. In vitro screening for inhibition of mycelial growth of phytopathogenic fungi by bacterial isolates is the first step in selecting putative bacterial biocontrol agents. Dual culture plate assay is the most common method involved in this first-line selection process. However, it needs independent agar plates to test antagonism by a specific bacterial isolate against each of the fungal phytopathogen. Two modified in vitro antagonism tests are proposed here. Antagonistic activity of a putative biocontrol bacterial strain against four different fungal phytopathogens could be assessed in a single agar plate simultaneously. A comparison of the new methods with conventional dual culture plate assay was also done. The proposed methods are easy to perform and results of antagonism are obtained rapidly. Results of fungal inhibition were qualitatively comparable with that generated through dual culture plate assay. Quantity of resources such as agar medium and plates required for the modified antagonistic assays is several folds less than that required for dual culture plate assay.

Ornamental plant Pachystachys lutea as a source of promising endophytes for plant growth and phytoprotective activity

Endophytes are growth-promoting agents capable of synthesizing phytohormones, uptaking nutrients, and controlling pathogens. There is a strong potential to exploit them in the agriculture field like biofertilizers and biocontrol agents. In this work, we aimed to evaluate endophytic fungi isolated from Pachystachys lutea for their potential to solubilize phosphate, synthesise indole acetic acid (IAA), antagonize phytopathogens, and promote plant growth under greenhouse conditions. The phosphate solubilization efficiency was assessed on Pikovskaya’s agar medium. For analysis of IAA production, mycelia plugs of endophytes were cultured in Potato Dextrose Broth medium supplemented with L-tryptophan, with Salkowski Reagent, and the absorbance of the culture was measured. The antagonism evaluation of strain Alternaria sp. PL75 against phytopathogens was performed using the paired-culture technique. The promotion of plant growth provided by Alternaria sp. PL75 was evaluated in tomato plants. All strains evaluated were able to solubilize phosphate; however, the strain Alternaria sp. PL75 was the most effective (4.29). Two strains, Nemania sp. PL27 and Alternaria sp. PL75, produced 1.86 and 1.73 μg mL-1 of IAA, respectively. In the antagonism assay, the endophyte Alternaria sp. PL75 and its fungal extract showed the best results against the pathogen Moniliophthora perniciosa. The greenhouse experiment result showed the endophyte Alternaria sp. PL75 increased the plantlets emergency speed index and the percentage of germination from 60 to 81.63%. It was also observed a statistical significance in the shoot length of the treated plants with the endophyte suspension (55.38 cm) compared to the control (41.67 cm).

Characterization of antibacterial activity of a N-acetylmuramoyl-L-alanine amidase produced by Latilactobacillus sakei isolated from salami.

Lactic acid bacteria are the predominant group within meat products, whose metabolites such as bacteriocins and peptidoglycan hydrolases inhibit pathogenic or spoilage bacteria. Fermented meat products, as a salami, is a good source to analyze the viable microbiota, due to these products present a low risk to consumer health. The aim of this work was to identify the lactic acid bacteria with broad antibacterial activity present in salami, purify the protein responsible for this activity, achieve antagonistic spectrum and perform the biochemical characterization. Five strains from salami were selected, isolated and identified by 16S rRNA gene sequencing. The antimicrobial activity was evaluated by antagonism assay and zymography, using spoilage microorganisms commonly found in meat products. The strain that showed a broad antibacterial activity was Latilactobacillus sakei and the antibacterial activity was given by a protein with 75-kDa of molecular mass, identified by LC/MALDI-TOF/TOF. The sequence analysis showed 67% of identity with a N-acetylmuramoyl-L-alanine amidase protein with five non-identical LysM domains. The purified protein showed an optimal pH of 8.0 and heat resistance at 80°C for 10min. L. sakei strain displayed antibacterial activity against Gram-negative and Gram-positive spoilage microorganisms. The results of this study provide the information to use Latilactobacillus sakei as a starter culture which will provide the necessary metabolites to combat undesirable microorganisms. Additionally, the conditions and properties for the best application and use of the antibacterial protein produced by this strain. This protein may have a potential use in the food industry as a new antibacterial agent.

The Banana Root Endophytome: Differences between Mother Plants and Suckers and Evaluation of Selected Bacteria to Control Fusarium oxysporum f.sp. cubense.

This study aimed to disentangle the structure, composition, and co-occurrence relationships of the banana (cv. Dwarf Cavendish) root endophytome comparing two phenological plant stages: mother plants and suckers. Moreover, a collection of culturable root endophytes (>1000) was also generated from Canary Islands. In vitro antagonism assays against Fusarium oxysporum f.sp. cubense (Foc) races STR4 and TR4 enabled the identification and characterization of potential biocontrol agents (BCA). Eventually, three of them were selected and evaluated against Fusarium wilt of banana (FWB) together with the well-known BCA Pseudomonas simiae PICF7 under controlled conditions. Culturable and non-culturable (high-throughput sequencing) approaches provided concordant information and showed low microbial diversity within the banana root endosphere. Pseudomonas appeared as the dominant genus and seemed to play an important role in the banana root endophytic microbiome according to co-occurrence networks. Fungal communities were dominated by the genera Ophioceras, Cyphellophora, Plecosphaerella, and Fusarium. Overall, significant differences were found between mother plants and suckers, suggesting that the phenological stage determines the recruitment and organization of the endophytic microbiome. While selected native banana endophytes showed clear antagonism against Foc strains, their biocontrol performance against FWB did not improve the outcome observed for a non-indigenous reference BCA (strain PICF7).