In vitro antifungal activity of cell-free supernatants from co-cultured Trichoderma spp. and Burkholderia spp. against Fusarium oxysporum f. sp. cubense causing banana wilt disease

The in vitro antagonistic activity of Bacillus velezensis LABIM40 (strain CMRP 4489) was assessed against Alternaria linariae, Botryotinia squamosa, Colletotrichum lindemuthianum, Gibberella zeae, and Rhizoctonia solani. An experiment was conducted using treated seeds under growth chamber conditions to determine the impact of various LABIM40 formulations on tomato seedling growth and the biocontrol of damping-off caused by R. solani. The treatments included the use of LABIM40 cell suspension, LABIM40 cell-free supernatant (CFS), 10 times concentrated CFS (10× CFS), commercial products based on Bacillus amyloliquefaciens (CP_1) and Bacillus subtilis (CP_2), and water. The effects of these products were assessed on tomato seedlings grown in sterile substrate or substrate inoculated with R. solani. In a dual culture test, B. velezensis LABIM40 inhibited the mycelial growth of the aforementioned fungal pathogens by 46.6%, 67.4%, 64.7%, 49.0%, and 54.4%, respectively. The minimum inhibitory concentration against each fungus was determined using varying concentrations of CFS in potato dextrose agar medium, followed by a regression analysis of mycelial growth inhibition. Except for A. linariae, the logarithmic model provided the best fit in all cases. Tomato seedlings from seeds treated with 10× CFS in inoculated substrate exhibited a survival rate 57% higher than that exhibited by the control treatment. However, no growth promotion was observed in tomato plants from seeds treated with LABIM40 cells or its CFS metabolites. In summary, these findings highlight the antagonistic activity of B. velezensis LABIM40 against A. linariae, B. squamosa, C. lindemuthianum, G. zeae, and R. solani, as demonstrated by dual culture and CFS diffusion tests. This suggests its potential as a biocontrol agent for damping-off in tomatoes.

Trichoderma spp. are widely distributed in natural habitats and have been evaluated as a potential biocontrol agent (BCA) for disease control and plant growth promotion. In this study, 1308 Trichoderma strains were obtained from the plant rhizosphere soil, above-ground plants, and decaying wood from natural habitats in China. Among them, 49 Trichoderma strains showed a good inhibitory effect, especially against Botrytis cinerea, Fusarium oxysporum, and Colletotrichum gloeosporioides with inhibition rate above 85% in the dual culture test. Among these 49 strains, the 13 strains with broad-spectrum inhibitory effects also significantly promoted the seed germination of five crops (rice, cucumber, tomato, melon, and pakchoi) and root growth of four crop seedlings (watermelon, tomato, eggplant, and chili). Furthermore, these strains showed effective colonization in the rhizosphere and root of cucumber. Trichoderma strains SC012 and NX043 showed the highest chitinase and β-1,3-glucanase activity among all strains. Based on the morphological characterization and phylogenetic analysis of the nuclear ribosomal internal transcribed spacer (ITS) and translation elongation factor 1 (tef1), twelve Trichoderma strains were identified as Trichoderma asperellum and one as Trichoderma afroharzianum. This study suggests that the 13 Trichoderma strains are promising BCAs and could be developed as biofertilizers and biological pesticides for agricultural applications.

Ginseng root rot caused by Fusarium oxysporum is serious disease that impacts ginseng production. In the present study, 145 strains of bacteria were isolated from the rhizosphere soil of healthy ginseng plants. Three strains with inhibitory activity against Fusarium oxysporum (accession number AF077393) were identified using the dual culture tests and designated as YN-42(L), YN-43(L), and YN-59(L). Morphological, physiological, biochemical, 16S rRNA gene sequencing and phylogenetic analyses were used to identify the strains as Bacillus subtilis [YN-42(L)] (accession number ON545980), Delftia acidovorans [YN-43(L)] (accession number ON545981), and Bacillus polymyxae [YN-59(L)] (accession number ON545982). All three isolates effectively inhibited the growth of Fusarium oxysporum in vitro and the antagonistic mechanism used by the three strains involved the secretion of multiple bioactive metabolites responsible for the hydrolysis of the fungal cell wall. All three biocontrol bacteria produce indoleacetic acid, which has a beneficial effect on plant growth. From our findings, all three antagonistic strains can be excellent candidates for ginseng root rot caused by the pathogenic fungus Fusarium oxysporum. These bacteria have laid the foundation for the biological control of ginseng root rot and for further research on the field control of ginseng pathogens.

Ginseng root rot caused by Fusarium oxysporum is serious disease that impacts ginseng production. In the present study, 145 strains of bacteria were isolated from the rhizosphere soil of healthy ginseng plants. Three strains with inhibitory activity against Fusarium oxysporum (accession number AF077393) were identified using the dual culture tests and designated as YN-42(L), YN-43(L), and YN-59(L). Morphological, physiological, biochemical, 16S rRNA gene sequencing and phylogenetic analyses were used to identify the strains as Bacillus subtilis [YN-42(L)] (accession number ON545980), Delftia acidovorans [YN-43(L)] (accession number ON545981), and Bacillus polymyxae [YN-59(L)] (accession number ON545982). All three isolates effectively inhibited the growth of Fusarium oxysporum in vitro and the antagonistic mechanism used by the three strains involved the secretion of multiple bioactive metabolites responsible for the hydrolysis of the fungal cell wall. All three biocontrol bacteria produce indoleacetic acid, which has a beneficial effect on plant growth. From our findings, all three antagonistic strains can be excellent candidates for ginseng root rot caused by the pathogenic fungus Fusarium oxysporum. These bacteria have laid the foundation for the biological control of ginseng root rot and for further research on the field control of ginseng pathogens.

Banana wilt disease caused by Fusarium oxysporum f.sp.cubense is considered as one of the most destructive diseases on banana plants in the tropical region. Biological control agents (BCA’s) have become a promising solution to overcome this disease. The objective of this study was to find potential BCA’s for wilt disease of banana plants. Bacteria and actinomycetes were isolated from banana’s rhizosphere in Lampung and Cianjur. As much as 64 actinomycetes and 142 bacteria isolates were obtained. Antagonistic test against F. oxysporum f. sp. cubense of those isolates showed that 21 bacteria and 10 actinomycete isolates have abilities to inhibit F. oxysporum f. sp. cubense. Actinomycetes showed relatively higher inhibition against F. oxysporum f. sp. cubense compared to bacteria. Isolates which have positive antagonistic activities against F. oxysporum f. sp. cubense were then tested for their protease, chitinase, and selulase activities qualitatively on specific medium. All actinomycetes which were tested had chitinase enzyme activities, while only 5 bacterial isolates had chitinase activities. On the other hand, 13 bacterial isolates showed protease activities and only 1 actinomycete showed protease activity. Two bacterial isolates (L.II.4.ND and L.A.I-5.DW) and 3 actinomycetes (L.A.I.DW, L.3.1.DW and Ci.I.A5.DW) which showed high inhibition against F. oxysporum f. sp. cubense and lyses enzymes activities were identified based on 16S rRNA genes. Analysis based on GenBank data, those isolates have 99% homology to Klebsiella pneumonia (L.II.4.ND), Burkholderia sp. (L.A.I-5.DW), Streptomyces sp. (L.A.I.DW), Streptomyces sp. (L.3.1.DW) and Streptomyces sp. (Ci.I.A5.DW).

Banana rhizosphere harbors a unique diversity of microbes including fungi that play critical roles in the growth of the plant host as well as might be important for biologically controlling the fungal soil-borne pathogens particularly Fusarium oxysporum f.sp. cubense (Foc), the causing agent of devastating Panama wilt. Among other fungi, we have succeeded to isolate a Trichoderma species from rhizosphere of healthy banana. Molecular identification revealed the isolate as Trichoderma virens InaCC F1030 (being collection of Indonesian Culture Collection or InaCC). Therefore, the aim of this study was to investigate the biological control of our isolate against Foc as well as plant growth promoting ability through its ability to produce auxin (indole-3-acetic acid/IAA). Two approaches were employed to evaluate the antagonism of our isolate against Foc, through direct confrontation test and volatile organic compounds (VOCs) producing. We found that our isolate was considered as antagonistic to the Foc, but not highly antagonistic according to direct confrontation assay. It was also revealed that our isolate produces the VOCs that inhibited around 50% of the mycelial growth of the test pathogen after six to seven days of exposure. Our isolate was able to produce the IAA in axenic submerged fermentation condition particularly in the presence of the precursor L-tryptophan. IAA production ability as well as the mycelial biomass of fungus were increased approximately 17% and 120% respectively as the effect of supplementation of 0.1% of L-tryptophan. These in vitro bioassays lead us to conclude that somehow our isolate T. virens InaCC F1030 has potency to be utilized as biocontrol and biofertilizer agent.

TWENTY-THREE different bacterial and fungal isolates were grown and screened for their capability to transform soy glycosides to their aglycone forms with higher titer of antioxidant activity compared to unfermented soy flour. Most of the bacterial isolates showed higher amounts of daidzein than of genistein, which are the aglycone products of daidzin and genistin. After fermentation of soybean flour using bacterial and fungal isolates, the content of isoflavone aglycones varied from 0.0 to 431.89 μg/g compared to unfermented autoclaved soybean flour. Extracellular β-glucosidase activity was ranged from 1.22 to 11.56 mU/mL and 0.3-534.3 U/mL for bacterial and fungal isolates, respectively, while, bacterial cell-bound β-glucosidase ranged from 44.72 to 128.89 mU/mL. Most of the bacterial isolates more efficiently transformed daidzin and genistin into the aglycones than fungal isolates. Among the tested bacterial isolates, the most potent one was selected, characterized according to the morphological and 16S rDNA sequence analysis and identified as Bacillus licheniformis NRC24.

Fusarium wilt caused by Fusarium oxysporum f.sp passiflorae is the most important disease on passion fruit that causes yield losses ranging from 50 to100%. The disease is difficult to control because the pathogen systematically infected plants and can survive up to five years in the soil in the absence of its hosts. The objective of this paper was to select potential antagonistic microbes in suppressing the growth of Fusarium oxysporum f.sp passiflorae (Fop) in vitro. Antagonists were isolated from the rhizosphere of purple passion fruit (Passiflora edulis form edulis) and sweet passion fruit (Passiflora sp) from Gowa and Makassar. To obtain the best isolates, their ability to inhibit the growth of Fop and their production of cellulase, chitinase, pectinase as well as toxine compound were tested in vitro. The results showed that out of 22 fungal and bacterial isolates tested, four and three isolates respectively, gave an excellent growth inhibition to Fop. Highest percentage of growth inhibition was provided by fungal isolate U1 (86.11%) and bacterial isolate Mb2 (76.44%). The highest cellulase, chitinase and pectinase enzyme production were observed on fungal isolate U1, followed by isolates U7, M1, M2 and M4. Only two bacterial isolates Ub1 and Mb3 showed highest cellulase, chitinase and pectinase enzyme production. The presence of toxin was detected by using a thin layer chromatography on fungal isolates U1, M4 and Mb3. HCN compound from bacterial isolates were obtained from isolates Ub1 and Mb3.

The main objectives of this study were to (i) identify the seed- and soil-borne fungal pathogens of okra (Abelmoschus esculentus L.) grown in the Hatay province of Türkiye, (ii) determine the in vitro biocontrol potential of bacterial isolates (BCAs) from healthy okra and closely related plants against common fungal pathogens, and (iii) characterize the antagonistic and plant growth-promoting (PGP) mechanisms involved in pathogen suppression and plant growth. Rhizoctonia solani, Macrophomina phaseolina, Sclerotinia sclerotiorum, and Fusarium oxysporum were determined as the most common soil and seed-borne disease agents on diseased seeds and plants. A total of 36 different endophytic and epiphytic bacterial isolates were selected and identified using MALDI-TOF MS, and their antagonistic potentials to inhibit mycelial growth of R. solani, M. phaseolina, S. sclerotiorum, and F. oxysporum were characterized in dual culture tests. Among BCA bacterial isolates, Bacillus cereus B1ep, B. cereus B2ep, Pseudomonas aeruginosa B3ep, B. cereus B11ep, and B. subtilis B12ep displayed high levels (≥70%) of antagonistic activity against all fungal agents tested. Enterobacter cloacae B10ep was identified as the highest siderophore producer, Microbacterium maritypicum X5 as the highest protease enzyme producer, Pseudomonas aeruginosa B3ep, and different Bacillus spp. as isolates producing hydrogen cyanide (HCN) and ammonia at the highest level. The suppression of mycelial growth was suggested to be associated with one or more antagonistic mechanisms. Enterobacter bugandensis B7ep was identified as the highest Indole-3 Acetic Acid (IAA) producer, and B. cereus P7en was identified as the most efficient phosphate-solubilizing bacterial isolate. In conclusion, BCA isolates belonging to Bacillus and Pseudomonas spp., which demonstrated strong antagonistic and PGP activities, have the potential to be developed as biopreparations against soil and seed-borne disease agents in okra plants.

Fusarium oxysporum is the pathogenic cause of the disease on chili plants that can reduce production in the cultivation process that needs to be done to control an environmentally friendly manner using endophytic fungi. This study aims to test the inhibition, the mechanism of antagonism, and pathogenicity of eleven endophytic fungi origin of pepper plants Bengkulu province against the pathogen Fusarium oxysporum in vitro. Research conducted in the Laboratory of Plant Protection, Faculty of Agriculture, University of Bengkulu from December 2020 to February 2021. The study consists of: the rejuvenation of the fungi isolates of endophytic and fungi pathogens Fusarium oxysporum, dual culture test, the mechanism of antagonism test, as well as pathogenesis endophytic fungi test. Endophytic fungi isolate obtained from the collection of the laboratory of Plant Protection, University of Bengkulu. The results of the research show from eleven endophytic fungi only 1 endophytic fungi which has a percentage of inhibition of 50% of the isolates of Rhizoctonia sp.2. The mechanism of antagonism occurs consisting of the competition between the space of nutrients and oxygen, hiperparasitisme, and antibiosis. patogenensitas test show only isolates of Rhizoctonia sp.2 that do not produce patches on the chili seeds.

Plant growth promoting and antifungal asset of indigenous rhizobacteria secluded from saffron (Crocus sativus L.) rhizosphere

Isolation and characterization of a bacterial isolate (strain FP10) from banana rhizosphere with innate potential as fungal antagonist and microbial adjuvant in micropropagation of banana. Bacterium FP10 was isolated from the banana rhizosphere and identified as Pseudomonas aeruginosa based on phenotypic, biochemical traits and sequence homology of partial 622-bp fragment of 16S ribosomal DNA (rDNA) amplicon, with the ribosomal database sequences. Strain FP10 displayed antibiosis towards fungi causing wilt and root necrosis diseases of banana. Production of plant growth hormone, indole-3-acetic acid (IAA), siderophores and phosphate-solubilizing enzyme in FP10 was determined. Strain FP10 tested negative for hydrogen cyanide, cellulase and pectinase, the deleterious traits for plant growth. Screening of antibiotic genes was carried out by polymerase chain reaction using gene-specific primers. Amplification of a 745-bp DNA fragment confirmed the presence of phlD, which is a key gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG) in FP10. The antibiotic produced by FP10 was confirmed as DAPG using thin layer chromatography, high performance liquid chromatography and Fourier transform infrared and tested for fungal antibiosis towards banana pathogens. Procedures for encapsulation of banana shoot tips with FP10 are described. Strain FP10 exhibited broad-spectrum antibiosis towards banana fungi causing wilt and root necrosis. DAPG by FP10 induced bulb formation and lysis of fungal mycelia. Encapsulation of banana shoot tips with FP10 induced higher frequency of germination (plantlet development) than nontreated controls on Murashige and Skoog basal medium. Treatment of banana plants with FP10 enhanced plant height and reduced the vascular discolouration as a result of Fusarium oxysporum f. sp. cubense FOC. Because of the innate potential of fungal antibiosis by DAPG antibiotic and production of siderophore, plant-growth-promoting IAA and phosphatase, the strain FP10 can be used as biofertilizer as well as a biocontrol agent.

Trichoderma species is one of the microorganisms with antagonistic properties as biological control agents. In the banana industry, Fusarium wilt disease caused by Fusarium oxysporum f. sp. cubense (Foc) has been practically managed using chemical pesticides that led to environmental disruptions, ineffective conditions and disease resistance. In preliminary study, T. asperellum gave better result compared to other species in inhibiting the growth of Foc in in vitro condition. Therefore, the aim of this study was to examine the effects of T. asperellum as a biological control of Fusarium wilt disease of banana. A total of 326 fungal isolates were isolated from soil samples obtained around Malaysia and identified as Trichoderma species based on phenotype characteristics. The species identity for the best candidates from dual culture test was confirmed based on internal transcribed spacers (ITS) and translation elongation factor 1 alpha (TEF-1α) sequence identity. In dual culture test, findings showed that three isolates with a high percentage inhibition of radial growth (PIRG) were observed in plates of T. asperellum isolates B1902 (84.85%), T2007 (77.78%) and C1667 (75.76%), which successfully inhibited the growth of F. oxysporum f. sp. cubense isolate 9888. Based on in vivo test, the best candidate was T. asperellum B1902 with lower disease severity index (DSI) value of 0.2 compared to the inoculated control with DSI of 3.6. As a conclusion, T. asperellum B1902 can be used as an alternative treatment in managing Fusarium wilt disease. Hence, future study should be focused on applying T. asperellum as a biocontrol agent in the field and controlling other plant diseases in the agricultural plantation.

This study investigated whether viable cells, dead cells or cell-free supernatants (CFS) were responsible for the biocontrol effect of strains from two important bacterial genera, Pseudomonas and Lactobacillus, known for their antifungal properties against plant pathogens and food spoilage microorganisms. Specifically, the capability of these strains to produce extracellular hydrolytic enzymes on specified media was assessed, along with their effectiveness in inhibiting the mycelial growth of several phytopathogenic fungi (Fusarium oxysporum, Botrytis cinerea, Pythium ultimum and Rhizoctonia solani) using dual culture plate assays. Results from these inhibition assays revealed that P. fluorescens PF05 and L. plantarum LMG 23520 strains were the most effective in suppressing fungal growth, especially F. oxysporum. Therefore, further experiments were carried out to investigate the antifungal potential of the viable cells, heat-killed cells (HKC) and CFS from these strains against the germination of F. oxysporum spores. The viable cell trial proved successful, whereas HKC from the two bacterial isolates were ineffective against fungal spore germination. Conversely, the CFS of L. plantarum LMG 23520 was able to prevent fungal spore development for up to six days. The CFS of P. fluorescens PF05, instead, did not yield positive results. Additional studies are required to evaluate the potential inhibitory effects of the CFS from P. fluorescens PF05 and the HKC from both strains.

This study was carried out to explore a non-chemical strategy for enhancing productivity by employing some antagonistic rhizobacteria. One hundred eighteen bacterial isolates were obtained from the rhizospheric zone of various crop fields of Gangwon-do, Korea, and screened for antifungal activity against Fusarium wilt (Fusarium oxysporum f. sp. lactucae) in lettuce crop under in vitro and in vivo conditions. In broth-based dual culture assay, fourteen bacterial isolates showed significant inhibition of mycelial growth of F. oxysporium f. sp. lactucae. All of the antagonistic isolates were further characterized for the antagonistic traits under in vitro conditions. The isolates were identified on the basis of biochemical characteristics and confirmed at their species level by 16S rRNA gene sequencing analysis. Arthrobacter sulfonivorans, Bacillus siamensis, Bacillus amyloliquefaciens, Pseudomonas proteolytica, four Paenibacillus peoriae strains, and Bacillus subtilis were identified from the biochemical characterization and 16S rRNA gene sequencing analysis. The isolates EN21 and EN23 showed significant decrease in disease severity on lettuce compared to infected control and other bacterial treatments under greenhouse conditions. Two bacterial isolates, EN4 and EN21, were evaluated to assess their disease reduction and growth promotion in lettuce in field conditions. The consortium of EN4 and EN21 showed significant enhancement of growth on lettuce by suppressing disease caused by F. oxysporum f. sp. lactucae respectively. This study clearly indicates that the promising isolates, EN4 (P. proteolytica) and EN21 (Bacillus siamensis), can be commercialized and used as biofertilizer and/or biopesticide for sustainable crop production.