Orchid‐associated endophytic Bacillus mediates Fusarium suppression and promotes in vitro regeneration of banana plantlets via culture supernatant

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), is one of the most severe banana diseases in the world. In this study, banana plants treated with endophytic bacteria Kocuria rhizophila showed increased PO enzyme activity, reaching the highest activity 72 h after inoculation in the shoots (0.1640 ± 0.0335 μmol/min) and 24 h after inoculation in the roots (0.0129 ± 0.0024 μmol/min). PPO enzyme activity increased significantly 24 h after inoculation in roots (0.0131 ± 0.0026 μmol/min) and 6 h after inoculation in shoots (0.0201 ± 0.0065 μmol/min). PAL enzyme activity on roots (1.776 μmol/min) and shoots (1.2170 μmol/min) inoculated with endophytic bacteria showed the highest value at 24 h. The highest total phenolic content in shoots treated with endophytic bacteria was at 72 h in roots (41.15384 mg GAE/g samples) and shoots (39.6102 mg GAE/g samples).

In Australia, banana tissue cultures are produced within a Quality Banana Approved Nursery Scheme (QBAN) to eliminate spread or introduction of pests and diseases such as Fusarium oxysporum f. sp. cubense (FOC) which is the current main disease threat to the Australian banana industry. Tropical Race 4 (TR4) of this persistent soil borne fungus was recently found in the major production area of North Queensland and attacks the commercial Cavendish cultivar ‘Williams’. Growers want evidence that FOC will not be carried in banana tissue culture and will not be suppressed due to culture conditions or an immune cultivar. To determine how FOC would grow in banana tissue cultures, two-week-old banana tissue cultures growing in Murashige and Skoog media were inoculated with FOC under commercial conditions, to determine survival of plants and rate of disease development. Tissue cultures of cultivars ‘Lady Finger’, susceptible to Race 1 (R1) and Subtropical Race 4 (STR4), ‘Williams’, immune to R1 but susceptible to STR4, and ‘Goldfinger’ immune to R1 and highly tolerant to STR4 were inoculated with R1 and Subtropical Race 4 FOC. Within three days the Fusarium had grown to cover more than 50% of the culture surface and within 10 days 100% of FOC inoculated cultures were fully colonised and within 2-3 weeks all plants were dead. All non-inoculated plants remained healthy. Both FOC Races grew rapidly in tissue culture with fluffy white mycelia covering the surface of media and plants. Fusarium growth was not restricted in banana tissue culture, where growth was rapid and very obvious. In all cultures inoculated with any FOC Race, the banana plantlets were rapidly invaded and killed, regardless if that cultivar was resistant to FOC under field conditions. Fusarium can be easily detected in banana tissue culture and rapidly kills plantlets before completion of one subculture cycle.

The global banana industry relies on tissue culture for uniform plants for better farm management, improved production and for prevention of pests and diseases otherwise transferred in soil or plant material. Banana tissue culture is also required by quarantine authorities to import/export banana plants and used in banana cultivar collections to safely maintain plantlets free of disease in the long term and protected against impacts of weather events or disease incursion. Production needs to be efficient to minimise costs and to deliver large quantities of plants on schedule. Both commercial tissue culture production and research applications involving long-term storage of banana tissue culture and cryopreservation are negatively impacted by bacterial contamination. Endophytic bacteria in tissue cultured plantlets that build up over time and adapt to culture conditions are a problem in banana tissue culture. While fungal contamination is relatively easy to identify and remove from the system, bacteria in tissue culture plants is not always apparent and can rapidly spread during plant micropropagation. Commonly bacteria may only become obvious after an extended culture time such as towards the end of a mass production cycle or in long term storage in a cultivar collection. The widespread bacteria contamination towards the end of a production cycle causes significant loss of plants and unreliable supply. Use of antibiotics rarely eliminates bacteria and can disadvantage plant growth and does not provide a contamination solution. This paper describes an effective assay allowing identification of explants used in banana tissue culture that contain culturable bacteria. Banana cultures initiated via meristem culture were indexed using a combination of four methods using modified nutrient agar that includes: streaking explant, plating macerated tissue taken adjacent explant into agar and broth, and observation of initiated cultures. Cultures initiated and indexed using this multi-culture assay have remained completely free from culturable bacteria for more than 12 years. This paper describes the bacterial screening assay and demonstrates its effectiveness by producing long-term bacterial free banana tissue cultures within the Australian banana germplasm collection.

Endophytic bacteria found in marine macroalgae have been studied for their potential antimicrobial activity, consequently, they could serve as a valuable source of bioactive compounds to control pathogenic bacteria, yeasts, and fungi. Algae endophytic bacteria were isolated from Caulerpa sp., Ulva sp., Ahnfeltiopsis sp., and Chondracantus chamissoi from Yacila and Cangrejo Beaches (Piura, Peru). Antimicrobial assays against pathogenic bacteria were evaluated using cross-culture, over-plate, and volatile organic compound tests. Afterward, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of selected crude extracts were determined, also ITS molecular analysis, antifungal activity, and PCR of iturin, fengycin, and surfactin genes were performed for bacteria strains exhibiting better activity. Forty-six algae endophytic bacteria were isolated from algae. Ten strains inhibited gram-positive pathogenic bacteria (Enterococcus faecalis, Staphylococcus epidermidis, S. aureus, and Listeria monocytogenes), and 12 inhibited gram-negative bacteria (Escherichia coli and Salmonella enteric sv typhimurium). Bacteria with better activity belong to Bacillus sp., Kluyvera ascorbata, Pantoea agglomerans, Leclercia adecarboxylata, and Enterobacter sp., which only four showed antifungal activities against Candida albicans, C. tropicalis, Colletotrichium sp., Fusarium sp., Fusarium oxysporum, and Alternaria sp. Furthermore, K. ascorbata YAFE21 and Bacillus sp. YCFE4 exhibited iturin and fengycin genes. The results indicate that the algae endophytic bacteria found in this study, particularly K. ascorbata YAFE21, Bacillus sp. YCFR6, L. adecarboxylata CUFE2, Bacillus sp. YUFE8, Enterobacter sp. YAFL1, and P. agglomerans YAFL6, could be investigated as potential producers of antimicrobial compounds due to their broad activity against various microorganisms.

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 (TR4) severely affects banana production worldwide. Thus, specific PCR primers have been developed to rapidly diagnose and monitor Foc TR4-related fusarium wilt outbreaks in bananas. However, evaluation of these primers revealed room for improvement in the accuracy. This study aimed to design highly specific PCR primers based on genome data for Foc TR4 downloaded from the National Center for Biotechnology Information database. The specificity of the primers was assessed using Foc TR4, Foc races 1 and 2, and 15 other formae speciales strains. The utility of the primers was verified by correctly detecting Foc TR4 in 7 out of 86 isolates of Fusarium spp. obtained from banana farms in the Philippines. The primers allowed for rapid detection in experimentally diseased tissues. We concluded that this novel primer set enables the simplified diagnosis of fusarium wilt caused by Foc TR4 in bananas.

An experiment was conducted during 2022 and 2023 at ICAR-National Research Centre for Banana, Tiruchirappalli, Tamil Nadu to isolate and assess native rhizospheric and endophytic bacterial strains for their potential biocontrol activities against Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4) causing wilt in banana (Musa spp.). A total of 75 bacterial strains, comprising 50 endophytic and 25 rhizospheric isolates, were obtained from various parts of banana plants and their rhizospheric soils. The experiment was laid out in a completely randomized block design (CRBD) with six treatments and five replications. In-vitro screening revealed that Bacillus subtilis (Ple-3 and Kr-2), Bacillus subtilis (Ykmr-6), Pseudomonas fluorescens (Por-4), and Bacillus velezensis (Toe2) significantly inhibited spore germination (93.43–100%) and mycelial growth (67–78%) of Foc TR4. These isolates demonstrated biocontrol activities, including HCN production, chitinase and protease activity, as well as phosphate solubilization, thereby enhancing their biocontrol potential. Molecular analysis confirmed their identity as Bacillus spp. and Klebsiella spp. and sequences were deposited in GenBank (OR472994 to OR472998). Combined soil application of Bacillus subtilis (Ykmr-6) + Pseudomonas fluorescens (Por-4) reduced internal wilt score in tissue-cultured banana plants. A lowest internal wilt score 0.8 (average of two seasons) was observed to be most effective in reducing internal wilt scores in tissue-cultured banana plants cv. Grand Nain. The study highlights the potential of bacterial strains, Bacillus subtilis (Ple-3 and Kr-2), Bacillus subtilis (Ykmr-6), Pseudomonas fluorescens (Por-4), and Bacillus velezensis (Toe2) as biocontrol agents, providing an alternative to chemical methods in managing Fusarium wilt in bananas and paving the way for integrated disease management strategies in banana cultivation.

Eight cm (approximately 3 inch) diameter Gleditsia triacanthos Inermis 'Imperial', Imperial honeylocust, and Quercus robur, English oak, were spring dug bare root and root pruned to one of four root configurations, standard, wide-deep, narrow-deep or wide-shallow, to simulate different ball sizes and shapes had the plants been balled and burlaped. The plants were placed in a healing-in area. Survival, leaf and shoot growth were followed for 18 months. All 40 honeylocust trees survived transplanting while three English oaks died. Honeylocust trees given the narrow-deep and wide-shallow root configurations had larger leaves and longer lateral shoots 18 months after transplanting than trees given standard and wide-deep configurations. English oak trees given wide-deep and wide-shallow root configurations had more shoot and leaf growth than did trees given standard or narrow-deep configurations. English oak recovered from transplanting more rapidly than did honeylocust. For both species, shoot and leaf growth during 1 986 were not significantly correlated with shoot and leaf growth in 1987.

Preface to the Series. Preface. List of Contributors. Dedication. 1. Historical Perspective: From Bacterization to Endophytes C. Elmerich 1. The Nitrogen Cycle: Heritage from the 19th Century 2. Nutritional Interactions between Bacteria and Plants 3. Associative Nitrogen-fixing Bacteria 4. Discovery of Nitrogen-fixing Endophytes 5. Cyanobacterial Associations 6. Concluding Remarks Acknowledgement References 2. Molecular Phylogeny and Ecology of Root-Associated Diazotrophic a- and ss-Protobacteria M. Schmid and A. Hartmann 1. Introduction 2. Tools for Molecular Phylogeny and in situ Localizationof Bacterial Isolates and Communities 3. Molecular Phylogeny and Ecology of Azospirillum and Other Nitrogen-fixing a-Subclass Protobacteria 4. Molecular Phylogeny and Ecology of Herbaspirillum, Diazotrophic Burkholderia spp., and Other Nitrogen-fixing ss-Protobacteria 5. Conclusions and Prospects for Future Studies Acknowledgements References 3. Regulation of Nitrogen Fixation and Ammonium Assimilation in Associative and Endophytic Nitrogen-fixing Bacteria F. O. Pedrosa and C. Elmerich 1. Introduction 2. Rhizospheric and Endophytic Bacteria: General Features 3. Structural Organization of nif Genes 4. Identification of RpoN and Its Involvement in Nitrogen Fixation 5. Thr Ntr System and Control of Nitrogen Metabolism and Nitrogen Fixation 6. Regulation of Nitrogen Fixation 7. Conclusions Acknowledgements References 4. Chemotaxis in Soil Diazotrophs: Survival and Adaptive Response G. Alexandre and I. B. Zhulin 1. Introduction 2. Gene-Expression Regulation and Chemotaxis as Adaptive Responses to Environmental changes 3. Molecular Mechanism of the Chemotactic Response: Learning from Escherichia coli 4. Directed Motility in Soil Diazotrophs 5. Future Studies References 5. Molecular Genetics of Rhizosphere and Plant-Root Colonization E. Vanbleu and J. Vanderleyden 1. Introduction 2. Motility of Associative Diazotrophs 3. Attachment to Plant Roots 4. Rhizosphere Competence 5. Conclusions Acknowledgement References 6. Microbial Production of Plant Hormones B. E. Baca and C. Elmerich 1. Discovery of Phytohormones 2. Production and Role of Phytohormones 3. Pathways for Plant Hormone Biosynthesis: Common Routes in Plants, Bacteria and Fungi 4. Major Routes for IAA synthesis in Pathogenic and Beneficial Nitrogen-fixing Bacteria Associated with Plants 5. Multiple Routes for IAA Synthesis in Azospirillum 6. Other Phytohormones Produced by Plant Pathogenic and Nitrogen-fixing Associated and Endophytic Bacteria 7. Plant Growth Promotion (PGP): Role of Bacterial Phytohormone Production, ACC-Deaminase, and the Use of Synthetic Auxins 8. Concluding Remarks Acknowledgement References 7. The Plant Growth-Promoting Effect and Plant Responses S. Dobbelaere and Y. Okon 1. N2 Fixation vs. Hormonal Effects: Historical Perspectives 2. Effects of Azospirillum and Other Diazotrophs on Root Morphology 3. Effects on Root Function 4. Effects on Plant Growth 5. Future Studies References 8. Biocontrol of Plant Diseases by Associative and Endophytic Nitrogen-fixing Bacteria R. Bally and C. Elmerich 1. Beneficial Plant-Associated Nitrogen-fixing Bacteria and Biocontrol of Plant Disease 2. Interactions within Microbial Communities: Competition 3. Biological Control against Soil-Borne Diseases 4. Regulation of Biocontrol Properties and Cell-Cell 5. Plant Response to Pathogens and Biological Control in the Rhizosphere 6. Concluding Remarks Acknowledgements References 9. Endophytic Associations of Azoarcus spp B. Reinhold-Hurek and T. Hurek 1. Introduction 2. The Rise of Interest in Diazotrophic Endophytes 3. Azoarcus spp. and related Genera: Strictly Plant-Associated vs. Soil Bacteria 4. Habitats and Ecophysiology 5. Interactions with Fungi 6. Infection of Roots by Endophytic Diazotrophs: An Active Specific Process? 7. Concluding Remarks References 10. Biological Nitrogen Fixation in Sugarcane V. Reis, S. Lee and C. Kennedy 1. Short History of the Sugarcane-Cropping System 2. Nitrogen-fixing Bacteria Colonizing Sugarcane: New Phylogenetic Data, Properties, and Endophytic Status 3. Contribution of BNF to the Sugarcane Crop 4. Effect of N Fertilization on BNF 5. Genes for Nitrogen Fixation and Their Regulation in G. diazotrophicus and H. seropedicae 6. Is Indole Acetic Acid Production an Important Factor in the Ability of G. diazotrophicus to Enhance Growth of Sugarcane? 7. Concluding Remarks Acknowledgements References 11. Heterocyst Differentiation and Nitrogen Fixation in Cyanobacteria R. Haselkorn 1. Early History of the Association of Nitrogen Fixation with Heterocysts 2. Cyanobacterial Nitrogenase and nif-Genes Organization 3. Pathway of N Assimilation 4. Carbon Metabolism in Heterocysts 5. Genetic Tools for Studying Cyanobacterial Nitrogen Fixation 6. Regulatory Genes Required for Heterocyst Differentiation 7. Prospects Acknowledgement References 12. Cyanobacterial Associations B. Bergman, A. N. Rai and U. Rasmussen 1. Introduction 2. Historical Aspects and Landmarks 3. Symbioses with Diatoms (Algae) 4. Symbioses with Fungi 5. Symbioses with Bryophytes 6. Symbioses with Pteridophytes 7. Symbioses with Cycads 8. Symbiosis with Gunnera 9. Creation of New Symbioses and Prospects Acknowledgements References 13. Prospects for Significant Nitrogen Fixation in Grasses from Bacterial Endophytes E. W. Triplett 1. Ultimate Objective of Nitrogen-fixation Research - Nitrogen Fixation in Maize, Wheat and Rice 2. Understanding the Basic Biology of Endophytic Colonization: Using K. pneumoniae 342 as the Model Diazotrophic Endophyute 3. Attributes Needed for a Model Diazotrophic Endophyte 4. Future Work Needed to Replace Nitrogen Fertilizer with Diazotrophic Endophytes References Subject Index

Fusarium oxysporum f.sp. cepae (FOCe) is a pathogenic fungus causing moler disease in shallots. One method of controlling this disease is by using endophytic bacteria. Endophytic bacteria are bacteria that live in plant tissues without causing disease symptoms. This research aims to obtain the most effective endophytic bacteria in inhibiting the growth of the pathogenic fungus FOCe. The study employs a Completely Randomized Design (CRD) consisting of 8 treatments with 3 replications each. The treatments include Bacillus sp. HI, Bacillus sp. SJI, Bacillus cereus P14, Bacillus cereus Se07, Bacillus subtilis, Serratia marcescens JB1E3, Serratia marcescens ULG1E4, and control. Tests conducted include the antibiosis test of endophytic bacterial cells using the dual culture method and the secondary metabolite test using media poisoning method. The observed parameters include inhibition zone, effectiveness of secondary metabolites, fresh weight, and dry weight of the FOCe fungus. All endophytic bacteria were capable of inhibiting the growth of FOCe. The most effective endophytic bacteria in inhibiting the growth of FOCe were found to be Serratia marcescens ULG1E4 and Serratia marcescens JB1E3. In the antibiosis test of endophytic bacterial cells, Serratia marcescens ULG1E4 exhibited an inhibition zone of 64.44%, while Serratia marcescens JB1E3 showed an inhibition zone of 61.11%. In the secondary metabolite compound test, the effectiveness values for Serratia marcescens ULG1E4 and Serratia marcescens JB1E3 were 95.31% and 95.03%, respectively.

Utilization of endophytic strain Bacillus sp. SBER3 for biodegradation of polyaromatic hydrocarbons (PAH) in soil model system

Agave tequilana Weber var. 'Azul' is grown for the production of tequila, inulin and syrup. Diverse bacteria inhabit plant tissues and play a crucial role for plant health and growth. In this study culturable endophytic bacteria were extracted from leaf bases of 100 healthy Agave tequilana plants. In plant tissue bacteria occurred at mean population densities of 3 million CFU/g of fresh plant tissue. Three hundred endophytic strains were isolated and 16s rDNA sequences grouped the bacteria into eight different taxa that shared high homology with other known sequences. Bacterial endophytes were identified as Acinectobacter sp., A. baumanii, A. bereziniae, Cronobacter sakazakii, Enterobacter hormaechei, Bacillus sp. Klebsiella oxytoca, Pseudomonas sp., Enterococcus casseliflavus, Leuconostoc mesenteroides subsp. mesenteroides and Gluconobacter oxydans. Isolates were confirmed to be plant growth promoting bacteria (PGPB) by their capacities for nitrogen fixation, auxin production, phosphate solubilization, or antagonism against Fusarium oxysporum AC132. E. casseliflavus JM47 and K. oxytoca JM26 secreted the highest concentrations of IAA. The endophyte Acinectobacter sp. JM58 exhibited the maximum values for nitrogen fixation and phosphate solubilization index (PSI). Inhibition of fungi was found in Pseudomonas sp. JM9p and K. oxytoca JM26. Bacterial endophytes show promise for use as bio-inoculants for agave cultivation. Use of endophytes to enhance cultivation of agave may be particularly important for plants produced by micropropagation techniques, where native endophytes may have been lost.

Endophytic bacteria have several advantages, including controlling plant pathogens and increasing plant growth. As a biological control, endophytic bacteria can suppress the development of plant pathogenic fungi. This study aimed to obtain endophytic bacteria and the consortium of endophytic bacteria that can inhibit the growth of C. oryzae. The study used a Completely Randomized Design (CRD) for studying the ability of endophytic bacteria using eight isolates: Bacillus sp HI, Bacillus sp SJI, B. cereus P14, B. cereus Se07, Serratia marcescens strain JB1E2, S. marces-cens strain JB3, S. marcescens strain ULG1E2 and S. marcescens strain ULG1E4, with three replications. For Studying the ability of endophytic bacteria consortium using six endophytic bacteria consortia and control with three replications. Observations were made on the inhibition growth of C. oryzae. The results showed that all endophytic bacteria and the endophytic bacteria consortium could inhibit C. oryzae, with inhibition of 58.50% – 75.00% and 38.00% – 77.00%. Endophytic bacteria which can significantly suppress the growth of C. oryzae were B. cereus Se07, Bacillus sp HI and Bacillus sp SJI with inhibition of 75.00%, 70.50%, and 70.00%. The endophytic bacteria consortium that had a high ability to suppress the growth of C. oryzae was the consortium of B (S. marcescens strain ULG1E4, S. marcescens strain JB1E3), D (Bacillus sp SJI, Bacillus sp HI, S. marcescenns JB1E3), and G (Bacillus sp SJI, S. marcescens strain ULG1E4), with inhibition of 77.00%, 72.33%, and 71.33%, respectively.

Towards consensus on the transfer of Fusarium oxysporum V5w2-enhanced tissue culture banana technology to farmers through public-private partnerships in East Africa

Fusarium Basal Root (FBR) disease locally known as "moler", caused by Fusarium oxysporum f.sp cepae (FOCe), poses a significant threat to Indonesian shallot cultivation, leading to yield losses of approximately 50%. Therefore, this research aims to identify endophytic bacteria with optimal capabilities to combat FOCe infection while improving shallot growth and yield. Randomized Completely Block Design (RCBD) consisting of seven treatments was used, including six different strains of endophytic bacteria (Bacillus cereus P14, Bacillus cereus Se07, Bacillus sp. HI, Bacillus sp. SJI, Serratia marcescens ULG1E2, and Serratia marcescens JB1E3) and a control, each treatment was replicated three times, and three plant units in each replication. The introduction of endophytic bacteria was performed through the bulb soaking method before planting, while FOCe suspension was inoculated to shallot growing media 4 weeks after planting. Parameters Variables such as moler disease development, plant growth, and shallot bulb weight were monitored. The results showed that endophytic bacteria effectively suppressed moler disease and boosted shallot growth and yield compared to the control. Specifically, S. marcescens JB1E3 showed 52.25% effectiveness in reducing moler disease severity, while S. marcescens ULG1E2 increased shallot yield by 65.50%.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), poses a significant threat to banana production globally, thereby necessitating effective biocontrol methods to manage this devastating disease. This study investigates the potential of Bacillus siamensis strain JSZ06, isolated from smooth vetch, as a biocontrol agent against Foc TR4. To this end, we conducted a series of in vitro and in vivo experiments to evaluate the antifungal activity of strain JSZ06 and its crude extracts. Additionally, genomic analyses were performed to identify antibiotic synthesis genes, while metabolomic profiling was conducted to characterize bioactive compounds. The results demonstrated that strain JSZ06 exhibited strong inhibitory activity against Foc TR4, significantly reducing mycelial growth and spore germination. Moreover, scanning and transmission electron microscopy revealed substantial ultrastructural damage to Foc TR4 mycelia treated with JSZ06 extracts. Genomic analysis identified several antibiotic synthesis genes, and metabolomic profiling revealed numerous antifungal metabolites. Furthermore, in pot trials, the application of JSZ06 fermentation broth significantly enhanced banana plant growth and reduced disease severity, achieving biocontrol efficiencies of 76.71% and 79.25% for leaves and pseudostems, respectively. In conclusion, Bacillus siamensis JSZ06 is a promising biocontrol agent against Fusarium wilt in bananas, with its dual action of direct antifungal activity and plant growth promotion underscoring its potential for integrated disease management strategies.