Endophytic Bacillus Research Articles

Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control.

The pinewood nematode (PWN) is responsible for causing pine wilt disease (PWD), which has led to the significant decline of conifer species in Eurasian forests and has become a globally invasive quarantine pest. Manipulating plant-associated microbes to control nematodes is an important strategy for sustainable pest management. However, it has proven difficult to find pine-associated bacteria that possess both nematocidal activity and the ability to colonize pine tissues. The stress experiments with turpentine and pine tissue extract were carried out to screen for the desired target strain that could adapt to the internal environment of pine trees. This strain was used to construct an engineered nematocidal strain. Additionally, a fluorescent strain was constructed to determine its dispersal ability in Pinus massoniana seedlings through plate separation, PCR detection, and fluorescence microscopy observations. The engineered nematocidal strain was tested in the greenhouse experiment to assess its ability to effectively protect P. massoniana seedlings from nematode infection. This study isolated a Bacillus toyonensis strain Bxy19 from the healthy pine stem, which showed exceptional tolerance in stress experiments. An engineered nematocidal strain Bxy19P3C6 was constructed, which expressed the Cry6Aa crystal protein and exhibited nematocidal activity. The fluorescent strain Bxy19GFP was also constructed and used to test its dispersal ability. It was observed to enter the needles of the seedlings through the stomata and colonize the vascular bundle after being sprayed on the seedlings. The strain was observed to colonize and spread in the tracheid after being injected into the stems. The strain could colonize the seedlings and persist for at least 50 days. Furthermore, the greenhouse experiments indicated that both spraying and injecting the engineered strain Bxy19P3C6 had considerable efficacy against nematode infection. The evidence of the colonization ability and persistence of the strain in pine advances our understanding of the control and prediction of the colonization of exogenously delivered bacteria in pines. This study provides a promising approach for manipulating plant-associated bacteria and using Bt protein to control nematodes.

Genomic analysis of endophytic Bacillus altitudinis strain VUMS1 from Kappaphycus alvarezii and its inhibitory effect against Vibrio parahaemolyticus

Genomic analysis of endophytic Bacillus altitudinis strain VUMS1 from Kappaphycus alvarezii and its inhibitory effect against Vibrio parahaemolyticus

Enhanced Cd activation by Coprinus comatus endophyte Bacillus thuringiensis and the molecular mechanism

Fungal endophytes not only tolerate and activate Cd in soil but also promote host growth, yet its Cd activation capacity and mechanism remain unrevealed. Our previous study isolated a robust endophyte Bacillus thuringiensis L1 from Coprinus comatus fruiting body with splendid Cd resistance and activation abilities under laboratory conditions. In this study, those peculiarities were investigated in the actual soil environment. L1 could significantly increase the soil bioavailable Cd content and effectively compensate for alkali-hydro nitrogen losses and microbial inhibition caused by Cd. Furthermore, L1 inoculation improved the soil's bacterial community structure and increased the relative abundance of Cd-resistant bacteria, such as Actinobacteria, Chloroflexi, Acidobacter, and Firmicutes, closely associated with the soil enzyme activity shift. The genome sequencing analysis revealed the presence of genes related to growth promotion, resistance to Cd stress, and Cd activation, which were significantly up-regulated under Cd stress. Notably, L1 mainly activates Cd in soil by secreting citric acid, succinic acid, siderophore, and soluble phosphorus substances to chelate with Cd or dissolve bounded Cd. Meanwhile, the metal-responsive transcription repressor (CadC) and the Cd-translocating protein P-type ATPase (CadA) can help the L1 to suppress the toxicity of Cd. Those results help to unveil the possible mechanism of L1 in Cd-contaminated soil remediation, providing a clear strategy for Cd bio-extraction from soil.

Multi-species endophytic Bacillus for improved control of potato soilborne and tuber-borne diseases in Tunisia: from laboratory to field conditions

BackgroundDue to the soilborne and tuber-borne nature of their causative agents, Fusarium wilt, Rhizoctonia root canker and black dot diseases are still leading to serious problems in potato production in Tunisia and worldwide. Among endophytic bacteria, the genus Bacillus is one of the most exploited microbial groups known as potent biocontrol agents against several potato diseases. In the present study, six strains belonging to five Bacillus species were screened for their abilities to inhibit root and wilt potato pathogens in vitro, in vivo and under natural conditions over three cropping seasons and to promote plant growth.ResultsBased on the dual-culture assays, the whole-cell suspensions of SV39 and SV104 (Bacillus tequilensis), SV41 (B. subtilis), SV44 (B. methylotrophicus) and SV65 (B. amyloliquefaciens subsp. plantarum) strains exhibited potent antifungal activity against important potato soilborne phytopathogens with ~ 65 to 70% inhibition rates. Significant inhibition rates were also induced by the cell-free culture filtrates, the butanolic and the chloroformic extracts depending on the target pathogens, the concentration used and the Bacillus strain tested. In pot experiment, a decrease in Rhizoctonia root canker severity, ranging from 43 to 65% compared to the inoculated and untreated control, was induced by all Bacillus spp. strain-based treatments, while SV39-, S42- and SV65-based treatments were the most effective in suppressing by 50–53 and 65–52%, black dot severity and the relative vascular discoloration extent induced by F. oxysporum f. sp. tuberosi, respectively, relative to positive control. This biocontrol potential was associated with an enhancement of potato growth parameters. Field studies indicated that soil treatment with the most of the Bacillus spp. strains had significantly controlled all the target fungal soilborne diseases and improved at the least two growth and/or production parameters depending on the strain used and the cropping seasons. SV39-, SV41-, SV44- and SV104-based treatments resulted in a significant increase in tuber yield in one cropping season.ConclusionThese Bacillus spp. strains could be used in combinations and/or introduced with other existing practices in order to provide supplemental control of target diseases and yield promotion under organic or conventional potato production systems.

Antifungal activity of bioactive compounds produced by the endophyte Bacillus velezensis NC318 against the soil borne pathogen Sclerotium rolfsii Sacc

Antifungal activity of bioactive compounds produced by the endophyte Bacillus velezensis NC318 against the soil borne pathogen Sclerotium rolfsii Sacc

Endophytic Bacillus sp. R1 and Its Roles in Assisting Phytoremediation and Alleviating the Toxicity of Aluminum Combined Phenanthrene Contaminations in Brassica napus.

The release of organic and inorganic contaminants into soil from industry, agriculture, and urbanization has become a major issue of international concern, particularly the heavy metals such as aluminum (Al) and the chemical phenanthrene (PHE). Due to their potential toxicity and non-biodegrade in the environment, efficient remediation methods are urgently needed. Recently, research has comprehensively discussed using plants and their endophytes in bioremediation efforts. Endophytic Bacillus sp. R1, isolated from Brassica napus permanently contaminated with Al and PHE, has growth-promoting properties and can efficiently detoxify these contaminants. The pot experiment indicated that compared to the Al combined PHE contaminated soil alone treatment, the R1 treatment led to increased Al accumulation in canola roots across different levels of PHE, Al, and combined PHE and Al contamination. However, Al accumulation in canola shoots and seeds remained unchanged for all treatments. Moreover, PHE in canola roots and shoots was decreased by R1 inoculation and thereby reducing 26.12-60.61% PHE translocated into canola seeds. Additionally, R1 inoculation significantly increased the proportion of extractable Al and, decreased the proportion of acid-soluble inorganic Al and humic-acid Al, but did not affect the concentration of organically complexed Al. In summary, endophyte R1 can degrade PHE, improve canola roots' Al uptake by increasing soil available Al, and scavenge the reactive oxygen species through production of antioxidant enzymes to help alleviate the toxicity of canola co-contaminated with aluminum and phenanthrene.

Biocontrol of Fusarium wilt disease by endophytic Bacillus xiamenensis BS10 isolated from the root nodules of Brassica nigra (mustard plant) and analysis of defence molecular biomarkers in the greenhouse and field trial

Endophytic bacteria possess positive effects on their host plants, including, secondary metabolite induction, disease-resistance improvement, and growth promotion. Bacillus spp. are considered to be an eco-friendly and bio-safe alternative to traditional chemical fungicides. In our study, a collection of 12 endophytic bacteria were isolated from Brassica nigra root nodules and analyzed for plant growth-promoting activities. The endophytic strain BS10 significantly solubilise phosphate (19 ± 1 mm halo zone), siderophore (17 ± 1 mm halo zone), HCN (18 ± 1 halo zone), and indole acetic acid (205.1 ± 2.8 μg/mL) significantly (p < 0.05) than other isolates. The antifungal activity of isolated endophytes was analyzed using a dual-culture screening method. The antifungal activity against Fusarium oxysporum revealed that the strain BS10 exhibited maximum activity against F. oxysporum and the inhibition rate ranged from 20 ± 1 to 70.2 ± 3.5 % after 4 days of treatment and increased after 8 days (36.9 ± 3.5–75.8 ± 3.2 %) among other endophytes. The cell-free extract decreased fungal growth and the inhibition rate varied between 12 % and 47 % (p < 0.05). The results revealed that the fungal infection of the mustard plants with wilt disease causing F. oxysporum significantly reduced the growth of the mustard plant, and root, shoot length, and dry weight compared with the control (p < 0.05). The infected plants treated with endophyte showed an improvement in the growth parameters (p < 0.05). The endophytes-treated plants showed fewer symptoms of rot disease in mustard plants. The chlorophyll content (2.27-fold), soluble leaf protein (1.22-fold), proline (26 ± 2.8 nM/g fresh weight) were improved in the plant leaves treated with endophyte. The biosynthesis of ascorbate peroxidase, phenylalanine ammonia-lyase, and catalase was improved in the endophyte-treated seeds compared with control seeds (p > 0.05). The isolated B. xiamenensis is an important resource to biocontrol Fusarium wilt diseases in the mustard plant.

Endophytic Bacillus aerophilus from the Leaves of Azadirachta indica as a Potential Biocontrol against Staphylococcus aureus

Aims: The study entailed isolation and identification of Bacillus aerophilus in the leaves of young Azadirachta indica plants, establishing the antimicrobial activity of the endophyte against Staphylococcus aureus, and screening for phytochemicals. The study investigated the potential of the endophytic Bacillus aerophilus in pathogen inhibition and phytochemical screening for bioactive compounds produced by the endophyte that can be used in drug development. The research contributes to the wider scientific goal of curbing rising cases of antibiotic resistance.&#x0D; Place and Duration of Study: Sample: collected from Kanyonyoo in Kitui County, between August 2022 and June 2023.&#x0D; Methodology: The bacteria were aseptically isolated and sub-cultured on nutrient agar. For identification, the bacteria underwent biochemical tests and molecular characterization. The 16S rRNA region was amplified and sequenced using universal primers 27F and 1492R. Bacterial isolate K1L003 was identified as Bacillus aerophilus. For antimicrobial testing, the agar disk diffusion method was used against pathogenic Staphylococcus aureus on Mueller Hinton Agar plates (MHA). The controls were doxycycline and vancomycin.&#x0D; Results: The endophytic isolate only inhibited Staphylococcus aureus (P &lt; .001), with an inhibition mean similar to doxycycline (26 mm), against the pathogen. The endophytic isolate produced terpenoids, saponins, alkaloids, and flavonoids.&#x0D; Conclusion: Endophytic Bacillus aerophilus has better antimicrobial inhibition potential than vancomycin, against Staphylococcus aureus and produces diverse bioactive secondary metabolites. The endophyte has the potential to produce compounds that are useful in treating drug-resistant Staphylococcal infections.

Potential utility of endophytic Bacillus altitudinis strain P32-3 as a biocontrol agent for the postharvest prevention of sweet potato black rot

Sweet potato black rot is a common postharvest disease caused by the pathogenic fungus Ceratocystis fimbriata. The toxins produced by infected tubers are potentially harmful to humans. Plant endophytic bacteria may serve as biocontrol agents for protecting crops (including vegetables) during postharvest storage without detrimental effects on humans or other animals. In this study, 11 bacterial strains of Bacillus, Priestia, Blautia, Pseudomonas, and Sphingomonas species were isolated from sweet potato storage roots, of which only one strain (P32-3), which was identified as Bacillus altitudinis, strongly inhibited C. fimbriata mycelial growth and spore germination. Furthermore, the volatile organic compounds and cell-free supernatant of strain P32-3 repressed C. fimbriata growth. Additionally, P32-3 treatments significantly decreased the disease spot diameter and depth on harvested sweet potato roots. A total of 981 differentially expressed genes were identified by comparing the transcriptomes of the control and P32-3-treated mycelia. Strain P32-3 significantly altered the expressions of genes related to cell wall integrity, the cell membrane, spore germination, energy metabolism, pathogenicity, and oxidative stress. In addition, RNA-seq results were validated by a quantitative real-time polymerase chain reaction analysis. Our results indicate that B. altitudinis strain P32-3 can inhibit C. fimbriata growth and development and protect postharvest sweet potato storage roots from C. fimbriata infection. The study findings provide the theoretical basis for future investigations on the utility of strain P32-3 for protecting sweet potato against black rot during the postharvest stage.

Role of Bacillus spp. Plant Growth Promoting Properties in Mitigating Biotic and Abiotic Stresses in Lowland Rice (Oryza sativa L.).

The ability of microorganisms to promote plant growth and mitigate abiotic and biotic stresses makes them an interesting tool for sustainable agriculture. Numerous studies aim to identify new, promising bacteria isolates. Traditional culture-based methods, which focus on selecting microorganisms with plant-growth-promoting traits, such as hormone production, nutrient solubilization, and antifungal properties, are widely used. This study aims to investigate the role of plant-growth-promoting properties in bacteria-mediated stress mitigation and the suitability of traditional culture-based methods as a screening tool for the identification of beneficial bacteria. To this end, we tested three endophytic Bacillus isolates, which have previously been shown to affect tolerance against iron toxicity in lowland rice, (a) for their effect on the resistance against brown spot disease, and (b) for plant-growth-promoting traits using common culture-based methods. Both B. pumilus isolates inhibited fungal growth in vitro and reduced brown spot disease in two of three rice cultivars in planta, although they tested negative for all plant-growth-promoting traits. While B. megaterium was negative for ACC deaminase activity and nutrient solubilization, it exhibited auxin production. Nevertheless, B. megaterium did not suppress brown spot disease in any of the three rice cultivars. This study shows that bacteria do not necessarily have to possess classical plant-growth-promoting properties in order to be beneficial to plants, and it emphasizes the limitation of common culture-based methods in effectively identifying beneficial bacteria. Moreover, our results highlight the significance of the interaction between bacteria and plant cultivars in determining the beneficial effects of Bacillus spp. on plants under biotic or abiotic stresses.

Enhancing defense against rice blast disease: Unveiling the role of leaf endophytic firmicutes in antifungal antibiosis and induced systemic resistance

Rice remains the primary staple for more than half of the world's population, yet its cultivation faces numerous challenges, including both biotic and abiotic stresses. One significant obstacle is the prevalence of rice blast disease, which substantially diminishes productivity and increases cultivation costs due to frequent fungicide applications. Consequently, the presence of fungicide residues in rice raises concerns about compliance with international maximum residue limits (MRLs). While host resistance has proven effective, it often remains vulnerable to new variants of the Magnaporthe oryzae pathogen. Therefore, there is a critical need to explore innovative management strategies that can complement or enhance existing methods. An unexplored avenue involves harnessing endophytic bacterial communities. To this end, the present study investigates the potential of eleven endophytic Bacillus spp. in suppressing Pyricularia oryzae, promoting plant growth, and eliciting a defense response through phyllobacterization. The results indicate that the secreted metabolome and volatilome of seven tested isolates demonstrate inhibitory effects against P.oryzae, ranging from a minimum of 40% to a maximum of 70%. Bacillus siamensis L34, B. amyloliquefaciens RA37, B. velezensis L12, and B. subtilis B18 produce antifungal antibiotics targeting P.oryzae. Additionally, B. subtilis S4 and B. subtilis S6 emerge as excellent inducers of systemic resistance against blast disease, as evidenced by elevated activity of biochemical defense enzymes such as peroxidase, polyphenol oxidase, and total phenol content. However, a balance between primary metabolic activity (e.g., chlorophyll content, chlorophyll fluorescence, and photosynthetic rate) and defense activity is observed. Furthermore, specific endophytic Bacillus spp. significantly stimulates defense-related genes, including OsPAD4, OsFMO1, and OsEDS1. These findings underscore the multifaceted potential of endophytic Bacillus in managing blast disease through antibiosis and induced systemic resistance. In conclusion, this study highlights the promising role of endophytic Bacillus spp. as a viable option for blast disease management. Their ability to inhibit the pathogen and induce systemic resistance makes them a valuable addition to the existing strategies. However, it is crucial to consider the trade-off between primary metabolic activity and defense response when implementing these bacteria-based approaches.

Endophytic Bacillus subtilis H17-16 effectively inhibits Phytophthora infestans, the pathogen of potato late blight, and its potential application.

As a highly prevalent epidemic disease of potato, late blight caused by Phytophthora infestans poses a serious threat to potato yield and quality. At present, Chemical fungicides are mainly used to control potato late blight, but long-term overuse of chemical fungicides may lead to environmental pollution and human health threats. Endophytes, natural resources for plant diseases control, can promote plant growth, enhance plant resistance, and secrete antifungal substances. Therefore, there is an urgent need to find some beneficial endophytes to control potato late blight. We isolated a strain of Bacillus subtilis H17-16 from potato healthy roots. It can significantly inhibit mycelial growth, sporangia germination and the pathogenicity of Phytophthora infestans, induce the resistance of potato to late blight, and promote potato growth. In addition, H17-16 has the ability to produce protease, volatile compounds (VOCs) and form biofilms. After H17-16 treatment, most of the genes involved in metabolism, virulence and drug resistance of Phytophthora infestans were down-regulated significantly, and the genes related to ribosome biogenesis were mainly up-regulated. Moreover, field and postharvest application of H17-16 can effectively reduce the occurrence of potato late blight, and the combination of H17-16 with chitosan or chemical fungicides had a better effect than single H17-16. Our results reveal that Bacillus subtilis H17-16 has great potential as a natural fungicide for controlling potato late blight, laying a theoretical basis for its development as a biological agent. This article is protected by copyright. All rights reserved.

A Review on Bio-Based Control of Post-Harvest Diseases

Bacillus subtilis non-pathogenic beneficial bacteria, promotes plant growth, disease resistance and tolerance to abiotic stresses. It produces bioactive substances with antibiotic properties and induces physiological features in plant metabolism without adverse effects on the environment or human health. Bacillus subtilis has been used to treat various postharvest diseases during handling, transportation and storage of various fresh fruits and vegetables. It is the first microorganism patented as a postharvest bio control agent for Brown rot of stone fruits, improving the post-harvest physiology of various fruit/vegetables. Bacillus strains AG1 and H110 have been shown to be effective against Vine wood fungal pathogens and post-harvest pathogens. They have been shown to reduce symptoms of Anthracnose in fruit caused by fungal pathogens Colletotrichum gloeosporioides and C. acutatum and White rot caused by Botryosphaeria dothidea. Endophytic Bacillus strains have been developed that can colonize plant tissues and live in the same ecological niches as pathogens, thus preventing post-harvest diseases and improving preservation during storage. Bacillus strains induce auxins, cytokinins, gibberellins, ABA, JA and SA in plants, which can stimulate plant growth under stressful conditions. Endophytic bacteria can induce ISR against pathogens and abiotic stressors, extending the shelf life of stored fruits and vegetables. Microbial antagonists can be applied after harvest to control fruit and vegetable diseases, but a single microbial strain cannot prevent all fruits/vegetables from decaying during storage. Combining diverse antagonistic microorganisms with diverse microbial activity and combining various bio-controlling characteristics can prevent post-harvest decay on fruits/vegetables.

The Potential of Hydroponic Seed Minituber Enrichment with the Endophyte Bacillus subtilis for Improving the Yield Components and Quality of Potato (Solanum tuberosum L.)

This study examined the potential of using the endophytic bacteria Bacillus subtilis (10-4 and 26D) to enrich hydroponically grown potato seed minitubers (Solanum tuberosum L. cv. Bashkirsky) to improve plant growth, photosynthetic pigments, yield, and quality parameters, including nutritional value (i.e., macro-/microelements, vitamin C, anthocyanins). Potato seed minitubers, obtained from in-vitro-grown microplants in a hydroponic system, were inoculated with endophytic B. subtilis and subsequently grown in pots under controlled conditions. The results demonstrated the successful colonization of seed minitubers by B. subtilis, with subsequent distribution into growing plants (roots, shoots). The endophytes accelerated the plant’s phenological shifts, resulting in earlier emergence of sprouts, budding, and flowering compared with control plants. They also had increased leaf photosynthetic pigments (chlorophyll (Chl) a, Chl b, and carotenoids), total leaf area, and positively influenced leaf proline contents. The height of plants and number of stems per plant did not change significantly upon endophyte treatment, but improved root growth was observed throughout the experiment. As a result of endophyte application, there was an increase in stolon weight, number and size of tubers, and overall tuber yield. There were no significant differences in terms of total dry matter and starch content of the tubers compared to the control group, but the sugar levels decreased and the size of the starch grains was larger in endophyte-treated tubers. Furthermore, endophyte treatment resulted in an increased accumulation of nutrients including N, P, K, Cu, and Fe, as well as vitamin C and anthocyanins in harvested tubers. These findings indicate that colonization of hydroponically grown potato seed minitubers with endophytic B. subtilis (10-4 and 26D) before planting has great potential as an eco-friendly approach to obtain higher-quality seeds and to increase tuber yield and nutritional value in field conditions.

Characterization of a Metallic-Ions-Independent L-Arabinose Isomerase from Endophytic Bacillus amyloliquefaciens for Production of D-Tagatose as a Functional Sweetener

D-Tagatose is a low-calorie sugar substitute that has gained increased attention as a functional sweetener owing to its nutraceutical and prebiotic properties. Traditionally, D-tagatose is produced via the enzymatic conversion of L-galactose to D-tagatose by L-arabinose isomerase (L-AI). Nonetheless, the most reported L-AI enzymes are ion-dependent enzymes requiring Mn2+ and/or Co2+ as cofactors for their reactions, which limits their application due to safety and health concerns. Herein, we addressed the facile bioconversion of L-galactose to D-tagatose using a novel recombinant metallic-ions-independent L-AI derived from endophytic Bacillus amyloliquefaciens CAAI isolated from cantaloupe fruits. The ORF (1500 bp) of the L-arabinose isomerase gene (araA) was cloned and over-expressed in Escherichia coli. The recombinant enzyme (BAAI) was purified to homogeneity using Ni-NTA affinity chromatography, yielding a single distinct band with an apparent molecular mass of approximately 59 kDa as deduced from SDS-PAGE analysis. The purified enzyme showed optimum activity at pH and temperature of 7.5 and 45 °C, respectively, with obvious enzymatic activity in the presence of ethylenediaminetetraacetic acid (EDTA), indicating the metallic-ions independence from BAAI. The Km values of BAAI for D-galactose and L-arabinose were 251.6 mM and 92.8 mM, respectively. The catalytic efficiency (kcat/Km) values for D-galactose and L-arabinose were found to be 2.34 and 46.85 mM–1 min–1, respectively. The results revealed the production of 47.2 g/L D-tagatose from D-galactose (100 g/L) with 47.2% bioconversion efficiency in a metallic-ions-free reaction system that could be implemented in safe-production of food-grade low-calorie sweetener, D-tagatose.

Endophytic Bacillus strains enhance biomass and bioactive metabolites of Gloriosa superba

Gloriosa superba L. is an economically important medicinal plant and variation in its colchicine content may be due to associated endo- and ectophytic beneficial microbes. Among them, endophytic bacteria contribute to enhancing plant growth, induce biotic and abiotic stress tolerance, and play a significant role in secondary metabolite synthesis. In the present study, 76 native endophytic bacteria were isolated from high and low-colchicine and gloriosine-containing chemotypes of G. superba rhizome and characterized for vegetative growth enhancement and abiotic stress mitigation. The screening has resulted in the selection of 23 plant growth-promoting (PGP) bacterial endophytes with abiotic stress tolerance. Most of these isolates belong to the Bacillus genera identification based on the 16S rRNA gene. Based on the significant enhancement in maize (Zea mays L.; as model host plant) under greenhouse conditions ten efficient bacterial strains were selected. Further, three endophytic bacterial strains, NBRI HYL5 and NBRI HYL8 (high colchicine and gloriosine containing chemotype), and NBRI LYN9 (low colchicine and gloriosine containing chemotype) were selected based on the survivability and enhanced 1-aminocyclopropane-1- carboxylate (ACC) deaminase activity, phosphate solubilization, biofilm, Indole acidic acid (IAA), exopolysaccharide, and alginate production abilities under different abiotic stress conditions. Finally, a greenhouse experiment was conducted to evaluate the effect of selected bacterial endophyte inoculation in G. superba for growth enhancement and secondary metabolite content. The results showed that plants treated with NBRI HYL5 and NBRI HYL8 significantly enhanced the colchicine content (97–98 %) and gloriosine content (70–73 %), respectively as compared with control. However, inoculation of NBRI LYN9 has demonstrated significant enhancement only in gloriosine content (79 %). In addition, NBRI HYL5 and NBRI HYL8 have demonstrated significant improvement in overall vegetative growth parameters, photosynthetic pigments, and total soluble sugar content. Colonization and persistence of selected endophytic bacteria in G. superba rhizome and shoot clearly depicted the involvement not only in enhancing the vegetative growth but also in enhancing the colchicine and gloriosine content. Overall, this study presents the beneficial role of endophytic Bacillus strains in enhancing the growth of G. superba and secondary metabolite content may lead to contribute in sustainable cultivation of economically important medicinal plants.

The Effects of Seed Pretreatment with Endophytic Bacteria Bacillus subtilis on the Water Balance of Spring and Winter Wheat Seedlings under Short-Time Water Deficit.

We investigated the effect of pre-sowing seed treatment with endophytic Bacillus subtilis 10-4 (B. subtilis) on spring and winter wheat (Triticum aestivum L.; cultivars Ekada-70 (Ek) and Scepter (Sc), respectively) growth and tolerance under 1-24 h of drought stress, modulated by 12% polyethylene glycol 6000 (PEG). The results showed that drought decreased transpiration intensity (TI), root relative water content (RWC), osmotic potential (Ψπ) of cell sap, and induced proline accumulation and electrolyte leakage (EL) in both wheat cultivars. It was revealed that Sc was more responsive to PEG and B. subtilis treatments than Ek. Under drought, Ek did not significantly change root length, shoot height, or dry biomass. The pretreatment of wheat plants with B. subtilis performed significantly better under drought conditions through the enhanced TI, RWC, and Ψπ of the cell sap in comparison with the plants treated with 12% PEG alone. B. subtilis also reduced stress-caused EL, especially in the Sc cultivar. Under water deficit wheat seedlings, pretreated with B. subtilis, have a higher proline accumulation in comparison to untreated stressed plants. Taken together, our results demonstrate the crucial role of endophytic B. subtilis in ameliorating the adverse effects of water stress on the water balance of both winter and spring wheat cultivars.

Evaluation of chitosan encapsulated salicylic acid, metal oxide, and endophytic Bacillus subtilis in amelioration of citrus decline

The citrus decline caused by ‘Candidatus Liberibacter asiaticus'(CLas), a fastidious phloem-restricted gram-negative bacterium, is spreading rapidly in many citrus-growing regions of Iran and has caused low crop productivity and quality and severe economic damage to citrus growers. In this experiment, the effect of chitosan-encapsulated endophytic Bacillus subtilis, salicylic acid, zinc, and copper oxide on the amelioration of citrus decline in Valencia orange trees (Citrus scinensis L. Osbeck) was investigated, during 2019 and 2022. The trial was conducted with two experimental factors contained first factor the foliar application of chitosan encapsulated salicylic acid; and second factor the foliar spray of chitosan encapsulated endophytic Bacillus subtilis, chitosan encapsulated zinc oxide, and chitosan encapsulated copper oxide. Results indicate that the effect of chitosan-encapsulated endophytic Bacillus subtilis was more durable and consistent in reducing CLas bacteria for the following years. Foliar spray with chitosan-encapsulated endophytic Bacillus subtilis, chitosan-encapsulated zinc oxide and chitosan encapsulated copper oxide had a significant effect on the reduction of visual disease severity. Foliar application of chitosan-encapsulated salicylic acid along with chitosan-encapsulated endophytic Bacillus subtilis significantly reduced the leaves CLas bacteria titer from 1.75 × 107 to about 1.62 × 103. This treatment had the highest interaction effect on the increment of fibrous root density (96.9%), root metabolic activity (98.8%), tree canopy volume (80.6%), leaf area index (50%) and leaf chlorophyll content (168.1%). Applying chitosan-encapsulated salicylic acid with chitosan-encapsulated endophytic Bacillus subtilis significantly improves the fruit quality and yield. This eco-friendly treatment was a long-term effective potential resistance inducer and more sustainable disease control of CLas-infected Valencia orange trees.

In Vitro Evaluation of Some Endophytic Bacillus to Potentially Inhibit Grape and Grapevine Fungal Pathogens

Romania has a long history of grapevine culturing and winemaking. However, like any agricultural sector, viticulture faces devastating biological threats. Fungi responsible for grapevine trunk diseases (GTDs) and grape spoilage lead to considerable yield losses and a decline in grapevine quality. In the actual context, many countries, including Romania, have reoriented their approaches to minimize chemical inputs, which have been proven to be toxic and to have negative impacts on the environment, and to replace them with sustainable biocontrol strategies for the wine-growing sector. Within biocontrol strategies, Bacillus spp. is a well-known plant-protective bacteria with antifungal properties. Within this paper, six endophytic bacteria from various plant sources were studied. The bacterial strains were identified as B. pumilus, B. subtilis, and B. velezensis by sequencing their 16S rDNA region. Regardless of the in vitro test methods (using living bacterial cells, bacterial-cell-free supernatant (CFS), and volatile active compounds (VOCs)), B. velezensis strains revealed strong and broad antifungal activity against grape and grapevine fungal pathogens such as Aspergillus spp., Botrytis cinerea, Penicillium expansum, Diplodia seriata, Eutypa lata, Fusarium spp., Clonostachys rosea, Neofusicoccum parvum, and Stereum hirsutum. The functional antifungal genes encoding for difficidin, fengycin, iturins, macrolactin, and mycosubtilin were molecularly detected, which could support the proven antifungal activity of the endophytic strains. Lytic enzymes involved in fungal growth inhibition, such as chitinase, cellulase, and proteases, were also revealed to be produced by some of these bacterial strains. Various other in vitro tests, such as phosphate and phytate solubilization, phytohormone synthesis, the production of enzymes involved in the polyamine biosynthetic pathway, and pH as well as temperature tolerance tests were carried out to reveal the plant-beneficial potential of these bacterial strains. These results revealed that the B. velezensis strains, especially BAHs1, are the most suitable endophytes for grapevine biologic control, which could lead to the future development of sustainable management strategies.

Endophytic Plant Growth-Promoting Bacterium Bacillus subtilis Reduces the Toxic Effect of Cadmium on Wheat Plants.

Heavy metal ions, in particular cadmium (Cd), have a negative impact on the growth and productivity of major crops, including wheat. The use of environmentally friendly approaches, in particular, bacteria that have a growth-stimulating and protective effect, can increase the resistance of plants. The effects of the pre-sowing seed treatment with the plant growth-promoting endophyte Bacillus subtilis 10-4 (BS) on cadmium acetate (Cd)-stressed Triticum aestivum L. (wheat) growth, photosynthetic pigments, oxidative stress parameters, roots' lignin content, and Cd ions accumulation in plants were analyzed. The results showed that the tested Cd-tolerant BS improved the ability of wheat seeds to germinate in the presence of different Cd concentrations (0, 0.1, 0.5, and 1 mM). In addition, the bacterial treatment significantly decreased the damaging effects of Cd stress (1 mM) on seedlings' linear dimensions (lengths of roots and shoots), biomass, as well as on the integrity and permeability of the cell walls (i.e., lipid peroxidation and electrolyte leakage) and resulted in reduced H2O2 generation. The pretreatment with BS prevented the Cd-induced degradation of the leaf photosynthetic pigments chlorophyll (Chl) a, Chl b, and carotenoids. Moreover, the bacterial treatment intensified the lignin deposition in the roots under normal and, especially, Cd stress conditions, thereby enhancing the barrier properties of the cell wall. This manifested in a reduced Cd ions accumulation in the roots and in the restriction of its translocation to the aboveground parts (shoots) of the bacterized plants under Cd stress in comparison with non-bacterized controls. Thus, the pre-sowing seed treatment with the endophyte BS may serve as an eco-friendly approach to improve wheat production in Cd-contaminated areas.