Abstract: This study aimed to isolate, identify, and evaluate the probiotic properties of Bacillus species from honey of the stingless bee Heterotrigona itama. Bacillus spp. were isolated from five different H. itama meliponicultures, and the isolates were characterized through Gram-staining and a catalase test. Tolerance to acidic conditions and bile salt (0.3%), hydrophobicity, and autoaggregation tests were performed to assess the probiotic properties of the selected isolates, B. amyloliquefaciens HTI-19 and B. subtilis HTI-23. Both Bacillus isolates exhibited excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria and possessed significantly high survival rates in 0.3% bile solution for 3 h. Their survival rates in acidic conditions were also comparable to a commercial probiotic strain, Lactobacillus rhamnosus GG. Interestingly, the hydrophobicity and autoaggregation percentage showed no significant difference from L. rhamnosus GG, a commercial probiotic strain. The results from this study suggest that B. amyloliquefaciens HTI-19 and B. subtilis HTI-23 isolated from stingless bee honey have considerably good probiotic properties. Therefore, more studies should be done to investigate the effects of these bacteria cultures on gastrointestinal health.

Abstract: Botrytis cinerea, a major phytopathogenic fungus, has been reported to infect more than 200 crop species worldwide, and it causes massive losses in yield. The aim of this study was to evaluate the inhibitory abilities and effects of Bacillus amyloliquefaciens RS-25, Bacillus licheniformis MG-4, Bacillus subtilis Z-14, and Bacillus subtilis Pnf-4 and their culture filtrates and extracts against the gray mold caused by B. cinerea on postharvest tomato, strawberry, and grapefruit. The results revealed that the cells of Z-14, culture filtrate of RS-25, and cells of Z-14 showed the strongest biocontrol activity against the gray mold on the strawberry, grape, and tomato fruit, respectively. All the strains produced volatile organic compounds (VOCs), and the VOCs of Pnf-4 displayed the highest inhibition values. Based on headspace solid-phase microextraction in combination with gas chromatography-mass spectrometry, esters accounted for the largest percentage of the VOCs produced by RS-25, MG-4, Z-14, and Pnf-4 (36.80%, 29.58%, 30.78%, and 36.26%, respectively). All the strains showed potent cellulase and protease activities, but no chitinase activity. RS-25, Z-14, and MG-4, but not Pnf-4, grew on chrome azurol S agar, and an orange halo was formed around the colonies. All the strains showed biofilm formation, fruit colonization, and lipopeptide production, which may be the main modes of action of the antagonists against B. cinerea on the fruit. This study provides the basis for developing natural biocontrol agents against the gray mold caused by B. cinerea on postharvest fruit.

Abstract: The results of this study indicate that the maize rhizosphere remains a reservoir for microbial strains with unique beneficial properties. The study sought to provide an indigenous Bacillus strain with a bioprotective potential to alleviate maize fusariosis in South Africa. We selected seven Bacillus isolates (MORWBS1.1, MARBS2.7, VERBS5.5, MOREBS6.3, MOLBS8.5, MOLBS8.6, and NWUMFkBS10.5) with biosuppressive effects against two maize fungal pathogens (Fusarium graminearum and Fusarium culmorum) based on 16S rDNA gene characterization and lipopeptide gene analysis. The PCR analysis revealed that lipopeptide genes encoding the synthesis of iturin, surfactin, and fengycin might be responsible for their antifungal activities. Few of the isolates also showed possible biosurfactant capability, and their susceptibility to known antibiotics is indicative of their eco-friendly attributes. In addition, in silico genomic analysis of our best isolate (Bacillus velezensis NWUMFkBS10.5) and characterization of its active metabolite with FTIR, NMR, and ESI-Micro-Tof MS confirmed the presence of valuable genes clusters and metabolic pathways. The versatile genomic potential of our Bacillus isolate emphasizes the continued relevance of Bacillus spp. in biological management of plant diseases.

Abstract: Bacillus species are common in the environment due to their spore-forming ability and nutritional versatility and cause food contamination. Bacilli play a significant role in foodborne illnesses and food spoilage. In this study, 52 Bacillus isolates from retail fish and ground beef were identified and differentiated based on 16S rRNA, gyrB, and rpoB gene sequencing. The presence of genes encoding emetic toxin (ces), hemolytic enterotoxin hemolysin BL (hbl), nonhemolytic enterotoxin (nhe) and cytotoxin K (cytK1) was assessed in all Bacillus isolates. The ability of the Bacillus isolates to produce several extracellular enzymes that contribute to pathogenicity and food spoilage was investigated. The 16S rRNA, rpoB, and gyrB gene sequence similarities of the Bacillus isolates tested were 96.1%, 83.2%, and 77.5%, respectively. The gyrB gene demonstrated a higher degree of sequence variation than the 16S rRNA and rpoB genes. The prevalence of Bacillus isolates producing at least two of the genes of the HBL and NHE complexes was 23.1% and 15.4%, respectively. Of the B. cereus isolates, 10 (41.7%) possessed two or more enterotoxin genes. None of the isolates carried the ces and cytK1 genes. All isolates were positive for the production of enzymes such as protease, lipase, gelatinase, and DNase. However, only 92.3% of the tested isolates were positive for amylase. In conclusion, our results revealed that the presence of genes involved in toxin production and enzyme production in meat-originated B. cereus and other Bacillus isolates may cause spoilage of food and pose a health risk for consumers. PRACTICAL APPLICATION: Bacillus species can be found in various foods due to their ubiquitous nature. Bacillus spp., especially B. cereus, are associated with food poisoning and other infections in humans. Toxins and many extracellular enzymes produced by Bacillus spp. are the causative agents of foodborne outbreaks, food spoilage, and low-quality food with significantly reduced edibility. This study highlights the characterization of Bacillus spp. and presence of potentially pathogenic Bacillus species in meats.

Abstract: A study was carried out to test direct and indirect antagonistic effect against Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC), and plant growth-promoting (PGP) traits of bacteria isolated from rhizosphere soils of chickpea (Cicer arietinum L.). A total of 40 bacterial isolates were tested for their antagonistic activity against FOC and of which 10 were found to have strong antagonistic potential. These were found to be Streptomyces spp. (five isolates) and Bacillus spp. (five isolates) in the morphological and biochemical characterisation and 16S rDNA analysis. Under both greenhouse and wilt sick field conditions, the selected Streptomyces and Bacillus isolates reduced disease incidence and delayed expression of symptoms of disease, over the non-inoculated control. The PGP ability of the isolates such as nodule number, nodule weight, shoot weight, root weight, grain yield and stover yield were also demonstrated under greenhouse and field conditions over the non-inoculated control. Among the ten isolates, Streptomyces sp. AC-19 and Bacillus sp. BS-20 were found to have more potential for biocontrol of FOC and PGP in chickpea. This investigation indicates that the selected Streptomyces and Bacillus isolates have the potential to control Fusarium wilt disease and to promote plant growth in chickpea.

Abstract: Since its isolation 20 years ago, many studies have been devoted to Bacillus velezensis FZB42 (former name Bacillus amyloliquefaciens subsp. plantarum FZB42), which has been gradually accepted as a model organism for Gram-positive rhizobacteria. FZB42 is different from another widely studied bacterial strain, Bacillus subtilis 168, in its many features that are closely associated with plants. FZB42 represents a large group of Bacillus isolates that are beneficial to plants and of great importance in agriculture. In this work a database for FZB42 named 'AmyloWiki' is built to integrate all information of FZB42 available to date. The information includes the genomic, transcriptomic, proteomic, post-translational data as well as FZB42 unique genes, protein regulators, mutant availability, publications and etc. The website is built up with PHP and MySQL with a function of keyword searching, browsing, data-downloading and other functions.

Abstract: Cellualse is one of the most important enzymes used in textile, detergent, paper, food and feed industries. Therefore, a study was undertaken to isolate Bacillus bacteria having the potential to produce cellulase from soil samples. 24 soil samples were analyzed and 54 presumptive Bacillus isolates were isolated after heating the soil samples at 80°C for 10 min. Among them 45 isolates showed enzyme activity ranging from 0.003 to 0.17 U/ml in test tubes containing 5 ml medium composed of (g/L) glucose 0.5 gm, peptone 0.75 gm, FeSO4 0.01 gm, KH2PO4 0.5 gm, and MgSO4 0.5 gm at 120 rpm, 37° C and pH 7. Among them 1RW, 2WS, 3YR, 4WT, 6 RR, and 9SS showed 0.17, 0.15, 0.14, 0.15, 0.147 and 0.14U/ml enzyme activities, respectively. Production of cellulase by these isolates was further scaled up to shake culture containing 50 ml medium similar to that used in test tube culture. Among the isolates 1 RW showed the maximum activity. This 1 RW was identified by API kit and showed that 59 % belongs to Bacillus licheniformis strain (51% confirmation) or Bacillus subtilis (31% confirmation). Further gene analysis is required to confirm the species. The genetic improvement study will make the isolate a good source of cellulase.

Abstract: Fusarium wilt of tomato is one of the most prevalent and economically important diseases of tomato worldwide especially in tropical regions. The aims of the present study were to isolate and charac...

Abstract: This study aims to isolate Bacillus that can fight the growth of Vibrio harveyi. Based on the results of the inter-Bacillus competition test show that Bacillus isolates was able to compete and grow with each other on the media of Sea Water Completed Agar (SWCA). The challenge test Bacillus bacterial to against Vibrio harveyi bacteria, that Bacillus did not yet produce anti-bacteria on the second day. In the joint culture test method between Bacillus and Vibrio harveyi that Bacillus were able to inhibit the growth of Vibrio harveyi bacteria on the 4th day.

Abstract: This study investigates the effect of metals (cadmium, lead, mercury, and tellurium) and organic pollutants (benzene, diesel, lindane, and xylene) on a dinoflagellate-Prorocentrum sigmoides Bohm-and its associated culturable bacteria. Two bacterial cultures (Bacillus subtilis strain PD005 and B. xiamensis strain PD006) were isolated from P. sigmoides and characterized by scanning electron microscopy, 16S ribosomal RNA sequencing, biochemical analyses, and growth curve studies. This study points to a mutualistic relationship between P. sigmoides and its associated Bacillus isolates. P. sigmoides enhanced the growth of its associated Bacillus spp., through the secretion of extracellular exudates. In return, both Bacillus isolates contributed to the resistance of P. sigmoides to metals and organic pollutants. P. sigmoides and both Bacillus isolates exhibited concentration-dependent responses to metals and organic pollutants. An intriguing feature was the similar response of P. sigmoides and its associated Bacillus isolates to mercury and cadmium, indicating a co-selection of mercury and cadmium resistance. This provides support to the "dinoflagellate host-phycosphere bacteria" behaving as a single functional unit. However, the sensitivity profiles of P. sigmoides and its associated Bacillus isolates are different with respect to metals versus organic pollutants. These aspects need to be addressed in future studies to unravel the effect of metal and organic pollutants on dinoflagellates, an important component of the phytoplankton community, and to discern the influence of associated "phycosphere" bacteria on the response of dinoflagellates to pollutants.

Abstract: Phytopathogenic fungi pose a serious problem around the world in the economically important plants. Chemical fungicides are widely used in current agriculture. However, excessive use of chemical fungicides has led to the deterioration of human health, environmental pollution, and development of pathogenic resistance to fungicides. A serious search is required to identify alternative methods for crop protection, which is less dependent on chemicals and more environmentally friendly. Microbial antagonists widely used for biocontrol of plant fungal disease. The success of biocontrol depends on the nature of antagonistic properties and mechanisms of action of the biocontrol agent against the phytopathogenic fungi. In the present study, 83 Bacillus isolates isolated from marine samples were screened on the colloidal chitin agar medium. Based on the chitinolytic activity and percentage inhibition against fungal phytopathogens by dual plate technique, isolate B26 is most potent as biocontrol agents. The Bacillus B26 inhibited the growth of Fusarium solani TISTR 3436 and Penicillium chrysogenum with percentage inhibition 69% and 46.6% respectively, while did not inhibit the growth of Aspergillus niger and Aspergillus flavus. Bacillus isolate B26 showed a positive result for the urease production, catalase test, starch hydrolysis, casein hydrolysis with most suitable growth condition at 37°C, pH 7-8, and could grow at 0-5% NaCl concentration.

Abstract: Bacillus is a diverse genus consisting of more than 200 species with extensive genetic diversity. Their beneficial effects in industrial shrimp farming have been well documented. However, little is known about the biodiversity of the Bacillus spp. in this aquaculture system. Taxonomic analysis by 16S rRNA sequencing does not always allow species-level identification of Bacillus spp. In this study, 26 Bacillus isolates from two industrial Litopenaeus vannamei shrimp ponds in Bac Lieu Province, Vietnam, were analyzed for their genetic diversity by multi-locus sequence typing (MLST). A total of 22 sequence types were identified and segregated into four distinct clusters, corresponding to B. subtilis, B. velezensis, B. siamensis, and B. licheniformis. Bacillus subtilis and B. velezensis accounted for more than 73% of the Bacillus isolates. Notably, the MLST scheme exhibited high discriminatory power and might be further simplified to be a convenient method to identify species of the genus Bacillus.

Abstract: Salt curing is the method most commonly utilized in the leatherindustry to prevent microbial growth on raw hides/skins. Despitethis processing, a wide diversity of microorganisms belonging toDomains Bacteria and Archaea have nevertheless been observedon salted hides/skins. In order to understand whetherhalotolerant bacterial species in salt contaminate hides/skinsduring the curing process, 30 salt samples collected from 14leather factories in Corlu and Tuzla (Turkey) were examined forhalotolerant bacteria. Total counts of halotolerant bacterialnumbers, pH values and moisture contents of the salt sampleswere respectively determined between 104 CFU/g and 106 CFU/g,6.23 and 7.22, 0.90 and 5.02. All isolates were able to grow onboth Nutrient Agar Medium without NaCl and Nutrient Agaredium containing NaCl at concentrations ranging from 2 to10%. The microorganisms isolated from the samples wereidentified using phenotypic characteristics and comparativepartial 16S rRNA gene sequence analysis. The phylogeneticanalysis, using more than 1300 base comparisons of 16S rRNAsequence data, revealed 83 halotolerant isolates that share highlysimilar identities (97.82-100%) with their closest phylogeneticrelatives. These isolates were assigned to 12 different Bacillusspecies (B. amyloliquefaciens, B.atrophaeus, B.halotolerans, B.licheniformis, B.mojavensis, B.paralicheniformis, B.pumilus,B.safensis, B.siamensis, B.subtilis, B.tequilensis, B.velezensis). Wedetected catalase and protease activities, as well as productionacid from fructose, in all Bacillus isolates. Fifty-five isolatesdemonstrated positive oxidase activities, and 50 isolates utilizedcitrate as a sole carbon source. While a fairly high percentage ofthe isolates produced acid from maltose, almost half of theisolates produced acid from myo-inositol. While 67% of the saltsamples contained 1-2 different Bacillus species, 33% of the saltsamples contained 3-4 different Bacillus species. AlthoughB.amyloliquefaciens, B.atrophaeus, B.safensis, B.siamensisspecies were detected at a few salt samples, B.paralicheniformis and B.halotolerans species were detected at more than half of thesalt samples. These results uphold the hypothesis that proteolytichalotolerant Bacillus species in the curing salts may contaminatehides/skins during curing process. Hence, we recommendsterilized salts be used in the preservation of the hides/skins toprevent economic losses in the leather industry.

Abstract: Microbial utilization of uncommon C4 dicarboxylate L-tartrate is largely anaerobic, with aerobic L-tartrate utilization known for few bacterial species including Rhodopseudomonas sphaeroides and Pseudomonas putida. Aerobic L-tartrate-utilizing microbes could be industrially relevant owing to the efficient nature of the bioprocess and catalytic versatility of tartrate dehydrogenase (TDH) responsible for aerobic catabolism of L-tartrate. Present work involves isolation and characterization of Bacillus strains capable of aerobic L-tartrate utilization and its correlation with occurrence of TDH activity. Two out of 37 isolates, IC1-G and IC1-Y were identified as Bacillus megaterium spp. showing efficient aerobic growth, utilizing ~3.7 and 2.8 mM L-tartrate respectively at the end of 48 h. Several organic acids possibly including oxalic, succinic and citric acids were secreted as by-products of L-tartrate metabolism. Utilization of L-tartrate directly correlated with induction of TDH activity by ~3.2 and 5.2 folds in IC1-G and IC1-Y respectively, when grown in presence of L-tartrate as compared to when grown on citrate. Overall, this study contributes Bacillus as only the third genus capable of aerobic, TDH mediated L-tartrate utilization. These Bacillus isolates thus offer potential targets to develop an industrially relevant bioprocess and biocatalyst.

Abstract: Wheat is grown on 20% of the cultivated land area of the world and is a main food resource for 40% of the world’s population (Braun et al., 2010). In 2019, the forecast of wheat production is at nearly 771 million tonnes, 5.6 percent high than previous year’s (FAO, 2019). In 2050, the world’s population is expected to reach 9 billion, thus it is estimated that cereal production needs to increase by 50% by 2030 (Alexandratos and Bruinsma, 2012). The wheat production is suffering substantial losses of biotic and abiotic stress factors (Elad and Pertot, 2014; Kan et al., 2017). PGPRs (Plant Growth Promoting Rhizobacteria) are a group of beneficial bacteria living in the rhizosphere, the phyllosphere, or in the plant tissues as entophytes (Ahemad and Kibret, 2014; Miliutė et al., 2015). PGPRs can promote plant growth against stresses by direct and indirect mechanisms or a combination of both (Ertürk et al., 2010; Siddikee et al., 2010; Kusek and Çınar, 2012; Imriz et al., 2014; Unlu and Aysan, 2016; Telek et al., 2019). Indirect mechanisms comprise the suppression of pathogens through the production of antibiotics and extracellular hydrolytic enzymes and the action of siderophores, Inducing Systemic Resistance (ISR), exo-polysaccharides production. Direct mechanisms involve making the natural nutrition source ready to use for plants including fixation of atmospheric nitrogen, solubilisation of phosphorus, potassium and iron; production of siderophores; producing phytohormones like auxins, cytokinins and gibberellins, (Ahemad and Kibret, 2014) or by the activity of 1-aminocyclopropane-1carboxylate (ACC)-deaminase, an enzyme which can hinder the “plant stress ethylene” that is typically arose by a number of environmental stresses such as flooding, extreme temperature, the presence of organic and inorganic toxicants, phytopathogens, drought or high salt concentrations (Cheng et al., 2007; Glick, 2014; Gamalero and Glick, 2015).

Abstract: Bacterial crown rot (BCR) is a re-emerging disease of papaya (Carica papaya) in Southeast Asia caused by a gram-negative, facultative anaerobic bacterium, known as Erwinia mallotivora. Ultrastructural observations using SEM revealed colonization of bacterial community common in a BCR-tolerant genotype, before onset of tisue regeneration or 9regrowth9. This observation led to the hypothesis that papaya-associated bacteria might play a role in BCR tolerance. Isolation, characterization and identification using 16S rRNA sequence of bacteria associated with the tolerant 9regrowth9 genotype resulted to the discovery of two endophytic bacteria, of genera Kosakonia (isolate EBW) and Sphingomonas (isolate EBY). Papaya root-associated Bacillus isolates were also identified to belong to the B. amyloliqufaciens clade (isolate BN, BS) and B. thuringiensis clade (isolate BT). Separate inhibition assays indicated that, papaya latex and autoclaved culture metabolites (ACMs) from isolated bacteria promptly (between 18-30h) and efficiently inhibited (60-65%) E. mallotivora proliferation in vitro. Moreover, when ACMs from BN and EBW were inoculated in surface-sterilized papaya seeds, it variably retarded seed germination (20-60% reduction), depending on plant genotype, but significantly stimulated plant growth and biomass accumulation, at around two-fold increase. Interestingly, ACMs from all isolates, especially EBW, significantly reduced BCR incidence and severity in susceptible genotype at around two-fold. Finally, these results indicated that tolerance of papaya against E. mallotivora infection is probably influenced by host innate properties such as tissue regrowth and pathogen inhibition by plant latex, and acquired properties including pathogen antagonism, growth promotion and defense biopriming through metabolites from beneficial endophytic and root-assocaited bacteria.

Abstract: Microbial compositions for application to plants, plant parts and plant seeds are provided for improvement of plant yield and/or other beneficial plant traits. Methods of making and applying microbial compositions or formulations to plants, plant parts or plant seeds or to growth media are further provided to increase or improve plant yield and/or other beneficial plant traits.

Abstract: Soil microorganisms, which are usually found in plant rhizosphere, have a wide spectrum of beneficial effects on the promotion of plant growth. The most comprehensively studied bacteria with these effects belong to the Bacillus genus. In this study, seven Bacillus isolates from Medicago sativa rhizosphere soils were isolated. Plant growth promoting characteristics of these isolates, such as production of indole-3aceticacid, siderophores and hydrogen cyanide were tested. The induction of wheat (Triticum aestivum) and barley (Hordeum jubatum) seed germination was evaluated in vitro. Isolate BMG2 produced the highest indole-3-aceticacid of 24.89 µg/mL. The length of roots of barley increased up to 60%, while the length of shoots of barley increased 2.23 times afterapplying isolatesPAZE-6 and BMG1, respectively. In addition, isolates BMG1, BMG2 and PAZE-6 improved germination of both types of seeds and showed ability to produce useful substances such as siderophores and indole-3-acetic acid. Further, these isolates could be used in the production of liquid crop additives that can improve the total yield of cultivated plants, especially barley.