Abstract: Objective To evaluate the ability of Bacillus spp. as direct-fed microbials (DFM) to biodegrade aflatoxin B 1 (AFB 1 ) by using an in vitro digestive model simulating in vivo conditions. Methods Sixty-nine Bacillus isolates were obtained from intestines, and soil samples were screened by using a selective media method against 0.25 and 1.00 μg/mL of AFB 1 in modified Czapek-Dox medium. Plates were incubated at 37 °C and observed every two days for two weeks. Physiological properties of the three Bacillus spp. candidates were characterized biochemically and by 16S rRNA sequence analyzes for identification. Tolerance to acidic pH, osmotic concentrations of NaCl, bile salts were tested, and antimicrobial sensitivity profiles were also determined. Bacillus candidates were individually sporulated by using a solid fermentation method and combined. Spores were incorporated into 1 of 3 experimental feed groups: 1) Negative control group, with unmedicated starter broiler feed without AFB 1 ; 2) Positive control group, with negative control feed contaminated with 0.01% AFB 1 ; 3) DFM treated group, with positive control feed supplemented with 10 9 spores/g. After digestion time (3:15 h), supernatants and digesta were collected for high-performance liquid chromatography fluorescence detection analysis by triplicate. Results Three out of those sixty-nine DFM candidates showed ability to biodegrade AFB 1 in vitro based on growth as well as reduction of fluorescence and area of clearance around each colony in modified Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identified the strains as Bacillus amyloliquefaciens , Bacillus megaterium and Bacillus subtilis . The three Bacillus strains were tolerant to acidic conditions (pH 2.0), tolerant to a high osmotic pressure (NaCl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No significant differences ( P > 0.05) were observed in the concentrations of AFB 1 in neither the supernatants nor digesta samples evaluated by high-performance liquid chromatography with fluorescence detection between positive control or DFM treated groups. Conclusions In vitro digestion time was not enough to confirm biodegradation of AFB 1 . Further studies to evaluate the possible biodegradation effects of the Bacillus -DFM when continuously administered in experimentally contaminated feed with AFB 1 , are in progress.

Abstract: Saputra R, Arwiyanto T, Wibowo A. 2015. Antagonistic activity assay of some isolates of Bacillus spp. against Bacterial Wilt Diseases (Ralstonia solanacearum) in some varieties of tomato and it’s identification. Pros Sem Nas Masy Biodiv Indon 1: 1116-1122. Ralstonia solanacearum is an important bacterial plant pathogen that frequently attacks in tomato plants and causes wilt disease. A great number effort has practiced minimizing this disease such as farming practices, the use of chemical pesticides and the development of resistant varieties, however, all showed limited success. Due to the limited effectiveness of some of these controls, yet bacterial wilt disease remains a serious problem economically. Therefore, biological control is still a great concern today. Of the many antagonistic bacteria, Bacillus spp. and Pseudomonas spp. are the most widely used in the biological control of plant diseases. The purposes of this study were to identify Bacillus spp. and investigate the capability of Bacillus isolates to suppress the development of bacterial wilt in tomato plants. The research was conducted at the Laboratory of Plant Bacteriology and greenhouse, Department of Plant Pest and Diseases, Faculty of Agriculture, UGM. Bacillus isolates were isolated from the rhizosphere of tomato plants. Based on the results of the study, six isolates of Bacillus showed diverse capabilities in suppressing the development of bacterial wilt disease, but Bacillus sp. isolate Ba-1 demonstrated the ability tends to be better than the other isolates. The biochemical identification results revealed that six isolates of Bacillus have similar properties. Homology of Bacillus sp. isolate Ba-1 by 16S rRNA gene of BLAST result showed this bacterium is closely related with Bacillus licheniformis strain DSM 13T (NR 118996) as the homology between them was 99%.

Abstract: In order to better understand the ecology and diversity of microbes in the rhizosphere of desert plants, we undertook a survey of Bacillus species isolated from soil around Rhazya stricta plants from the area around Jeddah, in The Kingdom, Saudi Arabia. We have sequenced the genomes of 8 Bacillus isolates representing four different species.

Abstract: Extreme environments merit special attention and significance because of the possible existence of thermophilic microorganisms in such ecological niches. Keeping this in mind indigenous stove ash samples were explored for extremophilic bacteria in term of their biodiversity. Accordingly, this study reports 37 bacterial isolates from the local wood run oven (Tandoor) ash samples. All the isolated strains belong to genus Bacillus on the bases of morpho-cultural and biochemical considerations. The average temperature tolerance profile was >45°C thereby, indicating towards the thermophilic nature of the isolated strains. The Bacillus isolates were screened for 10 different hydrolytic enzymes (cellulase, xylanase, amylase, pectinase, caseinase, keratinase, lipase, esterase, dextranase and β-galactosidase) by plate screening method using the medium incorporated with specific substrate(s). It was found that keratinase was produced by all the isolates while, 36 (97.2%) isolates showed caseinase and esterase production. Amylase was produced by 35(94.6%) isolates and 34 (91.8%) isolates were able to degrade Tween-80 and xylan as substrate for lipase and xylanase respectively. The enzyme, β-galactosidase was produced by 31 (89.1%) of the isolates. Cellulase and dextranase were produced by 26 (70.2%) and 22 (59.4%) isolates respectively. None of the isolates could (under the existing conditions) produce pectin-hydrolyzing enzyme. According to the Tukey's post hoc test, significant difference was found between the mean enzyme index of all the (screened) enzymes. Thus, the isolated bacterial strains with diverse hydrolytic potential may be of great value and relevance for the existing (national) industrial setups.

Abstract: Faba bean ( Vicia faba L.) is one of the most important pulse crops grown in eastern Africa. Black root rot ( Fusarium solani ) is known to cause great yield losses in faba bean, especially in the highlands of Ethiopia. The objective of this study was to evaluate the biological control ability of native Bacillus species on the basis of their antagonistic effects against F. solani . The study was conducted in vitro and in a greenhouse. All tested Bacillus isolates significantly (P<0.05) reduced radial mycelial growth of the pathogen. Seven bacterial isolates restricted growth of the pathogen to <14 mm diameter, and showed 39-44% efficacy over the positive control. Isolate BP048 was the most effective, with 43.6% efficacy. The lowest inhibitory effects, 15.5 and 27.8%, were recorded from isolates BS083 and BS0102, respectively. The culture filtrate of the bacterial isolates also inhibited F. solani spore germination. In vivo , the isolates significantly reduced severity of black root rot on artificially inoculated faba bean seedlings. The antagonist Bacillus isolates kept black root rot severity low with more than 50% disease suppression, compared to the untreated control. Keywords: Ethiopia, Fusarium solani , Vicia fabae

Abstract: In this study, the biocontrol potential of Bacillus isolates was investigated in laboratory and greenhouse experiments. Five out of 70 bacterial isolates from the root-zone of crops and a goat pasture caused second stage juvenile (J2) mortality greater than 50% in vitro after 24 hours.Three of the five selected isolates (BC27, BC29 and BC31) which were isolated from the root-zone of a goat pasture caused J2 mortality greater than 80% at 108spores ml-1in vitro after 24 hours, with BC27 causing100% J2 mortality after 3 hours. Seed treatment of soybean with Isolates BC27 and BC29 caused a reduction in rootknot galling and egg mass counts (P≤0.0001) and also caused a significant increase in shoot weight (P≤0.0001), when compared to the Control.Blast analysis revealed that the two select isolates, BC27 and BC29,hadsimilar sequences to Bacillus spp. T2 and Bacillus spp. KT18, as listed on the Gen-Bank, respectively.

Abstract: Our previous report demonstrated successful isolation of soil-borne bacteria that suppressed the potential of Rhizoctonia solani AG2-2 (IV) causing turfgrass large patch disease when applied to Korean lawngrass ( Zoysia japonica). The current study aimed to uncover the mechanisms of this antagonism of Rhizoctonia solani and to define culture conditions for the isolated microbes. We found that two Bacillus isolates, I-009 and FRIN-001-1 strains, produced cellulase and siderophore, but not chitinase, while the Pseudomonas YPIN-022 strain was found to release only siderophore, implying that three antagonistic bacteria commonly interrupt Fe uptake by the large patch pathogen. The I-009 and FRIN-001-1 isolates grew best at 35 and 30°C in growth medium of pH 5 to 8 for 32 and 28 h, respectively, while optimum growth for the YPIN-022 strain was found at 35°C at pH 5 to 9 for 24 h. Good growth of I-009 and YPIN-022 over 24 h was obtained in M9 minimal medium supplemented with 1% sucrose, 0.5% yeast extract and 0.1% potassium chloride. FRIN-001-1 grew well in M9 medium with 1% mannitol, 0.5% yeast extract and 0.1% potassium phosphate dibasic.Additional key words:

Abstract: Isolation of rhizosphere bacteria that facilitate plant growth of field pea (Pisum sativum L.) crop growing in Himachal Pradesh has resulted in 19 isolates. The present work deal with isolation and characterization of 12 Bacillus bacterial isolates with indole acetic acid (IAA) producing capacity. Optimization of IAA production was carried out at with varying media components. Partial purification of IAA was done and purity confirmed with thin layer chromatography. Since IAA production is one of a major property, isolates were tested by assay. Study concluded that isolate HPMI-10 was the best IAA producing bacteria, resulting in better % germination with higher vigorous index for promising varieties and may be used as an efficient bio-fertilizer, plant growth promoters (PGP) and bio-pesticide inoculants for healthy plants.

Abstract: Fifty five Bacillus isolates were isolated from compost, and alkaline silty clay soil (rhizosphere of potato plant) in Shambat, Khartoum North, Sudan, and screened using morphological tests, biochemical and molecular characterization using 16s rDNA analysis., Screening of cellulase producing isolates was done using carboxyl methyl cellulose (CMC) as a substrate at 25°C. Twenty six isolates were found to be cellulase producers. Among the isolates, four isolates, 9+, 23, 20 and 13 showed high potential in producing extracellular cellulase and had an average cellulase activity of 2.89, 3.12, 3.48 and 3.53 Unit/ml, respectively. Genetic distance between the four isolates with high cellulase activity was determined with RAPD analysis based on OPC-3 primer.

Abstract: This study evaluated the efficiency of 19 Bacillus isolates, obtained from the rhizosphere and rhizoplane of wild and cultivated castor bean plants, to control the pathogenic fungus Macrophomina phaseolina. Using in vitro assays, we examined the antifungal effects of volatile and non-volatile compounds, the production of siderophores and the activity of chitinase in these isolates. In vivo experiments were conducted to determine the potential of the Bacillus isolates to colonise castor bean plant roots and to control the fungus. In general, results showed that isolates from wild castor bean, compared with isolates from cultivated castor bean, were more efficient producers of antifungal compounds, better colonisers of plant roots and more effective protectors of plant seedlings against infection with M. phaseolina. Altogether, isolate RP 5, originating from the rhizoplane of wild castor bean, was the most promising candidate for future evaluation as a biological control agent of M. phaseolina.

Abstract: Purpose – The purpose of this paper is to investigate the ability of four microorganisms to reduce disease infection of root lesion nematode (Pratylenchus spp.) and Fusarium spp. the causal agent of wilt/root-rot disease complex in chickpea. Design/methodology/approach – A pot experiment was conducted for three consecutive winter seasons. A completely randomized design with five replicates was adopted. Two Bacillus isolates and Trichoderma harzianum and T. viride and their combinations were applied in infected soil. The effect on plant growth parameters, disease incidence and severity, root necrosis, weight of shoot and root, nematode population density and reproductive index were assessed. Findings – The applications significantly (p=0.05) reduced the wilt/root-rot diseases complex. The reduction was attributed to the decline of the population density of nematodes in soil and root, and the suppression of the disease complex compared to controls, represented by reproductive index and the disease intensity...

Abstract: Fusarium head blight (FHB) is economically one of the most important fungal diseases of wheat in the world. The aim of this research was to determine the antifungal metabolites of native Bacillus isolates. Bacil- lus strains isolated from soil of wheat and barley fields (Rural areas of southern Tehran, autumn 2012), and exhibited in vitro antagonistic activity against some Fusarium species isolated from infected wheat seeds (Wheat fields of Parsabad at Moghan, Ardebil Summer 2012). An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the selected Bacillus strain in Nutrient broth medium. High Performance Liquid Chromatography (HPLC) of partially purified extract of the strain showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (11.80 min) from Sigma-Aldrich. The structure was further confirmed by Fourier Transform- Infrared Spectrum (FTIR) and Liquid Chromatographic Mass Spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of fengycin with iturin A. The genome of the selected isolate of Bacillus had shown 99/9 percent similarity by B. aryabhattai and the genome of the selected isolates of Fu- sarium had shown 99 percent similarity by F. graminearum . According to the results of this experiment, the antifungal effects of native Bacillus strains and also their effects in the biological control of wheat FHB dis- ease were confirmed.

Abstract: This study was conducted to characterize the Bacillus populations associated with dead Rhynchophorus ferrugineus, to develop a biological control for the red palm weevil. Dead adult beetles, collected throughout Sicily, were used for isolating internal and external spore forming bacteria (SFB) microbiota. The isolates, preliminarily allotted to the Bacillaceae family, were tested at four concentrations (103 to 106 CFU/mL) for their ability to inhibit hatching of eggs of R. ferrugineus and were used at 106 CFU/mL to monitor their insecticidal activity against 10-day-old larvae. Total amounts of SFB measured outside the skeleton and in the inners part of the beetles were 5.59–6.94 and 5.17–7.05 Log CFU/g, respectively. Hatching was markedly inhibited by nine isolates, representing nine distinct strains of seven species (Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, and Lysinibacillus sphaericus), especially by the strains B. pumilus GC43 and GC51, which exhibited lethal concentrations 50 (LC50) values of 1.60 × 103 and 9.84 × 103 CFU/mL, respectively. Among all the strains tested, only B. licheniformis CG62 exhibited significant insecticidal activity against red palm weevil larvae. The Bacillus isolates characterized and tested in this study inhibited the hatching of red palm weevils in a contact-dependent manner. Thus, these isolates can be used as a preventive rather than as a curative treatment.

Abstract: Aims To detect and characterize broad-spectrum antipathogen activity of indigenous bacterial isolates obtained from potato soil and soya bean leaves for their potential to be developed as biofungicides to control soilborne diseases such as Fusarium crown and root rot of tomato (FCRR) caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl). Methods and Results Thirteen bacterial isolates (Bacillus amyloliquefaciens (four isolates), Paenibacillus polymyxa (three isolates), Pseudomonas chlororaphis (two isolates), Pseudomonas fluorescens (two isolates), Bacillus subtilis (one isolate) and Pseudomonas sp. (one isolate)) or their volatiles showed antagonistic activity against most of the 10 plant pathogens in plate assays. Cell-free culture filtrates (CF) of five isolates or 1-butanol extracts of CFs also inhibited the growth of most pathogen mycelia in plate assays. PCR analysis confirmed the presence of most antibiotic biosynthetic genes such as phlD, phzFA, prnD and pltC in most Pseudomonas isolates and bmyB, bacA, ituD, srfAA and fenD in most Bacillus isolates. These bacterial isolates varied in the production of hydrogen cyanide (HCN), siderophores, β-1,3-glucanases, chitinases, proteases, indole-3-acetic acid, salicylic acid, and for nitrogen fixation and phosphate solubilization. Gas chromatography–mass spectrometry analysis identified 10 volatile compounds from 10 isolates and 18 compounds from 1-butanol extracts of CFs of five isolates. Application of irradiated peat formulation of six isolates to tomato roots prior to transplanting in a Forl-infested potting mix and field soil provided protection of tomato plants from FCRR disease and enhanced plant growth under greenhouse conditions. Conclusions Five of the 13 indigenous bacterial isolates were antagonistic to eight plant pathogens, both in vitro and in vivo. Antagonistic and plant-growth promotion activities of these isolates might be related to the production of several types of antibiotics, lytic enzymes, phytohormones, secondary metabolites, siderophores and volatile compounds; however, any specific role of each needs to be determined. Significance and Impact of the Study Indigenous antagonistic bacterial isolates have the potential to be developed as biofungicides for minimizing early crop losses due to soilborne diseases caused by Fusarium and other soilborne pathogens.

Abstract: Vanilla beans were analyzed using biochemical methods, which revealed that glucovanillin disperses from the inner part to the outer part of the vanilla bean during the curing process and is simultaneously hydrolyzed by β-d-glucosidase. Enzymatic hydrolysis was found to occur on the surface of the vanilla beans. Transcripts of the β-d-glucosidase gene (bgl) of colonizing microorganisms were detected. The results directly indicate that colonizing microorganisms are involved in glucovanillin hydrolysis. Phylogenetic analysis based on 16S rRNA gene sequences showed that the colonizing microorganisms mainly belonged to the Bacillus genus. bgl was detected in all the isolates and presented clustering similar to that of the isolate taxonomy. Furthermore, inoculation of green fluorescent protein-tagged isolates showed that the Bacillus isolates can colonize vanilla beans. Glucovanillin was metabolized as the sole source of carbon in a culture of the isolates within 24 h. These isolates presented unique glucovanillin degradation capabilities. Vanillin was the major volatile compound in the culture. Other compounds, such as α-cubebene, β-pinene, and guaiacol, were detected in some isolate cultures. Colonizing Bacillus isolates were found to hydrolyze glucovanillin in culture, indirectly demonstrating the involvement of colonizing Bacillus isolates in glucovanillin hydrolysis during the vanilla curing process. Based on these results, we conclude that colonizing Bacillus isolates produce β-d-glucosidase, which mediates glucovanillin hydrolysis and influences flavor formation.