Application Of Cell Suspension Research Articles

Bacillus subtilis TE3: A promising biological control agent against Bipolaris sorokiniana, the causal agent of spot blotch in wheat (Triticum turgidum L. subsp. durum)

Bipolaris sorokiniana, the causal agent of wheat spot blotch, is an emerging phytopathogen in the Yaqui Valley, Sonora, Mexico. Currently, the unique method to control spot blotch on that region is employing synthetic fungicides with environmental and human health issues. Thus, the aim of this work was to identify and characterize native wheat-associated bacteria in order to find promising biological control strains that can be used as a sustainable alternative to control spot blotch. The identification of antagonistic strains was carried out by confrontation assay against B. sorokiniana. Antagonistic strains to B. sorokiniana were taxonomically classified by 16S rRNA gene sequencing, and characterized on important biological traits: (i) inhibition of the target phytopathogen, (ii) growth rate, and (iii) cytotoxic activity. Out 195 bacterial strains tested, 14 exhibited antagonistic activity against B. sorokiniana at different degree (1.6–8.0 mm of inhibition zone). The Antagonistic strains were taxonomically identified as members of Bacillus (eight strains), Paenibacillus (one strain), Pseudomonas (two strains), Achromobacter (one strain), Delftia (one strain), and Stenotrophomonas (one strain). The strain TE3, identified as Bacillus subtilis, showed promising biological control traits: (i) broad spectrum inhibition against B. sorokiniana, (ii) growth capacity in synthetic minimal medium, and (iii) non-cytotoxic activity to erythrocyte (γ-hemolysis). The application of cell suspension or cell-free culture filtrate of B. subtilis TE3 under highly conducive conditions (28 °C and 100% HR) for wheat spot blotch, resulted in a significant reduction of that disease. In conclusion, B. subtilis TE3 and its antifungal metabolites are promising effective treatment to control the causal agent of the wheat spot blotch.

First Report of Dickeya dianthicola Causing Blackleg on Potato (Solanum tuberosum) in Bulgaria.

Potato (Solanum tuberosum) is an important and widespread crop in Bulgaria. A new disease was observed on a single potato plot (Plovdiv region) without a history of potato cultivation in the spring of 2011. Initially, single lower leaves wilted on recently emerged plants (approx. 15% incidence) with subsequent desiccation of the leaf margins. The wilting progressed over time and eventually the whole stem became desiccated. A blackleg-like necrosis was noticed at the stem base when symptomatic plants were uprooted. Most diseased stems remained green above ground but pith tissue was heavily macerated and some of the stems became hollow as the pith dried out. Mother tubers were partially or entirely macerated. In most cases, the decay was initiated from the stolon end. Bacterial strains were obtained from symptomatic stems and tubers by dilution plating on King's B medium. The strains produced indigoidin pigment and induced a hypersensitive response 24 h after infiltration into tobacco and Sedum hybridum leaves (2). The strains were identified as Dickeya spp. by the production of the PCR amplicon of the pectate lyase ADE gene cluster (3) and of the pectate lyase I gene (4). The partial sequence of the fliC PCR amplicon (1) of strain SB2589 (GenBank Accession No. KF442436) displayed 100% homology with four whole genome shotgun sequences of Dickeya dianthicola in GenBank. Pectinolytic activity was demonstrated by inoculation of surface disinfested potato tubers of cv. Kondor. Conical core tissue was removed at the apical end and 100 μl bacterial suspension (107 CFU in sterile 10 mM phosphate buffer) was deposited in the cavity. The cap was reattached to the tuber and immobilized by Parafilm. Positive control tubers were inoculated with D. dianthicola reference strain GBBC 2039 (LMG 25864) and negative control tubers were inoculated with sterile 10 mM phosphate buffer. All tubers were incubated for 48 h at 28°C under micro-aerobic conditions reducing the air pressure to 90 mb in a vacuum incubator. The D. dianthicola reference strain and Bulgarian strains produced maceration of tuber tissue. Maceration was not observed in the negative control tubers. Potato plants cv. Kondor were grown from minitubers in sandy soil in plastic nursery containers. The plants were inoculated by root drenching (one application of cell suspension at 109 CFU/liter) when the stems were 15 to 20 cm high (tuber initiation stage). Plants were incubated at 25 to 28°C with regular watering. Wilting symptoms developed within 10 days of inoculation, followed by necrosis of the pith. Strains obtained from the inoculated stems were confirmed as D. dianthicola as described above. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the disease was caused by D. dianthicola and to our knowledge this is the first report of the pathogen on potato in Bulgaria. Furthermore, this incident warrants further surveys of pectinolytic bacteria causing blackleg-like symptoms in potato crops in Bulgaria.

Impact of time between field application of Bacillus subtilis strains SB01 and SB24 and inoculation with Sclerotinia sclerotiorum on the suppression of Sclerotinia stem rot in soybean

This study evaluated the impact of time between the application of cell suspensions or cell-free filtrates of Bacillus subtilis strains SB01 or SB24 on soybean plants under field conditions and inoculation with Sclerotinia sclerotiorum on their effectiveness for suppression of S. sclerotiorum. The results showed that the cell suspensions of two strains provided greater effectiveness than the cell-free filtrates, but the suppression effectiveness decreased as the time between application in the field and S. sclerotiorum inoculation increased. The B. subtilis cell suspensions applied on soybean leaves for up to 10 days under field conditions were able to provide a significant (P < 0.01) reduction in disease severity by approximately 20–90% at 5 days after the S. sclerotiorum inoculation. When rated 15 days after S. sclerotiorum inoculation, plants treated with bacterial cells for ≤6 days reduced Sclerotinia stem rot severity by 15–70%. Most effectiveness was provided by the cell suspensions present on soybean leaves for <3 days under field conditions, which significantly (P < 0.01) reduced disease severity by 40–70% over 15 days. In comparison, the cell-free filtrates remaining on leaves for <6 days significantly (P < 0.01) reduced disease severity during the first 5 days after the inoculation, while the best cell-free filtrate treatments were those with ≤1-day intervals, which significantly (P < 0.01) reduced disease severity by 10–40% during 15 days after the inoculation. The effectiveness of B. subtilis was reduced when it rained after application.

The influence ofBacillus subtilisRB14-C on the development ofRhizoctonia solaniand indigenous microorganisms in the soil

The effect of soil inoculation with an antagonistic strain Bacillus subtilis RB14-C on the development of Rhizoctonia solani and changes occurring in soil and rhizosphere microbial communities were studied. RB14-C was added to the soil as a water suspension of the cells or as a broth culture. Application of cell suspensions to non-planted soil reduced the number of culturable bacteria. The density of R. solani and the number of filamentous fungi were not significantly affected by RB14-C. A similar effect was observed in the rhizosphere of tomato plants growns in bacterized soil. Broth cultures of RB14-C suppressed R. solani 1 d after inoculation, but after 3 d there was no difference in the pathogen density between soil amended with broth culture and control soil. In microcosm studies, cell suspensions of RB14-C also did not inhibit growth of R. solani on filters buried in soil. However, an inhibitory effect was obtained when a broth culture of the bacterium was used. The effect of RB14-C on fungal biomass was also estimated by measurement of ergosterol concentration in soil. It was found that ergosterol was mostly derived from R. solani and that there were no significant differences in its content between untreated soil and soil treated with RB14-C. The results suggest that suppression of Rhizoctonia damping-off by B. subtilis RB14-C probably is not related to the reduction of the pathogen population in the soil.

Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable?

This article reviews the experimental and clinical strategies currently in use or under development for the treatment of articular cartilage lesions. The vast majority of protocols under investigation pertain to the treatment of full-thickness defects (i.e., those which penetrate the subchondral bone and trabecular–bone spaces) rather than partial-thickness ones (i.e., those which are confined to the substance of articular cartilage tissue itself). This bias probably reflects the circumstance that partial-thickness defects do not heal spontaneously whereas full-thickness ones below a critical size do, albeit transiently. And it is, of course, a seemingly easier task to manipulate a process which is readily set in train than it is to overcome an induction-problem which Nature herself has not solved. Indeed, the reasons for this inert state of partial-thickness defects have only recently been elucidated, and these are briefly discussed. However, the main body of this review deals with the various transplantation concepts implemented for the repair of full-thickness defects. These fall into two broad categories: tissue-based (entailing the grafting of perichondrial, periosteal, cartilage or bone-cartilage material) and cell-based (utilizing chondroblasts, chondrocytes, periochondrial cells or mesenchymal stem cells). Cell-based systems are further subdivided according to whether cells are transplanted within a matrix (biodegradable, non-biodegradable or synthetic) or free in suspension. Thus far, the application of cell suspensions has always been combined with the grafting of a periosteal flap. The strengths and weaknesses of each concept are discussed.