Optimizing a Bacillus subtilis isolate for biological control of sugar beet cercospora leaf spot

Bacillus spp. have been used to control a number of leaf spot and post harvest diseases. Their capacity to form endospores facilitates long-term storage and relatively easy commercialization. This study focuses on optimizing a Bacillus subtilis isolate, BacB, for the control of sugar beet Cercospora leaf spot, caused by Cercospora beticola Sacc., by examining application timing, biocontrol agent (BCA) concentration, use of the selective nutrient substrate β-glucan, and the form of the BCA at time of application. A method for germinating endospores prior to spraying, without active aeration, is described. Examining the effects of varying β-glucan concentrations and levels of BacB at application demonstrated a complex interaction between β-glucan, BCA population, and disease control. In the 1998 field season, disease severity was significantly decreased, as compared to the control, at an application rate of 1×10⁶ CFU/ml, or higher, with 0% β-glucan. In 1999, there was less disease pressure, and all treatments reduced disease severity. Growth chamber experiments demonstrated that applying the bacteria as vegetative cells instead of spores or applying the BCA 1–5 days before infection could significantly increase disease control. Laboratory experiments demonstrated the ability to induce germination and vegetative growth of BacB from a spore formulation, without shaking or fermentation equipment. This shows promise for optimizing Bacillus sp. for biological control. In field trials the vegetative cells did not perform better than the spore application, though the potential for β-glucan to increase disease was demonstrated.     

Programmed cell death in Blastocystis hominis occurs independently of caspase and mitochondrial pathways

We demonstrated previously that a cytotoxic monoclonal antibody (MAb) 1D5 elicits a programmed cell death (PCD) response in Blastocystis hominis and showed that caspase-3-like protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that mitochondrial dysregulation played a role in cell death. In the current study, we further analyzed the signaling pathways involved in PCD mediated by MAb 1D5. B. hominis cells were treated with MAb 1D5 or control MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial membrane potential (delta psi(m)), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and mitochondria-independent cell death pathways in a protozoan parasite.     

Spatial and temporal population dynamics of a phyllosphere colonizing Bacillus subtilis biological control agent of sugar beet cercospora leaf spot

This study examines the spatial and temporal variation of populations of the biological control agent (BCA) BacB, a Bacillus subtilis isolate, in the field and growth chamber in the presence of the fungus, Cercospora beticola, the causal agent of Cercospora leaf spot of sugarbeet. The use of the selective BCA support substrate β-glucan, applied at 0, 0.5, and 1.0% of the spray solution, did not influence differences in total population numbers (spores + vegetative cells) of a spontaneous rifampicin resistant isolate of BacB (Rif+) over a 14 day spray period. BacB Rif+, applied as a spore formulation, declined from 10,000 CFU/cm² on day 0.5–100 CFU/cm² on day 14 at the three levels of β-glucan tested. Distribution of BacB Rif+ populations was modeled on a leaf scale, with and without β-glucan. Higher populations of vegetative cells were more likely at 14 days with 1% β-glucan than with 0% β-glucan. BacB populations were more aggregated without β-glucan than with the nutrient substrate. There was no correlation between BacB density and Cercospora leaf spot disease severity, indicating that neither antibiosis nor parasitism is likely an important mechanism of disease control.     

Potential for biological control of Heterodera schachtii by Pochonia chlamydosporia var. chlamydosporia on sugar beet

The biological control potential of an isolate of Pochonia chlamydosporia var. chlamydosporia against Heterodera schachtii was examined by assessing the percentage of females and cysts that became infected on water agar, the effect of culture filtrate on juvenile mobility, and the effects of the fungus on the final population of the nematode on sugar beet under greenhouse conditions. After 3 weeks at 20°C, 74 and 95% of the eggs within cysts and females, respectively, were colonised by the fungus on water agar. The full concentration of the fungal filtrate from cultures in malt extract broth killed only 12% of the juveniles after 24 h at 25°C. In the greenhouse experiment, adding 16,000 chlamydospores of the fungus per gram of soil as either colonised barley grains or spores reduced the final number of females on roots of sugar beet by 50 and 66%, respectively, after 3 months. The reproduction factor was reduced to ×2 in spore-treated soil compared with ×5 in the untreated control, and 18% of the eggs in spore-treated soil were colonised by fungal mycelium. Generally, P. chlamydosporia var. chlamydosporia was more efficient at reducing the nematode population when applied as spores without any substrate than when used as colonised barley grains.     

A signaling pathway, independent of the oxidative burst, that leads to hypersensitive cell death in cultured tobacco cells includes a serine protease

To examine the role of reactive oxygen species (ROS) in the signal transduction that leads to hypersensitive cell death, we used a previously established system in which a xylanase from Trichoderma viride (TvX) induces an oxidative burst and cell death in a culture of tobacco cells. Diphenylene iodonium and N-Acetyl-L-cysteine known as an inhibitor of NADPH oxidase and a scavenger of superoxides, respectively, and catalase inhibited the oxidative burst but did not inhibit the induction of cell death. We also found that inhibitors of serine proteases inhibited TvX-induced cell death. These results suggest that there is a signaling pathway in which a serine protease might be responsible for the signal transduction, which is independent of the oxidative burst, that leads to the hypersensitive cell death of tobacco cells.     

Evaluation of Bacillus mycoides isolate BmJ and B. mojavensis isolate 203-7 for the control of anthracnose of cucurbits caused by Glomerella cingulata var. orbiculare

Bacillus mycoides isolate BmJ (BmJ) and Bacillus mojavensis isolate 203-7 (203-7) were tested in the greenhouse for their ability to control Glomerella cingulata var. orbiculare the causal agent of anthracnose of cucumber by induced systemic acquired resistance (SAR). BmJ and 203-7 delayed disease onset and reduced total (43% and 56%) and live spore production (38% and 49%) per mm² of lesion area when used to induce SAR in cucumber. 203-7 also reduced lesion diameter. Induction by G. cingulata conidia resulted in delayed disease onset, reduction of number of lesions per leaf and lesion diameter. Assays of cucumber apoplastic proteins extracted 6 days after induction showed that BmJ increased β-glucanase activity by 135%, and 203-7 increased β-glucanase activity by 72% and peroxidase activity by 79% when compared to the water control. Acibenzolar-S-methyl induced the highest (P = 0.05) levels of chitinase (950%) and peroxidase (420%) activity compared to water controls. Field experiments (2004 and 2005) evaluated applications of BmJ and fungicides for the control of anthracnose in cucumber (var. ‘General Lee’) and cantaloupe (var. ‘Athena’). BmJ was compared to full and half labeled rate alternate applications of azoxystrobin and chlorothalonil, and BmJ with half rate of azoxystrobin and chlorothalonil. BmJ applied seven days before inoculation reduced disease severity by 41% in cucumber in 2004 and by 24–21% in cantaloupe for both years compared to water controls which was statistically equal to the fungicide treatments. The full and half rate fungicide program provided 97–37% disease reduction compared to water controls. BmJ applied one week before inoculation significantly reduced AUDPC (P = 0.05) in cucumber compared to the water control in 2004 on cantaloupe for both years while the full and half rate fungicide program were equivalent and provided the lowest AUDPC. No yield reduction was noted as a result of the disease or treatment for either cantaloupe or cucumber.     

Spatial distribution and antifungal interactions of a Bacillus biological control agent on wheat surfaces

The proximity of a biological control agent and its associated anti-microbial metabolites to pathogens on plant surfaces can determine the outcome of disease control. In this study we investigated whether deficiencies in inoculum deposition and localization could explain the inability of the biological control agent Bacillus amyloliquefaciens strain TrigoCor to consistently control Fusarium head blight in the field, despite producing effective and consistent disease control in greenhouse settings. Using epifluorescent stereomicroscopy and confocal laser scanning microscopy, we visualized the coverage of wheat spike surfaces by Bacillus post-application in greenhouse and field environments, and determined that there are large unprotected areas on wheat spikes sprayed with commercial-scale field equipment, as compared to typical greenhouse applications. Additionally, we found that in conditions of low relative humidity, antifungal compounds produced by Bacillus were not able to diffuse across wheat surfaces in biologically relevant amounts, further suggesting that the inadequate coverage of wheat surfaces by Bacillus could be directly limiting disease control. Bacillus cells were easily rinsed off wheat surfaces within 8 h of application, indicating that rainfastness might be an additional limitation of biological control in field settings. Finally, we observed the inhibition of Fusarium graminearum spore germination by TrigoCor inoculum on wheat surfaces, confirming this as a mode of action for TrigoCor biocontrol. Future optimization efforts for biological control agents applied to above-ground plant parts should focus on enhancing the rainfastness, quantity, and spatial coverage of the inoculum on plant surfaces.     

Rearrangements in sugar beet mitochondrial DNA induced by cell suspension,callus cultures and regeneration

Structural alterations in mitochondrial DNAs (mtDNAs) from a plant of a sterile sugar beet line, callus derived from it, suspension-cultured cells and plants regenerated from the callus were studied. BamHI restriction analysis revealed that structural alterations between the mtDNAs of the callus and the control plant had occurred. Multiple rearrangements were also demonstrated in the mtDNA from the suspension culture, of which some were similar to those appearing in the callus, and others had arisen de novo. Rearrangements were also identified by means of blot hybridization of BamHI-digested mtDNA from suspension-cultured cells with the genes encoding subunit II of cytochrome oxidase (cox II) and subunit 1 of NADH-dehydrogenase (Nd1). No alterations were observed in the mitochondrial genome of the callus and regenerants. The location of the genes for the -subunit of F1-ATPase (atpA) and apocytochrome b (cob) in the mtDNA remained unchanged.Our salient finding was of a plant with an altered mitochondrial genome as judged by EcoRI and BamHI restriction analysis. This exceptional plant had retained the sterile phenotype like all of the other regenerants and the parent. The set of plasmid-like molecules of mtDNA remained the same as that in the control plant and in all of the regenerants, callus and suspension-cultured cells. The only type of plasmid-like molecule found in all of the DNAs was the 1.6-kbp minicircle, which is a feature of sterile cytoplasms. These structural changes in mtDNA were obviously a consequence of somaclonal variation during the in vitro cultivation of the sugar beet cells.     

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN(-/-)) mice were treated in vitro with H(2)O(2) to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN(-/-) cells but was increased to only 20% in WT cells. In contrast, after 1-8 h of treatment with H(2)O(2), the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN(-/-) cells. Electron microscopy of WT cells treated with H(2)O(2) showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H(2)O(2)-treated OPN(-/-) cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN(-/-) and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN(-/-) cells by approximately 30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN(-/-) cells was not altered. Restoration of OPN expression in OPN(-/-) fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H(2)O(2) treatment. Thus H(2)O(2)-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.     

Processing, disulfide pattern, and biological activity of a sugar beet defensin, AX2, expressed in Pichia pastoris.

AX2 is a 46-amino-acid cysteine-rich peptide isolated from sugar beet leaves infected with the fungus Cercospora beticola (Sacc.). AX2 strongly inhibits the growth of C. beticola and other filamentous fungi, but has little or no effect against bacteria. AX2 is produced in very low amounts in sugar beet leaves, and to study the protein in greater detail with respect to biological function and protein structural analysis, the methylotrophic yeast Pichia pastoris was used for large-scale production. The amino acid sequence, processing of the signal peptide, disulfide bridges, and biological activity of the recombinant protein were determined and compared with that of the authentic AX2. In P. pastoris, the protein was expressed with an additional N-terminal arginine. The disulfide bonding was found to be identical to that of the authentic AX2. However, when tested in in vitro bioassay, the biological activity of the recombinant protein was slightly lower than that measured for the authentic protein. Furthermore, the recombinant protein was significantly more sensitive to Ca(2+) than the authentic protein. This is most probably due to the extra arginine, since no other differences between the two proteins have been found.     

A cross-polarization, magic-angle-spinning, 13C-nuclear-magnetic-resonance study of polysaccharides in sugar beet cell walls

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T_1ρ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.     

Localization of new peptidoglycan at poles in Bacillus mycoides, a member of the Bacillus cereus group

Bacillus mycoides is a sporogenic Gram-positive soil bacillus of the B. cereus group. This bacillus, which forms hyphal colonies, is composed of cells connected in filaments that make up bundles and turn clock- or counterclockwise depending on the strain. A thick peptidoglycan wall gives the rod cells of these bacilli strength and shape. One approach used to study peptidoglycan neoformation in Gram positives exploits the binding properties of antibiotics such as vancomycin and ramoplanin to nascent peptidoglycan, whose localization in the cell is monitored by means of a fluorescent tag. When we treated B. mycoides strains with BODIPY-vancomycin, we found the expected accumulation of fluorescence at the midcell septa and localization along the cell sidewall in small foci distributed quite uniformly. Intense fluorescence was also observed at the poles of many cells, more clearly visible at the outer edges of the cell chains. The unusual abundance of peptidoglycan intermediates at the cell poles after cell separation suggests that the construction process of this structure is different from that of B. subtilis, in which the free poles are rarely reactive to vancomycin.     

Control of Pythium damping-off of sugar beet by seed treatment with crop straw powders and a biocontrol agent

Seed treatment with non-sterilized powdered straws from 39 crops was tested for the control of Pythium damping-off of sugar beet. Four straws, including flax, coriander, pea, and lentil were effective in controlling the disease in soil artificially infested with Pythium sp. “group G.” Sterilizing flax and pea straws eliminated the efficacy of these straws. Wheat straw powder coated on sugar beet seeds increased the incidence of Pythium damping-off but this effect was reversed by the co-inoculation of wheat straws with the biocontrol agent Pseudomonas fluorescens 708. Coating sugar beet seeds with P. fluorescens 708 and flax or pea straws also increased the efficiency of the bacterial strain for the control of Pythium damping-off. Pea straws and to a lesser extent lentil straws produced volatile substances that affected mycelial growth of Pythium sp. “group G” on potato dextrose agar in Petri plates when the straws were mixed with water and left to ferment for two days. Fermentation of pea straws led to the accumulation of volatile ammonia, which was produced by the reduction of the large amount of nitrate stored in the straw. Reduction of nitrate and therefore the release of volatile ammonia did not occur in sterilized pea straws. However, fermenting sterile pea straws with bacteria from different genera restored nitrate reduction and the release of volatile ammonia, suggesting that microorganisms associated with pea straws are responsible for the conversion of nitrate into volatile ammonia which in turn control Pythium damping-off disease in sugar beet.     

Properties of Bac W42, a bacteriocin produced by Bacillus subtilis W42 isolated from Cheonggukjang

Ten Bacillus strains with antimicrobial activities were isolated from Cheonggukjang produced at different parts in Korea. They all inhibited Listeria monocytogenes ATCC 19111 and nine inhibited Bacillus cereus ATCC 14579. Four isolates (W42, H27, SKE 12, and K21) showing strong inhibiting activities were identified as B. subtilis. B. subtilis W42 was the most inhibiting strain. The antimicrobial activity of culture supernatant from B. subtilis W42 was destroyed completely by proteinase K treatment, indicating that a bacteriocin was the responsible agent. The bacteriocin, Bac W42, was most stable at pH 7 and stable between pH 3-6 and 8-9. Bac W42 was stable up to 80°C. BHI (brain heart infusion) and TSB (tryptic soy broth) were the best media for the activity (320 AU/ml) followed by LB (160 AU/ml). Bac W42 was partially purified by column chromatographies. The specific activity was increased from 1,151.2 AU/ml to 9,043.5 AU/ml and the final yield was 26.3%. Bac W42 was 5.4 kDa in size as determined by SDS-PAGE. Bac W42 showed bactericidal activity against L. monocytogenes ATCC 19111.     

Resource regulation of an invasive tree by a classical biological control agent

The invasive tree Melaleuca quinquenervia experienced substantial declines in growth and reproduction in response to chronic herbivory by the defoliating weevil Oxyops vitiosa. Plants subjected to unrestricted defoliation replaced leaves that were more suitable for feeding by the next generation, a process envisioned by the Resource Regulation Hypothesis which posits that attack by one generation increases the amount of the preferred host resources for the next, resulting in a positive feedback loop for the herbivore. The production of juvenile replacement leaves stimulated additional bouts of oviposition and feeding by O. vitiosa, which ultimately produced positive effects for the herbivore with negative consequences for the plant. The addition of water resources to the plant prolonged the positive feedback loop such that more than twice as many insects were produced on irrigated versus non-irrigated trees. In a more simple, reassembled food web on M. quinquenervia, the lack of biotic constraints like parasitoids may have prevented the earlier termination of the feedback loop and thus increased the impact of the biological control agent on the target. The overall effectiveness of this classical biological control program can be attributed, in part, to the phenomenon of the target plant’s induced susceptible response to a herbivore.