The class IId bacteriocin thuricin-17 increases plant growth.

The mechanisms by which many plant growth promoting rhizobacteria (PGPR) affect plants are unknown. We recently isolated a rhizosphere bacterium (Bacillus thuringiensis NEB17), that promotes soybean growth and screened the liquid growth medium in which it grew for plant growth stimulating materials. We have also shown that it produces a bacteriocin (named by us as thuricin-17 and a member of the recently described class IId bacteriocins). Here we show that application of this bacteriocin to leaves (spray) or roots (drench) directly stimulates the growth of both a C(3) dicot (soybean) and a C(4) monocot (corn). This growth stimulation is similar in nature to that previously seen when plants are treated with Nod factors. Strain NEB17 contains three copies of the gene for thuricin 17 that code for identical amino acid sequences. These two lines of evidence suggest that the dual functions of these proteins may have constrained their evolution. This is the first report of direct plant growth enhancement by a bacteriocin.

Chitinases produced by Paenibacillus illinoisensis and Bacillus thuringiensis subsp. pakistani degrade Nod factor from Bradyrhizobium japonicum.

Chitinases are enzymes that hydrolyze internal beta-1,4-N-acetyl-D-glucosamine linkages of chitin. Since the backbone of Nod factors is a chitin oligomer, we investigated whether chitinases produced by soil bacteria Paenibacillus illinoisensis KJA-424 and Bacillus thuringiensis subsp. Pakistani HD 395 are able to degrade Nod factor produced by Bradyrhizobium japonicum, a phenomenon that could disrupt B. japonicum-soybean signaling and nodule establishment when chitinases are present. Purified Nod factor [LCO Nod Bj-V (C(18:1), MeFuc)] was isolated from Bradyrhizobium japonicum and incubated with crude chitinases isolated from KJA-424 and HD395, with or without acetate buffer. After 15 h of incubation, Nod factor in the resulting solution was quantified by HPLC. Degradation was greatest following treatment with KJA-424 (91.9%) and HD395 (86.5%) chitinases in acetate buffer. Treatments that included acetate buffer had higher levels of degradation than those without. For all treatments degradation was greater than 77%.

Stability and antibacterial activity of bacteriocins produced by Bacillus thuringiensis and Bacillus thuringiensis ssp. kurstaki.

Bacteriocins are antimicrobial peptides that are produced by bacteria and toxic to bacterial strains closely related to the producer strain. It has previously been reported that Bacillus thuringiensis strain NEB17 and Bacillus thuringiensis subsp. kurstaki BUPM4 produce the bacteriocins thuricin 17 (3,162 Da) and bacthuricin F4 (3,160.05 Da), respectively. Here, we demonstrate that these bacteriocins have functional similarities and show a similar spectrum of antimicrobial activities against indicator strains. We also studied the effects of sterilization methods on the recovery and biological activities of these bacteriocins. They were completely degraded by autoclaving and the two were similarly affected by the tested filter membranes. Polyvinylidene fluoride (PVDF), polyestersulfone (PES), and cellulose acetate (CA) are suitable for filter sterilization of these bacteriocins. The two bacteriocins were stable across a range of storage conditions. These data will facilitate their utilization in food preservation or agricultural applications.

Nod Bj-V (C18:1, MeFuc) production by Bradyrhizobium japonicum (USDA110, 532C) at suboptimal growth temperatures.

Nod factors (Lipo-chitooligosaccharides, or LCOs) act as bacteria-to-plant signal molecules that modulate early events of the Bradyrhizobium-soybean symbiosis. It is known that low root zone temperature inhibits the early stages of this symbiosis; however, the effect of low soil temperature on bacteria-to-plant signaling is largely uninvestigated. We evaluated the effect of low growth temperatures on the production kinetics of Nod factor (LCO) by B. japonicum. Two strains of B. japonicum, 532C and USDA110, were tested for ability to synthesize Nod Bj-V (C(18:1), MeFuc) at three growth temperatures (15, 17 and 28 degrees C). The greatest amounts of the major Nod factor, Nod Bj-V (C(18:1), MeFuc), were produced at 28 degrees C for both strains. At 17 and 15 degrees C, the Nod factor production efficiency, per cell, of B. japonicum 532C and USDA110 was markedly decreased with the lowest Nod factor concentration per cell occurring at 15 degrees C. Strain 532C was more efficient at Nod factor production per cell than strain USDA 110 at all growth temperatures. The biological activity of the extracted Nod factor was unaffected by culture temperature. This study constitutes the first demonstration of reduced Nod factor production efficiency (per cell production) under reduced temperatures, suggesting another way that lower temperatures inhibit establishment of the soybean N(2) fixing symbiosis.

Nod factor induces soybean resistance to powdery mildew.

Plants possess highly sensitive perception systems by which microbial signal molecules are recognized. In the Bradyrhizobium-soybean (Glycine max (L.) Merr.) symbiosis, recognition is initiated through exchange of signal molecules, generally flavonoids from soybean and lipo-chitooligosaccharides (Nod factors) from the microsymbiont. Application of the Nod factor Nod Bj-V (C18:1, MeFuc) induced soybean resistance to powdery mildew caused by Microsphaera diffusa. Addition of Nod factor (concentrations ranging from 10(-6) to 10(-10) M) to soybean root systems led to reductions in disease incidence. The lowest disease incidence was caused by Nod factor treatment at 10(-6) M. The effect of Nod factor application on fungal growth and development was measured at 4, 12, 48, and 96 h after inoculation. Colony diameter and number of germ tubes per conidium were decreased by 10(-6) M Nod factor. Phenylalanine ammonia lyase (PAL, EC.4.3.1.1.) is the first enzyme of the phenyl propanoid pathway, and is commonly activated as part of plant responses to disease. Treatment of soybean seedlings with Nod factor, through stem wounds, induced PAL activity; the most rapid increase followed treatment with 10(-6) M Nod factor. These data show that soybean plants are able to detect root applied LCO and respond by increased disease resistance.

Induction of defense-related enzymes in soybean leaves by class IId bacteriocins (thuricin 17 and bacthuricin F4) purified from Bacillus strains.

We have recently discovered a new class of bacteriocin (class IId) which stimulates plant growth in a way similar to Nod factors. Nod factors have been shown to provoke aspects of plant disease resistance. We investigated the effects of bacteriocins [thuricin 17 (T17) and bacthuricin F4 (BF4)] on the activities of phenylalanine ammonia lyase (PAL), guaiacol peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and polyphenol oxidase (PPO). Bacteriocin solutions were fed into the cut stems of soybean (Glycine max L. Merr. cv. OAC Bayfield) seedlings at the first trifoliate stage. PAL activity in T17 treated leaves was the highest at 72h after treatment and was 75.5% greater than the control at that time. At 72h after treatment POD activities in T17 and BF4 treated leaves increased by 72.7 and 91.3%, respectively, as compared with the control treatment. APX activity was 52.3 and 49.6% respectively, greater than the control in T17 and BF4 treated leaves at 72h after treatment. SOD activity in T17 treated leaves was the highest at 72h after treatment and was 26.0% greater than the control at that time. SOD activity was 70.5 and 60.2% greater, respectively, than the control in T17 and BF4 treated leaves, at 72h. Using PAGE we found that one APX isozyme (28kDa isoform) showed the strongest induction in all bacteriocin treated leaves at 72h. Activity of the seven SOD isozymes was increased by both bacteriocins, relative to the control treatment. The 33kDa PPO isozyme was induced strongly by both bacteriocins, relative to the control treatment. These results indicate that class IId bacteriocins can act as an inducer of plant disease defense-related enzymes and may be acting through mechanisms similar to Nod factors.

Effects of Pseudomonas aureofaciens 63-28 on defense responses in soybean plants infected by Rhizoctonia solani.

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.