KB Medium Research Articles

KARAKTERISASI DAN UJI AKTIVITAS BAKTERI RIZOSFER LAHAN ULTISOL SEBAGAI PEMACU PERTUMBUHAN TANAMAN DAN AGENSIA HAYATI CENDAWAN PATOGEN TULAR TANAH SECARA IN VITRO

Characterization and activities assay of rhizosphere bacteria from ultisol land for plant-growth promoting and biocontrol agents of soil-borne fungus pathogens under in vitro test. Although many studies have been conducted to identify the specific traits of the plant growth-promoting and bioprotecting rhizobacteria (PGPBR), they were limited to studying specific PGPBR isolates from ultisol lands. We selected 273 isolates from bulk soil and plant rhizosphere and examined them for a wide array traits that might inhibit the growth of plant pathogens and increase early cucumber growth in ultisol soil. A subsample of 25 isolates, all positively produce chitinase and sellulose enzymes, 18 positively produce protease and 7 were fluorescens on KB medium under UV lighting. All isolates could produce IAA and be able to solubilize phosphor in vitro test, 10 exhibited low level of nitrogenase activity. Futher test showed that out of 25 isolates, 12 inhibited F. oxysporum, P. capsici, R. solani and S. rolfsii in vitro. All isolates increased seed germination, but only 5 isolates significanty increased early cucumber growth in ultisol soil. The results suggest that rhizobacteria be able to produce extracelluler enzymes, siderophore, ACC deasiminase, and IAA or those which are able to solubilize phosphor in vitro may be potential to be uses as biofertilizer and biological control agents in ultisol land.

SCREENING SYSTEMIC RESISTANCE-INDUCING FLUORESCENT PSEUDOMONADS FOR CONTROL OF BACTERIAL BLIGHT OF COTTON CAUSED BY XANTHOMONAS CAMPESTRIS pv. MALVACEARUM

Iron-regulated metabolites, such as siderophores are important determinants of fluorescent pseudomonads in induction of systemic resistance (ISR) of plants. In this work, King’s medium B supplemented with 120 ppm 8-hydroxyquinoline (8-HQ) chelator was used for screening of pseudomonads from the rhizosphere of cotton with a high capacity to produce siderophores. Use of this medium resulted in isolates with a high ISR capability equal or greater than the reference strain P. aeruginosa 7NSK2. All strains except 13Q and EQ significantly decreased the percentage of infected area on leaves. Siderophore production by both the strains 35Q and 16Q, that were the fastest growing strains on KB media supplemented with 8HQ, was significantly higher than that of the reference strain 7NSK2. In hydroponic cultivation assays, ISR ability of 35Q depended on one or a complex of several iron regulated metabolite/s, as its ISR was significantly reduced in pots with iron excess. Nonetheless, ISR by 35Q was not completely abolished under iron excess suggesting a possible role for other determinants.

Assessment of strains of Pseudomonas syringae pv. tomato from Tanzania for resistance to copper and streptomycin

Fifty-six strains of Pseudomonas syringae pv. tomato (P.s. pv. tomato) were collected from tomato-producing areas in Tanzania and assessed for resistance to copper and antibiotics. The collection was done from three tomato-producing regions (Morogoro, Arusha and Iringa), representing three different ecological conditions in the country. After isolation and identification, the P. s. pv. tomato strains were grown on King's medium B (KB) amended with 20% copper sulphate (w/v). The strains were also assessed for resistance to antibiotics. Results indicated that there was widespread resistance of the P. s. pv. tomato strains to copper sulphate. The highest level of resistance was recorded from the Arusha region (Northern Tanzania), 83.3% of the P. s. pv. tomato strains from that region showed resistance to copper sulphate. This was followed by Iringa region (Southern Tanzania), from where strains of the pathogen were moderately resistant to copper sulphate, such that 54.0% of them were able to grow on the KB medium amended with 20% (w/v) of the copper compound. Out of seven strains of P. s. pv. tomato from Morogoro region (Central Tanzania) included in the study, five (71.5%) were resistant to copper sulphate. The only strain of P. s. pv. tomato from the Dodoma region (Central Tanzania, but with a different ecological condition from the Morogoro region) included in the study was unable to grow on the medium containing 20% copper sulphate. None of the P. s. pv. tomato strains in the four regions included in the study were resistant to streptomycin sulphate. These results suggest that in the Arusha and Iringa regions of Tanzania, there might be possibilities of excessive use of copper compounds in tomato production, such that strains of P. s. pv. tomato strains in the areas have become resistant to the compounds.

A novel method for isolation of Campylobacter spp. from environmental samples, involving sample processing, and blood- and antibiotic-free medium.

To develop a method that involves sample processing, and blood- and antibiotic-free medium for isolation and enumeration of Campylobacter spp. from environmental samples. The sample processing (preT) was standardized to minimize the population of competing bacteria. A blood- and antibiotic-free differential, Kapadnis-Baseri medium (KB medium) was formulated and tested for isolation of Campylobacter spp. in comparison with CAT medium. PreT-KB method was evaluated in comparison with the conventional viable count method and with the conventional most probable number (C. MPN) method for enumeration of Campylobcater from environmental samples. The results indicated that sample processing significantly reduced population of competing bacteria. The KB medium selected Gram-negative bacteria and differentiated Campylobacter from lactose-fermenting competing bacteria. The population of Campylobacter detected by preT-KB method was similar to that by conventional viable count method. While, the population of Campylobacter spp. determined by preT-KB method was higher than that by C. MPN method. In addition, the preT-KB method detected antibiotic sensitive campylobacters. The preT minimizes population of competing bacteria and the KB medium selects Gram-negative bacteria and differentiates Campylobacter from them. Therefore, Campylobacter can be isolated from environmental samples without using antibiotics. The preT-KB method is simple and facilitates isolation of antibiotic sensitive and enumeration of Campylobacter in the environmental samples. Therefore, the new method will be useful for isolation and enumeration of Campylobacter from water, food and sewage samples. Besides, it would also detect antibiotic-sensitive campylobacters, which are not detected by conventional viable count and MPN methods.

Disease suppression and crop improvement through fluorescent pseudomonads isolated from cultivated soils

Three fluorescent pseudomonads isolated from rhizosphere/rhizoplane of crop plants showed in vitro antibiosis against seven fungal and two bacterial plant pathogens on iron-deficient KB medium. Seed bacterization of chick- pea (Cicer arientinum L.), egg plant (Solanum melongena L.), soybean (Glycine max Merr.) and tomato (Lycopersicon esculentum Mill.) with these organisms showed an increased seed germination, shoot height, root length, fresh weight, dry weight and yield. Seed bacterization with one of these strains, RB 8, reduced the number of chick-pea wilted plants in wilt-sick (Fusarium oxysporum f.sp. ciceris) soil. Addition of iron into the soil eliminated the disease suppression. The disease suppression and/or growth enhancement along with the positive root colonization by these organisms indicate their possible use as plant growth-promoting rhizobacteria (PGPR)/biocontrol agents against chick-pea wilt.

Growth patterns of bacterial communities in the rhizoplane and rhizosphere of white clover ( Trifolium repens L.) and perennial ryegrass ( Lolium perenne L.) in long-term pasture

Rhizoplane bacteria in white clover roots (0–5 cm), perennial ryegrass roots (0–5 and 5–10 cm) and surrounding rhizosphere soil from a long-term pasture were studied for the rate of colony development over a 10 day period. Serial dilutions were inoculated onto 0.1 strength tryptose soy agar (TSA) and 0.1 strength modified King's medium B (KB). Bacterial counts were made eight times over 10 days. Bacterial colony development ( CD) and eco-physiological ( EP) indices were calculated from these data. There were no significant differences in CD and EP indices of fluorescent pseudomonads growing on KB medium. After 5 days, the number of colonies developing on KB as a proportion of total colonies appearing over 10 days was lower for ryegrass roots than for soil ( P < 0.05). The community structure derived from the colonies growing on 0.1 strength TSA showed that the rhizoplanes of white clover and ryegrass had a greater proportion of K-strategists and a smaller proportion of r-strategists than did the rhizosphere soil ( P < 0.001). CD values for roots were significantly lower than those for soil ( P < 0.001), while EP values were not significantly different. Randomly selected bacterial isolates were identified using the Microlog ™ system. The fastest growing (24 h) strains belonged to the genera Pseudomonas and Bacillus spp. Other genera identified included Acinetobacter, Sphingomonas, Streptococcus, Cellulomonas, Comamonas, Xanthomonas and Erwinia. The slowest growing bacteria belonged to the genera Cellulomonas, Rhizobium, Xanthomonas and Erwinia.

Differentiation of the Soft‐Rotting Erwinias (the Carotovora Group) by Fatty Acid Composition

AbstractAbout 90 % of total cellular fatty acids in E. carotovora, grown on KB medium for 1 day at 28°C, were the saturated, even‐carbon straight chains 12 : 0 (5.9%), 14 : 0 (1.7%) and 16 : 0 (30.1 %), and the unsaturated 16 : 1 (36.6%) and 18 : 1 (15.0%) fatty acids. Other components were the hydroxy‐substituted 3‐OH 14 : 0 (5.3%) and 21 minor fatty acids each occurring less than 0.1 % of the total — 14 of them reported herein for the first time in Erwinia. The ratio of 16 : 1/18 : 1 in KB‐grown cells was as useful in differentiating subspecies of E. carotovora as previously reported by other workers for TSA‐grown cells. A comparison of fatty acid profiles of E. carotovora on 4 different media, KB, TSA, NA and PDA, indicated that on KB there was the greatest proportion of Class A and C fatty acids, and the highest number of detectable components. Significant differences were noted in the 5 major fatty acids and in cyclic fatty acids among the 4 species of the carotovora group –E. carotovora, E. chrysanthemi, E. rhapontici and E. cypripedii. These differences could be expressed as algorithms that, when used in sequential dichotomous steps, could differentiate the 4 species.

Enumeration of oligotrophic rhizosphere pseudomonads with diluted and selective media formulations

Diluted and selective media formulations were evaluated for the recovery of oligotrophic pseudomonads from the rhizosphere of the cucumber plant (Cucumis sativus L.). Full-strength King's B medium (KB) and 250-fold diluted medium (KB/250) were used as the basal formulations. The selective media formulations were: (1) Novobiocin, penicillin, and cycloheximide; (2) crystal violet and nitrofurantoin; and (3) Irgasan. On average, more rhizobacteria were recovered on the KB/250 than on the KB medium. For the recovery of pseudomonads, media made selective with either the novobiocin-penicillin-cycloheximide or the crystal violet and nitrofurantoin formulations were superior to the Irgasan formulation. Attempts to identify isolates using phospholipid fatty acid profiling met with limited success. The test isolates yielded mostly low similarity indices or “no matches” with the Microbial Identification System (aerobic library version) used for this study. All oligotrophic isolates were facultative and no evidence was obtained for the presence of obligate oligotrophs in these plating experiments.

Low resting potentials in single isolated heart cells due to membrane damage by the recording microelectrode

Single myocytes from adult rat hearts were prepared following the method of Powell and co-workers (9, 10, 11). Low resting potentials (Em) could be improved by three techniques. (i) Elevation of Cao to 7.2 mM which, however, mostly resulted in spontaneity and irreversible contracture. (ii) Pre-incubation in a "KB medium" (6). (iii) Use of suction pipettes instead of tapered microelectrodes for intracellular recordings (2). It is concluded that low Em measured previously (11) were due to membrane damage upon microelectrode impalement accompanied by insufficient healing of the membrane around the electrode insertion.

Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium".

1. Ventricular myocytes were isolated by means of collagenase and hyaluronidase. Hearts of rats or guinea pigs were perfused in a retrograde manner; bovine ventricular tissue was incubated in from of tissue chunks. 2. In “low Ca medium” (aCa=1 μM), the myocytes beat spontaneously. Resting potentials of −4 mV and input resistances of 1 MΩ, associated with a fast decay of aKi, indicated “hyperpermeability” of the cell membrane. Within 2 h, the cells aged: they granulated (swelling of mitochondria), contracted, rounded up and finally disaggregated. When the superfusate was changed to Tyrode solution (3.6 mM CaCl2), 90–98% of those cells responded with the “Ca paradox”. Initially, aCai increased from 0.2 μM to >10 μM, and the sarcomere length (SL) shortened to <1.5 μm. Within the following 3 min aCai renormalized but the contracture was sustained. After another 5 min, the SL shortened to <1.2 μm, but a delay of 1–2 min passed before aCai started to increase towards aCa0. The 2–10% Ca tolerant cells beat spontaneously. Their resting potentials were between −10 and −70 mV, their input resistances between 4 and 20 MΩ. Repolarization by anodal current flow could restore the sodium spike but not the plateau of the action potential. 3. Freshly prepared myocytes were pre-incubated in a KB medium composed of 85 mM KCl, 30 mM K2HPO4, 5 mM MgSO4, 5 mM Na2ATP, 5 mM pyruvate, 5 mM succinate, 5 mM β-OH-butyrate, 5 mM creatine, adjusted with KOH to pH 7.2 and with EGTA to pCa 7.5. The minimum time of preincubation was 1 h, the maximum 5 days when the cells were stored at 5°C. The conditioning with the KB medium improved both Ca tolerance and electrophysiology significantly. About 70% of the rod shaped myocytes were Ca tolerant. These cells had resting potentials between −75 and −90 mV, the membrane resistances were around 7 kΩ×cm2. The action potentials showed well pronounced plateaus and lasted for 125 ms (rat), 300 ms (guinea pig) or 400 ms (bovine). Their shape showed the typical species related pecularities. 4. We discuss that both ageing and Ca intolerance of the un-conditioned cells result from Ca overload through the hyperpermeable membrane, and that Ca overload causes ATP depletion via mitochondrial disfunction. The constituents of the KB medium may act to prevent these processes.