Inocula For Use Research Articles

Construction of Geminivirus Infectious Clones for Agroinoculation into Plants.

The production of geminiviral infectious clones providesa standardized inoculum for use in several host-virus studies. Geminiviruses present either one (monopartite) or two (bipartite) circular single-stranded DNA components, which commonly range from 2.6 to 2.8kb. Cloning of a monomeric genome is useful for obtaining its precise sequence. For infectious clones, however, it is essential that more than one copy of the genome, more specificallyof the origin of replication, is present in order to guarantee the production of full-length progeny DNA. Here, the complete process of preparing infectious geminiviral clones is described starting from the DNA extraction and selection of restriction endonucleases followed by two protocols for constructing dimeric clones: restriction endonuclease digestion and ligation (1) and Gibson Assembly (2).

Plant growth promoting activities of Pseudomonas sp. and Enterobacter sp. isolated from the rhizosphere of Vachellia gummifera in Morocco.

The Moroccan endemic Vachellia gummifera grows wild under extreme desert conditions. This plant could be used as an alternative fodder for goats, and camels, in order to protect the Argan forests against overgrazing in Central and Southwestern Moroccan semiarid areas. With the aim to improve the V. gummifera population's density in semiarid areas, we proposed its inoculation with performing plant growth-promoting bacteria. Hence, 500 bacteria were isolated from the plant rhizosphere. From these, 291 isolates were retained for plant growth-promoting (PGP) activities assessment. A total of 44 isolates showed the best phosphates solubilization potential, as well as siderophore and auxin production. The combination of REP-PCR (repetitive extragenic palindromic-polymerase chain reaction) fingerprinting, PGP activities, and phenotypic properties, allowed the selection of three strains for the inoculation experiments. The three selected strains' 16S rRNA sequencing showed that they are members of the Enterobacter and Pseudomonas genera. The inoculation with three strains had diverse effects on V. gummifera growth parameters. All single and combined inoculations improved the plant shoot weight by more than 200%, and the root length by up to 139%, while some combinations further improved protein and chlorophyll content, thereby improving the plant's forage value. The three selected strains constitute an effective inoculum for use in the arid and semiarid zones of southern Morocco.

73 Impact of storage conditions on the viability of microbial inoculum for estimating in vitro digestibility in the horse

This study evaluated the effect of storage conditions of equine fecal material on the viability of derived microbial inoculum used for in vitro equine digestibility trials. Fecal material collected via rectal palpation from 3 mature quarter horse geldings was stored at 4 storage temperatures: 39°C for 15 min (control), 22°C for 6h, 3°C for 6h, and −18°C for 24h. Utilizing pooled microbial inoculum from stored fecal material, the in vitro digestion of 6 forage samples (NDF range 41 to 62%) consisting of 3 alfalfa and 3 mixed grass sample were digested in the Ankom Daisy II Incubator to evaluate interactions between storage condition of microbial inoculum and chemical composition of digested forage samples. After determination of dry matter digestibility (DMD), an Ankom2000 Fiber Analyzer was used to measure neutral detergent fiber digestibility (NDFD) and acid detergent fiber digestibility (ADFD). A mixed linear model was used to analyze differences in DMD, NDFD, and ADFD, with forage and treatment as class statements with date included as a random effect. No significant interaction of forage x treatment was observed ( P = 0.81 for DMD). Further analysis with a mixed linear model was used to evaluate the effect of forage quality parameters crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) as covariates for DMD, NDFD, and ADFD results. Significant differences were observed for DMD ( P < 0.0001 ), NDFD ( P < 0.01 ), and ADFD ( P < 0.001 ) between 39°C and 3°C, 39°C and −18°C, 22°C and 3°C, and 22°C and −18°C. No differences ( P > 0.05 ) were observed in DMD, NDFD, or ADFD between 39°C and 22°C or 3°C and −18°C. Covariance analysis found no difference ( P > 0.05 ) in slope between any of the 4 storage condition levels when CP, NDF, and ADF were modeled as covariates for DMD, NDFD, and ADFD. Results support that fecal material stored for up to 6 h in 22°C temperature conditions provide a viable alternative to fresh fecal material for the formation of microbial inoculum. When used to harvest inoculum for use in the Daisy II incubator fecal material that had been previously chilled or frozen did not result in DMD, NDFD or ADFD values of forage samples that were as high as when inoculum from the other 2 storage treatments were used. Further research is needed to determine the reason for the decline in DMD, NDFD, and ADFD when utilizing inoculum from chilled and frozen fecal samples.

Impact of carrier agents and temperature during storage of dry inocula of Salmonella enterica: A contribution to the validation of low water activity foods processing interventions

In this study, dry inocula were developed using four different carriers [talc, fine sand (0.05–0.2 mm), regular sand (0.2–0.6 mm), and calcium carbonate] for validation of inactivation steps of low moisture foods against Salmonella enterica. Besides, the changes in aw and the survival of S. enterica in the dry inocula made with the four carriers were evaluated throughout the storage period of up to 150–180 days at 4 and 25 °C. Among the carriers evaluated fine and regular sand seem to constitute the best carrier agents for longer preservation of S. enterica cells to be employed in dry inocula. Besides, the storage of inoculated carrier agents under refrigeration (4 °C) enhanced the stability of the dry inocula prepared, maintaining S. enterica counts unaltered for ~ six months (p < 0.05). Overall, based on the results, it has been found that the aw variation during storage did not directly influence S. enterica survival in the dry inocula. However, considering only the aw of the dry inocula obtained using the different carrier agents, it is possible to propose combinations between carrier agents and aw of low moisture foods (LMF). The findings of this study will be highly relevant for the development of stable dry inocula for use in the validation of the inactivation steps of LMF.

Bacteria immobilized electrospun polycaprolactone and polylactic acid fibrous webs for remediation of textile dyes in water

In this study, preparation and application of novel biocomposite materials for textile dye removal which are produced by immobilization of specific bacteria onto electrospun nanofibrous webs are presented. A textile dye remediating bacterial isolate, Clavibacter michiganensis, was selected for bacterial immobilization, a commercial reactive textile dye, Setazol Blue BRF-X, was selected as the target contaminant, and electrospun polycaprolactone (PCL) and polylactic acid (PLA) nanofibrous polymeric webs were selected for bacterial integration. Bacterial adhesion onto nanofibrous webs was monitored by scanning electron microscopy (SEM) imaging and optical density (OD) measurements were performed for the detached bacteria. After achieving sufficient amounts of immobilized bacteria on electrospun nanofibrous webs, equivalent web samples were utilized for testing the dye removal capabilities. Both bacteria/PCL and bacteria/PLA webs have shown efficient remediation of Setazol Blue BRF-X dye within 48 h at each tested concentration (50, 100 and 200 mg/L), and their removal performances were very similar to the free-bacteria cells. The bacteria immobilized webs were then tested for five times of reuse at an initial dye concentration of 100 mg/L, and found as potentially reusable with higher bacterial immobilization and faster dye removal capacities at the end of the test. Overall, these findings suggest that electrospun nanofibrous webs are available platforms for bacterial integration and the bacteria immobilized webs can be used as starting inocula for use in remediation of textile dyes in wastewater systems.

Comparison of intestinal contents from different regions of the equine gastrointestinal tract as inocula for use in an in vitro gas production technique

The aim of the experiment reported here was to compare equine faeces with inocula obtained from equine caecum, ventral colon and dorsal colon for use in the in vitro gas production technique. Freeze-dried grass (FDG), high-temperature dried grass (HDG) and unmolassed sugar beet pulp (BP) were incubated with either caecal fluid (C), dosal colonic fluid (D), ventral colonic fluid (V) or faeces (F) as the source of inoculum. Substrate/inocula combinations were fermented using an in vitro gas production (GP) technique. Mathematical analysis of cumulative gas production curves revealed an interaction (P<0.001) between inocula and substrate for all of the in vitro parameters measured. However, similar extents of gas production were seen for inocula obtained from caecal fluid and faeces across all substrates, with lower values obtained from bottles incubated with colonic fluid. Thus, results indicate that faeces are a suitable alternative to caecal fluid; however, it would appear that they are less effective in modelling fermentation in the colon. Consequently, further work is required to determine the use of faeces as an alternative to caecal and colonic fluid and to further investigate inocula/substrate interactions.

Innundative biocontrol of water hyacinth (Eichhornia crassipes (Mart.) Solms. Laubach) using zonate leaf spot (Acremonium zonatum Sawada Gams) fungal agent

Innundative approach of biocontrol involves culturing spore inoculum for use as a concentrated formulation able to withstand storage, handling and application in weed control the same way as a chemical herbicide. A trial was set up in Maseno University glass house to test the effect of concentrating and formulating A. zonatum spores in vegetable oil, mineral oil, pure commercial grade glycerol and tween 80 for use in water hyacinth biocontrol. A. zonatum was isolated from infected plants collected from Winam gulf (Lake Victoria) Kenya, cultured on PDA under aseptic conditions. The isolate was later multiplied on several PDA plates and spores harvested when mature, concentrated and formulated in 4 different oil emulsions. Fresh healthy water hyacinth plants were then collected from Winam gulf, injured and inoculated in a glass house in RCBD design, replicated 3 times. The results show that concentrating and formulating A. zonatum in vegetable oil and mineral oil may enhance its efficacy as a mycoherbicide, evidenced by the extensive leaf infection, though new leaves continued to form. A. zonatum concentrated and formulated can be used in the control of water hyacinth in the Winam gulf and L. Victoria in general. Suitability of an integrated approach with other insects should be investigated.

The effect of freezing on the fermentative activity of equine faecal inocula for use in an in vitro gas production technique

Two in vitro experiments were conducted to determine the effect of freezing on the fermentative activity of equine faecal inocula for use in an in vitro gas production technique. In Experiment 1, high-temperature dried alfalfa (HTA) and mature grass hay (GH) were incubated with an inoculum prepared either fresh (0 d) or following storage at −20°C for 7 days (7 d). In Experiment 2, HTA, GH, high-temperature dried grass (HTG), and unmolassed sugar beet pulp (SB) were incubated with an inoculum prepared either fresh (0 d) or following storage at −20°C for 24hours (24h), 48hours (48h) or 72hours (72h). Substrate/inocula combinations were fermented using an in vitro gas production (GP) technique. In both experiments, analysis of cumulative gas production values revealed an interaction (P<0.05) between inocula and substrate. Gas production from bottles containing HTA as the substrate was less affected by inocula source (fresh or frozen; 7 d, 24h, 48h and 72h) compared to all other substrates. In Experiment 1, total GP was reduced for GH inoculated with frozen faeces (7 d) compared to fresh faeces, and unaffected for HTA. In Experiment 2, total GP was also unaffected by inocula source in bottles containing HTA, whilst reduced in those containing HTG and SB and HTA, respectively when inoculated with frozen (24h) compared to fresh faeces. However, the rate of gas production was also affected by inocula source in both experiments across all substrates. In conclusion, preservation of equine faecal inocula by freezing for 7 d or 24, 48 or 72h has a marked effect on the extent and rate of substrate fermentation in vitro. Moreover, the extent of these effects appears to be dependent upon the nature of substrate used in the in vitro incubations. Consequently, further work is required on the use of frozen faeces as inocula for the in vitro gas production technique before this preservation treatment can be used routinely as an alternative to using fresh faeces.

Scarification and Environmental Factors that Enhance Carpogenic Germination of Sclerotia of Sclerotinia sclerotiorum.

Ascospores of Sclerotinia sclerotiorum are the primary source of inoculum for disease epidemics in many economically important crops. Mass production of ascospores under laboratory conditions is required to prepare inoculum for use in selection of genotypes with resistance against Sclerotinia diseases. A study was undertaken, first, to investigate the effect on carpogenic germination of scarifying sclerotia from two S. sclerotiorum isolates taken from canola (Brassica napus) and, second, to identify environmental factors that enhance carpogenic germination. Seven different environmental treatments were applied to scarified and unscarified sclerotia: (i) sterilized distilled water for 4 months at 15°C, (ii) aerated water for 4 months at 4°C, (iii) constant rinsing with tap water for 8 weeks at 4°C, (iv) cold-conditioning for 4 weeks at 4°C and subsequent transfer into moist unsterilized compost at 15°C, (v) incubation in sterilized river sand at 15°C, (vi) air drying for 2 weeks followed by subsequent transfer into sterilized moist river sand at 15°C, or (vii) placed into 0.5% water agar and incubated at 15°C. Carpogenic germination of scarified sclerotia was significantly greater (P < 0.05) than for unscarified sclerotia. There was significant interaction (P < 0.001) between scarification and the different environmental treatments in relation to the carpogenic germination. Carpogenic germination of scarified sclerotia was enhanced by incubation of sclerotia in compost or in sterilized river sand. Further, overall carpogenic germination of both scarified and unscarified sclerotia occurred to the greatest extent when sclerotia of either of the two isolates were subjected to constant rinsing with tap water. We believe this to be the first report of both the enhanced carpogenic germination by scarification in S. sclerotiorum and the environmental factors we report that enhance carpogenic germination of scarified sclerotia. The progression of carpogenic germination in all the environmental treatments was also monitored as a part of this study across the two consecutive years for the same two isolates. The majority of sclerotia of both isolates germinated between the months of June and September in both years, a period which coincides with the main part of the cropping season when Sclerotinia stem rot is normally observed in rainfed canola in Western Australia. These data suggested the existence of a seasonal rhythm-like pattern in relation to the carpogenic germination of this pathogen.

Performance Of Aerobic Bioreactors During Treatment Of Tunning Industry Wastewater

The performance of aerobic bioreactors loaded with novel biomass support (the KMTi?½ support particles) during the treatment of tanning industry wastewater is presented in this paper. The wastewater samples were collected from TANWAT Company in Njombe, which extracts tannins from barks of the wattle tree. The wastewater generated from TANWAT is very acidic with high loading of organic compounds, which are very complex and difficult to biodegrade using conventional methods. The paper introduces a new technology for treatment of heavily loaded industrial wastewater, like tannins, that is the Three-Phase Fluidized Bed Bioreactor (TPFBB) which utilizes the KMTi?½ support particles. Bench scale bioreactors (BSBs) were used to establish the suitable inoculum for use in the TPFBB. To establish the effect of the KMTi?½ support particles, the BSBs were operated with and without the biomass support particles (BSPs). The BSBs were also inoculated with different types of inoculum (waste stabilization ponds (WSP) and rumen fluid (RF) inoculums) to establish the suitable source of microorganisms. Control samples were also implemented accordingly. The results show that the WSP was a suitable inoculum for which high chemical oxygen demand (COD) removal efficiency and tannins concentration reduction efficiency (TCRE) were observed. Tests conducted in the TPFBB with initial COD of about 13,200 ppm shows that the reactor is capable of biodegrading the tannins (at 85% efficiency) after aeration times of up to 7 days in recirculation mode. The final COD values of 1,000 ppm were reached in most cases. For final discharge, however, such a final COD necessitates the use of sub-surface flow constructed wetland (SSFCW) coupled to the TPFBB for final smoothing of the effluent. The TPFBB is recommended for treatment of tannin wastewater to minimize chemical pollution in the receiving water bodies.

Development of a rapid method for determining the infectious dose (ID) 50 of Orientia tsutsugamushi in a scrub typhus mouse model for the evaluation of vaccine candidates

The infectious dose (ID) of an inoculum for which 50% of susceptible mice will become infected (ID 50) with Orientia tsutsugamushi is usually determined by rechallenging mice that have already been challenged with O. tsutsugamushi to determine the lethal dose (LD) 50 titer of the inoculum. Those mice not killed by the initial challenge and which survived a rechallenge with 1000 LD 50 were considered immune and to have been initially infected with O. tsutsugamushi. Mice that succumbed to the rechallenge were considered not to have been initially infected. We have developed a more rapid method of determining the ID 50 of inocula for use in our vaccine studies based upon the observation that mice surviving initial challenge and that go on to survive rechallenge produced detectable IgG to O. tsutsugamushi antigens by enzyme-linked immunosorbent assay (ELISA). Mice that did not survive rechallenge, and therefore did not receive an initial infectious inoculum did not produce detectable IgG to O. tsutsugamushi antigens. Both original LD 50 and ID 50 titers determinations require observation of mice for 21 days post-challenge. Our new ID 50 determination does not require mice or the additional 21-day observation period for rechallenge and therefore is more rapid and cost-effective than the previous standard method of determining ID 50 titer necessary for the evaluation of vaccine candidates.

The use of faecal inocula for estimating the in vitro digestibility of horse feeds

There is a dearth of information available on the effects of donor animal on the fermentative capacity of equine faecal inocula for use in in vitro digestibility determinations. Furthermore, there is little knowledge of the degradation characteristic of feedstuffs incubated with equine faecal inocula. As such this study aimed to elucidate the effect of donor animal on the fermentation of feedstuffs in vitro and to assess the in vitro degradation characteristics of three commonly fed components of horse diets incubated with equine faecal inocula.

Fresh or frozen rumen contents as sources of inocula to estimate in vitro degradation of ruminant feeds

Fresh rumen contents are the most common source of inoculum for use to estimate in vitro degradation of ruminant feeds. However, the need to routinely access fistulated or slaughtered cattle to obtain rumen contents limits the availability of such inoculum and hence the applicability of in vitro methods by the commercial laboratories. Therefore, it would be advantageous, if rumen contents are preserved in sufficient quantity and used as a source for inoculum for use when there is a need to do so to estimate degradability of ruminat feeds. This study compared the suitability of frozen rumen contents as a source of inoculum to estimate in vitro rumen degradation of rapeseed meal (rapeseed) and grass nuts at various times.

In sacco dry matter and nitrogen degradation and their relationship with in vitro dry matter digestibility of Napier grass (Pennisetum purpureum Schumach.) as influenced by height of plant at cutting

In vitro dry matter digestibility (IVDMD) as well as in sacco rumen dry matter degradability (DMD) and nitrogen degradability (ND) studies of Napier grass (Pennisetum purpureum) were carried out using a 3 3 factorial experiment arranged in a randomised complete block design with 3 replications. The treatments were 3 levels of nitrogen (N) fertiliser (0, 46, bacterium Synergistes jonesii is known to degrade these compounds and inoculation of the bacterium into the rumen protects cattle against mimosine and DHP toxicity. We report on the development, production, and storage of an in vitro, fermentor-based, mixed bacterial inoculum, which includes S. jonesii, for administration to cattle. This technique may be applicable to the production and delivery of other live, anaerobic, bacterial inocula for use in animal agriculture.

Effect of temperature and soil moisture on the survival and symbiotic effectiveness of Frankia spp.

Comparison of the effects of temperature on the growth in culture (increase in protein) of Frankia showed that three strains isolated from Casuarina were more tolerant of high temperature (45°C) than a strain from Alnus rubra. Optimal temperatures for growth of the Casuarina strains were in the range 25–30°C. Growth of the Alnus strain was good at 25°C but poor at 37°C. High temperatures (35–40°C) during storage for 7 months of these Frankia strains in sand, inoculated initially with liquid culture or with Frankia incorporated into alginate beads and permitted to dry, resulted in substantial loss of infectivity for the host plant species. Loss in infectivity was greater with an Alnus Frankia strain than strains from Casuarina cunninghamiana, C. equisetifolia and C. junghuniana. Three Frankia strains from C. equisetifolia were incorporated into a sand/perlite mixture with three different moisture regimes (field moisture capacity – wet: watered and maintained at field capacity; watered to field capacity but then allowed to dry – moderately wet; or watered to half field capacity and then permitted to dry – dry) and then stored for 12 weeks at 25°C and 35°C. Assessment by the most probable number (MPN) technique of the infectivity of the sand mixture for nodulation of C. equisetifolia showed significant interactions between Frankia strain, temperature and soil moisture content. The infectivity of Frankia strains ORS020607 and UGL020602q was not affected by incubation in wet sand at 25°C but fell by more than half after 12 weeks in moderate and dry conditions. Changes in infectivity were similar when incubation was at 35°C. By contrast, the infectivity of UGL020603q fell substantially under all moisture conditions and at both temperatures. The data show the importance of screening for tolerance of both temperature and moisture content when selecting strains for preparation of inoculum for use in hot climates.

Evaluation of manufactured inocula for use in the BOD test

While the BOD 5 test remains a major water quality measurement, its repeatability is poor. A dehydrated microbial seed was compared with seeds taken from various effluent treatment works using a standard glucose/glutamic acid solution. The repeatability estimate for the dehydrated microbial seed was significantly better than that attained using either raw or treated effluents as seed. However the accuracy of the test was not significantly improved by the use of the dehydrated seed, compared with fresh wastewater seeds, when seven replicates were used.

Effect of culture medium and carbon dioxide concentration on growth of anaerobic bacteria commonly encountered in clinical specimens.

Representative strains of anaerobic bacteria from human infections were used to evaluate broth media, gas mixtures, and inocula for use in developing a procedure for performing minimal inhibitory concentration antimicrobic susceptibility tests. Nine commercially available media, including two that were chemically defined, were tested. Tests were performed in atmospheres with carbon dioxide concentrations between 2.5 and 10% and also in the GasPak system (BBL) that had a disposable hydrogen-carbon dioxide generator. Growth curves on each organism grown in schaedler broth and a 5% carbon dioxide atmosphere were used to determine growth characteristics, equate time of the particular growth phases to turbidity readings, and determine the numbers of viable organisms present in the culture. Schaedler broth proved to be most advantageous in combination with an atmosphere of 5% carbon dioxide, 10% hydrogen, and 85% nitrogen. The growth curve studies yielded valuable data on the rapidity and quantity of growth under these conditions. We believe these data have provided information which can be used as the basis for developing a standardized procedure for antimicrobic susceptibility testing for anaerobic bacteria.

Effect of Culture Medium and Carbon Dioxide Concentration on Growth of Anaerobic Bacteria Commonly Encountered in Clinical Specimens

Representative strains of anaerobic bacteria from human infections were used to evaluate broth media, gas mixtures, and inocula for use in developing a procedure for performing minimal inhibitory concentration antimicrobic susceptibility tests. Nine commercially available media, including two that were chemically defined, were tested. Tests were performed in atmospheres with carbon dioxide concentrations between 2.5 and 10% and also in the GasPak system (BBL) that had a disposable hydrogen-carbon dioxide generator. Growth curves on each organism grown in schaedler broth and a 5% carbon dioxide atmosphere were used to determine growth characteristics, equate time of the particular growth phases to turbidity readings, and determine the numbers of viable organisms present in the culture. Schaedler broth proved to be most advantageous in combination with an atmosphere of 5% carbon dioxide, 10% hydrogen, and 85% nitrogen. The growth curve studies yielded valuable data on the rapidity and quantity of growth under these conditions. We believe these data have provided information which can be used as the basis for developing a standardized procedure for antimicrobic susceptibility testing for anaerobic bacteria.

Experience with a bacterial inoculum for use in respirometric tests for oxygen demand

Attempts have been made to develop a rapid, general test for waste waters, based on the oxygen uptake during a few hours in the presence of a specially prepared inoculum. The inoculum was obtained freshly as required by the aeration of settled sewage with organic additives, and was developed with the requirements of reproducibility, high activity, and low endogenous demand in mind. Preparation of the inoculum and its application were carried out at 30°C in the presence of inorganic nutrients and 0·04 M phosphate buffer, pH 7. The concentration of inoculum applied was usually in the range 100–400 mg 1 −1. The inoculum degraded a wide range of pure compounds in a few hours with good reproducibility. In the case of substances of relatively low biodegradability, some degree of adaptation was obtained by growing the inoculum in the presence of the substance in question. The oxygen uptake with settled sewage in 6 h was usually about 60–70 per cent of the BOD and 30–40 per cent of the COD. Promising results were obtained in tests carried out with industrial waste waters, but more work would be needed before the method could be recommended as a general one.