Inoculum Mixture Research Articles

Pathogenicity of a Subgroup C Feline Leukemia Virus (FeLV) Is Augmented when Administered in Association with Certain FeLV Recombinants

There is evidence to suggest that infectious feline leukemia viruses (FeLVs) may be altered biologically because of homologous recombination with non-infectious endogenous FeLV (enFeLV) sequences in the infected cells. To evaluate the role of such recombination events in FeLV pathogenesis, a molecular clone of subgroup C FeLV, Sarma strain (FSC), was tested for induction of aplastic anemia in the absence or presence of mixtures of recombinants between FSC and an enFeLV element. In the recombinants, FSC sequences in the viral surface glycoprotein (SU) protein were variably replaced by the corresponding sequences of the enFeLV. The results showed that the virus mixtures varied in their infectivity to neonatal specific pathogen-free cats. One group of mixtures, although exhibiting relatively reduced infectivity, represented the most acute disease-inducing agents. The presence of recombinants in this mixture significantly accelerated the development of erythrocyte aplasia compared to cats infected with FSC alone. In addition, infected cells appeared to be distributed differently in various hematopoietic organs with respect to infection with FSC versus viral mixture. Viral recombinants which were present in this inoculum mixture, however, could not be detected in the plasma or infected tissues of the cats at the end stage of the disease, although their presence in the plasma at the early stages could be detected. Clearly, parental FSC outgrew the recombinants in the infected animals, since its detection was prominent at all stages of the progression of the disease. Therefore, we hypothesize that recombinants initially present in the infected animals, while only poorly replicated compared to FSC in the host, might have had the opportunity to infect certain target cells (potentially erythroid progenitor cells) and then disappeared with the associated cytopathic effect.

Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge.

Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above.

Selection of a sterilization method for the study of cereal grain digestion

Experiments were initiated to select a sterilization method(s) that minimizes alterations in the digestive properties of cereal grains and, thus, would be suitable for the study of cereal grain digestion by pure cultures of ruminal bacteria. The following five treatments were examined: unsterilized (U), autoclaving with buffer (AB), autoclaving without buffer (AD), ethylene oxide (E), and gamma irradiation (I). Solubility of DM, starch, and CP was determined by soaking grain in buffer for 1 h followed by filtration through Whatman #54 filter paper. Ground corn and wheat from each treatment were placed in vials with a 1:1 mixture of Bryant's medium and ruminal inoculum. Vials were incubated for 4, 8, 12, 24, and 48 h and analyzed for starch content. Bacterial growth was not evident in sterilized, uninoculated samples. The AD treatment decreased the disappearance of CP in wheat and corn, whereas AB caused an increase in the disappearance of DM, CP, and starch in wheat (P less than .001) compared with U. Rates of microbial starch digestion for corn were 1.3, 1.5, 3.3, 14.7, and 3.5%/h and for wheat were 1.3, 3.4, 4.6, 17.1, and 4.6%/h for AD, E, I, AB, and U, respectively. Contrasts indicated that AD and AB differed (P less than .001) from U for both corn and wheat. It is likely that gelatinization of cereal starch enhanced microbial starch digestion in AB and the formation of Maillard products reduced starch digestion in AD. Corn and wheat sterilized with E or I had digestive properties that closely resembled those of U grain, and either sterilization method was suitable for studying cereal grain digestion.

Should VAM inocula contain single or several fungal species?

In a greenhose trial cassava was grown in an unsterilized acidic inceptisol which contained a high concentration of infective indigenous VAM propagules. At planting, VAM inoculum which contained Glomus manihotis (MAN), G. occultum (OCC) or Entrophospora colombiana (COL) as single species or in combination with each other was applied. MAN was the most effective and competitive fungus. Inoculum mixtures which contained 50% MAN and 50% of either medium (COL) or non-effective (OCC) endophytes were also highly effective. A single VAM species within the inoculum is preferable.

In Vitro Degradation of Choline from Selected Foodstuffs and Choline Supplements1,2

The objective of this experiment was to determine the extent of in vitro degradation of choline from barley, corn, corn gluten meal, cottonseed meal, fish meal, soybean meal, alfalfa hay, timothy hay, choline chloride, and choline stearate. During four individual fermentation runs, samples were incubated in vitro for .25, 1, 3, 6, 12, 18, 24, 36, and 48h with an inoculum mixture containing rumen fluid obtained from a rumen-fistulated dairy cow fed 17.5% corn silage and 28.7% grass silage and 53.8% concentrate diet. Because of their low choline content (less than .68 mg/g) corn, corn gluten meal, alfalfa hay, and timothy hay gave erratic values for choline disappearance for different fermentation runs and times of incubation although disappearance tended to increase with time. Data for the rest of the feeds and choline supplements were analyzed using nonlinear regression procedure to obtain estimates of potentially degradable choline, rapidly degradable choline, and the rate of choline degradation in vitro. The mean estimates of rumen degradable choline (%) were 79.4, 84.7, 82.9, 83.8, 98.0, and 98.6 for barley, cottonseed meal, fish meal, soybean meal, choline stearate, and choline chloride, respectively. The results suggest diat incorporating choline-rich feedstuffs in diets can only marginally increase the postruminal flow of choline in dairy cows.

Interstrain Competition between Representatives of Indigenous Serotypes of Rhizobium trifolii

The symbiotic characteristics of Rhizobium trifolii strains 1-01 and 2-01 were evaluated both individually and in various combinations on two cultivars (Mt. Barker and Woogenellup) of subterranean clover (Trifolium subterraneum L.). Nodules were observed on day 8 independent of cultivar or strain. Cultivar differences were measured in nodulating efficiency by 1-01 since 54% of the primary nodules were formed on cv. Mt. Barker and only 15% were formed on cv. Woogenellup in the zone above, or 1 cm below, the root tip location at the time of inoculation. The percentage of nodules formed in this zone by 2-01 was similar on both cultivars (31 to 32%). When mixtures of strains 1-01 and 2-01 (230:1 and 1:20) were used to inoculate plants, >90% of the nodules on both cultivars were occupied by the more abundant strain in the inoculum regardless of sampling date (4 or 8 weeks). In contrast, large percentages of nodules on 4-week-old plants of both cultivars exposed to a 5:1 inoculum mixture were doubly occupied (64 and 74%). By week 8 these values had decreased significantly (P </= 0.01) and were accompanied by large increases in the percentage of nodules occupied by either strain 1-01 alone (1 to 65%) on cv. Mt. Barker or 2-01 alone (4 to 49%) on cv. Woogenellup. The superior (cv. Mt. Barker) and inferior (cv. Woogenellup) symbiotic performance of plants inoculated with the 5:1 mixture correlated more closely with the 8-week than the 4-week nodule occupancy data. Primary nodule occupancy by 1-01 and 2-01 was significantly influenced by changes in the inoculum ratios of 1-01/2-01 from 5.7:1 to 0.67:1 on cv. Mt. Barker and from 1.9:1 to 0.67:1 on cv. Woogenellup. Despite evidence for extensive proliferation of the inoculant strains on the rhizoplanes, no evidence was obtained for either interstrain antagonism or selective proliferation as a valid reason to explain the outcome of primary nodulation.

Groundnut nitrogen fixation by three serologically and morphologically distinct rhizobia

Three cowpea miscellany rhizobia, a commercial strain (TAL 309) and two field isolates from Sudan (Wad Medani and Kadugli strains), were chosen for detailed studies because they could be separated by serological or morphological characteristics. The strains were screened in the greenhouse on three groundnut ( Arachis hypogaea L.) cultivars, “Ashford” and “MH383” (both Virginia types), and “Barberton” (a Spanish type). Whenever the TAL 309 strain was included in inoculum mixtures, it consistently occupied the majority of nodules with all three groundnut cultivars. Although significant differences were found in top dry-matter weights, color ratings, nitrogenase activities, and total nodule number among the three cultivars tested, there were no host-genotype-by-strain interactions either in growth measurements or in nodule occupancy. Correlation matrices indicated that several of the measures made could serve as reliable indicators of rhizobial efficiency. Both top dry-matter weight and plant color, and top dry-matter weight and C 2H 2 reduction were related at the 0.001 level of probability.

Nitrogen Fixation Estimates for Some Native and Introduced Legumes, Forbs, and Shrubs

Nitrogen Fixation Estimates for Some Native and Introduced Legumes, Forbs, and Shrubs

Evaluation of competitive ability of Rhizobium meliloti strains for nodulation in alfalfa

A method was developed for determination of the competitive ability of selected Rhizobium meliloti strains based on recovery from nodules and identification with commercially available antibiotic sensitivity disks. Four strains isolated from soil were used as inocula for alfalfa seedlings under controlled conditions in all possible two-, three-, and four-way combinations and alone as controls. One of the test strains readily formed nodules, but had low expressed nitrogenase activity when used alone. In contrast, another strain with high nitrogenase activity was found to be distinctly noncompetitive with the other strains, based on frequency of recovery from nodules. Nitrogenase activities of groups of plants resulting from growth with some inoculum mixtures showed evidence of linear additivity of the activities based on frequency of recovery of the strains from nodules and the activity of control plants inoculated with single strains. Synergistic responses were found for other inoculum mixtures. Likewise, competitive ability of inoculum strains in the four-way mixtures was not wholly predictable from results obtained in two- and three-way tests, indicating some modification of competitive abilities in multiple-inoculum mixtures. Occupancy of nodules by two strains was found to occur at very low frequency with multiple-strain inocula. The noncompetitive strain was never found in double occupancy with the other strains.

Competition Among Rhizobium leguminosarum Strains for Nodulation of Lentils ( Lens esculenta )

Thirty-one cultures of Rhizobium leguminosarum were screened for effectiveness (C(2)H(2) reduction) on lentils (Lens esculenta). Fluorescent antibodies prepared against three of the most effective strains (Hawaii 5-0, Nitragin 92A3, and Nitragin 128A12) exhibited a high degree of strain specificity; the antibodies reacted strongly with their homologous rhizobia in culture and with bacteroids in nodules. They did not cross-react with one another, and only weakly with 5 of the 47 other R. leguminosarum cultures tested. In competition studies in the growth chamber, whenever strain Nitragin 92A3 was included in the inoculum mixture, it consistently (but not always significantly, P = 0.05) occupied the majority of nodules on all four cultivars used. However, some degree of strain X cultivar interaction was apparent: Hawaii 5-0 was of equal competitiveness (P = 0.05) with Nitragin 92A3 on three of the varieties (Commercial, Tekoa, and Benewah), but inferior (P = 0.01) on the Chilean variety; Nitragin 92A3 completely dominated (P = 0.01) Nitragin 128A12 on all cultivars; and Hawaii 5-0 was of equal competitiveness (P = 0.05) to Nitragin 128A12 on the Chilean variety and more competitive (P = 0.01) on the commercial variety and less so on the other two varieties. In field experiments, Hawaii 5-0 proved of equal competitiveness (P = 0.01) with Nitragin 92A3 in one soil (an Inceptisol) and superior (P </= 0.05) to it in another (an Oxisol). Incidence of double-strain occupancy of nodules varied from 0 to 36% in vermiculite, depending on the strains in the mixture and the host variety, and from 0 to 38% in the field, depending on the strains in the mixture and the soil type. The results suggest a close relationship between the competitiveness of a strain and its occurrence in doubly infected nodules.

Competitive saprophytic colonization of pigeon-pea substrate by Fusarium udum in relation to environmental factors, chemical treatments and microbial antagonism

The competitive saprophytic colonization (CSC) of pigeon-pea substrates by Fusarium udum was studied in relation to environmental factors, chemical treatments and microbial antagonism. CSC was best at 22°C, good at 30°C, but at 40°C the substrate was vigorously colonized by Coprinus lagopus and Aspergillus nidulans which suppressed colonization by F. udum. High competitive saprophytic ability of F. udum was noted at pH 7 and 9 at soil moisture contents between 5 and 30%. At pH 4 and 5 F. udum was replaced by A. flavus and A. nidulans. The CSC of F. udum decreased in the soil-sand inoculum mixture amended with the fungicides bavistin, dithane Z-78 and difolatan. The herbicides 2,4-D and machete also reduced CSC by F. udum whereas urea promoted it. Colonization of pigeon-pea substrate by F. udum was highly suppressed by antagonism from Pénicillium citrinum, A. niger, Micromonospora gtobosa, A.flavus, A. terreus and Trichoderma viride when these were used in inoculum mixtures with F. udum or when substrates had already been colonized by them. Otherwise F. udum was shown to have a high competitive saprophytic ability.

Relative ability of two strains of Pucccinia graminis tritici to survive when mixed

SUMMARYThe relationship between relative survival ability and the number of genes for virulence in two strains of Puccinia graminis tritici was investigated using a differential variety to determine the proportion of each race in the mixture. The strain with the widest host range (21 Anz‐2,3,7) became predominant after a number of uredial generations, regardless of its proportion in the original inoculum mixture. However, despite the higher relative survival ability of strain 21 Anz‐2,3,7, strain 21 Anz‐2,7 persisted at c. 1% frequency after four successive uredial generations. The longer the period between successive inoculations, the more rapidly strain 21 Anz‐2,3,7 became predominant in the mixture. The variety on which the mixture was cultured did not influence the relative survival ability of the two strains.

The Agar Layer Method for Determining the Activity of Diverse Materials against Selected Protozoa*†

SYNOPSIS. An agar overlay technique developed to investigate the effect of various substances on growth of a selected group of protozoa is described. Test systems employing Crithidia fasciculata (culex), Ochromonas malhamensis, Tetrahymena pyriformis W and Trichomonas foetus were devised to evaluate the effect of known compounds and to assay the activity of antibiotic fermentation beers. This disc‐plate method can be summarized as follows: preparation of a foundation layer; overlaying of a mixture of agar, nutrients, protective antibiotics, and inoculum; placing of paper discs with the test material on the overlay followed by incubation and subsequent observation of the plates for zones of inhibition of growth. The concentration of agar in the overlay sufficient to restrict movement of the protozoa without inhibiting their growth, nutrients required for adequate growth and continued viability of the inoculum, and the concentration of penicillin and streptomycin which will not inhibit the assay protozoa but beeffective against the microorganisms in the fermentation beers are discussed. This technique offers a method not only for primary screening for cytotoxic substances but also one which may be adaptable to other studies of protozoa.