Citrus juice, a concentrate manufactured by the Jeju Provincial Corporation, was converted into vinegar orderly by alcohol and acetate fermentation. The juice with 6 folds dilution by distilled water was used as the sole nutrient source through out experiments. Diluted juice contained $12.96^{\circ}Brix$ of total sugar, $0.632\%$ of total acid and $20.23{\mu}g/m{\ell}$ of hesperidin. Naringin was not detected from the juice. Citrus wine having $5.6\~6.3\%$ alcohol was produced from diluted juice by 3 days of fermentation at $28^{\circ}C$. A kind of malomelo yeast CMY-28 was used for wine fermentation. The wine was succeedingly fermented for 8 days at $30^{\circ}C$ after inoculation of seed vinegar which contained active cells of acid producing bacteria CV1. Inoculum size of seed vinegar was controlled to $10\%$(v/v) of citrus wine. The wine converted into vinegar by the fermentation. Citrus vinegar, the final product of fermentation, was colored with very thin radish-yellow and transparent. It's acidity ranged between $5.8\~6.2\%$ as acetic acid. The vinegar got the best score by sensory test among several natural fruit vinegars. It was clear from the results that citrus vinegar in high quality could be produced from concentrated citrus juice, however fermentation conditions should be improved to reduce the amount of reducing alcohol.
Objective: To improve the feeding value of rice straw (RS), this study evaluated the potential of rice straw fermented with Pleurotus sajor-caju (FRS) as dairy goat feed. Methods: Spent Pleurotus sajor-caju mushroom substrate was used as fungi inoculum to break the lignocellulose linkage of rice straw, which was solid-fermented at 25℃ to 30℃ for 8 weeks. The ruminal degradation of pangolagrass hay (PG), FRS, and RS were measured in situ for 96 hours in three dry Holstein cows, respectively. Effect of fungi fermented RS on milking performance was studied in feeding trials. A total of 21 Alpine goats a trial were divided into 3 groups: a control group in which PG accounted for 15% of the diet dry matter, and FRS or RS was used to replace the PG in the control group. Goats were fed twice a day under two 28-day trial in individual pens. Meanwhile, a 3×3 Latin square trial (14 days/period) was conducted to study the rumen digestion of three diets by using three fistulated dry goats. Rumen contents were collected for metabolite analyses every one to three hours on the last two days. Results: In situ study showed that fermentation could elevate the rumen degradable fraction and effective degradability of RS (p<0.05). Effective degradability of FRS dry matter was significantly increased from 29.5% of RS to 41.7%. Lactating trial results showed that dry matter intake and milk yield in the PG group and FRS group were similar and higher than those in RS group (p<0.05). The concentration of propionic acid and total volatile fatty acid in the RS group tended to be lower than those in PG group (p<0.10). There were no differences in rumen pH value and ammonia nitrogen level among the groups tested. Conclusion: Fermentation of rice straw by spent Pleurotus sajor-caju mushroom substrate could substantially enhance its feeding value to be equivalent to PG as an effective fiber source for dairy goat. The fermented rice straw is recommended to account for 15% in diet dry matter.
Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that 2. tenezla first penetrate into the mucosal intraepithelial Iymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope labelled uracil ($^3H-uracil$) . Third, the E. tenella sporozoites viability was assayed after preincubation of them with thicken spleen cells. E. tenella oocysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (I) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schisogonic cycle of E. tenella in 3~4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merogoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48~60 hours, and decreased thereafter. The uptake amount of $^3H-uracil$ depended not only upon the inoculum sixte of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.
We investigated the effect of silicate coating of rice seeds on bakanae disease incidence and the quality of seedlings raised in seedling boxes and transplanted into pots. The silicate-coated rice seed (SCS) was prepared as follows. Naturally infested rice seeds not previously subjected to any fungicidal treatment were dressed with a mixture of 25% silicic acid at pH 11 and 300-mesh zeolite powder at a ratio of 50 g dry seed - 9 mL silicic acid - 25 g zeolite powder. The following nursery conditions were provided : Early sowing, dense seeding in a glass house with mulching overnight and no artificial heating, which were the ideal conditions for determining the effect on the seed. The nursery plants were evaluated for Gibberella. fujikuroi infection or to determine the recovery to normal growth of infected nursery plants in the Wagner pot. Seedlings emerged 2-3 days earlier for the SCS than they did for the non-SCS control, while damping-off and bakanae disease incidence were remarkably reduced. Specifically, bakanae disease incidence in the SCS was limited to only 7.8% for 80 days after sowing, as compared to 91.6% of the non-SCS control. For the 45-days-old SCS nursery seedlings, the fresh weight was increased by 11% and was two times heavier, with only mild damage compared to that observed for non-SCS. Even after transplanting, SCS treatment contributed to a lower incidence of further infections and possibly to recovery of the seedlings to normal growth as compared to that observed in symptomatic plants in the pot. The active pathogenic macro-conidia and micro-conidia were considerably lower in the soil, root, and seedling sheath base of the SCS. In particular, the underdeveloped macro-conidia with straight oblong shape without intact septum were isolated in the SCS ; this phenotype is likely to be at a comparative etiological disadvantage when compared to that of typical active macro-conidia, which are slightly sickle-shaped with 3-7 intact septa. A active intact conidia with high inoculum potential were rarely observed in the tissue of the seedlings treated only in the SCS. We propose that promising result was likely achieved via inhibition of the development of intact pathogenic conidia, in concert with the aerobic, acidic conditions induced by the physiochemical characteristics associated with the air porosity of zeolite, alkalinity of silicate and the seed husk as a carbon source. In addition, the resistance of the healthy plants to pathogenic conidia was also important factor.
Three species of Fusarium, F. fujikuroi, F. verticillioides and F. proliferatum, are known to be associated with bakanae disease of rice [1, 2]. F. fujikuroi infects rice flowers and survive in endosperm and embryo of the seeds. Infected seed is an important source of primary inoculum of pathogens [3]. Seeds of rice (Oryza sativa cv. Boramchan) collected from bakanae-infected field were found to be 96% infected with Fusarium sp., 52% with F. fujikuroi, 42% with F. verticillioides, and 12% with F. proliferatum as determined by incubation method and species-specific PCR assays. F. fujikuroi was detected at lemma/palea, endosperm and embryo whereas F. verticillioides and F. proliferatum were recovered only from lemma/palea by means of component plating test. Seed disinfection methods have been developed to control bakanae disease and prochloraz has been most widely used for rice seeds. Two chemicals formulated with prochloraz (PC 1) and prochloraz + hexaconazole (PC 2) that inhibit biosynthesis of ergosterol strongly reduced the incidence of Fusarium spp. on selective media to 4.7% and 2.0%, respectively. Disease symptoms of rice seedlings in nursery soil were alleviated by chemical treatment; seedlings with elongated leaves or wide angle between leaf and stem were strikingly reduced from 15.6 to 3.2% (PC 1) and 0 (PC 2), stem rots were reduced from 56.9 to 26.2% (PC 1) and 32.1% (PC 2), and normal seedling increased from 0.4 to 13.3% (PC 2). Prochloraz has some disadvantages and risks such as the occurrence of tolerant pathogens [4] and effects on the sterol synthesis in animals and humans [5]. For these reasons, it is necessary to develop new disinfection method that do not induce fungal tolerance and are safe to humans and animals. Chlorine dioxide ($ClO_2$), that is less toxic, produces no harmful byproducts, and has high oxidizing power, has been reported to be effective at disinfection of several phytopathogenic fungi including Colletotrichum spp. and Alternaria spp. [6]. Gaseous $ClO_2$ applied to rice seeds at a concentration of 20 ppm strongly suppressed mycelial growth of Fusarium fujikuroi, F. verticillioides and F. proliferatum. The incidence of Fusarium spp. in dry seed with 8.7% seed moisture content (SMC) tended to decrease as the concentration of $ClO_2$ increased from 20 to 40 ppm. Applying 40 ppm $ClO_2$ at 90% relative humidity, incidence was reduced to 5.3% and resulted in significant reduction of disease symptoms on MS media. In nursery soil, stem rot was reduced from 56.9 to 15.4% and the number of normal seedlings increased from 0.4 to 25.5%. With water-soaked seeds (33.1% SMC) holding moisture in the endosperm and embryo, the effectiveness of disinfection using $ClO_2$ increased, even when treated with only 20 ppm for four hours. This suggests that moisture was a key element for action of $ClO_2$. Removal of the palea and lemma from seeds significantly decreased the incidence of Fusarium spp. to 3.0%. Seed germination appeared to decrease slightly by water-soaking at $30^{\circ}C$ because of increased SMC and by physical damage of embryos from hulling. These results indicate that the use of gaseous $ClO_2$ was effective as a means to disinfect rice seeds infected with Fusarium spp. and that moisture around the pathogens in the seed was an important factor for the action of $ClO_2$. Further investigations should be conducted to ascertain the best conditions for complete disinfection of Fusarium spp. that infect deep site of rice seeds.
Some microorganisms are capable of leaching Mn(II) from nonsulfidic manganese ores indirectly via nonenzymatic processes. Such reductive dissolution requires organic substrates, such as glucose, sucrose, or galactose, as a source of carbon and energy for microbial growth. This study investigated characteristics of Mn(II) leaching from manganese nodules by using heterotrophic Bacillus sp. strain MR2 provided with corn starch as a less-expensive substrate. Leaching of Mn(II) at 25.6 g Mn(II) $kg^{-1}$ nodule $day^{-1}$ was accompanied with cell growth, but part of the produced Mn(II) re-adsorbed onto residual $MnO_2$ particles after 24 h. Direct contact of cells to manganese nodule was not necessary as a separation between them with a dialysis tube produced similar amount [24.6 g Mn(II) $kg^{-1}$ nodule $day^{-1}$]. These results indicated an involvement of extracellular diffusible compound(s) during Mn(II) leaching by strain MR2. In order to optimize a leaching process we tested factors that influence the reaction, and the most efficient conditions were $25\sim35^{\circ}C$, pH 5~7, inoculum density of 1.5~2.5% (v/v), pulp density of 2~3 g/L, and particle size <75 ${\mu}m$. Although Mn(II) leaching was enhanced as particle size decrease, we suggest <212 ${\mu}m$ as a proper size range since more grinding means more energy consumption The results would help for the improvement of bioleaching of manganese nodule as a less expensive, energy-efficient, and environment-friendly technology as compared to the existing physicochemical metal recovery technologies.
This experiment was conducted to find out the effects of fertilizer-N and co-inoculation of the Rhizobium japonicum and the Azospirillum lipoferum on nodulation, $N_2$-fixation, and growth of soybean under in situ conditions. The results obtained were summarized as follows: 1. The yield of soybean dry matter was significantly greater in the R. japonicum alone, and the mixed inoculation of R. japonicum and A. lipoferum than those of un-inoculation. But inoculum applied by different strains did not significant effect on plant growth. The effects of nitrogen applied on soybean dry matter were higher in the ammonium sulfate than potassium nitrate, and decreased with increasing rates of two forms of nitrogen applied regardless of nitrogen source. 2. Acetylene redution activity was more increased in a single inoculation of R. japonicum than those of the mixed inoculation of the R. japonicum and the A. lipoferum, in cases of Danyeup cultivar, regardless of the form of combined nitrogen used. 3. Nodule mass and total nitrogenase activity per plant showed the positively significant effect in the interrelationship between dry matter of soybean and some factors related to nitrogen fixation efficiency. 4. The highest symbiotic effect in Danyeup cultivar was obtained when a single R. japonicum 84 Dy-1 strain was inoculated and fertilized with 18 mM potassium nitrate.
Attempts to search infection period, infection speed in the tissue of neck blast of rice plant, location of inoculum source and effects of several conditions about the leaf sheath of rice plants for neck blast incidence have been made. 1. The most infectious period for neck blast incidence was the booting stage just before heading date, and most of necks have been infected during the booting stage and on heading date. But $Indica{\times}Japonica$ hybrid varieties had shown always high possibility for infection after booting stage. 2. Incubation period for neck blast of rice plants under natural conditions had rather a long period ranging from 10 to 22 days. Under artificial inoculation condition incubation period in the young panicle was shorter than in the old panicle. Panicles that emerged from the sheath of flag leaf had long incubation period, with a low infection rate and they also shown slow infection speed in the tissue. 3. Considering the incubation period of neck blast of rice plant, we assumed that the most effective application periods of chemicals are 5-10 days for immediate effective chemicals and 10-15 days for slow effective chemicals before heading. 4. Infiltration of conidia into the leaf sheath of rice plant carried out by saturation effect with water through the suture of the upper three leaves. The number of conidia observed in the leaf sheath during the booting stage were higher than those in the leaf sheath during other stages. Ligule had protected to infiltrate of conidia into the leaf sheath. 5. When conidia were infiltrated into the leaf sheath, the highest number of attached conidia was observed on the panicle base and panicle axis with hairs and degenerated panicle, which seemed to promote the infection of neck blast. 6. The lowest spore concentration for neck blast incidence was variable with rice varietal groups. $Indica{\times}Japonica$ hybrid varieties were infected easily compared to the Japonica type varieties, especially. The number of spores for neck blast incidence in $Indica{\times}Japonica$ hybrid varieties was less than 100 and disease index was higher also in $Indica{\times}Japonica$ hybrid than in Japonica type varieties. 7. Nitrogen content and silicate content were related with blast incidence in necks of rice plants in the different growing stage changed during growing period. Nitrogen content increased from booting stage to heading date and then decreased gradually as time passes. Silicate content increased from booting stage after heading with time. Change of these content promoted to increase neck blast infection. 8. Conidia moved to rice plant by ascending and desending dispersal and then attached on the rice plant. Conidia transfered horizontally was found very negligible. So we presumed that infection rate of neck blast was very low after emergence of panicle base from the leaf sheath. Also ascending air current by temperature difference between upper and lower side of rice plant seemed to increase the liberation of spores. 9. Conidial number of the blast fungus collected just before and after heading date was closely related with neck blast incidence. Lesions on three leaves from the top were closely related with neck blast incidence, because they had high potential for conidia formation of rice blast fungus and they were direct inoculum sources for neck blast. 10. The condition inside the leaf sheath was very favorable for the incidence of neck blast and the neck blast incidence in the leaf sheath increased as the level of fertilizer applied increased. Therefore, the infection rate of neck blast on the all panicle parts such as panicle base, panicle branches, spikelets, nodes, and internodes inside the leaf sheath didn't show differences due to varietal resistance or fertilizers applied. 11. Except for others among dominant species of fungi in the leaf sheath, only Gerlachia oryzae appeared to promote incidence of neck blast. It was assumed that days for heading of varieties were related with neck blast incidence.
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