The Influence of Nutrition on Conidial Production by Rhynchosporium alismatis and on Their Subsequent Infectivity to Alisma lanceolatum

Nutrient composition of liquid-shake cultures significantly influenced conidial production by five isolates of Rhynchosporium alismatis after 6 days at 25 C. Lima bean broth at pH 7.5 produced the most conidia (4.99 x 10 7 /ml). The zwitterionic buffers PIPES and HEPES did not significantly affect broth pH, but did significantly affect yield of conidia. pH per se did not appear to affect yield directly. The medium in which an isolate was grown had a significant effect on the virulence of the resulting conidia as measured by disease severity scores in leaf discs of Alisma lanceolatum after 3 and 13 days. There was a significant difference between isolates, produced in the same medium, in the subsequent rate of disease development. The correlation between image analysis of diseased leaf discs with visual assessment was low, and it was considered that automatic assessment of disease severity was not cost-effective with the equipment used. Isolate DAR 73158 and lima bean broth are considered to be the combination of choice for further studies to explore the fitness of conidia produced in small-scale biofermentors.     

Factors Affecting Disease Development by Rhynchosporium alismatis in Starfruit (Damasonium minus), a Weed of Rice

The fungal pathogen Rhynchosporium alismatis is being developed for biological control of starfruit (Damasonium minus), an important aquatic weed in Australian rice fields. The development of R. alismatis in starfruit differs between juvenile and adult plants. Juvenile starfruit plants are stunted as a result of fungal infection, while in adult plants, the main effect is necrosis and chlorosis of floating leaves. A conidial concentration of 1×10⁴ conidia mL^-1 was adequate to cause disease symptoms on floating leaves, but the stunting effect on juveniles was caused by concentrations of at least 1×10⁵ conidia mL^-1. To successfully inoculate juvenile plants, the water must be drained before inoculation to expose plants to the inoculum. The artificial addition of dew periods did not enhance disease development in plants. The stunting of juvenile starfruit plants caused by the infection of R. alismatis may give rice plants a competitive advantage over the weed at the seedling stage     

Production of Monascus-Pigment in a Submerged Culture

The production of pigment by the molds belonging to the genus Monascus in a submerged culture was examined. The extracellular pigment was mainly studied. Monascus sp. No. 2 was found to be the most potent pigment producer. The optimum cultural conditions were: pH of the medium, 6.5; the temperature, 25°C; carbon sources, glucose or ethyl alcohol; nitrogen sources, polypeptone, yeast extract, monosodium glutamate or casamino acids. Glycine, l-threonine, l-arginine, l-alanine and l-tyrosine were found to be the most effective substances promoting pigment production.

Mycelial forms of this strain were correlated with pigment formation in submerged culture. As it grew into pellet type, the yield of pigment was at high level.

The Monascus-pigment in the fermentation liquid seemed to be firmly bound to the protein-like substances which made the pigment apparently soluble.     

Early Sprouting and First Stages of Growth of Rice Seeds Exposed to a Magnetic Field

The effects of a stationary magnetic field on the germination of rice seeds (Oryza sativa, L.) and on the initial stages of growth of rice plants have been evaluated. In both tests, the seeds were exposed to one of two magnetic field strengths (125 or 250 mT) for different times (1 min, 10 min, 20 min, 1 h, 24 h, or chronic exposure) as 12 separate treatments (doses D1–D6 for 125 mT and doses D7–D12 for 250 mT). Nonexposed seeds were used as controls (C). The mean germination time (MGT) was significantly reduced compared to control when seeds were exposed to magnetic field (54.00 h for D5 and D11, and 58.56 h for control). The parameters T₁ and T₁₀, times required to obtain 1 and 10% of germinated seeds, were also reduced. The T₁₀ of control seeds was 44.40 h, while treatments D5, D6, D11, and D12 gave rise to values of 36.00, 36.96, 32.64, and 39.36 h, respectively. The higher germination rate of treated seeds obtained in the germination test is in agreement with the higher lengths and weights of rice plants exposed to a magnetic field recorded on the growth tests. All the parameters measured were over the control ones, although the highest lengths and weights of rice plants were obtained for chronic exposure to magnetic field (doses D6 and D12). Stem length of control plants (45.36 mm) measured at the tenth day was significantly lower than that obtained for doses D6 (58.58 mm) and D12 (80.63 mm); the same behavior was observed on total length, stem weight, and total weight. Our finding indicates that this type of magnetic treatment clearly affects germination and the first stages of growth of rice plants.     

Influence of Agricultural By-products in Liquid Culture on Chlamydospore Production by the Potential Mycoherbicide Fusarium oxysporum Foxy 2

Economically feasible inoculum mass production methods are required for successful application of Fusarium oxysporum Foxy 2 as a potential mycoherbicide. Therefore, different substrates (agricultural by-products) and the factors that influence the production of spores, especially chlamydospores, of Foxy 2 were investigated in liquid cultures. The substrates tested were cotton seed cake, maize stover, wheat and triticale stillage. The presence of plant fibers in the medium of unfiltered cotton seed cake (2.5%, w/v) significantly enhanced chlamydospore, micro- and macroconidia production by 150, 185, and 300%, respectively, compared to the filtered (fiber-free) medium. Regardless of the type of substrate tested, Foxy 2 was able to produce abundant chlamydospores (0.14-2.7×10⁷ mL^-1) in all growth media. Generally, increasing the concentrations of cotton seed cake and maize stover in the medium significantly increased chlamydospore formation; however, this was not the case for wheat-based stillage. To optimize conditions required for chlamydospore production of Foxy 2, the effect of near ultra-violet (NUV) light, substrate combinations (synergism), level of agitation, incubation time and their interaction were studied. A liquid culture of 2.5% (w/v) filtered cotton seed cake, exposed to continuous NUV for 15 days, doubled the yield of chlamydospores (4.7×10⁶ mL^-1) and macroconidia (5×10⁵ mL^-1), and increased microconidial production by one-third (1×10⁸ mL^-1) compared to natural light. An apparent synergistic effect of substrate combination was observed, since the addition of 20% (v/v) of either wheat or triticale-based stillage to maize stover medium (1%, w/v) increased the number of chlamydospores produced by 16 or 12 times, respectively, compared to maize stover alone (1.4×10⁶ mL^-1), and 2 times more than either of the stillages. A significantly positive effect between a high level of agitation tested during incubation and chlamydospore production of Foxy 2 was recorded. All in all, substantial chlamydospore production of Foxy 2 (4.3×10⁷ mL^-1) was successfully achieved within 12 days of incubation in a single-step liquid fermentation through the combination of 0.5% (w/v) maize stover plus 20% (v/v) wheat-based stillage and a high level of agitation (200 rpm).     

Effect of Aeration on Growth and Aflatoxin Production by Aspergillus flavus in Submerged Culture

Aspergillus flavus ATCC 15517 produced up to 212 mg per liter of total aflatoxin in submerged culture in aerated (3,000, 6,000, 9,000, and 12,000 ml/min) and agitated medium in 14-liter fermentors with 10 liters of medium consisting of 2% yeast extract and 10% sucrose. Aflatoxin production increased with time. A maximum of 212 mg/liter was produced at 9,000 ml/min aeration, whereas the yield decreased substantially at the lower aeration rates. Two other strains of A. flavus synthesized aflatoxin in smaller quantities.     

Effect of Stocked Fish on Rice Leaffolder Cnaphalocrocis medinalis and Rice Caseworm Nymphula depunctalis Populations in Intensive Rice Culture

It is often claimed that fish stocked in rice fields have a significant impact on the rice pest population. However, there is little experimental evidence for this. We used the data of six experiments to find out whether a polyculture of silver barb Barbodes gonionotus (Bleeker), common carp Cyprinus carpio (L.) and Nile tilapia Oreochromis niloticus (L.) has any effect on the population of rice leaffolders Cnaphalocrocis medinalis (Guenée) and rice caseworms Nymphula depunctalis (Guenée), two common pests in intensively cultured rice fields. Fish had no effect on the rice leaffolder population, but significantly lowered the number of rice caseworm adults and larvae. We concluded that fish have some effect on the arthropod pest population in rice fields, although, most probably, only on sluggish pests living in the water or on the water surface.     

Genetic diversity of <Emphasis Type="Italic">Rhynchosporium secalis</Emphasis> in Tunisia as revealed by pathotype,AFLP, and microsatellite analyses

Genetic variability among 122 Rhynchosporium secalis isolates collected from barley in three regions of Tunisia was investigated using host differentials, amplified fragment length polymorphism (AFLP), and microsatellite markers. The isolates were collected from a widely grown scald-susceptible barley cultivar Rihane and a range of local landrace cultivars in geographically distinct regions with different agroclimatic conditions. Pathotypic diversity (the proportion of unique pathotypes) was high in R. secalis populations from the high (100% diversity), moderate (95%), and low (100%) rainfall areas of Tunisia, and from both Rihane (which is the sole variety grown in the high rainfall region) and local landraces (which predominate in the low rainfall area). This may reflect a general adaptability for aggressiveness and suggests that the widely grown cultivar Rihane has exerted little or no selection pressure on the pathogen population since its release in 1983. Genotypic diversity (GD), defined as the probability that two individuals taken at random had different genotypes, was high for populations from Rihane, local landraces, and different agro-ecological zones (GD = 0.96–0.99). There was low genetic differentiation among pathogen populations from different host populations (G _ST ≤ 0.08, θ ≤ 0.12) and agro-ecological zones (G _ST ≤ 0.05, θ ≤ 0.04), which may be partly explained by gene flow due to the movement of infected stubble around the country. There was no correlation (r = 0.06, P = 0.39) between virulence phenotype and AFLP haplotype. A phenetic tree revealed groups with low bootstrap values that did not reflect the grouping of isolates based on host, pathotype, or agro-ecological region. The implications of these findings for R. secalis evolutionary potential and scald-resistance breeding in Tunisia are discussed.     

Agricultural By-products as Substrates for Growth, Conidiation and Chlamydospore Formation by a Potential Mycoherbicide, Fusarium oxysporum Strain EN4

Characteristics, such as mycelial growth, conidiation and chlamydospore formation, of three distinct variants (EN4-FT, EN4-FF and EN4-S), of the potential mycoherbicide, Fusarium oxysporum, strain EN4 were studied in liquid cultures. Agricultural by-products, such as cornmeal, corn cob, soya bean hull fiber (Dietfiber-Soyfiber), cotton seed embryo flour (Pharmamedia), cane molasses yeast extract and potato dextrose medium were used as substrates. The mycelial growth and conidia and chlamydospore formation were different for the three strains and varied with the substrate used. The quantity of conidia and chlamydospores produced depended on the concentration of the substrates. Irrespective of carbon:nitrogen ratios, chlamydospores were formed readily in liquid media and in greater amount on substrates with low utilizable carbon content (aqueous extracts of soya bean hull fiber and corn cob) than on those with higher utilizable carbon content (potato dextrose broth, aqueous extracts of cotton seeds and molasses yeast extract broth). In liquid cultures, increases in the concentration of substrates high in utilizable carbon resulted in reduced chlamydospore formation; however, this was far less in those with a lower carbon content. The effect on fungal growth characteristics due to the presence or absence of plant fibers in various concentrations of unfiltered and filtered extracts of soya bean hull fiber depended on the fungal variant tested. However, it had a greater effect on viable and microconidial counts than on chlamydospore and macroconidial counts. The viable and microconidial counts were significantly higher in unfiltered than in filtered extracts. The final pH of the liquid cultures (4.1-8.5), after 10 days of growth, was affected by the fungal variant, the substrate used and by the substrate concentration. In liquid cultures with 1% soya bean hull fiber, chlamydospore formation corresponded to an increase in the pH of the medium from 6.0 to 7.7. The interaction between fungal variants, the substrate used and the substrate concentration was highly significant for all the variables tested, indicating that optimization of the production of fungal propagules is complex. Overall, for all three variants, the best substrates for optimum conidiation and chlamydospore formation were aqueous extracts of soya bean hull fiber and corn cob at concentrations of 1-2.5%.     

Media and culturing protocol using a full 25 factorial design for the production of submerged aggregates by the potential bio-herbicide Plectosporium alismatis against weed species of Alismataceae

The potential bio-herbicide Plectosporium alismatis produces drying and UV tolerant micro-sclerotia-like structures named aggregates, effective against the weed Alisma plantago-aquatica. In this study, we evaluated (i) optimal liquid culture conditions that supported the high dry weight, conidia and aggregate yields and (ii) stress-tolerance and effectiveness of aggregates. Using a full factorial 2⁵ design, we studied the impact of agitation (A), glucose concentration (B), Tween 80 concentration (C), malt extract concentration (D), and inoculum density (E), on P. alismatis growth yields. The inoculum density (E), the agitation rate (A), and 3-factor interactions involving E and/or A had significant effects on aggregate yields (average 213 aggregates mL^?1; main effect of E: +112.87; main effect of A: ?74.81), but no significant effect on conidial yields and dry weight. The agitation was maintained at 100?rpm and increasing numbers of conidia were inoculated into the culture media. Maximal aggregate yields (3.6?×?10³ aggregates mL^?1) were obtained with an initial conidial density of 4?×?10⁶ conidia mL^?1. While freshly-harvested, dried and/or UV exposed aggregates germinated at high rates (respectively: 100%; 99%; 76%; 85%), sporogenesis significantly decreased after stress exposure. In bioassays using leaf discs of A. plantago-aquatica, chlorosis and/or necrosis was observed after 8 days incubation, regardless of whether aggregates had been exposed to desiccation and/or UV-radiations prior to application on leaf discs. These data provide a cultural protocol for the production of high numbers of UV and drying resistant aggregates effective against weed species of Alismatacae.     

Production of Mushroom Mycelium as a Protein and Fat Source in Submerged Culture in Medium of Vinasse

Of ten mushroom cultures investigated, only Agaricus campestris, Boletus indecisus, and Tricholoma nudum were capable of growing in submerged culture in medium of vinasse with added salts. Higher fermentative efficiencies were found under these conditions than in medium containing molasses or waste sulfite liquor. A. campestris showed a better capacity to produce protein but, since B. indecisus is capable of developing greater mycelium weight, its fermentative efficiencies are comparable. Both microorganisms could be grown in medium of vinasse with greatly varied amounts, producing higher mycelial weight in media with greater vinasse. The capacity of B. indecisus and A. campestris to utilize the noncarbohydrate fraction in total solids, instead of the total carbohydrates when they are in smaller amount, was observed in medium containing vinasse. B. indecisus and A. campestris were easily separated by filtration from the medium, although T. nudum was difficult to separate by this procedure. In experiments with A. campestris, the adaptative capacity of the organism to vinasse was demonstrated.     

Production, Survival and Evaluation of Liquid Culture-produced Inocula of Coniothyrium minitans Against Sclerotinia sclerotiorum

Growth of Coniothyrium minitans on potato dextrose broth was compared with that on an inexpensive molasses-yeast liquid medium at 18-22°C in static culture. Biomass and conidial production were, in general, similar, although the rate of biomass production was quicker and conidial production was slightly greater per unit volume of medium in the molasses-yeast medium. Air-dried biomass from molasses-yeast liquid culture containing mycelia, pycnidia and conidia of C. minitans was mixed (12%, w/w) with kaolin to give a kaolin-biomass dust. The ability of C. minitans to survive and subsequently infect and reduce the viability of sclerotia of Sclerotinia sclerotiorum from this kaolin-biomass dust was found to be little affected by storage for 48 weeks between 4 and 15°C but was decreased by higher storage temperatures. The kaolin-biomass dust preparation did not differ from a standard maizemeal-perlite inoculum of C. minitans in its ability to infect sclerotia of S. sclerotiorum or reduce their viability or carpogenic germination in glasshouse and field pot bioassays. Further, when either inoculum was applied once to glasshouse soil naturally infested with S. sclerotiorum prior to planting three successive crops of lettuce, the pattern of disease control, reduction of sclerotial numbers/ plot, infection of sclerotia, reduction of sclerotial viability and survival in soil were similar for both inocula. The potential for the commercial development of liquid-culture-produced inocula of C. minitans is discussed.     

碳源和氮源对彩绒革盖菌液体发酵合成漆酶的影响

研究了碳源、氮源对彩绒革盖菌液体发酵合成漆酶的影响。结果表明,在所选的几种碳、氮源中,麸皮为试验菌株合成漆酶的较好碳源;酵母浸膏、酒石酸铵、蛋白胨均是比较理想的氮源。不同的碳氮比对彩绒革盖菌漆酶的产量有着显著的影响,高碳高氮是其生产漆酶的最佳条件。在适宜的培养条件下该菌株能合成高活力的漆酶,其酶活力可达298 U/mL以上,产酶周期为6~7 d。     

适合飞虱虫疠霉菌丝生产的液体培养基组分及发酵条件

蚜虫、飞虱、叶蝉等是威胁农业生产的刺吸式口器害虫,长期以来依赖化学农药防治,严重影响农产品的安全性和农业生态环境.探讨这类害虫的生物防治途径,减轻农产品化学农药的残留污染,一直受到国内外学者的关注.虫霉(En-tomophthorales)的许多种类具有优异的诱病杀虫能力,尤其通过主动弹射孢子而在害虫种群中高强度流行的特征,可在短期内迅速压低虫口密度,是极其重要的杀虫微生物资源[1～3].     

Large Scale Production and Crystallization of Acid Carboxypeptidase from Submerged Culture of Penicillium janthinellum,and Stability of the Crystalline Enzyme

Acid carboxypeptidase of Penicillium janthinellum IFO–8070 was produced effectively in submerged culture on a medium of 4 ~ 5% rice bran. The enzyme production was enlarged to volume cultivation of 150-liters in a 200-liters jar fermentor, and the yield of acid carboxypeptidase per milliliter filtrate reached to the maximum 3 days after inoculation.

Acid carboxypeptidase of low molecular weight (M.W. = 51,000) produced in the liquid culture broth was purified and crystallized in a large scale. Purification steps include Amberlite CG–50 treatment, ammonium sulfate precipitation, dialysis using “Diaflow,” activated charcoal treatment, and condensation using collodion-bag, or condensation and dialysis using “Diaflow.”

The crystals of the acid carboxypeptidase suspended in 50 mm acetate buffer (pH 3.7) were completely stable for six months at 5°C. On the other hand, at low enzyme concentration (0.01 U/ml) in 50 mm acetate buffer (pH 3.7), crystallized enzyme was somewhat labile, whereas, this inactivation was completely depressed by covering enzyme solution with toluene.     

Production of Thermostable alpha-Amylase, Pullulanase, and alpha-Glucosidase in Continuous Culture by a New Clostridium Isolate

The production of alpha-amylase, pullulanase, and alpha-glucosidase and the formation of fermentation products by the newly isolated thermophilic Clostridium sp. strain EM1 were investigated in continuous culture with a defined medium and an incubation temperature of 60 degrees C. Enzyme production and excretion were greatly influenced by the dilution rate and the pH of the medium. The optimal values for the formation of starch-hydrolyzing enzymes were a pH of 5.9 and a dilution rate of 0.075 to 0.10 per h. Increase of the dilution rate from 0.1 to 0.3 per h caused a drastic drop in enzyme production. The ethanol concentration and optical density of the culture, however, remained almost constant. Growth limitation in the chemostat with 1% (wt/vol) starch was found optimal for enzyme production. Under these conditions 2,800 U of pullulanase per liter and 1,450 U of alpha-amylase per liter were produced; the amounts excreted were 70 and 55%, respectively.     

Production of trout,Salmo trutta,in a Danish stream

Synopsis The numbers of trout,Salmo trutta, in Granslev », Denmark, were estimated by the removal method on 18 dates from March 1974 to March 1976. Populations density varied from 0.39 to 0.74 trout m^–2 in 1974–1975 and from 0.36 to 0.59 m^–2 in 1975–1976 and at all times four or five year classes were present. The age structure of the population was unstable and the variable natural survival, immigration into and emigration from the study site could not be separated. An annual growth cycle with the most rapid growth for all year classes taking place from May to early August was found. Statistically significant differences between different years occurred in the growth of the 0,I and II age groups, but no evidence of density-dependent growth was found. The biomass ranged from 35.4 to 9.5 g m^–2. The total mean annual biomass was 22.8 and 14.7 g m^–2 in the two years and the II group made the greatest contribution, 44 and 48%, respectively. During 1975–1976 the mean annual biomass of each year class only was about two-thirds of that in 1974–1975. Annual production in the two years was 25.7 (range 24.7–28.5) and 12.6 g m^–2 (range 11.7–15.0) and the II group accounted for about 46 and 38% of the production. In addition eel,Anguilla anguilla, produced about 0.5 g m^–2 yr^–2. The unstable age structure of the trout population was compared with trout populations from other streams. The importance of immigration as a recruitment process in middle and lower reaches of streams and of migrations as a mechanism to optimise utilization of the total stream habitat, as well as temperature as a factor controlling the growth rate are discussed.     

渗透胁迫下水稻种子萌发特性及抗旱性鉴定指标研究

用聚乙二醇（PEG）-6000作为渗透剂模拟干旱胁迫,探讨在4种不同浓度的渗透胁迫下水稻种子的萌发特性,并确立间接评价水稻苗期抗旱性的萌发期鉴定指标。结果表明,高浓度渗透液对种子萌发的抑制作用明显大于低浓度,萌发率的干旱胁迫反应指数随渗透液浓度的降低和萌发进程而增加。在不同浓度的渗透胁迫处理中,20％浓度下萌发6d后的萌发率表现较大的变异幅度和变异系数以及较均衡的品种频率分布,较好地反映了水稻种质对渗透胁迫的不同反应,认为20％／6d条件较适合作为萌发率鉴定的渗透液模拟干旱胁迫;经反复干旱后的幼苗存活率与萌发胁迫指数和20％胁迫浓度下萌发8d后芽鞘长的干旱胁迫反应指数（DRI）表现极显著或显著正相关,认为萌发胁迫指数和芽鞘长的DRI可作为评价水稻苗期抗旱性的间接鉴定指标。