In vivo propagation of a microsporidan pathogenic to insects

Equivalent numbers of spores were produced when the microsporidan Nosema necatrix was propagated in either Trichoplusia ni or Heliothis zea. Maximum spore production was obtained at an inoculum level of 1 × 10⁵ spores/ml. Larvae inoculated 5 days post-hatching contained 1.6 × 10⁹ spores/gram larva after an incubation period of 21 days. Temperature optima for the parasite are 21–26°C in both hosts.     

Virulence of entomopathogenic Fungi (Fungi imperfecti) for the adults of Acanthoscelides obtectus (Coleoptera: Bruchidae)

Tests with the bean weevil, Acanthoscelides obtectus, in which the hosts were exposed indirectly to various dilutions of conidia of four entomopathogenic fungi showed that mortality was a function of the concentration of the inoculum. In these tests a given spore suspension was sprayed on the internal surfaces of a Petri dish. Adult weevils of a known age were placed in the dish, held there for 24 hr, then removed and kept at 20°C. After 20 days, the host mortality was determined. From the data obtained, it was possible to trace a probit regression line of the mortality in relation to the increasing spore concentration. Infection was observed in hosts exposed to a concentration of approximately 5 × 10⁶ spores/ml up to a maximum of about 1 × 10⁹ spores/ml. The A. obtectus was susceptible to infection by spores of Beauveria bassiana, B. tenella, Metarrhizium anisopliae, and Paecilomyces fumoso-roseus.     

Laboratory studies on the fungus Verticillium lecanii, a larval pathogen of the large elm bark beetle (Scolytus scolytus)

From 1972 to 1974, estimates of the natural larval mortality (> second instar) of elm bark beetles caused by pathogenic organisms were always below 7'5 % of the beetle population. The pathogenic fungus Verticillium lecanii was frequently isolated from field-collected dead larvae, and in the laboratory all larvae were killed in 5 days when exposed to spore concentrations of 4·5 × 10⁶ spores/ml. V. lecanii begins to lose its pathogenicity after prolonged culture on artificial media. The time taken for V. lecanii to kill Scolytus scolytus larvae when exposed to a logarithmic series of spore dilutions from 9·1 × 10⁷/ml to 9·1 × 10³/ml increased with decreasing amounts of inoculum. Even at spore concentrations as low as 9·1 × 10³/ml the mortality of treated larvae was greater than that of untreated individuals. At 100% r.h. all treated larvae were killed over a temperature range of 5–30 °C; those maintained at 25 °C were killed most rapidly and those kept at 5 °C the slowest.     

A greenhouse assay to screen sunflower for resistance to Alternaria helianthi

A greenhouse assay to screen sunflower for resistance to Alternaria helianthi is described. A comparison of conditions led to the following standard conditions being recommended. The first or second pair of leaves of seedling plants at the V8 growth stage are inoculated using inoculum grown on sunflower leaf extract agar for 5–10 days at an inoculum density of 1–2 spores cm² of leaf tissue. A 48 h dew period should be applied to plants covered by a plastic tent. A dew period temperature of 26/26°C night/day and a post-dew period temperature relative to that experienced under local growing conditions should be applied. Lesions are measured 7 days after inoculation, and mean lesion size per plant is calculated. Mean lesion size of lines being tested is expressed as a proportion of the mean lesion size of a susceptible standard included in each screening experiment.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.     

A rapid method to estimate the resistance of cold stored carrot roots to Botrytis cinerea

Inoculating whole carrot roots at 4°C with mycelial/agar discs of the grey mould fungus Botrytis cinerea gives a measure of their resistance and hence storage potential to this pathogen, but results are not obtained for at least 33 days. In the present investigation a more rapid method was used which involved inoculating root slices with spore suspensions containing 5 × 10^3–5 × 10⁶ spores/cm³ at 24°C. Resistance was assessed visually and by a chitin estimation 48 h after inoculation. Both methods were used to measure the resistance of cold stored carrot roots during two storage seasons, 1976/77 and 1977/78. In each season there was a particular inoculum level which most clearly recorded the increasing susceptibility of roots with time in store. In 1976/77 this was 1 × 10⁵ spores/cm³ whereas in 1977/78 it was the lower inoculum concentration of 5 × 10⁴ spores/cm³, suggesting the roots were generally more susceptible in 1977/78 than the previous season. This was in accord with the results from the whole root method of assessment. A chitin estimation proved to be the more sensitive method of assessment for inoculum potential experiments.     

Effect of plant stage, <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> dose,and use of herbicide on control of <Emphasis Type="Italic">Matricaria perforata</Emphasis>

Colletotrichum truncatum (Ct) was examined in a tank mix with the herbicide 2,4-D, clopyralid plus MCPA (Caurtail M^®), or metribuzin (Sencor^®) for control of scentless chamomile at 8- (younger) and 11-leaf stages (older) under controlled conditions. In initial trials, Ct at 7 × 10⁶ spores/ml (200 l/ha) reduced the fresh weight of scentless chamomile only slightly. However, its combinations with herbicides improved the efficacy variably depending on the herbicide used and stage of the weeds. Ct plus 2,4-D reduced the fresh weight by about 50% at both leaf stages of scentless chamomile when compared to untreated controls but no plants were killed. The fungus plus Curtail M consistently killed younger but not older plants, and the efficacy was substantially greater than that of the herbicide alone. The herbicide Sencor was highly effective on younger plants, and adding Ct did not achieve additional benefits. On older plants, however, Ct plus Sencor was substantially more effective than the herbicide alone, causing 76% fresh-weight reduction when compared to controls and killing 9 out of 16 older plants in four trials. Sencor applied alone reduced the fresh weight of older plants by 65%, but no plants were killed. Tested at doses ranging from 2 × 10⁶ to 20 × 10⁶ spores/ml, Ct plus Curtail M was most effective at the highest fungal inoculum dose, consistently killing younger but not older plants. In comparison, Ct at a medium dose (7 × 10⁶ spores/ml) plus Sencor killed the majority of older chamomile plants (7 out of 12), whereas the herbicide alone did not cause plant mortality. Further increasing fungal inoculum dose from this medium level did not enhance the weed control by Ct plus Sencor.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

Mycelium of Alternaria alternata as a potential biological control agent for Eupatorium adenophorum

The fungus, Alternaria alternata (Fr.) Keissler Strain 501, has been evaluated as a bioherbicide for control of Eupatorium adenophorum Spreng., but the biology of the pathogen–host interaction and the optimal environmental conditions for disease development and effective weed control are unknown. Disease development of A. alternata Strain 501 mycelia on E. adenophorum was assessed under several factors including pathogen inoculum concentration, plant age, dew period duration, post-dew temperature, storage temperature and duration. The minimum inoculum concentration required to kill E. adenophorum seedlings was 3.2×10⁶ mycelial fragment mL^?1. E. adenophorum seedlings at the four-leaf-pair stage were more susceptible than the older plants, especially those at the older than seven-leaf-pair stage. With a dew period of at least 14 h, 100% mortality occurred. The optimal post-dew temperature for disease development was 18–25°C. Storage at <4°C maintained the infectivity of A. alternata strain 501 mycelia on E. adenophorum longer. Using light and scanning electron microscopy to examine the infection process of A. alternata Strain 501 mycelia, it was shown that the time from initiation to completion of infection with mycelia was much shorter (14 h) than with conidia (72 h). It was further shown that mycelial infection occurred predominately through direct penetration at intercellular junctions, while conidial infection occurred predominately through stomatal penetration. This suggests that mycelia are more suitable as infection propagules for A. alternata strain 501 in a bioherbicide for the control of E. adenophorum.     

Effects of Leaf Age, Inoculum Dose and Freezing on Development of Chocolate Spot (Botrytis fabae) Lesions on Field Bean (Vicia faba) Leaves

Botrytis fabae spore suspensions containing c. 1, 10, 10², 10³, 10⁴, 10⁵, or 10⁶ spores/ml were used to inoculate 5, 17 or 30-day-old field bean leaves. The percentages of the leaf areas covered by, chocolate spot lesions and the percentages of the leaf areas bearing conidiophores were assessed 1, 6, 12, 14, and 19 days after inoculation. The percentage of the area covered by lesions and the percentage of the area bearing conidiophores (logit-transformed) increased linearly with increasing spore concentration (log₁₀-transformed). The proportions of leaf areas covered by lesions and bearing conidiophores were both greater on 17 and 30-day-old leaves than on 5-day-old leaves. The rate of lesion growth increased with both increasing inoculum dose and increasing leaf age. Generally there was no interaction between the effects of leaf age and the effects of inoculum dose on either lesion growth or sporulation. Two days after inoculation with suspensions of either 10⁴ or 10⁶ spores/ml, 7-day-old leaves grown at 15°C were transferred to –16°C or 2.5°C or kept at 15°C for 4 days. Two days later more spores had been produced on leaves which had been frozen (–16°C) than on, leaves kept at 2.5°C.     

Aflatoxin B1, zearalenone and deoxynivalenol production by Aspergillus parasiticus and Fusarium graminearum in interactive cultures on irradiated corn kernels

The influence of inoculum size in the production of aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) was determined when Aspergillus parasiticus NRRL 3000 and Fusarium graminearum ITEM 124 were cultured alone and in pairs on irradiated corn kernels at 28 °C and 0.97 water activity (aw). The highest levels of AFB1 produced by A. parasiticus were produced at the lowest levels of the inoculum (103 spores/ml). No significant differences were observed in ZEN and DON production at any inoculum level during the experimental period. When A. parasiticus was co-inoculated with F. graminearum both to the same inocula (106 spores/ml), AFB1 inhibition percentage were 60, 72 and 56% at 10, 20 and 35 days of incubation respectively, while at 106 spores/ml the percentages of inhibition were 34, 84 and 93% at 10, 20 and 35 days. In the mixture cultures A. parasiticus 103 × F. graminearum 106 spores/ml the percentage of inhibition of AFB1 oscillated in 99% during all the incubation. In the interaction A. parasiticus 106 spores/ml × F. graminearum 103 spores/ml the accumulation of AFB1 decreased in 80, 94 and 86% at 10, 20 and 35 days of incubation respectively. In single culture F. graminearum was inoculated with 10³ or 106 spores/ml and the highest levels of ZEN and DON were detected at 35 days of incubation. The levels oscillated in 538–622 μg/kg for ZEN and 870–834 μg/kg for DON respectively. In paired cultures there were no significant differences in the levels regardless of the spore concentrations during the incubation time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Increased alkalotolerant and thermostable ribonuclease (RNase) production from alkaliphilic Streptomyces sp. M49-1 by optimizing the growth conditions using response surface methodology

Total of 171 alkaliphilic actinomycetes were evaluated for extracellular RNase production and Streptomyces sp. M49-1 was selected for further experiments. Fermentation optimization for RNase production was implemented in two steps using response surface methodology with central composite design. In the first step, the effect of independent fermentation variables including temperature, initial pH and process time were investigated. After identification of carbon and nitrogen sources affecting the production by one variable at a time method, concentrations of glucose and yeast extract and also inoculum size were chosen for the second central composite design. A maximum RNase activity was obtained under optimal conditions of 4.14 % glucose concentration, 4.63 % yeast extract concentration, 6.7 × 10⁶ spores as inoculum size for 50 ml medium, 42.9 °C, 91.2 h process time and medium initial pH 9.0. Optimum activity of the enzyme is achieved at pH 11 and temperature 60 °C. The enzyme is highly stable at pH range 9.0–12.0 and at 90 °C after 2 h. Statistical optimization experiments provide 2.25 fold increases in the activity of alkalotolerant and thermostable RNase and shortened the fermentation time compared to that of unoptimized condition. The members of Streptomyces can be promising qualified RNase producer for pharmaceutical industries.     

Bioherbicidal potential of Xanthomonas campestris for controlling Conyza canadensis

The effects of environmental parameters on bioherbicidal activity of the bacterium Xanthomonas campestris, against glyphosate-resistant and – susceptible Conyza canadensis (horseweed), were studied under greenhouse conditions. Rosette leaf-stage plants were more susceptible than older plants, and increasing inoculum from 10⁵ to 10⁹ cells/mL caused significantly greater plant mortality and biomass reduction of plants in both the rosette and bolting growth stages. A dew period at 25°C was required to cause an 80% and 60% mortality of plants in the rosette and bolting growth stages, respectively. Results indicate that X. campestris can infect and kill horseweed, demonstrating its bioherbicidal potential.     

The susceptibility ofTribolium castaneum [Col.: Tenchrionidae] toFarinocystis tribolii [Protozoa: Schizogregarinida]

The LT₅₀ ofFarinocystis tribolii Weiser to larvae ofTribolium castaneum (Herbst) increased with the age of the insect indicating that older larvae were relatively more tolerant to the infection though there was 100 % mortality ultimately. The adults were less susceptible than larvae and between sexes, females were more susceptible than males. The number of spores produced increased with the stage of the larvae, but there was no variation in the size of spores in the different instars. The LC₅₀ on 20th and 40th day of inoculation were 1.4×10⁷ and 2.1×10⁶ respectively. Mortality-time due toF. tribolii was shorter at 35 °C than at 25 °C. Sporulation occurred earlier at 35 °C than at 25 °C.     

Evaluation of aflatoxin B1 and ochratoxin A in interacting mixed cultures of Aspergillus sections Flavi and Nigri on peanut grains

The influence of inoculum size on the colony-forming units, production of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was determined when Aspergillus flavus and A. niger aggregate strains were cultured alone and in pairs on irradiated peanut grains at 28°C and 0.97 water activity (a_W). The results showed a marked influence of inoculum factor on fungal counts, AFB₁ and OTA production in single and paired cultures. Fungal counts of the A. niger aggregate strain in interacting cultures at 7, 14 and 21?days of incubation were significantly higher than those observed in the A. flavus strain, except in the mixed culture with 10² spores/ml of both strains. In all mixed culture assays, the AFB₁ production was significantly reduced in comparison with the accumulation of mycotoxin in single cultures. A total inhibition in AFB₁ production was observed in some interactions as 10² spores/ml of A. flavus and 10³ spores/ml of A. niger aggregate strain at 7 and 14?days, among others. With regard to OTA production, a stimulation in the interacting cultures was observed at all inoculum sizes and incubation period. The highest levels of OTA accumulation were observed at 14?days for all interacting cultures. The maximum level was reach in the culture 10³ spores/ml of A. niger aggregate and 10⁴ spores/ml of A. flavus (p?<?0.001). These results suggest that, under optimal environmental conditions in peanut grains, the interaction between A. flavus and A. niger aggregate strains could result in an inhibition of AFB₁ and in a stimulation of OTA production.     

Host and environmental effects on the infection of maize by Puccinia sorghi. II. Post-penetration development

The numbers of pustules which subsequently developed on maize seedlings incubated at 5, 10, 15, and 20 °C and 100% r.h. were directly related to the temperature and length of incubation. The generation time was 16 days at 10 °C, 10 days at 15 °C, 7 days at 20 °C and 5 days at 25 °C. Increase in urediniospore production was observed with increase in temperature. Increase in the inoculum concentration up to 5 × 10⁴ urediniospores/ml increased the number of resultant uredinia. However, further increases did not increase uredinia-formation.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Patterns of primary spore discharge of Entomophthora spp from the blue green aphid,Acyrthosiphon kondoi

Experiments were conducted to study the effects of time, temperature, and light regime on primary spore formation at 100% RH for the three major pathogens of Acyrothosiphon kondoi. Only small differences were detected between the continuous light and continuous dark regimes. Entomophthora obscura produced between 6 and 10 × 10³ primary spores mostly during the first 48 hr. Total primary spore production was similar at the five temperatures tested from 5° to 25°C. Entomophthora planchoniana produced large numbers of primary spores (about 5 × 10⁴ per aphid) only at temperatures between 10° and 20°C. The majority of primary spores were formed during the first 24 hr. Primary spore production with Entomophthora nr. exitialis ranged from about 10⁵ per aphid at 5° and 10°C to 3 or 4 × 10⁵ at 15° to 25°C, with most spores being formed during the first 48 hr. It is suggested that rainfall is more likely to be important for transmission of E. obscura and E. nr. exitialis than for transmission of E. planchoniana, and that E. obscura is likely to be the most important pathogen during cool or cold weather.     

Trypanosoma acomys (Wenyon, 1909): Continuous Culturing with a Mouse Fibroblast Cell-Line (A9)

The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37° C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 × 10⁴, 1.5 × 10⁵ and 7.4 × 10⁵ parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 × 10⁶ parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.     

The use of a microinjection procedure for large-scale production of the microsporidian Nosema eurytremae in Pieris brassicae

Nosema eurytremae, a microsporidian parasite of Malaysian trematodes, was injected at the rate of 1 × 10⁴ spores/larva into Pieris brassicae. The larvae, which subsequently pupated, were incubated at 25 to 26°C and on harvesting 19 days later yielded an average of 6 × 10⁸ spores/pupa. This was equivalent to 60,000 times the initial dose. Purity of filtered, washed spore suspensions ranged from 80 to 99% with up to 20% host debris.