Hydroponic Seedling Bioassay for the Bioherbicides Colletotrichum truncatum and Alternaria cassiae

A rapid bioassay was developed to measure the bioherbicidal efficacy of spore preparations of the pathogens Colletotrichum truncatum (Schwein.) Andrus and W. D. Moore and Alternaria cassiae Jurair and Khan on hemp sesbania (Sesbania exaltata) and sicklepod (Cassia obtusifolia), respectively. The system uses 4-day-old dark-grown seedlings (grown hydroponically in paper towel cylinders) which were sprayed with spore suspensions. Shoot lengths were monitored non-destructively, and recorded over time under conditions of dark growth, 90-100% relative humidity and 25 C. Shoot growth inhibition and stem collapse (mortality) were directly related to the spore concentration applied. Generally, at 10 3 - 10 4 spores ml-1, these pathogens caused significant shoot growth inhibition within 25-30 h and seedling death within 40-50 h. This bioassay has been used to study herbicide-pathogen interactions, and may be extended to determine the bioherbicidal efficacy of different pathogen isolates, pathovars or spore formulations. This technique is more rapid, uses a lower inoculum volume, requires less space and is performed under more controlled conditions than conventional greenhouse bioassay methods. The data obtained are more quantitative than those obtained from bioassays relying on visual rating systems.     

Quantification of Infection by Alternaria cassiae Using Leaf Immuno-Autoradiography and Radioimmunosorbent Assays

The severity of fungal infection is usually estimated visually as a rating of injury. A more quantitative method was needed to ascertain the effect of additives used to enhance the virulence of mycoherbicidal preparations. A sensitive and accurate serological method is presented for quantitative measurement of Alternaria cassiae infection of Cassia obtusifolia. An antiserum was prepared against a homogenate of mycelium of A. cassiae. A fast and simple procedure of leaf immuno-autoradiography was developed for the visualization of A. cassiae mycelium on inoculated leaves. A radioimmunosorbent assay (RISA) was developed for quantification of the extent of fungal infection. As little as 1. 6 ng/ ml dry weight equivalent of mycelium could be detected by RISA. There was a linear relationship between the logarithm of the RISA values and the logarithm of fungus concentration up to 300 ng/ml. Reduced sensitivity of the assay was pronounced in extracts containing 400 μ g/ml or more fresh weight of leaves. There was 30–40 % cross-reactivity of the antiserum with two other species of Alternaria, as well as with Monilinia fructicola, and almost no reactivity was found with three other fungi tested.     

Inducing sporulation in Alternaria solani I. Effect of water treatment

The sporulation was induced when fully grown cultures were given dip or spray treatment with distilled water (cold or hot) and thereafter, kept partially covered at different temperatures. Cultures dipped in cold water (4° C) for 4 minutes or sprayed with cold water (4° C) or hot water (58° C) and thereafter incubated at room temperature (13–26° C) in diffused sunlight, produced maximum number of spores within 60 hours. Incubating water treated cultures in diffused sunlight or complete darkness and age and scraping of the cultures had a considerable effect upon intensity of sporulation. The cultures yield a number of subsequent crops of spores when scraped and given dip treatment with cold or hot water, after obtaining each crop of spores.     

Effect of carbon dioxide on the Oriental cockroach Blatta orientalis

Exposure to high concentrations of carbon dioxide (CO2) killed adult and nymphal stages of the oriental cockroach Blatta orientalis L. (Dictyoptera: Blattidae) with LT50 values of 11.5-16.2 h for 60% CO2 in air and 5.7-7.1 h for 100% CO2 at 20 degrees C; corresponding LT50s at 28 degrees C were 2.8-4.6 h for 60% CO2 in air and 2.3-3.6 h for 100% CO2. Complete kill of mobile stages was obtained within 24 h using 60% CO2 at 20 degrees C. Survivors of treatments with 100% CO2 at 28 degrees C remained completely paralysed for up to 3 days post-treatment and took up to 5 days to regain normal movement, but adult females then resumed production of oothecae with no significant loss in fecundity. Oothecae 5 or 30 days after deposition required 60-84 h exposure to 60% CO2 at 20 degrees C to prevent emergence of nymphs but less time using 100% CO2 at 28 degrees C. At 28 degrees C, when adult females were treated with 100% CO2 and 52% r.h. for 6 h (giving 100% mortality) loss of weight was significantly greater than that following treatment with air at 52% r.h. for 6 h (giving no mortality). However, significantly greater weight loss also occurred when they were treated with dried air (< 10% r.h.) for 6 h, also with no mortality. The toxicity of CO2 to mobile stages of the oriental cockroach appeared to result from irreversible effects on the nervous system, rather than from water loss during exposure.     

Control of Chenopodium album by soil application of Ascochyta caulina under greenhouse conditions

Effects of soil application of Ascochyta caulina spores on seedlings of Chenopodium album and five cultivated plant species were investigated under greenhouse conditions as a part of a study on biological control of C. album. Application of A. caulina spores to soils resulted in disease development on C. album and to a lesser degree on Spinacia oleracea seedlings, but not on Beta vulgaris subspecies vulgaris, Zea mays, Triticum aestivum and Pisum sativum seedlings. Affected C. album seedlings had an abnormal olive-green colour or necrotic spots on cotyledons and hypocotyls, and were stunted or died. Affected S. oleracea seedlings were pale in colour or had necrotic spots on the cotyledons, but did not die. Time courses of disease incidence and of mortality of C. album could be described by a monomolecular model. Effects of spore density, sowing depth, soil water content, soil type and time of sowing on disease development were examined. Disease incidence and mortality were influenced by spore density, soil water content and soil type, but not by sowing depth. Spores in a moist soil maintained infectivity at least 2 wk. Spore densities of 10⁹ to 10¹⁰ spores m^-2 were required for 50% mortality of emerged C. album plants. Aspects of the development of A. caulina into a soil-applied mycoherbicide are discussed.     

Effect of carbon dioxide on photorespiration

The isotopic CO₂ technique for measuring photorespiration was shown to be a valid technique for measuring the unidirectional inward and outward fluxes of CO₂ from a sunflower (Helianthus annuus L.) leaf in the light. The rate of photorespiration was decreased little as the CO₂ concentration was increased from 20 to 1,150 microliters per liter. This finding contradicts the widely held assumption that photorespiration is suppressed at high CO₂ concentrations. Some discussion regarding this apparent conflict is presented.     

Effect of plant age and turfgrass competition on the efficacy of the Sclerotinia minor granular bioherbicide on broadleaf plantain and prostrate knotweed

Broadleaf plantain and prostrate knotweed are important weeds of turfgrass systems. The fungus Sclerotinia minor Jagger (IMI 344141) has been registered as a biological herbicide (Sarritor?) for dandelion (Taraxacum officinale) in Canadian turfgrass habitats. The objective of this study was to evaluate the effect of plant age and turfgrass environment on the efficacy of S. minor against two additional weeds; broadleaf plantain and prostrate knotweed. The turfgrass environment alone exerted significant reduction of above and below ground biomass of broadleaf plantain, to the same magnitude as the S. minor treatment in a grass-free environment. Prostrate knotweed biomass, however, was not reduced to this extent by competition with turfgrass. In the presence of grass, S. minor caused a significant biocontrol effect on all studied variables with more than 90% above ground damage on both weed species. Severe damage occurred on 3–6-week-old plantains with 100% above and below ground reduction, although smaller dry weight reductions were observed on older plantains. Treatment with S. minor reduced the dry matter of 3–5-week-old prostrate knotweed by 65–85%, but less damage occurred on older prostrate knotweed. The bioherbicide fungus is destructive for both species, but variation in area of contact due to different growth forms, growth rates and resource allocation patterns due to different life forms resulted in different biocontrol efficacy on the two species. Control of broadleaf plantain was effective – similar to that previously reported for dandelion – but control of prostrate knotweed was only partial.     

Inactivation of Alternaria brassicicola spores by supercritical carbon dioxide with ethanol entrainer

Supercritical carbon dioxide (SC-CO₂) was used to inactivate fungal spores of Alternaria brassicicola. The inactivation conditions were optimized using response surface methodology (RSM). When the SC-CO₂-entrainer (ethanol) system was applied to fungal spores, the treatment time required for the complete inactivation of fungal spores was substantially reduced.     

Chondrostereum purpureum and Fusarium tumidum independently reduce regrowth in gorse (Ulex europaeus)

An experiment was conducted in two gorse populations (Ulex europaeus) in which Chondrostereum purpureum was applied each month as mycelial-agar cultures to the wounds of decapitated stems of previously untreated gorse plants to determine its potential as a mycoherbicide. Summer-autumn (Feb-May) or late winter-early spring (Aug-Sept) applications were effective in both populations, halving stem stump survival (from an average of 56 to 29%). Another experiment in the same gorse populations evaluated the combined effects of C. purpureum applied in May to decapitated stems, and Fusarium tumidum applied as spores in an invert emulsion to regenerative shoots 5-6 months later. There was no evidence of synergism between the two fungi; each pathogen independently reduced the density of regenerative shoots on the decapitated stems by 39-63% averaged over the 12 months following their respective applications. It is concluded that both pathogens have potential as mycoherbicides for gorse regenerating after stem decapitation.     

Screening of phytotoxicity of Alternaria macrospora MKP1 against Parthenium hysterophorus L.

Parthenium hysterophorus L. an exotic, pernicious weed is considered as one of the most troublesome weeds for agricultural sector by virtue of its high ecological amplitude and adaptability. Microbes and their by-products are now proved to be a worthy alternative to toxic chemicals used for weed management. Alternaria macrospora MKPI was isolated from the parthenium leaves infected with leaf blight and found pathogenic to the weed. The herbicidal potential of cell free culture filtrate of A. macrospora MKP1 has been tested against parthenium by employing detached leaf bioassay and seed germination bioassay and a significant damage was exhibited by the cultural filtrate of pathogen to the parthenium leaves and seeds.     

Effect of carbon dioxide on nitrification rates

Lab-scale ideal mixed, aerated reactors were employed to test the influence of carbon dioxide (CO(2)) on the growth rate of a nitrifier community. The buffer medium used did not contain any carbon sources. Reactors were inoculated alternatively with sludge from a nitrifying membrane assisted bioreactor, reflecting autotrophic material, or with sludge from a plant having denitrification and nitrification steps, which reflects mixed heterotrophic and autotrophic material. CO(2) was added as a gas with the intake air supply. Nitrification rates were related to the CO(2) in the intake air as well as to the total inorganic carbon in the medium. The batch experiments show a relationship between CO(2) concentration and growth rate. The optimum growth rate occurred at 5 mg CO(2)/L, corresponding to 0.4% (V/V) CO(2) in the inlet air. Different CO(2) optima for autotrophic and mixed sludges were found. In the case of the autotrophic sludge, the observed optimum growth rate was about 0.47/d and the optimum for the mixed sludge was about 0.75/d. Higher CO(2) concentrations lead to a decreasing growth rate. The first part of the kinetic graph can be described by Monod kinetics. Overall, the resulting graph can be described by Haldane kinetics.     

The infection process, sporulation and survival of Alternaria helianthi on sunflower

Electron microscopy was used to study the infection of sunflower leaves by Alternaria helianthi. Conidia germinated by producing one to many germ tubes which grew across the leaf surface before forming appressoria. The fungus directly penetrated its host through the cuticle and epidermis. Entry into the host through wounds and stomates was also observed. Extracellular sheaths were found to be associated with germ tubes and intercellular hyphae of A. helianthi. Conidiophores developed through collapsed stomates, from leaf veins, trichomes and also from mycelium growing across the host leaf surface. Microcylic conidia were produced directly from parent conidia under certain conditions. Studies using a volumetric spore trap showed that the airborne spore concentration followed a distinct periodicity with peaks occurring between 0900 and 1100 h each day. Laboratory studies showed that safflower, noogoora burr and bathurst burr could serve as alternative hosts for A. helianthi. The pathogen was readily isolated from sunflower crop debris from a diseased crop that had been harvested 1 yr earlier.     

Effect of carbon dioxide on yeast growth and fermentation

Inhibition of yeast function by ethanol and by high substrate concentrations is well recognized and, to a limited extent, quantified. The role of carbon dioxide in affecting yeast metabolism (particularly growth processes) is not clear although inhibition is generally found at moderate to high concentrations of the dissolved gas. A similar situation exists with other microorganisms and with other fermentation systems. An understanding of the role of carbon dioxide, and particularly of its inhibitory effects on enzyme action and membrane function, is required if the observed global inhibition of yeasts and other fermentation systems is to be partitioned to its appropriate causes.     

A retrospective analysis of known and potential risks associated with exotic toadflax-feeding insects

To date, eight exotic toadflax-feeding insect species have been accidentally or intentionally introduced to North America. Reports on their establishment and impact have been recorded for more than 60 years. Environmental risks linked to biological control of toadflax were identified in terms of host resources and undesirable impacts on the target species through the critical review of this record. Data gaps revealed during this retrospective analysis are addressed through suggestions for future research and associated experimental methodologies. Known and potential impacts of toadflax-feeding insects on both invasive toadflax and non-target species are examined. Recent programmatic demands for demonstrated agent efficacy and stringent host selectivity during the prerelease screening process clearly illustrate that classical biological control of invasive toadflax in North America is progressing beyond the so-called lottery approach.