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.     

Hemp sesbania (Sesbania exaltata) control in rice (Oryza sativa) with the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene formulated in an invert emulsion

In greenhouse and field experiments, an invert emulsion (MSG 8.25) was tested with dried, formulated spores of the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene, a highly virulent pathogen of the leguminous weed Aeschynomene virginica (northern jointvetch), but considered ‘immune’ against another more serious leguminous weed, Sesbania exaltata (hemp sesbania). A 1:1 (v/v) fungus/invert emulsion mixture resulted in 100% infection and mortality of inoculated hemp sesbania seedlings over a 21-day period under greenhouse conditions. In replicated field tests of the fungus/invert formulation conducted in Stuttgart, AR, and Stoneville, MS, hemp sesbania was controlled 85 and 90%, respectively. These results suggest that this invert emulsion expands the host range of C. gloeosporioides f. sp. aeschynomene, with a concomitant improvement of the bioherbicidal potential of this pathogen.     

Water Activity and Other Factors that Affect the Viability of Colletotrichum truncatum Conidia in Wheat Flour-Kaolin Granules ('Pesta')

Optimization of shelf-life is critically important for biocontrol products containing living microorganisms. Conidia of Colletotrichum truncatum, a fungal pathogen of the weed, hemp sesbania (Sesbania exaltata), were produced in shake flasks (corn meal-soya flour medium) and on Emerson Yp Ss agar and formulated in wheat flour-kaolin granules ('Pesta'). The granules were conditioned at water activities of 0, 0.12, 0.33, 0.53 and 0.75 during storage at 25 C over desiccant or saturated salt solutions. The longest shelf-life (conidial inoculum viability) was found in samples in the water activity range 0-0.33, where the water was bound by the matrix and not readily available to the fungus. At a water activity of 0.12, granules were 100% viable (on water agar) for at least 24 weeks, and were 87% viable after 1 year. Sucrose (5% w/w) partially counteracted the detrimental effect of high water activity on the shelf-life of C. truncatum when incorporated in the granules.     

Survival of Rhizoctonia solani AG‐1 IA,the Causal Agent of Rice Sheath Blight,under Different Environmental Conditions

The ability of Rhizoctonia solani AG‐1 IA, the causal agent of rice sheath blight, to survive in diseased rice straw and as sclerotia and mycelia was investigated. After storage for 10 months at 4°C, 25°C and non‐air‐conditioned natural room temperature (NRT, temperature range from 6°C to 35°C), sclerotia placed inside a desiccator, soaked in sterile water or immersed in wet paddy soil were viable. In contrast, only 15% of sclerotia in dry paddy soil survived. Survival of mycelia was severely affected by temperature and humidity. After 10 months in a desiccator at 4°C, 55% of mycelia samples could survive, whereas at 25°C and NRT, mycelial samples survived for only 7 and 5 months, respectively. However, mycelia stored in sterile water at constant temperatures (4°C or 25°C) survived for 10 months. A certain amount of UV radiation had no obvious effect on the survival of sclerotia or mycelia. The survival rate of the fungus in diseased rice straw stored for 16 months could reach 100% at 4°C, 50% at 25°C and 35% at NRT. The survival rates of the pathogen in diseased rice straw buried in dry, wet and flooded paddy soils after 10‐month storage at NRT were 75, 100 and 100%, respectively, indicating that soil humidity is a crucial factor for the survival of this fungus.     

Myrothecium verrucaria isolates and formulations as bioherbicide agents for kudzu

The fungus Myrothecium verrucaria (MV) has previously been shown to have potential as a bioherbicide for kudzu (Pueraria lobata) control. It has also been shown that MV wild-type (MV-wt) often forms sectors, when grown on various nutrient media. Experiments compared MV-wt and MV sector efficacy when grown on agar or on rice grains. In greenhouse evaluations of sectors, applied as foliar sprays in water or in other formulations (corn oil, surfactant, and corn oil plus surfactant) for efficacy against kudzu seedlings, some sectors possessed bioherbicidal activity equal that of MV-wt, but others exhibited lower activity. Without a dew period, aqueous formulations of MV-wt, a yellow sector, and a white sector provided zero control, but all three isolates were active without a dew period when formulated in corn oil, Silwet L-77 surfactant, and in surfactant plus corn oil. Generally, the yellow sector was less effective than the other two isolates in any formulations, and the MV-wt and white sector provided approximately 100% mortality of the test plants. Dew (10 h) increased weed control to 100, 33, and 65%, respectively, for MV-wt, the yellow sector and the white sector. All isolates provided nearly 100% control in the oil and surfactant formulations with a dew period compared to treatments receiving no dew. Soil incorporation studies were also performed to compare MV-wt efficacy of preparations grown on agar versus growth on rice grains. Higher efficacies (1.75-3.3-fold increase) were obtained from rice grain preparations compared to preparations grown on agar, when preparations were incorporated at several rates into soil prior to planting. Cell-free extracts of the MV-rice cultures were also phytotoxic to kudzu seedlings up to the eight- to 10-leaf growth stage. Thus, formulation, growth media, and the application method are important determinants in the efficacy of MV and MV sectors on kudzu seedlings.     

Stability of microsclerotial inoculum of Colletotrichum truncatum encapsulated in wheat flour–kaolin granules

Maintaining adequate viability of microorganisms in products for biocontrol is critically important for commercial reasons. Microsclerotia (MS) of the mycoherbicide agent, Colletotrichum truncatum, are its hardy, over-wintering fungal structures. Microsclerotial inoculum at 2, 7, and 23 MS/granule were matrix-encapsulated in wheat flour–kaolin granules (Pesta), in which the flour provided gluten for the matrix and a food base for the fungus. Pesta granules were dried to a water activity of 0.18–0.29. After storage for 52 weeks at 25°C, granules containing 7 and 23 MS were 100% viable and granules with 2 MS were 95% viable. Granules with 7 MS were 50% viable after 36 weeks at 35°C. Pesta granules (440 granules/g) with conidial inoculum at 3.3×105 c.f.u./g were less storage-stable than granules at the 2 MS/granule level. At all MS inoculum levels, granules stored for up to 2years produced 108 c.f.u./g in vitro when incubated on water agar. High water activity was detrimental to long-term viability. In the greenhouse, 7 MS/granule samples controlled 94% of hemp sesbania when incorporated into the soil pre-planting. The strategy of encapsulation of the naturally stable C. truncatum MS and drying to a favourable water activity led to excellent shelf-life for a live biocontrol agent.     

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.     

Enhancement of disease caused by Colletotrichum truncatum in Sesbania exaltata by coinoculating with epiphytic bacteria

Colletotrichum truncatum isolate NRRL 13737 (ARS patent culture No. 18434) is being evaluated for development as a mycoherbicide against the problematic weed Sesbania exaltata. Studies were conducted to determine whether selected phylloplane microorganisms, used as coinoculants, could increase the severity of disease incited by C. truncatum in S. exaltata. Hemp Sesbania seedlings were grown in a variety of soils and environments, sprayed with conidia of C. truncatum, and the formation of appressoria was examined on leaves using epifluorescence microscopy. From hemp sesbania plants that supported high levels of appressoria formation, over 200 phyllosphere microorganisms were isolated. Fifteen of 73 microbial isolates assayed stimulated appressoria formation in vitro on cellophane membranes. Five of 8 superior isolates from the in vitro assay also enhanced disease symptoms induced by C. truncatum on S. exaltata compared to seedlings treated with conidia only. Populations of three selected superior isolates remained high on leaves during plant exposure to dew. The superior isolates initiated no apparent symptoms and rarely decreased seedling growth parameters in the absence of C. truncatum. This is the first instance of utilizing phylloplane microorganisms to increase the level of disease incited by a mycoherbicide agent. Microbial facilitators may provide a means of improving the weed control efficacy of mycoherbicides.     

<Emphasis Type="Italic">Sesbania exaltata</Emphasis> biocontrol with <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> microsclerotia formulated in ‘Pesta’ granules

The weed Sesbania exaltata (Raf.) Rydb. ex A.W. Hill (hemp sesbania) was effectively controlled in soybean [Glycine max (L.) Merr.] field test plots over a 2-year testing period (1995–1996) with microsclerotia of the bioherbicidal fungus, Colletotrichum truncatum, formulated in wheat gluten-kaolin granules called ‘Pesta’. Weed control averaged 84% and 88%, respectively, in plots treated pre-plant incorporated (PPI) at ‘Pesta’ rates of 168 or 336 kg ha⁻¹, and 71% and 78%, respectively, in plots treated pre-emergence (PE) at ‘Pesta’ rates of 168 or 336 kg ha⁻¹ over the testing period. Post-emergence (POE) control averaged 30% and 50%, respectively, for the 168 and 336 kg ha⁻¹ treatments, and was significantly less effective than either PE or PPI treatments. Although pathogenesis and mortality occurred in hemp sesbania tissues, satisfactory weed control was not achieved in plots treated at rates of 17 or 84 kg ha⁻¹ with any of the application methods. Soybean yields were significantly greater in test plots treated PPI or PE, as compared to yields from test plots treated either POE, with inert ‘Pesta’ granules, or from untreated controls. Microsclerotia formulated in ‘Pesta’ granules exhibited excellent shelf-life, retaining high viability after storage for 10 years at 4 °C. These results suggest that microsclerotia of C. truncatum formulated in ‘Pesta’ granules offers an effective method for controlling this important weed and preserving the activity of this bioherbicide.     

Biological control of sheath-blight of rice in india with antagonistic bacteria

Strains of fluorescent and nonfluorescent bacteria that were isolated from rice rhizospheres of Southern India and showed antagonism towardsRhizoctonia solani were evaluated for biological control of rice sheath-blight (ShB). Efficient strains of bacteria inhibited mycelial growth ofR. solani, affected sclerotial viabilityin vitro and protected IR 20 and TKM 9 rice seedlings from infection byR. solani in greenhouse tests. Pretreatment of sclerotia in bacterial suspensions resulted in reductions in ShB lesion sizes up to 31 to 44% in IR20 and 58 to 74% in TKM 9 rice. In field plots, IR 50 and TKM 9 rice plants raised from bacterized seeds had 65 to 72% less ShB than those plants from untreated seeds.     

Reproduction of Heterodera zeae and Its Suppression of Corn Plant Growth as Affected by Temperature

Reproduction of the corn cyst nematode (Heterodera zeae) and its effect on growth of corn (Zea mays) was studied in plant growth chambers at 24, 27, 30, 33, and 36 C. Reproduction of H. zeae increased directly with increase in temperature from 24 to 36 C. Fifteen-cm-d pots of corn seedlings inoculated with a mixture of 5,000 eggs + J2 and maintained for 8 weeks in growth chambers contained an average of 7,042 cysts + females at 36 C, but only 350 cysts + females at 24 C. Fresh weights of plants without nematodes were highest at 27 C and lowest at 36 C. Nematodes suppressed plant fresh weight by an average of 30% at 27 C and by 27% at 33 C but did not suppress plant weight at 36 C. Heterodera zeae has the highest reported temperature optimum for reproduction of any cyst nematode.     

Factors affecting germination, mycoparasitism, and survival of Sporidesmium sclerotivorum.

Macroconidia of Sporidesmium sclerotivorum, a mycoparasite of Sclerotinia spp., germinated after 3 days in soil adjacent to sclerotia of S. minor and on membrane filters placed on soil containing sclerotia. Germination increased with time up to 18 days and with concentration of sclerotia. Conidia as distant as 9 mm from single sclerotia germinated. Germination of conidia was maximum on a sclerotial agar medium in the range of pH 5 to pH 7. Cultivation of S. sclerotivorum parasitically on living sclerotia proceeded optimally in moist, fine quartz sand amended with 1 to 2% (w/w) sclerotia and 0.07% (w/w) CaCO3, at 25 degrees C. Infection of sclerotia in sand reached 100% by 5 weeks. Conidia production paralled infection resulting in logarithmic increase in numbers; a maximum of 3 x 10(5) to 4 x 10(5) conidia/g was reached in 6 to 12 weeks. Viability of air-dried sand-sclerotial cultures of S. sclerotivorum was reduced after 1 and 6 days, but viability was undiminished in air-dried soil. Sporidesmium sclerotivorum survived in moist and air-dried soils stored at room temperature for 15 months.     

Factors Affecting Biological Control of Sclerotinia sclerotiorum by Fungal Antagonists

Studies were conducted to determine the effects of soil moisture (9, 16 or 24% w/w) and temperature (5, 15, 20 or 25°C) on the control of sclerotia of Sclerotinia sclerotiorum by five fungal agents in sterile and natural field soil. All five biocontrol agents were effective in reducing the survival of sclerotia of S. sclerotiorum in sterile soil under dry (9% moisture) or wet (24% moisture) conditions at 20°C, but only Coniothyrium minitans was effective in natural soil. Coniothyrium minitans was the most effective in reducing sclerotial viability at the temperature range of 15–25°C. Trichoderma virens was effective against sclerotia of S. sclerotiorum to a lesser extent than C. minitans , and in non-autoclaved soil, it performed best at 25°C. Although Epicoccum purpurascens , Talaromyces flavus and Trichothecium roseum were effective against sclerotia of S. sclerotiorum in some instances, they were less effective than C. minitans and T. virens . Sclerotia of S. sclerotiorum conditioned for myceliogenic germination were more vulnerable to attack by the biocontrol agents than dormant sclerotia. The implications are discussed with respect to enhancement of biological control of crop diseases caused by S. sclerotiorum in different geographic regions.     

Effectiveness of Different Frankia Cell Types as Inocula for the Actinorhizal Plant Casuarina

The soil bacterium Frankia of the Actinomycetales, capable of forming N₂-fixing symbiotic root nodules on a diverse array of actinorhizal plants, has several morphological forms when grown in pure culture. Fresh hydrated preparations of whole cells, hyphae, and spores were all infective on seedlings of Casuarina at different dilutions. Desiccated hyphae showed no infection capacity, while desiccated spores remained infective, although at a reduced level. On the basis of most-probable-number statistics, spore suspensions were 3 orders of magnitude more infective than hyphae.     

Survival of Heterodera zeae in Soil in the Field and in the Laboratory

Eggs and (or) second-stage juveniles (J2) inside cysts of Heterodera zeae survived over winter in the field with no detectable mortality at all six depths to 30 cm from which soil samples were collected between corn stubble in the row at 4-8-week intervals. Few or no free J2 were recovered from soil collected in January-April from the top 5 cm, but some were recovered at all samplings from soil collected at greater depths. Emergence of J2 from cysts and numbers of females developing on corn roots in bioassays of cysts increased substantially between January and April. Cyst numbers in a fallow area of the corn field did not decline at any depth to 30 cm during 20 months. Free soil J2, J2 emerged from cysts, and females from the bioassay of cysts were highest at the first soil sampling in July after 10 months of fallow; numbers of nematodes in all three categories declined thereafter, but a few were still detectable after 20 months of fallow. Some cysts were still being recovered after 51 months from naturally infested field soil stored moist in the laboratory at 2 C and 24 C. Females were produced in the bioassays of cysts recovered from soil stored for 38 months at 24 C and for 32 months at 2 C. No free J2 were recovered from soil after 1 month of storage at -18 C, but even after 7 months storage J2 emerged from cysts that were recovered and many females developed in bioassays of those cysts.     

Isolation, Pathogenicity and Safety of Curvularia eragrostidis Isolate QZ-2000 as a Bioherbicide Agent for Large Crabgrass (Digitaria sanguinalis)

A pathogen isolated from lesions on blighted leaves of crabgrass in three different locations of China was identified as Curvularia eragrostidis. Isolate QZ-2000 was the most virulent of six isolates tested. Experiments on morphology, pathogenicity, effect of environmental factors, and host-range of isolate QZ-2000 were conducted in the laboratory, greenhouse and field to assess the potential of this isolate as a biocontrol agent for grassy weeds. Pathogenicity was quantitatively determined based on mortality and dry-weight reduction of infected large crabgrass. Inoculum concentration, rapeseed oil concentration in formulaton, post-inoculation dew temperature and duration, and plant growth age all significantly influenced the efficacy of the isolate. A total of 85-100% control of large crabgrass was obtained when inoculum concentrations were ≥1×10⁶ conidia mL^-1, oil concentrations ≥0.9% (v/v), dew period ≥24 h and air temperatures 20-30°C in the greenhouse. A total of 51 plant species in 20 families were screened against isolate QZ-2000 in host-range studies. Six other species of grassy weeds were susceptible to isolate QZ-2000, but no mortality or significant dry-weight reduction was observed for maize (Zea mays), rice (Oryza sativa), soya bean (Glycine max), cotton (Gossypium hirsutum), or any other economically important crops and plants. In field trials, with 5×10⁶ conidia mL^-1 inoculum density, 60-7% reduction in dry weight was achieved for large crabgrass seedlings under natural dew-free conditions. These results indicate that isolate QZ-2000 is a potential microbial bioherbicide for control of large crabgrass in crops such as corn, soybean, cotton, water-melon, and peanut.     

Effect of Epidemiological Factors on the Impact of the Fungus Plectosporium tabacinum on False Cleavers ( Galium spurium )

The fungus Plectosporium tabacinum has been evaluated as a bioherbicide for control of false cleavers ( Galium spurium ), but the limiting factors and optimal conditions for successful control using this pathogen are not known. False cleavers mortality and dry weight reductions caused by P. tabacinum were assessed under single or combined factors, including pathogen inoculum concentration, plant growth stage, dew period (duration, frequency, and timing), and dew period temperature. The minimum inoculum concentration required to kill false cleavers seedlings was 1 ×10 7 conidia mL -1 at an application rate of 150 mL m -2 . False cleavers seedlings in the cotyledon or 1-whorl growth stage were the most susceptible. Increasing inoculum concentration increased weed control efficacy on older false cleavers seedlings. When an adequate dew period was provided, 100% mortality and dry weight reduction occurred. The minimum dew period to achieve 100% mortality was 16 h. Delaying the initiation of the dew period by 24 h significantly reduced disease development. Short, repetitive dew periods only improved false cleavers control to a limited extend and the length of initial dew is a critical factor influencing false cleavers mortality due to P. tabacinum . The optimal dew period temperature for disease development was above 15°C.     

Difference of molecular association in two types of curdlan gel

The molecular association in a curdlan gel formed by neutralizing an alkaline solution of curdlan with carbon dioxide was compared with those in gels obtained by heating aqueous suspensions of curdlan at various temperatures.

The neutralized and 60°C-set preparations were soluble in 0·01 sodium hydroxide, whereas preparations set at above 90°C were soluble only in concentrations of sodium hydroxide above 1 . The absorption of Aniline blue or Congo red to the preparations decreased with an increase in the temperature of heat treatment and the adsorption to a gel heated at 120°C for 4 h was about 30% of that for the unheated neutralized gel. Seventy-three per cent of the heated preparation was resistant to treatment with 32% sulfuric acid at 32°C for 30 days, whereas none of the neutralized gel was resistant. An electron micrograph of the resistant part of the curdlan showed that it had a pseudocrystalline form. X-ray studies showed a much higher crystalline structure in the resistant part than in the preparation without heat treatment. The X-ray patterns were almost the same for preparations treated with 32% sulfuric acid or (1 → 3)-β-glucanase.     

生物炭调控盐胁迫下水稻幼苗耐盐性能

土壤盐渍化降低土壤生产力.探索生物炭对盐胁迫下水稻幼苗耐盐性能的影响,对调控盐渍区水稻生产潜力具有重要意义.本研究通过生物炭介入盐胁迫稻田土壤的盆栽试验,调查了生物炭对盐胁迫下土壤环境和水稻幼苗耐盐性能的影响.盐胁迫设置4个水平,分别为0 g NaCl·kg-1土(S0),1 g NaCl·kg-1土(S1),2 g ...     

Freeze-Dehydration for Permanent Mounts after Aceto-Orcein Stain

Fresh smears of mammalian material were stained 5 min (only) by La Cour's (1941) aceto-orcein method at 60°C, briefly rinsed and drained, and then dehydrated in a prechilled equal-parts mixture of absolute alcohol and anhydrous acetone held 2 hr or more at — 20°C or lower by means of dry ice. The preparations were differentiated 30 sec at room temperature in 95% alcohol containing 1% HCl, counterstained (optional) in a 0.01% solution of fast green FCF in absolute alcohol for 10 sec, passed through 2 changes of xylene and mounted in HSR or other synthetic resin.