Adjuvants enhance the biological control potential of an isolate of Colletotrichum gloeosporioides for biological control of sicklepod (Senna obtusifolia)

In greenhouse experiments, unrefined corn oil, Silwet L-77, and an invert emulsion were tested as adjuvants with the mycoherbicidal fungus Colletotrichum gloeosporioides, a weakly virulent pathogen of sicklepod (Senna obtusifolia). A 1:1 (v/v) fungus/corn oil tank mixture containing 0.2% (v/v) Silwet L-77 surfactant reduced the dew period requirements for maximum weed infection and mortality from 16 to 8 h, and delayed the need for free moisture for greater than 48 h. This formulation also resulted in the ability of the pathogen to infect and kill weeds in larger (>5 leaf) growth stages. The invert emulsion resulted in similar effects upon these parameters. These results suggest that invert emulsions, unrefined corn oil and Silwet L-77 surfactant greatly improve the bioherbicidal potential of this pathogen for control of sicklepod, a serious weed pest in the southeastern US.     

Biological Control and Use of Adjuvants against Multiple Seeded Cocklebur (Xanthium strumarium) in Comparison with Several Other Cocklebur Types

Common cocklebur has several biotypes including multiple seeded cocklebur (MSC), NCC-TX, and NCC-MS. Alternaria helianthi applied at 2.5×10⁴ conidia mL^-1 in a 50% micro-emulsion of unrefined corn oil (MESUCO) or 0.2% Silwet L 77 caused 60-75% mortality on NCC-TX and MSC. Increasing the conidial concentration to 5×10⁴ mL^-1 increased mortality to 100% on MSC and NCC-TX, and 75% on NCC-MS. At 10×10⁴ conidia mL^-1, A. helianthi caused 100% mortality in all three biotypes. No mortality occurred in any biotype at inoculation rates of 2.5 and 5×10⁴ conidia mL^-1 when applied in water. Increasing the dew period from 0 to 12 h increased mortality from 0 to 100% on all three biotypes at a rate of 2.5×10⁴ conidia mL^-1 in Silwet and MESUCO. MSC appears to be the most sensitive biotype.     

Factors Influencing the Efficacy of the Potential Microbial Herbicide Alternaria alternata (Fr.) Keissler on Amaranthus retroflexus (L.)

Alternaria alternata , applied to run-off at 107 conidia ml -1 and given a 6 h dew period, caused an 88.5% reduction in dry weight of 4 to 5 true-leaf plants of Amaranthus retroflexus . This level of control was achieved whether the inoculated plants were grown in the glasshouse or outside. Formulation of the pathogen in oilseed rape emulsion reduced its dew period requirement from 24 h or more to 8 h or less. For example, with 106 conidia ml -1 , equivalent effects were found with 8 h dew with emulsion formulation compared to 48 h dew with the Tween formulation. Allowing spray deposits to dry on inoculated plants for ca 7 h, before exposing to dew increased the efficacy of the pathogen.     

Efficacy of Bauveria bassiana for red flour beetle when applied with plant essential oils or in mineral oil and organosilicone carriers

The carriers mineral oil and Silwet L-77 and the botanical insecticides Neemix 4.5 and Hexacide were evaluated for their impacts on the efficacy of Beauveria bassiana (Balsamo) Vuillemin conidia against red flour beetle, Tribolium castaneum (Herbst), larvae. The dosages of liquid treatments were quantified by both conidia concentration in the spray volume and conidia deposition on the target surface. The latter approach allowed comparison with dry, unformulated conidia. The median lethal concentrations of B. bassiana in 0.05% Silwet L-77 solution or without a carrier were approximately double that for conidia in mineral oil. Carriers had highly significant effects on the efficacy of B. bassiana. The lower efficacy of conidia in aqueous Silwet L-77 may have been the result of conidia loss from the larval surface because of the siloxane's spreading properties. Neemix 4.5 (4.5% azadirachtin) delayed pupation and did not reduce the germination rate of B. bassiana conidia, but it significantly reduced T. castaneum mortality at two of four tested fungus doses. Hexacide (5% rosemary oil) caused significant mortality when applied without B. bassiana, but it did not affect pupation, the germination rate of conidia, or T. castaneum mortality when used in combination with the 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.     

Effect of row spacing on biological control of sicklepod (Senna obtusifolia) with Colletotrichum gloeosporioides

In field experiments conducted over 3 years, the mycoherbicidal fungus Colletotrichum gloeosporioides, formulated either in 20% (v/v) unrefined corn oil and 0.2% Silwet L-77 surfactant or with an invert emulsion, provided season-long control of Senna obtusifolia in narrow (51 cm) rows of soybean. However, in wide (102 cm) rows, one application of either formulation failed to provide season-long control of S. obtusifolia, and two applications were required to achieve season-long weed control. In narrow (51 cm) rows, one application of the fungus either in unrefined corn oil or an invert emulsion controlled S. obtusifolia an average of >90%, and a second application was not required for season long weed control. Soybean yields in wide-row plots treated with two applications of either the fungus/corn oil or fungus/invert emulsion, or with a single application of the fungal treatments in narrow-row soybean plots, were not significantly different from weed-free control plots, or from plots treated with the herbicide chlorimuron. These results suggest that row spacing can affect mycoherbicidal efficacy of this fungus for controlling S. obtusifolia.     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.     

Potential of oil-based formulations of Beauveria bassiana to control Triatoma infestans

The in vitro development of Beauveria bassiana conidia was monitored when immersed in six concentrations of seven non-ionic (MP 6400, MP 600, Renex 60, Renex 95, Span 80, Tween 20 and Tween 80) and three anionic (DOS 75, Hostapaval BVQ 9 and Surfax 220) surfactants and 11 vegetable oils (linseed, soybean, groundnut, rapeseed, thistle, sunflower, olive, sesame, corn, castor, and babassu). The influence of the oils on the settling behavior of Triatoma infestans nymphs and the activity of an oil–water formulation of the fungus against this vector under laboratory and simulated field conditions were also determined. With exception of DOS 75 and Surfax 220 germination of conidia on complete medium was >98% at 24 h after exposure to surfactants up to 10%. Elevated rates of germination (>25%) were observed in 10% corn, thistle and linseed oil 8 days after incubation. Pure oils had a significant repellent effect to T. infestans. Repellency decreased generally at 10% of the oil and some oils showed some attractiveness for nymphs when tested at 1%. Nymphs were highly susceptible to oil–water formulated conidia, even at unfavorable moisture for extra-tegumental development of the fungus on the insect cuticle.     

Efficacy of Trematophoma lignicola Petrak as a Biological Control Agent for Amaranthus retroflexus L.

The pathogenicity of Trematophoma lignicola on Amaranthus retroflexus was increased when its conidia were formulated in oilseed rape emulsion. However, this formulation did not reduce the dew period required to allow conidial germination and infection of the host, nor did it protect the conidia from desiccation before the onset of dew. The fungus gave effective control of A. retroflexus plants up to the 4-true-leaf stage. Plants with 5 to 6-true-leaves were infected and their dry weight reduced, but plants with more than 6-true-leaves were unaffected. Plants grown in warm conditions (18-21°C/12-15°C, day/night) were more likely to be successfully controlled than those grown in the cold (10-12°C/7-8°C; day/night). Significant dry weight loss of A. retroflexus plants were achieved at application volumes down to 100 l ha -1 at 5 ×10 6 conidia ml -1 . More effective control was achieved by application of conidia of T. lignicola than mycelial or pycnidial applications.     

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.     

The mechanism of the mycoinsecticide diluent on the efficacy of the oil formulation of insecticidal fungus

Adverse conditions, including low humidity, UV irradiation, and high temperature, appreciably affect the efficacy of mycoinsecticides. Oil formulation increased the virulence of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against locusts and grasshoppers by reducing the dependence on saturated water. A mycoinsecticide diluent (a water-in-oil emulsion) has been widely used to dilute the oil formulation of M. anisopliae in China. The aim of our study was to elucidate the mechanism by which the mycoinsecticide diluent improves the virulence of M. anisopliae. We investigated the effects of the mycoinsecticide diluent on the virulence, invasion speed, and viability of the conidia under various adverse conditions. The results demonstrated that the mycoinsecticide diluent significantly improved the virulence of conidia at low humidity (68, 75, and 84%). In particular, at an RH of 68%, the LT₅₀ for locusts treated with the emulsion was 5.4 days and was 31.6% lower than the value for locusts treated with an oil formulation. In addition, the concentration of the hyphal bodies found in the haemolymph of locusts treated with emulsion was about 27-fold higher than that in locusts treated with oil formulation four days after inoculation. This result was further confirmed by determining the concentration of M. anisopliae var. acridum DNA in locust haemolymph using quantitative PCR. The percentage germination of conidia in the emulsion was also significantly higher than that in oil at 68% RH. There was no significant difference in percentage germination between conidia treated with the emulsion and oil when exposed to irradiation with ultraviolet-B (UV-B) or high temperature. These results demonstrate that the mycoinsecticide diluent enhances the virulence of M. anisopliae formulated in oil at low humidity by providing adequate water for germination without interfering with the UV tolerance and thermotolerance of the conidia.     

Production of thermotolerant entomopathogenic Isaria fumosorosea SFP-198 conidia in corn-corn oil mixture

Low thermotolerance of entomopathogenic fungi is a major impediment to long-term storage and effective application of these biopesticides under seasonal high temperatures. The effects of high temperatures on the viability of an entomopathogenic fungus, Isaria fumosorosea SFP-198 (KCTC 0499BP), produced on different substrates amended with various additives were explored. Ground corn was found to be superior in producing the most thermotolerant conidia compared to yellow soybean, red kidney bean, and rice in a polyethylene bag production system. Using ground corn mixed with corn oil as a substrate resulted in only 7% reduction in germination compared to ground corn alone (67% reduction) after exposure of conidia to 50°C for 2 h. Corn oil as an additive for ground corn was followed by inorganic salts (KCl and NaCl), carbohydrates (sucrose and dextrin), a sugar alcohol (sorbitol), and plant oils (soybean oil and cotton seed oil) in ability to improve conidial thermotolerance. Unsaturated fatty acids, such as linoleic acid and oleic acid, the main components of corn oil, served as effective additives for conidial thermotolerance in a dosage-dependant manner, possibly explaining the improvement by corn oil. This finding suggests that the corn-corn oil mixture can be used to produce highly thermotolerant SFP-198 conidia and provides the relation of unsaturated fatty acids as substrates with conidial thermotolerance.     

Trematophoma lignicola (Petrak): A Potential Microbial Herbicide for Control of Amaranthus retroflexus (L.)

Trematophoma lignicola (Petrak), sprayed on leaves at 106 conidia ml -1 in oil emulsion, kills Amaranthus retroflexus plants after a 16 h dew period. Application as a soil drench kills seedlings of this species.     

Investigation of Spray Application of Microbial Herbicides Using Alternaria alternata on Amaranthus retroflexus

Inclusion of Alternaria alternata conidia in a spray formulation affected the distribution pattern on the target. The dry weight of Amaranthus retroflexus plants was reduced by more than 83% when A. alternata conidia (107 ml -1 ) were applied at 200 L ha -1 or greater and when given a 24 h dew period. At low application volumes (25 or 50 L ha -1 ) plant dry weight was reduced by only 29 or 54%. After 7-8 h dew period, conidial germination on the leaf surface was 11-19%. This increased to 62-91% after 24 h dew period. Counts of conidia on leaves indicated that up to 86% of the conidia sprayed were not retained on the target plant, or did not reach it. This is reflected in lesion numbers per unit area being only 3-5% of the calculated theoretical numbers. The results cast doubt on the suitability of A. alternata as a microbial herbicide for the control of Am. retroflexus .     

A Proposed Mode of Action of Oil-Based Formulations of a Microbial Herbicide

Formulation of some microbial herbicides in emulsions of vegetable oil can significantly reduce their dependency on long dew periods to allow infection and control of the host weed. Examination of leaves of Viola arvensis , by a range of microscopy techniques, after spraying with an oilin-water emulsion formulation of the potential microbial herbicide Mycocentrospora acerina , suggests that the effect arises as a result of inversion of water into both oil droplets on the leaf surface and oil that penetrates into the leaf tissue. This oil probably mixes with aqueous components from the leaf tissue and, in doing so, forms an invert emulsion. Wesuggest that this supplies the water required by the fungus to initiate and sustain growth so that infection can result.     

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.     

Evaluation of the bioherbicide Myrothecium verrucaria for weed control in tomato (Lycopersicon esculentum)

An isolate of the fungus Myrothecium verrucaria was evaluated for its biocontrol potential against common purslane, horse purslane, spotted spurge, and prostrate spurge, all serious weed pests in commercial tomato fields in the southeastern US. In greenhouse and field tests, M. verrucaria was highly virulent against these weeds when applied as conidial sprays formulated in 0.2% Silwet L-77 surfactant, even in the absence of dew. In field test plots naturally infested with these weeds, seedlings in the two-to-three leaf growth stage treated with M. verrucaria at 2×10⁷ conidia mL^-1 in 0.2% Silwet, exhibited leaf and stem necrosis within 24 h following inoculation, with mortality occurring within 96 h. After 7 days, M. verrucaria had killed 90-95% of both purslane species and 85-95% of both spurge species. Tomatoes that were transplanted into plots treated with M. verrucaria remained healthy and vigorous throughout the growing season. Since M. verrucaria effectively controlled several common weeds under field conditions, this fungus appears to have potential as an effective bioherbicide for pre-plant weed control in production systems with transplanted tomato.     

The Potential of Stagonospora sp. as a Mycoherbicide for Field Bindweed

Field bindweed ( Convolvulus arvensis L.) is one of the 12 most important weeds worldwide. Stagonospora sp. Isolate LA39 was isolated from diseased field bindweed plants collected in Europe. No crop tested was susceptible to the fungus, but disease symptoms were observed on other Convolvulaceae species. On field bindweed, the fungus induces disease symptoms (i.e. lesions) mainly on leaves and less severely on stems. The application of spores in an oil emulsion (10% oil in water) enhanced the disease on field bindweed plants compared with spores suspended in a 0.1% aqueous solution of the surfactant agent, Tween 80. The necrotic leaf area of inoculated plants increased as the length of exposure to 100% relative humidity (RH) and the spore density applied increased. Severe disease developed on plants inoculated with 1 107 spores/ml in oil emulsion, even in the absence of exposure to 100% RH. A delay of exposure to 100% RH (up to 8 h) did not have a significant eVect on disease severity. Field bindweed was susceptible to the fungus at all growth stages tested, but older plants were more susceptible than younger ones. It was concluded that isolate LA39 has potential as a biocontrol agent of field bindweed, especially when applied in an oil emulsion. The oil emulsion maintains the aggressiveness of the pathogen during a dew-free period and provides a favourable microenvironment during the infection process.     

Spray retention for liquid and mycoherbicide inoculum in three weed-biocontrol systems

Spray retention is often used to measure herbicide delivery to optimize application parameters, but little is known about retention characteristics of mycoherbicide inoculum applied for weed biocontrol. This study examined inoculum retention of three mycoherbicide agents, Pyricularia setariae, Colletotrichum truncatum and C. gloeosporioides f. sp. malvae, on their respective weed targets: green foxtail, scentless chamomile and round-leaved mallow. Conidium suspensions of these fungal pathogens containing a sodium-fluorescein tracer dye were applied at 500, 1000 and 2000 L ha^-1 using a cabinet sprayer, and the liquid volume and number of conidia retained on the plants were quantified. On all three weed species, liquid and conidium retention showed a high degree of correlation at varying application volumes although slight differences existed depending on the weed species. Based on the analysis of regression slopes, liquid retention reflected conidium retention most closely on green foxtail but slightly overestimated the number on scentless chamomile and round-leaved mallow, possibly due to different plant morphology and spray run-off at extremely high application volumes. Liquid retention can generally be used as an indicator in studying effects of spray quality on mycoherbicide retention for improved delivery and biocontrol in these weed-biocontrol systems.     

Enhancing survival and subsequent infectivity of conidia of potential mycoherbistats using UV protectants

Ultraviolet radiation (UV) can reduce the effectiveness of fungi used for biological control; therefore, this study examined the photostabilising effect of water- and oil-soluble UV protectants on conidium germination of Plectosporium alismatis and Colletotrichum orbiculare, pathogens with potential as biocontrol agents, and the ability of conidia of C. orbiculare to cause disease. Formulation in riboflavin (1%), proline (1%), propyl gallate (1%), melanin (0.1%) and ascorbic acid (5%) increased the germination of UVB-exposed conidia of P. alismatis to levels found in the dark control without causing a delay in germination. Formulation in (a) pyridoxin (5%), (b) an nC24 mineral oil (5%), and (c) ECCO 1422 (5% in the mineral oil) also resulted in germination similar to the control but germination was delayed. Protection was provided to conidia of C. orbiculare treated with 1% aqueous solutions of proline and folic acid in vitro. Formulation of conidia of C. orbiculare in a 5% aqueous emulsion of the mineral oil and aqueous solutions of melanin (0.01%), proline and tyrosine (both at 1%) significantly increased anthracnose development above control levels on leaf discs of Xanthium spinosum exposed to UVB dose of 16.7 kJ m^-2. After exposure to natural sunlight at a UVB dose of 2.2 kJ m^-2, anthracnose development was greater on leaf discs inoculated with conidia of C. orbiculare formulated in 1% aqueous solutions of ascorbic acid (1%), proline (1%), tyrosine (1%) and melanin (0.01%), or in 5% aqueous emulsions of a canola-derived oil and the mineral oil than by conidia formulated in water alone. Therefore, a range of compounds can provide conidia with protection from UVB. Of these, propyl gallate and oils similar to the mineral oil are likely to be cost effective. Such formulations can be combined with suitable application times to increase mycoherbisitat efficiency.