Formulation

It is believed that many of the environmental challenges, particularly long dew period or leaf wetness requirements, can be tackled to a large extent with formulation technologies (Boyetchko et al. 1999; Greaves et al. 2000; Green et al. 1998). Formulation is essentially the blending of microbial propagules with a range of carriers or adjuvants to produce a form that can be effectively delivered to target weeds. For microbial agents, formulation may enhance pathogen survival and infection as well as extend propagule stability and product shelf life. Depending on the type of organism, mode of action, and available spray equipment, formulation ingredients vary substantially. For instance, foliar-applied agents may be exposed to rain-wash, UV irradiation, and desiccation prior to germination and penetration (Rhodes 1993). Therefore, various adjuvants with adhesive, sun-blocking, or humectant properties have been suggested to alleviate the negative impact by these factors (Schisler et al. 1995; Womack and Burge 1993). Formulations that increase moisture-retaining properties, reduce the rate of evaporation and/or enhance the rate of infection of the mycoherbicide agent should be explored to address dew limitations. In the literature, emulsions and hydrophilic polymers are reported most frequently to improve the performance of foliar-applied mycoherbicide agents. Formulation research has focused particularly on desiccation and dew requirements of fungal agents during the infection process and incremental to drastic improvements have been seen in different studies (Auld 1993b; Connick et al. 1990; Lawrie et al. 2000; Shabana 1997). There is a growing belief that innovations in formulation will be a vital component to the success of the next generation of bioherbicides, especially for foliar-applied products (Greaves et al. 1998). For best results, formulations should predispose weeds to infection by pathogens and buffer pathogen propagules against environmental extremes while promoting disease development. Nutrient supplements, including simple sugars, amino acids, pectins, salts, and plant extracts have been added to formulations to stimulate the infection process and protect germinating propagules, but these nutritional effects are often agent specific (Bothast et al. 1993; Schisler et al. 1995; Womack and Burge 1993). Exogenous nutrients may stimulate germination and growth of many fungi, but frequently appressorial initiation is even more important to plant penetration and infection. Oversupply of nutrients can lead to excessive growth of germlings, delaying, or even reducing appressorial formation and penetration (Takano et al. 1997). Tremendous efforts have been made on developing various emulsions to alleviate moisture constraints, thereby enhancing field performance. Invert emulsions showed the most impressive results by reducing or even eliminating the need for dew with several fungal agents (Connick et al. 1991b; Yang et al. 1993). Bioherbicidal control of hemp sesbania using C. truncatum in an invert emulsion was significantly enhanced under field conditions (Boyette et al. 1993). Invert emulsions consist of water droplets suspended in oil and evaporation of the trapped water is dramatically reduced and microbial propagules held in the water are, therefore, protected (Daigle et al. 1990; Womack and Burge 1993). Despite the apparent improvement, invert emulsions can be very complex, difficult to apply using existing spray equipment due to extreme viscosity and may exhibit phytotoxicity in many plant species (Boyette 1994; Womack et al. 1996). Although these invert emulsions have been used to expand the host range of mycoherbicides (Yang and Jong 1995), this change may also affect nontarget crops. High oil content also makes invert emulsions more costly, especially when high spray volumes are required. Being nonevaporative, oils were considered a compatible carrier with ultra-low volume application techniques for the mycoinsecticide M. anisopliae under extremely dry conditions (Bateman and Alves 2000). In contrast to invert emulsions, oil suspension emulsions are considered to be more practical because they have significantly lower oil content and can be applied with most existing spray equipment (Green et al. 1998). Fungal propagules can be first suspended in oils, then mixed with a much larger volume of water containing an emulsifier to make stable emulsions

(Auld 1993b). Klein et al. (1995) used suspension emulsions of C. orbiculare made from two vegetable and mineral oils that were mixed and applied with water ranging in concentrations from 0.5 to 10% for control of Xanthium spinosum under field conditions. These formulations enhanced weed control in several field trials compared to water as a carrier sprayed at similar application volumes. Oils may have variable effects on propagule germination and performance of mycoherbicide agents. Paraffin oils were toxic to spores of Ascochyta pteridis, a mycoherbicide candidate for bracken, Pteridium aquilinum (Womack et al. 1996), while unrefined corn oil enhanced spore germination of C. truncatum in emulsions containing 10-50% of oil (Boyette 1994; Egley and Boyette 1995). The mechanisms by which the oil suspension emulsions enhance mycoherbicidal activity are not well understood. Emulsions may help maintain the stability and infectivity of fungal propagules prior to onset of dew (Green et al. 1998). Spray retention is likely enhanced, reducing the spore dose required for effective weed control. Moisture retention has also been demonstrated with hydrophylic polymers such as Kelgin® HV, MV, LV, Kelzan® xanthan gum, Gellan gum, N-Gel™, Metamucil®, and Evergreen®500 (Shabana et al. 1997). These polymers enhanced viability, germination, and efficacy of the mycoherbicide agents A. cassiae and A. eichhorniae. Humectants such as psyllium (e.g., Metamucil®) are known to have high moisture retention properties and reduce the rate of moisture loss (Greaves et al. 2000). Coformulation of the mycoherbicide agent, A. caulina, with the skinning agent polyvinyl alcohol and Metamucil® enhanced control of Chenopodium album under reduced dew conditions. For mycoherbicides applied to the soil, encapsulation of the fungi in solid matrices is more suitable than liquid formulations. Calcium alginate has been used to mix fungal spores with a variety of carriers such as kaolin clay, ground oatmeal, soy flour, and cornmeal (Boyette and Walker 1986; Walker and Connick 1983; Weidemann and Templeton 1988). Conidial production and field efficacy can be enhanced by amending the mixture with various nutrients (Daigle and Cotty 1992; Weidemann 1988). "Pesta" has also been used as a type of granular formulation where fungal propagules are entrapped in a wheat-gluten matrix consisting of semolina flour, kaolin, and fungal biomass (Connick et al. 1991a). Further development of this process has resulted in the formation of uniform granules using a twin-screw extruder and by controlling the moisture content through fluid-bed drying (Connick et al. 1998).

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