Modifications were investigated to improve shelf-life or long-term survival upon storage of an extruded Oryza sativa L. (rice) flour : gluten : clay: oil formulation (C7) of a mycoherbicide, Fusarium oxysporum Schlechtend: Fr. f. sp. erythroxyli strain EN4, that causes vascular wilt in Erythroxylum coca var. coca (coca). Fermentor-produced biomass, which contained abundant desiccation-resistant chlamydospores, was incorporated into various adaptations of C7 and stored at room temperature (22 to 25 C) under moderately high (50 to 60%) and low (0 to 5%) relative humidities (RHs). The effect of RH on shelf-life was not significant up to 4 mo of storage, while the presence of oil, added to improve its extrusion, reduced viability significantly. Addition of Gossypium hirsutum L. (cotton) embryo flour or complete elimination of oil from the formulation improved shelf-life from 3 mo to > 12 mo. Shelf-life was further improved by removing the binding agent gluten in the formulation and replacing it with autoclaved O. sativa flour. Ability of the formulations to produce secondary propagules, tested on 1% water agar, indicated that, while adding oil had no effect, G. hirsutum embryo flour increased desiccation-resistant chlamydospore counts but lowered macroconidial counts. Autoclaved rice flour (MR) significantly improved both macroconidial and microconidial counts without affecting chlamydospore counts. None of the formulations affected the total viable propagule counts. When compared with the original formulation (C7), the modification (MRRP7), with MR, G. hirsutum embryo flour, and without oil, was found to have improved shelf-life at higher temperature and RHs and enhanced potential for secondary chlamydospore formation. These characteristics are important for survival of the formulated F. oxysporum under less expensive storage conditions and, once applied, for survival in the soil.

Biomass abundant in chlamydospores obtained by liquid fermentation of mycoherbicidal strains of Fusarium oxysporum was incorporated into alginate prills with various food substrates and granular formulations such as corn flour : starch, wheat flour : kaolin, rice : wheat flour, and rice : wheat gluten formulations. These fungal strains cause vascular wilts in coca (Erythroxylum coca var. coca) and in poppy (Papavar somniferum). Fungal strain, formulation method, and interaction of these two parameters significantly affected the shelf life of the formulations at room temperature as well as the ability to form secondary propagules on 1% water agar. In bioassays, there were no significant differences among F. oxysporum strain EN4-S formulations in their ability to colonize root tissue or the rhizosphere of E. coca seedlings. The study indicates that most of the formulations meet at least three of the four criteria important for biological control agents to be effective: low losses in viability during the formulation process, satisfactory shelf life at room temperature, abundant secondary chlamydospore formation, and rhizosphere colonization.

The fungus Pleospora papaveracea is a potential biocontrol agent for opium poppy. The objective of this study was to characterize the growth and production of propagules of P. papaveracea on various substrates and determine their infectivity on opium poppy. Pleospora papaveracea was grown on agar media containing wheat bran, corn cobs, soy fiber, cottonseed meal, rice flour, cornstarch, pectin, dextrin, or molasses, all with the addition of brewer's yeast (BY). Maximum radial growth of P. papaveracea occurred on molasses, soy fiber, and wheat bran media. Pleospora papaveracea produced chlamydospores on dextrin–BY and cornstarch–BY only. Pleospora papaveracea growth in liquid media with 1% (wt/v) dextrin, cornstarch, soy fiber, or wheat bran resulted in the production of greater than 106 colony-forming units (cfu) ml−1 within 3 to 5 d of incubation. Pleospora papaveracea produced less than 105 chlamydospores ml−1 after 10 d of incubation in wheat bran–BY and soy fiber–BY liquid media compared with the production of greater than 105 chlamydospores ml−1 after 5 d of incubation in dextrin–BY or cornstarch–BY liquid media. Fewer cfu were produced by P. papaveracea in 0.25% dextrin or 0.25 and 0.50% soy fiber liquid media than with 1 or 2% substrate. Greater than 107 chlamydospores g−1 dry weight and 108 cfu g−1 dry weight of P. papaveracea were produced in dextrin–BY liquid media in a commercial bench-top fermentor. After air drying biomass for 6 d, propagules of P. papaveracea remained infective on opium poppy. Mycelia and chlamydospores of P. papaveracea grew and formed appressoria during the infection process. Air-dried biomass, when rehydrated in 0.001% Tween 20, caused necrosis within 48 h after application to detached opium poppy leaves. At least 94% of the propagules from air-dried biomass that germinated and infected detached opium poppy leaves were of mycelial origin.