Location: Biological Control of Pests Research
Project Number: 6066-22000-093-000-D
Project Type: In-House Appropriated
Start Date: Jan 21, 2021
End Date: Jan 20, 2026
1. Develop new safe, effective augmentative bioherbicide products using techniques and methods to maximize virulence factors that promote pathogen specificity and efficacy on weeds, such as giant salvinia. [NP304, C1, PS1B and PS1C] 2. Develop, implement and evaluate weed biological control agents through coordinated and collaborative efforts with stakeholders and the public that will promote registration and commercialization of safe, effective bioherbicides. [NP304, C2, PS2A and PS2B]
In Objective 1, we will evaluate mycelial formulations of the fungus Botryosphaeria rhodina (Br) for managing giant salvinia (Salvinia molesta; Sm). Br kills Sm within 12 h, indicating production of virulence factors, e.g., the phytotoxic enzyme laccase. We will assay for increased laccase production in our Br strain using an assay described by Dias et al. Briefly, standard 6 mm blank paper discs impregnated with ABTS will be placed on microplates loaded with an aliquot from each Br sample. Discs containing laccase will become green-bluish colored allowing for visual laccase detection. In vitro laccase activity in cultures and cell-free extracts will be measured spectrophotometrically using a multicopper oxidase (laccase) from cyanobacteria. Protein concentration will be determined via Bradford’s reagent with bovine serum albumin standard. Laccase will be increased (via methods described) to include effect of: A) Inoculum; B) Time-point of veratryl alcohol addition; C) Growth media initial pH; D) Media carbon/nitrogen (C:N) ratio; E) N sources (organic and inorganic); F) Tween 60 and 80 and soybean oil; G) Copper. Laccase will be assayed using methods previously described. Cell-free filtrates from Br cultures with increased laccase levels (determined and quantitated as described previously) will be generated in enzymatic assays. Sm leaf disks (20; 4 mm dia.) will be placed in petri dishes containing a buffer, sucrose (1%), and the test compound. Controls will contain only buffer and sucrose. Conductivities will be determined with a conductivity meter at 0 to 12 h, every 3 h. Sm (1° and 2° growth stages) will be transferred to plastic containers and acclimated 3 d (greenhouse, 28-30° C, 16 h photoperiod). Inoculations will be made using aerosol sprayers. Controls will consist of distilled water. Treatments include Br-WT, mycelial formulations, and cell-free filtrates of Br-WT for comparison. Disease will be monitored at 3 d intervals for 12 d via a 0 to 5 disease rating scale. Percent control and biomass reductions will be determined after 12 d. The experiments will be repeated over time and data averaged. An RCBD will be utilized. The mean percentage of plant mortalities, height and biomass reductions will be calculated for each treatment, and data transformed as required. Transformed data will be compared using ANOVA at p = 0.05. Dr. R.E. Hoagland (CPSRU, Stoneville, MS) will collaborate in Objective 1. In Objective 2, in collaboration with various Game and Fish Commissions, experiments will be established to evaluate efficacies of Br formulations in areas recently infested with Sm. Formulations evaluated will include inocula applied at the most effective rate (as determined from the greenhouse). Quadrants of floating plastic frames composing an area of 1.0 x 1.0 m2 will be utilized to serve as test plots. Disease monitoring, weed control percentages, and dry weight determinations will be evaluated on Sm within the quadrants. Controls will consist of untreated and SW treated test plots. All experiments will be repeated over 3 y. If warranted, pilot-scale evaluations will be conducted to determine economic feasibility.