Location: Biological Control of Pests Research
2022 Annual Report
Objectives
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]
Approach
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.
Progress Report
Research with an Ascomycetous fungus Botryosphaeria rhodina (Br) to control giant salvinia continuesd. Highly infective, buoyant (floating) granular formulations infested with Br were developed. Research continued with an anthracnose-forming pathogen isolated from wild geranium (Geranium dissectum). The pathogen was identified as Colletotrichum gloeosporioides and was effective in controlling this weed over a wide range of environmental conditions. This weed can be problematic in early season row crops in the mid-south, but more importantly serve as overwintering hosts for certain cotton insect pests such as corn earworm (Helianthis zea) and tobacco budworm (H. virescense). In collaborative work, it was shown that increased pectinase activity resulted from an infectivity mechanism that degrades pectic polymers (important for sicklepod cell wall integrity) during pathogenesis of the bioherbicidal fungus Alternaria cassia infecting sicklepod. These studies provide important information on some biochemical interactions that may be useful for improvements to biological weed control programs utilizing plant pathogens (Hoagland, R.E. & Boyette, C.D. 2021, J. Fungi, 7, 1032). Consultations with ARS researcher (Agricultural Research Initiatives) and a local organic rice and soybean grower were conducted concerning host range expansion of the commercial bioherbicide Colletotrichum gloeosporioides f.sp. aeschynomene (LockDown™). Personal, innovative, and original research in accordance with the Project Plan and promoted cooperative research with other projects and labs continued, as well as collaboration with non-ARS entities (Agricultural Research Initiatives; Louisiana Fish & Wildlife; various landowners in Louisiana and Arkansas).
Accomplishments
1. Liquid formulations for biocontrol of invasive aquatic weeds. Giant salvinia, (Salvinia molesta) is an aquatic floating fern native to South America, but has become invasive in some areas in Africa, Asia, Australia, and in subtropical and warmer temperate areas of the U.S. ARS researchers in Stoneville, Mississippi, conducted experiments to determine the shelf-life of a liquid formulation of the fungus Botryosphaeria rhodina (Br) when stored under refrigeration (4° C). It was discovered that after 5 years the refrigerated formulation had lost little or no efficacy in the ability of Br to control giant salvinia under greenhouse conditions. The development of commercially-viable microbial pesticides for invasive weeds such as giant salvinia could provide growers, land managers and homeowners with safe, cost-effective, non-chemical control tools for use in agriculture, chemically-sensitive environments, and natural ecosystems. Furthermore, the results indicated that the Br liquid formulations can be inexpensively mass produced and stored for future use in field trials.
2. Buoyant granular gormulations for biocontrol of invasive aquatic weeds. Invasive aquatic weeds, such as giant salvinia (Salvinia molesta) are difficult to control using conventional weed control measures. ARS researchers in Stoneville, Mississippi, conducted experiments to determine the weed control efficacy of two buoyant (floating) formulations of the fungus Botryosphaeria rhodina (Br) for bioherbicidal control of giant salvinia under controlled conditions. One formulation consisted of Br mycelium-infested popcorn, which provided >60% control when direct contact of the popcorn granules to giant salvinia occurred. A second formulation consisted of Br mycelium incorporated into sodium alginate granules, using unrefined corn oil as the buoyant component. Upon application, the granules were hydrostatically attracted to the giant salvinia plants, and the corn oil component served as an adherent for the granules to the weeds, resulting in severe infection and >90% control of weeds under controlled conditions. The development of commercially-viable buoyant microbial pesticides for aquatic invasive weeds such as giant salvinia could provide growers, land managers and homeowners with safe, cost-effective, non-chemical control tools for use in agriculture, chemically-sensitive environments, and natural ecosystems. These results indicate that the buoyant Br formulations can be inexpensively mass produced and stored for future use in field trials.
Review Publications
Hoagland, R.E., Boyette, C.D. 2021. Effects of the fungal bioherbicide, Alternaria cassia on peroxidase, pectinolytic and proteolytic activities in sicklepod seedlings. The Journal of Fungi. https://doi.org/10.3390/jof7121032.
Duke, S.O., Pan, Z., Bajsa Hirschel, J.N., Boyette, C.D. 2022. The potential future roles of natural compounds and microbial bioherbicides in weed management in crops. Advances in Weed Science. 40(spe1):e020210054. https://doi.org/10.51694/AdvWeedSci/2022;40:seventy-five003.
Weaver, M.A., Mizra, N., Boyette, C.D., Brown, S.P., Mandel, J.R. 2022. Whole genome sequence and draft assembly of the biocontrol fungal pathogen Albifimbria verrucaria CABI-IMI 368023. Microbiology Resource Announcements. 11:1. https://doi.org/10.1128/MRA.00909-21.
Weaver, M.A., Hoagland, R.E., Boyette, C.D., Brown, S.P. 2021. Taxonomic evaluation of a bioherbicidal isolate albifimbria verrucaria, formerly myrothecium verrucaria. The Journal of Fungi. 7(9):694. https://doi.org/10.3390/jof7090694.
