Hemp Sesbania Seedlings Research Articles

Biological Control of the Weed Hemp Sesbania (Sesbania exaltata) in Rice (Oryza sativa) by the Fungus Myrothecium verrucaria

In greenhouse and field experiments, a mycelial formulation of the fungus Myrothecium verrucaria (IMI 361690) containing 0.20% Silwet L-77 surfactant exhibited high bioherbicidal efficacy against the problematic weed hemp sesbania. Infection and mortality levels of 100% of hemp sesbania seedlings occurred within 48 h after fungal application in the greenhouse. In rice field tests conducted over a three year period, M. verrucaria at an inoculum concentration of 50 g L−1 (dry mycelium equivalent) controlled 95% of ≤20 cm tall hemp sesbania plants. M. verrucaria also controlled larger plants (≥60 cm tall) using this high inoculum concentration. This level of weed control, as well as rice yields from plots where weeds were effectively controlled, were similar to those which occurred with the herbicide acifluorfen. These results suggest that a mycelial formulation of M. verrucaria has potential as a bioherbicide for controlling hemp sesbania in rice.

Adjuvant and Refined Corn Oil Formulation Effects on Conidial Germination, Appressorial Formation and Virulence of the Bioherbicide, Colletotrichum truncatum

Several surfactants, plant extracts, and fatty acids were tested for stimulation of conidial germination and appressorial formation of Colletotrichum truncatum, a bioherbicide of the weed, hemp sesbania (Sesbania exaltata). The commercial surfactants (Tweens® 40, 60, 80, 85 and Myvatex® 60) at concentrations of 0.25 to 1.0 % (v/v), stimulated germination, but these effects were not exclusively related to their hydrophylic-lipophylic balance values. The stimulatory surfactants were also tested for germination and virulence of C. truncatum when formulated with the conidia and applied to hemp sesbania seedlings. Conidia formulated in either water, a surfactant or, an emulsion of refined corn oil were ineffective on the plant in the absence of dew or when dew was delayed. However, formulations of conidia combined with surfactant in emulsified refined corn oil did exhibit bioherbicidal activity when adequate dew or free-moisture was unavailable. This is important since previous reports indicated that refined corn oil did not enhance bioherbicidal activity, whereas unrefined corn oil promoted pathogen germination and efficacy of C. truncatum. The fatty acids tested had little or no effect on conidial germination but, aqueous extracts of several plant species including pathogen hosts and non-hosts, stimulated germination. Appressorial formation influenced by the surfactants did not necessarily reflect the disease rating on hemp sesbania seedlings. Overall, results show that formulations containing an emulsion of conidia, a surfactant and refined corn oil have potential for enhancement of the efficacy of C. truncatum.

Hemp sesbania (Sesbania exaltata) control in rice (Oryza sativa) with the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene formulated in an invert emulsion

In greenhouse and field experiments, an invert emulsion (MSG 8.25) was tested with dried, formulated spores of the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene, a highly virulent pathogen of the leguminous weed Aeschynomene virginica (northern jointvetch), but considered ‘immune’ against another more serious leguminous weed, Sesbania exaltata (hemp sesbania). A 1:1 (v/v) fungus/invert emulsion mixture resulted in 100% infection and mortality of inoculated hemp sesbania seedlings over a 21-day period under greenhouse conditions. In replicated field tests of the fungus/invert formulation conducted in Stuttgart, AR, and Stoneville, MS, hemp sesbania was controlled 85 and 90%, respectively. These results suggest that this invert emulsion expands the host range of C. gloeosporioides f. sp. aeschynomene, with a concomitant improvement of the bioherbicidal potential of this pathogen.

Induction of infection in Sesbania exaltata by Colletotrichum gloeosporioides f. sp. aeschynomene formulated in an invert emulsion

In greenhouse experiments, an invert emulsion (MSG 8.25) was tested with spores of the mycoherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene, a highly virulent pathogen of the leguminous weed Aeschynomene virginica (northern jointvetch), but considered “immune” against another leguminous weed, Sesbaniaexaltata (hemp sesbania). A 1:1 (v/v) fungus/invert emulsion mixture resulted in 100% infection and mortality of inoculated hemp sesbania seedlings over a 21 day period. Microscopic examinations revealed that the fungus proliferated within the cells of hemp sesbania and produced anthracnose lesions containing acervuli on infected stems. The fungus was reisolated and found to infect and kill northern jointvetch seedlings, thus fulfilling Koch’s postulates for disease identification. These results suggest that this invert emulsion expands the host range of C. gloeosporioides f. sp. aeschynomene, and possibly improves the bioherbicidal potential of this pathogen.

Enzyme‐Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures, Extracts, and Plant Tissues

A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzyme‐linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross‐reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L‐2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of Myrothecium verrucaria (MV) in cell cultures, in plant tissues (hemp sesbania and kudzu) treated with purified roridin A, or ethyl acetate fractions of MV cultures. Evaluations of ELISA assays showed linear responses for standards of verrucarin A and roridin A over a concentration range of 0.2 to 20 ppb. Ethyl acetate or aqueous extractions were used to obtain samples from MV cultures and plant tissues for testing. Trichothecenes were detected in conidia and mycelia of MV, and in agar upon which wild‐type MV was grown, indicating secretion into the growth media. Two MV sectors (morphological variants of wild type) also tested positive for trichothecenes. Purified roridin A and concentrated extracts containing trichothecenes from MV spore cultures exhibited phytotoxicity (growth inhibition or necrosis) when applied to excised shoots of hemp sesbania seedlings and intact kudzu leaf tissues. Evidence of some translocation of trichothecenes from the application point in kudzu was found, but translocation to the upper shoot portion of hemp sesbania was not detected at the lowest limit of detection in this assay (0.14 ppb). This assay is also being employed to identify induced mutants and/or other naturally occurring sectors deficient in trichothecene mycotoxin production. Results indicated that ELISA is a sensitive and rapid assay method to quantify trichothecenes produced by this bioherbicidal fungus and in certain plant tissues treated with trichothecenes.

Solid Substrate Formulations of the Mycoherbicide Colletotrichum truncatum for Hemp Sesbania ( Sesbania exaltata ) Control

Colletotrichum truncatum was grown on kernels of eight different grains for 3 or 4 weeks at room temperature (22-24°C). Fresh preparations of conidia as well as fungus-infested corn and rice suspensions resulted in 100% mortality of hemp sesbania seedlings when sprayed postemergence with a 14 h dew period. Fresh preparations of mycelia and fungus-infested sorghum suspensions resulted in 90 and 65% mortality of hemp sesbania seedlings, respectively. Lower mortality ( h 15%) occurred with the other ground fungus-infested grain suspensions. Fresh preparations of conidia, fungus-infested corn, rice and sorghum, and mycelia, when applied to soil pre-emergence, resulted in 100, 94, 100, 83 and 71% mortality of hemp sesbania seedlings 14 days after application, respectively. Lower mortality ( h 23%) occurred with the other ground fungus-infested grain preparations. Freshly-prepared C. truncatum at 6.25, 12.5, 25 and 50 mg fungus-formulated rice cm -2 of soil surface, applied pre-emergence or at the time of planting, killed 97, 100, 100 and 100% of hemp sesbania, respectively. After storage at 22-24°C for 6 to 24 months, the rice formulation caused 67 to 93% mortality after 6 months, 39 to 81% after 12 months, and h 2% after 24 months, respectively. When C. truncatum was refrigerated at 4-6°C, the rice formulation retained good efficacy through 24 months, and when frozen, for up to 8 years. C. truncatum formulated on rice stored under all the above conditions contained mainly sclerotia at 2.4 x 10 5 sclerotia g -1 . C. truncatum killed hemp sesbania seedlings with a single soil application through 4 plantings on the same soil. These results indicate that rice and possibly corn are excellent solid substrates for the formulation of C. truncatum . This is a simple and effective method for enhancing the activity of C. truncatum against hemp sesbania.

Twin-screw extrusion of ‘Pesta’-encapsulated biocontrol agents

'Pesta' granules in which fungal propagules are encapsulated in a wheat gluten matrix were prepared in multi-pound quantities by twin-screw extrusion and fluid bed drying. Dough formulations for extrusion contained wheat flour and kaolin, or wheat flour, kaolin and rice flour, plus water and fungal inoculum. Conidial inoculum of Colletotrichum truncatum, a pathogen of the weed hemp sesbania (Sesbania exaltata), survived laboratory scale dough preparation [100% retention of colony-forming units (c.f.u.)] better than dough preparation for twin-screw extrusion (8-10% c.f.u. retention). The loss in viability was linked to the lower water content of dough used in the twin-screw extruder. Fluid bed drying reduced viability further to 1%. Retention of viability after twin-screw extrusion and fluid bed drying at 35-50 °C was 35% with Alternaria conjuncta/infectoria, a pathogen of swamp dodder (Cuscuta gronovii). Retention was 86-100% with atoxigenic strains of Aspergillus flavus and Aspergillus parasiticus used as biocompetitors to reduce aflatoxin levels in peanuts. In the greenhouse, twin-screw-extruded granules containing C. truncatum (at about 5 x 10 4 c.f.u. g -1 ) caused high levels of infection and mortality in hemp sesbania seedlings.

Formulation ofColletotrichum truncatumMicrosclerotia for Improved Biocontrol of the Weed Hemp Sesbania (Sesbania exaltata)

Submerged cultures of the biocontrol fungusColletotrichum truncatumproduce high concentrations of microsclerotia (MS) when grown under specific nutritional conditions. Previous studies have shown that MS survived drying, remained viable following long-term storage at 4°C, and killed hemp sesbania seedlings when incorporated into potting soil. In this study, dried preparations ofC. truncatumMS were encapsulated in wetted formulations of pregelatinized corn flour, pregelatinized cornstarch, or a mixture of the two (1:1). Germination rates of microsclerotia immediately after formulation and drying were not significantly different. After storage at 4°C for 18 months, MS germination rates were significantly lower for all formulations compared to nonformulated MS. Dried MS were capable of producing mycelia or spores upon germination when plated on water agar. After 3 days of growth on Noble agar, the MS–flour formulations produced significantly more conidia than those made with starch, starch–flour, or nonformulated MS. By the 10th day of incubation, significantly more conidia were produced in formulations containing starch–flour (≈10-fold) and flour (≈50-fold) than in MS–starch formulations and nonformulated MS. Bioassays showed that all MS preparations incorporated into potting soil incited disease in emerging hemp sesbania seedlings. A significantly higher incidence of disease was seen in seedlings grown in potting soil containing flour-formulated MS. These results suggest that corn flour formulations ofC. truncatummicrosclerotia can be used to increase the effectiveness of this bioherbicide for controlling the weed hemp sesbania and that increased conidial production may play a role in improving biocontrol efficacy.

Liquid culture production of microsclerotia of Colletotrichum truncatum for use as bioherbicidal propagules

Colletotrichum truncatum NRRL 18434 is a host-specific pathogen of hemp sesbania (Sesbania exaltata), a weed in cotton, soybeans and rice. The bioherbicidal potential of C. truncatum microsclerotia (MS) was assessed based on the feasibility of producing and storing microsclerotia and the efficacy of microsclerotia in controlling hemp sesbania. Microsclerotia were produced in highly aerated liquid cultures grown at 28 °C in a basal salts medium with 80 g glucose and 13·2 g Casamino acids l−1. Hyphal aggregation to form microsclerotial initials began on day 4 with maximum concentrations (6 × 106 MS l−1) being attained by day 6. Melanization started on day 6 and was completed by day 10. After air-drying at 22°, microsclerotia: diatomaceous earth preparations were stored at 4°. After storage for 7 months 90% of the dried C. truncatum MS germinated. Efficacy studies showed that the incorporation of either fresh or 1-month-old microsclerotia into potting soil (156 microsclerotia cm−3 soil) killed more than 95% of hemp sesbania seedlings after emergence. Microsclerotia-infested potting mix incited disease of a similar magnitude in a second crop of hemp sesbania seedlings planted 28 d after the first. These studies demonstrate that C. truncatum microsclerotia may have potential for use as an alternative to conidia for biocontrol since they can be produced in submerged culture, remain viable as a dry formulation and effectively control hemp sesbania seedlings when incorporated into potting medium prior to seeding.

Water-Corn Oil Emulsion Enhances Conidia Germination and Mycoherbicidal Activity ofColletotrichum truncatum

A water-corn oil (unrefined) emulsion has promise as a carrier for application of the mycoherbicide,Colletotrichum truncatum.We investigated this formulation (normal emulsion; oil in water) for influences on conidia germination, appressoria formation, and mycoherbicidal activity ofC. truncatum.Germination of conidia after application to hemp sesbania leaves was increased from 30% in water alone to 92% in the water-corn oil emulsion (water:oil, 1:1, by vol). The emulsion did not affect appressoria formation. of the several other oils tested, safflower seed oil enhanced in vitro germination but did not enhance germination and mycoherbicide activity when the conidia-emulsion formulation was applied to hemp sesbania leaves. Application ofC. truncatumconidia in the unrefined corn oil emulsion to hemp sesbania seedlings resulted in 89 to 97% control of the weed after a 24 to 72 h dew delay. The emulsion enhanced mycoherbicide efficacy by stimulating conidia germination by protecting the conidia during a dew-free period, hence, increasing weed infection when a dew occurred.

Biological Control of Hemp Sesbania (Sesbania exaltata) Under Field Conditions withColletotrichum truncatumFormulated in an Invert Emulsion

In greenhouse experiments, conidia ofColletotrichum truncatumapplied in an invert emulsion formulation controlled hemp sesbania 100% in the absence of a dew treatment. In field experiments, hemp sesbania control averaged 95 and 97% in 1989 and 1990, respectively, when this formulation was applied to hemp sesbania seedlings using tractor-mounted, air-assist nozzles. This level of weed control was comparable to that achieved from the herbicide acifluorfen. These results indicate thatC. truncatumhas excellent potential as a mycoherbicide for controlling hemp sesbania and that this potential can be augmented by formulating the pathogen as an invert emulsion.

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.

Host Range and Virulence ofColletotrichum truncatum,a Potential Mycoherbicide for Hemp Sesbania (Sesbania exaltata)

A previously unreported anthracnose disease incited by Colletotrichum truncatum was discovered on hemp sesbania seedlings in Washington County, Mississippi, in 1987. Hemp sesbania inoculated with 2×10 7 spores per milliliter in the greenhouse were killed within 7 days. Soybean cvs. Bragg, Bedford, and Hill developed small, necrotic leaf spots, but no further symptoms occurred after 3 wk. Several cultivars of cotton, rice, and 23 other plant species representing eight plant families were immune to disease, as measured by visual assessment(.)