Interaction of a Myrothecium verrucaria Mycelial Preparation and a Glyphosate Product for Controlling Redvine (Brunnichia ovata) and Trumpet Creeper (Campsis radicans)

A mycelial formulation of the bioherbicidal fungus Myrothecium verrucaria (Alb. & Schwein.) Ditmar: Fr. (MV) was tested alone and in combination with a commercially available glyphosate [N-(phosphonomethyl)glycine] (GLY) product for controlling the invasive vines, redvine [Brunnichia ovata (Walt.) Shinners], and trumpet creeper [Campsis radicans (L.) Seem. ex Bureau] in field experiments conducted near Stoneville, MS. Several application timing regimens were evaluated (Fall, Spring, Fall + Spring, and Spring + Fall). We found that a Fall + Spring application of MV + GLY controlled redvine and trumpet creeper by 95%, 12 days after the second treatment, through a synergistic interaction of the fungus and glyphosate. Disease symptomatology was characterized by rapid necrosis of leaf and stem tissues, with mortality occurring within 72 h. Neither glyphosate alone, nor MV alone, effectively controlled either weed species under any application timing regimen. No visual disease or herbicide damage occurred on glyphosate-resistant soybean plants in the treated test plots. These results suggest that some formulations of glyphosate, mixed with the bioherbicide MV, can effectively control redvine and trumpet creeper, two of the most troublesome weeds in the row crops of the Mississippi Delta region in the mid-southern U.S.


Introduction
Redvine [Brunnichia ovata (Walt.) Shinners] and trumpet creeper [Campsis radicans (L.) Seem. ex Bureau] are native perennial, deciduous, woody, dicotolydenous, vines capable of growing several meters in length [1]. These weeds are distributed extensively in the lower Mississippi Alluvial Plain area (Mississippi Delta) of the southern U.S., often found in dense populations in cultivated and fallowed fields, wastelands, fence rows, yards, riverbanks, swamps, and forests.
These weeds have been rated among the ten most troublesome weeds in the row crops of the Mississippi Delta region, since they reduce crop yield and quality, and interfere with cultivation and harvest operations [1].
Glyphosate with approximately 90% of soybean acres sown to glyphosate-resistant varieties [2]. Glyphosate is a broad-spectrum post-emergence herbicide with some herbicidal activity on redvine and trumpet creeper [3] [4] [5]. However, control of these weeds with glyphosate alone, even at rates two-to-four times the rates recommended in non-GMO soybeans, is temporary at best and when used alone cannot satisfactorily control these weeds [5]. The use of fungi and bacteria as inundative biological control agents (bioherbicides) has been recognized as both a viable method for controlling agronomic weed pests and a significant technological alternative to chemical herbicides [6]- [11]. Considerable interest exists worldwide regarding bioherbicides [9] [10] [11] [12] and there is an acute need to develop non-chemical or bioherbicidal weed management tools and strategies as alternatives to chemical weed control as summarized in several reviews [8]- [18].  [25]. Although redvine and trumpet creeper were not included in MV host range tests encompassing many plant species [19], later experiments revealed that both redvine and trumpet creeper were susceptible to infection by spores of this fungus, but not at levels that would provide adequate weed control [26]. In subsequent research conducted under field conditions near Stoneville, MS, USA, MV spore formulations applied alone and in combination with a glyphosate product (Touchdown™) controlled natural infestations of redvine and trumpet creeper 88% and 90%, respectively, through a synergistic interaction of the fungus and glyphosate [27]. Neither glyphosate alone, nor MV alone, controlled these weeds at commercially acceptable levels (80%).
Although MV can potentially control several weed species, its production of macrocyclic trichothecenes (mycotoxins) by fungal spores [28] [29] [30] [31] is an issue that limits its practical usage. Thus, a biologically effective, mycotoxin-free formulation could greatly expand the bioherbicidal potential of this bioherbicide. Experiments in our laboratory had previously demonstrated that a spore-free liquid culture of MV, comprised of mycelia fragments (void of trichothecenes), exhibited high bioherbicidal activity against the weed kudzu under field conditions [32]. Due to the high efficacy exhibited by MV mycelia against several weeds [20] [33] [34] [35] [36], we hypothesized that a mycelial formulation of MV, used in conjunction with a compatible glyphosate product, may also have potential for controlling redvine and trumpet creeper. Thus, the objectives of this present study were to determine the effects of combined applications of a glyphosate product (Touchdown™) and a mycelial-based MV formulation on biological control of redvine and trumpet creeper in field plots where greenhouse-grown seedings of these weeds had been transplanted, and to characterize possible combined effects of glyphosate and this bioherbicide as additive, antagonistic, or synergistic interactions. (mycelial preparation grown in shake-flasks). The shake-flask medium (soy flour-corn meal) was inoculated with a 10 mm agar plug (~10 6 spores•mL −1 ) from a petri dish of MV spores. The flask was incubated on a rotary shaker (185 -200 rpm, 28˚C, 7 days) and mycelial fungal growth proceeded without spore production. The MV mycelial product produced via fermentation for 48 -72 h was harvested and stored at 4˚C until use. Concentrations of the mycelial formulations used in these tests were based on percent (v/v basis) of the fermentation batch as described elsewhere [36]. That procedure consisted of determining the viable propagule density (colony forming units; cfu) of the MV mycelial fermentation product in diluted samples (1.0 mL product:1.0 L sterile H 2 O) after thoroughly mixing under sterile conditions, then plating aliquots of the mixture onto PDA in petri dishes, incubation of plates (28˚C), and lastly counting colonies after 48 h growth. Dry weight analysis of freshly prepared MV mycelium revealed that ca 50 g•L −1 were produced. Appropriate dilutions were made to obtain a concentration of 2.0 × 10 7 cfu•mL −1 for further studies.

MV Source and Production
The MV mycelial product used in these studies was analyzed for trichothe-cenes as described [32]. Samples of raw culture fermentation product (mycelium and unspent medium) were extracted by shaking in ethanol [1:1 culture product:ethanol (v:v)] at 22˚C for ≥ 2 h at 125 rpm. The mixture was centrifuged (14,000 RCF, 10 min, to remove particulate matter, and 1.0 mL of the clarified supernatant was transferred to amber glass vials. Trichothocenes were detected by UV absorbance after separation on a Polar Advantage II monolithic column (3 µm, 4.6 × 50 mm, Dionex, Sunnyvale, CA, USA) using a Dionex HPLC system. The mobile phase was: (A) 0.1% acetic acid in water and (B) 0.1% acetic acid in acetonitrile. Binary gradient elution starting conditions were: 35% B for 2 min, followed by an increase to 50% B at 4.5 min; 58% at 6 min; 70% at 7.29 min; 80% at 7.84 min; 95% at 8.5 min, holding for 0.5 min before returning to initial conditions. Roridin A and verrucarin A (Sigma Chemical Co., St. Louis, MO, USA) were used as standards. The limit of detection in this protocol was 2 µg•mL −1 [32].   Immediately prior to treatment, plants were mowed to uniform height (~15 cm).

Bioherbicide and Herbicide Treatments of Field Plants
Glyphosate was applied at a rate of 1.12 kg a.i. ha −1 . All applications were applied at 100 L•ha −1 using a pressurized backpack sprayer. Twenty-five plants of each species were randomly selected and tagged prior to treatment for assessment of plant injury and weed control days after treatment (DAT). The extent of plant injury was based on symptom expression from 0 to 1.0, where 0 represented unaffected plant tissues and 1.0 represented plant mortality. The selected plants were monitored over 12 days for injury development. Plant injury was considered "severe", and weeds were considered "controlled" at ratings of 0.80 to 1.0.
After this data was collected, the above-ground plant material from the selected plots was harvested and dried at 80˚C for 72 h and biomass values were recorded for each sample. For treatments receiving two treatments, mortality and dry weight determinations were conducted 12 days after the second treatment. Following the Spring treatment applications, soybeans (DP 5915RR) were planted in the treated areas 40 DAT, soybean plant heights were measured and compared to soybean plant heights in the untreated plots. A randomized complete block experimental design with four replications was utilized. The mean percentages of plant mortalities and biomass reductions were calculated for each treatment and subjected to Arcsin transformation. The transformed data were statistically compared using analysis of variance (ANOVA) (P = 0.05). Results were back-transformed to the original measurements (percentages) for presentation.
Data were analyzed via the PROC MIXED function of SAS v9.3 (SAS Institute, Cary, NC, USA) using the least significant difference of 0.05. In the plant injury kinetic studies, data were analyzed using standard mean errors and best-fit regression analysis. The experiment was repeated, and data over the two years were averaged, followed by subjection to Bartlett's test for homogeneity of variance [37].

Quantification of Interactions
Interactions between components in mixtures were analyzed according to Colby [38], using the formula E = X A Y B /100 in which X A and Y B represent weed control as a percentage of the control, with herbicide A (glyphosate) used at dosage p and (bio)herbicide B (MV) used at dosage q, respectively. E is the expected survival as a percentage of the control for mixture A and B at dosages p and q. The observed response is obtained experimentally by comparing the activity of single compounds with mixtures containing the same rate of the constituents as applied singly. A deviation from the expected response, as calculated from the level of interaction R between the expected and the observed response of the two compounds, could indicate synergism or antagonism. By definition, interactions are classified as additive when R = 1.0, synergistic when R > 1.0 and antagonistic when R < 1.0. Due to the inherent biological variability of the test systems, synergistic and antagonistic interactions are considered statistically significant when R ≥ 1.5 and R ≤ 0.5, respectively and additive interactions are significant at R values between 0.5 and 1.5 [39].

Results and Discussion
Redvine and trumpet creeper were each controlled 95% in the Fall + Spring timing regimen 12 DAT when MV mycelia preparations were tank-mixed with glyphosate ( Figure 1(a) & Figure 2(a)). The Fall + Spring application regimen provided significantly greater control of trumpet creeper (95%) than that from the other timing regimens (85% for Fall, Spring, or Spring + Fall). However, a single Fall treatment of MV + GLY + SW controlled redvine at levels that were not statistically different than levels achieved by a Fall + Spring treatment (90% and 95%, respectively) (Figure 1(a)). Neither glyphosate + SW alone, nor MV + SW alone, effectively controlled either weed under any of the timing regimens ( Figure 1(a) and Figure 2(a)). Dry weight reductions of plants followed a similar trend (Figure 1(b) & Figure 2(b)). Because the expected survival and observed survival ratios of both redvine and trumpet creeper receiving an application of MV + GLY + SW were greater than 1.5 [39], it is concluded that these interactions were synergistic (Table 1). In the plant injury, kinetic studies, only data from plots receiving the MV + SW, MV + GLY, and MV + GLY + SW treatments are presented. In the Fall application regimen, redvine plants treated with MV + SW, GLY + SW, or MV + GLY + SW, a 2nd degree polynomial regression curve provided the best fit for each curve (R 2 = 0.96, 0.98, and 0.96, respectively) (Figure 3(a)). Similarly, 2nd degree polynomial regression curves also provided the best fit for each curve (R 2 = 0.96, 0.98, and 0.98, respectively) for those treatments applied to trumpet creeper in the Fall regimen ( Figure   3(a)). In the Spring regimen, redvine plants treated with MV + SW, GLY + SW, or MV + GLY + SW, a 2nd degree polynomial regression curve provided the best fit for each curve (R 2 = 0.96, 0.98, and 0.96, respectively) (Figure 3(b)).
When trumpet creeper plants were treated with MV + SW, GLY + SW, or MV + SW + GLY in the Spring regimen, 2nd degree polynomial regression curves also provided the best fit for each curve (R 2 = 0.98, 0.96, and 0.98, respectively) ( Figure 3(b)). Soybeans planted in treated plots emerged normally and no reductions in plant height occurred (data not shown).
We have previously shown that, under controlled environmental conditions, a synergistic effect occurred when MV spores were applied two days after  Louis, MO 63167) to redvine and trumpet creeper, while an antagonistic interaction occurred when glyphosate and MV spores were applied simultaneously to plants [26]. Subsequent field experiments revealed that MV spore formulations  applied in combination with a glyphosate product (Touchdown™) controlled natural infestations of redvine and trumpet creeper 88% and 90%, respectively, through a synergistic interaction of the fungus and glyphosate [27]. Neither glyphosate alone, nor MV alone, controlled these weeds at commercially acceptable levels (80%). Other plant pathogens have also been shown to exhibit synergistic interactions with glyphosate [40] [41] [42] [43] and application of MV plus glyphosate resulted in a synergistic interaction for hemp sesbania control [44].
Other research in our laboratory has shown that different proprietary glyphosate formulations, as well as other commonly used chemical pesticides, have dramatically varying effects on conidial germination, radial growth, and the biocontrol efficacy of MV, suggesting that variations in glyphosate formulations (e.g., surfactants) may have interacted negatively with the bioherbicide [45].
In these present studies, we conclude that there was a synergistic effect upon weed control of redvine and trumpet creeper when a MV mycelial product and the glyphosate product Touchdown TM were tank-mixed. Because weed control was not increased by additional surfactant (SW), it was concluded that the increased weed control was due to synergy between MV mycelia and glyphosate. These findings are corroborated by other research using high purity glyphosate (without adjuvants/surfactants). In those studies, technical-grade glyphosate was not toxic to MV growth and sporulation at concentrations up to 2.0 mM when grown on agar supplemented with the herbicide, and an interaction of glyphosate plus MV mycelia was synergistic in controlling Palmer amaranth (Amaranthus palmeri S. Wats.) [36]. The bioherbicidal potential of this strain of MV has been thoroughly established, based on findings both in our laboratory and elsewhere [19] [36]. The results reported herein suggest that it is possible to enhance the bioherbicidal potential of a mycelial formulation of MV through synergistic interactions with compatible chemical herbicides, such as glyphosate and to control aggressive and established weeds such as redvine and trumpet creeper under field conditions.