Efficacy Improvement of a Bioherbicidal Fungus Using a Formulation-Based Approach

Greenhouse experiments were conducted to determine the effects of an invert (water-in-oil) emulsion (IE) on dew period duration and dew delay of Colletotrichum coccodes for biocontrol of the problematic weed, eastern black nightshade (Solanum ptycanthum). Dew periods of 4, 8, or 12 h provided 10%, 25%, and 40% control of eastern black nightshade plants, respectively, when C. coccodes (Strain NRRL 15,547) spores were applied in water + Tween 80 surfactant 12 days after inoculation, but a minimum of 16 h of dew was required to achieve ~95% plant mortality. In contrast, at these same intervals of dew, 95%, 100% and 100% mortality occurred, respectively, when fungal spores were formulated in the IE. Even in the absence of dew, 60% mortality and 70% dry weight reductions of plants was achieved with the fungus/IE formulation Delaying dew by 2 h after inoculation did not significantly reduce weed control or plant dry weight reductions when plants were inoculated with the fungus either in the aqueous or in the IE formulation. However, when dew was delayed for 4, 8, or 12 h, only 60%, 50%, and 25% mortality, respectively, of plants receiving the aqueous spore treatment occurred. In contrast, 95%, 90%, and 90% weed mortality occurred after the same dew delays of plants receiving the fungus/IE treatments. These results demonstrate that formulating C. coccodes spores in an invert emulsion greatly improves the bioherbicidal potential of this fungus. Furthermore, results suggest that this formulation may render pathogens previously rejected for development as bioherbicides due to restrictive dew requirements more efficacious for use in controlling their target weeds.


Introduction
Strategies using plant pathogens as bioherbicides to manage weeds have been examined as demonstrated in several reviews [1]- [10].In order to achieve greater predictability and acceptability, it is necessary to maximize the capability of a bioherbicidal pathogen to infect, kill, or reduce the competitiveness of the weed host.Many fungi, including most bioherbicidal fungi, have a requirement for free water, such as dew, for germination and infection to occur [7] [11] [12].Increasing the infectivity (ability to infect host) of a bioherbicidal fungus in any given dew period, theoretically will increase the effectiveness of pathogen [13].
One strategy to reduce lengthy dew requirements of bioherbicidal fungi is the use of selected adjuvants including surfactants, stickers, humectants, and anti-evaporation agents [7] [9] [14].Previous research in our laboratory and elsewhere has shown that invert (water-in-oil) emulsions and various vegetable oil-in-water emulsions provide a method to retard evaporation and trap water around fungal spores in the spray mixture, thereby decreasing the amount of additional free-moisture (dew) required for spore germination and infection [15]- [21].We have previously reported greenhouse data that show a 1:1 (v/v) fungus/invert emulsion could reduce the dew period requirements for maximum weed infection and mortality of sicklepod [Senna obtusifolia (L.) H.S. Irwin & Barneby] with the fungus Colletotrichum gloeosporioides (Penz.)Penz.& Sacc, from 16 h to 8 h, and could also delay the need for free moisture for >48 h [22].Emulsions of unrefined corn oil and spore suspensions of C. truncatum (Schw.) Andrus & Moore also reduced the dew period requirements for maximum infection and mortality of hemp sesbania [Sesbania exaltata (Raf.)Rydb.ex A.W. Hill] from 12 h to 2 h, and delayed the need for free moisture for >72 h [18].
In addition to dew period requirements, the length of time following bioherbicide application and the onset of dew can have negative effects upon infection and control of weeds.For example, dew treatment delays of only 1 h resulted in a 60% reduction of hemp sesbania control by C. truncatum spores in an aqueous formulation [23].A dew treatment of 8 h within two days after inoculation resulted in 95% control of sicklepod inoculated with spores of Alternaria cassia Jurair & Khan formulated in 0.02% nonoxynol (0 -10 POE) [α-(p-nonylphenyl)-ω-hydroxypoly (oxyethylene)] surfactant [24].
Previous research indicated that a strain (NRRL 15,547) of the fungus Colletotrichum coccodes (Wallr.)S. Hughes had bioherbicidal potential for controlling eastern black nightshade (Solanum ptycanthum Dun.) [25] [26], a problematic weed native to the Americas, and commonly found throughout the United States, east of the Rocky Mountains [27].However, spores of this fungus required a lengthy dew period (≥16 h) in order to germinate, establish infection, and cause disease, and thus it was concluded that this extensive dew period requirement limited the practical use of this pathogen as a mycoherbicide [25].Various studies of the effects of dew period on the efficacy of commercial and other potential bioherbicides, and methodologies to overcome unrealistically long dew period requirements have been reported [7].The objective of the present investigations was to determine if the dew period requirements of C. coccodes to infect and kill eastern black nightshade could be significantly reduced and delayed by formulating fungal spores in an invert emulsion.This research could have important implications since it may also be applicable to certain other fungi that have been considered impractical for use as bioherbicides because of lengthy dew period requirements.

Culture and Inoculum Preparation of C. coccodes
Cultures of C. coccodes (NRRL 15547) were re-generated from soil-tube [28] stock cultures from the USDA-ARS Southern Weed Science Laboratory Fungal Repository (currently the USDA-ARS Biological Control of Pests Fungal Repository) and grown on half-strength Emerson's yeast-starch agar [Yeast extract (4.0 g/L), soluble starch (15.0 g/L), Na 2 HPO 4 (1.0 g/L), MgSO 4 (0.5 g/L), agar (15.0 g/L), distilled water (1000.0ml); pH adjusted to 6.8] in petri dishes that were inverted on open-mesh wire shelves in an incubator (Precision Scientific Inc., Chicago, IL, USA) at 25˚C.Photoperiods (12 h) were provided by 2, 20-W cool white fluorescent lamps positioned 12 cm above each shelf to provide 200 μE•m −2 •s −1 .Spores were harvested by rinsing the cultures with distilled water and filtering debris through 4 layers of cheesecloth.Freshly harvested spores were used as inoculum for all experiments.The spore concentration in test preparations was estimated using hemacytometers.Long-term storage of the fungus was achieved in screw-capped tubes containing sterilized soil, maintained at 4˚C [28].

Culture of Plants
Eastern black nightshade seeds were obtained from C.T. Bryson (USDA-ARS, Stoneville, MS, USA).Seeds were surface-sterilized in 0.05% NaOCl for 5 min, rinsed with sterile distilled water, and germinated on moistened filter paper in petri dishes.After the seeds germinated (~48 h), they were planted in a commercial potting mix (Jiffy-mix; Jiffy Products of America, Batavia, IL, USA) contained in peat strips.Each strip contained 12 plants.The potting mix was supplemented with a controlled-release (14:14:14, NPK) fertilizer (Osmocote; Grace Sierra Horticultural Products, Milpitas, CA, USA).The plants were placed in sub-irrigated trays mounted on greenhouse benches.Greenhouse temperatures ranged from 25˚C to 30˚C with 40-90% relative humidity (RH).The photoperiod was approximately 14 h, with 1800 μE•m −2 •s −1 (PAR) as measured with a light meter (LI-COR, Inc., Lincoln, NE, USA) at midday.Seedlings at the 3 -4 leaf stage were used in all experiments.

Dew Period Duration and Dew Delay Studies
Following treatments, seedlings were placed either directly on greenhouse benches (0 h dew) or in darkened dew chambers (Model I-36 DL; Percival Sci.Ind., Perry, IA, USA) at 25˚C, 100 RH for 4, 8, 12, 16, 20, or 24 h, and then placed on greenhouse benches.To study the effect of dew treatment delays, plants were either placed directly in a dew chamber for 16 h (under conditions as described) or subjected to dew delays of 0, 2, 4, 8, 12, 16, 20, 24, 48, or 72 h before being subjected to a 16 h dew treatment.Plants were monitored at 3-day intervals for disease kinetic studies, and determinations of mortality and dry weight reductions over a 12-day period after treatment.A subjective visual disease severity rating scale (per plant basis) [19] was used to estimate disease progression where 0 = no disease, 1 = 1% -25% disease, 2 = 26% -50% disease, 3 = 51% -75% disease, 4 = 76% -99% disease, and 5 = plant death.Disease ratings ≤ 2.0 were considered "slight", 2.1 -3.9 were considered "moderate", and ≥ 4.0 were considered "severe".Surviving plants were excised at the soil line, oven-dried for 48 h at 85˚C, weighed, and the percent biomass reduction was determined.Treatments were replicated four times, for a total of 48 individual plants per treatment.The experiment was repeated over time, and data were averaged following Bartlett's test for homogeneity of variance [29].A randomized complete block experimental design with four replications was utilized.The mean percentage of plant mortalities and biomass reductions were calculated for each treatment, and were subjected to Arcsin transformation.The transformed data were statistically compared using analysis of variance (ANOVA) at the 5% probability level.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 a least significant difference of 0.05.In the dew period and disease kinetic studies, best-fit regression analysis was also utilized.

Results
Dew periods of 4, 8, or 12 h provided 10, 25, and 40% control of eastern black nightshade plants, respectively, when C. coccodes spores were applied in water + Tween 20, but at least 16 h of dew was required to achieve ~95% mortality of plants.Some mortality and dry weight reductions occurred with 0 h of dew, which was attributed to the wet conditions of the potting mix (Figure 1(A) & Figure 1(B)).In contrast, at these same dew period lengths, 95%, 100% and 100% mortality occurred, respectively, when spores were formulated in the invert emulsion.Even with no dew (0 h), 60% mortality and 70% dry weight reductions of plants was achieved when the fungus was formulated in the invert emulsion 72 h after inoculation (Figure 1(A) & Figure 1(B).Minimal (10% -15%) mortality and dry weight reductions were observed on plants treated with the invert formulation alone.
In the delayed dew studies, a 2 h dew delay did not significantly reduce weed control or dry weight reductions when plants were inoculated with C. coccodes in either the aqueous or in the invert emulsion treatment (Figure 2(A) & Figure 2(B).However, when dew was delayed for 4, 8, or 12 h, only 60%, 50%, and 25% control, respectively, of eastern black nightshade plants receiving the aqueous spore treatment was observed.
In contrast, 95%, 90%, and 90% weed mortality occurred after the same dew delays to plants receiving the fungus-invert emulsion treatments (Figure 2(A)).Dry weight reductions followed similar trends (Figure 2(B)).In the disease kinetic studies, after 3 days, plants that received the aqueous spore treatment were only slightly infected (disease rating 2.0), while plants that were treated with the fungal-invert emulsion treatment were severely infected (disease rating 4.5) after this time period (Figure 3).After 6 days, plants receiving the aqueous fungal treatment were still only moderately infected (disease rating 2.9), while 100% mortality occurred to plants treated with the fungus-invert formulation; 9 days were required before severe (disease rating 4.8) levels of disease were achieved on plants receiving the aqueous fungal treatment (Figure 3).
Although it has been suggested that dew period requirements ≥ 16 h could limit the practical use of fungi as biological control agents for weeds [3], through innovative use of formulations, such as invert emulsions, it may be possible to render such bioherbicidal fungi more useful for controlling their target weeds.Minimal (5% -15%) mortality and dry weight reductions were observed on eastern black nightshade plants treated with the invert formulation alone, which could have facilitated fungal entry and infection.This invert emulsion can also cause injury to some other species of weeds [36] and caution should be taken to avoid potential damage to crops associated with the target weed.These results provide further evidence that bioherbicidal efficacy can be improved using bio-based formulation approaches.Further research is needed to test this formulation for control of eastern black nightshade under field conditions.

Conclusion
The unpredictability of the occurrence of dew (or other free-moisture events) is a major factor influencing the efficacy of bioherbicidal fungi.Thus, the ability to mitigate environmental factors, such as requirements for lengthy dew periods, that restrict or limit a bioherbicidal microorganism's efficacy, can significantly improve their bioherbicidal potential.This research could have important implications since it may also be applicable to certain other fungi that have been considered impractical for use as bioherbicides because of lengthy dew period requirements.

Figure 2 .
Figure 2. Effects of delayed dew period on mortality (A) and dry weight reduction (B) of eastern black nightshade treated with C. coccodes formulated either in water + Tween 80 (0.05% w/v) surfactant or an invert emulsion, 12 days after inoculation.Treatments: 1) C. coccodes in water + Tween 80 (0.05% w/v) surfactant (gray bars); 2) C. coccodes in invert emulsion (white bars); water + Tween 80 control (red bars); 3) invert only control (blue bars); 4) untreated control (green bars).All plants were subjected to a 16-h dew period immediately, or after each delay time, and mortality and dry weight reductions were determined 12 days after inoculation.Error bars denoted with the same letter are not significant at p = 0.05 according to FLSD.