A population of seeds of the weed, Palmer amaranth, was collected from a site near Stoneville, MS, USA [14]. This population contained two distinct biotypes that exhibited resistance to the herbicide glyphosate, one biotype that produced the typical red-pigmented Palmer amaranth plants (Red biotype), and one biotype (Green biotype) that produced no visible red pigment as reported earlier [14]. Seeds were planted in a commercial potting soil under greenhouse conditions (22˚C - 25˚C; 16 h photoperiod). When seedlings were about 30 mm tall (cotyledon: first true-leaf stage), they were transplanted into individual pots (~75 L × 55 W × 50 D mm) containing potting soil and grown under greenhouse conditions to various growth stages for testing. Plants were watered with de-ionized water and fertilizer [N:P:K (13:13:13)] was provided routinely.

M. verrucaria (IMI 368023) originally isolated from sicklepod (Senna obtusifolia (L.) H.S. Irwin & Barneby) was cultured in Petri dishes on potato dextrose agar (PDA) (Difco Laboratories, Inc., Detroit, MI, USA). Inoculum from these dishes was used to generate mycelial cultures in a fermenter as described elsewhere [44]. This fungal product, consisting of mycelium and unspent growth medium without spores, was substantially lower in trichothecene content compared to spore cultures of this fungus [44]. The mycelial formulation from the fermenter was used full-strength (typically ~1.0 × 10⁷ cfu∙mL⁻¹) and a surfactant (Silwet L-77) was added to achieve a formulation containing 0.20% (v/v) surfactant.

Ten to twelve uniform-sized seedlings of each biotype at two growth stages (3-week and 6-week-old) were sprayed with MV plus Silwet (0.20%) mycelial formulation using compressed air spray canisters (Crown Spra-Tool, North American Professional Products, Woodstock, IL, USA). Plant leaves were sprayed until run-off, corresponding to an application rate of ~300 L∙ha⁻¹. Control plants received applications of Silwet only (0.20% in H₂O). All spray applications were performed in a biosafety cabinet (NuAire, Model No. NU-425-400, Plymouth, MN, USA). Immediately following treatment, plants were moved to a dew chamber (Percival Scientific, Model No. 1-35 DL, Boone, IA, USA) maintained at 25˚C for 16 h. At the end of the dew period, plants were placed under greenhouse conditions [27˚C - 31˚C, 45% - 65% RH, and a 14 h day, at 1650 - 1825 μE∙m⁻²∙s⁻¹ (photosynthetically active radiation) PAR] and observed and/or sampled as required for measurements.

Plants were visually examined for injury symptoms at various times after MV application. The fresh weights and dry weights of plant shoots (excised at the soil surface) were measured 5 days after MV application to assess MV effects on growth. Dry weight determinations were made on shoot tissue placed in paper bags and dried in an oven (90˚C to 98˚C, 48 h).

Progression or severity of the disease incited by MV spray application [MV mycelial fermentation product containing ~1.0 × 10⁷ cfu∙mL⁻¹] to these biotypes (6-week-old) after MV was applied as a spray prepared in 0.20% Silwet was monitored at several intervals over a 5-day period. A disease rating scale (modified from Horsfall and Barratt [45]) was used to visually evaluate bioherbicide efficacy. The numerical scale defined 0 as equivalent to no infection, and values of 1.0, 2.0, 3.0 and 4.0 represented 20%, 40%, 60%, and 80% leaf/stem lesion coverage and/or injury, respectively. Plant mortality was set at a value of 5.0. Disease ratings of 3.0 to 5.0 were considered “severe”. Standard errors of means and regression analyses were used to evaluate and pattern the data.

The experimental design was a randomized complete block and each treatment comprised 10 to 12 plants of each age. The treatments were performed in triplicate and all experiments were repeated in time. The data were compared using analysis of variance (P = 0.05%). Values shown are means of replicated experiments. When significant differences were detected (F-test), means were separated (Fisher’s protected LSD; P = 0.05). Error bars presented in graphics are ±1 SEM (standard error of the mean).

Visual appearance of the two Palmer amaranth biotypes used in this study is strikingly different (Figure 1). Pigmentation differences in the biotypes are discernable upon emergence of seedlings from the soil and at about 6 to 8 days after emergence (2- to 3-leaf stage; Figure 1, top) red pigmentation typically occurs in stems and leaf petioles of the red biotype, while no red pigments are visibly apparent in the green biotype. Examination of abaxial surfaces of these young leaves and cotyledons, again demonstrates red pigmentation in red biotype, but green biotype tissues are green (Figure 1, bottom). At later growth stages (Figure 2) and through seed production, each biotype retained their respective colorations, with the red usually deepening in color and the green often becoming more yellowish-green with time as shown in Figure 2 (bottom), comprised of excised stems from several more mature plants. The upper portion of the red biotype has much less red pigmentation than in its stems and petioles. Examination of mature roots of the green biotype also indicated no appearance of red pigmentation (data not presented). The red pigment is a betalain (betacyanin) and the green biotype may be void of betalains (red and yellow classes), or it may produce only betalain(s) belonging to the yellow class. Concurrent studies on the comparative characterization of these two biotypes are underway that will be published elsewhere.

Both Palmer amaranth biotypes exhibited some bioherbicidal effects of MV inoculation as early as 16 h after inoculation, when plants were removed from a high humidity cabinet and placed in a greenhouse (data not shown). These early effects were exemplified as some epinastic effects, slight wilting and initial development of lesions on leaf and stems. Generally, the younger (3-week-old) plants tended to be more sensitive to the MV inoculation than the older plants (6-week-old). Much more bioherbicidal damage occurred with time and at 48 h after inoculation with this fungus, effects were more severe (Figure 3). Epanastic curvature of stems, wilting/dehydration, necrosis and leave-drop were apparent effects in both biotypes. At this time point, the green biotype appeared slightly more damaged by the fungus than the red biotype. This effect was supported by fresh weight analysis from plants at the 48-h time point (Figure 4). Severe loss of fresh weight occurred in both biotypes compared to that of untreated their re-spective control plant shoots. The green biotype was slightly more damaged with

respect to fresh weight biomass.

Three-week-old green biotypes were generally more susceptible to infection by MV than were red biotype plants at this age (Figure 5). Severe infection of 3-week-old biotypes (disease rating of 3.8) occurred after 48 h, and increased to 4.5 after 96 - 120 h. In comparison, a disease rating of only 2.6 occurred on 3-week-old red biotype plants after 96 h, but this increased to a disease rating of 3.5 after 120 h (Figure 5). In the biotypes, MV disease progression and severity were generally greater on 3-week-old plants as compared to 6-week-old plants (Figure 5).

The green biotype might be compromised under natural environmental conditions and stress since betalains have been shown to be involved in plant photoprotection mechanisms. For example, photosynthetic capacity damage was reduced in red-pigmented versus green leaves after exposure to excess light [46] [47] [48]. Betalain synthesis in a related plant, Amaranthus tricolor, has been shown to be under photocontrol [49]. Other reports show that betalain production in plants is upregulated after exposure to light or UV radiation [50] [51] [52].

There was a definite trend that the green biotype was somewhat more susceptible to MV when treated under greenhouse conditions. This may suggest that lack of the red betacyanin pigment renders the green biotype less resistant to pathogen attack. As outlined and presented earlier, betacyanin has been implicated in disease resistance. Further testing of the effects of MV on these two biotypes under field conditions and expanded characterization of the pigment contents and traits of these two biotypes (work in progress) will help to clarify the interaction of the bioherbicide, Myrothecium verrucaria on these two glyphosate-resistant Palmer amaranth biotypes.