Emergence of Walnut Husk Maggot Adults in Central Illinois and Potential for Control with Metarhizium brunneum

Abstract

The walnut husk maggot, Rhagoletis sauvis (Loew) (Diptera: Tephritidae), causes damage to walnuts when maggots feed inside the husk. September applications of the entomopathogenic fungi Metarhizium brunneum F52 as microsclerotia laced granules to the soil in Illinois were evaluated for pest control based on adult emergence during the following summer. Over 3 yr in central Illinois, adult emergence began near 1 July, peaked before 23 July, and emergence extended as late as 23 August. One summer application of fungus (30 June) when pupae were present, did not reduce fly emergence. Of two September applications that targeted maggots as they move to the soil to pupate, one significantly reduced the number of flies emerging from treated plots when compared with untreated plots for one 7-d sample collected 29 July 2020. Emergence trap data show a defined peak adult emergence in July for central Illinois while September applications of granules containing Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) show shows potential to reduced subsequent fly emergence.

Black walnut (Juglans nigra L.) trees are an economical commodity for many mid-western states in the United States by providing both nuts and lumber. Walnut trees are recognized to suffer from a variety of insect pests. The most important pests tend to be leaf feeding caterpillars along with various wood boring and nut feeding beetles, which may slow tree growth, threaten tree survival, and reduce nut yields. Fruit flies in the Sauvis group of the genus Rhagoletis (Diptera: Tephretidae) are considered pests that damage nut production. Gravid females lay eggs in the husks of the nuts and the developing maggots feed while protected inside the husk. Maggot feeding reduces nut weight, nut protein content, and cause undesirable dark kernels (Baric et al. 2015, Solar et al. 2019, 2020) such that fly control may be warranted. Current controls rely on cultural control by removing fallen walnuts before larvae pupate in the soil and chemical insecticide applications targeting adults to prevent egg laying (Gibson and Kearby 1978). Success of these insecticide treatments is dependent on accurate timing to correspond with fly emergence. Although the maggots are protected while feeding, they are briefly exposed when they exit the husk of a fallen nut to pupate in the soil, which may provide an additional exposed opportunity for control of this pest.

The walnut husk fly (Rhagoletis completa Cresson) has been the topic of recent research because of introductions to western states of Oregon (Kasana and Aliniazee 1996) and California (VanSteenwyk et al. 2014), and Europe (Duso and Lago 2006, Baric et al 2015, Verheggen et al. 2017). Though less reported, the walnut husk maggot [Rhagoletis sauvis (Loew)] is the dominant species infesting black walnuts in midwestern states like Missouri (Gibson and Kearby 1978) and Illinois (Nisar et al. 2019). These two species have similar annual life cycles and cause the same damage to walnuts.

A 3-yr study was conducted to determine whether augmenting entomopathogenic fungi in the soil beneath walnut trees could control larval and/or pupal stages to reduce the number of emerging flies. This study recorded adult emergence data for a population in central Illinois that varied from a population reported in the neighboring state of Missouri (Gibson and Kearby 1978). The fungal treatment was based on laboratory studies that showed larvae to be susceptible to infection by Metarhizium brunneum (Nisar et al. 2019). Treatment evaluations were based on adult emergence as determined by emergence traps that were constructed using commercially available hardware supplies with commercially available sticky traps.

Materials and Methods

Field experiments were conducted in Tazewell County, IL (latitude 48.647730; longitude −89.424620) and this site was selected because it provided larvae used in the study reported by Nisar et al. (2019). The site habitat consisted of mowed turf under large black walnut trees. Four plots (9.3 m²) were staked at each of three locations (12 plots total) within the drip line of five trees ranging from 55 to 85 cm diameter at breast height. The three locations were spaced about 30 m apart. Two plots at each location were untreated and two plots were treated with an experimental granule formulation containing microsclerotia of Metarhizium brunneum strain F-52 (Behle et al. 2015) produced by liquid culture (Behle and Jackson 2014, Jackson and Payne 2016). The fungus was filtered from spent broth, mixed with montmorillonite clay (K-10, Sigma Aldrich, St. Louis, MO) at a rate of 25 g/liter of culture, extruded through a 0.8 mm die to form uniform granules, air dried to <0.2 water activity, vacuum packaged and stored refrigerated until use. Dry granules consisted of 50% fungus and 50% clay by weight and were expected to provide 3.5 × 10⁹ conidia g^-1 when applied to soil (R.W.B., data not shown). Granules were applied to plots using a lawn spreader (Model 42, Gandy Company, Owatonna, MN). The actual amounts of granules applied for each application was determined by measuring the mass of granules before and after application. Granules applications were 5.5, 11.2, and 8.7 g per plot on 30 June 2018 targeting pupae, 10 September 2018 targeting larvae, and 24 September 2019 targeting larvae, respectively.

Bucket emergence traps were placed in plots on 9 July 2018, 10 July 2019, and 24 June 2020. A bucket trap consisted of a 5-gallon white plastic bucket inverted over a Pherocon AM yellow sticky trap (Trécé Inc., Adair, OK) that was supported vertically above the turf with a wire. The wire was from a 50 cm wire stem flag with the plastic flag removed. One end of the wire was bent twice to form a hook that was used to hold the top of the sticky trap. The wire also extended through prefabricated holes in the bottom of the trap for more secure support. A brick was placed on each bucket to prevent wind from blowing it over. The area of soil covered by the bucket was 0.07 m². Four bucket traps were placed next to each other near the center of each plot for a total of 48 traps. The number of flies on each sticky trap were recorded at 7-d intervals after trap placement until no new flies were caught.

Fly emergence data were subjected to analysis of variance comparing treated with untreated plots for each week and for the total flies collected for each season. Each year was analyzed separately. Treated and untreated means were compared using the MEANS option with LSD (SAS 9.4 Software).

In 2020, 10 sticky traps were hung from tree branches to provide a comparison with results of Gibson and Kearby (1978). Unbaited Pherocon AM yellow sticky traps were hung from low hanging tree branches with wire at about 2.2 m above the ground on 22 July. The number of WHM flies stuck to traps were recorded daily and traps were replaced weekly. No lure was used.

Results

In 2018, plots were treated with Metarhizium fungal granules 10 d prior to trap establishment. Significantly more flies emerged from treated plots during the first week of sampling (F_1,36 = 4.26, P = 0.046) compared with untreated plots. Numbers of flies emerging for each of the other weeks of sampling were not significantly different (F_1,36 ≤ 2.00, P ≥ 0.166) as well as the total number of flies captured (F_1,36 = 1.34, P = 0.254) (Table 1). The greatest number of flies were captured during the week ending 23 July (Fig. 1). The total number of flies caught by emergence traps were 39 in untreated plots and 54 in treated plots and calculated to 31.85 flies/m² based on all trap data combined.

Table 1.

Total number of walnut husk maggot adults per emergence trap (mean ± SE) comparing untreated plots and plots treated with Metarhizium brunneum F-52 granules containing microsclerotia

Treatment date	Trapping year	Untreated	Treated
30 June 2018	2018	1.63 ± 0.37	2.25 ± 0.61
10 Sept. 2018	2019	1.21 ± 0.42	0.79 ± 0.23
24 Sept. 2019	2020	4.17 ± 0.72	2.71 ± 0.57

Fig. 1.

Weekly emergence of walnut husk maggot (Rhagoletis sauvis) adults combined for treated and untreated plots in central Illinois determined by inverted bucket emergence traps (2018, 2019, and 2020) and daily catch by sticky traps in the lower tree canopy (2020, Sticky Trap). Error bars represent standard error of the mean.

For the 2019 season, 29 flies were captured by emergence traps in untreated plots and 19 were captured in plots treated with Metarhizium granules the previous fall. The numbers of flies emerging from treated and untreated plots were not significantly different for each week of sampling (F_1,36 ≤ 3.57, P ≥ 0.067) or between the total numbers of flies emerging from treated and untreated plots (F_1,36 = 2.46, P = 0.126) (Table 1). The greatest number of flies were captured during the week ending 17 July, the first week of sampling in 2019 suggesting that the peak may have been earlier in July. The total number of emerging flies captured was calculated to 15.29 flies/m².

In 2020, fewer flies emerged from plots treated (65 flies) with Metarhizium the previous fall compared with untreated plots (100 flies). Significantly (F_1,36 = 6.74, P = 0.014) fewer flies emerged from treated plots (Table 1) for the week after peak emergence ending 29 July, demonstrating that the fall fungus treatment reduced the number of pests surviving to emerge as adults. Weekly emergence numbers were not significantly different between treated and untreated plots for all other sample dates (F_1,36 ≤3.00, P ≥ 0.092) and the total for the year (F_1,36 =3.14, P = 0.085). Overall, more flies were caught by emergence traps in 2020 compared with the two previous years. The entire emergence season was recorded as no flies were captured for the first 7 d after trap placement and evaluations continued until no additional files were captured for a 7-d sample. Fly emergence for 2020 totaled 48.81 flies/m²and the peak emergence aligned closely with that of the 2018 season.

Sticky traps hung in trees did not correspond with emergence trap data in 2020. More flies were captured on these traps during late August, well after emergence had subsided. Ten traps captured 115, 47, 54, 118, 211, and 103 flies for the weeks ending 29 July, 5 August, 12 August, 19 August, 26 August, and 2 September, respectively. Thus, flies remain active in the tree well after emergence from the soil.

Discussion

Gibson and Kearby (1978) reported on extensive studies to evaluate trap captures for walnut groves in Missouri. They concluded that baited Pherocon AM insect traps were most successful and that peak fly emergence occurred in mid- to late-September. This information prompted establishment of emergence traps in July for this study and resulted in missing portions of early fly emergence for the first two seasons. Emergence data presented here clearly show the peak fly emergence occurs in mid-July and may extend to late-August. Sticky traps hung in trees during 2020 showed a peak catch in late August, confirming the observations of Gibson and Kearby (1978), though their report of ‘emergence’ may better be described as adult flight activity. Most of the flies captured by sticky traps in this study were captured by two traps placed in a single tree that was observed to be barring a large crop of nuts relative to other trees, suggesting flies were attracted to this tree for oviposition sites and/or may have been more active, which resulted in higher trap captures. Adult flight prediction models of the R. complete varied significantly between California and Oregon (Emery and Mills 2019) and parameters affecting emergence included latitude, leaf out time, orchard age, and year. These data and models are appropriate for timing of insecticide applications for control of adults but are beyond the scope of the data presented here.

Studies of other Tephritid fruit flies (non-Sauvis group) have shown preimaginal stages to be susceptible to infection by entomopathogenic fungi (Ekesi et al. 2002, 2005, 2011; Cossentine et al. 2010, 2011). Among these studies, Metarhizium isolates were more virulent than Beauveria isolates to late third instar larvae (Ekesi et al. 2002), exposing larvae to soil treated with Metarhizium reduced adult emergence (Ekesi et al. 2011), fungal infection was dosage dependent (Cossentine et al. 2010), and that infection of Metarhizium induced mortality decreased with pupal age (Ekesi et al. 2002). In this study, application of Metarhizium fungus provided mixed results that may be dependent on the timing of application and/or environmental conditions. The walnut husk maggot spends a large portion of its annual life cycle in the soil, from late fall through summer emergence. When considering that older pupae were less susceptible to fungal infection (Ekesi et al. 2002), it seems unlikely that a pupae would become infected, especially for overwintered pupae exposed to the June 2018 application when the insects were about to emerge as adults. The application conditions required for emerging adults to contact sufficient fungal spores to initiate infection have yet to be determined. Similar to other Tephritid flies, walnut husk maggot larvae are susceptible to fungal infection (Nisar et al. 2019), leaving them as a likely target for control as they exit decaying walnut husks to move to the soil for pupation in the fall. Both fall fungal applications in this study, reduced fly emergence nearly 35% and although only one weekly evaluation showed a significant reduction (P < 0.05) for the 2020 adult emergence data.

In conclusion, this study identified peak adult emergence for the walnut husk maggot in central Illinois to occur in July based on simple soil covering bucket traps. This observation differed from tree-hung sticky trap captures that peaked in late August 2020, confirming observations reported for Missouri sites that were also based on tree-hung sticky traps (Gibson and Kearby 1978). The general reduction of adult emergence from plots treated with Metarhizium granules (including one significant weekly observation) suggest additional research is warranted to further develop this biological strategy in support of integrated fruit fly management.