SUGARBEET ROOT MAGGOT CONTROL WITH METARHIZIUM ANISOPLIAE

L. G. Campbell¹, G. A. Smith¹, J. D. Eide¹, and L. J. Smith² ¹USDA, Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND and
²Northwest Experiment Station, University of Minnesota, Crookston, MN

Root yield losses attributable to sugarbeet root maggot (Tetanops myopaeformis von Röder) damage can be significant in a number of sugarbeet (Beta vulgaris L.) production areas (Yun and Sullivan, 1980; Blickenstaff et al., 1981), including the Red River Valley. In the absence of control measures, yield losses of 40% would be common in portions of Minnesota and eastern North Dakota (Campbell et al., 1988). The primary control method is the application of a granular insecticide at planting time. Two organophosphate insecticides, Counter (terbufos) and Lorsban (chlorpyrifos ), are used extensively throughout the region (Dexter, et al., 1998). This almost exclusive use of a few similar chemicals is conducive to the development of insecticide resistant root maggot strains. Organophosphate resistance has been confirmed in other species, including other Diptera (Bisset, et al., 1990; Cilek, et al., 1991). Alternative root maggot control strategies also would be required if current insecticides were no longer available because of environmental concerns.

Metarhizium anisopliae (Metschnikoff) Sorokin , one of approximately 700 species of entomopathogenic fungi (Roberts, 1989), has received attention as a biocontrol agent in numerous agricultural applications (Samson et al., 1994; Kaaya and Munyinyi, 1995; Rath et al., 1995; Booth and Shanks, 1998). Identifying characteristics and descriptions of Metarhizium are readily available (Humber, 1997) as are guidelines for isolating, propagating, and evaluating survival in the field (Goettel and Douglas, 1997). The environmental conditions favoring Metarhizium have been characterized (Walstead et al., 1970; Li and Holdom, 1995). The use of Metarhizium to control insects began in the late 1800's (Metchnikoff, 1879), or earlier. Interest in biocontrol solutions continued until the development of efficient chemical pesticides in the 1940's. Recent concerns regarding the long-term effects of chemical pesticides has prompted renewed interest in the utilization of biocontrol agents as components of integrated pest management schemes that may continue to include some chemical pesticides. Increased consumer demand for organically produced food also has stimulated development of biocontrol solutions to pest problems.

This report summarizes results from preliminary field evaluations of Metarhizium anisopliae as a biocontrol agent for sugarbeet root maggot. It encompasses two years of exploratory research at Hillsboro and Saint Thomas, North Dakota plus three years of more detail trials at Crookston, Minnesota.

Field trials were conducted near Crookston, Minnesota. The experimental design was a randomized complete block with 6 replicates. Field plots were 770 ft²and consisted of 12 rows (22-inch spacing). Plots were planted with a commercial planter on May 15, 5, and 6 in 1996, 1997, and 1998, respectively and thinned to 31,000 plants / acre when plants were in the seedling stage. Weeds were controlled with herbicides, cultivation, and hand weeding. Cercospora leaf spot (Cercospora beticola Sacc.) was controlled with fungicides, when necessary. Harvest dates in 1996, 1998, and 1997 were September 25 and 23, and November 5, respectively. Plants were defoliated with a mechanical defoliator and the center two rows harvested with a commercial type harvester on the same day. A 10-15 beet sample from each plot was sent to the American Crystal Sugar Company tare laboratory for sucrose and quality analysis.

Autoclaved barley inoculated with M. anisopliae provided the inoculum for the field trials. The dried inoculated barley was spread evenly over the center six rows of field plots with a fertilizer spreader at a rate of 0.75 tons/acre (per application) and incorporated with a field cultivator. Spore (conidia) concentrations at the time of application ranged from 2.2 ´ 10¹³to 3.7 ´ 10¹³ spores / acre. Inoculum for the 1996 sugarbeet crop was applied in the fall proceeding the crop (13 October 1995), in the spring prior to planting (14 May 1996), or both in the fall and spring. The Metarhizium treatments for the 1997 sugarbeet crop consisted of fall (3 October 1996) and spring (5 May 1997) applications, a double rate (1.5 ton inoculated barley / acre ) spring application, and a fall plus spring application; all with an additional Metarhizium application (23 May 1996) prior to planting the barley crop proceeding the sugarbeets. Treatments for the 1998 sugarbeet crop consisted of fall (21 October 1997), spring (24 April 1998), and fall plus spring applications to the sugarbeet crop plus Metarhizium applications to either the proceeding (1997) barley crop (6 May 1997) or to both the (1996) wheat (14 May 1996) and barley (1997) crops proceeding sugarbeet in the 3-year crop rotation. Metarhizium treatments were compared with Lorsban (Chlorpyrifos; 1.5 lbs / acre ) applied at planting and no insecticide for maggot control.

Root maggot damage was assessed in late July or early August. Damage ratings for individual plots were the mean of 10 roots rated on a 0 to 9 scale where 0 = no root maggot feeding scars; 1 = 1 - 4 small scars (pin head size); 2 = 5 - 10 small scars; 3 = up to 3 large scars or scattered small scars; 4 = a few large scars and/or numerous small scars; 5 = several large scars and/or heavy feeding on lateral roots; 6 = numerous scars with up to 1/4 of root scared; 7 = 1/4 - 1/2 of root blackened by scars; 8 = 1/2 - 3/4 of root blackened by scars; and 9 = more than 3/4 of root surface blackened. Roots were hand dug from the 5th and 8th rows of each plot, washed, and immediately evaluated. All evaluations were dependent upon natural root maggot infestations at the site.

The M. anisopliae applied in all the field trials was derived from a strain obtained from the American Type Culture Collection (No. 22099). This culture is native to Israel and was characterized as "virulent on larvae of many species". Mortality of third instar root maggot larvae exposed to this strain in the laboratory was 15, 94, and 100% for 7, 15, and 29 days, respectively. Mortality at 29 days for larvae not exposed to the fungi was 3% (Smith and Eide, 1995). The Metarhizium used in the field trial was isolated from root maggot larvae prior to the large scale increase on autoclaved barley, to assure virulence to the maggot.

Preliminary field studies with Metarhizium began in 1994. Inoculated barley was placed in the seed furrow along with the sugarbeet seed at Hillsboro and St. Thomas, North Dakota. The ratio of inoculated barley to sugarbeet seed was about 20 to 1 (by weight). At Hillsboro, the Lorsban treatment yielded 5.4 tons/acre more than the untreated check but the Metarhizium treatment was not significantly better than the untreated check. Similar relationships were observed at St. Thomas where there was only a 2 ton/acre yield difference between the Lorsban treatment and no insecticide. Broadcast applications of Metarhizium were first examined in 1995 in a single trial near Hillsboro. In this trial the Lorsban treatment produced 21.7 tons/acre, compared to 14.6 tons/acre for plots with no insecticide. Root yields of the Metarhizium treatments ranged from 18.9 to 14.8 tons/acre with the highest observed in plots receiving both a fall (1994) and spring (1995) application.

A fall plus spring Metarhizium application reduced root maggot damage ratings and produced root yields equal to a planting-time Lorsban application in 1996 at Crookston (Table 1). When no insecticide was applied, root yields were 4.3 tons/acre lower than when Lorsban was used and 4.1 tons/acre lower than the fall plus spring Metarhizium treatment. A single Metarhizium application, either spring or fall, resulted in some reduction in root maggot damage and a corresponding root yield increase, but was inferior to the fall plus spring treatment. A measurable root yield response from the single fall application indicated that the introduced Metarhizium survived the winter of 1995-96.

The 1997-98 trials examined the possibility of improving root maggot control by allowing Metarhizium to increase and disperse during the non-sugarbeet crop (wheat and barley) years of the rotation. The 1997 responses were similar to those observed in the 1996 trial. Root yield of the Lorsban treated plots was 25.7 tons/acre, compared to 21.8 tons/acre with no insecticide. The plots receiving three Metarhizium (spring 1996 + fall 1996+ spring 1997) applications had root yields equal to the Lorsban treated plots and all other Metarhizium treatments produced higher yields than the no insecticide treatment. The root yield of the "spring 1996 + fall 1996" treatment indicated that the Metarhizium remained effective over the winter of 1996-97 and provided some root maggot control in the 1997 sugarbeet crop. While our intent was to apply excess Metarhizium inoculum with each application, the nearly equal root yield for the "spring 1996 + fall 1996 + spring 1997" and the "spring 1996 + spring 1997-double rate" treatments suggest that the reduced control observed in the other two Metarhizium treatments (Table 1) could be a dosage effect and not a time of application effect.

Treatment differences for root maggot damage ratings and root yields were small in 1998. Although the differences were not statistically significant, they appeared to follow the trends observed in the 1996 and 1997 trials. Sterile barley (without Metarhizium) had no effect on root yield in a separate trial at Crookston in 1998 in which differences in root maggot damage among treatments were not significant. The spring of 1998 was extremely wet resulting in saturated soils for extended periods. The effects of these conditions upon Metarhizium survival and dispersal or on the root maggot are not known. Root maggot damage was more severe and treatment differences more apparent in a nearby insecticide trial.

Treatment differences in sugar concentration were small and nonsignificant in two of the three years at Crookston As a result differences in recoverable sugar per acre are primarily a reflection of root yield decreases associated with increased root maggot damage. In the year (1997) that significant differences were detected, the response did not appear to be related to the amount of inoculum applied. The highest sugar treatment (spring 1996 + fall 1996 + spring 1997) and the lowest sugar treatment (spring 1996 + spring 1997 - double rate) had similar root yields (25.7 and 25.2 tons/A, respectively) and both received the same amount of Metarhizium inoculum (applied in different sequences).

Table 1.	Sugarbeet root maggot damage ratings and yield of sugarbeet treated with Metarhizium, Crookston,Minnesota, 1996 - 1998.

Sugarbeet crop year	Treatment / application time(s)	Damage rating	Root yield	Sugar	Recoverable sugar

		-- 0 - 9–	Tons/A	-- % --	Lbs/ton	Lbs/A
1996	Metarhizium:
	Fall 1995 + Spring 1996	2.9	26.2	17.1	308	8074
	Fall 1995	3.4	23.0	16.4	286	6597
	Spring 1996	3.6	22.7	16.6	296	6729
	No insecticide	4.1	22.1	16.4	286	6338
	Lorsban	3.0	26.4	16.8	294	7745
	LSD (0.10)	0.1	0.6	ns	22	584

1997	Metarhizium:
	Spring 1996 + Fall 1996 + Spring 1997	4.0	25.7	15.8	279	7167
	Spring 1996 + Fall 1996	4.5	23.5	14.8	256	6043
	Spring 1996 + Spring 1997	4.2	23.0	15.0	261	6035
	Spring 1996 + Spring 1997 (double rate)	4.4	25.2	14.6	250	6312
	No insecticide	4.6	21.8	15.1	263	5725
	Lorsban	3.6	25.7	15.1	262	6766
	LSD (0.10)	0.2	1.2	0.4	10	331

1998	Metarhizium:
	Spring 1996 + Spring 1997 + Fall 1997 + Spring 1998	3.5	28.1	16.5	289	8110
	Spring 1996 + Spring 1997 + Fall 1997	4.0	28.4	16.4	290	8255
	Spring 1996 + Spring 1997 + Spring 1998	4.1	27.8	15.9	269	7490
	Spring 1997 + Fall 1997 + Spring 1998	3.5	28.5	15.7	274	7814
	Spring 1997 + Fall 1997	3.9	27.8	15.8	269	7491
	Spring 1997 + Spring 1998	3.9	28.2	15.6	263	7433
	No insecticide	3.5	25.6	16.0	279	7114
	Lorsban	3.8	28.3	15.8	272	7711
	LSD (0.10)	ns	ns	ns	ns	ns

The results presented in this report, observations from laboratory studies, and other field observations indicate that Metarhizium merits further study as a possible sugarbeet root maggot biocontrol agent. The need for testing at more sites and under different conditions is recognized, but was not feasible with the current inoculum production and application methods. Our trials were designed to confirm laboratory studies suggesting the potential of Metarhizium as a root maggot biocontrol agent. As such, they provide only limited information on application rates and timing. A formulation with a uniform, known concentration that could be applied with, at most, minor modification of conventional application equipment is required for the extensive testing necessary for establishment of recommendations for commercial use. Some commercial formulations (Schwarz, 1995; Moscardi, 1989; Booth and Shanks, 1998) may be available but it is not known if these strains would be as effective as the strain tested. Even if commercial strains were found to be less effective than the strain used in these trials, the technology for producing current commercial strains most likely could adapted to other Metarhizium strains. We have tested a few Metarhizium strains in the laboratory and most appear to be effective against the root maggot and superior to a commercial Beauveria bassiana strain that was also examined. In addition to killing the root maggot, perspective Metarhizium strains must be adapted to the soil and climatic conditions of the sugarbeet production areas where root maggot is a problem.

Persistence in the soil and consistency of control are important considerations with any biopesticide. The extent Metarhizium will increase or survive for extended periods under cultivation in the Red River Valley is not known. Metarhizium anisopliae appeared to be more tolerant of agricultural disturbances than three other naturally occurring entomopathogenic fungi and was the only species found in sugarbeet fields in Finland (Vänninen, 1995). Although Metarhizium is capable of surviving on organic matter in the absence of a suitable insect host, it is not clear to what extent this saprophytic capability would contribute towards maintaining fungal populations in the field. In some environments, other saprophytic microorganisms associated with the soil and plant residues inhibit Metarhizium germination. In these situations the Metarhizium is restricted to insects for development (Walstad, et al., 1970; Quintela and McCoy, 1998). In the 1995 trial at Hillsboro, North Dakota and the 1996 and 1997 trials at Crookston some reduction in root maggot damage was obtained from fall applications indicating that, at least some of the applied Metarhizium survived the winters of 1994-95, 1995-96, and 1996-97.

In a 1997 trial at Crookston, Lorsban was mistakenly applied to five plots that had already received two or three Metarhizium applications. In this trial, the Lorsban by itself produced 23.9 tons/acre, compared to 20.7 tons/acre for no insecticide. Plots receiving Metarhizium plus Lorsban averaged 26.6 tons/acre, producing from 0.9 to 3.9 tons/acre more than adjacent plots with Lorsban alone. Conclusions based upon this limited information are very tenuous; however, the possibility that a combination of Metarhizium and chemical insecticide would provide optimal control has been examined in other situations (Ferron, 1978). For example, combining Metarhizium or Beauveria with Imidacloprid (Gaucho) reduced the time to mortality of citrus root weevil (Diaprepes abbreviatus L.) in Florida (Quintela and McCoy, 1997).

Although biocontrol agents are generally considered to be safer than most chemical insecticides they should not be handled recklessly. Metarhizium can cause eye irritation and the need for eye protection when handling inoculum became apparent early in our research program. While toxicity to healthy humans, and other mammals, is low, Metarhizium has been implicated as a complicating factor in individuals with malfunctioning immune systems (Burgner et al., 1998). The broad insect host range of many Metarhizium strains may be both a benefit through control of other crop pests and a detriment in reducing populations of beneficials and other nontarget organisms (Genthner and Middaugh, 1992). As with all pesticides it should be used judiciously and with appropriate precautions.