Prevalence and Distribution of Naturally Occurring Beauveria bassiana in San Joaquin Valley Populations of Lygus hesperus (Heteroptera: Miridae) 1

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1 Prevalence and Distribution of Naturally Occurring Beauveria bassiana in San Joaquin Valley Populations of Lygus hesperus (Heteroptera: Miridae) 1 Michael R. McGuire USDA-ARS N. Shafter Ave. Shafter, California J. Agric. Urban Entomol. 19(4): (October 2002) ABSTRACT Lygus hesperus Knight is a particularly damaging pest of cotton, and control methods rely on synthetic chemical pesticides. A brief survey in November 2000 revealed the presence of Beauveria bassiana (Balsamo) Vuillemin in L. hesperus populations. To determine the extent of B. bassiana prevalence, both geographically and seasonally, samples of L. hesperus were obtained several times through the spring, summer, and fall of 2001 from six counties in the San Joaquin Valley, California. Lygus hesperus were held live to determine percentage infection. B. bassiana was found in all counties throughout the sampling period except for a spring sample in one county. Prevalence of B. bassiana ranged from 0 50%, and no other pathogens or parasites of L. hesperus were found. In addition, weekly samples were obtained from each of several alfalfa fields in Kern County, California, over a 2-year period. In 2001, six fields were sampled, and in 2002, four fields were sampled. Beauveria bassiana was present in all fields but at different levels. In 2001, significant differences existed with respect to prevalence of B. bassiana and L. hesperus. Fields in their first year of production had lower levels of B. bassiana than fields in their second or third year of production. In 2002, these differences were not present. Linear regressions showed a positive but weak relationship between infection level and L. hesperus density in In 2002, a significant linear relationship existed between sample date and percentage infection suggesting a buildup of pathogen over time. A significantly higher percentage of adults were infected than nymphs. These data suggest that B. bassiana is present throughout the San Joaquin Valley and exists in the environment even in the hottest part of summer when temperatures routinely exceed 40 C. KEY WORDS Lygus hesperus, Heteroptera: Miridae, Beauveria bassiana, Deuteromycotina: Moniliales, alfalfa, cotton, epizootiology The western tarnished plant bug, Lygus hesperus Knight, is one of the primary pests affecting cotton in the San Joaquin Valley (SJV) in California. Estimates of loss and costs to control L. hesperus approach $30 million per year. Current control recommendations for L. hesperus call for applications of broad-spectrum 1 Accepted for publication 14 April Prod. #03-02

2 238 J. Agric. Urban Entomol. Vol. 19, No. 4 (2002) pesticides early and in the middle of the season as L. hesperus moves into cotton fields. These applications also affect natural enemies (Cave & Gutierrez 1983), which normally keep aphid, spider mite, and caterpillar populations in check. Without the natural enemy complex, populations of these other pests can increase later in the season and require further chemical applications. In addition to cotton, L. hesperus can survive on more than 100 host plants representing 24 families, including alfalfa, strawberries, and carrots (Scott 1977, Goodell 1998). Management of forage alfalfa, where L. hesperus is not an economic pest, to provide preferred habitat is strongly encouraged where available (Stern et al. 1964). Some success with the use of trap or buffer crops has been reported (Goodell & Eckert 1998), but this practice is not reliable under sustained and widespread L. hesperus movement. In cotton, there is no known host plant resistance to L. hesperus, and selective or reduced-risk insecticides are not effective at high population densities. Entomopathogenic fungi such as Beauveria bassiana (Balsamo) Vuillemin may play an important role in natural regulation of L. hesperus populations. Although no systematic search for naturally occurring B. bassiana attacking L. hesperus has been performed in California, 40 years ago, Dunn & Mechalas (1963) proposed using B. bassiana for control of L. hesperus. They reported 100% kill of laboratory-reared L. hesperus but did not report field studies. Characteristics of the B. bassiana strain, such as the host or locality from which it was isolated, were not revealed. More recently, Noma & Strickler (2000) demonstrated laboratory efficacy of Mycotrol, a commercially available B. bassiana product, against L. hesperus, but efficacy in field trials was low (Noma & Strickler 1999). The strain of B. bassiana used in Mycotrol was originally isolated from a chrysomelid beetle in the Northeast United States. It has been suggested (Fargues & Remaudiere 1977) and it makes empirical sense that a strain isolated from the intended target insect in the environment where it is to be controlled should be more efficacious than a strain isolated from another insect in a different locality. Work by Steinkraus & Tugwell (1997) supports this theory, as they determined that Mycotrol was significantly less effective in the field against Lygus lineolaris than a strain of B. bassiana isolated from L. lineolaris in Arkansas. The study reported here is the first part of an effort to isolate additional strains of B. bassiana that might better control L. hesperus than the currently available commercial strains. In addition, it is important to know the background infection rates of B. bassiana in L. hesperus populations before release of B. bassiana as a control strategy. To initiate this study, preliminary collections of L. hesperus made in November 2000 revealed evidence of B. bassiana infections. These infections were confirmed by R. Humber, curator of the USDA-ARS Entomopathogenic Culture Collection, Ithaca, New York, and several strains are deposited in the collection. In one of the fields sampled, 65% of the population was infected (McGuire et al. 2001). This surprising level of infection led us to question how widespread and prevalent B. bassiana is in the cotton production region of California and whether it is affecting population dynamics of L. hesperus. The purpose of the work reported here is 2-fold: 1) determine the geographic distribution and abundance of B. bassiana in L. hesperus populations and 2) determine the impact of the fungus on L. hesperus populations.

3 MCGUIRE: Prevalence of Beauveria in California Lygus Populations 239 Materials and Methods Distribution and prevalence of B. bassiana in Lygus spp. populations. Six cotton-producing counties in the SJV were selected for sampling (Fig. 1). At least four fields in each of these counties were sampled three times from April through September Global positioning coordinates were recorded for each site. Samples were typically taken in alfalfa fields, although the early spring samples consisted of roadside vegetation (mostly brassicaceous weeds) because Lygus spp. were not yet present in the alfalfa. Attempts were not necessarily made to return to previously sampled fields. Forage alfalfa was chosen because Lygus spp. are typically not economically important in this crop and populations can build to relatively high levels. In addition, the permanent nature of alfalfa should lend itself to a buildup of soil dwelling pathogens, such as B. bassiana. To estimate population density in each sample, 50 sweeps were made with a 35.5-cm (15-inch) diameter sweep net passed in a 180 degree arc from one side of the sampler to the other. After 50 sweeps, the net was opened and the total Lygus spp. (adults and nymphs) were counted as they crawled up the net. Attempts were made to collect 50 adults and 50 nymphs from each field on each sampling date to estimate prevalence of B. bassiana. In the field, sweep samples were examined and live insects were moved via aspirator to small glass vials containing a piece of fresh green bean (Phaseolus vulgaris L.). Insects were held individually to eliminate horizontal transmission of B. bassiana after collection. Vials were placed on ice and returned to the laboratory where they were held at room temperature for 2 weeks on a laboratory bench. Beans were changed every 2 to 3 days and dead insects were examined for emergence of B. bassiana. If no fungus emerged within 3 days of bug death, the vials were discarded. Historically the Lygus spp. collected in this manner were believed to be mostly L. hesperus. Recent work by S. Mueller (personal communication) demonstrated that the Lygus population is composed of approximately 7% L. elisus and 93% L. hesperus. Attempts were not made to determine the species of each individual collected and, for the purposes of this article, we refer to L. hesperus as the insect collected. Impact of B. bassiana on L. hesperus populations. Forage alfalfa fields in Kern County, California were chosen for weekly sampling. In 2001, six fields were sampled; three fields (# 5, 44, 55) were planted in October 2000 (first-year fields), one field (#56) was planted in October 1999 (second-year fields), and two fields (#3, 54) were planted in October 1998 (third-year fields). In 2002, four fields were chosen, including one (#101) first-year, two (#44, 102) second-year, and one (#103) third-year fields. One of the second-year fields (#44) was the same field as a first-year field from the 2001 samples; otherwise, all fields were different. Fields were harvested on a 28 days cutting cycle followed by flood irrigation. Field 44 was strip cut (i.e., only half the field was harvested every 2 weeks); all other fields were completely harvested on a given date. At weekly intervals, each field was sampled as described above for estimating Lygus spp. density. In addition, attempts were made to collect and hold 50 adults and 50 nymphs from each field on each sampling date for fungal emergence. Results were analyzed by analysis of variance and means among fields were separated by protected least significant difference (Statistix 1997). Contrasts were used to determine differences among fields of different ages and linear regression analysis was performed to determine

4 240 J. Agric. Urban Entomol. Vol. 19, No. 4 (2002) Fig. 1. Locations in California sampled for Lygus spp. and prevalence of Beauveria bassiana (2001). See text for sampling protocols and site selection. if percentage infection was related to population density. Paired t tests were used to determine whether differences occurred in percentage infection between nymphs and adults. Results Distribution and prevalence of B. bassiana in Lygus spp. populations. The geographic and seasonal prevalences of B. bassiana were determined in L. hesperus populations in each of six SJV cotton-growing counties in the spring, summer, and fall, 2001 (Table 1). Beauveria bassiana was present in every county sampled and, with the exception of one county, was present at all sampling times. Prevalence of B. bassiana ranged from 0% in several fields across all counties up to 50% in one field in Tulare County from a fall sample. In most instances, infection levels were highest in the fall as were L. hesperus populations. Across all counties, populations of L. hesperus averaged 5.2, 14.5, and 13.3 per 50 sweeps for the spring, summer, and fall samples, respectively. Percentage infection of nymphs and adults combined averaged 2.4, 3.0, and 6.7 for the three sampling periods. Typically, infected individuals succumbed to B. bassiana within 3 7 days

5 MCGUIRE: Prevalence of Beauveria in California Lygus Populations 241 Table 1. Prevalence of Beauveria bassiana in Lygus spp. populations throughout the San Joaquin Valley, California, County Season a sampled No. of fields Avg. no. Lygus/50 sweeps Average percentage infection Adults Nymphs b Kern Spring Summer Fall Fresno Spring N/A Summer Fall Kings Spring Summer Fall Madera Spring N/A Summer Fall Merced Spring N/A Summer Fall Tulare Spring N/A Summer Fall a Spring samples were taken in March and April; summer samples were taken in June and July; fall samples were taken in October. b N/A, No nymphs were present in the fields. but some infected individuals survived more than 10 days. No other pathogens or parasites were observed in any of the samples. Impact of B. bassiana on L. hesperus populations. To determine the impact of B. bassiana on L. hesperus populations, individual alfalfa fields were sampled intensively through each of two growing seasons (Figs. 2 and 3). In 2001, significant differences occurred among fields with respect to population size (F 9.85; df 5,137; P < 0.001), total percentage infection (F 3.37; df 5,137; P 0.007), and percentage infection of adults (F 3.12; df 5,137; P 0.01; Table 2). Percentage infection of nymphs was not significantly different among fields (F 0.52; df 5,112; P 0.76). Statistical contrast tests (df 1,137) suggested that fields in their third year of production had higher percentage infection (9.05%) than first-year fields (5.1%; t 2.65; P 0.009). Similarly, the field in its second year of production had higher prevalence of B. bassiana (13.41%) than the first year fields (t 4.41; P < 0.001). However, the second-year field also had higher prevalence of B. bassiana than the third-year fields (t 2.18; P 0.031). Although significant differences in population size were observed among fields, no significant differences occurred with respect to years in production (t 0.33, 1.49, and 1.16 for first versus third, first versus second, and

6 242 J. Agric. Urban Entomol. Vol. 19, No. 4 (2002) Fig. 2. Results from weekly samples, Left axis represents number of Lygus spp. per 50 sweeps (clear bars) and right axis represents percentage infection of collected Lygus (broken bars). Each pair of bars represents a collection. Field numbers are in the upper right-hand corner of each chart; note difference in scale for field 44. second versus third, respectively). A significant linear regression existed between population size and percentage infection (F 10.77; df 1,141; P ) but the amount of variation explained by the regression model was low (r ). A significant (F 7.33; df 1,141; P 0.008) but weak (r ) linear relationship also existed between collection date and population size, but no such relationship existed between date and percentage infection (F 3.41; df 1,141; P 0.07; r ). Across all fields and all samples, a significantly higher percentage of adults were infected than nymphs (paired t 8.09; P < 0.001). Adult infection averaged 8.7% while nymph infection averaged 2.3%. In 2002, there were no significant differences among fields with respect to average population density (F 2.04; df 3,94; P 0.11), total percentage

7 MCGUIRE: Prevalence of Beauveria in California Lygus Populations 243 Fig. 3. Results from weekly samples, Axes and bars are the same as Figure 2. infection (F 1.54; df 3,94; P 0.21), percentage infection of adults (F 1.16; df 3,94; P 0.33), nor percentage infection of nymphs (F 1.0; df 3,86; P 0.40). Similarly, there was only a very weak linear regression between density and percentage infection (F 3.87; df 1,94; P 0.051; r ). However, when regressed against collection date, there was a significant linear relationship for percentage infection (F 20.54; df 1,94; P < 0.001) and the model explained 18% of the variation. These results suggest a buildup of pathogen through the season. No such relationship existed for population density (F 1.04; df 1,94; P 0.31). Similar to 2001, adults had significantly higher infection rates than nymphs (paired t 7.74; P < 0.001). Adult infection averaged 6.2% whereas nymph infection averaged 1.5%. Discussion These data suggest that B. bassiana is widespread across the area sampled and that the fungus may play a role in the landscape ecology of L. hesperus population dynamics. Alfalfa production has increased in the SJV over the past several years. Because L. hesperus does not cause economic damage in forage alfalfa, populations can build to quite large numbers (e.g., Figure 2, field 44, 2001). In addition, forage alfalfa in the SJV typically does not incur economic damage

8 244 J. Agric. Urban Entomol. Vol. 19, No. 4 (2002) Table 2. Average Lygus population and percentage Beauveria bassiana infection based on weekly samples. Year Field no. Field age (years) Lygus per 50 sweeps Percentage infection Adults Nymphs Total c 6.64 bc bc a 4.92 c c bc 6.49 bc c bc a a b 8.77 bc bc b ab ab SE (Mean) SE (Mean) Means followed by the same letter in year 2001 not significantly different (protected least significant difference, P < 0.05). Means in year 2002 not significantly different. from many other insects and is rarely treated with insecticides. L. hesperus typically infests cotton from neighboring hosts and large populations can migrate quickly as these hosts senesce or become less habitable, e.g., when alfalfa is harvested (Leigh & Matthews 1994). The discovery of B. bassiana in alfalfa fields and roadside weeds harboring Lygus spp. populations suggests that innocula is present throughout the year and the observation of prolonged infection cycles suggests that the fungus can be moved from area to area by infected adults. Although significantly more adults were infected than nymphs, it is not necessarily safe to conclude that adults are more susceptible to B. bassiana. Because adults are able to fly, a sweep net may preferentially sample adults over nymphs. In addition, nymphs may be feeding within the canopy whereas adults may be in the upper portions of the canopy seeking mates. It is also possible, and currently under investigation, that infected individuals may go through a behavioral change similar to that observed in grasshoppers (Carruthers et al. 1992, Inglis et al. 1996) where they move to the top of a plant thus increasing body temperature. These increased temperatures may help the insect overcome the infection because the fungi typically cannot grow at higher temperatures. Adults may also be more heavily infected simply because they have been in the field longer and may have become infected as nymphs. Similarly, this study was not designed to determine the cause of the relationships between population density and percentage infection. These relationships are very complex (Carruthers & Soper 1987) and often involve many years of study and manipulation of the environment. Beauveria bassiana is a cosmopolitan facultative entomopathogenic fungus that infects more than 500 species of insects across several orders (Fargues & Remaudiere 1977). Therefore, it is not surprising that B. bassiana is found natu-

9 MCGUIRE: Prevalence of Beauveria in California Lygus Populations 245 rally occurring in Lygus spp. in California. What may be surprising is the prevalence of the fungus in some locations. McGuire et al. (2001) reported a field with 65% of the collected L. hesperus infected and within the study presented here, more than one sample contained approximately 50% infected individuals (Fig. 2). These levels of infection may have a profound local impact on L. hesperus population densities. Weekly surveys demonstrated occurrence of fungus throughout the summer when daily high temperatures often exceed 40 C. Although most collections resulted in 10% or less infection, the presence of any B. bassiana in the heat of the summer is significant. Many of these isolates have been collected and research is progressing on identifying highly infectious isolates that can survive at high temperatures. Development of these isolates may lead to a more viable and effective Lygus control product (Fargues et al. 1997). Acknowledgment This work was made possible by the excellent technical assistance of Danny Ballard, Neal Hudson, Maria Garcia, Monica Rodriquez, Vikki Gutierrez, and Leslie Tiner (USDA- ARS, Shafter, CA). Richard Humber kindly and quickly provided verification of Beauveria bassiana in our initial samples. The manuscript was helped by reviews from J. Bancroft, USDA-ARS, Shafter, CA and P. Goodell and S. Mueller, University of California, Kearney Research and Extension Center. References Cited Cave, R. D. & A. P. Gutierrez Lygus hesperus field life table studies in cotton and alfalfa (Heteroptera: Miridae). Can. Entomol. 115: Carruthers, R. I., T. S. Larkin & H. Firstencel Influence of thermal ecology on the mycosis of a rangeland grasshopper. Ecology 73: Carruthers, R. I. & R. S. Soper Fungal diseases, pp In J. R. Fuxa & Y. Tanada [Eds.], Epizootiology of insect diseases. John Wiley and Sons, New York, 555 pp. Dunn, P. H. & B. J. Mechalas The potential of Beauveria bassiana (Balsamo) Vuillemin as a microbial insecticide. J. Insect. Pathol. 5: Fargues, J. & G. Remaudiere Considerations on the specificity of entomopathogenic fungi. Mycopathology 62: Fargues, J., M. S. Goettel, N. Smits, A. Ouedraogo & M. Rougier Effect of temperature on vegetative growth of Beauveria bassiana isolates from different regions. Mycologia 89: Goodell, P. B Biology, ecology, and host plants of Lygus lineolaris and Lygus hesperus. Proc. Beltwide Cotton Conf. 2: Goodell, P. B. & J. W. Eckert Using buffer crops to mitigate Lygus migration in San Joaquin Valley cotton. Proc. Beltwide Cotton Conf. 2: Inglis, G. D., D. L. Johnson & M. S. Goettel Effects of temperature and thermoregulation on mycosis by Beauveria bassiana in grasshoppers. Biol Cont. 7: Leigh, T. F. & G. A. Matthews Lygus (Hemiptera: Miridae) and other Hemiptera, pp In G. A. Matthews and J. P. Tunstall [Eds.], Insect pests of cotton. CAB Intl., Wallingford, UK, 593 pp. McGuire, M. R., K. E. Godfrey & D. C. Steinkraus Entomopathogenic fungi for control of aphids and Lygus in California. Proc. Beltwide Cotton Conf. 2: Noma, T. & K. Strickler Factors affecting Beauveria bassiana for control of Lygus bug (Hemiptera: Miridae) in alfalfa seed fields. J. Agric. Urban. Entomol. 16:

10 246 J. Agric. Urban Entomol. Vol. 19, No. 4 (2002) Noma, T. & K. Strickler Effects of Beauveria bassiana on Lygus hesperus (Hemiptera: Miridae) feeding and oviposition. Environ. Entomol. 29: Scott, D. R An annotated listing of host plants of Lygus hesperus Knight. Bull. Entomol. Soc. Am. 23: Statistix for Windows User s manual. Analytical Software, Tallahassee, Florida. Steinkraus, D. C. & N. P. Tugwell Beauveria bassiana (Deuteromycotina: Moniliales) effects on Lygus lineolaris (Hemiptera: Miridae). J. Entomol. Sci. 32: Stern, V., R. van den Bosch & T. F. Leigh Strip cutting alfalfa for lygus bug control. Calif. Agric. 18: 4 6.