KEY WORDS Sugarcane, biological control, Scarnbaeidae, grubs, Metarhjzju,", Coleoptera.

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1 Pathogenicity of Metarhizium anisopliae var. major (Metschnikoft) Sorokin to a Sugarcane Grub Ligyrus subtropicus (Blatchley) (Coleoptera: Scarabaeidae)1 R N. Raid and R. H. Cherry University of Florida Institute of Food and Agricultural Sciences Everglades Research and Education Center P.O. Box 8003, Belle Glade, Florida 33430,I. Agric. Entomol. 9(1): (January 1992) ABSTRACf Three Meto.rhizium arlisoplio.e vaf. major (Metschnikoff) Sorokin isolates, each collected from 8 different sugarcane white grub species, were tested for pathogenicity to third-instar larvae of Ligyrus subtropicus (Blatchley). Only the isolate originating from L. subtropicus proved to be pathogenic under the conditions of this study, resulting in 78% and 92% mortality in two separate tests. Results from a field survey investigating natural incidence of M anisopliae in L. subtropicus larvae, in conjunction with field observations in associated studies, suggest that M. anisop/iae occurs at very low levels in L. subtropicus populations in sugarcane fields. The high mortalities observed in our pathogenicity tests indicate that the isolate originating from L. subtropicus warrants further consideration for biological control of that grub species. KEY WORDS Sugarcane, biological control, Scarnbaeidae, grubs, Metarhjzju,", Coleoptera Sugarcane is Florida's most valuable field crop and is primarily grown in the Everglades area of southern Florida. The crop is attacked by two major groups of soil insect pests, wireworms (Elateridae), which cause damage to newly planted sugarcane (Hall 1985), and several species of white grubs (Scarabaeidae). In Florida, grubs are considered pests of ratoon sugarcane (Cherry 1988), feeding on the roots of sugarcane plants. Of the grubs, Ligyrus sublropicus (Blatchley) is the I>rimary species of economic importance (Gordon and Anderson 1981). Yield reductions of up to 39% have been reported in areas of high infestation (Sosa 1984). Currently, there is no known chemical control for white grubs in Florida sugarcane, and flooding of grub-infested fields is sometimes used for control (Cherry 1984). Very little has been published on biological control of grubs in Florida sugarcane. Sosa and Beavers (1985) demonstrated that entomophilic nematode species were capable of infecting L. subtropicus under laboratory conditions. Boucias et a1. (1986) reported that Bacillus popilliae Dutky, the causal agent of milky disease, was the major pathogen found in third instar grubs of L. subtropicus in Florida sugarcane fields nnd that the bacterium was widespread in the sugarcane producing region. Ligyrus sublropicus was also found infected by two entomogenolls fungi, Melarhizium anisopliae var. major (MetschnikofO Sorokin and Beauueria I Heceived (or publication 7 May 1989: accepted 20 June

2 12 J. Agric. Entomol. Vol. 9, No. 1 (1992) bassiana (Balsamo) Vuillemin, although much less frequently. Illingworth (1921) reported a naturally occurring epidemic caused by a green muscardine fungus, presumably M. anisopliae, in Australian sugarcane grub populations. The ease of propagation (Barnes et al. ]975) makes M. anisopliae an excellent candidate fol' biological control. In Australia, laboratory results using M anisopliae to control several grub species have demonstrated enough promise to extend the research to field experiments (Samuels et al. 1990). The objectives of this study were to determine more fully the incidence of M. anisopliae infection in L. subtropicus under field conditions, and to investigate the pathogenicity of several M. anisopliae isolates to L. subtropicus under controlled conditions. Materials and Methods Field Swvey. First-, secondo, and third-instar larvae were collected at monthly intervals for 9 mo and observed for natural incidence of M. anisopliae infection. Collections were initiated \11th first instar larvae dw-ing July 1988, continued with second-instar larvae in August 1988, and concluded with third-instar Illlvae from September 1988 to April Twenty-five lalvae were collected from each of five commercial ratoon sugarcane fields in the Everglades Agricultural Area (EAA) each month. L31vae were located by digging beneath sugarcane plants. Grubs were placed into individual containers when found in the field by placing a metal specimen box beneath the grub and manipulating the glub into the box using the box lid to avoid cross contamination due to handling. First- and second-instar larvae were placed into 1.5- by 5 cm diam boxes while larger third-instar Imvae were placed into 2- by 8-cm diam boxes. Only live grubs not damaged during field collection were selected for sampling. Approximately 8-10 g of moist (1 ml of water!1 g air-dried soil), fumigated organic soil (muck), along with a slice of raw carrot for food, were added to each container on the date of collection. Boxes containing grubs were maintained at room temperature (23 27 C) with lids closed to maintain high relative humidity. In earlier tests, we had successfully induced M. anisopliae infection of L. subtropicus larvae under the temperatures and moisture conditions previously mentioned, with rnoliality normally occurring within 2 wk of inoculation. Thus, field-collected l8lvae were obselved for symptoms of disease development after 7 and 14 d of incubation. Pathogenicity Studies. A single isolate of M. anisopliae was obtained from each of three different species of sugarcane grubs: L. subtropicus, Cyclocephala pw allcla Casey, and Euphoria sepulchralis (F.). [solates were obtained from grub cadavers (third-instal' larvae) found covered with the fungus in Florida sugarcane fields during 1987 and 1988, and were designated as MA-LS, MA-CP, and MA-ES, respectively. Isolates were maintained by peliodic single-spore transfers onto Sabouraud maltose agar. Voucher specimens are to be maintained by the American Type Culture Collection (ATCC), Rockville, Maryland. Using Koch's postulates, the pathogenicity of each isolate (at a concentration of 1 X 10' conidida!g of air-dried fumigated organic soil) was demonstrated, on the grub species from which it was originally recovered, by following the pathogenicity test procedures described below.

3 RAID and CHERRY: Metarhizium in Sugarcane Grubs 13 Inoculum for the pathogenicity tests was produced by spreading 0.5-ml spore suspensions of each isolate onto the agar surface of Petri dishes (loo by 15 mm) containing 20 ml of Sabouraud maltose agar and incubating at 27 C for 10 d in darkness. Conidia were harvested using a rubber spatula and sterile deionized water (SDW) containing 0.5% Tween 20. Subsequent to passing through a double layer of cheesecloth, concentrations of conidial suspensions were adjusted to approximately conidia/ml of water using a stage haemocytometer. Aliquots of 300 ml of conidial suspension were used to infest 300 g (air-dried wt.) of fumigated organic soil. The inoculated soil was placed in transparent plastic boxes (23 by 30 by 10 cm high) with lids. Expermental controls consisted of soil receiving 300 ml of SDW in place of conidial suspensions. Ten third-instar L. subtropicus larvae (mean wt 4.1 g) were placed into each box containing soil infested with conidia of one of the three fungal isolates 01' the sterile water control. Raw carrot slices were placed into the bottom of each container as 8 food source. Each box was considered a replicate and five replications of each isolate and the control were incubated simultaneously, with containers being arranged in randomized complete blocks within an incubator maintained at 22 C. The experiment was initiated on 13 Jan Grubs were held for 25 d and observed for symptom development and mortality due to M. anisopliae. Data were analyzed utilizing Scheffe's multiple comparison procedure (SAS 1982). Pathogenesis. M. anisopliae isolate MA-LS was selected for further observations based upon its demonstrated pathogenicity to L. subtropicus in our initial pathogenicity study. During February 1989, five replications of 10 third-instar L. subtropicus larvae were incubated in non-infested soil and soil infested with the MA-LS Melarhizium isolate in the manner previously described. Grubs were obser...ed at 2 d intervals for symptom development and mortalit.y for 25 d. Results and Discussion Field Survey. A total of t,125 field-collected larvae (125, 125, and 875 first, second, and third-instar larvae. respectively) were observed for infection by M. anisopliae. Milky disease, caused by B. popilliae, developed in several grubs. M. anisopliae infection of L. sublropicus was not detected in this study. However, dead third instar larvae of L. subtropicus were occasionally observed colonized by the entomopathogen in sugarcane fields during other studies. Boucias et al. (l986) reported B. popilliae as the primary pathogen found in L. subtropicus grubs in Florida sugarcane fields although M. anisopliae was also detected. Our results support their findings. In studies of a sugarcane grub, Lepidiola albohirtum (Waterhouse), in Australia (lllingworth 1921), found the highest incidence of M. anisopliae to be under moist field conditions. Abnormally dry conditions during the major portion of this study may have been a factor contributing to the failure to detect the disease in the field survey. Young (1974) suggested t.hat low soil moisture was a factor in keeping M. ani.'lopliae at low levels in Tonga. Pathogenicity Studies. Results of the pathogenicity study are shown in Table 1. Differences among treatments in mortality due to causes other than M. anisopliae infection were not significant (P> 0.05). MA-LS, the isolate originating from L. subtropicus, was the only isolate causing mortlliity of L. subtropicus under the conditions of this study. Others (McCauley et al. 1968, Daoust and Roberts 1983) have demonstrated that certain strains of M. allisopliae are capable of infecting

4 14 J. Agric. Entomol. Vol. 9. No. 1 (1992) Table 1. Mortality of L. Bubtropicus third-instar larvae exposed to various isolates of M. anisopliae. Isolate (original host) MA-LS (L. subtropicus) Total 96a % Mortality" Metarhizium 78 a Other causes 18 a MA-CP (C. paral/ela) MA-ES (E. sepulchralis) 16 b Ob 16 a lob Ob 10 a Control 26 b Ob 26a a Percent mortality based upon observations of 50 larvae per isolole. Means within columns followed by the same letter are not significantly different (Scherre's multiple comparison procedure; P> D.Oa). several insect species, even those from different orders. The fact that the MA-CP and MA-ES isolates had earlier demonstrated pathogenicity on C. parallela and E. sepulchralis. respectively, suggests that the M. anisopliae isolates used in this investigation may be more specific. It is also possible that these results reflect only the relative virulence of the three isolates under our test conditions, and the MA-CP and MA-ES isolates could prove to be pathogenic to L. subtropicus under different conditions. Further research on this aspect is needed. Pathogenesis. Percentage accumulated mortality of third-instar larvae using the MA-LS isolate is presented in Figure 1. No mortality was observed in the uninoculated checks. The high mortality (92%) due to the MA-LS isolate agrees with the mortality (78%) observed in the pathogenicity test. The mortality rate due to M. anisopliae was greatest d after inoculation, and 96% of the M. anisopliae casualties were dead within 19 d. Observations on symptomatology showed a decrease in grub responsiveness to probing approximately 8-10 dafter inoculation. In contrast to the controls, the integument of newly-infected larvae was discolored and generally exhibited an orange tint. Approximately h later, infected larvae usually succumbed with the body becoming rigid but exhibiting no external fungal signs. External white mycelial growth was generally evident within an additional h. Characteristic external green sporulation covering the integument was observed within 24 h after the development of external mycelium. In summary, our observations showed M. anisopliae to occur naturally in Florida sugarcane fields, but at extremely low levels. It does not appear to be a major grub mortality factor at the present time. However, the MA-LS isolate of M. anisopliae was proven to be pathogenic to third-instar larvae. The third-instar is the longest-enduring (Cherry 1985) and most destructive stage (Cherry 1991). Additional research is needed to determine the factors presently limiting M. anisopliae effectiveness in F101ida sugarcane fields, and to investigate the feasibility and efficacy of augmentative releases of the entomopathogen for controlling L. subtropicus.

5 RAID and CHERRY: Metarhizium in Sugarcane Grubs 15 ;::: 100 -J ,,----,1, ~ 80 ~6J Cl ~ 40~ 20 «1 o 1="==::+::=~, --._------~ DAYS SINCE INOCULATION Fig. 1. Percent accumulated mortality of third-instar L. subtropicus larvae following exposure to M. anisopliae (MA-LS isolate). Acknowledgment We are grateful to numerous Florida sugarcane growers for open access to their land and to the Florida Sugar Cane Leab'Ue for grant. support. Florida Agricultural Experiment Station Journal series no References Cited Barnes, G. L., D. J. Boethel, R. D. Eikenbary, J. T. Criswell, and C. R. Gentry Growth and sporulation of Melarhizium allisopliae and Beauueria bassiana on media containing various peptone sources. J. Invertebr. Pathol. 25: Boucias, D. G., R. H. Cherry, and D. L. Anderson Incidence of Bacillus popillae in Ligyros subtropicus and Cyclocephaia paraiieia (Coleoptera: Scnrabaeidae) in Florida sugarcane fields. Environ. Entomol. 15: Cherry, R. H Flooding to control the grub LigynlS subtropicus (Colcoptem: Scarabaeidae) in Florida sugarcane. J. Ecan. Entomol. 77: Seasonal phenology of white grubs (Coleoptera: Scarabaeidae) in Florida sugarcane fields. J. Econ. Entomol. 78: Correlation of crop age with populations of soil insect. pests in Florida sugarcane. J. Agric. Entomol. 5: Feeding rates of different larval insters of a sugarcane grub. Ligyrus subtropicus Blatchley (Coleoptera: Scarabaeidae). J. Agric. Entomol. 8: Daoust, R. A. and D. W. Roberts Studies on the prolonged storage of Metarhizium anisopliae conidia: cffect of growth substrate on conidial survival and virulence against mosquitoes. J. Invcrtebr. Pathol. 41:

6 16 J. Agric. Entomo!' Vol. 9, No. I (1992) Gordon, R. A., and D. L. Anderson The species of Scarabneidae (Coleoptera) associated with sugarcane in south Florida. Fla. Entomol. 64: Hall, D. G Damage by the corn wirewonn, Melanotus communis (Gly.) to plant cane during gennination and early growth. J. Am. Soc. Sugar Cane Tech. 4: Ulingworth, J. F A study of natutal methods of control for white grubs. Australian Bureau of Sugar Experiment Station (BSES). Division of Entomology, BulJetin No. 12, Brisbane, 20 pp. McCauley, V. J. E., R. Y. Zacharuk, and R. D. Tinline Histopathology of green muscardine in larvae of four species of Elatcridae (Coleoptera). J. Invertebr. Pathol. 12: Samuels, K. D. Z., D. E. Pinnock, R. M. Bull Scarabeid larvae control in sugarcane using Metarhizium allisopliae. J. Invertcbr. Pathol. 55: SAS Institute SAS user's guide: statistics, 1982 edition. SAS Institute, Cary, Nortb Carolina. 584 pp. 8058, 0., Jr Effect of wbite grub (Coleoptera: Scarabaeidae) infestation on sugarcane yields. J. Econ. Entomol. 77: S088, 0., Jr., and J. B. Beavers Entomogenous nematodes as biological control organisms for Ligyrus subtropicus (Coleoptera: Scarabacidae) in sugarcane. Environ. Entomol. 14: Young, E. C The epizootiology of two pathogens of the coconut palm rhinoceros beetle. J. Invertebr. Pathol. 24: