Problems with in vitro production of spores of Bacillus popilliae for use in biological control of the Japanese beetle

JOURNAL

OF INVERTEBRATE

PATHOLOGY

60,

283-291

(19%)

Problems with in Vitro Production of Spores of Bacillus popilliae for Use in Biological Control of the Japanese Beetle D.P. *Department

of Microbiology, Horticultural

STAHLY*!~ AND M.G.

College of Medicine, University of Zowa, Iowa Insects Research Laboratory, USDA, Agricultural

KLEIN?

City, Iowa Research

52242; and tApplication Technology Research Service, OARDC, Wooster, Ohio 44691

Unit,

Received September 13, 1991; accepted January 30, 1992

cells rapidly

lose viability

in soil and possess question-

Bacillus popilliae spores have been used in the United able virulence. Despite considerable research on in States for the biological control of the Japanese beetle for vitro sporulation, very low levels of sporulation have over 50 years. Lack of efficient sporulation in vitro has been been attained (Stahly et al., 1992a). a major limiting factor in expanding the use of this bacteFrom 1939 to 1953, a massive program involving rium. Previously, commercial spore powders were produced federal and state governments was carried on to lower by maceration of milky disease infected larvae containing populations of Japanese beetle larvae (Fleming, 1968). large numbers of spores. Recently, (since 1985)a commerSpores used in this program were produced in uiuo by cially available spore powder produced by a patented in vitro infection of Japanese beetle larvae procedure has been marketed. However, most batches of the the artificial (Dutky, 1942). B. popilliae develops in the hemolymph in u&o spore powder and primary fermentation products of infected larvae causing a fatal bacteremia. During that we examined contained a bacterium identified asBacilthe last phase of the infection the bacteria sporulate; lus polymyxa, but no B. popilliae. We also examined four may be as high cultures submitted to the American Type Culture Collection the number of spores in the hemolymph (St. Julian et al., 1970). (Rockville, MD) at the time of patent application as repre- as 5 x lOi’/ml Fairfax Biological Laboratory, Inc. (Clinton Corners, sentative of the strains of B. popilliae produced in uitro. One spores for of these, referred to as the preferred strain, was identified as NY) continues to obtain in uiuo-generated their commercial product, Doom. An apparent breakB. polymyxa. The other ATCC strains were identical in their characteristics to Bacillus amyblyticus. Both B. polymyxa through in the in vitro sporulation of B. popilliae was and B. amylolyticus spores,and in u&-o-produced milky dis- documented in a patent approved in April, 1989, by easespore powder containing these bacteria rather than B. personnel formerly associated with Reuter Laboratopopilliae, causedno milky diseasein Japanesebeetle larvae. ries, Inc. (Haymarket, VA) (Ellis et al., 1989). Ringer The patented procedure should be reexamined since there is Corporation purchased Reuter Laboratories (Minneapno evidence that it is effective in producing infective B. po- olis, MN) and their products in 1989 and continued pilliae spores. 63 1992 Academic Press, Inc. marketing the in vitro product, Grub Attack. Even KEY WORDS: Bacillus popilliae; Bacillus polymyxa; Bacilthough the patent was not approved until 1989, it is lus amylolyticus; biological control; Popillia japonica; Grub our understanding that Grub Attack has contained priAttack; Doom; spores. marily in vitro-produced spores since it was first mar-

keted in 1985. Following the demonstration by one of us (D.P.S.) of a lack of B. popilliae and the presence of another Bacillus species in a Grub Attack sample purchased in Connecticut in 1990, we reexamined previous bioassays of Grub Attack, conducted new bioassays of spores produced in vitro and spore powder, and identified the bacteria present. We found that the in uitroproduced bacteria in recent Grub Attack primary fermentation powder samples and Grub Attack formulations is Bacillus polymyxa rather than B. popilliae. Infectivity studies reported here showed that B. polymyxa spores fed to or injected into Japanese beetle larvae did not cause milky disease or larval death.

INTRODUCTION

Spores of Bacillus popilliae, the causal agent of milky disease, have been used for the biological suppression of Japanese beetle larvae, Popillia japonica, for more than 50 years (Klein, 1992). The lack of efficient sporulation in vitro has been the major factor preventing greater use of these bacteria. Only spores are useful for biological control efforts, since vegetative 1 To whom correspondence

should be addressed. 283

All

Copyright 0 1992 rights of reproduction

0022-2011192 $4.00 by Academic Press, Inc. in any form reserved.

284

STAHLY MATERIALS

AND

METHODS

Bacterial Cultures and Products Containing Bacteria

Table 1 includes a description of the various bacterial cultures and products used in this study. These included type strains of three Bacillus species and four American Type Culture Collection (ATCC, Rockville, MD) cultures submitted with the patent application. Products tested included one formulation of B. popilZiae spores produced by M. Klein, 4 batches of Doom, 10 batches of Grub Attack, and 3 primary fermentation products used to formulate Grub Attack. Media and Procedures Used for Identification of Bacteria

Media and procedures used for identification of bacteria in this study are described by Gordon et al. (1973), Claus and Berkeley (19861, and Norris et al. (1981). Starch and gelatin hydrolysis were determined using petri dishes containing 10 ml of nutrient agar (Difco) supplemented with 0.4% starch or gelatin. Tween 80 hydrolysis was determined as described by Breuil and Gounot (1972). Urea hydrolysis was assessed after growth on slants of urea agar base (Difco) plus 1.5%

AND KLEIN

agar. Growth at various temperatures and in the presence of NaCl and lysozyme was determined using meat infusion broth (media kitchen, Department of Microbiology, University of Iowa) as the basal medium. Growth at pH 5.6 as ascertained by use of Sabouraud dextrose agar (Difco). Unless otherwise specified, incubation was at 30°C. Media used for routine maintenance of cultures and/ or for obtaining sporulation were MYPGP agar (Dingman and Stahly, 1983) and nutrient agar (Difco). Use of MYPGP agar supplemented with 150 pg/ml of vancomycin . HCl (Vm) (Stably et al., 199213) permitted detection of B. popilliae in the presence of B. polymyxa and/or B. amylolyticus. Only B. popilliae was Vmresistant. Fatty Acid Analysis of Cultures

Cultures of the bacterial strain isolated from Grub Attack purchased in Connecticut (GA . CT; Table l), the four ATCC cultures (53256, 53257, 53258, and 532591, and B. amylolyticus (NRRL B-377) were submitted to Five Star Laboratories (Branford, CT) for fatty acid analysis. Cells for fatty acid analysis were grown on trypticase soy broth agar (BBL; Becton Dick-

TABLE 1 Bacterial Cultures and Products Used in This Study A. Bacterial cultures Baciilus popilliue NRRL” B-230~the type strain Bacillus polymyxu BGSCb 25Al-the type strain Bacillus amylolyticus NRRL B-377-equivalent to the type strain, NRRL NRS-290 ATCC’ 53256, 53257, 53258, and 53259-the cultures submitted to ATCC at the time of patent application (Ellis et al,, 1989) B. Products Standard-AKJ-Doom Lot AKJ obtained from Fairfax Biological Laboratory, Inc. in August, 1983 Klein-85-Milky disease powder produced by M. Klein from infected Japanese beetle larvae in June, 1985 DM . GB-Doom purchased by the Greenbrier, White Sulphur Springs, WV in 1989 DM . KY-Doom Lot ALM supplied to Dr. D. Potter by Fairfax Biological Laboratory in 1988 DM . AZ-Doom purchased by USDA in March, 1990 for use in the Azores Japanese Beetle Suppression Program GA. BN-Granular Grub Attack supplied to Bills Nursery, Gettysburg, PA, in 1990 GA . AZ-Grub Attack purchased by USDA in Feb., 1990 for use in the Azores Japanese Beetle Suppression Program GA N-Grub Attack wettable powder suplied to Dr. H. D. Niemczyk, OARDC, in August, 1990, by Ringer Corp. GA. CT-Grub Attach purchased in 1990 in Connecticut by Dr. D. W. Dingman Granular GA-Granular Grub Attack supplied to Dr. H. D. Niemczyk, OARDC, in March, 1990, by Ringer Corp. GA . Ch-Spores produced in vitro in October, 1990, by Dr. F. D. Obenchain from B. popilliae isolated from Cyclocephala hiti in California A28-Milky disease spore powder formulated by M. Klein from primary fermentation product received from Ringer Corp. in March, 1990 ARBlO6-Milky disease spore powder formulated by M. Klein from primary fermentation product received from Ringer Corp. in March, 1990 K290-Milky disease spore powder formulated by M. Klein from primary fermentation product received from Ringer Corp. in March, 1990 GA. KY-Grub Attack supplied to Dr. D. Potter, University of Kentucky, in March, 1988, by Dr. F. D. Obenchain. Reuter Laboratories Lot 2-83-736. GA. 136Grub Attack Lot 136 supplied by Reuter Laboratories in May, 1987 GA .102-Grub Attack Lot 102 supplied by Dr. F. D. Obenchain in May, 1985 GA .89--Grub Attack Lot 89 purchased in Wooster, OH, in May, 1985 a NRRL, Northern Regional Research Laboratory (Peoria, IL). b BGSC, Bacillus Genetic Stock Center (Columbus, OH). ’ ATCC, American Type Culture Collection (Rockville, MD).

IN

VITRO

PRODUCTION

inson Microbiology Systems, Cockeysville, MD) at 28°C for 24 hr. Approximately 40 mg of bacterial cells (wet weight) were placed into a 13 x 100 mm test tube with a PTFE-lined cap. Saponification of fatty acids was accomplished by addition of 1 ml of 15% NaOH in 50% aqueous methanol and heating at 100°C for 30 min. The fatty acids were then methylated by addition of 2 ml of 3.25 N HCl in 46% aqueous methanol and heating at 80°C for 10 min. The methylated fatty acids were extracted with 1.25 ml of a mixture of hexane and tert-butyl methyl ether (l:l, v/v). The extraction mixture was rotated for 10 min after which the aqueous phase was removed and discarded. The organic phase was washed with 3 ml of 1.2% NaOH for 5 min and then was placed in a vial for analysis using the MIDI Microbial Identification System (Newark, DE). Quantitution

of Bacillus

Species in Product Samples

Aqueous suspensions (3 ml) of product (1 g) were heated at 60°C for 15 min to kill most vegetative cells. Suspensions were diluted in H,O and 0. l-ml portions of appropriate dilutions were plated onto MYPGP agar and MYPGP + Vm agar (Stahly et al., 199213). Identification of the species was accomplished by observation of colonial and cell morphology and performance of key biochemical tests. The number of spores of B. popilliae per gram of product was estimated by assuming that only about 1% of B. popilliae spores visible by phase contrast microscopy germinate and grow to produce colonies on MYPGP agar (D. Dingman, personal communication). No adjustment was made in the B. polymyxa spore counts; the plating efficiency in two determinations was 95.2 and 105%. Preparation of Spores of B. polymyxa B. amylolyticus for Bioassay

and

The Ringer Corporation preferred production strain (obtained from GA . CT), that we identified as B. polymyxa, was grown as a confluent lawn on MYPGP agar. After 4 days at 3o”C, sporulation was complete and the culture consisted almost entirely of free spores. The spores were suspended in sterile Ha0 and washed five times by alternate centrifugation and resuspension of the pellet in H,O. The spore titer was determined by use of the spread plate technique on MYPGP agar. Spores of ATCC 53259, that we identified as B. amylolyticus, were obtained by growth as a confluent lawn on nutrient agar. After 4 days at 30°C sporulation was complete. The spores were washed and titered as described above. Procedures Used for Bioassay of Spore Preparations

All bioassays to examine infectivity of bacteria by feeding (per OS) were conducted in the classic manner (Dutky, 1941) by incubating field-collected third instar

OF Bacillus

285

popilliae

Japanese beetle larvae in a spore-soil mixture in 3-0~ tins at 27°C. The spor+soil mixture consisted of airdried Wooster silt loam, water added to 60% of the ball point, 10 g of a 1:l (wt/wt) mixture of perennial ryegrass seed and white Dutch clover seed per kilogram of dry soil, and the spore-talc formulation. The recommended standard spore dosage for in vivo-produced spores is 1.0 x 10’ spores per kilogram of dry soil. Therefore, for the Doom batches, a dosage range of 0.58.0 X 10’ spores (5-80 g of spore powder) per kilogram of dry soil was used in the bioassays. Ellis et al. (1989) indicated that in vitro-produced spores were about 6.4 times less infective than in vivo-produced spores and Grub Attack is formulated to reflect this difference. Thus, for the Grub Attack batches a dosage range of 3.2-51.2 x 10’ spores (5-80 g of spore powder) per kilogram of dry soil was used. Grub Attack samples were first bioassayed in the fall of 1987. During 1990, we conducted five additional bioassays. Forty larvae per dosage (2 per tin) were used in the first four tests and 30 per dosage (1 per tin) were used in the last two tests. Larvae were examined twice a week for the duration of each test (6 weeks) and hemolymph from larvae which appeared infected or were dead was examined with a phase contrast microscope at 400 x to confirm the presence or absence of B. po pilliae spores and rods. The percentage of milky diseased larvae in each test was corrected for milky disease in the control by use of Abbott’s formula (Abbott, 1925). Third instar Japanese beetle larvae were also injected with spores of B. polymyxa at rates of 0, 1000, 10,000, and 100,000 spores per larva (20 larvae per dosage). Injected larvae were examined for symptoms of milky disease as described above. RESULTS

First Evidence that Grub Attack Did Not Contain

B. popilliae The Grub Attack sample identified in Table 1 as GA . CT was suspended in H,O, the suspension was heated as described under Materials and Methods, and dilutions were plated on MYPGP agar and MYPGP + Vm agar in an attempt to isolate a new culture of B. popilliae for another project. However, no typical B. popilliae colonies developed on either medium after incubation at 30°C for up to 13 days. Colonies very different from B. popilliae colonies appeared only on MYPGP agar. They appeared within 1 day at 30°C and developed to a diameter of about 1 cm within 1 week. The culture was catalase ( + 1. Typically, B. popilliae colonies arising from spores first become visible on MYPGP agar in 2 to 4 days at 30°C and develop to a diameter of 2-3 mm after incubation for 1 to 2 weeks. (B. popilliae colonies first become visible on MYPGP +

286

STAHLY

Vm agar after incubation for about 5 days.) B. popilliae is catalase (-1 (Gordon et al., 1973). The culture from GA . CT sporulated efficiently within 2 days. Sporulating cells consisted of oval spores in swollen sporangia without parasporal bodies. Typically, B. popilliae cultures exhibit little to no sporulation on MYPGP agar and, when they do sporulate, parasporal bodies are always evident. Sporangia of the GA. CT-derived culture lysed late in the sporulation process, liberating free spores. B. popilliae sporangia typically do not lyse late in sporulation (Gordon et al., 1973; Bulla et al., 1969). The titer of the bacteria in GA . CT was 8.4 x lo7 spores/gram. Identification of the Four ATCC Cultures At the time of patent application (Ellis et al., 1989), four strains reported to be B. popilliae were deposited with ATCC. These strains were obtained and identified. All four strains grew on nutrient agar and were catalase-positive, characteristics not shared by B. popilliae. ATCC 53256, the preferred production strain (Ellis et al., 19891, sporulated readily on MYPGP agar. The cells without spores were cl.0 km in width. Oval spores were produced within distinctly swollen sporangia and parasporal bodies were not observed. From cellular and colonial morphology, ATCC 53256 appeared identical to the strain isolated from GA. CT. ATCC 53257, 53258, and 53259 sporulated efficiently on nutrient agar but not on MYPGP agar. The cells without spores were cl.0 p,rn in width. Oval spores were produced within slightly swollen sporangia. Parasporal bodies were not observed. Table 2 lists other cultural and physiological characteristics of the four ATCC strains. Also shown for comparative purposes are characteristics of B. polymyxa, B. amylolyticus, and B. popilliae. The characteristics of the above three organisms were previously reported (Gordon et al., 1973; Nakamura and Swezey, 1983; Claus and Berkeley, 1986; Norris et al., 1981; Krieg, 1981; Stahly et al., 1992a; Nakamura, 1984; Milner, 1974; Steinkraus and Tashiro, 1967). In addition, some characteristics of the type strains of B. polymyxa (strain BGSC 25Al), B. amylolyticus (strain NRRL B-3771, and B. popilliae (strain NRRL B-2309) determined in this study are included. The characteristics of ATCC 53256 match perfectly with B. polymyxa. The characteristics of ATCC 53257, 53258, and 53259 are all the same and match closely with B. amylolyticus. In contrast, the characteristics of the four ATCC cultures are very different from those of B. popilliae. A partial characterization of the strain isolated from GA . CT was performed. In addition to the characteristics enumerated in a previous section, it was determined that the culture grew on nutrient agar. Other test results are shown in Table 2. All of these results were the same as those obtained with ATCC 53256 and B. polymyxa.

AND KLEIN

Another characteristic that is sometimes used as an aid in bacterial identification is the cellular fatty acid composition. Table 3 shows the fatty acid profiles of GA . CT and the four ATCC cultures compared to those from B. polymyxa, and from the type strains of B. amylolyticus, and B. popilliae. (The fatty acid profile of B. polymyxa represents the library average of the type strain and several other strains [A. Poirier, Five Star Laboratories, personal communication].) As with the cultural and physiological tests reported above, the fatty acid profiles of GA . CT and ATCC 53256 matched closely with that of B. polymyxa. The fatty acid profiles of GA . CT and ATCC 53256 were very different from that of B. popilliae. The fatty acid profiles of ATCC 53257, 53258, and 53259 were similar to each other and matched most closely with B. amylolyticus. Major differences were apparent between the fatty acid profiles of the ATCC 53257,53258, and 53259 cultures and that of B. popilliae (Table 3). Thus, conclusions derived from fatty acid analysis were consistent with those derived from anatomical, cultural, and physiological characteristics. Analysis of Different Batches of Grub Attack and Primary Fermentation Powder Table 4 shows the results of an analysis of 13 batches of Grub Attack including three primary fermentation powder samples (A28, ARB106, and K290). The year in which the products were obtained is also shown. All of the 1990 batches contained only B. polymyxa, with the possible exception of GA * BN which contained a low concentration of an unidentified Bacillus species. The one 1988 batch, GA. KY, contained B. polymyxa, B. popilliae, and B. amylolyticus. After obtaining these results, we were told by F. Obenchain (formerly with Reuter Laboratories) and J. Miko (Ringer Corp.) that some older batches of Grub Attack were formulated with different mixtures of the four ATCC cultures and that in a few batches (including GA * KY), B. popilliae spores produced in uiuo were included. GA . 102, obtained in 1985, also has B. popilliae in addition to B. polymyxa and an unidentified Bacillus species. The one 1987 batch analyzed contained B. thuringiensis in addition to B. polymyxa. The B. thuringiensis was identified by its typical colonial and cellular morphology. Of particular diagnostic significance were the parasporal bodies readily observable by phase contrast microscopy. As another example of the variability of the product, GA * 89, obtained in 1985, contained only B. amylolyticus. Not shown in Table 4 is the result of an analysis of one batch of Doom, Standard-AKJ (see Table 1). Since it was derived from macerated milky diseased larvae, many unidentified Bacillus species were present on MYPGP agar plates spread with O.l-ml portions of lower dilutions. Use of higher dilutions revealed only

IN VITRO PRODUCTION

287

OF Bacillus popilliae

TABLE 2 Cultural and Physiological Characteristics of the Four Cultures Deposited with ATCC at the Time of Patent Application and the Culture Isolated from GA . CT Compared to the Characteristics of B. polymyxa, B. amylolyticus, and B. popilliae Characteristic Catalase Anaerobic growth VogesProskauer test pH in V-P broth 4.5 Fermentation of D-glucose L-arabinose D-xylose D-IUaIIni~Ol

ATCC 53256

ATCC 53257

ATCC 53258

ATCC 53259

B polymyxa

+ + + +

+ + + +

+ + +

+ + +

+ + +

+ + + v

+ + +

AG AG

AG AG AG AG AG AG AG AG AG AG AG AG AG AG -

A A A A A A A A A A A A A A -

A A A A A A A A A A A A A A -

A A A A A A A A A A A A A A -

AG AG AG AG AG” AG” AG” AG” AG” AG” AG” AG” AG” AG” -= -=

A A A A A A A A A A A A A A -

+ +

+ +

N’D -

N+D -

+= + -

GA.CT

AG

cellobiose D-frUCt,OSe

D-galactose lactose maltose D-mannose melibiose D-ribose sucrose trehalose D-sorbitol L-rhamnose Hydrolysis of casein gelatin starch Tween-80 urea Utilization of citrate propionate acetate succinate fumarate malate Nitrate reduced to nitrite Formation of indole H,S production Growth at or in 3% NaCl 5% NaCl 7% NaCl 0.001% Lysozyme pH 5.6 30°C 37°C 45°C 50°C

+ +

-

+ -

+ + (no gas) -

+ (no gas) -

+ + + + + -

+ + + + + + + -

+ (no gas) -

+ (no gas) -

-= -= -= -= +” + (no gas) -a += V

+ + + V

-

B. amylolyticus

B. popilliae + A -= i -= A A -= -= r; -=

V

V

V

+ (no gas) -

ND ND ND ND ND ND ND (no gas) ND ND + + -

u Reaction not included in previous publications (see text). The reaction shown is that obtained in this study with the type strain. Note. + , positive reaction; - , negative reaction; ND, not determined; A, acid; G, gas; v, strain variation.

B. popilliae colonies were present on plates of MYPGP + Vm agar spread with all dilutions. The concentration of B. popilliae spores determined by counting colonies on both selective and nonselective media was essentially the same; 4.1 x 10s spores/g on MYPGP agar and 4.7 x 10’ spores/g on MYPGP + Vm agar.

B. popilliae colonies.

Bioassays

The 1987 bioassay (Table 5) showed that only one lot of Grub Attack (GA . 102) was able to cause high levels of milky disease in Japanese beetle larvae. In the first 1990 test (3/27/90 in Table 51, using larvae collected in the fall of 1989, we did not get the infectivity we ex-

288

STAHLY

AND KLEIN

TABLE 3 Fatty Acid Profiles of the Four ATCC Cultures and the Culture Isolated from GA * CT Compared to Those of B. polymyxa, B. amylolyticus, and B. popilliue Percentage of total Fatty acid

GA.CT

ATCC 53256

ATCC 53257

ATCC 53258

ATCC 53259

1l:O anteiso 13:0 anteiso 13:0 is0 14:0 is0 14:o 15:O is0 15:0 anteiso 15:o 16:l isoE 16:0 iso 16:l A 16:0 17:l isoE 17:0 is0 17:0 ant&so

0 0 0 1.98 1.37 5.54 58.61 0.85 0.60 10.85 2.25 7.53 0 2.28 7.61

0 0 0 1.81 2.26 5.25 59.76 0 0 9.04 1.39 10.19 0 2.46 7.84

0 0.38 0 2.84 4.11 8.08 46.97 0.52 0 5.63 9.23 14.67 0.43 3.53 3.61

0.32 0.34 0.30 2.54 4.50 6.34 46.10 0.47 0 5.37 5.52 20.90 0 3.53 4.07

0 0 0 3.74 4.04 8.96 41.15 0.57 0.38 6.94 8.51 16.61 0.5 4.86 3.45

petted with

our standard material (Doom, Lot AKJ B*R, from Fairfax Laboratories; Standard-AKJ) that had given high levels of infectivity in the 1987 test, indicating that the larvae were not feeding well or were of poor nutritional quality. One would expect 5080% milky larvae at the dosages we were using (Dutky, 1963). A second test (4/27/90 in Table 5) with larvae collected in spring gave higher levels of milky disease TABLE 4 Bacteria Detected in Different Batches of Grub Attack

Year obtained

Product GA.BN

1990

GA.AZ GA.N GA.CT Granular GA.Ch A28 ARB106 K290 GA.KY

1990 1990 1990 1990 1990 1990 1990 1990 1988

GA

GA. 136

1987

GA. 102

1985

GA.89

1985

Species detected P. polymyxa unidentified Bacillus sp. B . polymyxa B . polymyxa B . polymyxa B . polymyxa B . polymyxa B . polymyxa B. polymyxa B. polymyxa B. polymyxa B. popilline B. amylolyticus B. polymyxa B. thuringiensis B. popilliae B . polymyxu unidentified Bacillus sp. B. amylolyticus

Approximate concentration of spores/g of product 5.6 x 10’ 1.3 x 104 1.1 1.9 8.4 6.0 1.5 4.9 1.7 1.1 1.8 2.6 2.1 6.0 5.6 4.6 1.4 4.2

x x x x x x x x x x x x x x x x

107 108 lo7 lo7 109 10’ 108 108 10’ 10’ 106 lo7 lo* 10’ 108 lo7

3.5 x lo5

B. Polyw~ 0 0 0 1.98 1.98 7.50 61.65 0 0 7.92 2.25 7.53 0 2.71 6.65

B. amylolyticus 0 0 0 1.66 3.95 9.53 44.03 0.57 0.57 5.62 7.13 19.01 0.60 3.02 4.31

B. popilliae 0 0 0 1.00 4.89 11.22 39.43 0 0 2.40 0 30.72 0 7.03 2.17

with the Standard-AKJ, but was complicated by abnormally high levels of milky disease in the controls. In this test, it appeared that the three batches of Grub Attack exhibited some infectivity, although much less than the three batches of Doom, which contained B. popilliae spores produced in vivo. Because the spring tests (3/27/90 and 4/27/90) were inconclusive, we ran additional tests in fall and winter with Doom, Klein-85 (in viva), and Grub Attack (in vitro) spore powders. On the basis of these tests (10/26/ 90,12/28/90, and 3/B/91; Table 5) there is no evidence of appreciable infectivity by either the primary fermentation powders or commercially formulated Grub Attack batches. In contrast with results reported in the patent for the in vitro process (Ellis et al., 19891, we found neither their levels of infectivity nor a doseresponse relationship for any of the in vitro-produced bacterial products. Table 5 also shows that neither the spores of B. polymyxa (the strain isolated from GA . CT) or B. amylolyticus (strain ATCC 53259) were infective per OS for Japanese beetle larvae. J. Hanula and D. Dingman (Connecticut Agricultural Experiment Station, New Haven, CT) were also unable to observe any per OS infectivity of Japanese beetle larvae by B. polymyxa spores isolated from GA . CT (personal communication). We also tested the effect of injection of B. polymyxa spores on Japanese beetle larvae. Uninjected control larvae and those injected with water had 15 and 40% mortality, respectively. Mortality of larvae injected with 103, 104, and lo5 B polymyxa spores was 30, 40, and 25%, respectively; values equal to or less than that of the water-injected larvae. No evidence of milky disease or of septacemia due to B. polymyxa was

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PRODUCTION

1990-1991 Year obtained

Material Standard-AKJ

1985 1989

DM.KY

1988

DM.AZ

0.5 1.0 2.0 4.0 8.0 8.0 1.0 4.0 0.5 1.0 2.0 4.0 0.5 1.0 2.0 4.0 3.2 6.4 12.8 25.6 3.2 6.4 12.8 25.6 51.2 6.4 25.6 3.2 12.8 51.2 3.2 12.8 51.2 3.2 12.8 51.2 3.2 6.4 12.8 25.6 51.2 3.2 12.8 51.2 3.2 12.8 51.2 3.2 12.8 51.2 6.4 25.6 12.8 51.2

1990

GA.AZ

1990

GA.N

GA.CT

1990

A28

1990

ARBlO6

K290

1988

GA.KY

GA.

136

1987

GA.

102

1985

GA.89

Bacillus

1985

polymyxa

(from GA . CT) spores Bacillus amylolyticus ATCC 53259 spores Control

1

X 10’

289

popilliae

TABLE 5 DiseaseBioassays

Milky

No. milky

Spores/kg soil”

1983

Klein-85 DM.GM

OF Bacillus

10/14/87

3127190

4127190

20 (49)

4 (10)

14 (29)

27 (67)

8 (20)

21(51)

(% milky)b 10/26/90

12128190

318191

ll(27) ll(37) 15 (37) 29 (72)

12 (40) 13 (43)

20 (67)

7 (16) 16 (40) 15 (32) 10 (25) 14 (29) 16 (40) 15 (32) 9 (21) 16 (35) 11 (27) 5 (0) 2 (3) 10 (16) 010) l(3)

5 (0)

l(3)

10 (16)

1 (0) 0 (0)

0 (0) 0 (0) 0 (0) l(3) 0 (0)

0 (0) 0 (0) 3 (8)

0 (0) 2 (5) 0 (0)

0 (0) 10 (16) 1 (0) 9 (13) 4 (8) l(3) 4 (8) 2 (3) 3 (5) 17 (41) 19 (46) 29 (72) 3 (5) l(0) 4 (8)

3 5 1 1

(10) (17) (3) (3)

l(0) 2 (3) 0 (0) 5(-)

l(-)

0(-l

0 (0) 0(-j

a The number of spores/kg of soil was determined assuming that the concentration in the products was as specified by the manufacturers; x 10*/g for Doom and Klein-85 and 6.4 x 10s for Grub Attack and its primary fermentation product.

* Percentage 30 larvae/dosage

milky corrected for milky on 12128190 and 318191.

larvae

in the control

by Abbott’s

formula.

40 larvae/dosage

on 10114187,

3/27,

4127, and 10126190;

290

STAHLY

detected in any of the larvae in this test. About 50% of larvae injected with lo4 spores of B. popilliae would be expected to die from milky disease (Dutky, 1963). DISCUSSION

Our results suggest that the methods presented by Ellis et al. (1989) with regard to in vitro sporulation of B. popilliae should be reexamined. The four putative B. popilliae cultures capable of in vitro sporulation that they submitted to American Type Culture Collection at the time of patent application were identified in this study. One of these cultures, identified in the patent as the preferred strain, is B. polymyxa. The other three strains are B. amylolyticus. The identification of the above cultures was based on anatomical, cultural, and physiological characteristics and also on cellular fatty acid composition. Kaneda (1967,1969,1977) was one of the first to make a systematic study of the fatty acid composition of Bacillus species. He divided 22 Bacillus species into six different groups (Kaneda, 1977). Group B consists of B. polymyxa, B. larvae, B. lentimorbus, and B. popilliae. Members of this group have an “insignificant or very small” percentage (less than 3%) of unsaturated fatty acids and the predominant fatty acid is the anteiso-Cl5 acid (3962%). The range of chain length is 14-17. The data of Table 3 relating to B. popilliae, B. polymyxa, and ATCC 53256 confirm placement in Kaneda’s Group B. Several major differences between B. polymyxa and B. popilliae in the fatty acid profiles were noted, however. The percentages of the anteiso-&:o and C,,:, acids especially were significantly different. The profile of ATCC 53256 matched much closer to B. polymyxa than to B. popilliae. The fatty acid profile of B. amylolyticus had not been determined before this study. Based on the fatty acid profile, B. amylolyticus does not clearly fit in any of the six groups of Bacillus species proposed by Kaneda (1977). B. amylolyticus possesses 8.3% unsaturated fatty acids and the predominant fatty acid is the anteiso-C,,:, acid (44.03%). The range of chain length is 14-17. A good match was observed between the fatty acid profiles of B. amylolyticus and those of ATCC 53257, 53258, and 53259. A small quantity of fatty acids of chain length shorter than 14 was found in ATCC 53257 and 53258 but the quantity of the shorter fatty acids was less than 1% of the total. Samples of the product Grub Attack and primary fermentation powder used in formulation of Grub Attack were also analyzed for bacterial content. All of the batches received in 1990 with one possible exception (GA. BN) contained B. polymyxa only. Batches received in 1985 to 1988 were variable in composition. One (GA * 89) contained only B. amylolyticus. Two contained B. popilliue in addition to B. polymyxa and other Bacillus species (GA * KY, B. amylolyticus; GA . 102, an unidentified Bacillus species). One contained B.

AND KLEIN

thuringiensis in addition to B. polymyxa. As stated in the Results, the spores of B. popilliae (at least those in GA - KY) were produced in uivo (personal communication, F. Obenchain and J. Mike). Why in uiuogenerated B. popilliae spores were added to only certain lots of Grub Attack is unknown. It is possible that B. amylolyticus was present in other lots containing high concentrations of B. polymyxa. It is difficult to detect a few B. amylolyticus colonies in the presence of a much larger number of B. polymyxa colonies. Spores derived from pure cultures of B. polymyxa and B. amylolyticus did not produce milky disease or death of third instar larvae of Japanese beetles. Also, there was no evidence of significant infectivity by either primary fermentation powders or commercially formulated Grub Attack batches obtained between 1985 and 1990, with the exception of GA - 102. GA * 102 exhibited reasonably good infectivity in the 1987 bioassay. The explanation for the low level of infectivity of GA . 102 in the 3/8/91 bioassay (10 and 17%) is unknown. In any event, the relatively high concentration of B. popilliae spores in GA . 102 (4.6 X 10s spores/g) would account for the infectivity seen in the 1987 bioassay; the other in uitro-produced spores in GA * 102 were not responsible for the infectivity. The low level of infectivity by GA . KY, the other sample containing B. popilliae, is probably due to the low titer of B. popilliae present (2.6 x lo7 spores/g). Our levels of infectivity with GA . KY were somewhat lower, but roughly comparable, to those reported by Redmond (1990) with the same material. In uiuo-produced spores of B. popilliae (Doom and Klein-851 gave moderate levels of infectivity per OSexcept for the bioassay performed on 3/27/90 (Table 5). The levels of infectivity observed on that date were much lower than those observed historically when dosages of 0.2-0.5 x 10’ spores per kilogram of soil were used (Dutky, 1963). However, several factors including the nutritional status of the host can influence the infection process (Milner, 1981). In addition, the doseresponse relationships described by Dutky (1963) are the cumulative result of numerous tests conducted over many years. Results from individual tests can vary widely, but patterns of infectivity can be established. On May 7, 1991, Ringer Corporation announced a voluntary recall of Grub Attack produced for the 1991 season. However, they have acknowledged only that the product manufactured for the 1991 season exhibited performance problems. Our results suggest that compositional and performance problems with Grub Attack products lacking in uiuo-produced B. popilliae spores have existed since 1985, the year that the product was introduced. In 1989, Reuter Laboratories, Inc., obtained their patent for the in vitro process (Ellis et al., 1989) and sold those rights as well as the product name, Grub Attack, to Ringer Corporation. There is a

IN VITRO PRODUCTION

need for the in vitro production of high-quality infective spores of B. popilliue (Klein, 1992) and additional research efforts should be directed toward that goal. ACKNOWLEDGMENTS

Thanks to J. J. Moyseenko (USDA, Wooster, OH) for technical support in conducting bioassays. We thank D. Dingman and J. Hanula (Connecticut Agricultural Experiment Station, New Haven, CT) for conducting an independent analysis of the infectivity of B. polymyxa. The cooperation of F. Obenchain (formerly of Reuter Corp., now with HollyCroft Laboratories, Manassas, VA) and J. Miko (Ringer Corporation, Minneapolis, MN) is gratefully acknowledged for supplying information, cultures, and some of the product samples used in this study. Dr. A. R. Poirier (Five Star Laboratories, Branford, CT) provided help beyond that paid for in supplying information relating to the fatty acid analysis of cultures. Thanks to L. Nakamura (USDA, Peoria, IL) for reviewing the data relating to identification of B. amylolyticus and for supplying B. amylolyticus NRRL B-377. We also thank D. A. Potter and C. T. Redmond (University of Kentucky, Lexington, KY) for supplying samples of Doom and Grub Attack for our analysis and acknowledge their concerns about the infectivity of the in vitro-produced material. We are grateful for the secretarial assistance provided by Marcia Reeve. REFERENCES

Abbott, W. S. 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265-267. Breuil, C., and Gounot, A. M. 1972. Recherches preliminaries sur les batteries lipolytiques psychrophiles des sols et des eaux. Can. J. Microbial. 18, 1445-1451. Bulla, L. A., St. Julian, G., Rhodes, R. A., and Hesseltine, C. W. 1969. Scanning electron and phase-contrast microscopy of bacterial spores. Appl. Microbial. 18, 496495. Claus, D., and Berkeley, R. C. W. 1986. Genus Bacillus Cohn 1872. In “Bergey’s Manual of Systematic Bacteriology” (P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, Eds.), pp. 1105-1207. Williams and Wilkins, Baltimore. Dingman, D. W., and Stahly, D. P. 1983. Medium promoting sporulation of Bacillus laruae and metabolism of medium components. Appl. Environ. Microbial. 46, 860-869. Dutky, S. R. 1941. Testing the possible value of milky diseases for control of soil-inhabiting larvae. J. Econ. Entomol. 34, 217-218. Dutky, S. R. 1942. Method for the preparation of spore-dust mixtures of Type A milky disease of Japanese beetle larvae for field inoculation. U.S. Bur. Entomol. and Plant Quar. ET-192. Dutky, S. R. 1963. The milky diseases. In “Insect Pathology, an Advanced Treatise” (E. A. Steinhaus, Ed.), Vol. 2, pp. 75-115. Academic Press, New York. Ellis, B. J., Obenchain, F., and Mehta, R. 1989. “In Vitro Method for Producing Infective Bacterial Spores and Spore-Containing Insecticidal Compositions.” United States Patent Number 4,824,671. Fleming, W. E. 1968. “Biological Control of the Japanese Beetle”.

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