Milky disease development in field-infected Japanese beetle larvae

JOCRSbL

OF

INVER’TEBRhTE

Milky

P.ITHOLOGT

Disease

20,

(1972)

lo%-113

Development

in

Beetle (;liAxT

ST.

JULIAN,

.Vorthern

RegiorLal

~IEE

A.

Research Received

Field-Infected

Japanese

Larvae

BULLA,

-JR.,

Laboratory,’ January

AND

C+ORDON

I,.

Peoria,

Illinois

61604

ADAMS

SO, 1972

By correlated visual and microscopical examination, milky disease of field-infected, third-instar Japanese beetle larvae is cat)egorixed into four phases. The phases are described as sequential disease symptoms I through IV. All four phases persist simultaneously throughout experimental incubation. Larvae die during all phases of the disease; however, the largest percentage of death is at phase II and III of the infectious process. At phase II, 90’0 of the total population of cell types in the infected hemolymph are vegetative cells; in phase III 65-767; are vegetative cells with 17-28”; spores. The massive spore population (95@’,0 of population) that characterizes milk) disease is designated phase IV; less than 30y0 of larvae reach this phase of the disease.

The name “milky

disease” refers to the of the hemolymph of the Japnnesr bcctlc (Popillia jnporlica) larv:a after it becomw hcwily infested \vith spores of either Bacillus popilliae or Bacillus lejliiwwbus (Dutky, 1940). Sports of (lither bscterium :w c6wtivc insecticidal agents against the: .Japanesebeetle and some ot’hcr ausceptiblc hosts (Rcurd, 1945; Dutky, 1963; St. Julian and Hall, 196s; Tnshiro, 1957). Previously- \VC det’ermincd the number of sporesneeded t’r) infect larvae in soil and also outlined the pattern of dwelopmcnt of B. pqilliae within the larvae (St. .Juli:m ct al., 1970). TIN, infectious process in these lab~nilk~-

:Lppcarance

These data suggest important differences bekeen laboratory- and field-infected Inrvw. Awarcncss of the differences could have :L direct influence 011 the method and extent of sport application to field plots. ~IATERIALS

AND

RIETH~Ds

Lawae. Third-instar larvae of tile Japnnest beetle, P. japu2ica, WTC c*ollectecl by the Plant Protection Division, Agricultural Research Srrviw, C 3. Dcpnrtnwnt of Agriculture, from diseased areas in midwestern statrs. Their care and handling have been described by St’. *Julian ct al. (1963). Data presented lwrc summarize four separate csperimcnts that involved 2 total Of 12,000 larvae. The larvae n-we collcct~edin the fall or:ltor~-iIlfc~~t(‘d larvae 0ccurrc.d in four of 1969 and spring of 1970. phases, as tlctwmined by Iliicr~JsCOpical (ISErpwii?len tal prowrluw. Larvacx T~PI’LL nminntion c)f t hr. diwased h~molymph. Wck maintained in n-oodcn bows (25 X 13 X S have no\v cstablishcd (using t’hc prcviousll inches i.d.) containing finely sift,ed moistened clcscribed ph:ws of tllc infwtious process Roil to t’he depth of 6 inches. The soil came CL.< a reference) n C~x-rd:lt~i~J~l between visual from fic~lcl plots inhabited by the diseased gross s~mpt~oms and microscopic appe>arancn larvae. Healthy larvae as controls were of diseasrtl hemol\-mph of field-infected maintained in noninfected soil. Three thoularvae. sand larvae each wcrc placed into four boxes 1 This is a laboratory of the Northern Marketing and incubated at 25~23°C for a total of 30 and Nutrition Research Division, Agricultural days. From each of three storngc bows, 150 Research Service, U. S. Department of Agricullive larvae n-cre remowd 011 days 1, 7, 14, ture. Copyright 111 rights

@ 1972 by Academic of reproduction in any

Press, inc. form

reserved

100

110

ST.

JULIAN,

BULLA,

FIG. 1. Phase I through IV of the infectious process of milky disease in hemolymph of fieldinfected Japanese beetle larvae. Phase I contains blood cells (bc), transparent hemolymph (h); phase II, vegetative cells (vc) in hemolymph; phase III, vegetative cells (vc), prespores (p), and spores (s) in hemolymph; phase IV, spores of Bacillus popilliae in hemolymph. Viewed under phase-contrast optics at 1250X.

21, and 30. The live larvar then were examined for gross symptoms of milky disease. All dead larvae found at these time intervals were also examined for disease symptoms. The larvae were separated into four groups based on hemolymph t’urbidity. Each group of larvae were lvashed and bled as previously described (St. Julian et al., 1970). Differential microscopical count of cell types were made from the pooled hemolymph of each group \vi-ith a Petroff-Hausser2 bacteria counter with phase-contrast optics at 1250X. The fourth storage box was allowed to incubate undisturbed for 30 days at which time all live and dead larvae were removed and examined as mentioned above. Recovery of spores from field soil. At the termination of the experiments, soil from the four storage boxes was combined and mixed thoroughly. Samples of 1, 10, and 100 g of combined soil were placed separately in Erlenmeyer flasks containing 3X (w/v) of sterile distilled water. The flasks containing soil and water were shaken at 300 rpm for about 3 hr. Flasks then were allowed to stand about 1 min and the supernatant 2 Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.

JR.,

AND

ADAMS

water decanted away from settled soil; the supernatant then was centrifuged for 10 min at 1,800 y. The centrifugate was esamined microscopically for R. popilliue spores. Control soil containing 1 X lo9 spores/g of soil was examined in the same manner. Soil (1 X lo9 spores/g) previousl) has been shown to infect 50% of larvae (St.. Julian et al., 1970). The extent of infection Disease symptoms. in an intact larva can be det’ermined visually by gently pressing the larva posterior toward its anterior and observing the opacity and color of its hemolymph at the posterior. RESULTS

Nilk y Disease Developmell t Visual observation enables the disease process to be categorized as four distinct phases. Phase I, the beginning of bacterial invasion into the hemolymph, is characterized by transparent hemolymph. At phase II of the disease the hemolymph is slightly gray ; it is off-whit’c at phase III, and milk white at phase IV of the infection. Microscopical appearance of larval hemolymph throughout, the infectious process of milky disease is shown in Fig. 1. In phase I, the hcmolymph c+ont8ains only a few invading vegetative cells. Predominant’ vegetative proliferation in the hemolymph with limited spore formation indicates phase II of the infection. Intermediate phase III is characterized by concomitant vegetative growth and sporulation. In the t’erminal phase IV of the disease, there occurs massive accumulation of spores and typical milky white hemolymph. The percent of larvae in each of the four phases of the infectious process during a 30-day observation period is shown in Table 1. As incubation time increased, the percent of larvae in the later phases of the disease increased. However, it is evident from these data that all phases occur throughout the incubation period. Death occurs at all phases

PHASES

OF

MILKY

of t’hc disease; the largest percent die during phases II and III of the infectious process. Significantly, most larvae (dead and live) exhibit the intermediate phase of infection (III), at which time spores do not predominate in the cell population. Tables 2 and 3 show the quant’itative pattern of B. popilliae development in moribund and dead larvae, respectively. Vegetative growth and sporulation occur concomitantly so t’hat spores form continuously and, ultimately, accumulate in large numbers. Total vegetative populations never esccrd 3-4 X log/ml of hemolymph. How ever, vegetative cell numbers seem to be higher in dead than in moribund larvae (Table 3, phase III); the contrary is true for spore numbers (Table 2, phase IV). Vegetative cells in phase II constit’ut’e ossentially 100% of the total population. In phase III 65-76s are vegetative cells with 17-2S70 spores. Less than 10% of cells are presporcs. Hemolymph cxontains about 99% spores at pllasc IV.

Of t#lie10y sporesper gram of cont’rol soil, tdle average number of spores recovered per gram was about 1 X 103. Spores could be isolated only from the 100-g samples of the test soil, and then the average number of spores recovered per gram of test soil was lessthan 1.

IJntil now it was presumed that most field-infected larvae died at phase IV and contained about 5 X log spores. Our dat’a show that lessthan 30 % of larvae examined survive to phase IV although the microscopical pattern of growth and sporulation of B. popilliae in the field-infected larvae is similar to larvae infected in the laboratory (St,. Julian et al., 1970). Therefore, we belirve that the sudden death of field-infected larvae may be due to rapid proliferation of

111

DISEASE

TABLE VISUAL

1

OBSERVATION OF THF: INFECTIOUS P~00s5s OF MILKY Drs~~sa IN JAPANESE BEETIX

Percent Time of incubation (days)a

1 7 14 21 30 3oe

Live

16 76 60 9 1 36

LSRV~~E

of larvae in designated phase of infectior?

larvaec

0.i 3 27 68 67 39

Dead I

II

0 39 10 3 2 0

0 50 37 11

larvaed III

IV

0 0 11 0 47 5 80 7 2 85 11 28 1 71 a Larvae incubated at 25-28°C in field soil from which they were collected; day 1 represents the first day larvae were placed in laboratory condition. * Phase I = no visual evidence of infect,ion, hemolymph is transparent. Phase II = hemolymph slight gray ttubidity; vegetative cell proliferation. Phase III = off-white turbidity; concomitant vegetative growth, prespore formation and sporulation. Phase IV = milky white hemolymph containing massive spore population. (- On each designated day, 450 live larvae were examined. d All dead larvae found each designated day were examined. e Observed only after 30-day incubation at which time all larvae found dead or alive were examined.

-I

vegetative cells rather t,han t,o accumulation of spores. A wide variety of st’rains develop from B. popilliae spores (Sharpe et al., 1970). From the standpoint of larval inferkion, in vitro grown vegetative cells are of two types: (1) those that grow slowly in t,he hemolymph and cause diseasephases I through IV and (2) cells that grow rapidly and kill larvae at phasesII or III before massive spore produotion. The same substrains seem to develop in field-infected larvae. Injection of lOO5,000 in vit’ro grown vegetative cells per larva is required to obtain 50 % infectivity (Pridham et al., 1964). A challenge dose of B. popilliae

112

ST.

~~1c~osc0~1c.41,

JULIAN,

BULLA,

TABLE popilliae

OF Bacillus

OHSICKV.\TIONS

JH.,

BEETLE

Cells/ml Time of incubation (days)”

hemolymph

from

0.0001 0.003 j 0 0 0 ‘0 0.00001~

1 7 1-l 21 30 30"

Spores

0 0 0 0 0

i

~ p:;

0 0 0

-

Veg

larvae

5.5 2.x 5.9

OIGIS~VATIONS

Cells/ml

OF ~~OIZIBL~ND

at phases I-IV

Spores

0. OOOG 0.03 0.47 0.01 0.01 0.03

0.02 0.04 0.05 0.01 0.01 0.00003

hemolymph

from

Veg

Prespores

Spores

~ 0.0001 1 0.02 ~ 0.0001 i 0.0006 ‘0 I

0 0.0004 0 0 0

0 0 0 0 0

Vets

J.IPANESE

(count

X 1o8)b

-

made optics

31 13 8.3 33 7.2 10

Pre-

Spores

12 1.1 0.5 I.6 0.4 0.3

from pooled at 1250s.

Prepore: 3 Ispores

Veg

larval

I

i-

3 1 3.3 1 3 5

180 I60 130 180 150

1 x:: ~ 0.32 0. 6 0.G

hemolymph

with

Petroff-

3

dead larvae

Spore:

IV

!

IN

HEMOLYMPH

at phases I-IV

II

Veg

of infection

$&

!

OB Bacillus popilliae BEETLE L.uwM~:

IC 1. ~

HsMoLrMPrf

III

TABLE

Time of incubation (day+

IX

Prespores

a Same as footnote a, Table 1. b Average differential microscopical counts Hawser bacteria counter under phase-contrast c same as footnote b, Table 1. d Same as footnote e, Table 1.

MICROSCOPIC.~L

2

II

Prespores

Vet3

ADAMS

LARV-413:

moribund

IC

AND

OF DUD

of infection

Japan-ESE

(count

X lOa)*

III Spores

Veg

0.0000~ 0.02 0.04 0.03 0.02

35 26.2 23 13.2

IV

Prespores

Spores

1.6 0.9 1.2 0.4 0.6

5 5.3 G.3 3.3 2.5

Veg

Prespores

-I1 7 1-L 21 30 30" 0 Same 6 Same c Same d Same

-

as as as as

-

footnote footnote footnote footnote

a, b, b, e,

Table Table Table Table

7.4 7 6.2 22 9

0.1 0.08 0.03 0.02 0.04

8

-

-

-

-

0.6 0.4 2.4 0.3

0.4 0.5 0.3 0.000:

113 113 167 90

1. 2. 1. 1.

approsimatcl\5,000-10,000 viable vegetutive cells of B. popiilliae kills larvae within 3-5 days without the development of millq disease symptoms or microscopical evidence that spores were formed in the larvae. The dead larvae contain a massive vegetative population. By comparison, a high incidence of milky disease (90%) can be obtained by

injecting lo6 R. popilliae spores per larva. In addition, challenge doses up to 4 X lo6 spores per larva never causes the nonspecific death associated with the injection of large numbers of vegetative cells (St. Julian and Hall, 1968). The number of spores needed for oral infection is also high. Concentrations of 1 X log spores per gram of soil result in

30 c/, infxtion (St. Julian c% al., 1970). It is our contention that such large numbers of spores aw ncedcd to infect larvae brcausc of the> cstremrly Ion- germination and outgrowth rate of the bactwial sporw. Spores of B. popilliae do not wspond to common gcwninuting agents, and the germinnt,ion rate and subsequent out8grow-th of t8hesc sporrs range from only 2 to 10 ‘,‘h (St. Julian rtm al., 1967). Our data indicnt’e that’ the natural buildup of B. ~qpilliae spores in field plots from distrawl larvw does not occur as rapidly as ~~1s previously assumed. Therefore, UY’ bclicw that, to effcctivcly control ,Japnncsc~ bcc:t,lc lnrvw, hwvy conccntrutions of U. l~opilliue spores (> 1 X log spores per square inch of land surface) must bc applied in such manner as to become immediately available to t.hc larvae. In addit.ion, annual or semiunnual application \vith availnblc R. popillinc! strains is recommended.

S. R. 1940. Two new spore-forming bacteria causing milky disease of Japanese beetle larvae. J. Agr. Res., 61, 57-08. L)wK~-, S. R. 1963. The milky diseases. In “Insect Pathology” (E. A. Steinhaus, cd.), Vol. 3, pp. 75-115. Academic Press, n’ew E’ork. PIUDHAM, T. CT., sr. JuLI.~, c:., JK., .4o.\Ms, (;. L., H.LLL, II. IT., .\ND J \CKSOS, 1:. W. lYti4. Infect,ion of Popillia japonica Newman larvae with vegetative cells of L
E. S. Sharpe graphs.

SH.UIP52,

supplied

the accompanying

photo-

REFERENCES R. L. 1945. Studies on the milky Japanese beetle larvae. Conn. .4gr. Yew Haven Bull., 491, 505-583.

BE.IRL),

disease of Ezp. Sta.

DU'I'KY,

ST.

JuLI.~,

G.,

JK.,

PRIDH.\M,

T.

G.,

.\KD

HALL,

H. H. lQtj3. I+Xect of diluents on viabilit,y of Popilliu japonicu Newman larvae, Becillzts popilliar Uutky and HaGl1lL.s lm/im!~rbxs Dutky. J. Imect Pathol., 5, 440-450. ST.

JuLI.~,

G.,

1967. intact Dutky. ST.

PKLDH.IM,

T.

G.,

.\SIJ

HALL,

J-I.

Preparation and charact,erization of and free spores of Bnciltus pttpilliczr! Can. J. Mic~biol., 13, L’iY-285.

JULI.\N,

G.,

HH.WPE,

Ii:.,

\NII

RHODIGS,

It.

.k.

lY70. Growth pattern of Bacillus popilliae in Japanese beetle larvae. J. Z~e~lshr. Pathol., 15, 340-M. k:.

s.,

ST.

JULIAS,

G.,

.11\D

('l
c.

1970. Characteristics of a new strain of Nacillus popilliae sporogenic in vitro. -I ppl. Nimc Dial., 19, 681~GSX. TASHIKO, H. lY57. Susceptibility of 15uropran chafer and Japanese beetle larvae fo different strains of milky disease organisms. b. Eco7b. Entfmol., 50, 350-353.