The pattern of sporulation of Bacillus popilliae in colonies

JOURNAL OF INVERTEBRATE PATHOLOGY 21, 9-15 (1973)

The

Pattern

of Sporulation

of Bacil/os

popilliae

in Colonies’

E. S. SHARPE AND R. il. RHODES Northern

Regionnl Research Laboratory, CfS. Depnrtment of ilgriculture,

Agticulturcrl Resewch Peak, Illi~iois 61GO/,

Sewice,

Spores accumulate periodically in colonies of Brtci2l~s popillirte after 3 days of vegrt,ative growth on solid medium. Sporulation occurs on the surface and primarily in a ring causing slight changes in colony contour. The formation of mature near the periphery, spores and their acquisition of resistance to drying and to heat occur in a stepwise manner. A high level of prcspore forms persists in mature colonies. Sporulation in colonies is as efficient as early stages of sporulation in larvae. but efficiency in viva must increase as milky disease progresses.

yeast extract for sporulation of B. popilline Biological control of the *Japanesebeetle, var. melol’onthae 4263. We have conPopillia japonica, will be realized when tinued our studies of B-2309M because spores of Bacillus popilliae can be inexpen- of the ease and regularity of colonial sively produced in vitro. We recently de- sporulation. Sufficient spores were produced with beetle larvae scribed a new sporogenic strain of the for feeding t&s (Schwartz and Sharpe, 1970). Also, sporubacillus, NRRL B-2309M (Sharpe et al., lation in colonies has been confirmed by 1970). The M strain consistently sporulates Costilow and Coulter (1971), who have exat a frequency of 20-30s on solid medium, amined the physiological properities that and for this reason we have examined accompany in vitro sporulation of the closely its growth and sporulation pattern strain. In the present communication, we in colonies. Other systems for sporulating describe the kinetics of sporulation of M B. popilliae in liquid medium involve either strain in colonies and compare them to activated carbon (Haynes and Rhodes, growth and sporulation of the pathogen 1966) or a shaken and still culture procedure as described for the M strain (Sharpe within its insect host. et al., 1970). Each system now appears to MEDIUM AND METHODS require specific lots of yeast extract for optimal sporulation (W. C. Haynes, pers. Filter-sterilized MYPT medium and B. comm.). Wyss (1971) has also confirmed popilliae strain NRRL B-2309M were used the superiority of certain lots of Difco? exclusively (Sharpe et al., 1970). MYPT medium contained 1% Difco yeast extract ‘Presented in part at the 67th Annual Meeting of the American Society of Microbiology, New (Control No. 492496), 1% Difco MuellerYork City, April 1967. Hinton broth solids, 0.3% K,HPO,, 0.05% ‘Mention of firm names or trade products does trehalose, and 2% (w/v) Bacto agar. A not imply that they are endorsed or recommended flask fitted with a delivery head was used by the Department of Agriculture over other firms to pour plates (92 mm X 15 mm) of conor similar products not mentioned. 9 Copyright

@ 1973 by Academic

Press, Inc.

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SHARPE

AND

stant volume (30 ml) and depth of medium (6 mm). Plates must be inoculated within 48 hr of preparation, for optimal growth and maximal sporulation. The random location and variable number of colonies obtained by standard dilut’ion and spread plate techniques were inadequate for optimal and reproducible sporulation of B. popilliae. Careful placement of inoculum is essential so that colonies develop only at predetermined points on the agar surface. The highest yields of spores result when 1~12 colonies develop equidistant from one another and from the plate rim. To reproduce this pattern, a multiple inoculation tool was devised of 10 straight pins partiaIly embedded in a No. 13 rubber stopper (Fig. 1). This tool (Fig. 1C) was used in conjunction with a reservoir of spore inoculum (Fig. 1R) preparcd by distributing drop-

RHODES

wise 2 ml of a water suspension of spores (Fig. 1A) on a sterile filter paper fitting the bottom of a sterile petri dish. Plates were inoculated by touching the pin heads (arranged in a single plane) to the moist filt~er paper and then gently to the agar surface. Each plate so inoculated cventually developed 10 mature colonies at properly spaced stations (Fig. 1E). Concentrated spore suspensions (108/ml) were used to overcome the low germination rate characteristic of B. popillicre spores and to ensure formation of colonies at each station. Consequently, each mature colony usually developed from the fused outgrowth of several germinated spores. Preparation of inoculum. Selected sporulating colonies were suspended in water and placed in sterile SO-ml beakers covered with milk-filter discs (Fig. 1A). These were vacuum d&d at’ room temperature to in-

FIG. 1. Inoculation technique to achieve sporulating sion of vacuum-dried spores, (B) filter paper wetted lation tool, (D) sterile agar plate, and (E) resultant on agar surface.

colonies of Bac~Xus pop&e; (A) with spore suspension, (C) multiple uniform pattern of 10 sporulating

Suspeninocucolonies

SPORULATION

activate vegetative cells. Dried inoculum was stored for 30 days or longer at room temperature to minimize the appearance of asporogenic sectors in resultant colonies. Drying and aging are preferred because repeated heat treatments as low as 50°C for 15 min are detrimental to the spore-forming capability of B-2309M (Sharpe et al., 1970). Higher temperatures such as 80°C for 15 min inactivate many spores and do not eliminate asporogenic sectors in resultant colonies. The tendency toward asporogenicity is carried in the spores. The viability of vegetative cells and spores from colonies was determined by standard plating techniques. Microscopic counts were made with a Petroff-Hauser bacteria counter and a phase-contrast microscope. Heat resistance was determined by heating a sealed tube of cell suspension in a water bath for 20 min at 50°C before plating. Values reported are average counts of three colonies from each of three plates. RESULTS

Morphology

of Colonial Spodation

Characteristically, spores of B. popilliae strain B-2309M are concentrated in a ring near the periphery of the colony (Fig. 2). A microloop of material from the sporulation ring, detailed B, is shown at higher magnification in photomicrograph b. Likewise, areas A and C of the colony are shown in greater detail in corresponding photomicrographs a and c. It is evident that the highest concentration of spores (about 50% of cells present) occurs in area B. Few spores are found in A, the oldest portion of the colony. This area contains primarily granular, dead vegetative cells, and cellular debris. The area of most recent vegetative growth, C, contains mostly phase-dark, viable vegetative cells and some prespore forms ; appreciable sporulation does not occur in this peripheral zone. Normally, the ring of sporulation forms the area of greatest colony depth. Spores are concentrated in the upper layers of

IN

COLONIES

11

ceils. Swelling of cells during formation of prespores and spores increases their volume (three or four times) and thus raises the level of the sporulation ring. These colonial features are depicted in Fig. 3 by a drawing and by an oblique photograph of the same colony as in Fig. 2. Kinetics of Growth and Sporulation The pattern of sporulation in colonies is shown in Fig. 4. Vegetative cells were the only cell form observed microscopically during the first 48 hr of colony growth. Prespore forms appeared shortly after 48 hr when the rate of cell division was diminishing. Sporulation then occurs stepwise as indicated by the following microscopic determinations. Mature refractile spores appeared in samples taken after 3 days of colony development and 8 X 10F spores were formed per colony during Days 4 and 5. An inactive period of 2 clays was followed by a second wave of sporulation during Day 8 when 12 X lo6 spores accumulated in an average colony. A second idle period on Day 9 was followed by a third increase of about 20 X 10F spores per colony on Day 10. A third idle period on Day 11 preceded later sporulation, but we were unable to detect any furt’her periodic sporulation. Total viability by plate count closely approximates microscopic cell count at first but declines soon after spores appear. Outgrowth of germinable mature spores contributes only slightly to the total viable count because B. popilliae spores germinate poorly. Resistance to Heat and Drying Acquisition of heat resistance by mature, refractile spores lags behind acquisition of resistance to drying. Approximately 0.2% of the spores present at 4 days resist drying, but none survive heat treatment. At 10 clays 5% are resistant to drying and about 2% survive heating. At 14 days spores are equally resistant to both heat and drying, but germinable forms constitute only about 2.5% of the microscopic

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SHARPE

A?;D

spore count. Between 14 and 17 days 10’ spores accumulate per colony. Three percent of these resist drying at 17 days but not heat The three periods when the number of presporcs decline coincide well with periods when mature spores increase (see Fig. 4) ; the continued high level of presporce during the remainder of the incuba-

FIG. 2. Anatomy of a sporulating periphery of recent vegetative growth. and (c) of cells from t.hr corresponding

colony:

RHODES

tion period probably indicates that conditions are inadequate for their maturation. Some forms counted as prespores possibly were germinating spores; it is difficult to distinguish between the two. However, outgrowth stages of germination did not of B. popilliae appear, and germination spores is low (St. Julian et al., 1967). Con-

(A)

Colony

10 X magnification.

colony

areas

(A),

center, (B) sporulation ring, and (C) Below are photomicrographs (a), (b), (B), and (C). X2376, phase contrast,

SPORULATION

IN

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COLONIES

Periphery of recent vegetative growth Concentration of spores at surface Oldest vegetative growth

Young colony 6 days old 2 to 3 mm.. dia.

FIG.

graph

3. Drawing of young of same rolony shown

Mature sporulating colony 15 days old 7 to 9 mm. dia.

rolony and as Fig. 2.

mature

sequently, we feel that germination in colonies does not cause any significant error in counts. Further evidence for the periodicity of spore formation in colonies is presented visibly in Fig. 5. Colonies such as the one shown with two (and sometimes three) discrete sporulation rings often occur when the rate of radial growth is accelerated and prolonged by increased concentration of carbohydrate in the medium and bv using vegetative inoculum. The sporulation rings normally are not separated in colonies that develop more slowly. Sporulation is poor (l-5%) in colonies with multiple rings, and

sporulating

2

colony

and

4

6

an oblique

6 10 Time, days

photomic~ro-

12

14

,6

Fm. 4. The lInttern of suorulation of Bncillw popilliae in colonies. The curves showing quantities of vegetative cells. prrspore forms and mature spores are microscopic counts (labeled “micro”). Total viability, viability after drying, and viabilit)- after drying and heat shock arc plate counts.

14

SHARPE

AND

RHODES

B. popilliae is a facultative aerobe, which can be adapted to anaerobic growth. However, sporulation appears to be exclusively an aerobic process because spores form only in surface layers of the colony. Sporulation is depressed in atmospheres containing limited oxygen. The net result of growth kinetics, nutrient diffusion, and oxygen requirement is sporulation in one or more rings on the surface of colonies. To our knowledge, there are no reports in the literature on the pattern of sporulation of bacteria in colonies. Therefore, we cannot say whether this CYclic phenomenon is general. Spore formation by B. popilliae does not occur sufficiently in liquid to permit a comparison. FIG. 5. Colony with two sporulation rings. However, B. popilliae does sporulate extensuch colonies are not uniform in size, ap- sively in the host insect, and the pattern pearance, or degree of sporulation. of development there has been elaborated. At terminal stage of the disease, about DISCUSSION 5 X lOlo spores are found per milliliter of Our study shows that ,sporulation in larval hemolymph ; spores constitute 99% colonies of B. popi2liae occurs periodically of cell forms then present. However, at 5 rather than continuously and that acquisi- days only 20% of the cells growing in tion of resistance to heat and drying hemolymph has formed spores, while 50% parallels this stepwise pattern. Sporulation of the remaining vegetative cells is granuin a restricted area of a colony indicates lar and incapable of proliferation or sporuthe location of a microenvironment that lation (St. Julian et al., 1970). Since spores satisfies the requirements of the bacterium. constitute 2%30% of cell forms in The depressed appearance of the central B-230931 colonies, sporulation in vivo durarea of sporulating B. popilline colonies ing early stages of milky disease is no may result from extensive leakage of vege- more efficient than in mature colonies. tative cells along with limited cell lysis. Either sporulation becomesmore efficient It is tempting to speculate that products as t,he disease progresses,or large numbers of this activity diffusing outward are re(2 X loll) of dead vegetative cells would quired for the sporulation process and have to be removed by phagocytosis or somehow promote the cyclic pattern. These lytic processes in the hemolymph. B. nut’rients may be vegetative cell constitupopilliae cells are not markedly autolytic ents or, possibly, some met,abolic modi- in vitro, and attempts to demonstrate a fication of medium components. Also, lytic system in vivo, attributable either to sporulation apparently requires the close the insect or the bacterium, have failed. juxtaposition of cells in colonies so that In addition, phagocytosis has not been witlabile compounds or nutrients in low con- nessedmicroscopically and blood cells poscentration are preserved. However, it has sibly capable of phagocytizing B. popilliae been impossible to stimulate sporulation by disappear from hemolymph as the disease deliberately including mechanically dis- progresses. Nevertheless, changes in the rupted vegetative cells or media in which bacterial population in diseased larvae (St. vegetative growth had occurred. *Julian et al., 1970) indicate that at least

SPORULATION

a few million dead vegetative cells are somehow removed. Perhaps some low level lytic or phagocytizing activity has gone undetected. However, the rapid accumulation of spores during the latter half of the infection points to an increase in efficiency of sporulation, and we propose this efficiency increase as the principal change from the situation in vivo at 5 days. Now that the pattern of growth and sporulation in colonies has been cstablished, comparisons can be made with vegetative cell proliferation in liquid cultures of B. popilliae (Sharpe, 1966). Production of vegetative cells is more rapid and efficient in liquid than on solid medium; one billion cells are produced per milliliter of liquid medium in 17 hr vs the 3.5 X 10’ total cell-forms per milliliter (not per colony) found on solid medium at 17 days. However, the cells in liquid culture normally die within 48 hr ; whereas viability of some vegetative cells in colonies persists much longer (2 or 3 weeks), and sporulation is achieved on plates far more readily than in liquid. Perhaps a slow rate of growth with increased longevity of vegetative cells is conducive to sporulation. Haynes and Rhodes (1969) reported that activated carbon in liquid cultures prolongs viability and permits limited sporulation. Current research (unpubl.) involving growth of B. popilliae in continuous culture where growth rate can be selected and maintained, indicates biochemical differ-

IN

1d,-

COLONIES

ences in cells from widely different growth rates and eventually may afford some clue to better in vitro sporulation. REFERENCES R. N.? AND COULTER, W. H. 1971. Physiological studies of an oligosporogenous strain of B. popilline. Appl. Microbial., 22, 1076-1084. HA~NES, W. C., .4x11 RHODES, I,. .J. 1966. Sport formation by Bacillus popilline in liquid medium containing activated carbon. J. Bactcriol., 91, 227k-2274. HAYNES W. C.. AND RHODES, I,. J. 1969. Course of sporulation of Bacillus popilline in liquid medium containing activat,cd carbon. J. Invertebr. Pathol., 13, 161-166. rS~~w~~~~~, P. H., AND SHARP& R. S. 1976. Jnfectivit,!: of spores of Bncillus popilline produced on a laboratory medium. J. Invertebr. Pathol., 15, 126128. S.T. JULIAN, G., JR., PRIDHAM, T. G., AND HALL, H. H. 1967. Preparation and characterization of intact and free spores of Bacillus popilliae Dutky. Cm. J. Microbial., 13, 279-285. ST. JULIAN, G.. JR., SHARPE, E. S.. AND RHODES, R. A. 1970. Growth pattern of Bacillus popillitre in Japanese beetle Iarvnc. J. Znvertebr. Pathol., 15, 240-246. SHARPE, E. S. 1966. Propagation of Bacillus popilliae in laboratory fermenters. Biotechnol. Bioeng., 8, 247-258. SHARPE, E. S., ST. JULIAN, G.. JR., AND CROWELL. C. 1970. Characteristics of a new strain of Bacillus popilline sporogcnic in ?dro. Appl. Microbial., 19, 681-688. WYSS, C. 1971. Experiments on the sporulation of three vnrietics of Bacillus popilliae Dutky. Zentrdbl. Bokterinl. Parositenk. Injektionskr. Ilz~g. Abt. :‘, 126, 461.-492. COSTILOW,