Induction and inhibition of germination in Neozygites fresenii (Entomophthorales: Neozygitaceae) zygospores by various time-temperature stimuli

JOURNAL

OF INVERTEBRATE

PATHOLOGY

55,

l-10

(1990)

Induction and inhibition of Germination in Neozygites fresenii (Entomophthorales: Neozygitaceae) Zygospores by Various Time-Temperature Stimuli I. S. BEN-ZE'EV,~ Department

of Plant

Pathology

S. BITTON,*

and Microbiology, Faculty P.O. Box 12, Rehovot

AND R.G. of Agriculture, 76-100, Israel

KENNETH Hebrew

University

of Jerusalem,

Received August 16, 1988; accepted April 3, 1989 Neozygites fresenii is the most prevalent entomopathogen of the aphid pest of citrus in Israel, Aphis citricola. Observations in nature and germination experiments in vitro with zygospores of N. fresenii corroborated the indications of our previous report in 1979, that their overwintering is by

an endogenous dormancy mechanism, and that after dormancy breaks, germination accumulates logarithmically in a steep, sigmoidal curve. Germination measured following various timetemperature regimes of dry storage and moist incubation indicated a sequence of requirements: (i) a “maturation” period of ca. 20 days at So-23°C; (ii) a “vernalization” period of at least 14-15 days at 5”-14”C, which can overlap or coincide with maturation; (iii) an “activation” period after which germination starts, which in nature takes 200 or more hours if the time-temperature ratio is at least 4:l (hours above 10”C:hours below 10°C); activation was shorter in vitro when the temperature was held constant above 14°C (e.g., 72-96 hr at 23°C). Temperatures around 5°C deactivated the process, causing a reversible return to dormancy. Other findings were as follows: (i) Insufficient vernalization or maturation at temperatures above those needed for vernalization caused germination to accumulate as a linear slope rather than as a steep, sigmoidal curve drawn by normally vernalized spores. Additional vernalization of such spores returned germination to a steep sigmoidal accumulation; (ii) germination accumulated faster when after vernalization the temperature was raised gradually rather than abruptly; none occurred above 27°C. A possible model of dormancy breaking in N. fresenii zygospores is presented. Reversible deactivation is assumed to be a built-in safeguard against premature activation and germination of zygospores during periods of several days of warm day time weather which occur from time to time in mid-winter in Israel. o 1990 Academic

Press, Inc.

WORDS: Entomophthorales; Neozygites fresenii; Aphis citricola; zygospore maturation: zygospore dormancy; zygospore germination; zygospore vernalization; zygospore deactivation. KEY

need to be known (e.g., Soper, 1977; Soper et al. 1975). Germination of resting spores (RS) of entomopathogenic Entomophthorales has been observed and published for a limited number of species (see Soper et al., 1975; Bitton et al., 1979; Perry and Latge, 1982). Only about 10 species have been studied in some detail, especially as regards RS overwintering, breaking of dormancy, and germination. Periods of RS maturation and/or vernalization at temperatures which varied among species (mostly around 4”C), and elevated temperatures for germination, whether or not coupled with changes in photoperiod, have been demonstrated or implied as necessary in most of the follow-

INTRODUCTION

If mass-produced propagules of the Entomophthorales, including resting spores, are to be used as the active ingredient of mycoinsecticide formulations, the exact conditions for their storage and germination

’ Present address: Dept. of Plant Pathology, Institute of Plant Protection, Agricultural Research Organization, The Volcani Center, Bet Dagan 50-250, Israel. Correspondence and requests for reprints should be directed to the author at Hebrew University of Jerusalem, Faculty of Agriculture, Dept. of Plant Pathology and Microbiology, P.O. Box 12, Rehovot 76-100, Israel. ’ Present address: Israel Ministry of Agriculture, Extension Service, Bet She’an, Israel. 1

0022-2011/90 $1.50 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

BEN-ZE’EV,

BITTON,

ing species: (1) Erynia canadensis Humber 8z Ben-Ze’ev, attacking the woolly pine needle aphid, Schizolachnus piniradiatae; (2) Erynia radicans Humber, Ben-Ze’ev & Kenneth, attacking lepidopteran hosts; (3) Erynia bullata Thaxter 8z MacLeod in Humber, attacking Sarcophagidae and Calliphoridae (Diptera); (4) Erynia crustosa Humber & Ben-Ze’ev, attacking the forest tent caterpillar, Malacosoma disstria Hbn.; (5) Erynia phytonomi Humber, Ben-Ze’ev & Kenneth, attacking the alfalfa weevil, Hypera postica; (6) Conidiobolus obscurus Remaudiere & Keller; and (7) Conidiobolus thromboides Drechsler (= Entomophthora virulenta) on various aphids; (8) Conidiobolus destruens Ben-Ze’ev, attacking mosquitoes; (9) Neozygites fresenii Remaudiere & Keller, on aphids; (10) Entomophthora planchoniana Cornu on aphids (1: Grobler

et al., 1962; Tyrrell and MacLeod, 1975; Tyrrell et al., 1976; Wallace et al., 1976; 1978; Payandeh et al., 1978; 2: Gilliat, 1925; Dustan, 1927; Perry et al., 1982; 3,4,5: Perry, 1988; 6: Latge et al., 1978; Krejzova, 1978; Perry and Latge, 1982; 7: Hall and Halfhill, 1959; Soper et al., 1975; Matanmi and Libby, 1976; Krejzova, 1978; 8: Krejzova, 1978; 9: Wilding, 1970; Bitton et al., 1979; 10: Perry and Latge, 1982). Wilding (1970) has shown that N. fresenii RS can be germinated by a short incubation in high humidity at 20°C. Bitton et al. (1979) have shown that N. fresenii overwinters as RS on weeds and citrus leaves in Israel and resumes activity by sporulative germination (capilliconidia produced directly on the RS) in spring, in synchronization with the build-up of the population of its host, Aphis citricola (=A. spiraecola) on citrus. The data implied that RS dormancy was broken by a time-temperature relationship and it therefore appeared to be of an endogenous type. During most winter days in the coastal plain of Israel there are 4-6 hr when temperatures exceed 10°C and sporadic periods of a few days when temperature maxima exceed 20°C (Fig. 1). However, N. fre-

AND

KENNETH

RS did not germinate in nature following such periods although the threshold temperature for N. fresenii RS germination was found (Bitton et al., 1979) to be near 10°C in vitro. The goals of this study were (i) to clarify further the seasonal dynamics of N. fresenii RS production; (ii) to clarify the timetemperature requirements for N. fresenii RS germination; and (iii) to test the hypothesis that a built-in safeguard in its dormancy mechanism prevents germination during or following warm periods in midwinter. senii

MATERIALS AND METHODS N. fresenii zygospores were collected from populations of aphids, Aphis fabae and Aphis citricola. Germination experi-

ments were performed on cellophane squares floating on water, as described by Bitton et al. (1979). Controlled temperatures and photoperiods were obtained in various incubators and growth chambers. A standard illumination was used for all experiments, whether continuously or as part of a dark photoperiod. This was provided by one 25-W incandescent bulb placed 40 cm above each treatment. Subtreatments incubated at 23” + 1°C received additional daylight from a nearby, northern window. Preliminary data (Bitton, Kenneth and BenZe’ev, unpubl.) indicate that N. fresenii RS can germinate in complete darkness. Illumination was used because light could not be avoided during microscopical examinations. Each treatment or subtreatment consisted of lOOO-2OtlOspores and was replicated three times on separate pieces of cellophane. Germination was monitored every 48-72 hr under the x 10objective of a Nikon binocular microscope. Microscopical observations in each replicate were always initiated at the same point and followed the same direction, so that the same 500 spores were monitored for their cumulative germination throughout the duration of the experiment. A germinated spore is one which produced a germ capilliconidium. Germina-

Neozygites

fresenii

Dec.1977 -Temp. ; ( cl -30

I=

3

ZYGOSPORE GERMINATION

’

1’

Jan.1978

cl.5 II

--

Idd -- 05 10 15 20 25

‘I Rain I

-25

I

I

5

(mm.

I

1

II

m

15

20

25

28

5

?a

15

20

25

31

FIG. 1. Daily minimum, maximum, and average temperatures and precipitation measured hourly in the survey area (Meteorological Service Station, Bet Dagan) during December 1977-March 1978 (d, dripping).

tion percentages given are averages of three replicates, totaling 3 X -500 spores. Contamination with other fungi sometimes caused a reduction in the number of spores per replicate or reduced the number of replicates to two. Observations

in Nature

The seasonal occurrence of N. fiesenii in populations of A. citricola on citrus trees was monitored weekly in citrus groves (Ben-Ze’ev et al., 1984). Several hundred aphids were examined each time. Aphid carcasses and aggregates of N. fresenii RS

on citrus leaves were regularly brought to the laboratory and checked for germination under the microscope immediately after collection. Percentages of germination in nature (on leaves) are averages of three fields, each with 2500 spores/carcassor aggregate. Germination

at Constant

Temperatures Carcasses of freshly dead Aphis fabae filled with N. fiesenii RS were collected on January 31 on Solarium nigrum and were

stored on the leaves, in paper bags, at 4” +1°C. Three treatments were carried out

4

BEN-ZE’EV,

BITTON,

with RS from this batch (time-flow diagram in Fig. 2). Treatment a consisted of spores stored at 4” ? 1°C for 4 days after collection, then incubated for germination at 23” 2 1°C for 18 days. Spores in treatment b were stored at 4°C for 55 days after which they were divided into four subtreatments; each was incubated for 5 days as follows: (bl) at 13”-14°C; (b2) at 23” -C 1°C; (b3) at 27” 2 1°C; and (b4) at 30” 2 2°C. Spores in treatment c were stored at 4” +- 1°C for 63 days; then they were divided into two subtreatments and incubated together for 12 days at 9”-10°C. Then cl was incubated further for 5 days at 9”-10°C and c2 was incubated for 5 days at 23” ? 1°C. All treatments and subtreatments in this experiment were incubated under constant illumination Vernalization

for 30 days as shown in Figure 3. All four treatments were exposed for the duration of the experiment to a 12-h dark photoperiod beginning at 12 PM. Deactivation

Resting spores were collected in l- to 2month-old, dried carcasses of A. citricola or as aggregates around disintegrated cadavers, on leaves of an orange tree in a citrus grove, on January 1. They were stored on the leaves, in paper bags, at 23” +. 1°C. After 21 days of storage the RS were divided into four treatments and incubated INCUBATION: 15 DAYS AT 23°C

Experiments

RS were from the same batch as in the previous experiment, stored at 23” + 1°C for 17 days. Four treatments were incubated for the first 12 days at 9”-lO”C, under constant illumination, after which all four treatments were exposed to a 12-h dark photoperiod beginning at 12 PM for the rest of the experiment and to different timetemperature regimes as follows (see diagrams in Fig. 4): (h) constant temperature control incubated for 33 days at 20” 2 1°C; this treatment was started 5 days after the others (together with the vernalization experiments). (i) 19 days at constant 13”-14°C. Due to very low germination the treatment was transferred for the next 17 days to constant 20” + 1°C to determine the spores’ viability. (k) 16 days under alternating conditions; 5 hr at 5°C (in darkness) then 19 hr (illuminated for 12 hr) at 20°C.

Experiments

STORAGE: 4 DAYS AT4OC

AND KENNETH

t3 DAYS INCUBATION 23tl’ C 7 % GERMINATION

AT:

(a) +5 DAYS 13-140~ 7.4%

GERMINATION:

STORAGE:

55

DAYS

INCUBATION

23wc 3.1%

AT 4O C

AT:

27f1=x 0.6%

,

3awc 0

f

(b) + 4 DAYS INCUBATION GERMINATION: STORAGE:

63

IO

20

DAYS

AT 4’C

INCUBATION:

9-IO’C 9.5% 12DAYSAT S-10-c

AT:

23fl’C 45% I’

I

(cl

0

30

40

TIME

(days)

50

60

70

80

FIG. 2. Time-flow diagrams a, b, and c showing germination percentages of Neozygites fiesenii zygospores from Aphisfabae incubated under moist conditions at constant temperatures after diierent periods of dry storage at 4°C.

Neozygites

fresenii

cr-(DAYSt----6-------------(d) (e) v) fg)

ZYGOSPORE

12-m---

----------------------------------2o ----20 * l~C--j--lO ----2o 2 l”C--j --------------43 f l~C--]---lO

_____ ‘_

f

l”C--

1 10

2

1°C ----

f

--------

---ml8

--------

20

2

5

_______

-------24-------------3O

l”C--------------------------------->

-- ------------

1°C~--1

GERMINATION

l”C---

20 +I------_-__ 1 ----IO

t

l”C-

------------

20 l”C---

2 1

---->

l”C--------->

-40

*

l”C--->

FIGURE 3

(I) 27 days with alternation: 12 hr (darkness) at 5°C and 12 hr (illuminated) at 20°C. Due to very low germination the treatment was transferred for the next 12 days to constant 20” + 1°C to check the spores’ viability. RESULTS Observations

AND DISCUSSION

in Nature

N. fresenii resting spores were found in A. citricola in Israel usually at the beginning of November; the earliest observed time is in September 1977 (Ben-Ze’ev et al., 1984). The observations reported here were carried out during 197f$-1979. The first RS were found in one aphid on November 22, 1978. During the first half of December RS

32 2 E

=0 24

were found in increasing numbers of dead aphids, but the conidial stage was still prevalent. The proportion changed during the second half of December. By January 3. 1979, living aphids were very scarce; in dead ones RS were more frequent than the conidial stage, but none were germinating. The first natural RS germinations on citrus leaves, 0.7%, were observed on February 2nd, increasing to 8.5% on February 20th and to over 45% on March 1st. The winter of 1979 was an unusually short one and was followed by an early spring which started at the end of January. Around February 2nd the daily temperatures ratio were similar to those recorded as favorable for the germination of N. fresenii RS in the middle of March 1978 (Fig. 1) (see Bitton et al., 1979).

ALL TREATMENTS INCUBATED FOR THE FIRST 12 DAYS

19 DAYS W CONSTANT --------__------

13-14

Oc

G

TERNATING:12h-5°C: RNATING:12h-5°C:12h-20 l?h-20 -__-__-_--_----_ _--_----_

TIME

(days)

FIG. 4. Cumulative germination curves h, i, k, and 1 of Neozygites fresenii zygospores from Aphis incubated under moist conditions in different time-temperature regimes (time-flow diagrams), after dry storage at 23°C (Deactivation Experiments). Curve h represents also those of treatments d, e, f, and g (Vernalization Experiments; see time-flow diagrams in Fig. 3). citricola

6

BEN-ZE’EV,

BITTON,

The population of A. citricola was already building on citrus leaves during the first half of February 1979, vs in mid-March 1978. Germination

at Constant

Temperatures

In treatment a (Fig. 2) germination began only after 15 days and attained 7% after 3 more days, whereas Bitton et al. (1979) obtained first germinations under similar conditions after only 3 days, and recorded 7.5% germination on the 4th day. The difference between treatment a and that of Bitton et al. appears to be mainly the age of the RS; those used in treatment a were produced very recently whereas those used by Bitton et al. were ca. l-month-old RS. The 4 days of storage plus the 15 days of incubation at 23” +- 1°C that elapsed before the onset of germination in treatment a seem to indicate that freshly produced RS need a “maturation” period before they are ready to germinate. This maturation appears to be independent of moisture conditions, as spores maturing in nature are subject to wetting and drying (dew, rain, wind) while those which matured in storage (treatments b and c) were kept dry. Maturation occurred in spores stored at 4” ? 1°C and at 23” + 1°C (vernalization experiments), but occurs in nature at day/night alternating temperatures. In treatment b the RS were ca. 2 months old, and germination started, as expected for mature spores, after 3 days. The germination, recorded after 5 days at all temperatures, was nil at 30°C and increased with lower temperature, indicating a possible enhancing effect of incubation at 13”-14°C (Fig. 2). This effect was retested in treatment c, as a “preincubation” period at 9”-10°C. Germination started after 12 days at this temperature, and after 4 more days high germination percentages were recorded at 23” + 1°C (cl), but remained very low at Y-10°C (~2) (Fig. 2). These results corroborated the conclusions of Bitton et al. (1979), that the threshold temperature for RS of N. fresenii pro-

AND

KENNETH

duced in A. fabae is slightly below 9”-10°C and that time, as a maturation period, is involved in the predisposition of RS to germination. Furthermore, the results led us to hypothesize that a certain period of “vernalization” at or below the threshold temperature was needed in order to allow massive germination when temperature was raised to 20” or 23°C. This appears consistent with the gradual rise in temperatures at the end of winter and beginning of spring. No evidence that RS density influenced germination was found in this study or in that by Bitton et al. (1979). Vernalization

Experiments

It had been expected that one or more treatments (e,J and g) would show a higher germination percentage, due to vernalization at 10°C in comparison with treatment d which did not receive vernalization. However, the daily cumulative germination averages of all four treatments were not significantly different (Duncan’s multiple range test, P = 0.05) from each other and from those of treatment h of the deactivation experiments. The cumulative germination curves all 5 of these treatments were almost identical and are therefore represented by curve h in Fig. 4. Apparently, 6-12 days of vernalization at 9”-lo”C, accumulated by treatments e, f, g, and h were not sufficient to change their germination pattern, which remained similar to that of treatment d, which had no vernalization at all. Thus, the vernalization hypothesis could not be proved by the outcome of this experiment. Additional support for it, however, was obtained from the deactivation experiments. Deactivation

Experiments

Considering that N. fresenii RS germinated only at temperatures above 9°C and not before 72 hr at 23” + 1°C or 288 hr at 9”-10°C the simplest germination mechanism would be one activated by the accumulation of a certain amount of degree/ hours above the threshold. Tabulated tem-

Neozygites

fresenii

ZYGOSPORE

peratures measured hourly at the Meteorological Service Station at Bet Dagan, in the coastal plain, a few kilometers from our survey site, allowed us to estimate that such an accumulation in 1977 was completed in mid-December. Adding to this 20 days for maturation, deduced from treatment a, and 10 more days as an extra margin, germination should have proceeded in nature by mid-January 1978. This did not happen in spite of 5 consecutive warm days in mid-January with barely a few hours below 10°C (Fig. 1). Nevertheless, the RS had obviously accumulated all the degree/hours needed, as indicated by their high germination percentage (53.3%) obtained in the laboratory after only 3 days of incubation at 23” 2 1°C (Bitton et al., 1979, Fig. 6). This led us to a hypothesis of “deactivation” as found in Neurospora (Sun and Sussman, 1960), as it seems to apply here: the process leading toward germination (after a period of maturation) is promoted by temperatures above 9”-10°C but is reversed by lower ones. The selective advantage of such a mechanism could obviously be the prevention of germination following short periods of warm weather during winter. With such a mechanism the RS could germinate only when a certain ratio between low temperature-hours and high temperature-hours was attained in nature. It seemed reasonable to look for such a time-temperature ratio, with the germination threshold temperature of 9”-10°C as the balancing point. We divided the period of December 1, 1977, to March 30, 1978, into lo-day intervals and calculated the ratio of hours above 10°C to hours below 10°C. The beginning of germination in nature coincided with a ratio of 4: 1, attained in the first half of March 1978. The RS used in this experiment were from the same batch as those used in the former one, stored dry, at 23” t_ 1°C since collection. Treatments h and i (Fig. 4) were designed as constant temperature controls, looking also for an optimal temperature reference. Treatment k was designed to have the 4: 1 ratio of time above 10”C:time below

GERMINATION

7

10°C (actually Y’C), deduced in the “Observations in Nature” section to be conducive to germination. Treatment 1 was designed with a 1: 1 ratio of time above 10”C:time below 10°C to test the “deactivation” hypothesis. Individual treatments were terminated at different dates (Fig. 4), when contamination with other fungi precluded further examination. Germination started in treatment h after 3 days, as expected, but did not accumulate in the sigmoidal shape obtained by Bitton et al. (1979) with RS kept at 4” +_ 1°C throughout storage. Instead, it accumulated to ca. 35% in an approximately linear way, which was nearly identical to the cumulative germinations of all four subtreatments of the vernalization experiments. Its unusually high germination during incubation at 9”-10°C was probably due to frequent power shortages that occurred in that building for several days. Treatment i showed very low germination at the end of the 12 days vernalization period, as expected, but germination increased only very slightly during 19 more days of incubation at 13”-14°C. This result cast doubt on the viability of the RS, and the treatment was therefore incubated for 17 more days at 2O”C, whereby germination accumulated in a logarithmic way, as shown by the sigmoidal curve i (Fig. 4). The outcome of this treatment indicates that (i) the threshold temperature for RS germination of the N. fresenii isolate tested here is actually a range of -9”-14°C and (ii) incubation at 13”-14°C for 19 days acted as additional vernalization, allowing logarithmic rather than linear accumulation of germination. In treatment k germination was insignilicant after 12 days of vernalization, and started to climb after 10-12 days X 19 hr/ day at 20°C ( = 190-228 hr at 20°C) (Fig. 4) and additional vernalization for 50-60 hr at 5°C. Assuming that germination was triggered by the mere accumulation of 20°C hr, the climb in germination of treatment 1 could be predicted to occur in 16-19 days

8

BEN-ZE’EV,

BITTON,

12 hr/day at 20°C). If, on the other hand, deactivation occurred at Y’C, proportionally with incubation time, as found in Neumspora (Sun and Sussman, 1960), then germination in 1 would occur later than expected or not at all. Germination in 1 did not start to climb at the predicted date, nor after 10 more days (= 190 more hr at 20°C). On day 39, treatment 1 was transferred to incubation at constant 20” + 1°C. After this, germination accumulated as in treatments i and k (Fig. 4), showing that the RS were not dead but had been reversibly deactivated. The results of treatments i, k, and 1 vs the slower, linear accumulation of germination in treatments a and h and d, e,f, and g show that N. fiesenii RS require vernalization, as storage or incubation at temperatures between 5 and 14”C, for more than 12 days in order to achieve logarithmic cumulative germination. The results of treatment k indicate a minimal vernalization requirement of 14-15 days at the temperatures tested. These results also indicate that N. fresenii RS can germinate without vernalization after a maturation period (i.e., treatments a and h), but vernalization induces a faster and more uniform germination. In this respect the term vernalization, as used here, is not entirely parallel with its usage in higher plants. The differences between the results of treatments k and 1 were exactly those expected if deactivation was part of the overwintering mechanism, serving as a safeguard against germination at an improper time, such as during warm periods in winter, when the temperatures at night are at or below the deactivating time-temperature values. (X

A

Maturation & Vernalization ?-4123°C ?414”C -----------o------------m-, Deactivation ?-S-?“C --> FIGURE

AND KENNETH

General Discussion

Germination of fungal spores, whether triggered by low or high temperatures, depends on enzymatic reactions. Endogenous or constitutive dormancy is based (i) on germination inhibitor(s) which have to be enzymatically worn off, (ii) on metabolic lesion(s) (missing compound(s)) which have to be synthesized by enzymes, or (iii) on combinations of the two. Both processes depend on certain temperatures which affect the working velocities of the enzymes involved (Sun and Sussman, 1960; Sussman and Halvorson, 1966). The model in Figure 5 is modified from a model of vernalization and devemalization of flower buds (Wareing and Phillips, 1970) and provides a likely explanation for the observed behavior of N. fresenii RS. A is either an endogenous inhibitor or a precursor of compound B; in both cases A -+ B is a time-consuming process of one or more steps. B is either a modified, but still active, inhibitor, or a precursor processed further into the missing compound C in -3 days at 23°C. C is either a compound needed in a certain concentration for germination or the inactivated inhibitor (in the latter case, germination begins when the concentrations of A and B are lowered to a certain value). D is a “safeguard” product which has to be reprocessed into B before activation is possible again. The results of treatment 1 in the deactivation experiments indicate that the reaction B --j D has a higher velocity at -5°C than the reverse reaction D + B, thus shifting the direction of the process back to dormancy whenever temperatures of -5°C occur for more than

Activation Germination (9-lo)-27°C (9-lo)-27°C B - ---------> C - - - _ - - -> Reactivation Jr <-(9-lo)-27°C D 5

Neozygites fresenii

ZYGGSPORE GERMINATION

20% of the diurnal cycle. Other models are possible as well. N. fresenii and E. canadensis differ in their dormancy mechanisms with regard to the triggers of germination. In the first, the trigger was found to be a time-temperature ratio, while the role of light remains unknown but is probably minimal at best. The jet-black episporium of N. fresenii RS shields the spore from penetration of light, although some of the light might be absorbed and converted into chemical energy. In the second species the photoperiod is the trigger, temperature playing a secondary role. Several properties are shared by these two insect pathogens: both overwinter as endogenously dormant RS (for E. canadensis: Wallace et al., 1978) which germinate in spring in synchronization with the build-up of their aphid hosts, sometimes even causing double infections in S. piniradiatae (MacLeod et al., 1979). The maximal thresholds and the optimal temperatures for germination are similar for both fungi. These species show considerable variability in their RS population regarding the factors affecting germination: A small proportion of E. canadensis RS can germinate in darkness (Wallace et al., 1976); a small proportion of N. fresenii RS can germinate after a short period of maturation, or at the low temperature of 9”-10°C. In both fungi variability is displayed also by the sigmoida1 curves of cumulative RS germination or by the slower (in N. fiesenii) linear cumulative germination of mature, nonvernalized spores. Other species were not studied in sufftcient detail to compare with N. fresenii or E. canadensis. The germination requirements of Israeli isolates of N. fresenii studied here appear to be well adapted to the Mediterranean climatic conditions of the Israeli coastal plain. No difference was found in the behavior of RS collected from A. fabae and those from A. citricola, the latter being a fairly recent introduction in Israel. N. fresenii attacking it is probably an indigenous pathogen (Bit-

9

ton et al., 1979) known here since before the appearance of A. citricolu as a pathogen of various Aphis spp. and other aphid genera (Kenneth, unpubl.; Kenneth et al.. 1971). ACKNOWLEDGMENTS We thank Drs. Robert P. Jaques, Agriculture Canada, and Richard S. Soper, United States Department of Agriculture, for their constructive criticism of the manuscript. Note added in proof. A detailed study dealing with time/temperature requirements for zygospore germination in another entomophthoralean species was published while this paper was in press: Perry, D. F. and Fleming, R. A. 1989. Erynia crustosa zygospore germination. Mycologia 81, 154-158.

REFERENCES BEN-ZE’EV, I. S., KENNETH, R. G., BITTON, S., AND SOPER, R. S. 1984. The Entomophthorales of Israel and their arthropod hosts: Seasonal occurrence. Phyfoparasitica, 12, 167-176. BITTON, S., KENNETH, R. G., AND BEN-ZE’EV, I. 1979. Zygospore overwintering and sporulative germination in Triplosporium fresenii (Entomophthoraceae) attacking Aphis spiraecola on citrus in Israel. J. Znvertebr.

Pathol.,

34, 295-302.

DUSTAN, A. G. 1927. The artificial culture and dissemination of Entomophthora sphaerosperma Fres., a fungous parasite for the control of the European apple sucker (Psy//a ma/i Schmidb.). J. Econ. Entomol., 20, 68-75. GILLIAT, F. C. 1925. Some new and unrecorded notes on the life history of Entomophthora sphaerosperma. Proc. Acadian Entomol. Sot., 10, 46-54. GROBLER, J. H., MACLEOD, D. M., AND DELYZER, A. J. 1962. The fungus Empusa aphidis (Hoffman) parasitic on the woolly pine needle aphid, Schizolachnus pini-radiatae (Davidson). Canad. Entomol.,

94, 4U9.

HALL, I. M., AND HALFHILL, J. C. 1959. The germination of resting spores of Entomophthora virulenta Hall and Dunn. J. Econ. Entomol., 52, 30-35. KENNETH, R., WALLIS, G., OLMERT, Y., AND HALPERIN, J. 1971. A list of entomogenous fungi of lsrael. Zsr. J. Agric. Res., 21, 63-66. KRWZOVA, R. 1978. Germination process in resting spores of some Entomophrhora species and pathogenicity of spore material for lepidopterous larvae. Z. Ang.

Entomol.,

85, 42-52.

LATGE, J. P., PERRY, D., PAPIEROK, B., COREMANSPELSENEER, J., REMAUDIERE, G., AND REISINGER, 0. 1978. Germination d’azygospore d’Ento-

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