Cytophotometric determination of the nuclear DNA content of 23 Mexican and 18 non-Mexican isolates ofPhytophthora infestans

EXPERIMENTALMYCOLOGY

11,

19-26 (1987)

Cytophotometric Determination Mexican and 18 Non-Mexican

of the Nuclear DNA Content Isolates of Phytophthora ~nfe~t~~~

PAUL W. TOOLEYANDC.DALETNERRIEN* Foreign Disease- Weed Science Research, USDA-ARS. Ft. &trick Building 1301, Frederick, Mavland 21701, and *Department of Biology, Pennsylvania State Universiry, Univevsily Park, Pennsylvania 16802 Accepted for publication November 6, 1986 TOOLEY, P. W., AND THERRIEN, C. D. 1987. Cytophotometric determination of the nuclear DNA content of 23 Mexican and 18 non-Mexican isolates of Phytophthora infestans. Experimental Mycology 11, 19-26. The nuclear DNA content of zoospores of Phytophthora infestans was determined by Feulgen cytophotometry. Isolates from Mexico, where the sexual stage of the fungus is common, were compared with isolates from the United States and Europe. The mean nuclear DNA content of 23 Mexican isolates was 0.59 arbitrary units (a.u.; range, [email protected]), while that of 18 non-Mexican isolates was 0.92 a.u. (range, 0.56- 1.1 I). Mexican isolates are very likely diploid, as indicated by cytological studies of other workers. The non-Mexican group, by contrast, appears to include isolates that are diploid, triploid, tetraploid, and aneuploid. Of three non-Mexican isolates with DNA contents similar to those of the Mexican isolates (appar.ent diploids), two originated from Southern California and may be of recent Mexican origin. % 1987 Academic Press, Inc.

INDEX

DESCRIPTORS:

Phytophthora

infestans: Feulgen cytophotometry;

polyploidy; DNA con-

tent.

The fungus Phytophthora infestans (Mont.) de Bary (class Oomycetes) causes potato late blight, a destructive disease of potato (Solarium tuberosum L.) throughout the world. The P. infestans:Solanum pathosystem is believed to have originated in Mexico (Niederhauser, 1961) where the sexual stage of the fungus is commonly found in nature (Gallegly and Galindo, 2958; Niederhauser, 1956). Controversy has existed regarding the ploidy of somatic hyphae in Phytophthora (Shaw, 1983). Some early workers believed that the fungus had a haploid somatic phase (Gallegly, 1968; Timmer et al., 1970). Based on more recent evidence from genetic, cytological, and cytophotometric studies (Shaw and Khaki, 1971; Mortimer and Shaw, 1975; Brasier and Sansome, 1975) it is now generally recognized that Phyt~phthor~ spp. have gametangial meiosis and a diploid somatic phase. Polyploidy has been observed in some P~lyt~~~thora species (Sansome and Bra-

sier, 1974; Sansome, 1977), but too few isolates have been examined to know how commonly it occurs. Mexican P. iuafes2aPis isolates examined cytologically were found to be diploid (Sansome, 1977; Sanso Brasier, 1973) while polyploidy was observed among British isolates (Sansome, 1977). Sansome (1977) suggested that tetraploidy may be prevalent in P. infestans isolates from temperate regions. If such ploidy differences exist in populations of P. infestans, they could be important in establishing levels of population genetic diversity. It is known, for example. that Mexican isolates of P. infestarzs are more virulent and exhibit more variatio isozyme loci than isolates from other areas (Tooley et al., 1985, 1986). A study of ploidy in Mexican and non-Mexican iscalates of P. infestans is needed to determine whether ploidy differences may be lying the differences in virulence an characters noted between these populations ~ 19 0147-5975187 $3.00 Copyright 0 1987 by Academic Press, inc. All righta of reproduction m any km reacrved.

20

TOOLEY

AND

Knowledge of the ploidy of P. infestans isolates is also essential for genetic studies (Shattock et al., 1986a, b). To properly interpret genetic data, the ploidy of isolates to be used as parents in genetic crosses must be known. Poor oospore germination and low viability of progeny could result if parents are used which differ in ploidy. Although chromosome counts are the most direct measure of ploidy, Phytophthova chromosomes are very small and difficult to count (Sansome and Brasier, 1973; Shaw, 1983). However, measures of relative DNA content reflect ploidy levels and have been used as quantitative estimates of ploidy in Oomycetes and other classes of fungi (Bell and Therrien, 1977; Bryant and Howard, 1969; Howard and Bryant, 1971; Mortimer and Shaw, 1975; Peabody et al., 1978; Therrien, 1966; Whisler et al., 1983). Our objectives were to use cytophotometry in conjunction with Feulgen staining to assess nuclear DNA content of P. infestans, and to determine whether there are consistent ploidy differences between isolates from Mexico and those from the United States and Europe. MATERIALS

AND

METHODS

All isolates of P. infestans used in this investigation were maintained in the culture collection of the U.S. Department of Agriculture Foreign DiseaseWeed Science Research Unit, Ft. Detrick, Frederick, Maryland. The isolates were collected and maintained in the manner described previously (Tooley et al., 1985). Tables 1 and 2 give the origin and accession numbers of the isolates used in this study. Zoospores were produced as described by Caten and Jinks (1968). Zoospores were fixed at 5°C in a solution of 4% formalin in 0.25 M sucrose, pH 7.0, for a period of at least 12 h, then postfixed at 5°C in 70% ethanol for 12-18 h. DNA was stained by the Feulgen reaction as modified by Therrien and Yemma (1974). Stained specimens

THERRIEN

were dehydrated in a graded ethanol series, cleared in xylene, and mounted in Cargille’s index of refraction oil (R.I. 1.532).’ Cytophotometric measurements were taken with a Zeiss universal microscope equipped with a gradation interference filter monochromator and a PMI-1 photometer indicator. The two-wavelength method (Patau, 1952) was used for all FeulgenDNA (F-DNA) determinations. Since the extinction coefficient for the F-DNA complex was omitted from all calculations, the nuclear DNA values are reported only as arbitrary units. The reliability of Feulgen cytophotometry for accurate assessment of fungal ploidy has been established in prior studies in which myxamoebal (haploid) and plasmodial (diploid) stages of Didymium iridis were compared (Collins and Therrien, 1976; Collins et al., 1978; Therrien et al., 1977). Chicken erythrocyte nuclei, which have a genomic DNA content of 2.41 pg (Vaughn and Lacy, 1969), were utilized as an internal DNA standard (Dhillon et al., 1977). Chicken blood was drawn in a heparinized syringe, smeared on approximately onethird of an acid-alcohol-cleaned microscope slide, then fixed and postfixed as described above. Suspensions of fixed P. infestans zoospores were added to the opposite end of the slides on which the blood smears had been made and these preparations were stained by the Feulgen method. The protocol for Feulgen staining was as described for the P. infestans preparations alone, except that the oil used to mount the chicken erythrocytes had a refractive index of 1.544. The absolute genomic DNA content of P. infestans was determined by using the chicken erythrocytes to calculate the extinction coefficient for the F-DNA complex. This value was determined to be 0.28 ’ Abbreviations used: R.I., index F-DNA, Feulgen-DNA; a.u., arbitrary

of refraction; unit.

DNA CONTENT

OF Phytophthora

TABLE 1 Accession Numbers and Sources of Non-Mexican Isolate No. 111 (ATCC 127 (ATCC 128 (ATCC 134 (ATCC 135 (ATCC 136 (ATCC I37 (ATCC 140 141 I42 144 146 147 150 152 160 161 163

48720)” 48723) 48724) 52012) 52009) 52010) 52011)

infestuns

Isolates of Phytophfhora

21

infestam

Source

Year isolated

Albany Co., NY, USA Spencerport, NY, USA Steuben Co., NY, USA Portage Co., WI, USA Portage Co., WI, USA Langlade Co., WI. USA Langlade Co.. WI. USA Ireland Wales California, USAb California, USAb Wales Groton, NY, USA Auburn, NY. USA Erie Co.: NY, USA Freeville, NY, USA Latina, Italy The Netherlands

1979 1982 1982 1982 1982 1982 1982 Unknown I972 Unknown Unknown 1982 1984 1984 1984 I984 1985 Unknown

a American Type Culture Collection accession number. b Isolated from tomato; all other isolates originated from potato.

pg/t.r,m2 and was substituted for k in the Patau (1952) equation (where DNA = kAL,C) to calculate the absolute amount of DNA per P. in&tans nucleus. RESULTS

The mean nuclear DNA content of 23 Mexican isolates (Table 3 and Figs. 1 and 2) was 0.59 a.u. (range 0.48-0.79) while that of 18 non-Mexican isolates (Table 4 and Figs. 3-8) was 0.92 a-u. (range 0.56-l .ll).

Mexican isolates thus contained about onehalf the DNA of many no lates and thus are very lik shown by cytological studies (Brasier and Sansome, 1975; Sansome, 1977). In terms of absolute DNA content the mean value for all Mexican isolates was 0.52 pg per nucleus. Three non-Mexican isolates were ap ently diploid as well. United States isolates 142 and 144 (Table 4). and isolate ‘963 frozpl

TABLE 2 Accession Numbers and Sources of Mexican Isolates of Phytophthora Isolate No. 501, 502, 503, 504. 508, 512, 514. 517, 518,519, 522.525, 526 529 543 550 551 560, 561 562, 565, 568, 570

Source Toluca: international

late blight screening trial

Atizapan: international late blight screening trial La Puerta: commercial potato field Tenango: wild Solarium species El Tecolote: wild Solarium species Chapingo: Solarium cavdiophyllum Chapingo: potato research plot

infestans Year isolated 1983 1983 I983 1983 1983 1983

I984

TOOLEY

0.4

0.8

1.2 1.6 Nuclear

0.4 DNA

0.8

I.2

AND THERRIEN

I.6

Content

FIGS. l-8. Histograms representing relative nuclear Feulgen-DNA (F-DNA) distributions (arbitrary units) for selected Mexican and non-Mexican isolates of Phytophthova infestam. Isolate number, origin, mean nuclear F-DNA, and presumed ploidy are as follows: (1) Isolate 508, Mexican, 0.54, diploid. (2) Isolate 562, Mexican, 0.79, triploid. (3) Isolate 160, USA, 1.01, tetraploid. (4) Isolate 128, USA, 0.76, aneuploid. (5) Isolate 163, The Netherlands, 0.56, diploid. (6) Isolate 141. UK, 0.88, tetraploid. (7) Isolate 150, USA, 0.78, triploid. (8) Isolate 147, USA, 1.05, aneuploid. Isolate numbers are shown on the figures and broken vertical lines represent mean nuclear F-DNA values for each isolate.

The Netherlands (Table 4, Fig. 5) had mean nuclear F-DNA values that were clearly more similar to the diploid Mexican isolates than to the remainder of the non-Mexican isolates. When compared with Student’s t test, non-Mexican isolates 142, 144, and 163 did not differ significantly (P > 0.40) from Mexican isolates 526, 522, and 501 respectively. The mean nuclear F-DNA values for the remainder of the non-Mexican isolates varied from 0.76 to 1.11 au. of DNA (Table 4). In terms of absolute DNA content, these values are equivalent to 0.66 and 0.98

pg of DNA per nucleus. The non-Mexican group therefore appears to include isolates that are aneuploid, triploid (predicted mean DNA = 0.83 a.u.), and tetraploid (predicted mean DNA = 1.10 a.u.>. British isolate 141 (formerly B25) in which Sansome (1977) observed polyploidy was found to have a mean DNA content of 0.88 a.u. (Table 4, Fig. 6). Not all isolates which contained the mean tetraploid F-DNA content appear to be true tetraploids. For example, U.S. isolate 147 (Fig. 8) showed an unusually broad and somewhat bimodal distribution which suggests ploidal variation within this isolate. Mexican isolate 562 (Fig. 2) showed an intermediate mean nuclear F-DNA value which differed significantly (P < 0.001) from that of other Mexican isolates, but not from that of U.S. isolate 150 (P > 0.5). The mode of the nuclear F-DNA distribution for isolate 562 is shifted considerably to the right of the apparent diploid Mexican isolate 508 (Fig. l), but to the left of the apparent tetraploid U.S. isolate 160 (Fig. 3). We hypothesize that this isolate may be triploid. The mean F-DNA values for Mexican isolate 562 and U.S. isolates 128 and 150 were almost equivalent (Tables 3 and 4). However, since the DNA content histograms of Mexican isolate 562 and U.S. isolate 150 (Figs. 2 and 7) show unimodal rather than bimodal distributions, we hypothesize that these isolates are true triploids whereas U.S. isolate 128 which showed a somewhat bimodal distribution (Fig. 4) may be of mixed ploidy or aneuploid. DISCUSSION

Our results indicate that P. infestam isolates from Mexico are largely diploid, while isolates from the United States and Europe represent a range in ploidy and probably include diploids, triploids, tetraploids, and aneuploids. These findings support results

DNA CONTENT

Nuclear Feulgen-DNA

OF Phytophthom

23

infestnns

TABLE 3 Values in Mexican Isolates of Phyrophthova infestans

Isolate No.

Mean DNA (n = 50)

SE”

Isolate

Mean DNA (n = 50)

SE”

501 502

0.54 0.63 0.57 0.54 0.54 0.58 0.66 0.58 0.58 0.69 0.63 0.62

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

526 529 543 5.50 551 560 561 562 565 568 570

0.65 0.59 0.64 0.62 0.62 0.48 0.58 0.79 0.57 0.51 0.54

0.0' o.oi 0.01 0.01 0.01 Q.01 0.01 0.01 0.01 0.01 0.01

503 506 508 512 514 517 518 519 522 525

a Standard error of the mean.

of cytological studies by Sansome (1977) which indicated that Mexican isolates of P. infestans are diploid while non-Mexican (British) isolates may be tetraploid or diploid-tetraploid heterokaryons. Of the three non-Mexican isolates with DNA contents similar to those of the Mexican isolates, two originated from Southern California and may be of recent Mexican origin. Our results contrast with those of Rutherford and Ward (1985) who found uniform diploidy among nine races of P. megasperma f. sp. glycinea. However, P. megasperrna is a homothallic species in which sexual reproduction occurs commonly. In contrast, P. infestans is heterothallic and its sexual stage has not been observed outside of Mexico until very recently (Hohl and Iselin, 1984; Shaw et al., 1985). The common occurrence of sexual reproduction in a Phytophthora population may select against polyploid or aneuploid individuals, which may produce sterile or nonviable progeny upon mating with diploids. Thus, oes not seem surprising that polyploidy or aneuploidy would be more common in the asexual, non-Mexican P. infestans population compared with the sexual (Mexican) population of P. infestans or with P. megaspermn.

Another possible explanation for the DNA content variation observed among non-Mexican P. irzfestans isolates is the existence of diploid-polyploid heterokaryons (Sansome, 1977). Isolates containing mixtures of diploid and tetraploid nuclei should show mean DNA contents intermediate between those of diploid an tetraploid isolates. Although such beterokaryons might be expected to show bimodality in distribution of nuclear F-DNA content if sufficiently large numbers of both diploid and tetraploid zoospores are produced by such heterokaryons, our sample size (n = 50 per isolate) may have been too small to detect bimodality~ Some suggestion of bimodality is seen for U.S. isolates 128 and 147 (Figs. 4 and 8, respectively). Occasionally, individual nuclei existe that contained approximately twice the DNA content of the other nuclei of ticular isolate (Figs. 1 3 2, and 6) ~ could represent rare nuclei of higher ploidy, or nuclei in which DNA replication has occurred. Legrand-I’ernot and Pellegrin (1976), using gamma radiation killing curves and DNA cytophotometry, demonstrated that zoospore rmclei of P. p&m& parasitica were in the 61 stage

24

TOOLEY

Nuclear Feulgen-DNA Isolate No.

Mean DNA (n = 50)

111

0.81 1.11

127 128 134 13.5 137

0.76 0.84 1.09 0.94 0.95

140

1.03

141

0.88

136

AND THERRIEN

TABLE 4 Values in Non-Mexican Isolates of Phytophthoru SE”

Isolate

0.03 0.04 0.02

142 144 146 147

0.01 0.01

150 1.52

0.03 0.02 0.02

160 161 163

0.03

Mean DNA (fl = 50)

infestuns

SE”

0.66 0.62 0.92 1.05 0.78 0.90

0.02 0.02 0.01 0.03

1.01

0.01

0.83 0.56

0.03 0.02

0.01 0.02

L1Standard error of the mean.

of the mitotic interphase. Occasional zoo- chromosome counts are desirable to conspore nuclei of higher ploidy may have al- firm our results from DNA content meaready undergone DNA replication and be surement in P. infestam. Such studies with in interphase G2 prior to mitosis and germ other Phytophthora species have shown tube formation. Occasional nuclei of higher that chromosome number (Hansen et al., ploidy have also been observed in anther1986) and chromosome size (Sansome et idia and oogonia of Saprolegnia ferrestvis al., 1975) can vary substantially within a (Bryant and Howard, 1969). single Phytophthora species complex. FurWe estimated the average genome size of ther studies are needed to determine diploid P. infestans nuclei to be 0.52 pg whether P. infestans exhibits similar variaDNA per nucleus. This is about 2.5 times tion in chromosome number and/or chrothe genome size reported for P. megamosome size and to clarify such relationsperma f. sp. glycinea (Rutherford and ships among P. infestans isolates from difWard, 1985), 1.3 times that reported for Sa- ferent geographic regions. prolegnia sp. (Tanaka et al., 1982), and about lo-20 times that reported for a vaACKNOWLEDGMENTS riety of other fungi (Murrin et al., 1986; We acknowledge the excellent technical assistance Table 2). provided by Abhi Pathak. We also thank M. J. VillarIsozyme analysis of the isolates we have real and A. Rivera for assistance in collecting Mexican examined revealed banding patterns char- isolates of Phytophthora infesfuns. Finally, we acacteristic of diploids (Tooley et al., 1985). knowledge those who provided us with P. infestans isolates used in this study including J. Carroll, M. This suggests that tetraploid P. infestans Coffey, W. E. Fry, J. Galindo, R. Heisey, B. P. May, are functionally diploid. Cytological eviR. C. Shattock, W. R. Stevenson, and J. A. Sweigard. dence supports this hypothesis, as bivalents rather than tetravalents or trivalents REFERENCES were observed forming at meiosis in tetraploid British isolates (Sansome, 1977). BELL, W. R., AND THERRIEN, C. D. A cytophotomeRogers (1973) has suggested that polytric investigation of the relationship of DNA and RNA synthesis to ascus development in Sordariafiploidy may be more common in fungi than micoln. Canad. J. Genet. Cytol. 19: 359-370. generally recognized, with polyploids staBRASIER, C. M., AND SANSOME, E. 1975. Diploidy bilized so that they function as diploids in and gametangial meiosis in Phytophthora cinnameiosis. momi, P. infestans, and P. dreschleri. Trans. Brit. Cytological studies which yield direct Mycol. Sot. 65: 49-65.

DNA CONTENT

OF Phytophtkora infestam

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2.5

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THERRIEN, C. D., BELL, W. R., AND COLLINS, 0. R. 1977. Nuclear DNA content of myxamoebae and plasmodia in six nonheterothallic isolates of a myxomycete, Didymium iridis. Amer. J. Bot. 64: 286-291. THERRIEN, C. D., AND YEMMA, J. J. 1974. Comparative measurements of nuclear DNA in a heterothallic and a self-fertile isolate of the myxomycete Didymiam iridis. Amer. J. Bot. 61: 400-404. TIMMER, L. W., CASTRO. J., ERWIN, D. C., BELSER, W. L., AND ZENTMYER, G. A. 1970. Genetic evidence for zygotic meiosis in Phytophtho~a cupsici. Amer. J. Bat. 57: 1211-1218. TOOLEY, P. W., FRY, W. E., AND VILLARREAL GONZALEZ, M. J. 1985. Isozyme characterization of

THERFUEN

sexual and asexual Phytophthora infestans populations. J. Hered. 76: 431-435. TOOLEY P. W., SWEIGARD, J. A., AND FRY, W. E. 1986. Fitness and virulence of Phytophthora infestans isolates from sexual and asexual populations. Phytopathology 16: 1209- 1212. VAUGHN, J. C., AND LOCY, R. D. 1969. Changing nuclear histone patterns during development. III. The deoxyribonucleic acid content of spermatogenic cells in the crab Emerita analoga. J. Histochem. Cytochem. 17: 591-600. WHISLER, H. C., WILSON, C. M., T~AVLAND, L. B., OLSON, L. W., BORKHARDT, B., ALDRICH, J., THERRIEN, C. D., AND ZEBOLD, S. F. 1983. Meiosis in Coelomomyces. Exp. Mycol. 7: 319-327.