JOURNALOF
INVERTEBRATE
PATHOLOGY
%,96-105(1991)
DNA Restriction Polymorphism in Wild Isolates of Spodoptera frugiperda Nuclear Polyhedrosis Virus’ D. I. SHAPIRO,*
J. R. FUXA,*
H. D. BaAYMER,t
AND D. P. PASHLEYS
*Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana Agricultural Center, Baton Rouge, Louisiana 70803: tDepartment of Microbiology, University, Baton Rouge. Louisiana 70803; and SDepartment of Entomology, Louisiana Baton Rouge, Louisiana 70803
State University Louisiana State State University,
Received June 13, 1990; accepted October 9, 1990 Restriction endonuclease analysis was used to examine variation in DNA of 22 wild isolates of fiugiperda nuclear polyhedrosis virus (SfNPV). Eleven of the 15 isolates from Louisiana were distinguishable based on restriction fragment profiles from the enzymes BamHI, HindHI, and EcoRI. There was significant genetic variation in SfNPV isolates within single agricultural fields. Nucleotide sequence divergence values, based on restriction fragment profiles, indicated that genetic variation among isolates foreign to Louisiana (Ohio, Ecuador, Mexico, Georgia, Colombia, and Venezuela) was greater than that among the Louisiana isolates. However, certain foreign isolates were similar to or identical with Louisiana isolates. Genetic variation of the viral DNA was not influenced by the insect’s host plant species. o 1991 Academic press, IK. KEY WORDS: Spodoptera frugiperda; baculovirus; Spodoptera frugiperda NPV; restriction enzyme; viral variants; virus, geographical isolates. Spodoptera
INTRODUCTION The potential of baculoviruses for biological control has led to numerous studies of variation among viral isolates. Some methods used to characterize nuclear polyhedrosis viruses (NPVs) have included gel electrophoresis of viral structural proteins, serological reactions of proteins, and viral infectivity in cell culture (Maruniak et al., 1984). Rohrmann (1986) proposed a phylogenetic tree relating baculoviruses on the basis of their polyhedrin amino acid sequences. Variation has been reported in the survival time among isolates of Heliothis NPV (Hughes et al., 1983). Variation among NPVs also has been documented in terms of virulence (Williams and Payne, 1984; Hamm and Styer, 1985) and host range (Bilimoria, 1983). Restriction endonuclease (REN) analysis has been an efficient and exact method of distinguishing genotypic variants of NPVs (Lee and Miller, 1978; Miller and Dawes,
1978; Miller et al., 1980; Vlak and Groner, 1980; Gettig and McCarthy, 1982; Kislev and Edelman, 1982). Most REN studies of genotypic variation in virus isolates have been done over a large geographical scale. For example, Crawford et al. (1986) analyzed 12 geographical isolates of Oryctes BV from several islands in the Pacific Ocean, and Loh et al. (1982) found differences among Spodoptera frugiperda (Sf) BV isolates from different states in the United States. Variation also has been documented within a SfNPV population in a single host insect (Maruniak et al., 1984). There has been only one study of genetic variation over a relatively localized geographic area, involving Spodoptera littoralis NPV (SlNPV) in Israel (Cherry and Summers, 1985), and there has been no research of variation in viral populations on a single farm or within an agricultural field. Similarly, there have been no studies comparing the degree of variation among these various ecological levels, though there is little doubt that such variation exists. A major question arising from such studies of variation concerns the grouping of
’ Approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript number 90-17-4080. 96 0022-2011/91 $1.50 Copyright AII tights
0 1991 by Academic Press. Inc. of reproduction in any form reserved.
GENETIC
VARIANTS
OF
viral variants. How much difference must there be between REN digests of viral genomes before those viruses can be classified as significantly different groups, or even species? How can this be determined objectively? Cherry and Summers (1985) subjectively divided 21 isolates of SlNPV into two groups based on REN digests, but such subjective groupings cannot be used indefinitely. The purposes of this study were to: (1) compare genotypic variation of wild SfNPV isolates at several levels (over a wide geographical area, within a local agroecosystern, and among insects collected from different host plants), and (2) find a way of objectively grouping viral variants. MATERIALS Sources
AND METHODS
of 5” and NPV Isolates
Wild isolates of NPV were passed through laboratory reared S. frugiperda colonies. The S. frugiperda were originally collected from corn or grass in Hammond, Louisiana, or from grass fields in Baton Rouge, Louisiana. Nuclear polyhedrosis virus isolates were obtained by collecting S. frugiperda larvae
TABLE SOURCESOFWILD
Geographical location
Host plant
Tifton, Georgia” Ohio Medellin, Colombia Espinol, Colombia Araqua. Venezuela Merida, Mexico Ecuador Idlewild, Louisiana Ben Hur, Louisiana Hammond, Louisiana Hammond, Louisiana Hammond, Louisiana
Unknown Unknown Unknown Unknown Corn Bermuda grass Unknown Bermuda grass Bermuda grassb Comb Sorghum’ Signal grassb
S. frugiperda
97
NPV
from a farm in Hammond, Louisiana, or from experiment stations: Idlewild near Clinton, or Ben Hur near Baton Rouge, Louisiana. One field was selected for sampling each of three S. frugiperda host plants: sorghum, Sorghum vulgare; signal grass, Brachiaria platyphylla; and corn, Zea mays. Two bermuda grass, Cynodon dactylon, fields were selected. The fields were systematically split into three or four sections depending on size and shape of the field, except at Clinton where only one isolate was obtained. Virus from one infected larva from each section of a field constituted an isolate. Sections were approximately 300 m* at Hammond and 576 m’ at Baton Rouge. Larvae were collected from a sample site in an area ranging from 0.5 -3 m* in the center of each section. The fields sampled in Hammond were less than 2 km apart. A total of 15 Louisiana isolates (three each from corn and signal grass, five from bermuda grass, and four from sorghum) were analyzed, and seven isolates originating outside Louisiana (“foreign” isolates) were analyzed (Table 1). Virus Production
and Purification
Virus was purified from each of the 22 isolates and reproduced for DNA isolation.
1 SfNPV ISOLATES
______.---
Abbreviation D7 OH CM CE VZ MX EC LB5 LBl, LCl. LSl, LGI,
a A plaque purified isolate (Knell and Summers, 1981). b All isolates for each host plant were sampled from the same field.
LB2, LC2, LS2, LG2,
LB3, LB4 LC3 LS3, LS4 LG3
Date of collection 1963 1977 1978 1978 1981 l-30983 611984 711983 6-8/1986 7-80988 7-811988 7-8/1988
98
SHAPIRO
Each isolate (infected larva) was macerated, diluted to IO’ polyhedral inclusion bodies (PIBs), and spread on artificial velvetbean caterpillar diet. After the diet was dry 5-10 Sf larvae were fed the diet and incubated at 26.7 + 3” until death. The virus from these 5-10 larvae was purified and used to produce more virus by inoculation. Larvae were crudely homogenized in a Sorvall omni mixer for 3 min. The homogenate was tiltered through two-ply cheese cloth, and the suspension was centrifuged for 30 min on a table top centrifuge at 1600g. The white (viral portion) section of the precipitate was removed, diluted, and further purified on a cesium chloride step gradient of 3 1,42, and 54%. The suspensions were centrifuged in a SW41Ti rotor for 2 hr at 35,000 rpm. A single viral band was pipetted from each tube and dialyzed against 2 liters of distilled water for l-2 hr. A minimum of 1 x IO9 PIBs per insect was used for virus production by inoculation. The inoculation procedure was adapted from Stairs (1980). The PIBs were dissolved in an alkaline solution (0.1 M Na,CO,), and the released virions were injected into fifth instars with a Hamilton syringe (701 RN). The inoculated larvae were incubated at 26.7 ? 3°C until 2 days after death. For each isolate NPV was grown in 60-100 infected larvae, purified as described above, and used to isolate DNA. DNA
Isolation
Approximately 5 x 10” PIBs were puritied for DNA isolation from each viral isolate. Polyhedral inclusion bodies were dissolved in sodium carbonate as described in Loh et al. (1981). DNA was then extracted in a procedure adapted from Summers and Smith (1987). The released virions were layered on a 9-ml continuous sucrose gradient, 25-60%. The gradients were centrifuged for 1.5 hr at 28,000 rpm in a SW41Ti rotor. Multiple viral bands were removed with a Pasteur pipette. The purified virus was then diluted approximately four times
ET AL.
with 0.1 x TE (1 x TE = 10 mM Tris-HCl: 1 mM EDTA, pH 7.6) and centrifuged in a SW41Ti rotor for 40 min at 28,000 rpm. The viral precipitate was resuspended in 2.5 ml of extraction buffer (0.1 M Tris:0.2 M KC1:O.l M EDTA) and digested with 200400 pg of proteinase K (Tritirachium album, type XI.) at 50°C for 1.5 hr. To aid in protein degradation, 0.25 ml of 10% sarkosyl solution was then added, and digestion was continued at 50°C for 2 hr. The DNA was extracted twice with phenol:chloroform:isoamly alcohol (25:24: 1). The DNA was dialzyed against three changes of TE as follows: 3-4 hr with 0.1 x TE: 0.05 M KCI, 9-l 1 hr with 0.1 x TE: 0.01 M KCl, and 3 hr with 0.05~ TE. The DNA was then placed in Eppendorf tubes and concentrated with a Savant speed vat and refrigerated condensation trap to a volume of 100-300 ~1. Restriction
Endonuclease
Analysis
DNA (l-3 pg) from each sample was digested by BamHI, EcoRI, and Hind111 for 3-5 hr. The DNA was loaded onto a 0.6 or 0.7% agarose gel and electrophoresed at 35 V overnight. The gel was stained with ethidium bromide (0.5 kg/ml) for 20 min then destained twice with distilled water for 15 min each. The restriction fragments were visualized with an uv illuminator and photographed with an MP4 land camera. The sizes of DNA fragments were estimated by comparison to the standard molecular weight marker A Hind111 or a high molecular weight marker (BRL, Life Technologies, Inc., Gaithersburg, Maryland). Sequence divergence values (see equation 20 and Fig. I in Nei and Li, 1979) based on the number of shared fragments (all three enzymes) between two isolates were used to compare the variation among isolates. Sequence divergence or P values were calculated as pairwise comparisons between restriction fragment profiles. Greater P values indicate less similarity between the isolates being compared. A matrix of all P values was used to generate a phenogram
GENETIC
VARIANTS
OF S. frugiperda
showing relatedness among isolates (Kitsch program in Felsenstein, 1986). RESULTS The REN analysis detected differences among viral isolates in Louisiana (e.g., Fig. 1) as well as among foreign isolates (e.g., Fig. 2). Schematics based on the REN gels were then constructed (Figs. 3, 4, 5). Each fragment was assigned a letter, and the gels of each viral isolate were scored based on the presence or absence of each fragment. The estimated kilobase sizes for Hind111 fragments are presented in Table 2 to give an estimate of genome size. Sizes were not estimated for EcoRI because the lightest fragments were not distinguishable, and they were not estimated for BumHI due to the potential error in measuring distances of the heaviest fragments. The BumHI digests (Fig. 3) differed only in the presence or absence of three fragments (a, b. and g). Most of the Hind111 digests (19 out of 22) differed only in the presence or absence of fragment e (Fig. 4). Restriction fragment profiles of SfNPV DNA had the greatest variation when digested with EcoRI (Fig. LLLLLLLLLLL ssssCcceBse M23413212314A
FIG. 1. Restriction endonuclease digestions (BumHI) of DNA from 11 SfNPV isolates. See Table 1 for explanations of isolate abbreviations. M = high molecular weight marker (fragment molecular weights = 48,502. 38.416, 33,498. 29,942, 24,776, 22,621. 19,399, 17,057, 15,004, 12.220, 10,086. 8612, and8271). The last lane is A phage cut with Hind111 (fragment molecular weights = 27.491. 23,130. 9416. 6557, 4361, 2322, and 2027).
99
NPV EcoRl
0 H
m
VMOVMOV ZXHZXHZ
BarnHI
M Xl
FIG. 2. Restriction endonuclease digestions (BarnHI, EcoRI. and HindIII) of DNA from three SfNPV isolates. See Table 1 for explanaticn of isolate abbreviations. The last lane is A phage cut with Hind111 (fragment molecular weights = 27.491. 23,130, 9416. 6557, 4361. 2322. and 2027).
5); only 9 of the 20 fragments were common to all isolates. EcoRI fragments smaller than those represented in Fig. 5, ca. 3.3 kilobases, were not scored because they were not resolved in all gels. Measurements of the degree of variation among isolates were arranged in a sequence divergence matrix (Table 3). The greater the sequence divergence or P value, the less related were the isolates. Of the 15 Louisiana isolates, 11 were distinguishable from one another, and of the 22 total isolates 16 were genotypic variants. The following groups include identical isolates: CE and CM; OH, LCI, and EC: LC2 and LB5; LS2 and LGl; LG2 and LG3. Isolates LB2, D7, and VZ had diagnostic fragments. LB4 was missing a fragment that all other isolates had (Hind111 fragment j, Fig. 4). Mean sequence divergence values were calculated and analyzed statistically to examine the influence of host plant and geographic location on SfNPV variation (Table 4). Each mean P value includes all possible pairwise comparisons. For example, the mean P value for comparison “S vs. S” was calculated from the six pairwise comparisons of bermuda grass isolates to other ber-
100
SHAPIRO OH D7 VZ MX CE
CM EC
-----
LB4
LB1
LB3
LB2
ET AL. LC2
LCl
-----
;: c
-
d
------
-------
e
------
----
f
-------
g
-
h
----__
LC3
LSl
_ -
LS4
LS2
LS3
IGl
L&2
-
LB5
-------
------
--
--
---
1133
--
-----
----
--
----
----
--
----
----
-----
--
-----
FIG. 3. Schematic representation of BarnHI restriction fragment profiles of DNA from all the SfNPV isolates. See Table 1 for explanation of isolate abbrevations. Fragments are designated by letters on the left margin.
The mean sequence divergence values for isolates from corn and for those from signal grass were significantly different from each other but not from the other comparisons. The overall mean of within hostplant comparisons was not significantly dif-
muda grass isolates, and the mean sequence divergence for all foreign vs. Louisiana comparisons was calculated from 90 P values. CM was not used in the calculations because the two Colombian isolates were identical. OH D7 -
vz
MX
CE
-
CM EC
LB4
LB1
LB3
LB2
LC2
-
LCl
LC3
LSl
LS4
Is2
Ls3
LGl
LG2
LG3
LB5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
FIG. 4. Schematic representation of Hind111 restriction fragment profiles of DNA from all the SfNPV isolates. See Table 1 for explanations of isolate abbreviations. Fragments are designated by letters on the left margin.
GENETIC
OH D7 ;: :
---
e f g
------
VZ MX CE CM EC LB4 ----_ --
---------------
--
VARIANTS
LB1
LB3
LB2
-----
OF
LC2
-
1 m
------
LS2
LS3
-
------
-
--
---------
---
--
---
---
----
----
---
---
ferent from the overall mean of between host-plant comparisons (32.2 and 32, respectively). Hence, plant species had no effect on genotypic variation of SfNPV. This is confirmed in the phenogram (Fig. 6) because there was no clumping of viral isolates in relation to host plant. The average P values for comparisons among isolates separated by large geographic distance was sig-
KILOBASE OF
Fragment iIt : e f g h i .i k I m n 0
P 9
TABLE 2 SIZES OF Hind111 FRAGMENTS SfNPV DNA Kilobase size 18.5 16.5 14.7 13.9 12.6 10.7 10.0 9.4 8.2 6.9 6.0 5.6 5.4 3.7 2.9 2.7 2.3
LG2
LG3
LB5
---------
* ----__
-----_
n Cl p,q ----__------------s,t -----------FIG. 5. Schematic representation of EcoRI fragments profiles of DNA *Represents a submolar fragment. See Table I for explanations of isolate designated by letters on the left margin.
ESTIMATED
LGl
--
----
--
L.S.1 LS4
------
---
LC3
101
NPV
-----
------
i 2
LCl
--
------
h
S. frugiperda
------
-----from all the SfNPV isolates. abbreviations. Fragments are
nificantly greater than the average P value for the isolates within Louisiana (Table 4). Certain foreign isolates were similar to, or identical with, Louisiana isolates (Fig. 6). An additional test examined a potential problem with this study. Originally, S. fiugiperdu from both bermuda grass and corn were used for virus production, and different strains (possibly different sibling species) of S. fiugiperdu prefer these two host plants (Pashley, 1986). Thus, it is possible, though not likely, that inoculation of one viral isolate might activate a latent infection of another isolate (McKinley et al., 1981). Therefore, a signal grass NPV isolate (LGl) and a bermuda grass isolate (LBl) were both grown in S. frugiperdu from the two colonies. LB1 and LGl had identical restriction patterns (BumHI, data not shown) regardless of the S. fiugiperdu population in which they were produced, and in each case the isolate that was ingested was the one that replicated in the host. DISCUSSION
As the body of information on genetic variation of baculoviruses increases, the need for standardized criteria for the separation of variants into viral groups will become important. Variants may be defined
102
SHAPIRO ET AL. TABLE SEQUENCE
Oh Dl VZ MX CE CM EC LB4 LB1 LB3 LB2 LC2 LCl LC3 LSl LS4 LS2 LS3 LGl LG2 LG3 LB5
DIVERGENCE
OH
D7
VZ
0 57 101 84 73 73 0 46 55 66 52 46 0 75 17 27 36 70 36 53 53 46
0 84 87 55 55 57 48 37 48 73 28 57 57 75 28 37 73 37 55 55 28
0 70 60 60 101 93 62 73 95 73 101 43 118 73 81 58 81 60 60 73
MATRIX
MX
CE
CM
EC
0 43 43 84 96 64 55 79 75 84 45 81 75 84 60 84 62 62 75
0 0 73 45 17 26 50 26 73 17 70 45 35 33 35 17 17 26
0 73 45 17 26 50 26 73 17 70 45 35 33 35 17 17 26
0 46 55 66 52 46 0 75 17 27 36 70 36 53 53 46
BASED
LB4
LB1 --
0 27 37 43 18 46 46 45 18 9 43 9 26 26 18 .~
0 9 34 9 55 18 53 27 18 34 18 17 17 9
3
ON RESTRICTION LB3
LB2
LC2
0 43 18 66 27 64 37 27 43 27 26 26 18
0 43 52 52 50 43 34 49 34 33 33 43
0 46 27 45 18 9 43 9 26 26 0
U Nei and Li, (1979). Each number represents a pairwise comparison 1 for explanation of isolate abbreviations. The sequence divergence
as isolates with very similar genomes that can be distinguished only by minor differences in REN patterns (Gettig and McCarthy, 1982; McIntosh et al., 1987). Of the 15 Louisiana SfNPV isolates in this study 11 were distinct variants, and of the 22 total isolates 16 were variants. The phenogram based on sequence divergence (Fig. 6) indicated that the VZ and MX isolates were the
SEQUENCE
DIVERGENCE
(BASED
ON SIMILARITY SfNPV
Within-plant
average@
Between-plant
N
Mean
SE
Comparison
N
B vs B cvsc GVSG
6
32.2 ab 49.3 a 11.3 b 36.0 ab
5.2 14.0 5.6 5.9
B vs C BvsG c vs G c vs s B vs S SvsG
12 12 9 12 16 12
s vs s
LC3
0 75 17 27 36 70 36 53 53 46
0 73 46 36 34 36 17 17 27
LSl
0 45 35 50 35 52 52 45
OF SfNPV
LS4
LS2
LS3
0 9 43 9 26 26 18
0 34 0 17 17 9
0 34 16 16 43
between two isolates. Zero values are multiplied by lO4.
ISOLATES~ LGl
0 17 17 9
LG2
LG3
LB5
0 0 26
0 26
0
= no divergence.
See Table
4 IN REN
PROFILES)
FOR COMPARISONS
AMONG
ISOLATES
Comparison
3 3 6
LCI
PROFILES
most distinct from the other isolates, but whether these variants represent separate virus groups is unclear. The total length of the branches connecting any two sites in the phenogram (Fig. 6) indicates the degree of genetic differentiation between those two viral isolates. In other words, short branch lengths such as between LB1 and LB3 indicate very similar isolate pairs,
TABLE MEAN
DIGEST
average&’ Mean 37.5 24.3 30.3 37.8 37.1 25.0
ab ab ab ab ab ab
L-F averages’ SE
Comparison
5.3 2.2 5.2 5.6 3.6 4.6
F vs F F vs L L vs L
N
Mean
SE
17 90
68.6 a 53.8 b
6.6
31.3
1.6
105
c
2.4
a Comparisons followed by the same letter in both columns are not significantly different (Tukey’s multiple range test), P < 0.05. b B = bermuda grass, C = corn, G = signal grass, S = sorghum. Because isolate LB5 was collected from a separate field than the other bermuda grass isolates it was not included in the analysis. c Comparisons followed by the same letter are not significantly different (Tukey’s multiple range test), P < 0.01. F = isolates foreign to Louisiana, L = Louisiana isolates.
GENETIC
VARIANTS
vz Imx Ls3 cm LG2
-LB1
La LGI ’
Is2 Ls4 LB2 D? lcl
EC \
OH
--
FIG. 6. A phenogram generated by the Kitsch program (Felsenstein, 1986) displaying similarities among SfNPV isolates based on sequence divergence. See Table 1 for explanation of abbreviations. Isolates LC 1, EC, and OH were identical to one another, as were LGl to LSZ, LB5 to LC2, LG2 to LG3, and CM to CE.
whereas VZ is not similar to any other isolate. Sequence divergence values or phenograms have been used for analysis of genotypic variability of the NPVs of plusiine hosts (Bilimoria, 1983), characterization of bacterial genomes (Hartung and Civerolo, 1989), and characterization of mitochondrial DNA from eukaryotes (Avise, 1986; Avise et al., 1987). Variation among Louisiana isolates was, on average, less than that between foreign and Louisiana isolates (Table 4), though certain foreign isolates were similar to or identical with Louisiana isolates. The VZ and MX isolates were somewhat distinct, but foreign isolates generally were interspersed with Louisiana isolates in the cluster analysis (Fig. 6). Thus, SfNPV variation is not dependent on geographic location alone. Some of the sequence divergence values between Louisiana isolates were as high as those between foreign and Louisiana isolates (Table 3). It is possible that the similarity between certain foreign and Louisiana SfNPV isolates could be due to viral transport by S. frugiperda moths; the moths migrate over long distances (Pair et al., 1986), and SfNPV can be vertically transmitted (J. R. Fuxa, A. R. Richter, and
OF
S. frugiperda
NPV
103
E. H. Weidner, unpublished data). In another study, variation in the genome of the Oryctes baculovirus was proportionate to the distance between isolates (Crawford et al., 1986). However, there was no relationship between geographic location and SlNPV variants (Cherry and Summers, 1985). There have been other studies of genetic variation in populations of baculoviruses, but such studies have been restricted to one level of variation (i.e., within a single host insect or among states or countries). Variation in SfNPV has been found within a single host (Knell and Summers, 198 1; Maruniak et al., 1984). Therefore, variation among viral isolates within a single locality is to be expected, though the comparative degree of variation at different levels has not been studied. There has been little other research of variation within small regions such as within fields or between fields on the same farm. Cherry and Summers (1985) found variation in SlNPV within regions of Israel. However, variation within a single field was not examined, and distances between isolates were not documented. Loh et al. (1982) distinguished single SfNPV isolates from each of four states in the United States. The possibility that two sibling species of S. frugiperda exist (Pashley, 1986, 1988) raises questions about SfNPV variation. Genetic differentiation in S. fvugiperda populations is associated with host plant usage: one S. fiugiperda strain prefers corn and sorghum and the other prefers rice and grasses. Due to the host specificity of NPVs (Fuxa, 1987), it would not be unexpected if SfNPV isolated from S. frzcgiperda on corn and bermuda grass would vary more between host plants than within fields. However, the results of this study indicate that variation in SfNPV isolates is not correlated with host plant species (Table 4). The viral isolates from S. frugiperdu in bermuda grass were not, as a group, distinct from those from S. frugiperdu in corn (Fig. 6).
104
SHAPIRO ET AL.
The causes of variation in the REN protiles are unknown. Variation in restriction enzyme patterns of NPVs may result from three mechanisms: insertions of host DNA into the viral genome, duplications of viral sequences, and point mutations (Brown et al., 1985). The BamHI fragments b and g in isolates OH, EC, LSI, and LCl (Fig. 3) may have resulted from an additional cutting site in fragment a. The presence of fragments a and g (rather than b and g) in isolates MX and LB2 (Fig. 3) may be explained by a duplication or by an insertion in fragment b. A duplication or insertion in fragment b would increase the number of base pairs to a genome size close to that of fragment a. The NPV genome can acquire portions of host DNA (Miller and Miller, 1982; Fraser et al., 1983). However, changes in restriction profiles resulting from host DNA pickup by NPV have been documented only after numerous passages of the virus through cell culture. Croizier et al. (1985) reported such a difference in REN profiles only after 25 passages of Mamestra brassicue NPV, and McIntosh and Ignoffo (1986) found a difference in REN profiles in Heliothis NPV after 20 passages. Smith and Summers (1978) found no difference in Autographa californica NPV isolates after sixteen serial passages. The acquisition of host DNA has not been demonstrated in vivo. Hence, the possibility that NPV acquired host DNA during the current study is not likely to have affected restriction enzyme patterns. Further studies of variation and groupings within baculovirus “species” will be fundamental to their ecology, to their development for microbial control, and perhaps to an understanding of their evolution. ACKNOWLEDGMENTS
The authors thank J. R. Adams (U.S. Department of Agriculture, Beltsville, Maryland), J. J. Hamm (U.S. Department of Agriculture, Tifton, Georgia), J. D. Harper (North Carolina State University),
J. A. Jimenez (Instituto Colombiano Agropecuario, Bogota, Colombia), S. J . Johnson (Louisiana State University), F. L. Mitchell (Texas Agricultural Experiment Station, Stephenville), and H. W. Wassink (Universidad Central de Venezuela) for providing certain viral isolates or the insects from which isolates were obtained. REFERENCES AVISE, J. C. 1986. Mitochondral DNA and the evolutionary genetics of higher animals. Philos. Trans. R. Sot. London B., 312, 325-342. AVISE. J. C., ARNOLD, J., BALL, R. M., BERMINGHAM, E.. LAMB, T., NEIGEL, J. E., REEB. C. A., AND SAUNDERS, N. C. 1987. Intraspecific phylogeography: The mitochondrial DNA bridge between population genetics and systematics. Annu. Rev. Ecol. Sysr., 18, 489-522. BILIMORIA, S. L. 1983. Genomic divergence among single-nucleocapsid nuclear polyhedrosis viruses of plusiine hosts. Virology, 127, 15-23. BROWN, S. E.. MARUNIAK, J. E., AND KNUDSON, D. L. 1985. Baculovirus (MNPV) genomic variants: Characterization of Spodoptern exempta MNPV DNAs and comparison with other Autographa californica MNPV DNAs. J. Gen. Virol., 66, 24312441. CHERRY, C. L.. AND SUMMERS, M. D. 1985. Genotypic variation among wild isolates of two nuclear polyhedrosis viruses isolated from Spodoptera littoralis. J. Inverrebr. Pathol., 46, 289-295. CRAWFORD, A. M., ZELAZNY. B.. AND ALFILER, A. R. 1986. Genotypic variation in geographical isolates of Ovcfes baculovirus. J. Gen. Viral.. 67,949952.
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KISLEV, N., AND EDELMAN, M. 1982. DNA restriction-pattern differences from geographic isolates of Spodoptera littoralis nuclear polyhedrosis virus. Virology,
119, 219-222.
KNELL, J. D., AND SUMMERS, M. D. 1981. Investigation of genetic heterogeneity in wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus by restriction endonuclease analysis of plaquepurified variants. Virology, 112, 190-197. LEE, H. H., AND MILLER, L. K. 1978. Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus. J. Virol., 27, 754-767. LOH, L. C.. HAMM, J. J.. AND HUANG, E. 1981. Spodoptera frugiperda nuclear polyhedrosis virus genome: Physical maps for restriction endonucleases BamHl and HindIII. J. Virol., 38, 922-931. LOH, L. C.. HAMM, J. J.. KAWANISHI, C.. AND HUANG. E. 1982. Analysis of the Spodoptera frugiperda nuclear polyhedrosis virus genome by restriction endonucleases and electron microscopy. J. Virol.,
44, 747-751.
MARUNIAK. J. E.. BROWN, S. E., AND KNUDSON, D. L. 1984. Physical maps of SfMNPV baculovirus DNA and its genomic variants. Virology, 136, 221234.
MCINTOSH. A. H., AND IGNOFFO. C. M. 1986. Restriction endonuclease cleavage patterns of commercial and serially passaged isolates of Heliothis baculovirus. Int. Virol., 41, 172-176. MCINTOSH, A. H.. RICE, W. C.. AND IGNOFFO, C. M. 1987. Genotypic variants in wild-type populations of baculoviruses. In “Biotechnology in Invertebrate Pathology and Cell Culture” (K. Maramorosch, Ed.), pp. 305-325. Academic Press, San Diego.
OF
NPV
S. frugiperda
105
MCKINLEY, D. J., BROWN, D. A., PAYNE, C. C., AND HARRAP, K. A. 1981. Cross-infectivity and activation studies with four baculoviruses. Entomophaga, 26, 79-80.
MILLER, D. W., AND MILLER, L. K. 1982. A virus mutant with an insertion of a copia-like transposable element. Nature, 299, 562-564. MILLER, L. K., AND DAWES, K. P. 1978. Restriction endonuclease analysis for the identification of baculovirus pesticides. Appl. Environ. Microbial., 35, 411-421. MILLER, L. K., FRANZBLAU, S. G., HOMAN, H. W., AND KISH, L. P. 1980. A new variant ofAutographa californica nuclear polyhedrosis virus. J. Invertebr. Pathol.,
36, 159-165.
NEI, M., AND LI, W. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA., 76, 5269-6273.
PAIR, S. B., RAULSTON, J. R., SPARKS, A. N., WESTBROOK, J. K., AND DOUCE, G. K. 1986. Fall armyworm distribution and population dynamics in southeastern states. F/a. Entomol., 69, 468-486. PASHLEY, D. P. 1986. Host-associated genetic differentiation in fall armyworm (Lepidoptera: Nocutuidae): A sibling species complex? Ann. Entomol. Sot.
Am.,
79, 898-904.
PASHLEY. D. P. 1988. The current status of fall armyworm host strains. Flu. Entomol., 71, 227-234. ROHRMANN, G. F. 1986. Polyhedrin structure. J. Gen. Virol., 67, 14991513. SMITH, G. E., AND SUMMERS, M. D. 1978. Analysis of baculovirus genomes with restriction endonucleases. Virology, 89, 517-527. STAIRS, G. R. 1980. Comparative infectivity of nonoceluded virions, polyhedra, and virions released from polyhedra for larvae of Galleria mellonella. J. Invertebr. Pathol., 36, 281-282. SUMMERS, M. D., AND SMITH, G. E. 1987. Viral DNA isolation. In “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures.” Texas Agricultural Experiment Station, Bull. No. 1555, College Station, Texas. VLAK, J. M.. AND GRONER, A. 1980. Identification of two nuclear polyhedrosis viruses from the cabbage moth Mamestra brassicae (Lepidoptera: Noctuidae). J. Invertebr. Pathol., 35, 269-278. WILLIAMS, C. F., AND PAYNE, C. C. 1984. The susceptibility of Heliothis armigera larvae to three nuclear polyhedrosis viruses. Ann. Appl. Biol., 104, 405412.
INVERTEBRATE
PATHOLOGY
%,96-105(1991)
DNA Restriction Polymorphism in Wild Isolates of Spodoptera frugiperda Nuclear Polyhedrosis Virus’ D. I. SHAPIRO,*
J. R. FUXA,*
H. D. BaAYMER,t
AND D. P. PASHLEYS
*Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana Agricultural Center, Baton Rouge, Louisiana 70803: tDepartment of Microbiology, University, Baton Rouge. Louisiana 70803; and SDepartment of Entomology, Louisiana Baton Rouge, Louisiana 70803
State University Louisiana State State University,
Received June 13, 1990; accepted October 9, 1990 Restriction endonuclease analysis was used to examine variation in DNA of 22 wild isolates of fiugiperda nuclear polyhedrosis virus (SfNPV). Eleven of the 15 isolates from Louisiana were distinguishable based on restriction fragment profiles from the enzymes BamHI, HindHI, and EcoRI. There was significant genetic variation in SfNPV isolates within single agricultural fields. Nucleotide sequence divergence values, based on restriction fragment profiles, indicated that genetic variation among isolates foreign to Louisiana (Ohio, Ecuador, Mexico, Georgia, Colombia, and Venezuela) was greater than that among the Louisiana isolates. However, certain foreign isolates were similar to or identical with Louisiana isolates. Genetic variation of the viral DNA was not influenced by the insect’s host plant species. o 1991 Academic press, IK. KEY WORDS: Spodoptera frugiperda; baculovirus; Spodoptera frugiperda NPV; restriction enzyme; viral variants; virus, geographical isolates. Spodoptera
INTRODUCTION The potential of baculoviruses for biological control has led to numerous studies of variation among viral isolates. Some methods used to characterize nuclear polyhedrosis viruses (NPVs) have included gel electrophoresis of viral structural proteins, serological reactions of proteins, and viral infectivity in cell culture (Maruniak et al., 1984). Rohrmann (1986) proposed a phylogenetic tree relating baculoviruses on the basis of their polyhedrin amino acid sequences. Variation has been reported in the survival time among isolates of Heliothis NPV (Hughes et al., 1983). Variation among NPVs also has been documented in terms of virulence (Williams and Payne, 1984; Hamm and Styer, 1985) and host range (Bilimoria, 1983). Restriction endonuclease (REN) analysis has been an efficient and exact method of distinguishing genotypic variants of NPVs (Lee and Miller, 1978; Miller and Dawes,
1978; Miller et al., 1980; Vlak and Groner, 1980; Gettig and McCarthy, 1982; Kislev and Edelman, 1982). Most REN studies of genotypic variation in virus isolates have been done over a large geographical scale. For example, Crawford et al. (1986) analyzed 12 geographical isolates of Oryctes BV from several islands in the Pacific Ocean, and Loh et al. (1982) found differences among Spodoptera frugiperda (Sf) BV isolates from different states in the United States. Variation also has been documented within a SfNPV population in a single host insect (Maruniak et al., 1984). There has been only one study of genetic variation over a relatively localized geographic area, involving Spodoptera littoralis NPV (SlNPV) in Israel (Cherry and Summers, 1985), and there has been no research of variation in viral populations on a single farm or within an agricultural field. Similarly, there have been no studies comparing the degree of variation among these various ecological levels, though there is little doubt that such variation exists. A major question arising from such studies of variation concerns the grouping of
’ Approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript number 90-17-4080. 96 0022-2011/91 $1.50 Copyright AII tights
0 1991 by Academic Press. Inc. of reproduction in any form reserved.
GENETIC
VARIANTS
OF
viral variants. How much difference must there be between REN digests of viral genomes before those viruses can be classified as significantly different groups, or even species? How can this be determined objectively? Cherry and Summers (1985) subjectively divided 21 isolates of SlNPV into two groups based on REN digests, but such subjective groupings cannot be used indefinitely. The purposes of this study were to: (1) compare genotypic variation of wild SfNPV isolates at several levels (over a wide geographical area, within a local agroecosystern, and among insects collected from different host plants), and (2) find a way of objectively grouping viral variants. MATERIALS Sources
AND METHODS
of 5” and NPV Isolates
Wild isolates of NPV were passed through laboratory reared S. frugiperda colonies. The S. frugiperda were originally collected from corn or grass in Hammond, Louisiana, or from grass fields in Baton Rouge, Louisiana. Nuclear polyhedrosis virus isolates were obtained by collecting S. frugiperda larvae
TABLE SOURCESOFWILD
Geographical location
Host plant
Tifton, Georgia” Ohio Medellin, Colombia Espinol, Colombia Araqua. Venezuela Merida, Mexico Ecuador Idlewild, Louisiana Ben Hur, Louisiana Hammond, Louisiana Hammond, Louisiana Hammond, Louisiana
Unknown Unknown Unknown Unknown Corn Bermuda grass Unknown Bermuda grass Bermuda grassb Comb Sorghum’ Signal grassb
S. frugiperda
97
NPV
from a farm in Hammond, Louisiana, or from experiment stations: Idlewild near Clinton, or Ben Hur near Baton Rouge, Louisiana. One field was selected for sampling each of three S. frugiperda host plants: sorghum, Sorghum vulgare; signal grass, Brachiaria platyphylla; and corn, Zea mays. Two bermuda grass, Cynodon dactylon, fields were selected. The fields were systematically split into three or four sections depending on size and shape of the field, except at Clinton where only one isolate was obtained. Virus from one infected larva from each section of a field constituted an isolate. Sections were approximately 300 m* at Hammond and 576 m’ at Baton Rouge. Larvae were collected from a sample site in an area ranging from 0.5 -3 m* in the center of each section. The fields sampled in Hammond were less than 2 km apart. A total of 15 Louisiana isolates (three each from corn and signal grass, five from bermuda grass, and four from sorghum) were analyzed, and seven isolates originating outside Louisiana (“foreign” isolates) were analyzed (Table 1). Virus Production
and Purification
Virus was purified from each of the 22 isolates and reproduced for DNA isolation.
1 SfNPV ISOLATES
______.---
Abbreviation D7 OH CM CE VZ MX EC LB5 LBl, LCl. LSl, LGI,
a A plaque purified isolate (Knell and Summers, 1981). b All isolates for each host plant were sampled from the same field.
LB2, LC2, LS2, LG2,
LB3, LB4 LC3 LS3, LS4 LG3
Date of collection 1963 1977 1978 1978 1981 l-30983 611984 711983 6-8/1986 7-80988 7-811988 7-8/1988
98
SHAPIRO
Each isolate (infected larva) was macerated, diluted to IO’ polyhedral inclusion bodies (PIBs), and spread on artificial velvetbean caterpillar diet. After the diet was dry 5-10 Sf larvae were fed the diet and incubated at 26.7 + 3” until death. The virus from these 5-10 larvae was purified and used to produce more virus by inoculation. Larvae were crudely homogenized in a Sorvall omni mixer for 3 min. The homogenate was tiltered through two-ply cheese cloth, and the suspension was centrifuged for 30 min on a table top centrifuge at 1600g. The white (viral portion) section of the precipitate was removed, diluted, and further purified on a cesium chloride step gradient of 3 1,42, and 54%. The suspensions were centrifuged in a SW41Ti rotor for 2 hr at 35,000 rpm. A single viral band was pipetted from each tube and dialyzed against 2 liters of distilled water for l-2 hr. A minimum of 1 x IO9 PIBs per insect was used for virus production by inoculation. The inoculation procedure was adapted from Stairs (1980). The PIBs were dissolved in an alkaline solution (0.1 M Na,CO,), and the released virions were injected into fifth instars with a Hamilton syringe (701 RN). The inoculated larvae were incubated at 26.7 ? 3°C until 2 days after death. For each isolate NPV was grown in 60-100 infected larvae, purified as described above, and used to isolate DNA. DNA
Isolation
Approximately 5 x 10” PIBs were puritied for DNA isolation from each viral isolate. Polyhedral inclusion bodies were dissolved in sodium carbonate as described in Loh et al. (1981). DNA was then extracted in a procedure adapted from Summers and Smith (1987). The released virions were layered on a 9-ml continuous sucrose gradient, 25-60%. The gradients were centrifuged for 1.5 hr at 28,000 rpm in a SW41Ti rotor. Multiple viral bands were removed with a Pasteur pipette. The purified virus was then diluted approximately four times
ET AL.
with 0.1 x TE (1 x TE = 10 mM Tris-HCl: 1 mM EDTA, pH 7.6) and centrifuged in a SW41Ti rotor for 40 min at 28,000 rpm. The viral precipitate was resuspended in 2.5 ml of extraction buffer (0.1 M Tris:0.2 M KC1:O.l M EDTA) and digested with 200400 pg of proteinase K (Tritirachium album, type XI.) at 50°C for 1.5 hr. To aid in protein degradation, 0.25 ml of 10% sarkosyl solution was then added, and digestion was continued at 50°C for 2 hr. The DNA was extracted twice with phenol:chloroform:isoamly alcohol (25:24: 1). The DNA was dialzyed against three changes of TE as follows: 3-4 hr with 0.1 x TE: 0.05 M KCI, 9-l 1 hr with 0.1 x TE: 0.01 M KCl, and 3 hr with 0.05~ TE. The DNA was then placed in Eppendorf tubes and concentrated with a Savant speed vat and refrigerated condensation trap to a volume of 100-300 ~1. Restriction
Endonuclease
Analysis
DNA (l-3 pg) from each sample was digested by BamHI, EcoRI, and Hind111 for 3-5 hr. The DNA was loaded onto a 0.6 or 0.7% agarose gel and electrophoresed at 35 V overnight. The gel was stained with ethidium bromide (0.5 kg/ml) for 20 min then destained twice with distilled water for 15 min each. The restriction fragments were visualized with an uv illuminator and photographed with an MP4 land camera. The sizes of DNA fragments were estimated by comparison to the standard molecular weight marker A Hind111 or a high molecular weight marker (BRL, Life Technologies, Inc., Gaithersburg, Maryland). Sequence divergence values (see equation 20 and Fig. I in Nei and Li, 1979) based on the number of shared fragments (all three enzymes) between two isolates were used to compare the variation among isolates. Sequence divergence or P values were calculated as pairwise comparisons between restriction fragment profiles. Greater P values indicate less similarity between the isolates being compared. A matrix of all P values was used to generate a phenogram
GENETIC
VARIANTS
OF S. frugiperda
showing relatedness among isolates (Kitsch program in Felsenstein, 1986). RESULTS The REN analysis detected differences among viral isolates in Louisiana (e.g., Fig. 1) as well as among foreign isolates (e.g., Fig. 2). Schematics based on the REN gels were then constructed (Figs. 3, 4, 5). Each fragment was assigned a letter, and the gels of each viral isolate were scored based on the presence or absence of each fragment. The estimated kilobase sizes for Hind111 fragments are presented in Table 2 to give an estimate of genome size. Sizes were not estimated for EcoRI because the lightest fragments were not distinguishable, and they were not estimated for BumHI due to the potential error in measuring distances of the heaviest fragments. The BumHI digests (Fig. 3) differed only in the presence or absence of three fragments (a, b. and g). Most of the Hind111 digests (19 out of 22) differed only in the presence or absence of fragment e (Fig. 4). Restriction fragment profiles of SfNPV DNA had the greatest variation when digested with EcoRI (Fig. LLLLLLLLLLL ssssCcceBse M23413212314A
FIG. 1. Restriction endonuclease digestions (BumHI) of DNA from 11 SfNPV isolates. See Table 1 for explanations of isolate abbreviations. M = high molecular weight marker (fragment molecular weights = 48,502. 38.416, 33,498. 29,942, 24,776, 22,621. 19,399, 17,057, 15,004, 12.220, 10,086. 8612, and8271). The last lane is A phage cut with Hind111 (fragment molecular weights = 27.491. 23,130. 9416. 6557, 4361, 2322, and 2027).
99
NPV EcoRl
0 H
m
VMOVMOV ZXHZXHZ
BarnHI
M Xl
FIG. 2. Restriction endonuclease digestions (BarnHI, EcoRI. and HindIII) of DNA from three SfNPV isolates. See Table 1 for explanaticn of isolate abbreviations. The last lane is A phage cut with Hind111 (fragment molecular weights = 27.491. 23,130, 9416. 6557, 4361. 2322. and 2027).
5); only 9 of the 20 fragments were common to all isolates. EcoRI fragments smaller than those represented in Fig. 5, ca. 3.3 kilobases, were not scored because they were not resolved in all gels. Measurements of the degree of variation among isolates were arranged in a sequence divergence matrix (Table 3). The greater the sequence divergence or P value, the less related were the isolates. Of the 15 Louisiana isolates, 11 were distinguishable from one another, and of the 22 total isolates 16 were genotypic variants. The following groups include identical isolates: CE and CM; OH, LCI, and EC: LC2 and LB5; LS2 and LGl; LG2 and LG3. Isolates LB2, D7, and VZ had diagnostic fragments. LB4 was missing a fragment that all other isolates had (Hind111 fragment j, Fig. 4). Mean sequence divergence values were calculated and analyzed statistically to examine the influence of host plant and geographic location on SfNPV variation (Table 4). Each mean P value includes all possible pairwise comparisons. For example, the mean P value for comparison “S vs. S” was calculated from the six pairwise comparisons of bermuda grass isolates to other ber-
100
SHAPIRO OH D7 VZ MX CE
CM EC
-----
LB4
LB1
LB3
LB2
ET AL. LC2
LCl
-----
;: c
-
d
------
-------
e
------
----
f
-------
g
-
h
----__
LC3
LSl
_ -
LS4
LS2
LS3
IGl
L&2
-
LB5
-------
------
--
--
---
1133
--
-----
----
--
----
----
--
----
----
-----
--
-----
FIG. 3. Schematic representation of BarnHI restriction fragment profiles of DNA from all the SfNPV isolates. See Table 1 for explanation of isolate abbrevations. Fragments are designated by letters on the left margin.
The mean sequence divergence values for isolates from corn and for those from signal grass were significantly different from each other but not from the other comparisons. The overall mean of within hostplant comparisons was not significantly dif-
muda grass isolates, and the mean sequence divergence for all foreign vs. Louisiana comparisons was calculated from 90 P values. CM was not used in the calculations because the two Colombian isolates were identical. OH D7 -
vz
MX
CE
-
CM EC
LB4
LB1
LB3
LB2
LC2
-
LCl
LC3
LSl
LS4
Is2
Ls3
LGl
LG2
LG3
LB5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
FIG. 4. Schematic representation of Hind111 restriction fragment profiles of DNA from all the SfNPV isolates. See Table 1 for explanations of isolate abbreviations. Fragments are designated by letters on the left margin.
GENETIC
OH D7 ;: :
---
e f g
------
VZ MX CE CM EC LB4 ----_ --
---------------
--
VARIANTS
LB1
LB3
LB2
-----
OF
LC2
-
1 m
------
LS2
LS3
-
------
-
--
---------
---
--
---
---
----
----
---
---
ferent from the overall mean of between host-plant comparisons (32.2 and 32, respectively). Hence, plant species had no effect on genotypic variation of SfNPV. This is confirmed in the phenogram (Fig. 6) because there was no clumping of viral isolates in relation to host plant. The average P values for comparisons among isolates separated by large geographic distance was sig-
KILOBASE OF
Fragment iIt : e f g h i .i k I m n 0
P 9
TABLE 2 SIZES OF Hind111 FRAGMENTS SfNPV DNA Kilobase size 18.5 16.5 14.7 13.9 12.6 10.7 10.0 9.4 8.2 6.9 6.0 5.6 5.4 3.7 2.9 2.7 2.3
LG2
LG3
LB5
---------
* ----__
-----_
n Cl p,q ----__------------s,t -----------FIG. 5. Schematic representation of EcoRI fragments profiles of DNA *Represents a submolar fragment. See Table I for explanations of isolate designated by letters on the left margin.
ESTIMATED
LGl
--
----
--
L.S.1 LS4
------
---
LC3
101
NPV
-----
------
i 2
LCl
--
------
h
S. frugiperda
------
-----from all the SfNPV isolates. abbreviations. Fragments are
nificantly greater than the average P value for the isolates within Louisiana (Table 4). Certain foreign isolates were similar to, or identical with, Louisiana isolates (Fig. 6). An additional test examined a potential problem with this study. Originally, S. fiugiperdu from both bermuda grass and corn were used for virus production, and different strains (possibly different sibling species) of S. fiugiperdu prefer these two host plants (Pashley, 1986). Thus, it is possible, though not likely, that inoculation of one viral isolate might activate a latent infection of another isolate (McKinley et al., 1981). Therefore, a signal grass NPV isolate (LGl) and a bermuda grass isolate (LBl) were both grown in S. frugiperdu from the two colonies. LB1 and LGl had identical restriction patterns (BumHI, data not shown) regardless of the S. fiugiperdu population in which they were produced, and in each case the isolate that was ingested was the one that replicated in the host. DISCUSSION
As the body of information on genetic variation of baculoviruses increases, the need for standardized criteria for the separation of variants into viral groups will become important. Variants may be defined
102
SHAPIRO ET AL. TABLE SEQUENCE
Oh Dl VZ MX CE CM EC LB4 LB1 LB3 LB2 LC2 LCl LC3 LSl LS4 LS2 LS3 LGl LG2 LG3 LB5
DIVERGENCE
OH
D7
VZ
0 57 101 84 73 73 0 46 55 66 52 46 0 75 17 27 36 70 36 53 53 46
0 84 87 55 55 57 48 37 48 73 28 57 57 75 28 37 73 37 55 55 28
0 70 60 60 101 93 62 73 95 73 101 43 118 73 81 58 81 60 60 73
MATRIX
MX
CE
CM
EC
0 43 43 84 96 64 55 79 75 84 45 81 75 84 60 84 62 62 75
0 0 73 45 17 26 50 26 73 17 70 45 35 33 35 17 17 26
0 73 45 17 26 50 26 73 17 70 45 35 33 35 17 17 26
0 46 55 66 52 46 0 75 17 27 36 70 36 53 53 46
BASED
LB4
LB1 --
0 27 37 43 18 46 46 45 18 9 43 9 26 26 18 .~
0 9 34 9 55 18 53 27 18 34 18 17 17 9
3
ON RESTRICTION LB3
LB2
LC2
0 43 18 66 27 64 37 27 43 27 26 26 18
0 43 52 52 50 43 34 49 34 33 33 43
0 46 27 45 18 9 43 9 26 26 0
U Nei and Li, (1979). Each number represents a pairwise comparison 1 for explanation of isolate abbreviations. The sequence divergence
as isolates with very similar genomes that can be distinguished only by minor differences in REN patterns (Gettig and McCarthy, 1982; McIntosh et al., 1987). Of the 15 Louisiana SfNPV isolates in this study 11 were distinct variants, and of the 22 total isolates 16 were variants. The phenogram based on sequence divergence (Fig. 6) indicated that the VZ and MX isolates were the
SEQUENCE
DIVERGENCE
(BASED
ON SIMILARITY SfNPV
Within-plant
average@
Between-plant
N
Mean
SE
Comparison
N
B vs B cvsc GVSG
6
32.2 ab 49.3 a 11.3 b 36.0 ab
5.2 14.0 5.6 5.9
B vs C BvsG c vs G c vs s B vs S SvsG
12 12 9 12 16 12
s vs s
LC3
0 75 17 27 36 70 36 53 53 46
0 73 46 36 34 36 17 17 27
LSl
0 45 35 50 35 52 52 45
OF SfNPV
LS4
LS2
LS3
0 9 43 9 26 26 18
0 34 0 17 17 9
0 34 16 16 43
between two isolates. Zero values are multiplied by lO4.
ISOLATES~ LGl
0 17 17 9
LG2
LG3
LB5
0 0 26
0 26
0
= no divergence.
See Table
4 IN REN
PROFILES)
FOR COMPARISONS
AMONG
ISOLATES
Comparison
3 3 6
LCI
PROFILES
most distinct from the other isolates, but whether these variants represent separate virus groups is unclear. The total length of the branches connecting any two sites in the phenogram (Fig. 6) indicates the degree of genetic differentiation between those two viral isolates. In other words, short branch lengths such as between LB1 and LB3 indicate very similar isolate pairs,
TABLE MEAN
DIGEST
average&’ Mean 37.5 24.3 30.3 37.8 37.1 25.0
ab ab ab ab ab ab
L-F averages’ SE
Comparison
5.3 2.2 5.2 5.6 3.6 4.6
F vs F F vs L L vs L
N
Mean
SE
17 90
68.6 a 53.8 b
6.6
31.3
1.6
105
c
2.4
a Comparisons followed by the same letter in both columns are not significantly different (Tukey’s multiple range test), P < 0.05. b B = bermuda grass, C = corn, G = signal grass, S = sorghum. Because isolate LB5 was collected from a separate field than the other bermuda grass isolates it was not included in the analysis. c Comparisons followed by the same letter are not significantly different (Tukey’s multiple range test), P < 0.01. F = isolates foreign to Louisiana, L = Louisiana isolates.
GENETIC
VARIANTS
vz Imx Ls3 cm LG2
-LB1
La LGI ’
Is2 Ls4 LB2 D? lcl
EC \
OH
--
FIG. 6. A phenogram generated by the Kitsch program (Felsenstein, 1986) displaying similarities among SfNPV isolates based on sequence divergence. See Table 1 for explanation of abbreviations. Isolates LC 1, EC, and OH were identical to one another, as were LGl to LSZ, LB5 to LC2, LG2 to LG3, and CM to CE.
whereas VZ is not similar to any other isolate. Sequence divergence values or phenograms have been used for analysis of genotypic variability of the NPVs of plusiine hosts (Bilimoria, 1983), characterization of bacterial genomes (Hartung and Civerolo, 1989), and characterization of mitochondrial DNA from eukaryotes (Avise, 1986; Avise et al., 1987). Variation among Louisiana isolates was, on average, less than that between foreign and Louisiana isolates (Table 4), though certain foreign isolates were similar to or identical with Louisiana isolates. The VZ and MX isolates were somewhat distinct, but foreign isolates generally were interspersed with Louisiana isolates in the cluster analysis (Fig. 6). Thus, SfNPV variation is not dependent on geographic location alone. Some of the sequence divergence values between Louisiana isolates were as high as those between foreign and Louisiana isolates (Table 3). It is possible that the similarity between certain foreign and Louisiana SfNPV isolates could be due to viral transport by S. frugiperda moths; the moths migrate over long distances (Pair et al., 1986), and SfNPV can be vertically transmitted (J. R. Fuxa, A. R. Richter, and
OF
S. frugiperda
NPV
103
E. H. Weidner, unpublished data). In another study, variation in the genome of the Oryctes baculovirus was proportionate to the distance between isolates (Crawford et al., 1986). However, there was no relationship between geographic location and SlNPV variants (Cherry and Summers, 1985). There have been other studies of genetic variation in populations of baculoviruses, but such studies have been restricted to one level of variation (i.e., within a single host insect or among states or countries). Variation in SfNPV has been found within a single host (Knell and Summers, 198 1; Maruniak et al., 1984). Therefore, variation among viral isolates within a single locality is to be expected, though the comparative degree of variation at different levels has not been studied. There has been little other research of variation within small regions such as within fields or between fields on the same farm. Cherry and Summers (1985) found variation in SlNPV within regions of Israel. However, variation within a single field was not examined, and distances between isolates were not documented. Loh et al. (1982) distinguished single SfNPV isolates from each of four states in the United States. The possibility that two sibling species of S. frugiperda exist (Pashley, 1986, 1988) raises questions about SfNPV variation. Genetic differentiation in S. fvugiperda populations is associated with host plant usage: one S. fiugiperda strain prefers corn and sorghum and the other prefers rice and grasses. Due to the host specificity of NPVs (Fuxa, 1987), it would not be unexpected if SfNPV isolated from S. frzcgiperda on corn and bermuda grass would vary more between host plants than within fields. However, the results of this study indicate that variation in SfNPV isolates is not correlated with host plant species (Table 4). The viral isolates from S. frugiperdu in bermuda grass were not, as a group, distinct from those from S. frugiperdu in corn (Fig. 6).
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The causes of variation in the REN protiles are unknown. Variation in restriction enzyme patterns of NPVs may result from three mechanisms: insertions of host DNA into the viral genome, duplications of viral sequences, and point mutations (Brown et al., 1985). The BamHI fragments b and g in isolates OH, EC, LSI, and LCl (Fig. 3) may have resulted from an additional cutting site in fragment a. The presence of fragments a and g (rather than b and g) in isolates MX and LB2 (Fig. 3) may be explained by a duplication or by an insertion in fragment b. A duplication or insertion in fragment b would increase the number of base pairs to a genome size close to that of fragment a. The NPV genome can acquire portions of host DNA (Miller and Miller, 1982; Fraser et al., 1983). However, changes in restriction profiles resulting from host DNA pickup by NPV have been documented only after numerous passages of the virus through cell culture. Croizier et al. (1985) reported such a difference in REN profiles only after 25 passages of Mamestra brassicue NPV, and McIntosh and Ignoffo (1986) found a difference in REN profiles in Heliothis NPV after 20 passages. Smith and Summers (1978) found no difference in Autographa californica NPV isolates after sixteen serial passages. The acquisition of host DNA has not been demonstrated in vivo. Hence, the possibility that NPV acquired host DNA during the current study is not likely to have affected restriction enzyme patterns. Further studies of variation and groupings within baculovirus “species” will be fundamental to their ecology, to their development for microbial control, and perhaps to an understanding of their evolution. ACKNOWLEDGMENTS
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