1
Characterization of a Costa Rican granulovirus and adaptation to
2
Phthorimaea operculella and Tecia solanivora through serial passage
3
Y. Gómez-Bonillaa,b,c, M. López-Ferberd, P. Caballerob,e, X. Léryc, and D.
4
Muñozb*
5
6
7
a
Departamento de Producción Agraria, Universidad Pública de Navarra, 31006
8
Pamplona, Spain; bIRD (UR072), Centre de Recherche, av. Gén. de Gaulle,
9
30380, St Christol-les-Alès and Université Paris Sud 11, 91405, Orsay,
10
France; cInstituto Nacional de Investigación y Transferencia de Tecnología
11
Agropecuaria (INTA), San José, Costa Rica; dÉcole des Mines d´Alès, 6
12
Avenue de Clavières, 30319 Alès cedex, France; eInstituto de
13
Agrobiotecnología, CSIC-Gobierno de Navarra, 31192 Mutilva Baja, Spain
14
15
16
17
* Corresponding author at: Departamento de Producción Agraria, Universidad
18
Pública de Navarra, 31006 Pamplona, SPAIN. Tel.: +34 948 169716; fax: +34 948
19
169169
1
1
ABSTRACT
2
The most important potato pests in Costa Rica, Phthorimaea operculella (Zeller)
3
(Lepidoptera: Gellechiidae) and Tecia solanivora (Povolny) (Lepidoptera:
4
Gellechiidae), urgently require control methods alternative to chemicals. The
5
granulovirus (Baculoviridae) isolated from P. operculella (PhopGV) has already
6
been used successfully as a biological control agent. In this study, a novel
7
granulovirus strain from Costa Rica (PhopGV-CR1) has been isolated and
8
identified by restriction endonuclease analysis as a novel PhopGV strain. PCR
9
amplification of four specific variable genomic regions yielded multiple amplicons
10
for ORF84 and ORF129, revealing the presence of different genotypic variants
11
within the virus population. Biologically, PhopGV-CR1 was five fold more
12
pathogenic for P. operculella than for T. solanivora. P. operculella colonies from
13
Costa Rica and France were equally susceptible to PhopGV-CR1. Serial passage of
14
PhopGV-CR1 over four generations of T. solanivora increased its pathogenicity by
15
five fold in three generations, indicating a rapid adaptation to its alternate host.
16
17
Keywords: Phthorimaea operculella, Tecia solanivora, microbial control,
18
granulovirus, adaptation to host
19
20
2
1
1. Introduction
2
3
Two of the most important pests of potatoes are the lepidopterans Phthorimaea
4
operculella (Zeller) (Lepidoptera: Gellechiidae) and Tecia solanivora (Povolny)
5
(Lepidoptera: Gellechiidae). The mining larvae of both these species cause severe
6
damage, of up to 100% in some instances, to tubers in the field and in storage
7
(Niño, 2004; Raman et al., 1987; von Arx et al., 1987).. P. operculella is a
8
cosmopolitan species and the most damaging pest of potatoes in the tropical and
9
subtropical regions worldwide (Rondon, 2010). T. solanivora, originated from
10
Central America, has rapidly established in different Iberoamerican countries in the
11
last 20 years and has become a major potato pest in the Andean region (Pollet et
12
al., 2003). In 2000, this pest was reported to have invaded the Canary Islands
13
(CAB International, 2000). The control of P. operculella with chemical pesticides
14
during the last fifty years has caused the development of multiple resistances to
15
several organophosphorous and synthetic pyrethroids (Dogramaci and Tingey,
16
2008; Saour, 2008; Shelton et al., 1981; Symington, 2003). All this boosted the
17
development of alternative control methods for these pests that should also protect
18
their natural enemies and help maintain the ecological balance (Amonkar et al.,
19
1979).
20
Integrated Pest Management (IPM) programs for P. operculella and T.
21
solanivora have been developed and implemented in different countries (Zeddam
22
et al., 2008). They are based on a handful of similar practices (Das et al., 1992),
23
being the Phthorimaea operculella granulovirus (PhopGV) (Baculoviridae;
24
Betabaculovirus) their main component. Different geographical isolates of
3
1
PhopGV have proved their efficiency as biopesticides in rustic storage conditions
2
as well as during field outbreaks in countries such as Peru, Colombia, Bolivia,
3
Ecuador, Tunisia, Venezuela, India, Yemen, and Australia (Reed and Springett,
4
1971; Raman et al., 1987; Kroschel et al., 1996; Lagnaoui et al., 1996; Setiawati et
5
al., 1999). The different PhopGV isolates show distinct activity against alternate
6
hosts (Zeddam et al., 1994).
7
In spite of the existence of a wide array of efficient PhopGV strains, the search
8
for novel, indigenous isolates is always desirable since they are better adapted than
9
foreign ones to their natural environment (Cory et al., 2005). In addition, novel
10
strains may become excellent tools for managing insect resistance, as has occurred
11
with an Iranian strain of Cydia pomonella granulovirus (CpGV), highly infectious
12
to the codling moth populations from Europe that had developed resistance against
13
the traditionally commercialized CpGV strain from Mexico (Eberle et al., 2008).
14
Finally, novel strains with efficient insecticidal properties against a certain
15
complex of hosts may offer a better alternative to chemicals than those specialized
16
in a single host (Moura Mascarin, 2010; Zeddam et al., 2003). In Costa Rica, such
17
an isolate would be ideal to control simultaneously P. operculella and T.
18
solanivora, whose populations overlap spatial and temporally in Zarcero and
19
Cartago, the two Costarican regions where potato is mostly cultivated
20
(Anonymous, 2010).
21
Our aim in this work was to characterize novel PhopGV strains from Costa
22
Rica with efficient insecticidal activity against both P. operculella and T.
23
solanivora that will allow their inclusion in IPM programs in the short or medium
24
term.
4
1
2. Materials and Methods
2
3
2.1. Insect rearing
4
5
Two different P. operculella laboratory colonies were reared. One of them,
6
named P. operculella-FR, originated from an Egyptian population (kindly provided
7
Dr. Abol-Ela (Faculty of Agriculture, Cairo, Egypt) and maintained at the IRD
8
laboratory (St Christol-Les-Alés, France). The other colony, named P. operculella-
9
CR, originated from Costa Rica and was reared at the Research Center Carlos
10
Durán (Cartago, Costa Rica). Both colonies were maintained under constant
11
environmental conditions: 27ºC, 60% relative humidity and 16:8 light:dark
12
photoperiod. The colony of T. solanivora was established from a natural
13
population collected in Oreamuno (Cartago, Costa Rica) and reared under similar
14
environmental conditions.
15
16
Larvae were fed on potato tubers previously treated with chlorine solution.
17
Adults were fed with a 30% (p/v) solution of honey or sugar. Females laid their
18
eggs on filter papers which were collected every 24-48 hours, incubated in a
19
chamber at 27ºC until they darkened and then placed on potato tubers. Under these
20
conditions, an entire life cycle of both these insect species varied between four to
21
five weeks.
22
23
2.2. Viruses
24
5
1
The Costa Rican isolate (PhopGV-CR1) was isolated from field-collected
2
larval cadavers in 2006 and amplified on P. operculella-FR for two passages.
3
Infected larvae were homogenized in distilled water and this suspension was
4
spread onto potato surfaces to a concentration of approximately 100 to 500 larval
5
equivalents per liter. Between 15 and 20 neonate larvae were then placed on each
6
potato and these were incubated for 3 to 4 weeks at 27ºC, after which time the
7
infected larvae were collected and used as inoculum for another passage in larvae
8
using the same procedure. The other isolates used in this work were: i) PhopGV-
9
1346 from Tunisia (kindly provided by Dr. El Bedewi, IPC, Egypt, and multiplied
10
during several years in Egypt), ii) PhopGV-1390.9 from Kayra, Peru (kindly
11
provided by Dr. J. Cory, Oxford, United Kingdom) (Vickers et al., 1991), and iii)
12
PhopGV-4.2, a clone of the PhopGV 1346 isolate (Lery et al., 1998; Vickers et al.,
13
1991), whose genome has been completely sequenced (GeneBank NC004062)
14
(INRA/CNRS/Université de Montpellier II, Saint Christol les Ales, France). All
15
three reference isolates were amplified in 100 P. operculella-FR neonates as
16
described above.
17
To purify occlusion bodies (OBs), ca. 50 larval cadavers were collected and
18
homogenized in 10 ml 0.01 M Tris-HCl pH 7.5 using a Potter-Elvehjem
19
homogenizer (USA) and centrifuged at 664 g for 5 min. at 4 ºC. Supernatants were
20
centrifuged at 20,000 g for 20 min., pellets resuspended in 1 ml bidistilled H2O and
21
then placed on a continuous 30%-70% (w/v) sucrose gradient and centrifuged al
22
20,000 g for 20 min. OBs were collected with a Pasteur pipette, resuspended in 1
23
ml 1x TE buffer (0,1 M Tris-HCl, pH 7.5, and 10mM EDTA, pH 8.0), centrifuged
24
at 20,000 g for 20 min. and stored at -20 ºC. OB concentration was determined
6
1
with a spectrophotometer and calculated using the following formula: 6.8 x 108 x
2
OD450 x dilution = Number of granules/ml (Zeddam et al., 2003).
3
4
2.3. DNA Extraction and Restriction endonuclease analysis
5
Purified OB suspensions were incubated with 25 μl vol. of 2M Na2CO3 for 5
6
min and DNA was extracted using a phenol/chloroform/isoamyl alcohol protocol
7
and then precipitated with ethanol, as described in previous works (Muñoz et al.,
8
1998).
9
Between 0.25 to 0.5 μg of viral DNA were incubated with ten units of each of
10
these restriction enzymes: Sma I, BamHI, HindIII, NruI, MluI, HpaI, NsiI, NdeI,
11
DraIII, BstEII, BstApI (Promega, Charbonnières-les-Bains, France) at the
12
conditions specified by the supplier, according to the results obtained previously
13
(Lery et al., 1998; Vickers et al., 1991). After addition of loading buffer (0.25%
14
bromophenol blue, 40% w/v sucrose in water), samples were loaded in 1% agarose
15
gels with TAE buffer (40 mM Tris-acetate, 1mM EDTA, pH 8.0) and subjected to
16
electrophoresis at 80V. Ethidium bromide stained gels were then photographed on
17
a UV transilluminator. The REN fragment molecular weights were determined by
18
comparison with the corresponding sequenced PhopGV-4.2 fragments and the
19
lambda DNA marker fragments. Marker fragments for the PhopGV-CR1 isolate
20
were labeled using the same letter as the closest larger fragment of PhopGV-1346
21
in lower case, and with a sub index in instances when different sized marker
22
fragments shared the same letter.
23
24
2.4. Analysis of DNA by PCR
7
1
2
Previous RFLP analysis on the genetic diversity of PhopGV isolates
3
originating from various countries allowed to determine four variable regions
4
(affecting ORFs 46, 84, 109 and 129) (Léry et al., 2005). A fifth region (affecting
5
ORFs 90 and 91) was recently found in isolates coming from Colombia (Léry et
6
al., 2008). Primers encompassing these variable regions were designed using the
7
complete PhopGV sequence (NC004062). In addition, primers for lef-4 and
8
granulin genes were included as controls.
9
PCR reactions were carried out in a total volume of 25µl, containing 10 to 100
10
ng of DNA, 1 pmol of each primer, 3.5 mM MgCl2 and 0.5 vol. of a mixture
11
prepared by the supplier containing dNTP´s and Taq polymerase (Promega,
12
Charbonnières-les-Bains, France). Amplifications were carried out in a
13
thermocycler under the following conditions: a first cycle of 94ºC/4 min, continued
14
by 30 cycles of 94ºC/1min, 50ºC/1 min. and 72ºC/1 min., and a final cycle of
15
72ºC/5 min. The PCR products (amplicons) were electrophoresed in 2% agarose
16
gels at 150 volts. The PhopGV-1346 strain was used in all PCR reactions as a
17
reference.
18
19
2.5. Bioassays
20
21
22
The bioassays to determine the mean lethal concentration (LC50) were carried
out with neonate larvae (L1) as described by Espinel-Correal et al. (2010).
23
To assess the LC50 (an expression of the pathogenicity) of PhopGV-CR1 in P.
24
operculella-FR, and P. operculella-CR, and in T. solanivora, the viral inoculum
8
1
was previously amplified in each host colony. Six different viral concentrations
2
were prepared (1 x 105 to 1 x 1010 OBs/ml in 2 ml of water) and applied
3
homogeneously on the surface of the potato tuber using a nebulizer (Carrera et al.,
4
2008). Two tubers of ca. 5 cm diameter were used for each concentration and ten
5
neonate larvae were placed on each. For each concentration, three to five replicates
6
were arranged. The final concentrations applied on the potato surface were 0.1, 1,
7
10, 100, 1000 and 10,000 OBs/mm2. Since most larvae leave the tubers before
8
dying, the numbers of dead and infected larvae were recorded daily for three
9
weeks. At the end of the experiment tubers were opened to register the eventual
10
dead and infected larvae remaining inside the galleries. Mortality results were
11
subjected to probit analysis (Finney, 1971) using the POLO-PC program (LeOra
12
Software, 2002).
13
14
2.6. Successive passages of OBs in vivo
15
16
OBs obtained from all P. operculella larvae in the first bioassay described
17
above were pooled, amplified separately in P. operculella-CR and T. solanivora
18
and designated passage zero. These OBs were used as inocula to infect five groups
19
of 60 larvae. Dead larvae were pooled to obtain enough inoculum (designated
20
passage I) for the next subsequent passage (II), and the same procedure was
21
followed for two further passages (III, and IV).
22
To assess the pathogenicity of PhopGV-CR1 throughout four generations of
23
larvae, a bioassay identical to that described above was performed with five
24
different viral concentrations: 5 x 105, 5 x 106, 5 x 107, 5 x 108, and 5 x 109
9
1
OBs/ml. PhopGV-1346 was used as a reference. This bioassay was replicated three
2
times.
3
4
3. Results
5
6
3.1. Molecular characterization of PhopGV-CR1
7
8
PhopGV-CR1 genomic profiles obtained with 10 different restriction
9
endonucleases (RENs) and by PCR with four different sets of primers were
10
compared to those of PhopGV-1346, PhopGV-4.2 and PhopGV-1390.9.
11
REN profiles produced with NdeI were unique for the three isolates compared,
12
with five restriction fragment length polymorphisms (RFLP), allowing profile
13
discrimination (Table 2). With BamHI and BstEII, PhopGV-CR1 showed a novel
14
RFLP that distinguished this isolate from PhopGV-4.2 and PhopGV-1390.9, which
15
were identical. HpaI, MluI and NsiI did not differentiate PhopGV-CR1 from
16
PhopGV-4.2 but they were useful enzymes to tell apart PhopGV-4.2 from
17
PhopGV-1390.9. With NruI, PhopGV-CR1 and PhopGV-1390.9 showed identical
18
profiles but they differed by one fragment from that of PhopGV-4.2 (Table 2).
19
Finally, no polymorphisms were observed between REN profiles produced by
20
SmaI, HindIII, or DraIII . Submolar fragments were observed in both field isolates,
21
PhopGV-CR1 and PhopGV-1390.9, with several enzymes (Table 2).
22
By PCR, only the set of primers encompassing ORF-84 generated a PhopGV-
23
CR1 profile which differed from those of the reference strains, PhopGV-1346 and
24
PhopGV-1390.9 (Table 3). Using the set of primers for ORF129, PhopGV-CR1
10
1
appears similar to PhopGV-1390.9, both differing from PhopGV-1346 by the
2
presence of amplicons of 1023 and 869 bp and the absence of one of 723 bp (Table
3
3)
4
A single amplicon was amplified with the sets of primers ORF1, ORF83,
5
ORF87 and ORF109, with identical size for the three isolates analyzed. These
6
amplicons were not further analysed.
7
8
3.2. Pathogenicity of PhopGV-CR1
9
10
The dose-mortality responses of PhopGV-CR1 for the two P. operculella
11
populations and for T. solanivora were fitted with a common slope; the interaction
12
between host populations and log e [virus dose] was not significant (2= 3.82; df=
13
2; P= 0.148).
14
The pathogenicity (expressed as LC50) of PhopGV-CR1 for the two P.
15
operculella biotypes is not significantly different, indicating that this PhopGV
16
strain is equally pathogenic against biotypes of its homologous host as distant as
17
those from Costa Rica and Egypt (Table 4). However, the pathogenicity of
18
PhopGV-CR1 for T. solanivora was four fold lower than for P. operculella from
19
France, as indicated by the 95% fiducial limits of the relative potency, which did
20
not encompass number one (Robertson and Preisler, 1992).
21
11
1
3.3. Pathogenicity of PhopGV-CR1 upon serial passage in P. operculella and T.
2
solanivora
3
The pathogenicity of PhopGV-CR1 was significantly enhanced at the second
4
and third passages in P. operculella (by six fold) and T. solanivora, (by five fold),
5
respectively, as indicated by the potency 95% fiducial limits, which were above
6
one (Robertson and Preisler, 1992). LD50 values did not vary thereafter upon serial
7
passage (Table 5), indicating that the virus got quickly adapted to its host species.
8
What is more, the final pathogenicity of the viruses, once adapted to one or the
9
other host, is not statistically different, and this for each of the virus isolates tested
10
(Table 5). The pathogenicity of PhopGV-1346 was also increased, by 7.5 and 13.1
11
fold, at the second passage in P.operculella and T. solanivora, respectively (Table
12
5).
13
14
4. Discussion
15
16
The present study describes the molecular and biological characterization of a
17
granulovirus isolated from P. operculella in Costa Rica and named PhopGV-CR1.
18
REN analysis showed a high similarity of PhopGV-CR1 to the reference strains,
19
revealing that the novel Costa Rican isolate is a geographical strain of this viral
20
species, namely PhopGV. Different studies have shown that variations observed
21
among restriction patterns of PhopGV isolates collected in very distant locations,
22
(Australia, Peru, Yemen, Indonesia) are limited, although at least several of these
23
virus populations have probably been isolated for quite long periods. The reason
12
1
for such a strong conservation of restriction sites in viral sequences is unclear but
2
this fact is not unique among GVs (Zeddam et al., 1999).
3
The presence of submolar fragments in several REN profiles (Table 2) and the
4
existence of multiple fragment amplification with the same set of primers (Table 5)
5
strongly suggest the presence of different genotypes within PhopGV-CR1.
6
Genotypic heterogeneity within the same virus strain has also been observed for
7
other PhopGV isolates (Lery et al., 1998), is very common among NPV and GV
8
populations (Burden et al., 2006; Smith and Crook, 1988; Caballero et al., 1992;
9
Figueiredo et al., 2009) and denotes the existence of a functional diversity among
10
the different genotypes. Indeed, purified genotypes with significantly different
11
pathogenicity, virulence, or OB yield to that of the wild-type mixture have been
12
described (Hodgson et al., 2001; Muñoz et al., 2000; Simón et al., 2008). This
13
constitutes an evidence of the importance of genetic diversity in the capacity of a
14
pathogen to adapt to its environment, in particular to its hosts.
15
The adaptation effect of a virus isolate to different host colonies was tested
16
using the same methodology. The LC50 of PhopGV-CR1 was determined in two
17
populations of P. operculella from France and Costa Rica and also in a Costa
18
Rican colony of the alternate host, T. solanivora. The pathogenicity of PhopGV-
19
CR1 in its homologous host is similar to that obtained with the reference strain,
20
PhopGV-1346, its purified clone PhopGV-4.2, and two isolates from Colombia
21
and Peru, and lower than that of two other Colombian isolates (Espinel-Correal et
22
al., 2010). Comparison of these values with those of strains from Yemen (Kroschel
13
1
et al., 1996) or Indonesia (Zeddam et al., 1999) is difficult because of the different
2
methodologies employed for the bioassays.
3
The pathogenicity of PhopGV-CR1 was four times lower for its heterologous
4
host, T. solanivora. Similarly, for Colombian T. solanivora, a non adapted virus
5
strain is less efficient (Espinel-Correal et al., 2010). Larvae of T. solanivora have
6
been found infected by baculoviruses naturally (Niño, 2004; Villamizar et al.,
7
2005; Zeddam et al., 2003). The viruses they contain appeared to be related to
8
PhopGV. However, the process of adaptation was not explored, and the efficacy of
9
these isolates in the control of potato tuber moths was not indicated.
10
GV strains with good insecticidal performance are desirable for an efficient
11
control of both pests. This may be achieved for example, by thoroughly screening
12
isolates from different geographical regions. Indeed, three Colombian PhopGV
13
strains recently isolated by Espinel-Correal et al. (2010), have been found up to 50
14
fold more pathogenic than the Costa Rican or the Peruvian PhopGV strains,
15
showing LC50 values as low as 1.16 OBs/mm2. These highly pathogenic strains
16
were isolated from T. solanivora in regions where both hosts coexist; whereas the
17
Peruvian strain had not had previous contact with this host. T. solanivora origin
18
has been tracked back to Central America. It is thus likely that virus populations
19
infecting potato tuber moths in these regions, like PhopGV-CR1, had previously
20
been in contact with both host species, and retain the ability for quick adaptation to
21
their hosts. Increasing pathogenicity of the virus adaptation to its host has been
22
observed to occur upon serial passage of genotypically heterogeneous virus
23
populations (Berling et al. 2009; Kolodny-Hirsch & Van Beek, 1997). Both the
14
1
known genome plasticity of the baculoviruses and the genotypic heterogeneity of
2
baculovirus populations play a role in this adaptation. Given the genotypic
3
heterogeneity of PhopGV-CR1, it was passaged serially for four host generations
4
being its pathogenicity significantly increased, reaching LC50s as low as 3.0
5
OBs/mm2 for P. operculella and 5.4 OBs/mm2 for T. solanivora. These values are
6
strikingly similar to some of the field-adapted Colombian PhopGV strains
7
(Espinal-Correal et al., 2010). Likewise, fifteen-fold increased pathogenicity was
8
registered for AcMNPV against Plutella xylostella when passaged 20 times in this
9
host (Kolodny-Hirsch & Van Beek, 1997). More recently, an analogous
10
observation was made on the CpGV NPP-R1 strain that was passaged four times in
11
a colony of C. pomonella resistant to a commercialized CpGV strain from Mexico
12
(Berling et al. 2009). In this case, genotype selection accounted, at least partially,
13
for host adaptation, since the proportion of a genotype similar to the one dominant
14
in the Mexican CpGV strain was sharply reduced in this strain after only four serial
15
passages (Berling et al. 2009). It is likely that the adaptation of PhopGV-CR1 to T.
16
solanivora can also be explained by genotype selection, but molecular
17
characterization of the genotypes composing the PhopGV-CR1 strain from the first
18
and fourth serial passage in P. operculella and T. solanivora are needed to confirm
19
this hypothesis. Surprisingly, the reference strain, PhopGV-1346 also got quickly
20
adapted upon contact with the Costa Rican populations of both the original and the
21
heterologous hosts. This constitutes a further evidence of the ability of this virus to
22
get adapted to its hosts. In addition, the fact that PhopGV-1346 shows LC50 values
23
similar to those of the T. solanivora field-adapted Colombian strains seems to
24
invalidate a suggested link between pathogenicity and a 86 bp genomic insertion in
15
1
the egt gene (observed as a 1023 bp fragment by PCR). This insertion occurs in the
2
Colombian isolates and also in PhopGV-CR1, which led to this genotype-
3
phenotype hypothesis, but is absent in the PhopGV-1346 strain.
4
In conclusion, PhopGV-CR1 has great potential for the control of both P.
5
operculella and T. solanivora, in particular after passage in the same host species
6
to control.
7
8
Acknowledgements
9
Yannery Gómez-Bonilla was the recipient of scholarships from the Spanish
10
National Institute of Agricultural Research (INIA) and the Costa Rican Ministry of
11
Science and Technology (CONICIT-MICIT).
12
13
16
1
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papa
Phthorimaea
operculella
24
22
y Tecia
solanivora
(Lepidoptera:
1
2
Table 1: Forward and reverse primers used for PCR amplification of the different
PhopGV isolates.
3
Primers
Name
Sequence
Forward: 83-1
ATGTAGACGCGTCGTTAACCTGGGTGTA
Reverse: 83-2
ATGAACTGTTAAACGGCTTGAGTGAGCG
Forward: 84-1bis
CCGCGCCGATTACCAACAGCAGCACTAT
Reverse: 84-2
CCTTGTAGCGTAACACTGTTTGTGTCTC
Forward: 109-1
CGGTAGACGTGTAGATAATGTGCGCGTT
Reverse: 109-2
TCATCAATGATACCATAAGGCCCGGGCG
Forward: 129-1
GCGATGATGAGAATGGGAATGTGAAGAC
Reverse: 129-2
TGCCTGCTGTGCTCGACAACAATAGACC
Forward: Po 1
GAGATTAGACGAGTTCATCCAGAC
Reverse: Po2
TTGTTGTCGCTTTGGAGCTAGTAC
Forward: Po 5
CTGTCAGGACGTTCTTTGATTACT
Reverse: Po6
CTGCTATACGCGTACATGTCACCA
4
5
6
23
AT
PhopGV
CpGV
(°C)
ORF
ORF
63
83
ODV 25
63
84
ORF 92
64
109
Lef 9
64
129
EGT
70
1
Granuline
68
ORF 87
Lef 4
1
Table 2. Polymorphic fragments present in the REN profiles of PhopGV-1390.9
2
and PhopGV-CR1 with the endonucleases BamHI, NruI, MluI, HpaI, NsiI,
3
NdeI, and BstEII with respect to the cloned genotype PhopGV-4.2. Their
4
approximate molecular weight (in bp) is indicated in brackets.
5
REN
PhopGV-4.2
PhopGV-1390.9
PhopGV-CR1
I (6114)
+
+*
-
-
i1 (ca. 6000)
-
g1* (ca. 5000)
-
-
m1* (ca. 3400)
-
-
-
c1 (ca. 14500)
G (6746)
-
-
-
-
g1 (ca. 6500)
I (5994)
-
+
-
-
j1 (ca. 5800)*
BstEII
-
-
g1 (6400)
MluI
-
l1 (3500)
-
I (6104)
-
-
-
h1 (6300)
+
-
i1 (2600)
-
BamHI
HpaI
NdeI
NruI
NsiI
6
a
+/-: presence/absence of fragment; *: submolar fragment
24
1
2
Table 3. PCR amplicon sizes (bp) of genomic regions ORF129 and ORF84
amplified for PhopGV-1346, PhopGV-1390.9, and PhopGV-CR1.
ORF of region amplified
PhopGV-
PhopGV-
PhopGV-
(gene function)
1346
1390.9
CR1
-
1023
+
937
+
+
-
869
+
723
-
-
-
-
330
241
+
+
ORF129 (egt)
ORF84
3
4
5
25
1
2
Table 4. LC50 values of PhopGV-CR1 obtained from two colonies of P.
operculella and the Costa Rican population of T. solanivora.
3
Fiducial
LC50
Insect population
Relative
limits 95%
Regression line
(OBs/mm2)
potency
lower upper
P. operculella-FR
0.48x + 4.54
17.0
1
-
-
P. operculella-CR
0.48x + 4.53
17.9
0.95
0.42
2.15
T. solanivora
0.48x + 4.24
69.1
0.25
0.10
0.59
4
5
6
26
1
Table 5. LC50 values of PhopGV-1346 and PhopGV-CR1 obtained from P.
2
operculella and T. solanivora upon serial passage.
3
Fiducial limits
Virus
Host
Po
Regression
LC50
line
(OBs/mm2)
Passage
95%
Potency
lower
upper
I
0.56x+4.26
21.1
1
-
-
II
0.56x+4.35
2.8
7.54
2.54
22.58
III
0.56x+4.58
3.0
7.09
2.39
21.58
I
0.48x+4.18
47.7
1
-
-
II
0.48x+4.73
3.6
13.09
4.75
38.20
III
0.48x+5.85
2.0
23.38
5.32
110.69
I
0.45x+4.42
18.8
1
-
-
II
0.45x+4.78
3.0
6.28
2.43
16.94
III
0.45x+4.75
3.6
5.19
1.86
15.15
IV
0.45x+4.74
3.7
5.13
1.55
17.87
I
0.50x+4.27
28.9
1
-
-
II
0.50x+4.41
14.6
1.97
0.79
4.98
III
0.50x+4.63
5.4
5.35
2.00
14.71
IV
0.50x+4.65
4.9
5.91
2.22
16.29
1346
Ts
Po
CR1
Ts
4
5
27