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Perbanyakan Massal Virus sebagai Agens
Antagonis
Oleh Irda Safni
Kuliah ke-11
Virus sebagai agens antagonis
patogen tumbuhan
Ada beberapa cara virus dapat digunakan untuk mengendalikan patogen tumbuhan, yaitu :
1. Virus dapat membunuh atau mengurangi patogenisitas bakteri patogen dan jamur patogen tumbuhan.
2. Virus dapat membunuh vektor invertebrata virus pada tanaman tingkat tinggi sehingga mencegah penyebarannya.
3. Strain yang agak lemah yang menyebabkan sedikit atau tidak menimbulkan dapat melindungi tanaman dari infeksi laten oleh strain yang lebih parah.
• Stanway (1985) mendeteksi infeksi virus sebanyak 126 dari 157 isolat
jamur take-all pada tanaman gandum (Gaeumannomyces graminis
var. tritici).
• Kultivar gandum yang terinfeksi Helminthosporium victoriae (penyakit
hawar) adalah penyakit penting gandum di Amerika pada 1947
dan1948.
• Lindberg (1959) menemukan beberapa koloni jamur H. victoriae
menjadi memendek, ditandai dengan daerah pada pinggir koloni,
miselium udara menjadi lisis dan menghambat perkembangan koloni.
• Penyakit ini juga ditransmisikan ke kultur yang sehat oleh anastomosis
hifa dan mungkin disebabkan oleh 1 atau 2 dsRNA virus yang biasa
dijumpai pada H. victoriae (Ghabrial 1986).
• Tanaman gandum yang dijumpai isolat jamur tidak menyebabkan
kehilangan hasil yang besar, kemungkinan disebabkan penyebaran
virus pada populasi H. victoriae, karena isolat menghasilkan sedikit
toksin victorin dan menjadi kurang patogenik dibanding isolat yang
normal (Lindberg 1960).
• Isolat kultur Rhizoctonia solani mengandung 3 segmen virus
dsRNA, sedangkan tidak dijumpai virus dsRNA pada ujung hifa
yang sehat dari isolat yang mengifeksi.
Virus sebagai entomopatogen
Penyakit yang disebabkan virus entomopatogen mulai
diketahui sejak abad ke-16.
Penyakit yang disebut Jaundice o graserrie, sekarang
diidentifikasi sebagai nucleopolyhedrosis, ditemukan pada
ulat sutra (Bombyx mori) oleh Vida pada tahun 1524 dan
kemudian juga diisolasi dari lebah madu (Apis mellifera).
Pada tahun 1856, dua orang ahli Italia (Maestri dan
Cornalia) menjelaskan occlusion bodies (OBs) ulat sutra
nucleopolyhedrosis.
Pada tahun 1926 Paillot mendeskripsikan Granulovirus
(GVs) pertama sekali.
Pada tahun 1934 Ishimori menjelaskan jenis baru
polyhedrosis di dalam ulat sutra OBs dibentuk didalam
sitoplasma sel yang diinfeksi (bukan pada asam nukleat)
sekarang dikenal dengan Cypovirus.
Sejak tahun 1950 s/d 1970, Steinhaus dan koleganya menguji
baculovirus sebagai agens hayati di lapangan dengan
mengaplikasi nucleopolyhedrovirus (NPV) untuk mengendalikan
ulat alfalfa (Colias eurytheme Boisduval; Lepidoptera: Pieridae).
Bioinsektisida komersil berbahan aktif virus pertama
dikembangkan pertama sekali pada tahun 1975 oleh
Perusahaan Sandoz (dengan nama dagang Elcar) untuk
mengendalikan Heliothis/Helicoverpa Lepidoptera: Noctuidae).
Selama tahun 1979 s/d 1980, penemuan penting pada
genetika virus entomopatogen, khususnya baculovirus.
Hingga saat ini studi genetika virus entomopatogen difokuskan
pada studi genom lengkap telah ada 29 sekuensing genom
lengkap virus entomopatogen.
Keuntungan dan Kerugian Penggunaan Virus untuk
mengendalikan hama
Keuntungan
1. Selektif dan efektif terhadap hama sasaran.
2. Aman bagi serangga dan organisme bukan sasaran serta tidak menyebabkan
resistensi.
3. Persisten dan tidak meninggalkan residu beracun di alam.
4. Tidak menyebabkan pencemaran lingkungan.
5. Efektif menginfeksi ulat yang telah terkena insektisida kimia.
6. Tidak menyebabkan peningkatan populasi hama sekunder.
7. Dapat ditularkan oleh parasitoid dan predator ke inang yang sehat.
8. Dapat mengendalikan ulat instar V-VI.
9. Tidak menyebabkan penyakit virus pada tanaman.
10.Kompatibel dengan teknik pengendalian yang lain, termasuk insektisida kimia.
11.Mudah diproduksi dengan teknik sederhana (menggunakan alat semprot
standar).
12.Berpotensi sebagai pengendali hama jangka panjang.
13.Dapat beradaptasi dengan teknologi modifikasi secara genetik (bioteknologi).
Kerugian
1. Hanya spesifik terhadap hama sasaran.
2. Kemungkinan berbahaya bagi serangga bukan sasaran.
3. Waktu aplikasi harus tepat untuk memaksimalkan efektivitas.
4. Memerlukan pendistribusian secara merata pada kanopi tanaman
untuk meningkatkan kontak dengan hama sasaran.
5. Daya bunuh lambat.
6. Rentan terhadap pengaruh lingkungan.
7. Kehilangan virulensi dan patogenitas jika diperbanyak secara terus
menerus (jika tidak dilakukan penggantian inang baru).
8. Infektivitasnya di lapangan singkat dan membutuhkan penanganan
tertentu.
9. Kekhawatiran masyarakat terhadap kemungkinan
patogenik/menyebabkan alergi.
10. Menurunkan dosis perlakuan melalui bioteknologi menyebabkan
resurgensi hama atau serangga bukan sasaran.
Saran untuk aplikasi virus entomopatogen
Virus tidak dapat diaplikasikan sendiri, tetapi kerkonjugasi dengan teknik
pengendalian yang lain.
Virus entomopatogen bersifat spesifik, sehingga serangga target farus
diidentifikasi secara benar.
Lahan dicangkul terlebih dahulu sebelum aplikasi dilakukan, dan virus
diaplikasikan pada serangga target sewaktu masih mudah tetapi aktif makan.
Virus yang Menginfeksi Invertebrata
Virus DNA
Double stranded DNA
- Poxviridae
- Iridoviridae
- Baculoviridae: NPV & GV
- Polydnaviridae
Single stranded DNA
- Parvoviridae
Virus RNA
Double stranded RNA
- Rheoviridae: Cypovirus
Single stranded RNA (-)- Rhabdoviridae
- Bunyaviridae
Single stranded RNA (+)
- Picornaviridae, Togaviridae,
Tetraviridae, Flaviridiae,
Nodaviridae
FAMILY NUCLEIC
ACID
NUCLEOCAPSID
SIMETRY
OCCLUSION
BODY
Baculoviridae dsDNA Baciliform +
Reoviridae dsRNA Isometric +
Poxviridae dsDNA Ovoid +
Iridoviridae dsDNA Icosahedral -
Parvoviridae ssDNA Isometric -
Picornaviridae ssRNA Spherical -
Ascoviridae dsDNA Allantoid -
Polydnaviridae dsDNA Ovoid -
Rhabdoviridae ssRNA Baciliform -
Nodaviridae ssRNA Icosahedral -
Rhabdoviridae ssRNA Baciliform -
NON-CLASSIFIED RNA VIRUSESs
Divided genome ssRNA Isometric -
Nodaurelia ssRNA Isometric -
Kelply group ssRNA Isometric -
5-virus group ssRNA Isometric -
Minivirus ssRNA Isometric -
Ovoid virases ssRNA Ovoid -
Drosophila X Virus dsRNA Isometric -
Tabel 1. Kelompok Virus Entomopatogen
ds= double-stranded, ss= single-stranded.
6
2 microns
Baculoviruses
Spodoptera littoralis
From Hunter-Fujita et al
7
Baculoviruses -
Mode of action
From Hunter-Fujita et al
8
Found only in invertebrates
No member of the family is known to infect plant or
vertebrate
Most have narrow host insect range, and infectivity is
restricted to the original host genus or family
Baculoviruses
Susceptibility of Alternative Hosts
9
Baculoviruses
Toxicity studies - mammals
Toxicity test results from 1970s/80s of 29 NPVs
indicated no toxicity or pathogenicity. Doses were
generally 10 – 100 x the “per acre” (1 acre = 0.45ha)
field rate equated to a 70kg person.
Heliothis zea NPV most extensively tested for toxicity in
humans and led to registration of “Elcar” by Sandoz in
USA.
10
Baculoviruses
Toxicity studies – mammals cont.
No effects of HzNPV found in:
Acute toxicity-pathogenicity tests in mouse, rat, guinea pig, rabbit,
monkey and man at 6x109 – 3 x 1012 OB / kg.
Skin irritation sensitivity tests in guinea pigs, rabbits and man at 106
and 107 OB / mm2skin.
Eye irritation tests in rabbits with 105 and 2x106 OB / eye
Subacute toxicity-pathogenicity tests and subcutaneous injection into
mice, rats, dogs and rhesus monkeys.
Teratogenicity and carcenogenicity studies in rats and mice at 109 –
3.5x1012 OB / kg.
Similar but less extensive results for many other NPVs from the
1970s/80s
11
BaculovirusesToxicity studies – wildlife
Birds
Able to pass NPV through the alimentary tract unaffected
No deleterious effects
Aquatic organismsNo adverse effects
Beneficial insects
No direct effect on parasitoids, predators and pollinators
Indirect effects on parasitoids resulting from host death
12
Toxicity tests designed for testing effects of chemicals on
vertebrates are insufficient
Results reported in Gröner (1986) indicate no virus induced
antibody production in test mammals and chicken.
No cytogenetic effects of baculoviruses in mammalian cells either
in vivo or in vitro.
BaculovirusesPathology studies
13
AcNPV inoculated into vertebrate cells can be taken-
up and the degree of up-take depends on cell type,
temperature, time and viral phenotype.
BUT, none of the human and nonhuman vertebrate
lines tested showed evidence of viral replication.
NPVs unable to activate retroviruses in mammalian
cell lines
BaculovirusesVirus-cell interactions in vitro
15
Baculoviruses
a list of the baculoviruses regulated as pesticideactive ingredients by the US EPA Office of PesticidePrograms as of May 2005
Anagrapha falcifera NPV
Cydia pomonella GV
Douglas fir tussock moth NPV
Gypsy moth NPV
Helicoverpa zea NPV
Indian meal moth GV
Mamestra configurata NPV (pending)
Spodoptera exigua NPV
16
Baculoviruses – US EPA fact sheet
III. ASSESSING RISKS TO HUMAN HEALTH
These viruses infect only the target insect larvae and closely related
species. Toxicity tests show that the viruses pose no risk to the public.
Workers wear protective clothing to prevent possible irritation from
handling and applying the product.
IV. ASSESSING RISKS TO THE ENVIRONMENT
Tests show that the GV and NPVs that EPA has registered as pesticide
active ingredients specifically infect only certain species of moth larvae.
The viruses do not harm other organisms, including plants, beneficial
insects, other wildlife, or the environment. These viruses occur naturally
in their insect hosts.
17
Cypoviruses: Mode of action
Polyhedra ingested and dissolved in larval midgut
Virions released and attach to midgut columnar cells
Viral core enters cell cytoplasm
RNA transcription and replication
RNA occluded in capsules
Virus capsules occluded by virogenic stroma to form occlusion
bodies
18
Cypoviruses (Rheoviridae)
No CPV has been found infecting vertebrates or plants
(Belloncik, 1989)
Dendrolimus spectabilis CPV registered in Japan in
1974. Safety test results generally negative.
– Katagiri, K. (1981) Pest control by cytoplasmic polyhedrosos viruses. In:
Microbial control of pests and plant diseases 1970-1980. (Ed Burges, H.D.)
Academic Press.
Poxviridae:Entomopoxvirus
Member of the family of Poxviridae has a wide host,
including vertebrates and invertebrates.
Chicken pox and Small pox virus belong to this family.
The show allantoid – to brick-shaped virions, occluded within
ovoid OBs called Spheroids.
Entomopoxvirus has been isolated from 27 orthopterans,
lepidopterans, dipterans and coleopterans.
The subfamily Poxvirinae includes three genera, i.e.
Entomopoxvirus A, Entomopoxvirus B, and Entomopoxvirus C
Entomopoxvirus A infects only coleopteran species;
Entomopoxvirus B infects lepidopteran and coleopteran
species; Entomopoxvirus C infects only dipteran species.
The fourth group, group D, has been proposed by ICTV
which only attacks hymenopterans.
Poxviridae:Entomopoxvirus
Anomala cuprea (Coleoptera) larvae infected with an
entomopoxvirus show the symptoms of the infection
such as a whitish appearance and underdevelopment
(left, infected larva; right, healthy one).
Ascoviridae: Ascovirus
Members of the family of Ascoviridae are double strande
DNA (dsDNA) viruses that infect lepidopteran insects and
cause the unique pathology of forming virion containing
vesicles in the hemolymph of infected hosts.
The presence of the vesicles gives the hemolymph
a milky white appearance, which is a major characteristic
of the disease.
A few species of Ascovirus has been isolated only from
insects, specifically from Lepidopterans (Noctuidae).
Enveloped virions of ascoviruses are bacilliform, ovoid or
allantoid in shape, and occluded within vesicle-like OBs.
An ascovirus-infected caterpillar
Ascovirus symptoms
In cases where a Microplitis wasp has both parasitised a caterpillar and
infected it with ascovirus, the symptoms seen are those of the disease rather
than of the parasitoid. When ascovirus kills the caterpillar, it also kills the
developing Microplitis larva.
Caterpillars infected with ascovirus will generally stop eating within two
days. They stop growing, but can live for weeks in a lethargic state before
they die.
The blood of an ascovirus-infected caterpillar is white and creamy,
whereas the blood of a healthy caterpillar is clear. Blood colour gives the
best diagnosis in the laboratory and can be tested by splitting or pricking the
caterpillar.
Ascoviridae: Ascovirus
Iridoviridae: Iridovirus
Invertebrate Iridescent Viruses (IIVs) (family Iridoviridae)
are known to infect a number of agricultural pests,
medically important insect vectors, and terrestrial isopods that
live in damp or aquatic habitats.
The major characteristic of this family is the presence of
iridescent blue, green, orange, or purple coloration in heavily
infected individuals.
The small iridovirus tend to display colors from violet to
turquoise.
The viral structure is non-enveloped, non-occluded,
isocahedral viral particles.
Iridoviridaeviridae: Iridovirus
Although some iridoviruses infect frogs and fishes, those
infecting insects belong to two genera: Iridovirus, whose viral
particles fluctuate between 120 to 130 nm in size.
They mostly infect arthropods, particularly insects, in damp
or aquatic habitats worldwide (see complete list of
invertebrate hosts.
They are highly infectious by injection but have low
infectivity by ingestion.
Horizontal transmission can occur by cannibalism or
predation of patently infected individuals, or the virus may
even be vectored by nematodes and parasitoid wasps that
introduce viral particles into the host insect during the act of
penetration or oviposition.
Iridoviridaeviridae: Iridovirus
Iridovirus infected (blue) larva of Aedes aegypti
next to a healthy larva.
Polydnaviridae
Polydnaviridae only infects endoparasitic Hymenoptera.
Member of this family show non-occluded, ovoid virions,
containing multipartite dsDNA
ICTV recognizes two genera within this family, including
Ichneovirus and Bracovirus.
Life cycle of of parasitoid wasps and Polydnaviruses (PDVs) parasitizing a
lepidopteran larval host
Perbanyakan Virus
A. Perbanyakan in vivo
Walaupun produksi skala besar virus di dalam serangga hidup
membutuhkan tenaga kerja yang banyak, perbanyakan secara in vivo
masih layak digunakan.
Bagi laboratorium skala kecil, memberi makan virus diikuti dengan
memanen serangga terinfeksi adalah standar produksi stok virus.
Perbanyakan virus di dalam kultur sel akan beresiko kehilangan
kekhasan genetik pada sistem in vitro .
1. Propagation of Cydia pomonella granulovirus
(CpGV) in C. pomonella larvae
1.Rear neonate codling moth larvae for 10 days on virus-free artificial diet.
2.After 10 days, larvae reach instar L4eL5.
3.Pipette 1 ml virus suspension containing 1000 OBs on the surface of a
small piece of diet (about 2 × 2 ×2 mm).
4.Keep larvae single in a well containing one piece of contaminated diet
and incubate them for 24 h at 26○C.
5.Transfer larvae, which have eaten the contaminated
diet completely, to virus-free diet.
6.Incubate larvae at 26○C until infection is visible (usually after 5e6 days)
and monitor daily.
7.Collect infected larvae before tissue rupture.
8.Collected larvae can be frozen at —20○C until virus purification
Virus propagation follows the same protocol for small- and large-scale
production. Infection of about 200 codling moth larvae following this
protocol will usually result in 5 ml purified virus suspension with a
concentra- tion of about 1011 OB/ml.
It is recommended to feed more larvae, because not every larva will
eat its diet plug completely or show symptoms of virus infection.
Quality control of in vivo virus propagation
Perbanyakan virus di dalam tubuh serangga hidup dapat
menyebabkan variasi produk dalam hal komposisi dan kemurnian
– mikroorganisme lain yang hadir di dalam serangga dapat
menyebabkan kontaminasi produk akhir.
Oleh karena itu ―quality control" yang baik sangat diperlukan.
Stok virus yang digunakan untuk perbanyakan harus
menggunakan strain virus yang sudah dikarakterisasi sebelumnya
dengan analisa DNA restriction analysis untuk menentukan
apakah isolat tunggal atau campuran genotip.
Purifikasi inokulum dengan meminimalisasi resiko kontaminasi
dengan protozoa atau spora bakteri yang dapat mempengaruhi
replikasi virus.
Setiap virus yang dihasilkan harus diestimasi dengan
penghitungan atau metode lain yang sesuai dan diuji aktivitas
biologinya dengan bioassay.
B. Isolasi virus dari serangga
Isolasi virus dari inang yang terinfeksi biasanya tahapan lanjut
setelah perbanyakan virus.
Sangat diperlukan suspensi virus yang sangat murni.
Untuk uji bioassay, suspensi virus harus bebas dari mikroorganisme
yang mempengaruhi proses infeksi.
1. Homogenization and filtration
Serangga mati di-homogenisasi di dalam deterjen anionic yang rendah
konsentrasi untuk memfasilitasi lepasnya jaringan tubuh serangga dan
melepaskan partikel.
Membekukan larva sebelum homogenisasi juga membantu untuk merusak
sel.
Suspensi homogenisasi masih mengandung residu makanan rering, kapsul
kepala, dan bagian besar integumen, sehingga perlu disaring dengan is
therefore filtered through saringan kain (cheese-cloth / gauze).
2. Centrifugation
Suspensi virus yang disaring masih mengandung lemak, bakteri dan
partikel halus non-virus lainnya.
Dengan proses sentrifugasi beberapa kali virus dapat terpisah dari
kontaminan ini.
3. Purifikasi
Figure 1. Example of a NPV [Agrotis segetum NPV Oxford strain (AgseNPV-
B)] under the light microscope using an improved Neubauer hemocytometer
(depth 0.1 mm). The edge of each small square is 0.05 mm × 40 (for further
information see text). B. A granulovirus [Cydia pomonella granulovirus
(CpGV)] as seen by dark-field illumination under the light microscope. For
observation a PetroffeHausser counting chamber (depth 0.02 mm) is used.
The edge of each small square is 0.05 mm × 40.
Figure 2.. Basic bioassay procedures for LD50 and LC50 determination. A. For the diet plug method, a known
dosage of virus suspension is pipetted on a small piece of diet and fed to one test larvae each until full
consumption. Larvae are then reared on virus-free diet. B. Droplet feeding: single droplets of virus suspension
containing a known dosage of virus mixed with food dye are fed to single larvae. Larvae which have ingested the
droplet are then reared on virus-free diet. C. For surface contamination, virus suspension is added to a known
unit of artificial diet to cover the surface. After a short time of feeding, larvae are transferred to virus-free diet.
Concentration is given per mm2. D. Diet incorporation: virus suspension is mixed directly to a measured volume
of artificial diet. Test larvae feed on the contaminated diet until the end of the bioassay.
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