Kuliah 11_Perbanyakan virus sebagai agens antagonis_2018

  Perbanyakan Massal Perbanyakan Massal Virus sebagai Agens Virus sebagai Agens Antagonis Antagonis Oleh Irda Safni 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

• 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 dan1
• 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

• 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

   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

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).

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.

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 Virus RNA

  



Double stranded RNA

   Double stranded DNA

• Rheoviridae: Cypovirus
• Poxviridae

  



- Iridoviridae - Rhabdoviridae - Baculoviridae: NPV & GV

  Single stranded RNA (-)

• Bunyavir
• - Polydnaviridae

  



Single stranded RNA (+)

   Single stranded DNA

• Picornaviridae, Togaviridae,

  

Tabel 1. Kelompok Virus Entomopatogen

• 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 - ssRNA Isometric  Nodaurelia Kelply group ssRNA Isometric - 5-virus group
• ssRNA Isometric - Minivirus ssRNA Isometric Ovoid virases
• ssRNA Ovoid

  2 microns Baculoviruses

  Spodoptera littoralis

• Baculoviruses Mode of action

Baculoviruses

Susceptibility of Alternative Hosts

  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

• Toxicity studies mammals

  Toxicity test results from 1970s/80s of

  29 NPVs indicated no toxicity or pathogenicity. Doses were 10 100 x the “per acre” (1 acre = 0.45ha) – generally 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.

  

Baculoviruses

– Toxicity studies mammals cont.

  No effects of HzNPV found in: Acute toxicity-pathogenicity tests in mouse, rat, guinea pig, rabbit,

  9

  12 monkey and man at 6x10 – 3 x 10 OB / kg.

  10

  6 Skin irritation sensitivity tests in guinea pigs, rabbits and man at

  7

  2 and 10 OB / mm skin.

  

5

  6 Eye irritation tests in rabbits with 10 and 2x10 OB / eye Subacute toxicity-pathogenicity tests and subcutaneous injection into mice, rats, dogs and rhesus monkeys.

  9 Teratogenicity and carcenogenicity 10 – studies in rats and mice at

  12 3.5x10 OB / kg.

  Baculoviruses

• – Toxicity studies wildlife

  Birds Able to pass NPV through the alimentary tract unaffected No deleterious effects

  Aquatic organisms No adverse effects

  Beneficial insects

  Baculoviruses Pathology studies

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.

  Baculoviruses

Virus-cell interactions in vitro

  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

  Baculoviruses a list of the baculoviruses regulated as pesticide active ingredients by the US EPA Office of Pesticide Programs 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

• – Baculoviruses US EPA fact sheet

  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.

  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

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

  Dendrolimus spectabilis CPV registered in Japan in 1974. Safety test results generally negative.

  Cypoviruses (Rheoviridae) No CPV has been found infecting vertebrates or plants (Belloncik, 1989)

• – 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

  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

  Ascoviridae: Ascovirus

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.

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.

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

  

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.

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 ₁₁ concentra- tion of about 10 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

  

  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.

  

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.

  

_{containing a known dosage of virus mixed with food dye are fed to single larvae. Larvae which have ingested}

_{consumption. Larvae are then reared on virus-free diet. B. Droplet feeding: single droplets of virus suspension}

_{dosage of virus suspension is pipetted on a small piece of diet and fed to one test larvae each until full}

Figure 2.. Basic bioassay procedures for LD and LC determination. A. For the diet plug method, a known

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