Technical Bulletin 15 ments for pruning, weeding ses that are present in high Technical Bulletin 15 Guidelines 2) IV, TRANSMISSION OF VIRUSES IN THE LABORATORY ‘Transmission of viruses in the laboratory is necessary for their isolation from diseased field plan's, fer their dentiication and sometimes for their separation from plants infected with several viruses. A. Sap Transmission Sap transmission oF mechanical inoculation i the application of virus-contsining plant extracts ¢ inoculum) to the leaf surface of healthy plants. For vires particles to penetrate the catcle and epidermis of a healthy Leaf, leaf surfaces mus be ‘pulfeally wounded. When the inoculated plant is susceptible, the following reactions may oveur: «Virus movement is restricted symptoms usually appear as local lesions on the inoculated leaves. «The virus spreads systemically to other area of te host. Systemic symptoms appear, suchas mot, see ye let deformation, lesions, necrase, ec. which are usually distributed throughout the plant. + There are no symptoms: “Aitough te vius has invaded the plant and is multiplying no host reaction is visible, The pant dither tolerant of the virus or the symptoms are masked by environmental conditions ‘Aihoughthe vrashas entered the pan itis at multiplying and invading othe partsof he plan. an so systemic sympeoms are produced. The plant is highly esstant or hypersensitive to the virus ‘When the virus has not entered the plant, itis immune to the virus. [Not all viruses can be transmitted mechanically. Viruses that in nature persis: in the vector such as semiperssint and persistent aphid-transmied viruses and many ofthe Yeahopper- and whitefly- transmitted viruses are not usually transmitted by sap. 1. Indleator Host Plants Indicator hosts react diagnostically to certain vimses. They ca be used to distinguish among these ‘inuses, usally by showing immunity to one and susceptibility t0 the other. “The most commonly used indicator plants arc: ‘Chenopodivon amaranticolor (susceptible in more than 40 different viruses) Chenopodium quinoa Cucumis sativus Datura stramontum Gomphrena globosa Nicotiana benthamiana Nicociana gluinosa ‘Nicotiana tabacum "Kanthi’ Nicotiana tabacum ‘Samsun’ Phaseolus vulgaris “Pinto!Technical Bulletin 15 22 Guidelines Vieia faba Vigna unguiculata 1. Seeds ‘Seeds of indicator plants can be ‘obtained from: Plant Introduction ‘Germplasm Resources Laboratory Agricultural Research Center Belisville, MD 20705 USA from instittions atively engaged in applied virus rescarch. only asmall amount of seeds is sent ‘ritially, fist propagate the seeds in an insect-proof greenhouse b, Greenhouse Keep indicator plans in an inect-free greenhouse or sereenhouse Wo ensure sufficient light for optimal growth. Maintain heli stock plans separately (om inoculated plans, preferably ina separa foot "spray the greenhouse regulary with an insecticide wo avoid buildup of et populations, Use eee edek of varying nature to avoid development of resistance, The use of biological control gens, including predators or sicky traps has also become popals. ‘They are commercially (vailable forthe control of thrips, whiteflies and aphids. Fungicides should be spplied as necessary ©. Soil “To inactivate microbial pathogens and soil-nhabiting viruses and virus vectors, steam-sterlize the soil at about 90°C for 30 minutes. 4. Mumination Reduced ight intensity is known increase the suscepiility of ome pans wo cenain Vier Fe eats inthe dark or several hours ayspriortoinoeuiaontoincrease thelr suscep 2, Inoculum Preparation Inoculum isthe sap extracted from diseased plats used in wansmiting the vius- ‘The following points should be kept in mind when choosing viru snfectd leaf Hssue for inoculum ‘preparation: «Virus content often but not always correlates with the severity ofthe symptoms 7 Young tissues often have the highest virus content « Some viruses can only be transmitted at certain times of the year. «Roots or petals may sometimes be good sources of inoculum. fa. Maceration of virus-infected tissue Grind one pat virus-infected issue ina small sterilized (autoclaved for 0 minute 120°C or boiled cin omtrwo hours) mortar with 20 5 pans buffer (generally 0.01 M phosphate buffer, pH 7.0) Keep the inoculum coo! and use it immediately. belo: have ‘The sele 3, Mech Routine Grind 4 sterilize ofhealt water af a Indice compari ». Pret Keep in suscepti «Plant Use yor 4. Time PlantsTechnical Bulletin 15 ‘all amount of seeds is sent § ensure sufficient light for {erably ina separate 100m. of insect populations. Use ‘use of biological control Gr. They are commercially § vectors, steam-steritize the | Ineo certain viruses: Keep Jnereae hei suscepsbiiy. \ the virus. {ed leaf tissue for inoculum _ymptoms. ) minutes at 120°C or oiled { phosphate buffer, pH 7.0). i ‘echnical Bulletin 15 Guidelines 23 Prepare the buffer inthe following way: Solution A: 1.36 g KH,PO ta 1000 ml HO Solution B: 1.78 g Na,HPO~2H,0 in 1000 mal H,0 Mix 51.0m{of olution B with 49.0 m1 of solution Ato get 00 mlof0.01 Mphosphate buffer solution p70. ». Inoculum additives + Abrasives ‘Abrasives are necessary for succesful inoculation; their use increases nfes 808 by providing sats for the entry of virus particles. The most commonly used ae ‘earborundum (silicon owns 00-600 mess) and Celt (Satomaceous cat), Dust the abrasive ver the leaf surface tefore inoculation or suspend in the inoculum (0.5-1% w/v). - Stabilizing additives Many plants contain inhibitors that may inactivate the virus, esTeast inhibit it infectivity or Many Panfth is ransssion. The following compounds, when added Ye, ‘inoculum, are ine Mea stabilizing effecton vrsesinplantextratscontainingsuchinhibors ‘Theyalso have a stabilizing effect on unstable viruses. disodium ethylenediaminetetrancetate (EDTA) 0.0005 - 0.1M Sodium diethyl-dithiocarbamate (DIECA) oot - 002M + ascorbie acid (vitamin C) 002 -0.17M “sodium sulfite (Na,SO,) Ol -03% * polyvinyl-pyrrolidone (PVP) (MW 10000) 1 -2% “ thioglycollic acid (TGA) Ol 05% + 2-mercaptoethanol (ME) 02-10% bovine serum albumin (BSA) on1% ‘tues compounds are genealy added to the inoculum in he coneention nde A, The These com compound and concentration depends on the parila virusNox Plant == 43, Mechanical Inoculation Routine Inoculation Method Grind approximately 5g of virw-infctd caves with 101020 mi phosphate buffer (pH 7.0) in a Sertized mortar. AA EDTA or DIECA asastabiizing agent, Gently rub the suspension onthe leaves aerseny indicator plants which have ben dusted wih caborundam or Cel Rinse the leaves with water after te inoculation. a. Indicator planes Thosulate at eat swo plants of every species. St aside one control plan of each species for tater comparison of symptoms. ». Pre-inoculation treatment Keep indicator plans in the dark for several hours or days prio wo inosuation 19 increase theit susceptibility. ¢.Plamtage tee young plans since they are generally more susepbe to ins infecon than colder plants. Time of inoculation Plans are generally more sustepible to virus infection in the afternoon.24 Guidelines Technical Bulletin 15 e-Inoculation site Inocutate the upper leaf surface. Legumes: Tnoculate the primary leaves. ‘Cucumber: Tnoculate the cotyledons. Chenopodium: Inoculate the fourth to eighth leat. ‘Tobacco: Tnoculate ay leaf above the thied or fourth leaf Datura: Tnoculate when the first o second leaf pai has developed. £. Glassware se sterilized plassware; autoclave for 30 minutes at 120°C or ell in wales for two hours, Abrasives Biter add to inocelum or apply to leaves prior wo inoculation bh. Application of inoculuen ‘Apply the inoculum gently tothe laf surface withaccoton swab, apadof cheesecloth, a glass rod with a flatiened end or with your finger. i. Postinoculation weatments ‘ings te inoculated leaves with waterto remove natal oxinsin he noes which interfere with Fe pe, and wo rece injury fom chemicals which have been added io the inoculum. Rinsing also facilitates later observation of symptoms. + Light reduction Keep the plans inthe dark for several hours after inoculation to increase the susceptibility of virus jdicator plants and to promote betler symptom expression. + Drying Quickly dey eaves with an atomizer or with bloting paper. 4, Symptom Development and Recording « copgerve plans daily for several weeks (in some cass fr sever mendes Os transmission of eben woody plants) and compare them with contol plants ofthe same Be. «Many ost plans wil develop local eon, but oer symptoms can sso 4H "Dhanush between local reaction on te inoculated leaves and systemic eacuon o8 the non- inoculated leaves. «+ Record the symptoms and their sequence, «Some ofthe common symbols used for recording symptoms are LL: local lesions = LL: necrotic local lesions CLL + chlorotic loca! lesions Ve: vein clearing, M —: mosaic a Mo: mote SN: systemic necrosis ‘D. Appro: ‘Mal: malformation ‘Cut the s E : etching Chooses RS: Hngspot back the is syste