Practicall
I~entification of Disease Symptoms Symptoms.. 1. To fa fami mili liar ariz izee wi with th di diff ffer eren entt ty type pess of dise diseas asee sy symp mpto toms ms
Objectives:
2. To disti disting ngui uish sh di dise seas ased ed pl plan ants ts fr from om he heal alJh Jhy y pl plan ants ts 3. To detect detect di dise seas asee sy symp mpto toms ms ea earl rly y as poss possib ible le
Specimens:
Differ Dif ferent ent typ types es of dise disease ase Sym Sympto ptoms ms 1. Bl Blas astt di dise seas asee in ric ricee 2.
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Anthra Ant hracno cnose se
in chi chilli lli''
3. Po Powd wder ery y mi mild ldew ew in gr grap apes es 4. Do Down wny y mi mild ldeew in gr graape pess 5. Ca Cank nker er in ci citr trus us 6. Ru Rust st in gr grou ound ndnu nutt 7. Le Leaf af spot spot in co cowp wpea ea 8. Cl Club ub
ot in
bb
Practi Pra ctical cal 2 Preparation 'of Nutrient Nutrient Media
Materials: Potato Potato,, Dex Dextro trose, se, Aga Agarr, Dis Distil tilled led Wat Water, er, Buc Buchne hnerr fun funnel nel,, Aut Autocl oclave ave,, Con Conica icall flas fl ask, k, Co Cott tton on wo wool ol an and d Al Alum umin iniu ium m fo foil il
Compos Com positi ition on for 250m 250mll of medi media a Potato
50g
Sucrose
5g
Agar
5g
Distilled water
250ml
Procedure: •
Peel potato, cu cutt in into to sm smal alll pi piec eces es an and d wa wash sh in wat water. er.
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Add Ad d po pota tato to in 125 125 ml ml of wat water er an and d boil boil un unti till the the po pota tato to be beco come me ve very ry so soft ft..
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Stra St rain in the the po pota tato to using using..
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Mean Me an wh whil ilee pl plac acee th thee weigh weighed ed ag agar ar in a 500 500ml mlfl flas ask k
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Boil this ov over a' a 'wat water er bat bath h unt until il aga agarr dis dissol solves ves
with wi th 12 125 5 ml ml of of wate water. r.
Pract Pr actica icall 3 Pour Po urin ing g of nutri nutrien entt me medi dia a in to to petri petri di dish shes es
Objectives:: Objectives
1. To famili familiarize arize wit ith h nu nutr trie ient nt me med dia pou pourin ring g tec techni hnique quess 2. To know know ab abou outt pr prec ecau auti tion onss du durr ing po pour urin ing g of nut nutri rien entt me medi diaa
Procedure:
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Star St artt lamin laminaa fl flow ow 30 mi minu nute tess be befo fore re''the ope operat ration ion..
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Take Ta ke steril steriliz ized ed PD PDA A media media aw away ay from from autoc autocla lave ve or re refe ferg rger erat ator or..
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If it is in the the so soli lid d st stag age, e, bo boil il it in the the wa wate terr ba bath th un unti till re reac ach h 70 70°C °C te temp mper erat atur ure. e. "
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Stop St op la lami mina na fl flow ow and and sp spra ray y 90 90% % al alco coho holl in insi side de the the lamin laminaa fl flow ow..
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Was ash h the the ha hand ndss wi with th 70% 70% alc alcoh ohol ol..
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Keep th the co conical f las ask k co cont ntai aini ning ng me medi diaa in insi side de the lamin laminaa fl flow ow..
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Add ant ntib ibio ioti ticc ta tab ble lett (Pe Peni nici cill llin in or Strep Strepto tomy myci cin) n) to med media ia at 50° 50°C C.
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Add Ad d 50 500m 0mg g ta tabl blet et fo forr 1 li lite terr of of me medi dia. a.
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Ligh Li ghtt the spr sprit it lamp lamp insid insidee the lam lamin inaa fl flow ow..
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Heat He at the the ne neck ck of the the con conic ical al fl flas ask k be befo fore re po pour urin ing g.
Practi Pra ctical cal 4 Artifi Art ificia ciall ino inocul culati ation on of Pat Pathog hogens ens fro from m dis diseas eased ed pla plant nt par parts ts to nut nutrie rient nt med media ia \
.
Materials: Ino Inocul culati ation on nee needle dle,, for forcep ceps, s, Pet Petri ri dishes dishes,, al alcoh cohol ol (90%
70%),, & 70%)
beaker bea kerss and
lamina lami na flow
Procedure: •
Star St artt the the lamin laminaa fl flow ow 30 min minut utes es be befo fore re the ope opera rati tion on..
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Red Re d heat heat the the scis scisso sors rs,, fo forc rcep epss an and d inocu inocula lati tion on ne need edle less I
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Cutt the aff Cu affec ecte ted d po port rtio ion n in to sm smal alll pi piec eces es..
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Traans Tr nsfe ferr it int nto o the the 70% al alcoh cohol ol for 30 seco seconds nds (surfa (surface ce ster steriliz ilizati ation). on).
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Then Th en tran transf sfer er it in into to di dist stil ille led d wa wate ter. r.
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Mob Mo b the the spec specim imen en wi with th filte filterr pape paperr.
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Stop St op lam lamiina fl flow ow and and spr spray ay 90% alc alcoho oholl ins inside ide the lami lamina fl flow ow..
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Wash Wa sh th thee han hands ds wi with 70% alcohol alcohol..
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Tran Tr ansf sfer er th thee spec specim imen en in into to th thee petr petrii dish dish which which'' con conta taini ining ng nut nutrie rient nt med media ia..
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Seal Se al the the petr petrii di dish sh with with celot celotap ape. e.
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Labe La bell th thee petri petri dish dish and keep keep in insi side de the the incuba incubato tor. r.
Practi Pra ctical cal 5: Isol Is olat atio ion n an and d iden identi tifi fica cati tion on
of pa path thog ogen en fr from om dise diseas asee af affe fect cted ed pl plan antt pa part rtss
usin us ing g
Celot Ce lotape ape Im Impre pressi ssion on Met Method hod
I. To fami famili liar ariz izee wi with th isolat isolatio ion n an and d id iden enti tifi fica cati tion on of pa path thog ogen enss as asso soci ciat ated ed wi with th
Objectives:
disease
Diseased sed speci specimens mens,, Celotape Celotape,, La Lact cto o ph phen enol ol co cott tton on bl blue ue (s (sta tain in), ), Sc Scis isso sors rs,, Sl Slid ides es Materials: Disea and Micro Microscope scope
.
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Impr Im pres esss pi piec ecee of ce celo lota tape pe in di dise seas ased ed sy symp mpto tom m an and d remo remove ve fr from om it. it .
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Add Ad d on onee drop drop of sta stain in into into that that cel celot otap apee pi piec ece. e.
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Theen re Th re-i -im mpr preess ssiin
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Obse Ob serv rvee un unde derr the mi micr cros osco cope pe..
sliide sl de..
Observation: I. Shapes Shapes of the the fu fung ngal al sp spor ores es
Practi Pra ctical cal 6 Gram Gr am staining staining (Do (Doubl ublee or Diff Differe erenti ntial al Sta Staini ining) ng)
Equipments:
Sprit Sp rit lamp, lamp, alcohol alcohol-cl -clean eaned ed
micro mi crosco scope pe sli slide de,,
Reagents:
1.. Cry Crysta stall Vi Viole olett (th (thee Pri Prima mary ry St Stain ain)) 2.
Iodine Iod ine Solut Solution ion (th (thee Morda Mordant) nt) (bI (bIKI KI Sol Soluti ution) on)
3.·· Deco 3. Decoloriz lorizer er (Eth (Ethanol anol I Eth Ethyl yl al alccohol) 4.
Safran Saf ran in (th (thee Counter Counter St Stain ain))
5. Wat Water er (prefer (preferabl ably y'in
a sq squi uirt rt bottl bottle) e)
citrus' ' (Xantho~onas (Xantho~onas citri) Cank nker er di dise seas asee in citrus Specimens: Ca Procedure: Prepare Prepa re a slide slide sme smear ar Sp\ea Sp\ ead d the cultur culturee wi with th an inocula inoculatio tion n loo loop p to an an even even thi thin n fil film m on a slide. slide. Ai Air-d r-dry ry the cultur culturee (1 to to 2 min minut utes es)) an and d fi fixi xitt or over over a gent gentle le fl flam ame, e, wh whil ilee mo movi ving ng th thee sl slid idee in in''a ci circu rcular lar fas fashio hion n to avo avoid id lo loca cali lize zed d ov over erhe heat atin ing. g. Th Thee ap appl plie ied d he heat at helps helps th thee ce ceil il ad adhe hesi sion on on the the gl glas asss sl slid idee to make ma ke po poss ssib ible le th thee su subs bseq eque uent nt ri rins nsin ing g of the the smear smear wi with th wa wate terr wi with thou outt a sign signif ific ican antt lo loss ss of the culture.. He culture Heat at can als also o be appli applied ed to facili facilitat tatee dry drying ing the smear. smear. Gram staining staining I. Pl Plac acee sl slid idee on a slide slide ho hold lder er or a rack rack.. Fl Floo ood d (c (cov over er co comp mple lete tely ly)) th thee en enti tire re sl slid idee wi with th crysta cry stall vio violet let.. Le Lett th thee cr crys ysta tall vi viol olet et st stan and d fo forr ab abou outt 60 second secondss. Wh When en th thee ti time me ha hass elap el apse sed, d, ge gent ntly ly ri rins nsee th thee ex exce cess ss st stai ain n wi with th a strea stream m of wate waterr fr from om a tap tap or a pl plas asti ticc ,wa wate terr bo bott ttle le fo forr 5 seco second nds. s. No Note te th that at the objec objecti tive ve of this this st step ep is to wash wash of offf th thee st stai ain, n, nott the fixed no fixed cu cult ltur ure. e. Th Thee sp spec ecim imen en sh shou ould ld ap appe pear ar bl blue ue-v -vio iole lett wh when en ob obse serv rved ed wi with th th thee
Practi Prac tica call 7 Thee Ru Th Rust st Di Dise seas asee on Grou Ground ndnu nutt Obje Ob ject ctiv ives es::
1. To ide ident ntif ify y sy symp mpto toms ms pr prod oduc uced ed by ru rust st fun fungi gi 2. To To id iden enti tify fy di diff ffer eren entt st stag agees of rust fungi 3. To familia familiariz rizee wit with h con contro trolJT lJTIea Ieasur sures es
Introduction Leaf Le af ru rust st of groun ground d nu nutt is wo worl rld d wi wide de in di dist stri ribu buti tion on an and d af affe fect ctss gr grou ound ndnu nutt wh wher eree it is is grow grown n. Thee le Th leaf af rust rust fungus fungus at atta tack ckss al alll the the ab abov ovee gr grou ound nd pa part rtss an and d ca caus uses es lo loss sses es by redu reduci cing ng fo folia liage ge,, root ro ot develo developm pmen ent, t, yi yiel eld d an and d qu qual alit ity y of grou ground ndnu nutt. Symptom Thee sy Th symp mpto toms ms on grou ground nd ap appe pear ar fi firs rstt as oran orange ge~y ~yel ello low w powd powder ery y sp spot otss on the the lowe lowerr si side de of thee le th leav aves es.. Th Thee sp spot otss ar aree ci circ rcul ular ar an and d sm smal alll. Th Thee ce cent nter er of tthe he sp spot otss ev even entu tual ally ly be beco come me dr dry, y, turn tur n bro browni wnish sh and lea leaff fal falls ls pre prema matur turely ely.. Control • Cro Crop p rota rotatio tion n (ce (cerea real-c l-cere erealal-gro ground undnut nut). ). • •
.Ap Appl plic icat atio ion n of we wett ttab able le su sullph pher er (l (lkg kg pe perr ac acre re). ). Appl Ap plic icat atio ion n of ofch chlo loro roth thal alan anil il (4 (400 00g g pe perr acr acree mix mix with with 200 200 li litr tree of of wa wate ter) r)
•
Usee re Us resi sist stan antt va vari riet etie iess
Practi Prac tica call 7 Thee Ru Th Rust st Di Dise seas asee on Groun Groundn dnut ut Obje Ob ject ctiv ives es::
1. To id ident entif ify y sy symp mpto toms ms pr produ oduce ced d by ru rust st fung fungii 2. To To id iden enti tify fy di diff ffer eren entt st stag ages es of rust rust fungi fungi 3. To To fam famili iliari arize ze wit with h cont control rol;ne ;neasu asures res
Introduction Leaf Le af ru rust st of grou ground nd nu nutt is wo worl rld d wi wide de in dis distr trib ibut utio ion n an and d af affe fect ctss gr grou ound ndnu nutt wh wher eree it is is grown grown.. Thee le Th leaf af rust rust fu fung ngus us at atta tack ckss al alll the the ab abov ovee gr grou ound nd pa part rtss an and d ca caus uses es lo loss sses es by reduc reducin ing g fo foli liag age, e, root ro ot develo developm pment ent,, yi yiel eld d an and d qua quali lity ty of grou groundn ndnut ut.. Symptom Thee sy Th symp mpto toms ms on grou ground nd ap appe pear ar fi firs rstt as oran orange ge-: -:-y -yel ello low w po powd wder ery y sp spot otss on the lowe lowerr si side de of thee le th leav aves es.. Th Thee sp spot otss ar aree ci circ rcul ular ar an and d sm smal alll. Th Thee ce cent nter er of the the spot spotss ev even entu tual ally ly be beco come me dr dry, y, turn tur n bro browni wnish sh and lea leaff fal falls ls pre premat mature urely. ly. Control •
Crop Cro p rotat rotation ion (cere (cerealal-cer cereal eal-gr -ground oundnut nut). ).
•
.Ap Appl plic icat atio ion n of wet wetta tabl blee su sulp lphe herr (1 kg kg per acr acre) e)..
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Appl Ap plic icat atio ion n of ofch chlo loro roth thal alan anil il
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Usee re Us resi sist stan antt va vari riet etie iess
(400 (4 00g g pe perr acr acree mix mix with with 200 200 li litr tree of of wat water er))
Practical Practi cal 8 Club Root Disease in Crucifers Object Obj ective ives: s: 1. To identif identify y sym sympt ptom omss pr prod oduc uced ed by clu club b ro root ot fungi fungi
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2. To familia familiariz rizee with contro controll mea measur sures es
Specimens: Clu Club b roo roott disease diseased d cab cabbag bagee pla plants, nts, Per Perman mantt siid siidee of dise disease ased d spe specim cimen en
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Club Clu b roo roott fungi affect affect cru crucif cifero erous us pla plants. nts.
Symptoms: Infe In fect cted ed pla plant ntss ~a ~ave ve pa pale le gr gree een n to yell yellow owish ish le leav aves es . .. Sh Show ow wi wilti lting ng in th thee middle middle of hot, hot, su sunn nny y da days ys bu butt may may re reco cove verr dur during ing nig night ht.. Youn Yo ung g pla plants nts ma may y be kille killed d by the the diseas diseasee wi with th in a shor shortt tim timee af afte terr in infe fect ctio ion, n, w~ w~ile ile old older er.. plants pla nts rem remain ain ali alive. ve. Thee ch Th char arac acte teri rist stic ic sy symp mpto tom m ap appe pear ar on roots roots and somet sometim imee on the the under under gr groun ound d pa part rt of stem. stem. Symp Sy mpto tom m co cons nsist istss of smal small, l, spi spind ndle le lik like, e, spher spheric ical al,, kn knob obby by or cl clubb ubbed ed sha shape ped d sw swel elli ling ng on th thee roots roo ts and roo rootle tlets. ts.
Control: 1. Gr Grow owin ing g cr cruc ucif ifer erou ouss cr crop opss in fi fiel elds ds kn know own n to be be inf~s inf~ste ted d sh shoul ould d be avoi avoide ded; d; 2. Ca Cabba bbage ge sho shoul uld d be pla plant nted ed in we well ll-d -dra rain ined ed so soil il with with the PH 7.2 7.2 by addi adding ng hy hydr drat ated ed lime.
Practi Pra ctical cal 9 Isolat Iso lation ion of nem nemato atodes des fro from m inf infest ested ed soi plants nts soill and pla
Nematode odess inf infes este ted d so soil il,, Si Siev eves es 30 300/ 0/ll m and and 38 /l m mesh mesh,, Materials:: Nemat Materials Petrii dish Petr dishes es,, dis distill tilled ed wate waterr, glass glass..-rod and squa squash sh bot bottle tle
Ihee mes Ih mesh h, fil filter ter pap paper, er,
Procedure: Procedure: • Th Thre reee me meth thod odss us used ed for for is isol olaation of nema nemattodes. 1. Sei Seiving ving met method hod 2. Ba Baer erman man fu funne nnell me meth thod od 3. Ce Cent ntrif rifug ugee fl flot otat ation ion me meth thod od
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Take Ta ke kn know own n am amou ount nt (l (l00 00 g, of in infe fest sted ed so soil il in to th thee be beak aker er..
• •
Add wat water and cru crussh it well "wit with h glass glass rod. Filt lter er so soiil sus suspe pensi nsion on th throu rough gh 30 300/ 0/ll I ? mes mesh h seive. seive.
• •
Discar Disc ard d debr debris is.. _ Filt Fi lter er the the filt filtra rate te fr from om 300/ 300/ll m me mesh sh sei seive thr hrou ough gh 38 /l m mesh mesh se seiv ivee.
• •
Nemato Nema tode dess re reta tain in in th thee fil filte terr. Wash Wa sh the the nemat nematod odes es fr from om the the seiv seivee to bea beake kerr.
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.pla pla~ ~e filt filter er pa pape perr abo above ve th thee fi fine ne mesh. mesh.
•
Keep Ke ep the the setu setup p in th thee petr petrii dish dish.. Add Ad d nemat nematod od
ntai nt aini ni
luti lu ti
into in to fi
sh
Practi Pra ctical cal 9 Isol Is olat atio ion n of nem nemat atod odes es fr from om in infe fest sted ed so soiil and plant plantss
Materials:: Nemat Materials Nematod odes es in infe fest sted ed so soil il,, Sieves 300ll m and 38 Ilm mesh, mesh, fih fihee mes mesh h, filter paper paper,, Petri Pet ri dishes, dishes, dis distil tilled led wate waterr, glas glasss rod and squash squash bot bottle tle Procedure: Procedur • Th Thre reee me meth thod odss us used ed for for iso isola lati tion on of ne nema mattodes. I. Seiving me meth thod od 2. Ba Baer erma man n fu funn nnel el me meth thod od 3. Cen Centrif trifuge uge flo flotati tation on met method hod
•
Take Ta ke kn know own n am amou ount nt (1 (100 00 g, of inf infes este ted d so soil il in to th thee be beak aker er..
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Add Ad d wa wate terr an and d cr crus ush h it we well ll "w "wit ith h gl glas asss ro rod. d.
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Filt Fi lter er soi soill sus suspe pens nsio ion n th thro roug ugh h 300ll I? mes mesh h sei seive. ve.
• •
Discar Disc ard d debr debris is.. Fil ilte terr the the fil filtr trat atee fr from om 300ll m mesh mesh se seiv ivee th thro roug ugh h 38 Ilm me mesh sh se seiv ive. e.
• •
Nemato Nema tode dess re reta tain in in the the fil filte terr: Wash Wa sh the the nema nemato tode dess fr from om the the sei seive ve to to bea beake kerr.
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Plaqe fil filte terr pa pape perr ab abov ovee th thee fi fine ne mesh mesh.
• •
Keep the Keep the setu setup p in th thee petr petrii di dissh. Add Ad d nemat nematod odes es co cont ntai aini ning ng so solu luti tion on in into to fin finee mesh mesh ..
•
Keep for for 24 24 hours.
.
Objectives: To famili familiari arize ze wit with h post post harvest harvest dis diseas eases es To identify identify con contro troll mea measur sures es.. Specimens: 1. Ca Carr rrot ot ro rott - Erwi Erwinia nia carot carotovora ovora 2. Ant Anthra hracno cnose se in chi chill ll..i - Colletotrichum sp 3. An Anth thra racn cnos osee in ba bana nana na - Colletotrichum sp 4. Ba Bact cter eria iall br brow own n sp spot ot in bea beans ns - Pseud Pseudomona omonass syrin syringae gae
Observation: Identi Ide ntific ficati ation on of the charact characteri eristi sticc
featur fea tures es of the diff differe erent nt dis diseas easee sym sympto ptoms ms
