BRUVUUUOUOUUHUKVTVVTKEHYYU YY NF ¥ yen x VV ) h Plank Disease Diognestics~ Dllevent approsehes used tov sua) Symphems diagnosis of ploat discoses based 00 ) Plant diseases gre cused by distinct qroup af organisms, Predeminantty tongiy baceviay mollfeutes, viruses, viroids, Nematodes ond protegoa: “1 Rapid detection and acceipate fdentifieetfon ef the Pathogens 1s vey essential clo elledtive moaaqement of cep cliseases Symptoms ¢ symptoms are Yesponse of ne host ptant to Pathogen and x}er +o the appearence of infected Plants ey plant tissues. Symptoms mney result rom Over prectuistfon , underproduclfoa | Necro sis. “i signse signs af plant pathogens ave any portion of ‘the Pathogen or iis produtls A sign may alto be the product ch a pathegen such as bacteria) oo3e or odor ewhich indicates 4 pathogenis Presence. THentificaton of. syenptoms ond signs ¢- The following ave different symp-toms and signS induced by Pathegens in plants. \ Weewsis © death a} cells} -Hesues visualized ag brown] black aveas on atteded plants BAU Lose ab -turgidity of Jeaves and Shoots agseeitted with vasactar broconing [spoke localized Neewolie testers on host leaves Jarignk apd ard extensive Neerwsis o| -the infected plantLut aU , TLUULCELLLE! % , Tt » y coon) " IMI a bunk appeaveate Rote Veewsis of pavenchyma eoltenchy roa and pith tissues, wheve neansis ic not joatiged: Damping Veevesis a}, basal portion e| stem | ¢rous veqion of Seedlings Conkeve- Covey neensis in -the woody sHissues which is deep seated: Seabe Weeresis is usually superficted anol is vesticted to epidermal veqfon: Streakst-chlowti c | necrotic aweas aleng dhe veing in monocots- Dit backe Excessive necresis al -tewiqa beeqinning at the Hips and advancing towerds +ney base ST: Hypevplastice- over development of issues due hyperplasia and hypertrophy GrallsHumeye Enlorged sac Like qrowlh on stem, heaves ete ware small enlarged Shuctures | proturbevances on thers ond stem enactions eRIdges af dove green lea} Uke shuctaves jusualty on-the ventral suxface of the velo - Bidet Fastey growing qreen ports In mosaic affected Jeoves ov fruits fom Histers: witehy nant profes upusord branchiag af eaigs- Lar sryeplagic ondersprourth ef meted plan organs: [prestige Sunts of plans due -to echephy.Decay mildeoe pull ewhiley superficial quaths af myceticr Spovengiophores ond spevengia on uindey anfate of loves: Forder) Milde ceuhite , pastay, superficial gqrovaths a} mycelia and conidiophores on surface of leaves , Stems» Ptewers ond faut Acervulust- A flat Gv) saucer shaped bed of sherk conidiopheres evith conidia growing ander the epidermis] cutide of hast plant Percnicliume Flash shaped dove bedly that produces lerge number af usually partially buried in diseased -Hssue- coloured! asercudl -hulting conidia and body ium t- Brows to bade , Compact, herd vesting wotth a vind Like covering: Sclevo: at certain -tangt Cleistothectume Completely Closed ball Uke shuctuve cotth asd clishibuded at different levels: soris(pustulyA compart moss ol spores, oy o cluster ef sperengia ov rust, produced in oy on the host by fungi: e@uudate] ooze! Dreptels of. baclevia | -lengal uscal lyf mhted with hesd cell decomposition products found 00 surface of lesions. Nemedodyi—Reot @noty galls.cw Abnommeatities 1 _plont_pas ¢- Fem tea} and shoe shy dlatelee] lamina beleveea veing jg vedueed| and ts often coveted into bendy dike Streeluves. lea} roi upword cuwting of leo] trom the margins leo] carl Dawwneword auvling af teaves fren margins Cpinashyy- Download bending ef teal due to fost grat of eof surface or pediole -than vest o| the tee}. dee Reset stunted ‘plants with small) chlootic | pale qvee? » hwisted ond distorted lealtels. IZ. Colour deviation im leaves 1 stems, duils £ towers He Hesaitt Mosaics ove choracteriged by light green, Yellow white aveas intemingied with normal colour of foliage) uits: Hottlee A the discoloured pateh of a voriegated leoves ove younded ; the variegattbo ic usually designated 4s motting + Yellow mosal Scemtive leaf tums geiden Yellovo ing spots chinolic | necietic wings on foliage wein Heeeingt- 09 the batk af -the fea} Covresponcling sto yan cleaved aveoS ave obsewed vein deoringtveins ond veinlels eim Yellow -lorming net like pallern : 5 ; wetion a}. Vein bonding ¢Aeeuene of. dow 4*eeo bonds along veinsSwe ee ee Se ee ee eee ee AY” Vv 4 > Vv - “dillerent detection ffon_ of. the 2 Deledion al plant “pathosens methods and tools used an ide mii fie “pathogens Ievalved io disease se samples PDIGari delestion eetncds tor fungal tdeatittcat? @ > €xaminaten and Companised with drawings in iiteratare a whi ging reys Tn Viteratuve in reratuye sources 4 serological , moleaslay bests Ly Pish — Flevesceace insity hybridisation J HubHplex tandem per SS LANP- Loop -medlated isothermal) ampli Heaton D dius cence Imaging \ PD volattie organic compounds detestion using Gc-MS Metinods tor Barteria) _sdentitiea tore 4 o03¢ test S Biochemical tests =) corbohy dvate utfligaton § Caloto ey —) Faby Actd Hethyl ester Analysis FAM) ~) Molecular methods , Mavieers. _) Nuelete auld based Methods ~ serological sfests fq) ELIGA prolein Based Methods I—) bor host diseases and disorders | | |Methods Joy vinises aden! fication ¢ — Host hangs stesting J staining ineLuston bodies - vintses 2 serological desis — Tmmunsassays -foy vinises 3 Eleehon micrascepy I) Molecular metnods [> Smmuno-Hu rescence [> Flew tytemety [) MuHiplex nested per I) Pot blot assay I) tatera) flow device Dentification of nematodes 6 -) Sdraetton from gell and plat gelation ustag ceatthage! Hoadedion -fey sofl aad dissde n voot | stem materta) + soil 1+ @aeman -funnel techni gues L) Horst chamber eration cov nematodes i issue > pived- exami nation of nematodes yn issue:> > > > > > 5 3 3 > > > > 5 2 > 2 5 - 2 a 2 2 3 Coltuyal audics — Pave culbuve techniques of - > Morvorganisms in natuve occur only as mixed eultuves- A culluve that is grow hom a stagle cll fs called as puve cubhive: the pure cultuve fs necessary , -to study the colony ) Meycetial chavacterisHe morphology , biochemicad chavactetis Hes and immunological ef a parifeutar backeturns tung? and ackinemycetes and -flo maintain its physical ond genetic purity. isolation ¢— — Y Sfagle 5 i> Single sporing ‘nvolves -the dransfer af a single germinated conidfum -to obtain pure culture- L>Place an jneculum af qoeres in a tube Containing tom! af sterile water > Streak this spore suspension aleng a marked ne on tthe suvface af a thin tap watery agar medium , and nebate ak 22°C 5 After gubys ineulation, select germinated spoves casing 4 Glewoseple mlcroscepe and strans}ey one spore ab a -ime to another agav plate: this method is suitable -foy species of duagel genera shat produce spores tq cubtuve. #q1- Fusarium, Collelettchum, Alemaria, Slemphytiuny Bporaris VerHailtium ,Pnoma ete. ifi PY sched tie. enethed 6 > Hyphal -Hp methed tevolves the ‘lransfer of a hgphal ip to ebtalo puye culture: [? tecate the gqrewing -He a} the mycelium and them -foy hyphal -Hp under microscope ink on -the plate toy a single spore > cut the marked avea rnovie and maxe it with isolation > with a micomanipulatov ov A statle cove borer I) find gtranstey onto poA slant cov petidish- I) Gnuibale them @ 2e°%e I this methed 1s suftable 4oy species of toagal georra such as. Phustophthova, Rkigoctonia, Selevotum and selevoltaias(heehhbhhbhbhinhhninbmbRRRBPeRneesue { Puilicatfon af bacteria _by diltevent methods 9) Streak plate _methed 6- Purification af bacteria by streak plate method can be done by -wo methods. ) Simple streck method 2) Giss cross method Simple sheale metned ¢- I) Sterilized loop ts dipped frto ~the mixed culture suspension and -then streaked onthe suvjace of the alveady solidified agav plate by pavaltel and non ~ evevlapping streaks. fp Ges cues eee -) streale on pebi plates containing NA medium , elther jo criss aces GY porallel manner: Procedure ee fj Melt the nubient agay and distibute jn test lube and plug 1 wlth nen absorbant cotton and stenlige- + €eep the test-tube fn slanting position and allow it Lo ent + using “the tnoestatfon needle tale a top -hal) hom the isolated bacterfal colony previously developed: «streak fb en the quttent agar slant and dnaibate for growth af bacteria.| ‘The pour plate method The spread plate method roerotmt fh orm rene “| a + Bacterial diletion | ess itandet” EES Dole =e @=™ di st at ree yaoe ee eee See eee eee. vvvwv v e 9) Gov plate meted « [> fo -this Ne guecessive dilulten of the Inoculum is Prepared tm etthey is sterile cee ee and cooled (uste) ager mediunn sehen tn Hubes I) Betley distribution af colonies is also obtained in a coc] ~made pour plate and isolations ave made easily done- Procedure t- ID Take a joopted trom -the bacterial suspension and transfer |} foto Jest tube containing tom! sterile eater: Ly Hix properly and dranstey 4mi jnto tubes containing al el stevie water and dilution is upto 16% —) Pour into Pehi plates and dishibuted evenly: > pdd NA medium evenly and allow dor the solidi-ficaton i) Gneubate the plates (28 bate) spread plate method & Lo the microorganisms ave spread over -the golidi-lied 0997 medium with a sterile plates “U! shaped gies yods while ‘the pebialish is rept on the Spum tables LS when «tne petridish spuns, the cells get separated thom each other to allow the colonies +o develop from each other Procedure @ > Prepave aga plate containing 15-Rem) of -the NA: > place the ovlml of the bocterial cultuve suspension in the center of -the WA plate.ee ee rode oe Ue ote ee 8 eee T > Dip «rhe bent gless yod tn 484+ alcohol taken in a bealcer 26 seconds ond gently > Remove the glass yod -tor Qo- and Pace the stele glass yod over -the agay suvface ISp49 the petiplates on its ocon axis tor 5 -to min- [> Replace the petyidish cover and immerse the bent red in aleone) and flame it +o sterilize it L_) Theubale “the plates in Inverted positon doy Qu-y7 hs5: Use of selective_media ctor isolation of pathogens, Presewotion of singel and bacterial cultures by 4sing didfevent methods. aoe Selective. necktie > T is possible to isolate speditic plat pathogens by using selecive media iyvespective We “the category of the medium. Sublatle selective medium hag to be identified tor ditterent pathegens - fer tung’ e PDA & Potete Dextose Agar Composition Petia, = 2609 Dexhose — Reg aay a Reg Distilled Heo — \wooml cp'-a) Add oatibodies +o prevent bacterfab gree just before pouring -the plate: ma~ > =~ ~ ~ => > => > => > > => > => > J > =) > —) > = — —_ — = yy | 4 vvvv»e Foy_bactewae wa = Nutrient Agar composition ~ Peptone -84 Yeas) extroct - 2g sedfum chiorider 6a Agar Sioa Dist#Hed 4120 — [ecom! Cpl ey) Fing's B Medium 3- Pseudomenas -+Huresceace compestiion t Protease peptore — 20g K2HP oy — "S49 Hgsey Hae = — "Sg only cero) ons ane Age 883 pistitted #20 _— jecom) oat meal Agar 6- compesi-Hon oats Rolled ~ 304 Ager ~ 18g distited tye - te0om)LYesewa tone To maintain pure cubtave doy extended Periods 10 a Viable condftiens , whhout any geneHe change is Velerredt as preserwaton’ The method of presewation is mainly to types Namely — short-term methods long term methods short feaee s jethods 2 Y perviectic dranster_en agav| in Liquid _medtumt- > 5h nis cattuve can be maintained by perfodically Preparing a deesh euttare chrom “the previous stede cabte this is -the simplest method af presenatton tout also have gome disadvantages dike development of vavfous stapes of contamination ete: b) Precerwation of bacteia using Glycerol Baderia can be degen using 6%!" Glycerol is diued to elt and 09 equal amount of gqycere! and aulduve oth ave mixed , dispensed nto chlbes and “theo -loy drogen at —W0'e » The viability of organiem vartes coll + fe # merths:, ey stage ‘by _drying_metned & spores of. some members which ave -ceasi-tHve | +0 freee drying can be prepaved ying | Ugutd oft Wee yather than -+tegen state:eee eer a ee ee ee ee Dilevent daytng method inetude paper dise , éreladion dise , L~ doying Cdvied over 0S under vaceum)- A port fom Anis organisms ave also dvied over cats tn vacuum and ave store wn veprigerator: fq evartoy d) Storage by _Re | ee ced) we mieooes co ce stored tor lehot duration (2-3 weeks) ot 4 slemperature af ote uve wo clown the medabolfe activity + Refigeration a0 ste +o stop comp tet ely + Grout sometimes , tt does nol able cndinues , nubients ave etfliged and waste products ove Yeleosed fo medium and eltimately ead to death: long -term storage Methods 6 Y sclevotigation & Sclevoria preduclag -liagi ave presewed usually abasic as AN isa geod way “to presewe the sungal Shalns a5 YW vematns lable -lor 2-5 Years b) under mineral ofl & cubtuve ave qroven on agar lant and covered te a depth af tem above -the top af “the slant wh sterile minered off > Hycetial ors el. phytephtheva and pythum spy survive ee when dreated ‘im this way. 1) Fusarium canned be stored well onder oil+ gy silica gel & 11 @ ewoled spore suspension tn sor skimmed milk {5a eae a pee ene Ma Oe rr argv sere cee reece Bu 6 1s poured 0 ~t0 precooled silica gel ja screw cap bottles and allowed 46 doy out at wom temperature udtil the oystal ceparal- ‘The caps ave sevewed down and the boites gtoved ino Yebrigerator over indicator silica gel ab u-e’e. when required a fan crystals ave seatteved on an agay plate . sunival is upto about years according to sps- ®) storage In_watey ¢- Agay culture dises oy bacteral cells ave submerged fo glass vials halt ited eith etertle distilled satey shot can be sealed ov plugged eotth cotton and stored in rebagevetor ov at room ctemperatuyes Jean be stored Wn sterile waley der 2-3 Years: e) sollte 1A spore suspension is poured into sterile soil and alowed -to grow ter abot to days The cultaves cad linen be stored as ter agay slants: |) cuttuves dyealed t9 this way remain viable dor teng pertods: y Page some plant pathogens sumive toy ong periods when dried in Infected hast -tiasues or agay cultures: rine dungus cultuved on wice seeds, POA ov Ceaper's agay jo cutluve plates. when -the colonies ave fully developed » yemeve the CuHuye ‘hom she medtum witha sterileScalpel and place belween -koo sheels af sterile btoller and dry tog dest eeator cootatatng carla: Alte avytag place -them io eealed containevs and store Im Yelrigevators or at room -temperatuve at eo humidity: 9) Porcelain bead technique ~ > Fer growth of the organism » ungtaged porcelain beads impregnated wlth cell af the organism ave stored ovey sillea ge —) smal! gcrew caped glass boltles with silica gel oystals eotth indicator dye ave covered with -rgem thick cotter web and sterilized wlth dry heat for trays @ 160" |? Beads ove sterilized tn clase containers by dry heat 1) Cubtave -the -lingus on a suitable medium and pepave la dense spore suspension In tele cedium qlutamate fn ttguid catture broth can be used divectty i) Baeterial growth can be used direetly+ |) Add 20-30 @Y move sterile beads fnte +he cel) suspension } Shake -oy 6-10 minuies and invert -the -fube allewing suspension 40 -itey trough the colon plug. _> peur beads in fo the container cotth stifea gel and cotton wad: Simmediately cap and store vedeigerotoy @ ue a) ehib destecatfoo , the be silica gel “anes pinte pout as tong aS some onysteds vemain blue, the cultuve fs ees wel) when all Gystal tums pinky otto reettaving ,ee ew a mae 2a =) | SSepritigerton IM is a vacuim sublimation fechnigue- Process of drying sample fa which water is sublimed Cremoved) fhem the substance afer ft & dregen by the principle of Sublimation = “the lyophilization is done by the instrument called as (yop hiliger- — lyophilized caltuves ave stored fa dave at ue fo refrigerator: This can be preserved upto ge yeors| more: z) Oyopreservaton be Storage WW Uguid nitrogen is an ettective way +0 presewe: This method of preservation can be used -for -thase erganicms which enonet be lyophitised : =the culture is stoved | hogened io liquid attregeo et -tqee In pressure and stablliging agents such as gtycere! cor) DMSO Cadimethy! sulphextde) -that prevent the cell damage due +e formation # ice crystals and promote cell survive) + guch cattuves ave viable choy to~30 Years:| | 6 Pathogencity test Techniques of pleat pathegent testing itn Koch's pastalatss Y the suspected organism | eaucal ogcat must be preset Wn every dliseasee! ergentsin examined sa) agent onust in in puye cultuve 2) The suspected cau ve feotaked chon diseased organism ond ge suspected causa) agent Is ZB when a pure eattave of hast 1 the hesk | Jnceedated jnto a healthy susceptible must veproduce speific disease: cate must be recovered ogein to W the same causal °7 inocalated and infecbed hast ye. vecovered Srperimen-teelly ractenists as organism " agent must have the same chor step 2 Jechnigues oer patreencty taity arse 1) visual olgewartion X qPsotatioo 3) Groening uy Tnotulatoo 6) Ye isolation: