Diseases and Management of Crops under Protected Cultivation

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(Diseases and Management of Crops under Protected Cultivation) and affect product concentration. Early attempts at quantization involved stopping the PCR reaction at various points to generate standard curves, which resulted in a laborious, lowthroughput process. In TaqMan ® probe an oligonucleotide probe sequence of approximately 25 -30 nucleotides in length is labeled at the 5’ end with a fluorochrome (6- carboxyfluorescein - 6- FAM) and a quencher fluorochrome (carboxytetramethyl- rhodamine -TAMRA), at the 3’ end. The probe is degraded by the 5’-3’ exonuclease activity of the Taq polymerase as it extends the primer during each PCR amplification cycle and the fluorecent chromophore released. The amount of fluorescence is monitored during amplification cycle and it propotional to the amount of PCR product generated. Real-time reaction monitoring with specific instruments and fluorescent probes combines amplification, detection and quantification in a single step. TaqMan probes consist of single-stranded oligonucleotides that are complementary to one of the target strands. A fluorescent dye adorns the 5’ end and a quencher is bound to the 3’ end. Fluorescence occurs when the polymerase replicates a template on which a TaqMan probe is bound and the 5’ exonuclease activity cleaves the probe. R. solanacearum. 100 cfu/ ml of pure culture of R. solanacearum was detected by using TaqMan ® and the 7700 sequence detection system using either a genus- specific 16 S probe or a biovar 2 specific probe. However, when spiked potato tuber extracts were assayed, the threshold for detection dropped significantly 10 5 and 10 7 cfu/ml with the species specific and biovar 2 specific probes respectively in tuber extracts diluted by 1:10 and in undiluted extract, the threshold was 10 7 cfu/ml because of presence of PCR inhibitors. iv. Bio- PCR In this PCR, combine the viable enrichment of mostly growth media with an enzymatic amplification, which is known as BIO-PCR. The target bacterium is enriched in liquid or solid media and detected at extremely low levels in seeds and other propagative materials. The BIO- PCR assay includes the following simple steps as- i) extracting a sample ii) plating a sample onto agar media (half are retained for visual recovery or adding sample to liquid medium iii) incubating for 15 -72h, depending on the growth of target bacterium; iv) washing plates to remove bacteria or centrifuge liquid medium; and using 1 or 10 µl for direct PCR. The samples can be stored at – 20 o C. This techniques has several advantages over tradition PCR including increased sensitivity of the primer, elimination of PCR inhibition, and detection of viable cells only. It can be used in conventional - PCR or real time PCR. The key to developing a successful real –time BIO-PCR protocol are choose suitable semi-selective or selective liquid or solid medium for bacterium and determine precisely the time for enrichment. In solid medium only pin point size of colony of bacterium may be selected for PCR because larger size (1 mm) of colony may allow to grow other saprophytic bacteria. The most plant pathogenic bacteria are 24 -48 h to grow pin point colonies. However, R. solanacearum growing fast and require only 10 -15h. A single pinpoint size colony normally contains over 1000 cells/ ml, which is the threshold for the PCR. Mucoid colony of - 51 -
(Diseases and Management of Crops under Protected Cultivation) bacterium should be avoided for PCR. In Bio –PCR, semi selective medium as SM-1 was developed and further modifying the medium as MAS-1 used for R. solanacearum. The detection limit of this medium for through this technique is 100 cfu/ml from soil based on tested artificially or naturally infested soils. R. solanacearum was detected from soil of weed rhizoshphere, weed root and water with high sensitivity of detection 1.9 cfu/ ml and 17 cfu/g of soil using bio-PCR. Management of Bacterial wilt diseases of solanaceous crops Use disease free tubers and seeds. Treat tubers with streptocycline 500 ppm concentration. Grow bacterial wilt resistant varieties of following crops – Brinjal: Arka Keshav (long type), Arka Nidhi (long type), Arka Neelkanth (long type), Pusa Anupam (round type)and Hissar Shyamal (round type), Singnath. Tomato: Palam Pink, Palam Pride and F1 7711, Arka Rakshak, Arka Abha, Swarna Sampada Chilli: Surajmukhi Disinfecting the cutting knives with suitable bactericide. Avoidance of surface water for irrigation, cutting of potato seeds and education. The incidence of wilt diseases in tomato was reduced by applying panchagavya which contains 20 ml of cow ghee, 50 ml each of cow milk, and curd, 40 ml of cow urine, 400g of cow dung, 20 g of common salt and 10g bakers yeast. Biocontrol agent Pseudomonas fluorescens and Bacillus subtilis found effective to control the disease. Crop rotation is not effective as the pathogen can survive for a long period (several years) in the soil and also attack a wide range of crops and solanaceous weeds. Crop rotation 2-3 years with wheat and Crotolaria juncea Do not grow crops in soil where bacterial wilt has occurred. Rogue out wilted plants from the field to reduce spread of the disease from plant to plant Control root-knot nematodes since they could facilitate infection and spread of bacterial wilt Where feasible, extended flooding (for at least 6 months) of the infested fields can reduce disease levels in the soil Soil amendments (organic manures) can suppress bacterial wilt pathogen in the soil Good results have been encountered by using biofumigation as soil treatment for bacterial wilt. Application of bleaching powder 12 -15 kg/ ha in the furrow during transplanting/ sowing tubers in the field. Actigard (acibenzolar-s-methyl) has shown to be effective, but only when inoculum densities are low. In India, CRA 66 rootstocks were used in grafting of tomato to reduce bacterial wilt in - 52 -
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CENTRE OF ADVANCED FACULTY TRAINING
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CONTENTS Sl. No. Title Speaker Page
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REMARKS by Dr. K.S. Dubey Director
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and extension literature that have
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Water Requirement (lpd/plant) (Dise
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clear (Boulard et al, 1989). (Disea
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4. High value off season vegetable
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ANNEXURE-I CENTRE OF ADVANCED FACUL
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9. Dr. (Mrs.) T.K.S. Latha Assistan
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TRAINING ON ANNEXURE-III DISEASES A
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ANNEXURE-IV CENTRE OF ADVANCED FACU
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14:30-17:00 hrs Visit to VRC for pr
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Sunday 23.9.2012 Monday 24.9.2012 1
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