Diseases and Management of Crops under Protected Cultivation

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(Diseases and Management of Crops under Protected Cultivation) dependent on the performance on the PCR assay itself, but also on the efficiency of the procedure employed to extract the nucleic acids from the plant material. To check for substances that may interfere with the amplification process, internal controls can be designed for each pair of primers, or real-time PCR can be employed. PCR efficiency is controlled by many parameters, such as polymerase type, buffer composition and stability, purity and concentration of dNTPs, cycling parameters as well as the characteristics of the starting template. Several expensive commercial integrated systems allow for the automated extraction and analysis of nucleic acids from microorganisms, but they are not efficient with all types of plant material and need to be evaluated before they can be adopted for routine detection. The specific primers have been designed based on either the amplification of specific genes from the chromosome or plasmids ( Table 3) or on different approaches such as sequences selected from RAPD differential bands obtained by subtractive hybridization. Some specific primers have been developed for detection of R. solanacearum from tuber, seeds and planting material, soil as well as irrigation is given in Table 2 & 3. Table 2. Detection Ralstonia solanacearum from seed and planting materials by using PCR based techniques. PCR Genomic Host Detected Race / biovar of References assay from Bacteria RAPD Brinjal, chilli and - R. solanacearum race James et al. 2003 tomato 3 16S r RNA Tomato - R. solanacearum race Singh et al. 2010. 1 biovar 3 and 4 Fli C - soil R. solanacearum, R. Schonfeld et al. 2003 pickettii Nested- PCR Banana, tomato, Soil R. solanacearum Khakvar et al. 2008 chili, eggplant and tobacco hrp gene, PCR Tomato, potato Plant and R. solanacearum Poussier et al. 1999 tobacco, eggplant, tuber pepper Bio-PCR Tomato, melon soil R. solanacearum race 1 biovar 3 16 r RNA, PCR - - 16 r RNA, PCR Potato 16 r RNA, Multiplex PCR Multiplex –PCR (16S-23S r RNA ITS) Real Time Bio- PCR Potato tuber R. solanacearum subdivision 2a R. solanacearum R. solanacearum biovar1, 2, N2, 3 , 4 & 5 Potato Tuber R. solanacearum (Bertolini et al., 2003) Potato Tuber and plant R. solanacearum race 3 biovar 2 Lin et al., 2009 Boudazin et al. 1999 Pastrik and Maiss, 2000, Seal et al. 1999 Pastrik et al. 2002 Weller et al. 1999; Weller et al. 2000 16S r(NASBA)RNA Potato Tuber, irrigation water R. solanacearum race 3 biovar2 van der Wolf et al. 2004 - 49 -
(Diseases and Management of Crops under Protected Cultivation) IC-PCR Tomato, pepper, weeds ( Physalis minima, Amaranthus spinosus and Cytochromec1 signal peptide Plant and soil Euphorbia hirta) Tomato Soil and plants R. solanacearum race 1 R. solanacearum race 1 & 3 biovars 1, 2, 3 &4 Dittapongpitch Surat, 2003 Kang et al., 2007 and ii. Nested -PCR Sensitivity and specificity problems associated with conventional PCR and RT-PCR can be reduced by using nested PCR-based methods, based on two consecutive rounds of amplification. Usually, the products of the first amplification are transferred to another tube before the nested PCR is carried out using one or two internal primers. The potential of nested-PCR in plant pathology has been already reported (Pradhanang et al. 2000). Sensitivity is increased by two orders of magnitude reaching about 10 2 bacterial cells/ml of extract. However, the two rounds of amplification in different tubes also increase the risk of contamination, especially when the method is used on routine in a large scale. One limitation of the nested PCR approach concerns the need to accurately establish the ratio between external and internal primers and the use of limiting amounts of external primers to avoid interference during the second amplification. ii. Co-operational -PCR It is a new PCR, which has high sensitivity for the amplification bacterial targets from plant material. The Co-PCR (co-operational amplification) technique can be performed easily in a simple reaction based on the simultaneous action of four or three primers. The reaction process consists of the simultaneous reverse transcription of two different fragments from the same target, one internal to the other, the production of four amplicons by the combination of the two pairs of primers, one pair external to the other, and the co-operational action of amplicons for the production of the largest fragment. Metal block and capillary air thermal cyclers have been employed for the detection of a bacterium, but by using only three primers, which shows the possibilities of this new approach. The low amount of reagents (ten times less than in conventional PCR) probably increases susceptibility to inhibitors. However, this step was not necessary when analyzing the presence of R. solanacearum in water. Co-PCR requires only one reaction, minimizing manipulation and reducing risk of contamination. iii. Real-time (quantitative) – PCR Quantitative real time PCR has become possible by the development of detectors that can measure fluorescence that is emitted during the PCR cycle. This method is based on the 5’-- > 3’ exonuclease activity of the Taq DNA polymerase, which results in cleavage of fluorescent dyelabeled with different probes (TaqMan ® ) during PCR. The exponential nature of PCR in theory allows the amount of starting material to be calculated from the amount of product at any point in the reaction. In practice, however, reaction conditions can interfere with exponential amplification - 50 -
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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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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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Diseases and Management of Crops under Protected Cultivation

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