biology

The biology essay below has been submitted to us by a student in order to help you with your studies.

Fast And Direct Detection And Identification Of Xanthomonas Biology Essay

Bacterial Leaf Blight caused by Xanthomonas oryzae pv oryzae (Xoo) previously known as Xanthomonas campestris pv oryzae, is one of the most important bacterial diseases of padi. Padi leaves and stems showing symptoms of blight were collected from Penang, Kedah, Serdang, and Melaka between 2008 and 2010. For fast detection of bacteria, infected tissue of leaves and stems were crushed and suspended by blinder were done using CTAB buffer2% and contained in 1.5 ml microfuge tubes at 60°C. The entire mixture was held at 60°C for 20 min. after the incubation, add 600 ml of chloroform: is amyl alcohol (25:24:1; pH 7.6) was added, which they were mixed by vortexing for 1 min and centrifuged at 13000rpm for 8 min. The aqueous phase was Transferred to new tube adds an equal volume of ice cold isopropanol, and place the mixture in ice for 10 min. The solution centrifuged at14000rpm for 15min. DNA pellets were washed with 70% Ethanol, air-dry, and resuspend in 50–100 ml of water. This preparation was used in PCR. Samples of leaves and stem collected and then were identified as Xoo. Strains of bacteria were separated in to 470bp using pair of primers XOR-F and XOR-R2. Comparison with the Gene Bank database in NCBI using BLAST indicated 98-100% homology with Xoo accession number APOO8229 from Japan. We identified causal agent of bacterial blight of rice as X. oryzae pv. oryzae less 5 hour, there fore Direct DNA Extraction from plant tissue with CTAB (modified method) and PCR are considered as the less time consuming, cost effective, and rapid method for the detection of Xoo.

Key words: Xanthomonas oryzae pv oryzae, bacterium, CTAB, PCR and Peninsular Malaysia

Introduction

Rice (Oryza sativa) is one of the most important food crops in the world, feeding about half of the word population (Akhtar et al. 2008). Rice is the second major crop of Malaysia after oil palm. Bacterial Leaf Blight (BLB) caused by Xanthomonas oryzae pv oryzae (Xoo) is an important disease in many rice-growing areas of the world (Nino-Liu 2006). Xoo was first found in Japan in 1884, but was only identified in 1922 by Mew et al, 1993. The disease is found to occur in five continents of the world except Europe (OEPP/EPP, 2007 and Ghasemie et al, 2008).Yield losses of 10–20% are common and losses of 50–70% have been recorded in severely infected fields (Mew et al. 1993). Serious Bacterial leaf blight (BLB) outbreaks in the region was reported in Malaysia in 1988 and 1994, where more than 40% of the planted acreage in the region was BLB infected with an estimated yield loss of about 10-50%. (Saad et al 2000. Bacterial blight enters through wounds or water pores (hydathodes) in the leaf and travels systemically through the plant xylem. the commonly used methods such as isolation on semi-selective medium,(mXOS) using Peptone sucrose agar (PSA), Nutrient Broth Yeast Extract agar medium (NBY), Growth Factor (GF) agar Serological methods (ELISA), pathogenicity tests on host plants, biochemical tests and PCR method for diagnosis.(OEPP/EPP2007). ). Polymerase chain reaction (PCR) methods for amplifying a unique nucleotide sequence from the chromosome of targeted phytopathogenic bacteria have been reported (Adachi, 2000 and Li 1995).The present research focus direct detection of DNA extraction Xoo in samples in leaf and stem which we identified with asset primer XOR-F and XOR-R2 .

MATERIALS AND METHODS

Sample collection: Samples were collected with symptoms of blight infected tissue of leaves and stems from rice fields in Penang, Kedah, Selangor and Melaka in Peninsular Malaysia. Five rice fields were surveyed from each area. Ten symptomatic samples were collected from each field. The samples were labeled alphabetically and kept at 4°C in plastic bags on ice and were transferred to the laboratory.

Direct DNA Extraction from tissue plant with CTAB (modified method)

All samples rice were crushed and suspended using CTAB buffer (cetyltrimethylammonium bromide) (800 ml) (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris–HCl, pH 8.0, 0.2% mercaptoethanol) and contained in 1.5 ml microfuge tubes at 60°C. The entire mixture was held at 60°C for 20 min. During incubation, the mixture was briefly vortexed several times. after the incubation, add 600 ml of chloroform: isoamyl alcohol (24:1; pH 7.6) was added, which they were mixed by vortexing at the maximum speed for 1 min and centrifuged at 13000rpm for 8 min an Eppendorf Centrifuge model 5414. The aqueous phase was Transferred to new tube adds an equal volume of ice cold isopropanol, and place the mixture in ice for 15 min. The solution centrifuged at14000rpm for 15min. DNA pellets were washed with 70% Ethanol, air-dry, and resuspend in 50–100 ml of water. This preparation was used in PCR. (Modified method Zhang Y.P 1998).

PCR primer and conditions: XOR-F(5′-GCATGACGTCATCGTCCTG-3′) and XOR-R2 (5′-CTCGGAGCTATATGCCGTGC-3′) were made from the 16s-23s rDNA spacer region of Xanthomonas oryzae pv oryzae (Adachi, 2000), with a predicted PCR product of 470 bp. PCR reaction was performed in a 25 μl PCR mixture, PCR master mix(2X) is an optimized ready to use PCR mixture of Taq DNA polymerase (0.05unit / µl) Taq DNA in reaction buffer,mgcl2(4mM) and DNTP(0.4DATP,0.4mMDCTP,0.4mMDGTP and 0.4mMDTTP).1μl volume of DNA bacterial cells, 1 μl volume of primer forward, 1 μl volume of primer reverse was added to 12.5μl master mix ferments (2X) and 8.5 μl Water(PCR grade). Samples were amplified through 29 cycles, each consisting of 45 sec at 95 °C, 1 min at 51 °C, (modified angling temperature) 2 min at 72 °C, with initial denaturation of 2 min at 95 °C and final extension of 10 min at 72 °C . (Adachi, 2000).5μl of each amplified PCR product was fractionated on a 1.5% agarose gel in TBE buffer1X, Gel was stained with ethidium bromide and photographed under UV light (312 nm). (Martins et al. 2005).

Sequencing: PCR products were be purified by DNA purification kit (QIAGEN) and send for sequencing.

Result:

Isolates (34 of leaf and 8 of stem) are presented in Table 1 .

Table 1: characteristics of Xanthomonas oryzae pv oryzae used in this search

Number

strain

Isolation part

Geographic origin

variety

1

PXO6

leaf

Penang

MR84

2

PXO9

Leaf

Penang

MR84

3

PXO16

leaf

Penang

MR219

4

PXO21

stem

Penang

VR2320

5

PPXO26

leaf

Penang

MR220

6

PXO36

Leaf

Penang

MR232

7

PXO39

Seed

Penang

MR219

8

PXO41

Leaf

Penang

MRQ84

9

PXO43

Stem

Penang

MRQ84

10

PXO44

Seed

Penang

Red rice

11

PXO48

Stem

Penang

Local

12

KXO11

Stem

Kedah

MR84

13

KXO5

leaf

Kedah

Red rice

14

KXO219

Leaf

Kedah

MR219

15

KXO1

Leaf

Kedah

MR219

16

KXO3

Leaf

Kedah

Local

17

KXO9

Leaf

Kedah

Local

18

KXO12

Leaf

Kedah

Local

19

KXO14

leaf

Kedah

Local

20

KXO162

Leaf

Kedah

Red rice

21

KXO182

Leaf

Kedah

Red rice

22

KXO324

Leaf

Kedah

Local

23

KXOM

Stem

Kedah

MR219

24

SXO1

Leaf

Serdang

EMR219

25

SXO4

Leaf

Serdang

EMR219

26

SXO5

Leaf

Serdang

EMR220

27

SXO6

Leaf

Serdang

EMR219

28

SXO7

Leaf

Serdang

EMR219

29

SXO8

Leaf

Serdang

EMR220

30

MXO1

Leaf

Melaka

MR220

31

MXO5

Leaf

Melaka

Red rice

32

MXO6

Leaf

Melaka

MR220

Detection of X. oryzae pv. oryzae by direct PCR All DNA samples of X. oryzae pv. oryzae were identified by specific primers XOR-F. and XOR-R2 On 1.5%. agarose gel electrophoresis, isolates produced a band of 470 bp. (Figure 1 and 2).

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Agarose gel electrophoresis of PCR products, with specific primers of XOR-F and XOR-R2: Lane19 is negative control (distilled water); lane 1 until l8, showing the amplification of approximately 470 bp, isolates from rice Penang and Kedah in Peninsular Malaysia .the molecular size marker is 100bp.

.

19 20 21 222324 25 26 27 27 28 29 30 31 3233 34 35 36 37 M

Figure 2: Agarose gel electrophoresis of PCR products, with specific primers of XOR-F and XOR-R2: lane 19 until 37, showing the amplification of approximately 470 bp, isolates from rice Kedah, Selangor and Melaka in Peninsular Malaysia .the molecular size marker is 100bp

Discussion:

Based on PCR using specific primers XOR-F and XOR-R2 and after sequencing, we identified causal agent of bacterial blight of rice as X. oryzae pv. oryzae. According to Ochiai et al. (2000) RFLP analysis with the repetitive DNA element revealed a high level of polymorphism in the Sri Lankan isolates. Lee et al. (1999) and Ghasemie, et al(2008) characterized a large number of X.oryzae pv. oryzae isolates by genotypic analysis. Direct DNA Extraction from tissue plant with CTAB (modified method) with other techniques is very short, easy and is obtaining good quality DNA. PCR techniques with specific primers of XOR-F and XOR-R primers can be applied to detect and identify Xanthomonas oryzae pv oryzae. Direct DNA Extraction from tissue plant with CTAB (modified method) and PCR are considered as the less time consuming, cost effective, and rapid method for the detection and identification of pathogenic bacteria, although many improved methods (biochemical test, serological assays, fatty acids, and metabolic profiling) have been developed so far. There, Direct DNA Extraction and the PCR assay using a primer set primers of XOR-F and XOR-R were made from the 16s-23s rDNA spacer region of Xanthomonas oryzae pv oryzae (Adachi, 2000),of X. o. pv. oryzae will be a useful tool for the fast detection and identification of X. oryzae pv.. The development of varieties with different genes for resistance is the best option to reduce incidence in the field and this securing is an environmentally and economically acceptable solution to the problem. Potential incidence of Xoo was reduced if MR 84 could be replaced with other improved varieties. However, sustainability of any released varieties will be influenced by the resistance level of the genes involved, and the pathotypes is present at the locality. (Saad, A. and Habibuddin, H. 2002).Until now we have detected 43 different strains BLB from rice variety such as MR219 in Peninsular Malaysia that indicate this disease exists in different places in Malaysia but variety with genetic diversity for resistance became inhibition epidemic BLB in Malaysia like in 1988 -1994 .


Request Removal

If you are the original writer of this essay and no longer wish to have the essay published on the UK Essays website then please click on the link below to request removal:

Request the removal of this essay


More from UK Essays