QualiPlate kit for Detection of Soybean Rust. Murali Bandla EnviroLogix Inc

Transcription

1 QualiPlate kit for Detection of Soybean Rust Murali Bandla EnviroLogix Inc

2 Putting Science to the Test

3 EnviroLogix Inc. WHAT DO WE DO? We develop, manufacture and market immunoassay test kits for the detection of certain chemicals, agrochemicals, toxins, molds, transgenic crop traits and plant pathogens that may be found in water, soil, air, building materials, food, plant and crop matrices

4 Test Kit Formats ELISA Quantitative 1-22 hours Strip or micro-plate reader Lateral Flow Device Yes/no qualitative results Typically 5 minutes or less Simple procedure for lab or field Semi-quantitative with reader

5 The use of immunoassay for SBR detection ELISA Lateral Flow Devices

6 The use of immunoassay for SBR detection Scouting Monitoring Sentinel plots Confirmation (Primary) Aid for visual detection Spore traps IPM and risk management

7 ELISA Anti-SBR conjugate Antibody to SBR AOAC Midwest 2005

8 Development of ELISA test kit for SBR Unv of Free Orange State Bloemfontein, SA

9 Development of ELISA test kit Preparation of antigens Immunizations and antibody development Characterization of antibodies Development of Immunoassay Validation for SBR

10 Sample Preparation Snap a suspected leaf spot, mash in tube with Extraction Buffer

11 Sample Preparation-Whole leaf Add one leaf to a mesh bag Break up leaf by rubbing a hard object across it (pen, wooden pestle, etc.)

12 Sample Preparation-Whole leaf Add 20 drops buffer Rub again with a hard object to mix and extract Pipette or pour extract into a vial

13 ELISA Procedure Add samples Mix and incubate one hour

14 ELISA Procedure Add ready-to to-use conjugate Mix and incubate one hour

15 ELISA Procedure Wash plate with Wash Buffer Add Substrate

16 ELISA Procedure Incubate 30 minutes Blue color will begin to develop in positive wells Optional Stop

17 Characterization of antibodies Specificity

18 Soybean rust can be confused with other soybean diseases Septoria Brown Spot Bacterial Pustule Bacterial Blight Downy Mildew Frogeye Leaf Spot Cercospora Leaf Blight Photos courtesy

19 Organism/disease P. pachyrhizi P. meibomiae Fusarium sp. Rhizoctonia sp. Puccinia triticina Puccinia graminis Puccinia striiformis Puccinia helianthi Uromycces appendiculatus Aspergillus sp. Botrytis sp. Bean mosaic virus Pseudomonas savastanoi pv glycinea Xanthomonas axonopodis pv glycinea Uninfected soybean leaf

20 Characterization of antibodies Specificity Sensitivity

21 Sensitivity of SBR ELISA test 1 Absorbance (450 nm) spores 0 Single pustule Infected leaves Extraction buffer Uninfected leaves Samples

22 How early the infection can be detected?

23 Inoculation of soybean seedlings with urediniospores showed that high inoculum levels were detectable immediately after spore deposition whereas lower concentrations were positively detected in leaves five days post-inoculation. Time trial with high inoculum levels Time trial with low inoculum levels Absorbance (450 nm) Visible lesions Absorbance (450 nm) Visible lesions Visible lesions Days post infection 0 0:preinfection 0:postinfection 0:preinfection 0:postinfection Days post infection Undiluted infected tissue 1/2 diluted infected tissue 1/4 diluted infected tissue 1/8 diluted infected tissue 1/16 diluted infected tissue 1/32 diluted infected tissue Undiluted uninfected tissue Extraction buffer

24 Asymptomatic leaves 4 d.p.i. (high inoculum level [10 to 15 infection sites/cm²]) Symptoms 11 d.p.i. (low inoculum level [1 to 2 pustules/cm²]) Spore Appresorium The average colony size of P. pachyrhizi 5 d.p.i.

25 Validation

26 Following the detection of rust-infected kudzu (Pueraria lobata) at a new site in Mpumalanga, South Africa, both the ELISA and PCR (Frederick et al., 2001) tests confirmed the identity of the pathogen as P. pachyrhizi. Uninfected soybean leaf Soybean leaf infected with P. pachyrhizi Soybean leaf infected with Kudzu rust From top to bottom: Undiluted infected leaf sample ½ diluted infected leaf sample ¼ diluted infected leaf sample 1/8 diluted infected leaf sample 1/16 diluted infected leaf sample 1/32 diluted infected leaf sample Undiluted uninfected soybean leaf sample Extraction buffer 200 bp 100 bp P. pachyrhizi specific amplification P. meibomiae specific amplification P. pachyrhizi and P. meibomiae specific amplification

27 Dr. Carrie Lapaire Harmon, Assistant Coordinator, SPDN University of Florida/IFAS Department of Plant Pathology

28 Stephen R. Vann, PhD. Extension Plant Pathologist, Assistant Professor Plant Disease Clinic, University of Arkansas R = Plants inoculated in greenhouse, no recognizable pustules observed at the time sample was processed for ELISA, however "specks/flecks" visible to leaves. A = Senescent leaf samples (w/ pustules) collected and refrigerated until processing.

29 Test performed by crop research scientists Florida April 2005

30 Current programs at EnviroLogix Improving the sensitivity Monoclonal antibody development Polyclonal antibody development in goats Development of LFD

31 Dr. Botma Visser, Dr. Zack Pretorious University of Free State Bloemfontein, SA

32 Dr. Alan Henn, Mississippi State University. Dr. Phil Harmon and Carry Harmon, University of Florida. Dr. Stephen R. Vann, University of Arkansas. David Davison, Florida Dept of Ag. & Consumer services.