Tests for detection of Oomycete root and stem rot pathogens Tim P - - PowerPoint PPT Presentation

Tests for detection of Oomycete root and stem rot pathogens

Tim P Tim Pettit ettitt Univ Univer ersity sity of

• f

Wor

• rce

ceste ster

Main oomycete root and stem rot pathogen genera are Pythium, Phytophthora & Aphanomyces

• At least 90 species recorded worldwide on horticultural crops grown in UK
• Of these 28 are serious in UK and a further 26 pose a significant threat

Detection and diagnosis of oomycetes

Testing What? Why? When? Detection a) Conventional approaches b) More recent approaches AHDB CP136 Developing rapid and precise detection for use on nurseries

Testing

Main categories

1 Water treatment efficacy 2 Problem-specific - Are these plants infected by x?

• Is x present in this water/substrate?
• Is x present on the surfaces?

3 Health checks

• Are my water storage and distribution clean?
• Are my containers/production facilities clean?
• Is this incoming planting material clean?
• Water checks – should I treat this before using?

Detection

‘Conventional’ Baiting and ‘floats’ Direct plating More recent Immunodiagnostics Nucleotide based techniques e.g. PCR

Pythium (2-3 days)

Baiting A - Detection in water

• Limited capacity for quantification
• Used since the 1960s for Pythium and Phytophthora detection
• Confirmation of live pathogen presence/absence
• Takes time and skill = too slow to assist growers in useful disease management

decisions

Baits floated in water and transferred to antibiotic agar Lupin seedlings floated in water and

• bserved directly under microscope

Baiting B - Detection in soil/media

• Isolation of oomycete ‘damping off’ species :

damp soil incubated with bait seeds for 2-5

• days. Plate seeds on selective agar. Pythium/

Phytophthora will grow out in 2-3 days.

• Isolation of oomycetes species : Cover soil to

a depth of about 2 cm in water. Float baits

• n water for 1-7 days. Often a fringe of

sporangia will de accumulate around the bait – sometimes possible to identify from this,

• therwise baits plated as above.

☺ Large quantities of soil/media can be tested – can detect low live pathogen

densities

 Identification to the species level requires additional time and skilled

specialist work

Direct isolation A: Water

☺ Straightforward, accurate and robust  Takes time and requires skill in interpretation Membrane filtration – colony plating

Direct isolation B: from soil, plant tissue and other materials

• Soil or other material plated on a selective agar – colonies after 2 – 7

days.

• Plant tissue surface sterilised and plated on selective agar medium

mycelium growth after 2 – 7 days.

• To identify the species involved, isolation and sub culturing is needed.

Sometimes a bioassay can speed this up.

Immunoassays - general

• Use antibodies as probes that recognise target species at the

molecular level e.g. able to bind selectively to a specific protein, carbohydrate , lipid or glycoprotein complex.

• Tests can be qualitative (+/-) or quantitative.
• Antibody probes have been developed to discriminate at the

genus, species and stage of an oomycetes life cycle (zoospore).

• Immunoassay tests can be run as a single or multiplex test
• Immunoassay tests have been developed for the laboratory and
• n-site usage.
• Immunoassays used in a wide range of environmental samples

e.g. air, water and soil

• On site tests retail at approx.. £8-10/test.

Estimation of amounts of Phytophthora infestans mycelium in leaf tissue by ELISA. Harrison et

• al. Plant Pathology Volume 39, Issue 2, p ages 274–277, June 1990
• Often used in a 96 well format for high volume sample testing
• Can provide measurement of multiple targets in a single sample
• Requires laboratory analysis and skilled technical staff
• 3 -4 hours

Types of immunoassays used in diagnosis of

• omycetes

ELISA (Enzyme-Linked Immunosorbent Assay)

• Water samples filtered through a cellulose nitrate

membrane

• Glucose and antibiotics added – germinates target

spores

• Specific antibody-based stain highlights target

germlings

• ZTI Found to be more sensitive than conventional
• membrane filtration-dilution methods, dip stick

immunoassays

• and conventional bait tests

Types of immunoassays used Zoospore trapping immunoassay (ZTI)

• Cellulose nitrate membranes

attached to acetate sheet “handles”

• “Handles” facilitate suspension of

membrane in water

• Air dried and probed antibody stain

(as with ZTI – can germinate spores too)

• Dipsticks operate rather the same

way as baits – similar sensitivity

Types of immunoassays used Dipstick assays

• On-site usage
• Results within 10 minutes
• Specificity currently at genus level –

not species

• Cross reactivity (false positives) ?
• No information on viability
• Have been combined with baiting

PO/HNS 188

Types of immunoassays used Lateral flow devices (LFD)

Molecular tests (DNA /RNA)

• Based on specific amplification of a pathogen(s) genetic code
• Can be used to detect (PCR) or quantify (qPCR) oomycetes
• Tests developed for many plant pathogens (incl.

Phytophthora ramorum, P. infestans)

• Can be designed to detect at genus, species or pathotype

level

• Can be used in all types of sample (plant, water, soil, air)

PCR and qPCR

Benefits:

• Widely used in research labs
• Highly specific
• Can be very sensitive
• Wide range of substrates (soil, water, plant tissue)

Limitations:

• Highly trained operator
• Relatively high cost
• Relatively small sample volume
• Generally only performed in lab (but advances in

portability)

Development and testing of diagnostic devices for rapid and precise early detection

• f oomycete root and collar rot pathogens

AHDB Project CP 136

Project aims:

• Develop a new generation of antibody-based

diagnostic tests for pathogenic species of Phytophthora and Pythium

• A hierarchy of specificity: Phylum, Genus and

ultimately species specificity would be ideal although clade specificity would still be an excellent result

• A test for oomycete viability
• Multiplex tests

Nurseries tested to date = 55

Sectors represented: Protected edibles 8 Protected ornamentals 15 HNS & Trees 16 Soft Fruit 7 Field vegetables 2 Herbs 2 Botanical collections 5

• 110 isolates of pathogens and background species

described and collected

• Similar number of isolates have been tentatively

identified and subsequently discarded

• 39% from water samples
• 45% from plant tissue samples
• 4% from swab tests
• 2% directly from growing media

Plant samples

Phytophthora clades 1, 2, 4, 8 Pythium clades A, B1, B2, D, F, G, H, I, J

Water samples

Phytophthora clades 1, 2, 6, 8 Pythium clades A, B2, F, H, I

Media samples

Phytophthora clades 8 Pythium clades B2, D, F

Swab samples

Phytophthora clades 8 Pythium clades B2, F, I

14 Phytophthora species - 11 = serious pathogens 21 Pythium species - 8 = serious pathogens PLUS A representative range of other commonly-encountered species: Other oomycetes – most commonly Saprolegnia species Fungi: Mortierella, Mucor, Penicillium, Cylindrocarpon, Fusarium

Trichoderma

From these collections, isolates were used to raise antibodies and selected as representatives in cross-reactivity and validation tests

0.20 0.70 1.20 1.70 2.20 2.70

Antibodies 2B5, 5A9, 4F3, 6C8, 3B6, 1A10, 5F3, 1A11 & 5D3

2B5 G2 F3 5A9 H2 E1 4F3 E2 G1 6C8 A1 H2 3B6 A3 E1 1A10 G3 C6 1A11 G2 A10 5F3 H1 G2 5D3 C5

Cross-reactivity tests can often be rather disappointing

Nevertheless, this figure illustrates 2 things very well: (1) how important testing is and (2) just how much testing is often necessary to yield useful antibodies (this is just the anti-Phytophthora sets)

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 Phytophthora cactorum Clade… Phytophthora pgchlamydo… Phytophthora pgchlamydo… Phytophthora gonapodyides… Phytophthora rubi… Phytophthora rubi… Phytophthora rubi clade 7 Phytophthora cinnamomi… Phytophthora cryptogea clade… Pythium aphanidermatum… Pythium utonaiense Clade B Pythium dissotocum Clade B Pythium kashmirense Clade B Pythium oligandrum Clade D Pythium rostratum Clade E Pythium irregulare Clade F Pythium pectinolyticum Clade F Pythium violae Clade G Pythium ultimum var… Pythium nunn Clade J Saprolegnia ferax Saprolegnia parasitica Mucor circinelloides?

• C. confragosa

Motierella zychae (CC296) Fusarium (C276A/PR UV) Fusarium C352/OB3) C325 Butsa Trichoderma C366/1 Trichoderma (C371/3F2) Trichoderma C355/3/SSF Cladosporium cladosporoides Phoma lingham

3H7 H3

Over 30 MAbs raised to Phytophthora antigens

Of these, 2 have been selected for further development although we have other promising candidates

Genus – specific probe for Phytophthora

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 Phytophthora cactorum Clade 1a Phytophthora pgchlamydo Clade 6 Phytophthora pgchlamydo Clade 6 Phytophthora gonapodyides… Phytophthora rubi Clade 7 Phytophthora rubi Clade 7 Phytophthora rubi clade 7 Phytophthora cinnamomi Clade 7 Phytophthora cryptogea clade 8 Pythium aphanidermatum Clade A Pythium utonaiense Clade B Pythium dissotocum Clade B Pythium kashmirense Clade B Pythium oligandrum Clade D Pythium rostratum Clade E Pythium irregulare Clade F Pythium pectinolyticum Clade F Pythium violae Clade G Pythium ultimum var… Pythium nunn Clade J Saprolegnia ferax Saprolegnia parasitica Mucor circinelloides?

• C. confragosa

Motierella zychae (CC296) Fusarium (C276A/PR UV) Fusarium C352/OB3) C325 Butsa Trichoderma C366/1 Trichoderma (C371/3F2) Trichoderma C355/3/SSF Cladosporium cladosporoides Phoma lingham

3C4 A3

Over 30 MAbs raised to Phytophthora antigens

This is the second of these to be selected for immediate further development for its stability and its very useful clade 7/8 specificity

Species/clade – specific probes

Phytophthora clades 7/8

Tests

Suspected by morphology

LFD positive Confirmed by ITS Mycelium tests 65 7 9 9 Tissue baits 36 (10) 9 9 Membranes and swabs 23 (4) 4 4

Results of initial ‘in vitro’ tests of 3H7 H3 LFD test strips

Next phase of project Starting nursery evaluations

Sectors:

• Soft fruit – strawberries
• Protected ornamentals
• Trees (some samples already)

Approach: Still being developed, but I know we have to move fast!