Interim Report Form · 2020. 3. 18. · FOL4 by PCR / genome analysis 31/10/19 31/10/19 10.2. Develop and test qPCR / LAMP diagnostics for FOL4 31/10/19 31/10/19 10.3. Develop method

Horticultural Fellowship Awards

Interim Report Form

© Agriculture and Horticulture Development Board 2019. All rights reserved. 2

Project title: Maintaining and developing capability in

vegetable crop pathology

Project number: CP 113

Project leader: Dr John Clarkson, Warwick Crop Centre,

University of Warwick

Report: Annual Report, October 2019 (Year 6)

Previous report: Annual Report, October 2018 (Year 5)

Fellowship staff: Dr John Clarkson & Dr Andrew Taylor

(“Trainees”)

Location of project: Warwick Crop Centre

Industry Representative: n/a

Date project commenced: 1st November 2013

Date project completed

(or expected completion date):

28th February 2021


DISCLAIMER

While the Agriculture and Horticulture Development Board seeks to ensure that the

information contained within this document is accurate at the time of printing, no warranty is

given in respect thereof and, to the maximum extent permitted by law the Agriculture and

Horticulture Development Board accepts no liability for loss, damage or injury howsoever

caused (including that caused by negligence) or suffered directly or indirectly in relation to

information and opinions contained in or omitted from this document.

© Agriculture and Horticulture Development Board 2019. No part of this publication may be

reproduced in any material form (including by photocopy or storage in any medium by

electronic mean) or any copy or adaptation stored, published or distributed (by physical,

electronic or other means) without prior permission in writing of the Agriculture and

Horticulture Development Board, other than by reproduction in an unmodified form for the

sole purpose of use as an information resource when the Agriculture and Horticulture

Development Board or AHDB Horticulture is clearly acknowledged as the source, or in

accordance with the provisions of the Copyright, Designs and Patents Act 1988. All rights

reserved.

All other trademarks, logos and brand names contained in this publication are the trademarks

of their respective holders. No rights are granted without the prior written permission of the

relevant owners.


AUTHENTICATION

We declare that this work was done under our supervision according to the procedures

described herein and that the report represents a true and accurate record of the results

obtained.

Dr Andrew Taylor

Research Fellow

Warwick Crop Centre, University of Warwick

Signature Date: 28/10/19

Report authorised by:

Dr John Clarkson

Reader

Warwick Crop Centre, University of Warwick

Signature Date: 28/10/19


CONTENTS

Progress Against Objectives..................................................................................... 1

Objectives................................................................................................................... 1

Summary of Progress ................................................................................................ 4

Milestones not being reached.................................................................................... 4

Do remaining milestones look realistic? .................................................................... 5

Training undertaken ................................................................................................... 5

Expertise gained by trainees ..................................................................................... 5

Other achievements in the last year not originally in the objectives ........................ 6

Changes to Project ..................................................................................................... 7

Are the current objectives still appropriate for the Fellowship? ................................ 7

Grower Summary ........................................................................................................ 8

Headline ..................................................................................................................... 8

Background ................................................................................................................ 8

Summary .................................................................................................................... 8

Financial Benefits ..................................................................................................... 11

Action Points ............................................................................................................ 11

Science Section......................................................................................................... 11

Introduction .............................................................................................................. 12

Materials and methods ............................................................................................ 14

Results ..................................................................................................................... 20

Discussion ................................................................................................................ 37

Conclusions .............................................................................................................. 39

Knowledge and Technology Transfer ..................................................................... 40

Glossary ................................................................................................................... 40

References ............................................................................................................... 40


Progress Against Objectives

Objectives

N.B. Grey shading refers to this reporting period

Objective

Original Completion Date

Actual Completion Date

Revised Completion Date

1.1 Determine pathogenicity of a range of Fusarium oxysporum isolates on onion and complete DNA sequencing of a range of housekeeping genes.

31/10/15 31/10/15

1.2 Extract DNA, prepare libraries and carry out whole genome sequencing of F. oxysporum f.sp. cepae (FOC) isolates

31/10/17 31/10/17

1.3 Bioinformatic analyses of FOC isolate genomes and identification of potential primers for FOC diagnostics.

31/10/17 31/10/17

1.4 Test FOC diagnostic primers in vitro 31/10/18 31/10/18

1.5 Test FOC diagnostic primers using soil and bulb samples.

31/10/18 31/10/18*

1.6 Test published PCR diagnostic for Sclerotium cepivorum

31/10/16 31/10/16

1.7 Check existing Pythium violae specific primers using contemporary isolates / soil samples from carrot fields

31/10/14 31/10/14

1.8 Develop qPCR for P. violae using WCC Roche Lightcycler

31/10/14 31/10/14

1.9 Quantify P. violae in soil samples from AHDB Horticulture project FV 405 and other samples where available.

31/10/15 31/10/16

1.10 Identify potential primers for Itersonilia diagnostics from existing gene sequences (or whole genome sequence).

31/10/16 31/10/16

1.11 Test Itersonilia primers in vitro. 31/10/17 31/10/17

1.12 Test the newly developed Itersonilia diagnostic test on infected parsnip seed lots and compare with the industry standard agar plate test.

31/10/17 31/10/17

1.15 Test a range of S. cepivorum isolates for the presence of published pathogenicity genes

31/10/17 31/10/17

1.16 Test the ability of sclerotia to germinate for a range of S. cepivorum isolates using an

31/10/17 31/10/17


Objective




established assay based on diallyl disulphide (DADS)

2.1 Collect new isolates of Sclerotium cepivorum, Peronospora destructor (onion downy mildew), Botrytis squamosa (botrytis leaf blight) and Botrytis allii (neck rot of onion)

31/10/15 31/10/15

2.2 Confirm identity and characterise isolates from 2.1 by gene sequencing

31/10/16 31/10/16

2.3 Develop appropriate plant infection tests and confirm pathogenicity of isolates from 2.1

31/10/17 31/10/17

3.1. Gain experience with lettuce pathogens such as B. cinerea and B. lactucae through a work programme to be developed with Katherine Denby and Eric Holub.

31/10/18 31/10/18

3.2. Gain experience with brassica pathogens such as Turnip Mosaic Virus, Albugo candida Hyaloperonospora brassicae and Xanthomonas campestris through a work programme to be developed with Eric Holub and John Walsh.

31/10/18 31/10/18

3.3. Gain experience of other pathogens such as Pythium ultimum, Oidium. neolycopersici through existing projects (John Clarkson)

31/10/17 31/10/17

4.1. Synthesise Dez Barbara’s unpublished work on carrot/parsnip viruses

31/10/15 31/10/15

5.1. Attend relevant research project meetings.

Ongoing Ongoing

5.2. Present a poster at an industry meeting or event.

31/10/16 31/10/16

5.3. Give a talk at an industry meeting or event. 31/10/17 31/10/17

5.4. Work-shadowing of at least one industry collaborator.

31/10/17 31/10/17

6.1. Contribute to writing at least one research proposal

31/10/17 31/10/17

6.2. Initiate at least two research proposals and obtain funding for one.

31/10/18 31/10/18

7 Test published (and unpublished) methods for extraction of DNA from larger quantities of soil.

31/10/18 31/10/18

8 Isolate and confirm identity of the causal agent of onion pink root disease

31/10/17 31/10/17

Added milestone (9) - Molecular characterisation of F. oxysporum f. sp. narcissi isolates. This will involve PCR amplification and sequencing of effector genes in a range of isolates.

31/10/16 31/10/16


Objective




Additional milestones added in 2018

10.1. Identify SIX genes and other effectors in FOL4 by PCR / genome analysis

31/10/19 31/10/19

10.2. Develop and test qPCR / LAMP diagnostics for FOL4

31/10/19 31/10/19

10.3. Develop method to produce chlamydospores of FOL4

31/10/19 31/10/19

10.4. Test and develop lettuce differentials to confirm their utility in identifying FOL4

31/10/19 31/10/19

10.5. Carry out preliminary resistance screening for FOL4 using Warwick lettuce diversity set

31/10/19 31/10/19

10.6. Preliminary examination of DNA longevity in soil following FOL4 death / cell lysis

31/10/19 31/10/19

11. Repeat dose response experiment for Narcissus bulbs with FON

31/10/19 31/10/19

12. Discuss with Andy Richardson / Tom Will the commercial potential of FOC diagnostic assay (developed in 1.4)

31/10/19 31/10/19

13. Give talks at relevant industry events such as the UK Brassica and Leafy salad conference

Ongoing Ongoing

14. Investigate the feasibility of published (and unpublished) methods for extraction of DNA from larger quantities of soil. Liaise with University of Idaho and FERA

31/10/20

15. Develop bioinformatics skills and resources with Clubroot genomes

31/10/20

16. Test FOC isolates which have had effector genes knocked-out for pathogenicity against onion bulbs

31/10/20

*additional work carried out in 2019


Summary of Progress

Milestone 1.5: Test FOC diagnostic primers using soil and bulb samples. The FOC

qPCR assay was tested using 39 sub-samples from a single soil sample from the high-

disease pressure field at Wellesbourne and produced consistent positive results thus further

validating both the assay and the optimised method for DNA extraction from soil.

Milestone 10.1 / 10.2: Identify SIX genes and other effectors in FOL4 by PCR / genome

analysis and develop and test qPCR / LAMP diagnostics for FOL4. SIX genes and other

putative effectors were identified in FOL1 and FOL4 by PCR and genome analysis.

Differences in effector complement / sequence were observed between FOL1 and FOL4,

allowing the development of a specific qPCR assay for FOL4 which appears to be highly

sensitive and specific. A LAMP assay which detects both FOL1 and FOL4 was also

developed and initial testing produced robust results and suggested that this assay is highly

specific. These assays have been used to help track FOL4 outbreaks in the UK are being

used in experiments being carried out in AHDB project FV PE 458 to quantify FOL4 in soil

and lettuce roots.

Milestone 10.3: Develop method to produce chlamydospores of FOL4. Various methods

of FOL chlamydospore production were tested and a method based on sterile soil culture was

found to be highly effective, yielding high numbers of spores.

Milestone 10.4 / 10.5: Test and develop lettuce differentials to confirm their utility in

identifying FOL4 and carry out preliminary resistance screening for FOL4 using

Warwick lettuce diversity set. The Warwick lettuce diversity set was screened for resistance

to FOL1 and FOL4 using previously developed inoculation procedures and lines with high

levels of resistance to one or both of the races were identified. This also provided some new

‘differential’ lines for distinguishing FOL1 from FOL4.

Milestone 10.6: Preliminary examination of DNA longevity in soil following FOL4 death

/ cell lysis. It was shown that DNA from dead FOL4 spores degraded rapidly in soil and as

such the presence of dead pathogen should not significantly affect the results of molecular

diagnostic assays.

Milestone 11: Repeat dose response experiment for Narcissus bulbs with FON. An

experiment to determine the effect of FON inoculum levels on disease development in FV

POBOF 452 was repeated as a high background level of disease on the bulbs used

confounded results. The repeat experiment resulted in a clear dose-response and a critical

FON inoculum level for disease development was established.


Milestone 12: Discuss with Andy Richardson / Tom Will the commercial potential of

FOC diagnostic assay. Following discussions with Andy Richardson, a small pilot project

was started in order to test the FOC assay on commercial root and soil samples.

Milestone 13: Give talks at relevant industry events such as the UK Brassica and Leafy

salad conference. Presentations were given at the UK Brassica and Leafy Salads

Conference, the Hutchinsons Veg Conference and the International Allium Conference

(details below).

Milestones not being reached

n/a

Do remaining milestones look realistic?

New milestones need to be developed with AHDB

Training undertaken

• Attended and gave talk (Understanding and combatting Fusarium diseases of onion

and lettuce) at VeGIN meeting at Warwick Crop Centre (27th Nov 2018)

• Attended BSPP Presidential meeting at Warwick University (10th-11th Dec 2018)

• Completed ‘Preparing to Teach in Higher Education’ course (Dec 2018)

• Completed APP-PGR course leading to the award of an Associate Fellowship of the

Higher Education Authority (Jan – Aug 2019)

• Attended and gave talk (Lettuce Fusarium wilt in the UK) at the UK Brassica and Leafy

Salads Conference in Peterborough (23rd Jan 2019)

• Attended and gave talk (Research on Fusarium basal rot of onion and other vegetable

diseases) at Hutchinsons Veg Conference in Peterborough (26th Feb 2019)

• Hosted guest seminar by Sascha Mooney, University of Nottingham (7 th March 2019)

• Attended the Journal of Horticultural Science and Biotechnology conference in

Charlecote (9th May 2019)

• Hosted visit and guest seminar by Louise Thatcher from CSIRO, Australia (17th June

2019)


• Attended and gave an invited talk (Creation and characterisation of an onion diversity

set and identification of accessions with resistance to Fusarium basal rot and

improved seedling vigour) at the International Allium Conference in Madison, USA

(23rd – 27th July 2019)

• Visited lettuce grower to discuss Fusarium issues (26th Sept 2019)

• Attended Fusarium meeting in Utrecht and met with researchers at Wageningen (29 th

– 31st Oct 2019)

Expertise gained by trainees

• Improved communication skills

• Improved understanding of the lettuce industry

• Improved understanding of plant pathology

• Greater understanding of worldwide Allium research

• Expertise in Fusarium wilt of lettuce

• Greatly improved teaching skills including a formal qualification

Other achievements in the last year not originally in the objectives

• Obtained a formal teaching qualification as an Associate Fellow of the HEA

• Inoculated polytunnels with FOL4 and maintained these facilities for future projects

• A paper titled: ‘Assembly and characterisation of a unique onion diversity set identifies

resistance to Fusarium basal rot and improved seedling vigour’ was published in

Theoretical and Applied Genetics

• A paper titled: ‘First report of Fusarium oxysporum and Fusarium redolens causing

wilting and yellowing of wild rocket (Diplotaxis tenuifolia) in the UK’ was published in

Plant Disease.

• A paper titled: ‘First report of Fusarium oxysporum f. sp. lactucae Race 4 causing

lettuce wilt in England and Ireland was published in Plant Disease.

• A paper titled: ‘Draft genome sequence of an onion basal rot Isolate of Fusarium

proliferatum’ was published in Microbiology Resource Announcements.

• Took on the role of Associate Editor for the Journal of Horticultural Science and

Biotechnology


• Awarded 2 travel grants (Vegetable Research Trust and GCRI) totalling £2500 to

attend the International Allium Conference in Madison, USA.

• Carried out commercially funded research projects on Fusarium basal rot in onion

Changes to Project

Are the current objectives still appropriate for the Fellowship?

New objectives need to be set with AHDB


GROWER SUMMARY

Headline

Molecular diagnostic assays were developed and validated for Fusarium oxysporum f. sp.

lactucae (FOL), the cause of lettuce Fusarium wilt. These assays will be of great benefit for

testing soil and plant material for this important recently emerged pathogen. Lettuce lines

with resistance to FOL were identified, providing promise for future breeding programmes.

Background

Fusarium oxysporum and Fusarium basal rot of onion

Onion (Allium cepa) is an important horticultural crop which is cultivated by every agricultural

nation. Soilborne diseases caused by Fusarium oxysporum formae speciales (isolates

adapted to specific hosts, f. spp.) are major constraints to the production of many horticultural

food crops worldwide including onion, leek, lettuce, tomato, brassicas, asparagus, cucurbits,

peppers, coriander, spinach, basil, beans, peas, strawberry, watermelon and banana, and

also affect economically important non-food crops such as carnation and narcissus (Leslie

and Summerell 2006) and stocks. F. oxysporum was recently identified as the 5th most

important plant pathogenic fungus based on its economic and scientific impact (Dean et al,

2012). F. oxysporum f.sp. cepae (FOC) is one of the most important pathogens of onion crops

and infects the roots and/or basal plate at any stage of plant development (Cramer, 2000;

Taylor et al, 2013). This causes a damping-off symptom on seedlings and a basal rot on more

mature plants resulting in severe pre and/or post-harvest losses. In the UK, FOC is

recognised mainly as being a problem at harvest and in store but in severe cases entire crops

can be lost in the field. Economic losses due to FOC on onion are estimated at up to £20

million per year. FOC infection is favoured by warm temperatures and is predicted to get

worse in Europe due to climate change (Cramer, 2000). It produces long-lived

chlamydospores that survive in the soil for many years and hence control approaches have

previously relied on the use of soil sterilisation, chemical fumigation, drenches with fungicides

or seed treatments. These approaches have in some cases been unsuccessful, have

undesirable environmental effects and have been banned or are threatened by legislation

governing restrictions in pesticide use. In the past it has been difficult to distinguish f. spp. of

F. oxysporum and identification has relied on pathogenicity tests. However, work from F.

oxysporum f. sp. lycopersici, the f. sp. infecting tomato (Lievens et al, 2009; Ma et al, 2010)

has identified a set of pathogenicity related genes which are conserved in FOC (Taylor et al,


2016). Differences in the compliment or sequences of SIX (secreted in xylem) genes between

forma speciales of F. oxysporum can potentially be utilised to develop diagnostic assays

which can be utilised to test soil and plant material for presence of the pathogen. As part of

this fellowship, an assay for FOC was developed and validated (see year 5 annual report)

which will allow seed, soil, roots and bulbs to be tested for the presence of FOC.

Fusarium wilt of lettuce

Fusarium wilt of lettuce, caused by Fusarium oxysporum f. sp. lactucae (FOL), is a problem

in most production areas globally, causing severe economic losses in protected and field

crops. Initial symptoms are stunting and yellowing, particularly on older leaves (Taylor &

Clarkson 2018). A black/brown/red discoloration of the vascular tissue can be observed and

infection ultimately leads to plant death. There are 4 known races of FOL, the most

widespread being race 1 which affects both field and protected crops (Gilardi et al, 2017).

Races 2 and 3 are only found in Japan and Taiwan. FOL was only very recently first reported

in the UK in October 2017 (Taylor et al, 2019) with initial outbreaks affecting protected lettuce

crops in Lancashire and Ireland. However, since then there has been local spread within

these areas to other growers and also confirmed reports of FOL in Cambridgeshire (2018)

and Yorkshire (2019). Genetic analysis has confirmed the causal agent as FOL race 4 (FOL4,

Taylor et al, 2019). FOL4 was first reported in the Netherlands in 2013 (Gilardi et al, 2017)

and so far all cases have been confined to protected crops. It is also causing severe losses

for protected lettuce growers in Belgium. Currently, rapid spread of FOL4 is being prevented

by hygiene measures imposed by the industry while affected growers are mitigating disease

impact through use of the soil fumigant Basamid (dazomet), removal of contaminated soil or

by abandoning affected growing areas. Although there is no widespread availability of

resistant lettuce cultivars, these are under development. Developing a molecular test for

FOL4 would mean soil, seed and other plant material could be tested for the pathogen.

Narcissus basal rot

Daffodil (Narcissus spp.) is one of the most widely cultivated bulb crops of temperate regions.

The major production areas are the UK, Netherlands and USA although smaller areas are

cultivated across the world (Hanks, 2002). In the UK, bulbs are particularly prone to infection

by soil-borne pathogens due to the standard biennial growing system employed (Hanks,

2002). The most damaging pathogen is Fusarium oxysporum. f.sp. narcissi (FON), the cause

of narcissus basal rot (Linfield, 1994). The symptoms include pale yellow leaf tips, soft bulbs,

root rot and ultimately a bulb rot. Infected bulbs may not sprout and produce few or no flowers.


Controlling FON is challenging due to the production of chlamydospores as discussed above.

It is not known what concentration of FON is required for infection to occur.

Do molecular techniques detect dead pathogens?

There is some debate in the literature about how long after death a pathogen can be detected

by molecular techniques due to continued survival of DNA, particularly in soil. For example,

research carried out on Gaeumannomyces graminis (a fungus causing take-all disease of

cereals) suggests that fungal DNA is broken down rapidly in soil, reaching an undetectable

level after 8 days (Herdina et al, 2004). However, other work has suggested that DNA can

bind to soil particles or humic acids, protecting it from degradation (Alvarez et al, 1998;

Crecchio et al, 1998). If molecular assays detect dead pathogen then this could affect any

disease predictions based on DNA quantities in soil. Therefore, it is important to begin to

understand the persistence of DNA in soil following death of the pathogen.

Summary

• A highly specific and reproducible molecular diagnostic assay (qPCR) was developed

for FOC in year 5. This assay can be used to test seed, plant material and soil for

FOC and is fully quantitative.

• The FOC qPCR assay was further validated by testing 39 sub-samples from a single

soil sample collected from the FOC inoculated field at Wellesbourne and all samples

gave similar positive results indicating that the assay and the DNA extraction method

is highly reproducible.

• SIX genes and other predicted effectors were identified in FOL1 and FOL4 by PCR

and genome analysis. Differences between FOL1 and FOL4 were observed, allowing

the development of a specific qPCR assay for FOL4 which appears to be highly

sensitive and specific.

• A rapid assay similar to PCR (loop-mediated isothermal amplification, LAMP), which

detects both FOL1 and FOL4 was also developed and initial testing produced robust

results and suggested that this assay is highly specific. This assay gives a diagnosis

in under 1 hour and can potentially be used at grower sites.

• A method of chlamydospore production was developed for FOL4 which will allow

testing of heat / disinfectant treatments against the pathogen in another AHDB project.


• The Warwick lettuce diversity set was screened for resistance to FOL1 and FOL4 and

lines with high levels of resistance to one or both of the races were identified. This

will allow for future breeding of resistant cultivars.

• Clear lettuce ‘differential’ lines for distinguishing FOL1 from FOL4 were identified,

allowing definitive differentiation of races alongside molecular testing

• It was shown that DNA degrades rapidly in soil and as such dead pathogen should

not significantly affect molecular diagnostic assays.

• An experiment to determine the effect of FON inoculum levels on disease

development in FV POBOF 452 was repeated as a high background level of disease

on the bulbs used confounded results. The repeat experiment resulted in a clear dose-

response observed and the critical inoculum level for disease established. Further

work will now utilise a FON-specific qPCR assay to relate DNA levels to inoculum rate

and disease development. This will be an important first step in assessing the utility

of the PCR tests for assessing disease risk in the field.

Financial Benefits

None to report

Action Points

None to report


SCIENCE SECTION

Introduction

Fusarium basal rot of onion

F. oxysporum f.sp. cepae (FOC) is one of the most important pathogens of onion crops and

infects the roots and/or basal plate at any stage of plant development (Cramer, 2000; Taylor

et al, 2013). This causes a damping-off symptom on seedlings and a basal rot on more mature

plants resulting in severe pre and/ or post-harvest losses. In the UK, FOC is recognised

mainly as being a problem at harvest and in store but in severe cases entire crops can be

lost in the field. Economic losses due to FOC on onion are estimated at £10-11 million per

year and FOC infection is favoured by warm temperatures and is predicted to get worse in

Europe due to climate change (Cramer, 2000). FOC produces long-lived chlamydospores

that survive in the soil for many years and hence control approaches have previously relied

on the use of soil sterilisation, chemical fumigation, drenches with fungicides or seed

treatments. These approaches have in some cases been unsuccessful, have undesirable

environmental effects and have been banned or are threatened by legislation governing

restrictions in pesticide use.

FOC is one of more than 120 Fusarium oxysporum formae speciales (isolates adapted to

specific hosts) (Michielse & Rep, 2009) which are major constraints to the production of many

horticultural food crops worldwide including onion, leek, lettuce, tomato, brassicas,

asparagus, cucurbits, peppers, coriander, spinach, basil, beans, peas, strawberry,

watermelon and banana and also affect economically important non-food crops such as

carnation and narcissus (Leslie and Summerell, 2006). F. oxysporum was recently identified

as the 5th most important plant pathogenic fungus based on its economic and scientific impact

(Dean et al, 2012).

The genetically heterogeneous nature and lack of reliable morphological characters in this F.

oxysporum complex means that distinguishing between different pathogenic f. spp. and also

and between pathogenic and non-pathogenic isolates is difficult and can only be done using

laborious and time consuming pathogenicity tests on different hosts. The factors which

determine the host specificity and pathogenicity of different F. oxysporum f. spp. are poorly

understood although recent studies have identified the role of secreted effector proteins (SIX

genes) and mobile pathogenicity chromosomes in F. oxysporum f. sp. lycopersici, the f. sp.

infecting tomato (Lievens et al, 2009; Ma et al, 2010). The genetic basis for pathogenicity

appears to be partially conserved in FOC with 7 of the 14 known SIX genes identified and

predicted to have an important role in pathogenicity (Taylor et al, 2016). Differences in the

compliment and sequences of SIX genes between formae speciales of F. oxysporum


including FOC has allowed for the development of specific molecular (qPCR) diagnostic

assays. As part of this fellowship, an assay for FOC was developed and validated (see year

5 annual report) which will potentially allow seed, soil, roots and bulbs to be tested for the

presence of this pathogen.

Fusarium wilt of lettuce

Fusarium wilt of lettuce, caused by Fusarium oxysporum f. sp. lactucae (FOL), can be found

in most production areas globally, causing severe economic losses in protected and field

crops. Initial symptoms are stunting and yellowing, particularly on older leaves (Taylor &

Clarkson 2018). A black/brown/red discoloration of the vascular tissue can be observed and

infection ultimately leads to plant death. There are 4 known races of FOL, the most

widespread being race 1 which affects both field and protected crops (Gilardi et al, 2017).

Races 2 and 3 are only found in Japan and Taiwan. FOL was only very recently first reported

in the UK in October 2017 (Taylor et al, 2019) with initial outbreaks affecting protected lettuce

crops in Lancashire and Ireland. However, since then there has been local spread within

these areas to other growers and also confirmed reports of FOL in Cambridgeshire (2018)

and Yorkshire (2019). Genetic analysis has confirmed the causal agent as FOL race 4 (FOL4,

Taylor et al, 2019). FOL4 was first reported in the Netherlands in 2013 (Gilardi et al, 2017)

and so far all cases have been confined to protected crops. It is also causing severe losses

for protected lettuce growers in Belgium. Currently, rapid spread of FOL4 is being prevented

by hygiene measures imposed by the industry while affected growers are mitigating disease

impact through use of the soil fumigant Basamid, removal of contaminated soil or by

abandoning affected growing areas. Although there is no widespread availability of resistant

lettuce cultivars, these are under development.

Developing a molecular test for FOL4 would mean soil, seed and other plant material could

be tested for the pathogen. Whilst a FOL4 PCR assay has been developed (Gilardi et al,

2017), this is not a quantitative test and was shown to cross-react with other F. oxysporum

formae speciales (Taylor & Clarkson 2018). More recently, a loop-mediated isothermal

amplification (LAMP) assay was developed for FOL (Ortega et al, 2018) and this is available

as a commercial kit from Optigene Ltd, allowing rapid testing of plant samples for FOL. This

assay detects both FOL1 and FOL4.

Narcissus basal rot

Daffodil (Narcissus spp.) is one of the most widely cultivated bulb crops of temperate regions.

The major production areas are the UK, Netherlands and USA although smaller areas are

cultivated across the world (Hanks, 2002). In the UK, bulbs are particularly prone to infection

by soil-borne pathogens due to the standard biennial growing system employed (Hanks,


2002). The most damaging pathogen is Fusarium oxysporum. f.sp. narcissi (FON), the cause

of narcissus basal rot (Linfield, 1994). The symptoms include pale yellow leaf tips, soft bulbs,

root rot and ultimately a bulb rot. Infected bulbs may not sprout and produce few or no flowers.

Controlling FON is challenging due to the production of chlamydospores as discussed for

FON. It is not known what concentration of FON is required for infection to occur.

Do molecular techniques detect dead pathogens?

There is some debate in the literature about how long after death a pathogen can be detected

by qPCR due to continued survival of DNA, particularly in soil. For example, research carried

out on Gaeumannomyces graminis (take-all disease) suggests that fungal DNA is broken

down rapidly in soil, reaching an undetectable level after 8 days (Herdina et al, 2004).

However, other work has suggested that DNA can bind to soil particles or humic acids,

protecting it from degradation by nucleases (Alvarez et al, 1998; Crecchio et al, 1998). If

qPCR assays detect dead pathogen then this could affect any disease predictions based on

DNA quantities in soil. Therefore, it is important to begin to understand the persistence of

DNA in soil following death of the pathogen.

Materials and methods

1.5. Test FOC diagnostic primers using soil and bulb samples.

Whilst this milestone was completed in 2018, some additional work was undertaken in 2019

to test both the FOC qPCR assay and the repeatability of the soil extraction method. A soil

sample was taken from a quarantine field at Wellesbourne which was previously artificially

infected with FOC. This sample was taken by collecting ~50g of soil at 5 points (W formation)

in one bed which was used as an untreated control in a trial. The samples were pooled,

sieved / dried and mixed as described in the year 5 report. DNA was then extracted from 39

replicate 0.5 g samples taken from the pool using the method described in the year 5 report.

qPCR was carried out using a QuantStudio 5 (384-well) machine (Applied Biosystems) using

20 µl reactions containing both primers (final concentration 0.5 µM), 10 µl Power SYBR™

Green PCR Master Mix (Applied Biosystems) and 1 µl of DNA. All samples were run in

triplicate and an average taken. Cycling conditions were as listed in Table 1.


Table 1. Cycling conditions used for FOC qPCR assay

Temp (°C) Time (s) Cycles

95 120 1

95 3 45

60 30

Melt curve:

95 15 1

60 60 Increasing by 0.075°C/s 95 15


The genomes of FOL isolates AJ516 (race 4, Lancashire) and AJ520 (FOL1, Italy) were

sequenced, assembled and putative effectors identified by NIAB-EMR. Using the software

package Geneious, these genomes were queried specifically for the presence of SIX genes

(SIX 1-14). In addition, a DNA panel consisting of FOL isolates and putative non-pathogenic

F. oxysporum isolates from lettuce (England, Ireland and the Netherlands) as well as F.

oxysporum isolates pathogenic on rocket was assembled (Table 2) and screened for the

presence of SIX genes using PCR assays as described by Taylor et al, (2016).

Where a positive SIX gene amplicon was obtained, this was purified using a QIAQuick PCR

purification kit (Qiagen) and sequenced by GATC. For SIX8, sequences were aligned with

publicly available data using MEGA version 7 (Kumar et al, 2016) and a phylogenetic tree

constructed using the Jukes-Cantor method (gamma distributed) and 1000 bootstrap

replicates.

Table 2. F. oxysporum / FOL isolates screened for the presence of SIX genes.

Sample Origin FOL race

1 Co. Dublin site 1 4


3 Lancashire site 1 4



6 Lancashire site 3 N/A











16 Cambs site 1 4





21 Israel 1

22 Cambs site 2 4







29 Italy 1

30 Netherlands 4

31 Netherlands 4

32 UK, pathogenic on rocket N/A

33 UK, pathogenic on rocket N/A

34 France, isolated from soil; non-pathogenic isolate Fo47

N/A


This work was carried out to provide molecular tools for AHDB project FV PE 458 to quantify

FOL4 in soil and lettuce roots.

qPCR assays

Following initial genome analysis at NIAB-EMR, putative effector gene targets for diagnostic

primers were identified. FOL4-specific primers were designed by hand and conventional PCR

initially used to test them. PCRs were carried out in 20µl reactions containing primers (0.5

µM), 5µl of RedTaq (Sigma) and 1µl of DNA with the following conditions: 1 cycle of 94°C for

2 mins followed by 35 cycles of 94°C for 45s, 60°C for 30s and 72°C for 30s followed by 1

cycle of 72°C for 5 mins. PCR products (5 µl) were run on a 1.2% agarose gel. As a

preliminary specificity test, 8 DNA samples from other F. oxysporum formae speciales were

also included (f.sp. lycopersici race 1, f.sp. vasinfectum, f.sp. pisi race 1 and 5, f.sp.

limonii,f.sp. matthiolae and f.sp. narcissi). Four primer pairs were then selected for qPCR


testing using the methods described for FOC. A dilution series of FOL4 isolate AJ516

genomic DNA was included for each primer pair.

LAMP assays

Initially, a published FOL LAMP assay (Ortega et al, 2018; Optigene PK-F.oxy_lac-050W)

was tested. All reactions contained 15µl of isothermal mastermix, 5µl of primer mix and 5µl

of DNA. Firstly, the LAMP assay was tested against FOL1 (isolate AJ520 from Italy) and

FOL4 (isolate AJ516 from Lancashire) genomic DNA extracted from cultures, as well as crude

DNA extracts from FOL4 infected lettuce roots, extracted using the Optigene plant material

DNA extraction kit, following the manufacturer’s guidelines. Following this, ten soil DNA

extracts from a FOL4 infested polytunnel at Wellesbourne were tested as well as diseased

plant samples from FOL4 infected lettuce sent by a commercial grower. Finally, a small scale

specificity test was carried out using DNA extracted from other formae speciales of F.

oxysporum (see results section).

Due to some cross-specificity and the cost of the Optigene kit, new FOL LAMP primers were

designed based on effector gene targets identified from the whole genome sequences of

FOL1 and FOL4. Primers were designed using LAMP designer (Premier Biosoft) with

assistance from Matt Dickinson (University of Nottingham) and a primer mix prepared as

shown in Table 3. LAMP reactions were set up containing 5µl of DNA, 2.5µl of primer

mastermix, 15µl of isothermal mastermix ISO-001 (Optigene) and 2.5µl of sterile water in strip

cap tubes (OP-0008, Optigene). Following initial optimisation, strips were placed in a Genie

II LAMP machine and run at 64°C for 40 mins followed by the anneal curve analysis (98°C

for 1 min reducing by 0.05°C/s to 80°C). Initially, primers were tested using genomic DNA of

FOL1 and FOL4. Following this, primers were tested using soil DNA extracts from the FOL4

infested tunnel and a specificity test using DNA from several F. oxysporum f.spp. carried out

using the method described for the published LAMP assay.

Table 3: Primer mix for new FOL LAMP assay.

Primer* Conc. In 25 µl reaction

(µM)

Volume for 100 reactions

(µl)

F3 0.2 5

B3 0.2 5

FIP 2.0 50

BIP 2.0 50

LoopF 1.0 25

LoopR 1.0 25

Sterile water 90



Preliminary testing was carried out to evaluate three different methods for chlamydospore

production using FOL4 isolate AJ516.

1. Talc method: FOL4 isolate AJ516 was grown on PDA for 14 days at 20°C and a highly

concentrated spore suspension prepared by adding sterile water and gently removing

spores with a sterile spreader. Four millilitres of spore suspension (1.8 x 107 spore/ml)

was added to 20g sterile talc, 1ml at a time to allow mixing, and left for 6 weeks.

Chlamydospore production was assessed by microscopy and concentrations

calculated using dilution plating on PDA.

2. Salt solution method: (Navas-Cortes et al, 2006). A FOL4 spore suspension was

prepared as above and used to inoculate a conical flask containing 400ml of a

saturated sodium chloride solution. This was placed at 25°C in the dark for 4-6 weeks

and chlamydospore production assessed by microscopy.

3. Soil method: soil (sandy-loam) was taken from a field with no history of lettuce

production, sieved to 2mm and air-dried. 40g was then placed in 100ml bottles and

autoclaved twice on consecutive days. A FOL4 spore suspension was prepared as

described for the talc method and 8ml (1 x 107 spores/ml) added to each bottle.

Bottles were incubated at 20°C for 1 month and chlamydospore production assessed

by microscopy and concentrations calculated using dilution plating on PDA.



Previous work at Warwick had indicated that the current published lettuce differentials for

FOL4 (Gilardi et al, 2017) do not give a completely clear resistant / susceptible phenotype.

Lettuce lines (54) were selected from the Warwick lettuce diversity set in order to screen for

resistance to FOL1/ FOL4 and identify new differentials as part of the Defra Vegetable

Genetic Improvement project (VeGIN). Due to the size of the experiment, separate

experiments were conducted for FOL1 and FOL4. Lettuce seed was sown in peat blocks and

placed in a glasshouse for two weeks before use in experiments. FOL1 isolate AJ520 and

FOL4 isolate AJ516 were grown in a compost / bran mixture as described by Taylor et al.

(2013) and used to infest M2 compost at a rate of 1 x 106 cfu/g. Lettuce plants (3 replicates)

of each line were then transplanted into 9cm square pots containing either infested compost

or clean compost. Pots were positioned in a randomised block design in a glasshouse set to

25°C day, 18°C night with a 16-hour daylength. Wilting due to FOL was scored twice weekly


on a scale of 0-5 and after 39 days plants were cut longitudinally and the degree of vascular

browning scored on a 0-4 scale. Lettuce fresh and dry weights were also recorded.


Initially, a system was established for killing FOL4 spores in vitro. The FOL4 isolate AJ516

was grown on PDA for 14 days at 20°C and a conidial suspension prepared by adding 10ml

sterile distilled water gently removing conidia using a sterile spreader. Conidia were filtered

through 3 layers of sterile mira cloth (to remove mycelium), counted on a haemocytometer

and adjusted to 1 x 104 spores/ml. Spore suspensions (500 μl, in 2ml tubes) were then

exposed to temperatures of 20°C, 40°C, 50°C, 60°C and 70°C for 5, 15 and 60 min using

heat blocks. After each heat / duration treatment, 100 μl of spore suspension (undiluted and

diluted 1 in 10 in sterile water) was spread on PDA plates (three replicates). After incubating

plates at 25°C for two days, colonies were counted and the number of viable FOL4 spores

per ml calculated. The experiment was repeated once.

A Wellesbourne soil sample was taken (Wharf ground, sandy loam inceptisol, ’Wick’ series)

from a field with no history of lettuce production. This sample was sieved and mixed and 0.5g

subsamples weighed into 2ml DNA extraction tubes. Soil samples were then spiked with

either dead (treated at 60°C for 15 mins) or live conidia at a concentration of 1 x 106 spores/g.

The effectiveness of the heat treatment in killing spores was confirmed by plating 100μl (3

replicates) of spore suspension on PDA pre-soil spiking. Tubes containing the soil / dead

spores / live spore treatments were all incubated at 20°C and DNA extracted at 0, 3, 7, 14,

21, 28 and 35 days after spiking (3 replicate extractions per time point). FOL4 qPCR was

then carried out on diluted DNA (1 in 6 in TE) as described above.


In 2017, an experiment was set up to assess the effect of FON inoculum concentration on

disease development in Narcissus (AHDB project FV POBOF 452). Unfortunately, due to the

very high background level of basal rot in the bulbs used, no clear dose-response was

observed. In November 2018, the experiment was repeated (following the same method,

Clarkson et al., 2019) using a batch of bulbs (cv. Carlton) from Scotland which were largely

disease-free. Narcissus bulbs were planted (5 per pot, 28 replicate pots per treatment) in

FON infested compost on 5th November 2019 at concentrations ranging from ranging from 0

to 1 x 106 cfu g-1, placed in a frost-free glasshouse and on 4th June 2019 bulbs were bisected

and the level of Fusarium basal rot scored on a 1-10 scale.


Results

1.5. Test FOC diagnostic primers using soil and bulb samples

As a validation of both the FOC qPCR assay and the DNA extraction protocol, DNA was

extracted from 39 sub-samples taken from a pooled soil sample from the FOC infested field

at Wellesbourne. Whilst there was some variation in the qPCR cycle threshold value (Ct)

when significant amplification of FOC DNA was observed (Ct range 32.6-36.1), FOC was

nonetheless successfully detected in every sample providing validation that the method is

robust (Fig. 1). The majority (30) of the 39 samples (77%) varied by only 1.5 Ct, indicating a

high level of reproducibility.

Fig. 1: Amplification of FOC from 39 replicate DNA extractions from a single soil sample


PCR screening of DNA from FOL1 and FOL4 revealed that only two of the 14 SIX genes

(SIX8 and SIX9) are present in FOL4 whilst only a single SIX gene (SIX9) was found in FOL1

(Table 4) and this was confirmed in the genome analysis. Genome data analysis also

revealed that there were three DNA sequence variants of SIX9 in race 4 and two variants in

race 1. Preliminary genome analysis also identified a number of additional effectors present

in either FOL1 or FOL4 or in both that will provide a basis for future work to understand the

genes involved in FOL1 / FOL4 virulence.


Table 4. Identification of SIX genes in FOL1 and FOL4 using PCR and genome analysis.

FOL

Isolate Race

SIX8

PCR

SIX8

genome

SIX9

PCR SIX9 genome

AJ516 4 + + + + (3 variants)

AJ520 1 - - + + (2 variants)

When a wider panel of F.oxysporum isolates from lettuce and rocket was screened for SIX

genes, a consistent pattern was observed whereby all FOL4 isolates contained both SIX8

and SIX9 whereas the two FOL1 isolates only contained SIX9 (Table 5). Non-pathogenic

isolates contained no SIX genes with the exception of a single isolate which contained SIX2.

Therefore, a strong correlation between SIX8 and SIX9 gene presence and pathogenicity

against lettuce was evident. However, both F. oxysporum isolates which were pathogenic on

rocket also contained SIX8 but with a different sequence to the FOL4 SIX8 gene (Fig. 2).

Table 5. Screening a panel of F. oxysporum isolates from lettuce and rocket for the presence

of SIX genes.

Sample Origin FOL race SIX 2 SIX 8 SIX 9 other SIX genes

1 Co. Dublin site 1 4 - + + -


3 Lancashire site 1 4 - + + -



6 Lancashire site 3 N/A - - - -










16 Cambs site 1 4 - + + -





21 Israel 1 - - + -


22 Cambs site 2 4 - + + -




26 Lancashire site 4 N/A + - - -



29 Italy 1 - - + -

30 Netherlands 4 - + + -

31 Netherlands 4 - + + -

32 UK, pathogenic on rocket N/A - + - -

33 UK, pathogenic on rocket N/A - + - -

34 France, isolated from soil non-pathogenic Fo47 - - - -

Phylogenetic analysis of the SIX8 gene sequences grouped the FOL4 isolates into a single

clade but two sequence types were observed (Fig. 2). All isolates from Ireland and

Lancashire as well as one site in Cambridgeshire shared the same sequence type. An isolate

from a second FOL4 infected site in Cambridgeshire shared the same sequence type as the

two isolates from the Netherlands. The difference between the two FOL4 SIX8 sequence

types was three nucleotides, resulting in a change in two amino acids. The two F. oxysporum

isolates pathogenic on rocket also had two different sequence types which placed them in

separate clades. (Fig. 2).

FOL4 Ireland (all) FOL4 Cambs1 FOL4 Lancs (all)

FO rocket (FR3) KX435046 FO passiflorae strain BRIP28044

HQ260604 FO from Brassica oleracea FO mathiolae stocks4 PHW815 FO raphani FOL4 Cambs2 FOL4 NL1 FOL4 NL2

FO rocket (FR4) KP964974 FO phaseoli

FJ755837 FO lycopersici KY296401 FO cubense isolate FoxPDb1 KX435033 FO medicaginis strain BRIP5189 KY296403 FO cubense isolate FoxPDa3

FO statice KX435014 FO cubense strain BRIP44012 KF548064 FO cubense isolate 22615

II5 FO cubense MH752354 FO cubense isolate Png3-2 KX435025 FO cubense strain BRIP59161

MH636942 FO palmarum isolate PLM928A KX435039 FO niveum strain NTDPI36955

66

65

65

55

86

63

555291

23

20

29

0 .050


Fig. 2: Maximum likelihood phylogenetic tree of F.oxysporum isolates based on SIX8.

Numbers represent bootstrap values (1000 replicates). Scale bar indicates 0.05 substitutions

per site.


FOL4 qPCR assay

Four primer pairs for FOL4 produced promising results in the specificity test, resulting in good

amplification of FOL4 DNA and no amplification for FOL1 or the other F. oxysporum f.spp.

tested (Fig. 3).

Fig. 3. Gel electrophoresis showing PCR amplicons for four primer pairs designed to amplify

DNA from FOL4. 1=Fusarium oxysporum (FO) f. sp. lycopersici race 1; 2=FO vasinfectum;

3=FO pisi race 1; 4=FO from statice; 5=FO mathiolae; 6=FO narcissi; 7=FO pisi race 5;

8=FOL1; 9=FOL4; 10=water.

When tested as a qPCR assay, all four FOL4 primers worked well and produced a single melt

peak (Fig. 4). In terms of efficiency, primer pair 3 appeared to be the best (Table 6) and will

be further optimised and tested. All primer pairs gave a positive result at the lowest

concentration of FOL DNA tested which was 1pg/μl.

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

Primer pair 1 Primer pair 2

Primer pair 3 Primer pair 4


Fig. 4: qPCR standard curves and melt curves for four sets of FOL4 primers (top to bottom =

pair 1- pair 4).


Table 6: Efficiency of 4 primer pairs designed to amplify FOL4 when tested against a dilution

series of FOL4 genomic DNA.

Primer pair Slope R2 Efficiency (%)

1 -3.55 0.998 91.4

2 -3.53 0.997 92.1

3 -3.48 0.999 94.0

4 -3.50 0.997 92.9

FOL LAMP assay

The published FOL LAMP assay amplified DNA extracted from FOL1 and FOL4 cultures and

FOL4 infected lettuce roots and showed a strong, early pattern of amplification with a positive

result after 7 minutes and a single melt peak (Fig. 5).

When DNA extracts from FOL4 infested soil were tested using LAMP, (Fig. 6), all ten extracts

gave a positive result although sample 7 resulted in late amplification indicating a lower

amount of pathogen DNA. When diseased lettuce plant samples from a FOL4 infected

commercial site were tested, the LAMP assay clearly detected FOL4 in symptomatic material

with no amplification from tissue with no disease symptoms (Fig. 6b).

The newly developed in-house FOL LAMP assay amplified DNA extracted from cultures of

both FOL1 and FOL4 and showed a strong, early pattern of amplification with a positive result

after 7 minutes and a single melt peak (Fig. 7). The optimum assay temperature was found

to be 64°C (data not shown).

When DNA extracted from FOL4 infested soil was tested using the new FOL LAMP assay,

all ten extracts gave a positive result with a consistent time to amplification (Fig. 8a). The

new FOL LAMP assay also detected FOL4 in plant samples from FOL4 inoculated plants,

clearly amplifying DNA from symptomatic material with no amplification from tissue with no

disease symptoms (Fig. 8b).

The new FOL LAMP assay was also highly specific with no amplification of DNA from any

non-target fungi tested (Table 6). In contrast, the published LAMP assay showed cross-

reaction with non-pathogenic F. oxysporum isolates as well as isolates pathogenic on rocket

and statice. Of particular concern is the positive amplification from the statice isolate as the

time to positive is the same as for FOL1. Preliminary genome analysis has revealed that

FOL1 and FOL4 share a high level of sequence similarity with F. oxysporum from statice.


(a)

(b)

Fig. 5: Published LAMP assay results for FOL showing a) anneal curve and b) amplification

over time for FOL1 / FOL4 genomic DNA extracted from cultures and FOL4 infected roots.


(a)

(b)

Fig. 6: Published FOL LAMP assay results for DNA extracted from a) FOL4 infested soil

samples and b) commercial lettuce plants with / without symptoms of FOL4 infection.


(a)

(b)

Fig. 7: New FOL LAMP assay results showing a) the anneal curve and b) the amplification

over time for genomic DNA extracted from cultures of FOL1 and FOL4.


(a)

(b)

Fig. 8: New FOL LAMP assay results for DNA from a) FOL4 infested soil samples and b)

FOL4 inoculated lettuce plants with / without symptoms of disease.

Non-inoculated controls

Infected plants


Table 6: Comparison of specificity of the published and newly developed LAMP assay for

FOL.

Fusarium species Forma speciales Host Time to positive (mins)

Ortega new assay

F. oxysporum lactucae race 4 lettuce 7 7

F. oxysporum lactucae race 1 lettuce 7.25 7

F. oxysporum statice 7.25 negative

F. oxysporum lettuce (NP) 13.5 negative

F. oxysporum rocket 18.75 negative

F. oxysporum lettuce (NP) 18.75 negative

F. oxysporum (Fo47) soil 19.25 negative

F. oxysporum lettuce (NP) late amp negative

F. oxysporum pisi race 1 pea late amp negative

F. oxysporum lycopersici race 2 tomato negative negative


F. oxysporum phaseoli lettuce (NP) negative negative


F. oxysporum mathiolae stocks negative negative

F. oxysporum rocket negative negative

F. oxysporum pisi race 2 pea negative negative

F. oxysporum pisi race 5 lettuce (NP) negative negative

F. oxysporum lettuce (NP) negative negative




F. oxysporum cepae onion negative negative

F. oxysporum narcissi daffodil negative negative

F. langsethiae wheat negative negative

F. solani pea negative negative

Setophoma terrestris onion negative negative

F. equiseti rocket negative negative

F. redolens rocket negative negative



Talc method

Microscopic examination of FOL4 talc cultures were inconclusive due to the presence of very

fine talc particles which meant that although some chlamydospores could be observed, other

spore types such as micro and macro conidia could also have been present (Fig. 9). Use of

fungal stains did not improve the microscopy as some talc particles also absorbed the stains

tested. This method was therefore not taken forward.

Fig. 9: Microscopic image of FOL talc cultures showing a possible chlamydospore.

Salt method

Cultures of FOL4 grew effectively in the saturated salt solution but even when flasks were left

for longer than the recommended four week period, only small numbers of chlamydospores

were observed with the majority of spores being microconidia. Therefore, this method was

not deemed suitable for FOL4.

Possible chlamydospore


Soil method

Two FOL4 soil cultures were examined one month after inoculation and microscopy

confirmed that the majority of spores present were chlamydospores with very few

microconidia (Fig. 10). After dilution plating, concentrations were found to be 5.4 x 106 and

4.7 x 106 cfu/g respectively, which would be suitable for use in future experiments to test the

effect of heat and disinfectants on chlamydospore survival.

Fig. 10: Chlamydospores (stained with lactophenol blue) observed in soil cultures of FOL4

isolate AJ516. Chlamydospores can be identified by their distinctive double wall.



Separate glasshouse pot experiments to identify resistance to FOL within lines selected from

the Warwick lettuce diversity set were carried out for FOL1 and FOL4 and a high level of

disease observed by the end of both assays after 39 days. Lines 12, 4, 33, 36, 40, 41 and

42 were highly resistant to both FOL1 and FOL4 (Figs. 11 & 12) while many lines

(predominantly butterhead types) were highly susceptible to both races (e.g. 17, 44 and 22).

Potential differentials for distinguishing FOL1 from FOL4 were also identified with lines 1, 3,

5, 14, 31, 35 and 38 all highly susceptible to FOL1 but resistant to FOL4. Lines 10 and 46

were highly susceptible to race 4 but resistant to race 1. The published differentials did not

produce a clear phenotype. Line 54 (Banchu Red Fire, resistant to FOL4, susceptible to


FOL1) showed some vascular browning and was not completely resistant to FOL4. Line 32

(Costa Rica, resistant to FOL1, susceptible to FOL4) was highly susceptible to FOL4 but also

showed a very low level of vascular browning when inoculated with FOL1.

Fig. 11: Vascular browning scores for 54 lettuce lines from the Warwick diversity set 39 days

after transplanting into FOL1 or FOL4 infested compost.

Fig. 12: Symptoms of FOL infection on line 31 (susceptible to FOL1 but resistant to FOL4).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

41 40 3 14 18 16 20 13 21 50 53 15 19 6 43 44 17 22 54 26 42 51 49 11 25 35 33 47 48 10 34 27 28 29 30 31 7 36 23 4 24 45 2 9 32 46 5 39 52 12 38 37 8 1

Vas

cula

r b

row

nin

g sc

ore

Lettuce diversity set line

FOL race 4 FOL race 1

Batavian Butterhead Latin Leaf / Cutting Romaine WildIceberg

Oils

eed

Ste

m /

stl

ak

FOL1

CONTROL

FOL4

CONTROL

FOL1

INOC

FOL4

INOC



Initially, heat treatments were tested for their ability to kill FOL4 conidia. It was found that

treating spores at 60°C for 15 minutes was sufficient for 100% kill (Fig. 13). After just a 5

minute exposure time, almost all conidia were killed at 60°C. Lower temperatures had little

or no effect on the viability of conidia.

Fig. 13: Survival of FOL4 conidia after different heat treatments for 5, 15 or 60 minutes.

When used for the spore killing experiment, it was shown that the heat treatment was 100%

effective (Fig. 14). Based on the results of a controlled lab experiment, it seems that DNA

from dead spores is rapidly degraded in soil (Table 7). The Ct values of DNA extracted from

the killed spores was 35 after 7 days compared to 28 at time 0. When DNA concentration is

extrapolated from this value, it shows that there was a 99% reduction in the quantity of

detectible DNA after only 7 days. At all timepoints after 7 days, DNA was undetectable with

00 00 00

1

2

3

4

5

6

7

8

9

1 2 3 4 5

Spo

res

pe

r m

l (1

03)

Temperature (oC)

5

15

60

00 00 00

1

2

3

4

5

6

7

8

9

1 2 3 4 5

Spo

res

per

ml (

10

3)

Temperature (oC)

5

15

60

20 40 50 60 70

20 40 50 60 70


the exception of a single replicate at day 21. This shows that, in this particular soil, DNA is

rapidly degraded following cell death.

Fig. 14: The effect of heat treatment (60°C for 15 minutes) on the viability of FOL4 conidia.

No growth was observed following heat treatment.

Table 7: Detection of FOL spores by qPCR following a fatal heat treatment. Standard error

refers to the standard error of the mean (3 replicates). Colours indicate a low Ct (high

detection level, green), a medium Ct (orange) or a high Ct (low level detected, red).

Treatment Ct values Standard errors

Time (days)

No spores

Live spores

Killed spores

Live spores

Killed spores

0 negative 27.4 27.7 0.11 0.12

3 n/a 27.0 31.3 0.10 0.15

7 n/a 28.7 35.1 0.23 0.58

14 n/a 29.6 negative 0.24 n/a

21 n/a 30.4 36.0* 0.49 n/a



*only detected in 1 out of the 3 biological replicates


Following inoculation with FON at five different concentrations, a very clear dose-response

was observed in relation to final basal rot levels on the bulbs, with no significant disease

developing below 10,000 cfu g-1 (Fig. 15, P < 0.001). A low level of background bulb infection

present in the bulb used was observed as very low disease levels in the non-inoculated

Heat treatedUntreated


controls. There was a very strong correlation (R2 = 0.95) between FON inoculum

concentration and final basal rot disease score (Fig. 15).

Fig. 15: Effect of FON dose on the development of Narcissus basal rot. LSD refers to the

least significant difference following ANOVA analysis (5% level).

0

1

2

3

4

5

6

0 100 1000 10000 100000 1000000 LSD

Me

an b

asal

ro

t d

isea

se s

core

FON inoculum concentration (cfu g-1)

y = 1.1599x - 1.5519R² = 0.9489

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7

mea

n b

asl r

ot

dis

ease

sco

re

FON inoculum concentration (Log+1 cfu g-1)


Discussion

1.5. Test FOC diagnostic primers using soil and bulb samples

The FOC qPCR assay alongside an optimised soil DNA extraction method developed

previously has been shown to be highly specific and able to detect the pathogen in bulb, root

and soil samples (Clarkson et al., 2019). However, one concern was whether detection using

a soil sample of 0.5g was sufficiently representative of a larger soil sample of up to 500g

taken across a field. The consistent positive results that were observed for 39 sub-samples

from a larger field soil sample indicates that the method of mixing, drying and sieving provided

a homogenous sample from which 0.5g can be reliably used for qPCR.


SIX genes, originally identified in F. oxysporum f. sp. lycopersici, (Lievens et al, 2009; Ma et

al, 2010) appear to play an important role in F. oxysporum pathogenicity in numerous plant

hosts (Taylor et al, 2016). Moreover, reports have shown that F. oxysporum races can also

be defined by the presence / absence of SIX genes or sequence variation within a SIX gene

(Meldrum et al, 2012; Fraser-Smith et al, 2014). Here it was shown that FOL1 and FOL4

isolates vary in the presence / absence of SIX8 as well as the number of copies of SIX9.

Additional effectors, differing between FOL1 and FOL4, were also identifed following genome

analysis thus providing targets for development of diagnostics (10.2) and a basis for future

work to understand the genetic basis for pathogenicity in FOL.


As F. oxysporum isolates have a very high level of sequence similarity in their core genomes,

molecular tools are now targeting pathogenicity-related genes rather than core genome

genes (Edel-Hermann & Lecomte, 2019). This approach has been successful for f. spp.

cubense, lycopersici and phaseoli (Fraser-Smith et al, 2014, Lievens et al, 2009, de Sousa

et al, 2015) and with the rapid increase in availability of genome sequences, this is now

feasible for a greater number of f. spp. Using this approach for following FOL genome

analysis, qPCR primers were designed based on an effector gene that has a sequence

specific to FOL4. Assay efficiency was high and initial specificity testing suggested that these

primers are highly specific to FOL4. Further validation is being carried out as part of AHDB

project FV PE 458. Once validated, they can be used to test soil and plant material for FOL4.


These are a clear improvement on previously published primers (Gilardi et al, 2017) which

are not suitable for qPCR and showed some cross-reaction with other F. oxysporum f.spp.

Based on the FOL genome analysis, new LAMP primers were designed which efficiently

amplify DNA from both FOL1 and FOL4. These effectively detected FOL from infected plant

material following a crude DNA extraction which takes only 5 minutes. Therefore, using this

assay, it is possible to provide a molecular disease diagnosis within an hour of receiving

samples. The Genie II LAMP machine also has the advantage of being portable and could

potentially be used at grower sites. Initial specificity testing has been carried out and will be

completed in AHDB project FV PE 458. However, preliminary results suggested that the

assay is more specific than the published FOL LAMP assay (Ortega et al, 2018). The other

drawback with the published assay is that it can only be purchased as a kit which is much

more expensive. Once fully validated, the new LAMP assay will be a useful tool for rapid

diagnosis of FOL.


Isolates of F. oxysporum appear to vary in their ability to produce chlamydospores and it is

therefore critical that methods are optimised for each isolate. Of the methods tested here, a

soil culture method was the most effective for FOL4 and will be used in AHDB project FV PE

458. The method is simple and produces large numbers of chlamydospores in only four

weeks.



Fusarium wilt poses a serious threat to the UK protected lettuce industry (Taylor and Clarkson

2018). Control is challenging due to the accumulation and long-term survival of

chlamydospores in the soil. Therefore, identification of resistant varieties is an important

strategy for control the pathogen and has been successfully used to reduce the impact of

FOL1 in the USA (Scott et al, 2010). We have identified lettuce lines (from a diversity set)

that show a high level of resistance to FOL1 and FOL4 which will be verified in future

experiments with an increased level of replication. Further work will also be carried out on

interactions between FOL and lettuce in a recently funded iCASE PhD supported by AHDB.

Lettuce differential lines, to distinguish FOL1 and FOL4, were also identified. These lines

produced a clearer phenotype than the published differentials (GIlardi et al, 2017) and will


therefore be useful in future research. Ideally, new isolates of FOL should be identified using

a combination of molecular tools and plant testing.


The question of detecting dead fungal pathogen material in soil due to the survival of DNA is

often debated and conflicting reports are present in the scientific literature. Based on an initial

experiment using FOL spores killed by a heat treatment, it appears that the DNA released is

degraded rapidly in soil. This may vary between soil types particularly as reports suggest that

DNA will bind to clay particles (Alvarez et al, 1998). Therefore, this experiment should be

repeated using a range of soil types.


This experiment determined the critical level of FON inoculum required to cause significant

disease development. Further work will now utilise a FON-specific qPCR assay to relate

qPCR values to inoculum rate and disease development. This will be an important first step

in assessing the utility of the PCR tests for assessing disease risk in the field.

Conclusions

• A FOC qPCR diagnostic assay was shown to be reliable and reproducible

• Genome sequencing of FOL1 and FOL4 showed that they differ in their effector gene

compliment and sequence. In contrast to FOL1, SIX8 was present in all FOL4 isolates

tested, suggesting an important role in pathogenicity

• qPCR and LAMP assays were developed for FOL which will be of great value for

testing soil and plant material. The LAMP assay has the benefit that it produces results

very rapidly

• An efficient method of chlamydospore production was identified for FOL4

• A high level of resistance to both FOL1 and FOL4 was identified in a lettuce diversity

set as well as differential lines for distinguishing the two races

• It was found that FOL4 DNA is degraded rapidly in soil; hence the presence of dead

pathogen is unlikely to result in false positives.

• The critical level of FON inoculum required for disease was determined


Knowledge and Technology Transfer

• Attended and gave talk (Understanding and combatting Fusarium diseases of onion

and lettuce) at VeGIN meeting at Warwick Crop Centre (27th Nov 2018)

• Attended BSPP Presidential meeting at Warwick University and discussed our

research with people from around the world (10th-11th Dec 2018)

• Attended and gave talk (Lettuce Fusarium wilt in the UK) UK Brassica and Leafy

Salads Conference in Peterborough (23rd Jan 2019)

• Attended and gave talk (Research on Fusarium basal rot of onion and other vegetable

diseases) at Hutchinsons Veg Conference in Peterborough (26 th Feb 2019)

• Hosted guest seminar by Sascha Mooney, University of Nottingham (7 th March 2019)

• Attended the Journal of Horticultural Science and Biotechnology conference in

Charlecote and discussed our research with potential collaborators (9th May 2019)

• Hosted visit and guest seminar by Louise Thatcher from CSIRO, Australia (17 th June

2019)

• Attended and gave an invited talk (Creation and characterisation of an onion diversity

set and identification of accessions with resistance to Fusarium basal rot and

improved seedling vigour) at the International Allium Conference in Madison, USA

(23rd – 27th July 2019)

• Visited lettuce grower to discuss Fusarium issues (26th Sept 2019)

• Attended Fusarium meeting in Utrecht and met with researchers at Wageningen (29 th

– 31st Oct 2019)

• Multiple visits / phone conversations with a rocket grower to discuss Fusarium issues

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Documents

Interim Report Form · 2020. 3. 18. · FOL4 by PCR / genome analysis 31/10/19 31/10/19 10.2. Develop and test qPCR / LAMP diagnostics for FOL4 31/10/19 31/10/19 10.3. Develop method