• Grower summary


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    • Abstract: HDC project PC 186 (O’Neill, 2002) forewarned UK growers of a new form of verticillium wilt ... Grower estimates suggest a yield loss of 10-15% where wilting is obvious in a ...

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Horticultural
Development
Council
Grower summary
PC 186a
Tomato: Epidemiology and
control of Verticillium wilt in
hdyroponic and soil-grown
crops
Final report 2006
© 2007 Horticultural Development Council
Disclaimer
Whilst reports issued under the auspices of the HDC are prepared from the best available
information, neither the authors nor the HDC can accept any responsibility for inaccuracy or
liability for loss, damage or injury from the application of any concept or procedure
discussed.
The results and conclusions in this report may be based on an investigation conducted over
one year. Therefore, care must be taken with the interpretation of the results.
Use of pesticides
Only officially approved pesticides may be used in the UK. Approvals are normally granted
only in relation to individual products and for specified uses. It is an offence to use non-
approved products or to use approved products in a manner that does not comply with the
statutory conditions of use, except where the crop or situation is the subject of an off-label
extension of use.
Before using all pesticides check the approval status and conditions of use.
Read the label before use: use pesticides safely.
Further information
If you would like a copy of the full report, please email the HDC office ([email protected]),
quoting your HDC number, alternatively contact the HDC at the address below.
Horticultural Development Council
Tithe Barn
Bradbourne House
East Malling
Kent
ME19 6DZ
Tel: 01732 848 383
Fax: 01732 848 498
The contents of this publication are strictly private to HDC members. No part of this
publication may be copied or reproduced in any form or by any means without prior written
permission of the Horticultural Development Council.
© 2007 Horticultural Development Council
Headline
• This project has shown that a very low inoculum of Verticillium albo-atrum can result in
infection of tomato seedlings, that above- ground movement of the fungus is possible
and that there can be many symptomless infected plants in a crop at the end of a
season.
• Root drench treatment with carbendazim (eg Delsene 50 Flo or Cleancrop Curve)
reduced symptom development in infected plants, including plants which already
showed symptoms at the time of application and Unifect G (glutaraldehyde + QAC) was
the most effective disinfectant in preventing growth of V. albo-atrum mycelium when
tested in the presence of peat and soil.
Background and expected deliverables
HDC project PC 186 (O’Neill, 2002) forewarned UK growers of a new form of verticillium wilt
that is a potentially serious risk to the economic production of tomatoes. The problem
affected at least 10 nurseries in 2001 and was confirmed on a similar number in 2002,
affecting crops in England, Wales and Scotland. Previously, a verticillium wilt resistance
gene (Ve), incorporated in to many commercial European tomato varieties and rootstocks
since the late 1970s, had provided good control of verticillium wilt in crops. HDC project PC
186a, in conjunction with Defra project HH3222SPC, is intended as pre-emptive research to
devise a solution before the disease becomes more widespread and damaging.
Key points from the 2002 review were:
• The causal fungus is slow-growing and not always easy to detect in affected plants.
• Many varieties and rootstocks have been affected.
• The disease is a 'slow wilt' in tomatoes and may be confused with wilting caused by
root rotting fungi (e.g. Pythium).
• Growers estimated yield losses at 10 - 15%.
• The disease is difficult to eradicate from a nursery; it tends to occur in the same
glasshouses each year.
• Sources of the disease and how it spreads are unknown.
• Peppers and cucumbers are also susceptible; two lettuce varieties tested were
unaffected.
• No sources of resistance to verticillium, other than the Ve gene, are currently available in
either tomato varieties or rootstocks.
© 2006 Horticultural Development Council
This project aims to increase our understanding of the cause and spread of verticillium wilt
in tomato crops, of crop cultural and management practices that influence disease
expression, and to devise best methods of disease control before the disease becomes very
widespread. The associated Defra-funded project (HH3222SPC) investigated detection,
molecular characterisation and natural suppression of V. albo-atrum in tomato (O'Neill,
2006).
Summary of the project and main conclusions
Literature review
The fungus Verticillium albo-atrum, its life cycle, the infection process, host resistance,
disease epidemiology and diagnosis have been reviewed (Year 1 report, November 2004).
Symptoms and yield loss
The new verticillium wilt disease affecting resistant (Ve) varieties of tomato is essentially a
'slow wilt'. Symptoms differ from those of classic verticillium wilt. Thin stems, stunted
growth and reduced leaf size are the most common manifestations of the problem; leaf
wilting at this stage is rare. As the disease progresses, wilting may develop, and eventually,
but not always, the plant may die. At this late stage the pathogen may sporulate on the
stem. Grower estimates suggest a yield loss of 10-15% where wilting is obvious in a
rockwool crop, with occasional dead plants; and 20-25% where plant death is widespread
by September. The problem has been confirmed in rockwool, NFT and soil-grown crops.
Varieties affected
Many resistant (Ve+) varieties have been affected including Carousel, Cloe, Conchita, Eloise,
Encore, Espero, Ferrari, Romalina, Rosa, Sarena and Solairo. Both Espero and Carousel
grafted onto Beaufort and Maxifort rootstocks were found to be infected after 10 weeks but
apart from slight vascular discolouration the scions did not show symptoms (i.e. these
rootstocks are susceptible to Verticillium although in the presence of the pathogen they may
offer advantages over ungrafted plants because of enhanced root vigour).
© 2006 Horticultural Development Council
Causal fungus
The Verticillium species isolated most consistently from wilt-affected plants was V. albo-
atrum. V. dahliae, V. nigrescens and V. tricorpus were isolated occasionally in recent
outbreaks (isolate identity confirmed by DNA sequence analysis by D Barbara, Warwick
HRI). Previous inoculation studies (O'Neill, 2002a) confirmed that V. albo-atrum isolated in
1999 from cv. Espero (Ve+) in the UK was equally damaging to susceptible (cv. Shirley) and
resistant plants (cv. Trio and a numbered variety).
Occurrence of Verticillium in commercial crops
In October 2005 at the end of cropping, the incidence of Verticillium was determined in the
stem bases of 14 crops. Verticillium was confirmed in 12 crops with up to 89% of stems
infected. The mean incidence of stem infection was no greater in crops where verticillium
wilt was considered to be present (7) than in crops where there were no symptoms of the
disease (7). Affected plants were found in all counties where crops were examined (Isle of
Wight, Kent, Lancs, Norfolk, Yorks), and in crops grown in the soil, on rockwool slabs and in
NFT. These results suggest that infection of tomato stem bases by Verticillium is relatively
common at the end of cropping, and that infection is often symptomless.
In November 2006 at the end of the growing season, the distribution of infected plants was
examined in 10 adjacent rows of a crop grown on rockwool slabs. There were no obvious
symptoms of verticillium wilt in the crop. Verticillium was found in the stem base of 45% of
120 plants. The number of infected plants on either side of the double rows of plants were
similar (28/60 and 26/60), with no clear evidence of along-the-row spread. In only two of the
30 slabs were all four plants infected (there were two plants per propagation block and two
blocks per rockwool slab). A further seven slabs had three positive, eight had two positive,
none had one positive and four had no positives. These figures indicate that movement of
Verticillium within a slab is not guaranteed even when there is more that one plant infected.
Comparing the two plants in the same propagation block (where roots are presumably
intermingled), 12 out of 60 blocks had both plants positive, but 30 had only one and a
further 18 had neither infected. These results strongly suggest that both plants did not
become infected when one of them became infected. Possible explanations for this failure
of both plants in a block to be infected (where one is infected) are a low inoculum, different
times of infection or a means of spread other than through the roots. If there is a means of
spread above the root zone, possible methods are by airborne spores, insects and through
handling plants.
© 2006 Horticultural Development Council
Sources of V. albo-atrum on a nursery
Tomato crops on some nurseries and in certain glasshouse become infected by V. albo-
atrum each year despite apparently thorough nursery hygiene and intensive disinfection
between crops. Tests were undertaken to try and identify possible areas where V. albo-
atrum might persist on a nursery between crops.
A molecular method based on PCR (Polymerase Chain Reaction) for the specific and
sensitive detection of V. albo-atrum, developed in a parallel Defra-funded project
(HH3222SPC), was used to test various potential sources of the fungus. The detection limit
of the PCR test is around 1,000 spores. The fungus was not detected on tomato seed, in
rockwool taken from slabs, insects on sticky traps, or on drip pegs, all collected from an
affected crop. Samples of crop debris, volunteer tomato seedlings, fallen leaves and fruit,
and pathway sweepings were also negative. Further testing of samples (e.g. seed) is
considered warranted when a detection method with a lower sensitivity is developed.
An inoculation experiment did not support the hypothesis that infected seed are a source of
verticillium wilt in tomato. Tomato seeds inoculated with conidia of V. albo-atrum
germinated normally and, when grown on for 4 weeks, no symptoms of verticillium wilt
developed. However, further seed testing is needed before a firm conclusion can be drawn
regarding seed transmission of V. albo-atrum in tomato.
Spread of V. albo-atrum
The current literature indicates that infection by V. albo-atrum invariably occurs via the
roots. There are no reports of aerial dissemination of V. albo-atrum spores in tomato crops.
Nevertheless, there appears to be potential for above-ground spread. Stem lesions bearing
sporulating V. albo-atrum sometimes develop on tomato plants that have died following
infection by the fungus, especially late in the season. Also, sporulation of V. albo-atrum has
been observed on the undersurface of infected leaves if they are maintained in a humid
chamber, and on tomato debris in soil. Season-long spore trapping was therefore
undertaken on a nursery in Kent with a history of the disease to examine whether aerial
dissemination of the fungus occurs.
© 2006 Horticultural Development Council
Air was aspirated onto Vaseline-coated slides, the Vaseline removed and tested for V. albo-
atrum by PCR. V. albo-atrum was detected in the 2003/04 and 2004/05 crops (Table 1). V.
albo-atrum was not detected in 2005/06 even though the end-of-season level of Verticillium
in the crop was very similar to those of the earlier years. In 2003/04, the fungus was
detected before symptoms were seen in the crop. These results indicate that above-ground
spread of V. albo-atrum may account for outbreaks of the disease and spread within a
glasshouse crop. The fungus was not detected in the air on a nursery in Norfolk where
spore trapping was undertaken from March to May 2005 following occurrence of verticillium
wilt symptoms in a few plants in February.
Table 1: Detection of V. albo-atrum in Vaseline on spore trap slides at monthly intervals in
tomato crops on a nursery in Kent over three successive seasons.
V. albo-atrum detected (+):
Month 2003/04 2004/05 2005/06
December + - -
January + - -
February + + -
March - - -
April - - -
May + - -
June + + -
July + - -
August - + -
September - - -
October - - NT
November + NT NT
+ Vaa detected, - Samples tested and no V. albo-atrum found; NT – not tested
Each year the old crop was removed in early November and the new crop was planted in December.
Detection and monitoring of V. albo-atrum in commercial crops
Damp incubation and microscopic examination of leaf petioles slices was found to be a
useful method for detection of Verticillium in tomatoes without destroying the plant. This
method was used to examine the occurrence of the disease within commercial crops. In
2004, in a crop of cv. Encore where a few plants at a row end were visibly affected by
verticillium wilt, 15% of apparently healthy plants along the row were also found to be
infected. None of these plants developed obvious symptoms of verticillium wilt in the
subsequent 8 weeks.
© 2006 Horticultural Development Council
On a nursery in Kent with a history of verticillium wilt, rockwool crops were monitored for the
disease at intervals throughout 2004, 2005 and 2006. Wilting was first seen in the crops, at
a very low level, at the end of January 2004, and Verticillium was confirmed. There was little
visual affect of the disease for most of the season, although the plants thinned in the head
in midsummer. The incidence of infected plants of cv. Encore, determined by testing leaf
petioles, increased from 6.5% in March to 21.2% in October. When stem bases were
tested at crop pull-out in late October, 65% of stems were found to be infected. In 2005,
the incidence of Verticillium in petioles of a crop of cv. Encore at this site increased from
1.8% in March to 50% by early August; 92% of stem bases were infected by crop pull-out
on 15 October; verticillium wilt was not obvious in the crop. In 2006, the incidence of
Verticillium in petioles of a crop of cv. Encore at this site was 22% in early September; 59%
of stem bases were infected at crop pull-out in early November. Again, verticillium wilt was
not obvious in the crop.
Inoculum level and plant age
An inoculum level of just 100 spores applied as a drench to tomato seedlings at two, four
and six true leaves growth stages, resulted in development of wilt symptoms after around 5
weeks irrespective of seedling age. Seedlings inoculated with a higher inoculum of
1,000,000 conidia developed wilt symptoms after 4 weeks. Not all inoculated plants had
developed symptoms by 10 weeks after inoculation. The severity of symptoms increased
with spore concentration from 100 to 1,000,000 spores/plant.
Detection of V. albo-atrum within stems following root and leaf scar inoculation
Ten weeks after inoculation of plants by drenching spores into the root zone, V. albo-atrum
was confirmed by a specific PCR test in vascular tissue at the stem base and 50 cm above
the stem base. Using this method, V. albo-atrum DNA (either mycelium or spores) was
detected in plants inoculated with 106 spores/ml but not with the lower inoculum levels even
though these treatments showed wilt symptoms.
Following inoculation of V. albo-atrum spores onto fresh leaf scars of tomato seedlings, the
fungus was detected by PCR in surface sterilised stem tissue 1 cm above and 1 cm below
the inoculation point. This result indicates V. albo-atrum is present within the stem to this
extent.
© 2006 Horticultural Development Council
Effect of plant growth and environmental factors on symptom expression
Measures which reduce water stress on a tomato crop appear to slow the development of
verticillium wilt, and those which increase water stress enhance development of the disease.
In the Netherlands, for example, removal of some fruit from trusses is suggested as a
control measure. In chrysanthemum, verticillium wilt symptom expression is affected by
growth stage and tends to show as plants come into flower. In field-grown tomatoes,
verticillium wilt is reported to be worse when root growth is restricted (eg by a soil pan). A
series of experiments was therefore undertaken to investigate the effect of various plant
growth and environment factors on symptom expression of tomato wilt caused by V. albo-
atrum.
Experiments on tomatoes grown in peat bags indicated that the severity of verticillium wilt
symptoms is affected by fruit load and the extent of sideshoot growth. Wilt severity was
reduced by reducing the fruit load and by regular removal of sideshoots.
Following drench inoculation with a standard inoculum of V. albo-atrum spores, plants
grown in compost that was kept moist developed verticillium wilt symptoms whereas plants
grown dry did not. Possibly this was due to death of conidia in the plants watered
infrequently and allowed to dry out. A subsequent experiment on the effect of air-drying on
spore viability showed that wetted conidia of V. albo-atrum survived drying for 6h but not for
24h. Previous studies indicated that conidia of V. dahliae are capable of surviving for up to
2 weeks in soil (Green, 1969).
Eight weeks after inoculation, pot-grown plants simultaneously inoculated with
Phytophthora cryptogea, a pathogen which reduces root function, and V. albo-atrum, were
significantly reduced in height, by 14-20 cm, compared with plants inoculated with either
fungus alone.
The effect of daylength on expression of verticillium wilt symptoms was examined by
growing inoculated plants, cv. Espero, in growth rooms with 8 h and 16 h days. No
consistent effect was observed.
© 2006 Horticultural Development Council
Effect of rootstock on wilt development
The basis of resistance to Verticillium (the Ve gene) is the same in rootstocks as in F1
varieties. However, expression of resistance in rootstocks compared with F1 varieties may
be affected by modifier genes, the genetic background or the greater vigour of rootstocks.
The susceptibility of grafted and ungrafted plants was compared by drench inoculation of V.
albo-atrum spores onto the roots of potted plants. No verticillium wilt symptoms had
developed after 6 weeks. Surprisingly, a significantly greater incidence of stem base
infection was detected in grafted than in ungrafted plants. Only a very low incidence of
Verticillium infection was detected in stems of both grafted and ungrafted plants at 10 cm or
more above the stem base. No conclusions on the effect of grafting onto a rootstock on V.
albo-atrum in stems could be drawn because of the lack of symptoms and the low
incidence of stem infection above the base.
Fungicide efficacy
Six fungicide treatments were evaluated for control of verticillium wilt in tomato plants cv.
Shirley. The fungicides were applied as a drench to the compost surface before and/or after
inoculation with 106 conidia of V. albo-atrum in water. Amistar was phytotoxic and plants
were severely stunted. Verticillium wilt symptoms (leaf sector yellowing) were seen 28 days
after inoculation in all but the Amistar treated plants. By 44 days after inoculation,
significantly fewer leaves and plants were infected in the fungicide-treated plants than in the
inoculated control (Fig 1). At this time there was significantly better control (% leaves
affected) with two carbendazim drenches (Delsene 50 Flo) than with a single application.
Infection within stems was confirmed in all of the fungicide treatments.
In a further experiment on cv. Shirley, the efficacy of carbendazim was evaluated as a
curative treatment. Delsene 50 Flo applied as a drench significantly reduced symptom
development in infected plants, including plants already showing symptoms at the time of
application (Table 2).
Five isolates of V. albo-atrum obtained from tomato between 2000 and 2005 were tested for
their sensitivity to carbendazim (as Delsene 50 Flo) in agar plate tests. Mycelial growth of all
isolates was completely inhibited at concentrations of 2 mg/L or greater (i.e. there was no
evidence of fungicide resistance).
© 2006 Horticultural Development Council
8
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Fig 1. Effect of fungicide drench treatments on the percentage of leaves affected with
verticillium wilt
Chemical disinfectants
Six disinfectants (Harvest Wash, Jet 5, sodium hypochlorite, Trigene, Unifect G and Virkon
S) were all fully effective against conidia of V. albo-atrum when used at their standard rate
and with a contact time of 5 minutes or greater. Citric acid had no effect even after 8 hours.
When tested against V. albo-atrum mycelium grown on filter paper discs, only Panacide M
and Unifect G eliminated the fungus following a 5 minutes contact time. Sodium
hypochlorite was effective after 30 mins, Trigene after 1 hr and Jet 5 and Virkon S after 8
hours. Citric acid and Harvest Wash were ineffective. Unifect G was the most effective
disinfectant at preventing growth of V. albo-atrum mycelium in the presence of peat and
soil.
© 2006 Horticultural Development Council
Table 2: Effect of fungicide treatment after inoculation with V. albo-atrum on the
development of verticillium wilt symptoms.
Inoculation with Symptoms Delsene 50 % plants affected
V. albo-atrum present on drench
(15 Feb & 11 Apr) 5 May Applied 5 May 25 May
1. - - - 0 0
2. - - √ 0 0
3. √ √ - 23 62
4. √ √ √ 33 43
5. √ - - 0 31
6. √ - √ 0 17
Contamination and disinfection of hands
Spores of V. albo-atrum were found to be surprisingly persistent on hands and were
relatively difficult to remove by washing. Once contaminated by touching a sporulating stem
lesion, a thumb was still transmitting V. albo-atrum after 160 sequential contacts with an
agar plate. Even after washing with warm water and soap for 30 seconds, transmission of V.
albo-atrum occurred for over 50 sequential contacts.
Bare hands were effectively cleaned of V. albo-atrum by rubbing with Med Gel for 1 minute;
washing in warm water and soap for 1 minute was not effective. Latex gloves also
transmitted V. albo-atrum but were easier to clean; both Med Gel (1 minute) and warm water
and soap (1 minute) were effective.
Financial benefits
The UK area of protected tomatoes in 2003 was estimated at 179 ha (4 ha unheated), with a
total farm-gate value of around £80.1 million (Defra – Basic Horticultural Statistics for the
United Kingdom). Assuming that 5% of the cropped areas suffers a 10% loss due to
verticillium wilt, this equates to £400,425 per annum. Development of an effective strategy
to control verticillium wilt will thus have a significant financial benefit.
© 2006 Horticultural Development Council
Action points for growers
• Become familiar with the symptoms of 'slow wilt' caused by V. albo-atrum in
resistant (Ve+) varieties. These are illustrated and described in HDC Factsheet 15/01.
• If you suspect plants are affected by verticillium wilt, have affected plants examined
by a plant pathologist or tested at a diagnostic laboratory. The incidence of
verticillium in a crop can be assessed in a non-destructive manner by tests on leaf
petioles.
• Carefully and promptly remove severely wilted or dead plants that are infected by V.
albo-atrum. Sporulation of V. albo-atrum can occur on stem lesions of dying plants
late in the season, and there is evidence of aerial dissemination of these spores.
Possibly sporulation may also occur on leaf debris, earlier in the season. Although no
sporulation of Verticillium was found on leaves in affected crops we examined,
abundant sporulation developed when affected leaves were incubated in a humid
environment.
• Do not re-use rockwool slabs from an affected crop, either for tomato or another
verticillium-susceptible crop.
• Do not handle stem lesions. After touching a sporulating Verticillium stem lesion,
hands will be contaminated with spores of the fungus and they could spread the
disease (e.g. via fresh de-leafing scars) to healthy plants
• Hands or latex gloves contaminated with V. albo-atrum spores can be disinfected by
rubbing in Med Gel for 1 minute.
• Keep up with sideshoot trimming in an infected crop to reduce the risk of verticillium
wilt symptom development.
• Apply measures to reduce stress on infected plants, such as use of shade screen in
very bright conditions immediately after dull weather, adjusting heating and
ventilation, and reducing a very high fruit load.
© 2006 Horticultural Development Council
• Consider treatment with an approved carbendazim fungicide (eg Cleancrop Curve,
Delsene 50 Flo) if verticillium wilt is confirmed in a crop. A root drench treatment
reduced symptom development in infected plants, including plants already showing
symptoms at the time of application.
• Take precautions to minimise the risk of other diseases affecting the crop. Grower
observations, and research in the Netherlands, indicate rapid development of wilt
and plant death may occur when a crop is affected by both Pepino mosaic virus and
verticillium wilt. There is also evidence that severe reductions in growth occur when
a plant is affected by both verticillium wilt and Phytophthora root rot.
• Clean and disinfect the glasshouse and all associated equipment after an outbreak
of the disease. See HDC Factsheet 15/01 for more details.
• The disinfectants Harvest Wash, Jet 5, sodium hypochlorite, Trigene, Unifect G and
Virkon S are all fully effective against conidia of V. albo-atrum when used at their
standard rate and with a contact time of 5 minutes or greater.
• Only Panacide M and Unifect G eliminated V. albo-atrum mycelium grown on filter
paper discs following a 5 minutes contact time. Sodium hypochlorite was effective
after 30 mins. Consider using Unifect G to disinfect areas contaminated with V.
albo-atrum and soil or organic matter.
© 2006 Horticultural Development Council


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