HDC Project Self Assessment And Report Form



|Project Number: |SF 98 |

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|Project Title: |Sustainable management of Mucor and Rhizopus in strawberry |

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|Project Leader: |Dr Angela M Berrie, EMR |

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|Contractor: |East Malling Research |

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|Industry Representative: |Mr John Clark |

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|Report: |Final Report June 2011 |

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|Publication Date: | |

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|Start Date: |1 May 2009 |

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|End Date: |30 April 2011 |

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|HDC Cost: | |

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|Total Cost: | |

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|Key words: |Strawberry, raspberry, Mucor, Rhizopus, fungicide, biocontrol agent |

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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 Angela M Berrie

Research Leader

East Malling Research

Signature ............................................................ Date ............................................

Report authorised by:

Dr Christopher Atkinson

Head of Science

East Malling Research

[pic]

October 2011

Signature ............................................................ Date ............................................

CONTENTS

GROWER SUMMARY 1

Headline 1

Background and expected deliverables 1

Summary of the project and main conclusions 2

Financial benefits 4

Action points for growers 4

SCIENCE SECTION 6

Introduction 6

Summary of the project and main conclusions from the first year 7

Overall aim of project 7

Specific Objectives 8

Materials and methods 8

Objective 1 – Incidence and importance of Mucor and Rhizopus 8

Objective 2 – Management practices and disease risk 9

Objective 3 – Laboratory evaluation of efficacy of fungicides and alternative products - in vitro testing 9

Objective 4 – Identification of the importance of fruit flies in the spread and incidence of the soft rot diseases 15

Results and discussion 16

Objective 1 – Incidence and importance of Mucor and Rhizopus 16

Objective 2 – Management practices and disease risk 16

Objective 3 – Laboratory evaluation of efficacy of fungicides and alternative products In Vitro testing 18

Objective 4 – Identification of the importance of fruit flies in the spread and incidence of the soft rot diseases 27

Conclusions 30

Technology transfer 30

References 30

Acknowledgements 31

APPENDICES 32

GROWER SUMMARY

Headline

The fungicides Switch (cyprodonil + fludioxonil), Signum (pyraclostrobin + boscalid) and Thianosan (thiram) were shown in laboratory tests to be active at relatively low concentrations against Mucor and Rhizopus soft rots

Background and expected deliverables

Mucor and Rhizopus both cause soft rots (also called leak disease) in raspberries and strawberries and losses can be significant when conditions are favourable resulting in rapid spread of the fungi in harvested fruit. They are primarily post-harvest rots but also occur on ripe fruit in the field. Both fungi cause similar symptoms on fruit. Infected fruit are initially slightly discoloured, gradually turning pale brown. The tissue rapidly softens and collapses and, under humid conditions, the fruit is soon covered with a dense, fluffy white mycelium which bears stiff, black-headed sporangia (pin mould). Both fungi survive in soil and crop debris between seasons and produce thick walled resting bodies (zygospores) which serve as long-term resting spores. Some circumstantial evidence suggests that the rots may be encouraged by green manures.

Mucor and Rhizopus are both wound pathogens and favoured by wet, moderately warm conditions (around 18oC). The black spores are easily spread by wind, rain and pickers hands. Rhizopus is inhibited by temperatures below 6oC whereas Mucor can grow and infect fruit at 0oC as well as it can at higher temperatures. This means that the former can be controlled in soft fruit by cool chain management whereas Mucor will continue to develop during cold storage and marketing.

Neither fungus is well controlled by fungicides and there is evidence that the occurrence of these rots may be increased by the use of certain fungicides such as Rovral (iprodione) to control botrytis rot. Currently control of these soft rots is based on cultural measures including removal of all ripe fruit at harvest.

In order to develop improved methods of managing these rots to minimise losses it is important first to establish the relative incidence of Rhizopus and Mucor in soft fruit crops. Although fungicides appear to be relatively ineffective against these fungi there may be alternative chemicals such as those used in the food industry that suppress Mucor or Rhizopus and could be used near harvest.

Over two years, this project aimed to identify the relative incidence of Mucor and Rhizopus in soft fruit plantations. This will enable management systems for minimising losses to be developed. In addition, laboratory studies were done to identify potential alternative chemicals active against these rots.

Summary of the project and main conclusions

In 2009, 131 samples of soft rotted berries were received from fruit farms in England and Scotland. Mucor spp. was the more frequently isolated from strawberry and raspberry soft rots than Rhizopus. 74% of isolates from strawberry and 88% from raspberry were identified as Mucor spp. All samples except for 6 (open field Elsanta) were obtained from tunneled crops whether raspberry or strawberry. All isolates from the open field crop were Mucor spp. From this data it is not possible to draw any definitive conclusions on the effect of tunnels on the incidence of Mucor and Rhizopus. Very little information on management practices was collected from the growers that provided the rot samples so it was not possible to investigate the relation between practice and rot incidence. Management of fruit and rots at harvest did vary on the farms visited. Most removed all rots and non-marketable fruit from the plantation at harvest which would tend to reduce the soft rot inoculum and hence the risk of infection by Mucor and Rhizopus.

In 2010, 16 lots of fruit samples (13 strawberry samples, 3 raspberry samples) were received from growers and consultants. These were mainly from Kent (9 samples) but also from Staffordshire, Yorkshire and Cambridgeshire. None were received from Scotland.

Isolations were made from 164 berries, yielding 115 fungal isolates with characteristics typical of members of the Mucorales. Of these, 80 were positively identified as Mucor spp. and 35 as Rhizopus spp.

Thirteen of the Mucor spp. isolates and 15 of the Rhizopus isolates were recovered from raspberry. Similarly, 67 of the strawberry soft rot associated fungi were Mucor spp. and 20 were Rhizopus. That is, 77% of the strawberries with symptoms of soft rot had Mucor spp. as the causal agent, which are similar results to 2009 (74%). In contrast to 2009 (88%), 46% of the soft-rotted raspberries were infected by Mucor spp. The raspberry samples came from two farms in Kent of cvs. Maravilla and Octavia, and so the sample size was rather small.

The efficacy of a range of chemicals against Mucor and Rhizopus was tested in the laboratory using PDA (Potato Dextrose Agar) amended with the chemical under test at concentrations of 0, 1, 10, 100, 1000, 5000 ppm (in vitro tests). Growth of mycelial plugs of the test fungus was measured after 1 or 2 days. Standard isolates of Mucor piriformis, Mucor mucedo and Rhizopus stolonifera were used as well as isolates of Mucor and Rhizopus obtained during the survey. In the in vitro plate tests Signum (pyraclostrobin + boscalid), Switch (cyprodonil + fludioxonil), HDC F 13 (experimental) and Thianosan (thiram) were most effective in inhibiting mycelial growth of Mucor and Rhizopus. Sodium bicarbonate, potassium bicarbonate, potassium sorbate and sodium benzoate all inhibited growth at high concentrations, below that at which the products are used, and so may be worth further evaluation. Sulphur, Amistar (azoxystrobin), Frupica (mepanipyram) and ascorbic acid were ineffective.

Chemicals and biocontrol agents (BCAs) were also tested on tomatoes inoculated with either Mucor or Rhizopus spp. (in vivo tests). Fungal rots spread more slowly in tomatoes which makes it easier for scientists to assess the effect of fungicides. In these tests, Thianosan significantly reduced rotting on tomato fruit inoculated with Mucor spp. Switch, Signum, HDC F 13, Thianosan and the BCA Boni Protect forte all significantly reduced rotting on tomatoes inoculated with Rhizopus spp.

The results are summarised in Table 1a. The most effective products will be evaluated in field trials in strawberry in 2011 as part of the SCEPTRE Horticulture LINK project (HL 01109, CP 77).

In laboratory studies to investigate whether fruit flies were involved in the spread of Mucor in strawberries, fruit flies obtained from a laboratory supply company were placed in plastic boxes containing strawberries previously inoculated with Mucor. After two days the flies were transferred to healthy strawberries and plated on to PDA. The strawberry fruit in the plastic boxes where the flies were transferred rapidly developed soft rots with Mucor sporulating on the affected fruit. Similarly flies plated out onto PDA rapidly developed colonies of Mucor. This study shows that fruit flies are able to spread Mucor.

Financial benefits

Currently soft rots caused by Mucor and Rhizopus are managed by cultural methods only or not at all and are often overlooked as problems. Recently the incidence of these rots has increased, particularly after the last two seasons when the weather has been wet and favourable for spread. The incidence is high, especially after harvest during marketing when rot spread in punnets can be unacceptable to the retailers. Once the disease is present in harvested fruit, spread can be very rapid especially Mucor, which continues to grow at the temperatures used in cold storage and cool chain marketing. For developing strategies to control these rots it is important to know the relative incidence of Mucor and Rhizopus in soft fruit crops. If Mucor were the predominant species then additional control measures would need to be considered pre-harvest to control the problem. Such measures would not be needed for Rhizopus which would be suppressed by the temperatures used in cool chain marketing. Failure to recognise soft rots as a potential problem and take steps to manage them could result in the problem arising during marketing leading to rejection of the consignment with financial loss. This project has established the relative incidence of the two rots in soft fruit plantations, and identified chemicals or biocontrol agents that could be used to control the problem.

Action points for growers

• Most of the soft rots were due to Mucor spp. which can grow at the low temperatures at which raspberries and strawberries are held after picking and during marketing. Therefore it is important to minimise the risk of Mucor infection of fruit during harvest.

• The fungicides Switch, Signum and Thianosan which are approved for use on strawberry showed activity at low concentrations against Mucor and Rhizopus in laboratory tests and could be used to help reduce the soft rots in commercial crops.

• Signum and Switch could similarly be used in raspberries.

• Fruit flies were shown to spread Mucor in strawberry fruit. Measures to minimise fruit fly infestations in plantations and in the packhouse must be considered as part of the management strategy for control of soft rots.

Table 1a. Chemicals and BCAs evaluated in the laboratory in vitro (Plate tests) or in vivo ( Tomato tests) for efficacy against Mucor spp. or Rhizopus spp. in 2010

|Test Product |Active ingredient |

|Mucor piriformis (IMI190877) |CABI |

|Mucor mucedo (IMI133298) |CABI |

|Rhizopus stolonifera var. stolonifera (IMI340068) |CABI |

|Mucor spp. (SC195-106) |EMR, Kent |

|Rhizopus spp. (Fav2) |Faversham, Kent |

|Rhizopus spp. (K1) |Wickhambreaux, Kent |

Table 2. Chemicals and BCAs evaluated in the laboratory in vitro or in vivo for efficacy against Mucor spp. or Rhizopus spp. in 2010

|Test Product |Active ingredient |Rate/ha |Rate/ml |In vitro testing|In vivo testing|

|Amistar |azoxystrobin |1 L |1 ml |X | |

|Frupica |mepanipyram |0.9 L |0.9 ml |X | |

|Scala |pyrimethanil |2.0 L |2 ml |X | |

|Signum |pyraclostrobin (6.7%) + boscalid |1.8 kg |1.8mg |X |X |

| |(26.7%) | | | | |

|Switch |cyprodonil (37.5%)+ fludioxonil |1.0 kg |1.0mg |X |X |

| |(25%) | | | | |

|Headland Sulphur |sulphur |600 ml/100 L |0.006 ml |X | |

|Teldor |fenhexamid |1.5 kg |1.5 g |X | |

|Thianosan |thiram |3 kg/1000L |3 g |X |X |

|HDC F 13 |experimental |0.8 L |0.8 ml |X | |

|Vitamin C |ascorbic acid |- |- |X | |

|Chitoplant |chitosan |1 g/L |1 mg | | |

|Potassium bicarbonate |potassium bicarbonate |5 kg |5.0mg |X |X |

|Potassium sorbate |potassium sorbate |10 g/L |10.0mg |X | |

|Sodium benzoate |sodium benzoate |10 g / L |10 mg |X | |

|Sodium bicarbonate |sodium bicarbonate |20 g/L |20 mg |X | |

|Wetcit |alcohol ethoxylate |2 ml/L |0.002 ml | |X |

|Boni Protect forte |Aureobasidium pullulans |1.2 kg (at 2000 |0.6 g | |X |

| | |L/ha) | | | |

|Prestop |Gliocladium catenulatum |100 g/20 L |5 g | |X |

|Serenade |Bacillus subtilis |10 L |10 ml | |X |

Table 3. Stock solution preparation for Amistar

|Amount of Amistar (33.4% ai) |Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|product per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.43 ml |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|43.5 ml |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 4. Stock solution preparation for Frupica

|Amount of Frupica (45% ai) product|Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.22 ml |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|22 ml |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 5. Stock solution preparation for Scala

|Amount of Scala (40% ai) product |Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.245 ml |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|24.5 ml |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 6. Stock solution preparation for Signum

|Amount of Signum (33.4% ai) |Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|product per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.294 g |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|29.42 g |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 7. Stock solution preparation for Switch

|Amount of Switch (62.5% ai) |Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|product per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.16 g |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|16.0 g |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 8. Stock solution preparation for Sulphur

|Amount of Sulphur (80% ai) product|Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.125 ml |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|12.5 ml |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 9. Stock solution preparation for Teldor

|Amount of Teldor (50% ai) product |Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.2 g |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|20 g |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 10. Stock solution preparation for Thianosan

|Amount of Thianosan (80% ai) |Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|product per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.125 g |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|12.5 g |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 11. Stock solution preparation for HDC F13

|Amount of HDC F 13 (50% ai) |Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|product per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.2 ml |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|20 ml |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Table 12. Stock solution preparation for ascorbic acid, potassium bicarbonate, potassium sorbate, sodium bicarbonate and sodium benzoate (100% ai)

|Amount of Chemical (100% ai)+ |Concentration of fungicide ai in |Volume of stock solution (ml) to |Final concentration of fungicide |

|product per 100 ml water |stock solution |be added to 250 ml PDA |(ai) in agar (ppm) |

|0.1g |1,000 (A) |0.25 |1 |

| | |2.5 |10 |

| | | | |

|10 g |100,000 (B) |0.25 |100 |

| | |2.5 |1,000 |

| | |12.5 |5,000 |

Inhibition of mycelial growth

PDA plates were amended with six concentrations of the test chemicals (0, 1, 10, 100, 1000, 5000 ppm). Plates were inoculated with a 5 mm diameter plug from one or two-day old cultures of selected isolates of Mucor and Rhizopus. Colony diameters were measured after 1-2 days incubation at 20oC. Two replicate plates were included for each isolate/chemical concentration.

Objective 3 – Laboratory evaluation of efficacy of fungicides and alternative products - in vivo testing

Several fungicides, alternative chemicals and biocontrol agents (BCAs) were evaluated in the laboratory for their effects on infection, sporulation and germination of isolates of Mucor and Rhizopus using inoculated fruits.

Inoculum preparation

For evaluation of effects of treatment on infection and growth of Mucor and Rhizopus in vivo isolates from different origins were used (Table 1). Selected isolates were grown on PDA to produce sporing cultures. Mucor or Rhizopus cultures were combined to produce spore suspension of Mucor spp. or Rhizopus spp. For these tests Mucor mucedo cultures were excluded. A spore suspension was made from two plates of each isolate of Mucor spp. and three plates of each isolate of Rhizopus sp. Each plate was washed in distilled water and a sterile glass rod used to dislodge spores. Spore concentration was measured on a haemocytometer. Spore concentrations of approximately 3 x 102 spores per ml were used for Rhizopus spp. and 3 x 103 spores per ml for Mucor spp. The spore suspensions were made up to 200ml of each. Each tomato was inoculated by spraying the spore suspension for 1-2 sec.

Choice of fruit for in vivo tests

Preliminary tests were conducted to identify the best fruit on which to conduct the in vivo tests. Strawberry fruits, grapes and tomatoes were inoculated with spore suspensions of Mucor spp, incubated in sterile dishes at ambient temperature and assessed for time taken to rot and the ease of measuring lesion development. Tomatoes were subsequently selected for the tests as rotting was slower compared to grapes and strawberries and the fruit remained intact. It was also possible to measure lesions.

Tomato tests

In preparation for the tests tomatoes were surface sterilized in 5% bleach for 30 sec, rinsed twice in sterile distilled water and dried. Each tomato was wounded using a sterile needle to a depth of 1cm. Unwounded tomatoes were left as a control. One tomato was used per treatment and 5 replicate tomatoes in total. Tomatoes were placed in individual lidded plastic pots.

Preparation of test chemicals or BCAs for spraying

The test chemical or BCA (Table 2) was made up in 200ml of water in a hand sprayer. Each tomato was sprayed for 5 sec (to run-off). The test chemical or BCA was applied to tomatoes before or after inoculation with the spore suspension of Mucor spp. or Rhizopus spp. to test the products for both their protectant and/or eradicant effects.

Assessments

The tomatoes were checked daily for lesion development. After two days the lesion diameter was measured in two directions and the presence/absence of sporulation recorded.

Objective 4 – Identification of the importance of fruit flies in the spread and incidence of the soft rot diseases

Cultures of Mucor spp. (isolates SC195-106 and IMI190877) were subbed on to PDA and incubated at ambient temperature in the laboratory. Four punnets of strawberries cv Elsanta were obtained from J Sainsburys Ltd. Ten strawberry fruits were placed in each of four previously sterilised sandwich boxes. A spore suspension was prepared from the two plates of each isolate of Mucor sp. by washing each plate in distilled water and using a sterile glass rod to dislodge spores. The spore concentration was measured using a haemocytometer and the spore suspension made up to 200 ml. The strawberries in the sandwich boxes were inoculated by making two wounds in each strawberry using a sterile needle and then spraying each strawberry for 1 sec with the spore suspension. The fruit was left to incubate at ambient temperature for one day.

Four tubes of wild-type fruit flies (Drosophila melanogaster), containing adults, larvae and pupae, were obtained from Blades Biological Ltd. Five adult flies were removed from each tube and plated on to PDA, one fly per plate to check whether the flies were already contaminated with Mucor. One of each of the tubes with the remaining flies was placed in a sandwich box with the inoculated strawberries which were now sporulating with Mucor. The flies were left to infest the strawberries for two days.

After two days four further punnets of strawberries of cv. Elsanta were purchased from J Sainsbury Ltd. Ten fruits were placed in each of four sterile sandwich boxes. A pouter was used to transfer adult flies from the Mucor-infested boxes to the new fruit in the sandwich boxes. Each of the infested boxes was in turn opened inside an insect proof cage and the flies collected from the roof of the cage. By using this technique the risk of transferring actual spores of Mucor directly was minimized. The cage was emptied of flies before opening each new sandwich box. Five flies from each box were also plated onto PDA, one fly per plate. The PDA plates and new strawberries were monitored for signs of Mucor infection.

Results and discussion

Objective 1 – Incidence and importance of Mucor and Rhizopus

Sixteen lots of fruit samples (13 strawberry samples, three raspberry samples) were received from growers and consultants. These were mainly from Kent (nine samples) but also from Staffordshire, Yorkshire and Cambridgeshire. None were received from Scotland.

Isolations were made from 164 berries (Table 13), yielding 115 fungal isolates with characteristics typical of members of the Mucorales order. Of these, 80 were positively identified as Mucor spp. and 35 as Rhizopus spp.

Thirteen of the Mucor spp. isolates and 15 of the Rhizopus isolates were recovered from raspberry. Similarly, 67 of the strawberry leak disease associated fungi were Mucor spp. and 20 were Rhizopus. That is, 77% of the strawberries with soft rots had Mucor spp. as the causal agent, which are similar results to 2009 (74%). In contrast to 2009 (88%), 46% of the soft-rotted raspberries were infected by Mucor spp. The raspberry samples came from two farms in Kent of cvs. Maravilla and Octavia, and so the sample size was rather small.

Objective 2 – Management practices and disease risk

As in 2009 all of the samples except for one (open field Elsanta) were obtained from tunneled crops whether raspberry or strawberry. Both Mucor spp. and Rhizopus were present in the samples from the open field. From these data it is not possible to draw any conclusions on the effect of tunnels on the incidence of Mucor and Rhizopus.

Very little information on management practices was collected from the growers that provided the rot samples so it was not possible to investigate the relation between practice and rot incidence. Management of fruit and rots at harvest did vary on the farms visited. Most removed all rots and non-marketable fruit from the plantation at harvest which would tend to reduce the soft rot inoculum and hence the risk of infection by Mucor and Rhizopus. Work on cultural measures to control Mucor and Rhizopus will be conducted as part of the Sceptre project.

.

Table 13. Details of origin of fruit samples, numbers and identification of isolates in 2009

|Source |Grower |Date collected |Crop |Cultivar |Cultivation method |

|Location | | | | | |

|Mucor piriformis |0 |24.5 |24.5 |24.5 |24.5 |

|Isolate | | | | | |

|IMI190877 | | | | | |

| |1 |14 |9.5 |25 |27 |

| |10 |5.5 |10.5 |22 |25 |

| |100 |3.5 |1.5 |24.5 |19 |

| |1,000 |0 |0 |8.5 |1.5 |

| |5,000 |0 |0 |0 |0.5 |

| |

|Rhizopus |0 |75 |75 |75 |75 |

|Stolonifera | | | | | |

|Isolate IMI340068 | | | | | |

| |1 |39.5 |1.5 |73.5 |72 |

| |10 |15 |0 |72.5 |71 |

| |100 |18.5 |0 |56 |45 |

| |1,000 |1 |0 |25.5 |7.5 |

| |5,000 |0 |0 |3 |0 |

| |

|Mucor mucedo |0 |17 |17 |17 |17 |

|Isolate | | | | | |

|IMI133298 | | | | | |

| |1 |4 |10.5 |19.5 |19.5 |

| |10 |0 |3 |20 |20 |

| |100 |0 |0 |13.5 |17.3 |

| |1,000 |0 |0 |6.5 |7 |

| |5,000 |0 |0 |1 |0 |

| |

|Mucor spp |0 |57.3 |57.3 |57.3 |57.3 |

|SC195-106 | | | | | |

| |1 |36 |1 |50.8 |59.5 |

| |10 |18.5 |0 |48 |62 |

| |100 |8.5 |0 |46.8 |46 |

| |1,000 |1 |0 |3.5 |0 |

| |5,000 |0.5 |0 |0.5 |0 |

| |

|Rhizopus spp-Fav2 |0 |77 |77 |77 |77 |

| |1 |37.5 |1.5 |69 |78 |

| |10 |11.3 |0 |73.3 |76.8 |

| |100 |5.5 |0 |77 |0 |

| |1,000 |2.8 |0 |47 |0 |

| |5,000 |0 |0 |5 |0 |

| |

|Rhizopus spp – K1 |0 |42.3 |42.3 |42.3 |42.3 |

| |1 |29.5 |1.5 |56 |55.8 |

| |10 |6.5 |0 |44.5 |47.5 |

| |100 |0 |0 |49.5 |30.3 |

| |1,000 |0 |0 |28 |0 |

| |5,000 |0 |0 |1 |0 |

Table 15. Mean colony diameter (mm) of various isolates of Mucor or Rhizopus on PDA amended with various concentrations (ppm) of Signum (pyraclostrobin + boscalid), Switch (cyprodonil + fludioxonil), potassium bicarbonate and potassium sorbate tested in Experiment 2 22 October 2010

|Isolate |Concentration |Amistar |Sulphur |Ascorbic acid |Sodium bicarbonate |

| |ppm | | | | |

|Mucor piriformis |0 |75.8 |75.8 |75.8 |75.8 |

|Isolate | | | | | |

|IMI190877 | | | | | |

| |1 |60.0 |75.5 |72.0 |73.3 |

| |10 |53.0 |72.3 |75.5 |74.5 |

| |100 |41.8 |73.3 |72.3 |67.3 |

| |1,000 |38.0 |57.8 |73.0 |31.8 |

| |5,000 |33.0 |15.3 |76.0 |1.0 |

| |

|Rhizopus |0 |80.0 |80.0 |80.0 |80.0 |

|Stolonifera | | | | | |

|Isolate IMI340068 | | | | | |

| |1 |63.8 |80.0 |80.0 |80.0 |

| |10 |76.0 |80.0 |80.0 |80.0 |

| |100 |49.3 |80.0 |80.0 |80.0 |

| |1,000 |41.5 |62.5 |80.0 |33.8 |

| |5,000 |34.5 |34.3 |80.0 |3.0 |

| |

|Mucor mucedo |0 |37.8 |37.8 |37.8 |37.8 |

|Isolate | | | | | |

|IMI133298 | | | | | |

| |1 |8.0 |37.0 |38.0 |37.3 |

| |10 |8.0 |37.3 |36.5 |39.3 |

| |100 |7.3 |35.5 |35.8 |37.8 |

| |1,000 |1.0 |30.0 |36.5 |25.5 |

| |5,000 |1.0 |7.0 |40.8 |1.0 |

| |

|Mucor spp |0 |77.5 |77.5 |77.5 |77.5 |

|SC195-106 | | | | | |

| |1 |34.8 |76.0 |73.0 |79.5 |

| |10 |49.5 |75.5 |78.3 |78.8 |

| |100 |25.5 |76.0 |78.8 |77.8 |

| |1,000 |21.0 |52.0 |75.8 |79.5 |

| |5,000 |15.3 |16.3 |79.3 |1.0 |

| |

|Rhizopus spp-Fav2 |0 |80.0 |80.0 |80.0 |80.0 |

| |1 |76.5 |80.0 |80.0 |80.0 |

| |10 |77.3 |80.0 |80.0 |80.0 |

| |100 |50.0 |80.0 |77.5 |80.0 |

| |1,000 |54.8 |80.0 |80.0 |66.5 |

| |5,000 |36.8 |41.0 |80.0 |67.5 |

| |

|Rhizopus spp – K1 |0 |78.0 |78.0 |78.0 |78.0 |

| |1 |68.0 |79.0 |79.8 |69.3 |

| |10 |69.5 |78.5 |80.0 |80.0 |

| |100 |63.8 |80.0 |78.8 |80.0 |

| |1,000 |68.0 |79.8 |78.0 |72.5 |

| |5,000 |40.0 |36.3 |79.5 |64.8 |

Table 16. Mean colony diameter (mm) of various isolates of Mucor or Rhizopus on PDA amended with various concentrations (ppm) of Frupica (mepanipyram), Scala (pyrimethanil), Teldor (fenhexamid), Thianosan (thiram), HDC F 13 (experimental fungicide) and sodium benzoate tested in Experiment 3 on 3 December 2010

|Isolate |

|Rhizopus |

|Stolonifera |

|Isolate IMI340068 |

|Mucor mucedo |

|Isolate |

|IMI133298 |

|Mucor spp |

|SC195-106 |

|Rhizopus spp-Fav2 |

|Rhizopus spp – K1 |0 |70.3 |70.3 |

| | |

| |Protectant |Eradicant |Overall mean |

|Water |6.5 |6.0 |6.3 |

|Signum |8.4 |7.6 |8.0 |

|Switch |11.7 |7.3 |9.5 |

|Potassium bicarbonate |9.5 |9.9 |9.7 |

|Potassium sorbate |7.4 |9.3 |8.3 |

| | | | |

|F Probability |0.912 |0.839 |

|SED (20 df) |3.30 |4.67 |

|LSD (p= 0.05) |6.88 |9.74 |

Table 19. Mean diameter (mm) of rot growth on tomatoes treated with various chemicals and biocontrol agents before (protectant) or after (eradicant) inoculation with Rhizopus Test 1

|Treatment |Mean rot diameter mm |

| |Protectant |Eradicant |Overall mean |

|Water |12.8 |8.2 |10.5 |

|Signum |3.9 |1.1 |2.5 |

|Switch |3.2 |4.3 |3.7 |

|Potassium bicarbonate |10.4 |7.7 |9.1 |

|Potassium sorbate |10.0 |12.5 |11.2 |

| | | | |

|F Probability |0.771 |0.035 |

|SED (20 df) |4.49 |3.18 |

|LSD (p= 0.05) |9.37 |6.62 |

Table 20. Mean diameter (mm) of rot growth on tomatoes treated with various chemicals and biocontrol agents before (protectant) or after (eradicant) inoculation with Mucor Test 2

|Treatment |Mean rot diameter mm |

| |Protectant |Eradicant |Overall mean |

|Water |5.0 |4.9 |4.9 |

|HDC F 13 |5.0 |2.5 |3.7 |

|Switch |4.1 |5.5 |4.8 |

|Serenade |7.8 |3.7 |5.8 |

|Chitosan |6.4 |5.3 |5.9 |

| | | | |

|F Probability |0.122 |0.303 |

|SED (40 df) |1.541 |1.090 |

|LSD (p= 0.05) |3.115 |2.203 |

Table 21. Mean diameter (mm) of rot growth on tomatoes treated with various chemicals and biocontrol agents before (protectant) or after (eradicant) inoculation with Rhizopus Test 2

|Treatment |Mean rot diameter mm |

| |Protectant |Eradicant |Overall mean |

|Water |15.0 |10.2 |12.6 |

|HDC F 13 |7.6 |2.2 |4.9 |

|Switch |5.2 |0 |2.6 |

|Serenade |15.0 |8.8 |11.9 |

|Chitosan |13.5 |13.2 |13.3 |

| | | | |

|F Probability |0.493 | ................
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