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Case Study

A potential bio-control agent for Sooty Mould in Kiwifruit

Karin Watson.

BioCulture Laboratories.


  • Triple X- A potential bio-control for SootyMould in Kiwifruit
  • The following is an overview of the work carried out by BioStart and BioCulture Laboratories looking at various actives and their ability to control sooty mould of Kiwifruit.
  • Sooty mould growing on honey dew excreted from sucking insects (cicadas & passionvine hopper) is a significant problem in the kiwifruit industry in a dry growing season. Sooty mould fungi grow on the honey dew excretions on the surface of fruit and cause export fruit loss by lowering the visual appearance of the fruit.
  • All the actives tested produce antimicrobial compounds that have shown varying degrees of activity against sooty mould. The actives are all GRAS and have a nil withholding period.


1. In vivo inhibition study

  • This trial was carried out in April 2010. Fruit with clear symptoms of sooty mould infection were dipped in diluted TripleX and water. After 2 minutes in the solutions the fruit were gently rubbed and assessed.

Water control

Before (below):


After (below):




Before (below):



After (below):



  • The sootymould could be removed easily fromthe fruit dipped in TripleX, while the fungal structures were still difficult to rub off from the fruit just dipped in water.


While this test showed TripleX has good activity against sooty mould, it isn’t practical for the packhouses to dip and rub fruit. Therefore, further research was focused on preventing the sooty mould from establishing on the fruit in first place.

To establish intrinsic preventative activity of mainly TripleX and also other Bio-Start products against Sooty Mould, a number of in vitro studies were conducted:

2. In vitro inhibition study 424.

This trial was carried out in July 2010 by BioCult Cambridge. Agar dishes were first inoculated with Sooty Mould spores, and then with a cell suspension of the active in TripleX (Bacillus amyloliquefaciens BS1b).

The bacterial organism completely inhibited any growth and development of Sooty Mould.

The test showed Bacillus amyloliquefaciens BS1b has a clear and very strong activity against
sooty mould.

3. In vitro inhibition study 469 A

  • This trial was carried out in January 2011 by BioCult Cambridge. The test compared the inhibition zones produced by TripleX, Foliacin,Mycorrcin and Terracin. The products were applied to agar plate centre wells in their neat form, to observe activity along a concentration gradient via diffusion in the test agar. TripleX was also applied at 1:100, as it is known that the active inoculant (Bacillus amyloliquefaciens BS1b) remains dormant in the neat product, and only emerges into its active form when TripleX is diluted.


Activity against Sooty Mould of Kiwifruit:

Mean ZOI day 1 (mm) ( from 3 replicates each)Mean ZOI day 7 (mm) ( from 3 replicates each)
TripleX 1:10005
Untreated Control00
  • All tested products showed good initial knockdown activity in the first assessment, which however, became mostly overgrown by the fungal mycelium after 7 days. This is a normal occurrence in this test method, as the test products becomemore and more diluted in the agar over time.
  • Foliacin showed the best knock down activity from neat.
  • As the Bacillus amyloliquefaciens BS1b emerged out of the TripleX at 1:100, a lasting zone of inhibition developed, which grew through the existing mycelium, clearing a 5 mm radius around the centre wells by day 7. (Note: Due to the rapid diffusion gradient, the 1:100 dilution in this test system is not expected to produce a notable ZOI at day 1, as seen in this test, too. The ZOI appearing later is solely due to the BS-1b.)


All BioStart product formulations are providing a good level of direct inhibition activity against SootyMould. Furthermore, the beneficial bacterium Bacillus amyloliquefaciens BS1b in TripleX is able to provide a lasting inhibitory effect against the Sooty Mould.

Note: Parallel to Sooty Mould of Kiwifruit in this test 469 A, similar studies were also carried out against two other Kiwifruit pathogens – Botrytis cinerea (Test 469 C) and Sclerotinia (Test 469 H). Excellent results against both pathogens were shown by the product formulations and by the TripleX active Bacillus amyloliquefaciens BS1b.

4. In vitro spore germination inhibition test 470

  • In this trial carried out in January 2011 by BioCult Cambridge TripleX was tested for its ability to inhibit spore germination and following mycelial growth of sooty mould. The sooty mould spores were suspended in a 1:200 dilution of TripleX for 12 hours. Subsequently, droplets of the spore/test solutions were plated out onto fresh agar plates.


Activity against Sooty Mould of Kiwifruit:

Microscopy % spore germination 21.01.11Visual % Mycelium growth 22.01.11Visual % Mycelium growth 24.01.11
TripleX 1:200850 (Slight BS-1b growth)25 (Good BS-1b growth)
Untreated Control100100100


TripleX at 1:200 provided good initial knockdown activity on spore germination. This was then first becoming more overgrown by the aggressive surviving sootymould mycelium, but once the Bacillus amyloliquefaciens BS1b started to kick in, the sooty mould development has been halted by 75%. This activity remained unchanged for much longer.


5. In vitro Zone of Inhibition test 471 – Activity at lowand high disease pressure

  • This trial was carried out in January 2011 by BioCult Cambridge. TripleX was tested for its activity against Sooty Mould of Kiwifruit under low and high disease pressure.


Strong SootyMould Inoculum- Activity against SootyMould of Kiwifruit:

Test A: Strong Sooty Mould InoculumMean ZOI day 2 (mm) ( from6 replicates each)Mean ZOI day 3 (mm) ( from6 replicates each)Mean ZOI day 5 (mm) ( from6 replicates each)
TripleX neat853
TripleX 1:200534
Untreated Control000

Day 3:
Neat: Large ZOI
1:200: Smaller ZOI


Day 5:
Neat: Reduced ZOI
1:200: Increased ZOI


Weak SootyMould Inoculum- Activity against SootyMould of Kiwifruit:

Test B: Weak Sooty Mould InoculumMean ZOI day 2 (mm) ( from6 replicates each)Mean ZOI day 3 (mm) ( from6 replicates each)Mean ZOI day 5 (mm) ( from6 replicates each)
TripleX neat973
TripleX 1:200557
Untreated Control000


Day 3:
Neat: Large ZOI
1:200: Smaller ZOI


Day 5:
Neat: Reduced ZOI
1:200: Increased ZOI


Good and lasting activity of BS-1b in the 1:200 dilution of Triplexwas seen atweak and strong SootyMould disease pressure.


TimingApplication Rate
Prior to periods of high infestation of Passion Vine Hoppers&Cicadas fromJanuary onwards.3lt/ha TripleX.Water rate 1,500lt/ha. Add spreader sticker
7-10 days after infestation apply 2nd application.3lt/ha TripleX.Water rate 1,500lt/ha. Add spreader sticker.
Continued applications throughout the season, at 18 to 21 day intervals.3lt/ha TripleX.Water rate 1,500lt/ha. Add spreader sticker.

Recommended Programfor TripleX

Note : Recommended to add non-ionic sticker/spreader to aid coverage.(Duwett,Hywett,Nufilm,Filmstar, Spreadwet 1000) at label rates with all applications. Add TripleX to the tank first ,mix well, then add adjuvant.

Withholding period: One day

BioStart would like to thank DMS Progrowers Te Puna for their co-operation in conducting this trial.

This report is for information purposes only and does not constitute label claims for the use of the TripleX product to control Sooty Mould.

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