Inoculum production for field

experiments with artificial

Fusarium inoculation

M. Herrmann

Material:

Oat cv. Jumbo or Pergamon, or other (wheat or triticale may also be suitable); about 15kg (3 kg per Fusarium isolate)

Fusarium isolates: (from the former BBA-

collection – now JKI)Species Substrate Substrate

Remarks Country Year of

Isolation Fusarium sporotrichioides var. minus Wollenweber

Avena sativa L. rotting halm base

Germany, Berlin

1965

Fusarium avenaceum (Corda : Fries) Saccardo

Hordeum vulgare

Seeds from variety: 'Frisia'

Germany 1981

Fusarium culmorum (W. G. Smith) Saccardo

Secale L. ear Germany 1994

Fusarium langsethiae Torp & Nirenberg

Avena grain Austria 1999

Fusarium graminearum Schwabe

Triticum aestivum L.

variety: 'Trimaran' (Triticale)

Germany 2003

Transfer the cultures to SNA-agar for multiplication of the different isolates.

Preparation of seeds for inoculum production

•Soaking a few minutes and boiling of oat seeds

for 15 min with water. •Transferring the boiled seeds with less water into

autoclaving bags and autoclaving it two times.•Cool off the bags to room temperature.•Under sterile conditions: Put some peaces of agar

with the Fusarium strain into the autoclaved seeds

in the bag. Do not close the bag completely, to

permit some gas exchange.

•Let the fungus grow under dark conditions and

room temperature until the whole seedlot is

completely occupied by the fungus.•Put the seeds with mycelium to cleaned basins

for a slowly drying (over a few weeks). Seeds

with mycelium should be covered by cloths, to

slow down speed of drying. •Mixing the samples by hand all two days.

•Dryed samples can be stored in refrigerator for

several years•Counting of spore density after washing the

inoculum substrate in de-ionised water and tween-20•Adjust the spore amount for each Fusarium isolate

to get a inoculum density of 1 Mio spores per ml

Spore counting and calculation in 2008:

Species Number of spores found for each isolate

Fusarium sporotrichioides 2.578.796.550

Fusarium avenaceum 120.111.728

Fusarium culmorum 8.686.408.502

Fusarium langsethiae 6.521.739.000

Fusarium graminearum 24.812.462

In Romania and Germany 60l inoculum (3 dates with each 20l) with about 300000 Sporen/ml

•Analysis of the mycotoxin content of the 2008 inoculum

mixture (eurofins, LC/GC; HPLC-MS/MS):

Vomitoxin (DON), NIV, 3-ADON, 15-ADON, DAS, FX,

<20 μg/ kg

Zeralenone (ZON) 6000 μg/ kg

HT-2 8500 μg/ kg

T2 80000 μg/ kg

Neosolaniol 600 μg/ kg

Are the Fusarium strains multiplied mycotoxin producer?

•Conclusions:

DON- and NIV-producing Fusarium strains need to be involved.

Inoculum production method have to be improved.

Many thanks to the sponsors:

The European Commission’s ( Council Regulation

(EC) no. 870/2004),

Peter Koelln KGaA,

Emco spol. s r. o.,

Gemeinschaft zur Förderung der privaten

deutschen Pflanzenzüchtung e.V. (GFP)

Meeting additionally supported by the Association of the

Friends and Promotors of the Julius Kühn-Institute

(GFF) and Peter Koelln KGaA