Detection of Microdochium and Fusarium on wheat seeds – suitability of

traditional and molecular methods

Birgitte Henriksen (Kimen Seed Testing Laboratory) and Guro Brodal (Bioforsk)

ISTA Seminar: Molecular tools for seed quality & Seed health Montevideo June 2015

- Background

- Fusarium and Microdochium disease complex/FHB (species, symptoms, damage)

- Fusarium mycotoxins

- Severe epidemics (re-emergence) of FHB / mycotoxin contaminations reported from all continents recent years – changes in species composition (ex from Norway), poor seed quality

- A preliminary study to differentiate between Fusarium and Microdochium in wheat seed (two media, two temperatures, two laboratories), a first step of a validation procedure

- Comparison of seed incubation on PDA and molecular tests (qPCR) for different Fusarium and Microdochium species

Outline of presentation

Norway

Grassland 67%

Cereals (for

feed)25 %

Cereals for food 6 %

Main Fusarium and Microdochium spp. on wheat

Fusarium avenaceum, F. culmorum, F. equiseti,

F. graminearum, F. langsethiae, F. poae,

F. sporotrichioides, F. tricinctum,

Microdochium nivale og M. majus

Grain from blighted fields may have reduced value as a seed crop

– reduced germination/poor seed quality

– blighted seedlings/reduced emergence

Mycotoxins (DON, T2/HT-2, ZEA ….)

– a serious challenge for the cereal grain industry

Fusarium head blight caused by Fusarium spp, one of the worlds most important diseases as it may produce mycotoxins that pose a treath to human and animal health. Growing oat in Norway: challenges caused by high levels of mycotoxins especially in oats

Orange sporodochia may be seen on the

surface (heavy attacs)

Fusarium infection may lead to: • Yield loss (>50% loss reported from other countries due to

– Forced maturity and poor grain filling – Reduced germination – Poor establishment of plants, poor tillering

• Reduced grain quality – Protein degradation / gluten (reduced nutrient value and

baking quality) – Gushing of beer – Mycotoxin content

Leaf spots on winter wheat caused by Microdochium nivale

Pink snow mould (M. nivale)

Important Fusarium toxins (incomplete table)

DON NIV ZEA T2/HT2 MON ENNs BEA FBs

F. graminearum X X X

F. culmorum X X X

F. avenaceum X X X

F. tricinctum X X X

F. sporotrichioides X X X X

F. poae X X X X X

F. langsethiae X X

F. verticillioides X

Type A trichothecenes: T-2, HT-2 Type B trichothecenes: DON (vomitoxin), NIV Zearalenon (ZEA=ZON=ZEN) Moniliformin (MON) Enniatins (ENNs) Beauvericin (BEA) Fumonisins (FBs)

During the years 1970-1989, varying numbers of representative/selected cereals samples were tested for Fusarium/Microdochium infection frequencies. Since 1990 all cereal seed lots in Norway have been tested

A total of 79 423 samples: - 39 061 barley - 24 704 oats - 15 658 spring wheat

including both certified and farm-saved seed from all cereal growing areas

100 or 200 seeds / sample

Survey of Fusarium /Microdochium infection levels in Norwegian cereal seeds during 45 years

Orange sporodochia on barley (freezing

blotter method) Brown discoloration of oat roots

(blotter/Doyer method)

Colonies of Fusarium and Microdochium

(wheat on PDA)

Detection methods used

Fusarium/Microdochium infections were recorded as % infected seeds in each sample and average infection % were calculated each year for each cereal species

Fusarium /Microdochium infection in Norwegian cereal seed lots and July rainfall (1970-2014)

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Precipitation July Barley Oats Spring wheat

Data from State Seed Testing Station, Norwegian Agricultural Inspection Service and KIMEN (a total of 79 423 samples)

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Increased prevalence of F. graminearum in wheat

(Data fra Kosiak et al 2003, Elen O., KIMEN)

144 40 704 621 519 517 479 733

Fusarium spp. (% infecteed seeds) and mycotoxins (ppb) in wheat samples from experimental fields, Norway 2001-2004

(average of approx 110 samples each year)

Species 2001 2002 2003 2004

F. avenaceum 18.7 9.3 4.6 6.8

F. culmorum 1.3 0.6 0.9 0.4

F. tricinctum 2.3 0.4 0.2 0.4

F. equiseti 0.06 0 0 0.01

F. poae 0.1 0.5 0.4 0.7

F. graminearum 0.02 0.1 0.3 2.2

F. sporotrichioides 0.01 0.01 0.02 0.03

F. spp 0 0.1 0 0.05

Fusarium in total 22.5 11.1 6.4 10.6

DON 64 37 153 268

MON 116 61 27 49

Germination % and Microdochium /Fusarium in spring wheat seeds 2000-2007

% germination % infected seeds

Year Number of

samples

Untreated and after fungicide test treatment

Michro-dochium

Fusarium

2000 796 86 / 92 13 5

2001 1130 80 / 83 3 23

2002 755 89 / 93 5 12 2003 603 89 / 94 4 9

2004 671 86 / 92 5 13

2005 673 89 / 94 5 15

2006 622 87 / 92 1 17

2007 502 80 / 93 24 16 (incl. 3 % F. gram)

Fusarium – (conclusions) - The infection levels (% infected seeds) more than 100 % higher during the

last decade compared to the previous decades

- A significant increase in precipitation last decade, longer growing season

- The infection levels were positively correlated with precipitation during flowering and maturation

- Increased inoculum potential due to reduced tillage and limited crop rotations

- A change in the Fusarium species composition was recorded, with F. graminearum becoming more abundant.

- Significant changes in population frequencies among Norwegian F. graminearum have been observed (Aamot et al, 2015)

Routine testing of seeds for detecting levels of Fusarium and

Microdochium infections

- suitability of methods

Preliminary method study

- A method to detect different Fusarium species and Microdochium simultaneously on wheat seed is requested

- Results from a study comparing incubation on PDA with use of a selective medium (CZID)

- qPCR is run on some of the same material

Three wheat seed lots with different infection levels of both Fusarium and Microdochium present were tested on two agar media (PDA and CZID).

– Potato dextrose agar (PDA) is used in the current ISTA method for detection of Microdochium spp (7-022).

– Czapek–Dox Iprodione Dichloran agar (CZID) is a Fusarium selective medium. (used by the Nordic Committee on food analysis/NMKL)

– surface disinfection (10 min in 1% NaOCl), – incubation (7 days) at two temperatures (20 and 25 oC) - under

alternating light conditions (12 hours NUV+ white light/12 hours darkness).

- Two different laboratories carried out the test, - Replicated twice (in time) using 2 x 100 seed for each treatment both

times. - The samples were also analyzed using a qPCR method.

Materials included in the study

Colonies of Fusarium and Microdochium

(wheat on PDA)

Detection methods used

Fusarium spp and Microdochium spp were recorded as % infected seeds in each sample

Fusarium selective medium; Czapek Dox Iprodion Dichloran Agar

Microdochium Fusarium

Fusarium spp. (only) detected on PDA (Sept 2013) and by qPCR (May 2014)

Seed lot

% infected seeds (PDA)

Amounts fungal DNA (pg/µg) (qPCR)

Sept 2013

March 2014

F. gram. F. aven. F. culm.

1 7 2 0 2,402 0

2 30 24 69,724 42,241 0

3 25 7 153,166 2,097 0

Conclusions • Temperature and medium type, and interactions between

these, influenced on the level of Microdochium and Fusarium detected.

• PDA at 20 oC was the most suitable method for routine testing of Fusarium and Microdochium (separate recordings) in the same test, when the purpose is to assess the suitability of seed lots for sowing (seed quality) and possible need for seed treatment.

• However, if the purpose would be to distinguish between different Fusarium spp., the CZID medium at 25 oC appears to have advantages.

• It is likely that a PCR test would be more specific and sensitive; however, it will also pick up dead inoculum.

Average amounts of fungal DNA (pg/µg) in wheat seed lots harvested in Norway 2012

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Fusarium and Microdochium after incubation on PDA and in qPCR tests, and germination, of wheat seed harvested in Norway 2012 Seed lot

% infected seeds (PDA) Sept 2012

Amounts fungal DNA (pg/µg) in qPCR Dec 2013

% germin-ation

Sept 2012 Fus Microd majus nivale F gr F av Fc Fl Fp

1 6 56 1684 26 247 23 0 0 0 88/95

2 1 67 4450 82 174 39 0 0 0 86/93

3 1 57 2407 43 0 0 0 0 0 75/87

4 16 68 2190 299 626 100 0 2 0 73/88

5 3 62 4405 63 374 15 0 0 18 80/92

6 7 38 1555 77 1824 100 0 0 0 83/97

7 3 49 2097 38 95 3 0 0 0 69/92

8 9 50 5241 28 953 118 0 0 0 79/94

9 15 39 1735 26 3147 40 0 0 0 77/93

10 6 49 4535 137 1745 47 0 0 0 75/90

11 5 34 1202 29 51 5 0 0 0 86/97

12 0 10 414 17 179 4 0 1 0 96/99

Fusarium spp. (without Microd.) detected on PDA and by qPCR , and DON content, in wheat seed harvested in Norway 2012

Seed lot

% infected seeds pg F. graminearum DNA/µg plant DNA

(Dec 2013)

µg DON/kg (Dec 2013)

Sept 2012 Dec 2013

1 6 3 247 210

2 1 3 174 100

3 1 1 0 100

4 16 3 626 100

5 3 2 374 218

6 7 7 1824 628

7 3 0 95 203

8 9 2 953 480

9 15 11 3147 1109

10 6 5 1745 452

11 5 1 51 211

12 0 0 179 219

Suitability of a method is strongly depending on the goals of the pathogen detecting system

What do we want to achieve from routine testing of seed lots for Fusarium and Microdochium

– Presence and level of the pathogen(s) in a seed lot (Seed quality)

– Viability of the pathogen; ability to affect germination – Detecting more than one pathogen in one analysis – High through-put – Fast and accurate detection – Identification on species level? – (Produce data suitable for further studies?

Statistics/development over years? Comparisions?)

Traditional methods/agar method • May detect and quantify more than one pathogen in one

analysis • Detect viable pathogens! • Quantification • Variability in levels of accuracy and sensitivity (depending on

skills and training, equipment etc.) • Identification of species possible - may be time consuming • Easy to implement as routine analysis • Gives «understandable» results/counts («image» of the

mycoflora in a sample) – thus more control and monitoring possibilities for laboratory personel,

also picking up on new and different looking colonies/fungi.

Real-time PCR (as main relevant molecular method for detection of plant pathogens)

• Detection of a pathogen without prior culturing • Fast turn-around time • High throughput • Increased possibility for multiple pathogen detection • High specificity (specificity: the capability to detect the organism of interest in

the absence of false positives and negatives)

• High sensitivity (sensitivity: relates to the lowest number of pathogens reliably detected per assay or sample)

• BUT Does not discriminate viable from dead cells • Expensive equipment • Need for trained personel • Miss the «sample/fungal monitoring» possibility

Real-time PCR and suitability for routine seed testing analysis:

• «Great advantages of real-time PCR for diagnostic purposes»

• «Still major challenges of technical and economic nature that

need to be addressed to ensure reliable detection systems for routine applications» (Alemu, 2014: Real-Time PCR and Its Application in Plant Disease Diagnostics)

• «The lack of discriminating viable from dead cells is a pitfall common to the nucleic acid based detection systems including microarrays and diagnostic PCR»

(Keer and Birch, 2003)

• Expensive for routine diagnostics

Thank you for your attention!