Trichoderma atroviride SC1 can prevent infections of

Phaeoacremonium and Phaeomoniella in nurseries

Ilaria Pertot1, Luca Pasini1, Daniele Prodorutti1, Andrea Nesler1,2

Fondazione Edmund Mach1, BiPA2

Brief overview of the epidemiology of the tracheomycosis

• Infections: pathogens’ conidia released during the growing season colonize pruning wounds

• Most of the infections take place in the vineyards, however in many cases plants are already infected at the time of planting

• Risk of infections in the propagation process in the nurseries

• Infections from mother plants (mainly rootstock, relevance of the scion is still debated)

Mother plants

Nursery

Vineyard

High risk of infection by Phaeomoniella chlamydospora and Phaeoacremonium minimum (syn. P. aleophilum) during plant propagation in nurseries (conidia found in water, tanks, tools, substrate, soil, etc.)

MOTHER PLANTSRootstocks - Scions

www.viten.net

ROOTINGCALLUSINGGRAFTINGHYDRATIONRootstocks - Scions

FIELD

Infected plants Conidia in the water Conidia in the water

Conidia in the environment

Conidia in the substrate

Conidia in the water

Options to control infections in the nurseries

• The most promising treatment in the propagation process: hot water over 50 °C (Gramaje et al. 2009)

• On some varieties it negatively affect the quality of plants, in some contexts is not practicable

• Chemicals: not available (not registered, not effective)

• Trichoderma spp.: effective in protecting pruning wounds in the vineyard against the pathogens (Di Marco et al., 2000; Fourie et al., 2001; Fourie and Halleen, 2006)

• Trichoderma spp.: effective in protecting plant in nurseries (Fourie et al., 2001; Hunt et al., 2001; Di Marco et al., 2002; 2004; Fourie and Halleen, 2004; 2006; Veronesi et al., 2006; Di Marco and Osti 2007)

Trichoderma species/strain: how relevant is?

Very important for the treatments of the pruning wounds

Also important in the nursery treatment

• Fast germination and growth rate (efficient and fast colonization)

• High competition at the colonization site (successful and stable colonization)

• Temperature range (persistent colonization, especially at high T°C)

• Endophytic habit (not yet confirmed)

The strain must persist until the end of the season (full-year protection)

Trichoderma atroviride SC1

Result of a screening process (started in 2004) based on:

• Good technological properties (fast growth and conidiation, high conidia harvest in fermentation)

• Germination and colonization a low T°C (applied in winter)

• Good, fast and persistent colonization of wood (isolated from hazelnut dead wood)

• High growth rate and competition (niche exclusion for pathogens and other MOs)

• Production of lytic enzymes and toxins (good biocontrol activity)

Protocol:

Application of T. atroviride SC1: 4 times in nursery

Materialcollection

Hydratation prestorage

Cold storage

Hydratation pre-grafting

Graftedcuttings

Stratification in sawdust

Waxing Forcing

Rooting

Rooting

Planting

Grafting

Jan

uar

y

Mar

ch

May

Ap

ril

Feb

ruar

y

2nd

Application3rd

Application4th

Application1st

Application

• Vintec (Belchim; 2 x 1010

conidia g-1 formulated

product) at 2 g l-1

• Artificial infection (after

grafting)

• Trials repeated, data pooled

b

b

a a

b b

0

10

20

30

40

50

60

70

Pal Pch Pal Pch Pal Pch

Standard T. atroviride SC1 Untreated

Infe

cte

d p

lants

(%

)

Efficacy against Pal and Pch

Standard treatment (8-hydroxyquinoline sulphate, Cryptonol), many growers still use it

Treatment in T1, T2, T3, T4 with T. atroviride SC1 (water or sawdust)

Assessment of Pal and Pch(microbiological, with a subsample with PCR for validation) at the grafting union (no infections at the root side)

Kruskal–Wallis test: v2 = 29.47; df = 5; p<0.0001; multiple bilateral comparison at p<0.01

Although not significant, an increasing trend of infections was observed with hydroxyquinoline sulphate: tissue damages, reduction of natural biocontrol can be hypothesized to explain this slight increase

c

a a

c

b

0

10

20

30

40

50

60

70

80

90

Untreated T1 T2 T3 T4

Infe

ce

dp

lan

ts (

%)

c

a

a

ab

bc

0

10

20

30

40

50

60

70

80

90

Untreated T1 T2 T3 T4

What is the most effective treatment?

Hydration before cold storage (T1) and hydration before grafting (T2) are the most effective (treatment of sawdust and at rooting, T3, T4 are less effective) At each time point new wounds are created the full program may contribute to a safer protection

Pal Pch

(ANOVA Pal: F = 65.30; df = 4, 48; p<0.00001; ANOVA Pch: F = 16.33; df = 4, 35; p><0.00001; Tukey’s test at a = 0.05)

T. atroviride SC1 colonization

Better colonization at T1 and T2 Relation between colonization and efficacy (regression significant, p<0.01) (ANOVA: F = 71.30; df = 9, 30; p<0.00001; Tukey’s test at a = 0.05)

a a

cc

c c

ab

bab

b

0

20

40

60

80

100

120

UN

T-P

al

UN

T-P

ch

T1

-Pal

T1

-Pch

T2

-Pal

T3

-Pch

T3

-Pal

T3

-Pch

T4

-Pal

T4

-Pch

Tri

ch

od

erm

a s

pp

. c

olo

niz

ati

uo

n (

%)

Effect of water temperature

• At 2 g/l

• Viability >80%

• 10° and 15°C

• Always optimal colonization of grafting point (at planting time)

0

20

40

60

80

100

120

Pal Pch Pal Pch 10°C 15°C

10°C 15°C Untreated

T.

atr

ivir

ide

SC

1 o

lon

izati

on

(%)

Growth promotion?

T. atroviride SC1 was not selected as growth promoter (main action as biocontrol agent), however the treatment in T2 improves the quality of the plants

ab abb

a

e

d

c

b bab

0

20

40

60

80

100

120

140

Pch Pal Pch Pal Pch Pal Pch Pal Pch Pal

Untreated T1 T2 T3 T4

Sh

ho

t le

ng

ht

(cm

)

ab abbc

a

e

d

c

bc bc ab

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

Pch Pal Pch Pal Pch Pal Pch Pal Pch Pal

Untreated T1 T2 T3 T4

Dia

me

ter

of

the m

idd

el in

tern

od

e

(cm

)

(ANOVA shoot length: F = 152.14; df 9, 30; p\0.00001; ANOVA diameter: F = 70.29; df = 9, 30; p\0.00001; Tukey’s test at a = 0.05)

Practical questions from growers

Recommendation: to prepare the water suspension just before use

• Can I prepare the suspension in advance? What is the risk of keeping the suspension for some time before treatment?

Recommendation: not to reuse the suspension

• Can I reuse the suspension? Is the conidia concentration/viability reduced with the reuse of the suspension?

Viability of conidia in the suspension

Preparation of the suspension in advance is not recommended: however it may happen in the practice

Good survival until 72 h if the suspension is prepared in distilled water and kept from 5 to 15°C

0

20

40

60

80

100

120

24h 48h 72h 24h 48h 72h 24h 48h 72h

5°C 5°C 5°C 10°C 10°C 10°C 15°C 15°C 15°CC

on

idia

via

bil

ity (

% t

o T

0)

Neither temperature (F = 0.27; df = 2, 27; p = 0.76) nor time (F = 0.11; df = 2, 27; p = 0.89) nor temperature 9 time (F = 0.35; df = 4, 27; p = 0.84) are significant at the two-way ANOVA.

Recycling the suspension

Recycling of the suspension is not recommended: however in some nurseries growers soak plant material in the same suspension (suspension cannot be discarded each time)

Good survival until 72 h if the suspension is prepared in distilled water and kept from 5 to 15°C

0

20

40

60

80

100

120

140

14h 24h 38h 48h 14h 24h 38h 48h

10°C 10°C 10°C 10°C 15°C 15°C 15°C 15°CC

on

idia

via

bil

ity (

% t

o T

0)

(ANOVA F = 1.57; df = 9, 30; p = 0.17).

Nursery: efficacy trials

• GEP trials (dossier for registration)

• 14 trials DE, FR, IT, ES, HU

• 36 set of data GEP and DEMO (FR)

• 2012-14

Courtesy of BiPA/Belchim

Colonization

Efficacy trials in FR, meta-analysis (T. atroviride SC1 treated plants, n=18)

Very high colonization by SC1

No presence of Pal and Pch(efficacy = 100%)

Some presence of Botryosphaeriaceae (no distinction between pathogen and saprophytic)

0

10

20

30

40

50

60

70

80

90

100

SC1 PAL PCH BOC

olo

nia

zati

on

(%

)

Courtesy of BiPA/Belchim

Colonization and efficacy

• Meta-analysis of colonization by SC1 and PAL or PCH in efficacy trials (treated and untreated, n=36), loss of control below 20% of SC1 colonization at the end of the season

0

5

10

15

20

25

0 20 40 60 80 100 120

Pal p

rese

nce

(%

)

Trichiderma spp. colonization (%)

0

5

10

15

20

25

0 20 40 60 80 100 120

Pch

pre

sen

ce (

%)

Trichoderma spp. colonization (%)

Courtesy of BiPA/Belchim

Conclusions

• Treatments with T. atroviride SC1 in the propagation process in nurseries con prevent colonization of Pal and Pch (and most probably Bot)

• Colonization efficiency by Trichoderma spp. is crucial to achieve efficacy

• T. atroviride SC1: robust strain (flexibility in the treatment conditions), fast colonizer, good competitor at the site of infections

• Can only prevent infections in the plant propagation (healthy mother plants are crucial)

Thank you for your attention!