Phytotoxins from Botryosphaericeae associated with grapevine

Eliane Abou-Mansour

Plant Biology Department

University of Fribourg,

Switzerland

WG3- Plant-Pathogen interactions – 12-13 May 2015 Athens

manaGTD Sustainable control of grapevine trunk diseases

Introduction

• Fungi are the major causal agents of diseases in crop plants.

• Filamentous fungi are prolific producers of bioactive small molecules, known as secondary metabolites (SMs).

• In plant pathogenic fungi, the SMs often play an important role in plant infection and virulence.

• Phytotoxins : SMs that lead to the damage or killing of plants ; with diverse mode of action.

• Some of these phytotoxins are host specific while others have activities against a broader range of plant hosts.

• Some phytotoxins are not directly involved in damaging the plants but could play important role : • in protection of the fungi against environmental stress (melanin)

• mineral uptake (siderophore)

• interferring host hormone signalling (giberrellins)

• and supressing plant defence (supprescins, brefeldin)

• Pathogenic fungi and their hosts are not isolated in the environment, but interacting with other organisms,

• microbes,

• endophytes

• and fungivores

Introduction

• Therefore, many SMs from plant pathogenic fungi also possess antibacterial, antifungal and cytotoxic activities.

• Although there are increasing numbers of SMs being isolated and characterised from plant pathogenic fungi,

• Our understanding of SMs production and their functional roles in these pathogens their target in the host are still largely incomplete.

• This is especially true for many grapevine trunk pathogens which are currently the focus of the research in our laboratory.

• Here, we review SMs isolated from Botryosphaeriacea associated with grapevine diseases and provide a review of the biological activity and the biosynthetic pathway of the identified compounds.

• Grapevine (worldwide) : trunk, stem • Neofusicoccum parvum, N. australe, N. ribis, N. luteum, B. dothidea, Diplodia seriata, D. mutila, D.

porosum, Lasidipodia theobroma, Fusicoccum viticlavatum and F. vitifusiforme

• Blueberry (Nz): roots and leaves but not fruit • N. luteum, N. parvum, N. australe, and D. seriata

• Prunus species (SA): plum, peach, nectarine and apricot • D. seriata , N. australe, N. vitifusiforme, D. pinea, D. mutila, D. africana sp., Lasiodiplodia plurivora sp.,

and Dothiorella viticola

• Apples, pears, persimmon and kiwi fruit (Jp): stem cankers • B. dothidea, B. obtusa

• Olive (Gr): • B. dothidea

• Almonds (Es): trunk • B. dothidea, D. olivarum, D. seriata, N. australe, N. parvum

• Date Palm (Ir) : decline • B. dothidea, D. mutila

• Araucariaceae (Southern hemisphere): seedling • N. parvum

Botryosphaeriaceae spp. assosiated fruit tree diseases

• Botryosphaeria described from grapevines are known from other hosts in different parts of the world.

• It has not been established whether the difference in pathogenicity is due to the presence of more virulent strains in North America, or whether the conditions there are more conducive

• In England and New Zealand it is a weak secondary pathogen, but not necessarily the primary cause of cankers, leaf spots and fruit rots.

• In North America B. obtusa is reported to cause an important leaf spot, canker and fruit rot of apple

KTFREC, West Virginia University web page

• For many years, Botryosphaeriaceous species have been mostly considered saprophytes or secondary colonisers in grapevine (Phillips 2002)

Foliar symptoms of the mild form Symptoms in the wood

P. Larignon

Botryosphaeria dieback on Sauvignon

Diplodia seriata / Botryosphaeria obtusa

D. seriata / B. obtusa

• Ten strains of D. seriata/ B. obtusa were used for phytotoxins study (P. Larignon)

• a Isolates from the collection of INRA Bordeaux, Fr, except (*) • BDA ; Black Dead Arm, SD: Syrah decline, YGD: Young grapevine decline

Identified compounds

• Venkatasubbaiah et al. 1991 B. obtusa, black rot of apple fruit

R2 R1 R3

Mellein H H H

H OH H (3R,4R)-cis-hydroxymellein

(R)-(-)-5-hydroxymellein H H OH

Tyrosol

• Djoukeng et al. 2009 B. obtusa F-98-1, Syrah decline

(R)-(-)-7-hydroxymellein H H OH

(4,7)-(R)-(-)-dihydroxymellein OH H OH

• Glauser et al. 2009 B. obtusa in confrontation with Eutypa lata

R2 R1 R3

8-methoxymellein H H H

H OH H (3R,4S)-4-hydroxy-8-methoxymellein

(3R,4R)-4-hydroxy- 8-methoxymellein H H OH

(R)-5-hydroxy-8-methoxymellein OH H H

D. seriata culture on grapevine wood

• Canes of grapevine cv. Gamay were dried and grounded into powder.

• 400 g of wood powder wetted with 150 ml of water and placed in a plastic bag equipped with a filter to allow gas exchanges, sealed and sterilized.

• Kept in the dark 25°C during 40 days.

• Extracted individually with MeOH, followed by liquid/liquid extraction with heptane

• Complete degradation of resveratrol

• Melleine was detected in heptane extract by LC-ESI-MS,

• Unable to detect the remaining isocoumarins.

Djoukeng et al-2015

Neofusicoccum australe

• In the south west coast of Western Australia, N. australe induce significant decline in Eucalyptus gomphocephala (Taylor et al.2009)

• In southern Italy N. australe associated with drupe rot of olives (Lazziera et al. 2008)

• In New Zealand isolated from grapevine and broom (Cytisus scoparius) (Aponsah et al. 2009)

• Andolfi et al. 2012:

From a strain of N. australe BOT48 of (haplotype H4) associated with grapevine cordon dieback

Botryoxide showed necrosis on grapevine leaves by leaf puncture assay at 250 g/ml

cyclobotryoxide Tyrosol 3-methyl catechol

Neofusicoccum parvum

• Thirteen strains of Neofusicoccum parvum were analysed for phytotoxins

Isolate name Isolated from plants

aF003. Decline

aBW2BB Decline

aCharentes 11G Nursery plant

aVaucluse 14-PG1 Nursery plant

aBourgogne 2S-16 Nursery plant

aSainte Victoire Decline, BDA

aCharentes 26S Nursery plant bBt2 Esca; Esca-like symptoms bBt19 Decline, black streaks, hard necrosis bBt67 Esca; Esca-like symptoms bB75 BDA bBt80 Decline, black streaks, hard necrosis bBt85 BDA

A Collection of the Institut Français de la Vigne et du Vin, Rodilhan, France, P. Larignon b Single spore collection of the Instituto Superior de Agronomia , Universidade de Lisboa, Portugal, C. Rego

0

10

20

30

40

50

60

% n

ecro

tic

leaf

are

a

control F003 Charentes 11G Bourgogne Ste Victoire Bt2 Bt67

control

Bt85 Bt75l

Figure 2 : Necrosis induced by fungal extracts at 200 mg/ml after 48 hours. Quantification of necrose in leaves of A. thaliana Col0 after 48 h (n = 3 ;

SD).

Experiment was repeated 2 times with similar results

A representative picture of necrosis caused by N. parvum extract was placed above each histogram as a visual illustration.

N. parvum crude extracts leaf assay

Known compounds from N. parvum

• Evidente et al. 2010 N. parvum from a cankered branch of grapevine cv. Parellada, Catalonia All isolated compound showed phytotoxic activity against tomato cuttings at 50 g/ml

(3R,4R)-(-)-4-hydroxymellein

(3R,4S)-(-)-4-hydroxy-mellein

R1 R2

OH H

H OH

Tyrosol isosclerone

Isolated compounds

6-(2-hydroxypropyl)-salicylic acid 6-methyl salicylic acid R1 R2 R3

(3R,4R)-(-)-4-hydroxymellein OH H H

(3R,4S)-(-)-4-hydroxy-mellein H OH H

(R)-3-hydroxymellein OH H H

mellein H H H

(+)-asperlin (+)-(6R, 7S)-dia asperlin

4-hydroxysuccinate-terrem-hydrate 4-chloro-terrem hydrate (-)-Terremutin (+)-Terrem hydrate (+)-Epi-sphaeropsidone Terredionol

Figure 2 : Necrosis induced by seven of the isolated phytotoxins at 100 and 200 g.mL-1 48 hours post inoculation. Quantification of necrosis in leaves of V.

vinifera cv. Chardonnay after 48 h (n = 3 ;

SD). Experiment was repeated 2 times with similar results. (-)-terremutin (1); (+)-epi-sphaeropsidon (3); (+)-(6R,7S)-

dia-asperlin (7); (-)-mellein (8), (-)-(3R,4S)-trans-4-hydroxymellein (9), (-)-(3R,4R)-cis-4-hydroxymellein (10); (-)-(R)-3-hydroxymellein (11).

A representative picture of necrosis caused by the toxin is placed above each histogram as a visual illustration.

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

control compound (1) compound (3) compound (7) compound (8) compound (9) compound (10) compound (11)

% le

af d

isc

nec

rosi

s

100 ug 200 mg

N. parvum pure compounds leaf assay

Symptomatic and asymptomatic wood analysis

BDA symptoms: Esca symptoms

Green shoots

Mellein std Terremutin std

M-GSB extract

M-GSB extract

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time [min]

2000

4000

6000

8000 Intens.

111.0

139.0

0

200

400

600

Intens.

60 80 100 120 140 160 180 m/z

(2) e)

g)

h)

0.85

0.90

0.95

1.00

5 x10 f)

+MS2(157.4), 8.2-8.4min

M1-BW.d: EIC 50-157 +MS2(157.1)

M1-BW.d: EIC 157-157.5 +All MS

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time [min] 0.0

0.5

1.0

1.5

2.0

2.5

4 x10 Intens.

a)

b)

c)

d)

M-GS-B_3_01_3069.d: EIC 179-179.5 ±All MS

M-GS-B_3_01_3069.d: EIC 50-180 +MS2(179.2), 0.0

0.5

1.0

1.5

2.0

2.5 5 x10

Intens.

1

2

3

4

5

6

4 x10

10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 Time [min]

105.1

133.0

151.0

161.0 +MS2(179.3), 12.4-12.6min)

0

2000

4000

6000

Intens.

60 80 100 120 140 160 180 m/z

10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 Time [min] 0.0

0.2

0.4

0.6

0.8

5 x10 Intens.

Brown stripes from green shoots

b) EIC MS m/z 179 [M+H]+ c) MS2 (179) d) [M+H]+ of mellein

f) EIC MS m/z 157 [M+H]+ g) MS2 (157) h) [M+H]+ of terremutin

white asymptomatic wood

+ traces of the signal identified only by MS, ++, and +++ signal identified by MS and UV.

Table 2: Identification of (-)-terremutin (1) and (-)-mellein (8) in wood of grapevine.

Sample name Dry

weight

(g)

Description, wood origin Terremutin

m/z 155 [M+H]+

MS2 138, 115, 110

Mellein

m/z 179 [M+H]+

MS2 161, 151, 133, 105

CH1-BB 3.5 Brown stripes wood from trunk of plant showing Botryospharia

dieback cv. Chardonnay

+ +

CH1-BW 3.0 White asymptomatic wood from trunk of plant showing

Botryospharia dieback cv. Chardonnay

+ nd

CH-2-TSB 0.2 Black streaked trunk wood of plant showing leaf discoloration

cv. Chardonnay

+ +

CH2-TSW 0.2 White asymptomatic trunk wood of plant showing leaf

discoloration cv. Chardonnay

nd nd

CH3-TSB 0.2 Black streaked trunk wood of plant showing <50% of leaf

discoloration cv. Chardonnay

+ +

CH3-TSW 0.2 White asymptomatic trunk wood from plant showing <50% of

leaf discoloration cv. Chardonnay

nd nd

M1-BB 0.5 Brown stripes wood from trunk of plant showing Botryospharia

dieback cv. Mourvèdre

+ ++

M1-BW 1.5 White asymptomatic wood from trunk of plant showing

Botryospharia dieback cv. Mourvèdre

+ +

M1-GSB 2.0 Brown stripes from green shoots of plant showing

Botryosphaeria dieback cv. Mourvèdre

+ +++

Bioactivity and Biosynthesis considerations for Mellein and terremutin

Isocoumarin from Botryosphaeria spp.

dihydroisocoumarin

6

7

4 3

1 8

5

R-(-)-mellein (3R,4R)-cis-4,7-dihydroxymellein (R)-7-hydroxymellein

(R)-3-hydroxymellein

(3R,4S)-4-hydroxymellein (3R,4R)-4-hydroxymellein

(R)-5-hydroxymellein

R-(-)-methoxymellein

(3R,4S)-8-methoxy-4-hydroxymellein

(3R,4R)-8-methoxy-4-hydroxymellein

(R)-8-methoxy-5-hydroxymellein

6-(2-hydroxypropyl)-salicylic acid

Mellein / Ochracin

• 1933 : Aspergillus ochraceus (Yabuta & Sumiki) Ochracin, A. melleus (Nishikawa), Mellein.identified as

the lactone of 6-[α(or β) -hydroxypropyl]salicylic acid.

• 1955 : structure identification (Blair & Newbold)

• 1968 : absolute configuration determination (Arakawa)

• Mellein and its dihydroisocoumarins derivatives are over represented in fungal plant pathogens and

endophytes

• Mellein is widespread in fungi : all Dothideomycetes and two Sodariomycete genera (Chooi et al. 2015)

• Found also in actinomycetes (Sun et al. 2012) and insects (Bestmann et al. 1992) .

Bioactivities of Mellein

Mellein displays a myriad of bioactivities:

• Weak to moderately active against bacteria (Burton 1955) and fungi (Botrytis cinerea EC50

48ug/ml)(Wang et al. 2014) (Paecilomyces varioti 20ug/ml ) (Thines et al. 1997)

• Exhibit antiviral activity against hepatitis C virus (in vitro at 35 uM/L). (Dai et al. 2001)

• Antiparasitic activity against Schistosoma mansoni. (200 ug/ml mellein and 50 ug/ml cis-

hydroxymellein) (Ramos et al. 2013)

• Larvicidal activity against Aedes aegypti at 3rd and early 4th instars (LC50 of 1.4 and 4.3 ppm at 24 h)

(Kendagor et al. 2013)

• Zootoxic activity Artemia salina larvae (200 ug/ml) (Parisi et al.1993)

• Trail pheromone of ants (Krohn et al. 1991, Kern et al. 1997)

• Non-host-specific toxicity

• Venkatasubbaiah et a. 1991 reported from D. seriata:

Mellein and 4-hydroxymellein induce necrosis on apple (10ug)

Treatment of detached leaves or apple seedlings with mellein at 20ug => no increase in lesions.

No evidence of the role of mellein in the infection process.

Mellein et 4-hydroxymellein identified in the conidia fluid

Conidia from highly virulent isolate produce more lesion, but less virulent isolate produce more mellein

• Cabras et al. 2006 reported from Sphaeropsis sapinea:

Mellein induce chlorosis after 7 days and necrosis after 15 days on pine needles, and wilting on tomato cutting after 48h at 0.1mg/ml

4-hydroxymellein did not induce symptoms neither on pine needles or tomato cutting

• strong anti-germinative effect of O-methylmellein against garden cress (10 g/ml) Glauser et al. 2009

• Mellein inhibit the growth of wheat embryo (50 g/ml) and 200 ug/ml wheat (Chooie et al. 2014)

Phytotoxic activities of Mellein and derivatives

• Djoukeng et al. 2009 reported from D. seriata :

Mellein , 4-hydroxymellein and 4,7-dihydroxymellein showed an MIC that MIC of 3µg/mL by the leaf-puncture bioassay on Chasselas.

• Ramirez-Suero et al. 2014 reported from D. seriata :

Mellein induce induce partial necrosis on calli at 500 gml-1 after 6 days

Mellein induce two pathways (VvGST1) cellular detoxification and (VvPR6, VvGLU, VvCHIT4c and VvPR10.1)

Mellein, is not responsible for the toxicity of the extracellular compounds produced by N. parvum

Calli subcultured with mellein at 500 μg.ml-1 after 6 days of contact

Defence gene expression in calli of V. vinifera cv. Chardonnay subcultured in Petri dishes with 200 (A) and 500 μg ml−1 (B) mellein

Phytotoxic activity of Mellein derivatives on grapevine

• Chooi et al. 2014 & 2015 in Parastagnospora nodorum

• Transcrptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 (SN_477) highly upregulated during infection of wheat

• Disruption of the gene resulted in the loss of mellein and O-methymellein

• No significant difference in virulence between the wild type and SN477 mutant against wheat

• Mellein at 200 g/ml inhibited the germination of wheat seeds.

PR-PKS responsible of the synthesis of mellein

Terremutin

• 1968 : Aspergillus terreus mutant (Miller), structure and complete stereochemistry

Epoxyquinoids

Epoxyquinone Epoxyquinol

• Terremutin showed only moderate radical scavenging activity (Dewi et al. 2012)(1/13 papers SciFinder)

• Venkatabsubbaiah 1992:

Epoxydon and desoxy-epoxydon induce necrosis on detached leaves with 50 ug of toxins on detached leaves of rhubarb and other weed species.

Broad microbial toxicity

Terremutin Desoxy-epoxydon Epoxydon Terreic acid

• Abou-Mansour et al. 2015

Terremutin showed 35% and 60% of necrosis on grapevine leaf disc assay at 100 and 200 g/ml

Terremutin induce necrosis at 3 and 6 dpi at 200 g/ml on grapevine callus.

Terremutin induce the expression of genes involved in the cellular detoxification (VvGST1), jasmonic (VvAOS), phenylpropanoid (VvPAL) and flavonoid pathways (VvF3H)

Terremutin did not induce genes encoding enzymes involved in phytoalexin biosynthesis, such as VvSTS1

Bioactivity of Terremutin on grapevine

Terremutin induces cell death after 3 days by a signalling cascade that differs from a defence-related pathway leading to necrosis

Defence gene expression in calli of V. vinifera cv. Chardonnay subcultured with 200 gmL-1 terremutin at 6 dpi

Terremutin Biosynthesis considerations

• Read & Vining 1968 from Aspergillus terreus investigate the biosynthesis pathway of terreic acid

Biosynthesis of terremutin involves 6-MSA as intermediate

6-MSA undergoes decraboxylation and two hydroxylation reactions, followed by quinol ring oxydation and epoxide formation to yield terreic acid which changed to terremutin hydrate as end products through terremutin

The epoxide oxygen originates from the atmospheric oxygen

Terreic acid Compound unrelated to terreic acid Terremutin acid

Genes involved in the biosynthesis of terreic acid

Terremutin Biosynthesis considerations

• Guo et al. 2014 in Aspergillus terreus NIH 2624 characterised the molecular genetic pathway of terreic acid :

Genome-mining approach => atX gene a 6-methyl salicylic synthase (6-MSAS) (Fuji et al. 1996)

Deletion of the gene ATEG_06275.1 (atX) => elimination of terreic acid

atA , atC , atE produce intermediates and shunt metabolites

atC strain accumulated terremutin as stable metabolite

Terremutin Biosynthesis pathway proposal

• 1rst step synthesis of 6MSA by 6-MSAS AtX .

• The domain achitecture of ATX includes

• -ketoacety synthase (KS)

• Acetyl transferase (AT)

• Thioester hydrolase (TH)

• Ketoreductase (KR)

• Acyl carrier protein (ACP)

6MSA 3-methyl catechol terremutin terreic acid

Conclusion

• Botryosphaeriaceae associated with grapevine produce the well known compounds derivatives from dihydoisocoumarins and epoxyquinols.

• Isocoumarins and epoxyquinols are produced by wide spectre of fungal species and induces necrosis on wide range of plants => non-host specific

• Isocoumarins and epoxyquinols have a wide spectrum of biological activities.

• Isoucoumarins in Botryosphaeriaceae are most likely to have auxiliary roles in virulence against vine,

• The majority of SMs reported of these fungi come from endophytes

• Many fungal SM pathways are known to be silent and only expressed in response to specific environmental stimuli

• The mode of action of the isolated phytotoxins at the cellular level need to be accurately determined

Acknowledgments

Jean-Pierre Métraux

Université de Fribourg Université de Haute Alsace

Group of Christophe Bertsche

Montserrat Ramírez-Suero

Mélanie Bénard-Gellon

Université de Reims Champagne-Ardennes

Florence Fontaine

Maryline Magnin-Robert

Alessandro Spagnolo

Institut Français de la vigne et du vin

Philippe Larignon

Instituto Superior de Agronomia, Portugal

Cécilia Rego Jean-Luc Débieux

Floriane L’Haridon

Mario Serrano

Jules Djoukeng

Financial support • National Centre of Competence in Research (NCCR) Plant Survival • COST Action 858 the Swiss Secrétariat d’Etat à la Formation, à la

Recherche et à l’Innovation • The French Ministère de l’Agriculture, de l’Agroalimentaire et de la

Forêt (CASDAR)