Workshop Maximização dos serviços do ecossistema vinha. UTAD, 13 e 14 de Novembro de Lav Sharma and Guilhermina Marques CITAB, UTAD

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1 Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas Workshop Maximização dos serviços do ecossistema vinha. UTAD, 13 e 14 de Novembro de 2013 Diversity of entomopathogens (fungi) in Douro Demarcated Region vineyards. Lav Sharma and Guilhermina Marques CITAB, UTAD Co-financiamento: Programa de Desenvolvimento Rural Ministério da Agricultura, Mar, Ambiente e Ordenamento do Território Fundo Europeu Agrícola de Desenvolvimento Rural A Europa investe nas zonas rurais

2 PESTS IN VINEYARDS Phylum Arthropoda in vineyards, approximately 150 insects pests of grapes. a) Larva* Key Lepidopteran tortricids : 1) Eupoecilia ambiguella (Hübner) and, 2) Lobesia botrana (Denis & Schiffermüller) or European Grapevine Moth (EGVM). b) Pupa* Highly polyphagous (Gallardo et al., 2009). Economic losses (Carlos et al., 2011). c) Adult Lobesia botrana

3 CONTROLLING EVGM Several methods insecticide sprays: chitin synthesis inhibitors (flufenoxuron); growth regulators (fenoxycarb & methoxyfenozide); chlorantraniliprole; indoxacarb Biocontrol Strategies: 1) Introduction 2) Augmentation 3) Conservation a) mating disruption (Carlos et al., 2010) and, b) parasitoids, for e.g., Brachymeria sp.; Campoplex capitator (Aubert) (Carlos et al., 2011); biopesticide: Bacillus thuringiensis; Saccharopolyspora spinosa (spinosad).

4 ENTOMOPATHOGENS Organisms killing insect pests. For e.g., bacteria, fungi, nematodes and virus. Pesticides Market size at distributer Level* ($ US million) PRODUCT TYPE Chemical pesticides Biopesticide overall Plant extracts, others Bacillus thuringiensis Bacillus subtilis Trichoderma spp Beauveria spp Nematodes

5 ENTOMOPATHOGENIC FUNGI (EPF) Over 700 species from 90 different genera; around 170 products from 12 different species are commercialised (Vega et al., 2009). Species for e.g., Beauveria sp. (B. bassiana ; B. pseudobassiana, B. brongniartiii); Metarhizium sp. (M. anisopliae, M. acridum, M. robertsii); Isaria sp. and Lecanicillium sp. (Rehner et al., 2011). Fusarium spp. (Ali-Shtayeh et al., 2003; Oliveira et al., 2012). Trichoderma gamsii (Razinger et al., 2012). Bionectria ochroleuca (Oliveira et al., 2012). Beauveria bassiana: Infect 750 insect species approximately (Ghikas et al., 2010).

6 PROJECT Enhancing ecosystem services in Alto Douro Region vineyards to improve the management of arthropod pests. Aim: 1) To study the effects of the landscape and ground cover vegetation on the diversity of entomopathogenic fungi from different farm of Douro Demarcated Region. 2) To study the potential of these EPFs as biological control agent against Lobesia botrana.

7 SOIL ISOLATION SITES Aciprestes Arnozelo 6 Farms in study Carvalhas a) Raposeira b) Placas Granja Cidro S. Luis 110 soil samples were taken during Fall 2012 from farms mentioned above.

8 MATERIALS AND METHODS- Isolation Soil collection Incubating 6 Tenebrio molitor (Col: Tenebrionidae) Incubating larvae cadavers in cotton culture Surface sterilisation and subsequent pure culturing.

9 MATERIALS AND METHODS- Screening Preliminary pathogenicity test (Kochs Postulates) Fresh culture Potato Dextrose Agar Fungal suspensions. 3 final instar T. molitor (Col: Tenebrionidae) and Galleria mellonella (Lep: Pyralidae) larvae were dipped. Incubation at 22 C. Surface sterilisation and culturing on selective media (Ali-Shtayeh et al, 2002 and Goble et al., 2010)

10 MATERIALS AND METHODS- Molecular Identification PDA cultures with cellophane sheets. DNA extraction (Marques, G. 2003). Gel electrophoresis. PCR amplification of ITS region of 28S rdna using ITS1+ ITS4 primers (biology.duke.edu) DNA digestion (RFLPs), enzymes Hinf1 and Mbo1. Strains grouping according to the mol. wt profiles DNA sequencing

11 RESULTS: Restrcition fragment length polymorphism (RFLP) 11 Lav-PRESENTATION_RFLP_EXCEL.xlsx 231 isolates in total 140 Fusarium oxysporum species complex

12 30 F. solani 14 F. redolens

13 25 Bionectria ochroleuca

14 13 Beauveria genus species

15 2: Trichoderma gamsii 2: Purpureocillium lilacinum 9: Others

16 RESULTS: ITS phylogeny [Glimpses into species placement] P 1465+ITS1 2 P 1372B+ITS1 2 P 1159+ITS P 1336+ITS1 2 P 1699+ITS1 2 P 1924+ITS1 2 F. oxysporum clade 99 P 1711+ITS1 2 gi gb DQ Fusarium oxysporum f. sp. cyclaminis ATCC P 1899+ITS1 2 P 1234+ITS1 2 gi emb X Fusarium redolens P 1511+ITS1 2 P 1258+ITS1 2 F. redolens clade P 1053+ITS1 2 gi gb HQ Fusarium solani ATCC P 1930A+ITS1 2 P 1712+ITS1 2 P 1458+ITS1 2 F. solani clade 94 P 1176+ITS1 2 P 1013B+ITS P 1303+ITS1 P 1537+ITS1 gi gb HQ Beauveria bassiana ARSEF gi gb HQ Beauveria brongniartii ARSEF 617 P 1257+ITS1 P 1518+ITS1 Beauveria genus clade 96 P 1527B+ITS1 P 1531+ITS1 gi gb AY Beauveria pseudobassiana ARSEF 3405 P 1022+ITS gi AF Bionectria ochroleuca CBS P 1255+ITS1 2 P 1887+ITS1 2 Bionectria ochroleuca clade P 1673+ITS1 0.02

17 FARM TYPE DISTRIBUTION F. oxysporum F. solani F. redolens B. ochroleuca Beauveria sp. P. lilacinum Trichoderma gamsii Carvalhas S. Luis Granja Arnozelo Aciprestes Cidro Total (% Occurrence) 140 (60.6 %) 29 (12.5 %) 14 (6.1 %) 25 (10.8 %) 13 (5.69%) 2 (0.86%) 2 (0.86%)

18 40 35 Fusarium oxysporium 30 Fusarium solani Fusarium redolens Bionectria ochroleuca Beauveria sp. Purpureocillium lilacinum Trichoderma gamsii

19 Trichoderma gamsii 2 (100%) 0 HERBICIDE USAGE SPECIES HERBICIDE No. of isolates (share %) W/O HERBICIDE No. of isolates (share %) Fusarium oxysporum 72 (51.42%) 68 (48.57%) Fusarium solani 13 (44.82%) 16 (55.17%) Fusarium redolens 7 (50.0%) 7 (50.0%) Bionectria ochroleuca 13 (52.0%) 12 (48%) Beauveria sp (100%) Purpureocillium lilacinum 0 2 (100%)

20 No Hebicide

21 OTHER FACTORS Effect of ground cover and landscape CONCLUSION Entomopathogenic fungi are widely distributed within DDR. Fusarium sp. are quite dominant; Distribution of Bionectria ochroleuca and Beauveria sp. is evident. Usage of herbicide affects Beauveria sp. negatively.

22 FUTURE WORKS Maintaining a Geographic Information System; Infectivity analysis; Selection of few isolates for biocontrol of Lobesia botrana

23 THANK YOU