2ª REUNIÃO DA SOCIEDADE PORTUGUESA DE FITOPATOLOGIA. OEIRAS 24 E 25 DE SETEMBRO DE 19982ª REUNIÃO DA SOCIEDADE PORTUGUESA DE FITOPATOLOGIA. OEIRAS 24 E 25 DE SETEMBRO DE 1998

USE OF GLOMUS AGGREGATUM AND GLOMUS DESERTICOLA AS BIOCONTROL AGENTS AGAINST

PHYTOPHTHORA CINNAMOMI IN CHERRY

USE OF GLOMUS AGGREGATUM AND GLOMUS DESERTICOLA AS BIOCONTROL AGENTS AGAINST

PHYTOPHTHORA CINNAMOMI IN CHERRY

OBJECTIVEOBJECTIVE

INTRODUCTIONINTRODUCTION

MATERIAL AND METHODSMATERIAL AND METHODS

RESULTSRESULTS CONCLUSIONSCONCLUSIONS

Certain Zygomycetes of the Order Glomales are able to colonize the roots of a wide range of plants and form a mutualistic symbiosis known as arbuscular mycorrhiza (AM). Unlike other fungi, those forming arbuscular mycorrhiza do not trigger plant defense mechanisms when their infection structures get in contact with root cells of the epidermis and hypodermis (Gianinazzi-Pearson et al.,1996), and have been proved to enhance plant growth and mineral nutrition (Hayman, 1983). Several studies have also shown that arbuscular mycorrhizas may play an important role as biological control agents against soil-borne root diseases.

Certain Zygomycetes of the Order Glomales are able to colonize the roots of a wide range of plants and form a mutualistic symbiosis known as arbuscular mycorrhiza (AM). Unlike other fungi, those forming arbuscular mycorrhiza do not trigger plant defense mechanisms when their infection structures get in contact with root cells of the epidermis and hypodermis (Gianinazzi-Pearson et al.,1996), and have been proved to enhance plant growth and mineral nutrition (Hayman, 1983). Several studies have also shown that arbuscular mycorrhizas may play an important role as biological control agents against soil-borne root diseases.

In this work, we investigated the effects of the fungicide Fosetyl-Al and/or two arbuscular mycorrhizal (AM) fungi, namely Glomus aggregatum and Glomus deserticola, against Phytophthora cinnamomi infection in Prunus cerasifera.

In this work, we investigated the effects of the fungicide Fosetyl-Al and/or two arbuscular mycorrhizal (AM) fungi, namely and , against infection in .

Glomus aggregatum Glomus deserticolaPhytophthora cinnamomi Prunus cerasifera

Cuttings from Prunus cerasifera were inserted in a rooting bed of 3:1 peat:perlite in a greenhouse.Well-rooted cuttings were transplanted to 1 L pots filled with a quartz sand:peat mixture with the corresponding treatments, one plant being established per pot. The following factors and their interactions were studied: inoculation with Phytophthora cinnamomi, inoculation with the AM fungus G. aggregatum, inoculation with the AM fungus G. Deserticola and application of the fungicide Fosetyl-Al. A control treatment was also established. All these treatments were made at transplantation, but also another complete series of treatments was prepared to inoculate P. Cinnamomi seven days after transplantation. There were four replicates per treatment.

Cuttings from Prunus cerasifera were inserted in a rooting bed of 3:1 peat:perlite in a greenhouse.Well-rooted cuttings were transplanted to 1 L pots filled with a quartz sand:peat mixture with the corresponding treatments, one plant being established per pot. The following factors and their interactions were studied: inoculation with Phytophthora cinnamomi, inoculation with the AM fungus G. aggregatum, inoculation with the AM fungus G. Deserticola and application of the fungicide Fosetyl-Al. A control treatment was also established. All these treatments were made at transplantation, but also another complete series of treatments was prepared to inoculate P. Cinnamomi seven days after transplantation. There were four replicates per treatment.

The isolate of P. Cinnamomi was originally collected in Pontevedra (NW Spain) from Vitis vinifera var. Albariño. The fungus was grown on PDA medium P. cinnamomi inoculum was obtained by triturating the culture block from each petri dish with 100 mL sterile water. For P. Cinnamomi-inoculated treatments, plant roots were immersed for five minutes in this suspension before transplantation (Tello et al., 1991).

The isolates of G. aggregatum and G. deserticola were obtained from sand:vermiculite (1:1) pot cultures of Trifolium pratense.

In the corresponding treatments, cutting roots were immersed for one hour in a water solution of the -1fungicide Fosetyl-Al 80% w/p (2.5 g.L ) prepared from the commercial product Aliette (Rhône-Poulenc

Agro, S.A), before transplantation.

The number of green leaves per pot was periodically recorded after transplantation. Six months after cuttings transplantation, shoots were removed and roots washed free of substrate and then cleared (Philips & Hayman, 1970) and stained with 0.05% acid fuchsin in lactoglycerol to determine the percentage of root length colonized by G. aggregatum and G. deserticola (Ambler & Young, 1977). Samples of roots and rhizospheric soil were taken to reisolate P. Cinnamomi and evaluate the pathogen infection. Roots were cut in 1-2 cm pieces and plated onto V8 medium, modified in order to study fungal mycelium characteristics, while a trap technique was applied to the rhizospheric soil to detect the presence of Phytophthora sporangia (Mansilla et al., 1993).

The isolate of P. Cinnamomi was originally collected in Pontevedra (NW Spain) from Vitis vinifera var. Albariño. The fungus was grown on PDA medium P. cinnamomi inoculum was obtained by triturating the culture block from each petri dish with 100 mL sterile water. For P. Cinnamomi-inoculated treatments, plant roots were immersed for five minutes in this suspension before transplantation (Tello et al., 1991).

The isolates of G. aggregatum and G. deserticola were obtained from sand:vermiculite (1:1) pot cultures of Trifolium pratense.

The number of green leaves per pot was periodically recorded after transplantation. Six months after cuttings transplantation, shoots were removed and roots washed free of substrate and then cleared (Philips & Hayman, 1970) and stained with 0.05% acid fuchsin in lactoglycerol to determine the percentage of root length colonized by G. aggregatum and G. deserticola (Ambler & Young, 1977). Samples of roots and rhizospheric soil were taken to reisolate P. Cinnamomi and evaluate the pathogen infection. Roots were cut in 1-2 cm pieces and plated onto V8 medium, modified in order to study fungal mycelium characteristics, while a trap technique was applied to the rhizospheric soil to detect the presence of Phytophthora sporangia (Mansilla et al., 1993).

In the corresponding treatments, cutting roots were immersed for one hour in a water solution of the fungicide Fosetyl-Al 80% w/p (2.5 g.L ) prepared from the commercial product Aliette (Rhône-Poulenc Agro, S.A), before transplantation.

-1

When P. cinnamomi and AM fungal inoculation were simultaneously made, neither G. aggregatum nor G. deserticola were effective to protect Prunus plants against the pathogen. Even more, inoculation with either AM fungus, particularly with G. aggregatum, seemed to increase disease severity. However, our results indicate that AM fungi may be effective to help Prunus plants against Phytophthora cinnamomi if they are inoculated prior to root contact with the pathogen.

We find also experimental support to suggest that some AM fungi may show this effectivity in the short time while others in longer terms. G. desertícola was more effective up to 76 days growth,while G. aggregatum became more effective from that time on. Thus research on selection of AM fungi for plant protection against soil-borne fungal pathogens must consider AM fungal

When P. cinnamomi and AM fungal inoculation were simultaneously made, neither G. aggregatum nor G. deserticola were effective to protect Prunus plants against the pathogen. Even more, inoculation with either AM fungus, particularly with G. aggregatum, seemed to increase disease severity. However, our results indicate that AM fungi may be effective to help Prunus plants against Phytophthora cinnamomi if they are inoculated prior to root contact with the pathogen.

We find also experimental support to suggest that some AM fungi may show this effectivity in the short time while others in longer terms. G. desertícola was more effective up to 76 days growth,while G. aggregatum became more effective from that time on. Thus research on selection of AM fungi for plant protection against soil-borne fungal pathogens must consider AM fungal

Acknowledgements

This work was funded with grant XUGA26111B96 (Xunta de Galicia, Spain) and the Consejo Superior de Investigaciones Científicas (CSIC).We thank Dr. Carmen Salinero for her collaboration and helpful advice.

REFERENCES

Ambler, J.R. & Young, J. L. 1977. Techniques for determining root length infected by vesicular-arbuscular mycorrhizae. Soil Science Society of America Journal 41: 551-556.Gianinazzi-Pearson, V., Gollote, A., Cordier, C. & Gianinazzi, S. 1996. Root defense responses in relation to cell and tissue invasion by symbiotic microorganisms: Cytological investigations. In: Histology, Ultrastructure and Molecular Cytology of Plant-Microorganism Interactions. Ed. M. Nicole & V. Gianinazzi-Pearson, Kluwer Academic Publishers, Dordrecht, The Netherlands. Pp. 127-191.Hayman, D.S. 1983. The physiology of vesicular-arbuscular endomycorrhizal symbiosis.. Canadian Journal of Botany 61: 944-962.Mansilla, J. P., Pintos, C. & Salinero, C. 1993. Aislamiento e identificación en la provincia de Pontevedra de Phytophthora cinnamomi Rands. Como patógeno de viña. Boletín de Sanidad Vegetal Plagas 19: 541-549.Phillips, J. M. & Hayman, D.S. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assesment of infection. Transactions of the Bristish Mycological Society 55: 158-160.Tello, J., Vares, F. & Lacasa, A. 1991. Pruebas de patogeneicidad. In: Manual de laboratorio. Diagnóstico de hongos, bacterias y nemátodos fitopatógenos. Ed. Dirección General de Sanidad de la Producción Agraria, Ministerio de Agricultura, Pesca y Alimentación, Madrid. Pp. 79-82.

Acknowledgements

This work was funded with grant XUGA26111B96 (Xunta de Galicia, Spain) and the Consejo Superior de Investigaciones Científicas (CSIC).We thank Dr. Carmen Salinero for her collaboration and helpful advice.

J. L. 1977. Techniques for determining root length infected by vesicular-arbuscular mycorrhizae. Soil Science Society of America Journal 41: 551-556.

. 1996. Root defense responses in relation to cell and tissue invasion by symbiotic microorganisms: Cytological investigations. In: Histology, Ultrastructure and Molecular Cytology of Plant-Microorganism Interactions. Ed. M. Nicole & V. Gianinazzi-Pearson, Kluwer Academic Publishers, Dordrecht, The Netherlands. Pp. 127-191.

. The physiology of vesicular-arbuscular endomycorrhizal symbiosis.. Canadian Journal of Botany 61: 944-962.

1993. Aislamiento e identificación en la provincia de Pontevedra de Rands. Como patógeno de viña. Boletín de Sanidad Vegetal Plagas 19: 541-549.

1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assesment of infection. Transactions of the Bristish Mycological Society 55: 158-160.

. 1991. Pruebas de patogeneicidad. In: Manual de laboratorio. Diagnóstico de hongos, bacterias y nemátodos fitopatógenos. Ed. Dirección General de Sanidad de la Producción Agraria, Ministerio de Agricultura, Pesca y Alimentación, Madrid. Pp. 79-82.

REFERENCES

Ambler, J.R. & Young,

Gianinazzi-Pearson, V., Gollote, A., Cordier, C. & Gianinazzi, S

Hayman, D.S. 1983

Mansilla, J. P., Pintos, C. & Salinero, C.

Phillips, J. M. & Hayman, D.S.

Tello, J., Vares, F. & Lacasa, A

Phytophthora cinnamomi

The mycorrhizal inoculum consisted in rhizospheric soil containing spores, external mycelium and colonized root fragments.

The mycorrhizal inoculum consisted in rhizospheric soil containing spores, external mycelium and colonized root fragments.

Reisolation of P. Cinnamomi from roots and soil at the end of the experiment.Reisolation of P. Cinnamomi from roots and soil at the end of the experiment.

Phytophthora cinnamomi grown in PDA medium.Phytophthora cinnamomi grown in PDA medium.

1 2 3 1,3 2Pintos, C. ; Vilariño, A. ; Sainz, M.J. ; Mansilla, J.P. ; González-Penalta, B.

1Estación Fitopatológica “Do Areeiro”. Servicio Agrario. Excma. Diputación Provincial de Pontevedra. Subida a la Robleda, s/n. Pontevedra. Galicia. España.2Departamento de Fisiología Vegetal. Instituto de investigaciones Agrobiológicas de Galicia, CSIC. Santiago de Compostela. A Coruña. Galicia. España.

3Departamento de Producción Vegetal Universidad de Santiago de Compostela. Lugo. Galicia. España.

1 2 3 1,3 2Pintos, C. ; Vilariño, A. ; Sainz, M.J. ; Mansilla, J.P. ; González-Penalta, B.

1Estación Fitopatológica “Do Areeiro”. Servicio Agrario. Excma. Diputación Provincial de Pontevedra. Subida a la Robleda, s/n. Pontevedra. Galicia. España.2Departamento de Fisiología Vegetal. Instituto de investigaciones Agrobiológicas de Galicia, CSIC. Santiago de Compostela. A Coruña. Galicia. España.

3Departamento de Producción Vegetal Universidad de Santiago de Compostela. Lugo. Galicia. España.

T R E A T M E N T R o o t S o i l

P h y +F o s -

A M -u n i n o c u l a t e d

— ( + )

— ( + )

— ( + )

( + ) ( + )

G l o m u sa g g r e g a t u m

— ( + )

— ( + )

— ( + )

— ( + )

G l o m u sd e s e r t i c o l a

— ( + )

— ( + )

— ( + )

( + ) ( + )

T R E A T M E N T R o o t S o i l

P h y +F o s -

A M -u n i n o c u l a t e d

— ( + )

— ( + )

— ( + )

( + ) ( + )

G l o m u sa g g r e g a t u m

— ( + )

— ( + )

— ( + )

— ( + )

G l o m u sd e s e r t i c o l a

— ( + )

— ( + )

— ( + )

( + ) ( + )

T R E A T M E N T R o o t S o i l

P h y -F o s -

A M -u n i n o c u l a t e d

— —

— —

— —

— —

G l o m u sa g g r e g a t u m

— —

— —

— —

— —

G l o m u sd e s e r t i c o l a

— —

— —

— —

— —

T R E A T M E N T R o o t S o i l

P h y -F o s -

A M -u n i n o c u l a t e d

— —

— —

— —

— —

G l o m u sa g g r e g a t u m

— —

— —

— —

— —

G l o m u sd e s e r t i c o l a

— —

— —

— —

— —

T R E A T M E N T R o o t S o i l

P h y -

F o s +

A M -u n i n o c u l a t e d

— —

— —

— —

— —

G l o m u sa g g r e g a t u m

— —

— —

— —

— —

G l o m u sd e s e r t i c o l a

— —

— —

— —

— —

T R E A T M E N T R o o t S o i l

P h y -

F o s +

A M -u n i n o c u l a t e d

— —

— —

— —

— —

G l o m u sa g g r e g a t u m

— —

— —

— —

— —

G l o m u sd e s e r t i c o l a

— —

— —

— —

— —

T R E A T M E N T R o o t S o i l

P h y +F o s +

A M -u n i n o c u l a t e d

( + ) ( + )

— ( + )

( + ) —

— —

G l o m u sa g g r e g a t u m

— —

( + ) —

( + ) —

— —

G l o m u sd e s e r t i c o l a

— —

— —

— —

— —

T R E A T M E N T R o o t S o i l

P h y +F o s +

A M -u n i n o c u l a t e d

( + ) ( + )

— ( + )

( + ) —

— —

G l o m u sa g g r e g a t u m

— —

( + ) —

( + ) —

— —

G l o m u sd e s e r t i c o l a

— —

— —

— —

— —

T R E A T M E N T R o o t S o i l

P h y +F o s -

A M -u n i n o c u l a t e d

— ( + )

— ( + )

— ( + )

— —

G l o m u sa g g r e g a t u m

— ( + )

— —

— —

— ( + )

G l o m u sd e s e r t i c o l a

— ( + )

— —

— —

— —

T R E A T M E N T R o o t S o i l

P h y +F o s -

A M -u n i n o c u l a t e d

— ( + )

— ( + )

— ( + )

— —

G l o m u sa g g r e g a t u m

— ( + )

— —

— —

— ( + )

G l o m u sd e s e r t i c o l a

— ( + )

— —

— —

— —

T R E A T M E N T R o o t S o i l

P h y +F o s +

A M -u n i n o c u l a t e d

— —

— —

— —

— —

G l o m u sa g g r e g a t u m

— —

— —

— —

— ( + )

G l o m u sd e s e r t i c o l a

— —

— —

— —

— ( + )

T R E A T M E N T R o o t S o i l

P h y +F o s +

A M -u n i n o c u l a t e d

— —

— —

— —

— —

G l o m u sa g g r e g a t u m

— —

— —

— —

— ( + )

G l o m u sd e s e r t i c o l a

— —

— —

— —

— ( + )

Inoculated 7 days after transplantationInoculated 7 days after transplantation