Studies on differential susceptibility of ‘Rocha’ pear clones and Portuguese varieties of pear and apple to fire blight - tools for the sustainability of fruit chain production

Suscetibilidade diferencial de clones de pereira ‘Rocha’ e de variedades autóctones de pereira e macieira à doença do Fogo Bacteriano em Portugal demonstram potencial como alternativas para a sustentabilidade da fileira

Leonor Cruz^1,2*, Joana Cruz^1,2 and Rui Sousa³

¹ Instituto Nacional de Investigação Agrária e Veterinária, Unidade de Investigação e Serviços de Sistemas Agrários e Florestais e Sanidade Vegetal, Laboratório de Fitobacteriologia, Oeiras, Portugal

² Universidade de Lisboa, Faculdade de Ciências, Instituto de Biossistemas e Ciências Integrativas (BioISI), Lisboa, Portugal

 ³ Instituto Nacional de Investigação Agrária e Veterinária, Polo de Alcobaça, Alcobaça, Portugal

(*E-mail: leonor.cruz@iniav.pt)

ABSTRACT

Fire blight was officially identified for the first time in Portugal, in 2011, affecting pear cultivar ‘Rocha’, the most important Portuguese variety of pear, with high economic impact in the production chain and trade. Since then the bacterium has destroyed new orchards in several areas of pear and apple production in the country. Nurseries and phytogenetic resources of several important in vivo collections have also been affected and are considered endangered. Severe disease symptoms on branches, flowers and fruits are also present in several other autochthones pear and apple varieties, leading to the death of trees under the climatic and cultural conditions prevalent in Portugal. Coordinated efforts from all fruit production chain actors, including farmers, phytosanitary authorities and research institutions, produced tools to overcome disease impact and build economic and social sustainability of this activity. Within the frame of the national research project Proder InovPomo (2014-2017), the phytogenetic resources of two official collections of pear and apple trees were assessed towards susceptibility to fire blight disease, to select and preserve the individuals combining the best agronomic features as well as higher tolerance to fire blight. The results achieved are essential for selection and preservation of Portuguese phytogenetic resources and biodiversity, as well as for delivering high quality planting materials to farmers for the establishment of new orchards, especially in fruit production areas where fire blight disease is present.

RESUMO

A doença do Fogo Bacteriano das Rosáceas foi oficialmente identificada pela primeira vez em Portugal em 2011, afetando com grande impacto económico devido a quebras provocadas na produção e no volume de exportações, os pomares de pereira da cultivar ‘Rocha’, a mais importante variedade portuguesa. Desde então a bactéria tem vindo a destruir novos pomares em várias áreas de produção de peras e maçãs em Portugal. Os viveiros e importantes coleções in vivo de recursos fitogenéticos autóctones também foram afetados, encontrando-se em risco. Na generalidade observam-se sintomas muito característicos da doença nos ramos, flores e frutos de distintas variedades autóctones, que conduzem à morte das árvores afetadas, nas condições climáticas prevalentes nas regiões mais importantes de produção de pomóideas em Portugal. Os vários intervenientes da cadeia de produção, incluindo fruticultores, autoridade fitossanitária e instituições de investigação implementaram ferramentas para mitigar o impacto económico e social causado pela doença e conduzir à sustentabilidade desta atividade económica. O trabalho desenvolvido no âmbito do projeto de investigação nacional Proder InovPomo (2014-2017), permitiu identificar e avaliar a suscetibilidade de duas coleções vivas de recursos fitogenéticos autóctones de macieira e pereira, em risco, avaliando a sua suscetibilidade a Erwinia amylovora, com o objetivo de selecionar e preservar os indivíduos com as melhores características agronómicas, evidenciando maior tolerância à doença do Fogo Bacteriano das Rosáceas.

Palavras-chave: Controlo fitossanitário, Fogo bacteriano, Erwinia amylovora, patogenicidade

INTRODUCTION

Fire blight disease has a severe impact in the production and trade of Portuguese apple and pear, namely in what concerns pear from cultivar ‘Rocha’.

Since 2011, Erwinia amylovora has destructed nurseries and orchards from distinct production areas of the country (Cruz, 2011). Nurseries and phytogenetic resources are endangered as most of them are settled in these affected areas (Cruz, 2012).

Similar problems aroused in the main European pomme production areas where apple and pear genetic resources were also assessed (Thibault et al., 1987; Szobiczewski et al., 2011; Gassmann et al., 2014) with very promising results, identifying accessions to be included in new breeding programs as tolerant parents, or displaying good qualities for industry purposes, contributing moreover to maintain biodiversity.

Within the framework of Proder InovPomo Project (PA49448) (2014-2017), the phytogenetic resources from two official in vivo collections of autochthones varieties of pear and apple were assessed towards the presence of E. amylovora and further studied to identify the existence of differential susceptibility to fire blight disease, with the objective of selecting and preserving individuals displaying higher levels of tolerance to the disease.

The results gathered during the time lapse of this project allowed identifying distinct levels of susceptibility to E. amylovora within the 80 pear ‘Rocha’ clones tested under confinement conditions as well as several national varieties of pear and apple able to serve as alternatives for sustainability of the fruit chain in the main production areas, adapted to Portuguese climatic conditions. This work also allowed the preservation of the important endangered phytogenetic resources and perspective the study molecular mechanisms associated to specific plant immunity.

Bacterial strains

Erwinia amylovora strain CPBF 1307, isolated in Portugal and collected from Pyrus communis cv. ‘Rocha’, in 2010, in the Alcobaça area, previously characterized and conserved at -80ºC, was used as positive control for diagnostic analysis as well as for susceptibility trials implemented under environmental controlled and appropriate confinement conditions.

The detection and identification of the bacterium was performed according to EPPO standard PM 7/21(2) (EPPO, 2013). Susceptibility tests were performed according to Parprstein et al. (2014) and EPPO standard PM 7/21(1) (EPPO, 2004).

The bacterial strain selected was grown in KMB agar (King et al., 1954), for 48hours at 24ºC, streaked to check for purity and further tested using conventional PCR, following Obradovic et al. (2007) modified by Gottsberg (PM 7/21(2)) (EPPO, 2013). The bacterial inoculum was prepared in phosphate buffer saline 10mM at a concentration of approximately 10⁸ cells/ml (OD=0.1 at 600nm).

Biological tests

Biological tests were performed along the three years of the project and repeated for confirmation of preliminary results achieved.

Young branches of the year from 72 clones of pear cv. ‘Rocha’ collected at Quinta de S. Gonçalo (Caldas da Rainha) and eight clones collected at Quinta do Olival Fechado (INIAV, Alcobaça) were firstly tested to determine the presence of disease, and used further to evaluate susceptibility to Erwinia amylovora.

Comparative analysis of susceptibility towards fire blight disease was also tested among 15 Portuguese pear varieties, other than ‘Rocha’ and 16 autochthones apple varieties.

Ten branches from each of the selected trees genomically characterized for identity confirmation, with ca. 30cm long were collected at the end of spring, wrapped in paper and transported to the laboratory, refrigerated, in closed polyethylene bags. Four replicate branches from each clone/variety were surface disinfected with 70% ethanol for 30seconds and further washed for three times in sterile distilled water, and dried. All branches were cut aseptically at the bottom and at the tip with a sterile scalpel, immediately introduced in micropropagation tubes containing 0,1% agar for inoculation (EPPO standard PM 7/21(1), 2004).

RESULTS AND DISCUSSION 

The selected plants were confirmed as free of E. amylovora according to the diagnostic procedures used, and by this reason used for branch inoculation.

Results from branch inoculations revealed significantly distinct susceptibility levels of the 80 pear clones tested to the bacterium (p<0,05) (Figure 1).

Comparative analysis of susceptibility among 15 Portuguese varieties of pear, other than ‘Rocha’, highlighted a differential response to E. amylovora, revealing that four of them, ‘Pera Bonita’, ‘São Bartolomeu’, ‘Carvalhal’ and ‘Aguinha’, presented medium to high susceptibility to the bacterium. Inversely, the remaining 11 varieties, displayed lower susceptibility levels, showing to be potential alternatives to pear ‘Rocha’ (Figure 2). This variety displayed levels of disease susceptibility higher than the average of the 15 pear varieties tested, after assessing the average of all clones tested (Figure 4).

Similarly, 16 apple varieties, also evaluated towards fire blight susceptibility, displayed, in general, lower levels of disease symptoms three weeks after inoculation, when compared to pear varieties, showing low to medium susceptibility under our trial conditions (p<0,05) (Figure 3). Eight of these apple varieties showed no visible symptoms at 21 DAI. In addition, viable cells of the bacterium were not recovered after re-isolation from the inoculated branches. Among the authoctones apple varieties, the most susceptible was ’Bravo Esmolfe‘, one of the most popular in the country.

Global comparative analysis allowed to underline distinct levels of susceptibility to fire blight agent – Erwinia amylovora – and to demonstrate the potential of Pyrus sp. and Malus sp. phytogenetic resources as alternatives for the establishment of new orchards, controlling high levels of basal inoculum in areas contiguous to contaminated delimited areas where the disease is known to occur (Figure 4). Parallel studies in other European countries lead to the identification of similar results, with distinct levels of susceptibility for different sets of accessions and autochthones varieties of pear and apple (Paprstein et al., 2014; Sedlak et al., 2014).

CONCLUSIONS

Recent studies confirm the benefits of this approach as a tool to identify resistance sources, and to identify alternatives to commercial varieties preventing dispersal of the bacterium and sustainability of the fruit production chain in a short time lapse (Szobiczewski et al., 2011; Gassmann et al., 2014).

Evaluation of the presence of Erwinia amylovora in these in vivo collections allowed the sanitation and preservation of relevant ecological biodiversity and long-term conservation at the national germplasm bank (Banco Português de Germoplasma Vegetal – BPGV), by in vitro propagation.

Differential susceptibility of this resources towards the causal agent of fire blight disease, also allowed identifying the existence of climate adapted autochthones varieties of pear and apple, as well as pear ‘Rocha’ clones as solid alternatives for the sustainability of Portuguese fruit chain production.

References

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Acknowledgements

This work was funded by Project Proder InovPomo (PA49448) – ‘Melhoramento de peras e maçãs através da conservação e caracterização do material vegetal’ (2014 - 2017). Joana Cruz is a INIAV fellowship supported by Project Proder InovPomo (PA49448). Support from Biosystems and Integrative Sciences Institute (BioISI, FCT/UID/Multi/04046/2013) is also acknowledged.

Accepted/aceite: 2018.10.08