Survey and Geographic Distribution of Chestnut Blight in Portugal

Survey and geographic distribution of Chestnut Blight in Portugal

Prospecção e distribuição geográfica do cancro do castanheiro em portugal

Helena Bragança1, Sofia Simões1, Miguel Capelo1, José Marcelino2, Natércia Santos1

Abstract of C. parasitica isolates has been established and the study of the molecular variability of Cryphonectria parasitica, the fungus that C. parasitica in Portugal is underway. causes chestnut blight disease, was first de- scribed in Portugal (1929) on bark samples Key-words: Cryphonectria parasitica, Cas- of Castanea crenata, collected in Beira Inte- tanea sativa, Portugal rior province. After this report, only in 1989 two disease foci were found on native Euro- pean chestnut, C. sativa, in Trás-os-Montes Resumo e Alto Douro Province. To assess the current distribution of chestnut blight in Portugal Cryphonectria parasitica, o fungo respon- (mainland, Azores and Madeira) a nation- sável pelo cancro do castanheiro, foi des- wide monitoring program was implemented. crito pela primeira vez em Portugal (1929) A major cooperative effort, involving all the em amostras da casca de Castanea crenata, Portuguese Forest and Agricultural Services, provenientes da Beira Interior. Depois da was created for this endeavor and coordinat- detecção inicial, apenas em 1989, dois focos ed by the National Forestry Station. A total da doença foram encontrados em castanheiro of 191 chestnut stands was surveyed across Europeu, C. sativa, na província de Trás-os- the entire country. C. parasitica was isolated Montes e Alto Douro. Para determinar a dis- from 56.5% of the sampling sites (108 sites). tribuição actual do cancro do castanheiro em Sampled parcels were digitally geo-refer- Portugal (continente e Regiões Autónomas enced and mapped. In addition, a collection da Madeira e Açores), foi implementado um programa nacional de monitorização da do- ença. Para o efeito houve intensa cooperação entre os Serviços Florestais e Agrícolas do país, com coordenação da Estação Florestal 1  Instituto Nacional de Recursos Biológicos, I.P. Nacional. Foram visitadas, em todo o país, Edifício da ex-Estação Florestal Nacional, Quinta 191 parcelas de castanheiro. C. parasitica do Marquês 2784-505 Oeiras, Portugal. foi isolado em 56,5% da amostragem (108 2 Plant and Soil Science Department, University of parcelas). As parcelas amostradas foram di- Vermont, 661 Spear Street, Burlington VT 05405, USA. gitalmente georeferenciadas e cartografadas. Communication presented in 5º Congresso da Sociedade Uma colecção de isolados de C. parasitica Portuguesa de Fitopatologia, Coimbra, 2007. foi estabelecida encontrando-se em curso o Corresponding author: [email protected] estudo da variabilidade genética da doença em Portugal.

Palavras-chave: Cryphonectria parasitica, Recepção/Reception: 2008.02.19 Aceitação/Acception: 2008.10.07 Castanea sativa, Portugal Survey and geographic distribution of Chestnut Blight in Portugal 149

Introduction all counties. In the Chestnut Protected Deno- mination of Origin zone (PDO) of “Castanha In Portugal, Cryphonectria parasitica da Padrela”, one of the most important in the (Murrill) Barr was first described by Câmara country, 61 528 trees were monitored with an (1929), on bark samples collected on bran- infection rate of 100% reported in some loca- ches from young plants of Castanea crenata lities (Anastácio, 2001). Sieb. & Zucc, in the Beira Interior Province, In subsequent years, the disease spread near the village of Alcaide, Fundão. Forest by natural pathways and/or by the influence Services Publications reported in 1949 that of man, intensifying chestnut monoculture “no focus of chestnut blight was found in due to increasing economical interests and Portugal until now, although we are not cer- investments in chestnut orchards and forest tain that it does not exist since many Japanese stands (Abreu & Martins, 2002). Overall, the chestnuts have been imported in the last ye- balance of the program was that the eradi- ars” (Fernandes, 1949). The initial infection cation objectives were not totally achieved. was probably successfully eradicated, since Although the implementation of eradication only in 1989 two disease foci were found on measures allowed the decrease of the levels European chestnut (Castanea sativa Mill.) in of infection. In addition, the improvement of the Trás-os-Montes e Alto Douro province, good cultural practices by the growers, also the most important portuguese chestnut-gro- implemented during the program, was an im- wing region in the Northeast of the country, portant factor preventing the fast spread of in Carrazedo de Montenegro (Serra da Pa- the disease. drela -Valpaços) and Parada village, Terra The Portuguese archipelago of Azores Fria – Bragança, (Abreu, 1992). (“Região Autónoma dos Açores”) reported After the 4th meeting of the International chestnut blight for the first time in 1993. Two Commission for Chestnut, in 1958, Yugosla- internal reports from S. Miguel and Terceira via, Portugal is included within the range of Islands made by the Estação de Fruticultu- the fungus and in the geographic distribution ra Vieira Natividade in Portugal and Insti- of the disease in the Commonwealth My- tute Nationale de Recherche Agronomique cological Institute (CMI) records (Caetano, (INRA) in France, described the occurrence 1990). In 1990 the disease was of mandatory of the disease. In addition, Ormonde (1994) report by governmental ordinance (“Portaria describes that symptomatic trees in the Ter- 847/90, 18 Setembro”). In spite of this fact ceira Island were not significantly affected by and in opposition to the general rule giving the disease, thus, hypothetically the hypovi- priority to eradicate critical quarantine dise- rulence could also exist at that time and later ases, no financial support was allocated for Abreu and Martins (2002) referred the exis- this purpose. This measure was only taken in tence of an hypovirulent culture from Azo- 1998 (“Despacho Conjunto 117/98, 18 Feve- res. That situation was also observed a pos- reiro”) in compliance with the establishment teriori and referred by Bragança et al (2004). of an eradication program which included lo- In Madeira Island (“Região Autónoma da gging and incineration of diseased branches Madeira”), chestnut blight was detected for or the entire tree (Anastácio, 2001). the first time in 2002 by the Direcção Re- During the three years of the program, a gional de Agricultura da Madeira authorities total of 82 000 chestnut trees were monitored (DRAM), in chestnut stands of “Comissão de in Northern Portugal by the Direcção Regio- Levadas do Curral e Castelejo”, Curral das nal de Trás-os-Montes e Alto Douro services Freiras village. Laboratorial confirmation of (DRATM). The overall rate of infection at the disease was performed in the Laboratório that time was approximately 10%, although Agrícola da Madeira (Maia, 2003). major differences among localities were ob- To assess the distribution of chestnut blight served. However, the disease was present in and the variability of C. parasitica in Portugal, 150 REVISTA DE CIÊNCIAS AGRÁRIAS it was crucial to put into action a nationwide sia”) with an area >0.5 ha was done. Subse- monitoring program comprising all chestnut quently, these areas were monitored for the growing regions in Portugal. The experimen- presence of C. parasitica. In each district tal design of this work would have to assure parish, as a rule, sites with previously repor- both the accurate estimate of the distribution ted presence of chestnut blight were always of chestnut blight, which previously was pre- monitored. For the remaining selected areas, sumed to be centralized only in the Northeast monitoring sites were randomly selected and of the country, and the characterization of all then inspected for the presence of symptoms the existent populations of the fungus. The and/or signs of the blight. study of the genetic diversity in populations Cartographic localization of sampling are- of C. parasitica and the research for hypovi- as was performed using military cartography rulent isolates were required to implement, in and orthophotomaps (paper based and, when the nearest future, effective actions preventing available, digital materials). Sampling sites further spread of the disease. and localities were digitally geo-referenced In the aim of this work all chestnut gro- and mapped using GIS analysis tools throu- wing regions in Portugal were visited and a gh ArcView® 3.2 software (Environmental national collection of C.parasitica isolates Systems Research Institute, Inc.). The num- has been established. Considering the impor- ber of sites monitored per region, as well as, tance of the chestnut area and the incidence the number of C. parasitica isolates per site, of chestnut blight in Trás-os-Montes e Alto varied largely according to the distribution Douro, some areas in this province were of chestnut in Portugal and the incidence of most intensively prospected. In the course of chestnut blight, respectively. Distinct data the survey it was possible to observe signs from the sites (e.g. signs and symptoms of and symptoms not only of C. parasitica but disease, stand characterization, environmen- of other chestnut diseases as well. Although tal conditions, etc.) and individual tree des- not a primary goal of this study, additional criptions were gathered in order to find cor- information on other fungi found in chestnut relations with the disease. stands is also briefly presented. Field procedures involved visual assess- ment of all the trees inside the sites in order to detect chestnut blight symptoms: branch Material and methods or tree mortality with shrivelled, dried lea- ves, chestnut blight cankers with longitudi- Experimental design nal bark fissures and presence of fungal stro- The selection of the sites to be monitored mata or mycelial fans. Based on Robin et al. for the presence of chestnut blight sought to (2000) each sampling site was approx. 1 ha cover all the area of chestnut in Portugal with in size and one canker per tree was sampled the inclusion of the possible highest number for a maximum of ten different trees per site of sites previously known to present the di- (sporadically, in larger sites more than ten sease. trees were sampled). Sample sites were selected considering the information given by the Agriculture and Isolation and identification Forest Services and the map of chestnut dis- Using a knife, bark samples (approx. 2 x tribution area in the country. All provinces 2 cm) were removed from the cankers and in continental Portugal, Madeira Island, and brought to the laboratory. Small pieces (ap- three Islands of the Azores were surveyed for prox. 2 x 2 mm) were cut out from the bark chestnut blight in the course of the monito- samples and surface-disinfected by dipping ring program. for 15s in 70% ethanol. Then, the samples In each of the regions, assessment of all were rinsed in sterile water, blotted on fil- chestnut sites (by district parish – “fregue- ter paper and placed on potato dextrose agar Survey and geographic distribution of Chestnut Blight in Portugal 151

(PDA, Difco, Sparks, MD, USA). The plates confirmation of the phytopathogenic agent of were incubated at 25ºC in the dark and C. genus level. In some cases, soil analyses were parasitica cultures were transferred to new performed in the forest pathology laboratory PDA plates. One isolate per canker was used to confirm the presence of Phytophthora sp. for further analysis. as described by Campbell (1949).

Long-term storage of C. parasitica isolates Before stocking, the isolates were grown RESULTS on PDA medium placed in Petri dishes and McCartney bottles, then stored at 25ºC in A total of 191 chestnut stands were moni- 24h dark for seven days. Subsequently both tored across the entire country comprising containers were transferred to a laboratory 55 district parishes in 34 counties from all bench with natural light during 10 to 14 Regional Agricultural Department areas in days to allow profuse production of conidia. Portugal mainland, and 25 district parishes C. parasitica isolates were stored at 6ºC on from 13 municipality areas in the Archipela- McCartney bottles (three bottles per isola- goes). (Fig. 1, Table 1). The results obtained te). In parallel, the isolates were also frozen herein, and presented in Table 1 and Figs. 1 in glycerol and stored at -80ºC as follows: to 7 indicate that chestnut blight has spread 3-4 pieces of PDA (5x5mm) were cut from into most of the chestnut growing area of sporulating cultures and immersed in 800µL Portugal. Signs and symptoms of the disease of a 22% sterile glycerol solution (Riedel- have been detected in six Regional Agricul- deHaën, Seelze-Hannover, Germany) allo- tural Department areas of the mainland and cated in Cryo-vials tubes (Tenak™), mixed C. parasitica was isolated from four of these heavily by vortexing and finally frozen in six regions. The disease was also found in the liquid nitrogen before storage. Azores and Madeira archipelagoes. A total of 617 C. parasitica isolates were obtained Detection of other phytopathogens from 108 sampling sites (mainly orchards) Chestnut sample tissue with signs of other distributed across four mainland provinces diseases was observed and if necessary pro- (Minho, Trás-Montes, Beira Interior, Alen- cessed in laboratory through slide preparations tejo), the Azores Islands (Pico, Terceira, S. for microscopic observation and posterior Miguel), and the Madeira Island. 152 REVISTA DE CIÊNCIAS AGRÁRIAS

Figure 1 – Portugal mainland: distribution of European chestnut tree and chestnut blight survey by district parish. Survey and geographic distribution of Chestnut Blight in Portugal 153

Table 1 – Survey and distribution of chestnut blight disease in Portugal.

* Includes sites with signs and symptoms of chestnut blight. 154 REVISTA DE CIÊNCIAS AGRÁRIAS

Figure 2 – Geographic distribution of chestnut blight in Portugal mainland (by district parish). Survey and geographic distribution of Chestnut Blight in Portugal 155

Figure 3 – Geographic distribution of chestnut blight - Valpaços detail (in Trás-os-Montes e Alto Douro).

Figure 4 – Geographic distribution of chestnut blight - Beira Interior detail. 156 REVISTA DE CIÊNCIAS AGRÁRIAS

Figure 5 – Geographic distribution of chestnut blight - Alentejo detail.

Figure 6 – Geographic distribution of chestnut blight – Azores Islands detail (lines inside the maps represent District parish borders). Survey and geographic distribution of Chestnut Blight in Portugal 157

Figure 7 – distribution of chestnut blight – Madeira Island detail.

Regarding the survey of the other diseases, red past human interventions, although at a the following phytopathogenic fungi were much smaller rate than the orchards. Con- identified: Coryneum sp., Cytospora sp. and versely, the vast majority of the forests wi- Phytophthora sp.. Symptoms of ink disease thout conspicuous human intervention and were observed in all visited regions, diffuse management were found to be disease-free, cankers caused by Coryneum sp. were obser- with the exception of only two places, both ved mainly in Trás-os-Montes e Alto Douro in Beira Interior province. Furthermore, im- region and Cytospora sp. was found affec- portant portuguese ecological hotspots were ting young plants in São Mamede Natural also sampled during this survey, i.e., the Ser- Park in Alentejo region. ra de São Mamede Natural Park and Serra da Estrela Natural Park. These zones represent vulnerable ecosystems, which constitute a Discussion and Conclusions valuable ecological patrimony in Portugal. Chestnut stands in both areas were found to Portuguese chestnut stands monitored du- be extensively affected by chestnut blight. ring this work varied in structure and com- The results reported here constitute a pio- position. In Trás-os-Montes e Alto Douro, neer and baseline work from which several the most important chestnut area in Portu- other studies have been incorporated aiming gal, most chestnut areas form a continuous to: 1) Determine the diversity of vegetative surface of orchard stands. In Beira Interior, compatibility types and mating types of C. the type of chestnut stand is heterogeneous, parasitica in Portugal, as well as, the inci- with both small and extended orchards, high dence of natural hypovirulence (Bragança forest and coppice areas. Overall, the high et al. 2007). This information is required to forest and coppice stands where the disease evaluate the potential for biological control was observed were known to have suffe- of chestnut blight, following strategies im- 158 REVISTA DE CIÊNCIAS AGRÁRIAS plemented in other regions of the world, and do Castanheiro. In: Jornadas Transfronteiri- constitutes an essential step in order to block ças do Castanheiro. Bragança, 11 Maio. the dissemination of the disease; 2) Analyse Bragança, H.; Simões, S.; Santos, N.; Mar- the genetic diversity of C. parasitica in Por- tins, J.; Medeiros, A; Maia, A.; Sardinha, tugal mainland and archipelagoes to discern D.; Abreu, F.; Nunes, N. & Freitas. T. as to the phylogenetic history of C. parasiti- (2004) - Prospecção do cancro do casta- ca in the country; 3) Assess the presence of nheiro nas Regiões Autónomas dos Açores the sexual form of the pathogen, the perithe- e da Madeira. In: Sociedade Portuguesa de cia, elucidating about its reproductive stra- Fitopatologia Editor, Actas do 4º Congres- tegy in infected chestnut areas; 4) Ultimate- so da Sociedade Portuguesa de Fitopato- ly, to evaluate the biotic and abiotic factors logia. Universidade do Algarve, Faro, pp. which may be directly or indirectly involved 155-159. in the incidence of the disease. Bragança, H.; Simões, S.; Onofre, N.; Tenrei- Our results suggest that the current dis- ro, R. & Rigling D. (2007). Cryphonectria tribution and severity of chestnut blight in parasitica in Portugal - Diversity of vege- Portugal is of serious concern and that it is tative compatibility types, mating types, imperative to pursue the development of a and occurrence of hypovirulence. Forest holistic management strategy, capable of Pathology. 37: 391–402. blocking the spread of the disease into the Caetano, M.F. (1990) - O cancro do casta- last non-affected chestnut stands, and to op- nheiro – Uma ameaça já presente nos sou- timize the management of the currently in- tos e castinçais portugueses. Vida Rural 5: fected areas. 26-30. Câmara, M.S. (1929) - Micotes aliquot novi allique in mycoflora lusitaneae ignoti. Re- Ackowledgments vista Agronómica 17: 7. Campbell, W.A. (1949) - A method of iso- This research was supported by PIDDAC lating Phytophthora cinnamomi directly 204, PIDDAC 206 and AGRO 219 projects. from soil. Plant Disease Reporter 33: 134- We thank all the people (particularly from 135. Forest and Agricultural Services) who con- Fernandes, C.T. (1949) - Doenças do casta- tributed to this work. We also thank Rogério nheiro. A Endothia parasitica (Murr.) A. & Tenreiro and Daniel Rigling for suggestions A. Direcção Geral dos Serviços Florestais along this study and critical comments on the e Aquícolas, Alcobaça, 8 pp. manuscript. Maia, A. (2003) - Importância do Castanhei- ro – frutos e seus derivados. In: Jornal da Madeira - Boletim Agrícola 30 de Outubro, References pp. 4-5. Ormonde, C.J. (1994) - Contribuição para o Abreu, C. (1992) - A hipovirulência como estudo da cultura do castanheiro (Casta- forma de luta natural contra o cancro do nea sativa Miller) na Ilha Terceira (Aço- castanheiro. Revista das Ciências Agrárias res). Relatório de Estágio da Licenciatura 15: 167-171. em Engenharia Agrícola, Universidade dos Abreu, C. & Martins, M.L. (2002) - O homem Açores, Angra do Heroísmo, 143 pp. e o castanheiro: uma relação em crise. In: Robin, C.; Anziani, C. & Cortesi, P. (2000) XI Jornadas Luso-Galaicas de Ciencias e - Relationship between biological con- Desarrollo. Santiago de Compostela, pp. trol, incidence of hypovirulence, and di- 93-104. versity of vegetative compatibility types Anastácio, D.; Mesquita, M. & Ponteira,. D. of Cryphonectria parasitica in France. (2001) - Programa de Erradicação do Cancro Phytopathology 90: 730-737.