Phaeoacremonium Species Associated with Olive Wilt and Decline in Southern Italy

Eur J Plant Pathol (2015) 141:717–729 DOI 10.1007/s10658-014-0573-8 Phaeoacremonium species associated with olive wilt and decline in southern Italy Antonia Carlucci & F. Lops & F. Cibelli & M. L. Raimondo Accepted: 7 December 2014 /Published online: 20 December 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Six Phaeoacremonium species (spp.) were iso- Keywords Phaeoacremonium spp . Actin . β-tubulin . lated from symptomatic wood of olive trees (Olea Decline . Olive europea) in Apulia (southern Italy) that showed crown wilt and twig and branch dieback. These Phaeoacremonium spp. were identified according to their morphological char- Introduction acteristics and by analyses of partial sequences of the actin and β-tubulin genes. Combining these cultural, morpho- In historical times, the olive tree (Olea europea L.) logical and molecular data, three Phaeoacremonium spp. spread from its native Asia Minor, from the Paleolithic were isolated that are already known to be responsible for period (Salavert 2008) through the early Neolithic peri- severe decline of olive in Apulia, Phaeoacremonium od (Galili et al. 1997). Olive cultivation is considered to aleophilum, Phaeoacremonium alvesii and have then begun during the Chalcolithic period (Neef Phaeoacremonium parasiticum, together with three other 1990; Zohary and Spiegel-Roy 1975). Studies conduct- Phaeoacremonium spp. that are associated for the first ed by Van Zeist (1980) suggested that olive cultivation time with wilt, decline and dieback of olive orchards in gradually moved from the east to the west until it Italy and worldwide: Phaeoacremonium italicum, reached the Mediterranean area, and was taken to Phaeoacremonium sicilianum and Phaeoacremonium Greece around 2500 BC by the Phoenicians. scolyti. To understand and to confirm their involvement Subsequently, during the Roman Empire, the Romans in wilt and decline of olive trees, pathogenicity assays promoted the diffusion of the olive tree into some re- were performed on shoots of young olive plants. The data gions of southern Italy, including Apulia (Terral et al. indicate that all six of these Phaeoacremonium spp. can 2004). After the discovery of the Americas, the spread cause discolouration, necrotic wood, and death of shoots, of olive cultivation continued into Peru, Argentina, although different levels of virulence were observed, with Chile and Uruguay, and northwards to the coastal re- Pm. italicum, Pm. aleophilum and Pm. sicilianum pro- gions of Mexico and to the United States, where it found ducing greater necrotic lesions than the other an ideal environment in southern California. As the Phaeoacremonium spp. investigated here. olive fruit and oil have been always more appreciated and as their consumption has increased, olive cultivation became more widespread in South Africa and along the : : : coast of Australia, where it was introduced by Italian A. Carlucci (*) F. Lops F. Cibelli M. L. Raimondo and Greek immigrations, and also in New Zealand and Dipartimento di Scienze Agrarie, degli Alimenti e China. Hence, the olive tree is of major economic im- dell’Ambiente, Università degli Studi di Foggia, Via Napoli, 25, 71122 Foggia, Italy portance in many countries. The European Union is the e-mail: [email protected] leading world producer of olive oil and drupes, while 718 Eur J Plant Pathol (2015) 141:717–729 also being the leading consumer of about 70 % of the by Rhouma et al. (2010) in Tunisia. Botryosphaeriaceae world produce (Food and Agriculture Organisation of spp. have been reported to be associated with olive branch the United Nations 2013). dieback, canker and decline, although they are primarily Agosteo (2011) investigated olive diseases through a involved in olive fruit rot (Chattaoui et al. 2011;Moral historical account, and reported that in De Res Rustica, et al. 2008; Phillips et al. 2005). The main Columella (4–70 D.C.) described the olive as a frugal, Botryosphaeriaceae spp. from olive wood are: resistant and imperishable plant. This account further Botryosphaeria dothidea, Diplodia mutila, D. seriata, indicated that when left to itself, as often happened over Dothiorella iberica, Neofusicoccum mediteraneum,and the centuries, the olive tree was able to survive, and that Lasiodiplodia theobromae in California (Moral et al. with the use of charcoal burners at the base of the trunk, 2010; Úrbez-Torres et al. 2013); Botryosphaeria ribis in it could returned to its spontaneous state, as with the Spain (Romero et al. 2005); D. seriata in Croatia wild olive in the Mediterranean woodland, where it (Kaliterna et al. 2012); and D. mutila, L. theobromae and emulated oleaster and was found together with oak, Neofusicoccum parvum in Italy (Carlucci et al. 2013b). lentisk, broom and rosemary. During this period, agri- Another fungus, Pleurostomophora richardsiae,was cultural problems occurred with olive cultivation, such also recently associated with brown wood streaking and as wilting, loss of flowers, and lack of fruit production, canker of olive trees in Italy for the first time (Carlucci et al. although these were initially not investigated. In Italy in 2013b). Recently, a severe decline of olives occurred in 1936 (Agosteo 2011), three reasons were given regard- Italy characterized by a rapid dieback of shoots, twigs and ing the agricultural problems of olive plants: the cultural branches, and dark yellowing and browning of the leaf tips lack of care of the olive orchards; their spontaneous that was followed by death of the olive plant. In southern origin; and the long age and size of the trees. The age Italy, Nigro et al. (2013) isolated the following from symp- and the perpetual lack of care had made the olive tree a tomatic tissues: Phaeomoniella chlamydospora, plant in a constant state of disease. Phaeoacremonium aleophilum, Phaeoacremonium Investigations into plant pathology to identify the alvesii, Phaeoacremonium parasiticum and causal agents of olive diseases began to take their first Phaeoacremonium rubrigenum. The last three of these steps as an independent science only in the second half fungi were reported for the first time from olives in Italy. of the 1800’s, when it was necessary to find a remedy to The aim of the present study was to identify and protect the agricultural yields. Although olive trees were characterise the fungi associated with vascular considered to be very resistant to environment injuries, discolouration observed in xylem tissues of declining including biotic agents, several biotic entities were able olive plants in Apulia, and to use pathogenicity tests to to damage the healthy olive tree. The main fungal dis- determine if they are responsible for olive decline. eases that affected olives included: leaf diseases such as peacock spot, caused by Spilocaea oleaginaea;infec- tious leaf burn, caused by Martamyces panizzei (De Materials and methods Not.) Minter; cercospora leaf spot, caused by Pseudocercospora cladosporioides (Sacc.) (Braun Fungal isolations 1993); and vascular diseases such as tracheomycosis, caused by Verticillium spp.; and anthracnose, caused by During a field survey carried out from March 2012 to Colletotrichum species (spp.) and Botryosphaeriaceae December 2013 across several olive orchards of the spp. (Talhinhas et al. 2005; Moral et al. 2008). Foggia province in southern Italy, generalised wilt and More recently, decline, dieback and canker diseases decline symptoms were seen, and a large number of on olives have acquired great importance worldwide. In samples from the base of the trunk, and the trunks and the literature, different fungal species have been branches were collected. As the olive orchards visited associated with olive decline and dieback. Rumbos were of different ages (from 24 to over 100 years old), (1988, 1993)reportedonEutypa lata and Cytospora the samples were distinguished on the basis of age of the oleina in Greece, and Tosi and Natalini (2009)andTosi trees, while no distinction was made regarding the cul- and Zazzerini (1994)reportedonPhoma incompta and tivar. The samples were collected from a total of 62 E. lata in Italy. Phoma incompta was also associated symptomatic olive plants (33 plants over 50 years old, with olive dieback by Ivic et al. (2010)inCroatia,and and 29 plants under 50 years old), which showed Eur J Plant Pathol (2015) 141:717–729 719 external and internal symptoms on the olive trunks, necrotic areas with brown discolouration (in cross sec- branches and stems: first, wilting and dieback of entire tion) (Fig. 1). and/or part of the crown, with depressed browning areas The samples were transported into the laboratory for and brown streaking under the bark of the trunk and analysis and were subjected to surface sterilisation ac- branches (in longitudinal section); and second, extended cording to Fisher et al. (1992). Small pieces (2–5mm) Fig. 1 Disease symptoms observed on olive trees. a Generalised decline and die-back occurred on the tree. b Wiltandbrowningofleaves. c–d Discolouration of wood evolving in cankers. e–f Wilting of shoots after artificial inoculation caused from Pm. sicilianum 720 Eur J Plant Pathol (2015) 141:717–729 from discoloured wood of xylematic areas of the base of Amplification and sequencing the trunk, the trunk and the branches were placed on 2 % malt extract (Oxoid Ltd., UK) with 2 % agar (Difco, A fragment of 600 bp of the β-tubulin (TUB)genewas USA), supplemented with 300 mg/l streptomycin sul- amplified using the primers T1 (O’Donnell and Cigelnik phate (Sigma-Aldrich, USA) (MEAS), and incubated at 1997) and Bt2b (Glass and Donaldson 1995), and a 23 °C (±2 °C) in the dark. fragment of 300 bp of the actin (ACT) gene was ampli- All of the fungal colonies isolated were grown until fied using the primers ACT-512F and ACT-783R they sporulated, and then a conidial suspension was (Carbone and Kohn 1999) for 40 isolates as representa- spread onto agar plates. After 24 to 36 h of incubation, tive of the MSP-PCR profiles. The PCR reactions were single germinating conidia were transferred to fresh performed according to Raimondo et al.

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