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A Fast and Reliable Method for Diplodia Seriata Inoculation of Trunks And Phytopathologia Mediterranea Firenze University Press The international journal of the www.fupress.com/pm Mediterranean Phytopathological Union Research Papers A fast and reliable method for Diplodia seriata inoculation of trunks and assessment of Citation: Arrigoni E., Oliveira Longa C.M., Angeli D., Soini M., Pertot I., fungicide efficacy on potted apple plants under Perazzolli M. (2019) A fast and reliable method for Diplodia seriata inoculation greenhouse conditions of trunks and assessment of fungicide efficacy on potted apple plants under greenhouse conditions. Phytopatholo- gia Mediterranea 58(1): 163-173. doi: Elena ARRIGONI1,2, Claudia Maria OLIVEIRA LONGA1, Dario ANGE- 10.14601/Phytopathol_Mediterr-23764 LI1, Matteo SOINI1, Ilaria PERTOT1,3,#, Michele PERAZZOLLI1,#,* Accepted: December 6, 2018 1 Department of Sustainable Agro-ecosystems and Bioresources, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all’Adige, Italy Published: May 15, 2019 2 Department of Agricultural and Environmental Sciences, University of Udine, via delle Copyright: © 2019 Arrigoni E., Oliveira Scienze 206, 33100 Udine, Italy 3 Longa C.M., Angeli D., Soini M., Per- Center Agriculture Food Environment (C3A), University of Trento, via E. Mach 1, tot I., Perazzolli M. This is an open 38010 San Michele all’Adige, Italy access, peer-reviewed article published # These authors jointly supervised this work by Firenze University Press (http:// *Corresponding author: [email protected] www.fupress.com/pm) and distribuited under the terms of the Creative Com- mons Attribution License, which per- Abstract. Diplodia seriata is a polyphagous and widespread pathogen that infects mits unrestricted use, distribution, and trunks, shoots, fruit and leaves of apple plants. Fungicides applied in integrated pest reproduction in any medium, provided management programmes can act against D. seriata. However, introduction of scab- the original author and source are resistant apple cultivars and the consequent reduction in fungicide applications under credited. low input disease management may increase the incidence of disease caused by D. Data Availability Statement: All rel- seriata. Despite potential outbreaks of trunk canker, no fast and reproducible proto- evant data are within the paper and its cols for artificial inoculation of D. seriata and fungicide efficacy tests are available for Supporting Information files. apple plants. In this study, protocols for mycelium plug- and conidium suspension- inoculation of apple stems and shoots were optimised; canker disease assessments were Competing Interests: The Author(s) carried out on potted apple plants under greenhouse conditions and coupled with D. declare(s) no conflict of interest. seriata DNA quantification with quantitative PCR (qPCR). Efficacy tests of commercial Editor: Josep Armengol Forti, Universi- fungicides showed that captan, dithianon and fluazinam inhibited D. seriata mycelium dad Politécnica de Valencia, Spain. growth and conidium viability in vitro, while penconazole and ziram did not. However, dithianon spray applications did not reduce trunk canker severity and amount of D. seriata DNA in artificially inoculated plants under greenhouse conditions. This opti- mised protocol for fast and precise assessment of fungicide efficacy is suitable for fur- ther investigating the effects of other fungicides against D. seriata. Keywords. Botryosphaeria obtusa, Malus domestica, apple stem canker, Diplodia infec- tion protocol, qPCR quantification. INTRODUCTION Diplodia seriata (synonym Botryosphaeria obtusa) is a plant pathogen in the Botryosphaeriaceae (Ascomycota) that has been isolated from several Phytopathologia Mediterranea 58(1): 163-173, 2019 ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.14601/Phytopathol_Mediterr-23764 164 E. Arrigoni et alii different hosts worldwide (Phillips et al., 2007; Úrbez- Diplodia seriata black rot causes significant losses in Torres and Gubler, 2009; Mondello et al., 2017; Pou- organic orchards (up to 10% losses estimated in north- zoulet et al., 2017; Spagnolo et al., 2017). In particular, ern Germany), but damage is less frequent when inte- D. seriata causes trunk canker and shoot dieback, fruit grated pest management (IPM) is applied (Beer et al., black rot and leaf frog-eye spot on apple plants (Phil- 2015). Thus, it has been hypothesised that fungicides lips et al., 2007). Trunk canker is characterised by red- applied to control apple scab, flyspeck or sooty blotch dish brown lesions that each turn smoky and develop can also act against D. seriata (Brown-Rytlewski and series of alternate rings, rapidly becoming elliptical in McManus, 2000; Beer et al., 2015). Fungicides (benomyl, shape (Naqvi, 2007). The bark surface becomes rough kresoxim-methyl and trifloxystrobin) directly applied as and cracked with possible callus depositions around topical wound treatments reduced the incidence of D. the wounds and reddish brown stains on the underly- seriata trunk canker in apple trees (Brown-Rytlewski ing wood tissues (Naqvi, 2007). Diplodia seriata fruit and McManus, 2000), and fungicide sprays against apple infection (black rot) initially appears as small raised scab can control sooty blotch and flyspeck (Weber et al., purplish lesions on young fruits and develops into 2016). This is most probably related to side effects of fun- large, brown, firm lesions on ripening apples that may gicides applied against the main pathogen (i.e. Venturia later rot the entire fruit (Venkatasubbaiah et al., 1991). inaequalis), which can reduce the inoculum of some sec- Leaf infections (frog-eye leaf spot) initially appear as ondary pathogens. However, the introduction of scab- reddish-brown flecks that develop into circular brown resistant apple cultivars can reduce fungicide applica- lesions, often each surrounded by a purple halo, fol- tions compared with susceptible cultivars (Simon et al., lowed by leaf chlorosis and abscission (Venkatasub- 2011; Didelot et al., 2016), and the consequent reduction baiah et al., 1991). In addition, D. seriata is a polypha- in fungicide applications under low-input disease man- gous pathogen and causes pear (Choudhury et al., 2014), agements of scab-resistant apple cultivars may cause grapevine (Úrbez-Torres et al., 2008), olive (Kaliterna outbreaks of several secondary emerging pathogens, D. et al., 2012) and mulberry trunk canker (Arzanlou and seriata included (Ellis et al., 1998). An increasing risk Dokhanchi, 2013), olive (Moral et al., 2008) and loquat of canker outbreaks was also hypothesised due to cli- rot (Palou et al., 2013), and canker of ornamental plants mate change, which exposes plants to the risk of abiotic (e.g. Cotoneaster salicifolius) (Bobev et al., 2008) and stresses (Slippers and Wingfield, 2007), indicating that forest plants (e.g. Castanea sativa) (Dar and Rai, 2017). specific fungicide applications may be required to con- Trunk cankers and infected fruit left in orchards are the trol canker agents in the future (Ellis et al., 1998). Although the risk of emerging secondary pathogens main sources of pathogen inoculum (Beer et al., 2015), is real, no fast and reproducible protocols are available as well as infected tissues of other plant species in close to assess the efficacy of conventional spray applications proximity (Cloete et al., 2011). Thus, the removal of dis- against D. seriata canker under greenhouse conditions. eased shoots and fruit (Brown-Rytlewski and McManus, Most of the studies on D. seriata infection and disease 2000; Beer et al., 2015) and the use of resistant cultivars management have focused on apple black rot (Biggs and (Biggs and Miller, 2004) are recommended to limit the Miller, 2004; Beer et al., 2015) and grapevine trunk dis- spread of D. seriata. ease (Úrbez-Torres and Gubler, 2009; Pitt et al., 2012; Conidia of D. seriata spread from inoculum sourc- Mondello et al., 2017). Trunk canker diseases are rarely es to healthy trunks, shoots, fruit or leaves during rain studied due to the long incubation times and difficulties and penetrate these through natural openings, such as in reproducing infection under controlled conditions. wounds, lenticels and stomata (Naqvi, 2007). Trunk The aim of the present study was to optimise a fast and and shoot infections can occur mainly through pruning reliable protocol to inoculate and assess D. seriata on wounds in winter or summer, when trees are more vul- potted apple plants and to evaluate fungicide efficacy nerable to canker development due to possible drought against trunk canker, using conventional spray applica- stress (Brown-Rytlewski and McManus, 2000). However, tions under greenhouse conditions. Diplodia spp. have consistently been found at low levels in asymptomatic apple and pear bark, acting as potential sources of new infection (Arrigoni et al., 2018). Likewise, MATERIALS AND METHODS fungi in the Botryosphaeriaceae have been found as endophytes and latent pathogens in woody plants (Slip- Fungal isolates pers and Wingfield, 2007), and endophytic Botryospha- eriaceae can rapidly cause disease when their hosts are Two strains of D. seriata (S and VT) were isolated as under stress (Slippers and Wingfield, 2007). single conidium cultures from trunks of Malus domesti- Diplodia seriata inoculation of apple plants 165 ca plants with canker symptoms in the ‘Scurelle’ (lati- 15 g L-1 technical agar (PDB+A, Oxoid), and iv) 2.4 g L-1 tude, N46.0647636; longitude, E11.510519599999952; potato dextrose broth
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