Tropical Plant Pathology (2020) 45:25–33 https://doi.org/10.1007/s40858-019-00314-y ORIGINAL ARTICLE Neonectria ditissima physiological traits and susceptibility of ‘Gala’ and ‘Eva’ detached apple fruit Jhulia Gelain1 & Silvio Andre Meirelles Alves2 & Rafaele Regina Moreira1 & Louise Larissa May De Mio1 Received: 7 February 2019 /Revised: 9 September 2019 /Accepted: 23 September 2019 /Published online: 31 January 2020 # Sociedade Brasileira de Fitopatologia 2019 Abstract European Canker and Neonectria fruit rot (NFR), caused by Neonectria ditissima, has become a major problem for the produc- tion of apples (Malus domestica) in Brazil. This study characterized environmental factors affecting in vitro growth and germi- nation of Neonectria ditissima as well as infection, colonization and reproduction, ex vivo, on ‘Gala’ and ‘Eva’ fruit. Temperatures between 17 to 20 °C were optimum for mycelial growth, and minimal growth occurred at 35 °C. Micro/ macroconidia sporulation ratio was lower at 10 °C for most of the isolates tested, and no macroconidia were produced at 30 °C. More than 70% of conidia germinated after 12 h of exposure to constant humidity incubation and a temperature of 25 °C. Germination rate as high as 90% occurred after 48 h regardless of the temperature. Incubation of 2 h with high humidity is not sufficient for germination of 10% of conidia at any temperature between 10 to 30 °C. The cultivars Eva and Gala did not differ in relation to incubation period. A minimum of 25 days was necessary for sporulation on fruit incubated at 16.8–21.7 °C. Unwounded fruit did not present symptoms even 40 days after inoculation on intact epidermis, demonstrating the need of wounds for NFR development. Keywords Cylindrocarpon heteronema . Malus domestica . Nectria galligena . Basic biology . Postharvest disease Introduction southernmost states Rio Grande do Sul and Santa Catarina in 2012, and five years later, the disease was detected in two The production of apples (Malus domestica Borkh) is municipalities of Paraná state. Currently, the pathogen is clas- constrained by several factors including fungal diseases that sified as an A2 quarantine pest in Brazil, which means that the are damaging to the crop. Among them, European Canker, organism is already present in the country but exhibits local- caused by Neonectria ditissima (Tul. & C. Tul.) Samuels & ized distribution and is targeted by an official control program Rossman (syn. Nectria galligena Bres), anamorph (Brasil 2013). Cylindrocarpon heteronema (Berk. & Broome) Wollenw., is N. ditissima causes canker-like lesions in twigs and trunks, a disease of worldwide occurrence and damaging to apple while in fruit it causes a symptom of soft rot, which may be production in the most important production regions of pre-harvest (in the orchard) or post-harvest (during storage) Europe, North America, Chile, Australia, New Zealand, (Xu and Robinson 2010). The disease can cause serious dam- Japan and South Africa (Beresford and Kim 2011). In age due to fruit rot in orchards with high inoculum on twigs Brazil, the disease was officially reported to occur in the and trunks. However, even though the infection of woody tissues has generally been well elucidated, few studies have investigated the infection of fruit by the fungus, and most of Section Editor: Juan Antonio A. Navas-Cortés the existing studies were performed by Swinburne in the 1970s (Brown and Swinburne 1971; Swinburne 1971, 1973, * Jhulia Gelain [email protected] 1974, 1975) using apple cultivars that are not grown in Brazil. In addition, studies on the behavior of the pathogen in fruit 1 under the climatic conditions of the southern region of Brazil Universidade Federal do Paraná, Setor de Ciências Agrárias, are lacking. In this region the disease occurs at more severe Curitiba, Paraná 80035-050, Brazil 2 levels when compared with other countries, probably due to Embrapa Uva e Vinho, Vacaria, Rio Grande do Sul 95200-000, the more favorable climatic conditions or to the greater Brazil 26 Trop. plant pathol. (2020) 45:25–33 amount of inoculum in orchards, in addition to a lack of ex- The objectives of the present study were to evaluate i) the perience in the disease management (Alves and Czermainski effect of temperature on mycelial growth and sporulation of 2015). N. ditissima, ii) the effect of temperature and incubation peri- The literature is inconsistent regarding the name of the od on the germination of N. ditissima conidia, and iii) the disease affecting fruit. When the symptoms occur during the susceptibility of wounded and unwounded ‘Eva’ and ‘Gala’ pre-harvest period, the disease is often called ‘eye rot’, ‘stylar- fruit to N. ditissima and the incubation and latent periods of end rot’, ‘dry eye rot’ or ‘calyx rot’, which are generic terms NFR on both cultivars. used for symptoms that are caused by many fungal pathogens in apple (Weber and Dralle 2013). Furthermore, during the post-harvest period, Brown and Swinburne (1971), Swinburne (1974), and Noble and Drisdale (1983) referred Material and methods to the disease as ‘Nectria galligena apple rot’;Xuand Robinson (2010) named the disease as ‘Nectria rot’.Sutton Isolates et al. (2014) used the term ‘Neonectria galligena fruit rot’ and Maxin et al. (2014) used the term ‘Neonectria rot’. In the Four isolates of N. ditissima were used in this study. These present study, the disease is referred to as ‘Neonectria fruit were collected in 2012 (Codes 32 and I8, from Embrapa col- rot’ (NFR), and we propose this name to be used as a reference lection, Genbank accession numbers MK564529 and for the disease. MK564532, respectively) and 2016 (Codes FRR01 and ‘Gala’ and its clones are widely cultivated in the world and EMB, Genbank accession numbers MK564531 and represent ~65% of apple production in Brazil, which was es- MK564530, respectively). They were isolated from symptom- timated at 1.30 million tons in 2017 (FAOSTAT 2014). atic twigs of apple trees cv. Gala from commercial and exper- Meanwhile, ‘Eva’ apple, which was developed by the imental orchards of the city of Vacaria, in Rio Grande do Sul Instituto Agronômico do Paraná (Iapar) as a cross between state, southern Brazil. ‘Anna’ and ‘Gala’, is being grown at a large scale in Paraná Twigs were cut into pieces of about 10 cm, and had their state, owing to its high productivity, low chill requirements, cortex removed with sterilized blade. The area of transition of and early production, when compared with ‘Gala’ and ‘Fuji’ the healthy and the darkened diseased tissue was cut into apples, and adaptation to the tropical and subtropical climates pieces of about 5 mm and these were disinfested by immersion that are predominant in Brazil (Hauagge and Tsuneta 1999). in 70% alcohol solution for 1 min, followed by a 3-min im- Another reason for the expansion of ‘Eva’ apple is the low mersion in sodium hypochlorite 1% solution and three rinses susceptibility of the cultivar to Glomerella leaf spot in sterilized distilled water. The pieces were then transferred to (Colletotrichum spp.), a yield-limiting disease of ‘Gala’ ap- Petri dishes containing water agar (WA) medium and incubat- ples grown at regions of tropical and subtropical climates. The ed at 20 °C in the dark. When colonies started to form, myce- cultivar is also gaining market due to its color, taste, and wide lial plugs were transferred to Petri dishes with potato dextrose acceptance by consumers (Oliveira et al. 2011). agar (PDA) medium and incubated for 15 days at 20 °C in the Even though previous studies have reported the suscepti- dark. Isolates were confirmed as pathogenic to wounded bility of ‘Gala’ apples to European Canker (Alves and Nunes ‘Gala’ apple fruit, as this is a known susceptible cultivar to 2017) and NFR (Xu and Robinson 2010), the monocyclic NFR (data not shown). After growth, the fungi were removed components of fruit rot in this cultivar are yet to be investigat- from the culture medium, preserved in 2-mL microtubes con- ed. For example, there is no information in the literature re- taining silica and kept at 4 °C. garding the susceptibility of ‘Eva’ fruit to the pathogen or regarding the pathogen’s incubation and latent periods. The infectivity-related physiological traits of the pathogen, Effect of temperature on mycelial growth such as mycelial growth, sporulation at different temperatures, and sporulation and production of macroconidia and microconidia (Weber 2014) have not been extensively studied in isolates from Mycelial growth was measured on PDA plates, with three Brazil. Those studies are important to better understand the replicates. The colony diameter was measured after the fungi behavior of the pathogen under different environmental con- had been incubated at different temperatures (7, 10, 15, 20, 25, ditions. Latorre et al. (2002)reportedthatthein vitro germi- 30 and 35 °C) for 14 days in Biochemical Oxygen Demand nation of the species’ conidia occurred at 6–32 °C, provided growth chambers (BOD) with 12-h photoperiod to assess ra- that sufficient moisture conditions were maintained, but no dial growth. A beta-generalized model (Bassanezzi et al., information about conidia germination of Brazilian isolates 1998) was fitted to mycelial growth data under different tem- at different temperatures and incubation periods are available. perature and is given by: Trop. plant pathol. (2020) 45:25–33 27 hi completely randomized factorial design consisting of 7 tem- b3ðÞTopt−Tmin − T Tmin b3 peratures and 9 constant humidity incubation periods with five t* Topt−Tmin Tmax−T YTðÞ¼Yop * ; replicates. The test was conducted twice. For the estimation of Tmax−Topt Tmax−Topt the equations the temperature data between 7 and 35 °C were used. The response surface graph was performed using the where, Yopt refers to the value of colony diameter (mm) at the SigmaPlot 11.0 software (Systat Software, Inc.).