T.C. Hollinger

First Report of Colletotrichum godetiae Causing Bitter Rot on ‘Golden Delicious’ Apples in the Netherlands

Apple (Malus domestica) is an important fruit crop in the Netherlands, with a total production of 418,000 tons in 2011. Symptoms of apple bitter rot were observed on ‘Golden Delicious’ apples in the Netherlands in July 2013 after 9 months of storage in a packing house at controlled atmosphere. Lesions were round, 1 to 5 cm in diameter, gray and dry with acervuli, producing orange spore masses in concentric rings. Fruit were rinsed with sterile water, and lesions were sprayed with 70% ethanol until droplet runoff. The skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto Potato Dextrose Agar (PDA). The PDA plates were incubated at 20°C in the dark, and single-spore isolates were propagated on PDA. The isolates were identified as Colletotrichum sp. based on culture morphology, having light gray to pale orange mycelium and, when viewed from the reverse side, ranged from pink to reddish orange. The cultures carried yellowish spore masses and dark melanized structures similar to acervuli that oozed orange conidia. Conidia were cylindrical to fusiform, pointed at one or both ends, and measured 8.0 to 17.0 μm × 3.5 to 5.0 μm. Both cultural and morphological characteristics of the pathogen were similar to those described for C. acutatum, causal agent of bitter rot of apple. A representative isolate (PPO-44377) was used for multilocus gene sequencing (Damm et al. 2012). Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the KingFisher method (Waltham, USA) and six loci were amplified and sequenced. Primer pairs ACT-512F + ACT-783R, CHS-354R + CHS-79F, GDF1 + GDR1, CYLH3F + CYLH3R, BT2Fd + BT4R, and ITS1 and ITS4 (White et al. 1990) were used for amplification of parts of the actin (ACT), chitin synthase (CHS-1) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3), beta-tubulin (TUB2) and ITS region of the rDNA gene, respectively. One sequence for each locus has been deposited in GenBank under Accession Nos. KR180290 (ACT), KR180292 (CHS-1), KR180293 (GAPDH), KR180294 (HIS3), KR180295 (TUB2), and KR180296 (ITS). MegaBLAST analysis revealed that the ITS sequences matched with 98.9 to 100% identity to Colletotrichum spp. belonging to C. acutatum species complex (including C. godetiae). The phylogenetic trees constructed using sequences of ACT, CHS-1, GAPDH, HIS3, and TUB2 of our strain (PPO-44377), and available sequences from GenBank confirmed the identity of this strain as C. godetiae. Koch’s postulates were performed on 15 ‘Golden Delicious’ apples. Surface-sterilized fruit were inoculated with 20 μl of a spore suspension (105 conidia/ml) prepared from a 15-day-old PDA culture after wounding with a needle. Inoculated fruits were sealed in a plastic bag and were incubated in darkness at 20°C. Symptoms appeared after 4 to 6 days on 80% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA had morphological characteristics that resembled the original isolate from the infected apples. There are few reports of symptoms associated with C. godetiae on apple in Europe (Baroncelli et al. 2014; Ivic et al. 2013; Munda 2014). This is the first report of bitter rot caused by C. godetiae on apple fruit in the Netherlands. Currently, bitter rot is not an important disease in apples in the Netherlands. However, it is ranges worldwide and is considered one of the most important diseases, causing considerable crop losses, and may become an emerging problem in the Netherlands in the near future.

First Report of Neofabraea kienholzii Causing Bull’s Eye Rot on Pear (Pyrus communis) in the Netherlands

Pear (Pyrus communis L.) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014, and ‘Conference’ is the main cultivar. In the Netherlands, pears are kept in controlled atmosphere cold storage up to 11 months after harvest. Symptoms of bull’s eye rot were observed in 2015 on ‘Conference’ pears in storage in the Netherlands. Bull’s eye lesions on apple and pear fruits are generally caused by four Neofabraea species: N. alba Jacks, N. malicorticis Guthrie, N. perennans Kienholz, and N. kienholzii Seifert, Spotts & Levesque (Gariepy et al. 2005). N. alba is the major pathogen causing bull’s eye rot on pear fruits in the Netherlands. Independent of the species, the symptoms appear as flat or slightly sunken lesions, which are brown, often lighter brown in the center (Spotts et al. 2009). To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto potato dextrose agar (PDA). PDA plates were incubated at 20°C in the dark, and single spores were transferred to fresh PDA plates. The isolates produced colonies with white-yellowish to brownish mycelium. Microconidia were produced on feathery fascicles of aerial mycelium, with a white, powdery, or sugary appearance on the surface of the agar colony. Microconidia were 2.5 to 6.5 × 1.5 to 2.5 µm, ellipsoidal, slightly asymmetrical to a curved form. The identity of a representative isolate (PPO 45010) was confirmed by means of multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin) in combination with the Kingfisher method (Waltham, MA). Segments of the internal transcribed spacer region (ITS), 28S ribosomal RNA (28S rRNA) and beta-tubulin (TUB2) loci were amplified, sequenced with primers ITS1/ITS4, LR0R/LR5, and Btub2Fd/Btub4Rd (Chen et al. 2016), and deposited in GenBank under accession nos. KX424942 (ITS), KX424941 (28S rRNA), and KX424940 (TUB2). MegaBLAST analysis revealed that the ITS, 28S rRNA, and TUB2 sequences matched with 99 to 100% identity to N. kienholzii isolates in GenBank (KR859082 and KR859083 [ITS], KR858873 and KR858874 [28S rRNA], KR859288 and KR859289 [TUB2]). Alcohol surface sterilized fruits were inoculated in pathogenicity tests in two ways: (i) with an agar disk (10 mm diameter) with actively growing mycelium of N. kienholzii prepared from a 14-day-old culture grown on PDA; and (ii) with 20 μl of a spore suspension (105 conidia ml-1) prepared from a 21-day-old PDA culture after wounding with a needle. Both experiments were performed on 10 ‘Conference’ pears. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared between 7 and 14 days. Mock-inoculated controls with water and PDA-only controls remained symptomless. Fungi isolated from the lesions had morphological characteristics that resembled the original isolates from infected pears. The identity of these isolates was confirmed as N. kienholzii by sequencing, thus completing Koch’s postulates. Bull’s eye rot of apple and pear is an important postharvest disease, occurring in major fruit growing areas of North America, Chile, Australia, and Europe (Henriquez et al. 2004; Spotts et al. 2009). N. kienholzii was reported twice on apple in Europe (Michalecka et al. 2016). To the best of our knowledge, this is the first report of N. kienholzii causing bull’s eye rot of pear in Europe.

First Report of Truncatella angustata Causing Postharvest Rot on ‘Topaz’ Apples in the Netherlands

In the Netherlands, about 30% of the organic apple (Malus domestica Borkh.) production consists of apple scab resistant cultivars, such as Topaz and Santana. However, organic ‘Topaz’ apples show a high incidence of fungal rot after storage. Hot-water treatment (HWT) of freshly harvested apple fruit prior to long-term storage is an important strategy for the control of postharvest diseases, especially in the organic production sector (Maxin et al. 2012). The recommended treatment temperatures and times vary according to the cultivar because of the risk of heat damage to the fruit peel. In January 2016, light peel damage caused by HWT was observed on ‘Topaz’ apples from an organic orchard. Also, up to 15% of the ‘Topaz’ apples showed typical rot lesions of an unknown causal agent. The lesions showed brown, irregular necrosis and were slightly sunken. To isolate the causal agent, fruits were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were transferred to fresh PDA plates. The colonies that appeared on PDA were cottony to woolly, dull white to brown in color, with black acervuli mainly in the center of the PDA plates. The isolates produced four-celled conidia, 16 to 19 × 7 to 9 µm, straight to slightly curved, with two brown to dark-brown median cells that had thick walls. More than one hyaline apical appendage, variable in size and branched dichotomically, were observed and a basal appendage was absent. The fungus was morphologically identical to Truncatella angustata (Pers.) S. Hughes (Sutton 1980). The identity of two representative isolates (PPO-45246 and PPO-45321) was confirmed by means of gene sequencing. To this end, DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, MA). Sequences of the ITS region were amplified using primers ITS1/ITS4, sequenced, and deposited in GenBank under accession numbers KX085227 and KX085228. MegaBLAST analysis revealed that both of our ITS sequences matched 99% with T. angustata isolates in GenBank (EU342216, JX390614, and KF646105). Koch’s postulates were fulfilled using 10 ‘Topaz’ apples. Surface sterilized fruits were inoculated with 20 μl of 10 5 conidiospores ml –1 in water, prepared from a 15-day-old PDA culture of the isolate PPO-45246, after wounding with a needle. Inoculated fruits were sealed in a plastic bag and incubated in darkness at 20°C. Symptoms appeared after 7 days on 100% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the inoculated isolates. The identity of the reisolations was confirmed as T. angustata by sequencing. T. angustata has a worldwide distribution and has also been reported to cause leaf spot on Rosa canina (Eken et al. 2009), canker and twig dieback on blueberry (Vaccinium spp.) (Espinoza et al. 2008), and fruit rot of olive (Olea europaea) (Arzanlou et al. 2012). To the best of our knowledge, this is the first report of T. angustata causing fruit rot of apples. Importantly, we note that the occurrence of this fruit rot may be enhanced by wounding, in this case as a result of hot water treatment.

First Report of Cadophora luteo-olivacea Causing Side Rot on ‘Conference’ Pears in the Netherlands

Pear (Pyrus communis) is an important fruit crop in the Netherlands. Symptoms of side rot disease of pear fruits were first observed in 2008 on cv. Conference in storage in the Netherlands. Typical round to oval, dark-brown, and slightly sunken spots (size 0.5 to 1.0 cm in diameter) appeared after six or more months of cold storage under controlled atmosphere. Lesions of rinsed pears were sprayed with 70% ethanol and tissue under the lesion was placed onto potato dextrose agar (PDA) at 20°C in the dark. Colonies obtained from single spores produced on PDA were flat, felty and cottony in the middle, with smooth margins, an even edge, and varying in color from white turning to gray/black-olivaceous. Under UV light, ellipsoid or elongate conidia were produced (2.2 to 2.3 × 4.9 to 6.5 µm). Both cultural and morphological characteristics of the pathogen were similar to those described for Cadophora sp. (Spadaro et al. 2011). Three representative isolates (PPO 11-1228, PPO 24-1234, and PPO 107-1267) were sequenced using primers ITS1/ITS4 and EF1-728F and EF1-986R (Carbone and Kohn 1999). MegaBLAST analysis revealed that the ITS sequences (GenBank accession nos. KT350591, KT350592, and KT350593) matched with 99.8 to 100% identity to Cadophora luteo-olivacea in GenBank (KU141394 and KU141395). The TEF1 sequences (KT350597, KT350598, and KT350599) were 100% identical with many other culture collection C. luteo-olivacea sequences in GenBank (HQ661071 and KF764576) and only 71 to 80% to other Cadophora species isolated from pear (KT350601 and KT350602). Alcohol surface sterilized fruits were inoculated in pathogenicity tests in two ways: i) with an agar disk (10 mm diameter) with actively growing mycelium of C. luteo-olivacea prepared from a 14-day-old culture grown on PDA (isolates PPO 11-1228, PPO 24-1234, and PPO 107-1267); and ii) with 20 µl of a spore suspension (105 conidia ml–1) prepared from a 21-day-old PDA culture after wounding with a needle (isolates PPO 11-1228 and PPO 107-1267). Both experiments were performed at 5 and 15°C, on 10 ‘Conference’ pears per isolate-temperature combination. Inoculated fruits were sealed in plastic bags and were incubated in darkness. Typical symptoms appeared 7 to 14 days and 4 to 6 weeks later, for fruits incubated at 15 and 5°C, respectively. Mock-inoculated controls with water and PDA-only controls remained symptomless. Fungi isolated from the lesions had morphological characteristics that resembled the original isolates from infected pears. The identity of the reisolations was confirmed as C. luteo-olivacea by sequencing, thus completing Koch’s postulates. Side rot of long-term stored pears has first been reported in Oregon, United States (Bertrand et al. 1977). The primary causal fungus was identified as C. malorum (syn. Phialophora malorum) (Sugar and Spotts 1992). Recently, a skin pitting disease of kiwifruit caused by C. luteo-olivacea has been reported from Italy (Spadaro et al. 2010). To our knowledge, this is the first report of side rot disease of pear fruits caused by C. luteo-olivacea.

First Report of Neonectria candida Causing Postharvest Decay on ‘Conference’ Pears in the Netherlands

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014, and ‘Conference’ is the main pear cultivar that occupies 75% of the total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage up to 11 months after harvest. Occasionally, storage rots are observed when storage crates are contaminated with orchard soil. In a storage trial (2012 to 2013), boxes with ‘Conference’ pears were amended with soil particles from the same orchard from which the pears were harvested (four orchards), and stored for 11 months. Boxes without amended soil were included as controls. In contrast to the control boxes, up to 15% of the pears stored in boxes with soil particles showed typical rot symptoms (lesions) of an unknown causal agent. The lesions showed brown and watery circular necrosis, were slightly sunken, and displayed whitish to yellowish mycelia covering the lesions. To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were transferred to fresh PDA plates. These isolates produced fast-growing colonies with white-yellowish mycelium. Conidia were hyaline, cylindrical, 1 to 3 septate, and 15.8 to 26.4 × 5.3 to 7.9 µm. The fungus was morphologically identical to Neonectria candida (syn. N. ramulariae; anamorph Cylindrocarpon obtusiusculum) (Lombard et al. 2015). The identity of a representative isolate (VTN10Bs3) was confirmed by means of multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Sequences of the ITS region, translation elongation factor 1-alpha (TEF1), and actin (ACT2) loci were amplified, sequenced, and deposited in GenBank under accessions KU588183 (ITS), KU588186 (TEF1), and KU588184 (ACT2). MegaBLAST analysis revealed that our ITS, TEF1, and ACT2 sequences matched with >99 to 100% identity to N. candida isolates in GenBank (KM249079 and JF735314 [ITS], JF735791 and HM054091 [TEF1], and KM231146 [ACT2]). Subsequently, Koch’s postulates were performed on 15 ‘Conference’ pears. Surface sterilized fruits were inoculated with 20 µl of a suspension of 105 conidiospores ml–1 water, prepared from a 15-day-old PDA culture, after wounding with a needle. Inoculated fruits were sealed in a plastic bag and incubated in darkness at 20°C. Symptoms appeared after 7 days on 100% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. Moreover, symptoms observed on artificially inoculated ‘Conference’ pear fruit were identical to the decay observed on ‘Conference’ pears that were obtained from the cold storage experiment. The identity of the reisolations was confirmed as N. candida by sequencing. N. candida (syn. N. ramulariae) is known as a globally distributed soilborne fungus (Domsch et al. 2007), but only few studies have identified the fungus as plant pathogen (Hirooka 2012). This is the first report of N. candida causing storage rot of pears. Importantly, we note that the occurrence of storage rots may be enhanced by contamination of storage crates or fruit with orchard soil.