M.E.C. Lemmers

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.

Fibulorhizoctonia psychrophila is the causal agent of lenticel spot on apple and pear fruit in the Netherlands

In a survey for postharvest diseases of apples and pears in the Netherlands, an unknown postharvest fruit rot was observed. The disease appeared to originate from infected lenticels. A fungus was consistently isolated from the decayed fruits. The fungal pathogen was isolated on potato dextrose agar, and at low temperatures development of a fast-growing whitish mycelium was observed. Growth of this fungus was observed between 1 and 20xa0°C with an optimum at 15xa0°C, while incubation of mycelium at 25xa0°C resulted in no growth. The isolates did not produce asexual or sexual spores. The isolates were characterized and identified by morphology and molecular phylogenetic analysis. Genomic DNA was isolated and amplified using ITS1-ITS4, EF1 and RPB2 primers, and BLAST searches in GenBank placed the fungus taxonomically in the genus Fibulorhizoctonia, with the highest matches to F. psychrophila. Pathogenicity of representative isolates from apple and pear fruit was confirmed under laboratory conditions. To the best of our knowledge this is the first report of F. psychrophila causing lenticel spot on apple and pear, and also the cause of a whitish mould on storage bins.

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.

Differences in infectivity and pathogenicity of two Plantago asiatica mosaic virus isolates in lilies

Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus in the family Alphaflexiviridae and has been isolated from a variety of host plants. In particular, PlAMV isolates from ornamental lilies (Lilium spp.) cause necrotic symptoms in these plants, which significantly reduces their commercial value. However, it has not been clear whether PlAMV isolates from other host plants differ in their infectivity and/or pathogenicity to ornamental lilies, and whether growth conditions affect infectivity and pathogenicity. In this study, we inoculated an edible lily species (Lilium leichtlinii) and seven varieties of ornamental lilies with two PlAMV isolates, an isolate from ornamental lily (PlAMV-OL) and an isolate from edible lily (PlAMV-Li1). We found that PlAMV-OL showed higher infection rates and exhibited necrotic symptoms more frequently in lilies than PlAMV-Li1. Moreover, we observed higher infection rates of PlAMV-OL in open field than in greenhouse, and higher rates of necrotic symptoms in autumn test than in spring test, suggesting that growth conditions and season affect infectivity and pathogenicity of PlAMV in lilies. Our study would provide important information for estimating the risk of necrotic disease caused by PlAMV, as well as for cultivation management preventing the occurrence of the disease.

Non-persistent TBV transmissions in correlation to aphid population dynamics in tulip flower bulbs

Virus transmission by aphids causes millions of Euro’s damage in the flower bulb sector annually. Mineral oils and pyrethroids are applied weekly during the growth season to decrease the virus transmission by aphids in flower bulbs. Currently, little is known about the dynamics of aphid populations during the growth season of tulips and the accompanying risk of the non-persistent virus transmission of for example Tulip breaking virus (TBV). Therefore, population dynamics of aphids in tulip fields was monitored during three growing seasons. Simultaneously, the period of TBV transmission by aphids was investigated experimentally by exposing small tulip plots during specified intervals to naturally occurring aphids. Finally, timing of virus spread was correlated with aphid population dynamics and weather conditions. In 2007, TBV transmission started in April while the first aphids were found in May. Apparently, the first aphids of the season already contributed in great extend to the virus transmission and virus transmission was observed very locally in the tulip plots. A second, distinct TBV isolate was identified which resulted in the design of an improved TBV detection assay. The main objective of this research project is to enhance the knowledge about the risk of non-persistent virus transmission in relation to aphid population dynamics. This knowledge should enable us to formulate crop protection guidelines that are better fine-tuned with the risk of virus transmission and thereby decreasing the environmental damage caused by chemical crop protection.