First report of Didymella macrostoma causing calyx-end rot of pear (Pyrus communis) in The Netherlands.

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 400,000 tons in 2020, and 'Conference' is the main pear cultivar that comprises 80% of total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage (-0.5°C) up to 11 months after harvest. Calyx-end rot incidences of 1% to 5% were observed on 'Conference' pears from different orchards in surveys from 2019-2021 in packing houses in the Netherlands. Infections showed 1 to 3 cm brown necrosis. Lesions were round, slightly sunken and next to or including part of the calyx. 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) (Oxoid, UK). The PDA plates were incubated at 20°C in the dark, and hyphal tip isolates were transferred to fresh PDA plates. Colonies on PDA were rosy-whitish to peach-colored. Colonies grown on oat meal agar (OA) under UV light were peach to red color, aerial mycelium sparse, and produced a pink to salmon colored conidial matrix. Conidia were irregular-ellipsoidal to allantoid, smooth, hyaline and usually with one or several gutulles. Conidia were sometimes one septate and measured 15.2±2.8 x 4.0±0.7 µm (n =14), but mostly aseptate and measured 7.9±1.7 x 3.2±0.6 µm (n =100). The fungus was morphologically identical to Didymella macrostoma (syn. Phoma macrostoma) (Boerema et al. 2004; Hou et al. 2020). The identity of four representative isolates, WURR-206, WURR-223, WURR-227 and WURR-308, from affected pears from four orchards in the Netherlands, was determined by 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, MA). Sequences of the internal transcribed spacer (ITS) region of ribosomal DNA, the large-subunit rRNA (LSU) region, partial sequences of beta-tubulin (TUB) and the translation elongation factor 1-alpha (TEF1) gene region were amplified with primers ITS1/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and EF1-983F/EF1-1567R (Rehner and Buckley 2005), respectively. Sequences were deposited under GenBank accession numbers ON077588-ON077591 (ITS), ON113487-ON113490 (LSU), ON098515-ON098518 (TUB) and ON098519-ON098522 (TEF1). MegaBLAST analysis revealed that the ITS, LSU, TUB sequences matched with 100% identity to culture collection sequences of Didymella macrostoma in GenBank MH854841 (ITS), MH866341 (LSU), MN983895 (TUB). The TEF1 sequences matched with 99.7% identity to TEF sequence of Didymella macrostoma MT454020. Subsequently, Koch's postulates were performed on 10 'Conference' pears per isolate (WURR-206, WURR-223, WURR-227 and WURR-308). Fruits wiped with 70% ethanol were inoculated in pathogenicity tests with an agar disk (5 mm diameter) of D. macrostoma prepared from the actively growing edge of 14-day-old cultures grown on PDA. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared 7-10 days after inoculation on all pears. PDA-only controls remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. The identity of the re-isolations was confirmed as D. macrostoma by sequencing, thus completing Koch's postulates. To the best of our knowledge, this is the first report of D. macrostoma causing calyx-end rot of pears. The identification of this causal agent is important knowledge necessary for developing control measures for postharvest diseases of pear.

First report of preharvest fruit rot of 'Pink Lady' apples caused by Colletotrichum fructicola in Italy.

In late summer 2019, a severe outbreak of fruit rot was observed in commercial 'Pink Lady' apple orchards (>20 ha in total) in the region Emilia-Romagna (Northern Italy). The symptoms on the fruit appeared as small circular red to brown lesions. Disease incidences of over 50% of the fruits were observed. To isolate the causal agent, 15 affected apples were collected and small portions of fruit flesh were excised from the lesion margin and placed on potato dextrose agar (PDA). The plates were incubated at 20°C in the dark, and pure cultures were obtained by transferring hyphal tips on PDA. The cultures showed light to dark gray, cottony mycelium, with the underside of the culture being brownish and becoming black with age. Conidia (n=20) were cylindrical, aseptate, hyaline, rounded at both ends, and 12.5 to 20.0 × 5.0 to 7.5 µm. The morphological characteristics were consistent with descriptions of Colletotrichum species of the C. gloeosporioides species complex, including C. fructicola (Weir et al. 2012). The identity of two representative isolates (PinkL2 & PinkL3) from different apples was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Molecular identification was conducted by sequencing the ITS1/ITS4 region and partial sequences of four other gene regions: chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and beta-tubulin (TUB). The sequences have been deposited in GenBank under accession numbers MT421924 & MT424894 (ITS), MT424612 & MT424613 (CHS), MT424616 & MT424617 (GAPDH), MT424614 & MT424615 (ACT), and MT424620 & MT424621 (TUB). MegaBLAST analysis revealed that our ITS sequences matched with 100% identity to Colletotrichum fructicola (Genbank JX010177). The CHS, GAPDH, ACT and TUB sequences of both isolates were 100% identical with C. fructicola culture collection sequences in Genbank (JX009807, JX009923, JX009436 and JX010400, respectively), confirming the identity of these isolates as C. fructicola. Koch's postulates were performed with 10 mature 'Pink Lady' apples. Surface sterilized fruit were inoculated with 20 µl of a suspension of 105 conidia ml-1 after wounding with a needle. The fruits were incubated at 20˚C at high relative humidity. Typical symptoms appeared within 4 days on all fruit. Mock-inoculated controls with sterile water remained symptomless. The fungus was reisolated and confirmed as C. fructicola by morphology and sequencing of all previously used genes. Until recently the reported causal agents of bitter rot of apple in Europe belong to the Colletotrichum acutatum species complex (Grammen et al. 2019). C. fructicola, belonging to C. gloeosporioides species complex, is known to cause bitter rot of apple in the USA, Korea, Brazil, and Uruguay (Kim et al. 2018; Velho et al. 2015). There is only one report of bitter rot associated with C. fructicola on apple in Europe (France) (Nodet et al. 2019). However, C. fructicola is also the potential agent of Glomerella leaf spot (GLS) of apple (Velho et al. 2015; 2019). To the best of our knowledge this is the first report of C. fructicola on apples in Italy. It is important to stress that the C. gloeosporioides species complex is still being resolved and new species on apple continue to be identified, e.g. C. chrysophilum that is very closely related to C. fructicola (Khodadadi et al. 2020). Given the risks of this pathogen the presence of C. fructicola in European apple orchards should be assessed and management strategies developed.

First report of Phytophthora chlamydospora causing postharvest fruit rot on apples and pears in the Netherlands.

Apple (Malus domestica) and pear (Pyrus communis) are important fruit crops in the Netherlands, with total production of 269,000 tons and 402,000 tons in 2018, respectively. In 2018 and 2019 postharvest fruit rots were observed on the apple variety Elstar (one observation) and pear varieties Conference and Doyenné du Comice (multiple observations). The symptoms were found after storage in controlled atmosphere storage facilities on fruits from different orchards across the Netherlands. Disease incidences up to 50% of the stored fruit were observed. The diseased fruits showed circular brown to black spots with irregular and diffuse margins that enlarged rapidly to form distinctive rings, typical of Phytophthora infection. Several Phytophthora species are currently known to cause fruit rot of pome fruit (Sanchez et al. 2019). To isolate the causal agent, small portions of fruit flesh from decayed fruit were excised from the lesion margin and placed on potato dextrose agar (PDA). The plates were incubated at 20°C in the dark, and pure cultures were obtained by transferring hyphal tips on PDA. The colonies were white with petaloid and rosette-shaped patterns. The isolates grown on PDA formed irregularly branched hyphae, produced persistent non-papillate sporangia, usually on unbranched sporangiophores and chlamydospores were produced. The characteristics were similar to those described for Phytophthora chlamydospora Brasier and Hansen sp. nov. (Hansen et al. 2015). The identity of three representative isolates (KP00219, WURR121 and WURR119) from two different pear cultivars (Conference and Doyenné) and one apple cultivar (Elstar), respectively, was confirmed by means of multilocus gene sequencing. Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Sequences of ITS region, COX and EF were amplified and sequenced. The sequences have been deposited in GenBank (Accession Nos. MT125889, MT125891, and MT125890 [ITS], MT153610, MT153612, and MT153611 [COX], MT153613, MT153615, and MT153614 [EF]. MegaBLAST analysis revealed that our ITS, COX and EF sequences matched with 100% identity to Phytophthora chlamydospora isolates in GenBank AF541901 and AF541902 (ITS), JF771548 and JF771549 (COX), JN936005 and JN936006 (EF). In order to perform Koch's postulates a pathogenicity assay was performed using mycelial plugs of the cultures KP00219, on pear cv. Conference, and WURR119 and WURR121, on apple cv. Elstar and pear cv. Doyenné du Comice. Ten apples and pears per cultivar were disinfected, and wounded using a sterile cork borer in the middle of the fruit surface area. A mycelial plug of a two weeks old fungal culture was then placed onto the fruit. Placement of a PDA plug without fungal growth was used as a control. The fruits were incubated at 18˚C at high relative humidity for 7 days. Symptoms appeared within 3 days on all fruits. Mock-inoculated controls remained symptomless. The fungus was reisolated and confirmed as P. chlamydospora by morphology and sequencing. P. chlamydospora is found in streams and wet soil worldwide, and has only rarely been recovered as a pathogen from ornamental and woody species (Blomquist et al. 2012; Ginetti et al. 2014; Türkölmez et al. 2016). To our knowledge, this is the first report confirming P. chlamydospora as a causal agent of fruit rot of commercially produced apple and pear cultivars in the Netherlands.

Pesticide Exposure of Residents Living Close to Agricultural Fields in the Netherlands: Protocol for an Observational Study.

BACKGROUND: Application of pesticides in the vicinity of homes has caused concern regarding possible health effects in residents living nearby. However, the high spatiotemporal variation of pesticide levels and lack of knowledge regarding the contribution of exposure routes greatly complicates exposure assessment approaches. OBJECTIVE: The objective of this paper was to describe the study protocol of a large exposure survey in the Netherlands assessing pesticide exposure of residents living close (<250 m) to agricultural fields; to better understand possible routes of exposure; to develop an integrative exposure model for residential exposure; and to describe lessons learned. METHODS: We performed an observational study involving residents living in the vicinity of agricultural fields and residents living more than 500 m away from any agricultural fields (control subjects). Residential exposures were measured both during a pesticide use period after a specific application and during the nonuse period for 7 and 2 days, respectively. We collected environmental samples (outdoor and indoor air, dust, and garden and field soils) and personal samples (urine and hand wipes). We also collected data on spraying applications as well as on home characteristics, participants' demographics, and food habits via questionnaires and diaries. Environmental samples were analyzed for 46 prioritized pesticides. Urine samples were analyzed for biomarkers of a subset of 5 pesticides. Alongside the field study, and by taking spray events and environmental data into account, we developed a modeling framework to estimate environmental exposure of residents to pesticides. RESULTS: Our study was conducted between 2016 and 2019. We assessed 96 homes and 192 participants, including 7 growers and 28 control subjects. We followed 14 pesticide applications, applying 20 active ingredients. We collected 4416 samples: 1018 air, 445 dust (224 vacuumed floor, 221 doormat), 265 soil (238 garden, 27 fields), 2485 urine, 112 hand wipes, and 91 tank mixtures. CONCLUSIONS: To our knowledge, this is the first study on residents' exposure to pesticides addressing all major nondietary exposure sources and routes (air, soil, dust). Our protocol provides insights on used sampling techniques, the wealth of data collected, developed methods, modeling framework, and lessons learned. Resources and data are open for future collaborations on this important topic. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/27883.