An Evaluation of Diaporthe Nitschke (anamorph Sacc. Sacc.) Infecting Spinach (Spinacia oleracea L.) Seed

Agency Contact: Epidemiology and Risk Analysis Laboratory Science and Technology Plant Protection and Quarantine Animal and Plant Health Inspection Service United States Department of Agriculture 1730 Varsity Drive, Suite 300 Raleigh, NC 27606 August 7, 2019 Version 2

Diaporthe on spinach seed

Diaporthe (ana. Phomopsis) on spinach (Spinacia oleracea L.) seed The importation of seeds for planting into the United States is regulated under Title 7 of the Code of Federal Regulations (7 CFR § 319, 7 CFR § 360, and 7 CFR § 361). The movement of spinach seed in commerce is a pathway for the distribution of plant pests. A seed is defined as the mature plant ovule containing an embryo intended for planting (IPPC, 2017), and a pathway as “any means that allows the entry or spread of a pest” (IPPC, 2016). Pests associated with seed may be seed-borne (found on the seed or within the seed coat, but do not necessarily transfer from the seed to the resulting plant) or seed-transmitted (seed-borne pests that do transfer from the seed to the resulting plant) (IPPC, 2017). Since 2003, the United States has imported over 49 million kg of spinach seeds, with the majority coming from the Netherlands and Denmark (approximately 33 million and 7 million kg respectively) (PPQ, 2019).

Early detection of seed-borne fungi, such as Diaporthe and its anamorph Phomopsis, can help avoid the spread of these pests through commerce. Such spread could result in economic losses in some crops from crop damage, increased application of fungicides, and a greater agrochemical load entering the environment. However, infected seeds are often difficult to detect because 1) they can be asymptomatic, 2) the pathogens may have a low population density, and 3) the infected seeds may not be distributed uniformly within the shipment. Also, Plant Protection and Quarantine (PPQ) Identifiers are limited to visual (i.e., morphological) identification of seed- borne fungi at U.S. ports-of-entry. As a result, most identifications can go no further than the genus level. Currently, the genus Diaporthe is actionable, and a quarantine action, such as destruction, deportation, or treatment (if available) is required if the is detected. From 2017 to 2019, about 295,000 kg of S. oleracea seeds were imported into the United States. Upon port-of-entry inspection, about 90,000 kg (from Denmark, Germany, and the Netherlands) were re-exported or destroyed due to the presence of Diaporthe or Phomopsis (ARM, 2019). The seed industry believes the Diaporthe spp. found on spinach seed present a negligible phytosanitary risk (du Toit et al., 2016) and the resulting quarantine actions are unwarranted. However, PPQ has maintained a policy of taking action on these interceptions because Diaporthe spp. can only be identified to genus at U.S. ports-of-entry and this genus contains quarantine pests. The Plant Epidemiology and Risk Analysis Laboratory (PERAL) was asked to assess the Diaporthe species associated with spinach seed. This evaluation will help inform the decision to maintain or adjust the PPQ policy for Diaporthe spp. intercepted on spinach seed.

Diaporthe - Genus Level Synopsis The genus Diaporthe has recently undergone taxonomic revisions. Since the abolition of dual nomenclature for fungi (Dayarathne et al., 2016), the generic names Diaporthe and Phomopsis are no longer used to distinguish different reproductive stages of this genus. Rossman et al. (2015) recommend using Diaporthe rather than Phomopsis.

Diaporthe taxonomy is moving to a combination of morphological and molecular characterization based on multiple specimens for each species, type material, and multi-locus

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sequencing (Dissanayake et al., 2017). The Diaporthe and Phomopsis species names included in this analysis are the ones currently listed in the U.S. National Fungus Collections Fungal Database (Farr and Rossman, 2019). This includes more species names than are found in the genus circumscription by Dissanayake et al. (2017) to ensure that the genus is adequately represented.

Biology and Ecology Diaporthe spp. vary in life histories. Some species, such as D. ampelina (Erincik et al., 2001; Úrbez-Torres et al., 2013a) and D. helianthi (Mathew et al., 2015) are plant pathogens. Others behave as nonpathogenic endophytes (Ferreira et al., 2017; Gao et al., 2017; Gomes et al., 2013; Huang et al., 2015) or as saprobes on dead tissue (Hyde et al., 2016; Senanayake et al., 2017; Thambugala et al., 2017). Some species can behave as both endophytes and pathogens. For example, D. biguttulata can survive endophytically in Citrus spp. but causes cankers on Juglans regia (Huang et al., 2015; Yang et al., 2018). Other Diaporthe species, e.g. D. bougainvilleicola, can cause medical problems in humans (Ozawa et al., 2019; Thompson et al., 2013). Some Diaporthe species survive in host plant material or on plant debris (Almeida et al., 2001). For example, D. ambigua can live in dormant Vitis cuttings (Clarke et al., 2004), while other species can survive for years on seeds in cold storage (Raeisi et al., 2011).

Host Range Diaporthe species can infect a wide range of plant species including economically important crops and trees such as Glycine max (L.) Merr. (soybean), Helianthus annuus L. (sunflower), Cucumis melo L. (cantaloupe), Vitis vinifera L. (grapevine) and Juglans regia L. (walnut). They cause various symptoms in their hosts, such as dieback, cankers, blight, and fruit rots, which can cause significant yield losses (Baumgartner et al., 2013; Beraha and O'Brien, 1979; Carter, 1981; Fan et al., 2018; Gomes et al., 2013; Li et al., 2019; Santos et al., 2011; Udayanga et al., 2015; Úrbez-Torres et al., 2013a).

Association with Spinacia oleracea Diaporthe spp. are associated with imported S. oleracea seed. Pycnidia of these fungi have been found on both internationally and domestically produced seed (AQAS, 2019; du Toit et al., 2016). However, during pathogenicity testing, D. viticola, D. ganjae, D. melonis, and D. rudis did not cause symptoms when inoculated on S. oleracea leaves (du Toit et al., 2016). Infestation of seed with Diaporthe pycnidia did not significantly reduce germination (du Toit et al., 2016). We also examined peer-reviewed literature and found no reports of economic losses in S. oleracea associated with Diaporthe spp., or of Diaporthe spp. spreading from spinach seed to other hosts.

Pest Risk Assessments We examined the peer-reviewed literature, including reports by subject matter experts, on 476 Diaporthe and Phomopsis species for their presence in the United States and for their association with S. oleracea and seed. Based on that information and on port interception data, eight Diaporthe spp. are associated with spinach seed. Of these, six are non-quarantine pests, one is a

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quarantine pest for Puerto Rico and the U.S. Virgin Islands, and one is an undescribed species, which must be assessed to determine its quarantine status (Table 1).

Table 1. Diaporthe spp. associated with Spinacia oleracea; shaded rows indicate quarantine pests

Species Associated with Associated with In U.S. S. oleracea S. oleracea seed Nitschke du Toit et al., 2016 AQAS, 2019; du Baumgartner et al., (syn. Phomopsis cotoneastri Toit et al, 2016 2013; Farr and Punith., P. oblonga (Desm.) Rossman, 2019; Traverso, P. phaseoli Lawrence et al., (Desm.) Sacc.) 2015 Diaporthe ganjae (McPartl.) du Toit et al., 2016 du Toit et al, Farr and Rossman, R.R. Gomes, C. Glienke & 2016 2019; McPartland, Crous 1984 (syn. Phomopsis ganjae McPartland) Diaporthe melonis Beraha & du Toit et al., 2016 du Toit et al., Beraha and O'Brien, M.J. O'Brien 2016 1979; Carter, 1981; Farr and Rossman, 2019 Diaporthe rudis (Fr. : Fr.) du Toit et al., 2016 AQAS, 2017; du Farr and Rossman, Nitschke Toit et al, 2016 2019; KC and (syn. D. viticola Nitschke; Rasmussen, 2019; Phomopsis rudis (Fr. : Fr.) Úrbez-Torres et al., Höhn.) 2013b. Actionable for Puerto Rico, U.S. Virgin Islands Diaporthe sojae Lehman du Toit et al., 2016 AQAS, 2019; du Farr and Rossman, (syn. D. melonis var. Toit et al, 2016 2019; Gerdemann, brevistylospora Tak. 1954; Li et al., 2010; Kobayashi & Tak. Ohsawa, Udayanga et al., D. phaseolorum var. sojae 2015 (Lehman) Wehm.; Phomopsis sojae Lehman, P. glycines Petr.) Diaporthe vaccinii Shear du Toit et al., 2016 du Toit et al, Ames et al., 1988; (syn. Phomopsis vaccinii 2016 Farr et al., 2002; Shear) Farr and Rossman, 2019 Diaporthe vexans (Sacc. & Survase, 2016 Survase, 2016 Farr and Rossman, P. Syd.) Gratz. 2019 (syn. Phomopsis vexans (Sacc. & P. Syd.) Harter)

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Diaporthe sp. Kennedy, 2019 Kennedy, 2019 No evidence found 1. Diaporthe rudis on Spinacia oleracea seed imported into Puerto Rico, and the U.S. Virgin Islands.

Climatic Suitability Diaporthe rudis has the following geographic distribution: Africa: South Africa; Asia: Japan, Korea; Europe: Austria, France, Germany, Greece, Italy (including Sicily), Latvia, Netherlands, Poland, Portugal, Serbia, Spain (including the Canary Islands), Sweden, Switzerland, United Kingdom, Ukraine; North America: Canada, United States (10 states); Oceania: Australia, New Zealand; South America: Brazil, Chile (Díaz et al., 2017; Farr and Rossman, 2019; Torres et al., 2016; Udayanga et al., 2014). Based on this information, we estimate that this fungus could establish in areas corresponding to Plant Hardiness Zones 2-12 (USDA, 2012). This fungus is a quarantine pest for Puerto Rico and the U.S. Virgin Islands (PestID, 2017). Diaporthe rudis may be in Hawaii; however, the synonymies are unclear (Udayanga et al., 2014).

Potential Hosts Diaporthe rudis has been reported from multiple hosts: Acer, Asphodelus albus, Aucuba japonica, Brugmansia, Castanea, Corylus, Dipsacus fullonum, Epilobium, Eucalyptus, Fagus, Fraxinus, Glycine, Holcus, Hydrangea, Ileostylis, Laburnum, Lupinus, Malus, Persea, Protea, Pyrus, Rosa, Sambucus, Salix, Vaccinium and Vitis vinifera (Díaz et al., 2017; du Toit et al., 2016; Petrović et al., 2016; Torres et al., 2016; Udayanga et al., 2014; van Niekerk et al., 2005). Of the hosts listed here, only Lupinus contains species on the Endangered and Threatened Species lists (U.S. Fish and Wildlife Services, 2019). None of these species occur in Puerto Rico or the U.S. Virgin Islands (Kartesz, 2019).

Economically Important Hosts Hosts at risk in Puerto Rico are Persea americana Mill. (avocado), and Glycine max (L.) Merr. (soybean) (Petrović et al., 2016; Torres et al., 2016; Vilsack and Reilly, 2014). Persea americana is also at-risk host in the U.S. Virgin Islands (Clark, 2009). Although earlier reports list Citrus and other plant species as D. rudis hosts (Farr and Rossman, 2019), recent taxonomic research has identified the species infecting citrus as D. citri (H.S. Fawc.) F. A. Wolf (Udayanga et al., 2014).

Pest Potential on Economically Important Hosts This fungus appears to be an endophyte in S. oleracea, and did not cause symptoms on spinach in pathogenicity tests (du Toit et al., 2016). Diaporthe rudis has been reported to occasionally cause stem end rot and branch canker on P. americana as part of a complex with other Diaporthe spp. and often when plants were stressed (Eskalen and McDonald, 2010; Torres et al., 2016). One isolate of D. rudis was reported to cause seed decay in G. max (Petrović et al., 2016), although no further evidence of pathogenicity was found. Isolates of D. rudis were recovered from a kiwi (Actinidia chinensis) packinghouse survey at an incidence of less than 5.2% and caused post-harvest fruit rot in A. chinensis; however, these isolates were often found with other common post-harvest pathogens (Díaz et al., 2017). Diaporthe rudis is associated with Phomopsis cane and leaf spot in V. vinifera and is considered a minor pathogen on this host (van Niekerk et al., 2005). Diaporthe rudis did not cause stem cankers on V. vinifera when

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pathogenicity tests were done in the United States (du Toit et al., 2016). We found one report of D. rudis causing fruit rot of pear (Pyrus communis) in the continental United States (KC and Rasmussen, 2019); disease incidence was higher (30 percent) in culled fruit than in recently harvested fruit (5 percent).

Consequences of Introduction Diaporthe rudis is reportedly an endophyte on S. oleracea (du Toit et al, 2016). This species was also recovered from naturally infected (decayed) G. max seed (Petrović et al., 2016), from stem end rot (Torres et al., 2016) and stem cankers (Eskalen and McDonald, 2010) on P. americana, and from post-harvest fruit rot on A. chinensis (Díaz et al., 2017). Based upon this evidence, D. rudis may pose a risk to G. max and P. americana in Puerto Rico, and the U.S. Virgin Islands.

2. Undescribed, Intercepted Diaporthe species on S. oleracea seed imported into the United States Aaron H. Kennedy, a Molecular Biologist with the USDA-APHIS-PPQ National Identification Services (Kennedy, 2019) compared the phylogenetic position of the intercepted Diaporthe sp. to all other Diaporthe species with publicly available sequence data. He reported that the two closest relatives of the unknown Diaporthe were D. ganjae (McPartl.) R. R. Gomes, C. Glienke & Crous and D. compacta Y.H. Gao & L. Cai.

Diaporthe ganjae Diaporthe ganjae is native to the United States and was first identified from stems and leaves of Cannabis sativa (McPartland, 1983). Recent molecular studies showed that D. ganjae may be associated with fruit rot of Solanum melongena (eggplant) (Roze et al., 2017), and is an endophyte of Cinchona calisaya (quinine) twigs (Hidayat et al., 2016). We found no reports of this species causing symptoms on S. oleracea or V. vinifera, and pathogenicity tests have confirmed that it does not cause disease in those species (du Toit et al., 2016).

Diaporthe compacta Diaporthe compacta has been reported as an endophyte in healthy leaves of Camellia sinensis (tea) in China (Gao et al., 2016). Conclusions • Spinacia oleracea (spinach) seeds have been imported into the United States for decades. We found no evidence to suggest that these importations have resulted in any new diseases caused by Diaporthe spp. in spinach. We also found no evidence of Diaporthe spp. spreading from spinach to another host.

• The majority of Diaporthe species reported in the literature associated with S. oleracea seed are established in multiple states in the continental United States and do not meet the IPPC definition of a quarantine pest.

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• Diaporthe rudis may pose a risk to G. max and P. americana in Puerto Rico and the U.S. Virgin Islands. This fungus is a minor pathogen that can cause post-harvest fruit rot and canker in P. americana and seed decay in G. max. Diaporthe rudis is not a quarantine pest for the continental United States as it is widespread.

• The unknown Diaporthe sp. is most closely related to D. ganjae, which is native to the United States, and to D. compacta, which is an endophyte in healthy Camellia sinensis leaves in China.

• Our assessment shows that S. oleracea seed is a low risk pathway for the introduction of quarantine pests of the genus Diaporthe into the continental United States.

Authors Gary L. Cave, Ph.D., Senior Risk Analysta Nathan Miller, MS, NCSU-USDA Cooperatorb Sofia Pinzi, MS, NCSU-USDA Cooperatorb

aPlant Epidemiology and Risk Analysis Laboratory, USDA-APHIS-PPQ, Raleigh, NC bNorth Carolina State University, Department of Entomology and , Raleigh, NC

Acknowledgments: We thank Aaron H. Kennedy, Ph.D. (Molecular Biologist, National Identification Services, USDA-APHIS-PPQ), for conducting the Diaporthe sp. cladistic analysis.

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