HORTSCIENCE 53(10):1447–1452. 2018. https://doi.org/10.21273/HORTSCI13319-18 All the monitored V. pallidum accessions were highly susceptible (E. Babiker, unpub- lished data). In contrast, leaf rust symptoms Reaction of Different Vaccinium on cultivated blueberries was detected in the first 2 weeks of May 2017 and increases in Species to the Blueberry Leaf Rust leaf rust severity were associated with warm and humid weather (E. Babiker, unpublished Pathogen Thekopsora minima data). This suggest that the urediniospores of T. minima spread from infected leaves of Ebrahiem M. Babiker1, Stephen J. Stringer, Barbara J. Smith, V. pallidum to the newly emerging leaves of and Hamidou F. Sakhanokho southern highbush cultivars. United States Department of Agriculture, Agricultural Research Service, Development of disease-resistant culti- vars relies on the characterization and in- Thad Cochran Southern Horticulture Laboratory, 810 Highway 26W, corporation of genes for resistance. The Poplarville, MS 39470 native diploid Vaccinium species are impor- Additional index words. Vaccinium, Thekopsora minima, blueberry, rust resistance tant sources of adaptive traits. Southern highbush blueberry cultivars possess genes Abstract. Blueberry leaf rust caused by Thekopsora minima is a serious threat to introduced from several Vaccinium species, blueberry production. To investigate the host range and characterize new sources of including V. darrowii Camp, V. angustifo- resistance, 15 southern highbush accessions (Vaccinium corymbosum), two interspecific lium Ait., V. virgatum Ait., V. elliottii hybrids (V. elliottii 3 V. pallidum and V. corymbosum 3 V. pallidum), and accessions from Chapm., V. tenellum Ait., V. pallidum Ait., five diploid Vaccinium species were inoculated with an isolate of T. minima. Of 15, only V. myrsinites Lam., and V. stamineum Lam. two southern highbush accessions displayed resistance, whereas both accessions of V. (Brevis et al., 2008; Ehlenfeldt et al., 1995; arboreum displayed immunity against T. minima. Accessions of V. darrowii exhibited Hancock et al., 1995; Yousef et al., 2014). necrosis but with limited sporulation, indicating a high level of resistance. Sporulating Relatively little is known about the reaction lesions and brown spots were observed in accessions of V. elliottii and V. tenellum. Brown of commercial blueberry cultivars and native lesions, large pustules, and abundant sporulation were observed on V. pallidum diploid Vaccinium species to T. minima. accessions and their interspecific hybrids. As the lesions expanded, defoliation was To address this concern, this study was observed in V. pallidum accessions. When tested against rabbiteye (V. virgatum) and conducted to 1) determine whether T. minima southern highbush blueberries, urediniospores of T. minima from overwintering leaves of collected from overwintering leaves of V. V. pallidum were found to be virulent, suggesting that T. minima overwinters on V. pallidum is pathogenic on rabbiteye and pallidum. Based on symptoms and scanning electron microscopy (SEM) of uredinio- southern highbush blueberries; and 2) inves- spores, we hypothesize that V. elliottii, V. tenellum, V. pallidum, and V. corymbosum tigate the reactions of V. darrowii, V. elliottii, exhibit no host specificity to T. minima. V. tenellum, V. pallidum, V. arboreum, and southern highbush accessions to an isolate of T. minima collected from southern highbush The United States has more than 75,000 Georgia (Keith et al., 2008; Sato et al., 1993; blueberry. Information about host resistance acres of cultivated blueberries. One-third of Shands et al., 2018; Wiseman et al., 2016). In will help us to identify genes for resistance this production is in the Southeastern region, addition, leaf rust has been reported in many from different Vaccinium species. Genes for which is on track to be a major hub of U.S. countries, including South Africa, Mexico, resistance from the native diploid Vaccinium production within the next few years. Two Spain, Argentina, Australia, and China species could be used to develop disease- types of blueberries, rabbiteye (V. virgatum (Barrau et al., 2002; Dal Bello and Perello, resistant cultivars. Aiton. 2n =6x = 72) and southern highbush 1998; McTaggart et al., 2013; Mostert et al., (species complex between V. corymbosum L. 2010; Rebollar-Alviter et al., 2011; Zheng Materials and Methods 2n =4x = 48 and V. darrowii Camp 2n =2x = et al., 2017). T. minima is a heteroecious 24), are grown in the region. Leaf rust caused fungus requiring both primary and alternate Light and SEM. To examine the morpho- by the fungus Thekopsora minima P. Syd. & host plants to complete its life cycle logical characteristics, size, and shape of Syd, previously known as Pucciniastrum (Hiratsuka 1965). In the northern United urediniospores, small leaf segments bearing vaccinii (Pfister et al., 2004; Sato et al., States, the disease cycle begins in early urediniospores of T. minima were floated on 1993), infects blueberry leaves and causes summer when the windblown aeciospores glass slides, covered with coverslips, pressed defoliation throughout the season. This may spread from hemlocks (Tsuga spp.), an alter- gently, and examined with a bright-field light reduce plant vigor and lead to poor fruit nate host, to infect young blueberry leaves. In microscope at ·40 (Olympus, Center Valley, production. The incidence of the disease has the southeastern United States, the uredinio- PA). To verify the presence of urediniospores been increasing in the United States, where spores of T. minima are believed to survive and investigate the spore surface morphol- the pathogen has been reported in several the winter in a broad range of evergreen plant ogy, leaves of V. elliottii, V. tenellum, V. states, including Delaware, New York, species, including native Vaccinium species. pallidum, and V. corymbosum were collected Michigan, Oregon, Hawaii, California, and Since the alternate host is not present in the from naturally infected plants at Poplarville, southeastern states, further investigation is MS, and placed in a fixative solution consist- needed to identify the inoculum source in this ing of 2.5% glutaraldehyde and 2% para- Received for publication 20 June 2018. Accepted area. Several southern highbush cultivars formaldehyde buffer. The tissues were then for publication 13 Aug. 2018. retain mature leaves through the winter sea- treated with 1% osmium tetraoxide, dehy- This research was supported by the USDA-ARS son to support developing berries in spring drated in graded series of ethanol before project no. 6062-21000-010-00D. (Lyrene, 2005). However, most of the south- critical point drying, and examined using We are very grateful to Valerie Lynch-Holm at ern highbush cultivars flower in late winter FEI Quanta 200F SEM (FE-SEM; FEI Com- Washington State University’s Franceschi Micros- before the plant produces a new leaf canopy pany, Hillsboro, OR). copy and Imaging Center for her technical assis- to support developing berries (Lyrene, 2005). Pathogenicity of T. minima isolate from tance. 1Corresponding author. E-mail: Ebrahiem.Babiker@ In previous field scouting in 2017, we de- V. pallidum on cultivated blueberries. Ure- ars.usda.gov. tected leaf rust symptoms on overwintering diniospores of T. minima were collected from This is an open access article distributed under the leaves of the Blue Ridge blueberry (V. pustules on the lower leaf surface of V. CC BY-NC-ND license (http://creativecommons. pallidum), which is native to the southeastern pallidum accession B0339 using a vacuum org/licenses/by-nc-nd/4.0/). parts of the United States (Ballington, 2001). pump (Gamut, Chicago, IL). The spores were HORTSCIENCE VOL. 53(10) OCTOBER 2018 1447 collected into a gelatin capsule, diluted in per accession. Leaf rust severity was assessed a light mineral oil (Soltrol 170; Chevron 28 d after inoculation using the rating scale Phillips, The Woodlands, TX) to a concentra- described previously. tion of 3 · 105 spore/mL, and sprayed Statistical analysis. Analysis of variance homogenously with a portable air-pump was calculated using proc GLM in SAS 9.4 sprayer onto fully expanded leaves of two (SAS Institute, Cary, NC). The two experi- rabbiteye accessions (‘Tifblue’ and MS ments were conducted twice and means of 1408) and two southern highbush accessions disease rating displayed by different Vacci- [‘Suziblue’ (NeSmith, 2010) and MS 1425]. nium species were compared using Fisher’s The experiment was conducted in 2017 and protected least significant difference at P # 2018 and arranged in a complete randomized 0.05. block design with two replicates and four plants per accession. After inoculation, the Results leaves were allowed to dry for 60 min and Fig. 1. Urediniospores of Thekopsora minima with incubated for 48 h in a growth chamber Light and SEM. Light microscopy showed dense spinules collected from the lower leaf surface maintained at 70% relative humidity at that the urediniospores were obovate with an of Vaccinium pallidum. Bar represents 20 mm. 20 °C under 8 h of fluorescent lighting. An echinulate wall and measured 17.1–27.2 · ultrasonic humidifier was used to maintain 12.3–17.3 mm (Fig. 1). No teliospores were a high humidity level in the growth chamber. detected on the examined samples. FE-SEM After incubation, plants were moved to observation showed that the lower leaf sur- a growth chamber programmed for a 16-h face of V. pallidum was colonized by uredi- photoperiod at 22 °C/18 °C day/night tem- niospores of T. minima