Botany Screening of lupine germplasm for resistance against Phytophthora sojae Journal: Botany Manuscript ID cjb-2019-0163.R1 Manuscript Type: Article Date Submitted by the 22-Jan-2020 Author: Complete List of Authors: Beligala , Gayathri ; Bowling Green State University, Biological Sciences Michaels , Helen ; Bowling Green State University, Biological Sciences Phuntumart, Vipaporn; Bowling Green State University, Biological Sciences Draft <i>Phytophthora sojae</i>, <i>Lupinus </i>sp., Soybean, Keyword: Pathogenicity Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 36 Botany Screening of lupine germplasm for resistance against Phytophthora sojae Gayathri U. Beligala, Helen J. Michaels and Vipaporn Phuntumart* Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403 Gayathri U. Beligala. [email protected] Helen J. Michaels. [email protected] Vipaporn Phuntumart. [email protected] * Corresponding Author Dr. Vipaporn Phuntumart Draft Department of Biological Sciences 129 Life Sciences Building Bowling Green State University Bowling Green, OH 43403 Tel.: 419 372-4097 Fax: 419 372-2024 1 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 36 Abstract Phytophthora sojae is a major pathogen in cultivated soybeans world-wide. Although incorporating resistance genes has been an effective management tool for soybean breeders, surveys of soybean fields in the Midwest US indicate that some P. sojae strains are capable of overcoming all known resistance genes. While P. sojae is known to have a very narrow host range, it can also infect Lupinus (lupine), varieties of which may provide potential sources for novelDraft resistance genes that can be genetically engineered into soybean. The chemotactic behavior of zoospores and pathogenicity of P. sojae strain P6497 towards 17 lupine lines were explored. The two soybean varieties Williams and Williams 82 that are susceptible and resistant against P. sojae P6497, respectively, were used as controls. Chemotaxis assays showed that there was no coherent pattern between the number of zoospores colonizing the root surface and plant tolerance or resistance to phytophthora root rot. Pathogenicity tests identified two of 17 lupine lines tested (LAB 18 and LL 35) were resistant to P. sojae infection. Phylogenetic analysis of these two resistant lupine lines with Old World lupines of the Mediterranean and North African regions, and New World lupines of America, indicated that they originated from the Old World. Keywords: Phytophthora sojae, Lupinus sp., Soybean, Pathogenicity 2 https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 36 Botany Introduction Draft Oomycetes are a distinct group of eukaryotic, fungus-like filamentous microorganisms belonging to Kingdom Stramenopila (Thines 2014). Phytophthora is a genus of destructive plant-damaging oomycetes (Tyler et al. 2006; Kamoun et al. 2015). First identified in North America in the 1950s (Kaufmann and Gerdemann 1958), Phytophthora sojae is the causal agent of root and stem rot in soybean (Glycine max L.) leading to yield reductions of approximately 35 million bushels in 28 soybean-producing states in the US and Ontario, Canada in 2014 (Allen et al. 2017). Infection by P. sojae primarily occurs at the pre- emergence- and seedling stage of soybeans (Schmitthenner 1985; Dorrance et al. 2007; Tyler 2007). Chemical control of oomycete pathogens, including P. sojae, is challenging because of difficulties in treating the affected underground parts of the plant (Tyler 2007). Hence, breeding for resistance is the most economic and effective tool of managing phytophthora root rot. Two main sources of resistance against P. sojae have been utilized; i) race-specific resistance mediated by single dominant Rps (resistance to P. sojae) genes and ii) partial/broad-spectrum resistance, also known as tolerance (Sugimoto et al. 2012). When partial resistance becomes ineffective under high disease pressure, race specific resistance has been 3 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 36 considered as an alternative approach (Dorrance et al. 2003). In soybean, however, continuous usage of Rps genes has been shown to lead to development of more virulent P. sojae pathotypes (Stewart et al. 2014) and the efficacy of an Rps gene depends on the population of P. sojae it is exposed to. Therefore, it was suggested that both Rps-mediated and partial resistance should be employed to efficiently develop the soybean plants with increased resistance to P. sojae (Burnham et al. 2003). Phytophthora sojae has a narrow host range and infection is limited primarily to soybean (Tyler 2007). However, a study by Jones and Johnson (1969) showed that P. sojae infected some lupine species including L. albus, L. angustifolius, L. luteus, L. bicolor, L. succulentus and L. densiflorus. Furthermore, Jones (1969) expanded the previous study by including more cultivated and wild lupine species and reported that most species of Lupinus were highly susceptibleDraft to P. sojae infection. Similary, Erwin and Ribeiro (1996) reported that 26 species of the genus Lupinus are susceptible to P. sojae infection. Jones and Johnson (1969) suggested P. sojae originated as a pathogen of lupines in North America prior to the introduction of soybean as a crop. The genus Lupinus is a diverse group of papilionoid legumes consisting of approximately 280 annual and perennial species (Eastwood et al. 2008). They can grow in a wide variety of climates (Drummond et al. 2012) and are divided into an ancestral Mediterranean and African “Old World” group and the more species-rich American “New World” group. It has been suggested that lupines originated in the Mediterranean region and the separation of Old World and New World lupines was caused by the continental drift (Wink et al. 1999). To date, the Old World group in Europe and North Africa consists of 13 annual species (Gladstones 1984; Pascual et al. 2006; Nevado et al. 2016) and is further subdivided into two categories on the basis of seed coat texture: smooth- and rough-seeded. The New World lupines are evolutionary more diverse than the Old World lupines and the processes that are responsible for the rapid diversification of the New World group are poorly understood (Nevado et al. 2016). Phylogenetic analyses suggest that the diversity of the Old World is centered in the Mediterranean 4 https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 36 Botany and northern and eastern African regions, whereas New World species have two centers of diversity: (1) Atlantic region of South America and (2) North and central America and Andes (Ainouche and Bayer 1999; Wink et al. 1999; Wolko et al. 2011). At present, lupines are distributed in a wide range of ecological habitats across lowland and montane environments (Drummond et al. 2012; Nevado et al. 2016). Whole genome duplications and triplications that occurred, particularly in the papilionoid legumes and the genistoid lineage, may have contributed to the genomic complexities and novelties that enabled adaptation to a wide array of biotic and abiotic challenges (Cannon et al. 2015; Hane et al. 2017). Like other legumes, lupine plants can obtain nitrogen via nitrogen-fixing bacteria (Jarabo-Lorenzo et al. 2003; Beligala et al. 2017;). These beneficial bacteria are attracted to legumes via root exudates (Porter et al. 1985; Banfalvi et al. 1988; Kosslak et al. 2006;Draft Sugiyama 2019). In soybean, these exudates function not only to attract rhizosphere bacteria, but also as a zoospore attractant (Morris and Ward 1992; Tyler et al. 1996; Sugiyama 2019). In saturated soil conditions, zoospores are released from sporangia and swim towards plant roots to infect and cause root-rot diseases in susceptible plants (Tyler 2007). A number of studies document that zoospore attachment and pathogenicity are correlated (Chi and Sabo 1978; Erb et al. 1986), whereas other reports show no such relationship (Tippett et al. 1976; Halsall 1978; Raftoyannis and Dick 2006). The first aim of this study is to clarify the correlation between zoospore attachment and pathogenicity of P. sojae race 2, strain P6497 (Tyler et al. 2006) in lupine, using two soybean lines as controls. The second aim is to identify lupine lines that are resistant to phytophthora root rot. Lastly, phylogenetic analysis of internal transcribed spacer (ITS) regions of the ribosomal RNA genes was carried out to both confirm the identities of the 17 lupine lines used and to help identify the geographic origins of resistant lupines. To date, this is the first study to explore the zoospore attachment, pathogenicity, and disease severity of P. 5 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 6 of 36 sojae P6497 towards lupine lines including annuals and perennials. Materials and Methods Plant and pathogen materials Seventeen germplasms (referred to as ‘lines’) of lupine were selected for this study based on their availability (Table 1). Ten lines were obtained from the USDA-ARS grain legume collection at Pullman, WA, USA), five lines were purchased from Plant World Gardens & Nursery (Newton Abbot, Devon, UK), one line was purchased from Marde Ross & Company (Glen Ellen, CA, USA), and one line was supplied by Dr. Helen J. Michaels (Bowling Green State University, OH, USA). Phytophthora sojae race 2, strain P6497 was kindly provided by Brett Tyler, OregonDraft State University (Tyler et al. 2006). The virulence formula for P. sojae race 2 is 1b, 7 (Yang et al. 1996). Additionally, two soybean cultivars served as controls: Williams 82 (SB W82), which carries a resistance gene (Rps1k) against P. sojae P6497, as a resistant control and Williams (SB W) as a susceptible (rps) control (Bernard and Lindahl 1972; Bernard and Cremeens 1988; Dorrance et al. 2004). Soybean seeds were obtained from Dr. Paul Morris (Bowling Green State University, OH, USA). Preparation of zoospores Lupine seeds (35-60) of each line were surface sterilized by submerging in 2.5% sodium hypochlorite solution and then in 80% ethanol (each for 10 min) followed by rinsing three times with sterile deionized water.