Sweet basil is one of the most popular culinary herbs in the United States (Dhar, 2002; Putievsky and Galambosi, 1999; Vieira et al., 2003). Basil is sold in fresh, dried, and frozen forms and is an important source of income for vegetable and herb growers (Simon et al., 1990, 1999; Vieira et al., 2003). In recent years, BDM has become one of the most destructive diseases of sweet basil globally (Belbahri et al., 2005; McGrath et al., 2010). BDM, caused by the pathogen P. Belbahrii, was first reported in Uganda in 1932 as Peronospora sp. and later in 1937 as Peronospora lamii (Hansford, 1933, 1938). BDM was not reported again until 2001 in Switzerland (Heller and Baroffio, 2003). After this initial confirmation, other countries throughout Europe, the Mediterranean, and continents across the world reported BDM for the first time (Belbahri et al., 2005; Kofoet et al., 2008; Lefort et al., 2008; McGrath, 2011). This pathogen was first reported in the United States in 2007 in southern Florida (Roberts et al., 2009). Since then, the disease has spread across the continental United States and Hawaii (Wyenandt et al., 2015). Although the epidemiology of the pathogen is not completely understood, BDM appears to have been spread globally via infested seed as well as through wind currents (Thines et al., 2009; Wyenandt et al., 2015).
Over 5000 ha of sweet basil is grown in the United States on an annual basis (J.E. Simon, personal communication). Although multiple sources of resistance have been identified (Pyne et al., 2015; Wyenandt et al., 2015), there are no commercial sweet basils available that are resistant to BDM (McGrath, 2011; Mersha et al., 2012; Pyne et al., 2014; Römer et al., 2010; Wyenandt et al., 2015) creating a high risk for continued and significant crop losses (Raid et al., 2010; Wyenandt et al., 2010). Sweet basil cultivars among all basils (Ocimum sp.) are the most susceptible to P. belbahrii (Wyenandt et al., 2010). Varying degrees of potential resistance were also identified in other Ocimum species including the citrus, spice, and holy basils with symptoms and sporulation of BDM either nonexistent or significantly less than that observed in O. basilicum (Wyenandt et al., 2010, 2015). Although breeding for BDM has shown that heritable genetic resistance can be introduced into sweet basil without the issue of sterility barriers (Pyne et al., 2015), use of visual markers to aid in breeding for BDM resistance is still lacking.
Although there are a few fungicides currently registered for controlling BDM, significant crop loss and expenses can still occur with weekly preventative fungicide applications (Homa et al., 2014). Without effective fungicide control, BDM can cause 100% crop loss (Garibaldi et al., 2007; Wyenandt et al., 2010).
The main diagnostic sign of the pathogen is the production of purplish-gray sporangia that appear on the abaxial surface of the leaves. Symptoms include chlorosis, cupping, and eventual necrosis of leaf tissue (Thines et al., 2009; Wyenandt et al., 2010). Because several exotic species were identified as being resistant or highly tolerant to BDM (Pyne et al., 2014; Wyenandt et al., 2010), we hypothesized that morphological characteristics such as stomata density and leaf curvature influence infection of P. belbahrii in different Ocimum species, and if so, could be effective visual markers for screening in plant breeding.
The large morphological variations in the wide range of annual and perennial herbs and shrubs in the genus Ocimum (Carović-Stanko et al., 2010; Simon et al., 1990) are mainly due to geographic differences, polyploidy, interspecific hybridization, and generic description changes (Nurzyńska-Wierdak, 2007; Paton et al., 1999). Basils vary in many characteristics including vigor, shape, plant height, branching, pubescence, leaf size, leaf shape, leaf texture, leaf color, leaf dimension, plant color, flower color, flowering time, flavor, and aroma (Marotti et al., 1996; Morales et al., 1993; Simon et al., 1990, 1999; Singh et al., 2002). Given that BDM infection requires entrance into the stomata, stomatal density and leaf morphology are likely to be important factors in disease development.
Stomata provide a natural opening for the entrance of some pathogens to inner leaf tissues (Allègre et al., 2009; Alonso-Villaverde et al., 2011; Melotto et al., 2008). This favors the pathogen since host cells are not directly invaded and resistance responses by the plant are not encountered. The stoma serves as an entry and exit point for the pathogen (Ayres, 1981). Direct germination of downy mildew sporangia, entrance through stomatal openings, and sporangiophore emergence from stomata 48 h after inoculation has been recorded in basil (Koroch et al., 2013). An increase in stomatal density is usually positively correlated with disease severity because high stomatal density could provide for an increased chance for the pathogen to enter and exit the leaf (Stenglein et al., 2005).
Leaf curvature significantly influences microclimatic conditions on the abaxial surface of the leaf where P. belbahrii sporulates. For this reason, leaf curvature may be an important factor in BDM incidence and severity (Simon et al., 1999; Wyenandt et al., 2010). Although the epidemiology of BDM is not completely understood, the pathogen spreads during periods of high humidity and poor air circulation (Garibaldi et al., 2005; Spencer, 1981; Wyenandt et al., 2010), mild temperatures of 20 °C, at least 6 to 12 h of leaf wetness immediately after inoculation, and at least 24 h of leaf wetness after symptom appearance for sporulation (Garibaldi et al., 2007). Leaf curvature with heavily downward curved leaves can exacerbate sporulation in basil.
The objectives of the following study were to examine morphological characteristics in basil including stomatal density, stomatal length, and leaf curvature and determine if these characteristics were associated with BDM development.
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