Screening Strawberry Clones for Anthracnose Disease Resistance Using Traditional Techniques and Molecular Markers

Cultivated strawberry, Fragaria x ananassa Duchesne, is host for many pathogens. One of the most destructive diseases of strawberry is anthracnose, whose symptoms include fruit rot, leaf and petiole lesions, crown rot, wilt, and death. Three species of Colletotrichum are considered causative agents of anthracnose diseases of strawberry. Colletotrichum acutatum causes anthracnose fruit rot, has a broad host range, and occurs in most areas of the world where strawberries are grown. Colletotrichum fragariae, the primary causal fungus of anthracnose crown rot, may infect all aboveground parts of the strawberry plant, and has restricted host and geographic ranges. Colletotrichum gloeosporioides causes symptoms indistinguishable from those caused by C. fragariae but has much broader host and geographic ranges. Plant breeding programs generally require many years to produce commercially acceptable disease resistant cultivars; however, molecular tools may be used to identify genes that convey disease resistance and decrease the overall time required to develop new disease resistant cultivars. Two disease resistance genes have been identified in strawberry through controlled crosses: Rpf1 for resistance to Phytophthora fragariae and Rca2, a dominant gene that has been suggested to provide resistance to pathogenicity group 2 (q.v.) of C. acutatum (Van de Weg et al., 1997; Denoyes-Rothan et al., 2005). Two sequence characterized amplified region (SCAR) markers for the Rca2 resistance allele were found in several European and U.S. cultivars and, although there was not a perfect association with anthracnose resistance, these SCAR markers provide a unique set of tools to use in screening for anthracnose-resistant genotypes in strawberry breeding programs. The primary goal of this research was to establish the resistance or susceptibility of 81 strawberry germplasm lines to all three anthracnose-causing Colletotrichum species and to determine the degree to which an association exists between the resistance or susceptibility of these plants to the presence or absence of the Rca2 resistant allele SCAR markers. A secondary goal of this research was to determine if a detached leaf assay could be used to define the resistance or susceptibility of strawberry plants to anthracnose, thus providing an efficient, non-destructive method to screen strawberry germplasm for anthracnose resistance

Survey of Blueberry (\u3ci\u3eVaccinium\u3c/i\u3e spp.) Problems in the Gulf South

The blueberry (Vaccinium) industry in Mississippi has been steadily increasing since the early 1980s, but some plants in older fields are now in decline. The root rot pathogen, Phytophthora cinnamomi, is endemic in the southeastern United States and has caused severe losses to blueberries in North Carolina and Arkansas. Because the warm, humid climate of Mississippi, with periods of high precipitation, provides a favorable environment for Phytophthora root rot disease, it was suspected to be the cause of plant decline in older blueberry fields. Members of the Gulf South Blueberry Growers Association were mailed surveys to determine cultural practices and the extent of losses in their blueberry fields. The survey was written in partial, open-ended question format with 33 questions pertaining to cultivars, number of hectares planted, cultural practices, overall health of the blueberry plants, and losses due to diseases, insects, or other problems. Fifty-eight of 146 surveys (40%) were completed and returned. 89% of respondents grew rabbiteye (Vaccinium ashei) cultivars and the balance grew southern highbush (V. corymbosum hybrids) cultivars. The average number of plants per hectare was 1523 with the majority of plants 10 - 20 years old. 79% of the growers described the overall condition of their plants as average, healthy, or vigorous. The most common cause of plant death cited was environmental with damage caused by the 2004 and 2005 hurricanes at the top of the list. No major diseases were reported by 36% of the respondents, while mummy berry (Monilinia vaccinii-corymbosi [Reade] Honey), leaf spots (many causal organisms), and root rot (Phytophthora cinnamomi Rands) were noted as problems by 22, 15, and 10% of the growers, respectively. Phytophthora species were isolated from root and soil samples collected from symptomatic plants on three farms. Weeds were listed as problems on 100% of the surveys. 47% of the respondents plan to increase their blueberry plantings mainly with rabbiteye cultivars

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Evaluation of Detached Strawberry Leaves for Anthracnose Disease Severity Using Image Analysis and Visual Ratings

Inoculation of detached strawberry leaves with Colletotrichum species may provide an accurate, rapid, nondestructive method of identifying anthracnose-resistant germplasm. The purpose of this study was to statistically compare two methods (visual and image analysis) of evaluating disease severity of strawberry germplasm screened for anthracnose resistance. Detached leaves of 77 susceptible and resistant strawberry clones were inoculated with one Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and two C. fragariae A. N. Brooks isolates. Anthracnose disease symptoms on each leaf were assessed quantitatively via computer-based image analysis to determine percentage lesion area and qualitatively by two independent raters using a visual disease severity rating scale (0 = no symptoms to 5 = entire leaf dead). The two visual raters’ average disease severity ratings (n = 3413) were in substantial agreement with a weighted Cohen’s kappa coefficient (k) of 0.80 [95% confidence interval (CI) 0.79–0.82]. There was a strong positive correlation between percent lesion area determined by image analysis and the visual disease scores of the two raters (rp = 0.79). Image analysis provided a precise measurement of percent lesion area of infected leaves while visual assessment provided more rapid results. Our results indicate that detached leaf inoculations can be used as a rapid preliminary screen to separate anthracnose-susceptible from -resistant germplasm in large populations within breeding programs. It also may be used for assessing the resistance/susceptibility of parental breeding lines to various Colletotrichum species and isolates, for mapping germplasm for resistance genes, and in pesticide development studies

Evaluation of Detached Strawberry Leaves for Anthracnose Disease Severity Using Image Analysis and Visual Ratings

Inoculation of detached strawberry leaves with Colletotrichum species may provide an accurate, rapid, nondestructive method of identifying anthracnose-resistant germplasm. The purpose of this study was to statistically compare two methods (visual and image analysis) of evaluating disease severity of strawberry germplasm screened for anthracnose resistance. Detached leaves of 77 susceptible and resistant strawberry clones were inoculated with one Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and two C. fragariae A. N. Brooks isolates. Anthracnose disease symptoms on each leaf were assessed quantitatively via computer-based image analysis to determine percentage lesion area and qualitatively by two independent raters using a visual disease severity rating scale (0 = no symptoms to 5 = entire leaf dead). The two visual raters’ average disease severity ratings (n = 3413) were in substantial agreement with a weighted Cohen’s kappa coefficient (k) of 0.80 [95% confidence interval (CI) 0.79–0.82]. There was a strong positive correlation between percent lesion area determined by image analysis and the visual disease scores of the two raters (rp = 0.79). Image analysis provided a precise measurement of percent lesion area of infected leaves while visual assessment provided more rapid results. Our results indicate that detached leaf inoculations can be used as a rapid preliminary screen to separate anthracnose-susceptible from -resistant germplasm in large populations within breeding programs. It also may be used for assessing the resistance/susceptibility of parental breeding lines to various Colletotrichum species and isolates, for mapping germplasm for resistance genes, and in pesticide development studies

Comparison of Whole Plant and Detached Leaf Screening Techniques for Identifying Anthracnose Resistance in Strawberry Plants

Anthracnose is a destructive disease of strawberry caused by several Colletotrichum species including C. acutatum, C. fragariae, and C. gloeosporioides. Identification of anthracnose resistant strawberry germplasm has commonly relied on inoculation of whole plants with isolates of these pathogens. In this study, whole plants and detached leaves from 81 germplasm lines were inoculated with a conidial suspension of isolates of C. acutatum, C. fragariae, and C. gloeosporioides, incubated in the dark at 30°C, 100% relative humidity, for 48 h, and assessed for disease severity based on symptoms on inoculated petioles and leaves. The correlation between the disease severity ratings of the whole plants rated 30 days after inoculation and detached leaves rated 5 days after inoculation was determined. Based on leaf symptoms and petiole lesions, the association between the whole plant leaf disease severity rating (DSR) and detached leaf DSR was positive (rp = 0.70), and the association between the whole plant DSR and the detached leaf DSR was also positive (rp = 0.66). Whole plant and detached leaf DSRs were used to assign each germplasm line to a resistance category, and a posthoc Tukey’s test showed that the whole plant DSR means and the detached leaf DSR means for each resistance category differed significantly at p \u3c 0.05. This research was used to develop a strawberry detached leaf assay which can reliably and quickly determine the degree of resistance of strawberry germplasm to anthracnose

Comparison of Anthracnose Resistance With the Presence of Two SCAR Markers Associated With the \u3ci\u3eRca2\u3c/i\u3e Gene in Strawberry

Strawberry anthracnose diseases are caused primarily by three Colletotrichum species: C. acutatum J.H. Simmonds, C. fragariae A.N. Brooks, and C. gloeosporioides (Penz.) Penz. & Sacc. Molecular markers are being used in breeding programs to identify alleles linked to disease resistance and other positive agronomic traits. In our study, strawberry cultivars and breeding germplasm with known anthracnose susceptibility or resistance to the three anthracnose-causing Colletotrichum species were screened for two sequence characterized amplified region (SCAR) markers linked to the Rca2 gene. The Rca2 resistant allele SCAR markers were associated with varying degrees of significance for a strawberry plant’s anthracnose resistance to C. fragariae but not to C. acutatum or C. gloeosporioides. Although the presence or absence of the markers associated with the Rca2 resistance gene is an imperfect indicator of anthracnose resistance, it may serve as a useful starting point in selecting germplasm for breeding programs. In the southeastern United States, anthracnose diseases of commercial strawberry (Fragaria ×ananassa Duch.) are caused primarily by three Colletotrichum species: C. acutatum, C. fragariae, and C. gloeosporioides (Maas, 1998). Colletotrichum acutatum incites two major anthracnose diseases on strawberry: anthracnose fruit rot and root necrosis (Howard et al., 1992; Mertely and Peres, 2012; Mertely et al., 2005), and has been found to remain latent on symptomless strawberry plants (Leandro et al., 2001). The devastating Colletotrichum crown rot (anthracnose crown rot) may be caused by C. gloeosporioides or C. fragariae (Howard et al., 1992; Peres et al., 2007; Smith, 1998; Smith and Black, 1987; Ureña-Padilla et al., 2002). Both C. gloeosporioides and C. fragariae may also cause anthracnose symptoms on all aerial parts of the strawberry plant. Fungicides are used routinely to control anthracnose diseases; however, frequent use of the same fungicides has resulted in their failure to control anthracnose as a result of pathogen resistance (Forcelini and Peres, 2018; LaMondia, 1995; Smith and Black, 1992, 1993). The need for fungicides can be reduced by growing strawberry cultivars resistant to anthracnose diseases. Strawberry breeders and plant pathologists are working to develop strawberry germplasm resistant to anthracnose crown rot and fruit rot diseases using traditional and classic techniques (Denoyes and Guerin, 1996; Salinas et al., 2018; Whitaker et al., 2017), and are exploring native germplasm for sources of anthracnose resistance in strawberry (Lenne and Wood, 1991; Lewers et al., 2007). Various protocols have been developed to screen strawberry seedlings and mature plants for resistance to the different Colletotrichum species. Denoyes and Guerin (1996) screened for resistance to C. acutatum by immersing whole plants into a conidial suspension of the pathogen. Horowitz et al. (2004) developed a foliar-dip method for large-scale screening of 12- and 15-week-old strawberry seedlings for resistance to C. gloeosporioides and C. acutatum. A protocol for identifying strawberry germplasm with resistance to C. fragariae based on petiole and crown symptoms was developed at the U.S. Department of Agriculture–Agricultural Research Service, Poplarville, MS (Smith and Black, 1987; Smith and Spiers, 1982) and was used to select anthracnose-resistant germplasm with desirable horticultural characteristics. Selections from this program were given the prefix MSUS, and one anthracnose resistant cultivar, ‘Pelican’ (Smith et al., 1998), and four anthracnose resistant breeding lines—US70, US159, US292, and US438 (Galletta et al., 1993)—developed in this program were released. Recently the anthracnose resistance/susceptibility of 31 strawberry cultivars was compared with that of 50 anthracnose-resistant MSUS selections developed at Poplarville, MS (Miller-Butler et al., 2018). Most strawberry cultivars are octoploids, which presents difficulties in determining their genetics in classic breeding programs (Anciro et al., 2018; Hancock et al., 2008). Disomic inheritance for at least part of the strawberry genome has been demonstrated by some researchers (Folta and Davis, 2006; Lerceteau-Köhler et al., 2003), which allows breeders to identify dominant and recessive alleles for these loci. The advent of molecular genetics provided plant breeders with molecular markers to use for identification of alleles linked to disease resistance and other positive agronomic characteristics (Longhi et al., 2014). Genetic marker-assisted detection of resistance genes has been used in various Fragaria–pathogen systems, including the following: locating the Rpc1 gene that provides resistance to crown rot disease caused by Phytophthora cactorum in Fragaria vesca (Davik et al., 2015); screening strawberry seedlings in field trials for resistance to V. dahlia, the causative agent of verticillium wilt (Antanaviciute et al., 2015); identifying the Rpf1 gene for resistance to red stele root rot caused by Phytophthora fragariae (Van de Weg et al., 1997); locating a quantitative trait locus (QTL) conferring resistance to C. gloeosporioides (Anciro et al., 2018); and detecting the subgenome-specific locus FaRCa1 conferring resistance to C. acutatum (Salinas et al., 2018). SCAR markers were used to demonstrate a resistant:susceptible segregation ratio of 1:1 in strawberry progeny from crosses between red stele-susceptible × -resistant germplasm (Gel Vonauskienė et al., 2007). Several studies have tested strawberries for resistance to C. acutatum, which has been classified as either pathogenicity groups 1 or 2, based on its pathogenicity on five strawberry cultivars (Denoyes and Baudry, 1995). Classification of additional C. acutatum isolates was performed by Denoyes-Rothan et al. (2003), who conducted pathogenicity tests on two strawberry cultivars—Belrubi (resistant to group 2 and susceptible to group 1) and Elsanta (susceptible to both groups)—using a subset of 34 European C. acutatum isolates and the American C. acutatum isolate Goff. Denoyes-Rothan et al. (2005) examined the inheritance of high- and intermediate-level plant resistance to C. acutatum isolates of pathogenicity group 2 and found that the inheritance of a dominant gene (Rca2) controlled strawberry resistance to that group. Lerceteau-Köhler et al. (2005) identified two SCAR markers and used them to screen European and American strawberry genotypes, reported in the literature as resistant or susceptible, for the presence of the Rca2 gene. They found 13 of the 28 resistant genotypes had both SCAR markers and none of the 14 susceptible genotypes had either of the SCAR markers. They considered their results to be indicative of monogenic control for resistance to C. acutatum pathogenicity group 2. In our study we investigated the possibility that strawberry germplasm containing the Rca2 gene with resistance to group 2 C. acutatum isolates might also confer a level of resistance to C. fragariae and C. gloeosporioides, as well as to C. acutatum isolates for which a pathogenicity group has not been determined. The objective of our research was to screen 31 strawberry cultivars and 50 MSUS selections for the presence or absence of the two SCAR markers linked to the Rca2 gene and to determine whether their presence or absence in each germplasm line correlates with the resistance/susceptibility to each of the three Colletotrichum species as reported by Miller-Butler et al. (2018). Knowledge of the presence or absence of the Rca2 gene and a germplasm’s resistance to anthracnose should improve breeders’ decisions concerning which strawberry germplasm is most desirable to incorporate into breeding programs