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

Abstract

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 (r = 0.70), and the association between the whole plant DSR and the detached leaf DSR was also positive (r = 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 < 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.

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

“…Two isolates of C. fragariae (= C. theobromicola), Cf63 and Cf75, and one isolate of C. gloeosporioides (= C. fructicola), Cg162, were used as inoculum (Chang and Smith, 2007;Miller-Butler et al, 2018;Munir et al, 2016;Smith and Black, 1990). Each isolate was initiated from silica gel cultures and grown in petri dishes on oatmeal agar:potato dextrose agar (1:1) (Miller-Butler et al, 2018) under fluorescent lights with a 12-h photoperiod at a temperature of 20 to 28°C.…”

“…Two isolates of C. fragariae (= C. theobromicola), Cf63 and Cf75, and one isolate of C. gloeosporioides (= C. fructicola), Cg162, were used as inoculum (Chang and Smith, 2007;Miller-Butler et al, 2018;Munir et al, 2016;Smith and Black, 1990). Each isolate was initiated from silica gel cultures and grown in petri dishes on oatmeal agar:potato dextrose agar (1:1) (Miller-Butler et al, 2018) under fluorescent lights with a 12-h photoperiod at a temperature of 20 to 28°C. Inoculum was prepared as a conidial suspension from 7-to 14-d-old cultures by flooding each culture plate with sterile deionized water, gently scraping the agar surface with a glass rod to remove conidia, filtering the suspension through sterile cotton bandage gauze with 1 mm 2 openings (Johnson & Johnson, New Brunswick, NJ), and adjusting to a concentration of 1.5 · 10 6 conidia/mL by diluting with sterile deionized water containing one drop of the surfactant Tween-20 (Sigma Chemical Co., St. Louis, MO) per 1 L of water.…”

“…Detached leaf inoculations. Following the protocol reported by Miller-Butler et al (2018), young, fully developed, blemishfree leaves were removed from plants no more than 4 h before inoculation and rinsed in tap water; the petiole of each leaf was then inserted into sterile deionized water in a 10 · 150 mm test tube. Detached leaves from each of the 77 clones were inoculated separately with a conidial suspension of each of three isolates, C. fragariae (isolates Cf63 and Cf75) and C. gloeosporioides (isolate Cg162) by misting the adaxial surface of the leaf blades with a hand-pump sprayer to the point of runoff.…”

“…Inoculated and noninoculated leaves were immediately placed in a dew chamber at 100% relative humidity (RH) and 30°C, incubated in the dark for 48 h, and transferred to sealed, clear plastic containers at 100% RH and 28°C with continuous fluorescent light for an additional 3 d before assessing disease symptoms. Disease symptoms on the blade of each leaf were visually assessed by two independent, trained raters using a disease severity rating (DSR) scale of 0 = no visible disease symptom on any leaflet, 1 through 4 = increasing disease symptom severity, and 5 = total area of leaflets necrotic (Miller-Butler et al, 2018).…”

“…Screening strawberry germplasm for disease resistance using detached leaf assays is an alternative to inoculating whole plants that allows plant disease response to be determined without destroying the plant, reduces the time between inoculation and disease assessment, and confines the pathogen to the laboratory, which allows breeders to test for pathogens or races of pathogens from other geographic areas without transferring the pathogen to the field or risking its introduction to the industry. Inoculating detached leaves with a conidial suspension of Colletotrichum species was shown to be an accurate nondestructive method of identifying anthracnose-resistant germplasm by Miller-Butler et al (2018).…”