Abstract
Outbreaks of bitter rot disease occurred in Illinois apple (Malus ×domestica) orchards during 2010–20. This study was conducted to assess the incidence of bitter rot in commercial apple orchards in Illinois, identify pathogen species that cause bitter rot, and evaluate the efficacy of fungicides for managing the disease. Orchard surveys conducted during 2019–21 showed that fruits with bitter rot were present in most of the orchards in southern and central Illinois, whereas only a few orchards in the northern part of the state had symptomatic fruits. A total of 270 isolates of the pathogens were collected from symptomatic fruits of 14 cultivars, and pathogen species were identified based on the morphological and molecular characteristics of the isolates. GAPDH gene sequence analyses identified species of the pathogens as Colletotrichum fioriniae, C. siamense, and C. chrysophilum. Laboratory and orchards studies were conducted to evaluate the effectiveness of fungicides for managing bitter rot disease. Laboratory studies showed averages of 10.3, 9.6, and 0.24 mg⋅L−1 for the 50% effective concentrations (EC50) of benzovindiflupyr, captan, and fluxapyroxad + pyraclostrobin fungicides, respectively. Orchard experiments involving ‘Honeycrisp apples’ were conducted in 2019, 2020, and 2021. Benzovindiflupyr, captan, and fluxapyroxad + pyraclostrobin prevented bitter rot development in the treated plots.
Bitter rot of apples (Malus ×domestica) is a disease caused by Colletotrichum species including Colletotrichum acutatum and Colletotrichum gloeosporioides complexes (Braganca et al. 2016; Dowling et al. 2020; Gonzalez and Sutton 2004). It is currently considered the most important apple disease in Illinois because fruit losses of 100% for some apple cultivars have been recorded in several orchards in the state (Babadoost 2022).
Molecular methods have been widely used to accurately identify pathogen species that cause bitter rot of apples (Freeman et al. 1998; Sreenivasaprasad and Talhinhas 2005). Multilocus phylogenetic analyses are reliable for addressing challenges in the identification of Colletotrichum species. Investigators first used primers that target the internal transcribed spacer of the rDNA region (Martinez-Culebras et al. 2003; Mills et al. 1992; Sreenivasaprasad et al. 1994). However, it was concluded that the internal transcribed spacer sequences alone could not fully differentiate Colletotrichum spp. from each other (Weir et al. 2012). Therefore, sequence analyses of this genetic region and a combination of other genes that code for actin, calmodulin, glyceraldehyde-3-phosphate dehydrogenase, glutamine synthetase, β-tubulin, and histone have been used to accurately identify Colletotrichum species (Faedda et al. 2011; Lubbe et al. 2004; Prihastuti et al. 2009; Talhinhas et al. 2002; Wikee et al. 2011).
Although some apple cultivars are less susceptible to bitter rot (i.e., Red Delicious) than others (i.e., Golden Delicious), no highly resistant apple cultivar is commercially available. Thus, chemical management of the disease remains the dominant method for controlling bitter rot of apples. The availability of only a few effective fungicides to control Colletotrichum pathogens and the persistent threat of fungicide resistance development in the pathogens make management of bitter rot of apples a difficult task (Dowling et al. 2020). Previous studies have reported variability in fungicide efficacy against bitter rot, which is, to a large extent, related to differences in fungicide sensitivity among Colletotrichum species and efficacy among fungicide active ingredients (Chechi et al. 2019; Khodadadi et al. 2020; Martin et al. 2022; Munir et al. 2016). Differences in fungicide sensitivity among species from a single geographical location may be inherent or may have emerged because of fungicide selection pressure influenced by the species-specific inoculum frequency and density and the influx of wild-type phenotypes from nearby hosts (Chen et al. 2016). Excessive use of fungicides poses the threat of fungicide resistance of the pathogens.
Currently, single-site fungicide groups are commonly recommended in the United States for managing Colletotrichum spp. (Dowling et al. 2020). These fungicide groups are demethylation inhibitors [DMIs; FRAC 3 (Fungicide Resistance Action Committee)], fluazinam (FRAC 29), methyl benzimidazole carbamates (MBCs; FRAC 1), phenylpyrroles (PPs; FRAC 12), polyoxins (FRAC 19), quinone-outside inhibitors (QoIs; FRAC 11), and succinate dehydrogenase inhibitors (SDHIs; FRAC 7). MBC fungicides affect the fungal cytoskeleton by directly binding to β-tubulin subunits and inhibiting the assembly of microtubules (Angelini et al. 2015). Insensitivity of C. acutatum to MBC fungicides has been reported (Chung et al. 2006). The DMI fungicides are registered as either solo or mixture products for various crops. However, their efficacy as solo products against Colletotrichum spp. is poor (DeFrancesco et al. 2017). QoI (strobilurin) fungicides target the cytochrome b (cytb) gene and inhibit fungal mitochondrial respiration (Bartlett et al. 2002; Sauter et al. 1999; Ypema and Gold 1999). Widespread resistance of C. acutatum to QoI fungicides in strawberry (Fragaria ×ananassa) (Forcelini and Peres 2018) and apple (Chechi et al. 2019; Martin et al. 2022) have been reported. The PP fungicides are useful because there are only a few reported cases of resistance development in fungi to this fungicide group (Kilani and Fillinger 2016). Polyoxin has inconsistent and limited efficacy against Colletotrichum spp. on fruit crops (McManus and Perry 2017; Pscheidt and Bassinette 2016; Su and Gubler 2009). Fluazinam works by disrupting oxidative phosphorylation, thus inhibiting mitochondrial respiration. This class of fungicide has shown high efficacy against C. acutatum and C. gloeosporioides species complexes in vitro (Gang et al. 2015; Leroux 1996). Benzovindiflupyr fungicide (of the SDHI group) has been registered for the management of bitter rot of apples in the midwestern states of the United States and has good efficacy against C. acutatum (Beckerman et al. 2023; Dowling et al. 2020). The multisite fungicide captan provides satisfactory management of C. acutatum; however, it does not have any curative effect (Dowling et al. 2020; Martin et al. 2022; Mertely et al. 2010).
The objectives of this study were to conduct surveys to assess the incidence of bitter rot disease in commercial orchards in Illinois, identify species of pathogens that cause bitter rot disease, and evaluate the efficacy of fungicides for managing bitter rot of apples.
Materials and methods
Incidence of bitter rot disease in commercial orchards
Orchard surveys were conducted in 2019, 2020, and 2021 to assess the incidence of bitter rot of apples in commercial orchards in Illinois. Surveys included 24, 30, and 33 orchards in 2019, 2020, and 2021, respectively (Table 1, Fig. 1). The surveyed orchards were selected based on major apple-producing locations throughout the state. Seventeen cultivars, including Braeburn, Cortland, Empire, Fuji, Gala, Ginger Gold, Golden Delicious, Golden Supreme, Goldrush, Granny Smith, Honeycrisp, Jonagold, Jonathan, McIntosh, Red Delicious, Snow Sweet, and an unknown cultivar, were evaluated to determine the presence of fruits with bitter rot symptoms (Table 2). For each cultivar block at each orchard, five trees were randomly selected and 60 fruits (five fruits in each of the upper, middle, and lower canopies of eastern, northern, western, and southern sides) of each tree were examined to observe symptoms. The size of each cultivar block ranged from 0.25 to more than 2 ha. In each orchard, all existing cultivar blocks were visited. Orchards with no fruit symptomatic of bitter rot were recorded as “orchards without bitter rot”; orchards with at least one cultivar block with bitter rot symptoms were considered “orchards with bitter rot.” Five to 10 symptomatic fruits were collected from each cultivar block. The collected fruits were carried in a cooler and kept at 4 °C until associated fungi were isolated within 72 h of collection (Table 3).
Commercial apple orchards surveyed to determine the incidence of bitter rot of fruits in northerni, centralii, and southerniii Illinois.
Locations of surveyed commercial apple orchards in Illinois in 2019, 2020, and 2021.
Citation: HortTechnology 34, 2; 10.21273/HORTTECH05377-23
Apple cultivars in Illinois commercial orchards surveyed to determine the incidence of bitter rot disease during 2019–21.
Number of Colletotrichum isolates collected from symptomatic apple fruits in commercial orchards in three different areas of Illinois during 2019–21.
Isolation and identification of pathogen species
Using sterile blades, small pieces (diameter, 5 mm) of fruit skin and pulp were cut from the outer margins of each lesion of each fruit. The pieces were surface-disinfested by dipping in 95% ethanol for 60 s and then washed three times (1 min each) with sterile distilled water (SDW). Surface-sterilized pieces were dried between sterilized paper tissues and placed on potato dextrose agar (PDA) medium in petri plates (100 mm × 15 mm). The plates were incubated at 24 °C for 5 d in the dark. Pure cultures were obtained by performing single-spore isolation according to the methods reported by Du et al. (2005). Isolated fungal colonies were screened to determine the colony color, conidial shape and size, and growth rate of the colony on PDA. Two single-spore cultures of each isolate were examined after 7 d of growth on PDA. Colony colors were named according to the mycological color chart (Rayner 1970). The size and shape of conidia (fusiform with pointed ends or cylindrical with rounded ends) were examined using a light microscope (Olympus BX41; Olympus Life Science, Tokyo, Japan). The colony growth rate of isolates at 24 °C in the dark was determined by measuring the colony diameter of each isolate daily for 7 d. Isolates were organized into three general groups (morphotypes) based on the colony morphology and color.
All isolates were grown on PDA for 7 d, and genomic DNA was extracted using an E.Z.N.A. MicroElute Genomic DNA kit (Omega Bio Tek, Norcross, GA, USA) according to the manufacturer’s protocol. The polymerase chain reaction (PCR) primer pairs GDF1 (5′-GCCGTCAACGACCCCTTCATTGA-3′) and GDR2 (5′-GGGTGGAGTCGTACTTGAGCATGT-3′) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) amplification (Templeton et al. 1992) was used for PCR amplification. A 25-μL reaction was set up using the GoTaq Green Kit, including 3 μL of genomic DNA, 12.5 μL of GoTaq Green Master Mix, 7.5 μL of DNA-free water, and 1 μL of each of 10 μM GAPDH primers. The PCR amplification was performed using a C1000 Touch thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) with initial denaturation at 95 °C for 2 min, followed by 34 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, and final extension for 10 min at 72 °C. From each PCR product, 3 µL was examined in a 1% (weight/volume) agarose gel stained with EtBr in 1× TAE buffer electrophoresed at 96 V for 30 min. Then, PCR product purification was performed using a Wizard SV Gel and PCR Clean-Up system (Promega Corporation, Madison, WI, USA), and template DNA was used in 10 μL sequencing reactions with BigDye® Terminator version 3.1 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) using the PCR primers. Sequences were generated using an Applied Biosystems 3730XL high-throughput capillary sequencer at the Roy J. Carver Biotechnology Center (University of Illinois, Urbana, IL, USA). Sequences were assembled with Sequencher 5.4 (Gene Codes Corp., Ann Arbor, MI, USA), and their identification was confirmed by comparing type strain sequences using the BLASTn database from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA).
Pathogenicity of the isolates
Identified Colletotrichum isolates from ‘Empire’, ‘Golden Delicious’, and ‘Honeycrisp’ apple fruits were tested to determine their pathogenicity in a laboratory (Table 4). Apple fruits used for this experiment were harvested and stored at 4 °C in the dark for up to 2 weeks before inoculating. Fruits were washed with liquid detergent and tap water to ensure that no fungicide residue remained on the surface. Then, the fruits were surface-disinfested by soaking in 0.5% sodium hypochlorite for 2 min, rinsed in SDW twice (1 min each time), and air-dried on sterile paper towels on a laboratory bench at room temperature (24 °C). Ten fruits of each cultivar were inoculated with each of the identified Colletotrichum isolate. Fruits were wounded using syringe needles to produce a puncture depth of 0.5 mm (Velho et al. 2014, 2015). The wounded fruits were inoculated by adding 6 µL of a conidial suspension (1 × 104 mL−1 conidia in SDW water, determined using a hemocytometer) to each wounded spot. The conidial suspension was prepared from 7-d-old PDA cultures (Munir et al. 2016). Control fruits were wounded and treated with SDW. The fruits were placed on moist paper towels in translucent plastic boxes and incubated at room temperature (24 °C) with 12 h of light and 12 h of darkness. The diameters of the developed lesions were measured at 3, 6, 9, 12, and 15 d after inoculation.
Mean lesion diameter of Colletotrichum isolates on three apple cultivars 15 d after inoculation in the laboratory.
Fungicide efficacy for managing bitter rot
Effects of fungicides on colony development of Colletotrichum isolates in the laboratory
Three isolates of each of Colletotrichum chrysophilum, Colletotrichum fioriniae, and Colletotrichum siamense collected from commercial apple orchards in Illinois were included in this study. Isolates of C. fioriniae and C. siamense were collected from northern (latitudes: 40.90°–42.50°), central (latitudes: 39.20°–40.90°), and southern (latitudes: 37.00°–39.20°) Illinois (one isolate from each area), and all three isolates of C. chrysophilum were from central Illinois (the only location where it was collected).
Fungicide sensitivity of the isolates was evaluated against six fungicides registered for use on apple trees. The fungicides were benzovindiflupyr (Aprovia 0.83SC; Syngenta Corp., Greensboro, NC, USA), captan (Captan 80WDG; Loveland Products Inc., Greeley, CO, USA), fluxapyroxad + pyraclostrobin (Merivon 4.18SC; BASF Corp., Research Triangle Park, NC, USA), potassium phosphite (ProPhyt 4.2SC; Luxembourg-Pamol, Inc., Woodbridge, VA, USA), trifloxystrobin (Flint Extra 4.05SC; Bayer CropScience, St. Louis, MO, USA), and zinc dimethyldithiocarbamate (Ziram 76DF; United Phosphorus, Inc., King of Prussia, PA, USA) (Table 5). For the commercial Merivon 4.18SC fungicide, the rates of fluxapyroxad and pyraclostrobin were equal (21.26% each).
Concentration of selected fungicides for the EC50i of colony development of Colletotrichum spp. from apple orchards in Illinois on PDAii medium amended with the fungicides.
Isolates were grown on PDA in petri plates at 24 °C in the dark for 5 d. A mycelial plug (diameter, 3 mm) was cut from the actively growing part of each colony and transferred onto amended PDA in each petri plate. Concentrations of active ingredients in amended PDA were as follows: 8.3, 41.5, 83, and 830 mg⋅L−1 for benzovindiflupyr; 0.8, 8, 80, 400, and 800 mg⋅L−1 for captan; 0.02, 0.11, 2.13, 10.63, 21.26, and 212.6 mg⋅L−1 for fluxapyroxad + pyraclostrobin; 27.25, 54.5, 545, 818.5, and 1090 mg⋅L−1 for potassium phosphite; 4.26, 21.3, 42.6, and 426 mg⋅L−1 for trifloxystrobin; and 7.6, 38, 76, and 380 mg⋅L−1 for zinc dimethyldithiocarbamate. Control PDA plates contained no fungicide. Four replicate plates were included for each fungicide–isolate treatment, and the tests were repeated twice.
After analyzing the data, we used a linear regression model, Yi = β0 + β1Xi, to determine the 50% effective concentration (EC50) values of the fungicides. In this model, Y was the value of the ith dependent variable, β0 was the intercept parameter, β1 was the slope parameter, and Xi was value of the ith independent variable. The percent inhibition of the colony diameter was the dependent variable, and the fungicide concentration was the independent variable.
Efficacy of fungicides on managing bitter rot in the orchard
Orchard experiments were conducted in 2019, 2020, and 2021, to evaluate the effectiveness of the selected fungicides for managing bitter rot and other summer diseases of ‘Honeycrisp’ apples. The experiments were conducted at the University of Illinois Fruit Research Farm in Urbana, IL, USA (lat. 40°4.91′ N, long. 88°12.96 W, elevation 222 m). The trees were 10 years old in 2019; they were planted in 24 rows, with each row comprising 10 trees. Bitter rot had occurred in orchards during the previous 5 years.
In 2019, eight fungicides (Table 6) in 12 treatments were evaluated. For each treatment, two rows with up to 20 trees were included. To control spring diseases (powdery mildew, rusts, scab), the trees were sprayed with mancozeb (Manzate PRO Stick 0.75WP; United Phosphorus, Inc.) plus difenoconazole + cyprodinil (Inspire Super 2.82SC; Syngenta Corp.) and alternated with mancozeb plus penthiopyrad (Fontelis 1.67SC; Corteva Agriscience LLC, Indianapolis, IN, USA) at weekly intervals from the green-tip growth stage to 1-week before petal fall. Applications of the fungicides (Table 6) for managing summer diseases began at petal fall on 20 May and ended on 19 Aug (21 d before harvesting fruits). Fungicides were spray-applied at 7-, 10-, and 14-d intervals with an SR 450 motorized backpack sprayer (STIHL Corp., Virginia Beach, VA, USA) using 2 L of spray suspension per tree.
Fungicides tested in 2019 for the management of bitter rot caused by Colletotrichum spp. in the Honeycrisp apple orchard in Illinois.
Four trees in each treatment were randomly selected to evaluate the incidence of summer diseases. The incidence of diseases was determined on 28 Jul and 9 Sep. The percentage of symptomatic fruits in each tree was determined by examining 60 fruits, including five fruits from each of the upper, middle, and lower canopies on each of the northern, eastern, southern, and western sides. A total of 240 fruits in each treatment were examined for disease symptoms.
In 2020 and 2021, experiments were conducted at the same orchard block of 2019. Management of spring diseases was as described for the 2019 experiment. Selected fungicides for evaluations and spray intervals were considered after analyzing the results of the experiment in 2019.
Tested fungicides were benzovindiflupyr, captan, fluxapyroxad + pyraclostrobin, potassium phosphite, thiophanate-methyl (Topsin-M 70WSB; United Phosphorus, Inc.), trifloxystrobin, and zinc dimethyldithiocarbamate (Tables 7 and 8). The experiment was performed using a randomized complete block design with four replications. Each plot had two trees. Fungicides were spray-applied at 10- and 14-d intervals. Applications of the fungicides for managing summer diseases in 2020 began at petal fall on 18 May and ended on 26 Aug (17 d before harvesting fruits). Applications of the fungicides in 2021 began at petal fall on 14 May and ended on 20 Aug (17 d before harvest). Spray applications of fungicides were as described for the 2019 experiment. One tree in each plot with a good fruit load was selected to evaluate disease symptoms. In 2020, the incidence of diseases was assessed on 25 Jul; 1, 8, 15, 22, and 29 Aug; and 5 and 12 Sep. In 2021, the incidence of diseases was assessed on 11, 18, and 25 Jul; 1, 8, 15, 22, and 29 Aug; and 5 and 12 Sep. The percentage of symptomatic fruits in each tree was determined by examining 60 fruits per tree, as previously described.
Fungicides tested in the orchard in 2020 and 2021 to determine their effectiveness for the management of Colletotrichum spp. and incitants of bitter rot disease of apples in Illinois.
Efficacy of selected fungicides for the management of bitter rot of ‘Honeycrisp’ apples in Illinois.
Results
Incidence of bitter rot disease in commercial orchards
Bitter rot incidence was recorded in 14 of 24 orchards visited in 2019, 20 of 30 orchards visited in 2020, and 21 of 33 orchards visited in 2021 (Fig. 1). During the three years of surveys, fruits with bitter rot were observed in 55 of 87 (63.22%) of the commercial orchards (Table 1). Overall, 18.52%, 73.33%, and 86.67% of visited apple orchards in northern, central, and southern Illinois, respectively, had fruits with bitter rot symptoms (Table 1). The incidence of bitter rot in the orchards ranged from 0% to 63% in 2019, and from 0% to 100% in both 2020 and 2021. The highest percents of fruits (100%) with bitter rot symptoms were observed for ‘Honeycrisp’ in central Illinois and ‘Empire’ in southern Illinois in 2019, for ‘Golden Delicious’ and ‘Empire’ in southern Illinois, and for ‘Honeycrisp’ and ‘Empire’ in central Illinois in 2020 and 2021. Overall, the incidence of fruits with bitter rot symptoms of Empire, Golden Delicious, Honeycrisp, and Jonathan apples was higher than that of other apple cultivars (Table 2).
Identification of pathogen species
The GAPDH sequences of 270 bp confirmed that all isolates tested were Colletotrichum species. Based on the colony morphology, 270 isolates were divided into morphotype groups 1, 2, and 3. Morphotype group 1, which comprised 136 isolates (Table 3), produced a salmon to pink colony on PDA after 7 d that was visible on the top and bottom of the colony (Fig. 2A and B). Conidia produced by isolates in this group were fusiform with acute ends. Based on these morphological characteristics, isolates in this group corresponded to C. fioriniae in the C. acutatum complex. Morphotype group 2, which comprised 129 isolates (Table 3), produced a white to light gray colony with light grey centers on PDA (Fig. 2C and D). Conidia were cylindrical with rounded ends. Colonies of isolates in morphotype group 2 grew faster than colonies of isolates in morphotype group 1. These characteristics of morphotype group 2 were consistent with descriptions of C. siamense in the C. gloeosporioides complex. Morphotype group 3, which comprised five isolates (Table 3), produced a dark gray colony on PDA (Fig. 2E and F). These isolates produced cylindrical conidia with rounded ends. Colonies of this group grew faster than isolates in morphotype groups 1 and 2. Colony characteristics of this group were consistent with descriptions of C. chrysophilum, which is also in the C. gloeosporioides complex. C. fioriniae produced salmon to pink coloration on PDA, whereas neither of the other two species produced any pigment.
Colonies of Colletotrichum species identified in fruits with bitter rot symptoms in commercial apple orchards in Illinois during 2019–21. Cultures on potato dextrose agar in petri plates were 15 d old. (A, C, and E) The top of the culture plates of Colletotrichum fioriniae, C. siamense, and C. chrysophilum, respectively. (B, D, and F) The bottom of the culture plates of Colletotrichum fioriniae, C. siamense, and C. chrysophilum, respectively.
Citation: HortTechnology 34, 2; 10.21273/HORTTECH05377-23
One hundred representative isolates from the three morphotype groups were selected for the molecular study. These represented the different geographical locations of the orchards and variations in apple cultivars. Sequence similarity searches of only type strains using BLASTn indicated that all isolates in morphotype group 1 matched with C. fioriniae, that those in morphotype group 2 matched C. siamense, and that those in morphotype group 3 matched C. chrysophilum. The ranges of sequence variations within each species compared with the type sequence were 0% to 2.1% for C. fioriniae (JQ948622), 0% to 2.8% for C. siamense (JX009924), and 0% to 0.9% for C. chrysophilum (KX094183). Five unique genotype sequences were deposited in NCBI for C. fioriniae (OR140159–OR140163), four were deposited for C. siamense (OR137130–OR137133), and two were deposited for C. chrysophilum (OR142761–OR142762).
Pathogenicity of the isolates
All 270 isolates of Colletotrichum species were pathogenic and produced typical bitter rot symptoms on inoculated apple fruits of ‘Empire’, ‘Golden Delicious’, and ‘Honeycrisp’. The symptoms exhibited noticeably sunken lesions with orange or dark acervuli from infections by C. fioriniae (Fig. 3). In the cross-section, necrotic V-shaped symptomatic tissues were observed beneath the surface lesions. We conducted the mean separation procedure using Fischer’s least significant difference to compare diameters of lesions developed following inoculation of apple fruits. There was a significant (P = 0.015) difference between the mean lesion diameters of C. siamense and C. chrysophilum, a significant (P = 0.05) difference between those of C. fioriniae and C. chrysophilum, and no significant (P > 0.05) difference between those of C. siamense and C. fioriniae (Table 4). C. chrysophilum produced deeper lesions on ‘Empire’ and ‘Golden Delicious’ fruits than the other two pathogens. Only isolates of C. fioriniae produced acervuli on the lesions (Fig. 3). Pathogens were re-isolated from the lesions and re-identified as Colletotrichum spp. using the aforementioned methods. Fruits treated with SDW did not develop any symptoms.
Symptoms of bitter rot disease on inoculated apple fruits of ‘Empire’ (A), ‘Golden Delicious’ (B), and ‘Honeycrisp’ (C) in the laboratory. Colletotrichum fioriniae (left), C. chrysophilum (center), and C. siamense (right). Acervuli were produced by C. fioriniae on ‘Empire’, ‘Golden Delicious’, and ‘Honeycrisp’ apples.
Citation: HortTechnology 34, 2; 10.21273/HORTTECH05377-23
Fungicide efficacy
Laboratory fungicide evaluations
The mean EC50 values of benzovindiflupyr, captan, fluxapyroxad + pyraclostrobin, potassium phosphite, trifloxystrobin, and zinc dimethyldithiocarbamate were 8.3, 8.0, 0.11, 818.5, 76.1, and 37.6 mg⋅L−1, respectively, for all nine isolates tested (Table 5).
Orchard evaluations
During the orchard experiments, bitter rot was the only disease that developed in the plots. In 2019, the disease was first observed on 21 Jul, and its incidence increased as the season progressed. Benzovindiflupyr, captan, and fluxapyroxad + pyraclostrobin were the most effective fungicides for preventing the development of bitter rot on fruits. Benzovindiflupyr and captan were effective at all three spray intervals (7, 10, and 14 d). Benzovindiflupyr was effective at the 7-d interval (only sprayed interval) for protecting against disease development. Potassium phosphite and monopotassium and dipotassium phosphite (Reliant; Quest Products, LLC, Greeley, CO, USA) were the least effective fungicides for preventing disease development. The incidence of bitter rot of fruits was significantly (P = 0.01) higher in untreated plots than in the treated plots. Detailed results of the experiment in 2019 are not presented because some fungicides did not effectively protect fruits against bitter rot disease (Table 6), and there were no significant differences in the applications of benzovindiflupyr, captan, and fluxapyroxad + pyraclostrobin at 7-, 10-, and 14-d intervals.
In 2020, bitter rot was observed only in untreated plots. The disease was first observed on 25 Jul in two untreated plots, and its incidence in untreated plots increased as the season progressed. The disease incidence in untreated plots was 100% on 12 Sep. Bitter rot and other diseases of apples were not observed in the treated plots. All fungicide combinations, applied either at 10-d or 14-d intervals, were effective for preventing disease development (Table 8).
In 2021, fruits with bitter rot were first observed on 11 Jul in two untreated plots, and its incidence in untreated plots increased toward the end of the season. Bitter rot and other diseases of apple were not observed in treated plots until 5 Sep (16 d after the last fungicide application). Symptomatic fruits were observed in some treated plots on 5 Sep (Table 8). On 12 Sep (21 d after the last fungicide applications), bitter rot was observed in most of the plots. The lowest incidence of symptomatic fruits (0.5%) was observed in the plots sprayed with fluxapyroxad + pyraclostrobin plus captan (Table 8). More rainfall than usual occurred from 20 Aug to 15 Sep in 2021.
Discussion
This was the first field survey to assess the incidence of bitter rot disease in commercial apple orchards in Illinois. The results of our studies showed that some apple cultivars (Empire, Golden Delicious, Honeycrisp, and Jonathan) in Illinois are more susceptible than other cultivars. Studies in other apple-growing areas have also shown that ‘Honeycrisp and ‘Golden Delicious’ are more susceptible to bitter rot pathogens (Biggs and Miller 2001). Additionally, the disease was more prevalent in southern Illinois (with warmer and more humid conditions) than in northern Illinois (with cooler and less humid conditions), consistent with the results of Martin et al. (2021), who also reported that warmer temperatures and moist conditions are more conducive for bitter rot development.
In previous studies, combinations of morphology and molecular methods have been used to achieve accurate identification of Colletotrichum species (Gonzalez and Sutton 2004; Mills et al. 1992). The results of our study confirmed that using both colony morphology and molecular methods are necessary for the accurate identification of Colletotrichum species.
Our research identified Colletotrichum species that cause bitter rot of apples in Illinois orchards belong to the following three species: C. chrysophilum (1.85% of isolates), C. fioriniae (50.37% of isolates), and C. siamense (47.78% of isolates). These species belong to the C. acutatum and C. gloeosporioides species complexes and have been caused bitter rot of apples in other apple-growing areas in the world (Dowling et al. 2020; González et al. 2006; Martin and Peter 2021; Munir et al. 2016; Park et al. 2018; Peres et al. 2007). In 2021, C. chrysophilum was identified as a causal agent of bitter rot in only one orchard in central Illinois. This finding represents the first report of C. chrysophilum as a cause of bitter rot of apples in the Midwestern United States and the third report of C. chrysophilum as a pathogen of apples in the United States. C. chrysophilum was first reported as an incitant of bitter rot of apples in New York in 2020 (Khodadadi et al. 2020). C. fioriniae was the dominant species in the northern area, whereas C. siamense was more prevalent in the central and southern parts of the state. Both species were found within the same orchard and on the same fruit in several locations across the state. Overall, C. fioriniae was the most prevalent species identified. This is consistent with the findings of previous studies in Kentucky (Munir et al. 2016) and New York (Khodadadi et al. 2020), which reported C. fioriniae as the dominant species in both states.
All three identified Colletotrichum species caused bitter rot in inoculated apple fruits in the laboratory. On average, C. siamense produced larger lesions than C. fioriniae and C. chrysophilum on all three apple cultivars tested. This result is similar to that of previous studies in Kentucky (Munir et al. 2016) and South Korea (Oo et al. 2018). C. fioriniae isolates were the only ones to consistently exude red pigment on PDA, thus serving as a reliable morphological feature used for C. fioriniae identification.
Effective management of bitter rot requires timely cultural practices and applications of effective fungicides. Because species of bitter rot pathogens may vary in their sensitivity to fungicides (Chechi et al. 2019; Dowling et al. 2020; Gonzalez and Sutton 2004; Khodadadi et al. 2020; Martin et al. 2022; Wang et al. 2015), we evaluated the effectiveness of the potential fungicides in the laboratory against isolates of three identified species of Colletotrichum that had been identified in Illinois. Both laboratory and field studies showed that benzovindiflupyr, captan, and pyraclostrobin + fluxapyroxad were highly effective for managing Colletotrichum species that cause bitter rot disease of apples. Similar results have been reported by other studies of the management of bitter rot of apples (Chechi et al. 2019; Chen et al. 2016; Khodadadi et al. 2020; Martin et al. 2022; Munir et al. 2016) and Colletotrichum spp. on different crops (Adaskaveg and Hartin 1997; Bernstein et al. 1995; Freeman et al. 1998; Peres et al. 2007; Valero et al. 2010; Velho et al. 2014, 2015).
Results from our orchard trials revealed that benzovindiflupyr, captan, and fluxapyroxad + pyraclostrobin applied at either 10- or 14-d intervals were highly effective for managing bitter rot disease on ‘Honeycrisp’ apple. We did not have access to other apple cultivars susceptible to bitter rot for additional orchard experiments. Investigating the effectiveness of these fungicides for managing bitter rot and other summer diseases of apples such as ‘Empire’, ‘Golden Delicious’, and ‘Jonathan’ would provide more information regarding the chemical management of bitter rot disease of apples. However, commercial apple growers in Illinois who used the findings of our studies during 2020–2023 regarding various apple cultivars expressed their high satisfaction with the results.
Preharvest intervals of benzovindiflupyr (FRAC 7), captan (FRAC M4), and fluxapyroxad + pyraclostrobin (FRAC 7 + 11) are 30, 0, and 0 d, respectively. Thus, we suggest using benzovindiflupyr during the early summer season. Mixed sprays of captan with trifloxystrobin (FRAC 11), a phosphonate compound (i.e., potassium phosphite or monopotassium and dipotassium phosphite; FRAC P07), thiophanate-methyl (FRAC 1), or zinc dimethyldithiocarbamate (FRAC M) were effective for protecting apple fruits against bitter rot and other summer diseases. Spray applications of any of these mixed compounds in alternation with fluxapyroxad + pyraclostrobin plus captan are effective. For some of the treatments used for our orchard experiments, the numbers of applications and/or maximum rates of fungicides per season were more than those recommended by the labels. These procedures were performed for research purposes to determine the effectiveness of the fungicides. The label recommendations regarding the numbers of applications and total amount per season for each fungicide must be followed. During the past 3 years, we have been monitoring applications of the recommended fungicides according to the label directions in commercial apple orchards in Illinois. The recommended treatments in commercial orchards have been highly effective for managing bitter rot and other diseases of apples in Illinois.
Conclusions and recommendations
The results of our study and those of studies by other investigators have shown that bitter rot disease caused by Colletotrichum spp. is one of the most serious diseases of apples in areas with moist conditions. To develop effective management of bitter rot in each apple-growing area, the following actions are necessary: determine the susceptibility of the cultivars; identify the species of the causal agent(s); test the potential fungicides to determine their efficacy; and, after considering preharvest intervals specified on the fungicide labels, spray trees with effective fungicides at 14-d intervals until a maximum of 2 weeks before fruit harvest. All apple growers in Illinois who conducted timely cultural practices (removed mummified fruit, dead wood, and fire-blighted twigs before bloom) and applied the recommended fungicides in a timely manner reduced the incidence of bitter rot in their orchards from up to 100% fruit losses to 0% or negligible losses in most of the orchards with a history of bitter rot.
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