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Managing Colletotrichum on Fruit Crops: A “Complex” Challenge

    Affiliations
    Authors and Affiliations
    • Madeline Dowling1
    • Natalia Peres2
    • Sara Villani3
    • Guido Schnabel1 †
    1. 1Department of Plant and Environmental Sciences, Clemson University, Clemson, SC 29634
    2. 2Department of Plant Pathology, University of Florida, Gulf Coast Research and Education Center, Wimauma, FL 33598
    3. 3Department of Entomology & Plant Pathology, North Carolina State University, Raleigh, NC 27695
    Published Online:14 Jul 2020https://doi.org/10.1094/PDIS-11-19-2378-FE

    Abstract

    The fungal genus Colletotrichum includes numerous important plant pathogenic species and species complexes that infect a wide variety of hosts. Its taxonomy is particularly complex because species’ phenotypes and genotypes are difficult to differentiate. Two notable complexes, C. acutatum and C. gloeosporioides, are known for infecting temperate fruit crops worldwide. Even species within these complexes vary in traits such as tissue specificity, aggressiveness, geographic distribution, and fungicide sensitivity. With few effective chemicals available to control these pathogens, and the persistent threat of fungicide resistance, there is a need for greater understanding of this destructive genus and the methods that can be used for disease management. This review summarizes current research on diseases caused by Colletotrichum spp. on major fruit crops in the United States, focusing on the taxonomy of species involved, disease management strategies, and future management outlook.

    Species from the genus Colletotrichum cause destructive diseases on a wide variety of fruit crops. In 2012, the genus was voted one of the top 10 fungal plant pathogens because of its broad host range, devastation of essential crops, and importance as a postharvest pathogen (Dean et al. 2012). Species of Colletotrichum are also important because of their use as model organisms (Cannon et al. 2012). In the United States, Colletotrichum spp. can cause severe losses on diverse and economically important temperate fruit crops such as apple, peach, grape, blueberry, cranberry, and strawberry (Bernstein et al. 1995; Daykin 1984b; González and Sutton 2004; Howard 1972; Munir et al. 2016). This review will often highlight strawberry anthracnose because it has been extensively studied due to its importance and prevalence. Despite the importance of Colletotrichum spp. as pathogens, the vast complexity and diversity of this genus make it difficult to understand and study.

    Colletotrichum has been termed “a catalogue of confusion,” due to its perpetually changing taxonomy (Bernstein et al. 1995; Hyde et al. 2009b). Obstacles to systematizing the genus are numerous: limited morphological differentiation among species, rare presence of sexual stages, and variation in pathogenicity and cultural morphology (Cannon et al. 2012; Johnson 2018). In addition, hosts and infection courts of different species frequently overlap. Multiple species have been detected infecting the same fruit, and even within the same lesion on apple (Munir et al. 2016). Historically, the two most important Colletotrichum species infecting temperate fruit crops were reported as Colletotrichum acutatum Simmonds ex Simmonds and Colletotrichum gloeosporioides (Penzig) Penzig & Saccardo (Adaskaveg and Hartin 1997; Bernstein et al. 1995; Cannon et al. 2012; Dean et al. 2012; Freeman et al. 1998). However, the advent of sequencing and molecular identification revealed that these species, along with other species of Colletotrichum, are actually “species complexes,” composed of numerous diverse and distinct species (Damm et al. 2012; Weir et al. 2012). Some researchers have even suggested classifying C. siamense, a species within the C. gloeosporioides complex, as a complex within a complex (Sharma et al. 2013; Vieira et al. 2014)! With new advances in sequencing technology, older systematics for naming Colletotrichum spp. became obsolete, resulting in decades of publications containing inaccurately identified species and genetic sequences (Hyde et al. 2009b).

    Outbreaks caused by these species can be devastating. For example, epidemics of bitter rot of apple, caused by fungi in the C. acutatum and C. gloeosporioides species complexes, can result in loss of an entire crop during favorable conditions (S. Villani, personal communication). Prior to the use of synthetic fungicides, a bitter rot outbreak in the United States resulted in losses equivalent to approximately $400 million in today’s currency based on relative income, and an agricultural bulletin declared, “There is no other disease so enormously destructive to the apple fruit as is the one commonly called bitter rot.” (Burrill 1902). More recently, epidemics of strawberry anthracnose have been reported within major strawberry producing states, such as Florida and North Carolina as well as internationally in Israel, Brazil, and Bulgaria (Adaskaveg and Hartin 1997; Delp 1980; Ellis and Omer 2016; Freeman and Katan 1997; Henz 1992; Jelev et al. 2008). Anthracnose outbreaks have also occurred on peach and almond in California (Adaskaveg and Hartin 1997). In tropical and temperate fruit crops, postharvest outbreaks may also result in substantial financial losses to growers (Adaskaveg and Hartin 1997; Arauz 2000; Brown 1975; Prusky 1993; Rosenberger and Cox 2016). These recent outbreaks occurred despite the use of chemical control measures. The limited number of effective fungicides registered for control and the increasing prevalence of fungicide resistance limits chemical management options for diseases caused by Colletotrichum spp.

    Symptoms and Signs

    Anthracnose is the most common name given to diseases caused by Colletotrichum spp., though a variety of other names are used as well, including bitter rot on apple and cranberry, and ripe rot on grape and blueberry. The name anthracnose, literally “coal disease,” refers to the dark, necrotic lesions typically caused by Colletotrichum spp. (Oxford English Dictionary, n.d.). Within these necrotic lesions, acervuli may form under favorable conditions, producing pink to orange mucilaginous masses of conidia (Fig. 1). Some Colletotrichum spp., most notably those in the C. gloeosporioides species complex (Glomerella spp.) may form perithecia on the tissue of temperate fruit hosts.

    Fig. 1.

    Fig. 1. Symptoms and signs of Colletotrichum spp. causing anthracnose fruit rot on strawberry: A, ripening strawberry infected by C. acutatum; B, microscopic image of C. acutatum sticky spore masses on anthracnose lesion; C, microscopic image of C. acutatum spores. Reprinted with permission of Taylor & Francis Ltd.

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    Fruit rots are often the most obvious and economically important symptoms of diseases caused by Colletotrichum spp. (Fig. 2). However, various species, often in the C. gloeosporioides complex, infect leaves, crowns, stems, twigs, and petioles as well (Fig. 3). On apple, 75% of an orchard may be defoliated before harvest due to infections by fungi in the C. gloeosporioides complex (González and Sutton 1999; González et al. 2006). All parts of strawberry plants may be infected, including fruit, petioles, crowns, roots, stolons, and leaves (Freeman and Katan 1997; Freeman et al. 2001; Mertely and Legard 2004; Smith 2008; Ureña-Padilla et al. 2002). Highbush blueberry blossoms and fruiting wood may also be severely affected by C. gloeosporioides spp., and similar infections of vegetative cranberry tissue such as leaves and stems have also been reported (Doyle et al. 2013; Hartung et al. 1981; Oudemans et al. 1998).

    Fig. 2.

    Fig. 2. Symptoms of diseases caused by Colletotrichum spp. on representative temperate fruits: peach, apple, grape, and strawberry.

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    Fig. 3.

    Fig. 3. A comparison of Colletotrichum acutatum and C. gloeosporioides on temperate fruit crops.

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    Symptoms do not always appear when plants are colonized by Colletotrichum spp. For example, C. acutatum has been detected germinating and sporulating on symptomless strawberry and blueberry leaves (Leandro et al. 2001; Yoshida et al. 2007). Quiescent infections on immature fruit are also common (Fig. 3).

    Disease Cycle

    Nearly every crop worldwide serves as a host for at least one species of Colletotrichum (Dean et al. 2012). Colletotrichum spp. vary in their infection strategies on different hosts, and species and may preferentially infect different tissues (Fig. 3) (De Silva et al. 2017; González et al. 2006; Ureña-Padilla et al. 2002). However, disease cycles of Colletotrichum spp. infecting temperate fruit crops share several overarching similarities.

    First, Colletotrichum spp. can overwinter in mummified fruit, infected vegetative tissue, or on symptomless plants. For example, in muscadine grape, C. gloeosporioides was found overwintering in mummies, pedicels, and fruit spurs. On apple, both sexual and asexual stages of species in the C. gloeosporioides complex overwinter in dead wood and perithecia develop on infected fallen leaves. During warm spring rains, ascospores and conidia are released to initiate primary infections on emerging foliage and fruit (Sutton 1983). Though not technically “overwintering,” C. acutatum may germinate and even sporulate on strawberry leaves, through a process called secondary conidiation, without producing symptoms. In annual production systems, where plants are initially grown in nurseries, this allows it to be unwittingly transferred into the field, providing a continuous source of inoculum at the beginning of the season (Leandro et al. 2001). Weeds have also been shown to harbor C. acutatum that can lead to strawberry infections (Freeman et al. 2001) (Fig. 4). However, a multicrop study in Florida indicated that isolates of C. acutatum may be host-specific and pose little threat to other crops (MacKenzie et al. 2009b).

    Fig. 4.

    Fig. 4. Representative Colletotrichum disease cycle: disease cycle of anthracnose on strawberry caused by C. acutatum. Reprinted with permission of Taylor & Francis Ltd.

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    Second, the pathogen is typically waterborne, and spreads through rain-splash (Sutton 1983; Yang 1990), and infection is generally favored by warm and moist conditions (Brook 1977; Samuelian et al. 2012; Smith 2008).

    Third, quiescent infections of immature fruit, resulting in postharvest disease are common in temperate fruit crops, including peach, grape, strawberry, and apple (Biggs 1995; Daykin 1984b; King et al. 1997; Zaitlin et al. 2000). Quiescent infections on apple may even enlarge during cold storage (Biggs 1995). On grape, C. gloeosporioides can penetrate via appressoria, then become dormant, or spores remain dormant on the fruit surface until conditions favor infection (Daykin 1984a).

    Lastly, once infection, colonization, and symptom development occur, acervuli or perithecia often appear in lesions, producing inoculum and creating a continuous cycle of disease. Perithecial production has not been detected outside of the laboratory for many fruit-infecting Colletotrichum spp., most notably C. acutatum (Cannon et al. 2012). However, the sexual stage of C. gloeosporioides, formerly called Glomerella cingulata, occurs more frequently, and is generally present on plant debris (Peres et al. 2005). Because of the current mycological emphasis on “one fungus, one name,” we will refer to all Colletotrichum spp. by their asexual names in this review (Miller et al. 2011).

    Other reviews have provided excellent summaries of the lifestyles (De Silva et al. 2017; Peres et al. 2005), taxonomy (Damm et al. 2012; Hyde et al. 2009b, a), and biology (Wharton and Diéguez-Uribeondo 2004) of Colletotrichum spp. on fruit crops and other crop systems. With the increasing presence of fungicide resistance and the variable response of different species to management methods, this review will emphasize management of diseases caused by Colletotrichum spp. on major fruit crops in the United States, focusing on the species involved, disease management strategies, and future outlooks for disease management.

    Species Within the C. acutatum and C. gloeosporioides Complexes Responsible for Disease on Fruit Crops

    Four complexes: C. truncatum, C. boninense, C. acutatum, and C. gloeosporioides have been reported from temperate fruit crops (Table 1). Of these complexes, C. acutatum and C. gloeosporioides are by far the most prevalent in literature (Adaskaveg and Hartin 1997; Bernstein et al. 1995; Damm et al. 2012; Dean et al. 2012; Freeman et al. 1998) (Table 1). C. acutatum and C. gloeosporioides species complexes have been traditionally distinguished by C. acutatum’s fusiform conidia with acute ends (Simmonds 1966). Unfortunately, this trait varies widely among Colletotrichum species and even among isolates to such a degree that it is unreliable for diagnosis (Damm et al. 2012). However, the ITS region, commonly used for fungal identification, can distinguish the Colletotrichum complexes (Cannon et al. 2012; Hyde et al. 2009a) (Fig. 5). Morphological and molecular evidence for subgroups below the complex level has been described in C. acutatum for over 20 years (Guerber et al. 2003; Lardner et al. 1999). However, accurate differentiation below the complex level is difficult and currently requires a multigene sequencing approach (Damm et al. 2012; Weir et al. 2012) (Table 2; Fig. 5). Fortunately, researchers have made tremendous progress classifying species using multigene sequencing combined with “polyphasic characters,” important life traits such as physiology, pathogenicity, and cultural characteristics (Cai et al. 2009). Using these methods, C. gloeosporioides and C. acutatum sensu lato were separated into 22 and 29 species, respectively, in 2012 (Damm et al. 2012; Weir et al. 2012). While this excellent study has benefited our understanding of C. acutatum and C. gloeosporioides, there is still more to be done. For example, as one paper states, “there is no consensus as to which loci should be used to infer phylogeny” (Cannon et al. 2012). Some studies use up to nine genes, while others use only two genes (Cannon et al. 2012). This inconsistency and confusion combined with the difficulty and cost of sequencing multiple genes for identification of each isolate makes researchers less apt to distinguish species below the complex level. Vieira et al. (2020) performed an analysis to find a group of markers that would distinguish all species of Colletotrichum. Unfortunately, they concluded that such markers do not exist. Markers that were most effective for one complex were least effective for another. However, they propose that for most complexes, three highly variable genes can provide excellent differentiation to the species level (Table 2). To perform initial determination of species complex, Vieira et al. (2020) suggest that a single gene, such as GAPDH or TUB2, be used, and further differentiation be performed using other genes chosen based on complex identity (Fig. 5). However, the large number of species that may infect a single host make experimental design complicated. For example, our literature analysis revealed 21 species of Colletotrichum causing bitter rot on apple, 15 causing anthracnose on strawberry, and 12 causing ripe rot on grape worldwide (Table 1).

    Table 1. List of Colletotrichum spp. on major fruit crops compiled from literature reports. See Supplementary Table S1 for literature sources.

    Fig. 5.

    Fig. 5. Levels of complexity in distinguishing Colletotrichum spp.

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    Table 2. Primers to amplify multilocus regions for identification of Colletotrichum spp. on fruit crops

    Though distinguishing species is often challenging, it is necessary due to marked differences in their lifestyle traits (Peres et al. 2005). Here we review the differences between and within the C. gloeosporioides and C. acutatum species complexes on temperate fruit crops and highlight the importance of accurate species differentiation below the complex level.

    Plant organ specificity.

    C. gloeosporioides and C. acutatum have long been known to differ in host organ preference on fruit crops. For example, on strawberry, C. gloeosporioides is most often associated with crown rot infections, while C. acutatum is considered the main fruit rot pathogen, though both species are capable of infecting both crowns and fruit (Maas 1998; Ureña-Padilla et al. 2002). A similar organ preference occurs on apple, where species in the C. gloeosporioides complex are most often associated with Glomerella leaf spot symptoms, while species in both complexes are known to cause fruit rot (González et al. 2006; Johnson 2018). Below the complex level, little is known about host organ preferences, though one recent report on apple did not detect organ specificity among species within complexes infecting apple in North Carolina (Hoge 2017).

    Pathogen aggressiveness and symptom severity.

    On a single host organ type, species within each complex often vary in aggressiveness. Within the C. gloeosporioides complex, C. siamense infections on apple fruit consistently produced deeper and larger lesions than C. fructicola or C. theobromicola (Munir et al. 2016). Differences among species within the C. acutatum complex also occurred on apple, where C. fioriniae created more severe lesions than C. nymphaeae (Munir et al. 2016). A similar phenomenon was observed on peach fruit within the C. gloeosporioides species complex, where C. fructicola was more aggressive than C. siamense (Hu et al. 2015b). Interestingly, aggressiveness may even vary within a species within a species complex. Two isolates of C. nymphaeae on strawberry caused significantly larger lesions on fruit than other C. nymphaeae isolates, and these two isolates clustered apart from other isolates of the same species in a phylogenetic tree (Wang et al. 2019). Characterizing species and their aggressiveness on specific tissues is important for both laboratory research and field application. In laboratory work, results can be greatly altered depending on the aggressiveness of species used to inoculate plants or detached fruit. In the field, it is critical that management methods target the species causing disease, particularly when fungicide sensitivities and inoculum source may vary among species.

    Species frequency/distribution (United States).

    To date, studies using multigene methods to differentiate species within the C. gloeosporioides and C. acutatum complexes indicate that C. nymphaeae and C. fioriniae are the most commonly reported pathogens infecting many temperate fruit crops in the United States. C. nymphaeae is considered the major species within the C. acutatum complex infecting strawberry plants in the United States, while C. fioriniae has been reported as a major pathogen of grape, apple, and blueberry in the Mid-Atlantic and Southeastern United States, and C. fructivorum, within the C. gloeosporioides complex, is considered the major pathogen on cranberry (Johnson 2018; Kepner and Swett 2018; Munir et al. 2016; Shi 1996; Waller et al. 2018; Wang et al. 2019). However, other species within both complexes have been implicated causing disease on these crops to a lesser extent, indicating possible host, cultivar, or geographic preference that allows certain species within a complex to dominate over others (Biggs and Miller 2001; Grammen et al. 2019a; López-Moral et al. 2019).

    Species frequency/distribution (international).

    Geographically, there is variation in the species within each complex that is involved in temperate fruit crop diseases. For example, C. fructicola is considered the most important species causing apple bitter rot in Uruguay and Brazil, but both C. fructicola and C. fioriniae are the species most commonly detected in the Mid-Atlantic and Southeastern United States, depending on cultivar, while C. godetiae was most commonly found in a study in Belgium (Grammen et al. 2019b; Johnson et al. 2018; Munir et al. 2016; Rockenbach et al. 2016; Shi 1996). Possibly, this variation is due to differences in sampling and identification methods. It may also be due to differences in cultivars surveyed, environmental factors, climate, and management strategies. Because only limited research has yet been performed involving Colletotrichum spp. below the complex level, it is difficult to pinpoint the reason for differing reports. Currently, literature provides examples of Colletotrichum spp. not reported in the United States that are regularly detected internationally (Table 1). For example, C. godetiae is commonly found on apple, strawberry, and grape in Europe, but has never been reported in North or South America (Baroncelli et al. 2014; Grammen et al. 2019a; Munda 2014; Wenneker et al. 2016; Zapparata et al. 2017). Also, C. viniferum was the most common species detected in a study in China, and was also detected in Korea and Brazil, but was not detected in a study of Colletotrichum spp. causing ripe rot conducted in the Mid-Atlantic United States (Kepner and Swett 2018; Peng et al. 2013). Though these geographical differences in species distribution and frequency may be due to a variety or combination of factors, they are important to consider when performing research using Colletotrichum spp. since species-specific management practices in one geographical location may be irrelevant in another. Regional and national surveys on multiple crops are necessary to better understand the geographic distribution of species within each complex as there are significant gaps in our knowledge of species distribution for many crops that are regularly devastated by anthracnose.

    Fungicide sensitivity.

    Understanding fungicide sensitivity differences among species of Colletotrichum is critical for developing appropriate management recommendations. Growers have reported variability in fungicide efficacy against bitter rot of apple (Munir et al. 2016) that is likely due to differences in fungicide sensitivity among Colletotrichum species and species complexes. Overall, Colletotrichum spp. on fruit crops often differ in sensitivity to fungicides such as quinone-outside inhibitors (QoI; FRAC 11), methyl benzimidazole carbamates (MBC; FRAC 1), and demethylation inhibitors (DMI, FRAC 3), though both complexes showed limited sensitivity to the succinate dehydrogenase inhibitor fungicides boscalid, fluxapyroxad, and fluopyram (Chen et al. 2016, 2018; Chung et al. 2006; Ishii et al. 2016; Meazza et al. 2003).

    Differences in fungicide sensitivity among species from a single geographical location may be inherent or may have emerged as a result of fungicide selection pressure influenced by species-specific inoculum frequency and density, and influx of wildtype phenotypes from nearby hosts. On peach, multiple isolates of the species C. nymphaeae were resistant to the DMI fungicides flutriafol and fenbuconazole while other species tested, including C. fioriniae, were sensitive (Chen et al. 2016). A report of C. fructicola and C. siamense in commercial peach orchards in South Carolina revealed multiple isolates of C. siamense were resistant to QoI fungicides and some even dual-resistant to QoI and MBC fungicides, while C. fructicola isolates were sensitive (Hu et al. 2015b). A similar study of apple bitter rot in Japan provided opposite results, with C. fructicola isolates frequently resistant to QoI fungicides and dual-resistant to MBC and QoI fungicides, while no C. siamense isolates in the study were fungicide resistant (Yokosawa et al. 2017). Knowing fungicide resistance profiles of species is important for providing appropriate management recommendations to growers to ensure that only effective fungicides are being applied.

    These examples highlight the necessity for differentiating C. gloeosporioides and C. acutatum spp. further than the complex level. Much work is still necessary to understand the geographic distribution of these species and learn more about their unique and shared traits. Multigene analysis has helped resolve some of the confusion within the Colletotrichum genus. However, the high diversity it has revealed within each complex leads to further questions about whether the genes currently used are enough to resolve species with markedly different lifestyles. On strawberry, Wang et al. (2019) discovered C. nymphaeae isolates that caused more severe symptoms than other representatives of the C. nymphaeae species. These isolates were also genetically different, as evidenced by phylogenetic trees. A similar phenomenon was observed by Chen et al. (2016) on peach, where some “C. fioriniae-like” isolates were detected clustering in a separate group from C. fioriniae and had different fungicide resistance profiles than typical C. fioriniae, but were not differentiated by the molecular markers used. While some light is dawning on the “catalogue of confusion,” it is also revealing a wealth of diversity that has not been explored.

    Managing Colletotrichum spp. on Fruit Crops

    Though a variety of integrated management methods have been attempted over the years to combat Colletotrichum spp. infecting fruit crops, chemical control remains the major method for managing these diseases. This section will review general cultural and biological control methods, but will focus on chemical control of Colletotrichum spp., specifically the chemical classes available for management and current reports of fungicide resistance in Colletotrichum spp.

    Cultural and biological control.

    Various cultural practices are routinely employed to manage diseases caused by Colletotrichum spp. on temperate fruit crops. These vary with the disease cycle, but in general seek to adjust conditions to decrease inoculum production and spread. For example, in annual strawberry production, management methods involve generating and using clean planting stock, limiting rain-splash and overhead irrigation, adjusting soil nutrients, managing weeds, and using cultivars with limited susceptibility to the disease (Coelho et al. 2008; Freeman 2008; Maas 1998; Walter et al. 2008). Production of clean planting stock is paramount, but also difficult since symptomless infections are frequent and difficult to detect (Debode et al. 2015; Leandro et al. 2001, 2003). Methods such as LAMP and quantitative PCR assays are being refined that allow detection of symptomless infections (Rahman et al. 2019; Zhang et al. 2016). However, application at the large scale of strawberry plant production is challenging. Symptomless infections by members of the C. acutatum species complex may also occur on apple leaves, providing an additional source of inoculum (Børve and Stensvand 2017). For most fruit crops, removing diseased and mummified fruit and symptomatic vegetative tissue is generally recommended (Feil et al. 2003; Stensvand et al. 2017; Sutton et al. 2014; Wilcox et al. 2015). Removing whole sections of strawberry fields affected by anthracnose crown rot is recommended to decrease the spread of disease in nurseries or commercial production fields (Rahman et al. 2015). However, in regions with warm temperatures such as Florida, sanitation of strawberry fields may not be helpful since berries on the orchard floor are not considered a major source of inoculum (Ureña-Padilla et al. 2001). Instead, alternate hosts are the major source of inoculum for C. gloeosporioides strawberry crown infections in Florida, and high temperatures prevent the inoculum from over-summering (Ureña-Padilla et al. 2001). Breeding for host-resistance is an environmentally friendly and cost-effective method for managing disease if the resistant cultivars have other desired traits. However, while several cultivars of apple, strawberry, and blueberry have moderate resistance to C. acutatum or C. gloeosporioides, none of the commercially marketable cultivars provide complete protection against the disease. A case study discussing host resistance breeding against diseases caused by Colletotrichum spp. is provided later in this review.

    Many biological controls and biofungicides have been tested against Colletotrichum spp. on fruit crops, but none have reliably provided field efficacy. Numerous organisms have been tested including fungi such as Trichoderma spp., avirulent strains of Colletotrichum, and yeasts, as well as bacteria such as Bacillus subtilus and Streptomyces lydicus (Chalfoun et al. 2011; Conway et al. 2004; Freeman et al. 2004; Kim and Hwang 2007; Salazar et al. 2007, 2013). The most promising organisms, biofungicides, and systemic acquired resistance inducers have been formulated for testing in field trials, but they have provided inconsistent control even when combined with traditional chemical products (Cosseboom et al. 2018; Mertely and Peres 2013; Mertely et al. 2011, 2015, 2017; Shuman and Bratsch 2004; Yoder et al. 2017). Therefore, though most commercial growers use applicable cultural controls, they mainly rely on chemical control to manage Colletotrichum spp.

    Chemical control.

    Multisite protectant fungicides, including mancozeb, thiram, ziram, captan, and chlorothalonil, have historically provided moderate to good efficacy against plant diseases, including diseases caused by Colletotrichum spp. However, increasingly stringent export regulations for these fungicides and restrictions on applications despite wetter and longer growing seasons has necessitated the incorporation of modern locally systemic fungicides into Colletotrichum management programs.

    There are currently seven single single-site chemistries recommended in United States’ spray guides for Colletotrichum control (Table 3): methyl benzimidazole carbamates (MBC; FRAC 1), demethylation inhibitors (DMI; FRAC 3), succinate dehydrogenase inhibitors (SDHI; FRAC 7), quinone-outside inhibitors (QoI; FRAC 11), phenylpyrroles (PP, FRAC 12), polyoxins (FRAC 19), and fluazinam (FRAC 29). Of this list, MBCs, DMIs, SDHIs, QoIs, polyoxins, and fluazinam are available as solo products.

    Table 3. Currently registered chemicals for management of diseases caused by Colletotrichum spp. on fruit crops

    QoI (FRAC 11) fungicides.

    While there are many single-site options registered for controlling Colletotrichum spp. on fruit crops, QoI fungicides are arguably the most consistently effective mode of action registered against Colletotrichum spp. Ten of the 22 commonly used single-site fungicides and mixtures registered for managing Colletotrichum spp. on fruit crops are QoIs or QoI mixtures (Table 2), and there is at least one QoI or QoI mixture registered for every major fruit crop.

    Unfortunately, this high efficacy has produced a dependence on QoI fungicides that has led to selection for fungicide resistance. Colletotrichum spp. resistant to FRAC 11 (QoI) fungicides have already been reported on multiple commercial temperate fruit crops in the United States including apple, strawberry, peach, and blueberry (Chechi et al. 2019; Forcelini et al. 2016; Hu et al. 2015b; Johnson 2018). Widespread field resistance resulting in lack of control and disease outbreaks has occurred on strawberry, apple, and peach crops (Chechi et al. 2019; Forcelini et al. 2016; Hu et al. 2015a).

    QoIs act by binding to the cytochrome bc1 complex and blocking electron transfer, thus inhibiting mitochondrial respiration (Bartlett et al. 2002). Practical resistance to QoI fungicides is typically caused by mutations in the cytochrome b gene (CYTB) which prevent fungicide binding (Ma and Michailides 2005). The most commonly reported mutation, G143A, results in qualitative resistance to QoI fungicides, whereas other mutations, such as F129L, typically result in moderate or partial resistance (Fernandez-Ortuno et al. 2008; Fisher et al. 2004). The G143A mutation has been reported to confer high resistance to isolates of C. acutatum and C. gloeosporioides collected from apple, strawberry, blueberry, and peach in the United States (Chechi et al. 2019; Forcelini et al. 2016; Hu et al. 2015b; Johnson 2018), whereas the F129L mutation was reported in isolates of C. acutatum infecting strawberry (Forcelini et al. 2016). Interestingly, C. siamense (C. gloeosporioides complex) from apple fruit from Illinois lacked any mutation in the CYTB gene but was resistant to the QoI fungicide azoxystrobin with an EC50 value greater than 100 μg/ml (Chechi et al. 2019). Reduced sensitivity to QoI fungicides and resistance in the absence of CYTB mutations has also been reported in other pathogens, indicating that other resistance mechanisms may be involved (Gisi et al. 2000; Olaya et al. 1998).

    MBC (FRAC 1) fungicides.

    In the past, reports stated that MBC fungicides provided good efficacy against C. gloeosporioides spp. (MacKenzie et al. 2009a). As mentioned previously, fungi in the C. acutatum complex have inherent resistance or insensitivity to MBC fungicides (Chung et al. 2006). However, resistance to MBCs has also developed in C. gloeosporioides, threatening to make this chemical class ineffective as a chemical control option (Table 3).

    MBC fungicides attack the fungal cytoskeleton by directly binding to β-tubulin subunits and inhibiting the assembly of microtubules (De Miccolis Angelini et al. 2015). Mutations in the β-tubulin gene can decrease the ability of the fungicide to bind, resulting in fungicide resistance (Davidse 1986; De Miccolis Angelini et al. 2015). Resistance to this fungicide class is widespread in many plant pathogens, and though many resistance mutations have been reported in various fungi, the most frequently reported mutations that confer resistance are E198A, E198G, E198K, and F200Y (De Miccolis Angelini et al. 2015). Low, intermediate, and high MBC fungicide resistance levels have been reported for C. gloeosporioides on pear in Japan (E198A, F200Y); peach (E198A), blueberry (E198A), apple (E198A), and strawberry (E198A) in the United States; and grape in Korea and China (F200Y) (Chechi et al. 2019; Chen et al. 2013; Chung et al. 2010; Hu et al. 2015a; Hwang et al. 2010).

    DMI (FRAC 3) fungicides.

    A variety of DMI fungicides are registered as solo and mixture products for different fruit crops. Solo products show poor to good efficacy against Colletotrichum spp., though they do not typically perform as well as QoI fungicides (Defrancesco et al. 2018). However, on blueberry in the southeastern United States, metconazole is reported as having excellent efficacy (Burrack et al. 2019). Most of the mixtures available are combinations with QoI fungicides and perform well against diseases on registered crops. A recent in vitro study on C. nymphaeae and C. fioriniae indicates that mixtures of two DMI fungicides can have a synergistic effect against Colletotrichum spp., presumably due to differential binding to paralogous CYP51 proteins (Chen et al. 2020). An in vitro study of C. truncatum from peach revealed that this species had resistance or reduced sensitivity to most DMI fungicides registered for anthracnose management (Chen et al. 2018). Fortunately, C. truncatum is currently not commonly found on temperate fruit crops.

    SDHI (FRAC 7) fungicides.

    Several SDHI fungicides are registered for use on fruit crops either as solo products or in mixtures. Benzovindiflupyr is registered for the suppression of apple bitter rot in the Midwestern U.S. and reported as having excellent efficacy against the disease caused by members of the C. acutatum species complex (Beckerman et al. 2020). However, the same product showed only moderate efficacy against bitter rot and Glomerella leaf spot on apple caused by C. fructicola in the southeast (Johnson et al. 2018; Ritchie et al. 2016). In 2020, benzovindiflupyr is not yet registered for anthracnose fruit rot of strawberry, but has showed good efficacy against the disease in preliminary field trials (Mertely et al. 2018, 2019). In laboratory studies, C. gloeosporioides s.l. and C. acutatum s.l. isolates were naturally resistant to boscalid, fluxapyroxad, and fluopyram (Ishii et al. 2016) (Table 3). However, in field trials, a mixture of pyraclostrobin (a QoI fungicide) and fluxapyroxad was consistently one of the most effective compounds against bitter rot and Glomerella leaf spot on apple, performing better than the products containing just pyraclostrobin or fluxapyroxad. That suggests that fluxapyroxad may have at least some activity against Colletotrichum spp. in the field, or that the two fungicides may function synergistically (Brannen et al. 2018; Villani et al. 2018). An older SDHI fungicide, penthiopyrad, demonstrated moderate to good efficacy in vitro and in field trials for management of Glomerella leaf spot and bitter rot caused by C. fructicola, and was granted a special registration for apple in certain states (Villani et al. 2019). Incomplete cross-resistance among some SDHI fungicides has been reported in several fungal species (Ishii et al. 2011). However, the fungicide resistance action committee does not recommend rotating multiple chemicals within this class since selection for mutations conferring resistance to multiple SDHIs (such as P225F in the SDHB gene) has occurred in other crops (Hermann and Stenzel 2019). Development of newer SDHIs such as benzovindiflupyr provides opportunities for greater intrinsic efficacy against pathogens.

    PP (FRAC 12).

    The combination of the FRAC 12 fungicide, fludioxonil, and the FRAC 9 fungicide, cyprodinil, is registered for controlling Colletotrichum spp. causing strawberry crown rot and fruit rot as well as blueberry anthracnose. This mixture has shown similar field efficacy to the QoI fungicide pyraclostrobin for anthracnose management on strawberry (Wedge et al. 2007). Preplant strawberry dip treatments using this chemical were also effective (Haack et al. 2018; Mertely et al. 2010). The combination of pydiflumetofen and fludioxonil is registered for grape ripe rot. In 2019, a special registration of this combination was enacted for blueberry and strawberry, and it showed consistently high efficacy against C. acutatum crown rot on strawberry in dip treatments, the same or better than that of azoxystrobin against sensitive isolates in preplant dip tests (Haack et al. 2018). However, this activity is likely due to fludioxonil, since solo pydiflumetofen did not typically provide significant control of the disease compared with the untreated control (Haack et al. 2018; Mertely et al. 2010). Phenylpyrrole fungicides are particularly useful since very few instances of resistance development have occurred in this class of fungicides (Kilani and Fillinger 2016).

    Polyoxin (FRAC 19) fungicides.

    Though it is registered for use against anthracnose diseases on most fruit crops and showed promising results during in vitro tests (Hao et al. 2017), field tests of polyoxin d zinc salt indicate that it has inconsistent and often limited efficacy against Colletotrichum spp. on these crops (McManus and Perry 2017; Pscheidt and Bassinette 2016; Su and Gubler 2009).

    Fluazinam (FRAC 29).

    Fluazinam is registered for use on apple, blueberry, and highbush cranberry. In addition, extension specialists have requested a special registration of this product for strawberry nurseries (Forcelini and Peres 2018). Fluazinam acts by uncoupling oxidative phosphorylation, thereby inhibiting mitochondrial respiration, and has shown efficacy against C. gloeosporioides and C. acutatum species complexes in vitro (Gang et al. 2015; Leroux 1996). It also has particularly low risk of resistance development.

    On apple, it performed well in multiple field trials, providing similar levels of control to the commercial mixture of pyraclostrobin and fluxapyroxad (Brannen et al. 2018; Ritchie et al. 2016). On blueberry and strawberry, its efficacy was moderate, comparable to captan (Beckerman et al. 2020; Mertely et al. 2018).

    Outlook for Management of Colletotrichum spp. on Fruit Crops

    While seven single-site chemistries are available for control of Colletotrichum spp. on fruit crops as a whole, the loss of QoI and MBC fungicides to resistance would leave few effective single-site chemistries available for resistance management on several important crops (Table 3). For example, on apple, only SDHI fungicides and fluazinam would be available, while blueberry anthracnose management would be limited to DMIs and an SDHI/PP mixture. The limited number of effective fungicide chemical classes, the threat of fungicide resistance, the limitations of sanitation practices, and lack of reliability of biological control agents justify the development of more tools for an integrated disease management approach. These efforts include host resistance breeding, registration of new chemistries, and resistance monitoring. This section will focus on the current status and future directions of fungicide resistance management in Colletotrichum spp.

    Resistance breeding (strawberry as a case study).

    One of the most promising, environmentally friendly, and potentially cost-effective means to reduce infection pressure and manage fungicide resistant Colletotrichum isolates is to develop resistant cultivars. While some cultivars of blueberry, apple, and strawberry are reported to be “less susceptible” or “resistant” to Colletotrichum spp., chemical control is still a necessity. In addition, cultivars “resistant” to anthracnose are often susceptible to other diseases or have other characteristics that limit their marketability. Ideally, breeding programs can overcome some of these obstacles to create fruit with resistance to disease without a wide variety of negative traits attached. Here we will review the history of breeding efforts for strawberry anthracnose at the University of Florida (UF).

    For more than 70 years, the UF strawberry breeding program has used recurrent phenotypic selection to develop strawberry cultivars with high yields (Whitaker et al. 2011), desirable fruit quality traits, and disease resistance (Whitaker et al. 2012b) including resistance to C. acutatum and C. gloeosporioides. Broad variability for resistance to anthracnose fruit rot (AFR) exists in the UF breeding germplasm, with most cultivars being moderately to highly resistant (Chandler et al. 2006; Seijo et al. 2008; Whitaker et al. 2012a, b, 2015, 2018). A single locus, FaRCa1, appears to account for the variability in resistance to C. acutatum in the UF breeding program (Salinas et al. 2019). This locus explained at least 50% of phenotypic variance for AFR incidence in two distinct QTL discovery populations and was validated in advanced selections and cultivars (Salinas et al. 2019). For C. gloeosporioides, a quantitative trait locus that accounts for most of the genetic variation for resistance in the discovery sets was found and named FaRCg1 (Anciro et al. 2018). For both diseases, high-throughput marker assays have been developed for the most significant SNPs that correlated with the mode of the QTL region. The discovery and characterization of these loci has been used to develop molecular tools that are used by the breeding program to achieve increased genetic gains for resistance. The cultivars developed by the UF breeding program with moderate to high levels of resistance to anthracnose fruit and crown rots have been successfully adopted by the Florida strawberry growers, in combination with cultural and chemical control methods, to manage the diseases caused by Colletotrichum spp. However, these cultivars might not always perform well in other climates.

    Fungicide resistance management strategies.

    One means to avoid selection of resistant isolates is to reduce the number of fungicide applications, especially single-site fungicide applications. MacKenzie and Peres (2012) developed a threshold for timing fungicide applications based on leaf wetness duration and the temperature during that period from Wilson et al. (1990). The web-based Strawberry Advisory System (StAS) was launched in 2009 and provides growers with SMS and/or emails advising when to apply fungicides as well as recommended products (Pavan et al. 2011). Following the recommendations by StAS has reduced the number of fungicide applications for control of anthracnose as well as gray mold on strawberry in Florida by about half (Cordova et al. 2017, 2018b; Zhang et al. 2019). Adoption in some other states has been challenging, though. The model determines past infection risk and depends on growers to apply fungicides as soon as possible after infection has occurred. While on Florida’s fast-draining, sandy soils this may be feasible, getting a tractor back into fields with soil of higher clay proportion may be a challenge or even impossible. Nevertheless, where applicable, the system should reduce selection for resistance to fungicides prone to that problem and could potentially be applied in other crops.

    Registration of unique chemistries.

    Natamycin (FRAC 48), an active ingredient historically used for food preservation, has shown excellent potential against Colletotrichum spp. on fruit crops. This chemistry is particularly interesting due to its seemingly limited risk of resistance development. Natamycin acts by binding to ergosterol and disrupting the fungal membrane (te Welscher et al. 2008, 2010). It has such low toxicity in mammals that it has a long history of use as a food and beverage preservative and is used as an antifungal medication for human ophthalmic mycoses (Aparicio et al. 2016). Since its introduction as a preservative in 1967, no resistance has been reported in agricultural crops (Aparicio et al. 2016). It is currently registered as a postharvest treatment for citrus, pome, and stone fruits. It has also shown good efficacy as a preplant dip treatment against strawberry anthracnose crown rot caused by C. acutatum in field trials in both California and Florida, has been registered as a dip treatment in these states, and registration will likely be expanded soon (Cordova et al. 2018a; Cosseboom et al. 2018). When used in dip applications, its efficacy was excellent on both QoI resistant and sensitive isolates of C. acutatum, comparable to industry standard QoI fungicides on sensitive C. acutatum isolates (Haack et al. 2018). Limited information is currently available about the efficacy of natamycin against C. gloeosporioides on field applications since the currently available formulation is UV sensitive. However, in three separate human fungal eye infection cases, C. gloeosporioides isolates showed intermediate resistance to natamycin, indicating that this species may have inherent or acquired resistance (Shiraishi et al. 2011). Interestingly, each of these three mycoses resulted from injury in an agricultural setting, leading clinical researchers to believe that the fungi originated from infected plants, since C. gloeosporioides is not a common human pathogen (Shiraishi et al. 2011).

    Fungicide resistance monitoring.

    Location-specific resistance monitoring allows for rapid assessment of resistance risk. Monitoring can aid in the design of resistance management programs, prolonging the functional lifetime of chemicals, and preventing the use of fungicides rendered ineffective by resistance. Timely monitoring requires a rapid testing method for determining resistance, an effective sampling method for collecting isolates, and a reliable communication channel for providing resistance management advice to producers. Pilot monitoring programs using bioassays have been extremely useful for other diseases such as gray mold on strawberry (Botrytis cinerea) and grapevine powdery (Erysiphe necator) and downy (Plasmopara viticola) mildews (Corio-Costet 2015; Schnabel et al. 2015). A similar program has been developed for QoI resistance monitoring of C. acutatum on strawberry in the United States. This program has already provided many important insights about the distribution of resistance within the United States, the frequency of resistance, and the importance of nurseries to resistance development (Forcelini and Peres 2018).

    Rapid and accurate disease diagnosis and detection of fungicide-resistant isolates are important for effective monitoring. To inform strawberry growers of disease diagnostic results in time for effective deployment of chemical control practices, a multiplex high-resolution melting (HRM) assay was developed to detect strawberry crown rot pathogens and differentiate Colletotrichum from other pathogens (Forcelini et al. 2018). The HRM assay showed excellent specificity in the presence of DNA from individual isolates, with each primer pair generating the specific melting peak corresponding to the target pathogen and without any primer interactions. An end point assay was further developed to differentiate C. acutatum isolates resistant and sensitive to QoI fungicides (Forcelini et al. 2018). In the future, it may be possible to evaluate isolates in a specific planting and advise growers of the presence of resistance early in the season.

    Though much of the current focus of monitoring involves QoI fungicide resistance, it is equally important to monitor resistance and sensitivity to other fungicides with efficacy against Colletotrichum species, such as fluazinam, fludioxonil/cyprodinil mixture, and natamycin.

    Conclusions

    Effective disease management requires an understanding of the host, pathogen, and environment. Unfortunately, for Colletotrichum spp., each of these aspects is complex and multidimensional (Fig. 6). While it is difficult to prescribe a way to examine all three aspects, we propose focusing on three critical “need areas” that can improve our understanding of the Colletotrichum pathogen and ultimately lead to improved management: 1) pathogen identification, 2) larger-scale field sampling, and 3) fungicide sensitivity testing. Pathogen identification is the first and most critical step to designing appropriate disease management strategies. Studying complexes without differentiating individual species will result in skewed data and inaccurate comparisons due to species-specific sensitivity profiles. Simple but reliable techniques must be developed and employed for accurate species differentiation. Secondly, while many studies have researched traits of small groups of select Colletotrichum isolates, relatively few have investigated larger collections of field isolates. Studies using isolates that more closely represent field populations provide more accurate information for the development of science-based management strategies. Lastly, Colletotrichum spp. sensitivity testing has historically focused on QoI and MBC fungicides. An expansion of fungicide sensitivity information to include other promising single-site fungicides would advance the design of better management strategies. It would also expose potential threats of reduced sensitivity or resistance to other fungicides and inherent resistance in individual species.

    Fig. 6.

    Fig. 6. Complexity of managing Colletotrichum spp. explained using disease cycles.

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    Ultimately, no single method can fully control anthracnose. However, a better understanding of Colletotrichum species involved and the efficacy of different management tools will enable growers to make more successful integrated management decisions. Improving existing methods for species identification, applying resistance management practices, registering new modes of action, and breeding tolerant cultivars will further increase the available options for integrated management.

    Acknowledgments

    Technical contribution number 6851 of the Clemson University Experiment Station. Figures 1 and 4 were supported by the Southern IPM Center (Project number 215-2012523) from the USDA National Institute of Food and Agriculture as part of a grant received by Dr. Dowling and Dr. Schnabel. We thank Dr. Mengjun Hu for sharing his excellent image of grape ripe rot for Figure 2. We thank Dr. Hideo Ishii for valuable input and suggestions and P. K. Bryson (Clemson University) for technical support.

    The author(s) declare no conflict of interest.

    Literature Cited

    The author(s) declare no conflict of interest.

    Madeline Dowling

    Madeline Dowling is a postdoctoral scientist in the Department of Plant and Environmental Sciences at Clemson University. Her research involves fungicide resistance of fruit pathogens, particularly B. cinerea, Colletotrichum spp., and M. fructicola. For her research work, she was named Clemson’s graduate student researcher of the year in 2017 and one of the “Schroth Faces of the Future” by the American Phytopathological Society in 2019. She is also combining extension with her previous experience in graphic design and photography to create scientific illustrations, photographs, and animations explaining complex scientific concepts to a general audience. She has received grants, fellowships, and awards for this work, including a grant from the Southern IPM Center and the R.J. Tarleton Fellowship from the American Phytopathological Society. She aspires to be like one of her favorite plant pathologists, George Washington Carver, with whom she shares faith in God, love for art, and passion for extension. Like Carver, she hopes to encourage growers to further utilize scientific concepts in the field, resulting in application of more effective management practices in the future.

    Natalia Peres

    Dr. Natalia Peres is a professor at the University of Florida Gulf Coast Research and Education Center (GCREC). She has an active applied research and extension program focused primarily on the management of fungal diseases of strawberry. She is also responsible for the Diagnostic Clinic at GCREC serving the strawberry, ornamental, and vegetable industries in the area. Her research and extension programs focus on the development of integrated management approaches to reduce losses to growers in Florida, but recommendations extend across regions affecting many growers, including strawberry nurseries. She has developed the widely adopted web-based Strawberry Advisory System, which provides recommendations for strawberry growers on the need for fungicide applications based on the weather conditions. Her program is internationally known and has attracted students and visiting scientists from many countries. She has authored or co-authored over 100 peer-reviewed articles in scientific journals as well as technical extension bulletins and published multiple book chapters. In addition to numerous awards from the University of Florida, she received the APS William Boright Hewitt and Maybelle Ellen Ball Hewitt Award (2007), the APS Lee Hutchins Award for Excellence in Fruit Research (2014), and was recently recognized as an APS Fellow (2020).

    Sara Villani

    Dr. Sara Villani is an assistant professor and extension specialist in the Department of Entomology and Plant Pathology at North Carolina State University. She received a B.S. degree in chemistry from SUNY Geneseo in 2005 and a Ph.D. in plant pathology from Cornell University in 2016. Dr. Villani’s current research interests include understanding mechanisms driving practical fungicide and antibiotic resistance, understanding the effect of abiotic stressors on disease development, and the development of chemical, biological, and cultural strategies for the management of economically important diseases on fruit and woody ornamentals in the southeastern United States. Dr. Villani authored or co-authored over 20 peer-reviewed articles and book chapters. Her recent extension activities include co-developing apple disease content for the MyIPM smartphone app, developing the NC Appalachian Apples Extension Portal, and developing an apple bitter rot and Glomerella leaf spot management program for NC apple growers. For these efforts, Sara was the recipient the NC Cooperative Extension 2019 Dr. Joseph and Mrs. Lisé Zublena Program Impact Award.

    Guido Schnabel

    Dr. Guido Schnabel is a professor and extension plant pathologist at Clemson University with over 20 years of experience working with pathogens of fruit crops. He has an active research and extension program focused on management of small fruit and stone fruit diseases. His basic research program investigates the occurrence and molecular mechanisms of fungicide resistance in fruit crop pathogens, and his applied research program includes the improvement of fruit disease management, fungicide resistance management, cultural disease management, and identification and management of abiotic disorders such as peach skin bronzing. He is the creator of the MyIPM smartphone app that, with the help of many of his colleagues, provides practical pest and disease diagnostics and management information for fruit growers. His involvement in USAID-funded outreach in Indonesia, the Philippines, and Cambodia helped improve the lives of dozens of farm families. He has authored or co-authored over 100 peer-reviewed articles in premier scientific journals and published multiple book chapters. He received the APS Lee Hutchins Award for Excellence in Tree Fruit Research (2011), the APS Excellence in Extension Award, the Clemson University Godley-Snell Award for Excellence in Agricultural Research (2015), and the Clemson University Centennial Professorship Award (2017).