Strawberry Anthracnose: Progress toward Control through Science

Abstract

Anthracnose crown rot has been a destructive disease of strawberries in the southeastern U.S. since the 1930s. The causal fungus, C. fragariae, infects all above-ground plant parts; however, disease is most severe when the fungus infects the crown causing crown rot, wilt, and death. Colletotrichum gloeosporioides can also cause crown rot and has been a problem since the late 1970s. The anthracnose fruit rot pathogen, Colletotrichum acutatum, was first reported on strawberry in the U.S. in 1986. Scientific investigations of anthracnose have concentrated on its epidemiology, differences among the three causal Colletotrichum spp., their infection processes, and pathogenicity. Results from these many studies have improved control of this disease.

Keywords:

• Colletotrichum acutatum
• C. fragariae
• C. gloeosporioides
• Fragaria x ananassa
• disease control
• fungicides

INTRODUCTION

Colletotrichum species cause serious diseases of many fruit and vegetable crops worldwide. Colletotrichum acutatum, C. fragariae, and C. gloeosporioides (Howard et al.,. 1992; Smith, 1998a, 1998b, 1998c), cause anthracnose diseases of strawberry. The presence of the anthracnose fruit rot pathogen, C. acutatum, was first reported on strawberry in the U.S. in 1986 (Smith and Black, 1986) and is now widespread causing major economic losses in production fields. The fungus had previously been reported causing anthracnose fruit rot of strawberry in Queensland, Australia (Simmonds, 1965) and now has been reported on strawberries in most areas of the world where they are grown. Colletotrichum fragariae and C. gloeosporioides cause anthracnose crown rot, a destructive disease in strawberry nurseries and fruit production fields in the southeastern U.S. (Smith, 1998a). Both species also cause petiole lesions and leaf spots. Anthracnose diseases are increasing in importance and have caused major economic loss to strawberry growers worldwide. The objective of this report is to provide examples of anthracnose-related research that has led to a better understanding of these diseases and their control.

PATHOGEN IDENTIFICATION

Cultural characteristics, conidia, appressoria, and setae are used to distinguish Colletotrichum species (Smith and Black, 1990). In culture, C. acutatum isolates develop white, pink, orange, rose, or beige colonies. They produce fusiform conidia tapered on both ends, do not form setae, and rarely form the ascigerous state. C. fragariae isolates develop beige to olive to dark gray colonies. They produce cylindrical conidia sharply tapered on one end and rounded on the other end, and do not form the ascigerous state. C. gloeosporioides isolates are very similar to C. fragariae except they may form the Glomerella cingulata ascigerous state in culture and their cylindrical conidia usually are rounded on both ends. Isolates of both C. fragariae and C. gloeosporioides produce dark black setae in acervuli in culture and on petiole, stolon, and fruit lesions. The growth rate of C. acutatum in culture is slower than the other two species, especially at higher temperatures. It is difficult to identify Colletotrichum species by their morphological and cultural characteristics alone because of overlapping ranges in various characteristics. A number of molecular methods (Freeman et al., 2000a; Lewis Ivey et al., 2004; Smith et al., 2007), including arbitrary primed polymerase chain reaction (ap-PCR), restriction fragment length polymorphisms (RFLPs) of PCR-amplified ribosomal DNA (rDNA), nucleotide sequences of the internal transcribed spacer (ITS) regions of rDNA, and species-specific primers based on the ITS regions, have been used to differentiate these species.

The greatest economic loss due to anthracnose on strawberry is from fruit rot caused by C. acutatum. This pathogen also infects many other fruit and vegetable crops, including blueberry, blackberry, grapes, tomatoes, peaches, and peppers (Bernstein et al., 1995; Howard et al., 1992, Smith, 2002). Crown infections of strawberry plants by C. acutatum often result in stunted plants rather than plant death. Infected plants usually do not thrive after transplant and produce few berries at harvest.

Colletotrichum fragariae was first identified in Florida in 1931 (Brooks, 1931), and it spread throughout the southeast where it was responsible for crown rot and death of many plants in strawberry nurseries in the 1970s (Horn et al., 1972). This species has a narrow host range and is rarely found outside the southeastern U.S. It generally causes more severe petiole and crown symptoms than those caused by C. acutatum, and is considered by some to be a host-specific or con-specific form of C. gloeosporioides (Howard, 1983; Howard et al., 1992; Sutton, 1992).

Colletotrichum gloeosporioides was identified as the causal agent of a crown rot identical to that caused by C. fragariae (Howard et al., 1992) in the late 1970s and destroyed the strawberry industry in several states. This species has a wide host and geographic range causing diseases of many plants worldwide. It may cause major plant death in the fall when plants are transplanted to the production fields.

Colletotrichum acutatum is the most common anthracnose fruit rotting pathogen, whereas C. fragariae and C. gloeosporioides are more often associated with petiole and stolon lesions and crown rot; however, all three species may cause similar symptoms and are sometimes found on the same plant (Howard et al., 1992). Isolates of Colletotrichum spp. have been disseminated world-wide, probably through international plant exchanges as their genetic polymorphism and geographical origins are not related (Denoyes-Rothan et al., 2003; Sreenivasaprasad and Talhinhas, 2005). Colletotrichum acutatum may have evolved into a subgroup that is highly virulent and host specific to strawberries. Denoyes-Rothan et al. (2003) characterized a group of Colletotrichum isolates using various molecular methods and pathogenicity tests. Random amplified polymorphic DNA (RAPD) polymorphism and internal transcribed spacer 2 sequence data provided genetic evidence of two subgroups within C. acutatum: (1) CA-clonal, that included only isolates from strawberry and exhibited identical RAPD patterns and nearly identical ITS2 sequence, and (2) CA-variable, that included isolates from various hosts and exhibited variable RAPD patterns and divergent ITS2 sequence. Based on these molecular analyses, they proposed that the CA-clonal subgroup contained closely related, highly virulent C. acutatum isolates that may have developed host specialization to strawberry.

ANTHRACNOSE DISEASE SYMPTOMS

Often the first indication of anthracnose crown rot infection, caused by either C. fragariae or C. gloeosporioides, of plants in the field is the wilting of the youngest leaves (Smith, 1998a). The wilted leaves may appear to recover and become turgid in the evenings; however, most will wilt and die after a few days. A red discoloration then appears within the crown tissue, and the causal pathogen may be isolated from the discolored tissue. C. acutatum also may cause crown death; however, typically a single side of the crown is infected rather than the entire crown, and infected plants are stunted but do not die. Any of the three Colletotrichum spp. may cause petiole and stolon lesions, which are dark brown or black and sunken and often girdle the petiole or stolon. Pink masses of conidia are usually visible near the center of the lesions. All three species also cause leaf spots (Howard et al., 1992; Maas and Palm, 1997; Smith, 1998c). Black leaf spot, typically caused by C. fragariae and C. gloeosporioides, is characterized by grey or light black spots, peppered across the top surface of strawberry leaflets. These spots usually are not necrotic. C. acutatum more typically causes irregular leaf spots whose primary symptom is necrotic black lesions at the tip of the leaflets. It also causes root lesions. All three Colletotrichum spp. also incite flower blights and fruit rots (Smith, 1998b). Fully open flowers are much more susceptible than closed buds (Smith, 2007). Infected green fruit are often hard and brown, and mummified rather than ripened. Anthracnose lesions on ripe fruit are firm, slightly sunken, and covered with pink spore masses.

COLLETOTRICHUM INFECTION PROCESS AND DISPERSAL

The time from infection of the strawberry by Colletotrichum spp. to first sporulation (the latent period) is an important factor in the speed at which anthracnose may spread within a field and depends on the temperature and can range from 2 to 3 days at 25°C to 6 to 17 days at 5°C (King et al., 1997). Appressoria and secondary conidia produced by C. acutatum on symptomless foliage may be a significant source of inoculum for fruit infections (Leandro et al., 2001) and may also contribute to the availability of inoculum throughout the growing season (Leandro et al., 2003). Conidial germination, appressorial production, and secondary conidiation are all favored by longer periods of wetness than the 4 hr required for secondary conidia to form. C. acutatum may survive up to 8 weeks on leaves (Leandro et al., 2003), up to 5 weeks on fabric (Norman and Strandberg, 1997) and up to 12 months in soil and plant debris under dry conditions; but conidia and sclerotia die rapidly under moist conditions, i.e., soil moisture ≥12% (Feil et al., 2003; Urena-Padilla et al., 2001).

Rain splash is the primary means by which conidia of Colletotrichum spp. are spread from plant to plant in the field. Madden and Boudreau (1997) found that anthracnose fruit rot incidence generally declined as plant density increased and concluded that plant density reduced the amount of rain that penetrated the plant canopy, thus reducing the amount of splash. Most fruit infection occurs in a 25-cm radius of the source of the inoculum, which is usually an infected fruit (Madden and Wilson, 1997).

Using light and electron microscopy, Curry et al. (2002) studied the infection process of strawberry petioles and stolons by C. acutatum and C. fragariae. The fungi penetrated the cuticle via an appressorium and their hyphae grew within the cuticle and cell walls of epidermal, subepidermal, and subtending cells. After a brief biotrophic phase in which the hyphae invaded living cells, they entered an extended necrotrophic phase in which they proliferated among dead cells. Acervuli erupted through the cuticle and released conidia. Invasion of the vascular tissue typically occurred after acervuli matured but remained minimal.

ANTHRACNOSE CULTURAL CONTROL MEASURES

Since the primary source of infection in most fruiting fields is infected transplants, the planting of disease-free plants is the best control method. However, obtaining anthracnose-free plants has become more difficult as the geographic range of the causal pathogens has expanded to most areas where strawberry plants are produced. McInnes et al. (1992a) demonstrated that anthracnose-free transplants could be produced in the southeastern U.S. where the pathogens are endemic by locating plant production nurseries away from commercial fruit production fields.

Colletotrichum spp. infect many other hosts, therefore, it is sometimes assumed that the primary infection in strawberry fields is from infected plants of these hosts growing near the strawberry field. To test the hypothesis that Colletotrichum spp. may move from other fruit or vegetable crops to strawberry, Smith (2002) wound-inoculated the leaves and stems of strawberry, blueberry, blackberry, muscadine grape, tomato, and pepper with 37 Colletotrichum isolates, representing 9 species collected from 12 hosts. Isolates of Colletotrichum fragariae were the most aggressive causing lesions at an average of 38% of inoculation sites on all hosts except pepper. The percentage of infection for the other eight Colletotrichum species ranged from 5% to 25%. Strawberry was the most susceptible host with 58% of petiole and 14% of leaf infection with all isolates resulting in lesion development. Pepper was the most resistant host with no symptom development on leaves or stems following inoculation with any isolate. These results suggest that primary anthracnose infections in strawberry production fields are most often from infected strawberry transplants and only rarely from anthracnose infected fruit or vegetable crops adjacent to the strawberry fields. However, C. acutatum and C. gloeosporioides have both been shown to move from infected host plants near the fields to strawberry production fields (Freeman et al., 2000b).

Anthracnose spreads within a field by splashing water. Living mulches (such as wheat, rye, or rye grass) in row middles have been shown to reduce disease spread within a field. Organic mulches, such as wheat straw or pine needles, will also reduce splash and result in lower incidence of anthracnose compared to rows mulched with plastic (Coelhoa et al., 2008; Madden, 1992; Smith and Spiers, 1986). Anthracnose is less severe when water is supplied to plants using drip rather than overhead irrigation (Coelhoa et al., 2008; Madden, 1992; Smith and Spiers, 1986).

Anthracnose crown rot is less severe in commercial fields when strawberries were grown on soils with low nitrogen fertility (Howard et al., 1992). In greenhouse studies, strawberries grown in soils with high levels of nitrogen, especially from ammonium sources, were more susceptible to anthracnose than plants grown in soils with lower nitrogen levels or those with high levels of calcium nitrate (Smith, 2009a, 2009b). In field studies, Nam et al. (2006) showed that higher levels of nitrogen and potassium increased anthracnose crown rot disease severity compared to phosphorus and calcium. Anthracnose fruit rot was less severe on fruit from greenhouse-grown plants receiving drench or foliar applications of calcium sulfate than on fruit from plants receiving water, calcium chloride, or calcium nitrate treatments. Fruit from plants receiving foliar applications of CaCl₂ developed less fruit rot than those from plants receiving soil applications of CaCl₂ (Smith and Gupton, 1993).

STRAWBERRY BREEDING PROGRAMS TO DEVELOP ANTHRACNOSE RESISTANT CULTIVARS

In the early 1980s, when anthracnose became a major disease of strawberries in the southeastern U.S., breeding programs to develop anthracnose-resistant cultivars adapted to the strawberry-growing areas in the southeastern U.S. were instituted by the USDA, ARS in Mississippi and Maryland, and at state experiment stations in Florida, Louisiana, and North Carolina (Ballington et al., 2002; Galletta et al., 1997; Smith and Spiers, 1982; Smith et al., 1990). The USDA-ARS program resulted in over 1500 anthracnose resistant selections that were made from the seedlings field-tested in Mississippi. Four of these anthracnose-resistant strawberry clones were released as breeding lines (Galletta et al., 1993) and one was released as the cultivar Pelican, which is highly resistant to anthracnose crown rot caused by C. fragariae and to anthracnose fruit rot caused by C. acutatum (Smith et al., 1998). ‘Pelican’ is also resistant to five races of red stele caused by Phytophthora fragariae. Other selections have been used as parent lines in several breeding programs. To increase the genetic diversity in the strawberry germplasm used in breeding programs, anthracnose resistant clones have been identified in native plant collections and are being used as parent lines (Lewers et al., 2007).

The resistance of strawberry plants to C. fragariae is influenced by environmental conditions after inoculation. Plants incubated at a high temperature (35°C) for 48 hr at a relative humidity near 100% developed more severe disease symptoms than plants incubated at 25 or 30°C (Smith and Black, 1987). Further incubation at 32°C results in more severe symptoms than incubation at 25°C. Tissue culture-induced (somaclonal) variation is another strategy that has been pursued for generating disease-resistant genotypes (Hammerschlag et al., 2006).

Planting resistant cultivars, such as Sweet Charlie, Florida Radiance, and Florida Elyana, has reduced losses due to anthracnose fruit rot in Florida, possibly because C. acutatum lacks the genetic diversity to overcome this resistance (Mertely et al., 2009). However, when moderately susceptible cultivars (e.g., Strawberry Festival) or highly susceptible cultivars (e.g., Camarosa and Treasure) (Chang and Smith, 2007) are grown, regular applications of fungicides are often needed to suppress the disease.

CHEMICAL CONTROL

Unless highly resistant cultivars are grown, growers must rely on a combination of chemical applications and cultural practices to reduce losses due to anthracnose. Failure of fungicides to control anthracnose epidemics in the past may have been due to the development of fungicide resistance in the Colletotrichum spp. population. In the early 1970s, benomyl was shown to effectively reduce the incidence of anthracnose crown rot (Horn et al., 1972; Howard, 1971) and was used intensively by strawberry growers for years to control anthracnose and other diseases. However, the anthracnose pathogens developed resistance to it and other benzimidazole fungicides, and benomyl was no longer effective. In vitro and greenhouse trials have been used to identify fungicides to which the Colletotrichum pathogens have developed resistance (LaMondia, 1993, 1995; McInnes et al., 1992a, 1992b; Smith and Black, 1991, 1992, 1993). Fungicide labels now address the issue of pathogen resistance to fungicides by restricting the number of consecutive applications and the number of total applications of a product per season. For example, the use of the fungicides, Abound (azoxystrobin, Syngenta, Greensboro, NC), Cabrio (pyraclostrobin, BASF, Research Triangle Park, NC), and Pristine (pyraclostrobin and boscalid, BASF), are limited to four or five applications per season with no more than two consecutive applications before alternating with a fungicide with a different mode of action. Natural products have also been evaluated as alternate fungicides with different modes of action, lower toxicity, and increased efficacy (Abril et al., 2008).

The efficacy of fungicides for anthracnose control is routinely evaluated in field studies. For example, 16 different fungicide treatments were evaluated in 5 fungicide studies conducted at Hammond, LA, and Poplarville, MS, during the 2002, 2003, and 2005 fruiting seasons (Wedge et al., 2007). In these trials, the most frequent fruit rots at harvest were anthracnose fruit rot, stem end rot (caused by Gnomonia comari), and gray mold (caused by Botrytis cinerea). Compared to the nontreated control treatment, less anthracnose fruit rot occurred on berries from the pyraclostrobin + boscalid, cyprodinil + fludioxonil, azoxystrobin, pyraclostrobin, captan + fenhexamid, and captan treatments. Studies conducted in North Carolina indicated that a fungicide spray program with only four applications [one application of a mixture of captan and Topsin M (thiophanate-methyl, Cerexagri-Nisso, King of Prussia, PA), two applications of Pristine, and one application of CaptEvate (fenhexamid and captan, Arysta, Cary, NC)] provided a similar level of disease control as a season-long spray program with eight applications (Rahman et al., 2009). Each of the applications in the shorter program actually contained two fungicides since Pristine and CaptEvate each contains two fungicides.

Based on field trials in Florida (Mertely et al., 2009), the recommendation for anthracnose control is weekly applications of a broad-spectrum protectant fungicide (captan) at the lowest label rate early in the season when weather conditions are less favorable for anthracnose development. Later, when anthracnose-infected flowers and fruit may be present in the fields, the recommendation is to increase the rate of the protectant fungicide to the highest label rate. Warm, rainy weather in early spring often leads to epidemics during which additional fungicides may be needed. Growers are encouraged to scout their fields regularly for anthracnose symptoms, such as blighted flowers or black spots on green fruit. If symptoms are found, a strobilurin fungicide (Abound or Cabrio) is recommended to be applied with the regularly used protectant fungicide. During the main bloom period, in late January and early February, fungicides containing two active ingredients (one for anthracnose control and one for Botrytis fruit rot control), such as Captevate, Pristine, or Switch, are recommended. Tank mixes or higher rates of protectant fungicides should be continued until the end of the harvest season or until dry weather completely suppress the disease.

The effectiveness of fungicides can be improved and their cost reduced when applications are based on environmental models rather than on a calendar schedule. Equations to predict the severity of both anthracnose and Botrytis fruit rots have been developed in Florida based on temperature and leaf wetness duration (Pavan et al., 2009). This information was used to develop a Web-based decision support system for growers to make recommendations of the timing of fungicide applications for control of fruit rot diseases. Field tests of the systems showed that the number of fungicide applications could be reduced by half without any significant loss of yield or quality.

CONCLUSIONS

As our knowledge of the anthracnose pathogens and the epidemiology of anthracnose diseases have increased, so has our ability to control these diseases. Changes in cultural practices have resulted in reduced levels of disease. At the same time, development of more effective fungicides and their registration for use on strawberries have greatly reduced losses due to anthracnose crown rot and fruit rot. Partly-resistant cultivars also have reduced economic losses due to these diseases. Even so, growers may sustain severe losses when environmental factors are highly favorable for anthracnose development.

Increased losses due to anthracnose fruit and crown rots may be related to the shift from matted row culture to the annual plasticulture production system in the U.S. Breeding for resistance to anthracnose and the development of resistant cultivars is a primary means for reducing economic losses due to this disease and would result in reduced use of fungicides.

Notes

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