1. Introduction
Strawberries are one of the most popular dessert berries in the world due to their pleasant taste and aroma, as well as the fact that they contain many useful bioactive components [
1]. Strawberry is the most important small fruit crop in Lithuania, produced on 560 hectares. With the growing consumption of fresh fruit and berries, it is becoming increasingly important not only to expand the range but also to extend the growing season of berries such as strawberries. As in other parts of the world, the strawberry business is expanding in Lithuania. Therefore, new varieties are being introduced from warmer climate zones. Strawberry growing is expanding not only outdoors, but also in greenhouses. Growing strawberries in greenhouses facilitate the spread of pathogens in tropical and subtropical climates.
One of the most common strawberry diseases is anthracnose. Strawberry anthracnose causes yield losses of up to 50% and up to 80% of plant death in nurseries [
2]. The
Colletotrichum species-genus comprises about 190 species that cause plant disease in various plant crops worldwide. Complexes of several
Colletotrichum species cause strawberry anthracnose:
C. acutatum J. H. Simmonds, brooks and
C. gloeosporioides (Penz.) Penz. and Sacc. [
3,
4]. Anthracnose infects strawberry roots, crowns, petioles, leaves, runners, buds, flowers and fruits. The
Colletotrichum species complex has several development stages and infects different plant parts during their vegetation period [
5,
6,
7].
C. fragariae mainly cause crown rot and lesions in vegetative tissue, and
C. acutatum causes fruit rot [
8]. The ability of these pathogens to attack different plant parts is due to the species complex involved in strawberry anthracnose [
9,
10,
11].
Previously, strawberry anthracnose pathogen was considered to be a warmer climate pathogen, where temperatures ranging from 15 to 30 °C and optimal at 25 °C are needed for disease development. In comparison, the spread of
C. fragariae is optimal between 26.7–32 °C. Temperature is a key factor for anthracnose development and occurrence [
12,
13,
14,
15].
Colletotrichum spp. mostly causes plant disease in tropical, subtropical and temperate regions around the world, as these meteorological conditions are most optimal for pathogen development [
16]. Temperature influences the development of
Colletotrichum spp. and appressoria formation [
17]. Therefore, it is mostly not relevant in northern countries, except for warmer climate periods during berry harvesting and high rainfall [
12,
14,
15]. However,
C. acutatum causes berry infections at temperatures from 20 °C [
13,
18] and when there is a period of more than 12 h of leaf wetness [
19]. The optimal temperature for anthracnose development is 12–27 °C and leaf wetness for more than 12 h [
17,
19]. Lithuania generally has temperate climatic conditions, hence the impact of
Colletotrichum spp. should be minimal. According to our ongoing studies in Lithuania, the
Colletotrichum spp. pathogen is not widespread, but due to climate change and its extremes, the occurrence of this disease is now more noticeable.
Due to climatic conditions, the battle against plant disease presents new challenges, and new strategies are required for plant protection. The prediction of meteorological conditions and the occurrence of plant diseases is a complicated science as it involves determining the relationship between meteorological conditions and plant disease risk. At the same time, host development may be influenced by climate change [
20]. The growing demand for new solutions to optimise chemical pesticide usage and disease warning systems will not only help to monitor disease risk but also to predict disease epidemics. Meteorological parameters used to predict crop disease occurrence include air temperature, leaf wetness, precipitation and relative humidity. Disease forecasting models determine the critical timing of fungicide application when the conditions are most favourable for disease development. This also avoids unnecessary spraying and applications are made when the conditions are favourable for disease. Disease forecasting models are based on the impact of temperature and leaf wetness duration [
7,
19,
21,
22]. There are various disease forecasting models for specific plant diseases. This study summarises the meteorological conditions for
C. acutatum to determine the risk of strawberry anthracnose infection [
8]. The anthracnose fruit rot (AFR) model shows that disease control based on an advisory system could significantly reduce the number of sprayings [
23]. Similarly, the StAS web-based forecasting system has the potential to reduce spraying times compared with fixed-interval applications [
13,
24]. However, all forecasting models are based on counting the favourable periods for disease development. In Lithuania, IMETOS
® meteorological stations with forecasting models were used for a while [
21,
22,
25,
26]. The disease forecasting models are not only beneficial for the farmer (more precise application) but also for the environment (fewer pesticides, accurate applications).
The sources of a fungal infection include previous crops, contaminated soil, irrigation water and farmworkers. Soilborne pathogens can be species-specific, while resistance to anthracnose differs among strawberry varieties. Soilborne pathogens cause up to 20–30% yield losses in strawberry crops and can survive in the soil for several years [
27,
28,
29]. Strawberry disease control generally relies upon several fixed-interval (7–10 days) applications of fungicides from the time of leaf emergence until harvest, thus requiring significant amounts of fungicides [
7,
22,
30], which are costly and ideally should be applied when the risk of infection is high. The growing resistance to pesticide use and their adverse environmental effects are leading to new, environmentally safer disease control strategies [
7,
31,
32]. The selection of resistant cultivars (cv.) to anthracnose reduces the inoculum levels; additionally, meteorological conditions’ monitoring could assist in controlling the spread of
C. acutatum. This study aims to determine the meteorological conditions for anthracnose development, evaluate the susceptibility of different strawberry cultivars and detect the distribution of strawberry pathogens in temperate climate conditions.
3. Results
3.1. iMETOS® Meteorological Station Conditions
A comparison of meteorological conditions was performed for four farm locations in 2018 and 2019 during May, June, July and August. Meteorological conditions for
C. acutatum infection development are provided in
Figure 1. In 2018 and 2019 in Lithuania, strawberry vegetation seasons had quite similar climatic conditions, a relatively warm temperature with low precipitation and low air humidity. However, evaluating the data from iMETOS
® meteorological stations in 2018 and 2019 indicated different conditions for strawberry
C. acutatum.
The iMETOS
® meteorological conditions for strawberry anthracnose of 2018 are provided in
Figure 1. In 2018, the risks of infection in Farm 3 (
Figure 1C) lasted for ten days: one day in May, four days in July and 5 days in August (
Table 2). The air temperature, precipitation and leaf wetness parameters did not create favourable conditions for the spread of anthracnose for three months in Farm 4 (
Figure 1E). Analysis of the iMETOS
® data records showed that conditions for strawberry anthracnose in 2018 were favourable in August; 5–7 days were suitable for the spread of infection (Farms 4 and 3) (
Table 2). The following factors determined that the risk periods in 2018 for infection: air temperature between 12.0–25.1 °C and a leaf wetness period lasting more than 720 min.
The analysis of iMETOS
® meteorological station data showed that, in 2019, favourable conditions for the spread of
C. acutatum infection occurred during strawberry flowering at the end of May (for 3–4 days) in Farms 1, 2 and 3. (
Table 3). The meteorological station data for 2018 were different compared to 2019. The highest air temperature was in June, but the amount of precipitation and leaf wetness was low. Therefore, the risk of infection lasted 2–3 days but did not occur in Farm 4. However, sufficient precipitation, the temperature and leaf wetness conditions in July created favourable conditions for the spread of
C. acutatum (favourable for 8–11 days). The risk of infection lasted 4–7 days in August (
Table 3). The following factors determined the risk periods for infection: air temperature between 12–25.8 °C and a leaf wetness period lasting more than 720 min.
Meteorological data for 2019 showed that the most favourable conditions for the spread of anthracnose occurred at the end of May and in the first ten days of July. However, the prevailing temperature was below the optimum. Meteorological data showed that the favourable conditions for strawberry anthracnose spread varied in different years. Sufficient precipitation and long periods of leaf wetness provided suitable conditions for the spread of C. acutatum infection. Therefore, it can be stated that, in a colder climate, the temperature favourable for the spread of anthracnose is between 15.0–22.0 °C. According to the meteorological data obtained in Farms 1 and 2, the conditions for the spread of strawberry anthracnose differ very slightly in 2019. More favourable conditions for the spread of C. acutatum infection occurred at the beginning of strawberry vegetation.
3.2. Strawberry Contamination
A study of the contamination of different parts of strawberry plants with pathogenic fungi showed that infestation of strawberry leaves and stems was similar (
Figure 2). The results from Farm 1 showed that the highest contamination with pathogenic fungi was observed in cv. ‘Malvina’ (75.3% and 65.8%, stems and leaves, respectively), with the contamination being evenly distributed on leaves and stems. The lowest contamination was observed in cv. ‘Vibrant’ plant parts. Contamination of plant parts was similar in cultivars in Farm 2. Cv. ‘Rumba’ grown in Farm 3 had a high infestation in its stems and leaves (69% and 47.2% respectively).
The results differed between leaves and the stem in different strawberry cv. The contamination was highest when comparing the obtained results in the same cvs., for example, ‘Rumba’ and ‘Malvina’, grown in different farms (
Figure 2).
Evaluation of strawberry plant parts indicated that strawberry leaves and stems were similarly contaminated with pathogenic fungi. Differences between strawberry cultivars were observed. The highest contamination with pathogenic fungi was observed in ‘Malvina’ stems and leaves in two strawberry farms. Contamination of ‘Rumba’ plant parts with pathogenic fungi was lower than ‘Malvina’ in Farms 1 and 4. However, results obtained in Farm 3 showed contamination of the stems and leaves with pathogenic fungi in cv. ‘Rumba’ was relatively high.
Our data indicate (
Table 4) that the isolated fungi were
Mycosphaerella spp.,
Alternaria spp.,
Fusarium spp.,
Colletotrichum spp.,
Mucor spp.,
Penicillium spp.,
Phytophthora spp.,
Botrytis spp. and that
Trichoderma spp. was the most frequently isolated fungus from the leaf and stem samples of strawberry plants collected from cv. ‘Rumba’, ‘Asia’, ‘Florence’, ‘Malvina’ and ‘Senga Sengana’. The
Colletotrichum spp. pathogen was most frequently found in cv. ‘Malvina’ compared with other tested cultivars. Identification was carried out according to the cultural and morphological characteristics.
3.3. Soil Contamination
Soil samples from leading strawberry farms were collected to determine the prevalence of pathogens in the soil, in which the different strawberries cultivars were grown. During our research, 571 bacteria and only 99 fungal samples were isolated from the soil. The species of fungi detected were:
Colletotrichum spp.,
Fusarium spp.,
Penicillium spp.,
Aspergillus spp.,
Alternaria spp.,
Botrytis spp.,
Mucor spp. and others. The bacteria CFU was higher than fungi in all strawberry cultivars. The total amount of colony-forming units detected in the soil is presented in
Table 5.
The lowest CFU of bacteria was determined in a soil sample taken from Farm 4 (2.87 × 103 CFU/g−1), where various strawberry cv. were grown, but this was particularly low for the ‘Malvina’ cv. However, the CFU of fungal pathogens was one of the highest (3.60 × 103 CFU/g−1). An increase in bacteria CFU was found in ‘Sonata’ (4.49 × 103 CFU/g−1) and ‘Deluxe’ (4.49 × 103 CFU/g−1) from Farm 4 soil samples. Farm 4 ‘Sonata’ and Farm 3 ‘Polka´ had the lowest CFU of fungi (CFU 2.70 × 103 CFU/g−1). Among all evaluated soil samples from different farms, the highest CFU of fungi were found in Farm 4 ‘Elkat’ (3.72 × 103 CFU/g−1). The soil contamination of bacteria ranged from 3.92 to 4.14 × 103 CFU/g−1, and fungal pathogens ranged from 3.24 to 3.60 × 103 CFU/g−1 among different strawberry cv. in Farm 1. In this study, we found that ‘Rumba’ had similar contaminations of bacterial and fungal pathogens in different growing locations. However, in Farm 1, ‘Asia’ had significantly higher contamination of bacterial and fungal pathogens than those cultivated in Farm 4. The ‘Polka’ in Farm 3 was the least infected with soil pathogens compared with other cultivars. The lowest CFU of soil pathogens was detected in Farm 3 compared with Farms 1, 2 and 4. Soil contamination indicates that there were higher concentrations of pathogens. Colletotrichum spp. was observed in only three soil samples, that is, one sample from Farm 4 and 2 samples from Farm 1. From our results, it can be concluded that the anthracnose that causes Colletotrichum spp. was not equally prevalent in the soil of all strawberry farms. Our data show that bacterial and fungal pathogens distribution varies in different cultivars and locations. However, as some cultivars are more susceptible to some pathogens, their distribution was higher.
3.4. Effect of Temperature on Colletotrichum acutatum Growth
Temperature is one of the essential factors influencing Colletotrichum spp. growth. The isolates of C. acutatum showed significant differences in fungal growth under different temperature conditions. The highest colony growth rate was observed at 25 °C, and the lowest at 5 °C. The optimal temperature range was 20–25 °C, and the maximum growth rate was from 4.41 to 6.01 cm, respectively. Significant differences were observed between temperatures of 20–25 °C at 7 DPI. C. acutatum mycelium growth differs by only 0.03 cm at 5 °C after 4 and 7 DPI. The mycelium of C. acutatum was able to grow at 5 and 10 °C, but growth was slower compared with other temperatures. Furthermore, no visible colonies were observed at 2 DPI at 5 and 10 °C. Our data revealed that at 10 °C (1.20 cm), C. acutatum mycelium grew faster compared with 5 °C (0.6 cm) at 4 DPI. The mycelium diameter was 0.81, 1.61 and 2.73 cm after 2, 4 and 7 DPI at 15 °C, respectively. No significant differences were observed at 2 and 4 DPI. However, the highest growth was observed at 25 °C after 2, 4 and 7 DPI (1.76, 3.35 and 6.01 cm, respectively).
The results of our experiment indicate significant differences in fungal growth under different temperature conditions. In vitro temperature tests revealed that the optimal temperature for
C. acutatum development was between 20–25 °C. However, the most suitable temperature for
C. acutatum development was 25 °C (
Table 6).
3.5. Susceptibility of Strawberries to C. acutatum
The detached strawberry leaf assay was developed to determine the susceptibility of strawberry cv. to
C. acutatum. The results revealed that
C. acutatum isolate from strawberry was able to cause anthracnose in tested cv. and were pathogenic. Clear
C. acutatum necrotic (black) areas developed around mycelial plugs on inoculated leaves, however, no necrotic lesion was observed on control leaves. The ‘Elkat’ and ‘Deluxe’ cv. showed the highest susceptibility (4.64 and 4.33, respectively). The cultivars ‘Malvina’ and ‘Sonata’ had a lower disease severity index (3.77 and 2.89, respectively) and were not significantly different. The ‘Rumba’ (1.50) and ‘Asia’ (1.83) cv. had a significantly lower disease severity index (
Figure 3).
4. Discussion
Climate change is promoting the spread of plant disease, and the spread of the anthracnose pathogen is noticeable. It is becoming complicated to predict meteorological conditions as well as disease development. The variation between rainfall, temperature and leaf wetness affects plant diseases [
20]. The temperature and relative humidity are potentially significant factors affecting the development of
Colletotrichum species complexes. We evaluated the meteorological conditions in a temperate climate for
C. acutatum development. Our study showed that meteorological conditions have become more favourable for
C. acutatum [
7,
8,
19,
21,
22,
35]. Disease forecasting models help predict disease risks and reduce fungicide application by 50%. The anthracnose advisory system based on the AFR model showed that it could significantly reduce the number of sprayings [
23,
38]. Rasiukevičiūtė et al. [
22] evaluated the iMETOS
® strawberry
Botrytis cinerea forecasting model. Their results were very promising, as the pesticide applications were reduced based on the forecasting model. Our results show that meteorological conditions vary in different areas and years. We found that there were an optimal 12 days in 2018 and 26 days in 2019 for the development and spread of anthracnose in strawberry plants. The anthracnose infection risk in Farm 3 in 2018 lasted for 10 days in May-August and for seven days in Farm 4, only in August. Meanwhile, in 2019, in Farms 1 and 4 it lasted 21 days in May-August, in Farm 2 for 18 days and 15 days in Farm 3.
Meteorological conditions influence the conditions for disease development. However, there is always a source of infection. The primary source of infection is contaminated soil and previous crops. Soilborne pathogens reduce strawberry yield by up to 20–30% [
27,
28,
29]. We conducted a series of experiments to evaluate soil and the contamination of strawberry plant parts by pathogens. Soil contamination shows that there are higher concentrations of pathogens. In our research, 571 bacteria and only 99 fungal samples were isolated from the soil. The number of bacteria in the soil was higher than that of fungal pathogens. However,
Colletotrichum spp. was not equally prevalent in the soil at all tested farms.
Strawberry plant part evaluation indicated that the contamination of strawberry leaves and stems was similar. The highest level of contamination by pathogenic fungi was observed in cv. ‘Malvina’ and ‘Rumba’ stems and leaves. According to the results obtained, it can be stated that ‘Asia’ leaves and stems were least infected with pathogenic fungi. Wagner et al. [
6] indicated that ‘Florence’ was more susceptible to
C. acutatum than other strawberry cultivars, for instance, ‘Darselect’. The susceptibility of various cv. was unequal.
A source of inoculum is also essential for disease development. The susceptibility of cv. is important for anthracnose development. Furthermore, the strawberry flower is more susceptible compared to immature fruit [
13]. Jacobs et al. [
39] found that there are no significant differences between
Colletotrichum species, but a significant difference among strawberry genotypes. The cv. ‘Elkat’ and ‘Deluxe’ showed the highest susceptibility; however, ‘Rumba’ and ‘Asia’ had a lower disease severity index. Casado Diaz et al. [
10] and Wagner et al. [
6] observed that ‘Camarosa’ was very susceptible to
C. acutatum. We observed a slight trend between soil and plant parts contamination with pathogenic fungi and susceptibility to
C. acutatum in two strawberry cvs. The highest soil contamination of fungi was found in cv. ‘Elkat’, also cv. ‘Elkat’ was distinguished as the most susceptible to
C. acutatum among all studied cv. The
Colletotrichum spp. pathogen was most frequently found in cv. ‘Malvina’, soil and plant parts contamination by pathogenic fungi was also found of the highest levels, furthermore, cv. ‘Malvina’ showed high susceptibility to
C. acutatum.Temperature plays an essential role in
Colletotrichum species complex development, spore germination and appressorial formation [
17]. In the literature, it is considered that strawberry anthracnose is a warmer climate zone pathogen with an optimal temperature of 26.7–32 °C for
C. fragariae and around 20 °C for
C. acutatum [
12,
13,
14,
15,
18]. We evaluated the influence of temperature on anthracnose mycelium development. Our data confirmed the results of Feil et al. [
5] that
C. acutatum mycelium was able to grow at 5 and 10 °C. He et al. [
15] observed that colony growth was fastest in the temperature range of 25–28 °C; however, we found that the optimal temperature for
C. acutatum development was between 20–25 °C.