Plant Pathology (2015) 64, 385–395
Doi: 10.1111/ppa.12247
Latent entry and spread of Colletotrichum acutatum (species complex) in strawberry fields J. Debodea*, W. Van Hemelrijckb, X.-M. Xuc, M. Maesa, P. Creemersb and K. Heungensa Plant Sciences Unit – Crop Protection, Institute for Agricultural and Fisheries Research (ILVO), Burg. van Gansberghelaan 96 bus 2, 9820 Merelbeke; bResearch Centre for Fruit Cultivation (pcfruit), Fruittuinweg 1, 3800 Sint-Truiden, Belgium; and cEast Malling Research, Pest and Pathogen Ecology for Sustainable Crop Management, West Malling, Kent, ME19 6BJ, UK
a
Anthracnose, caused by Colletotrichum acutatum (species complex), has become a troublesome problem in strawberry production worldwide. This paper reports (i) an optimized sampling method combined with a real-time PCR technique to detect the latent presence of C. acutatum in cold-stored strawberry plants used as planting material in several European countries, and (ii) a study of the spread of C. acutatum following a point inoculation under field conditions. Screening of different parts of planting material suggested that C. acutatum is most likely to be present on runners and old petioles. In addition, in seven out of nine batches of planting material from different nurseries, latent infection by C. acutatum was detected in at least one of five replicate samples. Field experiments in 2009 and 2010 showed extensive latent within-field spread of the pathogen on strawberry leaves, with a within-row dispersal distance up to at least 175 m in 1 week. A straw ground cover between the rows did not decrease C. acutatum spread, probably because introduced (and/or subsequent) inoculum was confined to the plant bed (within the row) and was not present between the beds. Moreover, the number of C. acutatum spores on the symptomless leaves, as estimated using a real-time PCR method, was significantly (P < 005) correlated with the incidence of fruit rot at harvest and post-harvest (r = 056–066). These results illustrate the importance of detecting latent infections in planting material and strawberry leaves in the field. Keywords: latency, planting material, point inoculation, real-time PCR
Introduction Anthracnose, caused by Colletotrichum spp., has become a troublesome problem in strawberry production worldwide. Symptoms include fruit rot (black spot), crown rot and spotting of petioles and runners. These symptoms are associated with three Colletotrichum species: C. acutatum, C. fragariae and C. gloeosporioides (Freeman et al., 1998). Colletotrichum gloeosporioides and C. fragariae usually cause petiole and stolon lesions and crown rot, but they occasionally also produce symptoms on fruit (Peres et al., 2005). Colletotrichum acutatum (species complex) is most important as a fruit rot pathogen but also infects petioles and crowns (Peres et al., 2005). In Europe, C. acutatum is the most prevalent species complex, while the occurrence of C. gloeosporioides is sporadic, and C. fragariae has not yet been reported (Garrido et al., 2008; Van Hemelrijck et al., 2010). Characterization of C. acutatum populations revealed that this pathogen is genetically highly heterogeneous
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Published online 22 June 2014 ª 2014 British Society for Plant Pathology
and can be classified into nine subgroups (A1–A9; Sreenivasaprasad & Talhinhas, 2005; Whitelaw-Weckert et al., 2007), and therefore C. acutatum has long been regarded as a species complex. To solve this problem, Damm et al. (2012) recently proposed to split this species complex into 31 species. Strawberry isolates fall into six of the nine subgroups, namely A2, A3, A4, A5, A7 and A9, or six species: C. simmondsii, C. nymphaeae, C. fioriniae, C. godetiae, C. acutatum s. str. and C. salicis (Damm et al., 2012). All further mentions of C. acutatum in this paper refer to the complex and not to the sensu lato as defined by Damm et al. (2012). Currently, control of C. acutatum on strawberry mainly relies on the use of fungicides, which is not sustainable given the increased consumer demand for high quality fruit free of pesticide residues, and the acquired resistance of several strawberry fungal pathogens to benzimidazole or dicarboximide fungicides. Several strawberry pathogens are currently controlled by fungicides such as the quinone outside inhibitors (QoI), fungicides that are rated as ‘high risk’ for resistance development (Wedge et al., 2007). For all of these reasons, strategies must be developed to reduce pesticide input. Identification and elimination of inoculum sources and limiting the within-field spread of C. acutatum are two important components of an integrated strategy for management of C. acutatum. 385
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Planting material that is latently infected with C. acutatum is believed to be the main inoculum source (Sreenivasaprasad & Talhinhas, 2005; Smith, 2008; Calleja et al., 2013). Colletotrichum acutatum has been detected on strawberry planting material in Switzerland (Bosshard, 1997), Finland (Parikka & Lemmetty, 2004) and Belgium (Debode et al., 2009). However, these studies were largely based on sampling at a single time point and/or a limited number of samples. For example, Debode et al. (2009) and Parikka & Lemmetty (2004) extracted DNA from 100 mg plant tissue taken from only one plant; a sample of 100 mg plant tissue may not be representative of the whole plant and one single plant is unlikely to represent all plants in the batch. Therefore, a protocol appropriate for testing large batches of planting material to detect the presence of C. acutatum is needed to reliably estimate the extent of fungal presence on a batch of plants (Calleja et al., 2013). Planting material in Belgium and the Netherlands typically consists of cold-stored plants of the cultivar Elsanta (Lieten et al., 1995). These plants are lifted from the field in December and cold-stored at 15°C. Subsequently, they can be planted from January until the end of August, which greatly extends the season (Lieten & Baets, 1991). These cold-stored plants mostly consist of the crown and roots but also contain parts of old petioles/runners and one to three green leaves. Most Belgian strawberry growers do not propagate their own planting material but rather obtain it from propagation nurseries specialized in the production of planting material. Belgian growers mainly buy from nurseries located in Belgium and the Netherlands, but also from other countries (Spain, Italy, the USA and others). The spread of C. acutatum in the field is normally via rain-splash dispersal of conidia (Peres et al., 2005); these conidia may be dispersed to new infection sites in a series of consecutive splash-dispersal events on the ground and in the canopy. Studies of conidial dispersal based on simulated rain showed that most new infections occur within a 25 cm radius of the inoculum source and are greatly influenced by rainfall intensity and ground cover (Yang et al., 1990a,b; Madden et al., 1993; Ntahimpera et al., 1997, 1998; Smith, 2008). Laboratory experiments suggested that symptomless strawberry leaves could be an important source of inoculum for fruit infection (Leandro et al., 2001, 2003) but this has yet to be confirmed under field conditions. Various sampling and detection methods have been used to detect C. acutatum in strawberry plants; these methods differ in their sensitivity and specificity. For example, Cook (1993) and Mertely & Legard (2004) described methods for detecting C. acutatum on symptomless strawberry plants. Petioles or leaves are first killed using paraquat or freezing to enhance fungal sporulation. After 5–7 days of incubation under humid conditions and ambient temperature, the spore masses can then be assessed visually. However, it is difficult to identify the exact species of Colletotrichum based on the morphology of these spore masses (Bosshard, 1997),
which can also be confused with those of other strawberry pathogens such as Pilidium concavum (Debode et al., 2011). To increase the specificity of detecting latent presence of Colletotrichum spp. on strawberry, molecular techniques have been developed (Sreenivasaprasad et al., 1996; Martinez-Culebras et al., 2002, 2003; Perez-Hernandez et al., 2008; Debode et al., 2009; Garrido et al., 2009). These molecular techniques are not only species-specific, but are also very sensitive, which enables reliable detection of latent infections. However, the sampling protocol must be revised before these methods can be used to test large samples of planting material or to examine the spread of the pathogen under field conditions. The results presented in this paper form part of a study on the epidemiology of C. acutatum on strawberry. The four objectives were as follows: first, the development of a sampling and detection method based on a previously published TaqMan real-time PCR (Debode et al., 2009). The aim was to determine the relative importance of plant parts (green leaves, crown, and runners/old petioles) in carrying C. acutatum inoculum as well as their importance in detecting the latent presence of C. acutatum on batches of strawberry plants. Secondly, the use of this sampling and detection method to quantify the extent of the latent presence of C. acutatum on batches of fresh and cold-stored strawberry plants from various growers. Thirdly, the execution of field experiments to determine whether latent spread of C. acutatum on the leaves was found and if so, what the rate of this spread was. The spread of C. acutatum was determined through point inoculation in the field and subsequent real-time PCR quantification of C. acutatum on symptomless leaves and assessing the incidence of fruit rot at harvest and post-harvest. Finally, in the same field experiments, it was investigated whether the spread of latent C. acutatum was affected by row direction and ground (straw) cover between beds. The main hypotheses were that (i) the level of latent entry of C. acutatum inoculum in the field via symptomless planting material is extensive; (ii) under conducive conditions (rain), within-field spread of C. acutatum on symptomless leaves is fast and extensive; and (iii) a straw soil cover does not reduce latent spread of C. acutatum in the field if the source of inoculum originates from within the plant bed.
Materials and methods Three plant parts distinguished Experiments were conducted to determine the relative importance of each part of the planting material as a carrier of latent infection of C. acutatum. Commercial fresh and cold-stored planting material (Elsanta) was bought from two growers, one located in the Netherlands (grower 1, Table 1a) and the other in Belgium (grower 2, Table 1a). Three parts of the planting material were distinguished per plant: crown, old petioles and runners, and green leaves. The number of old petioles and runners,
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Table 1 Origin of the strawberry planting material and log value of the Colletotrichum acutatum spore equivalents (LS) per plant in various plant parts (two replicates) (a) or in petioles and runners (five replicates) (b) LS per plant
Grower
Geographic origin
Year of harvest
Cold storage
(a) 1 2
Netherlands Belgium
2008 2008
No Yes
Green leaves
Crown
1
2
Mean
1
2
Mean
1
2
Mean
55 60
56 60
55 01 60 00
79 62
77 75
78 01 69 09
68 65
65 54
67 02 59 08
Grower
Geographic origin
Year of harvest
Cold storage
(b) 2 3
Belgium Belgium
4
Belgium
5 6 7 8
Netherlands Italy Spain USA
2008 2009 2009 2007 2008 2009 2011 2011 2011
Yes No Yes Yes Yes No No No No
a
Petioles/Runners
LS per plant 1
2
3
4
5
Mean
63 00a 00a 00a 00a 00a 58 00a 58
76 00a 00a 00a 59 00a 62 00a 66
75 00a 00a 00a 00a 00a 57 00a 54
66 00a 00a 00a 00a 00a 57 00a 55
64 54 53 00a 60 53 54 00a 53
69 19 19 00 60 19 58 00 57
06 20 19 00 01 19 03 00 05
00 means a Ct-value > 40, corresponding to a detection limit of a log value of 44 spore equivalents.
and green leaves varied greatly among individual plants, usually ranging from 1 to 10 old petioles and runners and one to three green leaves per plant. All three of these plant parts from all plants in the experiment were sampled. Each replicate sample consisted of 10 randomly chosen plants per grower; all three parts of these 10 plants were then pooled for testing. There were two replicate samples per grower. From each replicate sample, the old petioles and runners were combined and blended using a professional blender (Grindomix GM 200; Retsch) either with or without adding 100 mL 01 M Tris.HCl buffer (pH 8) to obtain a homogenate. The same procedure was done for the pooled crowns and again for the pooled green leaves. After blending each sample, the blending equipment was rigorously cleaned with water, treated with DNA-Erase (MP Biomedicals) and rinsed with distilled water. A 100 mg subsample of each homogenate was used for DNA extraction and subsequent realtime PCR-based quantification of C. acutatum DNA. When buffer was added prior to blending, a volume of homogenate was taken that corresponded to 100 mg plant material after centrifugation and only the pellet was used for subsequent processing. (To determine the effect of the buffer, five additional test samples of petioles and runners were analysed with and without buffer.) DNA extraction followed the method of Parikka & Lemmetty (2004). This method uses a slightly modified DNA extraction based on the commercially available DNeasy kit (QIAGEN) and also involves adding PVPP to the lysis buffer. According to authors, this results in good quality DNA from strawberry plant tissue, free of PCR inhibitors. The C. acutatum DNA in these samples was quantified using the previously published TaqMan real-time PCR method of Debode et al. (2009) (see below).
Latent presence of C. acutatum in planting material Nine samples of planting material were obtained from seven growers and assessed for the latent presence of C. acutatum
Plant Pathology (2015) 64, 385–395
(Table 1b). There were 50 plants in each sample (five replicate samples, 10 plants each). For five growers (grower 2, 5, 6, 7 and 8), only a single sample was obtained. Grower 3 provided two samples from the same year (2009): one cold-stored and the other fresh planting material. Grower 4 provided two coldstored samples, each from a different year. This yielded a total of nine samples: four fresh and five cold-stored. Because the results indicated that C. acutatum was present in lower amounts on green leaves or crowns, only old petioles and runners were tested for latent presence of C. acutatum.
Within-field spread of C. acutatum on strawberry leaves and fruit To study the within-field latent spread of C. acutatum on strawberry plants, two experiments were conducted in 2009 and 2010 at two experimental fields in Sint-Truiden (Belgium). The two experimental fields were located 30 m apart. In both experiments, cold-stored plants of the highly susceptible cultivar Elsanta (Lieten et al., 1995; Denoyes-Rothan et al., 1999) were planted in the beginning of May 2009. At the end of the production season in autumn 2009, the plants were mowed and this material was removed from the field. In 2010, the plants therefore had new leaves. Plots of 35 9 4 m were marked in the field and each plot included three double raised-beds with 11 pairs of plants per bed (i.e. 22 plants per bed in each plot; Fig. 1). The distance between the centres of two neighbouring beds was 13 m. To introduce inoculum to the plots, a strawberry fruit with symptoms and sporulating C. acutatum lesions was placed under the central pair of plants in the middle bed of each plot. This fruit was obtained from artificial inoculation of unripe fruit with a mixed conidial suspension of two C. acutatum isolates (PCF223 and PCF541), as described by Van Hemelrijck et al. (2010). These two isolates belong to the two most prevalent subgroups in Belgium: PCF223 belongs to the A2 subgroup or C. nymphaeae species and PCF541 belongs to A4 or
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(a)
(b)
Figure 1 Plot design of the field experiment in 2009 (a) and 2010 (b). Plots lined with a yellow border represent plots with straw between the beds. Red dots represent plants with infected fruit placed under them. Each set of three beds is labelled 0, 1 or 2.
C. godetiae species (Van Hemelrijck et al., 2010; Damm et al., 2012). Preliminary experiments showed that a single inoculated strawberry fruit may contain c. 2 9 108 conidia. To detect latent infection on leaves, symptomless leaves were sampled from all of the plants in all plots on three occasions in both 2009 and 2010 (times 0, 1 and 2; see below). The fruits were also sampled at harvest and tested for C. acutatum symptoms (in 2010 only). A leaf sample consisted of two leaflets per plant taken from each pair of plants within each bed, giving four leaflets per sample; the four leaflets were sealed in a labelled plastic BIOREBA extraction bag. Samples were then stored at 20°C until they were homogenized using a Homex 6 macerator (BIOREBA). DNA extraction and C. acutatum quantification using real-time PCR were performed as described by Debode et al. (2009) (see below). Daily rainfall and air temperature during the experimental period were recorded near the field. The daily prevailing wind direction in 2010 recorded at a weather station located at 30 km distance from the field was obtained from the Royal Meteorological Institute (RMI) in Belgium. In 2009, there were two plots (A and B) located in the same three beds; the two plots were separated by 45 m (Fig. 1a). Inoculum was introduced on 8 August, i.e. 39 days after flowering (=10% of the flowers were open); this was 3 days after starting harvesting of the fruits. Leaves were sampled 1 day before the infected fruit was introduced in each plot (time 0) and then again at c. 1 week (time 1) and 5 weeks post-inoculum introduction (wpi) (time 2). There was no ground cover between beds; each bed was covered with a standard plastic cover. In 2010, there were six plots (A–F). Plots A, C and E were in the same three beds; plots B, D and F were in another
three beds. The distance between neighbouring plots was 35 m. The ground cover within a bed was plastic, whereas the ground between beds was either not covered (plots A, C and F) or covered with straw (plots B, D and E; Fig. 1b). Inoculum was introduced on 7 May, 5 days before flowering. Leaves were sampled 1 day before the inoculum was introduced (time 0) and then again c. 2 and 4 wpi (times 1 and 2). The number of fruit with symptoms (SF) per pair of plants was also visually assessed at harvest (5–6 wpi). Fruit without symptoms at harvest were incubated in a moist chamber at 6°C for 10 days and then assessed for rot symptoms (SF postharvest).
Real-time PCR To detect C. acutatum on planting material and on symptomless leaves collected during the field experiments, the previously published TaqMan real-time PCR with the C. acutatum-specific ITS-based primers CaITS_F701/R699/R815 and probe CaITS_P710 was used (Debode et al., 2009). All DNA samples were diluted to 5 ng lL 1; 5 lL of this dilution was used per reaction. For each sample, the Ct values were converted to the amount of genomic DNA (gDNA; 50–5 9 106 fg) using the standard curve determined during each real-time PCR run (Debode et al., 2009). The amounts of gDNA were used to calculate the log value of the spore number equivalents (LS) per plant, taking the dilution factor and the relationship between the amount of C. acutatum gDNA and the number of spores into account (Debode et al., 2009). Briefly, DNA extracted from a 10-fold dilution series of C. acutatum spores (25–25 000 conidia) was analysed in the same real-time PCR run as a 10-fold dilution
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series of C. acutatum gDNA (50–5 9 106 fg), resulting in the standard curve: log (number of spores) = (gDNA 4029)/ 357 (R2 = 0987).
Statistical analysis Data were analysed using either GENSTAT (v. 14; VSN International) or STATISTICA (v. 11; Statsoft). The real-time PCR failed to detect any presence of C. acutatum on symptomless leaves 1 day before inoculum introduction (time 0), so the incidence of latent presence of C. acutatum on symptomless leaves at times 1 and 2 represented latent spread of the pathogen from the introduced inoculum source. Analysis of variance (ANOVA) was used to assess the differences in the severity of latent disease on each pair of plants (quantified as LS by the real-time PCR method) between the two assessment times. To establish whether the latent pathogen spread could be described in terms of a dispersal gradient, an exponential model was used to describe the spatial relationship of pathogen development on leaves (LS) or fruit (SF) with the distance to the initial inoculation (i.e. inoculum introduction) point in the inoculated bed only. This can be described using the equation ln (y + 1) = a + bx, where y is LS or SF and x is the distance to the inoculation point. The direction in relation to the inoculation point was included in the model fitting as a factor with two levels: either down or up the row. A strategy of comparing nested models was used to assess whether parameters a and b were affected by the direction. In the present study it was not possible to fit a model to describe pathogen spread in two dimensions at a given time (and hence to compare between-beds and within-bed pathogen spread) because the number of beds was too small. Instead, generalized linear models (GLM) were used to compare the incidences of symptomless leaf samples with C. acutatum DNA detected (LS) between the central bed (containing the introduced infected fruit) and the other beds (Fig. 2). In this analysis, the pathogen incidence data were assumed to follow a binomial distribution and a logit link function was used. Similarly, GLM analysis was used for SF data (number of fruit with the disease) assuming a Poisson distribution for the data to compare between-beds and within-bed pathogen spread. In 2010, ground cover was included as a factor in the GLM analysis to assess whether ground cover between beds affected latent disease spread. In addition to the GLM, disease variables in the non-inoculated bed were expressed as the percentage of corresponding disease variables of the inoculated bed in the same plot; then ANOVA was applied to the data to assess whether there were effects of ground cover on latent disease spread. Non parametric correlation coefficients (Spearman) between the variables (LS and SF at various time points) were calculated to assess whether a temporal relationship in the amount of quantified pathogen could be determined at each sampling point (time 1 or 2).
Results Three parts distinguished Analysing different parts of planting material (i.e. old petioles and runners, crown and green leaves) from growers 1 and 2 showed that C. acutatum was mainly present on the runners and old petioles, with the esti-
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mated number of spores ranging from c. 106 to 108 spores per plant (Table 1a). Therefore, only the runners and old petioles of the nine additional samples of planting material obtained from the seven growers mentioned above were screened for the latent presence of C. acutatum (see below). A preliminary assay showed that the variability between the 100 mg subsamples of the homogenate was low. The average LS per plant of two replicate samples from the same grower, each based on DNA extraction and real-time PCR of three subsamples of 100 mg homogenate, was 35 04 and 34 02 (average standard deviation (SD)). This within-homogenate variability was very low, thus it was decided to analyse more replicate samples per grower (five instead of two) for the nine additional samples of planting material obtained from the seven growers (see below). Using the pellet from a centrifuged suspension of planting material blended with buffer provided results comparable to those of samples processed without buffer. Based on five replicates, the mean (SD) LS per plant values were 35 03 and 33 01, respectively. They were not statistically different (P > 005).
Detection of the latent presence of C. acutatum on planting material In seven of the nine samples, latent infection by C. acutatum was detected in at least one of the five replicate samples, with the number of estimated spores per plant ranging from c. 105 to 108 (Table 1b). For plant material from three growers (2, 6 and 8), C. acutatum was present in all five replicate samples. Colletotrichum acutatum DNA was detected in three of the four long-term coldstored samples (from growers 2, 3 and 4) and in four out of five fresh material samples (from growers 3, 5, 6 and 8; Table 1b). All water controls were blank (negative = Ct value > 40), indicating absence of C. acutatum DNA.
Within-field spread of C. acutatum on strawberry leaves and fruit Pathogen development on leaves and fruit in 2009 and 2010 field plots is shown in Figure 2. In both years, no C. acutatum DNA was detected in the water controls or on the leaves of all plants in the plots 1 day before the introduction of the infected fruit (time 0), as indicated by Ct values > 40. Although no symptoms were observed on leaves at sampling times 1 and 2 in either year, high LS values were obtained on these leaves based on the real-time PCR method. This indicated the latent presence of C. acutatum on the leaves during both years. In 2009, only 1 week after the introduction of an infected fruit, the pathogen DNA was already detected on the leaves at the end of the same bed (Fig. 2a). At time 1, 36% of the samples had quantifiable C. acutatum DNA (i.e. LS ≥35 in Fig. 2a) compared with 55% at time 2. LS values on the two sampling occasions in
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(a) plot A
plot B
bed 1
