Temporal Dynamics of Incidence of Shot Hole Disease Affected by Training Systems and Cultivar Susceptibilities in an Integrated Plum Orchard

Shot hole disease (SHD) can cause severe epidemics in plum orchards, depending on cultivar susceptibility and training system; however, the combined effect on the progress of temporal disease and on the possible reduction in SHD in the disease management was not investigated. The aim of this 3-year study was (i) to monitor and analyze the temporal dynamics of SHD progress under four training systems (4 × 1.5, 4 × 2, 5 × 2.5 and 6 × 3 m) and on four plum cultivars (‘Čačanska lepotica’, ‘Bluefre’, ‘Stanley’ and ‘President’) in an integrated plum orchard; (ii) to identify those time periods when training system and cultivar combinations can reduce the disease development. Both SHD incidences and the area under the disease progress curves (AUDPC) were significantly affected by the training system, cultivar and year. Plum cultivars with high or mid–high susceptibility to SHD showed continuous SHD development from May to November, while cultivars with low susceptibility to SHD showed no symptoms until mid-summer and then progressed slowly until November. High (4 × 1.5 m) vs. low (6 × 3 m) density training systems reduced SHD incidence and AUDPC consistently for three cultivars (‘Čačanska lepotica’, ‘Stanley’ and ‘President’) in September, October and November, compared to the high-density training system. Only cv. ‘Bluefre’ showed no effect either on disease incidence or AUDPC, due to very high disease incidences in all training systems from September to November. In conclusions, combinations of training system and cultivar can significantly reduce SHD incidence, which may be successfully used as a part of the integrated pest management approach during the establishment new plantations.

Symptoms of SHD occur on the leaves, shoots and fruits of most cultivated stone fruit species [6]. In the case of plum, the leaf symptom of SHD is the most common symptom type [7][8][9][10]. Leaf symptoms appear as tiny light spots that gradually turn brown. Later, a purple-brown border develops around the spots. The middle of the spots die and fall out and the 'shot hole' symptom appears [11][12][13]. Under favorable weather conditions, SHD becomes severe and the leaves of the tree fall before harvest, resulting in an early defoliation of the tree [14]. Due to early leaf fall, the health of trees reduces year by year which is also reflected in yield reductions [6,[15][16][17][18].

Orchard Site, Plant Material, Expermental Desingn and Orchard Management
A three-year study (2017, 2018, and 2019) was performed in an integrated plum orchard in Eastern Hungary. The orchard was established at the University of Debrecen Experimental Station, Debrecen-Pallag (47 • 31 60 N, 21 • 37 60 E) in the spring of 1997 with 4 plum cultivars ('Čačanska lepotica', 'Bluefre', 'Stanley' and 'President'). Cultivar characteristics including pedigree, origin, susceptibility to SHD and harvest time are given in Table 1. The trees were grafted on myrobalan 'C 359' rootstock. The trees of each cultivar were planted in four training systems containing a 0.25 ha plot of each cultivar. The four training systems were designed as high, mid, low to mid, and low densities with tree spacings of 4 × 1.5, 4 × 2, 5 × 2.5 and 6 × 3 m, respectively. The trees were pruned to slender spindle for the training system that was spaced at 4 × 1 m (to free the spindle for the training systems that were spaced at 5 × 2.5 and 6 × 3 m) and to a combination of slender and free spindle for the training system that was spaced at 4 × 2. The experimental design was a split plot, where the three years were referred to as blocks, the four training systems as main plots (replicated four times) and the four cultivars as subplots.
The orchard soil type was Lamellic-Brunic Arenosol soil with alternating layers of clay [32]. Bare soil was maintained mechanically with a distiller in the spacings between rows, and 0.5 m wide straw mulch was used in the rows. Tree pruning, nutritional management and spray schedules against shot hole disease were prepared according to the integrated fruit production guidelines. Table 1. Plum cultivar characteristics used in this study including pedigree, origin, susceptibility to shot hole disease (SHD) and harvest time in an integrated plum orchard at Debrecen-Pallag, East Hungary (2017-2019).

Cultivar
Origin Pedigree SHD Susceptibility Harvest Time Reference mid-high End July-early August [30,31] 'Bluefre' USA 'Stanley' × 'President' mid-high End August-early September [31] 'Stanley' USA 'Ageni' × 'Grand Duke' low End August-early September [31] 'President' UK Developed by English breeders mid mid-September [31] Trees in the high and mid training systems received an annual summer pruning in July, and a supplementary winter pruning was performed every 2nd and 3rd year for removing the twig part of the trees that were older than 4 years. Trees in the low to mid and low training systems received a winter pruning in March of each year and no summer pruning was performed.
The orchard relied on the annual application of a nitrogen-phosphorus-potassium (NPK) complex fertilizer (Péti Kevert NPK Műtrágya, Nitrogénművek GmbH, Pétfürdő, Hungary) at the beginning of March at a dosage of 100 kg ha −1 active ingredient with 10:15:15 N-P-K ratio for nutrient supply. The orchard was not irrigated.
Sprays against SHD started at the dormant bud stage; copper hydroxide was used (0.1%; Funguran-OH 50 WP, 77%, Spiess-Urania Chemicals GmbH, Hamburg, Germany) and then additional sprays were applied during the season with fungicide active ingredients of: penconazole, tebuconazole, prochloraz, mancozeb, captan and copper hydroxide from mid-April (white flower bud) to the end of September (after harvest) ( Table 2). All the sprays were applied with a Kertitox 2000 axial blower spray machine (Debreceni Gépgyár B.V., Debrecen, Hungary) with a ceramic hollow cone at 1.1-1.2 MPa with a volume of 1000 L ha −1 .

Meteorological Data
During the 3-year periods, a Metos Compact agrometeorological station (Pessl Instrument GmbH, Weiz, Austria) was operated to measure rainfall (mm day −1 ) and the mean daily temperature ( • C day −1 ) from 15 April to 15 October in 2017, 2018 and 2019.

Shot Hole Disease Assessment
Disease assessments were performed in the middle 10 trees of each cultivar subplot in each year for the four cultivars and four training systems. A total of 4 × 100 leaves were assessed in each tree, thus the trees were divided into four quadrants. The presence of SHD on the leaves of each quadrant were determined in each year for the four cultivars and four training systems. Seven assessments were conducted in each year on the first decade of May, June, July, August, September, October and November. A leaf was considered diseased if at least one visible SHD lesion was observed. SHD incidence was calculated as values from the quadrants were averaged to obtain the percentage of diseased leaves.

Data Analyses
SHD incidences from the four replicates were averaged to obtain a single value for each year, training system, cultivar and assessment date. In addition, SHD incidences of the last assessment date (final SHD incidence-Y f ) were separately analyzed with a single value for each year, training system and cultivar. Moreover, the area under the disease progress curve (AUDPC) was calculated for each year, training system and cultivar. AUDPC as percent days was calculated as: where 'n' is the total number of assessments, 'y i ' is SHD incidence at the 'i'th assessment date and the term of t i+1 − t i is the time duration between two assessments. Then, the SHD incidences, final SHD incidence and AUDPC data were analyzed by a split-plot analysis of variance (ANOVA) using the statistical package of Statistical Analysis System v. 8.1; SAS Institute Inc., Cary, NC. Means were separated by the least significance difference (LSD) test using LSD 0.05 values. Significant F tests (p = 0.05) were followed by an LSD test for a comparison of the means of the training systems, cultivars or assessment dates using LSD 0.05 values. Prior to the analyses, SHD incidences data were arcsine-square root transformed in order to make the data normally distributed. Standard errors and LSD 0.05 values for the differences are given in the figures and tables as appropriate.
In order to visualize the time periods when training system and cultivar combination could reduce the disease development, significant F tests (p = 0.05) followed by LSD tests were prepared for each assessment date for each training system vs. cultivar combination using LSD 0.05 values.

Environmental Monitoring
The daily mean temperature ranged from 5.9 to 27.5

Cultivar 'Čačanska lepotica'
In the case of plum cultivar 'Čačanska lepotica', SHD incidences were the highest in 2017 in the 4 × 1.5 m training system (ranging between 8.1 and 89.4%) and the lowest in 2019 in the 6 × 3 m training system (ranging between 5.9 and 62.1%, Figure 1). SHD progress in the four training systems started before the first assessment date (the first day of May) and increased with various progress speeds until the last assessment date (in November) in all years ( Figure 1).
In 2017, the SHD incidence values were the highest in the mid-density training system (4 × 2 m) from May to July, which were significantly higher compared with the training system of 5 × 2.5 and 6 × 3 m in May and June, and in the training systems of 4 × 1.5 and 5 × 2.5 in July ( Figure 1A). The SHD incidence values were the highest in the high-density training system (4 × 1.5 m) from August to November, which were significantly higher compared with the training system of 5 × 2.5 m in August, in the training systems of 4 × 2, 5 × 2.5, and 6 × 3 m in September and October, and in the training systems of 5 × 2.5 and 6 × 3 m in November.
In 2018, the SHD incidence values were the highest in the high-density training system (4 × 1.5 m) in all assessed months, which were significantly higher compared with the other three training systems (4 × 2, 5 × 2.5 and 6 × 3 m) with the exception of October when the values in the high-density training system (4 × 1.5 m) were significantly different from the values of the training systems of 4 × 2 and 6 × 3 m ( Figure 1B).        In 2019, the SHD incidence values were the highest in the high-density training system (4 × 1.5 m) in all assessed months, which were significantly higher than the values in the training system of 6 × 3 m in May, in the training systems of 4 × 2, 5 × 2.5, and 6 × 3 m in June and August, and in the training systems of 5 × 2.5 and 6 × 3 m in July, September, October and November ( Figure 1C).

Cultivar 'Bluefre'
In the case of plum cultivar 'Bluefre', SHD incidences were the highest in 2019 in the 4 × 1.5 m training system (ranging between 30.8 and 100%) and the lowest in 2018 in the 6 × 3 m training system (ranging between 11.1 and 90.8%, Figure 2). SHD progresses in the four training systems started before the first assessment date (first decade of May) except for the training system of 6 × 3 m in 2017 ( Figure 2). In 2017 and 2019, the disease progress rapidly increased until September when it levelled off (Figure 2A,C). In 2018, SHD incidences increased with various progress speeds until the last assessment date (in November) in all years ( Figure 2B).
In 2017, the SHD incidence values were the highest in the mid-density training system (4 × 2 m) from May to July, which were significantly higher compared with the training systems of 4 × 1.5, 5 × 2.5 and 6 × 3 m (Figure 2A). In August, the SHD incidence values were similar in the training systems of 4 × 2, 5 × 2.5 and 6 × 3 m, which were significantly different from the values of the training system of 4 × 1.5 m. The SHD incidence values were the highest in the high-density training system (4 × 1.5 m) from September to November but these values were not significantly different from the other three training systems.
In 2018, the SHD incidence values were the highest in the high-density training system (4 × 1.5 m) in all assessed months with the exception of June when the highest values were reached in the training system of 5 × 2 m ( Figure 2B). The SHD incidence values in the high-density training system (4 × 1.5 m) were significantly higher than the values in the training system of 6 × 3 m in May and September, and in the training systems of 4 × 2, 5 × 2.5 and 6 × 3 m in July and August. (Figure 2B). In October and November 2018, the SHD incidence values were not significantly different among the four training systems.
In 2019, the SHD incidence values were the highest in the high-density training system (4 × 1.5 m) in all assessed months ( Figure 2C).The SHD incidence values in the high-density training system (4 × 1.5 m) were significantly higher than the values in the training system of 6 × 3 m in May, in the training systems of 5 × 2.5 and 6 × 3 m in June and August, and in the training systems of 4 × 2, 5 × 2.5 and 6 × 3 m in July ( Figure 2C). In September, October and November 2019, the SHD incidence values were not significantly different among the four training systems.

Cultivar 'Stanley'
In the case of plum cultivar 'Stanley', SHD incidences were the highest in 2019 in the 4 × 1.5 m training system (ranging between 0 and 29.1%) and the lowest in 2017 in the 5 × 2.5 m training system (ranging between 0 and 4.3%, Figure 3).
In 2017, SHD progress began in mid-July in the training system of 4 × 1.5 m and in mid-September in the other three training systems ( Figure 3A). The SHD progress of the four training systems started int mid-August in 2018, and in mid-June in 2019 (Figure 3). Following this, the disease increased with various progress speeds until the last assessment date (in November) in all years and in all training systems (Figure 3).
In 2017, the SHD incidence values were the highest in the mid training system (4 × 2 m) from September to November, which were significantly higher compared with the training system of 5 × 2.5 and 6 × 3 m in September and October, and in the training systems of 6 × 3 m in November ( Figure 3A).
In 2018, the SHD incidence values were the highest in the high-density training system (4 × 1.5 m) in September, which were significantly different from the values in the training systems of 5 × 2.5 and 6 × 3 m ( Figure 3B). The SHD incidence values were the highest in the mid-density training system (4 × 2 m) in October and November, which were significantly different from the values in the training systems of 5 × 2.5, and 6 × 3 m in September and October and in the training system of 6 × 3 m in November ( Figure 3B).
In 2019, the SHD incidence values were the highest in the mid-density training system (4 × 2 m) in August, which were significantly different from all the other three training systems ( Figure 3C). The SHD incidence values were the highest in the high-density training system (4 × 1.5 m) from September to November, which were significantly different from the values in the training systems of 5 × 2.5 and 6 × 3 m in September and in all the other three training systems (4 × 2, 5 × 2.5 and 6 × 3 m) in October and November ( Figure 3C). In July 2019, the SHD incidence values were not significantly different among the four training systems.

Cultivar 'President'
In the case of plum cultivar 'President', SHD incidences were the highest in 2018 in the 4 × 1.5 m training system (ranging between 11.8 and 91.2%) and the lowest in 2017 in the 6 × 3 m training system (ranging between 0 and 65.3%, Figure 4). SHD progresses in the four training systems started before the first assessment date (first decade of May) with the exceptions of the training systems of 5 × 2.5 and 6 × 3 m in 2017. Following this, the disease progressed continuously until the last assessment date (in November) in all years and in all training systems ( Figure 4).
In 2017, the SHD incidence values were the highest in the high-density training system (4 × 1.5 m) in May and from August to November, which were significantly higher compared with the training systems of 4 × 2, 5 × 2.5, and 6 × 3 m in May, August and October, and the training systems of 5 × 2.5, and 6 × 3 m in September and November ( Figure 4A). The SHD incidence values were the highest in the mid-density training system (4 × 2 m) in June, which were significantly different from the values of all the other three training systems ( Figure 4A). The SHD incidence values were the highest in the low to mid density training system (5 × 2.5 m) in July, which were significantly different from the values of all the other three training systems ( Figure 4A).
In 2018, the SHD incidence values were the highest in the high-density training system (4 × 1.5 m) from May to July and from October to November, which were significantly different from the training system of 5 × 2.5 in May, and from the training systems of 5 × 2.5 and 6 × 3 m in June, July, October and November ( Figure 4B). The SHD incidence values were the highest in the mid-density training system (4 × 2 m) in August and September, which were significantly different from the values of the three training systems of 5 × 2.5 and 6 × 3 m ( Figure 4B).
In 2019, the SHD incidence values were the highest in the high-density training system (4 × 1.5 m) from June to July and from October to November, which were significantly different from the training systems of 5 × 2.5 in June, July, October and November ( Figure 4C). The SHD incidence values were no different from each other in the four training systems in May, August and September ( Figure 4C).

Final Disease Incidence
Analyses of variance for the final disease incidences of SHD indicated significant (p < 0.05) differences amongst years, training systems and cultivars (Table 4). There were no significant interactions among the treatment factors.
According to the results of the ANOVA, the final disease incidences of SHD were shown separately for years, training systems and cultivars ( Table 5). The values of the final disease incidence were 2 to 20 times lower on cv. 'Stanley' compared to the other three cultivars in all years, which was significantly different (p < 0.05). In general, the values of the final disease incidence increased in the order of high, mid, mid-low and low training systems.  The lowest final disease incidence value was 4.8% in the 5 × 2.5 m training system for cv. 'Stanley' in 2017, while the highest one was 100% for cv. 'Bluefre' in the training systems of 4 × 1.5, 4 × 2, and 5 × 2 m for cv. 'Bluefre' in 2017, and for all training systems in 2019 ( Table 5). The overall years for the final disease incidences were significantly different only for cvs. 'Stanley' and 'President' when all cultivars were combined. Analyses of the overall training systems showed that the values of the final disease incidence in the training system of 4 × 1.5 m were significantly different from the training systems of 5 × 2.5 and 6 × 3 m when all years and all cultivars were combined ( Table 5).
Analyses of each cultivar showed that the final disease incidence varied among training systems and years (Table 5). In case of cv. 'Bluefre', the values of the final disease incidence were not significantly affected by years and training systems. The final disease incidence values of the training system of 4 × 1.5 m were significantly different from the training systems of 4 × 2, 5 × 2.

AUDPC
Analyses of variance for the AUDPC values of SHD indicated significant (p < 0.001) differences amongst years, training systems and cultivars (Table 4). There were no significant interactions among the treatment factors.
According to the ANOVA, the AUDPC values of SHD were shown separately for years, training systems and cultivars ( Table 6). The AUDPC values were the lowest for cv. 'Stanley' compared to the other three cultivars.   Table 5.  system ( Figure 5). Only cv. 'Bluefre' showed no effect either on disease incidence or AUDPC, due to a very high disease incidence in all training systems from September to November (Figure 2).

Reduce the Disease Development
High (4 × 1.5 m) vs. low (6 × 3 m) density training systems reduced the shot hole incidence and AUDPC in each of the assessed months depending on cultivar susceptibility to shot hole ( Figure 5). The disease reduction effect of low vs. high training systems were various from May to August among the cultivars, and cv 'Stanley' showed no effect due to a low disease incidence (Figure 3). The low-density training system reduced AUDPC and SHD incidence consistently for three cultivars ('Čačanska lepotica', 'Stanley' and 'President') in September, October and November, compared to the high-density training system ( Figure 5). Only cv. 'Bluefre' showed no effect either on disease incidence or AUDPC, due to a very high disease incidence in all training systems from September to November (Figure 2).

Figure 5.
Time periods when high-vs. low-density training systems and cultivar combinations can reduce the disease development of shot hole disease incidence and area under the disease progress curve (AUDPC) in an integrated plum orchard at Debrecen-Pallag, Eastern Hungary. Low-and high-density training systems are 6 × 3 m and 4 × 1.5 m, respectively. Years were combined in the data analyses. Different color boxes represent significant differences at p = 0.05 level between lowand high-density training systems at a given month for the four plum cultivars. White color represents 'no significant differences' and black color represents 'significant differences at p = 0.05 level. Grey box represents no or below 3% shot hole incidence for cv. 'Stanley'.

Discussion
In this study, we evaluated the effect of four training systems and four cultivars with various SHD susceptibility on shot hole temporal epidemics in an integrated plum orchard. In general, the SHD incidences and AUDPC of individual cultivars were lower on trees under the low (6 × 3 m) density training systems, compared to trees under the high (4 × 1.5 m) density training system, depending on the susceptibility of the cultivars and the annual weather conditions. Years were combined in the data analyses. Different color boxes represent significant differences at p = 0.05 level between low-and high-density training systems at a given month for the four plum cultivars. White color represents 'no significant differences' and black color represents 'significant differences at p = 0.05 level. Grey box represents no or below 3% shot hole incidence for cv. 'Stanley'.

Discussion
In this study, we evaluated the effect of four training systems and four cultivars with various SHD susceptibility on shot hole temporal epidemics in an integrated plum orchard. In general, the SHD incidences and AUDPC of individual cultivars were lower on trees under the low (6 × 3 m) density training systems, compared to trees under the high (4 × 1.5 m) density training system, depending on the susceptibility of the cultivars and the annual weather conditions.
The results of this study showed a great annual variation in the SHD incidences of the evaluated four plum cultivars, which are in agreement with previous studies on various fruit species, e.g., [4,6,7,9,15,21,25,[27][28][29][30][31]. Cultivars 'Čačanska lepotica' and 'Bluefre' showed high, SHD incidences, cv. 'Bluefre' and 'President' showed mid-high SHD incidences, and 'Stanley' showed low SHD incidences (91-100%, 62-91%, and 8.4-30%, respectively) at harvest in all years, independently of a training system (Table 5). In agreement with the previous research of Benedek et al. [30], Romanazzi et al. [33], Bubici et al. [7] and Khromykh et al. [34], our results indicate that SHD resistance has a great influence on the disease's development; therefore, the successful incorporation of the promising SHD-resistant plum cultivars into the growing practice is essential in those plum-growing areas where SHD is endemic. In addition to the SHD susceptibility of a plum's genotype, the nitrogen and potassium content of the leaves [35] and the growth habits of the trees (e.g., a dense type of canopy or an open type of canopy) can also be factors that cause differences in the observed final SHD incidences [31]. Tutida et al. [35] showed that plum cultivars with a higher leaf content of nitrogen and potassium reduced SHD infections. In addition, plum trees with open canopies allowed better sunlight penetration and thus better photosynthetic activities in the canopy, compared to cultivars with dense canopies [31]. Moreover, variations in growth habit can also affect the spray depositions in the canopy which can influence the temporal dynamics of S. carpophila infection during the season.
The SHD incidences and/or AUDPC of the investigated cultivars were lower under the low (6 × 3 m) density training systems, compared to the high (4 × 1.5 m) density training system, except for AUDPC on cv. 'Bluefre' (Tables 5 and 6; Figure 5). The effect of training systems on annual SHD progress was not previously evaluated in stone fruit orchards, but on similar diseases (such as leaf spot, which causes early leaf defoliation) that were studied in sweet cherry orchards [24]. Our results were in contrast with the study of Vámos and Holb [24] on sweet cherry vs. leaf spot pathosystems, as leaf spot incidences were lower in the higher density (4 × 1 m) orchard, compared to the lower density (5 × 2 m) one. Vámos and Holb [24] concluded that the depositions of spray droplets have better distribution within the tree canopy of the higher density orchard compared to the lower density one, which resulted in lower numbers of leaf spot infections during the season In this plum study, despite the larger trees being in the low (6 × 3 m) density training systems, they had a more open tree canopy (due to different pruning actions) compared to the high (4 × 1.5 m) density training system. This resulted in increasing sunlight penetration and air movement within the tree canopy of the low-density training system. Thus, not the tree volume but the airy component of the canopy may help to reduce SHD and enable better distributions of the spray droplets within the tree canopy.
Our study clearly demonstrated that the low-density training system reduced AUDPC and SHD incidence consistently for the cultivars with high or mid-high susceptibility to SHD, compared to the high-density training system. Low or no effects were seen on the low susceptibility cultivar ( Figure 5). These results indicate that certain combinations of training systems and cultivars can significantly reduce the temporal development of SHD during the season and the accumulation of inoculum sources by the end of the season. This information may be successfully used for the most suitable selection of training system vs. cultivar combinations in those regions where SHD can cause severe epidemics. However, it is important to note that our results on SHD may need to be adjusted in regions with more humid and/or colder climates than Central Europe.

Conclusions
Our study showed that both training system and cultivar susceptibility can significantly influence the temporal epidemics of SHD in an integrated plum orchard. More specifically: (i) Plum cultivars with high or mid-high susceptibility to SHD showed continuous SHD development from May to November, while cultivars with low susceptibility to SHD showed no symptoms until mid-summer and then progressed slowly until November. (ii) The annual disease incidences and AUDPC of SHD on plum cultivars with high or mid-high susceptibility to SHD showed more sensitivity to training systems, compared to cultivars with low susceptibility to SHD.
Certain combinations of training system and cultivar can significantly reduce the temporal development of SHD during the season and the accumulation of inoculum sources (AUDPC) by the end of the season. This may be successfully used as a part of the integrated pest management approach during establishing new plantations.