The investigations of this study revealed four Botryosphaeria species associated with disease symptoms such as organ blight, dieback, and gummosis in almond fruit trees grown extensively in the Yozgat Province of Turkey. D. seriata, L. theobroma, B. dothidea and N. parvum isolates were characterized by combining morphological, pathological, and molecular data. In addition, these four species were detected for the first time in almond trees in Yozgat Province of Turkey with this study as well as B. dothidea is the first report of this species in almond in Turkey. Based on literature reports, these species have a cosmopolitan distribution and a wide host range (Gure et al. 2005; Slippers et al. 2007; Abdollahzadeh et al. 2010). In previous studies, D. seriata has been reported as a pathogen in woody plants such as peach (Endes et al. 2016), apricot (Smith and Stanosz 2006; Damm et al. 2007; Liu et al. 2015), plum (Phillips et al. 2012; Endes and Kayım 2022a), pear Kurbetli and Demirci (2014) and vineyard (Akgül et al. 2015), almond (Olmo et al. 2016; Gharbi et al. 2017; Holland et al. 2021). In this study, D. seriata was determined as the dominant species. Similarly, the isolation rate of D. seriata from host plants was reported to be higher than other Botryosphaeriaceae species (Slippers et al. 2007; Damm et al. 2007). Moreover, this species is quite common in walnuts (Chen et al. 2014) and almonds (Inderbitzin et al. 2010) in California and vineyards in Australia (Pitt et al. 2013). In contrast, L. theobromae was the least isolated species compared to the other three species. This can be explained that L. theobromae is more pathogenic in woody plants in tropical regions (Abdollahzedeh et al. 2010; Munirah et al. 2017). Although the Eastern Mediterranean Region of Turkey generally has subtropical climatic conditions, L. theobromae was reported as a pathogen in peach orchards and plums in Adana Province of the Eastern Mediterranean Region (Endes et al. 2016; Endes and Kayım 2022a). L. theobromae also was isolated from grafted walnut in the southeastern Turkish with temperate climate conditions (Çiftçi et al. 2023) In Aegean Region of Turkey's, which is warmer and closer to tropical climate conditions compared to the Eastern Mediterranean Region, highly aggressive isolates of L. theobromae were found in fruit species such as figs (Çeliker and Michailides 2012), vineyards (Akgül et al. 2014) and strawberries (Yildiz et al. 2014). Moreover, Burges et al. (2006) reported that L. theobromae is mostly pathogenic in tropical regions, and N. parvum is pathogenic in hot climate conditions. However, L. theobromae was also isolated from a temperate region such as Southern-east Anatolia (Özer et al. 2022) and Southern Iraq (Al-Saadoon et al. 2012). N. Parvum, obtained from infected almond trees, has been reported as a pathogen in peach, mango, pistachio, orange and horticultural crops such as vineyards in Australia (Cunnington et al. 2007); on Syzygium cordatum tree in South Africa (Pavlic et al. 2009); mango tree in Italy (Ismail et al. 2013); peaches in Greece (Thomidis et al. 2011); walnuts and olives in Spain (Moral et al. 2010); and in almond, vineyards, and plum in Turkey (Kayım et al. 2015; Akgül et al. 2015; Endes and Kayım 2022a).
In morphological characterization studies, there was no overlap between the characters such as color, division, length, and width of the conidia as well as the mycelial development of the species. The optimum radial mycelial growth temperature was in the range of 26–29°C for all species (Table 3). L. theobromae showed faster radial mycelial growth at 25°C than the other three species. Similarly, Thomidis et al. (2011) reported that the temperature required for optimum radial mycelial growth of N. parvum was 25°C and (Ismail et al. 2013) reported that Neofusicoccum isolates (N. parvum and N. australe) grow at a minimum of 10°C, an optimum of 25°C and a maximum of 35°C. Copes and Hendrix (2004) reported that the optimum growth temperature of B. obtuse was between 20°C and 26°C; although it grew in a temperature range of 8°C to 36°C and its development stopped at 4°C; and B. rhodina grew from 15°C to 35°C, the optimum growth temperature was between 25°C and 35°C; in 10°C and 40°C the growth was stopped, or the growth was inconsiderable. In addition to these previous studies, Wang et al. (2011) found that L. theobromea had a faster radial mycelial growth rate than D. seriata at 25°C on PDA medium; Chen et al. (2014) reported that N. parvum, D. seriata, and L. theobromea showed optimum mycelial growth on PDA medium at 25°C and 30°C, respectively. The results obtained in this study were in accordance with mentioned literature.
In this study, all species produced specific colony morphology. D. seriata formed the only one colony with olive-gray color. Contrary, Moral et al. (2010) reported that D. seriata isolates formed two different groups based on their colony characteristics. The first group was initially pastel-gray but turned greenish-gray as it matured; the isolates in the second group produced abundant aerial mycelium and were initially whitish-gray but turned greenish-gray or black as they matured. Similarly, Akgül et al. (2015) reported that D. seriata isolates formed a single type of colony. However, in both studies, D. seriata isolates easily formed pycnidium on PDA medium. In this study, D. seriata produced oval, ellipsoid, or cylindrical-shaped brown conidia, some with septa (Gure et al. 2005; Endes et al. 2016). Reports from Akgül et al. (2015) and the data obtained in this study are quite similar. However, Wang et al. (2011) reported that this species produced abundant overhead hyphae with non-septa and dark brown mature conidia. The colony characteristic of N. parvum with aerial mycelium on whitish gray and bubble-shaped masses of hyphae, producing a very small amount of pycnidium compared to L. theobromae and D. seriata isolates (Amponsah et al. 2008); and fusoid shaped, colorless and non-septa conidia with 1–2 divisions turning brown after a long time (Ismail et al. 2013; Phillips et al. 2013; Akgül et al. 2015) showed that this species is culturally different from L. theobromae and D. seriata isolates. L. theobromae had the fastest radial mycelial growth on PDA medium at 25°C and formed large pycnidia, as well as ellipsoidal or cylindrical shaped, with septa, straight lines on brown mature conidia and paraphyses, which is a useful character in distinguishing Lasiodiplodia species from each other, therefore leads to easy distinguish of this species from D. seriata and N. parvum species (Burgess et al. 2006; Alves et al. 2008; Abdollahzadeh et al. 2010; Wang et al. 2011; Chen et al. 2014; Akgül et al. 2015; Endes et al. 2016). B. dothidea showed the maximum temperature for optimum mycelial growth compared to the other three species. Similarly, the optimum temperature range for mycelial growth of B. dothidea was determined in the range of 25 − 32°C in previous studies (Luo et al. 2022; Nazerian et al. 2019).
Recent PCR-based studies have shown that fungi of Botryosphaeria are generally associated with anamorphic genera such as Fusicoccum and Diplodia (Crous et al. 2006; Slippers et al. 2007; Phillips et al. 2013). Studies proved significant differences between the morphological characters of these two genera of Botryosphaeriaceae (Wang et al. 2011). Slippers and Wingfield (2007) reported that fusicoccum-like species form hyaline (colorless), narrow (˂ 10 µm) conidia and have thin conidia walls (˂ 0.5 µm); Diplodia-like species, on the other hand, have wider conidia (˃10 µm) and thicker conidia walls (0.5–2 µm), with colored conidia over time in mature form, the other important anamorphic genus of Botryosphaeria fungi, Lasiodiplodia, have always been grouped separately from these two genera. In characterization studies similar to this study, the morphology of colonies and conidia of the species were supported by phylogenetic studies. Fusicoccum-like species generally form colorless and fusoid-shaped conidia, and Diplodia-like species, which form brown oval, ellipsoid, and cylindrical-shaped conidia, were grouped into two distinct clades (N. parvum and Diplodia-like species). Subsequently, Diplodia-like species forming colored conidia grouped in two different clades within themselves, one containing isolates of D. seriata and the other isolates of L. theobromae. Pathogenicity tests (Table 4; Fig. 3) determined D. seriata, L. theobromae and N. parvum as pathogens in almond trees in the Eastern Mediterranean Region. The results of cut branch pathogenicity and seedlings pathogenicity overlap entirely with each other. However, in cut branch pathogenicity studies, while no isolate formed gum in the bark tissue of 25 cm long almond branches, it formed gum in the inoculation areas of the trunk of almond saplings. The pathogenicity test results of all isolates, L. theobromae was determined as the most virulent species by forming longer lesion length and more abundant gum than the other two species, which is in accordance with other studies (Britton and Hendrix 1989; Wang et al. 2011). In the cut-branch pathogenicity studies of D. seriata isolates, virulence levels were determined to be statistically different (Laundon 1973; Brown-Rytlewski and McManus 2000; Úrbez-Torres and Gubler 2009). This difference can be explained by the resistance mechanisms of host plant against each isolate in species with a wide host range, such as D. seriata (Lv et al. 2012; Li et al. 2014). In addition, D. seriata produced less gum but longer lesions in trunk tissue than N. parvum compared to the other two species. However, Akgül et al. (2015) reported that N. parvum was the most virulent species among the Botryosphaeriaceae species isolated from the Vineyards in the Aegean Region of Turkey. Similar to their study, in this study, it was demonstrated that N. parvum is at least as important as L. theobromae and D. seriata in the Eastern Mediterranean Region, considering the length of the lesion formed in the trunk as well as exuding gum from bark tissue. As far as our knowledge, this is the first study that deals with field survey, morphology, phylogeny, and pathogenicity of L. theobromae, D. seriata and N. parvum that cause wilt, gummosis, trunk and branch canker of almond trees in Turkey.
This study also draws attention to the development of effective control strategies for these three species that cause wilt, gum disease, and dead tissue on the trunk and branches of apricot trees. Because these three species are among the potential risk factors for citrus, vineyard, pome, and stone fruit trees in the Eastern Mediterranean Region, which is one of the most important fruit production centres of Turkey, therefore, to avoid or prevent diseases caused by Botryosphaeria species on almonds and other host plants, good care of fruit trees as well as the application of protective fungicide, especially after pruning, can be a good prevention approach.