Identification and pathogenicity of fungal species associated with grapevine trunk diseases in Khorasan-Razavi province, Iran

Fungal isolation and morphological identification

In this study, a total number of 258 fungal isolates were obtained. Isolation, frequency and geographical distribution of the isolates recovered from diseased grapevines in Khorasan-Razavi province are shown in Table 1. Based on colony appearance, culture characteristics, and microscopic structures, the main fungal isolates were divided in three groups.

Group one: One hundred and fifty-four isolates (59.7% of total isolates) were classified in this group. These isolates were characterized by beige medium brown, pale to pale brown to reddish brown flat slow-growing cultures on PDA. Sporulation was abundant, conidia were hyaline and aseptate, hyphae were single or fasciculate, and three types of phialides (I, II, and III types), that were different in size and shape were observed in these isolates. All of these isolates were tentatively identified as Phaeoacremonium (Crous et al. 1996, Mostert et al. 2006, Spies et al. 2018).

Group two: Forty-eight isolates (18.6 % of total isolates) as 'pestalotioid fungi' were placed in this group. These isolates were characterized by white to medium-brown, margin uneven and flat slow-growing cultures on PDA. Hyphae hyaline, straight, branched, and septate. Conidiomata pycnidioid, solitary or aggregate, dark, 95−345 μm diam. Conidiophores 7−18 × 2−3 μm,Conidia abundant, single,obovoid to fusiform, straight to slightly curved, 12−19 × 4−6 μm, with 3 dark brown transverse septa, hyaline to sub-hyaline basal and apical cell and two hyaline, unbranched and short flexuous appendages (Fig. 2). These morphological features were compatible with the description of Seimatosporium spp. (Senanayake et al. 2015, Wijayawardene et al. 2015, Lawrence et al. 2018).

Group three: Fifty-six isolates (21.7 % of total isolates) were classified in this group. These isolates were characterized by white and medium-growing mycelium on PDA (28 mm at 25 within 8 days) that became yellow-orange and brown in color with age. These morphological features were not sufficient to identify these isolates at the generic level; however, they were considered as basidiomycetous fungi (Sigler & Abbott 1997, Markakis et al. 2017. Brown et al. 2019).

Molecular identification and phylogenetic analysis

To confirm the identification based on morphology and cultural characteristics, BLASTn searches in GenBank showed that ITS sequences of basidiomycetous isolates had 100 % identity with Fomitiporia mediterranea (isolate: CB6, accession no.  EU477476). Beta tubulin sequences of Seimatosporium isolates were also 100 % identical to that of Seimatosporium vitis Napa776 (accession no. KY706252). For phylogenetic analyses, the BT sequences of 17 suspected Phaeoacremonium isolates were aligned with 34 sequences of reference isolates and Pleurostoma richardsiae (CBS270.33) was used as out-group. The alignment consisted of 628 characters (including gaps), of which 215 were constant and 292 parsimony informative. Maximum parsimony analysis resulted in 4 equally most parsimonious trees (TL=1303, CI=0.475, RI=0.765, RC=0.363). Analysis of the BT clearly separated Iranian isolates in 4 clades including P. iranianum (52 isolates), P. tuscanum (8 isolates), P. minimum (75 isolates) and P. parasiticum (19 isolates). One of the ML trees is shown in Fig. 3 with bootstrap support values at the nodes.

Pathogenicity tests

Mean lengths of wood discolorations caused by inoculated isolates on rooted grapevines are shown in Fig 4. All the inoculated isolates produced wood lesion lengths were significantly different from those in the control plants (P<0.05). Phaeoacremonium minimum was the most aggressive fungal species and produced the longest necrotic wood lesions (29.8 mm) on the inoculated shoots followed by S. viticola (27.8 mm) and F. mediterranea (12.1 mm). No significant difference was observed between P. minimum and S. viticola. Re-isolation percentages were between 60.0 % (F. mediterranea) and 100 % (P. minimum and S. viticola) on inoculated plants, and no fungal isolateswere isolated from control treatments.

DISCUSSION

Nowadays, GTDs are considered as the most destructive diseases of grapevine (Gramaje et al. 2018, Guerin-Dubrana et al. 2019) and to date, more than 138 fungal species have been associated with GTDs (Gramaje et al. 2018, Raimondo et al. 2019) worldwide. The results of this study showed that grapevines in Khorasan-Razavi province are infected with known fungi causing decline diseases. In this province, morphological and molecular data provided evidence that at least Phaeoacremonium spp., F. mediterranea and S. vitis occur on grapevines in this region. In addition to these taxa, several isolates of Aspergillus, Penicillium, Alternaria, Fusarium, Bipolaris and Coniothyrium species, were also isolated from affected grapevines in this province, which were not considered in this paper. Phaeoacremonium isolates were the most abundant fungal groups isolated from affected trees. More than 60 species of Phaeoacremonium have been isolated and reported from different woody plant hosts around the world, among which grapevine is known as one of the most important host of this group of fungi (Crous et al. 1996, Mostert et al. 2006, Essakhi et al. 2008, Graham et al. 2009, Ariyawansa et al. 2015, Gramaje & Di Marco 2015, Crous et al. 2016, Da Silva et al. 2017, Spies et al. 2018). Throughout this work, four Phaeoacremonium species, including P. parasiticum, P. minimum, P. iranianum and P. tuscanum were recovered from grapevines showing decline symptoms. The first three species are relatively widespread and have been reported from grapevine in various countries around the world (Mostert et al. 2006, Spies et al. 2018), while, P. tuscanum is reported only from grapevines in Italy (Essakhi et al. 2008) and Iran (Mohammadi 2012, Arabnezhad & Mohammadi 2014). 

In this study, P. minimum was the dominant Phaeoacremonium species obtained from diseased grapevines. This is in agreement with the previous studies on grapevine in France (Larignon & Dubos 1997), Italy (Mugnai et al. 1999), Australia (Pascoe & Cottral 2000), South Africa (Groenewald et al. 2001), Spain (Armengol et al. 2001), Argentina (Dupont et al. 2002), Chile (Auger et al. 2005) as well as Iran (Mohammadi & Banihashemi, 2007, Arabnezhad & Mohammadi, 2012. Mohammadi et al. 2013a, Arabnezhad et al. 2013). All of the four species of Phaeoacremonium isolated during this study, have previously been reported from grapevines and other woody plants in various provinces of Iran. For instance, P. minimum has previously been reported affecting grapevines in Fars, Hamadan, Isfahan, Yazd, Kerman, Kohgiluyeh and Boirahmad, and West and East Azerbaijan provinces (Arabnezhad & Mohammadi, 2012, 2014, Arzanlou et al. 2013, Mohammadi et al. 2013a, Narmani et al. 2014, Faraji & Mohammadi, 2015). Phaeoacremoniumparasiticum was associated with grapevine trunk diseases in Fars and Kerman provinces and stone and pome fruit trees (Arzanlou et al. 2014, Sami et al. 2014, Soltaninejad et al. 2017), cypress (Mohammadi et al. 2014), date palm (Mohammadi, 2014), poplar trees (Hashemi & Mohammadi, 2016, Kazemzadeh Chakusary et al. 2017) and some other forest trees (Kazemzadeh Chakusary et al. 2017) in Iran. Phaeoacremonium iranianum was isolated and recorded from grapevines in Fars and South Khorasan and also from alder (Kazemzadeh Chakusary et al. 2017), hawthorn, quince(Sami et al. 2014), apple (Arzanlou et al. 2014, Sami et al. 2014) and pomegranate (Kazemzadeh Chakusary et al. 2017) trees in various provinces of this country. Phaeoacremonium tuscanum has previously been reported affecting grapevines in Kohgiluyeh and Boirahmad province (Arabnezhad & Mohammadi 2012) and peach trees in Kerman province (Soltaninejad et al. 2017). Therefore, the current study is the first report on the occurrence of these species on grapevine in Khorasan-Razavi province.

Fomitiporia mediterranea was another fungal species that was isolated from grapevine in this study. Wood-rotting basidiomycete fungi, including Fomitiporia spp., are known as grapevine trunk pathogens (Bertsch et al. 2013, Brown et al. 2019). Fomitiporia mediterranea introduced by Fischer (2002) and as a fungal trunk pathogen on grapevine is widespread in the Mediterranean area (Fischer & Kassemeyer 2003). The importance of thisbasidiomycete as a fungal trunk pathogen has been widely investigated (Mugnai et al. 1999, Fischer 2002, Fischer & Binder 2004). This fungus is the main wood-decaying pathogen in some wine-growing areas of southern Italy (Ciccarone et al. 2004) and Greece (Markakis et al. 2017). In Iran, this taxon has been previously reported to affect grapevines in Northern Khorasan province (Farashiyani et al. 2010, Rajaiyan et al. 2013), Russian olive (Ahmadyusefi Sarhadi & Mohammadi, 2019) and elm trees (Mirsoleymani & Mostowfizadeh Ghalamfarsa 2019) in Fars province. To the best of our knowledge, this study is the first report of the occurrence of F. mediterranea on grapevine in Khorasan-Razavi province.

The genus Seimatosporium Corda, with S. rosae Corda as the type species, was first described and introduced by Corda (1833). Various Seimatosporium species have been isolated and reported from symptomatic or dead parts of grapevines in different countries (Diaz et al. 2012, Vaczy 2017, Farr & Rossman, 2018, Lawrence et al. 2018, Camele & Mang, 2019, Liu et al. 2019). In this study, 48 isolates of S. vitis were obtained from necrotic wood tissues of grapevines in Khorasan-Razavi province. This species has also been reported from symptomatic grapevines in Hungary (Vàczy 2017), North Carolina, USA (Lawrence et al. 2018) and Italy (Camele & Mang, 2019). To date, there has only been one report of the sexual morph of S. vitis on a dead branch of Vitis sp. in Iran (Mehrabi et al. 2017). Therefore, this study is the first report of this species from necrotic wood tissues of grapevine in Iran.

The results of the pathogenicity tests showed that all the inoculated species on the rooted cuttings of the grapevines were pathogenic and produced wood discoloration on inoculated shoots, developing from the point of inoculation. Phaeoacremonium minimum showed the longest lesions and were the most virulent species on the rooted cuttings of the grapevines. The findings of this study are in accordance with the results previously reported by other researches on grapevine (Feliciano et al. 2004, Halleen et al. 2007, Aroca & Raposo 2009, Gramaje et al. 2010). Mohammadi et al. (2013a) reported P. minimum causing significantly longer lesions than the other Phaeoacremonium species tested on grapevine shoots under field conditions in Iran. Similar results were also reported for this fungus on green shoots of grapevine under greenhouse conditions in this country (Mohammadi et al. 2014). Our findings for the first time in Iran, indicated that S. vitis is involved in the grapevine trunk-disease complex. Relatively, little is known regarding the importance of this species on grapevine. However, pathogenicity tests in Hungary (Vaczy 2017), Italy (Camale & Mang 2019) and the USA (Lawrence et al. 2018) have also shown that S. vitis can be considered as a fungal trunk pathogen on grapevine. Overall, all the fungal species were identified in this study, have previously been isolated and reported from grapevine in other provinces of Iran with the exception of S. vitis, which was reported here for the first time associated with wood necrosis of grapevine in this country. Further research is required to provide better understanding of the role of various fungal species on grapevine GTDs in Iran especially Khorasan-Razavi province.