First Report of root rot of White Mulberry Caused by Simultaneous Infections of Phytophthora megasperma and P. multivora in Italy

White mulberry (Morus alba L., Moraceae) is a deciduous shrub or tree native to East Asia where it is cultivated for feeding silkworms. In many temperate to subtropical regions of the Northern Hemisphere it is wild and cultivated; in Italy it is cultivated for its edible fruits and for landscaping and gardening. In summer 2015, symptoms of leaf blight, chlorosis, wilting, defoliation, trunk cankers with resin exudates and root rot were observed on scattered trees in home gardens near Catania (Sicily). Overall 15 out of 100 trees were symptomatic. Two homothallic Phytophthora species were isolated on BNPRAH selective medium singularly from infected roots of 3 and 5 trees, respectively, while both species were isolated from roots of 7 out of 15 trees; both species were found in the rhizosphere of all 15 symptomatic trees with carob leaf as baits (Jung et al. 2016). Pure cultures were obtained by single-hypha transfer. Isolates of first species formed petaloid colonies on potato dextrose agar (PDA) and had an optimum growth for temperature of 25°C (6 mm/day). On V8 juice agar, they produced nonpapillate, noncaducous sporangia and oogonia with paragynous antheridia. Mean diameter of oospores was 37.8 ± 0.4 μm (range 30 to 43 μm). Second species formed on PDA petaloid felty colonies, had an optimum growth of 25°C; it produced ovoid or obpyriform, semipapillate and persistent sporangia and oogonia with paragynous antheridia. Oospores (mean diameter 22.7 ± 1.3 µm; range 19–31 µm) were nearly plerotic with a thick wall (2.6 ± 0.5 µm). Internal transcribed spacer region, (ITS)1 5.8S-ITS2 (Van Tri et al. 2015) and a fragment of cytochrome oxidase subunit 1gene (COI) from mitochondrial DNA (Robideau et al. 2011) of all isolates were sequenced. Sequences of representative isolates were submitted to GenBank (P. megasperma, Accession N°. KX370824 and KX370823 for ITS and COI, respectively; P. multivora, KX370826 and KX370825 for ITS and COI, respectively). ITS fragments had a 100% similarity with reference isolates of P. megasperma (GU259356) and P. multivora (KU146526), respectively. COI amplicons of mulberry isolates of two species had 99% similarity with reference isolates of P. megasperma (HQ261362) and P. multivora (HQ708344) retrieved from GenBank. On the basis of morphological and molecular characters the species were identified as P. megasperma s.s. and P. multivora. Pathogenicity of two representative isolates (one per each species, separately and in combination) was determined by a soil infestation test using inoculum produced on kernels and ten 18-month-old white mulberry rooted cuttings for each isolate (Salamone et al.2011). Ten rooted cuttings were used as a control. Cuttings were grown in a greenhouse (20-32°C). In combination trials, soil was infested with a mixture of inoculum of both isolates. Within 40 days, all cuttings in infested soil showed leaf chlorosis, wilting and root rot. Controls remained healthy. The two Phytophthora species were re-isolated solely from inoculated roots thus fulfilling Koch’s postulates. Presently the economic significance of this disease is limited as silk production in Italy has been dramatically reduced. However, to our knowledge, this is the first report of Phytophthora root rot on Morus species worldwide.