Neopestalotiopsis rosae Causing Black Spot on Leaf and Fruit of Pecan (Carya illinoinensis) in China.

Pecan (Carya illinoinensis K. Koch) is very productive and brings good economic returns for mountain dwellers. In recent years, symptoms were observed on both leaves and fruits of pecan in orchards, Fuyang, Anhui Province, and Jiande, Zhejiang Province. On the leaf, spots, rust-colored, long shuttle to irregular shaped, and 1-3 mm in size, firstly appeared between veins. The color of area around the spot gradually changed from green to yellow making a yellow halo. Then, some spots often merged in to a big lesion. On the fruit, symptoms firstly appear as irregular and black spots. The spots gradually spread to most part of the fruit and the core of the fruit turned black at the late stage of pathogenesis. Normally, the leaf infection rate was less than 10%, whereas infection rate of fruit could reach 5-15% which leads to drastic quality reduction and significant yield losses. To isolate the pathogens, fresh infected tissues were cut from samples, disinfected and cultured at 2% water agar at 28 oC till colony could be spotted. Fungal isolates were obtained by mycelia tip transferring. Similar cultural and morphological characters showed on 39 of the total 45 isolates. The mycelia grew rapidly in PDA plates, and colony diameter could reach 8.0 cm after culture at 25 oC in dark for 5 days. Mycelia on surface of the colony were cotton-like and showed obvious growth rings, and turn yellow from the middle on aged cultures. Pycnidia, brown to dark in color, began to form on PDA after 15 days' culture in the dark. Conidia were fusoid, straight to slightly curved, 4-septate, 23.5-26.3×7.6-9.0μm, average 24.9×8.3μm (n=60) in size. The basal cell was hyaline, conic with a truncate base and thin-walled, average 4.4-5.1μm long (n=60). The three median cells were doliiform and brown with darker septa. The apical cell was hyaline, cylindrical, with 2-3 tubular apical appendages arising from the apical crest, unbranched, filiform, average 21.7-29.2 μm(n=60) long. The cultural and morphological characters of the strains matched the previous descriptions of Neopestalotiopsis sp. (Maharachchikumbura et al., 2014). Their 3 partial genes, ITS, β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1-α), were amplified and sequenced with primers (Lee et al., 2006; Maharachchikumbura et al., 2014). These sequences were submitted in GenBank with accession number of MW504791 (ITS), MW633956 (TEF1-α), and MW633958 (TUB2), seperately. The BLAST results against the GenBank database showed more than 99% identity with sequences claimed from Neopestalotiopsis sp. The multilocus phylogenetic analysis of the three genes showed the isolate was closest with N. rosae. The strains were finally identified as N. rosae by morphological and molecular analysis. Pathogenicity testing was performed by spreading 20μl conidia suspension drop (1 × 106 conidia ml-1) on surface of wounded or unwounded leaves and fruits. The pecan variety MaHan was used for inoculation. Wounds were made by scratching with sterile needles, and then a drop of 20μl conidia suspension was spread on the wounded area. For unwounded inoculation, a drop of 20μl conidia suspension was spread without wound procedure. The same amount of water drops were spread on the surface of wounded and unwounded tissues as controls. The plant parts were wrapped with PE films to keep moisture after drop spreading. Ten trees were selected for inoculation, and 10 leaves and fruits were randomly selected separately for wounded and unwounded inoculation and corresponding controls in each tree. The similar symptoms appeared from 3 dpi on leaves and 9 dpi on fruits, and finally showed on more than 90% wounded inoculated leaves or fruits and on less than 50% after unwounded inoculation, while controls remained symptomless. N. rosae was consistently re-isolated from inoculated parts with symptoms, while not from the control. Therefore, N. rosae was confirmed as the pathogen causing black spot on leaf and fruit of pecan (C. illinoinensis). To the best of our knowledge, this is the first report of N. rosae causing black spot disease of pecan (C. illinoinensis). In recent years, both the infection area and severity of this disease grow rapidly, which means the potential of the disease to become a big problem in local orchards.