First Report of Diaporthe cercidis and D. nobilis Causing Leaf Blotch on Populus davidiana × P. bolleana in China.

Shanxin yang (Populus davidiana × P. bolleana) is a commercially valuable hybrid poplar that is widely planted in northern China. Efficient genetic transformation and gene-editing systems have been established for this hybrid poplar (Wang et al., 2011; Wang et al., 2020). However, records of fungal diseases on Shanxin yang are very limited. In July 2020, large necrotic lesions were observed on 16 one-year-old Shanxin yang seedlings planted in a greenhouse of Nanjing Forestry University, Nanjing, China. The disease symptoms appeared mostly on the leaves and not on the stems. Symptoms first manifested as differently sized and shaped brown spots, having clearly demarcated margins. As the disease progressed, the spots coalesced, and large lesions were present on the leaves. Severe infections resulted in whole-plant defoliation and eventually plant death. Small leaf samples (5 mm2) cut from lesion margins were surface sterilized with 75% ethanol for 30 s, followed by 1% NaClO for 90 s and then washed three times with sterile distilled water. After drying on sterilized filter paper, the cut tissues were plated on potato dextrose agar (PDA) supplemented with ampicillin (100 μg/mL) and incubated at 25°C in the dark. Three isolates (named as SX-1, SX-2 and SX-3, respectively) were obtained after 5 days. The isolation frequency was low, which might be due to the greenhouse microclimate that was unfavorable for pathogen spread. Mycelial plugs (5 mm) cut from the leading edge of the mycelia were transferred onto fresh PDA and synthetic nutrient-poor agar (SNA) plates to obtain pure cultures. On both media, colonies of the isolates were white on the front and light yellowish on the back, with concentric zonation. Abundant aerial mycelia developed; the hyphae were hyaline, non-septate, and approximately 0.794-2.961 µm in diameter. On the SNA medium, SX-1 and SX-3 produced globose to subglobose, black pycnidia after 18 days under a 12 h photoperiod. The alpha conidia were fusoid, aseptate, hyaline, smooth, and 6.4 ± 1.2 × 2.4 ± 0.6 µm (n = 50) in size. Under the same conditions, SX-2 produced pycnidia after 20 days, and the conidia were 2.8 ± 0.7 × 7.5 ± 1.3 µm. Beta conidia were not observed on any colony. Based on the morphological characteristics, the isolated mycelia resembled Diaporthe spp. (Gomes et al., 2013). To determine the species identity, genomic DNA from each isolate was extracted, and five loci were amplified, namely, part of the internal transcribed spacer (ITS) amplified with primers ITS1/ITS4 (White et al. 1990); part of the translation elongation factor 1-alpha (EF1-α) with primers EF1-728F/EF1-986R (Carbone and Kohn, 1999); part of the calmodulin (CAL) with primers CAL-228F/CAL-737R (Carbone and Kohn, 1999); part of the β-tubulin (β-tub) with primers Bt2a/Bt2b (Glass and Donaldson, 1995), and part of the histone H3 (HIS) with primers CYLH3F/H3-1b (Glass and Donaldson 1995, Crous et al., 2004). The obtained sequences were deposited in GenBank (accession numbers are listed in Table S1). BLAST analyses showed that the all the amplified fragments were highly homologous to Diaporthe spp. (Table S1). Based on concatenated sequences of the amplicons, a phylogenetic tree was constructed by using Maximum-likelihood and Bayesian inference methods. The multi-locus phylogenetic analyses distinguished SX-1 and SX-3 as D. cercidis, and SX-2 as D. nobilis. To complete Koch's postulates, the pathogenicity of SX-1, as well as SX-2, was tested on both detached and attached leaves of one-year-old Shanxin yang seedlings grown under greenhouse conditions. Healthy leaves were pierced with a sterile needle and then inoculated independently with 5-mm mycelial plugs cut from the edge of the 4-day-old colonies of SX-1 and SX-2 colonies. Controls were inoculated with noncolonized PDA plugs. Three replicates were prepared for each isolate. For the in-vitro tests, detached leaves were placed on wet filter paper in parafilm-sealed Petri dishes and cultured at 25 °C in the dark. For the attached leaf assays, the plants were kept in an 85% humidity chamber immediately after inoculation. All the inoculated leaves developed dark or brown necrotic lesions at 5 days after inoculation, whereas the control leaves showed no symptoms. D. cercidis and D. nobilis were separately reisolated from the inoculated leaves. The former was first described by Yang et al. (2018) as occurring on twigs and branches of Cercis chinensis, and very recently, this pathogen was reported to cause leaf blotch on Acer pictum subsp. mono (Wan et al., 2021). The latter infects some fruit trees (Yu et al., 2018; Sun et al., 2019; Ma et al., 2019) and chestnut (Zhang et al., 2018). All of these studies were conducted in China where there is a great diversity of Diaporthe species (Yang et al., 2018). To our knowledge, this is the first report of both D. cercidis and D. nobilis causing leaf blotch on poplar. The identification of these pathogens is essential for understanding the range of their host species and to manage the resulting fungal diseases, which may cause severe economic damage.