First Report of <i>Alternaria alternata</i> Causing Leaf Spot on <i>Broussonetia papyrifera</i> in China

Paper mulberry (Broussonetia papyrifera) belongs to the Moraceae family and originates from East Asia. It serves as source material for papermaking, livestock feed, and traditional Chinese medicine (Peng et al. 2019). In September 2023, a serious leaf spot occurred in the agricultural zone in Laohe town (32°56’31” N, 112°25’27” E), Henan Province of China. A total of 200 plants were inspected over an area of about 50 acres and the incidence rate reached 79%. The disease initially presents as small black spots at the edge of the blade, which gradually extend toward the center. Subsequently, dark spots form around the lesions, leading to yellowing of the affected area. Five diseased leaves were collected from each of ten individual plants (n = 50). All disease leaves exhibited typical necrotic lesions covering between 5% and 10% of the leaf surface area and were excised into small sections at the interface between infected and healthy tissues. The samples were surface sterilized using 75% ethanol for 30 seconds, followed by 1% NaClO for 1 minute. After rinsing with sterile water, the tissues were placed on potato dextrose agar (PDA) plates and incubated at 25°C for 5 days. A total of 33 purified fungal strains were obtained. Three isolates (GS-11, GS-34, and GS-51) were selected for further investigation. The colony displayed abundant aerial mycelium, with colors ranging from dark green to greyish brown on the reverse side. Conidia were borne in unbranched chains of 4 to 11 conidia, obclavate or spheroidal, dark brown, with 2 to 5 transverse septa and 0 to 3 longitudinal septa, measuring 12.5 to 27.8 × 5.9 to 11.9 μm (n=100). The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (tef1), glyceraldehyde 3-phosphate dehydrogenase gene (gapdh), and RNA polymerase II second largest subunit (rpb2) were amplified for further molecular identification. The obtained sequences were deposited in GenBank under the following accession numbers: PP839931 to PP839933 (ITS), PP842671 to PP842673 (tef1), PP842674 to PP842676 (gapdh), and PP842677 to PP842679 (rpb2). BLASTn analysis of isolate GS-11 showed that ITS region revealed 99.81% identity (522/523 bp) with Alternaria alternata strain accession No. ON973891, and 100% identity with PP351934 (230/230 bp), OP946448 (566/566 bp), OR343386 (872/872 bp). A phylogenetic tree was constructed using neighbor-joining method based on the combined genes sequences (ITS, tef1, gapdh, and rpb2), along with sequences from type strains. The three isolates formed a clade with the strains CBS 130265 of A. alternata in phylogenetic trees, being separated from other Alternaria spp. The molecular analyses supported the isolates as members of A. alternata (Woudenberg et al. 2015). To confirm pathogenicity, 2 ml of a 1 × 106 conidia ml-1 suspension per plant was sprayed onto the unwounded leaves of five plants, while five additional plants were sprayed with sterile water as mock-inoculated controls. The plants were incubated in environmental chambers maintained a 12 h light / 12 h dark cycle at 28°C with 90% relative humidity. Seven days post-inoculation, lesions resembling those observed under natural conditions were detected, whereas control plants remained asymptomatic. Subsequent reisolation yielded cultures that were morphologically and molecularly identical to the original strains, thereby fulfilling Koch's postulates. Witches’-Broom and bacterial leaf spot of B. papyrifera caused by phytoplasma and Pseudomonas syringae have been previously reported in China (Liu et al. 2004; Li et al. 2021). This study presents the first report of A. alternata as the causal agent of leaf spots on B. papyrifera in China. Determining the pathogen is essential for applying effective disease control measures, particularly in the selecting of appropriate pesticide treatments.