First report of Neocosmospora pisi causing blight on Ammopiptanthus mongolicus in China.

Ammopiptanthus mongolicus is the only evergreen broad-leaved shrub in the desert region of Northwest China, which is one of the dominant species in the desert vegetation of the region, playing an important role in maintaining the stability of the local desert ecosystem. A. mongolicus is also very hardy and drought resistant and can survive extreme temperatures (Liu et al. 2013; Yang et al. 2022). The large-scale death of A. mongolicus could cause desertification in the region. Two months after the discovery of Fusarium verticillioides causing blight on A. mongolicus in Etuoke county, Inner Mongolia Autonomous Region in September 2023 (Yang et al. 2024), a large number of A. mongolicus plants with symptoms of blights were found in Lingwu city, Ningxia Hui Autonomous Region, China (106.442368°E, 37.734026°N) in November 2023. The incidence of diseased plants in this field was about 30%. The field symptoms in Lingwu city were similar to those observed in Etuoke county. The diseased leaves initially turned yellow, then wilted and dehisced, eventually resulting in plant death (Figure 1). The roots of the diseased plants were cut diagonally and the central cylinder showed a brown color (Figure 2). In order to investigate whether the death of A. mongolicus was caused by the same pathogen as those identified previously, 30 roots were collected from 10 diseased plants. After rinsing and surface sterilization (70% ethanol for 3 min and 2.5% NaClO for 5 min, rinsed 3 times with sterile distilled water), diseased tissues (10×10 mm) were placed on potato dextrose agar (PDA) (3 pieces per plate) and incubated from 3 to 5 days at 25°C. The strain AmP5 was isolated and used for further study. After 3 days on PDA medium, fungal colonies were white to milky, the undersides of the cultures were yellowish to orange-brown (Figure 3). After 7 days on synthetic nutrient-poor agar (SNA), microconidia were ovoidal or with a rounded apex and truncate base, 10.5 ± 1.5 μm × 1.6 ± 0.2 μm (×400). The macroconidia were slightly curved or arcuate, 40.5 ± 3.5 μm × 5 ± 0.5 μm (×400) (Figure 4) (Šišić et al. 2018). The pathogen was confirmed to be Neocosmospora pisi by multigene phylogenetic analysis of TEF, RPB1 and RPB2 genes using primers EF1/EF2, F5/G2R and 5F2/11AR, respectively (O'Donnell et al. 2022). The sequences of PCR products were deposited in GenBank with accession numbers OR944631 (RPB1), OR988086 (TEF) and OR988087 (RPB2), respectively. The results of pairwise alignment in Fusarioid-ID database (Crous et al. 2021) showed 99.84% similarity and 83.96% overlap of the EF1-α sequence to the corresponding sequence LR583636 of ex-epitype CBS 123669 of Neocosmospora pisi (syn. Fusarium solani f. sp. pisi), 99.72% similarity and 85.66% overlap of the RPB1 sequence to the corresponding sequence MW834242 of ex-epitype CBS 123669 of N. pisi, and 99.47% similarity and 78.26% overlap of RPB2 sequence to the corresponding sequence LR583862 of ex-epitype CBS 123669 of N. pisi. Moreover, the result of polyphasic identification in the Fusarioid-ID database also showed EF1-a, RPB1, and RPB2 sequences had 99.15% similarity to the corresponding sequences of CBS 1233669. The pathogenicity of AmP5 was tested on potted 64 days old seedlings A. mongolicus plants. The roots of 3 seedlings were inoculated with conidial suspension (1×106 /ml), and another 3 used as controls were inoculated with sterile water, by gently peeling off the soil around the roots during inoculation, and pouring the conidial suspension around the roots (10 ml/seedling). All plants were placed in a growth chamber at 18-25℃ (10 h light; 14 h dark). After incubation for 3-5 days, the symptoms similar to those observed in the field (Figure 5), including brown rot of steles (Figure 6), developed on plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The same pathogen was reisolated from inoculated roots and confirmed as N. pisi based on morphological and molecular analyses (TEF, RPB1 and RPB2). To our knowledge, this is the first report of blight on A. mongolicus caused by N. pisi in China. This study also indicates that blight on A. mongolicus can be caused by different fungal pathogens. Blight caused by different pathogens may have different in terms of control measures and pathogenic mechanisms, so the study of blight caused by different pathogens is of profound value.