Fusarium and Neocosmospora Species Associated with the Decline of Metasequoia glyptostroboides in China.

During surveys conducted in 2020, severe symptoms associated with death and decline were observed on >30-year-old Metasequoia glyptostroboides (Chinese redwood) trees in the shelter-forests along Yangtze River in Jingzhou city, Hubei province, China. A previous study showed that Phytophthora acerina was one of the causal agents of the decline of the Chinese redwood. In this study, a total of 147 fungal isolates were obtained from the diseased roots and xylem of trunks of declining M. glyptostroboides trees. Through morphology and multi-locus phylogenetic analysis, these isolates were identified as eight species belonging to the genera Fusarium and Neocosmospora including F. fujikuroi, F. irregulare, F. odoratissimum, F. reticulatum， N. falciformis, N. keratoplastica, N. solani, and N. tonkinensis. Single inoculation and co-inoculation with P. acerina assays of these Fusarium and Neocosmospora species were then performed to test pathogenicity on three-year-old seedlings of M. glyptostroboides. Lesions (i.e., on seedling stems) caused by species of the genera Neocosmopora and Fusarium were smaller than those caused by P. acerina. Co-inoculation of F. fujikuroi and P. acerina, as well as the co-inoculation of F. reticulatum and P. acerina caused larger lesions than inoculations with P. acerina alone. All these species of Fusarium and Neocosmospora were shown to have the potential to be pathogenic to M. glyptostroboides. This study provided evidence that the decline of M. glyptostroboides in Jingzhou is a disease complex.

First Report of Root Rot Caused by Phytophthora acerina on Metasequoia glyptostroboides in China.

Metasequoia glyptostroboides Hu & W. C. Cheng (Taxodiaceae), commonly called the Chinese redwood or dawn redwood, is a well-known "living fossil" and rare relict plant species endemic to China, which has been successfully cultivated throughout the world (Ma 2007). In July to September 2020, trees of Chinese redwood which were more than thirty years-old, showed symptoms of decline and death associated with branch dieback, root and collar rot (Fig. 1) in Yangtze River shelter-forests of Jiangling County in Hubei Province, China (112°15'19″E, 30°11'56″N; 40m). Diseased roots and rhizosphere soils were collected in September 2020 and April 2021. Using the baiting method, a homothallic Phytophthora sp. was recovered consistently from diseased roots and soil samples of Chinese redwood. All the isolates of this Phytophthora sp. formed similar colonies on V8 agar and corn meal agar (Fig. 2), and then three representative isolates (L4-5-4, L4-5-5 and L4-5-6) were randomly selected for morphological and molecular identification. In distilled water, semipapillate persistent sporangia were borne in simple sympodial branched sporangiophores. Sporangia were predominantly ovoid (Fig. 3a, d and f), but other shapes were observed including subglobose (Fig. 3b), limoniform (Fig. 3c) or distorted shapes (Fig. 3e), averaging 44.1 ± 7.7 µm (n=102) in length and 32.8 ± 5.2 µm (n=102) in width, with narrow exit pores of 8.0 ± 1.4 µm (n=93) and a length/breadth ratio of 1.3 ± 0.10 (n=102). Chlamydospores were not observed. Oogonia were globose or subglobose, 20.51 to 40.15 µm (av. 33.1 ± 3.9 µm) (n=119) in diameter, with smooth walls and paragynous antheridium (Fig. 3g-i). Oospores were globose or subglobose in elongated oogonia with medium wall thickness of 1.9 ± 0.5 µm (n=36), aplerotic or plerotic and 16.9 to 32.6 µm in diameter (av. 26.6 ± 3.8 µm) (n=40). According to the above morphological characteristics, this Phytophthora sp. was placed in Waterhouse's (1963) group III. The sequences of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA of each isolate (GenBank Accession No. OK087320, OK087321 and OK087322) was 760 bp and had identity of 99.84% with three P. acerina isolates (JX951285, JX951291 and JX951296), while the 800 bp ß-tubulin (BTUB) sequences (OK140540, OK140541 and OK140542) showed 99.97% homology to the sequence of P. acerina (KC201283) (Ginetti, Moricca and Squires 2014) (Table 1). The ML phylogenetic trees were established by comparing ITS and BTUB sequences of three Phytophthora strains (L4-5-4, L4-5-5 and L4-5-6) with reference sequences of isolates of Phytophthora in ITS and BTUB in GenBank (Fig. 4-5). Based on the morphological and molecular characteristics, the strains were identified as namely P. acerina. In addition, pathogenicity assays were performed with one of the three strains (L4-5-4) on M. glyptostroboides using both one year old and three years old seedlings. Inoculum was prepared by subculturing agar plugs from edges of CMA cultures into V8 medium plates, incubating at 20 ℃ in darkness for 10 days. Six seedlings planted in pots filled with sterilized soil were inoculated by mycelium plug at root collar and stem wounded by a 8 mm diameter puncher. Six control seedlings were inoculated in the same manner as above, and sterile agar plugs were used. After 35 days, inoculated seedlings all had necrotic lesions at the inoculation sites, and some seedlings had the symptoms of foliage blight and dieback, whereas control seedlings remained healthy (Fig. 6). The number of fibrous roots after inoculation was significantly less than the control, and the roots of inoculated seedlings blackened or even rotted, while there were no obvious symptoms in the control (Fig. 7). Phytophthora isolates recovered from the symptomatic tissues of artificially inoculated plants were identical to isolate L4-5-4 in morphological characters and ITS sequencing. This is the first report of P. acerina causing root rot on the Chinese redwood in China. As only the seedlings were inoculated, further research is needed to address the epidemiology and pathogenicity of P. acerina to adult trees of Chinese red wood. References: Ginetti, B. et al. 2014. Plant Pathology, 63(4): 858-876. Ma, J. S. 2007. Bulletin of the Peabody Museum of Natural History, 48(2): 235-253. Waterhouse, G. M. 1963. Mycological Papers 92:1-22.