First report of leaf spot on Magnolia grandiflora caused by Lasiodiplodia theobromae in Jiangsu.

Magnolia grandiflora linn, with large and fragrant flowers, is widely planted in the south of Yangtze River valley in China. It is an excellent street tree and a beautiful ornamental tree for landscaping. In October 2021, a new leaf spot disease was observed on M. grandiflora seedlings and mature trees growing in Nanjing Botanical Garden, Jiangsu Province, China. According to statistics, about 300 M. grandiflora trees were planted here, and approximately 60% of M. grandiflora trees suffered from the disease. In the beginning, small black spots appeared on the leaf of M. grandiflora, and then the disease spots were connected into coalesced, and eventually lead to a large area of leaf dead (Figure S1A). To isolate the pathogen, ten diseased leaves were collected from ten plants distributed in different five areas of the botanical garden. The leaf sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% alcohol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in the darkness. Pure cultures were obtained by monosporic isolation. Twenty-three isolates were obtained (the isolate rate of 72%), and identified as Lasiodiplodia sp.. A representative isolate, G-H-1 was used for the further study. The colony of G-H-1, growing on PDA was cotton-like. The primary mycelia was gray and white in the early stage of culture. It gradually turned black gray in the later stage, and the reverse was similar in color (Figure S1B). The pycnidia (fruiting body) was black and appeared over PDA plates after 15 days (Figure S1C). The hyphae of G-H-1 were dark brown, and the conidia were monospora, oval or elliptic, with a size of (9.6 ~ 13.3) µm× (5.7 ~ 8.0) µm (mean 11.7×6.6 µm, n=35) (Figure S1D). In the pycnidia, the conidiophores were inside and produced conidia (Figure S1E). In the early stage, the conidia of G-H-1 were colorless transparent, then gradually turned dark brown with a septum in the center (Figure S1F). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The regions of ITS, translation elongation factor 1-alpha (TEF1α) and β-tubulin (TUB) genes (GenBank Accession No.OM698339, No.OM942757, and No.OM942756, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995). The obtained sequences showed 99.05-99.81% similarity with those from L. theobromae accessions in GenBank. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate G-H-1 clustered in the L. theobromae clade with 96% bootstrap support (Figure S2). To test pathogenicity, three one-year-old M. grandiflora seedlings that previously had been wounded with a sterile needle were inoculated with 20 μL conidia suspension (1×106 spores/mL) on the left sides of leaves. Inoculation with 20 µL sterile water was treated as the control, which were inoculated on the right sides of leaves. All plants were covered with clear polyethylene bags to keep moisture. And inoculated detached leaves were incubated in a greenhouse (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, typical black spots were found on the left sides of all inoculated leaves and the right sides did not have any leaf spot symptoms (Figure S1G-H). After 25 days of inoculation, perforation occurred at the black spots on the leaves of the inoculated plants, resulting in incomplete leaf (Figure S1I), which is identical disease symptoms to those observed in garden. The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased spots of the inoculated leaves to complete Koch,s postulates. The pathogen has a very wide host range. For example, it has been reported to cause dieback and sooty canker on Ficus trees (Abo Rehab et al. 2014), infected trunk of sultana seedless (Tang et al. 2021) and castor bean (Akgul et al. 2015), root of Citrus (Al-Sadi et al. 2014), date palm, and Mango (Al-Sadi et al. 2013) and Cassia fistula (Deng et al. 2015). But, according to nt.ars-grin.gov, there are no other reports of L. theobromae on M. grandiflora in the world. So, this would be the first one. This study provides an important reference for the biology, epidemiology.