Lasiodiplodia pseudotheobromae causes stem canker of Chinese hackberry in China

Chinese hackberry (Celtis sinensis Pers.) is an adaptable species widely growing in southern China. The symptoms of canker on stems of seedlings were discovered mid-July 2017 in Shuyang, Jiangsu Province. The diseased portions of the stems were dark brown due to discoloured xylem. Some seedlings showed symptoms of wilting, leaf fall, twig dieback, and tissue discolouration. The outbreak period was concentrated in July and August, suggesting that the disease spread during summer months. Possible fungal causal agents were isolated from naturally infected canker tissue and discoloured xylem. The isolate from xylem tissue with a high frequency (> 50%) was named Ls7 type. Pathogenicity tests were carried out on 4-year-old seedlings. The symptoms of canker began to develop 20 days after inoculation with Ls7 isolate and by day 35, there were dark, enlarged longitudinal lesions. A phylogenetic tree of the isolate was developed using the internal transcribed spacer, elongation factor-1α (tef1-α), β-tubulin gene (TUB) and RNA polymerase II subunit primer genes (RPB2). Based on morphological features and phylogenetic information, the pathogen was identified as Lasiodiplodia pseudotheobromae. This is the first report of L. pseudotheobromae causing canker on Chinese hackberry stems in China.


Introduction
Chinese hackberry (Celtis sinensis Pers.) is an important and widely planted ornamental tree species in China, Japan and Korea (Lee et al. 2019) and is tolerant of dry and nutrientpoor soils. The tender leaf, roots and bark of hackberry can reduce swelling, relieve pain, clearing away heat and toxic materials. It is also used as industrial materials and various furniture. Its rhizome fibers are strong and used in paper making, making ropes, artificial cotton and artificial fiber. The species has significant value in medicine and industry (Cao 2017). However, with increased planting, Chinese hackberry diseases are increasing and becoming more and more serious (Cacciola 2000;Berbegal et al. 2012;Luongo et al. 2015). Common diseases of Chinese hackberry are powdery mildew, stem and root rot, twig blight and leaf spot.
The Botryosphaeriaceae is a fungal family containing pathogens that cause canker disease of trees (Pavlic 2004). Lasiodiplodia species are members of the Botryosphaeriaceae and are widely found in tropical and subtropical regions . Lasiodiplodia species causing disease of Chinese hackberry have not been found in China.
In the summer of 2017 in Shuyang, Jiangsu Province, a severe outbreak of Chinese hackberry disease appeared. The slightly sunken grey-black lesions enlarged longitudinally and the associated weakening of tree vigor affected the ornamental and commercial value of the species.
Abstract Chinese hackberry (Celtis sinensis Pers.) is an adaptable species widely growing in southern China. The symptoms of canker on stems of seedlings were discovered mid-July 2017 in Shuyang, Jiangsu Province. The diseased portions of the stems were dark brown due to discoloured xylem. Some seedlings showed symptoms of wilting, leaf fall, twig dieback, and tissue discolouration. The outbreak period was concentrated in July and August, suggesting that the disease spread during summer months. Possible fungal causal agents were isolated from naturally infected canker tissue and discoloured xylem. The isolate from xylem tissue with a high frequency (> 50%) was named Ls7 type. Pathogenicity tests were carried out on 4-yearold seedlings. The symptoms of canker began to develop 20 days after inoculation with Ls7 isolate and by day 35, there were dark, enlarged longitudinal lesions. A phylogenetic tree of the isolate was developed using the internal transcribed spacer, elongation factor-1α (tef1-α), β-tubulin gene (TUB) and RNA polymerase II subunit primer genes (RPB2). Based on morphological features and phylogenetic information, the pathogen was identified as Lasiodiplodia 1 3 The objectives of this study were to: (1) discover the epidemiology of canker occurring on Chinese hackberry; (2) prove the pathogenicity of the fungal pathogen using Koch's Postulates Test; and (3) identify the causal agent using morphological and molecular techniques.

Sampling and fungal isolations
In mid-July 2017, symptoms of stem canker were found in commercial nurseries located in Shuyang County, Suqian City, Jiangsu Province (118°36′48′′E; 34°15′25′′N). The entire infected 4-year-old seedlings were brought to the laboratory for isolation of the pathogen.
Two ends of the cankers with spots in the middle were cut to form a small segment of 50 mm, and treated under aseptic conditions. The samples were sterilized in 75% alcohol for 3 min, dried quickly under a flame and rinsed three times with aseptic water for 30 s. Aseptic filter paper was used to absorb the water on the surface. The tissues were cut into 4 × 4 mm pieces between symptomatic and healthy tissues and cultured on fresh potato dextrose agar (PDA) medium containing 100 μg mL −1 ampicillin (Dai et al. 2007;Correia et al. 2016).
Samples were cultured at 25 °C in a dark incubator. Colony and spore morphologies were recorded every 24 h using a Nikon DS-Ri1 camera, Stereo Discovery, V20, Zeiss. The length and width of conidia were measured and the means, standard deviations and 95% reliable intervals computed from 50 samples.

Pathogenicity tests
Sixty fungal samples were divided into different groups according to colony morphology and ITS (internal transcribed spacer) sequences. All the isolate groups were tested for pathogenicity on the seedling stems. Each group was cultured for 7 days at 25 °C in darkness. Ten healthy Chinese hackberry seedlings (from 'Yanguan chunxiao farm'; Jiaxing City, Zhejiang Province 30°27′21′′N, 120°32′40′′E) were inoculated with each type of fungal isolates, respectively. Each plant was stabbed by placing a 4-mm-diameter mycelium plug of PDA placed face-down on each puncture wound. An additional ten seedlings were inoculated with sterilized PDA agar blocks as controls. All inoculated seedlings were kept in a greenhouse at 25 °C under ambient conditions of 70-80% humidity, with 24 h light every day. The experiment was carried out during the summer of 2018.

DNA extraction and sequence amplification
Fungal isolates were cultured in PDA medium for 5 days at 25 °C. Genomic DNA was extracted from mature mycelia according to Zhang et al. (2008). The procedure was: (1) mycelium grown for 3-4 days was placed in 2 mL test tubes with 500 µL of 2 × CTAB (containing 1% β-mercaptoethanol), rapidly cooled for 30 s in liquid nitrogen, and then heated immediately to 65 °C for 30 s. This was repeated three times; (2) a 1/2 volume of glass beads was added to each test tube before placing on a vortex shaker for 4-5 min, followed by heating to 65 °C for 20 min and shaking once every 10 min. Equal amounts of phenol: chloroform: isoamyl alcohol (25:24:1, v:v:v) were added and the tubes centrifuged at 12,000/min for 10 min; (3) two volumes of pre-cooled absolute ethanol was added to the supernatant, mixed well and the tubes placed in stand still for 20-30 min at 20 °C followed by centrifuging for 10 min at 12,000 r/ min. The supernatant was poured out and 200 µL of 70% alcohol was added to wash the DNA pellet, which was then dried at room temperature. One hundred µL TE was added to completely dissolve the DNA; and, (4) 100 µL of RNase A (10 mg/mL) was attached to each tube and dried at 65 °C for 30 min.
Step 3 was repeated and 30 µL of ddH 2 O was added to obtain a solution of the DNA.
The PCR reaction procedure had the following steps: (1) initial denaturation at 95 °C for 5 min; (2) denaturation at 95 °C for 30 s, (3) annealing at 55 °C for 30 s and extending at 72 °C for 33 cycles; and (4) extending at 72 °C for 10 min. However, the annealing temperature of TUB and RPB2 was 52 °C (Alves et al. 2004).
DNA amplification and sequencing were carried out with a GStorm Gradient PCR (Mastercycler nexus GSX1) using a program according to Zhang et al. (2008). The PCR products were sequenced by GenScript Biotechnology in Nanjing, China. To avoid errors in sequencing, DNA strands were spliced using forward and reverse primers (Two-way measurement: Sequencing Complete Sequences from Two Directions).

Phylogenetic analyses
The resulting ITS, tef1-α, TUB and RPB2 gene sequences were blasted. Other sequences needed were obtained from the GenBank database of NCBI (https ://www.ncbi.nlm.nih. gov). These sequences were aligned using MEGA version 5.0. Ambiguously aligned regions were excluded and gaps were treated as missing data. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) approach (Tamura et al. 2011), which was validated using bootstrap analyses with 1000 repetitions (Saitou and Nei 1987). The phylogenetic tree was based on the concatenated sequences of ITS, tef1-α, TUB and RPB2.

Incidence of the disease and symptoms
In the summer of 2016, a small amount of Chinese hackberry canker disease occurred but did not attract attention in Suqian City. However, by mid-July 2017, the incidence of Chinese hackberry canker was high with symptoms of the disease in over 21% of the 6000 m 2 of plantations of the species. The canker disease reappeared again in late July and August as well as spread rapidly.
The initial sites of infection appeared as watery reddish brown spots (Fig. 1a), and the top young leaves on twigs were wrinkled and brittle due to dehydration (Fig. 1b). With the development of the disease, slightly concave canker spots extended and gradually turned black-brown (Fig. 1c).
Transverse sections of xylem tissue were also black-brown (Fig. 1d). Eventually, the twigs wilted.
In mid-June 2018, each of the seven types of fungi was inoculated to 4-year-old seedlings with 10 replicates. After 20 days, eight seedlings inoculated Ls7 isolate showed disease symptoms similar to those that appeared in the field, including local, spindle-shaped necrosis and slightly concave red-brown spots (approximately 25 mm × 5 mm) (Fig. 2b). Two seedlings inoculated with Ls7 isolate developed canker spots on days 23 and 27 after inoculation, respectively. After 35 days, the lesions on these seedlings spread longitudinally on the stem, gradually changed shape from round to fusiform and became slightly concave. The lesions were approximately 98 mm × 6 mm (Fig. 2c, d). These dark brown lesions contained pycnidia densely packed with conidia, and leaves on the twigs began to wilt (Fig. 2c). The xylem was also dark brown (Fig. 2e). Seedlings inoculated with sterile PDA showed no symptoms of canker (Fig. 2a) and resembled the Ls1-Ls6 isolates.
The fungus was recovered from the symptomatic inoculated tissues and the culture's characteristics and morphology were similar to the Ls7 isolate. Therefore, the Ls7 isolate was determined to be the pathogen, although the seedlings infected with Ls7 isolate did not die.

Morphological characteristics of pathogen
Colonies of the Ls7 isolate grew rapidly, and after 3 days of culturing at 25 °C, were grayish-white with uneven edges (Fig. 3a). The colonies turned dark gray after 5 days (Fig. 3b), at which stage aerial hyphae were thick and fluffy. Following 2 weeks of treatment under UV light, the colonies produced pycnidia (Fig. 3c, d). There were many short conidiophores on the inner walls and conidia on the conidiophores. The pycnidia were nearly spherical, approximately 220-240 µm diameter, and formed scattered, unilocular, dark-brown to black areas on the stem and host epidermis (Fig. 4a). Conidiogenous cells were hyaline, cylindrical and holoblastic (Fig. 4c). Conidiophores were inflated at their base with one diaphragm which reduced to conidiogenous cells (Fig. 4d, e). Conidia were oval, both ends round and  (Fig. 4f). These had one septum and became puce after maturity; melanin appeared in the conidia and the surface was striped (Fig. 4g, h). [A condium accompany with partial germ tube ( Fig. 4i)]. The sizes of conidia were 22.2-28.6 × 12.1-17.4 μm (95% confidence limits = 25.2 ± 2.6 × 14.4 ± 2.4) and the average conidia length-width ratio was approximately 1.7 (Table 1). The paraphyses were hyaline and upward cylindrical. (Fig. 4b).
In addition, the Ls7 isolate grew at 10 °C and produced dark pink colonies at 35 °C (Fig. 5). The morphological characteristics of the Ls7 isolate fit those of L. pseudotheobromae ) and therefore confirmed as L. pseudotheobromae causing Chinese hackberry canker.
Before the establishment of phylogenetic trees with four groups of gene links, two tree-building methods were used to sequence and analyze individual gene sequences. There were no obvious conflicts in a single gene phylogeny and the ITS, tef1-α, TUB and rpb2 datasets can be amalgamated. Sequences downloaded from NCBI (National Center for Biotechnology Information) were then used to carry out   (Dou et al. 2017). The Diplodia mutila isolate CMW7060 was selected as an out-group (Table 2).
A phylogenetic tree based on ITS, tef1-α, TUB and RPB2 sequences was constructed by cluster analysis of the gene sequences of the test pathogen and other Lasiodiplodia species complex strains (number of sites = 410, bootstrap replicates = 1000). The results show that isolates of Lasiodiplodia sp. were clearly divided into several clades or groups with a common ancestor representing different species within Lasiodiplodia. The test isolate showed high homology with L. pseudotheobromae (Fig. 6). Therefore, the isolate was confirmed to be L. pseudotheobromae on the grounds of molecular identification. f Hyaline, aseptic conidia. f-h Septate, melanin deposits on the inner surface of the wall arranged longitudinally giving a striate appearance to the conidia. i A condium with partial germ tube (Bars = 10 µm)

Discussion
Based on available knowledge, this is the first report of Chinese hackberry stem canker disease caused by L. pseudotheobromae in China. The twigs and stems of all infected seedlings became cankerous and turned black, which was devastating to Chinese hackberry plantations. Lasiodiplodia pseudotheobromae is important in agriculture and forestry as it causes cankers, stem-end rot, dieback and fruit rot (Sakalidis et al. 2011;Ismail et al. 2012;Marques et al. 2013). These diseases lead to bark discolouration, browning of medullary tissue, a decline in growth, and drying of leaves on upper branches. In severe cases, they may even lead to plant death (Alves et al. 2004), resulting in considerable economic losses. The pathogen overwinters in the soil, and in the summer of the following year, conidia and conidiomata are spread by wind and rain (Yee et al. 2019).
Worldwide, this species has a wide range. L. pseudotheobromae is an aggressive species in Australia, Cameroon, Egypt, Brazil, Mexico, China and other countries (Begoude et al. 2010;Sakalidis et al. 2011;Ismail et al. 2012;Maricarmen et al. 2013;Coutinho et al. 2016). In China, it is mainly distributed in central and eastern portions of the country, including Beijing, Jiangsu, Hubei, Sichuan, and Shandong .
This study focused on a Chinese hackberry canker disease outbreak between July and August. The maximum temperature tolerated by L. pseudotheobromae was 54 °C this resistance to high temperatures enables it to survive during the summer months. The disease spreads rapidly and is most severe during summers when temperatures reaches 30 °C and above (Zhang 2012). In other seasons the disease is less aggressive (Wu et al. 2018).
Conidia exist all year-round but are rarely detected in winter (Xu et al. 2014). The pathogen overwinters in the diseased twigs or soil and becomes the source of infection the following year, with spores travelling by means of wind, rain and insects (Wu et al. 2001). The pycnidia or fruiting body of the fungus is produced near the canker before spreading to neighboring hosts. Pathogens commonly enter the plant through wounds left by pruning of twigs and this may be the main reason for the spread of the disease. The presence of the disease cannot be ignored as it leads to significant economic losses in forestry and agriculture (Wang 2016). Therefore, control measures should be carried out in the early stage of discovery of the disease.
In the genus Lasiodiplodia, L. pseudotheobromae is phylogenetically close to L. theobromae ). Although they are closely related, the size and shape of L. pseudotheobromae conidia differ from those of L. theobromae which are larger and have orbicular ends . In addition, L. pseudotheobromae can also grow at 10 °C and form deep-pink colonies at 35 °C but L. theobromae does not (Marques et al. 2013;Netto et al. 2014). The fungal samples categorized as Ls7 type in this study showed similar features with L. pseudotheobromae and was consistent with these characteristics.
Based on the phylogenetic relationship of β-tubulin, Cheng (2012) concluded that the sexual stage of L. pseudotheobromae may be Botryosphaeria rhodina. In 2016, Tennakoon et al. (2016) found the sexual morph of L. pseudotheobromae collected from dead leaves of Plukenetia volubilis L. (Euphorbiaceae) in Yunnan Province, China. Using phylogenetic analyses based on ITS and tef1-α sequence data, they authenticated the sexual-asexual connection in L. pseudotheobromae. It was the first evidence in molecular genetics of a sexual morph for this species. However, the sexual morph of the Ls7 isolate was not found in this study.

Conclusions
This is the first report of hackberry canker in China and Chinese hackberry (C. sinensis) is a new host of L. pseudotheobromae. The occurrence of the disease should be closely monitored and preventative measures put in place to avoid the spread of the disease. This study may provide reference value for the prevention of Chinese hackberry canker.
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